Asymmetric reduction of cyclic imines catalyzed by a whole-cell biocatalyst containing an (S)-imine reductase

Article abstract of DOI:10.1002/cctc.201300539

Biocatalytic imine reduction: A whole-cell recombinant E. coli system, producing an (S)-selective imine reductase (IRED) from Streptomyces sp. GF3546, is developed. This biocatalyst is used for the enantioselective reduction of a broad range of substrates such as dihydroisoquinolines and dihydro-β- carbolines as well as iminium ions. Copyright

Full text of DOI:10.1002/cctc.201300539

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DOI: 10.1002/cctc.201300539
Asymmetric Reduction of Cyclic Imines Catalyzed by
a Whole-Cell Biocatalyst Containing an (S)-Imine
Reductase
[
a]
Friedemann Leipold, Shahed Hussain, Diego Ghislieri, and Nicholas J. Turner*
In view of the importance of chiral amines as building blocks
for biologically active pharmaceutical drugs, considerable
effort has been devoted to the development of asymmetric
catalytic methods for their preparation. Notable advances have
[99% enantiomeric excess (ee)], although it displayed no activi-
[10]
ty towards related imine substrates,
whereas the (S)-IRED
from Streptomyces sp. GF3546 showed similarly good activity
towards 1a with a much lower level of activity towards six-
[
1a]
[11]
been made by using transition-metal catalysis, organocataly-
membered ring imines. Recently, the structure of the (R)-
[
1b]
[1c]
[12]
sis, and artificial transfer hydrogenases. However, biocata-
IRED from Streptomyces kanamyceticus was reported. Other
[2]
1
lytic approaches have emerged as being especially important,
IREDs have also been described; for example, D -piperideine-2-
[
3]
1
including those based upon transaminases, monoamine oxi-
carboxylate/D -pyrroline-2-carboxylate reductases have been
[
4]
[13]
dases, and recently engineered NADH-dependent l-amino
acid dehydrogenases (NADH is the reduced form of NAD =
reported in several Pseudomonas strains.
+
To assess the potential for using IREDs as biocatalysts in
preparative synthesis, we report the cloning, overexpression,
and kinetic characterization of the (S)-selective imine reduc-
tase, (S)-IRED, from Streptomyces sp. GF3546. The (S)-IRED gene
has also been overexpressed in E. coli to yield a whole-cell bio-
catalyst that is able to utilize glucose for cofactor recycling.
Substrate specificity studies revealed that the (S)-IRED is able
to reduce a much broader range of substrates than previously
recognized, including five-, six- and seven-membered ring con-
taining imines. In most cases, these reductions proceeded with
both high activity and enantioselectivity to yield a broad range
of structurally different amines of (S) configuration.
[
5]
nicotinamide adenine dinucleotide). These methods are com-
plementary in terms of the required substrates (amine, ketone)
and also the amine that they generate (primary, secondary, or
tertiary). In some cases, these biocatalytic processes have been
successfully demonstrated at an industrial scale for the manu-
[
6]
facture of recently launched drugs. However, there are no
general biocatalytic strategies available that are based upon
asymmetric reduction of imines as a route to enantiomerically
pure amines.
Imine reductases (IREDs) catalyze the reduction of imines to
amines by utilizing NADH or NADPH as a cofactor (Scheme 1,
+
NADPH is the reduced form of NADP =nicotinamide adenine
The gene sequence of the (S)-IRED was initially codon-opti-
mized for expression in E. coli (by using the OptimumGene al-
[
7]
dinucleotide phosphate).
gorithm) with the addition of an N-terminal His tag to enable
6
purification by metal ion affinity chromatography. Expression
in E. coli BL21 (DE3) by using a pET-28a (+)-based vector con-
struct containing the (S)-IRED gene yielded an active recombi-
nant enzyme suitable for initial activity studies. Biotransforma-
tions were performed by using resting E. coli cells, and glucose
was added to regenerate the cofactor. Conversion of 10 mm
Scheme 1. Asymmetric reduction of imines by using an imine reductase.
