Direct Alkylation of Amines with Primary and Secondary Alcohols through Biocatalytic Hydrogen Borrowing

Article abstract of DOI:10.1002/anie.201705848

The reductive aminase from Aspergillus oryzae (AspRedAm) was combined with a single alcohol dehydrogenase (either metagenomic ADH-150, an ADH from Sphingobium yanoikuyae (SyADH), or a variant of the ADH from Thermoanaerobacter ethanolicus (TeSADH W110A)) in a redox-neutral cascade for the biocatalytic alkylation of amines using primary and secondary alcohols. Aliphatic and aromatic secondary amines were obtained in up to 99 % conversion, as well as chiral amines directly from the racemic alcohol precursors in up to >97 % ee, releasing water as the only byproduct.

Full text of DOI:10.1002/anie.201705848

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
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Accepted Article
Title: Direct alkylation of amines with primary and secondary alcohols
via biocatalytic hydrogen borrowing
Authors: Nicholas John Turner, Sarah Montgomery, Juan Mangas-
Sanchez, Matthew Thompson, Godwin Aleku, and Beatriz
Dominguez
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
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201705848
Angew. Chem. 10.1002/ange.201705848
Link to VoR: http://dx.doi.org/10.1002/anie.201705848
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Angewandte Chemie International Edition
10.1002/anie.201705848
COMMUNICATION
Direct alkylation of amines with primary and secondary alcohols
via biocatalytic hydrogen borrowing
[
a]
[a]
[a]
[a]
Sarah L. Montgomery, Juan Mangas-Sanchez, Matthew P. Thompson, Godwin A. Aleku,
[
b]
[a]*
Beatriz Dominguez and Nicholas J. Turner
Abstract: The reductive aminase from Aspergillus oryzae
AspRedAm) has been combined with single alcohol
stereoselectivity. This discovery prompted us to consider the
application of AspRedAm in hydrogen borrowing amination of
alcohols.
(
a
dehydrogenase (either metagenomic ADH-150, an ADH from
Sphingobium yanoikuyae (SyADH), or a variant of the ADH from
Thermoanaerobacter ethanolicus (TeSADH W110A)) in a redox-
neutral cascade for the biocatalytic alkylation of amines using
primary and secondary alcohols. Aliphatic and aromatic secondary
amines have been obtained in up to 99% conversion, as well as
chiral amines directly from the racemic alcohol precursors in up to
>97% ee, releasing water as the only by-product.
Amines are important targets for synthesis and are present in a
myriad of organic compounds ranging from adhesives and
[1,2]
Figure 1. Methods for the asymmetric conversion of alcohols to amines via
biocatalytic hydrogen borrowing.
resins to agrochemicals and pharmaceuticals.
Consequently
the development of new methodologies that enable access to
this class of compound is a major research goal in synthetic
organic chemistry. In particular, the activation of alcohols under
mild conditions for nucleophilic substitution has been identified
We and subsequently others have reported a method for the
conversion of alcohols to primary amines via biocatalytic
[29–31]
hydrogen-borrowing.
An alcohol dehydrogenase (ADH) was
[3]
as a key area for method development. Hydrogen borrowing or
autotransfer is an elegant, redox-neutral approach for the
formation of C-C, C-O and C-N bonds. This strategy has high
atom economy as both oxidation and reduction steps are
combined in a single pot, removing the need for sacrificial
combined with an amine dehydrogenase (AmDH) and catalytic
NAD , establishing proof-of-concept but also highlighting
important challenges for further development. Firstly, the
oxidation of racemic chiral secondary alcohols required the use
of two enantiocomplementary alcohol dehydrogenases (ADHs)
to convert racemic secondary alcohols (Figure 1). Secondly, the
+
[4–9]
reagents that produce significant waste.
chemical methods exist for mediating hydrogen borrowing,
frequent limitation is a lack of stereocontrol during the reductive
While a number of
[10,11]
a
initial system employed
an NADH-dependent amine
dehydrogenase (AmDH) for the amination step and hence is
limited to the formation of primary amines. Herein we describe
the combination of single non-selective, NADPH-dependent
ADHs with AspRedAm to generate redox-neutral cycles for the
conversion of alcohols into primary and secondary amines
[12]
amination step.
