Synthesis and preliminary pharmacological evaluation of novel derivatives of l-β-threo-benzylaspartate as inhibitors of the neuronal glutamate transporter EAAT3

Article abstract of DOI:10.1016/j.bmc.2008.07.001

A series of β-benzylaspartate derivatives were prepared from N-trityl-l-aspartate dimethyl ester and evaluated as inhibitors of neuronal glutamate transporter EAAT3. The result of the structure-activity studies suggests that the position occupied by the aromatic ring of β-benzylaspartate within the binding site of EAAT3 may be different from that occupied by comparable groups in previously identified inhibitors, such as l-threo-benzyloxy aspartate (TBOA). Further, halogen substitutions at the 3-postition of the aromatic ring of β-benzylaspartate can increase the potency with which the analogues inhibit EAAT3.

Full text of DOI:10.1016/j.bmc.2008.07.001

Bioorganic & Medicinal Chemistry 16 (2008) 7740–7748
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and preliminary pharmacological evaluation of novel derivatives
of L-b-threo-benzylaspartate as inhibitors of the neuronal glutamate
transporter EAAT3
*
Terri L. Mavencamp, Joseph F. Rhoderick, Richard J. Bridges, C. Sean Esslinger
NIH-COBRE Center for Structural and Functional Neuroscience, Department of Biomedical & Pharmaceutical Sciences, The University of Montana, Missoula, MT 59812, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of b-benzylaspartate derivatives were prepared from N-trityl-L-aspartate dimethyl ester and
Received 11 April 2008
Revised 1 July 2008
Accepted 2 July 2008
Available online 8 July 2008
evaluated as inhibitors of neuronal glutamate transporter EAAT3. The result of the structure–activity
studies suggests that the position occupied by the aromatic ring of b-benzylaspartate within the binding
site of EAAT3 may be different from that occupied by comparable groups in previously identified inhib-
itors, such as L-threo-benzyloxy aspartate (TBOA). Further, halogen substitutions at the 3-postition of the
aromatic ring of b-benzylaspartate can increase the potency with which the analogues inhibit EAAT3.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Pharmacophore
Bound conformation
Diastereoselective
Lipophilic pocket
1. Introduction
transporter function and the physiological consequences of de-
creased activity.
L
-Glutamate is the most abundant excitatory neurotransmitter
Five distinct glutamate transporters (EAATs 1–5) have been
identified by molecular cloning that, along with the sodium-
dependent neutral amino acid transporters ASCT1-2, comprise a
novel gene family (i.e., SLC1 in the Human Genome Organization
HUGO nomenclature).7 Uptake through the EAATs occurs via an
alternate access mechanism that is electrogenic and driven by ionic
gradients across the cell membrane. In this manner, the transport
of one molecule of glutamate into a cell is stoichiometrically cou-
pled to the import of three Na+ ions and one H+, and to the counter
transport of one K+8 While the EAATs share a similar mechanism
and ionic dependence, each exhibits a distinct localization.4 EAAT1
and EAAT2 are primarily considered to be glial transporters that
exhibit a preferential distribution in the cerebellum and forebrain,
respectively. In contrast EAAT3 is present on neurons and is en-
riched in forebrain areas. EAAT4 is localized to purkinje neurons
in the cerebellum, and EAAT5 is restricted to the retina. To a large
degree the EAATs can be pharmacologically distinguished from one
another based upon the comparative actions of a number of sub-
strates and inhibitors, although EAAT2 stands alone with respect
to readily available, well-characterized subtype-selective inhibi-
tors.5,9 Not surprisingly, considerable effort has been focused on
the development of inhibitors that can be used to selectively mod-
ulate the activity of the other individual transporters.
in the mammalian CNS and, as such, contributes to neuronal sig-
naling and cognitive function through its activation of a wide vari-
ety of ionotropic and metabotropic excitatory amino acid (EAA)
receptors.1 If extracellular glutamate concentrations in the CNS be-
come excessive, it can lead to the over-activation of EAA receptors
and the triggering of numerous neuropathological pathways.1–3
Termed excitotoxicity, glutamate-mediated neuronal injury is be-
lieved to contribute to CNS pathology in acute insults (ischemia,
traumatic injury), as well as chronic neurological disorders (e.g.,
ALS, Alzheimer’s disease, epilepsy, and Huntington’s disease).
Excitatory amino acid transporters (EAATs) present on neurons
and glia play a critical role in regulating extracellular levels of glu-
tamate and are thereby positioned to influence (i) the access of
neurotransmitter to synaptic and extrasynaptic EAA receptors,
(ii) the recycling of the neurotransmitter, and (iii) the accumula-
tion of excitotoxic levels of glutamate.4,5 The expression of EAATs
has also been found to be altered in neurological disorders such
as epilepsy, ischemia, spinal cord injury, amyotrophic lateral scle-
rosis (ALS), Alzheimer’s and schizophrenia.6 Much of our under-
standing of the function of the EAATs has been dependent upon
the development of substrates and inhibitors with which to probe
Toward this goal our laboratories prepared
-b-BA) and identified it as one of the few EAAT inhibitors
that exhibits a marked selectivity for EAAT3.10 The more potent
L-b-benzylaspartate
(L
* Corresponding author. Tel.: +1 406 243 4713; fax: +1 406 243 5228.
E-mail address: christopher.esslinger@umontana.edu (C.S. Esslinger).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.07.001

T. L. Mavencamp et al. / Bioorg. Med. Chem. 16 (2008) 7740–7748
7741
enantiomer,
L
-threo-b-benzylaspartate ((2S,3S) 3-benzylaspartate,
this step, yields of the mixture ranged from 13.5% to 87% following
chromatography. Yields generally improved with greater equiva-
lents of substituted-benzylic bromide, Table 1: 5 versus 6, as well
as higher temperatures, 2 versus 3, though diastereoselectivity de-
creased with increasing temperature. We found that the ratio of
SS:SR increases to 1:11 if the temperature is decreased to ꢀ55 °C
and the reaction is quenched at this cold temperature (entries 5
and 6, Table 1). If the reaction temperature is allowed to rise to
0 °C after addition of substituted-benzylic bromide the ratio of
SS:SR decreases to 1:2 to 1:1. Interestingly we found that adding
DMPU to the starting material reverses the stereochemistry to give
SS:SR in a 3:1 mixture of diastereomers.16
When KHMDS was used in place of LiHMDS we observed a
switch in diastereoselectivity similar to that observed by Hum-
phrey et al., with N-benzyl-N-9-phenylfluoren-9-yl dimethyl aspar-
tate.14 We propose that Li complexes tightly to the enolate but may
be forming a loose cyclic chelate between the enolate oxygen and
the nitrogen (Fig. 1), similar to that suggested by Fernàndez-Megía
and Sardina.15 This is supported by the decrease in selectivity ob-
served at higher temperatures, as well as the reversal of diastere-
oselectivity observed upon addition of DMPU.
L-t-b-BA), shows an approximate 10-fold preference for blocking
EAAT3 compared to either EAAT1 or EAAT2 in C17.2 cells express-
ing the human isotypes. This contrasts with activity of the closely
related and widely used inhibitor
L-threo-benzyloxy aspartate
(TBOA) which has been reported to more potently inhibit the activ-
ity of EAAT2 than EAAT3.11,12,14 In the present study a new series of
L
-b-benzylaspartate derivatives has been prepared from the easily
accessible precursor N-trityl- -aspartate dimethyl ester. The
analogues were evaluated as inhibitors of 3H-
-aspartate uptake
L
D
in C17.2 neuroprogenitor cells transiently transfected to express
human EAAT3. We report that structure–activity profile of these
inhibitors is quite different from TBOA-based analogues suggesting
that subtle variations in size, substituents and orientation of the
aromatic ring appended to the b-position of aspartate can notice-
ably influence activity at the EAATs. In particular, substitutions at
the 3-position of the aromatic ring of
for imparting potency at EAAT3.
L-t-b-BA are most favorable
2. Results and discussion
This reversal of selectivity suggests that the metal is being com-
plexed by DMPU, thereby disrupting the nitrogen–oxygen chelate
and allowing the molecule more rotational flexibility. This would
increase the probability that the Si face is open for attack. We
2.1. Preparation of b-substituted aspartates
The starting material N-trityl- -aspartate dimethyl ester (2) was
L
prepared following a general procedure. The aspartate dimethyl es-
ter was formed using thionyl chloride in methanol. N-Tritylation is
achieved with triphenylmethyl chloride and triethylamine.13 The
resulting starting material was purified by two subsequent crystal-
lizations from methanol. The triphenylmethyl protecting group
was chosen due to ease of preparation, mild deprotection condi-
tions, and the protection against alpha-carbon deprotonation it im-
parts to the molecule to avoid alpha-racemization (Scheme 1).
