Kinetic Resolution and Deracemization of Racemic Amines Using a Reductive Aminase

Article abstract of DOI:10.1002/cctc.201701484

The NADP(H)-dependent reductive aminase from Aspergillus oryzae (AspRedAm) was combined with an NADPH oxidase (NOX) to develop a redox system that recycles the co-factor. The AspRedAm-NOX system was applied initially for the kinetic resolution of a variety of racemic secondary and primary amines to yield S-configured amines with enantiomeric excess (ee) values up to 99 %. The addition of ammonia borane to this system enabled the efficient deracemization of racemic amines, including the pharmaceutical drug rasagiline and the natural product salsolidine, with conversions up to >98 % and >99 % ee Furthermore, by using the AspRedAm W210A variant it was possible to generate the opposite R enantiomers with efficiency comparable to, or even better than, the wildtype AspRedAm.

Full text of DOI:10.1002/cctc.201701484

Accepted Article
Title: Kinetic resolution and deracemisation of racemic amines using a
reductive aminase
Authors: Nicholas John Turner, Godwin Aleku, Juan Mangas-Sanchez,
Joan Citoler, Scott France, Sarah Montgomery, Matthew
Thompson, and Rachel Heath
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.201701484
Link to VoR: http://dx.doi.org/10.1002/cctc.201701484
A Journal of
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10.1002/cctc.201701484
ChemCatChem
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Kinetic resolution and deracemisation of racemic amines using a
reductive aminase
Godwin A. Aleku, Juan Mangas-Sanchez, Joan Citoler, Scott P. France, Sarah L. Montgomery, Rachel
S. Heath, Matthew P. Thompson and Nicholas J. Turner. *
Abstract: The NADP(H)-dependent reductive aminase from
Aspergillus oryzae (AspRedAm) has been combined with an NADPH
oxidase (NOX) to develop a redox system that recycles the co-factor.
The AspRedAm-NOX system has been applied initially for the kinetic
resolution of a variety of racemic secondary and primary amines to
yield (S)-configured amines with enantiomeric excess (e.e.) up to
99%. Addition of ammonia borane to this system enabled the
efficient deracemisation of racemic amines, including the
pharmaceutical drug rasagiline and the natural product salsolidine,
with conversions of up to >98% and e.e.’s >99% e.e. Furthermore,
using the variant AspRedAm W210A it was possible to generate the
opposite (R)-enantiomers with efficiency comparable to, or even
better than, the wild-type AspRedAm.
borohydride (NaBH4)[5c], resulting in yields that are comparable
to asymmetric synthetic methods (Figure 1a).
Recently we reported the discovery of an NADPH-dependent
reductive aminase (RedAm) from Aspergillus oryzae
(AspRedAm) and demonstrated the application of this
biocatalyst in the synthesis of mostly (R)-configured chiral
amines from the corresponding ketones.[12] For certain
amine/ketone combinations, AspRedAm is able to catalyse
reductive amination with ratios as low as 1:1. RedAms are part
of the IRED enzyme family and thus can also catalyse the
reverse reaction, namely the oxidation of amines to imines at the
expense of NADP+. We envisaged exploiting this reverse activity
as a means of gaining access to the opposite (S)-enantiomers
using AspRedAm. Indeed IREDs have previously been reported
to catalyse the kinetic resolution of racemic amines although
with low efficiency (9% conv; 10% e.e.).[13] Herein we report the
application of AspRedAm for both the kinetic resolution and
deracemisation of racemic amines (Figure 1b).
A significant proportion of small molecule therapeutics contain
one or more chiral amine functional groups and indeed a recent
evaluation of novel drug approvals highlights the prominence of
these moieties as building blocks of pharmaceuticals.[1]
Consequently, a number of amine-forming synthetic methods,
utilising transition metal catalysis, organocatalysis or biocatalysis,
have been developed.[2] Enzymatic transformations are often
highly chemo-, regio- and stereoselective, and operate under
mild and environmentally-friendly conditions.[3] As a result,
preparative biocatalytic routes to chiral amines employing
lipases,[4] amine oxidases,[5] amine transaminases (ATAs),[2c, 6]
ammonia lyases,[7] amine dehydrogenases (AmDHs)[8] and imine
reductases (IREDs),[9] or engineered variants of these
biocatalysts, have received considerable attention.
