Rapid chemoenzymatic route to glutamate transporter inhibitor l-TFB-TBOA and related amino acids

Article abstract of DOI:10.1039/c7ob00305f

The complex amino acid (l-threo)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (l-TFB-TBOA) and its derivatives are privileged compounds for studying the roles of excitatory amino acid transporters (EAATs) in regulation of glutamatergic neurotransmission, animal behavior, and in the pathogenesis of neurological diseases. The wide-spread use of l-TFB-TBOA stems from its high potency of EAAT inhibition and the lack of off-target binding to glutamate receptors. However, one of the main challenges in the evaluation of l-TFB-TBOA and its derivatives is the laborious synthesis of these compounds in stereoisomerically pure form. Here, we report an efficient and step-economic chemoenzymatic route that gives access to enantio- and diastereopure l-TFB-TBOA and its derivatives at multigram scale.

Full text of DOI:10.1039/c7ob00305f

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DOI: 10.1039/C7OB00305F
Rapid Chemoenzymatic Route to Glutamate Transporter Inhibitor
L-TFB-TBOA and Related Amino Acids
Haigen Fu,[a] Sabry H. H. Younes,[a] Mohammad Saifuddin,[a] Pieter G. Tepper,[a]
Jielin Zhang,[a] Erik Keller,[b] André Heeres,[b] Wiktor Szymanski,[c] and
Gerrit J. Poelarends*[a]
[a] H. Fu,+ Dr. S. H. H. Younes,+ Dr. M. Saifuddin, P. G. Tepper, J. Zhang, Prof.
Dr. G. J. Poelarends
Department of Chemical and 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
Homepage: http://www.rug.nl/staff/g.j.poelarends/
[b] Dr. E. Keller, Dr. A. Heeres
Syncom BV, Kadijk 3, 9747 AT Groningen, The Netherlands
[c] Dr. W. Szymanski
Department of Radiology, University of Groningen, University Medical Center
Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands
[+] These authors contributed equally to this work.
1

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DOI: 10.1039/C7OB00305F
Abstract
The complex amino acid (L-threo)-3-[3-[4-(trifluoromethyl)benzoylamino]
benzyloxy]aspartate (L-TFB-TBOA) and its derivatives are privileged compounds for
studying the roles of excitatory amino acid transporters (EAATs) in regulation of
glutamatergic neurotransmission, animal behavior, and in the pathogenesis of
neurological diseases. The wide-spread use of L-TFB-TBOA stems from its high
potency of EAAT inhibition and the lack of off-target binding to glutamate receptors.
However, one of the main challenges in the evaluation of L-TFB-TBOA and its
derivatives is the laborious synthesis of these compounds in stereoisomerically pure
form. Here, we report an efficient and step-economic chemoenzymatic route that gives
access to enantio- and diastereopure L-TFB-TBOA and its derivatives at multigram
scale.
2

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Introduction
L-Glutamate is the major excitatory neurotransmitter in the mammalian central
nervous system (CNS) and mediates numerous neuronal communications in the
brain.1 However, accumulation of high levels of extracellular glutamate may lead to
over-activation of glutamate-gated ion channels and, consequently, neuronal injury.2
Synaptic glutamate concentrations are strictly kept below levels of neurotoxicity by
the family of excitatory amino acid transporters (EAATs) expressed on neurons and
surrounding glial cells.3 Dysfunction of EAATs has been implicated in many
neurological disorders, such as Alzheimer’s disease, epilepsy, amyotrophic lateral
sclerosis, and Huntington’s disease.4
L-Aspartate derivatives with aryloxy substituents at the C3 position, exemplified
by L-threo-3-benzyloxyaspartate (1, L-TBOA, Scheme 1), were identified as the first
class of nontransportable EAAT inhibitors.5 The importance of these L-aspartate
derivatives as tools in neurobiological research was further highlighted by the
identification
of
the
most
potent
and
widely
used
blocker,
(L-threo)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate
(2a,
L-TFB-TBOA, Scheme 1), which has nanomolar affinity to EAAT1 and EAAT2 and
lacks affinity towards other glutamate receptors and transporters.6 However, the
asymmetric synthesis of enantiopure L-TFB-TBOA and related compounds proved to
be extremely challenging. Shimamoto and coworkers reported the asymmetric
synthesis of enantiopure 2a through an elaborate 20-step synthetic procedure.7
Although a concise synthesis of 2a based on Sharpless aminohydroxylation with
3

