Inhibition of Helicobacter pylori aminoacyl-tRNA amidotransferase by chloramphenicol analogs

Article abstract of DOI:10.1016/j.bmc.2010.09.045

Genomic studies revealed the absence of glutaminyl-tRNA synthetase and/or asparaginyl-tRNA synthetase in many bacteria and all known archaea. In these microorganisms, glutaminyl-tRNA^Gln (Gln-tRNA^Gln) and/or asparaginyl-tRNA^Asn

Full text of DOI:10.1016/j.bmc.2010.09.045

Bioorganic & Medicinal Chemistry 18 (2010) 7868–7872
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Inhibition of Helicobacter pylori aminoacyl-tRNA amidotransferase by
chloramphenicol analogs
Christian Balg a, Maria De Mieri c, Jonathan L. Huot b, Sébastien P. Blais b, Jacques Lapointe b,
Robert Chênevert a,
⇑
a Département de Chimie, PROTEO, Faculté des Sciences et de Génie, Université Laval, Québec, Canada G1V 0A6
b Département de Biochimie et de Microbiologie, PROTEO, Faculté des Sciences et de Génie, Université Laval, Québec, Canada G1V 0A6
c Dipartimento di Chimica, Biochimica e Biotecnologie per la Medicina, Università degli Studi di Milano, 20133 Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 July 2010
Revised 14 September 2010
Accepted 18 September 2010
Available online 1 October 2010
Genomic studies revealed the absence of glutaminyl-tRNA synthetase and/or asparaginyl-tRNA synthe-
tase in many bacteria and all known archaea. In these microorganisms, glutaminyl-tRNAGln (Gln-tRNAGln
)
and/or asparaginyl-tRNAAsn (Asn-tRNAAsn) are synthesized via an indirect pathway involving side chain
amidation of misacylated glutamyl-tRNAGln (Glu-tRNAGln) and/or aspartyl-tRNAAsn (Asp-tRNAAsn) by an
amidotransferase. A series of chloramphenicol analogs have been synthesized and evaluated as inhibitors
of Helicobacter pylori GatCAB amidotransferase. Compound 7a was identified as the most active compet-
Keywords:
itive inhibitor of the transamidase activity with respect to Asp-tRNAAsn (Km = 2
27 M.
lM), with a Ki value of
Chloramphenicol
Amidotransferase
Aminoacyl-tRNA
Transamidation
GatCAB
l
Ó 2010 Elsevier Ltd. All rights reserved.
Helicobacter pylori
Puromycin
1. Introduction
as a Glu-tRNAGln amidotransferase and is found only in archaea.5
Crystal structures of GatCAB and GatDE have been determined re-
cently.6–9
The specific aminoacylation of tRNAs is essential for the faithful
translation of the genetic information into proteins. Aminoacyl-
tRNA synthetases (aaRS) are the enzymes that catalyze the esterifi-
cation reaction between an amino acid and its cognate tRNA.1,2 In
the cytoplasm of eukaryotic cells, the pairing of glutamine and
asparagine with their corresponding tRNA proceeds through a direct
pathway involving a glutaminyl-tRNA synthetase (GlnRS) and an
asparaginyl-tRNA synthetase (AsnRS), respectively (Fig. 1A). Geno-
mic studies revealed the absence of GlnRS and/or AsnRS in all known
archaea, in many bacteria, and in some organelles. The alternative
indirect pathway for the formation of Gln-tRNAGln and Asn-tRNAAsn
involves the misacylation of tRNAGln with Glu (or tRNAAsn with Asp)
by a nondiscriminating aminoacyl-tRNA synthetase (ND-aaRS) fol-
lowed by the transamidation of the misacylated aa-tRNA by an
amidotransferase (AdT) (Fig. 1B).3,4
The transamidation of the misacylated Glu-tRNAGln and/or Asp-
tRNAAsn by the GatCAB-type aminoacyl-tRNA amidotransferase
(AdT) involves three distinct events (Fig. 2). Glutamine is hydrolyzed
in the GatA subunit serine-based glutaminase site to generate
ammonia, which reaches GatB through a molecular tunnel. In the
GatB subunit, the side-chain carboxyl group of the misacylated-
tRNA is activated by ATP to form a phosphoryl-Glu-tRNAGln or a
phosphoryl-Asp-tRNAAsn intermediate (kinase step). In the final
transamidase step, the enzyme-bound ammonia reacts with the
high-energy mixed anhydride to yield the corresponding amide
side-chain (Gln-tRNAGln or Asn-tRNAAsn). The small GatC subunit
Two types of AdT are found in nature,
a heterotrimeric
(GatCAB) and a heterodimeric protein (GatDE). GatCAB uses both
Glu-tRNAGln and Asp-tRNAAsn as substrates and is present in bac-
teria, archaea and some organelles, while GatDE functions solely
⇑
Corresponding author. Tel.: +1 418 656 3283; fax: +1 418 656 7916.
