Enantioselective Cascade Biocatalysis for Deracemization of Racemic β-Amino Alcohols to Enantiopure (S)-β-Amino Alcohols by Employing Cyclohexylamine Oxidase and ω-Transaminase

Article abstract of DOI:10.1002/cbic.202000491

Optically active β-amino alcohols are very useful chiral intermediates frequently used in the preparation of pharmaceutically active substances. Here, a novel cyclohexylamine oxidase (ArCHAO) was identified from the genome sequence of Arthrobacter sp. TYUT010-15 with the R-stereoselective deamination activity of β-amino alcohol. ArCHAO was cloned and successfully expressed in E. coli BL21, purified and characterized. Substrate-specific analysis revealed that ArCHAO has high activity (4.15 to 6.34 U mg^?1 protein) and excellent enantioselectivity toward the tested β-amino alcohols. By using purified ArCHAO, a wide range of racemic β-amino alcohols were resolved, (S)-β-amino alcohols were obtained in >99 % ee. Deracemization of racemic β-amino alcohols was conducted by ArCHAO-catalyzed enantioselective deamination and transaminase-catalyzed enantioselective amination to afford (S)-β-amino alcohols in excellent conversion (78–94 %) and enantiomeric excess (>99 %). Preparative-scale deracemization was carried out with 50 mM (6.859 g L^?1) racemic 2-amino-2-phenylethanol, (S)-2-amino-2-phenylethanol was obtained in 75 % isolated yield and >99 % ee.

Full text of DOI:10.1002/cbic.202000491

Combining Chemistry and Biology
Accepted Article
Title: Enantioselective Cascade Biocatalysis for Deracemization of
Racemic β-Amino Alcohols to Enantiopure (S)-β-Amino Alcohols
Employing Cyclohexylamine Oxidase and ω-Transaminase
Authors: Jiandong Zhang, Ya-Wen Chang, Rui Dong, Xiao-Xiao Yang,
Li-Li Gao, Jing Li, Shuang-Ping Huang, Xing-Mei Guo, Chao-
Feng Zhang, and Hong-Hong Chang
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemBioChem 10.1002/cbic.202000491
Link to VoR: https://doi.org/10.1002/cbic.202000491
01/2020

10.1002/cbic.202000491
ChemBioChem
COMMUNICATION
Enantioselective Cascade Biocatalysis for Deracemization of
Racemic β-Amino Alcohols to Enantiopure (S)-β-Amino Alcohols
Employing Cyclohexylamine Oxidase and ω-Transaminase
Jian-Dong Zhang,*[a] Ya-Wen Chang,[b] Rui Dong,[a] Xiao-Xiao Yang,[a] Li-Li Gao,[c] Jing Li,[a] Shuang-
Ping Huang,[a] Xing-Mei Guo,*[b] Chao-Feng Zhang[a] and Hong-Hong Chang[a]
Dedication ((optional))
[a]
[b]
[c]
Dr. J. D. Zhang, R. Dong, X. X. Yang, Dr. J. Li, Dr.S. P. Huang, Dr. C. F. Zhang, Dr. H. H. Chang
Department of Biological and Pharmaceutical Engineering,
College of Biomedical Engineering, Taiyuan University of Technology
79 West Yingze Street, Taiyuan 030024, Shanxi (P.R. China)
E-mail: zhangjiandong@tyut.edu.cn
Y. W. Chang, Dr. X. M. Guo
Department of Environmental Engineering
Taiyuan University of Technology
79 West Yingze Street, Taiyuan 030024, Shanxi (P.R. China)
E-mail: guoxingmei@tyut.edu.cn
Dr. L. L. Gao
Department of Environmental Engineering
Taiyuan University of Technology
79 West Yingze Street, Taiyuan 030024, Shanxi (P.R. China)
Supporting information for this article is given via a link at the end of the document.((Please delete this text if not appropriate))
Abstract: Optically active β-amino alcohols are very useful chiral
strategies have been developed for the synthesis of these chiral
intermediates frequently used in the preparation of pharmaceutically
chemicals.[6] Such as Overman’s aminolysis of epoxides,[7]
active substances. Herein,
a
novel cyclohexylamine oxidase
Sharpless asymmetric aminohydroxylation of alkenes,[8] and
[9]
(ArCHAO) was identified from the genome sequence of Arthrobacter
sp. TYUT010-15 with the (R)-stereoselective deamination activity of
β-amino alcohol. ArCHAO was cloned and successfully expressed in
E. coli BL21, purified and characterized. Substrate specific analysis
revealed that ArCHAO has high activity (4.15 to 6.34 U mg-1 protein)
and excellent enantioselectivity toward the tested β-amino alcohols.
