Stereochemical Control of Enzymatic Carbon–Carbon Bond-Forming Michael-Type Additions by “Substrate Engineering”

Article abstract of DOI:10.1002/ejoc.201601126

The enzyme 4-oxalocrotonate tautomerase (4-OT) promiscuously catalyzes the Michael-type addition of acetaldehyde to β-nitrostyrene derivatives to yield chiral γ-nitroaldehydes, which are important precursors for pharmaceutically active γ-aminobutyric acid

Full text of DOI:10.1002/ejoc.201601126

A Journal of
Accepted Article
Title: Stereochemical Control of Enzymatic Carbon-Carbon Bond-Forming Michael-
type Additions by ’Substrate Engineering’
Authors: Yufeng Miao; Pieter Tepper; Edzard Geertsema; Gerrit Jan Poelarends
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201601126
Link to VoR: http://dx.doi.org/10.1002/ejoc.201601126
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European Journal of Organic Chemistry
10.1002/ejoc.201601126
Stereochemical Control of Enzymatic Carbon-Carbon Bond-
Forming Michael-type Additions by ‘Substrate Engineering’
Yufeng Miao,[a] Pieter G. Tepper,[a] Edzard M. Geertsema[a,b] and Gerrit J. Poelarends*[a]
[a]
Dr. Y. Miao, Mr. P.G. Tepper, Dr. E.M. Geertsema, Prof. Dr. G.J. Poelarends
Department of Chemical and 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
Web: http://www.rug.nl/staff/g.j.poelarends/
[b]
Present address: Institute for Life Science and Technology, Hanze University of
Applied Sciences, Zernikeplein 11, 9747 AS Groningen
1
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European Journal of Organic Chemistry
10.1002/ejoc.201601126
Abstract
The enzyme 4-oxalocrotonate tautomerase (4-OT) promiscuously catalyzes the Michael-
type addition of acetaldehyde to β-nitrostyrene derivatives yielding chiral γ-nitroaldehydes,
which are important precursors for pharmaceutically active γ-aminobutyric acids. In this study,
we investigated the effect of different substituents at the aromatic ring of the Michael acceptor
on catalytic efficiency and stereoselectivity of the 4-OT-catalyzed acetaldehyde addition
reactions. Highly enantioenriched (R)- and (S)-γ-nitroaldehydes and 4-substituted chroman-2-
ol can be obtained with good to excellent yields by applying different substituents at
appropriate positions of the aromatic substrate. The stereochemical control of these enzymatic
Michael-type additions by ‘substrate engineering’ allows enantioselective synthesis of
valuable γ-aminobutyric acid precursors. In addition, the results suggest a novel enzymatic
synthesis route towards precursors for chromans and derivatives, which are valuable scaffolds
for preparing biologically active natural products.
2
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European Journal of Organic Chemistry
10.1002/ejoc.201601126
Introduction
γ-Aminobutyric acid (GABA) is the primary inhibitory neurotransmitter widely distributed in
the mammalian central nervous system to regulate neuronal excitation.[1] GABA deficiency
has been associated with several neurodegenerative disorders such as Parkinson’s disease,[2]
Huntington’s disease[2b, 3 ] and Alzheimer’s disease,[ 4 ] and psychiatric disorders including
depression,[5] alcoholism[6] and anxiety.[5b-c, 7] Over the past few decades, a number of GABA
derivatives showing biological activities have been developed as potential drugs for the
treatment of neurological disorders.[8, 9] It has been shown that the biological activities of
these compounds are largely dependent on the configuration of the chiral center. For example,
only the (S)-enantiomer of Vigabatrin,[10] the (R)-enantiomer of Baclofen,[11] and the (S)-
enantiomer of Pregabalin[ 12 ] are the active pharmaceutical ingredients. The asymmetric
synthesis of GABA derivatives has therefore attracted considerable attention and several
methodologies using metal-, organo- and biocatalysts, or their combinations, to construct
these chiral molecules have been established.[8a]
We have recently reported a biocatalytic approach for the asymmetric Michael-type
addition of acetaldehyde to nitroalkenes to afford enantioenriched γ-nitroaldehydes,[13] which
can be readily converted into chiral GABA derivatives by two simple chemical steps. This
methodology utilizes the catalytic promiscuity of the enzyme 4-oxalocrotonate tautomerase
(4-OT).[14] In this study, we investigated the effect of different substituents at the aromatic
ring of the Michael acceptor on catalytic efficiency and stereoselectivity of the 4-OT-
catalyzed Michael-type addition reactions. We report the asymmetric 4-OT-catalyzed
Michael-type addition of acetaldehyde (1, Scheme 1) to a series of mono-substituted β-
nitrostyrene derivatives (2c-m) yielding enantioenriched (R)- or (S)-γ-nitroaldehydes (3c-m),
as well as the 4-OT-catalyzed Michael-type addition of 1 to trans-2-hydroxy-β-nitrostyrene
(2b), which, after cyclization, affords 4-(nitromethyl)-chroman-2-ol (5, Table 1).
