5'2-(N -aminoacyl)-sulfonamido-5'2-deoxyadenosine: Attempts for a stable alternative for aminoacyl-sulfamoyl adenosines as aaRS inhibitors

Article abstract of DOI:10.1016/j.ejmech.2015.02.010

Synthesis of aminoacyl-sulfamoyl adenosines (aaSAs) and their peptidyl conjugates as aminoacyl tRNA synthetase (aaRS) inhibitors remains problematic due to the low yield of the aminoacylation and the subsequent conjugation reaction causing concomitant formation of a cyclic adenosine derivative. In an effort to reduce this undesirable side reaction, we aimed to prepare the corresponding aminoacyl sulfonamide (aaSoA) analogues as more stable alternatives for aaSA derivatives. Deletion of the 5'2-oxygen in aaSA analogues should render the C-5'2 less electrophilic and therefore improve the stability of the aminoacyl sulfamate analogues. We therefore synthesized six sulfonamides and compared their activity against the respective aaSA analogues. However, except for the aspartyl derivative, the new compounds are not able to inhibit the corresponding aaRS. Possible reasons for this loss of activity are discussed by modeling and comparison of the newly synthesized aaSoA derivatives with their parent aaSA analogues.

Full text of DOI:10.1016/j.ejmech.2015.02.010

European Journal of Medicinal Chemistry 93 (2015) 227e236
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
0
0
5
-(N-aminoacyl)-sulfonamido-5 -deoxyadenosine: Attempts for a
stable alternative for aminoacyl-sulfamoyl adenosines as aaRS
inhibitors
*
Bharat Gadakh, Simon Smaers, Jef Rozenski, Mathy Froeyen, Arthur Van Aerschot
Medicinal Chemistry, Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 December 2014
Received in revised form
Synthesis of aminoacyl-sulfamoyl adenosines (aaSAs) and their peptidyl conjugates as aminoacyl tRNA
synthetase (aaRS) inhibitors remains problematic due to the low yield of the aminoacylation and the
subsequent conjugation reaction causing concomitant formation of a cyclic adenosine derivative. In an
effort to reduce this undesirable side reaction, we aimed to prepare the corresponding aminoacyl sul-
4
February 2015
Accepted 7 February 2015
Available online 9 February 2015
0
fonamide (aaSoA) analogues as more stable alternatives for aaSA derivatives. Deletion of the 5 -oxygen in
0
aaSA analogues should render the C-5 less electrophilic and therefore improve the stability of the
aminoacyl sulfamate analogues. We therefore synthesized six sulfonamides and compared their activity
against the respective aaSA analogues. However, except for the aspartyl derivative, the new compounds
are not able to inhibit the corresponding aaRS. Possible reasons for this loss of activity are discussed by
modeling and comparison of the newly synthesized aaSoA derivatives with their parent aaSA analogues.
© 2015 Elsevier Masson SAS. All rights reserved.
Keywords:
Amino acids
Nucleosides
Antibacterial
tRNA synthetase
Sulfonamides
Inhibitors
1. Introduction
reached the clinic for treatment of urinary tract infections by sys-
temic use, but suffered from resistance issues [10]. Several other
Aminoacyl tRNA synthetases (aaRS) comprise a family of en-
zymes which are the key players in translating the genetic code [1].
These enzymes catalyze the ligation of the correct amino acids to
their cognate tRNA and the obtained conjugates are further used in
protein synthesis. The aaRS herein proved vital in obtaining the
necessary accuracy of translation [2,3]. Despite the conserved
functionality of all 20 aaRS, these enzymes display significant
divergence between prokaryotic and eukaryotic enzymes. Given
their indispensible role in translation and their lack of sequence
homology between prokaryotes and eukaryotes, these enzymes are
considered as a promising and clinically validated target for
development of antimicrobial agents [4]. This has been further
exemplified by the clinical use of mupirocin (IleRS inhibitor) which
aaRS inhibitors for different tRNA synthetases likewise are under
study [11e13]. However, selective inhibition of pathogen aaRSs
over their human counterparts remains
considering aaRS as a drug target.
a bottleneck while
Various aaRS inhibitors from natural sources likewise illustrate
the possibility of selective inhibition of pathogen aaRSs, although
none of them (except for mupirocin) reached the clinic [14,15].
Therefore, efforts over the last decade have been directed towards
rational design of aaRS inhibitors based on either a substrate mimic
or a mimic of aminoacyl-adenylate (aa-AMP, 1, Fig. 1) being the
common intermediate in aminoacylation of tRNA [16]. The later
idea also derived from natural examples. Herein, microcin C
[17e19] (2a, Fig. 1) and agrocin 84 [20e22] (3a, Fig. 1) are aaRS
inhibitors from natural sources where the labile acyl-phosphate
linkage is replaced with a relatively stable phosphoramidate,
whereas ascamycin [23,24] (4, Fig. 1) possesses a sulfamate linkage.
The former two are in fact prodrugs which are processed to the
non-hydrolysable aa-AMP mimics 2b and 3b following internali-
zation. In general, the aa-AMP intermediate has two or three orders
of magnitude of higher affinity for the enzyme as compared to the
substrate amino acid or ATP. Consequently, non-hydrolysable
mimics of aa-AMP where the activated phospho ester linkage is
®
is marketed by GSK as Bactroban [5,6]. In addition, being an
editing site inhibitor for LeuRS, Tavaborole (formerly AN2690, trade
name Kerydin™) from Anacor Pharamaceuticals was cleared for
human use by the FDA in July 2014 for the treatment of onycho-
mycosis [7e9]. Another Anacor allosteric inhibitor (AN3365)
*
Corresponding author.
E-mail address: arthur.vanaerschot@rega.kuleuven.be (A. Van Aerschot).
http://dx.doi.org/10.1016/j.ejmech.2015.02.010
223-5234/© 2015 Elsevier Masson SAS. All rights reserved.
0

228
B. Gadakh et al. / European Journal of Medicinal Chemistry 93 (2015) 227e236
Fig. 1. Structures of the aminoacylation reaction intermediate (aa-AMP, 1), and of the natural antibiotics microcin C (2a), agrocin 84 (3a) and ascamycin (4) as examples of non-
hydrolysable mimics of aa-AMP from natural sources.
replaced with a chemically stable linkage are expected to show
excellent potency. Most of the aa-AMP modifications are aimed to
tackle issues like chemical stability, tight binding, in vivo efficacy
and selectivity. To date, aminoalkyl-phosphate (5) [4,25,26], esters
aminoacyl-sulfamoyl adenosines by conjugation with either trihy-
droxamate siderophore or McC hexapeptide, we consistently
observed the formation of a cyclic adenosine derivative as a side
product (9, Fig. 3) [17].
