Stereo-controlled asymmetric bioreduction of α,β-dehydroamino acid derivatives

Article abstract of DOI:10.1002/adsc.201100042

α,β-Dehydroamino acid derivatives proved to be a novel substrate class for ene-reductases from the 'old yellow enzyme' (OYE) family. Whereas N-acylamino substituents were tolerated in the α-position, β-analogues were generally unreactive. For aspartic aci

Full text of DOI:10.1002/adsc.201100042

UPDATES
DOI: 10.1002/adsc.201100042
Stereo-Controlled Asymmetric Bioreduction of
a,b-Dehydroamino Acid Derivatives
Clemens Stueckler,a Christoph K. Winkler,a Mꢀlanie Hall,a Bernhard Hauer,b,c
Melanie Bonnekessel,b Klaus Zangger,a and Kurt Fabera,*
a
Department of Chemistry, Organic & Bioorganic Chemistry, University of Graz, Heinrichstrasse 28, A-8010 Graz, Austria
Fax : (+43)-316-380-9840; e-mail: Kurt.Faber@Uni-Graz.at
BASF SE, GVF/B-A030, 67056 Ludwigshafen, Germany
Current address: Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.
b
c
Received: January 17, 2011; Published online: April 28, 2011
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/adsc.201100042.
Abstract: a,b-Dehydroamino acid derivatives
proved to be a novel substrate class for ene-reduc-
tases from the ꢁold yellow enzymeꢂ (OYE) family.
Whereas N-acylamino substituents were tolerated
in the a-position, b-analogues were generally un-
reactive. For aspartic acid derivatives, the stereo-
chemical outcome of the bioreduction using OYE3
could be controlled by variation of the N-acyl pro-
tective group to furnish the corresponding (S)- or
(R)-amino acid derivatives. This switch of stereo-
preference was explained by a change in the sub-
strate binding, by exchange of the activating ester
group, which was proven by 2H-labelling experi-
ments.
Inspired by our recent success using a-alkoxy-sub-
stituted enones as substrates,[8] we aimed to apply this
method to the production of non-racemic amino acids
via the bioreduction of a,b-dehydroamino acid deriva-
tives. While metal-based catalysts[9] and artificial met-
alloenzymes[10] are known for the stereoselective hy-
drogenation of N-protected dehydroamino acids, a
biocatalytic equivalent has not been reported so far.
The reduction of amino acid precursors, such as b-ni-
troacrylates by OYE1 from Saccharomyces pastoria-
nus (formerly Saccharomyces carlsbergensis) followed
by chemical conversion to b-amino acids[11] and the
reduction of b-cyanocinnamic acid derivatives leading
to g-amino acids using whole cells of anaerobic bacte-
ria was reported recently.[12]
Here we report the scope and limitations of the re-
duction of a,b-dehydroamino acid derivatives as a
new substrate class using ene-reductases OYE1–3
from Saccharomyces sp.,[13] OPR1 and OPR3 from
Lycopersicon esculentum,[14] YqjM from Bacillus sub-
tilis[15] and NCR-reductase from Zymomonas mobi-
lis.[16]
Keywords: asymmetric bioreduction; dehydroamino
acids; ene-reductase; old yellow enzyme; stereocon-
trol
The asymmetric reduction of C=C bonds creates (up
In order to avoid extractive and analytical problems
to) two chiral carbon centres and is thus one of the associated with highly polar substrates bearing free
most widely employed strategies for the production of amino and/or carboxy groups, we chose N-acyl-pro-
chiral materials.[1,2] The biocatalytic variant, which is tected methyl esters as substrates (Scheme 1). Since
applicable to activated alkenes bearing an electron- the (E/Z)-configuration of alkenes may have dramatic
withdrawing substituent, is catalysed by ene-reductas- consequences on the stereochemical course of the
es,[3,4] which are members of the ꢁold yellow enzymeꢂ bioreduction using OYEs,[7b,d,e,16] special care was
(OYE) family.[5] During the past few years, increasing taken to use well-defined stereochemically pure (Z)-
attention has been devoted to these flavo-proteins in alkenes as substrates. The most simple dehydroamino
view of their substrate scope, encompassing a,b-unsa- acid derivative, methyl 2-acetamidoacrylate (1a),
turated carbonyl compounds (such as enals and which was selected as test candidate, was reduced at
enones), as well as carboxylic acids and derivatives poor to moderate rates by five of the seven ene-re-
thereof (such as esters, cyclic imides, nitriles, lactones) ductases investigated (OYE1–3, YqjM, OPR1), albeit
and nitroalkenes.[6,7]
with good stereoselectivity (Table 1). Under standard
conditions, YqjM gave best conversion (41%) with ex-
Adv. Synth. Catal. 2011, 353, 1169 – 1173
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Clemens Stueckler et al.
