BIOMIMETIC G-QUARTET COMPOUNDS

Abstract

A compound of formula I:

##STR00001##

wherein A is present or absent; X1, X2, X3 and X4 are, independently from each other, an alkyl;

Y1, Y2, Y3 and Y4 are independently from each other a C1-C10 alkyl, -Z1, Z2, Z3 and Z4 are independently from each other a C1-C5 linear alkyl; R1 is a group allowing to carry out bioorthogonal reactions; and R2 is group including a N.

Claims

1.-12. (canceled)

13. A compound of formula I: ##STR00065## wherein A is absent or is a metallic cation, in particular a lanthanide used for optical imaging, in particular Eu.sup.3+, Tb.sup.3+, Dy.sup.3+, or Yb.sup.3+; X1, X2 and X4 are, independently from each other a C1-C3 linear alkyl; X3 is a C2-C3 alkyl, substituted by R1; Y1, Y2, Y3 and Y4 are independently from each other a C1-C10 alkyl, saturated or not, substituted or not by an acid group, possibly with a substitution of at least one carbon atom by N, P, a phenyl, a C═O group, phosphonate group, or a triazole group; Z1, Z2, Z3 and Z4 are independently from each other a C1-C5 linear alkyl; R1 is (CH.sub.2).sub.p-L-T, wherein p varies from 0 to 5; and L is a linear or branched, saturated or not, C1-C12 alkyl, possibly with a substitution of at least one carbon by: a C═O group or an heteroatom, an aryl group possibly substituted, a triazole group, or a diazirin group; T is a group allowing to carry out bioorthogonal reactions, in particular the following groups: azide, tetrazine substituted or not, alkyne, constrained alkyne such as cyclooctyne or cyclononyne, and in particular dibenzocyclooctyne, bicyclononyne, constrained cycloalkenes, such as trans-cyclooctene, norbornene, cycloproprene, and R2 is (CH.sub.2).sub.m-NHRx, wherein m varies from 1 to 4 and Rx is H or a protecting group such as Boc, Fmoc, Carboxybenzyl, or a guanidinium group, or a salt or a solvate thereof.

14. The compound according to claim 13, wherein Y1, Y2, Y3 and Y4 are independently from each other a C5-C10 alkyl, saturated or not, substituted or not by an acid group, possibly with a substitution of at least one carbon atom by N, P, a phenyl, a C═O group, phosphonate group, or a triazole group, and wherein A, X1, X2, X3, X4, Z1, Z2, Z3, Z4, R1 and R2 are as defined in claim 1.

15. The compound according to claim 13, said compound being of formula II: ##STR00066## wherein B′ is —(CO)—(CH2).sub.m-T, m varying from 1 to 6, and Y1, Y2, Y3, Y4, T and R2 are as defined above.

16. The compound according to claim 13, said compound being of formula III: ##STR00067## wherein: B′ is —W—(CH2).sub.m-T, m varying from 1 to 6, W═CO, C(═O)—NH, C(═S)—NH, or a group squaraine preferably CO, and Y1, Y2, Y3, Y4, T and R2 are as defined above.

17. The compound according to claim 13, of formula IV: ##STR00068## wherein B′ is —(CO)—(CH2).sub.m-T, m varying from 1 to 6, and R2 are as defined above, in particular the compound of formula IVa: ##STR00069##

18. The compound according to claim 13, of formula V: ##STR00070## wherein B′ is —(CO)—(CH.sub.2).sub.m-T, m varying from 1 to 6, and R2 are as defined above, in particular the compound of formula Va: ##STR00071##

19. The compound according to claim 13, said compound having one of the following formula: ##STR00072## ##STR00073## ##STR00074## ##STR00075## ##STR00076## ##STR00077##

20. The compound according to claim 13, coupled by click chemistry to: fluorescent imaging probes, such as cyanine, fluorescein, rhodamine, bodipy derivatives, a biotin derivative, a biological support such as for example entire antibody type biological molecules, fragments of antibodies, peptides, oligonucleotide, sugar moieties, or a solid support such as graphene or biochips.