E-mail address: robert.chenevert@chm.ulaval.ca (R. Chênevert).
Figure 1. Glutaminyl-tRNA and asparaginyl-tRNA biosynthetic pathways. ND-
aaRS = nondiscriminating aminoacyl-tRNA synthetase; AdT = amidotransferase.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.09.045

C. Balg et al. / Bioorg. Med. Chem. 18 (2010) 7868–7872
7869
Figure 2. Reaction mechanism of GatCAB aminoacyl-tRNA amidotransferase. A = Adenine; aa = Asn, n = 1; aa = Gln, n = 2. For each of the three reactions represented here, the
catalysis takes place in the subunit whose symbol (A or B) is underlined in GatCAB.
wraps around the interface of the A- and B-subunits, suggesting that
it may stabilize the complex.
2. Results and discussion
For bacteria lacking a GlnRS or AsnRS, AdT activity is essential
for protein biosynthesis.10 Therefore, inhibition of this enzyme
could provide a novel basis for the development of selective anti-
microbial agents.11 Only a few AdT inhibitors have been reported
so far. Some analogs of ATP were useful to study the reaction
2.1. Synthesis of chloramphenicol derivatives (7a–f)
The synthesis of chloramphenicol analogs 7a–f is outlined in
Scheme 1. Amines 4a–f were condensed with commercially avail-
able N-Boc-methionine-sulfone 5 under standard conditions using
mechanism.12 Glutamyl-
c-boronate derivatives were evaluated as
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride
serine inactivators in the AdT-glutaminase active site; these inhib-
itors provided potent inhibition in vitro and displayed antibacterial
activities for many AdT-dependent bacteria.13 Recently, we re-
ported the inhibition of Helicobacter pylori GatCAB aminoacyl-tRNA
amidotransferase by puromycin (1, Fig. 3) analogs.14,15 Compound
2 was found to have the most potent inhibitory activity against
(EDC)/N-hydroxysuccinimide in DMF. Amino-alcohol 4a–d are
commercially available whereas 4e and 4f were prepared from
4a and 4d, respectively using a literature procedure.17 Treatment
of 6a–f with 4 M HCl/dioxane resulted in cleavage of the N-tert-
butoxycarbonyl group to provide the corresponding amides 7a–f.
These compounds were isolated after precipitation in Et2O as the
hydrochloride salts.
GatCAB (Ki = 4 l
M with respect to Asp-tRNAAsn). Our strategy
was based on the premise that the sulfone moiety mimics the tran-
sition state in the transamidation reaction (last step in Fig. 2).
The antibiotic chloramphenicol 3 inhibits protein synthesis by
binding to the peptidyl transferase region of the ribosome, and
overlaps the binding site of puromycin.16 In the search of novel
AdT inhibitors, this similarity and the structural relationship of
2.2. Inhibition of H. pylori GatCAB amidotransferase (AdT)
Chloramphenicol 3 and analogs 7a–f were evaluated for in vitro
inhibitory activity against H. pylori GatCAB aminoacyl-tRNA
amidotransferase (Table 1). Enzyme production and kinetic
the two compounds led us to explore the potential of L-methio-
nine-sulfone derivatives of chloramphenicol as inhibitors of Gat-
CAB aminoacyl-tRNA amidotransferase.
