Synthesis of β-ketophosphonate analogs of glutamyl and glutaminyl adenylate, and selective inhibition of the corresponding bacterial aminoacyl-tRNA synthetases

Article abstract of DOI:10.1016/j.bmc.2006.09.056

The aminoacyl-β-ketophosphonate-adenosines (aa-KPA) are stable analogs of the aminoacyl adenylates, which are high-energy intermediates in the formation of aminoacyl-tRNA catalyzed by aminoacyl-tRNA synthetases (aaRS). We have synthesized glutamyl-β-ketop

Full text of DOI:10.1016/j.bmc.2006.09.056

Bioorganic & Medicinal Chemistry 15 (2007) 295–304
Synthesis of b-ketophosphonate analogs of glutamyl and glutaminyl
adenylate, and selective inhibition of the corresponding bacterial
aminoacyl-tRNA synthetases
b,
a
b
Christian Balg,a, Sebastien P. Blais, Stephane Bernier, Jonathan L. Huot,
´
´
Manon Couture,b Jacques Lapointeb,* and Robert Cheˆneverta,*
aDe´partement de chimie, Centre de recherche sur la fonction, la structure et l’inge´nierie des prote´ines (CREFSIP),
Faculte´ des sciences et de ge´nie, Universite´ Laval, Que´bec, Canada G1K 7P4
bDe´partement de biochimie et de microbiologie, Centre de recherche sur la fonction, la structure et l’inge´nierie des
prote´ines (CREFSIP), Faculte´ des sciences et de ge´nie, Universite´ Laval, Que´bec, Canada G1K 7P4
Received 4 August 2006; revised 21 September 2006; accepted 26 September 2006
Available online 29 September 2006
Abstract—The aminoacyl-b-ketophosphonate-adenosines (aa-KPA) are stable analogs of the aminoacyl adenylates, which are high-
energy intermediates in the formation of aminoacyl-tRNA catalyzed by aminoacyl-tRNA synthetases (aaRS). We have synthesized
glutamyl-b-ketophosphonate-adenosine (Glu-KPA) and glutaminyl-b-ketophosphonate-adenosine (Gln-KPA), and have tested
them as inhibitors of their cognate aaRS, and of a non-cognate aaRS. Glu-KPA is a competitive inhibitor of Escherichia coli glut-
amyl-tRNA synthetase (GluRS) with a Ki of 18 lM with respect to its substrate glutamate, and binds at one site on this monomeric
enzyme; the non-cognate Gln-KPA also binds this GluRS at one site, but is a much weaker (Ki = 2.9 mM) competitive inhibitor. By
contrast, Gln-KPA inhibits E. coli glutaminyl-tRNA synthetase (GlnRS) by binding competitively but weakly at two distinct sites
on this enzyme (average Ki of 0.65 mM); the non-cognate Glu-KPA shows one-site weak (Ki = 2.8 mM) competitive inhibition of
GlnRS. These kinetic results indicate that the glutamine and the AMP modules of Gln-KPA, connected by the b-ketophosphonate
linker, cannot bind GlnRS simultaneously, and that one Gln-KPA molecule binds the AMP-binding site of GlnRS through its AMP
module, whereas another Gln-KPA molecule binds the glutamine-binding site through its glutamine module. This model suggests
that similar structural constraints could affect the binding of Glu-KPA to the active site of mammalian cytoplasmic GluRSs, which
are evolutionarily much closer to bacterial GlnRS than to bacterial GluRS. This possibility was confirmed by the fact that Glu-KPA
inhibits bovine liver GluRS 145-fold less efficiently than E. coli GluRS by competitive weak binding at two distinct sites (average
Ki = 2.6 mM). Moreover, these kinetic differences reveal that the active sites of bacterial GluRSs and mammalian cytoplasmic
GluRSs have substantial structural differences that could be further exploited for the design of better inhibitors specific for
bacterial GluRSs, promising targets for antimicrobial therapy.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
Aminoacyl-tRNA synthetases (aaRSs) play a key role in
protein biosynthesis in all living organisms. These
enzymes catalyze the esterification of amino acids to
cognate tRNA in a two-step process1–3 (Scheme 1).
Keywords: Aminoacyl-tRNA synthetases; Inhibitors; Aminoacyl
adenylates; Analogs; b-Ketophosphonates.
*
Corresponding authors. Tel.: +1 418 656 3283; fax: +1 418 656 7916
(R.C.); tel.: +1 418 656 2965; fax: +1 418 656 7176 (J.L.);
Scheme 1.
e-mail
chenevert@chm.ulaval.ca
addresses:
jacques.lapointe@bcm.ulaval.ca;
robert.
First, ATP reacts with the amino acid (aa) to form
an enzyme-bound aminoacyl adenylate intermediate
(aa-AMP) with displacement of pyrophosphate (PPi).
The two authors contributed equally to this work. C. B. for the
organic synthesis part, and S. P. B. for the enzyme kinetics part.
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.09.056

296
C. Balg et al. / Bioorg. Med. Chem. 15 (2007) 295–304
In aminoacyl adenylates, the high-energy mixed anhy-
dride bond activates the carboxyl group of the amino
acid. In the second step, the aminoacyl group is trans-
ferred to the 20- or 30-OH at the 30 end of the tRNA,
generating aminoacyl-tRNA (aa-tRNA) and adenosine
monophosphate (AMP).
cytoplasmic GluRS, and we discuss its interest for the
design of novel antibiotics targeting bacterial GluRSs.
