Substituted diazenylanilines as fluorescence quencher and use thereof

A process for the preparation of compound of general formula I, nucleic acid conjugates, complexes, and salts thereof,

• wherein R is selected from the group consisting of hydrogen and halogen;
• R1 and R2 are independently selected from the group consisting of hydrogen, substituted or unsubstituted (C1-C4) alkyl and (C1-C6) alkoxy;
• R3 is selected from the group consisting of hydroxy, halogen, (C1-C6) alkoxy, substituted or unsubstituted (C1-C4) alkyl, thioalkyl (SC1-C6), methylamino and dimethylamino;
• R4 is selected from the group consisting of hydrogen, hydroxy, halogen, (C1-C6) alkoxy, and substituted or unsubstituted (C1-C4) alkyl;
• m and n are independently selected from 0 to 3;
• Y1 and Y2 are independently selected from the group consisting of hydrogen, (C1-C6) alkyl, glycol, substituted or unsubstituted alkylaryl, oxo-alkanoic acid, epoxy, N-hydroxysuccinimide ester, N-hydroxybenztriazole ester, acid halide, acyl imidazole, thioester, p-nitrophenyl ester, alkyl ester, alkenyl ester, alkynyl ester, aromatic ester, phosphoramidite, mononucleotide unit, two or more mononucleotide units with or without separate phosphate or polyphosphate groups linked by nucleoside groups, comprising the steps of:
• a. reacting a solution of unsubstituted or substituted 4-nitroaniline in HCl with a solution of sodium nitrite in distilled water to form diazonium salts followed by reaction with substituted aniline to form a compound having general formula S1;
• b. reacting a mixture of substituted aniline with substituted alkylhalide in the presence of base to form a compound having general formula S2;
• c. reacting a compound having general formula S1 in HCl with a solution of sodium nitrite to form a diazonium salt, which was then reacted with a compound having general formula S2 in the presence of NaOAc buffer to obtain a compound having general formula I and
• d. isolating the compound of general formula I from the reaction mixture and purifying by washing with organic solvents or by chromatography.

A process for the preparation of conjugate compound of general formula I wherein Q is the quencher compound of formula 1 comprising the steps of:

• a. coupling of a compound of formula S4 with the amine functionality of the solid support CPG beads of formula S5 to produce S6 followed by deprotection of DMT group to obtain S7;
• b. reacting S7 with nucleotide phosphoramidites to form oligonucleotide S8 followed by 5′-modiciation with hexynyl-phosphoramidite to afford product S9 and
• c. treating S9 with abase for cleaving the oligonucleotide from the solid support to obtain S10 and
• d. reacting S10 with fluorescent dye azides to furnish oligonucleotides probes of general formula S12.