Identification of Novel Bacterial Members of the Imine Reductase Enzyme Family that Perform Reductive Amination

Article abstract of DOI:10.1002/cctc.201701408

Reductive amination of carbonyl compounds constitutes one of the most efficient ways to rapidly construct chiral and achiral amine frameworks. Imine reductase (IRED) biocatalysts represent a versatile family of enzymes for amine synthesis through NADPH-mediated imine reduction. The reductive aminases (RedAms) are a subfamily of IREDs that were recently shown to catalyze imine formation as well as imine reduction. Herein, a diverse library of novel enzymes were expressed and screened as cell-free lysates for their ability to facilitate reductive amination to expand the known suite of biocatalysts for this transformation and to identify more enzymes with potential industrial applications. A range of ketones and amines were examined, and enzymes were identified that were capable of accepting benzylamine, pyrrolidine, ammonia, and aniline. Amine equivalents as low as 2.5 were employed to afford up to >99 % conversion, and for chiral products, up to >98 % ee could be achieved. Preparative-scale reactions were conducted with low amine equivalents (1.5 or 2.0) of methylamine, allylamine, and pyrrolidine, achieving up to >99 % conversion and 76 % yield.

Full text of DOI:10.1002/cctc.201701408

Accepted Article
Title: Identification of Novel Bacterial Members of the Imine Reductase
Enzyme Family that Perform Reductive Amination
Authors: Scott P France, Roger M Howard, Jeremy Steflik, Nicholas J
Weise, Juan Mangas-Sanchez, Sarah L Montgomery, Robert
Crook, Rajesh Kumar, and Nicholas John Turner
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemCatChem 10.1002/cctc.201701408
Link to VoR: http://dx.doi.org/10.1002/cctc.201701408
A Journal of
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10.1002/cctc.201701408
ChemCatChem
COMMUNICATION
Identification of Novel Bacterial Members of the Imine Reductase
Enzyme Family that Perform Reductive Amination
Scott P. France,[a] Roger M. Howard,[b,c] Jeremy Steflik,[b] Nicholas J. Weise,[a] Juan Mangas-
Sanchez,[a] Sarah L. Montgomery,[a] Robert Crook,[c] Rajesh Kumar*[b] and Nicholas J. Turner*[a]
Abstract: Reductive amination of carbonyl compounds constitutes
one of the most efficient ways to rapidly construct chiral and achiral
amine frameworks. Imine reductase (IRED) biocatalysts represent a
versatile family of enzymes for amine synthesis via NADPH
mediated imine reduction. The reductive aminases (RedAms) are a
sub-family of IREDs which have recently been shown to catalyse
imine formation as well as imine reduction. Herein, a diverse library
of novel enzymes were expressed and screened as cell-free lysates
for their ability to facilitate reductive amination, in order to expand
the known suite of biocatalysts for this transformation and to identify
more enzymes with potential industrial applications. A range of
ketones and amines were examined and enzymes were identified
that were capable of accepting benzylamine, pyrrolidine, ammonia
and aniline. Amine equivalents as low as 2.5 were employed to
afford up to >99% conversion and for chiral products, up to >98%
e.e. could be achieved. Preparative-scale reactions were conducted
with low amine equivalents (1.5 or 2.0) of methylamine, allylamine
and pyrrolidine, achieving up to >99% conversion and 76% isolated
yield.
As an alternative to traditional chemical methods, biocatalysis is
becoming an increasingly desirable option for industry due to the
benefits of sustainable catalyst supply, milder reaction
conditions and the enantioselectivity offered by enzymes. One of
the more recent additions to the suite of biocatalysts available
for chiral amine synthesis are imine reductases (IREDs) that
catalyse the asymmetric reduction of prochiral imines using the
cofactor NADPH (Figure 1a).[13–16] It has also been shown that,
in addition to reduction of preformed imines, certain enzymes of
this class can also enable reductive amination between a
carbonyl and amine substrate.[17–22] Furthermore, we have
recently identified enzymes from a sub-group of the IRED
enzyme family that catalyse imine formation as well as imine
reduction. These reductive aminases (RedAms) from fungi are
able to utilise stoichiometric or near-stoichiometric ketone:amine
equivalents to achieve high conversions (Figure 1b).[23] Other
enzyme classes that catalyse reductive amination include amino
acid dehydrogenases (AADHs) and the engineered amine
dehydrogenases (AmDHs)[24,25] as well as N-methyl-amino acid
dehydrogenases (NMAADHs),[26] although, to date, the latter are
limited to ammonia or methylamine as the amine coupling
partner.[27] Opine dehydrogenases (OpDHs) catalyse reductive
amination between an amino acid and keto acid and have been
engineered to broaden their substrate scope and application by
Codexis.[28]
Secondary and tertiary amines are motifs present in many
natural products, active pharmaceutical ingredients (APIs) and
agrochemicals. Unsurprisingly, the synthesis of these
frameworks has been the focus of considerable research efforts.
