Synthesis of β-Chiral Amines by Dynamic Kinetic Resolution of α-Branched Aldehydes Applying Imine Reductases

Article abstract of DOI:10.1002/cctc.201900806

Imine reductases (IREDs) allow the one-step preparation of optically active secondary and tertiary amines by reductive amination of ketones. Until now, mainly α-chiral amines have been prepared by this route. In this study, we explored the possibility of synthesizing β-chiral amines, a class of compounds which is also frequently found as structural motif in pharmaceuticals but much more challenging to prepare due to the following reasons: (i) The aldehyde substrate already contains the chiral center and needs to be racemized to enable full conversion. (ii) Because the intermediate imine bears the stereo center two carbon atoms remote to the imine nitrogen, it is more challenging to achieve high enantioselectivity compared to α-chiral amine synthesis. For investigating the proof of concept, we first confirmed that different IREDs are able to convert a variety of α-branched aldehydes when combined with five different amine substrates. The IRED from Streptomyces ipomoeae was a suitable enzyme facilitating the dynamic kinetic resolution of 2-phenylpropanal and a substituted 2-methyl-3-phenylpropanal: the corresponding N-methylated β-chiral amines were obtained with '95 % conversion and 78 and 95 %ee. Other amines were formed with low to moderate enantiomeric excess. This exemplifies the potential of IREDs for the one-step synthesis of secondary β-chiral amines, but also the challenge to identify highly selective enzymes for a desired amine product.

Full text of DOI:10.1002/cctc.201900806

Accepted Article
Title: Synthesis of β-Chiral Amines by Dynamic Kinetic Resolution of α-
Branched Aldehydes Applying Imine Reductases
Authors: Philipp Matzel, Sebastian Wenske, Simon Merdivan,
Sebastian Günther, and Matthias Höhne
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Link to VoR: http://dx.doi.org/10.1002/cctc.201900806
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10.1002/cctc.201900806
ChemCatChem
COMMUNICATION
Synthesis of β-Chiral Amines by Dynamic Kinetic Resolution of
α-Branched Aldehydes Applying Imine Reductases
Philipp Matzel[a], Sebastian Wenske[a] Simon Merdivan[b], Sebastian Günther[b] and Matthias Höhne*[a]
Imine reductases (IREDs) allow the one-step preparation of
optically active secondary and tertiary amines by reductive
amination of ketones. Until now, mainly α-chiral amines have
been prepared by this route. In this study, we explored the
limited to aromatic amine donors such as p-methoxyaniline and
therefore require deprotection of the products to obtain the free
primary amines. Alternatively, the optically pure aldehyde
precursors can be prepared in
a first step by catalytic
possibility of synthesizing β-chiral amines,
a
class of
processes[5] or by crystallization-induced deracemization,[6]
followed by a non-selective reductive amination as the second
step. Finally, (iii) asymmetric synthesis can also be employed to
synthesize other optically pure precursor molecules, which are
then transformed by functional group interconversion into
amines. Examples of such precursors include optically active
nitro,[7] azido,[8] or cyano[4] compounds.
compounds which is also frequently found as structural motif
in pharmaceuticals but much more challenging to prepare due
to the following reasons: (i) The aldehyde substrate already
contains the chiral center and needs to be racemized to enable
full conversion. (ii) Because the intermediate imine bears the
stereo center two carbon atoms remote to the imine nitrogen, it
is more challenging to achieve high enantioselectivity
compared to α-chiral amine synthesis. For investigating the
proof of concept, we first confirmed that different IREDs are
NH
O
R
O
OH
N
N
H
N
F3C
able to convert
a variety of α-branched aldehydes when
NBoc
N
H
F
SO2Et
combined with five different amine substrates. The IRED from
Serotonin agonist
Renin inhibitor
Antitumor agent
HO
Streptomyces ipomoeae was a suitable enzyme facilitating the
dynamic kinetic resolution of 2-phenylpropanal and
a
substituted 2-methyl-3-phenylpropanal: the corresponding N-
methylated β-chiral amines were obtained with >95%
conversion and 78 and 95%ee. Other amines were formed with
low to moderate enantiomeric excess. This exemplifies the
potential of IREDs for the one-step synthesis of secondary β-
chiral amines, but also the challenge to identify highly selective
enzymes for a desired amine product.
