Opposite enantioselectivity in the bioreduction of (Z)-β-aryl-β-cyanoacrylates mediated by the tryptophan 116 mutants of old yellow enzyme 1: Synthetic approach to (R)- and (S)-β-aryl-γ-lactams

Article abstract of DOI:10.1002/adsc.201500206

The Trp 116 mutants of Old Yellow Enzyme 1 that catalyse the reduction of (Z)-β-aryl-β-cyanoacrylates give the opposite enantioselectivity according to the nature of the amino acid in position 116. Small amino acids (e.g., alanine) make the substrate bind

Full text of DOI:10.1002/adsc.201500206

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DOI: 10.1002/adsc.201500206
Opposite Enantioselectivity in the Bioreduction of (Z)-b-Aryl-b-
cyanoacrylates Mediated by the Tryptophan 116 Mutants of Old
Yellow Enzyme 1: Synthetic Approach to (R)- and (S)-b-Aryl-g-
lactams
Elisabetta Brenna,a,b,* Michele Crotti,a Francesco G. Gatti,a Daniela Monti,b
Fabio Parmeggiani,a Robert W. Powell III,c Sara Santangelo,a and Jon D. Stewartc
a
Dipartimento di Chimica, Materiali ed Ingegneria Chimica “Giulio Natta” Politecnico di Milano, Via Mancinelli 7,
I-20131, Milano, Italy
Fax: (+39)-02-2399-3180; phone: (+39)-02-2399-3077; e-mail: mariaelisabetta.brenna@polimi.it
Istituto di Chimica del Riconoscimento Molecolare, C.N.R., Via Mario Bianco, 9, 20131, Milano, Italy
Department of Chemistry, University of Florida, 126 Sisler Hall, Gainesville, Florida 32611, USA
b
c
Received: February 27, 2015; Revised: April 13, 2015; Published online: May 13, 2015
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201500206.
Abstract: The Trp 116 mutants of Old Yellow acids (e.g., tryptophan in the wild-type) the reduc-
Enzyme 1 that catalyse the reduction of (Z)-b-aryl- tion does not occur. The enantiomerically enriched
b-cyanoacrylates give the opposite enantioselectivity cyanopropanoates thus prepared can be converted
according to the nature of the amino acid in position into the corresponding (S)- and (R)-b-aryl-g-lactams,
116. Small amino acids (e.g., alanine) make the sub- precursors of inhibitory neurotransmitters belonging
strate bind to the enzyme’s active site in a “classical” to the class of g-aminobutyric acids, by a simple
orientation, affording the (S)-enantiomer of the re- functional group interconversion in a sequential one-
duced product. When the size of the amino acid in- pot procedure.
creases (e.g., leucine), a “flipped” binding mode is
adopted by the substrate, which is converted into the Keywords: chirality; enantioselectivity; enzyme cat-
corresponding (R)-derivative. With bulky amino alysis; nitriles; reduction
Introduction
catalysed reactions with a well-defined substrate
scope, and to understand how they can be combined
A constant need of fine chemicals manufacturers, es- with classical chemical transformations in the most ef-
pecially in the pharmaceuticals field, is to develop ficient and productive way.[4]
more sustainable and cost-effective synthetic proce-
The enantioselective reduction of suitably substitut-
dures without impairing the quality and purity of the ed C=C double bonds, for example, represents a key
final products.[1] A possible solution is the replace- chemical transformation for the creation of stereogen-
ment of traditional chemical reactions with biocatalyt- ic centres in chiral building blocks to be used for fine
ic processes, that generally provide very pure products chemicals production. A very efficient biocatalysed
with improved step and atom economy,[2] under safe variant of this reaction is based on the use of enzymes
conditions, at a reduced cost, by exploiting the typical called ene-reductases (EC 1.6.99.1, ERs), most of
high chemo- and stereoselectivity of enzymes. More- which belong to the family of Old Yellow Enzymes
over, during the last decade, advancements in protein (OYEs).[5] Many efforts have been devoted not only
engineering strategies, in particular directed evolu- to isolate new ERs,[6] in order to enlarge the collec-
tion, have made available robust engineered biocata- tions of the available wild-type catalysts, but also to
lysts, that are able to outperform traditional stoichio- improve considerably their performance by protein
metric and chemocatalytic methods.[3]
To harness this great potential of enzymes in the strate scope or opposite enantioselectivity.
development of manufacturing processes, it is essen-
Currently, the mechanism[8] of OYE-mediated hy-
tial to enlarge the portfolio of synthetically useful bio- drogenations and the stereoelectronic effects of the
engineering,[7] affording variants with broader sub-
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Elisabetta Brenna et al.
