Synthesis of (6R)- And (6S)-5,10-dideazatetrahydrofolate oligo-γ-glutamates: Kinetics of multiple glutamate ligations catalyzed by folylpoly-γ-glutamate synthetase

Article abstract of DOI:10.1039/b505907k

Folylpoly-γ-glutamate synthetase (FPGS, EC 6.3.2.17) catalyzes the ATP-dependent ligation of glutamic acid to reduced folates including (6S)-5,6,7,8-tetrahydrofolate (H₄PteGlu), as well as to anticancer drugs such as 5,10-dideaza-5,6,7,8-tetrahydrofolate ((6R)-DDAH ₄PteGlu₁, (6R)-DDATHF, Lometrexol^(tm)). Synthesis of unlabeled mono- and polyglutamates, DDAH₄PteGlu _n (6R, n = 1-6; 6S, n = 1-2), as well as (6R)-DDAH ₄Pte[¹⁴C]Glu₁, was effected from (6R)- or (6S)-5,10-dideazatetrahydropteroyl azide and glutamic acid, H-Glu-γ- Glu_n-y-Glu-OH (n = 0-4), or [¹⁴C]glutamic acid, respectively. These compounds were evaluated as FPGS substrates to determine steady-state kinetic constants. Michaelis-Menten kinetics were observed for (6-R)-DDAH₄PteGlu₁, the isomer corresponding to H ₄PteGlu, whereas marked substrate inhibition was observed for (6S)-DDAH₄PteGlu_n (n = 1-2) and (6R)-DDAH ₄PteGlu_n (n = 2-5), but not (6.R)-DDAH ₄PteGlu₆. Multiple ligation of glutamate renders a quantitative analysis of these data difficult. However, approximate values of K_M = 0.65-1.6 μM and K₁, = 144-417 μM for DDAH ₄PteGl_n were obtained using a simple kinetic model. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b505907k

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A R T I C L E
Synthesis of (6R)- and (6S)-5,10-dideazatetrahydrofolate
oligo-c-glutamates: Kinetics of multiple glutamate ligations
catalyzed by folylpoly-c-glutamate synthetase†
John W. Tomsho,a John J. McGuireb and James K. Coward*a
a Departments of Medicinal Chemistry and Chemistry, University of Michigan, 3813 Chemistry,
930 N. University, Ann Arbor, MI, 48109-1055, USA. E-mail: jkcoward@umich.edu;
Fax: 734-647-4865
b Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, NY, 14263
Received (Pittsburgh, PA, USA) 26th April 2005, Accepted 20th July 2005
First published as an Advance Article on the web 15th August 2005
Folylpoly-c-glutamate synthetase (FPGS, EC 6.3.2.17) catalyzes the ATP-dependent ligation of glutamic acid to
reduced folates including (6S)-5,6,7,8-tetrahydrofolate (H4PteGlu), as well as to anticancer drugs such as
5,10-dideaza-5,6,7,8-tetrahydrofolate ((6R)-DDAH4PteGlu1, (6R)-DDATHF, LometrexolTM). Synthesis of unlabeled
mono- and polyglutamates, DDAH4PteGlun (6R, n = 1–6; 6S, n = 1–2), as well as (6R)-DDAH4Pte[14C]Glu1, was
effected from (6R)- or (6S)-5,10-dideazatetrahydropteroyl azide and glutamic acid, H-Glu-c-Glun-c-Glu-OH (n =
0–4), or [14C]glutamic acid, respectively. These compounds were evaluated as FPGS substrates to determine
steady-state kinetic constants. Michaelis–Menten kinetics were observed for (6R)-DDAH4PteGlu1, the isomer
corresponding to H4PteGlu, whereas marked substrate inhibition was observed for (6S)-DDAH4PteGlun (n = 1–2)
and (6R)-DDAH4PteGlun (n = 2–5), but not (6R)-DDAH4PteGlu6. Multiple ligation of glutamate renders a
quantitative analysis of these data difficult. However, approximate values of KM = 0.65–1.6 lM and KI =
144–417 lM for DDAH4PteGlun were obtained using a simple kinetic model.
to polyglutamate forms has also been observed with purified
Introduction
hFPGS12 and in the mouse.13 It is an excellent substrate for
Folylpoly-c-glutamate synthetase (FPGS, EC 6.3.2.17) is the
enzyme responsible for the intracellular poly-c-glutamylation
of natural folates as well as many antifolates (Scheme 1). This
purified mammalian FPGS with kinetic constants similar to the
best natural substrates, 10-formyltetrahydrofolate (10-formyl-
H4PteGlu) and tetrahydrofolate (H4PteGlu).8,10,14,15 The (6R)-
poly-c-glutamylation leads to increased intracellular retention
and (6S)-diastereomers of DDAH4PteGlu1 have KM values
and (often) affinity for the target enzymes.1–3 The antifolate (6R)-
with human FPGS of 1.7 and 1.0 lM respectively. Substrate
5,10-dideaza-5,6,7,8-tetrahydrofolate ((6R)-DDAH4PteGlu1,
inhibition was observed, but only by the (6S)-diastereomer.10 It
(6R)-DDATHF, LY264618, LometrexolTM) has been evaluated
was also found that the poly-c-glutamate forms are much more
in clinical trials as a treatment for breast and non-small cell
potent inhibitors of GARFT, with KI values for DDAH4PteGlu1
lung cancers as well as melanoma, sarcoma, and others.4–7
and DDAH4PteGlu5 being 39 nM and 0.39 nM, respectively.16
DDAH4PteGlu1 is an inhibitor of de novo purine biosynthesis
that specifically inhibits the reaction catalyzed by glycinamide
This increase in potency for the target enzyme upon polyglu-
tamylation has been observed for many anti-folate compounds.17
ribonucleotide formyltransferase (GARFT, EC 2.1.2.1).8 The
Resistance of cancer cells to DDAH4PteGlu1 has been ob-
(6R)- and (6S)-diastereomers of DDAH4PteGlu1 are equally
served to arise from a decrease in polyglutamylation.11,18,19
effective at inhibiting the growth of cancer cell lines in vitro and
These data strongly suggest that the polyglutamylated form of
tumors in vivo.9,10 The finding that the antiproliferative effect of
DDAH4PteGlu1 is responsible for its cytotoxic activity.
DDAH4PteGlu1 is due to inhibition of GARFT sparked much
DDAH4PteGlu1 was first synthesized as a mixture of di-
interest in this compound as a drug candidate.4,8 In contrast, two
astereomers about the C-6 position as reported by Taylor
well known antifolates, aminopterin and methotrexate, both act
by inhibition of dihydrofolate reductase (DHFR, EC 1.5.1.3).
et al.14 Since that time a number of other racemic syn-
theses have been reported.20,21 Access to the individual di-
Early work with DDAH4PteGlu1 indicated that cells treated
astereomers of DDAH4PteGlu1 was originally obtained by
with (6R)-DDAH4PteGlu1 rapidly convert the drug to its pen-
fractional crystallization or chiral HPLC of DDAH4PteGlu1
taglutamate and hexaglutamate metabolites.11 Rapid conversion
diethyl ester, d-10-camphorsulfonic acid salt, followed by
careful saponification.10,22 An asymmetric synthesis of (6R)-
DDAH4PteGlu1, the diastereomer selected for clinical trials, has
† Electronic supplementary information (ESI) available: Experimental
details. See http://dx.doi.org/10.1039/b505907k
been reported.23
Scheme 1 The reaction of FPGS with (6R)-DDAH4PteGlu1 (1). The structure of (6S)-tetrahydrofolic acid is shown for comparison.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 3 8 8 – 3 3 9 8 T h i s j o u r n a l i s T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
3 3 8 8
©

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In this paper, we report the syntheses of (6R)-DDAH4Pte-
Glu1–6, (6S)-DDAH4PteGlu1–2, and (6R)-DDAH4Pte[14C]Glu1
via a 5,10-dideaza-5,6,7,8-tetrahydropteroyl azide (DDAH4Pte–
N3) intermediate that allows for the incorporation of completely
unprotected L-glutamate, poly-c-glutamate peptides, or L-[U–
14C]glutamate, respectively, as the final step in the synthesis.24,25
The (6R)- and (6S)-isomers of DDAH4Pte–N3 were derived from
the individual enantiomers of methyl 2-pivaloyl-5,10-dideaza-
5,6,7,8-tetrahydropteroate, which, in turn, were purified from
the racemic mixture26 by chiral HPLC. The only previously re-
ported synthesis of radiolabeled (6R)-DDAH4PteGlu1 involved
a multi-step procedure in which the unstable isotope (3H) was
incorporated to form a racemic precursor midway through
the synthesis.27 Resolution of racemic [3H]DDAH4PteGlu1 was
required to obtain the desired single diastereomer for use in
metabolic studies.11 In contrast, in the synthesis described herein,
the 14C label is introduced into a single isomer, via (6R)-
DDAH4Pte–N3, in the final step.
The full range of poly-c-glutamate forms of individual
folates and antifolates have been synthesized previously by
both solid-phase28–30 and solution-phase29,31–35 chemistries. In
this research, the poly-c-glutamate peptides were synthesized
by solid-phase peptide synthesis utilizing L-glutamate protected
as the N-Fmoc and a- or c-tert-butyl ester. The procedure
included a capping step that prevented further elongation of
any unreacted N-terminal amines. The biochemical evaluation
of these compounds as substrates for hFPGS was then pursued.
