JAMES MACLEAN COULL, Purdue University


Fast atom bombardment mass spectrometry was useful for the identification of impurities in phosphoramidite monomer preparations obtained from the reaction of 5'-O-dimethoxy-trityl-N-acyl-deoxyribonucleosides with N,N-diethylaminomethoxychloro-phosphine. Many impurities were due to detritylation, oxidation of phosphorus containing species, and incomplete reaction of the starting nucleoside. Addition of potassium hydroxide to the glycerol sample matrix improved the reproducibility and intensity of the ion signals. The enzyme E. coli ribonuclease H cleaves the RNA strand of a RNA/DNA duplex and was used to develop an assay for the detec- tion of exposed single-stranded regions in RNA molecules. Synthetic oligodeoxyribonucleotides 5 to 6 residues in length, complementary to ten sites within the sequence of E. coli MRE600 5S RNA, were combined individually with the purified A and B conformers of the RNA and RNase H. Cleavage at sites in the RNA showed that the nucleotides of those sites were not involved in intramolecular base pairing. The data were used to predict a secondary structural model for the B conformer. The synthesis of a new phosphoramidite reagent, N-trifluoro-acetyl-2-aminoethyl-(2-cyanoethyl)-N,N-diisopropylaminophos- phoramidite, useful for the introduction of a primary aliphatic amino group at the 5' terminus of synthetic oligonucleotides is also described. The reagent was used in a solid-phase phosphorami- dite protocol to synthesize H(,2)NCH(,2)CH(,2)O-(pT-T(,4)-T). The amino-derivatized oligomer was quantitatively reacted with the N-hydroxy- succinimide ester of d-biotin to demonstrate the utility of the new reagent for the synthesis of oligonucleotide-small molecule adducts. Lastly, the high yield synthesis of non-ionic oligonucleotide analogue is presented. Reaction of 5'-O-dimethoxytritylthymidine with 1,1'-carbonyldiimidazole gave a 97% yield of the 3'-O-carbonyl- imidazolide, which was condensed in high yield with 5'-amino-5'-deoxythymidine to produce a dinucleoside containing a 3'-O-5'-N-carbamoyl linkage. Four repetitions of 3'-O-carbonylimidazolide formation and condensation produced, upon detritylation, a carbamate-linked hexamer of thymidine. Hydrolytic and thermal denaturation studies did not indicate the presence of intramolecular base stacking and the molecule did not interact in 10 mM sodium phosphate, pH 7.0, with A(pA)(,5) or d(pA)(,5) as determined by ultraviolet spectroscopy.



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