Initially, beta addition was achieved by the addition of lithium
hexamethyldisilyl amide (LiHMDS, 2 equiv in THF) to a 1 M solu-
tion of 2 in anhydrous THF at ꢀ32 °C under inert atmosphere. This
was followed by quenching with the desired substituted-benzylic
bromide (2–3 equiv) and allowing the temperature to rise to
0 °C.14 Substituted-benzylic bromides are shown in Table 2 as R,
A–J. The reaction was terminated by the addition of 2 N ammo-
nium chloride. This produced a mixture of 3, SS:SR with SR as the
major component in a 1:2 to 1:1 mixture of diastereomers. This
was verified by detailed NMR analysis and compared to similar
systems with similar reaction conditions reported that were
accompanied by in-depth stereochemical determinations.15 HPLC
hypothesize that potassium forms a cyclic enolate-a-ester chela-
tion as previously proposed14,15, giving the (S,S) product (Fig. 1).
Deprotection was achieved by heating the alkylated product (3)
in 6 N HCl to afford the chloride salt of the product. The reaction
was then neutralized to a pH of 7, and the mixture was loaded
on an anion exchange resin (acetate form, 10 g resin per gram
product), and eluted using a gradient of 1–5 N acetic acid. The
zwitterion was obtained by concentrating the ninhydrin positive
fractions followed by chasing the product with deionized water
several times. Alternatively, the product can be precipitated out
of ethyl acetate then water to give final product.
2.2. Inhibitory activity at EAAT3
The compounds were evaluated as inhibitors of EAAT3 by quan-
tifying their ability to reduce the uptake of 3H-
D-aspartate in stan-
dard competition assays.10 In each instance the analogues (SS:SR
ratio of approximately 1:1) were added simultaneously with the
radiolabel to achieve a final concentration of 100 lM and 25 lM,
respectively. The results are summarized in Table 2 as % of Control
activity, that is, uptake in the absence of any inhibitor. The replace-
separation of
L-b-BA diastereomers 4A gave NMR spectra consis-
tent with previous reports of similar aspartate syntheses.14–17 At
ment of the benzyl group on
resulted in a marked reduction of inhibitory activity. Interestingly,
when an analogous substitution is made with -TBOA to produce
-threo-b-(1-naphthyl)methyloxaspartate ( -TNOA1) and -threo-
b-(2-naphthyl)methyloxaspartate ( -TNOA2), the inhibitory activ-
L-b-BA with a naphthyl moiety (4B)
L
CO2H
CO2
base, E+
1) MeOH, SOCl2
2) TrCl, Et3N
CO2Me
CO2Me
L
L
L
THF
conditions
H3N
L
TrN
H
ity was retained (or increased) as reflected by a decrease in the
1
2
IC50 with which the compounds blocked the uptake of 14C-gluta-
mate into COS-1 cells expressing EAAT3 (i.e.,
TNOA1, 4.8 M; -TNOA2, 6.5
M).11 This would suggest that the
proposed lipophilic regions adjacent to the substrate binding do-
main on EAAT3 with which the benzyl groups of either -TBOA or
-t-b-BA interact can only accommodate a larger naphthyl group
when its orientation is dictated by the methoxy linkage of TBOA
and not the methylene linking group of -t-b-BA. Recent structural
L-TBOA, 7.0 lM; L-
MeO2C
HO2C
H3N
4i (S,S
Ar
Ar
l
L
l
TrN
H
CO2Me
1) HCl
CO2
L
L
)
3i (S,S
+
)
+
2) ion exchange
L
MeO2C
HO2C
Ar
Ar
insight into these substrate binding domains has emerged from
crystallographic studies in which the archaeal aspartate trans-
porter Gltph from Pyrococcus horikoshii has been crystallized in
TrN
H
CO2Me
3ii (S,R
H3N
CO2
)
4ii (S,R
)
the presence of either L-aspartate or L
-TBOA.18 It was concluded
that the binding site is positioned between two hairpin loops that
extend from opposite sides of the membrane and likely participate
Scheme 1. Synthesis of b-benzylaspartates. Tr, triphenyl methyl. See Table 1 and
Section 4 for base, E+, and conditions.

7742
T. L. Mavencamp et al. / Bioorg. Med. Chem. 16 (2008) 7740–7748
Table 1
Conditions used and results obtained in the benzylation reactions. S,S:S,R ratios as determined by 1H NMR
N-Protecting group + additives
Temperature (°C)
Base: E+
Time of rxn (h)
E+
S,S:S,R (i:ii)
% Yield
1. Trityl
2. Trityl
3. Trityl
4. Trityl DMPU
5. Trityl
6. Trityl
7. Trityl
8. Trityl
9. Trityl
ꢀ35 ? 0
ꢀ55
2:2 LiHMDS
2:2 LiHMDS
2.5:3 LiHMDS
2:3 LiHMDS
2:2 LiHMDS
4
6
6
21
21
21
21
21
21
3F
3F
3F
3Cl
3Cl
3Cl
3Nitro
3F
1:2
1:11
1:2
57
55
65
74
68
76
51
58
88
ꢀ55 ? 0
ꢀ55
3:1
ꢀ65
1:11
1:11
99:1
99:1
2.5:1
ꢀ65
2:3 LiHMDS
ꢀ55
2.1:2.3 KHMDS
2.1:2.3 KHMDS
2.1:2.3 KHMDS
ꢀ55
ꢀ55 ? 0
3Br
Re
Si
OMe
O
H
H
H
MeO
O
H
H
MeO2C
N
Li
K
NH
O
OMe
Lithium Enolate
Figure 1. Low temperature chelation controlled diastereomeric outcome of enolates using lithium and potassium bases.
Potassium Enolate
in the gating of substrate movement. While non-substrate inhibi-
tors such as -TBOA or -t-b-BA can fit into this site normally occu-
pied by -glutamate, interaction with nearby lipophilic residues in
the vicinity of the HP2 loop may preclude subsequent conforma-
tional movements that are necessary for substrate translocation,
such as the closure of the external HP2 gate or providing access
of external sodium to its requisite binding site. Interestingly, the
ability of an inhibitor to interact with EAAT3 in this manner may
increase its potency as an inhibitor, but decrease its ability to act
as an alternative substrate. Comparisons between the naphthyl
with both regional sidechain R groups and backbone peptide
bonds.19
To illustrate the diastereomeric preference of EAAT3 for ligand
binding as well as highlight the diastereoselectivity of the benzyla-
tion reaction depending on base used, the individual diastereomers
L
L
L
(S,S)4Gi and (S,R)4Gii of
uated as inhibitors of EAAT3. Competitive binding curves were ob-
tained using 3H-
-asparate at and by varying the
L-b-3-F-BA 4G were synthesized and eval-
D
1 lM
concentration of inhibitor (Fig. 2). Curve fitting using GraphPad
Prism 4.0 software to the equation Y ¼ 1=ð1 þ 10ðXꢀlog IC ÞÞ, where
50
derivatives of
L
-TBOA or
L
-b-BA suggest, however, that the exact
Y is the fractional % of control and X is the log[inhibitor] in M,
IC50 values were obtained for the two diastereomers ((S,S)-3-F-b-
placement of the aromatic rings within the binding site of EAAT3
is probably distinct and may additionally point to structural differ-
ences between the EAAT3 and EAAT2 subtypes.
The addition of a methyl (4D) or nitro (4I) group to the 3-posi-
tion of L-b-BA, as well as a methyl group to both the 3- and 5-posi-
BA IC50 = 2.46 0.9 lM, (S,R)-3-F-b-BA IC50 = 21.1 9.2 lM). In
agreement with that reported previously, the (S,S) diastereomer
is the more potent10,11, and in this case (S,S)4Gi exhibits a 10-fold
higher affinity for EAAT3 than (S,R)4Gii.
tions (4C), resulted in a retention of some inhibitory activity,
although to a lesser degree than found in the parent molecule
(e.g., about 30% of Control uptake vs 4%). Again, this may be indic-
ative of a more constrained binding arena surrounding the aro-
2.3. Molecular modeling
The recently published crystallographic study in which L-TBOA
matic ring of
L
-b-BA when compared to
L
-TBOA. In contrast to
is bound to the archaeal aspartate transporter Gltph provides a
strategy to conformationally compare these two inhibitors.18 The
similarities are readily apparent when the bound conformation of
these compounds, the di-substitution of chloro groups at the 2-
and 6-positions (4E) produced an almost complete loss of inhibi-
tory activity. In this instance the decreased ability to bind to EAAT3
may be attributable to steric clashes with the transporter itself or,
given the locations on the aromatic ring, between the substituents
and the carbon backbone of aspartate when it assumes the requi-
site conformation for binding. Comparisons among additions made
at the 3-position revealed a rank order of inhibitory activity of
L
-TBOA is extracted from crystal structure and overlaid with a com-
putationally minimized10 conformation of
-threo-b-BA (Fig. 3).
