Enantioselective Enzymatic Reduction of Acrylic Acids

Article abstract of DOI:10.1021/acs.orglett.0c02959

An ene-reductase (ERED 36) with broad substrate specificity was identified, and optimization studies led to the development of an enzymatic protocol for the reduction of α,β-unsaturated acids under mild, aqueous conditions. The substrate scope includes aromatic- A nd aliphatic-substituted acrylic acids, as well as cyclic α,β-substituted acrylic acids, yielding chiral α-substituted acids with exquisite levels of enantioselectivity (>99% ee).

Full text of DOI:10.1021/acs.orglett.0c02959

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Letter
Enantioselective Enzymatic Reduction of Acrylic Acids
Chihui An,* Megan H. Shaw,* Annika Tharp, Deeptak Verma, Hongming Li, Heather Wang,
and Xiao Wang
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ABSTRACT: An ene-reductase (ERED 36) with broad substrate
specificity was identified, and optimization studies led to the
development of an enzymatic protocol for the reduction of α,β-
unsaturated acids under mild, aqueous conditions. The substrate
scope includes aromatic- and aliphatic-substituted acrylic acids, as
well as cyclic α,β-substituted acrylic acids, yielding chiral α-
substituted acids with exquisite levels of enantioselectivity (>99%
ee).
hiral α-substituted carboxylic acids are found in a wide
array of biologically active molecules, including non-
simple unsaturated substrates.11−14 However, the use of
expensive transition metals, safety hazards associated with
the use of hydrogen gas and chemoselectivity challenges,
render this approach nonideal. Over the past decade,
biocatalysis has come to the forefront of synthetic chemistry,
enabling mild, green, and highly enantio- and chemoselective
syntheses of diverse compounds. Herein, we outline the
development of a green synthetic approach to deliver
enantioenriched carboxylic acids via enzymatic reduction of
readily accessible acrylic acid derivatives.
C
steroidal anti-inflammatory compounds such as ibuprofen,
naproxen, and indan-1-carboxylic acids (Figure 1).1−4 Recent
Ene-reductases (EREDs) are a class of enzymes that catalyze
the reduction of activated alkenes such as α,β-unsaturated
aldehydes, ketones, esters, and nitroalkenes, often with
exquisite stereoselectivity (Scheme 1).15−17 Although the
synthetic applicability of nicotinamide-dependent flavoproteins
from the “Old Yellow Enzyme” (OYE) family is well-
established, the use of these enzymes for reduction of less
activated CC bonds (e.g., α,β-unsaturated carboxylic acids)
remains underexplored. Mechanistically, it is understood that
the activated alkene binds in the active site of the enzyme and
undergoes a stereoselective anti-reduction via hydride con-
jugate addition from a flavin mononucleotide (FMNH2)
cofactor, followed by protonation from a tyrosine residue.18,19
Given that carboxylic acids are poor Michael acceptors, it is
perhaps unsurprising that EREDs possess limited activity on
unactivated acids. Indeed, previous literature reports are
restricted to enzymatic reduction of activated bisacids,20−22
employ EREDs that require anaerobic conditions,23−26 or
display narrow scope and low activity (typically on 2-
Figure 1. Chiral substituted carboxylic acids and derivatives.
reports outlining the discovery of 2-benzylpropanoic acid
derivatives with important biological properties highlight the
importance of developing enantioselective methods that
provide synthetic chemists with simple and efficient
approaches for accessing this structural motif.5,6
Despite the importance of these scaffolds, many current
methods fail to uphold green chemistry principles: low step
count, sustainable and environmentally benign conditions, and
minimal associated safety risks.7 Large-scale syntheses often
rely on chiral auxiliaries8 or kinetic resolution,9,10 approaches
that require additional steps or limit the theoretical yield to
50%. Asymmetric hydrogenation protocols, with finely tuned
catalyst systems, can be employed to provide chiral acids in
excellent yields and enantioselectivities from comparably
Received: September 4, 2020
https://dx.doi.org/10.1021/acs.orglett.0c02959
© XXXX American Chemical Society
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a
Scheme 1. Ene-Reductase-Mediated Synthesis of Chiral
Acids
Table 1. pH, Temperature, and Cosolvent Screening
b
c
entry
pH
temperature
cosolvent
yield
1
2
3
4
5
6
7
8
9
6.0
6.5
7.0
7.5
6.5
6.5
7.0
7.0
7.0
7.0
7.0
25 °C
25 °C
25 °C
25 °C
30 °C
35 °C
30 °C
30 °C
30 °C
30 °C
30 °C
none
none
none
none
none
none
DMSO
DMF
IPA
MeOH
MeCN
30%
97%
96%
66%
98%
43%
94%
6%
48%
88%
3%
10
11
a
Reaction conditions: 2-benzylacrylic acid 1a (0.1 M), sodium
phosphite dibasic pentahydrate (1.5 equiv), ERED 36 (50 mg/mmol
arylpropanoic acids).27,28 Scrutton and co-workers circum-
vented the poor reactivity of ene-acids by subjecting more
reactive enals to a biocatalytic reduction/oxidation cascade
sequence.29 Considering the scarcity of the literature on the
enzymatic reduction of unsaturated acids and the value this
synthetic strategy could offer industrial chemists, we were
motivated to investigate this transformation.
