Enzymatic synthesis of chiral γ-amino acids using ω-transaminase

Article abstract of DOI:10.1039/c3cc44864a

In this study, we successfully synthesized enantiomerically pure (R)- and (S)-γ-amino acids (>99% ee) using ω-transaminase (ω-TA) through kinetic resolution and asymmetric synthesis respectively. The present study demonstrates the high potentiality of ω-TA reaction for the production of chiral γ-amino acids.

Full text of DOI:10.1039/c3cc44864a

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Enzymatic synthesis of chiral c-amino acids using
x-transaminase†
Cite this: Chem. Commun., 2014,
50, 12680
Minsu Shon,‡a Ramachandran Shanmugavel,‡b Giyoung Shin,a Sam Mathew,a
Sang-Hyeup Lee*b and Hyungdon Yun*c
Received 28th June 2013,
Accepted 22nd August 2014
DOI: 10.1039/c3cc44864a
www.rsc.org/chemcomm
In this study, we successfully synthesized enantiomerically pure of chiral g-amino acids have not been studied well. Recently, o-TA
(R)- and (S)-c-amino acids (499% ee) using x-transaminase (x-TA) catalyzed reaction has gained a lot of attention due to its ability to
through kinetic resolution and asymmetric synthesis respectively. generate enantiomerically pure amines and unnatural amino acids.9
The present study demonstrates the high potentiality of x-TA Desirable characteristics like broad substrate specificity, high enantio-
reaction for the production of chiral c-amino acids.
selectivity, high turnover number and non-requirement for regenera-
tion of external cofactors have made o-TA an attractive biocatalyst.9
Non-natural g-amino acids have attracted a great deal of atten- Some o-TAs such as a o-TA from Polaromonas sp. (o-TAPO)
tion because of their important role in the design and synthesis showed a broad substrate specificity toward amines, L-amino
of bioactive molecules and in the application of hybrid pep- acids and b-amino acids, suggesting that those enzymes may
tides.1 For example, g-amino acids are key components of also be reactive towards g-amino acids.10,11 Here, we report an
natural products such as hapalosin,2 dolastatin,3 caliculins4 alternative method for the synthesis of (R)- and (S)-g-amino
and of various enzyme inhibitor g-aminobutyric acid analo- acids via kinetic resolution and asymmetric synthesis using
gues.1 Given the significance of g-amino acids, their efficient o-TAs, respectively, (Scheme 1).
synthesis in optically pure form has become an attractive
The genes of o-TAPO11 and a newly identified o-TA from
challenge to organic chemists and biologists. Lately, a number Burkholderia graminis C4D1M (accession #; ZP_02885261,
of strategies have been developed for the chemical synthesis o-TABG) were cloned into the vector pET24ma with a C-terminal
of enantiomerically pure g-amino acids.5 Recently, peptide- His6-tag and effectively expressed in Escherichia coli BL21 (DE3).
catalyzed asymmetric Michael addition of nitromethane to The enzymes were then purified on a Ni-NTA affinity column
b-disubstituted a,b-unsaturated aldehydes5a and enantioselective (Fig. S1, ESI†). The purified enzymes were then subjected to activity
g-amination of a,b-unsaturated acyl chlorides with azodicarboxyl- assay which was performed with 10 mM (S)-b-phenylalanine and
ates5b were developed to synthesize chiral g-amino acid derivatives. 20 mM pyruvate (a typical amino acceptor) in 200 mM Tris/HCl
Enzyme-catalyzed chemical transformations are now buffer (pH 7.0) at 37 1C. The specific activities of o-TAPO and
widely recognized as practical alternatives to traditional organic o-TABG were 5.6 and 13.6 U mgÀ1, respectively (Fig. 1).