Scheme 1. Chemoenzymatic synthesis of pregabalin.
substituents on the starting alkene,[6,9] have been ex-
tensively investigated, in order to define the limits
and the potential of the reactions in the field of or-
ganic synthesis. It has been established that at least
one of the two olefinic carbon atoms must be con-
nected to an electron-withdrawing group (EWG), typ-
ically the carbonyl function of aldehydes and ketones,
or a nitro group, or an imido functionality.[8b,10] The
presence of a CN moiety as the sole EWG of the
alkene derivative has been found to promote the bio-
Scheme 2. Synthesis and bioreduction of substrates (Z)-1a–g
and (E)-1a.
reduction only in the case of 2-arylacrylonitriles[11] report herein on the investigation of the ER-mediated
and 2-aryl-2-butenenitriles.[12] Generally, unsaturated hydrogenation of compounds 1a–g, and on the exploi-
esters are not reduced by OYEs, unless a halogen tation of the corresponding 3-aryl-3-cyanopropa-
atom is linked in the a position,[13] or another EWG noates 2a–g by means of simple functional group ma-
(e.g., an ester,[14] a nitrile[8d,e,15] or a nitro group[16]) is nipulation.
linked to the b olefinic carbon atom.
Indeed, the presence of two EWGs on the C=C
double bond makes the bioreduction of these alkenes Results and Discussion
of particular interest, because it affords chiral building
blocks with two functional groups that can be manip- Synthesis of Substrates (Z)-1a–g and (E)-1a
ulated, broadening the spectrum of the final products
that can be prepared. For example, the reduction of The most convenient synthetic procedure to arylcya-
cyano ester (E)-I (Scheme 1) mediated by ERs was noacrylates 1a–g was found to be the condensation of
carefully investigated by Pfizer in order to prepare the suitable arylacetonitrile derivative with glyoxylic
the (S)-enantiomer of the reduced product II on acid and potassium carbonate in methanol solution,
a preparative scale.[17] For this particular cyano ester first reported by Dean et al. in 1993,[18] to give only
the best results were achieved by using OPR1 (from the (Z)-stereoisomer of the corresponding cyanoacryl-
Lycopersicon esculentum). Derivative II (ee >99%) ic acid potassium salt (Scheme 2). The esterification
was submitted to saponification of the ester group was then performed by reaction with methyl iodide in
and reduction of the CN moiety, to afford the g-ami- DMF. In the case of compound 1a, the (E)-isomer
nobutyric acid analogue pregabalin (IV), developed was obtained by Wittig reaction of benzoyl cyanide
for the treatment of central nervous system disorders. and
methyl
(triphenylphosphoranylidene)acetate
The data collected in the literature on the bioreduc- (Scheme 2): after column chromatography, a sample
tion of cyano esters of type I are limited to com- containing 20% of (Z)-stereoisomer was recovered.
pounds bearing an alkyl substituent at the prostereo-
The only data concerning the bioreduction of these
genic carbon atom.[8d,e,15] Thus, we decided to extend derivatives were described for the potassium salts of
the investigation to those having an aromatic ring in compounds (Z)-1a, b, c and e,[19] and for the free car-
this position, i.e., substrates 1a–g (Scheme 2), in order boxylic acids of derivatives (Z)-1a, and c.[20] The po-
to enrich the knowledge of the substrate scope of the tassium salts had been reduced by the crude cell ex-
reaction. We also envisaged the possibility to convert tracts of the anaerobic bacteria Clostridium sporo-
the resulting reduced compounds into b-aryl-g-lac- genes (DSM 795), Ruminococcus productus (DSM
tams, the synthetic precursors of the inhibitory neuro- 3507), and Acetobacterium woodii (DSM 1030), under
transmitters belonging to the class of g-aminobutyric an H2 atmosphere to afford the (S)-enantiomer of the
acids (GABA), such as baclofen and phenibut. We reduced products in quantitative yields and high
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Opposite Enantioselectivity in the Bioreduction of (Z)-b-Aryl-b-cyanoacrylates
Table 1. Biotransformations of compounds (Z)- and (E)-1a
with wild-type OYE1–3.
(Z)-1a
ee [%][b]
(E)-1a
ee [%][b]
ER
c [%][a]
c [%][a]
OYE1
OYE2
OYE3
5
2
8
–
–
–
99
99
99
82 (R)
84 (R)
70 (R)
[a]
Conversion, calculated on the basis of GC analysis of the
crude mixture after 24 h reaction time.
Calculated on the basis of GC analysis on a chiral sta-
tionary phase.
[b]
enantiomeric excess values in 3 days reaction time.
The carboxylic acids of (Z)-1a, and c had been con-
verted into the corresponding (R)-saturated com-
pounds in 80% yields and 98 and 94% ee values, re-
spectively, by the whole cells of Achromobacter sp.
JA81 in 48 h.
OYE1-3-Mediated Reductions of Compounds (Z)-1a–
g and (E)-1a
When substrates (Z)-1a–g were submitted to OYE1-
3-mediated reductions in the presence of glucose and
glucose dehydrogenase (GDH) for NADPH regener-
ation, either very low or no conversion was observed
[Table 1 for (Z)-1a, Table S1 in the Supporting Infor-
mation for (Z)-1b–g]. On the contrary, when com-
pound (E)-1a (de=60%) was employed, the (E)-
alkene was completely reduced to afford (R)-2a (ee=
70–84%) (Table 1) and the (Z)-stereoisomer was left
unreacted. The absolute configuration of compound
2a was assigned by comparison with the specific opti-
cal rotation value reported for (S)-2a in the litera-
ture.[19]
In order to understand the marked preference of
OYE1-3 for the reduction of (E)-1a with respect to
the (Z)-stereoisomer, we investigated the stereochem-
ical course of the reaction. First of all, deuteration ex-
periments were performed in order to establish
whether the CN or the ester moiety was the activating
EWG, i.e., the one establishing hydrogen bonds with
1
1
Figure 1. a) H NMR spectrum of rac-2a; b) H NMR spec-
trum of (R)-2a-d1 obtained from (E)-1a (de=60%) by
OYE1-mediated reduction in D2O in the presence of stoi-
chiometric NADH (c=80%, ee=80%); the singlet at
3.90 ppm is due to the COOMe of unreacted (Z)-1a. All
spectra are recorded in CDCl3.