We report steady-state kinetics data (KM, Vmax, V/K) for all
newly synthesized polyglutamates, and also document marked
substrate inhibition by all compounds with the exception of
(6R)-DDAH4PteGlu1 and (6R)-DDAH4PteGlu6.
by centrifugation. 5,10-Dideaza-5,6,7,8-tetrahydropteroyl azide
(9) was formed by the treatment of 8 with ethyl chloroformate
in the presence of triethylamine in DMSO to produce the mixed
anhydride, followed by displacement with azide anion. The
product was then recovered by precipitation with aqueous HCl
followed by centrifugation. HPLC analysis of the reaction mix-
ture prior to work-up consistently indicated 95–100% conversion
to product. However, during the acidic aqueous workup, a
significant portion of the product (5–10%) was hydrolyzed back
to 8. Since other work-up options provided no improvement in
product purity and 8 would not interfere in the following steps,
9 contaminated with a small amount of 8 was carried through
the subsequent transformations.
Initially, the synthesis of 9 was attempted by treating 8
with diphenylphosphoryl azide, as has been previously reported
for similar antifolates.25 Unfortunately, this reaction proceeded
very slowly, with less than 50% conversion after prolonged
reaction (9 days) based on HPLC analysis, in contrast to 85%
conversion of 4-deoxy-4-amino-10-methylpteroic acid to the
corresponding azide over a much shorter reaction time (2 days).
This marked difference in reaction rate is presumably due to
decreased nucleophilicity of the conjugate base of the carboxylic
acid 8 (pKa = ca. 4.3) vs. that of the corresponding pteroic
acid analogue (pKa = ca. 5.0).36 Therefore, formation of a
mixed anhydride followed by reaction with azide ion37 was
investigated and proved to be effective in converting 8 to 9
in nearly quantitative yield. The coupling reactions between 9
and L-glutamate or poly-c-glutamates to produce 5,10-dideaza-
5,6,7,8-tetrahydrofolic acid (1) or its polyglutamates (2 through
6) were performed in DMSO in the presence of Et3N (vide infra).
The optimal reaction conditions consisted of two equivalents of
9 per equivalent of amino acid or peptide with a reaction time of
two days at room temperature. Any of the parent acid, 8, formed
from excess 9 due to the presence of adventitious water in the
reaction mixture, or during workup, could be recovered.
Results and discussion
Chemistry
The poly-c-glutamates required for the preparation of
DDAH4PteGlun were synthesized by standard solid-phase
Fmoc-peptide synthesis procedures.38 Two differently pro-
tected Fmoc-L-glutamic acid building blocks were utilized in
the solid-phase synthesis. N-a-Fmoc-L-glutamic acid a-tert-
butyl ester (Fmoc-Glu-(OH)Ot-Bu) was required to obtain
the desired c-linkages, while N-a-Fmoc-L-glutamic acid c-tert-
butyl ester (Fmoc-Glu(Ot-Bu)–OH) was used to load the
p-benzyloxybenzyl alcohol (Wang) resin (Scheme 3). Acetic
anhydride and dimethylaminopyridine (DMAP) in DMF were
used in a capping step to acylate any free N-termini remaining
after each peptide coupling step, thereby preventing further
elongation of any unreacted peptide. Fmoc-Glu(Ot-Bu)–OH
was chosen for resin loading because it has been reported that
the c-linkage to the resin is more labile than the standard a-
linkage.28 Also, as this is the least efficient step (40–50% yield),
Synthesis of (6RS)-5,10-dideaza-5,6,7,8-tetrahydrofolic acid
(6RS-1) was achieved via the route illustrated in Scheme 2. This
route is based on the work of Taylor et al.,26 but is modified to
allow the incorporation of unprotected glutamate, radiolabeled
glutamate, or poly-c-glutamate peptides as the final synthetic
step in the synthesis. This was achieved through the use of
an acyl azide, 9, derived from (6RS)-methyl 2-pivaloyl-5,10-
dideaza-5,6,7,8-tetrahydropteroate (6RS-7). The racemic ester,
6RS-7, was separated into its individual enantiomers by chiral
HPLC, enabling ready access to either the (6R)- or (6S)-isomer
of DDAH4PteGlu1 and DDAH4Pte polyglutamates.
Deprotection of 7 was achieved by basic hydrolysis in 1 M
NaOHaq. Isolation of 5,10-dideaza-5,6,7,8-tetrahydropteroic
acid (8) involved acidification of the basic product solution with
HCl to precipitate the insoluble salt, which was then collected
Scheme 2 Synthesis of DDAH4PteGlu1 (1) from protected DDAH4Pte–OH (7).
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 3 8 8 – 3 3 9 8
3 3 8 9

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Table 1 Kinetic constants of the C-6 stereoisomers of DDAH4PteGlu1
Michaelis–Menten fita
Substrate inhibition fitb
max/lM h−1
M/lM
V
max/lM h−1
K
M/lM
V
K
KI/lM
6R
6S
3.15 0.11
3.31 0.15
1.93 0.23
1.06 0.20
NAc
4.01 0.20
NAc
1.56 0.23
NA
76 10
a Values obtained using the Michaelis–Menten equation to fit the data in Fig. 1A. b Values obtained using eqn (2) to fit the (6S)-isomer data in
Fig. 1B. c KM = 3.67 0.55 lM and Vmax = 3.94 0.10 lM h−1 were obtained when the Michaelis–Menten equation was used to fit the data for the
(6R)-isomers in Fig. 1B.
folate concentrations up to at least ∼15 × KM (Fig. 1A). The
kinetic constants that were obtained (Table 1) agree with the
reported values of 1.7 and 1.0 lM for the (6R)- and (6S)-
diastereomers, respectively.10
Scheme 3 Solid-phase synthesis of poly-c-glutamate peptides.
the fact that the Fmoc-Glu(Ot-Bu)–OH is five times less costly
than Fmoc-Glu-Ot-Bu makes its use in this reaction more
attractive. This method allows for the facile synthesis of any
c-glutamyl peptide on a 100 mg scale in a few days using
manual methods. Purification is achieved by reversed-phase,
semi-preparative HPLC followed by lyophilization to yield the
trifluoroacetate salt of the peptide. Purified c-glutamyl peptides
were coupled to 5,10-dideaza-5,6,7,8-tetrahydropteroyl azide (9)
under the optimized conditions described above (Scheme 4).
Scheme
4
Synthesis of DDAH4Pte polyglutamates (2–6) from
DDAH4Pte–N3 (9). Synthesis of 6R-2 and 6S-2 utilized crude
H-Glu-c-Glu-OH peptide in the coupling reaction. All product yields
are determined by UV/vis spectroscopy.
Biochemical studies
The biochemical studies utilized an end-point assay that quan-
titated the total amount of L-[3H]glutamic acid ligated to the
folate substrate by FPGS. Assays were performed at fixed,
saturating concentrations of ATP (5 mM, ca. 100 × KM) and L-
glutamic acid (2 mM, ca. 10 × KM) with varying concentrations
of the DDAH4PteGlun being characterized. Both diastereomers
of DDAH4PteGlu1 exhibited Michaelis–Menten kinetics at
Fig. 1 Steady-state kinetics: hFPGS-catalyzed ligation of L-glutamate
to each of the C-6 stereoisomers of DDAH4PteGlu1, (6R)- (᭹), and
(6S)- (᭺). hFPGS was expressed in E. coli and partially purified. Each
assay was done in duplicate and the data points shown are the average
values range. A. Curves are the best fit of the data in the non-inhibitory
concentration range to the Michaelis–Menten equation. B. Curves are
the best fit of all data to the Michaelis–Menten equation (6R), or eqn
(2) (6S).
3 3 9 0
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 3 8 8 – 3 3 9 8

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However, as is apparent from Fig. 1B, the data for the (6R)-
diastereomer at concentrations >30 lM deviate significantly
from the best fit to the Michaelis–Menten equation for a single
E·S complex. A model which invokes the formation of two
different E·S complexes (eqn (1)) yielded a better fit to the entire
range of data for the (6R)-diastereomer (Fig. 1B) and resulted in
values of KM1 and KM2 that differed by two orders of magnitude
(KM1 ꢀ KM2, ca. 1 and 100 lM respectively). However, the value
V
max [S]
V
max [S]
v =
+
(1)
KM1+ [S] KM2 + [S]
of KM2 differs markedly from the steady state KM value
determined for the (6R)-DDAH4Pte–Glu2 species (vide infra),
the presumed substrate involved in the second E·S complex. This
model does not appear to be adequate to explain the deviation
from Michaelis–Menten kinetics and was not pursued further.
At higher concentrations of (6S)-DDAH4PteGlu1 (30–
300 lM), substrate inhibition was noted (Fig. 1B). This is
consistent with what is known in the literature.8,10 In order to
obtain KM, KI, and Vmax values from these data, a substrate
inhibition equation for a simple system, eqn (2), was used.39
Fig. 2 Steady-state kinetics: hFPGS-catalyzed ligation of L-glutamate
Vmax
to each of the C-6 stereoisomers of DDAH4PteGlu2, (6R)- (᭹), and
(6S)- (᭺). hFPGS was expressed in E. coli and partially purified. Each
assay was done in duplicate and the data points shown are the average
values range. Curves shown are the best fit of all data to eqn (2). Data
in the non-inhibitory range were fit to the Michaelis–Menten equation.
v =
(2)
K
[S]
M
1 +
+
[S]
K
1
It was expected that the (6R)- and (6S)-diastereomers of the
poly-c-glutamate conjugates would exhibit similar properties as
was observed for the respective monoglutamate diastereomers
in terms of substrate inhibition. However, as can be seen in
Fig. 2, both the (6R)- and (6S)-isomers of the diglutamate,
DDAH4PteGlu2, exhibit striking substrate inhibition that oc-
curred at much lower relative concentrations (∼5 × KM) than
that observed for (6S)-DDAH4PteGlu1. Due to the low concen-
tration at which the substrate inhibition became significant, the
Michaelis–Menten equation was used for fitting only the data
in the non-inhibitory region (≤5 lM). The kinetic constants for
the diastereomers of DDAH4PteGlu2 are summarized in Table 2.
