Biocatalytic Asymmetric Michael Additions of Nitromethane to α,β-Unsaturated Aldehydes via Enzyme-bound Iminium Ion Intermediates

Article abstract of DOI:10.1021/acscatal.9b00780

The enzyme 4-oxalocrotonate tautomerase (4-OT) exploits an N-terminal proline as main catalytic residue to facilitate several promiscuous C-C bond-forming reactions via enzyme-bound enamine intermediates. Here we show that the active site of this enzyme c

Full text of DOI:10.1021/acscatal.9b00780

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Letter
Biocatalytic asymmetric Michael additions of nitromethane to #,#-
unsaturated aldehydes via enzyme-bound iminium ion intermediates
Chao Guo, Mohammad Saifuddin, Thangavelu Saravanan, Masih Sharifi, and Gerrit J. Poelarends
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.9b00780 • Publication Date (Web): 10 Apr 2019
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Biocatalytic Asymmetric Michael Additions of Nitromethane to α,β-
Unsaturated Aldehydes via Enzyme-bound Iminium Ion
Intermediates
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Chao Guo,† Mohammad Saifuddin,† Thangavelu Saravanan, Masih Sharifi, and Gerrit J.
Poelarends*
Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of
Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.
ABSTRACT: The enzyme 4-oxalocrotonate tautomerase (4-OT) exploits an N-terminal proline as main catalytic residue to
facilitate several promiscuous C-C bond-forming reactions via enzyme-bound enamine intermediates. Here we show that
the active site of this enzyme can give rise to further synthetically useful catalytic promiscuity. Specifically, the F50A mutant
of 4-OT was found to efficiently promote asymmetric Michael additions of nitromethane to various α,β-unsaturated
aldehydes to give γ-nitroaldehydes, important precursors to biologically active γ-aminobutyric acids. High conversions,
high enantiocontrol and good isolated product yields were achieved. The reactions likely proceed via iminium ion
intermediates formed between the catalytic Pro-1 residue and the α,β-unsaturated aldehydes. In addition, a cascade of three
4-OT(F50A)-catalyzed reactions followed by an enzymatic oxidation step enables assembly of γ-nitrocarboxylic acids from
three simple building blocks in one pot. Our results bridge organo- and biocatalysis, and emphasize the potential of enzyme
promiscuity for the preparation of important chiral synthons.
KEYWORDS: biocatalysis • Michael addition • asymmetric synthesis • enzyme catalysis • protein engineering
-H2
O
γ-Nitroaldehydes are important chiral building blocks for
the preparation of biologically active γ-aminobutyric acids.
The asymmetric synthesis of γ-nitroaldehydes from simple
starting materials has become feasible due to outstanding
developments within the organocatalysis field, particularly
fueled by aminocatalysis.[1] This is nicely illustrated by the
work of Hayashi and co-workers, who reported that
diphenylprolinol silyl ether can promote the asymmetric
synthesis of γ-nitroaldehydes through alternative Michael-
type reactions: enamine-mediated addition of aldehydes to
nitroalkenes, and nitroalkane addition to α,β-unsaturated
aldehydes activated as iminium ions.[1d-f]
A
4-OT
R
4-OT
N
N
+
H
H
2
O
O
O
O
NO2
2-steps
OH
H
nitroalkene
NH2
NO2
R
R
acetaldehyde
4-OT
*
*
-aminobutyric
acids (GABAs)
-nitroaldehydes
O
H 2
-H2
O
+
B
4-OT
N
N
H
R
O
NO2
nitromethane
R
,-unsaturated
aldehyde
Scheme 1. Proposed mechanisms for the 4-OT catalyzed
Inspired by these developments in the organocatalysis
field, work from our laboratory focused on the
development of a biocatalytic procedure for asymmetric
synthesis of γ-nitroaldehydes. We reported that 4-
oxalocrotonate tautomerase (4-OT), which utilizes a
unique N-terminal proline as key catalytic residue, can
promiscuously catalyze the Michael addition of
acetaldehyde (as well as various other aldehydes) to
nitroalkenes yielding enantioenriched γ-nitroaldehydes
(Scheme 1A).[2] The catalytic mechanism involves the
formation of an enamine species between acetaldehyde
and the Pro-1 residue (pKa ~6.4).[3,4] Hilvert and coworkers
have reported a highly engineered computationally
designed artificial aldolase, RA95.5-8, which can catalyze
the asymmetric synthesis of γ-nitroketones (but
Michael additions of acetaldehyde to nitroalkenes (A) and
nitromethane to α,β-unsaturated aldehydes (B) to yield γ-
nitroaldehydes.