2-methyl-1-pyrroline (1a) into 2-methylpyrrolidine (2a) was
IREDs from different Streptomyces strains have been shown
to reduce 2-methyl-1-pyrroline (1a) to corresponding pyrroli-
achieved (57%) after 6 h. Subsequently, the (S)-IRED was puri-
2
+
fied to homogeneity by Ni -affinity chromatography by using
a HisTrap column (Table S1 and Figure S5, Supporting Informa-
tion) to yield a pure protein with a molecular weight of 32 kDa
[
8]
dine 2a, and the sequences of two enantiocomplementary
imine reductases from Streptomyces sp. GF3587 and Streptomy-
[
9]
À1
ces sp. GF3546 have been previously reported. The (R)-IRED
from Streptomyces sp. GF3587 was able to convert 1a into 2a
and a specific activity of 0.03 Umg .
To gain insight into the effect of ring size and also the range
of cyclic imines able to undergo reduction, the kinetic con-
stants of the (S)-IRED were determined towards a small panel
of 2-substituted-1-pyrrolines 1a–d together with imines 3, 5, 7,
and 11 as well as iminium ion 9 (Table 1; Figures S6–S13, Sup-
porting Information).
[a] Dr. F. Leipold, S. Hussain, D. Ghislieri, Prof. N. J. Turner
School of Chemistry
University of Manchester
Manchester Institute of Biotechnology
1
31 Princess Street
For previously reported substrate 1a, the turnover number
Manchester, M1 7DN (UK)
Fax: (+44)161-445-0093
À1
(
kcat) was 0.024 s with a Michaelis constant K =11.82 mm. In-
m
terestingly, the (S)-IRED displayed higher catalytic activity and
significantly lower Km values towards six-membered cyclic
imines 3, 7, 9, and 11. For imine 7 and iminium ion 9, the cata-
E-mail: Nicholas.Turner@manchester.ac.uk
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cctc.201300539.
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Table 1. Kinetic constants determined for (S)-imine reductase substrates.
Table 2. Whole-cell biocatalysis with various imine reductase substrates.
Conversions after 1 and 6 h as well as enantioselectivities after 6 h are
given.
Substrate
K
m
V
max
k
[s
cat
À1
k
cat/K
m
À1
À1
À1
[
mm]
[Umg
]
]
[s mm
]
Substrate
% Conversion
% ee
Product
1
1
1
3
5
7
9
a
b
d
11.82
0.54
0.99
5.50
8.19
0.63
0.60
1.05
0.044
0.006
0.009
0.242
0.069
0.807
0.876
0.072
0.024
0.004
0.005
0.137
0.039
0.445
0.483
0.040
0.002
0.007
0.005
0.025
0.005
0.708
0.801
0.038
1 h
6 h
6 h
[
[
a]
a]
7
9
>98
>90
>98
48
>98
69
21
29
29
>98
>90
>98
92
>98
>98
69
n/a
n/a
98
>98
>98
>98
98
>98
>98
8
10
1
1
(S)-12
(S)-14
(S)-16a
(S)-16b
(S)-16c
(S)-16d
(S)-18
1
1
1
1
1
1
3
5a
5b
5c
5d
7
11
97
50
lytic efficiency (k /K ) of the (S)-IRED was >100-fold higher
cat
m
[
a] n/a=not applicable.
than that for 2-methyl-1-pyrroline (1a). Indeed, the activity to-
[11]
wards 11 was much higher than previously reported. Our
data suggest that six-membered, rather than five- or seven-
membered, cyclic imines may represent natural substrates for
the (S)-IRED. Interestingly, bulky and rigid aromatic groups in
the 2-position of the pyrroline significantly decreased the ve-
locity of the reaction, whereas the enzyme exhibited increased
affinity (Km).
