Tural Products
Tural Products
Tural Products
SURFACE
12-2011
Recommended Citation
Pinto, Atahualpa, "Synthesis and Biosynthesis of Polyketide Natural Products" (2011). Chemistry -
Dissertations. 181.
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Abstract
organic chemistry and biochemistry have found in the last two decades a fertile common
ground in the area pertaining to the biosynthesis of natural products. Both disciplines
field. Our contributions to this interdisciplinary pursuit have been confined to the
validate complex metabolic processes. One such example pertained to the uncertainty
pathway of the marine polyketide spiculoic acid A. The molecule's key intramolecular
and tested its viability to undergo a mild chemical transformation to spiculoic acid A. In
outcome of the enzyme's activity is divided between hydrolysis and macrocyclization and
four enantioenriched NAC-thioester analogs and assayed them with overexpressed DEBS
TE, the canonical thioesterase of polyketide natural products, to gain insight into the
effect key structural and stereochemical elements of the polyketide substrate have in the
By
Atahualpa Pinto
Department of Chemistry, Syracuse University
B.S. Chemistry, SUNY College of Environmental Science and Forestry, Syracuse, NY
DISSERTATION
Submitted in partial fulfillment of the requirements for the
Doctor of Philosophy in Chemistry in the
Graduate School of Syracuse University
December 2011
Copyright© 2011
by
Atahualpa Pinto
All rights reserved.
For my sisters
iv
Table of Contents
v
2.2.2.2. Strategy II 41
2.2.2.3. Strategy III 42
2.2.2.4. Strategy IV 43
2.2.3. Syntheses of 2.39, the putative polyketide intermediate of spiculoic 44
acid A
2.2.3.1. Strategy I 44
2.2.3.2. Strategy II 48
2.2.3.3. Strategy III 50
2.2.3.4. Strategy IV 52
2.3. Conclusions 55
2.3.1. A spontaneous cycloaddition reaction is not operative 55
2.3.2. A non-canonical domain is likely responsible for the olefin 58
regioisomerization
2.4. References 60
2.5. Experimental section 67
2.5.1. General methods 67
2.5.2. Experimental procedures 68
2.5.3. References 94
2.5.4. Selected 1D and 2D NMR spectra 95
vi
3.2.3. Enzymatic assay of enantioenriched NAC-thioester substrates 134
3.2.4. Hydrolysis of macrolactone standard 3.42 136
3.3. Conclusions 137
3.3.1. Absolute stereochemistry is a key factor driving enzyme specificity 138
3.3.2. Applications of DEBS TE in synthesis and biosynthesis 139
3.4. References 142
3.5. Experimental section 148
3.5.1. General methods 148
3.5.2. Experimental procedures 150
3.5.2.1. Macrolactone standards 150
3.5.2.2. Enantioenriched NAC thioesters 169
3.5.2.3. Transformation, expression and purification of wild-type 194
DEBS-TE
3.5.2.4. Kinetic analysis of the DESB TE 195
3.5.2.5. Hydrolytic assay of anti-3.42 with DEBS TE 196
3.5.3. References 196
3.5.4. Selected 1D and 2D NMR spectra 197
List of Figures
Figure 1.1. Structural diversity of the alkaloid class of natural products 3
Figure 1.2. Daphniphyllum alkaloids 3
Figure 1.3. Proposed biosynthetic hypothesis of daphniphyllum alkaloids 5
Figure 1.4. Synthesis of secodaphniphylline alkaloids 1.11 and 1.12 6
Figure 1.5. Mevalonic acid pathway and the biosynthesis of terpenoid natural 7
products
Figure 1.6. The furanocembranoid terpenoids 8
Figure 1.7. Proposed biosynthesis of furanocembranoid terpenoids 9
Figure 1.8. Biomimetic conversion of 1.13 to 1.14 by an Achmatowicz-[3+2] 9
dipolar cycloaddition sequence
Figure 1.9. Polyketide natural products are complex and structurally diverse 10
Figure 1.10. PKS chain elongation of a malonyl-CoA derivative displaying the 11
consensus mechanism for the essential KS-AT-ACP catalytic triad
Figure 1.11. Biosynthetic proposals for the origins of polyether biotoxins (e.g. 13
monensin A (1.18)). (A) Cane-Celmer-Westley and (B) Townsend-
vii
Basak-McDonald hypotheses
Figure 1.12. Precursor directed biosynthesis of erythromycin analogs 15
Figure 1.13. CurM, the termination module of the curacin A PKS, contains a 16
sulfotransferase domain (ST) flanked by an ACP and a TE domain
Figure 1.14. Substrate studies of curacin A's sulfotransferase (ST) and 17
thioesterase domains
Figure 1.15. Synthesis of phosphonate 1.25, an irreversible inhibitor of the 17
pikromycin thioesterase domain
Figure 1.16. Crystal structure of phosphonate 1.25 (circled) bound to the active 18
site of the pikromycin thioesterase domain
Figure 2.1. A) Diels-Alder reaction B1) Orbital correlation diagram of a 25
normal electron demand Diels-Alder reaction. B2) Orbital
correlation diagram of a inverse electron demand Diels-Alder
reaction. The orbitals in red represent the frontier molecular
orbitals. C1) Diastereo- and regioselectivity of a normal electron
demand Diels-Alder reaction. C2) Diastereo- and regioselectivity
of an inverse electron demand Diels-Alder reaction
Figure 2.2. Total syntheses of natural products featuring the versatility of the 28
Diels-Alder reaction
Figure 2.3. Polyketide natural products with cyclic elements derived from a 29
putative Diels-Alder reaction
Figure 2.4. SpnF is the first example of a putative Diels-Alderase that 29
exclusively yields a single diastereomer
Figure 2.5. trans-Hydrindane polyketides 30
Figure 2.6. Spiculoic Acid A (2.11) 31
Figure 2.7. Spiculoic acid A linear chain and PKS extender unit framework. 32
Thioesterase (TE) hydrolysis is hypothesized to be followed by an
intramolecular Diels-Alder cycloaddition
Figure 2.8. Spiculoic acid A IMDA cyclization may take place during 33
polyketide chain extension
Figure 2.9. Hypothetical biosynthetic routes: A) Abnormal PKS-bound 34
dehydration. B) Normal dehydrated product catalyzed by canonical
DH domains
Figure 2.10. Synthetic strategies employed for the formation of spiculoic acid 35
A’s trans-hydrindane framework
Figure 2.11. An intermediate regioisomerization step may take place prior to 36
cyclization by a putative cyclase
Figure 2.12. Steps involved in the conversion of linear polyketide chains to 2.11. 37
A) IMDA reaction of putative intermediate 2.17 directly yields
viii
2.11. B) IMDA reaction of 2.18 may produce an enol intermediate
2.19 that after equilibration may yield product 2.11
Figure 2.13. Cyclic endo products from the IMDA cycloaddition of 2.18. 39
Dienophile-diene facial selectivity has the potential of producing
two diastereomers which are expected to undergo an enol-keto
tautomerization to products
Figure 2.14. Inseparable isomers of dienyl compounds 2.28-2.30 were identified 46
by 1D and 2D NMR spectroscopy
Figure 2.15. Inseparable isomers of trienyl compounds 2.41-2.43 were identified 46
by 1D and 2D NMR spectroscopy
Figure 2.16. Key olefination strategies in the synthesis of pinnaic acid 52
Figure 2.17. Modeled analogs employed for the orbital coefficient analysis of 56
the IMDA of linear precursor (E)-2.68 in route to spiculoic acid A
Figure 2.18. Ab initio analysis of regioisomerization of linear precursor 2.39 57
Figure 2.19. Canonical mechanism of dehydration in PKS DH domains. 58
Figure 2.20. Modules 7 and 9 from the rhizoxin D biosynthetic pathway 59
Figure 3.1. Macrolactones can be synthesized by numerous (A) acid activation 114
and (B) alcohol activation methods
Figure 3.2. Macrolide polyketides share a common biosynthetic origin which is 115
capable of generating molecules of great structural diversity and
assorted therapeutic applications
Figure 3.3. Erythromycin biosynthesis is performed by a polyketide synthase 116
(PKS). Domains represented: KS (ketosynthase), ACP (acyl carrier
protein), AT (acyltransferase), DH (dehydratase), KR
(ketoreductase), (KR) (inactive ketoreductase), ER
(enoylreductase), and TE (thioesterase)
Figure 3.4. DEBS TE crystal structure (PDB ID: 1KEZ). (A) Secondary 117
structure representation of the homodimer. (B) Van der Waals
space-filling model. The substrate channel traversing the enzyme is
conspicuous. (C) Topographical map of DEBS TE. The
connectivity of the enzyme's α-helices (red rods) and β-strands
(yellow arrows) is shown. The catalytic residue (red dots) and their
approximate location is also shown
Figure 3.5. Canonical mechanism of the TE domain. Common among α/β 118
hydrolase family, it involves a classic charge relay mechanism.
The active site serine is deprotonated in the loading step and a new
O-acyl-enzyme intermediate is formed upon its attack on the
upstream S-acyl-ACP. The conformation of the substrate in the
channel may lead to either a regioselective intramolecular
macrocyclization or intermolecular attack by a molecule of water to
yield a seco-acid. The latter has been observed as an occurrence of
ix
in vitro experiments
Figure 3.6. DEBS TE competently loads a wide range of linear chain lengths 118
Figure 3.7. Putative side reactions of SNAC 6dEB substrate 3.10 meant to 120
illustrate the difficulties associated with its use as a probe for
thioesterase macrocyclization. Under thermodynamic control
hemiketalization (structures 3.8 and 3.9) would likely take place.
In addition, formation of a δ-lactone would be kinetically favored
to yield structure 3.11
Figure 3.8. Substrate analogs designed by Wang et al. to probe the kinetics of 121
the loading step with wild-type polyketide thioesterases (Deb,
Pim, Epo). 7-Aminoheptanoate (3.12) is highlighted
Figure 3.9. 6dEB analog 3.13 designed to explore the DEBS TE 121
regiochemistry of cyclization. Linear 6dEB is shown for ease of
comparison with our synthetic target
Figure 3.10. Treatment of isolated DEBS TE with analog 3.13 generated a 122
mixture of products (as monitored by LCMS)
Figure 3.11. We propose that the absolute stereochemistry of the terminal O- 123
nucleophile (highlighted in red) determines the outcome of the TE-
catalyzed macrolactonization
Figure 3.12. LC traces and product distribution from enzymatic assays with wild 135
type DEBS TE and enantioenriched NAC substrates
Figure 3.13. Results of the enzymatic hydrolysis assay of macrolactone standard 137
3.42 by the DEBS TE. (A) Standard anti-seco-3.41; (B)
Hydrolysis assay at180 min; (C) negative control with no enzyme
List of Tables
Table 3.1. Calculated specificity constants (k cat /K M ) for the reaction of 195
enantioenriched substrates ent-3.29, ent-3.30, 3.29 and 3.30 with
the DEBS TE (values represent the mean ± standard error)
List of Schemes
Scheme 2.1. Strategy I: Chiral retrosynthetic strategy to 2.11 40
Scheme 2.2. Strategy II: Achiral retrosynthetic strategy to 2.11 42
Scheme 2.3. Strategy III: Convergent and achiral retrosynthetic strategy to 2.11 43
Scheme 2.4. Strategy IV: Redesigned convergent and achiral retrosynthetic 44
strategy to 2.11
Scheme 2.5. Synthesis of aldehyde 2.43 45
Scheme 2.6. Asymmetric methodologies employed in forming the C4-C5 bond 47
x
with aldehyde 2.43
Scheme 2.7. Synthetic studies towards the C3 chain extension of aldehyde 2.43 48
Scheme 2.8. Synthesis of carboxylic acid 2.57 50
Scheme 2.9. Synthesis of advanced intermediates to employ for the olefination 51
of 2.30
Scheme 2.10. Synthesis of 2.39 53
Scheme 2.11. Conditions attempted for the IMDA cyclization of aldehyde 2.39 to 54
2.11. The molecule’s equilibrium will be dominated by the
presence of its Z or E enolate tautomers. Only the E enolate will be
reactive towards an IMDA. Formation of the bicyclic IMDA
adduct will be dependent on accessing the conjugated E enol as this
conformation will favor the intramolecular approach of the reactive
moieties
xi
List of Abbreviations
AT Acyltransferase
ATP Adenosine-5'-triphosphate
CoA Coenzyme A
DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene
DCM Dichloromethane
DH Dehydratase
DIPEA Diisopropylethylamine
DMAP 4-Dimethylaminopyridine
DMF Dimethylformamide
DTP 2,2'-Dithiopyridine
EB Elution buffer
xii
EI Electron impact ionization
ER Enoylreductase
HSNAC N-Acetylcysteamine
IC Inhibitory concentration
IR Infrared
KS Ketosynthase
KR Ketoreductase
LB Luria-Bertani/Lysing broth
xiii
LLS Longest linear sequence
MOM Methoxymethyl
OD Optical density
PA para-Anisaldehyde
PhMe Toluene
PhH Benzene
PMB para-Methoxybenzyl
PMP para-Methoxyphenyl
xiv
TBAF Tetrabutylammonium fluoride
TBS tert-Butyldimethylsilyl
TE Thioesterase
TEA Triethylamine
THF Tetrahydrofuran
TIPS Triisopropylsilyl
TMP 2,2,6,6-Tetramethylpiperidine
UV Ultraviolet
xv
1
1.1. Introduction
and serendipitous discoveries, has been inexorably linked to the understanding of the
chemical processes of organisms. This connection however was not born of a natural
progression but of the separate growth of the fields of organic chemistry and
biochemistry from pioneering works of 19th century scientists such as Friedrich Wöhler
and Eduard Buchner.2 Their discoveries regarding the synthesis of urea3 and cell-free
fermentations,4, 5
respectively, had profound repercussions to the established view of
vitalism and its élan vital so championed by the likes of Berzelius and Pasteur.6, 7
It was these momentous scientific developments that set the tone and laid the
foundation for research endeavors in the 20th century to gradually develop free from the
confines of doctrine. For the chemical and biochemical disciplines this path would allow
eventual work by Hans Krebs and Kurt Henseleit to identify the metabolic origins and
The maturation of the synthetic chemistry discipline owes to the isolation and
both prokaryote and eukaryote natural sources. Limited access to sufficient quantities of
2
compound from their biological sources, and the associated difficulties in providing
sustainable growing and culturing conditions in the laboratory have historically been the
motivation for the development of synthetic routes and scale up methodologies for
underlying motivation amongst synthetic chemists has been the remarkable creative
potential provided by the enormous structural diversity found within the various
relationship with the biosynthesis of natural products has opened new avenues for
exploration by the synthetic chemist. This chapter will review some notable strategies
and contributions synthetic chemists have had on the advancement of the biosynthetic
field with a particular focus on the formulation and testing of biosynthetic hypotheses and
the development of biosynthetic precursors for the in depth study of individual enzymatic
mechanisms.
natural products is the ideal path to take to decipher their assembly, realistically the
process is time consuming and complex endeavor. Given this, the unavailability of a
natural product's biosynthetic gene cluster has not hampered the attuned chemist from
proposing reasonable steps to rationalize its origins. It is the purpose of this section to
highlight the complementary role synthetic experiments and/or chemical hypotheses have
1.2.1. Alkaloids
diverse pathways, unique building blocks, and a plethora of strategies9, each tailored to
produce exquisitely complex structures (Figure 1.1). The origin of nitrogen incorporation
transamination.10
plant family Daphniphyllaceaea (Figure 1.2). Suzuki et al. were first to scrutinize the
precursor. Beyond this however they were unable to provide a satisfactory hypothesis to
explain the steps leading to the formation of its characteristic polycyclic scaffold.
This challenge was seized 15 years later by the Heathcock laboratory.19-23 With the
sought inspiration from the elegant simplicity of Nature. They reasoned a pathway from
dialdehyde 1.1, which would undergo the formation of azadiene 1.2 by amination with
amino acid residue localized in the enzyme's active site, to yield the reactive and
reactions24, compounds such as enamine 1.4 may undergo conversion to 1.5, after which
an aminal hydrolysis and rearrangement of the bicycle leads to key azadiene 1.7. This
structural motifs in the compound class. The biosynthetic hypothesis informed and
served as a blueprint for the design of a synthetic strategy. Their approach was simplified
The decision would permit direct access to a reactive enamine, eliminating the
intermediate tautomerization steps and setting the stage for the ensuing intramolecular
cyclization cascade. Noteworthy was their chance discovery of the contrasting effects an
alkylamine and ammonia showed in the mechanism leading to the structure of the
isopropenyl side chain. Whereas with ammonia a 1,5 proton transfer resulted in the
expected isopropenyl side chain of 1.11, methyl amine however resulted in the isolation
mechanism to be operative.
for daphniphyllum biosynthesis have not yet been isolated and characterized, yet the
7
sound mechanistic insight and elegance in design that resulted from these studies will be
hard to ignore and will likely remain as the point of reference for future analyses.
1.2.2. Terpenoids
Terpenoids9 are an enormous and highly varied family of natural products that have
traditionally been associated with multicellular organisms including plants and animals.
These compounds are not only of great interest to researchers for their physiologically
active properties but for the tremendous complexity produced from the cationic
isopentenyl pyrophosphate (IPP). In animals these key metabolites are the products of
FIGURE 1.5. Mevalonic acid pathway and the biosynthesis of terpenoid natural products.
1.2.2.1. Furanocembranoids
bipinnatin J and intricarene by the Trauner laboratory.25, 26 The work perfectly illustrates
8
and how organisms generate enormous structural diversity and complexity through
choice modifications of key metabolites. The strategy is proposed to yield a few reactive
1.6).
structurally related macrocyclic diterpenoids isolated from gorgonian corals of the genus
remained an elusive, unexplored topic. That is until the elegant work of Trauner and co-
regioselective epoxidation of the furan with mCPBA mediated by the adjacent furfuryl
grants access to oxidopyrillium dipole 1.17 which, by heating to 150 °C, initiates the
expected [3+2] dipolar cycloaddition thus granting access to 1.14 in 26% yield. The high
10
1.2.3. Polyketides
FIGURE 1.9. Polyketide natural products are complex and structurally diverse.
known as a polyketide synthase (PKS). 36 Several varieties of PKSs have been identified,
antibiotics and polyether biotoxins to name a few. The process takes place by iterative
domains arranged into repetitive units or modules: ketosynthase (KS), acyl transferase
11
(AT) and acyl carrier protein (ACP); the number of modules present in the PKS dictates
the length of the carbon chain of the end product. In addition, the modules may also
hydroxy units (ketoreductase – KR); conjugated E alkenes (dehydratase – DH); and fully
saturated products (enoyl reductase – ER). Release from the enzyme complex is
respectively.
FIGURE 1.10. PKS chain elongation of a malonyl-CoA derivative displaying the consensus mechanism for
the essential KS-AT-ACP catalytic triad.
12
chemically reasonable pathways that have gone without independent corroboration from
among experimentally supported hypotheses, the best and only solution in discriminating
between these is the characterization of the natural product's biosynthetic pathway. This
rationalize the molecule's origins as the product of an elegant intramolecular epoxide ring
biosynthetic step is its application in the facile synthesis of complex polyether natural
confirmed the potential of their proposed step to the synthesis of 1.18 by employing PCC
FIGURE 1.11. Biosynthetic proposals for the origins of polyether biotoxins (e.g. monensin A (1.18)). (A)
Cane-Celmer-Westley and (B) Townsend-Basak-McDonald hypotheses.
Refutal of one of these hypotheses required the isolation, sequence and expression of
intermediates in the pathway. Work by Leadlay and coworkers44 provided the first
1.3. Precursor and analog design for enzymology of polyketide natural products
Another key area in the field of biosynthesis where synthetic chemists have made
profound contributions has been that of enzymology and the understanding of the
The high point of the application of the experimental possibilities has been reached with
biosynthetic pathways.".45 Aside from the obvious goal of this concept which is to
products was designed by the Khosla group in 1997.46 Employing the canonical
erythromycin PKS pathway, they generated a null mutant by deleting the ketosynthase
15
domain of module 1 (KS1), rendering the pathway dependent on the exogenous supply of
The study served to underline the capacity of the pathway to accept and produce
interesting analogs, and moreover to reveal the tolerance of downstream enzymatic steps
and their products. Chemical synthesis is an excellent tool in the elucidation of these
thioesterase domain (TE). These catalytic domains mediate the cleavage of the thioester
bond of the upstream ACP-bound mature polyketide and concomitantly produce either a
by a terminal olefin moiety in place of the canonical seco-acid (Figure 1.13). The
molecule's gene cluster was isolated and sequenced by Gerwick and coworkers to reveal
a termination module containing a putative sulfotransferase domain (ST) among the ACP
and TE domains.48
FIGURE 1.13. CurM, the termination module of the curacin A PKS, contains a sulfotransferase domain
(ST) flanked by an ACP and a TE domain
From analysis of the predicted mature polyketide the ST was hypothesized to act on
to formation of 1.21 however, remained unclear. To determine ST domain's role and its
timing within the termination of the mature polyketide leading to 1.21, Sherman and
biochemical analyses of 1.22 with recombinant ST and TE domains ratified the sequence
prior to the action of the thioesterase domain which catalyzes a hydrolysis reaction and
FIGURE 1.14. Substrate studies of curacin A's sulfotransferase (ST) and thioesterase domains.
represents a potential new and useful tool for the inclusion of terminal olefins in
gain structural information that would guide their desired rational engineering of a PKS
FIGURE 1.15. Synthesis of phosphonate 1.25, an irreversible inhibitor of the pikromycin thioesterase
domain.
18
The structural characteristics of the enzyme that stabilize the substrate in the channel
remained unclear. Initially thought to be due to various hydrophilic residues lining the
(Figure 1.16) where they observed that the co-crystallized phosphonate inhibitor made
few specific contacts with the channel, suggestive of hydrophobic interactions being key
water molecules in the channel's exit was suggested to be the underlying cause of the
FIGURE 1.16. Crystal structure of phosphonate 1.25 (circled) bound to the active site of the pikromycin
thioesterase domain.
1.4. Conclusion
Natural product biosynthetic studies have experienced enormous growth in the last 20
years by the swift technological advances and invaluable contributions of the synthetic
chemistry and biochemistry disciplines. These, have been employed effectively in the
showcase the seamless relationship the chemical sciences, whether synthetic chemistry or
specificity and mechanism of the DEBS-TE, the thioesterase involved in the cyclization
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Spencer, J. B.; Staunton, J.; Leadlay, P. F., Accumulation of an E,E,E-triene by the
monensin-producing polyketide synthase when oxidative cyclization is blocked. Angew.
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Sherman, D. H.; Gerwick, W. H., Biosynthetic Pathway and Gene Cluster Analysis of
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Decarboxylative Chain Termination Preceded by O-Sulfonation in Curacin A
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24
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25
2.1. Introduction
The Diels-Alder reaction, discovered by Otto Diels and Kurt Alder in the early 20th
century,1 has become a powerful tool in the synthetic chemist's collection of reactions.
FIGURE 2.1. A) Diels-Alder reaction B1) Orbital correlation diagram of a normal electron demand Diels-
Alder reaction. B2) Orbital correlation diagram of a inverse electron demand Diels-Alder reaction. The
orbitals in red represent the frontier molecular orbitals. C1) Diastereo- and regioselectivity of a normal
electron demand Diels-Alder reaction. C2) Diastereo- and regioselectivity of an inverse electron demand
Diels-Alder reaction.
