Copper Bioinorganic Chemistry From Health To Bioinspired Catalysis (Simaan J.A., Réglier M. (Ed.) )

Copper Bioinorganic
Chemistry
From Health to Bioinspired Catalysis
Copper Bioinorganic
Chemistry
Editors
A Jalila Simaan • Marius Réglier
Centre Nationale de la Recherche Scientifique, France &
Aix Marseille Université, France
World Scientific
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Preface
Bioinorganic chemistry is a booming field of research that has its source

at the frontiers of chemistry, biochemistry, molecular biology, medicinal
chemistry, all disciplines that are in perpetual mutual enrichment. While
in the late 1970s bioinorganic chemistry focused mainly on metals such as
iron (hemoproteins, iron/sulfur proteins …) or zinc (aminopeptidases …),
over the years it has gradually developed towards other metals including
copper. One reason for the initial low interest in copper biochemistry was
the few known copper-containing biological systems. In the early 1980s,
most studies on copper proteins concerned those on electron transfer pro-
teins (azurin, plastocyanin, …). Another reason was probably due the fact
that within the two oxidation states of copper, i.e. Cu(I) and Cu(II)
involved in biological systems, only the Cu(II) oxidation state was acces-
sible by spectroscopic methods commonly used in these years (UV-vis.
and EPR) while Cu(I) oxidation state remained invisible. Over the past
few decades, advances in chemistry, spectroscopic methods and systems
biology have enabled several discoveries about the role of copper in
human pathologies (e.g. Menkes, Wilson and Alzheimer diseases) as well
as on the molecular functioning of copper-containing systems. This was
accompanied by growing discovery of copper-containing biological sys-
tems, shedding a new light on this metal ion.
Copper is an essential micronutrient for most living beings including
bacteria. This is mainly due to essential roles of catalytic Cu-centers in
enzymes. However, an excess of Cu is toxic and it has thereby been used
over centuries as a powerful antimicrobial agent. Bacteria developed sev-
eral systems to detect Cu and protect themselves from high intracellular
Cu concentration, namely via Cu-extrusion and/or Cu- storage. Chapter 1
entitled “Ligands as a tool to tune the toxicity of Cu on bacteria: from
v
vi Preface
boosting to silencing” by Peter Faller, Marianne Ilbert et al. addresses this

topic focusing on Cu-detoxification systems and progress in understand-
ing the molecular mechanisms of Cu-toxicity for bacteria.
Tyrosinase is an iconic quasi-ubiquitous mono-oxygenase/oxidase
studied since the early 1980’s. Tyrosinase is involved in the biosynthetic
pathway of melanin pigments that is associated with cutaneous pigment
disorders essentially linked to (i) the hypopigmentation of the skin such as
in albinism and vitiligo or (ii) the hyperpigmentation of the skin such as in
melanoma, melasma and solar lentigo. In addition, tyrosinase is overex-
pressed during tumorigenesis and has been demonstrated to be a sensitive
marker for melanoma. Since the reduction/suppression of melanogenesis
is supposed to restore the sensitivity of cancer cells to immuno-, radio- or
chemotherapies, the use of selective inhibitors of tyrosinase and related
proteins as adjuvants represents a realistic strategy for melanoma therapy.
This aspect of the tyrosinase function is addressed by Catherine Belle,
Marius Réglier et al. in Chapter 2 entitled: “Transition State Analogue
Molecules as Mechanistic Tools and Inhibitors for Tyrosinase.”
The understanding of tyrosinase functioning is intimately linked to
the chemistry of biomimetic models. Indeed, it is thanks to the pioneering
work of Kitajima,1 Solomon,2 Karlin3 and Tolman4 on the dioxygen
chemistry of binuclear copper complexes that we precisely understand
Tyrosinase’s mechanism of action. Two complementary chapters illustrate
this aspect of the chemistry of tyrosinase biomimetic models. Chapter 3
by Rabindranath Mukherjee et al. is entitled: “Modeling Tyrosinase
Activity Using m-Xylyl-based Ligands. Ring Hydroxylation, Reactivity
and Theoretical Investigation” and Chapter 4 by Felix Tuczek et al. is
entitled: “Monooxygenation of phenols by small-molecule models of
tyrosinase: Correlations between structure and catalytic activity”. Both
chapters report on discoveries on bioinspired catalysts that have contrib-
uted to a better understanding of tyrosinase mechanism, in particular
thanks to reactivity studies, intermediate trapping, spectroscopic methods,
and theoretical calculations. Achievements in the development of catalytic
bioinspired systems for phenol or catechol oxidation are also described.
The past two decades have seen the emergence of two new copper-
containing monooxygenases having a peculiar interest in sustainable
Preface vii
chemistry, i.e. the mysterious particulate methane mono-oxygenase

(pMMO)5 and the Lytic Polysaccharide monooxygenase (LPMO). While
pMMO which converts methane into methanol is far from having revealed
all its secrets, LPMOs are well characterized mononuclear copper-con-
taining enzyme including their uses in biotechnology for the development
of biofuel production and biosourced chemicals. In an open debate on
LPMO active copper/oxygen species, the synthesis of small copper mim-
ics reproducing LPMO structure and activity is in full development and
the subject of several publications in the literature. Chapter 5 by Ivan
Castillo entitled “Inorganic Models of Lytic Polysaccharide
Monooxygenases” addresses this topic.
Studies of copper enzymes mechanism are closely linked to the iden-
tification of reactive species.6 The development of spectroscopic methods
coupled with electrochemistry, including at low temperature, is of high
importance for the study of the redox properties of transient copper-oxy-
gen species relevant for intermediates in biological processes. These tech-
niques allow accessing important thermodynamical parameters. Chapter 6
from Nicolas Le Poul on “Electrochemistry and spectroelectrochemistry
of copper-oxygen adducts” illustrates recent achievements in the field.
Finally, inspired by copper enzymes, chemists have developed peptide
mimics to bind metal ion while adopting more complex three-dimensional
folds. In particular, peptoids — N-substituted glycine oligomers- are
bioinspired biopolymers capable of folding into well-defined three-
dimensional structures in solution. Chapter 7 entitled “Structure and
Function of Cu-Peptoid Complexes” and written by Galia Maayan et al.
describes the emergence of peptoids as multifaceted ligands for copper
and the use of such peptoid-Cu complexes for bioinspired catalysis.
This volume aims to provide interested readers with a selected sum-
mary of advances in bioinorganic copper chemistry. We hope that the
selection of topics will inspire researchers who wish to undertake research
in the bioinorganic field of copper.
A Jalila Simaan
Marius Réglier
July 2023
viii Preface
References
1. Kitajima, N., Fujisawa, K., Morooka, Y., Toriumi, K. m-η2:η2-Peroxo binuclear copper
complex, [Cu(HB(3,5-(Me2CH)2pz)3)]2(O2). J. Am. Chem. Soc. 111, 8975–8976
(1989).
2. Solomon, E. I., Sundaram, U. M., Machonkin, T. E. Multicopper Oxidases and
Oxygenases. Chem. Rev. 96, 2563–2606 (1996).
3. Karlin, K. D., Kaderli, S., Zuberbühler, A. D. Kinetics and Thermodynamics of
Copper(I)/Dioxygen Interaction. Acc. Chem. Res. 30, 139–147 (1997).
4. Halfen, J. A. et al. Reversible cleavage and formation of the dioxygen O-O bond within
a dicopper complex. Science 271, 1397–1400 (1996).
5. Koo, C. W., Tucci, F. J., He, Y., Rosenzweig, A. C. Recovery of particulate methane
monooxygenase structure and activity in a lipid bilayer. Science 375, 1287–1291
(2022).
6. Mirica, L. M., Ottenwaelder, X., Stack, T. D. P. Structure and spectroscopy of copper-
dioxygen complexes. Chem. Rev. 104, 1013–1045 (2004).
Contents
Preface v
1 Ligands as a Tool to Tune the Toxicity of Cu on Bacteria:

from Boosting to Silencing 1
Lisa Zuily, Nora Lahrach, Enrico Falcone,
Merwan Bouraguba, Vincent Lebrun, Elisabeth Lojou,
Marie-Thérèse Giudici-Orticoni, Peter Faller,
and Marianne Ilbert
I. Introduction 2
II. Copper, a Biocidal Compound 3
III. Copper Homeostasis Systems in Bacteria 5
IV. Copper Toxicity: A Phenomenon Dependent on
the Bioavailability of Copper and on Bacteria 7
V. Specificity of Copper Chemistry 8
A. Cu Coordination Chemistry 8
B. Cu Reactivity: The Case of ROS Production 9
C. In Vivo Complexation of Cu 10
VI. Mechanisms of Cu Toxicity: Macromolecules Targeted by
Copper10
A. Membranes 11
B. DNA 12
C. Proteins 13
VII. Chemistry of Copper Complexes 14
A. Reactivity of Cu Complexes 15
B. Stability of the Cu-L Complex 16
ix
x Contents
C. Kinetics of the Cu-L Complex 17

D. Redox Potential 17
VIII. Impact of Ligands on the Biological Activity of Copper 17
A. Localization 18
B. Presence in Biology of Potentially Competing
Ligands for Cu(II) 18
C. Presence in Biology of Potentially Competing
Ligands for Cu(I) 19
D. Reactivity of Cu-L in Bacteria 20
E. Reactivity of the Free Ligand in Bacteria 21
IX. Copper Complexes as Antimicrobial Agents, a
Non-Exhaustive List 24
A. Dithiocarbamates Compounds 24
B. 8-Hydroxychinoline (8-HQ) Compounds 25
C. Phenanthrolines 27
D. Pyrithione 29
E. Bis-Thiosemicarbazones (atsm, gtsm) 30
X. Peptides-Based Cu-Chelators 33
XI. Recent Advances Toward the Future Use of Copper in
Medicine36
XII. References 38
2 T
ransition State Analogue Molecules as Mechanistic
Tools and Inhibitors for Tyrosinase 45
Clarisse Faure, Amaury du Moulinet d’Hardemare,
Hélène Jamet, Catherine Belle, Elisabetta Bergantino,
Luigi Bubacco, Maurizio Benfatto, A. Jalila Simaan,
and Marius Réglier
List of Abbreviations 46
I. Introduction 47
II. Biological Functions of Melanins 47
A. Melanin as Color Pigment 48
B. Melanin as a Defensive Barrier 50
Contents xi
III. Tyrosinase 51
A. Structures of the Tyrosinase Active Sites 51
B. Catalytic Mechanism of Tyrosinase 53
C. Tyrosinase-Related Proteins TRP1 and TRP2 55
IV. Dysfunction in Tyrosinase Activity 58
A. Pathologies Linked to TYR Dysfunction 58
B. Skin Whitening 59
V. Tyrosinase Inhibition 59
A. Variation in TYR Sources 60
B. Transition-State Analogue (TSA) Inhibitors 60
1. L-Mimosine 63
2. Kojic acid and derivatives 63
3. Tropolone and derivatives 67
4. HOPNO inhibitor 68
5. HOPNO derivatives 73
VI. Conclusions 74
Acknowledgment76
VII. References 76
odeling Tyrosinase Activity Using m-Xylyl-Based

3 M
Ligands: Ring Hydroxylation, Reactivity, and
Theoretical Investigation 81
Puneet Gupta and Rabindranath Mukherjee
I. Introduction 81
A. General Considerations 81
B. Scope of the Review 83
II. Dicopper Proteins — Brief Overview 84
A. Hemocyanins 85
B. Tyrosinases 85
C. Catechol Oxidase 86
III. Three Cu2O2 Core Structures 87
IV. Biomimetic Studies on Tyrosinase 88
A. Intramolecular m-Xylyl and Aromatic Ring
Hydroxylation88
xii Contents
B. Intramolecular m-Xylyl and Aromatic Ring

Hydroxylation89
C. Ring Hydroxylation Reactions with Non-m-Xylyl-Based
Ligands95
D. Oxidation of External Substrates by Dicopper Systems 95
V. Theoretical Studies on Tyrosinase Models 102
A. Frontier Molecular Orbitals of the Cu2O2 Cores 102
1. μ-ηη2:ηη2-Peroxo-dicopper(II); {CuII2(μμ-ηη2:ηη2 O2)}2+
{Cu2PS}102
2. Trans-μμ-1,2-peroxo-dicopper(II):
{CuII2(μμ-ηη1:ηη1-O2)}2+ {Cu2PE}103
3. Bis(μμ-oxido)dicopper(III): {CuIII2(μμ-O)2}2+ {Cu2O2}104
B. Interconversion Between {Cu2PS} and {Cu2O2} Cores:
A Torture Track for Computations 106
S E
C. {Cu2P }, {Cu2P }, and {Cu2O2} Motifs in C−H
Oxidation: DFT Studies 107
S
1. Aromatic C−H hydroxylation via {Cu2P } motifs 108
2. Aromatic C−H hydroxylation via {Cu2O2} 111
E
3. Aromatic C−H hydroxylation via {Cu2P }116
VI. Conclusions 117
VII. References 118
4 M
onooxygenation of Phenols by Small-molecule Models
of Tyrosinase: Correlations Between Structure and
Catalytic Activity 123
Alexander Koch, Tobias A. Engesser, Ramona Jurgeleit,
and Felix Tuczek
I. Introduction 123

II. Model Systems of Tyrosinase 124
A. Model Systems Performing Ligand Hydroxylation 125
B. Model Systems Exhibiting Reactivity Toward External
Substrates126
III. Catalytic Tyrosinase Models with Bidentate Ligands 129
A. Mechanistic Cycle of the Tyrosinase-like
Monooxygenation of External Substrates 129
Contents xiii
B. Substitution of Pyridine in the Lpy1 Ligand by

N-Heterocycles132
C. Substitution of Imine in the Lpy1 Ligand by
N-heterocycles: Symmetric Bidentate Ligands 134
D. Hybrid Ligands Composed of Different N-Heterocycles138
E. Steric Influence of Substituents on the Supporting
Ligands142
F. Variation of Substrates: Electronic and Steric Factors 143
IV. Summary 148
V. Acknowledgment 148
VI. References 149
5 E
lectrochemistry and Spectroelectrochemistry of
Copper-Oxygen-Relevant Species 153
Nicolas Le Poul
I. Introduction 153

II. Electrochemistry of Peroxide and Superoxide
Copper–Oxygen Species 156
III. Electrochemistry of Copper(II) Hydroxo, Alkoxo,
Carboxylato, and Oxo Precusors 162
IV. Electrochemistry of Copper Hydroperoxide and
Alkoxoperoxide Species 168
V. Electrochemistry of Copper(II)-Phenoxide Species 172
VI. Electrochemistry of Nitroso and Sulfide Analogues of
Copper–Oxygen Complexes 177
VII. Summary 181
VIII. References 182
6 I norganic Models of Lytic Polysaccharide

Monooxygenases187
Ivan Castillo
I. Introduction 187

II. Lytic Polysaccharide Monooxygenases 188
III. Copper Complexes as Hydrolase Mimics 190
xiv Contents
A. Polysaccharide Hydrolysis 190

B. Copper Complexes 190
IV. Copper Complexes as LPMO Mimics 192
A. Copper(II)/Bis(Benzimidazolyl)Amine System 192
B. Copper(III)/Bis(Carboxamido)Pyridine System 196
C. Copper(II)/Copper(I)/(Pyridyl,Imidazolyl)Amine
System199
D. Copper(II)/(Pyridyl)Diazepane Complexes 200
E. Copper(II)/Bis(Imidazolyl)Amine System 203
F. Copper(II)/Bis(Picolyl)Amine System 205
V. Concluding Remarks 206
VI. References 207
7 Structure and Function of Cu–Peptoid Complexes 211

Anastasia E. Behar, Pritam Ghosh, and Galia Maayan,
I. Introduction 212

A. Peptoid Synthesis 213
B. Secondary Structure of Peptoid Oligomers 214
II. Structural Aspects of Cu(II)–Peptoid Complexes 216
A. Structural Design of Cu(II)–Peptoid Complexes 216
B. Choice of the Metal-Binding Ligands 217
C. Characterization of Cu(II)–Peptoid Complexes 219
1. Evaluation of Cu(II) binding and synthesis of
Cu(II)–peptoid complexes 219
2. Determining the association constants and evaluating
the selectivity of the peptoid chelators to Cu(II) 221
3. Structure and coordination sphere of Cu centers
within Cu(II)–peptoid complexes 222
III. Effect of Cu(II) Binding on the Structure of Peptoid 223
A. Cu(II) Binding as a New Approach for Peptoid Folding 223
B. Cu(II)-Mediated Peptoids Self-Assembly 229
xv

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https://doi.org/10.1142/9789811269493_0001
1 Ligands as a Tool to Tune the

Toxicity of Cu on Bacteria:
from Boosting to Silencing
Lisa Zuily,* Nora Lahrach,* Enrico Falcone,†

Merwan Bouraguba,† Vincent Lebrun,† Elisabeth Lojou,*
Marie-Thérèse Giudici-Orticoni,* Peter Faller,†,‡ and
Marianne Ilbert*,‡
*Aix-Marseille University, CNRS, BIP, UMR 7281, 31 Chemin

Aiguier, 13009 Marseille, France
†
Biometals and Biological Chemistry, Institut de Chimie (CNRS
UMR7177), Université de Strasbourg, 4 rue B. Pascal, 67081
Strasbourg, France
‡
[email protected]
‡
[email protected]
Abstract
Copper is an essential micronutrient for most living beings including
bacteria. This is mainly due to essential roles of catalytic Cu-centers in
enzymes. However, an excess of Cu is toxic and thereby used over cen-
turies as a powerful antimicrobial agent. Bacteria developed several
systems to detect Cu and protect themselves from high intracellular
Cu concentration, namely via Cu-extrusion and/or Cu-storage. Several
Cu-detoxification systems have been described and new ones are still
1
2 Zuily et al.
being discovered. Progress has also been made in understanding the

molecular mechanism of Cu toxicity by identifying target macromole-
cules. Nevertheless, the importance of each mechanism is bacteria- and
environment-dependent paving the way for new findings.
The chemical reactivity of Cu can be modulated by its coordination
environment in a complex with a ligand (L) and this opens large oppor-
tunities to tune the Cu-ligand complexes (Cu-L) via ligand design. For
instance, boosting the toxicity of Cu ions toward bacteria was promoted
by several small organic ligands. These ligands can make Cu-L com-
plexes with different properties in terms of coordinating atoms (S, N,
and O), Cu to L stoichiometry, ligand denticity, thermodynamic stability,
kinetic inertia, and redox potential. However, one common feature
shared by Cu-L complexes that efficiently boost Cu toxicity is the ability
of Cu-L to cross the two bacterial membranes and to release the Cu in
the cytosol. If this property is required to improve bacteria killing is not
clear yet, but considering the ever-growing resistance of bacteria, it
would be interesting in the future to try to boost Cu toxicity via unprec-
edented mechanisms.
I. Introduction
Before the Great Oxygenation Event, copper (Cu) was found as copper
sulfide minerals such as chalcocite (Cu2S) and chalcopyrite (CuFeS2),
impairing its bioavailability. In this anoxic environment, earliest anaerobic
prokaryotes were not using copper as a metal center for their enzymes to
operate.1 Apparition of oxygen on earth led to the oxidation of copper,
increasing its solubility, hence turning Cu into a bioavailable metal.2,3
Living organisms had to adapt to this drastic modification of their environ-
ment and they subsequently modify their metallome. For instance, in cur-
rent aerobic organisms, key enzymatic reactions like the reduction of
dioxygen into water are performed with copper-containing proteins.4
However, such benefits came with its part of inconveniency. Indeed,
while being a key element, Cu-chemical properties render this metal
highly toxic for living organisms. Living cells had to face this paradox of
an essential yet toxic element. General strategies have been developed by
bacteria to remove any excess of copper. Nonetheless, saturation of these
Ligands as a Tool to Tune the Toxicity of Cu on Bacteria 3
systems induces cell death. Such powerful antimicrobial activity enables

beneficial use of Cu in several applications. A promising strategy is the
development of synthetic copper complexes (Cu-ligands, referred herein
as Cu-L) in which the ligand can drastically modulate the properties of
copper ions and their mode of action. In this chapter, we will mainly
review research exploring the impact of copper and copper complexes on
bacteria, and their mode of action. Cu-binding peptides are also attractive
options as antimicrobial agents that we will further discuss. Overall, accu-
mulation of recent studies in this field opens new avenue toward potential
use of Cu-based compounds in medicine as antibacterial agents.
II. Copper, a Biocidal Compound

The potential use of Cu-related compounds as antimicrobial agents is not
a recent idea. Indeed, four thousand years ago, Egyptians already used Cu
to sterilize water. Other historical documents revealed that during antiq-
uity, Cu was also prescribed to treat some diseases. A more detailed over-
view of historical facts revealing utilization of Cu as biocidal compound
can be found in other reports.5–7
Nowadays, Cu is still worldly employed for its biocidal effect to pre-
vent microorganisms spread: materials containing copper are used in
wound dressing, filters, hygienic medical devices, and many others
(Figure 1).8
Potable water
Wound
dressings Filters
Agriculture Clothes
Paints Intra-body
compounds
(contraception,
Hospitals
dentistry)
surfaces
Figure 1. Current applications of copper in our society due to its antimicrobial

properties.
4 Zuily et al.
As often, the use of the antimicrobial property of copper is not an

original human invention, as natural copper-based defense systems were
evolved to struggle against invading intracellular organisms. It is now
clearly established that increased amount of copper (up to 400 µM) were
measured in phagolysosomes after bacteria engulfment.9–11 In contrast to
other metals like iron or manganese that are withhold, the immune system
induces production and stimulation of copper pumps (Ctr1 and ATP7A)
leading to a copper burst to destroy pathogens (Figure 2).11,12 Silencing of
the gene encoding for ATP7A clearly attenuated bactericidal activity of
macrophages.13 Accordingly, the deletion of copA gene (a bacterial gene
encoding a pump to export any intracellular copper excess, see section
“Copper Homeostasis Systems in Bacteria”) renders Escherichia coli
hypersensitive to macrophage copper-dependent killing.13
Cu(I)
CTR1
ATOX1
NAD+ NADH
ATP7A
Macrophage
Cu(II) Cu(I)
O2 O2
Phagolysosome
H2O2 OH
CueO
Figure 2. Cu-dependent strategies of the immune system to kill invading microbes.

After pathogenic bacteria engulfment, expression of the gene encoding for Ctr1
pump is stimulated, leading to Cu accumulation in the phagolysosome via the met-
allochaperone Atox1 and the ATPase ATP7A. Pathogenic bacteria (in yellow) will
protect themselves from this increase in Cu content by overexpressing specific sys-
tems that pump out, transform, or store Cu metals. In this scheme, the CopA pump
and the multicopper oxidase CueO are represented as examples.
III. Copper Homeostasis Systems in Bacteria

Copper traffic in cells is tightly controlled because on one hand it is essen-
tial for the function of key enzymes but on the other hand it will severely
impact cell viability in case of intracellular excess. Cu trafficking occurs
mainly in the periplasmic/membrane environment of Gram-negative bac-
teria where most copper enzymes (superoxide dismutase, cytochrome
oxidase, and multicopper oxidase) are localized. How the Cu enters the
cells is still unknown as no specific copper transporter has been identified
so far. Passive diffusion via the outer membrane is suggested but needs to
be further confirmed.14 In some bacteria, the metallophores yersiniabactin,
methonobactin or staphylopin are secreted to the extracellular environment
where it can bind metals, like copper.15,16 Such metallophores could play
an active role in specific strains to carry metals to the periplasmic compart-
ment.17 Periplasmic proteins called metallochaperones will then coordi-
nate any periplasmic Cu ions with the dual goal first to carry Cu to specific
enzymes that require copper for their function, and second to prevent
unwanted Cu reactions with other biomolecules (BMs).18,19 CusF is, for
example, a well-known periplasmic metallochaperone that limits the
amount of free Cu and releases it to pumps for Cu export outside the
cells.20
Any excess of Cu in the environment may lead to an increase of intra-
cellular Cu. To prevent Cu-accumulation and maintain copper homeosta-
sis, specific Cu pumps are overproduced such as CopA or the CusABC
system that export Cu out of the different compartments (Figure 3).18,20
The expression of Cu-specific defense mechanisms is tightly controlled
via two regulatory systems, one detecting Cu concentration in the peri-
plasmic environment (CusS/CusR two-component systems in E. coli,
Figure 3), the other detecting an increase in free copper in the cytoplasm
(CueR in E. coli, Figure 3).21,22 Other defense mechanisms exist such as
the periplasmic enzyme CueO which oxidizes Cu(I) into Cu(II) to prevent
the production of toxic reactive oxygen species (ROS) (see section « Cu
Reactivity: The Case of ROS Production” for Cu reactivity).23 Cu-storage
proteins have also been discovered. The protein Csp binds up to 80 Cu
atoms per tetramer and can be found in the periplasmic space and in the
6 Zuily et al.
Outer membrane
CusC
CueO Cu(I) CusF
CusB
CusB
Cu(II)
Periplasm
CusA
CopA CusS
CopA
Inner membrane
P Cytoplasm
ATP ADP+Pi
CusR
CopZ
CueR
P
CusR
CueR CueR
cueO copA(Z) cusS cusR cusC cusF cusB cusA
Figure 3. Copper homeostasis systems in E. coli. Increase of copper in the periplas-

mic compartment activates the CusS/CusR two-component systems and leads to the
induction of the CusR regulon. CusABC system exports periplasmic copper in
the extracellular environment. CusF metallochaperone carries any copper excess to
the Cus system to further remove any intracellular copper. Any increase in cytoplasmic
intracellular Cu content will activate CueR regulator leading to the overexpression of
copA(Z) encoding for the inner membrane pump CopA and the cytoplasmic metal-
lochaperone CopZ which bind cytoplasmic copper and exports it to the periplasmic
compartment via CopA. CueR also induces the expression of cueO gene encoding for
a periplasmic enzyme, CueO, which catalyzes the oxidation of Cu(I) into Cu(II).
cytoplasmic compartment of some bacteria.24,25 In addition, non-protein

molecules play a central role to maintain copper homeostasis. The cyto-
plasmic tripeptide glutathione (GSH) not only maintains a reducing envi-
ronment but also interacts with copper in excess and confers additional
copper tolerance.26 All these systems found in E. coli (Figure 3) are a
non-exhaustive list of proteins known to be involved in copper homeosta-
sis in bacteria. Other specialized systems are indeed found in other organ-
isms. For example, in Rubrivivax gelatinosus, copI is a gene encoding a
protein belonging to the cupredoxin family, usually known to be involved
in electron transfer.27 However, in this case, copI deletion leads to a strong
sensitivity of the strain to copper.28 The protein CopG recently found in
several organisms including E. coli and Pseudomonas aeruginosa is
involved in the cellular protection toward Cu stress.29 Even in some

strains of E. coli, a genomic island encoding for the pco systems (with an
additional multicopper oxidase (PcoA), other periplasmic chaperones
(PcoC and PcoE) and a two-component copper sensor (PcoSR)) signifi-
cantly increases the bacterial resistance to copper.18,30
IV. Copper Toxicity: A Phenomenon Dependent on

the Bioavailability of Copper and on Bacteria
Copper can be encountered in different habitats as a trace element.
However, excess of this metal can be found in contaminated environments
after mining process or extensive agriculture treatments. In such cases,
environmental bacteria are directly impacted.31 Even though high level of
copper can be measured in such environment, its toxicity will be related
to its bioavailability, which is governed by kinetics and thermodynamics.
Cu can bind to substances present in a medium or in the soil. Such com-
plexation of the metal renders difficult to estimate the toxicity of an envi-
ronment based on metal quantification. In soil, for example, the total
amount of Cu measured by conventional methods such as inductively
coupled plasma–mass spectrometry (ICP-MS) or inductively coupled
plasma–optical spectroscopy (ICP-OES) will not provide clear informa-
tion concerning the amount of bioavailable copper in the sample tested.
Pathogenic bacteria may also encounter high concentrations of copper
upon infection of a host. Recent studies on the Bordetella pertussis strain
have shown that specific copper homeostasis systems have been con-
served to withstand copper excess and peroxide stress found in the phago-
lysosomes.32 Depending on the complexation properties of the available
compounds in its environment, several Cu-derived species may form, with
different antimicrobial activities. This may be observed in a simple exam-
ple: to kill E. coli cells, mM concentrations of CuCl2 or CuSO4 are
required in Luria Broth medium while only µM are sufficient in a minimal
medium (mainly composed of phosphate buffer). Therefore, the most
toxic environments correspond to the highest concentration of bioavaila-
ble copper which may be quite different from the highest amount of total
amount of copper.
In addition, different bacteria species do not survive the same concen-
tration of bioavailable copper. Increasing number of experimental evidences
8 Zuily et al.
highlight distinct strategies explaining such results. As an example, acido-

philic organisms used in bioleaching process can resist copper concentra-
tion higher than 100 mM CuSO4;33,34 in comparison, E. coli survives 3–5
mM of CuSO4 in aerobic conditions and in Luria Broth medium.35 The
origin of such resistance has been explained by the duplication of known
Cu-resistance genes, new copper-chaperones, surface proteins acting as
repellent, and an abundant reserve of inorganic polyphosphate (polyP).34,36
V. Specificity of Copper Chemistry

Even though most of the essential d-block metal ions are in the first row
(4th period), Cu differs from other metal ions. According to Irving–
Williams series, complexes of Cu(II) have the highest stability compared
to the other divalent metal ions. Another characteristic is the occurrence
of monovalent state Cu(I) (much less common for other biological essen-
tial metals like Fe, Mn, etc.), which implies a very high thiophilicity. In
biological systems, Cu ions are mainly found as Cu(I) (Cu+, or reduced
cuprous state) or Cu(II) (Cu2+ or oxidized cupric state). Here, we will
rapidly introduce Cu coordination in pure water, coordination geometries
found in cells at neutral pH, and then Cu reactivity in aerobic conditions.
A. Cu Coordination Chemistry
Cu(I) and Cu(II) are cations soluble in polar solvents. In water, Cu(II) is
the main form encountered, and is coordinated with six water molecules.
At higher pH, copper hydroxide Cu(OH)2 is formed, with a very low solu-
bility in water. Cu(I) is not stable in pure water: either it is readily oxi-
dized to Cu(II) by dioxygen in aerobic conditions, or it disproportionates
into Cu(II) and Cu(0) under anaerobic conditions. Hence, to exist as Cu(I)
in aqueous solvent, it needs to be stabilized by appropriate ligands.
Interestingly, Cu(I) and Cu(II) prefer different coordination geometries, as
referred in Table 1, which means that each of these two forms can be sta-
bilized by different sets of ligands.37,38 Worthy of note, Cu(I) is a soft
cation (the softest of the essential metal ions) and hence is very thiophilic.
In addition, due to the difference in coordination chemistry of Cu(II) and
Cu(I), the redox potential can be tuned over a wide range.39,40
Table 1. Key coordination properties of Cu(I) and Cu(II) to understand their behav-
ior in biological environment.
Cu(I) Cu(II)
Preferred geometry Digonal, trigonal, tetrahedral, Square planar, with one or
or penta-coordinated two weaker axial ligands
Preferred ligands Thiolates > thioether, amines > Amines, sulfur > oxygen
oxygen
Stability in aerobic Easily oxidized Stable
condition
Stability in water Not stable Stable
(pH 7) disproportionation
Solubility in water KsCuOH = 1.2 10–6(a) (KsCu(OH)2 = 2.2 × 10–20)b
Interaction with Can bind strongly to thiols Is reduced to Cu(I) by
thiols thiols
a
K s CuOH =
[Cu+ ] × [OH − ]
[CuOH]
[Cu2+ ] × [OH − ]
2
b
Ks Cu ( OH )2
=
[Cu ( OH )2 ]
B. Cu Reactivity: The Case of ROS Production

Cu, like Fe, is a metal ion catalyzing the Fenton reaction. Cu(I) will cata-
lyze the formation of highly toxic hydroxyl radicals (HO•) from hydrogen
peroxide, a by-product of the respiratory chain. The Cu(II) generated can
be reduced by reducing agent (e.g., ascorbate, glutathione (GSH) or any
other thiol found in cells). Cu(I) will then further react with either hydro-
gen peroxide or directly O2, as described in the following series of reac-
tions (“red” denotes a reducing agent and “ox” its oxidized form):
Cu ( I ) + H 2O2 → Cu ( II ) + HO • + HO −
Cu ( II ) + red → Cu ( I ) + ox
Cu ( I ) + O2 → Cu ( II ) + O2• −
Cu ( I ) + O2• − + 2 H + → H 2O2 + Cu( II )
Non-controlled Cu amount can thus be a source of reactive oxygen

species (ROS).41 To prevent such reaction to occur, cells maintain at a low
level both Cu(I) and Cu(II) species.
10 Zuily et al.
C. In Vivo Complexation of Cu
In cell, both Cu(I) and Cu(II) are present, in tight interaction with BMs
(mainly proteins), which can either stabilize Cu(I) or Cu(II) or cycle
Cu(I)/Cu(II) in redox active enzymes. Cu(II) and Cu(I) are found in the
periplasmic environment of Gram-negative bacteria, whereas Cu(I)
predominates in the cytosol. The presence of thiols (as GSH) in mM
concentration explains why copper can be maintained in its reduced
form.42 Under growth condition with low level of Cu in the media,
intracellular free Cu(I) is estimated to be extremely low. Around 10–21
M Cu(I) is found in the bacteria cytoplasm, compared to 10–15 M
Zn.43,44 Actually, keeping intracellular free copper concentration as low
as possible constitutes one of the main strategies to prevent intracellular
Cu toxicity.
VI. Mechanisms of Cu Toxicity:

Macromolecules Targeted by Copper
The bactericide property of copper is well-established. However, how
exactly copper leads to cell death still requires intensive investigation.
A pleiotropic impact of copper emerged as the most likely scenario
(Figure 4). In vivo and in vitro studies gathered several evidences that Cu
acts on any type of biological macromolecules. As previously mentioned,
in aerobic conditions Cu is known to produce via the Fenton reaction
highly toxic hydroxyl radicals, well known to lead to severe damages on
proteins, but also on DNA and lipids. For long time, Cu toxicity was
believed to be mainly mediated by ROS production. However, it became
difficult to explain bacteria sensitivity toward Cu under anaerobic condi-
tions. Remarkably, Imlay et al. were able to demonstrate in E. coli that
excess of Cu under anaerobic conditions lead to mismetallation of key
enzymes.45 Fe–S clusters are particularly vulnerable to Cu-transmetallation,
in agreement with the high thiophilicity of Cu(I). Nonetheless, it starts to
be accepted that accumulation of several intracellular damages (see
Figure 4, and explained below) explains the high level of Cu toxicity
toward all cell’s kind.
Cu(II)
Cu(II) Cu(I)
Fenton reaction
DNA
+H2O2
S
Cu(I) +OH. Proteins
Fe Fe Fenton reaction
S Lipides
Metalloproteins
inactivation GSH Cu(I)
S
Cu Cu GSH-Cu+ complex
S
increase Misfolding
M Aggregation
Figure 4. Intracellular Cu impacts. Copper enters the bacterial cell via an unknown
pathway. In aerobic conditions, copper can be found in the periplasmic compartment
as Cu(I) or Cu(II). However, in the reducing environment of the cytoplasm, Cu(II) will
be rapidly reduced into Cu(I). Cu(I) in both compartments can be involved in Fenton-
type reactions to produce hydroxyl radicals that are highly reactive and toxic to mac-
romolecules. Copper can also have a direct impact on lipids, proteins, and nucleic
acid by altering their structure. Free copper or probably the accumulation of glu-
tathione-copper complexes will interfere with metalloproteins by the displacement of
the physiological metal and the perturbation of the metallocenter assembly.
A. Membranes
Cellular membrane is, in fact, the first component Cu encounters, and
lipids have been described as one of the Cu targets. Nevertheless, the
action of Cu on membranes appears to be related to indirect effect via
ROS production or via cysteine-binding to proteins, hence, leading to the
modification of proteins and/or lipids that will end in the destabilization
of the membrane integrity.
Membrane phospholipids such as phosphatidyl ethanolamine (PE) or
phosphatidyl serine (PS) have been shown to coordinate Cu(II) ions via
reactive groups such as the primary amine of PE found at its head group.46,47
Hence, the production of ROS is locally increased explaining the high level
of lipid oxidation in presence of Cu(II).47 Other reports confirm that Cu ions
bound to the membrane will induce the production of hydroxyl radicals,
which will react with lipids and induce lipid peroxidation of the double
12 Zuily et al.
bonds of unsaturated fatty acids.48 This phospholipid bilayer modification

will further lead to a loss of membrane integrity.
A recent study described the impact of Cu on lipoprotein maturation
by impairing its acylation.49 Cu binding to the cysteine involved in the
acylation process, prevented lipoprotein trafficking to the outer mem-
brane. This mislocalization was proposed to additionally damage the
membrane integrity and consequently its permeability.
The binding of Cu to other key cysteines was also shown to impair
peptidoglycan maturation which, per se, perturbs cell envelop and renders
it more sensitive to detergents and other compounds.50
B. DNA
Cu toxicity has long been attributed to DNA damages. Indeed, several
experimental evidences demonstrate in vitro a clear denaturation of DNA
fragments in presence of increasing amount of Cu(II).51 By binding to
phosphate and nitrogen ligands, Cu prevents the correct folding of
DNA.52,53 Once bound, Cu further reacts and induces DNA break via the
production of ROS.54 However, all these earlier studies were performed in
a non-cellular environment. In an environment containing millimolar
GSH concentrations, the affinity of DNA to the relevant redox state Cu(I)
is low compared to Cu(I) binding to GSH and to other thiolate or sulfide
containing proteins (such as those containing FeS clusters). The stronger
complex DNA-Cu(II) would never form in vivo as Cu(II) will be reduced
immediately by GSH. In addition, in E. coli, most of Cu ions are found in
the periplasm. Very little amount may enter in the cytoplasm where the
formation of hydroxyl radicals is unlikely given the high concentration of
GSH in this compartment and its potency to stabilize Cu(I). In accord-
ance, Macomber et al. demonstrated the absence of severe DNA damages
in E. coli after Cu treatment.35 In fact, the Cu binding capacity of DNA
might be used by bacteria to scavenge extracellular Cu ions and prevent
Cu intracellular damages. Dalecki et al.55 proposed that such strategy
could be used by bacteria pathogens to protect themselves against copper
by the release of extracellular DNA once inside eukaryotic cells56 or once
they formed biofilm matrix notably composed of extracellular DNA.57
Further studies might be of interest to better define the role played by
DNA among bacterial strategies to survive copper excess.
C. Proteins
Proteins are one of the most sensitive targets to Cu overload in bacteria
cells. Cu ions bind to O-, N-, and S- donors, elements that are found in the
protein backbone as well as in several amino acid side chains (such as
cysteine, methionine, histidine, etc.). Such variability of interactions leads
to a multitude of impact on proteins and, in fine, on cells.
In aerobic conditions, intracellular Cu excess leads to severe oxidative
damage linked to hydroxyl radical production via the Fenton reaction, as
well as to the oxidation of cysteine residues that may form unwanted
disulfide bonds. Deletion of periplasmic repair system known to fix abnor-
mal disulfide bonds, decreases drastically E. coli survival upon Cu stress in
aerobic conditions.58 This was not observed under anaerobic conditions,
underlying the different mechanism of action of Cu under anaerobic versus
aerobic conditions.59 Cu might also inhibit the reduction of disulfide bonds
in the periplasm, a process required to maturate cytochrome c and this has
been shown to perturb key pathways impairing cell survival.28 Recently,
methionine oxidation of the periplasmic protein CusF has been reported.60
These results confirm the production of ROS in the periplasmic environ-
ment of E. coli. Under these conditions, periplasmic methionine sulfoxide
reductase repairs the oxidized methionines of CusF, demonstrating the
importance for cells to keep their copper homeostasis systems active by
repairing the oxidation of their residues.
In addition, Cu(II) induces protein misfolding in vitro and this was
recently demonstrated in vivo.59,61,62 As described earlier, Cu(II) metal ion
possesses a strong affinity for proteins compared to other metals, reflecting
its place in the Irving–Williams series. Furthermore, Cu(I) shows a high
affinity for thiols, such as cysteines. Hence, any intracellular concentration
increase of Cu could lead to a strong perturbation of Zn-, Fe-, or other
metalloproteins, as intracellular concentration of metals have been shown
to dictate some proteins metalation.63,64 Under anaerobic conditions, direct
inactivation of metalloproteins by Cu has been demonstrated in vivo by
Imlay et al.45 The authors clearly showed that the iron–sulfur enzyme (iso-
propylmalate dehydratase) is one of the first Cu-target in E. coli. Cu-induced
protein inactivation leads to cell death unless amino acids were added to the
media. Inactivation of iron–sulfur enzymes or other metalloproteins, as
well as perturbation in iron–sulfur biosynthesis or heme biosynthesis, has
been further confirmed by other authors in other microorganisms.65–68
14 Zuily et al.
Intriguingly, under anaerobic conditions, a lower concentration of Cu

in the medium is required to kill E. coli cells compared to aerobic treat-
ment.35 This was at first unexpected as Cu toxicity was thought to act
mainly via ROS production. Instead, this result further accentuates the
adverse impact of intracellular Cu on proteins in a ROS-independent man-
ner. Recently, a correlation between a higher intracellular copper content
observed under anaerobic conditions and an increased level of intracellu-
lar aggregated-proteins has been clearly demonstrated.59 This result
emphasizes the direct impact of copper on protein folding. Molecular
chaperones have been shown to maintain the cellular proteostasis under
copper stress conditions highlighting a central role of this family of pro-
teins to protect cell against copper toxicity.59
To summarize, the proposed model to explain bacteria death by Cu
is more complexed than initially thought. Under aerobic conditions,
intracellular copper excess as well as ROS-generated by Cu will interfere
with all macromolecules and destabilize several pathways. Accordingly,
a recent study highlighted several genes involved in the resistance of E.
coli toward a prolonged copper exposure confirming the multiple impact
of copper on the cell.69 Under anaerobic conditions, Cu will destroy cells
by inducing protein misfolding and/or by mismetalating catalytic sites
and this will also lead to cell death. As mentioned in a recent paper by
O'Hern et al.: “a fundamental understanding of Cu speciation and avail-
ability in cells is essential to uncover the cellular consequences of Cu
cytotoxicity”.70
Overall, Cu toxicity in bacterial cells is likely to be multifactorial:
ROS production, mismetalation, and binding to key amino acids might be
the main features that lead to global cell defect in bacteria. However, in
the abovementioned experimental studies, high Cu salt concentration
(CuCl2 or CuSO4) was required to observe cell death. With the objective
of using Cu as medical treatment, Cu-cytotoxicity needs to be improved.
With such aim, Cu-complexes appear as appealing compounds and we
will next develop the advances made in this domain.
VII. Chemistry of Copper Complexes

The emergence of antibiotic-resistant bacteria has urged scientists to find
alternative killing strategies in replacement to classic compounds.
Developing new antibiotic agents is challenging, time consuming, and

might end with the apparition of new resistance mechanisms. Cu is cur-
rently reconsidered as therapeutic agent in complex or in combination
with antibiotics.55
A variety of ligands (hereafter designated as L) can form complexes
with Cu ions through the formation of coordination bonds. Inducing dras-
tic changes in the properties of Cu, a ligand can be rationally designed to
obtain complexes for specific applications. For example, depending on its
chemical groups, the global charge of Cu-L complexes can be negative,
positive, or neutral, hence changing the solubility in hydrophilic or hydro-
phobic environments as well as its affinity toward intracellular compo-
nents. In this chapter, we will give a rapid overview of the chemical
properties of Cu-L that can be tuned by modifying the ligand.
A. Reactivity of Cu Complexes
Since Cu-cytotoxicity can arise from different chemical mechanisms (see
Section 1.6), ligands (L) are a good tool to tune it and/or control its speci-
ficity. Indeed, ligands will promote or prevent some of the mechanisms
listed below.
(a) Redox-based: ligands modulate Cu redox potential (see section

“Redox Potential”) modifying Cu redox reactivity. Moreover, a redox
reaction often involves the binding of the substrate, replacing a labile
ligand (e.g., H2O), prior to the electron transfer. In that case, which is
considered common for Cu-L complexes, the ligand (L) might influ-
ence the interaction with the substrate, from hindering it, to attracting
or directing/orientating a potential substrate.
(b) Catalytic redox-independent: Cu(II) (but not Cu(I)) being a quite
strong Lewis acid, biological relevant reactivity could include non-
redox reactivity, such as hydrolytic cleavage of BMs via the modula-
tion of the pKa of Cu-bound water. In this case, for instance, a ligand
can further change the pKa. Of note, non-redox catalysis cannot be
excluded as a potential mechanism involved in Cu toxicity, but is
often considered less likely.
(c) Pure coordination: the type and structure of the ligand (L) in Cu-L can
influence how Cu binds to a BM, often a protein (Figure 5). This is
16 Zuily et al.
(a) (b)
(c) (d)
Figure 5. Influence of ligand L on the interaction of Cu with biomolecule: (a) ionic

Cu (blue balls) can bind to several coordination sites. L (in yellow) can confer selec-
tivity to Cu binding via ternary complex by different means; (b) by steric hindrance
of the L that confers selectivity to certain Cu-binding sites, e.g., Cu-L cannot bind to
buried sites in a protein; (c) by the interaction of the ligand with the target protein
together with a Cu-coordination site of the protein; or (d) by binding of the Cu-L via
weak interactions (e.g., H-bonds) but without coordination bond between Cu and
target protein.
especially relevant for ternary complexes L-Cu-BM. A ligand can

modulate the interaction with BM via steric hindrance or ligand–
protein interactions (Figure 5). As a result, some BMs will be pre-
ferred targets based on the nature (i.e., chemical function) and
exposition (i.e., accessibility) of their coordinating groups.
B. Stability of the Cu-L Complex

The stability of metal-complexes against dissociation is a key parameter
to define their intracellular behavior. Complex stability is often expressed
as apparent dissociation constants (Kd), which are measured under a cer-
tain condition (pH, buffer, concentration, etc.). To compare the affinity
among different complexes, these apparent Kd have to be measured at the
same pH (often being pH 7.4) without any competitor (such as buffer).
Besides, one must care of the stoichiometry of the complexes before com-
paring their Kd. When appreciating apparent dissociation constant of
complexes, important concepts have to be considered, such as chelate
effect, Pearson’s principle (hard and soft acids and bases [HSAB]), coor-
dination geometry, and charges (including partial and electronic densi-
ties). Of course, another important concern is the physiological context in
which the metal-complex will be used. All of this needs to be taken into
account in in vitro measurements to get a better idea of Cu-L complex
intracellular stability and consequently intracellular behavior.
C. Kinetics of the Cu-L Complex

The inertia of the Cu-L complex, i.e., how fast L can dissociate from a
Cu-L complex and/or how fast an external ligand LE can bind to Cu-L, can
also play a role. Cu(II) and Cu(I) are quite labile metal ions. They can be
exchanged quite rapidly in Cu-L, unless the affinity is very high or the
ligands are very bulky and shield Cu. The latter is common in some pro-
teins, but more difficult to obtain with smaller ligands. Here again, the
design of the ligands will allow to tune the kinetics of dissociation for the
purpose expected.
D. Redox Potential
Cu ions in a Cu-L complex can have very different redox potentials. The
main biological redox couple is Cu(I)/Cu(II). Depending on the properties
of the ligand (geometry, number, and type of coordination atoms (O, N, S,
etc.) and charge or electron density of the ligand (see Table 3), the redox
potential can vary from below –0.4 up to +0.8 V versus NHE. This means
that a range of potentials can be reached in which Cu can switch from
quasi-inert behavior in biological environment to a high redox activity.
VIII. Impact of Ligands on the Biological

Activity of Copper
As previously mentioned, modification of the ligands may change signifi-
cantly the intrinsic properties of Cu. Drastic intracellular changes and
impact on cell survival are expected. Four main strategies drive the
18 Zuily et al.
development of new ligands toward the tuning and control of the killing
efficiency of copper-based drugs: (i) improving membrane permeability,
(ii) improving the specificity of the reaction, (iii) tuning the compartment
targeted, or (iv) tuning the mechanism of action. In this chapter, we will
discuss the different intracellular routes Cu-complexes could undertake.
A. Localization
Cu-L cellular localization is largely influenced by its charge: (i) hydro-
phobic Cu-L might localize into the membranes, (ii) less hydrophobic (yet
not too hydrophilic) ones could cross the membranes, and (iii) highly
charged ones are expected not to passively penetrate membranes.
Noteworthy, a hydrophobic complex (neutral global charge, hydrophobic
ligand) might have low solubility in aqueous media. Therefore, experi-
menters must care about precipitation.
B. Presence in Biology of Potentially Competing

Ligands for Cu(II)
As mentioned earlier, biological media possess high amount of potential
metal ligands such as glutathione, proteins, etc. Concerning the extracel-
lular and the periplasmic environments, where Cu(II) is stable, weak Cu-L
can dissociate rapidly. In such case, any biological effects will be due to
Cu and/or ligand only. To maintain intracellular Cu-L complex, a strong
affinity is required to prevent dissociation. For instance, if a Cu-L is con-
ceived to target bacteria in humans via the blood, the Cu-L has to resist to
Cu-binding proteins present in that fluid, such as serum albumin (Cu(II)
Kd = 10–13 M).71
As a general rule of thumb, monodentate and bidentate ligands are
generally too weak and will be dissociated easily, unless a stable ternary
complex with an external ligand is formed (see section “Reactivity of the
Free Ligand in Bacteria”, Table 2). To keep the integrity of the initial Cu-L
complex, stronger ligands, tridentate or tetradentate, should be favored. It
is noteworthy that ligands coordinating metals via only oxygen atoms
have generally a moderate affinity for Cu(II).
While in the periplasm Cu(II)-L remains oxidized, Cu(II)-L entering

the cytosol will be reduced in this compartment, which contains high
amount of thiols, e.g., 1–10 mM of reduced GSH. Thiols are very efficient
reducing agents for Cu(II) and Cu(II)-L and quite strong ligands for Cu(I)
(apparent Kd of low µM Cu(I) to 1–10 mM GSH is ~10–16 M39). Actually,
most of the Cu-L used as antimicrobial agents are relatively strong Cu(II)
ligands, stable in the periplasmic compartment. Once such complex enters
the cytosol, Cu-L will be reduced and dissociated if L is a weak ligand for
Cu(I). As the coordination chemistry of Cu(II) and Cu(I) are very differ-
ent, a ligand that strongly binds Cu(II) can be a weak ligand for Cu(I).
Ligands that enable such transport of Cu into the cytosol have been
referred to as ionophores (Figure 6). Here, the toxicity of the Cu-L will be
in fact due to the Cu(I) and ligand released into the cytoplasm rather than
directly to the Cu-L complex.
Highly stable Cu(II)-L complexes do exist. They are even maintained
in the cytosol in presence of thiol compounds. However, in such a case,
they have no redox activity and are less toxic72 (e.g., Cu-atsm, see section
“Bis-Thiosemicarbazones (atsm, gtsm)”).
Furthermore, the formation of ternary complexes can occur already
outside the bacteria, in the cell medium or in the blood of the host, for
example. This can even more modulate the fate and activity of Cu-L.
Indeed, extracellular ligands (e.g., amino acids and proteins) may either
act as “scavenger/trap” of Cu-L, weakening its effect, or enhance its cel-
lular uptake and hence its activity.
C. Presence in Biology of Potentially Competing

Ligands for Cu(I)
Cu(I)-L complexes are unstable in presence of O2. They can be rapidly
oxidized extracellularly or in the periplasm. In such case, dissociation of
the Cu-L complex will occur because strong Cu(I)-ligands are often
weaker Cu(II)-ligands. However, O2-stable Cu(I)-L exists, and if they are
hydrophobic, they can passively enter in the periplasm and/or cytosol.
They can be stable in the cytosol. The requirement is a stability constant
high enough to resist GSH (Kd < fM) or to prevent the activation of the
20 Zuily et al.
Cu (I/II) - Complexes
Ligand alone
1. Localisation change/ 2. New Reactivity 3. Quench / Loss of

Ionophore Gain of function function
Cu removal
Substrate Product
Substrate Product
Substrate Product
Cu
toxicity
Reductive
dissociation Metal interference
Ligand Cu+ Cu2+ Other metal Cu-binding

Cu enzymes
Protein
Figure 6. Possible routes and activities of Cu bound to ligands. The left part of the
panel describes the activity of a Cu(I/II)-L encountering the bacteria with three con-
ceptual routes. On the right part of the panel, the Cu-free ligand (L) encounters the
bacteria, but once inside the cells, it will interact with endogenous Cu enzymes or
interfere with intracellular metal homeostasis. For Cu-L, route 1 describes the iono-
phore activity (also called “mechanism I” in this review). The role of the ligand is to
help the Cu to pass the membranes hence increasing the intracellular Cu concentra-
tion. Very often the Cu dissociates from the ligand triggered by Cu(II) reduction. The
released ligand could bind other metals or interfere with other targets as well the Cu.
Route 2 consists of a new function of the Cu-L. Classically, this would be a Cu-L
catalysing a reaction, most likely the production of ROS (also called “mechanism II”
in this review). In route 3, the Cu-L inhibits an essential function (loss of function)
such as binding to an essential cysteine in a cytoplasmic or periplasmic enzyme.
transcription factor CueR (Kd < zM) and the consequent expression of the
Cu extrusion machinery. Such complex might not have strong intracellular
toxic effect. Otherwise, Cu(I)-L can dissociate in the cytosol, exerting an
ionophore activity.
D. Reactivity of Cu-L in Bacteria

Based on several experimental evidences, some Cu-L show a clear
increased bacterial toxicity in comparison to free copper (see section
“Copper Complexes as Antimicrobial Agents, a Non-Exhaustive List”).