Using the purified ArCHAO, a wide range of racemic β-amino alcohols
were resolved, (S)-β-amino alcohols were obtained in >99% ee.
Deracemization of racemic β-amino alcohols was conducted by
ArCHAO catalyzed enantioselective deamination and transaminase
catalyzed enantioselective amination, affording (S)-β-amino alcohols
in excellent conversion (78-94%) and enantiomeric excess value
(>99%). Preparative scale deracemization was carried out with 50 mM
Jacobsen’s ring-opening of epoxides
were the most typical
methods. However, in most cases, these methods often require
harsh reaction conditions, laborious processes, and expensive
transition metals, making the methods inconvenient and
unsustainable.
During the past two decades, biocatalysis offers a green and
sustainable strategy for the synthesis of chiral β-amino alcohols.
For example, chiral amino ketones asymmetric reduction,[10]
racemic amino alcohols kinetic resolution,[11] and α-hydroxy
ketones asymmetric reduction amination.[11h,12] Very recently,
enantioselective aminohydroxylation of styrenyl olefins catalyzed
by an engineered hemoprotein.[13] In addition, several cascade
biocatalysis strategies combining different enzymes were also
reported for chiral vicinal amino alcohol synthesis.[14] However,
the limited scope of substrates, unsatisfactory product yield and
enantiomeric excess, and the protection and de-protection
processes made some of these methods inconvenient, alternative
strategies remain of great interest.
Cyclohexylamine oxidase (CHAO) was first described in
Brevibacterium oxydans IH-35A belongs to the flavin
monooxygenase family,[15] the reaction of this enzyme only
requires aerial oxygen with no need for additional co-factors. It
has been utilized in the preparation of enantiopure amines via
kinetic resolution or deracemization.[16] However, its synthetic
application was not fully exploited, especially, the synthesis of
optically active β-amino alcohols by CHAO was limited. Prompted
by the wide-ranging applications of enantiopure β-amino alcohols
in the pharmaceutical industry, we were interested in the
exploitation of novel enzymes and new methods for the synthesis
of these important chemicals. We recently isolated a new
(6.859
g
L‒1) racemic 2-amino-2-phenylethanol, (S)-2-amino-2-
phenylethanol was obtained in 75% isolated yield and >99% ee.
Optically active β-amino alcohols are very useful chiral
intermediates frequently used in the preparation of
pharmaceutically active substances.[1] Important examples
include Xanthine analogues as phosphodiesterase type 5 (PDE5)
inhibitors for the treatment of male erectile dysfunction (ED).[2]
Cycloalkanediamine derivatives as activated factor X (fXa) for the
therapy of thrombosis and related diseases.[3] Indinavir, an
important biologically active compound as
a
human
immunodeficiency virus (HIV) protease inhibitor.[4] In addition,
Ethambutol (EMB), an important anti-tubercular drug,[5] can be
prepared directly from (S)-(+)-2-amino-1-butanol. The importance
of chiral β-amino alcohols has attracted continuous and significant
attention from the synthetic groups, and a variety of chemical
1
This article is protected by copyright. All rights reserved.

10.1002/cbic.202000491
ChemBioChem
COMMUNICATION
Arthrobacter strain from the soil sample with (R)-selective
deamination activity of β-amino alcohols.[17] Herein, a novel
cyclohexylamine oxidase (ArCHAO) was successfully identified
from the genome sequence of Arthrobacter sp. TYUT010-15.
