Imine Reductase-Catalyzed Intermolecular Reductive Amination of Aldehydes and Ketones

Article abstract of DOI:10.1002/cctc.201500764

Imine reductases (IREDs) have emerged as promising biocatalysts for the synthesis of chiral amines. In this study, the asymmetric imine reductase-catalyzed intermolecular reductive amination with NADPH as the hydrogen source was investigated. A highly chemo- and stereoselective imine reductase was applied for the reductive amination by using a panel of carbonyls with different amine nucleophiles. Primary and secondary amine products were generated in moderate to high yields with high enantiomeric excess values. The formation of the imine intermediate was studied between carbonyl substrates and methylamine in aqueous solution in the pH range of 4.0 to 9.0 by ¹H NMR spectroscopy. We further measured the kinetics of the reductive amination of benzaldehyde with methylamine. This imine reductase-catalyzed approach constitutes a powerful and direct method for the synthesis of valuable amines under mild reaction conditions. IRED all about it: The intermolecular asymmetric reductive amination of carbonyls catalyzed by a stereoselective imine reductase produces chiral amines in high yields with high enantioselectivities. The reaction efficiency is attributed to its remarkable tolerance to high concentrations of amine nucleophiles, high pH values, high chemoselectivity towards imines, and high stereoselectivity of the biocatalyst.

Full text of DOI:10.1002/cctc.201500764

DOI: 10.1002/cctc.201500764
Communications
Imine Reductase-Catalyzed Intermolecular Reductive
Amination of Aldehydes and Ketones
[
a]
Philipp N. Scheller, Maike Lenz, Stephan C. Hammer, Bernhard Hauer, and Bettina M. Nestl*
Imine reductases (IREDs) have emerged as promising biocata-
lysts for the synthesis of chiral amines. In this study, the asym-
metric imine reductase-catalyzed intermolecular reductive ami-
nation with NADPH as the hydrogen source was investigated.
A highly chemo- and stereoselective imine reductase was ap-
plied for the reductive amination by using a panel of carbonyls
with different amine nucleophiles. Primary and secondary
amine products were generated in moderate to high yields
with high enantiomeric excess values. The formation of the
imine intermediate was studied between carbonyl substrates
and methylamine in aqueous solution in the pH range of 4.0
vents that favored reversible imine formation by shifting the
equilibrium towards the condensation product.
The application of biocatalysis is an important complement
to chemical catalysis for chiral amine synthesis in water. To
date, two main types of enzymes have been investigated for
the transformation of carbonyl groups into amines: Amino
acid dehydrogenases and w-transaminases. Amino acid dehy-
drogenases catalyze the amination of carbonyl compounds,
usually a-keto acids and a-keto esters with NADH as the hy-
[
8]
drogen source. Using existing amino acid dehydrogenase
scaffolds, the group of Bommarius successfully altered the sub-
strate specificity through several rounds of protein engineering
1
to 9.0 by H NMR spectroscopy. We further measured the kinet-
[
9,10]
ics of the reductive amination of benzaldehyde with methyl-
amine. This imine reductase-catalyzed approach constitutes
a powerful and direct method for the synthesis of valuable
amines under mild reaction conditions.
to create amine dehydrogenases.
However, the application
of amine dehydrogenases still suffers from a poor substrate
scope. In contrast, w-transaminases catalyze the transfer of
amino groups from co-substrates to carbonyl compounds to
form new chiral amines. These enzymes have proven to be
powerful catalysts and are frequently used in the synthesis of
[
11–13]
Chiral amines are an important class of organic compounds
that serve as key intermediates in the synthesis of a variety of
a broad range of chiral amines.
Challenges associated with
w-transaminases are the disfavored reaction equilibrium for re-
ductive aminations of ketones and inhibition by the formed
byproduct. Both w-transaminases and amine dehydrogenases
are inherently restricted to the transfer of ammonia as amine
[1]
biologically active molecules. The most straightforward strat-
egy for the generation of chiral amines comprises the forma-
tion of C=N bonds by the condensation of carbonyls and
amines, followed by reduction of the in situ formed imine. The
chemoselectivity of the reducing agent, however, is crucial, as
it should efficiently reduce the C=N bond while leaving the po-
tentially reducible carbonyl compound unaffected. Tremen-
dous efforts have been made in the areas of organometallic
catalysis, organocatalysis, and biocatalysis to develop efficient
chemo-, regio-, and enantioselective strategies for this pro-
[
14]
substrate and thus, do not offer access to pharmaceutically
important chiral secondary and tertiary amine building blocks.
In this light, imine reductases represent a promising alternative
and extension to reported biocatalysts.
Previously, imine reductases were investigated for the asym-
metric reduction of C=N bonds with NADPH as the hydride
[
15–24]
donor.
To date, both (R)- and (S)-selective reductases have
[
2,3]
cess.