Only a handful of examples have been
reported involving enantioselective transformations with modest
[13–15]
enantiomeric excess.
Biocatalysis offers a variety of methods for the asymmetric
synthesis of amines including transaminases (TAs), monoamine
oxidases (MAO-N), phenylalanine ammonia lyases (PALs),
amine dehydrogenases (AmDHs) and imine reductases
(
Figure 2) with water as the sole by-product.
[16–24]
(
IREDs).
These enzymes have also been combined in
cascades to carry out multiple transformations in one pot, often
[
25–
with oxidation and reduction steps taking place concurrently.
.
2
7]
Recently we reported the discovery of a new enzyme, a
reductive aminase from Aspergillus oryzae (AspRedAm),
[28]
which catalyses the reductive amination of a variety of carbonyl
compounds at low amine loadings with good to excellent
[
[
a]
b]
S. L. Montgomery, Dr. J. Mangas-Sanchez, M. P. Thompson, Dr. G.
A. Aleku, Prof. N. J. Turner
School of Chemistry, University of Manchester
Manchester Institute of Biotechnology
Figure 2. ADH/RedAm catalytic cycle for the biocatalytic alkylation of primary
amines with primary and secondary alcohols.
131 Princess Street, Manchester M1 7DN (UK)
E-mail: nicholas.turner@manchester.ac.uk
Dr. B. Dominguez
Johnson Matthey Catalysis and Chiral Technologies
In order to establish the feasibility of the H-borrowing cascade,
we selected a variant of the ADH from Thermoanaerobacter
[32]
28 Cambridge Science Park
ethanolicus (TeSADH W110A)
with known activity for the
Milton Road, Cambridge CB4 0FP (UK)
oxidation of cyclohexanol 1, and examined its amination using
[28]
benzylamine
5
in the presence of AspRedAm.
A 17%
Supplementary information for this article is given via a link at the
end of the document.
conversion to N-benzylcyclohexylamine 12 was observed, which
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Angewandte Chemie International Edition
10.1002/anie.201705848
COMMUNICATION
was significantly lower than for the corresponding conversion for
reductive amination of cyclohexanone with AspRedAm (82%).
To optimise the conversion, the AspRedAm loading (1 – 2 mg
(91%).. Several other amines were then studied with
cyclohexanol 1, benzyl alcohol 2, 3-phenylpropan-1-ol 3 and 1-
hexanol 4. These transformations proceeded in moderate to
high conversion (Table 1), except in the case of 2 where
substrate inhibition of the ADH lowered the conversion. This
problem was alleviated to some degree by running the
biotransformations as biphasic systems with 50% v/v
cyclohexane, which is well tolerated by AspRedAm. However,
TeSADH W110A still showed significantly lower conversions
than ADH-150 and the ADH from Sphingobium yanoikuyae
-
1
+
mL ), amine concentration (20
– 250 mM) and NADP
concentration (0.5 – 3.0 mM) were varied in biotransformations
-
1
also containing 5 mM 1 and 1 mg mL of a different ADH, ADH-
50, with monitoring of the conversion to both cyclohexanone
1
and the amine product 12. The amine concentration showed the
largest effect on conversion to 12, with increases in conversion
of up to 71% with 100 mM of 5 followed by a decline, reaching
negligible conversion at 250 mM amine (Supplementary
Information Figure S1).
[33,34]
(SyADH).
[a]
Table 1. Bioalkylation of amines with cyclohexanol and primary alcohols.
