Identification of an Imine Reductase for Asymmetric Reduction of Bulky Dihydroisoquinolines

Article abstract of DOI:10.1021/acs.orglett.7b01274

A new imine reductase from Stackebrandtia nassauensis (SnIR) was identified, which displayed over 25- to 1400-fold greater catalytic efficiency for 1-methyl-3,4-dihydroisoquinoline (1-Me DHIQ) compared to other imine reductases reported. Subsequently, an efficient SnIR-catalyzed process was developed by simply optimizing the amount of cosolvent, and up to 15 g L^-1 1-Me DHIQ was converted completely without a feeding strategy. Furthermore, the reaction proceeded well for a panel of dihydroisoquinolines, affording the corresponding tetrahydroisoquinolines (mostly in S-configuration) in good yields (up to 81%) and with moderate to excellent enantioselectivities (up to 99% ee).

Full text of DOI:10.1021/acs.orglett.7b01274

Letter
pubs.acs.org/OrgLett
Identification of an Imine Reductase for Asymmetric Reduction of
Bulky Dihydroisoquinolines
Hao Li,† Ping Tian,‡ Jian-He Xu,*,† and Gao-Wei Zheng*,†
†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
‡Shanghai Institute of Organic Chemistry, Chinese Academy of Science, 345 Lingling Road, Shanghai 200032, P. R. China
S
* Supporting Information
ABSTRACT: A new imine reductase from Stackebrandtia
nassauensis (SnIR) was identified, which displayed over 25- to
1400-fold greater catalytic efficiency for 1-methyl-3,4-dihydro-
isoquinoline (1-Me DHIQ) compared to other imine reductases
reported. Subsequently, an efficient SnIR-catalyzed process was
developed by simply optimizing the amount of cosolvent, and up
to 15 g L−1 1-Me DHIQ was converted completely without a
feeding strategy. Furthermore, the reaction proceeded well for a
panel of dihydroisoquinolines, affording the corresponding tetrahydroisoquinolines (mostly in S-configuration) in good yields
(up to 81%) and with moderate to excellent enantioselectivities (up to 99% ee).
ptically pure tetrahydroisoquinolines (THIQs) as an
important class of chiral scaffolds exist widely in
of dihydroisoquinolines (DHIQs) to chiral THIQs has been
developed rapidly as an attractive approach owing to its
environmental friendliness and 100% theoretical yield.13 An
(S)-IRED from Streptomyces sp. GF354611a and an (R)-IRED
from Streptomyces sp. GF358711e that are capable of the
asymmetric reduction of 1-Me DHIQ were first identified.
Although good to high enantioselectivities (>98% ee for (S)-
IRED and 71% ee for (R)-IRED) were achieved, the two
enzymes displayed relatively low catalytic efficiency (kcat/Km =
0.038 s−1 mM−1 for (S)-IRED and 1.22 s−1 mM−1 for (R)-
IRED). Recently, another (S)-selective IRED named AoIRED
from Amycolatopsis orientalis and its mutant Y179A were
described, which showed the highest catalytic efficiency for 1-
Me DHIQ so far (kcat/Km of 2.03 s−1 mM−1 for mutant
Y179A).12d
However, to date, the reported IREDs rarely possess
sufficient activity for preparative synthesis of chiral THIQs at
high concentrations. Although asymmetric reduction of 10 g/L
of 1-Me DHIQ (69 mM) had been carried out by an (R)-IRED,
the reaction had to be performed using a substrate feeding
strategy (twice) to avoid possible substrate inhibition.11f
Furthermore, to the best of our knowledge, reported IREDs
were mainly employed for asymmetric reduction of model
substrates, such as 1-Me DHIQs 1a and 1g. For more bulky 1-
substituted DHIQs, particularly the pharmaceutically relevant
imines, such as 1-phenyl-3,4-DHIQ, an important scaffold for
the pharmaceutical solifenacin (Figure 1), have not been tested.
Therefore, the exploration of new IREDs that allow efficient
synthesis of valuable chiral THIQs remains highly desirable.
