Imine Reductase-Catalyzed Enantioselective Reduction of Bulky α,β-Unsaturated Imines en Route to a Pharmaceutically Important Morphinan Skeleton

Article abstract of DOI:10.1002/adsc.201801326

The morphinan skeleton is an important sub-structure in many medicines such as dextromethorphan, and can be constructed from 1-benzyl-1,2,3,4,5,6,7,8-octahydroisoquinoline (1-benzyl-OHIQ) derivatives. 1-Benzyl-3,4,5,6,7,8-hexahydroisoquinolines (1-benzyl-HHIQs), the precursors of 1-benzyl-OHIQs, constitute a type of bulky α, β-unsaturated imines. Until now, the application of imine reductases (IREDs) to α, β-unsaturated imines has only rarely been reported. In this study, through evaluation of 48 IREDs, both enantiomers of 1-(4-methoxybenzyl)-1,2,3,4,5,6,7,8-octahydroisoquinoline (1-(4-methoxybenzyl)-OHIQ) were obtained in high yield and excellent optical purity. Among the enzymes, the most steric hindrance-tolerant IRED from Sandarearacinus amylolyticus (IR40) was able to convert various phenyl substituted 1-benzyl-HHIQ to the corresponding 1-benzyl-OHIQ derivatives with excellent enantiometric excess. These results provide an effective route to synthesize these important compounds via enantioselective reduction of bulky α, β-unsaturated imine precursors, which can be readily prepared from 2-(1-cyclohexenyl)ethylamine and corresponding aryl acetic acids. (Figure presented.).

Full text of DOI:10.1002/adsc.201801326

Title: Imine Reductase-Catalyzed Enantioselective Reduction of
Bulky α, β-Unsaturated Imines en Route to a Pharmaceutically
Important Morphinan Skeleton
Authors: Peiyuan Yao, Zefei Xu, Shanshan YU, Qiaqing Wu, and
Dunming Zhu
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To be cited as: Adv. Synth. Catal. 10.1002/adsc.201801326
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10.1002/adsc.201801326
Advanced Synthesis & Catalysis
FULL PAPER
DOI: 10.1002/adsc.201((will be filled in by the editorial staff))
Imine Reductase-Catalyzed Enantioselective Reduction of
Bulky α, β-Unsaturated Imines en Route to a Pharmaceutically
Important Morphinan Skeleton
Peiyuan Yao,a,b,‡ Zefei Xu,a,b,‡ Shanshan Yu,b Qiaqing Wu,a,b,* and Dunming Zhua,b,*
a
University of Chinese Academy of Sciences, 19(A) Yuquan Road, Shijingshan District, Beijing 100049, People’s
Republic of China
b
National Engineering Laboratory for Industrial Enzymes, Tianjin Engineering Research Center of Biocatalytic
Technology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 Xi Qi Dao, Tianjin
Airport Economic Area, Tianjin 300308, People’s Republic of China
Guangdong Provincial Key Laboratory of Fermentation and Enzyme Engineering, South China University of
c
Technology, Guangzhou 510006, People's Republic of China
Fax: (+86) 22-24828703; e-mail: zhu_dm@tib.cas.cn, wu_qq@ tib.cas.cn
These authors contributed equally
‡
Received: ((will be filled in by the editorial staff))
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201######.((Please
delete if not appropriate))
Abstract. The morphinan skeleton is an important sub- hindrance-tolerant
IRED
from
Sandarearacinus
structure in many medicines such as dextromethorphan, and amylolyticus (IR40) was able to convert various phenyl
can be constructed from 1-benzyl-1,2,3,4,5,6,7,8- substituted 1-benzyl-HHIQ to the corresponding 1-benzyl-
octahydroisoquinoline (1-benzyl-OHIQ) derivatives. 1- OHIQ derivatives with excellent enantiometric excess.