O
N
O
N
H
OH
R
N
H
N
Propanolol, β-blocker
T226296, anti obesity
Faxeladol, analgesic
O
H
N
N
NH2
N
Cl
Phenpromethamine, stimulant
N
β-Methylfentanyl, opioid
DB07860, protein kinase inhibitor
Chiral amines are indispensable building blocks for the
preparation of a variety of pharmaceutically active ingredients.[1]
Drugs such as tapentadol or the fungizide fenpropidine belong to
a subgroup of chiral amines having the stereo center in β-
position to the amine nitrogen (see Scheme 1). Beside classical
N
N
O
tBu
fungicide
Fenpropidine, fungicide
Fenpropimorph, fungicide
O
kinetic resolution of amine racemates,
a large variety of
H
chemical methods have been devised to prepare optically active
β-chiral amines, which can be grouped in three different
approaches: (i) asymmetric alkylation installs the β-branch of the
carbon sceleton by starting with an activated nitrogen precursor,
such as chiral hydrazones or amide enolates.[2] (ii) Reductive
amination of aldehydes can be carried out in two variants:
Dynamic kinetic resolution (DKR) starts from the corresponding
racemic precursor aldehyde. Chiral Brønsted acids as catalysts[3]
or Lewis-base organocatalysts[4] render the reactions
O
N
HO
N
N
N
O
H
O
H
R
S
Tapentadol, analgesic
Levopromazine, antipsychotic Somatostatin receptor antagonist
Scheme 1. Examples of physiologically active β-chiral amines.
All these processes are multi-step reactions and require
further transformations to prepare the secondary or tertiary
amine of choice. Enzymatic approaches represent a promising
alternative: Amine transaminases give the primary β-chiral
amine in a DKR directly, as demonstrated in the synthesis of
niraparib or pregabalin.[9] Additionally, IREDs and reductive
aminases have been explored for the one-step asymmetric
synthesis of secondary[10] or tertiary amines from ketones.[11]
Until now, these reductive aminations have been investigated
only to prepare α-chiral amines. Interestingly, there are a few
examples from natural biosynthesis pathways demonstrating
that IREDs also mediate imine reductions with β-selectivity:
plants produce the cyclic β-chiral amine metabolites
stereoselective,
and
racemization
via
imine/enamine
tautomerization facilitate high conversions. These reaction are
[a]
Institute of Biochemistry, University Greifswald, 17487 Greifswald,
Germany
Philipp Matzel, Sebastian Wenske, Dr. Matthias Höhne
+49 3834 420 4417
Matthias.Hoehne@uni-greifswald.de
Dr. S. Merdivan, Prof. Dr. S. Günther
Institut of Pharmacy,
[b]
University of Greifswald
Friedrich-Ludwig-Jahn-Str. 17, 17489 Greifswald, Germany
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.

10.1002/cctc.201900806
ChemCatChem
COMMUNICATION
festuclavine[12], tetrahydroalstonine[13], and vitrosamine[14], which
are obtained by reducing an intramolecularly formed imine.
reactions with methyl- and ethylamine gave best activities, but
also dimethylamine and pyrrolidine were converted to yield the
corresponding tertiary amine products. The short aliphatic 2-
methylpentanal 4, its methoxy analogue 5, and the cyclic
aldehyde 6 were converted with all amines. Other functional
groups, e.g. a trifluorosubstituent or hydroxygroups in 11 and 12
prevented the reaction. A similar tendency was obtained with β-
phenyl-bearing aldehydes: aldehyde 7 with its methyl branch is
converted (only with IR-SIP), but activity is lost for aldehydes 13
- 15 where the methyl substituent is replaced by a cyano group,
a second aldehyde function, or by a second bulky phenyl group.
Activity is increased, if the phenyl substituent is moved away
from the α-branch by one methylene group (aldehyde 8).
Interestingly, the 2-methyl substituent of 8 increased activity of
imine reduction with IR-Sip compared to the linear 3-
phenylpropanal 3 up to 6-fold, but activity declines when the
saturated cyclohexyl group replaces the phenyl moiety in 9. The
more bulky aldehyde 10 is only converted by IR-SIP, and
interestingly shows the highest activities in the whole screening
when combined with methyl- or ethylamine as nucleophiles (1.4
and 1.1 U/mg). In contrast to our prediction, IR 32 and 33 were
less active for most substrate combinations, but gave best
activities in the amination of 4-6 with dimethylamine and
pyrrolidine.
