Methods of synthesis and characterization of an octadeoxyribonucleotide and a non-ionic hexadeoxyribonucleotide analog

Dean Vincent Carlson, Purdue University

Abstract

This thesis describes three studies on various aspects of the synthesis and characterization of oligodeoxyribonucleotides and a non-ionic DNA analog. First, the synthesis of the octadeoxyribonucleoside heptaphosphate d(TGACGTCA) by solid phase phosphoramidite chemistry is described. Preliminary NMR studies on this molecule included phase-sensitive COSY, DQF COSY, and NOESY. Most of the non-exchangable protons were assigned. The data indicates that the molecule exists predominantly in the duplex form at 24$\sp\circ$C, that the bases are all in the anticonformation, that the predominant sugar conformation of each residue is the S-form, and that all the backbone phosphates exist in a similar conformations. From these preliminary results, it was concluded that the duplex exists in a B-family helical conformation in solution. Second, the development of a modified solid support for oligonucleotide synthesis is described. This support offers a simplified route to 3$\sp \prime$-derivatives of oligonucleotides. Two $\gamma$-keto-esters, 4-oxopimelic acid and 3-acetoglutaric acid, were used to link 5$\sp \prime$-DMTr-dT to LCAA CPG. Comparison of the hydrazinolysis rates of these two linkages showed that the pimelate reacted roughly twice as fast as the glutarate. The cleavage of the ketopimelate was c. 65% complete within 10 min; in the same time period the glutarate reaction gave only 28% cleavage. Finally, the synthesis of 2$ \sp \prime $-deoxythymidinyl-($ O\sp{3 \sp \prime} \to N \sp{5 \sp \prime} $-carbamoyl)-tetra-(5$ \sp \prime $-amino-2,5$ \sp \prime$ -dideoxythymidinyl-($O \sp{3 \sp \prime} \to N \sp{5 \sp \prime} $-carbamoyl)) -5$ \sp \prime $-amino-2$ \sp \prime $,5$ \sp \prime $-dideoxythymidine, a non-ionic DNA analog, is described. The compound was prepared to test the possible biological activity of oligonucleoside polyurethanes and to study the importance of a charged backbone in base stacking phenomena. Synthesis of the compound from DMTr-dT and 5$ \sp \prime $-amino-2$ \sp \prime $,5$ \sp \prime $-dideoxythymidine involved the use of $N,N\sp \prime $-carbonyldiimidazole as the carbamate carbonyl synthon. The molecule exhibits a very low solubility in water and most organic solvents, but is soluble in basic aqueous solutions. Preliminary optical studies in a low ionic strength buffer show no interaction between the carbamate polymer and dA-(pdA)$\sb4 $-pdA or A-(pA)$\sb4 $-pA. In contrast to the parent compound dT-(pdT)$\sb4 $-pdT, the uncharged molecule exhibits no thermal or polymerization hypochromicity. These observations indicate that the thymine rings in the carbamate analog are not stacked.

Degree

Ph.D.

Advisors

Weith, Purdue University.

Subject Area

Organic chemistry

COinS