In the last several decades, most work has focused on
been screened for the transformation of five-, six-, and seven-
membered cyclic imines, dihydroisoquinolines, b-carbolines,
and iminium ions. They were shown to possess high catalytic
activities and stereoselectivities. This makes them promising
biocatalysts for chiral amine synthesis. However, whereas intra-
molecular reductions with the use of imine reductases have
been reported, only a few intermolecular ones have been de-
veloped. Evidently, intermolecular processes would represent
an important advancement in this methodology. Recent work
showed that the (S)-selective imine reductase from Streptomy-
ces sp. GF3546 catalyzed the conversion of 4-phenyl-2-buta-
none with methylamine into the corresponding product with
the transition-metal-catalyzed asymmetric hydrogenation of
imines with particular attention paid to the reductive amina-
tion of carbonyl compounds with primary amines. Several re-
agents and combinations have been reported through the
years with modified borohydride reagents playing a predomi-
[
4]
nant role. Inspired by biological systems in nature that use
NAD(P)H, the Hantzsch ester was employed as a hydrogen
donor in combination with chiral phosphoric acids for the
enantioselective reductive amination of aliphatic ketones to
[5–7]
furnish amines with excellent enantioselectivities.
In most
cases, these reactions were performed in different organic sol-
[
24]
8
.8% conversion and 76%ee.
Furthermore, Codexis/Merck
[
a] P. N. Scheller, M. Lenz, Dr. S. C. Hammer, Prof. Dr. B. Hauer, Dr. B. M. Nestl
Institute of Technical Biochemistry
Universität Stuttgart
Allmandring 31, 70569 Stuttgart (Germany)
E-mail: bettina.nestl@itb.uni-stuttgart.de
have developed and patented an engineered opine dehydro-
genase from Arthrobacter sp. possessing imine reductase activi-
ty for the conversion of various ketone and amine substrates
into secondary and tertiary amines under industrially applica-
[
25]
ble conditions. Despite these preliminary works, biocatalytic
reductive aminations to afford chiral amines in high yields with
Supporting Information for this article is available on the WWW under
http://dx.doi.org/10.1002/cctc.201500764.
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Communications
Table 1. Conversion of benzaldehyde (1) into amination products 3a–d
[a]
with amine nucleophiles 2a–c by using purified imine reductase.
Scheme 1. Imine reductase-catalyzed asymmetric reductive amination.
high enantiomeric excesses in one step are scarcely reported.
Recently, we described that the (R)-selective imine reductase
from Streptosporangium roseum (R)-IRED-Sr is a promising
novel biocatalyst for the asymmetric transfer hydrogenation of
cyclic imines to produce amines with excellent enantioselectiv-
Carbonyl
acceptor
Amine
nucleophile
Nucleophile
[equiv.]
Formed product
[%]
8 h
1 h
24 h
[b]
[b]
[b]
[b]
[b]
[b]
[b]
[b]
1
1
1
1
1
1
1
2a
2a
2a
2b
2b
2b
2c
1
<1
<1
<1
6
33
58
4
1/<1
7/2
51/3
61/1
51
66
69
[
[
b]
b]
[b]
10
50
1
21/<1
[b]
[15]
48/<1
ities. As a result of continuing efforts towards the develop-
ment of selective imine reductase-catalyzed reactions, we now
report the reductive amination of a set of carbonyl substrates
with various amines by using this enzyme (Scheme 1).
35
72
73
13
10
50
1
25
To approach the challenge of the chemo- and enantioselec-
tive formation of new carbon–nitrogen bonds, a substrate
panel comprising aromatic and cyclic carbonyls and aliphatic
and aromatic amines was chosen. As carbonyl compounds,
benzaldehyde (1), acetophenone (4), and cyclohexyl methyl
ketone (6) were selected. The set of amine nucleophiles con-
sisted of ammonia (2a), methylamine (2b), and aniline (2c).
Although general features of imine-forming reactions are
well understood, the available data from the scientific litera-
[
a] Reaction conditions: carbonyl substrate 1 (10 mm), purified IRED
À1
(2.5 mgmL ) and cofactor regeneration system in Tris HCl buffer (50 mm,
pH 8.0). To compare conversion rates and to evaluate formation of by-
products, all reactions were performed with the same batch of purified
biocatalyst. Reduction of 1 to benzyl alcohol depended on the nature
and the excess amount of the amine nucleophile. Further details are
given in the Supporting Information. [b] Equivalents of nucleophile 2a
used in the reaction setup with product 3a itself acting as an additional
nucleophile. Higher levels of product 3a were obtained; however, 3a re-
acted further with benzaldehyde (1) to give the dialkylation product di-
benzylamine (3d).