The dihydro-b-carbolines used in this study were all synthe-
[14]
sized according to previously reported methods. In contrast
[11]
to a recent report, we were able to obtain high levels of con-
version by using this whole-cell system. In the absence of glu-
cose, conversions were found to be lower, although the reac-
tions eventually went to completion. All six- and seven-mem-
bered ring substrates tested showed good to excellent conver-
sions after 6 h (ranging from 50 to 99%) with excellent levels
of enantioselectivity (>98%ee). Remarkably, for the series of
dihydro-b-carbolines, substitutions with bulky aliphatic chains
and alicyclic rings on both the imine (i.e., 15a–d) and the
indole ring (i.e., 17) were tolerated with relatively little change
in activity. In all cases, the products obtained (i.e., 12, 14,
16a–d, and 18) were shown to possess the (S) configuration
by comparison with known standards. Tetrahydroisoquinolines
and tetrahydro-b-carbolines with the (S) configuration are im-
portant building blocks for the synthesis of biologically active
To address the challenge of developing a preparative biocat-
alyst for asymmetric imine reduction, we examined the use of
a whole-cell system by using resting E. coli cells expressing the
(
S)-IRED, together with the addition of glucose, to enable
in situ cofactor recycling. These reactions were performed at
a substrate concentration of 5 mm and were monitored by
HPLC on a chiral stationary phase to determine both conver-
sion and enantioselectivity of the reduction. Control reactions,
in which the E. coli cells contained an empty pET-28a (+) plas-
mid instead of the (S)-IRED-containing vector, showed no con-
version with any of the substrates. In light of the high in vitro
activity observed for the reduction of six-membered ring
imines (Table 1), we focused on the activity and enantioselec-
tivity of the (S)-IRED towards substituted dihydroisoquinolines
[15,16]
amines.
(S)-1-Methyltetrahydroisoquinoline (12) has previ-
ously been prepared biocatalytically by lipase-catalyzed kinetic
[17]
resolution, and (R)-12 has been synthesized by monoamine
[18]
7
, 9, 11, and 13 together with a series of 1-substituted dihy-
oxidase mediated deracemization.
offers an alternative third approach.
The current method
dro-b-carbolines 15a–d and 17 (Table 2).
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To further optimize the biotransformation of 11, reactions
were performed at a higher substrate loading. At a substrate
concentration of 20 mm, the reactions proceeded to comple-
tion, although higher concentrations up to 100 mm led to in-
complete conversion. A preparative-scale biotransformation
tive scale, and this points the way towards future applications
of these IREDs. Despite the unexpected broad specificity of the
wild-type enzyme, there is undoubtedly further scope for engi-
neering these enzymes to enhance their properties for future
applications in the synthesis of chiral amines.
(200 mg) was successfully performed at 20 mm by using the
whole-cell biocatalyst. Conversion was observed to proceed to
Experimental Section
>
98% for amine (S)-12, which was isolated in 87% yield.
We also explored the effect of substitutions at the 2-position
The (S)-IRED biocatalyst was produced by using E. coli BL21 (DE3)
bearing a pET-28a (+) plasmid with an inserted codon-optimized
of pyrrolines (i.e., 1a–d) and also the effect of ring size on the
level of conversion and ee (i.e., 3 and 5; Table 3). For the series
(
S)-IRED gene cloned by using NdeI and XhoI restriction sites. Culti-
vation was performed in lysogeny broth (LB) medium (500 mL) at
78C and 250 rpm to OD600 nm =0.6–0.8 (OD=optical density) and
3
recombinant protein expression was induced by using 0.2 mm iso-
propyl b-d-1-thiogalactopyranoside (IPTG). Cells were harvested
after a further cultivation at 208C and 250 rpm for 18–22 h. Cell
disruption was performed by ultrasonication of a suspension of
E. coli BL21 (DE3) expressing the (S)-IRED in sodium phosphate
buffer (100 mm, pH 7.0).
Table 3. Whole-cell biocatalysis with cyclic imine reductase substrates of
different ring sizes. Conversions after 6 and 18 h as well as enantioselec-
tivities after 18 h are given.
Substrate
% Conversion
18 h
% ee
Product
6
h
18 h
2
+
1
1
1
1
3
5
a
b
c
57
55
36
17
42
>98
>98
>95
87
>98
98
>98
>98
(S)-2a
The (S)-IRED was purified by Ni -chelating affinity chromatogra-
phy by using a HisTrap column whereby elution was performed by
using sodium phosphate buffer (100 mm, pH 7.0) containing
[
a]
28
14
25
>98
>98
(R)-2b
[
a]
(R)-2c
[
a]
d
(R)-2d
(S)-4
(S)-6
3
00 mm NaCl and 300 mm imidazole.