26
breakthroughs which laid down the sophisticated mechanistic foundation necessary for its
the Diels-Alder is typified by a cyclic transition state where, in a single concerted step,
three π bonds are broken concomitantly forming two σ bonds and a π bond (Figure 2.1-
A). The key orbital interactions involving the reaction's components are their highest
occupied and lowest unoccupied molecular orbitals (HOMO and LUMO respectively) as
changes the energetic landscape of the reaction as regio- and stereoselective pathways to
electronic effects in the transition state effectively eliminates the majority of these
putative pathways. On account of the reaction being concerted and stereospecific, only
thermodynamically controlled product, obtained from an exo transition state, may be also
isolated owing to favorable steric interactions of reactants. Of note is also the marked
effect the polarizing substituents have on the orbital coefficients of the reaction's
27
components and thus its regiochemical outcome. This effect creates geometric
asymmetries in the key frontier molecular orbitals which affect the synchronicity of bond
formation at the transition state.6 The favored transition state will then be that which
The flow of electrons determines how the Diels Alder reaction is classified. A
rich diene and an electron-poor dienophile and, as expected, the HOMO of the diene and
the LUMO of the dienophile are its key frontier molecular orbital interactors (Figure 2.1-
B1 and C1). The opposite polarization is also applicable and the reaction is known as an
Shown in Figure 2.2, are a few relevant cases where a Diels-Alder cycloaddition
FIGURE 2.2. Total syntheses of natural products featuring the versatility of the Diels-Alder reaction.
containing the familiar scaffold set in motion the intense search for the enzymatic step
believed to be involved in each individual pathway. The few results thus far have been
subject to intense debate. In vitro assays with enzyme preparations (crude or otherwise)
macrophomate and lovastatin synthases, have shown product mixtures consistent with a
FIGURE 2.3. Polyketide natural products with cyclic elements derived from a putative Diels-Alder reaction.
Most recently, a study on the cyclization of the polyketide spinosyn A led by Liu et
al. successfully cloned and isolated gene product SpnF, a putative Diels-Alderase that
catalyzes an exclusive transannular cyclization of 2.7 in vitro (Figure 2.4) and produced a
FIGURE 2.4. SpnF is the first example of a putative Diels-Alderase that exclusively yields a single
diastereomer.
The clear implication of this discovery is that a concerted reaction mechanism may be
taking place. Carefully crafted mechanistic studies and high resolution structural data
indeed the the mechanism is found to be pericyclic, the often-quoted enzyme catalysis
ionophore antibiotic indanomycin has been isolated,30-32 its study has yet to shed light on
the chemistry involved in trans-hydrindane formation from the putative linear polyketide
precursor.
Figure 2.6), a natural product isolated from tissue extracts of the Caribbean sponge
product33. The nature of this enzyme-bound linear chain remains a subject of debate
since its biosynthetic gene cluster has yet to be isolated, sequenced and characterized; the
complexity of both the sponge microbiome and the concomitant metagenomic mining
The linear chain of 2.11 is hypothesized33 to be the product of a type I PKS chain
FIGURE 2.7. Spiculoic acid A linear chain and PKS extender unit framework. Thioesterase (TE) hydrolysis
is hypothesized to be followed by an intramolecular Diels-Alder cycloaddition.
The timing of the cyclization leading to spiculoic acid A remains unknown. This step
intramolecular cyclizations42, 43
of PKS-bound intermediates poses the question of
whether this step may take place while bound to the PKS enzyme. In this case, the lower
would be more reactive in a Diels-Alder reaction than the seco-acid of the hydrolyzed
FIGURE 2.8. Spiculoic acid A IMDA cyclization may take place during polyketide chain extension.
polyketides and fatty acids is known to consistently produce double bonds in conjugation
with the acyl-enzyme intermediate. If this logic is applied to the biosynthesis of the
2.11 (Figure 2.9-A). The HOMO diene -LUMO dienophile energy gap of the mature enzyme-
Consistent with this view have been the elevated temperature employed to effect the
IMDA in the synthetic studies and total syntheses of 2.1150-53 (Figure 2.10). Baldwin et
al. showed that under the reaction conditions selected to install the molecule's dienophile
react at elevated temperatures (70 °C). These studies suggest a couple of interpretations:
either the HOMO diene -LUMO dienophile energy gap54 of the intermediate is too large to
allow for a spontaneous IMDA and/or the energy of activation of the IMDA is
exceedingly high given the entropic and enthalpic cost of organizing the sterically
FIGURE 2.10. Synthetic strategies employed for the formation of spiculoic acid A’s trans-hydrindane
framework.50
consequence, we expect the intermediate to remain unreactive towards the IMDA. The
olefins defining the conjugated diene in the system would have to undergo a
contained in the putative cyclase or in the active site of a non-canonical PKS domain
(Figure 2.11).55-58
36
FIGURE 2.11. An intermediate regioisomerization step may take place prior to cyclization by a putative
cyclase.
the favorable electronics of the extended enol or enolate intermediate. The increased
electron density of the diene provided by the extended conjugation of the enol moiety
will decrease the HOMO diene -LUMO dienophile energy gap54 of the system.
herein we describe our efforts in the development and synthesis of a novel biomimetic
acyclic polyketide intermediate of 2.11 and its ability to undergo an IMDA under a
envisioned the results would provide insight into the equilibrium conformation energies
of putative key intermediates and stereoisomers populating the potential energy surface
of the reaction coordinate to 2.11. Unlike the single concerted step to product shown in
Figure 2.12-A, we anticipated the path from linear intermediate 2.18 to cyclic 2.11 to go
through a transient cyclic enol such as 2.19, which in route to product would require an
FIGURE 2.12. Steps involved in the conversion of linear polyketide chains to 2.11. A) IMDA reaction of
putative intermediate 2.17 directly yields 2.11. B) IMDA reaction of 2.18 may produce an enol
intermediate 2.19 that after equilibration may yield product 2.11.
In addition, the outcome of the IMDA reaction will be biased by the endo vs. exo
selectivity and facial approach of the molecule's dienophile. Synthetic precedence50, 51, 53
suggests that the cyclization will take place preferentially through an endo transition state
produced by a β-face approach of the dienophile on the diene. Assuming our acyclic
38
analog 2.39 (see Scheme 2.4) displays similar endo selectivity, we hypothesize that the
facial approach will be dictated by the energy profile of the reaction's transition state
conformation. The congested structure of the cyclic enol stereoisomers, suggests a late
transition state. Thus, we expect key steric interactions present in the products to reflect
We have modeled and optimized the endo cyclic enol products of the IMDA reaction
of 2.18 (Figure 2.13) and the products from their C6 tautomerization. The energies of the
putative products are shown relative to the energy of spiculoic acid A. Unexpectedly
2.21 was lower in energy by 2.4 kcal/mol relative to 2.19, implying the potential presence
conformation from the more strained boat conformation required for the IMDA
state energies. Analysis of the structures resulting from tautomerization of C6 show the
anti epimer 2.11 to be 9.2 kcal/mol lower than the syn epimer 2.20, likely due to the
absence of the 1,3 diaxial interaction between the alkyl groups at C4 and C6. On the
contrary, the epimers originating from 2.21 show a great degree of strain from 1,3-diaxial
interactions between the C2 and C4 ethyl side chains. Noteworthy from these separate
energy C4 and C6 anti product, which act as a thermodynamic sink, corresponds with the
FIGURE 2.13. Cyclic endo products from the IMDA cycloaddition of 2.18. Dienophile-diene facial
selectivity has the potential of producing two diastereomers which are expected to undergo an enol-keto
tautomerization to products.
40
2.2.2 Retrosyntheses
2.2.2.1. Strategy I
that would emulate our hypothetic biosynthetic pathway of PKS-mediated linear chain
tetraene 2.46, the route’s key linear target. Upon the retrosynthetic reduction and
silylation of the secondary alcohol at C5, the disassembly of compound 2.46 can begin by
cleavage at the C2-C3 olefin. The step reveals chiral β-hydroxy aldehyde 2.45; related in
The rationale of this methodology lies in our desire to obtain an intermediate that, by
control of the oxidation state at C5, would enable us to test various IMDA conditions to
efficiently access the scaffold of 2.11. The syn relationship between the C4 and C5
substituents in 2.45 leads our analysis to β-hydroxy intermediate 2.44, in which either an
selectivity. Disconnection between C4-C5 of 2.44 yields key trienal 2.43. This
aldehyde to the carboxylic acid oxidation state, followed by an olefin cleavage. This
spiculoic acid A’s biosynthetic starter unit62. In the forward sense, the aldehydes may be
Regrettably, at the crux of our strategy, the Evans aldol reaction failed to provide
proclivity of 2.43 to enolize under the reaction conditions, which prevented us entry to
the chiral β-hydroxy target in the manner proposed. The results, which will be discussed
2.2.2.2. Strategy II
experienced while implementing our initial strategy, the design was simplified to include
construction of rac-2.46 (Scheme 2.2). We envisioned 2.11 to arise directly from the
acid-catalyzed deprotection of ketal intermediate 2.54. It was hoped that the ketalization
of the C5 ketone would permit the isolation and spectroscopic characterization of the
IMDA precursor. In keeping with the linear theme of Strategy I, disconnection of 2.54 at
the C2-C3 olefin exposes aldehyde 2.53 accessible by oxidation of the alcohol derived
from reduction of the C3 ester of 2.50. Formation of the C4-C5 bond in ester 2.50 was
During this synthesis, ketalization of the C5 ketone that resulted from the oxidization
attempts to circumvent this obstacle, the chemistry remained uncooperative and forced us
A convergent approach was chosen that would incorporate and recycle some elements
of our prior designs. As per Scheme 2.3, retrosynthetic bond cleavage of the C6-C7
trisubstituted olefin in rac-2.46 yields dienal 2.30 and the novel phosphonate 2.59 which
may be crafted from the addition of commercial diethyl ethylphosphonate (2.31) to the
cleavage of the C2-C3 olefin and modification of the oxidation state of C5. This acid is
SCHEME 2.3. Strategy III: Convergent and achiral retrosynthetic strategy to 2.11.
The synthesis of fragment 2.59 was smoothly carried out from 2.34, however, we
section Section 2.2.3.3. Based on these results, we once again redesigned our synthesis
to follow a similar strategy while avoiding the unstable phosphonate coupling partner.
2.2.2.4. Strategy IV
Our final route relied on the use of an aldol coupling to access a similar olefination
product as envisioned in the prior strategy. This route also exploited the symmetry
element of malonate 2.31 (Scheme 2.4). The strategy's most salient feature, however,
was the incorporation of the C6-C7 double bond of fragment 2.39 by a Lewis acid-
SCHEME 2.4. Strategy IV: Redesigned convergent and achiral retrosynthetic strategy to 2.11.
The C5-C6 bond can be formed from a Grignard alkylation of conjugated Weinreb
amide 2.36, in turn formed from a nucleophilic displacement of the conjugated ester
sequence would provide us with our most advanced intermediate, and enable us to test
2.2.3.1. Strategy I
Scheme 2.5 outlines the synthesis of aldehyde 2.43 starting with commercially
a
Reagents and conditions: (a) 2.40, toluene, reflux, overnight; (b) DIBAL-H, CH 2 Cl 2 , 0°C (or toluene, -
78°C), 1hr; (c) IBX, DMSO, r.t., 1.5hrs; (d) 2.47, benzene, reflux, overnight.
The sequence began with the olefination of aldehyde 2.24 with known ylide 2.4069, 70
in refluxing toluene. The resulting conjugated ester 2.25 was obtained in 77% yield with
excellent E selectivity (E/Z = 17:1 by 1H NMR). The stereoselectivity of this step has
been discussed in the literature; the authors rationalize the result through analysis of the
orbital, the expected scenario when the oxaphosphetane's bulkiest groups are anti with
respect to each other. Reduction of ester 2.25 by dropwise addition of DIBAL-H carried
out in an ice-cold solution of DCM furnished alcohol 2.26 in 100% yield. Oxidation of
alcohol 2.26 to aldehyde 2.27 was attempted using a variety of methods including PCC71,
TCCA/TEMPO72, manganese dioxide73 and IBX74. Aldehyde 2.27 (97% yield) was
limit decomposition63, aldehyde 2.27 was immediately olefinated with ylide 2.40 in
2.28 and 2.28a respectively in 33% yield (Figure 2.14), as well as unreacted starting
46
FIGURE 2.14. Chromatographically inseparable isomers of dienyl compounds 2.28-2.30 were identified by
1D and 2D NMR spectroscopy.
Ester mixture 2.28 and 2.28a was reduced with DIBAL-H at 0°C in DCM to furnish
chromatographically inseparable alcohol isomers 2.29 and 2.29a as a 1:1 ratio (52%
yield). Oxidation of this mixture with IBX in DMSO at room temperature to aldehydes
2.30 and 2.30a (1:1 ratio, 93% yield), followed by immediate olefination with
commercially available ylide 2.47 provided trienyl esters 2.41 and 2.41a as a 1:1 mixture
(89% yield). The terminal carbonyl in 2.41 and 2.41a was reduced with DIBAL-H at -
78°C in toluene to alcohols 2.42 and 2.42a (1:1 ratio, 87% yield), which in turn were
oxidized with IBX in DMSO to aldehydes 2.43 and 2.43a (1:1 ratio, 81% yield). Notable
among compounds 2.28-2.30 and 2.41-2.43 was the presence of a double bond pattern
analogous to that of dienyl ester mixture 2.28 and 2.28a. Although the increased length
NMR data confirmed the mixtures consisted only of two isomers (Figure 2.15).
FIGURE 2.15. Chromatographically inseparable isomers of trienyl compounds 2.41-2.43 were identified by
1D and 2D NMR spectroscopy.
SCHEME 2.6. Asymmetric methodologies employed in forming the C4-C5 bond with aldehyde 2.43.a
a
Reagents and conditions: (a) 2.48, nBu 2 BOTf, iPr 2 NEt, CH 2 Cl 2 , -78°C; (b) TBSOTf, 2,6-lutidine, THF,
0°C; (c) Auxiliary cleavage – DIBAL-H; HN(Me)OMe; LiOH; (d) 2.49, TiCl 4 , (-)-Sparteine, CH 2 Cl 2 , -
78°C.
Initially, an Evans chiral auxiliary (2.48) was employed and, to our delight, it
furnished alcohol 2.44 in 62% yield. Unfortunately, after alcohol silylation, the amide
conditions. Attempts to scale up the chiral aldol for further analysis proved challenging,
as the aldol reaction was highly irreproducible, leading us to examine the Crimmins61
(2.49) chiral aldol reaction. Regrettably, under the reaction conditions, 2.43 remained
unreactive, allowing for its full recovery upon quenching. We believe the combination of
a highly oxophilic Lewis acid (TiCl 4 ) and an enolization-prone aldehyde such as 2.43
quickly led to suppression of the C5 electrophilic center prior to its reaction with the
2.2.3.2. Strategy II
We shifted our focus towards synthesizing the linear chain in a non-selective manner
(Scheme 2.7).
SCHEME 2.7. Synthetic studies towards the C3 chain extension of aldehyde 2.43.a
a
Reagents and conditions: (a) 2.55, Zn°, benzene, reflux, 4hrs; (f) LAH, Et 2 O, 0°C, 1hr; (g) PivCl, Et 3 N,
DMAP. CH 2 Cl 2 , -78°C, 5 h; (h) TBSOTf, 2,6-lutidine, CH 2 Cl 2 , 0°C, 5 min.
effectively yield syn and anti β-hydroxy esters 2.50a/b, and their respective olefin
regioisomers, in a combined 78% yield (45% and 33% yield respectively). The expected
of protecting group manipulations and modifications in the oxidation state of key carbon
49
atoms on 2.50a/b would permit access to rac-2.46. Yet the molecule remained
unreactive upon the multiple attempts to reach our goal. Our initial plan was to protect
C5 at the keto oxidation state, a move that would subsequently allow us to extend the
chain at C3. Oxidation of 2.50a with IBX followed by ethylene glycol ketalization under
acidic and refluxing conditions (Scheme 2.7-A) failed to yield product. Access to the C3
electrophile was again denied after silylation of 2.50a and 2.50b (Scheme 2.7-B). As
with TBS-protected 2.44, and likely due to steric crowding, the molecule proved
amidation with the Weinreb amine. Treatment with LAH effectively reduced C3, but
esters 2.50a/b with LAH: 1,3 diols 2.51a/b (Scheme 2.7-C1). The sequence, attempted
on diol 2.51a, involved pivalate acylation and silylation of the primary and secondary
and olefination would effectively lead to the protected dihydro-2.52 and to the route’s
nucleophilic attack, and reduction were attempted, and the moiety was either unreactive,
aldehydes and ketones, we hoped that double oxidation of diols 2.51a/b followed by an
olefination reaction might lead to target rac-2.46 (Scheme 2.7-C2). Oxidation of diol 13a
with 2 equivalents of IBX quickly led to formation of the enone, but left the primary
50
decomposition.
The route was redesigned to include the synthetic convergence by a HWE olefination
(Scheme 2.8). Synthesis of 2.59 began with the hydride reduction of commercially
available diethyl ethylmalonate (2.31) in refluxing THF to produce 2.32 in 82% yield.
diol 2.32 and TBSCl were dissolved in a binary solvent system comprised of hexanes and
acetonitrile, and vigorously stirred overnight to furnish monoprotected diol 2.33 (56%
yield). Oxidation of alcohol 2.33 under Swern conditions76 furnished aldehyde 2.34 in
64% yield, which was easily converted via a Wittig reaction and deprotection to ester
a
Reagents and conditions: (a) LAH, THF, r.t. to reflux, 36 h, 82%; (b) TBDPSCl, 1:4 MeCN-Hexanes,
Et 3 N, r.t., 72 h, 56%; (c) (COCl)2, DMSO, Et 3 N, -60 to -78°C to r.t., 5 h, 64%; (d) 32, benzene, reflux,
overnight; 10%HCl/MeOH, r.t., 3h, 39% over two steps; (e) K 2 Cr 2 O 7 , H 2 SO 4 , Acetone, 0°C, 2.5 h, 27%;
(f) 32, benzene, reflux, overnight, 74%.
Jones oxidation77 of alcohol 2.56 to converted it to carboxylic acid 2.57 in 27% yield.
51
Initially, several attempts to access phosphonate 2.59 were met with inconsistent results
and side reactions. However, careful study of the reaction conditions of alkyl
phosphonate addition and the later substitution of the electrophile in the reaction to ester
SCHEME 2.9. Synthesis of advanced intermediates to employ for the olefination of 2.30.a
a
Reagents and conditions: (a) 2.60, n-BuLi, THF, -78°C to -40°C, 3 h, 60%.
As was previously touched upon, the conditions employed for the HWE reaction
failed to yield the expected linear chain. It was observed that the phosphonate, under the
uncharacterized mixture (by 1H NMR) as that found for the HWE reaction, suggesting
Heathcock et al. used a similar approach and phosphonate 2.66 towards the synthesis
of the halichlorine family of natural products.64 They reported difficulty with the HWE
olefination; their best yield (27%) was achieved under the Masamune-Roush conditions.
Without additional insight into the basis of their results, they replaced the phosphonate
2.66 with the phosphorane equivalent 2.67 (Figure 2.16) and found an increase in yield
(66%).
The latter step proved ineffective in the synthesis of our system, precipitating our
2.2.3.4. Strategy IV
Our fourth and final strategy was conceived to circumvent our setback in strategy 3
and retain the simplicity of the route (Scheme 2.10). The feature of our scheme is the
Lewis acid-mediated aldol reaction between dienal 2.30 and ketone 2.37. We converted
ester 2.35 to Weinreb amide 2.36 by a nucleophilic addition of the deprotonated Wenreib
amine in THF (61%). The product was treated with ethyl Grignard to smoothly furnish
ketone 2.37 in 62% and unreacted 2.36. With both key fragments in hand, we proceeded
to couple them using an aldol methodology. Generation of the enolate was effected by
53
activated intermediate with Hunig's base. Dienal 2.30 was diluted in DCM and slowly
added by syringe pump overnight to produce the β-hydroxyketone adduct, which was
treated with MsCl/Et3N in THF to form the C6-C7 olefin, and immediately followed by
deprotection of the C1 silyl ether with TBAF in THF to isolate the stable hydroxyketone
a
Reagents and conditions: (a) i-PrMgCl, HCl·HN(OMe)Me, THF, -20°C, 2 h, 61%; (b) EtMgBr, THF,
0°C, 2 h, 62%; (c) TiCl4, (i-Pr) 2 NEt, CH 2 Cl 2 , -78°C, overnight; MsCl, Et 3 N, 0°C, overnight; TBAF, THF,
r.t., overnight, 6% over three steps; (d) (COCl) 2 , DMSO, Et 3 N, -78°C, overnight, 100%.
The end game towards the synthesis of 2.11 began with the oxidation of alcohol
mixture 2.38 using the Swern conditions which furnished tetraene aldehyde 2.39 as a
mixture of diastereomers. Gratifyingly, the C3-C4 double bond from conjugation with
the C5 ketone to conjugation with the newly formed C1 aldehyde was unambiguously
(p-TSOH) in CDCl 3 , we expected proton transfer and the concomitant enolization to lead
to product. The 1H NMR showed full conversion to the iminium ion upon treatment with
the catalyst. After five days and slow heating from room temperature to reflux, the
increase the reactivity of the acyclic intermediate, we opted to employ MeAlCl 2 , a strong
Lewis acid common in Diels-Alder reactions.79-81 The acyclic intermediate 2.39 was
treated with the Lewis acid at -78°C in DCM and was slowly warmed up to room
temperature overnight. After aqueous workup, we were surprised to discover the intact,
SCHEME 2.11. Conditions attempted for the IMDA cyclization of aldehyde 2.39 to 2.11.a The molecule’s
equilibrium will be dominated by the presence of its Z or E enolate tautomers. Only the E enolate will be
reactive towards an IMDA. Formation of the bicyclic IMDA adduct depends on accessing the conjugated
E enol, as this conformation will favor the intramolecular approach of the reactive moieties.
a
Reagents and conditions: (1) p-TSOH, MacMillan's catalyst, r.t. to reflux, 5 d; (2) MeAlCl 2 , CH 2 Cl 2 , -
78°C to r.t., overnight; (3) H 2 O, d-DMSO, 80°C, 5 d.
86
The substrate was dissolved in d-DMSO and 10% H 2 O, and heated to 80°C for five
days. The use of water as a co-solvent was favored over its deuterated counterpart to
prevent proton exchange and better follow formation of precedented bicyclic aldehyde
2.69 by 1H NMR.53 However, intermediate 2.39 remained unreactive under the reaction
2.3. Conclusions
biosynthesis of the polyketide chain and its relationship with the trans-hydrindane
scaffold of spiculoic acid A. We were concerned that the initial biosynthetic hypothesis
of spiculoic acid A33 did not adequately address the double bond regiochemistry in the
confirm it by the synthesis of a linear polyketide polyene consistent with the canon of
PKS dehydration, with the belief it might under mild, enzyme free conditions, grant
access to the spiculoic acid A framework. Our convergent route was effective in
generating the full carbon skeleton of our linear putative biomimetic intermediate.
However, we were unable to coax the linear precursor to cyclize under either mild or
Our hypothesis for formation of the trans-hydrindane scaffold under mild reaction
conditions did not prove correct. We have rationalized our results by analyzing the
orbital coefficients of the reactive centers in our linear enol precursor to spiculoic acid A.
We expect coefficient overlap of the reactants at the transition state to lead to the
56
preferred route to products7. For our analysis we designed, modeled and optimized two
component of the IMDA reaction. The A semi-empirical extended Hückel method was
employed to generate the frontier molecular orbitals of each analog. Our goal was to
extract from the frontier orbitals calculated a qualitative description of the influence the
asynchronicity of the reaction7. Our results clearly show the incompatibility of the
analog's orbital coefficients (Figure 2.17). Although we did not observe enolization
cycloaddition of (E)-2.68.
FIGURE 2.17. Modeled analogs employed for the orbital coefficient analysis of the IMDA of linear
precursor (E)-2.68 in route to spiculoic acid A.
In addition, the stability of 2.39 under the various reaction conditions employed was
high barrier for enolization, likely due to a buildup of 1,3-allylic strain. Accordingly, we
set to understand the energy landscape and key structural characteristics of the steps
involved in the conversion of 2.39 to 2.70, the compounds involved in the reaction
FIGURE 2.18. Ab initio analysis of regioisomerization of linear precursor 2.39. The C10 proton (blue) is
coplanar to the C8-C9 and C11-C12 olefins, which results in its decreased acidity and likely prevents the
regioisomerization of the molecule's olefin system.