[26]
ture on imine formation in water is rather limited. The posi-
tion of the equilibrium for imines formed from carbonyl com-
pounds and amines by elimination of water is expected to be
[27]
highly disfavored in aqueous buffer systems. Hence, we first
studied the reduction of the commercially available imine N-
benzylidenemethylamine (corresponding imine species of 3b
shown in Table 1). In the initial screening, enzyme activity was
detected, but the imine also rapidly decomposed at pH values
below 8. To ensure activity of the biocatalyst at elevated pH
levels, the pH profile of the enzyme was recorded by using the
cyclic imine substrate 2-methylpyrroline. The activity of the
IRED was maximum at pH 7.0 (Figure S7, Supporting Informa-
tion); however, at pH 8.0 the conversion of N-benzylideneme-
thylamine to the amine was approximately 75% (Table S4). The
hydrolytic product benzaldehyde was also reduced to the cor-
responding benzyl alcohol byproduct (<6%) in these biotrans-
formations.
in the reduction of N-benzylidenemethylamine no amine prod-
uct was observed with the mutant; however <3% of benzyl
alcohol was detected. Although we cannot exclude the possi-
bility that IREDs possess weak promiscuous activities towards
carbonyls, it seems very likely that byproduct formation results
from contaminations of co-purified ketoreductases in the
enzyme preparations.
To prove the applicability of the (R)-selective IRED also for in-
termolecular reductive aminations, carbonyl 1 was treated with
the three different amine nucleophiles. In control reactions
with the inactive enzyme as well as bovine serum albumin
(BSA) by using the nicotinamide cofactor regeneration system,
no amine products were detected.
Benzaldehyde (1) was readily converted by the purified
imine reductase into the corresponding amination products
with all three amine nucleophiles. Depending on the nature of
the amine nucleophile, moderate to good conversions were
obtained. In these biotransformations, a definite trend was
suggested by the increasing nucleophilicity of the amines lead-
All IREDs reported so far demonstrated high chemoselectivi-
ties towards C=N bonds. Attempts to detect activities for alde-
[18,19,21]
hydes, ketones, and keto acids failed.
To ensure that al-
cohol formation was not catalyzed by the IRED, biotransforma-
tions with N-benzylidenemethylamine were performed with
a previously described IRED mutant. In the variant (R)-IRED-Sr
D191A, the proposed catalytically important amino acid is re-
placed by alanine. In comparison to the wildtype enzyme, the
mutant displays strongly decreased reduction rates in whole-
[
28]
ing to higher formations of the amination products (Table 1).
Further, the direct comparison of transformations by using nu-
cleophiles 2b and 2c also indicates the substrate specificity of
the applied IRED. The preference of (R)-IRED-Sr towards smaller
amines and imines might be responsible for the fact that the
formation of product 3b was twofold higher than that of 3c
after 24 h under identical conditions.
[15]
cell biotransformations. In purified form, it showed activity
for the reduction of cyclic 2-methylpyrroline that was approxi-
mately 100-fold lower than that of the wildtype (Figure S6 and
Table S3). Both wildtype and variant possess the same level of
purity of approximately 90%, as judged by size-exclusion chro-
matography (SEC) analysis on HPLC (Figures S1–S5). Moreover,
By increasing the amine equivalents from an equimolar ratio
to 10-fold and 50-fold excess amounts, reaction rates strongly
increased, and overall, much higher conversion rates were ob-
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tained. It is assumed that the excess amount of the amine
boosts the equilibrium of the reaction intermediate towards
imine formation, which thus leads to higher saturation of the
biocatalyst with its substrate.
Table 2. Conversion of acetophenone (4) and cyclohexyl methyl ketone
(
6) into their different amination products 5a–b and 7a with amine
[a]
nucleophiles 2a and 2b by using purified imine reductase.
During the reductive amination of benzaldehyde (1) with
2
a, minor reductions to the alcohol byproduct and a small
amount of dialkylation product dibenzylamine (3d) were ob-
served.
Next, the generality of the developed reductive amination
protocol was explored for reactions with aromatic and cyclic
ketones. In contrast to aldehydes, the direct reductive amina-
tion of ketones provides access to chiral amines in a single
step. However, they are far less reactive with aromatic unsatu-
Carbonyl
acceptor
Amine
nucleophile
pH
Formed
product [%]
ee
[%]
[b]
[c]
4
4
4
4
4
4
4
4
6
6
6
6
2a
2a
2a
2a
2b
2b
2b
2b
2a
2a
2a
2a
8
8.5
9
9
8
8.5
9
9
8
8.5
9
5
7
97 (R)
98 (R)
98 (R)
97 (R)
87 (R)
86 (R)
86 (R)
87 (R)
74 (R)
78 (R)
77 (R)
78 (R)
10
16
9
15
19
39
16
18
19
53
[d]
[d]
[d]
[
29]
rated ketones being described to be challenging substrates.
To explore the transformation of such compounds, ketones 4
and 6 differing in their reactivity were selected.
