Deracemization of Racemic Amines to Enantiopure (R)- and (S)-amines by Biocatalytic Cascade Employing ω-Transaminase and Amine Dehydrogenase

Article abstract of DOI:10.1002/cctc.201900080

A one-pot deracemization strategy for α-chiral amines is reported involving an enantioselective deamination to the corresponding ketone followed by a stereoselective amination by enantiocomplementary biocatalysts. Notably, this cascade employing a ω-transaminase and amine dehydrogenase enabled the access to both (R)-and (S)-amine products, just by controlling the directions of the reactions catalyzed by them. A wide range of (R)-and (S)-amines was obtained with excellent conversions (>80 %) and enantiomeric excess (>99 % ee). Finally, preparative scale syntheses led to obtain enantiopure (R)- and (S)-13 with the isolated yields of 53 and 75 %, respectively.

Full text of DOI:10.1002/cctc.201900080

Accepted Article
Title: Deracemization of Racemic Amines to Enantiopure (R)- and (S)-
amines by Biocatalytic Cascade Employing ω-Transaminase and
Amine Dehydrogenase
Authors: Sanghan Yoon, Mahesh D. Patil, Sharad Sarak, Hyunwoo
Jeon, Geon-Hee Kim, Taresh P. Khobragade, Sihyong Sung,
and Hyungdon Yun
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To be cited as: ChemCatChem 10.1002/cctc.201900080
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ChemCatChem
10.1002/cctc.201900080
COMMUNICATION
Deracemization of Racemic Amines to Enantiopure (R)- and (S)-
amines by Biocatalytic Cascade Employing ω-Transaminase and
Amine Dehydrogenase
†
†
Sanghan Yoon, Mahesh D. Patil, Sharad Sarak, Hyunwoo Jeon, Geon-Hee Kim, Taresh P.
Khobragade, Sihyong Sung, and Hyungdon Yun*
Abstract: A one-pot deracemization strategy for α-chiral amines is
reported involving an enantioselective deamination to the
corresponding ketone followed by a stereoselective amination by
enantiocomplementary biocatalysts. Notably, this cascade employing
a ω-transaminase and amine dehydrogenase enabled the access to
both (R)-and (S)-amine products, just by controlling the directions of
the reactions catalyzed by them. A wide range of (R)-and (S)-amines
was obtained with excellent conversions (>80%) and enantiomeric
excess (>99% ee). Finally, preparative scale syntheses led to obtain
enantiopure (R)- and (S)-13 with the isolated yields of 53 and 75%,
respectively.
has been shown to efficiently deracemize racemic amines.[9]
Yasukawa et al. generated R-stereoselective amine oxidase from
porcine kidney D-amino acid oxidase and used it for the
deracemization of α-methylbenzylamine.[ Recent years have
evidenced effective deracemization protocols for the synthesis of
10]
[
11]
important pharmaceuticals such as mexiletine,
salsolidine,[ to name a few.