3
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European Journal of Organic Chemistry
10.1002/ejoc.201601126
Scheme 1. Michael-type addition of acetaldehyde (1) to β-nitrostyrene derivatives (2a-m)
Results and Discussion
The 4-OT-catalyzed reaction of donor 1 with a series of nitrostyrene derivatives (2b-l)
as potential acceptors (Scheme 1) was examined. In separate analytical-scale experiments, 2b-
l and 1 were incubated with 4-OT in NaH2PO4 buffer containing an appropriate co-solvent
(Table S1). The reactions were followed by monitoring the change in absorbance at λmax of
2b-l by UV-spectroscopy. During all reactions, a decrease in the absorbance of 2b-l was
observed in course of time, which indicated depletion of these nitroolefins (Figure S1-S10).
Incubations of 1 with 2b-l under the same conditions but in the absence of 4-OT showed
negligible decreases in absorbances at λmax, 2b-l, indicating that 4-OT is responsible for
catalysis (Figure S1-S10).
Next, preparative-scale experiments were performed to allow product identification by
NMR spectroscopy and hence to confirm that the 4-OT-catalyzed additions of 1 to 2b-l give
Michael-type addition adducts 3b-l. In addition, the enzymatic preparation of 3m from 2m
4
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European Journal of Organic Chemistry
10.1002/ejoc.201601126
and 1 was investigated. In separate experiments, 2b-m, 1, and 4-OT were incubated in
NaH2PO4 buffer with an appropriate co-solvent (Table 1, Table S2). The progress of each
reaction was monitored by UV-spectroscopy (Figure S11-S14). After the reaction was
completed, work-up and purification procedures were carried out to isolate the enzymatic
products. NMR spectroscopic analysis of the purified products confirmed the formation of γ-
nitroaldehydes 3c-m (Figure S16-S25). Intriguingly, the expected γ-nitroaldehyde 3b (from 1
and 2b) was not observed. Instead, the formation of 4-(nitromethyl)-chroman-2-ol (5) as a
result of hemiacetalization of 3b was established by NMR spectroscopy (Figure S15) and
comparison with literature data.[ 15 ] The formed 4-substituted chroman-2-ol (5) is a
synthetically valuable intermediate, which can be easily converted into chromans and their
derivatives with antimicrobial and anticancer properties.[16] Good to excellent yields (up to
96%) were achieved for products 3c-f, 3h-i, 3m and 5 (Table 1). The somewhat lower yields
of 3j-l (31-37%) were caused by the low conversion rates with nitrostyrene derivatives 2j-l
and the non-enzymatic hydration of 2j-l in aqueous reaction media.
The enzymatically obtained γ-nitroaldehydes 3c-m were reduced to the corresponding
alcohols 4c-m for the determination of their enantiomeric excesses (ee) by chiral phase HPLC.
Good to excellent ee values (between 82% and 97%) were achieved for 3c-m, indicating that
4-OT is highly stereoselective during the catalytic process (Table 1; Figure S46-S55). The
absolute configuration of 3c-m was determined by optical rotation. Interestingly, comparison
with literature data revealed that the meta- and para-substituted γ-nitroaldehydes (3d, 3f, 3g,
3i, and 3j) have the (S)-configuration, whereas the ortho-substituted γ-nitroaldehydes 3e and
3k have the (R)-configuration (Table 1, Table S3). The observed negative optical rotation of
meta-substituted γ-nitroaldehydes 3c, 3l and 3m could not be compared with literature data
since, to the best of our knowledge, these compounds have not been reported in the literature
so far. We assume the major enantiomers of 3c, 3l and 3m to have the (S)-configuration
5
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European Journal of Organic Chemistry
10.1002/ejoc.201601126
because they showed the same negative optical rotation and elution order in HPLC
chromatograms as observed with the meta-substituted products 3f and 3i. Similarly, the
absolute configuration of the major enantiomer of 3h is tentatively assigned as R by
comparing its optical rotation data with those of 3e and 3k.
