Mapping the aspartic acid binding site of Escherichia coli asparagine synthetase B using substrate analogs

Article abstract of DOI:10.1021/jm9601009

Novel inhibitors of asparagine synthetase, that will lower circulating levels of blood asparagine, have considerable potential in developing new protocols for the treatment of acute lymphoblastic leukemia. We now report the indirect characterization of th

Full text of DOI:10.1021/jm9601009

J . Med. Chem. 1996, 39, 2367-2378
2367
Ma p p in g th e Asp a r tic Acid Bin d in g Site of Esch er ich ia coli Asp a r a gin e
Syn th eta se B Usin g Su bstr a te An a logs
Ian B. Parr,† Susan K. Boehlein,‡ Anthony B. Dribben,† Sheldon M. Schuster,‡,§ and Nigel G. J . Richards*,†
Department of Chemistry, University of Florida, Gainesville, Florida 32611, Department of Biochemistry and
Molecular Biology, College of Medicine, University of Florida, Gainesville, Florida 32610, and Interdisciplinary
Center for Biotechnology Research, University of Florida, Gainesville, Florida 32610
Received February 2, 1996X
Novel inhibitors of asparagine synthetase, that will lower circulating levels of blood asparagine,
have considerable potential in developing new protocols for the treatment of acute lymphoblastic
leukemia. We now report the indirect characterization of the aspartate binding site of
Escherichia coli asparagine synthetase B (AS-B) using a number of stereochemically, and
conformationally, defined aspartic acid analogs. Two compounds, prepared using novel reaction
conditions for the stereospecific â-functionalization of aspartic acid diesters, have been found
to be competitive inhibitors with respect to aspartate in kinetic studies on AS-B. Chemical
modification experiments employing [(fluorosulfonyl)benzoyl]adenosine (FSBA), an ATP analog,
demonstrate that both inhibitors bind to the aspartate binding site of AS-B. Our results reveal
that large steric alterations in the substrate are not tolerated by the enzyme, consistent with
the failure of previous efforts to develop AS inhibitors using random screening approaches,
and that all of the ionizable groups are placed in close proximity in the bound conformation of
aspartate.
In tr od u ction
asparagine synthetase for which nucleotide sequences
have been reported.11-13 AS-B is a member of the purF
family of glutamine-dependent amidotransferases,14
which also includes glutamine phosphoribosylpyrophos-
phate amidotransferase (GPA)15 and glutamine fructose-
6-phosphate amidotransferase (GFAT).16 Although the
crystal structure of an inactive form of GPA complexed
with AMP has been reported,17 no corresponding struc-
tural information has been obtained for any other purF
enzyme. Functionalized aspartic acid analogs have
proven useful tools in the elucidation of structure-
function relationships associated with the NMDA18 and
glutamate19 receptors and for the preparation of chiral
intermediates in the synthesis of a number of complex
natural products.20 We have therefore prepared a series
of â-functionalized aspartates as probes of the specificity
and stereochemical preferences of the AS-B active site.21
We now describe the first well-characterized, albeit
weak, inhibitors of AS-B that are competitive with
respect to L-aspartic acid and demonstrate their interac-
tion with the AS-B aspartic acid binding in chemical
modification experiments using [(fluorosulfonyl)benzoyl]-
adenosine (FSBA).22 Our results are consistent with a
model of the bound conformation for aspartate in which
all ionizable groups are placed on the same face of the
substrate.
The enzyme L-asparaginase1 is often employed in the
treatment of acute lymphoblastic leukemia (ALL), its
mechanism of action being to deplete the level of
circulating asparagine synthesized by the liver.2 In-
deed, resistance of human neoplasms to protocols in-
volving L-asparaginase arises from the synthesis of
endogenous asparagine in these malignant cells.3 There
is also evidence to suggest that blood asparagine
concentration may also play a role in T-cell response.4
Inhibitors of asparagine synthetase (AS), the glutamine-
dependent amidotransferase that converts aspartate to
asparagine in the liver,5 therefore represent targets with
potential application in treating leukemia and in ex-
ploring cellular mechanisms of immunosuppression.6
Extensive screening studies employing hundreds of
analogs of aspartic acid, glutamine, and ATP have failed
to yield any well-characterized, reversible AS inhibi-
tors,7,8 although several glutamine analogs capable of
covalent attachment to the enzyme have been de-
scribed.9 These irreversible inhibitors, however, possess
little selectivity, modifying all cellular glutamine-de-
pendent amidotransferases. In efforts to develop AS
inhibitors using rational methods, we have cloned,
expressed, and purified the asparagine synthetase
encoded by the asnB gene of Escherichia coli (AS-B).10
This bacterial enzyme catalyzes the following reactions:
Ch em istr y
L-Gln + L-Asp + L-ATP f L-Asn + L-Glu + AMP + PPi
The N-protected diester 1 (Scheme 1) was readily
prepared in large scale from (S)-aspartic acid in three
steps.23,24 Alkylation of the dianion of 1 had been shown
to proceed without racemization at the chiral carbon
using a wide variety of electrophiles.23,25,26 Treatment
of 1 with lithium hexamethyldisilazide (LiHMDS) in
THF at -75 °C, followed by addition of neat methyl
iodide, gave the desired methylated diester as a 3:1
mixture of diastereoisomers 2 and 3 after column
chromatography. No N-methylated material was ob-
tained, and the lack of stereoselectivity in this reaction
was consistent with previous observations.24-27 Recov-
ered diester 1 had an unchanged optical rotation,
(1)
NH3 + L-Asp + L-ATP f L-Asn + AMP + PPi
L-Gln f L-Glu + NH3
(2)
(3)
In E. coli, asnB is one of two unlinked genes encoding
* Corresponding author: Department of Chemistry, P.O. Box
117200, University of Florida, Gainesville, FL 32611-7200. Phone:
(904) 392-3601. Fax: (904) 846-2095.
† Department of Chemistry.
‡ Department of Biochemistry and Molecular Biology.
§ Interdisciplinary Center for Biotechnology Research.
X Abstract published in Advance ACS Abstracts, May 15, 1996.
S0022-2623(96)00100-8 CCC: $12.00 © 1996 American Chemical Society

2368 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12
Parr et al.
Sch em e 1a
a
Z ) PhCH2OCO. (i) LiHMDS, THF, -78 °C; MeI, THF, -78 °C; (ii) CF3CO2H, CH2Cl2, rt; (iii) C18 RP-HPLC separation of
diastereoisomers; (iv) LiOH, MeOH, H2O; (v) 10% Pd/C, C6H10, MeOH, reflux; (vi) LiHMDS, THF, -78 °C; H2CdCHCH2Br, THF, -78 °C;
(vii) cat. OsO4, NaIO4, MeOH, H2O, rt; (viii) CF3CO2H, Et3SiH, CHCl3, reflux.
suggesting deprotonation at C-2 had not occurred under
these reaction conditions. In addition, the dianion of 1
was quenched with D2O and the product examined by
NMR. Deuterium was incorporated at C-3 but not at
the R-carbon of diester 1. At this stage in the synthesis
we were unable to separate diesters 2 and 3, and so the
mixture was reacted with anhydrous trifluoroacetic acid
(TFA)28 giving mono-acids 4 and 5. Further treatment
with lithium hydroxide (LiOH) in aqueous MeOH29
yielded two diacids 6 and 7, that could be separated by
reverse-phase HPLC on a C18 column. No epimerization
at either chiral center was assumed to occur in these
deprotection steps given that the ratio of 6 to 7 was
identical to that determined for the mixture of mono-
methylated aspartates 2 and 3. Deprotection of the
major product 6 was effected using catalytic hydrogen
transfer,30 giving only (2S,3S)-2-amino-3-methylsucci-
nate (8) by examination of the behavior of its specific
rotation as a function of solvent.31-33 Thus, in agree-
ment with previous findings,32 the specific rotation
associated with 8 was -12° in aqueous solution and
+13.6° in 1 M HCl. Likewise, diacid 7 was deprotected
to yield only (2S,3R)-2-amino-3-methylsuccinate (9).
These assignments for 8 and 9 imply that the relative
stereochemistry of compounds 2-7 is as shown (Scheme
1).
In contrast to the methylation of 1, we were able to
develop conditions for reaction of allyl bromide with the
dianion of 1 that gave allylated diester 10 as a single
diastereoisomer.34 By analogy to the major methylation
product, functionalized diester 10 was tentatively as-
signed to be the (2S,3R) diastereoisomer. The trans-
formation of 10 into the functionalized pyrrolidine 11
was carried out by double-bond cleavage using sodium
periodate and a catalytic amount of osmium tetraoxide
(Scheme 1).35 Pyrrolidine 11 was formed in 83% yield
as a single diastereoisomer, suggesting that cyclization
proceeded with complete stereocontrol at C-5. The
aldehyde 12, presumably an intermediate in this cy-
clization reaction, was also isolated in 6% yield. The
relative stereochemistry of 11 was established using
NOE difference spectroscopy36 and by examination of
scalar coupling constants.37 Thus, the cis-arrangement
of the ester substituents was suggested by the scalar
coupling, J 23 ) 8.4 Hz,37 and supported by a strong NOE
between the protons at C-2 and C-3 (Figure 1A).
Assuming no epimerization at C-3, other NOEs were
consistent with 11 being the (2S,3R,5R) diastereoiso-
mer. So as to confirm that the conformational proper-
ties of 11 had not led to a misassignment of the relative
stereochemistry at C-2 and C-3, we prepared a mixture
of 10 and its epimer at C-3 by â-functionalization using

Mapping the AS-B Aspartate Binding Site
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12 2369
F igu r e 1. (A) Schematic representation of interproton NOEs observed for 11. (B) Global energy minimum for 11 obtained using
the AMBER* parameters and potential energy functions. Shading represents atom type. C: fully shaded; H: white; N: high
density dots; O: low density dots.
literature conditions.23,25,26 Cyclization of this mixture
using osmium tetraoxide/sodium periodate then gave 11
and its C-3 epimer. Although these compounds were
not separated, the relevant C-2 proton was identified
isomers. The major product 21 could be obtained,
however, by chromatography and fractional recrystal-
lization. The relative stereochemistry of 21 was tenta-
tively assigned by analogy to alkylation products 2 and
10. Cleavage of the double bond and subsequent
cyclization was accomplished using sodium periodate
and catalytic OsO4, giving substituted pyrrolidines 22
and 23 in a 9:1 ratio. As these diastereoisomers proved
difficult to separate, the mixture was reduced by reac-
tion with triethylsilane forming the desired cis-diester
24 as a single product. Removal of the benzyl protecting
groups to yield the target proline analog 25 was suc-
cessfully accomplished using catalytic hydrogen transfer
in reasonable overall yield.30,42 As the usefulness of this
route was limited, however, by difficulties in purifying
large quantities of 21 from the â-allylation reaction
product mixture, we investigated purification of the
diastereoisomeric products at a later stage in the
synthesis (Scheme 2B). Hence, cyclization of a mixture
of 21 and its C-3 epimer, followed by sequential treat-
ment with OsO4/NaIO4 and Et3SiH/TFA gave a 2.5:1
mixture of the pyrrolidines 24 and 26. These could be
separated by column chromatography and indepen-
dently converted to the desired aspartic acid analogs 25
and 16, respectively.
1
using H NMR and the key coupling constant found to
be J 23 ) 3.9 Hz, confirming our assignment of 11. Given
that the stereospecific formation of 11 as the (2S,3R,5R)
diastereoisomer, a complete conformational analysis of
11 and its C-5 epimer was carried out using a stochastic
search algorithm38 and the AMBER* force-field39 as
implemented in MacroModel V5.0 (Figure 1B).40 This
study indicated that 11 was more stable than its C-5
epimer by 7 kJ /mol, a result consistent with the
hypothesis that the cyclization proceeds under thermo-
dynamic control. Removal of the hydroxyl group from
11 was accomplished using triethylsilane to give 13,41
and subsequent deprotection of the tert-butyl ester
proceeded smoothly in anhydrous TFA, yielding the acid
14 in good overall yield.28 Unfortunately, removal of
the methyl ester under basic conditions gave the (2S,3S)-
diacid 15, as the predominant product due to epimer-
ization at C-3. Removal of the N-protecting group by
catalytic hydrogen transfer gave conformationally con-
strained aspartate analog 16 in excellent yield.42
Having defined the stereochemistry of allylated as-
partate 10 unambiguously, we converted it into diacid
18 by sequential reaction with TFA and then LiOH in
aqueous methanol. Unfortunately, all efforts to remove
the N-protecting group, such as reaction with 48% HBr
in acetic acid,43 failed to give products that were easily
separable, presumably due to side-reactions involving
the double bond. Removal of the N-carbobenzoxy pro-
tecting group, however, under standard catalytic hy-
drogen transfer conditions, proceeded with concomitant
reduction of the double bond to give 3-n-propyl aspartate
19 (Scheme 1).30
Biologica l Resu lts a n d Discu ssion
The inhibitory effects of a series of aspartic acid
analogs on glutamine-dependent asparagine synthesis
were determined using recombinant, wild-type AS-B.9
Our initial studies focused upon the commercially
available compounds 27-32 (Figure 2). Standard reac-
tion solutions in these experiments contained 1 mM
glutamine, 1 mM ATP, 1 mM aspartic acid, and 10 mM
MgCl2 in TrisHCl, pH ) 8.9 Asparagine production was
assayed by measuring the amount of pyrophosphate
formed under steady state conditions.9 The sulfinate
27 proved to be an effective, competitive inhibitor of
aspartate (KIS ) 1.45 mM), consistent with the results
In order to obtain large amounts of material, we next
examined an alternate preparation of the diacid 25 from
allylated aspartate derivative 21 in which the key step
was â-functionalization of dibenzyl ester 20 (Scheme
2A). Standard reaction conditions for the alkylation of
the dianion of 20 with allyl bromide gave the desired
â-functionalized diester as a 6:1 mixture of diastereo-

