Biocatalytic Synthesis of Chiral N-Functionalized Amino Acids

Article abstract of DOI:10.1002/anie.201806893

N-Functionalized amino acids are important building blocks for the preparation of diverse bioactive molecules, including peptides. The development of sustainable manufacturing routes to chiral N-alkylated amino acids remains a significant challenge in the pharmaceutical and fine-chemical industries. Herein we report the discovery of a structurally diverse panel of biocatalysts which catalyze the asymmetric synthesis of N-alkyl amino acids through the reductive coupling of ketones and amines. Reactions have been performed on a gram scale to yield optically pure N-alkyl-functionalized products in high yields.

Full text of DOI:10.1002/anie.201806893

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Accepted Article
Title: Biocatalytic Synthesis of Chiral N-Functionalized Amino Acids
Authors: Julia Hyslop, Sarah L Lovelock, Peter Sutton, Kristin K Brown,
Allan J. B. Watson, and Gheorghe-Doru Roiban
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201806893
Angew. Chem. 10.1002/ange.201806893
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10.1002/anie.201806893
Angewandte Chemie International Edition
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Biocatalytic Synthesis of Chiral N-Functionalized Amino Acids
Julia F. Hyslop,[a,b] Sarah L. Lovelock,[b,c] Peter W. Sutton,[d,e] Kristin K. Brown,[f] Allan, J. B. Watson,*[a,g]
and Gheorghe-Doru Roiban*[b]
Abstract: N-Functionalized amino acids are important building
blocks for the preparation of diverse bioactive molecules including
peptides. The development of sustainable manufacturing routes to
chiral N-alkylated amino acids remains a significant challenge in the
pharmaceutical and fine chemical industries. Herein we report the
discovery of a structurally diverse panel of biocatalysts which
catalyze the asymmetric synthesis of N-alkyl amino acids via the
reductive coupling of ketones and amines. Reactions have been
reductive amination strategies have also been reported[6]
including biomimetic routes,[7] however they are limited by their
narrow substrate range, the use of heavy metals, and the
requirement to pre-form imine intermediates to avoid undesired
ketone reduction.[5a] Biocatalytic approaches to chiral amino acid
synthesis provide a ‘greener alternative’ to traditional chemical
methods. Ammonia lyases[8] and amino acid dehydrogenases[9]
have been successfully employed in the synthesis of a broad
range of primary amino acids, however, they are limited to the
use of ammonia as a nitrogen source. In recent years, a family
of NAD(P)+ dependent oxidoreductases including imine
reductases (IREDs),[10] opine dehydrogenases,[11] and reductive
aminases,[12] have emerged as useful tools for the asymmetric
synthesis of a broad range of amines. Despite the substrate
promiscuity of these classes of enzymes, there are few
examples of catalysts with activity towards keto acids for the
synthesis of N-functionalized amino acids.[12, 13]
N-Methylamino acid dehydrogenases (NMAADHs, EC
1.5.1.1 and EC 1.5.1.21) (also known as dPkAs or Δ1-piperidine-
2-carboxylate (Pyr2CR)/ Δ1-pyrrolidine-2-carboxylate reductases
(Pip2CR)),[14] ketimine reductases (KIREDs, EC 1.5.1.25)[15] and
Δ1-pyrroline-5-carboxylate reductases (P5CR, EC 1.5.1.2)[16] are
structurally unrelated enzyme classes which catalyze the
reduction of cyclic imino acids in Nature (Figure 1).[17]
performed on
a gram scale to yield optically pure N-alkyl
functionalized products in high yields.