Because a variety of pharmaceuticals represent acyclic β-chiral
amines (Scheme 1), we wanted to explore the potential of
IREDs for intermolecular reductive amination by dynamic kinetic
resolution of α-branched aldehydes (Scheme 2). A highly
enantioselective enzyme is required for a successful dynamic
kinetic resolution. In addition, the reaction needs to be carried
out under conditions that allow the remaining enantiomer to
racemize, e.g. via keto-enol tautomerism of the non-consumed
aldehyde enantiomer.
Imine formation
R3–NH2
Imine reduction
IRED
R1
R3
R1
R1
R3
N
H
N
N
O
R2
R2
R2
NADPH NADP+
GDH
H2O
R3–NH2
R
OH Racemisation
R'
Cofactor
regeneration
R1
R1
R3
O
R2
R2
glucose gluconic acid
H2O
Scheme 2. Dynamic kinetic resolution to β-chiral amines applying IREDs. For
the IREDs employed in this study we assume that imine formation occurs in
the reaction solution outside of the enzyme, and imine reduction takes place in
the enzyme’s active site.
Ammonia
Methylamine
Ethylamine Dimethylamine Pyrrolidine
a
b
c
d
e
Aldehyde
IRED: 22 32 33 Sip 22 32 33 Sip 22 32 33 Sip 22 32 33 Sip 22 32 33 Sip
15]
In previous work we[11] and others[10a,
demonstrated that
190
15
20
0
253
12
83
403
299
153
0
0
139
15
17
50
38
45
0
163
0
0
0
0
0
0
0
0
0
0
0
408
34
31
78
98
71
6
246
120
124
149
54
108
54
103
106
547
54
100
361
232
27
0
147
457
154
simple achiral aldehydes are converted by IREDs and reductive
aminases[10a] (RedAms; this subgroup of IREDs catalyze imine
formation directly in the active site rather than using the pre-
formed imine out of the reaction solution and are hence
especially efficient in reductive amination because they require
only stoichiometric amounts or a small excess of the amine
nucleophile substrates). Employing the IRED from Streptomyces
ipomoeae, aliphatic aldehydes are converted with similar
efficiency or outperform the reaction with the model substrate
cyclohexanone with some amine nucleophiles such as
pyrrolidine. Additionally, the RedAm from Aspergillus oryzae
shows activity for benzenepropanal and benzaldehyde.[10a]
Based on previous results, we selected four IREDs due to their
different substrate specificities: IR-22 (Streptomyces sp.)
accepts larger imines than those constructed with methylamine,
and IR-Sip (Streptomyces ipomoeae) demonstrated activity in
reactions with a range of secondary amines and has shown to
be a useful catalyst for preparative reactions in our lab
previously.[12] IR-32 and IR-33 (both from Aeromonas veronii)
convert acetophenone–a ketone having a carbon branch (the
aromatic ring) at the α-position. Therefore we assumed that
these enzymes might be suitable for reactions with α-branched
aldehydes as well.
1
O
O
0
0
0
0
0
153
0
0
0
0
0
16
16
0
2
3
4
O
0
0
0
0
29
35
27
34
0
113
0
0
26
55
46
63
0
18
15
69
12
74
34
18
0
176
41
51
0
10
17
28
291
100
230
208
719
207
41
60
0
81
113
0
291
141
O
O
O
0
0
0
0
60
128
0
0
82
164
5
O
0
0
62
211
28
0
97
110
6
7
8
9
0
0
0
0
0
0
123
88
0
1
1
10
30
0
258
109
46
15
O
0
0
0
47
45
28
18
0
33
573
35
6
3
30
33
O
0
0
0
0
0
0
0
0
0
121
0
0
0
0
0
O
O
O
0
5
0
69
0
7
5
0
4
0
0
0
37
0
5
4
10
O
0
20
40
60
80
100
120
140
160
180
200
220
300
350
400
450
500
Sp. Activity [mU/mg] 0 20 40
80
120
160
200
300
400
500 >1000
O
O
O
F3C
HO
O
O
Cl
Non-reactive:
OH
O
CN
12
11
13
14
15
Figure 1. Heat map representing the specific activities of four IREDs in the
conversion of (mainly) α-branched aldehydes. Activities were determined in a
photometric screening. The reaction was composed of 200 mM of the desired
amine substrate (also serving as the buffering agent) at pH 9.5 , 1 mM
aldehyde substrate, NADPH (0.5 mM) and purified enzyme (1 mg/mL or
diluted if neccessary) and measured at 340 nm and 30°C for 10 min. The
signal from the control reaction (without enzyme) was subtracted prior to
activity calculations. We also performed control reactions without the amine
substrate and ruled out that the IREDs reduced the aldehyde substrates to
from the corresponding alcohols.