synthesis.6 A number of biocatalytic routes were developed for
rac-g-Amino acids (1a–g) were prepared from their corre-
the synthesis of chiral a- and b-amino acids using enantio- sponding, commercially available g-keto acids via a two step
selective enzymes such as transaminase (TA), hydantoinase, dehy-
drogenase, aminoacylase, penicillinG amidase, b-aminopeptidase,
lipase, phenylalanine aminomutase, and Baeyer–Villiger
monooxygenase.7,8 However, biocatalytic routes for the synthesis
a School of Biotechnology, Yeungnam University, Gyeongsan, Gyeongbuk,
South Korea
b Department of Life Chemistry, Catholic University of Daegu, Gyeongbuk 700-443,
Korea. E-mail: leeshh@cu.ac.kr
c Department of Bioscience & Biotechnology, Konkuk University, Seoul 143-701,
Korea. E-mail: hyungdon@konkuk.ac.kr; Fax: +82-2-450-0686; Tel: +82-2-450-0496
Scheme 1 Synthesis of enantiomerically pure g-amino acid by using
o-TA. (a) Kinetic resolution of g-amino acids. (b) Asymmetric synthesis of
g-amino acids.
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c3cc44864a
‡ These authors contributed equally to this work.
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Table 1 Kinetic resolution of g-amino acidsa
o-TAPO
o-TABG
Sub. Time (h) Conv. (%) eeR (%) Time (h) Conv. (%) eeR (%)
1a
1b
1c
1d
1e
24
24
24
24
24
50.1
50.3
50.1
n.c.d
50.1
49.6
46.5
50.1
499
499
499
—
499
98
24
24
24
24
24
24
36
24
50.0
50.5
50.2
n.c.
50.2
50.2
45.6
50.2
499
499
499
—
499
499
84
1fb,c 24
1g
36
24
87
499
1gc
499
Fig. 1 Substrate scope for deamination of o-TAPO and o-TABG. Enzyme
reactions were carried out in 1 mL of 200 mM Tris/HCl buffer (pH 7.0)
containing 10 mM racemic substrate (1a–g), 20 mM pyruvate, 0.1 mM PLP
and o-TA (0.02 mg mLÀ1) for 30 min at 37 1C. One unit is defined as the
amount of enzyme that catalyzes depletion of 1 mmol of pyruvate, beta-
Phe and (S)-b-phenylalanine.
a
Enzyme reactions were carried out at 37 1C in 1 mL of 200 mM Tris/HCl
buffer (pH 7.0) containing 100 mM g-amino acid, 200 mM pyruvate,
DMSO (10%), and 1 mM PLP by using o-TAPO (0.2 mg mLÀ1) or o-TABG
(0.4 mg mLÀ1); conv. and ee were determined by HPLC. 50 mM 1f.
b
c
d
0.68 mg mLÀ1 of o-TA was used. No conversion.
To carry out the enzyme reaction at higher concentration,
dimethylsulfoxide (DMSO) was added into the reaction mixture
for increasing the solubility of the substrates. The kinetic
resolution of g-amino acids was carried out in 1 mL of 10%
DMSO (v/v) solution containing 100 mM g-amino acids
(Table 1). In the case of 1f, 50 mM 1f was used for the reaction
due to its low solubility. Except for 1d, all aromatic g-amino
acids were successfully resolved into the (R)-form by both
enzymes with a high ee value and ca. 50% conversion. In the
case of 1d, 10 mM rac-1d was successfully resolved into a (R)-g-
amino acid (499%) by both enzymes (Table S3, ESI†), however,
both enzymes did not show any reactivity in the presence of
100 mM 1d which may be due to severe substrate inhibition.
(S)-g-Amino acids were also produced using g-keto acids
(2a–g) as initial substrates via asymmetric synthesis (Scheme 1b).
