Biocatalytic N-Alkylation of Amines Using Either Primary Alcohols or Carboxylic Acids via Reductive Aminase Cascades

Article abstract of DOI:10.1021/jacs.8b11561

The alkylation of amines with either alcohols or carboxylic acids represents a mild and safe alternative to the use of genotoxic alkyl halides and sulfonate esters. Here we report two complementary one-pot systems in which the reductive aminase (RedAm) from Aspergillus oryzae is combined with either (i) a 1° alcohol/alcohol oxidase (AO) or (ii) carboxylic acid/carboxylic acid reductase (CAR) to affect N-alkylation reactions. The application of both approaches has been exemplified with respect to substrate scope and also preparative scale synthesis. These new biocatalytic methods address issues facing alternative traditional synthetic protocols such as harsh conditions, overalkylation and complicated workup procedures.

Full text of DOI:10.1021/jacs.8b11561

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Communication
Biocatalytic N-Alkylation of Amines using either Primary
Alcohols or Carboxylic Acids via Reductive Aminase Cascades
Jeremy Ramsden, Rachel S. Heath, Sasha Derrington, Sarah Louise
Montgomery, Juan Mangas-Sanchez, Keith R. Mulholland, and Nicholas J Turner
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b11561 • Publication Date (Web): 02 Jan 2019
Downloaded from http://pubs.acs.org on January 3, 2019
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Journal of the American Chemical Society
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Biocatalytic N-Alkylation of Amines using either Primary Alcohols or
Carboxylic Acids via Reductive Aminase Cascades
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Jeremy I. Ramsden1, Rachel S. Heath1, Sasha R. Derrington1, Sarah L. Montgomery1, Juan Mangas-
Sanchez1, Keith R. Mulholland2 and Nicholas J. Turner1*
1School of Chemistry, University of Manchester, Manchester Institute of Biotechnology, 131 Princess Street, Manchester,
M1 7DN, United Kingdom.
2Chemical Development, AstraZeneca, Silk Road Business Park, Macclesfield, SK10 2NA, United Kingdom.
ABSTRACT: The alkylation of amines with either alcohols or carboxylic acids represents a mild and safe alternative to the use of
genotoxic alkyl halides and sulfonate esters. Here we report two complementary one-pot systems in which the reductive aminase
(RedAm) from Aspergillus oryzae is combined with either (i) a 1o alcohol/alcohol oxidase (AO) or (ii) carboxylic acid/carboxylic
acid reductase (CAR) to effect N-alkylation reactions. The application of both approaches has been exemplified with respect to
substrate scope and also preparative scale synthesis. These new biocatalytic methods address issues facing alternative traditional
synthetic
protocols
such
as
harsh
conditions,
over-alkylation
and
complicated
work-up
procedures.
Primary and secondary amines constitute important classes of
compound for the chemical industry, with applications ranging
from surface coatings, resins, polymers, dyestuffs, resins as
well as building blocks for the synthesis of pharmaceuticals
and agrochemicals. The two most widely employed methods
for their preparation involve either N-alkylation or reductive
amination; however, these methods are far from ideal and have
problems associated with their use.1–4 Alkylating agents (e.g.
alkyl halides, sulfonate esters) are highly genotoxic, and their
application is further limited by poor chemoselectivity due to
the formation of byproducts resulting from over-alkylation.
Reductive amination of carbonyl compounds with primary
amines requires an excess of the amine reagent in order to
prevent over-alkylation which lowers the efficiency of the
process and complicates work-up procedures. Both methods
also utilize highly reactive and often non-renewable
feedstocks.
Aldehydes usually need to be prepared in situ and are unstable
to aerobic oxidation and other side reactions including
condensation and aldol type processes. These limitations have
prompted the recent development of synthetic methodologies
that employ alcohols and carboxylic acids as surrogate
reagents for amine alkylation. The use of alcohols requires
homogenous transition-metal catalysis, in an overall redox
neutral transformation, where water is the sole by-product.
