One-step asymmetric synthesis of (R)- and (S)-rasagiline by reductive amination applying imine reductases

Article abstract of DOI:10.1039/c6gc03023h

Imine reductases (IREDs) show great potential as catalysts for reductive amination of ketones to produce chiral secondary amines. In this work, we explored this potential and synthesized the pharmaceutically relevant (R)-rasagiline in high yields (up to 81%) and good enantiomeric excess (up to 90% ee) from the ketone precursor. This one-step approach in aqueous medium represents the shortest synthesis route from achiral starting materials. Furthermore, we demonstrate for the first time that tertiary amines also can be accessed by this route, which provides new opportunities for eco-friendly enzymatic asymmetric syntheses of these important molecules.

Full text of DOI:10.1039/c6gc03023h

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One-step asymmetric synthesis of (R)- and
S)-rasagiline by reductive amination applying
imine reductases†‡
(
Cite this: DOI: 10.1039/c6gc03023h
Received 2nd November 2016,
Accepted 16th December 2016
a
b
a
DOI: 10.1039/c6gc03023h
P. Matzel, M. Gand and M. Höhne*
www.rsc.org/greenchem
Imine reductases (IREDs) show great potential as catalysts for
reductive amination of ketones to produce chiral secondary
amines. In this work, we explored this potential and synthesized
the pharmaceutically relevant (R)-rasagiline in high yields (up to
8
1%) and good enantiomeric excess (up to 90% ee) from the
ketone precursor. This one-step approach in aqueous medium rep-
resents the shortest synthesis route from achiral starting materials.
Furthermore, we demonstrate for the first time that tertiary amines
also can be accessed by this route, which provides new opportu-
nities for eco-friendly enzymatic asymmetric syntheses of these
important molecules.
Introduction
Scheme 1 Enzymatic approaches provide a “green” alternative for syn-
thesis of chiral amines. Route (a): Imine hydrogenation requires multiple
steps. Route (b): Enzymatic preparation of amines with imine reductase
Chiral amines are key components of a broad spectrum of
pharmaceuticals and agrochemicals, and widely used in chem-
1
,2
istry, e.g. as chiral auxiliaries and ligands.
Numerous
(IRED), amine transaminase (ATA) and amine dehydrogenase (ADH). Only
synthesis approaches to optically pure amines have been devel- IREDs are suitable for obtaining sec-amines in one step. Some IREDs
1
also accept ammonia to yield primary amines.
oped. In view of efficiency and atom economy, asymmetric
syntheses starting from prochiral ketones as precursors are the
preferred choice. Classical transition metal-catalyzed enamine
or imine hydrogenation methods require 2–4 steps for synthe-
Compared to many other catalysts, enzymes can be produced
3
,4
sizing primary amines (Scheme 1, route a). An important
requirement is to find a catalyst that specifically reduces the
imine bond but not the carbonyl to the corresponding
from renewable resources. Toxic reagents or catalysts contain-
ing heavy metals are avoided, as well as extensive protection
and deprotection steps, which otherwise guarantee regio-
selectivity. Shorter routes offer savings of reagents and solvents
that otherwise are consumed during intermediate product
isolation and purification steps, which also often reduce the
overall yield.
5
alcohol.
Biocatalysis is increasingly applied in developing green
6
–8
chemistry for chemical synthesis at the industrial scale.
Enzymes often provide excellent stereo- and regioselectivity
and facilitate to work under mild and aqueous conditions.
Many established enzymatic routes for the synthesis of
9
–13
chiral amines start from the amine racemate,
but more
a
interesting are enzymes that transform prochiral carbonyl
compounds into amines: transaminases, amine dehydrogen-
ases, and imine reductases (IREDs) (Scheme 1, route b).
Transaminases transfer an amino group from a donor
substrate to an acceptor compound utilizing the cofactor
Institute of Biochemistry, Greifswald University, Felix-Hausdorff-Str. 4,
1
b
7487 Greifswald, Germany. E-mail: Matthias.Hoehne@uni-greifswald.de
Biocenter Klein Flottbek, University of Hamburg, Ohnhorststr. 18, 22609 Hamburg,
Germany
†
This manuscript is dedicated to Professor Romas J. Kazlauskas on the occasion
of his 60th birthday.
Electronic supplementary information (ESI) available. See DOI: 10.1039/
1
4
pyridoxal-5-phosphate (PLP). This type of reaction enables
kinetic resolution of a racemic amine as well as asymmetric
‡
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1
5
30
synthesis from a ketone substrate. Dehydrogenases catalyze ment of Parkinson’s disease, rasagiline, selegiline and pra-
the NAD(P)H-dependent reductive amination of carbonyl com- mipexole (Scheme 2b).