2370 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12
Parr et al.
Sch em e 2a
a
Z ) PhCH2OCO. (i) LiHMDS, THF, -78 °C; H2CdCHCH2Br, THF, -78 °C; (ii) chromatography followed by fractional recrystallization;
(iii) cat. OsO4, NaIO4, MeOH, H2O, rt; (iv) CF3CO2H, Et3SiH, CHCl3, reflux; (v) 10% Pd/C, C6H10, MeOH, reflux.
of previous studies on aspartate transcarbamoylase.44
On the other hand, compounds 28-32 exhibited only
low levels of inhibition even at 10 mM concentration.
The inability of malic acid to inhibit asparagine syn-
thesis clearly shows that the hydroxyl cannot act as an
isostere of the charged amino group, consistent with
results reported for the asparagine synthetase present
in RADA1, a murine leukemia.45 It therefore appears
that AS-B requires the presence of all of the ionizable
groups in aspartate for substrate binding. The signifi-
cance of the â-carboxylate in recognition is underscored
by the failure of L-proline to inhibit AS-B, within the
limits of the assay, at concentrations up to 50 mM.
Having established that all three ionized functional
groups were essential to the recognition of aspartate by
AS-B, we then assayed the â-functionalized aspartic acid
analogs 8, 9, 16, 19, and 25. We note that in previous
experiments on asparagine synthetase isolated from
asparaginase-resistant Novikoff hepatomas,8 27% AS
F igu r e 2. Aspartic acid analogs 27-34 used in these and
inhibition was observed when synthetase activity was
assayed in the presence of the racemate of erythro-â-
hydroxyaspartate 33 (Figure 2). While no reduction in
AS-B activity was observed for 3-n-propyl aspartate 19,
the remaining analogs were weak AS-B inhibitors
(Table 1). Furthermore, there was a significant effect
of stereochemistry at C-3 upon the level of AS-B
inhibition. Again, this observation is consistent with
reports that threo-â-hydroxyaspartate 34 (Figure 2)
cannot inhibit AS isolated from Novikoff hepatomas.8
A Dixon plot of the reciprocal of the initial velocity, 1/vo,
against inhibitor concentration, [I], for AS-B in the
presence of analog 16, gave an apparent KI of 60 mM
for this compound (data not shown). In contrast, 25,
differing only in its C-3 configuration, was competitive
with respect to aspartate, having a KIS of 2.65 mM. A
previous inhibition studies, together with the irreversible
inhibitor FSBA (35).
similar dependence of inhibitory properties upon C-3
stereochemistry was also observed for the methylated
analogs 8 and 9 (Table 1). Detailed kinetic analysis
confirmed that 9 and 25 were competitive only with
respect to aspartate, suggesting that these compounds
did indeed bind to the same form of the enzyme
(probably the E‚ATP complex46) as aspartic acid, pre-
venting the natural substrate from binding and taking
part in subsequent chemical transformations.47
Given the conformationally restricted nature of 16
and 25, and the observation that these compounds are
only competitive with respect to aspartate, it is reason-
able to assume that 25 defines the bound conformation

Mapping the AS-B Aspartate Binding Site
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12 2371
Ta ble 1. Inhibition Constants for Selected Aspartic Acid
Analogs in the Glutamine-Dependent Synthetase Activity of
AS-B
substrate
varied
a
a
inhibitor
inhibition pattern Kis (mM) Kii (mM)
8
8
8
9
9
aspartate
glutamine
ATP
aspartate
glutamine
ATP
aspartate
glutamine
ATP
noncompetitive
noncompetitive
noncompetitive
competitive
noncompetitive
noncompetitive
competitive
uncompetitive
noncompetitive
competitive
18.0
93.0
8.0
>50.0
16.5
15.0
na
0.25
1.62
0.45
2.65
na
2.65
1.45
1.71
3.28
na
2.57
4.80
na
9
25
25
25
27
aspartate
a
Kis and Kii are the inhibition constants computed from the
intercepts and slopes, respectively, of the double reciprocal plots
of 1/vo versus 1/[aspartate] obtained at various concentrations of
b
the inhibitor.51 na ) not applicable.
of aspartic acid. In addition, the identical correlation
between C-3 stereochemistry and the ability to inhibit
AS-B observed for the diastereoisomers of â-methylas-
partate suggests that 9 adopts a bound conformation
on the enzyme similar to that of 25. Both 9 and 25
possess a carboxyl group capable of undergoing reaction
with ATP to form reactive intermediates cognate to
â-aspartyl-AMP. The turnover of pyrophosphate by
AS-B was not observed, however, in control experiments
in which AS-B was incubated with either 9 or 25 in the
presence of glutamine and ATP. We therefore became
concerned about the possibility that these aspartate
analogs were binding to a region of the enzyme other
than the aspartate binding site. In order to address this
question, the ability of â-methylaspartate 9 to behave
in an identical manner to aspartic acid in chemical
modification experiments was examined.
5′-O-[p-(Fluorosulfonyl)benzoyl]adenosine (FSBA) (35)
(Figure 2) is an ATP analog that covalently modifies
proteins by reaction with hydroxyl or thiol groups22 and
has been widely used in studies of ATP-dependent
enzymes.48 When FSBA is incubated with AS-B in the
absence of substrates or substrate analogs, the enzyme
is covalently modified, resulting in a linear decrease of
activity with respect to time when the incubated mater-
ial is exposed to a mixture of glutamine, aspartate, and
ATP. That FSBA indeed bound within the AS-B ATP
binding site was demonstrated by a decreased rate of
covalent modification when ATP was present in the
incubation mixture (Figure 3). In contrast, when AS-B
was incubated with FSBA in the presence of aspartate
or analog 9, there appeared to be little effect on the rate
of covalent modification (Figure 3). The formation of
â-aspartyl-AMP as a reaction intermediate in AS-
catalyzed asparagine synthesis has been shown using
isotopic labeling, implying that the ATP and aspartate
binding sites are located close together in the enzyme.
This is consistent with the observation that when ATP
and aspartic acid were both present with AS-B and
FSBA in the incubation mixture, the rate of enzyme
inactivation was significantly decreased. Although this
result might imply that â-aspartyl-AMP is tightly bound
by AS-B, the presence of both substrates is also likely
to enhance the stickiness of ATP with the free enzyme.
In agreement with the latter proposal, similar decreases
in the ability of FSBA to modify AS-B covalently were
observed when either mixtures of aspartate/ATP or
9/ATP were present during incubation. Hence, although
F igu r e 3. Effects of added substrates and aspartic acid analog
9 on the rate of AS-B inactivation by covalent modification by
FSBA. At sufficiently high ATP concentrations, FSBA cannot
inactivate AS-B. In these experiments, however, the ATP
concentration employed was chosen so as to indicate any
differential ability of aspartic acid and 9 to protect against
FSBA inactivation. Enzyme activity was assayed by measuring
the amount of pyophosphate released by the incubated enzyme
in the presence of glutamine, aspartate, and ATP. Each
experiment differed only in the compounds present in the
incubation solution with AS-B. (O) FSBA only; (b) FSBA +
ATP; (0) FSBA + Asp; (9) FSBA + 9; (2) FSBA + ATP + Asp;
(1) FSBA + ATP + 9.
no reaction between ATP and 9 takes place to release
pyrophosphate, analog 9 almost certainly occupies the
same site on the enzyme as aspartate. In the absence
of detailed structural data upon the complexes between
AS-B and these aspartate analogs, we also propose that
25 binds within the same pocket as 9, especially given
the correlation between C-3 stereochemistry and inhibi-
tion for the aspartate analogs.
It is reasonable to suggest that aspartic acid binds,
at least initially, to AS-B in a conformation that is
identical to that of the rigid analog 25. In this shape,
all of the polar functionality is placed upon one face of
the molecule, with the hydrogen atoms defining a
hydrophobic surface that makes contact with the en-
zyme (Figure 4A). Given that the methylated analog 9
also interacts with this site, there is some flexibility in
the protein that can be used to accommodate the larger
substitutent. On the other hand, the inability of 19 to
inhibit AS-B synthetase activity argues that this pocket
in the enzyme cannot be distorted significantly.
There are now three examples of aspartate binding
sites for which the X-ray structures have been re-
ported.49 In all cases, arginine residues interact with
the carboxylate groups of aspartate, although the num-
ber employed is variable. Given that both carboxylates
are placed in close proximity in our model of the bound