Chiral non-natural amino acids represent a class of molecules
which have gained increased attention within the pharmaceutical
and agrochemical industries in recent years. They are key
components of many active pharmaceutical ingredients (APIs)
and have been incorporated into various biologically active
peptides[1] such as cyclosporins,[2] vancomycin,[3] and
actinomycins,[4] to generate analogues that can display
enhanced bioavailability and metabolic stability. As a result, the
development of sustainable and cost-effective methods for the
synthesis of functionalized α-amino acid derivatives is highly
desirable. Current chemical methods for the synthesis of N-
functionalized amino acids rely on either N-alkylation processes,
which often require toxic reagents and hazardous solvents, or
reductive amination which utilizes stoichiometric quantities of
hazardous hydrides and requires complex work-up procedures
which generate significant waste.[5] Numerous asymmetric
[a]
[b]
Dr J. F. Hyslop
Department of Pure and Applied Chemistry, University of
Strathclyde, 295 Cathedral Street, Glasgow G1 1XL,UK
Dr J. F. Hyslop , Dr S. L. Lovelock, Dr G.-D. Roiban
Advanced Manufacturing Technologies, GlaxoSmithKline, Medicines
Research Centre, Gunnels Wood Road, Stevenage SG1 2NY, UK
E-mail: gheorghe.d.roiban@gsk.com
[c]
Dr S. L. Lovelock
Current address: Manchester Institute of Biotechnology, School of
Chemistry, University of Manchester, 131 Princess Street,
Manchester, M1 7DN, UK
[d]
[e]
Dr P. W. Sutton
API Chemistry, GlaxoSmithKline, Medicines Research Centre,
Gunnels Wood Road, Stevenage SG1 2NY, UK
Dr P. W. Sutton
Current address: Department of Chemical Engineering, Universitat
Autꢀnoma de Barcelona, 08193 Bellaterra, (Cerdanyola del Vallꢁs),
Catalunya, Spain
Figure 1. Examples of reductive aminations catalysed by: imine reductases
(IREDs), N-methylamino acid dehydrogenases (NMAADHs), ketimine
reductases (KIREDs), amino acid dehydrogenases (AADHs), fungal Δ1-
pyrroline-5-carboxylate reductases (P5CR), and opine dehydrogenases
(ODHs).
[f]
Dr K. K. Brown
Molecular Design, Computational and Modeling, Sciences,
GlaxoSmithKline, 1250 S. Collegeville Road, Collegeville,
Pennsylvania 19426, USA
[g]
Dr A. J. B. Watson
EaStCHEM, School of Chemistry, University of St Andrews, North
Haugh, St Andrews, Fife, KY16 9ST, UK
E-mail: aw260@st-andrews.ac.uk
NMAADHs have also been reported to promote the
intermolecular coupling of α-keto acids with methylamine,[18]
however, their widespread use as biocatalysts for the synthesis
of enantiopure N-methyl amino acids has been limited by their
requirement for large excesses of the amine coupling partner
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.201806893
Angewandte Chemie International Edition
COMMUNICATION
and their narrow substrate specificity. Herein, we have evaluated
a panel of diverse NMAADHs KIREDs and P5CRs for the
reductive amination of α-keto acids. We have shown that
together these enzymes can accept a broad range of both
aliphatic and aromatic amines and we demonstrate their
synthetic utility in the synthesis of N-functionalized amino acids
on a preparative scale. Although NMAADHs, KIREDs and
P5CRs catalyze similar reactions in Nature, there is low
sequence homology between clusters (Figure 2 and Figure S8).
coli, purified and the activity towards the reductive coupling of
phenyl pyruvate 1a and amines 2a-g was evaluated using HPLC
analysis. Interestingly, the different classes of enzymes
displayed complementary reactivities and together were able to
tolerate a range of primary amine substrates (Table 1). Under
optimized reaction conditions (200 mM Tris HCl buffer pH 8.5)
using 10 equivalents of methylamine (2a), the NMAADHs (En01-
En03) proceed with almost complete conversion to the product
3a. En01-En03 also accept amines 2b-2c and have good activity
While all exist as dimers and contain an NADPH binding domain, towards propargylamine (2d) and cyclopropylamine (2e). The
they have diverse folding architectures.
KIREDs (En04-En06) have the broadest substrate tolerance and
are active towards larger amines containing aromatic
functionality such as 2f despite of reduced activity for 2g. Unlike
the NMAADHs and KIREDs, P5CR En07 has a preference for
propargylamine (2d) over methylamine (2a) and the product 3d
is formed with 82% conversion. n-Propylamine, i-propylamine,
dimethylamine, and aniline derivatives were not tolerated by any
of the enzymes evaluated. All biotransformations were (S)-
selective and yielded secondary amine products with excellent
ee as confirmed by chiral HPLC and comparison to optically
We selected seven enzymes (En01-En07) from three distinct
clusters for evaluation. En01-En03 (from Pseudomonas
putida,[14c] P. syringae[19] and P. fluorescens,[20] respectively)
belong to the NMAADH cluster, En04-En06 (from Rattus
norvegicus,[21] Homo sapiens,[15c] and Bos taurus[16a]
respectively) are KIREDs, and the fungal enzyme En07 (from
Neurospora crassa[16a]) is from the P5CR superfamily. The
selected enzymes are not only structurally diverse but also
originate from different species (mammalian, fungal, and
bacterial) (See Table S1). En01-07 were overexpressed in E.
