Chemoenzymatic asymmetric synthesis of pregabalin precursors via asymmetric bioreduction of β-cyanoacrylate esters using ene-reductases

Article abstract of DOI:10.1021/jo302484p

The asymmetric bioreduction of a library of β-cyanoacrylate esters using ene-reductases was studied with the aim to provide a biocatalytic route to precursors for GABA analogues, such as pregabalin. The stereochemical outcome could be controlled by substrate-engineering through size-variation of the ester moiety and by employing stereochemically pure (E)- or (Z)-isomers, which allowed to access both enantiomers of each product in up to quantitative conversion in enantiomerically pure form. In addition, stereoselectivities and conversions could be improved by mutant variants of OPR1, and the utility of the system was demonstrated by preparative-scale applications.

Full text of DOI:10.1021/jo302484p

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Article
Chemoenzymatic Asymmetric Synthesis of Pregabalin-
Precursors via Asymmetric Bioreduction of beta-
Cyano-Acrylate Esters Using Ene-Reductases
Christoph K. Winkler, Dorina Clay, Simon Davies, Pat O’Neill, Paul McDaid,
Sebastien Debarge, Jeremy Steflik, Mike Karmilowicz, John W Wong, and Kurt Faber
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/jo302484p • Publication Date (Web): 14 Jan 2013
Downloaded from http://pubs.acs.org on January 17, 2013
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Chemoenzymatic Asymmetric Synthesis of Pregabalin-Precursors via Asymmetric
Bioreduction of β-Cyano-Acrylate Esters Using Ene-Reductases
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Christoph K. Winkler,a Dorina Clay,a Simon Davies,b Pat O’Neill,b Paul McDaid,b Sebastien
Debarge,b Jeremy Steflik,c Mike Karmilowicz,c John W. Wong,c,* Kurt Fabera,*
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a
Department of Chemistry, Organic & Bioorganic Chemistry, University of Graz,
b
Heinrichstrasse 28, A-8010 Graz, Austria; Pfizer Global Process Development Centre,
c
Loughbeg, Co. Cork, Ireland; Pfizer Chemical R&D, Eastern Point Road, Groton, CT
06340, USA.
Keywords Pregabalin, ene-reductase, cyanoacrylate ester, bioreduction, flavoprotein.
Graphical Abstract - Table of Contents
Abstract
The asymmetric bioreduction of a library of β-cyano-acrylate esters using ene-reductases was
studied with the aim to provide a biocatalytic route to precursors for GABA analogs, such as
pregabalin. The stereochemical outcome could be controlled by substrate-engineering through
size-variation of the ester moiety and by employing stereochemically pure (E)- or (Z)-isomers,
which allowed to access both enantiomers of each product in up to quantitative conversion in
enantiomerically pure form. In addition, stereoselectivities and conversions could be
improved by mutant variants of OPR1 and the utility of the system was demonstrated by
preparative-scale applications.
* Corresponding author information: <John.W.Wong@Pfizer.com>, phone +1-860-441-6882;
<Kurt.Faber@Uni-Graz.at>, phone +43-316-380-5332.
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Introduction
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In recent years ene-reductases became prominent for providing a green alternative for the
highly selective production of chiral molecules.1 The enzymes are members of the flavin-
dependent Old Yellow Enzyme (OYE) family and catalyse the asymmetric reduction of a
broad variety of activated alkenes at the expense of a nicotinamide cofactor.2,3 Ene-reductases
have been utilized for the synthesis of diverse enantiomerically pure products, including
asymmetric building blocks such as chiral acyloins4 and the 'Roche-ester'.5 Further examples
encompass enantiopure α- and β-amino acid derivatives,6,7 molecules for pharma-
applications,8 and compounds of commercial importance, such as the fragrances LilialTM or
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LysmeralTM 9
.
Pregabalin [(S)-3-aminomethyl-5-methylhexanoic acid], the active ingredient in LyricaTM, is a
β-substituted γ-amino acid belonging to the class of drugs known as GABA analogs. The
manufacture of pregabalin begins with the enzymatic resolution of a β-cyanodiester,10
followed by decarboxylation to give the penultimate intermediate, (S)-ethyl 3-cyano-5-
methylhexanoate (S)-4b. Reduction of the nitrile moiety gives pregabalin.11 While this
chemo-enzymatic process achieved a large increase in efficiency over the initial
manufacturing protocol,12 we were interested in evaluating stereoselective approaches
independent on kinetic resolution protocols with the objective of further increasing process
efficiency. We postulated that asymmetric bioreduction of a β-cyanoacrylate ester by an ene-
reductase could provide access to (S)-4b, thus enabling a novel route to pregabalin, and set
out to evaluate the feasibility of this potential strategy (SCHEME 1). This approach could also
be useful for the stereoselective synthesis of related β-substituted γ-amino acids (Baclofen) or
derivatives (Brivaracetam).
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SCHEME 1. Asymmetric bioreduction of β-cyanoacrylic esters.
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Results and Discussion
β-Cyanoacrylate esters (E)-1a–5a and (Z)-1a-5a were prepared from the appropriate β-keto-
ester via enol triflate precursors using the procedure described by Frantz et al.13 (SCHEME
2). The selectivity for (Z)-enol triflates was excellent with no trace of (E)-isomers present at
the end of the reaction and (Z)-enol triflates were isolated in 80-92% yield after
chromatography. The conditions to generate (E)-analogs gave poorer selectivity (>50:1 to
5:1), as the steric bulk of the R1 group increased from Me to i-Bu. Furthermore, lower isolated
yields (54-71%) were observed for the (E)-enol triflates, which was due to less effective
extraction of the product from the Me4N+OH-/water mixture going in hand with competing
hydrolysis of triflic anhydride. Treatment of (E)- or (Z)-enol triflates with Zn(CN)2 in the
presence of a palladium(0) catalyst using the procedure of Tebben et al.14 generated the
desired β-cyanoacrylate esters in 51-86% yield. Substrates (Z)-6a and (E)-7a were obtained
by transesterification from (Z)-3a and (E)-5a, respectively.
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SCHEME 2. Synthesis of substrates (E)- and (Z)-1a-5a
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Since the (E/Z)-configuration of alkenes has been shown to have a dramatic impact on the
stereochemical outcome of ene-reductions,15-18 stereochemically pure substrates 1a-4a were
used. In addition, the size of the alcohol moiety of the (activating) ester group was varied in
order to test its influence on the substrate-docking mode of substrates 5a-7a within the active
site.4,6,18 All substrates were tested using a set of native ene-reductases, which have shown to
possess a broad substrate spectrum on acrylic ester derivatives,5,6,15,18,19 (SCHEME 1, TABLE
1).
We envisaged that the appropriate combination of substrate- and enzyme-engineering would
enable us to access both stereoisomers of GABA-precursors with high conversion and perfect
stereoselectivity. Interestingly, in line with previous reports,15-18 (E)-β-cyanoacrylate esters
generally were converted to the (S)-products while the bioreduction of (Z)-analogs gave the
enantiomeric (R)-β-cyano esters. Accordingly, (S)-1b was obtained from (E)-1a in perfect
conversion and stereoselectivity using OYE2, OYE3 and EBP1, while (R)-1b was formed
from (Z)-1a using NCR (e.e. >99, c. 94%). The same enzymes showed similar activities
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towards (E)-2a and (Z)-2a: OYEs 1-3 and NCR produced (S)-2b from (E)-2a (e.e. >99, c.
>99%) and (R)-2b could be obtained with perfect e.e. from (Z)-2a using OPR3, XenB and
NCR. In analogy, (S)-3b could be produced from (E)-3a (e.e. >99, c. >99%) and the reduction
of (E)-4a afforded (S)-4b with perfect stereoselectivity in up to 48% conversion using OYE1,
OYE2 and NCR. However, with increasing steric demand of the substrates, the conversion of
(Z)-β-cyanoacrylate esters dropped, resulting in low reactivity towards (Z)-3a and no activity
towards (Z)-4a at all. In contrast, decreasing the size of the ester moiety from ethyl to methyl
had a strongly positive impact on reaction rates: By applying methyl (E)-β-cyanoacrylate (Z)-
6a instead of the ethyl ester (Z)-3a, the activity of EBP1 forming the (R)-enantiomer could be
improved from 5% conversion (85% e.e.) to 64% conversion and perfect stereoselectivity. In
the same way, ethyl ester (Z)-4a was not accepted by any of the enzymes, but the
corresponding methyl ester (Z)-5a was converted with modest rates (OYE3, c. 29%). For the
bulkier substrates 4a, 5a and 7a bearing the i-butyl pharmacophor side chain of Pregabalin,
the stereorecognition was not as homogeneously conserved as for the smaller analogs.
Whereas the less bulky E-configured substrates strictly yielded S-products with the majority
of enzymes in up to quantitative conversion, the stereopreference of OYE3 switched to R (c.