O
numerous alkaloids, possessing exquisite biological activity
and pharmaceutical properties.1 The representative examples
include 1-methyl-1,2,3,4-tetrahydroisoquinoline (1-Me THIQ)
with a parkinsonism-preventing effect,2 salsolidine with an
inhibition effect on monoamine oxidase,3 and solifenacin for
treating overactive bladder syndrome (Figure 1).4 Therefore,
Figure 1. Structures of 1-Me THIQ, salsolidine, and solifenacin.
the highly efficient methods for synthesis of chiral THIQs,
especially asymmetric hydrogenation and asymmetric transfer
hydrogenation, have attracted increasing interest in recent
years.5 Various metal and organic catalysts have been developed
for the asymmetric synthesis of THIQs in good yields.6
However, despite the high catalytic efficiency of chemical
catalysts, their stereoselectivity remains a challenge in these
methods. To date, only relatively few examples with excellent
enantioselectivity for 1-Me THIQ have been reported by the
Xiao group (S-configuration),6a Zhou group (S-configura-
tion),6e and Chan group (R-configuration).6h Recently, artificial
imine reductases7 and a cascade deracemization method
combined with monoamine oxidase8 were also developed for
the synthesis of enantiomerically pure THIQs.
In recent years, with the continuous development of imine
Received: April 27, 2017
reductases (IREDs),9−12 the enzymatic asymmetric reduction
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.7b01274
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Table 1. Screening of IREDs
a
b
entry
IRED
source
accession no.
specific activity (U/mg)
ee (%)
1
2
3
SnIR
SeIR
AdIR
S. nassauensis
S. espanaensis
A. decaplanina
WP_013019548.1
WP_015105194.1
2.9
1.9
1.6
99 (S)
98 (S)
99 (S)
WP_007032738.1
a
b
Specific activity was determined at pH 7.0 and 30 °C using purified enzyme. Enantiomeric excess was determined by chiral HPLC.
As part of our ongoing research on IREDs-catalyzed
To evaluate the synthetic capability of SnIR, preparative
asymmetric reduction of 100 mM 1a was performed in a 10 mL
reaction system. However, only 18% of 1a was converted within
24 h when 5% DMSO was added, which might be attributed to
severe substrate inhibition, thus significantly decreasing the
conversion. Interestingly, 10 mM 1a could be reduced
efficiently into 2a with >99% conversion when 1% DMSO
was utilized. We presumed that lower concentration of DMSO
probably led to the drop of soluble substrate in aqueous
solution, alleviating substrate inhibition of SnIR. Therefore, the
reactions with varied amounts of DMSO were systematically
investigated at a substrate concentration of 20 mM. The results
summarized in Table 3 clearly show that the reaction is
asymmetric reduction of cyclic imines,14 herein we report an
S-preferential IRED (designated as SnIR) from Stackebrandtia
nassauensi, which possesses the highest catalytic efficiency (up
to 54.0 and 79.7 s−1 mM−1 for 1a and 1b) compared to other
IREDs reported so far in the literature. Up to 100 mM (15 g/
L) of 1a was converted completely by SnIR to the
corresponding 1-Me THIQ, with >99% ee under the optimized
reaction conditions. Moreover, for other 1-substituted-DHIQs,
even bulky 1-phenyl-3,4-DHIQ (1f), SnIR was also able to
reduce.
Initially, an IRED library constructed previously in our lab14a
was explored for the asymmetric reduction of the model
substrate 1a. As a result, three (S)-IREDs exhibited good
activities and excellent enantioselectivities (Table 1). Among
them, SnIR from Stackebrandtia nassauensis displayed the
highest specific activity (2.9 U/mg) and enantioselectivity
(>99% ee). Subsequently, to further assess their catalytic
efficiency, the kinetic parameters of these three IREDs toward
1a were determined. As shown in Table 2 (entries 1−3), SnIR
Table 3. Optimization of Reaction Conditions for the
a
Asymmetric Reduction of 1a
b
entry
concn [mM]
DMSO [%]
time [h]
conversion [%]
1
2
3
4
5
6
7
8
100
10
5
1
5
2
1
0
1
1
24
0.5
12
1
18
>99
>99
>99
>99
>99
>99
>99
20
a
20
Table 2. Kinetics Parameters of SnIR and Other IREDs
20
0.5
2
20
50
2
100
4
a
The reaction mixture (10 mL) contains 0.1−1 mmol substrate, 100
mg of SnIR (cell-free extract), 10 mg of BmGDH (cell-free extract),
1.5 mmol of glucose, 5 μmol of NADP+, 0−0.5 mL of DMSO, and
9.5−10 mL of potassium phosphate buffer (100 mM, pH 7.0) shaken
IRED-
Km
Ki
kcat
kcat/Km
entry
substrate
[mM]
[mM]
[s−1
]
[s−1 mM−1
]
b
1
2
3
4
5
6
SnIR-1a
SeIR-1a
AdIR-1a
SnIR-1b
SnIR-1c
SnIR-1d
0.056
0.048
0.113
0.041
0.045
0.068
1.5
8.6
2.0
1.9
3.4
2.7
3.02
1.22
1.26
3.27
1.37
0.21
54.0
25.5
11.1
79.7
30.4
3.07
at 30 °C, 200 rpm. Conversion was determined by HPLC.