In the optimized, lowest energy structure of 2.39, the coplanarity of the C10 proton to
the C8-C9 and C11-C12 olefins's π system would minimize its acidity and clearly prevent
(+10.8 kcal/mol) and 2.39 may explain the reason for the latter's stability in solution.
Interestingly, the (E)-2.68 necessary for an IMDA failed to converge in our attempts to
find its energy minimum. The structure appeared to have been caught oscillating
between two energy minima, a telling result that strengthens the likelihood the structure
E1cb syn elimination promoted by the catalytic histidine contained in the enzyme's
signature HX 3 GX 3 P motif. The acidic pro-S α-proton is transferred to the general base
elimination of the β-hydronium ion, a step that ensures that conjugation with the ACP-
thioester is preordained.87, 88
Given this, an unusual non-canonical step must take place to regioisomerize the
olefins of our linear polyketide on its way to spiculoic acid A. Biosynthetic gene clusters
have recently been identified of a novel class of polyketide natural products containing
modifications during the assembly of the linear chain.55, 58 For example, modules 7 and 9
of the rhizoxin D's biosynthetic pathway (Figure 2.20) contain abnormal DH domains
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Reactions were carried out under an argon atmosphere with dry solvents and oven-
dried glassware under anhydrous conditions unless specified otherwise. All reactions
were carried out under an inert argon atmosphere with dry tetrahydrofuran, diethyl ether,
and DCM solvents by passing them through activated alumina columns. Toluene,
benzene, and triethylamine were freshly distilled over calcium hydride. Reagents were
purchased at the highest commercial quality and used without further purification, unless
out on 0.25 mm E. Merck silica gel plates (60F-254) using UV light as a visualizing
agent and/or ceric ammonium molybdate (CAM), p-anisaldehyde (PA), and potassium
performed with E. Merck silica gel (60, particle size 0.040-0.063 mm). Preparative thin-
layer chromatography (PTLC) separations were carried out on 0.25 mm E. Merck silica
Data are reported as follows: chemical shifts in ppm (δ); multiplicities are indicated by
spectra (IR) were obtained on NaCl discs on a Nicolet IR200 series FT-IR spectrometer.
Low resolution atmospheric pressure chemical ionization (APCI) mass spectra were
Spectrum Centre).
O
O 1
O
10 3
11
6
2.24 2.25
Phenylacetaldehyde (2.24, +90% purity) (2.87 mL, 23.0 mmol) and ylide 2.40 (10.6
g, 28.2 mmol, 1.1 equiv) were dissolved in toluene (20 mL) and gently refluxed for 10 h.
The reaction mixture was purified directly and without work-up by silica gel
chromatography (3% EtOAc-hexanes) affording ester 2.25 (5.02 g, 23.0 mmol, 100%) as
a yellow oil.
M.W.: 218.29
Reference: Tanaka, T.; Hiramatsu, K.; Kobayashi, Y.; Ohno, H., Chemo- and
stereoselectivity in titanium-mediated regioselective ring-opening reaction
of epoxides at the more substituted carbon. Tetrahedron 2005, 61 (28),
6726-6742
a
Ester 3 was identical to its previously reported spectroscopic values
2-Ethyl-4-phenyl-but-2-en-1-ol (2.26)a
O 1
OH
O 10 3
11
6
2.25 2.26
Ester 2.25 (4.09 g, 18.7 mmol) was dissolved in dry dichloromethane (DCM, 20 mL)
(DIBAL-H, 1.0 M in toluene, 37.5 mL, 37.5 mmol, 2.0 equiv) was added dropwise over 5
min. The mixture was slowly warmed-up to room temperature and stirred for 1 h. Upon
completion (as per TLC) the reaction was cooled to 0°C and quenched by the slow,
stepwise addition of water (1.52 mL), aqueous 15% NaOH solution (1.52 mL), and water
(3.8 mL). The white, gel-like precipitate was filtered and the mother liquor extracted
with ethyl acetate (EtOAc, 3 × 100 mL). The organic layers were combined and washed
with brine (150 mL), dried over Na 2 SO 4 and filtered. The solvent was removed in vacuo
and the residue was purified by silica gel chromatography (10% EtOAc-hexanes)
M.W.: 176.25
Reference: Tanaka, T.; Hiramatsu, K.; Kobayashi, Y.; Ohno, H., Chemo- and
stereoselectivity in titanium-mediated regioselective ring-opening reaction
of epoxides at the more substituted carbon. Tetrahedron 2005, 61 (28),
6726-6742
a
Alcohol 2.26 was identical to its previously reported spectroscopic values.
2-Ethyl-4-phenyl-but-2-enal (2.27)a
H
1
OH O
10 3
11
6
2.26 2.27
Alcohol 2.26 (1.54 g, 8.68 mmol) and iodoxybenzoic acid (IBX, 4.87 g, 17.4 mmol,
room temperature for 1 h 20 min. Upon completion, the mixture was purified without
M.W.: 174.10
71
IR: (Film)
2970, 1691, 1493, 1454, 1376, 1224, 1065, 796, 745, 699, 519
1
H NMR: (300 MHz, CDCl 3 )
9.41 (s, 1 H, H1), 7.29 (m, 5 H, PhH), 6.57 (t, J = 7.5 Hz, 1 H, H3), 3.71
(d, J = 7.5 Hz, 2 H, H4), 2.40 (q, J = 7.5 Hz, 2 H, H11), 1.05 (t, J = 7.5 Hz,
3 H, H12)
13
C NMR: (75 MHz, CDCl 3 )
195.1 (C(1)), 152.1 (C(2)), 145.5 (C(3)), 138.4 (C(Ph)), 129.0 (C(Ph)),
128.6 (C(Ph)), 126.9 (C(Ph)), 35.0 (C(4)), 17.5 (C(11)), 13.6 (C(12))
O 1 O 1
H O O
O
12 12
5
+ 5
13 13
15 15
8 8
2.27 2.28 2.28a
Aldehyde 2.27 (1.46 g, 8.38 mmol) and ylide 2.40 (5.04 g, 13.4 mmol, 1.6 equiv)
were dissolved in benzene (21 mL), and the pale yellow solution was refluxed for 20 h.
Purification was performed directly without work-up by silica gel chromatography (4%
EtOAc-hexanes) affording an inseparable mixture of ester isomers 2.28 and 2.28a (400
mg, 1.47 mmol, 37%) as well as unreacted starting material (470 mg, 2.7 mmol, 32%)
M.W.: 272.38
IR: (Film)
3027, 2968, 2934, 2875, 1711, 1640, 1494, 1236, 1128, 746, 698
1
H NMR: (500 MHz, CDCl 3 )
7.38-7.34 (m, 5 H, PhH), 7.27 (s, 1 H, H3), 5.82 (t, J = 7.5 Hz, 1 H, H5),
4.45-4.35 (q, J = 7 Hz, 2 H, H17), 3.67 (d, J = 7.5 Hz, 2 H, H6), 2.61 (q, J
= 7.5 Hz, 2 H, H15), 2.46 (q, J = 7.5 Hz, 2 H, H13), 1.46 (t, J = 7 Hz, 3 H,
H18), 1.23-1.17 (m, 6 H, H14 & H16)
13
C NMR: (125 MHz, CDCl 3 )
168.62 (C(1)), 141.27 (C(3)), 140.68 (C(4)), 138.88 (C(Ph)), 136.07 (C(2)),
129.88 (C(5)), 129.36 (C(Ph)), 128.70 (C(Ph)), 126.26 (C(Ph)), 60.61
(C(17)), 34.33 (C(6)), 23.72 (C(13)), 21.17 (C(15)), 14.49 (C(18)), 14.06
(C(16)), 13.57 (C(14))
M.W.: 272.38
IR: (Film)
3027, 2968, 2934, 2875, 1711, 1640, 1494, 1236, 1128, 746, 698
1
H NMR: (500 MHz, CDCl 3 )
7.47-7.44 (m, 5 H, PhH), 6.78 (d, J = 10 Hz, 1 H, H3), 6.53 (d, J = 16 Hz, 1
H, H6), 6.25 (dd, J = 15.5, 7.5 Hz, 1 H, H5), 4.44-4.36 (q, J = 7 Hz, 2 H,
H17), 3.30 (m, 1 H, H4), 2.54 (q, J = 7.5 Hz, 2 H, H13), 1.80-1.67 (m, 2 H,
H15), 1.46 (t, J = 7 Hz 3 H, H18), 1.23-1.17 (m, 3 H, H14), 1.10 (t, J = 7.5
Hz, 3 H, H16)
13
C NMR: (125 MHz, CDCl 3 )
168.48 (C(1)), 143.26 (C(3)), 135.24 (C(Ph)), 134.02 (C(2)), 131.67 (C(5)),
130.20 (C(6)), 128.62 (C(Ph)), 128.46 (C(Ph)), 126.26 (C(Ph)), 60.88
(C(17)), 44.17 (C(4)), 28.43 (C(15)), 21.00 (C(13)), 14.49 (C(18)), 14.73
(C(14)), 12.03 (C(16))
2,4-Diethyl-6-phenyl-hexa-2,4-dien-1-ol (2.29)
2,4-Diethyl-6-phenyl-hexa-2,5-dien-1-ol (2.29a)
1 1
O O OH OH
O O
12 5 12 5
+
13 13
15 15
8 8
Esters 2.28 and 2.28a (1.35 g, 4.96 mmol) were dissolved in toluene (25 mL) and
cooled in an acetone-dry ice bath to -78 °C under argon. DIBAL-H (1.0 M in toluene,
19.8 mL, 19.8 mmol, 4.0 equiv) was added by syringe over 15 min. The solution was
warmed up slowly to room temperature and stirred for 1 h. Quenched the mixture by the
slow, stepwise addition of water (0.79 mL), aqueous 15% NaOH solution (0.79 mL), and
water (1.98 mL). The gel-like precipitate was filtered and the mother liquor extracted
with ethyl acetate (EtOAc, 2 × 20 mL), washed with brine (20 mL), dried over Na 2 SO 4
and filtered. The solvent was removed in vacuo and the residue purified by silica gel
M.W.: 230.35
IR: (Film)
3315, 3026, 2964, 2873, 1494, 1454, 1029, 744, 695
1
H NMR: (500 MHz, CDCl 3 )
7.20-7.00 (m, 5 H, PhH), 5.74 (s, 1 H, H3), 5.30 (t, J = 7.5 Hz, 1 H, H5),
3.95 (s, 2 H, H1), 3.31 (d, J = 7.5 Hz, 2 H, H6), 2.17-1.99 (m, 4 H, H13 &
H15), 0.98-0.84 (m, 6 H, H14 & H16)
13
C NMR: (125 MHz, CDCl 3 )
142.03 (C(4)), 141.43 (C(2)), 139.13 (C(Ph)), 128.75 (C(Ph)), 128.45
(C(Ph)), 127.65 (C(3)), 126.17 (C(5)), 125.99 (C(Ph)), 66.62 (C(1)), 34.22
(C(6)), 24.23 (C(15)), 21.98 (C(13)), 13.71 (C(14)), 13.52 (C(16))
74
M.W.: 230.35
IR: (Film)
3315, 3026, 2964, 2873, 1494, 1454, 1029, 744, 695
1
H NMR: (500 MHz, CDCl 3 )
7.20-7.00 (m, 5 H, PhH), 6.20 (d, J = 16 Hz, 1 H, H6), 5.95 (dd, J = 16, 7.5
Hz, 1 H, H5), 5.14 (d, J = 9.5 Hz, 1 H, H3), 3.93 (s, 2 H, H1), 2.90 (m, 1 H,
H4), 2.17-1.99 (m, 2 H, H13), 1.50-1.20 (m, 2 H, H15), 0.98-0.84 (m, 3 H,
H14), 0.77 (t, J = 7.5 Hz, 3 H, H16)
13
C NMR: (125 MHz, CDCl 3 )
141.11 (C(2)), 137.90 (C(Ph)), 133.76 (C(5)), 128.91 (C(6)), 128.61
(C(Ph)), 128.35 (C(Ph)), 128.20 (C(3)), 126.61 (C(Ph)), 66.71 (C(1)), 43.03
(C(4)), 28.87 (C(15)), 21.53 (C(13)), 13.39 (C(14)), 12.02 (C(16))
2,4-Diethyl-6-phenyl-hexa-2,4-dienal (2.30)
2,4-Diethyl-6-phenyl-hexa-2,5-dienal (2.30a)
1 1
O O
OH OH
12 5 12 5
+
13 13
15 15
8 8
Alcohols 2.29 and 2.29a (790 mg, 3.43 mmol) and IBX (1.92 g, 6.86 mmol, 2.0
equiv) were dissolved in DMSO (9 mL) by vigorously stirring at room temperature for 1
h and 20 min. Purified the mixture directly and without prior work-up by silica gel
M.W.: 228.33
IR: (Film)
3027, 2968, 2875, 2711, 1685, 1613, 1454, 1377, 1189, 1060, 967, 910,
793, 747, 697
1
H NMR: (500 MHz, CDCl 3 )
9.40 (s, 1H, H1), 7.39-7.23 (m, 5 H, PhH), 6.72 (s, 1 H, H3), 5.94 (t, J = 7.5
Hz, 1 H, H5), 3.59 (d, J = 7.5 Hz, 2 H, H6), 2.48-2.37 (m, 4 H, H13 &
H15), 1.12-1.03 (m, 6 H, H14 & H16)
13
C NMR: (125 MHz, CDCl 3 )
195.26 (C(1)), 152.98 (C(3)), 145.03 (C(4)), 143.51 (C(2)), 139.02 (C(7)),
134.53 (C(5)), 128.89 (C(Ph)), 127.71 (C(Ph)), 126.38 (C(Ph)), 34.59
(C(6)), 23.25 (C(15)), 18.35 (C(13)), 13.92 (C(14)), 13.68 (C(16))
M.W.: 228.33
IR: (Film)
3027, 2968, 2875, 2711, 1685, 1613, 1454, 1377, 1189, 1060, 967, 910,
793, 747, 697
1
H NMR: (500 MHz, CDCl 3 )
9.44 ( s, 1 H, H1), 7.39-7.23 (m, 5 H, PhH), 6.45 (d, J = 16 Hz 1 H, H6),
6.33 (d, J = 10 Hz, 1 H, H3), 6.13 (dd, J = 16, 7.5 Hz, 1 H, H5), 3.36 (m, 1
H, H4), 2.36-2.31 (m, 2 H, H13), 1.74-1.61 (m, 2 H, H15), 1.12-1.03 (m, 3
H, H14), 0.98 (t, J = 7.5 Hz, 3H, H16)
13
C NMR: (125 MHz, CDCl 3 )
196.04 (C(1)), 155.54 (C(3)), 152.08 (C(2)), 137.30 (C(7)), 130.97 (C(6)),
130.62 (C(5)), 128.81 (C(Ph)), 128.54 (C(Ph)), 126.53 (C(Ph)), 44.30
(C(4)), 28.33 (C(15)), 17.84 (C(13)), 13.84 (C(14)), 11.92 (C(16))
16 16
8 18 8 18
10 10
2.30 2.30a 2.41 2.41a
Aldehydes 2.30 and 2.30a (760 mg, 3.33 mmol) and ylide 2.47 (3.02 g, 8.33 mmol,
2.5 equiv) were dissolved in benzene (20 mL) and refluxed overnight. The mixture was
affording a mixture of inseparable esters 2.41 and 2.41a (880 mg, 2.82 mmol, 85%) as a
M.W.: 312.45
IR: (Film)
3061, 3026, 2964, 2874, 1713, 1631, 1453, 1367, 1248, 1114, 1031, 964,
912, 746, 696
1
H NMR: (500 MHz, CDCl 3 )
7.20-7.00 (m, 5 H, PhH), 6.96 (s, 1 H, H3), 5.72 (s, 1H, H5), 5.36 (t, J = 7.5
Hz, 1 H, H7), 4.04 (q, J = 7.5 Hz, 2 H, H20), 3.33 (d, J = 7.5 Hz, 2 H, H8),
2.10-2.05 (m, 4 H, H16 & H18), 1.83 (d, J = 1.5 Hz, 3 H, H15), 1.15 (t, J =
7.5 Hz, 3 H, H21), 0.88-0.76 (m, 6 H, H17 & H19)
13
C NMR: (125 MHz, CDCl 3 )
169.13 (C1)), 142.27 (C(3)), 142.05 (C(4)), 139.19 (C(6)), 138.35 (C(Ph))
134.29 (C(5)), 131.55 (C(2)), 128.52 (C(Ph)), 127.25 (C(7)), 126.26
(C(Ph)), 60.81 (C(20)), 34.40 (C(8)), 24.31 (C(16)), 24.04 (C(18)), 14.53
(C(21), 14.34 (C(15)), 14.18 (C(17)), 13.46 (C(19))
M.W.: 312.45
IR: (Film)
3061, 3026, 2964, 2874, 1713, 1631, 1453, 1367, 1248, 1114, 1031, 964,
912, 746, 696
1
H NMR: (500 MHz, CDCl 3 )
7.20-7.00 (m, 5 H, PhH), 6.96 (s, 1 H, H3), 6.20 (d, J = 16 Hz, 1 H, H8),
5.94 (dd, J = 16, 7.5 Hz, 1 H, H7), 5.18 (d, J = 10 Hz, 1 H, H5), 4.04 (q, J
= 7.5 Hz, 2 H, H20), 2.96 (m, 1 H, H6), 2.15 (q, J = 7.5 Hz, 2 H, H16), 1.82
(d, J = 1.5 Hz, 3 H, H15), 1.47-1.30 (m, 2 H, H18), 1.15 (t, J = 7.5 Hz, 3 H,
H21), 0.88-0.76 (m, 6H, H17 & H19)
13
C NMR: (125 MHz, CDCl 3 )
169.06 (C(1)), 141.87 (C(3)), 141.25 (C(4)), 137.86 (C(Ph)), 135.29 (C(5)),
133.16 (C(7)), 131.38 (C(2)), 129.35 (C(8)), 128.97 (C(Ph)), 128.70
(C(Ph)), 126.14 (C(Ph)), 60.81 (C(20)), 43.67 (C(6)), 28.90 (C(18)), 24.76
(C(16)), 14.53 (C(21)), 14.29 (C(15)), 13.69 (C(17)), 12.14 (C(19))
4,6-Diethyl-2-methyl-8-phenyl-octa-2,4,6-trien-1-ol (2.42)
4,6-Diethyl-2-methyl-8-phenyl-octa-2,4,7-trien-1-ol (2.42a)
O O
1 1
O O OH OH
5 5
15 15
14 + 14
16 16
8 8
18 18
10 10
2.41 2.41a 2.42 2.42a
Esters 2.41 and 2.41a (880 mg, 2.82 mmol) were dissolved by vigorously stirring in
toluene (5 mL) and cooled in an acetone-dry ice bath to -78 °C under argon. DIBAL-H
(1.0 M in toluene, 26.3 mL, 26.3 mmol, 9.0 equiv) was added dropwise over 15 min. The
reaction was warmed-up to room temperature and stirred an additional 2.5 h. The
reaction was quenched by the slow, stepwise addition of water (0.6 mL), aqueous 15%
NaOH solution (0.6 mL), and water (1.5 mL). The precipitate was filtered and the
mother liquor washed with brine (15 mL), dried over Na 2 SO 4 and filtered. The solvent
78
was removed in vacuo and the residue purified by silica gel chromatography (10%
EtOAc-hexanes) affording alcohols 2.42 and 2.42a (662 mg, 2.45 mmol, 87%) as a
colorless oil.
M.W.: 270.41
IR: (Film)
3323, 3025, 2963, 1494, 1452, 1376, 1070, 963, 908, 745, 695
1
H NMR: (500 MHz, CDCl 3 )
7.20-7.00 (m, 5 H, PhH), 5.70 (s, 1 H, H3), 5.57 (s, 1 H, H5), 5.32 (t, J =
7.5 Hz, 1 H, H7), 3.91 (s, 2 H, H1), 3.32 (d, J = 7.5 Hz, 2 H, H8), 2.11-1.98
(m, 4 H, H16 & H18), 1.65 (d, J = 1.5 Hz, 3 H, H15), 0.89-0.77 (m, 6 H,
H17 & H19)
13
C NMR: (125 MHz, CDCl 3 )
139.63 (C(6)), 139.51 (C(4)), 138.53 (C(2)), 136.44 (C(Ph)), 130.76 (C(5)),
128.55 (C(Ph)), 127.73 (C(3)), 126.13 (C(Ph)), 125.93 (C(7)), 125.80
(C(Ph)), 69.10 (C(1)), 34.28 (C(8)), 25.10 (C(18)), 24.49 (C(16)), 15.48
(C(15)), 13.98 (C(19)), 13.44 (C(17))
M.W.: 270.41
IR: (Film)
3323, 3025, 2963, 1494, 1452, 1376, 1070, 963, 908, 745, 695
1
H NMR: (500 MHz, CDCl 3 )
7.20-7.00 (m, 5 H, PhH), 6.21 (d, J = 16 Hz, 1 H, H8), 5.99 (dd, J = 16, 7
Hz, 1 H, H7), 5.75 (s, 1 H, H3), 4.99 (d, J = 7 Hz, 1 H, H5), 3.91 (s, 2 H,
H1), 2.94 (m, 1 H, H6), 2.11-1.98 (m, 2 H, H16), 1.67 (d, J = 1.5 Hz, 3 H,
H15), 1.50-1.35 (m, 2 H, H18), 0.89-0.77 (m, 6 H, H17 & H19)
13
C NMR: (125 MHz, CDCl 3 )
79
4,6-Diethyl-2-methyl-8-phenyl-octa-2,4,6-trienal (2.43)
4,6-Diethyl-2-methyl-8-phenyl-octa-2,4,7-trienal (2.43a)
H H
1 1
OH OH O O
5 5
15
+ 15
14 14
16 16
8 8
18 18
10 10
2.42 2.42a 2.43 2.43a
Alcohols 2.42 and 2.42a (662 mg, 2.45 mmol) and IBX (1.37 g, 4.90 mmol, 2.0
equiv) were dissolved in DMSO (22 mL) and vigorously stirred at room temperature for
45 min. The mixture was purified directly and without prior work-up by silica gel
2.43 and 2.43a (536 mg, 2.0 mmol, 82%) as a yellow oil.