UPDATES
Scheme 1. Asymmetric bioreduction of N-acetylamino acryl-
ic esters 1a–3a.
Scheme 2. Asymmetric bioreduction of N-acylamino fumaric
diesters 4a–9a.
cellent optical purity (ee 97%). All active enzymes
yielded the (S)-enantiomer of the alanine derivative
1b with an ee of 85–97%.
Much to our disappointment, the a-amino acid de- [(Z)-6a] and butanoyl [(Z)-7a] gave increased activi-
rivative 2a bearing an additional methyl group at Cb ties again for OYE3, YqjM and both OPR enzymes,
and the b-dehydroamino acid derivative 3a showed while the (S)-stereopreference remained intact for all
no conversion with all enzymes tested. In order to en- enzymes except for OYE3, which switched to (R)
hance the reactivity of the substrate, we chose a-de- with an ee of 68%. Surprisingly, NCR (previously in-
hydroamino acid derivatives bearing an additional active on monoester and diester derivatives bearing
(activating) ester group (Scheme 2).
small formyl and acetyl protecting groups) displayed
The additional activating effect of a second ester reasonable activity and high stereoselectivity with
group in the substrate proved to be dramatic: in con- both (Z)-6a and (Z)-7a (ee 90% and 95%, respective-
trast to the N-acetyl-protected monoester 1a, diester ly) For the N-benzoyl derivative 8a, the stereoselec-
(Z)-4a bearing a small N-formyl group was converted tivity was perfect with OYEs 1–3 and YqjM, and (S)-
quantitatively by OYE1 and OYE3, and perfect ste- 8b was formed in >99% ee in acceptable yields rang-
reoselectivity for the (S)-enantiomer was achieved ing from 12% to 47%. Interestingly, an analogous
with OYE1, OYE2, YqjM and both OPR enzymes. In (R)-switch was detected with the N-phenylacetyl sub-
order to investigate the steric constraints for substrate strate 9a with OYE3 (ee 92%), while OPR1 yielded
recognition, a variation of the N-protective group was the (S)-enantiomer in >99% ee. For the remaining
introduced. Increasing the N-protecting group from enzymes, substrate 9a was obviously too bulky to be
formyl to acetyl had again drastic effects on both con- accepted.
version and stereoselectivity. While OYE1, OYE2 and
Since both large N-benzoyl (Z)-8a and N-phenyl-
OYE3 were highly active with the N-formyl protected acetyl (Z)-9a substrates gave opposite enantiomeric
substrate 4a, OYE1 and OYE2 displayed no activity products [(S)-8b, (R)-9b] with OYE3, the switch in
with the N-acetyl equivalent 5a and OYE3 showed stereopreference could not be explained by a ꢁright/
strongly reduced levels of stereoselectivity (ee re- leftꢂ-flipped orientation of the substrates induced by
duced from 88% to 23%) and conversion (reduced an increase in the size of the N-acyl protective group,
from >99% to 36%). YqjM activity was not affected. but rather in a reversed ꢁbottom/topꢂ orientation by a
Further extension of the amide-chain to propionyl switch of the activating ester group (cf. Scheme 3).