One of the most powerful ways to construct these architectures
is via reductive amination of a carbonyl compound with an amine
because it can enable the coupling of two smaller fragments
directly with concomitant formation of a stereogenic centre. A
number of advances have recently been made in the field of
catalytic asymmetric reductive amination processes[1–5] although
there are still considerable challenges such as the
chemoselectivity of the catalyst for imine functionality, the
disfavoured equilibrium of imine formation in water and the
enantioselectivity of the reaction.[6] Alternatively, preformation of
the imine followed by asymmetric transition metal-catalysed
hydrogenation is an option,[7–12] although it is a two-step process
and brings with it additional safety considerations around the
use of hydrogen. Furthermore, achiral secondary and tertiary
amine motifs are also present in many bioactive compounds and
their synthesis by reductive amination is often advantageous
over N-alkylation which requires toxic alkylating agents and can
easily lead to over-addition products.
Figure 1: Imine reductase (IRED) and reductive aminase (RedAm)
biocatalysts for amine synthesis. a) IRED-catalysed reduction of preformed
imines. b) RedAm-catalysed imine formation and subsequent reduction.
In light of the recent identification of fungal RedAms, we decided
to investigate new bacterial IREDs as biocatalysts for reductive
amination with particular emphasis on operating at near-
stoichiometric amounts of ketone and amine reagents.
A group of 45 bacterial IREDs was selected using the Basic
Local Alignment Search Tool (BLAST) which included 27 novel
enzymes and 18 enzymes previously disclosed[17,29–35] (see
Supporting Information). The protein sequences of the known
IREDs from Pseudomonas putida (3L6D),[30] Streptomyces sp.
GF3546 (BAM99301)[33,34] and an undisclosed IRED of industrial
interest, all of which have broad substrate specificity, were used
as query sequences. Genes were synthesised, subcloned into
pET28b, expressed in E. coli BL21 (DE3) and applied as cell-
free lysates (see Supporting Information). Preliminary screening
with cell-free lysate containing the reductive aminase from
Aspergillus oryzae (AspRedAm)[23] suggested successful
reductive amination could be achieved by using a crude
biocatalyst preparation (see Supporting Information).
[a]
Mr S. P. France, Dr. N. J. Weise, Dr. J. Mangas-Sanchez, Miss S. L.
Montgomery, Prof. N. J. Turner
School of Chemistry, University of Manchester, Manchester Institute
of Biotechnology, 131 Princess Street, Manchester M1ꢀ7DN, UK.
E-mail: nicholas.turner@manchester.ac.uk
[b]
[c]
Dr. R. M. Howard, Mr. J. Steflik, Dr. R. Kumar
Groton Laboratories, Pfizer Worldwide Research and Development,
445 Eastern Point Road, Groton, Connecticut, 06340, US.
E-mail: Rajesh.Kumar3@pfizer.com
Dr. R. M. Howard, Mr. R. Crook
Sandwich Laboratories, Pfizer Worldwide Research and
Development, Discovery Park, Sandwich, Kent, CT13 9NJ, UK.
This article is protected by copyright. All rights reserved.

10.1002/cctc.201701408
ChemCatChem
COMMUNICATION
Ketone 1 was accepted by the majority of enzymes in the library
with modest to high conversions achieved. Interestingly, in the
conversion of the prochiral ketones 2-4, a large proportion of the
enzyme library afforded (S)-configured products. These
examples constitute valuable additions to the toolbox of
biocatalysts for reductive amination since they are
enantiocomplementary to the previously described AspRedAm.
The N-protected pyrrolidinone substrates 5 and 6 were generally
more challenging substrates. However, interestingly IR48
afforded high conversion with the Cbz-protected analogue 5.
The products 5a and 6a are industrially relevant since the N-
methylpyrrolidin-3-amine framework is found in a number of
biologically active compounds described in the academic and
patent
literature.[36–39]
Substrates
α-tetralone
7
and
acetophenone 8 showed little to no conversion across the entire
library of enzymes screened.
Figure 2: Reductive amination ketone and amine substrate panel for IRED
To explore the substrate scope of reductive amination with
respect to the amine substrate, cyclohexanone 1 was chosen as
the model compound and screened against a panel of amines b-
f. This panel encompassed primary and secondary amines
including ammonia f as well as bulkier amines and those with
chemical handles for further synthesis. With larger and more
expensive amine donors, it is clearly important that this
component is added in a low number of equivalents relative to
the ketone substrate to lower costs and reduce product
contamination, thereby minimising downstream processing.
However, when using ammonia f, for example, the volatility of
screening.