>99, e.e. >99) for (E)-5a. This exceptional behaviour of OYE3 has been observed before6 and
depends on the steric demand of the ester group, going from absolute R-selectivity for the
methyl ester (E)-5a to moderate S-preference for the ethyl ester (E)-4a (e.e. 38%). In order to
push the incomplete S-selectivity, the size of the ester group was increased to n-propyl [(E)-
7a], which successfully resulted in (S)-7b as the sole product. Overall, OPR3, XenA and
YqjM showed poor activities towards all tested substrates and NerA and XenB accepted only
the smaller substrates 1a-3a. Using OYE2 in combination with a recently developed ADH-
based NADH-cofactor recycling system,20 (S)-1b-3b could be produced on preparative scale
(>1 g) with perfect e.e. and good isolated yields (up to 83%), which nicely demonstrates the
applicability of this bioreduction protocol to asymmetric syntheses.
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TABLE 1. Asymmetric bioreduction of β-cyanoacrylic esters using native OYEs.
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Substrate
O
OPR1 OPR3 OYE1 OYE2 OYE3 XenA XenB YqjM EBP1 NerA NCR
c.
e.e.
c.
13
50
(S)
22
>99
(R)
8
n.c.
91
>99
(S)
>99
>99
(S)
>99
>99
(S)
n.c.
14
95
(S)
56
>99
(R)
4
n.c.
>99
>99
(S)
9
45
90
(S)
97
(S)
N
C
OEt
-
-
-
(E)-1a
O
9
N
C
25
>99
(R)
15
>99
(R)
16
>99
(R)
33
88
(R)
>99
10
>99
(R)
4
>99
(R)
54
92
(R)
>99
27
94
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>99
(R)
n.c.
>99
(R)
77
OEt
e.e.
c.
(Z)-1a
n.c.
>99
>99
n.c.
n.c.
O
91
(S)
>99
(S)
>99
(S)
>99
(S)
>99
(S)
>99
(S)
>99
(S)
N
C
OEt
e.e.
-
-
-
-
(E)-2a
C
N
O
c.
e.e.
c.
15
94
(R)
10
5
n.c.
-
n.c.
-
8
n.c.
-
4
>99
(R)
4
n.c.
-
16
78
(R)
34
n.c.
28
>99
(R)
>99
(R)
57
(R)
93
OEt
-
(Z)-2a
n.c.
>99
>99
n.c.
n.c.
3
>99
O
>99
(S)
>99
(S)
>99
(S)
86
(S)
>99
(S)
>99
(S)
>99
(S)
>99
(S)
N
C
N
OEt
OEt
e.e.
-
-
-
(E)-3a
C
O
c.
e.e.
c.
n.c.
-
n.c.
-
n.c.
-
n.c.
-
4
n.c.
-
n.c.
n.c.
-
5
85
(R)
9
n.c.
n.c.
-
76
(S)
39
-
-
(Z)-3a
10
n.c.
34
39
n.c.
3
n.c.
3
48
O
>99
(S)
>99
(S)
>99
(S)
38
(S)
>99
(S)
>99
(S)
>99
(S)
>99
(S)
e.e.
-
-
-
N
C
N
OEt
OEt
(E)-4a
C
O
c.
e.e.
c.
n.c.
-
n.c.
-
n.c.
-
n.c.
-
n.c.
-
n.c.
-
n.c.
-
n.c.
n.c.
-
n.c.
-
n.c.
-
-
(Z)-4a
O
11
n.c.
30
48
>99
n.c.
n.c.
3
11
n.c.
76*
46
(S)
43
(S)
76
(S)
>99
(R)
>99
(S)
>99
(S)
>99
(S)
e.e.
-
-
-
-
N
C
OMe
OMe
OMe
(E)-5a
C
N
O
c.
e.e.
c.
9
>99
(S)
5
79
(S)
12
n.c.
-
3
n.c.
-
29
95
(S)
96
>99
(S)
18
n.c.
-
n.c.
n.c.
-
n.c.
-
n.c.
5
>99
(S)
3
2
(S)
47
64
(R)
7
50
(R)
30
-
n.c.
-
-
n.c.
-
(Z)-5a
C
N
O
n.c.
-
7
55
(R)
5
n.c.
-
n.c.
-
64
>99
(R)
11
e.e.
c.
(Z)-6a
n.c.
n.c.
3
<1
2
O
>99
(S)
>99
(S)
>99
(S)
>99
(S)
>99
(S)
>99
(S)
>99
(S)
>99
(S)
>99
(S)
e.e.
-
-
N
C
OPr
(E)-7a
c. = conversion [%]; e.e. = enantiomeric excess of products 1b-7b [%]; n. c. = no conversion; *double enzyme
and half substrate amount: >99% conversion, >99 e.e. (S).
In order to improve stereoselectivities of native enzymes, mutant variants were
constructed.21,22 A basic sequence alignment of OPR1 identified many potential sites for
mutation of which positions Cys20, Ile287 and His245 were selected for investigation. His245
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(neutral at pH 7.5) was mutated to a negatively charged Asp to enhance the polarity around
the active site entrance for improved substrate binding. The sequence alignment showed a
high variability of 14 amino acids at position Ile287, which is located in a flexible loop close
to the active site entrance extending from Arg279 to Thr290. In order to make this region
more rigid, lipophilic Ile287 was exchanged by a more polar His.43 Cys20 located at the
surface opposite to the active site was exchanged by a Tyr to probe whether such a distant
mutation had any effects on the chiral recognition process.44
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Results from bioreduction screens on OPR1 variants (TABLE 2) showed improved
conversions for variant Cys20Tyr with (E)-4a and (E)-5a compared to wild-type OPR1 while
Ile287His did not give improved conversions for any of the substrates. However, Cys20Tyr
and Ile287His both gave greatly enhanced stereoselectivities in the bioreduction of (E)-5a and
slight improvements for (E)-2a and (Z)-2a. Variant His245Asp gave the highest increase in
selectivity for the bioreduction of (E)-5a and also showed the highest improvements in
conversion for several of the substrates.
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TABLE 2. Asymmetric bioreduction of β-cyanoacrylic esters using wild-type and
mutant variants of OPR1.
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6
Substrate
Cys20Tyr Ile287His His245Asp
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8
9
c.
e.e.
c.
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8
57 (S)
18
12
41 (S)
37
O
N
C
OEt
(E)-1a
58 (S)
18
10
11
12
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N
C
O
OEt
(Z)-1a
e.e. >99 (R)
>99 (R)
2
>99 (R)
7
c.
e.e.
c.
11
>99 (S)
16
O
N
C
OEt
(E)-2a
(Z)-2a
>99 (S)
11
>99 (S)
29
N
C
O
OEt
e.e. >99 (R)
>99 (R)
5
>99 (R)
13
c.
e.e.
c.
10
>99 (S)
n.c.
-
O
N
C
OEt
(E)-3a
>99 (S)
n.c.
-
>99 (S)
n.c.
N
C
O
OEt
(Z)-3a
e.e.
c.
-
17
10
24
O
e.e.
c.
>99 (S)
n.c.
-
>99 (S)
n.c.
-
>99 (S)
n.c.
N
C
OEt
OEt
(E)-4a
(Z)-4a
N
C
O
e.e.
c.
-
18
9
37
O
e.e.
c.
72 (S)
13
78 (S)
9
90 (S)
19
N
C
OMe
OMe
(E)-5a
(Z)-5a
N
C
O
e.e.
>99 (S)
>99 (S)
>99 (S)
c. = conversion [%]; e.e. = enantiomeric excess of products 1b-5b [%]; n. c. = no conversion.
Conclusion
The asymmetric bioreduction of β-cyano-acrylate esters using ene-reductases provides an
elegant alternative to metal-dependent hydrogenation protocols for the asymmetric synthesis
of precursors for GABA analogs, such as pregabalin. This concept was successfully applied to
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the production of a library of chiral β-cyano esters. The stereochemical outcome could be
switched via substrate-engineering through size-variation of the alkoxy-moiety and by
employing stereochemically pure (E)- or (Z)-isomers, which allowed to access both
enantiomers of each product with up to quantitative conversion in enantiomerically pure form.