ArCHAO was heterologously expressed in the E. coli BL21,
purified and characterized. By using the purified ArCHAO, a
variety of racemic β-amino alcohols were resolved (Scheme 1) for
the first time. Since the maximum theoretical yield of the kinetic
resolution was only 50%, deracemization as an attractive strategy
oxidase (CHAO) from Brevibacterium oxydans. Then, the name
of the cloned enzyme was designated as ArCHAO (GenBank
accession number: MT862133). While cyclohexylamine oxidase
was reported for chiral amine synthesis, it is the first time that a
cyclohexylamine oxidase has been identified from Arthrobacter sp.
with the enantioselective deamination activity of β-amino alcohols.
Recombinant E. coli (ArCHAO) was induced with IPTG (0.5
mM) at the temperature of 20oC for 12 h. SDS-PAGE analysis
revealed that the cloned recombinant ArCHAO produced in E. coli
was in soluble form, and the enzyme was purified by using a Ni-
NTA affinity column chromatography (Figure S2). The purified
enzyme showed the expected molecular size of about 55 kDa.
The activity of purified ArCHAO toward the 2-aminocyclohexanol
1b was tested, the results showed that the ArCHAO has the
activity of 6.0 U mg-1 toward (1R, 2R)-1b, and 2.5 U mg-1 toward
(1S, 2S)-1b, demonstrated that the ArCHAO was an (R)-selective
amine oxidase toward β-amino alcohol. Methylbenzylamine was
also tested, a switch in enantiopreference to methylbenzylamine
was observed, this could be explained by Cahn-Ingold-Prelog
rules.[19] Then, the purified ArCHAO was characterized, the
optimum pH of purified ArCHAO toward the trans-2-
aminocyclohexanol 1b was determined using standard assay
conditions at different pHs. ArCHAO showed the maximum
activity at pH 7.0 (Figure 1a). The pH stability of ArCHAO was
investigated by storing the ArCHAO in 100 mM buffer with various
pH values (6.0-11.0) at 20oC. The results showed that the
ArCHAO was stable between pH7.0 and 10.0, >80% of residual
activities were observed over a period of 24 h (Figure 1c). The
optimum temperature for the deamination reaction at 30oC (Figure
1b). The temperature stability of the enzyme was investigated by
storing the enzyme in sodium phosphate buffer (100 mM, pH7.0)
at the temperature from 4 to 50oC for 24 h. As shown in Figure 1d,
the purified ArCHAO was stable at the temperature from 4 to 30oC,
and >70% residual activities of ArCHAO were observed at these
temperatures over a period of 24 h. When the temperature
increased to 50oC, only 10% residual activity of the enzyme was
detected after incubation for 24 h.
[18]
was adopted for the synthesis of optically active β-amino
alcohols in 100% theoretical yield. The deracemization process
was conducted by ArCHAO catalyzed enantioselective
deamination and transaminase catalyzed enantioselective
reduction amination (Scheme 2), affording the (S)-β-amino
alcohols in excellent conversions and ee values.
Scheme 1. Cyclohexylamine oxidase (ArCHAO) catalyzed kinetic resolution of
racemic β-amino alcohols
Scheme 2. Deracemization of racemic β-amino alcohols by ArCHAO combined
with transaminase
In order to acquire the target enzyme in the Arthrobacter sp.
TYUT010-15. Cell-free extract of Arthrobacter sp. TYUT010-15
was utilized to deamination of β-amino alcohol with or without
additional amine acceptor (pyruvate). However, no difference of
substrate conversions was observed. Interestingly, hydrogen
peroxide as the main by-product was detected from the reaction
mixture. We speculated that an amine oxidase from this strain
might be responsible for the deamination of β-amino alcohol.
Based on the genome sequence of Arthrobacter sp. TYUT010-15,
four amine oxidase genes were identified and cloned from
Arthrobacter sp. TYUT010-15 (Table S1-2, Figure S1). One of the
amine oxidases (Gene 4149) with significant deamination activity
of 2-aminocyclohexanol was discovered (Table S3). Nucleic acid
sequence alignment revealed that the discovered amine oxidase
has 99% identity to a FAD-dependent oxidoreductase from
Arthrobacter sp. TB 26 and 99% identity to a cyclohexylamine
Figure 1. Effect of pH and temperature on the activity and stability of ArCHAO.
Kinetic parameters of ArCHAO were determined at various
substrate concentrations with the use of Lineweaver-Burk plots
and nonlinear regression analysis. As shown in Table 1, although
2
This article is protected by copyright. All rights reserved.