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DOI: 10.1039/C7OB00305F
chiral ligand (DHQD)2PHAL was recently reported by Leuenberger et al., this
procedure provided 2a with only 40% enantiomeric excess (ee).8 Efficient asymmetric
synthesis of enantiopure L-TFB-TBOA and its derivatives would facilitate the
further discovery of EAAT subtype-selective inhibitors, which are highly useful tools
for the elucidation of the exact physiological roles of distinct EAATs in glutamate
accumulation9, synaptic transmission6c and animal behavior6a. Hence, alternative
procedures that provide efficient and more step-economic access to 2a and related
compounds are in great demand. Herein, we report a rapid chemoenzymatic route that
gives convenient access to enantiopure L-TFB-TBOA and its derivatives at multigram
scale. This method uses the late functionalization of a common precursor to provide a
convenient way of divergent preparation of L-aspartate derivatives with large aryloxy
substituents at the C3 position (Scheme 1).
Results and Discussion
Retrosynthetic analysis suggests that L-TFB-TBOA (2a) could be derived from
dimethyl (L-threo)-N-Boc-3-hydroxyaspartate (4) and substituted benzyl bromide 5a
via O-alkylation and subsequent deprotection (Scheme 1). The key challenge was the
formation of chiral building block 4 due to the possible difficulties in constructing the
required L-threo configuration at vicinal chiral centers with a 1,2-aminoalcohol
motif.10 We envisioned that this key precursor 4 could be readily generated from 1,
which has the desired L-threo configuration, via protection and hydrogenolysis steps.
While the chemical synthesis of 1 is a highly challenging 11-step procedure,5c
a
4

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DOI: 10.1039/C7OB00305F
straightforward three-step chemoenzymatic methodology for the asymmetric synthesis
of 1 (de >99%, ee >99%), starting from commercially available dimethyl
acetylenedicarboxylate 3, has recently been reported (Scheme 2A).11
Scheme 1. Chemoenzymatic retrosynthesis of L-TFB-TBOA (2a).
To enable efficient multigram-scale (see SI) synthesis of 1, we first optimized the
previously used procedure for the small-scale (150 mg) synthesis of 1.11b The addition
of benzyl alcohol to 3 yields a mixture of cis and trans product isomers (Scheme 2A,
step a). After ester hydrolysis of this isomeric mixture (step b), we purified the
trans-2-benzyloxyfumaric acid (6) by recrystallization (37% yield over two steps).
This provided a higher yield of 6 when compared to the previously reported column
chromatography method for isomer separation.11b In addition, the efficiency of the
methylaspartate ammonia lyase (MAL-L384A)-catalyzed amination of 6 (step c) was
enhanced by using NH3 (instead of NH4Cl) and pH 9.5 (instead of pH 9.0), leading
to >98% conversion and affording product 1 in 80% isolated yield.
5