Table 1. Preparative-scale asymmetric 4-OT-catalyzed Michael-type additions of
acetaldehyde 1 (50 eq.) to β-nitrostyrene derivatives 2a-n (1.5-2.0 mM) in NaH2PO4 buffer
(pH 5.5) yielding γ-nitroaldehydes 3a-n or chroman-2-ol 5.
β-
entry Nitro-
styrene
Co-
solvent
(v/v)
t
Conversion Yielda
eeb
[%]
Abs.
4-OT
Product
[h]
[%]
[%]
Conf.c
[mol %]d
EtOH
10%
1e
0.3
n.d.
65
51
S
5.6
2a
3a
n.d.i
syn/anti: n.d.
1.8/1
EtOH
10%
2f
6.1
0.7
3.0
96
98
99
95
94
85
1.0
1.0
1.0
2b
5
EtOH
10%
3
4
95
94
S
S
2c
3c
3d
DMSO
40%
2d
DMSO
40%
5
6
1.0
0.7
2.5
99
92
93
81
51
97
96
69
R
S
S
1.0
1.0
2.8
2e
2f
3e
3f
DMSO
40%
DMSO
45%
7g
n.d.
2g
3g
EtOH
25%
8
9
2.8
3.9
98
93
94
93
82
84
R
S
1.0
1.0
2h
2i
3h
3i
EtOH
25%
6
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European Journal of Organic Chemistry
10.1002/ejoc.201601126
DMSO
40%
10
3.3
83
37
87
S
1.0
2j
3j
DMSO
40%
11
12
3.7
4.0
48
84
31
35
94
95
R
S
1.0
1.0
2k
2l
3k
3l
DMSO
40%
DMSO
40%
13
1.2
2.0
98
96
70
92
81
S
S
1.0
1.4
2m
3m
3n
EtOH
10%
2n
(R=H)
14h
n.d.
[a] Isolate yield. [b] Determined by HPLC analysis with a chiral stationary phase. [c] Absolute
configurations of major enantiomers of 3c-m were determined by optical rotation and comparison with
literature data (see Table S3 in the Supporting information). [d] Compared to β-nitrostyrene
derivatives 2a-n. [e] Previous results, reaction performed with 1: 50 mM, 2a: 1.3 mM, 4-OT: 73 µM
1
in phosphate buffer (pH 7.3).[16a] [f] Diastereomeric ratio (dr) was determined by H NMR and
comparison with literature data;[18] enantiomeric excess of 5 was not determined. [g] Previous results,
reaction performed with 1: 65 mM, 2g: 1.3 mM, 4-OT: 36 µM in phosphate buffer (pH 5.5).[16c] [h]
Previous results, reaction performed with 1: 50 mM; 2n: 2 mM; 4-OT: 28 µM in phosphate buffer (pH
5.5).[16b] [i] n.d. = not determined.