not γ-nitroaldehydes) via acetone addition to
nitrostyrenes, and nitroalkane addition to conjugated
ketones.[5] However, a biocatalytic methodology for
nitroalkane addition to α,β-unsaturated aldehydes to yield
enantioenriched γ-nitroaldehydes is an as yet unmet
challenge.
Here, we report that the F50A mutant of the 4-OT
enzyme can efficiently promote asymmetric Michael
additions of nitromethane to α,β-unsaturated aldehydes,
yielding various γ-nitroaldehydes with high enantiopurity
1
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(e.r. up to >99:1) and in high isolated yield (61-96%). The
catalytic mechanism appears to involve formation of
enzyme-bound iminium ion intermediates in a manner
reminiscent of organocatalysis (Scheme 1B).
Page 2 of 6
allowing the production of optically pure (R)-3a (e.r. 99:1)
in high isolated yield of 92% (Table 1, entry 1; Figure S2-S4).
These results underscore the potential of the highly
promiscuous 4-OT enzyme for evolutionary optimization.
At semi-preparative scale, the 4-OT(F50A) catalyzed
Michael addition of 1 (50 mM, 152 mg in 50 mL) to 2a (25
mM, 157 mg in 50 mL) gave product (R)-3a (96%
conversion in 11 h) in good isolated yield (204.7 mg, 85%
yield) and with high e.r. of 98:2 (Figure S34, S35).
1
2
3
4
5
6
7
8
We previously reported that 4-OT catalyzes the aldol
condensation of acetaldehyde with benzaldehyde to yield
cinnamaldehyde.[3,6] Considering that the active site of 4-
OT can accommodate cinnamaldehyde, this aromatic α,β-
unsaturated aldehyde was tested as potential Michael
acceptor substrate. Cinnamaldehyde was expected to react
with Pro-1, the catalytic amine, to form a covalently bound
iminium ion intermediate, which could be attacked by
nitromethane (Scheme 1B). This Michael reaction was
performed in the presence of wild-type 4-OT in HEPES
buffer (pH 7.3), containing 5% (v/v) EtOH, 200 mM of
nitromethane (1, Scheme 2) and 3 mM of cinnamaldehyde
(2a), and reaction progress was monitored by following the
depletion of 2a by UV-VIS spectrophotometry. Under
these conditions, 50% of substrate 2a was consumed in 24
h and the corresponding product 3a was obtained in 35%
9
Notably, the mutation F50A was previously found to
improve the aldol condensation activity of 4-OT.[6a] This
mutation makes the active site pocket of 4-OT more
accessible to the outside aqueous environment, without
changing the pKa of Pro-1 too much, and likely enhances
the aldol condensation activity of 4-OT by promoting the
final hydrolysis step in which product is released from Pro-
1, which has been suggested to be rate-limiting.[6a] Similarly,
the F50A mutation may increase the Michael addition
activity of 4-OT by making the active site more amenable
for hydrolytic cleavage of the covalent enzyme-product
intermediate.
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1
isolated yield (as confirmed by H NMR spectroscopy).