In addition, stereoselectivities and conversions could be improved by mutant variants of
OPR1. Finally, the utility of the system for preparative-scale applications was demonstrated
by the isolation of gram-amounts of β-cyano esters with perfect e.e. Upscaling to industrial
production should be feasible as the procedure utilized inexpensive co-substrate to recycle the
nicotinamide cofactor, improvement of the volumetric efficiency would be feasible by
development of more active enzymes via enzyme engineering and/or screening natural
diversity. Further process efficiency could be achieved by the implementation of a hydrolase-
reductase cascade process:45 In the case of pregabalin synthesis, the addition of a hydrolase to
the bioreduction of (E)-4a could result in a one-pot process to (S)-3-cyano-5-methylhexanoic
acid, the penultimate precursor to pregabalin,11 thus eliminating an additional step for
hydrolysis of (S)-4b. In conclusion, this study demonstrates the applicability of ene-
reductases as a general synthetic tool by meeting the demand for green and highly
stereoselective synthetic methods.31
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Experimental Section
General
GC analyses were carried out using a Chiraldex G-TA column (30 m x 0.25 mm id, 0.12 ꢀm
film) or a Hydrodex-β-TBDAc capillary column (250 m x 0.25 mm id). NMR spectra were
obtained on a 400 MHz or a 300 MHz NMR spectrometer. Chemical shifts are reported
relative to TMS (δ 0.00) and coupling constants (J) are given in Hz. High resolution mass
spectra were obtained either on a oa-TOF mass spectrometer with direct insertion and electron
impact ionization (70eV) or on a Ion Trap FT-MS using electrospray ionization in positive
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mode or APCI ionization in negative mode. TLC plates were run on silica gel and compounds
were visualized by spraying with phosphomolybdic acid stain or by UV (254 nm).
Lactobacillus brevis alcohol dehydrogenase was obtained from X-Zyme.
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Source of enzymes
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12-Oxophytodienoate reductase isoenzymes OPR1 and OPR3 from Lycopersicon esculentum
and the OYE homologue YqjM from Bacillus subtilis were overexpressed and purified as
reported.23-25 The cloning, purification and characterisation of OYE isoenzymes from yeast
(OYE1 from Saccharomyces pastorianus, OYE2 and OYE3 from Saccharomyces cerevisiae)
and nicotinamide-dependent cyclohexenone reductase (NCR) from Zymomonas mobilis were
performed according to literature methods.26,27 Xenobiotic reductases XenA and XenB from
Pseudomonas putida and P. fluorescens, respectively, and glycerol trinitrate reductase NerA
from Agrobacterium radiobacter were obtained as recently published.28-30 Lycopersicon
esculentum OPR1 variants Cys20Tyr, Ile287His, and His245Asp were constructed in pET26b
using QuikChange® Lightning Site-Directed Mutagenesis Kits (Stratagene, La Jolla, CA) and
were expressed as HIS-tagged proteins in E. coli BL21 cells. The cloning and purification of
EPB1 from Candida albicans and of Lycopersicon esculentum OPR1 variants Cys20Tyr,
Ile287His, and His245Asp can be found in the supporting information.
General procedure for enzymatic bioreduction
An aliquot of enzyme (OPR1-wt, OPR3, OYE1-3, XenA, XenB, YqjM, EBP1, NerA or NCR;
protein concentration in biotransformations: 75 - 125 µg/mL) was added into a 1.5 mL
microcentrifuge tube containing a Tris-HCl buffer solution (0.8 mL, 50 mM, pH 7.5), the
substrate (10 mM) and the cofactor (NADH, 15 mM). The mixture was shaken at 30 °C and
120 rpm. After 24 h the products were extracted with ethyl acetate (0.6 mL). The organic
phase was dried over Na2SO4 and analysed on GC to determine enantiomeric excess and
conversion. For every screening a control experiment without the addition of enzyme was
included.
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Determination of absolute configuration of products 1b-3b.
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Absolute configurations of 1b, 2b and 3b were determined by preparation of (S)-1b, (S)-2b
and (S)-3b via bioreductions of (E)-1a, (E)-2a, and (E)-3a at preparative scale and subsequent
conversions to suitable derivatives and comparison of specific rotations of the latter with
literature values. For determination of the absolute configuration of 4b both reference
materials (R)- and (S)-4b were prepared. For the assignment of the absolute configurations of
5b, 6b and 7b, enantiomerically pure (S)-isomers were prepared by transesterification of the
corresponding ethyl-esters (S)-4a and (S)-3a.
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Synthesis and characterisation of substrates (E)- and (Z)-1a-5a
General procedure A: Synthesis of keto-esters.
The ketone (1.0 equiv) was added slowly, over 15 min, to a stirred suspension of NaH (60%
in mineral oil, 1.2 equiv) in dry THF (500 mL) at 0°C. After the addition was complete the
cooling bath was removed and the resulting mixture was stirred at rt for 40 min. The reaction
was cooled to 0 °C and the carbonate ester (1.5 equiv) was added slowly over 1 h. After the
addition was complete the reaction was allowed to slowly warm to rt with stirring (18 h). The
reaction was quenched and acidified to pH ~6 by careful addition of acetic acid (60 mL).
Water (400 mL) was added and the resulting mixture was extracted with Et2O. The organic
extracts were washed with brine, dried over Na2SO4 and concentrated under reduced pressure
to give the crude β-ketoester.
Ethyl 3-oxopentanoate.32 Using general procedure A, 2-butanone (51.6 mL, 41.5 g, 576
mmol), NaH (60% in mineral oil, 27.6 g, 691 mmol) in dry THF (500 mL) and diethyl
carbonate (105 mL, 102 g, 864 mmol) gave the crude product as a clear yellow oil, which was
distilled twice under reduced pressure (60 °C, 4 mbar) resulting in a 2:1 mixture of the desired
product and the regioisomer ethyl 2-methyl-3-oxobutanoate as a clear colourless oil (31.9 g,
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38%). H NMR (300 MHz, CDCl3): δ 1.09 (t, J = 7.5 Hz, 3H), 1.28 (t, J = 7.0 Hz, 3H), 2.56
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(q, J = 7.5 Hz, 2H), 3.43 (s, 1H), 4.19 (q, J = 7.0 Hz, 2H); ethyl 2-methyl-3-oxobutanoate: 1H
NMR (300 MHz, CDCl3): δ 1.28 (t, J = 7.0 Hz, 3H), 1.34 (d, J = 7.0 Hz, 3H), 2.24 (s, 3H),
3.49 (q, J = 7.0 Hz, 1H), 4.19 (q, J = 7.0 Hz, 2H).
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Ethyl 3-oxohexanoate.33 Using general procedure A, 2-pentanone (61.5 mL, 49.6 g, 576
mmol), NaH (60% in mineral oil, 27.6 g, 691 mmol) in dry THF (500 mL) and diethyl
carbonate (104.9 mL, 102.1 g, 864 mmol) gave the crude product as a clear yellow oil, which
was distilled under reduced pressure (60 °C, 4 mbar) yielding the desired product as a clear
colourless oil (39.9 g, 44%). 1H NMR (300 MHz, CDCl3): δ 0.93 (t, J = 7.5 Hz, 3H), 1.28 (t, J
= 7.0 Hz, 3H), 1.56-1.70 (m, 2H), 2.51 (t, J = 7.0 Hz, 2H), 3.42 (s, 2H), 4.19 (q, J = 7.0 Hz,
2H).
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Ethyl 5-methyl-3-oxohexanoate.34 Using general procedure A, 4-methyl-2-pentanone (608.0
mL, 487.0 g, 4.86 mol), NaH (60% in mineral oil, 256.7 g, 1.32 eq.) in dry THF (4.87 L) and
diethyl carbonate (884.0 mL, 1.5 eq.) gave the crude product as an orange oil. The crude
product was distilled under reduced pressure (64-82°C, 7-10 mbar) the desired product as a
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pale yellow oil (661.2 g, 79% isolated yield). H NMR (300 MHz, CDCl3): δ 0.86 (d, J = 6.4
Hz, 6H), 1.21 (t, J = 7.2 Hz, 3H), 2.09 (J = 6.7 Hz, 1H), 2.35 (d, J = 6.9 Hz, 2H), 3.34 (s, 2H),
4.12 (q, J = 7.2 Hz, 2H).
Methyl 5-methyl-3-oxohexanoate.35 Using general procedure A, 4-methyl-2-pentanone (72.1
mL, 57.7 g, 576 mmol), NaH (60% in mineral oil, 27.6 g, 691 mmol) in dry THF (500 mL)
and dimethyl carbonate (72.8 mL, 77.8 g, 864 mmol) gave the crude product as a cloudy
yellow oil. The crude product was distilled under reduced pressure (60 °C, 4 mbar) to give the
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desired product as a clear colourless oil (61.9 g, 68%). H NMR (300 MHz, CDCl3): δ 0.93
(d, J = 6.5 Hz, 6H), 2.09-2.22 (m, 1H), 2.41 (d, J = 7.0 Hz, 2H), 3.42 (s, 2H), 3.73 (s, 3H).
General procedure B: Synthesis of (Z)-triflates.
Lithium triflate (2.0 equiv) was added to a stirred solution of the keto-ester (1.0 equiv) in dry
CH2Cl2 (150 mL). The resulting mixture was cooled to 0 °C and diisopropylethylamine (1.1
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equiv) was added dropwise over 10 min. After the addition was complete the mixture was
stirred at 0 °C for 20 min before triflic anhydride (1.1 equiv) was added slowly over 30 min.
