Chemical synthesis of d(GC)₄,d(GC)₅ and d(GGTGGACCTC) by continuous flow solid phase phosphotriester method

Abstract

A simplified protocol for the synthesis of oligodeoxyribonucleotides by phosphotriester approach on controlled pore glass resins using a manualdna synthesiser is presented. The main features of this method are: (i) a single system of solvents (acetonitrile:dichloromethane, 8 : 2) is used in the assembly procedure reducing the number of mechanical manipulations, (ii) dichloroacetic acid is used as a good compromise between the efficiency of deprotection and minimal depurination and (iii) it competes effectively with the phosphite method in terms of speed, efficiency and ease. All the required protected mononucleotides and functionalised resins were home-made and detailed procedures are reported. The utility of the procedure is demonstrated by the actual synthesis of sequences d(G-C)₄, d(G-C)₅ and d(GGTGGACCTC) required for biophysical studies in our laboratory. The oligonucleotides were purified by the recently introduced method of fast protein liquid chromatography which gives good resolutions in shorter time periods as compared to the high performance liquid chromatography technique.