Figure 3. Chemical structures of puromycin 1, AdT inhibitor 2 and chloramphen-
icol 3.
Scheme 1. Reagents and conditions: (a) EDC, N-hydroxysuccinimide, DMF, 24 h (b)
4 M HCl/dioxane, 40 min.

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C. Balg et al. / Bioorg. Med. Chem. 18 (2010) 7868–7872
Table 1
Inhibition of H. pylori GatCAB aminoacyl-tRNA amidotransferase (AdT) by chloram-
phenicol and several analogs
Figure 4. Analogy between a putative transition state of the transamidase step (A)
and the sulfone side chain of inhibitors 7a–f (B).
amide functionalities.22 The p-nitro group and the (1R,2R) stereo-
chemistry of chloramphenicol combined with the L-methionyl-
Compound
R
Stereochemistry
Ki (
1850 550
27
120 24
400 100
160 20
lM) standard error
Chloramphenicol 3
NO2
NO2
SO2CH3
H
NO2
NO2
NO2
(1R,2R)
(1R,2R)
(1R,2R)
(1R,2R)
(1S,2S)
(1S,2R)
(1R,2S)
sulfone moiety appeared to be optimal in this series. Given the much
smaller number of possible contacts between 7a and GatCAB when
compared to those possible for Asp-tRNAAsn, it is noteworthy that
the Ki for 7a is only ten times greater than the Km for the natural
substrate.
Chloramphenicol analogs 7a–f represent a new class of AdT
inhibitors and may provide the basis for the design of other low-
molecular weight inhibitors. Further investigation is currently
underway to find more potent inhibitors of GatCAB amidotransfer-
ase based on compound 7a.
7a
7b
7c
7d
7e
7f
6
2800 500
370 40
Standard errors were calculated by curve-fitting of our data, using KALEIDAGRAPH 4.0
(Synergy Software). Values calculated from averages of two distinct experiments
(four, for 7a), weighting by inverse variance.18
experiments were carried out as previously described.14 Competi-
tive inhibition with respect to Asp-tRNAAsn was observed for all
compounds.
3. Experimental
3.1. General
The parent compound chloramphenicol 3 is a very weak inhibitor
of GatCAB (Ki ꢀ 1.9 mM). Replacement of the dichloroacetyl moiety
Chemical reagents were purchased from Aldrich–Sigma Chemi-
cal Company. Flash column chromatography was carried out using
40–63 lM (230–400 mesh) silica gel. Optical rotations were mea-
sured using a JASCO DIP-360 digital polarimeter (c as g of com-
pound per 100 mL). Infrared spectra were recorded on a Bomem
MB-100 spectrometer. NMR spectra were recorded on a Varian Ino-
va AS400 spectrometer (400 MHz). Mass spectra were obtained on
an Agilent 6210 ESI TOFMS.
of chloramphenicol by L-methionyl-sulfone considerably enhances
the activity and 7a was identified as the most active inhibitor with
a Ki value of 27 M. Replacement of the para-nitro group by a sulfone
(7b, related to thiamphenicol) or a hydrogen (7c) decreased the abil-
ity to inhibit GatCAB (Ki = 120 and 400 M, respectively). In order to
l
l
complete this structure–activity relationship study, we evaluated
the effect of the configurations at C-1 and C-2 stereogenic centers
on the activity. The (1S,2S) enantiomer 7d exhibited a moderate five-
fold decrease in activity (Ki = 160
Diastereoisomers (1S,2R)-7e and (1R,2S)-7f were less active deriva-
tives (Ki = 2.8 mM and 370 M, respectively) and, in particular, the
loss in potency was more marked for (1S,2R) analog than for the
(1R,2S) isomer.
lM for 7d versus 27 lM for 7a).
3.2. Typical procedure for preparation of amides 6a–f
l
Amine 4a (25.0 mg, 118
(36.5 mg, 130 mol), N-hydroxysuccinimide (14.9 mg, 130
and EDC (27.1 mg, 141 mol) were dissolved in anhydrous DMF
lmol), N-Boc-L-methionine-sulfone 5
l
lmol)
l
The structural similarity of puromycin, chloramphenicol and the
aminoacyl-adenosyl terminus of tRNA has already been stressed.