the amino acids residues within the enzyme binding hydrogen atom at C-3 highlights the incorporation of
pocket. OYE1-mediated reduction of (E)-1a was car- a deuterium atom in this position.
ried out in D2O in the presence of a stoichiometric
We drew the conclusion that the CN moiety was
quantity of NADH to afford (R)-2a-d1 (Figure 1). the activating EWG in the OYE-mediated reduction
These reaction conditions are known[21] to promote of (E)-1a. The following step was to define the pro-
the delivery of a HÀ to the olefinic carbon atom in ductive binding mode adopted by the substrate within
the b position to the activating EWG, and a D+ to the the enzyme active site during the reaction. There are
one in the a position. In Figure 1 the proton NMR two possible substrate orientations described in the
spectrum of (R)-2a-d1 (Figure 1b) is compared to that literature: the “classical” (or “normal”) binding mode
of the unlabelled racemic compound 2a prepared by and the “flipped” binding mode (Scheme 3).
NaBH4 reduction (Figure 1a): the nearly complete
disappearance of the multiplet at 4.28 ppm due to the cyclohexenone from the crystal structure of oxidised
The former was inferred by Massey et al.[8a] for 2-
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Scheme 4. OYE-meditated reduction of (E)-1a in a flipped
binding mode with the CN as the activating EWG, affording
(R)-2a.
based on the hypothesis of an anti hydrogen addition
(a hydride from below the plane of the alkene to Cb
and a proton from above the plane to Ca) and on the
evidence that the stereogenic centre was created in
(R) configuration with the CN involved in the hydro-
gen bonds within the active site.
Scheme 3. Substrate binding modes in OYE active site. G is
the substituent linked to the C=O moiety of the EWG;
a and b are the substituents in alpha and beta position with
respect to the EWG; S and L stand for small and large, re-
spectively.
Then, the marked preference for the OYE-mediat-
OYE1 with the inhibitor para-hydroxybenzaldehyde ed reduction of (E)-1a with respect to the (Z)-stereo-
bound to the active site, by assuming that the carbon- isomer could be explained by the fact that the hin-
yl group would be positioned in an analogous way to drance of the phenyl ring favoured a flipped orienta-
the phenolate oxygen of the aromatic aldehyde. The tion, which was optimal for the (E)-stereoisomer, af-
latter[9b] was deduced by a 1808 rotation of the sub- fording the (R) enantiomer of the reduced product.
strate about the axis passing through the oxygen atom
of the carbonyl group and the carbon atom of the
double bond in the b position to the activating OYE1-W116X-Mediated Reductions of Compounds
moiety. The experimental data collected in the litera- (Z)-1a–g
ture for acyclic trisubstituted olefins activated by an
EWG linked to the prostereogenic carbon atom and From a synthetic route planning perspective, the aryl
efficiently converted by OYE1-3 enzymes, have al- cyano esters 1a–g were much more easily obtained as
ready shown a connection[8c,d] between the preferred (Z)- rather than (E)-stereoisomers, so it was more ad-
reactive binding mode and the relative size of the vantageous to find a way to get the former accepted
groups shown as G and a in Scheme 3. If G is bulky by the enzyme, e.g., by protein engineering, rather
(large, L) and a has a modest steric hindrance (small, than struggling to synthesise the latter. The use of Trp
S), a classical binding mode is adopted and the alkene 116 mutants of S. pastorianus OYE1 represented an
stereoisomer best reduced is the one with the large attractive option,[7c] because literature data, based
substituent (L) at Cb on the same side of the EWG. If also on crystallographic studies,[22] had shown that the
G is small, and a is large, then a flipped binding Trp 116 residue (conserved in OYE2–3) played a criti-
mode is preferred, and the favoured alkene stereoiso- cal role in the stereochemistry of OYE-mediated
mer is the one with the large substituent (L) at Cb on alkene reductions by influencing the substrate orien-
the opposite side of the EWG. These considerations tation within the active site.
could be also extended to alkenes for which the acti-
Therefore, we decided to investigate the transfor-
mation of cyano esters (Z)-1a–g by using the com-
vating EWG is a CN moiety without a G group.[8e]
As for compound (E)-1a, the information on the plete set of Trp 116 mutants of OYE1. The results of
activating EWG was combined with the absolute ste- the biocatalysed reductions highlighted the possibility
reochemistry of the reduced compound, which defines to organise the twenty variants in three different
the stereoheterotopic face of the alkene on which the groups: (A) Ala, Cys, Gly, Ile, Asn, Gln, Ser, Thr and
addition of the proton has occurred. A flipped bind- Val mutants, affording (S)-2a–g (Table 2); (B) His,
ing mode could be inferred for (E)-1a (Scheme 4) Leu and Met mutants giving (R)-2a–g derivatives
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Opposite Enantioselectivity in the Bioreduction of (Z)-b-Aryl-b-cyanoacrylates
Table 2. Biotransformations of compounds (Z)-1a–g affording (S)-2a–g.
