Biocatalytic Michael-type additions of acetaldehyde to nitroolefins with the proline-based enzyme 4-oxalocrotonate tautomerase yielding enantioenriched γ-nitroaldehydes

Article abstract of DOI:10.1002/chem.201302351

Call me Michaelase: The enzyme 4-oxalocrotonate tautomerase (4-OT) promiscuously catalyzes the Michael-type addition of acetaldehyde to a collection of aromatic and aliphatic nitroolefins with high stereoselectivity producing precursors of γ-aminobutyric

Full text of DOI:10.1002/chem.201302351

COMMUNICATION
DOI: 10.1002/chem.201302351
Biocatalytic Michael-Type Additions of Acetaldehyde to Nitroolefins with
the Proline-Based Enzyme 4-Oxalocrotonate Tautomerase Yielding
Enantioenriched g-Nitroaldehydes
Edzard M. Geertsema, Yufeng Miao, Pieter G. Tepper, Pim de Haan,
Ellen Zandvoort, and Gerrit J. Poelarends*[a]
g-Nitroaldehydes are versatile and practical precursors for
chiral g-aminobutyric acids (GABAs). In particular, promi-
nent GABA analogues, such as marketed pharmaceuticals
phenibut[1] (GABAB receptor agonist, anxiolytic), pregaba-
lin[2] (anticonvulsant), baclofen[3] (GABAB receptor agonist,
anti-alcoholism), and rolipram[4] (type IV phosphodiesterase
inhibitor, antidepressant) can be readily obtained from di-
verse chiral g-nitroaldehydes by two, well-precedented,
chemical synthesis steps.[5] One of the most important strat-
egies to construct g-nitroaldehydes is the Michael-type addi-
tion of unmodified aldehydes to nitroolefins.[6] Following
this approach, construction of the appropriate g-nitroalde-
hyde precursors for above-mentioned, pharmaceutically
active GABA analogues would require the Michael-type ad-
dition of acetaldehyde to various nitroolefin acceptors
(Scheme 1). The Michael-type addition of unmodified alde-
hydes to nitroolefins has recently become viable by the de-
velopment of proline- and peptide-based organocatalysts.[7,8]
However, examples including acetaldehyde as the donor are
scarce since acetaldehyde is a relatively reactive and difficult
to tame chemical and 10–20 mol% of organocatalyst is typi-
cally applied.[9] Alternative procedures for the asymmetric
synthesis of g-nitroaldehydes from acetaldehyde and nitroo-
lefins are therefore of great interest. Although a few exam-
ples of enzyme-catalyzed carbon–carbon bond-forming Mi-
chael-type additions are known, these do not involve acetal-
dehyde as the donor and mainly exhibit low stereoselectivi-
ties.[10]
We herein report that the enzyme 4-oxalocrotonate tauto-
merase (4-OT),[11] which carries a nucleophilic amino-termi-
nal proline residue (Pro1), promiscuously catalyzes the
asymmetric Michael-type addition of acetaldehyde to vari-
ous aromatic and aliphatic nitroolefins yielding chiral g-ni-
troaldehydes (Scheme 1) with high stereoselectivities. In
combination with our previously described 4-OT-catalyzed
addition of linear aldehydes (acetaldehyde up to octanal) to
trans-nitrostyrene,[12,13] this is the first example of enzyme-
catalyzed carbon–carbon bond-forming Michael-type addi-
tions that includes a range of linear aldehyde donors and
a series of aromatic and aliphatic nitroolefin acceptors.[14]
Furthermore, we found that catalytic activity of 4-OT is pre-
served in aqueous solvent systems containing up to 50% (v/
v) of DMSO as co-solvent. The ꢀMichaelaseꢁ activity of 4-
OT and preservation of this activity in the presence of 50%
(v/v) of an organic co-solvent are two important steps
toward our aim of developing versatile and robust proline-
based biocatalysts for carbon–carbon bond-forming Mi-
chael-type addition reactions.