First screening results with nucleophiles 2a and 2b and the
reaction conditions previously employed for benzaldehyde in-
dicated only low product formation (<2%) with acetophenone
9
(
4). Owing to the preference for smaller nucleophiles in the re-
ductive aminations with benzaldehyde (1), aniline 2c was not
employed in this set of reactions. The best conversion rates
were obtained with a 50-fold excess amount of amine donors
[a] Reaction conditions: carbonyl substrate 4 or 6 (10 mm), purified IRED
(2.5 mgmL ), amine nucleophiles 2a and 2b (500 mm) and cofactor re-
À1
generation system in Tris HCl buffer (50 mm, pH 8.0 to 9.0). To compare
conversion rates and to evaluate the formation of byproducts, all reac-
tions were performed with the same batch of purified biocatalyst. Trace
amounts of the alcohol reduction products of carbonyls 4 and 6 were de-
tected (<1% for all reactions). [b] Conversions were determined after
2
9
a and 2b, which resulted in 5% of amination product 5a and
% of 5b, respectively. To further increase product formations,
conditions were again adjusted to raise the concentration of
the imine intermediate at its equilibrium. This was done by
slightly increasing the pH value, which thereby enhanced the
concentration of the reactive amine species.
24 h by GC analysis. [c] Enantiomeric excess was determined after deriva-
tization with acetic anhydride by GC on a chiral stationary phase as de-
tailed in the Supporting Information. [d] Reactions were performed with
a fourfold increased enzyme loading (0.78 mol% catalyst to 3.1 mol%).
The stepwise increase in the pH of the reaction medium re-
sulted in enhanced transformations of acetophenone (4) into
amination products 5a and 5b with approximately 50% more
amination product formed. Remarkably, the imine reductase-
catalyzed formation of the new CÀN bond proceeded for prod-
ucts 5a and 5b with very high to excellent enantioselectivities
To obtain further insight into this process, the condensations
of benzaldehyde (1) as well as acetophenone (4) with methyl-
1
[30]
amine (2b) were monitored by H NMR spectroscopy. For
benzaldehyde (1) at pHꢀ8.2 (corresponds to a pDꢀ8.6), sub-
stantial imine formation was observed (Figure 1, see also Fig-
ures S19–S24). The course of the condensation reactions was
followed by the appearance of the CH signal of the imine
(
up to 98%ee) under all conditions (Table 2). In control reac-
tions with the inactivated enzyme or BSA, no amine products
were detected. Furthermore, alcohol formation was observed
only in trace amounts (<1%) with ketone substrates. In con-
trast, the amination of nonaromatic ketone 6 to give cyclohex-
ylethylamine (7a) did not require extensive optimization, as at
the starting conditions considerable amounts of the amination
product were already formed with good selectivity. Reductive
amination of ketones 4 and 6 was then also explored by em-
ploying slightly higher catalyst loadings (increase from 0.78 to
1
bond in the H NMR spectrum, which is shifted downfield with
respect to the signal of the parent aldehyde. The molar ratio
of imine was determined by integration of the resonances of
the aldehyde and imine moieties. However, imine formation
for acetophenone (4) was not detectable (Figure S25). Consid-
ering that the reductive amination of 4 resulted in good con-
versions, at least some levels of the imine intermediate are ex-
pected to be formed. Estimating the detection limit of the
3
.1 mol%). This ultimately increased the conversion rates by
1
approximately twofold to 16, 39, and 53% for products 5a, 5b,
and 7a, respectively, while maintaining the high selectivity
H NMR spectrometer to be <500 mm, it becomes clear that
(R)-IRED-Sr is a very effective catalyst in withdrawing already
low levels of imine intermediates from the equilibrium while
leaving the potential reducible carbonyl compound un-
touched.
(
Table 2).
As demonstrated by the optimization of our amination
setup, the formation of imines represents a potential bottle-
neck for enzyme-catalyzed intermolecular reductive aminations
in an aqueous environment. First, the amount of nucleophile
in the mixture was varied and then the pH was also raised. We
reasoned that increasing the nucleophile concentration and
pH should result in higher amounts of the condensation prod-
ucts.
In addition, we measured the kinetics for the reductive ami-
nation reaction of benzaldehyde (1) with a 50-fold excess
amount of 2a at pH 8 and at pH 9. The limited imine availabili-
1
ty that was also assigned by H NMR spectroscopy is reflected
by an apparent affinity in the low millimolar range (Table S11).
As shown by the Michaelis–Menten plots (Figures S9 and S11),
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Keywords: asymmetric synthesis · biocatalysis · chiral amines ·
imine reductase · reductive amination
[
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[
(
FP7/2007-2013) and EFPIA companies’ contribution in kind.
Received: July 8, 2015
Published online on August 31, 2015
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3242
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim