Stereochemical outcome of the biocatalysed reduction of activated tetrasubstituted olefins by old yellow enzymes 1-3

Article abstract of DOI:10.1002/adsc.201100504

The ene-reductase-mediated reactions of (E)- and (Z)-stereoisomers of tetrasubstituted activated alkenes were performed by means of isolated old yellow enzymes (OYEs) 1-3. The comprehension of the resulting data required a careful analysis of the stereochemical course of this kind of reaction. The investigation of the bioreduction of tetrasubstituted alkenes allowed us to appreciate the contribution of several factors (configuration of the starting alkene, mechanism of hydrogen addition, substrate binding mode) to the overall stereochemistry of the reaction.

Full text of DOI:10.1002/adsc.201100504

FULL PAPERS
DOI: 10.1002/adsc.201100504
Stereochemical Outcome of the Biocatalysed Reduction of
Activated Tetrasubstituted Olefins by Old Yellow Enzymes 1–3
Elisabetta Brenna,a,* Francesco G. Gatti,a Daniela Monti,b Fabio Parmeggiani,a
and Stefano Serrab
a
Politecnico di Milano, Dipartimento di Chimica, Materiali, Ingegneria Chimica, Via Mancinelli 7, I-20131 Milano, Italy
Fax : (+39)-02-2399-3180; phone: (+39)-02-2399-3077; e-mail: elisabetta.brenna@polimi.it
Istituto di Chimica del Riconoscimento Molecolare – CNR, Via Mario Bianco 9, I-20131 Milano, Italy
b
Received: June 29, 2011; Revised: September 9, 2011; Published online: January 12, 2012
Dedicated to Professor Claudio Fuganti with esteem and affection.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/adsc.201100504.
Abstract: The ene-reductase-mediated reactions of tion of several factors (configuration of the starting
(E)- and (Z)-stereoisomers of tetrasubstituted acti- alkene, mechanism of hydrogen addition, substrate
vated alkenes were performed by means of isolated binding mode) to the overall stereochemistry of the
old yellow enzymes (OYEs) 1–3. The comprehension reaction.
of the resulting data required a careful analysis of
the stereochemical course of this kind of reaction.
The investigation of the bioreduction of tetrasubsti- Keywords: asymmetric catalysis; bakerꢀs yeast; bio-
tuted alkenes allowed us to appreciate the contribu- transformations; enzyme catalysis; reduction
Introduction
two diastereoisomeric reduction products (2R,3S)-
and (2S,3S)-3 in 3/7 and 4/6 ratios, respectively, and
The stereoselective reduction of activated C=C that, starting from the two possible diastereoisomers
double bonds mediated by ene reductases (ER), be- of the alkene, saturated alcohols showing the same
longing to the old yellow enzyme (OYE) family,[1] is absolute configuration were obtained. The concomi-
currently receiving great attention,[2] because the reac- tant formation of two stereogenic centres allowed us
tion can be exploited in the development of biocata- to infer the mechanism of hydrogen addition from the
lysed syntheses of enantiopure biologically active analysis of the relative configurations of the reduction
molecules.[3] This research activity is supported by the
plethora of data collected in the past on the same
type of reactions performed by using microbial cell
systems, especially by bakerꢀs yeast (BY) fermenta-
tions.[4] Investigation of the synthetic versatility of this
bioreduction[5] and elucidation of mechanicistic as-
pects (e.g., through deuterium labelling[6]) are funda-
mental to understand the scope and limitations of
OYE-mediated biotransformations.
Recently, we prepared[7] the two unsaturated alde-
hydes (E)- and (Z)-1 (Scheme 1), and submitted them
to BY reduction. These substrates have not been
deeply investigated, because the synthesis of a tetra-
substituted double bond in a definite configuration is
quite a challenging task. The results of the BY bio-
transformations showed that the reaction occurs with
low diastereoselectivity, giving rise to mixtures of the Scheme 1. BY reduction of unsaturated aldehydes (ref.[7]).
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action was due to a different stereoselectivity of the
ERs active in the whole-cell systems, or if it was a
characteristic of the bioreduction of this kind of sub-
strate. We also submitted nitroalkenes (E)- and (Z)-2
(Scheme 2) to the same investigation, their BY reduc-
tions had been already reported in the literature to
occur with low diasteroselectivity,[9] affording the
same enantiomers of the two diastereoisomeric nitro-
alkanes 4. The same authors investigated the stability
of the reduction products under fermentation condi-
tions, in order to exclude a contribution to the diaste-
reoisomeric ratio due to the epimerisation of the Ca
stereogenic centre.