1
6
pounds, usually α-keto acids, but also ketones. However,
Especially the tertiary amine selegiline is challenging, as it
especially for secondary and tertiary amines, asymmetric syn- requires the acceptance of a secondary amine by the IRED,
thesis is quite challenging due to the natural catalytic limit- which has not yet been shown so far.
ations of these enzymes. Transaminases are limited to the
transfer of a (primary) amino group because an N-alkyl substi-
tuent of a secondary amine substrate would cause a steric
clash with the 3′-OH-group of the PLP cofactor when the
Results and discussion
planar quinoid intermediate is formed during catalysis. Also
21
We screened the IRED library provided by Roche, and
1
7
amine dehydrogenases are currently limited to ammonia as
a nitrogen donor due to steric restrictions of the ammonia
binding pocket of the enzymes. Recently, several groups
investigated asymmetric reductive amination catalyzed by
31
enzymes from our group (enzymes summarized in Table S1‡)
for conversion of the corresponding ketones (for selegiline we
used 4-fluoro-phenyl acetone due to the chemical regulations)
and amines to yield the desired pharmaceutical products. Only
for two IREDs out of this enzyme library, we found an initial
activity giving a conversion of 21% for IR-14 (91% ee (R)-rasagi-
line) and 12% for IR-Sip (71% ee (S)-enantiomer of rasagiline).
As for most of the enzymes these substrate combinations
did not yield detectable product formation, we were interested
whether the corresponding ketone or amine substrates are
causing the limitation. The ketones 2–4 (Scheme 3) are larger
compared to the successfully converted ketones reported
1
8–20
IREDs
substrates,
been formed in small quantities by condensation of a ketone
(Scheme 2a). Besides reduction of cyclic imine
2
1–26
27–29
IREDs also reduce acyclic imines
that have
2
7
and an amine directly in the aqueous reaction medium. This
facile reaction setup is possible due to the outstanding chemo-
selectivity of the IREDs that reduce selectively the imine, but
not the excess ketone substrate. Thus, this one-step enzymatic
conversion represents an interesting alternative and bears
large potential as an environmentally benign approach. Due to
the unfavorable equilibrium of imine formation under
aqueous conditions, reductive amination turned out to be
challenging. The feasibility of reductive amination was demon-
27
recently. It was also shown that larger amines such as butyl-
amine are only accepted by few IREDs. Besides propylamine c,
amines a and b (Scheme 3) might be difficult substrates
because of steric demands (rigid geometry at the triple bond,
secondary amine). In our previous study, the combination of
cyclohexanone and methylamine was especially well accepted
2
8,29
strated recently at the analytical scale.
consortium focusing on IREDs,
In our joint research
suitable enzymes from
2
7
Roche’s in-house collection were identified that convert several
model ketones such as (cyclo)hexanone with methyl- or butyl-
amine or ammonia. After optimizing the reaction conditions,
increased conversions and reduced enzyme loads allowed the
preparative scale conversion of 1% (w/v) substrate to the
corresponding amines with high yields and enantiomeric/
27
by almost all IREDs investigated in the library. Thus, we
decided to combine the challenging ketones 2–4 with methyl-
amine d and to react the desired amines a–c with cyclohexa-
none 1 to explore the enzyme’s limitations.
In our screening we employed the 14 most promising IREDs.
When using the well-accepted model ketone cyclohexanone,
we identified various enzymes that accepted propargyl- and
propylamine to yield the secondary amines 1a and 1c with
high conversion (Table 1). Most interestingly, a significant for-
mation of the tertiary amine 1b with >20% conversion could
be observed by two IREDs. When we reacted the pharma-
ceutically relevant ketones 2 and 3 with methylamine, we
obtained conversions of up to 70% for 1-indanone and >80%
2
7
diastereomeric excess. This finding provides a new biocata-
lytic opportunity for the straightforward synthesis of chiral sec-
ondary amines.
In this study, we wanted to investigate the scope of IREDs
as catalysts for reductive amination for the preparation of
pharmaceutically relevant amines. We have selected three con-
ceivable candidates which are used in the application of treat-
Scheme 2 (a) IRED-catalyzed reductive amination of the model sub-
strates cyclohexanone and methylamine. Whether the imine forms in
the bulk solution or inside the active site of the enzyme is not yet
known. (b) Three pharmaceutical target molecules are subjects of this
study.
Scheme 3 Selected compounds for the substrate screening of IREDs.
1–4: ketone substrates, a–i: amine substrates.
