Glutamyl adenylate analogues are inhibitors of glutamyl-tRNA synthetase

Article abstract of DOI:10.1006/bioo.1998.1082

Glutamol adenylate 10 was a competitive inhibitor (K(i) = 3 μM) of glutamyl-tRNA synthetase from Escherichia coli. The N⁶-benzoyl adenine derivative 9 was also an inhibitor (K(i) ~ 60 μM). Replacement of adenine by other bases (purine, cytosine

Full text of DOI:10.1006/bioo.1998.1082

BIOORGANIC CHEMISTRY 26, 1–13 (1998)
ARTICLE NO. BH981082
Glutamyl Adenylate Analogues Are Inhibitors of
Glutamyl-tRNA Synthetase
Michel Desjardins,* Sylvie Garneau,* Julie Desgagnes,* Lucille Lacoste,†
´
Fu Yang,† Jacques Lapointe,† and Robert Chenevert*,1
ˆ
*De´partement de chimie et †De´partement de biochimie, Faculte´ des sciences et de ge´nie,
Universite´ Laval, Que´bec, Canada G1K 7P4
Received June 29, 1997
Glutamol adenylate 10 was a competitive inhibitor (Ki ϭ 3 ȐM) of glutamyl-tRNA synthetase
from Escherichia coli. The N6-benzoyl adenine derivative 9 was also an inhibitor (Ki ȁ
60 ȐM). Replacement of adenine by other bases (purine, cytosine, dihydrocytosine, uridine)
resulted in a more than 1000-fold loss in activity, indicating the important contribution of the
adenine ring to the enzyme binding.
1998 Academic Press
In the first stage of protein biosynthesis, the 20 standard amino acids are esterified
to their cognate transfer RNA (tRNA) by the action of a class of enzymes, the
aminoacyl-tRNA synthetases (aaRS). It has been established that this reaction is
a two-step event (1, 2). In the first step, the amino acid reacts with ATP, with
displacement of pyrophosphate (PPi) to form an enzyme-bound mixed anhydride
(aminoacyl adenylate). In this intermediate, the high-energy anhydride bond acti-
vates the carboxyl group of the amino acid. In the second step, the activated
amino acid is transferred to the CCA end of the corresponding tRNA to form the
aminoacyl-tRNA and AMP. This transfer is a nucleophilic attack of the 2Ј or 3Ј
ribose hydroxyl group of the terminal AMP residue at the 3Ј end of the tRNA on
the activated carboxyl group of the intermediate. The two steps are the following:
Mg2
ϩ
Amino acid ϩ ATP ϩ aaRS
(aa-AMP)aaRS ϩ tRNA
(aa-AMP)aaRS ϩ PPi ,
aa-tRNA ϩ AMP ϩ aaRS.
The resulting aminoacyl-tRNAs are the major activated forms of amino acids in
the living cells, and they are used mostly for protein biosynthesis on the ribosomes.
Selective inhibition of bacterial aaRS has proved to be a successful strategy for the
production of anti-bacterial agents (3). Pseudomonic acid (generic name: mupi-
rocin), isolated from Pseudomonas fluorescens, is a highly potent inhibitor of bacte-
rial isoleucyl-tRNA synthetases (4, 5). This antibiotic shows a very high selectivity
in favor of prokaryote forms of this enzyme and plays an important clinical role.
1 To whom correspondence should be addressed. Fax: (418) 656-7916. E-mail: robert.chenevert@chm.
ulaval.ca.
1
0045-2068/98 $25.00
Copyright 1998 by Academic Press
All rights of reproduction in any form reserved.

2
DESJARDINS ET AL.
SCHEME 1. Reagents: (a) NaHCO3 , CbzCl, H2O : THF (1 : 1); (b) Et3N, ethyl chloroformate (Ϫ10ЊC),
NaBH4 (0ЊC), H2O, THF.
Synthetic analogues of aminoacyl adenylates are inhibitors of the aminoacyl-
tRNA synthetase (6–12). Prolylsulfamoyladenosine is a potent inhibitor of both
human and Escherichia coli prolyl-tRNA synthetase (6). This compound is a mimic
of the mixed anhydride intermediate in which the phosphate group is replaced by
the isosteric but more stable sulfamoyl linkage (7, 8). Replacement of the amino acid
by the corresponding amino alcohol produced stable esters (aminoalkyl adenylates)
which are good inhibitors of the cognate aaRS (9, 10). Aminophosphonyl adenylates
are also inhibitors of aaRS (11, 12).
Glutamyl-tRNA synthetase (GluRS) has the characteristic, which is common
also to the glutaminyl- and arginyl-tRNA synthetases from E. coli and all other
organisms in which these enzymes have been studied, of requiring the presence of
its cognate tRNA to catalyze the activation of its amino acid substrate. With the
aim of obtaining inhibitors of GluRS, we have synthesized analogues of glutamyl
adenylate and tested their properties in the aminoacylation reaction of tRNAGlu
catalyzed by E. coli GluRS.
RESULTS AND DISCUSSION
Synthesis of Glutamyl Adenylate Analogues
The commercially available -glutamic acid Ͳ-benzyl ester 1 was converted into
L
the corresponding N-benzyloxycarbonyl (N-Cbz) derivative 2 under standard condi-
tions. Reduction of 2 via a mixed carbonic anhydride with sodium borohydride
gave alcohol 3 (Scheme 1).
N6-Benzoyl-2Ј,3Ј-O-isopropylideneadenosine 4a was prepared by benzoylation
of commercially available 2Ј,3Ј-isopropylideneadenosine. The phosphoramidite-
phosphite triester approach was used for the condensation between alcohol 3 and
adenosine derivative 4a (Scheme 2). Compound 4a was first phosphorylated with
N,N-diisopropylmethylphosphonamidic chloride in the presence of N,N-diiso-
propylethylamine in dry CH2Cl2 to give 5a. Phosphoramidite 5a was coupled with
3 in dry acetonitrile using 1H-tetrazole as an activating agent and then the phosphite
triester was oxidized to phosphate triester 6a by treatment with iodine. This phos-
photriester was deprotected by sequential treatment with wet trifluoroacetic acid
(hydrolysis of the isopropylidene acetal group) to give 7a and then with sodium
iodide in butanone to give phosphodiester 8a. Other compounds (4b, c, d to 8b, c,
d) were prepared according to the same reaction sequence (Scheme 2).