(
6a) [27e29], amide (6b) [27], N-alkoxy-hydroxamate (6c),
hydroxamate (6d) [27e29], aminoacyl-sulfamate (7a)
4,25,30e32], sulfamides (7b) [4], N-alkoxysulfamides (7c) and N-
2. Compound design
[
hydroxy sulfamide (7d) [33] surrogates have been used as a
replacement for the labile aminoacyl-phosphate (Fig. 2). Among
them, the aminoacyl-sulfamate (aaSA) bioisoster yielded the most
potent inhibitors with improved hydrolytic stability compared to
aminoalkyl and aminoacyl-adenylate. Unfortunately, these ana-
logues (aaSAs) could not reach the clinic due to their lack of
selectivity (in view of high structural similarity with aa-AMP) and
poor in vivo efficacy (due to lack of efficient cell-penetration).
Moreover, chemical synthesis of aaSAs is cumbersome due to
difficult purification and poor stability of aaSAs under acidic con-
ditions. Indeed, in our attempts to improve the in vivo efficacy of
As mentioned above, among the different non-hydrolysable
mimics of aa-AMP, the aminoacyl-sulfamoyl adenosines proved
to be excellent inhibitors of the corresponding aaRS in vitro.
However, these analogues could not be pursued further due to
their lack of selectivity and poor cell penetration. Several modi-
fications have been attempted to address these issues. For
example, a series of aryl-tetrazole containing sulfamate de-
rivatives reported by Cubist Pharmaceuticals showed 1000-fold
selectivity for pathogen aaRS over human aaRS. Despite the
high selectivity and excellent inhibitory potency in vitro, these
analogues could not reach the clinic due to high serum albumin
Fig. 2. Representative structures of various synthetic non-hydrolysable mimics of aa-AMP.

B. Gadakh et al. / European Journal of Medicinal Chemistry 93 (2015) 227e236
229
synthesized in almost quantitative yield in coupling thioacetic acid
via a Mitsunobu reaction [42] followed by deprotection of the
thioacetyl moiety by ammonolysis (Scheme 1). The obtained thiol
(13) upon oxidative chlorination using 1,3-dichloro-5,5-
dimethylhydantoin (DCDMH) in a mixture of acetonitrile: acetic
acid: water afforded the sulfonyl chloride [43] which without pu-
rification, was reacted with aqueous ammonia to yield the desired
sulfonamide (14) as key intermediate in relatively poor yield (32%).
The oxidative chlorination itself forms an excellent alternative to
fruitless attempts for chlorination of nucleoside sulfonates.
Fig. 3. Cyclic adenosine derivative formed during synthesis of transport module-aaSA
conjugates or upon acidic treatment.
Having the sulfonamide in hand, aminoacyl-sulfonamides were
synthesized analogously to a literature procedure [17] but using
orthogonal protecting groups for the side chain with respect to the
binding and poor cell penetration. In our previous report, aryl-
tetrazole containing sulfamates were coupled to either trihy-
droxamate or the McC hexapeptide (as transport modules) in an
attempt to improve their in vivo efficacy by a Trojan-horse
mechanism [34]. Unexpectedly, these conjugates failed to cross
the cell membrane. We therefore concluded that the adenine
base may be playing a vital role in recognition by the transporter
a-amino group. The aminoacyl-sulfonamides 15aef were obtained
by condensation of the respective N-hydroxy succinimide ester of
appropriately protected amino acids with the key sulfonamide 14
using DBU as a base in DMF. The Boc, tBu and acetonide protecting
groups were cleaved by acidolysis followed by hydrogenolysis of
the intermediates in a mixture of methanol-water containing cat-
0
(
being either an iron channel or the YejABEF peptide transporter
alytic acetic acid affording compound 15aef. Syntheses of 5 -O-(N-
in the above examples). Hence, conjugates of different
aminoacyl-sulfamoyl adenosines would seem an interesting
target, as they should be recognized by a transporter and once
internalized could act by a Trojan-horse mechanism. However,
trihydroxamate-aaSA conjugates could not be synthesized due to
instability of theses derivatives. Moreover, synthesis of McC
hexapeptide-aaSA conjugates proved low yielding [34,35] in part
as of formation of a cyclic degradation product by nucleophilic
attack of the adenine N on the sugar 5 -carbon. Similar problems
were encountered by Van de Vijver et al. when studying dipep-
tidyl-sulfamoyl adenosine as potential antibiotics [36], and aaSA
analogues as immunosuppressant [37] and by Vondenhoff et al.
while synthesizing hexapeptidyl conjugates of aaSA [35]. We
therefore aimed to improve the stability of the aaSA analogues
L-aminoacyl)-sulfamoyl adenosines have been well described
before [44e47].
4. Antibacterial assay
The antibacterial activities of all newly synthesized aa-
sulfonamides against Escherichia coli K-12 BW28357, Staphylo-
coccus aureus (ATCC6538), Sarcina lutea (ATCC 9341) and Candida
albicans CO11 were determined by measuring the optical density
reached by the cell suspension in the wells of microtiter plates in
the presence of various concentrations of the respective inhibitors.
These four strains were selected to cover the spectrum of activity
going from Gram-negative (E. coli) to Gram positive strains
(S. aureus and S. lutea), and to fungi (C. albicans). All strains were
grown on LB medium. As can be seen from Fig. 4, only the
aspartyl-sulfonamide 15f was active only against E. coli K-12
(panel F). The likely reason(s) for inactivity could be the lack of
cell-penetration of these analogues (similar to aaSA analogues) or
3
0
0
and hypothesized that deletion of the 5 -oxygen would render
0
3
the C-5 less electrophilic and prone to attack by N of adenine. In
general, sulfonamides are known to exhibit enzyme inhibitory
activity in view of their non-hydrolysable properties [38].
0
0
Therefore, deletion of the 5 -oxygen could yield aminoacyl-
loss of affinity of the compound due to deletion of the 5 -oxygen
sulfonamide with improved stability and hopefully equal po-
tency as compared to aaSAs.
resulting in reduced distance between the respective amino acid
and the adenine base. It has been reported in the literature that
ascamycin (4) could not penetrate the cell membrane whereas its
dealanyl analogue is able to penetrate the cell membrane and
showed broad-spectrum antibacterial activity. Thus, only the
AspSoA analogue 15f showed selective toxicity against E. coli, but
this is indicative that these analogues apparently can be trans-
ported across the cell membrane. Hence, loss of recognition by the
respective aaRS enzymes could be the likely reason for inactivity.
The results of the different broth dilution tests are summarized in
Fig. 4 and Table 1.
To understand the rationale for this disappointing lack of anti-
bacterial activity of all newly synthesized aaSoAs and to investigate
the probable mode of action of AspSoA 15f, in vitro aminoacylation
experiments were performed. Hereto, the ability of all compounds
to inhibit the corresponding aaRS in E. coli wt extract was deter-
mined. As shown earlier for aaSA analogues, the intracellular target
for the respective aaSA analogue is determined by the amino acid
moiety attached to sulfamoyl adenosine. The respective aaSA ana-
logues were used as the control compounds (Fig. 5).