Benzyl-3,4,5,6,7,8-hexahydroisoquinolines
(1-benzyl- These results provide an effective route to synthesize these
HHIQs), the precursors of 1-benzyl-OHIQs, constitute a type important compounds via enantioselective reduction of
of bulky α, β-unsaturated imines. Until now, the application bulky α, β-unsaturated imine precursors, which can be
of imine reductases (IREDs) to α, β-unsaturated imines has readily prepared from 2-(1-cyclohexenyl)ethylamine and
only rarely been reported. In this study, through evaluation corresponding aryl acetic acids.
of 48 IREDs, both enantiomers of 1-(4-methoxybenzyl)-
1,2,3,4,5,6,7,8-octahydroisoquinoline (1-(4-methoxybenzyl)-
OHIQ) were obtained in high yield and excellent optical
purity. Among the enzymes, the most steric
Keywords: Imine reductase; α, β-unsaturated imine;
enantioselectivity; chemoselectivity; biocatalysis;
octahydroisoquinoline
access their optically pure forms,[4] but it is
economically and ecologically unattractive due to the
Introduction
The isoquinoline nucleus is an important molecular
scaffold in natural products and pharmacologically
active compounds.[1] Within this class of compounds,
the
1-benzyl-1,2,3,4,5,6,7,8-octahydroisoquinoline
(1-benzyl-OHIQ) derivatives are of particular interest
as starting materials in the synthesis of the morphinan
skeleton. For example, dextromethorphan (1), which
is widely used as a non-opioid antitussive for over 50
years and has anticonvulsant and neuroprotective
properties,[2] could be prepared from (S)-1-(4-
methoxybenzyl)-N-formyl-OHIQ (2) by Grewe
cyclization and reduction of the N-formyl group
(Scheme 1).[3] Therefore, the development of
efficient methods for the synthesis of optically pure
1-benzyl-OHIQ derivatives is highly desired.
Scheme 1. Synthesis of dextromethorphan (1).
maximum yield of 50%. Transition-metal complexes
such as iridium and ruthenium have been reported to
catalyze the asymmetric transfer hydrogenation of the
corresponding (Z)-enamide or iminium salts in high
ee values and yields.[5] However, harsh reaction
conditions such as high pressure and flammable
hydrogen were used. As an alternative to traditional
chemical methods, biocatalysis deserves special
Various methods for the synthesis of these
compounds have been previously reported. Kinetic
resolution of the racemic amines has been used to
1
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10.1002/adsc.201801326
Advanced Synthesis & Catalysis
attention because of its mild reaction conditions,
nontoxicity and high stereoselectivity.[6] But
biocatalytic methods for the synthesis of 1-benzyl-
OHIQ derivatives are limited. Deracemization of 1-
(4-methoxybenzyl)-OHIQ (5h) to (S)-5h in 78%
isolated yield and 99% ee has been achieved in our
laboratory by using the mutant Y321I of a
overexpressed in E. coli BL21 (DE3). The resulting
cell-free extracts were used to examine their activity
and enantioselectivity towards substrate 4h, and the
results are presented in Table 1.
As shown in Table 1, eighteen out of the 48
phylogenetically different IREDs were found to
catalyze the asymmetric reduction of 4h to give 5h.
cyclohexylamine
oxidase
(CHAO)
from
Table 1. Enantioselectivity of IREDs towards 4ha,b)
Brevibacterium oxidans IH-35A.[7] In this
deracemization process, the chemical reducing agent
(ammonia borane) was added in 8-fold excess amount.