Analysis of product 3a by chiral HPLC revealed high
enantiocontrol at the site of addition with formation of the
(R)-configured product (e.r. of 86:14). Interestingly, we
earlier reported that wild-type 4-OT catalyzes the Michael
addition of acetaldehyde to trans-β-nitrostyrene to yield
(S)-3a with an e.r. of 95:5.[2a] Hence, 4-OT catalyzes the
Having established that 4-OT(F50A) can efficiently
promote the asymmetric Michael addition of 1 to 2a, a set
of α,β-unsaturated aldehydes was prepared (see Support
Information for details) and tested as Michael acceptor
substrates. The results demonstrate that the 4-OT(F50A)
enzyme has a broad substrate scope, accepting both
aromatic and aliphatic Michael acceptor substrates, and
catalyzes the addition of 1 to the α,β-unsaturated
aldehydes 2b-k to yield the corresponding γ-
nitroaldehydes 3b-k with excellent enantiopurity (e.r. up
to >99:1) and in good isolated yield (61-96%) (Table 1,
Figure S5-S33). Interestingly, the enzymatic Michael
synthesis
of
γ-nitroaldehyde
3a
via
two
enantiocomplementary Michael reactions: enamine-
mediated addition of acetaldehyde to trans-β-nitrostyrene,
and nitromethane addition to cinnamaldehyde likely
activated as iminium ion.
O
O
H
4-OT WT
reactions
with
meta-
and
para-substituted
NO2
NO2
H
(R)
20 mM HEPES/EtOH 95:5,
pH 7.3, RT
cinnamaldehydes (2c,d and 2f-j) provided the
corresponding products as the (R)-configured enantiomers,
while those with the ortho-substituted cinnamaldehydes
(2b and 2e) yielded the (S)-configured product
enantiomers (Table 1, entries 2 and 5). This suggests that
positioning substituents on the ortho position of the
substrate promoted steric effects, which caused either
substrate relocation in the enzyme active site or a
stereofacial shielding effect. Notably, the consequence of
ortho-substituents on the stereochemical outcome of
organo- and biocatalytic reactions with aromatic aldol and
Michael acceptor substrates has been observed before.[2d,8]
1
2a
3a
(R)-
Scheme 2. Wild-type 4-OT catalyzed Michael addition of
nitromethane (1) to cinnamaldehyde (2a) to yield γ-
nitroaldehyde (R)-3a.
Encouraged by these initial findings, a systematic
mutagenesis approach was applied to enhance this
promiscuous Michael addition activity of 4-OT. For this, an
earlier constructed collection of 4-OT genes coding for
almost all possible single-mutant variants of 4-OT was
used.[7] Improved variants (>2-fold increase in activity)
were identified by monitoring the depletion of 2a in a
spectrophotometric kinetic assay in multi-well plates.
Given that several mutations at positions Met-45 and Ala-
46 (M45G, M45H, M45S, A46H and A46S) result in a slight
improvement in activity (~3-fold), three mutations at
position Phe-50 (F50I, F50V and F50A) significantly
enhanced the Michael addition activity. Assays with the
purified mutant enzymes showed a 6-fold, 8-fold and 15-
fold increase in activity for F50I, F50V and F50A,
respectively (Figure S1). Further characterization of the
Michael reaction between 1 and 2a catalyzed by the best 4-
OT variant (F50A) showed that besides increased activity,
this mutant enzyme also has enhanced stereoselectivity,
We next investigated whether the mechanism of the 4-
OT(F50A) catalyzed Michael reaction proceeds via
iminium ion formation between Pro-1 and the α,β-
unsaturated aldehyde substrate. A 4-OT(F50A) sample
modified by 2a in the presence of NaBH4 and an
unmodified 4-OT(F50A) sample were digested with Glu-C
(an endoproteinase from Staphylococcus aureus V8), and
the generated peptides were characterized by LC-MS
(Figure S43-S45). Comparing the peaks of the modified 4-
OT(F50A) sample to those of the nonmodified 4-OT(F50A)
sample revealed
a
modification of the fragment
PIAQIHILE by a species with a mass of 116 Da. This
corresponds to labeling by one cinnamaldehyde molecule.