While the aromatic rings are positioned in a similar direction it
also appears that the benzyl group of -threo-b-BA is considerably
closer to the carbon backbone of the amino acid than that of
-TBOA. In this respect the aromatic rings of the two analogues
L
L
L
methyl (4D) ꢁ nitro (4I) < bromo (4F) < fluoro (4G). Lastly,
F-BA (4G) and -b-4-F-BA (4H) inhibited EAAT3-mediated uptake
to a similar or greater extent than observed with either -b-3-Br-
BA or the parent -b-BA. This would be consistent with the steric
argument that the lipophilic region interacting with the benzyl
group of -b-BA in the binding site of EAAT3 is spatially confined,
as well as reflects the a ability of fluoro groups to favorably interact
L
-b-3-
are likely interacting with subtly different lipophilic regions within
the binding domain on EAAT3. Our SAR data would suggest that in
L
L
the instance of
accommodating of steric bulk than the comparable region delin-
eated by -TBOA. Further, it is tempting to speculate that the ability
of -threo-b-BA to interact with these portions of the binding site
may be a factor in its selectivity for EAAT3.
L-threo-b-BA, this region of the protein is less
L
L
L
L

T. L. Mavencamp et al. / Bioorg. Med. Chem. 16 (2008) 7740–7748
7743
Table 2
trometry. Intermediates were treated with trifluoroacetic acid
prior to mass spectrometry to remove the trityl group, so the signal
of the molecular ions could be detected. Proton and carbon NMR
were taken on a Varian 400 MHz NMR in CDCl3, D2O or DMSO with
TMS as the internal standard. NMRs were reported as mixtures of
SS:SR with each diastereomer being identified where possible. Dia-
stereomers on carbon NMR are indicated in parentheses where car-
bon peaks of each molecule are resolved. Chirality was verified by
optical rotation on a Perkin-Elmer 241 Polarimeter in 1 N HCl un-
less otherwise specified in a 1.0 dm tube. Reagents were purchased
from Sigma (St. Louis, MO) and were used without further purifica-
tion. Microanalyses were performed for C, H, and N (Midwest
Microlab, Indianapolis, IN) and were within 0.4% of theoretical
Percent of control uptake in the presence of L-b-benzylaspartate derivatives (control
uptake with no inhibitor present is 100%)
Compound
R-Group
EAAT3 uptake
3H- -Asp (25
(% of control)
D
lM)
+H3N CO2-
R
HO2C
L
L
L
-b-Benzyl-asp 4A
4
2
1
-b-Benzyl-asp S,S-4i
-b-Methyl-2-napthyl-asp 4B
1
values. Compounds showing potency equal to or greater than L-
68
6
7
b-benzyl-aspartate (approximate 1:1 S,S:S,R mixture) were re-
solved by semi-preparative reverse phase HPLC. The detection
method was a Waters 486 tunable absorbance detector set at
254 nm.
CH3
L
-b-3,5 Dimethyl-benzyl-asp 4C
36
H3C
4.1. N-Trityl aspartate dimethyl ester (2)
L
L
-b-3-Methyl-benzyl-asp 4D
33
86
3
5
H3C
Cl
L(+)-Aspartic acid (26 g, 100 mmol) was stirred in a round-bot-
tomed flask in a 1 M solution of methanol (200 ml). Thionyl chlo-
ride (22 ml, 140 mmol) was added dropwise. The solution was
stirred at room temperature for 48 h. The resulting dimethyl ester
aspartate hydrochloride was washed three times with methanol,
three times with methylene chloride, two times with toluene,
and once with water. The aspartate dimethyl ester was then sus-
pended in methylene chloride (200 ml) and dried with magnesium
sulfate. Triphenylmethyl chloride (53 g, 95 mmol) was added fol-
lowed by the dropwise addition of triethylamine (83.6 g,
300 mmol). The reaction was stirred overnight, then diluted with
ether and filtered through a silica plug with a 30:70 solution of
ethyl acetate/hexanes. The filtrate was concentrated and rinsed
two times with methylene chloride and recrystallized from meth-
anol to yield N-trityl aspartate dimethyl ester 2 (70%). 1H NMR
(400 MHz, CDCl3) d: 7.54–7.48 (m, 6H), 7.32–7.15 (m, 9H), 3.77–
3.71 (m, 1H), 3.69 (s, 3H), 3.28 (s, 3H), 2.99–2.91 (s, 1H), 2.73–
2.62, (m, 1H), 2.58–2.48 (m, 1H). 13C NMR (100 MHz, CDCl3) d:
Cl
-b-2,6-Dichloro-benzyl-asp 4E
L
L
L
L
L
-b-3-Br-Benzyl-asp 4F
-b-3-F-Benzyl-asp 4G
-b-4-F-Benzyl-asp 4H
-b-3-Nitro-benzyl-asp 4I
-b-4-Nitro-benzyl-asp 4J
8
3
0
3
Br
F
0
7
F
29
5
O2N
65
8
173.86, 170.95, 145.61, 128.71, 127.83, 126.48, 71.13, 53.63,
21
NO2
C17.2 cells assayed for their ability to block 25
presence of 100 M inhibitor, n P 3. Inhibitors in an approximate 1:1 ratio of
51.91, 51.71, 40.16. [
a]
13.3° (ethyl acetate).
D
l
M D aspartate uptake in the
3H-
l
4.2. General procedure for alkylation
(S,S):(S,R) unless specified.
Compound 2 was placed in a flame dried round-bottomed flask
equipped with a stir bar. Anhydrous THF was added under argon
and the solution was cooled. Once cooled 1 M LiHMDS in THF
(2–3 equiv) was added via needle and syringe. After approximately
20 min the desired substituted-benzylic bromide (2–3 equiv) was
added. The temperature was then allowed to rise to 0 °C (unless
specified) and the reaction mixture was stirred until quenched
(4–24 h) with 2 N NH4Cl. Water and ethyl acetate (or ether) were
added for separation. The water layer was subsequently washed
three times and the organic layers were combined and concen-
trated. Flash chromatography, when needed, through silica gel
using hexane/ethyl acetate in an approximate 85:15 ratio gave
the desired protected substituted benzylaspartate in good to mod-
erate yields.
3. Conclusions
A series of
L-b-benzylaspartate derivatives have been prepared
from N-trityl-L-aspartate dimethyl ester in which diastereomeric
control was achieved using either lithium hexamethyldisilazide
to yield predominantly the (2S,3R) adduct in one example or potas-
sium hexamethyldisilazide to yield predominantly the (2S,3S) ad-
duct in one example and evaluated as inhibitors of the neuronal
glutamate transporter EAAT3. SAR data, including the inactivity
of
of
L
-b-methyl-2-naphthyl-aspartate (4B) and the increased activity
L
-b-3-F-benzylaspartate relative to the lead analogue -b-benzyl-
L
aspartate, suggest a confined interaction between lipophilic do-
mains of EAAT3 and the aromatic moiety of -b-benzylaspartate
that is distinct from previously characterized inhibitors, such as
-TBOA. These finding will be further explored in pursuit of more
L
L
4.3. N-Tritylamino dimethyl ester b-benzylaspartate (3a)
potent and selective ligands for the EAAT subtypes.
To 2 (1.55 g, 3.84 mmol) in 1 M THF under argon at ꢀ23 °C was
added LiHMDS (9.6 ml, 11.53 mmol). After 15 min benzyl bromide
(1.15 ml, 9.6 mmol) was added and temperature allowed to rise to
0 °C at which time it was stirred for a subsequent 2.5 h. The reac-
tion was quenched and ether was added for separation followed by
4. Experimental section
In order to verify the purity and composition of the compounds
product analysis was performed by high resolution mass spec-

7744
T. L. Mavencamp et al. / Bioorg. Med. Chem. 16 (2008) 7740–7748
4.5. N-Tritylamino dimethyl ester b-3,5 dimethyl-benzylaspar-
tate (3c)
IC50 Curves for S,S-3-F-βBA
and S,R-3-F-βBA
1.00
0.75
0.50
0.25
0.00
S,S-βBA
To 2 (3.2 g, 8.02 mmol) in 1 M THF under argon at ꢀ55 °C was
added LiHMDS (24 mmol). Twenty minutes later 3,5-dimethylben-
zyl bromide was added (6.4 g, 32.1 mmol) as a solid all at once.
Temperature was allowed to rise to 0 °C, at which time it was stir-
red for an additional 1.5 h before being quenched with 2 N NH4Cl
(12 ml). Ethyl acetate was added for separation, followed by silica
column 85:15 hexane/ethyl acetate (61%, 3.9 g). 1H NMR
(400 MHz, CDCl3) d: 7.47 (m, 6H), 7.29–7.20 (m, 9H), 6.85–6.76
(m, 3H), (3.86–3.83 (m, .66H, S,S), 3.72–3.68 (m, .33H, S,R)), (3.63
(s, 1H, S,S), 3.60 (s, 2H, S,R)), (3.25 (s, 1H, S,S), 3.20 (s, 2H, S,R)),
3.03–2.74 (m, 3H), (2.29 (5H app. s, S,S), 2.17 (1H app. s, S,R)).