M.W.: 268.39
IR: (Film)
3341, 2963, 2710, 1686, 1494, 1378, 1198, 1014, 912, 746
1
H NMR: (500 MHz, CDCl 3 )
9.27 (s, 1 H, H1), 7.20-6.99 (m, 5 H, PhH), 6.58 (s, 1 H, H3), 5.99 (s, 1 H,
H5), 5.44 (t, J = 7.5 Hz, 1 H, H7), 3.34 (d, J = 7.5 Hz, 2 H, H8), 2.29 (q, J
= 7.5 Hz, 2 H, H18), 2.12 (q, J = 7.5 Hz, 2 H, H16), 1.77 (d, J = 1.5 Hz, 3
H, H15), 0.93-0.79 (m, 6 H, H17 & H19)
13
C NMR: (125 MHz, CDCl 3 )
80
195.99 (C(1)), 153.95 (C(3)), 139.19 (C(2)), 138.91 (C(6)), 138.62 (C(4)),
138.05 (C(5)), 137.02 (C(Ph)), 128.70 (C(Ph)), 128.49 (C(7) & (C(Ph))),
126.26 (C(Ph)), 34.43 (C(8)), 24.20 (C(16) & C(18)), 14.48 (C(17)), 13.50
(C(19)), 11.06 (C(15))
M.W.: 268.39
IR: (Film)
3341, 2963, 2710, 1686, 1494, 1378, 1198, 1014, 912, 746
1
H NMR: (500 MHz, CDCl 3 )
9.28 ( s, 1 H, H1), 7.39-7.23 (m, 5 H, PhH), 6.59 (s, 1 H, H3), 6.22 (d, J =
16 Hz 1 H, H8), 5.94 (dd, J = 16, 7.5 Hz, 1 H, H7), 5.47 (d, J = 9.5 Hz, 1 H,
H5), 3.02 (m, 1 H, H6), 2.21 (q, J = 7.5 Hz, 2 H, H16), 1.78 (d, J = 1.5 Hz,
3 H, H15), 1.48-1.36 (m, 2 H, H18), 0.93-0.79 (m, 6 H, H17 & H19)
13
C NMR: (125 MHz, CDCl 3 )
196.00 (C(1)), 153.58 (C(3)), 140.91 (C(2)), 139.73 (C(5)), 137.64 (C(Ph)),
137.49 (C(4)), 132.38 (C(7)), 129.85 (C(8)), 128.70 (C(Ph)), 128.49
(C(Ph)), 126.26 (C(Ph)), 43.91 (C(6)), 28.81 (C(18)), 23.53 (C(16)), 13.99
(C(17)), 12.06 (C(19)), 10.96 (C(15))
(4E,6E,8E)-Ethyl 2,6,8-triethyl-3-hydroxy-4-methyl-10-phenyldeca-4,6,8-trienoate
(2.44)
EtO EtO
H H O O
O O
OH OH
To a well stirred solution of aldehyde 2.43 and 2.43a (107 mg, 0.40 mmol) in
benzene (0.40 mL), zinc metal (29 mg, 0.44 mmol, 1.1 equiv) and 2-3 crystals of iodine
81
were added. Ethyl 2-bromobutyrate (2.55, 62 μL, 1.05 equiv) was added over 5 minutes
and heated mixture to reflux. After 10 minutes, dark green solution had turned ochre.
Continued to reflux for a total of 3 hours and solution was then cooled to room
temperature. Once cool, quenched reaction with 10% aqueous H 2 SO 4 (1 mL) and
extracted with EtOAc (3 x 1 mL), dried organic phase over Na 2 SO 4 and filtered.
Evaporated solvent in vacuo and purified residue by silica gel chromatography (10%
EtOAc-hexanes) affording alcohols 2.50 (68 mg, 0.18 mmol, 45%) and 2.50a (51 mg,
M.W.: 384.27
IR: (Film)
3462, 2965, 2930, 1731, 1459, 1375, 1262, 1179, 1031, 746, 696
1
H NMR: (500 MHz, CDCl 3 ) - Regioisomer I
7.18-6.97 (m, 5 H), 5.76 (s, 1 H), 5.49 (s, 1 H), 5.26 (t, 1 H), 4.07 (d, J =
6.5 Hz, 1 H), 4.03-3.92 (m, 2 H), 3.30 (d, J = 7.5 Hz, 2H), 2.40 (m, 1 H),
2.40 (m, 1 H), 2.06-1.93 (m, 2 H), 1.60 (s, 3 H), 1.56-1.27 (m, 4 H), 1.11-
1.01 (m, 3 H), 0.85-0.66 (m, 9 H)
1
H NMR: (500 MHz, CDCl 3 ) - Regioisomer II
7.18-6.97 (m, 5 H), 6.18 (dd, J = 15.5, 7,5 Hz, 1 H), 5.94 (m, 1 H), 5.74 (s,
1 H), 4.91 (d, J = 9.5 Hz, 1 H), 4.07 (d, J = 6.5 Hz, 1 H), 4.03-3.92 (m, 2
H), 2.90 (quintet, J = 8.0 Hz, 1 H), 2.40 (m, 1 H), 2.06-1.93 (m, 2 H), 1.61
(s, 3 H), 1.56-1.27 (m, 4 H), 1.11-1.01 (m, 3 H), 0.85-0.66 (m, 9 H)
M.W.: 384.27
IR: (Film)
3461, 2964, 2931, 2874, 1733, 1459, 1376, 1267, 1178, 1028, 746, 695
82
1
H NMR: (500 MHz, CDCl 3 ) – Regioisomer I
1
H NMR of Regioisomer I is inconclusive.
1
H NMR: (500 MHz, CDCl 3 ) – Regioisomer II
7.17-7.00 (m, 5 H), 6.19 (dd, J = 16, 4.5 Hz, 1 H), 5.94 (ddd, J = 16, 4.5,
2.0 Hz, 1 H), 5.72 (s, 1 H), 4.93 (d, J = 9.5 Hz, 1 H), 4.00 (m, 3 H), 2.90
(quintet, J = 8.0 Hz, 1 H), 2.42 (m, 1 H), 2.08-1.93 (m, 2 H), 1.58 (s, 3 H),
1.46-1.28 (m, 4 H), 1.12-1.07 (m, 3 H), 0.84-0.69 (m, 9 H)
2-(Hydroxymethyl)butanol (2.32)
O O
3 1
2
EtO OEt HO OH
4
5
2.31 2.32
Diethyl ethylmalonate 2.31 (941 mg, 5.00 mmol) was added dropwise to a solution of
lithium aluminum hydride (LAH, 285 mg, 7.53 mmol, 30.0 equiv) vigorously stirred in
tetrahydrofuran (THF, 50 mL) at 0°C. The resulting reaction was warmed up to r.t. and
stirred overnight. To bring the reaction to completion, solution was refluxed for an
additional 24 h. After cooling the mixture, it was quenched with 0.4 ml H 2 O, followed
by 0.4 mL 15% NaOH(aq) and lastly 1.2 mL H 2 O. Precipitated aluminum salts were
filtered and the oil was purified by silica gel chromatography (30% EtOAc-hexanes)
affording diol 2.32 (427 mg, 4.10 mmol, 82%) as a clear oil.
M.W.: 104.15
2-((tert-Butyldiphenylsilyloxy)methyl)butan-1-ol (2.33)
6
7
3 1
2 Si
HO OH HO O
4
5
2.32 2.33
Diol 2.32 (521 mg, 5.00 mmol) was dissolved in a 1:4 biphasic solution of
0.84 mL, 1.2 equiv) and finally tert-butyldiphenylsilyl chloride (TBDPSCl, 1.3 mL, 5.0
mmol, 1.0 equiv) were added. Stirred at r.t. for 72 h, quenched with a saturated NH 4 Cl
solution, and extracted with EtOAc (3 x 20 mL), dried organic phase over Na 2 SO 4 and
filtered. Solvent was evaporated in vacuo and the residual oil purified by silica gel
chromatography (5% EtOAc-hexanes) affording monoprotected diol 2.33 (962 mg, 2.81
M.W.: 342.55
2-((tert-Butyldiphenylsilyloxy)methyl)butanal (2.34)
6
7
3 1
2 Si
HO OTBDPS O O
4
5
2.33 2.34
In a round bottomed flask (r.b.f.) oxalyl chloride ((COCl) 2 , 0.27 mL, 3.1 mmol, 3.5
Dimethylsulfoxide (0.31 mL, 4.4 mmol, 5.0 equiv) was added dropwise at this
temperature, and immediately upon addition gas generation was observed. The solution
was stirred for an additional 30 min at -60°C, at which point the temperature was lowered
to -78°C. A precooled (-78°C) solution of monoprotected diol 2.33 (300 mg, 0.88 mmol)
in DCM (3 mL) was added dropwise by cannula and the white and opaque mixture was
stirred for 1 h. Triethylamine (1.22 mL, 8.8 mmol, 10 equiv) was added dropwise and
the thick slurry was slowly warmed to r.t. after which distilled water (3mL) was added to
quench. Extracted with EtOAc (3 x 10 mL), dried organic phase over Na 2 SO 4 and
filtered. After evaporation of solvent the oil was purified by silica gel chromatography
(5% EtOAc-hexanes) affording aldehyde 2.34 (192 mg, 0.56 mmol, 64%) as a clear oil.
M.W.: 340.53
85
Reference: Smith, A. B., III; Cox, J. M.; Furuichi, N.; Kenesky, C. S.; Zheng, J.;
Atasoylu, O.; Wuest, W. M., Total Synthesis of (−)-2-epi-Peloruside A.
Org. Lett. 2008, 10(24), 5501-5504
a
Aldehyde 2.34 was identical to its previously reported spectroscopic values.
2.34 2.35
Aldehyde 2.34 (670 mg, 1.96 mmol) and ylide 2.40 (1.5 g, 4.0 mmol, 2.0 equiv) were
dissolved in benzene (20 mL, 0.1M) and stirred vigorously while it refluxed for 24 h.
The mixture was cooled to r.t. and the solvent evaporated in vacuo. The mixture was
filtered without work-up through a silica gel plug (100% hexanes), and the residual oil
M.W.: 438.67
7.81-7.61 (m, 4 H, PhH), 7.43-7.33 (m, 6 H, PhH), 6.51 (d, J = 10.5 Hz, 1
H, H3), 4.18 (m, 2 H, H10), 3.55 (dq, J = 9.9, 3.7 Hz, 2 H, H1), 2.52 (m, 1
H, H2), 2.25 (m, 2 H, H6), 1.67 (m, 1 H, H8'), 1.27 (m, 1 H, H8''), 1.27 (t, J
= 7.1 Hz, 3 H, H11), 1.01 (s, 9 H, H12), 0.94 (t, J = 7.4 Hz, 3 H, H7), 0.83
(t, J = 7.4 Hz, 3 H, H9)
13
C NMR: (100 MHz, CDCl 3 )
167.83 (C(5)), 143.35 (C(3)), 135.65 (2 C, Ph), 135.63 (2 C, Ph'), 135.30
(C4)), 133.66 (C(Ph)), 133.63 (C(Ph')), 129.62 (C(Ph)), 129.61 (C(Ph')),
127.63 (4 C, Ph, Ph'), 66.58 (C(1)), 60.29 (C(10)), 43.13 (C(2)), 26.80 (3 C,
C(12)), 24.11 (C(8)), 20.43 (C(6)), 19.26 (C(13)), 14.30 (C(11)), 14.29
(C(7)) , 11.85 (C(9))
HRMS: (EI) m/z calcd for C 23 H 29 O 3 Si [(M-C 4 H 9 )+] 381.1880, found 381.1855
2.35 2.56
Ester 2.35 (327 mg, 0.96 mmol) was treated with a 10% HCl/MeOH (5 mL) solution
and stirred at r.t. for 3 h. The solution was slowly quenched with a saturated sodium
bicarbonate solution, diluted with H 2 O and EtOAc and extracted thrice with EtOAc. The
organic layers were combined, dried with Na 2 SO 4 and, after evaporation, the oil was
M.W.: 200.27
13
C NMR: (100 MHz, CDCl 3 )
167.6 (C(5)), 142.4 (C(3)), 136.8 (C(4)), 65.8 (C(1)), 60.5 (C(10)), 43.4
(C(2)), 24.0 (C(8)), 20.5 (C(6)), 14.3 (C(11)), 14.2 (C(7)), 11.8 (C(9))
2.56 2.57
Alcohol 2.56 (67 mg, 0.33 mmol) was pre-dissolved in an acidic solvent system of
aqueous 10% H 2 SO 4 (0.3 mL) and acetone (3 mL) while stirring at 0°C under an argon
atmosphere. Prior to addition, the Jones reagent was freshly prepared by vigorously
mixing in a vial potassium dichromate (294.8 mg, 1 mmol), conc. H 2 SO 4 (0.2 mL)
followed by distilled water (1 mL). The bright orange mixture (0.43 mL, 0.36 mmol, 1.1
equiv) was slowly syringed into the alcohol solution and stirred for 2.5 h at which time
the chromium's oxidation state change was evidenced by a concomitant color change
saturated solution of NaHCO 3 (4 mL) and dilution with EtOAc (1 mL). The aqueous
layer was acidified by an aqueous 0.1M HCl solution until the pH reached ~2.0. After
extraction with EtOAc (3x5 mL), drying of organic layers with Na 2 SO 4 and evaporation
solvent in vacuo, the oil was purified by column chromatography (20% EtOAc-hexanes)
M.W.: 214.26
6.60 (d, J = 10.4 Hz, 1 H, H3), 4.18 (q, J = 7.2 Hz, 2 H, H10), 3.30 (dt, J =
6.8, 3.6 Hz, 1 H, H2), 2.34 (m, 2 H, H6), 1.88 (m, 1 H, H8'), 1.63 (m, 1 H,
H8''), 1.29 (t, J = 7.2 Hz, 3 H, H11), 1.02 (t, J = 7.6 Hz, 3 H, H7), 0.94 (t, J
= 7.6 Hz, 3 H, H9)
13
C NMR: (100 MHz, CDCl 3 )
177.8 (C(1)), 167.2 (C(5)), 137.0 (C(3) & C(4)), 60.7 (C(10)), 46.3 (C(2)),
25.8 (C(8)), 20.5 (C(6)), 14.2 (C(11)), 14.0 (C(7)), 11.7 (C(9))
(E)-Diethyl 6-((tert-butyldiphenylsilyloxy)methyl)-4-ethyl-3-oxooct-4-en-2-
ylphosphonate (2.61)
O O O
EtO
P
EtO OTBDPS EtO OTBDPS
2.35 2.61
Triethyl ethylphosphonate 2.60 (100 µL, 0.62 mmol, 2.5 equiv) was dissolved in
THF (1.5 mL) and cooled to -78°C while stirring under argon. After 10 min n-BuLi (250
µL, 0.63 mmol, 2.5 equiv) was added and warmed up to -40°C over 1.5 hrs. Ester 2.35
(109 mg, 0.25 mmol) dissolved in THF (1 mL) was cooled to -40°C and transferred by
(1 mL, 10%), diluted with water (0.5 mL) and extracted with EtOAc (3 x 2 mL). Dried
with MgSO 4 and evaporated solvent in vacuo. The viscous oil was purified by column
1.4:1 ratio) of phosphonate diastereomers 2.61 (84 mg, 0.15 mmol, 60%).
M.W.: 558.76
7.64-7.59 (m, 4 H), 7.42-7.33 (m, 6 H), 6.41 (d, J = 10.1 Hz, 1 H), 6.29 (d,
J = 10.1 Hz, 1 H), 4.07 (m, 4 H), 3.83 (m, 1 H), 3.60 (m, 2 H), 2.62 (m, 1
H), 2.26 (m, 1 H), 1.74 (m, 1 H), 1.31 (m, 1 H), 1.15 (t, J = 7.1 Hz, 3 H),
1.03 (s, 9 H), 1.00 (s, 9 H), 0.85 (m, 6 H)
13
C NMR: (100 MHz, CDCl 3 )
197.94, 197.89, 197.47, 197.43, 145.79, 144.31, 144.25, 144.22, 144.19,
144.17, 135.59, 135.58, 135.54, 130.89, 129.775, 129.71, 129.58, 127.73,
127.70, 127.61, 66.54, 66.14, 62.59, 62.57, 62.52, 62.50, 62.37, 62.31,
62.26, 62.19, 43.69, 43.66, 40.63, 40.18, 39.29, 38.87, 36.49, 26.86, 26.79,
24.29, 24.24, 19.66, 19.64, 19.27, 16.47, 16.45, 16.42, 16.37, 16.32, 14.07,
13.97, 13.93, 13.12, 13.06, 12.81, 12.74, 11.85, 11.77
(E)-4-((tert-Butyldiphenylsilyloxy)methyl)-2-ethyl-N-methoxy-N-methylhex-2-
enamide (2.36)
12
O O 13
11 3 1
4 2 Si
EtO OTBDPS N 5 O
O 6 8
10 7 9
2.35 2.36
Ester 2.35 (343 mg, 0.78 mmol) and N,O-dimethylhydroxylamine hydrochloride (117
mg, 1.2 mmol, 1.5 equiv) were stirred vigorously in THF (4 mL, 0.2M) and cooled under
argon to -20°C. i-PrMgCl (1.2 mL, 2.4 mmol, 3.0 equiv) was added over 10 minutes and
warmed up to 0°C. After 2 hrs, quenched with NH 4 Cl (2 mL) and extracted with EtOAc
(2 x 5 mL). Dried with MgSO4 and after evaporation of solvent in vacuo the crude was
mg, 0.47 mmol, 61%) while also recovering unreacted ester 2.35 (130 mg, 0.30 mmol).
M.W.: 453.69
7.64-7.62 (m, 4 H, PhH), 7.43-7.32 (m, 6 H, PhH), 5.48 (d, J = 10.1 Hz, 1
H, H3), 3.58 (s, 3 H, H10), 3.54 (d, J = 6.2 Hz, 2 H, H1), 3.20 (s, 3 H,
H11), 2.46 (m, 1 H, H2), 2.67 (dq, J = 7.6, 0.9 Hz, 2 H, H6), 1.69 (m, 1 H,
H8'), 1.23 (m, 1 H, H8''), 1.03 (s, 9 H, H12), 0.94 (t, J = 7.5 Hz, 3 H, H7),
0.85 (t, J = 7.4 Hz, 3 H, H9)
13
C NMR: (75 MHz, CDCl 3 )
171.96 (C(5)), 138.33 (C(3)), 135.59 (2 C, Ph), 135.57 (2 C, Ph'), 133.73
(C(4)), 133.68 (C(Ph)), 133.62 (C(Ph')), 129.60 (C(Ph)), 129.58 (C(Ph')),
127.61 (4 C, Ph, Ph'), 66.70 (C(1)), 60.82 (C(10)), 42.30 (C(2)), 34.13
(C(11)), 26.84 (3 C, C(12)), 24.30 (C(8)) , 21.80 (C(6)), 19.23 (C(13)),
13.21 (C(7)), 11.73 (C(9))
HRMS: (EI) m/z calcd for C 23 H 30 NO 3 Si [(M-C 4 H 9 )+] 396.1989, found 396.2013
(E)-6-((tert-Butyldiphenylsilyloxy)methyl)-4-ethyloct-4-en-3-one (2.37)
12
O O 13
11 3 1
10 4 2 Si
N OTBDPS 5 O
O 6 8
7 9
2.36 2.37
Amide 2.36 (80 mg, 0.18 mmol) was dissolved by stirring in THF (1.8 mL, 0.1M)
cooled under argon to 0°C. After 10 min, EtMgBr (0.14 mL, 0.42 mmol, 3.0 equiv) were
added and the solution stirred for another 2 hrs at 0°C. Quenched with NH 4 Cl (0.5 mL),
diluted with water (0.3 mL) and extracted with EtOAc (3 x 2 mL). Dried with MgSO4
and after evaporation of solvent in vacuo the crude was purified by column
chromatography (20% EtOAc-hexanes) isolating ketone 2.37 (47 mg, 0.11 mmol, 62%)
while also recovering unreacted amide 2.36 (20 mg, 0.044 mmol).
M.W.: 422.67
7.64-7.60 (m, 4 H, PhH), 7.43-7.33 (m, 6 H, PhH), 6.33 (d, J = 10.2 Hz, 1
H, H3), 3.63 (m, 2 H, H1), 2.60 (m, 3 H, H2, H10), 2.25 (dq, J = 7.4, 1.1
Hz, 2 H, H6), 1.68 (m, 1 H, H8'), 1.23 (m, 1 H, H8''), 1.07 (t, J = 7.3 Hz, 3
H, H11), 1.01 (s, 9 H, H12), 0.88 (t, J = 7.4 Hz, 3 H, H7), 0.84 (t, J = 7.5
Hz, 3 H, H9)
13
C NMR: (100 MHz, CDCl 3 )
202.36 (C(5)), 144.11 (C(4)), 143.36 (C(3)), 135.62 (2 C, Ph), 135.58 (2 C,
Ph'), 133.56 (2 C, Ph, Ph'), 129.71 (C(Ph)), 129.68 (C(Ph')), 127.67 (4 C,
Ph, Ph'), 66.59 (C(1)), 43,28 (C(2)), 30.60 (C(10)), 26.81 (3 C, C(12)),
24.27 (C(8)), 19.38 (C(6)), 19.27 (C(13)), 14.25 (C(7)), 11.85 (C(9)), 8.94
(C(11))
HRMS: (EI) m/z calcd for C 23 H 29 O 2 Si [(M-C 4 H 9 )+] 365.1931, found 365.1925
(4E,7E,9E,12E)-5,9,11-Triethyl-3-(hydroxymethyl)-7-methyl-13-phenyltrideca-
4,7,9,12-tetraen-6-one (2.38)
O O O
11 9 7 3 1
12 10 8 6 4 2
+ OTBDPS 5 OH
13 15 17 18 20
14 16 19 21
Aldol. Ketone 2.37 (386 mg, 0.92 mmol) was dissolved in DCM (15 mL, 0.03M)
and cooled under argon to -78°C. After stirring for 20 min, to the clear solution, titanium
tetrachloride (TiCl 4, 121 µL, 1.1 mmol, 1.2 equiv) was added dropwise over 15 min. The
light yellow solution was stirred for an additional 30 min at -78°C at which time N,N-
diisopropylethylamine (DIPEA, 192 µL, 1.1 mmol, 1.2 equiv) was added, immediately
red/purple hue. Stirred the solution for 1 hr at -78°C, after which a solution of aldehyde
2.30/2.30a (158 mg, 0.69 mmol, 0.75 equiv) dissolved in DCM (20 mL) was added by
syringe pump (0.7 mL/hr) overnight. Quenched with MeOH (10 mL), and slowly
warmed up to r.t. The solution was diluted with water (10 mL) and extracted with EtOAc
(3 x 10 mL). Dried the organic phase with MgSO 4 , evaporated solvent in vacuo, and
partially purified the crude oil by column chromatography (5% EtOAc-hexanes) to obtain
92
β-hydroxy ketone (272 mg) which was mesylated without further purification.
mmol), DMAP (114 mg, 0.93 mmol, 2.5 equiv) were dissolved in DCM and cooled at -
20°C while stirring under argon. Slowly added TEA (780 µL, 5.6 mmol, 15 equiv)
followed by methanesulfonyl chloride (MsCl, 147 µL, 1.9 mmol, 5.0 equiv). The
solution was slowly warmed up to r.t. and stirred overnight. Quenched with a saturated
solution of NaHCO 3 (5 mL), diluted with H 2 O (10 mL), and extracted with EtOAc (3 x
10 mL). Dried the organic phase with MgSO 4 , evaporated solvent in vacuo, and partially
purified the crude oil by column chromatography (5% EtOAc-hexanes). The oil obtained
was deprotected by addition of TBAF (2 mL, 2.0 mmol, 5.5 equiv) to a solution in THF
(10 mL) while stirring at r.t. under argon overnight. Quenched by addition of H 2 O and
extracted with EtOAc (3 x 5 mL). The organic phase was dried with MgSO 4 , evaporated
solvent in vacuo, and purified the crude oil by column chromatography (10% EtOAc-
hexanes) to furnish alcohol 2.38 (20 mg, 0.051 mmol, 6% over three steps) and its
M.W.: 394.59
201.98 (C(5)), 144.69 (C(8)), 143.03 (C(7)), 139.70 (C(6)), 139.68 (C(3)),
137.56 (C(4)), 135.24 (C(9)), 135.11 (C(Ph)), 132.84 (C(11)), 129.15
(C(12)), 128.48 (2 C, Ph), 127.05 (C(Ph)), 126.01 (2 C, Ph), 65.95 (C(1)),
43.62 (C(10)), 43.23 (C(2)), 28.68 (C(13)), 24.28 (C(18)), 24.11 (C(20)),
21.22 (C(15)), 14.31 (C(17)), 13.51 (C(19)), 13.48 (C(16)). 11.91 (C(21)),
11.88 (C(14))
HRMS: (EI) m/z calcd for C 19 H 23 O [(M-C 8 H 15 O)+] 267.1740, found 267.1743
(2E,6E,8E,11E)-2,4,8,10-Tetraethyl-6-methyl-5-oxo-12-phenyldodeca-2,6,8,11-
tetraenal (2.39)
O O O
11 9 7 3
12 10 8 6 4 2
OH 5 1 H
13 15 17 18 20
14 16 19 21
2.38 2.39
Oxalyl chloride ((COCl) 2 , 16 µL, 0.18 mmol, 3.5 equiv) was dissolved in
dichloromethane (2.0 mL) and cooled to -78°C. Dimethylsulfoxide (17 µL, 0.26 mmol,
5.0 equiv) was added dropwise and the solution stirred for an additional 30 min at -78°C.