For our screening, we chose a collection of 15 aldehydes
(Figure 1), where the majority will lead to β-chiral amine products.
They reflect the structural diversity found in physiologically
active compounds (compare with Scheme 1). Pleasingly, with
several of the α-branched aldehydes we noted enzyme activity
in our screening: from out of 75 combinations, for 50 at least
one enzyme showed activity >20 mU/mg. Regarding the
preference for particular amine nucleophiles, we observed a
similar trend in our screening compared to the pattern found in
reductive amination of ketones: ammonium was less tolerated,
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10.1002/cctc.201900806
ChemCatChem
COMMUNICATION
The next step was to conduct exemplary biocatalytic
reactions to investigate conversion and enantiomeric excess of
the produced amine. For the five amines 4b, 7b, 8b, 10b, 10c
we were able to obtain separation of the enantiomers by chiral
GC/HPLC. In addition we investigated the methylamination of
aldehyde 16 (Scheme 3), which is a precursor to the fungicides
fenpropidine and fenpropimorph. We selected IR-Sip, because it
showed the highest activities for most of the substrate
combinations, except for amine 4b, where we employed IR-22.
20 mM of the aldehyde substrate was reacted with 10 equiv. of
the amine nucleophile at pH 9.5, which on the one hand is
optimal for the enzymatic reaction and additionally will promote
the racemization of the aldehyde substrates, as observed
previously.
with methylamine. The obtained optical purities varied greatly:
Product 4b, 8b, 10c and 16b showed only a low to moderate
enantiomeric excess, but for 7b and 10b good and excellent
enantiomeric purities of 78%ee and >95%ee were observed.
The last two examples clearly demonstrate the proof of concept
for a dynamic kinetic resolution: As IR-Sip catalyzes these imine
reductions with high enantioselectivity, conversions of >50%
would not be possible without the concurrent racemization.
Furthermore, we synthesized amines 4b and 10b at preperative
scale. To avoid substrate and product inhibitions, we chose a
reaction volume to obtain a maximum product concentration at
quantitative conversion of 100 mM, and employed 500 mg of 4
and 1 g of 10, which we added in four batches during the
reaction. 4b was isolated with 56% yield (86%c, 28%ee) and
81% yield was obtained after quantitative conversion of 10
(Table 2).
Table 1. Imine reductase-catalyzed stereoselective reductions to β-chiral
amines. Reaction conditions: 1 mL reaction volume, 200 mM amine, pH
adjusted to 9.5, 20 mM aldehyde, 0.5 mg/ml IRED (IR-Sip, IR-22 for product
4b), 0.5 mM NADPH, 0.1 mg/ml GDH, 60 mM glucose, 24 h at 30°C and 800
rpm. Enantiomeric excesses were determined for product 4b, 7b, 8b, 10b, 10c,
16b, 16f, 16g via GC and products 4b, 10b, 16f, 16g were isolated (for details
see experimental section).
Finally, we wanted to apply IR-Sip for industrially relevant
compounds. The two fungicides fenpropidine 16f and
fenpropimorph 16g (Scheme 2) were selected as suitable
candidates, due to the high conversions observed with
aldehydes 8 and 10. These fungicides are currently applied in
their racemic form, although the (S)-enantiomer is the
physiologically active one.[16] Most of the reported chemical
syntheses are either very efficient but yield the racemic
compounds[17], or multistep procedures to obtain the desired
enantiomers[6, 18]. When the p-tert-butyl derivative of aldehyde 8
was combined with methylamine, it gets converted nearly
quantitatively in reactions with IR-Sip to give amine 16b.
