Biocatalytic Asymmetric Synthesis of N-Aryl-Functionalized Amino Acids and Substituted Pyrazolidinones

Article abstract of DOI:10.1021/acscatal.9b01748

N-arylated α-amino acids and pyrazolidin-3-ones are widely being used as chiral building blocks for pharmaceuticals and agrochemicals. Here we report a biocatalytic route for the asymmetric synthesis of various N-arylated aspartic acids applying ethylenediamine-N,N′-disuccinic acid lyase (EDDS lyase) as a biocatalyst. This enzyme shows a broad substrate scope, enabling the addition of a variety of arylamines to fumarate with high conversions, yielding the corresponding N-arylated aspartic acids in good isolated yields and with high enantiomeric excess (ee > 99%). Furthermore, we developed a chemoenzymatic method toward the synthetically challenging chiral 2-aryl-5-carboxylpyrazolidin-3-ones, using arylhydrazines as bis-nucleophilic donors in the EDDS lyase catalyzed hydroamination of fumarate followed by an acid-catalyzed intramolecular amidation, achieving good overall yields and high optical purity (ee > 99%). In addition, we successfully combined the EDDS lyase catalyzed hydroamination and acid-catalyzed cyclization steps in one pot, thus providing a simple chemoenzymatic cascade route for synthesis of enantiomerically pure pyrazolidin-3-ones. Hence, these biocatalytic methods provide convenient alternative routes to important chiral N-arylated aspartic acids and difficult 2-aryl-5-carboxylpyrazolidin-3-ones.

Full text of DOI:10.1021/acscatal.9b01748

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Article
Biocatalytic Asymmetric Synthesis of N-Aryl-functionalized
Amino Acids and Substituted Pyrazolidinones
Haigen Fu, Alejandro Prats Luján, Laura Bothof, Jielin Zhang, Pieter G. Tepper, and Gerrit J. Poelarends
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.9b01748 • Publication Date (Web): 27 Jun 2019
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Biocatalytic Asymmetric Synthesis of N-Aryl-functionalized
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Amino Acids and Substituted Pyrazolidinones
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Haigen Fu,1 Alejandro Prats Luján,1 Laura Bothof,1 Jielin Zhang,1 Pieter G. Tepper,1 and
Gerrit J. Poelarends*,1
1Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of
Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The
Netherlands.
*Address correspondence to Prof. Dr. Gerrit J. Poelarends. Tel: +31-50-3633354; Fax: +31-50-
3633000; E-mail: g.j.poelarends@rug.nl.
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ABSTRACT
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N-arylated α-amino acids and pyrazolidin-3-ones are widely being used as chiral building
blocks for pharmaceuticals and agrochemicals. Here we report a biocatalytic route for the
asymmetric synthesis of various N-arylated aspartic acids applying ethylenediamine-N,N'-
disuccinic acid lyase (EDDS lyase) as biocatalyst. This enzyme shows a broad substrate scope,
enabling the addition of a variety of arylamines to fumarate with high conversions, yielding the
corresponding N-arylated aspartic acids in good isolated yields and with high enantiomeric
excess (ee >99%). Furthermore, we developed a chemoenzymatic method towards synthetically
challenging chiral 2-aryl-5-carboxylpyrazolidin-3-ones, using arylhydrazines as
bisnucleophilic donors in the EDDS lyase-catalyzed hydroamination of fumarate followed by
an acid-catalyzed intramolecular amidation, achieving good overall yields and high optical
purity (ee >99%). In addition, we successfully combined the EDDS lyase-catalyzed
hydroamination and acid-catalyzed cyclization steps in one pot, thus providing a simple
chemoenzymatic cascade route for synthesis of enantiomerically pure pyrazolidin-3-ones.
Hence, these biocatalytic methods provide convenient alternative routes to important chiral N-
arylated aspartic acids and difficult 2-aryl-5-carboxylpyrazolidin-3-ones.
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KEYWORDS
Asymmetric synthesis, biocatalysis, EDDS lyase, unnatural amino acids, pyrazolidinones,
cascade synthesis.
