159

2.1.5 Addition to C=N Bonds

A. Ilari, A. Bonamore, and A. Boffi

General Introduction

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The Pictet–Spengler reaction consists of a Mannich-type cyclization in which an electron-
rich aromatic carbon attacks a C=N bond, in the form of an electrophilic iminium ion,
thus yielding a heterocyclic scaffold and generating a new asymmetric center.[1,2] The for-
mation of the iminium species must be considered mandatory for the subsequent cycliza-
tion. As such, the Pictet–Spengler reaction is commonly considered as a two-step reaction.
Typically, an electron-rich (-arylalkyl)amine and an aldehyde first condense to form an
iminium species and then an electrophilic aromatic substitution reaction occurs in which
the regiocompetent carbon atom of the aryl group of the (arylalkyl)amine attacks the elec-
trophilic iminium ion (Scheme 1). Each step of the reaction may be thought of as a con-
certed acid–base process in which a proton is exchanged from/to a donor/acceptor species,
be it a protic solvent or a catalyst. Thus, each reaction step can be conveniently promoted
by a bifunctional catalyst, able to provide and abstract a proton to/from the participating
reactants.[3] In nonenzymatically catalyzed reactions, however, two stereoisomers are typ-
ically formed as a result of the generation of a new asymmetric carbon center.

Scheme 1 Pictet–Spengler Cyclization[1,2]

H+

NH2 − H2O N − H+ ∗ N
H H
H
O R 1 R1
R1

2.1.5.1 Pictet–Spenglerases

The identification and isolation of enzymes capable of catalyzing diastereoselective Pic-

tet–Spengler reactions has represented an important breakthrough in enzyme-based
chemical synthesis. Pictet–Spenglerases thus identified turn out to be key enzymes in im-
portant plant secondary metabolisms such as benzylisoquinoline and monoterpene in-
dole alkaloid biosynthetic pathways. Strictosidine synthase from Rauvolfia serpentina
(RsSTS; EC 4.3.3.2), the first isolated Pictet–Spenglerase,[4] is the central enzyme in the bio-
synthesis of monoterpene indole alkaloids, whereas norcoclaurine synthase from Thalic-
trum flavum (TfNCS; EC 4.2.1.78)[5] catalyzes the key step in benzylisoquinoline alkaloid
pathways. Both enzymes belong to the lyase family according to the EC (enzyme commis-
sion) classification and have been shown to be capable of catalyzing diastereoselective
Pictet–Spengler cyclization according to the general acid–base bifunctional catalysis out-
lined in the General Introduction. More recently, other Pictet–Spenglerases have been
identified and isolated and novel putative enzymes belonging to this class have been in-
ferred from metabolic pathways. Taken together with the possibility of active-site engi-
neering by mutagenesis, Pictet–Spenglerase enzymes provide precious tools for the ste-
reoselective synthesis of tetrahydroisoquinolines and terpene indole derivatives as well

for references see p 175

160 Biocatalysis 2.1 C—C Bond Formation

as novel heterocyclic compounds. Within this review, the substrate scope and limitations
of the best known Pictet–Spenglerases are described in order to pave the way for develop-
ment of a general biocatalytic strategy for stereoselective addition to C=N bonds.

2.1.5.1.1 Biosynthesis of Benzylisoquinoline and Indole Alkaloids

Benzylisoquinoline alkaloids (BIAs) comprise a wide array (>2500) of plant secondary me-
tabolites endowed with a variety of biological activities and pharmacological proper-
ties.[6,7] Narcotic analgesics, such as morphine and codeine, or muscle relaxants, such as
tubocurarine and papaverine, are just a handful of examples of medicines derived from
plant benzylisoquinoline alkaloids. The biosynthetic pathways leading to benzylisoquin-

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oline alkaloid biosynthesis originate from tyrosine through a lattice of decarboxylations,
hydroxylations, and deaminations that generates a species-specific mosaic of diverse sec-
ondary metabolites that have evolved as a plant defense system against pathogens or as
deterrents against herbivores.[7] The common central pathway among different species
evolves from l-tyrosine as precursor and comprises the condensation reaction between
dopamine (1) and (4-hydroxyphenyl)acetaldehyde (2) (both derived from tyrosine metab-
olism) to yield the alkaloid (S)-norcoclaurine (3), catalyzed by the enzyme norcoclaurine
synthase (NCS) (Scheme 2). (S)-Norcoclaurine is subsequently converted into (S)-reticuline
by O- and N-methyltransferases and a P450 hydroxylase. (S)-Reticuline is a branch-point
intermediate in the biosynthesis of many other benzylisoquinoline alkaloids, leading to
protoberberine alkaloids, such as berberine and palmatine, and morphinan alkaloids,
such as morphine and codeine. Details of the relevant biosynthetic pathways are being
extensively updated and reviewed.[6,7]
 2.1.5 Addition to C=N Bonds 161

Scheme 2 Pictet–Spenglerases within Alkaloid Pathways

H
HO

2 HO

norcoclaurine
NH
synthase HO

HO
3

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NH2 benzylisoquinoline
alkaloids
HO