2372 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12
Parr et al.
C18 and C8 reverse-phase columns (250 mm × 4.6 mm or 250
mm × 21.4 mm), monitoring at 226 nm with
a Rainin
Dynamax UV-1 absorbance detector. HPLC grade solvents
were obtained from Fisher Scientific and were used without
further purification. Tetrahydrofuran (THF) was distilled
from sodium metal/benzophenone ketyl. Aspartate analogs
27-32 and 35 were purchased from Sigma and used without
further purification.
Gen er a l P r oced u r e for th e Alk yla tion of Asp a r ta te
Diester 1. LiHMDS was prepared in situ by the dropwise
addition of n-butyllithium (2.1 equiv as a solution in hexane)
to hexamethyldisilazane (2.2 equiv) dissolved in dry THF (5
mL), under a stream of dry N2 so as to maintain the reaction
temperature at 0 °C. After 60-90 min at 0 °C, the resulting
solution was cooled to -78 °C before the addition of 1 (1 equiv)
as a solution in dry THF (5 mL) to give a pale-yellow reaction
mixture. After stirring at -35 °C for a further 150 min to
ensure dianion formation, the solution was recooled to -78
°C, and neat electrophile (5-6 equiv) added at a rate so as to
maintain the temperature below -75 °C. After stirring at -75
°C, for between 6 to 16 h, depending on the electrophile, the
reaction was quenched by pouring the cold solution directly
into 1 M HCl (60 mL) at rt. The resulting mixture was
extracted with Et2O (4 × 20 mL), and the organic extracts were
dried (MgSO4) before evaporation of the solvent under reduced
pressure. The crude material was purified by “flash” chro-
matography (eluant: 4:1 petroleum ether/EtOAc).
F igu r e 4. (A) Newman projection of the bound conformation
of L-aspartate in the AS-B active site based on the constrained
analog 25. (B) Schematic model for the aspartic acid binding
site in AS-B.
aspartate conformation, it is possible that only a single
arginine residue will be present in the binding site of
AS-B. Interactions that stabilize the protonated amine
of L-aspartate, however, seem less predictable. For
example, the amino group is placed over the face of a
tryptophan ring in adenylosuccinate synthetase, while
in the bacterial aspartate receptor, backbone carbonyl
groups are used to bind the amine. Our data suggest
that while two of the three protons in aspartate are
placed within a well-defined pocket on the surface of
the enzyme, there is room in the site to accommodate a
small hydrophobic substituent in place of the pro-(R)
hydrogen at C-3 (Figure 4B).
ter t-Bu tyl (2S,3R)-2-(Ben zyloxyca r bon yla m in o)-3-ca r -
bom eth oxybu ta n oa te (2) a n d ter t-Bu tyl (2S,3S)-2-(Ben -
zyloxycar bon ylam in o)-3-car bom eth oxybu tan oate (3). The
methylation of the diester 1 (100 mg, 0.3 mmol) was carried
out according to the general procedure outlined above, using
hexamethyldisilazane (0.16 mL, 0.74 mmol), n-butyllithium
(0.25 mL of a 2.48 M solution in hexane, 0.86 mmol) and neat
methyl iodide (0.12 mL, 1.8 mmol). After 17 h at -75 °C,
followed by quenching, workup, and purification by chroma-
tography, the methylated product was obtained as a 3:1
mixture of diastereoisomers 2:3 based on 1H NMR, as a clear
oil: 67 mg, 64%; IR (neat) ν 3431, 3359, 2980, 1730, 1504, 1456,
Con clu sion
â-Alkylation of the aspartate derivative 1 has allowed
the preparation of a number of stereochemically defined
aspartate analogs suitable for probing the molecular
features of the aspartic acid binding site in AS-B. The
enzyme appears to be extremely selective, being able
to discriminate between metabolites that have similar
structures to aspartic acid, which is clearly important
in terms of cellular metabolism. On the other hand, the
protein residues that are responsible for mediating this
selectivity remain to be defined by site-directed mu-
tagenesis or X-ray crystallography. Although it appears
that AS-B can tolerate only relatively minor structural
alterations in the aspartic acid substrate, these data
suggest that functionalized aspartate analogs can be
developed that are selective, tight-binding AS inhibitors.
1370, 1219, 1157 cm-1 13C NMR (CDCl3, 75.4 MHz) δ 12.66
;
(q), 27.82 (q), 41.84 (d), 51.86 (q), 56.26 (d), 68.58 (t), 82.53
(s), 127.72 (d),128.01 (d),128.31 (d), 136.29 (s), 156.39 (s),
169.36 (s), 173.77 (s); MS (CI: CH4) m/e (relative intensity)
352 (MH+, 100), 296 (89), 252 (10), 206 (6), 91 (6); exact mass
calcd for MH+ C18H26NO6 requires 352.1760, found 352.1705
(CI).
2: 1H NMR (CDCl3, 300 MHz) δ 1.26 (3 H, d, J ) 8.0 Hz),
1.46 (9 H, s), 2.96 (1 H, m), 3.69 (3 H, s), 4.51-4.61 (1 H, m),
5.10 (2 H, s), 5.50 (1 H, d, J ) 9.0 Hz), 7.35-7.39 (5 H, m).
3: 1H NMR (CDCl3, 300 MHz) δ 1.20 (3 H, d, J ) 8.0 Hz),
1.42 (9 H, s), 3.20 (1 H, m), 3.69 (3 H, s), 4.51-4.61 (1 H, m),
5.12 (2 H, s), 5.63 (1 H, d, J ) 9.0 Hz), 7.35-7.39 (5 H, m).
(2S ,3R )-2-(Be n zyloxyca r b on yla m in o)-3-ca r b om e t h -
oxybu ta n oic Acid (4) a n d (2S,3S)-2-(Ben zyloxyca r bon -
yla m in o)-3-ca r bom eth oxybu ta n oic Acid (5). TFA (1.48
mL, 19.3 mmol) was added to a solution of diesters 2 and 3
(342 mg, 0.97 mmol) in CH2Cl2 (40 mL) and stirred at rt for
72 h. During this period, a further portion of TFA (1 mL, 13
mmol) was added. Upon complete consumption of starting
diester, removal of the solvent under reduced pressure yielded
an oily residue which was redissolved in EtOAc (30 mL). This
solution was extracted using 10% (w/v) aqueous NaHCO3 (3
× 15 mL). After removal of the organic phase, the aqueous
solution was acidified to pH 1 using 1 M HCl and then
reextracted with EtOAc (4 × 20 mL). The organic phases were
combined and dried (MgSO4), and the solvent was removed
under reduced pressure to give the desired acids as a 3:1
mixture of diastereoisomers 4:5 in quantitative yield. This
material was used without further purification in subsequent
reactions: IR (neat) ν 3730-2696, 3331, 2602, 1732, 1713,
Exp er im en ta l Section
Melting points were recorded using a Fisher-J ohns melting
point apparatus, and are uncorrected. Optical rotations were
measured using a Polyscience Model SR-6 polarimeter. 1H and
13C NMR spectra were obtained on General Electric QE-300,
Bruker AM-360, or Varian Unity-500 spectrometers. Chemical
shifts are reported in ppm (δ) downfield of tetramethylsilane
as an internal reference (δ 0.0). Splitting patterns are
abbreviated as follows: s, singlet, d, doublet, t, triplet, q,
quartet, and m, multiplet. Infrared spectra were recorded on
a Perkin-Elmer 1600 FT-IR instrument. Combustion analyses
for C, H, and N were determined in the Microanalysis Facility
in the Department of Chemistry, University of Florida. EI,
CI, and FAB mass spectra were recorded upon a VG Analytical
7-250-SE (low resolution) or a Finnigan MAT 25Q (high
resolution) spectrometer. Ammonia or isobutane were used
in the CI measurements. Analytical thin layer chromatrog-
raphy (TLC) was performed on silica gel 60F-245 plates. Flash
chromatography was performed using standard methods50 on
Davisil grade 633 type 60A silica gel (200-425 mesh). Ana-
lytical and preparative HPLC was carried out on Dynamax
1519, 1456, 1408, 1384, 1092, 1061 cm-1 13C NMR (CDCl3,
;
75.4 MHz) δ 13.90 (q), 41.55 (d), 52.16 (q), 55.58 (d), 67.65 (t),
127.79 (d), 127.96 (d),128.36 (d), 135.96 (s), 156.93 (s), 174.41
(s), 175.11 (s); MS (EI) m/e (relative intensity) 295 (M+, 8),
220 (3), 206 (8), 160 (8), 108 (35), 91 (100); exact mass calcd
M+ for C14H17NO6 requires 295.1056, found 295.1053 (EI).

Mapping the AS-B Aspartate Binding Site
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12 2373
4: 1H NMR (CDCl3, 300 MHz) δ 1.28 (3 H, d, J ) 4.0 Hz),
3.05 (1 H, m), 3.69 (3 H, s), 4.75 (1 H, m), 5.13 (2 H, s), 5.75 (1
H, d, J ) 9.0 Hz), 7.35-7.39 (5 H, m), 10.58 (1 H, s).
5: 1H NMR (CDCl3, 300 MHz) δ 1.25 (3 H, d, J ) 4.0 Hz),
3.30 (1 H, m), 3.69 (3 H, s), 4.59-4.65 (1 H, dd, J ) 9.0, 3.5
Hz), 5.13 (2 H, s), 5.86 (1 H, d, J ) 9.0 Hz), 7.35-7.39 (5 H,
m), 10.58 (1 H, s).
(relative intensity) 148 (46), 115 (100); exact mass calcd for
MH+ C5H10NO4 requires 148.0610, found 148.0620 (FAB).
ter t-Bu tyl (2S,3R)-2-(Ben zyloxyca r bon yla m in o)-3-ca r -
bom eth oxyh ex-5-en oa te (10). The allylated diester 10 was
prepared by the general alkylation procedure using hexa-
methyldisilazane (1.41 mL, 6.67 mol), n-butyllithium (2.24 mL
of a 2.48 M solution in hexane, 5.6 mmol), diester 1 (900 mg,
2.6 mmol), and allyl bromide (1.35 mL, 15.6 mmol). After 18
h at -75 °C, the standard workup procedure gave the allylated
(2S,3R)-2-(Ben zyloxyca r b on yla m in o)-3-m et h ylsu cci-
n ic Acid (6) a n d (2S,3S)-2-(Ben zyloxyca r bon yla m in o)-3-
m eth ylsu ccin ic Acid (7). The mixture of diastereoisomeric
acids 4 and 5 (285 mg, 0.97 mmol) was dissolved in 4:1 MeOH/
H2O so as to give an 0.1 M solution. After the addition of 4
equiv of LiOH‚H2O, the reaction was stirred at room temper-
ature until the starting esters were completely consumed. The
reaction mixture was concentrated, under reduced pressure,
to approximately 20% of its initial value, and poured into an
excess of 1 M HCl. After adjustment of the solution pH to
1-2, the mixture was extracted with EtOAc. The combined
organic phases were dried (MgSO4), and the solvent was
removed under reduced pressure. Purification of this crude
mixture of diacids was accomplished using reverse phase
HPLC with gradient elution (C18 column; solvent flow rate 20
mL/min; 80:20 H2O/CH3CN + 1% TFA to 66:34 H2O/CH3CN
+ 1% TFA over a period of 28 min), with monitoring at 226
nm. The minor product, diacid 7 (ret time: 19.6 min), eluted
first and was finally obtained as a white, hygroscopic solids
by freeze-drying overnight: 29 mg, 11%; IR (CHCl3) ν 3264-
2719, 1714, 1514, 1455, 1416, 1343, 1061 cm-1; 1H NMR (CD3-
OD, 300 MHz) δ 1.21 (3 H, d, J ) 8.0 Hz), 3.12 (1 H, m), 4.49
(1 H, d, J ) 5.0 Hz), 5.12 (2 H, s), 7.30-7.42 (5 H, m); 13C
NMR (CD3OD, 75.4 MHz) δ 12.74 (q), 41.02 (d), 55.77 (d), 66.44
(t), 127.92 (d), 128.66 (d), 129.05 (d), 136.65 (s), 157.31 (s),
174.14 (s), 177.79 (s); MS (EI) m/e (relative intensity) 281 (M+,
11), 263 (6), 192 (4), 146 (7), 108 (52), 91 (100); exact mass
calcd for M+ C13H15NO6 requires 281.0899, found 281.0887
(EI).
diester 10 as a clear oil: 600 mg, 60%; [R]23 +15.4° (c ) 1.3,
D
CHCl3); IR (CH2Cl2) ν 3421, 3056, 2978, 1728, 1641, 1506,
1264, 1156, 922 cm-1; 1H NMR (CDCl3, 300 MHz) δ 1.42 (9 H,
s), 2.23-2.34 (1 H, m), 2.43-2.54 (1 H, m), 3.09-3.17 (1 H,
m), 3.64 (3 H, s), 4.51-4.56 (1 H, dd, J ) 10.1, 4.1 Hz), 5.04-
5.09 (2 H, m), 5.12 (2 H, s), 5.70-5.85 (1 H, m), 5.78 (1 H, d,
J ) 10.1 Hz), 7.25-7.36 (5 H, m); 13C NMR (CDCl3, 75.4 MHz)
δ 27.04 (q), 31.83 (t), 45.94 (d), 51.04 (q), 53.85 (t), 66.17 (d),
81.54 (s), 117.13 (t), 127.28 (d),127.32 (d), 127.71 (d), 133.72
(d), 135.71 (s), 155.70 (s), 168.95 (s), 172.32 (s); MS (CI: H+)
m/e (relative intensity) 378 (MH+, 41), 322 (100), 278 (41), 232
(9); exact mass calcd for MH+ C20H28NO6 requires 378.1917,
found 378.1911 (CI).
ter t-Bu tyl (2S,3R,5S)-1-(Ben zyloxyca r bon yl)-3-ca r bo-
m eth oxy-5-h yd r oxyp yr r olid in e-2-ca r boxyla te (11). OsO4
(1.7 mL of a 2.5% solution in 2-propanol, 0.14 mmol) and NaIO4
(1.28 g, 6.0 mmol) were added to a solution of diester 10 (750
mg, 2.0 mmol) in MeOH (44 mL) and water (22 mL). The
resulting reaction mixture was stirred at rt for 75 min. After
halving the solvent volume under reduced pressure, the
solution was poured into water (140 mL) and extracted with
EtOAc (6 × 50 mL). The organic extracts were dried (MgSO4),
and the solvent was removed under reduced pressure to yield
the crude product as a brown oil. This material was purified
by flash chromatography (eluant: 30% EtOAc/petroleum ether)
to yield, as the first material from the column, the aldehyde
12 as a clear oil: 46 mgmg, 6%; IR (CHCl3) ν 3433, 3020, 1724,
1508, 1455, 1439, 1395, 1369, 1342, 1296,1155 cm-1; 1H NMR
(CDCl3, 300 MHz) δ 1.46 (9 H, s), 2.60 (1 H, m), 2.93 (1 H, m),
3.69 (3 H, s), 3.72 (1 H, m), 4.55-4.61 (1 H, dd, J ) 9.0, 3.5
Hz), 5.12 (2 H, s), 5.56 (1 H, d, J ) 8.0 Hz), 7.32-7.39 (5 H,
m), 9.77 (1 H, s); 13C NMR (CDCl3, 75.4 MHz) δ 27.81 (q), 41.43
(d), 41.61 (t), 52.17 (q), 54.73 (d), 67.27 (t), 128.12 (d), 128.25
(d), 128.53 (d).
Further elution gave the diastereoisomeric diacid 6 (ret
time: 22.0 min), which was also obtained as a white, hygro-
1
scopic solid following lyophilization: 123 mg, 45%; H NMR
(CD3OD, 300 MHz) δ 1.17 (3 H, d, J ) 8.0 Hz), 2.98 (1 H, m),
4.60 (1 H, d, J ) 6.5 Hz), 5.11 (2 H, s), 7.28-7.39 (5 H, m); 13
C
NMR (CD3OD, 75.4 MHz) δ 12.47 (q), 41.35 (d), 56.02 (d), 67.58
(t), 127.92 (d), 128.66 (d), 129.05 (d), 136.65 (s), 157.31 (s),
174.14 (s), 177.79 (s); MS (EI) m/e (relative intensity) 281 (M+,
2), 263 (3), 146 (4), 108 (39), 91 (100); exact mass calcd for M+
C13H15NO6 requires 281.0899, found 281.0887 (EI).
Continued elution then gave the desired pyrrolidine deriva-
tive 11 as an oil which solidified upon standing: 625 mg, 83%;
mp 56-58 °C; IR (CHCl3) ν 3464, 3009, 2980, 1744, 1708, 1498,
1410, 1367, 1345, 1306, 1128, 1067, 1031 cm-1 1H NMR
;
(2S,3R)-2-Am in o-3-m eth ylsu ccin ic Acid (9). The N-
protected aspartic acid derivative 7 (780 mg, 2.77 mmol) was
dissolved in MeOH (50 mL) together with freshly distilled
cyclohexene (5.18 mL). After the addition of Pd/C (240 mg)
the suspension was refluxed for 2.5 h, allowed to cool, and then
poured into water (300 mL) and stirred for 1 h. Filtration
through through Celite, followed by reduction of the solvent
under reduced pressure, and freeze-drying yielded the aspartic
(CDCl3, 300 MHz) δ 1.36 (9 H, s), 2.05 (1 H, m), 2.50 (1 H, m),
3.65 (1 H, m), 3.72 (3 H, s), 4.20 (1 H, br s), 4.55 (1 H, d, J )
8.5 Hz), 5.14 (2 H, m), 5.70 (1 H, d, J ) 6.0 Hz), 7.3-7.4 (5 H,
m); 13C NMR (CDCl3, 75.4 MHz) δ 27.72 (q), 34,46 (t), 44.27
(d), 52.06 (q), 61.50 (d), 67.38 (t), 81.24 (d), 82.31 (s), 128.28
(d), 128.02 (d),128.58 (d), 135.67 (s), 154.46 (s), 168.47 (s),
170.17 (s); MS (FAB) m/e (relative intensity) 379 (MH+, 5),
362 (21), 262 (34), 155 (24), 133 (100), 91 (33); exact mass calcd
for MH+ C19H26NO7 requires 380.171, found 380.166 (FAB).
Anal. (C19H25NO7) C, H, N, requires C 60.15, H 6.64, N 3.69,
found C 60.12, H 6.68, N 3.64.
acid analogue 9 as a hygroscopic solid: 310 mg, 76%; [R]23
D
+5.8° (c ) 3.1, H2O); IR (nujol) ν 3645-2692, 1712, 1632, 1514,
1416, 1096 cm-1; 1H NMR (D2O, 300 MHz) δ 1.29 (3 H, d, J )
7.4 Hz), 3.18 (1 H, dq, J ) 7.4, 4.3 Hz), 4.01 (1 H, d, J ) 4.2
Hz); 13C NMR (D2O, 75.4 MHz) δ 12.87 (q), 39.95 (d), 56.04
(d), 171.95 (s), 176.92 (s); MS (FAB: glycerol + 5% TFA) m/e
(relative intensity) 148 (100), 115 (67), 74 (40), 63 (40); exact
mass calcd MH+ for C5H10NO4 requires 148.061, found 148.060
(FAB).
ter t-Bu tyl (2S,3R)-1-(Ben zyloxycar bon yl)-3-car bom eth -
oxyp yr r olid in e-2-ca r boxyla te (13). Alcohol 11 (150 mg,
0.39 mmol) and Et3SiH (0.1 mL, 0.59 mmol) were dissolved in
dry CHCl3 (2 mL). Neat TFA (0.3 mL, 3.9 mmol) was then
added dropwise to this solution with vigorous stirring. Upon
completion of the addition, the reaction mixture was stirred
at rt for a further 40 min before the solvent volume was
reduced under reduced pressure. This gave an oily residue
which was redissolved in EtOAc (20 mL). After washing with
5% aqueous NaHCO3 (3 × 5 mL), the organic layer was dried
(MgSO4) and the solvent removed to yield 13 as an oil which
(2S,3S)-2-Am in o-3-m eth ylsu ccin ic Acid (8). The N-
protected aspartic acid derivative 6 (360 mg, 1.28 mmol) was
treated as for the corresponding diastereoisomer 7 for 1 h.
Filtration through Celite, followed by reduction of the solvent
under reduced pressure, and freeze-drying yielded the desired
aspartic acid analogue 8 as a white hygroscopic solid: 184 mg,
98%; decomp at 275 °C; IR (CHCl3) ν 3264-2766, 3119, 1710,
1610, 1494 cm-1; [R]23 -12.0° (c ) 0.5, H2O); [R]23 +13.6° (c
solidified on standing: 107 mg, 74%; mp 81-82 °C; [R]20
D
+17.5° (c ) 0.71, CHCl3); IR (CHCl3) ν 3019, 2982, 1741, 1703,
1498, 1455, 1420, 1369, 1347, 1215, 1175, 1155 cm-1; 1H NMR
(CDCl3, 300 MHz) δ 1.38 (9 H, s), 2.10 (1 H, m), 2.40 (1 H, m),
3.25 (1 H, m), 3.46 (1 H, m), 3.72 (3 H, s), 3.77 (1 H, m), 4.52
(1 H, d, J ) 8.3 Hz), 5.10-5.16 (2 H, m), 7.30-7.40 (5 H, br
m); 13C NMR (CDCl3, 75.4 MHz) δ 25.56 (t), 27.80 (q), 45.50
D
D
1
) 0.46, 1 M HCl); H NMR (D2O, 300 MHz) δ 1.20 (3 H, d, J
) 7.4 Hz), 3.18 (1 H, dq, J ) 7.4, 3.5 Hz), 4.01 (1 H, d, J ) 3.5
Hz); 13C NMR (D2O, 75.4 MHz) δ 11.63 (q), 39.93 (d), 55.97
(d), 172.45 (s), 177.93 (s); MS (FAB: glycerol + 5% TFA) m/e