INHIBITION OF GLUTAMYL-tRNA SYNTHETASE
3
SCHEME 2. Reagents: (a) N,N-diisopropylmethylphosphonamidic chloride, i-Pr2NEt, CH2Cl2; (b)
(i) 3, tetrazole, MeCN, (ii) I2 , H2O : THF : pyr (1 : 1 : 1); (c) CF3CO2H : H2O (9 : 1); (d) Nal, 2-butanone.
Hydrogenolysis of the benzyl ester and the benzyloxycarbonyl group of 8a in the
presence of a 10% palladium-on-charcoal catalyst provided compound 9 with the
N-benzoyl protecting group on the purine base. This group was removed by treat-
ment with NH4OH to give compound 10 (Scheme 3). With compounds 8b–8d,
reduction of the bases was observed during the cleavage of protecting groups with
molecular hydrogen. For instance, hydrogenation of 8b with hydrogen followed by
aminolysis of the N-benzoyl group gave the dihydrocytosine derivative 11 as the sole
product (Scheme 4). Transfer hydrogenation with 1,4-cyclohexadiene as hydrogen
donor was used in the purine, cytosine, and uracyl series (8b–8d to 12–14, Schemes
4 and 5).
In structures 5, 6, and 7, the phosphorus is a center of chirality and these com-
pounds were obtained as a mixture of diastereoisomers. The nonequivalence (chemi-
cal shift difference of some groups) was observed in 1H, 13C, and 31P NMR spectra.
Inhibition of GluRS by Compounds 9–14
L
-Glutamol-AMP 10 inhibits E. coli GluRS in a competitive fashion (Fig. 1),
since the maximum velocity Vmax is unchanged, whereas the apparent Kmapp is in-
creased for increasing concentrations of this inhibitor [I]. The replot of Kmapp versus

4
DESJARDINS ET AL.
SCHEME 3. Reagents: (a) H2 , Pd/C, H2O; (b) NH4OH.
[I] gives the Ki value, which is 3 Ϯ 1 ȐM for the two substrates studied, glutamate
and ATP (Fig. 2). There is no inhibition of E. coli glutaminyl-tRNA synthetase by
this compound.
Other compounds (9, 11–14) inhibit E. coli GluRS slightly. The Ki values are
listed in Table 1. The N6-benzoyl adenine derivative 9 was also an inhibitor (Ki ȁ
SCHEME 4. Reagents: (a) (i) H2 , Pd/C, H2O, (ii) NH4OH; (b) (i) Pd/C, 1,4-cyclohexadiene, EtOH,
(ii) NH4OH.

INHIBITION OF GLUTAMYL-tRNA SYNTHETASE
5
SCHEME 5. Reagents: (a) H2 , 1,4-cyclohexadiene, EtOH.
FIG. 1. Determination of the apparent Km for glutamate of E. coli GluRS in the presence of different
fixed concentrations of inhibitor 10: 0 ȐM (᭺), 4 ȐM (ᮀ), 6 ȐM (᭛), and 8 ȐM (᭝).

6
DESJARDINS ET AL.
FIG. 2. Determination of the Ki values of inhibitor 10 for E. coli GluRS, with respect to glutamate
(A) and ATP (B).
60 ȐM). Replacement of adenine by dihydrocytidine (11), cytidine (12), purine
(13), and uridine (14) resulted in a three order of magnitude loss in activity (0.63
mM Ͻ Ki Ͻ 16.7 mM), indicating the important contribution of the adenine ring
to the enzyme binding.
In addition to gaining mechanistic information about E. coli GluRS, specific
inhibitors such as 10 should facilitate the crystallization of the enzyme.
EXPERIMENTAL
Synthesis of Inhibitors
N-Benzyloxycarbonyl-5-O-benzyl-
L-glutamic acid (2). To a mixture of Ͳ-benzyl
L-glutamate (1) (2.46 g, 10.4 mmol) and NaHCO3 (1.76 g, 20.8 mmol) in 20 mL of
water was added a solution of benzyl chloroformate (1.57 mL, 11 mmol) in 20 mL
TABLE 1
Inhibition of E. coli GluRS by Glutamyl
Adenylate Analogues
Compound (base)
Ki (ȐM)
9 (N6-benzoyladenine)
10 (adenine)
60
3
11 (dihydrocytidine)
12 (cytidine)
16,700
630
13 (purine)
14 (uridine)
4,100
2,750