10.1002/cbic.202000491
ChemBioChem
COMMUNICATION
Table 2. Kinetic resolution of racemic β-amino alcohols by ArCHAO. [a]
the affinities of ArCHAO toward (1S, 2S)-trans-1a-b (with the KM
values of 0.9 mM and 1.0 mM) were higher than (1R, 2R)- trans-
1a-b (with the KM values of 1.1 mM and 1.3 mM), the kcat/KM
values of the enzyme toward (1S, 2S)-trans-1a-b (1.8 s-1 mM-1
protein and 1.5 s-1 mM-1) were lower than (1R, 2R)-trans-1a-b (5.1
s-1 mM-1 and 4.5 s-1 mM-1). For the substrate 1c, the kcat/KM values
were 8.2 s-1 mM-1 toward (1R,2S)-cis-1c and 1.7 s-1 mM-1 toward
(1S,2R)-cis-1c with the KM values of 0.6 mM and 0.7 mM,
respectively. These results revealed that ArCHAO is more active
toward (R)-enantiomer than (S)-enantiomer, further demonstrated
the (R)-selective of the enzyme toward β-amino alcohol.
Sub.
Sub.
[mM]
50
Time
Specific activity
[U mg-1]
6.10
Conv.
[%][b]
56.2
58.1
53.4[d]
55.4
53.6
50.5
50.4
50.2
50.2
Sub. ee [%][c]
[h]
2
2
4
3
3
4
6
7
8
1a
1b
1c
1d
1e
1f
1g
1h
1i
>99.0 (1S,2S)
>99.0 (1S,2S)
>99.0(1S,2R)[e]
>99.0 (S)
>99.0 (S)
>99.0 (S)
>99.0 (S)
>99.0 (S)
>99.0 (S)
50
30
20
20
50
20
20
20
6.23
4.87
4.76
4.15
6.34
5.91
5.58
5.77
Table 1. Kinetic parameters of ArCHAO toward cyclic β-amino alcohols.[a]
Entry
Enantiomer
KM [mM]
Vmax [μmol
kcat [s-1]
kcat/KM [s-1
mM-1]
5.1
[a] Reaction conditions: rac-1 (20-50 mM), ArCHAO (10 mg mL-1), phosphate
buffer (100 mM, pH 7.0), shaking at 30°C for 2-8 h. [b] Determined by GC. [c]
Determined by chiral GC analysis. [d] Determined by HPLC. [e] Determined by
chiral HPLC analysis. Error limit: <2% of the state values.
mg-1]
6.2
1.8
6.4
1.7
5.4
1.3
1
2
3
4
5
6
(1R,2R)-1a
(1S,2S)-1a
(1R,2R)-1b
(1S,2S)-1b
(1R,2S)-1c
(1S,2R)-1c
1.1
0.9
1.3
1.0
0.6
0.7
5.6
1.6
5.8
1.5
4.9
1.2
1.8
4.5
1.5
8.2
1.7
[a] Reaction conditions: sodium phosphate buffer (100 mM, pH7.0) and six
concentrations (0.5-8 mM) of substrates, shaking at 30°C for 5 min. Kinetic
analysis with initial rate data obtained at the conversion lower than 5%.