21. The compound according to claim 20. having one of the following formula: ##STR00078## ##STR00079## ##STR00080## ##STR00081##

22. A kit comprising a compound according to claim 13, and in association with a compatible compound allowing click chemistry.

23. A method for identifying molecules for purifying molecules comprising a G quadruplex, said method comprising a step of contacting a molecule to be identified/purified with a compound according to claim 13.

24. A method for identifying molecules for in vitro and/or ex vivo imaging molecules comprising a G quadruplex structure, said method comprising: a first step of contacting a molecule comprising a G quadruplex structure to be identified with a compound according to claim 13, and a step of coupling said compound with a fluorophore, a biotin derivative, solid or biological support.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0083] FIG. 1 represents FRET-melting curves of experiments performed with the doubly labelled quadruplex F21T (0.2 μM) in Caco.K buffer without (black curves-A) or with 5 molar equivalents of TASQ (gray curves: .sup.PNADOTASQ (D): R=—H, X=—CONH—; BioTASQ (C): R=—CH.sub.2—NHCO—(CH.sub.2).sub.4-C.sub.5H.sub.7N.sub.2OS; X=—CONH; and MultiTASQ (B): R=—CH.sub.2—NHCO—(CH.sub.2).sub.3-CC; X=—CH.sub.2—CH.sub.2—. Thermal stabilization, expressed as ΔT.sub.1/2(in ° C.) are indicated for each TASQ (BioTASQ: 2.2° C., MultiTASQ: 8.9° C. and .sup.PNADOTASQ: 9.1° C.). Y-axis: Normalized FAM expression; X-axis: Temperature in ° C. ***: FRET.

[0084] FIG. 2 represents optical images of MCF-7 cells untreated (control: 1) or live-incubated with MultiTASQ (100 μM, 2-4: fields 1, 2 and 3; a-d: zooms from fields 1, 2 and 3) after fixation (PFA) and in situ click reactions performed with AlexaFluor488-azide® (1 μM) catalyzed by copper (CuSO.sub.4, 4 mM). Nuclei are counterstained with DAPI (light gray); accumulation of MultiTASQ highlighted by arrows in zooms a-d.

[0085] FIG. 3 represents a graph showing the normalized FAM emission of a probe FAM-myc-TAMRA (F-myc-T) over the temperature (° C.; X-axis) of the probe (A), Multi TASQ-compound VII (B) and MultiTASQ-compound VIII (C).

[0086] FIG. 4 represents a graph showing the normalized FAM emission of a probe FAM-duplex-TAMRA (F-duplex-T) over the temperature (° C.; X-axis) of the probe (A), MultiTASQ-compound VII (B) and MultiTASQ-compound VIII (C).

[0087] FIG. 5 represents a graph showing the normalized FAM emission of a probe FAM-VEGF-TAMRA (F-VEGF-T) over the temperature (° C.; X-axis) of the probe (A), MultiTASQ-compound VII (B) and MultiTASQ-compound VIII (C).

[0088] FIG. 6 represents three graphs showing the HPLC traces (monitored at 488 nm) of AlexaFluor488-azide® alone (upper panel, retention time=3.86 min), of MultiTASQ-compound VII alone (central panel, no absorbance) and of the mixture of the two compounds in presence of CuSO.sub.4 (a new product is formed with a retention time=2.89 min).

EXAMPLES

Example 1—Synthesis of Compounds According to the Invention

[0089] A-MultiTASQ-compound VII

##STR00055##

[0090] To a solution of Boc-.sup.PNAG(Z)—OH (1 g, 1.84 mmol) in methanol (50 mL) was added Pd/C (39.1 mg, 20 mol %). The suspension was stirred overnight at room temperature under H.sub.2. The solid was then filtered over dicalite and washed with methanol. The residue was concentrated under reduced pressure to afford Boc-.sup.PNAG-OH as a white solid (540 mg, 1.31 mmol, 72%).

[0091] ESI-LRMS: [M+H]+m/z=410.82 (calcd. for C.sub.16H.sub.23N.sub.7O.sub.6:410.40). .sup.1H NMR (500 MHz, d6-DMSO): b 1.37 (s, 9H), 3.20-3.80 (m, 4H), 3.99 (s, 2H), 4.85 (s, 2H), 6.46 (s, 2H), 6.95 (m, 1 H), 7.51 (s, 1H), 10.59 (s, 1H).

