Structure of the product of intracomplex alkylation of target octanucleotide pd[TGTTTGGC] by means of 4-[N-methyl-N-(2-chloro-ethyl)-amino]benzyl-5′-phosphamido derivative of heptanucleotide pd[CCAAACA]: Two-dimensional1H-NMR spectroscopy and restrained molecular mechanics refinement

The spatial structure of a covalent adduct — the product of intracomplex alkylation at N-3-position of dC-8 nucleoside residue of target octanucleotide pd[TGTTTGGC] by means of 4-[N-methyl-N-(2-chloroethyl)amino]benzyl-5′-phosphamido derivative of heptanucleotide pd[CCAAACA] — has been investigated in aqueous solution by two-dimensional (2D)1H-NMR spectroscopy and restrained molecular mechanics calculations. By using COSY, COSY-DQF and NOESY experiments the assignment of oligonucleotide protons as well as protons of modifying group was carried out. The correlation times of benzylamide fragment protons and those of neighbouring nucleotide residues dC-1 and dC-8 were shown to be equal and shorter thanτ c of intraduplex nucleotides. The analysis of proton-proton coupling constants for H1′, H2′a, H2′b and H3′ protons showed all sugar residues to be in 2′-endo conformation. The distances between protons closed in the space were determined by means of a set of one-dimensional (1D) NOE experiments. The experimental distances were used as the constraints for energy minimization by molecular mechanics calculations. Eight conformations of benzylamide fragment of the covalent adduct differed with orientations of 4-N-methyl residue of the alkylating group and 1-methyleneamide linker function were constructed as starting structures in energy minimization procedure. Among of the resulting optimized structures only one S(Alk)8* was found to satisfy both experimental data and energetic criteria. The benzylamide fragment in conformation S(Alk)8* has been shown to localize in the region of lacked nucleoside residue of the duplex at the end of heptanucleotide chain less than 5 A apart from the residues dC-1 and alkylated dC-8.