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Table 1 Screening results for the reductive amination with IREDs
ents larger than a methyl group were not accepted. In contrast,
we were pleased to find nearly quantitative conversion with
pyrrolidine i. This calls for a systematic in-depth characteriz-
ation of secondary amines as substrates, which is planned for
a future study.
Next, we conducted preparative scale experiments for the
asymmetric reductive amination. Of special interest was the syn-
thesis of rasagiline considering the pharmaceutical relevance of
this compound. To increase the initial conversion of only 21%,
we optimized the reaction conditions in small-scale experi-
ments: ketone and amine concentrations and enzyme con-
centrations were varied as described in detail in the ESI (Fig. S4–
S7‡). Under the optimal conditions, rasagiline 2a was obtained
with 86% conversion employing IR-Sip. Furthermore, prepara-
tive-scale experiments were performed to yield 4-fluoro meth-
amphetamine 3d and the tertiary amine N-methyl-N-propargyl
cyclohexanamine 1b. For rasagiline 2a, two reactions employing
IR-14 and IR-Sip yielded both enantiomers (Scheme 4).
The products were precipitated as HCL-salt, yielding 70 mg
(
58%) for (R)-2a with 90% ee; 98 mg (81%) for (S)-2a with 72% ee;
01 mg (83%) for 3d with 52% ee and 73 mg (59%) for 1b. This
one-step approach is an interesting alternative to recently reported
–5 step syntheses relying on the preparation of enantiopure pre-
cursors 1-aminoindane or indane-1-ol followed by an alkynylation
1
3
3
2
a
step. These precursors can be prepared by (dynamic) kinetic
IRED screening 1: Cyclohexanone with three amines 1a–1c; and
33
34
methylamine with three ketone substrates 2d–4d. Screening con- resolution, or asymmetric synthesis via C–H-amination.
−1
ditions: 500 mM amine, 10 mM ketone, pH 9.5, 0.5 mg mL purified
Synthesis approaches for chiral primary amines featuring
an outstanding eco-efficiency were developed by utilizing
sodium hydroxide (end concentration 1.7 M). Apparent conversion amine transaminases, for instance the synthesis of sitag-
enzyme, 0.5 mM NADPH, 0.1 mg mL− glucose dehydrogenase
1
(Codexis GDH-105), 60 mM D-glucose, 20 h, 30 °C, stopped with
b
3
5
36,37
was calculated based on the peak areas of product and substrate.
liptin or nor(pseudo)ephedrine.
Imine reductases clearly
c
1
Substrate precipitated during time, kinetic H-NMR measurements
showed keto–enol tautomerization and a decrease of substrate concen-
d
tration to 79% during 20 h (ESI Fig. S3a–S3d). IRED screening 2:
Cyclohexanone with six secondary amines b, e–i. Compared to screen-
ing 1, the reaction time was elongated to 90 h.
for 4-fluoro-phenyl acetone, in contrast to product 4d, which
could not be detected and thus ketone 4 seems not to be a sub-
strate for the IREDs investigated. This experiment shows that
although an IRED might accept a desired ketone or amine (if
combined with well-known model substrates), this does not
guarantee that arbitrary combinations of these (accepted) sub-
strates are converted efficiently.
The formation of the tertiary amine 1b demonstrates for
the first time that IREDs also accept secondary amines as
nucleophiles. As asymmetric synthesis of tertiary amines is
challenging, we explored the amination of cyclohexanone 1
using the two enzymes (IR-14, IR-Sip) that were positive hits
for the formation of the tertiary amine product 1b and selected
the secondary amines e–i as model substrates. The reaction
conditions were similar to the first screening except for a pro-
longed reaction time (4 d). Compared to the conversion with
amine b, the analogous saturated amine e showed a signifi-
cantly lower conversion. How the triple bond of substrate b
increases the activity of the IRED is not understood at the
Scheme 4 Preparative-scale reductive aminations catalyzed by IREDs.
Reaction conditions: 100 mg substrate (1-indanone, 4-fluoro-phenyl
acetone or cyclohexanone, 10 mM), amine buffer pH 9.5 (propargy-
−1
lamine, N-methyl propargylamine 0.4 M; methylamine 1 M), 1.5 mg mL
moment. Substrate amines g and h having both alkyl substitu- purified IRED, 7 days at 30 °C.
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tiary amines, if the following issues are optimized in future
studies: identifying or creating IREDs that (i) are highly active
and stable to decrease the enzyme load and (ii) convert high
ketone concentrations with preferentially one equivalent of the
amines. Increasing efficiency was also a challenge when IREDs
were first investigated for reduction of cyclic imines. The
characterization of a large number of IREDs showed that
specific activities and enantioselectivities vary considerably. A
few enzymes could be identified that are highly active, e.g. the
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