The resulting mixture was stirred at 0 °C for 1 h before carefully being quenched by addition
of saturated aqueous NH4Cl (60 mL). The reaction was allowed to warm to rt and an
additional portion of CH2Cl2 (30 mL) was added. The layers were separated and the organic
layer was washed with aqueous HCl (1 M, 2 x 30 mL), water (30 mL) and brine (30 mL). The
organic layer was dried over Na2SO4 and concentrated under reduced pressure. Et2O (60 mL)
was added to the resulting semi-solid and the mixture was filtered. The solids were extracted
with more Et2O (2 x 20 mL) and the combined ether extracts were concentrated under reduced
pressure to give the crude product.
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(Z)-Ethyl 3-(trifluoromethylsulfonyloxy)but-2-enoate.13 Using general procedure B,
lithium triflate (24.0 g, 154 mmol), ethyl acetoacetate (9.7 mL, 10.0 g, 76.8 mmol) in dry
CH2Cl2 (150 mL), diisopropylethylamine (14.7 mL, 10.9 g, 84.5 mmol) and triflic anhydride
(14.22 mL, 23.9 g, 84.5 mmol) gave the crude product as a clear yellow oil (18.6 g, 92%)
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which was used without further purification. H NMR (300 MHz, CDCl3): δ 1.30 (t, J = 7.0
Hz), 2.16 (s, 3H), 4.24 (q, J = 7.0 Hz, 2H), 5.75 (q, J = 1.0 Hz, 1H); 13C NMR (100MHz,
CDCl3): δ 14.2, 21.1, 61.4, 113.0, 118.5 (q, J = 319.8 Hz, CF3), 155.2, 162.5.
(Z)-Ethyl 3-(trifluoromethylsulfonyloxy)pent-2-enoate.13 Using general procedure B,
lithium triflate (21.6 g, 139 mmol), ethyl 3-oxopentanoate (10.0 g, 69.4 mmol) in dry CH2Cl2
(150 mL), diisopropylethylamine (13.3 mL, 9.9 g, 76.3 mmol) and triflic anhydride (12.8 mL,
21.5 g, 76.3 mmol) gave the crude product as a clear dark orange oil. Purification by flash
chromatography (silica gel, cyclohexane/EtOAc, 9:1) afforded the desired product as a clear
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pale yellow oil (17.7 g, 92% yield). H NMR (300 MHz, CDCl3): δ 1.18 (t, J = 7.5 Hz, 3H),
1.31 (t, J = 7.0 Hz, 3H), 2.44 (qd, J = 7.5, 1.0 Hz, 2H), 4.25 (q, J = 7.0 Hz, 2H), 5.75 (t, J =
1.0 Hz, 1H).
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(Z)-Ethyl 3-(trifluoromethylsulfonyloxy)hex-2-enoate. Using general procedure B, lithium
triflate (19.7 g, 126 mmol), ethyl 3-oxohexanoate (10.0g, 63.2 mmol) in dry CH2Cl2 (150
mL), diisopropylethylamine (12.1 mL, 9.0 g, 69.5 mmol) and triflic anhydride (11.7 mL, 19.6
g, 69.5 mmol) gave the crude product as a clear dark red oil (17.0 g, 93% yield) which was
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used without further purification. H NMR (300 MHz, CDCl3): δ 0.99 (t, J = 7.5 Hz, 3H),
1.30 (t, J = 7.0 Hz, 3H), 1.55-1.69 (m, 2H), 2.37 (t, J = 7.5 Hz, 2H), 4.25 (q, J = 7.0 Hz, 2H),
5.74 (t, J = 1.0 Hz, 1H). 13C NMR (100MHz, CDCl3): δ 13.3, 14.2, 19.5, 36.5, 61.5, 112.1,
118. 6 (q, J = 319.8 Hz, CF3), 158.9, 162.7. HRMS (EI+) m/z calcd for C9H13F3O5S [M]+
290.0439, found 290.0436.
(Z)-Ethyl 5-methyl-3-(trifluoromethylsulfonyloxy)hex-2-enoate.34
A 20L jacketed reactor was charged with CH2Cl2 (2.45L), ethyl 3-oxo-5-methylhexanoate
(163.3 g; 1eq) and lithium triflate (295.86 g; 2eq) under an inert atmosphere. The resulting
white suspension was stirred and cooled to 0°C. N,N-Diisopropylethylamine (134.8 g; 182 ml;
1.1 eq) was added over 10 min at <10°C. The suspension was stirred for 20 min at 0°C, then
triflic anhydride (294.28 g; 175 ml; 1.1eq) was added slowly to the mixture over 30 minat
<10°C. The suspension was stirred at 0°C for 1 hour. The mixture then was allowed to warm
from 0 to 25°C over 1hr whilst quenching with sat. NH4Cl solution (980 ml).
Dichloromethane (490 ml) was added and the mixture was stirred for 5 min, then allowed to
settle and the layers were separated. The aqueous layer was extracted with CH2Cl2 (490 ml).
The combined organic phases were then washed with 1M HCl (2 x 490 ml), water (490 ml)
and 20% brine (490 ml), then stirred for 30 min with MgSO4 (~165 g) for 15-30 min. The
solids were removed by filtration and washed with CH2Cl2 (165 ml). The filtrate was
concentrated under vacuum at ≤40°C to give an orange/brown oil containing crystalline
solids. The crude product was triturated with MTBE (980 ml). The mixture was filtered and
the solids were washed with MTBE (2 x 165 ml). The filtrate was concentrated under vacuum
at ≤40°C to give the title product as an orange/brown oil: overall yield 271 g (94%; corrected
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for 2.1% w/w MTBE content); 87.7% area purity by GC. 1H NMR (400MHz, CDCl3): δ 0.94
(6H, d, J= 6.7 Hz), 1.24 (3H, t, J= 7.2 Hz), 1.89 (1H, hept, J= 6.8 Hz), 2.17 (2H, d, J= 7.2
Hz), 4.18 (2H, q, J= 7. 2 Hz), 5.67 (1H, s).
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(Z)-Methyl 5-methyl-3-(trifluoromethylsulfonyloxy)hex-2-enoate. Using general procedure
B, lithium triflate (19.7 g, 126 mmol), methyl 5-methyl-3-oxohexanoate (10.0 g, 63.2 mmol)
in dry CH2Cl2 (150 mL), diisopropylethylamine (12.1 mL, 9.0 g, 69.5 mmol) and triflic
anhydride (11.7 mL, 19.6 g, 69.5 mmol) gave the crude product as a clear orange oil.
Purification by flash chromatography (silica gel, cyclohexane/EtOAc, 9:1) afforded the
desired product as a clear pale yellow oil (14.7 g, 80%). 1H NMR (300 MHz, CDCl3): δ 0.99
(d, J = 6.6 Hz, 6H), 1.98 (dp, J = 13.6, 6.8, 1H), 2.27 (d, J = 7.2 Hz, 2H), 3.80 (s, 3H), 5.76 (s,
1H). 13C NMR (75 MHz, CDCl3): δ 21.9, 25.8, 43.7, 51.9, 112.4, 118. 4 (q, J = 318.0 Hz,
CF3), 158.3, 162.8. HRMS (ES+) m/z calcd for C9H14F3O5S [M+H]+ 291.0514, found
291.0519.
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General procedure C: Synthesis of (E)-triflates.
A mixture of Me4N+OH- (25% in H2O, 5.0 equiv) and water (72.5 mL) was added dropwise,
over 30 min to a vigorously stirred solution of the keto-ester (1.0 equiv) in heptane (300 mL)
at 0°C. After the addition was complete the mixture was stirred at 0 °C for 15 min before
triflic anhydride (2.5 equiv) was added dropwise over 1 h. After the addition was complete the
reaction was stirred for another 1 h at 0 °C before being quenched by careful addition of water
(75 mL). The mixture was allowed to warm to rt and the layers were separated. The aqueous
layer was extracted with EtOAc (3 x 100 mL). The combined organic extracts were washed
with water (75 mL) and brine (75 mL), dried over Na2SO4 and concentrated under reduced
pressure to yield the crude product.
(E)-Ethyl 3-(trifluoromethylsulfonyloxy)but-2-enoate.13 Using general procedure C,
Me4N+OH- (25% in H2O, 137 mL, 140 g, 384 mmol) and water (72.5 mL), ethyl acetoacetate
(9.7 mL, 10.0 g, 76.8 mmol) in heptanes (300 mL) and triflic anhydride (32.3 mL, 54.2 g,
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192.1 mmol) gave the crude product as a clear colourless oil (14.4 g, 71%), which was used
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without further purification. H NMR (400 MHz, CDCl3): δ 1.31 (t, J = 7.0 Hz, 3H), 2.51 (s,
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3H), 4.22 (q, J = 7.0 Hz, 2H), 5.95 (s, 1H).
