Deracemization of Phenyl-Substituted 2-Methyl-1,2,3,4-Tetrahydroquinolines by a Recombinant Monoamine Oxidase from Pseudomonas monteilii ZMU-T01

Article abstract of DOI:10.1002/cctc.201701995

A monoamine oxidase (MAO5) from Pseudomonas monteilii ZMU-T01 was first heterologously expressed in Escherichia coli BL21(DE3) and then used as a biocatalyst for the deracemization of racemic 2-methyl-1,2,3,4-tetrahdroquinoline derivatives to yield the unreacted R enantiomer with up to >99 % ee. Sequence alignment revealed that MAO5 shared 14.7 % identity toward the well-studied monoamine oxidase (MAO-N).

Full text of DOI:10.1002/cctc.201701995

Accepted Article
Title: Deracemisation of phenyl substituted 2-methyl-1,2,3,4-
tetrahydroquinolines by a recombinant monoamine oxidase from
Pseudomonas monteilii ZMU-T01
Authors: Guozhong Deng, Nanwei Wan, Lei Qin, Baodong Cui, Miao
An, Wenyong Han, and Yongzheng Chen
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To be cited as: ChemCatChem 10.1002/cctc.201701995
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10.1002/cctc.201701995
ChemCatChem
COMMUNICATION
Deracemisation of phenyl substituted 2-methyl-1,2,3,4-
tetrahydroquinolines by a recombinant monoamine oxidase from
Pseudomonas monteilii ZMU-T01
Guozhong Deng,[a,b] Nanwei Wan,[a,b] Lei Qin,[a,b] Baodong Cui,[a,b] Miao An,[a,b] Wenyong Han,[a,b] and
Yongzheng Chen*[a,b]
Abstract:
A monoamine oxidase (MAO5) from Pseudomonas
monteilii ZMU-T01 was firstly heterologously expressed in
Escherichia coli BL21(DE3), and used as an biocatalyst for
deracemisation of racemic 2-methyl-1,2,3,4-tetrahdroquinoline
derivatives to yield unreacted (R)-enantiomer with up to >99% ee.
Sequence alignment revealed MAO5 shared 14.7% identity toward
the well-studied MAO-N.
Figure1. Representative examples of pharmaceuticals and natural
products containing chiral tetrahydroquinolinescaffold.
Enantiomerically pure amines are important scaffolds in
pharmaceutical compounds and widely used as chiral auxiliaries
and resolving agents in organic synthesis. In the past decades,
considerable efforts have been devoted to developing
asymmetric routes to produce chiral amine derivatives with both
highly chemical and enantiomeric purity. The chemocatalytic
methods such as reductive amination and imine reduction have
been well discussed in several reviews.[1] An alternative
methodology for the synthesis of chiral amines via the toolkit of
biocatalysts has been developed involving different classes of
enzymes, including ω-transaminases, ammonia lyases, amine
dehydrogenases, NADPH-dependent imine reductases, Pictet-
Spenglerase and flavin-containing monoamine oxidases
(MAOs).[2] MAOs could catalyse oxygen-dependent conversion
of one enantiomer of amines into imines, thereby providing the
corresponding chiral amines. MAO enzyme family include
monoamine oxidase N (MAO-N) from Aspergillus niger, bacterial
cyclohexylamine oxidase from Brevibacterium oxydans IH-35A
(CHAO)[3] and 6-hydroxy nicotine oxidase (6-HNO).[4] The MAO-
N is usually S selective and always has a broad substrate scope
that has been demonstrated by Turner'swork, involving directed
evolution forgenerating many genetic variants for the
deracemisation of primary, secondary and tertiary amines.[5]
Optically pure tetrahydroquinoline (THQ) derivatives are an
important class of chiral amines, which serve as building blocks
for asymmetric synthesis of pharmaceuticals, agrochemicals,
and natural products,[6] such as antibacterialdrug (S)-flumequine
and bioactive alkaloids (+)-Galipimine and (+)-Cuspareine
(Figure1).[7] The transition- metal catalysed asymmetric
hydrogenation and asymmetric transfer hydrogenations of
quinolines are the usually efficient and atom-economic methods
for synthesis of the chiral tetrahydroquinolines.[8]Recently, we
have exploring the biocatalytic routes for the synthesis of chiral
[9]
THQ derivatives.
The previous study revealed that strain
Pseudomonas monteilii ZMU-T01 could specifically mediate
oxidative resolution of racemic 2-substituted-THQ derivatives,
but non activity were found toward the substrates with
[9c]
substitution on phenyl ring.