A panel of model ketones 1-8 and amines a-f was selected to
study the reductive amination activity of the novel enzymes. The
IRED library was first screened with the panel of ketones 1-8
and methylamine a. For the purposes of initial testing, 50
equivalents of amine a were used to maximise the chances of
identifying hits. However, with ketone substrates 1 and 2 that
have recently been demonstrated to work well with near
stoichiometric quantities of amine coupling partner,[23] reactions
were conducted at 2.5 amine equivalents. The results of
biotransformations with a selection of the best performing
enzymes are presented in Table 1 (for full screening results see
the Supporting Information).
the amine necessitates
a higher loading but it is also
inexpensive and can be easily removed during work up.
Therefore, for the library screening presented in Table 2, 2.5
equivalents of amines b-e were used while 20 equivalents were
added in the case of ammonia f.
Table 1: Biotransformation data for screening of a subset of the best performing enzymes for reductive amination of a panel of ketones 1-6 with methylamine a.
Apparent conversion to product/%, e.e./% (R or S)
Enzyme
1a
2a
3a
4a
5a
6a
IR44
IR46
IR47
IR48
IR50
IR51
IR56
IR58
IR59
IR61
IR62
IR66
IR67
IR68
IR69
IR70
IR77
IR91
94
95
98
96
98
95
97
98
13
79
31
99
70
19
18
97
98
22
45, >98 (S)
>99, >98 (S)
19, >98 (S)
53, >98 (R)
41, 18 (S)
22, >98 (S)
19, >98 (S)
44, >98 (R)
12
0
0
25
0
14
38
4
0
4
0
0
16
5
0
0
0
0
23
0
6
6
>99, 26 (R)
>99, >98 (S)
>99, >98 (S)
1
>99, >98 (S)
2
56, >98 (S)
99, >98 (S)
1
19, 78 (S)
41, >98 (S)
46, >98 (S)
58, 89 (S)
89, 96 (S)
23
0
0
0
0
27
0
2
0
2
4
43, >98 (S)
7, 53 (R)
7, >98 (S)
32, >98 (S)
51, >98 (S)
1
2
1
66, 7 (R)
50, 51 (S)
10, >98 (S)
3
10, >98 (S)
10, >98 (S)
18, >98 (S)
74, >98 (S)
9, 50 (R)
8, 62 (S)
0
43, >98 (S)
0
2
3
0
5
0
1
0
22
1
43, >98 (S)
>99, >98 (S)
30, 76 (S)
0
85
Reaction conditions: Ketone (1, 2: 10 mM; 3-6: 5 mM), methylamine a (for reactions with 1, 2: 25 mM; for 3-6: 250 mM), glucose (50 mM), GDH CDX-901 (0.3
mg/mL), NADP+ (0.4 mM), enzyme lysate in Tris buffer (100 mM, pH 9.0). Incubated at 25°C, 250 rpm for 24 h. For full screening results, see the Supporting
Information. Conversion determined by GC-FID and e.e. by chiral phase GC-FID or HPLC.
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10.1002/cctc.201701408
ChemCatChem
COMMUNICATION
Table 2: Biotransformation data for screening of
a
subset of the best
loadings for preparative reductive aminations with IREDs
described in the literature. Intensification efforts for the synthesis
of (S)-5a were limited by the solubility of substrate 5.
performing enzymes for reductive amination of 1 with amines b – f.
Apparent conversion to product/%
Enzyme
IR44
IR46
IR47
IR48
IR50
IR51
IR56
IR58
IR59
IR61
IR62
IR66
IR67
IR68
IR69
IR70
IR77
IR91
1b
1c
79
1
66
79
76
4
81
1
61
78
31
2
32
23
43
1
1d
11
0
30
9
20
0
16
0
1
10
0
1
0
5
0
0
23
1
1e
1f
0
0
5
0
0
0
0
10
0
0
0
20
0
0
0
2
0
0
92
11
72
83
80
15
87
74
24
65
35
84
62
10
46
78
84
30
99
15
83
91
99
13
99
78
14
83
2
79
1
7
4
55
97
8
78
63
Reaction conditions: 1 (10 mM), amine (b - e: 25 mM; f: 200 mM), glucose (50
mM), GDH CDX-901 (0.3 mg/mL), NADP+ (0.4 mM), enzyme lysate in Tris
buffer (100 mM, pH 9.0). Incubated at 25°C, 250 rpm for 24 h. For full
screening results, see the Supporting Information. Conversion determined by
GC-FID.
Scheme 1: Preparative-scale reductive amination biotransformations.
Reaction conditions: Enzyme lysate (10% v/v), glucose (1.1 eq.), NADP+ (0.5-
0.6 mg/mL) and GDH (CDX901, 0.5-0.6 mg/mL) in Tris buffer (100 mM), 30oC,
pH 9, 350 rpm for 24 h. Maintained at pH 8.0 with titration with 2N NaOH.
