Kinetic resolution of aromatic β-amino acids by ω-transaminase

Article abstract of DOI:10.1039/c1cc11528f

Racemic aromatic β-amino acids have been kinetically resolved into (R)-β-amino acids with high enantiomeric excess (>99%) by a novel ω-TA with ca. 50% conversion.

Full text of DOI:10.1039/c1cc11528f

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COMMUNICATION
Kinetic resolution of aromatic b-amino acids by x-transaminasew
Han-Seop Bea,a Hye-Jeong Park,b Sang-Hyeup Lee*b and Hyungdon Yun*a
Received 17th March 2011, Accepted 24th March 2011
DOI: 10.1039/c1cc11528f
Racemic aromatic b-amino acids have been kinetically resolved
into (R)-b-amino acids with high enantiomeric excess (>99%)
by a novel x-TA with ca. 50% conversion.
acid or an a-keto acid.11 Nowadays, o-TAs have gained
increasing interest owing to their high utility in the production
of chiral amines through kinetic resolution and asymmetric
synthesis.10–12 Recently, it was reported that o-TAs from
Alcaligenes denitrificans2 and Mesorhizobium sp. LUK9 can
catalyze the transamination of aliphatic and aromatic b-amino
acids, respectively. The asymmetric synthesis of (S)-3-amino-
3-phenylpropanoic acid 1a from ethyl 3-oxo-3-phenyl-
propanoate has been carried out by using an o-TA/lipase
coupled system with 20% yield from 10 mM substrate.9
However, the kinetic resolution of various aromatic b-amino
acids with o-TA has not been reported. In this study, we
identified a novel o-TA that is reactive towards various
aromatic b-amino acids and successfully demonstrated the
kinetic resolution of various aromatic b-amino acids 1a–e into
(R)-b-amino acids with >99% ee (Scheme 1).
To isolate the novel o-TA, we performed a BLAST search
(http://blast.ncbi.nlm.gov/Blast.cgi) and identified a candidate
gene using the protein sequence of o-TA from Mesorhizobium
sp. LUK. Among the homologue genes, the putative glutamate-
1-semialdehyde 2,1-aminomutase (protein_id = ‘‘ABE43415.1’’)
from Polaromonas sp. JS666 (54% identity and 99% query
coverage) was selected. In this communication, the enzyme has
been newly renamed as Po o-TA (vide infra). Po o-TA
contains the conserved residues in o-TA, such as K278 (Schiff
base formation with PLP), D251 (a hydrogen bond to PLP)
and R403 (a salt bridge with a-carboxylate group).9,10
Optically active a- and b-amino acids are the fundamental
building blocks for the preparation of pharmaceutical and
agrochemical target molecules, such as peptides, proteins and
many other natural products.1,2 In particular, the biologically
active b-peptides have great potential because of their
enhanced stability towards proteolytic enzymes, and this offers
new possibilities in the creation of drugs that are not rejected
or degraded by the human body.3 Given the significance of the
b-amino acids, their efficient synthesis in optically pure form
has become an attractive challenge to organic chemists and
biologists in recent years.1 Biocatalytic methods for the
production of chiral b-amino acids fall into several classes
depending on the enzymatic reactions: (i) aminoacylase-
catalyzed hydrolysis of N-acetylated b-amino acids,4 (ii) PenG
amidase-catalyzed acylation of b-amino acid and deacetylation
of N-phenylacetylated b-amino acids,5 (iii) b-aminopeptidase-
catalyzed kinetic resolution of aliphatic b-amino acid amides,6
(iv) lipase and esterase-catalyzed deacylation of b-amino acid
esters,7 (v) phenylalanine aminomutase-catalyzed enantio-
selective addition of ammonia to substituted cinnamic acids,8
(vi) Baeyer–Villiger monooxygenase-catalyzed formation of
b-amino acids3 and (vii) transaminase (TA)-catalyzed kinetic
resolution of b-amino acids2 and asymmetric synthesis from
b-keto esters.9
The gene of Po o-TA was cloned into the vector pET24ma
with N-terminal His6-tag and effectively expressed in
Escherichia coli BL21 (DE3).10 The enzyme was then purified
on a Ni-NTA affinity column (Fig. S1, ESIw). The purified
enzyme was then subjected to o-TA activity assay performed
with 10 mM (S)-a-methylbenzylamine (MBA, a typical
substrate for o-TA) and 10 mM pyruvate in 100 mM phosphate
buffer (pH 7.0). The purified enzyme displayed high activity
(2.4 U mgÀ1) which confirmed that Po o-TA falls into a o-TA
class.11 The Po o-TA showed the highest activity at pH 8.5.
TAs have been studied for the production of chiral amino
acids because they generally show rapid reaction rates, broad
substrate specificity, and high enantioselectivity.10 Generally
TAs can be classified into a-TAs and o-TAs according to the
relative position of the amino group that is to be transferred
with respect to the carboxyl group of the substrate. o-TAs are
defined as enzymes that transfer an amino group from an
amino donor onto a carbonyl moiety of an amino acceptor,
whereby at least one of the two substrates is not an a-amino
a School of Biotechnology, Yeungnam University, Gyeongbuk,
712-749, Korea. E-mail: hyungdon@ynu.ac.kr;
Fax: +82-53-810-4769
b Department of Life Chemistry, Catholic University of Daegu,
Gyeongbuk 712-702, Korea. E-mail: leeshh@cu.ac.kr;
Fax: +82-53-850-3781
w Electronic supplementary information (ESI) available: Experimental
details, preparation of the enzyme, HPLC analysis. See DOI: 10.1039/
c1cc11528f
Scheme 1 The kinetic resolution of aromatic b-amino acids using
Po o-TA.
c
5894 Chem. Commun., 2011, 47, 5894–5896
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Table 1 Substrate specificity of Po o-TA
presence of 10 mM pyruvate. The apparent Km and kcat for
(S)-1a were 0.83 mM and 292 minÀ1, respectively (Fig. S3,
ESIw). Then, the kinetic parameters of the enzyme towards
pyruvate were analyzed in the presence of 10 mM (S)-1a,