2374 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12
Parr et al.
(t), 46.52 (d), 52.00 (q), 61.19 (d), 67.07 (t), 82.05 (s), 127.83
(d), 127.95 (d), 128.38 (d), 135.30 (s), 154.09 (s), 168.89 (s),
170.29 (s); MS (EI) m/e (relative intensity) 363 (M+, 3), 307
(21), 262 (39), 218 (49), 91 (100); exact mass calcd for M+
C19H25NO6 requires 363.1682, found 363.1686 (EI). Anal.
(C19H25NO6) C, H, N, requires C 62.80, H 6.93, N 3.85, found
C 63.06, H 7.03, N 3.77.
X2 anion exchange resin (5 g, wet weight) by elution with 1%
AcOH.42 Ninhydrin positive fractions were collected and
freeze-dried to give 16 as a white solid: 26 mg, 76%; mp >
300 °C dec (lit.42 333 °C dec); IR (KBr) ν 3600-2800, 1702,
1618, 1460, 1102 cm-1; 1H NMR (D2O, 300 MHz) δ 2.19-2.40
(2 H, m), 3.32-3.51 (3 H, m), 4.46 (1 H, d, J ) 5.6 Hz); 13C
NMR (CD3CN, 75.4 MHz) δ 28.11 (t), 45.33 (t), 47.34 (d), 62.91
(d), 172.64 (s), 176.06 (s); MS (FAB: glycerol + 5% TFA) m/e
(relative intensity) 160 (MH+, 10), 154 (15), 133 (100), 93 (41);
exact mass calcd for MH+ C6H10NO4 requires 160.0610, found
160.044 (FAB). Anal. (C6H9NO4) C, H, N, requires C 45.28,
H 5.70, N 8.80, found C 45.25, H 6.02, N 8.57.
(2S,3S)-1-(Ben zyloxyca r bon yl)-3-ca r bom eth oxyp yr r o-
lid in e-2-ca r boxylic Acid (14). Neat TFA (1.1 mL, 1.4 mmol)
was added to a solution of diester 13 (230 mg, 0.7 mmol)
dissolved in CH2Cl2 (10 mL), and the resulting solution was
stirred at -5 °C for 30 min. After warming to rt, the reaction
was stirred for a further 20 h. Removal of the solvent under
reduced pressure then yielded the desired mono-acid 14 as an
oil of sufficient purity for use in subsequent reactions, in
(2S,3R)-2-(Ben zyloxyca r bon yla m in o)-3-ca r bom eth oxy-
h ex-5-en oic Acid (17). A solution of the diester 10 (1.01 g,
2.7 mmol) dissolved in CH2Cl2 (20 mL) was cooled to -8 °C
before the dropwise addition of TFA (4.13 mL, 53.7 mmol) with
vigorous stirring. After 70 h at this temperature, the reaction
mixture was warmed to 15 °C over a period of 24 h, before
being extracted with 5% (w/v) aqueous NaHCO3
quantitative yield: [R]20 -11.2° (c ) 1.34, CHCl3); IR (neat)
D
ν 3557-2736, 1738, 1704, 1670, 1498, 1434, 1360, 1305, 1269,
1
1133, 1090, 1030 cm-1; H NMR (CDCl3, 300 MHz) δ 2.15 (1
H, m), 2.40 (1 H, m), 3.31 (1 H, m), 3.50 (1 H, m), 3.68 (3 H, s),
3.75 (1 H, m), 4.67 (1 H, d, J ) 8.5 Hz), 5.10-5.20 (2 H, m),
7.25-7.38 (5 H, m), 10.00-10.25 (1 H, s); 13C NMR (CDCl3,
75.4 MHz) δ 26.32 (t), 45.76 (t), 46.65 (d), 52.32 (q), 60.54 (d),
67.65 (t), 127.82 (d), 128.11 (d), 128.47 (d), 136.03 (s), 155.50
(s), 170.39 (s), 170.50 (s); MS (EI:) m/e (relative intensity) 307
(M+, 8), 262 (7), 218 (29), 172 (35), 128 (21), 91 (100); exact
mass calcd for M+ C15H17NO6 requires 307.1056, found 307.1062
(EI).
(3 × 100 mL).
The aqueous phases were combined, acidified to pH 1 using 1
M HCl, and extracted using EtOAc (5 × 100 mL). The organic
extracts were dried (MgSO4
), and removal of the solvent under
reduced pressure yielded a clear oil which solidified on
standing. The desired ester 17 was obtained as a white solid
after recrystallization from EtOAc/petroleum ether: 763 mg,
89%; mp 93-94 °C; [R]20
+72.6° (c ) 0.31, CHCl3); IR (CH2-
D
Cl2) ν 3672-2732, 1728, 1512, 1266, 1223, 738 cm-1; 1H NMR
(CD3OD, 300 MHz) δ 2.30 (1 H, m), 2.45 (1 H, m), 3.16 (1 H,
m), 3.54 (3 H, s), 4.52 (1 H, m), 5.04 (2 H, m), 5.10 (2 H, m),
5.70-5.86 (1 H, m), 7.25-7.38 (5 H, m); 13C NMR (CD3OD,
75.4 MHz) δ 32.55 (t), 46.53 (d), 51.12 (q), 53.86 (d), 66.50 (t),
116.93 (t), 127.48 (d), 127.66 (d), 128.07 (d), 134.38 (d), 136.61
(s), 157.11 (s), 172.61 (s), 173.13 (s); MS (CI: H+) m/e (relative
intensity) 322 (MH+, 37), 278 (40), 219 (44), 91 (100); exact
mass calcd for MH+ C16H20NO6 requires 322.1291, found
322.1292 (CI).
(2S,3S)-1-(Ben zyloxyca r b on yl)-p yr r olid in e-2,3-d ica r -
boxylic Acid (15) a n d (2S,3R)-1-(Ben zyloxyca r bon yl)-
p yr r olid in e-2,3-d ica r boxylic Acid (36). The methyl ester
14 (196 mg, 0.6 mmol) was dissolved in 4:1 MeOH/H2O so as
to give an 0.1 M solution. After the addition of 4 equiv of
LiOH‚H2O, the reaction was stirred at room temperature until
the starting esters were completely consumed. The reaction
mixture was concentrated, under reduced pressure, to ap-
proximately 20% of its initial value and poured into an excess
of 1 M HCl. After adjustment of the solution pH to 1-2, the
mixture was extracted with EtOAc. The combined organic
phases were dried (MgSO4), and the solvent was removed
under reduced pressure to give the crude diacid as a buff-
colored solid. Purification by reverse phase HPLC with
gradient elution (C18 column; solvent flow rate 20 mL/min;
from 80:20 H2O:CH3CN + 1% TFA to 65:35 H2O:CH3CN + 1%
TFA over a period of 30 min), with monitoring at 226 nm, gave
diacid 15 (ret time: 18.7 min) as a clear oil after freeze-drying
overnight: 95 mg, 51%; IR (neat) ν 3742-2719, 3413, 3019,
(2S,3R)-2-(Ben zyloxyca r bon yla m in o)-3-ca r boxyh ex-5-
en oic Acid (18). Methyl ester 12 (200 mg, 0.33 mmol) was
dissolved in MeOH (30 mL) and water (30 mL) together with
NaOH (400 mg, 10.2 mmol) and the resulting solution refluxed
for 1.5 h. After cooling to rt, the reaction mixture was poured
into 1 M HCl (60 mL) and extracted using EtOAc (4 × 40 mL).
The combined organic phases were dried (MgSO4), and the
solvent was removed under reduced pressure. The crude
diacid was then purified using reverse phase HPLC with
gradient elution (C8 column; solvent flow rate 12 mL/min; 50:
50 H2O/CH3CN + 1% TFA to 10:90 H2O/CH3CN + 1% TFA
over a period of 20 min), with monitoring at 226 nm. Diacid
1
2590, 1715, 1499, 1424, 1360, 1129 cm-1; H NMR (CD3CN,
300 MHz) δ 2.12-2.24 (2 H, m), 3.18-3.26 (1 H, m), 3.46-
3.58 (2 H, m), 4.56 (1 H, d, J ) 3.6 Hz), 5.15-5.24 (2 H, m),
7.30-7.42 (5 H, m), 7.80-8.30 (2 H, br s); 13C NMR (CD3CN,
75.4 MHz) δ 27.03 (t), 45.49 (t), 46.24 (d), 60.90 (d), 66.63 (t),
127.32 (d), 127.66 (d), 128.14 (d), 136.53 (s), 154.76 (s), 172.05
(s), 172.64 (s); MS (LSIMS: glycerol + 5% TFA) m/e (relative
intensity) 294 (MH+, 3), 284 (18), 264 (20), 133 (100) 93 (92);
exact mass calcd for MH+ C14H16NO6 requires 294.0978, found
294.1350 (LSIMS: glycerol + 5% TFA).
Continued elution also yielded the diastereoisomeric diacid
36 (ret time: 21.0 min) as a clear oil after lyophilization: 52
mg, 28%; IR (neat) ν 3542-2531, 1742, 1710, 1651, 1499, 1428,
1361, 1147 cm-1; 1H NMR (CD3CN, 300 MHz) δ 2.12 (1 H, m),
2.22 (1 H, m), 3.32 (1 H, m), 3.40 (1 H, m), 3.63 (1 H, m), 4.54
(1 H, d, J ) 9.2 Hz), 5.06-5.15 (2 H, m), 6.30-6.60 (2 H, s),
7.30-7.40 (5 H, m); 13C NMR (CD3CN, 75.4 MHz) δ 26.41 (t),
45.53 (t), 46.30 (d), 60.38 (d), 66.74 (t), 127.56 (d), 127.90 (d),
128.37 (d), 136.83 (s), 171.14 (s); MS (LSIMS: glycerol + 5%
TFA) m/e (relative intensity) 294 (MH+, 100), 250 (60), 158
(7) 91 (89); exact mass calcd for MH+ C14H16NO6 requires
294.0978, found 294.1060 (LSIMS: glycerol + 5% TFA).
(2S,3S)-P yr r olid in e-2,3-d ica r boxylic Acid (16). The
N-protected diacid 15 (64 mg, 0.22 mmol) was dissolved in
MeOH (10 mL) together with dry cyclohexene (5 mL). Pd/C
(10%) (37 mg) was added and the reaction mixture refluxed
for 1 h. Filtration through Celite, followed by removal of the
solvent under reduced pressure, yielded the crude aspartic acid
18 (ret time: 8.1 min) was obtained as a clear gum: 58%; [R]20
D
+52.5° (c ) 0.67, CHCl3); IR (CHCl3) ν 3660-2308, 3418, 3056,
2980, 1722, 1514, 1419, 1226 cm-1 1H NMR (CD3CN, 300
;
MHz) δ 2.3 (1 H, m), 2.5 (1 H, m), 3.14 (1 H, m), 4.51 (1 H, dd,
J ) 10.0, 4.0 Hz), 4.98 (2 H m), 5.12 (2 H, s), 5.70-5.80 (1 H,
m), 6.10 (1 H, d, J ) 10.0 Hz), 7.20-7.30 (5 H, m), 8.00-8.50
(2 H, br s); 13C NMR (CD3CN, 75.4 MHz) δ 32.55 (t), 45.81 (d),
53.45 (d), 66.45 (t), 117.29 (t), 127.67 (d), 127.94 (d), 128.43
(d), 134.70 (d), 135.95 (s), 156.45 (s), 171.81 (s), 173.73 (s); MS
(EI) m/e (relative intensity) 307 (M
+, 2), 289 (6), 262 (3) 218
(9), 172 (6), 108 (22), 91 (100); exact mass calcd for MH+ C15H18
NO6 requires 308.1134, found 308.1133 (CI, C4H8).
-
(2S,3R)-2-Am in o-3-n -p r op ylsu ccin ic Acid (19). The N-
protected diacid 18 (88 mg, 0.29 mmol) was dissolved in MeOH
(5 mL) together with dry cyclohexene (2.5 mL). Pd/C (10%)
(38 mg) was added and the reaction mixture refluxed for 75
min. Filtration through Celite, followed by removal of the
solvent under reduced pressure, yielded the desired aspartic
acid analogue 19 as an off-white gum: 21 mg, 42%; IR (CHCl3)
ν 3668-2698, 3408, 3017, 2625, 1714, 1634, 1516, 1505, 1410
cm-1 1H NMR (D2O, 300 MHz) δ 0.93 (3 H, t, J ) 7.2 Hz),
;
1.35-1.48 (2 H, m), 1.52-1.65 (1 H, m), 1.68-1.70 (1 H, m),
3.03-3.11 (1 H, m), 3.94 (1 H, d, J ) 4.3 Hz); 13C NMR (D2O,
75.4 MHz) δ 12.93 (t), 12.95 (t), 19.93 (d), 19.95 (d), 30.61 (q),
177.55 (s), 182.52 (s); MS (FAB: glycerol + 5% TFA) m/e
(relative intensity) 176 (MH+, 100), 155 (21), 130 (9), 93 (19);
FAB HRMS exact mass calcd for MH+ C7H14NO4 requires
176.0923, found 176.0920 (FAB, glycerol + 5% TFA).
analog 16. This hygroscopic solid was redissolved in
a
minimum amount of water and purified using Bio Rad Ag-1-

