One-pot one-step deracemization of amines using ω-transaminases

Article abstract of DOI:10.1039/c3cc43348j

In this study, we developed a one-pot one-step deracemization method for the production of various enantiomerically pure amines using two opposite enantioselective ω-TAs. Using this method, various aromatic amines were successfully converted to their (R)-forms (>99%) with good conversion.

Full text of DOI:10.1039/c3cc43348j

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One-pot one-step deracemization of amines using
x-transaminases†
Giyoung Shin,a Sam Mathew,a Minsu Shon,a Byung-Gee Kimb and
Hyungdon Yun*a
Cite this: Chem. Commun., 2013,
49, 8629
Received 6th May 2013,
Accepted 11th June 2013
DOI: 10.1039/c3cc43348j
www.rsc.org/chemcomm
In this study, we developed a one-pot one-step deracemization racemization of amine using two opposite enantioselective
method for the production of various enantiomerically pure o-TAs have been reported.7 As a more general method, derace-
amines using two opposite enantioselective x-TAs. Using this mization using a one-pot two-step reaction was developed, in
method, various aromatic amines were successfully converted to which the kinetic resolution of the racemic amine was per-
their (R)-forms (>99%) with good conversion.
formed using an o-TA, followed by asymmetric synthesis of
the corresponding ketone using an opposite enantioselective
Enantiomerically pure amines are important building blocks for o-TA.8 One drawback of this method is the need to inactivate
preparing bioactive compounds for use in pharmaceutical and the o-TA used in the kinetic resolution before starting asym-
chemical industries.1 Several chemical methods have been devel- metric synthesis. Subsequently an opposite enantioselective
oped for the production of chiral amines, such as hydrogenation o-TA and its amino donor were added to the reaction mixture.
of imines and enamines, alkylation of imines, aminohydroxyla- To improve this process, immobilized o-TA was employed in
tion, and reductive amination.2,3a Recently, deracemization pro- the first step and the immobilized o-TA was separated easily by
cesses have gained considerable attention due to their ability to filtration or centrifugation after the first step.9 Here, we report a
produce enantiomerically pure amines from racemates with a general method, a one-pot one-step deracemization method,
maximum theoretical yield of 100%. Deracemization of amines for production of various enantiomerically pure (R)-amines
via dynamic kinetic resolution using lipase and racemization using two opposite enantioselective o-TAs.
using transition metal complexes such as Pd and Ru catalysts
To deracemize rac-a-methylbenzylamine (MBA, model com-
have been well reported.3 Also deracemization of amines via pound) to (R)-a-MBA, (S)-a-MBA is converted to the corres-
enantioselective oxidation using evolved amine oxidase and ponding ketone by (S)-o-TA using an amino acceptor, after
non-selective reduction using an ammonia–borane complex have which the corresponding ketone is converted back to (R)-a-MBA
been employed to produce optically pure amines.4,5f
by (R)-o-TA with an amino donor (Scheme 1). In the second step
Among different biocatalysts, o-transaminases (o-TAs) have of the reaction ((R)-o-TA reaction), (R)-o-TA has negligible
recently received a great deal of attention as promising catalysts activity toward the amino acceptor, which is used in the (S)-o-
due to their ability to produce a wide range of optically pure TA reaction. If not, the asymmetric reaction of (R)-o-TA will not
amines and unnatural amino acids.5 Chiral amines are gener- proceed efficiently because (R)-a-MBA will be converted to
ally produced by o-TAs through asymmetric synthesis from ketone by (R)-o-TA using the amino acceptor.
ketones and kinetic resolution from rac-amines. However,
We selected a pair of o-TAs to carry out this reaction.
deracemization of amines using o-TAs has, until recently, been Specifically, the (R)-selective o-TAs from Mycobacterium vanbaalenii
a less-explored area. Deracemization using an o-TA was first ((R)-o-TAMV)1a and Neosartorya fischeri ((R)-o-TANF),1a and
reported to produce an enantiomerically enriched amine using
spontaneous racemization of a ketone substrate (4-oxo-3-phenyl-
butyric acid ethyl ester).6 In addition, deracemization of a
homoalanine using D-amino acid oxidase and an o-TA, and
a School of Biotechnology, Yeungnam University, Gyeongsan, Gyeongbuk, South Korea.
E-mail: hyungdon@ynu.ac.kr; Fax: +82-53-810-4769; Tel: +82-53-810-3044
b School of Chemical and Biological Engineering, Seoul National University,
Seoul 151-742, South Korea
Scheme 1 Deracemization of rac-amine (e.g. rac-a-MBA) into (R)-amine by (S)-
and (R)-selective o-TAs.