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INTRODUCTION
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Optically pure functionalized α-amino acids are highly valuable as tools for biological research
and as chiral building blocks for pharmaceuticals, nutraceuticals, and agrochemicals.1–3 In
particular, N-arylated α-amino acids are part of the core structures of a number of medicinally
important agents, such as fibrinogen receptor antagonist Lotrafiban (1, Figure 1a)4 and protein
kinase C activator Indolactam-V (2, Figure 1a).5,6 Despite their broad applications, the direct
synthesis of chiral N-arylated α-amino acids remains a challenge. Current chemical strategies
for the synthesis of enantioenriched N-arylated α-amino acids and their esters are mainly based
on extending the existing free amino group of the Cα-stereocentre through Cu-catalyzed
Ullmann-type coupling reactions6–9, Pd-catalyzed N-arylation10–13, and hypervalent iodine
chemistry (Figure 1a).14 However, they are limited by their poor atom economy, the use of
heavy metals, and harsh reaction conditions that may result in partial or complete racemization
of the α-stereocentre. Biocatalysis provides a valuable alternative route to chiral unnatural
amino acids.15–18 Previously reported enzymatic asymmetric synthesis of N-alkyl-
functionalized α-amino acids were primarily based on two types of carbon-nitrogen bond-
forming reactions: (i) conjugate addition of amines to the double bond of α,β-unsaturated acids
catalyzed by various types of carbon-nitrogen lyases, including aspartate ammonia lyases
(aspartases)19,20, methylaspartate ammonia lyases (MALs)21,22, and the recently reported
ethylenediamine-N,N'-disuccinic acid lyase (EDDS lyase)23,24; and (ii) reductive amination of
α-keto acids with amines catalyzed by a number of oxidoreductases, such as reductive aminase
(RedAm)25, opine dehydrogenases (OpDHs)26,27, N-methyl-amino acid dehydrogenases
(NMAADHs)28,29, ketimine reductases (KIREDs)29,30, and Δ1-pyrroline-5-carboxylate
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reductases (P5CRs)29,31. However, to the best of our knowledge, no enzymatic route has been
reported for the synthesis of N-aryl-functionalized α-amino acids. Thus, the development of an
efficient and sustainable biocatalytic methodology to enantiomerically pure N-arylated α-amino
acid derivatives would be particularly desirable.
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Pyrazolidin-3-ones and related five-membered dinitrogen-fused heterocycles are widely
found as core framework in dyes, agrochemicals, and pharmaceutically active molecules, such
as the very first synthetic analgesic and antipyretic drug Phenazone (3, Figure 1b)32,
lipoxygenase inhibitor Phendione (4, Figure 1b)33, and anti-Alzheimer agents (5, Figure 1b).34
In addition, chiral pyrazolidine-3-ones can also function as efficient catalysts in promoting
Diels-Alder reactions35, and catalyzing kinetic resolution of secondary alcohols and axially
chiral biaryl compounds.36 Due to their broad application in drug development, as well as in
synthetic methodologies, several chemical methods have been developed for the synthesis of
enantiomerically pure pyrazolidinones and related heterocycles, including chemical35,37 or
kinetic resolution38, 1,3-dipolar cycloaddition39–41, Lewis acid catalyzed conjugate addition42,
and Pd-catalyzed asymmetric hydrogenation.43 However, creating a biocatalytic methodology
as alternative route to chiral pyrazolidinones is an as yet unmet challenge.
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H3C
CH3
H
N
Previous work
1) Pd (or Cu)-catalyzed N-arylation
2) hypervalent iodine chemistry
(a)
O
H3C
CH3
O
N
OH
N
N
O
This work
biocatalytic hydroamination
Ar
N
CO2H
H
N
H
NH
HN
(R = H or Me)
COOR
1
2
(Lotrafiban)
fibrinogen receptor antagonist
(Indolactam-V)
ROOC
protein kinase C activator
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Previous work
1) chemical or kinetic resolution
2) 1,3-dipolar cycloaddition reaction
3) Lewis acid catalyzed conjugate addition
4) Pd-catalyzed asymmetric hydrogenation
(b)
Ar
CH3
N
N
H3C
N
O
N
N
NH
This work
one-pot chemoenzymatic route
O
O
N1
N2
O
5
3
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5
(Phenazone)
analgesic and antipyretic drug
(Phendione)
lipoxygenase inhibitor
R
3
4
anti-Alzheimer agent
O
Figure 1. Synthetic strategies for preparation of noncanonical amino acids that are part of the core
structures of biologically active compounds. (a) Biologically active molecules containing an N-arylated
amino acid (left panel), and previous synthetic strategies and our biocatalytic strategy towards N-
arylated aspartic acids (right panel). (b) Biologically active molecules containing pyrazolidine-3-one
(left panel), and established synthetic strategies and our chemoenzymatic route towards pyrazolidine-3-
ones (right panel).