Table 1. Comparison of enzymatic reductive amination activity between phenyl pyruvate (1a) with different amines (2).[a]
Entry
Amine
Amine
Equiv.
Product
En01
En02
En03
En04
En05
En06
En07
conv.[b] ee[c] conv.[b] ee[c]
conv.[b] ee[c]
conv.[b] ee[c]
conv.[b] ee[c] conv.[b] ee[c] conv.[b] ee[c]
1
2a, R=Me
10
1
3a
3a
3b
3b
3c
3c
3d
3d
3e
3e
3f
97%
55%
16%
2%
>99% 98%
>99% 47%
>99% 6%
>99% 98%
>99% 51%
>99% 81% >99% 32% >99% 33%
>99% 23% >99% 10% >99% 8%
>99%
>99%
nd[d]
2
2a, R=Me
>99% 50%
>99% 5%
>99% 14%
3
2b, R=Et
10
1
>99% 5%
>99% 9%
>99% 2%
nd[d]
2%
--[e]
[e]
[e]
[e]
4
2b, R=Et
nd[d]
>99% 6%
nd[d]
1%
1%
nd[d]
97%
nd[d]
1%
5%
1%
nd[d]
-
-
-
5
2c, R=Allyl
10
1
12%
2%
>99% 16%
nd[d]
3%
>99% 30% >99% 10% >99% 23% >99%
6
2c, R=Allyl
nd[d]
>99% 24% >99% 26% >99% 82%
nd[d] nd[d] nd[d]
3% 2% 21%
5%
>99% 2%
nd[d]
3%
nd[d]
7
2d, R=Propargyl
2d, R=Propargyl
2e, R=Cyclopropyl
2e, R=Cyclopropyl
2f, R=Benzyl
2f, R=Benzyl
2g, R=CH2CH2Ph
2g, R=CH2CH2Ph
10
1
92%
20%
59%
32%
>99% 52%
>99% 9%
>99% 27%
>99% 11%
>99% 58%
>99% 10%
>99% 29%
>99% 11%
>99% 19%
>99% 2%
>99% 43%
>99% 11%
18%
>99%
>99%
nd[d]
8
9
10
1
>99% 79% >99% 29% >99% 2%
[e]
10
11
12
13
14
>99% 23% >99% 1%
>99% 17% >99% 7%
>99% 18% >99% 4%
nd[d]
-
-
-
[e]
[e]
[e]
[e]
[e]
10
1
-
-
-
>99%
>99%
nd[d]
[e]
[e]
[e]
3f
-
-
-
7%
[e]
[e]
[e]
10
1
3g
3g
-
-
-
3%
nd[d]
nd[d]
5%
3%
>99% 1%
nd[d]
2%
2%
1%
nd[d]
nd[d]
[e]
[e]
[e]
-
-
-
3%
nd[d]
[a]Reaction parameters: keto acid 1a (10 mM), amines 2a-g (10 mM or 100 mM), NADPH (1 mM), En01-07 (0.1 µM), GDH (1 mg/mL), D-glucose (30 mM), Tris
HCl buffer (200 mM, pH 8.5), 25 °C, 24 h. [b] Conversion to the (S)-product determined by HPLC. [c]Determined by chiral HPLC and absolute configuration
assigned in comparison to racemic and authentic (S)-product standards . [d]Not determined due to low conversion. [e]No conversion to the product observed.
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10.1002/anie.201806893
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pure standards. Biotransformations performed using equimolar
ratios of ketone and amine gave reduced conversions to the
amino acid products, due to a combination of increased recovery
of keto acid starting materials and low levels of competing
enzyme-catalyzed ketone reduction (< 15%). Nevertheless, in
favourable cases substantial conversions could be acheived (up
to 55%) using stoichiometric substrate ratios, with considerable
potential to further optimize these processes through
combinations of reaction engineeing and enzyme evolution.