Recently, imine reductases (IREDs) have been shown
to catalyze the asymmetric reduction of a wide range
of five-, six-, and seven-membered cyclic imines with
good conversions and high enantioselectivity.[8]
However, the IRED-catalyzed chemo- and
enantioselective reduction of α, β-unsaturated imine,
IRED ee(%) IRED
ee(%)
IRED
ee(%)
IR1
IR2
NDc)
NDc)
ND c)
NDc)
ND c)
NDc)
ND c)
ND c)
NDc)
NDc)
NDc)
NDc)
NDc)
NDc)
NDc)
NDc)
IR17
IR18
IR19
IR20
IR21
IR22
IR23
IR24
IR25
IR26
IR27
IR28
IR29
IR30
IR31
IR32
NDc)
NDc)
73(S)
92(S)
NDc)
91(S)
90(S)
38(S)
NDc)
60(R)
NDc)
NDc)
NDc)
96(S)
35(S)
64(S)
IR33
IR34
IR35
IR36
IR37
IR38
IR39
IR40
IR41
IR42
IR43
IR44
IR45
IR46
IR47
IR48
NDc)
NDc)
IR3
IR4
IR5
IR6
95(R)
97(R)
87(R)
87(R)
76(R)
>99(R)
NDc)
IR7
IR8
IR9
especially
the
bulky
1-benzyl-3,4,5,6,7,8-
hexahydroisoquinoline (1-benzyl-HHIQ) has been
less developed. Turner et al. reported that 2-
isopropenyl-3,4,5,6-tetrahydropyridine could be
IR10
IR11
IR12
IR13
IR14
IR15
IR16
NDc)
NDc)
chemoselectively reduced by
a
IRED from
>99(R)
84(R)
NDc)
Streptomyces sp. GF3587 at the carbon-nitrogen
double bond with no detectable reduction of the
alkene in 96% ee.[8i]
NDc)
Herein, through systematically exploring the
activity of a collection of 48 IREDs, we successfully
identified a group of highly hindrance-tolerant
enzymes that could efficiently convert a series of
phenyl substituted 1-benzyl-HHIQ derivatives into
the corresponding 1-benzyl-OHIQs with high
enantioselectivity and conversion (Scheme 2).
71(R)
a)
The reaction mixture (1.0 mL) contained 10 μmol of
substrate in 20 μL of DMSO, 5 U of GDH, 20 μmol of
glucose, 0.5 mg of NADP+, and 980 μL cell-free extracts
of IREDs (50 mg wet cells weight) in sodium phosphate
buffer (100 mM, pH 7.5), and was shaken at 30ºC, 200
b)
rpm for 18 h. Enantiomeric excess was determined by
chiral HPLC analysis. c) No desired product was detected.
IR19, IR20, IR22-IR24, and IR30-IR32 are S-
selective, while IR26, IR35-IR40, IR44, IR45, and
IR48 are R-selective towards 4h. Among them, IR30
from Sciscionella marina, IR40 from Sandaracinus
amylolyticus and IR44 from Rhizobium sp. LCM
4573 displayed the highest enantioselectivity with
different stereopreferences. IREDs are sometimes
classified into R-selective IRED and S-selective
IRED according to their stereopreference.
A
conservation analysis of all residues in the proposed
substrate binding cleft shows that the amino acid
residues at positions 139 and 194 of S-selective
IREDs are proline and phenylalanine, respectively,
while R-selective IREDs have a hydrophobic residue
(valine, threonine or isoleucine) at position 139 and a
methionine or leucine at position 194.[12] The multiple
sequence complete alignment of IREDs in this study
reveals that the residue at position 139 is a valine for
S-selective IREDs and a threonine for R-selective
IREDs, except for the R-selective IRED IR26 from
Nocardiopsis chromatogenes which has a valine at
position 139. For the residue at position 194, all the
active IREDs towards 4h have a methionine or
leucine (Figure S2 in the Supporting Information). It
is worth noting that the residue at position 225 (W210
for IR18) is conserved and differs in R- and S-
selective IREDs, which has dramatic effects on the
Scheme 2. Asymmetric Reduction of α, β -unsaturated
imines catalyzed by IREDs.
Results and Discussion
The protein sequences of the known pteridine
reductase from Leishmania major (IR1)[9] and
reductive aminase from Aspergillus oryzae (IR18)[10]
were respectively used as a query sequence in Basic
Local Alignment Search Tool (BLAST) searches.