Production of enantiopure amines can be achieved via
biocatalytic kinetic resolution (KR) orderacemisation (DR) of
racemic amines. Although KRs using lipases and transaminases
are widely used, they possess the inherent limitation of a
maximum yield of 50%. This issue may be addressed by the
conversion of KRs to deracemisation methods such as dynamic
kinetic resolutions (DKR) in which an additional biocatalyst or
chemical reagent is added to racemise in situ the unreacted
enantiomer.[10,11] Another important deracemisation approach
involves the interconversion of the two enantiomers of the amine,
usually via the corresponding imine. For example, the
deracemisation of racemic amines employing amine oxidases
has been achieved with repeated cycles of enzyme-catalysed
enantioselective amine oxidation and non-selective chemical
imine reduction using ammonia borane (NH3.BH3)[5b] or sodium
Figure 1. Biocatalytic deracemisation of racemic amines via (a) monoamine
oxidase (MAO)-catalysed route and b) Aspergillus reductive aminase
(AspRedAm)-catalysed route.
In order to assess the potential for the application of AspRedAm
in the kinetic resolution (KR) of racemic amines, we initially
examined the oxidative deamination activity towards a panel of
amine substrates comprising cyclic and acyclic 1o, 2o and 3o
amines. Initial reaction rates were measured at pH 10.5 and
revealed the preference of AspRedAm for secondary amines,
with the highest specific activity of 6.5 U mg-1 observed towards
1-methyl-1,2,3,4-tetrahydroisoquinoline 1 (Figure 2).
Dr. G. A. Aleku, Dr. J. Mangas-Sanchez, J. Citoler, S. P. France, S. L.
Montgomery, Dr. R. S. Heath, M. Thompson, Prof. N. J. Turner .
School of Chemistry, University of Manchester,
Manchester Institute of Biotechnology,
131 Princess Street, Manchester M1 7DN, UK.
Email: Nicholas.Turner@manchester.ac.uk
Supporting information for this article is given via a link at the end of the
document.
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10.1002/cctc.201701484
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Based upon this initial assessment we then applied AspRedAm
for the KR of racemic amines N-(4-phenylbutan-2-yl)butan-1-
amine 8 and rasagiline 16. The enantioselective deamination of
these racemic amines led to the accumulation of the (S)-
enantiomer in excellent e.e. of >99% and conversion of 49%
when a stoichiometric amount of NADP+ was used. The
deamination was also observed to be highly regioselective, e.g.
HPLC and GC analysis of biotransformations for the
deamination of racemic amines 8 and 16 and 28 confirmed that
hydride abstraction occurred at the stereogenic carbon atom
rather than from the N-alkyl group (Supporting Information,
Section 1.3).
Specific ac)vity (U/mg)
0.1
0
0.05
0.15
32
31
30
29
28
27
26
25
24
23
22
21
20
19
18
17
16
15
14
13
12
11
10
9
We next addressed the issue of in situ co-factor recycling and
evaluated the incorporation of an NADPH oxidase, an enzyme
which mediates the transfer of electrons from NADPH to
molecular oxygen, generating hydrogen peroxide as the by-
product.[14] Using two model amine substrates
8 and 1-
aminoindane 25, the optimal reaction pH and buffer conditions
were determined. The AspRedAm-NOX coupled system
tolerated pH values between 8.0 and 11.0, however the best
conversions (up to 50%) were achieved at pH values between
10.0 and 10.5 (Supporting Information Section 1.1). Control
experiments featuring all reaction conditions including NOX but
lacking either AspRedAm or the cofactor did not result in any
conversion.
Primary axis
Secondary axis
8
7
6
5
4
3
2
1
0
2
4
6
8
Specific ac)vity (U/mg)
Figure 2. Substrate specificity of AspRedAm in the NADP+-dependent
oxidative deamination of amines 1-32. Two y-axes have been used to allow
representation of high (blue) and moderate (red) specific activities. Activities
were measured in triplicate and the error bars represent standard deviation
from the mean specific activity.