Then, the purified ArCHAO was employed to kinetic resolution
of racemic β-amino alcohols 1a-i. As shown in Table 2, ArCHAO
has high activity toward the cyclic β-amino alcohols (±)-trans-1a
and (±)-trans-1b, with the activity of 6.1 U mg-1 and 6.23 U mg-1,
respectively. For (±)-cis-1c, the activity of the enzyme was
relatively low, and 4.87 U mg-1 was detected. Kinetic resolution of
racemic trans-1a-b and cis-1c were conducted by ArCHAO with
30-50 mM substrate concentration, (1S, 2S)-trans-1a, (1S, 2S)-
trans-1b and (1S, 2R)-cis-1c were obtained in >99% ee and 53-
58% conversion rates at 2-4 h, respectively. Four β-amino
alcohols 1f-i with different para substituent groups on an aromatic
ring were tested as substrates, high activities (from 5.77 U mg-1
to 6.34 U mg-1) of the enzyme toward these substrates were
detected, kinetic resolution of (±)-1f-i resulted in about 50%
conversion rate, leave the (S)-1f-i in >99% ee. Racemic 1d and
1e as two typical aliphatic β-amino alcohols were then tested,
relative low activities (4.76 U mg-1 and 4.15 U mg-1) were obtained,
kinetic resolution of these substrates resulted in 55.4% and 53.6%
conversion, leave the (S)-1d and (S)-1e in >99% ee. In addition,
two racemic amines phenethylamine and 1,2,3,4-tetrahydro-1-
naphthylamine were also resolved by ArCHAO, (R)-
phenethylamine and (R)-1,2,3,4-tetrahydro-1-naphthylamine
were obtained in >99% ee and 50% conversion (Figure S12 and
Figure S13), respectively. The time courses for the kinetic
resolution 50-400 mM of racemic trans-1b were shown in Figure
2. ArCHAO exhibited strong tolerance against high substrate
concentration of 1b, 55-56% conversion could be obtained when
kinetic resolution 50-200 mM of 1b at 3-12 h, even if the
concentration of 1b was increased to 400 mM, racemic trans-1b
could be completely resolved by ArCHAO at 30 h. The synthetic
potential of ArCHAO was then demonstrated by kinetic resolution
of racemic trans-1b (400 mM) at a 50 mL-scale, (1S, 2S)-trans-1b
obtained in 26.3% isolated yield (790.2 mg) and >99% ee. To our
best knowledge, (1S, 2S)-trans-1b can be used as a building
block in the synthesis of “Peptoid” cholecystokinin (CCK-B)
receptor antagonists (CI-1015) for the treatment of anxiety and
panic disorder.[1b]
Figure 2. The time course for the kinetic resolution of racemic trans-1b by
ArCHAO.
Since the highest yield of the kinetic resolution was only 50%,
deracemization was chosen as an efficient process to access the
chiral β-amino alcohols in a 100% theoretical yield. Herein we
reported a new deracemization strategy for the preparation of
chiral β-amino alcohols, the strategy was conducted by ArCHAO
catalyzed enantioselective deamination and transaminase
catalyzed enantioselective reduction amination (Scheme 2).
While many amines have been deracemized by amine oxidases
combined with various chemical reducing agents,[20] studies on
the deracemization of racemic β-amino alcohols have been
limited. As shown in Scheme 2, the ArCHAO mainly oxidizes the
(R)-enantiomer to the corresponding α-hydroxy ketone 2, which is
then reduced back to the (S)-enantiomer 1 by an (S)-selective
transaminase (MVTA).[11h] The cascade biocatalysis process
results in eventual accumulation of the (S)-enantiomer in high
yield and ee. To decrease the cost of enzyme preparation, the
whole cells of E. coli (ArCHAO) and E. coli (MVTA) were used in
the deracemization process. Six racemic β-amino alcohols (1d-i)
were subjected to the new deracemization protocol using the
whole cells of E. coli (ArCHAO) (10 g cdw L-1) and E. coli (MVTA)
(10 g cdw L-1) (Table 3). (S)-1d and (S)-1f-g could be obtained in
91.3-92.9% conversions and >99% ee at 6-8 h with 20 mM (±)-1d,
(±)-1f and (±)-1g, respectively. Increasing the (±)-1f to 50 mM,
92% conversion of 1f could be obtained at 12 h. In the case of (±)-
1e and (±)-1h-i, (S)-1e and (S)-1h-i were obtained in 78.5-85.7%
conversions and >99% ee. To demonstrate the synthetic potential
3
This article is protected by copyright. All rights reserved.

10.1002/cbic.202000491
ChemBioChem
COMMUNICATION
3272; e) D. A. Erlanson, J. W. Arndt, M. T. Cancilla, K. Cao, R. A.
Elling, N. English, J. Friedman, S. K. Hansen, C. Hession, I. Joseph,
G. Kumaravel, W. C. Lee, K. E. Lind, R. S. McDowell, K. Miatkowski,
C. Nguyen, T. B. Nguyen, S. Park, N. Pathan, D. M. Penny, M. J.
Romanowski, D. Scott, L. Silvian, R. L. Simmons, B. T. Tangonan, W.
Yang, L. Sun, Bioorg. Med. Chem. Lett. 2011, 21, 3078–3083.