According to the sequence similarity, 48 IREDs were
selected. These included 44 new enzymes and 4
previously disclosed ones (Table S1 and Figure S1
in the Supporting Information).[10-11] Genes encoding
these IREDs were synthesized based on the codon
bias of E. coli, cloned into pET28a or pET21a, and
2
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10.1002/adsc.201801326
Advanced Synthesis & Catalysis
stereoselectivity, as demonstrated in the reductive
amination of several ketones.[10] Therefore, although
some substrate-binding residues exert significant
control on the stereoselectivity of IREDs, the
connection of certain residues with a particular
enantiopreference, and thus classification of these
enzymes as (R) or (S) are not straightforward.[8a]
control of a strong inducible promoter (T7/lac) and
all genes carried the same ribosome binding site
(RBS). Then the recombinant plasmids pRSFduet-
GDH-IR30
and
pRSFduet-GDH-IR40
were
transformed into E. coli BL21 (DE3) for
overexpression. Analysis of the E. coli BL21(DE3)
cells harboring pRSFduet-GDH-IR30 and pRSFduet-
GDH-IR40 by protein gel electrophoresis of cell-free
extracts showed that all three enzymes were
expressed (Figure S6 in the Supporting Information).
The activities of co-expression strains were then
measured. The activities of IR30 and GDH for
pRSFduet-GDH-IR30 were 1.2 U/g wet cells and 740
U/g wet cells, while those of pRSFduet-GDH-IR40
were 1.0 U/g wet cells and 860 U/g wet cells,
respectively.
Under the optimized conditions, preparative
asymmetric reduction of 4h to (R)- or (S)-5h was
carried out by using 50 g/L of wet E. coli BL21(DE3)
cells harboring pRSFduet-IR30-GDH at 10 mM
substrate concentration or pRSFduet-IR40-GDH at 25
mM substrate concentration. 4h was completely
converted within 18h, affording (S)-5h in 74% yield
and 98% ee, and (R)-5h in 80% yield and >99% ee,
respectively. As such, the stereo-complementary 5h
were prepared in good yields and excellent optical
purity by employing the enantio-complementary
IREDs.
After evaluation of the enantioselectivity and
relative activity, IR30 and IR40 with different
stereopreferences were selected for further study. The
enantioselectivity of IR30 towards 4h (96% ee
values) was not high enough, so we tried to optimize
the
reaction
condition
to
improve
its
enantioselectivity. The effect of reaction pH on the
enantioselectivity of 4h (10 mM) to 5h by using the
cell-free extracts of 5.0 wt% wet cells was
investigated at 25ºC (Figure S3 in the Supporting
Information). IR30 displayed activity from pH 5.5 to
pH 9.5. As the pH value increased, the
enantioselectivity became higher and higher. The
optimal pH values was approximately 9.0 using
tris(hydroxymethyl)aminomethane
hydrochloride
(Tris-HCl) buffer (50 mM), and the ee value was
98%. The effect of reaction temperature was also
examined. As shown in Figure S4 in the Supporting
Information, higher stereoselectivity was observed at
lower reaction temperature. As the temperature
increased from 20 to 37ºC, the ee values decreased
from 98% to 96%, and dramatically dropped to 27%
at 45ºC. Therefore, all subsequent biotransformation
for IR30 was performed in Tris-HCl buffer (50 mM,
pH 9.0) at 25ºC.
Table 3. Asymmetric Reduction of 1-benzyl-HHIQs
(4a-h) catalyzed by IR30- and IR40a)
isolated
ee
The N-terminal His6-tagged IR30 and IR40 were
purified by nickel-affinity chromatography (Figure
S5 in Supporting Information). The kinetic
parameters of IR30 and IR40 towards 4h were
obtained by measuring the initial velocities of the
enzymatic reaction at varied substrate concentrations
and curve-fitting according to the Michaelis–Menten
equation. The Vmax of IR30 and IR40 towards 4h
proved to be 0.035 U/mg and 0.087 U/mg,
respectively. The data of the catalytic rate (kcat) and
catalytic efficiency (kcat/Km) are summarized in Table
2. IR40 showed a 2.6-fold and 4.5-fold greater kcat
and kcat/Km, respectively, towards 4h than IR30.