2. Results and discussion
2.1. Synthesis of b-ketophosphonates 4, 9, and 15
AaRSs are classified into two main groups of 10 en-
zymes each on the basis of common structural and
mechanistic features.3 GluRS and GlnRS belong to class
I, whereas AspRS is a member of class II. GluRS and
GlnRS have the characteristic, shared only by ArgRS
and class I LysRS, of requiring the presence of their cog-
nate tRNA to catalyze the activation of the amino acid
substrate.1
We recently reported the synthesis of ketophosphonate
4.9 This synthesis relied upon the activation of phos-
phonic monoester 2 with p-tosyl chloride in the presence
of pyridine as a base, followed by condensation with iso-
propylideneadenosine 1 to give ketophosphonate 3
(Scheme 3). Although this condensation–deprotection
reaction provided pure 3, reaction times were long (14
days) and yields were low (30%). We sought to improve
the synthesis of b-ketophosphonates, so we explored
alternative strategies for the activation of phosphonic
esters. The synthesis of glutamic ketophosphonate 9 is
outlined in Scheme 4. Condensation of known glutamic
ester 511 with the lithium salt of dimethyl methylphosph-
onate provided b-ketophosphonate 6. Phosphonic
monoester 7 was obtained by selective demethylation
of 6 with tert-butylamine.12 The phosphonic ester 8
was obtained by esterification of 7 with isopropylidene-
adenosine 1 in the presence of O-benzotriazol-1-yl-
Several natural products with various chemical struc-
tures have been identified as inhibitors of aaRSs.4,5
These enzymes have been subjected to significant evolu-
tionary divergence, and selective inhibition of bacterial
enzymes has been observed. Pseudomonic acid, a sec-
ondary metabolite from Pseudomonas fluorescens, is
the sole aaRS inhibitor currently used as an antibiotic.6
This natural product is a potent (Ki = 6 nM for E. coli
IleRS) and selective (104-fold selectivity for bacterial
versus mammalian IleRS) inhibitor of bacterial IleRS.7
N,N,N0,N0-tetramethyluronium
hexafluorophosphate
AaRSs are amenable to high throughput screening of
compound libraries.8 Rationally designed synthetic
inhibitors of aaRS are typically stable analogs of amino-
acyl adenylates (aa-AMP on Scheme 1).4,5 The stability
is achieved by replacement of the labile mixed anhydride
function by non-hydrolyzable bioisosteres. Several
aminoacylsulfamoyladenosines or aminoalkyl adeny-
lates have been synthesized and shown to be inhibitors
of corresponding aaRSs. The b-ketophosphonate func-
tion can also serve as a stable surrogate for the reactive
acylphosphate subunit of aminoacyl adenylates in the
design of aaRS inhibitors (Scheme 2).9,10
(HBTU) as a coupling reagent, followed by demethyla-
tion of the phosphonic methyl ester with pyridine/water
at 55 ꢁC.13 Reaction of 8 with wet trifluoroacetic acid
provoked the removal of the three remaining protecting
groups (tert-butyl ester, N-Boc, and isopropylidene ace-
tal) to give glutamic b-ketophosphonate 9.
The synthesis of glutamine ketophosphonate 15 is sum-
marized in Scheme 5. Glutamine methyl ester 11 was
prepared by the reaction of the cesium salt of glutamine
derivative 10 with CH3I in DMF.14 Condensation of es-
ter 11 with the lithium anion of dimethyl methylphosph-
onate afforded dimethyl b-ketophosphonate 12.15
Selective monodemethylation of 12 with PhSH/Et3N
provided monoester 13. Esterification of phosphonic salt
13 with adenosine derivative 1 by the HBTU procedure,
followed by demethylation of the phosphonic methyl es-
ter, gave phosphonic ester 14. We have observed that the
intermediate phosphonic methyl ester is unstable but the
corresponding salt 14 was easily isolated. The overall
yield for this key two-step sequence was 57%. Treatment
of 14 with CF3COOH/H2O resulted in simultaneous
cleavage of the Boc, trityl, and isopropylidene groups
to provide glutamine b-ketophosphonate 15. This
compound is stable in the solid state but decomposes
slowly in solution.
Herein we report the synthesis of b-ketophosphonate
analogs of glutamyl and glutaminyl adenylates, and
their interaction with their cognate aaRSs. We discuss
the structural significance of the unusual kinetics of E.
coli GlnRS inhibition by Gln-b-ketophosphonate and
of a mammalian GluRS by Glu-b-ketophosphonate;
we also underline the high specificity of Glu-b-keto-
phosphonate for a bacterial GluRS versus a mammalian
2.2. Inhibition of E. coli GluRS by Glu-b-ketophospho-
nate-adenosine 9 (Glu-KPA) and specificity of this
inhibition
The comparison of the double-reciprocal plots of the
aminoacylation kinetic data at various concentrations
of Glu-KPA (Fig. 1A) shows that it is essentially a
competitive inhibitor of E. coli GluRS with respect to
glutamate, as revealed by the intersection of the lines
Scheme 2.

C. Balg et al. / Bioorg. Med. Chem. 15 (2007) 295–304
297
Scheme 3. Reagents and conditions: (a) TsCl, pyridine, rt, 14 days (b) CF3COOH/H2O, rt, 15 min.
Scheme 4. Reagents and conditions: (a) LiCH2P(O)(OCH3)2, THF, ꢀ78 ꢁC, 1 h; (b) i—t-BuNH2, reflux, 24 h; ii—HCl; (c) i—HBTU, i-Pr2NEt, 20,30-
isopropylideneadenosine 1, DMF, rt, 8.5 h; ii—pyridine/H2O, 55 ꢁC, 10 h; (d) CF3COOH/H2O, rt, 15 min.
Scheme 5. Reagents and conditions: (a) i—Cs2CO3, MeOH/H2O; ii—MeI, DMF, rt, 1 h; (b) LiCH2P(O)(OCH3)2, THF, ꢀ78 ꢁC, 2 h; (c) PhSH,
Et3N, THF/DMF, rt, 48 h; (d) i—HBTU, DMAP, 20,30-isopropylideneadenosine 1, DMF, 3.5 h; ii—PhSH, Et3N, DMF, rt, 24 h; (e) CF3COOH/
H2O, rt, 15 min.
near the axis 1/[S] = 0. A Ki value of 18 lM was ob-
tained from a replot of the slopes of the above-men-
tioned lines versus [inhibitor]16 (Fig. 1B). This
competitive character of Glu-KPA inhibition with re-
spect to glutamate is also revealed by the fitting of the
kinetic relation vi/v0 = f ([inhibitor]) with the competitive
inhibition equation 1 (Fig. 2A, left curve; see Section 4).
This curve-fitting procedure led to the same Ki value of
18 lM Glu-KPA (Fig. 1) for E. coli GluRS, and thus
was used for identifying the other cases of competitive

298
C. Balg et al. / Bioorg. Med. Chem. 15 (2007) 295–304
Figure 1. Inhibition of Escherichia coli GluRS by Glu-KPA 9: (A) determination of the apparent Km for glutamate in the presence of several
concentrations of this inhibitor: 0 lM (Ç), 10 lM (h), 40 lM (m), 80 lM (·), 120 lM (d), and 200 lM (+). (B) Determination of the Ki value for this
inhibitor with respect to glutamate, from the linear relation between the slopes of the reciprocal plots presented in A, as a function of the inhibitor
concentration.