This approach, termed “chemocatalytic hydrogen borrowing”,
involves the in situ activation of an alcohol to its
corresponding carbonyl compound prior to imine formation
and subsequent reduction to yield the amine (Figure 1a).5–9
The ‘hydrogen borrowing’ principle has been exploited using
both homogeneous and heterogeneous10 catalysis and has
recently been applied on kilogram scale by Pfizer in the
synthesis of an API for the treatment of schizophrenia.11
(a) Established Chemical Methods
(b) This Work
Chemical "Borrowing Hydrogen"
O
O
R2
Oxidase
R1
N
H
R1 OH
R1
H
H
O2
30oC
R2NH2
[Ru] / [Ir]
>110oC
Sustainable
Renewable
Feedstocks
O
R2
Reductive Aminase
R1
N
R2NH2
NADPH
30oC
R1
H
H
Carboxylic Acid
Reductase
Carboxylic Acid Reductive Amination
O
R2
R1
N
H
R1
R1 OH
R2NH2
[Ru], H2
160oC
ATP, NADPH
30oC
in situ Aldehyde
in situ Aldehyde
Generation
Generation
Figure 1: A comparison of (a) previously reported chemocatalytic methods of amine alkylation using sustainable feedstocks via in
situ aldehyde generation with (b) the one-pot biocatalytic cascades demonstrated in this work.
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Despite the favorable atom economy of these reactions, their
Page 2 of 7
Herein we present two new complementary methods for the
direct biocatalytic N-alkylation of amines through the simple
one-pot combination of AspRedAm with either an engineered
choline oxidase variant (AcCO6)27 or the WT carboxylic acid
reductase from Segniliparus rugosus (CARsr) (Figure 1b).28
The ability of these enzymes to generate aldehydes
irreversibly allows for N-alkylation to be achieved using either
alcohol or carboxylic acid substrates in high conversion and
isolated yield.
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green credentials are somewhat limited by requirements for
high temperatures, long reaction times and precious metal
catalysts, which may be poisoned by either the substrate or
product amine (or both). Carboxylic acids represent attractive
alternatives as reagents for alkylation reactions in view of their
availability from renewable feedstocks. However, implicit in
their use is the selective reduction to aldehydes for application
in the alkylation of amines. Two complementary methods
utilizing ruthenium catalysis have been reported by Beller et
al.12–14 and a metal free method using a boron catalyst was
reported by Fu et al.15 These approaches elegantly demonstrate
the concept of alkylation of amines with carboxylic acids
although their suitability for large scale application may be
limited by the requirement for hydrogen gas or silanes as a
hydride source.
In order to establish the basis for a combined oxidase/RedAm
coupling of amines and alcohols, we initially examined the
AspRedAm mediated reductive amination of several aldehyde
substrates which can be generated from the corresponding
primary alcohol using the recently reported engineered choline
oxidase variant AcCO6.31 The results of these
biotransformations (see Supporting Information) demonstrated
a clear overlap between the product scope of the oxidase and
the substrate scope of the reductive aminase. Although all
aldehydes tested proved to be substrates for AspRedAm, a side
reaction was observed in which some of the aldehyde was
oxidized to the corresponding carboxylic acid in up to 10%
conversion, likely as a result of stirring under an O2
atmosphere.
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In recent years, the principles of green chemistry have been
increasingly applied to chemical synthesis and process
design,16 leading to the emergence of enzymes as alternative
catalysts due to their ability to perform a wide range of
transformations with outstanding selectivity under benign
conditions. Biocatalysis also represents a key enabling
technology in the pursuit of more sustainable synthesis, with
the inherent ability of enzymes to better utilize natural
feedstocks providing more opportunities for renewable
synthesis as manufacturing seeks to transition from non-
renewable fossil reserves to a bio-based economy.17
Next we examined the direct coupling of amines 1a-8a and
alcohols i-v using a combination of AcCO6 and AspRedAm
together with NADPH (cat.) and glucose/GDH for in situ
cofactor recycling (Table 1). Purified AcCO6 and AspRedAm
were applied at 1 mg mL-1 with the alcohol concentration kept
constant (10mM) whilst amine loading was varied (20-
100mM). In general high conversions (>99%) were obtained
using only 2 equiv. of amine. Where quantitative conversion
was achieved at lower amine loadings, higher concentrations
were not examined. Previously we have observed that for
AspRedAm catalyzed reductive amination reactions, an
increase in amine concentration generally leads to higher
conversion to product.24 However, this phenomenon was not
generally observed in this cascade, possibly due to inhibition
of the oxidase AcCO6 at higher amine loadings. In fact the
conversions obtained were typically higher those achieved
from direct AspRedAm mediated reductive amination with the
equivalent aldehyde, presumably as a result of elimination of
the aerobic oxidation side reaction by in situ formation of the
aldehyde. Benzyl alcohol 7a gave lower conversions with
formation of the conjugated imine often observed.
