Site-specific photomodification of single-stranded DNA targets by arylazide and perfluoroarylazide derivatives of oligonucleotides.

Highly efficient site-specific photomodification of single-stranded DNA targets was achieved with oligonucleotide reagents bearing aromatic azido groups (R (R1 = p-azidotetrafluorobenzoyl, R2 = 2-nitro-5-azidobenzoyl, R3 = p-azidobenzoyl) at either the terminal phosphate or at the C5 position of deoxyuridine at the end or inside of the oligonucleotide chain. The extent of modification strongly depends on the reagent type. It does not exceed 5% in the case of the reagent with R3. It was 25%-50% and 60%-70% for the reagents with R2 and R1 depending on the target structure. The reagent with perfluoroarylazido group R1 appeared to be most efficient. The extent of covalent adduct formation amounts to 70% for all reagents bearing a perfluoroarylazine group at the end of the oligonucleotide chain, independently of whether it was attached to the 3'- or 5'-phosphate or to the C5 of deoxyuridine. The reagents with the reactive group within the chain provided fewer cross-links (50%-55%). The reagents with R1 and R2 were found to be sensitive to the nucleotide structure of the target. Guanine and cytosine residues were modified preferentially when adjacent to the R1 or R2 group of the reagent, respectively.

Oligonucleotide derivatives bearing reactive and stabilizing groups attached to C5 of deoxyuridine.

Oligonucleotides bearing an aliphatic amino group at the C5-position of deoxyuridine (ULNH2TCCCA, TULNH2CCCA, ULNH2CCACTT, where L = -CH2-, -CH2OCH2CH2- or -CH2NHCOCH2CH2-) have been synthesized. The photoactive (p-azidotetrafluorobenzamido, 2-nitro-5-azidobenzamido, or p-azidobenzamido), alkylating [4-[N-(2-chloroethyl)-N-methylamino]benzyl], or intercalating [N-(2-hydroxyethyl)phenazinium] groups were attached to the amino linker of oligonucleotides. The Tm values were determined for the duplexes formed by the above oligonucleotide derivatives. The alkylating group does not change the melting temperature of the corresponding duplex. The duplex stability is increased a little in the case of photoactive groups. The influence of the phenazinium residue on the duplex stability strongly depends on its location in the oligonucleotide. The spacer length between the C5 atom of deoxyuridine and the photoactive or phenazinium group was shown to influence the complementary duplex stability.

The reactivity of phosphomono-and phosphodiester groups in oligonucleotides.

The rate constants were estimated by phosphorus NMR spectroscopy for the reactions of alcohols (Tr-dT, 2-cyanoethanol) in pyridine with the main types of the reactive phosphorylating intermediates formed by treatment of pdT-Ac, pdTpdT-Ac, Tr-dTpdT-Ac, Tr-dTpdTpdT-Ac with 2, 4, 6-triisopropylbenzene-sulfonyl chloride (TPS): 1) B type derivatives with phosphomono ester (PME) group converted to a phosphoryl pyridinium residue; 2) C type derivatives with PME and phosphodiester (PDE) groups converted to trisubstituted pyrophosphate; 3) D type derivatives with PDE groups converted to tetrasubstituted pyrophosphate. The two latter types are partially present as cyclic intramolecular pyrophosphates Ci and Di. The reactivity of the intermediates decrease in the series B greater than Ci approximately Di greater than C approximately D. The Ci derivative of pdTpdT-Ac when obtained in dimethylformamide was found to be rather stable to hydrolysis and could be separated from the other dinucleotide derivatives by ion-exchange chromatography. The Arrhenius parameters of all steps of the conversion of PME group of pdT-Ac to B derivative and of the reaction of TPS with PDE group of dinucleoside phosphate Tr-dTpdT-Ac were measured.