Old Yellow Enzyme-mediated reduction of β-cyano-α,β- unsaturated esters for the synthesis of chiral building blocks: Stereochemical analysis of the reaction

Article abstract of DOI:10.1039/c3cy20804d

Baker's yeast and Old Yellow Enzyme-mediated reduction of the carbon-carbon double bonds of β-cyano-α,β-unsaturated esters was investigated, in order to broaden the applicability of this kind of reaction in the field of preparative organic chemistry. The synthetic significance of the enantioselective reduction of these difunctionalised substrates was shown by considering the conversion of saturated chiral cyanoesters into γ²-amino acid derivatives for foldamer chemistry applications. The stereochemical outcome of the biotransformations was carefully analysed by means of deuterium labeling experiments. The results of this analysis were employed to rationalise the effects of substrate-control on the stereoselectivity of a certain class of ene-reductase-mediated reduction reactions. A simple model was developed to describe the structural prerequisites for the optimal arrangement of the substrates within the binding site of OYE1-3 enzymes.

Full text of DOI:10.1039/c3cy20804d

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Old Yellow Enzyme-mediated reduction of b-cyano-a,b-
unsaturated esters for the synthesis of chiral building
blocks: stereochemical analysis of the reaction†
Cite this: Catal. Sci. Technol., 2013,
3, 1136
Elisabetta Brenna,*a Francesco G. Gatti,a Alessia Manfredi,a Daniela Montib and
Fabio Parmeggiania
Baker’s yeast and Old Yellow Enzyme-mediated reduction of the carbon–carbon double bonds of
b-cyano-a,b-unsaturated esters was investigated, in order to broaden the applicability of this kind of
reaction in the field of preparative organic chemistry. The synthetic significance of the enantioselective
reduction of these difunctionalised substrates was shown by considering the conversion of saturated
chiral cyanoesters into g2-amino acid derivatives for foldamer chemistry applications. The stereochemical
outcome of the biotransformations was carefully analysed by means of deuterium labeling experiments.
The results of this analysis were employed to rationalise the effects of substrate-control on the
stereoselectivity of a certain class of ene-reductase-mediated reduction reactions. A simple model was
developed to describe the structural prerequisites for the optimal arrangement of the substrates within
the binding site of OYE1-3 enzymes.
Received 21st November 2012,
Accepted 4th January 2013
DOI: 10.1039/c3cy20804d
www.rsc.org/catalysis
(OYE) family,3 is still an emerging chemistry.4 If the starting
olefin is suitably substituted, the reaction occurs with the
Introduction
Bioconversions, including biocatalytic processes with isolated
enzymes and biotransformations with whole cell systems, are
now becoming key components in the toolbox of the process
chemist for the synthesis of chiral compounds,1 with a place
alongside chemocatalysis and chromatographic separation.2
The importance given to the cost and profit margin in today’s
competitive market, and the growing societal concern for the
development of clean, or at least less polluting, chemical
processes have increased the need for novel, high-yield, short
process routes, characterised by high selectivity, low working
temperatures and pressures, and waste reduction. Biocatalysis
is best suited to satisfy these requirements.
creation of at least one stereogenic centre under high stereo-
chemical control, thus making this kind of reduction of great
value for the synthesis of enantiopure chiral compounds: the
first large scale application of ERs for the preparation of chiral
building blocks has been recently reported in the literature.5
It has been established that the double bond can be effec-
tively reduced by ERs, if it is made susceptible to the nucleo-
philic attack of a hydride, delivered by the reduced flavin
cofactor, in the presence of an electron-withdrawing substituent,
which is also able to establish hydrogen bonds with certain
amino acid residues within the active site of the enzyme.6
a,b-Unsaturated aldehydes and ketones together with
nitroalkenes are typical substrates for ERs, whereas the bio-
reduction of a,b-unsaturated carboxylic acids and derivatives is
quite a challenging task, because the presence of a single
carboxylic moiety generally does not promote ER-mediated
reduction: only a few exceptions are reported in the literature,
consisting of a-substituted a-methylenic esters.7 The structural
prerequisites, which are needed for the double bond reduction
of an a,b-unsaturated acid or ester, are still to be clearly
defined, in order to establish the synthetic usefulness of this
procedure and to include it into the ready-to-use reactions
available to the synthetic chemists in charge of manufacturing
process development.
Among known enzyme mediated procedures, the stereo-
selective reduction of carbon–carbon double bonds promoted
by ene-reductases (ERs), belonging to the Old Yellow Enzyme
a Politecnico di Milano, Dipartimento di Chimica, Materiali, Ingegneria Chimica,
Via Mancinelli 7, I-20131 Milano, Italy. E-mail: elisabetta.brenna@polimi.it;
Fax: +39 02 23993180; Tel: +39 02 23993077
b Istituto di Chimica del Riconoscimento Molecolare-CNR, Via M. Bianco 9,
I-20131 Milano, Italy
† Electronic supplementary information (ESI) available: Full product character-
isation, NMR data of deuterated compounds, analytical procedures for the
determination of conversion and enantiomeric excess values. See DOI: 10.1039/
c3cy20804d
c
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Fig. 1 b-Cyano-a,b-unsaturated esters subjected to ER-mediated reduction.
Scheme 1 Unsaturated esters already subjected to ER-mediated reduction.