The observed substrate inhibition could be associated with either
non-sequential kinetics and/or partial sites reactivity in an
oligomeric protein. However, there is no supporting evidence
for either of these scenarios for FPGS.
This substrate inhibition may be explained by two molecules
of substrate binding to a single hFPGS simultaneously. The first
molecule of substrate would bind in the active site correctly
oriented for turnover, while the second molecule would bind to
another site that inhibits catalysis. This second site could either
be an allosteric binding site involved in feedback inhibition or
an incorrect binding of the second folate substrate molecule
in the active site. Based on the crystal structure of a binary
complex (L. casei FPGS·ATP), the C-terminal domain of FPGS
has a very similar secondary structure to that of DHFR from
several species,41 indicating a possible role in folate binding.
Preliminary docking studies performed based on the crystal
structure of a ternary complex (L. casei FPGS·AMPPCPP·5,10-
CH2-H4PteGlu1)42 indicates the possible presence of a second
folate substrate binding site on the N-terminal domain of
the protein (Dr Heather Carlson, University of Michigan,
personal communication). Recent work by Mathieu et al.
supports this finding. A second folate substrate binding site
with dihydropteroate bound was observed when a structure of
the bifunctional E. coli FolC enzyme, harboring dihydrofolate
synthetase and folylpolyglutamate synthetase activities, was
determined.43
The trend of substrate inhibition carried through the entire
series of (6R)-DDAH4PteGlun (n = 3–5, Fig. 3). Of the
polyglutamate compounds, only the hexaglutamate exhibited
Michaelis–Menten behaviour. Table 3 lists the kinetic constants
obtained from the (6R)-DDAH4PteGlun compounds.44 As glu-
tamate chain-length increased (n = 1–5), the V/K values first
increased approximately 2.5-fold when going from the mono- to
diglutamate species. This difference arises from a much lower
KM value for the latter. The tri-, tetra-, and pentaglutamates
exhibited much lower, but similar, V/K values than those
observed for the shorter chain length substrates due to much
reduced Vmax values. These data follow the same general pattern
reported by Chen et al. for pteroyl and (6S)-tetrahydropteroyl
poly-c-glutamaten (n = 1–5).45 A similar trend has been reported
for FPGS from L. casei, an enzyme for which a crystal structure
is available.42 Analysis of this structure indicates that the c-
carboxyl group of the monoglutamate substrate does not reach
the ATP and is therefore incapable of forming the c-glutamyl
phosphate46 without significant, and energetically costly, con-
formational changes. However, the diglutamate is perfectly
accommodated in the active site.47 There is no indication of how
longer chain length products could be accommodated within
the active site. In the current work, a very low value of V/K
Table 2 Kinetic constants of the C-6 stereoisomers of DDAH4PteGlu2
Michaelis–Menten fita
Substrate inhibition fitb
V
max/lM h−1
K
M/lM
V
max/lM h−1
K
M/lM
KI/lM
6R
6S
1.84 0.24
2.83 0.29
0.87 0.33
1.16 0.35
1.64 0.11
2.65 0.15
0.65 0.17
0.95 0.20
157 25
92 20
a Values obtained using the Michaelis–Menten equation to analyze the data in the non-inhibitory region (≤5 lM). Similar values were obtained when
the data were analyzed utilizing the Hanes linear transform.40 b Values obtained using eqn (2).
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 3 8 8 – 3 3 9 8
3 3 9 1

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a
Table 3 Kinetic constants of (6R)-DDAH4PteGlun
n
V
max/lM h−1
K
M/lM
KI/lM
(V/K)/h−1
1
2
3
4
5
6
3.94 0.10
1.64 0.08
0.30 0.03
0.71 0.07
0.22 0.01
0.59 0.02
3.67 0.55
0.65 0.12
0.96 0.43
1.60 0.55
1.05 0.24
14.5 1.9
NA
1.07 0.16
2.52 0.48
0.31 0.14
0.44 0.16
0.21 0.05
0.04 0.01
157 25
179 91
144 56
417 152
NA
a Values obtained using the Michaelis–Menten equation (n = 1 and 6)
or eqn (2) (n = 2–5).
Fig. 3 Steady-state kinetics: hFPGS-catalyzed ligation of L-glutamate
to (6R)-DDAH4PteGlun (n = 3–6). hFPGS was expressed in bac-
culovirus-infected SF9 insect cells and partially purified. Each assay was
done in duplicate and the data points shown are the average values
range. n = 3 (ꢀ), 4 (ꢁ), 5 (ꢂ), and 6 (ꢁ). Curves are the best fit of all
data to the Michaelis–Menten equation (n = 6) or eqn (2) (n = 3–5).
for (6R)-DDAH4PteGlu6 was determined, due primarily to a
significantly increased value of KM.
One caveat in these analyses is that multiple glutamate
ligations are occurring during the reactions, i.e., the di-, tri-,
tetra-, etc. glutamate products are formed since each product
is also a substrate for further ligation. This complexity makes
the following assumptions in the standard Michaelis–Menten
analysis invalid: (1) only a single substrate and single enzyme–
substrate complex are involved, (2) the enzyme–substrate com-
plex breaks down directly to form free enzyme and product,
and (3) enzyme, substrate, and enzyme–substrate complex are
at equilibrium, i.e., the dissociation of ES to E + S is rapid in
comparison to the formation of E + P.48
To establish that multiple glutamate ligations were occurring
to a single folate substrate, product distribution analysis was
done at varied (6R)-DDAH4Pte[14C]Glu1 concentrations. When
these reactions were analyzed, it was determined that multiple
products were produced and that the product distribution varied
with changing concentrations of (6R)-DDAH4PteGlu1 (Fig. 4).
The observed trend is that progressively shorter chain length
products are produced with increasing concentrations of (6R)-
DDAH4PteGlu1. The results of this experiment are very similar
to those observed for rat liver FPGS with (6RS)-H4PteGlu as
the substrate.49 In that case, multiple glutamate ligations to
(6RS)-H4PteGlu at 5 lM (∼KM) yielded H4PteGlu4–5 as the
predominant products, following initial formation of H4PteGlu2
and H4PteGlu3 that built up to a low level and subsequently
Fig. 4 Product distribution of reactions of (6R)-DDAH4Pte[14C]Glu1
with purified hFPGS expressed in bacculovirus-infected SF9 insect cells.
The concentrations of the glutamate and ATP substrates were 5 mM and
10 mM respectively. Reactions contained the following concentrations
of (6R)-DDAH4Pte[14C]Glu1: A. 2.5 lM. B. 25 lM. C. 250 lM.
decreased. In contrast, (6RS)-H4PteGlu at 35 lM (∼10 × KM)
yielded predominately H4PteGlu2 and H4PteGlu3; H4PteGlu4–5
products were only observed at longer reaction times.
The product distribution results at a low concentration,
2.5 lM, agree well with what has been observed in whole
CCRF-CEM cells11 and in the livers of mice13 that were
treated with (6R)-DDAH4PteGlu1. In cells treated with 10 lM
(6R)-DDAH4PteGlu1, polyglutamate products (n = 2–6) were
observed even at the earliest time point, 4 h, with the pen-
taglutamate product predominating at longer incubation times.
3 3 9 2
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 3 8 8 – 3 3 9 8

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Analysis of the livers of mice dosed with 0.72 mg kg−1 of
(6R)-DDAH4PteGlu1 indicated that the pentaglutamate product
was formed relatively quickly with no detectable diglutamate
product and smaller amounts of tri-, tetra- and hexaglutamate
products.
Experimental
Materials
Common solvents and reagents were obtained from commercial
sources and were of the highest purity available. L-[2,3-3H]-
Glutamic acid (NET-395) and L-[U-14C]glutamic acid (NEC-
290E) were obtained from Perkin–Elmer Life Sciences. p-
Benzyloxybenzyl alcohol (Wang) resin, N-a-Fmoc-L-glutamic
acid a-tert-butyl ester (Fmoc-Glu(OH)–Ot-Bu), N-a-Fmoc-
L-glutamic acid c-tert-butyl ester (Fmoc-Glu(Ot-Bu)–OH), N-
hydroxybenzotriazole–H2O (HOBt), benzotriazole-1-yloxytris-
(pyrrolidino)phosphonium hexafluorophosphate (PyBOP),
and 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU) utilized for solid phase peptide
synthesis were obtained from NovaBiochem. CDCl3, D2O,
d-TFA, and NaOD were from Isotec and d6-DMSO was from
Cambridge. (6R)-5,10-Dideaza-5,6,7,8-tetrahydrofolic acid
sodium ((6R)-DDAH4PteGlu1–Na, LometrexolTM sodium,
Lot 235MH8) and (6RS)-5,10-dideaza-5,6,7,8-tetrahydro-
pteroyl polyc-glutamates (DDAH4PteGlu2, Lot 266251 and
DDAH4PteGlu5, Lot 266578) were kind gifts of Dr Chuan Shih,
Eli Lilly & Co, Indianapolis, IN. These compounds were used
only in initial studies on assay development and for comparison
of physical properties with newly synthesized compounds.