the apparent Km and kcat for pyruvate were 47.4 mM and
1190 minÀ1, respectively (Fig. S4, ESIw). The kinetic resolution
of 10 mM 1a–e was carried out with 1 mL of 100 mM Tris/HCl
buffer (pH 8.5) containing Po o-TA (15.7 mg mLÀ1) and
10 mM pyruvate. The conversion was analyzed using a
Crownpak CR column (Daicel Co., Japan), and the ee values
of 1a–e were determined by a C18 Symmetry column (Waters,
MA) after the derivatization with 2,3,4,6-tetra-O-acetyl-b-D-
glucopyranosyl isothiocyanate (GITC)2 (Table S1, ESIw). The
ee values of 1a–e reached >99% with a conversion of about
50–51% after 3 h (Table S2, ESIw). The enantioselectivity (E)
was calculated using the equation E = ln[(1 À c)(1 À ees)]/
In[(1 À c)(l + ees)]14 and was found to be >100%. This
indicates that Po o-TA is a suitable biocatalyst for the kinetic
resolution of various aromatic b-amino acids.
Enzyme inhibition by the keto product was the major hurdle
to overcome and carry out efficient o-TA reactions.10 To
investigate the product inhibition, Po o-TA activity was
analyzed with acetophenone, which is one of the most well
reported carbonyl compounds that inhibits o-TA activity.10 It
is well known that 2a (deaminated product of 1a) is easily
converted to acetophenone by spontaneous decomposition
(decarboxylation). Thus, the product inhibition was examined
in the presence of 10 mM 1a and 10 mM pyruvate with
different concentrations of acetophenone (0–10 mM). VF
o-TA lost almost 70% of its activity in the presence of
10 mM acetophenone.10 However, there was no product
inhibition up to 10 mM acetophenone, which indicates that
Po o-TA can be useful for the kinetic resolution of high
concentrations of aromatic b-amino acids (Fig. S6, ESIw).
Thus, the kinetic resolution of 1a–e was carried out with
40 mM concentration of substrates, which was close to the
maximum solubility of the b-amino acids in this reaction
media. All aromatic b-amino acids 1a–e were successfully
resolved into (R)-form with a high ee (>99%) (Table 2).
Generally, enzymatic reactions are carried out under
conditions in which the substrate is completely dissolved in
the reaction media. Usually for higher concentration reactions
(in terms of solubility of the substrate), the reaction media is
optimized by adding organic solvent to increase the solubility of
the substrate. Increasing the solubility of aromatic b-amino acids
Substrate
Specific activityc/U mgÀ1
Amino donora
(S)-1a
rac-1b
3.92
5.53
1.19
0.84
0.67
9.73
2.24
9.66
16.4
rac-1c
rac-1d
rac-1e
rac-b-homoalanine
rac-b-homoleucine
(S)-a-MBA
b-Alanine
Amino acceptorb
Pyruvate
9.66
8.19
10.43
o0.05
2.03
a-Ketoglutarate
Benzaldehyde
Trimethylpyruvate
2-Oxobutyrate
Fluoropyruvate
3.22
a
Reaction conditions: 1 mL reaction volume, 10 mM amino donor,
10 mM pyruvate, Po o-TA (0.32 mg mLÀ1), Tris/HCl buffer (100 mM,
b
pH 8.5), incubation at 37 1C for 30 min. Reaction conditions: 1 mL
reaction volume, 10 mM amino acceptor, 10 mM (S)-a-MBA,
Po o-TA (0.32 mg mLÀ1), Tris/HCl buffer (100 mM, pH 8.5), incubation
c
at 37 1C for 30 min. One unit is defined as the amount of enzyme that
catalyzes the formation of 1 mmol of L-alanine or acetophenone.
This slightly alkaline pH for the optimal enzyme activity was
also observed with o-TA from Vibrio fluvialis JS17 (VF o-TA,
most extensively studied one),10 which was likely due to the
increase in the effective concentration of the deprotonated
amino donor that enhances the formation of an external
aldimine.
To explore the applicability of Po o-TA for the kinetic
resolution of aromatic b-amino acids, the substrate specificity
of Po o-TA was examined. The racemic compounds 1a–e were
synthesized by a modified Rodionov synthesis from the
corresponding aromatic aldehydes by condensation with
malonic acid in the presence of ammonium formate, the
mixture was then heated at reflux in EtOH.13 Nine amino
donors were analyzed among which b-alanine (16.4 U mgÀ1
)
showed the highest reactivity (Table 1). Given that b-amino
acids are good substrates for Po o-TA, its amino acceptor
specificity was investigated with six carbonyl compounds (five
a-keto acids, one aldehyde). It should be noted that this
enzyme showed similar reactivity towards pyruvate and
a-ketoglutarate because TA generally showed a clear one side
preference to either pyruvate or a-ketoglutarate/oxaloacetate.10
Interestingly, 2-oxobutyric acid and fluoropyruvate are good
amino acceptors for Po o-TA, which indicates that the enzyme
is applicable for the production of valuable L-2-aminobutyric
acid and L-fluoroalanine. Indeed, HPLC analysis showed that
only the L-form of amino acids was produced. However,
trimethylpyruvate was almost inert, suggesting that the
active site of the enzyme has a steric constraint that cannot
accommodate the bulky side chains of a-keto acids. In
contrast to a-TAs, which exclusively use a-keto acids as an
amino acceptor, o-TAs showed distinctive reactivity towards
aldehydes. Indeed, Po o-TA showed high reactivity towards
an aromatic aldehyde (benzaldehyde).
Table 2 Preparation of (R)-1a–e using Po o-TA-catalyzed kinetic
resolution of racemic substratea
Substrate
t/h
Conv.b (%)
eec (%)
E
1a
1b
1c
1d
1e
3
3
6
6
6
50.7
50.5
50.8
50.9
51.2
>99
>99
>99
>99
>99
>100
>100
>100
>100
>100
a
Reaction conditions: 1 mL reaction volume, 40 mM rac-1a–e, 40 mM
pyruvate, Po o-TA (63 mg mLÀ1), Tris/HCl buffer (100 mM, pH 8.5)
b
and incubation at 37 1C. Conversion was determined by a Crownpak
CR column. ee was determined by C18 Symmetry column after
GITC derivatization.
c
For kinetic analysis, the initial rate of enzyme was analyzed
with various concentrations of (S)-1a (0–5 mM) in the
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 5894–5896 5895