This can be explained by our previous description of the reactivity of
intracellular copper complexes, which can be summarized as follows
(Figure 6):
(a) Cu-L ionophore activity: the complexation of Cu serves to increase

Cu transport efficiency through the membranes. Higher intracellular
accumulation of free released Cu can easily explain the higher level of
toxicity. In addition, the dissociated ligand could also perturb intracel-
lular pathways. Most of the Cu-L characterized so far, belong to this
category (see section “Copper Complexes as Antimicrobial Agents, a
Non-Exhaustive List”).
(b) Gain of function via redox reactions: some Cu-L can undergo redox
reactions, like reducing O2 to form ROS or oxidizing essential BMs
(cysteines, GSH, NADH, etc.). To have a high redox activity, the
redox potential should be between ~0 and 0.3 V versus NHE, and the
Cu-L has to rapidly cycle between Cu(I) and Cu(II). In this case, there
is tradeoff between stability and reactivity. Indeed, to obtain a stable
and highly reactive Cu-L complex, the ligand needs to bind strongly
both redox states (Cu(I) and Cu(II)). This represents a real challenge
as the two oxidation states prefer drastically different coordination
spheres (in terms of geometry mainly, but also composition).72
(c) Loss of function via binding: a Cu-L complex may be stable enough
to react/bind, for instance, to an essential residue of an enzyme, such
as cysteine (for Cu(I)-L or Cu(II)-L) or histidine (most likely for
Cu(II)-L), which will lead to a loss of function. In that case, one of the
ligands of the initial complex must be labile to be replaced by the
targeted protein residue. It is also possible that Cu-L binds to a target
via weak bonds (e.g., hydrophobic interaction and hydrogen bonds)
in a pocket of its target and inactivates it (see case of Bis-
thiosemicarbazones (bTSC) below, section “Bis-Thiosemicarbazones
(atsm, gtsm)”).
E. Reactivity of the Free Ligand in Bacteria

Instead of providing the Cu-L complex directly to the cell, it could be
possible to form it intracellularly by adding the ligands in the media that
22 Zuily et al.
would bind endogenous Cu in the bacteria (Figure 6). This may have bio-
logical activity via two possible mechanisms:
(a) Such interaction could perturb intracellular copper homeostasis and

destabilize essential metabolic pathways, in addition to the intrinsic
activity of Cu-L. An important prerequisite is the strong affinity of
the ligand L for Cu. In the cytosol this is very challenging, as intrin-
sic protein (CueR) has already a very strong affinity (zM range43),
and GSH binds Cu(I) in the low µM range.42 In the periplasmic com-
partment, strong competitors are available. Indeed, even proteins that
do not bind Cu under physiological conditions can have quite strong
affinity for Cu(II) to compete against L. As an example, proteins
containing a His in 2nd or 3rd position (from the first amino acid in
N-terminal) which are quite common in biology (albumin, histatin,
antimicrobial peptides (AMPs), etc.) have Kd in the range of 10–12 M
to 10–14.5 M.73 Therefore, the design of L may be quite challenging,
as its affinity must be suited to compete against endogenous Cu(II)- or
Cu(I)-ligands.
(b) An interesting and underexplored case is the formation of a ternary
complex L-Cu-protein. Here, a ligand added to the media could, after
cellular insertion, bind to a copper center found in an essential
enzyme. This will impair the function of the enzyme and kill the cells.
Noteworthy, this strategy converges with previously described one,
consisting in inhibiting an enzyme by targeting a cysteine or histidine
in its active site with Cu-L. In this strategy, it is important to consider
the denticity of the ligand and the favored stoichiometry of Cu/L
complex(es) (Table 2). For, Cu(II), a tetradentate L has no vacant
equatorial coordination site available for a strong bond, and hence
ternary L-Cu-protein is not stable. In contrast, a bidentate ligand to
Cu(II) can form two strong equatorial bonds in L-Cu-protein. This
could happen also after the release of one L in a Cu(II)-L2, for exam-
ple in Cu(II)-1,10-Phen2 (see section “Phenanthrolines”).
As just discussed, several parameters can be modified to specifically

modulate the properties of a Cu-complex. Even though several Cu-L have
already been designed and show interesting antibacterial properties, the
next chapter will provide some relevant examples, all the possibilities
Table 2. Cu(II)-L complexes general properties.

Ligand type Bidentate Tridentate Tetradentate
Complexes formed
with Cu(II)
Thermodynamic stability
Kinetics (ligand exchange)
Formation of ternary
complexes (e.g., with
proteins)
Reduction by physiological
reducing agent
Example Cu(II)-1,10-Phen2 Cu-terpya Cu(II)-atsm

(for more details see section Cu(II)-HQ2 Cu(II)-gtsm
“Copper Complexes as
Antimicrobial Agents, a Non-
Exhaustive List”)
In green LE correspond to external ligand. External ligands are classically water (or other sol-
vents) or anions during the preparation of the complexes (e.g., halides). Upon addition in cell
culture medium or application to living organisms, they are supposed to be exchanged easily
with small molecules (water, amino acid, and anions) or with proteins and hence form the
ternary complex (L-Cu-BM).
a - .74
24 Zuily et al.
presented above have not yet been fully exploited and further efforts are
required to improve our knowledge on the impact of diverse Cu-L on
bacteria.
IX. Copper Complexes as Antimicrobial Agents,

a Non-Exhaustive List
Hereafter, we will discuss some of the most prominent class of Cu-complexes
(Cu-L) used as antibacterial agent. Some of these complexes are already
used to treat cancer or neurogeneration but they appear well-suited to fight
microbes as a very low amount is sufficient to kill them.
A. Dithiocarbamates Compounds
Dithiocarbamates (DTC) are a class of metal chelators that can be formed
from thiuram disulfides by a two-electron reduction (Figure 7). They bind
Cu(II) bidentately and can form Cu(II)-DTC and Cu(II)-DTC2 complexes.
Cu(II)-DTC2 is neutral and the redox potential is quite low (Table 3).
Thiuram disulfides have a much lower affinity for copper ions than DTCs.
Thus, in terms of copper binding, thiuram disulfides are prodrugs, and
need to be activated by reduction, which is supposed to occur mainly
intracellularly by thiols. The most prominent example is the antabuse drug
disulfiram (a neutral thiuram disulfide) that upon reduction yields the
active DTC drug, diethyldithiocarbamate (DEDTC).
Thiuram disulfides, or most likely the intracellular compound DTC,
have been shown to have antibacterial activity on Staphylococcus aureus,75
on Mycobacterium tuberculosis,76 and on P. aeruginosa.77 A link
Figure 7. Reduction of thiuram disulfides to dithiocarbamates and subsequent

Cu(II)-chelation. Several derivatives are studied depending on the R/R’. (R=R’=
-CH2-CH3 for DETC, R/R’ = cyclic -CH2-CH2-CH2-CH2- for pyrrolidinedithiocarba-
mate (PDTC)).
between the antimicrobial activity and DTC ability to bind copper was
proposed but not clearly demonstrated. As a thiol agent, DTC might react
with cysteine from key enzymes to form a thioester.77 DTC might also be
able to bind Cu(II) in the oxidizing periplasmic compartment of Gram-
negative bacteria and per se perturbs Cu trafficking toward Cu-enzymes.
Last, DTC might transport Cu(II) into the cytoplasm where it is expected
to be released as Cu(I) due to the high thiol content.72 Coherently, in this
reducing environment of bacteria cytoplasm, DTC might not form a com-
plex with Cu(I) as it will not compete with GSH found in mM
concentration.72
Instead of adding only the ligand DTC, direct addition of Cu(II)-
DTC2 complex in the media has a strong antimicrobial effect. This activity
can be explained by intracellular accumulation of Cu due to ionophoric
activity (mechanism I). Accordingly, strong Cu-dependent antimicrobial
activity of disulfiram/DTC was reported against M. tuberculosis, with a
minimum inhibitory concentration (MIC) around 0.3 mM,78 and against
S. pneumoniae.79 The authors further showed the ability of Cu(II)-DTC2
complex to cross bacteria membranes and inactivate cytoplasmic
enzymes.78 In such condition, intracellular Cu stress response is induced.
This confirms that Cu(II)-DTC2 entering the cytoplasm might release
high Cu(I) concentration which perturbs metabolic pathways. Such
Cu-dependent growth inhibition of disulfiram/DTC has also been observed
recently on mollicutes.80 The addition of a Cu(II) chelator in the media
completely modifies the MIC values of disulfiram from nanomolar to
micromolar range. This result further confirms the critical role of Cu for
disulfiram antimicrobial activity.
Besides, Cu(II)-DTC2 has been shown to interact with zinc finger
proteins in cancer cells, and to oxidize Zn(II)-thiolates to form disulfide.81,82
Even though this mechanism has not been reported in bacterial cells, its
occurrence cannot be ruled out.
B. 8-Hydroxychinoline (8-HQ) Compounds

8-HQs are neutral quite hydrophobic compounds able to cross membranes
passively.83,84 They have been described as antimicrobial agent and have
26 Zuily et al.
been studied on several organisms demonstrating their efficiency.83,85 The

5-chloro-7-iodo-8-HQ, called clioquinol, was developed in 1899 as an
antiseptic and later used as an amebicide, but withdrawn from the market
in the 70s due to side effects.86 A high-throughput physiological screen
made against M. tuberculosis identified 8-HQ compounds as efficient
molecules.87 Although the antimicrobial mechanism is multifaceted, 8-HQ
interaction with metal ions including Cu seems to play a role. Addition of
Cu ions triggered killing fungal pathogen Cryptococcus neoformans by a
8-HQ, and further addition of the strong Cu-chelator BCS forming a
membrane-impermeable Cu–BCS2 complex reversed the toxicity, in line
with an important contribution of a Cu-ionophore activity of 8-HQ.83
8-HQ can form Cu(II)-HQ and Cu(II)-HQ2 complexes (Figure 8). The
affinity of Cu(II) for HQ2 is quite high at neutral pH, whereas the metal
ions binding via a phenolate (hard base) suggests a relative low affinity for
Cu(I) (soft acid). Despite the quite low redox potential of Cu(II)-HQ2, it
can be readily reduced by the highly concentrated GSH tripeptide found
in the cytoplasm, likely via the formation of a ternary complex.
Cu-complexes formation with 8-HQs will consequently depend on the
bacteria compartment, i.e., possible with Cu(II) in the periplasm or in the
environment, but not with Cu(I) in the cytoplasm. Consequently, 8-HQ is
also well suited to serve as a ionophore (mechanism I). Such a mechanism
of action was indeed proposed by Festa et al.83 to explain 8-HQ and
Cu-8-HQ toxicity on C. neoformans. The authors observed an increase in
intracellular bioavailable Cu content which can then affect intracellular
pathway either by acting directly on proteins or by generating toxic ROS.
Figure 8. Structure of 8-HQs and their Cu(II)-complexes.

However, 8-HQ toxicity varies depending on the organism tested.83

Species-specific targets and different Cu resistance strategies may explain
this diversity, requiring further investigations.
It is also important to mention that 8-HQ with their hard phenolate
ligand are good Fe(III) binders compared to DTC. Dissociation of Cu(II)
might induce some cross talk with Fe(III) pathways which could also be
a process involved in 8-HQ toxicity.
C. Phenanthrolines
Phenanthrolines (Phen) are bidentate ligands forming both 1:1 and 1:2
complexes with Cu(I) and Cu(II) (see Figure 9). 2,9-unsubstituted
Figure 9. Structure of phenanthroline, its derivatives and their Cu(II)-complexes.

Structural difference between the ligands are highlighted in green.
28 Zuily et al.
phenanthrolines (e.g., 1,10-phenanthroline, bathophenanthroline) have

generally a high affinity for both Cu redox states.
In contrast to 8-HQs and DTCs, these phenanthrolines can undergo
rapid redox-cycling between Cu(I) and Cu(II) and are potentially very
competent in ROS production (mechanism II). However, in vivo, they
likely act only as ionophores (mechanism I) rather than pro-oxidant. For
instance, the Cu(I) and Cu(II)-complexes with neutral phenanthrolines
can passively cross the membranes, despite the positive charge of the
complex formed. Then, Cu-(Phen)2 is rapidly reduced by GSH, at least
one Phen dissociates and a ternary complex Phen-Cu(I)-GSH might
form.88 However, other proteins with stronger Cu(I) affinity than GSH
might be able to totally subtract Cu(I) from Cu-phenanthroline com-
plexes. This has been shown for mammalian metallothioneins,72 but might
be also the case for bacterial metallothioneins or for copper storage pro-
teins such as Csp3.25
It is important to distinguish phenanthrolines according to the substi-
tution at positions 2 and 9 (Figure 9). Methyl (or larger) groups at these
two positions, such as in bathocuproine (BC), neocuproine (NC), cuproin,
or bathocuproine disulfonate (BCS), stabilize Cu(I) over Cu(II) in the
Cu-Phen2. In other words, they have a stronger Cu(I)-affinity, but lower
Cu(II)-affinity compared to 2,9 unsubstituted phenanthrolines. The bulky
methyl-groups force the two phenanthrolines into a tetrahedral coordina-
tion of the metal ion, a geometry suited for Cu(I) but not for Cu(II).
Related to that, Cu(I)-complexes with two 2,9-substituted phenanthrolines
(e.g. Cu(I)-BC2) are difficult to oxidize and are generally air-stable.
It is a general feature that the apparent affinity for a metal ion in a
1:2 (metal:ligand) complex has a more drastic dependence on the ligand
concentration compared to a 1:1 complex. Hence, 2,9 substituted phen-
anthrolines that form a 1:2 complex can become stronger than Cu(I)-
proteins (that often are 1:1 complexes) at higher concentrations
> 0.1 mM.89 However, at lower ligand concentrations, Cu(I) might dis-
sociate from 1:2 complexes as shown for 10 µM Cu(I)-BCS2 which dis-
sociated at about 50% in 3 mM GSH and totally with mammalian
metallothioneins.72 Therefore, Cu(I) complexes with two neutral
2,9-substituted phenanthrolines (like BC or NC) can also have ionophore
activity at lower concentrations.
The antimicrobial activity of some of these compounds has been dem-

onstrated, which is highly dependent on their ability to cross the mem-
brane. NC exhibited efficient M. tuberculosis growth inhibition in
presence of increasing amount of copper.90 Such an effect was also
observed on Mycoplasma and Ureaplasma species.80 In contrast, bathocu-
proine disulfonate is membrane impermeable and does not show any
effect on M. tuberculosis growth.
Even if some of these compounds cannot be used as antimicrobial
agent, their ability to tightly bind Cu(I), as NC and BC, have made them
widely used as chelating agents in vitro or in vivo for several experimental
purposes.
D. Pyrithione
Pyrithione (PT) is a neutral ligand which can bind Cu(II) as a Cu(II)-PT2
complex, which is neutral as well, due to the loss of the two hydroxy-
lamine protons (Figure 10). Thus, in terms of charge, PT has properties
suited for ionophore activity (mechanism I), as Cu(II)-PT2 can enter the
cytoplasm and PT exit it. The affinity of Cu(I) to PT is unknown, but
according to Pearson theory, Cu(I) has a low affinity for N-OH. This
means that once reduced in the cytoplasm, Cu(I) will dissociate from PT.
This was further confirmed by a recent study,62 where the authors dem-
onstrated the intracellular increase of copper in E. coli after treatment
with a mix of Cu and PT and in comparison, with Cu alone. Cu-induced
Figure 10. Structure of PT and its Cu(II)-complexes.

30 Zuily et al.
cell death is amplified by Cu-PT2 which most likely might overwhelm

copper homeostasis systems. Cu-PT2 treatment was also tested on
Neisseria gonorrhoeae which is highly sensitive to this complex with a
MIC similar to MIC observed for Cu-gtsm compounds (see section “Bis-
Thiosemicarbazones (atsm, gtsm)).91 On Klebsiella pneumoniae, the
addition of PT with either Zn or Cu increases the antibiotic activity of
amikacin. Here, the authors propose an ionophore activity of this
compound.92
Of note, the structure of Cu-PT is reminiscent of the natural copper-
containing antimicrobial agent Fluopsin C, in which Cu is coordinated by
two units of N-methylthiohydroxamate. Interestingly, both Cu-PT and
Fluopsin C show broad antimicrobial activity against Gram-negative and
Gram-positive bacteria, as well as multidrug-resistant strains. By contrast,
they are not effective against P. aeruginosa PAO1, which produces
Fluopsin C to cope with excess Cu stress.93
E. Bis-Thiosemicarbazones (atsm, gtsm)

Bis-thiosemicarbazones (bTSC) form strong Cu(II)-complexes at neutral
pH (log K of the bTSC 3-ethoxy-2-oxobutyraldehyde-bis-thiosemicarba-
zone [compound called kts] was determined to be 19 at pH 794). They
form much stronger 1:1 complexes with Cu(II) than DTC or 8-HQ, even
compared to the 1:2 complex of DTC and 8-HQ in the relevant concentra-
tion of low micromolar or below. This is mainly due to the chelate effect
as they are tetradentate ligands (Figure 11). Using a drug screen assay,
Speer et al.90 demonstrated that bTSC induces a copper hypersensitivity
phenotype in M. tuberculosis and proposed that copper complexation was
the potential mechanism.
Cu-bTSC complexes are quite stable in biological environments. They
form neutral, lipophilic compounds that enter the cell by passive mem-
brane crossing. They are weaker Cu(I) than Cu(II) chelators but still have
a log K of around 13 at pH 7 for Cu(I).95 Once inside the cells, Cu(II)
bound to bTCS could be reduced to Cu(I) and transferred from bTCS to
GSH. However, depending on the complex formed and on its redox poten-
tial, such reduction can be very slow (Table 3, Figure 11). For instance,
the Cu(II)-atsm complex has been shown to not be reduced in vitro even
Figure 11. Structure of bis-thiosemicarbazones (bTSC) and their Cu(II)-complexes.

Structural difference between the ligands are highlighted in green.
in the presence of millimolar of GSH, in line with Cu(II)-atsm low redox

potential (see Table 3, Figure 11).72,95 In E. coli, Cu seems to be released
from Cu-atsm, but with a much slower rate than from Cu-gtsm.91 The dif-
ference observed between Cu(II)-atsm and Cu(II)-gtsm in terms of redox
potential and reactivity is reflected in term of level of toxicity. Cu(II)-gtsm
exerts a higher level of toxicity on bacteria than Cu(II)-atsm. This differ-
ence can be explained by the fact that Cu-gtsm most likely acts as an
ionophore (mechanism I): it enters the cells and Cu is rapidly released by
the action of intracellular thiols (GSH and others), the fast accumulation
of intracellular Cu(I) exerting toxicity. In contrast, Cu(II)-atsm is less
toxic, which is consistent with the fact that Cu(II)-atsm is more stable,
releases Cu(I) only very slowly and this gives more time to the bacteria to
react and to switch on the defense system.91 Toxicity of cytoplasmic Cu(I)
released from Cu(II)-gtsm was further confirmed by the increased sensi-
tivity of E. coli strain deleted of copA gene, encoding the exporter which
removes Cu cytoplasmic excess. In contrast, the deletion of cueO gene,
encoding a periplasmic copper oxidizing enzyme (Figure 3), has no effect
on cell survival toward Cu(II)-gtsm.91 This finding suggests that dissocia-
tion of Cu(II)-gtsm occurs specifically in the cytosol.
A recent study from Totten et al.80 defined the impact of different
compounds toward Mycoplasma and Ureaplasma species. In their assays,
they observed a bactericidal activity of gtsm compound, however, not in a
Table 3. Thermodynamic and kinetics properties of Cu-L complexes.
32
Affinity (pH 7.4)
in log Kd for
E° Cu(II)/Cu(I) Intracellular reactivity
Compound class Representative Cu(II) Cu(I) (V/NHE) (cytoplasm) Denticity
Cu alone 0.16 – Fast reduction of Cu(II) —
– Cu(I) binds to GSH
Dithiocarbamate DETC – Fast reduction 2
(DTC) Cu + DETC 11.7c – Complete dissociation
Cu-DETC + DETC 12.2c –0.14k – Cu(I) binds to GSH
8-hydroxyquinoline Clioquinol (CQ) – Fast reduction 2
(HQ) Cu + CQ 10.8f – Dissociation
Zuily et al.
Cu + HQ 9.75g –0.36h – Cu(I) binds to GSH
Cu-CQ + CQ 9.2f
Cu-HQ + HQ 8.65g
Phenanthrolines Cu + Phen 9.1l 10.3l – Fast reduction 2
Cu-Phen + Phen 6.8l 5.5l – Partial dissociation
Cu + 2 Phen 15.9l 15.8l 0.17i – Cu(I) binds to GSH
BCS
Cu + 2BCS 12.5d 20.8d 0.62j
Pyrithione Cu + PT > 8.5e – Fast reduction 2
Cu(II)-PT + PT 5.9e – Dissociation
– Cu(I) binds to GSH
Bis- Cu + Atsm ~13a –0.40a – Stable 4
thiosemicarbazone Cu + Gtsm ~13a –0.24a – Slow
Cu + Kts ~19b –0.12b (pH 6.6) – Dissociation
a 95 b. 94 c
; ; in methanol;97; d 98 e 99 f
; ; in 80% Methanol and 20% H2O, 100 g 101 h 102 i 103 j 104 k 105 l 106
; ; ; ; ; ;
copper dependent manner. Still, the use of Cu(II) chelator in the media
decreased gtsm antibacterial activity, in line with the fact that small
amount of Cu in the media is sufficient to boost the growth inhibitory
effect of gtsm.
The higher toxicity of Cu-gtsm compare to Cu-atsm applies to other
bacteria (Gram-positive and -negative).91,96 However, the susceptibility of
the bacteria is very different, and is directly related to the bacterial physi-
ology. Bacteria with a high developed Cu-detoxification system like
E. coli are quite resistant, other with a poorer Cu extrusion system, like
N. gonorrhoeae are much more susceptible (IC 50 < µM). This can be
explained by the fact that N. gonorrhoeae have only one exporter, CopA,
whose expression is not inducible by Cu. Moreover, in N. gonorrhoeae an
additional toxicity mechanism was described related to the inhibition of
dehydrogenases in the respiratory chain.96 The results suggested that
Cu-bTSC complexes bind to membrane proteins, likely in hydrophobic
pockets where they interfere with ubiquinol. This mechanism is independ-
ent of Cu release or redox activity, in line with experiments showing that
Zn-bTSC showed similar activity.96 However, other mechanisms may
explain the toxicity of Cu-gtsm, as bacteria growing under fermentation
(S. pneumoniae and E. coli) are also highly sensitive to Cu-gtsm.91
X. Peptides-Based Cu-Chelators
Antimicrobial peptides (AMPs) are described as peptides of about 10 to
100 amino acids that can be either naturally produced by the immune
system to combat invading pathogens or chemically synthesized with non-
natural sequences. For certain natural AMPs, the binding of copper or
other metal ions was proposed to be required for their activity. The main
idea was that Cu-binding to AMPs may improve the antimicrobial activity
either through Cu-catalyzed ROS production or Cu-induced conforma-
tional changes enhancing the interaction of the AMP with its target. Two
main approaches are reported: (i) identification of native AMPs contain-
ing a high affinity-Cu-binding site, (ii) engineering such a site in a native
or artificial peptide (recent review on AMPs107).
Short peptides are often dynamic molecules lacking a well-defined
3D structure. Hence, Cu-binding to peptides is generally weaker than to
34 Zuily et al.
Figure 12. Structure of Cu(II)-XZH and Cu(II)-XH complexes.
Cu-proteins with defined 3D-structures, due to entropic penalty for

arranging the peptidic ligands in a constrained coordination sphere around
the metal ion. To date, no simple Cu(I)-binding peptide motifs with sub-
femtomolar affinity (unlike Cu(I)-proteins) are known. Instead, two natu-
rally occurring His-containing Cu(II)-binding peptide motifs can attain a
sub-picomolar affinity at pH 7.4. They require a His residue at either
position 2 or 3 in a peptide with a free N-terminus, i.e., NH2-Xxx-His-
Peptide (XH) or NH2-Xxx-Zzz-His-Peptide (XZH, also known as ATCUN
motif)73 (Figure 12). Most studies focused on these motifs as they show
comparable affinity to endogenous Cu(II)-carriers (such
as serum albumin in human blood), which makes them apt to bind Cu(II)
in biological environments. Antimicrobial activities of several native
AMPs containing a XZH motif have been compared with and without Cu
(reviewed in108). Generally, the impact of Cu was quite low, with a similar
antimicrobial activity (measured as MIC values) whether Cu was added to
AMPs or not. The modest Cu effect could be explained by the redox prop-
erties of Cu bound to XZH which induces a very low ROS production.109
However, ATCUN peptides bearing an additional Cu(I)-stabilizing site
show higher antimicrobial activity. For instance, the salivary peptide
Histatin-5 (Hist-5) shows antimicrobial activity against the yeast
C. albicans and P. gingivalis108 (Table 4). Hist-5 binds Cu(II) in a XZH
motif, but has in addition a vicinal Cu(I)-binding bis-His (-HH-) motif
which might favor the redox cycling and hence the ROS production.
Remarkably, the mutation of Cu-binding His residues from the HH motif
Table 4. Peptides mentioned

in this chapter and their
Cu-binding motifs (italic for
Cu(II), underlined for Cu(I), and
bold for both).
PEPTIDE Cu binding motifs
Hist-5 DSHAKRHH…
Piscidin 1 FFHH…
Piscidin 3 FIHH…
Ixosin GLHKVM…
significantly affects the antimicrobial activity of Hist-5.110,111. Likewise,

ATCUN-bearing AMPs such as Piscidins and Ixosin could show higher
ROS production than simple ATCUN peptides thanks to their Cu(I)-
binding motifs, i.e., -HH- and -HXZM-, respectively108 (Table 4).
Nevertheless, the impact of such Cu(I)-binding sites on the ROS produc-
tion by ATCUN-AMPs has never been assessed quantitatively.112,113 On
the other hand, in the cytosol, Cu(II)-XZH complexes are slowly reduced
and dissociated by GSH.114,115 In this case, a Cu(I)-binding site could lead
to a faster dissociation of Cu from Cu(II)-XZH, via the faster reduction to
Cu(I) and subsequent dissociation. Overall, this implies that (i) the poten-
tial pro-oxidant activity might take place only in the extracellular or peri-
plasmic compartments (mechanism II) and (ii) these complexes might act
as ionophores (mechanism I), even though no report has yet shown any
AMP with an efficient ionophore activity.
Recently, the shorter XH motif in an AMP peptide (XH-AMP) was inves-
tigated and compared to XZH-AMP and AMP only. Cu(II)-XH is slightly
more active in ROS production than Cu(II)-XZH, although still less com-
pared to Cu in buffer or in a well redox active Cu-complex such as Cu-Phen2.72
Nevertheless, the same antimicrobial activity as pure AMP against E. coli was
observed with Cu-bound or unbound XH- or XZH-AMP.116
Besides, structural changes upon Cu-binding to XH and XZH motifs
are likely not drastic as these motifs are short and located at the
N-terminus.
36 Zuily et al.
XI. Recent Advances Toward the Future

Use of Copper in Medicine
Copper is a potent antimicrobial agent and even though used since thou-
sands of years, it regains interest as a promising compound to increase
therapeutic efficiency toward recalcitrant microbial infections. Indeed, as
increasing amount of copper in bacteria environment leads to cell death,
such property is even used by macrophages to kill invading pathogens.
Cu-mechanism of action to explain toxicity as well as strategies developed
by cells to survive such thread start to be better understood and these fun-
damental knowledges will pave the way toward the discovery of copper-
related compounds with application in medicine. Copper-complexes, as
described in this chapter, show interesting properties demonstrating their
potential for clinical treatment. They present a higher level of bactericide
activity compared to Cu or the ligand alone. For most of them, Cu-L per-
mit to cross bacteria membranes with a higher efficiency than “free” Cu
and once in the reducing cytoplasmic compartment they dissociate and the
released Cu accumulates and perturbs intracellular pathways. This iono-
phore activity has been mainly described for most of the Cu-complexes.
Other mechanisms of action could occur, however, such as a gain of func-
tion to induce ROS production by the Cu-L once inside the periplasmic
compartment. This requires further experimental evidences to be
confirmed.
Cu-gtsm is a promising Cu-complex, highly effective toward multid-
rug-resistant bacteria, giving some hope to cure disease related to highly
resistant strains. Very interestingly, antimicrobial Cu-gtsm doses used to
kill the extracellular mucosal pathogen N. gonorrhoeae did not show sig-
nificant toxicity toward cervical epithelial cells.91 Recently, other mole-
cules, like the 1-hydroxy-5-R-pyridine-2(1H)-thiones (with R: COOMe,
COOEt, CF3), have been shown to efficiently inhibit M. tuberculosis at
very low amount and in a copper-dependent manner.117
However, the next challenge in this area of study will be to develop
specific antibacterial compounds to target pathogenic bacteria but neither
host cells nor non-pathogenic bacteria. Even if some complexes might be
interesting to further study for clinical applications, most of the
Cu-complexes described in this issue have no selectivity toward bacteria
versus mammalian cells. Some of these compounds are even used to treat
cancer and are known to negatively impact eukaryotic cells.118,119 To
increase selectivity, several strategies can be envisioned.
Festa et al.83 used a compound that gets activated once inside mac-
rophages. They took advantage of the high level of ROS as well as of
copper in macrophages to transform a non-toxic compound into a highly
toxic one. In particular, oxidation of a pro-drug released the 8-HQ ligand
that complexed Cu and then acted as ionophore to aid the immune
system to kill the pathogens. They were able to show that such condi-
tional activation worked in vitro and in mouse model on the pathogen
C. neoformans.83
Another strategy followed by Wolschendorf and coworkers was to
screen different compounds in combination with copper against
S. aureus.120 They were able to highlight the potential of new copper-
dependent molecules such as thiourea inhibitors which were able to
inhibit efficiently S. aureus growth and seemed tolerated in cell culture.
They also demonstrated the toxicity of PZP-915 (5-Benzyl-3-(4-
chlorophenyl) -2-methyl-4H,7H-pyrazolo [1,5-a] pyrimidin-7-one) toward
S. aureus resistant strains with a certain selectivity toward bacteria over
eukaryotic cells.121
The use of peptide with Cu-binding motifs is another way to obtain
higher selectivity toward bacteria as it is exemplified by the natural AMPs.
Peptides could also be used for targeting a Cu-site to compartments or
proteins. Accordingly, cytoplasm-targeted peptides could act as iono-
phores by adding a Cu(II)-site to the peptide. This strategy would need
optimization of the peptide sequence toward cell-penetration, so far, an
area of research poorly explored. However, it is not clear whether peptides
can be as efficient as small organic ligands. For the antimicrobial strategy
consisting in employing a Cu-site on an AMP to catalyze ROS production,
reports are mainly limited to Xxx-Zzz-His motifs. This motif is quite
redox inert as it stabilizes Cu(II) and hence ROS production is low. Other
high affinity Cu-binding sites are needed to increase ROS production.
This seems to be very challenging for short peptides, and protein-like
folding and/or organic ligands are needed to reach the balance between
stability and redox reactivity. Moreover, peptides form a scaffold which
38 Zuily et al.
can be conjugate to other entities of interest, such as fluorophore for track-

ing other antimicrobial molecules.
Synergic effect of copper/copper derivatives with antibiotics is also an
alternative way of finding highly specific molecules toward pathogenic
bacteria.55 ATP-6K in presence of copper and in combination with ampi-
cillin was shown to restore ampicillin antimicrobial activity against resist-
ant strains without affecting eukaryotic cells.122
These strategies among others should allow to overcome the limitation
of copper use in medical applications. Indeed, study on copper and its
derivatives is of high interest to develop novel efficient antimicrobial agents.
However, the use of metals to kill pathogenic bacteria has to be carefully
thought. Indeed, feeding animals by copper in industry lead to the spread of
resistance mechanism such as the pco genomic island in E. coli.123 as well
as of a functional cus locus in S. enterica that increases its resistance toward
metal.124 This raises the concern of the presence of metal in food which acts
as a disseminating agent of resistance mechanisms.
The development of Cu-dependent inhibitors might impose selection
pressure to bacteria and lead to the development of metal resistance strate-
gies. Efficient copper pumps, for instance, could help bacteria to resist
Cu-complexes ionophore activity. However, as described in this review,
copper induces bacteria cell death by targeting a variety of cellular pro-
cesses. These multiple targets make Cu-dependent inhibitors very attrac-
tive compared to some antibiotics which target a single process that
bacteria can easily overcome. A good balance is required: the use of small
amount of copper specifically dedicated to kill invading microorganisms
should be clearly envisioned whiles decreasing the excessive use of cop-
per as food additive and biocide.
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2 Transition State Analogue

Molecules as Mechanistic Tools
and Inhibitors for Tyrosinase
Clarisse Faure,* Amaury du Moulinet d’Hardemare,*

Hélène Jamet,* Catherine Belle,*,|| Elisabetta
Bergantino,† Luigi Bubacco,† Maurizio Benfatto,‡
A. Jalila Simaan§ and Marius Réglier§,¶
*University of Grenoble Alpes, CNRS-UGA UMR 5250, DCM,

CS 40700, 38058 Grenoble Cedex 9, France
†
Department of Biology, University of Padova, Via Ugo Bassi
58b, 35131 Padova, Italy
‡
Laboratori Nazionali di Frascati — INFN, Via E. Fermi 44,
00044 Frascati, Italy
§
Aix Marseille Université, CNRS, Centrale Marseille, iSm2,
Marseille, France
||
[email protected]
¶
[email protected]
45
46 Faure et al.
List of Abbreviations
Ab Agaricus bisporus
Ao Aspergillus oryzae
AUS Aureusidin synthase
Bm Bacillus megaterium
CO Catechol oxidase
DHI Dihydroxyindole
DHICA Dihydroxyindole carboxylic acid
DNA Desoxyribonucleic acid
ESEEM Electron spin echo envelope modulation
EXAFS Extended X-Ray Absorption Fine Structure
H-BPEP 2,6-Bis[(bis(2-pyridylethyl)amino)
methyl]-4-methylphenol
H-BPMEP 2-[(Bis(2-pyridylmethyl)amino)methyl]-6-[(bis
(2-pyridylethyl)amino)methyl]-4-methylphenol
H-BPMP 2,6-Bis[(bis(2-pyridylmethyl)amino)
methyl]-4-methylphenol
Hc Hemocyanins
HOPNO 2-Hydroxypyridine N-oxide
Hs Homo sapiens
HSPNO 2-Pyridinethiol N-oxide
HYSCORE Hyperfine sublevel correlation
Ib Ipomoea batatas
Jr Juglans regia
KA Kojic acid
L-DOPA L-3,4-dihydroxyphenylalanine
Mim L-mimosine
Mj Marsupenaeus japonicas
MM Molecular mechanics
Ms Manduca sexta
Transition State Analogue Molecules as Mechanistic Tools 47
MXAN Minuit XANES

PTU Phenylthiourea
QM Quantum mechanics
ROS Reactive oxygen species
Sc Streptomyces castaneoglobisporus
SP Square-based pyramid
TBP Trigonal bipyramid
Thuj Thujaplicin
Trop Tropolone
TRP1 Tyrosinase related protein 1
TRP2 Tyrosinase related protein 2
TSA Transition-state analogues
TSC Thiosemicarbazone
TYR Tyrosinase
XAS X-ray absorption spectroscopy
I. Introduction
Tyrosinases (TYRs) are copper-containing redox enzymes present in
prokaryotes and eukaryotes (plants, arthropods, fungi, and mammals)
where they are involved in tissue pigmentation, wound healing, radiation
protection, and primary immune response. Acting in phenol oxidation,
TYRs belong to the class of polyphenol oxidases (PPOs). The TYR-
catalyzed oxygenation consists of two consecutive reactions, namely the
ortho-hydroxylation of phenol to catechol (cresolase or monophenolase
activity, EC 1.14.18.1) and its subsequent oxidation into ortho-quinone
(catecholase or diphenolase activity, EC 1.10.3.1) (Figure 1). In organ-
isms, TYR is involved in the biosynthesis of melanin pigments.
II. Biological Functions of Melanins

Melanin pigments are amorphous polymers of high molecular weight.1,2
Their structures remain poorly defined and differ according to the
48 Faure et al.
O 2 + 2 H+ H 2O 1/2 O2 H2O
HO HO O
R R R
TYR HO TYR O
Phenol Cresolase Catechol Catecholase ortho-Quinone

activity activity
Figure 1. TYR-catalyzed oxygenation reaction.
organisms. The common feature of all melanins is that they are formed by
the polymerization of phenolic and indolic compounds catalyzed by TYR
or TYR-like enzymes.
A. Melanin as Color Pigment

In humans, the color of the skin, hair, and eyes mainly depends on the
concentration and ratio of eumelanin and pheomelanin. Human melanin is
produced in melanosomes, which are organelles within cells called mel-
anocytes found in the epidermis of the skin. Human melanocytes produce
two chemically distinct types of melanin pigments, the brown-black
colored pigment eumelanin, and the yellow-red colored pigment pheomela-
nin3,4 (Figure 2). With broad optical absorption, eumelanin exhibits colors
ranging from black to brown. Not only does eumelanin absorb all frequen-
cies of visible light, but its optical absorption spectrum extends to the
ultraviolet (UV) and infrared (IR) regions. With this characteristic, some
consider eumelanin as “darker than black,” a base for the “outrenoir” of
the French painter Pierre Soulages. Other melanin pigments such as
pheomelanin exhibit colors ranging from yellow to orange depending on
their structures. The mixture of these melanin pigments is responsible for
the coloring of the integuments in the animal kingdom.
The major role of melanins is to protect the skin from the harmful
effects of UV rays and thus to prevent the development of skin cancers.
In addition, eumelanin and pheomelanin play an important detoxification
role within melanocytes and keratinocytes due to their ability to bind ions
and various chemicals (Ca, Zn, Cu, Fe, orthoquinone).5–7 Eumelanin is a
Figure 2. Eumelanin and pheomelanin biosynthesis from L-tyrosine (L-Tyr).

50 Faure et al.
highly heterogeneous copolymer consisting of dihydroxyindole (DHI)

and dihydroxyindole carboxylic acid (DHICA) units in reduced or oxi-
dized form. Pheomelanin is mainly composed of benzothiazine deriva-
tives. The first steps in eumelanin and pheomelanin biosynthesis are
catalyzed by TYR, the hydroxylation of L-tyrosine into L-DOPA
(monophenolase or cresolase activity) followed by its subsequent oxida-
tion into dopaquinone (diphenolase or catecholase activity). The pheomela-
nin pigments are the result of a cascade of non-catalyzed reactions, which
include the nucleophilic addition of a L-cysteinate onto dopaquinone
followed by a heterocyclization into 1,4-benzothiazine and polymeriza-
tion (Figure 2).
Many animals produce melanin, such as birds (feather coloring) and
some protozoa. Melanin is also found as a pigment in fungi (mycete) in
which it colors and strengthens the cell wall.
B. Melanin as a Defensive Barrier

In all organisms, melanins act as protective pigments against stresses that
involve cellular damage such as sun UV rays and reactive oxygen species
(ROS). Melanins are also known to protect against ionizing radiations. In
radiotrophic fungi, melanin has a protecting role against γ rays and at the
same time the received energy is harnessed for the growth of fungi.
Melanin can also act as a defense against pathogen attack. In inverte-
brates, the innate immune defense system against invading pathogens
involves melanin. Within minutes after infection, the pathogen is encap-
sulated in a cocoon of melanin and the generation of ROS during the
formation of this cocoon leads to the destruction of the pathogen. Finally,
melanin is found in the ink used by many cephalopods as a defense
mechanism against predators.
In addition to these defense-related roles, melanin can be used by
some pathogens as an attack agent. It is indeed reported that some human
and plant pathogenic microorganisms, such as Cryptococcus neoformans
(fungus), melanin plays an important role in the virulence and pathogenic-
ity of the microorganism by protecting it against the immune responses of
the host.8
III. Tyrosinase
A. Structures of the Tyrosinase Active Sites
TYR belong to type 3 copper-containing proteins, which encompass
hemocyanins (Hc), catechol oxidase (CO, EC 1.10.3.1), and aureusidin
synthase (AUS, EC 1.21.3.6).9–11 The active site of TYR had been identi-
fied very early as a structural analogue12,13 of that of CO14,15 and Hc16 for
which 3D X-ray structures were available. It was not until 2006 that
Matoba et al. confirmed this analogy with the resolution of the 3D struc-
ture of the recombinant TYR from Streptomyces castaneoglobisporus
bacteria (ScTYR).17 In type 3 copper-containing proteins, the common
feature is an active site composed of two copper ions at a distance of about
3.6 Å both coordinated by three N-imidazoles of histidine residues.18 In
fungi19–22 and plant23 TYRs known to date (Table 1), one of the six histi-
dine residues forms an unusual thioether bond with an adjacent cysteine
residue (Figure 3a). The role of this post-translational modification that
does not significantly modify the structure of the active site is not clear.
Several hypotheses have been made such as the stabilization of the binu-
clear copper site to facilitate the electron transfer during the catalytic
reactions24 or a role in the copper incorporation process into the apo-
TYR.19 However, despite this post-translational modification, the active
sites of all TYRs are similar and nearly superimposable (Figure 3b).
No structure of human TYR is yet available with a good resolution.28
However, two homology models based on ScTYR and Ipomoea batatas
Table 1. TYR 3D structures known to date.