of 1a), NADP+ (5 mol %), PDH (8 mg/mmol 1a), H2O (0.1 M),
b
c
cosolvent (5 vol %), 24 h. pH is adjusted with 1 N NaOH. Yield
determined by HPLC.
We selected 2-benzylacrylic acid 1a as a model substrate for
our investigations. To the best of our knowledge, enzymatic
reduction of 2-benzylacrylic acid derivatives has not been
reported, likely due to insufficient activation of the alkene
toward hydride addition. To this end, we evaluated two panels
of commercially available EREDs (Codexis and Prozomix
panels, see Supporting Information Table S1) for the
enantioselective reduction of acid 1a. From over 130 ene-
reductases screened, only three enzymes displayed appreciable
activity (>2% conversion). Catalysis by Prozomix ERED 36
delivered chiral acid (R)-2a with the highest level of
conversion (52%) and >99% ee, with the minor (S)-
enantiomer undetectable by chiral GC.
With this result in hand, we conducted further optimization
of the reaction conditions using ERED 36 (Table 1).
Regeneration of the reduced flavin cofactor bound within the
ERED active site requires the addition of a second reducing
cofactor nicotinamide adenine dinucleotide phosphate
(NADPH). However, nicotinamide cofactors are extremely
costly, and using these reagents on scale in stoichiometric
quantities is not feasible. As such, we introduced a second
enzyme, phosphite dehydrogenase (PDH), to regenerate
NADPH.30 This recycling system uses inexpensive sodium
phosphite as the stoichiometric reductant, generates innocuous
inorganic phosphate as the byproduct, and has no requirement
for active pH control (unlike glucose dehydrogenase recycling
systems).31 Using the outlined enzyme system, we established
that temperature and pH are critical parameters for achieving
high activity (entries 1−6). ERED 36 was most active at
slightly acidic to neutral conditions (pH 6.5−7), and outside
this range a dramatic reduction in rate was observed. Likewise,
while excellent yields could be achieved by performing the
reaction between 25 and 30 °C, the limited thermostability of
ERED 36 led to a considerably decreased reaction rate at 35
°C. The low solubility of lipophilic organic molecules in water
is a key challenge for the application of enzymatic trans-
formations in industrial chemical synthesis; therefore, we
investigated organic cosolvent tolerance as a key reaction
parameter (entries 7−11). Pleasingly, ERED 36 remains highly
active in the presence of 5 vol % of DMSO or MeOH. The use
of other cosolvents, including DMF, IPA, and MeCN, resulted
in a significant reduction or complete loss of catalytic activity.
Lastly, to ensure synthetic utility, we demonstrated gram-scale
synthesis and isolation of acid 2a in 97% yield and >99% ee.
With optimized conditions in hand, we next investigated the
substrate scope of ERED 36 by exploring differentially
substituted 2-benzylacrylic acids 1b−1h (Table 2). Ortho-
and para-substituted benzyl acrylic acids 1b−g were well
tolerated (42−88% yield of 2b−g, >99% ee), although higher
enzyme loading was required (vs unsubstituted 2-benzylacrylic
acid, 1a) with more sterically encumbered substrates. The
exquisite functional group tolerance offered by enzymatic
reduction protocols was demonstrated through formation of
product 2g containing a ketone substituent (2g, 42% yield,
>99% ee), functionality that would likely suffer from
competitive reduction when subjected to transition metal-
catalyzed hydrogenation. Under standard conditions, no
reactivity was observed for substrate 1h containing an
unprotected indole. We noted that acid 1h was poorly soluble
in the aqueous reaction medium and postulated that increased
B
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a
Table 2. Substrate Scope of Enzymatic Reduction with ERED 36
a
Reaction conditions unless otherwise stated: acid 1 (1.5 mmol), ERED 36 (50−300 mg/mmol of 1), NADP+ (5 mol %), PDH (8 mg/mmol of 1),
sodium phosphite dibasic pentahydrate (1.5 equiv), H2O (0.1 M), 18−24 h. Isolated yields reported. ee values and absolute configuration for 2a
and 2i were determined by chiral SFC or GC analysis. Unless otherwise stated, the absolute stereochemistry of other products was assigned by
b
c
d
analogy. Reaction performed on 1.6 g (10 mmol) scale. 10 vol % of DMSO. ee value and absolute stereochemistry were determined by
conversion to 2-methylsuccinic acid and subsequent analysis by chiral SFC.