Several different classes of enzyme have demonstrated
potential as biocatalytic tools for amine synthesis including
transaminases (TAs)18, amine dehydrogenases (AmDHs)16,
Pictet-Spenglerases17, cytochrome P411s18, phenylalanine
ammonia lyases19, amine oxidases20–22, and imine reductases
(IREDs).22,23
We have previously reported the
characterization of a reductive aminase from Aspergillus
oryzae (AspRedAm).24 AspRedAm was shown to be capable of
not only catalyzing imine reduction, but also the more
challenging step of imine formation through condensation of
amine and carbonyl substrates in aqueous media,25
highlighting the potential of this biocatalyst and other related
members of the sub-class to be developed further for synthetic
application.
We, and others, have also previously described the bio-
catalytic amination of alcohols through one pot enzymatic
systems that mimic chemocatalytic hydrogen borrowing, in
which two complementary oxidoreductases perform hydrogen
autotransfer through a nicotinamide cofactor. In these studies
the conversion of alcohols to primary amines was achieved by
the coupling of alcohol dehydrogenases (ADH) with
AmDHs26,27 and a redox self-sufficient combination of an
ADH, TA and alanine dehydrogenase (AlaDH).28 Recently, we
reported a system in which AspRedAm was coupled with a
panel of non-enantioselective ADHs to demonstrate the first
example of primary amine alkylation directly from alcohols
using a biocatalytic hydrogen borrowing system.29 However,
while these systems allow for redox neutral amine synthesis,
the reversibility of both the ADH and AspRedAm enzyme
catalyzed reactions leads to conversions that ultimately are
We next examined a complementary cascade using carboxylic
acids as substrates instead of alcohols (Table 2). CARsr was
selected, due to its wide substrate scope, and combined with
AspRedAm together with glucose/GDH and ATP. As in
previous work involving CAR mediated cascades,33–36 the
enzyme was initially as applied as a lyophilized whole cell
biocatalyst. However,
a significant amount of alcohol
byproduct was observed from over-reduction of the carboxylic
acid, likely due to endogenous reductase enzymes in E. coli
intercepting the aldehyde intermediate. Therefore CARsr was
instead applied as a purified enzyme alongside a previously
described
ATP
recycling
system
(AdK,
PAP,
polyphosphate).37 Three carboxylic acid substrates 1b, 6b, 8b
were tested with three amine donors i, ii and v. AspRedAm
was again applied at 1 mg mL-1 and the other enzymes at a
concentration of 0.25 mg mL-1. For this cascade, both acid and
amine loading were kept constant at 10 mM and 20 mM
respectively, whilst glucose concentration was increased to
limited by reaction thermodynamics. This leads to
a
requirement for a large excess of amine substrate and is more
pronounced in the synthesis of secondary amines, where the
equilibrium is less favourable.30
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100mM to reflect the requirement for two glucose
dehydrogenase turnovers for each alkylation reaction.
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2
3
4
Table 1. Biocatalytic N-alkylation of amines using primary alcohols.
5
6
7
8
H2N
R2
A
B
C
= 2 equiv, = 4 equiv, = 10 equiv
O
AcCO6
AspRedAm
H
R1
N
R1 OH
R2
R1
H
9
O2
H2O2
NADP+
NADPH
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Gluconolactone
Glucose
GDH
H2N
H2N
H2N
H2N
H2N
iii
iv
v
i
ii
N
n=4
N
H
N
H
N
H
n=4
n=4
H
n=4
N
n=4
OH
OH
n=4
H
1iii
1v
1iv
1a
1ii
1i
A:
A:
A:
A:
>99% conv.
>99% conv.
A:
A:
>99% conv.
A:
A:
>99% conv.
A:
A:
>99% conv.
N
n=5
N
N
N
n=5
n=5
H
n=5
N
H
H
n=5
H
n=5
H
2iii
2v
2iv
2a
2ii
2i
>99% conv.
>99% conv.
>99% conv.
>99% conv.
>99% conv.
N
n=6
H
N
N
N
n=6
n=6
n=6
N
H
H
OH
OH
OH
n=6
H
n=6
H
3ii
3iii
3v
3iv
3a
A:
B:
C:
89% conv.
78% conv.
70% conv.
3i
A:
A:
>99% conv.
>99% conv.
A:
>99% conv.
A:
>99% conv.
N
n=7
N
N
n=7
H
n=7
H
H
N
n=7
N
H
n=7
n=7
4iii
4iv
H
4ii
A:
B:
C:
4v
69% conv.