TYRs Organisms PDB ID Cys-His Ref.
ScTYR Streptomyces castaneoglobisporus 1wx2 No 17
BmTYR Bacillus megaterium 3nm8 No 25
AoTYR Aspergillus oryzae 3w6w Yes 19
AbTYR Agaricus bisporus 2y9w Yes 20
MsTYR Manduca sexta 3hhs No 26
MjTYR Marsupenaeus japonicas 3wky No 27
JrTYR Juglans regia 5ce9 Yes 23
52 Faure et al.
(a) (b)
Figure 3. (a) Post-translational modification involving cross-linking of a His ligand

with a Cys residue in AbTYR and (b) Active site alignments for ScTYR (salmon),
BmTYR (magenta), and AbTYR (green).
Figure 4. Overlay of the homology model of HsTYR (green) based on TRP130 and
the one obtains from AlphaFold AI approach (AF-P14679-F1 model_v2) (pink).
Copper ions are in blue.
catechol oxidase (IbCO) crystal structures,29 and on the TYR-related pro-

tein (TRP1) crystal structure (vide infra)30 have been generated. More
recently, with Alphafold AI interface,31 a model was also generated that
overlaps well with the one based on TRP1 (Figure 4).
The type 3 copper-containing proteins have been shaped by evolution
to bind and activate dioxygen: (i) O2 transporters in mollusks and arthro-
pods (Hc) where the bound dioxygen is shielded by the protein matrix and
(a) (b)
(c) (d)
Figure 5. Structures of the TYR active site from Streptomyces castaneoglobisporus

bacteria (ScTYR) showing: (a) the dicopper(I) deoxy form (PDB ID: 2ahl), (b) the
dicopper(II) oxy form (PDB ID: 1wx4), (c) the dicopper(II) met1 form (PDB ID:
2zmx), and (d) the dicopper(II) met2 form (PDB ID: 2zmy) that are involved in the
catalysis. Cu(I) are displayed as yellow balls and Cu(II) are displayed as blue balls.
therefore not reactive, (ii) mono-oxygenases in ortho-hydroxylation of

phenols (TYR and AUS) and (iii) di-oxygenases in catechol oxidation
(TYR and CO) in which activated dioxygen can react with the substrate.
From all the collected spectroscopic data and 3D structures from diverse
TYR sources, three different forms have been identified, the met, oxy, and
deoxy forms (Figure 5).
B. Catalytic Mechanism of Tyrosinase

The TYR resting state is composed by 85% of the met1 form, in which a
hydroxyl ligand holds the two Cu(II) ions approximately 3.2–4.0 Å apart.
Recent QM/MM calculations seem to suggest that an aqua (H2O) bridging
54 Faure et al.
ligand might be energetically more favorable than a hydroxido ion.32 In

addition, with ScTYR, a met2 form having two hydroxido or aqua bridging
ligands was also identified and characterized.33 From X-ray absorption
spectroscopy, Bubacco et al.34 reported that the met2 form is predominant
in solution. The remaining 15% of the TYR resting state is composed by
the oxy form where a (m-η2:η2)-peroxo ligand bridges the two Cu(II) ions.
While the met forms are only able to react with catechol in a catecholase
cycle, the oxy form can react both with catechol and phenol in an inter-
mixed catecholase and cresolase cycles (Figure 6). The catecholase and
cresolase cycles both converge to the deoxy form where the two copper
ions are in the formal Cu(I) oxidation state. The deoxy form reacts with
dioxygen to produce the active oxy form and the cycle continues.
The structure of the (m-η2:η2)-peroxo ligand of the oxy-TYR is now
well documented. Several X-ray structures of model compound35,36 and
of the oxy-TYR confirm this structure.17 Regarding the reactivity of the
oxy-form, Karlin’s pioneering work on chemical model complexes
has revealed the electrophilic behavior of the (m-η2:η2)-peroxo ligand.37
The same feature was observed also with the oxy-TYR. Thanks to a
HO
2 H+ OH
His His
II O II HO
Cu Cu
His His
O O O
H+
II O His His HisI
His II oxy
Cu Cu O2
His O His
His His
His His
Catecholase Cresolase O
I His
activity CuI Cu activity O
3 H+ His His His II
II
Cu Cu
His His His
His O
deoxy His
H2O
H2O His
O
O
H+
His H His
II O O O-atom transfer
II O His
Cu Cu O
II O II
His His His
Cu Cu
His His His His
I O
I
met H His
His
2 H+
HO
OH
Figure 6. General mechanism for the cresolase and catecholase TYR activities.
kinetic study of the TYR catalyzed phenol hydroxylation, Itoh et al. have
shown that the rate-determining step is the O-atom transfer occurring
through an electrophilic aromatic substitution mechanism.38
With data from different groups, the TYR mechanism is quite well
established39 (Figure 7). The first step is the formation of a phenolate
bond on the CuA site of the oxy-TYR. Two mechanisms have been pro-
posed for the formation of the phenolate bond on the CuA site. The first
was proposed by Decker and Tuczek on the base of crystallographic data
of ScTYR.40 The authors proposed a pre-orientation of the phenolate by
p-staking interaction with the H194 of the CuB site followed by a pheno-
late sliding toward CuA (Figure 7A). With AbTYR, a similar proposal
involving a pre-orientation of the phenolate by a p-staking interaction
with a histidine residue of the CuB site was proposed by Dijkstra.41 Based
on crystallographic data on AoTYR, Itoh et al. proposed another mecha-
nism for the formation of the CuA-phenolate. While Decker and Tuczek
propose a shift from phenolate toward CuA, Itoh proposes an opposite
movement, that of CuA toward phenolate located in a stabilizing enzy-
matic pocket42 (Figure 7B). This latter proposal is supported by several
strong evidences obtained with AoTYR mutants, copper-depleted or zinc-
replaced AoTYR.
Both mechanisms lead to phenolate binding to CuA site in trans posi-
tion relative to H63 for ScTYR with a concomitant elongation of the
H63-CuA distance (H103 for AoTYR). Phenolate binding impulses a 90°
rotation of the O–O plan of the peroxo group toward the aromatic ring
that allows the peroxide σ* orbital to overlap with the p orbital of the
phenolate ortho-position thus facilitating the electrophilic attack of the
peroxide by the phenolate.39,40 This process leads to the formation of a
catecholate bridging the two copper centers. The structure of this cat-
echolate that is not well defined could be a κ2-catecholate on CuA (a), or
a m-1,4-catecholate (c) or the combination of both, that is (η2:η1)-
catecholate (b) (Figure 7C).
C. Tyrosinase-Related Proteins TRP1 and TRP2

Two other enzymes, TRP1 and TRP2, closely related to TYR, are
involved in the biosynthesis of eumelanin pigments.30 TRP1 and TRP2
share ~70% amino acid homology, suggesting that they originate from a
56 Faure et al.
Figure 7. Mechanisms proposed by (A) Decker and Tuczek39 on ScTYR and (B) Itoh
on AoTYR42 with putative structures (a)–(c) for the catecholate-TYR form.
common ancestral gene. While TRP2 was very early identified as zinc-
containing analogue involved in DHIC formation, TRP1 was firstly identi-
fied as copper-containing analog having a catecholase activity and
involved in the oxidation of DHI and DHICA into the eumelanin pigments
precursors.43 However, the recent 3D structure of TRP1 solved by Soler-
López and Dijkstra et al.44 revealed, against all expectations, the presence
of Zn(II) instead of Cu(II) ions in the active site thus ruling out the pos-
sible role of TRP1 in DHICA oxidation. Since this publication, TRP1
function has become enigmatic, and several hypotheses have been formu-
lated such as a role in stabilization of HsTYR as proposed by Dolinska
et al.45 or its contribution in increasing the ratio between eumelanin and
pheomelanin by promoting the synthesis of eumelanin as proposed by
Ishikawa et al.46 But an unresolved question still remains: “could TRP1
integrate Cu(II) ions in vivo?” Indeed, ex vivo experiments have shown
that catecholase activity (DHIC oxidation) can be partially conferred on
the human TRP1 when its Zn(II) ions are replaced by Cu(II) ions.44
Another possibility is that like TRP2 (Figure 8b), TRP1 may have an
(a)
(b)
Figure 8. Reaction catalyzed by TRP2 (a) and reaction which may be catalyzed by
TRP1 (b).
58 Faure et al.
activity linked to the Lewis acid property of the Zn(II) ions such as the
decarboxylation of Dopachrome into DHI that is supposed to be non-
catalyzed (Figure 8b).
IV. Dysfunction in Tyrosinase Activity

A. Pathologies Linked to TYR Dysfunction
Being involved in the biosynthesis of melanin, TYR is associated with
cutaneous pigment disorders essentially linked to (i) the hypopigmenta-
tion of the skin such as in albinism and vitiligo or (ii) the hyperpigmenta-
tion of the skin such as in melanoma, melasma, and solar lentigo.47
Overexpressed during tumorigenesis,48 TYR has been demonstrated
to be a sensitive marker for melanoma.49 Melanoma, which is the most
serious type of skin cancer, is a malignant tumor arising from melano-
cytes. During the tumorigenesis process, melanocytes lose their interac-
tions with epidermis cells (keratinocytes) that result in uncontrolled
proliferation followed by an invasion of malignant cells. Melanoma is the
deadliest form of skin cancer (20–25% mortality), ranked 17th among
cancers of all genders with more than 150,000 new cases of melanoma in
2020.50
It was observed that an increase in melanogenesis may affect immune
responses to chemotherapy and radiotherapy for melanoma. A high level
in melanogenesis shortens overall survival in patients with metastatic
melanoma. Inhibition of melanogenesis appears a rational approach to the
therapy of metastatic melanoma.51 In that sense, TYR DNA vaccines have
been widely developed for the treatment of melanoma since they are much
more stable than conventional vaccines and since they have less severe
side effects.52 Treatments by TYR DNA vaccines are generally associated
with adjuvants to induce the innate immune system. Because of its over-
expression mainly confined in melanocytes during tumorigenesis, TYR is
a potential molecular target for the development of specific inhibitors that
could be used as adjuvant to TYR DNA vaccine in melanoma therapy.53,54
This is supported by recent observations suggesting that melanin secreted
from melanoma cells would be responsible for the immunomodulation of
tumor microenvironment. An increase of the level of secreted melanin
would promote tumor angiogenesis, thus sustaining melanoma progression

and metastasis.55,56 Since the reduction/suppression of melanogenesis is
supposed to restore the sensitivity of cancer cells to immuno-, radio-, or
chemotherapies, the use of selective inhibitors of TYR and TRP1,2 as
adjuvants represents a realistic strategy for melanoma therapy.57–59
B. Skin Whitening
Depigmenting agents are commonly used in dermatology in the treatment
of hyperpigmentary disorders.60 Kojic acid in association with hydroqui-
none is prescribed in Europe under medical supervision, in the treatment
of post-inflammatory hyperpigmentation61 and in melasma.62
Unfortunately, the effectiveness of these agents has led to their use being
diverted, toward non-therapeutic purposes, in voluntary depigmentation
with the objective of lightening the natural complexion of the skin.63,64
This practice, which is widespread in sub-Saharan Africa, has become a
real public health problem as it leads to serious dermatological and sys-
temic complications. The ingredients historically encountered in cosmetic
products intended to lighten the skin are hydroquinone, mercury deriva-
tives, and corticosteroids. Recent studies have demonstrated the harmful
effects of these active ingredients for human health with the development
of ochronosis, skin complications, addiction to steroids, periorbital hyper-
chromia, facial lesions, or even kidney failure.65,66 Although better con-
trolled, this practice is also rampant in Asia (China, Japan, and India)
where it is estimated that these products represent nearly 10% of the
cosmetics market.
In this context, measures must be taken to curb this tendency. Facing
the difficulty of fighting these dangerous practices through laws, the
development of harmless and inexpensive bleaching agents accessible to
all could be a solution to this problem.
V. Tyrosinase Inhibition
Because inhibition of TYR is a well-established approach to control
in vivo melanin production, the development of inhibitors has a huge eco-
nomic and industrial impact in medicine and cosmetics (vide supra) but
60 Faure et al.
also in agriculture to reduce the browning of fruits and vegetables.67 This

impact is assessed by the increasing number of review articles on TYR
inhibitors reported in the recent literature.68–74 A large part of this litera-
ture is devoted to naturally occurring compounds such as flavonoids and
polyphenolic compounds,71 triterpenes, alkaloids, as well as mixture
extracts from plants73–75 and peptides.72
A. Variation in TYR Sources

Due to the commercial availability of the fungal AbTYR, most TYR inhi-
bition studies have been conducted on this TYR as a model for the human
form (HsTYR). The molecules resulting from these tests are often incor-
rectly designated as potential TYR inhibitors that can be used in human
pathologies.76 Although the structure of the first coordination sphere of
the dinuclear active site is conserved in all the TYRs, they present signifi-
cant structural and cellular localization differences.11 In addition, although
there are very strong intra-species sequence homologies, the inter-species
differences are important. For example, within a region of 48–49%
sequence coverage, fungal TYRs have only 22–24% identity with mam-
malian ones. It should be mentioned that the fungal enzyme AbTYR is a
cytosol-soluble heterodimer whereas HsTYR is a 529–amino acid glyco-
protein anchored to the membranes of melanosome vesicles by a 21–
amino acid transmembrane domain. Some differences reside also at the
level of the second coordination sphere of the metal center with residues
of polar or hydrophobic character controlling the accessibility to the
active site and substrate’s selectivity (activity controllers).77 As it can be
seen in Figure 9, recent 3D structures highlight the huge differences in
active site accessibility between AbTYR, ScTYR, and HsTYR.
B. Transition-State Analogue (TSA) Inhibitors

One way to selectively inhibit TYR activity is to target its copper binuclear
active site that is unique within human metalloenzymes. Some of TYR
inhibitors such as phenylthiourea (PTU),78 thiosemicarbazones (TSC),79–89
L-mimosine (Mim),90,91 Kojic acid (KA),92,93 tropolone (Trop)94 and
thujaplicin (Thuj),95,96 2-hydroxypyridine N-oxide (HOPNO),97 and
(a)
(b)
(c)
Figure 9. Accessible surface of TYR structures with longitudinal sections showing

the accessibility of the copper active site; (a) AbTYR (PDB ID 2y9x), (b) ScTYR (PDB
ID 2ahl), and (c) HsTYR (Homology model).30
2-pyridinethiol N-oxide (HSPNO)98 fulfil this objective. These inhibitors

are chelators that all target the dinuclear copper site of TYR (Figure 10).
Among them, kojic acid, L-mimosine, tropolone, thujaplicin, HOPNO, and
HSPNO are also TSAs of the O-atom transfer reaction. Indeed, they
present structural analogies with catechol, but they are not oxidizable
(Figure 10b). These molecules present a double interest, they are both capa-
ble of modulating TYR activity (medicinal interest) and they are likely to
62 Faure et al.
(a)
(b)
Figure 10. TYR Inhibitors that target the copper dinuclear active site: (a) metal
chelating by the thiourea moieties in blue and (b) TSAs.
Table 2. Inhibition constant (KI) for selected TSA compounds.
Fungus Bacterial Mammalian

Inhibitors AbTYR SaTYR BmTYR HsTYR
99 100
Mim C, 8 mM C, 54 mM nd C, 10.3 mM100
KA C, 4.3 mM101 C, 109 mM100 M, 3.5 and M, 350 and
150 mM102 730 mM100
HOPNO C, 1.8 mM97 C, 7.7 mM103 nd C, 128 mM104
C, competitive; M, mixed; nd, not determined.
provide information on the structure of the catecholate-TYR intermediate

(structural and mechanistic interest).
Kojic acid, L-mimosine, and HOPNO inhibitors for which KI con-
stants are available on three sources of TYR, fungus (AbTYR), bacterial
(SaTYR and BmTYR), and human (HsTYR) have been used as reference
to better understand the mechanism of TYR inhibition by TSA com-
pounds. As expected, the selected TSA compounds behave as competitive
inhibitors for the substrate L-DOPA (Table 2). Kojic acid behaves as
mixed inhibitor for the HsTYR and BmTYR but with a competitive com-
ponent much lower than the uncompetitive one. The inhibitory properties
estimated from the KI reveal significant differences between the three
sources of TYR, and the best inhibitions are always obtained with the
fungal AbTYR. However, L-mimosine, which is the best inhibitor for the
(a) (b)
Figure 11. TRP1 active site with (a) L-mimosine (PDB ID: 5m8n) and (b) kojic acid
(PDB ID: 5m8m).44 The coloring of the cycles in yellow shows the stabilizing
p-stacking interactions.
HsTYR, shows comparable inhibitory properties for the fungal and the
human TYRs.
1. L-Mimosine
A TRP1 structure with L-mimosine as ligand has been recently solved by
Soler-López and Dijkstra et al.44 Surprisingly, this structure reveals that
L-mimosine does not directly interact with the Zn(II) ions but that the two
O-atoms of L-mimosine are H-bonded to the water molecule bridging the
two Zn(II) ions (Figure 11a). The structure also points out additional inter-
actions such as p-stacking with the His381 and H-bonding of one of the
O-atom with Ser394.
2. Kojic acid and derivatives

Based on ESEEM (electron spin echo envelope modulation),105 HYSCORE
(Hyperfine sublevel correlation),106 and XAS (X-ray absorption spectros-
copy)34 coupled to MXAN simulations,107 all performed on SaTYR,
Bubacco et al. proposed a model where kojic acid is binding in a η2:η1-KA
coordination mode, that is, as bidentate ligand to CuB with one O-atom
axially coordinated and the second bridging the two copper ions in an
equatorial coordination position in place of one of the two water (OH)
bridging O-atoms (Figure 12a).
64 Faure et al.
(a) (b) (c)
Figure 12. Kojic acid bound to a binuclear copper center, (a) SaTYR-KA adduct
according to X-ray absorption spectroscopy studies34 (ScTYR numbering); (b) X-ray
crystal structure with a copper model complex,108 and (c) QM/MM dynamics simu-
lations for ScTYR-KA adduct.108 Stable position is obtained after 2.5 ps of QM/MM
dynamics simulations. The coloring of the cycles in yellow shows the stabilizing
p-stacking interaction and the dotted red line the H-bond interaction between S206
and KA.
The η2:η1-KA coordination mode has been also reported in model

chemistry (vide infra) by Belle et al.108 With a binuclear Cu(II) com-
plex109 known to be a structural and functional model of TYR diphenolase
activity, an adduct with kojic acid that exhibits this binding mode was
obtained and characterized by X-ray diffraction (Figure 12b).
While the XAS studies identify a η2:η1-KA coordination mode for
kojic acid,34 it is not obvious on which Cu-ion (CuA vs. CuB) kojic acid is
bidentatety bound. QM/MM simulations performed by Jamet et al.108
have tried to clarify the situation. Since no X-ray structure of the SaTYR
was available, QM/MM simulations were performed using the structure of
ScTYR that shares more than 82% sequence identity with SaTYR.
Therefore, both possibilities were tested in dynamic QM/MM simula-
tions. Kojic acid as bidentate ligand to CuA was found to be more stable
than as bidentate ligand to CuB. Moreover, the simulations highlight
p-stacking interaction with the His388 and H-bonding interaction of
one of the O-atoms with Ser206, interactions already observed with
L-mimosine in TRP1 (vide supra). During the simulation with kojic acid
as bidentate ligand to CuA, the His63 coordinated to CuA, moves to a
distance around 3.76 Å from the copper leading to a penta-coordinated
CuA-ion (Figure 12c). This agrees with EXAFS (Extended X-Ray

Absorption Fine Structure) data,34 that support histidine decoordination in
SaTYR-KA complex. During the simulation with kojic acid as CuB biden-
tate ligand, the CuB–O3 distance increases until decoordination and the
O3-atom shift toward CuA to become a CuA-bidentate ligand.
More recently 3D X-ray structure of the BmTYR in complex with
kojic acid was reported by Fishman et al.25 Surprisingly, in this structure,
kojic acid is oriented with its hydroxymethyl group in the direction of the
active site at a relatively remote distance of 7 Å, which is the opposite of
what was reasonably expected and observed with SaTYR (Figure 13b).
Kojic acid is stabilized by interactions with Phe197, Pro201, Asn205, and
Arg209. The same group reported later a novel structure where kojic acid
is oriented as expected toward the active site (Figure 13a). But in this
structure, kojic acid does not directly interact with the Cu(II) ions102
(Figure 13a). The hydroxyl and carbonyl groups are found at distances
greater than 3.3 Å from the CuA center, which does not allow to consider
coordination bonds. However, kojic acid is stabilized by p-stacking inter-
action with His208 and H-bonding interaction with the water molecule
bridging the two Cu(II) ions, a situation already encountered for
L-mimosine in interaction with TRP1. Furthermore, Soler-López and
(a) (b)
Figure 13. BmTYR active site with kojic acid (a) oriented to the active site (PDB ID:
5i38)102 and (b) in entrance to the active site (PDB ID: 3nq1).25 The coloring of the
cycles in yellow shows the stabilizing p-stacking interaction.
66 Faure et al.
Dijkstra et al. reported a structure of TRP1 with kojic acid in which

the same features are retrieved44 (Figure 11b). These two orientations of
kojic acid support the mixed inhibition observed for BmTYR (Table 2).
Kojic acid in the active site precludes the substrate’s fixation while in the
other position, it reduces substrate accessibility and product release.
Apart from the binding mode of kojic acid to the met-TYR, these lat-
est results raise fundamental questions about the hydroxylation mecha-
nism catalyzed by TYR, namely: (i) if the substrate binds directly to the
metal center or if the oxidation takes place without binding and (ii) if the
substrate binds to the metal center on which site does it bind, to CuA or
CuB? Until now, all the mechanistic discussions agreed on the hypothesis
that the substrate had to be fixed to the metal center, by a yet not eluci-
dated mechanism (Figure 7a vs. 7b),39,42 to induce the rotation of the
peroxo group, which triggers the reaction.40
Crystallization results with inhibitors were achieved by soaking TYR
crystals in an inhibitor solution. These conditions are however far from
those used during catalysis, which raises questions about the relevance of
these structures for understanding the catalytic mechanism of TYR.
Nevertheless, they can be seen as inspiration for the development of
increasingly effective inhibitors. Starting from the observation of two dif-
ferent binding sites for kojic acid, ditopic inhibitors have been synthe-
sized and studied (KA-TSA110 and bis-KA111) (Figure 14). These
inhibitors were designed to interact with both high and low affinity
KA-sites in BmTYR, and probably in HsTYR. Unfortunately, they dis-
played only slightly improved inhibitory properties as compared to kojic
acid.
Figure 14. Ditopic inhibitors based on kojic acid.

(a) (b)
Figure 15. Tropolone in interaction with (a) AbTYR active site (PDB ID: 3y9x)20 and
(b) showing the hydrophobic pocket above the active site where tropolone is
stacked.
3. Tropolone and derivatives

Tropolone is a slow-binding, reversible AbTYR inhibitor that only inhib-
its oxy-TYR.112,113 The first crystal structure of AbTYR in interaction
with tropolone was reported by Dijkstra et al.20 In this structure, tropolone
binds to a large cavity (Figure 15b) without the need of conformational
change of the protein to allow inhibitor’s entrance. Since the O-atoms of
tropolone are quite away from the copper center (>3.5 Å, Figure 15a), it
was concluded that this tropolone-binding mode represents a pre-Michae-
lis complex that foreruns dioxygen fixation.
TRP1 also binds tropolone in a κ2-coordination mode on the ZnA ion
as shown by crystal structures.44 Like other TSA inhibitors, tropolone
binding occurs by aromatic p-stacking interactions with H381, ligation of
the keto- and hydroxy groups to the ZnA ion, and H-bonding interaction
with S394 (Figure 16).
a, b, and g-thujlaplicins, naturally occurring compounds containing a
tropolone framework, exhibit interesting inhibition properties of AbTYR
and HsTYR (Figure 17).114,115 In the case of AbTYR, with the exception
of a-thujlaplicin, which isopropyl group precludes correct positioning in
the active site, both b and g-thujlaplicins exhibit IC50 in the nanomolar
range. They are therefore 700-fold more active than the kojic acid
68 Faure et al.
Figure 16. Tropolone in interaction with the TRP1 active site (PDB ID: 5m80).44
The coloring of the cycles in yellow shows the stabilizing p-stacking interaction.
Figure 17. Thujlapines as TYR-inhibitors.
reference. For HsTYR, the tendency is the same; g-thujlaplicin exhibits

IC50 in micromolar range, 500-fold more active than the kojic acid
reference.
4. HOPNO inhibitor
In the absence of 3D structures of TYR with the HOPNO, XAS spectra
were recorded on SaTYR in complex with HOPNO (Figure 18).
Significant modifications were observed in the XANES part of the XAS
spectra upon addition of HOPNO to the SaTYR indicating an interaction
of HOPNO involving a rearrangement around the coordination sphere of
Figure 18. XAS of SaTYR alone (green) and with KA (blue) and HOPNO (red).
the Cu(II) ions. The same behavior was also observed upon addition of
kojic acid, but significant differences indicate that OPNOa and kojic acid
bind differently the copper center.
The biomimetic approach in developing small molecular complexes
reproducing the main structural characteristics of the active site and the
reactivity of metalloenzymes allows to better understand their structure–
function relationships [Chapters 3 and 4].116 With this strategy, Belle
et al. has reported the synthesis and characterization of a copper complex
based on H-BPMP ligand that reproduces: (i) the copper coordination
with 3 N-atoms and a m-phenolate and m-hydroxy groups allowing a copper–
copper distance of 2.966 Å and (ii) the catecholase activity of TYR and
CO109 (Figure 19).
On the contrary of the kojic acid adduct to BPMP(Cu2)OH complex
that features a η2:η1-KA coordination mode107 (vide supra), it was
observed a HOPNO adduct to BPMP(Cu2)OH complex featuring a
κ2-OPNO coordination mode on only one of two copper ions.97 This high-
lights the differences between inhibitors interaction at a dinuclear active
site and the difficulty to draw general conclusions for all TSA inhibitors.
a
Since the pKa for the N–OH group of HOPNO is 6.07 [98], in the conditions used
(pH = 7) HOPNO is mainly in the deprotonated form OPNO.
70 Faure et al.
Figure 19. Biomimetic strategy in TYR study.
An important factor in the formation of TSA adducts is the geometry

around the Cu(II) ions. Although the coordination around the two Cu(II)
ions is the same in all TYR active sites, there are significant differences
in their accessibility but also by their geometry, which reflects their reac-
tivity. The geometry of penta-coordinated complexes, that is, square-
based pyramid (SP) versus trigonal bipyramid (TBP) can be characterized
by the t factor, which is determined by taking the difference of the two
largest angles in the coordination sphere of the metal divided by 60.
A t = 1 is characteristic of a pure TBP, whereas a t = 0 is of a pure SP
geometry.117 In the met2-form of ScTYR, the two Cu(II) ions present
distorted SP geometries as reflected by their t factors (CuA, t = 0.3 vs.
CuB, t = 0.5, Figure 20a). The distortion is more pronounced for CuB than
is between SP and TBP geometries.
A similar distortion is also observed in the model complex BPMP(Cu2)
OH, in which one Cu(II) ion is in a pure TBP geometry while the other
exhibits a distorted SP geometry (Figure 20b). The distortion in coordina-
tion can be controlled by the size of the metallacycle of the chelate ligands
around the Cu(II) ions. The size of the metallacycle depends on the num-
ber of CH2 group (methano vs. ethano bridge) between the N-tertiary
amino group and the pyridine nucleus. The dinuclear complex BPEP(Cu2)
OH featuring four 6-membered ring metallacycles, has one Cu(II) ion in
a pure SP geometry (t = 0) while the other is in distorted SP geometry
(t = 0.3) (Figure 20c). The HOPNO adduct features a m-1,4-OPNO
(a)
(b) (c) (d)
Figure 20. OPNO ligand binding modes on dicopper(II) center. (a) met2-ScTYR
active site (PDB ID: 2zmy17) with the t factor117 for the two copper centers; (b,c) the
three different coordination mode obtained with model complexes.
coordination mode.98 The dinuclear complex BPMEP(Cu2)OH featuring

two 6- and two 5-membered ring metallacycles, has one Cu(II) ion in pure
SP geometry (t = 0) while the other exhibits a distorted SP geometry (t =
0.53) (Figure 20d). Its HOPNO adduct features a third coordination
mode, that is, η2:η1-OPNO.103 With three slightly different ligands that
impose a certain coordination geometry around the Cu(II) ions, it is pos-
sible to control the coordination mode of the HOPNO inhibitor. These
72 Faure et al.
(a) (b)
(c) (d)
Figure 21. The two minimized structures (a) the m-1,4-OPNO adduct and (b) the
κ1-OPNO adduct. (b) XAS simulation of the m-1,4-OPNO adduct and (d) of the
κ1-OPNO adduct. XAS of SaTYR (black plain line) and simulation (red dotted line).
results emphasize that the coordination mode of OPNO is sensitive to

small changes in the environment of the metal center.
To decipher the binding mode of OPNO ligand onto TYR, Jamet
et al. performed QM/MM calculations using data from the model com-
plexes exhibiting the three different OPNO binding modes. Starting from
these binding modes, the systems converge to two structures, the m-1,4-
OPNO and at 10 kcal/mol lower the one with OPNO as monodentate
ligand that establishes a H-bond with a S206 residue (Figure 21a and b).
From these structures, the simulation of the XANES spectra have been
performed using MXAN software.107 The simulation for the m-1,4-OPNO
adduct led to a plot (Figure 21c, red dotted line) that does not overlap
correctly with the measured XAS of the SaTYR-OPNO adduct
(Figure 21c, black plain line). Better results were obtained with the simu-
lation of the κ1-OPNO adduct. In that case the simulation better repro-
duces the measured XAS of the SaTYR-OPNO adduct (Figure 21d red
Figure 22. HOPNO-embedded aurones used as TYR inhibitors.
dotted line vs. black plain line). These results tend to confirm a dynamic
behavior of the OPNO ligand in the TYR active site, with probably sev-
eral species in equilibrium such as the κ1-OPNO and m-1,4-OPNO
adducts.
5. HOPNO derivatives
Naturally occurring aurone compounds have been reported in the litera-
ture as inhibitors in melanin biosynthesis in human melanocytes.118 The
true effect of aurones is controversial since some compounds of this fam-
ily act as alternative substrates rather than inhibitors.119,120 Nevertheless,
aurones display strong affinities for TYR, which encourages their integra-
tions in TSA moieties such as HOPNO. Therefore, several HOPNO-
embedded compounds have been reported in the literature for TYR
inhibition.104,119,121
The HOPNO-embedded aurones I–III were evaluated for the inhibi-
tion of three forms of TYR, one mammalian (HsTYR), one bacterial
(SaTYR) and one fungal (AbTYR) (Table 3). HOPNO-embedded aurones
I–III exhibit better inhibition properties than the parent compound
HOPNO, although the effect is less pronounced for AbTYR. In the case
of SaTYR, the substitution of the aurone moieties seems does not signifi-
cantly modify the inhibition properties since the three compounds I–III
exhibit KI in the range 0.1 µM. On the contrary for the HsTYR, this effect
is more important, with a KI = 0.35 µM for compound I and KI = 1.20 µM.
for compound III.
The HOPNO-embedded aurones I–III were also tested in the inhibi-
tion of melanin biosynthesis in a human-integrated cellular model (MNT-1
74 Faure et al.
Table 3. Inhibition constant KI (µM) on purified TYRs of HOPNO-embedded

aurones I–III.
HsTYR SaTYR AbTYR
Inhibitors KI type Ref. KI type Ref. KI type Ref.

104 103 97
HOPNO 128 C 7.7 C 1.8 C
104 122 119
I 0.35 C 0.13 C KIC = 1.27 M
KIU = 1.62
104 122 122
II 1.02 C 0.15 C KIC = 0.34 M
KIU = 0.90
104 122 122
III 1.20 C 0.16 C KIC = 2.9 M
KIU = 2.5
Table 4. IC50 (μM) of HOPNO-embedded aurones I–III in TYR

inhibition in human MNT1 melanoma cells.104
Inhibitors Purified HsTYR Lysate Whole cells Cytotoxicity
HOPNO 128 1300 150 > 200
I 0.35 16.6 85.3 > 500
II 1.02 30 120 80
III 1.2 34 119 > 500
cells). Results from MNT-1 cell lysates conﬁrmed the tendency observed
with purified HsTYR (I > II ~ III >> HOPNO). However, they displayed
a low capacity to reduce melanogenesis in MNT-1 whole cells. Except
compound II, the others exhibit a low cytotoxicity (Table 4).
HOPNO-embedded aurones have a great inhibition potency. However,
the lower efficiency in suppressing melanogenesis in MNT-1 human
whole cells remains to be addressed.
VI. Conclusions
From the medicinal point of view, the TYR TSA compounds are promis-
ing inhibitors because they target the binuclear copper active site, which
is unique in humans. Their potential lies in the fields of antifungals, anti-

bacterials, and in diseases linked to disorders of human melanocytes.
Among the TSA compounds, HOPNO derivatives present the highest
potential due to the possibility of decorating the pyrimidone ring to
improve inhibitory properties, cell penetration, and inter-species
selectivity.
From the mechanistic point of view, TSA compounds are molecular
tools capable of providing functional information on TYR. On one hand,
functional studies on the mechanism of TYRs converge on a consensus in
which the phenolate binds to the CuA site of the m-η2:η2-peroxo species to
trigger the O-atom transfer reaction (Figure 7A and B). On the other hand,
all the 3D structures of the TSA inhibitors (L-mimosine, kojic acid, and
tropolone) with TRP1 and AbTYR show that the inhibitors do not bind to
the binuclear copper active site but are positioned through p-stacking and
H-bonds interactions. This raises the following question: is it possible that
the O-atom transfer takes place without the phenolate being bound to the
CuA (Figure 23)? This is an interesting question that would deserve to be
addressed.
Figure 23. O-atom transfer to phenolate without binding to copper ions.

76 Faure et al.
Acknowledgment
The authors gratefully acknowledge the Cosmethics project, an
“Investissements d’Avenir” program (ANR-15-IDEX-02) and the French
national synchrotron facility Soleil (proposal number 20120202).
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Oct. 2012.
3 Modeling Tyrosinase Activity

Using m-Xylyl-Based Ligands:
Ring Hydroxylation, Reactivity,
and Theoretical Investigation
Puneet Gupta¶ and Rabindranath Mukherjee†,‡,§
¶
Department of Chemistry, Indian Institute of Technology Roorkee,
Roorkee, Uttarakhand 247 667, India
†
Department of Chemistry, Indian Institute of Technology Kanpur,
Kanpur, Uttar Pradesh 208 016, India
‡
Present address: Department of Chemistry and Chemical Biology,
Indian Institute of Technology (Indian School of Mines) Dhanbad,
Dhanbad, Jharkhand 826 004, India
§
[email protected]
I. Introduction
A. General Considerations
Dioxygen is crucial for aerobic organisms, serving as a primary source of
energy with its thermodynamically favorable reduction to water. It is used
as an oxidant in nature. However, the use of dioxygen as an oxidant is not
straightforward. Dioxygen is a diradical species and hence its triplet
ground state electronic structure makes it unreactive, under ambient con-
ditions, toward most organic substrates, which are generally diamagnetic
81
82 Gupta and Mukherjee
and hence have singlet ground state. This is due to spin-forbidden nature
of the reaction.1,2 In nature, metalloenzymes activate O2 and its further
processing is done by reduction for oxidation reactions.3 The reduced
forms of O2 such as O22− (peroxide) or O2− (oxide) exist in singlet ground
states, therefore they can easily react with singlet organic molecules.
In addition, O atom incorporation into biological substrates occurs by
functionalization through mono- or dioxygenation processes mediated by
metalloenzymes. The corresponding metalloenzymes are called monoox-
ygenases and dioxygenases, respectively. The former one incorporates
one O atom to the substrate and the other O atom of dioxygen is reduced
to H2O. The latter one incorporates both the O atoms to the substrate.
Molecular-level understanding of basic chemical principles and
mechanisms of O2 activation at metal sites and its subsequent processing
for oxygenations and oxidations of organic molecules have been of inter-
est for a few decades now. The continued interests are because of their
potential relevance to biological processes.4–16 Due to its generally acces-
sible Cu(II) and Cu(I) states and bioavailability, copper plays a wide
variety of roles in nature.1a,4
In the domain of synthetic modeling approach involving biomimetic
studies,17 many factors affect the reactivity of low-molecular-weight
metal complexes with tailor-made organic ligands toward dioxygen.1–16,17b
Since the reaction causes one-electron oxidation of the metal center with
reduction of dioxygen, the reduction potential of MII/MI (for M = Cu) or
MIII/MII (for M = Fe) redox couples is one of the important factors gov-
erning the reaction between the metal complex and dioxygen.9c The redox
potentials of metal complexes vary dramatically depending upon the
ligand environment. The solvent exerts a strong effect as well. One-
electron reduction of dioxygen is a thermodynamically unfavorable pro-
cess, since for O2 + 1e– = O2– redox process the Eo = −0.35 V versus NHE
at pH 7. However, for the two-electron, O2 + 2H+ + 2e– = H2O2 reduction
process (Eo = 0.28 V vs. NHE at pH 7) and for four-electron, O2 + 4H+ +
4e− = 2H2O reduction process (Eo = 0.82 V vs. NHE at pH 7) are favorable
reactions. Although the reduction potential is a good predictor of the reac-
tivity of the metal complexes toward dioxygen, it is not directly correlated
with the feasibility for the formation of the dioxygen adduct, since the
Modeling Tyrosinase Activity Using m-Xylyl-Based Ligands 83
redox potentials are defined for outer-sphere-type electron-transfer

reactions.9c On the other hand, metal-dioxygen interaction is an inner-
sphere-type electron transfer process.
The feasibility of formation of the “M–O2” or “M–O2–M” adduct is
associated with both thermodynamic and kinetic factors.9c The former
factor is directly related to the stability of the resultant metal-dioxygen
adduct. The contributors to stability of the metal complex due to the ligand
environment include the ligand donor set, geometry, and substituent-
induced electronic and steric effects, and chelate-ring size.17b The availa-
bility of an open coordination site at the metal center to bind dioxygen
and the steric bulk of the ligand substituent groups are also important,
having a striking kinetic effect on the binding rate of dioxygen to the
metal center.
B. Scope of the Review

Copper proteins/enzymes participate in activities such as dioxygen bind-
ing, dioxygen activation, and oxidation and reduction processes of the
substrates.4 The activities of copper enzymes triggered by dioxygen can
be broadly classified as oxidase, monooxygenase, or dioxygenase.5a,15
These reactivity aspects have long interested inorganic chemists. The
protein of interest in the context of this article is that of antiferromagneti-
cally coupled dicopper-containing monooxygenase, tyrosinase, in its
oxygenated state.1–14,17a The structural and spectroscopic modeling of
tyrosinase (monophenolase and diphenolase activity; see below) active
site is an important and challenging topic of bioinorganic chemistry of
copper. It has been well-documented that coordination chemists can make
significant contributions to reactivity studies and mechanisms.1a,2,3,5–17b,c
The closely related members of this family of proteins are hemocyanin
and catechol oxidase.4,9c,d,14 The basic structural and electronic structural
properties of these type 3 copper proteins/enzymes9d,17a are the necessary
reference for any attempt to reproduce in biomimetic systems key aspects
of the protein structure and associated reactivity.
A large volume of outstanding contributions has been made in the
copper–dioxygen chemistry by synthetic chemists.1a,2,3,5–16 The success
story on the synthetic front in structural and spectroscopic characteriza-

tion of three Cu2O2 cores (side-on peroxo, bis-oxido, and end-on peroxo)
have not only enriched but also sharpened our knowledge on dicopper
proteins and enzymes. The outcome of an in-depth mechanistic studies of
tyrosinase-like reactivity on suitable substrates using these Cu2O2 reactive
intermediates is considered as a breakthrough in molecular-level under-
standing of the functioning of tyrosinase.
The focus of this article has been divided into two main parts. The
first section describes how ligand modifications8a,11 can often result in
drastically different dioxygen reactivity of the dicopper(I) complexes. We
will examine several examples to highlight the ligand aspects such as
chelate-ring size, electronic and steric effects, nitrogen donor type, and
their consequences on the reactivity of the copper(I) complexes toward O2.
The ligand systems we have primarily paid attention to are m-xylyl-based
dinucleating chelating ligands of varying denticity. We will emphasize
the importance of such ligands accommodating two copper ions, initially
as Cu(I) and after activation of O2 as Cu(II)/Cu(III), the subsequent
reactivity of the Cu2O2 intermediates (spectroscopically characterized
or even not characterized) toward the aromatic ring to afford the final
intramolecular m-xylyl ring hydroxylated product as m-phenoxo m-hydroxo-
bridged dicopper(II) complex. Examples are also chosen to exhibit gen-
eral aromatic ring hydroxylation and formation of di-m-phenoxo-bridged
dicopper(II) complex. In the second part of the article we will discuss the
electronic structure of the reactive intermediates (Cu2O2 adducts) and
understanding of their reactivity properties.
It is the purpose of this article to highlight the biomimetic studies on
tyrosinase. Our own research efforts have focused on both the aspects.
It is admitted that this report is not exhaustive, and interested readers are
referred to excellent review articles.1–16
II. Dicopper Proteins — Brief Overview

Hemocyanin, tyrosinase, and catechol oxidase belong to the class of type
3 copper proteins.14c An overview of the structure and functioning of these
dicopper-containing proteins/enzymes is briefly discussed here.
Figure 1. Schematic drawing of the structures of deoxy- and oxy-state of Hc.
A. Hemocyanins
Hemocyanins (Hc) are the oxygen-carrier protein of molluscs and arthro-
pods. Hemocyanin, unlike hemoglobin, has no heme group; the copper is
bound directly to the protein side chain. Reversible binding of dioxygen
is depicted in Figure 1. X-ray structures of both deoxy- and oxy-forms of
Hc have been determined.4,14b,18
B. Tyrosinases
Polyphenol oxidase is a generic term for the group of enzymes that cata-
lyze the oxidation of phenolic compounds to produce brown color on cut
surfaces of fruits and vegetables.4,14c,19 The enzyme tyrosinase (Tyr)
catalyzes the hydroxylation of monophenols (tyrosine) to o-diphenols
(L-3,4-dihydroxyphenylalanine, L-DOPA; monophenolase activity) and
subsequent two-electron oxidation to o-quinones (L-DOPA-quinone;
diphenolase activity), which constitutes the first step of melanin biosynthe-
sis through a series of spontaneous, non-enzymatic reactions (Figure 2).
Tyrosinase appears to be ubiquitous in living organisms. It is widely
distributed in plants, fungi, bacteria, mammalians, and animals. When
potatoes, apples, bananas, sweet potatoes, or mushrooms are injured they
turn brown.4,19 This is due to the conversion of tyrosine to the pigment
melanin, by the sequence of reactions shown in Figure 2. The same process
causes skin to turn brown, following exposure to ultraviolet radiation. The
enzymatic reactions are catalyzed by tyrosinase. The enzyme is present in
Figure 2. Tyrosinase and catechol oxidase activities.
Figure 3. Active site of oxy-tyrosinase, depicting a m-η2:η2-peroxo-dicopper(II) core.
the interior of the plant material and, since the reaction requires molecular
oxygen, the pigmentation does not occur until the interior is exposed.
Since melanin is a key pigment of some phenomena such as suntan, skin
disorder, and bruising of fruits, tyrosinase has practical and economic
importance, and thus attracting much attention from cosmetology, medi-
cine, and agriculture to control the synthesis of the melanin pigment.19
Tyrosinase is an O2-activating enzyme. The active site of tyrosinase
has a dinuclear copper center with each copper coordinated by three his-
tidines. The oxy-state of the enzyme contains a O22− ion bound to two
copper(II) centers in a η2-fashion (Figure 3).19a The X-ray structure of
oxy-Tyr is very similar to oxy-Hc (Figure 1), the only difference being the
active site of Tyr is more exposed to external substrate than in Hc.
C. Catechol Oxidase
Catechol oxidase (Cat Ox) is an enzyme that catalyzes the two-electron
oxidation of catechols to o-quinones (diphenolase activity/catecholase
Figure 4. Structure of the met-form of catechol oxidase.
activity).14c,20 In contrast to tyrosinase, which performs both the hydroxy-

lation and the oxidation step, catechol oxidase is only able to mediate the
latter reaction (Figure 2), without acting on phenols. Catechol oxidases
are found in plant tissues and in some insects and represent a group of
ubiquitous oxidases in plants. The mechanism of its function is closely
associated with that of tyrosinases. The active site structure of its physi-
ologically inactive met-form is shown in Figure 4.
III. Three Cu2O2 Core Structures

The X-ray structure of oxy-Hc and met-form of catechol oxidase are
shown in Figures 1 and 4, respectively. The X-ray structure of oxy-Tyr
(Figure 3) is very similar to oxy-Hc.
For the dicopper sites, three basic Cu2O2 core structures (Figure 5)
arising from the reactions of discrete Cu(I) complexes and O2 have been
widely investigated by synthetic chemists. One of the highlights of the
success story of synthetic chemists is the structural characterization of
these dicopper cores. A specific Cu2O2 core signifies a particular spectro-
scopic and chemical properties.1–3,5–16
Side-on peroxo-dicopper(II)/{CuII2(m-η2:η2-O2)}2+/{Cu2PS} and bis
(m-oxido)dicopper(III) {CuIII2(m-O)2}2+/{Cu2O2} cores show electrophilic
character and can mediate tyrosinase-like phenolate ortho-hydroxylation
reactions. End-on trans-peroxo-dicopper(II) {CuII2(m-η1:η1-O2)}2+/
{Cu2PE} core exhibits nucleophilic and basic behavior. The tyrosinase-
like reactivity has recently been observed for end-on {CuII2-(m-η1:η1-O2)}2+
core as well (see below). It should be noted that in {Cu2PS} and {Cu2PE}
binding modes the copper centers are in +2 oxidation state and O2 is in
its two-electron reduced form (O22−); whereas in {Cu2O2} mode both
Distances
(Å)
{Cu2PS} {Cu2O2} {Cu2PE}
d(Cu…Cu) 3.51 2.80 4.36
d(O–O) 1.42 2.32 1.43
d(Cu–O) 1.92 1.82 1.85
Figure 5. Three isoelectronic Cu2O2 binding modes, their shorthand notations, and
geometrical parameters. L: supporting ligands.
coppers are in +3 state and O2 is in its four-electron reduced (O2−) form.

The three motifs are electronic isomers.
Excellent review articles are available in the literature on molecular,
structural, spectroscopic, electronic structural, and reactivity aspects of
these three cores.1–3,5–16 In-depth discussion on the mechanism of the
functioning of tyrosinases1–16,19 and catechol oxidases20 are available in
the literature. The intricate structure-directed functional properties of
three dicopper-containing proteins/enzymes Hc, Tyr, and Cat Ox are
noteworthy.
IV. Biomimetic Studies on Tyrosinase

A. Intramolecular m-Xylyl and Aromatic Ring Hydroxylation
Fascinated by the biological role of tyrosinase, several synthetic models
have so far been made.2,5–11,15,16,21–26 The reactions of Cu(I) complexes of
simple monodentate/bidentate and tailor-made chelating ligands with O2
and the oxidative properties of the resulting Cun–O2 species has attracted
much interest over the past several decades. Excellent review articles have
appeared in the literature, which has addressed various aspects of this
field.1–16,19,21–24 Due to the substitution lability of Cu(I) and Cu(II) ions,
the ligand coordination subtly controls which species is formed and their
stability.8a In fact, the ligand architecture defines the Cun–O2 structure to
be attained during oxygenation and subsequent reactivity pattern. It has
been well documented that even seemingly minor alterations in the ligand
dramatically affect the oxygenation reactions, thus providing a direct
mechanistic probe.8a
In what follows, we highlight some of the biomimetic studies aimed

at generating and characterizing (structurally and/or spectroscopically)
the copper–dioxygen intermediates and subsequent reactions of such reac-
tive species to bring about hydroxylation/oxidation of copper-bound
ligand and/or of externally added substrates of relevance to tyrosinase
activity. Also included are the systems where ligand hydroxylation/oxida-
tion has occurred, as revealed by X-ray crystallographic and/or spectro-
scopic analysis of the final product, even though direct proof of the
formation of copper–dioxygen intermediate could not be provided. Apart
from m-xylyl-based ligands a few systems that had undergone aromatic
ring hydroxylation/oxidation by reactive copper–dioxygen intermediate
have also been considered.
B. Intramolecular m-Xylyl and Aromatic Ring Hydroxylation

The earliest examples of biomimetic oxygenation reactions (tyrosinase-
like activity) came from the outstanding studies performed by Karlin’s
group on aromatic hydroxylation reaction occurring in the m-xylyl-based
ligand (Figure 6), upon oxygenation of dicopper(I) complexes.5,11,21
Moreover, the most significant insights into the chemical activation of
dioxygen by dinuclear copper(I) sites, where the two steps of dioxygen
binding and ligand oxygenation were separately identified.21b
Specifically, Karlin’s group reported the synthesis of a dinucleating
ligand system (1: Scheme 1), which provides three nitrogen donors
((2-pyridyl)ethylamine unit to each copper ion), separated by a m-xylyl
spacer.5,21 Upon reaction with O2 at low temperature (193 K), the dioxy-
gen adducts form reversibly and these subsequently yield 2-xylylene-
hydroxylated products, which are phenoxo- and hydroxo-bridged
dicopper(II) complexes (Scheme 1). The products have been characterized
via the X-ray structure of the complex with R = H and by their UV–visible
Figure 6. Schematic representation of m-xylyl ring hydroxylation.