The nitrile moiety is particularly versatile, and it can be
easily converted into other functional groups either by classical
or biocatalysed organic reactions. Thus, the enantiomerically
enriched 3-cyanoalkanoates recovered from ER-mediated bio-
reduction of substrates 4 and 5 represent useful chiral synthons
for organic synthesis: their conversion into g2-amino acids for
foldamer chemistry applications is herein considered. The
results of a detailed mechanistic analysis, carried out by means
of deuteration experiments to explain the stereochemical
outcomes obtained with these cyanoesters, are also shown
and employed to establish general criteria for the identification
of optimal substrates for this kind of biocatalysts.
Utaka et al.8 first observed that the presence of a chlorine
atom on the same carbon atom bearing the carboxylic moiety
promoted CQC reduction by baker’s yeast (BY) fermentation.
The same effect was not observed when the chlorine atom was
either absent (e.g. in (E)-2-heptenoic acid and in its methyl
ester) or on the b carbon atom. The successful biocatalysed
reduction of methyl citraconate and mesaconate9 further
supported the hypothesis that the presence of a second electron-
withdrawing group was necessary to make the double bond of
a,b-unsaturated esters electrophilic enough to undergo hydride
attack in ene-reductase biotransformations.
Results and discussion
Biocatalysed reduction of cyanoesters 4 and 5
We have recently investigated the OYE-mediated reduction
of a-haloalkenoates (Z)-1 (Scheme 1),10 of 2-substituted methyl
fumarates and maleates (E)- and (Z)-2, and of alkyl mesaconates Compounds (E)- and (Z)-4a–i and (E)- and (Z)-5a–c were
and citraconates (E)- and (Z)-3,11 in order to evaluate the effects prepared by Wittig condensation of the suitable ketoester with
due to the stereochemistry of the starting alkene and the nature 2-(triphenylphosphoranylidene)acetonitrile. The (E)- and (Z)-stereo-
of substituents. High values of enantioselectivity and conver- isomers were separated by column chromatography, and subjected
sions were obtained with (Z)-1, (E)-2, and (Z)-3, affording, separately to bioreduction, employing either fermenting cells of BY
respectively (Scheme 1), the (S)-, (S)-, and (R)-enantiomers of or isolated OYE1-3 (with in situ regeneration of the NAD(P)H
the corresponding reduced products. Compounds (Z)-2 and cofactor with a glucose dehydrogenase and glucose as a sacrificial
(E)-3 were found to be completely unreactive.
cosubstrate). The results are collected in Table 1.
For substrates (Z)-1 a great variety of alkyl and aryl sub-
The analysis of these data highlighted that the bioreduction
stituents could be accommodated at the carbon atom in posi- of methyl (E)-cyanomethylenealkanoates 4 occurred with higher
tion b;10 either a short chain alkyl group or a phenyl ring could enantioselectivity and more satisfactory yields than those of the
be present at the a carbon atom on methyl esters (E)-2;11 the use corresponding (Z)-stereoisomers, except for the least hindered
of alkyl chains other than methyl groups at the ester moieties in substrates (E)- and (Z)-4a. No reaction occurred with substrates
derivatives 3 made (Z)-stereoisomers the most favourable sub- (E)- and (Z)-4d and f, bearing a branched chain at the carbon
strates for the reduction, with a modification of the most atom in position 2.
reactive binding mode with respect to compounds (E)-2 and,
consequently, a change in the stereochemical outcome of the (Scheme 2) were converted into derivatives (S)-6 (Scheme 2),
reaction.11
whereas the corresponding (Z)-stereoisomers gave reduced
The (E)-stereoisomers of compounds 4a–c, e, g, and h
With the aim of expanding the applications of this kind of compounds of opposite configuration, with the exception of
reduction in the field of preparative organic chemistry, we now compound (Z)-4b, which afforded very poorly enriched
report the effect produced by a nitrile group in the b position (S)-reduction products. It should be noted that for derivative
with respect to the ester functionality on the reduction of the 6h (Scheme 2) a switch of Cahn–Ingold–Prelog priority of the
double bond of methyl 2-cyanomethylenealkanoates (E)- and groups linked to the stereogenic carbon atom occurs: thus, the
(Z)-4 and alkyl 3-cyanomethacrylates (E)- and (Z)-5 (Fig. 1). Up to (R)-configuration of derivative 6h corresponds to a spatial
now, only a few examples of enzymatic carbon–carbon double distribution of substituents which is identical to that of the
bond reduction reactions of unsaturated nitriles have been (S)-enantiomers of the other reduced compounds of type 6.