[0092] 5-((Tert-Butoxycarbonyl)Amino)Pentyl Methanesulfonate

##STR00056##

[0093] To a solution of 5-aminopentanol (1 g, 9.7 mmol) in CH.sub.2Cl.sub.2 (100 mL) was added triethylamine (1.5 mL, 10.7 mmol, 1.1 equiv.) and the solution was cooled down to 0° C. with an ice bath. Di-tert-butyl dicarbonate (2.114 g, 9.7 mmol, 1 equiv.) was added portion-wise and the solution was allowed to warm to RT and stir overnight at RT. The reaction completion was monitored and the reaction was added silica and the solvent was evaporated. The residue was purified by silica gel chromatography (CH.sub.2Cl.sub.2/MeOH 4% to 6%) to afford 5-((tert-butoxycarbonyl)amino)pentanol in 93% yield. 5-((tert-butoxycarbonyl)amino)pentanol (1 g, 4.5 mmol) was then dissolved in a mixture of THE (40 mL), pyridine was added (2 mL, 45.2 mmol, 10 equiv.), and MsCl (2 mL, 45.2 mmol, 10 equiv.) was added dropwise. The solution was allowed to stir over 48 h at RT. The THF was evaporated under vacuo, the mixture was then added 30 mL of ethyl acetate and 60 ml of acidified brine (30 mL of aq solution HCl 1M and 30 mL of brine). The aqueous phase was extracted 3 times with ethyl acetate (30 mL each), the organic phases were pooled together, dried over MgSO.sub.4, filtered, and dried under vacuo. The residue was purified by silica gel chromatography (CH.sub.2Cl.sub.2/MeOH 4% to 6%) to afford 5-((tert-butoxycarbonyl)amino)pentyl methylsulfonate in 57% yield (non optimised).

[0094] 1H NMR (500 MHz, Chloroform-d) b 4.16 (t, J=6.4 Hz, 2H), 3.06 (t, J=6.4 Hz, 2H), 2.94 (s, 3H), 1.80-1.60 (m, 2H), 1.49-1.42 (m, 2H), 1.38 (s, 9H).

[0095] Aminomethylcyclene Derivative

##STR00057##

[0096] To a solution of hexynoic acid (136 mg, 134 L, 1.2 mmol, 1 equiv.) and DIPEA (315 μL, 2.4 mmol, 2 equiv.) in DMF (4 mL) was added TSTU (404 mg, 1.3 mmol, 1.1 equiv.) and the solution was allowed to stir 1 h until complete conversion of the starting material. To a solution of AMC (aminomethylcyclene) in DMF at 0° C. was added dropwise the latter reaction mixture containing N-hydroxysuccinimide hex-5-ynoate (1 mL/3 hrs). The reaction was carefully monitored by RP-HPLC and quenched by addition of TFA (466 μL, 5 equiv.) as soon as disubstituted AMC appeared in the chromatogram. The solution was then concentrated under vacuum, added water and purified by RP-HPLC in a H.sub.2O/MeCN+0.1% TFA mixture (gradient of 1 to 25% over 20 minutes). After evaporation of the solvents, the AMC derivative was obtained (259 mg, 0.876 mmol, 72% yield) (exact amount of TFA not quantified).

[0097] .sup.1H NMR (500 MHz, D.sub.2O): δ3.26 (d, J=5.6 Hz, 1H), 3.14-2.61 (m, 15H), 2.26 (t, J=7.4 Hz, 2H), 2.21 (t, J=2.7 Hz, 1H), 2.09 (td, J=7.0, 2.7 Hz, 2H), 1.65 (td, J=7.4, 7.0 Hz, 2H)..sup.13C NMR (151 MHz, D.sub.20): δ177.04, 162.83, 162.60, 119.09, 117.16, 115.23, 84.58, 69.91, 52.26, 46.42, 44.34, 44.18, 43.89, 42.67, 42.08, 40.51, 39.01, 34.38, 23.75, 17.04. ESI-HRMS: [M+H]+m/z=296.2434 (calcd. for C.sub.15H.sub.30N.sub.5O: 296.2450).