Scheme 1. A copper monooxygenase (aromatic m-xylyl ring hydroxylation) model

system of Karlin and coworkers.
spectral features. Like monooxygenases, one O atom of O2 is inserted into

C–H bond to form C–OH (Figure 6) and the other O atom is reduced to
OH– ion. The involvement of the side-on dicopper(II)-peroxo intermediate
{CuII2(m-O2)}2+ {Cu2PS}, formed upon reaction of dicopper(I) complex
of the m-xylyl ligand and dioxygen, was firmly established.21b,c
Mechanistically, electrophilic attack (see below) of the intermediate
was proved by the substituent effect on the 2- and 5-positions of the
m-xylyl ring.21b,d For this ligand system, coordination of the CuII/CuI ions
by three N donors at each arm gives rise to the formation of six-membered
chelate rings. This confers stability not only to Cu(I) ions but also allows
the Cu(II) ions to attain tetragonal geometry. Karlin’s Cu2–O2-mediated
N N
N N
Figure 7. The ligands that failed to demonstrate intramolecular m-xylyl ring

hydroxylation.
intramolecular m-xylyl ring hydroxylation reaction (Scheme 1) occurs

due to electrophilic attack (for details see Section 1.5.3.1) of {Cu2PS}
species.
A small perturbation of ethylpyridine to methylpyridine arm in the
ligand system (Figure 7), with six-membered (1) to five-membered (2)
chelate ring formation, leads to irreversible oxidation of Cu(I) to bis(m-
hydroxo)dicopper(II) complex, that is, without xylyl-hydroxylation, fol-
lowing a drastic change in the path of Cu(I)–O2 reactivity (4Cu(I) + O2 +
4H+ = 4Cu(II) + 2H2O).5e
Inspired by the success story of Karlin’s aromatic ring hydroxylation
reaction,5,11,21 systematic modifications of the original m-xylyl ligand
system (1) have been done by many researchers.22–25 The ligand varia-
tions include:
(i) tridentate arms with macrocyclic N3 donor

(ii) tridentate N3 five-membered chelate ring (methyl substituents near
donor site of the pyridyl rings) (steric effect)
(iii) N3 donor with one (2-pyridyl)ethylamine and one (2-pyridyl)meth-
ylamine arm
(iv) unsymmetrical arms with one tridentate and the other bidentate
(v) tridentate N3 to bidentate N2 arm (two to one (2-pyridyl)ethylamine
arm) of non-Schiff base and Schiff base variety
(vi) bidentate Schiff base variety with other donors instead of pyridine
(vii) replacing heterocycles by aliphatic amines of non-Schiff base and
Schiff base variety
(viii) macrocyclic systems (two N donor as well as three N donor arms)
with Schiff base and non-Schiff base variety.
The protocol followed by other groups for intramolecular aromatic

m-xylyl ring hydroxylation reactions is like that of Karlin and cowork-
ers.11,21 Dissolving the isolated yellowish dicopper(I) complex, obtained
from the reaction between the ligand and appropriate copper(I) salt in
suitable solvents (CH2Cl2, MeOH, MeCN, acetone, DMF, or THF), under
anaerobic conditions, or anaerobic solution–generated dicopper(I) com-
plex in solution, followed by exposure to dry O2 at low temperatures
(183–213 K) or even at room temperature (298 K). Attempts were made
to spectroscopically identify the intermediate formed, with characteristic
absorption and resonance Raman spectroscopic signatures,5,11,21 at low
temperatures and then warming the solutions to room temperature. Efforts
have also been made to spectroscopically characterize the final green solu-
tions and/or isolate the solid in pure form for its X-ray crystallographic
structural analysis. Green solutions of µ-phenoxo µ-hydroxo-bridged
dicopper(II) complexes display phenoxo-to-Cu(II) and hydroxo-to-Cu(II)
ligand-to-metal charge-transfer (LMCT) transition at ~350 nm.
Ligand variation types (i) and (ii) (see above) afforded 3 and 4,
respectively,25a,b and (iii) and (iv) resulted in the isolation of 5 and 6,
respectively,25c,d and (v) afforded 7 and 8, respectively25c,e,f (Figure 8).
Changing a six-membered chelate ring forming ligand 8 to a five-
membered chelate ring forming ligand 9 (Figure 7) notably stops intramo-
lecular ring hydroxylation.25f Similar behaviors were observed for ligands
2 and 9. Ligand variation (v) afforded 10.25g Ligand variation type (vi) led
to the generation of species 11–17.25h–l The presence of six-membered
chelate rings seems crucial for the stability of binuclear copper(I) counter-
parts of 12–14, without phenolic group, since all attempts to prepare
dicopper(I) complexes containing five-membered chelate rings were
unsuccessful.25i,j Generation of 13 was also accomplished independently,25l
which belongs to this category. Ligand variation type (vii) led to the isola-
tion of 18–2025m–o and 21.25p
The ligand design variation type (viii) was considered to change from
open-chain to macrocylic m-xylyl-based systems. The 18-atom tetra
Schiff base dinucleating macrocycle led to intramolecular m-xylyl ring
Figure 8. m-Xylyl ring hydroxylation model systems, using a large variety of

m-xylyl-based ligands.
hydroxylation with concomitant hydrolysis of the macrocycle (22,

Scheme 2).25q However, no hydrolysis of macrocycles was observed for
similar reactions with 24-atom tetra Schiff base25r,s and 28-atom25t,u dinu-
cleating macrocycles (Scheme 3); aromatic ring hydroxylation was
observed in the case of 2325r,s and 24.25t,u
Scheme 2. Intramolecular ring hydroxylation with concomitant hydrolysis of the

macrocycle.
Scheme 3. Intramolecular ring hydroxylation with intact macrocyclic ligand

systems.
C. Ring Hydroxylation Reactions with Non-m-Xylyl-Based

Ligands
Now we discuss examples of aromatic ring hydroxylation reactions with
non-m-xylyl-based open-chain ligands. The reactivity of {Cu2O2} core
was evidenced by Tolman and coworkers.26a The systems laid the founda-
tions for a mechanistic understanding of the tyrosinase reaction. This
reactivity was reinvestigated by Schindler, Tuczek, Holthausen, and
coworkers.26b Thus, dinuclear copper complexes exhibiting bis-µ-oxido
structure have been discovered to be able to mediate aromatic hydroxyla-
tion reactions in analogy to tyrosinase. The complexes 25 and 26 were
obtained (Scheme 4). Although a bis-µ-oxido intermediate has not been
observed in the enzyme as a stable intermediate, it may be relevant to the
reactivity of tyrosinase. Stack and coworkers with simple diamine sys-
tems proposed an alternative aromatic hydroxylation mechanism.27 Using
a m-xylyl-derived macrocyclic ligand, the complex 27 was obtained.28
Notably, using a tailor-made ligand with an appended phenol group, the
formation of 28 was observed.29 It is the first example provided by Tuczek
and coworkers, mediating the monooxygenation of a phenol by the
{Cu2O2} core, in the absence of an external base.
D. Oxidation of External Substrates by Dicopper Systems

Initially, mainly aromatic m-xylyl ring hydroxylation reactions by
{Cu2PS} and {Cu2O2} cores were investigated. Subsequently, the focus
of interest shifted into hydroxylation of external mono- and diphenolic
substrates, with an additional challenge to develop catalytic tyrosinase-
model systems.
Using a m-xylyl-based ligand system (Scheme 1), Karlin and cowork-
ers were the first to demonstrate spectroscopically at low temperature the
formation of side-on peroxodicopper(II).21 Casella and coworkers also
showed the existence of {Cu2PS} with 29 (R = Me).30a Costas, Casella,
Rybak-Akimova, Que, and coworkers provided evidence for the forma-
tion of {Cu2O2} 30.30b,c Using an unsymmetrical ligand, Costas, Luis,
Ribas, Que, Casella, and coworkers demonstrated the existence of an
{Cu2PE} core 31.30d The formation of {Cu2O2} 3230e and {Cu2PE} 3330d
Scheme 4. Intramolecular ring hydroxylation with non-m-xylyl-based ligand

systems.
were also reported with symmetrical Schiff base macrocyclic ligands. The
intermediates 29–32 are schematically displayed in Figure 9. The cores 30
and 31 have been shown to mediate tyrosinase-like phenolate ortho-
hydroxylation reactions (Figure 9). Apart from abovementioned intramo-
lecular aromatic ring hydroxylation reactions, a couple of biomimetic
R 2+ 2+
N N N N
N N O
II
N Cu CuII N O
N O N N CuIII CuIII N
N N O
N N
R = H, Me
29 30
2+
2+
N O N
N N CuIII CuIII
N Cu II O O
N HN N
N O H
N N
CuII
N N N N
31 32
2+
N O O N
CuII CuII
N
N
N NN
N
33
Figure 9. Formation of {Cu2PS}, {Cu2O2}, {Cu2PE}, and {Cu2O2} cores.
binuclear copper complexes were synthesized and tested for their capabil-
ity to convert external monophenolic substrates to o-diphenols or
o-quinones.
Réglier et al. reported31a that a mixture of a ligand, containing pyri-
dylethylimine sidearms bridged by a biphenyl spacer (Figure 10) placed
(a) (b) (c)
Figure 10. (a) Réglier’s ligand with biphenyl spacer, (b) tridentate analogue of the
Casella’s ligand used in 29 (Figure 9), and (c) bidentate analogue of Réglier’s biphe-
nyl spacer ligand.
with two equiv of Cu(I) salt, 100 equiv of 2,4-di-tert-butylphenol

(DTBP–H), and 200 equiv of Et3N led to the catalytic generation of qui-
none with a turnover number (TON) of 16 (Figure 10a). The progress of
the reaction was monitored by the appearance of the optical absorption
band of 3,5-di-tert-butyl-o-quinone (DTBQ) at ~400 nm. After 1h, the
reaction stopped, presumably by the formation of an oxo-bridged
dicopper(II) complex. A mechanism was proposed for this reaction,
involving the formation of a {Cu2PS} and a catecholate-bridged
dicopper(II) intermediate. Stoichiometric hydroxylation of phenols to
quinones has also been shown for a mononuclear Cu(I) complex sup-
ported by the tridentate analogue (Figure 10b) of the dinucleating ligand
present in 29 (see Figure 9). In this reaction an involvement of the
{Cu2PS} intermediate was demonstrated.31b
Tuczek and coworkers reported the first catalytic tyrosinase model
system, based on a mononuclear four-coordinate Cu(I) complex supported
by a bidentate ligand (a modified version of Réglier ligand) (Figure 10c)
and two MeCN.31c Oxygenation of a mixture of this Cu(I) complex with
a phenolate (ArO–) and Et3N with 50 equiv of DTBP–H and 100 equiv of
Et3N in CH2Cl2 was found to catalytically generate DTBQ, with a TON
of 18. Oxygenation initially formed the {Cu2PS} intermediate, containing
two coordinated phenolates (Figure 11). In this complex only one of the
bound phenolates was hydroxylated, leading to an asymmetrically coordi-
nated m-catecholato-m-hydroxo-bridged intermediate 34 (Figure 11). In a
stoichiometric mode it allowed the two consecutive stages of the tyrosi-
nase reaction, phenol hydroxylation, and product release as quinone, to be
addressed individually.
Figure 11. Intermediates (a) containing two coordinated phenolates and (b) asym-
metrically coordinated m-catecholato m-hydroxo-bridging.
Scheme 5. Catalytic system forming bis-phenol and diphenoquinones, using F in

the 2-position of the xylyl ring of the ligand used in 8 (Figure 8).
Oxygenation of a dicopper(I) complex of a m-xylyl-based ligand used

by one of us in the synthesis of 8,25e,f with a selective substitution of a fluo-
rine atom in the 2-position of the ring, which underwent ring hydroxylation
in 8, proved to be a catalytic system in the promotion of oxidative carbon–
carbon coupling of hindered phenols, which leads to the bis-phenol,
3,3′,5,5′-tetra-tert-butyl-2,2′-dihydroxybiphenyl and diphenoquinones,
3,3′,5,5′-tetra-tert-butyl-4,4′-diphenoquinone and 3,3′,5,5′-tetramethyl-
4,47-diphenoquinone (Scheme 5). The reaction stopped with the formation
of a dihydroxo-bridged dicopper(II) complex.32 Unfortunately, we could not
detect any copper–dioxygen intermediate.
Using 2,4-di-tert-butylphenolate as the substrate, the {Cu2O2} inter-
mediate generated (THF, 183 K) with the ligand present in 21 (Figure 8),
we demonstrated25p the formation of C–C bonded bis-phenol in 42%
HO OH
HO CO2Me
CO2Me
HO OH O O
HO
Figure 12. Tyrosinase-like chemical reactivity of 29 (R = H).
yield, catechol product in 38% yield (radical-coupled product; see

Scheme 5), and quinone (Figure 12) in 8% yield.
The dicopper(I) complex of the ligand present in 29 (R = H; Figure 9)
exhibits tyrosinase-like activity on exogenous phenols in the presence of
dioxygen (Figure 12). With easily oxidizable phenols the reaction is cata-
lytic but eventually leads to a complex mixture of products, as does tyrosi-
nase (Figure 2). With more oxidation-resistant phenols the reaction is
stoichiometric and stops at the level of catechol.33a The reaction suffers
some limitation in the operation conditions in that a rigorously anhydrous
and non-protic medium is required and formation of a phenolate adduct of
the Cu(I) complex prior to the reaction with O2 is also required, otherwise
simple copper(I) oxidation occurs.5e
Experiments performed based on in situ generation of the phenolate
showed that the dicopper(I) complexes of the following ligands (Scheme 6)
in MeCN at 298 K exhibit tyrosinase-like monooxygenase activity in the
presence of O2. Yield of isolated catechol (~20–40%), in terms of chelate-
ring size, follows the order: 6,6 > 5,6 > 5,5 > 5′,6 (5′ stands for five-
membered chelate ring forming pyridine ring) of the ligand systems
(Scheme 6). The catechol is the only product of phenol hydroxylation,
when the reaction is carried out at 233 K.33b
It has been demonstrated that a {Cu2O2} core 30 (Figure 9) is com-
petent to bind and hydroxylate phenolates (Figure 13b(i)).30c Exclusive
formation of bis-oxido species is observed, before and after phenolate
binding to the Cu2O2 site.30c
An asymmetric end-on peroxo-dicopper(II) complex 31 (Figure 9),
with available coordination site, selectively binds phenolates and mediates
Scheme 6. Different dinucleating ligand systems for phenol hydroxylation.
HO OH
(a) NaO CO2Me
CO2Me
HO OH
(b) NaO X
X
(i) acetone; 183 K; X = Cl, F, CO2Me, CN
(ii) acetone; 183 K; X = Cl, F, Me, H
Figure 13. Tyrosinase-like activity by 29–31 intermediates: (a) with 29, (b) with 30,
and (c) with 31.
its ortho-hydroxylation (Figure 13b(ii)), thus functionally mimicking

tyrosinase activity.30d This report was the first to demonstrate electro-
philic character of the end-on {Cu2PE} core, which was not observed for
symmetric analogues.30d DFT computations were performed to assess the
stability of different isomers ({Cu2PE}, {Cu2PS}, and {Cu2O2}), and it
was found that the {Cu2PE} is the most stable out of three species. So, the
authors concluded that the {Cu2PE} core is involved in aromatic hydroxy-
lation reaction. Not many DFT studies are available on {Cu2PE}, com-
pared to {Cu2PS} and {Cu2O2} cores (see below).
V. Theoretical Studies on Tyrosinase Models

Now we turn attention to the discussion on electronic structural properties
from theoretical calculations. A brief discussion on the structure of oxy-
Tyr and three Cu2O2 cores, identified and structurally and spectroscopi-
cally characterized, has already been done in Section 1.3. In this section,
we will discuss the frontier molecular orbitals (FMOs) of the three Cu2O2
cores.8a
A. Frontier Molecular Orbitals of the Cu2O2 Cores

1. μ-ηη2:ηη2-Peroxo-dicopper(II): {CuII2(μμ-ηη2:ηη2 O2)}2++{Cu2PS}
During the {Cu2PS} core formation, the two Cu(I) centers interact with
O2 resulting in the oxidation of Cu(I) to Cu(II) and reduction of O2 to
O22−. Each Cu(II) center would have an unpaired electron. The two
unpaired spins of Cu could exist either in parallel (triplet) or anti-parallel
(open-shell singlet) configuration. The {Cu2PS} cores found in tyrosinase
and its biomimetic complexes are EPR silent, thus reflecting that the two
unpaired spins of Cu are anti-parallel and strongly antiferromagnetically
coupled. For a better understanding of the bonding picture in {Cu2PS},
the FMOs are presented (Figure 14), in which half-filled Cu(dx2–y2)
orbitals are shown to interact with O22− valence MOs (in-plane pip*, out-
of-plane pop*, and σ*). Here, pip* and pop* are doubly occupied and σ* is
a vacant MO.
The FMO diagram of {Cu2PS} displays that the pop*(O−O) orbital of
2−
O2 is perpendicular to the Cu2O2 plane, thus pop*(O−O) brings a non-
bonding interaction with Cu d-orbitals and forms HOMO-1. The pip*(O−O)
orbital lying in the Cu2O2 plane overlaps with the Cu(dx2–y2) orbitals and
provides a bonding (HOMO-2) and an antibonding (LUMO) FMOs. The
bridging pip*(O−O) orbital in HOMO-2 provides a super-exchange path-
way to achieve strong antiferromagnetic coupling. Thus, tyrosinase and
synthetic inorganic complexes containing {Cu2PS} cores are EPR-silent.
The HOMO in {Cu2PS} is formed due to back-bonding from filled
Cu(dx2–y2) orbitals to the empty σ*(O−O) orbital. The back-donation from
Cu(dx2–y2) to σ*(O−O) weakens the σ(O−O) bond of {Cu2PS}, compared
to a typical peroxide ion (O22−).
Figure 14. Frontier molecular orbitals of a {CuII2(m-η2:η2-O2)}2+ core.
rans-μμ-1,2-peroxo-dicopper(II): {CuII2(μμ-ηη1:ηη1-O2)}2++
2. T
{Cu2PE}
Similar to {Cu2PS} (Section 1), {Cu2PE} cores also contain two Cu(II)
centers bound to a O22− ligand; however, unlike a η2-binding mode as in
{Cu2PS}, {Cu2PE} cores have a η1-binding mode between the two Cu(II)
and O22− ions (Figure 5). In {Cu2PE}, the in-plane pip*(O−O) orbital of
O22− overlaps with the Cu(dz2) orbitals to form a bonding (HOMO-2) and
an antibonding (LUMO) FMOs (Figure 15). The Cu(dz2) orbitals attain a
head-to-head overlap with pip*(O−O). In {Cu2PS} cores, the Cu(dx2–y2)
and pip*(O−O) orbitals do not have head-to-head overlap like in {Cu2PE}
cores. Thus, the Cu−O distances in {Cu2PS} (~1.92 Å) are longer com-
pared to {Cu2PE} (~1.85 Å).
The O−O distance in {Cu2PS} (~1.42 Å) and {Cu2PE} (~1.43 Å) are
comparable as in both the cores only the O2 p-bond is broken and the
σ-bond is still intact. The {Cu2PS} core is more compact than {Cu2PE} as
the distance between the two Cu in {Cu2PS} (3.51 Å) is smaller than in
{Cu2PE} (4.36 Å). Thus, {Cu2PE} species are preferred in large-size
ligands, where due to the steric crowding of the ligands the two Cu cannot
achieve a shorter distance. The inorganic complexes containing {Cu2PE}
Figure 15. Frontier molecular orbitals of a {Cu2(m-η1:η1-O2)}2+ core.
cores do not show EPR peaks, suggesting that the two Cu(II) centers are
antiferromagnetically coupled. This antiferromagnetic interaction is
achieved by following a super-exchange pathway in the bonding FMO
(HOMO-2).
3. Bis(μμ-oxido)dicopper(III): {CuIII2(μμ-O)2}2++ {Cu2O2}

A {Cu2O2} core has two Cu(III) centers bound to two bridging O2−
ligands (Figure 5). The {Cu2O2} cores can be derived from {Cu2PS} by
the oxidation of Cu(II) to Cu(III) and the reduction of O22− to 2O2−
(oxides). The σ-bond of dioxygen in {Cu2O2} is completely broken.
Therefore, the distance between the two oxygens in {Cu2O2} is longer
(~2.3 Å) than the O−O distance in {Cu2PS}/{Cu2PE} (~1.4 Å) and O2
(~1.2 Å) (Figure 5).
The FMO diagram of {Cu2O2} demonstrates the interaction of
vacant Cu(dx2–y2) orbitals with filled oxide orbitals (pip*, pop*, and σ*)
(Figure 16). As for {Cu2PS} and {Cu2PE}, the out-of-plane pop* orbital
Figure 16. Frontier molecular orbitals of a {CuIII2(m-O)2}2+ core.
and Cu(dx2–y2) orbitals interact to form a non-bonding HOMO-1 FMO.

The in-plane pop* orbital overlaps with Cu(dx2–y2) orbitals to produce a
bonding (HOMO-2) and an antibonding (LUMO+1) FMOs. Unlike
{Cu2PS} and {Cu2PE}, the σ* orbital lies at a similar energy level as that
of Cu(dx2–y2) orbitals. Thus, an effective overlap between the two orbitals
takes place resulting in HOMO and LUMO FMOs and a stable Cu−O
covalent bond. The Cu−O distance in {Cu2O2} is ~1.82 Å, which is
shorter than the Cu−O distance in {Cu2PS} (~1.85 Å)/{Cu2PE} (~1.92
Å), indicating a strong σ(Cu−O) bond in {Cu2O2}. Due to elongation
between the two oxygens in {Cu2O2}, the core has a compact motif
(d(Cu…Cu) ~2.80 Å) than the other two isomers {Cu2PS} (d(Cu…Cu)
~3.51 Å)/{Cu2PE} (d(Cu…Cu) ~4.36 Å). So, sterically demanding bulky
ligands do not support the formation of {Cu2O2} motifs.
We discussed previously that {Cu2O2} cores can be derived from
{Cu2PS} by pulling the electron density from two Cu(II) centers to
σ*(O−O) orbital, thus breaking the dioxygen bond of O22−. So, an inter-
conversion between the two cores is possible. Indeed, such interconver-
sions have been detected in synthetic laboratories7b and studied using
various computational methods.
B. Interconversion Between {Cu2PS} and {Cu2O2} Cores:

A Torture Track for Computations
Experimentally in 1996, Tolman and coworkers established that an inter-
conversion between {Cu2PS} and {Cu2O2} cores is operative in solu-
tion.34 It was found that this interconversion is sensitive to counter-anions,
solvents, and temperature used in the reaction. Theoretically, such core
transformations were studied in detail by Cramer,35 Siegbahn,36 Neese,37
and Holthausen38 groups. Based on their computational studies, these
groups showed that the interconversion between the two isomers is not
trivial for computational methods such as DFT. Cramer has named this
conversion a “torture track” for theoretical chemists,39 and even after
10 years of Cramer’s findings, Herres-Pawlis still named the conversion a
“torture track” for computations.40 Thus, the interconversion has become
an interesting problem for theoretical studies. Various benchmark studies
have been performed to find out the best computational method for the
relative stability of the two isomers. To benchmark DFT methods for such
interconversions, scientists chose either experimental results or high-level
computational methods as references.
Cramer and coworkers constructed {Cu2PS} and {Cu2O2} computa-
tional models supported by ammonia ligands for DFT benchmark stud-
ies.35,39 Ab initio multi-reference configuration interaction (MRCI)41 and
renormalized coupled-cluster [CR-CC-(2,3)]42 methods were used as ref-
erence calculations to perform benchmark DFT studies. Cramer group
found that GGA density functionals (such as BLYP)43,44 within the
closed-shell singlet approach were the best in agreement with the chosen
reference calculations. However, the drawback of this study was the very
small model system with NH3 as terminal ligands.
Later, Siegbahn and coworkers used a larger model system for the
computations and taken experimental relative stability data of {Cu2PS}
and {Cu2O2} for the reference.36 The chosen model systems ({Cu2PS}
and {Cu2O2}) were supported by iPr3-TACN (1,4,7-tri-isopropyl-
triazacyclononane) ligands. In their study the authors found that the
B3LYP* density functional45 (with 15% Fock exchange) within the bro-
ken-symmetry DFT approach works well for relative stability comparison
of the two isomers. They also included dispersion and relativistic effects
in the computations and their results were in good agreement with

experiments.
Computational studies by the Cramer group have suggested to use the
closed-shell singlet approach with GGA density functionals (0% Fock
exchange), whereas Siegbahn found that B3LYP* (15% Fock exchange)
functional works well but broken-symmetry solutions should be consid-
ered.36 So, Neese and coworkers re-investigated this isomerization prob-
lem using {Cu2PS} and {Cu2O2} motifs, supported by ethylenediamine
ligands. To generate an accurate reference data for the relative stability of
the two isomers, the group used ab initio single reference local pair natu-
ral orbital coupled cluster (LPNO-CCSD)46 theory. The authors found that
the B3LYP (20% Fock exchange)47 density functional within the closed-
shell solution, including dispersion and relativistic effects, is the best
method for studying this isomerization.
All the benchmark studies presented so far were based on either
experimental references or high-level computational references for the
relative stability of the two isomers. In 2017, Holthausen and coworkers
chose both experimental and high-level computational methods as refer-
ences for their benchmark studies.38 The experimental relative stability
data was taken from the work of Stack’s group and the computational
reference was generated from the CCSD(T) calculations. The authors
showed that GGA functionals works best but it is also important to include
closed-shell solutions for the two isomers to compare their relative stabil-
ity. Moreover, DFT calculation must include dispersion, solvent, and rela-
tivistic corrections to get the best comparison with references. Moreover,
Holthausen and coworkers also did a DFT benchmark study for C−H
oxidation step and proposed that GGA functionals also work well for the
hydroxylation steps. Hence, at present GGA functionals are the best
choice for studying the interconversion between {Cu2PS} and {Cu2O2},
and subsequent C−H oxidation step.
C. {Cu2PS}, {Cu2PE}, and {Cu2O2} Motifs in C−H Oxidation:

DFT Studies
Tyrosinase utilizes a {Cu2PS} intermediate for performing aromatic C−H
oxidation of tyrosine. However, in synthetic biomimetic studies
interconversion between {Cu2PS} and {Cu2O2} cores were detected.

Hence, out of the two, which core is active for C−H oxidation/hydroxyla-
tion is always an open question. Similarly, {Cu2PE} could also be a poten-
tial candidate for performing C−H hydroxylation. In this regard,
computational mechanistic studies have been very successful. Here, we
will present DFT-based mechanistic studies on aromatic C−H hydroxyla-
tion via {Cu2PS}, {Cu2PE}, and {Cu2O2} motifs. These models duplicate
the reactivity of the tyrosinase enzyme.
1. Aromatic C−H hydroxylation via {Cu2P S} motifs

Holthausen and Schindler groups carried out a combined experimental
and computational work on a copper complex supported by a m-xylyl-
based dinucleating ligand (Figure 17) (structural drawing 20; Figure 8).25o
In this work, the authors were successful in performing intramolecular
Figure 17. Intramolecular aromatic ligand hydroxylation via a {Cu2PS} core.

C−H hydroxylation at the 2-position of the ligand. DFT studies revealed

that the {Cu2PS} core approaches toward 2-position of the m-xylene
spacer and forms a 3-center transition state, in which O−O bond cleavage
and C−O bond formation take place in a single step to produce an are-
nium-like σ complex. From this σ complex, two pathways leading to a
single hydroxylated product were computed. Along Path-I, hydrogen
present at the 2-position of the m-xylene ring is abstracted by the bridging
μ-oxo atom of the Cu2O2 motif to generate the hydroxylated (m-phenoxo
m-hydroxo) product in a single step. In Path-II, the σ complex converts into
the hydroxylated product in two steps: first, the σ complex rearranges into
a dienone intermediate via a 1,2-H shift across the aromatic ring. Such a
hydrogen shift was earlier proposed by Karlin and denoted as the NIH
shift.48 In the second step, hydrogen present at the 1-position of the ring
is abstracted by the bridging m-oxo atom to yield the desired hydroxylated
product. Out of the two mechanisms the second one, which proceeds via
a dienone intermediate, is preferred based on the lower transition state
barriers along the reaction steps. In this study, the authors showed that the
{Cu2PS} core is directly involved in the aromatic ligand hydroxylation,
without the formation of a {Cu2O2} intermediate. The 3-center transition
state connected to {Cu2PS} species and arenium-like σ complex is found
to be the rate-limiting step.
In a more recent DFT-based mechanistic study by Hong and cowork-
ers used Karlin’s tricoordinated dicopper(I) complex (Scheme 1)21a,50 to
49
study intramolecular ligand hydroxylation in presence of O2 (Figure 18).

Karlin’s group observed m-xylyl ring hydroxylation at the 2-position. The
group also studied the influence of para-substituent effect on the reaction
rate. They concluded that an electron-donating group at para position
increases the rate of hydroxylation, whereas electron-withdrawing groups
decrease the reaction rate. To identify intermediate steps of this hydroxy-
lation, Hong’s group explored two mechanisms, which were earlier pro-
posed by Holthausen and coworkers for aromatic ligand hydroxylation
(structural type 19) (Figure 17). They showed in their DFT study that
Path-II (multi-step mechanism via a dienone intermediate) is preferred
over Path-I (single-step direct hydrogen abstraction from bridged oxy-
gen), based on lower transition state barriers along Path-II. The rate-
limiting barrier is extracted from the very first step in which the {Cu2PS}
Figure 18. Intramolecular aromatic ring hydroxylation via a XYL-supported

(Scheme 1) {Cu2PS} core. Detailed reaction paths are not shown, as they are like that
shown in Figure 17.
motif attacks at the 2-position and builds a multi-center transition state. In

the rate-determining transition step the O−O bond cleavage and C−O
bond formation take place in a concerted fashion to yield an arenium-like
σ complex. Like the Holthausen group, Hong and coworkers did not see a
{Cu2O2}-type of species in their mechanistic investigations. The group
also demonstrated theoretically the p-substituent effect by computing
reaction barriers for the rate-determining step.
Based on DFT studies carried out by the Holthausen group and later
by the Hong group, it can be concluded that dinuclear copper species sup-
ported by m-xylyl type ligands prefer aromatic ligand hydroxylation via
dienone intermediate. However, such intermediate has not been detected
in experimental findings. Their studies also suggest that {Cu2PS} is the
active site and {Cu2O2} species does not exist in such cases.
Solà and coworkers used a large macrocyclic ligand-supported dicop-
per complex to study the intramolecular aromatic ligand hydroxylation
(Figure 19).25u The said ligand contains two closely placed m-xylyl rings.
In their DFT study they found that the two isomers ({Cu2PS} and
{Cu2O2}) are not significantly different in energies. So, both cores are
potential candidates for aromatic hydroxylation. The group studied intra-
molecular aromatic hydroxylation only via the {Cu2PS} intermediate;
however, they also provide a note that the pathway via the {Cu2O2} core
cannot be ruled out. Thus, in the initial step of the mechanism {Cu2PS}
core attacks at the 2-position of the ring to yield a σ complex. In the next
steps the other m-xylyl ring assists the hydrogen transfer from the
Figure 19. Intramolecular aromatic ligand hydroxylation via {Cu2PS} core to the
product (24).
2-position of the first ring to the bridged oxygen and eventually yield a
µ-phenoxo µ-hydroxo product. Hence, Solà and coworkers showed the use
of a second m-xylyl ring for hydrogen transfer, such a mechanism could
be envisioned in tyrosinase since histidine moieties close to tyrosine may
assist the hydrogen transfer in aromatic C−H bond oxidation.
2. Aromatic C−H hydroxylation via {Cu2O2}

In this section, we discuss DFT studies on {Cu2O2}-mediated aromatic
C−H hydroxylation reactions. Holthausen, Schindler, and Tuczek groups
reported {Cu2O2}-mediated ligand-based aromatic hydroxylation.26b
In this study these groups revisited Tolman’s aromatic hydroxylation sys-
tem26a and provided a detailed mechanistic study of this reaction. They
also considered a new ligand in which the copper(I) complex on oxygen-
ation-afforded salicylaldehyde due to ring hydroxylation/oxidation (26).
Figure 20. Formation of the σ-complex 3 from the {Cu2O2} intermediate in Tolman
model.
DFT-based mechanism for Tolman’s aromatic hydroxylation system is

displayed in Figure 20.
DFT computations showed that in Tolman’s system the {Cu2O2}
intermediate 2 (it is not complex 2) is significantly stable than its {Cu2PS}
isomer 1 (Figure 20). Thus, the {Cu2O2} species would clearly be the
dominant motif in the reaction mixture. The {Cu2O2} intermediate 2
attacks the nearest carbon of the phenyl ring to yield a σ-complex 3. From
the intermediate 3 (1, 2, and 3 are not complex numbers) two pathways
branch off.
Out of the two pathways the one that carries multiple steps and pro-
ceeds via a dienone intermediate is found to be the preferred one due to
low activation barriers along the pathway (Figure 21). The rate-limiting
transition state in the overall reaction is the 1,2-H shift (transition-state
TS34), which results in a dienone intermediate 4. Hence, the nature of the
rate-limiting step is different in the {Cu2O2}-mediated aromatic hydroxy-
lation than in {Cu2PS} hydroxylation (Figures 17, 18, and 20). In Path-I,
TS36 provides direct path to the hydroxylated product.
Holthausen and coworkers also computed theoretically the p-substit-
uent effect on the rate of the reaction. They found that the groups CF3,
NO2, CHO, and so on, which deactivate the benzene ring increase the
barrier, whereas the groups OCH3, OH, CH3, and so on, which activate the
Figures 21. Two pathways leading to the hydroxylated product from the σ-complex
3 in the model complex of Tolman.
benzene ring decrease the barrier. Hence, the authors proposed that
{Cu2O2}’s attack on the aromatic ring is an electrophilic substitution
reaction.26b
The groups of Holthausen, Schindler, and Tuczek showed an intramo-
lecular aromatic ligand hydroxylation via {Cu2O2} species. Blomberg
and coworkers employed DFT to investigate aromatic hydroxylation of an
external phenolate via a {Cu2O2} species (Figures 22).51 For this purpose,
the Blomberg group chose an experimental reaction established by the
Stack group. In this reaction, Stack and coworkers used a DBED (DBED =
N,N′-di-tert-butyl-ethylenediamine) supported copper/O2 complex.27
DFT-computed energetics of DBED-supported {Cu2O2} and {Cu2PS}
systems confirmed that the two isomers are in equilibrium. However, after
the addition of the external phenolate substrate in the reaction mixture, the
equilibrium is shifted toward the phenolate-bound {Cu2O2} isomer. In
the next step, {Cu2O2} core at attacks 2-position of the ring carbon (of
phenolate) to yield a σ complex. Subsequently, the bridged m-oxido
abstracts H atom from 2-position of the ring to yield the hydroxylated
Figures 22. Hydroxylation of an external phenolate as substrate by a {Cu2O2} core.
product. Thus, the Blomberg group showed in their computations that

{Cu2O2} cores are capable of hydroxylating aromatic C−H bond of exter-
nal substrates.
In a recent study, Costas, Company, Luis, and coworkers utilized
Stack’s {Cu2O2(DBED)2}2+ system27 to selectively activate a strong C−F
bond of a phenolate substrate (Figure 23).52 The authors also observed in
their experimental studies that the ortho-hydroxylated product is minor
whereas the ortho-defluorinated hydroxylated product is the major prod-
uct. This finding was indeed non-intuitive as C−F bonds are stable than
C−H bonds. So, activation of a C−F bond in the presence of a relatively
weaker C−H bond was unclear (Figures 23). To gain insights of this find-
ing the authors computed reaction pathways for the ortho-defluorinated
hydroxylated product and the ortho-hydroxylated product.
Figure 23. Selective C−F bond activation catalyzed by a {Cu2O2} core:

{CuIII2O2(DBED)2}2+.
Figure 24. Equilibrium between {Cu2PS} and {Cu2O2} cores (before and after phe-
nolate binding).
In their DFT calculations, these authors computed that the {Cu2PS}

motif supported by DBED ligands is more stable than its corresponding
{Cu2O2} motif. So, the equilibrium between the two cores would shift
toward the {Cu2PS} species (Figure 24). However, 2-fluorophenolate
bound {Cu2O2(DBED)2}2+ system is more stable for the {Cu2O2} core.
Thus, after the addition of 2-fluorophenolate, {Cu2O2} species would
dominate the reaction mixture. Similar results were also seen in Blomberg’s
DFT investigation (Figure 22). From here, two pathways branch off.
First pathway leads to the ortho-hydroxylated product (minor) and
other provides a pathway to reach the ortho-defluorinated hydroxylated
product (major) (Figure 25). The first pathway is like the one discussed in
Figure 17. In Path-II, the phenolate substrate containing fluorine interacts
with less-coordinated Cu(III). The reason for this interaction is the
Figure 25. Pathways leading to ortho-hydroxylated product (minor in Path-I) and

ortho-defluorinated hydroxylated product (major in Path-II).
attraction between a lone pair of F and Cu(III). Due to Cu…F interaction

the C−F bond is found to be pre-activated. Such a pre-activation is not
possible in ortho C−H hydroxylation as H does not contain lone pair.
Thus, the attack of the {Cu2O2} core at the carbon of the C−F bond is
favorable over the carbon of the C−H bond. This results in a lower barrier
for Path-II (Figure 25).
3. Aromatic C−H hydroxylation via {Cu2P E}

Garcia-Bosch et al. found that {Cu2PE} cores are also capable of per-
forming aromatic C−H hydroxylation (Figure 26; cf. Figure 9).30d They
reacted the asymmetric CuI2(m-xylN3N4) species with O230d and detected
a {Cu2PE} intermediate (structural drawing 31). In the next step, sodium
p-chlorophenolate was added in the reaction mixture with subsequent
workup steps to yield p-chlorocatechol. DFT computations were per-
formed on this reaction to compute the stability of different isomers
Figure 26. Aromatic ligand hydroxylation by a {Cu2PE} core.
({Cu2PE}, {Cu2PS}, {Cu2O2}), and it was found that the {Cu2PE} is the
most stable species out of the three. So, the authors concluded that the
{Cu2PE} core is involved in aromatic hydroxylation. Not many DFT
studies are available on {Cu2PE} as compared to {Cu2PS} and {Cu2O2}
cores.
VI. Conclusions
The use of dioxygen as an oxidant in organic transformations would very
much benefit from fundamental knowledge on the mechanisms of its acti-
vation. Copper enzymes have been illustrated to process dioxygen in an
efficient and purposeful manner. Modeling copper–dioxygen chemistry
occurring in enzymes has attracted the attention of synthetic inorganic
chemists. A powerful tool to extract mechanistic information on copper-
catalyzed biochemical transformations, such as the tyrosinase reaction, is
the study of small-molecule complexes, which mimic the active site struc-
ture of their biological counterparts. An additional challenge in copper–
dioxygen chemistry has been the synthesis of catalytic model systems of
tyrosinase. A significant challenge is the development of selective cata-

lytic systems that use O2 as the oxidant and avoid deleterious side reac-
tions. Biological systems have unique capabilities in this regard through
the control of the spatial and/or temporal distribution of substrates and
oxidants. Such control has yet to be exerted in model systems and needs
to be addressed, perhaps through more sophisticated biomimetic design
strategies than those used so far. Further discoveries are likely to be made
as mechanistic understanding of the enzymes accrues, new types of reac-
tive intermediate in the biological and synthetic model systems are uncov-
ered and applications toward catalysis are explored. Developing efficient
synthetic catalysts to perform these transformations for industrial pro-
cesses are central goals in chemical research.
In summary, this chapter briefly addresses the developments that have
happened in the biomimetic front in benchmarking ligand design aspect to
model tyrosinase-like activity and how theoretical calculations have
strengthened our understanding from rationalization point of view.
Copper-based oxidants and greater insights into biochemical oxidation
processes with O2 is a hot topic in bioinorganic chemistry of copper. From
a humble beginning of intramolecular m-xylyl-based binucleating ligand
hydroxylation first reported by Karlin and coworkers in early eighties, the
biomimetic studies have enriched to advance our understanding of tyrosi-
nase activity. The versatile reactivity properties of {Cu2PS}, {Cu2O2},
and {Cu2PE} cores toward biorelevant substrates have been systemati-
cally investigated. The results have forced us to pay a new look at this area
of research with sharpened direction.
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4 Monooxygenation of Phenols
by Small-molecule Models of
Tyrosinase: Correlations
Between Structure and
Catalytic Activity
Alexander Koch*, Tobias A. Engesser*,

Ramona Jurgeleit*, and Felix Tuczek*,†
*Institut für Anorganische Chemie, Christian-Albrechts-Universität

zu Kiel, Max-Eyth Straße 2, 24118 Kiel, Germany
†
[email protected]
I. Introduction
Copper enzymes are involved in important biochemical processes in ani-
mals or plants such as dioxygen transport and metabolism or electron
transfer and are present in the three domains of life.1,2 They are often
categorized by classes (type I–IV, CuA, CuB, CuZ, and Cu0), which derive
from their structural environment or the spectroscopic properties of the
metal center(s).1,3 Type III refers to a (oxygen-binding) binuclear copper
protein family comprising tyrosinase (Ty), tyrosinase-related proteins,
catechol oxidase (CO), and hemocyanin.1,3–5
Tyrosinase is a phenoloxidase and catalyzes the conversion and oxidation
of mono- and diphenols into the corresponding ortho-quinones whereas the
catechol oxidase only exhibits the latter reactivity.1,6–8 These enzymes mediate
123
124 Koch et al.
Scheme 1. Overview of the reactivities of tyrosinase (Ty) and catechol oxidase

(CO).6–8
the oxygenation of L-tyrosine and, respectively, oxidation of L-DOPA to

L-dopaquinone, which in turn polymerizes to melanin (Scheme 1).
II. Model Systems of Tyrosinase

For a better understanding of the reactivity and the functional differences of
these enzymes and in addition to insights already obtained by structural and
molecular biology, mechanistic investigations on small-molecule models can
be beneficial.7,9–11 In full analogy with their biological counterparts, the
synthetic model systems of phenoloxidases can be divided into those exhibit-
ing catechol oxidase and those exhibiting tyrosinase activities.3,12–17 A fur-
ther important distinction relates to the question of whether these systems are
catalytic or not. Whereas the former in most cases applies to catechol oxidase
models, most of the existing tyrosinase models perform monooxygenation
reactions of aromatic substrates in a stoichiometric fashion.7 By contrast, the
number of catalytic model systems of tyrosinase is still very limited.6,7,18
An important factor in the investigation of copper-catalyzed oxidation
and oxygenation reactions relates to the presence or absence of base.
Whereas catechol oxidase reactions occur in the absence of base,
monooxygenation reactions on external phenolic substrates in general
require the addition of base to deprotonate phenolic substrates.8
In order to explore this correlation in more detail, several studies on
inorganic model systems, which catalyze the monooxygenation of
Monooxygenation of Phenols by Small-molecule Models of Tyrosinase 125
phenolic substrates, have been published in the past. This has provided
insight into relevant steps of the monophenolase cycle, that is, the forma-
tion of the peroxo complex, the interaction of the substrate with the peroxo
complex leading to a coordinated catecholate (including the role of base in
this reaction), the two-electron oxidation of the bound catecholate and the
release of the ortho-quinone product. As this chapter focuses on tyrosinase
activity, small-molecule models of catechol oxidase will not be discussed
here. Excellent reviews on the latter topic can be found elsewhere.3,12–16
A. Model Systems Performing Ligand Hydroxylation

First information on the mechanism of tyrosinase was obtained by studies
referring to the hydroxylation of an aromatic, but non-phenolic part in the
employed ligand framework, starting with the XYL-based system by
Karlin et al. in the mid-eighties (Figure 1).19 Following studies on differ-
ent model systems by Tolman et al., Réglier et al., Schindler et al., and
again by Karlin et al. gave further insight into the mechanism of these
ligand hydroxylation reactions, indicating that a µ-η2:η2-peroxo/bis-µ-oxo
intermediate typically formed upon exposure of a Cu(I) precursor to
dioxygen mediates electrophilic attack on the ligand framework.11,19–27
These investigations provided significant information toward a mechanis-
tic understanding of tyrosinase activity.
Given the fact that tyrosinase specifically mediates the monooxygena-
tion of phenols (and not non-phenolic aromatics), a further development
Figure 1. The XYL system by Karlin et al., which is able to perform the hydroxyla-
tion of the aromatic ring in the ligand backbone and the L4-H system that performs
hydroxylation of the phenolic residue.19,28
126 Koch et al.
of these systems toward mediating the hydroxylation of a phenolic residue

appended to the ligand framework appeared of interest. Apart from Karlin
et al. and Itoh et al., only few studies have been published on this issue so
far.29–32 In 2015, our workgroup presented a model system exhibiting the
described reactivity. The tridentate L4-H system is composed of a bis(2-
pyridylmethyl)amine unit (Figure 1), which is complemented with a phe-
nol residue to a tertiary amine.28 In the presence of dioxygen at low
temperatures, the mononuclear copper(I) complex of L4-H mediates an
aromatic hydroxylation of the appended phenol, that is, the phenol residue
in the ligand backbone is ortho-hydroxylated and oxidized to the corre-
sponding CuL4quinone derivative upon reaction of the Cu(I) precursor
with dioxygen at low temperatures.28
Apart from tyrosinase models that are able to hydroxylate an aromatic
part of the ligand framework,6,7,19–33 model systems have been developed,
which are able to convert external monophenolic substrates to the corre-
sponding ortho-quinones in a stoichiometric or a catalytic fashion.6,7,9,34–53
In the remainder of this review we will focus on systems converting exter-
nal substrates.
B. Model Systems Exhibiting Reactivity Toward External Substrates

Synthetic model systems with binuclear copper sites that catalyze the con-
version of external monophenols to the corresponding ortho-quinones have
been studied for a long time.6,7,54 Bulkowski et al. took the first steps in the
field of catalytic model systems for tyrosinase in 1985 with the develop-
ment of the necessary conditions for a catalytic monooxygenation of phe-
nolic substrates in an enzyme-like fashion.35 He used copper(I) complexes
supported by dinucleating, macrocyclic tetraamine ligands (Figure 2) as
catalysts. For the conversion of phenols to ortho-benzoquinones in the pres-
ence of these complexes and dioxygen, an excess of the external monophe-
nol (100 eq.) and triethylamine (200 eq.) was employed. The base was used
for deprotonation of the phenol, leading to a better coordination to the cop-
per ions, a step that is necessary for the following ortho-hydroxylation.35
In the following, the conditions established by Bulkowski et al. were
applied in further catalytic studies and were improved by Réglier and
coworkers in 1990 with their Cu(I)2(BiPh(impy)2) system (Figure 2).36
Figure 2. Early model systems with the ability to catalytically convert monophenols
to ortho-quinones.35,36,55
The reaction of this copper(I) catalyst with 2,4-di-tert-butylphenol