To test our hypothesis, six aminoacyl-sulfonamides 15aef were
synthesized and tested for their ability to inhibit the corre-
sponding aaRS. Herein, sulfonamides 15aec are targeting IleRS,
LeuRS and TyrRS, respectively from class I of the tRNA synthetases,
whereas sulfonamides 15def target GlyRS, SerRS and AspRS
belonging to class II of the synthetases. Isoleucyl- (15a) and leucyl
(15b) sulfonamides were selected for their straight forward syn-
thesis while tyrosyl sulfonamide (15c) was selected for its aro-
matic side chain. All three aaRSs have been used as target in the
past of different medicinal chemistry efforts for inhibition of
various microorganisms. Glycyl-sulfonamide 15d was selected for
its small size and specifically for the considerable sequence
divergence of the hetero-tetramer as found in eubacteria
compared to the dimeric structure of human GlyRS [39]. Seryl-
(15e) and Aspartyl-(15f) sulfonamide were selected for their
polar side chain and as they were subject already of our previous
efforts for developing antibiotics based on either microcin C or
albomycin [34].
From the in vitro experiments, it can be concluded that deletion
0
of the 5 -oxygen of the sulfamate leads to a significant reduction in
3
. Chemistry
the inhibitory activity of these analogues as compared to the
respective aaSA analogue (with notable exception as found for the
AspSoA, 15f). Removal of the amino acid part of the aaSoA and aaSA
analogues provided the plain adenosyl sulfonamide and sulfamate
Following protection of adenosine (10) by an isopropylidene
moiety to afford compound 11 [40,41], the thiol (13) was

230
B. Gadakh et al. / European Journal of Medicinal Chemistry 93 (2015) 227e236
0
0
ꢀ
Scheme 1. Synthesis of 5 -aminoacyl-5 -deoxy-nucleoside sulfonamide. Reagent and conditions: (i) acetone, PTSA, DMP, rt, 24 h; (ii) PPh
3
, DEAD, thiolacetic acid, THF, 0 C, 1.5 h;
ꢀ
ꢀ
(
iii) methanolic ammonia/aq. ammonia (1:1) 0 C to room temperature (rt), overnight; (iv) (a) CH
3
CN/AcOH/H
2
O (40:1.5:1v/v), DCDMH, 1 h; (b) liquid ammonia, 0 C to rt, 1 h; (v)
2
Boc/Cbz-aa-(tBu/Bn)-OSu, DBU, DMF, 6e8 h; (vi) TFA/water (5/2 v/v), rt, 2.5 h (vii) Pd/C, methanol, cat. acetic acid, H atm. rt, overnight.
respectively, but these lead to further reduction in inhibitory ac-
tivity against all tested aaRSs (not shown).
activity. Moreover, replacement of the linking oxygen by a methy-
lene or by a nitrogen atom reduced the potency dramatically. They
concluded that the stereoelectronic properties of the sulfamate are
optimal to mimic the acyl-phosphate intermediate in the amino-
acylation reaction [4]. Furthermore, replacement of the sulfamate
oxygen with eNHOe or substituting the sulfamate linkage with an
ester or amide groups yielded analogues with either increased or
decreased distance between the sugar and the amino acid, both
leading to a significant decrease in potency [28,33]. Moreover,
replacement of the sulfamate oxygen with nitrogen also lead to a
significant loss of activity implying that sulfamide analogues
although having the same length, failed to mimic the negative
charge density of the acyl phosphate of aaAMP. Our results are in
good agreement with the literature, although it remains unclear
whether it is the overall length or the charge around the sulfon-
amide linkage which are the main culprit for the affinity loss of
5
. Discussion
All newly synthesized analogues except for AspSoA 15f which
showed selective toxicity against E. coli wt only. In view of AspSoA
showing growth inhibitory activity in a whole cell based assay, lack
of activity due to the lack of cell penetration is unlikely. Therefore
we assume that these analogues apparently can be transported
across the cell membrane and loss of affinity is the most probable
reason for the inactivity of these analogues. This has been further
confirmed by in vitro aminoacylation experiments. As can be seen
from these experiments (Fig. 5), the newly synthesized sulfon-
amide analogues failed to inhibit the corresponding aaRS. The most
likely reason could be the shortened linker length as compared to
the respective aaSA analogues which in turn could lead to the loss
these analogues. Therefore, the study of homoadenosine analogues
0
(
replacing the 5 -oxygen with a methylene moiety) occupying the
of H-bonding with the
recently also established that the
a
-amino group of the new compounds. We
-amino group is an important
same length as that of aaSA analogues is warranted.
a
To our surprise, only aspartic acid derivative 15f displayed some
selective toxicity against E. coli wt in a whole-cell assay and also
showed AspRS inhibition in the in vitro aminoacylation experiment.
In order to get some insight into the binding mode of the AspSoA in
the active site of AspRS, we performed some molecular simulations
on E. coli AspRS (PDB code 1c0a) [49]. We introduced AspSoA in the
active site and forced its nucleic base to coincide with the position
of the base in the AspSA structure. No steric clashes were observed
when entering of the AspSoA within the active site of AspRS. From
the superposition of AspSoA and AspSA in Fig. 6 we can conclude
that AspSoA and AspSA bind in a very analogous mode. Not all
models are available for comparison of the different inhibitors, but
inspection of the published crystal structure for the Aspartyl ade-
nylate in complex to AspRS [50] shows several ionic interactions
from the aspartyl side chain to different enzymatic residues. In the
AspSoA model these strong ionic interactions are still present and
may compensate better for the small change in position for the
aspartyl moiety of the new inhibitor compared to the situation for
the other tRNA synthetases used in this study (Fig. 7).
recognition point in the active site of aaRSs with N-methylation
annihilating the inhibitory activity [48]. Alternatively, deletion of
the 5 -oxygen provides aminoacyl sulfonamides which possibly fail
0
to mimic the stereoelectronic properties of the sulfamate apart
from the different overall length of the inhibitor. It has been re-
ported in the literature that the distance between the amino acid
and the sugar moiety is crucial for tight binding [4]. Elimination of
0
the 5 -oxygen leads to a decreased distance between the sugar and
the amino acid which therefore may result in steric clashes at the
amino acid binding site of aaRS. Moreover, the degree of puckering
in the ribose ring also plays an important role to achieve the desired
accuracy in the aminoacylation reaction [30].