HO
1
HO

deacetylipecoside NH
HO
synthase H
OGlc
H
MeO O

O
O H
5
OGlc
ipecac alkaloids
O O

OMe
4

strictosidine OGlc
NH
synthase
O
N H
H H
O
OMe
NH2
7

N
H monoterpene
indole alkaloids
6

HO
HO
O O OH
Glc =
OH

Terpenoid indole alkaloids (TIAs) comprise a large family of more than 2000 compounds
that include structurally diverse medicinal compounds such as the antineoplastic agents
vinblastine and camptothecin, the antimalarial drug quinine, and other bioactive sub-
stances such as yohimbine, ajmaline, and strychnine, and that are found in a wide variety
of plant species.[6] As in the case of benzylisoquinoline alkaloids, terpenoid indole alka-
loids play a role in the defense of plants against pests and pathogens. The general struc-
ture of terpenoid indole alkaloids consists of an indole moiety derived from tryptophan
and a monoterpenoid component belonging to the iridoid family. Terpenoid indole alka-

for references see p 175

162 Biocatalysis 2.1 C—C Bond Formation

loid biosynthesis thus represents a most interesting metabolic coupling between trypto-
phan metabolism and the iridoid monoterpene pathway. Iridoid monoterpene biosynthe-
sis is a unique metabolic pathway starting from the hydroxylation of geraniol to 10-hy-
droxygeraniol by an NADPH dependent P450 monooxygenase, followed by subsequent ox-
idations and a unique redox-dependent cyclization catalyzed by the enzyme iridoid syn-
thase to yield the cis-trans-nepetalactol bicyclic scaffold with three asymmetric carbons.[8]
Subsequent enzymatic steps, up to the glycosylated iridoid loganin, are as yet only partial-
ly characterized. The conversion of loganin into secologanin (4) represents the last step in
the iridoid pathway and is also catalyzed by a P450 dependent enzyme.[6] The monoter-
pene-derived seco-iridoid -d-glucoside secologanin (4), is thus the precursor for all mono-
terpene indole alkaloids. Secologanin (4) is a highly functionalized molecule, character-

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ized by the presence of three stereogenic centers on its dihydropyran core. Only the
2S,3R,4S-isomer is produced biosynthetically. Tryptamine (6), produced through trypto-
phan metabolism, and secologanin (4) are then condensed by strictosidine synthase
(STS), a typical Pictet–Spenglerase, to form strictosidine (7), a -carboline containing the
tetrahydro--carboline (tryptoline) scaffold that represents the common precursor to ter-
penoid indole alkaloids (Scheme 2). Starting from strictosidine, all other terpenoid indole
alkaloids can be produced, though the relevant enzymatic pathways are fully understood
only for a few selected compounds.[6]

2.1.5.1.2 Structural Basis of Norcoclaurine and Strictosidine Synthases

The key enzymes of the benzylisoquinoline alkaloid and terpenoid indole alkaloid path-
ways, namely (S)-norcoclaurine synthase and strictosidine synthase, respectively, have
been fully characterized in terms of structure and catalytic properties. The X-ray structure
of strictosidine synthase from Rauvolfia serpentina, in complex with its natural substrates
tryptamine (6) and secologanin (4), provides details of the observed substrate preference
and identifies residues lining the active-site surface that contact the substrates.[9,10] Struc-
tural analysis and site-directed mutagenesis experiments demonstrate the essential cata-
lytic role of Glu309, which most likely represents the key proton donor/acceptor neces-
sary for the bifunctional acid–base catalysis (Scheme 3). A specific role for substrate ac-
commodation/recognition has been inferred for Val208, Phe226, and His277 residues.[10,11]

Scheme 3 Pictet–Spenglerase Active Site: Strictosidine Synthase[9–11]

Glu309

HO
HO
O
H O
Val208 NH H O OH

O HO OH
N H
H H
O
OMe HN N
7

His277
 2.1.5 Addition to C=N Bonds 163

The X-ray structure of (S)-norcoclaurine synthase from Thalictrum flavum reveals that this
enzyme harbors a relatively wide substrate-binding site (Scheme 4), in which the aromatic
(4-hydroxyphenyl)acetaldehyde (2) and dopamine (1) appear to stack together.[12] The
Lys122 residue appears to interact directly with the aldehyde functional group, and resi-
dues Tyr108 and Glu110 are at hydrogen-bonding distance with the hydroxy groups of the
catechol moiety. On the basis of structural data and enzyme kinetic measurements, sev-
eral mechanistic models have been proposed that account for the high stereoselectivity of
the enzyme.[12,13]

Scheme 4 Pictet–Spenglerase Active Site: (S)-Norcoclaurine Synthase[12]

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Tyr108
OH
HO

NH
HO

OH
H2N
O HO Lys122
Glu110
3

2.1.5.1.3 Other Pictet–Spenglerases

Further enzyme activity, other than that exhibited by norcoclaurine and strictosidine syn-
thases, entails the Pictet–Spengler condensation between dopamine (1) and secologanin
(4) to generate tetrahydroisoquinoline 5 (see Scheme 2, Section 2.1.5.1.1) and thus tetra-
hydroisoquinoline monoterpene skeletons (emetine-type or ipecac alkaloids). The en-
zymes involved in the formation of deacetylipecoside (5) have been identified and isolat-
ed in Alangium lamarckii. The two enzymes apparently lead to the formation of the R- and
S-epimers, respectively.[14] Based on their activities, the two enzymes have been called de-
acetylipecoside synthases (DIS).[15] These most interesting enzymes, however, are at pre-
sent unsequenced.
Lastly, complex Pictet–Spengler reactions have been found to be catalyzed within
non-ribosomal peptide synthesis. In particular, the saframycin synthase modular com-
plex (SfmC) catalyzes a seven-step transformation of dipeptidyl substrates with long acyl
chains into the saframycin scaffold.[16] Isolation of the Pictet–Spengler domain out of the
modular complex, however, has not been considered for synthetic purposes in favor of
direct utilization of the bacterial strain within biotransformations leading to tetrahydro-
isoquinoline antibiotics.

