ω-Transaminase-catalyzed kinetic resolution of chiral amines using l-threonine as an amino acceptor precursor

Article abstract of DOI:10.1039/c2gc35615e

Kinetic resolution of chiral amines using l-threonine as a cosubstrate was demonstrated by a biocatalytic strategy in which (S)-selective ω-transaminase (ω-TA) was coupled with threonine deaminase (TD), eliminating the need to use an expensive keto acid as an amino acceptor. The coupled enzyme reaction enabled simultaneous production of enantiopure (R)-amine and l-homoalanine which are pharmaceutically important building blocks. To extend the versatility of this strategy to production of both enantiomers of chiral amines, (R)-selective ω-TA coupled with TD was employed to produce (S)-amine.

Full text of DOI:10.1039/c2gc35615e

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ω-Transaminase-catalyzed kinetic resolution of chiral amines using
L-threonine as an amino acceptor precursor†
M. Shaheer Malik, Eul-Soo Park and Jong-Shik Shin*
Received 6th February 2012, Accepted 22nd May 2012
DOI: 10.1039/c2gc35615e
synthesis of chiral amines starting with prochiral ketones is
Kinetic resolution of chiral amines using L-threonine as a
cosubstrate was demonstrated by a biocatalytic strategy in
which (S)-selective ω-transaminase (ω-TA) was coupled with
threonine deaminase (TD), eliminating the need to use an
expensive keto acid as an amino acceptor. The coupled
enzyme reaction enabled simultaneous production of enan-
tiopure (R)-amine and L-homoalanine which are pharmaceu-
tically important building blocks. To extend the versatility of
this strategy to production of both enantiomers of chiral
amines, (R)-selective ω-TA coupled with TD was employed to
produce (S)-amine.
usually preferred over kinetic resolution of racemic amines
owing to a 2-fold higher maximum yield. However, several
drawbacks of the asymmetric synthesis, typically very low
amino acceptor reactivity of ketones and unfavorable reaction
equilibrium,16,18,25,26 render the kinetic resolution approach
often more suitable for practical preparation of enantiopure
amines using the ω-TA. Indeed, the kinetic resolution approach
benefits by much faster reaction rate, high solubility of amine
substrate at neutral pH, use of stoichiometric amounts of co-
substrate and favorable reaction equilibrium.20
When setting up a kinetic resolution process, choice of an
amino acceptor is crucial to the feasibility of industrial process
scale-up. Considering amino acceptor reactivity, α-keto acids
such as pyruvate and aliphatic aldehydes such as propanal would
be good cosubstrate options.25 We tested propanal, in compari-
son with pyruvate, for kinetic resolution of α-methylbenzyl-
amine (α-MBA) with (S)-selective ω-TA from Ochrobactrum
anthropi,23 because propanal shows reactivity as good as pyru-
vate (i.e. 94% relative activity toward propanal compared with
pyruvate)‡ but is much cheaper. Although the amino acceptor
reactivities based on initial rate measurements were similar, pro-
panal turned out to be an inefficient amino acceptor for the
kinetic resolution (Fig. 1). Enantiomeric excess (ee) of (R)-
α-MBA reached >99% with pyruvate at 3 h, whereas ee was
only 57% with propanal even after the reaction for 24 h. This
result is ascribed to severe enzyme inactivation by propanal.§
Production of optically pure chiral compounds has attracted
growing attention in the pharmaceutical industry owing to strict
regulatory requirements for preparation of single enantiomer
drugs.1–4 Among the chiral compounds, chiral amines are used
as indispensable building blocks for a number of pharmaceutical
drugs including (S)-rivastigmine for treatment of Alzheimer’s
disease,5,6 dilevalol as an antihypertensive drug,7 sitagliptin as
an antidiabetic drug8 and mexiletine as an antiarrhythmic and
antimyotonic drug.9–11 Owing to the pharmaceutical importance
of the chiral amines, biocatalytic approaches to prepare the
enantiopure amines such as kinetic resolution,12–14 asymmetric
synthesis6,15–18 and deracemization11,19 have been extensively
studied for developing greener alternatives to chemical
processes4 including preferential crystallization and asymmetric
catalytic hydrogenation. ω-Transaminase (ω-TA) has been
proven to be a promising enzyme enabling all the three prepara-
tive approaches.20 ω-TA displays high turnover rate, stringent
enantioselectivity, high stability and no requirement of external
cofactor regeneration, which renders the enzyme attractive for
industrial process development.20,21 In addition to the chiral
amines, applicability of the ω-TA reactions has been extended
to the preparation of unnatural amino acids of pharmaceutical
interest, which is well summarized in a recent review paper.22
In the ω-TA reaction, an amino group of primary amines (i.e.