1a, R=H
ee
[%][a] [%][b] [%][a]
1b, R=p-OMe
1c, R=p-Cl
ee
[%][a] [%][b] [%][a] [%][b] [%][a] [%][b]) [%][a] [%][b] [%][a] [%][c]
1d, R=p-Br
1e, R=p-F
1f, R=m-F
1g, R=o-F
c ee
Variant
c
c
ee
c
c
ee
c
ee
c
ee
[%][c]
W116A 100
W116C 60
W116G 31
48
70
84
rac
50
36
6
77
32
45
68
76
38
40
39
95
56
rac
38
60 (R)
28
64 (R)
28
34 (R)
24
92
40
44
86
79
68
25
23
28
60
50
6
42
42
26
64
54
48
95
95
81
90
82
68
58
95
88
68
68
70
30
60
rac
rac
60
50
94
41
33
88
25
64
51
82
90
90
72
94
30
26
rac
73
60
70
100
100
50
100
65
94
100
88
56
48
60
rac
40
16
56
60
56
48
74
29
65
66
81
47
86
100
68
66
66
52
60
6
64
74
72
W116I
23
W116N 80
W116Q 70
W116S 42
W116T 58
W116V 85
58
70
100
[a]
Conversion calculated on the basis of GC analysis of the crude mixture after 24 h reaction time (isolation yields are re-
ported in the Supporting Information).
Calculated on the basis of GC analysis on a chiral stationary phase.
[b]
[c]
Calculated on the basis of HPLC analysis on a chiral stationary phase. Reactions were performed in pH 7 buffer solution.
Table 3. Biotransformations of compounds (Z)-1a–g affording (R)-2a–g.
1a, R=H
ee
[%][a] [%][b] [%][a]
1b, R=p-OMe
1c, R=p-Cl
ee
[%][a] [%][b] [%][a] [%][c] [%][a] [%][b] [%][a] [%][b] [%][a] [%][c]
1d, R=p-Br
1e, R=p-F
1f, R=m-F
1g, R=o-F
Variant
c
c
ee
c
c
ee
c
ee
c
ee
c
ee
[%][c]
W116H 42
W116L 91
W116M 83
74
88
29
44
95
79
99
99
84
13
84
17
67
66
14
40
100
95
54
66
40
18
93
25
99
80
2
5
100
100
–
60
rac
7
100
86
–
64
46 (S)
[a]
Conversion calculated on the basis of GC analysis of the crude mixture after 24 h reaction time (isolation yields are re-
ported in the Supporting Information).
Calculated on the basis of GC analysis on a chiral stationary phase.
[b]
[c]
Calculated on the basis of HPLC analysis on a chiral stationary phase. Reactions were performed in pH 7 buffer solution.
(Table 3); (C) Trp (wild-type), Asp, Glu, Phe, Lys,
The absolute configuration of compounds 2a, b, c,
Pro, Arg and Tyr mutants characterised by very low and e was established by comparison with the specific
or no conversions (Table S2, Supporting Information). optical rotation values reported in the literature; it
As for the enantioselectivity of the bioreductions was assigned by chemical correlation to compound
with enzymes of groups A and B, the only exceptions 2d, and by analogy to derivatives 2f and 2g (see the
were observed in the reactions of the para-methoxy Supporting Information).
derivative (Z)-1b, catalysed by the W116I, W116Q
We determined the activating group for substrate
and W116T variants which afforded compound (R)- (Z)-1a in the reactions with W116L and W116A, char-
2b, although with poor selectivity, and in the reduc- acterised by opposite enantioselectivity, by perform-
tion of ortho-fluoro cyano ester (Z)-1g with the ing the bioreductions in deuterated water in the pres-
W116M mutant giving derivative (S)-2g.
ence of stoichiometric NADH. In both cases the pres-
As for the efficiency of the reactions with the mu- ence of a deuterium atom at the carbon atom bearing
tants of group C, exceptions were observed with the the CN group was highlighted by the analysis of the
1
para-bromo derivative, which was reduced by these corresponding H NMR spectrum (Figure 2).
enzymes, even if with modest yields and very low
enantiomeric excess values.
Thus, a classical binding mode (Scheme 5a) could
be deduced for the W116A mutant (or any of the var-
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Elisabetta Brenna et al.
Scheme 5. Binding modes of (Z)-1a in the active site of
OYE1-W116A (a) and OYE1-W116L (b) deduced by analy-
sis of the bioreduction results. The flavin prosthetic group
lies below the plane of the substrate, while the catalytic resi-
due Y196 lies above.
the collected X-ray data of the crystals showed the
presence in the active site of an electron density best
fit, respectively, by substrates (Z)-1a (PDB code:
4YNC) and (Z)-1e (PDB code: 4YIL). The resulting
orientations of these two substrates in the active site
were found to be very similar, and in both cases they
showed features suggesting that they did not repre-
sent catalytically productive arrangements (Figure 3).