INHIBITION OF GLUTAMYL-tRNA SYNTHETASE
7
of THF. The mixture was stirred for 18 h at room temperature. The organic solvent
(THF) was removed under reduced pressure. The aqueous layer was washed with
EtOAc, acidified to pH 3 with 10% aqueous citric acid, and then extracted with
EtOAc. The combined organic extracts were washed with brine, dried, and concen-
trated in vacuo. The crude product was recrystallized from CCl4 to yield 2 as a
white solid (3.40 g, 88%). IR (KBr) 3600–2800, 3300, 1730–1690, 1535, 1460, 1440,
1
1390, 1330, 1315, 1250, 1170, 1085, 1065, 1040, 950, 735, 690 cmϪ ; 1H NMR (CDCl3)
2.05 (m, 1H), 2.25 (m, 1H), 2.46 (m, 2H), 4.42 (m, 1H), 5.07 (s, 4H), 5.64 (d, J ϭ
7.9 Hz, 1H), 7.30 (ls, 10H), 10.67 (ls, 1H); 13C NMR (CDCl3) 27.14, 30.19, 53.12, 66.57,
67.17, 128.02, 128.16, 128.22, 128.45, 128.46, 135.52, 135.90, 156.21, 172.89, 175.89.
Benzyl 4-[(benzyloxycarbonyl)amino]-5-hydroxypentanoate (3). Ethyl chloro-
formate (1.26 mL, 13.26 mmol) was added at 10ЊC to a stirred solution of acid 2
(2.46 g, 6.63 mmol) and Et3N (0.95 mL, 6.63 mmol) in dry THF (22 mL). The
mixture was stirred at 10ЊC for 30 min. Then, the precipitate was filtered, and the
filtrate was added during 15 min at 0ЊC to a solution of NaBH4 (744 mg, 19.9 mmol)
in 22 mL of H2O : THF (1 : 4). The mixture was stirred for 4 h at room temperature.
The mixture was acidified with 1 N HCl, and THF was evaporated. The aqueous
layer was extracted with EtOAc. The organic layer was washed with saturated
NaHCO3 , water, and brine; dried (MgSO4); and concentrated in vacuo. The crude
product was purified by flash chromatography (CH2Cl2 : EtOAc, 3 : 1) to yield 3 as
a white solid (1.90 g, 80%). IR (KBr) 3600–3200, 3060, 3020, 2940, 1730–1680, 1525,
1
1450, 1240, 1160, 1065, 905, 725, 690 cmϪ ; 1H NMR (CDCl3) 1.85 (m, 2H), 2.45 (t,
J ϭ 7.2 Hz, 2H), 2.60 (m, 1H), 3.50–3.80 (m, 3H), 5.06 (s, 2H), 5.09 (s, 2H), 5.18
(d, J ϭ 8.3 Hz, 1H), 7.30 (m, 10H); 13C NMR (CDCl3) 26.13, 30.71, 52.56, 64.57, 66.39,
66.70, 127.94, 128.02, 128.13, 128.18, 128.40, 128.46, 135.64, 136.25, 156.54, 173.44.
Compound 5a. N6-Benzoyl-2Ј,3Ј-isopropylideneadenosine (4a) (4.84 g, 10 mmol)
was dissolved in dry CH2Cl2 (45 mL) containing i-Pr2EtN (8 mL, 46 mmol). N,N-
Diisopropylmethylphosphonamidic chloride (3.6 mL, 18 mmol) was added rapidly
with a syringe to the magnetically stirred solution at room temperature under dry
atmosphere. After 12 min the solution was poured into EtOAc. The organic solution
was washed with brine, dried (MgSO4), and evaporated. The crude product was
purified by flash chromatography on silica gel (hexane : CHCl3 : Et3N, 12 : 8 : 1) to
yield 5a (4.98 g, 74%). IR (KBr) 3000–2800, 2235, 1700, 1605, 1580, 1505, 1450,
1
1
1240, 1205, 1175, 1150, 1070, 1020, 970, 905, 790, 725, 640 cmϪ ; H NMR (CDCl3)
1.08–1.17 (m, 12H), 1.41 and 1.66 (2s, 6H), 3.36 and 3.41 (2d, JP-H ϭ 13.2 Hz),
3.50–3.60 (m, 2H), 3.82 (m, 2H), 4.56 (m, 1H), 5.02 (m, 1H), 5.32 (m, 1H), 6.26 and
6.29 (2d, J ϭ 2.8 Hz, 1H), 7.50–7.65 (m, 3H), 8.02 (d, J ϭ 7.4 Hz, 2H), 8.34 and
8.40 (2s, 1H), 8.84 and 8.85 (2s, 1H), 8.98 (ls, 1H); 13C NMR (CDCl3) 24.50, 25.20,
27.09, 42.62, 50.40, 50.64, 63.05, 63.38, 81.60, 81.80, 84.58, 84.71, 86.19, 86.31, 91.30,
91.40, 114.05, 114.16, 123.13, 123.21, 127.77, 128.59, 132.50, 133.64, 141.79, 141.86,
149.43, 151.22, 151.33, 152.60, 164.53; 31P NMR (CDCl3) 150.24, 150.29.
Compound 5b. Yield 78%. IR (KBr) 2950, 2225, 1650, 1550, 1370, 1300, 1250,
1
1060, 970, 900, 865, 780, 720, 640 cmϪ ; 1H NMR (CDCl3) 1.04–1.13 (m, 12H), 1.30
(s, 3H), 1.53 (s, 3H), 3.31 and 3.36 (2d, JP-H ϭ 13.5 Hz, 3H), 3.40–3.56 (m, 2H),
3.70–3.96 (2m, 2H), 4.44 (m, 1H), 4.75 (d, J ϭ 3.0 Hz, 2H), 5.95 (d, J ϭ 6.1 Hz,
1H), 7.39–7.56 (m, 4H), 7.83 (d, J ϭ 7.3 Hz, 2H), 8.13 and 8.15 (2d, J ϭ 3.7 Hz,