amino donor) is transferred to a carbonyl group of keto acids,
aldehydes or ketones (i.e. amino acceptor).23,24 Asymmetric
Department of Biotechnology, Yonsei University, Shinchon-Dong 134,
Seodaemun-Gu, Seoul 120-749, South Korea.
E-mail: enzymo@yonsei.ac.kr; Fax: +82-2-362-7265;
Tel: +82-2-2123-5884
†Electronic supplementary information (ESI) available: Experimental
procedures and analysis details. See DOI: 10.1039/c2gc35615e
Fig. 1 Comparison of pyruvate and propanal as an amino acceptor for
the kinetic resolution of α-MBA. Reaction conditions were α-MBA
(100 mM), sodium pyruvate or propanal (80 mM), PLP (0.1 mM), phos-
phate buffer (50 mM, pH 7.5) and 5 U ml−1 ω-TA from O. anthropi at
37 °C.
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In addition to the severe enzyme inactivation, chemical toxi-
city of aldehydes to environments renders propanal not suited to
a green process. Therefore, α-keto acids such as pyruvate should
be a better option for the kinetic resolution process. Moreover,
exploitation of α-keto acids as an amino acceptor provides an
additional benefit to the kinetic resolution by enabling simul-
taneous production of two products of commercial interest rather
than just one valuable product.27,28 For example, kinetic resolu-
tion of α-MBA with pyruvate by (S)-selective ω-TA leads to pro-
duction of enantiopure (R)-α-MBA as well as L-alanine.
However, there is one serious problem for using α-keto acids as
a cosubstrate: α-keto acids are too expensive to allow develop-
ment of a cost-effective kinetic resolution process. To overcome
this limitation, it is highly demanded to design a new kinetic res-
olution process that implements generation of the α-keto acid
from a cheap precursor. The purpose of this study is to realize
the one-stone-two-birds strategy, permitting production of two
valuable products and exploitation of a cheap substitute for the
α-keto acid.
Scheme 1 One-pot conversion of racemic α-MBA and L-threonine to
(R)-α-MBA and L-homoalanine using (S)-selective ω-TA and TD.
Table 1 Amino donor reactivity of the racemic aminesa
Substrate
R1
R2
Relative reactivityb (%)
We searched for a keto acid allowing facile generation from a
cheap chemical as well as displaying high amino acceptor reac-
tivity. We envisioned that 2-oxobutyrate was such an ideal amino
acceptor based on the following reasons. First, 2-oxobutyrate can
be easily generated from L-threonine by threonine deaminase
(TD) as demonstrated in our previous report.29 Second, L-threo-
nine is one of the cheap amino acids that are produced on a
million-ton scale annually.30 Third, 2-oxobutyrate is as reactive
as pyruvate which is a typical amino acceptor for all known
ω-TAs. For example, the ω-TA from O. anthropi showed 78%
relative activity toward 2-oxobutyrate compared with pyruvate.23
Finally, L-homoalanine, the amination product of 2-oxobutyrate,
is a pharmaceutically important unnatural amino acid serving as
a key intermediate in the synthesis of antitubercular drug etham-
butol31 and antiepileptic drug levetiracetam.32 Owing to the
importance of L-homoalanine as a pharmaceutical intermediate, a
fermentative approach to produce L-homoalanine was recently
developed by redirecting a metabolic pathway of Escherichia
coli.33
We set out to explore the feasibility of the ω-TA reaction for
simultaneous production of enantiopure (R)-amine and L-homo-
alanine via kinetic resolution of racemic amine with concurrent
stereoselective amination of 2-oxobutyrate generated from
L-threonine by the action of TD. In the coupled enzyme reaction
employing racemic α-MBA (1a) and L-threonine (2) as substrates
(Scheme 1), (S)-selective ω-TA carries out oxidative deamination
of (S)-1a as well as reductive amination of 2-oxobutyrate (3),
leading to enrichment of (R)-1a and generation of L-homoalanine
(4) and acetophenone (5). 2 serves as a precursor of 3 which is
used as an amino acceptor for the ω-TA reaction and conse-
quently converted to 4.
1a
1b
1c
1d
1e
C6H5
p-F-C6H4
C6H5
CH3
CH3
CH2CH3
CH3
100c
91
18
C6H5(CH2)2
70
64 (153)d
1f
CH3CH2
CH3
CH3
CH3
CH3
CH3
CH3
54
63
29
146
60
63
1g
1h
1i
1j
1k
Cyclopropyl
CH3(CH2)2
CH3(CH2)5
CH3OCH2
HOCH2
a Reaction conditions: racemic amine (10 mM), sodium pyruvate
(10 mM), PLP (0.1 mM), phosphate buffer (50 mM, pH 7.5) and 0.125
U ml−1 (S)-selective ω-TA from O. anthropi at 37 °C for 10 min. One
unit of ω-TA activity is defined as the enzyme amount catalyzing the
formation of 1 μmole of 5 in 1 min at 10 mM (S)-1a and 10 mM
pyruvate. b Relative reactivity represents the initial reaction rate
normalized by that of 1a. L-Alanine was analyzed by HPLC to measure
the initial rates. c Initial reaction rate for 1a was 0.033 mM min−1 d The
.
value in the parenthesis represents the reactivity of (S)-1e (10 mM)
relative to that of (S)-1a (10 mM), which was taken from the literature.23
ω-TA reactions.25 In the previous study, we proposed that the
active site of ω-TA carries two binding pockets, i.e. large (L) and
small (S) pockets.23,24 The S pocket was found to display a
steric constraint prohibiting entry of a substituent larger than an
ethyl group, whereas the L pocket can accept a bulk substituent
such as a phenyl group of 1a through a hydrophobic inter-
action.23 Consistent with the two binding site model, 1c showed
a 5-fold decrease in amino donor reactivity compared with 1a
owing to the steric constraint in the S pocket. In contrast, amino
donors carrying the same R2 substituent as 1a (i.e., 1b and 1d)
showed modest reactivity decreases. It is notable that 1e showed
a reactivity lower than 1a although reactivity of (S)-1e was
higher than (S)-1a. The discrepancy of the relative reactivities,
depending on the enantiomeric purity of the amine substrate,
seems to result from the different degree of enzyme inhibition by
the (R)-enantiomer of the chiral amines. In the previous study,
To carry out the coupled reaction, we overexpressed and
purified (S)-selective ω-TA from O. anthropi and TD from
E. coli as described elsewhere.23,29 Using the purified enzyme
preparation of the ω-TA, we measured amino donor reactivity of
five arylalkyl amines (1a–1e) and six alkyl amines (1f–1k)
(Table 1). We chose pyruvate as an amino acceptor for measure-
ments of the amino donor reactivity, because pyruvate is one of
the most reactive amino acceptors and thereby has been widely
used as a standard substrate for the initial rate measurements of
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Table 2 Effect of enzyme concentrations in the coupled reactiona
Table 3 Kinetic resolution of chiral amines via ω-TA/TD coupled
reactions using 2 as an amino acceptor precursora
Entry ω-TA (U mL−1
)
TDb (U mL−1
)
4c (mM) ee of (R)-1ad (%)
Chiral
Entry amine
Reaction
time (h)
Conversionb
(%)
ee of (R)-amineb
(%)
1
2
3
4
5
1.25
2.50
3.75
1.25
1.25
9
9
9
18
27
15
33
47