13C (100 MHz, CDCl3) d: (173.05, 172.96), (172.80, 172.69),
145.52, (139.05, 138.93), (137.78, 137.69), (128.81, 128.69),
127.93, 127.87, 127.78, 127.17, 126.72, 126.58, 126.48, 126.37,
(71.16, 70.93), (58.19, 57.79), (52.86, 52.77), (51.78, 51.72),
S,R-βBA
-7.5
-6.5
-5.5
-4.5
-3.5
-2.5
log[I]
Figure 2. IC50 curves for (S,S)-3-F-b-BA and (S,R)-3-F-b-BA with [3H-
tate] = 1 M. (S,S)-3-F-b-BA IC50 = 2.46 0.9 M (S,R)-3-F-b-BA IC50 = 21.1 9.2
(IC50 SEM).
D-aspar-
l
l
lM
(51.65, 51.60), (33.93, 33.36), 21.21. HRMS m/e calcd for
+
C
13H18NO4 280.1549, found 280.1540.
a silica plug (85:15 hexanes/ethyl acetate) (82.7%: 1.57 g). 1H NMR
(400 MHz, CDCl3) d: 7.57–7.48 (m, 6H), 7.32–7.20 (m, 14H), (3.94–
3.90 (m, .5H, S,S), 3.75–3.72 (m, .5H, S,R)), (3.65 (s, 1.5H, S,S), 3.60
(s, 1.5H, S,R)), (3.28 (s, 1.5H, S,S), 3.23 (s, 1.5H, S,R)), 3.19–2.88 (m,
3H). 13C NMR (100 MHz, CDCl3) d: 172.77, 172.51, 145.43, (139.15,
138.96), 129.12, 128.72, 127.71, 126.40, 126.22, (71.07, 70.93),
(58.11, 57.67), 52.82, (51.65, 51.95), (51.53, 51.48), (34.05,
33.47). HRMS m/e calcd for C13H17NO4+ 252.1236, found 252.1225.
4.6. N-Tritylamino dimethyl ester b-3 methyl-benzylaspartate
(3d)
To 2 (1.86 g, 4.61 mmol) in 1 M THF under argon at ꢀ30 °C was
added LiHMDS (13.8 mmol) slowly. Twenty minutes later 3 methyl
benzyl bromide was slowly added (1.56 ml, 11.5 mmol). Tempera-
ture was allowed to rise to 0 °C, at which time it was stirred for an
additional 4 h before being quenched with 2 N NH4Cl (7 ml). Ethyl
acetate was added for separation, followed by silica column 85:15
hexane/ethyl acetate (1.05 g, 45%). 1H NMR (400 MHz, CDCl3) d:
7.48–7.45 (m, 7H), 7.32–7.13 (m, 10H), 7.03–6.93 (m, 2H), (3.86–
3.81 (m, .66H, S,R), 3.68–3.64 (m, p. obsc., .33H, S,S)), (3.63 (s, 1H,
S,S), 3.59 (s, 2H, S,R)), (3.24 (s, 1H, S,S), 3.19 (s, 2H, S,R)), 3.08–
2.77 (m, 3H), (2.33 (s, 2H, S,R), 2.30 (s, 1H, S,S)). 13C NMR
(100 MHz, CDCl3) d: 172.99, 172.74, 145.55, 139.15, 139.01,
129.71, 128.83, 127.81, 126.51, 125.82, (71.16, 70.98), (58.19,
57.79), (52.89, 52.86), (51.84, 51.77), (51.71, 51.65), (34.02,
4.4. N-Tritylamino dimethyl ester b-2-naphthylmethyl-
aspartate (3b)
To 2 (2.5 g, 6.2 mmol) in 1 M THF under argon at ꢀ35 °C was
added LiHMDS (18.6 mmol). Twenty minutes later 2-naphthyl
methyl bromide was added (3.4 g, 15.5 mmol) as a solid all at once.
Temperature was allowed to rise to 0 °C, at which time it was stir-
red for an additional 4 h before being quenched with 2 N NH4Cl
(9 ml). Ethyl acetate was added for separation, followed by silica
column 85:15 hexane/ethyl acetate (1.68 g, 50%). 1H NMR
(400 MHz, CDCl3) d: 7.84–7.74 (m, 3H), 7.66–7.59 (m, 1H), 7.47–
7.43 (m, 7H), 7.37–7.29 (m, 1H), 7.18–7.26 (m, 8H), 7.16–7.11
(m, 1H), 7.09–6.99 (m, 1H) (3.91–3.87 (m, .4H, S,S), 3.73–3.70 (m,
.6H, S,R)), (3.61 (s, 1.2H, S,S), 3.56 (s, 1.8H, S,R)), (3.27 (s, 1.2H,
S,S), 3.22 (s, 1.8H, S,R)), 3.19–2.90 (m, 3H). 13C (100 MHz, CDCl3)
d: 173.20, 172.93, 145.76, 135.32, 133.38, 129.80, 129.07, 129.01,
128.93, 128.19, 128.08, 128.04, 127.96, 127.00, 126.81, 126.72,
126.64, (71.41, 71.31), 60.63, 52.98, 52.02, 49.66, 34.30. HRMS m/e
+
33.46), 21.38. HRMS m/e calcd for C14H19NO4 266.1392, found
252.1225.
4.7. N-Tritylamino dimethyl ester b-2,6 dicloro-benzylaspartate
(3e)
To 2 (4.35 g, 10.78 mmol) in 1 M THF under argon at ꢀ35 °C was
added LiHMDS (32.3 mmol). After stirring at ꢀ35 °C for 15 min,
2,6-diclorobenzyl bromide was added by removing the septa and
+
calcd for C17H20NO4 302.1392, found 302.1388.
Figure 3. Overlay of
L-TBOA and L-threo-b-BA using the bound conformation of L-TBOA and similar conformation of L-threo-b-BA. Viewed by (A) looking down perpendicular
to the plane of the aromatic ring, and (B) looking parallel to the plane of the aromatic ring.

T. L. Mavencamp et al. / Bioorg. Med. Chem. 16 (2008) 7740–7748
7745
quickly adding the dry reactant (6.47 g, 26.95 mmol). Temperature
was allowed to rise to 0 °C, at which time it was stirred for an addi-
tional 5 h before being quenched with 2 N NH4Cl (15 ml). Ethyl
acetate was added for separation. No starting material was ob-
served by TLC, and NMR showed disappearance of starting material
with no breakdown to aspartic acid. 1H NMR (400 MHz, CDCl3) d:
7.53–7.49 (m, 6H), 7.29–7.15 (m, 11H), 7.10–7.06 (m, 1H), (3.94–
3.90 (m, .5H, S,R), 3.78 (m, .5H, S,S)), (3.63 (s, 1.5H, S,S), 3.62, (s,
1.5H, S,R)), 3.55–3.38 (m, 2H), (3.20 (s, 1.5H, S,S), 2.19 (s, 1.5H,
S,R)), 3.06–3.02 (m, 1H). 13C NMR (100 MHz, CDCl3) d: (172.72,
172.51), (172.31, 172.17), 145.46, 135.75, 135.29, 134.90, 129.40,
128.84, 127.74, 127.22, 127.02, 126.42, 126.34, (71.25, 70.90),
(58.08, 57.70), (51.86, 51.74), (51.68, 51.60), (50.11, 49.25),
ethyl acetate and hexanes 15:85 gave product in 58% yield
(.407 g). 1H NMR (400 MHz, CDCL3) d: 7.51–7.46 (m, 7H), 7.27–
7.20 (m, 11H), 7.00–6.86 (m, 1H), 3.69 (s, 3H), 3.64–3.60 (p. obsc.,
m, 1H-a), 3.28 (s, 3H), 2.95 (m, 1H-b); (2.69–2.65 (m, 1H), 2.54–
2.50 (m, 1H). 13C NMR (100 MHz, CDCl3) d: 173.04, 172.64,
161.81, 145.86, 142.09, 130.02, 129.05, 128.11, 126.82, 124.82,
(116.13, 115.91), (113.62, 113.41), 71.32, 58.30, 52.86, 52.08,
51.99, 33.33.