The 4-OT-catalyzed Michael-type addition with donor
acetaldehyde 1 was explored with a series of nitroolefin ac-
ceptors (2a–f) in separate analytical scale experiments
(Scheme 1). Nitroolefins 2a–f (0.7–3.0 mm)[15] were incubat-
ed with acetaldehyde 1 (25–150 mm)[16] and 4-OT (32–
150 mm)[16] in NaH2PO4 buffer (20 mm, pH 5.5) and a co-sol-
vent. A co-solvent was required to achieve sufficient solubil-
ity of nitroolefins 2a–f in an aqueous solvent system. Apart
from enhancing solubility of 2a–f, the co-solvent should be
water-miscible, should not impede catalytic activity of 4-OT,
and should not chemically react with any of the substrates
(1 and 2a–f). Screening the activity of 4-OT in 20 mm
NaH2PO4 buffer mixed with various amounts (5.0 to 72.5%
v/v) of EtOH, DMSO, dioxane, THF, MeCN, and DMF re-
vealed that EtOH (up to 10% v/v) and DMSO (up to 50%
Scheme 1. Michael-type addition of acetaldehyde 1 to nitroolefins 2a–g.
*=chiral center.
[a] Dr. E. M. Geertsema,+ Y. Miao,+ P. G. Tepper, P. de Haan,
Dr. E. Zandvoort, Prof. Dr. G. J. Poelarends
Department of Pharmaceutical Biology
Groningen Research Institute of Pharmacy, University of Groningen
Antonius Deusinglaan 1, 9713 AV Groningen (The Netherlands)
E-mail: g.j.poelarends@rug.nl
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201302351.
Chem. Eur. J. 2013, 19, 14407 – 14410
ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
14407

v/v) are suitable co-solvents that meet all above-mentioned
criteria.[17]
solvent system (Table 1) and reactions were followed by UV
spectroscopy. After disappearance of the absorbance at lmax
of 2a–f, standard workup and purification procedures were
carried out which afforded g-nitroaldehydes 3a–f as con-
firmed by 1H NMR spectroscopy. Yields between 49 and
74% were achieved for 3a–e, whereas 3 f was obtained in
26% yield. Products 3a–c,e are useful precursors for impor-
tant GABA analogues since 3a–b can be converted into ro-
lipram,[5c,20] 3c into pregabalin,[5b] and 3e into baclofen[5a,b,d–f]
in two or three chemical synthesis steps, respectively. Fur-
thermore, obtaining products 3a–f shows that 4-OT accepts
aromatic as well as aliphatic nitroolefins as substrates for
Michael-type addition reactions. The enantiomeric excesses
(ee) of 3a–f were determined by GC or HPLC analysis with
chiral stationary phases. Excellent ee values between 95 and
98% were established for 3a, 3c, and 3d meaning that the
enzyme 4-OT is highly stereoselective during the catalytic
process. Obtained ee values of 3b,e,f range from 69 to 81%.
The absolute configurations of the major enantiomers of
3a–f, respectively, were determined by HPLC and/or optical
rotation.[17] Comparison with literature data revealed that
The analytical scale reactions were followed by monitor-
ing the change of absorbance at lmax of 2a–f by UV-spec-
troscopy.[18] During all reactions, decrease of the absorbance
at lmax,2a–f was observed in course of time (20–120 min),[17]
which indicated almost complete depletion of nitroolefins
2a–f (see Figures S1–S6 in the Supporting Information).
Identical experiments with 4-OT and 2a–f, respectively, but
in the absence of acetaldehyde 1, showed negligible decreas-
es of absorbances at lmax,2a–f (except for compound 2c, vide
infra), which demonstrated that acetaldehyde 1 is involved
in the 4-OT-catalyzed conversions of 2a–f (see Figures S1–
S6 in the Supporting Information). These experiments also
confirmed that EtOH and DMSO solely act as co-solvents
and not as reagents. Three types of additional control ex-
periments were executed to confirm that the enzyme 4-OT
and its catalytic Pro1 residue are responsible for conversions
of 2a–f and 1 (see Figure S1–S6 in the Supporting Informa-
tion). First, incubation of 1 with nitroolefins 2a–f, respec-
tively, but in the absence of 4-OT did not result in any sig-
nificant decreases of absorban-
ces at lmax,2a–f, which indicated
that 4-OT is responsible for the
Table 1. Preparative scale 4-OT-catalyzed Michael-type addition reactions of acetaldehyde 1 (50–150 mm) to
nitroolefins 2a–g (2–5 mm) in NaH2PO4 buffer (pH 5.5) yielding chiral g-nitroaldehydes 3a–g.
catalytic activities. Second, ex-
periments with 1 and 2a–f, re-
spectively, in the presence of
the P1A mutant of 4-OT
showed no decreases of absor-
bances at lmax,2a–f, which implied
that the Pro1 residue is crucial
for the catalytic activities of 4-
OT. Third, 1 and 2a were incu-
bated with synthetic 4-OT[19]
and the rate of decrease of ab-
sorbance at lmax,2a was identical
to that observed with recombi-
nant 4-OT. Although highly pu-
rified recombinant 4-OT was
used in the analytical assays,
this finding eliminated the pos-
sibility that any contaminating
proteins from the expression
strain may be responsible for
catalysis.