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Starting from compound 1, the synthesis of key intermediate 4 was achDieOvI: e10d.10i3n9/C7OB00305F
three successive reactions (steps d-f in Scheme 2B). Diesterification of 1 with SOCl2
in dry methanol, followed by Boc-protection, delivered compound 8 without the need
for purification. Hydrogenolysis of 8 using HCOONH4/Pd gave 4 in excellent yield
(71% over three steps).12 Notably, exhaustive esterification of both carboxyl groups of
compound 1 needs high SOCl2 concentration (10 equivalents) under reflux, while
mono-esterification of the C4-carboxyl group of 1 could be easily accomplished under
ambient conditions with one equivalent of SOCl2. Another route (steps g-i in Scheme
2B) for preparing the chiral building block 4 was also designed by rearranging the
three reactions described above. We initiated the synthetic procedure with
debenzylation of 1, which provided 3-hydroxyaspartic acid (9), and which was
followed by esterification and Boc-protection to afford 4 in three steps with 73%
overall yield.
Scheme 2. Chemoenzymatic synthesis of L-TBOA (1) and chiral building block 4.
Reagents and conditions: a) BnOH, DABCO, DCM, rt, 4 h; b) NaOH (2 M), reflux,
2h, then HCl (1 M), two-step yield after recrystallization was 37%; c) MAL-L384A
(0.01 mol%), 5 M NH3/NH4Cl, 20 mM MgCl2, pH = 9.5, 24 h, conversion >98%,
6

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isolated yield 80%; d) SOCl2, MeOH, reflux, 6 h; e) di-tert-butyl dicarbonate, DIEA,
DCM, rt, 24 h; f) Pd/C, HCOONH4, MeOH, reflux, 45 min, 71% for 3 steps; g) Pd/C,
HCOONH4, MeOH, reflux, 45 min; h) SOCl2, MeOH, reflux, 6 h; i) di-tert-butyl
dicarbonate, DIEA, DCM, rt, 24 h, 73% for
3
steps. DABCO,
1,4-diazabicyclo[2.2.2]octane;
dichloromethane.
DIEA,
N,N-diisopropylethylamine; DCM,
With chiral building block 4 in hand, the synthesis of target molecule
L-TFB-TBOA (2a) could be accomplished through O-alkylation, followed by global
deprotection (steps a-c in Scheme 3). This strategy provides a general synthesis route
towards derivatives of L-threo-3-hydroxyaspartic acid, including valuable analogs of
L-TBOA and L-TFB-TBOA. To facilitate efficient O-alkylation, which is effected by a
nucleophilic substitution reaction of 4 and 5 (for synthesis details, see SI), the strong
base NaH was used to deprotonate the hydroxyl group of 4. A low temperature
(-20 °C) was needed to avoid epimerization, and the desired compound 11 (i.e.,
globally protected 2a) was obtained with an isolated yield of 45%. Subsequently,
global deprotection of 11 was conducted via treatment with TFA and followed by
hydrolysis with LiOH, providing the desired final product 2a (L-TFB-TBOA
hydrochloride) in a yield of 59% over two deprotection steps.
As anticipated, product 2a was identified as the desired threo isomer (de >98%)
1
by comparison of its H-NMR signals and J-coupling values to those of an authentic
standard (commercially available L-TFB-TBOA) and chemically synthesized
DL-threo and DL-erythro stereoisomers (Table S1). To determine the absolute
configuration of product 2a, chiral HPLC analysis was conducted by using the
7

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authentic standard L-TFB-TBOA and chemically synthesized DL-TFB-TBOAD(OsIe: 1e0.1S0I3)9/C7OB00305F
as reference molecules. This analysis revealed that the chemoenzymatically produced
2a is present as a single enantiomer with exclusively the L-threo configuration
(ee >99%, Table 1). The usefulness of our synthesis strategy was further demonstrated
by the preparation of optically pure 2a at multigram-scale (2.1 g; see SI). In addition,
two novel optically pure L-TFB-TBOA analogs, 2b (o-CF3, de >98%, ee >99%) and
2c (m-CF3, de >98%, ee >99%), were prepared using this newly developed
methodology (Table 1).
Scheme 3. Synthesis of L-TFB-TBOA and its derivatives. Reagents and conditions: a)
ArCH2Br (5, see SI), NaH, DMF, -20 °C, 4 h, 42% - 50%; b) TFA/DCM (2:5, v/v),
0 °C, 1.5 h; c) THF/H2O (1:1, v/v), LiOH, rt, 2 h, then HCl (1 M), 38% - 59%; d)
3-nitrobenzylbromide, NaH, DMF, -20 °C, 4 h, 61%; e) Pd/C, H2, MeOH, 25 min,
92%; f) acyl chlorides (14, see SI), TEA, DCM, rt, 2 h, 51% - 65%. TFA,
trifluoroacetic acid; THF, tetrahydrofuran; TEA, trimethylamine.
Medicinal chemists are highly interested in building a large collection of
meta-substituted analogs of L-TBOA to screen for potent and selective inhibitors of
8