[0098] Compound 19

##STR00058##

[0099] To a solution of previously prepared AMC derivative (47.2 mg, 0.16 mmol, 1 equiv.) in MeCN (1.16 mL) was added 5-((tert-butoxycarbonyl)amino)pentyl methanesulfonate (360 mg, 1.28 mmol, 8 equiv.) and K.sub.2CO.sub.3 (176 mg, 1.28 mmol, 8 equiv.) and the solution was allowed to stir for 48 h until complete conversion of the starting material. The crude mixture was filtered, and concentrated under vacuum, added water, and purified by RP-HPLC in a H.sub.2O/MeCN+0.1% TFA mixture (gradient of 1 to 100% over 20 minutes). After evaporation of the solvents, the compound 19 was obtained (75.2 mg, 0.07 mmol, 45% yield) (exact amount of TFA not quantified).

[0100] ESI-HRMS: [M+H]+m/z=1036.8098 (calc. for C.sub.55H.sub.105N.sub.9O.sub.9: 1035.8035).

[0101] Protected MultiTASQ-Compound VII

##STR00059##

[0102] A solution of compound 19 was stirred with 2 ml of TFA during 1 hour. After evaporation of the TFA, the unprotected compound 19 was obtained 78.5 mg, 0.072 mmol, 100% yield) (exact amount of TFA not quantified). HPLC-MS profiles page 33). Boc-.sup.PNAG-OH (145.55 mg, 0.35 mmol, 4.5 equiv.) and TSTU (107.04 mg, 0.35 mmol, 4.5 equiv.) were dissolved in DMF (1 mL), DIPEA was added (61 μL, 4 equiv.). After 1 hour, a solution of the deprotected AMC derivative (70.2 mg, 0.09 mmol, 1 equiv.) and DIPEA (61 μL, 4 equiv.) in DMF (1 mL) was added to the mixture. The mixture was stirred at RT for 3 days. The solution was then concentrated under vacuum, added water (2 mL), and purified by RP-HPLC in a H.sub.2O/MeCN+0.1% TFA mixture (gradient of 1 to 50% over 20 minutes). After evaporation of the solvents, the compound protected MultiTASQ was obtained (14.3 mg, 0.006 mmol, 8% yield) (exact amount of TFA not quantified).

[0103] ESI-HRMS: [M+H]+m/z=2202.2475 (calc. for C.sub.99H.sub.157N.sub.37O.sub.21: 2201.2427). .sup.1H NMR (500 MHz, DMSO-d.sub.6): b 10.80-10.62 (m, 4H), 8.09 (s, 3H), 7.86 (d, J=19.7 Hz, 2H), 7.59 (s, 3H), 6.48 (s, 8H), 4.95 (s, 3H), 4.79 (d, J=10.1 Hz, 3H), 4.17 (s, 2H), 4.10 (s, 2H), 3.90 (s, 2H), 3.86 (s, 3H), 3.71 (s, 2H), 3.61 (m, 4H), 3.46 (s, 3H), 3.26 (d, J=7.0 Hz, 2H), 3.21 (d, J=7.0 Hz, 5H), 3.13 (m, 7H), 3.03 (d, J=12.3 Hz, 8H), 2.97 (s, 4H), 2.88 (s, 3H), 2.79-2.70 (m, 5H), 2.68 (s, 2H), 2.60-2.51 (m, 9H), 2.17 (d, J=17.0 Hz, 4H), 1.72-0.98 (m, 64H).

[0104] MultiTASQ-Compound VII

##STR00060##

[0105] The protected MutiTASQ was stirred with 2 mL of TFA during 1 hour. After evaporation of the TFA, tituration in diethyl ether and drying under reduced pressure, the desired compound MultiTASQ was obtained 11 mg, 0.0064 mmol, 100% yield (exact amount of TFA not quantified). ESI-HRMS: [M+2H].sup.2+ m/z=901.0192 (calc. for C.sub.79H.sub.125N.sub.37O.sub.13: [M+2H].sup.2+=901.0128).