yield
[%]c)
Entry substrate
IRED
[%]b)
1
2
3
4
5
6
7
8
9
4a
4b
4c
4d
4e
4f
4g
4h
4f
IR40-GDH
IR40-GDH
IR40-GDH
IR40-GDH
IR40-GDH
IR40-GDH
IR40-GDH
IR40-GDH
IR30-GDH
IR30-GDH
>99 (R)
99 (R)
98 (R)
99 (R)
99 (R)
99 (R)
97 (R)
>99 (R)
94 (S)
98 (S)
78
90
73
95
85
82
47
80
70
74
10
4h
a) The reaction mixture contained 10 mM substrate, 20 mM
of glucose, 6 mg of NADP+ and 2% (v/v) DMSO in 100
mL Tris HCl buffer (50 mM, pH 9.0) for IR30-GDH, and
25 mM substrate, 50 mM glucose, 12 mg NADP+ and 2%
(v/v) DMSO in 50 mL sodium phosphate buffer (100 mM,
pH 7.5) for IR40-GDH. In all cases, 50 mg/mL wet cells
was added and the reaction mixtures were protected by
Table 2 Kinetics Parameters of IR30 and IR40 towards
4ha)
Vmax
kcat/Km
kcat
[s-1]
Km
[mM]
IRED
[μmol/min/
[s-1
mg]
mM-1]
b)
(3.5±0.1)
×10-2
(1.8±0.1) (33.3±2.4)
×10-2 ×10-2
(4.7±0.2) (19.5±1.5)
×10-2 ×10-2
5.4
nitrogen and shaken at 25ºC, 200 rpm. Enantiomeric
IR30
IR40
×10-2
24.1
×10-2
excess was determined by chiral HPLC analysis. The
absolute configurations of the products were assigned by
comparing the sign of the optical rotation with literature
data. c) Isolated yield.
(8.7±0.2)
×10-2
a)
Kinetic parameters were determined at pH7.5 for IR40
and pH9.0 for IR30 using purified enzymes.
To investigate the substrate scope of whole cell
biocatalysts carrying pRSFduet-GDH-IR30 or
pRSFduet-GDH-IR40, the asymmetric reductions of
4a-4g were also performed on a preparative scale
To further simplify the biotransformation process,
we constructed two co-expression vectors pRSFduet-
GDH-IR30 and pRSFduet-GDH-IR40 under the
3
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10.1002/adsc.201801326
Advanced Synthesis & Catalysis
The HPLC analysis was performed on an Agilent 1200
series HPLC system. 1H and 13C NMR spectra were
recorded on a Bruker Avance III 600 MHz NMR
spectrometer.
under the same conditions (Table 3). For pRSFduet-
GDH-IR40, 25 mM substrates were totally converted
within 18 h, affording the corresponding products in
good yields (73-95%) except 4g (47% isolated yield),
and excellent enantioselectivities (97−99% ee values)
(Table 3). These results suggest that IR40 is a steric
hindrance-tolerant enzyme accepting a wide range of
phenyl-substituted 1-benzyl-HHIQ derivatives as the
substrate. However, for the reaction of 1-(ortho-
substituted-benzyl)-HHIQs (4a – 4d) with pRSFduet-
GDH-IR30 as the biocatalyst, no desired product was
detected. The enantioselectivity and activity of
pRSFduet-GDH-IR30 were also not good towards the
1-(para-substituted-benzyl)-HHIQs 4e and 4g, and
(S)-5f was isolated from the reaction mixture of 4f in
70% yield and 94% ee (Table 3). These results
indicate that the substituent on the phenyl ring of 1-
benzyl-HHIQ derivatives greatly affect the activity
and enantioselectivity of IRED IR30.