Kinetic studies of the 4-OT-catalyzed addition of 1 to 2a-o (2o: R = para-fluoro) were
carried out to elucidate the influence of substituents on the kinetic parameters. To address the
influence of co-solvent and pH on kinetic parameters, the kinetic assays of the 4-OT-catalyzed
reaction with trans-β-nitrostyrene (2n) were performed at different pH values and in different
solvent systems (Table 2, entry 1-4). We have previously reported that lowering the pH of the
reaction media from 7.3 to 5.5 gives an ~4-fold increase in both kcat and KM, resulting in an
unchanged catalytic efficiency (kcat/KM) (Table 2, entry 1 and 2). Interestingly, switching the
co-solvent of the reaction medium from EtOH (10%, v/v) to DMSO (40%, v/v) also resulted
in a slight increase (up to 2-fold) in both kcat and KM (Table 2, entry 3 and 4, compare to entry
1 and 2). The kinetic data obtained with 2a in two different solvent systems showed similar
7
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European Journal of Organic Chemistry
10.1002/ejoc.201601126
changes in kcat and KM values (Table 2, entry 5-6), suggesting that this solvent effect on the
kinetic parameters is consistent for the different nitroolefins. This effect may be caused by a
small conformational change in 4-OT’s structure induced by the co-solvent DMSO.[17]
The kinetic parameters of the 4-OT-catalyzed additions of 1 to 2a-o were determined
and compared with those of 2n (R=H) measured under the same solvent and pH conditions
(Table 2). In case of para-substituted nitrostyrenes, an increase in kcat for substrates carrying
electron donating groups (hydroxyl and methoxyl group) and a decrease in kcat for the
substrate with a strongly electron withdrawing group (nitro group) were observed (Table 2,
compare entry 6-10 to entry 3). The kcat seems not to be significantly affected by fluoro- and
chloro-substituents. The obtained KM values suggest that the affinity of 4-OT for para-
substituted nitrostyrenes is affected by the hydrophilicity of the substituents. Substituents with
high hydrophilicity generally result in an increase in KM value, indicating a lower affinity of
4-OT towards the substrate (Table 2, entry 6, 8, 9, compare to entry 3). The highest KM values
were obtained with hydroxyl- and fluoro-substituted nitrostyrenes (Table 2, entry 6 and 8).
Results obtained with meta-substituted nitrostyrenes show similar electronic effects
caused by the substituents. The presence of electron donating groups (hydroxyl and methoxyl
group) enhances the kcat, while the presence of a strongly electron withdrawing group (nitro
group) shows the opposite effect. Furthermore, the affinity of 4-OT towards meta-substituted
nitrostyrenes seems to be influenced by the hydrophilicity of the substituents as well, as the
bromo- and chloro-substituted nitrostyrenes showed the lowest KM values (Table 2, entry 13-
14, compare to entry 3) while a more hydrophilic substituent (hydroxyl group) at the meta-
position leads to an increased KM value (Table 2, entry 11, compare to entry 3). The KM values
obtained with meta-substituted nitrostyrenes are generally somewhat lower than those
determined for the corresponding para-substituted substrates, suggesting that nitrostyrene
derivatives with a meta-substitution fit better to the activity site of 4-OT. The relatively low
8
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European Journal of Organic Chemistry
10.1002/ejoc.201601126
KM values obtained with meta-substituted nitrostyrenes also result in higher kcat/KM values
compared to that obtained with 2n (Table 2, entry 11-15, compare to entry 3).
Table 2. Apparent kinetic parameters for the 4-OT-catalyzed Michael-type addition of
acetaldehyde (1) to para-, meta-, and ortho-substituted β-nitrostyrenes (2a-o)
kcat
KM for 2
kcat/KM
(M-1 s-1)
68
Co-
solvent
entry
R
Substrate
pH
(s-1) × 103 (mM)
1[a]
2[b]
3[c]
4[d]
5
H
H
17
70
0.25
1.1
7.3 EtOH
5.5 EtOH
7.3 DMSO
5.5 DMSO
EtOH
2n
2n
2n
2n
2a
2a
2d
2o
2g
2j
66
no
H
33±1
118±12
60
0.46±0.06
1.84±0.29
1.6
68±3
64±6
37
substitution
H
p-OHa
p-OH
p-MeO
p-F
6
150±2
61±2
39±1
33±1
15±1
71±3
103±6
44±1
31±8
24±1
31±1
270±22
174±8
139±27
4.34±0.80
0.64±0.05
1.35±0.09
0.69±0.05
0.46±0.06
0.67±0.09
1.00±0.14
0.17±0.02
0.19±0.02
0.34±0.05
0.67±0.04
4.75±0.54
0.92±0.09
4.54±1.19
34±5
95±3
28±1
47±2
33±2
106±5
103±6
260±8
168±5
71±3
46±1
57±5
187±9
31±6
DMSO
7
DMSO
7.3
para-
substitution
8
DMSO
9
p-Cl
DMSO
DMSO
10
11
12
13
14
15
16
17
18
19
p-NO2
m-OH
m-MeO
m-Br
m-Cl
m-NO2
o-OH
o-MeO
o-Cl
2c
2f
meta-
substitution
7.3 DMSO
5.5e DMSO
2m
2i
2l
2b
2e
2h
2k
ortho-
substitution
o-NO2
[a] Previously reported data, assays were performed in EtOH/phosphate buffer (10/90, v/v) at pH
7.3.[16a] [b] Previously reported data, assays were performed in EtOH/phosphate buffer (10/90, v/v) at
pH 5.5.[16c] [c] Assays were performed in DMSO/phosphate buffer (40/60, v/v) at pH 7.3. [d] Assays
were performed in DMSO/phosphate buffer (40/60, v/v) at pH 5.5. [e] Assays were performed in
DMSO/phosphate buffer (40/60, v/v) at pH 5.5 to prevent nonenzymatic hydration of nitrostyrene
derivatives 2b, 2e, 2h and 2k in the aqueous solvent system.