Characterization of the remaining peaks revealed no
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by 1H NMR analysis; cIsolated yield (%). dDetermined by chiral
labeling of other fragments (Figure S44, S45). Within the
N-terminal fragment Pro-1 to Glu-9, the most probable
positions for alkylation are Pro-1 and His-6. To identify the
HPLC or GC. eThe absolute configuration was determined by
comparison of chiral HPLC or GC data with those previously
1
2
3
4
5
6
7
8
f
reported (see Supporting Information for details). Apparent
O
kinetic parameters determined with this substrate at a fixed
nitromethane concentration of 25 mM: kcat = 0.05 (±0.002) s-1;
Km = 367 (± 37) µM. gFurther purified using flash column
chromatography.
O
4-OT F50A
H
NO2
NO2
R
H
R
*
20 mM HEPES/EtOH 95:5,
pH 6.5, RT
-unsaturated
-nitroaldehydes
nitromethane
aldehyde
The spectrum of the ion corresponding to the unlabled
PIAQIHILE peptide showed the characteristic b5 ion
resulting from the peptide fragment PIAQI. MS/MS
analysis of the modified PIAQIHILE peptide revealed a
mass increase of 116 Da for this b5 ion. Therefore, it can be
concluded that Pro-1 is the sole site of modification by 2a.
This result supports the hypothesis that Pro-1 functions as
an amine catalyst in the enzymatic addition reaction,
increasing the electrophilicity of the Michael acceptor
through Schiff base formation (Scheme 1B). Replacement
of Pro-1 with an alanine in wild-type 4-OT led to a 32-fold
decrease in activity for the addition of 1 to 2a (Figure S1),
providing further support for this mechanism. Work is in
progress to determine the structure of 4-OT(F50A)
covalently modified by 2a.
1
2a 2k
-
3a-3k
9
2a
2b
2c
2d
2e
2f
2g
2h
2i
R = Ph
3a
3b
3c
3d
3e
3f
3g
3h
3i
(precursor phenibut)
(precursor baclofen)
R = o-Cl-Ph
R = m-Cl-Ph
R = p-Cl-Ph
R = o-MeO-Ph
R = p-MeO-Ph
R = p-NO2-Ph
R = p-OH-Ph
R = p-F-Ph
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3j
(precursor rolipram)
2j
2k
R = 3-OH-4-MeO-Ph
R = isobutyl
3k
(precursor pregabalin)
labeled residue, the modified and unmodified peptides
were analyzed by LC-MS/MS (Figure S46).
Table 1. 4-OT(F50A)-catalyzed nitromethane addition to
α,β-unstaturated aldehydes 2a-2k using optimized
reaction conditionsa
α,β-
Abs.
We have previously reported that 4-OT(F50A) can
catalyze the aldol condensation of acetaldehyde with
benzaldehyde to give cinnamaldehyde.[3,6] Here we show
that the three different activities observed for the 4-
OT(F50A) enzyme can be used to prepare γ-nitroaldehydes
in a biocatalytic cascade involving sequential aldol
addition of acetaldehyde to a suitable aromatic aldehyde,
dehydration, and Michael addition of nitromethane.
Inclusion of a suitable aldehyde dehydrogenase and
cofactor-recycling NADH oxidase[9] in the reaction mixture
enabled efficient one-pot synthesis of γ-nitrocarboxylic
acids (Scheme 3). Using acetaldehyde (4), benzaldehyde
(5a) and nitromethane (1) as starting substrates, product
(R)-7a was obtained in 53% isolated yield (65% overall
conversion) and with an excellent e.r. of 99:1 (Figure S36-
S38). Replacing substrate 5a with 5b, yielded product (S)-
7b in 80% isolated yield (>99% overall conversion) and
with an excellent e.r. of 99:1 (Figure S39-S42). This simple
and effective cascade further demonstrates the
tremendous potential of combining different enzymes to
construct simple synthetic routes for preparation of
important chemical products.