[0106] .fwdarw. Of note, Freshly prepared MultiTASQ aliquots must be prepared just before used.

[0107] Protected MultiTASQ is the privileged form for being stored.

[0108] B-MultiTASQ-Compound VIII

##STR00061##

[0109] To a solution of 3-(3-But-3-yn-1yl)-3H-Diazirin-3yl) propanoic acid (17.98 mg, 0.11 mmol, 1 equiv.) and DIPEA (40 μL, 0.22 mmol, 2.1 equiv.) in DMF (3 mL) was added TSTU (44 mg, 0.14 mmol, 1.3 equiv.) and the solution was allowed to stir 1 h until complete conversion of the starting material (monitored by HPLC-MS analyses). Then, this solution was added dropwise (3 hrs) to a solution of AMC (29.3 mg, 0.14 mmol, 1.3 equiv.) in DMF (3 mL) at room temperature. Again, the reaction was carefully monitored by HPLC-MS analysis. After completion, the solution was concentrated under vacuum and the crude mixture was purified by semi-preparative HPLC in a H.sub.2O/CH.sub.3CN+0.1% TFA mixture (Jupiter Proteo 4 μm 90 Å column, 250×21.2 mm; gradient of 2 to 35% over 35 minutes, retention time: 12.5 min). After evaporation of the solvents and lyophilization, the compound was obtained with 31% yield (27 mg, 0.03 mmol).

[0110] MALDI-ToF: [M+H]+m/z=350.276 (Calcd. For C.sub.17H.sub.32N.sub.7O: 350.266)

##STR00062##

[0111] HPLC-MS characterization (Phenomenex Kinetex C18 column, 2.6 μm, 2.1×50 mm; from 5% to 100% CH.sub.3CN/H.sub.2O+0.1% formic acid in 7 min): retention time=0.360 min; purity: >90% at 201 nm; m/z=350.3 [M+H]+.

[0112] To a solution of the previously prepared AMC derivative (34.2 mg, 0.04 mmol, 1 equiv.) in MeCN (1 μL) was added 5-((tert-butoxycarbonyl)amino)pentyl methanesulfonate (71.6 mg, 0.26 mmol, 6 equiv.) and K.sub.2CO.sub.3 (35.9 mg, 0.26 mmol, 6 equiv.). The solution was heated at 60° C. for 48 h and carefully monitored by serial HPLC-MS analyses. To improve the conversion, two additional equivalents of 5-((tert-butoxycarbonyl)amino)pentyl methanesulfonate were added and the mixture was let to stir for additional 24 h. After completion, the crude mixture was filtered, the resulting solution concentrated under vacuum and the residue purified by semi-preparative HPLC in a H.sub.2O/CH.sub.3CN+0.1% TFA mixture (Jupiter Proteo 4 μm 90 Å column, 250×21.2 mm; gradient of 5 to 60% over 50 minutes, retention time: 37 min). After evaporation of the solvents and lyophilization, the compound was obtained with 30% chemical yield (14.2 mg, 0.47 nmol).

[0113] ESI-HRMS: [M+H]+m/z=1090.83405 (calcd. for C.sub.57H.sub.105N.sub.11O.sub.8: 1090.83260).

[0114] HPLC-MS characterization (Phenomenex Kinetex C18 column, 2.6 μm, 2.1×50 mm; from 5% to 100% CH.sub.3CN/H.sub.2O+0.1% formic acid in 7 min): retention time=4.243 min; purity: >92% at 201 nm; m/z=1091.1 [M+H].sup.+.

##STR00063##

[0115] Protected MultiTASQ-Compound VIII

[0116] A solution of the previously prepared compound (15.1 mg, 0.014 mmol, 1 equiv.) was stirred in 500 μL TFA for 1 hour to deprotect the amines. After evaporation, the complete deprotection of the starting material was checked by HPLC-MS. The deprotected compound (9.66 mg, 0.014 mmol, 100% yield) was used without further purification. In the meantime Boc-.sup.PNAG-OH (22.5 mg, 0.55 mmol, 4 equiv.), TSTU (19.2 mg, 0.062 mmol, 4.4 equiv.) were dissolved in DMF (1 mL) and DIPEA was added (10 μL, 4 equiv.).