rasagiline and
9]
Various enzymes such as ω-transaminases (ω-TAs) and
amine dehydrogenases (AmDHs)[12] have emerged as viable
options for the biocatalytic syntheses of chiral amines. Although
ω-TAs have been well documented in deracemization;[
11,13]
the
utility of AmDHs in deracemization protocols to produce
enantiopure amines is still unexplored. Although, the synthesis of
Enantiopure chiral amines have been described as indispensable
constituents of nearly 50% small molecule pharmaceuticals.[1] The
chemical synthesis of enantiopure chiral amines is challenging
owing to the use of toxic intermediates, formation of large number
of by-products, thereby complicating the system and ultimately
increasing the overall costs.[2] In this context, biocatalytic methods
have been recognized as major ‘green technologies’ that provide
sustainable methods for the synthesis of enantiopure chiral
amines. Moreover, the judicial integration of chemical syntheses
and ‘greener’ enzymatic approaches is highly desirable for the
increasing demands of the enantiopure amines in pharmaceutical,
fine chemicals, and agricultural industries.[2,3]
The practical application of one of the widely used strategies
for the synthesis of chiral amines, i.e. kinetic resolution, is limited
by the maximum theoretical yield of 50%.[4] On the other hand,
deracemization is an attractive strategy, especially when racemic
amines can easily be accessed compared to their corresponding
prochiral ketones.[5] Deracemization strategies enable to achieve
(
R)-amines have been sucessfullyl carried out by easier
strategies such as AmDH-catalyzed reductive amination of
ketones.[1a,1d] Also, recent years have evidenced the improved
array of new (R)-seletive ω-TAs for the scalable synthetic
applications.[13d] Since AmDHs exhibit exclusive selectivity
[1d, 12a]
towards (R)-amines,
they open a new avenue of a class of
enzymes representing enantiocomplementarity to well reported
S)-selective ω-TAs. We envisaged to utilize (R)-AmDHs in
(
tandem with enantiomerically complementary (S)-ω-TA to
constitute a deracemization cascade enabling the access to both
the enantiomers [(R)- and (S)] of a range of chiral amines. We
herein report a one-pot deracemization strategy which proceeds
via two steps: 1) Enantioselective deamination of racemic amine
substrates to corresponding ketones and 2) Stereoselective
amination of the ketones generated in step 1 yielding enantiopure
amine products with theoretical yield of 100% [Scheme 1].
theoretical
complete
conversion
by
employing
two
stereocomplementary enzymes.[6] Since the successful
demonstration of lipase-catalyzed dynamic kinetic resolution of
amines,[7] various other biocatalysts have been explored for their
application in deracemization for the synthesis of chiral amines.
For instance, the group of Turner used the variants of monoamine
oxidase from Aspergillus niger in tandem with nonselective
chemical reductants for the deracemization of a wide range of
primary, secondary and tertiary amines.[8] Recently, coupling of a
nonselective chemical reducing agent with reductive aminases
[
a]
S. Yoon, Dr. M. D. Patil, S. Sarak, H. Jeon, G-H. Kim, T. P.
Khobragade, S. Sung and Prof. Dr. Hyungdon Yun
Department of Systems Biotechnology
Konkuk University, 120 Neungdong-ro, Gwanjin-gu, Seoul-050-29,
South Korea
Correspondence E-mail: hyungdon@konkuk.ac.kr
Sanghan Yoon and Mahesh D. Patil contributed equally
†
Scheme 1. Deracemization of racemic amines to enantiopure A) (R)-amines
and B) (S)-amines using a cascade employing (S)-ω-transaminase and (R)-
amine dehydrogenase
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.

ChemCatChem
10.1002/cctc.201900080
COMMUNICATION
It is worth emphasizing that both the enantiomers [(R)- and
S)-] of the amine products could be easily accessed, notably
reported to improve the solubility of the hydrophobic substrates
and thereby yields of the enzymatic syntheses.[17] The use of
DMSO in the present study improved the generation of (R)-7 and
(R)-13; and complete conversions were achieved when the
concentration of DMSO was increased up to 15 % (v/v) [Fig. S9
and Fig. S10].
(
using the same pair of enantiocomplementary biocatalysts, just
by controlling the directions of the constituent reactions catalyzed
by applied biocatalysts.
We commenced this study with the establishment of
deracemization protocol for the synthesis of (R)-amines [Scheme
The successful deracemization of racemic model substrates
1
A]. To examine the AmDH-catalyzed enantioselective amination, to enantiopure amine products encouraged us to check the
two (R)-AmDHs, Rs-AmDH (developed from L-phenylalanine
applicability of this system for the synthesis of wide range of (R)-
amines. A panel of 13 racemic amine substrates was tested,
among which complete conversion was achieved only for four
substrates [System 1 in Table 1]. Low conversions albeit of
excellent ee values obtained for the racemic substrates implied
that the activity of AmDH was inadequate. Furthermore, low
expression level of AmDH was identified as a limiting factor for
the modest conversions achieved for majority of the substrates
[Fig. S11].