Ent unsaturat Produ
t
Conv.b
[h] (Yield)c
e.r.d confi
g.e
ry
ed
aldehyde
ct
O
H
NO2
1
2a
2b
2c
2d
7
2
6
99 (92)
99 (90)f
98 (93)
99:1
R
S
(R)
3a
O
H
NO2
2
3
>99:1
98:2
(S)
3b
Cl
O
H
NO2
(R)
R
3c
Cl
O
H
4
5
6
7
8
9
24 98 (93)
10 99 (96)
18 90 (80)g
18 85 (75)g
20 98 (71)
98:2
86:14
98:2
98:2
97:3
R
S
NO2
(R)
3d
Cl
O
H
NO2
2e
2f
(S)
3e
O
O
H
NO2
R
R
R
R
(R)
3f
O
O
H
2g
2h
2i
NO2
(R)
3g
O2
N
O
O
OH
O
O
H
4-OT F50A
4-OT F50A
O
NO2
(R)
H
H
H
3h
R
R
R
HO
H
2a
2b
R = H
R = o-Cl
O
6a
6b
R = H
R = o-Cl
H
4
5a R = H
5b
8
97 (89)g
>99:1
NO2
(R)
3i
R = o-Cl
4-OT F50A
CH3NO2
F
O
1
H
10
11
2j
20 84 (73)g
18 95 (61)g
91:9
93:7
R
S
NO2
(R)
3j
O
O
MeO
OH
OH
NO2
ALDH
H
O
NO2


2k
R
R
NO2
(S)
3k
NAD+
NADH
O2
3a
(R)- R = H
3b
(S)- R = o-Cl
aAll the reactions were performed in buffer [20 mM HEPES/5%
(v/v) ethanol] at pH 6.5 with 4-OT F50A (72 µM, except for 2g
and 2i for which 36 µM enzyme was used), 1 (25 mM) and 2a-
k (3 mM, except for 2g which was used at 2 mM); bDetermined
7a
(R)- R = H
7b
(S)- R = o-Cl
H2O
NOX
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[1] a) Wiesner, M.; Revell, J. D.; Wennemers, H. Tripeptides as
Scheme 3. Four-step biocatalytic cascade synthesis of γ-
nitrobutyric acids 7a and 7b in one pot. The cascade
reactions were performed with 1 (50 mM), 4 (150 mM) and
either 5a or 5b (3 mM).
Efficient Asymmetric Catalysts for 1,4-Addition Reactions of
Aldehydes to Nitroolefins--A Rational Approach. Angew. Chem.
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Wennemers, H. Tripeptides of the Type H-D-Pro-Pro-Xaa-NH2 as
Catalysts for Asymmetric 1,4-Addition Reactions: Structural
Requirements for High Catalytic Efficiency. Chem. Eur. J. 2009, 15,
10103-10109; c) García-García, P.; Ladépêche, A.; Halder, R.; List, B.
Catalytic Asymmetric Michael Reactions of Acetaldehyde. Angew.
Chem. Int. Ed. 2008, 47, 4719-4721; d) Gotoh, H.; Ishikawa, H.;
Hayashi, Y. Diphenylprolinol Silyl Ether as Catalyst of an
Asymmetric, Catalytic, and Direct Michael Reaction of
Nitroalkanes with α,β-Unsaturated Aldehydes. Org. Lett. 2007, 9,
5307-5309; e) Hayashi, Y.; Gotoh, H.; Hayashi, T.; Shoji, M.
Diphenylprolinol Silyl Ethers as Efficient Organocatalysts for the
Asymmetric Michael Reaction of Aldehydes and Nitroalkenes.