In this work, we cloned and
heterologously expressed a monoamine oxidase (MAO5) from
the strain P.monteilii ZMU-T01, and further explored its
application in deracemisation of racemic 2-methyl-1,2,3,4-
tetrahdroquinoline (2-MTHQ) with different substitution on
phenyl ring (Scheme 1). Herein, we wish to disclose our
research progress on this subject.
We began our study by searching the complete genome
sequence of P. monteilii SB3101.[10] Five MAO sequences were
obtained, amplified and heterologously expressed in E. coli
BL21(DE3) with the expression plasmid pET-32a(+) (see
TableS1 and Figure S1). Activity assay was carried out using the
whole cells of recombinant MAO strains. The results
revealedthat only recombinant MAO5 showed the catalytic
activity toward (rac)-2-MTHQ. Sequence alignment showed the
amino acid sequence of MAO5 sharing low identity toward MAO-
N[3] (14.7%) and CHAO[5] (25.2%) (see Figure S2).
[a]
[b]
Generic Drug Research Center of Guizhou Province, School of
Pharmacy, Zunyi Medical University, Zunyi, 563000, China.
Green Pharmaceuticals Engineering Research Center of Guizhou
Province, School of Pharmacy, Zunyi Medical University, Zunyi,
563000, China.
*Prof. Dr.Yongzheng Chen.
E-mail: yzchen@zmc.edu.cn.
Supporting information for this article is given via a link at the end of
the document.
Scheme 1.Deracemisation of 2-substituted THQ derivatives using
P.monteilii ZMU-T01 or
a recombinant monoamine oxidase strain
(MAO5-ZMU6).
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Table 1. Catalytic activity and enantioselectivity of the strain MAO5-
ZMU6 on a series of 2-substituted THQ 1a-h.[a]
The expression conditions of MAO-5 in E. coli were optimized,
including the concentration of IPTG, induction temperature and
induction time (see Figure S3-S5). To further improve the
catalytic efficiency, several expression systems were applied to
heterologous expression of MAO5. From the results of Figure 2,
it could be found that strain MAO5-ZMU6, which expressed
MAO5 using pET-28b(+) in E. coli BL21(DE3) along with a
chaperone plasmid pG-KJE8, showed the relative highest
activity. The relative activity of strain MAO5-ZMU6 was 14-fold
higher than P. monteilii ZMU-T01 and 1.6-fold higher than strain
MAO5-ZMU1. Unfortunately, expression of MAO5 in Pichia
pastoris X-33 did not show activity with both shuttle plasmid
pPICZα-A and pPICZ-A. SDS-PAGE analysis indicated P.
pastoris X-33 was not applicable to heterologous express MAO5.
Subsequently, the pH and temperature effects of strain MAO5-
ZMU6 were investigated. The results indicated the relative
highest activity was found at pH 7.5 in Tris-H2SO4 buffer and 37
oC (see Figure S6-S7).
Based on the optimal reaction conditions, the activity and
enantioselectivity toward eight 2-substituted-THQ derivatives 1a-
h were investigated using the whole cells of strain MAO5-ZMU6
as biocatalyst (Table 1). Generally, the MAO5 was found to be
slightly active towards 1a-f, while inactive towards 1g and 1h.
Among these eight substrates examined in Table 1, strain
MAO5-ZMU6 showed specifically excellent resolution activity to
1a, giving >99% eeof (R)-1a.
Entry
1[d]
Conv.(%)[b]
51
ee (%)[b]/Config.[c]
>99/(R)-1a
Substrate
1
1a
1b
1c
2
3
56
64
14/(R)-1b
18/(R)-1c
36/(S)-1d
4
5
6
7
8
66
39
1d
1e
1f
27/(R)-1e
10/(R)-1f
N.D [e]
6
N
H
Trace
Trace
1g
1h
N.D.
Subsequently, the substrate specificity in 2-methyl-THQ
derivatives 1i-r with different substitution on the phenyl ring
wasfurther examined and the results were depicted in Table 2.In
general, regardless of electronic property or position of the
substitution on the phenyl ring of 2-methyl-THQ 1, strain MAO5-
ZMU6 showed overall excellent catalytic activity and the
corresponding (R)-2-methyl-THQ 1 were obtained with97%-
>99% ee and up to>200 E value.
[a]Unless otherwise noted, the reactions were performed in 5 mL Tris-
H2SO4 buffer (50 mM, pH 7.5) containing 20 dcw g/L of MAO5-ZMU6 and
2 mM substrate.[b]The conversion of 1a-h to 2a-h was calculated by HPLC
analysis.[c]Determined by comparison with the literature data or by
analogue. [d]The reaction was conducted for 6 h. [e]N.D. = not detected.
Table 2. Deracemisation of 2-methyl-THQ derivatives 1i-r using the
MAO5-ZMU6 strain.[a]
Conv.