reported in the literature.12
Furthermore, with compound (E)-4h the outcome of the
c
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Table 1 Results of BY and OYE1-3-mediated reduction of substrates (E)- and (Z)-4a–i and (E)- and (Z)-5a–c
BY
OYE 1
OYE 2
OYE 3
R
R0
Substrate
ca (%)
eeb (%)
cc (%)
eeb (%)
cc (%)
eeb (%)
cc (%)
eeb (%)
Me
Me
(E)-4a
(Z)-4a
(E)-4b
(Z)-4b
(E)-4c
(Z)-4c
(E)-4d
(Z)-4d
(E)-4e
(Z)-4e
(E)-4f
100
100
100
83
45
43
0
38 (S)
67 (R)
99 (S)
10 (S)
99 (S)
58 (R)
100
100
100
100
100
100
4
34 (S)
88 (R)
99 (S),
68 (S)
99 (S)
rac
50
53
100
45
44
45
0
40 (S)
98 (R)
99 (S)
38 (S)
99 (S)
47 (R)
100
77
100
55
87
46
1
97 (S)
52 (R)
99 (S)
rac
99 (S)
76 (R)
Et
Me
Me
Me
Me
Me
Me
Me
n-Pr
i-Pr
1
2
1
2
n-Bu
48
58
0
94 (S)
91 (R)
100
100
1
3
99
100
100
85
100
100
100
93
100
46
100
99 (S)
72 (R)
32
50
0
99 (S)
90 (R)
75
53
0
99 (S)
83 (R)
i-Bu
(Z)-4f
0
1
2
(CH2)2COOMe
Ph
(E)-4g
(Z)-4g
(E)-4h
(Z)-4h
(E)- + (Z)-4i
(E)-5a
(Z)-5a
(E)-5b
(Z)-5b
(E)-5c
(Z)-5c
68
22
26
6
85
10
83
2
78 (S)
35 (S)
28 (R)
34 (S)
94 (S)
32 (R)
99 (R)
16 (R)
40
36
58
24
88
45
100
26
100
10
60
94 (S)
58 (R)
99 (R)
22 (R)
100
94
97
51
100
21
96
12
94
12
97
94 (S)
46 (R)
99 (R)
7 (S)
2-furyl
Me
Me
Et
95 (R)
99 (R)
70 (R)
99 (R)
98 (R)
98 (R)
64 (R)
97 (R)
96 (R)
99 (R)
84 (R)
99 (R)
42 (R)
99 (R)
90 (R)
99 (R)
71 (R)
99 (R)
74 (R)
99 (R)
Me
Me
n-Bu
41
0
37
96 (R)
98 (R)
n-Hex
a
b
c = Conversion percentage: calculated by GC analysis of the crude mixture after 72 h reaction time. Calculated by GC analysis on a chiral
c
stationary phase. c = Conversion percentage, calculated by GC analysis of the crude mixture after 24 h reaction time; isolated yields are reported
in ESI.
Scheme 2 Products obtained by reduction of b-cyano-a,b-unsaturated esters 4 and 5.
reduction was sensibly different when performed by means of the two diastereoisomers by column chromatography: the
isolated enzymes or in the presence of BY. OYE1-3 reduced bioreductions proceeded nearly quantitatively to afford racemic
substrate (E)-4h completely and with high enantioselectivity, reduction products.
while the product obtained by BY fermentation showed a low
A different stereochemical outcome (Table 1) was observed
enantiomeric excess value, as a consequence of double bond for the biocatalysed reduction of 3-cyanomethacrylates (E)- and
isomerisation occurring in the fermenting medium, monitored (Z)-5, which were characterised by the presence of a methyl
by GC/MS analysis of the reaction. Substrate (Z)-4h was group on carbon atom 2, and by different alkyl chains in the
converted into poorly enriched or nearly racemic reduction ester moiety. The reduction of compounds (Z)-5a–c occurred
products, either by means of OYE1-3 biotransformation or with with higher yields and better enantioselectivity values than that
the use of BY. As for substrate 4i, it was impossible to separate of the (E)-stereoisomers, and both diastereoisomers were
c
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converted into the (R)-enantiomers of the corresponding alkyl for g-amino acids reported up to now do not involve g2
cyanopropanoates 7a–c (Scheme 2).
derivatives.
Our synthetic sequence (Scheme 3), based on the stereo-
Similar stereochemical outcomes have already been
obtained for the bioreduction of diesters 2 and 3 (Scheme 1),11 selective reduction of the double bond followed by chemical
structurally related to cyanoesters 4 and 5 by substitution of the reduction of the nitrile functionality, widens the choice of
nitrile with an ester moiety: (E)-2 and (Z)-3 were found to synthetic routes available for these valuable chiral synthons.
afford the (S)- and (R)-reduced products, respectively, just as Compound (S)-6b was obtained by BY fermentation of (E)-4b in
(E)-4 and (Z)-5.
72% isolated yield after column chromatography. Subsequently,
The configuration of the double bond of starting unsatu- the chemoselective reduction of the nitrile group by treatment
rated compounds 4 and 5 was determined by analysis of the 1H with a catalytic amount of NiCl2ꢀ6H2O and an excess of NaBH4 in
and 13C NMR spectra of the two stereoisomers. The absolute the presence of di-tert-butyl dicarbonate promoted the conver-
configuration of compounds 6a–c, g, h and 7a–c was estab- sion of (S)-6b into the corresponding Boc protected g2-amino
lished by chemical correlation to the corresponding dimethyl ester (S)-8 (70% after column chromatography).
esters: the enantiomerically enriched cyanoesters were heated
Stereochemical analysis of the ER-catalysed reductions
in methanol solution in the presence of sulphuric acid, to
afford the corresponding diesters of known absolute configu-
ration. Compound 6e was transformed into the corresponding
diacid of known absolute configuration by reaction in 37%
refluxing HCl solution.