[0117] After 1 hour, the complete activation of the acid was assessed by HPLC-MS and the mixture was added to the solution containing the previously prepared deprotected compound (9.66 mg, 0.014 mmol, 1 equiv.) followed by the addition of DIPEA (10 μL, 4 equiv.) in DMF (1 mL). The mixture was stirred at RT for 3 days. The solution was then concentrated under vacuum, solubilized in a mixture of water and CH.sub.3CN (50/50, 2 mL), and purified by semi-preparative HPLC in a H.sub.2O/CH.sub.3CN+0.1% TFA mixture (Jupiter Proteo 4 μm 90 Å column, 250×21.2 mm; gradient of 5 to 15% over 5 min, then from 15 to 65% over 50 min, retention time: 29 min). After evaporation of the solvents, the protected MultiTASQ* was obtained in 4% chemical yield (1.36 mg, 0.56 nmol) (exact amount of TFA not quantified).

[0118] ESI-HRMS: [M+H+Na].sup.2+ m/z=1139.12403 (calcd. for C.sub.101H.sub.160N.sub.39NaO.sub.21: 1139.12689).

[0119] HPLC-MS characterization (Phenomenex Kinetex C18 column, 2.6 μm, 2.1×50 mm; from 5% to 100% CH.sub.3CN/H.sub.2O+0.1% formic acid in 7 min): retention time=3.680 min; purity: >99% at 280 nm; m/z=1128.7 [M+2H].sup.2+.

##STR00064##

[0120] MultiTASQ-Compound VIII

[0121] The protected MultiTASQ-compound VIII (1.95 mg, 0.9 nmol) was dissolved in TFA (200 μL) and the complete deprotection was assessed via HPLC-MS analyses. After completion, the mixture was diluted in water and the compound was lyophilized to offer MultiTASQ compound VIII as a white powder (2.0 mg, 0.9 nmol, 100%).

[0122] ESI-HRMS: [M+2H]2+m/z=928.03214 (calcd. for C.sub.81H.sub.128N.sub.39O.sub.13: 928.03105).

[0123] HPLC-MS characterization (Phenomenex Kinetex C18 column, 2.6 μm, 2.1×50 mm; from 5% to 100% CH.sub.3CN/H.sub.2O+0.1% formic acid in 7 min): retention time=0.627 min; purity: >98% at 280 nm.

Example 2—Fret Melting Assay

[0124] Material and Methods

[0125] The lyophilized DNA strands (purchased from Eurogentec, Seraing, Belgium) were firstly diluted at 500 μM in deionized water (18.2 MΩ.Math.cm resistivity). The DNA batch was prepared in a Caco.K buffer, comprised of 10 mM lithium cacodylate buffer (pH 7.2) plus 10 mM KCl/90 mM LiCl.

[0126] The quadruplex structure was prepared by mixing 40 μL of the constitutive strand (500 μM) with 8 μL of a lithium cacodylate buffer solution (100 mM, pH 7.2), plus 8 μL of a KCl/LiCl solution (100 mM/900 mM) and 24 μL of water. The final DNA concentration was theoretically 250 μM. The actual concentration of the DNA was determined through a dilution to 1 μM theoretical concentration through UV spectral analysis at 260 nm (after 5 min at 90° C.) with the following molar extinction coefficient values: 268300 M.sup.−1.cm.sup.−1 (F21 T). The higher-order DNA structure was folded as follows: solutions were heated (90° C., 5 min), cooled on ice (7 h) and then stored at least overnight (4° C.). FRET-melting experiments were performed in a 96-well format using a Mx3005P qPCR machine (Agilent) equipped with FAM filters (λ.sub.ex=492 nm; λ.sub.em=516 nm) in 100 μL (final volume) of Caco.K buffer with 0.2 μM of labeled oligonucleotide and 1 μM of TASQ (.sup.PNADOTASQ, BioTASQ and MultiTASQ). After a first equilibration step (25° C., 30 s), a stepwise increase of 1° C. every 30 s for 65 cycles to reach 90° C. was performed, and measurements were made after each cycle. Final data were analyzed with Excel (Microsoft Corp.) and OriginPro® 9.1 (OriginLab Corp.). The emission of FAM was normalized (0 to 1), and T.sub.1/2 was defined as the temperature for which the normalized emission is 0.5; ΔT.sub.1/2 values are means of 3 experiments (FIG. 1).