(2,4-DTBP-H, 100 eq.) and 200 equivalents of triethylamine (from here
on called Bulkowski–Réglier conditions) in the presence of dioxygen led
to the formation of the corresponding 3,5-di-tert-butyl-ortho-quinone
(3,5-DTBQ) with a turnover number of 16 after one hour.36
In 1991, Casella and coworkers established a further system sup-
ported by the ligand L66, which was able to mediate the stoichiometric as
well as catalytic tyrosinase-like reactivity of external phenolic sub-
strates.18,55 Importantly, in the year 2000, the µ-η2:η2-peroxo intermediate
of the Cu(I)2L66 system could be generated reversibly and shown to be
the hydroxylating agent in monooxygenation reactions (Figure 2).56,57
Following studies by Garcia-Bosch, Karlin, and Solomon as well as
Herres-Pawlis et al. gave further important insight into the mechanism of
the tyrosinase reaction.38,43,58–61
128 Koch et al.
In most of the published studies on catalytic monooxygenations of

phenolic substrates, the combination of a significant excess of the sub-
strate and base was applied, and the obtained results indicate that the
excess of the added base is indeed important for the catalytic activ-
ity.6,7,35–43,55,56,58–61 By the addition of neutral phenols as substrates,
phenoxyl radicals are generated and only unphysiological radical cou-
pling products were obtained.6 This constitutes a remarkable difference to
the enzymatic system, which mediates the monooxygenation of phenols
in the absence of an external base and without side-products like C–C-
coupled molecules.
In order to increase the catalytic activity regarding the formation of
ortho-quinones and realize more biomimetic model systems, a range of
model systems was established by several workgroups during the last
years. Depending on the employed ligand framework, characteristic dif-
ferences in the monooxygenation activity toward various monophenols
were observed. Scheme 2 depicts the possible aspects according to which
these systems can be classified. An important distinction exists between
structural model systems, which are primarily designed to stabilize cop-
per–oxygen intermediates, and functional models with the ability to stoi-
chiometrically or catalytically mediate the tyrosinase reaction.11 Regarding
Scheme 2. Different aspects of tyrosinase model chemistry and scope of this

review (black).
the latter category, copper complexes supported by mono- or dinucleating

ligands can be distinguished in turn. Moreover, simple copper salts have
been found to catalyze the monooxygenation of phenols.[48,49,62] Describing
all of these systems would be beyond the scope of this chapter, so only
findings regarding catalytic systems supported by mononucleating, biden-
tate N-donor ligands are discussed in the following.
III. Catalytic Tyrosinase Models with Bidentate Ligands

A. Mechanistic Cycle of the Tyrosinase-like Monooxygenation of
External Substrates
In 2005, Stack et al. reported the first Cu(I) complex supported by a biden-
tate ligand exhibiting tyrosinase activity toward external substrates in a
stoichiometric fashion (Figure 3).34,47 By exposure to dioxygen, the Cu(I)
DBED complex (DBED = N,N’-di-tert-butyl-ethylenediamine) was
shown to form a m-η2:η2-peroxo dicopper(II) species. Addition of
(a) (b) (c)
(d)
Figure 3. The first mononucleating model system with tyrosinase activity, devel-
oped by Stack et al. and (a) the m-η2:η2-peroxo dicopper(II) complex, which is formed
upon coordination to copper(I) and oxygenation at low temperatures, (b) the ternary
adduct between a bis-µ-oxo dicopper(III) complex with bound phenolate, (c) the
catecholato adduct, and (d) the resulting copper(II)-semiquinone complex.34,47
130 Koch et al.
phenolate led to isomerization to a bis-m-oxo dicopper species, in which

attack on the bound substrate occurs, leading to a bound catecholato spe-
cies (and subsequently to a copper(II)-semiquinone complex). Importantly,
this system provided the first evidence for a ‘‘ternary intermediate” (Cu +
O2 + substrate), which gave additional hints on how this reaction might
occur in the enzyme. In 2014 Lumb et al. showed that the copper(I)
DBED system also exhibits catalytic tyrosinase activity if a slightly over-
stoichiometric amount of ligand is used. In this case the diamine ligand
also adopts the role of a base.48 Applying this protocol, Lumb et al.
described the catalytic conversion of several monophenols to the corre-
sponding ortho-quinones and other functionalized organic products,
respectively.50–53
The first catalytic tyrosinase model complex supported by a bidentate
ligand was presented in 2010.37 The employed ligand Lpy1 (Figure 4)
represents one-half of the dinucleating BiPh(impy)2 ligand (see above
and Figure 2) and contains a combination of a tert-butyl-terminated imine
and a pyridine.36,37 Employing Bulkowski-Réglier conditions, the Cu(I)
complex in the presence of dioxygen mediates the formation of 3,5-
DTBQ with a TON of 22 after six hours.37 This discovery was the starting
point for the development of other mononuclear catalytic model systems
of tyrosinase supported by bidentate N-donor ligands. The focus of this
research was mainly put on increasing the activity and the stability of the
corresponding catalysts.37,39,40,42,44,45,63
Based on comprehensive spectroscopic studies, a catalytic cycle was
derived for the Lpy1 system (Scheme 3).37 By addition of two equivalents
of 2,4-DTBP-H and triethylamine to the Cu(I)Lpy1 complex (1), Cu(I)
complex 2 is formed, which could be isolated and analyzed using UV/vis
and NMR spectroscopy. Through oxygenation of 2 a band at 340 nm
appears in the UV/vis spectrum indicating the formation of a µ-catecholato-
µ-hydroxo intermediate (4). Furthermore, an HCl quench following the
oxidation of 2 leads to 3,5-DTBQ, as well as 3,5-di-tert-butylcatechol
(3,5-DTBC-H2), deriving from the catecholato adduct in 4. Under
Bulkowski-Réglier conditions, 4 releases the bound catecholato adduct as
3,5-DTBQ. The protonation of the hydroxide group of 4 through proto-
nated triethylamine initiates the formation of 3,5-DTBQ, indicating that
triethylamine acts as a proton shuttle. The release of 3,5-DTBQ and water
Scheme 3. Catalytic cycle for the monophenolase reaction mediated by bidentate

N-donor model systems.7,37,39,40,42,45
results in a reduction of CuII to CuI and the reformation of 2, thus closing

the catalytic cycle.37
The isolation and characterization of the catecholato adduct 4 imply
the presence of a ternary phenolato-peroxo complex 3, which, however,
could not be detected in the Lpy1 system. Nevertheless, formation of the
µ-η2:η2-peroxo species could be evidenced at 185 K in acetone using UV–
vis spectroscopy, showing that this species is also mechanistically
relevant.
In 2014 Lumb et al. used Lpy1 derivatives for the conversion of 4-tert-
butylphenol to the corresponding coupled quinone, which was mediated
132 Koch et al.
(a) (b)
Figure 4. Model systems with an imine moiety and an N-heterocycle and the
obtained TONs under Bulkowski–Réglier conditions with 2,4-DTBP-H as substrate
and a catalyst concentration of 500 µmol/L. (a) The TONs were obtained using 0.1
mmol catalyst, 10 eq. of 4-tert-butylphenol and 5 eq. of NEt3.37,39,40,44,48
by Lpy1 derivatives.48 The substitution of the tert-butyl moiety of Lpy1

with 4-methoxybenzene and naphthalene led to L5 and L6, respectively.
For ligand L3 the alkyl chain of Lpy1 was shortened to only one CH2-unit
(Figure 4). In this study a 1:10 and thus higher catalyst-to-substrate ratio
was applied and the oxygenation was performed in the absence and pres-
ence of 4 Å molecular sieves. These conditions led to product yields of
57–58% (TON ≈ 3) without desiccant, and 93–96% (TON ≈ 5) with the
use of molecular sieves, respectively.48
B. Substitution of Pyridine in the Lpy1 Ligand by N-Heterocycles

To learn more about electronic and structural influences on the reactivity
of the Lpy1 system toward phenolic substrates, and eventually increase its
catalytic activity, the pyridine moiety was first substituted by other hetero-
cyclic groups. Notably, previous investigations had shown that Cu(I)
complexes supported by ligands, composed of two pyridine (DPM) and
two tert-butyl-terminated imine functions (L2), are catalytically inac-

tive.37,64 Therefore, the combination of an imine unit and a heterocycle
appeared to be superior to symmetric ligands composed of two heterocy-
cles/two imine units (see below).
In pursuit of the stated goals, analogs of Lpy1 were established, which
in particular contain (i) a benzimidazole (Lbzm1), (ii) a pyrazole (Lhpz1
and Lhpz2), or (iii) an imidazole (Limz1) moiety instead of the pyridine
ring (Figure 4).37,39,40,44 All of these systems showed catalytic monophe-
nolase activity toward 2,4-DTBP-H.39,40,44 By substitution of the pyridine
moiety of Lpy1 with pyrazole (Lhpz1) and 3,5-dimethylpyrazole (Lhpz2),
respectively, the catalytic activity of the systems increased from a TON of
22 to 29 and 23, respectively.40 The substitution of pyridine with benzimi-
dazole (Lbzm1) even resulted in a TON of 31, whereas the system with an
imidazole as N-heterocycle only exhibited a TON of 16.39,44 Thus, using
pyrazole and benzimidazole instead of pyridine increases the catalytic
activity whereas imidazole lowers the TON. In contrast to the parent Lpy1
system it was not possible to detect a µ-η2:η2-peroxo intermediate for any
of these modified systems.37,39,40,44
The obtained experimental results can be rationalized by considering
the bonding properties of the employed N-heterocyclic groups. In order to
mediate an electrophilic substitution (SE) on the arene ring of the coordi-
nated substrate, the side-on-bound peroxo ligand should transfer as much
electron density as possible into the two copper centers. This will in turn
be facilitated by employing weakly s-donating and/or strongly p-accept-
ing co-ligands. As a measure of s-donor strength, the pKa value of the
bound N-heterocycle (in its protonated form, cf. Table 1) may be
employed.65 Inspection of these values explains that replacement of pyri-
dine by, for example, pyrazole increases the catalytic efficiency. A further
increase of activity should be possible by going to triazole (see below). On
Table 1. pKa values of imines and different N-heterocycles (in their protonated
form).65,66,67
imine imidazole benzimidazole pyridine pyrazole triazole
pKa 7–8 7 5.6 5.2 2.5 1.17
134 Koch et al.
the other hand, exchange of pyridine by imidazole should lower the cata-
lytic activity as s-donation is increased, again in agreement with the
experimental observation.
Of particular interest is the large activity increase observed upon
replacing pyridine by benzimidazole. Based on the pKa values, this sub-
stitution should have little effect. However, benzannulation lowers both
the HOMO and the LUMO of imidazole.67,68 While the former explains
the decrease of s-donation (and concomitant decrease of pKa) upon going
from imidazole to benzimidazole (cf. Table 1), the latter acts to increase
the p-backbonding capability, that is, benzimidazole should be more back-
bonding than imidazole. Probably, the combination of both effects makes
benzimidazole superior to pyridine when employed as a donor group in
copper monooxygenation catalysts. This has already been exploited by
Casella whose L66- and L6-supported tyrosinase models, for example,
contained benzimidazoles as terminal N-donors (and not pyridines; see
above).55,56
C. Substitution of Imine in the Lpy1 Ligand by N-heterocycles:

Symmetric Bidentate Ligands
After replacement of the pyridine heterocycle in the ligand backbone by
other N-heterocycles, it was of interest to check whether the imine moi-
ety of the Lpy1 ligand can be replaced by an N-heterocycle as well.
First, symmetric ligands were considered. As the DPM system had been
found to be catalytically inactive (see above),37 the focus was first
put on (substituted) pyrazoles as N-heterocycles.42 Specifically, Cu(I)
complexes were prepared using the ligands 1,1’-methylenebis-1
H-pyrazol (BPM), 1,1’-methylenebis(3-methyl-1H-pyrazole) (mBPM)
and 1,1’-methylenebis(3,5-di-methyl-1H-pyrazole) (dmBPM; Figure
5).42 By investigating the model systems regarding the monooxygena-
tion of 2,4-DTBP-H, catalytic activity could be detected for all three
model systems. Specifically, the BPM-, mBPM-, and dmBPM-sup-
ported systems mediated the conversion to 3,5-DTBQ with TONs of 21,
19 and 11, respectively. Besides 2,4-DTBP-H various other substrates
were tested, for example small substrates like phenol (P-H), substrates
containing electron-drawing residues such as 4-methoxyphenol
Figure 5. Symmetric model systems for tyrosinase and their catalytic activity under
Bulkowski-Réglier conditions vs. 2,4-DTBP-H and a catalyst concentration of 500
µmol/L.37,42,44,64
(4-MeOP-H), or the biomimetic substrate N-acetyl-L-tyrosine ethyl

ester monohydrate (NATEE). The monophenolase reaction was cata-
lyzed by the model systems for all substrates, albeit with different
TONs.42 These findings allowed to derive substrate-specific factors
influencing their catalytic monooxygenation (see below). Interestingly,
it was possible to detect the µ-η2:η2-peroxo intermediate for the dmBPM
system at –90°C in acetone, whereas this was not possible for the BPM
and mBPM systems.42 This strongly indicates a steric influence on the
stability of the peroxo intermediate as well as on the reactivity of the
model systems (this point is discussed in more detail below).
By substitution of the pyrazole moieties with N-methylimidazoles, the
BIMZ ligand was obtained (Figure 5). Although a monophenolase activ-
ity towards 2,4-DTBP-H was detected, only a small TON of 9 was
observed.44 Note that the increase of catalytic activity upon going from
DPM to the pyrazole-based ligands BPM, mBPM and dmBPM corre-
sponds to the results obtained with the asymmetric LhetX ligands (het =
N-heterocycle, X = 1 or 2; cf. previous section), where replacement of
Lpy1 by the (less electron-donating) Lhpz1/Lhpz2 ligands was found to
increase the catalytic activity of the corresponding Cu(I) complexes.
Moreover, replacement of the pyrazole moieties in BPM by (more elec-
tron-donating) imidazoles led to a decreased catalytic activity of the Cu(I)
136 Koch et al.
complex supported by the BIMZ ligand, again in line with the results
obtained for the asymmetric ligands.
Overall, however, the catalytic activities of the Cu(I) complexes sup-
ported by the symmetric ligands are conspicuously lower than those of
their counterparts with asymmetric ligands. This result was surprising as
it is not compatible with the bonding properties discussed so far: based on
the corresponding pKa values, all of the employed heterocycles are
weaker s-donors than imine (cf. Table 1). From a purely electronic point
of view, replacement of imine by these moieties thus should increase (and
not decrease) the catalytic activity. The complete catalytic inactivity of the
DPM complex was even more striking; based on the activity sequence of
the asymmetric systems it should lead to a TON between that of the imi-
dazole and the pyrazole system.
In an attempt to understand the described findings, the formation and
possible constitution of the ternary intermediate 3 (cf. Scheme 3) are con-
sidered in more detail. Note that in a stoichiometric reaction mode (upon
which the mechanism depicted in Scheme 3 is based) the substrate is
bound to the Cu(I) precursor before oxygenation. In a catalytic run, how-
ever, the substrate is added to the Cu(I) precursor [Cu(L)(CH3CN)2]+
(L = bidentate ligand) and will bind to the µ-η2:η2-peroxo complex formed
upon oxygenation of the latter, generating ternary intermediate 3. In order
to get hydroxylated, the substrate has to bind within the Cu2O2 plane
(Scheme 4).6
Such a configuration, in which the peroxide s* orbital overlaps with
the p-system of the arene ring6 can in principle be reached by two path-
ways which correspond to an associative and a dissociative mechanism. In
the associative scheme initial binding of the substrate proceeds axially to
one of the Cu(II) centers of the planar µ-η2:η2-dicopper complex; e.g. CuB
N
N O N
Cu Cu
O N
O
Scheme 4. Overlap of a phenolate p-type orbital and the σ* orbital of the peroxo
ligand.6
Scheme 5. Associative and dissociative pathways of the substrate coordination for

a monooxygenation reaction mediated by a catalytic copper complex supported by
(top) symmetric and (bottom) asymmetric ligands.
(cf. Scheme 5 left). To reach an equatorial coordination, the ligand sphere

around CuB has to rotate around an axis connecting the two copper centers
(cf. Scheme 5, top). Such an axial → equatorial rearrangement has also
been discussed for the enzyme.4,6,69
In the frame of a dissociative mechanism, equatorial coordination of
the substrate in the Cu2O2 plane occurs after dissociation of one of the two
donor groups of the bidentate ligand from the peroxo complex (Scheme
5. bottom). Rebinding of that donor after coordination of the substrate at
the free equatorial position, may subsequently occur in axial position or
not depending on its donor/acceptor strength (if not, the bidentate ligand
would act as a hemilabile ligand).70 In the context of this (dissociative)
pathway, the function of a comparatively strong donor (such as imine)
would be to keep the bidentate ligand bound to the peroxo dicopper com-
plex and the ternary intermediate, respectively. On the other hand, such a
dissociative scenario would be mechanistically unfavorable for a symmet-
ric bidentate ligand with two comparatively weak donors as the ternary
138 Koch et al.
Table 2. Observed TOFs (min–1) after 15 min for different model systems upon
catalytic conversion of 2,4-DTBP-H.37,39,40,42,44
Lhpz1 BPM
Lpy1 Lbzm1 (Lhpz2) Limz1 (mBPM/dmBPM) BIMZ
TOF 0.41 0.84 0.85 0.38 0.27 0.17
(0–15 min) (0.62) (0.24/ 0.22)
intermediate would be less stabilized and total loss of the bidentate ligand
may occur. It is thus conceivable that mixed ligands with one imine group
(as a comparatively strong donor) and one (weaker coordinating)
N-heterocycle follow a dissociative pathway for the binding of the sub-
strate whereas symmetric ligands with two comparatively weak donors
follow an associative pathway. The latter pathway is a priori kinetically
disfavored compared to the former due to the fact that equatorial coordi-
nation of the substrate only occurs after an axial → equatorial rearrange-
ment whereas this is not necessary within the dissociative scheme. This
mechanistic difference may explain the overall lower activity of the sym-
metric ligands, which is also evident from lower observed TOFs as com-
pared to their asymmetric, imine-containing analogs
(cf. Table 2). The total lack of catalytic activity observed for the DPM
system, finally, may be associated with the fact that this is the only sym-
metric ligand that is composed of two six-membered rings as opposed to
the other symmetric ligands that contain two N-heterocycles with five-
membered rings. This renders the bite angle of DPM smaller than that of
the latter systems, which in turn could become relevant, e.g., in the course
of the axial → equatorial rearrangement.
D. Hybrid Ligands Composed of Different N-Heterocycles

In view of the fact that the imine function of Lpy1 and related systems is
unstable against hydrolysis, which might be one reason for limited TONs
of the corresponding copper catalysts,37,44 it also appeared of interest to
replace it by hydrolytically more stable oxazoline units. Combination with
imidazole groups led to the three asymmetric ligands LOL1, LOL2 and
LOL3 (Figure 6).44 Three different substituents; i.e., phenyl, methyl and
tert-butyl, provided electronic and steric variation of the ligands.
Figure 6. The LOLX model systems with the ability to mediate the tyrosinase reac-
tion in a stoichiometric fashion under Bulkowski-Réglier conditions vs. 2,4-DTBP-H
and a catalyst concentration of 500 µmol/L.44
Investigation of the catalytic activity, however, showed that the derived

Cu(I) complexes are inactive regarding the catalytic monooxygenation of
phenols and only mediate the conversion of 2,4-DTBP-H in a stoichiomet-
ric fashion.44 This aspect could be explained with the influence of struc-
tural factors like the bulky substituents of the oxazoline residues and a
lower flexibility of the ligand backbone. The oxazoline units, especially in
case of the LOL1 ligand, are more sterically demanding than the imine
function.44
In order to further explore whether bidentate ligands for copper
monooxygenase models can be assembled in a modular fashion, a
hybrid of the (catalytically inactive) dipyridylmethane (DPM) and the
(catalytically active) bispyrazolylmethane (BPM) system was investi-
gated in 2018. Specifically, the ligands pyrazolmethylpyridine (PMP)
and 3,5-dimethylpyrazolmethylpyridine (dmPMP) were prepared
(Figure 7),37,42,45 and corresponding copper(I) complexes were investi-
gated regarding their ability to catalyze the oxygenation of several
monophenolic substrates. Regarding 2,4-DTPB-H these complexes
exhibited TONs of 14 for PMP and 11 for dmPMP. Remarkably, the
TON of the PMP system is intermediate between that of the catalyti-
cally inactive DPM (TON = 0) and the medium-to-highly active BPM
model system (TON = 21),45 confirming the foregoing hypothesis.
Again, the methyl-substituted dmPMP system exhibits a lower activity
towards external substrates than its parent, unsubstituted analog. The
140 Koch et al.
Figure 7. Ligands containing different N-heterocycles and the TON of the corre-
sponding model systems under Bulkowski-Réglier conditions with 2,4-DTBP-H as
substrate and a catalyst concentration of 500 µmol/L.45,63
µ-η2:η2-peroxo intermediate could be observed for both complexes

upon exposure to dioxygen at low temperatures, although with low
yields (2.5% for the PMP and 3.3% for the dmPMP system).45
Interestingly, crystals grown from a solution of [Cu(PMP)(MeCN)2]
PF6 provided evidence for the formation of the homoleptic complex
[Cu(PMP)2]PF6. For the dmPMP system the analogous complex was not
isolated under these conditions, but it was possible to obtain it by direct
synthesis.45 These findings suggest the existence of an equilibrium
between the heteroleptic and the homoleptic complex in solution, poten-
tially rendering the mechanism of the catalytic monooxygenation reaction
more complicated. Density functional theory (DFT) was used to gain
more insights into the behavior of the two systems in solution. Importantly,
these calculations supported the presence of an equilibrium between the
heteroleptic and the homoleptic complex in solution for both systems.45
Furthermore, investigation of the PMP systems led to single crystals of a
dinuclear complex in which two Cu(II)-dmPMP units are bridged by a
fluoro ligand deriving from the counter ion PF6.45 This might correspond
to one of the decay products of PMP-type catalysts after prolonged reac-
tion times and give a hint on possible deactivation pathways for this type
of catalysts (see below).
As the electron-poor N-donor pyrazole in ligands such as PMP and

BPM led to medium-to-high catalytic activities (see above), using even
less electron-donating N-heterocycles such as triazoles was supposed to
further increase the reactivity towards external substrates (cf.
Table 1).42,45,63 Exchanging the pyrazole group in PMP by triazole led to
the TMP family of ligands containing a tolyl (TMP1), anisole (TMP2)
and tert-butyl (TMP3) residue at the triazole’s C4 position (Figure 7).
The ability of these systems to mediate the monophenolase reaction was
investigated using 2,4-DTBP-H, 3-TBP-H (3-tert-butylphenol) and
4-MeOP-H (4-methoxyphenol) as substrates. In fact, the corresponding
model systems exhibited high catalytic activities towards 2,4-DTBP-H
with TONs of 20, 22 and 24 for TMP1, TMP2 and TMP3, corroborating
the inverse relationship between the s-donor strength of the supporting
ligand and the catalytic monooxygenation activity of the derived Cu(I)
complex (see above). Employing the TMP3 model system, the highest
TON (48) yet observed for the copper-mediated monooxygenation of a
phenolic substrate (4-MeOP-H) was achieved.63 Interestingly and in con-
trast to the previous model systems, the influence of the residue on the
triazole heterocycle was found to be small. Thus, the TMP3 system with
the most sterically demanding tert-butyl residue exhibited the highest
catalytic activity. The difference towards the methyl-substituted systems
of BPM, PMP, and Lhpz is that the C3 and C5 positions are unsubstituted
for the TMP systems and the residue is bound in C4 position.42,45 This
results in a larger distance to the peroxo ligand, which leads to a smaller
steric influence. Therefore, although the residues for the TMP systems
are larger than for the previous systems, their steric influence is small. As
for the PMP model systems a µ-η2:η2-peroxo intermediate was also
detected for the TMP model systems, although only in small yields of
1.1% to 2.6%.63
Concluding this chapter, it should be mentioned that recently Herres-
Pawlis et al. presented another model system with a bidentate N-donor
ligand. Supported by a hybrid guanidine ligand the corresponding copper
complex forms a bis-µ-oxo dicopper(III) species when exposed to oxygen
at low temperatures. The copper-oxygen species catalytically converts dif-
ferent phenolic substrates into ortho-quinones with high product yields.61
142 Koch et al.
Figure 8. Different bidentate ligands with and without methyl residues.40,42,45
Table 3. Catalytic activity of different unsubstituted model systems and their

methyl substituted counterparts.40,42,45
Decrease of activity vs.
Model system TON vs. 2,4-DTBP-H unsubstituted system (in %)
Lhpz1 (Lhpz2) 29 (23) 21
BPM (mBPM/dmBPM) 21 (19/11) 10/48
PMP (dmPMP) 14 (11) 21
E. Steric Influence of Substituents on the Supporting Ligands

In the preceding sections the electronic influence on the catalytic activity
of copper catalysts supported by bidentate ligands has been considered in
detail. As already mentioned, the use of different residues altering the steric
bulk of the ligand adds another parameter influencing the catalyst’s activity.
This first became evident when investigating the Lhpz1 system, which
exhibited a TON of 29 towards 2,4-DTBP-H (Figure 8): using the 3,5-dime-
thyl substituted derivate Lhpz2, decreased the activity by 21% (TON = 23,
Table 3).40 For the symmetric BPM systems analogous results were
obtained throughout all examined substrates, with exception of phenol
(P-H) and 4-MeOP-H; i.e., the unsubstituted BPM shows the highest activ-
ity towards external substrates, while the TON is lowered for the 3-methyl
substituted mBPM and significantly decreased for the 3,5-dimethyl substi-
tuted dmBPM. The conversion of 2,4‑DTBP-H to 3,5‑DTBQ, e.g., is medi-
ated with a TON of 21 for BPM, while reduced by 10%/48% when using
mBPM/ dmBPM, respectively. The same observation was made for the
PMP systems: while the parent, unsubstituted system mediates the
conversion to 3,5-DTBQ with a TON of 14, the activity is lowered by 21%

for the dmPMP system, where a TON of 11 was obtained (Table 3).45
These findings can be explained by the fact that the presence of bulky
residues in the ligand backbone disfavors the coordination of the substrate
to the copper center in the peroxo intermediate (Scheme 3) as well as
electrophilic attack of the µ‑η2:η2‑peroxo ligand on the coordinated phenol
in ortho-position (Scheme 3).37 Thus, conversion of the substrate to the
ortho-quinone is hampered and the system’s catalytic activity reduced. On
the other hand, steric shielding of the peroxo ligand stabilizes the copper-
peroxo complex. Thus, detection of the µ‑η2:η2‑peroxo intermediate was
successful for the dmBPM system, while it could not be achieved for
BPM or mBPM.42 In agreement with this, the yield of the µ‑η2:η2‑peroxo-
intermediate detected for the dmPMP was larger than for the PMP sys-
tem.45 An important consequence of the lower reaction rate associated
with steric shielding may be that the significance of side reactions, deac-
tivating the catalyst, increases. One of these pathways is the reaction of
the Cu(II) catecholato complex (Scheme 3) with water, which is liberated
in the course of the catalytic cycle, leading to a bis-µ-hydroxo species.48
Other possible decay products are generated by reactions with anions
deriving from the counterion (cf. the µ-fluoro-bridged dinuclear Cu(II)
dmPMP; see above).45
F. Variation of Substrates: Electronic and Steric Factors

Apart from the electronic and steric properties of the ligands, which
have been considered in the previous sections, the product yields and
corresponding TONs of copper-mediated monooxygenation reactions
are significantly influenced by the nature of the substrate and its interac-
tion with the catalyst. The substrates which we have employed to inves-
tigate the tyrosinase activity of our model systems range from small or
low-activated (P-H, 4-MeP-H) to sterically demanding (2,4-DTBP-H,
NATEE) and highly activated (4-MeOP-H) phenols.7,37,42,44,45,63 In the
following the different properties of the used substrates and their influ-
ence on the outcome of the monooxygenation reaction are briefly
discussed.
144 Koch et al.
Scheme 6. The reactivity of the various investigated substrates.[37,42,48,49,63,72]
Although the substrate 2,4‑DTBP‑H carries two bulky tert-butyl resi-

dues and is therefore sterically hindered, it is commonly used in investi-
gations of monophenolase activity.7,44,45,63 Apart from increasing the
steric bulk of the substrate the residues induce a +I-effect, thus increasing
the electron-density in the aromatic system.7,42,71 This favors the electro-
philic attack by the peroxo ligand. Furthermore, the corresponding ortho-
quinone (Scheme 6 top, labs = 407 nm, e = 1830 L mol–1 cm–1) exhibits
a high stability, hence isolation and characterization of this product is
easy.37,44,71 Nevertheless, when using 2,4-DTBP-H as a substrate the
coupled biphenol 3,3’,5,5’-tetra-tert-butyl-2,2’-biphenol (BP-H) is
obtained as an unphysiological by-product. It is formed from phenoxyl
radicals generated by H-atom transfer to the peroxo complex via an oxi-
dative coupling reaction.49,71
An alternative to the “standard substrate” 2,4-DTBP-H is represented
by 3-TBP-H, which reacts under catalytic conditions to the coupled qui-
none 4-(tert-butyl)-5-[3-(tert-butyl)phenoxy]cyclohexa-3,5-diene-1,2-di-
one (Scheme 6 bottom, labs = 425 nm, e = 898 L mol–1 cm–1).7,44,48 Here,
only one tert-butyl residue is bound to the aromatic ring in meta-position,
thus leading to a less sterically encumbered coordination to the copper
Table 4. Overview of the obtained TONs with different bidentate model systemsa
and different substrates.
BPM TMP1
Lhpz1 (mBPM/ PMP (TMP2/
Lpy1 (Lhpz2) dmBPM) LIMZ1 BIMZ LOL1–3 (dmPMP) TMP3)
2,4-DTBP-H 18 29 21 16 9 <1 14 20
(23) (19 / 11) (11) (22 / 24)
3-TBP-H 22 20 6 25 20
(15 / 12) (13) (19 / 24)
4-MeOP-H 35 34 6 34 42
(10 / 15) (33) (45 / 48)
4-MeP-H 14
(12 / 10)
P-H 9
(9 / 9)
NATEE 18 25 0
(15 / 11)
a
DPM and L2 are catalytically inactive and therefore not listed
center in comparison to 2,4-DTBP-H.42,63 On the other hand, the elec-

tronic activation of the residue is smaller. Consequently for the most
model systems with this substrate, similar TONs are observed as for
2,4-DTBP-H (Table 4).42,44,63
In contrast to the substrates considered so far, 4-MeOP-H does not
contain bulky residues, but a methoxy group in para-position that acti-
vates the aromatic system due to the a +M-effect.42,44,45,63 In accordance
with the high activation and small steric bulk of the substrate, the observed
TONs for this substrate are very high. Thus, TONs between 42 and 48
were observed for the TMP model systems (Table 4).63 Under catalytic
conditions 4-MeOP-H is converted into the coupled quinone 4-methoxy-
5-(4-methoxyphenoxy)cyclohexa-3,5-diene-1,2-dione (Scheme 6 bottom,
labs = 418 nm, e = 524 L mol–1 cm–1).44,48,73 In the presence of water the
para-quinone 2-hydroxy-5-methoxy-1,4-benzoquinone is formed from
the coupled quinone.42,63,72
When investigating the monophenolase activities of the symmetric
BPM systems, three further substrates were tested.42 The para-methyl
146 Koch et al.
substituted 4-MeP-H is slightly activated due to the +I-effect of the methyl

group. The small activation leads to low turnover frequencies (TOFs) and
TONs (14‑10 for BPM systems, Table 4), despite the fact that the steric
interaction between the substrate and the copper is small. In addition to
the monophenolase reaction a side reaction occurred during the catalysis,
reducing the substrate concentration and thus the possible product yield.
Presumably, under catalytic conditions a copper semiquinone complex is
formed and subsequently hydrolyzed in the presence of water, that itself
is generated during the catalysis.42 The formation of the 4-methylquinone
is associated with an absorption band at 445 nm (Scheme 6 top, e = 1400
L mol–1 cm–1).42 The smallest and least sterically hindered substrate is
P-H; the corresponding ortho-benzoquinone exhibits an absorption band
at 398 nm (Scheme 6 top, e = 1417 L mol–1 cm–1).74 The lack of bulky
residues, however, leads to two disadvantages. On the one hand, the phe-
nol is not activated and therefore the resulting TOF is small. On the other
hand, the unsubstituted aromatic C-H groups provide a large number of
possible binding sites for side-reactions such as oxidative coupling or
polymerization reactions.49,51,75 Indeed for the BPM supported systems
only TONs of 9 and low TOFs were obtained (Table 4).42
The substrate NATEE, finally, is closely related to the natural sub-
strate L-tyrosine and therefore the best choice from a biomimetic point of
view. Under catalytic conditions it is converted into N-acetyl-L-
dopaquinone ethyl ester (NADQEE) with an absorption band at 392 nm
(Scheme 6 top, e = 1417 L mol–1 cm–1).76 With this substrate for the BPM
model systems as well as for Limz1 medium to high TONs were observed
using UV/vis spectroscopy, which could be attributed to the +I-effect of
the residue and the fact that the residue is bound in para-position and
therefore substrate coordination to the copper center is not sterically hin-
dered (Table 4).42,44 However, the absorption band corresponding to the
ortho-quinone decreased after a few hours and the isolation via an HCl
quench failed as well. Presumably the product is unstable in the presence
of water, that originates from the catalytic cycle and the substrate
itself.42,44
To conclude, it is possible to mimic tyrosinase activity with our model
systems employing a large range of monophenols. It was found that
especially the activation of the aromatic system through the electronic
Scheme 7. a: electronic activation of the aromatic system by the +I / +M-effect of

the residues, b: steric hindrance of the residues upon coordination on the copper
center.
influence of the residues greatly influences the resulting turnover num-

ber.7,42,44,45,63 Furthermore, due to steric hindrance it is favorable to use
substrates without residues in or in vicinity to the ortho-position to the
hydroxyl group. These findings are graphically summarized in Scheme 7,
showing the interplay between electronic activation and steric bulk. As
4-MeOP-H is not sterically encumbered and exhibits a high activation of
the aromatic system induced by the +M-effect of the methoxy group, usu-
ally the highest TONs are observed for this substrate (Table 4).42,44,45,63
For the biomimetic substrate NATEE, on the other hand, a high conversion
to the corresponding ortho-quinone is also observed; however, this system
suffers from the decay of NADQEE in the presence of water.42,44
148 Koch et al.
IV. Summary
Tyrosinase is a dinuclear copper protein that converts L-tyrosine to
L-dopaquinone. Besides structural and mechanistic investigations of the
natural enzyme relevant information about the underlying chemistry can
be obtained using small-molecule model systems. In the last decade sev-
eral catalytic tyrosinase models with bidentate N-donor ligands have been
established. Through detailed investigations of these systems, correlations
between the properties of the ligand and the substrate on the one side and
the observed catalytic activity on the other could be obtained. An impor-
tant aspect of these correlations is the s-donor/p-acceptor strength of the
employed N-donor groups. Moreover, asymmetric ligands are more effec-
tive than their symmetric counterparts, which can be attributed to the
kinetics of substrate binding.
Apart from the electronic influence of the N-donor groups the pres-
ence of bulky residues in the ligand backbone also influences the catalytic
activity of the system. While stabilizing the peroxo complex, sterically
demanding substituents may inhibit coordination of the substrate and
electrophilic attack by the peroxo group. On the other hand, small steric
hindrance in the vicinity of the ortho-position to the hydroxyl group and
an increased electron density within the aromatic system (e.g., through a
+M-effect of a methoxy group in 4-position) facilitate bonding of a phe-
nolic substrate to the copper-peroxo intermediate and subsequent electro-
philic attack by the peroxo group. The reactivity of the presented
tyrosinase model systems towards external monophenols thus is well
understood. Remaining challenges to be adressed refer to increasing the
turnover numbers, which are still low, and reducing the amount of base
needed for a catalytic activity of the investigated model systems.
V. Acknowledgment
The authors would like to thank M. Rolff, J. Schottenheim, J. Hamann, F.
Wendt, B. Herzigkeit, R. Schneider and other present and former mem-
bers of the copper workgroup for their contributions to the research
described in this review.
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5 Electrochemistry and
Spectroelectrochemistry of
Copper-Oxygen-Relevant
Species
Nicolas Le Poul
Laboratoire CEMCA, UMR CNRS 6521, Université de Bretagne

Occidentale, 29238 Brest, France
[email protected]
I. Introduction
Redox reactions involving molecular oxygen (O2) are ubiquitous in chem-
istry and biology.1 For instance, the hydroxylation of dopamine into nor-
epinephrine in the catecholamine synthetic pathway is catalyzed by the
copper-based DbM glycoprotein (DbM = Dopamine b Monooxygenase).
This redox enzyme, among others such as the particulate methane
monooxygenase (pMMO)2 or peptidylglycine α-hydroxylating monooxy-
genase (PHM),3 is able to cleave the O–O bond of O2 and to perform the
incorporation of one oxygen atom into a C–H bond at a specific position.
Alternatively, the four-electron reduction of dioxygen into water is carried
153
154 Le Poul
out at the biological scale by oxidases such as laccases or cytochrome

c oxidase, where dioxygen serves as a pure electron/proton acceptor, thus
inhibiting the generation of partially reduced oxygen species (PROS).4
For more than 40 years now, these enzymes have been a source of inspira-
tion for chemists5 aiming at developing efficient catalysts for synthetic
chemistry, fuel cell devices, solar photoelectrochemical cells, or organic
lithium–air batteries.6 Since dioxygen is kinetically inert toward organic
substrates, its activation is classically carried out by reduced metals
(M = Fe, Cu, Mn) in active synthetic and biological systems.1a,7
For instance, the reaction of one or several Cu(I) centers with O2 results
in the formation of mono- or polynuclear metal–oxygen adducts such as
copper superoxide, peroxides, or hydroperoxides (Figure 1). These tran-
sient species are particularly reactive and can evolve under certain
conditions (presence of acid and/or reductants) toward ultra-reactive high-
valent species.1b,5 The latter have attracted much attention these last
years because of their potential use as catalysts for hydrogen-atom
Figure 1. Schematic representation of the different copper–oxygen relevant species

including precursors and sulfur derivatives investigated by electrochemistry and
considered in this chapter. Abbreviations: ES: end-on superoxo; SP: side-on peroxo;
O: bis(µ-oxo); EP: end-on peroxo; OH: hydroxo; OA: alkoxo; OPh: phenoxo; OC:
carboxylato; Cu2O: µ-oxo; HP: hydroperoxo; AP: alkoperoxo; PhNOCu: nitrosoarene;
SH: hydrosulfido; SPh: thiophenolato; SS: side-on disulfido. *Precursors for reactive
species.
Electrochemistry and Spectroelectrochemistry 155
abstraction (HAA) of strong C–H bonds of alkanes (BDFE > 90 kcal mol–1,
BDFE = Bond Dissociation Free Energy).8 Significant progress has been
recently made on the elucidation of the different mechanistic pathways,
which can occur for HAA reactions involving Cun:O2 species. In particu-
lar, the concerted or stepwise features of the coupled electron–proton
reactions have been thoroughly investigated in both thermodynamics and
kinetics terms.9 This has often, but not always, required the determination
of redox potential values of the copper–oxygen adducts by either chemical
or electrochemical means.
Independently, many copper systems have also been studied as poten-
tial electrocatalysts for the oxygen reduction reaction (ORR) in fuel
cells10,11 and oxygen evolution reaction (OER) from water oxidation.12
Most of reported studies have been carried out by using voltammetric
methods, with the aim of quantifying the electrode kinetics from electro-
chemical analysis (Tafel slope, E/pH variation). More rarely, key active
copper–oxygen species have been characterized by decoupling electron
and proton transfers in presence of organic solvents.13
As a matter of fact, electrochemical and spectroelectrochemical char-
acterizations of Cun:O2 species have been seldom reported, although they
have been well investigated by UV–vis or resonance Raman spectrosco-
pies.5,14 One main reason is that these species are often unstable in the
conditions of experimental measurement (room temperature). As it will be
shown in the following sections throughout chosen examples, several
strategies have been proposed to overcome this issue: (i) modification of
the ligand topology by using a pre-organized framework to trap O2, or by
inserting H-donors to interact with O atoms; (ii) in situ electrochemical
reduction of stable Cu(II) species, which can further react with O2, or
electrochemical oxidation of stable Cu(II) precusors such as such as
copper(II) hydroxo, alkoxo, or carboxylato cores (Figure 1) into reactive
oxidized species; and (iii) decrease of the temperature of measurement to
increase the lifetime of the copper–oxygen adduct. These three main strat-
egies have been successfully applied (sometimes combined) and have
allowed electrochemical characterization of several species. An alterna-
tive approach based on the reaction of copper–oxygen adducts with
chemical reductants/oxidants has also been developed these last years.15
156 Le Poul
Nevertheless, this indirect procedure yields only approximated values of

thermodynamic and kinetic parameters.
From this statement, the purpose of this chapter is to gather electro-
chemical data existing on copper–oxygen relevant species (Figure 1) and
to demonstrate how electrochemistry and spectroelectrochemistry can
bring useful information and help for getting mechanistic insights on
dioxygen activation pathways. The chapter has been divided into four sec-
tions according to the different families of complexes shown in Figure 1.
The first section describes the electrochemistry of superoxides and perox-
ides species formed upon reaction of Cu(I) complexes with dioxygen. For
most of these complexes, low-temperature was necessary to allow full
stabilization and characterization by electrochemical and spectroelectro-
chemical techniques. The second section is focused on the redox proper-
ties of mono/dicopper(II) hydroxides, alkoxides, and carboxylates species,
which are precursors for reactive Cu(III)–oxygen adducts. The third sec-
tion aims at describing the electrochemistry of Cu(II) hydroperoxides and
alkoperoxides complexes, which can themselves act as reactive species
toward hydrogenated substrates, or lead upon O–O bond cleavage to high-
valent oxyl adducts. The fourth section discusses about the redox chemis-
try of copper(II) phenoxides complexes, which yield reactive phenoxyl
metal species upon oxidation. At last, the fifth section describes the recent
electrochemical studies of sulfides and nitrosoarene analogues of copper–
oxygen adducts. For each section, the most striking examples have been
chosen and illustrated.
II. Electrochemistry of Peroxide and Superoxide

Copper–Oxygen Species
As shown in Figure 2, mono or dinuclear superoxide and peroxide copper
species can be generated from the reaction of Cu(I) complexes with
dioxygen. For a great majority of cases, these copper–oxygen adducts are
highly unstable and require the use of low temperature to stabilize them,
making their experimental characterization quite difficult. Since Cu(I)
complexes are highly O2-sensitive, they can also be prepared in situ from
their Cu(II) analogues by using either chemical or electrochemical reduc-
tion under inert conditions. Once stabilized at low temperature, Cu(I) can
be reacted with O2 to generate Cu(II) superoxide or peroxide species.
Figure 2. Electron and proton–electron transfer reactions for mono and dinuclear
superoxo and peroxo copper species. Plain-line rectangles designate the species of
interest, here superoxo and peroxo Cu(II) cores. Dotted-line rectangles indicate reac-
tions that have not yet been evidenced by electrochemical methods.
As shown in Figure 2, monoelectronic reduction of Cu(II)-superoxides

leads to Cu(II)-peroxides. In presence of proton source, this monoreduction
produces Cu(II) hydroperoxide species, which may further undergo O–O
bond cleavage through further e−, H+ transfer. The resulting Cu(II)-oxyl
158 Le Poul
species have been postulated as being strong oxidant for HAA reactions.16
Figure 2 also displays the redox chemistry starting from end-on or side-on
dinuclear peroxo species. In particular, monoelectronic reduction of side-
on dicopper(II) peroxides may lead to O–O bond cleavage and formation
of mixed-valent Cu(II)–Cu(III) bis(µ-oxo) adducts. The latter have been
suggested to be key species in the oxidation of methane in pMMOs.17
First investigations on redox properties of peroxide/superoxide
copper–oxygen species were initiated in 1992 by Karlin and co-workers
for three different side-on peroxo dicopper(II) complexes 1–3 bearing two
linked bis-pyridyl(ethylamine) moieties (Figure 3).18 These adducts were
prepared at low temperature (193 K) in dichloromethane from the reaction
of bis-Cu(I) complexes with dioxygen. UV–visible spectroscopic analyses
supported by EXAFS studies of these adducts evidenced the formation of
bent-butterfly peroxo dicopper(II) species resulting from an
Figure 3. Schematic representation of peroxo and superoxo complexes 1–9.