Aminoacyl analogs of chloramphenicol were evaluated as inhibitors
of the peptide bond formation in a ribosomal cell-free system
derived from Escherichia coli.19–21 There is however a major
difference between the ribosomal peptidyl transferase and the
(1.0 mL). The mixture was stirred at room temperature for 24 h.
The solvent was coevaporated with toluene and the residue was
purified by flash chromatography (7–8% MeOH/CH2Cl2) to yield
6a (42.3 mg, 76%).
a
3.2.1. (1R,2R)-1-(4-Nitrophenyl)-2-(N -Boc-
L-methionyl-
amidotransferase reactions. In the peptidyl transfer, the ester (
a
-po-
sulfone-amido)-1,3-propadiol (6a)
sition) of the P-site peptidyl-tRNA is attacked by the -amino group
a
White solid; mp 77–79 °C; ½a D21
ꢁ
ꢂ43.5 (c 0.84, CH2Cl2); IR (KBr)
of the A-site aminoacyl-tRNA. In contrast, the aminoacyl-tRNA
3400, 2979, 1666, 1521, 1349, 1294, 1164, 1129 cmꢂ1 1H NMR
;
amidotransferase reaction is the simple conversion of the side chain
carboxylic acid (b-position for Asp, c-position for Glu) into an amide
(Asn or Gln) while the amino acid is still attached to a tRNA (pre-
(400 MHz, CD3OD) d 1.42 (s, 9H), 1.85–1.97 (m, 1H), 1.97–2.07
(m, 1H), 2.89 (s, 3H), 3.01 (t, J = 8.1 Hz, 2H), 3.55 (dd, J = 10.8 and
6.5 Hz, 1H), 3.74 (dd, J = 10.8 and 6.9 Hz, 1H), 4.06–4.15 (m, 2H),
5.07 (d, J = 2.0 Hz, 1H), 7.60 (d, J = 8.5 Hz, 2H), 8.14 (d, J = 8.8 Hz,
2H); 13C NMR (100 MHz, CD3OD) d 25.0, 27.5, 39.3, 50.6, 53.2,
56.5, 61.4, 70.4, 79.8, 123.0, 127.2, 147.3, 150.6, 156.4, 172.3;
HRMS (ESI) calcd for C19H29N3O9SNa [M+Na]+ 498.1517. Found:
498.1514.
translational modification). The enzyme is equally efficient in trans-
amidation of both Asp-tRNAAsn and Glu-tRNAGln 3
. Although the
detailed interactions of inhibitors 7a–f with GatCAB are yet to be
defined, thesulfone moietyof 7a canbe consideredas a stable analog
of the transition state in the last step of the transamidation process,
where the carbonyl to be attacked by ammonia is replaced by a
tetrahedral sulfur atom with a methyl group mimicking ammonia
(Fig. 4). Various sulfur or phosphorus-containing derivatives have
been proposed as analogs of the tetrahedral intermediate formed
during enzymatic reactions involving hydrolysis or formation of
a
3.2.2. (1R,2R)-1-(4-Methylsulfonylphenyl)-2-(N -Boc-
L
-
methionyl-sulfone-amido)-1,3-propadiol (6b)
White solid; yield 52%; mp 90–92 °C; ½a D21
ꢁ
ꢂ32.6 (c 0.60,
1H
MeOH); IR (KBr) 3370, 2931, 1668, 1523, 1299, 1149 cmꢂ1
;

C. Balg et al. / Bioorg. Med. Chem. 18 (2010) 7868–7872
7871
NMR (400 MHz, CD3OD) d 1.44 (s, 9H), 1.85–1.96 (m, 1H), 1.96–
2.07 (m, 1H), 2.90 (s, 3H), 2.98–3.06 (m, 2H), 3.07 (s, 3H), 3.53
(dd, J = 10.7 and 6.4 Hz, 1H), 3.72 (dd, J = 10.8 and 7.0 Hz, 1H),
4.05–4.13 (m, 2H), 5.04–5.08 (m, 1H), 7.63 (d, J = 8.2 Hz, 2H),
7.86 (d, J = 8.4 Hz, 2H); 13C NMR (100 MHz, CD3OD) d 24.9, 27.5,
39.4, 43.4, 50.7, 53.5, 56.5, 61.3, 70.4, 79.9, 127.0, 127.2, 139.6,
The solvent was coevaporated under reduced pressure with MeOH.