4.11. N-Tritylamino dimethyl ester b-3 fluoro-benzylaspartate
(3g S,R)
Trityl aspartate (.5 g, 1.24 mmol) was placed in a flame-dried
round-bottomed flask equipped with stir bar. Anhydrous THF
(5 ml) was added under argon and reaction mixture was cooled
to ꢀ55 °C. Once cooled LiHMDS in THF (2.5 ml) was slowly added,
and 20 min later 3-floro benzyl bromide was slowly added (.3 ml,
2.48 mmol). The reaction mixture was stirred for an additional
21 h before being quickly quenched with 2 N NH4Cl (4 ml). Sepa-
rate by adding water and ethyl acetate. Wash water layer two more
times with ethyl acetate, combine organic layers, and concentrate.
Purification by chromatography on silica gel using ethyl acetate
and hexanes 15:85 gave product in 55% yield (.35 g). 1H NMR
(400 MHz, CDCL3) d: 7.51–7.46 (m, 6H), 7.31–7.27 (m, 9H), 7.22
(d, 1H, J = 7.33), 7.00 (d, 1H, J = 7.33), 6.94–6.92 (m, 2H), 3.95 (m,
.1H, S,S), 3.68 (m, .9H, S,R), 3.65 (s, .3H, S,S), 3.60 (s, 2.7H, S,R),
3.27 (s, .3H, S,S), 3.22 (s, 2.7H, S,R), 3.08–2.85 (m, 3H).
+
(30.28, 29.62). HRMS m/e calcd for C13H15Cl2NO4 320.0456, found
320.0441.
4.8. N-Tritylamino dimethyl ester b-3 bromo-benzylaspartate
(3f)
To 2 (2.27 g, 5.63 mmol) in 1 M THF under argon at ꢀ35 °C was
added LiHMDS (16.9 mmol) slowly. Twenty minutes later 3-bro-
mo-benzyl bromide was added (3.5 g, 14.1 mmol) as a solid all at
once. Temperature was allowed to rise to 0 °C, at which time it
was stirred for an additional 2.5 h before being quenched with
2 N NH4Cl (5 ml). Ethyl acetate was added for separation, followed
by silica column 85:15 hexane/ethyl acetate (56%, 1.8 g). Spectra
S,R, separated via column chromatography. S,R comes off first fol-
lowed by S,S. Spectra reported for S,R 1H NMR (400 MHz, CDCl3)
d: 7.46–4.44 (m, 5H), 7.34–7.29 (m, 5H), 7.26–7.08 (m, 9H), 3.86–
3.82 (m, 1H), 3.58 (s, 3H), 3.21 (s, 3H), 2.99–2.78 (m, 3H). 13C
NMR (100 MHz, CDCl3) d: 173.04, 172.55, 145.68, 141.96, 132.15,
130.16, 129.72, 129.05, 128.11, 127.85, 126.82, 122.63, 71.34,
4.12. N-Tritylamino dimethyl ester b-4-fluoro-benzylaspartate
(3h)
To 2 (.536 g, 1.33 mmol) in 1 M THF under argon at ꢀ30 °C was
added LiHMDS (2.66 mmol) slowly. Twenty minutes later 4-fluoro-
benzyl bromide was added (.33 ml, 2.66 mmol) dropwise. Temper-
ature was allowed to rise to 0 °C, at which time it was stirred for an
additional 2 h before being quenched with 2 N NH4Cl (6 ml). Ethyl
acetate was added for separation, followed by silica column 85:15
hexane/ethyl acetate (.517 g, 76%). Found: C, 75.06; H, 6.00; N,
2.74. C32H30FNO4 requires C, 75.13; H, 5.91; N, 2.74. 1H NMR
(400 MHz, CDCl3) d: 7.48–7.44 (m, 6H), 7.29–7.15 (m, 9H), 7.13–
7.05 (m, 2H), 6.99–6.89 (m, 2H), (3.86–3.82 (m, .5H, S,R), 3.69–
3.65 (m, .5H, S,S)), (3.63 (s, 1.5H, S,S), 3.58 (s, 1.5H S,R)), (3.24 (s,
1.5H, S,S), 3.19 (s, 1.5H, S,R)), 3.09–2.78 (m, 3H). 13C NMR
(100 MHz, CDCl3) d: (172.83, 172.78), 172.51, (172.71, 160.28),
145.44, (134.85, 134.82), (134.65, 134.62), (130.45, 130.38),
(130.35, 130.27), 128.77, 128.65, 127.78, 126.52, 126.42, (115.26,
115.20), (115.05, 115.01), (71.13, 70.99), (58.05, 57.52), 53.00,
(51.89, 51.75), (51.72, 51.65), (33.26, 32.67).
+
58.32, 52.77, 51.96, 33.21. HRMS m/e calcd for C13H16BrNO4
330.0341, found 330.0336.
4.9. N-Tritylamino dimethyl ester b-3 fluoro-benzylaspartate
(3g)
To 2 (.5 g, 1.24 mmol) in 1 M THF under argon at ꢀ35 °C was
added LiHMDS (2.5 ml) slowly. Twenty minutes later 3-fluoro-ben-
zyl bromide was slowly added (.304 ml, 2.40 mmol). Temperature
was allowed to rise to 0 °C, at which time it was stirred for an addi-
tional 4 h before being quenched with 2 N NH4Cl (3 ml). Ethyl ace-
tate was added for separation, followed by silica column 85:15
hexane/ethyl acetate (57%, .363 g). 1H NMR (400 MHz, CDCl3) d:
7.47–7.43 (m, 6H), 7.32–7.18 (m, 10H), 6.98–6.83 (m, 3H), (3.86–
3.82 (m, .66H, S,R), 3.64–3.60 (m, .33H, S,S)); (3.64 (s, 1H, S,S),
3.59 (s, 2H, S,R)); (3.25 (s, 1H, S,S), 3.20 (s, 2H, S,R)); 3.05–2.87
(m, 3H). 13C NMR (100 MHz, CDCl3) d: 173.04, 172.64, 161.81,
145.86, 142.09, 130.02, 129.05, 128.11, 126.82, 124.82, (116.13,
115.91), (113.62, 113.41), 71.32, 58.30, 52.86, 52.08, 51.99, 33.33.
4.13. N-Tritylamino dimethyl ester b-3-nitro-benzylaspartate
(3i)
+
HRMS m/e calcd for C13H16FNO4 270.1142, found 270.1141.
To 2 (3.546 g, 8.8 mmol) in 1 M THF under argon at ꢀ23 °C was
added LiHMDS (26.4 mmol) slowly. Twenty minutes later 3-nitro-
benzyl bromide was added (4.7 g, 21.97 mmol) as a solid all at
once. Temperature was allowed to rise to 0 °C, at which time it
was stirred for an additional 4 h before being quenched with 2 N
NH4Cl (13 ml). Ethyl acetate was added for separation, followed
by silica column 85:15 hexane/ethyl acetate (13.5%, .64 g). 1H
NMR (400 MHz, CDCl3) d: 8.20–8.10 (m, 3H), 8.07–8.04 (m, 1H),
7.60–7.17 (m, H), (3.95–3.90 (m, .75H, S,R), 3.76–3.70 (m, .15H,
S,S)), (3.66 (s, .45H, S,S), 3.63, (s, 2.55H, S,R)), (3.28 (s, .45H, S,S),
3.25 (s, 2.55H, S,R)), 3.08–2.98 (m, 3H), 2.91 (s, .75H), 2.88 (s,
.25H). 13C (100 MHz, CDCl3) d: 172.68, 182.36, 145.32, 135.47,
135.40, 129.81, 129.25, 128.77, 128.63, 128.33, 128.18, 127.93,
127.87, 126.99, 126.69, 126.55, 123.88, 123.75, 121.64, 121.56,
4.10. N-Tritylamino dimethyl ester b-3 fluoro-benzylaspartate
(3g, S,S)
Trityl aspartate (.555 g, 1.38 mmol) was placed in a flame-dried
round-bottomed flask equipped with stir bar. Anhydrous THF
(5.5 ml) was added under argon and reaction mixture was cooled
to ꢀ35 °C. Once cooled 20% KHMDS in THF (3.2 ml) was slowly
added, and 20 min later 3-floro benzyl bromide was slowly added
(.39 ml, 3.16 mmol). The reaction mixture was stirred for an addi-
tional 21 h before being quickly quenched with 2 N NH4Cl (4 ml).
Separate by adding water and ethyl acetate. Wash water layer
two more times with ethyl acetate, combine organic layers and
concentrate. Purification by chromatography on silica gel using

7746
T. L. Mavencamp et al. / Bioorg. Med. Chem. 16 (2008) 7740–7748
71.23, (57.99, 57.59), 52.25, 52.12, 51.92, (33.71, 32.70). HRMS m/e
126.54, 71.02, 58.00, 52.48, 51.77, 51.71, 32.94. HRMS m/e calcd
calcd for C13H16N2O6+, 297.087, found 297.1076.
for C13H16ClNO4+ 286.0846, found 286.0840.