The enzyme ethylenediamine-N,N'-disuccinic acid lyase (EDDS lyase), from Chelativorans
sp. BNC1, naturally catalyzes a reversible two-step sequential addition of ethylenediamine to
two molecules of fumarate providing (S, S)-EDDS as final product (Figure 2a).44 We recently
demonstrated that EDDS lyase could accept a wide variety of amino acids with terminal amino
groups for regio- and stereoselective addition to fumarate, providing the natural product
aspergillomarasmine A and various related aminocarboxylic acids (Figure 2b).23 In addition,
EDDS lyase could also accept a number of (hetero)cycloalkyl-substituted amines, allowing the
asymmetric synthesis of (S)-N-cycloalkyl-substituted aspartic acids (Figure 2b).24 Therefore,
the remarkably broad nucleophile scope of EDDS lyase prompted us to further explore the less
nucleophilic arylamines as novel non-natural substrates in the asymmetric hydroamination of
fumarate, which would enable the production of chiral N-arylated aspartic acids as the
corresponding enzymatic products (Figure 2c). Moreover, we envisioned that chiral
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pyrazolidine-3-ones could be constructed by using arylhydrazines as bisnucleophilic donors in
the EDDS lyase-catalyzed regio- and stereoselective addition to fumarate followed by a simple
intramolecular amidation (Figure 2c).
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Herein, we report a biocatalytic methodology for the synthesis of optically pure (S)-N-
arylated aspartic acids in high conversions and isolated yields. Moreover, an efficient one-pot
two-step chemoenzymatic route towards chiral pyrazolidine-3-ones has been developed. These
strategies highlight a highly regio- and stereoselective hydroamination step catalyzed by EDDS
lyase, offering alternative synthetic choices to prepare chiral N-arylated α-amino acids as well
as chiral pyrazolidine-3-ones.
(a) Natural reaction catalyzed by EDDS lyase
HO2C
HO2C
CO2H
CO2H
CO2H
H
N
CO2H
H
N
NH2
HO2C
NH2
H2N
HO2C
N
EDDS lyase
EDDS lyase
H
CO2H
CO2H
ethylenediamine
N-(2-aminoethyl)aspartic acid
ethylenediamine-N,N'-disuccinic acid
(S, S)-EDDS
(b) Our previous work
CO2H
CO2H
H
N
R1
=
O
HO2C
N
n
H
CO2H
NH2
X
R1
(hetero)cycl
amine
CO2H
NH2
H
N
HN
HO2C
n = 1 (aspergillomarasmine A), 2
acids
X = CH2, O, S
X
CO2H
HO2C
oalkyl
n
s
amino
ref.
CO2H
ref.
CO2H
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H
n
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n = 1 (toxin A), 2, 3, 4
HO2C
N
CO2H
H
CO2H
n = 1 (aspergillomarasmine B), 2
X = CH2, O, NH, S,
NMe, CH2NH2
Aminocarboxylic acids
N-cycloalkyl-substituted aspartic acids
CO2H
EDDS lyase
HO2C
+
(c) This work
R3
H
R2
N
N
HO2C
arylhyd
HN
nes
razines
CO2H
HO2C
O
arylami
R2
=
R3
=
H, m-F, m-Me, m-OMe, p-X (F, Cl, Br, I),
p-Me, p-OMe, p-CO2H, p-Et, p-iPr, m,p-diMe.
H, o-F, m-F, m-Me, m-Cl, p-X (F, Cl, Br),
p-CN, p-Me, p-OMe, p-CO2H
Pyrazolidinones
N-arylated aspartic acids
Figure 2. Overview of reactions catalyzed by EDDS lyase. (a) Natural reaction catalyzed by EDDS
lyase. (b) EDDS lyase-catalyzed asymmetric addition of amino acids and (hetero)cycloalkyl-substituted
amines to fumaric acid to yield complex aminocarboxylic acids and N-cycloalkyl-substituted aspartic
acids, respectively. (c) Exploring the arylamines and arylhydrazines scope of EDDS lyase for the
(chemo)enzymatic asymmetric synthesis of N-arylated aspartic acids and pyrazolidinones, respectively.
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RESULTS
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Biocatalytic synthesis of N-arylated aspartic acids
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In contrast to aliphatic amines, aromatic amines are challenging substrates for biocatalytic
addition reactions due to their relatively low nucleophilicity. Our previous study demonstrated
that EDDS lyase could accept glycine as an unnatural substrate, facilitating the low nucleophilic
α-amino group of glycine to function as the nucleophile in the amination of fumarate (6).23 This
prompted us to start our investigation by testing aniline (7a, Table 1, entry 1) as potential
substrate in the EDDS lyase-catalyzed biotransformation. Remarkably, aniline (7a) was
efficiently converted by purified EDDS lyase to afford N-phenyl-substituted aspartic acid (8a),
with high conversion (91%) and good isolated yield (80%) using only 0.05 mol% biocatalyst
loading under optimized conditions (Table 1). To determine the stereochemistry of enzymatic
product 8a, HPLC analysis on a chiral stationary phase was conducted by using chemically
prepared authentic standards with known (R/S)- and (S)-configuration (Figure S89). This
analysis revealed that product 8a was present as a single (S)-configured enantiomer with
excellent enantiomeric excess (ee >99%, Table 1, entry 1).