In order to increase the expression levels of AmDHs, the
gene encoding AmDH in pET-duet vector was transferred to pET-
24ma vector, and that of FDH in pET-24ma was transferred to
PQE80L vector [Fig. S12]. The vector system used for the
expression of (S)-ω-TAPO was unaltered. This newly designed
vector strategy improved the expression levels of AmDHs [Fig.
S13]. Although expression level of FDH marginally reduced, the
limiting factor of poor AmDH expression was circumvented, which
ultimately resulted in excellent conversions (~80-100%) of all the
racemic substrates, except one, to their corresponding (R)-
amines with excellent enantioselectivities (>99 ee) [System 2 in
Table 1].
[14a]
dehydrogenase of Rhodococcus sp.)
and chimeric AmDH
14b]
(
Chi-AmDH)[
were cloned, expressed and purified [ESI]. Rs-
AmDH and Chi-AmDH have been reported to exhibit good activity
toward aromatic and aliphatic ketone substrates, respectively.[14]
Thus, 4-phenyl-2-butanone 7b and 2-heptanone 13b were used
as model substrates for Rs-AmDH and Chi-AmDH, respectively.
To select the operational pH, we examined the reactivity of
purified AmDHs at varying pH from 7.0 to 10.5. Rs-AmDH and
Chi-AmDH exhibited the maximum specific activity of 353 and 305
mU/mg toward 7b and 13b, respectively at pH 9.5 [Fig. S1 and
Fig. S2]. To ensure the efficient regeneration of NADH cofactor,
formate dehydrogenase (FDH) from Pseudomonas sp. 101
(
Uniprot ID. No. P33160.3) and glucose dehydrogenase (GDH)
[
14c]
from Bacillus subtilis (Uniprot ID. No. P12310-1)
systems were
tested, wherein FDH consistently performed better than GDH
system and >99% conversion of both the model substrates could
be achieved [Fig. S3 and Fig. S4].
Since whole-cell systems are advantageous in enabling
simpler handling and cost-effective biotransformations,[15,16] the
applicability of the AmDH-FDH reaction system was tested in
whole-cell transformations. A reaction was carried out with
varying amounts of cells (0.5 to 10 mgDCW/mL), wherein 5
mgDCW/mL of Rs-AmDH cells (One-cell two-vector system co-
expressing Rs-AmDH and FDH) achieved comparatively better
conversion (96%) than that by Chi-AmDH cells (One-cell two-
vector system co-expressing Chi-AmDH and FDH) (80%).
Nevertheless, 10 mgDCW/mL of both Rs-AmDH and Chi-AmDH
cells could successfully transform >99% of their corresponding
substrates in 24 h [Fig. S5 and Fig. S6].
Once the AmDH system was instituted, we turned our
attention on establishing the ω-TA system. For the selection of
efficient ω-TA catalyzing enantioselective deamination of racemic
amines, whole cells separately expressing four ω-TAs (3.33
mgDCW/mL each) were reacted with 20 mM rac-2-aminoheptane
Table 1. Deracemization of racemic amines to (R)-amines
using the whole-cell biocatalytic cascade employing (S)-ω-
TAPO and (R)-AmDH
(S)-ω-TAPO – (R)-Rs-AmDH
System 1[a] System 2[b]
(S)-ω-TAPO – (R)-Chi-AmDH
System 1[a] System 2[b]
Sub.
eeR
(%)
eeR
(%)
eeR
(%)
eeR
(%)
Conv
Conv
(%)
Conv
(%)
Conv
(%)
[c]
[c]
[c]
[c]
(%)
rac-1
rac-2
rac-3
rac-4
rac-5
rac-6
rac-7
rac-8
rac-9
rac-10
rac-11
rac-12
66.9
66
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
96.5
100
100
100
100
100
100
100
100
61.1
79.4
100
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
72.9
73
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
87.7
94.8
81.8
95.7
96.4
96.9
100
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
(
rac-13) and rac-4-phenyl-2-aminobutane (rac-7) in the presence
66.7
63.4
63.6
72.5
100
100
72.6
52.3
77.1
100
71.6
70.2
73
of 20 mM pyruvate and 0.1 mM PLP. While all the four ω-TAs
efficiently catalyzed the enantioselective deamination of 13 and
yielded 10 mM (50% conversion) (R)-13, only ω-TAVF and ω-
TAPO exhibited better activity towards 7 [Fig. S7 and Fig. S8].