Figure 2. Inhibition by Glu-KPA 9 (s) and by Gln-KPA 15 (h) of: (A) E. coli GluRS; and inhibition of a mammalian GluRS by Glu-KPA 9 (Æ);
(B) E. coli GlnRS.
inhibition reported here, and for measuring the Ki values
(Fig. 2, except for the left curve of Figure 2B). The relat-
ed compound Gln-KPA 15, which inhibits E. coli GlnRS
(see below), evolutionarily related to GluRS17, is a 170-
fold weaker inhibitor (Ki = 2.9 mM) of E. coli GluRS
(Fig. 2A, right curve, and Table 1) than is Glu-KPA.
inhibition, represented by the less tilted curve of
vi/v0 = f ([inhibitor]), but trace a steeper curve fitting cor-
rectly the data. As these aa-AMP analogs are made of
two modules (aminoacyl and AMP), we considered the
possibility that Gln-KPA binds at two sites on GlnRS,
possibly the glutamine and the AMP sites, through its
corresponding modules (Fig. 3). We thus used the mech-
anism of cooperative pure competitive inhibition by two
different non-exclusive inhibitors16 shown in Figure 4A,
adapted to our situation where the two inhibitory com-
ponents are part of the same Gln-KPA molecule. The
velocity equation for this system under rapid equilibri-
um conditions, and the corresponding relation for
2.3. Inhibition of E. coli GlnRS by Gln-b-ketophospho-
nate-adenosine 15 (Gln-KPA) and specificity of this
inhibition
The kinetics of E. coli GlnRS inhibition by Gln-KPA
(Fig. 2B) cannot be explained by a simple competitive
Table 1. Ki for the inhibition of various GluRS and of E. coli GlnRS by aa-AMP analogs
aa-AMP analog
E. coli GlnRS
E. coli GluRS
Mammalian liver GluRS
Ratio Ki mammalian/Ki E. coli
Ref.
Gln-KPA
Glu-KPA
Glu-AMS
0.65 mM
2.8 mM
ND
2.9 mM
18 lM
2.8 nM
ND
2.6 mM
70 nM
ND
145
25
This work
This work
25
ND, not determined.

C. Balg et al. / Bioorg. Med. Chem. 15 (2007) 295–304
299
The unusual inhibitory properties of Gln-KPA for E.
coli GlnRS (2 sites, and Ki of 0.65 mM) compared to
those of Gln-ol-AMP18 (1 site, Ki = 280 nM, structure
on Scheme 2) and of Gln-AMS19 (1 site and Ki =
1.32 lM, structure on Scheme 2) may be due to a steric
hindrance between the hinge, connecting the two mod-
ules of this inhibitor, and the active site area of GlnRS,
thus preventing the simultaneous binding of the two
modules to each of their sites on this enzyme. This
unusual Gln-KPA/GlnRS interaction, and the fact that
the two modules of Glu-KPA can bind simultaneously
to E. coli GluRS, reveals structural differences between
the active sites of GlnRS and of GluRS. Considering
that bacterial GlnRSs are evolutionarily closer to
eukaryotic GluRSs than to bacterial GluRSs,20–22
Glu-KPA could inhibit bacterial GluRSs much more
efficiently than mammalian GluRSs. This model is sup-
ported by the fact that Glu-KPA does not inhibit
significantly bovine liver GluRS (see below).
Figure 3. Schematic models of the binding of one molecule of aa-AMP
to an aaRS (left) and of two molecules of Gln-KPA 15 to E. coli
GlnRS (right).
2.4. Inhibition of a mammalian cytoplasmic GluRS by
Glu-KPA 9
Glu-KPA inhibits the cytoplasmic GluRS of calf liver,
competitively with respect to glutamate (Fig. 2A, Æ),
with a Ki of 2.6 mM. This inhibition is 145-fold weaker
than that of E. coli GluRS (Table 1).
3. Discussion
Among all the known stable analogs of aminoacyl ade-
nylates (aa-AMP) which inhibit aaRSs,4 Gln-KPA is the
only one for which the presence of two binding sites on
the cognate aaRS (GlnRS) was revealed by inhibition
kinetics. Considering the bimodular structure (aminoac-
yl and adenosine) of these inhibitors, this property of
Gln-KPA suggests that its two structural modules can-
not bind simultaneously to GlnRS. A possible model
is that one Gln-KPA molecule binds GlnRS via its ami-
noacyl module, whereas the other binds it via its adeno-
sine module (see Fig. 3). This model is consistent with
the fact that GlnRS inhibition by Gln-KPA is 40-fold
weaker than that of GluRS by Glu-KPA (Table 1)
whose two modules can bind simultaneously to GluRS
(Fig. 1). The joint binding of two covalently linked mod-
ules of a ligand to a protein is generally much stronger
than that of each of the corresponding free modules;
for instance, the covalent linking of the modules phenyl-
alanine and AMP to form phenylalanyl adenylate leads
to a much stronger binding of the latter (Kd = 4 nM) to
phenylalanyl-tRNA synthetase than that of phenylala-
nine (Kd = 30 lM) or of AMP (Kd = 1 mM).23
Figure 4. Cooperative pure competitive inhibition by two different
non-exclusive inhibitors: (A) scheme of this mechanism under rapid
equilibrium conditions; velocity equation for this system and the
corresponding relation for v1/v0 = f ([inhibitor]).
vi/v0 = f ([inhibitor]) are shown in Figure 4B. This equa-
tion 1 fits very well our experimental data (the steeper
curve in Figure 2B), which supports the above-men-
tioned two-site model.
This curve fitting yields a value of 0.42 mM2 for the
product aKi1Ki2, where Ki1 and Ki2 are the competitive
inhibition constants for the interaction of Gln-KPA
with GlnRS through its aminoacyl and AMP modules,
respectively, and where a expresses the cooperativity be-
tween the binding of Gln-KPA at these two sites. If we
assume that a = 1 (i.e., no cooperativity), and that
Ki1 = Ki2, then we obtain an average value of 0.65 mM
for the Ki of Gln-KPA with GlnRS (Fig. 2B). To obtain
information on each of these three parameters, we
sought to evaluate the inhibitory action of two inhibi-
tors which share with Gln-KPA either its aminoacyl
module or its AMP module. Glu-KPA fulfills the condi-
tions for binding to the AMP-binding site of GlnRS and
not to its amino acid-binding site; therefore, its Ki for E.
coli GluRS (2.8 mM; see Fig. 2A and Table 1) is an esti-
mate of Ki2. On the other hand, none of the glutamine
analogs that we tested inhibited GlnRS. Therefore, we
cannot estimate the value of a.
The two main types of stable aa-AMP analogs that have
been characterized as aaRS inhibitors are the aminoalkyl
adenylates (aa-ol-AMP) and the aminoacylsulfamoylade-
nosines (aa-AMS).4 In some cases, large differences be-
tween the Ki of the aa-ol-AMP and of the aa-AMS
specific to a given aaRS have been reported; for instance,
these values for E. coli GluRS are 3 lM and 2.8 nM,
respectively.24,25 The novel Glu-AMP analog presented
here, Glu-KPA, has a Ki of 18 lM. These differences be-

300
C. Balg et al. / Bioorg. Med. Chem. 15 (2007) 295–304
tween aa-AMP analogs which share the same aminoacyl
and adenosine modules but differ by the hinges linking
them may be due to the binding properties of their hinges
to the active site area, and/or their flexibility, including
their vibrational modes on the enzyme.26
added dropwise (30 min) n-BuLi in hexane (2.5 M,
1.51 mL, 3.78 mmol). A solution of ester 5 (600 mg,
1.89 mmol) in THF (5 mL) was added and the solution
was stirred at ꢀ78 ꢁC for 1 h. The reaction was
quenched by dropwise addition of satd aq NH4Cl
(30 mL) and THF was evaporated. The aqueous layer
was extracted with ethyl acetate (3· 50 mL) and the
organic layer was washed with satd aq NH4Cl and brine,
dried over MgSO4, and evaporated. The crude product
was purified by flash chromatography (gradient
The only other aminoacyl-KPA characterized so far as
an inhibitor is Asp-KPA.9 It inhibits E. coli aspartyl-
tRNA synthetase (AspRS) competitively with respect
to aspartate. It has one binding site on this enzyme,
and is much more efficient (Ki = 123 nM) than are
Glu-KPA with GluRS (18 lM) and Gln-KPA with
GlnRS (0.65 mM). As AspRS is a member of the class
II aaRSs, which have no evolutionary linkage with the
class I aaRSs22 to whom GluRS and GlnRS belong,
these inhibitory properties of Asp-KPA suggest that
the linker between its two modules does not interfere
with their optimal binding in the AspRS active site. It
remains to be determined if this is a general property
of the b-ketophosphonates (not yet synthesized) corre-
sponding to the nine other class II aaRSs.