19
13
28
65
90
24
26
1
2
3
4
5
6
7
8
1a
1b
1c
1c
1d
1e
1f
1g
1h
1i
3
49.9
50.5
35.1
50.1
50.9
50.0
50.2
50.1d
50.5
37.4
50.2
50.6
40.8d
>99
>99
52
>99
>99
>99
>99
>99d
>99
58
3.5
28
30c
5
7
8
a Reaction conditions: 1a (100 mM), 2 (60 mM), PLP (0.1 mM),
phosphate buffer (50 mM, pH 7.5) at 37 °C for 2.5 h. b One unit of TD
activity is defined as the enzyme amount that catalyzes the formation of
1 μmole of 3 in 1 min from 2 (50 mM). c Concentration determined by a
standard calibration method from HPLC analysis of the GITC derivative.
d Determined by HPLC analysis of the GITC derivatives.
8
9
9
10
11
12
13
24
24c
9
1i
1j
1k
>99
>99
66d
30c
we found that (R)-amine, of which the (S)-counterpart displays
higher reactivity, inhibits enzyme activity more strongly.34 This
accounts for the reversal of the relative magnitude of the reactiv-
ity of 1e and (S)-1e compared with 1a and (S)-1a, respectively.
In the case of alkyl amines except 1i, amino donor reactivities
were lower than 1a presumably owing to weakened hydrophobic
interaction in the L pocket.
a Reaction conditions: 1a–1k (100 mM), 2 (60 mM), PLP (0.1 mM),
phosphate buffer (50 mM, pH 7.5), 3.75 U ml−1 (S)-selective ω-TA and
9 U ml−1 TD at 37 °C. b Determined by a standard calibration method
from HPLC analysis of GITC derivatives of corresponding amines.
c 10 U ml−1 ω-TA and 9 U ml−1 TD were used. d Determined by HPLC
analysis employing Marfey’s reagent.
To optimize coupled enzyme reactions, it is desirable to
achieve reaction conditions where each enzyme reaction occurs
at a similar rate, so a bottleneck step that limits the overall reac-
tion is not present. For example, the ω-TA reaction occurring
much slower than the TD reaction must result in accumulation of
the reaction intermediate (3). In this case, increase in the ω-TA
concentration allows the overall reaction to proceed faster. We
examined which enzyme reaction is rate-determining by carrying
out the coupled reaction at varying concentrations of either
enzyme while keeping the concentration of the other one con-
stant (Table 2). In the coupled reaction, we used a 20% higher
concentration of 2 (60 mM) than the stoichiometric amount
required for complete kinetic resolution of 1a (100 mM).
Increasing ω-TA concentration at a constant TD concentration
(entries 1, 2 and 3) led to substantial enhancement in the pro-
duction of 4 and enrichment of (R)-1a. In contrast, varying TD
concentration at a constant ω-TA concentration (entries 1, 4 and
5) did not induce such a substantial change in the reaction
efficiency. This result indicates that the ω-TA reaction is a rate-
determining step within the enzyme concentration range used in
this study. Entry 3 (3.75 U ml−1 ω-TA and 9 U ml−1 TD) among
the 5 reaction conditions yielded the best reaction outcome
(47 mM of 4 with >99% ee corresponding to 94% of the theor-
etical maximum yield).