Y. Wang, S. Chackalamannil, Z. Hu, C. D. Boyle, C. M. Lankin, Y. Xia,
X. Ruo, T. Asberom, D. Pissarnitski, A. W. Stamford, W. J. Greenlee,
J. Skell, S. Kurowski, S. Vemulapalli, J. Palamanda, M. Chintala, P.
Wu, J. Myers, P. Wang, Bioorg. Med. Chem. Lett. 2002, 12, 3149–
3152.
T. Nagata, T. Yoshino, N. Haginoya, K. Yoshikawa, Y. Isobe, T.
Furugohri, H. Kanno, Bioorg. Med. Chem. Lett. 2007, 17, 4683–4688.
R. Sakurai, K. Sakai, Tetrahedron: Asymmetry 2003, 14, 411–413.
R. E. Lee, M. Protopopova, E. Crooks, R. A. Slayden, M. Terrot, C. E.
Barry III, J. Comb. Chem. 2003, 5, 172–187.
of this new deracemization process, a 100 mL-scale preparation
experiment was carried out with 50 mM racemic 1f (686 mg). (S)-
1f could be obtained in 90% conversion and >99% ee at 16 h.
After extraction and purification, (S)-1f was obtained in 75%
isolated yield (514 mg) and >99% ee (Figure S21).
[2]
[3]
Table 3. Deracemizatioin of racemic β-amino alcohols employing E. coli
(ArCHAO) and E. coli (MVTA).[a]
[4]
[5]
[6]
a) T. Kawabata, K. Yamamoto, Y. Momose, H. Yoshida, Y. Nagaoka,
K. Fuji, Chem. Commun. 2001, 24, 2700–2701; b) I. Schiffers, T.
Rantanen, F. Schmidt, W. Bergmans, L. Zani, C. Bolm, J. Org. Chem.
2006, 71, 2320–2331; c) C. H. Senanayake, L.M. Dimichele, J. Liu, L.
E. Fredenburgh, K. M. Ryan, E. F. Roberts, R. D. Larsen, T. R.
Verhoeven, P. J. Reider, Tetrahedron let. 1995, 36, 7615–7618; d) S.
Liu, J. H. Xie, W. Li, W. L. Kong, L X. Wang, Q. L. Zhou, Org. Lett.
2009, 11, 4994–4997; e) Q. Hu, J. Chen, Z. Zhang, Y. Liu, W. Zhang,
Org. Lett. 2016, 18, 1290–1293; f) M. J. McKennon, A. I. Meyers, J.
Org. Chem. 1993, 58, 3568–3571; g) A. W. Buesking, J. A. Ellman,
Chem. Sci. 2014, 5, 1983–1987.
Substrate
Sub. [mM]
Time [h]
6
7
6
12
8
10
10
Conv. [%][b]
91.3
85.4
93.6
92.0
92.9
85.7
78.5
Sub. ee [%][c]
>99.0 (S)
>99.0 (S)
>99.0 (S)
>99.0 (S)
>99.0 (S)
>99.0 (S)
>99.0 (S)
1d
1e
1f
20
20
20
50
20
20
20
1f
1g
1h
1i
[7]
[8]
L. E. Overman, S. Sugai, J. Org. Chem. 1985, 50, 4154–4155.
a) K. B. Sharpless, D. W. Patrick, L. K. Truesdale, S. A. Biller, J. Am.
Chem. Soc. 1975, 97, 2305–2307; b) G. Li, H. Chang, K. B. Sharpless,
Angew. Chem. Int. Ed. 1996, 35, 451–454.
a) L. E. Martinez, J. L. Leighton, D. H. Carsten, E. N. Jacobsen, J.
Am. Chem. Soc. 1995, 117, 5897–5898; b) J. A. Birrell, E. N.
Jacobsen, Org. lett. 2013, 15, 2895–2897.
[a] Reaction conditions: rac-substrates (20-50 mM), E. coli (ArCHAO) (10 g cdw
L-1), E. coli (MVTA) (10 g cdw L-1), (R)-phenethylamine (10-25 mM), phosphate
buffer (100 mM, pH7.0, 0.1 mM PLP), shaking at 30oC for 6-12 h. [b] Determined
by GC, defined as percentage ratio of the (S)-β-amino alcohol product (mM) to
initial racemic substrate (mM), error limit: <2% of the state values. [c]
Determined by GC analysis on a chiral phase.