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c3cc43348j
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(S)-selective o-TAs from Polaromonas sp. JS666 ((S)-o-TAPO)10 its reactivity was less than 10% of that for a1. Based on the
and Vibrio fluvialis JS177a ((S)-o-TAVF) were cloned into the reactivity (good substrate for (S)-o-TA, poor substrate for (R)-o-TA),
pET24ma vector with an N-terminal His6-tag and effectively a7, a8, and a9 can be used as amino acceptors for (S)-o-TA. Among
expressed in Escherichia coli BL21 (DE3).7a The enzymes were them, a9 was selected for deracemization because it is inexpensive
then purified on a Ni-NTA affinity column (Fig. S1 ESI†), after and has high reactivity. Additionally, glutamate (aminated a9) can
which the purified enzymes were subjected to an o-TA activity be easily removed by another coupling system (when needed).
assay performed with 10 mM a-MBA and 10 mM pyruvate in Therefore, one-step one-pot deracemization was carried out with
100 mM phosphate buffer (pH 7.0) at 37 1C. Depending on the (S)-o-TAPO and two (R)-o-TAs with a9.
stereo-selectivity of the enzyme, either 10 mM of (R)- or (S)-a-
Before performing the deracemization reaction, we carried
MBA was used. One unit of activity was defined as the amount out the kinetic resolution of rac-a-MBA (first step reaction) with
of enzyme that produced 1 mmol acetophenone per minute. The (S)-o-TAPO. 10 mM rac-a-MBA were completely resolved with
specific activities of (R)-o-TAMV, (R)-o-TANF, (S)-o-TAPO and 20 mM a9 into (R)-a-MBA for 12 h with high enantioselectivity
(S)-o-TAVF were 64.7, 39.0, 2.32 and 27.3 U mgÀ1, respectively (E > 100) (data not shown). The asymmetric synthesis of (R)-o-
(ESI†).
MBA from acetophenone (second step reaction) with (R)-o-TAs
To identify a good amino acceptor of (S)-o-TA, which is not a was performed using D-Ala as an amino donor, in which the
substrate for (R)-o-TA, acceptor specificities of the enzymes were co-product pyruvate was removed by lactate dehydrogenase
investigated using nine carbonyl compounds (seven a-keto acids, (LDH). The cofactor NADH required by LDH was recycled by
two esters) in the presence of 10 mM (S)-a-MBA (or (R)-a-MBA), employing glucose and glucose dehydrogenase (GDH).12 When
and the produced acetophenone was analyzed to measure the asymmetric synthesis was carried out in 1 mL of 100 mM Tris–
initial reaction rate (Fig. 1). Interestingly, (R)-o-TAs showed a HCl buffer (pH 8.5) containing 10 mM acetophenone, 50 mM
narrow substrate specificity when compared to (S)-o-TAs. (R)-o- D-Ala, 50 mM glucose, 1 mM NADH, LDH (5 U), GDH (5 U) and
TAs had no activity toward a6, a7, a8 and a9. Recently, Shin et al. 0.3 mg of (R)-o-TA at 37 1C, after 24 h, both (R)-o-TAs quantita-
examined the amino acceptor specificity of two (R)-o-TAs from tively converted acetophenone to (R)-a-MBA (ee > 99). Since the
Aspergillus terreus.11 These enzymes did not show any activity kinetic resolution with (S)-o-TAPO and the asymmetric synthesis
towards a6, a7, a8 or a9. Since (S)-o-TAs showed considerable with (R)-o-TAMV (or (R)-o-TANF) were carried out successfully,
reactivity towards a6, a7 and a8, the active sites of (R)-o-TAs might we carried out deracemization of rac-a-MBA by combining both
have a narrower binding pocket to accommodate the bulky side reactions simultaneously in one pot (Fig 2). Since PLP helps to
chains of keto acids than those of (S)-o-TAs. The relative activities stabilize the enzyme, 0.1 mM PLP was added to the reaction
of (S)-o-TAVF for a7 and a8 were 6.4% and 14.1% of that for a1 mixture; and for the efficient removal of a1, excess of LDH
(typical amino acceptor), respectively. In the case of (S)-o-TAPO, a9 (112 U) was used for the deracemization of 50 mM rac-a-MBA.
was a good amino acceptor, which showed 70% reactivity of a1. After two days of reaction, (S)-o-TAPO–(R)-o-TAMV and (S)-o-
Actually, it is the unique characteristic of (S)-o-TAPO that enables TAPO–(R)-o-TANF gave 49.5 mM and 48.9 mM of (R)-a-MBA
it to use a9 as an amino acceptor because TA generally shows a (>99%). The slightly higher conversion obtained for (S)-o-
clear one sided preference to either a1 or a9–oxaloacetate.10 Even TAPO–(R)-o-TANF might be due to the higher specific activity
though (S)-o-TAPO showed considerable reactivity for a7 and a8, of (R)-o-TAMV (64.7 U mgÀ1) compared to that of (R)-o-TANF
(39.0 U mgÀ1). It is noteworthy that in this reaction scheme,
deracemization can be performed without deactivating any enzyme.