The residue was dissolved in a minimum of EtOH (2.0 mL) and the
product was precipitated by the addition of Et2O (20 mL). The
product was collected by filtration and washed with Et2O to give
7a (30.3 mg, 84%).
149.4, 156.5, 172.3; HRMS (ESI) calcd for
C20H32N2O9S2Na
3.3.1. (1R,2R)-1-(4-Nitrophenyl)-2-(L-methionyl-sulfone-
[M+Na]+ 531.1441. Found: 531.1448.
amido)-1,3-propadiol (7a)
White solid; mp 140–143 °C (dec); ½a D21
ꢁ
14.6 (c 0.19, MeOH/H2O
1H
a
3.2.3. (1R,2R)-1-Phenyl-2-(N -Boc-
L
-methionyl-sulfone-amido)-
1:1); IR (KBr) 3386, 2926, 1683, 1520, 1351, 1286, 1133 cmꢂ1
;
1,3-propadiol (6c)
NMR (400 MHz, CD3OD) d 2.21–2.39 (m, 2H), 2.99 (s, 3H), 3.27 (t,
J = 8.0 Hz, 2H), 3.54 (dd, J = 10.9 and 7.4 Hz, 1H), 3.77 (dd, J = 10.9
and 5.4 Hz, 1H), 4.03 (t, J = 6.3 Hz, 1H), 4.17–4.25 (m, 1H), 5.02
(d, J = 3.7 Hz, 1H), 7.65 (d, J = 8.9 Hz, 2H), 8.17 (d, J = 8.8 Hz, 2H);
13C NMR (100 MHz, CD3OD) d 24.4, 39.6, 49.1, 51.5, 57.4, 61.6,
70.7, 123.1, 127.3, 147.5, 150.4, 167.8; HRMS (ESI) calcd for
White solid; yield 81%; mp 55–57 °C; ½a D21
ꢂ41.1 (c 0.38,
ꢁ
MeOH); IR (KBr) 3387, 2979, 1694, 1664, 1521, 1292, 1165, 1128,
1053 cmꢂ1 1H NMR (400 MHz, CD3OD) d 1.44 (s, 9H), 1.89–2.02
;
(m, 1H), 2.02–2.14 (m, 1H), 2.89 (s, 3H), 2.96–3.10 (m, 2H), 3.44
(dd, J = 10.0 and 6.4 Hz, 1H), 3.63 (dd, J = 10.2 and 6.2 Hz, 1H),
4.01–4.09 (m, 1H), 4.09–4.18 (m, 1H), 7.20 (t, J = 7.1 Hz, 1H), 7.29
(t, J = 7.4 Hz, 2H), 7.36 (d, J = 7.4 Hz, 2H); 13C NMR (100 MHz,
CD3OD) d 25.0, 27.5, 39.3, 50.6, 53.5, 57.1, 61.2, 71.4, 79.9, 126.2,
127.3, 128.1, 142.4, 156.5, 172.3; HRMS (ESI) calcd for
C
14H22N3O7S [M+H]+ 376.1173. Found: 376.1184.
3.3.2. (1R,2R)-1-(4-Methylsulfonylphenyl)-2-(L-methionyl-
19H30N2O7SNa [M+Na]+ 453.1666. Found: 453.1669.