First of all, the activating nitrile group was not di-
rected towards Ne2 of H191 and Nd2 of N194
(Figure 3), but it was positioned between Y82 and
Y375. The hydrogen bonds with H191 and N194 are
known to be essential for the catalytic turnover by
OYE1.[23] Then, the orientation of the alkene with re-
spect to the FMN cofactor was not optimal: the angle
formed between Cb of the cyano ester and the plane
of the FMN is 121.58 for 1a and 112.98 for 1e; the dis-
tance between Cb of the cyano ester and N-5 of FMN
is 4.70 Š for 1a and 4.78 Š for 1e. These values are
outside the range observed by Fraaije et al. in their
survey of flavoproteins bound to their respective sub-
strates and/or products.[24]
1
Figure 2. a) H NMR spectrum of (R)-2a-d1 obtained from
(Z)-1a by OYE1-W116L-mediated reduction in D2O in the
presence of stoichiometric NADH (c=65%, ee=90%); b)
1H NMR spectrum of (S)-2a-d1 obtained from (Z)-1a by
OYE1-W116A-mediated reduction in D2O in the presence
of stoichiometric NADH (c=32%, ee=65%). The singlet at
3.72 ppm is due to the COOMe of the reduced product, and
that at 3.90 ppm is due to the COOMe of the unreacted
starting compound. All spectra are recorded in CDCl3.
iants belonging to group A) in the reaction to (S)-2a,
No information could be obtained on the geometry
and a flipped one (Scheme 5b) for the W116L mutant of the hydrogen bonds accepted by the nitrogen atom
(or any of the variants belonging to group B) in the of the nitrile within the active site.
reduction affording (R)-2a.
We attempted to support these conclusions on the
reactive binding mode of b-aryl-b-cyano esters and to Understanding the Stereoselectivity of OYE1-W116X
obtain information on the coordination geometry of Variants in the Reactions with Cyano Esters (Z)-1a–g
the nitrile group by crystallographic studies. Soaking
experiments of (Z)-1a and (Z)-1e were successful In a previous work,[22] the stereochemistry of the bio-
only with crystals of OYE1-W116A. The analysis of reductions of (R)- and (S)-carvone with wild-type
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Opposite Enantioselectivity in the Bioreduction of (Z)-b-Aryl-b-cyanoacrylates
Figure 4. Structure of active site of oxidized OYE1 from the
crystal data of Fox and Karplus[25] (PDB code: 1OYA).
commodation is within the hydrophobic pocket in the
right side of the active site. The achievement of this
orientation, made accessible by small amino acids, im-
plies a classical binding mode for the substrate
(Scheme 5a). When larger amino acids, such as Leu,
His and Met, hinder the occupancy of this pocket,
a flipped orientation is adopted by the substrate with
the aromatic ring pointing towards the left side
(Scheme 5b).
We could make some considerations on these re-
sults by taking into account the different size of the
amino acid residues represented by the numerical
values of their Van der Waals volumes (Figure 5).[26]
Figure 3. Crystal structures of the complexes between
OYE1-W116A mutant and (a) compound (Z)-1a (PDB
code: 4YNC), or (b) compound (Z)-1e (PDB code: 4YIL).
The presence in position 116 of an amino acid char-
3
OYE1 and OYE1-W116X variants was successfully in- acterised by a side-chain volume higher than 130 Š
vestigated by solving the X-ray crystal structures of (W116F, K, Y and R variants and wild-type) inhibited
pseudo-Michaelis complexes formed in cristallo by the reduction of cyano esters (Z)-1a–g: the substrate
soaking each protein individually with the substrate. was allowed to bind the active site neither in a classi-
This study concluded that in the active site of these cal nor in a flipped orientation. The first was forbid-
enzymes there is a pocket lined entirely by hydropho- den by the size of the residue in position 116, the
bic residues (i.e., T37, M39, F74, Y82, A85, and L118) latter by the necessity to re-orient the side chains of
where large substituents of the substrate can be fa- some residues on the opposite side of the binding
vourably accommodated (Figure 4). The access to this pocket. The reactions did not occur also with W116D
pocket can be physically blocked by the presence of and E, in which the tryptophan was replaced by an
a bulky amino acid in position 116. In this case the acidic amino acid, and with W116P, characterised by
substrate adopts a different orientation in which the the presence of the sterically constrained ring of pro-
large substituent is projected into a portion of the line.
active site partially defined by the side-chains of
With amino acids showing a volume lower than
F250, F296, and Y375 (Figure 4). This accommodation 100 Š3, the reactive substrate binding mode was the
is generally less favourable because it induces a rear- classical one, affording the (S)-enantiomer of the re-
3
rangement of the side-chains of both F296 and Y375 duced products. 120 Š represents a kind of threshold
from their preferred positions in the apoprotein.[22]
for the transition from (S)-selectivity (with W116Q
These observations are crucial to understand the and I) to (R)-selectivity (with W116H, L and M).
stereochemical course of OYE-mediated reactions of Modest ee values were obtained with all the sub-
cyano esters (Z)-1a–g. For these substrates the bulki- strates and W116Q, H, I, and M variants, whereas the
est substituent is the aryl ring linked to the carbon presence of the leucine residue created the optimal
atom in the a position to the nitrile, whose best ac- situation for a flipped binding mode.
Adv. Synth. Catal. 2015, 357, 1849 – 1860
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Elisabetta Brenna et al.