We soon realised that the comprehension of the
data collected during these latter investigations re-
quired a careful analysis of the stereochemical course
of OYE-mediated bioreductions. We want to report
Scheme 2. BY reduction of nitroalkenes (ref.[9]).
products. Compound (2R,3S)-3 was the outcome of here on the results of this analysis and on the data
anti hydrogen addition to (E)-1 and of syn addition to concerning the OYE-catalysed reactions of (E)- and
(Z)-1. Compound (2S,3S)-3 was the diastereoisomer (Z)-isomers of compounds 1 and 2.
obtained by syn hydrogen addition to (E)-1 and anti
addition to (Z)-1.
With BY whole cells the concomitant reduction of Results and Discussion
the aldeyhde group to the corresponding primary al-
cohol by alcohol dehydrogenases could not be avoid- General Considerations on the Mechanism of ER-
ed.
These data were in contrast with the commonly ac-
Mediated Reactions
cepted ideas that the reaction consists in an anti hy- According to the currently postulated mechanism for
drogen addition to the activated carbon double the OYE-catalysed reduction of a,b-unsaturated alde-
bond,[2b] and that the stereochemistry of the starting hydes and ketones,[10] the substrate is located within
alkene plays a key role on the enantioselectivity of the active site of the enzyme with the carbonyl group
the reaction.[2b,8]
activated by the hydrogen bonds to His191 and
Thus, we decided to re-investigate the biocatalysed Asn194 (Figure 1).
reduction by using isolated OYEs, in order to verify
In this way the substrate is stacked above the re-
whether the global stereochemical outcome of the re- duced flavin with its b-carbon atom aligned to N-5
Figure 1. Postulated mechanism of OYE-mediated reduction of C=C double bonds. The priority rules employed to assign ste-
reochemical descriptors are the following ones: for double bonds CHO>R’’, R>R’; for stereoheterotopic faces CHO>=
CRR’>R’’.
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Stereochemical Outcome of the Biocatalysed Reduction of Activated Tetrasubstituted Olefins
Figure 2. Stereochemical outcome of bioreductions of (E)- and (Z)-tetrasubstituted olefins. The priority rules employed to
assign stereochemical descriptors are the following ones: for double bonds: CHO>R’’, R>R’; for stereogenic centres
(Ca,Cb): R>CHR’’CHO>R’, CHO>CHRR’>R’’; for stereoheterotopic faces: CHO>=CRR’>R’’.
which transfers the hydride. Tyr196 lies above the amples of syn reduction of activated carbon double
substrate and donates a proton to the a-carbon atom. bonds have been reported by using BY as a biocata-
The delivery of a hydride to Cb from below the plane lyst.[6e,f,9] Through deuterium labelling experiments, it
of the double bond, and of a proton to Ca from above was demonstrated[6b] that the chiral a,b-unsaturated
accounts for the anti mode of hydrogen addition, aldehyde perillaldehyde was reduced by bakerꢀs yeast
which is the stereochemical course usually observed in either a syn or anti mode according to its absolute
in this kind of reductions. In the model proposed by configuration.
Massey et al.[10c] the unsaturated carbonyl compound
As for the substrate binding mode, a 1808 flipping
is bound in such a way that the proton is delivered to of the olefin around the carbon double bond
the Ca-si face (Figure 1).
[Figure 2, (c) and (d)] has been assumed in some
If this binding mode is satisfied, and the addition cases[2d,11] to explain the experimental data. According
occurs with anti stereochemistry, the switch from (E)- to this flipped arrangement, hydrogen addition takes
to (Z)-alkenes will be characterised by inversion of place on the other stereoheterotopic face of the
configuration of Cb (Figure 1).
olefin, with delivery of the proton on the Ca-re and
When these expectations are disappointed, the fol- Ca-si faces in the anti and syn addition modes, respec-
lowing justifications may be given: (i) (E)/(Z) isomeri- tively. The enantioselectivity of this reaction is oppo-
sation occurs in the reaction medium; (ii) the location site to that occurring with the same substrate in the
of the electron-withdrawing group in the binding site classical binding mode [Figure 2 (a) vs. (c); Figure 2
is different from the one described for the carbonyl (b) vs. (d)], with inversion of configuration at Ca as
moiety of unsaturated aldehydes; (iii) the hydrogen well as at Cb, given the same stereospecificity of hy-
addition step is characterised by a different stereospe- drogen addition.