CH2Cl2/EtOAc, 3:2 to 1:1) to give 6 (555 mg, 72%) as
21
D
a colorless oil: ½aꢁ 1.1 (c 3.3, CHCl3); IR (neat) 3600–
3100, 2978, 2929, 1713, 1518, 1250, 1158, 1031 cmꢀ1
;
1H NMR (400 MHz, CDCl3) d 1.26 (s, 18 H), 1.59–
1.69 (m, 1H), 1.79–2.14 (m, 3H), 3.03 (dd, J = 22.0
and 14.4 Hz, 1H), 3.17 (dd, J = 22.6 and 14.4 Hz, 1H),
3.60 (d, J = 11.4 Hz, 3H), 3.61 (d, J = 11.4 Hz, 3H),
4.14–4.19 (m, 1H), 5.53 (d, J = 8.0 Hz, 1H); 13C NMR
(100 MHz, CDCl3) d 25.7, 28.0, 28.2, 31.2, 37.4 (d,
J = 130.4 Hz), 52.9 (d, J = 6.9 Hz), 53.0 (d,
J = 6.1 Hz), 59.6, 79.5, 80.3, 155.6, 172.0, 201.1 (d,
J = 6.1 Hz); 31P NMR (162 MHz, CDCl3) d 23.1;
HRMS (CI, NH3) calcd for C17H33NO8P (M+H)+
410.1944, found 410.1933.
The facts that Glu-KPA and Glu-AMS inhibit E. coli
GluRS much more efficiently than mammalian cytoplas-
mic GluRSs (Table 1) reveal the existence of important
structural differences between the active sites of these
orthologous enzymes. These differences make bacterial
GluRSs promising targets for antimicrobial therapy.
4.3. Methyl b-ketophosphonate 7
A solution of dimethyl b-ketophosphonate 6 (558 mg,
1.36 mmol) in tert-butylamine (30 mL) was heated at re-
flux for 24 h. The solvent was evaporated and the solid
residue was dissolved in CH2Cl2 (50 mL). A solution
of HCl in dioxane (4 M, 0.34 mL, 1.36 mmol) was added
and the mixture was diluted in ethyl acetate (100 mL).
The organic phase was washed with brine, dried over
MgSO4, and evaporated. Flash chromatography (gradi-
4. Experimental
4.1. General
Chemical reagents were purchased from Aldrich–Sigma
Chemical Company. Flash chromatography was carried
out using 40–63 lM (230–400 mesh) silica gel. Optical
rotations were measured using a JASCO DIP-360 digital
polarimeter (c as g of compound per 100 mL). Infrared
spectra were recorded on a Bomem MB-100 spectrome-
ter. NMR spectra were recorded on a Varian Inova
AS400 spectrometer (400 MHz).
ent EtOAc to MeOH/EtOAc, 2:3) provided 7 (507 mg,
21
D
MeOH), IR (KBr) 3600–3100, 2978, 2934, 1703, 1508,
94%) as a white solid: mp 103–105 ꢁC; ½aꢁ 0.2 (c 0.55,
1
1241, 1163, 1046 cmꢀ1; H NMR (400 MHz, CD3OD)
d 1.44–1.46 (m, 18H), 1.74–1.79 (m, 1H), 2.13–2.22 (m,
1H), 2.29–2.33 (m, 2H), 3.00 (dd, J = 21.2 and
12.8 Hz, 1H), 3.18 (dd, J = 21.8 and 12.8 Hz, 1H), 3.59
(d, J = 10.9 Hz, 3H), 4.39–4.44 (m, 1H); 13C NMR
(100 MHz, CD3OD) d 26.9, 28.5, 28.9, 32.7, 52.5 (d,
J = 5.6 Hz), 60.6, 80.9, 81.7, 158.1, 174.2, 206.8 (d,
J = 5.4 Hz); 31P NMR (162 MHz, CD3OD) d 16.6;
LRMS (ESI) 418.1 (M+Na)+.
For enzyme purification and assay, the following com-
pounds were of the highest quality available and were
purchased from the indicated companies: [14C]labeled
L-amino acids from PerkinElmer Life and Analytical
Sciences; unfractionated tRNA from E. coli MRE 600
´
and DNase I from Roche Diagnostics (Laval, Quebec);
4.4. Phosphonate 8
unlabeled L-amino acids, ATP, the reducing agents
2-mercaptoethanol (2-ME) and dithiothreitol (DTT),
and chicken egg white lysozyme from Sigma–Aldrich
Canada Ltd; isopropyl 1-thio-b-D-galactopyranoside
(IPTG), imidazole, and the buffers tris(hydroxymeth-
yl)aminomethane (Tris) and 4-(2-hydroxyethyl)pipera-
zine-1-ethanesulfonic acid (HEPES) from Laboratoire
To a solution of 7 (350 mg, 0.855 mmol) in anhydrous
DMF (2 mL) were added 20,30-isopropylideneadenosine
(408 mg, 1.32 mmol), HBTU (402 mg, 1.06 mmol), and
i-Pr2NEt (0.62 mL, 3.54 mmol), and the solution was
stirred at room temperature for 8.5 h. The reaction mix-
ture was diluted with AcOEt (120 mL), washed with 1 M
HCl, satd aq NaHCO3, brine, and the organic layer was
dried (MgSO4), and evaporated. The crude product was
purified by flash chromatography (EtOAc to 20%
MeOH/AcOEt) to give the coupled product partially
contaminated by 20,30-isopropylideneadenosine. This
mixture was stirred with pyridine/H2O (27 mL/3 mL)
at 55ꢁC for 10 h. The solvents were evaporated and
´
MAT (Beauport, Quebec). Ni–NTA agarose columns
from Qiagen Inc. (Canada).