After successful deracemization of rac-a-MBA into (R)-a-MBA
by the one-pot, one-step reaction, we were curious to find out
whether this reaction system would be applicable to other
amines and unnatural amino acids. Therefore, the amino donor
specificities of enzymes were examined with various compounds
Fig. 1 Amino acceptor specificities of o-TAs. Amino acceptors (A) and the
relative reaction rate (B). Reaction conditions: 10 mM (S)-a-MBA (or (R)-a-MBA),
10 mM amino acceptor, 100 mM Tris–HCl (pH 7.5) at 37 1C. The initial reaction
rate of the enzyme toward a1 was taken as 100%. In the case of inactive
substrates, the vertical bar is not visible in the graph.
Fig. 2 Deracemization of rac-a-MBA. Reaction conditions: 1 mL reaction
volume, 50 mM rac-a-MBA, 50 mM a9, 100 mM D-Ala, 100 mM glucose, 1 mM
NADH, LDH (112 U), GDH (5 U), 0.2 mM PLP, (S)-o-TAPO (0.93 mg), (R)-o-TA
(0.8 mg), 200 mM Tris–HCl (pH 7.5) at 37 1C.
c
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Table 1 Deracemization of various aromatic aminesa
(S)-o-TAPO–(R)-o-TAMV
were added into the reaction mixture due to their lower reactivity
((S)-o-TAPO; 1.0 mg, (R)-o-TA; 0.5 mg) (Fig. 3). All racemic
aromatic amines were successfully converted into (R)-amines
(>99%) with good conversion (Table 1). All substrates except d2
and d4 could be deracemized into (R)-amines (>99%) with >97%
conversion. In addition, (S)-o-TAPO–(R)-o-TAMV gave higher
conversion than (S)-o-TAPO–(R)-o-TANF (except for d5 and d6).
In summary, we successfully developed a one-pot one-step
deracemization method using (S)-o-TA and (R)-o-TA through
careful screening of amino acceptors for (S)-o-TA. This novel
deracemization method enabled various aromatic amines
(a-MBA, d1–d8) to be converted into the (R)-form (>99%) with
good conversion. We are currently developing a deracemization
method to produce useful unnatural amino acids using o-TAs.
This research was partially supported by the Basic Science
Research Program (2012044222) through the National Research
Foundation of Korea, Ministry of Education, Science and Tech-
nology, Korea. And this research was partially supported by a
grant (A10301712131560200) of Global Cosmetics R&D Project,
Ministry of Health & Welfare, Korea.
(S)-o-TAPO–(R)-o-TANF
Substrate
Conv. (%)
ee (%)
Conv. (%)
ee (%)
d1
d2
>99
82
98
87
90
>99
98
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
71
87
94
97
89
79
77
>99
>99
>99
>99
>99
81
d3
d4c
d5b
d6b
d7
>99
>99
d8
a
Reaction conditions: 1 mL reaction volume, 10 mM racemic substrate,
50 mM a9, 50 mM D-Ala, 50 mM glucose, 1 mM NADH, LDH (112 U),
GDH (5 U), 0.2 mM PLP, (S)-o-TAPO (1.0 mg), (R)-o-TA (0.5 mg), 200 mM
Tris–HCl (pH 7.5), 37 1C, 24 h reaction. Conv. and ee were determined
using a Crownpak CR(+) column. Conv. is the percentage of the
remaining amine concentration to the initial amine concentration.
b
c
(S)-o-TAPO (1.86 mg), (R)-o-TA (1.5 mg). ee was determined using
a C18 symmetry column after GITC derivatization.
Notes and references
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Fig. 3 Amino donor specificities of o-TAs. Amino donors (A) and the relative
reaction rate (B). Reaction conditions: 10 mM pyruvate (a1), 10 mM racemic
amino donor, 100 mM Tris–HCl (pH 7.5) at 37 1C. The initial reaction rate of the
enzyme towards rac-a-MBA was taken as 100%. In cases of inactive substrates,
the vertical bar is not visible in the graph.
(eight aromatic amines, two aliphatic amines, two unnatural
amino acids) in the presence of a 10 mM racemic amino donor
and 10 mM a1, and the consumption rate of a1 was analyzed
using an Aminex HPX-87H HPLC column (Bio-Rad, CA) with the
elution of 5 mM sulfuric acid solution. All enzymes showed
considerable reactivity toward aromatic amines (d1–d8) and
unnatural amino acids (d11). In the case of aliphatic amines
(d9, d10), (R)-o-TAMV and (R)-o-TANF showed considerable
activity, but (S)-o-TAPO did not show any reactivity. In the case
of beta-amino acid (d11), only (S)-o-TAPO showed reactivity.
These results suggested that aromatic amines can be derace-
mized well with o-TAPO–(R)-o-TAMV and (S)-o-TAPO–(R)-o-
TANF, but in the case of aliphatic amine and beta-amino acid,
the deracemization reaction could not be performed efficiently.
Finally, deracemization of aromatic amines (d1–d8) was car-
ried out with 10 mM substrate (Table 1). In the case of d5 and d6,
higher amounts of o-TAs ((S)-o-TAPO; 1.86 mg, (R)-o-TA; 1.5 mg)
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c
This journal is The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 8629--8631 8631