Recently, Pope et al. developed a binding model for mupirocin
bound to IleRS. Based on this model they have designed IleRS in-
hibitors combining the structural features of mupirocin and IleSA
[4]. They further optimized the length and polarity of the linker for
IleRS inhibition. According to their data, the distance between the
sugar and the Ile moiety is crucial for tight binding. An increased
distance herein resulted in significant reduction in inhibitory

B. Gadakh et al. / European Journal of Medicinal Chemistry 93 (2015) 227e236
231
Fig. 4. Broth dilution tests to determine MIC of newly synthesized aaSoA analogues.
6
. Conclusion
Several aaSoA analogues have been synthesized and evaluated
7. Experimental section
7.1. Materials and methods
for antibacterial activity. Unfortunately, no inhibitory activity was
observed for these aaSoA analogues (except for AspSoA 15f)
either in in vitro or in whole-cell assays. From the in vitro ex-
periments it can be concluded that the loss of affinity for the
target is the most likely reason for inactivity of these analogues.
However, whether it is the overall length or the charge around
the sulfonamide which is the main culprit for the affinity loss
remains unclear.
Reagents and solvents were purchased from commercial
suppliers (Acros, SigmaeAldrich, Bachem, Novabiochem) and
used as provided, unless indicated otherwise. DMF and THF
were of analytical grade and were stored over 4 Å molecular
sieves. All other solvents used for reactions were analytical
grade and used as provided. Reactions were carried out in oven-
dried glassware under a nitrogen atmosphere with stirring at

232
B. Gadakh et al. / European Journal of Medicinal Chemistry 93 (2015) 227e236
Table 1
MIC50 values of all new aaSoA analogues against different microorganisms.
SN
Compounda
MIC50 (mM)
E. coli wt
S. aureus
S. lutea
C. albicans
1.
2.
3.
4.
5.
6.
15a
15b
15c
15d
15e
15f
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
~50 mM
a
Maximum inhibitor concentration tested was 100 mM.
room temperature, unless indicated otherwise.
Fig. 6. Superposition of AspSoA and AspSA in the active site of E. coli AspRS high-
lighting the close resemblance of the respective interactions.
1
13
H and C NMR spectra of the compounds dissolved in CDCl
OD, DMSO-d or D O were recorded on a Bruker UltraShield
Avance 300 MHz, 500 MHz or 600 MHz spectrometer. The chemical
shifts are expressed as values in parts per million (ppm), using the
residual solvent peaks (CDCl
3
,
CD
3
6
2
10 min. To the resulting yellow suspension a solution of thioacetic
d
1
13
acid (1.0 mL, 14.32 mmol) in dry THF (5 mL) was added drop wise
3
: H, 7.26 ppm; C, 77.16 ppm; DMSO:
1
13
1
1
ꢀ
and stirring was continued for another 1.5 h at 0 C. During this
H, 2.50 ppm; C, 39.52 ppm; HOD: H, 4.79 ppm; CD
.31 ppm; 13C, 49.00 ppm) as a reference. Coupling constants are
given in Hertz (Hz). The peak patterns are indicated by the
following abbreviations: bs broad singlet, doublet,
m ¼ multiplet, q ¼ quadruplet, s ¼ singlet and t ¼ triplet. High
resolution mass spectra were recorded on a quadrupole time-of-
flight mass spectrometer (Q-Tof-2, Micromass, Manchester, UK)
equipped with a standard ESI interface; samples were infused in 2-
3
OD: H,
time the yellow suspension cleared, and an orange solution was
obtained. At the end of the reaction the solvent was removed under
reduced pressure, and the resulting yellowish residue was purified
by flash chromatography on silica gel. The column was eluted with
3
¼
d
¼
CHCl
CHCl
3
:THF (4:1 v/v) followed by gradient of 2e10% methanol in
3
. The product containing fractions were evaporated to afford
2
.35 g (6.43 mmol, 99%) of the title compound as a white solid.
1
ꢁ1
H NMR (300 MHz, CDCl
.34 (s, 3H, COCH
3
)
d
1.39 (s, 3H, CH
3
), 1.60 (s, 3H, CH
3
),
2
propanol/H O (1:1) at 3 mL min .
0
2
3
), 3.19 (dd, 1H, J ¼ 6.9 Hz and 13.5 Hz, H-5 a), 3.30
For TLC, precoated aluminium sheets were used (Merck, Silica
gel 60 F254). The spots were visualized by UV light at 254 nm.
Column chromatography was performed on ICN silica gel 60A
0
0
(
dd, 1H, J ¼ 6.9 Hz and 13.5 Hz, H-5 b), 4.35 (dt, 1H, J ¼ 3.3 Hz, H-4 ),
0
4
2
8
.98 (dd, 1H, J ¼ 3.0 Hz, H-3 ), 5.53 (dd, 1H, J ¼ 6.6 Hz and 2.1 Hz, H-
0
0
), 5.66 (bs, 2H, 6-NH
2
), 6.06 (d, 1H, J ¼ 2.1 Hz, H-1 ), 7.90 (s, 1H, H-
6
0e200. Final products were purified using a PLRP-S 100 Å column
1
3
), 8.37 (s, 1H, H-2); C NMR (75 MHz, CDCl
3
)
d
24.5 (CH
3
), 26.2
connected to a Merck-Hitachi L6200A Intelligent pump. Eluents
compositions are expressed as v/v. Purity was checked by analytical
HPLC on an Inertsil ODS-3 (C-18) (4.6 ꢂ 100 mm) column, con-
nected to a Shimadzu LC-20AT pump using a Shimadzu SPD-20A
UV-detector. Recordings were performed simultaneously at
0
0
0
0
(CH
3
), 29.7 (COCH
3
), 30.4 (C-5 ), 82.9 (C-3 ), 83.4 (C-2 ), 85.3 (C-4 ),
0
90.1 (C-1 ), 113.7 (C(CH
3
)
3
), 139.2 (C-8), 152.4 (C-2), 154.7 (C-6),
1
93.7 (CO), C-4 and C-5 not detected; HRMS for C15
20
H N
5
O
4
S
þ
([MþH] ) calcd: 366.1230 found 366.1227.
2
54 nm and 214 nm.
0
0
0
0
7
.1.2. 5 -Deoxy-2 ,3 -O-isopropylidene-5 -mercapto-adenosine (13)
0
0
0
0
7
.1.1. 5 -Deoxy-2 ,3 -O-isopropylidene-5 -thioacetyl-adenosine (12)
[42]
[
42]
To an ice-cold solution of triphenylphosphine (3.76 g,
4.32 mmol) in dry THF (20 mL), diethyl azodicarboxylate (2.2 mL,
4.32 mmol) was added over 5 min. After stirring for 30 min, 11
2.0 g, 6.51 mmol) was added, and stirring was continued for
Compound 12 (8.4 g, 22.99 mmol) was dissolved in a mixture of
methanolic ammonia: aqueous ammonia (1:1, 100 mL) and was
ꢀ
1
1
(
stirred at 0 C for 1 h. After 1 h, the ice-bath was removed and the
reaction mixture was stirred at room temperature for 24 h while
monitoring for completion. The solvent was evaporated under
reduced pressure and the product was purified by column chro-
matography to yield 6.9 g (21.34 mmol, 93%) of the title compound
as a foam.
