DOI: 10.1002/cctc.201501170
Communications
A Novel (R)-Imine Reductase from Paenibacillus lactis for
Asymmetric Reduction of 3H-Indoles
Hao Li, Guang-Xiang Zhang+, Liu-Mei Li+, Yu-Shi Ou+, Ming-Yang Wang+, Chun-Xiu Li,
Gao-Wei Zheng,* and Jian-He Xu*[a]
A novel (R)-imine reductase (PlRIR) from Paenibacillus lactis was
heterologously overexpressed in Escherichia coli, purified and
characterized. The purified PlRIR exhibited relatively high cata-
lytic efficiency (kcat/Km =1.58 sÀ1 mmÀ1) towards 2,3,3-tri-
methylindolenine. A panel of 3H-indoles and 3H-indole iodides
were reduced by PlRIR to yield the corresponding products
with good-to-excellent enantioselectivity (66–98% ee). In addi-
tion, PlRIR also possesses good activities toward other types of
imines such as pyrroline, tetrahydropyridine, and dihydroiso-
quinoline, indicating a reasonably broad substrate acceptance.
In a 100 mg scale preparative reaction, 100 mm 2,3,3-tri-
methylindolenine was converted efficiently to afford (R)-2,3,3-
trimethylindoline with 96% ee and 81% yield.
pounds.[6] Asymmetric reduction of aryl imines and b-carboline
imines has been performed with Candida parapsilosis
ATCC 7330 and Saccharomyces bayanus, respectively.[7] Howev-
er, the two enzymes responsible for the reduction reactions
have not been isolated, purified, and further studied. Recently,
the Mitsukura group identified two useful wild-type strains
from their stock microorganisms with R-selective (Streptomyces
sp. GF 3587) and S-selective (Streptomyces sp. GF 3546) IRED
activities by using 2-methyl-1-pyrroline (2-MPN) as the screen-
ing substrate.[8] Subsequently, both R- and S-selective IREDs
were purified and characterized, and their DNA sequences
were also reported.[9] Turner and co-workers overexpressed
these two IREDs in Escherichia coli, and expanded their sub-
strate scope to other cyclic imines, such as tetrahydropyridines,
dihydroisoquinolines, and b-carboline imines.[10] Furthermore,
the ability of IREDs to directly catalyze the reductive amination
of ketones was demonstrated, even though the conversion
was unusually low.[11] The X-ray crystal structures of R-selective
and S-selective IREDs have been solved and revealed the
active-site residues (Asp or Tyr).[11a,12]
Chiral amines represent the core structure of numerous natural
alkaloids, pharmaceuticals, and agrochemicals because of their
exquisite biological activity, and thus are increasingly in
demand in organic synthesis.[1] Traditionally, chiral amines are
synthesized by kinetic resolution of a racemate by using
a chiral acid as the resolving agent.[2] Compared with kinetic
resolution, which has a maximum possible yield of 50%, asym-
metric synthesis by chemical approaches has attracted consid-
erable attention in recent years because of high atom efficien-
cy, and includes asymmetric reduction of enamides, reductive
amination of ketones, and asymmetric reduction of imines.[3]
As an alternative method, biocatalysis has attracted attention
for the preparation of chiral amines because of the high ste-
reoselectivity and environmental friendliness of bioprocesses.[4]
Despite the significant development of biocatalytic ap-
proaches,[5] there is strong interest in the asymmetric reduction
of imines by using imine reductases (IREDs), which afford the
product in 100% yield and with 100% ee.
Because of the tremendous potential in organic synthesis,
the exploration of novel IREDs is very attractive. Hauer and co-
workers constructed several IREDs by sequence alignment and
characterized three representative IREDs.[13] Both reductive and
oxidative activities of three other IREDs towards imines and
amines were examined by the Hçhne group.[14] Iding and co-
workers identified 20 new IREDs by C-terminal domain cluster-
ing, further expanding the IRED toolbox.[15] Nonetheless, the
exploration of novel IREDs with both a broad substrate scope
and high enantioselectivity remains highly desirable for the
synthesis of chiral amines.
In our previous work, an S-selective IRED from Paenibacillus
lactis (PlSIR) was identified, and exhibited high activity and ex-
cellent enantioselectivity in the asymmetric reduction of 3H-in-
doles, yielding a series of (S)-chiral indolines.[16] 3H-indoles are
a class of cyclic imine reported frequently in asymmetric reduc-
tions promoted by chemical catalysts.[17] Herein, an R-selective
IRED from P. lactis (PlRIR) was identified through a genome
data-mining approach by using 2,3,3-trimethylindolenine as
the model substrate (Scheme 1). The biochemical properties
and substrate scope of this novel enzyme were investigated in
detail. Furthermore, the enzymatic synthesis of (R)-2,3,3-tri-
methyl indoline was performed at a preparative scale to verify
the practical capacity of PlRIR.
IREDs, a rapidly emerging class of enzymes, catalyze the
asymmetric hydrogenation of the prochiral C=N bond of
imines (especially cyclic imines) to form chiral amine com-
[a] H. Li, G.-X. Zhang,+ L.-M. Li,+ Y.-S. Ou,+ M.-Y. Wang,+ Dr. C.-X. Li,
Dr. G.-W. Zheng, Prof. Dr. J.-H. Xu
State Key Laboratory of Bioreactor Engineering
Shanghai Collaborative Innovation Center for Biomanufacturing
East China University of Science and Technology
130 Meilong Road, Shanghai 200237 (P.R. China)
E-mail: gaoweizheng@ecust.edu.cn
[+] These authors contributed equally to this work.
The PlRIR-encoding gene (WP_007130043.1) inserted into
plasmid pET-28a was successfully overexpressed in E. coli BL21
(DE3). The target protein was predominantly in the soluble
Supporting Information for this article is available on the WWW under
ChemCatChem 2016, 8, 724 – 727
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