cific mechanism; (iv) the alkene is arranged in the
binding site exposing the other stereoheterotopic face highlighted factors, i.e., configuration of the starting
to the reduced flavin. alkene, mechanism of hydrogen addition, and sub-
The effects on stereochemistry due to the three
As for the hydrogen addition mode, the protona- strate binding mode, can be summarised as follows:
tion step of the a-carbon atom can be provided also the other two factors being fixed, a switch from (E)-
by the solvent water, with delivery of the proton on to (Z)-alkenes causes inversion of configuration of Cb,
the Ca-re face from the same side of the hydride, thus a switch from anti to syn addition causes inversion of
allowing a hydrogen syn addition to the double bond. configuration at Ca, while a switch from classical to
The final product of syn addition [Figure 2 (a) for flipped binding mode causes inversion of configura-
(E)-olefins, Figure 2 (b) for (Z)-olefins] is character- tion both at Ca and Cb. Only the concomitant varia-
ised by opposite configuration at Ca, if compared with tion of all the three factors affords the same enantio-
the anti reduction product obtained from the same mer.
olefin arranged in the same binding mode. A few ex-
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Table 1. Stereochemical outcome of bioreductions of (E)- and (Z)-trisubstituted olefins. The priority rules employed to
assign stereochemical descriptors are the following ones: for double bonds: CHO>R’’, R>R’; for stereogenic centres: R>
CH2CHO>R’, CHO>CH2R’>R’’; for stereoheterotopic faces: CHO> =CRR’,=CHR’>R’’.
À
À
À
À
Entry Binding mode Substrate
Product CHO CH2 CHRR’
A) anti addition B) syn addition
Substrate
Product CHO CHR’’ CH2R’
C) anti addition D) syn addition
1
2
3
4
Classical
Classical
Flipped
Flipped
Cb-(S)
Cb-(R)
Cb-(R)
Cb-(S)
Cb-(S)
Cb-(R)
Cb-(R)
Cb-(S)
Ca-(R)
Ca-(R)
Ca-(S)
Ca-(S)
Ca-(S)
Ca-(S)
Ca-(R)
Ca-(R)
General Considerations on the Stereochemistry of
ER-Mediated Reduction of Trisubstituted Activated
Alkenes
The most common substrates for ER reductions are
trisubstituted olefins, in particular unsaturated alde-
hydes (Table 1). According to these considerations, in
the hypothesis of a classical binding mode of the sub-
strate, and of a hydrogen anti addition mechanism, it
is expected that (E)- and (Z)-stereoisomers of b,b-di-
Figure 3. Bioreduction of a-methylcinnamaldehyde.
ACHTUNGTRENNUNG
tries 1A and 2A), whereas (E)- and (Z)-stereoisomers syn addition in the flipped accommodation (Table 1,
of a,b-disubstituted unsaturated aldehydes will be entries 3D and 4D).
converted into derivatives showing the same configu-
For example, the ER-catalysed reduction of (E)-a-
ration at Ca (Table 1, entries 1C and 2C). As a matter methylcinnamaldehyde (Figure 3) either by isolated
of fact, only the configuration of Cb should be affect- OYEs or by bakerꢀs yeast, afforded, respectively, (S)-
ed by the change in the alkene stereochemistry.
2-methyl-3-phenylpropanal[12] (ee=94, 96, 90% with
In the case of (E)- and (Z)-b,b-disubstituted enals OYE1–3, respectively) and (S)-2-methyl-3-phenylpro-
the switch from anti to syn addition either in the clas- panol (ee=70%),[13] in contrast with the expectation
sical (Table 1, entries 1/2A vs. 1/2B) or flipped binding of Table 1 entry 1C. Recently, deuterium labelling ex-
mode (Table 1, entries 3/4A vs. 3/4B) will have no periments[7] showed a preferential (85:15) anti hydro-
effect on the absolute configuration of the products, gen addition to this substrate in BY reaction, thus a
because Ca is not a stereogenic centre. Given the flipped binding mode can be assumed to explain the
same addition mode, opposite enantioselectivity will enantioselectivity of the reduction (Table 1, entry 3C).
be obtained by a flipping of the substrate in the active
site (Table 1, entries 1A/B vs. 3A/B, and 2A/B vs.