2.1.5.2 Norcoclaurine Synthase Catalyzed Pictet–Spengler Reactions

The present knowledge of Pictet–Spengler reactions catalyzed by norcoclaurine synthase

relies on a single enzyme obtained as a recombinant protein from the relevant gene of
Thalictrum flavum (TfNCS).[5] The reaction catalyzed by the TfNCS enzyme can be conve-
niently exploited for synthetic purposes in view of its broad specificity toward aldehyde
substrates that contrasts with the strict requirement for the amine substrate, dopamine
(1).[17] Overall, this substrate specificity conforms with the reaction mechanism proposed

for references see p 175

164 Biocatalysis 2.1 C—C Bond Formation

by Luk and co-workers[13] that requires the presence of a hydroxy group at the C2 position
of the phenolic aromatic ring of the amine, necessary for the formation of a quinoid spe-
cies at this position. Most recently, several genes codifying for novel norcoclaurine syn-
thases from different plant species have been identified and are under screening.[18,19]
Recombinant TfNCS can be readily obtained in E. coli BL21(DE3) cells as a truncated
protein at the first 19 amino acids with a polyhistidine tag (His-tag) at either the C- or N-
terminus. C-Terminal tagging has been inferred as a preferential construct because of pos-
sible interactions of the tag with the active site upon modification of the N-terminal re-
gion. The TfNCS enzyme can be thus purified in high yields by standard procedures using
a nickel-chelating resin.[13] The enzyme-catalyzed reaction has been shown to proceed
with a reasonably high turnover rate at pH values around neutral. The reported KM values

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are 0.35 mM for dopamine (1) and 0.29 mM for (4-hydroxyphenyl)acetaldehyde (2), respec-
tively, at pH 7.5 and 37 8C. Saturating conditions for both substrates are reported to be
around 2 mM.[13]
TfNCS appears to exhibit good overall stability and has been used in recycling mea-
surements. However, excessive aldehyde substrate concentrations are better avoided due
to possible parasitic reactions with lysine side chains of the enzyme. Further concern
must be mentioned regarding the use of the appropriate buffer system in TfNCS-catalyzed
reactions. The use of tris(hydroxymethyl)aminomethane/hydrochloric acid (Tris-HCl)
buffer at pH 7.0 was initially proposed by Luk and co-workers[13] and further exploited by
OConnor and co-workers[17] (see the procedure detailed in Section 2.1.5.2.2). However, in-
terfering reactions between the Tris amine and the aldehydes have been reported that
might decrease aldehyde availability.[20] Also, the use of the phosphate buffer described
in Section 2.1.5.2.1 has been challenged due to the observed increased background reac-
tion that may lead to the formation of sizeable amounts of racemic products.[18,21] At pre-
sent, although other buffers have been investigated (HEPES at pH 7.0, maleic acid at
pH 6.7, and imidazole at pH 6.5), there are no conclusive studies on the optimal buffer
composition of the medium[20] nor on the possible use of cosolvents to increase substrate
solubility or eventually decrease product solubility.

2.1.5.2.1 One-Pot Preparation of (S)-Norcoclaurine

A one-pot, two-step synthesis has been set up starting from tyrosine (8) and dopamine (1)
in the presence of TfNCS (Scheme 5).[22] The major limiting step in the large-scale produc-
tion of (S)-norcoclaurine (3) is represented by the low stability of both dopamine (1) and
the aldehyde substrate 2 in aqueous solution at neutral pH values in view of possible auto-
oxidation and/or uncatalyzed background reaction. Thus, (4-hydroxyphenyl)acetaldehyde
(2) is conveniently generated by the oxidative decarboxylation of tyrosine (8) in aqueous
solution in the presence of an equimolar amount of hypochlorite,[23] followed by its imme-
diate use in the enzymatic reaction. The product yield for oxidative decarboxylation is es-
timated to be greater than 99%, with less than 0.1% of chlorinated byproducts (by GC/MS).
This reaction is carried out in the same phosphate buffer as needed for TfNCS catalysis,
thus suggesting that the whole (S)-norcoclaurine synthesis could be optimized as a one-
pot reaction by quickly adding dopamine (1) and TfNCS to the solution containing the
newly synthesized aldehyde 2. In order to prevent oxidation of dopamine (1) during the
reaction course, ascorbate is added to the reaction mixture in the absence of cosolvents.
Of the various reductants tested, ascorbate was observed to be the most efficient, easiest
to use, and least expensive. This strategy allows at least five-fold higher concentrations
(10 mM) of dopamine (1) to be used with respect to previously reported methods. The en-
zyme concentration is adjusted to achieve relatively fast reaction kinetics in order to limit
the competing non-enantioselective chemical coupling between dopamine (1) and (4-hy-
droxyphenyl)acetaldehyde (2), which would dominate the Pictet–Spengler reaction for
longer periods of incubation. The optimal enzyme concentration is, however, relatively
 2.1.5 Addition to C=N Bonds 165

high, corresponding to about 10 mg • L–1 enzyme in the solution. An extraction strategy

that avoids the use of organic solvents has been developed based on the use of activated
carbon. The adsorption/desorption properties of activated carbon toward phenolic com-
pounds have been widely characterized, and conditions for optimal binding/desorption
of (S)-norcoclaurine (3) have been investigated as a function of temperature and solvent
composition. The adsorption is achieved by shaking carbon granules directly added to
the aqueous phase at room temperature. Control experiments indicate that the dopamine
(1) substrate is not adsorbed onto the activated carbon beads, whereas the aldehyde sub-
strate is strongly adsorbed. The best desorption conditions for (S)-norcoclaurine are ob-
tained in ethanol at 40 8C in the presence of a slight molar excess of sodium hydroxide.
This procedure allows a very good yield of (S)-norcoclaurine (3) to be obtained without

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the presence of unreacted aldehyde contaminant, which remains adsorbed on the carbon
matrix. The procedure based on activated carbon has the notable advantage of allowing
the recovery of about 70% of the active TfNCS enzyme from the reaction mixture once the
carbon has been removed by filtration. In recycling experiments, the same enzyme solu-
tion could be used up to five times, with a steady decrease in overall activity. However,
further optimization may be necessary to make this recycling cost effective, for example
by enzyme immobilization. The current limitation of the process resides mainly in the
relatively low stability of the aldehyde substrate. In turn, the aldehyde might be conve-
niently generated in situ by means of an appropriate amino acid or amine oxidase. With
this aim in mind, several enzymes bearing appropriate specificities for tyrosine or tyr-
amine [4-(2-aminoethyl)phenol] are currently being tested.

