COMPARISON OF METHODS FOR PHOTOCHEMICAL PHOSPHORAMIDITE-BASED DNA-SYNTHESIS

Toward the goal of preparing surface-bound arrays of short oligonucleo tides using light-directed synthesis, we have studied novel photoremov able protecting groups for solid-phase DNA synthesis. We have protecte d the 5'-hydroxyls of all four nucleosides with our recently-described 3',5'-dimethoxybenzoin carbonate (DMB-carbonate) group and converted the products to (cyanoethyl)phosphoramidites. For comparison with anot her recently-described method for photochemical DNA synthesis, we have prepared the methylnitropiperonyl carbonate (MeNPOC) derivative of th ymidine and its amidite. To permit chain attachment to supports, the t hymidine DMB-carbonate succinoyl derivative was also prepared. High cy cle yields (based on release of the photoproduct dimethoxyphenylbenzof uran) were obtained in initial photochemical DNA synthesis studies wit h DMB-carbonates. An increase in the deprotection half-life was seen w ith increasing oligomer length, but this effect was scale-dependent an d should be minimal at the small scale at which most DNA arrays will b e prepared. The oligonucleotides resulting from these syntheses contai ned some impurities. It was shown that 350 nm irradiation does not pro duce thymine dimers but does damage benzoylcytidine. This problem is s olved through a change of protecting groups. However, other factors al so affect the quality of the DNA prepared by photochemical synthesis, since direct comparisons between short oligomers of thymine prepared w ith DMTr, DMB-carbonate, and MeNPOC protecting groups show that photoc hemical deprotection results in diminished cycle yields compared to ac id deprotection.