4A/B).
Analysis of the Stereochemical Outcome of the ER-
As for a,b-disubstituted enals, syn addition with the Mediated Reduction of Tetrasubstituted Activated
substrate located in a classical binding mode (Table 1, Alkenes
entries 1D and 2D), and anti addition on a flipped
substrate (Table 1, entries 3C and 4C) will afford In the analysis of the overall stereochemistry of biore-
products showing the same absolute configuration, ductions, the contribution due to the arrangement of
opposite to those obtained with anti addition in the the substrate within the binding site can be discrimi-
classical binding mode (Table 1, entries 1C and 2C) or nated experimentally from that due to the stereospe-
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Stereochemical Outcome of the Biocatalysed Reduction of Activated Tetrasubstituted Olefins
Table 2. Experimental data of BY and OYE-mediated reductions of tetrasubstituted alkenes (E)- and (Z)-1, and (E)- and
(Z)-2.
Substrate
Biocatalyst
Hydride on Cb-si face
Hydride on Cb-re face
anti/syn addition mode,[a]
conversion[a] [%]
anti addition
syn addition
anti addition
ee [%][b]
syn addition
ee [%][b]
ee [%][b]
ee [%][b]
BY[c]
OYE1
OYE2
OYE3
93
72
60
93
85
90
30/70,[d] 18[e]
45/55, 100
50/50, 100
75/25, 100
90
62
syn addition
anti addition
syn addition
anti addition
ee [%][b]
ee [%][b]
ee [%][b]
ee [%][b]
BY[c]
OYE1
OYE2
OYE3
33
0
33
0
60/40,[d] 19[e]
50/50, 62
50/50, 65
40
90
46
95
70/30, 100
anti addition
ee [%][b]
87
syn addition
ee [%][b]
83
anti addition
ee [%][b]
syn addition
ee [%][b]
BY
60/40, 54
40/60, 87
13/87, 37
30/70, 42
OYE1
OYE2
OYE3
88
96
87
76
16
50
syn addition
anti addition
syn addition
anti addition
ee [%][b]
ee [%][b]
ee [%][b]
ee [%][b]
BY
98
99
82
78
97
99
99
99
40/60, 72
40/60, 100
25/75, 100
30/70, 88
OYE1
OYE2
OYE3
[a]
Evaluated by GC analysis of the crude reaction mixture.
Evaluated by GC analysis on a chiral stationary phase.
[b]
[c]
Data for BY reduction are referred to saturated alcohols syn- and anti-3, obtained as a consequence of the presence of al-
cohol dehydrogenases within cells.[7]
From ref.[7]
[d]
[e]
Isolated yields from ref.[7]
cificity of hydrogen addition by investigating the ER- OYEs 2–3,[14] even if the presence of other enzymes
mediated reactions of tetrasubstituted olefins, in capable of the same reaction cannot be excluded.
which two stereogenic centres are created upon re-
BY reduction of (E)-1 (Scheme 1) showed a slight
duction. As a matter of fact, in this case tetrasubstitu- preference for syn addition with the alkene located in
tion allows the distinction between the products of order to offer the Cb-si face to HÀ attack.
syn and anti hydrogen addition to be made by the
analysis of their relative configuration, avoiding label- the BY reduction of (Z)-1. The absolute configuration
ling experiments. of both diastereoisomers can be explained by the ad-
An anti addition seemed to be slightly favoured in
The results of BY reductions of tetrasubstituted dition of the hydride on the Cb-si face, which is here
olefins (E)- and (Z)-1 and 2 shown in Scheme 1 and possible in a flipped binding mode. However, very
Scheme 2 can be now reconsidered under the light of poor enantioselectivity was observed.
the previously reported observations, on the assump-
As for nitroalkenes (Scheme 2), BY reduction of
tion that the ene-reduction activity of BY is due to both diastereoisomers was characterised by very
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scarce diastereoselectivity. However, rather high small extent: in particular, the effect could be that of
enantiomeric excess values were obtained especially lowering the enantiomeric excess values of the reduc-
for the two products of (Z)-2 biotransformation. Even tion products. However, in spite of this alteration,
in this case the absolute configuration of the nitroal- they were decidedly discordant with those obtained
kanes could be explained by assuming the exposure with BY. OYE1-3 afforded the reduction products ex-
of the Cb-si face to hydride addition for both (E)- and pected for HÀ attack on the Cb-re face in a flipped
(Z)-2, in a classical and flipped binding mode, respec- binding mode, except for syn-4 recovered from OYE2
tively.