Scheme 5 One-Pot Synthesis of (S)-Norcoclaurine[22]

OH NaOCl
50 mM potassium phosphate buffer (pH 7.0) H
O 37 oC, 1 h

NH2 99% O
OH OH
8 2

OH

NH2 HO
OH
1
NH
TfNCS, 37 oC, 30 min HO

HO

3 81%

(S)-Norcoclaurine (3); Typical Procedure for a One-Pot, Two-Step Synthesis:[22]

(4-Hydroxyphenyl)acetaldehyde (2) was prepared according to the method of Hazen and
co-workers:[23] A soln of tyrosine (8; 1.82 g, 10 mmol) was mixed with a soln of NaOCl
(0.752 g, 10.1 mmol) in 50 mM potassium phosphate buffer (pH 7.0; final volume 1 L) at
37 8C for 1 h. Once the tyrosine (8) had been completely converted into (4-hydroxyphenyl)-
acetaldehyde (2) (10 mM final concentration), dopamine (1; 1.53 g, 10 mmol), TfNCS
(10 mg, corresponding to a final concentration of 0.5 mM), and ascorbic acid (88 mg,
0.5 mmol) were added to the mixture, which was then incubated at 37 8C for 30 min.
Thereafter, activated carbon (Norit; 10 g) was added to the aqueous mixture. After
30 min shaking at rt, the mixture was filtered. The activated carbon adsorbent was recov-
ered and washed with distilled H2O (2  50 mL). It was then transferred into a conical flask
and treated with 0.005 M NaOH in 99% EtOH (100 mL). Desorption was carried out by shak-
ing for 2 h at 40 8C. The (S)-norcoclaurine-enriched organic fraction was neutralized with

for references see p 175

166 Biocatalysis 2.1 C—C Bond Formation

0.005 M HCl, concentrated to dryness under reduced pressure, and the resulting solid was
characterized by GC/MS, ESI/MS, 1H NMR, and 13C NMR; yield: 2.2 g (81%). The specific ro-
tations of MeOH solns of norcoclaurine were measured using a JASCO P-1030 polarimeter
at 25 8C. The enantiomeric excess of the product was determined by chiral HPLC using a
teicoplanin-based chiral selector.

2.1.5.2.2 Biocatalyzed Synthesis of 1-Substituted Tetrahydroisoquinolines

The stereoselectivity of the TfNCS-catalyzed reaction to yield S-configured products has

been thus probed toward a number of aldehyde substrates 10 including phenylacetalde-
hydes substituted with various electron-withdrawing or electron-donating groups, het-

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eroaromatic moieties, aromatic bicyclics, aliphatic (hetero)cycles, and aliphatic open-
chained compounds (Scheme 6).[17] All benzylic aldehydes are turned over at a comparable
rate whereas aliphatic compounds and unsubstituted phenylacetaldehyde are processed
at a slower rate. Small aldehydes, such as acetaldehyde and propanal, as well as Æ-substi-
tuted aldehydes and benzaldehyde, are not recognized as substrates. General procedures
for the synthesis of the aromatic- and aliphatic-substituted acetaldehydes comprise oxida-
tion of the corresponding alcohols 9 by chemical methods that require a final chromato-
graphic purification step and the use of dichloromethane as solvent. TfNCS-catalyzed re-
actions between dopamine (1) and aldehydes 10 were carried out on analytical scale and
were analyzed by quantitative chromatographic methods (HPLC-UV and LC/MS). No total
yields of isolated products 11 were reported.

Scheme 6 Synthesis of 1-Substituted Tetrahydroisoquinolines[17]

O
I OAc
AcO
OAc O
CH2Cl2
OH
R1 H
R1
9 10

R1 Aldehyde Generation Methoda Yield (%) Ref

[17]
4-HOC6H4 A 60
[17]
2-FC6H4 A 65
[17]
3-FC6H4 A 66
[17]
4-FC6H4 A 71
[17]
2-Tol A 66
[17]
3-Tol A 58
[17]
4-Tol A 57
[17]
4-MeOC6H4 B 69
[17]
3,4-(MeO)2C6H3 B 69
[17]
4-F3COC6H4 A 65
[17]
Ph commercially available 51
[17]
2-thienyl A 68
b [17]
1-naphthyl – 61
 2.1.5 Addition to C=N Bonds 167

R1 Aldehyde Generation Methoda Yield (%) Ref

[17]
Cy A 71

O O [17]
B 42

[17]
t-Bu commercially available 52
a
For details see procedures below for liquid alcohols (Method A)
and solid alcohols (Method B).
b
Synthesized by reduction of the corresponding Weinreb amide.

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HO NH2 O HO
TfNCS
+ H NH
HO R1 HO

R1
1 10 11

Aldehydes 10; General Procedure from Liquid Alcohols:[17]

Method A: The liquid alcohol 9 (R1 = 2-FC6H4, 3-FC6H4, 4-FC6H4, 2-Tol, 3-Tol, 4-Tol,
4-F3COC6H4, 2-thienyl, Cy; 1.0 equiv) was added to a suspension of Dess–Martin periodi-
nane (1.2 equiv) in CH2Cl2 (100 mM) under an argon atmosphere at rt. The mixture was
stirred for 0.5–20 h, while consumption of starting material was monitored by TLC. After
the reaction was complete, 1 M aq Na2S2O3 (equal volume to CH2Cl2) was added. After stir-
ring for 15 min, the phases were separated and the aqueous phase was extracted twice
with CH2Cl2. The combined organic layers were washed with 5% aq NaHCO3 and brine,
and dried (Na2SO4). The solvent was evaporated under reduced pressure and the residue
was purified by column chromatography to afford the analytically pure aldehyde.