A careful stereochemical analysis was necessary, in order to
explain the observed enantioselectivities of the bioreduction of
(E)- and (Z)-unsaturated cyanoesters 4 and 5. The generally
accepted mechanism6c of OYE-mediated reactions (Fig. 2)
consists first in the transfer of a hydride ion from the flavin
prosthetic group to the sp2 carbon atom in the b position with
respect to the activating substituent, which is involved in the
formation of hydrogen bonds with His191 and/or Asn194 in the
active site of the enzyme. A tyrosine residue then delivers a
Synthetic exploitation of the reduction products
To add practical synthetic value to our screening study, we
manipulated the nitrile functionality of saturated cyano methyl
ester (S)-6b, in order to obtain the g2-amino ester (S)-8
(Scheme 3).
g2-Amino acids are an important class of non-natural amino
acids: being analogues of the neurotransmitter g-aminobutyric
acid, they are of potential biomedical utility for the treatment of
neurological disorders.13 They are also useful building blocks
for g-peptides14 and heterogeneous backbone foldamers.15
Peptides with a homogeneous backbone, e.g. g-peptides, or
a,g- or b,g-hybrid peptides, are relevant synthetic targets, and
their availability greatly depends on the possibility of accessing
suitable g-amino acids. The synthesis of optically enriched
g2-amino acids is rather challenging: the known methods16
consist, for example, in resolution techniques based on the use
of (R)- and (S)-pantolactone,17a or (R)- and (S)-pantolactam,17b–d
or in procedures involving Evans oxazolidinone.18 Only a few
examples of stereoselective synthesis have been reported so far,
such as an enantioselective organocatalytic Michael addition of
aldehydes to nitroethylene,19 or an enantioselective addition of
methyl cyanoacetate to allylic acetates.20 Biocatalysed methods21
Fig. 2 Mechanism and possible substrate reactive binding modes for
OYE-mediated reduction of CQC double bonds. G is the substituent linked to
the CQO moiety of the withdrawing group; a, b1 and b2 are substituents of the
double bond (numbering according to PDB structure code 1OYB).
Scheme 3 Synthesis of g2-amino ester (S)-8 from cyano methyl ester (S)-6b.
c
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proton, which can be derived by exchange with the solvent, to Cyanoesters 4a represented the reference compounds of this
the a carbon atom, usually22 on the opposite stereoheterotopic series, with the minimum steric hindrance (a methyl group)
face of the alkene, thus affording anti hydrogen addition.
either on the double bond or at the ester moiety. The choice of
In the case of an electron withdrawing group containing a other substrates was motivated by the observation that the
carbon–oxygen double bond, the ‘‘classical’’ binding mode is reactions of the two diastereoisomers of compounds 4b and
considered to be that reported in Fig. 2a,6c,23a which has been 5b afforded the same enantiomer of the corresponding reduced
inferred from the structure of oxidised OYE1, bearing p-hydroxy- products, 6b and 7b, respectively, even though with different
benzaldehyde6c in the active site. Nonetheless, examples of enantioselectivity values: (S)-6b was obtained either from (E)- or
substrates binding in more than one active conformation have (Z)-4b; (R)-7b was the reduced product, starting either from
been described in the literature.4d,23 In particular, an alternative (E)- or (Z)-5b. Compound 4e was representative of the other
‘‘flipped’’ binding mode (Fig. 2b)4d,23a has been described: it can substrates of type 4, which gave opposite enantiomers upon
be obtained by a 1801 rotation of the substrate about the axis reduction of the two starting alkene diastereoisomers.
passing through the oxygen atom of the carbonyl group and the
The analysis of the 1H NMR spectra of the saturated pro-
carbon atom of the double bond in the b position to the ducts obtained starting from (E)- and (Z)-4a, (E)- and (Z)-4b,
activating moiety. In this way, the oxygen atom, involved in (E)- and (Z)-5b, and (E)-4e highlighted that the ester moiety was
hydrogen bonds with the amino acids of the binding pocket, and indeed the activating group for these substrates. In each
the b carbon atom, which has to be at a suitable distance spectrum the intensity of the multiplet due to the hydrogen
and orientation from the flavin cofactor to allow Hꢁ transfer, atom alpha to the ester group was very low, thus proving the
maintain their position.
Recently,24 a modification of the arrangement of the sub- the CH2CN fragment was reduced to an AB system.
strate in the enzyme active site induced by rotation of the Only in the reduction of (Z)-4e, and at a decidedly minor
delivery of a D+ to this position, and, accordingly, the signal of
alkenyl fragment on the Ca–CO single bond has been hypothe- extent in that of (Z)-4b, a reactive binding mode with the nitrile
sised. This conformational change produces only a different group acting as the activating functionality was deduced from
orientation of the substrate in the so called ‘‘flipped’’ binding the analysis of the 2H NMR spectra of monodeuterated
mode: the stereoheterotopic faces exposed to H+ and Hꢁ compounds.