Table 1. Conversion and enantiomeric excess of bioreduction products 1b–9b.[a]
Substrate Product
OYE1
OYE2
OYE3
YqjM
OPR1
OPR3
NCR
c.
ee
c.
ee
c.
ee
c.
ee
c.
ee
c.
ee
c.
ee
[%] [%]
[%] [%]
[%] [%]
[%] [%]
[%] [%]
[%] [%]
[%] [%]
1a
1b
2b
3b
4b
5b
6b
7b
8b
9b
30 (S) 95
6
(S) 85
n.c.
n.c.
15 (S) 95
41
(S) 97
n.c.
n.c.
16 (S) 91
n.c.
<1 n.d.
n.c.
<5 n.d.
(Z)-2a
(Z)-3a
(Z)-4a
(Z)-5a
(Z)-6a
(Z)-7a
(Z)-8a
(Z)-9a
n.c.
n.c.
n.c.
nc
n.c.
n.c.
n.c.
n.c.
>99 (S)>99 89 (S)>99 >99 (S) 88
<5 n.d. n.c. n.d. 36 (S) 23
n.d.
29 (S) 82
26
26
(S)>99 34 (S)>99 6
(S)>99 19 (S)>99 <5 n.d.
(S)>99 <1 n.d.
<5 n.d.
14 (S) 90
22 (S) 95
<5 n.d.
7
n.d.
6
>99 (R) 61 >99 (S)>99 58 (S)>99 <5 n.d.
24
34
(S) 60
98
(R) 68 95
(S)>99 12
(S)>99 66 (S) 89
(S)>99 <5 n.d.
45 (S) 95
<5 n.d.
(S)>99 35 (S)>99 47
<5 n.d. 41
<5 n.d.
(R) 92 <5 n.d.
16 (S)>99 n.c. n.d.
n.c. n.d.
[a]
Standard conditions: ene-reductase (75–125 mgmLÀ1 enzyme), substrate 1a–9a (10 mm), NADH (15 mM), Tris-HCl-buffer
50 mM, pH 7.5, 308C, 24 h; c.=conversion; n.d.=not determined; n.c.=no conversion.
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Adv. Synth. Catal. 2011, 353, 1169 – 1173

Stereo-Controlled Asymmetric Bioreduction of a,b-Dehydroamino Acid Derivatives
Scheme 3. Stereo- (ꢁright/leftꢂ) and regio-complementary (ꢁbottom/topꢂ) flip of substrate and switch of activating group in
the bioreduction of N-acylamino fumarate diesters using OYE3; residues His-191 and Asn-194 are involved in substrate
binding.[13c]
In order to prove this hypothesis, the position of in>99% and 92% ee, respectively. This switch of the
hydride attack (derived from NADH) was determined substrate activating group opens new perspectives for
2
by H-labelling experiments using substrates 4a, 6a, the asymmetric synthesis of b-amino acids.