A precooled (-78°C) solution of alcohol 2.38 (20 mg, 0.052 mmol) in DCM (0.5 mL) was
added by cannula and the white and opaque mixture was stirred for 1 h. Triethylamine
(73 µL, 0.52 mmol, 10 equiv) was added and the thick slurry was slowly warmed to r.t.
mL). The organic phase was dried over MgSO 4 and filtered. After evaporation in vacuo
mixture of aldehyde 2.39, and uncharacterized stereroisomers (20 mg, 0.052 mmol,
100%).
M.W.: 392.57
1
H NMR: (500 MHz, CDCl 3 )
9.38 (s, 1 H, H1), 7.17-7.35 (m, 5 H, PhH), 6.97 (d, J = 1.1 Hz, 1 H, H7),
6.46 (d, J = 10.5 Hz, 1 H, H3), 6.37 (dd, J = 16.2, 3.3 Hz, 1 H, H12), 6.08
(dd, J = 15.9, 7.4 Hz, 1 H, H11), 5.40 (d, J = 9.8 Hz, 1 H, H9), 4.29 (m, 1
H, H4), 3.13 (bp, J = 7.7 Hz, 1 H, H10), 2.28 (m, 4 H, H15, H20), 1.95 (d,
J = 0.9 Hz, 3 H. H17), 1.66-1.46 (m, 2 H), 1.25 (m, 2 H), 1.01-0.80 (m, 12
H)
2.5.3. References
O O
O O
2.28 2.28a
96
OH OH
2.29 2.29a
97
O O
2.30 2.30a
98
O O
O O
2.41 2.41a
99
OH OH
2.42 2.42a
100
O O
2.43 2.43a
101
EtO EtO
O O
OH OH
+
2.50 + 2.50a
Reigioisomer 1 Regioisomer 2
102
HO OH
2.32
103
TBDPSO OH
2.33
104
TBDPSO O
2.34
105
EtO OTBDPS
2.35
106
EtO OH
2.56
107
O O
HO OEt
2.57
108
O
O
N OTBDPS
2.36
109
OTBDPS
2.37
110
OH
2.38
111
112
2.39
113
3.1. Introduction
products has been a lively area of research.1 The great complexity and variety of these
These large structures are rife with synthetic challenges owing to case-specific
thermodynamic considerations of size, entropy and strain of the macrocycle and its
taken have commonly been classified by the mode of activation of the alcohol or acid
involved in the reaction. Both strategies involve chemoselective reactions that polarize
the targeted moiety and consequently increase their reactivity. Some common acid
and O-acylisoureas4 (3.3) (Figure 3.1-A). Although less prevalent, alcohol activation
methodologies are also employed, and they generally involve its conversion to leaving
FIGURE 3.1. Macrolactones can be synthesized by numerous (A) acid activation and (B) alcohol activation
methods.
process that lengthens routes and minimizes yields. Few compounds in the literature with
processes may provide a viable alternative towards this important objective, in particular
such as spiramycin and pimaricin have been clinically relevant in the treatment and
prevention of common human and animal microbial ailments.11-16 Others, such as the
epothilone family of natural products and their synthetic analogs, represent a class of
115
novel anticancer agents that exhibit greater effectiveness and lower toxicity than the
FIGURE 3.2. Macrolide polyketides share a common biosynthetic origin which is capable of generating
molecules of great structural diversity and assorted therapeutic applications.
pathways.2-8 Erythromycin, the canonical macrolide system, and its core precursor 6-
three large open reading frames each coding for the homodimeric megaproteins DEBS1,
DEBS2 and DEBS3.26 Each megaprotein includes separate modules with multiple
equivalents, and reductively tailor the newly formed keto functional group (Figure 3.3).
116
extension, is that catalyzed by the DEBS thioesterase domain (TE).27-29 The enzyme
assures substrate turnover from the PKS, delivering it in its bioactive conformation. Its
(Figure 3.4).30 The active site of the DEBS TE is contained within a hydrophobic
substrate channel that effectively traverses the monomer, a feature that is in contrast to
the homologous fatty acid thioesterases (FA TEs)31. The presence of a substrate channel
was shown to not be an isolated example, but a conserved element of PKS TEs, and has
117
been seen in structures of the picromycin32 and curacin33 thioesterases. The function of
the substrate channel is a topic of debate; a widely viewed hypothesis27 predicts the
polyketide from its ACP-bound state to the TE active site takes place at one end of the
channel, while exit of the macrocyclic or hydrolytic product occurs at the opposite end.
the DEBS-TE in close proximity to the substrate channel that presumably serves as a
A. B.
C.
FIGURE 3.4. DEBS TE crystal structure (PDB ID: 1KEZ). (A) Secondary structure representation of the
homodimer. (B) Van der Waals space-filling model. The substrate channel traversing the enzyme is
conspicuous. (C) Topographical map of DEBS TE. The connectivity of the enzyme's α-helices (red rods)
and β-strands (yellow arrows) is shown. The catalytic residue (red dots) and their approximate location is
also shown.
The DEBS TE active site is comprised of the conserved catalytic triad Ser-142, Asp-
169, His-259.34 In a classic charge-relay mechanism (Figure 3.5), key residue Ser-142 in
the active site is deprotonated to generate a strong O-nucleophile that attacks the
incoming S-acyl-ACP, cleaving the thioester bond.35 In vivo, this new O-acyl-TE
118
FIGURE 3.5. Canonical mechanism of the TE domain. Common among α/β hydrolase family, it involves a
charge relay mechanism. The active site serine is deprotonated in the loading step, and a new O-acyl-
enzyme intermediate is formed upon its attack on the upstream S-acyl-ACP. The conformation of the
substrate in the channel may lead to either a regioselective intramolecular macrocyclization or
intermolecular attack by a molecule of water to yield a seco-acid. The latter has been observed as an
occurrence of in vitro experiments.
Very little is known about the underlying factors that determine the regioselectivity of
FIGURE 3.6. DEBS TE competently loads a wide range of linear chain lengths.
interactions as the main stabilizing forces of the substrate in the channel,41 likely inducing
119
weak hydrophilic barrier at the PICS TE channel’s exit consisting of bulk water and a
glutamine residue has been proposed to explain the substrate’s bent arrangement in the
channel and the proximity of the proper O-nucleophile to the active site.32
A recent study of serine hydrolases and acyltransferases42, both belonging to the α,β-
fold class, correlated differences in the type of β-turn present between helix A and B in
configuration of this element, Kazlauskas et al. propose it can either increase the
make way for a nucleophile to gain access to the O-acyl enzyme intermediate. This turn
on sequence data.
No systematic study has been yet conducted with a full length substrate where the
combined with its challenging multistep synthesis,43-47 has been an impediment to this
in several structural variants for the examination of their interaction with the enzyme,
would be out of the question because of its intricacy and inefficiency. Furthermore, a
thioester such as 3.10 would be susceptible to multiple side reactions that would impede
their utility in such a study (Figure 3.7). For example, the δ-hydroxy ketone structure in
120
of a δ-lactone would be a highly favorable step, yielding structure 3.11. These examples
serve to highlight the important role the binding cavity has in stabilizing the
FIGURE 3.7. Putative side reactions of NAC-6dEB substrate 3.10 meant to illustrate the difficulties
associated with its use as a probe for thioesterase macrocyclization. Under thermodynamic control,
hemiketalization (structures 3.8 and 3.9) would likely take place. In addition, formation of a δ-lactone
would be kinetically favored to yield structure 3.11.
Our lab recently reported the ability of wild-type, heterologously expressed DEBS TE
FIGURE 3.8. Substrate analogs designed by Wang et al. to probe the kinetics of the loading step with wild-
type polyketide thioesterases (Deb, Pim, Epo). 7-Aminoheptanoate (3.12) is highlighted.
(3.7, Figure 3.9). The other half of our envisioned analog, a phenylhexanoic acid moiety
FIGURE 3.9. 6dEB analog 3.13 designed to explore the DEBS TE regiochemistry of cyclization. Linear
6dEB is shown for ease of comparison with our synthetic target.
Among the salient features in the design of the substrate is the inclusion of a phenyl
structural element,52 and its presence is expected to simplify the analysis of UV traces
obtained by LC/MS. Retaining the native substrate's 1,3 diol would be key in providing
would aid in correlating stereo- and regioselectivity of the enzyme. Lastly, the
native substrate.
Initial work with the racemic and diastereoisomeric mixture of isomers, substrate 3.13
including two peaks that suggested either the presence of macrolactone regioisomers 3.16
3.18
3.16 / 3.17
3.13
3.19
Time (min)
FIGURE 3.10. Treatment of isolated DEBS TE with analog 3.13 generated a mixture of products (as
monitored by LCMS).
contributions of all stereoisomers to the observed product distribution. For this purpose,
a stereocontrolled synthetic route was designed and a suitable hypothesis was formulated
to guide and inform our discoveries. We have based the latter on a stereochemical
pattern observed on numerous substrates ably cyclized by a DEBS TE. We believe that
123
FIGURE 3.11. We propose that the absolute stereochemistry of the terminal O-nucleophile (highlighted in
red) determines the outcome of the TE-catalyzed macrolactonization.
Herein we present: (1) the total synthesis of four enantioenriched substrates; (2) the
total synthesis of authentic macrolactone standards; (3) results of our enzymatic assays;
and (4) a brief synopsis of our future efforts towards this project.
3.2.1. Retrosyntheses
To evaluate the contribution each discrete stereoisomer of 3.13 made to the product
mixture observed upon treatment with the DEBS TE (Figure 10), we developed a general
stereoselective route that would allow us to efficiently synthesize them. The salient
SCHEME 3.1. Retrosynthesis of enantioenriched NAC substrates. For simplicity, only isomer 3.29 is
shown.
Our analysis begins with thioester 3.29, one of four stereoisomers necessary for our
convenient and broadly employed methodology for mimicking CoA and ACP-bound
thioesters, enabling the incorporation of substrates onto polyketide and fatty acid
synthases. Disconnection of 3.29 at the amide bond (shown), reveals the NAC-thioester
available HSNAC and seco-acid 3.33. The chiral dihydroxyacid coupling partner of
amine 3.15 can be obtained from terminal olefin 3.25 through oxidative cleavage of the
regioselective vinyl cuprate addition to chiral epoxide 3.22. The latter is a product of a
mixture derived from mCPBA epoxidation of the chiral olefin 3.21. Disconnection of
such as the Brown allylboration,54 to set the first stereocenter of the route.
would be possible only if a set of standards was also synthesized. Toward this goal, we
designed a route adapted from the stereoselective methodology employed in the synthesis
since only diastereopurity (not enantiopurity) was needed (Scheme 3.2). Two racemic,
diastereomerically pure 1,3-diols (syn and anti) are at the centerpiece of our design.
Cleavage of syn macrolactones syn-3.42 and syn-3.43 reveals diprotected methyl ester
conformational bias and large number of degrees of freedom of these linear molecules
would provide access to both ring sizes in about equal amounts.55 Macrolactonization
selectivity dependent on the conformation of the chains and energies of the transition
state. The enthalpy differences between 12- and 14-membered rings56 suggest of a slight
Disconnection at the bond shown yields intermediates syn-3.40 and 3.34, both easily
accessed from simple precursors and chemical steps. In the forward sense oxidative
cleavage of the terminal olefin of syn-3.40 is expected to procure the carboxylic acid
the separation of the diastereomers of olefin 3.38 would not be possible without prior
removal of the TBS protecting group. Removal afforded only limited success with the
preparative HPLC step to cleanly separate the mixture. At this juncture, olefin 3.38 would
be accessible even more readily than homochiral analog 3.25 (Scheme 3.1), starting from
SCHEME 3.2. Retrosynthesis of the racemic 12 and 14-membered macrolactone standards syn-3.42 and
syn-3.43. For simplicity, only the route of the syn-standards is shown.
127
3.2.2. Syntheses
complementary routes in under nine linear steps. Our enantioselective route was initiated
bath at -100 °C for 30 minutes (Scheme 3.3) furnishing homoallylic alcohols 3.21 and
ent-3.21 in 63% and 44% yield respectively. Epoxidation of each enantiomer with a
SCHEME 3.3. Synthesis of enantioenriched epoxides 3.22, 3.23, ent-3.22 and ent-3.23.a
a
Reagents and conditions: (a) (-)-Ipc 2 BAllyl, Et 2 O, -100°C, 30 min, 63%; (b) (+)-Ipc 2 BAllyl, Et 2 O, -
100°C, 30 min, 44%; (c) m-CPBA, NaHCO 3 , CH 2 Cl 2 , r.t., 12hr; TBSCl, Imidazole, DMF, r.t., 24hr;
(R,R)-Salen, H 2 O, THF, 16hr, 39% over three steps; (d) m-CPBA, NaHCO 3 , CH 2 Cl 2 , r.t., 12hr; TBSCl,
Imidazole, DMF, r.t., 24hr; (S,S)-Salen, H 2 O, THF, 16hr, 51% over three steps; (e) Bu 2 SnO, TsCl,
CH 2 Cl 2 , 4hr; NaH, THF, overnight, 100% over two steps; (f) Bu 2 SnO, TsCl, CH 2 Cl 2 , 4hr; NaH,
overnight, THF, 58% over two steps.
All four epoxide stereisomers were obtained by using the Jacobsen hydrolytic kinetic
128
3.23, and yielded enantioenriched epoxides 3.22 and ent-3.22 (50%, 38% yield
respectively), and triols 3.24 and ent-3.24 (42%, 36% yield respectively). The triols were
respectively (over two steps). This straightforward route provided access to our four key
With all four epoxide isomers in hand, we selected 3.22 to examine the steps leading
smoothly (85%) to produce olefin 3.25 in 85% yield. The final steps to our key acid
began by the silylation of alcohol 3.25, which was immediately followed by the oxidative
cleavage of its terminal olefin with a buffered KMnO4-NaIO4 mixture. This step, after
workup and aqueous extraction, gratifyingly yielded the expected acid as confirmed by
1
H NMR of the crude product mixture. The key acid's coupling partner, NAC-
Regrettably, standard deprotection conditions of 3.12’s terminal amino group (20% TFA
SCHEME 3.4. First attempt towards the synthesis of substrate 3.29.a Failure to cleanly deprotect 3.12
hindered our progress.
a
Reagents and conditions: (a) VinylMgBr, CuI, THF, -78°C, 85%; (b) HSNAC, EDC, DMAP, THF, r.t.,
87%.
To solve this problem, we examined a longer route relying on the amino acid methyl
ester 3.34 as our coupling substrate (Scheme 3.5). 3.34 was synthesized by esterification
followed by KMnO4/NaIO4 cleavage of olefin 3.25. The crude acid was immediately
treated with EDC and coupled with 3.34 to yield bis-silylated substrate 3.31 in 28% yield
over three steps. Hydrolysis with LiOH in MeOH/THF/H2O, followed by EDC coupling
with HSNAC yielded thioester 3.27 in 53% yield over two steps. Deprotection in an
SCHEME 3.5. End game for the synthesis of NAC enantioenriched substrate 3.29.a
a
Reagents and conditions: (a) TMSCl, MeOH, r.t., overnight, 100%; (b) TBSCl, Imidazole, DMF, r.t.,
overnight; KMnO 4 , NaIO 4 , 1:1 t-BuOH-buffer (pH 7), r.t., 1 h; 3.34, EDC, DMAP, CH 2 Cl 2 , r.t. overnight,
28% over three steps; (c) LiOH, 3:2:1 MeOH/H 2 O/THF, r.t., 6h; HSNAC, EDC, DMAP, CH 2 Cl 2 , r.t.,
overnight, 53% over two steps; (d) HF, Pyridine, MeCN, r.t., 5hrs, 35%.
To shorten our synthesis and improve our overall yield, we explored the literature for
We came across the work of Kokotos et al.60 that reported an efficient removal of this
protected amine 3.12 with this solution, we were pleased to observe clean and
presented in Scheme 3.6; it differs only from our first attempt in the use of amine 3.15 in
Reagents and conditions: a) VinylMgBr, CuI, THF, -78°C, 85%; (b) 3.12, HCl/Dioxane, r.t., 45 min;c)
TBSCl, Imidazole, DMF, r.t., overnight; KMnO 4 , NaIO 4 , 1:1 t-BuOH-buffer (pH 7), r.t., 1 h; 3.15, EDC,
DMAP, CH 2 Cl 2 , r.t. overnight; HSNAC, EDC, DMAP, CH 2 Cl 2 , r.t., overnight; (d) HF, Pyridine, MeCN,
r.t., 5hrs.
furnished epoxide 3.36 (77% yield) as evidenced by the loss of the signature olefinic
132
NMR signals between 5-6 ppm. Predictably, the NMR integration ratios point towards
a
Reagents and conditions: (a) AllylMgBr, Et 2 O, 0°C, 30 min, 100%; (b) mCPBA, CH 2 Cl 2 , r.t., 12hr,
77%; (c) TBSCl, Imidazole, DMF, r.t., 24hr, 100%; (d) VinylMgBr, CuI, THF, -20°C, 35%; (e) PTSA,
EtOH, r.t., overnight, 60%; (f) TBSCl, Imidazole, DMF, r.t., overnight; (g) KMnO 4 , NaIO 4 , 1:1 t-BuOH-
buffer (pH 7), r.t., 1 h; 3.34, EDC, DMAP, CH 2 Cl 2 , r.t; (h) LiOH, THF/H 2 O, r.t.; HF/MeCN/Pyr, 5h, r.t.;
(i) (PyS) 2 , PPh 3 , THF, r.t.; dilute by syringe pump addition to toluene at 80°C.
133
The regioselective vinylation of epoxide 3.36 required the protection of the alcohol
moiety. This was accomplished with TBSCl64 to produce epoxide 3.37 in quantitative
homoallylic alcohols in 35% yield. Attempts to separate 3.38 into its diastereomeric
components by reverse-phase PHPLC were initially unsuccessful. From many test runs,
a compromise was reached, requiring the removal of all protective groups from the
mixture by treatment with p-TsOH, to furnish the more polar 1,3-diol 3.39 in 60% yield.
chromatography (PHPLC). The mixture was found to separate well under the conditions
stated, providing pure diols syn-3.39 and anti-3.39, both in 32% yield. Their
stereochemistry was unambiguously established by 13C NMR from their respective syn-
and anti-acetonides (Scheme 3.8).66 Treatment of each diol with a solution of p-TsOH in
SCHEME 3.8. Rychnovsky's acetonide methodology was used to determine the relative stereochemistry of
racemic 1,3-diols separated by PHPLC.a
a
Reagents and conditions: (a) Acetone, PTSA, r.t., overnight.
both anti-3.39 and syn-3.39 to a sequence comprised of a silylation with TBSCl under
134
permanganate67 in tert-butanol, and lastly, EDC coupling with free amine 3.34 to provide
full length chain esters anti-3.41 and syn-3.41 in 32% and 36% yields, respectively
(Scheme 3.7). The endgame route to our macrocycle standards was initiated by the basic
hydrolysis of each anti-3.41 and syn-3.41 ester. The crude acids were individually
2-pyridinethioesters. These were slowly added by syringe pump to hot toluene (80 °C) to
effect the high dilution conditions necessary to minimize intermolecular reactivity. The
reactions were monitored by LC/MS and after no more than 10 days the products anti-
3.42, anti-3.43, syn-3.42, syn-3.43 were purified by PHPLC and isolated (2%, 0.1%,
18%, 7% respectively).
four simultaneous enzyme assays with recombinant DEBS TE and each of our
enantiopure NAC-thioester substrates. Wild type DEBS TE (213 μM) employed in our
assays was expressed and purified as previously described.51 Our four enzymatic
DEBS TE with 2.5 mM substrate in 50 mM phosphate buffer (pH 7.4) at 20°C with
DMSO (5% v/v) as a solubility additive. After 18 hrs, the reaction was diluted 4-fold and
FIGURE 3.12. LC traces and product distribution from enzymatic assays with wild type DEBS TE and
enantioenriched NAC substrates.
136
The results from syn NAC-substrates 3.29 and ent-3.29 are fully consistent with our
cyclizes substrate 3.29. Comparison of the retention time from the assay's macrocyclic
product and the syn-3.42 standard confirms that DEBS TE regioselectively generates the
14-membered ring macrolactone product from 3.29. Substrate ent-3.29 is not converted
However, our assay results for the anti NAC-substrates 3.30 and ent-3.30 did not
fully agree with our working hypothesis. While substrate ent-3.30 underwent hydrolysis
exclusively, consistent with our hypothesis, unexpectedly 3.30 also produced a seco-acid.
macrocyclic product of 3.30 to be highly analogous to the reaction's transition state, and
thus bind tightly in the active site. This will favor restoration of the acyl-enzyme
intermediate. Subsequent hydrolysis will rapidly release the seco-acid of 3.30 from the
macrolactone was competently loaded on to the Epo TE and hydrolyzed to form the seco
acid. We thus set to examine if this mode of reactivity was operative within the DEBS
TE.
(Figure 3.13). Hydrolysis was not observed even under reaction conditions and
incubation times identical to those used for macrocyclization formation. This result
137
unambiguously confirms the DEBS TE's inability to open the macrocycle. Furthermore,
FIGURE 3.13. Results of the enzymatic hydrolysis assay of macrolactone standard 3.42 by the DEBS TE.
(A) Standard anti-seco-3.41; (B) Hydrolysis assay at180 min; (C) negative control with no enzyme.
3.3. Conclusions
Our systematic study of our substrate analogs and their relationship with the DEBS
Our initial hypothesis stated that the absolute stereochemistry of the nucleophilic alcohol
138
the observation that all isolated macrocyclic products from the DEBS TE have
macrocyclization selectivity of the DEBS TE. The hydrolysis of substrate 3.30, was
analogous to the native substrate. This implies that additional interactions between the
enzyme and the native substrate not captured in our analog are required for the proper
enzyme intermediates of substrates 3.29 and 3.30. This system is expected to provide
Our data demonstrates that substrate 3.30 does not model all the native features of the
6dEB substrate. Previous work hypothesized the importance of the C9 carbonyl to effect
the bent conformation of the native substrate.32 In addition, our hypothesis underlines the
effect macrocyclization. Both these features are likely complementary, yet insufficient in
explaining the macrocyclization specificity of the DEBS TE. Previous examples show
that methyl side chains close to the O-nucleophiles are highly conserved.36-40 Due to the
hydrophobic nature of the substrate-enzyme interactions, these methyl side chains may
play an important role in dictating the conformation of the substrate in the enzyme active
site, and thus its reactivity. Ongoing work in our lab is focusing on introducing these key
side chains into our substrates, to evaluate if they are sufficient to make substrate 3.30
functional groups that are necessary and sufficient to predict the macrocyclization
The DEBS TE has been used as a critical feature in numerous engineered PKS
pathways to produce macrocycles. However, for each of these published success stories
there are countless examples of engineered chimeric systems which did not produce
activity suggests that its exquisite and as yet unpredictable substrate specificity may limit
because of its reliable hydrolytic activity, it is ideally suited for applications where a
carboxylic acid metabolite is desired. For example, a recent study relied on engineered
PKS enzymes terminating in the DEBS TE to hydrolyze key metabolite intermediates for
employed in the macrocyclization of natural products remain limited in their scope and
would be ideal in examples where macrolactonization steps have been problematic, such
approaches can be devised to find them. The first approach involves screening known
native TEs from a variety of PKS pathways in the hope of finding one with broad
have likely influenced TEs to retain stringent substrate selectivity, likely rendering this
structural data has been reliably used to modify substrate specificity71, 72, this approach is
perhaps best suited to producing broadly substrate tolerant TEs for multiple applications.