Aldehydes 10; General Procedure from Solid Alcohols:[17]

Method B: The solid alcohol precursor 9 [R1 = 4-MeOC6H4, 3,4-(MeO)2C6H3, 5,5-dimethyl-1,3-
dioxan-2-yl; 1.0 equiv] was dissolved in CH2Cl2 (200 mM) and the soln was added to a sus-
pension of Dess–Martin periodinane (1.2 equiv) in CH2Cl2 (200 mM) under an argon atmo-
sphere at rt. The mixture was stirred for 0.5–20 h (TLC monitoring). Then, Et2O (equal vol-
ume to CH2Cl2), 1 M aq Na2S2O3, and sat. aq NaHCO3 (both equal volume to CH2Cl2) were
added. After stirring for 15 min, the phases were separated and the aqueous phase was
extracted twice with Et2O. The combined organic layers were washed with sat. aq
NaHCO3 and brine, and dried (Na2SO4). The solvent was evaporated under reduced pres-
sure and the residue was purified by column chromatography to afford the analytically
pure aldehyde.

1-Substituted Tetrahydroisoquinolines 11; General Procedure:[17]

To a 100 mM soln of Tris buffer (pH 7.0) containing dopamine (1) (1 mM; prepared from
the addition of 3 L of a 100 mM stock in MeOH) and the aldehyde 10 (1 mM; prepared
from the addition of 3 L of a 100 mM stock in MeOH) was added norcoclaurine synthase
(15 L, 25.2 g; 15 L of boiled enzyme for the negative control) for a total volume of
300 L. The reaction was incubated at rt. After 30 min, 1 h, 2 h, and 3 h (for the negative
control, only the 3 h time point was taken), a 60-L aliquot was removed and quenched by
the addition of MeOH (15 L; containing 200 M naphthalen-1-ylacetic acid as an internal
standard). After centrifuging at 13 000 rpm for 5 min, a sample (27 L) was injected onto
the HPLC (5–81% MeCN over 8 min in 0.1% aq TFA).

for references see p 175

168 Biocatalysis 2.1 C—C Bond Formation

2.1.5.3 Strictosidine Synthase Catalyzed Pictet–Spengler Reactions

The Pictet–Spengler reaction between tryptamine derivatives and aldehydes yields the
tetrahydro--carboline moiety, a heterocyclic framework of paramount interest in a
wide range of natural products and synthetic pharmaceuticals. At present, three strictosi-
dine synthases obtained from Rauvolfia serpentina (RsSTS), Ophiorrhiza pumila (OpSTS), and
Catharantus roseus (CrSTS) have been employed in biocatalysis, and demonstrated to pos-
sess diverse catalytic properties.[24–26] Recombinant RsSTS is the most highly characterized
enzyme and has been shown to display a strict substrate specificity and complete enantio-
selectivity. Recognition properties of wild-type strictosidine synthase enzymes are limit-
ed to a few tryptamine derivatives and secologanin (4), leading exclusively to 3Æ-(S)-stric-

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tosidine.[26,27] RsSTS can be expressed as a recombinant protein in Escherichia coli cells and
improved expression methods for His-tagged protein at high yield have been devel-
oped.[27] Kinetic parameters of RsSTS for the natural substrate tryptamine, under saturat-
ing secologanin concentrations, indicate a high substrate affinity and relatively high turn-
over rate (average values: KM = 5 M and kcat = 100 min–1 in 50 mM phosphate buffer at
pH 7.0 and 25 8C).[11] The properties of CrSTS are almost superimposable with those ob-
served for RsSTS. However, one rationally designed active site RsSTS mutant (namely
RsSTSVal208Ala)[28] and two CrSTS mutants (CrSTSVal214Met and CrSTSPhe232Leu), ob-
tained in a saturation mutagenesis study,[29] have demonstrated broader substrate specif-
icity and the ability to process A-ring-substituted tryptamines in their reaction with seco-
loganin (4) (see Scheme 7, Section 2.1.5.3.1). More recently, strictosidine synthase from
Ophiorrhiza pumila (OpSTS) has emerged as a most interesting active site natural variant
with respect to RsSTS in that it lacks His283 (His277 in RsSTS), a residue that is involved
in the hydrogen bonding network including the glucose moiety of secologanin (4). OpSTS
demonstrates a broader, and therefore potentially more useful, aldehyde substrate scope
compared to wild-type CrSTS and RsSTS.[30]

2.1.5.3.1 Synthesis of Strictosidine Analogues with Substituents on the Indole Unit

Catalytic methodologies have been developed to generate chiral tetrahydro--carbolines

13 from substituted tryptamines 12 and secologanin (4). The transformation of substitut-
ed tryptamines is achieved using wild-type RsSTS or the engineered RsSTSVal208Ala mu-
tant and CrSTSVal214Met or CrSTSPhe232Leu mutants, as shown in Scheme 7. Trypt-
amines with substituents in the 4–7 positions of the six-membered A-ring have been syn-
thesized separately or are commercially available, as described in the relevant refer-
ences.[28,29] Secologanin is obtained from Lonicera tatarica using the procedure reported
by Bernhardt and co-workers.[29] Selected examples of synthetic procedures are presented
below.
 2.1.5 Addition to C=N Bonds 169

Scheme 7 Synthesis of Strictosidine Analogues with Substituents on the Indole Unit[28,29]