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Next, the substrate scope was investigated by examining a panel of electronically diverse
substituted anilines and heteroarylamines as unnatural substrates in the EDDS lyase-catalyzed
amination of fumarate, as monitored by 1H NMR spectroscopy (Table S1). We were pleased to
find that EDDS lyase displayed a broad arylamine substrate scope which was, as expected,
affected by the electron-withdrawing/donating nature, position and bulkiness of the substituents
on the aromatic ring (Table 1 and Table S1). Clearly, substitution of the aromatic ring at the
ortho-position was not tolerated by the enzyme, for which only o-fluoroaniline (7b) gave a very
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low conversion (6%, Table 1 and Table S1). Impressively, arylamines with meta-substituents,
including m-fluoroaniline (7c), m-toluidine (7d) and m-methoxyaniline (7e), were efficiently
accepted by EDDS lyase providing the respective products 8c-e (Table 1, entry 3-5). High
conversions (87-97%), good isolated product yields (53-84%), and excellent stereoselectivity
(ee >99%) were observed (Table 1, entry 3-5). Similarly, para-substituted arylamines, such as
p-fluoroaniline (7f), p-toluidine (7g), p-methoxyaniline (7h), p-ethylaniline (7i), m,p-
dimethylaniline (7j), and p-carboxylaniline (7k), were also well accepted by the enzyme, giving
high to excellent conversions (75-95%) and yielding the corresponding amino acids 8f-k (34-
85% isolated yields) as the (S)-configured enantiomers with >99% ee (Table 1, entry 6-11).
Noteworthy, para-halogenated anilines (7l-n) were also processed to deliver chiral (S)-N-
haloarylaspartic acids (8l-n) with 82-96% conversions and 52-69% isolated yields (ee >99% in
all cases, Table 1, entry 12-14), leaving the halogens available for potential downstream
synthetic manipulation. The larger nucleophile p-isopropylaniline (7o) was a poor substrate for
EDDS lyase, resulting in low conversion (17%, Table 1, entry 15). Arylamines bearing a strong
electron-withdrawing group (such as p-nitro or p-CF3) or electron-deficient heteroarylamines
(such as pyridine-2-amine, pyridine-4-amine, and thiazol-2-amine) were not accommodated as
substrates by EDDS lyase, most likely due to their diminished nucleophilicity (Table S1).
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Table 1. Enzymatic synthesis of (S)-N-arylated aspartic acids.
6
7
8
9
R
NH2
EDDS lyase
conditionsa
HOOC
HN
HOOC
+
R
COOH
COOH
6
7a-o
8a-o
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Time
[h]
Conv.b
eed
Entry Arylamine Product
R
(yieldc) [%]
[%]
1
7a
7b
7c
7d
7e
7f
8a
8b
8c
8d
8e
8f
H
48
72
48
24
48
24
48
48
48
72
24
48
48
48
72
91 (80)
>99 (S)f
2
o-F
m-F
6 (n.d.e)
91 (53)
97 (84)
87 (53)
92 (85)
75 (57)
92 (75)
76 (53)
90 (70)
95 (34)
96 (63)
95 (69)
82 (52)
17 (n.d.e)
---
3
>99 (S)g
>99 (S)g
>99 (S)g
>99 (S)f
>99 (S)f
>99 (S)f
>99 (S)g
>99 (S)g
>99 (S)g
>99 (S)g
>99 (S)f
>99 (S)g
---
4
m-Me
m-OMe
p-F
5
6
7
7g
7h
7i
8g
8h
8i
p-Me
p-OMe
p-Et
8
9
10
11
12
13
14
15
7j
8j
m,p-diMe
p-CO2H
p-Cl
7k
7l
8k
8l
7m
7n
7o
8m
8n
8o
p-Br
p-I
p-iPr
aConditions and reagents: the reaction mixture (15 mL) consisted of fumaric acid (6, 50 mM), arylamine substrates
7a-o (10 mM) and purified EDDS lyase (0.05 mol% based on arylamine) in buffer (50 mM NaH2PO4/NaOH, pH
8.5), with 5% DMSO as cosolvent at room temperature. A 5-fold excess of 6 (rather than an excess of amine) was
used to drive amine 7 to completion, simplifying product purification and preventing enzyme inhibition by a high
concentration of amine substrate. bConversions were determined by comparing 1H NMR signals of substrates and
c
d
corresponding products. Isolated yield after cation-exchange chromatography. Enantiomeric excess (ee) was
e
determined by HPLC on a chiral stationary phase using racemic standards. Not determined owing to low
conversion, the product formation was confirmed by comparison of 1H NMR data of a crude reaction mixture to
that of chemically prepared reference compound. fThe absolute configurations of 8a, 8f-h and 8m were determined
by chiral HPLC using chemically synthesized authentic standards with known (R/S) and (S) configuration
g
respectively. The absolute configurations of 8c-e, 8i-l and 8n were tentatively assigned the (S)-configuration
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based on analogy and according to chiral HPLC data.