8
DESJARDINS ET AL.
1H); 13C NMR (CDCl3) 24.45, 24.49, 24.53, 24.58, 24.68, 25.13, 25.17, 27.11, 42.68,
42.84, 50.40, 50.65, 62.94, 63.46, 80.37, 80.50, 85.93, 86.08, 87.08, 94.03, 94.12, 96.12,
113.74, 127.42, 128.86, 132.97, 133.08, 145.27, 147.41, 154.50, 162.19, 166.74; 31P NMR
(CDCl3) 150.28, 150.59.
Compound 5c. Yield 76%. IR (KBr) 2960, 1595, 1490, 1450, 1380, 1300, 1200,
1
1155, 1110, 1075, 1025, 975, 910, 865, 790, 730, 635 cmϪ ; 1H NMR (CDCl3) 1.04–1.16
(m, 12H), 1.39 (s, 3H), 1.64 (s, 3H), 3.34 and 3.39 (2d, JP-H ϭ 13.4 Hz, 3H), 3.48
(m, 2H), 3.74 to 3.95 (2m, 2H), 4.55 (q, J ϭ 3.1, 1H), 5.00 (2t, J ϭ 2.5 Hz, 1H),
5.27 (2t, J ϭ 5.9 Hz, 1H), 6.28 and 6.31 (2d, J ϭ 2.8 Hz, 1H), 8.33 and 8.47 (2s,
1H), 8.98 and 8.99 (2s, 1H), 9.13 and 9.14 (2s, 1H); 13C NMR (CDCl3) 24.61, 25.20,
27.12, 42.63, 50.52, 63.49, 81.60, 81.81, 84.78, 86.16, 86.32, 91.09, 91.21, 114.08, 114.17,
134.49, 144.04, 144.10, 148.50, 150.00, 152.62; 31P NMR (CDCl3) 150.31, 150.47.
Compound 5d. Yield 75%. IR (KBr) 3180, 3050, 2960, 1690, 1455, 1380, 1270,
1
1
1215, 1185, 1155, 1110, 1075, 1025, 975, 910, 855, 805, 730 cmϪ ; H NMR (CDCl3)
1.08–1.20 (m, 12H), 1.28 (s, 3H), 1.52 (s, 3H), 3.32 and 3.36 (2d, JP-H ϭ 13.4 Hz,
3H), 3.39–3.61 (m, 2H), 3.73 and 3.88 (2m, 2H), 4.32 (m, 1H), 4.67 (2m, 1H), 4.76
(2m, 1H), 5.61 and 5.64 (2d, J ϭ 1.2 Hz, 1H), 5.93 and 5.94 (2d, J ϭ 8.1 Hz, 1H)
7.65 and 7.70 (2d, J ϭ 8.1 Hz, 1H); 13C NMR (CDCl3) 24.47, 24.61, 25.15, 25.20,
27.06, 27.09, 42.61, 42.77, 50.39, 50.64, 62.92, 63.37, 80.55, 80.65, 84.72, 84.85, 85.47,
85.59, 91.70, 91.88, 102.13, 114.01, 114.03, 140.91, 140.99, 150.19, 163.43; 31P NMR
(CDCl3) 150.48, 150.59.
Compound 6a. Aminoalcohol 3 (0.426 g, 1.19 mmol), phosphoramidite 5a (1.338
g, 2.34 mmol), and tetrazole (0.404 g, 5.77 mmol) were dissolved in dry CH3CN.
The mixture was stirred at room temperature under dry nitrogen atmosphere for
5 h. Then a solution of iodine (0.206 g) in H2O : THF : pyridine (1 : 1 : 1, 3 mL) was
added until no decoloration was observed. The mixture was diluted with CH2Cl2 ,
and the organic layer was washed with brine, dried (MgSO4), and evaporated. The
crude product was purified by flash chromatography on silica gel (EtOAc : MeOH,
99 : 1) to give 6a (782 mg, 78%). IR (KBr) 3600–3100, 3040, 2960, 1725, 1605, 1575,
1
1510, 1445, 1250, 1080–1000, 855, 790 cmϪ ; 1H NMR (CDCl3) 1.38 (s, 3H), 1.60 (s,
3H), 1.84 (m, 2H), 2.41 and 2.45 (2d, J ϭ 7.0 Hz, 2H), 3.61 and 3.63 (2d, JP-H
ϭ
11.0 Hz, 3H), 3.85–4.20 (m, 5H), 4.48 (m, 1H), 5.00 (d, J ϭ 3 Hz, 1H), 5.04 (s, 4H),
5.44 (m, 1H), 6.20 (m, 1H), 7.26 (m, 10H), 7.46 (m, 2H), 7.57 (m, 1H), 8.16 and
8.18 (2s, 1H), 8.79 (s, 1H), 9.03 (m, 1H); 13C NMR (CDCl3) 25.16, 26.93, 25.98,
30.44, 50.49, 54.51, 66.29, 66.49, 66.57, 66.77, 69.18, 80.97, 81.42, 84.09, 84.25, 85.27,
85.36, 90.74, 90.90, 114.03, 114.58, 123.53, 123.60, 127.78, 127.87, 128.04, 128.10,
128.32, 128.39, 132.57, 135.58, 141.98, 142.51, 149.69, 150.16, 150.57, 151.14, 152.06,
152.55, 155.97, 164.81, 172.66; 31P NMR (CDCl3) 0.85, 0.87.
Compound 6b. Yield 89%. IR (KBr) 3250, 3040, 2940, 1700, 1660, 1625, 1550,
1
1
1480, 1380, 1320, 1250, 1165, 1025, 895, 860, 785, 730, 695 cmϪ ; H NMR (CDCl3)
1.31 (s, 3H), 1.54 (s, 3H), 1.92 (m, 2H), 2.53 (m, 2H), 3.67 and 3.71 (2d, JP-H ϭ 11.1
Hz, 3H), 3.96–4.14 (2m, 3H), 4.25–4.36 (2m, 2H), 4.90 (m, 1H), 5.00–5.10 (m, 5H),
5.63 (d, J ϭ 7.8 Hz, 1H), 5.80 and 6.01 (2m, 1H), 7.28 (m, 10H), 7.48–7.59 (m, 4H),
7.71 (d, J ϭ 7.3 Hz, 1H), 7.92 (d, J ϭ 3.7 Hz, 2H), 9.04 (s, 1H); 13C NMR (CDCl3)
26.11, 25.06, 25.13, 26.89, 26.96, 30.73, 50.70, 54.49, 66.29, 66.58, 66.90, 69.13, 69.44,
80.90, 81.08, 84.62, 86.71, 96.91, 97.54, 114.13, 127.71, 127.93, 128.10, 128.35, 128.40,