96% conv.
98% conv.
A:
B:
C:
88% conv.
93% conv.
>99% conv.
4a
A:
B:
C:
52% conv.
49% conv.
23% conv
4i
A:
>99% conv.
A:
>99% conv.
N
n=8
N
N
n=8
n=8
H
N
H
H
n=8
N
n=8
H
H
n=8
5iii
5v
5ii
5iv
A:
B:
C:
A:
B:
C:
5i
46% conv.
60% conv.
>99% conv.
96% conv.
93% conv.
79% conv.
5a
A:
B:
C:
30% conv.
53% conv.
21% conv.
A:
B:
66% conv.
>99% conv.
A:
89% conv.
>99% conv.
B:
N
H
N
H
N
H
N
H
N
H
OH
6iii
6iv
6i
6ii
6v
A:
B:
C:
A:
B:
C:
0% conv.
0% conv.
0% conv.
40% conv.
67% conv.
76% conv.
6a
A:
A:
A:
>99% conv
>99% conv
>99% conv.
N
H
N
H
N
H
N
H
N
H
OH
7ii
7iii
7v
7iv
7i
A:
B:
C:
A:
B:
C:
A:
B:
C:
38% conv.
74% conv.
>99% conv.
19% conv.
67% conv.
60% conv.
A:
B:
67% conv.
21% conv.
73% conv.
38% conv.
74% conv.
7a
A:
B:
C:
25% conv.
29% conv.
55% conv.
C:
>99% conv.
N
H
N
H
N
H
N
H
N
H
OH
8ii
8iii
8v
8iv
8a
8i
A:
A:
A:
>99% conv.
>99% conv.
>99% conv.
A:
>99% conv.
A:
>99% conv.
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Reaction conditions: 10mM alcohol, 20mM/40mM/100mM amine, 0.1mM NADP+, 50mM glucose, 1 mg mL-1 AspRedAm, 1 mg mL-1
AcCO6, 0.3 mg mL -1 CDX-901 GDH, 2% (v/v) DMSO, 100 mM pH 7.0 KPi buffer, 500 μL reaction volume, 30°C, 250 rpm, 24 h.
1
2
3
4
5
6
7
8
Table 2. Biocatalytic N-alkylation of amines using carboxylic acids.
H2N
R2
2 equiv
AspRedAm
O
O
CARsr
H
R1
N
R2
R1
H
R1 OH
9
NADPH
NADP+
NADP+
ATP
NADPH
AMP
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Gluconolactone
Glucose
Glucose
GDH
Gluconolactone
GDH
AdK
PAP
Polyphosphate
H2N
H2N
H2N
v
i
ii
O
N
n=4
H
N
N
n=4
n=4
H
H
OH
n=4
1i
1ii
1v
1b
97% conv.
97% conv.
>99% conv.
O
N
H
N
H
N
H
OH
6b
6v
(S)-
6i
6ii
(S)-
85% conv.
(S)-
96% conv.
>99% conv.
O
N
H
N
H
N
H
OH
8ii
8v
8i
>99% conv.
8b
96% conv.
>99% conv.
Reaction conditions: 10mM carboxylic acid, 20mM amine, 0.1mM NADP+, 100mM glucose, 100mM MgCl2, 4 mg mL-1 polyphosphate,
1 mg mL-1 AspRedAm, 0.25 mg mL-1 CARsr, 0.25 mg mL-1 Adk, 0.25 mg mL-1 PAP, 0.3 mg mL -1 CDX-901 GDH, 2% (v/v) DMSO, 100
mM pH 7.5 Tris buffer, 500 μL reaction volume, 30°C, 250 rpm, 24 h.
The use of purified CARsr significantly reduced the problem
of over reduction of the intermediate aldehyde to the
corresponding alcohol and resulted in the overall alkylation of
primary amines using carboxylic acids as substrates with
generally high conversions (>95%). In most case crude
reaction mixtures were sufficiently pure to enable further use
of amine products without purification if desired (see
Supporting Information).
yield (49-74%) through straightforward work-up procedures.
In examples where full conversion (>99%) to product was
observed by GC-FID, the reaction media was simply basified
(pH = 12) before extraction into ethyl acetate to yield the
product. In cases where reagents remained present, prior
acidification and extraction was used to remove impurities.
While this work-up procedure is suitable for the alkylation of
small low molecular weight amines, which when used in
excess may be removed under reduced pressure, it cannot
provide pure product when less volatile amines such as
benzylamine ii are used in excess. Therefore, in the
biocatalytic alkylation of benzylamine ii, the alkylating agent
hexanol 1a was used in excess (1.25 equivalents), allowing
pure product to again be obtained through acid-base
extraction. Although with many chemical methods, an excess
of alkylating agent risks byproduct formation through over-
alkylation, the intrinsic selectivity of these enzyme mediated
cascades avoids this issue thereby offering a distinct advantage
in terms of selectivity and mildness of reaction conditions.
Prior to carrying out preparative scale reactions, both cascades
were subsequently optimized with respect to substrate (i.e.
alcohol, carboxylic acid, amine concentration), biocatalyst
loading and form (i.e. crude lysate versus purified enzyme),
addition of co-solvent (DMSO), buffer concentration, cofactor
recycling enzymes (GDH, PAP, AdK), agitation speed and
reaction time. Reactions were performed on a 1 mmol scale
with a reaction volume of either 40 mL (alcohol to amine) or
100 mL (carboxylic acid to amine) (Table 3). For all of these
preparative reactions, high conversions were observed (84-
99%) and product of high purity (>95%) was isolated in good
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1
2
3
4
5
Table 3. Preparative Scale Reactions
6
7
8
9
b
a
H2N
H2N
R2
RedAm
R2
O
H
H
R1
N
R1 OH
or
R1
N
R2
R2
Asp
AcCO6
R1 OH
AspRedAm
CARsr
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Entry Alkylating Agent Amine
Amine Equivalents Product
Conversion (%) Yield (%)
OH
1
2
>99
67
n=4
N
H2N
H2N
n=4
H
N
n=4
OH
H
2
3
4
0.8
4
>99
>99
>99
72
62
70
n=4
n=6
OH
N
n=6
H2N
H
OH
N
H
H2N
4
O
5
6
2
4
>99
84
49
64
N
n=4
H2N
H2N
OH
n=4
H
O
N
n=6
H
OH
O
n=6
N
H
H2N
OH
7
4
93
74
aReaction conditions: 25mM alcohol, 0.2 mM NADP+, 50mM glucose, 1 mL DMSO, 0.2 mg mL-1 AspRedAm, Lysate formed from 3g
E. coli expressing AcCO6, 0.3 mg mL -1 CDX-901 GDH, 100 mM pH 7.0 KPi buffer, 40 mL reaction volume, 30°C, 250 rpm, 24 h.
bReaction conditions: 10mM acid, 0.08 mM NADP+, 100mM glucose, 100mM MgCl2, 4 mg mL-1 polyphosphate, 2 mL DMSO, 0.2 mg
mL-1 AspRedAm, 0.25 mg mL-1 CARsr, 0.25 mg mL-1 Adk, 0.25 mg mL-1 PAP, 0.3 mg mL -1 CDX-901 GDH, 100 mM pH 7.5 Tris
buffer, 100 mL reaction volume, 30°C, 250 rpm, 24 h.
experiments (PDF). This material is available free of charge via
the Internet at http://pubs.acs.org.
In summary we have demonstrated how the recently reported
reductive aminase from Aspergillus oryzae (AspRedAm) can
be combined with either an engineered primary alcohol
oxidase (AcCO6) or a carboxylic acid reductase (CAR) for the
development of two simple one-pot biocatalytic systems in
which N-alkylation of amines may be achieved using either
alcohol or carboxylic acid substrates as alkylating agents
respectively. Both of these novel cascades yield product in
high conversion and allow for isolation of pure product
through simple work-up procedures. The reductive aminase
displays remarkable complementarity with both of these
enzymes with in situ aldehyde generation by both cascades
leading to higher product conversion than direct biocatalytic
reductive amination of aldehyde substrates. The irreversibility
of aldehyde formation also offers a marked advantage over
previous biocatalytic systems reported for N-alkylation that
utilize similar feedstocks.26 The limitations of traditional
chemical approaches such as the requirement for harsh
conditions and over-alkylation are avoided by these systems
and with the recent discovery of new members of reductive
aminase enzyme family38 we envisage further applications of
the methodology reported herein for amine synthesis.
AUTHOR INFORMATION
Corresponding Author
*E-mail: nicholas.turner@manchester.ac.uk
Author Contributions
All authors have given approval to the final version of the
manuscript.
ACKNOWLEDGMENT
This research has received equal funding from The University of
Manchester and AstraZeneca. NJT is grateful to the ERC for the
award of an Advanced Grant (Grant number 742987).
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