4.2. Dimethyl b-ketophosphonate 6
To a solution of dimethyl methylphosphonate (0.43 mL,
3.97 mmol) in anhydrous THF (20 mL) at ꢀ78 ꢁC was

C. Balg et al. / Bioorg. Med. Chem. 15 (2007) 295–304
301
the residue was purified by flash chromatography (15%
MeOH/CH2Cl2 to 40% MeOH/CH2Cl2) to give 8
33.7, 52.5, 70.7, 80.2, 127.0, 128.2, 129.0, 144.7, 155.9,
170.9, 172.8.
21
D
(208 mg, 35%) as a white solid: mp 147 ꢁC (dec); ½aꢁ
ꢀ34.7 (c 0.65, MeOH); IR (KBr) 3600–3100, 1709,
4.7. Dimethyl b-ketophosphonate 12
1212, 1156, 1064, 856 cmꢀ1
;
1H NMR (400 MHz,
CD3OD) d 1.35–1.41 (m, 21H), 1.57 (s, 3H), 1.65–1.75
(m, 1H), 2.08–2.17 (m, 1H), 2.19–2.25 (m, 2H), 2.85–
3.18 (m, 2H), 4.04–4.08 (m, 2H), 4.32–4.37 (m, 1H),
4.44 (br s, 1H), 4.85–5.11 (m, 1H), 5.28 (dd, J = 6.0
and 3.2 Hz, 1H), 6.17–6.19 (m, 1H), 8.18 (s, 1H), 8.44
(s, 1H); 13C NMR (100 MHz, CD3OD) d 24.4, 25.5,
26.3, 27.2, 27.6, 31.3, 59.3, 59.4, 64.6, (d, J = 4.8 Hz),
64.7 (d, J = 4.8 Hz), 79.6, 79.7, 80.43, 80.45, 82.0, 82.1,
84.55, 84.56, 85.5 (d, J = 7.6 Hz), 90.6, 113.9, 114.0,
119.0, 140.3, 140.4, 149.2, 149.3, 152.8, 156.1, 156.7,
156.8, 172.9, 205.0; 31P NMR (162 MHz, CD3OD) d
18.0; HRMS (FAB) calcd for C28H44N6O11P (M+H)+
671.2805, found 671.2800.
To
a
solution of dimethyl methylphosphonate
(10.40 mL, 96.00 mmol) in anhydrous THF (70 mL) at
ꢀ78 ꢁC was added dropwise n-BuLi in hexane (1.6 M,
60.00 mL, 96.00 mmol) and the mixture was stirred for
45 min. A solution of ester 11 (2.01 g, 4.00 mmol) in
anhydrous THF (100 mL) was added and the solution
was stirred at ꢀ78 ꢁC for 1 h, then at ꢀ30 ꢁC for another
1 h. The reaction was quenched by the addition of satu-
rated aqueous NH4Cl (50 mL). The reaction mixture
was extracted with ethyl acetate and the organic phase
was washed with water, brine, dried over MgSO4, and
concentrated to 20 mL. Petroleum ether was added
and filtration after 24 h at room temperature provided
12 (1.92 g, 81%) as a white solid: mp 181–182ꢁC, lit.15
21
4.5. Glutamic ketophosphonate 9
183–184ꢁC; ½aꢁ ꢀ18.9 (c 1.08, MeOH); IR (KBr)
D
3600–3100, 3030, 2978, 2957, 1717, 1674, 1650, 1256,
A solution of compound 8 (40 mg, 59.6 lmol) in trifluoro-
acetic acid/water (9 mL/1 mL) was stirred at room tem-
perature for 15 min. The solvents were evaporated
under reduced pressure. The residue was co-evaporated
1035 cmꢀ1 1H NMR (400 MHz, CDCl3) d 1.43 (s,
;
9H), 1.73–1.90 (m, 1H), 2.13–2.29 (m, 1H), 2.30–2.51
(m, 2H), 3.06 (dd, J = 21.8 and 14.4 Hz, 1H), 3.24 (dd,
J = 22.2 and 14.5 Hz, 1H), 3.73 (d, J = 11.1 Hz, 3H),
3.75 (d, J = 11.3 Hz, 3H), 4.27–4.37 (m, 1H), 5.55 (d,
J = 7.1 Hz, 1H), 7.16–7.35 (m, 15H); 13C NMR
with water, then with MeOH/Et2O to give 9 (34 mg,
21
D
1.0, DMF); IR (KBr) 3600– 2800, 1684, 1201,
97%) as a white solid: mp 122 ꢁC (dec); ½aꢁ ꢀ21.4 (c
(100 MHz, CDCl3) d 27.0, 28.3, 33.1, 37.7 (d,
1
1130 cmꢀ1; H NMR (400 MHz, D2O) d 1.87–1.96 (m,
J = 134.1 Hz), 53.1, 60.1, 70.6, 80.3, 127.0, 127.9,
128.8, 144.7, 155.9, 171.2, 201.1 (d, J = 6.2 Hz); 31P
NMR (162 MHz, CDCl3) d 23.0.
1 H), 2.16–2.26 (m, 1H), 2.33–2.40 (m, 2H), 3.96–4.06
(m, 2H), 4.18–4.21 (m, 1H), 4.22–4.25 (m, 1H), 4.29–
4.33 (m, 1H), 4.55–4.58 (m, 1H), 6.00 (d, J = 5.0 Hz,
1H), 8.25 (s, 1H), 8.42 (s, 1H); 13C NMR (100 MHz,
D2O) d 23.6, 29.08, 29.12, 38.2–40.0 (m, C–D coupling,
H–D exchange with D2O), 58.8, 64.0, 64.1, 64.2, 69.9,
70.0, 74.51, 74.54, 83.7 (d, J = 7.6 Hz), 88.3, 88.4, 116.2
(q, J = 289.0 Hz, TFA), 118.53, 118.54, 142.48, 142.53,
144.7, 148.23, 148.24, 149.8, 162.6 (q, J = 35.0 Hz,
TFA), 175.8, 175.9, 200.9, 201.0; 31P NMR (162 MHz,
D2O) d 16.1; LRMS (ESI) 475.1 (M+H)+.
4.8. Methyl b-ketophosphonate 13
To a solution of phosphonate 12 (1.60 g, 2.70 mmol) in
THF/DMF (15 mL/5 mL) were added triethylamine
(2.00 mL) and benzenethiol (1.00 mL). The solution
was stirred at room temperature for 48 h. The solvents
were evaporated and then co-evaporated with toluene.
The crude product was purified by flash chromatogra-
phy (1% Et3N/10% MeOH/ 89% CH2Cl2) to give 13
21
(1.82 g, 98%) as a white solid: mp 156–158 ꢁC; ½aꢁ
4.6. Glutamine methyl ester 11
D
ꢀ0.5 (c 1.04, MeOH); IR (KBr) 3600–3100, 3058,
N-a-Boc-N-c-trityl-L-glutamine 10 (2.62 g, 5.36 mmol)
was dissolved in MeOH/H2O (10:1, 35 mL) and the solu-
tion was titrated to pH 7.0 with a 20% aqueous solution
of Cs2CO3. The solvents were evaporated and the resi-
due co-evaporated with toluene. The solid cesium salt
was stirred with methyl iodide (0.370 mL, 5.90 mmol)
in dry DMF (25 mL) for 1 h at room temperature.