pH and/or inclusion of an organic cosolvent could aid
dissolution and recover reactivity. Indeed, when increasing to
pH 7 and including 10 vol % of DMSO as a cosolvent, full
conversion to the desired saturated carboxylic acid was
observed (2h, 95% yield, >99% ee).
mechanism of enzymatic reduction. While there have been
reports of ene-reductases with limited activity on 2-aryl-
substituted acids,27,28 we were particularly surprised to observe
activity on less activated aliphatic and benzylic acrylic acids
(1a−1h and 1m−1o). We considered a mechanistic scenario
in which isomerization to the α,β-substituted ene-acid occurs
prior to reduction (Scheme 2A).32 Cinnamic acids can be
reduced by oxygen-sensitive ene-reductases,25,26 though
comparable activity with ene-reductases applicable to industrial
processes (e.g., oxygen-tolerant EREDs from the OYE family)
has not been demonstrated.28,29 To probe this hypothesis, we
tested ERED 36 on (E)-α-methylcinnamic acid and both
geometric isomers of cinnamic acid (Scheme 2A). Regardless
of the alkene geometry, we failed to observe reduction
products 2a and 2p, invalidating the intermediacy of these
conjugated acids along the reaction pathway.
Ene-reductase 36 catalyzes efficient and highly enantiose-
lective reduction of activated 2-phenylacrylic acids (2i−2l, 62−
79% yield). Here, we evaluated the influence of alkene
electronics on reactivity; substrates containing electron-with-
drawing and electron-donating substituents on the aryl ring
were both converted to optically pure acids in excellent yields.
Next, we explored the reactivity of aliphatic acrylic acids and
found that ERED 36 accepts substrates of varying steric bulk
with the reduction proceeding in a highly stereoselective
manner (2m, 2n, and 2o, 95%, 77%, and 69% yield,
respectively). Formation of chiral acid 2n highlights the
applicability of this method to the diastereoselective synthesis
of unnatural β-amino acids. Finally, for substrate 1o,
chemoselective alkene reduction in preference to 4-methox-
ybenzyl ester deprotection demonstrates a clear advantage over
hydrogenation protocols.
The outlined studies, in accordance with prior reports,28
highlight the challenge of identifying ene-reductases from the
OYE family capable of reducing β-substituted ene-acids and
prompted us to explore other substrates in this class (Scheme
2B). Initially, we probed the impact of alkene geometry on
enzymatic activity by evaluating the reduction of (E)- and (Z)-
2-methyl-2-butenoic acid. Interestingly, while ERED 36
exhibited minimal activity on the cis-alkene isomer (Z-1q),
trans-isomer E-1q undergoes reduction to deliver saturated
acid 2q with high enantioselectivity (98.4% ee) albeit in low
yield (Table S5). Having discovered that unhindered β-
Exploration of the substrate scope highlighted two key
features of ERED 36: (i) rare activity toward unactivated α,β-
unsaturated acids and (ii) broad substrate specificity, with
ERED 36 accepting substrates with electronically and sterically
diverse substituents. The discovery of this reactivity motivated
us to design further experiments probing the scope and
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atoms was trans, indicating that the reactivity conferred by
ERED 36 follows the typical anti-reduction mechanism.
Overall, the studies described above probing the reactivity of
ERED 36 toward β-substituted ene-acids (i) indicate that
products 2a−o (Table 2) are likely generated via direct anti-
reduction of the 1,1-disubstituted ene acids and (ii) further
highlight the reactivity of ERED 36 with respect to the
reduction of challenging ene-acid substrates.
Scheme 2. Reactivity of β-Substituted Unsaturated Acids:
(A) Experiments Probing the Viability of an Isomerization−
Reduction Mechanistic Pathway; (B) Reduction of β-
Substituted Unsaturated Acid Substrates; and (C)
Deuterium Labeling Experiment Probing the Reduction
a
Mechanism
In summary, enzymatic reduction of a wide range of
unsaturated acids was demonstrated with exceptional levels of
enantioselectivity. We identified a commercially available
ERED (Prozomix ERED 36) that reduces unactivated ene-
acids such as benzyl- and aliphatic-substituted acrylic acids.
Although α,β-substituted ene-acids are typically challenging
substrates for EREDs, we successfully demonstrated reduction
of cyclic α,β-substituted ene-acids. The utilization of an
environmentally benign catalyst under mild aqueous con-
ditions with unrivaled chemoselectivity illustrates clear
advantages of enzymatic reduction over asymmetric hydro-
genation. Finally, mechanistic studies indicated that ERED 36
reduces ene-acids via the canonical anti-reduction pathway.