Synthesis, Cloning, and Expression of IREDs
The amino acid sequence of IR-1, 18, 19 and 35 are the
same as the previously identified proteins PTR1,[9]
AspRedAm Q240A,[10] 95% identity with IR-09,[11b] IR-
01.[11b] Other IREDs were achieved from the National
Center for Biotechnology Information Database based on
homologous search of IR-1 and IR-18. The genes of these
enzymes including 44 novel entities (Table S1) and 4
known IREDs were all synthesized by GENEWIZ
(Genewiz Biotech Co., Ltd. China) with codons optimized
for expression in E. coli. Synthetic IRED genes (with CAT
and GAATTC in the 5’- and 3’- termini respectively of the
deposited sequences) were further cloned into the
NdeI/EcoRI sites of pET28a. and the resulting plasmids
pET28a-IR1-48 were transformed into E. coli BL21 (DE3)
for overexpression of N-terminal 6×His-tagged fusion
proteins (Table S1). IR1 was cloned into the NdeI/Xhol
sites of pET21a with His-6-tag in N terminal. The
recombinant cells were cultivated in LB medium
containing 50 μg/mL kanamycin at 37ºC with shaking at
180 rpm until OD600 nm reached 0.6‒0.8, and then the
IRED gene was induced by 0.5 mM isopropyl β-D-1-
thiogalactopyranoside (IPTG ) at 25ºC for 12 h. The cells
were harvested by centrifugation (6000 g, 10 min, 4ºC),
washed once by sodium phosphate buffer (100 mM, pH
7.5) and stored at -20C for further use.
Conclusion
In summary, through identification and evaluation of
a large collection of IREDs (44 new and 4 known
IREDs), two IREDs with high stereospecificity and
complementary enantiopreferences towards 1-benzyl-
HHIQ derivatives have been identified. In particular,
IR40 is able to efficiently convert para- and ortho-
substituted 1-benzyl-HHIQs into the corresponding 1-
benzy-OHIQ products with high yields and excellent
enantioselectivity. The possible applications of IR30
and IR40 in the synthesis of the dextromethorphan
intermediate (S)-5h and levallorphan intermediate
(R)-5h was demonstrated at preparative-scale. This
study provides a novel chemo-enzymatic approach to
prepare these important compounds from readily
Screening of IREDs’ Enantioselectivity with Crude
Enzyme
20 μL of 1-(4-methoxybenzyl)-3,4,5,6,7,8-hexahydro
isoquinoline hydrochloride (4h) in DMSO (500 mM, 10
mM of final concentration), 20 mM of glucose, 0.5 mg of
NADP+ and 2 mg of GDH (2.5 U/mg) were added to the
cell-free extracts of IREDs (50 mg wet weight) in 980 μL
of sodium phosphate buffer (100 mM, pH 7.5) in 2 mL
Eppendorf tube. The mixture was shaken at 200 rpm and
30°C on an orbital shaker for 18 h. Then the reaction
mixture was carefully adjusted to pH 10-11 with 5.0 M
NaOH. The aqueous layer was extracted with 1.0 mL of
tert-butyl methyl ether and the phase separation was
facilitated by centrifugation (12000 g, 1 min). The organic
phase was collected, dried over anhydrous sodium sulphate
and analyzed by chiral HPLC.
available
2-(1-cyclohexenyl)ethylamine
and
corresponding aryl acetic acids. Further protein
engineering of selected IREDs is underway in our
laboratory to reveal the substrate-binding and
stereoselective mechanisms, and to obtain highly
active and enantioselective mutant IREDs towards
these important substrates and other non-cyclic α, β-
unsaturated imines.
Determination of Specific activities and Kinetic
Constants
The N-terminal His6-tagged IR30 and IR40 were purified
from the clarified lysate by nickel-affinity chromatography
and analyzed by SDS-PAGE (Figure S5). Specific
activities and kinetic parameters for substrates were
determined using purified enzyme on PolarStar Omega 96
well plate reader by monitoring the decrease of NADPH at
340 nm (ε= 6220 L M−1cm−1) at 28ºC. The reaction volume
(200 µL) contained buffer and 150-200 μg pure enzyme,
0.5 mg/mL NADPH, 15%(v/v) dimethylsulfoxide and
imine substrate. Tris-HCl buffer (100 mM pH8.5）and
sodium phosphate buffer (100 mM, pH 7.5) were used for
IR-30 and IR-40, respectively. The reaction was performed
by adding the enzyme to the mixture. One unit of imine
reductase is defined as the amount of protein that oxidizes
1 μmol NADPH per minute. Kinetic parameters were
deduced by non-linear regression analysis based on
Michaelis–Menten kinetics using the program Origin 9.0.