4.14. N-Tritylamino dimethyl ester b-3-nitro-benzylaspartate
(3i, S,S)
4.17. General procedure for deprotection
The protected b-substituted aspartate was taken up in 6 N HCl
and refluxed for 3–12 h. The reaction mixture was then diluted
with water and washed three times with ether. The water layer
was concentrated and neutralized with NH4OH and loaded onto
an Ag 1X 8 acetate form ion exchange resin in the ratio of 1 g prod-
uct to 10 g resin. The product was eluted with varying concentra-
tions of acetic acid from .1 to 5 N. Product came off around 2 N.
Water was lyophilized 2–3 times to remove the acetic acid.
Trityl aspartate (.5 g, 1.34 mmol) was placed in a flame dried
round-bottomed flask equipped with stir bar. Anhydrous THF
(5 ml) was added under argon, and the solution was cooled to
ꢀ55 °C. Once cooled KHMDS (20% in THF, 2.6 mmol) was slowly
added, and 20 min later 3-nitro-benzyl bromide was added
(.634 g, 2.9 mmol) as a solid all at once. The reaction mixture
was stirred for an additional 21 h before being quenched with
2 N NH4Cl (6 ml). Ethyl acetate was added for separation. The
water layer was washed two more times, and the organic layers
were concentrated down for separation on silica in 15% ethyl
acetate, 85% hexanes (51%, .34 g). 1H NMR (400 MHz, CDCl3) d:
8.26–8.00 (m, 1H), 7.52–7.37 (m, 4H), 7.52–7.16 (m, 15H), 3.63
4.18. b-Benzylaspartate (4A)
The protected benzyl aspartate (1.496 g) was taken up in 6 N
HCl and refluxed for 3 h. Product came off at 2 N as indicated by
NMR (8.6%, .058 g). 1H NMR (400 MHz, D2O) d: 7.32–7.21 (m,
2H), 7.25–7.21 (m, 3H), (4.04 (d, .25H, S,S, J = 3.88), 3.96 (d, .75H,
S,R, J = 3.88), (3.40 (ddd .75H, S,R, J = 3.88, 6.47, 9.06), 3.24 (ddd,
.25H, S,S, J = 3.88, 6.47, 9.06) 3.09–3.01 (m, 1H), 2.95–2.89 (m,
1H). 13C (100 MHz, CDCl3) d: 172.69, 169.64, (139.69, 138.69),
(s, 3H), 3.58 (m, 1H-b), 3.30 (s, 3H), 3.27–3.25 (m, 1H-a), 3.22–
3.15 (m, 2H), 3.03–2.94 (m, 2H). 13C (100 MHz, CDCl3) d:
172.36, 145.34, 141.06, 135.47, 129.01, 128.77, 128.63, 127.87,
127.66, 126.55, 123.75, 121.64, 71.23, 57.59, 52.24, 52.10,
51.91, 33.71.
129.07, 128.42, 126.54, (54.85, 52.91), (49.66, 47.87), (33.46,
+
4.15. N-Tritylamino dimethyl ester b-p-nitro-benzylaspartate
(3j)
32.99). HRMS m/e calcd for
C
11H14NO4 224.0923, found
21
224.0923. [
a]
+29.8°.
D
Compound 2 (3.123 g, 7.74 mmol) in 1 M anhydrous THF under
argon was cooled to ꢀ40 °C. Once cooled 1 M LHMDS (23.2 mmol)
was added. After 20 min p-nitro benzyl bromide, dissolved in
anhydrous THF, was added (4.18 g, 19.35 mmol). The temperature
was then allowed to rise to 0 °C and the reaction mixture was stir-
red for 4 h at which time the reaction was quenched with 2 N
NH4Cl (10 ml). Water and ether were added for separation. The
water layer was subsequently washed three times, and the ether
layers were combined and concentrated down. Chromatography
through silica gel (90% hexanes, 9.5% ethyl acetate, and 0.5% trieth-
ylamine) loaded using methylene chloride gave 3 (3.63 g, 87%) as a
yellow solid. 1H NMR (400 MHz, CDCl3) d: (8.14 (d, 1.55H, S,R,
J = 8.06), 8.08 (d, .44H, S,S, J = 8.06)), 7.43 (dd, 6H, J = 7.33, 8.79),
7.34 (d, 2H, J = 8.79), 7.28 (dd, 2H, J = 7.33), 7.26 (m, 2H), 7.26 (d,
1.55H, S,R, J = 8.06), 7.22 (d, .44H, S,S, J = 8.06), 7.21 (d, 2H,
J = 7.33), 7.19, (d, 1H, J = 7.33). 3.90–3.87 (m, .8H, S,R), (3.63 (s,
.65H, S,S), 3.57 (s, 2.33H, S,R)), (3.25 (s, .65H, S,S), 3.24 (d, 2.33H,
S,R)), 3.03–2.94 (m, 3H). 13C (100 MHz, CDCl3) d: 172.65, 171.98,
145.34, 129.83, 128.77, 127.92, 126.69, 123.62, 58.03, (52.25;
51.92), (32.94, 30.88). HRMS m/e calcd for C13H16N2O6+, 297.087,
found 297.1076.
4.19. S,S -b-Benzylaspartate (4A)
L
The solvent system comprised of buffered solution of ammo-
nium acetate and water (0.1 M at pH 6.4). Reversed phase C18
3l analytical and 10l semi-prep columns were used with a reten-
tion time for S,S being 2.59 min and 16.00 min, respectively, and
for S,R 3.19 min and 21.00 min, respectively. 1H NMR (400 MHz,
D2O) d: 7.29–7.25 (m, 2H), 7.21–7.17 (m, 3H), 3.89 (d, 1H,
J = 3.24), 2.99 (ddd, 1H, J = 3.24, 4.53, 11.00), 2.79–2.66 (m, 2H).
13C NMR (400 MHz, D2O) d: 22.20, 33.46, 49.41, 55.96, 126.96,
25
128.96, 139.02, 176.693. S,S: [
a]
+14.11.
D
4.20. b-2 Naphthylmethylaspartate (4B)
The protected b-alkylated aspartate (1.68 g, 3.09 mmol) was ta-
ken up in 6 N HCl and refluxed for 4 h. Ethyl ether was added for
separation; the water layer was washed three times and concen-
trated. Product crashes out of solution upon neutralization with
NaOH. The pH was adjusted to about 12 and the product was pre-
cipitated out of methanol (.5 g, 51%). 1H NMR (400 MHz, D2O) d:
7.83–7.78 (m, 3H), 7.66 (app. s, 1H), 7.47–7.35 (m, 3H), 3.60 (m,
1H, S,S), 3.27–3.25 (m, 1H, S,R), 3.03–2.94 (m, 1H), 2.86–2.83 (m,
1H), 2.69–2.60 (m, 1H). 13C NMR (100 MHz, DMSO) d: (172.93.
172.82), (170.44, 170.12), 136.99, 133.65, 132.50, 129.93, 129.29,
128.29, 128.13, 127.70, 127.50, 126.65, 126.11, (55.27, 53.18)
(50.54, 48.62), (33.53, 31.39). 18.27 = acetic acid. HRMS m/e calcd
4.16. N-Tritylamino dimethyl ester b-3-chloro benzylaspartate
Trityl aspartate (.50 g, 1.2 mmol) was placed in a flame-dried
round-bottomed flask equipped with stir bar. Anhydrous THF
(5 ml) was added under argon and the solution was cooled to
ꢀ65 °C. Once cooled 1 M LHMDS in THF (2.5 ml) was slowly added,
and 20 min later 3-cloro-benzyl bromide was added (.47 ml,
3.6 mmol) at which time the reaction mixture was stirred for an
additional 21 h before being quenched with 2 N NH4Cl (6 ml). Ethyl
acetate was added for separation. The water layer was washed two
more times and the organic layers were concentrated down for sep-
aration on silica in 15% ethyl acetate, 85% hexanes (63%, .654 g). 1H
NMR (400 MHz, CDCl3) d: 7.46–7.42 (m, 6H), 7.25–7.22 (m, 7H),
7.19–7.16 (m, 5H), 7.06–7.05 (m, 1H), (3.85–3.83 (m, 1H), (3.61 (s,
.3H, S,S), 3.56 (s, 2.7, S,R)), (3.23 (s, .3H, S,S), 3.19 (s, 2.7, S,R)),
3.02–2.78 (m, 3H). 13C (100 MHz, CDCl3) d: 173.10, 172.66, 146.81,
145.37, 141.31, 134.05, 129.59, 128.93, 128.74, 127.81, 127.13,
+
21
for C15H16NO4 349.9889, found 349.9902. [
a
]
D
+9.8.