addition are indeed the same. What is really relevant is that
In the case of (Z)-4b, (S)-6b-d1 (ee = 58%) was obtained as the
the switching between the two possible binding modes, either reduction product (c = 67%): a modest contribution of a
by flipping or by rotation of a fragment, causes an exchange of binding mode characterised by the activation of the nitrile
the stereoheterotopic faces of the alkene on which the addition group was apparent from the corresponding 2H NMR spectrum
of either H+ and Hꢁ occurs, with formation of reduced products (Fig. 3a): besides the main broad singlet of CDCOOMe at
of opposite absolute configuration.‡
2.70 ppm, identical to that of the deuterium spectrum of
It is clear that the stereochemical outcome of the reaction is (S)-6b-d1 obtained from (E)-4b (Fig. 3b), a less intense signal
controlled by the arrangement of the sterereoheterotopic faces due to CHDCN at 2.65 ppm was detected. The low enantio-
of the olefin towards hydride and proton delivery, and by the selectivity observed in the reduction of (Z)-4b was thus tenta-
stereospecificity of the hydrogen addition step.22 When two tively attributed to the concomitant realization of this
electron withdrawing groups are present on the double bond, alternative binding mode, affording (2R,10S)-6b-d1. The configu-
the accommodation of the substrate within the catalytic site ration at C2 in this compound was derived as a consequence of
0
does not only depend on the steric and electronic effects of this assumption; that of C1 was determined by an anti
substituents, but also on which group acts effectively as the reduction mechanism, and it was confirmed by further
activating one.25 Thus, for the cyanoesters of this study it was deuteration experiments (see the next section).
necessary to establish whether the ester or the cyano function-
ality was the real activating group making bioreduction group as the activating one was found to be the most reactive:
feasible.
in the 1H NMR spectrum§ of the corresponding reduced
In the case of (Z)-4e the binding mode showing the nitrile
Identification of the activating electron withdrawing group product the signal of the hydrogen atom adjacent to the ester
of cyanoesters 4 and 5. The use of stoichiometric NADH and of moiety resulted to be a quartet, as a consequence of the
deuterium oxide as a solvent promotes the incorporation of a addition of a D+ to the carbon atom in the a position to the
hydrogen atom at Cb and of a deuterium atom at Ca with nitrile group. Derivative (2R,10S)-6e-d1 (ee = 72%) was recovered:
0
respect to the activating group, i.e. the one which is in the active the absolute configuration of C1 was determined by an anti
site of the enzyme. Derivatives (E)- and (Z)-4a, 4b, 4e and 5b reduction mechanism, which was later confirmed (see the next
were subjected to OYE1-mediated biotransformations in D2O section). The 2H NMR spectrum of this product (Fig. 3c) showed
solution in the presence of a stoichiometric quantity of NADH. a main singlet at 2.64 ppm for CHDCN, but also a less intense
signal at 2.73 ppm for CDCOOMe, identical to that of (S)-6e-d1
(Fig. 3d). Thus, the decrease in enantioselectivity for the
‡ In the work11 devoted to the investigation of unsaturated diesters 2 and 3, we
have established by means of deuterium labelling that the different stereo-
chemical outcome of their bioreduction was due to the fact that the proton was
§ The 1H NMR spectra of the monodeuterated compounds obtained from (Z)-4b
and (Z)-4e are reported as examples in ESI† as Fig. S1 and S2.
delivered, respectively, on the Ca-re and Ca-si face in substrates (E)-2 and (Z)-3.
c
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Fig. 3 (a) 2H NMR spectra of the 79/21 mixture (determined by GC analysis on a chiral stationary phase) of (S)-6b-d1 and (2R,10S)-6b-d1 obtained from (Z)-4b; (b)
2H NMR spectra of (S)-6b-d1 obtained from (E)-4b; (c) 2H NMR spectra of the 14/86 mixture (determined by GC analysis on a chiral stationary phase) of (S)-6e-d1 and
(2R,10S)-6e-d1 obtained from (Z)-4e; (d) 2H NMR spectra of (S)-6e-d1 obtained from (E)-4e.
reduction of (Z)-4e was assumed to be produced by an alter-
native active binding conformation, affording (S)-6e-d1.
Assessment of the stereochemistry of hydrogen addition and
considerations on the reactive binding modes of the substrates.
After the identification of the activating group, the stereospecificity
Scheme 4 syn chemical double deuteration of (Z)-substrates.
of hydrogen addition has to be assessed, in order to understand the
diverse stereochemical outcome of these bioreductions.
Thus, OYE1-mediated reduction of derivatives (E)- and produced by deuterium syn addition to the (Z)-double bond.
(Z)-4a, 4b, 4e and 5b was also performed in the presence of The 2H NMR spectra of the doubly deuterated compounds
(R)-NADPD (regenerated in situ by alcohol dehydrogenase and recovered from the enzymatic reduction of both diastereo-
i-PrOH-d8) in D2O solvent,26 in order to incorporate two deuter- isomers of substrates 4e and 5b are reported in Fig. 4b, c and
ium atoms. The 2H NMR spectra of the doubly deuterated e, f, respectively, and compared with those of the chemically
reduced compounds were compared with those of the deriva- reduced products obtained from (Z)-4e and (Z)-5b (Fig. 4a and d).
tives obtained upon reduction of the same starting substrates
2H NMR spectra showed that biocatalysed anti addition to
with deuterium gas in the presence of Pt/C in benzene solution. (E)-alkenes gave the same stereoisomers recovered from
Metal-catalysed syn addition of deuterium to (Z)-4a, 4b, 4e chemical syn deuteration of (Z)-substrates. Furthermore, it
and 5b afforded the (2RS,10RS) stereoisomer of the corre- was found that the deuterium atom introduced in the a position
sponding reduced compounds 6a, 6b, 6e, and 7b (Scheme 4): to the nitrile by anti addition to (Z)-compounds was charac-
with this relative configuration, the deuterium atom introduced terised by a higher chemical shift (dD = 2.67, 2.64, 2.64, 2.66 ppm,
in the a position to the nitrile was the one characterised by the respectively, for 6a, 6b, 6e, and 7b) than that inserted by syn
lowest chemical shift (dD = 2.54, 2.55, 2.55, 2.53 ppm, respectively, deuteration. The latter information also confirmed the configu-
0
for 6a, 6b, 6e, and 7b).
ration of C1 in compounds (2R,10S)-6b-d1 and (2R,10S)-6e-d1
In Fig. 4a and d the 2H NMR spectra of (2RS,10RS)-6e-d2 and depicted in Fig. 3.