Kinetic constants were determined from a liquid-phase spectro-
photometric assay by monitoring the decrease in NADPH at 340
e=6.22 mm cm ) or 370 nm (e=2.216 mm cm ). Reaction
[a] The formation of the (R)-amine is due to a change in the Cahn–
À1
À1
À1
À1
(
Ingold–Prelog assignment.
mixtures contained sodium phosphate buffer (100 mm, pH 7.0),
NADPH to an absorbance of 0.8–1 at the respective wavelength,
1
% (v/v) dimethyl sulfoxide, and the substrate at the desired con-
of pyrrolines, conversions were found to vary with the sub-
stituents (17–55%), although high ee values were generally ob-
tained (87–98%). Comparison of 1a, 3, and 5 revealed that the
centration. The reaction was started by adding the purified
enzyme to the mixture. One unit of the (S)-IRED is defined as the
amount of protein that oxidizes 1 mmol NADPH per minute.
(
S)-IRED is highly tolerant of ring size and indeed favors six-
Biotransformations were performed at 308C in sodium phosphate
buffer (100 mm, pH 7.0) by using resting cells of E. coli BL21 (DE3)
expressing the (S)-IRED at a final OD600 nm of 30. The reaction mix-
tures typically contained 5 mm of the substrate imine, 50 mm of
glucose, and 2% (v/v) dimethylformamide unless otherwise stated.
Samples were typically taken after 0, 1, 3, 6, 18, and 30 h, extracted
by using dichloromethane, and used directly for analysis. Negative-
control biotransformations were performed in the same way by
using cells harboring empty pET-28a (+) vector cultivated and in-
duced like the cells expressing the biocatalyst. A preparative-scale
biotransformation for the enantioselective reduction of 11 was per-
formed on a 200 mg scale at a substrate concentration of 20 mm
with 100 mM of glucose and cells added at a final OD600nm of 30
for 24 h. Conversion was >98% and (S)-12 was isolated in 87%
yield.
and seven-membered rings.
The data shown in Tables 2 and 3 may provide insight into
the exact role of imine reductases in the synthesis of cyclic sec-
ondary and tertiary amines. The biosynthesis of the natural
product eleagnine (16a) in pea seedlings from l-tryptophan
has been shown to proceed via imine 15a, which was itself
generated in situ by internal cyclization of an amine onto an
[
19]
aldehyde. Conversion of 15a into 16a was suggested to be
due to a putative imine reductase. In separate studies, it was
shown that the reduction of a series of dihydro-b-carbolines
could be effected by using the biocatalytic capabilities of dif-
[20a]
ferent eukaryotic organisms such as Saccharomyces bayanus
[
20b]
and Eisenia foetida.
The use of S. bayanus appears to be the
only report of imine reductase activity in a yeast, which are
well documented to exhibit high ketoreductase and ene
reductase activities.
Acknowledgements
In conclusion we have shown that the (S)-imine reductase
from Streptomyces sp. GF3546 is able to catalyze the highly
enantioselective reduction of five-, six-, and seven-membered
imines, including substituted dihydro-b-carbolines, which high-
lights the preference for six-membered imines as well as the
broad substrate scope of this enzyme. In addition, we demon-
strated that this (S)-IRED also catalyzes the reduction of imini-
um ions. Reactions performed with the use of E. coli whole-cell
biocatalyst supplemented with glucose gave excellent conver-
sions (>95%) and yields of the isolated products on a prepara-
The research leading to these results received funding from the
European Union’s Seventh Framework Programme FP7/2007–
2013 under grant agreement no. 266025 (BIONEXGEN, to F.L.). We
are also grateful for funding from the Marie Curie Initial Training
Network (Biotrains FP7-ITN-238531, to D.G.). We also acknowl-
edge support from EPSRC and AstraZeneca (to S.H.) and the
Royal Society for a Wolfson Research Merit Award (NJT).
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Keywords: amines · asymmetric synthesis · biocatalysis · imine
reductase · Streptomyces
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Received: July 5, 2013
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Biocatalytic imine reduction: A whole-
cell recombinant E. coli system, produc-
ing an (S)-selective imine reductase
F. Leipold, S. Hussain, D. Ghislieri,
N. J. Turner*
&& – &&
(IRED) from Streptomyces sp. GF3546, is
developed. This biocatalyst is used for
the enantioselective reduction of
a broad range of substrates such as di-
hydroisoquinolines and dihydro-b-car-
bolines as well as iminium ions.
Asymmetric Reduction of Cyclic
Imines Catalyzed by a Whole-Cell
Biocatalyst Containing an (S)-Imine
Reductase
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These are not the final page numbers!