The substrate promiscuity of En01-En07 was further evaluated
using α-keto acids 2-oxo-4-phenylbutanoic acid (1b) and 3-(4-
hydroxy-3-methoxyphenyl)-2-oxopropanoic acid (1c), with
methylamine (2a) and propargylamine (2d) as co-substrates
(Supporting Information). All biocatalysts tolerated substrates
with an increased linker length (1b), with product yields
comparable to reactions with substrate 1a. Keto acid 1c
containing substituted aromatic groups was also tolerated by all
enzymes screened.
To demonstrate the practical utility of these biocatalysts for the
synthesis of N-functionalized amino acids, selected reactions
were performed on a preparative scale using clarified lysates
containing an enzyme from each of the families evaluated.
Reactions catalyzed by En01 and En04 resulted in excellent
yields of N-methylphenylalanine (3a) and N-benzylphenylalanine
(3f) respectively. The conversion to 3f is higher than previously
reported using purified En04 (Table 1) and this can be attributed
to higher catalyst loading. The En07-catalyzed reaction between
1a and propargylamine (2d) resulted in a modest yield, due to
competing ketone reduction catalyzed by endogenous E. coli
enzymes present in the cell lysate.
82/80
98/9[d]
En01
En04
3.0/2a
0.2/2f
5
2
>99
>99
3a
3f
32/21
En07
0.2/2d
67
>99
3d
[a]Reaction parameters: Phenyl pyruvate 1a (10 mM), amine 2a (700 mM) or
2d, 2f (100 mM), En01 (60 mL cell free extract from 7.5 g wet cell pellet),
En04, En07 (40 mL cell free extract from 5.0 g wet cell pellet), NADPH (1
mM), GDH (1 mg/mL), D-glucose (20 mM), 25 °C, 24 h. Tris HCl buffer (200
mM, pH 8.5), 27 h for entry 1 and 24 h for entries 2-3. [b]% HPLC conversion
to desired product 3. [c]% HPLC conversion to 3-phenyl lactic acid. [d]Product
isolation was performed using preparative LC-MS (mass-directed
autopurification system, emphasizing purity over yield).
To gain insights into the origins of reaction selectivities, we
performed docking studies with N-methyl-L-phenylalanine (3a)
using available crystal structures of En01, En05 and En07,
representing each of the three oxidoreductase families
investigated. The models reveal that hydrogen bonding
interactions between the substrate/product carboxylate and a
conserved active site arginine, position the Re-face of the imine
intermediate adjacent to the nicotinamide ring of NADPH in a
productive conformation for hydride transfer. However in P5CR
En07, the carboxylate appears to interact with a threonine
(Thr209) and the backbone NH of Thr154. All proteins also
contain a conserved serine residue, which is suitably positioned
to serve as a proton donor (Figure 2).
Table 2. Preparative scale synthesis of N-alkyl amino acids 3a, d, and f.[a]
Product
conv
(%)[b]
/
isolated
yield (%)
Amount
of 1a
(g)/
3-Phenyl
lactic acid
conv
ee
(%)
(S)
Enzyme
Product
Amine
(%)[c]
A
B
C
Figure 2. Models of En01 (A), En05 (B) and En07 (C) with the product (S)-N-methylphenyalalanine, 3a (yellow) docked into the active site. Models of the
enzymes were generated using MOE modeling software and x-ray crystal structures available in the PDB, 2CWH (En01)[22], 4BVA (En05)[23] and 5BSF (En07)[24]
NADP+ is shown in cyan.
.
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In summary, we have identified and evaluated a panel of
biocatalysts from three distantly related enzyme classes for the
synthesis of N-functionalized amino acids. Our study
demonstrates the first examples of KIRED and P5CR catalyzed
reductive amination reactions, and shows that these enzymes
are useful biocatalysts with complimentary substrate specificities
to NMAADHs. The synthetic utility of this enzyme panel has
been demonstrated through gram-scale biotransformations.
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Keywords: N-methyl amino acid dehydrogenases • ketimine
reductases • α-keto amino acids • biocatalysis • reductive
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Julia F. Hyslop,[a,b] Sarah L. Lovelock,[b,c]
Peter W. Sutton,[d,e] Kristin K. Brown,[f]
Allan, J. B. Watson,*[a,g] and Gheorghe-
Doru Roiban*[b] Page No. – Page No.
Biocatalytic Synthesis of Chiral N-
Functionalized Amino Acids
Exploring the reductive amination space: The characterization of a new family of
biocatalysts for the asymmetric synthesis of N-functionalized amino acids.
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