The reaction mixture was diluted with EtOAc
(250 mL) and the organic phase was washed with satd
aq NaHCO3 and brine, dried over MgSO4, evaporated,
and then co-evaporated with toluene to remove residual
DMF. The crude product was purified by flash chroma-
2981, 2948, 2678, 2491, 1697, 1492, 1214, 1167, 1050,
701 cmꢀ1 1H NMR (400 MHz, CDCl3) d 1.27 (t,
;
J = 7.3 Hz, 9H), 1.42 (s, 9H), 1.78–1.94 (m, 1H), 2.21–
2.40 (m, 3H), 2.86 (dd, J = 20.9 and 12.1 Hz, 1H),
2.94–3.06 (m, 6H), 3.34 (dd, J = 21.9 and 12.2 Hz,
1H), 3.59 (d, J = 11.1 Hz, 3H), 4.30–4.40 (m, 1H), 6.77
(d, J = 8.0 Hz, 1H), 7.17–7.33 (m, 15H), 7.39 (s, 1H),
12.01 (br s, 1H); 13C NMR (100 MHz, CDCl3) d 8.5,
28.3, 28.4, 33.8, 41.0 (d, J = 112.4 Hz), 45.6, 52.3, 59.9,
70.4, 80.0, 126.8, 127.8, 128.8, 144.9, 156.3, 171.6,
204.8; 31P NMR (162 MHz, CDCl3) d 13.6; LRMS
(ESI) 682.3 (M+H)+.
tography (CH2Cl2/EtOAc, 10:1) to give 11 (2.37 g, 88%)
as a white solid: mp 153–154 ꢁC, lit.15 153–154 ꢁC; ½aꢁ
21
D
15.2 (c 1.01, CHCl3); IR (KBr) 3600–3100, 2971, 1746,
4.9. Phosphonate 14
1714, 1638, 1210, 710 cmꢀ1
;
1H NMR (400 MHz,
A solution of phosphonate 13 (0.830 g, 1.17 mmol), iso-
propylideneadenosine 1 (0.504 g, 1.64 mmol), DMAP
(0.162 g, 1.33 mmol), and HBTU (1.33 g, 3.51 mmol)
in anhydrous DMF (8 mL) was stirred at room temper-
ature for 3.5 h. The mixture was partitioned between
CDCl3) d 1.44 (s, 9H), 1.80–1.96 (m, 1H), 2.11–2.26
(m, 1H), 2.28–2.49 (m, 2H), 3.72 (s, 3H), 4.27–4.38 (m,
1H), 5.24 (d, J = 7.6 Hz, 1H), 7.13 (s, 1H), 7.17–7.39
(m, 15H); 13C NMR (100 MHz, CDCl3) d 28.3, 29.2,

302
C. Balg et al. / Bioorg. Med. Chem. 15 (2007) 295–304
ethyl acetate and saturated aqueous NH4Cl and the
organic layer was washed with brine, dried over MgSO4,
evaporated, and then co-evaporated with toluene. The
product was purified by flash chromatography
(2%MeOH in CH2Cl2 to 5%MeOH in CH2Cl2). To a
solution of this product in DMF (6 mL) were added tri-
ethylamine (0.50 mL) and benzenethiol (0.25 mL), and
the mixture was stirred at room temperature for 24 h.
The solution was evaporated and then co-evaporated
with toluene. The crude product was purified by flash
chromatography (1%Et3N/20%MeOH/79%CH2Cl2) to
Mini, EDTA-free protease inhibitor cocktail’ (Roche
Diagnostics). DNase I was added to the lysate and the
mixture stirred on ice until the viscosity was significantly
reduced; then, 1 mM 2-ME was added. The lysate was
centrifuged for 45 min at 17500g, and the supernatant
was clarified by passage through a 0.22 lm filter. The en-
zyme was purified by affinity chromatography on a 5 ml
Ni–NTA agarose column equilibrated in 20 mM Tris–
HCl (pH 7.9) and 5 mM imidazole. The column was suc-
cessively washed with this Tris buffer containing 5 and
25 mM imidazole. The enzyme was eluted with 1 M imid-
azole in the same buffer. The washing and elution buffers
also contain 1 mM 2-ME. The GluRS was concentrated
and dialyzed against the storage buffer (20 mM HEPES–
KOH, pH 7.2, 45% glycerol, and 5 mM 2-ME).
give phosphonate 14 (0.923 g, 57% for 2 steps) as a white
21
D
(KBr) 3600–3100, 3058, 2978, 2936, 2678, 1691, 1491,
solid: mp 169–171 ꢁC; ½aꢁ ꢀ25.0 (c 1.04, MeOH); IR
1
1214, 1164, 1064 cmꢀ1; H NMR (400 MHz, CDCl3) d
1.20 (t, J = 7.3 Hz, 9 H), 1.35 (s, 3H), 1.40 (s, 9H),
1.61 (s, 3H), 1.77–1.95 (m, 1H), 2.22–2.41 (m, 3H),
2.74–2.92 (m, 1H), 2.95 (q, J = 7.3 Hz, 6H), 3.20–3.38
(m, 1H), 3.98–4.12 (m, 2H), 4.29–4.43 (m, 1H), 4.45–
4.53 (m, 1H), 5.03–5.11 (m, 1H), 5.13–5.23 (m, 1H),
6.20–6.26 (m, 1H), 6.48 (br s, 2H), 7.10–7.32 (m, 15H),
7.44 (d, J = 9.2 Hz, 1H), 8.29 (s, 1H), 8.37 (s, 1H); 13C
NMR (100 MHz, CDCl3) d 8.5, 25.3, 27.2, 28.4, 29.7,
33.7, 41.5 (d, J = 113.6 Hz), 45.6, 59.9, 65.0, 70.4, 79.6,
81.7, 85.0, 85.8, 90.8, 114.0, 119.4, 126.8, 127.8, 128.8,
139.9, 144.8, 149.5, 152.3, 155.1, 156.3, 171.6, 204.9;
31P NMR (162 MHz, CDCl3) d 12.0; LRMS (ESI)
856.3 (M+H)+.
Glutaminyl-tRNA synthetase (GlnRS): The gene of the
E. coli His-tagged GlnRS cloned in the pQRST plasmid
(kindly provided by Prof. John Perona, Univ. Calif.