Whilst higher AspRedAm concentrations improved conversions,
-
1
enzyme loadings above
2
mg mL
gave diminishing
improvements in conversion to product, perhaps due to
decreased enzyme stability at higher concentration. The optimal
+
NADP concentration was found to be 1 mM, with higher
concentrations resulting in more ketone but no additional amine
product in the reaction mixture. The optimised reaction
conditions resulted in an improved conversion to amine 12 of
71%.
Next, a time point study was conducted to investigate the
relative rates of the oxidation and reductive amination steps
(
Supplementary Information Figure S2). The concentration of
cylohexanone reached a steady state within one hour, thereafter
being slowly consumed in the reductive amination step. A further
study of different temperatures (20°C and 30°C) and pHs (7.0
and 9.0) indicated that the transformation occured more slowly
at lower temperature and neutral pH but with a less noticeable
decline in rate (Figure 3). The final conversions after 3 days
were remarkably similar, indicating that the optimal conditions
required a compromise between enzyme stability and rate of
reductive amination.
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
pH 7,
20 °C
[b]
[b]
Entry
Product
Conversion (%)
Conversion (%)
(SyADH)
Conversion
[b]
(
ADH-150)
(%) (TeSADH
W110A)
pH 7,
3
0 °C
1
2
3
4
5
6
7
8
9
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
71
88
79
91
95
79
75
63
21
79
60
0
54
92
79
95
99
88
90
58
32
56
69
0
58
90
44
89
95
86
85
16
0
pH 9,
0 °C
2
pH 9,
0 °C
3
0
20
40
60
Time (hours)
10
11
12
13
14
15
16
17
12
6
Figure 3. Comparison of time points for the hydrogen borrowing bioalkylation
of A with 1a catalysed by ADH-150 and AspRedAm under different conditions.
0
95
99
99
99
99
99
83
94
99
96
82
99
96
86
79
The optimised conditions were then applied to a different amine,
propargylamine 6, resulting in 88% conversion to N-
propargylcyclohexylamine 13 with 6% cyclohexanone remaining
in the reaction mixture after 24 hours. Increasing the amine
loading to 250 mM resulted in a slightly improved conversion
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Angewandte Chemie International Edition
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COMMUNICATION
18
19
20
21
22
29
30
31
32
33
96
99
99
92
57
99
95
99
99
74
99
93
99
99
99
9 and 12). In the reaction of alcohol 34 with amine 8, after 24
hours we observed 92% ee in the starting material when
TeSADH was used and 42% ee when using SyADH. This
indicates that SyADH is less enantioselective, although both
ADHs will oxidise both enantiomers of the alcohol.
[
a] Reaction conditions: 5 mM alcohol, 100 mM amine. [b] Conversion to produc tP rior to studying the scalability of the system, further analytical
determined by GC-FID.
scale biotransformations were performed in order to intensify the
process. For the conversion of 1 to N-methylcyclohexylamine 17
Mixtures of products were observed in AspRedAm-catalysed
reductive aminations of 2 – 4 with ammonia 7 and methylamine
(
Table S5), it was observed that concentrations of 1 up to 50
mM were tolerated, but at 100 mM 1 precipitation of one or both
enzymes occurred. Up to 250 mM of 10 could be added with no
10, so these amines were excluded from the cascade screen
(
Table 1). The highest conversions (99%) were observed with 3
+
reduction in conversion. Lower concentrations of NADP were
and 4 using short-chain, linear amines. ADH-150 appears to
have the broadest substrate tolerance overall, although SyADH
and TeSADH W110A gave better conversions in some cases.
also well tolerated (95% conversion with 5 mol%), as were
-
1
decreased enzyme loadings (89% conversion with 0.5 mg mL
ADH-150 and AspRedAm).
[a]
Table 2. Bioalkylation of amines with racemic secondary alcohols.
