Adenylation Activity of Carboxylic Acid Reductases Enables the Synthesis of Amides

Article abstract of DOI:10.1002/anie.201707918

Carboxylic acid reductases (CARs) catalyze the reduction of a broad range of carboxylic acids to aldehydes using the cofactors adenosine triphosphate and nicotinamide adenine dinucleotide phosphate, and have become attractive biocatalysts for organic synthesis. Mechanistic understanding of CARs was used to expand reaction scope, generating biocatalysts for amide bond formation from carboxylic acid and amine. CARs demonstrated amidation activity for various acids and amines. Optimization of reaction conditions, with respect to pH and temperature, allowed for the synthesis of the anticonvulsant ilepcimide with up to 96 % conversion. Mechanistic studies using site-directed mutagenesis suggest that, following initial enzymatic adenylation of substrates, amidation of the carboxylic acid proceeds by direct reaction of the acyl adenylate with amine nucleophiles.

Full text of DOI:10.1002/anie.201707918

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Accepted Article
Title: Adenylation Activity of Carboxylic Acid Reductases Enables the
Synthesis of Amides
Authors: Sabine Flitsch, Alexander Wood, Nicholas Weise, Joseph
Frampton, Mark Dunstan, Michael Hollas, Sasha Derrington,
Richard LLoyd, Daniela Quaglia, Fabio Parmeggiani, David
Leys, and Nicholas Turner
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201707918
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10.1002/anie.201707918
Angewandte Chemie International Edition
COMMUNICATION
Adenylation Activity of Carboxylic Acid Reductases Enables the
Synthesis of Amides
Alexander J. L. Wood1, Nicholas J. Weise1, Joseph D. Frampton1, Mark S. Dunstan2, Michael A.
Hollas1, Sasha R. Derrington1, Richard C. Lloyd3, Daniela Quaglia1,4, Fabio Parmeggiani1, David Leys1,
Nicholas J. Turner1, and Sabine L. Flitsch1*
Abstract. Carboxylic acid reductases (CARs) catalyze the reduction
of a broad range of carboxylic acids to aldehydes using the co-
factors ATP and NADPH, and have become attractive biocatalysts
for organic synthesis. Here we exploit our mechanistic
understanding of CARs to expand their reaction scope, generating
biocatalysts for amide bond formation from carboxylic acid and
amine. After reaction engineering, CARs were found to have
amidation activity for various acids and amines. Optimization of
reaction conditions with respect to pH and temperature allowed for
the synthesis of the anticonvulsant ilepcimide with up to 96%
conversion. Mechanistic studies using site-directed mutagenesis
suggest that following initial enzymatic adenylation of substrates,
amidation of the carboxylic acid proceeds via direct reaction of the
acyl adenylate with amine nucleophiles.
composed of three distinct protein domains: the adenylation
domain, which itself is divided into core-domain and
a
subdomain,[7] the peptidyl carrier protein with the bound 4’-
phosphopantetheine (PPant) prosthetic group and a reductase
domain. The adenylation domain activates carboxylic acids by
catalyzing the formation of an acyl adenylate intermediate, at the
expense of ATP.[8] The activated substrate is then attacked by
the thiol of the enzyme-bound PPant group, generating a
thioester intermediate which is transferred to the reduction
domain. Here NADPH is expended to reduce the intermediate to
an aldehyde (Figure 1).[3] CARs have a broad substrate range
and a number of examples from different organisms are now
available, making them attractive for applications in single step
biotransformations,[9-10] as well as enzymatic cascades.[11-13]
Carboxylic acids are common reagents in organic synthesis, and
their interconversion to other functional groups generally
requires activation to reactive acyl intermediates as the key
step.[1] In Nature, there are many enzymes that catalyze
transformations of carboxylic acids using activated esters as
intermediates.[2] An interesting group are carboxylic acid
reductases (CAR)[3-4] which catalyze the reduction of carboxylic
acids to aldehydes via acyl adenylates and thioesters (Figure 1).