INHIBITION OF GLUTAMYL-tRNA SYNTHETASE
9
128.80, 133.07, 135.62, 147.07, 156.03, 162.06, 164.81, 169.12, 173.01; 31P NMR
(CDCl3) 0.27, 0.76.
Compound 6c. Yield 60%. IR (KBr) 2930, 2250, 1710, 1585, 1495, 1450, 1390,
1
1
1250, 1205, 1050, 905, 725 cmϪ ; H NMR (CDCl3 1.38 (s, 3H), 1.61 (s, 3H), 1.84,
(m, 2H), 2.42 and 2.47 (2d, J ϭ 7.4 Hz, 2H), 3.58 and 3.63 (2d, JP-H ϭ 11.3 Hz, 3H),
3.80–3.86 (m, 1H), 3.88–4.10 (m, 2H), 4.13–4.26 (m, 2H), 4.49 (m, 1H), 5.06 (m,
1H), 5.08 (s, 4H), 5.40 (d, J ϭ 6.2 Hz, 1H), 6.22 (d, J ϭ 4.5 Hz, 1H), 7.31 (m, 10H),
8.24 (d, J ϭ 4.8 Hz, 1H), 8.99 (d, J ϭ 1.9 Hz, 1H), 9.09 (d, J ϭ 8.0 Hz, 1H);
13C NMR (CDCl3) 25.18, 26.12, 26.97, 30.48, 50.49, 54.54, 66.54, 67.04, 69.33, 80.92,
81.05, 84.00, 84.19, 85.22, 90.57, 90.97, 114.69, 114.77, 127.96, 128.03, 128.10, 128.14,
128.39, 128.43, 134.52, 135.62, 136.18, 144.10, 148.94, 152.68, 155.99, 172.60; 31P NMR
(CDCl3) 0.91, 0.98.
Compound 6d. Yield 90%. IR (KBr) 3040, 2960, 1690, 1525, 1450, 1375, 1250,
1
1
1160, 1030, 900, 850, 725, 690 cmϪ ; H NMR (CDCl3) 1.30 (s, 3H), 1.52 (s, 3H),
1.87 (m, 2H), 2.46 (t, J ϭ 7.1 Hz, 2H), 3.68 and 3.71 (2d, JP-H ϭ 11.2 Hz, 3H),
3.86–3.98 (m, 1H), 4.02–4.26 (2m, 5H), 4.86 (m, 1H), 5.00 (d, J ϭ 5.2 Hz, 1H), 5.05
(s, 2H), 5.08 (s, 2H), 5.56 (m, 1H), 5.62 and 5.65 (2d, J ϭ 2.8 Hz, 1H), 5.67 and
5.69 (2d, J ϭ 8.1 Hz, 1H), 7.30 (m, 10H), 7.42 (d, J ϭ 8.1 Hz, 1H), 8.79 (s, 1H),
9.76 (s, 1H); 13C NMR (CDCl3) 25.05, 25.10, 26.11, 26.89, 27.11, 30.56, 50.60, 54.65,
62.35, 66.60, 69.39, 80.29, 80.70, 83.75, 84.16, 86.81, 95.22, 102.34, 102.59, 114.19,
114.38, 127.92, 128.03, 128.11, 128.16, 128.39, 128.44, 135.56, 136.21, 142.60, 150.02,
156.07, 163.27, 173.02; 31P NMR (CDCl3) 0.36, 0.52.
Compound 7a. Phosphate 6a (0.265 g, 0.314 mmol) was dissolved in 90% (v/v)
aqueous trifluoroacetic acid (10 mL) and the solution was stirred at room tempera-
ture for 10 min. The solvent was removed in vacuo and the residue was purified
by flash chromatography on silica gel (EtOAc : MeOH, 94 : 6) to yield 7a (236 mg,
1
93%). IR (KBr) 3700–2900, 1695, 1610, 1580, 1510, 1450, 1250, 1025, 630, 595 cmϪ ;
1H NMR (CD3OD) 1.68 and 1.81 (2m, 1H), 2.39 (m, 2H), 3.64 and 3.68 (2d, JP-H ϭ
11.3 Hz, 3H), 3.75–4.45 (m, 6H), 4.47 (m, 1H), 4.77 (m, 1H), 5.00 (s, 2H), 5.03 (s,
2H), 6.15 (m, 1H), 7.27 (m, 10H), 7.50 (d, J ϭ 7.5 Hz, 2H), 7.61 (m, 1H), 8.04 (m,
2H), 8.52 and 8.53 (2s, 1H), 8.69 (s, 1H); 13C NMR (CD3OD) 27.10, 31.55, 51.97,
52.05, 55.80, 67.52, 67.70, 68.47, 70.73, 71.43, 75.52, 84.18, 90.68, 90.76, 125.02, 128.95,
129.14, 129.70, 129.91, 134.94, 137.54, 138.24, 144.32, 151.11, 153.18, 153.42, 158.53,
168.04, 174.50; 31P NMR (CD3OD) 0.81, 0.83.
Compound 7b. Yield 96%. IR (KBr) 3620–3000, 2940, 2480, 1670, 1450, 1360,
1
1
1255, 1195, 1135, 1035, 835, 795, 715, 690 cmϪ ; H NMR (CD3OD) 1.72 and 1.89
(2m, 2H), 2.44 (t, J ϭ 4.3 Hz, 2H), 3.74 and 3.77 (2d, JP-H ϭ 11.1 Hz, 3H), 3.88 (m,
1H), 4.05 (m, 2H), 4.16–4.41 (m, 5H), 5.02 (m, 4H), 5.89 (d, J ϭ 2.6 Hz, 1H), 7.27
(m, 10H), 7.43–7.62 (m, 4H), 7.94 (d, J ϭ 7.5 Hz, 2H), 8.23 (2d, J ϭ 7.4 Hz, 1H);
13C NMR 26.98, 31.34, 51.99, 55.66, 67.41, 67.60, 67.73, 70.15, 70.78, 76.02, 83.50,
93.74, 98.76, 128.80, 129.03, 129.14, 129.18, 129.31, 129.55, 129.89, 134.32, 134.44,
137.50, 138.22, 146.31, 157.64, 158.56, 162.05, 162.53, 163.01, 163.48, 164.57, 169.12,
174.49; 31P NMR 0.82, 0.86.
Compound 7c. Yield 97%. IR (KBr) 3250, 2920, 1675, 1600, 1450, 1200, 1135,
1
1040, 835, 795 cmϪ ; 1H NMR (CD3OD) 1.69 and 1.84 (2m, 2H), 2.42 and 2.47 (2d,
J ϭ 7.4 Hz, 2H), 3.66 and 3.68 (2d, JP-H ϭ 11.2 Hz, 3H), 3.70–3.82 (m, 1H), 3.84–4.00