142
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148
Reactions were carried out under an argon atmosphere with dry solvents and oven-
dried glassware under anhydrous conditions unless specified otherwise. All reactions
were carried out under an inert argon atmosphere with dry tetrahydrofuran, diethyl ether,
and DCM solvents by passing them through activated alumina columns. Toluene,
benzene, and triethylamine were freshly distilled over calcium hydride. Reagents were
purchased at the highest commercial quality and used without further purification, unless
homogeneous materials.
out on 0.25 mm E. Merck silica gel plates (60F-254) using UV light as a visualizing
agent and/or ceric ammonium molybdate (CAM), p-anisaldehyde (PA), and potassium
performed with E. Merck silica gel (60, particle size 0.040-0.063 mm). Preparative thin-
layer chromatography (PTLC) separations were carried out on 0.25 mm E. Merck silica
Data are reported as follows: chemical shifts in ppm (δ); multiplicities are indicated by
Agilent 6890 fitted with a flame ionization detector (He carrier gas, 3 mL/min). The
column used was a J&W Scientific chiral Cyclosil B column (30m x 0.25mm x 0.25µm).
The injector temperature was 250°C, the detector temperature was 320°C. Retention
times (t R ) and integrated ratios were obtained from the Agilent Technologies GC
Chemstation software. The temperature program used for the analytical GC was as
Agilent 1100 series HPLC-DAD system equipped with a reversed-phase analytical scale
Gemini 250 × 21.2 mm column, particle size 10 µm. Preparative high pressure liquid
chromatography (PHPLC) was carried out on an Agilent 1200 series system comprising a
LC/MS/MS System (Applied Biosystems) equipped with a turbo-ion spray ESI probe
interfaced with a Prominence UFLC (Shimadzu) equipped with a reverse phase BDS
acid resin (Ni-NTA) from QIAGEN, while antibiotics and media/buffer components were
purchased from Fisher Scientific. Bacterial concentration (OD 600 ) was monitored with a
150
(BrandTech).
1-Phenylpent-4-en-2-ol (rac-3.21)
OH
2
5 3 1
O 4
3.20 rac-3.21
Phenylacetaldehyde (3.20, +90% purity) (1.12 mL, 10.0 mmol) was dissolved in
diethyl ether (Et 2 O, 100 mL, 0.1 M) and cooled to 0°C while vigorously stirring under an
argon atmosphere. After 20 minutes, allylmagnesium bromide (10.5 mL, 10.5 mmol,
1.05 equiv) was added dropwise over 5 min. The reaction was monitored by TLC and
after 30 min. it was quenched with an aqueous saturated solution of NH 4 Cl (50 mL).
Diluted mixture with EtOAc (50 mL) and extracted aqueous layer with EtOAc (4x30
mL). The combined EtOAc fractions were washed brine (2x100 mL) and subsequently
dried over anhydrous Na 2 SO 4 . The slurry was filtered through a plug of cotton and the
filtrate evaporated in vacuo. Crude oil was purified by silica gel chromatography (20%
clear oil.
M.W.: 162.23
7.38-7.24 (m, 5 H, PhH), 5.88 (m, 1 H, H2), 5.21 (m, 1 H, H1'), 5.16 (m, 1
H, H1''), 3.90 (m, 1 H, H4), 2.80 (m, 2 H, H5), 2.32 (m, 2 H, H3), 1.94 (bs,
1 H, OH)
13
C NMR: (75 MHz, CDCl 3 )
138.59 (C(Ph)), 134.87 (C(2)), 129.56 (2 C, C(Ph)), 128.61 (2 C, C(Ph)),
126.56 (C(Ph)), 118.10 (C(1)), 71.83 (C(4)), 43.41 (C(5)), 41.28 (C(3))
1-Oxiranyl-3-phenyl-propan-2-ol (3.36)
OH OH
O
4 2
5 3 1
rac-3.21 3.36
Alcohol rac-3.21 (1.62 g, 10.0 mmol) and mCPBA (3.20 g, 13 mmol, 1.3 eq) were
dissolved in dry dichloromethane (DCM, 100 mL) and stirred under an argon atmosphere
at room temperature. After 12 h., the reaction was quenched with an aqueous saturated
solution of NaHCO 3 followed by extraction with EtOAc (4x60 mL). The organic
fractions were combined and extracted in addition with NaHCO 3 (3x50 mL) and
repeatedly washed with brine (6x30 mL). The organic layer was dried over Na 2 SO 4 and
filtered, after which the solvent was evaporated in vacuo. The residual oil was purified
M.W.: 178.23
7.36-7.22 (m, 10 H, PhH), 4.08 (m, 2 H, H4), 3.19-3.10 (m, 2 H, H2), 2.89-
2.72 (m, 6 H, H5, H1'), 2.61 (dd, J = 2.8, 2.0 Hz, 1 H, H1''), 2.51 (dd, J =
2.8, 2.0 Hz, 1 H, H1''), 2.17 (d, J = 3.1 Hz, 1 H, OH), 2.14 (d, J = 3.1 Hz, 1
H, OH), 1.90 (m, 2 H, H3), 1.64 (m, 2 H, H3)
13
C NMR: (75 MHz, CDCl 3 )
138.16 (2 C, C(Ph)), 129.64 (2 C, C(Ph)), 129.62 (2 C, C(Ph)), 128.80 (4
C, C(Ph)), 126.81 (2 C, C(Ph)), 71.37 (C(4)), 70.37(C(4)), 50.53 (C(1)),
50.30 (C(1)), 47.16 (C(2)), 46.76 (C(2)), 44.33 (C(5)), 44.11 (C(5)), 39.10
(C(3)), 38.88 (C(3))
(1-Benzyl-2-oxiranylethoxy)-tert-butyldimethylsilane (3.37)
9
8
7
Si
OH O
O 6
O
4 2
5 3 1
3.36 3.37
1.5 eq) and imidazole (9.39 g, 32.1 mmol, 1.7 equiv) were dissolved in
dimethylformamide (DMF, 200 mL, 0.1 M) while stirring at room temperature for 1 h 20
min. Quenched with NH 4 Cl (100 mL) and extracted with EtOAc (4x100 mL). Washed
combined organic fractions with brine (6x60 mL) and dried with Na 2 SO 4 , followed by
the evaporation of the solvent in vacuo. Purified residual oil by silica gel
silylated epoxide diastereomers 3.37 (5.53 g, 18.9 mmol, 100%) as a colorless oil.
M.W.: 292.49
HRMS: (EI) m/z calcd for C 13 H 19 O 2 Si [(M-C 4 H 9 )+] 235.1149, found 235.0973
6-(tert-Butyldimethylsilyloxy)-7-phenylhept-1-en-4-ol (3.38)
9
11
8
Si 10
O OH
OTBS 2
O 7 5 3 1
6 4
3.37 3.38
A heterogeneous solution in THF (200 mL) of CuI (3.9 g, 21 mmol, 1 equiv) was
vinylmagnesium bromide (63 mL, 63 mmol, 3 equiv) was added and stirred for an
additional 30 min., producing a brown solution. Epoxide 3.37 (6.0 g, 21 mmol) was
dissolved in THF (100 mL) and added by canula over 20 min., and stirred vigorously for
2.5 hrs. Quenching with NH 4 Cl (50 mL), immediately produced a brown precipitate,
which was filtered and the organic layer extracted with excess water. Organic extract
was washed with brine (2x50 mL), dried over Na 2 SO 4 and the solvent evaporated in
vacuo. Purification of the residual oil was accomplished by silica gel chromatography
M.W.: 320.54
154
HRMS: (EI) m/z calcd for C 15 H 23 O 2 Si [(M-C 4 H 9 )+] 263.1462, found 263.1445
1-Phenylhept-6-ene-2,4-diols (syn-3.39/anti-3.39)
TBSO OH OH OH OH OH
2 2
7 5 3 1 7 5 3 1
6 4 6 4
A solution in EtOH (10 mL) of monoprotected diol 3.38 (1.3 g, 4.2 mmol) and PTSA
(5.6 g, 29 mmol, 7 equiv) was stirred under argon for 4 hrs. After checking by TLC, the
reaction was quenched with a saturated NaHCO 3 solution (5 mL) and extracted with
EtOAc (4 x 10 mL). Washed the organic extract with brine (2 x 20 mL), dried over
MgSO 4 and the solvent was evaporated in vacuo. Purification by silica gel
phase analytical scale Gemini 250 × 21.2 mm column, particle size 10 µm by applying
water with methanol, acetonitrile and their 50:50 combination as organic modifiers using
155
an Agilent 1100 series HPLC-DAD system. The final method with optimal binary linear
gradient conditions was transferred and upscaled on an Agilent 1200 series preparative
HPLC system comprising a binary pump (flow rate range 5-100 mL/min), an autosampler
with a 2 mL loop, a diode array detector with a flow cell (path length 3 mm and
maximum pressure limit 120 bar) and a fraction collector (40 mL collection tubes).
Preparative scale separations were performed on a reverse-phase Gemini Axia 250 × 21.2
mm column, particle size 10 µm (Phenomenex Inc., Torrance, CA) using a linear gradient
Fractions were collected, combined, and the solvent evaporated in vacuo to yield anti-
3.39 (163 mg, 0.79 mmol, 32%) as a crystalline solid and syn-3.39 (160 mg, 0.78 mmol,
M.W.: 206.13
HRMS: (EI) m/z calcd for C 6 H 11 O 2 [(M-C 7 H 7 )+] 115.0754, found 115.0772
M.W.: 206.13
1
H NMR: (400 MHz, CDCl 3 )
7.32-7.19 (m, 5 H, PhH), 5.84-5.73 (m, 1 H, H2), 5.14-5.08 (m, 2 H, H1),
4.05 (m, 1 H, H6), 3.87 (m, 1 H, H4), 2.89 (bs, 1 H, OH), 2.76 (d, J = 6.5
Hz, 2 H, H7), 2.22 (m, 2 H, H3), 1.69 (dt, J = 14.4, 2.3 Hz, 1 H, H5'), 1.54
(m, 1 H, H5'')
13
C NMR: (100 MHz, CDCl 3 )
137.97 (C(Ph)), 134.34 (C(2)), 129.46 (2 C, C(Ph)), 128.63 (2 C, C(Ph)),
126.60 (C(Ph)), 118.30 (C(1)), 73.72 (C(6)), 71.66 (C(4)), 44.52 (C(7)),
42.41 (C(5)), 41.82 (C(3))
HRMS: (EI) m/z calcd for C 6 H 11 O 2 [(M-C 7 H 7 )+] 115.0754, found 115.0768
anti-4-Allyl-6-benzyl-2,2-dimethyl-1,3-dioxane (anti-3.44)
8 9
OH OH O 10 O
2
7 5 3 1
6 4
In an acetone solution (0.1 mL, 1.4 mmol, 50 equiv), PTSA (1.6 mg, 0.008 mmol, 0.3
equiv) and anti-3.39 (5.5 mg, 0.027 mmol) were stirred overnight under an argon
atmosphere. Quenched with TEA (5.0 µL 0.04 mmol, 1.3 equiv) and after evaporating
M.W.: 246.34
HRMS: (EI) m/z calcd for C 15 H 19 O 2 [(M-CH 3 )+] 231.1380, found 231.1388
syn-4-Allyl-6-benzyl-2,2-dimethyl-1,3-dioxane (syn-3.44)
8 9
OH OH O 10 O
2
7 5 3 1
6 4
sy n-3.39 sy n-3.44
In an acetone solution (0.1 mL, 1.4 mmol, 50 equiv), PTSA (1.0 mg, 0.005 mmol, 0.2
equiv) and syn-3.39 (5.5 mg, 0.027 mmol) were stirred overnight under an argon
atmosphere. Quenched with TEA (5.0 µL 0.04 mmol, 1.3 equiv) and after evaporating
M.W.: 246.34
HRMS: (EI) m/z calcd for C 15 H 19 O 2 [(M-CH 3 )+] 231.1380, found 231.1375
158
anti-5-Allyl-7-benzyl-2,2,3,3,9,9,10,10-octamethyl-4,8-dioxa-3,9-disilaundecane (anti-
3.40)
9 13
11 15
10 Si Si 14
OH OH 8 O O 12
2
7 5 3 1
6 4
Diol anti-3.39 (155 mg, 0.75 mmol) was dissolved by stirring in anhydrous DMF (3.8
mL, 0.2M) at r.t. under an argon atmosphere. Imidazole (116 mg, 1.7 mmol, 2.2 equiv)
was added followed by TBSCl (256 mg, 1.7 mmol, 2.2 equiv) and the reaction was
allowed to proceed overnight. After checking by TLC for completion, extracted product
with hexanes (4x2 mL), dried over MgSO 4 and the solvent was evaporated in vacuo.
Purification of the residual oil was accomplished by silica gel chromatography (10%
EtOAc-hexanes) affording bis-silylated diol anti-3.40 (326 mg, 0.75 mmol, 100%) as a
M.W.: 434.30
HRMS: (EI) m/z calcd for C 21 H 37 O 2 Si 2 [(M-C 4 H 9 )+] 377.2327, found 377.2306
159
syn-5-Allyl-7-benzyl-2,2,3,3,9,9,10,10-octamethyl-4,8-dioxa-3,9-disilaundecane (syn-
3.40)
9 13
11 15
10 Si Si 14
OH OH 8 O O 12
2
7 5 3 1
6 4
sy n-3.39 sy n-3.40
Diol syn-3.39 (157 mg, 0.76 mmol) was dissolved by stirring in anhydrous DMF (3.8
mL, 0.2M) at r.t. under an argon atmosphere. Imidazole (116 mg, 1.7 mmol, 2.2 equiv)
was added followed by TBSCl (256 mg, 1.7 mmol, 2.2 equiv) and the reaction was
allowed to proceed overnight. After checking by TLC for completion, extracted product
with hexanes (4x2 mL), dried over MgSO 4 and the solvent was evaporated in vacuo.
Purification of the residual oil was accomplished by silica gel chromatography (10%
EtOAc-hexanes) affording bis-silylated diol syn-3.40 (330 mg, 0.76 mmol, 100%) as a
M.W.: 434.30
HRMS: (EI) m/z calcd for C 21 H 37 O 2 Si 2 [(M-C 4 H 9 )+] 377.2327, found 377.2318
160
7-Aminoheptanoic acid (3.35) (150 mg, 1.03 mmol) was dissolved in MeOH (1.0 mL,
24.7 mmol, 24 equiv) at r.t. under argon. To the solution, trimethylsilyl chloride
(TMSCl, 0.13 mL, 1.03 mmol, 1.0 equiv) was added and stirred overnight. The solvent
was evaporated in vacuo yielding crude methyl ester 3.34 (202 mg, 1.03 mmol, 100%) as
M.W.: 195.69
1
H NMR: (300 MHz, D 2 O)
3.55 (s, 3 H, H8), 2.85 (bt, J = 7.5 Hz, 2 H, H7), 2.26 (t, J = 7.5 Hz, m, 2
H, H2), 1.48 (m, 4 H, H3, H6), 01.22 (m, 4 H, H4, H5)
13
C NMR: (75 MHz, D 2 O)
177.60 (C(1)), 51.99 (C(8)), 39.31 (C(7)), 33.44 (C(2)), 27.51 (C(6)), 26.40
(C(4)), 25.09 (C(5)), 23.88 (C(3))
Reference: Gros, L.; Orenes Lorente, S.; Jimenez Jimenez, C.; Yardley, V.; Rattray,
L.; Wharton, H.; Little, S.; Croft, S. L.; Ruiz-Perez, L. M.; Gonzalez-
Pacanowska, D.; Gilbert, I. H., Evaluation of azasterols as anti-parasitics.
J. Med. Chem. 2006, 49 (20), 6094-6103
a
Aminoester 3.34 was identical to its previously reported spectroscopic values
Methyl 7-anti-3,5-bis(tert-butyldimethylsilyloxy)-6-phenylhexanamido)heptanoate
(anti-3.41)
16 20
18 22
Si Si 17 Si Si 21
O O 15 O O 19 O O
6 4 2 8 10 12
5 3 1 N 7 9 11 13 O 14
H
anti -3.40 anti -3.41
161
In a flask, NaIO 4 (780 mg, 1.4 mmol, 8.0 equiv) and KMnO 4 (34 mg, 0.22 mmol, 1.2
equiv) were dissolved in pH 7.24 phosphate buffer (1.0 mL, 0.05M) and stirred
vigorously at r.t. Olefin anti-3.40 (80 mg, 0.18 mmol) was dissolved in t-BuOH (1.8
mL) while stirring. After 15 min, the dark purple permanganate solution was transferred
by Pasteur pipette to the olefin solution and stirred overnight. Two buffer washes (0.4
mL) were employed to quantitatively transfer the oxidant mixture. Upon contact, the
saturated solution of sodium thiosulfate (1.0 mL), stirred for 30 min and extracted with
EtOAc (4x1.5 mL). The organic extract was washed with brine (2x2 mL), dried over
MgSO 4 and the solvent evaporated in vacuo. The isolated crude oil (42 mg) was
dissolved in DCM (1.9 mL, 0.05M) and, while stirring under argon, added: EDC (27 mg,
0.14 mmol, 1.5 equiv), DMAP (1.1 mg, 0.009 mmol, 0.1 equiv) and amine 3.34 (22 mg,
0.11 mmol, 1.2 equiv). After 5 min, TEA was added (31 μL, 0.22 mmol, 2.4 equiv) and
the solution was stirred overnight. Quenched with NH 4 Cl (1.0 mL) and extracted with
EtOAc (5x2 mL). Organic extracts were washed with brine (2x5 mL) and dried over
MgSO 4 . After solvent evaporation, the oil obtained was purified by flash column
chromatography (30% EtOAc-hexanes) furnishing amide anti-3.41 (34 mg, 0.058 mmol,
M.W.: 593.99
7.25-7.13 (m, 5 H, PhH), 6.33 (bt, J = 5.5 Hz, 1 H, NH), 4.04 (m, 1 H, H3),
3.87 (p, J = 6.3 Hz, 1 H, H5), 3.64 (s, 3 H, H14), 3.26-3.07 (m, 2 H, H7),
2.70 (d, J = 6.4 Hz, 2 H, H6), 2.50 (dd, J = 15.1, 4.0 Hz, 1 H, H2'), 2.28 (m,
3 H, H12, H2''), 1.68 (m, 2 H, H4), 1.58 (m, 2 H, H11), 1.41 (m, 2 H, H8),
1.28 (m, 4 H, H9, H10), 0.82 (s, 9 H, H22), 0.81 (s, 9 H, H18), 0.00 (s, 6 H,
H15, H19), -0.03 (s, 3 H, H16), -0.18 (s, 3 H, H20)
13
C NMR: (100 MHz, CDCl 3 )
174.08 (C(13)), 170.55 (C(1)), 138.55 (C(Ph)), 129.71 (2 C, C(Ph)), 128.17
(2 C, C(Ph)), 126.19 (C(Ph)), 71.30 (C(5)), 67.64 (C(3)), 51.44 (C(14)),
44.33 (C(4)), 44.15 (C(6)), 44.14 (C(2)), 39.14 (C(7)), 33.96 (C(12)), 29.36
(C(8)), 28.76 (C(9)), 26.68 (C(10)), 25.88 (3 C, C(18)), 25.74 (3 C, C(22)),
24.80 (C(11)), 17.98 (C(17)), 17.85 (C(21)), -4.52 (C(15)), -4.56 (C(19)), -
4.60 (C(18)), -4.68 (C(20))
HRMS: (EI) m/z calcd for C 28 H 50 NO 5 Si 2 [(M-C 4 H 9 )+] 536.3222, found 536.3251
Methyl 7-syn-3,5-bis(tert-butyldimethylsilyloxy)-6-phenylhexanamido)heptanoate
(syn-3.41)
16 20
18 22
Si Si 17 Si Si 21
O O 15 O O 19 O O
6 4 2 8 10 12
5 3 1 N 7 9 11 13 O 14
H
sy n-19 sy n-20
In a flask, NaIO 4 (780 mg, 1.4 mmol, 8.0 equiv) and KMnO 4 (34 mg, 0.22 mmol, 1.2
equiv) were dissolved in pH 7.24 phosphate buffer (1.0 mL, 0.05M) and stirred
vigorously at r.t. Olefin syn-3.40 (80 mg, 0.18 mmol) was dissolved in t-BuOH (1.8 mL)
while stirring. After 15 min, the dark purple permanganate solution was transferred by
Pasteur pipette to the olefin solution and stirred overnight. Two buffer washes (0.4 mL)
were employed to quantitatively transfer the oxidant mixture. Upon contact, the new
solution of sodium thiosulfate (1.0 mL), stirred for 30 min and extracted with EtOAc
(4x1.5 mL). The organic extract was washed with brine (2x2 mL), dried over MgSO 4
and the solvent evaporated in vacuo. The isolated crude oil (51 mg) was dissolved in
163
DCM (2.2 mL, 0.05M) and added while stirring under argon: EDC (33 mg, 0.17 mmol,
1.5 equiv), DMAP (1.2 mg, 0.01 mmol, 0.1 equiv) and amine 3.34 (25 mg, 0.13 mmol,
1.2 equiv). After 5 min, TEA was added (36 μL, 0.26 mmol, 2.4 equiv) and the solution
was stirred overnight. Quenched with NH 4 Cl (1.0 mL) and extracted with EtOAc (5 x 2
mL). Organic extracts were washed with brine (2x5 mL) and dried over MgSO 4 . After
solvent evaporation, the oil obtained was purified by flash column chromatography (30%
EtOAc-hexanes) furnishing amide syn-3.41 (34 mg, 0.065 mmol, 36% over two steps) as
an amber oil.
M.W.: 593.99
HRMS: (EI) m/z calcd for C 28 H 50 NO 5 Si 2 [(M-C 4 H 9 )+] 536.3222, found 536.3256
164
Hydrolysis. The methyl ester and LiOH (10 equiv) are dissolved and vigorously
stirred in a 5:1 solution of MeOH:H 2 O (0.02M) at r.t. for 6 hr. Solvent was evaporated
off and the crude was acidified to a pH of 3 units by the dropwise addition of 1M HCl.
Extracted with EtOAc (4x0.25 volumes), dried with brine and MgSO 4 , and evaporated in
vacuo. Silyl deprotection. In an Eppendorf tube the crude acid was dissolved in MeCN
(0.1 M) and vortexed for 10 sec to homogeneity. To this solution, pyridine (10 equiv)
and hydrofluoric acid (HF, 50 equiv) were added and, following mixing by vortexing 10
sec, the mixture was left to react 6 hr. The mixture was diluted with H 2 O, extracted with
EtOAc (5 x 0.5 volumes) and the organic phase was dried with MgSO 4 and evaporated in
equiv), and PPh 3 (1.5 equiv) were dissolved in THF (0.01 M) and stirred at r.t. for 5.5 hr.
The bright yellow solution was quantitatively transferred and diluted into a syringe by
portionwise addition of toluene (~0.005 M). The solution was transferred dropwise by
syringe pump (0.6 mL/hr) into a round bottom flask fitted with a reflux condenser
containing toluene (~0.0005 M) heated to 85°C under an argon atmosphere. The reaction
was stirred for 5 to 8 days while monitoring for completion by LC/MS, at which time the
solvent was evaporated in vacuo and the crude partially purified by chromatographic
presence of macrocycle in the eluted fractions was checked by LC/MS. After pooling the
reverse phase Eclipse XDB-C18 150 × 4.6 mm column, particle size 5 µm (Agilent
165
Technologies, Inc., Santa Clara, CA) using a linear gradient of 0-40% 95%
anti-2-Benzyl-4-hydroxy-1-oxa-7-azacyclotetradecane-6,14-dione (anti-3.42)
anti-2-(2-Hydroxy-3-phenylpropyl)-1-oxa-5-azacyclododecane-4,12-dione (anti-3.43)
O
13
OH
9 8 NH 12
Si Si 9
7 11 O
O O O 11 10 OH 6
10 8
O 13
12 5 O O HN
N 6 O 4
1
7
H 4
O 1 3
2 6
O 2 3 5
anti -3.41 anti -3.42 anti -3.43
Hydrolysis. Methyl ester anti-3.41 (117 mg, 0.20 mmol) was treated with LiOH
(84 mg, 2.0 mmol, 10 equiv) and dissolved in MeOH:H 2 O (12 mL, 0.02 M) at r.t. Upon
acidification of aqueous layer, extracted with EtOAc (4x3 mL) and dried with brine (1 x
1 mL) and MgSO 4 . Filtered and evaporated solvent in vacuo. Silyl deprotection. The
isolated crude oil (~114 mg) was dissolved in MeCN (2 mL) and vortexed to
homogeneity (10-20 sec). Pyridine (145 μL, 2.0 mmol, 10 equiv) followed by HF (174
μL, 10 mmol, 50 equiv) were added and after vortexing thoroughly, the reaction was left
undisturbed for 6 hr. Diluted with H 2 O (1.5 mL) and extracted with EtOAc (5x1 mL).