Table 4. Effect of pH on the bioreduction of (Z)-1a and
c mediated by OYE1-W116A and OYE1-W116L.
pH[a]
(Z)-1a, R=H
(Z)-1c, R=p-Cl
c [%][b]
ee [%][c]
c [%][b]
ee [%][c]
OYE1-W116A
4.0
5.0
6.0
7.0
8.0
9.0
0
–
0
–
43
100
100
96
93
86 (S)
90 (S)
48 (S)
40 (S)
rac
33
85
92
97
90
86 (S)
82 (S)
60 (S)
22 (S)
rac
OYE1-W116L
4.0
5.0
6.0
7.0
8.0
9.0
0
18
100
91
100
100
–
0
–
90 (R)
94 (R)
88 (R)
60 (R)
40 (R)
15
92
84
100
97
70 (R)
88 (R)
66 (R)
40 (R)
rac
Figure 5. Van der Waals volumes of amino acid residues vs.
outcome of the bioreduction catalysed by the corresponding
OYE1-W116X variant [grey bars: (S)-selective bioreduc-
tions; black bars: (R)-selective bioreductions; white bars: bi-
oreductions with low or no conversions].
[a]
Buffers: acetate for pH 4.0–5.0, phosphate for pH 6.0–
8.0, tris for pH 9.0.
Conversion, calculated on the basis of GC analysis of the
crude mixture after 24 h reaction.
Calculated on the basis of GC analysis on a chiral sta-
tionary phase.
[b]
[c]
Synthesis of (R)- and (S)-b-Aryl-g-lactams
One of the aims of this work is to optimise efficient
synthetic procedures to enantiopure chiral building
blocks for fine chemicals production. A two-step se-
quence, consisting in the biocatalysed hydrogenation
of the alkene followed by the chemical reduction of a selection of mutants. Conversions were in general
the nitrile, can be employed to convert substrates (Z)- lower, but the decrease was not substantial.
1a–g into chiral b-aryl-g-lactams,[27] whose structural
OYE1-W116A afforded the best results for the syn-
pattern is inserted in many active pharmaceutical in- thesis of (S)-2a, c–g in nearly quantitative yields and
gredients such as baclofen,[28] phenibut[29] and roli- ee values in the range 82–95% in 24 h reaction time.
pram.[30] For these drugs the (R)-enantiomer has been Derivative (S)-2b could be obtained only with poor
identified to be the most active one.
enantiomeric enrichment. OYE1-W116L was selected
The intermediate reduced compounds 2a–g are for the preparation of the (R)-enantiomers of deriva-
characterised by the presence of a benzylic stereogen- tives 2, in particular (R)-2a and (R)-2c, precursors of
ic centre which could be configurationally unstable the active forms of phenibut and baclofen.
because of the acidity of the hydrogen atom in the
The reduction of the nitrile by treatment with
a position to the CN group. In a first screening evalu- NiCl2·6H2O and NaBH4 (Scheme 6), already em-
ation the bioreductions of compounds (Z)-1a–g were ployed by Fryszkowska et al.,[19] was performed in this
performed in pH 7.0 buffer solutions, as it is usually case directly in the aqueous medium of the biocata-
reported in the literature. The dependence of the lysed reduction, without isolation of the intermediate
enantiomeric purity of the reduced compounds on the cyano esters 2, to afford, after extraction and isola-
reaction pH was investigated in the range 4–9 for sub- tion, the corresponding lactams (R)-3a and (R)-3c.
strates (Z)-1a and c, precursors of phenibut and ba-
This procedure represents a very effective route to
clofen, with two mutants of opposite enantioselectivi- the pharmacologically active enantiomers of phenibut
ty, OYE1-W116A and OYE1-W116L (Table 4). The and baclofen. The bioreductions, performed in pH 6.0
acidic medium inhibited the reduction, whereas basic buffer solution in the presence of OYE1-W116L,
conditions caused a significant loss of the enantiomer- afford (R)-2a (ee=94%) and (R)-2c (ee=90%), re-
ic purity of compounds 2a and c, as a consequence of spectively, in 2.7 and 2.0 gLÀ1·dÀ1 space-time yields,
the configurational lability of the stereocentre.
showing potential scalability. The use of the system
The optimal value was established to be pH 6.0, NiCl2/NaBH4 has the advantage to afford the reduc-
thus the screening of OYE1-W116X variants in the tion of the nitrile quantitatively and almost instanta-
reduction of cyano esters (Z)-1a–g was repeated at neously, and to be compatible with the water medium
this pH value (see Tables S3 and S4 in the Supporting of the enzymatic reactions. Thus, the two subsequent
Information for the complete set of data). Figure 6 reduction steps, the first catalysed by the enzyme and
gives at a glance the general increase of the enantio- the second promoted by the chemical reducing agent,
selectivity of the reaction of all the substrates with can be easily telescoped, without impairing the enan-
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Opposite Enantioselectivity in the Bioreduction of (Z)-b-Aryl-b-cyanoacrylates
Scheme 6. Synthetic procedure to the lactams (R)-3a and
(R)-3c, precursors of (R)-phenibut and (R)-baclofen.