Asymmetric synthesis has been of keen interest among researchers,
as it can theoretically generate two-fold higher yield (100%) than
that of the kinetic resolution. To identify the best amino donor for
o-TAs, the asymmetric syntheses of 1a from 10 mM 2a were carried
out with L-Ala (with or without a pyruvate removal system), b-Ala,
L-glutamic acid (2-ketoglutarate is a good amino acceptor for
o-TAPO),11 and (S)-a-methylbenzylamine (MBA, a typical substrate
process, oximation followed by catalytic hydrogenation of the
resultant oximes12 (ESI†). The amino donor specificities of
enzymes toward 1a–g were examined in the presence of 10 mM
rac-g-amino acids and 20 mM pyruvate (Fig. 1). Among 1a–g,
o-TAPO and o-TABG showed the highest reactivity for 1d
(5.5 U mgÀ1) and 1e (3.9 U mgÀ1), respectively. Both o-TAPO
(1.5 U mgÀ1) and o-TABG (0.36 U mgÀ1) showed the least
reactivity towards 1f. The lower activity of 1f and 1g towards
o-TAs may be due to the presence of bulky side chains which
cause steric hindrance in the active sites of the enzymes. o-TABG
showed about 2.4-fold higher specific activity towards (S)-b-
phenylalanine, while o-TABG showed only about 36% specific
activity towards 1a than that of o-TAPO. In addition, (R)-selective
o-TAs from Mycobacterium vanbaalenii13 and Neosartorya
fischeri,13 and (S)-selective o-TAs from Vibrio fluviallis JS1714 were
expressed in E. coli and purified. To examine the suitability of
these enzymes for the production of g-amino acids, enzyme
activities were measured using 1a as a representative g-amino
acid, and these enzymes did not show any reactivity towards 1a
(data not shown).
The kinetic resolution of 1a was carried out using o-TAPO
and o-TABG, and both enzymes converted only (S)-1a into 2a,
and enantiomerically pure (R)-1a (499% ee) was obtained
using both the enzymes in the presence of 10 mM rac-1a and
20 mM pyruvate (Fig. S2, ESI†). o-TAPO showed a faster reac-
tion profile than o-TABG. Subsequently, the kinetic resolution
of various g-amino acids (1a–g) using o-TAs was carried out
under similar conditions as described above (Table S3, ESI†).
All racemic substrates except 1f and 1g were successfully
resolved into (R)-g-amino acids (499% ee) with about 50% con-
version using o-TAPO (0.02 mg mLÀ1) and o-TABG (0.04 mg mLÀ1).
In the case of 1f and 1g, higher concentration of enzymes
(0.2 mg mLÀ1) was required to reach 499% ee due to its lower
reactivity towards these substrates. The enantioselectivities
(Es) of the enzymes were calculated using the equation E =
ln[(1 À c)(1 À ees)]/ln[(1 À c)(l + ees)]15 and were found to be
4100 for all substrates. This result indicates that o-TAPO and
o-TABG are suitable biocatalysts for the production of highly
enantiomerically pure g-amino acids.
Fig. 2 Asymmetric synthesis of 1a from 2a using various amino donors.
Enzyme reactions were carried out at 37 1C in 1 mL of 200 mM Tris/HCl
buffer (pH 7.0) containing 100 mM amino donor, 10 mM 2a, 0.1 mM PLP by
using o-TA (0.34 mg mLÀ1) for 16 h. Conversion was determined by HPLC.
In the case of L-Ala/LDH, 200 mM glucose, 1 mM NADH, lactate dehydro-
genase (LDH, 55 U), and glucose dehydrogenase (2.7 U) were added in the
L-Ala reaction solution for removal of pyruvate.
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for o-TA)9 (Fig. 2). o-TAs gave the highest conversion with (S)-a-MBA, 80 mL of 20 mM phosphate buffer (pH 7.0) containing 10 mM
and o-TAPO gave higher conversion (56.1%) than that of o-TABG 2a, 100 mM (S)-a-MBA, and 0.1 mM PLP using purified o-TAPO
(25.3%). Thus, (S)-a-MBA was selected as the best amino donor for (8 mg), and carefully 40 mL of iso-octane was added to 80 mL of
the asymmetric synthesis. When asymmetric synthesis was carried the aqueous reaction mixture. After 24 h of reaction, HPLC
out with 100 mM 2a, 100 mM (S)-a-MBA and o-TAPO (1 mg mLÀ1), analysis showed 93% conversion with 499% ee. 1a was isolated
the conversion was o3% after 24 h of reaction. Therefore, the from this reaction with 38% yield (ESI†).