Moreover, the concentration of desired (R)-enantiomer in the
case of ω-TAVF decreased over the time. Thus, ω-TAPO was
used in the subsequent experiments.
Having the individual AmDH and ω-TA whole-cell systems
in hand, we further checked their application in tandem for the
deracemization of two model substrates i.e. rac-7 using Rs-
AmDH - ω-TAPO and rac-13 using Chi-AmDH - ω-TAPO system.
Both, Rs-AmDH - ω-TAPO and Chi-AmDH - ω-TAPO systems
could achieve good conversions and yielded 84.4% (R)-7 and
72.5
100
100
68
100
89.5
75.7
96.9
100
69
87.8
100
86% (R)-13, respectively from their corresponding racemic
substrates in 24 h. Organic co-solvents such as DMSO have been
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ChemCatChem
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COMMUNICATION
Scheme 2. Deracemization of racemic amines to enantiopure (S)-amines using
an AHR recycling system
rac-13
100
>99
100
>99
100
>99
100
>99
Reaction conditions: 10 mM racemic substrate, 10 mM
pyruvate, 0.1 mM PLP, 3.3 mgDCW/mL ω-TAPO cells, 30
mgDCW/mL AmDH-FDH cells, 37 °C, 100 mM Glycine
buffer (pH 9.5) and 200 mM ammonium formate, 15% (v/v)
DMSO, 24 h
To check the applicability of the AHR system in Rs-AmDH-
catalyzed enantioselective deamination, a reaction was carried
out using 10 mM rac-7 in the presence of 10 mM benzaldehyde.
This reaction yielded 5 mM 7b (50% Conv.), implying that AHR
[
a] System 1: AmDH-pET-duet; FDH-pET-24ma; (S)-ω-
TAPO- pET-24ma
b] System 2: AmDH-pET-24ma; FDH-PQE80L; (S)-ω-
TAPO- pET-24ma
+
could efficiently regenerate NAD for the AmDH-catalyzed
[
enantioselective deamination [Fig. S14]. Following this, the
efficiency of AHR system was tested for another constituent
reaction of this deracemization protocol, i.e. (S)-ω-TA-catalyzed
enantioselective amination of prochiral ketones to their
corresponding (S)-amines. Three purified (S)-ω-TAs were
examined for their reactivity towards model ketone substrate 7b
using the AHR system and varying pH from 8.5-9.5. Among the
tested enzymes, (S)-ω-TAVF exhibited marginally better activity
irrespective of the reaction pH [Fig. S15]. (S)-ω-TAVF could
successfully transform 94.5% of 7b to (S)-7 at pH 8.5.