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Chemoenzymatic synthesis of chiral pyrazolidine-3-ones
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Enantioselective conjugate addition of bisnucleophilic donors (such as hydrazines) to electron-
poor acceptors provides convenient access to valuable small-ring heterocycles with potential
pharmaceutical applications.32-34,42 Encouraged by the exquisite stereoselectivity of the EDDS-
lyase-catalyzed biotransformation accepting a broad range of arylamines (7, Table 1), we
further questioned whether bisnucleophilic arylhydrazines (9, Table 2) could be processed as
substrates by this enzyme in the amination of fumarate (6). The corresponding enzymatic
products, N-(arylamino)aspartic acids (10), are not only valuable scaffolds in their own right,
but they also could serve as chiral precursors for the preparation of synthetically challenging
chiral pyrazolidine-3-ones (11) through an acid-catalyzed cyclization reaction (Table 2).
Remarkably, phenylhydrazine (9a), as the first chosen potential bisnucleophilic substrate, was
efficiently converted by EDDS lyase (0.1 mol%) to afford the single product N-
(phenylamino)aspartic acid (10a), as ascertained by 1H NMR in comparison with a chemically
prepared authentic standard. In the enzymatic semipreparative synthesis (0.20 mmol scale) of
compound 10a, excellent conversion (94%) and good isolated yield (80%, 36 mg) were
achieved (Table 2, entry 1). Note that it is necessary to perform the enzymatic reaction under
anaerobic conditions, otherwise the substrate phenylhydrazine could be oxidized by molecular
oxygen and thus lead to diminished conversion. Subsequently, the enzymatic product 10a was
cyclized smoothly under optimized conditions (1 M HCl, reflux for 3 h)37, affording the desired
heterocycle 2-phenyl-5-carboxylpyrazolidin-3-one (11a, 71% isolated yield) without
racemization of the potentially sensitive Cα stereogenic center (ee >99%, Table 2, entry 1). The
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chemoenzymatically prepared heterocycle 11a was identified as the (S)-configured enantiomer
by chiral HPLC analysis (Figure S102).
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To further illustrate the synthetic usefulness of this chemoenzymatic method, we first
determined that EDDS lyase has a broad substrate scope with respect to arylhydrazines (9),
enabling the addition of various arylhydrazines to fumarate (Table S2). Pleasingly, several
arylhydrazines with an ortho-substituent (o-fluoro, 9b) or meta-substituent (such as m-fluoro,
m-methyl, and m-chloro, 9c-e) were efficiently converted by EDDS lyase giving the respective
enzymatic products 10b-e with excellent conversions (92-98%) and good isolated yields (76-
85%, Table 2, entry 2-5). Notably, a number of arylhydrazines containing para-substituents,
such as p-fluoro, p-chloro, p-bromo, p-cyano, p-methyl, and p-carboxyl (9f-k), were also well
accepted by the enzyme giving the corresponding N-(arylamino)aspartic acids (10f-k) in high
to excellent conversions (71-97%) and 52-89% isolated yields (Table 2, entry 6-11). However,
p-(methoxyphenyl)hydrazine (9l) was not well accepted by EDDS lyase with unsatisfactory
conversion (28%, Table 2, entry 12); the reason for the low conversion of this substrate is not
known. Typically, arylhydrazines containing a strong electron-withdrawing group at the
aromatic ring (namely p-nitro or p-CF3), or a bulky naphthalen-2-yl group, failed to undergo
the EDDS-lyase-catalyzed hydroamination reaction (Table S2).
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With the precious enzymatically prepared intermediates (10b-k) in hand, we subsequently
performed the acid-catalyzed cyclization reaction to achieve the target 2,5-disubstituted
pyrazolidin-3-one products. Remarkably, all the intermediates (10b-k) could be cyclized
smoothly under the optimized conditions to provide the desired pyrazolidin-3-ones (11b-k)
with good isolated yield (46-80%, Table 2, entry 2-11). Moreover, all the tested
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chemoenzymatic products (11b-k) were assigned the (S)-configuration, with excellent
enantiomeric excess (ee >99%, Table 2, entry 2-11), using chiral HPLC analysis (Figures S103-
S111). As such, we have established an efficient two-step chemoenzymatic route towards chiral
2-aryl-5-carboxylpyrazolidin-3-ones (11a-k) with good overall yields and excellent
stereoselectivity (ee >99%).
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Table 2. Chemoenzymatic synthesis of chiral pyrazolidine-3-ones.