reaction, showing indeed a very low enantiomeric
The data of OYE-mediated biotransformations of excess. The anti addition was disfavoured with respect
olefins (E)- and (Z)-1, and (E)- and (Z)-2 are report- to the syn mode, even though nitroalkanes obtained
ed in Table 2. The regeneration of the catalytic by an anti reduction mode were characterised by
NADPH cofactor was performed by using glucose de- enantiomeric excess values higher than those of syn
hydrogenase (GDH) with glucose as a sacrificial sub- reduction products.
strate.
As a general remark, the enantioselectivity of these
All the starting tetrasubstituted alkenes were dia- reductions is not usually high, with some interesting
stereoiosomerically pure. The occurrence of double exceptions. The formation of the other enantiomer
bond isomerisation was monitored by GC/MS during can be due to a lack of selectivity in the binding
the biotransformations. No isomerisation was ob- mode of the diastereoisomeric olefin in the enzyme
served for (E)- and (Z)-1 and for (Z)-2. A low per- site, with the hydrogen addition occurring with the
centage of (Z)-2 was detected in the course of same mechanism. It is plausible that an increase in
OYE1–3 reductions of substrate (E)-2: in these reac- the steric hindrance of the substituents at Ca and/or
tions final conversion values were less than 100%, Cb could improve binding specificities and lead to
and the unreacted (E)-olefin showed the following higher enantioselectivities.
final values of diastereoisomeric excess: de=82%
(OYE1), 76% (OYE2), and 77% (OYE3).
The configurational stability of the saturated alde- Conclusions
hydes 5 was verified by incubating a sample of
(2R,3S)-5 under the bioconversion conditions. No ap- The stereochemical outcome of ER-mediated reduc-
preciable formation of the other diastereoisomer due tions of activated alkenes is influenced by the configu-
to epimerisation at Ca was detected after 24 h.
ration of the C=C double bond, the arrangement of
OYE1- and OYE2-mediated reductions of (E)-1 the substrate in the active site, and the stereospecific-
proceeded in agreement with the BY biotransforma- ity of the hydrogen addition step. If suitable tetrasub-
tion as regards the enantioselectivity, with addition of stituted olefins are employed as substrates, it is possi-
the hydride on the Cb-si face in a classical substrate ble to evaluate the contribution of each factor to the
arrangement, but without any diastereoselectivity. overall stereochemical course of the reaction.
The results obtained with OYE3 can be explained by
In the BY-mediated reactions of compounds 1 and
admitting a flipped binding mode of (E)-1 in the 2 the hydride attack occurs exclusively on the Cb-si
active site of the enzyme, promoting addition of HÀ face with both (E)- and (Z)-stereoisomers: a flipped
on the Cb-re face. A preference for anti addition substrate binding mode has to be assumed for (Z)-1
(75:25) was also shown in this case.
and (Z)-2.
BY and OYE1 catalysed the reduction of (Z)-1
When isolated OYEs 1–3 were employed, some
with poor and no enantioselectivity, respectively, af- cases of hydride addition to the Cb-re face were ob-
fording 1:1 mixtures of the two diastereoisomers: in served. This reduction stereochemistry requires a flip-
the case of BY, addition of HÀ on the Cb-si face in a ped arrangement for (E)-olefins, and a classical ac-
flipped binding mode is slightly prevailing. The results comodation for (Z)-isomers.
obtained by using OYE2 and OYE3 revealed addition
The reaction performed either with whole-cell
of HÀ on the Cb-re face, in a classical binding mode, system or with isolated enzymes is characterised by
with anti addition being a little more favoured than low values of diastereoselectivity: no definite prefer-
the syn mode in the case of OYE3.
ence for syn or anti addition was observed.