substrate inhibition of o-TA by 2a was examined with 50 mM
In conclusion, enantiomerically pure (R)- and (S)-g-amino
(S)-a-MBA and various concentrations of 2a (0–100 mM) (Fig. S3, acids (499% ee) were produced using o-TAs through kinetic
ESI†). The initial reaction rates of o-TAPO and o-TABG were the resolution and asymmetric synthesis respectively. The present
highest at 5 mM 2a, and thereafter decreased with an increase in study demonstrates the high potentiality of o-TA reaction for
the concentration of 2a. In the presence of 10 mM 2a, o-TAPO the production of chiral g-amino acids. We are currently devel-
and o-TABG showed, respectively, 74 and 87% activities of those oping an asymmetric synthesis method to overcome product
with 5 mM 2a.
inhibition by using a coupling reaction.
Next, the asymmetric syntheses were carried out with 10 mM
This research was partially supported by Basic Science
2a due to severe substrate inhibition at higher concentration of Research Program through the National Research Foundation
2a (410 mM). Here, (S)-a-MBA was used as the amino donor for of Korea (NRF) funded by the Ministry of Education, Science and
the asymmetric synthesis of g-amino acids. The product inhibi- Technology (2013R1A2A2A01068013). And this research was
tion by acetophenone (deaminated product of a-MBA) is well partially supported by a grant (A10301712131560200) of Global
reported.9 To overcome the product inhibition by acetophenone, a Cosmetics R&D Project, Ministry of Health & Welfare, Korea.
biphasic reaction system with iso-octane was introduced (Fig. S4,
ESI†).14,16 In the biphasic system, g-amino acid (1a), g-keto acid (2a),
and (S)-a-MBA existed in the aqueous solution due to their
Notes and references
´˜
electric charges at neutral pH while the inhibitory acetophenone
gets transferred to the iso-octane layer. In the biphasic reaction
system, the biphasic mixture was gently shaken in a shaking
incubator with 250 rpm at 37 1C. As expected, the biphasic
reaction system gave higher conversion of the product (97%)
than the monophasic reaction system (80%). Subsequently,
asymmetric syntheses of various g-amino acids were carried
out in monophasic and biphasic systems (Table 2). Both systems
gave enantiomerically pure (S)-g-amino acids (499% ee). For all
the substrates, o-TAPO gave higher conversion than o-TABG,
which indicates that o-TAPO is a more suitable catalyst than
o-TABG for the asymmetric synthesis of g-amino acids. In most
of the cases, the biphasic system gave a higher conversion. It is
notable that 1f and 1g were less reactive substrates for the
kinetic resolution, but in asymmetric synthesis, the conversion
of 2f and 2g with o-TAPO was higher than other substrates.
Finally a preparative scale reaction was carried out at 37 1C in
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´
´
´
´
Table 2 Asymmetric synthesis of g-amino acids
´
¨
8 For selected examples, see: (a) H. Groger, H. Trauthwein,
o-TAPO
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Monophasica
Biphasicb
Conv. eeS
Monophasic
Conv. eeS
Biphasic
Conv. eeS
Conv.
Sub. [%]
eeS
[%]
[%]
[%]
[%]
[%]
[%]
[%]
2a
2b
2c
2d
2e
2f
77.6
75.1
72.1
72.9
52.7
85.3
99.9
499
499
499
499
499
499
499
95.8
94.3
91.0
88.5
67.5
99.0
99.9
499 14.0
499 17.3
499 14.7
499
499
499
499 23.0
499 17.2
499 27.7
499 14.5
499
499 23.4
499 57.6
499
499
499
499
3.21
5.10
1.02
8.45 499
499
499
2g
499 48.9
a
Enzyme reactions were carried out at 37 1C in 1 mL of 200 mM Tris/
HCl buffer (pH 7.0) containing 10 mM substrate, 20 mM (S)-a-MBA,
0.1 mM PLP for 36 h by using o-TAPO (0.34 mg mLÀ1) or o-TABG
(0.68 mg mLÀ1); conv. and ee were determined by HPLC (see the ESI). b 1 mL
of iso-octane was added to 1 mL of the aqueous reaction mixture.
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