(6RS)-Methyl 2-pivaloyl-5,10-dideazatetrahydropteroate (6RS-
7) was synthesized as described by Taylor26 with significant
procedural modifications. Details may be found in the
Supplementary Information. Spin-X HPLC 0.2 lm nylon
microcentrifuge filters (spin filters) were obtained from Costar.
Human folylpolyglutamate synthetase was expressed in E.
coli or bacculovirus-infected SF9 insect cells12,45 and partially
purified as described previously.50 Homogenous hFPGS,
expressed in bacculovirus-infected SF9 insect cells and purified
as described previously,12 was a generous gift of Dr Richard
Moran. hFPGS concentration was determined by the method
of Bradford51 corrected for protein purity as determined by
quantitative PAGE.
In an attempt to explain these product distributions, a kinetic
modeling study was undertaken. This study utilized the KM and
V
max values listed in Table 3 and a minimum mechanism based on
sequential elongation of the substrate. Product release between
each catalytic step was assumed, i.e., a distributive mechanism,
so that the Michaelis–Menten kinetic equation could be utilized
to analyze these data. Substrate inhibition by the polyglutamates
was ignored because of the low concentrations of products
formed in the reactions. Modeling of this distributive mecha-
nism, while able to produce different product distributions with
differing initial monoglutamate substrate concentrations, was
unable to mimic the product distributions actually observed.
A model in which product release was not mandated between
each catalytic step, i.e. a processive mechanism, could not
be readily developed. However, a processive mechanism was
assessed directly by subsequent substrate-trapping and pulse-
chase experiments, the results of which will be published
separately.
Conclusions
In this paper, we report the synthesis of the antifo-
late drug LometrexolTM ((6R)-DDAH4PteGlu1), its poly-c-
glutamates ((6R)-DDAH4PteGlu2–6), and a radiolabeled form
((6R)-DDAH4Pte[14C]Glu1). In addition, the (6S)-isomers, (6S)-
DDAH4PteGlu1 and (6S)-DDAH4PteGlu2, were synthesized.
To obtain the single isomer materials, a chiral HPLC separa-
tion of (6RS)-methyl 2-pivaloyl-5,10-dideazatetrahydropteroate
(6RS-7) was developed. This separation was achieved on a
preparative scale (∼5 g) and resulted in material with an
isomeric purity of >98%. The isomerically pure materials were
subsequently used in the preparation of all DDAH4PteGlun
compounds as well as (6R)-DDAH4Pte[14C]Glu1. These com-
pounds were then evaluated as substrates for recombinant
human cytosolic FPGS, and the kinetic data augmented by
reaction product analysis.
The observed biochemical properties of the (6R)- and (6S)-
diastereomers of DDAH4PteGlu1, including the segregation of
substrate inhibition to the (6S)-diastereomer, are comparable
to that previously reported in the literature. The properties of
the single diastereomers of DDAH4PteGlu2 are reported for
the first time and show no such separation of the substrate
inhibition. Indeed, all of the (6R)-DDAH4PteGlu2–5 compounds
show substrate inhibition. (6R)-DDAH4PteGlu6 is the only
polyglutamate substrate not to exhibit substrate inhibition.
When the kinetic constants for the (6R)-DDAH4PteGlun com-
pounds are compared (Table 3), it is seen that the diglutamate has
the highest V/K value. The value for the diglutamate is over two-
fold greater than that obtained for monoglutamate indicating
that the diglutamate is the preferred substrate for hFPGS. This
is primarily due to the much lower KM for the diglutamate vs. the
monoglutamate which overcomes the lower Vmax of the former.
The rest of the poly-c-glutamates, tri- through hexaglutamate,
exhibit V/K values of less than half that of the monoglutamate,
indicating that they are much poorer substrates than the mono-
and diglutamate compounds.
General procedures
All synthetic reactions were carried out in oven-dried glassware.
Moisture-sensitive reactions were carried out under a dry
atmosphere of nitrogen or argon. Solvents and liquid reagents
were measured and dispensed in oven-dried glass syringes
or dry nitrogen-flushed disposable syringes with oven-dried
needles. Et3N, pyridine, MeOH, and CH3CN were distilled
from CaH2. THF was distilled from benzophenone and sodium.
1H and 13C NMR spectra were recorded on a Bruker 300
or 500 MHz instrument using either TMS or solvent peaks
as an internal reference. When an internal reference was not
available (e.g. a 13C NMR spectrum in D2O) the frequency
1
of the solvent deuterium was utilized. H NMR assignments
of diastereotopic protons are noted as, e.g. “1.86 (1 H, m,
C(8)H2), 2.07 (1 H, m, C(8)H2)” and signify that each of the two
methylene protons attached to carbon 8 have unique chemical
shifts of 1.86 and 2.07. All chemical shifts (d) are reported in
ppm. Assignments for nuclei in the c-glutamyl peptide portion
use the prime notation (e.g., C(9ꢂ)) to distinguish them from
nuclei in the didezatetrahydropteroyl portion (Supplementary
Information, Schemes S2 and S3). Assignment of peaks as
indicated was aided and confirmed by the acquisition of various
2D spectra (COSY, HETCOR, COLOC). Nominal and high
resolution mass spectra utilizing electrospray ionization with
positive detection utilized Na+ and/or H+ matrices. Nominal
mass spectral data are reported only for those fragments with a
relative intensity of ≥20% of the base peak. UV/visible spectra
were recorded on a Beckman Model 640B spectrophotometer.
Melting points were determined with a MEL-TEMP apparatus
and are reported uncorrected. Scintillation counting was carried
out with a Packard 1600 scintillation counter using Bio-Safe II
It is also apparent that the product distributions obtained
utilizing (6R)-DDAH4Pte[14C]Glu1 exhibit a significant depen-
dence on substrate concentration (Fig. 4). The experimentally
observed product distributions are similar to those observed in
cells and whole animals. This similarity of product distributions
(in vitro vs. in vivo) provides strong support for the validity of
applying conclusions obtained from this work to what may be
occurring in vivo.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 3 8 8 – 3 3 9 8
3 3 9 3

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scintillation cocktail from Research Products Inc. and 20 mL
glass scintillation vials with foil-lined lids or 5.5 mL polypropy-
lene multi vials from Life Science Products. The analytical HPLC
system used was manufactured by Varian and consisted of a
Model 410 auto-sampler, two Model 210 Prostar pumps, a
Model 330 photo diode array detector, and a Model 701 fraction
collector. The semi-preparative HPLC system was manufactured
by Rainin and consisted of two Rabbit Model HPX pumps fitted
with 50 mL min−1 pump heads, a dynamic mixer, and a single
wavelength UV detector. Varian Star v6.3 software controlled
both systems. All HPLC was run at ambient temperature.
Optical rotation was determined with a Perkin–Elmer Model
241 polarimeter using the sodium D line source (589 nm) with
an integration time of 5 s, at ambient temperature, a cell length
of 10.002 cm, and with a solution concentration of 10 mg
mL−1 unless otherwise noted. [a]D values are given in 10−1
deg cm2 g−1 and are uncorrected. All kinetics data were fitted
using KaleidaGraph v3.5, Synergy Software (Reading, PA).
B; 20 min, 60% B; 25 min, 100% B; 40 min, 100% B. Monitored
at 280 nm.
Ion-pair HPLC. Eluant A: 100% ddH2O. Eluant B: 65%
CH3CN, 35% ddH2O. Both eluants A and B contain 1 mM
KH2PO4, 5 mM tetrabutylammonium phosphate (TBAP),
7 mM NaCl, and 3 mM NaN3. Column: Vydac 218TP54,
5 lm, C18 protein and peptide, 4.6 × 250 mm. Flow rate:
1.0 mL min−1. Gradient: 0 min, 10% B; 5 min, 10% B; 10 min,
36% B; 20 min, 40% B; 50 min, 55% B; 53 min, 100% B, 58 min,
100% B. Monitored at 280 nm or by the scintillation counting
of fractions. This method was developed from a previously
published procedure.52
Ion-pairing buffers for HPLC were prepared as follows. 1 L
of a 10 × stock solution was prepared and consisted of 10 mM
KH2PO4, 50 mM TBAP, 70 mM NaCl, and 30 mM NaN3 in
ddH2O. For both eluants A and B, 100 mL of the 10 × stock
solution was measured using a graduated cylinder and added
to a 1 L volumetric flask. The graduated cylinder was rinsed
two times with 100 mL ddH2O and once with 50 mL ddH2O. The
eluants A and B were brought up to 1 L with ddH2O and CH3CN
respectively and filtered through a 0.2 lm nylon filter.
Equilibration of the HPLC column was achieved in two steps.
The first step was preparation of the column for the ion-pairing
buffers and consists of washing the column at 1 mL min−1 with
ddH2O (A) and CH3CN (B). Gradient: 0 min, 100% B; 5 min,
100% B; 8 min, 50% B; 10 min, 50% B. The flow was stopped
and the pumps were switched to ion-pair containing eluants A
and B. The flow was resumed at 1 mL min−1 and 100% B until
the operating pressure had been reached. Gradient: 0 min, 100%
B; 10 min, 100% B; 15 min, 10% B; 30 min, 10% B.
Chromatography methods
Size-exclusion chromatography. A 1.6 × 80 cm column was
packed with Bio-Gel P-2 (Bio-Rad, fine mesh) that had been
previously swelled and equilibrated with aqueous 50 mM ammo-
nium bicarbonate, pH 8.1. Flow through the column was gravity
controlled with a 10 cm column head whose level was main-
tained with a siphoning system. Fractions (150-drop, ∼3.4 mL)
of column eluant were collected beginning with column loading.
Elution of products was monitored by UV/visible absorbance,
scintillation counting, and/or ninhydrin staining.