[9]
[10] R. Patel, A. Banerjee, J. Howell, C. McNamee, D. Brozozowski, D.
Mirfakhrae, V. Nanduri, J. Thottathil, L. Szarka, Tetrahedron:
Asymmetry 1993, 4, 2069–2084.
In summary, a novel cyclohexylamine oxidase (ArCHAO) was
discovered from Arthrobacter sp. TYUT010-15 with the highly (R)-
selective deamination activity toward β-amino alcohols. A variety
of racemic β-amino alcohols were resolved by the purified
ArCHAO, excellent conversion and ee value were obtained.
Deracemization of racemic β-amino alcohols was conducted by
[11] a) A. Mitrochkine, G. Gil, M. Réglier, Tetrahedron: Asymmetry 1995
,
6, 1535–1538; b) A. Rouf, P. Gupta, M. A. Aga, B. Kumar, R. Parshad,
S. C. Taneja, Tetrahedron: Asymmetry 2011, 22, 2134–2143; c) A.
Maestro, A. Astorga, V. Gotor, Tetrahedron Asymmetry 1997, 8,
3153–3159; d) A. Luna, C. Astorga, F. Fülöp, V. Gotor, Tetrahedron:
Asymmetry 1998, 9, 4483–4487; e) J. González-Sabín, N. Ríos-
Lombardía, V. Gotor, F. Morís, Tetrahedron: Asymmetry 2013, 24,
1421–1425; f) P. Gupta, N. Mahajan, New J. Chem. 2018, 42, 12296–
12327; g) M. S. Malik, E. S. Park, J. S. Shin, Green Chem. 2012, 14,
2137–2140; h) J. D. Zhang, J. W. Zhao, L. L. Gao, H. H. Chang, W.
L. Wei, J. H. Xu, J. J. Biotechnol. 2019, 290, 24–32.
ArCHAO
catalyzed
enantioselective
deamination
and
transaminase catalyzed enantioselective reduction amination,
various racemic β-amino alcohols were deracemized to (S)-β-
amino alcohols in excellent conversions (78-92%) and ee values
(>99%). Preparation experiment of deracemization was
[12] a) A. Nobili, F. Steffen-Munsberg, H. Kohls, I. Trentin, C. Schulzke,
M. Höhne, U. T. Bornscheuer, ChemCatChem 2015, 7, 757–760; b)
F. F. Chen, S. C. Cosgrove, W. R. Birmingham, J. Mangas-Sanchez,
J. Citoler, M. P. Thompson, G. W. Zheng, J. H. Xu, N. J. Turner, ACS
Catal. 2019, 9, 11813−11818.
conducted with 50 mM (6.86
g
L‒1) racemic 2-amino-2-
phenylethanol, (S)-2-amino-2-phenylethanol was obtained in 75%
isolated yield and >99% ee. The remarkable features of ArCHAO
highlight its greater potential for the synthesis of chiral β-amino
alcohols. The designed deracemization process might be
generally applicable to access other important chiral amines by
employing appropriate enzymes.
[13] I. Cho, C. K. Prier, Z-J. Jia, R. K. Zhang, T. Görbe, F. H. Arnold,
Angew. Chem. Int. Ed. 2019, 58, 3138-3142.
[14] a) T. Sehl, H. C. Hailes, J. M. Ward, R. Wardenga, E. von Lieres, H.
Offermann, R. Westphal, M. Pohl, and D. Rother, Angew. Chem. Int.
Ed. 2013, 52, 6772–6775; b) J. D. Zhang, X. X. Yang, Q. Jia, J. W.
Zhao, L. L. Gao, W. C. Gao, H. H. Chang, W. L. Wei, J. H. Xu, Catal,
Sci, Technol. 2019, 9, 70–74; c) M. L. Corrado, T. Knaus, F. G. Mutti,
Green Chem. 2019, 21, 6246-6251.
[15] a) H. Iwaki, M. Shimizu, T. Tokuyama,Y. Hasegawa, Appl. Environ.