[0127] Results

[0128] The apparent affinity of each G4-ligands is quantified via fluorescence resonance energy transfer (FRET)-melting experiments. .sup.PNADOTASQ, BioTASQ and MultiTASQ were thus assayed against the dual-labeled F-21-T FAM-d[seq]-TAMRA, wherein [seq] is.sup.5′GGGTTAGGGTTAGGGTTAGGG.sup.3′ (SEQ ID NO: 1), in a dose-response manner (experiments were performed with 1 and 5 μM ligand versus 0.2 μM DNA, i.e. 5 and 25 mol. equiv. ligand). Results seen in FIG. 1 show that the quadruplex-stabilizing capacity (or apparent G-quadruplex affinity) of MultiTASQ (compound according to the invention) is restored as compared to BioTASQ (ΔT.sub.1/2=8.9 versus 2.2° C. for MultiTASQ and BioTASQ, respectively), in a manner that is comparable to the results obtained with the parent compound .sup.PNADOTASQ.

[0129] Additionally, MultiTASQ-compound VII and MultiTASQ-Compound VIII (MultiTASQ*) were assayed against the dual-labeled F-Myc-T FAM-d[seq]-TAMRA, wherein [seq] is .sup.5′GAGGGTGGGGAGGGTGGGGAAG.sup.3′ (SEQ ID NO: 2), F-duplex-T FAM-d[seq]-TAMRA, wherein [seq] is.sup.5′TATAGCTATATTTTTTTATAGCTATA.sup.3′ (SEQ ID NO: 3) and F-VEGF-T FAM-r[seq]-TAMRA, wherein [seq] is.sup.5′GGAGGAGGGGAGGAGGA.sup.3′ (SEQ ID NO: 4) at 5 μM concentration versus 0.2 μM DNA (i.e. 5 mol. equiv. ligand).

[0130] Results seen in FIGS. 3-5 show i-that the quadruplex-stabilizing capacity (or apparent G-quadruplex affinity) of both MultiTASQ and MultiTASQ* (two compounds according to the invention) are fully comparable (ΔT.sub.1/2=8.0 and 9.1° C. for MultiTASQ and MultiTASQ* with G-quadruplex-DNA; respectively; ΔT.sub.1/2=14.6 and 16.8° C. for MultiTASQ and MultiTASQ* with G-quadruplex-RNA, respectively); and ii-that both compounds are highly selective for G-quadruplex versus duplex (ΔT.sub.1/2 between 0 and 0.2° C.).

[0131] Example 3—Coupling MultiTASQ-Compound VII to a Fluorophore Via Click Chemistry

[0132] A 1:1 mixture (100 μM final concentration, in water) of AF488 azide (or Alexa Fluor™ 488 5-Carboxamido-(6-Azidohexanyl) bistriethylammonium salt) and MultiTASQ was stirred in presence of an excess of sodium ascorbate and CuSO.sub.4*5H.sub.2O for 1 h at RT. The complete conversion was assessed via HPLC analyses (Phenomenex Kinetex C18 column, 2.6 μm, 2.1×50 mm; from 5% to 100% CH.sub.3CN/H.sub.2O+0.1% formic acid in 7 min): retention time of the starting material=3.857 min; of the conjugate=2.713; conversion: >99% at 488 nm.

[0133] Results seen in FIG. 6 show that AF488 azide is fully converted into a higher polarity conjugate when mixed for 1 h at room temperature with MultiTASQ-CompoundVII in presence of copper catalyst (CuSO.sub.4) and sodium ascorbate, likely through an azide-alkyne Huisgen cycloaddition between the azide moiety of AlexaFluor488-azide® and the alkyne appendage of MultiTASQ-compound VII.