However, in combination with the more challenging amines
piperidine f or (2S,6R)-2,6-dimethylmorpholine g, we observed
only very slow product formations under prolonged reaction
times: after 96h, 9% of 16f was detected compared to 6% of 16g.
While the conversion of fenpropimorph stagnates at 6%, 16f
increased to 13% after 144 h. As the more bulky amine g
caused lower conversions compared to amine f, we speculate
that the low conversions are due to size limitations of the active
site when accommodating the intermediate imines. Both
products were isolated and verified by 1H-NMR (yield
fenpropidine: 8%, fenpropimorph: 3%, see SI).
Product
Conv. [%]
ee [%]
Yield [%]
4b[a]
7b
86
28
56
>95
>95
>95
78
51
-
-
8b
10b[a]
>95
81
In summary, we were able to give a proof of concept that
IREDs can be applied for reductive amination by dynamic kinetic
resolution of β-branched aldehydes to synthesize secondary β-
chiral amines. Amines 7b and 10b were formed with full
conversion and a good to excellent ee, showing that the method
is useful to apply IREDs in the synthesis of pharmaceutically
relevant compounds: amine structures constructed from
aldehyde 10 and various cyclic amines showed potential as
somatostatin receptor antagonists. Compared to previously
reported chemo-catalytic reductive aminations of aldehydes, our
approach is the first example that uses simple alkyl amines
directly as nitrogen source, rather then aromatic amines such as
p-methoxyaniline, and thus represents the shortest route to
directly access secondary β-chiral amines. However, our study
also showed that the employed IR-Sip is not an universal
catalyst with equal selectivity for different imines: biocatalytic
reactions employing similar compounds were not as successful
and resulted in lower conversions and/or lower enantiomeric
purities. This is especially evident when combining aldehyde 10
with methyl or ethyl amine: The enantiomeric purity drops from
>95%ee to 63%ee with the larger amine substrate. Similar
10c
16b
>95
>95
63
31
-
-
16f[b]
16g[b]
13
6
-
-
8
3
[a] Conditions: 50 mL reaction volume, initial aldehyde concentration: 25 mM,
additional addition of 1.25 mmol (25 mM) substrate for three times after 24 h
each, 0.2 M methylamine, 0.25 M CHES buffer 0.25 M pH 9.5, 0.2 M glucose.
[b] Conditions: 10 mM aldehyde of 10 mM, 100 mL reaction volume, reaction
time: 144h.
After 24 h, reactions with aldehyde 7 and 8 with methylamine,
and 10 with methyl or ethylamine showed full conversion
(Scheme 3). Aldehyde 4 was also converted efficiently (86%)
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10.1002/cctc.201900806
ChemCatChem
COMMUNICATION
The enantiomers were then separated on a Chiralpak AD-H column from
Daicel Chiral Technologies.
results have been obtained in approaches using amine
transaminases: these catalysts are known to feature excellent
stereoselectivities for a large range of α-chiral amines[19], but
when investigated for synthesizing β-chiral amines, many wild-
type
amine
transaminases
showed
only
modest
Acknowledgements
enantioselectivities.[9a] Besides enantioselectivity, activity and
catalyst stability have also to be improved, especially regarding
the synthesis of bulk products such as fenpropimorph and
fenpropidine. We expect that protein engineering[20] will also help
to overcome limitations of IRED-mediated catalysis to β-chiral
amines, as this single-step route represents the most atom
economic and direct approach to this class of molecules and has
a great potential to replace the current asymmetric multi-step
synthesis.
We would like to thank Gabriele Thede, Ina Menyes and
especially the Enzymicals AG for analytical support. We thank
Lukas Krautschick for his experimental assistance and Prof. Dr.
Uwe Bornscheuer for his continuous support and inspiring
discussions.
Keywords: Imine Reductase • β-chiral Amine• Biocatalysis •
Fungicide • Dynamic Kinetic Resolution
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[2]
a) M. Breuer, K. Ditrich, T. Habicher, B. Hauer, M. Kesseler, R.
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Schrittwieser, S. Velikogne, W. Kroutil, Adv Synth Catal 2015, 357,
1655-1685.