In general, the relative specific activities for the oxidation of
amine substrates follow similar trends to the activities observed
in the synthetic direction, as previously reported.[12] For example,
AspRedAm displayed higher activities of 0.9-2.3 U mg-1 for N-
alkyl-cyclohexylamines (2, 3 and 4) whereas the enzyme’s
activity towards cyclohexylamine 30 (0.010 U mg-1) and most
primary amines (0.003 – 0.028 U mg-1) were up to two orders of
magnitude lower. Interestingly, the type of N-alkyl substituent
appears to exert a significant effect on the catalytic rates, with
AspRedAm displaying higher specific activities in the oxidation
of N-allylamine, N-propargylamine, N-butylamine and N-
cyclopropylamine derivatives than with N-methyl derivatives. For
example, the initial rates for the oxidations of 6-9 (0.90 – 0.65 U
mg-1) were up to an order of magnitude greater than the
oxidation of 20 (0.05 U mg-1) even though these compounds
differ only by the N-alkyl substituents.
Figure 3. Biotransformation results of AspRedAm-catalysed kinetic resolution of
racemic amines employing an NADPH oxidase to recycle the cofactor. Resolution of
the racemic amines were achieved using either AspRedAm wild-type (WT) enzyme
(black), AspRedAm Q240A (blue) or AspRedAm W210A (red). Conversions were
determined by HPLC or GC-FID analysis. Reaction conditions: racemic amine (5 mM),
AspRedAm (0.5 mg mL-1), NADP+ (0.3 mM) and lyophilised lysate powder of prozomix
NADPH oxidase Pro-NOX001, (1 mg mL-1). Reactions were run at pH 10.0 (100 mM
glycine-NaOH buffer) unless stated otherwise. Reactions were incubated at 25 °C
with 250 rpm shaking for 24 h. [a] Reactions were run at pH 8.0 (100mM Tris-HCl
buffer). [c] Conversion not determined as only negligible amount of the ketone
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10.1002/cctc.201701484
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further sponstaneous oxidation of the resulting ketone.
formed from the AspRedAm-catalysed deamination was detected. This may be due to
and indanol (up to 34 %) were detected in amounts that
depended on the number of equivalents of NH3.BH3 used. This
limitation may be addressed by the use of other chemical
reducing agents for imine reduction as recently reported.[5c] To
demonstrate preparative scale application, we performed 100
mg scale biotransformations for the deracemisation of racemic
amines 1 and 16 (20 mM) using 4 equiv. and 10 equiv. of
NH3.BH3 respectively. Deracemisation of racemic 1 yielded (S)-1
(78% isolated yield and 93% e.e.) after 24 h, while (S)-16 was
obtained after 48 h (64% isolated yield and >98% e.e.) from
deracemisation of racemic amine 16.
The AspRedAm-NOX system was applied to the KR of a panel
of racemic secondary and primary amines (Figure 3), affording
>99% e.e. in most cases for secondary amines, and up to 83%
e.e. for primary amines. The KR of N-allyl-4-phenylbutan-2-
amine
6
and 4-phenyl-N-(prop-2-yn-1-yl)butan-2-amine
7
resulted in high conversion to ketone (up to 80%) due to the
poor enantioselectivity of the wild-type enzyme towards these
substrates. To address this issue, we employed a variant of
AspRedAm (Q240A) which displays improved (R)-selectivity,
affording 49% conversion and >99 e.e. for 6 and 7. Interestingly,
the Q240A variant also showed improved efficiency towards a
range of substrates (Figure 3, Supporting Information Section
1.2, Table S1), affording higher conversions and e.e.’s than the
wild type enzyme.
Table 1. Biotransformation results for the deracemisation of racemic amines
using the AspRedAm-NOX-NH3.BH3 cascade.