Given that both activated esters are shared with other enzyme
pathways, such as those of nonribosomal peptide synthetases
(NRPS),[5] we were interested in exploring the activity of CARs
towards other nucleophiles (Figure 1). Of specific interest were
amine nucleophiles that would lead to amide bond formation
from acid and amine, an important challenge in organic
chemistry.[6]
Figure 1. CARs are composed of three domains: an adenylation domain,
which is divided into a core-domain (A(core)) and a subdomain (A(sub)), a
peptidyl carrier protein (PCP) and a reduction domain (R). CARs catalyze
reduction of the carboxylic acid at the expense of ATP and NADPH via the
production of acyl adenylate and enzyme-thioester intermediates. Both
intermediates on the reaction pathway might potentially be intercepted by
nucleophiles (Nu) such as amines (cartoon adapted from previous work[7]).
Our recent structural work[7] has shown that CARs are
particularly good candidates for this approach: CARs are
[1]
A. J. L. Wood, Dr. N. J. Weise, J. D. Frampton, M. A. Hollas, Dr. S. R.
Derrington, Dr. D. Quaglia, Dr. F. Parmeggiani, Prof. D. Leys, Prof. N.
J. Turner, Prof. S. L. Flitsch*
School of Chemistry & Manchester Institute of Biotechnology
The University of Manchester
131 Princess Street, M1 7DN, Manchester, United Kingdom
E-mail: sabine.flitsch@manchester.ac.uk
Dr M. S. Dunstan
Manchester Centre for Synthetic Biology of Fine and Speciality
Chemicals (SYNBIOCHEM), Manchester Institute of Biotechnology,
The University of Manchester, Manchester M1 7DN, United Kingdom.
Dr. R. C. Lloyd
Dr. Reddy’s Laboratories (EU) Ltd.
410 Cambridge Science Park, Milton Road, Cambridge CB4 0PE UK
Dr D. Quaglia
The three-domain topology of the enzyme suggests that, in
the presence of ATP but the absence of NADPH, the substrate
carboxylic acid might still be activated, yielding the acyl
adenylate or thioester. Both intermediates could potentially react
with an external nucleophile, such as an amine, to generate
amides (Figure 1). Amidation would be of particular interest
because many natural amide synthetases such as NRPS[14] and
ATP-grasp enzymes[15-16] suffer from very narrow substrate
specificity, which limits their applications in biocatalysis.
Alternatively, hydrolases (such as lipases[17] and proteases[18]),
which are commonly used for amide formation,[2] require low
[2]
[3]
[4]
Chemistry Department
Université de Montréal
2900, Edouard-Montpetit, H3C 3J7, Montréal, Canada
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.201707918
Angewandte Chemie International Edition
COMMUNICATION
water systems, such as organic solvents to drive the reaction
towards amide bond formation.[19-22]
which either ATP or enzyme was omitted showed no amide
production. Table 1 shows that conversions of up to 25% were
observed, demonstrating clear evidence of CAR-dependent
amide production. Higher conversions were observed for the
cinnamic acid derivatives (2-3) compared to benzoic acid
derivatives (1,4-6) (Table 1). Investigations of the optimal
temperature of the reactions showed different profiles for the
four CARs (Table S2).
Next, amines other than ammonia were investigated for
amidation of 1, exploring both primary amines 13-14 and
secondary amine 15 (Table 2). Comparison of conversions for
all enzymes showed that amines 13 and 15 were better
reactants than ammonia, but propargylamine 14 only afforded
amide 17 with a conversion of 4% by CARmm.
Four CAR candidates (CARmm from Mycobacterium
marinum,[3] CARni from Nocardia iowensis,[4] CARtp from
Tsukamurella paurometabola,[10] and CARse from Segniliparus
rotundus[23]) were produced recombinantly in E. coli, with the co-
expressed gene for the B. subtilis phosphopantetheinyl
transferase (PPTase) (Sfp)[24], which is required for post-
translational addition of the PPant group.[4] These CARs were
purified (Figure S1) and the amidation of carboxylic acid
substrates 1-6 was tested. Instead of NADPH, ammonia was
added to the biotransformations in excess, with a ratio of 100:1
amine to acid, at neutral pH. However, only trace amounts of
amide were produced under these conditions (Table S1).
Table 2. CARs can generate secondary and tertiary amides 16-18 by reaction
Table 1. CARs can generate a range of primary amides 7-12 from acids 1-6
a
a
of 1 with ATP and amines 13-15.
by reaction with ATP and ammonia.
b
b
Conversion
Conversion
Acid
Amide
CARmm CARni CARtp CARse
Amine
Amide
CARmm CARni CARtp CARse
11%
22%
3%
1%
4%
11%
8%
25%
4%
12%
3%
14%
12%
c
c
c
–
–
–
25% 13%
6%
1%
2%
1%
15%
<1%
1%
8%
10%
13%
2%
3%
3%
8%
7%
2%
8%
a) 1 mM 1, 100 mM 13-15, 100 µg mL–1 purified CAR, 5 mM ATP, 10 mM
MgCl2, 100 mM sodium carbonate buffer, pH 9.0, 30°C (37°C for CARmm,
22°C for CARni), 24 h. b) Conversion determined by HPLC on a non-chiral
phase at 24 h. c) No activity detected.
8%
After establishing that various carboxylic acids and amines
could be used for amide production with this method, the CARs
were tested as biocatalysts for the formation of a commercially-
relevant target amide, ilepcimide 20, shown to possess
anticonvulsant properties.[25] Preparative scale CAR-mediated
production of ilepcimide 20 was carried out at 37°C, using cell
lysate containing CARmm, with 100 mM 15 (19 eq.), 15 mM
ATP, 20 mM MgCl2, 100 mg of 19, affording 25 mg of 20 (19%
yield).
Further optimization using purified CARs at pH 9.0 at 22°C,
30°C, and 37°C resulted in conversions of 71% by CARmm at
30°C, 68% by CARni at 22°C, 32% by CARtp and 7% by
CARse both at 37°C (Table 3). Lastly, conversion of 96% to
ilepcimide 20 by CARmm was achieved at 30°C, pH 9.0 when
the reaction was left for up to 72 h (Table 3). A pH profile was
a) 1 mM 1-6, 100 mM ammonia, 100 µg mL–1 purified CAR, 5 mM ATP, 10 mM
MgCl2, 100 mM sodium carbonate buffer, pH 9.0, 37°C (30°C for CARse), 24 h.
b) Conversion determined by HPLC on a non-chiral phase at 24 h.
Given that amidation might have very different
requirements to the native reduction reaction, a wider range of
conditions was investigated. Firstly, in preliminary work, a pH
range of pH 7.5-10.5 was investigated for the production of
primary amides, with pH 9.0 found to be optimal (Table S1).
Using pH 9.0, formation of primary amides 7-12 was clearly
detected by HPLC and products were identified by comparison
with authentic synthetic standards. Comparatively, controls in
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10.1002/anie.201707918
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COMMUNICATION
also conducted using CARmm for the production of 20 (Figure
S2), which confirmed that pH 9.0 was optimal for this reaction
with 71% conversion. However conversion of 21% could still be
observed at neutral pH, while at pH values higher than 9.0, the
conversion was also lower, with 40% conversion at pH 10.0.
These results suggest that both amine deprotonation and
decreased enzyme stability at higher pH may have an effect on
conversion. The specific activity of CARmm for the production of
20 was shown to be 12.22 ±0.15 mU mg–1 (Figure S3). As with
the other amide products, control reactions without enzyme or
ATP showed no conversion to 20.
and whole-cell systems, where the presence of NADPH would
normally suppress CAR-mediated amidation in favor of
reduction.[12]
A.
a
Table 3. Synthesis of ilepcimide 20 by CAR amidation.
b
Conversion
Temperature
22°C
CARmm
52%
CARni
68%
CARtp
5%
CARse
1%
30°C
71% (96%)c
50%
27%
4%
B.
37°C
17%
48%
32%
7%
a) 1 mM 19, 100 mM 15, 100 µg mL–1 purified CAR, 5 mM ATP, 10 mM MgCl2,
100 mM sodium carbonate buffer, pH 9.0, 22-37°C. b) Conversion determined
by HPLC at 24 h. c) Value determined at 72 h.
Finally, the mechanism of amide bond formation was
explored, in particular addressing the important question
whether the acyl phosphate or the thioester was intercepted by
the amine (Figure 1). To probe this question, the Ser689Ala
variant of CARni[4] (Figure 2A) was generated. This variant lacks
the Ser689 attachment site to the PPant group that is involved in
thioester formation and therefore would only allow the formation
of the acyl adenylate but not the thioester. Conversion to
ilepcimide 20 (79%) was still observed with this variant,
suggesting that adenylation activity alone is sufficient for
amidation, presumably via nucleophilic attack onto the acyl
adenylate (Figure 2B), a reaction which has been observed with
other adenylating and phosphorylating enzymes, including
NRPS and glutamine synthetase.[26-30] Our current results are
not able to establish if this reaction is enzyme catalyzed.
Figure 2. A) CARs are bound to a PPant group at serine (Ser689 on CARni or
Ser685 on CARmm) which is replaced with an alanine in mutant CARni
Ser689Ala. CARmm729-1175 lacks the reduction domain (images adapted
from previous work[7]). B) Mechanism proposed for the amidation reaction.
In conclusion, we have shown that in the absence of
NADPH, the adenylation activity of CARs can be exploited for
amide bond formation. The reaction was tolerant to a range of
carboxylic acids and amines and could be performed in an
aqueous medium using ATP as the driving force. The method
was applied to a target drug molecule, ilepcimide 20 with up to
96% conversion and preparative scale-up. Given that all four
CARs tested exhibited this activity, the range of enzymes which
could be used for this amidation reaction might be rapidly
expanded by tapping into the collection of known CAR enzymes
with differing and broad substrate specificities.[9-10] While our
method is limited by the use of the expensive co-factor ATP, this
may be overcome in future by established ATP regenerating
systems.[31] Two CAR variants were generated by the rational
redesign of the CARs, which possess no reduction activity but
retain amido synthetase activity. The success with these
To demonstrate that the reduction domain was not
required for amidation, and to remove the wild-type reduction
activity of the enzyme, a truncated form of CARmm was
constructed, which had been described before.[7] This construct
lacks the terminal reduction domain but retains the adenylation
domain and carrier protein (CARmm729-1175) (Figure 2A). In
the amidation assay, this variant gave a conversion of 69% to 20. variants suggests that the adenylation activity alone is sufficient
It should be noted that both the CARni mutant and the
CARmm truncation still retain amido synthetase activity, but
have lost reductase activity (even in the presence of NADPH),
and therefore present a new class of amido synthetase
biocatalysts with no residual CAR activity, i.e. a full switch of
activity. These variants might have application as amidation
catalysts in more complex enzyme cascades, both in cell-free
for activation of carboxylic acids for subsequent amidation, and
also lays the foundations for future work using this method in
more complex enzyme systems, such as in enzyme cascades or
whole-cell biotransformations.
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Keywords: • amides • amidation • carboxylic acid reductase •
biocatalysis • amido synthetase
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10.1002/anie.201707918
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COMMUNICATION
Entry for the Table of Contents
Layout 1:
COMMUNICATION
Carboxylic acid reductases can be
used as biocatalysts for the activation
and subsequent amidation of a range
of carboxylic acids with ammonia,
primary and secondary amines. The
enzymes were used for the synthesis
of ilepcimide.
Alexander J. L. Wood, Nicholas J.
Weise, Joseph D. Frampton, Mark S.
Dunstan, Michael A. Hollas, Sasha R.
Derrington, Richard C. Lloyd, Daniela
Quaglia, David Leys, Nicholas J. Turner
and Sabine L. Flitsch*
Page No. – Page No.
Adenylation Activity of Carboxylic
Acid Reductases Enables the
Synthesis of Amides
This article is protected by copyright. All rights reserved.