R
H
R
H
EDDS lyase
1 M HCl
N
N
HOOC
NH2
+
N
COOH
HOOC
R
NH
conditionsa
reflux, 3 h
HN
HOOC
step 1
step 2
O
COOH
6
9a-l
10a-l
11a-l
First enzymatic step
Second cyclization step
Aryl
Overall
yieldi
Conv.b
(Yieldc)
[%]
eee
Entry
R
Yieldc
hydrazine
Intermediate
Product
[%]
[%]
1
2
9a
9b
9c
9d
9e
9f
H
10a
10b
10c
10d
10e
10f
10g
10h
10i
94 (80)
98 (81)
98 (83)
92 (76)
98 (85)
85 (52)
80 (68)
91 (81)
92 (70)
71 (63)
97 (89)
11a
11b
11c
11d
11e
11f
11g
11h
11i
71
46
73
62
67
59
80
76
57
58
69
---
>99 (S)f
>99 (S)g
>99 (S)f
>99 (S)f
>99 (S)f
>99 (S)f
>99 (S)f
>99 (S)f
>99 (S)g
>99 (S)f
n.d.h
57
37
61
47
57
31
54
62
40
37
61
---
o-F
3
m-F
4
m-Me
m-Cl
p-F
5
6
7
9g
9h
9i
p-Cl
p-Br
p-CN
p-Me
8
9
10
11
12
9j
10j
10k
10l
11j
11k
11l
9k
9l
p-CO2H
p-OMe
28 (n.d.d)
---
aConditions and reagents: the reaction mixture (20 mL) consisted of fumaric acid (6, 50 mM), arylhydrazine
substrates 9a-l (10 mM) and purified EDDS lyase (0.1 mol% based on arylhydrazine, the amount of applied
enzyme was chosen such that reactions were completed within 24-96 h) in degassed buffer (50 mM
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NaH2PO4/NaOH, pH 8.5) under argon atmosphere at room temperature (24 h for 10k; 48 h for 10a, 10c, 10e, and
b
1
10h-i; 96 h for 10b, 10d, 10f-g, 10j and 10l). Conversions were determined by comparing H NMR signals of
substrates and corresponding products. cIsolated yield after purification. dNot determined owing to low conversion,
1
the product formation was confirmed by comparison of H NMR data of a crude reaction mixture to that of
e
chemically prepared reference compound. Enantiomeric excess (ee) was determined by HPLC on a chiral
stationary phase using chemically synthesized racemic standards. fThe absolute configurations of 11a, 11c-h, and
11j were determined by chiral HPLC using authentic standards with known (R/S) and (S) configuration. gThe
absolute configurations of 11b and 11i were tentatively assigned the (S)-configuration based on analogy and
according to chiral HPLC data. h The ee value was not determined. iIsolated yield over two steps.
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One-pot chemoenzymatic synthesis of chiral pyrazolidin-3-ones
Having established a stepwise chemoenzymatic route towards chiral pyrazolidin-3-ones (11),
we sought to combine the EDDS lyase-catalyzed biotransformation and acid-catalyzed
cyclization into one pot (Figure 3). In order to achieve high overall yield, as well as to effect
the second cyclization step in the one-pot synthesis of pyrazolidin-3-ones (11), high conversion
of the starting arylhydrazine substrates 9 in the first enzymatic step is required, preventing it
from reacting with the intermediate 10 during the subsequent acid-promoted amidation step.
Toward this end, the substrate phenylhydrazine (9a) that could be efficiently converted by
EDDS lyase with 94% conversion was chosen for our initial investigation to provide the
corresponding intermediate 10a (Table 2 and Figure 3). Without any purification, intermediate
10a was subjected to cyclization in the same pot, to which fuming hydrochloric acid (HCl) was
added to adjust the final concentration of HCl to 1 M, providing full conversion for the second
cyclization step after heating to reflux for 3 h. Pleasingly, the one-pot chemoenzymatically
prepared product (S)-2-phenyl-5-carboxylpyrazolidin-3-one (11a) was isolated with good
overall yield (68%) and excellent optical purity (ee >99%, Figure 3).
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9
To further demonstrate the synthetic usefulness of this one-pot two-step chemoenzymatic
strategy, we selected five other starting arylhydrazines (9c-e and 9h-i, Table 2), which proved
to be well accepted as substrates by EDDS lyase. The corresponding chemoenzymatically
prepared (S)-pyrazolidin-3-one derivatives (11c-e and 11h-i) were obtained with good overall
isolated yields (61-70%) and excellent enantiopurity (ee >99% in all cases, Figure 3). Therefore,
this one-pot chemoenzymatic synthesis route provides a simplified practical procedure towards
optically pure pyrazolidin-3-ones with higher overall isolated yields.