10
DESJARDINS ET AL.
(m, 2H), 4.26–4.40 (m, 3H), 4.47 (t, J ϭ 4.9 Hz, 1H), 4.81 (m, 1H), 5.02 (s, 2H),
5.06 (s, 2H), 6.17 (d, J ϭ 4.3 Hz, 1H), 7.29 (m, 10H), 8.65 (s, 1H), 8.94 (s, 1H), 9.07
(s, 1H); 13C NMR (CD3OD) 26.94, 31.33, 50.50, 54.55, 67.37, 67.54, 71.41, 75.02,
84.12, 84.22, 90.46, 90.55, 128.80, 128.99, 129.16, 129.46, 129.53, 135.57, 146.99, 149.05,
153.53, 156.00, 172.60; 31P NMR (CD3OD) 0.94, 0.98.
Compound 7d. Yield 97%. IR (KBr) 3600–3150, 3040, 2930, 1685, 1525, 1450,
1
1380, 1250, 1190, 1130, 1030, 825, 715, 690 cmϪ ; 1H NMR (CD3OD) 1.71 and 1.85
(2m, 2H), 2.45 (t, J ϭ 6.5 Hz, 2H), 3.73 and 3.75 (2d, JP-H ϭ 11.1 Hz, 3H), 3.86–3.95
(m, 1H), 4.00–4.30 (4m, 7H), 5.05 (s, 2H), 5.08 (s, 2H), 5.72 (d, J ϭ 8.1 Hz, 1H),
5.86 (d, J ϭ 3.0 Hz, 1H), 7.51 (m, 10H), 7.65 and 7.66 (2d, J ϭ 8.1 Hz, 1H); 13C
NMR (CD3OD) 27.00, 31.35, 51.92, 55.59, 62.31, 67.51, 70.76, 71.40, 75.01, 75.75,
83.39, 83.48, 86.43, 90.74, 91.61, 103.06, 128.88, 129.04, 129.21, 129.52, 129.56, 137.58,
138.29, 142.30, 142.73, 152.25, 158.56, 166.00, 174.43; 31P NMR (CD3OD) 0.93, 0.95.
Compound 8a. To a stirred solution of phosphate 7a (456 mg, 0.57 mmol) in 20
mL of dry 2-butanone was added NaI (262 mg, 1.75 mmol), and the reaction mixture
was refluxed for 4 h under dry atmosphere. After evaporation of the solvent in vacuo,
the residue was purified by flash chromatography on silica gel (acetone : MeOH, 4 : 1)
to yield 8a (363 mg, 79%). IR (KBr) 3700–2800, 1700, 1610, 1580, 1510, 1450, 1230,
1
1170, 1070, 850, 815, 790, 730, 690 cmϪ ; 1H NMR (CD3OD) 1.73 (m, 1H), 1.88 (m,
1H), 2.38 (m, 2H), 3.82 (m, 2H), 4.10 (m, 2H), 4.23 (m, 1H), 4.42 (m, 1H), 4.71 (t,
J ϭ 5.0 Hz, 1H), 4.96 (s, 2H), 5.02 (s, 2H), 6.22 (d, J ϭ 5.2 Hz, 1H), 7.24 (m, 10H),
7.52 (m, 2H), 7.62 (d, J ϭ 7.3 Hz, 1H), 8.06 (d, J ϭ 7.3 Hz, 2H), 8.69 (s, 1H), 8.78
(s, 1H); 13C NMR (CD3OD) 27.73, 31.70, 52.28, 52.33, 66.04, 67.37, 67.55, 72.09,
76.35, 85.74, 89.65, 124.52, 128.80, 128.99, 129.19, 129.54, 129.60, 129.85, 134.10,
134.68, 137.61, 138.85, 144.33, 150.90, 153.45, 153.61, 158.64, 168.32, 174.81; 31P NMR
(CD3OD) Ϫ0.94.
Compound 8b. Yield 78%. IR (KBr) 3600–3000, 2930, 1685, 1640, 1565, 1480,
1
1420, 1240, 1205, 1175, 1080, 780, 690 cmϪ ; 1H NMR (CD3OD) 1.76 (m, 1H), 1.93
(m, 1H), 2.43 (t, J ϭ 4.3 Hz, 2H), 3.87–4.23 (2m, 8H), 5.00 (s, 2H), 5.02 (s, 2H),
5.96 (d, J ϭ 2.7 Hz, 1H), 7.28 (m, 10H), 7.49–7.57 (m, 4H), 7.93 (d, J ϭ 7.6 Hz,
2H), 8.49 (d, J ϭ 7.2 Hz, 1H); 13C NMR (CD3OD) 27.72, 31.62, 52.00, 67.29, 67.54,
70.21, 70.75, 76.62, 83.47, 92.69, 98.96, 128.76, 128.94, 129.11, 129.23, 129.46, 129.51,
129.82, 134.10, 134.69, 137.59, 146.62, 158.12, 159.10, 164.70, 168.95, 174.71; 31P NMR
(CD3OD) 0.61.
Compound 8c. Yield 76%. IR (KBr) 3640–3000, 2930, 1710, 1590, 1530, 1495,
1
1
1450, 1405, 1350, 1220, 1080, 905, 830, 785, 730, 695 cmϪ ; H NMR (CD3OD) 1.67
and 1.92 (2m, 2H), 2.38 (t, J ϭ 7.4 Hz, 2H), 3.81 (m, 3H), 4.10 (m, 1H), 4.19 (m,
2H), 4.42 (t, J ϭ 4.4 Hz, 1H), 4.65 (t, J ϭ 5.3 Hz, 1H), 5.00 (s, 2H), 5.03 (s, 2H),
6.21 (d, J ϭ 4.2 Hz, 1H), 7.27 (m, 10H), 8.90 (s, 1H), 8.92 (s, 1H), 9.04 (s, 1H); 13
C
NMR (CD3OD) 27.56, 31.53, 50.51, 67.31, 67.66, 71.65, 76.03, 84.10, 84.23, 89.91,
128.75, 128.98, 129.12, 129.46, 129.52, 135.14, 146.92, 148.79, 153.54, 156.03, 174.64;
31P NMR (CD3OD) Ϫ1.57.
Compound 8d. Yield 80%. IR (KBr) 3620–3150, 3060, 2940, 1685, 1555, 1430,
1
1210, 1060, 820, 730, 690 cmϪ ; 1H NMR (CD3OD) 1.74 and 1.91 (2m, 2H), 2.43 (t,
J ϭ 7.5 Hz, 2H), 4.16–4.21 (3m, 8H), 5.04 (s, 2H), 5.08 (s, 2H), 5.78 (d, J ϭ 8.1 Hz,
1H), 5.95 (d, J ϭ 4.7 Hz, 1H), 7.32 (m, 10H), 7.93 (d, J ϭ 8.1 Hz, 1H); 13C NMR