Mapping the AS-B Aspartate Binding Site
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12 2375
Diben zyl (2S)-2-(Ben zyloxyca r bon yla m in o)su ccin a te
(20). The dibenzyl ester of L-aspartic acid, as its p-toluene-
sulfonate salt (1 g, 2 mmol) was added to a solution of dioxane
(16 mL) and water (32 mL) containing Na2CO3 (1.24 g, 10
mmol). After cooling the reaction mixture to 0 °C, neat benzyl
chloroformate (1.43 mL, 10 mmol) was added. The reaction
was warmed to rt and stirred for 18 h. Water (30 mL) was
added, and the resulting solution was extracted with Et2O (5
× 15 mL). The aqueous portion was then acidified to pH 1
with HCl and extracted using EtOAc (5 × 25 mL). The organic
extracts were then combined and dried, and the solvent was
removed under reduced pressure to give an oil. Excess benzyl
alcohol was then removed by vacuum distillation to give the
desired N-protected diester 20 as a clear oil which solidified
to a white solid on cooling: 871 mg; 95%; mp 61-63 °C (lit.52
oil. This material was purified by flash chromatography
(eluant: 30% EtOAc/petroleum ether) to yield a mixture of the
diastereoisomers 22 and 23, which solidified on standing (214
mg, 22%). 1H NMR analysis showed that this material was a
9:1 ratio of 23:22, which was used in the subsequent reduction
reaction. Careful column chromatography gave a small amount
of pure diester 23: mp 71-72 °C; [R]20 +31.3° (c ) 0.64,
D
CHCl3); IR (CHCl3) υ 3446, 3019, 1749, 1734, 1715, 1699, 1558,
1540, 1507, 1457, 1419 cm-1 1H NMR (CDCl3, 300 MHz) δ
;
2.05-2.15 (1 H, m), 2.48-2.62 (1 H, m), 3.65-3.80 (1 H, m),
3.83 (1 H, br s), 4.68-4.78 (1 H, m), 4.80-5.20 (6 H, m), 5.72
(1 H, d, J ) 5.6 Hz), 7.16-7.22 (5 H, m), 7.25-7.36 (10 H, m);
13C NMR (CDCl3, 75.4 MHz) δ 34.73 (t), 44.43 (d), 60.96 (d),
67.18 (t), 67.41 (t), 67.62 (t), 81.43 (d), 127.76 (d), 128.06 (d),
128.14 (d), 128.26 (d), 128.35 (d), 128.47 (d), 128.50 (d), 128.68
(d), 128.78 (d), 135.13 (s), 135.18 (s), 135.74 (s), 154.31 (s),
169.54 (s), 169.69 (s); MS (FAB NBA) m/e (relative intensity)
490 (MH+, 2), 472 (30), 428 (100), 338 (15); HRMS exact mass
calcd for MH+ C28H28NO7 requires 490.1866, found 490.1858.
Diben zyl (2S,3R)-1-(Ben zyloxyca r bon yla m in o)p yr r o-
lid in e-2,3-d ica r boxyla te (24). The mixture of diastereoiso-
meric diesters 22 and 23 (200 mg, 0.4 mmol) and Et3SiH (0.1
mL, 0.59 mmol) were dissolved in dry CHCl3 (5 mL). Neat
TFA (0.31 mL, 4.0 mmol) was then added dropwise to this
solution with vigorous stirring. Upon completion of the
addition, the reaction mixture was stirred at rt for a further
40 min before the solvent volume was reduced under reduced
pressure. This gave an oily residue which was redissolved in
EtOAc (20 mL). After washing with 5% aqueous NaHCO3 (3
× 5 mL), the organic layer was dried (MgSO4) and the solvent
61-63 °C); [R]22 +14.1° (c ) 6.9, CHCl3); IR (CHCl3) υ 3429,
D
3357, 3033, 1733, 1506, 1456, 1338 cm-1; 1H NMR (CDCl3, 300
MHz) δ 2.82-3.15 (2 H, ABX q, J AB ) 17.1 Hz, J AX ) 4.5 Hz,
J BX ) 4.5 Hz), 4.69 (1 H, m), 5.05 (2 H, s), 5.11 (2 H, s), 5.13
(2 H, s), 5.80 (1 H, d, J ) 8.4 Hz), 7.3-7.36 (15 H, m); 13C
NMR (CDCl3, 75.4 MHz) δ 36.76 (t), 50.57 (d), 66.83 (t), 67.12
(t), 67.54 (t), 126.97 (d), 127.56 (d), 128.06 (d), 128.19 (d),
128.25 (d), 128.33 (d), 128.44 (d), 128.52 (d), 128.59 (d), 131.27
(s), 135.15 (s), 135.32 (s), 140.99 (s), 156.00 (s), 170.50 (s); MS
(FAB NBA) m/e (relative intensity) 448 (MH+, 11), 404 (7), 181
(7), 136 (11), 91 (100); exact mass calcd MH+ for C26H26NO6
requires 448.1760, found 448.1700 (FAB, NBA). Anal. (C26H25
-
NO6) C, H, N, requires C 69.79, H 5.63, N 3.13, found C 69.70,
H 5.64, N 3.03.
Ben zyl (2S,3R)-2-(Ben zyloxyca r bon yla m in o)-3-ca r bo-
ben zoxyh ex-5-en oa te (21). A solution of LiHMDS (3.8 mL
of a 1.0 M solution in THF, 3.80 mmol) dissolved in dry THF
(10 mL) was cooled to -80 °C before the addition of diester 20
(0.77 g, 1.72 mmol) dissolved in dry THF (30 mL) to give a
pale-yellow solution. The reaction mixture was stirred at -78
°C for 30 min and then warmed slowly to -30 °C and stirred
for 2 h to ensure formation of the dianion. After recooling the
resulting orange solution to -78 °C, neat allyl bromide (0.75
mL, 8.6 mmol) was added at such a rate that the solution
temperature did not exceed -75 °C. Stirring was continued
for 16 h, at -78 °C, before the reaction was quenched by
pouring it into aqueous 1 M HCl (120 mL) at rt. After
extraction using Et2O (4 × 50 mL), the organic phases were
dried (MgSO4), and the solvent was evaporated under reduced
pressure. The crude material was purified by flash chroma-
tography (eluant: 70:30 40-60 °C petroleum ether/EtOAc) to
yield a clear oil, which was a mixture of epimers at C-3,
followed by starting diester 20 (236 mg, 31%). However, the
oil solidified on standing and careful recrystallization from
petroleum ether/EtOAc gave the desired allylated diester 21
removed to yield 24 as a pale-yellow oil: 171 mg, 89%; [R]20
D
+14.7° (c ) 4.0, CHCl3); IR (CHCl3) υ 3065, 3034, 2957, 2894,
1748, 1716, 1700, 1498, 1456, 1418, 1361, 1188 cm-1; 1H NMR
(CDCl3, 300 MHz) δ 2.08-2.20 (1 H, m), 2.46-2.52 (1 H, m),
3.24-3.40 (1 H, m), 3.40-3.54 (1 H, m), 3.74-3.86 (1 H, m),
4.71 (1 H, d, J ) 8.4 Hz), 4.75-5.15 (6 H, m), 7.18-7.35 (15
H, m); 13C NMR (CDCl3, 75.4 MHz) δ 26.55 (t), 45.68 (t), 46.83
(d), 60.73 (d), 66.99 (t), 67.13 (t), 67.22 (t), 127.83 (d), 127.98
(d), 128.39 (d), 128.45 (d), 128.51 (d), 135.24 (s), 136.74 (s),
136.87 (s), 154.11 (s), 169.77 (s), 169.89 (s); MS (FAB NBA)
m/e (relative intensity) 474 (MH+, 100), 430 (67), 338 (33), 294
(35), 181 (45); exact mass calcd for MH+ C28H28NO6 requires
474.1917, found 474.1942 (FAB, NBA).
(2S,3R)-P yr r olid in e-2,3-d ica r boxylic Acid (25). The
N-protected diester 24 (160 mg, 0.34 mmol) was dissolved in
a mixture of MeOH (10 mL) and cyclohexene (1 mL) together
with Pd/C (50 mg). After refluxing for 3 h the reaction was
cooled to rt, poured into water (100 mL), and stirred vigorously
for a further hour. The resulting reaction mixture was then
filtered through Celite. After repeated washing with water,
the filtrate was collected and its volume reduced to 30 mL
before lyophilization. This procedure yielded the crude diacid
25 as a hygroscopic white solid. This material was redissolved
in a minimum amount of water and purified by chromatog-
raphy on Bio Rad Ag-1-X2 anion-exchange resin (10 g wet
weight) by elution with 1% AcOH.42 Ninhydrin positive
fractions were collected and lyophilized to give the desired
diacid 25 as a white solid: mp > 300 °C dec (lit.42 332 °C dec);
IR (KBr) v 3600-2800, 1702, 1637, 1416, 1101 cm-1; 1H NMR
(D2O, 500 MHz) δ 2.10-2.20 (1 H, m), 2.25-2.30 (1 H, m),
3.30-3.45 (3 H, m), 4.20 (1 H, d, J ) 7.3 Hz); 13C NMR (D2O,
125.6 MHz) δ 26.38 (t), 42.09 (t), 43.80 (d), 61.95 (d), 169.70
(s), 174.62 (s); MS (FAB glycerol + TFA 5%) m/e (relative
intensity) 160 (MH+, 66), 130(17), 115 (100), 104 (25), 102 (37);
exact mass calcd for MH+ C6H10NO4 requires 160.061, found
160.058 (FAB, NBA).
Diben zyl (2S,3R)-1-(Ben zyloxyca r bon yl)p yr r olid in e-
2,3-d ica r boxyla te (24) a n d Diben zyl (2S,3R)-1-(Ben zyl-
oxyca r bon yl)p yr r olid in e-2,3-d ica r boxyla te (26). A mix-
ture of dibenzyl (2S,3R,5R)-1-(benzyloxycarbonyl)-5-hydroxy-
pyrrolidine-2,3-dicarboxylate, dibenzyl (2S,3R,5S)-1-(benzyl-
oxycarbonyl)-5-hydroxypyrrolidine-2,3-dicarboxylate, dibenzyl
(2S,3S,5R)-1-(benzyloxycarbonyl)-5-hydroxypyrrolidine-2,3-di-
carboxylate, dibenzyl (2S,3S,5S)-1-(benzyloxycarbonyl)-5-hy-
droxypyrrolidine-2,3-dicarboxylate (872 mg), and Et3SiH (0.45
mL, 2.82 mmol) were dissolved in dry CHCl3 (25 mL) at rt.
as a single diastereoisomer: 476 mg, 57%; mp 82-83 °C; [R]20
D
+29.4° (c ) 2.3, CHCl3); IR (CHCl3) υ 3428, 3019, 1733, 1507,
1
1456 cm-1; H NMR (CDCl3, 300 MHz) δ 2.26-2.38 (1 H, m),
2.47-2.58 (1 H, m), 3.21-3.29 (1 H, m), 4.68 (1 H, dd, J )
9.9, 3.7 Hz), 5.04-5.14 (8 H, m), 5.68-5.84 (2 H, m), 7.25-
7.36 (15 H, m); 13C NMR (CDCl3, 75.4 MHz) δ 32.86 (t), 46.56
(d), 54.12 (d), 66.93 (t), 67.14 (t), 67.39 (t), 118.35 (t), 128.00
(d), 128.06 (d), 128.13 (d), 128.22 (d), 128.40 (d), 128.46 (d),
128.51 (d), 128.55 (d), 128.60 (d), 133.99 (d), 135.29 (s), 135.33
(s), 136.34 (s), 156.55 (s), 170.76 (s), 172.76 (s); MS (FAB) m/e
(relative intensity) 488 (MH+, 100), 444 (58), 308 (19), 181 (96);
exact mass calcd for MH+ C29H30NO6 requires 488.2073, found
488.2082 (FAB). Anal. (C29H29NO6) C, H, N requires C 71.44,
H 6.00, N 2.87, found C 71.20, H 5.98, N 2.82.
Diben zyl (2S,3R,5S)-1-(Ben zyloxyca r bon yl)-5-h yd r oxy-
p yr r olid in e-2,3-d ica r b oxyla t e (22) a n d Dib en zyl (2S,
3R,5R)-1-(Ben zyloxyca r bon yl)-5-h yd r oxyp yr r olid in e-2,3-
d ica r boxyla te (23). OsO4 (1.8 mL of a 2.5% solution in
2-propanol, 0.144 mmol) and NaIO4 (1.39 g, 6.5 mmol) were
added to a solution of diester 21 (960 mg, 2.16 mmol) in MeOH
(40 mL) and water (20 mL). The resulting reaction mixture
was stirred at rt for 75 min. After halving the solvent volume
under reduced pressure, the solution was poured into water
(140 mL) and extracted with EtOAc (6 × 50 mL). The organic
extracts were dried (MgSO4), and the solvent was removed
under reduced pressure to yield the crude product as a brown