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(E)-Ethyl 3-(trifluoromethylsulfonyloxy)pent-2-enoate.13 Using general procedure C,
Me4N+OH- (25% in H2O, 124 mL, 26.4 g, 347 mmol) and water (72.5 mL), ethyl 3-
oxopentanoate (10.0 g, 69.4 mmol) in heptanes (300 mL) and triflic anhydride (29.2 mL, 48.9
g, 173.4 mmol) gave the crude product as a clear pale yellow oil. Purification by flash
chromatography (silica gel, cyclohexane/EtOAc, 19:1) afforded the desired product as a clear
colourless oil (10.6 g, 55%). 1H NMR (400 MHz, CDCl3): δ 1.21 (t, J = 7.5 Hz, 3H), 1.31 (t, J
= 7.0 Hz, 3H), 2.94 (q, J = 7.5 Hz, 2H), 4.22 (q, J = 7.0 Hz, 2H), 5.92 (s, 1H).
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(E)-Ethyl 3-(trifluoromethylsulfonyloxy)hex-2-enoate.36 Using general procedure C,
Me4N+OH- (25% in H2O, 113 mL, 115 g, 316.1 mmol) and water (72.5 mL), ethyl 3-
oxohexanoate (10.0 g, 63.2 mmol) in heptanes (300 mL) and triflic anhydride (26.6 mL, 44.6
g, 158.0 mmol) gave the crude product as a clear yellow oil (13.8 g, 75% yield), which was
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used without further purification. H NMR (300 MHz, CDCl3): δ 0.99 (t, J = 7.5 Hz, 3H),
1.30 (t, J = 7.0 Hz, 3H), 1.59-1.73 (m, 2H), 2.90 (t, J = 7.5 Hz, 2H), 4.22 (q, J = 7.0 Hz, 2H),
5.95 (s, 1H).
(E)-Ethyl 5-methyl-3-(trifluoromethylsulfonyloxy)hex-2-enoate.34
A 20L jacketed reactor was charged with hexane (7.83L) and ethyl 3-oxo-5-methylhexanoate
(261.0 g; 1eq) under an inert atmosphere. The solution was cooled to 5°C and stirred
vigorously whilst adding a mixture of 25% aq. tetramethylammonium hydroxide (2.76 kg;
2.76 L; 5eq) and water (1.89 L) over 30 min at 15°C. The biphasic mixture was stirred
vigorously for 10 min at 5-10°C, then triflic anhydride (1.07 kg; 637 ml; 2.5 eq) was added
dropwise to the mixture over 1.5 hours at <10°C. The mixture was stirred vigorously at 5°C
for a further 1 hour, then agitation was stopped and the layers allowed to settle before
sampling upper organic layer. The mixture was then allowed to warm from 5 to 25°C over 1
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hour whilst quenching with water (1.97 L), then stirred for 5 min. The mixture was allowed to
settle and layers were separated. The aqueous layer was extracted with EtOAc (3.92 L). The
combined organic layers were washed with water (1.97 L) then 20% brine (1.97 L), stirred
with MgSO4 (~300g) for 15-30 min, then filtered. The residue was washed with EtOAc (260
ml) and the filtrate was concentrated under vacuum at ≤40°C to give the E-vinyl triflate as a
brown oil (overall yield of 378 g, 82%; corrected for 8.4%w/w EtOAc content, 75.5% area
purity by GC). NMR analysis indicated that the isomeric ratio was 6:1.
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1H NMR (400 MHz, CDCl3): δ 0.92 (6H, d, J= 6.7 Hz), 1.24 (3H, t, J= 7.2Hz), 1.95 (1H,
hept, J= 6.8 Hz), 2.17 (0.33H, d, J= 7.2 Hz), 2.76 (2H, d, J= 7.3 Hz), 4.14 (2H, q, J= 7. 2),
4.18 (0.28H, q, J= 7. 2 Hz), 5.66 (0.14H, s), 5.91 (1H, s).
(E)-Methyl
5-methyl-3-(trifluoromethylsulfonyloxy)hex-2-enoate.
Using
general
procedure C, Me4N+OH- (25% in H2O, 113 mL, 115 g, 316 mmol) and water (72.5 mL), ethyl
3-oxo-5-methylhexanoate (10.0 g, 63.2 mmol) in heptane (300 mL) and triflic anhydride (26.6
mL, 44.6 g, 158 mmol) gave the crude product as a clear pale yellow oil. Purification by flash
chromatography (silica gel, cyclohexane-EtOAc, 19:1) afforded the desired product as a clear
colourless oil (10.0 g, 54%). 1H NMR (400 MHz, CDCl3): δ 0.99 (d, J = 6.5 Hz, 6H), 2.02 (m,
1H), 2.83 (d, J = 7.5 Hz, 2H), 3.76 (s, 3H), 5.99 (s, 1H). 13C NMR (125MHz, CDCl3): δ 21.8,
22.1, 26.5, 39.8, 51.9, 112.9, 118. 4 (q, J = 319.8 Hz, CF3), 164.5, 165.1. HRMS (ES+) m/z
calcd for C9H14F3O5S [M+H]+ 291.0514, found 291.0516.
General procedure D: Synthesis of α,β-dehydro cyanoesters.
The triflate (1.0 equiv) and Zn(CN)2 (0.6 equiv) were added to dry DMF (75 mL). The
mixture was purged with N2 for 15 min before Pd(PPh3)4 (0.01 equiv) was added and the
mixture was heated at 70 °C for 15 h. The reaction mixture was allowed to cool to rt and
toluene (125 mL) was added. The mixture was washed with aqueous ammonium hydroxide
(20%, 2 x 50 mL) and the aqueous washings were extracted with toluene (50 mL). The
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combined organic extracts were washed with brine (50 mL) dried over Na2SO4 and
concentrated under reduced pressure to give the crude product.
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(Z)-Ethyl 3-cyanobut-2-enoate [(Z)-1a].37 Using general procedure D, (Z)-ethyl 3-
(trifluoromethyl sulfonyloxy)but-2-enoate (12.3 g, 46.8 mmol), Zn(CN)2 (3.30 g, 28.1 mmol)
in DMF (75 mL) and Pd(PPh3)4 (0.54 g, 0.47 mmol) gave the crude product as a clear yellow
oil. Purification by flash chromatography (silica gel, cyclohexane/EtOAc 9:1) afforded the
desired product as a clear pale yellow oil (4.23 g, 65%). 1H NMR (400 MHz, CDCl3): δ 1.33
(t, J = 7.0 Hz, 3H), 2.16 (s, 3H), 4.28 (q, J = 7.0 Hz, 2H), 6.32 (s, 1H); 13C NMR (100 MHz,
CDCl3): δ 14.0, 22.0, 61.5, 116.4, 122.8, 133.1, 162.7.
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(E)-Ethyl 3-cyanobut-2-enoate [(E)-1a].37 Using general procedure D, (E)-ethyl 3-
(trifluoromethyl sulfonyloxy)but-2-enoate (12.3 g, 46.8 mmol), Zn(CN)2 (3.30 g, 28.1 mmol)
in dry DMF (75 mL) and Pd(PPh3)4 (0.54 g, 0.47 mmol) gave the crude product as a clear red
oil. Purification by flash chromatography (silica gel, cyclohexane/EtOAc, 9:1) afforded the
desired product as a clear pale yellow oil (4.52 g, 69%). 1H NMR (400 MHz, CDCl3): δ 1.31
(t, J = 7.0 Hz, 3H), 2.35 (s, 3H), 4.23 (q, J = 7.0 Hz, 2H), 6.41 (s, 1H); 13C NMR (100 MHz,
CDCl3): δ 14.1, 17.3, 61.2, 118.8, 125.9, 132.7, 163.8.
(Z)-Ethyl 3-cyanopent-2-enoate [(Z)-2a)]. Using general procedure D, (Z)-ethyl 3-
(trifluoromethyl sulfonyloxy)pent-2-enoate (17.66 g, 63.9 mmol), Zn(CN)2 (4.50 g, 38.4
mmol) in dry DMF (75 mL) and Pd(PPh3)4 (0.74 g, 0.64 mmol) gave the crude product as a
clear yellow oil. Purification by flash chromatography (silica gel, cyclohexane/EtOAc 9:1
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afforded the desired product as a clear pale yellow oil (7.27 g, 74%). H NMR (400 MHz,
CDCl3): δ 1.24 (t, J = 7.5 Hz, 3H), 1.34 (t, J = 7.1 Hz, 3H), 2.45 (qd, J = 7.4, 1.5 Hz, 2H),
4.29 (q, J = 7.1 Hz, 2H), 6.33 (t, J = 1.5 Hz, 1H); 13C NMR (100 MHz, CDCl3): δ 12.0, 14.0,
29.3, 61.5, 115.9, 129.2, 131.4, 162.9; HRMS (ESI) m/z calcd for C8H11NO2Na (M + Na+)
176.0682, found 176.0680.