[
c] Conversion was defined as percentage ratio of the (R)-
[
13a]
amine product to initial racemic amine substrate;
[
(푅)−amine product (mM)]
x 100
[
Initial racemic amine substrate (mM)]
…
Having successfully demonstrated the deracemization of
racemic amines to their corresponding (R)-amines, we further
decided to check the usability of this system for the
deracemization of racemic amine substrates to their
corresponding (S)-enantiomers. For the synthesis of (S)-amines,
the first step of enantioselective deamination was catalyzed by
Having successfully demonstrated the individual constituent
reactions i.e. AmDH-catalyzed enantioselective deamination of
racemic amine substrate and ω-TA-catalyzed enantioselective
amination of prochiral ketone, we further checked their application
in combination for the deracemization of model substrate rac-7 to
(
R)-AmDH, followed by the enantioselective amination by (S)-ω-
TA [Scheme 1B]. However, compared to the deracemization of
racemic amines to (R)-amines, the equilibrium of both the
constituent reactions in the synthesis of (S)-amines is highly
unfavourable. For instance, the first step of AmDH-catalyzed
enantioselective deamination for the synthesis of (S)-amines is
unfavorable since the pertinent function of AmDHs is the reductive
amination of ketones to amines and not the deamination of
racemic amines to their corresponding ketones.[12] Moreover, ω-
TA-catalyzed asymmetric synthesis of enantiopure amines from
prochiral ketones also suffers from a unfavorable reaction
equilibrium compared to that of kinetic resolution of racemic
amines.[18]
To establish the ω-TA system catalyzing the enantioselective
amination of prochiral ketone substrates to their corresponding
chiral amines, four purified (S)-ω-TAs were tested for their
reactivity towards model substrates 7b and 13b using alanine as
an amino donor. Alanine dehydrogenase (AlaDH) was used to
recycle pyruvate (deaminated alanine) back to alanine and to
regenerate nicotinamide cofactor used in the first step of AmDH-
catalyzed enantioselective deamination. However, AlaDH system
performed poorly when AmDH and (S)-ω-TA were combined for
the deracemization of racemic amines [Data not shown]. We thus
decided to use benzylamine as an amino donor, wherein the
deaminated benzylamine i.e. benzaldehyde is utilized by
(
S)-7. Although excellent conversion of 90% could be achieved,
mediocre enantiomeric excess only of 91% was attainable [Fig.
S16]. Unreacted amounts of (R)-7 in the reaction mixture
suggested that the activity of AmDH was inadequate and the poor
ee values obtained could be consequent to the poor drive of the
reaction. Ideally, generating a driving force for the forward
reaction is a key episode in the cascade reactions.[16] We
anticipated that the addition of trivial quantity of benzaldehyde
could help to drive the reaction in the forward direction, as more
substrate would be easily available for AHR and thereby efficient
regeneration of nicotinamide cofactor would be enabled. Thus,
0.2 mM benzaldehyde was added in the subsequent experiments.
As benzylamine serves as an amino donor, increasing
concentration of benzylamine was anticipated to drive the ω-TA-
catalyzed reaction towards the formation of (S)-amines. Thus,
further experiments were performed with the increasing amounts
of benzylamine ranging from 20 to 60 mM and fixed amount of
benzaldehyde (0.2 mM) [Fig. S17]. However, the increasing
concentration of benzylamine negatively affected the activity of
AHR [Fig. S18]. Thus, further experiment was performed by
varying the amounts of benzaldehyde and fixing the benzylamine
concentration at 20 mM. It was observed that increasing the
concentration of benzaldehyde beyond 0.2 mM decreased the ee
of the (S)-amine product. The best combination (20 mM
benzylamine and 0.2 mM benzaldehyde) resulted in the improved
conversion to 100% and ee to 95.4% [Fig. S19].
[
19]
aldehyde reductase (AHR) from Synechocystis sp.
to give
+
benzyl alcohol and regenerate NAD cofactor for AmDH [Scheme
2
].
NH2
R2
R1
I. Enantioselective deamination
II. Enantioselective amination
Following the successful improvements in conversion and ee
values using the purified enzyme system, we extended this
deracemization protocol to a whole-cell system expressing
AmDH-AHR and (S)-ω-TA. The addition of 1 mM of benzaldehyde
resulted in the excellent conversion of 100 and ee of >99% [Fig.
S20].
+
NH2
NH2
(
R)-AmDH
O
(S)--TAVf
R1
rac)-1-13
R2
R1
R2
R1
R2
(
(S)-1-13
1
b-13b
NADH
O
NH2
AHR
NAD+
OH
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ChemCatChem
10.1002/cctc.201900080
COMMUNICATION
The robustness of the established deracemization protocol
was evaluated for the synthesis of a wide range of (S)-amines. A
panel of 13 racemic amine substrates was tested, wherein this
deracemization protocol could successfully catalyze the synthesis
of (S)-amine products with excellent conversions (~80-100%) and
enantioselectivities (>99% ee) [Table 2].