(2RS,10RS)-7b-d2 are reported. The relative configuration of the
A stereospecific anti addition mechanism was confirmed for
stereogenic centres created upon insertion of two deuterium all the four pairs of diastereoisomers.
atoms by OYE1 biotransformation of substrates (E)- and (Z)-4a,
The information on the absolute configuration of C2 and
4b, 4e and 5b was obtained by analysis of their 2H NMR spectra, that on the activating group were combined, in order to
using as a reference the chemical shifts of the stereoisomer establish the preferred reactive binding mode for the
c
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The formation of (2R,10S)-6e-d2 (ee = 88%) from (Z)-4e
was explained by the fact that the nitrile group became the
activating one, and the substrate was arranged in the active site
in such a way that D+ was delivered on the si face of the olefinic
carbon atom linked to the CN group. The non-exclusive
formation of (2R,10S)-6e-d2 was explained by the possibility of
a binding mode being activated by the ester group with
addition of D+ on the re face of the adjacent carbon atom,
giving (2S,10R)-6e-d2.
For both substrates (E)- and (Z)-5b (Fig. 5d), the stereochemistry
of the two bioreductions could be justified by delivery of D+ to
the Ca-si face, with formation of (2R,10R)-7b-d2 (ee = 78%) and
(2R,10S)-7b-d2 (ee = 99%), respectively.
The non-exclusive formation of a single enantiomer in the
bioreduction of (E)-4a and (E)-5b could be attributed neither to
a binding mode activated by the nitrile nor to the contribution
of syn addition which were not highlighted by 2H NMR analysis.
Only a competition between the flipped and classical binding
modes of the substrate could account for the formation of the
minor enantiomer.
General considerations on the stereochemical course of OYE-
mediated reduction of unsaturated compounds
The data so far collected allowed us to provide some general
considerations on the stereochemical course of the bio-
catalysed reduction of a,b-unsaturated derivatives with a
carbonyl or carboxyl moiety as the activating group and a
substituent at Ca, which is described to occur with high values
of enantioselectivity. This analysis highlighted the structural
requisites necessary for the optimal arrangement of these
substrates in the binding site of OYE1-3 enzymes, which make
them adopt a single preferred reactive conformation.
2
Fig. 4 (a) and (d) H NMR spectra of (2RS,10RS)-6e-d2 and (2RS,10RS)-7b-d2
obtained from metal catalyzed deuterium syn addition to (Z)-4e and 5b; (b) and
(e) 2H NMR spectra of (2S,10S)-6e-d2 and (2R,10R)-7b-d2 obtained from enzymatic
reduction of (E)-4e and 5b; (c) and (f) H NMR spectra of (2R,10S)-6e-d2 and
(2R,10S)-7b-d2 obtained from enzymatic reduction of (Z)-4e and 5b.
2
For unsaturated methyl esters 4, showing an increasing
steric hindrance at the carbon–carbon double bond, the most
satisfactory values of conversion and enantioselectivity were
investigated substrates. These conclusions are collected in obtained for the (E)-stereoisomers. The experimental informa-
Fig. 5: exclusive arrangements affording bioreductions with tion, gathered within this work on the stereochemistry of their
enantioselectivity values higher than 99% are highlighted with reduction, showed that the H+ addition took place on the
shaded boxes, and the predominant activating group is shown stereoheterotopic face of the olefin established by a flipped
in bold on the left side.
binding mode, obtained either by flipping of the whole
The opposite enantioselectivity observed for the reduction substrate or by rotation of the alkene fragment (Table 2, entry 1).
of the two stereoisomers (E)- and (Z)-4a (Fig. 5a) could be This binding mode is very likely to be preferred also by haloalk-
attributed to a change in the preferred reactive binding mode enoates (Z)-1 (entry 2), affording (S)-enantiomers (ee = 94–99%),10
of the two substrates, with delivery of D+ on the Ca-re and Ca-si unsaturated esters (E)-2 (entry 3), converted into (S)-reduction
face, respectively.
products,11 a-methylenic methyl esters (entry 4), giving (R)-enantio-
For both the stereoisomers (E)- and (Z)-4b (Fig. 5b) the mers (ee >99% with OYE1-3),7a–c and some dimethyl N-acylamino
stereochemical course could be justified by the same arrange- fumarates (entry 5), affording reduction products with (S)-configu-
ment of the stereoheterotopic face of the alkene in the active ration (ee >99% with OYE1-3).25 The same flipped arrangement can
site, with addition of D+ to the Ca-re face. The low enantio- be deduced for a-alkylcinnamaldehydes (entry 6),27 and substituted
selectivity of the bioreduction of (Z)-4b was attributed to the benzylacrylaldehydes (entry 7),28 affording (S)- and (R)-enantiomers,
competition of a binding mode activated by the nitrile moiety, respectively.
affording the enantiomer (2R,10S)-6b-d2, with addition of a D+
to the si face of the olefinic carbon atom linked to the afforded the highest values of enantioselectivity. The stereo-
CN group. chemical analysis, herein performed for these substrates, high-
In the reduction of compound (E)-4e (Fig. 5c), addition of D+ lighted H+ addition on the stereoheterotopic face established
As for cyanoesters 5, the bioreduction of (Z)-stereoisomers
on the Ca-re face afforded (2S,10S)-6e-d2 (ee
=
99%). by a classical binding mode (entry 8). This arrangement was
c
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Fig. 5 Identification of the reactive binding modes of the investigated substrates.