The organic layers were combined, dried with MgSO 4 and, after evaporation in vacuo, a
crude white solid was isolated (~60 mg). Macrolactonization. Crude hydroxyacid (3.5
mg, 0.010 mmol) was dissolved in THF (0.8 mL, 0.01 M) followed by DTP (3.3 mg,
0.015 mmol, 1.5 equiv) and PPh 3 (3.9 mg, 0.015 mmol, 1.5 equiv). After 5.5 hr, diluted
with toluene (5 mL, 0.002 M) and added via syringe pump (0.6 mL/hr) to hot toluene.
After 5 days at 86°C, solvent was evaporated and the crude was purified by PHPLC.
Two fractions were collected and lyophilized to yield 1) spectroscopically pure anti-3.42
166
(0.5 mg, 2 μmol, 2% after three steps) and 2) a crude mixture of anti-3.42 and anti-3.43
(0.05 mg, 0.15 μmol, 0.1% after three steps) as white solids. The latter mixture was
repurified by LC/MS and regrettably its yield was insufficient for full spectroscopic
characterization.
M.W.: 333.42
Note: Protons on C3, C4, C5, C6 are covered by H 2 O peak and solvent.
Peaks were assigned from COSY and HSQC correlation spectra
13
C NMR: (75 MHz, CDCl 3 )
174.62 (C(1)), 171.80 (C(8)), 136.91 (C(Ph)), 129.11 (2 C, C(Ph)), 128.53
(2 C, C(Ph)), 126.78 (C(Ph)), 72.11 (C(12)), 66.29 (C(10)), 41.94 (C(9)),
40.98 (C(11)), 40.42 (C(13)), 39.11 (C(7)), 34.24 (C(2)), 29.12 (C(4)),
27.35 (C(6)), 25.59 (C(5)), 23.20 (C(3))
M.W.: 333.42
syn-2-Benzyl-4-hydroxy-1-oxa-7-azacyclotetradecane-6,14-dione (syn-3.43)
syn-2-(2-Hydroxy-3-phenylpropyl)-1-oxa-5-azacyclododecane-4,12-dione (syn-3.43)
O
13
OH
9 8 NH 12
Si Si 9
7 11 O
O O O 11 10 OH 6
10 8
O 13
12 5 O O HN
N 6 O 4
1
7
H 4
O 1 3
2 6
O 2 3 5
sy n-3.41 sy n-3.42 sy n-3.43
Hydrolysis. Methyl ester syn-3.41 (99 mg, 0.17 mmol) was treated with LiOH (71
mg, 1.7 mmol, 10 equiv) and dissolved in MeOH:H 2 O (12 mL, 0.014 M) at r.t. Upon
acidification of aqueous layer, extracted with EtOAc (4 x 3 mL) and dried with brine (1 x
1 mL) and MgSO 4 . Filtered and evaporated solvent in vacuo. Silyl deprotection. The
isolated crude oil (~94 mg) was dissolved in MeCN (1.6 mL) and vortexed to
homogeneity (10-20 sec). Pyridine (116 μL, 2.0 mmol, 10 equiv) followed by HF (140
μL, 10 mmol, 50 equiv) were added and after vortexing thoroughly, the reaction was left
undisturbed for 6 hr. Diluted with H 2 O (1.5 mL) and extracted with EtOAc (5 x 1 mL).
The organic layers were combined, dried with MgSO 4 and, after evaporation in vacuo, a
crude white solid was isolated (~50 mg). Macrolactonization. Crude hydroxyacid (30
mg, 0.085 mmol) was dissolved in THF (0.85 mL, 0.1 M) followed by DTP (29 mg, 0.13
mmol, 1.5 equiv) and PPh 3 (34 mg, 0.13 mmol, 1.5 equiv). After 5.5 hr, diluted with
toluene (10 mL, ~0.009 M) and added via syringe pump (0.6 mL/hr) to hot toluene. After
10 days at 85°C, solvent was evaporated and the crude was partially purified by FCC
obtained (~10 mg) was purified by PHPLC. Two fractions were collected and
168
lyophilized to yield 1) spectroscopically pure syn-3.42 (5.0 mg, 15 μmol, 18% after three
steps) and 2) syn-3.43 (2.0 mg, 6 μmol, 7% after three steps) as white solids.
M.W.: 333.42
M.W.: 333.42
42.91 (C(9)), 41.41 (C(11)), 37.37 (C(7)), 33.51 (C(2)), 25.81 (C(4)), 24.63
(C(5)), 23.99 (C(6)), 23.63 (C(3))
In a round bottomed flask (r.b.f.), Ipc 2 BAllyl (5 mL, 1M in pentanes) was cooled to -
slowly cannulated over 5 min. After 30 min and warming to -78°C, quenched with 2 mL
MeOH and slowly allowed mixture to warm up to r.t., at which point 2 mL of a 15%
NaOH solution was added followed by 4 mL of 30% H 2 O 2 . This mixture was equipped
with a water condenser and refluxed for 30 min. Purification was accomplished by
extracting with EtOAc (3x15 mL), and drying with brine and Na 2 SO 4 . The organic
fractions were combined and solvent evaporated in vacuo. The residual clear oil was
(S)-1-Phenylpent-4-en-2-ol (3.21)
OH
2
5 3 1
O 4
3.20 3.21
The procedure outlined above was employed in the conversion of aldehyde 3.20 to
homoallylic alcohol 3.21 (460 mg, 2.8 mmol, 63%, 90% ee) with (-)-Ipc 2 BAllyl.
M.W.: 162.23
1
H NMR: (300 MHz, CDCl 3 )
7.33-7.19 (m, 5 H, PhH), 5.81 (m, 1 H, H2), 5.16 (m, 1 H, H1'), 5.12 (t, J =
1.2 Hz, 1 H, H1''), 3.86 (m, 1 H, H4), 2.75 (m, 2 H, H5), 2.29 (m, 2 H, H3),
1.65 (d, J = 10 Hz, 1 H, OH)
13
C NMR: (75 MHz, CDCl 3 )
138.39 (C(Ph)), 134.70 (C(2)), 129.44 (2 C, C(Ph)), 128.57 (2 C, C(Ph)),
126.51 (C(Ph)), 118.18 (C(1)), 71.69 (C(4)), 43.32 (C(5)), 41.21 (C(3))
(R)-1-Phenylpent-4-en-2-ol (ent-3.21)
OH
2
5 3 1
O 4
aldehyde 3.20 to homoallylic alcohol ent-3.21 (320 mg, 1.97 mmol, 44%, 90% ee) with
(+)-Ipc 2 BAllyl.
M.W.: 162.23
rac-3.21
3.21
172
ent-3.21
General procedure for the synthesis of enantioenriched epoxides 3.22 and ent-3.22
The purified chiral Brown allylation product was treated to the following sequence:
Epoxidation. The chiral olefin was dissolved at 0°C in DCM (0.05M conc.) along with
mCPBA (2.1 equiv, 77% purity) and NaHCO 3(s) (4.2 equiv) and vigorously stirred
overnight and allowing the mixture to gradually warm up to r.t. Quenching was carried
with EtOAc, H 2 O and copious amounts of saturated NaHCO 3 solution. Dried organic
layer with Na 2 SO 4 and the solvent was evaporated in vacuo. The crude oil was filtered
through a plug of silica with EtOAc. Silylation. The crude oil was dissolved in DMF at
r.t. to which imidazole and TBSCl were added sequentially. The solution was vigorously
stirred for 24 hrs and subsequently quenched with NH 4 Cl and diluted with EtOAc and
H 2 O. Extracted 8-10 times with brine and after solvent evaporation, the residual oil was
filtered through a plug of silica with hexanes. Jacobsen's hydrolytic kinetic resolution.
method1. The epoxide and catalyst were dissolved in THF and cooled to 0°C while
173
stirring in a round bottomed flask. H 2 O was added and warmed up to r.t. while stirring
for 16 h. The solvent was evaporated and the oil purified by flash column
tert-Butyldimethyl((R)-1-((R)-oxiran-2-yl)-3-phenylpropan-2-yloxy)silane (3.22)
(2S,4R)-4-(tert-Butyldimethylsilyloxy)-5-phenylpentane-1,2-diol (3.24)
9 9 7
8
7 8 Si 6
Si
OH O O OH
6
O
+ OH
4 2 4 2
5 3 1 5 3 1
3.21 3.22 3.24
alcohol 3.21 (460 mg, 2.8 mmol), mCPBA (1.33 g, 5.9 mmol, 2.1 equiv, 77% pure),
NaHCO 3 (991 mg, 11.8 mmol, 4.2 equiv) in DCM (56 mL). The reaction yielded a crude
oil (273 mg, ~1.5 mmol) that was used without further purification on the next step.
Silylation. Crude epoxide (273 mg, ~1.5 mmol), imidazole (177 mg, 2.6 mmol, 1.7
equiv), TBSCl (347 mg, 2.3 mmol, 1.5 equiv) was stirred in DMF (30 mL). The crude
oil (341 mg, ~1.2 mmol) obtained was used in the hydrolytic kinetic resolution step.
Jacobsen's hydrolytic kinetic resolution. Crude silylated oil (341 mg, ~1.2 mmol),
R,R-Salen catalyst (692.8 mg/mmol, 17 mg, 0.024 mmol, 2 mol%), H 2 O (12 μL, 0.66
mmol, 0.55 equiv), THF (1 mL). After careful purification epoxide 3.22 (175 mg, 0.60
mmol, 90% de, 21% overall) and vicinal diol 3.24 (156 mg, 0.50 mmol, 18% overall)
were isolated. The three step sequence furnished 3.22 and 3.24 in a 39% overall yield.
M.W.: 292.49
1
H NMR: (400 MHz, CDCl 3 )
7.27-7.15 (m, 5 H, PhH), 4.02 (dt, J = 5.6, 1.2 Hz, 1 H, H4), 3.09 (m, 1 H,
H2), 2.83 (d, J = 2.4, 2 H, H5), 2.74 (m, 1 H, H1'), 2.40 (m, 1 H, H1''), 1.66
(t, J = 5.6 Hz, 2 H, H3), 0.83 (s, 9 H, H9), -0.05 (s, 3 H, H6), -0.22 (s, 3 H,
H7)
13
C NMR: (100 MHz, CDCl 3 )
138.87 (C(Ph)), 129.80 (2 C, C(Ph)), 128.20 (2 C, C(Ph)), 126.20 (C(Ph)),
72.00 (C(4)), 49.38 (C(2)), 46.71 (C(1)), 43.99 (C(5)), 39.81 (C(2)), 25.82
(3 C, C(9)), 18.02 (C(8)), -4.90 (C(6), -5.07 (C(7))
HRMS: (EI) m/z calcd for C 13 H 19 O 2 Si [(M-C 4 H 9 )+] 235.1149, found 235.0922
M.W.: 310.49
HRMS: (EI) m/z calcd for C 10 H 23 O 3 Si [(M-C 7 H 7 )+] 219.1411, found 219.1397
tert-Butyldimethyl((S)-1-((S)-oxiran-2-yl)-3-phenylpropan-2-yloxy)silane (ent-3.22)
(2R,4S)-4-(tert-Butyldimethylsilyloxy)-5-phenylpentane-1,2-diol (ent-3.24)
9 9 7
8
7 8 Si 6
Si
OH O O OH
6
O
+ OH
4 2 4 2
5 3 1 5 3 1
ent -3.21 ent -3.22 ent -3.24
alcohol ent-3.21 (320 mg, 1.97 mmol), mCPBA (928 mg, 4.14 mmol, 2.1 equiv, 77%
175
pure), NaHCO 3 (696 mg, 8.28 mmol, 4.2 equiv) in DCM (40 mL). The reaction yielded
a crude oil (301 mg, ~1.69 mmol) that was used without further purification on the next
step. Silylation. Crude epoxide (301 mg, ~1.69 mmol), imidazole (195 mg, 2.87 mmol,
1.7 equiv), TBSCl (383 mg, 2.54 mmol, 1.5 equiv) was stirred in DMF (34 mL). The
crude oil (405 mg, ~1.38 mmol) obtained was used in the hydrolytic kinetic resolution
step. Jacobsen's hydrolytic kinetic resolution. Crude silylated oil (405 mg, ~1.38
mmol), S,S-Salen catalyst (692.8 mg/mmol, 19 mg, 0.030 mmol, 2 mol%), H 2 O (14 μL,
0.76 mmol, 0.55 equiv), THF (0.3 mL). After purification epoxide ent-3.22 (152 mg,
0.52 mmol, 90% de, 26% overall) and vicinal diol ent-3.24 (151 mg, 0.49 mmol, 25%
overall) were isolated. The three step sequence furnished ent-3.22 and ent-3.24 in a 51%
overall yield.
M.W.: 292.49
HRMS: (EI) m/z calcd for C 13 H 19 O 2 Si [(M-C 4 H 9 )+] 235.1149, found 235.1110
M.W.: 310.49
1
H NMR: (400 MHz, CDCl 3 )
7.28-7.13 (m, 5 H, PhH), 4.21 (m, 1 H, H4), 4.15 (m, 1 H, H2), 3.57 (m, 1
H, H1'), 3.38 (m, 1 H, H1''), 2.93 (dd, J = 13.2, 6.8 Hz, 1 H, H5'), 2.80 (dd,
J = 13.6, 7.2 Hz, 1 H, H5''), 2.0 (m, 1 H, OH), 1.70 (m, 1H, H3'), 1.46 (m,
1H, H3''), 0.87 (s, 9 H, H9), 0.03 (s, 3 H, H6), -0.15 (s, 3 H, H7)
13
C NMR: (100 MHz, CDCl 3 )
138.41 (C(Ph)), 129.54 (2 C, C(Ph)), 128.45 (2 C, C(Ph)), 126.45 (C(Ph)),
72.95 (C(4)), 68.84 (C(2)), 67.13 (C(1)), 42.90 (C(5)), 36.98 (C(2)), 25.83
(3 C, C(9)), 17.95 (C(8)), -4.96 (C(6), -5.08 (C(7))
HRMS: (EI) m/z calcd for C 10 H 23 O 3 Si [(M-C 7 H 7 )+] 219.1411, found 219.1405
General procedure for the conversion of vicinal diols to epoxides 3.23 and ent-3.23
The purified vicinal diol was stirred along with dibutyltin oxide (Bu 2 SnO, 0.2 equiv),
p-toluene sulfonate chloride (TsCl, 1 equiv) and triethylamine (Et 3 N, 1 equiv) in DCM
(0.15M conc) at r.t. under an argon atmosphere. After 3h, filtered through a plug of
cotton and evaporated solvent. The oil was redissolved in THF (0.2M conc) and added
dropwise to an ice-cold suspension of sodium hydride (NaH, 2 equiv, 60% in oil) in THF
(0.4M conc). Stirred overnight and quenched with NH 4 Cl (1 mL). Extracted with
EtOAc (3x2 volumes), dried organic layer with brine and Na 2 SO 4 and after filtration and
tert-butyldimethyl((R)-1-((S)-oxiran-2-yl)-3-phenylpropan-2-yloxy)silane (3.23)
9
8
7
Si
TBSO OH O 6
O
OH
4 2
5 3 1
3.24 3.23
The above general procedure was employed with vicinal diol 3.24 (136 mg, 0.44
mmol), Bu 2 SnO (21.7 mg, 0.087 mmol, 20 mol%), TsCl (83.9 mg, 0.44 mmol, 1.0
177
equiv), Et 3 N (61.4 μL, 0.44 mmol, 1.0 equiv) in DCM (2.9 mL). The reaction furnished
a crude oil (205 mg, ~0.44 mmol) that was redissolved in THF (3.5 mL) and added to a
suspension of NaH ( 35.2 mg, 0.88 mmol, 2.0 equiv) in THF (1 mL). Isolated 3.23 as an
amber oil (129 mg, 0.44 mmol, 90% de, 100% over two steps).
M.W.: 292.49
HRMS: (EI) m/z calcd for C 13 H 19 O 2 Si [(M-C 4 H 9 )+] 235.1149, found 235.1057
tert-Butyldimethyl((S)-1-((R)-oxiran-2-yl)-3-phenylpropan-2-yloxy)silane (ent-3.23)
9
8
7
Si
TBSO OH O 6
O
OH
4 2
5 3 1
The general procedure was employed with vicinal diol ent-3.23 (151 mg, 0.49 mmol),
Bu 2 SnO (25 mg, 0.10 mmol, 20 mol%), TsCl (93 mg, 0.49 mmol, 1.0 equiv), Et 3 N (68
μL, 0.49 mmol, 1.0 equiv) in DCM (3.3 mL). The reaction produced a crude oil that was
redissolved in THF (4.0 mL) and added to a suspension of NaH (39 mg, 0.98 mmol, 2.0
equiv) in THF (1 mL). Isolated ent-3.24 as an amber oil (83 mg, 0.28 mmol, 90% de,
M.W.: 292.49
HRMS: (EI) m/z calcd for C 13 H 19 O 2 Si [(M-C 4 H 9 )+] 235.1149, found 235.1110
A mixture of purified epoxide, copper (I) iodide (CuI, 0.1 equiv) in THF (1.0M conc)
was vigorously stirred under argon at -78°C for 20 min. Vinyl magnesium bromide
(VinylMgBr, 1.3 equiv) was added dropwise and the mixture allowed to slowly warm up
to r.t. overnight. The dark brown solution was quenched with NH 4 Cl (0.5 volumes) and
extracted with EtOAc (3x2 volumes). After drying the organic layer with brine (2
volumes) and Na 2 SO 4 , the mixture was filtered and the solvent evaporated. Purification
(4S,6R)-6-(tert-Butyldimethylsilyloxy)-7-phenylhept-1-en-4-ol (3.25)
9
11
8
Si 10
OTBS O OH
O 2
7 5 3 1
6 4
3.22 3.25
The general procedure was employed with epoxide 3.22 (60.2 mg, 0.21 mmol), CuI
(4.4 mg, 0.023 mmol, 0.1 equiv), THF (0.14 mL) and vinylmagnesium bromide (0.27
179
mL, 0.27 mmol, 1.3 equiv). After work up and purification of the crude organic extract,
homoallylic alcohol 3.25 (57.2 mg, 0.18 mmol, 85%) was isolated as an amber oil.
M.W.: 320.54
HRMS: (EI) m/z calcd for C 12 H 25 O 2 Si [(M-C 7 H 7 )+] 229.1618, found 229.1514
(4R,6R)-6-(tert-Butyldimethylsilyloxy)-7-phenylhept-1-en-4-ol (3.26)
9
11
8
Si 10
Si
O O OH
O 2
7 5 3 1
6 4
3.23 3.26
The general procedure was employed with epoxide 3.23 (35.1 mg, 0.12 mmol), CuI
(2.3 mg, 0.023 mmol, 0.1 equiv), THF (0.12 mL) and vinylmagnesium bromide (0.16
mL, 0.16 mmol, 1.3 equiv). After work up and purification of the crude organic extract,
homoallylic alcohol 3.26 (20 mg, 0.062 mmol, 52%) was isolated as an amber oil.
M.W.: 320.54
1
H NMR: (400 MHz, CDCl 3 )
7.25-7.07 (m, 5 H, PhH), 5.78 (m, 1 H, H2), 5.09-5.00 (m, 2 H, H1), 4.19
(m, 1 H, H6), 4.10 (m, 1 H, H4), 2.90 (dd, J = 13.2, 6.4 Hz, 1 H, H7'), 2.79
(dd, J = 13.2, 7.2 Hz, 1 H, H7''), 2.13 (m, 2 H, H3), 1.54 (m, 2 H, H5), 0.86
(s, 9 H, H11), 0.02 (s, 3 H, H8), -0.16 (s, 3 H, H9)
13
C NMR: (100 MHz, CDCl 3 )
138.64 (C(Ph)), 134.92 (C(2)), 129.59 (2 C, (C(Ph)), 128.36 (2 C, (C(Ph)),
126.33 (C(Ph)), 117.39 (C(1)), 73.12 (C(6)), 67.73 (C(4)), 43.04 (C(7)),
42.37 (C(5)), 40.78 (C(3)), 25.87 (3 C, (C(11))), 17.97 (C(10)), -4.93
(C(8)), -5.07 (C(9))
HRMS: (EI) m/z calcd for C 12 H 25 O 2 Si [(M-C 7 H 7 )+] 229.1618, found 229.1617
(4R,6S)-6-(tert-Butyldimethylsilyloxy)-7-phenylhept-1-en-4-ol (ent-3.25)
9
11
8
Si 10
Si
O O OH
O 2
7 5 3 1
6 4
The general procedure was employed with epoxide ent-3.22 (152.0 mg, 0.52 mmol),
CuI (10.0 mg, 0.052 mmol, 0.1 equiv), THF (0.52 mL) and vinylmagnesium bromide
(0.68 mL, 0.68 mmol, 1.3 equiv). After work up and purification of the crude organic
extract, homoallylic alcohol ent-3.25 (39.3 mg, 0.12 mmol, 23%) was isolated as an
amber oil.
M.W.: 320.54
HRMS: (EI) m/z calcd for C 12 H 25 O 2 Si [(M-C 7 H 7 )+] 229.1618, found 229.1616
(4S,6S)-6-(tert-Butyldimethylsilyloxy)-7-phenylhept-1-en-4-ol (ent-3.26)
9
11
8
Si 10
Si
O O OH
O 2
7 5 3 1
6 4
The general procedure was employed with epoxide ent-3.23 (83.0 mg, 0.28 mmol),
CuI (6.0 mg, 0.030 mmol, 0.1 equiv), THF (0.28 mL) and vinylmagnesium bromide (0.36
mL, 0.36 mmol, 1.3 equiv). After work up and purification of the crude organic extract,
homoallylic alcohol ent-3.26 (42.6 mg, 0.13 mmol, 46%) was isolated as an amber oil.
M.W.: 320.54
HRMS: (EI) m/z calcd for C 12 H 25 O 2 Si [(M-C 7 H 7 )+] 229.1618, found 229.1604
182
Methyl 7-((3R,5R)-3,5-bis(tert-butyldimethylsilyloxy)-6-phenylhexanamido)
heptanoate (3.31)
16 20
18 22
Si 17 Si Si 21
O OH 15 O O 19 O O
6 4 2 8 10 12
5 3 1 N 7 9 11 13 O 14
H
3.25 3.31
mg, 0.36 mmol, 2.0 equiv) and imidazole (26.0 mg, 0.38 mmol, 2.1 equiv) were
temperature overnight. Diluted with water (1 mL) and extracted with hexanes (4x2 mL);
dried with Na 2 SO 4 and followed by evaporating solvent in vacuo. The crude bis-silylated
olefin (45.8 mg, ~0.11 mmol) was used in the next step without purification.