sulfone-amido)-1,3-propadiol (7b)
C
White solid; yield quant.; mp 135–137 °C (dec); ½a D21
ꢁ
8.3 (c 0.39,
1H NMR
MeOH); IR (KBr) 3434, 2924, 1679, 1286, 1147 cmꢂ1
;
a
3.2.4. (1S,2S)-1-(4-Nitrophenyl)-2-(N -Boc-
L
-methionyl-sulfone-
(400 MHz, CD3OD) d 2.21–2.40 (m, 2H), 2.99 (s, 3H), 3.09 (s, 3H),
3.28 (t, J = 7.9 Hz, 2H), 3.52 (dd, J = 10.9 and 7.3 Hz, 1H), 3.75 (dd,
J = 11.0 and 5.4 Hz, 1H), 4.05 (t, J = 6.3 Hz, 1H), 4.17–4.23 (m, 1H),
5.00 (d, J = 3.8 Hz, 1H), 7.67 (d, J = 8.3 Hz, 2H), 7.89 (d, J = 8.3 Hz,
2H); 13C NMR (100 MHz, CD3OD) d 24.4, 39.6, 43.2, 49.1, 51.5,
57.5, 61.5, 70.8, 127.2, 127.3, 139.8, 149.2, 167.8; HRMS (ESI) calcd
for C15H25N2O7S2 [M+H]+ 409.1098. Found: 409.1099.
amido)-1,3-propadiol (6d)
White solid; yield 87%; mp 82–85 °C; ½a D21
ꢂ0.58 (c 0.90,
ꢁ
MeOH); IR (KBr) 3409, 2979, 1665, 1521, 1350, 1294, 1164,
1128 cmꢂ1 1H NMR (400 MHz, CD3OD) d 1.42 (s, 9H), 1.70–1.83
;
(m, 1H), 1.93–2.05 (m, 1H), 2.78–2.97 (m, 5H), 3.57 (dd, J = 10.9
and 5.9 Hz, 1H), 3.74 (dd, J = 10.8 and 7.3 Hz, 1H), 4.08 (dd,
J = 8.5 and 5.2 Hz, 1H), 4.11–4.17 (m, 1H), 5.10 (d, J = 1.7 Hz, 1H),
7.61 (d, J = 8.6 Hz, 2H), 8.16 (d, J = 8.8 Hz, 2H); 13C NMR
(100 MHz, CD3OD) d 25.3, 27.5, 39.3, 50.6, 53.0, 56.6, 61.4, 70.1,
79.8, 123.1, 127.1, 147.4, 150.8, 156.4, 172.2; HRMS (ESI) calcd
for C19H29N3O9SNa [M+Na]+ 498.1517. Found: 498.1511.
3.3.3. (1R,2R)-1-Phenyl-2-(L-methionyl-sulfone-amido)-1,3-
propadiol (7c)
White solid; yield quant.; mp 120–125 °C (dec); ½a D21
ꢁ
0.65 (c
0.48, MeOH); IR (KBr) 3425, 2923, 1684, 1561, 1496, 1285, 1133,
1056 cmꢂ1 1H NMR (400 MHz, CD3OD) d 2.24–2.43 (m, 2H), 2.99
;
a
3.2.5. (1S,2R)-1-(4-Nitrophenyl)-2-(N -Boc-
L
-methionyl-
(s, 3H), 3.25–3.35 (m, 2H), 3.41 (dd, J = 10.9 and 7.2 Hz, 1H), 3.61
(dd, J = 11.0 and 4.6 Hz, 1H), 4.07 (t, J = 6.1 Hz, 1H), 4.12–4.19 (m,
1H), 4.81 (d, J = 5.9 Hz, 1H), 7.24 (t, J = 7.3 Hz, 1H), 7.31 (t,
J = 7.4 Hz, 2H), 7.39 (d, J = 7.3 Hz, 2H); 13C NMR (100 MHz, CD3OD)
24.3, 39.7, 49.2, 51.7, 57.9, 61.2, 72.1, 126.4, 127.6, 128.2, 142.2,
167.9; HRMS (ESI) calcd for C14H23N2O5S [M+H]+ 331.1322. Found:
331.1319.