Semi-preparative reversed-phase (RP)–HPLC. All separa-
tions utilized a Varian Dynamax 21.4 × 250 mm, Microsorb
60–8, C18 column with a flow rate of 21 mL min−1. Methods
1–4 utilized Eluant A consisting of 0.1% w/v TFA in ddH2O,
pH 1.8. Eluant B consisting of 0.1% w/v TFA in CH3CN, and
detection at 214 nm with gradient profiles as follows:
Method 1: 0 min, 1% B; 2 min, 1% B; 15 min, 10% B.
Method 2: 0 min, 1% B; 5 min, 1% B; 15 min, 10% B; 20 min,
100% B.
Method 3: 0 min, 5% B; 5 min, 5% B; 20 min, 15% B; 25 min,
50% B.
Method 4: 0 min, 2% B; 2 min, 2% B; 20 min, 15% B; 25 min,
50% B.
Methods 5–8 utilized an Eluant A consisting of 20 mM
ammonium acetate, pH 6.5, in ddH2O, an Eluant B consisting
of MeOH, and detection at 280 nm with gradient profiles as
follows:
Method 5: 0 min, 5% B; 25 min, 20% B; 30 min, 50% B; 35 min,
50% B.
Method 6: 0 min, 2% B; 25 min, 20% B; 30 min, 50% B; 35 min,
50% B.
Method 7: 0 min, 2% B; 2 min, 2% B; 25 min, 15% B; 30 min,
50% B; 35 min, 50% B.
Chiral-HPLC (analytical). Eluant A: ddH2O. Eluant B:
CH3CN. Column: Chiral Technologies (DAICEL Chemical
Industries, LTD) Chiralcel OJ–R, 4.6 × 150 mm. Isocratic at
40% B. Flow rate: 0.5 mL min−1. Monitored at 280 nm.
Chiral-HPLC (preparative). Eluant A: hexane. Eluant B:
EtOH. Column: Chiralcel OJ, 2 cm × 25 cm. Isocratic at
70% B. Flow rate: 30 mL min−1. Run time: 22 min. Sample
concentration: 1.25 mg mL−1 in 25% A: 75% B. Monitored at
240 nm.
(6R)- and (6S)-Methyl 2-pivaloyl-5,10-dideaza-5,6,7,8-tetra-
hydropteroate (6R-7 and 6S-7). (6RS)-Methyl 2-pivaloyl-5,10-
dideaza-5,6,7,8-tetrahydropteroate (6RS-7, 4.75 g) was resolved
into its individual isomers by preparative chiral-HPLC as
described above (see Supplementary Information, Figure S1).
This separation was done at the Analytical Development
Department, Pfizer Global Research and Development, Ann
Arbor, MI. The (6R)-isomer (6R-7) (1.6 g) was obtained in 98.2%
chiral purity (tr = 15.2 min) as determined by analytical chiral
HPLC (see Supplementary Information, Figure S2). [a]D
=
−44.5 (MeOH–CHCl3 1.5 : 98.5); m/z (ESI+) 435.2020 (M +
Na+. C22H28N4NaO4 requires 435.2008) 436 (25%), and 435
(100). The (6S)-isomer (6S-7) (1.8 g) was obtained in 98.2%
chiral purity (tr = 10.6 min) as determined by analytical chiral
Method 8: 0 min, 2% B; 2 min, 2% B; 25 min, 10% B; 30 min,
50% B; 35 min, 50% B.
HPLC (see Supplementary Information, Figure S2). [a]D
=
+39.7 (MeOH–CHCl3 1.5 : 98.5); m/z (ESI+) 435.2015 (M +
Na+. C22H28N4NaO4 requires 435.2008) 436(25%), and 435(100).
It is noted that the optical rotations of the isomers are not equal
and opposite but chiral HPLC analysis provides strong evidence
of extremely high stereochemical purity of both diastereomers.
Analytical RP-HPLC. Method 1:24 Eluant A: 50 mM
sodium phosphate buffer, pH 7.0. Eluant B: CH3CN. Column:
˚
Chrompack Kromasil 100 A, C18, 4.6 × 250 mm. Flow rate:
0.7 mL min−1. Gradient: 0 min, 2% B; 25 min, 50% B; 35 min,
50%B. Monitored at 280 nm or scintillation counting of 30 s
fractions.
(6R)-5,10-Dideaza-5,6,7,8-tetrahydropteroic acid (6R-8).
Method 2: Eluant A: 0.1% w/v TFA in ddH2O, pH 1.8. Eluant
B: 0.1% w/v TFA in CH3CN. Column: Vydac 218TP54, 5 lm,
C18 protein and peptide, 4.6 × 250 mm. Flow rate: 1.0 mL min−1.
Isocratic at 13% B. Monitored at 280 nm.
Method 3: Eluant A: ddH2O. Eluant B: CH3CN. Column:
Vydac 218TP54, 5 lm, C18 protein and peptide, 4.6 × 250 mm.
Flow rate: 1.0 mL min−1. Gradient: 0 min, 30% B; 1 min, 30%
(6R)-2-Pivaloyl-5,10-dideaza-5,6,7,8-tetrahydropteroic
acid
methyl ester (6R-7) (250 mg, 0.242 mmol) was suspended
in 6.1 mL 1 M NaOH. A reflux condenser was fitted to the
flask and the reaction was heated at reflux temperature for
30 min after the starting material had completely dissolved.
The flask was then cooled to ambient temperature, and the
solution was filtered through a paper filter, which was then
3 3 9 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 3 8 8 – 3 3 9 8

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about stability, 1H NMR spectra were not obtained. These data
washed with water. The pH of the filtrate was adjusted to
∼1 with 6.7 mL of 1 M HCl. The resulting suspension was
lyophilized to a small volume and resuspended in water for
centrifugation. The precipitate was collected by centrifugation
(15 min, 4 ◦C, 12 000g) to obtain the desired product as a
pellet. The pellet was then washed with ddH2O (2 × 10 mL),
CH3CN (2 × 10 mL), and Et2O (1 × 10 mL). The product
was dried under high vacuum in the presence of P2O5. Ion-pair
for racemic 9 are reported in the Supplementary Information.
Solid phase synthesis of glutamyl-c-glutamate peptides (H-Glu-
c-Glun-c-Glu-OH, n = 0–4). Typical procedure.38 L-Glutamyl-c-
L-glutamate (c-Glu2).
Resin loading. .714 g (4.03 mmol) Fmoc-Glu(Ot-Bu)–OH,
0.545 g (4.03 mmol) HOBt, and 2.06 g (3.95 mmol) PyBOP were
dissolved in 10 mL of anhydrous DMF in a dry test tube with
gentle mixing. After 15 min, 1.5 mL (8.64 mmol) of DIPEA
was added to the test tube with gentle mixing. After 5 min,
this solution was added to a reaction tube containing 0.905 g
(1.08 mmol) of swelled Wang resin. After 90 min of agitation by
inversion at ambient temperature, the reaction was stopped by
filtering the resin and washing with 3 × 10 mL DMF and 2 ×
10 mL CH2Cl2. Loading yield was determined by Fmoc quan-
tification as follows. Two samples of loaded resin (approximately
1 mg each) were accurately weighed out and placed in two test
tubes. A third tube lacking resin was used as the blank. 3 mL of
20% piperidine in DMF was added to each tube with agitation
for 3 min. The solutions were then placed in quartz cuvettes
and their absorbance at 290 nm measured. After subtracting
the blank absorbance, eqn (3)38 was used to determine that a
loading yield of 0.53 mmol g−1 (44%) was achieved.
HPLC indicated >99% product purity. (196 mg, 92%); [a]D
=
−49.5 (0.1 M NaOH) (lit.,23 [a]D = −48.3 (1 N NaOH, from
the acid hydrolysis of (6R)-DDAH4PteGlu1) and [a]D = −52.6
(1 N NaOH)); dH (300 MHz; 0.1 M NaOD in D2O) 1.60 (2 H,
m, C(10)H2), 1.73 (1 H, m, C(6)H), 1.97 (1 H, dd, J = 8.8,
15.5 Hz, C(5)H2), 2.53 (1 H, m, C(9)H2), 2.72 (2 H, m, C(5)H2
and C(9)H2), 2.85 (1 H, m, C(7)H2), 3.23 (1 H, m, C(7)H2),
7.30 (2 H, d, J = 8.1 Hz, C(12)H and C(16)H), 7.74 (2 H,
d, J = 8.1 Hz, C(13)H and C(15)H); m/z (ESI+) 315.1448
(M + H+. C16H19N4O3 requires 315.1457) 316(23%), 315(100),
and 274(28); Ion-pair HPLC, tr = 15.8 min. Spectral data are
identical to those reported for this compound in the literature.23
(6S)-5,10-Dideaza-5,6,7,8-tetrahydropteroic acid (6S-8). (6S)-
2-Pivaloyl-5,10-dideaza-5,6,7,8-tetrahydropteroic acid methyl
ester (6S-7) was used in a procedure identical to that described
for the synthesis of (6R)-5,10-dideaza-5,6,7,8-tetrahydropteroic
acid (6R-8). Ion-pair HPLC indicated >99% product purity.