Future studies will focus on understanding the remarkable
reactivity of ERED 36 toward ene-acids with the goal of further
expanding the scope of enzymatic reduction.
ASSOCIATED CONTENT
* Supporting Information
■
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.orglett.0c02959.
Experimental procedures, compound characterization,
NMR spectra, and chiral assays (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
Chihui An − Department of Process Research & Development,
MRL, Merck & Co., Inc., Rahway, New Jersey 07065, United
States; orcid.org/0000-0001-5403-6859;
a
Reaction conditions: acid 1 (1.5 mmol), ERED 36 (50−300 mg/
mmol 1), NADP+ (5 mol %), PDH (8 mg/mmol 1), sodium
phosphite dibasic pentahydrate (1.5 equiv), water (0.1 M). ee values
and absolute stereochemistry were determined by chiral SFC or GC
Email: chihui.an@merck.com
Megan H. Shaw − Department of Process Research &
Development, MRL, Merck & Co., Inc., Rahway, New Jersey
07065, United States; orcid.org/0000-0003-4981-1448;
Email: megan.shaw@merck.com
b
c
analysis. Conversion determined by NMR. Isolated yields reported.
substituents are tolerated (E-1q, Me group), we anticipated
that cyclic α,β-substituted ene-acids might display enhanced
reactivity due to the limited steric bulk of the constrained β-
substituent and relief of ring strain upon reduction. Indeed,
exposure of 1-cyclopentene carboxylic acid 1r and 1H-indene-
3-carboxylic acid 1s to ERED 36 resulted in formation of the
corresponding saturated acids in excellent yields (2r and 2s,
92% and 82% yield, respectively).
Since ERED 36 exhibits remarkable activity on unactivated
carboxylic acids generating saturated products with excellent
enantioselectivity, we utilized cyclic substrate 1s to probe
whether the reduction mechanism is consistent with the
canonical anti-reduction pathway of the OYE family. We
performed a deuterium labeling experiment with 1H-indene-3-
carboxylic acid 1s in the presence of D2O and in situ generated
NADPD (from NADP+, D-glucose-d7, and glucose dehydrogen-
ase) to produce bisdeuterated 2s-d2 (Scheme 2C).33 NMR
analysis (see Supporting Information Figures S2 and S3)
revealed that the relative stereochemistry of the deuterium
Authors
Annika Tharp − Department of Process Research &
Development, MRL, Merck & Co., Inc., Rahway, New Jersey
07065, United States
Deeptak Verma − Department of Process Research &
Development, MRL, Merck & Co., Inc., Rahway, New Jersey
07065, United States
Hongming Li − Department of Process Research &
Development, MRL, Merck & Co., Inc., Rahway, New Jersey
07065, United States
Heather Wang − Department of Process Research &
Development, MRL, Merck & Co., Inc., Rahway, New Jersey
07065, United States
Xiao Wang − Department of Process Research & Development,
MRL, Merck & Co., Inc., Rahway, New Jersey 07065, United
States; orcid.org/0000-0003-3238-5366
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.orglett.0c02959
D
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(14) Monteiro, A. L.; Zinn, F. K.; de Souza, R. F.; Dupont, J.
Asymmetric Hydrogenation of 2-Arylacrylic Acids Catalyzed by
Immobilized Rn-BINAP Complex in 1-n-Butyl-3-Methylimidazolium
Tetrafluoroborate Molten Salt. Tetrahedron: Asymmetry 1997, 8, 177−
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Author Contributions
All authors have given approval to the final version of the
manuscript.
Notes
́
(15) Winkler, C. K.; Tasnadi, G.; Clay, D.; Hall, M.; Faber, K.
The authors declare no competing financial interest.
Asymmetric Bioreduction of Activated Alkenes to Industrially
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381−389.
ACKNOWLEDGMENTS
■
(16) Toogood, H. S.; Scrutton, N. S. New Developments in ’Ene’-
Reductase Catalysed Biological Hydrogenations. Curr. Opin. Chem.
Biol. 2014, 19, 107−115.
We thank Pharmaron scientists Fan Zhang, Panfeng Wei,
Xiupeng Duan, Chunhiu Dang, Kailin Liu, and Longgang Kang
for the synthesis of acrylic acids. We thank the following
scientists from Merck & Co., Inc., Rahway, NJ, USA, Dan
DiRocco, Kevin Maloney, Jeff Moore, and Mark Huffman, for
helpful suggestions; Melodie Christensen and Jon Jurica for
help with the AmigoChem for time course experiments;
Wilfredo Pinto for HRMS analysis; Peter Dormer for
assistance with quantitative NMR analysis; and John McIntosh
for assistance with protein analysis.
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Activated C=C-Bonds and Beyond: Emerging Trends. Curr. Opin.
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