The use of ribonucleoside phosphoramidites protected at the 2$ sp prime$-hydroxyl with alkylsilyl ethers was developed for the solid phase synthesis of oligoribonucleotides. A series of pentadecameric oligomers of various base composition was synthesized using this methodology and a complete protocol for the deprotection of synthetic oligoribonucleotides was developed. The use of $ beta$-cyanoethyl phosphate protection was compared to that of methyl protection in the synthesis of pentadeca-uridylic acid sequences and no significant difference between the use of the two protecting groups was found. Concurrently a procedure for the derivatization of long chain alkylamine controlled pore glass beads with ribonucleosides was used to provide an improved solid phase support for the efficient automated synthesis of long oligomers. A side reaction on this support was identified and a method for its prevention developed. / A reaction occurring at the O$ sp6$-position of guanosine residues, which leads to chain cleavage, was identified. The basis of this reaction was characterized by both $ sp{31}$P nuclear magnetic resonance and the synthesis of oligoriboguanylate sequences. Three methods involving: protection of the O$ sp6$-position, the use of an alternate coupling cycle, and the use of a different phosphoramidite activator, were successfully applied to the resolution of this problem. The aforementioned procedures were utilized in the synthesis of a half E. Coli N- f - met tRNA molecule 43 units in length. / Finally the cumulative knowledge gained from the above studies was applied to the synthesis of an entire E. Coli N- f - met analogue 77 units in length along with a number of other very long sequences. Methods for the deprotection of these oligomers were investigated culminating in the isolation of oligoribonucleotides which were successfully characterized by polyacrylamide gel electrophoresis, enzyme degradation, enzymatic and chemical sequencing, terminal nucleotide analysis, and, in the case of the E. Coli f - met tRNA analogue, an aminoacylation assay.

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Written for the Dept. of Chemistry. Title from title page of PDF (viewed 2009/06/25). Includes bibliographical references.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009.

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The "modified triester" and phosphodichloridite procedures for oligoribonucleotide synthesis, using 2'- silylated nucleosides, were compared. Phosphodichloridite reagents were faster and more efficient than the arylsulphonyl reagents for both dinucleotide and mononucleotide phosphotriester synthesis. / The trichloroethyl group was better than tribromoethyl, p-nitrophenethyl or methyl protecting groups for solution-phase synthesis. Trichloroethyl dichlorophosphite was used in block condensations to prepare CCCGA and UCAUAA. / An automated RNA synthesizer was developed. Nucleoside methyl chlorophosphites were used with silica gel supports. Polystyrene, polyacryloylmorpholide and controlled pore glass supports were also used but were not as satisfactory. Oligoribonucleotides, up to 17 units long, were rapidly prepared in high overall yield. The sequences were purified by TLC and gel electrophoresis. Enzymatic degradations and HPLC confirmed the composition of the products.

<p> An improved method for the synthesis of oligoribonucleotides of defined sequence was developed. The general phosphotriester synthesis of Neilson and co-workers was modified by the introduction of a new condensing agent, mesitylenesulfonyl-1,2,4-triazole, and by the replacement of the bis(cyclohexylammonium) salt of mono-2,2,2-trichloroethyl phosphate with its acid salt. These modifications provided significant increases in the yields for the condensation of protected nucleosides - especially in the case of purine residues. Finally, modification of the three-step procedure for the deprotection of protected oligoribonucleotides resulted in the isolation of oligomers of exceptional purity and biological activity.</p> <p> Oligomers corresponding to natural sequences in transfer RNA molecules were obtained by this improved method of synthesis. These oligomers were then used to study: 1. The formation of short double-stranded RNA helices and 2. The interactions of aminoacyl-tRNA ligases with tRNA fragments.</p> / Thesis / Doctor of Philosophy (PhD)