Experimental Section
Photometric Screening
The specific activity of the investigated IREDs was determined by the
photometric NADPH Assay. The cofactor NADPH is oxidized during the
reaction. In contrast to the reduced form, NADP+ does not absorb at 340
nm, thus a decrease of absorption can be used for calculation of activity.
The reactions contained: amine-buffer (200 mM, pH 9.5), aldehyde
substrate (1 or 10 mM dependent on solubility, added as a 100 mM
solution in methanol) and NADPH 0.5 mM. The reaction was measured
over 10 min at 30°C and the enzyme concentration was adjusted in
accordance to the activity. Similar to our published assay procedure,[11]
we performed control reactions where either the enzyme, NADPH, amine
or aldehyde was replaced by buffer. The signals of the controls without
cofactor, amine or aldehyde were similar to that of the background
oxidation of NADPH (control without enzyme). Thus, this value was
subtracted from all of the reactions and the controls confirmed that the
decline of the NADPH concentration is not caused by side reactions such
as reduction to the alcohol.
a) D. Enders, H. Schubert, Angew Chem Int Edit 1984, 23, 365-
366; b) F. Colpaert, S. Mangelinckx, N. De Kimpe, J. Org. Chem.
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S. Gu, S.-F. Zhu, Q.-L. Zhou, ACS Catal. 2018, 8, 7351-7355.
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[6]
[7]
Biocatalytic Reaction:
Biocatalytic reactions were performed for elected substrate combinations.
The reactions contained: 200 mM amine buffer pH 9.5, 20 mM aldehyde,
0.5 mg/ml IRED, 0.5 mM NADPH, 0.1 mg/ml GDH, 60 mM glucose and
were incubated for 24h at 30°C and 800 rpm. Samples of 200 µL were
then extracted with 260 µl of DCM after adding 40 µL of 10 M NaOH.
Finally, the extracted samples were analyzed by GC-MS using the
column BPX5 from SGE and the ee was determined as described below.
[8]
[9]
Z.-K. Xue, N.-K. Fu, S.-Z. Luo, Chin. Chem. Lett. 2017, 28, 1083-
1086.
Preparative Syntheses and Product Isolation
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E. Scott, G. R. Humphrey, J. Limanto, D. C. Bachert, K. M.
Emerson, Org. Proc. Res. Dev. 2013, 18, 215-227; b) C. S. Fuchs,
J. E. Farnberger, G. Steinkellner, J. H. Sattler, M. Pickl, R. C.
Simon, F. Zepeck, K. Gruber, W. Kroutil, Adv Synth Catal 2017,
360, 768-778.
Product 4b and 10b were synthesized by continuous substrate feeding.
The total volume of the reaction was set to 50 ml with an initial substrate
concentration of 25 mM, methylamine 0.2 M, CHES buffer 0.25 M pH 9.5,
glucose 0.2 M, NADPH 0.5 mM and an enzyme load of 0.5 mg/ml. Three
times, 1.25 mmol substrate were added every 24 h, the overall reaction
time was 144 h. At several time points samples were taken and analyzed
by GC-MS, the datapoints are shown in the supporting information. The
fungicides 16f and 16g were synthesized with less substrate
concentration (10 mM) but 100 ml reaction volume: The products were
isolated by extraction: due to the de- and protonation of amines by basic
or acidic pHs the significant change of the polarity of the products was
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10.1002/cctc.201900806
ChemCatChem
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This article is protected by copyright. All rights reserved.

10.1002/cctc.201900806
ChemCatChem
COMMUNICATION
COMMUNICATION
NADPH + H+
NADP+, H2O
Philipp Matzel, Sebastian Wenske,
Simon Merdivan, Sebastian Günther and
Matthias Höhne*
Reductive
Amination
CH3
R
+
R
R
N
O
+
O
H2N
H
Imine reductase
OH
Page No. – Page No.
Racemization
Synthesis of β-Chiral Amines by
Dynamic Kinetic Resolution of α-
Branched Aldehydes Applying Imine
Reductases
R
From racemic aldehydes to optically active secondary amines via DKR: We
demonstrated the proof of concept for enzymatic β-chiral secondary amine
synthesis via dynamic kinetic resolution (DKR) employing imine reductases.
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