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This research was supported by Basic Science Research
Program through the National Research Foundation of Korea
(2010–0006343).
Notes and references
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Fig. 1 The kinetic resolution of rac-1a with an amount of substrate
above the solubility limit in the reaction media. Reaction conditions:
100 or 200 mM rac-1a, equivalent pyruvate, Po o-TA (0.16 mg mLÀ1
for 100 mM rac-1a and 0.24 mg mLÀ1 for 200 mM rac-1a), Tris/HCl
buffer (100 mM, pH 8.5), shaking at 37 1C.
is not easily achievable by adding organic solvent and it might
also be toxic to the enzyme. Therefore, rac-1a (100 or 200 mM)
was added as solid powder directly into the 5 mL reaction
media containing Po o-TA and equivalent pyruvate, and the
resulting cloudy mixture was shaken at 37 1C. During the
reaction, (S)-1a will be converted into 2a, and the remaining
substrate will be dissolved in the reaction media until its
solubility reaches to saturation state. Also, a dynamic equilibrium
exists between the dissolved and undissolved form of substrates.
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52% conversion (Fig. 1).
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In addition, the asymmetric synthesis was carried out with
10 mM ethyl 3-oxo-3-phenylpropanoate and 200 mM amino
donor (L-alanine or b-alanine) in 100 mM phosphate buffer
(10 mL, pH 7.0) containing lipase from Candida rugosa
(0.14 mg mLÀ1, Sigma, L1754) and Po o-TA (0.077 mg mLÀ1
)
at 37 1C.9 2a was produced from ethyl 3-oxo-3-phenyl-
propanoate by lipase-catalyzed hydrolysis and was converted
into 1a by Po o-TA (Fig. S7, ESIw). After a 24 h reaction,
5.2 and 1.8 mM (S)-1a (>99%) were produced with b-alanine
and L-alanine, respectively. b-alanine as amino donor showed
the higher yield than L-alanine and also gave 2.6-fold higher
yield than the reported result (20% yield from 10 mM
substrate).9 During the reaction, 3-oxo-propanoic acid
(deaminated product of b-alanine) would be spontaneously
decomposed into acetaldehyde and carbon dioxide. This might
drive the reaction in the forward direction.
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In summary, we demonstrated the kinetic resolution of
various aromatic b-amino acids and the asymmetric synthesis
of aromatic b-amino acids from b-keto esters using a novel
o-TA. Further development of asymmetric synthesis of chiral
amines and b-amino acids by Po o-TA is currently being
investigated.
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c
5896 Chem. Commun., 2011, 47, 5894–5896
This journal is The Royal Society of Chemistry 2011