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(E)-Ethyl 3-cyanopent-2-enoate [(E)-2a]. Using general procedure D, (E)-ethyl 3-
(trifluoromethyl sulfonyloxy)pent-2-enoate (10.62 g, 38.4 mmol), Zn(CN)2 (2.71 g, 23.1
mmol) in dry DMF (75 mL) and Pd(PPh3)4 (0.44 g, 0.38 mmol) gave the crude product as a
clear yellow oil. Purification by flash chromatography (silica gel, cyclohexane/EtOAc 9:1)
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afforded the desired product as a clear pale yellow oil (4.9 g, 84%). H NMR (400 MHz,
CDCl3): δ 1.23 (t, J = 7.6 Hz, 3H), 1.32 (t, J = 7.1 Hz, 3H), 2.81 (qd, J = 7.6, 1.2 Hz, 2H),
4.24 (q, J = 7.1 Hz, 2H) 6.39 (t, J = 1.2 Hz, 1H); 13C NMR (100 MHz, CDCl3): δ 12.4, 14.1,
23.6, 61.2, 118.0, 131.8, 132.6, 163.7; HRMS (EI) m/z calcd for C8H11NO2 (M+) 153.0790,
found 153.0798.
(Z)-Ethyl 3-cyanohex-2-enoate [(Z)-3a]. Using general procedure D, (Z)-ethyl 3-
(trifluoromethyl sulfonyloxy)hex-2-enoate (13.6 g, 46.8 mmol), Zn(CN)2 (3.30 g, 28.1 mmol)
in dry DMF (75 mL) and Pd(PPh3)4 (0.54 g, 0.47 mmol) gave the crude product as a clear
yellow oil. Purification by flash chromatography (silica gel, cyclohexane/EtOAc 9:1, product
from first column was re-purified on a second silica column) afforded the desired product as a
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clear pale yellow oil (4.56 g, 58%). H NMR (300 MHz, CDCl3): δ 0.98 (t, J = 7.4 Hz, 3H),
1.34 (t, J = 7.1 Hz, 3H), 1.68 (m, 2H), 2.37 (td, J = 7.5, 1.5 Hz, 2H), 4.29 (q, J = 7.1 Hz, 2H),
6.32 (t, J = 1.5 Hz, 1H); 13C NMR (100 MHz, CDCl3): δ 13.1, 14.0, 20.8, 37.7, 61.5, 115.9,
127.8, 132.3, 162.9; HRMS (ESI) m/z calcd for C9H13NO2Na (M + Na+) 190.0839, found
190.0838.
(E)-Ethyl 3-cyanohex-2-enoate [(E)-3a]. Using general procedure D, (E)-ethyl 3-
(trifluoromethyl sulfonyloxy)hex-2-enoate (13.6 g, 46.8 mmol), Zn(CN)2 (3.30 g, 28.1 mmol)
in dry DMF (75 mL) and Pd(PPh3)4 (0.54 g , 0.47 mmol) gave the crude product as a clear
yellow oil. Purification by flash chromatography (silica gel, cyclohexane/EtOAc, 19:1,
product from first column was re-purified on second silica column) afforded the desired
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product as a clear pale yellow oil (4.02 g, 51%). H NMR (400 MHz, CDCl3): δ 1.00 (t, J =
7.4 Hz, 3H), 1.32 (t, J = 7.1 Hz, 3H), 1.67 (dq, J = 14.8, 7.4 2H), 2.77 (td, J = 7.6, 1.1 Hz,
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2H), 4.23 (q, J = 7.0 Hz, 2H), 6.42 (s, 1H); 13C NMR (100 MHz, CDCl3): δ 13.3, 14.1, 21.3,
31.8, 61.2, 118.2, 131.2, 132.4, 163.7; HRMS (EI) m/z calcd for C9H13NO2 (M+) 167.0946,
found 167.0952.
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(Z)-Ethyl 5-methyl-3-cyanohex-2-enoate [(Z)-4a].34 Using General Procedure D, (Z)-ethyl-
5-methyl-3-(trifluoromethylsulfonyloxy)hex-2-enoate (271.38 g, 1 equiv), Zn(CN)2 (62.8 g,
0.6 equiv) in dry DMF (1.63 L) and Pd(PPh3)4 (10.31 g, 0.01 equiv) gave the crude product as
a yellow oil. Purification by chromatography on a Biotage apparatus (25.3g per run using a
65i cartridge (350 g silica) and 1-6% EtOAc in cyclohexane) gave the product as a yellow oil
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(67 g, 60% yield). H NMR (400 MHz, CDCl3): δ 0.90 (d, J = 6.6 Hz, 6H), 1.27 (t, J = 7.2
Hz, 3H), 1.99 (hept, J = 6.8 Hz, 1H), 2.19 (dd, J = 7.2 Hz, 1.2 Hz, 2H), 4.22 (q, J = 7.2 Hz,
2H), 6.21 (t, J = 1.2 Hz, 1H).
(E)-Ethyl 5-methyl-3-cyanohex-2-enoate [(E)-4a].34 Using general procedure D, (E)-ethyl-
5-methyl-3-(trifluoromethylsulfonyloxy)hex-2-enoate (378 g, 1 equiv), Zn(CN)2 (87.6 g, 0.6
equiv) in dry DMF (2.27 L), and Pd(PPh3)4 (14.4g; 0.01 equiv) gave the crude product as a
red oil (244 g, 108%). A portion of the crude product (111 g) was purified by chromatography
on a Biotage apparatus using a 65i cartridge (350 g silica) and 1-6% EtOAc in cyclohexane to
give the product as an yellow-orange oil (55 g, 53%). 1H NMR (400 MHz, CDCl3): δ 0.91 (d,
J = 6.7 Hz, 6H), 1.24 (t, J = 7.1 Hz, 3H), 1.95 (hept, J = 6.8 Hz, 1H), 2.61 (dd, J = 7.3, 1.1
Hz, 2H), 4.15 (q, J = 7.1 Hz, 2H), 6.37 (t, J = 1.1 Hz, 1H).
(Z)-Methyl 5-methyl-3-cyanohex-2-enoate [(Z)-5a].38 Using general procedure D, (Z)-
methyl 5-methyl-3-(trifluoromethylsulfonyloxy)hex-2-enoate (14.7 g, 50.8 mmol) and
Zn(CN)2 (3.58 g, 30.5 mmol) in dry DMF (75 mL) and Pd(PPh3)4 (0.59 g, 0.51 mmol) gave
the crude product as a clear yellow oil. Purification by flash chromatography (silica gel,
cyclohexane/EtOAc 9:1) afforded the desired product as a clear pale yellow oil (7.32 g, 86%).
1H NMR (400 MHz, CDCl3): δ 0.96 (d, J = 6.5 Hz, 6H), 2.05 (m, 1H), 2.20 (dd, J = 7.0, 1.0
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Hz, 2H), 3.82 (s, 3H), 6.29 (t, J = 1.0 Hz, 1H); 13C NMR (100 MHz, CDCl3): δ 21.9, 27.1,
44.8, 52.3, 116.0, 127.5, 132.6, 163.2.
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(E)-Methyl 5-methyl-3-cyanohex-2-enoate [(E)-5a].34 Using general procedure D, (E)-
methyl 5-methyl-3-(trifluoromethylsulfonyloxy)hex-2-enoate (9.96 g, 34.3 mmol), Zn(CN)2
(2.42 g, 20.6 mmol) in dry DMF (75 mL) and Pd(PPh3)4 (0.40 g, 0.34 mmol) gave the crude
product as a clear red oil. Purification by flash chromatography (silica gel,
cyclohexane/EtOAc 9:1, product from first column was re-purified on a second silica column)
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afforded the desired product as a clear pale yellow oil (4.24 g, 74%). H NMR (400 MHz,
CDCl3): δ 0.98 (d, J = 6.5 Hz, 6H), 2.02 (m, 1H), 2.69 (dd, J = 7.5, 1.0 Hz, 2H), 3.77 (s, 3H),
6.45 (t, J = 1.0 Hz, 1H); 13C NMR (100 MHz, CDCl3): δ 22.0, 28.0, 38.5, 52.0, 118.3, 131.1,
132.5, 164.2.
Synthesis and characterisation of substrates (Z)-6a and (E)-7a
General procedure E: Synthesis of α,β-dehydro cyanoesters (Z)-6a and (E)-7a.
To a solution of starting ester (1 mmol) in alcohol (methanol or n-propanol, 25 mL) in a round
bottom flask (100 mL) equipped with a reflux condenser hydrochloric acid (conc., 0.5 mL)
was added. The resulting mixture was stirred under reflux until all starting material was
consumed. After the reaction was complete a solution of NaHCO3 (saturated, 15 mL) was
added and the mixture was extracted three times with ethyl acetate. The combined organic
phases were dried over Na2SO4, and yielded the crude product after the solvent was removed
under reduced pressure.
