[6] Improved phosphotriester method for the synthesis of gene fragments

This chapter discusses an experimental study focusing on the improved phosphotriester method for the synthesis of gene fragments. During studies with a modified triester approach, it was observed that the pure, fully protected product was difficult to separate quantitatively by the conventional silica gel chromatography from the crude reaction mixture, especially from the components containing several guanine bases. It was found that thin-layer chromatography (TLC) on silanized silica gel (RP-2) and KC18 (RP-18) plates in an acetone–water solvent gave excellent separation of the components containing trityl and hydroxyl groups and also of those differing in sizes. The polar component (containing 3′ phosphodiester) generally moved to the solvent front, and the fully protected component (containing the trityl group) was the slowest. The mobility of a component containing a 5′-hydroxyl group was between that of the polar and nonpolar compounds. The purification of GGCA could not be achieved on silica gel as determined after complete deblocking and TLC on a polyethyleneimine (PEI), whereas purification by TLC on reverse-phase (RP-2) plates gave pure GGCA as analyzed on a PEI plate. These results indicated that reverse-phase chromatography of a triester oligonucleotide could yield a pure product at each step of synthesis.