Santa Barbara) was over-expressed in BL21[DE3]pLysS
by induction with 1 mM IPTG at A600 nm = 0.5 for 4 h at
37ꢁC. Cells were resuspended in 50 mM HEPES–KOH
buffer (pH 7.2) containing 0.5 M NaCl, 10 mM imidaz-
ole, and 15 mM MgCl2. The lysis and protein extract
preparation were performed as for the GluRS. The puri-
fication and storage of the enzyme were performed as
described.27
Mammalian GluRS and tRNA: A partially purified frac-
tion of bovine liver containing aaRSs was obtained from
Bio S&T Inc. (Montreal, Canada). Transfer RNA
from calf liver was obtained from Sigma–Aldrich Canada
Ltd.
4.10. b-Ketophosphonate 15
A solution of compound 14 (142 mg, 0.242 mmol) in tri-
fluoroacetic acid/water (5.4 mL/0.6 mL) was stirred at
room temperature for 15 min. The solvents were evapo-
rated under reduced pressure and the solid residue co-
evaporated with Et2O/MeOH. The crude product was
dissolved in a minimum of MeOH and precipitated by
the addition of Et2O (10 mL). The precipitate is filtered
4.12. Enzyme activity and inhibition assays
E. coli. GluRS: The tRNA glutamylation reactions were
carried out in 50 mM HEPES–KOH, pH 7.2, 16 mM
MgCl2, 2 mM ATP, 3 mM DTT, 5 lM tRNAGlu in E.
coli tRNA, and 50–400 lM L-[14C(U)]glutamate (adapt-
ed from Tremblay and Lapointe28). The reaction was ini-
tiated by adding the pre-incubated enzyme to a final
concentration of 2 nM. The amount of glutamyl-tRNA
formed was determined by measuring the radioactivity
present in 5% trichloroacetic acid precipitates of reaction
mixture aliquots, as previously described.29 The initial
rates of the reaction were determined by measuring the
amounts of [14C]glutamyl-tRNA formed in 40 ll ali-
quots taken at 2 min intervals over 12 min. The inhibitor,
Glu-KPA 9, freshly prepared as a 25 mM solution in
50 mM HEPES–KOH, pH 7.2 buffer, was added at var-
ious final concentrations (0–200 lM) just before pre-in-
cubating the reaction medium at 37 ꢁC for 2 min. No
significant decrease of inhibitory activity was observed
for Glu-KPA after 10 months in the above buffer at
4 ꢁC. Analysis of this fraction by electrophoresis on poly-
acrylamide gel in the presence of sodium dodecyl sulfate
(SDS–PAGE) (Fig. 5) indicates that this GluRS is pure,
as it migrates as a single band, and has the expected
molecular mass of about 55 kDa with the (His)6 tag.30
and washed with Et2O/MeOH to give 15 as a white solid
21
D
H2O); IR (KBr) 3600–2700, 2977, 2934, 1677, 1203,
(78 mg, 68%): mp 133–135 ꢁC (dec); ½aꢁ ꢀ8.2 (c 0.10,
1132, 1065 cmꢀ1 1H NMR (400 MHz, D2O) d 2.04–
;
2.17 (m, 1H), 2.27–2.53 (m, 3H), 3.03–3.45 (m, 2H),
4.16–4.24 (m, 2H), 4.36–4.44 (m, 2H), 4.49–4.55 (m,
1H), 4.72–4.84 (m, 1H), 6.17 (d, J = 5.1 Hz, 1H), 8.37
(s, 1H), 8.55–8.56 (2s, 1H); 13C NMR (100 MHz,
D2O) d 27.1, 33.1, 42.7 (d, J = 116.8 Hz), 61.7, 66.9,
72.9, 77.2, 86.8, 91.1, 121.5, 145.4, 147.6, 151.2, 152.8,
176.1, 204.0; 31P NMR (162 MHz, D2O) d 15.9; LRMS
(ESI) 474.1 (M+H)+.
4.11. Enzyme purifications
Glutamyl-tRNA synthetase (GluRS): The E. coli His-
tagged GluRS was produced in the E. coli strain
JP1449[DE3]pLysS containing the pET28cTXERS plas-
mid (Dubois, D.Y., unpublished results), by induction
with 0.5 mM IPTG at A600 nm = 0.4 and overnight cul-
ture at room temperature to avoid the formation of
inclusion bodies. Cells were resuspended in 20 mM
Tris–HCl buffer (pH 7.9) containing 5 mM imidazole.
The lysis was performed by the addition of lysozyme
from chicken egg white and one tablet of ‘Complete,
Mammalian GluRS: The aminoacylation reactions were
carried out in 50 mM HEPES–KOH, pH 7.2, 50 mM

C. Balg et al. / Bioorg. Med. Chem. 15 (2007) 295–304
303
at the amino acid concentration corresponding to Km,
in the presence of saturating concentrations of both
ATP and tRNA, and the ratio vi/v0 (where v0 is the rate
in the absence of inhibitor under the same substrate con-
centrations) was plotted as a function of [I]. Curve-fit-
ting of these data using the theoretical functions for
vi/v0 for various types of inhibition16 was made with
the software Kaleidagraph (version 4.0) and was used
to identify the types of inhibition and the Ki values.
For competitive inhibition with one binding site for the
inhibitor, the velocity equation for this system under
rapid equilibrium conditions, and the corresponding
relation for vi/v0 = f ([inhibitor]) are:
V max½Sꢁ
vi ¼
½Sꢁ þ Kmð1 þ K½IꢁÞ
i
vi
½Sꢁ þ Km
¼
½Sꢁ þ Kmð1 þ K½IꢁÞ
v0
i
Figure 5. SDS–PAGE (10%) of the purified fractions of E. coli GluRS
and GlnRS (5 lg per lane).
vi
2
When ½Sꢁ ¼ K
¼
Equation 2
m v0
½Iꢁ
KCl, 10 mM ATP, 8 mM MgCl2, 3 mM DTT,
0.1 mg/ml BSA, and 192 lM L-[14C(U)]glutamate (the
Km value). The partially purified fraction of bovine liver
aaRSs was added in a proportion of 2% v/v. The reac-
tion was initiated after a 20 min pre-incubation period
at 37 ꢁC, by the addition of 100 lM of pre-incubated,
unfractionated calf liver tRNA. Aliquots of 20 ll were
taken at 5 min intervals and treated as described for
the E. coli GluRS assay.
2 þ K
i
This last equation was used for the curve-fittings pre-
sented in Figure 2, except for the left curve of Figure
2B and for the curve in Figure 2A describing mammali-
an GluRS inhibition (Æ).