INHIBITION OF GLUTAMYL-tRNA SYNTHETASE
11
(CD3OD) 27.67, 31.61, 51.93, 65.73, 67.43, 71.37, 75.52, 84.92, 86.44, 90.06, 103.20,
128.84, 128.97, 129.14, 129.19, 129.48, 129.53, 137.64, 142.50, 152.61, 158.56, 166.17,
174.67; 31P NMR (CD3OD) 0.54.
Compound 9. Compound 8a (273 mg, 0.336 mmol), palladium (10%) on activated
carbon, and water (15 mL) were shaken under H2 in a Parr apparatus at 40 psi for
4 h. The mixture was filtered through Celite and concentrated in vacuo (T Ͻ 50ЊC)
to yield 9 as a white solid (163 mg, quant). IR (KBr) 3600–3000, 2900, 1680, 1600,
1570, 1510, 1450, 1390, 1340, 1280, 1250–1200, 1110–1010, 940, 860, 810, 780, 690
1
1
cmϪ ; H NMR (D2O) 1.70 (m, 2H), 2.14 (t, J ϭ 7.5 Hz, 2H), 3.30 (m, 1H), 3.78
(m, 1H), 3.87 (m, 1H), 4.06 (m, 2H), 4.28 (m, 1H), 4.42 (t, J ϭ 4.7 Hz, 1H), 4.69
(t, J ϭ 5.1 Hz, 1H), 6.09 (d, J ϭ 5.1 Hz, 1H), 7.35 (t, 7.6 Hz, 2H), 7.48 (t, J ϭ 7.3
Hz, 1H), 7.77 (d, J ϭ 7.6 Hz, 2H), 8.54 (s, 1H), 8.56 (s, 1H); 13C NMR (D2O) 28.05,
36.16, 54.48, 67.58, 69.36, 72.91, 76.88, 86.40, 90.51, 130.90, 131.60, 135.43, 136.15,
151.93, 154.73, 154.93, 175.05; 31P NMR (D2O) 0.49.
Compound 10. Compound 9 was dissolved in a saturated solution of aqueous
ammonia and the solution was stirred at room temperature for 48 h. The solvent was
evaporated and the product was purified by column chromatography on cellulose
(MeOH to MeOH/H2O 4 : 1) to yield 10 (133 mg, 82%). IR (KBr) 3700–2400, 1640,
1
1
1605, 1575, 1280–1180, 1160–980 cmϪ ; H NMR (D2O) 1.73 (m, 2H), 2.21 (t, J ϭ
7.4 Hz, 2H), 3.37 (m, 1H), 3.69 (m, 1H), 3.86 (m, 1H), 4.08 (m, 2H), 4.30 (m, 1H),
4.45 (t, J ϭ 4.7 Hz, 1H), 4.68 (t, J ϭ 5.1 Hz, 1H), 5.90 (d, J ϭ 5.1 Hz, 1H), 8.01 (s,
1H), 8.29 (s, 1H); 13C NMR (D2O) 27.55, 35.05, 54.22, 67.34, 67.60, 72.88, 76.77,
85.19, 90.02, 120.99, 142.48, 151.35, 154.72, 157.43, 182.31; 31P NMR (D2O) 0.40.
Compound 11. Compound 8b (100 mg, 0.13 mmol), palladium (10%) on activated
carbon, and water (6 mL) were shaken under H2 in a Parr apparatus at 40 psi for
4 h. The mixture was filtered through Celite and concentrated in vacuo. The residue
was dissolved in a saturated solution of aqueous ammonia and the solution was
stirred at room temperature for 48 h. The solvent was evaporated and the product
was purified by column chromatography on cellulose (MeOH to MeOH/H2O 4 : 1)
to yield 11 (60 mg, 80%). IR (KBr) 3600–3000, 2940, 1665, 1575, 1400, 1220, 1070,
1
1
820 cmϪ ; H NMR (D2O) 1.88 (q, J ϭ 7.4 Hz, 2H), 2.36 (td, J ϭ 7.6 and 2.9 Hz,
2H), 2.54 (t, J ϭ 7.4, 2H), 3.50 (m, 3H), 3.86 to 4.06 (m, 5H), 4.12 to 4.22 (m, 2H),
5.54 (d, J ϭ 7.3 Hz, 1H); 13C NMR (D2O) 27.67, 35.13, 39.24, 42.57, 51.67, 54.25,
67.35, 68.26, 72.81, 72.97, 84.32, 91.84, 163.67, 181.52; 31P NMR (D2O) 0.58.
Compound 12. Compound 8b (45 mg, 0.057 mmol), palladium (10%) on activated
carbon (90 mg), and 1,4-cyclohexadiene (1.5 mL) in 4 mL of EtOH : H2O (1 : 1)
were stirred at room temperature for 48 h. The mixture was filtered through Celite
and concentrated in vacuo. The residue was dissolved in aqueous ammonia and
the solution was stirred at room temperature for 48 h. The solvent was evaporated
and the product was purified by column chromatography on cellulose (MeOH to
MeOH/H2O 4 : 1) to yield 12 (22 mg, 85%). IR (KBr) 3700–3020, 2920, 1720, 1650,
1
1
1490, 1400, 1280, 1220, 1060, 780 cmϪ ; H NMR (D2O) 1.83 (q, J ϭ 7.4 Hz, 2H),
2.26 (t, J ϭ 7.4 Hz, 2H), 3.54 (m, 1H), 3.84 to 4.04 (m, 5H), 4.13 to 4.25 (m, 2H),
5.89 (d, J ϭ 3.1 Hz, 1H), 6.02 (d, J ϭ 7.5 Hz, 1H), 7.82 (d, J ϭ 7.5 Hz, 1H); 13C
NMR (D2O) 27.80, 35.40, 54.30, 67.18, 67.37, 67.44, 71.86, 76.77, 85.18, 92.69, 98.89,
144.76, 158.28, 167.35, 182.84; 31P NMR (D2O) Ϫ0.44.