R1
O H
R2
NH2 HO
R3 O OH
strictosidine synthase
+ O
N O O
R4 OH
H
R5 OMe OH
12 4

HO
R1 OH
HO

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2
R O OH
O
R3 NH

O
R4 N H
H H
R5
O
OMe
13

R1 R2 R3 R4 R5 Strictosidine Synthasea Ref

[29]
H H H H H CrSTSVal214Met, CrSTSPhe232Leu, CrSTSwt
[29]
H H Me H H CrSTSVal214Met
[29]
CH2OH H H H H CrSTSPhe232Leu
[29]
H H Cl H H CrSTSVal214Met
[29]
H H Br H H CrSTSVal214Met
[29]
H F H H H CrSTSwt
[29]
H Me H H H CrSTSwt
[29]
H OMe H H H CrSTSwt
[29]
H H F H H CrSTSwt
[29]
H H OH H H CrSTSwt
[29]
H H H F H CrSTSwt
[29]
H H H OMe H CrSTSwt
[29]
H H H H F CrSTSwt
[29]
H H H H Me CrSTSwt
[29]
H H H H OMe CrSTSwt
[28]
H H Me H H RsSTSVal208Ala
[28]
H H OMe H H RsSTSVal208Ala
[28]
H H F H H RsSTSVal208Ala, RsSTSwt
[28]
H H OH H H RsSTSVal208Ala, RsSTSwt
[28]
H H H Me H RSTSsVal208Ala, RsSTSwt
[28]
H H H OMe H RsSTSVal208Ala, RsSTSwt
[28]
H H H F H RsSTSVal208Ala, RsSTSwt
a
“wt” suffix denotes wild-type enzyme.

10-Methylstrictosidine (13, R1 = R2 = R4 = R5 = H; R3 = Me); Typical Procedure:[28]

5-Methyltryptamine hydrochloride (0.59 mg; 2.8 mol) and secologanin (4; 1.087 mg;
2.8 mol) were incubated in the presence of pure His-tagged RsSTSVal208Ala mutant
(560 g) in 50 mM potassium phosphate buffer (pH 7.0; 1.4 mL). The reaction was per-

for references see p 175

170 Biocatalysis 2.1 C—C Bond Formation

formed at 35 8C for 60 min. The mixture was then diluted with MeOH (3 mL), centrifuged,
and the supernatant was freeze-dried. H2O (3 mL) and 4.2 M Na2CO3 (1.5 mL) were added to
the residue. The mixture was saturated with N2 gas and heated for 4 h at 75 8C. NaCl was
added for saturation and the mixture was extracted with EtOAc (3  1 mL). The extracts
were combined and concentrated and the residue was dried.

(5R)-5-(Hydroxymethyl)strictosidine (13, R1 = CH2OH; R2 = R3 = R4 = R5 = H);

Typical Procedure:[29]
A soln of (R)-2-amino-3-(1H-indol-3-yl)propan-1-ol (190 mg, 1 mmol, 1 equiv) in MeCN
(25 mL; 5% v/v final concentration of organic solvent) and powdered secologanin (4;
388 mg, 1 mmol, 1 equiv) were added to stirred 25 mM aq sodium phosphate buffer

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(pH 7.0; 450 mL). Cell-extract containing CrSTSPhe232Leu[29] was then added (25 mL, 5%
v/v) and the reaction was continued at 30 8C for 46 h. Most of the H2O was removed using
a rotary evaporator and MeOH (two volumes) was added to precipitate the protein. Filtra-
tion and evaporation of the remaining solvent afforded a yellow oil. The oil was dissolved
in MeOH (40 mL) and purified by preparative HPLC (reversed-phase column, 1-mL injec-
tions, gradient 80:20 to 40:60 H2O containing 0.1% TFA/MeCN over 10 min). Analytically
pure (5R)-5-(hydroxymethyl)strictosidine was obtained as a pale yellow amorphous solid;
yield: 190 mg (35%); dr >99.5%.

10-Chlorostrictosidine (13, R1 = R2 = R4 = R5 = H; R3 = Cl); Typical Procedure:[29]

5-Chlorotryptamine hydrochloride salt (230 mg, 1 mmol, 1 equiv) and secologanin (4;
388 mg, 1 mmol, 1 equiv) were dissolved in stirred 25 mM aqueous sodium phosphate
buffer (pH 7.0; 450 mL). CrSTSVal214Met crude extract[29] was then added (25 mL, 5% v/v)
and the reaction was continued for 72 h at 30 8C. H2O was removed using a rotary evapo-
rator and MeOH (two volumes) was added to precipitate the protein. After sonication and
filtration, the solvent was evaporated to afford a yellow solid. The solid was dissolved in
MeOH (40 mL) and 10 mg of the product was purified by preparative HPLC (reversed-phase
column, 1-mL injections, gradient 80:20 to 10:90 H2O containing 0.1% TFA/MeCN over
15 min). Analytically pure 10-chlorostrictosidine was characterized by NMR; dr >99.5%.

2.1.5.3.1.1 Synthesis of Piperazinoindoles

RsSTS-mediated synthesis of novel strictosidine analogues containing the piperazinoin-

dole core instead of the tetrahydro--carboline (tryptoline) unit has been reported.[31] Pyr-
azino- and piperazino[1,2-a]indole systems deserve a special mention in view of their
prominent biological activities linked to advanced pharmaceutical product develop-
ment.[32] Current synthetic strategies for this class of compounds, especially for regio-
and enantioselective synthesis, require demanding chiral transition-metal catalysts.
Thus, the use of enzyme catalysis to gain access to the piperazinoindole scaffold is of
high potential interest. 2-(1H-Indol-1-yl)ethanamine (15, IEA) has been synthesized and
tested as a possible substrate.[31] Incubation of recombinant RsSTS wild-type protein with
this indole derivative, under neutral conditions in the presence of the monoterpenoid glu-
coside secologanin (4), results in excellent enzyme-dependent conversion (>95%), as
shown by HPLC (Scheme 8). In this transformation, the 3Æ-(S)-piperazinoindole strictosi-
dine analogue 16 [3Æ-(S)-PIS] is formed, in which the typical tetrahydro--carboline skele-
ton is replaced by the unusual piperazino[1,2-a]indole framework, representing the first
example of a piperazino monoterpenoid indole alkaloid. As is the case for the natural
RsSTS substrate tryptamine (6), the complete stereoselectivity of RsSTS delivers exclusive-
ly 3Æ-(S)-PIS (16) by condensation of 4 and 15. The use of the enzyme RsSTS presently pro-
vides exclusive access to 16.
 2.1.5 Addition to C=N Bonds 171