Table 1. Electrochemical data for side-on peroxo (SP), end-on peroxo (EP), bis(µ-
oxo) (O), and end-on superoxo (ES) complexes 1–9 (red: reduction; ox: oxidation).
Complex E1/2 / V vs. Fc+/Fc Conditions Adduct Ref.
a,b S
1 −1.77 (red) 193 K, CH2Cl2/NBu4PF6 P [18]
a,b S
2 −1.57 (red) 193 K, CH2Cl2/NBu4PF6 P [18]
a,b S
3 −1.87 (red) 193 K, CH2Cl2/NBu4PF6 P [18]
4 0.08 (red)c 195 K, CH2Cl2/NBu4(B(C6F5)4) S
P/O [20]
c S
5 −0.03 (red) 195 K, CH2Cl2/NBu4(B(C6F5)4) P/O [20]
E
6 −0.36 (ox) 193 K, CH2Cl2/NBu4ClO4 P [21]
7 −0.75 (red)a 293 K, CH2Cl2/NBu4PF6 S
P [19]
d E
8 −0.59 (ox) 273 K, CH3CN/NBu4PF6 P [9c]
E
9 < −0.90 (red) 213 K, Acetone/NBu4PF6 S [22]
a
Irreversible peak; bRecalculated versus Fc+/Fc by taking E0(Fc+/Fc) = 0.67 V versus Ag/AgCl
in this solvent; cRecalculated versus Fc+/Fc by taking E0(Fc+/Fc) = 0.47 V versus SCE in this
solvent; dRecalculated versus Fc+/Fc by taking E0(Fc+/Fc) = 0.08 V versus Ag/AgNO3 in this
solvent.
intramolecular O2 binding. Low-temperature CV of the peroxo complexes

performed under argon displayed an irreversible reduction peak varying
between −1.57 V and −1.87 V versus Fc+/Fc (Table 1), a potential value
close to that of free dioxygen.19 A comparative voltammetric analysis sug-
gested that the process was likely monoelectronic. No oxidation peak
could be detected until ca. 1.4 V versus Fc+/Fc, hence demonstrating the
difficulty to generate any superoxo bis-Cu(II) species.
Karlin and co-workers reported a few years later a voltammetric study
of peroxo complexes 4 and 5 (Figure 3).20 These adducts, generated by
reaction of mononuclear cuprous complexes [CuI(MeRPY2)]+ with dioxy-
gen, were previously shown to be efficient catalysts for HAA reactions for
a wide range of hydrogenated substrates. Low-temperature (195 K) CVs
were carried out in dichloromethane by using NBu4(B(C6F5)4) as support-
ing electrolyte. Noteworthy, both complexes 4 and 5 displayed a quasi-
reversible system upon reduction at +0.08 V and −0.03 V versus Fc+/Fc
(Figure 4 and Table 1), respectively, which was ascribed to the formation
of transient mixed-valent Cu2III/II bis(µ-oxo) species. From these thermo-
dynamic data and with the support of complementary studies, the authors
concluded that HAA of substrates by complexes 4 and 5 was likely occur-
ring through a concerted electron–proton transfer reaction (Figure 4).
160 Le Poul
(a) (b)
Figure 4. (a) CVs of complexes 4 (top) and 5 (bottom) (T = 195 K) in CH2Cl2/

NBu4(B(C6F5)4). (b) Concerted and stepwise mechanistic pathways considered for
HAA by these complexes. Adapted with permission from Ref. [20]. Copyright (2005)
American Chemical Society.
In 2017, the groups of Karlin and Le Poul on one side, as well as that
of Meyer on the other side reported the electrochemical properties of end-
on peroxo complexes 6 and 8.9c,21 For the complex 8 (Figure 3),9c cyclic
voltammetry analysis in acetonitrile at 0°C was sufficient to demonstrate
the reversibility of the peroxide/superoxide reaction at −0.59 V versus
Fc+/Fc (Table 1). This half-wave value was further utilized to calculate the
BDFE(O-H) (72 kcal mol–1) of the corresponding hydroperoxo species
knowing the pKa for the peroxo/hydroperoxo couple (22.2 in MeCN). For
the peroxo complex 6 (Figure 3), Lopez et al. investigated several aspects
of the peroxide/superoxide reaction upon oxidation of the complex at 193
K.21. A reversible monoelectronic system (Figure 5) was detected at a
slightly higher potential (−0.36 V vs. Fc+/Fc) than for complex 8 (Table 1).
Electrochemical impedance spectroscopy allowed the determination of
the electron transfer kinetics for this process. The relatively low inner-
sphere reorganizational energy (0.11 eV) was ascribed to an electron-
transfer process occurring exclusively on the O2 core. Noteworthy, in situ
UV–vis characterization of the superoxide was carried out at 193 K by a
cryo-spectroelectrochemical setup (Figure 5). Reduction of the superox-
ide allowed the back formation of the complex 6.
(a) (b)
Figure 5. (a) CVs of complex 6 in CH2Cl2/NBu4ClO4 for 183 < T < 203 K. (b) Low-
temperature (193 K) UV–vis in situ spectroelectrochemical monitoring of the perox-
ide oxidation (absorbance at 530 nm) leading to the formation of the superoxide
complex (absorbance at 406 nm). Adapted from Ref. [21] with permission from John
Wiley and Sons.
Electrochemical studies of the room-temperature stable side-on per-

oxo complex 7 was performed by Le Poul and co-workers in 2018.19 The
complex exhibited an irreversible two-electron reduction peak at −0.75 V
versus Fc+/Fc, which was assigned to the O–O bond cleavage.
Spectroelectrochemical measurements showed a complete disappearance
of the peroxo characteristic band at 365 nm upon reduction, while chemi-
cal analysis after exhaustive electrolysis indicated no formation of
hydrogen peroxide but merely a dicopper(II) bis(µ-OH) species. More
recently, de Leener et al. studied the reaction of a calix[6]trenamide
Cu(I) complex with dioxygen.22 Low-temperature spectroelectrochemis-
try in acetone showed the formation of a transient superoxo species
(complex 9, Figure 3) by in situ reduction of the Cu(II) complex.
Although no further reduction could be detected by CV, the data sug-
gested that reduction of the superoxide could only occur at lower poten-
tial values than −0.90 V versus Fc+/Fc. Noteworthy, room temperature
investigations demonstrated that the copper superoxo could perform self-
oxidation of the ligand (insertion of an oxygen atom) despite its low
oxidation potential value.
162 Le Poul
hydroxo, alkoxo, and carbonato copper species. Plain-line rectangles designate the
species of interest, here hydroxo, alkoxo, and carbonato Cu(II) cores. The dotted-line
rectangle indicates a reaction that has not yet been evidenced by electrochemical
methods.
III. Electrochemistry of Copper(II) Hydroxo,

Alkoxo, Carboxylato, and Oxo Precusors
Alternatively to the classical Cu(I)/O2 approach described above, a quite
recent strategy based on the electrochemical oxidation of Cu(II) hydroxo,
alkoxo, or carboxylato complexes has been reported. The principle is to
generate a Cu(III) oxygen species from a stable Cu(II) precursor, which
can be easily handled at room temperature. This simple approach allows
direct generation of high-valent copper oxygen adducts. In the case of
hydroxo complexes, further deprotonation of the Cu(III)-OH species may
yield Cu(III)-oxo core (Figure 6).
This strategy was first initiated by Tolman and co-workers in 2014. As
a first example, a dicopper(II) mono-µ-hydroxo complex bearing a
Figure 7. Schematic representation of hydroxo, alkoxo, carboxylate, and oxo pre-

cursors 10–31.
164 Le Poul
macrocyclic ligand with two pyridine(dicarboxamide) moieties was

reported (complex 10, Figure 7).23 This anionic and strongly donating
ligand was specifically chosen in order to stabilize the high-valent Cu(III)
states of the complex upon oxidation. Room temperature voltammetry
experiments in wet DMF exhibited two reversible systems at 0.18 and
Table 2. Electrochemical data for hydroxo (OH), alkoxo (OA), carboxylato (OC), and
µ-oxo (Cu2O) complexes 10–31 (red: reduction; ox: oxidation).
H
10 0.18 (ox1); 0.47 (ox2) 293 K, DMF/NBu4PF6 O [23]
H
11 −0.17 (ox) 293 K, Acetone or THF/ O [8a,24]
NBu4PF6
12 0.12 (ox) 293 K, DFB/NBu4PF6 OH [15b]
13 0.14 (ox) 293 K, DFB/NBu4BArF4 O H
[25]
H
14 −0.13 (ox) 293 K, DFB/NBu4PF6 O [25]
15 −0.26 (ox) 293 K, DFB/NBu4PF6 OH [15b]
16 −0.20 (ox) 293 K, THF/NBu4PF6 OH [24b]
A
17 0.04 (ox) 293 K, THF/NBu4PF6 O [24b]
18 −0.02 (ox) 293 K, THF/NBu4PF6 OA [24b]
C
19 0.23 (ox) 293 K, THF/NBu4PF6 O [26]
20 0.24 (ox) 293 K, THF/NBu4PF6 OC [26]
C
21 0.17 (ox) 293 K, THF/NBu4PF6 O [26]
C
22 0.15 (ox) 293 K, THF/NBu4PF6 O [26]
23 0.30 (ox) 293 K, THF/NBu4PF6 OC [26]
C
24 0.15 (ox) 293 K, THF/NBu4PF6 O [26]
a C
25 0.63 (ox) 293 K, DMF/NBu4PF6 O [27]
26 0.12 (ox1); 0.64 (ox2)a 213 K, DMF/NBu4ClO4 OH [28]
H
27 −0.48 (red);1.87 (ox) 243 K, MeCN/NBu4ClO4 O [9d,e]
H
28 0.54 (ox) 243 K, MeCN/NBu4ClO4 O [9d]
29 −1.05 (ox1);−0.28 293 K, DMF/NBu4PF6 OH [29]
(ox2)
30 1.26 (ox) 293 K, MeCN/NBu4PF6 OH [30]
Cu2
31 0.76 (ox1); 1.35 (ox2) 243 K, MeCN/NBu4ClO4 O [9d]
a
Irreversible peak.
0.47 V versus Fc+/Fc, which were ascribed to the generation of Cu2(III)/(II)

and Cu2(III)/(III) µ-hydroxo species (Table 2). From these values, low-T
chemical oxidation with appropriate oxidants led to the UV–vis and EPR
spectroscopic characterization of the mono- and bis-oxidized species.
With the support of TD-DFT, the authors suggested that the bridging
ligand remained as a hydroxo group (rather than an oxo one), and that
oxidation steps occurred effectively on the metal ions.
The same group then carried out similar studies with a series of mono-
nuclear Cu(II)-hydroxo complexes bearing one pyridine(dicarboxamide)
moiety (complexes 11–16, Figure 6). The subtle modification of the
ligand by various substituting groups allowed fine tuning of the Cu(II)–
Cu(III) oxidation potential from −0.26 V to 0.14 V versus Fc+/Fc accord-
ing to their room-temperature voltammetric measurements (Table 2).
Interestingly for complex 11, the electrochemical oxidation was found to
be reversible at moderate scan rate in acetone24a while it was irreversible
in THF.8a In complement, partial reversibility was recovered in deuterated
THF at 0.2 V s–1 (Figure 8),8a hence evidencing the reaction of the elec-
trochemically generated Cu(III) species with THF. This effect was further
analyzed in the frame of a HAA reaction for which THF acts as substrate
(BDFE = 92 kcal mol−1) through the formation of a Cu(III)-OH-THF
adduct (Figure 8).
(a) (b)
Figure 8. (a) Mechanistic pathway for THF oxidation mediated by the mono-
oxidized Cu(III)-OH form of complex 11. (b) Room-temperature CV at 0.2 V s–1 of
complex 11 in THF/NBu4PF6 (black) and THF-d8/NBu4PF6 (red). Adapted with per-
mission from Ref. [8a]. Copyright (2015) American Chemical Society.
166 Le Poul
More recently, various alkoxo (complexes 17–18, Figure 7) and car-

boxylato (complexes 19–24, Figure 7) Cu(II) adducts of this series were
prepared and studied by CV. For the carboxylate precursors, the oxidation
potential values were found to be more positive than for the hydroxo ana-
logues 11 and 17, as expected from their lower basicity. Furthermore, the
donating/withdrawing properties of the carboxylate ligand were shown to
impact significantly the redox potential value (150 mV variation over the
series; see Table 2), and consequently modify the HAA properties.
According to the authors, the experimental data evidenced an asynchro-
nous PCET oxidative mechanism for the reaction of the Cu(III)-
carboxylate species with tris-tertbutylphenol.
Other examples of hydroxo, alkoxo, or carboxylato complexes bearing
a different ligand backbone than the pyridine(dicarboxamide) moiety have
also been reported for the last five years (see complexes 25–31, Table 2,
and Figure 7). For instance, Thibon-Pourret et al. synthesized a dicopper(II)
hydroxide-phenoxide complex comprising an unsymmetrical ligand with
a N,N-bis(2-pyridyl)methylamine moiety on one side and a dianionic bis-
amide on the other side (complex 26, Figure 7).28 Electrochemical studies
of the complex at low temperature in DMF allowed the reversible oxida-
tion according to a monoelectronic process (Figure 9). In situ
(a) (b)
Figure 9. (a) Low-temperature (213 K) CV at v = 0.05 V s–1 of complex 26 in DMF/

NBu4ClO4. (b) UV–vis in situ spectroelectrochemical monitoring of the oxidation at
213 K leading to the formation of the mixed-valent CuIII/II hydroxide species (red
curve: i vs. t from CV plots). Adapted with permission from Ref. [28]. Copyright
(2018) American Chemical Society.
(a)
(b)
Figure 10. (a) X-ray structure of complex 30. (b) Electrocatalytic oxidation of SH by
the complex 30+ in presence of 2,6-lutidine (Lut). Adapted from Ref. [31] with per-
mission of the Royal Society of Chemistry.
time-resolved UV–vis spectroelectrochemistry at T = 213 K showed the

formation of a strong absorption band at 386 nm, which was further
ascribed to a phenoxido-to-Cu(III) LMCT transition for the mixed-valent
Cu2III/II µ(O(H)) species (Figure 9). The same research group published
two papers on the oxidation of a bis-µ(OH) dicopper(II) complex bearing
a naphthyridine-bridging ligand (complex 30, Figure 7).30–31 Noteworthy,
mono-oxidation of this complex at 1.2 V versus Fc+/Fc was shown to yield
a Cu2III/II bis-µ(OH) adduct at room temperature from UV-vis-NIR spec-
troelectrochemical characterization.30 Complementary works have dem-
onstrated that the latter exhibited electrocatalytic HAA oxidative properties
toward acetonitrile in presence of base (2,6-lutidine) through the forma-
tion of a putative Cu2III/II µ(O) µ(OH) species (Figure 10).31
Starting from a dicopper(II) µ-hydroxo complex 27 (Figure 7) based
on the TMPA ligand (TMPA = tris(methylpyridine)amine), Kieber-
Emmons and co-workers investigated either the mono-reduction9e or
mono-oxidation9d of this complex in order to generate CuII/I and CuIII/II
mixed-valent species, respectively. On one hand, low-T (243 K) CV in
reduction yielded a reversible system at −0.48 V versus Fc+/Fc, which was
assigned to a metal-centered process. From this value and knowing the
pKa (24.3) for the dicopper(II) hydroxo/oxo couple (complexes 27 and
31, Figure 7), the BDFE(OH) of the mixed-valent CuII/I hydroxide species
168 Le Poul
was found to be equal to 74.9 kcal mol–1. This value indicated that the
µ-oxo dicopper(II) complex 31 was a relatively poor oxidant for H-atom
abstraction. On the other hand, irreversible oxidation of complex 27 was
detected at high potential in dry and cooled (243 K) acetonitrile by square
wave voltammetry (SWV) (1.87 V vs. Fc+/Fc). Addition of a strong base
(in excess) allowed the generation of the unprotonated µ-oxo complex 31,
which displayed an oxidation peak at 0.76 V versus Fc+/Fc. According to
the authors, the system was reversible in the SWV timescale hence sug-
gesting the formation of the mono-oxidized oxo species, formally a CuIII-
O-CuII adduct. Further oxidation of this transient species was observed at
1.35 V versus Fc+/Fc by SWV, which was assigned to the dicopper(III)
µ(oxo) adduct. With the support of DFT calculations, and on the basis of
determined pKa values, the authors concluded that both mono and bis-
oxidized oxo adducts were strong oxidants for HAA (103.4 and 91.7 kcal
mol–1, respectively). Noteworthy, the better oxidative properties for the
mono-oxidized were ascribed to the linear feature of the Cu-O-Cu core,
which favored strong superexchange between Cu centers through the cen-
tral oxygen and was merely considered as a dicopper(II)-oxyl adduct.
Alternatively, Garcia-Bosch and co-workers carried out room-
temperature voltammetric studies of the mononuclear copper(II)-hydroxide
complex 29 bearing a redox-active ligand comprising a tridentateamine and
two ureanyl H-bonds donors (Figure 7).29 Two reversible monoelectronic
oxidation waves were detected at –1.05 V and −0.28 V versus Fc+/Fc
(Table 2). Noteworthy, UV–vis, EPR, and XAS spectroscopic methods
using chemical oxidants evidenced that the electron transfer processes were
essentially located on the ligand backbone and not on the metal. While the
generated semi-quinone Cu(II) hydroxide species was found to be a moder-
ate H-abstractor, the bis-oxidized quinone Cu(II) hydroxo complex exhib-
ited a significant HAA 2e−/2H+ activity according to two sequential steps.
IV. Electrochemistry of Copper Hydroperoxide

and Alkoxoperoxide Species
Copper(II) hydroperoxide and alkoxoperoxide species have recently
drawn more attention because of the potential implication of either Cu(II)
or Cu(III) hydroperoxide adducts in PCET processes occurring in
hydroperoxo or alkoxoperoxo copper species. Plain-line rectangles designate the
species of interest, here hydroperoxo and alkylperoxo Cu(II) cores. Dotted-line rec-
tangles indicate reactions that have not yet been evidenced by electrochemical
methods.
monooxygenases such as PHM. As shown in Figure 11, oxidation of a

Cu(II)-OOR core occurs on the metal center and leads preferentially to a
Cu(III)-OOR species. The latter can perform oxidation reactions or poten-
tially release OOR• by homolytic Cu–O bond cleavage.32 Accordingly,
1e−, 1H+ reduction of Cu(II)-OOH species may lead to high-valent
Cu(III)-oxo adducts, whereas 1e−, 1H+ oxidation may yield superoxo
Cu(II) cores (Figure 11). Nevertheless, none of these two reactions has
ever been reported so far by electrochemical approaches.
170 Le Poul
Figure 12. Schematic representation of hydroperoxide and alkylperoxide com-

plexes 32–34.
(a) (b)
Figure 13. Normalized-scan-rate CVs of (a) complex 32 in DFB/NBu4PF6 at 273 K,

and (b) complex 34 in THF/NBu4PF6 at 293 K. Adapted with permission from Refs.
[9b] and [32]. Copyright (2017 and 2019) American Chemical Society.
Indeed, while many examples have been described on the electro-

chemistry of Cu(II)-hydroxide complexes, these for Cu(II) alkylperoxo
and hydroperoxo remain scarce. Only three studies have been reported so
far, all by the group of Tolman for mononuclear complexes (see com-
plexes 32–34, Figure 12).9b,32 One main reason is that Cu(II)-OOH spe-
cies are themselves reactive species toward hydrogenated substrates, as
peroxide and superoxide copper complexes, and thus require low-
temperature conditions to be stabilized and characterized. Moreover, their
preparation based on the reaction of a Cu(II) hydroxo complex with
hydrogen peroxide in presence of a base, is usually not quantitative. This
renders the in situ electrochemical characterization not feasible.
Table 3. Electrochemical data for hydroperoxide (HP) and alkylperox-

ide (AP) complexes 32–34 (red: reduction; ox: oxidation).
a H
32 −0.21 (ox) 273 K, DFB/NBu4PF6 P [9b]
b A
33 −0.20 (ox) 293 K, THF/NBu4PF6 P [32]
c A
34 −0.15 (ox) 293 K, THF/NBu4PF6 P [32]
a
E1/2 value assumed from CV simulations. Irreversible peak found at −0.18 V
versus Fc+/Fc at v = 0.1 V s−1 Irreversible peak; bE1/2 value taken at v = 0.5 V s−1;
c
E1/2 value taken at v = 0.5 V s−1.
As shown in Figure 13, CV of the hydroperoxide complex 32

displayed an irreversible oxidation peak at −0.18 V versus Fc+/Fc in
1,2-difluorobenzene at 273 K. According to the authors, the process was
monoelectronic and led to the transient Cu(III)-OOH species. Increase of
the scan rate until 2 V s–1 did not reveal any reversibility, thus suggesting
that a fast chemical reaction followed the oxidation of the hydroperoxide
species (EC mechanism, E = electrochemical, C = chemical). Interestingly,
the peak potential value was close to that found for the Cu(II)-OH analo-
gous complex 11 (Table 2), but here oxidation was fully irreversible
whereas it was reversible for 11 in DFB and acetone. From simulated CVs
assuming an EC mechanism, a half-wave potential value equal to −0.21 V
versus Fc+/Fc was considered to calculate the BDFE(OH) of complex 32
knowing the pKa of the Cu(II)-superoxo/Cu(III)-hydroperoxo couple
(pKa = 19). The resulting high BDFE(OH) value (87 kcal mol–1) was
indicative of the relatively strong oxidative HAA properties of the super-
oxide species.
With the same pyridine(dicarboxamide) ligand, alkylperoxide Cu(II)
complexes 33 and 34 displayed slightly more positive oxidation potential
values than that obtained for complex 32 (see Table 3).32 However, for the
alkylperoxide complexes, reversibility was detected for v > 0.5 V s−1 at
room temperature hence evidencing that the alkyl groups stabilize the
Cu(III) redox state. The Cu(III) character of the mono-oxidized species
was confirmed by UV–vis and EPR spectroscopic studies by using chemi-
cal oxidants.
172 Le Poul
copper phenoxide species. Plain-line rectangles designate the species of interest,
here phenolato Cu(II) cores.
V. Electrochemistry of Copper(II)-Phenoxide Species

Phenoxide-based mono- and dicopper(II) precursors have been exten-
sively investigated by electrochemical methods. Indeed, their oxidation
affords the generation of phenoxyl Cu(II) radical species (Figure 14),
which are of interest for oxidation reactions in synthetic chemistry.
Moreover, these species are bio-relevant, since tyrosyl radicals participate
as co-factor (tyrosine residue) in biological reactions such as in occurring
in galactose oxidase. Many different ligands bearing one or two phenox-
ide moieties have been reported, and the readers are invited to refer to
several articles and reviews that gather spectroscopic and electrochemical
properties.33 In the following, a restricted list of complexes has been con-
sidered in the view of their remarkable redox behavior.
For instance, Tolman and co-workers investigated the redox properties
of a series of three monocopper(II) complexes bearing a phenoxide moi-
ety attached to a macrocyclic TACN ligand (TACN = triazacyclononane)
(complexes 35–38, Figure 15).34 These biomimetic complexes were stud-
ied at room temperature in dichloromethane. As shown in Table 4, their
monoelectronic oxidation occurred at potential values at ca. 0.20 V versus
Figure 15. Schematic representation of phenoxo complexes 35–55.
Fc+/Fc, which were dependent on both the substituents on the phenoxo

unit (120 mV increase from Me to iPr) and the exogenous ligand bound
to the Cu(II) (chloride or triflate anions). The Cu(II)-phenoxide radicals
of complexes 36 and 37 were also prepared by exhaustive electrolysis and
174 Le Poul
Table 4. Electrochemical data for phenoxo (OPh) copper(II) complexes 35–55 (red:
reduction; ox: oxidation).
a,b Ph
35 0.24 (ox) 293 K, CH2Cl2/NBu4PF6 O [34a]
b Ph
36 0.12 (ox) 293 K, CH2Cl2/NBu4PF6 O [34a]
b Ph
37 0.25 (ox) 293 K, CH2Cl2/NBu4PF6 O [34a]
b Ph
38 0.32 (ox) 293 K, CH2Cl2/NBu4PF6 O [34b]
39 0.03 (ox1)b; 0.31 (ox2)b 293 K, CH2Cl2/NBu4PF6 OPh [34b]
c c Ph
40 0.41 (ox1) ; 0.58 (ox2) 233 K, CH3CN/NBu4ClO4 O [35]
Ph
41 0.55 (ox) 233 K, CH2Cl2/NBu4PF6 O [36]
Ph
42 0.28 (ox) 233 K, CH2Cl2/NBu4PF6 O [36]
Ph
43 0.46 (ox) 293 K, CH2Cl2/NBu4ClO4 O [37]
Ph
44 0.45 (ox1); 0.65 (ox2) 293 K, CH2Cl2/NBu4ClO4 O [38]
Ph
45 0.15 (ox1); 0.54 (ox2) 293 K, CH2Cl2/NBu4ClO4 O [38a]
Ph
Ph
Ph
Ph
50 0.34 (ox1); 0.53 (ox2) 293 K, CH2Cl2/NBu4ClO4 OPh [39]
Ph
51 0.51 (ox) 293 K, CH3CN/NBu4PF6 O [33d,40]
a Ph
52 0.71 (ox) 293 K, CH3CN/NBu4PF6 O [40]
a Ph
53 1.20 (ox) 293 K, CH3CN/NBu4PF6 O [40]
54 1.04 (ox)a 293 K, CH3CN/NBu4PF6 OPh [40]
a Ph
55 0.90 (ox) 293 K, CH3CN/NBu4PF6 O [40]
a b + 0 +
Irreversible peak; Recalculated versus Fc /Fc by taking E (Fc /Fc) = 0.47 V versus SCE in this
solvent; cRecalculated versus Fc+/Fc by taking E0(Fc+/Fc) = 0.40 V versus SCE in this solvent.
characterized by UV–vis and EPR spectroscopies.34a The resulting spectra

exhibited a band around 410 nm, which was ascribed to a p-p* transition
of the phenoxyl radicals by analogy with analogous studies with free radi-
cals. In a further paper,34b the same research group synthesized a bis-
phenoxide complex of the same family of ligand (TACN) (complex 39).
CV studies displayed two reversible monoelectronic waves at 0.03 and
(a) (b)
Figure 16. (a) Room-temperature CVs at 0.1 V s−1 of complex 44 (black), 45 (red),
and 46 (blue) in CH2Cl2/NBu4ClO4 (R = tBu). (b) Schematic representation of the
mono-oxidized species 44+, 45+, and 46+. Adapted with permission from Refs. [38a]
and [38b]. Copyright (2018) Elsevier. Copyright (2008) American Chemical Society.
0.31 V versus Fc+/Fc, which were ascribed to the successive oxidation of

each phenoxo moieties by comparison with an analogous bis-phenoxide
Zn(II) complex and the phenoxo complex 36. Chemical generation of the
mono-phenoxyl species was achieved by reaction of complex 38 with a
Ce(IV) salt at 233 K.
Another interesting study of Cu(II)-phenoxide oxidation was also
performed by the group of Stack in 2003 by using salen ligand (N2O2
Schiff bases).38, 41 Two complexes differing by the nature of the bridging
moiety (complexes 44 and 46, Figure 15) were analyzed by electrochemi-
cal and spectroscopic methods. As shown in Table 1, a substantial
decrease of the oxidation potential by nearly 400 mV for both oxidation
events was observed when the imine groups were substituted by amines
(Figure 16). This result was consistent with the stronger basic properties
of the amine. However, complex 46 was shown to be unexpectedly better
than complex 44 for the catalytic oxidation of benzylic alcohol in pres-
ence of a chemical oxidant.38 More recently, Storr and co-workers
176 Le Poul
Figure 17. UV — vis spectroelectrochemical monitoring of the oxidation of com-

plex 55 in CH3CN/NBu4PF6. Adapted with permission from Ref. [40]. Copyright
(2017) American Chemical Society.
completed these investigations by determining the redox and catalytic

properties of complex 45 bearing one imino group and one amino moiety
(Figure 15).38a They found that mono-oxidation occurred on the ami-
nophenoxide side, leading to the formation of Cu(II)-phenoxyl species.
However, benzylic alcohol oxidation was found to be slower than for
complex 46.
Very recently, bis-phenoxide salen monoCu(II) complexes 47–50
(Figure 15) comprising distorted bridging ligands and various substituents
on the phenoxide moieties were studied by Thomas and co-workers39 in
the continuation of Stack’s complex 40.35 As shown in Table 4, a substan-
tial variation of the oxidation potential value for the generation of mono-
phenoxyl radical species was observed for both oxidation processes,
particularly by changing the donor properties of the substituting groups
(OMe vs. tBu) on the phenoxide moieties. The generated mono-Cu(II)
mono-phenoxyl radicals, which were all assigned to class II mixed-valent
compounds, displayed remarkable catalytic properties for the aerobic oxi-
dation of unactivated primary alcohols such as 2-phenylethanol.
Dicopper(II) complexes 51–55 comprising hydroxo and phenolato
bridging moieties as well as bis(alkylpyridyl)amine ligands were thor-
oughly studied by electrochemistry, spectroelectrochemistry, and DFT
calculations.40 Whereas complex 51 bearing a methoxy-phenolate ligand
displayed a reversible monoelectronic oxidation at 0.51 V versus Fc+/Fc,
the other Cu(II) complexes 52–55 exhibited an irreversible oxidation peak

at potential values higher than 0.71 V. A remarkable result was obtained
with the unsymmetrical complex 55 since a transient mixed-valent
Cu(II)–Cu(III) phenoxide species displaying a PhO− → Cu(III) charge
transfer band at 518 nm was detected by performing room-temperature in
situ and time-resolved spectroelectrochemical experiments (Figure 17).
This species evolved rapidly (t1/2 = 14 s at 20°C) toward a dicopper (II,II)
phenoxyl radical complex. The specific redox behavior of this complex
was ascribed to the unsymmetrical design of the ligand combined with the
chain length separating the amine from the pyridyl groups on one side of
the molecule. According to the authors, ethyl spacers (vs. methyl, see
complex 52, Figure 15) were sufficiently flexible to accommodate for the
geometrical arrangements required to stabilize the Cu(III) species.
VI. Electrochemistry of Nitroso and Sulfide Analogues

of Copper–Oxygen Complexes
Very recent works have shown that nitroso and sulfide Cu(II) adducts
could also be of interest for oxidation reaction, such as HAA, by analogy
with their copper–oxygen counterparts. Figure 18 displays the different
electron-transfer reactions that can be expected from such species. On one
hand, mono or dicopper(II) nitrisoarene complexes may undergo monoe-
lectronic reduction since they are isoelectronic analogues of copper super-
oxide adducts, the electron input taking place either on the metal or on the
nitrosoarene ligand. On the other hand, copper(II)-sulfide are analogues of
Cu(II)-hydroxo adducts. They can be thus employed as precursors for the
generation of high-valent Cu(III) species as for the copper(II)-hydroxo
case. In consequence, their redox behavior is expected to be similar to that
of Cu(II)-OH species. At last, side-on dicopper(II) bisulfides are ana-
logues of dicopper(II) peroxides, hence expecting that their reduction
would lead to reactive mixed-valent Cu(II)–Cu(III) bis(µ-S) adducts
(Figure 18).
The recent investigations performed by Ottenwaelder and co-workers
on copper-nitrisoarene complexes have shed light on the redox behavior
of this family of complexes depending on the electronic properties of the
nitrosobenzene ligand (complexes 56–59, Figure 19).42 These adducts
178 Le Poul
Figure 18. Electron transfer reactions for mono and dinuclear nitrosoarene and (bi)
sulfido copper species. Plain-line rectangles designate the species of interest, here
nitrosoarene, sufido, and bisulfido Cu(II) cores. The dotted-line rectangle indicates a
reaction that has not yet been evidenced by electrochemical methods.
were prepared by reaction of copper(I) complexes of N,N,N′,N′-

tetramethypropylenediamine ligand (TMPD) with a series of para-
substituted nitrosobenzene derivatives. Through adequate tuning of the
substituting groups on the arylnitroso moiety, various Cu-NO binding
modes were obtained and characterized at the solid state and in solution
by spectroscopic and electrochemical methods. Voltammetric studies of
Figure 19. Schematic representation of nitroso, disulfido, hydrosulfido, and thio-

phenolato complexes 56–63.
both the redox-active nitrosoarene ligands and their copper complexes

were carried out in dichloromethane. The ligands exhibited two succes-
sive quasi-reversible systems when scanning negatively, consistent with
an ECE mechanism comprising two monoelectronic electron transfers. In
strong contrast, all copper complexes displayed an irreversible reduction
peak at potential values varying between −0.72 V and −1.05 V versus Fc+/
Fc according to the donating/withdrawing properties of the substituent
(Table 5). These potential values were roughly 500 mV more positive than
those found for the nitrosoarene ligands. Exhaustive electrolysis indicated
that the reduction processes were monoelectronic and led to fast decom-
plexation, which released either the TMPD or nitrosoarene moieties. On
the other hand, electrochemical oxidation of the copper–nitrosoarene
complexes yielded a quasi-reversible system at a potential value varying
180 Le Poul
Table 5. Electrochemical data for nitrosoarene (PhNOCu), side-on disuldido (SS),

hydrosulfido (HS), and thiophenolato (PhS) complexes 56–63 (red: reduction; ox:
oxidation).
a a PhNO
56 −0.92 (red) ; 0.28 (ox) 293 K, CH2Cl2/NBu4OTf Cu [42]
a PhNO
57 −0.82 (red) ; 0.08 (ox) 293 K, CH2Cl2/NBu4OTf Cu [42]
58 −0.72 (red)a; 0.11 (ox) 293 K, CH2Cl2/NBu4OTf PhNO
Cu [42]
a PhNO
59 −1.05 (red) ; −0.36 (ox) 293 K, CH2Cl2/NBu4OTf Cu [42]
a S
60 −0.30 (red) 293 K, CH2Cl2/NBu4PF6 S [43]
61 −0.77 (red)a 293 K, CH2Cl2/NBu4PF6 S
S [43]
62 −0.21 (ox) 293 K, THF/NBu4PF6 HS [44]
63 −0.25 (ox) 293 K, THF/NBu4PF6 PhS [44]
a
Irreversible peak.
between −0.36 and 0.28 V versus Fc+/Fc, depending on the nature of sub-
stituting group. These complexes were shown to be capable to perform
H-atom abstraction on C–H bonds of dihydroanthracene (BDFE = 76 kcal
mol–1) but at low reaction rates.
Another electrochemical study of interest was carried out by Tolman
and co-workers in 2008 on side-on disulfido dicopper complexes,43 which
are structural models of side-on peroxide copper adducts. Voltammetry at
room temperature in dichloromethane displayed an irreversible reduction
peak at −0.30 V and −0.77 V versus Fc+/Fc for complexes 60 and 61,
respectively (Figure 19 and Table 5). The difference of potential was
ascribed to the donor ability of the neutral ligand (peralkylated bi- or tri-
amine). In contrast to complex 61, complex 60 was shown to be a two-
electron oxidant for substituted phenolates. More recently, the same group
conducted researches on sulfur-containing analogues of monocopper-
hydroxo species.44 Two Cu(II) complexes bearing a macrocyclic ligand
and a hydrosulfido and thiophenolato anion were synthesized (Figure 19,
complexes 62 and 63, respectively). Room-temperature voltammetric
studies of these complexes in THF exhibited a quasi-reversible 1e– system
in oxidation at −0.21 V and −0.25 V versus Fc+/Fc (Table 5). The slightly
more negative E1/2 value for the complex 63 versus 62 was consistent with
the better donor properties of the phenyl moiety compared to the hydrogen
atom. Analogously, complex 62 showed a significantly lower oxidation
potential value (−50 mV) than that obtained for its hydroxo counterpart
(complex 11), as a result of the higher electronegativity of S versus O.
Reactivity of complexes 62 and 63 with TEMPOH was much slower than
for the OH analogue (complex 11) in agreement with the electrochemical
data and the lower basicity of the SH- and SPh- ligands relative the
hydroxide.
VII. Summary
In conclusion, this chapter has reviewed the electrochemical and spectro-
electrochemical studies of relevant copper species, including unstable
copper oxygen adducts and their precursors. While the reported examples
remain still scarce, one can observe that the number of electrochemical
studies of these species has dramatically increased for the last five years.
One main reason is that electrochemical data (thermodynamics) can be
directly correlated to HAA reactivity, hence allowing the calculation of
BDFEs through the well-known Bordwell analysis knowing pKa values.
Moreover, under certain circumstances, kinetics of electron transfer have
been directly quantified from electrochemical measurements (CV or
EIS), giving the possibility to determine inner and outer reorganizational
energies with the help of heterogeneous electron transfer theories.
Another source of progress has been achieved with in situ UV–visible
cryo-spectroelectrochemical measurements that have afforded direct
characterization of transient species generated at the electrode surface at
low temperature21,28,45 hence avoiding exhaustive electrolysis and uncer-
tainty about the real nature of the generated transient species.
From all data given in this chapter, it seems difficult to provide defini-
tive conclusions and/or to predict any redox behavior for any or other
specific family of copper–oxygen adducts. The principal reason is that
subtle variation of the geometric or electronic properties of the ligand
itself can induce significant modification of the redox properties, such as
the half-wave potential value, or even move the location of the electron
transfer. This is best exemplified by the different UV–vis spectroelectro-
chemical responses that were obtained upon oxidation of the phenoxo-
based complexes 52 and 55, which only differ by the spacer chain length
on one side of the dinucleating ligand.
182 Le Poul
In the future, the employment of electrochemistry and spectroelectro-

chemistry for investigating the reactivity of copper–oxygen adducts may
become more systematic given the clear benefits provided by such meas-
urements. In complement, the development of new coupled in situ
spectroelectrochemical techniques adapted to this chemistry, such as EPR
and resonance Raman, may be of great help to better rationalize their
structure-reactivity relationships. Furthermore, the remarkable oxidative
properties of these species have not yet been fully exploited for the homo-
geneous and heterogeneous electrocatalytic oxidation of organic sub-
strates. Efforts in these directions should provide substantial progress
toward a better utilization of their remarkable properties.
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6 Inorganic Models of
Lytic Polysaccharide
Monooxygenases
Ivan Castillo*
*Instituto de Química, Universidad Nacional Autónoma de México,

Circuito Exterior, CU, 04510, Ciudad de México, México
[email protected]
I. Introduction
Global sources of energy and chemicals have depended heavily on petro-
leum-based platforms, which have been affordable thus far, at least from
an economic standpoint. It is evident, however, that sustainable practices
need to be prioritized to avoid serious environmental consequences,
beyond the ones we experience nowadays.1 To this end, efficient use of
renewable carbon feedstocks is paramount for the sustainable production
of fuels and chemical supplies.2,3
The aim of this chapter is to present a brief background of the
discovery of copper-dependent lytic polysaccharide monooxygenases
(LPMOs),4–6 within the context of inorganic and coordination chemistry.
Their discovery and characterization have led to the design of synthetic
systems that possess some of the properties of the enzymatic active sites.
Copper complexes thus developed will be discussed in terms of their capa-
bility to act as LPMO mimics, and to provide insight on the mechanism
187
188 Castillo
that allows the oxidative degradation of recalcitrant polysaccharides and

model substrates, starting with representative examples of copper-based
hydrolase mimics.
II. Lytic Polysaccharide Monooxygenases

The family of copper-dependent LPMOs was discovered among a consor-
tium of enzymes that act collectively to degrade recalcitrant polysaccha-
rides such as cellulose and chitin. Fungi and bacteria use such LPMO
enzymes to oxidatively cleave polysaccharide chains on their natural solid
substrates, in contrast to the more commonly described hydrolysis carried
out by numerous hydrolase enzymes.7,8
The active site of LPMOs consists of a type 2 copper center exposed
on the flat face of the enzymes that allows the interaction with its crystal-
line polymeric substrates.9 The coordination environment is defined by
three nitrogen donors, two of them from imidazole histidines and the third
one from the amino terminus that is part of one of the histidine residues.
This chelating motif has also been observed in copper-dependent particu-
late methane monooxygenase and is referred to as the “histidine brace,”
see Figure 1.
The electronic environment dictated by the T-shaped N3 donor set is
one of the key elements necessary to emulate the physicochemical proper-
ties of the LPMO active sites. These include among others the coordina-
tion geometry for both CuI and CuII, the redox potential, and ultimately
the reactivity toward O2 and H2O2 as oxidants to generate a yet to be
identified highly reactive copper–oxygen species. Aside from the T-shape
Figure 1. Schematic representation of the reduced (CuI) active site of LPMOs.

Inorganic Models of Lytic Polysaccharide Monooxygenases 189
environment that defines the equatorial coordination plane around copper

centers, and the bidentate histidine brace motif, another noteworthy aspect
of the LPMOs is the proximity of a phenylalanine or tyrosine residue to
one of the axial positions of the copper ions.4,10,11 Additionally, in fungal
enzymes, an unusual methylation of the amino-terminal histidine imida-
zole occurs at the N-atom.
Polysaccharide hydrolysis is hampered by their tightly packed crystal-
linity, and their recalcitrant nature is further reflected in the conditions
required to achieve oxidative degradation. In this regard, a potent oxidant
must be generated at the active site of LPMOs to activate the very strong
C–H bond at C1 or C4 of the polymer chains in the first step (Figure 2),12
which has an estimated strength of 95 kcal mol−1.13 This leads to aldonic
ketones and aldonolactones as products, with the latter being easily hydro-
lyzed to the corresponding aldonic acids in aqueous solution. These prod-
ucts constitute the oxidized end of the broken polymer chain, while the
remaining polymer should remain intact, as shown in Figure 2.
It has been speculated whether the natural oxidant employed by
LPMOs consists of atmospheric O2, or H2O2 generated by the microor-
ganisms.14 In the latter case, it might be worth considering renaming the
enzymes Lytic Polysaccharide Peroxygenases (LPPO). Furthermore, the
Figure 2. Oxidative mechanism of cellulose cleavage by LPMOs: top, aldonic

ketone formation after activation at C4; bottom, aldonolactone formation after acti-
vation at C1, followed by hydrolysis. R and R’ represent the polymer chains.
190 Castillo
nature of the reactive copper–oxygen species responsible for the initial

activation of the C–H bond at C1 or C4 of the monomer that approaches
the active site of the enzymes remains to be established.15 Both questions
may be answered by combining the insight gained from interrogating the
enzymes themselves, with the information that simple inorganic models
have provided in other copper–oxygen systems. A better understanding of
the mechanistic details of recalcitrant polysaccharide degradation should
lead to better catalysts for industrial applications to develop sustainable,
second-generation biofuels and chemicals from the abundant sources that
cellulose, chitin, and lignocellulose represent.
III. Copper Complexes as Hydrolase Mimics

A. Polysaccharide Hydrolysis
In contrast to the oxidative cleavage represented in Figure 2, initial poly-
saccharide degradation studies with copper complexes as catalysts
involved a glycoside hydrolysis. In this type of reactivity, carried out
enzymatically by hydrolases, attack of a molecule of water at either C1 or
C4 should result in the same products. Thus, no oxidation takes place at
either end of the broken polymer chains, as exemplified schematically for
chitin in Figure 3.
B. Copper Complexes
Studies regarding copper complexes as hydrolase mimics involve the
commonly employed p-nitrophenylpyranosides model substrates, with
cleavage of the p-nitrophenolate moiety conveniently monitored by
Figure 3. Hydrolytic mechanism of chitin cleavage; R and R’ represent the polymer

chains.
Figure 4. Top: binuclear CuII complexes 1–3, bottom: catalytic p-nitrophenyl-a-D-

galactopyranoside hydrolysis in aqueous 3-(cyclohexylamino)-1-propanesulfonic
acid (CAPS) buffer.
ultraviolet–visible spectroscopy (UV–vis) absorption spectroscopy at 410

nm (Figure 4). This strategy established that binuclear CuII complexes
with N2O donor sets are well suited for pyranoside hydrolysis at pH 10.5,
with a 11,000-fold increase relative to the background reaction at such pH
value.16
The binucleating scaffolds employed in these hydrolase mimics have
been exploited for the development of “microgel” particles that incorpo-
rate catalytic dicopper sites.17 Although the microgels display improved
hydrolase efficiency, likely due to the use of a carbohydrate template for
substrate recognition, the key component appears to be the bimetallic
nature of the copper centers. This is reflected in related dicopper systems
that display similar reactivity, with only minor modifications from N2O to
a N2O2 donor set relative to the original reports.18 Based on the evidence
provided by these dicopper systems, binuclearity appears as a requisite for
hydrolysis to occur, as opposed to the oxidative cleavage observed for
LPMOs and their models described below.
192 Castillo
Chemoselectivity for the hydrolysis of a- and b-glycopyranosides

featuring p-nitrophenolate and o-Cl-p-nitrophenolate as leaving groups
has been achieved with chiral versions of the binucleating ligand in
complex 2 (Figure 4).19 Moreover, a closer analogy to polysaccharide
cleavage was reported with this chiral system, resulting in catalytic
hydrolysis of the disaccharides maltose, cellobiose, and lactose at 60°C
and pH 8; notably, cellobiose was the most resilient of the disaccharides
toward hydrolysis under the conditions reported. In a related report, the
cyclic pseudo-peptide petallamide, isolated from blue algae, produces
dicopper(II) complexes with broad hydrolytic capacity that extends to
a- and b-glycosides.20 Interestingly, one of the structurally characterized
complexes features a bridging hydroxo ligand, which may be another
requisite for synthetic dicopper glycosidase mimics.
IV. Copper Complexes as LPMO Mimics

Reports concerning copper complexes specifically designed to emulate
some properties of the active site of LPMOs, including reactivity, have
slowly started to accumulate since the discovery of the enzymes. Such
LPMO model systems will be presented chronologically in the next sec-
tion. A related example of a copper-based system with hydrogen peroxide
as oxidant for the degradation of biomass feedstocks in alkaline media
exists.21 Nonetheless, the CuII/2,2′-bipyridine mixture employed was not
characterized, so that the nature of the complex formed in solution
remains to be established. It is hard to envision, however, that the species
formed may resemble the active site of LPMOs due to the bidentate nature
of bipyridine, and the lack of an aliphatic amine.
A. Copper(II)/Bis(Benzimidazolyl)Amine System
Structural analogs of the active site of LPMO enzymes were reported as
early as 2014, with the tridentate bis(benzimidazolyl)amine ligand 2BB
in Figure 5 providing the T-shaped N3 set that includes the amine as cen-
tral donor.22 An important feature of the ligands employed for LPMO
mimics is the size of the chelate defined by the cis-N2 moieties: the two
Figure 5. Left: 2BB; center: schematic representation of the T-shape active site of
AA9-11 LPMOs; right: Mercury diagram of [(2BB)Cu(H2O)2](OTf)2 at the 50% prob-
ability level; H atoms and triflate counterions omitted for clarity. Color code: C, gray;
N, blue; O, red; Cu, turquoise.
methylene linkers that connect the central amine to the 2-benzimidazolyl

fragments in 2BB give rise to six-member rings when coordinated to the
copper ion. Their relevance lies in the control of the redox potential that
the chelate size offers, where five-member rings favor low (more nega-
tive) and six-member rings favor high (more positive) potentials.23
Although electrochemistry was not explored in this initial report, the
solid-state structures of the complexes obtained with Cu(BF4)2(H2O)6,
CuCl2(H2O)2, and Cu(OTf)2(H2O)6 (OTf − = CF3SO3−) as CuII sources
consist of bimetallic [(2BB)Cu(m-F)2](BF4)2, as well as monometallic
[(2BB)CuCl2] and [(2BB)Cu(H2O)2](OTf)2.
Bond lengths in the monometallic complexes [(2BB)CuCl2] and
[(2BB)Cu(H2O)2](OTf)2, in particular, are comparable to those deter-
mined for the oxidized (CuII) forms of the enzymes.5,24 The Cu–N dis-
tances to the benzimidazole donors are almost constant, with an average
value of 1.99 Å, while the average Cu–N bond length to the central amine
is 2.11 Å; the corresponding Cu–N average distance to the imidazole
N-donors from histidine residues is 2.0 Å, and the distance to the primary
amines is 2.3 Å. These similarities extend to the T-shape provided by the
N3 donor set of 2BB, and the N–Cu–N angles involved: the trans angle
involving the cupric center and the benzimidazole N-atoms in the com-
plexes has an average of 170°, while the cis angle between the central
amine and the benzimidazole donors has a value of 92°. In the enzymes,
the values correspond to 165° and 97°, respectively. Despite these simi-
larities, [(2BB)CuCl2] and [(2BB)Cu(H2O)2](OTf)2 feature trigonal
194 Castillo
bipyramidal (TBP) geometries defined by the external Cl− and H2O

ligands, as opposed to the mostly square pyramidal (SP) geometry of the
active site of LPMOs, although in some cases intermediate structures have
been observed. The TBP geometries persist in acetonitrile solution, as
evidenced by low-temperature electron paramagnetic resonance (EPR)
spectroscopy at X-band frequency. The parameters determined for both
complexes are virtually identical, with g⊥ = 2.157 and g|| = 2.021, consist-
ent with a dz2 ground state; the A|| value could not be determined due to
poor spectral resolution.
This work was extended to the cuprous complex [(2BB)Cu]OTf,
which is monomeric in solution based on 1H nuclear magnetic resonance
spectroscopy (1H NMR) and electrospray ionization mass spectrometry
(ESI-MS) determinations.25 Confirmation was obtained by X-ray crystal-
lography, where the Cu(I) ion is coordinated by the T-shape N3 donor set
of 2BB at Cu–N distances characteristic of the reduced form of the
enzymes. This includes a wide N–Cu–N angle of 165° between the ben-
zimidazole donors, and an average of 97.5° between the benzimidazoles
and the central amine (Figure 6). For the previously described cupric
analog [(2BB)Cu(H2O)2](OTf)2, speciation studies in aqueous solution
revealed a stability constant of log K = 12.44, with the dicationic species
in the form of [(2BB)Cu(H2O)n]2+ (n = 1, 2) dominating from pH 4 to 8.
At higher pH values, a deprotonated species would be present in the form
of a cupric hydroxo dimer [(2BB)Cu(H2O)n-1]2(m-OH)2, see Figure 6.
Figure 6. Left: Mercury diagram of [(2BB)Cu]OTf at the 50% probability level; H

atoms (except H3 on the central amine) and triflate counterion omitted for clarity.
Color code: C, gray; N, blue; O, red; Cu, turquoise. Right: species distribution for
the aqueous 2BB/CuII system.
Deprotonation of [(2BB)Cu(H2O)n]2+ leading to the formation of a CuII–

OH species is relevant to LPMO and model systems: formulations of
alternative reactive copper-oxygen species contemplate a deprotonated
amine as anionic donor in the active site of LPMOs.26,27 However, com-
parison of the pKa values of CuII-coordinated water versus primary amine
favors deprotonation of the former, while access to amido donors under
the slightly acidic conditions for optimum LPMO catalytic activity
appears unlikely.28
Electrochemical determinations by cyclic voltammetry (CV) estab-
lished that the redox potential (E1/2) for the CuII/CuI couple of [(2BB)
Cu(H2O)2](OTf)2/[(2BB)Cu]OTf in aqueous solution from pH 4 to 7 has
a value of 226 mV versus the standard hydrogen electrode (SHE). This
value falls within those reported for LPMOs, which range from 150 to 370
mV.4,10,11,29 Thus, from an electrochemical perspective, the electronic
environment provided by 2BB mimics that of the active sites, and this is
further reflected in the reactivity with cellobiose as model substrate. The
use of the soluble disaccharide allowed to test the reactivity of the [(2BB)
Cu(H2O)2](OTf)2/[(2BB)Cu]OTf system in the presence of O2, KO2, and
H2O2/triethylamine (Et3N) as oxidants, instead of the insoluble natural
substrate cellulose. Analysis of the reaction mixtures by high-performance
liquid chromatography mass spectrometry (HPLC-MS) revealed in all
cases the formation of gluconic acid as the product of oxidative cleavage
of the disaccharide, together with a product assigned as doubly oxidized
glucose, see Figure 7.
Reactions of [(2BB)Cu]OTf with dioxygen in different solvents at
low temperature hinted at a copper–oxygen species that gives rise to an
absorption band at 360 nm and a d–d transition at 680 nm. Both [(2BB)
Cu(H2O)2](OTf)2 and [(2BB)Cu]OTf afforded inconclusive observations by
several spectroscopic techniques, including UV–vis, EPR, and ESI-MS upon
Figure 7. Oxidative cellobiose cleavage with [(2BB)Cu(H2O)2](OTf)2 or [(2BB)Cu]