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R
HOOC
H
COOH
R
H
N
1 M HCl
step 2b
6
N
NH2
NH
N
HOOC
R
HN
HOOC
EDDS lyase
step 1a
COOH
O
9
10
11
F
CH3
H
N
H
N
H
N
N
HOOC
N
N
HOOC
HOOC
O
O
O
11a
11c
11d
yieldc 68%
eed >99%, (S)e
yield 65%
ee >99%, (S)
yield 70%
ee >99%, (S)
Cl
CN
Br
H
H
H
N
N
N
N
N
N
HOOC
HOOC
HOOC
O
O
11h
O
11i
11e
yield 61%
ee >99%, (S)
yield 63%
ee >99%, (S)
yield 68%
ee >99%, (S)
Figure 3. One-pot two-step chemoenzymatic synthesis of chiral pyrazolidin-3-ones. Reagents and reaction
conditions: aarylhydrazine substrates 9 (10 mM), fumaric acid (6, 50 mM), and purified EDDS lyase (0.1 mol%
based on arylhydrazine) in degassed buffer (50 mM NaH2PO4/NaOH, pH 8.5) under argon atmosphere at room
b
temperature (48 h for 9a, 9c, 9e and 9h-i; 96 h for 9d). 1 M HCl, reflux for 3 h under nitrogen atmosphere.
d
cIsolated yield over two steps. Enantiomeric excess (ee) was determined by HPLC on a chiral stationary phase
e
using chemically synthesized racemic standards. The absolute configuration of the one-pot chemoenzymatic
products 11a, 11c-e and 11h were determined by chiral HPLC analysis using authentic standards with known (R/S)
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and (S) configuration. The absolute configuration of product 11i was tentatively assigned the (S)-configuration
based on analogy and according to chiral HPLC data.
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DISCUSSION
In contrast to previously reported chemical synthesis strategies for preparation of
enantioenriched N-arylated α-amino acids, such as metal-catalyzed N-arylation7-13 or
hypervalent iodine chemistry14, which mainly depend on extending the free amino group of the
starting chiral α-amino acids (or their esters), our biocatalytic method starts with a prochiral
α,β-unsaturated acid (fumarate) and creates the Cα-stereocentre of the target N-arylated amino
acids in a single asymmetric step with excellent stereocontrol (Figure 1a). We demonstrated
that EDDS lyase shows a broad scope of anilines, enabling the addition of a variety of aromatic
amines (7a-n) to fumarate, yielding optically pure (ee >99%) (S)-N-arylated aspartic acids (8a-
n) with high conversions and in good isolated yields (Table 1). Furthermore, we discovered that
EDDS lyase can accept a wide range of arylhydrazines (9a-k) in the hydroamination of
fumarate, yielding the corresponding N-(arylamino)-substituted aspartic acids (10a-k) with
high conversions and in good isolated yields (Table 2). Subsequently, these enzymatic products
(11a-k) could undergo a smooth acid-catalyzed cyclization to give the synthetically challenging
chiral (S)-pyrazolidin-3-one derivatives 11a-k with excellent enantiomeric excess (ee >99%,
Table 2). In addition, we successfully combined the EDDS lyase-catalyzed biotransformation
and acid-catalyzed cyclization into one pot, thus providing a rather simple two-step
chemoenzymatic route for the rapid synthesis of optically pure pyrazolidin-3-ones with good
overall isolated yields (Figure 3).
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Enantioselective addition of ammonia or amines to appropriate α,β-unsaturated carboxylic
acids catalyzed by carbon-nitrogen lyases represents an attractive strategy for the synthesis of
chiral unnatural amino acids. This enzymatic strategy makes use of readily available prochiral
α,β-unsaturated acids as starting substrates without a requirement for cofactor recycling,
circumvents tedious steps of protecting or activating carboxylic groups, gives 100% theoretical
yield, and normally provides high stereoselectivity under mild and potentially green reaction
conditions. Several synthetically useful carbon-nitrogen lyases, such as aspartate ammonia
lyases (DALs)16,17,20,45, methylaspartate ammonia lyases (MALs)16,17,21,46, phenylalanine
ammonia lyases (PALs)16,17 and phenylalanine aminomutases (PAMs)16,17, were successfully
used in the synthesis of optically pure unnatural α- or β-amino acids. However, with the
exception of an engineered mutant of MAL (MAL-Q37A), which accepts various alkylamines
as substrates in the addition to mesaconate21, these enzymes display a rather limited nucleophile
scope. In contrast, EDDS lyase has a very broad nucleophile scope, accepting a wide variety of
structurally distinct amines for stereoselective addition to fumarate, providing enzymatic access
to various aminocarboxylic acids including the natural products toxin A, aspergillomarasmine
A and aspergillomarasmine B23, N-cycloalkyl-substituted aspartic acids24, as well as difficult
N-arylated aspartic acid derivatives and substituted pyrazolidin-3-ones (this study, Figure 2).