dramatically affected by the amount of cosolvent DMSO. The
reduction of DMSO amount could remarkably shorten the
reaction time. When the amount of DMSO was reduced to 1%
from 5%, the time required for a full conversion dropped from
12 to 0.5 h. Nevertheless, when no DMSO was added, the
reaction proceeded at a slightly slow rate, which may be due to
the limitation of mass transfer of substrate. Hence, 1% DMSO
was chosen for reactions with a higher substrate loading and the
reduction of even 100 mM 1a could proceed well in 1%
DMSO, thereby providing a simple and efficient procedure for
preparative synthesis of THIQs.
Finally, to investigate the substrate scope of SnIR, the specific
activities of SnIR toward other DHIQs were also measured
(Table S1). The results showed that the activity decreased
relatively along with the increase of substituent size on the 1-
position of the DHIQ ring. Then asymmetric reductions of a
panel of DHIQs were performed on a preparative scale (1.0
mmol) under the optimized conditions (Table 4). For 1a, 1b,
and 1g, a 100 mM substrate could be converted totally within
a
Kinetic parameters were determined at pH 7.0 and 30 °C using
purified enzyme.
showed the highest catalytic efficiency (kcat/Km = 54.0 s−1
mM−1), which is over 1400-, 40-, and 25-fold greater than (S)-
IRED from Streptomyces sp. GF3546,11a (R)-IRED from
Streptomyces sp. GF3587,11e and AoIRED from Amycolatopsis
orientalis,12d respectively. The kinetic parameters of SnIR
toward other substrates 1b−1d were also investigated. The
highest turnover number (kcat) and catalytic efficiency (kcat/Km)
were observed for 1b, after which the kcat/Km significantly
decreased with the increase of steric hindrance of substrates
(Table 2, entries 4−6). However, different extents of substrate
inhibition (Ki) were observed in all cases.
B
DOI: 10.1021/acs.orglett.7b01274
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Table 4. Asymmetric Reduction of 3,4-DHIQ Derivatives by
SnIR
ASSOCIATED CONTENT
* Supporting Information
■
a
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.7b01274.
Experimental procedures; characterization of SnIR;
characterization data of the products; copies of NMR,
HPLC, GC spectra (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
concn
volume time
conv
c
yield
d
c
*E-mail: jianhexu@ecust.edu.cn.
entry substrate [mM]
[mL]
[h]
[%]
[%]
ee [%]
*E-mail: gaoweizheng@ecust.edu.cn.
ORCID
1
2
3
4
5
6
7
8
1a
1b
1c
1d
1e
1f
100
100
50
10
10
4
2
>99
>99
>99
>99
>99
>99
>99
>99
72
81
77
76
73
81
76
75
>99 (S)
93 (S)
8 (R)
20
3
Hao Li: 0000-0001-5630-7018
Notes
50
20
12
12
12
4
98 (S)
80 (R)
51 (S)
>99 (S)
87 (R)
10
100
200
10
The authors declare no competing financial interest.
5
1g
1h
100
10
ACKNOWLEDGMENTS
b
■
50
12
a
This work was financially supported by the National Natural
Science Foundation of China (Nos. 31200050, 21472045, and
21536004), the Fundamental Research Funds for the Central
Universities (22A201514043) and Shanghai Pujiang Program
(15PJ1401200).
The reaction mixture contained 5−100 mM substrate, 100 mg of
SnIR (cell-free extract), 10 mg of BmGDH (cell-free extract), 50−150
mM glucose, 0.5 mM NADP+, 1% (v/v) DMSO and potassium
b
phosphate buffer (100 mM, pH 7.0) shaken at 30 °C, 200 rpm. No
DMSO was added. Conversion and enantiomeric excess were
determined by chiral HPLC or GC. The absolute configurations of
c
the products were assigned by comparing the sign of the optical
rotation with literature data. Isolated yields.
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DOI: 10.1021/acs.orglett.7b01274
Org. Lett. XXXX, XXX, XXX−XXX