1H NMR (500 MHz, DMSO-d
)
d
1.32 (s, 3H, CH
), 1.52 (s, 3H,
6
3
0
CH
3
), 2.96 (dd, 1H, J ¼ 7.0 Hz and 13.5 Hz, H-5 a), 3.04 (dd, 1H,
0
J ¼ 7.5 Hz and 14.0 Hz, H-5 b), 4.33 (dt, 1H, J ¼ 2.5 Hz and 4.5 Hz, H-
0
0
4
), 5.01 (dd, 1H, J ¼ 2.5 Hz and 6.0 Hz, H-3 ), 5.50 (dd, 1H, J ¼ 2.0 Hz
0
0
and 6.0 Hz, H-2 ), 5.75 (bs, 1H, SH), 6.17 (d, 1H, J ¼ 2.0 Hz, H-1 ), 7.34
13
(
bs, 2H, 6-NH
2
), 8.17 (s, 1H, H-8), 8.30 (s, 1H, H-2); C NMR
0
(
3
125 MHz, DMSO-d
), 83.32 (C-2 ), 84.8 (C-4 ), 89.4 (C-1 ), 113.4 (C(CH
6
)
d
25.2 (CH
3
), 26.9 (CH
3
), 55.0 (C-5 ), 83.26 (C-
0
0
0
0
3
)
3
), 119.3 (C-5),
1
C
6
40.2 (C-8), 148.8 (C-4), 152.8 (C-2), 156.2 (C-6); HRMS for
þ
26
33
H N
10
O
6
S
2
(disulfide) ([MþH] ) calcd: 645.2020 found
45.2023.
0
0
0
0
Fig. 5. Inhibition of the in vitro aminoacylation reaction using E. coli wt S30 cell extract
at 50 M concentration of the respective inhibitors (sulfamate versus the corre-
sponding sulfon-amide congener) using the different tRNA synthetases in the order
IleRS, LeuRS, GlyRS, SerRS, TyrRS, AspRS, with bars showing the remaining % of amino
acid incorporation. For all experiments a control sample was used to set the respective
7.1.3. 5 -(sulfonamido)-2 ,3 -O-isopropylidene-5 -deoxyadenosine
(
m
14)
To an ice-cold solution of 13 (6.9 g, 21.34 mmol) in a mixture of
CN-HOAc-H O (104.0 mLe4.0 mLe2.7 mL) was added 1,3-
dichloro-5,5-dimethylhydantoin (6.31 g, 32.01 mmol) in two
CH
3
2
100% value (see experimental).

B. Gadakh et al. / European Journal of Medicinal Chemistry 93 (2015) 227e236
233
Fig. 7. Pictorial view (PoseView) of AspSoA docked in the active site highlighting the continued presence of the ionic interactions and most of the hydrogen bonds, compensating for
the slight positional shift compared to the known inhibitor AspSA. The green dots and dashed lines in addition display stacking of Phe229 with adenine, and a cationepi interaction
of adenine with Arg537, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
portions over 5 min. The cooling bath was removed and the reac-
tion mixture was allowed to warm to 20 C and stirred for 1 h. Next,
the coupled intermediate which was dissolved in a mixture of TFA/
water (5:2 v/v, 3.5 mL) at 0 C and stirred at room temperature for
ꢀ
ꢀ
the reaction mixture was added drop wise to ice-cold aqueous
ammonia over a period of 30 min and was stirred at 0 C for an
additional hour. The ice-bath was removed and the reaction
mixture was stirred at room temperature overnight. Next day, the
solvent was evaporated to dryness and the residue was subjected to
column chromatography to yield 2.53 g (6.84 mmol, 32%) of the
2 h. The volatiles were evaporated under reduced pressure fol-
lowed by coevaporation with toluene (2x) and ethanol (2x). The
yellow residue obtained was purified by column chromatography
and finally with RP HPLC using PLRP-S column to afford 24 mg
ꢀ
(0.06 mmol, 20%) of the title compound 15a as white solid.
1
H NMR (500 MHz, D
2
g
O)
-CH
-CH Hb), 1.79e1.88 (m, 1H, Ile-
d
0.75 (t, 3H, J ¼ 7.5 Hz, Ile-
d
-CH
-CH
3
), 0.82
Ha),
-CH), 3.57
title compound as a pale yellow solid.
(d, 3H, J ¼ 7.0 Hz, Ile-
3
), 1.01e1.11 (m, 1H, Ile-
g
b
2
1
H NMR (500 MHz, DMSO-d
6
)
d
1.33 (s, 3H, CH
3
), 1.54 (s, 3H,
1.32e1.41 (m, 1H, Ile-
g
2
0
CH
3
), 3.21 (dd, 1H, J ¼ 6.5 Hz and 14.0 Hz, H-5 a), 3.58 (dd, 1H,
(d, 1H, J ¼ 4.5 Hz, Ile-
a
-CH), 3.73 (dd, 1H, J ¼ 3.0 Hz and 15.0 Hz, H-
0
0
0
0
J ¼ 6.5 Hz and 14.0 Hz, H-5 b), 4.58 (dt, 1H, J ¼ 2.5 Hz and 6.0 Hz, H-
5 a), 3.84e3.91 (m, 1H, H-5 b), 4.44 (t, 1H, J ¼ 5.0 Hz, H-4 ),
0
0
0
0
4
), 5.17 (dd, 1H, J ¼ 3.0 Hz and 6.5 Hz, H-3 ), 5.50 (dd, 1H, J ¼ 2.0 Hz
4.49e4.53 (m, 1H, H-3 ), 6.07 (d, 1H, J ¼ 4.5 Hz, H-1 ), 8.23 (s, 1H, H-
13
0
0
and 6.0 Hz, H-2 ), 6.21 (d, 1H, J ¼ 2.0 Hz, H-1 ), 6.92 (s, 2H, SO
2
NH
2
),
8), 8.30 (s,1H, H-2); C NMR (125 MHz, D
2
O) -CH
d
10.8 (Ile-
d
3
),14.3
13
7
(
8
.36 (s, 2H, 6-NH
2
), 8.18 (s, 1H, H-8), 8.33 (s, 1H, H-2); C NMR
(Ile-
g
-CH
3
), 24.1 (Ile-
g
-CH
2
), 36.3 (Ile-
b
-CH), 54.6 (Ile-a-CH), 60.2
0
0
0
0
0
0
0
125 MHz, DMSO-d
3.3 (C-2 ), 84.1 (C-4 ), 89.6 (C-1 ), 113.2 (C(CH
6
)
d
25.2 (CH
3
), 26.9 (CH
3
), 57.4 (C-5 ), 81.9 (C-3 ),
), 119.3 (C-5), 140.3
(C-5 ), 72.9 (C-3 ), 73.0 (C-2 ), 79.3 (C-4 ), 88.2 (C-1 ), 118.9 (C-5),
0
0
0
3
)
3
140.4 (C-8), 148.9 (C-4), 152.9 (C-2), 156.6 (C-6), 175.3 (C]O);
ꢁ
(
(
C-8), 148.7 (C-4), 152.8 (C-2), 156.2 (C-6); HRMS for C13
[MꢁH] ) calcd: 369.0987 found 369.0985.