OD290
mmol g−1
=
(3)
mg re sin × 1.65
(197 mg, 93%); [a]D = +44.5 (0.1 M NaOH) (lit.,53,54 [a]D
=
+40.7 (1 N NaOH) and [a]D = +48.3 (1 N NaOH, from the
acid hydrolysis of (6S)-DDAH4PteGlu1)); dH (300 MHz; 0.1 M
NaOD in D2O) 1.59 (2 H, m, C(10)H2), 1.73 (1 H, m, C(6)H),
1.96 (1 H, dd, J = 8.8, 15.5 Hz, C(5)H2), 2.52 (1 H, m, C(9)H2),
2.71 (2 H, m, C(5)H2 and C(9)H2), 2.85 (1 H, m, C(7)H2), 3.23 (1
H, m, C(7)H2), 7.29 (2 H, d, J = 8.1 Hz, C(12)H and C(16)H),
7.73 (2 H, d, J = 8.1 Hz, C(13)H and C(15)H); m/z (ESI+)
315.1456 (M + H+. C16H19N4O3 requires 315.1457) 316(24%),
315(94), 305(48), 297(21), 275(24), 274(100), 268(50), 233(31),
and 79(40); Ion-pair HPLC, tr = 15.9 min.
Capping. A solution of 0.20 mL (2.16 mmol) acetic anhydride
and 0.132 g (1.08 mmol) DMAP in 10 mL of anhydrous DMF
was added to the resin. The mixture was agitated by inversion
for 60 min at ambient temperature. The reaction was stopped by
filtering the resin and washing with 3 × 10 mL DMF and 2 ×
10 mL CH2Cl2.
Deprotection. 50 mL of a 20% (v/v) solution of piperidine in
anhydrous DMF was prepared. 10 mL of this solution was added
to the resin with agitation. After 3 min, the resin was filtered and
the above deprotection steps were repeated four times. After the
final filtration, the resin was washed with 3 × 10 mL DMF and
2 × 10 mL CH2Cl2.
(6R)-5,10-Dideaza-5,6,7,8-tetrahydropteroyl azide (6R-9).
(6R)-5,10-Dideaza-5,6,7,8-tetrahydropteroic
acid
(6R-8)
Coupling. 0.808 g (1.90 mmol) Fmoc-Glu-Ot-Bu, 0.257 g
(1.90 mmol) HOBt, and 0.702 g (1.85 mmol) HBTU were
dissolved in 10 mL of anhydrous DMF in a dry test tube with
gentle mixing. After 15 min, 0.66 mL (3.80 mmol) of DIPEA
was added to the test tube with gentle mixing. After 5 min,
this solution was added to a reaction tube containing the resin
to which the C-terminal Glun was attached. After 90 min of
agitation by inversion at ambient temperature, the reaction was
stopped by filtering the resin and washing with 3 × 10 mL DMF
and 2 × 10 mL CH2Cl2.
(47 mg, 0.13 mmol) was added under Ar to a dry flask
containing a stir bar. The flask was sealed with a septum under
an atmosphere of Ar (balloon) before the addition of 1 mL
anhydrous DMSO. The solution was cooled in a 20 ◦C water
bath before the addition of Et3N (107 lL, 0.77 mmol). After
15 min, cold ethyl chloroformate (14 lL, 0.15 mmol) was added.
After 45 min, NaN3 (10.5 mg, 0.16 mmol) was then added. The
reaction was allowed to stir for 1 h at ambient temperature.
Reaction completion was verified by RP-HPLC, Method 1.
The reaction solution was diluted with 6 mL of 0.1 M HCl for
precipitation. The precipitate was collected by centrifugation
(15 min, 4 ◦C, 12 000g). The supernatant was removed and the
pellet was washed with: 1 × 10 mL ddH2O; 1 × 10 mL CH3CN;
and 1 × 5 mL Et2O. The pellet was allowed to air-dry for 15 min
before drying under high vacuum in the presence of P2O5 for
several hours. Total mass recovery, 49 mg, a mixture of 6R-9
(90.5%) and 6R-8 based on RP-HPLC (Method 1); Ion-pair
HPLC, tr = 30.6 min. This material was used in subsequent
coupling reactions without further purification. Because of
Capping and deprotection. As above.
Cleavage/deprotection. The resin was transferred to a 25 mL
round-bottomed flask and mixed with 9.5 mL TFA, 2.5 mL
ddH2O, and 2.5 mL TIS. After 90 min at ambient temperature
with occasional swirling, the solution was filtered and the resin
was washed with TFA. The combined filtrates were concentrated
in vacuo. The residue was then diluted with ddH2O and
lyophilized to remove trace TFA. The residue was dissolved
in ddH2O with the resulting solution being filtered with a
0.2 lm PTFE syringe filter prior to lyophilization. Mass spectral
analysis indicated the presence of a significant amount of the
mono-tert-butyl ester so the deprotection step was repeated as
above except utilizing 9.5 mL TFA, 0.25 mL ddH2O, and 0.25 mL
TIS. Lyophilization yielded the crude dipeptide (172 mg, 93%).
A portion of this material, 154 mg, was then purified by semi-
preparative RP-HPLC (Method 1), tr = 6.4 min, to obtain the
TFA salt of the dipeptide after lyophilization. Total yield 75 mg
(40% based on total resin loading, 45% based on fraction of
crude material purified). dH (500 MHz; D2O; numbering system
for Glu2–6, see Supplementary Information Scheme S2) 1.96 (1 H,
m, C(8ꢂ)H2), 2.14 (3 H, m, C(3ꢂ)H2 and C(8ꢂ)H2), 2.46 (4 H,
1
concerns about stability, H NMR spectra were not obtained.
These data for racemic 9 are reported in the Supplementary
Information.
(6S)-5,10-Dideaza-5,6,7,8-tetrahydropteroyl azide (6S-9).
(6S)-5,10-Dideaza-5,6,7,8-tetrahydropteroic acid (6S-8) was
used in a procedure identical to that described for the synthesis
of (6R)-5,10-dideaza-5,6,7,8-tetrahydropteroyl azide (6R-9).
Total mass recovery, 51 mg, a mixture of 6S-9 (89.6%) and
6S-8 based on RP-HPLC (Method 1); Ion-pair HPLC, tr =
30.4 min. This material was used in subsequent coupling
reactions without further purification. Because of concerns
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 3 8 8 – 3 3 9 8
3 3 9 5

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m, C(4ꢂ)H2 and C(9ꢂ)H2), 3.87 (1 H, dd, J = 4.0, 6.1 Hz, C(2ꢂ)H),
4.35 (1 H, t, J = 4.4 Hz, C(7ꢂ)H); dC (500 MHz; D2O) 26.1 (C8ꢂ),
consisting of (6R)-, or (6S)-5,10-dideazatetrahydropteroyl
azide·HCl (9) (2 equiv.) in anhydrous DMSO was added to the
tube with stirring followed by Et3N (1 equiv. for each COOH
of the amino acid or peptide, 1 equiv. for the amine salt (if
applicable) of the amino acid or peptide, and 4 equiv. for 9).
The reaction was monitored by HPLC and stopped after 2 days
by the addition of aqueous NH4HCO3 buffer (pH 8.1). The
reaction solution was then concentrated on a vacuum centrifuge
overnight with heating (35–40 ◦C) for product analysis (HPLC)
followed by purification by size-exclusion chromatography.
All DDAH4PteGlu2–6 compounds were further purified by
semi-preparative RP-HPLC, as detailed below, unless otherwise
noted. Product yields were determined by UV absorption
(272 nm, 0.1 M NaOH, e = 11 700 M−1 cm−1).10
26.2 (C3ꢂ), 30.5 (C9ꢂ), 31.5 (C4ꢂ), 52.7 (C7ꢂ), 53.7 (C2ꢂ), 173.3
+
=
=
=
=
(C O), 174.9 (C O), 175.7 (C O), 177.5 (C O), m/z (ESI )
277.1. (C10H17N2O7 requires 277.1). This material was identical
to a sample of L-Glu-c-L-Glu prepared by solution methods (see
Supplementary Information).
An identical procedure varying only in the number of cycles of
coupling, capping, and deprotection was used to synthesize all
other peptides of the series H-Glu-c-Glun-c-Glu-OH, n = 0–4.
Purification of poly(c-L-glutamate) peptides
The crude TFA salts of the peptides were dissolved in ddH2O to
yield solutions with a concentration of ∼12 mg per 50 lL. 50 lL
of these solutions were then individually used for purification
via semi-preparative RP-HPLC as follows:
(6R)-5,10-Dideaza-5,6,7,8-tetrahydrofolic acid, (6R)-DDAH4-
PteGlu1 (6R-1). Yield, 87%; kmax (0.1 M NaOH)/nm 242
and 272; [a]D = −29.3 (0.1 M NaOD in D2O) ([a]D
=
L-Glutamyl-(c-L-glutamate)2
(c-Glu3). Semi-preparative
−27.2 (0.1 M NaOD in D2O, from an authentic sample of
(6R)-DDAH4PteGlu1–Na); lit.,10 [a]D = −21.1 (1 N NaOH
in methanol, from the in situ saponification of the d-10-
(+)-camphorsulfonic acid salt of (6R)-DDAH4PteGlu1 diethyl
ester)); dH (500 MHz; 0.1 M NaOD/D2O) 1.65 (2H, dd, J =
7.6, 15.0 Hz, C(10)H2), 1.79 (1 H, m, C(6)H), 2.01 (2 H, dd,
J = 8.9, 15.4 Hz, C(5)H2, C(3ꢂ)H2), 2.15 (1 H, m, C(3ꢂ)H2), 2.30
(2 H, dd, J = 7.6, 7.9 Hz, C(4ꢂ)H2), 2.58 (1 H, dd, J = 4.9,
15.6 Hz, C(9)H2), 2.79 (2 H, m, C(5)H2 and C(9)H2), 2.89 (1 H,
m, C(7)H2), 3.28 (1 H, br d, J = 10.2 Hz, C(7)H2), 4.31 (1 H,
m, C(2ꢂ)H), 7.41 (2 H, d, J = 7.7 Hz, C(12)H and C(16)H), 7.74
(2 H, d, J = 7.7 Hz, C(13)H and C(15)H); dC (500 MHz; 0.1 M
NaOD/D2O) 26.3, 28.9, 31.5, 33.0, 34.7, 45.7, 56.4, 87.5, 127.8,
129.2, 131.4, 148.1, 161.0, 161.6, 170.6, 174.1, 179.4, 182.7; m/z
(ESI+) 444.1873 (M + H+. C21H26N5O6 requires 444.1883) 445
(25%), and 444 (100); Ion-pair HPLC, tr = 18.4 min. Spectral
RP-HPLC (Method 2) yielded 10.9 mg, 21.0 lmol, of the TFA
salt of the tripeptide after lyophilization. dH (300 MHz; D2O)
1.87 (2 H, m, C(8ꢂ)H2 and C(13ꢂ)H2), 2.07 (4 H, m, C(3ꢂ)H2,
C(8ꢂ)H2, and C(13ꢂ)H2), 2.33 (6 H, m, C(4ꢂ)H2, C(9ꢂ)H2, and
C(14ꢂ)H2), 3.93 (1 H, t, J = 6.4 Hz, C(2ꢂ)H), 4.23 (2 H, m,
C(7ꢂ)H and C(12ꢂ)H); Semi-preparative RP-HPLC (Method 2),
tr = 16.0 min; m/z (ESI+) 406.2 (C15H24N3O10 requires 406.2)
406 (23%), 304 (100), and 282 (54).