Entry Nitro-
olefin
Product (g-nitroaldehyde)
t
Yield[a]
[%]
ee[b]
[%]
Abs.
4-OT
Co-solvent
[h]
conf.[c]
[mol%][d] (v/v)
DMSO
40%
1
2
2a
2b
3a 2.5
3b 2.0
64
49
96
74
S
S
3.7
1.8
EtOH 10%
DMSO 5%
3
4
2c
2d
3c 0.4
3d 2.0
74
64
98
95
R
S
5.3
3.0
DMSO
40%
DMSO
45%
5
2e
3e 2.5
51
69
S
2.8
Preparative-scale experiments
were performed to allow unam-
biguous product identification
by 1H NMR spectroscopy and
thus to ascertain that 4-OT-cat-
alyzed conversions of 1 with
2a–f give Michael-type addition
adducts 3a–f (Table 1). Nitroo-
lefin (2a–f: 2–5 mm),[17] acetal-
dehyde (1, 50–150 mm),[16] and
4-OT (1.5–5.3 mol%)[16] were
DMSO
40%
6
2 f
2g
3 f 2.5
26
70
81
81
S
S
1.5
1.4
7[e]
3g 2.0
EtOH 10%
[a] Isolated yields. [b] Determined by GC or HPLC analysis with chiral stationary phase.[17] [c] Determined by
HPLC analysis with chiral stationary phase and/or optical rotation.[17] [d] Compared to nitroolefin. [e] Previous
incubated in the appropriate result.[13]
14408
www.chemeurj.org
ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 14407 – 14410

Biocatalytic Michael-Type Additions of Acetaldehyde to Nitroolefins
COMMUNICATION
the chiral centers of the major enantiomers of 3a–g, respec-
tively, all have identical geometry as depicted in Table 1.
This means that the stereocontrol of 4-OT in the catalytic
process of acetaldehyde addition to nitroolefins 3a–g is con-
sistent regardless of the R-substituent (Scheme 1) at the ni-
troolefin. The major enantiomers of 3a,b,d–g have an S con-
figuration, whereas 3c has an R configuration. The deviant
configuration of 3c is due to different prioritization of the
substituents at the chiral center relative to 3a,b,d–g. The
amounts of applied 4-OT (1.5–3.7 mol%) were adjusted
such that conversions of 2a,b,d–f were all completed within
2.5 h. Conversion of aliphatic substrate 2c was effected
within 25 min due to the presence of 5.3 mol% of 4-OT.
This amount of 4-OT was required to outcompete nonenzy-
matic water addition to 2c (giving racemic product 4-
methyl-1-nitropentan-2-ol). Indeed, the amount of water ad-
dition product, 4-methyl-1-nitropentan-2-ol, went down
from 4 to <2 mol% (compared to 3c) when 5.3 mol% of 4-
OT was used instead of 2.6 mol% as determined by GC
plied mol% of catalyst and reaction time is to assess effi-
ciency by the weight amount of product (in terms of milli-
grams) that is produced per weight amount of used catalyst
per unit of reaction time (mgproduct mgcatalystÀ1 hÀ1). Applying
the latter definition, the 4-OT-based biocatalytic methodolo-
gy and the most potent organocatalytic[9e] methodology, to
the best of our knowledge, are equally efficient for the Mi-
chael-type addition of acetaldehyde (1) to nitrostyrene
(2g).[9,17,21] This observation in combination with the broad
substrate scope of this new enzyme-based methodology to
prepare precursors of GABA analogues with high stereose-
lectivities inspired us to currently run protein engineering
studies with the aim to enhance the unnatural ꢀMichaelaseꢁ
activities of 4-OT. If successful, newly designed enzyme var-
iants can also be tested in a whole cell system based on re-
combinantly expressed 4-OT, which appears to be an effec-
tive biocatalyst for the asymmetric Michael-type addition of
acetaldehyde to a few selected aromatic b-nitrostyrenes.[22]
1
analysis and H NMR spectroscopy. In contrast to 2c, non-
enzymatic water addition to substrates 2a,b,d–f was not ob-
served under the conditions used.