In a r.b.f. sodium metaperiodate (NaIO 4 , 455.0 mg, 0.84 mmol, 8.0 equiv) and
potassium permanganate (KMnO 4 , 18.7 mg, 0.12 mmol, 1.1 equiv) were dissolved in pH
7.0 buffer (0.5 mL, 0.05M) and stirred vigorously at r.t. The crude olefin was dissolved
in t-BuOH (0.5 mL) while stirring. After 15 min, the dark purple permanganate solution
was transferred by Pasteur pipette to the olefin solution. Two buffer washes (0.1 mL)
were employed to quantitatively transfer of the oxidant. Upon contact, the mixture
changed coloration to a pale brown. The solution was stirred overnight. Quenched with
a saturated solution of sodium thiosulfate (0.5 mL), diluted with 1 mL of water and
extracted with EtOAc (3x1 mL). The organic extract was washed with brine (2x0.5 mL),
dried over Na 2 SO 4 and the solvent evaporated in vacuo. The isolated crude oil was used
Dissolved crude oil in DCM (1.0 mL) and added while stirring under argon:
EDC•HCl (29.9 mg, 0.16 mmol, 1.5 equiv), DMAP (1.2 mg, 0.010 mmol, 0.1 equiv) and
183
amine 3.34 (28.6 mg, 0.15 mmol, 1.4 equiv). After 5 min, TEA was added (21.7 μL, 0.16
mmol, 1.5 equiv) and the solution was stirred overnight. Quenched with NH 4 Cl (1.0 mL)
and extracted with EtOAc (3x1 mL). Organic extracts were washed with brine (2x1 mL)
and dried over Na 2 SO 4 . Upon solvent evaporation, the oil obtained was purified by flash
column chromatography (20% EtOAc-hexanes - 50% EtOAc) furnishing amide 3.31 (30
M.W.: 593.99
3.12 3.15 O
Boc-protected aminothioester 3.122 (102 mg, 0.29 mmol) was dissolved and stirred in
HCl/Dioxane (0.73 mL, 2.9 mmol, 10 equiv) at r.t. under argon. After 45 min, the
184
solvent was evaporated in vacuo and free amine 3.15 was isolated (82 mg, 0.29 mmol,
M.W.: 282.83
1
H NMR: (300 MHz, D 2 O)
3.24 (t, J = 6.2 Hz, 2 H, H3), 2.91 (t, J = 6.5 Hz, 2 H, H4), 2.85 (bt, J = 7.4
Hz, 2 H, H11), 2.52 (t, J = 7.4 Hz, 2 H, H6), 1.82 (s, 3 H, H1), 1.52 (m, 4
H, H7, H10), 1.23 (m, 4 H, H8, H9).
13
C NMR: (75 MHz, D 2 O)
204.70 (C(5)), 174.18 (C(2)), 43.20 (C(11)), 39.33 (C(3)), 38.60 (C(4)),
27.92 (C(6)), 27.31 (C(10)), 26.39 (C(1)), 25.10 (C(7)), 24.91 (C(8)), 21.75
(C(9))
S-2-Acetamidoethyl 7-((3R,5R)-3,5-bis(tert-butyldimethylsilyloxy)-6-
phenylhexanamido)heptanethioate (3.27)
19 23
21 25
Si Si 20 Si Si 24
O O O 18 O O 22 O O
H
O 6 4 2 8 10 12 15 N 16
N 6 5 3 1 N 7 9 11 13 S 14 17
H H
3.31 O 3.27 O
Hydrolysis. Ester 3.31 (30 mg, 0.051 mmol) and LiOH (21 mg, 0.51 mmol, 10
equiv) were dissolved in a 3:2:1 solution of MeOH:H 2 O:THF (0.6 mL, 0.4 mL and 0.2
mL respectively) and stirred vigorously at r.t. for 6 hr. Quenched with 1M HCl (0.5 mL),
EtOAc (6x0.5 mL) and dried with brine (3x0.5 mL) and MgSO 4 . Coupling. After
evaporating the solvent in vacuo, the crude acid was dissolved in DCM (0.5 mL) and,
while stirring under argon at r.t., added: EDC (14.8 mg, 0.077 mmol, 1.5 equiv), DMAP
(0.6 mg, 0.005 mmol, 0.1 equiv) and HSNAC (9.0 μL, 0.085 mmol, 1.7 equiv). After
stirring overnight, the solution was diluted with water (0.5 mL) and EtOAc (0.5 mL), and
extracted with EtOAc (3x1 mL). The organic phase was washed with brine (2x1 mL) and
185
Acetone-hexanes) furnishing NAC thioester 3.27 (18.4 mg, 0.027 mmol, 53% over two
M.W.: 681.13
2,6-lutidine (2.2 equiv) were dissolved in DCM (to a 0.1 M conc.) while stirring in an ice
bath overnight. The mixture was quenched with a saturated aqueous NH 4 Cl solution (1
volume) and extracted with hexanes (4x1 volumes); dried with brine and Na 2 SO 4 , and
olefin is dissolved in t-BuOH (0.05M) and stirred under argon at r.t. In a separate flask,
186
NaIO 4 (8.0 equiv) and KMnO 4 (1.2 equiv) were vigorously stirred in a 0.2M pH 7.0
phosphate buffer (0.05M) at r.t. After 15 min, the dark purple permanganate solution was
diluted with water and extracted thrice with EtOAc (1 volume). The organic extract was
washed twice with brine, dried over Na 2 SO 4 and the solvent evaporated in vacuo.
Coupling. The isolated crude oil was dissolved in DCM (0.1M) along EDC (1.5 equiv),
DMAP (0.1 equiv) and NAC thioester 3.15 (1.2 equiv) was added while stirring under
argon. After 5 min, TEA was added (1.5 equiv) and the solution stirred overnight.
Quenched with NH 4 Cl (0.5 volumes) and extracted thrice with EtOAc (1 volume). The
organic extract was washed twice with brine and dried over Na 2 SO 4 . Following solvent
evaporation, the crude product obtained was purified by flash column chromatography
(30% acetone-hexanes).
S-2-acetamidoethyl 7-((3S,5R)-3,5-bis(tert-butyldimethylsilyloxy)-6-
phenylhexanamido)heptanethioate (3.28)
19 23
21 25
Si 20 Si Si 24
O OH 18 O O 22 O O
H
6 4 2 8 10 12 15 N 16
5 3 1 N 7 9 11 13 S 14 17
H
3.26 3.28 O
alcohol 3.26 (20.0 mg, 0.062 mmol), TBSOTf (30 µL, 0.13 mmol, 2.1 equiv), and 2,6-
lutidine (22 µL, 0.19 mmol, 3.0 equiv) in DCM (0.62 mL, 0.1M). Oxidative cleavage.
The bis-silylated crude was dissolved in t-BuOH (0.6 mL) and oxidatively cleaved by
reaction with KMnO 4 (12.0 mg, 0.074 mmol, 1.2 equiv), NaIO 4 (270 mg, 0.5 mmol, 8.0
187
equiv) dissolved in pH 7.0 buffer (0.6 mL). Coupling. The crude acid obtained was
dissolved in DCM (1.2 mL, 0.05M) and combined with NAC aminothioester 3.15 (21.0
mg, 0.074 mmol, 1.2 equiv), EDC (18.0 mg, 0.093 mmol, 1.5 equiv), DMAP (1.0 mg,
0.006 mmol, 0.1 equiv), and TEA (13 µL, 0.093 mmol, 1.5 equiv). Following purification
NAC substrate 3.28 (9.0 mg, 0.013 mmol, 21% over three steps) was isolated.
M.W.: 681.13
S-2-Acetamidoethyl 7-((3S,5S)-3,5-bis(tert-butyldimethylsilyloxy)-6-
phenylhexanamido)heptanethioate (ent-3.27)
19 23
21 25
Si 20 Si Si 24
O OH 18 O O 22 O O
H
6 4 2 8 10 12 15 N 16
5 3 1 N 7 9 11 13 S 14 17
H
ent -3.25 ent -3.27 O
alcohol ent-3.25 (39.3 mg, 0.12 mmol), TBSOTf (30 µL, 0.13 mmol, 1.1 equiv), and 2,6-
188
lutidine (31 µL, 0.27 mmol, 2.2 equiv) in DCM (1.2 mL, 0.1M). Oxidative cleavage.
The bis-silylated crude (21.2 mg) was dissolved in t-BuOH (0.4 mL) and oxidatively
cleaved by reaction with KMnO 4 (9.2 mg, 0.058 mmol, 1.2 equiv), NaIO 4 (209 mg, 0.39
mmol, 8.0 equiv) dissolved in pH 7.0 buffer (0.4 mL). Coupling. The crude acid
obtained (18.3 mg) was dissolved in DCM (0.8 mL, 0.05M) and combined with NAC
aminothioester 3.15 (13.9 mg, 0.049 mmol, 1.2 equiv), EDC (11.7 mg, 0.061 mmol, 1.5
equiv), DMAP (0.5 mg, 0.004 mmol, 0.1 equiv), and TEA (8.5 µL, 0.061 mmol, 1.5
equiv). Following purification NAC substrate ent-3.27 (16.0 mg, 0.023 mmol, 19% over
M.W.: 681.13
S-2-Acetamidoethyl 7-((3R,5S)-3,5-bis(tert-butyldimethylsilyloxy)-6-
phenylhexanamido)heptanethioate (ent-3.28)
19 23
21 25
Si 20 Si Si 24
O OH 18 O O 22 O O
H
6 4 2 8 10 12 15 N 16
5 3 1 N 7 9 11 13 S 14 17
H
ent -3.26 ent -3.28 O
alcohol ent-3.26 (42.6 mg, 0.13 mmol), TBSOTf (34 µL, 0.15 mmol, 1.1 equiv), and 2,6-
lutidine (35 µL, 0.30 mmol, 2.2 equiv) in DCM (1.3 mL, 0.1M). Oxidative cleavage.
The bis-silylated crude was dissolved in t-BuOH (0.43 mL) and oxidatively cleaved by
reaction with KMnO 4 (25.0 mg, 0.16 mmol, 1.2 equiv), NaIO 4 (542 mg, 1.0 mmol, 8.0
equiv) dissolved in pH 7.0 buffer (0.87 mL). Coupling. The crude acid obtained (48
mg) was dissolved in DCM (1.1 mL, 0.1M) and combined with NAC aminothioester 3.15
(37.0 mg, 0.13 mmol, 1.2 equiv), EDC (33.0 mg, 0.17 mmol, 1.5 equiv), DMAP (1.3 mg,
0.011 mmol, 0.1 equiv), and TEA (24 µL, 0.17 mmol, 1.5 equiv). Following purification
NAC substrate ent-3.28 (59.5 mg, 0.088 mmol, 68% over three steps) was isolated.
M.W.: 681.13
13
C NMR: (100 MHz, CDCl 3 )
199.99 (C(13)), 170.65 (C(1)), 170.29 (C(16)), 138.55 (C(Ph)), 129.72 (2
C, C(Ph)), 128.21 (2 C, C(Ph)), 126.23 (C(Ph)), 71.32 (C(5)), 67.66 (C(3)),
44.30 (C(4)), 44.18 (C(6)), 44.12 (C(2)), 43.95 (C(12)), 39.70 (C(15)),
39.00 (C(7)), 29.30 (C(8)), 28.57 (C(14)), 28.42 (C(9)), 26.53 (C(10)),
25.90 (3 C, C(21)), 25.76 (3 C, C(25)), 23.23 (C(17)), 18.01 (C(20)), 17.88
(C(24)), -4.49 (C(18)), -4.54 (C(22)), -4.56 (C(19)), -4.66 (C(23))
The purified NAC substrate contained in an Eppendorf tube was dissolved in MeCN
(0.1M) at r.t.. To this solution pyridine (10 equiv) was added, followed by HF (50
equiv). The mixture was vortexed to homogeneity for 10 sec and the reaction was
allowed to proceed without stirring for 5 hr, at which time the reaction was diluted with
water (0.2 volumes) and extracted with EtOAc (5 x 0.2 volumes). The organic extracts
were pooled and dried with MgSO 4 , and the crude purified by flash chromatography (5%
MeOH-DCM).
S-2-Acetamidoethyl 7-((3R,5R)-3,5-dihydroxy-6-phenylhexanamido)heptanethioate
(3.29)
Si Si
O O O OH OH O O
H
SNAC 6 4 2 8 10 12 15 N 16
N 6 5 3 1 N 7 9 11 13 S 14 17
H H
3.27 O 3.29 O
The general procedure was employed as described with bis-silylated NAC substrate
3.27 alcohol (16 mg, 0.023 mmol), pyridine (14 µL, 0.23 mmol, 10 equiv), and HF (99
µL, 2.3 mmol, 100 equiv) in MeCN (800 µL, 0.03M). Following purification, free diol
M.W.: 452.61
HRMS: (EI) m/z calcd for C 16 H 29 N 2 O 5 S [(M-C 7 H 7 )+] 361.1792, found 361.1831
S-2-acetamidoethyl 7-((3S,5R)-3,5-dihydroxy-6-phenylhexanamido)heptanethioate
(3.30)
Si Si
O O O OH OH O O
H
SNAC 6 4 2 8 10 12 15 N 16
N 6 5 3 1 N 7 9 11 13 S 14 17
H H
3.28 O 3.30 O
The general procedure was employed as described with bis-silylated NAC substrate
3.28 alcohol (3 mg, 0.004 mmol), pyridine (3 µL, 0.04 mmol, 10 equiv), and HF (7 µL,
0.20 mmol, 50 equiv) in MeCN (40 µL, 0.1M). Following purification, free diol 3.30
M.W.: 452.61
7.32-7.18 (m, 5 H, PhH), 5.99 (bs, 1 H, NH), 5.89 (bs, 1 H, NHAc), 4.32
(m, 1 H, H3), 4.15 (m, 1 H, H5), 3.40 (q, J = 6.0 Hz, 2 H, H15), 3.22 (q, J =
6.6 Hz, 2 H, H7), 3.00 (t, J = 6.3 Hz, 2 H, H14), 2.78 (d, J = 6.6 Hz, 2 H,
H6), 2.55 (t, J = 7.5 Hz, 2 H, H12), 2.34 (m, 2 H, H2), 1.94 (s, 3 H, 17),
1.66 (m, 4 H, H4, H11), 1.51 (m, 2 H, H8), 1.31 (m, 4 H, H9, H10)
13
C NMR: (100 MHz, CDCl 3 )
200.13 (C(13)), 172.29 (C(1)), 129.34 (2 C, C(Ph)), 128.58 (2 C, C(Ph)),
126.53 (C(Ph)), 69.92 (C(5)), 66.39 (C(3)), 43.96 (C(6)), 43.78 (C(12)),
42.29 (C(2)), 41.59 (C(4)), 39.65 (C(15)), 39.03 (C(7)), 29.13 (C(8)), 28.50
(C(14)), 28.21 (C(9)), 26.23 (C(10)), 25.38 (C(11)), 23.21 (C(17))
S-2-acetamidoethyl 7-((3S,5S)-3,5-dihydroxy-6-phenylhexanamido)heptanethioate
(ent-3.27)
Si Si
O O O OH OH O O
H
SNAC 6 4 2 8 10 12 15 N 16
N 6 5 3 1 N 7 9 11 13 S 14 17
H H
ent -3.27 O ent -3.29 O
The general procedure was employed as described with bis-silylated NAC substrate
ent-3.27 alcohol (16.0 mg, 0.023 mmol), pyridine (17 µL, 0.23 mmol, 10 equiv), and HF
(42 µL, 1.15 mmol, 50 equiv) in MeCN (250 µL, 0.1M). Following purification, free
M.W.: 452.61
13
C NMR: (100 MHz, CDCl 3 )
200.11 (C(13)), 171.87 (C(1)), 170.43 (C(16)), 137.93 (C(Ph)), 129.45 (2
C, C(Ph)), 128.60 (2 C, C(Ph)), 126.58 (C(Ph)), 73.40 (C(5)), 69.48 (C(3)),
44.40 (C(6)), 43.84 (C(12)), 43.01 (C(2)), 41.59 (C(4)), 39.67 (C(15)),
39.06 (C(7)), 29.16 (C(8)), 28.52 (C(14)), 28.28 (C(9)), 26.30 (C(10)),
25.42 (C(11)), 23.21 (C(17))
HRMS: (EI) m/z calcd for C 16 H 29 N 2 O 5 S [(M-C 7 H 7 )+] 361.1792, found 361.1745
S-2-acetamidoethyl 7-((3R,5S)-3,5-dihydroxy-6-phenylhexanamido)heptanethioate
(ent-3.30)
Si Si
O O O OH OH O O
H
SNAC 6 4 2 8 10 12 15 N 16
N 6 5 3 1 N 7 9 11 13 S 14 17
H H
ent -3.28 O ent -3.30 O
The general procedure was employed as described with bis-silylated NAC substrate
ent-3.28 alcohol (22 mg, 0.032 mmol), pyridine (23 µL, 0.23 mmol, 10 equiv), and HF
(58 µL, 1.6 mmol, 50 equiv) in MeCN (320 µL, 0.1M). Following purification free diol
M.W.: 452.61
HRMS: (EI) m/z calcd for C 16 H 29 N 2 O 5 S [(M-C 7 H 7 )+] 361.1792, found 361.1783
employing standard protocols.4 Recovered cells were spread on LB+Amp plates and
incubated at 37°C for 12 hr. Expression. E. coli BL21(DE3) was grown in 400 mL of
°C. At OD 600 = 0.45, protein expression was induced by addition of 1 mM IPTG, and the
culture incubated at 20°C with shaking at 250 rpm for 12 h. Purification. Protein
sodium phosphate, 300 mM NaCl, 10% (v/v) glycerol, 1 mg/mL lysozyme, 1 µg/mL
pepstatin A, 1-2 µg/mL leupeptin, pH 8.0). The cells were lysed by sonication on ice and
the debris was removed by centrifugation at 1.16x104 g for 45 min. The clarified lysate
was gently mixed for 3 hr with 1 mL of nickel-nitrilotriacetic acid (Ni-NTA) resin and
transferred onto a purification column. The protein was eluted with wash buffer of
purified protein was exchanged into dialysis buffer (100 mM Tris, 300 mM NaCl, 30%
(v/v) glycerol, pH 7.43) and concentrated by centrifugation with an Amicon Ultra 5000
MWCO (Millipore) at 10°C. Upon concentration, the protein was flash-frozen in liquid
nitrogen and stored at -80°C. Protein concentration was determined by the Bradford
195
culture.
μM DEBS TE, 50 mM phosphate buffer (pH 7.37), 4 % (v/v) saturated solution of DTNB
where x is the variable amount) and 10 % (v/v) DMSO in total volume of 15 μL reaction.
The formation of free thiol was quantified by measuring the absorption at 412 nm using a
The reactions were monitored for 60 min at room temperature and data points were
collected at 1, 3, 5, 10, 30, and 60 min intervals. Initial velocities (v) were determined by
linear regression analysis of the data. All steady state kinetic assays were performed with
five different concentrations of substrates (0.1, 0.2, 0.5, 2, and 5 mM) in duplicate. The
kinetic parameter k cat /K M was calculated from the slope of v versus [S]. Because of
preparation was used for determination of the specificity constant for all enantioenriched
substrates.
Table 3.1. Calculated specificity constants (k cat /K M ) for the reaction of enantioenriched substrates ent-
3.29, ent-3.30, 3.29 and 3.30 with the DEBS TE (values represent the mean ± standard error).
phosphate buffer (pH 7.4) with 2.5 mM anti-3.42 macrolactone standard at 20°C. At
various time points (15, 30, 60, 120, 180, 1620 min), aliquots were taken, diluted 4-fold
with a 50% MeCN:H 2 O solution and analyzed for hydrolysis by analytical HPLC
3.5.3. References
2. Wang, M.; Opare, P.; Boddy, C. N., Polyketide synthase thioesterases catalyze
rapid hydrolysis of peptidyl thioesters. Bioorg. Med. Chem. Lett. 2009, 19 (5), 1413-
1415.
3. Gokhale, R. S.; Hunziker, D.; Cane, D. E.; Khosla, C., Mechanism and specificity
of the terminal thioesterase domain from the erythromycin polyketide synthase. Chem.
Biol. 1999, 6 (2), 117-125.
OH
rac-3.21
198
OH
O
3.36
199
OTBS
O
3.37
200
TBSO OH
3.38
201
OH OH
anti -3.39
202
OH OH
sy n-3.39
203
O O
anti -3.44
204
O O
sy n-3.44
205
OR OR
anti -3.40
R = TBS
206
OR OR
sy n-3.40
R = TBS
207
Cl H3N O
3.34
208
OR OR O O
N O
H
anti -3.41
R = TBS
209
OR OR O O
N O
H
sy n-3.41
R = TBS
210
NH
OH
O
anti -3.42
211
COSY
HSQC
212
HMBC
213
OH
O O HN
anti -3.43
214
NH
OH
O
sy n-3.42
215
COSY
HSQC
216
HMBC
217
OH
O O HN
sy n-3.43
218
COSY
HSQC
219
HMBC
220
OH
3.21
221
OH
ent -3.21
222
OTBS
O
3.22
223
TBSO OH
OH
3.24
224
OTBS
O
ent -3.22
225
TBSO OH
OH
ent -3.24
226
OTBS
O
3.23
227
OTBS
O
ent -3.23
228
TBSO OH
3.25
229
OR OR O O
N O
H
R = TBS
3.31
230
OR OR O O
H
N
N S
R = TBS H
O
3.27
231
OH OH O O
H
N
N S
H
O
3.29
232
TBSO OH
3.26
233
TBSO OH
ent -3.25
234
TBSO OH
ent -3.26
235
OR OR O O
H
N
N S
R = TBS H
O
3.28
236
OR OR O O
H
N
N S
R = TBS H
O
ent -3.27
237
OR OR O O
H
N
N S
R = TBS H
O
ent -3.28
238
OH OH O O
H
N
N S
H
O
3.30
239
OH OH O O
H
N
N S
H
O
ent -3.29
240
OH OH O O
H
N
N S
H
O
ent -3.30
241
e-mail: atpinto@syr.edu
E DUCATION
2011 Ph.D. in Chemistry – Syracuse University
Advisor: Dr. Christopher N. Boddy
Dissertation: Synthesis and biosynthesis of polyketide natural products
2006 B.Sc. in Chemistry – SUNY College of Environmental Science and Forestry
Magna Cum Laude with Honors
Concentration: Biochemistry and Natural Products
Honors Thesis: Progress towards the biomimetic synthesis of spiculoic acid A
P ROFESSIONAL E XPERIENCE
2006-2011 Syracuse University/University of Ottawa
NSF Fellow/Research Assistant – Advisor: Dr. Christopher N. Boddy
Synthesis of polyketide Spiculoic Acid A by a biosynthesis inspired route. Designed the
study to test our hypothesis regarding the regiochemistry of double bond incorporation and
ensuing cyclization in Spiculoic Acid A biosynthesis
Understanding thioesterase (TE) catalyzed macrocyclization of polyketides. Carried out the
first rigorous study of the role absolute stereochemistry has on the regio- and
stereoselectivity in TE-catalyzed macrocyclizations using synthetic chemistry and in vitro
biochemistry
Involved in the design, implementation and optimization of a short route towards a key
Research Assistant – Advisors: Dr. Arthur J. Stipanovic; Dr. José Giner; Dr. Paul
Caluwe
Provided support in the development of an algorithm capable of estimating the relative
samples
Synthesized, purified and characterized functionalized derivatives of polycarboxylic acids to
P RESENTATIONS
2011 BioMolar Network Meeting; Ottawa, ON Canada
Pinto, A., Boddy, C.N. Understanding thioesterase catalyzed macrocyclization of
polyketides
2008 ACS NERM; Burlington, VT
Pinto, A., Boddy, C.N. Polyketide substrate analogs: investigating the TE
regiochemistry of cyclization regiochemistry of cyclization
2007 Graduate Student Symposium; Buffalo, NY
Pinto, A., Boddy, C.N. Spiculoic Acid A: understanding its biogenesis through its
biomimetic total synthesis
2006 ACS NERM; Binghamton, NY
Pinto, A., Boddy, C.N. Towards the biomimetic synthesis of Spiculoic Acid A
243
P UBLICATIONS
Pinto, A., Boddy, C.N. Spiculoic Acid A: Testing an Alternate Biosynthetic Hypothesis
(Manuscript in preparation)
Pinto, A., Boddy, C.N. Investigating the Regio- and Stereochemical Factors Effecting
Cyclization in the DEBS TE (Manuscript in preparation)