Acknowledgments
This work was supported by Grant OGP 0009597 from
the Natural Sciences and Engineering Research Council
of Canada (NSERC, to J. L.) and by Grant PR-105-092
from the ‘‘Fonds de recherche sur la nature et les tech-
E. coli. GlnRS: The tRNA glutaminylation reactions were
carried out as described above for GluRS, except for the
following differences in the reaction medium: 50 mM
HEPES–KOH, pH 7.2, 10 mM Mg acetate, 2 mM ATP,
6 lM tRNAGln in E. coli MRE 600 unfractionated tRNA,
and 15–100 lM L-[3,4-3H(N)]glutamine (adapted from
Hong et al.31). The enzyme was added to a final concentra-
tion of 1 nM. The inhibitor, Gln-KPA 15, freshly pre-
pared as a 25 mM solution in 50 mM HEPES–KOH,
pH 7.2, buffer, was added at various final concentrations
(0–1.5 mM). Analysis of this fraction by SDS–PAGE
(Fig. 5) indicates that this GlnRS is pure, as it migrates
as a single band, and has the expected molecular mass
of about 64.2 kDa with the (His)6 tag.32
´
nologies, Quebec’’ (to R. C. and J. L.).
References and notes
1. Ibba, M.; So¨ll, D. Annu. Rev. Biochem. 2000, 69, 617.
2. Martinis, S. A.; Plateau, P.; Cavarelli, J.; Florentz, C.
Biochimie 1999, 81, 683.
3. Eriani, G.; Delarue, M.; Poch, O.; Gangloff, J.; Moras, D.
Nature 1990, 347, 203.
ˆ
4. Chenevert, R.; Bernier, S.; Lapointe, J. In Trans-
lation Mechanisms; Lapointe, J., Brakier Gingras,
L., Eds.; Landes Bioscience/Kluwer Academic:
Georgetown, TX/New York, 2003; pp 416–428.
5. Kim, S.; Lee, S. W.; Choi, E. C.; Choi, S. Y. Appl.
Microbiol. Biotechnol. 2003, 61, 278.
4.13. Determination of the inhibition type and constant
(Ki)
The Km and Kampp for the amino acid substrate were first
calculated from Lineweaver–Burk plots. The Km values
for the amino acids were, respectively, of 113 lM for
the GluRS and 25 lM for the GlnRS. The Ki values
were calculated from the Kampp vs [I] plot. The Ki values
for the inhibitors with respect for the amino acid
substrates were determined by measuring the apparent
Km for the amino acid in the presence of saturating
concentrations of both ATP and tRNA, and of various
fixed concentrations of the inhibitor. Alternatively, the
rate ‘vi’ of the aminoacylation reaction in the presence
of various inhibitor concentrations [I] was determined
6. Hurdle, J. G.; O’Neill, A. J.; Chopra, I. Antimicrob.
Agents Chemother. 2005, 49, 4821.
7. Hugues, J.; Mellow, G. Biochem. J. 1980, 191, 209.
8. Jarvest, R. L.; Berge, J. M.; Berry, V.; Boyd, H. F.;
Brown, M. J.; Elder, J. S.; Forrest, A. K.; Fosberry, A. P.;
Gentry, D. R.; Hibbs, M. J.; Jaworsky, D. D.; O’Hanlon,
P. J.; Pope, A. J.; Rittenhouse, S.; Sheppard, R. J.; Slater-
Radosti, C.; Worby, A. J. Med. Chem. 2002, 45, 1959.
ˆ
9. Bernier, S.; Akochy, P. M.; Lapointe, J.; Chenevert, R.
Bioorg. Med. Chem. 2005, 13, 69.
10. Southgate, C. C. B.; Dixon, H. B. F. Biochem. J. 1978,
175, 461.

304
C. Balg et al. / Bioorg. Med. Chem. 15 (2007) 295–304
11. Chauvel, E. N.; Coric, P.; Llorens-Cortes, C.; Wilk, S.;
Roques, B. P.; Fournie-Zalusky, M. C. J. Med. Chem.
1994, 37, 1339.
12. Gray, M. D. M.; Smith, D. J. H. Tetrahedron Lett. 1980,
21, 859.
13. Vercerkova, H.; Smrt, J. Collect. Czech. Chem. Commun.
1983, 48, 1323.
14. Wang, S. S.; Gisin, B. F.; Winter, D. P.; Makofske, R.;
Kulesha, I. D.; Tzougraki, C.; Meienhofer, J. J. Org.
Chem. 1997, 42, 1286.
21. Gagnon, Y.; Lacoste, L.; Champagne, N.; Lapointe, J.
J. Biol. Chem. 1996, 271, 14856.
22. Woese, C. R.; Olsen, G. J.; Ibba, M.; So¨ll, D. Microbiol.
Mol. Biol. Rev. 2000, 64, 202.
23. Lin, S. X.; Baltzinger, M.; Remy, P. Biochemistry 1983, 22,
681.
´
24. Desjardins, M.; Garneau, S.; Desgagnes, J.; Lacoste, L.;
Yang, F.; Lapointe, J.; Chenevert, R. Bioorg. Chem. 1998,
ˆ
26, 1.
25. Bernier, S.; Dubois, D. Y.; Habegger-Polomat, C.;
ˆ
Gagnon, L. P.; Lapointe, J.; Chenevert, R. J. Enzym.
Inhib. Med. Chem. 2005, 20, 61.
15. Brewer, H.; James, C. A.; Rich, D. H. Org. Lett. 2004, 6,
4779.
16. Segel, I. H. Enzyme kinetics; John Wiley: New York, 1975.
17. Dubois, D. Y.; Lapointe, J.; Sekine, S. I. In The
Aminoacyl-tRNA Synthetases; Ibba, M., Francklyn, C.,
Cusack, S., Eds.; Landes Bioscience: Georgetown, Texas,
2005, chapter 10.
26. Schramm, V. L. Curr. Opin. Struct. Biol. 2005, 15, 604.
27. Uter, N. T.; Gruic-Sovulj, I.; Perona, J. J. J. Biol. Chem.
2005, 280, 23966.
28. Tremblay, T. L.; Lapointe, J. Biochem. Cell. Biol. 1986, 64,
315.
18. Bernier, S.; Dubois, D.; Therrien, M.; Lapointe, J.;
29. Lapointe, J.; Levasseur, S.; Kern, D. Methods Enzymol.
1985, 113, 42.
30. Breton, R.; Papayannopoulos, I.; Biemann, K.; Lapointe,
J. J. Biol. Chem. 1986, 261, 10610.
ˆ
Chenevert, R. Bioorg. Med. Chem. Lett. 2000, 10,
2441.
19. Rath, V. L.; Silvian, L. F.; Beijer, B.; Sproat, B. S.; Steitz,
T. A. Structure 1998, 6, 439.
ˇ
31. Hong, K. W.; Ibba, M.; Weygand-Durasevic, I.; Rogers,
20. Lamour, V.; Quevillon, S.; Diriong, S.; N’Guyen, V. C.;
Lipinski, M.; Mirande, M. Proc. Natl. Acad. Sci. U.S.A.
1994, 91, 8670.
M. J.; Thomann, H. U.; So¨ll, D. EMBO J. 1996, 15, 1983.
32. Rould, M. A.; Perona, J. J.; So¨ll, D.; Steitz, T. A. Science
1989, 246, 1135.