These results are in agreement with the considera-
tions made on the importance of the relative size of
the G and a groups in establishing the reactive bind-
ing mode for acyclic alkenes with the EWG linked to
the prostereogenic carbon atom (Scheme 3). When
wild-type OYE1-3 are employed, a Trp occupies posi-
tion 116, and the favoured substrate orientation is the
one in which a small group, either G or a, is located
within the hydrophobic pocket in the right side of the
active site.
OYE1-W116A and OYE1-W116L were selected for
the preparation of the (S)- and (R)-enantiomer, re-
spectively, of derivatives 2a–g. We showed the possi-
bility to convert these reduced enantiomerically en-
riched intermediates into the corresponding (S)- and
(R)-b-aryl-g-lactams by a simple functional group in-
terconversion to be performed according to a sequen-
tial one-pot procedure by addition of the chemical re-
Figure 6. Effect of pH on the enantioselectivity of the biore-
duction of (Z)-1a–g with selected variants (grey bars: ee
values obtained at pH 7.0; black bars: ee values obtained at
pH 6.0).
tiomeric excess of the final compounds, with the ad- agent for the reduction of the CN group directly to
vantage of diminishing the number of purification op- the biotransformation medium.
erations, and contributing significantly to decrease
cycle time, solvent usage and waste.
Experimental Section
Conclusions
Sources of Enzymes
Wild-type Old Yellow Enzymes fused with a His6-tag
(OYE1 from Saccharomyces pastorianus, OYE2 and OYE3
from Saccharomyces cerevisiae), OYE1-W116X site-satura-
tion mutagenesis library fused with a GST tag (OYE1-
W116X from Saccharomyces pastorianus) and glucose dehy-
drogenase fused with a His6-tag (GDH from Bacillus mega-
terium) were overproduced in Escherichia coli BL21(DE3)
The reductions of cyano esters (Z)-1a–g catalysed by
OYE1-W116X mutants confirm the strategic role
played by the amino acid in position 116 on the ste-
reochemical course of these reactions by controlling
the access to a hydrophobic pocket that favourably
hosts bulky substituents.
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Elisabetta Brenna et al.
strains, harbouring the following plasmids: pET-30a-OYE1
containing the oye1 gene provided by Neil C. Bruce;[31] pET-
30a-OYE2 and pET-30a-OYE3 containing the oye2 and
oye3 genes cloned from S. cerevisiae BY4741;[32] pDJB5-
OYE1-W116X provided by J. D. Stewart;[7c] pKTS-GDH
containing the gdh gene cloned from B. megaterium
DSM509.[32]
duced product. A solution of the suitable cyano ester in i-
PrOH (1 mL, 250 mM) was added to a potassium phosphate
buffer solution (25 mL, 50 mM, pH 6.0 or pH 7.0) containing
the required OYE (4–5 mg), GDH (100 U), glucose
(1 mmol, 180 mg) and NADP+ (5 mmol, 3.7 mg). The reac-
tion was monitored by GC until complete conversion (gen-
erally 24 h). The mixture was then extracted with EtOAc
(3”10 mL), dried over anhydrous Na2SO4 and purified by
column chromatography (n-hexane with increasing amount
of EtOAc).
Overexpression of the Enzymes in E. coli
BL21(DE3)
LB medium (15 mL) supplemented with the appropriate an-
tibiotic (50 mgmLÀ1 kanamycin for pET-30a and pDJB5,
100 mgmLÀ1 ampicillin for pKTS) was inoculated with
a single colony from a fresh plate and grown overnight at
378C and 220 rpm. This starter culture was used to inoculate
1.5 L of LB medium, which was incubated at 378C and
220 rpm until OD600 reached 0.4–0.5, then enzyme expres-
sion was induced by the addition of IPTG (0.1 mM final
concentration). For the pKTS-GDH plasmid anhydrotetra-
cycline was also added (50 ngmLÀ1 final concentration).
After 5 h, the cells were harvested by centrifugation (5000”
g, 20 min, 488C), resuspended in 50 mL of lysis buffer
(20 mM potassium phosphate buffer pH 7.0, 300 mM NaCl,
10 mM imidazole), disrupted by sonication (Omni Ruptor
250 ultrasonic homogeniser, five sonication cycles, 15 s each,
50% duty) and centrifuged (20000”g, 20 min, 48C). The
cell-free extracts of GST-tagged OYE1-W116X mutants
were aliquoted and stored frozen at À808C. The cell-free ex-
tracts of His-tagged OYE1–3 and GDH, were purified by af-
finity chromatography on IMAC stationary phase (Ni-Se-
pharose Fast Flow, GE Healthcare) with a mobile phase
composed of 20 mM potassium phosphate buffer pH 7.0,
300 mM NaCl and a 10–300 mM imidazole gradient. Protein
elution was monitored at 280 nm, the fractions were collect-
ed according to the chromatogram and dialysed twice
against 1.0 L of 50 mM potassium phosphate buffer pH 7.0
(12 h each, 48C) to remove imidazole and salts. Purified
protein aliquots were stored frozen at À808C.
Bioreduction Procedure for the Preparation of
Monodeuterated Samples
A solution of the substrate in i-PrOH (100 mL, 500 mM) was
added to potassium phosphate buffer solution (5.0 mL,
50 mM in D2O, pH 7.0) containing glucose (20 mmol),
NADH (75 mmol) and the required OYE1 mutant (1–3 mg).
