A novel acyl protecting group for cytosine and adenine has been prepared from 4-( chloromethyl) benzoic acid. Reaction of the acid with morpholine produces 4-( 4-morpholinyl )methyl benzoic acid which is converted to it's acid chloride with thionyl chloride. This may be used to acylate cytidine and adenosine under standard conditions. This ionizable protecting group has the ability to solublize protected oligomers, which allows their purification with ion-exchange chromatography on S-Sepharose. Solid phase synthesis has been performed using this protecting group on morpholine nucleosides. Morpholine nucleoside carbamates were synthesized in high yields. The results revealed that high purities of the hexamers were obtained. These hexamers, which have the protecting groups intact, provide the potential for further segment condensation to make large size oligonucleotide analogues. The success of the solid phase synthesis was dependent upon use of a selectively cleavable anchor which allowed the finished oligomer to be released from the resin with protecting groups intact. Attempts to modify the anchor to make it more efficient were unsuccessful. It was found that DBU degrades derivatized polystyrene resin and should not be used at early stages in solid phase synthesis. / Graduation date: 1992

During the past two decades, oligonucleotide analogues have drawn considerable attention as potential therapeutic and diagnostic agents. Gene silencing through "RNA interference" (siRNA) or the more mature "antisense" technology (AONs) have proven to be powerful tools for studying gene functions. Chemical modifications of these compounds are generally required to improve their "drug-like" properties such as potency, selectivity and delivery, particularly in the development of oligonucleotide-based therapeutics. Aptamers are another emerging class of oligonucleotide therapeutics and diagnostics. / This thesis focuses on oligonucleotides containing 1-(2-deoxy-2-alpha-C-hydroxymethyl-beta- D-ribofuranosyl)thymine (2'-alpha-hm-dT, abbreviated as "H") and 2'-deoxy-2'-fluoroarabinonucleotides (2'F-araN), and their applications. A major component of this work focused on the synthesis of 2'-alpha-hm-dT (H) and the first investigation of oligoribonucleotides containing this nucleoside analogue. Specifically, 2'-CH2O-phosphoramidite and 3'-O-phosphoramidite derivatives of H were synthesized and incorporated into both 2',5'-RNA and RNA chains. Incorporation of 3',5'-linked H units into a DNA, 2',5'-RNA or RNA strand led to significant destabilization of duplexes formed with unmodified RNA targets. 2',5'-Linked H units into 2',5'-RNA or RNA caused significantly less destabilization, and in fact, they were shown to stabilize the loop structure of some RNA hairpins. These results were rationalized in terms of the "compact" and "extended" conformations of nucleotides. / A series of branched RNAs (Y-shaped) related to yeast pre-mRNA splicing intermediates were synthesized incorporating both natural (i.e., ribose) and nonnatural (i.e., H, and acyclic nucleoside) branch points in order to examine the effect of sugar conformation and phosphodiester configuration on yeast debranching enzyme (yDBR) hydrolytic efficiency. The results indicate that 2'-phosphodiester scission with yDBR occurs only with a ribose-phosphate backbone at the branch point, whereas some of the H-containing branched RNAs were found to competitively inhibit yDBR hydrolytic activity. / This thesis also examines the stabilization of DNA guanine-quadruplexes (G-quadruplexes) by replacing the deoxyribose sugar by a 2-deoxy-2-fluoroarabinose. The effect of this substitution was assessed in the well-known thrombin-binding DNA aptamer d(G2T2G2TGTG2T 2G2), the telomeric DNA d(G4T4G 4) sequence and a phosphorothioate octanucleotide PS-d(T2G 4T2), all of which are known to fold into G-quadruplex structures. Stabilization of the G-quadruplexes was possible provided that the arabinose sugar was introduced at guanosine residues adopting an anti N-glycosidic bond conformation. Some of the arabinose modified thrombin-binding aptamers not only exhibited superior thermal stability and nuclease resistance, but also maintained high thrombin binding affinity. / Finally, this thesis examines the ability of DNA polymerases to recognize and utilize 2'-deoxy-2'-fluoro-beta-D-arabinonucleoside 5'-triphosphates (2'F-araNTPs) as building blocks for the synthesis of 2'-deoxy-2'-fluoro-beta- D-arabinonucleic acids (2'F-ANA). The results obtained indicate that a few DNA polymerases can synthesize 2'F-ANA and 2'F-ANA-DNA chimeras on a DNA template. Conversely, certain enzymes were shown to catalyze 2'F-ANA template-directed DNA synthesis. While it was not possible to synthesize 2'F-ANA strands on a 2'F-ANA template, it is possible for some DNA polymerases to catalyze the formation of multiple 2'F-ANA:2'F-ANA base pairs within a DNA-FANA chimeric duplex. These results suggest that it should be possible to evolve FANA-modified aptamers via SELEX.

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