The preparative conversion of 1 to N-allylcyclohexylamine 15
was then investigated (Supplementary Information Scheme S1,
Table S6). Increasing the amine loading to 0.5 M did not further
improve conversion, thus the conditions from entry 1 were
deemed most promising for scale up, and were replicated on
100 mg scale, resulting in 98% conversion to amine 15 by GC-
FID analysis and an isolated yield of 61%.
In conclusion, the ADH/AspRedAm-catalysed alkylation of
amines with alcohols has enabled the preparation of a wide
range of secondary amines via biocatalytic hydrogen borrowing.
This redox-neutral cascade generates water as the sole by-
product and to our knowledge constitutes the first example of
biocatalytic hydrogen autotransfer for the direct preparation of
secondary amines. The use of chiral secondary alcohols has
permitted access to optically active amines in up to 84%
conversion with ee‘s up to >97%. The results reported here
further demonstrate the potential of reductive aminases
(RedAms) as powerful new biocatalysts for the preparation of
enantiopure amines under environmentally benign conditions.
[b]
Entry
Product
ADH
Conversion (%)
ee (%)
1
2
3
4
5
6
7
8
9
36
36
36
37
37
37
38
38
38
39
39
39
ADH-150
SyADH
8
n.d.
n.d.
n.d.
84
67
9
TeSADH
ADH-150
SyADH
[c]
44 (R)
38 (R)
42 (R)
[
c]
70
52
25
31
41
13
27
44
Acknowledgements
[c]
TeSADH
S.L.M. was supported by a CASE studentship from Johnson
Matthey. J.M.S was funded by grant BB/M006832/1 from the UK
Biotechnology and Biological Sciences Research Council. We
thank the industrial affiliates of the Centre of Excellence for
Biocatalysis, Biotransformations and Biomanufacture (CoEBio3)
for studentships to M.P.T. and G.A.A. N.J.T. also acknowledges
the Royal Society for a Wolfson Research Merit Award.
ADH-150
SyADH
96 (R)
96 (R)
95 (R)
>97
TeSADH
10
11
12
ADH-150
SyADH
>97
TeSADH
>97
[
a] Reaction conditions: 5 mM alcohol, 250 mM amine. [b] Conversions and
Keywords: biocatalysis
cascades • reductive aminase • synthetic methods.
• asymmetric amination • enzyme
enantiomeric excesses determined by GC-FID on a chiral stationary phase. [c]
Enantiomeric excesses determined by HPLC on a chiral stationary phase.
To examine the potential of this new process for the conversion
of racemic secondary alcohols into enantiomerically enriched
amines, 4-phenyl-2-butanol 34 and 1-(4-fluorophenyl)propan-2-
ol 35 were selected as substrates. Under the same conditions
used for the N-alkyation of amines with 1 – 4, initial yields in the
alkylation of allylamine 7 with 35 were relatively low. We
speculate that this could be due to the lower activity of the ADHs
with bulky alcohols. A lower ADH:AspRedAm ratio (1:4) afforded
higher conversions (Table 2). For 34, higher yields were
observed using SyADH (entries 2 and 5), while for 35, the best
results were obtained when TeSADH W110A was used (entries
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S. L. Montgomery, J. Mangas-Sanchez,
M. P. Thompson, G. A. Aleku, B.
Dominguez, N. J. Turner*
Page No. – Page No.
Direct alkylation of amines with
primary and secondary alcohols via
biocatalytic hydrogen borrowing
The reductive aminase from Aspergillus oryzae (AspRedAm) has been
combined with a single alcohol dehydrogenase (either metagenomic ADH-150,
an ADH from Sphingobium yanoikuyae (SyADH), or a variant of the ADH from
Thermoanaerobacter ethanolicus (TeSADH W110A)) in a redox-neutral cascade
for the biocatalytic alkylation of amines using primary and secondary alcohols.
Aliphatic and aromatic secondary amines have been obtained in up to 99%
conversion, as well as chiral amines directly from the racemic alcohol precursors
in up to >97% ee, releasing water as the only by-product.
This article is protected by copyright. All rights reserved.