PURIFICATION OF 5'-O-TRITYL-ON OLIGORIBONUCLEOTIDES - INVESTIGATION OF PHOSPHATE MIGRATION DURING PURIFICATION AND DETRITYLATION

Synthetic oligoribonucleotides (RNA) are efficiently prepared with 2'- O-tert-butyldimethylsilyl nucleoside 3'-O-phosphoramidites with labile base-protection; A(dmf) or A(pac), G(dmf), C-ibu, U. After cleavage f rom the polystyrene support, the exocyclic amine protecting groups are removed with cone. NH4OH : ethanol/3:1 by heating at 55 degrees C for 3-5 h. The 2'-O-silyl protecting groups are removed with tetra-n-buty lammonium fluoride in THF or more conveniently with neat triethylamine trihydrofluoride. To gain the advantages of increased capacity on rev erse phase HPLC and the convenience of cartridge based purification (O PC, Oligonucleotide Purification Cartridge), the 5' trityl was left on the RNA as the final protecting group to be removed. The mild conditi ons which are effective for trityl removal are shown to preserve 3'-5' phosphate linkage integrity in RNA. The absence of phosphate migratio n is demonstrated by model studies, utilizing N-4-isobutyryl 2'-O-(2-c yanoethyl-N,N-diisopropylphosphoramidite) as a control monomer and dig estion by 3'-5' selective P1 nuclease and alkaline phosphatase and HPL C analysis. Oligoribonucleotides were analyzed by MicroGel capillary e lectrophoresis, anion-exchange HPLC, and the enzymatic digest/HPLC met hod.