8a and 9a in D2O as solvent.[17] It is well established
The difference in reactivity within the series of sub-
that the attack of the hydride derived from the flavin strates 4a–7a is surprising and cannot be explained by
cofactor (which is ultimately provided by NADH) differences in the electron-withdrawing capacity of
always occurs on Cb, while concomitant proton-dona- the N-acyl substituent. Although OYE3 shares high
tion (derived from the solvent) occurs on Ca relative sequence identity with OYE1 and OYE2 (80.2% and
to the activating group.[3,18] NMR analysis of the prod- 81.5%, respectively) and identical substrate-binding
ucts 4b, 6b, 8b and 9b thus obtained with respect to residues (H191 and N194), phenylalanine F296 (in
the position of H/D revealed that hydride incorpora- OYE1 and OYE2) is replaced by serine S297 in
tion on 4a, 6a, 8a and 9a [yielding (S)-4b, (S)-8b and OYE3, which enlarges the binding pocket and thus
(R)-6b, (R)-9b] occurred at different carbon atoms allows different substrate conformations.[19] In addi-
(C-2/C-3), thus proving that the substrates were posi- tion, subtle changes in the binding pocket of OPR1
tioned in a flipped ꢁbottom/topꢂ orientation, which and OPR3 have already been shown to dramatically
was enabled by an exchange of the activating carbox- influence substrate binding and thus enzymatic activi-
ylic ester groups (Scheme 3). This picture was further ty.[6b]
verified by the observation that the low enantiomeric
Optically enriched N-acyl derivatives of alanine
excess of 6b (61% ee) perfectly matched to a deuteri- and aspartic acid esters were obtained by biocatalytic
um incorporation on C-2/C-3 of 18.5/81.5 which corre- reduction of their a,b-dehydroamino acid precursors.
sponds to an ee of 63%. This demonstrates that the A switch of stereopreference in the reduction of the
incomplete chiral recognition of (Z)-6a by OYE3 was aspartic acid precursor could be induced by variation
due to weak regio- rather than stereoselectivity. On of the size of the N-acyl protective group leading to
substrates bearing aromatic side chains, this effect the (S)- and (R)-enantiomer in up to >99% and 92%
was remarkably sensitive because the introduction of ee, respectively. The latter could be explained by an
a single methylene unit (N-benzoyl versus N-phenyl- exchange of the activating carboxylic acid ester group
ACHTUNGTRENNUNGacetyl) was sufficient to furnish (S)-8b and (R)-9b responsible for substrate binding.
Adv. Synth. Catal. 2011, 353, 1169 – 1173
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Clemens Stueckler et al.
UPDATES
References
Experimental Section
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TLC plates were run on silica gel Merck 60 (F254) and com-
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Source of Enzymes
12-Oxophytodienoate reductase isoenzymes OPR1 and
OPR3 from Lycopersicon esculentum and the OYE homo-
logue YqjM from Bacillus subtilis were overexpressed and
purified as reported.[6a,14,15] The cloning, purification and
characterisation of OYE isoenzymes from yeast (OYE1
from Saccharomyces pastorianus, OYE2 and OYE3 from
Saccharomyces cerevisiae) and nicotinamide-dependent cy-
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General Procedure for the Enzymatic Bioreduction
of 1a–9a
An aliquot of enzyme (OYE1–3, OPR1, OPR3, YqjM, and
NCR, protein concentration in biotransformations 75–
125 mgmLÀ1) was added to a Tris-HCl buffer solution
(0.8 mL, 50 mM, pH 7.5) containing the substrate (10 mM)
and the cofactor NADH (15 mM). Substrates 8a and 9a
were solubilised by addition of t-BuOMe (v:v 20%).[7c] The
mixture was shaken at 308C and 120 rpm. After 24 h prod-
ucts were extracted with EtOAc (2ꢄ0.5 mL). The combined
organic phases were dried over Na2SO4 and analysed on
achiral GC to determine the conversion and on chiral GC to
determine the enantiomeric excess.
Supporting Information
Syntheses of substrates 2a–9a, syntheses of racemic refer-
ence materials rac-1b, rac-4b–9b, syntheses of chiral refer-
ence materials for (S)-1b, (S)-4b–9b, analytical procedures
for the determination of conversion, the determination of
enantiomeric excess and absolute configuration of products,
and NMR spectra are given in the Supporting Information.
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[19] F296 and S297 are part of the binding pocket directly
above the si-face of the flavin and are thus very close
to the substrate (<4 ꢇ), as determined in the crystal
structure of OYE1 (PDB DOI:10.2210/pdb1oyb/pdb)
taking the docked inhibitor p-hydroxybenzaldehyde as
substrate mimic by using PyMol (The PyMOL Molecu-
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