(%)b
ee(%)[b]
E[d]
Entry
1
Substrate1
/Config.[c]
64
58
56
53
99/(R)-1i
>99/(R)-1j
99/(R)-1k
>99/(R)-1l
17
1i
2
3
4
31
39
80
1j
1k
1l
F
5
6
51
51
>99/(R)-1m
>99/(R)-1n
>200
>200
N
H
1m
1n
Figure 2. The relative activities toward (rac)-2-MTHQ of different strains.
The information of strains was listed in Table S2.
7
8
58
51
99/(R)-1o
97/(R)-1p
28
1o
1p
>200
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10.1002/cctc.201701995
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COMMUNICATION
9
65
51
>99/(R)-1q
98/(R)-1r
16
1q
10
122
1r
[a]Unless otherwise noted, the reactions were performed in 5 mL Tris-
H2SO4 buffer (50 mM, pH 7.5) containing 20 dcwg/L of MAO5-ZMU6
and substrate 1i-r (1-4 mM).[b] The conversion of 1i-r to 2i-r was
calculated by HPLC analysis.[c]Determined by comparison with the
literature data or by analogue. [d]The E values were calculated by
using the program selectivity.[11]
To understand the resolution process of 2-MTHQ
derivatives, kinetic resolution experiment was performed
using 4 mM of (rac)-1a with the cell pellet of strain MAO5-
ZMU6 as biocatalyst. The reaction time courses were
determined and showed in Figure 3A. In the kinetic
resolution process, (S)-1a was quickly converted to 2a for 5
h. At this moment, the ee of (R)-1a reached up to >99%,
remaining 1.94 mM unreacted (R)-1a. As time went by, the
concentration of (R)-1a was reduced slowly and
accompanied by the increase of the yield of 2a. The kinetic
resolution results revealed that the (R)-1a could also be
converted to 2a when the (S)-1a was depleted. The highest
49% analytical yield and >99% ee of (R)-1a were obtained
in this kinetic resolution process.
Dynamic kinetic resolution experiment toward 4 mM of
(rac)-1a was also carried out using 4 mM of NH3·BH3 as
non-selective reductant (Scheme 2). The reaction course
depicted in Figure 3B revealed the concentration of (R)-1a
could reach highest to 3.54 mM with only 96% ee for 10 h.
Furthermore, the ee of (R)-1a slowly increased to 97% for
18 h and 98% for 24 h, respectively, while the concentration
reduced to about 3.20 mM. In the dynamic kinetic resolution
process, up to 98% ee of (R)-1a was obtained with 83%
analytical yield. Obviously, this was the highest yield to be
Figure 3. The time courses for kinetic resolution (A) and dynamic kinetic
resolution (B) of (rac)-1a using the whole cells of strain MAO5-ZMU6.
obtained by deracemisation of (rac)-1a
.
In summary, we have developed an efficient biocatalytic
procedure for specific deracemisation of 2-methyl-1,2,3,4-
tetrahdroquinolines with different substitution on the phenyl
ring. With the recombinant monoamine oxidase from P.
monteilii ZMU-T01 as the biocatalyst, a series of (R)-2-
methyl-1,2,3,4-tetrahdroquinolines were provided with up
to >99% ee. Besides, dynamic kinetic resolution of (rac)-2-
MTHQ 1a with NH3·BH3 improved the yield of (R)-2-MTHQ
1a up to 83% with 98% ee. Further study focusing on
soluble expression and directed evolution of MAO5 will be
performed to increase substrate concentration.
Acknowledgements
We are grateful for financial support from the NSFC (No.
21562054), Guizhou Science and Technology Department (No.
QKHRCTD-2014-4002, QKHRC-2016-4029, QKHPTRC-2016-
5801), National First-Rate Construction Discipline of Pharmacy
(GNYL-2017-006), the Fifth Batch of Talent Base in Guizhou
Province, and Program for Outstanding Youth of Zunyi Medical
University.
Keywords:Monoamine oxidase • Deracemisation• 2-methyl-
1,2,3,4-tetrahydroquinoline• Biocatalysis
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Entry for the Table of Contents (Please choose one layout)
Layout 2:
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Guozhong Deng, Nanwei Wan,Lei Qin,
Baodong Cui, Miao An,Wenyong Han,
and Yongzheng Chen*
Page No. – Page No.
A novel monoamine oxidase (MAO5) was heterologously expressed in Escherichia
coli BL21(DE3). Deracemisation of eleven 2-methyl-1,2,3,4-tetrahydroquinoline
derivatives by the recombinant monoamine oxidase gave excellent ee values up to
>99% of unreacted (R)-enantiomer.
Deracemisation of 2-methyl-1,2,3,4-
tetrahydroquinoline derivatives by a
recombinant monoamine oxidase
from Pseudomonas monteilii ZMU-
T01
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