12
DESJARDINS ET AL.
Compound 13. Yield 94%. IR (KBr) 3660–2500, 1600, 1415, 1270–1180, 1160–990
1
1
cmϪ ; H NMR (D2O) 1.72 (m, 2H), 2.16 (t, J ϭ 7.5 Hz, 2H), 3.31 (m, 1H), 3.76
(m, 2H), 3.86 (m, 1H), 4.11 (m, 2H), 4.34 (t, 1H), 4.49 (t, J ϭ 4.8 Hz, 1H), 6.23 (d,
J ϭ 5.1 Hz, 1H), 8.73 (s, 1H), 8.91 (s, 1H), 9.09 (s, 1H); 13C NMR (D2O) 27.95,
35.79, 53.91, 54.02, 67.22, 67.29, 67.45, 67.52, 72.53, 76.41, 86.10, 89.98, 135.96, 147.56,
150.52, 153.38, 154.62, 183.43; 31P NMR (D2O) 0.40.
Compound 14. Yield 96%. IR (KBr) 3680–2500, 1690, 1560, 1410, 1205, 1080,
1
1
815, 650 cmϪ ; H NMR (D2O) 1.83 (q, J ϭ 7.3 Hz, 2H), 2.26 (t, J ϭ 7.9 Hz, 2H),
3.23–3.49 (m, 1H), 3.83–4.30 (m, 7H), 5.84 (d, J ϭ 8.1 Hz, 1H), 5.88 (d, J ϭ 4.8
Hz, 1H), 7.82 (d, J ϭ 8.1 Hz, 1H); 13C NMR (D2O) 27.68, 35.13, 54.30, 67.37, 67.44,
72.17, 76.40, 85.62, 91.80, 105.28, 144.35, 154.48, 168.94, 182.81; 31P NMR (D2O) 0.46.
Inhibition of E. coli Glutamyl-tRNA Synthetase by Analogues of
Glutamyl Adenylate
E. coli GluRS was purified to homogeneity as previously described (13). The
GluRS was assayed by measuring the rate of formation of [14C]glutamyl-tRNA as
given by the amount of [14C]glutamate incorporated into the trichloroacetic acid
precipitate after various incubation times at 37ЊC; 1 unit was defined as the amount
of enzyme that aminoacylates 1 nmol of tRNA specific for glutamic acid in 1 min
as previously described (14). Potential inhibitors of GluRS were added to the
reaction mixture in various concentrations and the percentage of inhibition was
calculated from the resulting loss in specific activity of the GluRS. For kinetic
measurements, all substrates, except the one studied whose concentration was
variable, were present at such concentrations that their specific binding sites on the
GluRS were saturated as previously described (14) with different concentrations
of the inhibitor such that a 10 to 75% decrease in GluRS activity was observed.
We tested two substrates, glutamate and ATP, whose concentrations varied from
50 to 200 ȐM for glutamate and 0.5 to 4 mM for ATP.
The kinetic constants KGmlu and KmATP were determined from Lineweaver–Burk
plots, whereas Ki were measured from the Kmapp vs [I] plot, according to the equation
Kmapp ϭ Km/Ki [I] ϩ Km , the Kmapp being calculated from 1/V vs 1/S reciprocal plots
for various [I]. To estimate the apparent Ki values of compounds 9 and 11–14, we
measured the rate of glutamyl-tRNA formation at saturating concentrations of
ATP and tRNA and at the Km value of 100 ȐM for glutamate; under these conditions,
Ki is the value of [I] such that v ϭ 0.33 Vmax , since for competitive inhibition,
v/Vmax ϭ S/[S ϩ Km (1 ϩ 1/Ki)].
ACKNOWLEDGMENTS
This work was supported by Grants OGP0009597 (to J.L.) and CPG0164048 (to R.C. and J.L.) from
the Natural Sciences and Engineering Research Council of Canada and by Grant 98-ER-2481 from the
´
‘‘Fonds FCAR du Ministere de l’Education du Quebec’’ (to R.C. and J.L.).
`
´

INHIBITION OF GLUTAMYL-tRNA SYNTHETASE
13
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