Angew. Chem. Int. Ed. 2005, 44, 4212-4215; f) Hayashi, Y.; Itoh, T.;
Ohkubo, M.; Ishikawa, H. Asymmetric Michael Reaction of
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Chem. Int. Ed. 2008, 47, 4722-4724; g) Jentzsch, K. I.; Min, T.;
Etcheson, J. I.; Fettinger, J. C.; Franz, A. K. Silyl Fluoride
Electrophiles for the Enantioselective Synthesis of Silylated
Pyrrolidine Catalysts. J. Org. Chem. 2011, 76, 7065-7075; h) Qiao,
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Acetaldehyde with Nitroolefins Catalyzed by Highly
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Highly Selective, Polymer-Supported Organocatalyst for Michael
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Functionalization of Fe3O4 Magnetic Nanoparticles for
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Hartikka, A.; Moghaddam, F. M.; Arvidsson, P. I. A. New
1
2
3
4
5
6
7
8
In summary, our results indicate that the active site of 4-
OT can give rise to synthetically useful promiscuous
activities. Like proline-based organocatalysts, 4-OT
utilizes a prolyl amine to attack diverse aldehydes forming
reactive enamine and iminium ion intermediates. Hence,
this natural enzyme with its unique catalytic amino-
terminal proline could possibly accelerate many of the
bond-forming reactions promoted by organocatalysts. We
have therefore initiated studies aimed at exploring
alternative nucleophiles for addition to α,β-unsaturated
aldehydes, which would allow for the enzymatic synthesis
of additional products.
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In contrast to difficult to prepare proline- and peptide-
based organocatalysts, the enzyme 4-OT can be produced
in large amounts by simple bacterial fermentation.
Moreover, the enzymatic reaction proceeds in eco-friendly
aqueous buffer rather than in organic solvent. In previous
work, we have demonstrated that 4-OT can be engineered
into a more efficient biocatalyst for the aldol condensation
of
acetaldehyde
with
benzaldehyde
to
yield
cinnamaldehyde, with a >5000-fold enhancement in
catalytic efficiency (kcat/Km) and a >107-fold change in
reaction specificity.[6b] It is therefore conceivable that the
promiscuous activity of 4-OT(F50A) for the Michael
addition of nitromethane to α,β-unsaturated aldehydes
can be optimized by directed evolution to generate novel
biocatalysts for practical synthesis of chiral precursors for
important pharmaceuticals.
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Organocatalyzed Asymmetric Conjugate
Catalyst
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for
of
Nitroalkanes to Aldehydes. Adv. Synth. Catal. 2007, 349, 740-748;
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Vera, S. Water-Compatible Iminium Activation: Organocatalytic
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AUTHOR INFORMATION
Corresponding Author
* Corresponding author. Tel.: +31503633354; E-mail:
g.j.poelarends@rug.nl; Web:
http://www.rug.nl/staff/g.j.poelarends/
Author Contributions
†Chao Guo and Mohammad Saifuddin contributed equally to
this work.
ASSOCIATED CONTENT
Supporting
Information.
Additional
experimental
procedures and compound characterization.
ACKNOWLEDGMENT
[2] a) Zandvoort, E.; Geertsema, E. M.; Baas, B. J.; Quax, W. J.;
Poelarends, G. J. Bridging Between Organocatalysis and
Biocatalysis: Asymmetric Addition of Acetaldehyde to β-
This research was financially supported by the European
Union’s Horizon 2020 Research and Innovation Programme
(grant 635595), the European Research Council (grant 713483)
and the Netherlands Organization of Scientific Research
(grant 724.016.002). We thank M.H. de Vries for support in
acquiring mass spectrometry data, and Lieuwe Biewenga for
insightful discussions.
Nitrostyrenes Catalyzed by
a Promiscuous Proline-Based
Tautomerase. Angew. Chem. Int. Ed. 2012, 51, 1240-1243; b) Miao,
Y.; Geertsema, E. M.; Tepper, P. G.; Zandvoort, E.; Poelarends, G.
J. Promiscuous Catalysis of Asymmetric Michael-Type Additions
of Linear Aldehydes to β-Nitrostyrene by the Proline-Based
Enzyme 4-Oxalocrotonate Tautomerase. ChemBioChem 2013, 14,
191-194; c) Geertsema, E. M.; Miao, Y.; Tepper, P. G.; de Haan, P.;
Zandvoort, E.; Poelarends, G. J. Biocatalytic Michael-Type
Additions of Acetaldehyde to Nitroolefins with the Proline-Based
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