As for (Z)-2, BY- and OYE1-3-mediated reactions
It is rather difficult to rationalise the data so far
gave reduced products with fairly high enantioselec- collected: probably the presence of steric hindrance
tivity, always adding the hydride to the Cb-si face in a on both Ca and Cb prevents the substrate from finding
flipped binding mode, and with syn addition prevail- an optimal binding mode within the enzyme active
ing over the anti mode.
site, which would be necessary to achieve higher ste-
The results achieved in the OYE-mediated bio- reoselectivity in the reduction process.
transformations of (E)-2 could be partially altered by
The detailed stereochemical analysis performed in
the occurrence of double bond isomerisation to a this work highlights all the parameters to be consid-
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Stereochemical Outcome of the Biocatalysed Reduction of Activated Tetrasubstituted Olefins
(Z)-2-Methyl-3-phenylbut-2-enal
[(Z)-1]:
1H NMR
ered, in order to explain the stereochemical outcome
(CDCl3, 400 MHz): d=1.92 (q, J=1.0 Hz, 3H), 2.27 (q, J=
1.0 Hz, 3H), 7.18–7.25 (m, 2H), 7.31–7.41 (m, 3H);
13C NMR (CDCl3, 100 MHz): d=11.1, 23.4, 128.2, 128.7,
134.4, 140.2, 157.4, 193.5; GC/MS (EI): tR =15.36 min: m/z
(%)=160 (M+, 57), 159 (100), 145 (22), 131 (17), 115 (32),
103 (4), 91 (25), 78 (10).
of ER-mediated reductions. The configuration of the
starting double bond is not always the only factor
controlling stereochemistry: the possibility of a syn
addition mechanism, and of a flipped binding mode in
the active site may play a crucial role.
(E)-(3-Nitrobut-2-en-2-yl)benzene
[(E)-2]:
1H NMR
(CDCl3, 400 MHz): d=2.09 (q, J=1.5 Hz, 3H), 2.29 (q, J=
1.5 Hz, 3H), 7.10–7.40 (m, 5H); 13C NMR (CDCl3,
100 MHz): d=16.1, 21.0, 126.2, 127.9, 128.5, 136.9, 139.5,
144.5; GC/MS (EI): tR =16.77 min; m/z (%)=176 (M+À1,
6), 160 (25), 115 (56), 91 (100).
Experimental Section
General Methods
TLC analyses were performed on Merck Kieselgel 60 F254
plates. All the chromatographic separations were carried out
(Z)-(3-Nitrobut-2-en-2-yl)benzene
[(Z)-2]:
1H NMR
(CDCl3, 400 MHz): d=1.99 (q, J=1.5 Hz, 3H), 2.12 (q, J=
1.5 Hz, 3H), 7.20–7.45 (m, 5H); 13C NMR (CDCl3,
100 MHz): d=16.9, 21.7, 127.0, 128.1, 128.5, 137.9, 139.7,
145.3; GC/MS (EI): tR =15.92 min; m/z (%)=176 (M+À1,
7), 160 (21), 115 (57), 91 (100).
1
on silica gel columns. H and 13C NMR spectra were record-
ed on a 400 MHz spectrometer (Bruker ARX 400). The
chemical shift scale was based on internal tetramethylsilane.
GC/MS analyses were performed using an HP-5MS column
(30 mꢂ0.25 mmꢂ0.25 mm). The following temperature pro-
gramme was employed: 60 8C (1 min)/68C/min/1508C
(1 min)/128C/min/2808C (5 min).
Compounds anti- and syn-4 and 5 were detected in the re-
action mixtures by GC/MS. The identity and absolute con-
figuration of anti- and syn-5 were established by GC/MS
and GC analysis on a chiral stationary phase, respectively,
by comparison with samples of (2R,3S)-5 and (2S,3S)-5, pre-
pared by Swern oxidation of the two mixtures of (2R,3S)-3
and (2S,3S)-3 obtained by BY reduction of (E)-1 and (Z)-1,
according to ref.[7] The identity and absolute configuration
of anti- and syn-4 were established by GC/MS and GC anal-
ysis on a chiral stationary phase, respectively, by comparison
with a 60/40 mixture of (2R,3R)-4 and (2R,3S)-4, obtained
by BY reduction of (E)-2, according to ref.[9] The GC/MS
data of the reduced products are as follows: anti-4 tR =
15.21 min, m/z (%)=179 (M+, 1), 132 (46), 117 (23), 105
(17), 91 (100); syn-4 tR =16.08 min, m/z (%)=179 (M+, 1),
132 (62), 117 (33), 105 (42), 91 (100); anti-5 tR =13.05 min,
m/z (%)=162 (M+, 17), 147 (4), 105 (100); syn-5 tR =
13.56 min, m/z (%)=162 (M+, 25), 147 (8), 105 (100).
