Chemical synthesis of oligonucleotides containing a naphthalene diolepoxide deoxycytidine adduct in solution and using a mixed chemistry semi-automated solid phase approach

Abstract

I have recently reported the first total synthesis of a naphthalene diolepoxide--deoxycytidine nucleoside adduct suitable for incorporation into synthetic oligonucleotides. The racemic synthesis yielded the modified deoxycytidine nucleoside as a mixture of diastereomers (1a,b). I now report the conversion of the modified nucleosides (1a,b) into their phosphotriester salts (2a,b) and subsequent incorporation at a specific site into synthetic oligodeoxyribonucleotides. These syntheses yielded oligonucleotides possessing the diastereomerically modified nucleosides, N4-[(+/-)-1(b),2(a),3(a)-trihydroxy-4(b)-1,2,3,4-tetrahydronaphthyl]-2'- deoxycytidine (3a,b = C*), at a specific position. A sample of the modified nucleosides (3a,b) was obtained by chemically removing the protecting groups from (1a,b) and purification on C-18 reverse-phase chromatography. The structure of the modified nucleosides (3a,b) was supported by 400 MHz 1H-NMR and fast atom bombardment MS. The modified tetramers C*pGpApT (9a,b) along with the parent sequence CpGpApT were synthesized using the phosphotriester chemistry in solution. Interestingly, the diastereomerically modified tetramers (9a) and (9b) could be resolved using C-18 reverse-phase chromatography. Using a mixed chemistry semi-automated approach on a solid support the modified oligonucleotide 24mers, ApApTpTpGpCpApApGpTpC*pCpApTpApTpGpGpApCpTpTpGpC (10a,b), were synthesized and purified by anion exchange chromatography. The nucleoside composition of all synthetic oligonucleotides was quantitatively examined by reverse-phase chromatography following enzymic digestion.