Microbiol. 1999, 65, 2232-2234; b) H. Leisch, S. Grosse, H. Iwaki, Y.
Hasegawa, P. C. K. Lau, Can. J. Chem. 2012, 90, 39–45.
[16] a) G. Li, J. Ren, P. Yao, Y. Duan, H. Zhang, Q. Wu, J. Feng, P. C. K.
Lau, D. Zhu, ACS Catal. 2014, 4, 903–908; b) X. Wu, Z. Huang, Z.
Wang, Z. Li, J. Wang, J. Lin, F. Chen, J. Org. Chem. 2020, 85, 5598–
5614.
Acknowledgements ((optional))
This study was financially supported by the National Natural
Science Foundation of China (Grant No. 21772141), the Shanxi
Province Science Foundation for Youths (grant No.
201701D221042).
[17] Y. W. Chang, J. D. Zhang, X. X. Yang, J. Li, L. L. Gao, S. P. Huang,
X. M. Guo, C. F. Zhang, H. H. Chang, J. H. Xu, Biotechnol. Lett. 2020
,
42, 1501–1511.
[18] a) M. Alexeeva, A. Enright, M. J. Dawson, M. Mahmoudian, N. J.
Turner, Angew. Chem. Int. Ed. 2002, 41, 3177–3180; b) C. C. Gruber,
I. Lavandera, K. Faber, W. Kroutil, Adv. Synth. Catal. 2006, 348,
1789–1805; c) D. Ghislieri, D. Houghton, A. P. Green, S. C. Willies,
N. J. Turner, ACS Catal. 2013, 3, 2869–2872; d) G. Y. Li, J. Ren, P.
Y. Yao, Y. T. Duan, H. L. Zhang, Q. Q. Wu, J. H. Feng, P. C. K. Lau,
D. M. Zhu, ACS Catal. 2014, 4, 903–908; e) S. W. Han, Y. H. Jang,
J. S. Shin, ACS Catal. 2019, 9, 6945–6954; f) S. Yoon, M. D. Patil, S.
Sarak, H. Jeon, G. H. Kim, T. P. Khobragade, S. Sung, H. Yun,
ChemCatChem 2019, 11, 1898–1902.
[19] I. V. Pavlidis, M. S. Weiβ, M. Genz, P. Spurr, S. P. Hanlon, B. Wirz,
H. Iding, U. T. Bornscheuer, Nat. Chem. 2016, 8, 1076–1082.
[20] a) C. J. Dunsmore, R. Carr, T. Fleming, N. J. Turner, J. Am. Chem.
Soc. 2006, 128, 2224–2225; b) V. F. Batista, J. L. Galman, D. C. G.
A. Pinto, A. M. Silva, N. J. Turner, ACS Catal. 2018, 8, 11889–11907.
Keywords: cascade biocatalysis • deracemization •
cyclohexylamine oxidase • transaminase • β-amino alcohols
[1]
a) S. C. Bergmeier, Tetrahedron 2000, 56, 2561–2576; b) B. K.
Trivedi, K. Janak, J. K. Padia, A Holmes, S. Rose, D. S. Wright, J. P.
Hinton, M. C. Pritchard, J. M. Eden, C. Kneen,; L. Webdale, N.
Suman-Chauhan, P. Boden, L. Singh, M. J. Field, D. Hill, J. Med.
Chem. 1998, 41, 38–45; c) T. Govindaraju, R. G. Gonnade, M. M.
Bhadbhade, V. A. Kumar, K. N. Ganesh, Org. Lett. 2003, 5, 3013–
3016; d) H. D. Arndt, B. Ziemer, U. Koert, Org. Lett. 2004, 6, 3269–
4
This article is protected by copyright. All rights reserved.

10.1002/cbic.202000491
ChemBioChem
COMMUNICATION
Entry for the Table of Contents
Insert graphic for Table of Contents here. ((Please ensure your graphic is in one of following formats))
Enantioselective cascade biocatalysis for deracemization of racemic β-amino alcohols was conducted by employing cyclohexylamine
oxidase and ω-transaminase. (S)-β-amino alcohols were obtained in excellent conversions and ee values.
Institute and/or researcher Twitter usernames: ((optional))
5
This article is protected by copyright. All rights reserved.