R3
R3
AspRedAm or variant
O
HN
HN
*
NH
R3NH2
+
+
R1 R2
R1 R2
rac-amine
R1 R2 R1 R2
NADP+
NADPH
Encouraged by the efficiency and improved selectivity of the
Q240A variant, we attempted to further expand the product
profile of the AspRedAm-NOX KR system. Previously, we
reported a variant of AspRedAm W210A which in several cases
displayed opposite enantioselectivity when compared to the
wild-type enzyme.[12] The (S)-selective AspRedAm W210A
yielded the corresponding (R)-configured products in good to
excellent e.e.s (Figure 3, Supporting Information Section 1.2,
Table S2). AspRedAm W210A catalysed KR allows access to
novel (R)-configured amine products, which are difficult to
access with MAO-N variants,[5d] and therefore expands the
substrate scope of (S)- selective KR biocatalysts.
H2O2
O2
NADPH oxidase (NOX)
H3N BH3
Racemic
amine
NH3.BH3
equiv.
AspRedAm
Conv.
[%]
e.e. [%]
(R or S)
Entry
variant
1
2
3
4
5
6
7
8
1[a]
2
4
AspRedAm wt
AspRedAm Q240A
AspRedAm W210A
AspRedAm wt
>98
>97
>97
>98
>98
>97
74[b]
>97
>98 (S)
96 (S)
6
Finally we explored the development of an AspRedAm-NOX
based deracemisation system in order to achieve yields of up to
100%. Previously we and others have shown that
deracemisation of amines with monamine oxidases[5] and
PALs[7b] can be achieved by the addition of a mild chemical
reducing agent such as ammonia borane (NH3.BH3) to reduce in
situ the corresponding imine. Repeated cycles of selective
oxidation of the reactive enantiomer catalysed by AspRedAm,
and non-asymmetric imine reduction by NH3.BH3, was envisaged
to lead to the complete deracemisation of racemic amines,
provided that the co-factor was also continuously recycled by
NOX. This cascade was evaluated by performing
biotransformations for the deracemisation of 1 and 8 at different
pHs (7.0-11.0). Interestingly, conversions of >95% were
achieved for 1 and 8 at pH values of 8.0 and 10.0 respectively
using 4 equiv. of NH3.BH3, hence subsequent biotransformations
were performed at pH 8.0 and pH 10.0 for cyclic and acyclic
amines respectively (Table 1). Control biotransformations were
performed and featured all reaction components and conditions
but lacking either AspRedAm or NADP+; in both cases the
racemic amine starting materials remained unconverted.
6
4
94 (R)
8
4
>99 (S)
>99 (R)
>99 (S)
93 (S)[b]
>99 (S)
8
4
AspRedAm W210A
AspRedAm Q240A
AspRedAm wt
15[a]
16
20
10
4
10
AspRedAm Q240A
Reactions were run at pH 10.0 (100 mM glycine-NaOH buffer) unless stated
otherwise. Reactions were incubated at 25°C with 250 rpm shaking for 24 h.
[a] Reactions were run at pH 8.0 (100mM Tris-HCl buffer). [b] 18% and 34%
indanol were detected with 10 equiv. and 5 equiv. of NH3.BH3 respectively.
wt = wild-type. Conv. = conversion.
In summary we have shown that the reductive aminase from
Aspergillus oryzae (AspRedAm) can be employed for both the
kinetic resolution and deracemisation of a range of cyclic and
acyclic racemic amines. A key element of both systems is the
use of an NADPH oxidase for in situ co-factor recycling of
NADP+. The AspRedAm-NOX-NH3.BH3 system enables
deracemisation of a variety of cyclic and acyclic racemic amines
to provide access to enantiomerically pure (S)-configured amine
products. Furthermore, switching to the rationally engineered
single point variant of AspRedAm W210A enables access to (R)-
configured products.
By using the one-pot AspRedAm-NOX-NH3.BH3 system, the
deracemisation of a number of racemic cyclic and acyclic
amines was achieved with conversion of up >98% and up to
>99% e.e (Table 1). In general the formation of alcohol side
product (resulting from imine hydrolysis and subsequent
reduction of the ketone by NH3.BH3) was not observed for most
of the substrates screened. This observation contrasts with that
found with the MAO-N based deracemisation system[15] and is
presumably a consequence of the ability of AspRedAm to
recycle the ketone back to the amine via the imine. However, for
the deracemisation of rasagiline 16, both indanone (up to 18%)
Experimental Section
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10.1002/cctc.201701484
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Details of experimental procedures are described in Supporting
Information Section 2.