2376 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12
Parr et al.
Ta ble 2. Substrate Concentrations Employed in Experiments
To Determine the Ability of AS-B-Selected Substrates and the
Aspartate Analog 7 To Protect the Enzyme against Covalent
Modification by FSBA
Neat TFA (1.4 mL, 18.2 mmol) was then added dropwise to
this solution with vigorous stirring. Upon completion of the
addition, the reaction mixture was stirred at rt for a further
90 min before the solvent volume was reduced under reduced
pressure. This gave an oily residue which was redissolved in
EtOAc (20 mL). After washing with 5% aqueous NaHCO3 (2
× 5 mL), the organic layer was dried (MgSO4) and the solvent
removed to give a mixture of the diastereoisomeric pyrrolidines
24 and 26 as a pale-yellow oil. These compounds could be
separated by chromatogrpahy on silica using a gravity column
(eluant: 3:1 40-60 °C petroleum ether/EtOAc). Diester 24
was obtained first from the column (401 mg, 47%) as a clear
oil, with identical properties to the material prepared from
purified 21. Further elution then gave the diastereoisomeric
substrate/analog concentration (mM)
experiment no.
ATP
Asp
7
FSBA
1
2
3
4
5
6
0.0
0.0
0.0
0.0
0.0
1.0
1.0
0.0
0.0
5.0
5.0
0.0
0.0
1.0
1.0
1.0
1.0
1.0
1.0
0.05
0.05
0.0
0.0
0.05
diester 26 as a clear oil: 177 mg, 20%; [R]20 +28.8° (c ) 1.7,
D
coupling reagent for the detection of pyrophosphate (Sigma
Bulletin No. B1-100) to a final volume of 140 µL. The reaction
buffer (10 mM Gln, 10 mM Asp, 10 mM ATP, 15 mM was
prewarmed to 37 °C. The ability of the FSBA-treated AS-B
to catalyze asparagine synthesis was then determined by
measuring the amount of pyrophosphate produced in 5 min
by monitoring the reduction of NAD+ to NADH by glutamate
dehydrogenase. In control solutions from which FSBA was
omitted, AS-B retained greater than 95% of its original activity
over the entire duration of the incubation experiments.
CHCl3); IR (neat) υ 3065, 3034, 2958, 2893, 1715, 1608, 1587,
1497, 1456, 984 cm-1; 1H NMR (CDCl3, 300 MHz) δ 3.20-3.32
(1 H, m), 3.66-3.74 (2 H, m), 4.85-4.92 (1 H, dd, J ) 18.8,
3.4 Hz), 5.1-5.3 (7 H, m), 7.15-7.25 (15 H, m); 13C NMR
(CDCl3, 75.4 MHz) δ 27.1 (t), 45.4 (t), 47.9 (d), 61.5 (d), 66.9
(t), 127.5 (d), 126.6 (d), 127.7 (d), 127.8 (d), 127.9 (d), 128.0
(d), 128.05 (d), 128.2 (d), 128.25 (d), 128.3 (d), 128.4 (d), 135.0
(s), 135.2 (s), 136.1 (s), 136.2 (s), 153.7 (s), 154.4 (s), 170.5 (s),
171.1 (s), 171.2 (s); MS (FAB NBA) m/e (relative intensity) 474
(MH+, 12), 430 (10), 338 (2), 294 (1), 181 (8); exact mass calcd
for MH+ C28H28NO6 requires 474.1917, found 474.1918 (FAB,
NBA).
(2S,3S)-P yr r olid in e-2,3-d ica r boxylic Acid (16). Pd/C
(10%) (50 mg) was suspended in a solution of the N-protected
diester 26 (175 mg, 0.32 mmol) and NH4OCHO (560 mg, 8.9
mmol) dissolved in MeOH (11 mL). After stirring at rt for 210
min, the suspension was filtered through Celite and the solvent
removed under reduced pressure to give an oily residue.
Lyophilization then gave the crude diacid 25, which was
purified using anion-exchange chromatography as described
above.
Gen er a l P r oced u r e for In h ibitor Assa ys. Inhibition
constants for 8, 9, 25, and 26 were determined using standard
methods. All assays were performed using 2.3-5 µg of wild-
type AS-B in a reaction buffer composed of 5 mM MgCl2 and
100 mM Tris-HCl, pH 8. In experiments to determine the
extent of AS-B inhibition with respect to aspartate, ATP, and
glutamine were fixed at 1 mM and the concentration of
aspartic acid varied (0.1-1.1 mM). Inhibitor concentrations
were the following: 8, 0, 2, 6, 10, 15, and 20 mM; 9, 0, 0.5, 1,
1.5, and 3 mM; 25, 0, 1, 2.5, and 5 mM; 26, 0, 0.5, 1, and 1.5
mM. In experiments to determine the extent of AS-B inhibi-
tion with respect to ATP, aspartate and glutamine were fixed
at 1 mM and the concentration of ATP varied (0.075-0.3 mM).
Inhibitor concentrations were the following: 8, 0, 2.5, 5, and
7.5 mM; 9, 0, 0.5, 1, and 1.5 mM; 25, 0, 1, 2.5, and 5 mM. In
experiments to determine the extent of AS-B inhibition with
respect to glutamine, ATP and aspartate were fixed at 1 mM
and the concentration of glutamine varied (0.1-0.85 mM).
Inhibitor concentrations were the following: 8, 0, 10, 15, and
20 mM; 9, 0, 0.5, 1, and 1.5 mM; 25, 0, 2.5, 5, and 7.5 mM.
Inhibition constants were obtained from double-reciprocal plots
using initial velocities that were the average of those measured
in duplicate assays. The rate of asparagine synthesis was
determined using a coupled assay for the detection of pyro-
phosphate (Sigma Bulletin No. B1-100).
F SBA In a ctiva tion of AS-B. A stock solution of 10 mM
FSBA in 1:1 EtOH/DMSO was used in all inactivation experi-
ments. The concentration was verified by measuring the
absorption of this solution at 259 nm (ꢀ 1.35 × 104 M-1 cm-1).
Enzyme inactivation was carried out by incubating AS-B (28
µg) in 100 mM Tris, pH 8, with FSBA at rt in solutions (total
volume 140 µL) containing substrate(s) and/or the aspartate
analogs (Table 2). Incubations proceeded in solutions contain-
ing 1 mM FSBA, 0.2 µg/µL AS-B, 8 mM MgCl2, and 100 MM
Tris-HCl, pH 8, together with 5% EtOH and 5% DMSO.
Control solutions had an identical composition except that
FSBA was omitted. At a given time-point, 20 µL aliquots were
withdrawn from each of the incubations and diluted into a
reaction buffer (10 mM L-aspartate, 10 mM glutamine, 10 mM
ATP, 8 mM MgCl2, and 100 mM Tris-HCl, pH 8) containing a
Ack n ow led gm en t. This work was supported by
Grant CA-28725 from the National Cancer Institute,
National Institutes of Health, DHSS. Partial support
was also provided by the American Cancer Society,
Florida Affiliate, Inc. I.B.P. also thanks the Science and
Engineering Research Council (U.K.) for the award of
a studentship.
Refer en ces
(1) (a) Broome, J . D. Evidence that the L-Asparaginase of Guinea
Pig Serum is Responsible for its Antilymphoma Effects. J . Exp.
Med. 1963, 118, 99-148. (b) Burchenal, J . H. A Summary of
the Clinical Status of Asparaginase. Recent Results Cancer Res.
1970, 33, 350-354.
(2) (a) Cooney, D. A.; Handschumacher, R. E. L-Asparaginase and
L-Asparagine Metabolism. Annu. Rev. Pharmacol. 1970, 10,
421-440. (b) Broome, J . D. Studies on the Mechanism of Tumor
Inhibition by L-Asparaginase. J . Exp. Med. 1968, 127, 1055-
1072.
(3) (a) Kiriyama, Y.; Kubota, M.; Takimoto, T.; Kitoh, J .; Tanizawa,
A.; Akiyama, Y.; Mikawa, H. Biochemical Characterization of
U937 Cells Resistant to L-Asparaginase: The Role of Asparagine
Synthetase. Leukemia 1989, 3, 294-297. (b) Broome, J . D.;
Schwartz, J . H. Differences in the Production of L-Asparagine
in Asparaginase-Sensitive and Resistant Lymphoma Cells. Bio-
chim. Biophys. Acta 1967, 138, 637-639.
(4) Hersh, E. M. Immunosuppression by L-Asparaginase and Related
Enzymes. Transplantation 1971, 12, 368-376.
(5) (a) Cedar, H.; Schwartz, J . H. The Asparagine Synthetase of
Escherichia coli. I. Biosynthetic Role of the Enzyme, Purification,
and Characterization of the Reaction Products. J . Biol. Chem.
1969, 244, 4112-4121. (b) Cedar, H.; Schwartz, J . H. The
Asparagine Synthetase of Escherichia coli. II. Studies on Mech-
anism. J . Biol. Chem. 1969, 244, 4122-4127. (c) Reitzer, L. J .;
Magasanik, B. Asparagine Synthetases of Klebsiella aerogenes:
Properties and Regulation of Synthesis. J . Bacteriol. 1982, 151,
1299-1313. (d) Mehlhaff, P.; Luehr, C. A.; Schuster, S. M.
Studies of the Ammonia-Dependent Reaction of Beef Pancreatic
Asparagine Synthetase. Biochemistry 1985, 24, 1104-1110. (e)
Van Heeke, G.; Schuster, S. M. Expression of Human Asparagine
Synthetase in Escherichia coli. J . Biol. Chem. 1989, 264, 5503-
5509. (f) Ray, P. N.; Siminovitch, L.; Andrulis, I. L. Molecular
Cloning of a cDNA for Chinese Hamster Ovary Asparagine
Synthetase. Gene 1984, 30, 1-9. (g) Lam, H. M.; Peng, S. S.;
Coruzzi, G. M. Metabolic Regulation of the Gene Encoding
Glutamine-Dependent Asparagine Synthetase in Arabidopsis
thaliana. Plant Physiol. 1994, 106, 1347-1357.
(6) (a) Goodbourn, S. Transcirptional Regulation in Activated T-
cells. Curr. Biol. 1994, 4, 930-932. (b) Clipstone, N. A.; Crabtree,
G. R. Identification of Calcineurin as a Key Signalling Enzyme
in T-Lymphocyte Activation. Nature 1992, 357, 92-94.
(7) (a) Cooney, D. A.; Driscoll, J . S.; Milman, H. A.; J ayaram, H.
N.; Davis, R. D. Inhibitors of L-Asparagine Synthetase In Vitro.
Cancer Treat. Rep. 1976, 60, 1493-1557. (b) Cooney, D. A.;
J ones, M. T.; Milman, H. A.; Young, D. M.; J ayaram, H. N.
Regulators of the Metabolism of L-Asparagine: A Search for
Endogenous Inhibitors. Int. J . Biochem. 1980, 11, 519-539.