Scheme 8 Synthesis of a Piperazino[1,2-a]indole Strictosidine Analogue[31]

Br CN LiAlH4
NaH, DMF Et2O

N N N
H

NC
NH2
14 15 22%

O H
HO
O OH

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O
O O
OH
OMe OH HO
4
OH
HO O
50 mM potassium phosphate buffer (pH 7.0) OH
O
RsSTS, 28 oC, 2 h N NH

>95% O
H
H
O
OMe
16

2-(1H-Indol-1-yl)ethanamine (15); Typical Procedure:[31]

CAUTION: Solid lithium aluminum hydride reacts vigorously with a variety of substances, and
can ignite on rubbing or vigorous grinding.
A mixture of NaH (60% dispersion in mineral oil; 600 mg, 15 mmol) and 1H-indole (1.17 g,
10 mmol) in dry DMF (15 mL) was stirred at rt for 30 min before the addition of bromoace-
tonitrile (1.39 mL, 20 mmol) in dry DMF (2.0 mL). The mixture was heated to 65 8C for
30 min, stirred at rt for 6 h, and then treated with ice. The separated solid was filtered
off and purified by flash chromatography to give 2-(1H-indol-1-yl)acetonitrile (14). A sus-
pension of 2-(1H-indol-1-yl)acetonitrile (14; 780 mg, 5 mmol) in dry Et2O (50 mL) was
added slowly to a well-stirred slurry of LiAlH4 (540 mg, 14.2 mmol) in dry Et2O (25 mL).
The mixture was refluxed for 4 h and then poured into ice water. 1 M aq NaOH (15 mL)
was added and the aqueous phase was extracted with EtOAc (3  30 mL). The collected or-
ganic layers were dried and concentrated. The crude product was purified; yield: 22%
(based on 1H-indole).

[(3R,4S)-5-(Methoxycarbonyl)-4-{[(S)-1,2,3,4-tetrahydropyrazino[1,2-a]indol-1-yl]methyl}-3-
vinyl-3,4-dihydro-2H-pyran-2-yl]-d-glucopyranose [16; 3Æ-(S)-PIS]:[31]
The incubation mixture (5.0 mL), containing RsSTS (1.0 mg), 10 mM secologanin (4), 10
mM 2-(1H-indol-1-yl)ethanamine (15), and 50 mM potassium phosphate buffer (pH 7.0),
was incubated for 2 h at 28 8C with shaking. The reaction was terminated by addition of
MeOH (2 mL), and the mixture was extracted with EtOAc (3  15 mL). The collected organic
layers were dried, concentrated, and purified by preparative TLC; yield: >95%.

2.1.5.3.1.2 Synthesis of Azaindole-Containing Strictosidine Analogues

Azaindoles represent important heterocycles that are isosteric with indoles but display
improved water solubility and unique hydrogen-bonding properties.[32] Azaindoles can
be thus considered as powerful pharmacophoric structures, whose biological properties
also find significant representation in several natural products.[33]

for references see p 175

172 Biocatalysis 2.1 C—C Bond Formation

Both strictosidine synthases RsSTS and CrSTS have been shown to display reactivity
toward azaindole derivatives, namely azatryptamines.[27,34] The enzymes exhibit greater
tolerance for substrates with nitrogen at the 4- and 7-positions of the indole ring com-
pared with substrates with nitrogen at the 5- and 6-positions (Scheme 9). Although the
overall reactivity of azatryptamines in the Pictet–Spengler reaction is expected to be im-
paired due to the electron-deficient character of aza-substitution, 4- and 7-azatryptamines
17 (X = CH; Z = N) and 17 (X = N; Z = CH), respectively, are converted into the correspond-
ing azatetrahydro--carboline products 18. No product formation is observed with 5- and
6-azatryptamines. Overall, the catalytic efficiency using 7-azatryptamine (17, X = N; Z =
CH) as substrate is approximately 100-fold lower compared with tryptamine (6), and
4-azatryptamine (17, X = CH; Z = N) is 280-fold lower relative to tryptamine (6).

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Scheme 9 Synthesis of Azastrictosidines[27,34]

NH2
O H
HO
Z O OH
strictosidine synthase
+ O
O O
X N OH
H
OMe OH
17 4

HO
OH
HO O OH
O
Z NH

O
X N H
H H
O
OMe
18

Azastrictosidines 18; General Procedure:[34]

To a soln of strictosidine synthase (CrSTS or RsSTS) and secologanin (4; 10 mg, 25.8 mol)
in 50 mM phosphate-buffered saline (PBS; pH 7.0; 3 mL) was added 4- or 7-azatryptamine
17 (4.6 mg, 28.5 mol) in 50 mM PBS (pH 7.0; 1 mL). The mixture was incubated at 30 8C for
48 h. The product 18 thus obtained was not isolated and was directly used in biotransfor-
mation with Catharantus roseus cell-culture suspensions in order to obtain directly the cor-
responding alkaloid compound.

12-Azastrictosidine (18, X = N; Z = CH); Typical Procedure Using Nickel Affinity Resin Im-
mobilized RsSTS:[27]
Prepurified His6-RsSTS was dissolved in 50 mM potassium phosphate buffer (pH 7.0) and
immobilized on the Ni-NTA column. 7-Azatryptamine (17, X = N; Z = CH; 0.4 g, 2.5 mmol)
and secologanin (4; 88% purity; 1.1 g, 2.5 mmol) were each dissolved in 50 mM potassium
phosphate buffer (200 mL) separately. Both solns were pumped through the column at a
speed of 0.5 mL • min–1 at 4 8C. After overnight running, the combined aqueous solns were
collected and freeze dried to yield a dry powder, which was extracted with MeOH (leaving
inorganic salts). The organic phase was concentrated under reduced pressure, and the
crude product (1.3 g) was subjected to preparative HPLC to afford pure 12-azastrictosidine
(18, X = N; Z = CH); yield: 0.7 g (50%).
 2.1.5 Addition to C=N Bonds 173