4.21. b-3,5 Dimethyl-benzylaspartate (4C)
The protected b-alkylated aspartate (1.29 g, 2.47 mmol) was ta-
ken up in 6 N HCl and heated for 24 h at 70 °C. Ethyl ether was
added for separation; the water layer was washed and concen-
trated. NH4OH was added to adjust the pH to 7 and the mixture
was loaded onto an anion exchange resin. Product came off at
2 N (.032 g, 8%). 1H NMR (400 MHz, D2O) d: (6.92 (app. s, .67H,
S,R.); 6.91 (app. s, .33H, S,R)), (6.88 (1.33H, app. s, S,R); 6.87
(.66H, app. s, S,S)), (4.08–4.07 (m, .33H, S,S), 4.00–3.99 (m, .66H,
S,R)), (3.45–3.40 (m, .66H, S,R), 3.26–3.21 (m, .33H, S,S)),

T. L. Mavencamp et al. / Bioorg. Med. Chem. 16 (2008) 7740–7748
7747
3.05–2.98 (m, 1H), 2.89–2.81 (m, 1H), (2.18 (br s, 5H), 2.12 (br s, 1H
S,S)). 13C (100 MHz, D2O) d: (172.54, 172.07), (169.43, 169.37),
(138.52, 138.20), (137.34, 137.14), (128.05, 127.87; 126.78,
(52.68, 52.36), (47.90, 47.46), (34.23, 32.03), 21.00. HRMS m/e calcd
added for separation; the water layer was washed three times
and concentrated. NH4OH was added until the pH reached 7 and
the mixture loaded onto an ion exchange resin where product
comes off around 2 N (.115 g, 36.2%). 1H NMR (400 MHz, DMSO)
d: 7.34–7.29 (m, 1H), 7.08–6.99 (m, 3H), (3.99 (d, .33H, S,S,
J = 4.40), 3.88 (d, .66H, S,R, J = 4.40)), (3.33 (ddd, .66H, S,R,
J = 4.40, 6.59, 9.52), 3.20 (ddd, .33H, S,S, J = 4.40, 5.86, 10.26)),
3.08–2.86 (m, 2H). 13C (100 MHz, DMSO) d: (172.54, 171.89),
169.17, (163.18, 160.77), (141.60, 141.53), (130.15, 130.03,
129.94), 125.05, (115.63, 115.42), (113.31, 113.16, 112.94),
(52.97, 52.48), (48.89, 47.25), (33.70, 32.03, 30.65). HRMS m/e for
+
21
for C13H18NO4 252.1236, found 252.1230. [
a]
+34.8°.
D
4.22. b-3 Methyl-benzylaspartate (4D)
The protected b-alkylated aspartate (.9 g, 1.77 mmol) was taken
up in 6 N HCl and heated for 24 h at 70 °C. Ethyl ether was added
for separation; the water layer was washed and concentrated.
NH4OH was added until the pH reached 6. THF was added for sol-
ubility and the product was loaded onto an Ag 1 ꢂ 8 acetate form
ion exchange resin in the ratio of 1 g product to 10 g resin. Product
was eluted with varying concentrations of acetic acid from .1 to 5 N
in a 4:1 ratio of water/THF. Product comes off around 2 N. (.0765 g,
18.3%). 1H NMR (400 MHz, D2O) d: 7.24–7.19 (m, 1H), 7.11–7.05
(m, 3H), (4.14–4.13 (m, .36H, S,S), 4.09–4.08 (m, .64H, S,R)),
(3.52–3.47 (m, .64H, S,R), 3.31–3.28 (m, .36H, S,S)), 3.12–3.07 (m,
1H), 2.98–2.88 (m, 1H), 2.25 (s, 3H). 13C NMR (100 MHz, CDCl3)
d: 174.89, 170.46, 139.13, 137.49, 129.78, 128.96, 127.93, 126.06,
52.86, (47.55, 46.88), (33.76, 33.65), 20.47. HRMS m/e calcd for
+
21
C
11H13FNO4 calcd 242.0829, found 242.0834. [
a
]
D
+40°.
The mixture of diasteromers was resolved by HPLC as well as
synthetically prepared in ratios of 99:1, S,S:S,R and 11:1 S,R:S,S.
For HPLC resolution the solvent system comprised of buffered solu-
tion of ammonium acetate and water (0.1 M at pH 6.4). Reversed
phase C18 3
l analytical and 10l semi-prep columns were used
with a retention time for S,S being 2.3 min and 18.48 min, respec-
tively, and for S,R 2.8 min and 27.22 min, respectively.
4.26. b-3 Fluoro-Benzylaspartate (4G, S,S)
C
12H16NO4 238.1079, found 238.1079. [
a]
+7.8° (in DI H2O,
1H NMR (400 MHz, D2O) d: 7.30–7.26 (m, 1H), 7.06–6.94 (m,
3H), 3.94 (m, 1H), 3.08–3.05 (m, 1H), 2.88–2.81 (m, 1H), 2.77–
2.73 (m, 1H). 13C (100 MHz, DMSO) d: 178.20, 172.02, 164.62,
+
21
D
adjusted to 7.5 with NaOH).
4.23. b-2,6 Dicloro-benzylaspartate (4E)
162.21, 143.35, 131.50, 126.19, 116.81, 114.49, 56.53, 49.31,
21
33.91. [
a
]
D
+18.3°.
The protected b-alkylated aspartate was dissolved in THF and
6 N HCl and refluxed for 5 h. The reaction mixture was washed
with ethyl acetate, and the water layers were combined and con-
centrated. The reaction was neutralized to pH 5 using NaOH; the
mixture loaded onto an anion exchange resin and eluted with
4:1 H2O/THF mixture followed by a gradient of acetic acid prepared
from concentrated acetic acid in the same 4:1 H2O/THF ratio to
give a gradient from .1 to 5 N. Product was isolated from 2 and
5 N acetic acid (1 g, 37.2%). 1H NMR (400 MHz, D2O) d: 7.37–7.32
(m, 2H), 7.21–7.14 (m, 1H), (4.05 (m, .4H, S,S), 3.96 (m, .6H, S,R)),
3.47–3.33 (m, 2H), 3.24–3.21 (m, .6H), 3.12–3.09 (m, .4H). 13C
NMR (100 MHz, CDCl3) d: 171.75, 169.40, 135.23, 134.03, 129.18,
4.27. b-3 Fluoro-Benzylaspartate (4G, S,R)
1H NMR (400 MHz, D2O) d: 7.32–7.25 (m, 1H), 7.05–6.95 (m,
3H), 3.64–3.59 (m, 1H), 3.07–3.02 (m, 1H), 2.96–2.88 (m, 2H). 13C
(100 MHz, DMSO) d: 179.81, 172.63, 163.22, 160.81, 140.12,
129.93, 125.11, 115.43, 112.90, 52.68, 49.74, 28.22. [a]
21 +38.23°.
D
4.28. b-4 Fluoro-benzylaspartate (4H)
The protected b-alkylated aspartate (.457 g, .893 mmol) was
taken up in 6 N HCl and heated for 24 h at 75 °C. Ethyl ether was
added for separation; the water layer is washed and concentrated.
NH4OH is added until the pH reached 7. THF 1:7 ratio was added
for solubility and the mixture was loaded onto an Ag 1 ꢂ 8 acetate
form ion exchange resin. Product is eluted with varying concentra-
tions of acetic acid in a mixture of water/THF in a ratio of 4:1. Prod-
uct comes off at 2 and 5 N acetic acid. Lyophilize off water 2–3
times to remove the acetic acid (.156, 73%). Found: C, 53.48; H,
4.83; N, 5.60. C11H11FNO4ꢃ1/3H2O requires C, 53.66; H, 4.78; N,
5.69. 1H NMR (400 MHz, D2O) d: 7.26–7.20 (m, 2H), 7.06–7.00
(m, 2H), (3.98 (d, .5H, S,S, J = 3.24), 3.88 (d, .5H, S,R, J = 3.24)),
(3.31 (ddd, .5H, S,R, J = 3.24, 7.12, 9.71), 3.18 (ddd, .5H, S,S,
J = 3.24, 5.18, 9.06)), 3.05–2.84 (m, 2H). 13C NMR (100 MHz, D2O)
d: (176.21, 176.00), (172.29, 171.81), (163.01, 162.93), (160.60,
160.53), (133.80, 133.39), (130.72, 130.68), (130.65, 130.59),
+
128.37, 52.80, 46.16, 28.07. HRMS m/e calcd for C11H12Cl2NO4
21
320.0456, found 320.0441. [
a]
+31.7°.
D
4.24. b-3 Bromo-benzylaspartate (4F)
The protected b-alkylated aspartate (.5 g, .873 mmol) was taken
up in 6 N HCl and heated for 24 h. Ethyl ether was added for sep-
aration; the water layer was washed three times and concentrated.