H
17
N
6
O
5
S
HRMS for C16
H N
24 7
O
6
S ([MꢁH] ) calcd: 442.1514 found 442.1518.
ꢁ
0
0
7
.1.5. 5 -(N-L-leucyl-sulfonamido)-5 -deoxyadenosine (15b)
0
0
7
.1.4. 5 -(N-L-isoleucyl-sulfonamido)-5 -deoxyadenosine (15a)
To a solution of sulfonamide 14 (100 mg, 0.27 mmol) in dry DMF
2 mL) were added DBU (102 L, 0.68 mmol) and Boc-Ile-OSu
177 mg, 0.54 mmol). The reaction mixture was stirred at room
This compound was synthesized analogously 15a. Yield: 48%.
1
H NMR (600 MHz, D
(d, 3H, J ¼ 6.6 Hz, Ile- -CH
(m, 1H, Ile- -CH Ha), 1.46e1.54 (m, 1H, Ile-
2
O)
), 1.03e1.10 (m, 1H, Ile-
-CH Hb), 3.51 (dd, 1H,
d
0.61 (d, 3H, J ¼ 6.6 Hz, Ile-
3
d-CH ), 0.75
-CH), 1.36e1.42
(
(
m
d
3
g
b
2
b
2
temperature for 8 h during which the reaction was monitored by
TLC. Next, the solvent was evaporated and the product was purified
by silica gel column chromatography (0e20% MeOH:DCM). The
fractions containing the desired product were evaporated yielding
J ¼ 4.2 Hz and 9.0 Hz, Ile-
a
-CH), 3.65 (dd, 1H, J ¼ 2.4 Hz and 15.0 Hz,
0
0
H-5 a), 3.94 (dd, 1H, J ¼ 9.6 Hz and 15.0 Hz, H-5 b), 4.40 (t, 1H,
0
0
J ¼ 4.8 Hz, H-4 ), 4.48e4.50 (m, 1H, H-3 ), 4.74 (t, 1H, J ¼ 4.8 Hz, H-
0
0
2 ), 6.08 (d, 1H, J ¼ 4.8 Hz, H-1 ), 8.24 (s, 1H, H-8), 8.29 (s, 1H, H-2);

234
B. Gadakh et al. / European Journal of Medicinal Chemistry 93 (2015) 227e236
13C NMR (150 MHz, D
O)
-CH
d
20.0 (Ile-
d
-CH
), 21.5 (Ile-
d
-CH
), 23.4
1H NMR (600 MHz, D
Asp- -CH
O)
d
2.53 (dd, 1H, J ¼ 9.0 Hz and 17.4 Hz,
-CH Hb),
2
3
3
2
0
0
(
7
Ile-
g
-CH), 39.9 (Ile-
b
2
), 53.7 (C-5 ), 53.9 (Ile-
a
-CH), 72.6 (C-3 ),
b
2
Ha), 2.77 (dd, 1H, J ¼ 3.6 Hz and 17.4 Hz, Asp-
b
2
0
0
0
0
0
2.7 (C-2 ), 78.9 (C-4 ), 87.6 (C-1 ), 118.4 (C-5), 139.9 (C-8), 148.4 (C-
3.72e3.82 (m, 2H, H-5 a and H-5 b), 3.89 (dd, 1H, J ¼ 3.6 Hz and
0
4
), 152.6 (C-2), 155.3 (C-6), 177.4 (C]O); HRMS for C16
H
24
N
7
O
6
S
8.4 Hz, Asp-
a
-CH), 4.43 (t, 1H, J ¼ 5.4 Hz, H-3 ), 4.53 (quin, 1H,
ꢁ
0
0
(
[MꢁH] ) calcd: 442.1514 found 442.1510.
J ¼ 4.2 Hz, H-4 ), 6.10 (d, 1H, J ¼ 4.8 Hz, H-1 ), 8.27 (s, 1H, H-8), 8.33
1
3
(
s,1H, H-2); C NMR (150 MHz, D
2
O)
d
35.9 (Asp-
b
-CH
2
), 52.6 (Asp-
0
0
0
0
0
0
0
7
.1.6. 5 -(N-L-tyrosyl-sulfonamido)-5 -deoxyadenosine (15c)
To a solution of 14 (200 mg, 0.54 mmol) in dry DMF (3 mL) were
added DBU (164 L, 1.08 mmol) and Boc-Tyr(Bzl)-OSu (379 mg,
.81 mmol). The reaction mixture was stirred at room temperature
a-CH), 53.7 (C-5 ), 72.55 (C-3 ), 72.58 (C-2 ), 79.0 (C-4 ), 87.9 (C-1 ),
118.6 (C-5), 140.3 (C-8), 148.3 (C-4), 151.3 (C-2), 154.4 (C-6), 174.7
þ
m
(C]O), 176.3 (b-COOH); HRMS for C14
H
20
N
7
O
8
S ([MþH] ) calcd:
0
446.1094 found 446.1090.
for 6 h during which the reaction was monitored by TLC. Next, the
solvent was evaporated and the product was purified by silica gel
column chromatography using MeOH:DCM as eluents. Fractions
containing the desired product were evaporated to yield interme-
0
0
7.1.10. 5 -(sulfonamido)-5 -deoxyadenosine (16)
Compound 14 (70 mg, 0.19 mmol) was dissolved in a mixture of
TFA and water (5:2 v/v, 1 mL) at 0 C and stirred at room temper-
ꢀ
diate which was dissolved in a mixture of TFA/water (5:2 v/v,
ature for 2.5 h. Next, the reaction mixture was co-evaporated with
toluene (3 times) and further co-evaporated with ethanol (3 times).
The residue was dissolved in ethanol, neutralized with TEA (0.5 mL)
and co-evaporated with toluene (2 times). The yellow residue ob-
tained was purified by column chromatography and finally with RP
HPLC using PLRP-S column to yield 24.5 mg (0.07 mmol, 40%) of the
title compound as a white solid.