2.1.5.3.2 Biocatalyzed Synthesis of -Carbolines Using Substituted Aldehydes

The X-ray structure of RsSTS in complex with strictosidine (Scheme 3, Section 2.1.5.1.2)
suggests that the glucose moiety forms hydrogen bonds with histidine residues 277 and
307. These interactions appear to govern the recognition of the substrate and account for
the high substrate selectivity of strictosidine synthases. However, glucose removal from
the dihydropyran scaffold has been shown to generate an array of enzyme-accessible se-
cologanin aglycone analogues. These analogues have been used to assess the stereochem-
ical preference of strictosidine synthase, demonstrating that the enzyme may accept
more than one unnatural aldehyde stereoisomer. Strictosidine synthase from Ophiorrhiza
pumila (OpSTS) has been demonstrated to be capable of utilizing a broad range of simple

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achiral aromatic and aliphatic aldehydes to form an array of novel tetrahydro--carbo-
lines.

2.1.5.3.2.1 Synthesis of Tetrahydro--carbolines Using Secologanin Aglycone Analogues

Synthesis or semi-synthesis of secologanin analogues has not been systematically investi-

gated. An interesting approach has been developed by Bernhardt and co-workers,[35] in
which compounds with alternate stereochemistry are used to obtain the des-vinyl agly-
cone O-analogues 19 of secologanin. Access to the acetal-protected des-vinyl O-analogues
19 is achieved by Tietzes tandem Knoevenagel–hetero-Diels–Alder reaction.[35]
The aldehydes 19 thus obtained have been assayed with strictosidine synthase in the
presence of tryptamine (6) and monitored for appearance of the products 20 by high-res-
olution electrospray ionization (ESI) mass spectrometry (Scheme 10). This investigation
demonstrates the capability of OpSTS to process these substrates independently of the
configuration at the C2 carbon. In fact, both the 2,4-trans configuration, found in the nat-
ural substrate secologanin (4), and the unnatural 2,4-cis configuration are accepted by
strictosidine synthase. For smaller O-substituents (R1 = Et, iBu, t-Bu), the trans/cis selectiv-
ity is low (5–10% de), whereas for 19 (R1 = Cy) the selectivity for the trans configuration is
significantly higher (>60%). Therefore, increased acetal substituent size appears to be cor-
related with a substrate preference for the natural trans stereochemistry.

Scheme 10 Synthesis of Tetrahydro--carbolines Using Secologanin Aglycone Analogues[35]

NH2
O H

OR1
sepharose-immobilized OpSTS
∗
+
O O
N
H
OMe
6 19

OR1
NH
∗
O
N H
H H
O
OMe
20
R1 = Et, iBu, t-Bu, Cy

for references see p 175

174 Biocatalysis 2.1 C—C Bond Formation

2.1.5.3.2.2 Synthesis of Tetrahydro--carbolines Using Achiral Aliphatic and

Aromatic Aldehydes

A representative array of achiral aldehydes 21 have been screened in the OpSTS-catalyzed

reaction with tryptamine (6) (Scheme 11).[36] All aldehydes tested are commercially avail-
able. The resulting tetrahydro--carboline products 22 were identified by mass spectrom-
etry and by comparison with a conveniently prepared racemic authentic standard. OpSTS
turns over aldehydes 21 although at a 1000-fold slower rate with respect to the natural
substrate. Kinetic analysis clearly indicates that OpSTS prefers aldehydes unsubstituted
at the Æ-position.[36] The enantiomeric excess of the products of the OpSTS-catalyzed reac-
tions is >98%, as evidenced by chiral HPLC. The absolute configuration was determined

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for tetrahydro--carboline 22 (R1 = Bu) with this reaction being carried out on a milligram
scale using OpSTS immobilized on a solid support and the product being compared to an
authentic (3S)-standard obtained by an organocatalytic method.

Scheme 11 Synthesis of -Carbolines Using Achiral Aliphatic and Aromatic

Aldehydes[36]

NH2

O
NH
OpSTS
+ H
N N R1
H R1 H
6 21 22

R1 ee (%) Ref
[36]
tetrahydropyran-4-yl >98
[36]
Cy >98
a [36]
iPr –
[36]
Ph >98
[36]
Bu >98
a
Not determined.

1-Substituted Tetrahydro--carbolines 22; General Procedure:[36]

OpSTS (2 U total activity, where 1 U = 1 mol strictosidine formed per minute) was immo-
bilized on cyanogen bromide activated Sepharose 4B (1.4 mL) according to the manufac-
turers instructions (71–7086–00 AD, GE Healthcare, Uppsala, Sweden). Immobilized
OpSTS was active, where 1 U total activity was immobilized on the resin (51% recovered
yield). Enzyme-catalyzed reactions (total volume 4 mL) contained 0.025 M sodium phos-
phate buffer (pH 7.0), tryptamine (6; 1 mM), aldehyde 21 (5 mM), and immobilized
OpSTS (0.02 U; based on strictosidine forming activity). After incubation for 4 d at 100
rpm and 30 8C, the Sepharose beads were settled and the supernatant was extracted with
CH2Cl2 (3 ) and with EtOAc (1 ). Immobilized OpSTS could be reused at least three times
with no noticeable loss of activity, similar to Zenks previous observations with CrSTS.[37]
The combined organic layers were concentrated under reduced pressure and the residue
was dissolved in MeCN/H2O (1:1; 2 mL). The tetrahydro--carboline product was isolated
by semi-preparative HPLC (gradient 30–100% MeCN in H2O containing 0.1% TFA over
20 min). The collected fractions were concentrated under reduced pressure and the resi-
due was separated by chiral HPLC. 1-Pentyltetrahydro--carboline (22, R1 = Bu) was isolat-
ed from a scaled-up reaction (mg-scale) using immobilized OpSTS.
 References 175

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