OTf as catalysts and O2, KO2, or H2O2/Et3N as oxidants.
196 Castillo
addition of KO2. Finally, evidence for the formation of a peroxodicopper(II)

species was obtained from the reaction of the CuII complex [(2BB)
Cu(H2O)2](OTf)2 with excess H2O2/Et3N in acetonitrile solution at −30°C,
formulated as {[(2BB)CuII]2(m-h2:h2-O2)}2+ based on the Ligand to Metal
Charge Transfer band (LMCT) at 365 nm (e ≧ 8000 M−1cm−1).30,31
Cryospray ionization mass spectrometry (CSI-MS) confirmed the presence
of a dimeric species assigned as {[(2BB)CuII]2(O2)}2+, although resonance
Raman (rR) spectroscopy afforded no isotope-sensitive features under the
conditions described earlier for the generation of {[(2BB)CuII]2(m-
h2:h2-O2)}2+ with either H216O2 or H218O2/Et3N.
Recently, closely related bis(benzimidazolyl)amine-derived copper
complexes carried out the oxidative degradation of cellulose.32 The cupric
complexes feature axial EPR spectra, as expected based on their SP geom-
etries in the solid state,32,33 with very similar parameters among them at
g⊥ = 2.053 and g|| = 2.380 (A|| = 511 MHz). Reactions with the H2O2/Et3N
oxidant mixture give rise to monocopper species, based on their EPR sig-
nals. The most informative UV–vis spectroscopic study with one of the
complexes afforded bands at 347 (e = 1280 M−1cm−1) and 610 nm (e = 570
M−1cm−1) in acetonitrile at −30°C. These optical transitions are consistent
with a hydroperoxo to CuII LMCT and d-d/LMCT combination bands,
based on their intensities and DFT calculations. Preparative scale reac-
tions with cellulose as substrate resulted in up to 67% conversion to the
water-soluble products cellobiose, levoglucosan, and aldonic acid, based
on the mass recovered of insoluble substrate and HPLC-MS data.
B. Copper(III)/Bis(Carboxamido)Pyridine System
Tolman and coworkers have exploited the dianionic N3 scaffold provided
by the ligand in Figure 8, featuring a central pyridine flanked by two car-
boxamido donors and sterically-encumbering N-aryl substituents, which
enforces meridional coordination.34 Its potential to mimic the active site
of LPMO was identified in 2015 in the form of a CuIII–OH complex (1)
as oxidant,35 which is capable of oxidizing strong C–H bonds by hydro-
gen atom abstraction (HAT). Although the dianionic ligand differs signifi-
cantly from the neutral donor set provided by the enzymes, its relevance
arises from the potential involvement of the invoked deprotonated primary
(a) (b)
(c)
Figure 8. Left: complex 1−, X = OH. Right: oxidative glycosidic cleavage catalyzed
by LPMO and proposed structures for active site oxidants (a–c), including a CuIII–OH
complex (c), featuring a deprotonated amine.
amine that is part of the histidine brace in LPMOs.26,27 Complex 1 was

obtained by chemical oxidation of anionic 1−; the latter features an axial
EPR spectrum consistent with tetragonal symmetry at the [CuII–OH]+
core, and a sharp n(OH) at 3628 cm−1 in the IR spectrum.
Solid-state characterization of 1− was hampered by compositional
disorder due to a mixture of chloride and hydroxide ligands at the X posi-
tion in Figure 8. Nonetheless, the Cu–N1 distance of 1.920(2) Å to the
central pyridine and the average of 2.003 Å to the carboxamido N-donors
were reliably established.34 This is the opposite trend to what is observed
in LPMO active sites, where the central amine has the longest Cu–N
bond distance. Recently, a closely related ligand scaffold that stabilizes
the formally CuIII species [CuIII–OR]2+ (R = H, CH2CF3) with an elec-
tron-donating methoxy group in para position relative to the pyridine
N-atom allowed its structural authentication, as shown in Figure 9.36 As
expected for the CuIII–OH complex, all Cu–N/O distances shorten
198 Castillo
Figure 9. (a) Representation of the solid-state structure at the 50% probability

level of a p-methoxy substituted ligand supporting a [CuIII–OH]2+ complex.36
(b) H-abstraction by 1 from dihydroanthracene to generate anthracene and
[CuII–OH2]2+ complex 2.35
relative to its CuII–OH counterpart by an average of 0.102 Å. Assignment

of 1 as a CuIII species was confirmed by Cu K-edge X-ray absorption
spectroscopy (XAS), with a pre-edge feature at an energy that is ~1.7 eV
higher relative to its CuII counterpart 1−. Support for a diamagnetic spe-
cies was obtained by 1H NMR spectroscopy and a silent EPR spectrum
at X-band frequency.
Conversion of 1− to 1 was probed by CV, exhibiting a pseudo-
reversible oxidation at E1/2 = −74 mV for the CuIII/CuII couple relative to the
ferrocenium/ferrocene (Fc+/Fc) couple in THF solution.35 Its optical spec-
trum is characterized by a strong absorption band at λ = 540 nm
(ε ~12,400 M−1 cm−1). Regarding its reactivity, the authors highlight the
importance of 1 in the activation of the strong C–H bond of THF that is in
the a position relative to an ether functionality, which is related to the gly-
coside cleavage catalyzed by LPMOs. H-abstraction was tested with sub-
strates that span a range of ca. 20 kcal mol−1, from dihydroanthracene to
cyclohexane with the strongest C–H bond at around 100 kcal mol−1;37 in all
cases, the reaction is driven by the formation of the O–H bond, as exempli-
fied by the [CuII–OH2]2+ complex 2 in Figure 9. Reactions were monitored
by UV–vis spectroscopy, following the decay of the absorption band of 1 at

563 nm in difluorobenzene at −25°C. Intermediate (c), in Figure 8, has some
resemblance to 1 due to the presence of an anionic donor, and it has been
proposed that it is stabilized by deprotonation of the central amine, although
as mentioned before the pKa value of a primary amine would make this
unlikely in the enzymatic system in aqueous solution.28
C. Copper(II)/Copper(I)/(Pyridyl,Imidazolyl)Amine System
The complexes reported by Concia and coworkers feature closely related
ligands, where the central nitrogen donor takes the shape of an amine
(LAM) or an imine (LIM).38 In both cases, a 2-pyridyl fragment is con-
nected to the central N-atom by an ethylene linker that results in six-
membered chelates upon binding of copper. On the other hand, the
2-imidazolyl moiety is connected by a single carbon atom bridge to the
central nitrogen, resulting in five-membered chelates in both LAM and
LIM (Figure 10).
Structural characterization of the corresponding copper complexes
resulted in pseudo-octahedral geometries, with both chelating ligands act-
ing as equatorial N3-donors complemented by a molecule of water, and
the perchlorate anions as weak axial O-donors. The Cu–N distances range
from 1.97 to 2.03 Å, also within the range of reported LPMO active
sites. Aqueous EPR spectra are consistent with axial geometry in solution
and a dx2–y2 ground state, g⊥ = 2.059, g|| = 2.260 (A|| = 530 MHz) for
[LAMCu(H2O)2](ClO4)2 (1), and g⊥ = 2.060, g|| = 2.265 (A|| = 530 MHz)
for [LIMCu(H2O)2](ClO4)2 (2). All parameters are comparable to those
reported for LPMOs.4,9–11,29 An important physicochemical property that
appears to be directly related to the reactivity of LPMOs for these types
Figure 10. Ligands LAM and LIM

200 Castillo
of systems is the redox potential, which was determined for both com-
plexes 1 and 2 by CV and redox titrations. The values determined range
from 5 to 50 mV versus SHE, and contrast with those reported for the
metalloenzymes.4,10,11,29
Complexes 1 and 2 represent functional models of LPMO on the
model substrate p-nitrophenyl-β-D-glucopyranoside that features the
p-nitrophenolate anion as leaving group. Thus, incubation of the com-
plexes with hydrogen peroxide as oxidant at pH 10.5 in carbonate buffer
resulted in p-nitrophenolate cleavage from the model substrate. The oxi-
dative nature of the reaction was confirmed by the low conversion in the
absence of H2O2, and by identification of gluconic acid as the product of
the initially formed gluconolactone by ESI-MS, see Figure 11. For both 1
and 2, analysis of the reaction with H2O2 in the presence of Et3N as base
in aqueous solution by UV–vis spectroscopy resulted in the detection of
an intense band at 305 nm, with a shoulder around 375 nm (Figure 12).
The intermediates were postulated as CuII–OOH species based on previ-
ous reports,39 the d–d band at 650 nm, and X-band EPR spectra that
resemble those of the parent monometallic complexes.
D. Copper(II)/(Pyridyl)Diazepane Complexes
Mayilmurugan and coworkers devised the pyridyl-appended ligands L1
through L3 from the 1,4-diazepane backbone (1-methyl homopiperazine)
for mimicking of the N3 coordination environment in LPMOs.40 While the
diazepane moiety provides a rigid framework for CuII binding, there are
differences among the ligands regarding the nature of the chelates formed
with the pyridyl substituents. L1 features a methylene linker between the
unsubstituted 2-pyridyl group and the diazepane N-atom, L2 has an
Figure 11. Oxidative cleavage of p-nitrophenyl-b-D-glucopyranoside with 1 and 2

as catalysts and H2O2 as an oxidant in aqueous buffer solution.
Figure 12. Room temperature absorption spectra of 0.25 mM aqueous buffer solu-
tion of [LAMCu(H2O)2](ClO4)2 (dotted line) and after addition of 25 mM H2O2 (solid
lines).
Figure 13. Schematic representation of ligands L1–L3.
ethylene linker, and L3 is characterized by a methylene linker and a

3,5-dimethyl-4-methoxy substitution pattern, see Figure 13. This results
in five-, six-, and five-member chelates for L1–L3.
The corresponding cupric complexes [Cu(Ln)(H2O)ClO4](ClO4)
(Ln = L1–L3, complexes 1–3) were characterized by standard techniques,
and in the case of 1 and 2 by X-ray crystallography. In both cases, dis-
torted SP structures around the CuII centers are defined at the basal plane
by the ligand N3-donors and a molecule of water, and a weak perchlorate
O-donor in the axial position. The distortion in 2 is more pronounced, as
expected for a more flexible ligand with an ethylene linker defining a six-
membered chelate. The Cu–N distances range from 1.99 to 2.02 Å, all
similar to those in LPMOs, with the distortion in 2 reflected in the overall
longer Cu–N bond lengths as well. EPR spectra acquired in methanol/
DMF mixtures are consistent with axial geometry and a dx2–y2 ground
202 Castillo
state, g⊥ = 2.02–2.08, g|| = 2.25–2.37 (A|| = 468–528 MHz); A⊥ were also

reported from 423 to 462 MHz. Thus, the EPR parameters for 2 and 3 are
comparable to those of LPMOs.4,9–11,29
CV studies of 1–3 in aqueous solution evidenced quasi-reversible
Cu /CuI redox processes, with differences between the anodic and
II
cathodic peaks DE from 145 to 163 mV. The redox potentials reported
have increasing values at E1/2 = 8, 41, and 112 mV relative to the normal
hydrogen electrode (NHE). However, this is inconsistent with the values
reported versus Ag/AgCl as reference, which correspond to the descend-
ing values −213, −246, and −317 mV for 1–3, respectively. Thus, further
analysis based on the structure of the complexes and ligands is not pos-
sible, as is a comparison of the values reported here with those of other
model systems and LPMOs.
With these systems, p-nitrophenyl-β-D-glucopyranoside was also
used as a model substrate, with 30% aqueous hydrogen peroxide and Et3N
as oxidant mixture. Complex 3 featuring electron-donating methyl and
methoxy groups on the pyridine donor exhibited the highest rate constant
for p-nitrophenolate cleavage, as evidenced by UV–vis spectroscopy. The
authors attribute this behavior to stabilization of the putative CuII–OOH
intermediate, but this is counterintuitive as it would result in a less elec-
trophilic species. An oxidative process is assumed to be operative,
although in preparative scale reactions the co-product was identified as
D-allose, see Figure 14. This result contrasts with the observation of glu-
conic acid as an oxidation product,25,38 whereas D-allose may be expected
in the case of hydrolysis. The reasonable doubt regarding the proposed
oxidative nature of the cleavage reaction is further supported by the lack
of activity of the most active catalyst 3 in the attempted oxidations of
benzyl alcohol and 1-phenylethanol.
Attempts to identify the cupric hydroperoxo intermediate with
10 equivs. of H2O2 in the presence of Et3N in aqueous solution by
UV–vis spectroscopy resulted in the detection of LMCT bands around
375 nm, accompanied by d–d transitions at ca. 630 nm; unfortunately, the
extinction coefficient for the LMCT bands was not reported. Addition of
tert-butyl hydroperoxide to solutions of 3 resulted in a band assigned as
LMCT at 380 nm, although the extinction coefficient reported is low for
such assignment (ε = 182 M−1cm−1). In the latter experiment, the authors
Figure 14. Proposed reaction scheme for the cleavage of p-nitrophenol from
p-nitrophenyl-b-D-glucopyranoside with complexes 1–3 as catalysts, with D-allose
as co-product.
measured solution FT-IR spectra of the species formed, and determined

Cu–O and O–O stretching frequencies for the proposed Cu(II)-OOtBu
species at 750 and 925 cm−1.
E. Copper(II)/Bis(Imidazolyl)Amine System
Modeling of the histidine brace of LPMO by the group of Itoh involved a
tridentate N3 donor that incorporates two imidazole groups and a central
amine.41 In addition to the T-shaped geometry, the authors deemed neces-
sary to have a large “twist” angle between the two imidazole planes, as is
the case in the active site of the enzymes, and acidic N–H protons at
the same N-heterocycles. These considerations notwithstanding, t-N-
methylation of imidazole moieties in N-terminal histidines of fungal
LPMOs is a common occurrence.5,42 The ligand (N-(2-(1H-imidazol-4-
yl)-benzyl)histamine) (LH3) and its cupric complex [Cu(LH3)(TFA)2]
(TFA = trifluoroacetate) were thus prepared. The authors mention that
under neutral or basic conditions, insoluble blue crystals were obtained
when Cu(ClO4)2 was added to solutions of the ligand, which they attribute
to deprotonation of the N–H group of the imidazolyl moieties and subse-
quent formation of coordination polymers, but potentiometric measure-
ments were not undertaken to determine their pKa values.
Structural characterization of [Cu(LH3)(TFA)2] revealed a SP geom-
etry (t5 = 0.06),43 with the basal plane defined by the N3-donor set and a
monodentate TFA counterion; the axial position is occupied by the second
204 Castillo
(a) (b)
Figure 15. Comparison of (a) bond lengths (Å) and (b) bond angles (°) of [Cu(LH3)
(TFA)2] (top) with those of oxidized LPMOs (bottom).
TFA O-atom. The corresponding Cu–N/O distances are similar to those

observed in LPMO active sites. As mentioned previously, the authors
highlight the angle between the two imidazole planes at 75°, which is
close to the average value of 72° determined for the active site of CuII
LPMOs, see Figure 15. Spectral characterization by UV–vis and EPR
spectroscopy revealed physicochemical parameters that are comparable to
those of the enzymes: a d–d absorption band at lmax = 662 nm (e = 83
M−1cm−1) typical of SP cupric complexes, and an axial EPR signal char-
acteristic of tetragonal CuII, g⊥ = 2.062, g|| = 2.266 (A⊥ = 35, A|| = 445
MHz). Electrochemical studies afforded a CuII/CuI half wave potential of
323 mV versus SHE, with quasi-reversible behavior. The authors stress
out that a large “twist” angle between imidazole donors is required to
achieve E1/2 values that are close to those reported for LPMOs, while
model systems with small angles exhibit redox potentials that are lower
than 100 mV. However, the CuII/CuI couple of the [(2BB)Cu(H2O)2]
(OTf)2/[(2BB)Cu]OTf system was incorrectly identified as having a redox
potential of 76 mV, while the actual reported value was 226 mV versus
SHE despite the angle being 14.5° in the cupric complex.25
Catalytic tests of [Cu(LH3)(TFA)2] with hydrogen peroxide (20 mM)
as an oxidant in carbonate buffer at pH 10 were also carried out with
p-nitrophenyl-β-D-glucopyranoside as substrate. As is the case with
[Cu(Ln)(H2O)ClO4](ClO4) (Ln = L1–L3, complexes 1–3) in Section IV.D,
an oxidative cleavage was reported, but the products observed correspond
to p-nitrophenolate and D-allose. Once again, the identification of
D-allose would correspond to hydrolysis rather than oxidation, raising
questions about the mechanism of model substrate cleavage.
F. Copper(II)/Bis(Picolyl)Amine System
The most recent example of a simple model system that mimics some
aspects of LPMO activity is the one reported by Cowan and coworkers.44
Thus, bis(picolyl)amine copper(II) chloride (1a) was employed along
with di- and tetranuclear analogs that will not be discussed, because the
enzymatic active sites are mononuclear. The preparation of 1a was previ-
ously reported,45 and its solid-state structure established that the Cu(II)
center has a distorted SP coordination environment, with all N-donors in
basal positions.46 Its redox potential was reported relative to the reversible
hydrogen electrode (RHE) at a value of E1/2 = 230 mV,47 within the values
reported for LPMOs.
The report focuses on glycosidase activity by oxidative cleavage of
p-nitrophenol conjugated glucose, galactose, and related disaccharides as
substrates in the presence of ascorbate and H2O2 under physiological con-
ditions.44 Similar to the earlier examples, the cleavage of p-nitrophenolate
was monitored by UV–vis spectroscopy, starting with ascorbate and
dioxygen, which resulted in ~10% activity when compared to hydrogen
peroxide as oxidant. The authors indicate that Fenton-like chemistry
might be responsible for the observed reactivity, with hydroxy radicals
promoting H-abstraction from the saccharides in the initial step of
p-nitrophenolate cleavage. Unfortunately, the identity of the saccharide
fragments after degradation was not established, leaving the open question
of whether the observed glycoside cleavage is indeed oxidative in nature.
206 Castillo
V. Concluding Remarks
The meridional donor set provided by the N3 scaffold in the form of the
histidine brace and an additional histidine imidazole in LPMOs is a req-
uisite that needs to be fulfilled to access functional model systems. The
effect of the angle between the imidazole rings remains to be understood
although it does not appear to be crucial to access the range of redox
potentials of the CuII/CuI couple in the enzymes. Moreover, the half wave
potentials may be a first approximation to the electronic environment
around the copper centers in the active sites of LPMOs, but this physico-
chemical parameter does not appear to be a strict requisite to develop
mimics, at least when model substrates are considered. Another observa-
tion that emerges is that hydrogen peroxide affords far superior results in
the degradation of model substrates and cellulose, which is in line with
recent studies that point to H2O2 as the oxidant in the enzymatic systems,
rather than O2. It may be time to consider renaming the enzymes as Lytic
Polysaccharide Peroxygenases.
While the minimum requirements appear to have been laid out for the
development of oxidative glycosidase model systems, key points remain
to be addressed: can model systems actually degrade the natural substrates
cellulose and/or chitin? This is as challenging to explore with model sys-
tems due to the insoluble and recalcitrant nature of such polysaccharides,
as it is in nature. Thus far, only the bis(benzimidazole)amine-based copper
complexes have demonstrated oxidative cleavage activity on cellulose.32
Another question that remains to be answered is whether the true oxidant
in the enzymatic systems is dioxygen or hydrogen peroxide, and simple
model complexes may provide relevant information. In this regard, LPMO
mimics may be active at oxidatively degrading model and natural sub-
strates, but they may operate through different reaction mechanisms. In
some cases, even the oxidative nature of the model substrate degradation
needs to be firmly established. Finally, perhaps the most fundamental
question that remains open to speculation is the nature of the copper–
oxygen intermediate responsible for the activation of the strong C–H bond
of insoluble substrates in LPMOs. Initial proposals considered cupric-
superoxide [CuII–O2]+ species, but recent experimental and theoretical
studies tend to favor a copper-oxyl [CuII–O]+ intermediate for H-abstraction
from strong C–H bonds. Model systems have added to the discussion the
potential involvement of a high-valent copper(III)-hydroxide [CuIII–
OH]2+. Fenton-type reactivity may also be invoked, whether it occurs in
the proximity of the copper complexes that may generate hydroxy radi-
cals, or in bulk solution after their release. The combined data obtained
from LPMOs themselves, and model systems inspired by the enzymes,
should provide answers to these fundamental questions in due course.
VI. References
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7 Structure and Function

of Cu–Peptoid Complexes
Anastasia E. Behar,* Pritam Ghosh,* and Galia

Maayan*,†
*Schulich Faculty of Chemistry, Technion — Israel Institute of

Technology, Technion City, 3200008 Haifa, Israel
†
[email protected]
The remarkable efficiency and selectivity of natural biopolymers in carry-

ing out valuable functions such as molecular recognition and catalysis
have been a source of inspiration for the design and synthesis of functional
chemical systems. Mimicking the capabilities of biopolymers requires the
ability to imitate their unique properties, such as their well-defined three-
dimensional structures and their high selectivity toward specific metal
ions that are required for their utility, as well as to imitate conceptual
features of their activity, such as intramolecular cooperativity. A promis-
ing approach to achieve biomimetic properties involves (i) facile synthe-
sis of a versatile oligomeric backbone that enables the incorporation of
several functional groups with sequence specificity and (ii) control over
structure formation including the ability to enforce folding and self-
assembly. These aptitudes should allow generating functional peptidomi-
metic systems in which various components act cooperatively toward a
211
212 Behar et al.
specific biomimetic function including selective recognition as well as

efficient and selective catalysis.
Copper ions are key elements in both the structure and function of
natural biopolymers, being employed as cofactors in many essential pro-
teins,1 facilitating folding or stabilizing specific conformations, and regu-
lating vital functions in living organisms,2 including oxygen transport,
cellular stress response, antioxidant defense, and more. In addition, Cu
ions are vital components of many enzymes, for example, the zinc–copper
superoxide dismutase.2 Despite the great importance of Cu within biologi-
cal systems, its overloading can be potentially toxic to living cells and
may cause oxidative stress, neurodegenerative diseases including
Alzheimer’s disease,3 and other disorders such as Wilson’s disease,4 and
cancer.4,5 A possible treatment of Cu intoxication is via the administration
of chelating molecules (chelation therapy),6,7 designed for the specific
binding of Cu ions.
In this chapter, we will describe peptide mimics called peptoids —
N-substituted glycine oligomers — that can either bind Cu ions with high
specificity or fold, change their conformation, and/or self-assemble via Cu
coordination. We will also present the applications of Cu–peptoid com-
plexes in intramolecular cooperative catalysis, a concept inspired by the
activity of enzymes.
I. Introduction
Biomimetic oligomers are synthetic molecules akin to natural biopoly-
mers, namely peptides, proteins, and oligonucleotides.8–10 Both natural
biopolymers and their artificial mimics are sequence-specific oligomers
capable of folding into well-defined three-dimensional structures in solu-
tion. One difference between them, however, is the identity of their back-
bone: while peptides and proteins are composed of a-amino acids,
biomimetic oligomers are assembled from non-natural monomers.11,12
Among the peptide mimics, peptoids are unique, as they combine proper-
ties of both biological materials and synthetic polymers; they are inert
toward many catalytic transformation13 and oxidative conditions14–16; and
possess better physiochemical and pharmacokinetic properties relative
to a-peptides, including stability in oxidative conditions13 and high
Structure and Function of Cu–Peptoid Complexes 213
temperatures,17 tolerance toward high salts concentration and various pH

conditions,13,18 enhanced proteolytic stabilities,19 and increased cellular
permeability.20 These advantages, taken together with their ease of syn-
thesis on solid support (see the following section), their biomimetic struc-
ture, and ability to control their sequence and structure, hold great
potential in developing peptoids as biomimetic functional materials
including selective chelators for metal ions and catalysts.
A. Peptoid Synthesis
Peptoid oligomers have identical backbone to that of polypeptides, but the
side chains are appended to the nitrogen rather than to the a-carbon
(Figure 1a). Consequently, peptoids lack backbone chirality and cannot
form hydrogen bonds. In addition, the amide bonds in peptoids are tertiary
and this allows isomerization between the cis and trans conformations
(Figure 2a),21 in contrast to the a-peptides, where the trans conformation
is preferable. Furthermore, the lack of amide protons restricts the stabili-
zation of secondary structures by backbone hydrogen bonding, as
observed in a-peptides. Nevertheless, peptoids have several properties that
make them superior over peptides, and one of the main advantages is the
ease of the peptoids synthesis. Peptoids can be synthesized efficiently on
solid support, using the submonomer approach22 (Figure 1b), which
involves two main steps: N, N-diisopropylcarbodiimide (DIC) mediated
(a)
(b)
Figure 1. (a) Generic structure of a peptoid compared to α-peptide. (b) Schematic

representation of peptoid submonomer synthesis.
214 Behar et al.
acylation with bromo- or chloroacetic acid, followed by amine displace-

ment with a primary amine to form N-substituted glycine. This two-step
sequence is repeated iteratively to obtain the desired oligomer, followed
by the cleavage of the oligomers from the resin. This synthesis can be
performed using any primary amine, which is either commercially avail-
able or synthesized; the synthesis typically does not include any protec-
tion/deprotection steps, and thus the submonomer approach allows to
introduce various functional groups within the peptoid sequence.23 The
submonomer approach makes the construction of peptoids relatively inex-
pensive compared to the synthesis of a-peptides.
B. Secondary Structure of Peptoid Oligomers

In contrast to peptides, where folding arises from backbone hydrogen
bonding interactions, the backbone nitrogen atoms in peptoids are substi-
tuted, and therefore lack the ability to form hydrogen bonding. This
results in highly flexible backbones and complicates the design of well-
defined secondary structures in peptoids. However, different methods
were developed to allow folding of peptoids into secondary structures by
the incorporation of the bulky chiral side chains within the oligomer
sequence, thus restricting backbone conformation.17 More specifically,
incorporation of branching bulky substituents induce steric repulsion
between side chains, leading to charge-charge repulsion with the back-
bone carbonyls; hydrophobic interactions or n → p* or p-p interactions
between aromatic bulky substituents play a role in peptoid folding.17
Peptoid oligomers have been found to be able to fold into loop,17
helix,17,21,24 or turn.14,25 Among them, one of the most studied secondary
structures of peptoids throughout the last decades is the helix, which typi-
cally resembles the polyproline type I (PPI) peptide helix, and depending
on the type and number of the bulky chiral side chains, have a pitch of
roughly three residues per helical turn.17 In contrast to peptides, in which
the amide bond is mostly in the trans conformation, the amide bond of
peptoids goes through rapid isomerization between the cis and trans con-
formations leading to significant conformational heterogeneity in solu-
tion. Nevertheless, a peptoid helical structure could be obtained even in
short oligomers (as short as pentamers).17 Interestingly, among various
chiral side chains that were shown to induce helicity within peptoids, a
few of them have been determined as the side chains that shift the cis/
trans isomerization toward a majorly of cis amide bonds, for example,
N-(S)-1-phenylethylglycine (Nspe), N-(R)-1-phenylethylglycine (Nrpe)
(S)-N-(1-naphthylethyl)glycine (Ns1npe)17,24,26 and some were even able
to lead to “all cis” helices, for example, (R)-(−)-3,3-dimethyl-2-butylamine
(Nr1tbe), (S)-(+)-3,3-dimethyl-2-butylamine (Ns1tbe)21,27 (Figure 2b).
Several sequences requirements for obtaining peptoid helices were estab-
lished. First, the peptoid helix can be stabilized when the majority of the
peptoid’s side chains (at least two-third) are bulky and chiral.28 Second,
placing a chiral bulky monomer at the C-terminal of the peptoids sequence
can induce folding of the peptoid into a helical structure, so-called “ser-
geant-soldier” effect; in this case, the overall number of chiral subunits
within peptoid scaffold could be reduced.28 Third, the longer the peptoid
sequence is, the more stable is the helical structure (peptoid decamers and
beyond).28
The secondary structure of the peptoids can be initially estimated by
circular dichroism (CD) spectroscopy. For example, the typical spectra of
Nspe-bearing peptoids show typical double minima indicating a negative
(a)
(b) (c)
Figure 2. (a) Isomerization of amides in the peptoid backbone. (b) Common

a-chiral monomers used to induce secondary structure in peptoids. (c) CD spectra of
the polypeptoids (Nspe)6 and (Nrpe)6. Peptoid concentration was 0.2 mg/ml (≈1 mM
residue molarity for all species) in 10 mM sodium phosphate (pH 7.0) at 25°C.28
216 Behar et al.
ellipticity near 218 nm and 202 nm, and a maximum indicating a positive
ellipticity near 190 nm (Figure 2c).17,28 These bands correspond to
n → p* transition of the amide chromophore and high- and low-wave-
length components of the exciton split p → p* transition. The similar,
mirror-like pattern of opposite chirality (double-maxima and minima,
correspondingly) is observed for Nrpe-bearing peptoids (Figure 2c).17 In
accordance with the helical peptoids having a pitch of about three residues
per turn,17 pendant groups located at positions i and i + 3 along the peptoid
backbone are facing the same side of the helix, and this can serve as a
handle for the design of peptoids as selective chelators for metal ions,
including Cu.
II. Structural Aspects of Cu(II)–Peptoid Complexes

A. Structural Design of Cu(II)–Peptoid Complexes
Over the last years the main focus of our group has been the synthesis of
metal-binding peptoids and their coordination to metal ions, specifically
Cu(II). Our main working hypothesis for designing peptoid oligomers
with both high affinity and high selectivity to Cu(II), was that the pre-
organization of specific coordinative ligands at positions i and i + 3 of
unstructured or structured (helical) oligomers will enhance their affinity to
Cu(II) compared with two such ligands tethered together via a simple
chemical bond or in any other unstructured oligomer, and may also enable
secondary structure stabilization. For the generation of helical metal-
binding peptoids, bulky chiral side chains (see previous section) were
incorporated in the other positions along the sequence, not occupied by
the metal-binding ligands, provided that these chiral bulky side chains
constitute the majority of the substituents along the peptoid scaffold.
These metal-binding peptoids are expected to form complexes of the
type PCu(II) (Figure 3).29–31 Peptoids bearing two metal-binding sites,
either identical (or similar) or different — having a significantly distinct
metal affinity that can bind metal ions in different coordination geome-
tries were developed for the selective intermolecular binding of either two
Cu(II) ions or one Cu(II) ion and one different metal ion, yielding com-
plexes of the type P2Cu2 or P2CuM, where M = Zn(II), Co(II), or Fe(III),
Figure 3. Generic representation of the different types of the metallopeptoid com-

plexes structures.
respectively29,30 (Figure 3). Controlling the interactions between these

two ligands via metal coordination could be further extended by placing
them at i and i + n positions where n ≠ 3. Taking this approach, we have
shown that it is possible to separate between two or more different coor-
dination sites and design oligomers capable of binding two ions, being
either Cu(II) ions or Cu(II) ion and another metal ion. The latter binding
was shown to proceed selectively in distinct sites forming various hetero
bimetallic peptoid complexes PCu(II)M (Figure 3).32 The use of peptoid
sequences as scaffolds for coordinative ligands enabled to tune their
chemical properties, such as lipophilicity and chirality, simply by modify-
ing the non-coordinative side chains, thus controlling the peptoids’ com-
patibility with various applications.30,31
B. Choice of the Metal-Binding Ligands

The coordination properties of the incorporated ligands were also consid-
ered in the design of the new selective metal-binding peptoids; two
ligands that are able to provide preferable coordination geometry to
Cu(II), for example, penta-coordination, were placed at positions i and
i + 3 to enable strong binding in an intramolecular fashion. According to
Hard and Soft Acids and Bases (HSAB) principle,33 Cu(II) is considered
borderline hard/soft metal ion; thus in nature, protein–copper(II) binding
sites are dominated by side chains containing borderline ligands that com-
bine coordination the atoms N and S and O.34 In addition, based on Ligand
Field Theory (LFT) and the ligand field stabilization energy (LFSE),35
218 Behar et al.
Figure 4. Metal-binding ligands for Cu(II) used in peptoids design.
Cu(II) is a d9 system, which exhibits geometric preferences based in part

on LFSE, and is found to be coordinated by 4, 5, or 6 ligands in square
planar, square pyramidal, or axially distorted octahedral geometries.
Considering preferences from both HSAB and LFT, versatile examples of
metal-binding ligands for Cu(II) ions that were incorporated within pep-
toid sequences are depicted in Figure 4. These mono-, bi-, and tridentate
ligands combine both N and O atoms and therefore are varied in their
electronic properties and in their possible geometrical coordination modes
thus enable the formation of either tetragonal-, pentagonal-, or hexagonal-
coordinated complexes. All these ligands are also known to bind
Cu(II) ions with high affinity (stability constants of Cu(II) complexes
Phen, Pam, Bipy, Terpy, HQ, and PicT are logK = 17.9, 10.2, 17.85, 6.97,
13.00, and 7.33 respectively, as obtained by potentiometric titrations at
25°C).36–40
Metal-binding (MB) ligands are introduced into the peptoid backbone
unprotected (thus avoiding additional protection and de-protection syn-
thetic steps), in the amine displacement step as a primary amine, which
are readily available — Phen and Pam, or could be synthesized according
to procedures developed by our group: HQ ligand has been previously
synthesized by a one-step hydrogenation reaction41 and Terpy and Bipy
Figure 5. Solid-phase synthesis of the peptoid oligomers by the submonomer

method and their copper(I)-catalyzed azide–alkyne [3 + 2] cycloaddition (click)
reaction.
by one-step nucleophilic substitution reaction.27,41 In addition, inclusion

of pendant functional groups within peptoid sequences can be also accom-
plished by the Cu(I)-catalyzed azide-alkyne (3 + 2) cycloaddition reaction
(“click”) on solid support by microwave irradiation at 60°C. (Figure 5).
By using this approach, we have recently exploited this synthetic path for
incorporating the pyridine-triazole ligands PyrT and PicT within four
helical peptoids.42
C. Characterization of Cu(II)–Peptoid Complexes

1. E valuation of Cu(II) binding and synthesis of Cu(II)–peptoid
complexes
The coordination capabilities of metal-binding peptoids to Cu(II) can be
evaluated using several analytical techniques. Initial assessment of Cu(II)
binding to the peptoid ligand(s) is usually done in solution via titrations
of Cu(II) solutions in known concentrations (in solvents such as acetoni-
trile, water, and methanol, where both the Cu(II) salts and the peptoids are
soluble) to peptoids solutions in known concentrations, followed by UV–
vis spectroscopy. Based on the obtained data, the Cu(II):peptoid stoichio-
metric ratio is determined from ratio plots and/or Job plots. Further
220 Behar et al.
confirmation of the stoichiometric ratio can be obtained from mass spec-

trometry (MS) techniques, specifically by isotopic analysis that can sup-
port a 1:1, 1:2, or 2:1 Cu(II):peptoid ratio (intramolecular complexes of
the type PCu/PCuM, PCu2 or P2CuM, respectively; see examples in
Figure 6)29–32 or a 2:2 Cu(II):peptoid ratio (intermolecular duplexes of the
type P2Cu2).29 Using the determined ratio, metal complexes are synthe-
sized in a larger scale and their identity is confirmed by MS. Cu–peptoid
complexes of the type PCu, PCu2, and P2Cu2 are typically prepared by
treating a peptoid solution with Cu(II) salt under stirring, following their
isolation either by precipitation with a counter ion or, in the case of neutral
complexes, by solvent evaporation and further purification (e.g., recrystal
lization).31 Heteronuclear metallopeptoids of the types P2CuM and PCuM
are usually prepared in two methods, under kinetic control and under
thermodynamic control. Following the first method, metal ions are added
step by step such that one metal is added first, stirred for some time, and
its binding to the peptoid solution is verified by UV–vis and MS spectros-
copies. Subsequently, the second metal is added, and the binding process
and analysis is repeated until the entire oligomer is bound.30–32 Following
the second method, a mixture of metal ions is added to the peptoid solu-
tion simultaneously, heated and/or stirred for several hours, isolated, and
characterized as described above.31
Detecting the binding of two different metal ions, namely one Cu(II)
ion and one other metal ion, to one peptoid could be also done by UV–vis
spectroscopy using the kinetic control approach. First, each metal ion
solution is added separately to the peptoid aiming to coordinate at its
designed site, and its UV–vis spectra is compared with the one previously
found in order to ensure that the binding indeed occurs in the desired site.
In addition, as each coordinative ligand exhibits different absorbance
band(s), the changes in each band upon the addition of each metal ion
solution is followed separately by UV–vis. This provides information
about the number and type(s) of ligands involved in the binding of each
metal ion. All the acquired data from the kinetic experiments was com-
pared with the data obtained from the binding of two metal ions from their
mixture solution. Overlap between different bands is possible, but in most
cases enough data is gained from non-overlapping bands, and the overall
information allows to precisely determine the coordination of different

metal ions to the same peptoid.
2. D
etermining the association constants and evaluating the
selectivity of the peptoid chelators to Cu(II)
One of the important parameters for evaluating the Cu(II) binding to pep-
toids is the association/dissociation constant of the Cu(II)–peptoid com-
plex. Association constants of metallopeptoids can be determined by one
of the following methods depending on the solubility of the peptoid and
the MB ligands. For peptoids with low water solubility UV–vis titrations
in aqueous solutions containing 10–20% methanol (the peptoid is dis-
solved in methanol prior to water addition). Low peptoid and metal ion(s)
concentrations are used in order to ensure low ionic strength. Association
constants are estimated by fitting the obtained data via non-linear regres-
sion using the appropriate kinetic equations.15 For water-soluble peptoids,
isothermal titration calorimetry (ITC) measurements can be used in buffer
solutions.42,43 Association constants and other physical parameters are
extrapolated directly, using the ITC software. In addition, in order to
verify the results (obtained in either method), competition experiments
can be conducted, either between the metal ions competing over the pep-
toid ligand, or between the peptoid ligand and a strong Cu(II) chelator
with a known association constant. Using the first competition method,
two different metal ions are titrated with a peptoid solution, followed by
UV–vis spectroscopy and MS. From these experiments, quantitative data
about the selectivity of one metal ion over the other is deduced. Using the
second competition method, the dissociation constant for Cu(II)–peptoid
complex could be determined by metal ions titrations with both chelators
followed by UV–vis spectroscopy. From the obtained UV–vis data, using
the appropriate equations and pH correction factors, dissociation constant
is found from the calculated slope.44
To determine the selectivity of the peptoids toward Cu(II), several
approaches could be applied. Typically, each peptoid is treated with a
mixture of different metal ions in different concentration and the solution
mixture is investigated. First, the solution mixture is analyzed by UV–vis
222 Behar et al.
and MS spectroscopies in order to determine the identity of the com-

plexes. The obtained spectra are compared to the spectra of each metal–
peptoid complex that was generated separately (not from a mixture).
Identity between the spectra obtained from the mixture solution and the
spectra of the Cu(II)–peptoid complex indicates that the peptoid is selec-
tive to Cu(II).31 Second, the association constants of the peptoids with
each metal ion from the tested mixture are compared in order to evaluate
which ion has the highest affinity to the peptoid; highest affinity to Cu(II)
supports selectivity to this ion. Finally, the precipitate formed from the
mixture solution is filtered, the filtrate solution is analyzed by inductively
coupled plasma optical emission spectroscopy (ICP-OES) technique and
the filtrate is analyzed again by UV–vis and MS. The absence of Cu(II)
from the filtrate and its sole presence in the precipitate indicates its selec-
tive binding and extraction by the peptoid.31
3. S tructure and coordination sphere of Cu centers within

Cu(II)–peptoid complexes
Electron paramagnetic resonance spectroscopy (EPR) and X-ray analysis
are usually used to characterize the structure and coordination geometry of
Cu(II)–peptoid complexes in solution and in the solid state. The experi-
mental EPR bands of solid or frozen solution samples are recorded in the
presence of an internal standard and the obtained spectra are simulated in
order to obtain the Hamiltonian parameters (g and A|| values). According
to these parameters, the coordination geometry of the Cu(II)–peptoid com-
plexes can be estimated. Typically, the EPR spectra of Cu(II)–peptoids
resemble tetra-30,42,45 or penta-31,46–49 coordination geometry of the Cu(II)
center. For tetra-coordinated Cu(II) complexes, g|| > g⊥ and the quotient
g|| /A|| (cm) is calculated.50 The value of this quotient indicates whether the
coordination geometry of the Cu(II) center is square planar (with quotient
g||/A|| range between 105 and 13542) or has a tetrahedral distortion (when
the quotient g||/A|| is higher than 13530,45). For penta-coordinated Cu(II),
typically two g values are obtained, with g|| > g⊥ > 2.0023 and A|| been any
measurable value, and then the geometry is determined between square-
pyramidal or trigonal-bipyramidal according to exact values of these
parameters.13,31,47 In some cases, the determination between tetra- or
penta-coordination only on the basis of the EPR Hamiltonian parameters

could be rather challenging, and thus should be supported by other analyti-
cal methods including X-ray, FT-IR, CD, and UV–vis.46 Finally, in some
cases the intermediate situations exhibiting three g values could be
observed, resulting in the so called “rhombic” spectrum; the coordination
of Cu(II) center in these cases is intermediate between square-pyramidal
and trigonal-bipyramidal.51 For complexes of this type, a parameter R
(where R = (gy–gz)/(gx–gy) with gx > gy > gz) should be calculated in order
to determine the predominance of the ground state among two possible,
and as a result, the prevalence of one of them could be established. If
R > 1, then the greater contribution to the ground state arises from dz2
(trigonal-bipyramidal), if R < 1, the greater contribution to the ground state
arises from dx2−y2 (square-pyramidal).47,49
In cases where crystals suitable for single crystal X-ray analysis are
obtained, X-ray analysis of the obtained crystals provides precise infor-
mation about the bond distances and the exact geometry of the Cu(II)
metal center, including second coordination sphere and full structure of
metallopeptoid in the solid state.46
To evaluate the influence of Cu(II) on the secondary structure of the
peptoid, CD spectroscopy measurements of both the peptoid and its Cu(II)
complex are typically performed in solution. The comparison between the
obtained CD spectrum of the peptoid and this of the Cu(II)–peptoid com-
plex indicate whether the peptoid had some secondary structure before
Cu(II) binding and if so, whether this structure is stabilized or destabilized
upon Cu(II) binding. In case the peptoid was not structured prior to Cu(II)
binding, the spectra provide information to whether Cu(II) binding can
enforce folding and eventually lead to some secondary structure of the
Cu(II)–peptoid.
III. Effect of Cu(II) Binding on the Structure

of Peptoid
A. Cu(II) Binding as a New Approach for Peptoid Folding
Since the first introduction of peptoids as a new class of peptidomimetic
oligomers 30 years ago, their folding in solution and the requirements for
224 Behar et al.
achieving well-defined secondary structures were established and

described in details (see section I.B). Folding of peptoids into a stable
helical secondary structure in solution generally requires incorporation of
chiral bulky side chains within peptoids sequence, such that they occupy
at least two thirds of the peptoids length.17 This strategy, although been
well known and commonly used in the field, restraints potential applica-
bility of the peptoid helices: currently, only several side chains are known
to enable folding, out of the numerous that exist, and these do not contrib-
ute to peptoid functionality as they only serve structural purposes.
Furthermore, the helix-inducing side chains should occupy the majority of
the peptoid structure, and therefore the introduction of functional groups
is limited. Thus, finding ways to control both the structure and function of
peptoids is required for the development of unique functional biomimetic
materials. Among a variety of interactions known to enforce folding
within biopolymers, including hydrogen bonding, solvophobic effects,
and metal–ligand interactions, the latter, and particularly in the case of
copper, are found to enable folding and function. As peptoid helices
resemble polyproline-type helices (PP-I and PP-II),17 with a helical pitch
of three residues per turn, we have suggested that the incorporation of MB
ligands in i and i + 3 positions of peptoid sequences might enforce peptoid
folding and eventually will result in peptoid helices upon metal binding.
The first Cu(II) peptoids were generated from helical peptoids having
MB ligands in i and i + 3 positions and Nspe side chains (that ensure
peptoids helicity) in the other positions along the peptoid sequence.15,42
These examples showed that Cu(II) binding has a minor effect on peptoids
initial secondary structure. On the other hand, Cu(II) binding to
HQ-containing peptoids resulted in new CD peaks between 240 nm and
280 nm, the region corresponding to the HQ p-p* transition, reflecting the
transmission of stereogenic character of the helical peptoids scaffold to
the metal center. Although this was a unique illustration of chiral induc-
tion to Cu(II) center in peptoids, it was not determined whether the chiral-
ity of the metal centers arises from the peptoid helicity or simply from the
fact that these oligomers are chiral.
In order to probe this point and also explore if it is possible to induce
folding within unfolded peptoid sequences by metal coordination only, a
peptoid heptamer having two HQ groups at positions 3 and 6 (from the
N-terminal), and chiral, non-bulky (S)-1-methoxy-2-propyl (Nsmp) groups

in the other five positions was synthesized (7mer-HQ2, Figure 6a).30 The
Nsmp groups are not secondary structure inducers, but are hydrophilic,
thus 7mer-HQ2 was not expected to have a helical or any other secondary
structure but was expected to be water soluble, and it was suggested that
folding might be enforced by Cu(II) binding to form water-soluble helical
Cu–peptoid.30 Although both UV–vis and ESI-MS of the peptoid after
Cu(II) binding were consistent with the formation of 1:1 intramolecular
(7mer-HQ2)Cu complex, no helix formation was observed upon binding
of Cu(II) to 7mer-HQ2, as revealed from the CD; the spectra shape in the
range of 190–230 nm for both, metal-free peptoid 7mer-HQ2 and its
Cu(II) complexes, suggested completely disordered structures.
Consequently, the peptoid dodecamer 12mer-HQ4, bearing four HQ
ligands that form two MB sites at positions i and i + 3 (Figure 6b) for the
formation of an intramolecular complex, of the type PCu2, was synthe-
sized and treated with Cu(II) ions. The formation of the complex (12mer-
HQ4)Cu2 was verified by UVvis and ESI-MS (Figure 6b). This time, the
CD spectra of 12mer-HQ4 after addition of Cu(II) ions revealed a simul-
taneous, although minor, decrease in the CD magnitude at 191 nm and
increase near 210 nm, suggesting a small increase in the conformational
order of the peptoid. Cu(II) binding to both 7mer-HQ2 and 12mer-HQ4
produced new peaks between 240 nm and 280 nm, implying that chiral
induction from the peptoid to the metal centers is attributed to the chirality
of the oligomers and not to their helicity. The EPR spectra of both the
Cu(II)–peptoid complexes suggested a tetrahedral distortion from
the expected square planar coordination geometry, indicating that not only
the chirality of the peptoid effects the Cu(II) center by establishing an
asymmetric environment about it, but also the coordination geometry of
the Cu(II) center is influenced by the peptoid backbone. Overall, it was
concluded that metal-binding interactions alone could not lead to peptoid
folding, suggesting that the presence of at least some chiral-bulky side
chains might be also required. The results also suggested that more than
one MB site is required to initiate peptoids folding.30
Therefore, in follow-up studies, the goal was to generate peptoid heli-
ces upon Cu(II) binding, utilizing the above conclusions. To do that, a set
of Cu(II)-binding peptoids having much less than two thirds of
226 Behar et al.
(a)
(b)
(c)
Figure 6. Schematic illustration of the peptoids 7mer-HQ2 (a) and 12mer-HQ4 and
(b) forming intramolecular complex (7mer-HQ2)Cu and (12mer-HQ4)Cu2 upon
binding to Cu ions. (c) UV–vis titrations of 7mer-HQ2 and 12mer-HQ4 with Cu(II)
ions. Insets: molar-to-ratio plots, suggesting the stoichiometric peptoid:Cu(II) ratio.
chiral-bulky substituents were designed based on the sequences of 7mer-