L-Glutamyl-(c-L-glutamate)3
(c-Glu4). Semi-preparative
RP-HPLC (Method 3) yielded 8.4 mg, 13.0 lmol, of the TFA
salt of the tetrapeptide after lyophilization. dH (300 MHz; D2O)
1.96 (3 H, m, C(8ꢂ)H2, C(13ꢂ)H2, and C(18ꢂ)H2), 2.17 (5 H,
m, C(3ꢂ)H2, C(8ꢂ)H2, C(13ꢂ)H2, and C(18ꢂ)H2), 2.45 (8 H, m,
C(4ꢂ)H2, C(9ꢂ)H2, C(14ꢂ)H2, and C(19ꢂ)H2), 3.97 (1 H, t, J =
6.4 Hz, C(2ꢂ)H), 4.32 (2 H, m, C(7ꢂ)H and C(12ꢂ)H), 4.38 (1 H,
m, C(17ꢂ)H); Semi-preparative RP-HPLC (Method 3), tr
=
1
data (UV/vis, H NMR, and 13C NMR) are identical to those
5.7 min; m/z (ESI+) 535.3 (C20H31N4O13 requires 535.2) 557
obtained with an authentic sample of (6R)-DDAH4PteGlu1.
(20%), and 535 (100).
(6S)-5,10-Dideaza-5,6,7,8-tetrahydrofolic acid, (6S)-DDAH4-
PteGlu1 (6S-1). Yield, 92%; kmax (0.1 M NaOH)/nm 242 and
272; [a]D = +29.2 (0.1 M NaOD in D2O) (lit.,10 [a]D = +31.1 (1 N
NaOH in methanol, from the in situ saponification of the d-10-
(+)-camphorsulfonic acid salt of (6S)-DDAH4PteGlu1 diethyl
ester); dH (500 MHz; 0.1 M NaOD/D2O) 1.65 (2H, dd, J = 6.7,
14.3 Hz, C(10)H2), 1.79 (1 H, m, C(6)H), 2.02 (2 H, dd, J =
7.4, 15.4 Hz, C(5)H2, C(3ꢂ)H2), 2.14 (1 H, m, C(3ꢂ)H2), 2.30
(2 H, dd, J = 6.7, 7.9 Hz, C(4ꢂ)H2), 2.58 (1 H, m, C(9)H2), 2.79
(2 H, m, C(5)H2 and C(9)H2), 2.89 (1 H, dd, C(7)H2), 3.28 (1 H,
br d, J = 11.7 Hz, C(7)H2), 4.31 (1 H, m, C(2ꢂ)H), 7.42 (2 H,
d, J = 8.2 Hz, C(12)H and C(16)H), 7.74 (2 H, d, J = 8.2 Hz,
C(13)H and C(15)H); dC (500 MHz; 0.1 M NaOD/D2O) 26.3,
28.8, 31.4, 32.9, 34.7, 45.7, 56.4, 87.4, 127.8, 129.1, 131.4, 148.0,
161.0, 161.6, 170.6, 174.1, 179.4, 182.7; m/z (ESI+) 444.1879 (M
+ H+. C21H26N5O6 requires 444.1883) 445 (23%), and 444 (100);
Ion-pair HPLC, tr = 17.7 min.
(6R)-[14C]5,10-Dideaza-5,6,7,8-tetrahydrofolic acid, (6R)-
DDAH4Pte[14C]Glu1. A 10 mL solution (ethanol–H2O, 2 : 98)
containing 1.0 mCi of L-[U-14C]glutamic acid was evaporated
to dryness on a vacuum centrifuge. This residue was used in
a reaction with 6R-9 generally as described above but with
special precautions in place to prevent the inadvertent release
of radioactive material. Two purifications by size-exclusion
chromatography were required to obtain product of the
desired radiochemical purity. Yield, 62%; Ion-pair HPLC, tr =
17.5–18.5 min.; Purity, 96%; Specific activity = 249 Ci mol−1.
L-Glutamyl-(c-L-glutamate)4
(c-Glu5). Semi-preparative
RP-HPLC (Method 4) yielded 8.9 mg, 11.4 lmol, of the TFA
salt of the pentapeptide after lyophilization. dH (300 MHz;
D2O) 1.96 (4 H, m, C(8ꢂ)H2, C(13ꢂ)H2, C(18ꢂ)H2, and C(23ꢂ)H2),
2.17 (6 H, m, C(3ꢂ)H2, C(8ꢂ)H2, C(13ꢂ)H2, C(18ꢂ)H2, and
C(23ꢂ)H2), 2.45 (10 H, m, C(4ꢂ)H2, C(9ꢂ)H2, C(14ꢂ)H2, C(19ꢂ)H2,
and C(24ꢂ)H2), 3.97 (1 H, t, J = 6.4 Hz, C(2ꢂ)H), 4.32 (3 H,
m, C(7ꢂ)H, C(12ꢂ)H, and C(17ꢂ)H), 4.39 (1 H, m, C(22ꢂ)H);
Semi-preparative RP-HPLC (Method 4), tr = 10.7 min; m/z
(ESI+) 664.4 (C25H38N5O16 requires 664.2) 664(100%).
L-Glutamyl-(c-L-glutamate)5
(c-Glu6). Semi-preparative
RP-HPLC (Method 4) yielded 7.7 mg, 8.5 lmol, of the TFA salt
of the hexapeptide after lyophilization. dH (300 MHz; D2O) 1.96
(5 H, m, C(8ꢂ)H2, C(13ꢂ)H2, C(18ꢂ)H2, C(23ꢂ)H2, and C(28ꢂ)H2),
2.17 (7 H, m, C(3ꢂ)H2, C(8ꢂ)H2, C(13ꢂ)H2, C(18ꢂ)H2, C(23ꢂ)H2,
and C(28ꢂ)H2), 2.46 (12 H, m, C(4ꢂ)H2, C(9ꢂ)H2, C(14ꢂ)H2,
C(19ꢂ)H2, C(24ꢂ)H2, and C(29ꢂ)H2), 3.96 (1 H, t, J = 6.4 Hz,
C(2ꢂ)H), 4.33 (4 H, m, C(7ꢂ)H, C(12ꢂ)H, C(17ꢂ)H, and C(22ꢂ)H),
4.39 (1 H, m, C(27ꢂ)H); Semi-preparative RP-HPLC (Method
4), tr = 11.4 min; m/z (ESI+) 793.5 (C30H45N6O19 requires 793.3)
794 (22%), and 793 (100).
5,10-Dideaza-5,6,7,8-tetrahydropteroylglutamyl-c-glutamates
(DDAH4PteGlun, n = 1–6) (1–6). An aqueous solution of
the L-glutamate or poly-c-glutamate (c-Glu2–6) was placed
into a 10 × 75 mm test tube and concentrated to dryness
on a vacuum centrifuge overnight at ambient temperature.
When the tube was removed from the vacuum, a small, dry
stir bar was placed in the tube and it was immediately sealed
with a rubber septum. The tube was then gently flushed for
15 min with a stream of dry Ar introduced with a needle
through the septum. The vent was then removed and the
Ar line was replaced with an Ar-filled balloon. A solution
(6R)-5,10-Dideaza-5,6,7,8-tetrahydropteroyl-L-glutamate-c-L-
glutamate (6R-2). Crude c-Glu2 was used for this reaction.
Purified using semi-preparative RP-HPLC (Method 5), tr =
20.0 min. Yield, 39%; kmax (0.1 M NaOH)/nm 242 and 272; m/z
(ESI+) 573.2 (M + H+. C26H33N6O9 requires 573.2); m/z (ESI+)
3 3 9 6
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 3 8 8 – 3 3 9 8

View Online
573.2305 (M + H+. C26H33N6O9 requires 573.2309) 574 (32%),
silanized Eppendorf tubes and were stopped by the addition of
573 (100), 349 (21), and 305 (48); Ion-pair HPLC, tr = 22.2 min.
1 mL of ice-cold buffer consisting of 5 mM glutamate, pH 7.5.