Mapping the AS-B Aspartate Binding Site
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12 2377
(8) (a) Mokotoff, M.; Bagaglio, J . F.; Parikh, B. S. Potential Inhibi-
tors of L-Asparagine Biosynthesis. 2. Chemistry and Biological
Activity of â-Hydroxyaspartic Acid and Its Derivatives. J . Med.
Chem. 1975, 18, 354-358. (b) Mokotoff, M.; Brynes, S.; Bagaglio,
J . F. Potential Inhibitors of L-Asparagine Biosynthesis. 3.
Aromatic Sulfonyl Fluoride Analogs of L-Asparagine and L-
Glutamine. J . Med. Chem. 1975, 18, 888-891.
(22) Colman, R. F. Site-Specific Modification of Enzyme Sites. In The
Enzymes, Vol. XIX, 3rd ed.; Sigman, D. S., Boyer, P. D., Eds.;
Academic Press: San Diego, 1990; pp 283-321.
(23) Baldwin, J . E.; Moloney, M. G.; North, M. Non-Proteinogenic
Amino Acid Synthesis: Synthesis of â,γ-Unsaturated R-Amino
Acids from Aspartic Acid. Tetrahedron 1989, 45, 6319-6325.
(24) (a) Coleman, D. Synthetic Polypeptides Part IV. Preparation and
Polymerization of Oxazolid-2,5-diones Derived From R-Amin-
odicarboxylic acids. J . Chem. Soc. 1951, 2294-2295. (b) Schwarz,
H.; Bumpus, F. M.; Page, I. H. Synthesis of a Biologically Active
Octapeptide Similar to Natural Isoleucine Angiotonin Octapep-
tide. J . Am. Chem. Soc. 1957, 29, 5697-5703.
(25) Baldwin, J . E.; Moloney, M. G.; North, M. Non-Proteinogenic
Amino Acid Synthesis: The â-Anion Derived from Aspartic Acid
and its Application to R-Amino Acid Synthesis. Tetrahedron
1989, 45, 6309-6318.
(26) Baldwin, J . E.; Moloney, M. G.; North, M. Asymmetric Amino
Acid Synthesis: Preparation of the â-Anion derived from As-
partic Acid. J . Chem. Soc., Perkin Trans. 1 1989, 833-834.
(27) Wolf, J .-P.; Rapoport, H. Conformationally Constrained Peptides.
Chirospecific Synthesis of 4-Alkyl-Substituted γ-Lactam-Bridged
Dipeptides from L-Aspartic Acid. J . Org. Chem. 1989, 54, 3164-
3173.
(28) (a) Anderson, G. W.; Callahan, F. M. tert-Butyl Esters of Amino
Acids and Peptides and Their Use in Peptide Synthesis. J . Am.
Chem. Soc. 1960, 82, 3359-3363. (b) Bryan, D. B.; Hall, R. F.;
Holden, K. G.; Huffman, W. F.; Gleason, J . G. Nuclear Analogues
of â-Lactam Antibiotics. 2. The Total Synthesis of 8-oxo-4-thia-
1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic Acids. J . Am. Chem.
Soc. 1977, 99, 2353-2355.
(9) (a) J ayaram, H. N.; Cooney, D. A.; Ryan, J . A.; Neil, G.; Dion,
R. L.; Bono, V. H. L-[RS, 5S]-R-Amino-3-Chloro-4,5-Dihydro-5-
Isoxazoleacetic Acid (NCS-163501): A New Amino Acid Antibi-
otic With the Properties of an Antagonist of L-Glutamine. Cancer
Chemother. Rep. 1975, 59, 481-491. (b) Handschumacher, R.
E.; Bates, C. J .; Chang, P. K.; Andrews, A. T.; Fischer, G. A.
5-Diazo-4-oxo-L-norvaline: Reactive Asparagine Analog with
Biological Specificity. Science 1968, 161, 62-63. (e) J ayaram,
H. N.; Cooney, D. A.; Milman, H. A.; Homan, E. R.; Rosenbluth,
R. J . DON, CONV, and DONV-I. Inhibition of L-Asparagine
Synthetase in vitro. Biochem. Pharmacol. 1976, 25, 1571-1582.
(10) (a) Boehlein, S. K.; Richards, N. G. J .; Schuster, S. M. Glutamine-
dependent Nitrogen Transfer in Escherichia coli Asparagine
Synthetase B. Searching for the Catalytic Triad. J . Biol. Chem.
1994, 269, 7450-7457. (b) Boehlein, S. K.; Richards, N. G. J .;
Walworth, E. S.; Schuster, S. M. Arginine-30 and Asparagine-
74 Have Functional Roles in the Glutamine Dependent Activities
of Escherichia coli Asparagine Synthetase B. J . Biol. Chem.
1994, 269, 26789-26795.
(11) Humbert, R.; Simoni, R. D. Genetic and Biochemical Studies
Demonstrating a Second Gene Coding for Asparagine Synthetase
in Escherichia coli. J . Bacteriol. 1980, 142, 212-220.
(12) Nakamura, M.; Yamada, M.; Hirota, Y.; Sugimoto, K.; Oka, A.;
Takanami, M. Nucleotide Sequence of the asnA Gene Coding
for Asparagine Synthetase of E. coli K-12. Nucl. Acids Res. 1981,
9, 4669-4676.
(13) Scofield, M. A.; Lewis, W. S.; Schuster, S. M. Nucleotide
Sequence of Escherichia coli asnB and Deduced Amino Acid
Sequence of Asparagine Synthetase B. J . Biol. Chem. 1990, 265,
12895-12902.
(14) (a) Zalkin, H. The Amidotransferases. Adv. Enzymol. Relat.
Areas Mol. Biol. 1993, 66, 203-309. (b) Buchanan, J . M. The
Amidotransferases. Adv. Enzymol. Relat. Areas Mol. Biol. 1973,
39, 91-183.
(29) Corey, E. J .; Szekely, I.; Shiner, C. S. Synthesis of 6,9R-oxido-
11R,15R-dihydroxyprosta-(E)5,(E)13-dienoic Acid, an Isomer of
PGI2 (Vane’s PGX). Tetrahedron Lett. 1977, 3529-3532.
(30) (a) Anantharamaiah, G. M.; Sivanandaiah, K. M. Transfer
Hydrogenation: A Convenient Method for Removal of Some
Commonly Used Protecting Groups in Peptide Synthesis. J .
Chem. Soc., Perkin Trans. 1 1977, 490-491. (b) J ackson, A. E.;
J ohnstone, R. A. W. Rapid, Selective Removal of Benzyloxycar-
bonyl Groups from Peptides by Catalytic Transfer Hydrogena-
tion. Synthesis 1976, 685-687.
(31) Seebach, D.; Wasmuth, D. Alkylation of Amino Acids without
Loss of Optical Activity: R- and â-Alkylation of an Aspartic Acid
Derivative. Angew. Chem., Int. Ed. Engl. 1982, 20, 971-971.
(32) Barker, H. A.; Smyth, D.; Wilson, R. M.; Weissbach, H. The
Purification and Properties of â-Methylaspartase. J . Biol. Chem.
1959, 234, 320-328.
(33) Bodanzky, M.; Marconi, G. G. Configuration of the â-Methylas-
partic Acid Residue in Amphomycin. J . Antibiot. 1970, 23, 238-
241.
(34) The high level of stereoselectivity under our reaction conditions
for the â-functionalization of aspartate derivative 1 is unusual.
Detailed experimental studies of the origins of this observation
will be published elsewhere.
(35) (a) Pappo, R.; Allen, D. S., J r.; Lemieux, R. U.; J ohnson, W. S.
Osmium Tetroxide-Catalyzed Periodate Oxidation of Double
Bonds. J . Org. Chem. 1956, 21, 478-479. (b) Inomata, K.;
Nakayama, Y.; Kotake, H. Quaternary Ammonium Periodate as
a New Oxidizing Agent. Bull. Chem. Soc. J pn. 1980, 53, 565-
566
(36) (a) Derome, A. E. Modern NMR Techniques for Chemistry
Research; Pergamon: Oxford, 1987. (b) Sanders, J . K. M.; Mersh,
J . D. Nuclear Magnetic Double Resonance; The Use of Difference
Spectroscopy. Prog. NMR Spectrosc. 1982, 4, 353-400.
(37) (a) Mauger, A. B.; Irreverre, F.; Witkop, B. The Stereochemistry
of 3-Methylproline. J . Am. Chem. Soc. 1966, 88, 2019-2024. (b)
Ibrahim, I.; Lubell, W. D. Synthesis of Enantiopure δ-Oxo
R-Amino Esters and Prolines via Acylation of N-(Phenylfluore-
nyl)glutamate Enolates. J . Org. Chem. 1993, 58, 6438-6441.
(38) (a) Chang, G.; Guida, W. C.; Still, W. C. An Internal Coordinate
Monte Carlo Method for Searching Conformation Space. J . Am.
Chem. Soc. 1989, 111, 4379-4386. (b) Saunders, M.; Houk, K.
N.; Wu, Y. D.; Still, W. C.; Lipton, M. A.; Chang, G.; Guida, W.
C. Conformations of Cycloheptadecane. A Comparison of Meth-
ods for Conformational Searching. J . Am. Chem. Soc. 1990, 112,
1419-1427.
(39) MacDonald, D. Q.; Still, W. C. AMBER* Torsional Parameters
for the Peptide Backbone. Tetrahedron Lett. 1992, 33, 7743-
7746.
(40) Mohamadi, F.; Richards, N. G. J .; Guida, W. C.; Liskamp, R. M.
J .; Lipton, M. A.; Caufield, C. E.; Chang, G.; Hendrickson, T.
F.; Still, W. C. MacroModel: An Integrated Software System for
Modelling Organic and Bioorganic Molecules Using Molecular
Mechanics. J . Comput. Chem. 1990, 11, 440-467.
(15) (a) Tso, J . Y.; Zalkin, H.; van Cleemput, M.; Yanofsky, C.; Smith,
J . M. Nucleotide Sequence of Escherichia coli purF and Deduced
Amino Acid Sequence of Glutamine Phosphoribosylpyro-phos-
phate Amidotransferase. J . Biol. Chem. 1982, 257, 3525-3531.
(b) Makaroff, C. A.; Zalkin, H.; Switzer, R.; Vollmer, S. J . Cloning
of the Bacillus subtilis Glutamine Phosphoribosylpyro-phosphate
Amidotransferase Gene in Escherichia coli. Nucleotide Sequence
Determination and Properties of the Plasmid-Encoded Enzyme.
J . Biol. Chem. 1983, 258, 10586-10593. (c) Tso, J . Y.; Hermod-
son, M. A.; Zalkin, H. Glutamine Phosphoribosylpyrophosphate
Amidotransferase from Cloned Escherichia coli purF. NH2-
Terminal Amino Acid Sequence, Identification of the Glutamine
Site, and Trace Metal Analysis. J . Biol Chem. 1982, 257, 3532-
3536.
(16) (a) Winterburn, P. J .; Phelps, C. F. Purification and some Kinetic
Properties of Rat Liver Glucosamine Synthase. Biochem. J . 1971,
121, 701-709. (b) Badet, B.; Vermoote, P.; Haumont, P. Y.;
Lederer, F.; LeGoffic, F. Glucosamine Synthase from Escherichia
coli. Purification, Properties, and Glutamine-Utilizing Site Loca-
tion. Biochemistry 1987, 26, 1940-1948. (c) Badet, B.; Vermoote,
P.; Le Goffic, F. Glucosamine Synthase from Escherichia coli:
Kinetic Mechanism and Inhibition by N 3-Fumaroyl-L-2,3-di-
aminopropionic Derivatives. Biochemistry 1988, 27, 2282-2287.
(d) Golinelli-Pimpaneau, B.; Le Goffic, F.; Badet, B. Glu-
cosamine-6-phosphate Synthase from Escherichia coli: Mecha-
nism of the Reaction at the Fructose 6-Phosphate Binding Site.
J . Am. Chem. Soc. 1989, 111, 3029-3034.
(17) Smith, J . L.; Zaluzec, E. J .; Wery, J .-P.; Nu, L.; Switzer, R. L.;
Zalkin, H.; Satow, Y. Structure of the Allosteric Regulatory
Enzyme of Purine Biosynthesis. Science 1994, 264, 1427-1433.
(18) Ornstein, P. L.; Arnold, M. B.; Augenstein, N. K.; Paschal, J .
W. 4-(Tetrazolylalkyl)piperidine-2-carboxylic Acids. Potent and
Selective N-Methyl-D-Aspartic Acid Receptor Antagonists with
a Short Duration of Action. J . Med. Chem. 1991, 34, 90-97.
(19) (a) For a review, see: Monaghan, D. T.; Bridges, R. J .; Cotman,
C. W. The Excitatory Amino Acid Receptors: Their Classes,
Pharmacology, and Distinct Properties in the Function of the
Central Nervous System. Annu. Rev. Pharmacol. Toxicol. 1989,
29, 365-402. (b) Bridges, R. J .; Stanley, M. S.; Anderson, M.
W.; Cotman, C. W.; Chamberlin, A. R. Conformationally Defined
Neurotransmitter Analogues. Selective Inhibition of Glutamate
Uptake by One Pyrrolidine-2,4-dicarboxylate Diasteromer. J .
Med. Chem. 1991, 34, 717-725.
(20) Gmeiner, P.; Feldman, P. L.; Chu-Moyer, M. Y.; Rapoport, H.
An Efficient and Practical Total Synthesis of (+)-Vincamine from
L-Aspartic Acid. J . Org. Chem. 1990, 55, 3068-3074.
(21) Presented in part at the 205th National American Chemical
Society Meeting, Denver, CO, March 28-April 2, 1993, Abstract
MEDI-36.
(41) West, C. T.; Donnelly, S. J .; Kooistra, D. A.; Doyle, M. P. Silane
Reductions in Acidic Media. II. Reduction of Aryl Aldehydes and
Ketones by Trialkylsilanes in Trifluoroacetic Acid. A Selective
Method for Converting the Carbonyl Group to Methylene. J . Org.
Chem. 1973, 38, 2675-2681.