Using the optimal enzyme concentrations (entry 3 in Table 2;
3.75 U mL−1 ω-TA and 9 U mL−1 TD), we carried out kinetic
resolution of 11 chiral amines listed in Table 1. As shown in
Table 3, the reaction time required to attain ee of (R)-amine
higher than 99% was inversely correlated with the amino donor
reactivity except 1i and 1k. All the arylalkyl chiral amines
except 1c could be completely resolved within 7 h with reaction
conversion close to 50%. 1c was not completely resolved even at
28 h (entry 3) presumably due to the drastic decrease in the
amino donor reactivity compared with 1a. Therefore, higher con-
centration of ω-TA (10 U mL−1) without varying the TD concen-
tration was employed to attain complete resolution of 1c, leading
to >99% ee of (R)-1c at 30 h (entry 4). Among the alkyl amines,
1f–h and 1j were successfully resolved within 9 h under the opti-
mized conditions (entry 3 in Table 2). Despite the amino donor
reactivity of 1i being higher than 1a, 1i led to incomplete resolu-
tion even after 24 h reaction (entry 10 in Table 3). Similar to 1c,
high ω-TA concentration was used to completely resolve 1i
(entry 11). In the case of 1k, the kinetic resolution reaction
occurred much slower than expected. 1k showed only 40.8%
conversion even after the prolonged reaction using the high
ω-TA concentration (entry 13). In all the cases, the other desired
product (4) was simultaneously formed in high enantiopurity
(>99% ee).
The results in Table 3 demonstrate that kinetic resolution of
chiral amines to yield (R)-amine can be successfully carried out
using L-threonine (2) as a substitute for α-keto acid. To produce
(S)-amine as well by this strategy, we investigated the coupled
enzyme reaction employing (R)-selective ω-TA. To this end, we
cloned, overexpressed and purified (R)-selective ω-TA from
Aspergillus terreus identified elsewhere.35 To render the coupled
reaction executable using the (R)-selective ω-TA, it is crucial that
the enzyme displays substantial activity toward 2-oxobutyrate
(3). We found that the (R)-selective ω-TA exhibited 42% relative
activity toward 3 compared with pyruvate.¶ Therefore, we
carried out kinetic resolution of 1a as a model substrate using 2
as a cosubstrate (Fig. 2). Under the reaction conditions, kinetic
resolution of 1a was completed in 5 h, resulting in >99% ee of
(S)-1a. Because enantioselectivity of the ω-TA from A. terreus is
opposite to that from O. anthropi, D-homoalanine of >99% ee
was formed in this coupled reaction.
In summary, we have developed a facile approach to the
kinetic resolution of chiral amines by coupling ω-TA with TD
to enable in situ generation of an expensive keto acid from a
readily available natural amino acid. This allowed production of
two valuable products (enantiopure (R)-amine and L-homoala-
nine) from cheap substrates (racemic amine and L-threonine).
Moreover, the feasibility of this strategy to obtain (S)-amine
is also demonstrated using an ω-TA displaying opposite
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Acknowledgements
This work was supported by the Advanced Biomass R&D
Center (ABC-2010-0029737) and the Basic Science Research
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Notes and references
‡Initial reaction rates for pyruvate and propanal were 92 and 86 μM
min−1, respectively. Reaction conditions were (S)-α-MBA (20 mM),
amino acceptor (20 mM), phosphate buffer (50 mM, pH 7.5),
0.1 U ml−1 (S)-selective ω-TA from O. anthropi for 10 min at 37 °C.
§When incubated in phosphate buffer (50 mM, pH 7.5) containing
80 mM propanal for 1 h at 37 °C, only 24% residual enzyme activity
was observed. In contrast, no significant decrease in the enzyme activity
was observed in the same buffer containing 80 mM pyruvate after 1 h.
¶Initial reaction rates for pyruvate and 2-oxobutyrate were 61 and
25 μM min−1, respectively. Reaction conditions were (R)-1a (20 mM),
amino acceptor (20 mM), phosphate buffer (50 mM, pH 7), 60 mU ml−1
(R)-selective ω-TA from A. terreus for 10 min at 37 °C.
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2140 | Green Chem., 2012, 14, 2137–2140
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