Interestingly, the enzyme library performed well with allylamine b
as well as the more bulky substrate benzylamine c. Furthermore,
several enzymes were identified that gave high conversions with
the secondary amine pyrrolidine e which must react via an
iminium ion intermediate to afford the tertiary amine product 1e.
Interestingly, AspRedAm is unable to catalyse the formation of
1e, further highlighting the utility of the enzymes described
herein. Aniline d was also a substrate for several of the
biocatalysts. Due to the lower nucleophilicity of arylamine
systems compared to simple alkylamines, enzymes that are
capable of utilising d in reductive aminations with ketones are of
particular interest with some recent advances in this area made
independently by Roiban et al.[22] during the course of our
investigations. Ammonia f did not give high conversions, despite
20 equivalents being employed, although IR66 afforded 20%
conversion to the product 1f.
Preparative-scale reactions were performed with 1 with 100 mM
or 125 mM substrate loading and 1.5 or 2 equivalents of amines
a, b and e, achieving high conversions and good isolated yields.
All these reactions showed good initial rates of reaction,
resulting in >60% conversion in the first 6 h before slowing down.
These larger-scale reactions revealed that the use of the
glucose/GDH cofactor recycling system resulted in a drop in
reaction pH, and therefore titration with 2N sodium hydroxide
solution was required to maintain a pH of 8 over the course of
the reaction. To date, these represent the highest substrate
The 45 enzymes reported in this study were then ranked based
on their conversions in reductive amination reactions across the
entire substrate set. For the top performing enzymes, their
active-site residues were compared to AspRedAm (Figure 3).
Interestingly, several of the best performing enzymes cluster
together in the cladogram (see Supporting Information) and
based on sequence alignments, show a conserved active site
aspartate at the position homologous to D169 in AspRedAm.
However, at the homologous positions to AspRedAm’s N96,
Y177 and Q240, there are different residues with serine,
tryptophan and histidine being the most commonly found
respectively. Homology models of IR48 from Rhizobium sullae
and IR91 from Kribbella flavida DSM 17836 were created and
their active sites overlaid with that of AspRedAm (Figure 3a).
IR48 is representative of the best performing enzymes that have
the conserved residues described above and IR91 is an enzyme
that has some significantly different active site residues, yet still
facilitates reductive amination. It should be noted that none of
the highlighted residues is completely conserved across the
three active-site architectures shown. There are also significant
differences in residue functional equivalence, for example
between the homologous Y177, W179 and L180 in AspRedAm,
IR48 and IR91 respectively. This analysis serves to highlight that
reductive amination catalysed by these enzymes may occur,
mechanistically, in different ways compared to that of the
recently discovered fungal RedAms, with different modes of
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10.1002/cctc.201701408
ChemCatChem
COMMUNICATION
binding of substrates. However, further experiments are required
to prove whether these enzymes are capable of catalysing imine
formation, and thus be confidently classified as RedAms.
Acknowledgements
S.P.F. acknowledges a CASE award from the UK Biotechnology
and Biological Sciences Research Council (BBSRC) and Pfizer.
J.M.S was funded by grant BB/M006832/1 from the BBSRC and
S.L.M. was supported by a CASE studentship from Johnson
Matthey. NJT is grateful to the ERC for an Advanced Grant.
AspRedAm
Pfizer IR48
Pfizer IR91
a)
D171
D169
S97
S95
T209
N93
Experimental Section
See supporting information.
Y172
M210
Keywords: biocatalysis • imine reductase • reductive amination
• amines • chirality
W210
W179
NADPH
Y177
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10.1002/cctc.201701408
ChemCatChem
COMMUNICATION
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COMMUNICATION
A diverse group of novel imine
Scott P. France, Roger M. Howard,
reductases (IREDs) were expressed
and screened as cell-free lysates for
their ability to facilitate reductive
amination. A range of ketones and
amines were examined and enzymes
identified that accepted benzylamine,
pyrrolidine, ammonia and aniline.
Amine equivalents as low as 2.5
afforded up to >99% conversion and
up to >98% e.e. Preparative-scale
reactions with low amine equivalents
(1.5 or 2.0) of methylamine, allylamine
and pyrrolidine, achieved up to >99%
conversion and 76% isolated yield.
Jeremy Steflik, Nicholas J. Weise, Juan
Mangas-Sanchez, Sarah L.
Montgomery, Robert Crook, Rajesh
Kumar* and Nicholas J. Turner*
Page No. – Page No.
Identification of Novel Bacterial
Members of the Imine Reductase
Enzyme Family that Perform
Reductive Amination
This article is protected by copyright. All rights reserved.