sulfone-amido)-1,3-propadiol (6e)
White solid; yield 84%; mp 171–173 °C (dec); ½a D21
ꢂ18.4 (c
ꢁ
0.96, CH2Cl2/MeOH 1:1); IR (KBr) 3426, 3342, 2982, 1681, 1658,
1522, 1348, 1285, 1163, 1129, 1051 cmꢂ1 1H NMR (400 MHz,
;
CD3OD) d 1.39 (s, 9H), 1.83–1.95 (m, 1H), 1.95–2.08 (m, 1H), 2.89
(s, 3H), 3.03 (t, J = 7.8 Hz, 2H), 3.64 (dd, J = 10.9 and 2.8 Hz, 1H),
3.78 (dd, J = 10.8 and 6.4 Hz, 1H), 4.00–4.14 (m, 2H), 4.80 (d,
J = 7.5 Hz, 1H), 7.60 (d, J = 8.2 Hz, 2H), 8.24 (d, J = 8.2 Hz, 2H); 13C
NMR (100 MHz, CD3OD) d 25.1, 27.4, 39.3, 50.6, 53.3, 56.6, 60.3,
72.3, 79.8, 123.0, 127.8, 147.5, 150.0, 156.4, 172.0; HRMS (ESI)
calcd for C19H29N3O9SNa [M+Na]+ 498.1517. Found: 498.1530.
3.3.4. (1S,2S)-1-(4-Nitrophenyl)-2-(L-methionyl-sulfone-amido)-
1,3-propadiol (7d)
White solid; yield 91%; mp 85–90 °C (dec); ½a D21
ꢁ
19.4 (c 0.53,
MeOH); IR (KBr) 3426, 2925, 1679, 1519, 1351, 1285, 1134 cmꢂ1
;
1H NMR (400 MHz, CD3OD) d 1.95–2.14 (m, 2H), 2.71–2.82 (m,
1H) 2.84–2.94 (m, 4H), 3.60 (dd, J = 10.9 and 6.3 Hz, 1H), 3.78
(dd, J = 10.9 and 6.5 Hz, 1H), 4.00 (t, J = 6.3 Hz, 1H), 4.23–4.30 (m,
1H), 5.09 (d, J = 3.2 Hz, 1H), 7.66 (d, J = 8.6 Hz, 2H), 8.19 (d,
J = 8.8 Hz, 2H); 13C NMR (100 MHz, CD3OD) d 24.3, 39.6, 49.2,
51.6, 57.0, 61.5, 70.2, 123.3, 127.1, 147.5, 150.8, 167.7; HRMS
(ESI) calcd for C14H22N3O7S [M+H]+ 376.1173. Found: 376.1167.
a
3.2.6. (1R,2S)-1-(4-Nitrophenyl)-2-(N -Boc-
L
-methionyl-
sulfone-amido)-1,3-propadiol (6f)
White solid; yield 72%; mp 175–176 °C (dec); ½a D21
ꢂ10.8 (c 1.70,
ꢁ
MeOH); IR (KBr) 3424, 3357, 2978, 1681, 1660, 1523, 1350, 1294,
1166, 1131, 1053 cmꢂ1 1H NMR (400 MHz, CD3OD) d 1.43 (s, 9H),
;
1.64–1.74 (m, 1H), 1.80–1.88 (m, 1H), 2.87 (s, 3H), 2.83–2.89 (m,
2H), 3.70 (dd, J = 11.2 and 3.7 Hz, 1H), 3.86 (dd, J = 11.3 and 5.5 Hz,
1H), 4.03 (dd, J = 8.5 and 5.1 Hz, 1H), 4.15–4.19 (m, 1H), 4.82 (d,
J = 8.1 Hz, 1H), 7.63 (d, J = 8.6 Hz, 2H), 8.20 (d, J = 8.8 Hz, 2H); 13C
NMR (100 MHz, CD3OD) d 26.5, 28.7, 40.6, 51.8, 54.4, 57.6, 61.9,
73.4, 81.1, 124.6, 129.3, 149.0, 151.4, 157.7, 173.1; HRMS (ESI) calcd
for C19H29N3O9SNa [M+Na]+ 498.1517. Found: 498.1505.
3.3.5. (1S,2R)-1-(4-Nitrophenyl)-2-(L-methionyl-sulfone-
amido)-1,3-propadiol (7e)
White solid; yield 81%; mp 185–190 °C (dec); ½a D21
ꢁ
18.0 (c 0.58,
MeOH); IR (KBr) 3351, 2927, 1683, 1519, 1351, 1285, 1133 cmꢂ1
;
1H NMR (400 MHz, CD3OD) d 2.20–2.40 (m, 2H), 2.98 (s, 3H),
3.27 (t, J = 8.0 Hz, 2H), 3.63–3.74 (m, 2H), 3.99 (t, J = 6.4 Hz, 1H),
4.15–4.22 (m, 1H), 4.88 (d, J = 6.4 Hz, 1H), 7.64 (d, J = 8.4 Hz, 2H),
8.20 (d, J = 8.8 Hz, 2H); 13C NMR (100 MHz, CD3OD) d 24.6, 39.6,
49.1, 51.6, 57.3, 59.8, 72.4, 123.2, 127.5, 147.6, 149.9, 167.6; HRMS
(ESI) calcd for C14H22N3O7S [M+H]+ 376.1173. Found: 376.1186.
3.3. Typical procedure for the cleavage of the N-tert-
butoxycarbonyl groups
A solution of compound 6a (41.9 mg, 88.1
dioxane (2.0 mL) was stirred at room temperature for 40 min.
lmol) in 4 M HCl/

7872
C. Balg et al. / Bioorg. Med. Chem. 18 (2010) 7868–7872
tested: chloramphenicol: 125–2000
1660 M; 7c: 100–1660 M; 7d: 60–1000
7f: 60–1000 M.
l
M; 7a: 5–1000
l
M; 7b: 10–
l
l
lM; 7e: 60–1000 l
M;
l
Acknowledgments
This work was supported by the Natural Sciences and Engineer-
ing Research Council of Canada (NSERC) and the ‘‘Fonds de recher-
che sur la nature et les technologies, Québec (FQRNT)”. C.B. and J.H.
thank NSERC and FQRNT for graduate scholarships, respectively.
References and notes
Figure 5. Compound 7a is a competitive inhibitor of H. pylori AdT with respect to
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H. pylori Asp-tRNAAsn. A Ki of 27
6 lM was obtained by fitting the data points to
the competitive inhibition equation described above for four experiments, and the
calculated values were weighted by inverse variance.
2. Translation Mechanisms; Lapointe, J., Brakier-Gingras, L., Eds.; Landes
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3.3.6. (1R,2S)-1-(4-Nitrophenyl)-2-(L-methionyl-sulfone-
amido)-1,3-propadiol (7f)
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White solid; yield 75%; mp 165–170 °C (dec); ½a D21
ꢂ1.8 (c 1.00,
ꢁ
MeOH); IR (KBr) 3409, 2925, 1683, 1520, 1352, 1286, 1135 cmꢂ1
;
1H NMR (400 MHz, CD3OD) d 1.88–2.09 (m, 2H), 2.70–2.90 (m,
2H), 2.92 (s, 3H), 3.72–3.86 (m, 2H), 3.90–3.99 (m, 1H), 4.23–
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3.4. Typical enzyme assay
Competitive inhibition of H. pylori AdT by compound 7a (Fig. 5),
with respect to Asp-tRNAAsn, was characterized in 50 mM Hepes-
KOH pH 7, 15 mM MgCl2, 25 mM KCl, 1 mM DTT, 2 mM ATP, and
1.28 mM L-glutamine, 0.50–2.00 l
M Asp-tRNAAsn and 6 nM AdT.
V0 is the uninhibited rate of product formation under these condi-
tions, Vmax the maximum rate, S the substrate concentration, Vi the
inhibited rate and I the inhibitor concentration. According to
Michaelis–Menten, in the absence of inhibitor, V0 = (VmaxS)/(S +
Km), while in the presence of a competitive inhibitor, the reaction
rate is Vi = (VmaxS)/(S + Km (1 + I/Ki)). Therefore, Vi/V0 = (S + Km)/
(S + Km (1 + I/Ki)). Curvefitting of the data to this equation was used
to identify the competitive nature of this inhibition with respect to
Asp-tRNAAsn, and to calculate the Ki value. In all cases (except for
compound 7e which was poorly soluble) the Ki values obtained
(Table 1) were within the range of the inhibitor concentrations
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