NH4OH is added until the pH is 7. THF is added for solubility and is
loaded onto an Ag 1 ꢂ 8 acetate form ion exchange resin in the ra-
tio of 1 g product to 10 g resin. Product is eluted with varying con-
centrations of acetic acid from .1 to 5 N in a mixture of water/THF
in a ratio of 4:1. Product comes off around 2 N. Lyophilize off water
2–3 times to remove the acetic acid (.1 g, 38.5%). 1H NMR
(400 MHz, DMSO) d: 7.47 (m, 1H), 7.41–7.38 (m, 1H), 7.28–7.22
(115.69, 115.58), (115.47, 115.37), 54.74, (48.19, 48.03), (33.82,
21
(m, 2H), 4.01 (br s, .85H,
a
S,S) 3.82, (br s, .15H,
a
S,R), 3.25–3.21
32.82). [
a]
+116.6° in DI H2O adjusted to 7.5 with NaOH.
D
(m, .15H, b S,R), 3.12–3.10 (m, .85H, b S,S), 3.04–3.00 (m, 1H),
3.28–2.74 (m, 1H). 13C NMR (100 MHz, DMSO) d: 171.80, 169.23,
141.69, 131.65, 130.42, 129.27, 128.22, 121.52, 52.68, (48.16,
47.35, b C), (31.62, 30.76, CH2), acetic acid 21.15, 172.33. HRMS
4.29. b-3 Nitro-benzylaspartate (4I)
The protected b-alkylated aspartate (.24 g, .446 mol) was taken
up in 6 N HCl and heated for 24 h at 65 °C. Ethyl ether was added
for separation; the water layer was washed and concentrated.
NH4OH was added until the pH reached 7. THF was added for sol-
ubility and the mixture was loaded onto an ion exchange resin.
Product was eluted with a mixture of water/THF in a ratio of 4:1.
Product came off around 2 N. Lyophilize off water 2–3 times to re-
move the acetic acid (.022 g, 18.4%). Product was sparingly insolu-
+
21
m/e calcd for C11H13BrNO4 302.0028, found 302.0014. [
a]
D
+26.9°.
4.25. b-3 Fluoro-Benzylaspartate (4G)
The protected b-alkylated aspartate (.678 g, 1.33 mmol) was
taken up in 6 N HCl and heated for 24 h at 70 °C. Ethyl ether was

7748
T. L. Mavencamp et al. / Bioorg. Med. Chem. 16 (2008) 7740–7748
ble in H2O. 1H NMR (400 MHz, D2O) d: 8.13–8.06 (m, 2H), 7.67–
7.61 (m, 1H), 7.54–7.48 (m, 1H), (3.99 (d, .6H, S,S, J = 3.88), 3.87
(d, .4H, S,R, J = 3.88)), (3.30 (ddd, .4H, S,R, J = 3.88, 5.18, 9.17),
3.21 (ddd, .6H, S,S, J = 3.88, 5.18, 9.06)), 3.11–3.05 (m, 1.4H), 2.97
(d, .45H, J = 5.18) 2.94 (d, .25H, J = 5.18). 13C (100 MHz, DMSO) d:
(173.92, 172.48), (169.50, 168.75), (146.79, 146.56), 139.37,
136.19, (131.76, 130.60), (123.72, 123.55), (122.23, 122.02),
52.92, 48.83, 29.69. HRMS m/e calcd for C11H13N2O6 269.0774,
tial amino acids solution. At 24 h after plating, cells were transfec-
ted using FuGENE6 Transfection Reagent (Roche, Indianapolis, IN)
in a ratio of 4 ll of FuGENE6 to 3 lg of purified plasmid DNA in
accordance with manufacturers instructions. The cells were used
in transport assays 24 h following transfection as described by
Esslinger et al.10 Briefly, transfected C17.2 cells were grown in
DMEM containing 10% FCS in a humid atmosphere of 5% CO2.
Near-confluent cells (plated at 5 ꢂ 104 cells/well) were rinsed with
found 269.0779. [c]21 +2.7° in DI H2O adjusted to 7.5 with NaOH.
a physiological buffer (138 mM NaCl, 11 mM D-glucose, 5.3 mM
D
KCl, 0.4 mM KH2PO4, 0.3 mM Na2HPO4, 1.1 mM CaCl2, 0.7 mM
MgSO4, 10 mM Hepes, pH 7.4) and allowed to pre-incubate at
37 °C for 5 min. Uptake was initiated by replacing the pre-incuba-
4.30. b-3 Nitro-benzylaspartate (4I, S,S)
The protected b-alkylated aspartate (.325 g, .603 mmol) is taken
up in 6 N HCl with a little acetone and heated for 24 h at 65 °C. The
reaction mixture is then concentrated down, washed with water
two times, and placed under vacuum. Ethyl acetate is added and
product precipitates out white powder. For ease this was centri-
fuged down for 10 min at 4000 rpm and the supernatant poured
off. The precipitate was dissolved in a small amount of water from
which it almost immediately crashed out as white crystals. This is
washed with water and concentrated down 2 more times (.045 g,
28%). 1H NMR (400 MHz, D2O) d: 8.10 (s, 1H), 8.06 (d, 1H,
J = 8.06), 7.64 (d, 1H, J = 7.33), 7.50 (t, 1H, J = 8.06, 7.33), 4.13
tion buffer with buffer containing 3H-
D-aspartate (25 lM) and
inhibitors at the concentrations indicated. Following a 5 min incu-
bation, the media were removed by rapid suction and the cells
rinsed three times with ice-cold buffer. The cells were dissolved
in 0.4 N NaOH for 24 h and analyzed for radioactivity by LSC and
protein by the BCA (Pierce) method. Transport rates were corrected
for background: that is, radiolabel accumulation at 4 °C. Initial
studies confirmed that uptake quantified in this manner was linear
with time and protein levels and that uptake in untransfected
C17.2 cells was indistinguishable from background.
(1H,
a
), 3.40–3.36 (m, 1H, b), 3.28–3.23 (m, 1H), 3.11–3.06 (m,
Acknowledgments
1H). 13C (100 MHz, D2O) d: 172.28, 169.29, 147.75, 141.07,
21
136.05, 129.69, 123.67, 121.64, 52.79, 47.28, 33.85. [
a
]
D
ꢀ20°.
The authors thank J. Gerdes, M. Kavanaugh, S. Patel, C. M.
Thompson, and T. Denton for their insightful advice regarding
the studies presented in this manuscript. Molecular modeling
studies were conducted in the Molecular Computational Core Facil-
ity of the Center for Structural and Functional Neuroscience, and
HRMS were obtained in the Proteomics and Mass Spec. Core Facil-
ity of the Center for Structural and Functional Neuroscience. This
work was supported in part by NINDS NS30570 (R.J.B.), NINDS
NS045704 (C.S.E.), and NCRR COBRE RR15583 (R.J.B., C.S.E.).
4.31. b-para Nitro-benzylaspartate (4J)
The protected b-alkylated aspartate (1.35 g, 2.51 mmol) was
placed in a round-bottomed flask with 6 N HCl and THF, to dis-
solve. Reaction mixture was refluxed for 3 h then washed with
ethyl acetate. The ethyl acetate layer was washed 2 more times
and the aqueous layer was concentrated down. 7.4 g AgX1-8 resin
200–400 mesh size was loaded into a column and washed with
two columns of water. Product was loaded and the column was
washed with a gradient of acetic acid from .1 to 5 N. Factions 1–
5 are lyophilized down (.378 g, 56%). 1H NMR (400 MHz, D2O) d:
8.04–8.00 (m, 2H), 7.39–7.34 (m, 2H), (3.99–3.88 (d, .65H, S,S),
3.92–3.91 (d, .35H, S,R)), (3.35–3.33 (m, .35H, S,R), 3.23–3.22 (m,
.65H, S,S)), 3.12–2.91 (m, 2H). 13C NMR (100 MHz, D2O) d:
(178.86, 178.41), (173.37, 172.64), (147.30, 146.96), (146.42,
146.35), (129.92, 129.83), (123.86, 123.80), (55.73, 55.67), (49.40,
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.07.001.
References
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48.99), (36.12, 33.29). HRMS m/e calcd for C11H14N2O6 calcd
21
252.1236, found 252.1225. [
a]
+18.52°.
D
4.32. b-3 Chloro-benzylaspartate S,R
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49.61, 32.14. [
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+25.7°.
D
4.33. EAAT3-Mediated transport in C17.2 cells
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EAAT3 was transiently expressed in C17.2 cells (obtained from
Dr. Evan Snyder, Burnham Inst., La Jolla, CA) using an AAV-based
vector (kindly provided by Dr. Mathew During, University of Auck-
land, NZ) pAM-CAG-EAAT3-WPRE as previously described.10 Cells
between passages 10–20 were seeded at 5 ꢂ 104 cells/well in 12-
well plates and grown in complete DMEM supplemented with
10% fetal bovine serum, 1 mM sodium pyruvate, 0.1 mM nonessen-