9
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European Journal of Organic Chemistry
10.1002/ejoc.201601126
The results of the kinetic assays with ortho-substituted substrates show that introducing a
methoxyl group to the ortho-position of nitrostyrene significantly increases the value of kcat,
while the presence of a hydroxyl group at this position results in a significant decrease in kcat
(Table 2, entries 16 and 17, compare to entry 4). The different KM values observed for the
different ortho-substituted nitrostyrenes may be related to steric effects.
Conclusion
4-OT catalyzes the asymmetric Michael-type addition of acetaldehyde to various ortho-,
meta-, and para-substituted β-nitrostyrenes. For most of the γ-nitroaldehydes excellent yields
(up to 96%) and high optical purities (ee values up to 97%) were achieved. Enzymatic access
to (R)-γ-nitroaldehydes was achieved with ortho-substituted nitrostyrenes, while (S)-γ-
nitroaldehydes were obtained with para- and meta-substituted nitrostyrenes. Apparently,
attaching substituents to the ortho-position of the aromatic substrate induces steric effects,
causing either substrate re-positioning in the active site of 4-OT or a stereofacial shielding
effect. Introducing electron donating groups at the meta- and para-position of nitrostyrene
derivatives could significantly enhance the catalytic rates (up to 5-fold improvement in kcat).
Together with the ongoing enzyme engineering studies in our group, this ‘substrate
engineering’ work is an important step towards our aim of developing novel “Michaelases”
for carbon-carbon bond-forming Michael-type addition reactions with high efficiency and
stereoselectivity. Finally, the 4-OT-catalyzed reaction between 1 and 2b does not give γ-
nitroaldehyde 3b as final product; instead, it yields 4-nitromethyl-chroman-2-ol (5) as a result
of hemiacetalization between the aldehyde and hydroxyl groups of presumed intermediate 3b.
This Michael-type addition-cyclization cascade reaction provides exciting options for the
enzymatic synthesis of precursors for chromans and derivatives, which are valuable scaffolds
for preparing biologically active natural products.
10
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10.1002/ejoc.201601126
Experimental Section
For experimental procedures and characterization of compounds, see Supporting Information.
Acknowledgement: This research was financially supported by the European Research
Council under the European Community's Seventh Framework Programme (FP7/2007-
2013)/ERC Grant agreement n° 242293 (to G. J. P.). We thank C. M. Jeronimus-Stratingh
(University of Groningen) for her expert assistance in acquiring mass spectrometry data and
Marcel P. de Vries (University of Groningen) for his expert assistance in acquiring exact MS
data.
11
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European Journal of Organic Chemistry
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European Journal of Organic Chemistry
10.1002/ejoc.201601126
Keywords
Catalytic promiscuity; Michael-type additions; substrate engineering; γ-nitroaldehydes; γ-
aminobutyric acids
Table of Contents Entry
Key Topic: Enzyme Promiscuity
Short text: The enzyme 4-oxalocrotonate tautomerase promiscuously catalyzes the Michael-
type addition of acetaldehyde to β-nitrostyrene derivatives. Highly enantioenriched (R)- or
(S)-γ-nitroaldehydes and 4-substituted chroman-2-ol can be obtained with good to excellent
yields by applying different substituents at appropriate positions of the aromatic substrate.
TOC graphic:
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