2378 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12
Parr et al.
(42) (a) Humphrey, J . M.; Bridges, J . M.; Hart, J . A.; Chamberlin,
A. R. 2,3 Pyrrolidinedicarboxylates as Neurotransmitter Con-
former Mimics: Enantioselective Synthesis via Chelate-Con-
trolled Enolate Alkylation. J . Org. Chem. 1994, 59, 2467-2472.
We note that this study, describing an elegant and more efficient
preparation of both 16 and 25, appeared just after the completion
of our syntheses of these proline analogs. This alternate route
is conceptually identical to the one described herein, employing
it is likely that the analogs are competing with aspartate for
the E. ATP complex.
(47) Segel, I. H. Enzyme Kinetics; Wiley-Interscience: New York,
1975; pp 100-160.
(48) (a) Kim, H.; Lee, L.; Evans, D. R. Identification of the ATP
Binding Sites of the Carbamyl Phosphate Synthetase Domain
of the Syrian Hamster Multifunctional Protein CAD by Affinity
Labeling with 5′-[p-(Fluorosulfonyl)benzoyl]adenosine. Biochem-
istry 1991, 30, 10322-10329. (b) Mei-Hsu, C.; Kaur, P.; Karkar-
ian, C. E.; Steiner, R. F.; Rosen, B. P. Substrate-Induced
Dimerization of the ArsA Protein, the Catalytic Component of
an Anion-Translocating ATPase. J . Biol. Chem. 1991, 266,
2327-2332.
(49) (a) Danishefsky, A. T.; Onnufer, J . J .; Petsko, G. A.; Ringe, D.
Activity and Structure of the Active-Site Mutants R386Y and
R386F of Escherichia coli Aspartate Aminotransferase. Bio-
chemistry 1991, 30, 1980-1985. (b) Yeh, J . I.; Biemann, H.-P.;
Pandit, J .; Koshland, D. E.; Kim, S.-H. The Three-dimensional
Structue of the Ligand-binding Domain of a Wild-type Bacterial
Chemotaxis Receptor. J . Biol. Chem. 1993, 268, 9787-9792. (c)
Wang, W.; Poland, B. W.; Honzatko, R. B.; Fromm, H. J .
Identification of Arginine Residues in the Putative L-Aspartate
Binding Site of Escherichia coli Adenylosuccinate Synthetase.
J . Biol. Chem. 1995, 270, 13160-13163.
(50) Still, W. C.; Kahn, M.; Mitra, A. Rapid Chromatographic
Technique for Preparative Separations with Moderate Resolu-
tion. J . Org. Chem. 1978, 43, 2923-2925.
(51) Segel, I. H. Enzyme Kinetics; Wiley-Interscience: New York,
1975; pp 125-136.
(52) De Tar, D. F.; Rogers, F. F.; Bach, H. Sequence Peptide Polymers.
III. Poly Asp(OH)-Ser(H)-Gly and Other Serine-Containing
Polymers. J . Am. Chem. Soc. 1967, 89, 3039-3045.
a
stereocontrolled â-alkylation of a protected aspartic acid
derivative. The N-phenylfluorenyl protecting group was em-
ployed in the synthesis. The relative stereochemistry of the
alkylated material was proved by X-ray crystallography. A direct
comparison of our intermediates with those described in this
reference was therefore precluded, although the spectroscopic
and chromatographic properties of 16 and 25 are identical to
those reported. We recommend this prior approach for the large-
scale preparation of 16 and 25. (b) Baldwin, J . E.; Adlington, R.
M.; Gollins, D. W.; Godfrey, C. R. A. The Stereospecific Synthesis
of (2S, 3R) 3-Carboxyproline and (2S, 3R) 3-Aminoproline.
Tetrahedron 1995, 51, 5169-5180.
(43) Haskell, B. E.; Bowlus, S. B. A New Synthesis of L-2-Amino-3-
oxalylaminoproprionic Acid, the Lathyrus sativus Neurotoxin.
J . Org. Chem. 1976, 41, 159-160.
(44) (a) Foote, J .; Lauritzen, A. M.; Lipscomb, W. N. Substrate
Specificity of Aspartate Transcarbamylase. Interaction of the
Enzyme with Analogs of Aspartate and Succinate. J . Biol. Chem.
1985, 260, 9624-9629. (b) Turnbull, J . L.; Waldrop, G. L.;
Schachman, H. K. Ionization of Amino Acid Residues Involved
in the Catalytic Mechanism of Aspartate Transcarbamoylase.
Biochemistry 1992, 31, 6562-6569.
(45) Horowitz, B.; Meister, A. Glutamine-dependent Asparagine
Synthetase from Leukemia Cells. Chloride Dependence, Mech-
anism of Action and Inhibition. J . Biol. Chem. 1972, 247, 6708-
6719.
(46) Recent isotope partitioning experiments (P.-H. Tari, unpublished
results) suggest that ATP binds before aspartic acid. Therefore
J M9601009