Table 2 Reactive arrangements of optimal substrates for ene-reductases and the suggested empirical model for their rationalisation
Entry
Arrangement
G
b1
b2
a
Empirical model
1
2
3
4
5
6
OMe
OMe
OMe
OMe
OMe
H
H
H
H
H
H
H
CN
R, Ar
Cl, Br
R, Ar
CH2OR
NHCOR
R
R, Ar
COOMe
H
COOMe
Ar
7
H
H
H
ArCH2
8
9
10
OR
OR
CH2CH2
CN
COOR
CH2
H
H
H
Me
Me
Me
found to be the preferred one also for unsaturated esters (Z)-3, group at Cb on the same side of the activating group; for a
transformed into (R)-reduction compounds (entry 9),11 and for classical binding mode the favoured stereochemistry of the
2-methyl-2-cyclohexenone (entry 10),9,23a converted into the double bond is the one with the smallest group at Cb on the
(R)-enantiomer of the reduced product.
same side of the substituent at Ca (Table 2).
For all these compounds, which are optimal substrates for
If the stereochemistry of the starting alkene is not the
OYE1-3 enzymes, with a carbonyl or carboxyl moiety as the optimal one for the particular binding mode which is induced
activating group and a substituent at Ca, the preferred reactive by the bulkiness of G and a, an exclusive arrangement within
binding mode seems to be determined by the relative size of the the active site cannot be achieved and enantioselectivity
groups labelled G and a (Table 2): if G has a modest steric decreases.
hindrance (S, small), and a is bulky or more hindered (L, large),
then a flipped binding mode is preferred; if G is large, and a is citraconate (diesters of type 3), G and a are a methyl and a
small, then a classical binding mode is adopted. methoxy group of similar dimensions. A flipped binding mode
In the case of (E)- and (Z)-4a, and of methyl mesaconate and
When (E)- and (Z)-derivatives are compared, one diastereo- is assumed by (E)-4a and by methyl mesaconate, and a classical
isomer is usually found to be more suitable for bioreduction binding mode by the corresponding (Z)-stereoisomers.
than the other one: for a flipped binding mode the favoured
As for compounds (Z)-4 and (E)-5, which were found to be
stereochemistry of the double bond is the one with the smallest less favourable substrates for ER-mediated reductions than the
c
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corresponding diastereoisomers, the spatial disposition of sub- Experimental sectionz
stituents prevented them from finding an exclusive optimal
General procedure for the OYE-mediated bioreduction
binding mode, of the types described in Table 2, and their
bioreduction proceeded with modest yields and enantioselec-
tivity. The competition between two binding modes assumed by
(Z)-4b and 4e, and by (E)-5b resulted in a less efficacious control
on the efficiency and the stereochemistry of the bioreduction.
The substrate (5 mmol) dissolved in DMSO (10 mL) was added to
a KPi buffer solution (1.0 mL, 50 mM, pH 7.0) containing
glucose (20 mmol), NADP+ (0.1 mmol), GDH (4 U) and the
required OYE (ca. 40 mg dissolved in 100–200 mL of H2O). The
mixture was incubated for 24 h in an orbital shaker (160 rpm,
30 1C). The solution was extracted with EtOAc (2 ꢂ 250 mL),
centrifuged after each extraction (15 000 g, 1.5 min), and the
combined organic solutions were dried over anhydrous Na2SO4.
Conclusions
The investigation of the effects of substituents on the activation
of CQC double bonds towards bioreduction is still of great
relevance, with the aim of broadening and better understanding
the applicability of the reaction in the field of preparative
synthetic chemistry. The experimental data collected in this
work have shown that b-cyano-a,b-unsaturated esters are useful
substrates for ER-mediated reactions. The cyano derivatives
obtained upon reduction can be easily converted into valuable
g2-amino acids by simple manipulation of the nitrile function-
ality, without altering the absolute configuration of the mole-
cule. This fact highlights the significance of this bioreduction
from the practical synthetic point of view.
Bioreduction procedure for the preparation of monodeuterated
samples
The substrate (50 mmol) dissolved in i-PrOH (100 mL) was added
to a KPi buffer solution (5.0 mL, 50 mM in D2O, pH 7.0)
containing glucose (20 mmol), NADH (75 mmol) and the
required OYE (ca. 250 mg, dissolved in 500–700 mL of H2O).
The mixture was incubated for 24 h in an orbital shaker
(160 rpm, 30 1C). The solution was extracted with EtOAc (3 ꢂ
5.0 mL), centrifuged after each extraction (3000 g, 1.5 min), and the
combined organic solutions were dried over anhydrous Na2SO4.