Strains
Bakerꢀs yeast from Lesaffre Italia (code number 30509) was
employed for the preparation of reference compounds. All
the enzymes employed were overexpressed in Escherichia
coli BL21 (DE3) strains harbouring a specific plasmid pre-
pared according to standard molecular biology techniques:
pET30a-OYE1 from the original plasmid kindly provided
by Neil C. Bruce,[15] pET30a-OYE2 and pET30a-OYE3
from Saccharomyces cerevisiae BY4741 and pKTS-GDH
from Bacillus megaterium DSM509 (detailed steps reported
in ref.[16]).
Overexpression of the Enzymes in E. coli BL21
(DE3)
LB medium (5 mL) containing the appropriate antibiotic
(50 mgmLÀ1 kanamycin for pET-30a, 100 mgmLÀ1 ampicillin
for pKTS) was inoculated with a single colony from a fresh
plate and grown 8 h at 378C and 220 rpm. This starter cul-
ture was used to inoculate 200 mL medium, which was incu-
bated for 8 h at the same conditions and used to inoculate
1.5 L medium. The latter culture was shaken at 378C and
220 rpm until OD600 reached 0.4–0.5, then enzyme expres-
sion was induced by the addition of 0.1 mM IPTG
(50 ngmLÀ1 anhydrotetracycline was also added in the case
of the pKTS-GDH plasmid). After 5–6 h the cells were har-
vested by centrifugation (5000 g, 20 min, 48C), resuspended
in 50 mL of lysis buffer (20 mM phosphate buffer pH 7.0,
300 mM NaCl, 10 mM imidazole) and homogenised (Haskel
high-pressure homogenizer). The cell-free extract, after cen-
trifugation (20000 g, 20 min, 48C), was chromatographed on
IMAC stationary phase (Ni-Sepharose Fast Flow, GE
Healthcare) with a mobile phase composed of 20 mM phos-
phate buffer, pH 7.0, 300 mM NaCl and a 10–300 mM imida-
zole gradient. Protein elution was monitored at 280 nm, the
fractions were collected according to the chromatogram and
dialysed twice against 1.0 L of 20 mM phosphate buffer
pH 7.0 (12 h each, 48C) to remove imidazole and salts. Puri-
fied protein aliquots were stored frozen at À808C.
The enantiomeric excess values were determined by GC
analysis, performed using a DAcTBSil.BetaCDX 0.25 mmꢂ
0.25 mmꢂ25 m column (Mega, Italy), according to the fol-
lowing conditions: (i) 608C (1 min)/58C minÀ1/938C/0.058C
minÀ1/978C/308CminÀ1/2208C: (2R,3S)-syn-4 tR =47.0 min;
(2S,3R)-syn-4 tR =53.9 min; (2R,3R)-anti-4 tR =65.4 min;
(2S,3S)-anti-4 tR =66.6 min; (ii) 708C (1 min)/0.308C minÀ1/
908C/308CminÀ1/2208C:
(2R,3S)-anti-5
tR =33.4 min;
(2S,3R)-anti-5 tR =35.4 min; (2S,3S)-syn-5 tR =40.3 min;
(2R,3R)-syn-5 tR =41.2 min.
Synthesis of Tetrasubstituted Alkenes
Aldehydes (E)- and (Z)-1 were prepared according to ref.[7]
Nitro derivatives (E)- and (Z)-2 were prepared according to
ref.[9]
(E)-2-Methyl-3-phenylbut-2-enal
[(E)-1]:
1H NMR
(CDCl3, 400 MHz): d=1.66 (q, J=1.4 Hz, 3H), 2.46 (q, J=
1.4 Hz, 3H), 7.15–7.20 (m, 2H), 7.29–7.43 (m, 3H);
13C NMR (CDCl3, 100 MHz): d=12.9, 19.3, 126.7, 127.8,
128.4, 133.2, 142.8, 155.3, 191.7; GC/MS (EI): tR =15.19 min:
m/z (%)=160 (M+, 50), 159 (100), 145 (22), 131 (16), 115
(32), 103 (4), 91 (24), 77 (10).
Adv. Synth. Catal. 2012, 354, 105 – 112
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
111

Elisabetta Brenna et al.
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Acknowledgements
Prof. Claudio Fuganti is warmly acknowledged for fruitful
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Department of Biosystems Science and Engineering, Basel)
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Adv. Synth. Catal. 2012, 354, 105 – 112