(Z)-Methyl 3-cyanohex-2-enoate [(Z)-6a]. Using general procedure E, (Z)-ethyl 3-cyanohex-
2-enoate [(Z)-3a] (300 mg, 1.79 mmol) in methanol (40 mL) and hydrochloric acid (conc., 0.5
mL) gave the product after 5 days reaction time. Purification by flash chromatography (silica
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gel, petroleum ether/EtOAc 20:1) afforded the product (173 mg, 63%). H NMR (300 MHz,
CDCl3): δ 6.33 (s, 1H), 3.83 (s, 3H), 2.38 (dd, J = 10.8, 4.1, 2H), 1.84 – 1.60 (m, 2H), 0.98 (t,
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J = 7.4, 3H); 13C NMR (75 MHz, CDCl3): δ 163.3, 131.9, 128.2, 115.9, 52.3, 37.7, 20.8, 13.1;
HRMS (EI) m/z calcd for C8H11NO2 (M+) 153.0790, found 153.0786.
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(E)-Propyl 3-cyano-5-methylhex-2-enoate [(E)-7a]. Using general procedure E, (E)-ethyl 3-
cyano-5-methylhex-2-enoate [(E)-5a] (150 mg, 0.82 mmol) in n-propanol (30 mL) and
hydrochloric acid (conc., 0.5 mL) gave the pure product (120 mg, 75%) after 5 days reaction
time. 1H NMR (300 MHz, CDCl3): δ 6.47 (s, 1H), 4.14 (t, J = 6.7, 2H), 2.70 (dd, J = 7.3, 1.0,
2H), 2.17 – 1.94 (m, 1H), 1.79 – 1.64 (m, 2H), 1.00 (d, J = 6.0, 6H), 0.98 (t, J = 9.0, 3H); 13C
NMR (75 MHz, CDCl3): δ 163.9, 133.0, 130.6, 118.4, 66.8, 38.5, 28.0, 22.0, 21.8, 10.4;
HRMS (EI) m/z calcd for C11H17NO2 (M+) 195.1259, found 195.1277.
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Synthesis and characterisation of racemic reference-compunds rac-1b-7b
General procedure F: Synthesis of racemic reference-compunds rac-1b-6b.
The starting acrylic ester (0.5 mmol) was dissolved in THF (5 mL) and hydrogenated under
H2 at atmospheric pressure at room temperature employing 10% Pd/C (5 mg) as catalyst by
stirring overnight at room temperature. Afterwards the reaction mixture was filtered through
celite and concentrated to yield the pure hydrogenated product in quantitative conversion.
rac-Ethyl 3-cyanobutanoate (rac-1b).10 Using general procedure F, hydrogenation of (Z)-
ethyl 3-cyanobut-2-enoate [(Z)-1a] (51 mg, 0.39 mmol) gave the pure product (97%, 50.3 mg,
0.35 mmol). 1H NMR (300 MHz, CDCl3): δ 4.22 (dq, J = 14.3, 7.2, 2H), 3.30 – 2.98 (m, 1H),
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2.63 (ddd, J = 56.4, 16.6, 7.2, 2H), 1.39 (d, J = 7.1, 3H), 1.29 (t, J = 7.1, 3H); C NMR (75
MHz, CDCl3): δ 169.6, 121.8, 61.3, 38.2, 21.7, 17.7, 14.1.
rac-Ethyl 3-cyanopentanoate (rac-2b).10 Using general procedure F, hydrogenation of (Z)-
ethyl 3-cyanopent-2-enoate [(Z)-2a] (50 mg, 0.33 mmol) gave the pure product (97%, 49.5
mg, 0.32 mmol). 1H NMR (300 MHz, CDCl3): δ 4.20 (q, J = 7.1, 2H), 3.14 – 2.86 (m, 1H),
2.63 (ddd, J = 23.3, 16.5, 7.2, 2H), 1.84 – 1.57 (m, 2H), 1.29 (t, J = 7.1, 3H), 1.12 (t, J = 7.4,
3H); 13C NMR (75 MHz, CDCl3): δ 169.8, 121.0, 61.3, 36.4, 29.1, 25.2, 14.1, 11.4.
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rac-Ethyl 3-cyanohexanoate (rac-3b).10 Using general procedure F, hydrogenation of (Z)-
ethyl 3-cyanohex-2-enoate [(Z)-3a] (51 mg, 0.31 mmol) gave the pure product (80%, 42 mg,
0.25 mmol). 1H NMR (300 MHz, CDCl3): δ 4.21 (q, J = 7.1, 2H), 3.03 (m, 1H), 2.82 – 2.46
(m, 2H), 1.82 – 1.34 (m, 4H), 1.34 (t, J = 9, 3H), 0.98 (t, J = 6.8, 3H); 13C NMR (75 MHz,
CDCl3): δ 169.8, 121.1, 61.3, 36.8, 33.8, 27.4, 20.2, 14.1, 13.4.
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rac-Methyl 3-cyano-5-methylhexanoate (rac-5b).34 Using general procedure F,
hydrogenation of (Z)-methyl 3-cyano-5-methylhex-2-enoate [(Z)-5a] (90 mg, 0.54 mmol)
gave the pure product (93%, 85 mg, 0.50 mmol). 1H NMR (300 MHz, CDCl3): δ 3.75 (s, 3H),
3.16 – 2.96 (m, 1H), 2.63 (ddd, J = 23.3, 16.6, 7.1, 2H), 1.98 – 1.76 (m, 1H), 1.65 (ddd, J =
13.5, 10.7, 4.8, 1H), 1.40 – 1.28 (m, 1H), 0.97 (dd, J = 6.6, 3.5, 6H); 13C NMR (75 MHz,
CDCl3): δ 170.2, 121.1, 52.3, 40.7, 36.9, 26.1, 25.8, 22.9, 21.2.
rac-Methyl 3-cyano-hexanoate (rac-6b).39 Using general procedure F, hydrogenation of (Z)-
methyl 3-cyano-hex-2-enoate [(Z)-6a] (40 mg, 0.24 mmol) gave the pure product (92%, 38
mg, 0.22 mmol). 1H NMR (300 MHz, CDCl3): δ 3.76 (s, 3H), 3.16 – 2.94 (m, 1H), 2.66 (ddd,
J = 23.4, 16.6, 7.2, 2H), 1.73 – 1.53 (m, 4H), 0.99 (t, J = 6.9, 3H); 13C NMR (75 MHz,
CDCl3): δ 170.3, 121.1, 52.2, 36.6, 33.8, 27.4, 20.2, 13.4.
Preparation of rac-ethyl 3-cyano-5-methylhexanoate (rac-4b).34 A 1:1 mixture of (R)- and
(S)-ethyl 3-cyano-5-methylhexanoate (4b) was prepared.
Synthesis of rac-propyl 3-cyano-5-methylhexanoate (rac-7b). Using general procedure E,
rac-ethyl 3-cyano-5-methylhexanoate (rac-4b) (30 mg, 0,17 mmol) in n-propanol (20 mL)
and hydrochloric acid (conc., 0.2 mL) gave the pure product (88%, 29 mg, 0,15 mmol) after
24 h reaction time. 1H NMR (300 MHz, CDCl3): δ 4.11 (t, J = 6.7, 2H), 3.07 (dt, J = 12.1, 7.0,
1H), 2.63 (ddd, J = 48.8, 16.5, 7.1, 2H), 1.98 – 1.56 (m, 4H), 1.41 – 1.28 (m, 1H), 1.04 – 0.85
(m, 9H); 13C NMR (75 MHz, CDCl3): δ 169.9, 121.2, 66.9, 40.8, 37.2, 26.1, 25.8, 22.9, 21.9,
21.2, 10.3; HRMS (EI) m/z calcd for C11H18NO2 ([M-H]+) 196.1338, found 196.1329.
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Preparative-scale bioreduction of β-cyanoacrylate esters and chiral characterisation of
(S)-1b-3b
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General Procedure for preparative-scale bioreductions of β-cyanoacrylate esters.20
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Biotransformations of (E)-1a, (E)-2a, and (E)-3a were carried out in 100 ml volumes at 30 °C
for 19 – 25 h. Reactions contained alkene substrate (10 mmol), 2-propanol (26 mmol),
Lactobacillus brevis alcohol dehydrogenase (3200 U), NADP+ (1 mmol), OYE2 (4 – 8 g wet
E. coli cells; OYE2 was constructed in pET28b and expressed in BL21 E. coli cells) and 100
mM potassium phosphate buffer (pH 7). Reactions were monitored by GC until conversion of
alkene substrates were complete. The reaction mixtures were centrifuged and the resulting
supernatants were extracted with EtOAc. The organic layers were dried over MgSO4,
concentrated under reduced pressure and the resulting crude products were purified by
Kugelrohr distillation.