In summary, we have successfully developed an efficient
biocatalytic cascade catalyzing the deracemization of racemic
amines by employing AmDH and (S)-ω-TA. This reaction enabled
the access to a range of chiral amine products, notably to both the
enantiomers, with excellent conversions ranging from 80-100%
and enantioselectivities (>99%). It is worth emphasizing that the
absolute configuration of the amine products could be easily
switched, just by controlling the directions of the constituent
reactions of the biocatalytic cascade catalyzed by the same pair
of enantiocomplementary enzymes. The broad substrate
specificity of both the enzymes i.e. ω-TA and AmDH imply that
the biocatalytic systems employing these enzymes, such as
demonstrated herein, would enjoy large applicability in
biocatalytic syntheses for chiral amines.
Table 2. Deracemization of racemic amines to (S)-amines using the whole-
cell biocatalytic cascade employing (R)-AmDH and (S)-ω-TAVF
(S)-ω-TAVF– (R)-Rs-
(S)-ω-TAVF– (R)-Chi-
AmDH
AmDH
Substrate
Conv (%)[a]
eeS (%)
Conv (%)[a]
eeS (%)
rac-1
rac-2
rac-3
rac-4
rac-5
rac-6
rac-7
rac-8
rac-9
rac-10
rac-11
rac-12
rac-13
100
100
100
100
100
100
100
100
100
61.4
76.0
98.0
100
>99
>99
>99
>99
>99
>99
>99
>99
>99
23
98
97
Experimental Section
100
100
100
100
100
100
100
100
80.4
85.0
100
100
>99
>99
>99
>99
>99
>99
>99
>99
61
Details of the experimental procedures are provided in the Supporting
Information.
Acknowledgements
This work was supported by Konkuk University (2016-A019-0507).
MDP gratefully acknowledges ‘2018-KU Brain Pool program’ of
Konkuk University, Seoul.
Keywords: Biocatalysis • chiral amines • deracemization •
transaminases • amine dehydrogenases
52
71
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Reaction conditions: 10 mM racemic substrate, 20 mM Benzylamine, 1 mM
Benzaldehyde, 0.1 mM PLP, 10 mgDCW/mL ω-TAVF Cells, 27 mgDCW/mL
AmDH-AHR Cells, 37 °C, 100 mM Tris buffer (pH 8.5), 15% (v/v) DMSO, 24
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a] Conversion was defined as percentage ratio of the (S)- amine product to
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Finally, to demonstrate the synthetic applicability of the
biocatalytic cascade, deracemization of a model substrate rac-13
was carried out on a preparative-scale [20 mM in 25 mL reaction
volume; 57.5 mg] [ESI, Section 4] using the reaction conditions
optimized for analytical-scale reactions. The results demonstrated
that complete deracemization of rac-13 substrate could be
achieved to obtain enantiopure (R)-13 and (S)-13 (ee >99%) [Fig.
S21 and Fig S22]. R)-13 and (S)-13 from these reactions were
isolated with the yields of 53 and 75% respectively [Fig. S23].
These results confirm that this biocatalytic cascade is promising
for the preparative scale deracemization of racemic substrates to
obtain valuable enantiopure amine compounds.
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Entry for the Table of Contents
COMMUNICATION
Novel
cascade,
one-pot
deracemization
amine
Sanghan Yoon, † Mahesh D. Patil, †
Sharad Sarak, Hyunwoo Jeon, Geon-
Hee Kim, Taresh P. Khobragade,
Sihyong Sung and Hyungdon Yun*
employing
dehydrogenase and ω-transaminase,
is reported for the synthesis of
enantiopure amines with excellent
conversions
Page No. – Page No.
Deracemization of Racemic Amines to
Enantiopure (R)- and (S)-amines by
Biocatalytic Cascade Employing ω-
Transaminase
and
Amine
Dehydrogenase
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