The mixture was incubated for 24 h in an orbital shaker
(160 rpm, 308C). The solution was extracted with EtOAc
(3”5.0 mL), centrifuging after each extraction (3000”g,
1.5 min), and the combined organic solutions were dried
over anhydrous Na2SO4.
In situ Preparation of (R)- and (S)-b-Aryl-g-
lactams[19]
General procedure: The enantioselective reduction of the
suitable (Z)-methyl 3-cyano-3-arylacrylate was performed
with the required OYE1-GST fusion protein mutant as it
has been already described for biotransformations on the
50-mg scale. The reaction was monitored by GC/MS. When
the reduction was complete, NiCl2·6H2O (1 equiv.) and
NaBH4 (3 equiv.) were added cautiously to the reaction mix-
ture under vigorous stirring. After 1 h, the mixture was ex-
tracted with EtOAc (3”10 mL) and purified by column
chromatography (n-hexane with increasing amount of
EtOAc).
(R)-4-Phenylpyrrolidin-2-one [(R)-3a]: From the corre-
sponding
unsaturated
compound
(Z)-1a
(50.0 mg,
General Procedure for Enzyme-Mediated
Biotransformations of Substrates 1a–g (Screening)
0.27 mmol) by reaction with OYE1-W116L in buffer solu-
tion (pH 6.0, followed by in situ nitrile reduction, derivative
(R)-3a was obtained; yield: 26.5 mg (61%); [a]D: À34.1 (c
0.55, MeOH) {lit.[33] [a]D: À36 (c 0.5, MeOH) for (R)-3a
with ee=99.9%}.1H NMR (400 MHz, CDCl3):[33] d=7.40–
7.30 (m, 5H), 3.72 (m, 1H), 3.65 (m, 1H), 3.36 (dd, J=9.0,
7.1 Hz, 1H), 2.68 (dd, J=17.0, 8.9 Hz, 1H), 2.45 (dd, J=
17.0, 9.0 Hz, 1H); 13C NMR (101 MHz, CDCl3):[33] d=177.3,
142.1, 128.9, 127.2, 126.7, 49.3, 40.4, 37.7; GC-MS (EI): tr =
21.56 min, m/z (EI)=161 (M+, 50), 133 (7), 104 (100).
(R)-4-(4-Chlorophenyl)pyrrolidin-2-one [(R)-3c]: From
the corresponding unsaturated compound (Z)-1c (50.0 mg,
0.23 mmol) by reaction with OYE1-W116L in buffer solu-
tion (pH 6.0), followed by in situ nitrile reduction, derivative
(R)-3c was obtained; yield: 30.4 mg (68%); [a]D: À34.7 (c
0.78, EtOH) {lit.[33] [a]D: À38.0 (c 1. EtOH) for (R)-3c with
ee=99.9%}; 1H NMR (400 MHz, CDCl3):[33] d=7.31 (m,
2H), 7.18 (m, 2H), 3.77 (m, 1H), 3.66 (m, 1H), 3.37 (dd, J=
8.5, 7.5 Hz, 1H), 2.72 (dd, J=17.0, 8.9 Hz, 1H), 2.45 (dd, J=
17.0, 8.0 Hz, 1H); 13C NMR (101 MHz, CDCl3):[33] d=177.1,
140.7, 133.0, 129.0, 128.1, 127.1, 49.3, 37.7; GC-MS (EI): tr =
24.34 min, m/z (EI)=195 (M+, 46), 138 (100), 103 (23).
A solution of the substrate in DMSO (10 mL, 500 mM) was
added to a potassium phosphate buffer solution (1.0 mL,
50 mM, pH 7.0) containing glucose (20 mmol), NADP+
(0.1 mmol), GDH (4 U) and the required purified or cell-
free extract OYE (80–120 mg). The mixture was incubated
for 24 h in an orbital shaker (160 rpm, 308C). The solution
was extracted with EtOAc (2”250 mL), centrifuging after
each extraction (15000”g, 1.5 min), and the combined or-
ganic solutions were dried over anhydrous Na2SO4. Two rep-
licates were performed for each biotransformation: no sig-
nificant differences (less than 5%) were observed for con-
version and enantiomeric excess values.
General Procedure for OYE-W116X Cell-Free
Extracts-Mediated Biotransformations
For substrates (Z)-1a–g a similar protocol was followed on
a larger scale (50 mg), employing the appropriate OYE1-
W116X mutant that provided the best conversion and/or ee,
in order to isolate and characterise the corresponding re-
1858
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FULL PAPERS
Opposite Enantioselectivity in the Bioreduction of (Z)-b-Aryl-b-cyanoacrylates
Catal. Sci. Technol. 2013, 3, 1136–1146; e) E. Brenna,
Supporting Information
M. Crotti, F. G. Gatti, A. Manfredi, D. Monti, F. Par-
meggiani, A. Pugliese, D. Zampieri, J. Mol. Catal. B:
Enzym. 2014, 101, 67–72.
Tables S1–S4, characterization data of cyano esters 1a–g and
reduced products 2a–g, analytical methods for the determi-
nation of conversion and enantiomeric excess, crystallo-
graphic data, and copies of the NMR spectra are given in
the Supporting Information.
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Adv. Synth. Catal. 2015, 357, 1849 – 1860