As such, EDDS lyase nicely complements the biocatalytic toolbox for the preparation of
noncanonical amino acids. In future work, we will focus our attention on extending the
electrophile scope of EDDS lyase, which was found to be highly specific for fumarate with
other α,β-unsaturated carboxylic acids (including crotonic acid, mesaconic acid, itaconic acid,
or 2-pentenoic acid) not being accepted as alternative electrophiles,44 by computational design
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and structure-guided protein engineering.
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METHODS
Enzymatic synthesis of (S)-N-arylated aspartic acids (8a-n)
Enzyme expression and purification were performed according to procedures described elsewhere (see
Supporting Information).23,44 The reaction mixture (15 mL) consisted of fumaric acid (50 mM) and an
arylamine substrate (7a-n, 10 mM) in 50 mM NaH2PO4-NaOH buffer (pH 8.5) with 5% DMSO as
cosolvent. The pH of the reaction mixture was adjusted to pH 8.5. The enzymatic reaction was started
by addition of freshly purified EDDS lyase (0.05 mol%). The reaction mixture was then incubated at
room temperature from 24 h to 72 h (Table 1). After completion of the reaction, the enzyme was
inactivated by heating to 70 °C for 10 min. The progress of the enzymatic reaction was monitored by
1H NMR spectroscopy by comparing signals of substrates and corresponding products.
The amino acid products were purified by cation-exchange chromatography. For a typical purification
procedure, the precipitated enzyme was removed by filtration (pore diameter 0.45 μm). The filtrate was
washed with ethyl acetate (10 mL x 3) to remove the remaining amines. The aqueous layer was acidified
with 1 M HCl to pH=1 and loaded slowly onto a cation-exchange column (5 g of Dowex 50W X8 resin,
100-200 mesh), which was pretreated with 2 M aqueous ammonia (5 column volumes), 1 M HCl (3
column volumes) and finally water (5 column volumes). The column was washed with water (3 column
volumes) to remove the remaining fumaric acid and eluted with 2 M aqueous ammonia until the desired
product was collected. The ninhydrin-positive fractions were collected, concentrated under vacuum and
lyophilized to provide the desired products (8a-n) as ammonium salts.
One-pot chemoenzymatic synthesis of pyrazolidine-3-ones (11)
Step 1: The reaction mixture (20 mL) consisted of fumaric acid (50 mM) and an arylhydrazine substrate
(9, 10 mM) in 50 mM NaH2PO4-NaOH degassed buffer (pH 8.5) under argon atmosphere. The pH of
the reaction mixture was adjusted to pH 8.5. The enzymatic reaction was started by addition of freshly
purified EDDS lyase (0.1 mol%). The reaction mixture was then incubated at room temperature from
48 h to 96 h (Figure 3). The progress of the enzymatic reaction was monitored by 1H NMR spectroscopy
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by comparing signals of substrates and corresponding products. Without purification of the enzymatic
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product 10, the reaction mixture was subjected to the next step immediately.
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8
9
Step 2: To the stirred reaction mixture from the previous step was added 1.6 mL of fuming hydrochloric
acid dropwise under cooling (ice-bath). After 5 min, the reaction mixture was heated to reflux for 3 h
under nitrogen atmosphere. After completion of the reaction, the reaction mixture was allowed to cool
down to room temperature. The reaction mixture was extracted with EtOAc (20 mL x 3), and washed
with brine (30 mL). The solvent was evaporated to provide crude product 11, which was purified by
C18 column chromatography (5% to 50% CH3CN in H2O as the eluent).
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ASSOCIATED CONTENT
Supporting Information
This information is available free of charge on the ACS Publications website. Detailed
experimental procedures, 1H NMR and 13C NMR spectra illustrating chemical structures, and
chiral HPLC analysis for (chemo)enzymatic products (PDF).
AUTHOR INFORMATION
Corresponding Author
*E-mail: g.j.poelarends@rug.nl. Tel.: +31 503633354.
ORCID
Gerrit J. Poelarends: 0000-0002-6917-6368
Haigen Fu: 0000-0002-4993-0570
Notes
The authors declare no competing financial interest.
Data availability
All data are available from the corresponding author upon reasonable request. Correspondence
and requests for materials should be addressed to G.J.P.
ACKNOWLEDGEMENTS
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We acknowledge financial support from the Netherlands Organization of Scientific Research
(VICI grant 724.016.002). Haigen Fu and Jielin Zhang acknowledge funding from the China
Scholarship Council.
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TOC
H
NH2
N
NH2
R
H
R
R
R
N
N
HN
HOOC
CO2H
EDDS lyase
HO2C
COOH
+
HOOC
O
one-pot procedure
N-Arylated Asp
Chiral Pyrazolidinones
Overall yield 61-70%
Conv. up to 97%
Yield up to 85%
ee >99%
ee >99%
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