All activities were measured in triplicates and error bars
indicate the standard deviation.
Experimental Section
Materials
N-(2-cyclohexenylethyl)-2-arylacetamide
(6a-h)
was
and
prepared from 2-(1-cyclohexenyl)ethylamine
corresponding aryl acetic acids by following the similar
experimental
procedure
of
literature.[13]
2-(1-
cyclohexenyl)ethylamine was purchased from Heowns
Biochem LLC. DifcoTM LB Broth, Miller (Luria-Bertani)
was purchased from Becton Dickinson and Company.
Ampicilin was purchased from Beijing Probe Bioscience
Co., Ltd. Isopropyl β-D-1-thiogalactopyranoside (IPTG)
was purchased from AMRESCO Inc. Other commercial
chemicals were purchased from Aladdin Industrial
Corporation, Sinopharm Chemical Reagent Co., Ltd and
Shanghai Macklin Biochemical Co., Ltd. The restriction
enzymes and other reagents for molecular biology were
supplied by Fermentas (Germany) and TaKaRa (Japan).
Asymmetric
Reduction
of
1-benzyl-3,4,5,6,7,8-
hexahydroisoquinoline derivatives Using Recombinant
Cells of IR30-GDH and IR40-GDH
4
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10.1002/adsc.201801326
Advanced Synthesis & Catalysis
General procedure for the asymmetric reduction of 1-
benzyl-3,4,5,6,7,8-hexahydroisoquinoline derivatives was
carried out as follows: the wet cell pellets of IR30-GDH
(5.0 g) or IR40-GDH (2.5 g) was resuspended in 100 mL
of 2% (v/v) DMSO and Tris HCl buffer (50 mM, pH 9.0)
or 50 mL 2% (v/v) DMSO and sodium phosphate buffer
(100 mM, pH 7.5). 10 or 25 mM substrate (due to the
instability in air, the HCl salts of the substrates were used),
20 mM or 50 mM of glucose, 12 mg or 6 mg of NADP+
were added to the reaction mixture. The mixture was
protected by nitrogen and shaken at 200 rpm and 25°C on
an orbital shaker for 18 h. Then the reaction mixture was
carefully adjusted to pH 10-11 with 5.0 M NaOH. The
aqueous layer was extracted three times with 100 mL of
ethyl acetate and the phase separation was facilitated by
centrifugation (6000 g, 15 min). The combined organic
layer was dried over anhydrous sodium sulfate and filtered.
Removal of the solvent and purification by column
chromatography over silica-gel with appropriate mixture of
dichloromethane and methanol gave the product. The
absolute configurations for the products were determined
by comparison of its optical rotation with the literatures.
[6] a) G. Torrelo, U. Hanefeld, F. Hollmann, Catal. Lett.
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Acknowledgements
This work was financially supported by Youth Innovation
Promotion Association of the Chinese Academy of Sciences
(Grant No. 2016166), and Tianjin Municipal Science and
Technology
Commission
(15PTGCCX00060
and
15PTCYSY00020). We thank Professor Peter C. K. Lau for his
constructive suggestions on the manuscript.
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5
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10.1002/adsc.201801326
Advanced Synthesis & Catalysis
FULL PAPER
Imine Reductase-Catalyzed Enantioselective
Reduction of Bulky α, β-Unsaturated Imines en
Route to
a
Pharmaceutically Important
Morphinan Skeleton
Adv. Synth. Catal. Year, Volume, Page – Page
Peiyuan Yao,a,b,‡ Zefei Xu,a,b,‡ Shanshan Yu,b
Qiaqing Wu,a,b,* and Dunming Zhua,b,*
6
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