The stereochemical course of these biotransformations has
been analysed carefully, providing a wealth of information
about possible stereocontrol strategies. The reduction of
cyanoesters 4 is favoured when the double bond has an
(E)-configuration, and it affords the (S)-enantiomers of the
corresponding saturated products. The reaction does not take
place when the alkyl chain in the a position contains a
branched carbon atom. The increase of the steric hindrance
of the alkyl residue of the ester moiety has a different effect: the
(Z)-stereoisomer becomes the best substrate, and the same
(R)-enantiomer is obtained starting from either stereoisomer.
These results further support the idea, already expressed in
recent literature,4,10,22,26 that the stereochemistry of bioreduc-
tions can be controlled by substrate engineering with great
efficacy.
The work also suggests a rationalization of the stereochemi-
cal outcomes of highly enantioselective bioreduction of acti-
vated unsaturated compounds having a carbonyl or a carboxyl
group as the activating moiety and a substituent at Ca. These
substrates are characterized by the exclusive preference for a
certain reactive arrangement within the enzyme active site,
which can be explained by using an empirical model. This
model takes into consideration both the bulkiness of the
groups linked to the CQO moiety and to Ca, and the stereo-
chemistry of the double bond. Up to now, the effects due to the
steric hindrance of substituents and to the configuration of the
alkene have been considered as affecting separately the course
of the bioreductions. This model shows that they are strictly
interconnected: each binding mode shows a favoured double
bond configuration related to the occupancy of the whole
enzyme active site. Useful indications can be obtained, to drive
the selection of optimal substrates for ER-mediated reactions,
and to exploit all the effects of substrate-control on the stereo-
chemical outcome of the reactions.
Bioreduction procedure for the preparation of doubly
deuterated samples
The substrate (50 mmol) dissolved in i-PrOH-d8 (100 mL) was
added to a KPi buffer solution (5.0 mL, 50 mM in D2O, pH 7.0)
containing NADP+ (15 mmol), TBADH (4 U, 3 mg) and the
required OYE (ca. 250 mg, dissolved in 500–700 mL of H2O).
The mixture was incubated for 24 h in an orbital shaker (160 rpm,
30 1C). The solution was extracted with EtOAc (3 ꢂ 5.0 mL),
centrifuged after each extraction (3000 g, 1.5 min), and the
combined organic solutions were dried over anhydrous Na2SO4.
General procedure for baker’s yeast fermentation
A mixture of baker’s yeast (100 g) and D-glucose (40 g) in tap
water (1 L) was prepared. After 10 min stirring at 30 1C the
suitable cyanoester (3.0 g) adsorbed on a hydrophobic resin (60 g,
polystyrene XAD-1180N) was added in one portion. The mixture
was kept under stirring for 72 h at r.t. and then filtered on a cotton
plug. The collected mass was washed with tap water repeatedly, to
remove most of the cells. The resin was then collected and extracted
twice in sequence with acetone (200 mL) and ethyl acetate
(200 mL). The organic phase was concentrated to 1/3 and then
washed with brine. The residue obtained upon evaporation of the
dried (Na2SO4) extract was chromatographed on a silica gel column
with increasing amounts of ethyl acetate in hexane.
Synthesis of the c2-aminoacid derivative: (S)-methyl
4-(t-butoxycarbonylamino)-2-ethylbutanoate [(S)-8]
To a stirring solution of (S)-6b (ee = 99%, 0.340 g, 2.4 mmol) in
MeOH (30 mL) Boc2O (1.05 g, 4.8 mmol) and a catalytic amount
of NiCl2ꢀ6H2O were first added; then, at
0 1C, NaBH4
(0.64 g, 16.8 mmol) was added portionwise over 30 min.
¶ GC methods are reported in ESI.†
c
1144 Catal. Sci. Technol., 2013, 3, 1136--1146
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W. M. F. Fabian, M. Hall, K. Ditrich and K. Faber,
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E. Brenna, F. G. Gatti, A. Manfredi, D. Monti and
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The reaction mixture was stirred for 24 h. After the usual
workup, column chromatography eluting with hexane and an
increasing amount of ethyl acetate gave compound (S)-8 (0.41 g,
70%): ee = 99%, [a]D20 + 16.5 (c 1.96 in CHCl3); 1H NMR
(400 MHz, CDCl3) d = 3.66 (3H, s, COOCH3), 3.09 (2H, m,
CH2NH), 2.31 (1H, m, CHCOOCH3), 1.80–1.45 (4H, m, 2CH2),
1.41 (9H, s, (CH3)3C), 0.86 (3H, t, J = 7.4 Hz, CH3); 13C NMR
(100.6 MHz, CDCl3) d = 176.1, 155.8, 79.1, 51.4, 44.6, 38.8, 32.0,
28.4, 25.3, 11.5; GC-MS (EI) tR = 20.0 min: m/z (%) = 189
(M+ ꢁ56, 7), 172 (13), 144 (27), 57 (100).
Acknowledgements
Prof. Claudio Fuganti and Prof. Giovanni Fronza are warmly
acknowledged for fruitful discussions. Prof. Neil C. Bruce
(Department of Biology, University of York) is kindly acknowl-
edged for the gift of plasmid pT7-OYE1. Prof. Sven Panke and
Christian Femmer (ETH Zu¨rich Department of Biosystems
Science and Engineering, Basel) are kindly acknowledged for
the help provided in the preparation of the plasmids and the
overexpressing strains.
13 J. S. Bryans and D. J. Wustrow, Med. Res. Rev., 1999, 19, 149;
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