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EAAT subtypes.6b, 13 In order to rapidly construct a library of L-TBOA analogs with
various groups at the meta-position, we envisioned that intermediate 13, with a free
m-NH2 group, would be a convenient precursor for fast structural diversification by
applying combinatorial chemistry methodologies. The synthesis of compound 13 was
accomplished through a nucleophilic substitution reaction between 4 and
m-nitrobenzylbromide, yielding 12, followed by conversion of the nitro group to an
amino group with Pd/C/H2, in a yield of 59% over two steps. To demonstrate the
utility of intermediate 13, we synthesized two L-TFB-TBOA analogs (16a-b) with a
longer alkyl spacer between the two phenyl rings (Scheme 3). Compound 15 was
formed via amidation of the amino group of 13 with acyl chloride 14. Target products
16a and 16b were obtained in optically pure form after two steps of deprotection
(Table 1).
DOI: 10.1039/C7OB00305F
Conclusion
In conclusion, we have managed to construct the complex amino acid L-TFB-TBOA
using only 9 steps with 6% overall yield, starting from commercially available
dimethyl acetylenedicarboxylate. Compared with the previously reported 20-step
synthesis of L-TFB-TBOA, this is a dramatic reduction in step count with fewer than
half the steps. This chemoenzymatic synthesis methodology can be easily up-scaled to
multigram-scale and gives convenient access to enantiopure derivatives of L-TBOA
and L-TFB-TBOA
9

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DOI: 10.1039/C7OB00305F
Table 1. Absolute configuration of chemoenzymatically obtained L-TFB-TBOA and
its derivatives.
Entry No.
R
n
de [%]a
ee [%]b
Absolute
configuration
1
p-CF3
o-CF3
m-CF3
p-CF3
p-CF3
0
0
0
1
2
>98
>98
>98
>98
>98
>99
>99
>99
>99
>99
(2S,3S)c
(2S,3S)
(2S,3S)
(2S,3S)
(2S,3S)
2a
2
3
4
5
2b
2c
16a
16b
a Diastereomeric excess (de) was determined by 1H NMR. b Enantiomeric excess (ee)
c
was determined by chiral HPLC.
Absolute configuration was determined
1
unambiguously by comparison of H NMR, chiral HPLC and optical rotation data to
those of an authentic sample of L-TFB-TBOA (2S,3S).
Acknowledgements
This research was financially supported by the Netherlands Organisation for Scientific
Research (KIEM grants 731.013.110 and 731.015.108). Haigen Fu and Jielin Zhang
acknowledge funding from the China Scholarship Council. The authors thank Dr.
Marianne de Villiers for insightful discussions.
10

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DOI: 10.1039/C7OB00305F
Keywords
Asymmetric synthesis, biocatalysis, amino acids, inhibitor, glutamate transport.
Experimental Section
For detailed experimental procedures and characterization of compounds, see
supplementary information.
Notes and References
1
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TOC text
Rapid route to L-TFB-TBOA: Using only nine chemical transformations, we have
managed to construct the complex amino acid L-TFB-TBOA starting from
commercially available dimethyl acetylenedicarboxylate. Compared with the
previously reported 20-step synthesis of L-TFB-TBOA, this is a dramatic reduction in
step count with fewer than half the steps. This methodology can be easily upscaled to
multigram-scale and gives convenient access to enantiopure derivatives of
L-TFB-TBOA.
TOC graphic
13