ꢀ
3
.5 mL) at 0 C and stirred at room temperature for 2 h. The volatiles
were evaporated under reduced pressure followed by coevapora-
tion with toluene (3x) and ethanol (3x). The yellow residue ob-
tained was purified by column chromatography. The fractions
containing the desired product were evaporated to yield interme-
diate which dissolved in a mixture of methanol-water (4:1 v/v,
1
0
0
5
7
mL) containing glacial acetic acid (0.5 mL). To this Pd/C (10%w/w,
0 mg) was added and stirred under H atmosphere at room
H NMR (300 MHz, D
2
O)
d
3.75e3.91 (m, 2H, H-5 a and H-5 b),
0
0
2
4.46 (t, 1H, J ¼ 5.4 Hz, H-4 ), 4.53e4.61 (m, 1H, H-3 ), 6.07 (d, 1H,
0
0
temperature for overnight. Next, the catalyst was filtered off and
washed with a mixture of methanol: water (1:1 v/v, 10 mL). The
solvent was evaporated under reduced pressure followed by
coevaporation with toluene (3x) and ethanol (3x) to remove traces
of acetic acid and water. The crude product was purified by column
chromatography and finally by RP-HPLC to yield 81 mg (0.17 mmol,
J ¼ 4.5 Hz, H-1 ), 8.17 (s, 1H, H-8), 8.25 (s, 1H, H-2), H-2 merged in
13
0
0
D O signal; C NMR (75 MHz, D O) d 56.4 (C-5 ), 72.3 (C-3 ), 72.4
2
2
0
0
0
(C-2 ), 78.5 (C-4 ), 80.1 (C-1 ), 139.9 (C-8), 148.3 (C-4), 152.4 (C-2),
þ
155.1 (C-6), C-5 not detected; HRMS for C10
H
15
N
6
O
5
S ([MþH] )
calcd: 331.0819 found 331.0822.
3
1%) of the title compound 15c as a white solid.
7.2. Biological evaluation
1
H NMR (500 MHz, D
-CH
2
O)
d
1.88 (dd, 1H, J ¼ 10.0 Hz and 14.5 Hz,
Tyr-
b
2
Ha), 2.67 (dd, 1H, J ¼ 3.5 Hz, 14.5 Hz, Tyr-
b
-CH Hb), 3.57
2
7.2.1. Whole-cell based assay
(
dd, 1H, J ¼ 2.0 Hz and 15.0 Hz, Tyr-
a
-CH), 3.65 (dd, 1H, J ¼ 4.0 Hz
The bacterial strains were grown overnight in LB medium and
cultured again the following day in fresh LB medium. Compounds
were titrated in a 96-well plate using LB-medium to dilute the
0
0
and 10.0 Hz, H-5 a), 3.80e3.90 (m, 1H, H-5 b), 4.35 (t, 1H, J ¼ 6.0 Hz,
0
0
0
H-4 ), 4.40e4.45 (m, 1H, H-3 ), 5.99 (d, 1H, J ¼ 4.0 Hz, H-1 ), 7.95 (s,
1
3
1
H, H-8), 8.29 (s, 1H, H-2); C NMR (125 MHz, D
2
O)
d
0
35.5 (Tyr-
b
-
compounds. To each well containing 5
was added 85 L LB-medium to obtain a total volume of 90
Next, 10 L of bacterial cell culture grown to a density OD600 of
0.1 was added. The cultures were next placed into a Tecan Infinite
m
L of inhibitor solution,
0
0
0
CH
2
), 53.4 (Tyr-
a
-CH), 57.0 (C-5 ), 72.1 (C-3 ), 72.5 (C-2 ), 78.4 (C-4 ),
m
m
L.
0
8
7.1 (C-1 ), 115.4 (Tyr-ortho-C), 118.2 (C-5), 125.7 (Tyr-ipso-C), 130.0
m
(
Tyr-meta-C), 139.6 (C-8), 148.4 (C-4), 152.5 (C-2), 154.5 (Tyr-para-
-
®
ꢀ
C), 155.0 (C-6), 175.3 (C]O); HRMS for C19
calcd: 492.1307 found 492.1310.
H
23
N
7
O
7
S ([MꢁH] )
M200 incubator and shaken at 37 C, subsequently the OD600
was determined after 18 h. The broth dilution tests were per-
formed in triplicate. Bacterial strains used for the evaluations
were E. coli wt, Staphylococcus aureus (ATCC 6538), Sarcina lutea
(ATCC 9341) and Candida albicans CO11. The antibacterial activ-
ities of all compounds were determined by monitoring the op-
tical density of suspensions of cell-cultures in presence of
different concentrations of the tested inhibitors.
0
0
7.1.7. 5 -(N-L-glycyl-sulfonamido)-5 -deoxyadenosine (15d)
This compound was synthesized analogously to 15a. Yield: 16%.
1
H NMR (300 MHz, D
2
O)
d
3.59 (s, 2H, Gly-
a
-CH
2
), 3.76e3.82 (m,
0
0
0
2
H, H-5 a and H-5 b), 4.42 (t, 1H, J ¼ 5.1 Hz, H-4 ), 4.51 (q, 1H,
0
0
J ¼ 5.4 Hz, H-3 ), 6.06 (d, 1H, J ¼ 4.8 Hz, H-1 ), 8.18 (s, 1H, H-8), 8.26
13
(
s, 1H, H-2); C NMR (125 MHz, D
2
O)
d
42.5 (Gly-
a
-CH
2
), 54.0 (C-
0
0
0
0
0
5
), 72.55 (C-3 ), 72.63 (C-2 ), 78.8 (C-4 ), 87.9 (C-1 ), 118.4 (C-5),
7.2.2. Aminoacylation experiments
1
39.8 (C-8), 148.3 (C-4),152.4 (C-2),155.1 (C-6),172.7 (C]O); HRMS
To assess the degree of inhibition of the aminoacylation reac-
tion, in vitro tests were performed using the relevant S30 cell
extracts.
ꢁ
for C12
H
16
N
7
O
6
S ([MꢁH] ) calcd: 386.0888 found 386.0894.
0
0
7.1.8. 5 -(N-L-seryl-sulfonamido)-5 -deoxyadenosine (15e)
This compound was synthesized similar to 15c. Yield: 9%.