Acknowledgements
This research was financially supported by the European Research Coun-
cil under the European Communityꢁs Seventh Framework Programme
(FP7/2007–2013)/ERC Grant agreement no8 242293 (to G.J.P.). We thank
M.P. de Vries (University of Groningen) for his expert assistance in ac-
All preparative-scale experiments of the 4-OT-catalyzed
acetaldehyde addition to nitroolefins 2a–f were repeated
under identical conditions but in the absence of 4-OT. In all
cases no g-nitroaldehyde product was observed (as con-
´
quiring exact MS data and W. Szymanski for his aid in obtaining optical
1
rotation data. We thank T. Chan, M. Herbrink, W. Huizinga, R. Kempers,
and A. Sulaiman for their contributions to this work. Chiral Technologies
Europe (Illkirch Cedex, France) is gratefully acknowledged for providing
a chiralpak IB column on loan for HPLC analysis.
firmed by H NMR spectroscopy), which demonstrated that
formation of 3a–f is solely the result of 4-OT-catalyzed Mi-
chael-type additions and not of nonenzymatic addition of
1 to 2a–f. In the case of 2c, nonenzymatic water addition re-
sulted in the formation of 4-methyl-1-nitropentan-2-ol as
1
Keywords: aminobutyric acid · biocatalysis · C–C bond
formation · enzymes · Michael-type addition
confirmed by H NMR spectroscopy and GC analysis.
Summarizing, this work presents a biocatalytic methodol-
ogy for asymmetric Michael-type additions of acetaldehyde
to a collection of aliphatic and aromatic nitroolefin accept-
ors. The Michael-type additions are promiscuously catalyzed
by the enzyme 4-OT and yield chiral g-nitroaldehydes that
are valuable precursors for GABA analogues. Yields up to
74% and ee values up to 98% were established, which dem-
onstrated that 4-OT exerts high stereoselectivity during the
catalytic process. Control experiments revealed that the ꢀMi-
chaelaseꢁ activity takes place in the active site of 4-OT. The
catalytic activity of 4-OT is preserved in aqueous solvent
systems containing up to 50% DMSO (v/v). This finding im-
plies that the substrate scope of our biocatalytic methodolo-
gy is not limited to water-soluble chemicals and allows uti-
lization of poorly water-soluble nitroolefins as substrates.
The employed amounts of catalyst of 1.4–5.3 mol% in our
methodology for Michael-type addition of acetaldehyde to
nitroolefins are lower, and reactions times of ꢀ2.5 h are
generally shorter, than in the scarce conventional organoca-
talytic methodologies for identical type of reactions.[9,17] De-
spite a relatively low molecular weight considering enzymes,
the molecular weight of 4-OT is still considerably higher
than those of organocatalysts[9] that are able to catalyze
acetaldehyde addition to nitroolefins. Bearing this in mind,
an alternative for defining efficiency on the basis of the ap-
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type additions is reminiscent of proline-based organocatalysis and
involves the formation of a nucleophilic enamine intermediate of
the Pro1 residue of 4-OT with acetaldehyde (see Scheme S1 in the
Supporting Information). This intermediate reacts with the double
bond of the nitroolefin acceptor creating a new carbon–carbon bond
after which the product is released from 4-OTꢁs Pro1 by hydrolysis.
[15] Concentrations of 2a–f were adjusted on the basis of their specific
emax values and solubility properties. See the Supporting Information
for details.
[16] Concentration of acetaldehyde and 4-OT was adjusted on basis of
concentration of nitroolefin (2a–f).
[17] See the Supporting Information for details.
[18] 2a–f have different lmax values. See the Supporting Information for
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23.2 mg of organocatalyst catalyzes the Michael-type addition of
acetaldehyde (1) to nitrostyrene (2g) to give 54.2 mg of product 3g
in 3 h of reaction time. This comes down to the production of
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d) G. A. Strohmeier, T. Sovic, G. Steinkellner, F. S. Hartner, A. An-
mgcatalystÀ1 hÀ1). In our methodology (Table 1 and reference [13])
´
3g
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Received: June 19, 2013
Published online: September 24, 2013
14410
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Chem. Eur. J. 2013, 19, 14407 – 14410