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(S)-Ethyl 3-cyanobutanoate [(S)-1b].10 Bioreduction of (E)-1a afforded 1.17 g (83%) of (S)-
1b as a clear colorless liquid (100%). Spectroscopic data were in agreement with data for rac-
1b; [α]23 +30.7 (c 1.07, MeOH); ee >99% determined by GC analysis (G-TA, 135°C -
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165°C temperature gradient) tr 5.09 min. For determination of absolute configuration, (S)-1b
(0.73 g, 5.2 mmol) was converted to the methyl ester by transesterification40 with Ti(OEt)4
(0.5 g, 2.2 mmol) and ethylene glycol (0.07 g, 1.1 mmol) in refluxing MeOH (25 ml) for 118
h. The reaction mixture was concentrated (~5 ml) under reduced pressure, diluted with 10 ml
of 4 N aqueous HCl and extracted with EtOAc. The organic extract was washed with water,
dried over magnesium sulfate, concentrated under reduced pressure and Kugelrohr distilled to
give 0.46 g (60%) of (S)-methyl 3-cyanobutanoate as a clear liquid (97%, contaminated with
~3% (S)-1b). Spectral data and specific rotation for (S)-methyl 3-cyanobutanoate were
consistent with literature values:12 [α]23D +33.2 (c 1.11, MeOH); ee >99% determined by GC
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analysis (γ-TA, 135°C - 165°C temperature gradient) tr 4.79 min; lit.41 [α]23 +35.3 (c 1.05,
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MeOH), 93% ee.
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(S)-Ethyl 3-cyanopentanoate [(S)-2b].10 Bioreduction of (E)-2a afforded 1.0 g (64%) of (S)-
2b as a clear colorless liquid (>99%). Spectroscopic data were in agreement with data for
racemic 2b. [α]23D +12.7 (c 1.03, MeOH); ee >99% determined by GC analysis (G-TA 135°C
- 165°C temperature gradient), tr 6.16 min. For determination of absolute configuration, (S)-
2b (0.73 g, 4.7 mmol) was converted to the methyl ester by transesterification according to
the procedure described for transesterification of (S)-1b. 0.56 g (84%) of (S)-methyl 3-
cyanopentanoate was obtained as a clear liquid (~97%, contaminated with ~3% (S)-2b).
Spectral data and specific rotation for (S)-methyl 3-cyanopentanoate were consistent with
literature values:41 [α]23D +14.9 (c 1.06, MeOH); ee >99% determined by GC analysis (G-TA,
135°C - 165°C temperature gradient) tr 5.82 min; lit.41 [α]24D +11.8 (c 1.00, MeOH), 98% ee.
(S)-Ethyl 3-cyanohexanoate [(S)-3b].10 Bioreduction of (E)-3a afforded 1.05 g (60%) of (S)-
3b as a clear colorless liquid (98%). Spectroscopic data were in agreement with data for
racemic 3b: [α]23D +2.2 (c 1.03, MeOH); ee >99% determined by GC analysis (G-TA, 135°C
- 165°C temperature gradient) tr= 7.18 min. For determination of absolute configuration, (S)-
3b (0.66 g, 3.9 mmol) was converted to the corresponding carboxylic acid by hydrolysis with
50 wt% NaOH (0.25 ml) in MeOH (2.5 ml) at room temperature for 3 h. The reaction mixture
was acidified with 2 ml of 4 N HCl, diluted with 10 ml of water and extracted with EtOAc.
The organic extract was dried over Na2SO4 and concentrated under reduced pressure to give
0.5 g (91%) of (S)-3-cyanohexanoic acid as a viscous liquid. Spectral data for (S)-3-
cyanohexanoic acid were consistent with literature values for (R)-3-cyanohexanoic acid and
specific rotation was consistent with literature value in magnitude but opposite in direction:39
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[α]23 -3.1 (c 1.18, CHCl3). For chiral GC analysis, the acid (0.25 mg in 0.5 ml EtOAc) was
D
converted to the methyl ester by treatment with TMS-diazomethane (0.02 ml) and MeOH
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(0.02 ml) for 30 min at room temperature; ee >99% determined by GC analysis (G-TA, 135°C
- 165°C temperature gradient) tr 6.66 min; lit.13 [α]25D +1.1 (c 1.1, CHCl3), 98% ee.
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Synthesis and characterisation of chiral reference-compounds (R)-4b, and (S)-4b-7b
(S)-Ethyl 3-cyano-5-methylhexanoate [(S)-4b]. Reference material for (S)-4b was
independently prepared from diethyl 2-(1-cyano-3-methylbutyl)malonate according to the
published procedure11 and purified by Kugelrohr distillation for characterization: 98.3 % ee
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determined by GC analysis (G-TA, 135°C - 165°C temperature gradient) tr= 7.53 min; [α]20
D
-10.9 (c 1.09, MeOH). Spectral data for (S)-4b were in agreement with data for rac-4b and
with previously reported data.42
(R)-Ethyl 3-cyano-5-methylhexanoate [(R)-4b]. Preparation of (S)-4b also gave (R)-diethyl
2-(1-cyano-3-methylbutyl)malonate as a by-product of the enzymatic hydrolysis step.11 For
preparation of (R)-4b, the published procedure was modified by running the enzymatic
hydrolysis step to >50% conversion to obtain (R)-diethyl 2-(1-cyano-3-methylbutyl)malonate
with higher enantiomeric purity. Partial hydrolysis of (R)-diethyl 2-(1-cyano-3-
methylbutyl)malonate was carried out with KOH in EtOH and H2O. Aqueous KOH (8.8 wt%,
13.6 g, 21.3 mmol) was slowly added over 1 h to an ice-cold solution of (R)-diethyl 2-(1-
cyano-3-methylbutyl)malonate (5.0 g, Kugelrohr distilled, 19.6 mmol, >99% ee) in EtOH (5.9
ml). The ice-bath was removed and the reaction was stirred for 18 h at rt. The reaction
mixture was extracted with toluene (3 x 10 ml) and the toluene layers discarded. The
remaining aqueous layer was stirred at 100°C for 4 h. The reaction mixture was extracted with
EtOAc, dried over MgSO4 and concentrated under reduced pressure to give the crude product.
Purification of the crude product by Kugelrohr distillation afforded (R)-4b as a clear
colourless liquid (>99% by GC, 1.18 g, 32.8%); 89.8 % ee determined by GC analysis (G-TA,
135°C - 165°C temperature gradient) tr 7.34 min; [α]20D +10.9 (c 1.08, MeOH). Spectral data
for (R)-4b were in agreement with data for rac-4b.
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(S)-Methyl 3-cyano-5-methylhexanoate [(S)-5b]. Using general procedure E, (S)-ethyl 3-
cyano-5-methylhexanoate [(S)-4b] (52 mg, 0.28 mmol) in methanol (5 mL) and hydrochloric
acid (conc., 0.2 mL) gave the pure product (73%, 35 mg, 0.21 mmol) after 24 h reaction time.
Spectral data for (S)-5b were in agreement with data for rac-5b.
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(S)-Methyl 3-cyano-hexanoate [(S)-6b]. Using general procedure E, (S)-ethyl 3-cyano-
hexanoate [(S)-3b] (11 mg, 0.07 mmol) in methanol (5 mL) and hydrochloric acid (conc., 0.1
mL) gave the pure product (54%, 7 mg, 0.04 mmol) after 24 h reaction time. Spectral data for
(S)-6b were in agreement with data for rac-6b.
(S)-Propyl 3-cyano-5-methylhexanoate [(S)-7b]. Using general procedure E, (S)-ethyl 3-
cyano-5-methylhexanoate [(S)-4b] (30 mg, 0.16 mmol) in n-propanol (20 mL) and
hydrochloric acid (conc., 0.5 mL) gave the pure product (99%, 32 mg, 0.16 mmol) after 24 h
reaction time. Spectral data for (S)-7b were in agreement with data for rac-7b.
Acknowledgements
We would like to acknowledge Almac Sciences (Nick Tyrrell & Scott Wharry, Craigavon,
Northern Ireland) and Celtic Catalyts (Johan Granander, Dublin, Ireland) for assistance in the
preparation of analogues 1a-5a. Financial support by the Austrian Science Fund (FWF,
Vienna, project P22722) is gratefully acknowledged. Klaus Zangger (Graz) is cordially
thanked for his help in NMR spectroscopy. Brian Jones and Robert Saf are thanked for their
help in obtaining HRMS analyses.
Supporting Information. 13C- and 1H-NMR for new compounds: (Z)-ethyl 3-
(trifluoromethylsulfonyloxy)hex-2-enoate,
(Z)-methyl
5-methyl-3-(trifluoromethyl-
sulfonyloxy)hex-2-enoate, (E)-methyl 5-methyl-3-(trifluoromethylsulfonyloxy)hex-2-enoate,
rac-1b-7b, (E)-2a, (Z)-2a, (E)-3a, (Z)-3a, (Z)-6a, (E)-7a and (S)-4b and chiral analytical
methods. Cloning and purification of EBP1 from Candida albicans and of Lycopersicon
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esculentum OPR1 variants Cys20Tyr, Ile287His, and His245Asp. This material is available
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free of charge via the Internet at http://pubs.acs.org.
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