Assay products were purified with DEAE-cellulose (DE-
52) ion-exchange chromatography using a buffer consisting
of 10 mM Tris, and 110 mM NaCl, conductivity ∼11.5 mS,
at pH 7.5 to remove unincorporated [3H]glutamate. Folate
products were eluted with 0.1 M HCl. The amount of tritium
incorporation into the folate products was determined by liquid
scintillation counting. All reactions were carried out in duplicate
and the blank reactions lacked the folate substrate.
(6S)-5,10-Dideaza-5,6,7,8-tetrahydropteroyl-L-glutamate-c-L-
glutamate (6S-2). Crude c-Glu2 was used for this reaction.
Purification was by size-exclusion chromatography only. Yield,
31%; kmax (0.1 M NaOH)/nm 242 and 272; m/z (ESI+) 573.2324
(M + H+. C26H33N6O9 requires 573.2309) 574 (29%), 573 (100),
305 (50), 274 (22), and 261 (34); Ion-pair HPLC, tr = 20.8 min.
(6R)-5,10-Dideaza-5,6,7,8-tetrahydropteroyl-L-glutamate-(c-L-
glutamate)2 (6R-3). Purified using semi-preparative RP-HPLC
(Method 5), tr = 15.0 min. Yield, 78%; kmax (0.1 M NaOH)/nm
242 and 272; dH (500 MHz; 0.1 M NaOD/D2O; numbering
system, see Supplementary Information, Scheme S3) 1.72 (2 H,
m, C(10)H2), 1.89 (3 H, m, C(6)H, C(8ꢂ)H2, and C(13ꢂ)H2), 2.07
(5 H, m, C(5)H2, C(3ꢂ)H2, C(8ꢂ)H2, and C(13ꢂ)H2), 2.23 (2 H,
dd, J = 7.8, 8.3 Hz, C(9ꢂ)H2), 2.31 (2 H, dd, J = 7.7, 8.2 Hz,
C(4ꢂ)H2), 2.48 (2 H, dd, J = 6.4, 7.8 Hz, C(14ꢂ)H2), 2.63 (1 H,
m, C(9)H2), 2.84 (2 H, m, C(5)H2 and C(9)H2), 2.95 (1 H, m,
C(7)H2), 3.34 (1 H, br d, J = 11.5 Hz, C(7)H2), 4.12 (2 H, m,
C(7ꢂ)H and C(12ꢂ)H), 4.40 (1 H, dd, J = 4.5, 8.9 Hz, C(2ꢂ)H),
7.46 (2 H, d, J = 7.9 Hz, C(12)H and C(16)H), 7.76 (2 H, d,
J = 7.9 Hz, C(13)H and C(15)H); m/z (ESI+) 724.2527 (M +
Na+. C31H39N7NaO12 requires 724.2554) 702 (100%); Ion-pair
HPLC, tr = 29.3 min.
(6R)-5,10-Dideaza-5,6,7,8-tetrahydropteroyl-L-glutamate-(c-L-
glutamate)3 (6R-4). Purified using semi-preparative RP-HPLC
(Method 6), tr = 12.0 min. Yield, 59%; kmax (0.1 M NaOH)/nm
242 and 272; m/z (ESI+) 853.2989 (M + Na+. C36H46N8NaO15
requires 853.2980) 832 (20%), 831 (100), and 427 (50); Ion-pair
HPLC, tr = 37.0 min.
(6R)-5,10-Dideaza-5,6,7,8-tetrahydropteroyl-L-glutamate-(c-L-
glutamate)4 (6R-5). Purified using semi-preparative RP-HPLC
(Method 7), tr = 13.5 min. Yield, 53%; kmax (0.1 M NaOH)/nm
242 and 272; m/z (ESI+) 982.3409 (M + Na+. C41H53N9NaO18
requires 982.3406) 960 (20%), 492 (30), 491 (63), and 381 (100);
Ion-pair HPLC, tr = 43.4 min.
Analysis of product formation. General conditions: As above,
except for final concentrations of 20 mM MgCl2, 5 mM
dithiothreitol, 10 mM ATP, and 5 mM glutamate. The con-
centrations of (6R)-DDAH4Pte[14C]Glu1 utilized were 2.5, 25,
or 250 lM with specific activities of 249, 24.9, and 2.49 Ci mol−1
respectively. A 5 min reaction time was utilized with a final
[hFPGS] = 1 lM and volume of 80 lL.
The solution was incubated at 37 ◦C for 5 min prior to
reaction initiation by addition of purified hFPGS. The reactions
were stopped by addition of 50% w/v trichloroacetic acid, with
mixing, to a final volume of 5% w/v. For the control reaction,
trichloroacetic acid was added immediately prior to the addition
of hFPGS and analysis showed no product formation. The tubes
were stored at −80 ◦C. The samples were clarified prior to HPLC
analysis by centrifugation (16 000g, 10 min).
Ion-pair HPLC method: A portion (75 lL) of each sample
was analyzed using the ion-pair HPLC method described above
except fractions (30 s) were collected over the range tr = 10–
60 min.
Liquid scintillation counting method: The 30 s fractions were
collected directly into 5.5 mL plastic scintillation vials. Bio-Safe
II scintillation cocktail (4.0 mL) was added to each vial before
capping and mixing by vortexing. Radioactivity (DPM) of each
fraction was determined by liquid scintillation counting.
Acknowledgements
This research was supported in part by grants from the
National Cancer Institute, CA 28097 (J.K.C.), CA43500
(J.J.M.), and CA16056 (Roswell Park Cancer Center Support
Grant). J.W.T. was a trainee of the Michigan Chemistry-
Biology Interface Training Program, supported in part by
a grant from the National Institutes of General Medical
Sciences (GM008597). J.W.T. was the recipient of a Fred
W. Lyons Fellowship from the College of Pharmacy, University
of Michigan, and a fellowship from the American Foundation
for Pharmaceutical Education. We thank Dr Chuan Shih, Eli
Lilly & Co, Indianapolis, IN, for the generous gift of (6R)-
DDAH4PteGlu1 (LometrexolTM), (6RS)-DDAH4PteGlu2, and
(6RS)-DDAH4PteGlu5 used in initial experiments. In addition,
we gratefully acknowledge the contribution to this research of
Dr Michael J. Lovdahl and Anne Akin, Analytical Development
Department, Pfizer Global Research and Development, Ann
Arbor, MI, for carrying out the separation of 6R-7 and 6S-7 by
chiral HPLC on a preparative scale. We thank Sanyo Tsai for
aid in the preparation of the poly-c-glutamate peptides, William
Haile for partial purification of hFPGS, Prof. Richard Moran
for a sample of purified hFPGS, Prof. Edwin Vedejs for the loan
of an analytical chiral HPLC column, and Prof. Bruce Palfey
for assistance with the kinetic modeling studies.
(6R)-5,10-Dideaza-5,6,7,8-tetrahydropteroyl-L-glutamate-(c-L-
glutamate)5 (6R-6). Purified using semi-preparative RP-HPLC
(Method 8), tr = 15.5 min. Yield, 55%; kmax (0.1 M NaOH)/nm
242 and 272; m/z (ESI+) 1111.3868 (M + Na+. C46H60N10NaO21
requires 1111.3832) 1089 (20), 556 (100), 545 (30), and 381 (41);
Ion-pair HPLC, tr = 48.4 min.
Preparation of DDAH4PteGlun solutions
Each DDAH4PteGlun was dissolved in 1–2 mL of 20–100 mM
NaOH. The resulting solutions were filtered through a spin-
filter. All solutions were then stored at −20 ◦C. Concentrations
of the solutions were determined based on absorbance at 272 nm
(0.1 M NaOH, e = 11,700 M−1 cm−1).10 All solutions of
DDAH4PteGlun were freshly prepared and purity confirmed by
HPLC. Extended storage (>2 months, −20 ◦C) of solutions with
[NaOH] > 20 mM led to partial hydrolysis of the polyglutamate
chain yielding a mixture of shorter chain compounds.
hFPGS Assays
Total glutamate incorporation.
Determination of steady-state kinetic constants49,55. General
conditions: Final concentrations of 100 mM Tris (pH 8.85),
10 mM MgCl2, 20 mM KCl, 10 mM NaHCO3, 0.5 mg mL−1
bovine serum albumin, 5 mM ATP, 2 mM L-[3H]glutamate
(specific activity ∼2.6 cpm pmol−1), 0–300 lM DDAH4PteGlun,
14 nM hFPGS, 250 lL reaction volume, 60 min reaction time,
and 37 ◦C. The solution was incubated at 37 ◦C for 5 min prior
to addition of partially purified hFPGS expressed in either E.
coli (n = 1–2) or bacculovirus-infected SF9 insect cells (n = 3–6)
to initiate the reaction. Reactions were carried out in 1.5 mL,
References and notes
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44 Additional reducing agent was not included in these reactions, as
early experiments with hFPGS expressed in E. coli indicated no
measurable effect of the presence of reducing agent on enzyme
activity or product distribution. However, the reactions that utilized
enzyme expressed in E. coli had a final concentration of 4 mM 2-
mercaptoethanol due to its presence in the enzyme storage buffer. In
contrast, the reactions in which enzyme expressed in SF-9 insect cells
had been used, the final concentration of 2-mercaptoethanol was
0.4 mM. Subsequently, it was determined that additional reducing
agent (100 mM 2-mercaptoethanol or 5 mM dithiothreitol) increased
the chain-length of the polyglutamate products produced only for the
hFPGS expressed in SF-9 insect cells. This observation suggests that
the addition of reducing agent to the assays shown in Fig. 3 would
intensify the observed substrate inhibition.
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