[0134] Example 4—In situ localization of G-quadruplex containing structures By using the compounds according to the invention, the inventors intended to detect, by fluorescent imaging, in situ localization of G-quadruplex containing molecules.

[0135] Material and Methods

[0136] Cell Culture and Fluorescence Microscopy

[0137] MCF7 cell line were obtained from the American Type Culture Collection (ATCC). Cells were cultured in 75 cm.sup.2 flasks (Corning) in DMEM (Life Technologies) supplemented with 5% synthetic feta bovine serum (FetalClone III, GE LifeSciences) and 100 U penicillin-streptomycin mixture (1.0 U.Math.mL.sup.−1 Pen/1.0 mg.Math.mL.sup.−1 Strep) at 37° C. in a humidified, 5% CO.sub.2 atmosphere-controlled incubator (HERAcell). The standard protocols were used for subculturing the cells: aspiration of medium, PBS (Gibco) wash, trypsinization in Trypsin-EDTA (0.25%) and reseeding in appropriate density. All cell counting was performed using the Coulter Counter (Beckman Coulter).

[0138] Click Imaging with MultiTASQ

[0139] MCF-7 cells were seeded on chambered coverglass (24 well-plate) and allowed to recover for 24 h. Cells were incubated with 100 μM MultiTASQ at 37° C. After 4 h, cells were fixed and permeabilized with paraformaldehyde (2% solubilized in 0.1% Triton X-100/PBS) for 5 min at room temperature. Treated cells were incubated during 30 min at room temperature with 1 μM alexafluor488-azide*+ Igepal*0,05% in PBS+4 mM CuSO.sub.4+10 mM Sodium ascorbate and rinsed with PBS 1×(thrice 5 min). Nuclei are counterstained with DAPI (1 μg/mL, 5 min). Cells were washed with PBS and mounted with Fluoromount-G (Southern Biotech). Confocal microscopy was performed either on a Zeiss LSM700 or on a Leica DMi8 microscope with the appropriate filters using the 63× objective. Foci quantification is done using Leica software.

[0140] G4RP Protocol with MultiTASQ

[0141] MCF7 cells were seeded at 3.5×10.sup.5 cells per 10-cm dish for 72 h. Cells were then crosslinked using 1% formaldehyde/PBS for 5 min at 25° C. and the crosslinking was then quenched with 0.125 M glycine for 5 min. Cells were scraped and resuspended in G4RP buffer (150 mM KCl, 25 mM Tris pH7.4, 5 mM EDTA, 0.5 mM DTT, 0.5% NP40, RNase inhibitor (Roche), homebrew protease inhibitor cocktail). Cells were then sonicated using Covaris m220 Ultrasonicator using default settings at 10% duty for 2 min. The sonicated fractions were then incubated with pre-clicked biotin conjugated beads (150 μM Biotin azide (Sigma), 5 mM E301 (Sigma), 4 mM CuSO4, 25 μM MultiTASQ (or 25 μM biotin for negative control)) overnight at 4° C. 10 μg of streptavidin-magnetic beads (Promega) was added and the extract was incubated for 2 h at 4° C. Magnetic beads were then washed 4 times in G4RP buffer for 5 min. The beads were then incubated at 70° C. for 1 h to reverse crosslink. TRIZOL was then used to extract the RNA from the beads using manufacturer's instructions.

RESULTS AND CONCLUSIONS

[0142] The in situ click images seen in FIG. 2 shown that MultiTASQ enters cells (live incubation) and accumulates in perinuclear regions (arrows) in a manner that was already described with the intrinsically fluorescent N-TASQ probe. More precisely, MultiTASQ interacts with accessible RNA G-quadruplexes within the cytoplasmic compartment, thereby altering their functionalities. As a consequence, ineffective RNA bound to TASQ accumulates in cytoplasmic granules known as processing bodies (or P-bodies), in which they will be either processed or degraded. This events thus triggers the accumulation of RNA/TASQ in clearly defined cytoplasmic foci, making them readily detectable via in situ click imaging after copper-catalyzed cycloaddition of alexafluor488-azide.