7.2.2.1. Preparation of S30 cell extracts. Cells were grown in 50 mL
LB-medium. After centrifugation at 3000 ꢂ g for 10 min the su-
pernatant was discarded and the pellet was resuspended in 40 mL
buffer containing: Tris.HCl or Hepes.KOH (pH ¼ 8.0) (20 mM),
1
0
0
H NMR (500 MHz, D
2
O)
d
3.58e3.64 (m, 2H, H-5 a and H-5 b),
0
3
.69e3.84 (m, 4H, Ser-
a
-CH, Ser-
b
-CH
2
and H-4 ), 4.41 (q, 1H,
0
0
J ¼ 5.4 Hz, H-3 ), 4.46e4.52 (m, 1H, H-2 ), 6.06 (d, 1H, J ¼ 2.4 Hz, H-
0
13
1
d
), 8.22 (s, 1H, H-8), 8.28 (s, 1H, H-2); C NMR (125 MHz, D
2
O)
2
MgCl (10 mM), KCl (100 mM). The cell suspension was centri-
0
0
0
54.0 (Ser-
a
-CH), 57.3 (C-5 ), 61.3 (Ser-
b
-CH
2
), 72.7 (C-2 and C-3 ),
fuged again at 3000 ꢂ g. This procedure was repeated twice. The
0
0
7
2
9.0 (C-4 ), 87.9 (C-1 ), 118.7 (C-5), 140.1 (C-8), 148.7 (C-4), 152.7 (C-
pellet was resuspended in 1 mL of the following buffer Tris.HCl or
-
), 155.4 (C-6), 175.8 (C]O); HRMS for C13
calcd: 416.0994 found 416.0998.
18
H N
7
O
7
S ([MꢁH] )
Hepes.KOH (pH ¼ 8.0) (20 mM), MgCl
2
(10 mM), KCl (100 mM),
DTT (1 mM) and kept at 0 C. Subsequently, the cells were soni-
ꢀ
ꢀ
cated for 10 s and left at 0 C for 10 min. This procedure was
0
0
7
.1.9. 5 -(N-L-aspartyl-sulfonamido)-5 -deoxyadenosine (15f)
repeated 5e8 times. The lysate was centrifuged at 15,000 g for
This compound was synthesized similar to 15c. Yield: 9%.
30 min at þ4 ꢀC.

B. Gadakh et al. / European Journal of Medicinal Chemistry 93 (2015) 227e236
235
7
.2.2.2. tRNA aminoacylation reaction. To 1
m
L of solution contain-
urinary tract infections, Antimicrob. Agents Chemother. 59 (2015) 289e298.
11] M. Teng, M.T. Hilgers, M.L. Cunningham, A. Borchardt, J.B. Locke, S. Abraham,
G. Haley, B.P. Kwan, C. Hall, G.W. Hough, K.J. Shaw, J. Finn, Identification of
bacteria-selective threonyl-tRNA synthetase substrate inhibitors by structure-
based design, J. Med. Chem. 56 (2013) 1748e1760.
[12] C.Y. Koh, J.E. Kim, A.B. Wetzel, W.J. de van der Schueren, S. Shibata,
R.M. Ranade, J. Liu, Z. Zhang, J.R. Gillespie, F.S. Buckner, C.L.M.J. Verlinde,
E. Fan, W.G.J. Hol, Structures of Trypanosoma brucei methionyl-tRNA synthe-
tase with urea-based inhibitors provide guidance for drug design against
sleeping sickness, PLoS Negl. Trop. Dis. 8 (2014) e2775.
[
ing inhibitor, 3 L of E. coli wt S30 extracts was added. Next, 16
the following aminoacylation mixture was added: Tris.HCl (30 mM,
pH 8.0), DTT (1 mM), bulk of E. coli tRNA (5 g/l), ATP (3 mM), KCl
m
mL of
14
(
30 mM), MgCl
2
(8 mM), and the specified, C-radiolabeled amino
acid (40 M, 200 mCi/mmol). The reaction products were precipi-
m
tated in cold 10% TCA on Whatman 3 MM paper, 5 min after the
aminoacylation mixture was added. The aminoacylation reaction
was carried out at room temperature. Depending on whether or not
processing was needed, variable time intervals were included be-
tween the addition of the cell extract and the addition of the
aminoacylation mixture. After thorough washing with cold 10%
TCA, the papers were washed twice with acetone and dried on a
heating plate. Following the addition of scintillation liquid (12 mL),
the amount of radioactivity was determined in a Tri-card 2300 TR
[
13] A. Abibi, A.D. Ferguson, P.R. Fleming, N. Gao, L.I. Hajec, J. Hu, V.A. Laganas,
D.C. McKinney, S.M. McLeod, D.B. Prince, A.B. Shapiro, E.T. Buurman, The role
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a novel auxiliary pocket in bacterial phenylalanyl-tRNA synthetase
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[
14] J. Tao, P. Schimmel, Inhibitors of aminoacyl-tRNA synthetases as novel anti-
infectives, Expert Opin. Investig. Drugs 9 (2000) 1767e1775.
[15] G.H.M. Vondenhoff, A. Van Aerschot, Aminoacyl-tRNA synthetase inhibitors as
potential antibiotics, Eur. J. Med. Chem. 46 (2011) 5227e5236.
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[
Bioscience, Georgetown, Texas, U.S.A, 2005.
14
liquid scintillation counter. C-Radiolabeled amino acids and
scintillation liquid were purchased from Perkin Elmer.
[17] P. Van de Vijver, G.H. Vondenhoff, T.S. Kazakov, E. Semenova, K. Kuznedelov,
A. Metlitskaya, A. Van Aerschot, K. Severinov, Synthetic microcin C analogs
targeting different aminoacyl-tRNA synthetases, J. Bacteriol. 191 (2009)
6
273e6280.
7
.3. Model building and analysis
[18] A. Tikhonov, T. Kazakov, E. Semenova, M. Serebryakova, G. Vondenhoff, A. Van
Aerschot, J.S. Reader, V.M. Govorun, K. Severinov, The mechanism of microcin
C resistance provided by the MccF peptidase, J. Biol. Chem. 285 (2010)
Pdb entry 1c0a with the E coli Asp-tRNA synthetase was used as
37944e37952.
0
template. The aspartyl-adenosine-5 -monophosphate was remod-
eled into a sulfonamide with AspSoA 15f by substituting the
phosphate group for a sulfonamide group as found in csd entry
[19] A. Metlitskaya, T. Kazakov, A. Kommer, O. Pavlova, M. Praetorius-Ibba, M. Ibba,
I. Krasheninnikov, V. Kolb, I. Khmel, K. Severinov, Aspartyl-tRNA synthetase is
the target of peptide nucleotide antibiotic microcin C, J. Biol. Chem. 281
(2006) 18033e18042.
0
BIVTAB (without O5 ). The Ade base and sugar are in the same
[20] S.F. Ataide, M. Ibba, Small molecules: big players in the evolution of protein
position as in the original 1C0A structure. The Asp-sulfonamide is
positioned by rotation of dihedral angles, so that the overlap with
the original substrate in the X-ray structure is optimized (no clashes
with the surrounding residues as verified by Chimera). Although
the sulfonamide linker is shorter due to the missing O5 , many of
the original hydrogen bonds of the aspartyl-adenosine-5 -mono-
synthesis, ACS Chem. Biol. 1 (2006) 285e297.
[
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Appendix A. Supplementary data
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