Parmeggiani, S. L. Lovelock, N. J. Weise, S. T. Ahmed, N. J.
Turner, Angew. Chem. Int. Ed. 2015, 54, 4608-4611.
[8]
a) L. J. Ye, H. H. Toh, Y. Yang, J. P. Adams, R. Snajdrova, Z. Li,
ACS Catal. 2015, 5, 1119-1122; b) M. J. Abrahamson, E.
Vazquez-Figueroa, N. B. Woodall, J. C. Moore, A. S. Bommarius,
Abstr. Pap. Am. Chem. Soc. 2012, 244; c) M. J. Abrahamson, E.
Vazquez-Figueroa, N. B. Woodall, J. C. Moore, A. S. Bommarius,
Angew. Chem. Int. Ed. 2012, 51, 3969-3972; d) T. Knaus, W.
Bohmer, F. G. Mutti, Green. Chem. 2017, 19, 453-463.
Acknowledgements
G.A.A. and M.P.T. were supported by the industrial affiliates
programme of the Centre of Excellence for Biocatalysis,
Biotransformations and Biocatalytic Manufacture (CoEBio3).
S.P.F. was supported by
a
CASE studentship from
BBSRC/Pfizer. J.M.S received funding by grant BB/M006832/1
from the UK Biotechnology and Biological Sciences Research
Council. S.L.M. was funded by a studentship from Johnson
Matthey. R.S.H. received funding by grant agreement no.
613849 supporting the project BIOOX from the European
Union’s Seventh Framework Programme for research,
technological development and demonstration. N.J.T. is grateful
to the Royal Society for a Wolfson Research Merit Award and
the ERC for an Advanced Grant.
[9]
a) J. H. Schrittwieser, S. Velikogne, W. Kroutil, Adv. Synth. Catal.
2015, 357, 1655-1685; b) G. Grogan, N. J. Turner, Chem. Eur. J.
2016, 22, 1900-1907; c) J. Mangas-Sanchez, S. P. France, S. L.
Montgomery, G. A. Aleku, H. Man, M. Sharma, J. I. Ramsden, G.
Grogan, N. J. Turner, Curr. Opin. Chem. Biol. 2016, 37, 19-25; d)
D. Wetzl, M. Gand, A. Ross, H. Müller, P. Matzel, S. P. Hanlon, M.
Müller, M., B. Wirz, M. Höhne, H. Iding, ChemCatChem, 2016, 8,
2023-2026; e) G. A. Aleku, H. Man, S. P. France, F. Leipold, S.
Hussain, L. Toca-Gonzalez, R. Marchington, S. Hart, J. P.
Turkenburg, G. Grogan, N. J. Turner, ACS Catal. 2016, 6, 3880-
3889; e) H. Li, P. Tian, J. H. Xu, G.W. Zheng, G. W. Org. Lett.
2017, DOI: 10.1021/acs.orglett.7b01274 ; f) Z. Maugeri, D. Rother,
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Pontini, S. Hussain, N. J. Turner, ChemCatChem 2016, 8, 117-120.
O. Verho, J.-E. Bäckvall. J. Am. Chem. Soc. 2015, 137, 3996-
4009.
Keywords: Biocatalysis • Chiral amines • Deracemisation •
Kinetic resolution • Reductive aminase
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Entry for the Table of Contents
COMMUNICATION
Dr. G. A. Aleku, Dr. J. Mangas-Sanchez,
J. Citoler, S. P. France, S. L.
Montgomery, Dr. R. S. Heath, M. P.
Thompson and Prof N. J. Turner*
Page No. – Page No.
Kinetic resolution and
deracemisation of racemic amines
using a reductive aminase
The reductive aminase from Aspergillus oryzae (AspRedAm) has been combined
with NADPH oxidase (NOX) to enable the kinetic resolution of a variety of racemic
amines yielding (S)-configured products. The corresponding (R)-enantiomers were
obtained by the use of a single amino acid variant AspRedAm W210A.
Furthermore, AspRedAm-NOX-NH3.BH3 cascade was constructed to allow the
efficient deracemisation of racemic amines.
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