DOI: 10.1002/cctc.201500318
Communications
Chemoenzymatic Synthesis of ortho-, meta-, and para-
Substituted Derivatives of l-threo-3-Benzyloxyaspartate,
An Important Glutamate Transporter Blocker
JandrØ de Villiers,[a] Marianne de Villiers,[a] Edzard M. Geertsema,[a] Hans Raj,[a, b] and
Gerrit J. Poelarends*[a]
A simple, three-step chemoenzymatic synthesis of l-threo-3-
benzyloxyaspartate (l-TBOA), as well as l-TBOA derivatives
with F, CF3, and CH3 substituents at the aromatic ring, starting
from dimethyl acetylenedicarboxylate was investigated. These
chiral amino acids, which are extremely difficult to prepare by
chemical synthesis, form an important class of inhibitors of ex-
citatory amino acid transporters involved in the regulation of
glutamatergic neurotransmission. In addition, a new chemical
procedure for the synthesis of racemic mixtures of TBOA and
its derivatives was explored. These chemically prepared race-
mates are valuable reference compounds in chiral-phase HPLC
to establish the enantiopurities of the corresponding chemo-
enzymatically prepared amino acids.
several neurological disorders such as amyotrophic lateral scle-
rosis, Alzheimer’s disease, epilepsy, and Huntington’s disease.[3]
Studies on EAATs, of which five subtypes (EAAT1–5) have
been identified, have been largely dependent upon the devel-
opment of selective and potent inhibitors that can be used to
probe the physiological roles of these transporters in the regu-
lation of glutamatergic neurotransmission or in the pathogene-
sis of neurological diseases.[1,3] Aspartate derivatives with large
aryl or aryloxy substituents at the C3 position form an impor-
tant class of inhibitors of EAATs.[3,4] This is exemplified by one
of the first EAAT inhibitors to be reported, l-threo-3-benzyloxy-
aspartate (l-TBOA), which is a widely used nontransportable
blocker for all five EAAT subtypes.[4a–c] However, the chemical
synthesis of l-TBOA, the enantiomer of TBOA with the most
potent inhibitory properties, is a highly challenging 11-step
procedure starting from (R)-Garner aldehyde.[4c,d] Therefore,
there is great interest in the development of alternative proce-
dures that provide simple and environmentally friendly access
to l-TBOA and derivatives of l-TBOA with various substituents
on the aromatic ring.
l-Glutamate is the major excitatory neurotransmitter in the
mammalian central nervous system and, as such, contributes
to neuronal signaling through its activation of a variety of glu-
tamate-gated ion channels.[1] Excitatory amino acid transport-
ers (EAATs) are responsible for the uptake of glutamate from
the synaptic cleft, which thereby terminates the glutamatergic
neurotransmitter signal.[1,2] Hence, EAATs present on neurons
and surrounding glia cells play a critical role in regulating syn-
aptic glutamate concentrations. Accumulation of excitotoxic
levels of extracellular glutamate may lead to overactivation of
glutamate-gated ion channels and, consequently, neuronal
injury. Glutamate-mediated neuronal injury has been linked to
Several research groups have explored ammonia lyases as
biocatalysts in the asymmetric amination of unsaturated acids
to yield chiral a-amino acids.[5] This is a very attractive strategy
for amino acid synthesis, because it makes use of readily avail-
able starting compounds without the requirement for recycling
of cofactors, implementation of dynamic kinetic resolution
strategies, or additional enzymes. The academic and industrial
interest in aspartate derivatives, combined with the potential
advantages of replacing chemical processes with biocatalysis,
has prompted us to focus our attention on methylaspartate
ammonia lyase (MAL).[6] This enzyme catalyzes the reversible
addition of ammonia to mesaconate to give l-threo-3-methyl-
aspartate and l-erythro-3-methylaspartate as products.[7] How-
ever, the wild-type enzyme has a narrow electrophile scope
and only displays amination activity towards fumarate and its
derivatives with a small substituent at the C2 position.[8–10] In-
terestingly, recent protein engineering work on MAL has
shown that a glycine or an alanine mutation at position
Leu384 resulted in mutant enzymes with the ability to aminate
fumarate derivatives with large substituents at the C2 posi-
tion.[10] The synthetic potential of the L384A mutant has been
demonstrated by its use as a biocatalyst in the asymmetric
synthesis of various 3-substituted aspartate derivatives, includ-
ing the important EAAT inhibitor TBOA.[10,11]
[a] Dr. J. de Villiers,+ Dr. M. de Villiers,+ Dr. E. M. Geertsema,+ Dr. H. Raj,
Prof. Dr. G. J. Poelarends
Department of Pharmaceutical Biology,
Groningen Research Institute of Pharmacy
University of Groningen
Antonius Deusinglaan 1
9713 AV Groningen (The Netherlands)
E-mail: g.j.poelarends@rug.nl
[b] Dr. H. Raj
Current address: Chr-Hansen A/S
Boge Alle 10-12
2970 Horsholm (Denmark)
[+] These authors contributed equally to this work.
Supporting Information for this article is available on the WWW under
ꢀ 2015 The Authors. This is an open access article under the terms of the
Creative Commons Attribution Non-Commercial NoDerivs License, which
permits use and distribution in any medium, provided the original work is
properly cited, the use is non-commercial and no modifications or adapta-
tions are made.
In this report, we describe the synthetic potential of the
L384A and L384G mutants of MAL in the selective preparation
ChemCatChem 2015, 7, 1931 – 1934
1931 ꢀ 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim