L. Micouin, C. Tisnꢀ et al.
pH 6.5. For the measurement of the dissociation constant (K
d
) between
by NMR spectroscopy, a sample that con-
in 10 mm phosphate buffer pH 6.5 was titrat-
ed by increasing concentrations of ligand in the same buffer: 2, 4, 5, 7,
0, 15 and 20 mm. K values were extracted by non-linear least-square fit-
ting of the variation of the imino proton chemical shifts to a single-site
Lys
compound 1 or 2 and tRNA
3
Lys
tained 0.55 mm of tRNA
3
1
d
[
22]
binding hyperbolic function by using the MC-fit program. For the mea-
surement of the dissociation constant between compound 34 (by NMR)
Lys
or compound 38 (by fluorescence) and tRNA
3
, see the Supporting In-
formation.
Lys
Sample preparation: Human tRNA
3
was expressed in vivo in E. coli
[11]
from a recombinant plasmid and purified as previously described.
Chemistry
Typical procedure for the synthesis of compounds rac-29–38:
Compound 25: Compound 24 (373 mg, 1.7 mmol), which was prepared in
its racemic form according to ref. [14], N-benzyloxycarbonyl sarcosine
(
0
252 mg, 0.79 mmol), dicyclohexylcarbodiimide (DCC; 164 mg,
.79 mmol) and 4-dimethylaminopyridine (DMAP; 156 mg, 1.27 mmol)
were stirred in CH Cl (25 mL) for 16 h. The mixture was filtered, and
CH Cl (10 mL) was added, followed by a sat. aq NH Cl solution
(20 mL). After extraction of the aqueous phase by CH Cl , the organic
layers were dried on MgSO , filtered, concentrated and purified by
column chromatography (two successive elutions: cyclohexane/EtOAc
:3 then 6:4) to give 25 (88%).
Compound 27: 10% Pd/C (25 mg), was added to a solution of 25
250 mg, 0.48 mmol) in EtOAc (13 mL), and the mixture was stirred
2
2
Scheme 3. a) NaN
3
, H
2
O/acetone; b) CuSO
4
, sodium ascorbate; c) HCl,
2
2
4
MeOH, then amberlyst resin, then NH
3
/MeOH.
2
2
4
7
by linking together a 1,3-diaminocyclopentanic moiety and
an aromatic appendage. NMR spectroscopic detection ex-
periments proved to be extremely powerful for each step of
this process, and provided not only insights for fragment de-
tection, but also for linker length estimation and binding
site localisation. In this study, the binding-site specificity
seems to be governed by the aromatic part of the ligands,
which act as a molecular anchor, whereas the choice of an
appropriate linker will select an additional binding site rec-
ognised by the diaminocyclopentanic fragment, which acts
as a deoxystreptamine surrogate that is able to bind specifi-
cally to different sites of the target. Furthermore, the choice
of the linker seems to allow a fine-tuning of binding selectiv-
(
under a H2 atmosphere overnight, diluted with MeOH (1 mL), filtered
over Celite and rinsed with MeOH. The solvent was evaporated and the
crude was purified by column chromatography (two successive elutions:
EtOAc then EtOAc/MeOH 9:1) to give compound 27 (96%).
Compound 29: Phenyl vinyl ketone (53 mg, 0.13 mmol) was added to a
solution of 27 (60 mg, 0.45 mmol) in anhydrous CH Cl (10 mL), and the
2 2
resulting mixture was stirred for 16 h. The solvent was evaporated and
the crude was purified by column chromatography (two successive elu-
tions: EtOAc/MeOH 95:5 then EtOAc/MeOH 9:1) to give compound 29
(
63%).
Compound 34: Compound 29 (30 mg, 0.1 mmol) was dissolved in EtOAc
8 mL) and the solution was saturated with HCl , stirred for 15 min, and
(
g
concentrated to give 34 (hydrochloride, quantitative).
Typical procedure for the synthesis of compounds 39a–h: a-Bromo-2’-
ity. The use of this modular strategy for the design of com-
acetonaphtone (225 mg, 2 equiv) and NaN3 (61 mg, 2.05 equiv) were
Lys
pounds with improved specificity for the D-arm of tRNA
stirred at room temperature in H
Alkyne (160 mg, 1 equiv) in acetone (1.5 mL) was then added, followed
by the addition of sodium ascorbate (0.5 equiv) and CuSO (0.5 equiv).
2
O/acetone (1:2; 4.5 mL) for 1 h.
3
(
known to interact with the nucleocapside protein during
[7c]
4
the annealing process) or another possible binding site of
this target (for instance, the T-arm is known to be involved
in the early stage of the annealing process) is underway in
our laboratories.
The resultant mixture was then stirred at room temperature until com-
plete consumption of the alkyne (2 d) and was monitored by TLC
(EtOAc/cyclohexane 1:1). The precipitate was filtered, rinsed with H
and dried under vacuum. The crude product was then stirred for 1 h in a
m solution of methanolic HCl (6 mL). After evaporation of the solvent,
2
O
2
Amberlyst 15 resin (380 mg, 4 equiv) and MeOH (6 mL) were added and
stirred overnight. The resin was filtered, rinsed with MeOH (5ꢂ5 mL),
3
and stirred for 3 h in a 2m solution of methanolic NH (6 mL). The resin
was filtered and rinsed with MeOH. Evaporation of the organic phase
Experimental Section
led to 39 f as an oil (46%).
NMR spectroscopy experiments: Experiments were recorded on
a
For other detailed experimental procedures and characterisation data of
compounds 9–22, 25–38 and 39a–h, see the Supporting Information.
Bruker Avance DRX 600 spectrometer equipped with a 3 mm triple-reso-
nance flow-injection probe. The probe was connected to a Gilson 215
liquid handler controlled by the NMR spectroscopy console (Bruker
BEST system). For tRNA–ligand mixtures, solvent suppression was
ACHTUNGTRENNUNGa chieved by using the “jump-and-return” sequence to avoid the satura-
[
19]
tion of imino protons. All NMR spectroscopy experiments were con-
ducted at 158C. Samples for 1D NMR spectroscopic screening contained
0
.3 mm of tRNA and 1.2 mm of ligand in 10 mm phosphate buffer pH 6.5,
in a total volume of 200 mL in 96-well plates. The injected sample vol-
umes were 160 mL. For ligands that were identified in the primary screen,
Acknowledgements
1
15
[20]
[21]
H- N HMQC spectra or TROSY spectra were recorded by using a
This work was supported by the French AIDS national agency (ANRS),
the 6th framework program of the European Union (FSG-V-RNA) and
the MRT (ACI jeune chercheur to L. M., PhD grant to F. Chung).
1
5
sample that contained 0.2 mm to 0.4 mm N-labelled tRNA for an equiv-
alent of ligand concentration (1, 2, 4 and 7) in 10 mm phosphate buffer
7114
ꢁ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 7109 – 7116