HQ2 and 12mer-HQ4. First, based the peptoid heptamer 7P1, which was
identical to 7mer-HQ2 except one Nsmp group at the 7th position that
was replaced by one Nspe group. This modification was expected to
slightly increase the conformational order of the peptoid and facilitate
folding upon Cu(II) binding. Indeed, the CD spectra of 7P1 revealed low-
intensity double minima, consistent with an initiation of a helix formation.
Upon Cu(II) binding, the intensity of this signal decreased, and this
decrease was attributed to structural changes within the peptoid, enforced
by the coordination geometry of the Cu(II)–peptoid complex (Figure 7).45
Therefore, additional modifications of Nsmp groups by Nspe side
chains were made, generating the set of peptoids 7P2–7P5 by keeping
Nspe at 7th position constant and replacing and additional Nsmp group by
an Nspe side chain in different positions along the peptoid scaffold. In
addition, we designed peptoid 7P6, having the two Nspe groups at the 3rd
and 4th positions (between the two HQ ligands), forming a “bulky core.”
All peptoids were shown to bind Cu(II) in an intramolecular fashion,
forming complexes of the type PCu, as revealed by UV–vis and ESI-MS.
Systematic studies of the Cu–peptoid complexes by CD and EPR spec-
troscopies revealed the following clear trend: the larger the angle between
the two HQ groups is (detected by the lower-exciton-coupled CD
intensity), the larger is the distortion of the metal center from square
planar geometry toward tetrahedral geometry (from EPR data) and the
more ordered the peptoid secondary structure is (as seen by near-UV
Figure 7. Schematic illustration of peptoid 7P6 forming a PP-I-like helix upon bind-
ing to Cu(II) ions. Changes in the CD spectra of free peptoid 7P6 (left) and (7P6)Cu
(right).
228 Behar et al.
signals in the CD spectra). Surprisingly, coordination of Cu(II) to 7P6

resulted in a CD signal completely different from that of the free peptoid,
exhibiting minima at 200 and 226 nm and maximum at 212 nm (Figure 7).
Interestingly, this different spectrum resembles the spectrum of a PP-I
peptide helix, similarly to previously reported CD spectra of the all-cis
Ns1npe- and Ns1tbe-based PP-I peptoid helices.17,21,24 We assumed that
the driving force for the formation of this exceptionally ordered (7P6)Cu
helix is the initial secondary structure of 7P6 as implied from the low-
intensity double minima observed in its CD spectra combined with the
coordination geometry of its Cu complex. Our findings reveal that the
effect of Cu(II) binding on the peptoid structure depends both on the pep-
toid sequence (leading to the initial secondary structure), and on the pre-
ferred coordination geometry of the Cu(II) ion.
Following the two studies described above, the sequence of 12mer-
HQ4 was modified to include two Nspe side chains instead of two Nsmp
side chains and a new set peptoid dodecamers was designed. This new set,
namely peptoids 12P1, 12P2, and 12P3, had Nspe groups in the 1st and
12th positions, 2nd and 12th position, or 1st and 2nd positions, respec-
tively, four HQ ligands in the 3rd and 6th positions and in the 8th and 11th
positions forming two distinct binding sites, and six Nsmp groups at other
positions along peptoid scaffold.32 Based on rigorous spectroscopic meas-
urements performed after the addition of Cu(II) to 12P1–12P3, all three
peptoids were shown to form diCu–peptoid complexes of the type PCu2
with Cu(II) metal center coordination geometry been pseudo-tetrahedral,
in accordance with previously reported results for HQ-bearing peptoids.
Importantly, results obtained from CD spectroscopy, solution NMR tech-
niques and high-level DFT calculations demonstrated that 12P1 and 12P2,
which were shown to be unstructured peptoids, can fold upon Cu(II) bind-
ing to form helical structures. The structure of (12P1)Cu2 is depicted in
Figure 8. These results represent the first demonstration of generating a
peptoid helix by metal coordination. Moreover, DFT-based calculated
spectra and structures of metallopeptoids were obtained in this study for
the first time. Interestingly, the peptoid 12P3, which was also shown to be
unstructured, did not fold upon Cu(II) binding. It was therefore concluded
that the Nspe group at the C-terminal of peptoid dodecamers plays a cru-
cial role in the stabilization of the helical secondary structure.
Figure 8. Side view of (12P1)Zn2 optimized at the TPSS-D3/def2-TZVP + COSMO

(methanol) level, assuming the right-handed cis PP-I helical structure. The main-
chain C, N, O atoms and acidic H atoms (HQ hydroxyl and amide NH groups) are
highlighted as grey, blue, red, and white balls, respectively.
B. Cu(II)-Mediated Peptoids Self-Assembly

In the previous section we have described our attempts to constrain the
conformational order of peptoids by stabilizing their secondary (helical)
structure in solution via their coordination to Cu(II) ions. Another known
effective strategy for establishing conformational order of peptoids is
macrocyclization.52,53 The first peptoid macrocycles were formed via
covalent bonds between pendent and/or backbone groups within the pep-
toid.52,53 Notably, in nature, formation of high-order structures is often
achieved via self-assembly rather than covalent interactions.46 Moreover,
metal–ligand coordination is a key interaction in the self-assembly of both
biopolymers and synthetic oligomers.46 Thus, the next step toward the
development of highly constrained three-dimensional architectures from
peptoids was to demonstrate that two or more peptoid segments can self-
assemble via metal coordination to form high-ordered structures such as
macrocycles, helical rods, nanotubes, and so on.
The first example of Cu(II)-mediated self-assembly of peptoids
described a series of three self-assembled Cu(II)-peptoids and their
unique crystal structures: highly symmetric macrocycles composed of two
peptoid trimers held together via coordination to two Cu2+ ions, which
230 Behar et al.
two of these macrocycles are bridged by a water molecule (Figure 9).46

The first macrocyclic duplex was assembled from two peptoid sequences
incorporating Bipy as a ligand for Cu(II) binding, an ethanol group as an
additional ligand, and non-coordinating benzyl group for structure direct-
ing and further stabilization of the macrocycle (Figure 9a). In attempts to
gain control over the self-assembly process we further modified the
ethanol group by a methoxy (Figure 9b) or an amine group (Figure 9c).
After treating the peptoids with Cu(II) in methanol, the Cu–peptoid com-
plexes were formed and crystallized. Single-crystal X-ray analysis
showed that all three metallopeptoids formed Cu–peptoid duplexes of the
type P2Cu2, while self-assembling into macrocyclic arrangements.
Interestingly, while the ethanol group participated in the coordination of
Cu(II) (Figure 9d), the methoxy and amine groups did not, and instead,
the two Cu(II) ions within their corresponding Cu–peptoid complexes,
bound one water molecule as an oxo-bridge between them, enabling
(a) (b) (c)
(d) (e) (f)
(g) (h) (i)
Figure 9. Peptoid sequences and representation of their corresponding complexes

with Cu(II). Each color indicates one binding peptoid.
aromatic ring stacking (Figure 9e, f), which leads to a unique assembly
resulting in the first metallopeptoid helicates (Figure 9h–i).46 Notably,
both experimental spectroscopic data and DFT calculations suggested that
the Cu–peptoid macrocycles assembled from the peptoids having ethanol
or methoxy groups are not present in solution but rather exist as mono-
meric complexes of the type PCu in acetonitrile, presenting a solid/solu-
tion state equilibrium. In contrast, the macrocycle assembled from the
peptoid having the amine group does exist in solution.46
Although the macrocycles described above represent the first example
of metal ions–mediated self-assembly of peptoids, they can still be
defined as distinct structures as they did not pack nor stack to form more
complex (and thus more biomimetic) supramolecular architectures.
Indeed, targeted/controlled self-assembly of subunits toward the forma-
tion of distinct/desired supramolecular architectures is one of the most
significant phenomena that happens in natural system and thus is highly
significant in chemistry and biology.
In natural processes, peptide-based secondary structures form versa-
tile turns, different helices, or multiple types of sheets by directed associa-
tion of the peptide to produce 3D well-defined frameworks that work as
membranes and/or channels.54 These supramolecular architectures play
significant role in several biochemical processes spanning recognition
and/or transportation of biologically important ions and molecules.55
Although there are several examples showing self-assembly of artificial
peptides upon metal binding,56–58 controlling this process toward a spe-
cific architecture is still challenging. Therefore, a worthy goal is control-
ling/directing the Cu(II)-mediated self-assembly of peptoids toward the
controlled formation of various architectures.
To this aim, a set of peptoid trimers having Bipy in the second posi-
tion within the sequence together with a pyridine (Pam) group in the
N-terminal that acts as coordinating group, and a non-coordinating group
at the C-terminus, were designed (Figure 10).47 The Pam group in the
N-terminal was included as it has the potential to interact with a benzyl or
Pam group from another P2Cu2 unit via p-p interactions, such that it might
enable further self-assembly to form supramolecular structures. For the
monomer in C-terminus, various aromatic and aliphatic groups were
232 Behar et al.
Figure 10. Peptoid sequences with varying substitution in C-terminus with Bipy in
the second and pyridine in the third substitution.
chosen in order to evaluate the effect of this group on the self-assembly

process, such that by controlling the type of this group (and thus the pep-
toid sequence) control over the self-assembled supramolecular architec-
ture will also be achieved. The hypothesis was that while intermolecular
interactions between an aromatic group and Pam might lead to one type
of architecture, such interactions will not be possible with aliphatic
groups, and as a result, different supramolecular structures might be
formed in these cases.
Accordingly, a set of four peptoids was prepared (Figure 10), of which
two have aromatic side chains, benzyl and naphthyl (PPh and PNph,
respectively), and two have aliphatic side chains, cyclohexyl and tertiary
butyl groups (PCy and PTBu, respectively) in the C-terminus. The pep-
toids were treated with Cu(II), resulting in Cu(II)–peptoid duplexes as
evident by X-ray analysis. Interestingly, these were stable in solution as
evident by ESI-MS study.
The crystals obtained for Cu(II)-PPh/PNph yielded a helical rod-like
architecture as evident from the X-ray analysis. For Cu(II)-PPh, one dis-
tinct metallopeptoid duplex is shown in Figure 11a, which was further
packed with other such duplexes via intermolecular p-p interactions
(a) (b)
(c)
Figure 11. (a) Folded single metallopeptoid duplex of Cu2+ with peptoid having
benzyl amine substitution. (b) Supramolecular rod-like helical strand formed by self-
assembly of several metallopeptoid duplexes. (c) Cryo-TEM analysis of a sample
crystal of Cu2+ with peptoid having benzyl amine substitution (100 mM).
between the non-coordinating benzyl moiety from one metallopeptoid and

the pyridine group from adjacent unit, eventually forming the obtained
supramolecular helical structure. The stability of metallopeptoid duplexes
in solution prompts us to investigate the stability of the supramolecular
architecture in solution. Therefore, for further insight regarding the solu-
tion state stability of the architectures, cryo-TEM analysis was performed
with Cu2+-PPh. Interestingly, similar helical structure as observed from
X-ray study was also observed from cryo-TEM analysis. Helical segments
were shown to aggregate into entangled fibrils and fold into bundles
(Figure 11c). The width of each fiber was found approximately 11 nm that
indicates stacking of several helices together. Crystallographic analysis of
Cu(II)-PNph shows identical packing and supramolecular architecture as
of Cu(II)-PPh.
Interestingly, in the cases of the Cu(II)–peptoids having an aliphatic
substitution (PCy and PTBu, Figure 10), the obtained crystal structures
234 Behar et al.
demonstrated architectures that mimic pore-forming protein framework

(Figure 12a, b). These metallopeptoids form nano channel–based struc-
ture with varying pore size. For Cu(II)-PCy the pore size was larger than
Cu(II)-PTBu as obtained from X-ray study. The crystal structure revealed
that all amide bonds of Cu(II)-PCy are oriented toward the pore, while in
the case of Cu(II)-PTBu, the amide bonds are oriented in an alternating
(“zigzag”) fashion leading to a much tighter packing that eventually
reduces the pore size. Cu(II)-PCy was chosen and analyzed by cryo-TEM
and the monographs showed white spheres (circled in yellow, Figure 12c)
with similar sizes to that of the pores obtained from the crystal structure.
(a) (b)
(c)
Figure 12. Spatial orientation of Cu(II) with peptoid having cyclohexyl amine sub-
stitution supramolecular cavity along the crystallographic axis in the absence (a) and
presence (b) of water (Color code — red: oxygen; blue: nitrogen; cyan: Cu(II); and
grey: carbon. Hydrogen atoms and perchlorate ions are removed for clarity). (c)
Cryo-TEM of the crystals (100 mM in water) with expanded view marked framed in
purple.
Thus these were suggested to be the channel pores. Overall, for the first
time, controlling the sequence by simply modifying the non-coordinating
group at the C-terminus lead to control over the supramolecular architec-
ture of metallopeptoids.
IV. From Structure to Function: Cu-Binding Peptoids

and Cu–Peptoids as Functional Materials
A. Selective Recognition of Small Molecules by Self-Assembled
Cu(II)–Peptoids
The Cu–peptoid nano-channels described in section III.B (Figure 12),
which were self-assembled from the peptoids PCy and PTBu (Figure 10)
were found to be stable in solution as evident from the cryo-TEM and ITC
experiments. This allowed further exploration of their (selective) recogni-
tion properties toward biologically relevant small molecules and anions.
The recognition and transport of ions and small molecules by biological
channels are important processes for regulation of chemicals within the
cells. Mimicking such processes is therefore a key to the development of
specific receptors for therapeutic applications.59,60 Selective recognition
of ions and small biomolecules requires control over the pore size of the
nano-channels and their ability to recognize guest ions and small mole-
cules by non-covalent interactions. As amide groups can interact with ions
and small biomolecules, the host–guest interactions and recognition abil-
ity of the two types of nano-channels, formed by either Cu(II)-PCy and
Cu(II)-PTBu toward the anions Cl−, H2PO4−, CO32−, PO43−, citrate, and
glutamate, and the neutral small biomolecules glucose, glycerol, arab-
inose, and mannose in solution were explored. The host–guest interac-
tions between Cu–peptoid nano-channels and anions were initially
monitored by following the changes in the vibrational stretching of the
amide bond (n = ~1600 cm–1) using IR spectroscopy. The results from
these experiments suggested that there is an interaction, and possible
binding, between Cu(II)-PCy and the smaller anions, while the larger
anions citrate and glutamate did not interact with the metallopeptoid host.
Interestingly, similar measurements using Cu(II)-PTBu resulted in the
expected shift only upon the addition of Cl−, while no change was
236 Behar et al.
detected upon the addition of H2PO4−. These results indicated that nano-
channels assembled from Cu(II)-PCy, having larger pores are a non-
selective host for Cl−, whereas the nano-channels assembled from
Cu(II)-PTBu interact with Cl− only.
The host–guest interactions between Cu–peptoid nano-channels and
anions were initially monitored by gradually increasing the concentration
of glycerol in the solution containing Cu(II)-PCy resulted in a gradual
shift of the vibrational stretching near 1605 cm−1 to a higher wavenumber
near 1616 cm−1 while gradual addition of glucose, arabinose, or mannose
did not lead to any shift in the FT-IR spectra. These results suggested that
there is a selective interaction, and possible selective binding, between
Cu(II)-PCy and glycerol, while the larger, sugar molecules, do not inter-
act with the metallopeptoid host. Similar titrations to the solution of
Cu(II)-PTBu did not lead to changes in the vibrational stretching of the
metallopeptoids, suggesting that the pore size of the corresponding nano-
channels is too small to host such biomolecules.
The interaction between the Cu–peptoid nano-channels and glycerol
was further verified by 1H-NMR study, in which Cu(II)-PCy or Cu(II)-
PTBu were added to D2O solutions of either glycerol or glucose. The
1
H-NMR spectra of glycerol and glucose were measured and monitored
before and after the addition of the Cu–peptoids. The spectra showed that
while there was no change in the chemical shift of glucose upon the addi-
tion of each Cu–peptoid solution or in the chemical shift of glycerol after
the addition of Cu(II)-PTBu (Figure 13a, b), a clear downfield shift was
observed after the addition of Cu(II)-PCy to glycerol (Figure 13c, d), sup-
porting the results obtained from the FT-IR analysis. Furthermore, the
sample solution containing the Cu(II)-PCy loaded with glycerol was lyo-
philized and the microcrystalline solid obtained was washed thoroughly
with methanol to remove residues of unbound dissolved glycerol (if any),
and dried in vacuum. The dry solid was solubilized in diluted hydrochloric
acid and treated with bicarbonate, and a sample from this solution was
analyzed by ESI-MS. The ESI-MS analysis showed a signal indicating the
mass of glycerol, indicating again the interaction of Cu(II)-PCy with
glycerol. Similar workup and analysis performed on solution sample of
Cu(II)-PTBu loaded with glucose did not reveal any trace of glucose in
(a) (c)
(b) (d)
Figure 13. 1H-NMR of glycerol in D2O (a) before and (b) after addition of Cu(II)-
PTBu and (c) before and (d) after addition of Cu(II)-PCy (host and guest concentration
is 10 mM).
the ESI-MS spectrum, supporting the results obtained from the FT-IR and
1
H-NMR experiments indicating no glucose recognition.
Overall, these results demonstrate that the self-assembled nano-chan-
nels can interact with biologically relevant anions or molecules and that
by controlling their pore size we can regulate interactions with specific
anions and small biomolecules toward the use of these channels as
238 Behar et al.
selective receptors. The selective binding of the anions and small biomol-
ecules to Cu(II)-PCy and Cu(II)-PTBu also validate the self-assembly of
the Cu–peptoids in solution.
B. Selective Recognition of Cu(II) by Peptoids Toward

Drug Design
Following the understanding regarding the structural aspects of Cu(II)–
peptoids and the sequence requirements for high affinity to Cu(II), selec-
tive chelators to Cu(II) could be designed toward selective extraction of
Cu(II) from biomimetic and biological media, as well as from Cu–pro-
teins. As Cu is an essential trace element, involved as a cofactor in many
proteins, its concentration level is typically tightly regulated within cells.
However, Cu could be also toxic due to its redox ability, and its misregula-
tion is usually associated with two rare genetic disorders: Wilson’s disease
(copper overload) and Menkes disease (copper deficiency).61,62 Moreover,
copper was found to play a role in several neurodegenerative diseases,
including Alzheimer’s and Parkinson’s diseases, and high levels of Cu
ions are usually found in serum and tissue samples of cancer tumors. One
of the therapeutic approaches to restore balance of copper is to extract the
excess copper by chelation, that is, molecules with high selectivity to Cu
ions. The field of copper chelation accounts for hundreds of known suit-
able molecules,63,64 however, many of those still do not meet all the
requirements for carrying out efficiently the recognition process. Thus,
there is still a demand for the new ligands to be developed. One of the
recent approaches is the development of peptidomimetics as selective
chelators for Cu, as they combine the advantages of currently known
ligands with improved bioavailability properties. Among peptidomimet-
ics, peptoids are specifically interesting due to their excellent pharma-
cokinetic properties, such as long half-lives and high membrane
permeability.
It was previously shown, already in the reported first example of
Cu(II)–peptoids,15 that the pre-organization of two HQ ligands at the i and
i + 3 positions of a helical (Nspe-based) peptoid, lead to a very large
Figure 14. A schematic summary of Cu(II) binding to the helical peptoid Helix i + 3,
which is capable of intramolecular binding of Cu(II) ions or intermolecular binding
of two different metal ions in a selective manner.
binding constant observed for the Cu(II)–peptoid complex (logK = 14, in

MeOH:H2O (4:1) solution15). This observation established the require-
ment for intramolecular binding of the Cu(II) by two MB ligands that are
pre-organized at the same site of the peptoid’s helix in order to achieve
exceptionally high affinity to Cu(II). Capitalizing on this requirement, the
helical Nspe-peptoid hexamer Helix i + 3, bearing the MB ligands HQ
and Terpy at the i and i + 3 positions (Figure 14), was designed and syn-
thesized.31 Upon Cu(II) binding, the (Helix i + 3)Cu(II) was generated,
and its spectroscopic analysis suggested a pentagonal binding environ-
ment about the Cu(II) and high association constant was evaluated (logK
= 13, in MeOH:H2O (4:1) solution). Moreover, in methanol, Helix i + 3
was shown to bind 1 equiv. of Cu(II), selectively, from a mixture of
excess up to 20 equiv. of other metal ions, as confirmed by UV–vis,
ICP-MS, and ESI-MS studies. The unique selectivity for Cu(II) was
shown to be a result of the combination of both the choice of the two
specific MB ligands and their pre-organization within the peptoid helix.
Interestingly, in aprotic solvents, such as acetonitrile, a heterobimetallic
peptoid duplex of the type P2CuM was obtained when Helix i + 3 was
treated with a solution mixture of Cu(II) and another metal ion such as
Zn(II) and Co(II) (Figure 14).
Unfortunately, the Nspe side chains are hydrophobic, thus limiting the
solubility of the peptoid in water due to its high overall hydrophobicity.
240 Behar et al.
As a result, possible utilization of Helix i + 3, with its remarkable selectiv-

ity to Cu(II), as a drug candidate, required further optimization of the
sequence. A possible solution was the incorporation of one or more pip-
erazine units within the backbone of hydrophobic peptoids, which ensures
their water solubility, while maintaining their sequence and structure.65
This should initiate opportunities for future biochemical studies with
modified Helix i + 3 toward selective chelation in the context of disease
therapeutics.
Another possibility for the design of water-soluble-selective peptoid
chelators was to use short peptoids with a different set of MB ligands.
Thus, the water-soluble peptoid trimer PCA-Nspe, having an Nspe side
chain at its C-terminal, followed by Bipy) and Npam (at the N-terminus)
as a MB ligands was recently synthesized and evaluated for metal coordi-
nation.48 PCA-Nspe was shown to selectively bind Cu(II) in the presence
of high excess of 40 equiv. of other metal ions, forming an intramolecular
complex of the type PCu, as confirmed by UV–vis, CD, EPR, and ESI-MS
analysis. Moreover, PCA-Nspe could extract Cu(II) from the natural
copper-binding protein metallothionein, as indicated from the CD spec-
troscopy (supported by LC-MS analysis) before and after the addition of
PCA-Nspe. Computational studies suggested the structure-directing Nspe
group plays the crucial role in pre-organization of the MB ligand in PCA-
Nspe, which results in high selectivity. Interestingly, replacing the Nspe
side chain with a non-chiral benzyl amine side chain, resulted in an inter-
molecular binding of Cu(II), forming a complex of the type P2Cu2. This
intriguing observation prompted further sequence–function relationship
studies aiming to understand the role of the non-coordinating side chain
in the selectivity to Cu(II).49 Consequently, a set of peptoids, varied in
their side chain at the C-terminal, using different aromatic and aliphatic
chiral and achiral side-chains, was synthesized and its metal coordination
was estimated and investigated. The results suggested that the substitution
at the C-terminal has a significant effect on the selectivity to Cu(II), and
that high selectivity requires not only chirality and bulkiness, but rather
the combination of both, together with a specific position of the non-
coordinating side chain along the peptoid sequence. Moreover, one of
these chelators, PC1, having an Ns1npe side chain instead of Nspe, could
Figure 15. A cartoon representation of Cu(II) chelation by PC peptoids from a

copper-containing protein (left) or selective recognition from the mixture of other
metal ions (right).
extract Cu(II) from metallothionein, as indicated from CD spectroscopy

(Figure 15).
C. Positive Allosteric Cooperativity in Metal Binding to

Cu(II)–Peptoid
The high selectivity of Helix i + 3 to Cu(II) (see section III.A), prompted
the idea to replace one of the HQ ligands in the peptoid 12P1 with a Terpy
ligand, forming a peptoid with two distinct binding sites. This was antici-
pated to enable selectivity to Cu(II) in the HQ/Terpy site leaving the
HQ/HQ site available for the binding of a different metal ion, resulting in
a peptoid complex of the type PCuM. Consequently, the peptoids 12P5
and 12P6 (Figure 16a) were synthesized, characterized, and treated with
Cu(II). CD spectroscopy of 12P5 revealed that coordination of Cu(II) to
the Terpy/HQ binding site initiated folding, followed by the pre-organiza-
tion of the HQ/HQ binding site, and by this facilitated the coordination of
Zn(II) or Co(II) ions to the HQ/HQ site, demonstrating positive allosteric
cooperativity in the binding of Zn(II) or Co(II) (Figure 16b).32 In contrast,
the unstructured 12P6 did not fold upon Cu(II) binding and the corre-
sponding Cu–peptoid complex did not facilitate the binding of a second
metal ion (Figure 16c). Overall, this study demonstrated for the first time
that a structural change in a peptoid, induced by the selective metal bind-
ing of Cu(II), leads to positive allostery.
242 Behar et al.
(a)
(b)
(c)
Figure 16. (a) Peptoid sequences of 12mer peptoids 12P5 and 12P6. (b) A cartoon
demonstrating positive allosteric cooperative binding to (12P5)Cu, compared to (c)
no allosteric cooperativity of (12P6)Cu.
D. C
u(II)–Peptoid Complexes as Versatile, Efficient, and
Selective Bio-Inspired Catalysts
Enzymatic catalysis is one of the most important functions carried out by
natural metalloproteins. The catalytic activity of enzymes is largely
dependent on the cooperativity between binding sites, creating catalytic
pockets, which enable enzyme specificity and efficiency. A promising

approach for the design of synthetic catalytic systems that can imitate
enzymatic efficiency is to mimic the intramolecular cooperativity of
enzymes and the secondary coordination sphere about the catalytic metal
site, which facilitate its activity. This can be potentially done by designing
artificial peptidomimetic catalytic systems, containing functional groups
responsible for catalytic activity, as well as structural elements, in close
proximity to each other within one oligomer. This should create a con-
fined catalytic pocket together with its second coordination sphere, simi-
lar to the ones observed in nature.
The first attempt to mimic such enzymatic cooperativity between two
catalytic groups within a peptoid, capitalized on the oxidation of alcohols
to aldehydes by a Cu-Bipy catalysts that had utilized (2,2,6,6-tetramethyl-
piperidin-1-yl)oxyl (TEMPO) as a co-catalyst, N-methyl imidazole as a
nucleophile, and molecular oxygen as an oxidizer (Figure 17a, left).66
Based on this intermolecular cooperative catalytic system, a set of peptoid
trimers incorporating 1,10-phenanthroline as a ligand for Cu at the
N-terminus, TEMPO in the respective position, and a non-catalytic aro-
matic or alkyl group at the C-terminus, as intramolecular cooperative cata-
lytic systems for aerobic oxidation of alcohols, in the same reaction
conditions as the intermolecular cooperative catalytic system but with
lower catalyst loading.67 All the peptoids showed catalytic activity, which
(a)
(b)
Figure 17. Oxidation of alcohols to aldehydes and oxidative coupling of alcohols

and amines to imines catalyzed by an intermolecular cooperative catalytic system
(a) or by the peptoid-based intramolecular cooperative catalytic system (b).
244 Behar et al.
was at least 10 times higher than the intermolecular cooperative catalytic

system or a control peptoid dimer (DI) that did not have the non-catalytic
group. Moreover, the position of the three groups relative to each other
also had a role in the catalytic activity. These results demonstrated that the
pre-organization of the catalytic groups within the peptoid, and the pres-
ence of a non-catalytic group have a crucial role in the activity, suggesting
that these enable the intramolecular mode of action similar to the intramo-
lecular cooperativity in enzymes. The trimer BT, having a benzyl group
at the C-terminus, combined with Cu, could catalyze the oxidation of vari-
ous benzylic, allylic, and aliphatic primary alcohols with a TON of up to
16 times higher than a mixture of the two catalytic groups or the peptoid
dimer that is lacking the non-catalytic group (Figure 17b, left).
Following studies revealed that the catalyst CuBT could also catalyze
the oxidative coupling of alcohols and amines for the production of ben-
zyl, aryl, heteroaryl, allylic, and aliphatic imines68 with a TON up to 45
times higher than this achieved when phenanthroline, Cu, and TEMPO are
mixed in solution69 (Figure 17a, b, right). Recently, in a more subsequent
research, resin-bound BT and DI (TG-BT and TG-DI) were presented as
the first insoluble and recyclable catalytic peptoids. It was shown that
although TG-BT also operates via the intra-peptoid cooperativity mode,
it is less efficient than BT, even after several cycles. On the other hand,
TG-DI is far more active than DI (than BT) and can be recycled several
times to enable a much higher turnover number. These studies revealed
that TG-DI operates via an intra-resin cooperativity mode, which enables
its high activity compared with DI, BT, and TG-BT (Figure 18).70
Figure 18. The three different cooperativity modes suggested for the catalytic activ-
ity of TG-DI (intra-resin and inter-resin) and of TG-BT (intra-peptoid).
An important task in the field of catalysis, which is also a very chal-

lenging one, is the development of efficient electro- and photocatalysts for
splitting water to molecular oxygen and hydrogen, the latter being a
renewable and sustainable energy source. The first step of this catalytic
reaction, water oxidation (2H2O → O2 + 4H+ + 4e−; ∆E° = 1.23 V, ∆G =
113 kcal mol−1), is the thermodynamically challenging step, both in natu-
ral and especially in artificial systems, due to the transfer of four protons
and four electrons in a relatively low potential (0.876 V at pH 6).71 In
nature, this transformation is catalyzed by the oxygen-evolving complex
(OEC) in photosystem II of green plants and cyanobacteria, utilizing solar
energy and a manganese-oxo complex that its oxidation toward water
oxidation is facilitated by amino acids such as tyrosine and histidine from
the second coordination sphere.72
Based on the catalytic capabilities of BT and of the Cu-Bipy com-
plexes that act as efficient electrocatalysts for water oxidation, albeit at
high pH, with high overpotential and most importantly which were unsta-
ble under the electrocatalytic conditions,72,73 a Cu–peptoid was recently
designed and reported as the first peptoid-based electrocatalyst for water
oxidation.13 This peptoid trimer (BPT) is composed of a Bipy group as a
(a) (b)
Figure 19. (a) Molecular structure of Cu(II)BPT(OH)2 and its geometry-optimized

structure (optimized by DFT-D3 calculations (considering the dispersion correction)
at the level of B3-LYP with def2-TZVP for Cu(II) and def2-SVP for the other atoms as
basis sets with unrestricted Hartree−Fock, using turbomole and ORCA (3.0.3) soft-
ware package. (b) Evolution of O2 and charge accumulation during nine runs (40
min. each) in phosphate buffer solution (0.1 M) at pH 11.5 with 0.5 mM Cu(II)
BPT(OH)2. Oxygen was measured with a fluorescence probe. Porous glassy carbon
electrode was used at 1.35 V versus NHE.
246 Behar et al.
ligand for Cu(II), an –OH group, as a tyrosine mimic, and a benzyl group;
the two non-catalytic groups were designed to act as a second coordina-
tion mimic aiming to stabilize the Cu(II)/Cu(III) center and facilitate its
activity. Upon treating the peptoid with Cu(II) in alkaline buffer, the
complex Cu(II)BPT(OH)2 was formed and both experimental and compu-
tational data revealed that Cu(II) is bound to BPT via Bipy and two
hydroxyl ions originating from the basic solution. At pH 11, the catalyst
was stable over at least 15 hours of electrolysis and could be reused for at
least nine times in 40-minute runs, resulting in an overall TON of ~56
within 6 hours, if the pH was readjusted after each run. Electrochemical
experiments revealed that the reversible CuIII/II oxidation wave, which is
a key for water oxidation, occurred at an unusually low E1/2 of +0.30 V
versus NHE in contrast to a peptoid analog, in which the ethanolic –OH
group was replaced by –OCH3 to give a potential of +0.50 V for the CuIII/
II
transition. Based on these as well as other electrochemical experiments,
spectroscopic data, and DFT calculations, a stable key peroxide interme-
diate was identified and an intramolecular cooperative catalytic pathway
was proposed, suggesting that the proximal –OH group and the etheric
oxygen atom attached to the Bipy moiety form strong hydrogen bonding
with the coordinated hydroxide groups, thus has a major role in the high
stability of the complex.
V. Summary
The incorporation of MB ligands within peptoids, first demonstrated in
2009 by Maayan et al. has opened up the new field of metallopeptoids.
Extensive research on Cu(II) coordination has expanded the field of pep-
toids and of peptidomimetics in general, beyond structure and structural
requirements and considerations, toward function. The broad understand-
ing regarding the role of Cu(II) in peptoid folding and self-assembly led
to the development of Cu(II)–peptoids as functional materials that are
inspired by natural Cu(II)-binding peptides and proteins. This includes the
ability to mimic complicated biological activities such as selective recog-
nition, allosteric cooperativity, and intramolecular cooperative catalysis
similar to the one performed by enzymes. Although exciting, these
achievements represent only the initial steps toward the many possibilities
to utilize Cu for obtaining functional peptoids. Further advances in this

field include studies on Cu(I) coordination to peptoids, utilization of Cu(I)
and Cu(II)-binding peptoids as therapeutics, and the development of
mono- and multi-Cu–peptoids as efficient and selective catalysts for vari-
ous transformations, including ones that are currently too challenging to
be performed by molecular catalysts.
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Index
affinity to Cu(II), 216, 222, 238, 239 bis-oxidized quinone Cu(II) hydroxo,
albinism, 58 168
alkoxo, 162, 166 bis-phenoxide, 175
alkoxoperoxide, 168 bis-phenoxide salen, 176
allostery, 241 bis-thiosemicarbazones (bTSC), 21,
antimicrobial peptides (AMPs), 22, 30
33 bis(μ-oxido)dicopper(III) /
applications, 212, 217, 235 {CuIII2(μ-O)2}2+ / {Cu2O2}, 104
aromatic C−H hydroxylation, 108, Bond Dissociation Free Energy
111, 116 (BDFE), 155, 160, 165,180, 181
aromatic ring hydroxylation, 84, 88,
89, 91, 94, 95, 96, 110 C−F bond, 114, 115, 116
association constants, 221, 222 C−H oxidation, 107, 108
aureusidin synthase (AUS), 51 calix[6]trenamide Cu(I) complex, 161
carboxylato, 166
backbone conformation, 214 carboxylato complexes, 162
bakcbone isomerisation inducers, catechol oxidase (CO), 51, 83, 84,
214, 215 86, 87
bathocuproine (BC), 28 cellobiose, 195, 196
bathocuproine disulfonate (BCS), 28 cellulose, 188, 190, 196, 206
benzimidazole, 194, 206 chalcocite (Cu2S), 2
benzylic alcohol, 175 chalcopyrite (CuFeS2), 2
bidentate ligand, 129, 130, 134, characterisation methods, 220
136–139, 142 chelators, 213, 216, 221, 238, 240
biomimetic, 70, 82–84, 88, 89, 96, chiral, 214–216, 224, 225, 227,
102, 107, 118, 212 240
biomolecules (BMs), 5 chitin, 188, 190, 206
Bipy, 218, 230–232, 240, 243, 245, circular dichroism (CD), 215,
246 223–225, 227, 228, 240, 241
251
252 Index
class II mixed-valent compounds, 176 Cu(II)-phenoxyl, 176

click reactions, 219 Cu(II) precursor, 162
competition experiment, 221 Cu-ligand complexes (Cu-L), 2
concerted electron–proton transfer, CV, 159–161, 165–167, 170, 174,
159 175, 181
cooperative catalysis, 212, 246 cytochrome c oxidase, 154
copper-containing proteins, 51
copper-containing redox enzymes, 47 dicopper(III) µ(oxo) adduct, 168
copper–dioxygen, 83, 89, 99, 117 dicopper(II)-oxyl, 168
copper(II) hydroperoxide, 168 dicopper proteins, 84
copper–oxygen, 190, 195, 206 diethyldithiocarbamate (DEDTC), 24
copper–oxygen adducts, 155, 182 dihydroxyindole (DHI), 50, 57, 58
coupled electron–proton reactions, dihydroxyindole carboxylic acid
155 (DHICA), 50, 57
cryo-spectroelectrochemical dithiocarbamates (DTC), 24
measurements, 181
cryo-spectroelectrochemical setup, 8-hydroxychinoline (8-HQ), 25
160 EIS, 181
cryo-TEM, 233–235 electrocatalysis, 245
Cu2III/II µ(O) µ(OH) species, 167 electrocatalytic HAA oxidative
Cu2O2, 84, 87, 88, 95, 97, 99–102, properties, 167
104–118 electrochemical impedance
Cu(II) alkylperoxo, 170 spectroscopy, 160
Cu(II) binding preferences, 218 electrochemistry, 182
Cu(II)–Cu(III) oxidation potential, electron spin echo envelope
165 modulation (ESEEM), 63
Cu(II) hydroxo, 162 electron transfer kinetics, 160
CuIII, 197, 198 electrophilic substitution, 133
CuIII-O-CuII adduct, 168 end-on peroxo complexes, 160
Cu(III)-OH species, 162 end-on trans-peroxo-dicopper(II) /
Cu(III)-OOH species, 171 {CuII2(μ-η1:η1-O2)}2+ /
Cu(III)-oxo adducts, 169 {Cu2PE}, 87
Cu(III)-oxo core, 162 EPR, 182, 222, 223, 225, 227,
Cu(III) oxygen species, 162 240
Cu(II)-oxyl, 157 ESI-MS, 225, 227, 232, 236, 237,
Cu(II)-peptoid complexes, 216, 219, 239, 240
222, 225, 242 eumelanin, 49
Cu(II)-phenoxide, 175 external substrates, 126, 129, 139,
Cu(II)-phenoxide radicals, 173 141, 142
Index 253
folding facilitation, 212, 227 inductively coupled plasma–mass

frontier molecular orbitals, 102–105 spectrometry (ICP-MS), 7
inductively coupled plasma–optical
galactose oxidase, 172 spectroscopy (ICP-OES), 7
gluconic acid, 195, 200, 202 inhibitors, 58–62, 66–69, 73–75
glutathione (GSH), 6, 9 intermolecular binding, 216, 239, 240
intramolecular binding, 239
hard and soft acids and bases IR spectroscopy, 235
(HSAB), 17 isothermal titration calorimetry
helix, 214–216, 224, 225, 227, 228, (ITC), 221, 235
239–241
hemilabile ligand, 137 kojic acid (KA), 59–69
hemocyanin, 83–85
hemocyanins (Hc), 51 laccases, 154
heterogeneous electron transfer, 181 L-DOPA, 50
high-valent species, 154 LE correspond to external ligand, 23
histidine brace, 188 ligand hydroxylation, 125
homolytic Cu–O bond, 169 ligand (L), 2
host-guest interactions, 235, 236 L-mimosine (Mim), 60
HQ, 218, 224, 225, 227–229, 238, loop, 214
239, 241 low temperature, 156
hydrogen atom abstraction (HAA), low-temperature CV, 159
155, 198, 206 Lpy1, 131–135, 138, 145
HAA properties, 166, 171 L-tyrosine, 50
HAA reactions, 158 lytic polysaccharide
HAA reactivity, 181 monooxygenases, 187, 188
hydrogen bonding, 213, 214, 224, lytic polysaccharide peroxygenases,
246 189, 206
hydrogen peroxide, 192, 200, 205,
206 (m-η2:η2)-peroxo ligand, 54
hydroperoxo, 170 macrocycles, 229, 230, 231
hyperfine sublevel correlation macrocyclic ligands, 96
(HYSCORE), 63 melanin, 47
hyperpigmentation, 58 melanoma, 58
melasma, 58
i and i+3, 216, 217, 224, 225, 238, metal-binding, 216–219, 225
239 metal binding ligands, 224, 231, 241
imidazole, 203, 204, 206 metal-ligand interactions, 224
254 Index
metal–oxygen adducts, 154 Nsmp, 225, 227, 228

minimum inhibitory concentration Nspe, 215, 224, 227, 228, 238–240
(MIC), 25
Minuit XANES (MXAN), O–O bond cleavage, 157, 161
mixed-valent Cu2III/II bis(µ-oxo) oxidation of methane, 158
species, 159 oxidative cleavage, 206
mixed-valent Cu2III/II µ(O(H)) oxygen evolution reaction (OER),
species, 167 155
mixed-valent Cu(II)–Cu(III) oxygen reduction reaction (ORR),
bis(µ-oxo) adducts, 158 155
mixed-valent Cu(II)–Cu(III)
phenoxide, 177 Pam, 218, 231, 232
molecular oxygen, 153 partially reduced oxygen species,
molecule recognition, 211, 235 154
mono- and diphenolic substrates, 95 particulate methane monooxygenase,
monooxygenases and 153
dioxygenases, 82 PCET, 168
monooxygenation reaction, 124, 127, peptide mimics, 212
137, 140, 143 peptidylglycine α-hydroxylating
monophenolase activity, 133, 135, 144 monooxygenase, 153
µ-hydroxo species, 165 peptoid duplex, 230, 232, 239
µ-oxo complex, 168 peptoid’s design, 216, 227
m-Xylyl, 81, 84, 88–96, 99, 108–111, peptoid structure, 224, 228
118 peptoid synthesis, 213
peroxide copper species, 156
nano-channels, 235–237 peroxo intermediate, 127, 133, 135,
neocuproine (NC), 28 140, 141, 143, 148
NIH shift, 109 phenanthrolines (Phen), 27, 218
nitrosobenzene derivatives, 178 phenol oxidation, 47
nitrosobenzene ligand, 177, 179 phenoxo complex, 173–175
N,N-bis(2-pyridyl)methylamine, 166 phenoxyl Cu(II) radical species,
N,N’-di-tert-butyl-ethylenediamine 172
(DBED), 129, 130 pheomelanin, 49
Nr1tbe, 215 phosphatidyl ethanolamine (PE),
Nrpe, 215, 216 11
Ns1npe, 215, 228, 240 phosphatidyl serine (PS), 11
Ns1tbe, 215, 228 PicT, 218, 219
Index 255
p-nitrophenyl-β-D-glucopyranoside, side-on peroxo-dicopper(II) /

200, 202, 205 {CuII2(μ-η2:η2-O2)}2+ /
polyphenol oxidases (PPOs), 47 {Cu2PS}, 87
polyproline type I (PPI), 214 skin whitening, 59
polysaccharides, 188 solar lentigo, 58
pore-forming framework, 234 solid support synthesis, 213, 219
pre-organisation, 216, 238–241, 244 spectroelectrochemistry, 176, 182
pyridine(dicarboxamide), 164, 171 strong superexchange, 168
pyrithione (PT), 29 submonomer approach, 213, 214
PyrT, 219 sulfide Cu(II) adducts, 177
super-exchange, 102, 104
QM/MM calculations, 53 superoxide, 156
superoxo species, 161
reactive oxygen species (ROS), 5, 9 supramolecular architectures, 231
recalcitrant polysaccharides, 188 synthesis, 211, 213, 214, 216,
redox-active ligand, 168 219
redox potential, 193, 200, 204, 205,
206 TEMPOH, 181
redox reactions, 153 Terpy, 218, 239, 241
reorganizational energies, 160, 181 tetramethypropylenediamine ligand
residues per helical turn, 214, 216, (TMPD), 178, 179
224 thujaplicin (Thuj), 60
resin-bound catalysis, 244 time-resolved spectroelectrochemical
resonance Raman, 182 experiments, 177
transition-state analogue (TSA),
secondary structure, 213–216, 60–62, 67, 69, 70, 73, 74
223–225, 227, 228, 231 tris-tertbutylphenol, 166
selectivity, 211, 216, 221, 222, tropolone (Trop), 60
238–241 two-electron reduction, 161
self-assembly, 211, 229–233, 238, 246 2-hydroxypyridine N-oxide
self-oxidation of the ligand, 161 (HOPNO), 60
semi-quinone Cu(II) hydroxide, 168 two metal binding sites, 216
side-chains, 240 2-pyridinethiol N-oxide
side-on dicopper(II) bisulfides, 177 (HSPNO), 61
side-on disulfido, 180 tyrosinase (TYR), 47, 55, 81, 83–89,
side-on peroxo, 161 95–103, 107, 108, 111, 117–119
side-on peroxo dicopper(II), 158 TYR-like enzymes, 48
256 Index
TYR-related protein 1 (TRP1), 52, vitiligo, 58

55, 57, 59, 63–68, 71
TYR-related protein 2 (TRP2), 55, 57 water oxidation, 245, 246
unsymmetrical complex, 177 X-ray, 222, 223, 230, 232–234

unsymmetrical ligand, 166 X-ray absorption spectroscopy
UV–vis spectroelectrochemistry, (XAS), 63, 64, 68, 69,
167 72
UV-Vis titrations, 221, 226 XYL system, 125

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Copper Bioinorganic

Library of Congress Control Number: 2023031038

British Library Cataloguing-in-Publication Data

COPPER BIOINORGANIC CHEMISTRY

ISBN 978-981-126-948-6 (hardcover)

For any available supplementary material, please visit

Typeset by Stallion Press

Bioinorganic chemistry is a booming field of research that has its source

boosting to silencing” by Peter Faller, Marianne Ilbert et al. addresses this

chemistry, i.e. the mysterious particulate methane mono-oxygenase

1 Ligands as a Tool to Tune the Toxicity of Cu on Bacteria:

C. Kinetics of the Cu-L Complex 17

 odeling Tyrosinase Activity Using m-Xylyl-Based

B. Intramolecular m-Xylyl and Aromatic Ring

I. Introduction 123

B. Substitution of Pyridine in the Lpy1 Ligand by

I. Introduction 153

6 I norganic Models of Lytic Polysaccharide

I. Introduction 187

A. Polysaccharide Hydrolysis 190

7 Structure and Function of Cu–Peptoid Complexes 211

I. Introduction 212

1 Ligands as a Tool to Tune the

Lisa Zuily,* Nora Lahrach,* Enrico Falcone,†

*Aix-Marseille University, CNRS, BIP, UMR 7281, 31 Chemin

being discovered. Progress has also been made in understanding the

systems induces cell death. Such powerful antimicrobial activity enables

II. Copper, a Biocidal Compound

Figure 1. Current applications of copper in our society due to its antimicrobial

As often, the use of the antimicrobial property of copper is not an

Figure 2. Cu-dependent strategies of the immune system to kill invading microbes.

III. Copper Homeostasis Systems in Bacteria

cueO copA(Z) cusS cusR cusC cusF cusB cusA

Figure 3. Copper homeostasis systems in E. coli. Increase of copper in the periplas-

cytoplasmic compartment of some bacteria.24,25 In addition, non-protein

involved in the cellular protection toward Cu stress.29 Even in some

IV. Copper Toxicity: A Phenomenon Dependent on

highlight distinct strategies explaining such results. As an example, acido-

V. Specificity of Copper Chemistry

B. Cu Reactivity: The Case of ROS Production

Non-controlled Cu amount can thus be a source of reactive oxygen

VI. Mechanisms of Cu Toxicity:

bonds of unsaturated fatty acids.48 This phospholipid bilayer modification

Intriguingly, under anaerobic conditions, a lower concentration of Cu

VII. Chemistry of Copper Complexes

Developing new antibiotic agents is challenging, time consuming, and

(a) Redox-based: ligands modulate Cu redox potential (see section

Figure 5. Influence of ligand L on the interaction of Cu with biomolecule: (a) ionic

especially relevant for ternary complexes L-Cu-BM. A ligand can

B. Stability of the Cu-L Complex

C. Kinetics of the Cu-L Complex

VIII. Impact of Ligands on the Biological

B. Presence in Biology of Potentially Competing

While in the periplasm Cu(II)-L remains oxidized, Cu(II)-L entering

C. Presence in Biology of Potentially Competing

1. Localisation change/ 2. New Reactivity 3. Quench / Loss of

Ligand Cu+ Cu2+ Other metal Cu-binding

D. Reactivity of Cu-L in Bacteria

“Copper Complexes as Antimicrobial Agents, a Non-Exhaustive List”).

1 Ligands as a Tool to Tune the Toxicity of Cu on Bacteria:

C. Kinetics of the Cu-L Complex 17

odeling Tyrosinase Activity Using m-Xylyl-Based

B. Intramolecular m-Xylyl and Aromatic Ring

I. Introduction 123

B. Substitution of Pyridine in the Lpy1 Ligand by

I. Introduction 153

6 I norganic Models of Lytic Polysaccharide

I. Introduction 187

A. Polysaccharide Hydrolysis 190

7 Structure and Function of Cu–Peptoid Complexes 211

I. Introduction 212

B. Presence in Biology of Potentially Competing

C. Presence in Biology of Potentially Competing

*University of Grenoble Alpes, CNRS-UGA UMR 5250, DCM,