Development of new routes to end-conjugated and phosphate-modified oligodeoxyribonucleotides

Keith Andrew Ray, Purdue University

Abstract

Methods for preparing modified oligodeoxyribonucleotides have been investigated. It was shown that 5$\sp\prime$-conjugates of oligonucleotides could be made using phosphoramidite reagents. This idea was tested with a phosphoramidite prepared from 2-(9-acridinyl) ethanol. The 3$\sp\prime$ end of an oligonucleotide was derivatized by both phosphoramidite and carbamate chemistry. Both methods rely on a modified solid support. This support incorporates a 4-oxopimelate linkage that allows the release, by hydrazinolysis, of an oligonucleotide with all its protecting groups intact, but having an exposed 3$\sp\prime$-hydroxyl. Low yields in subsequent derivatization reactions led to the identification of a side product where the 3$\sp\prime$-hydroxyl was blocked. Redesign of the solid support was undertaken to circumvent this problem and several approaches are described; the most promising is based on 5-bromolevulinic acid. This modified support also plays a prominent role in the synthesis of phosphate-methylated oligonucleotides. In contrast to other synthetic procedures in which the methyl phosphotriesters are formed en masse on a normal, phosphodiester-linked oligonucleotide, our method uses special conditions to make the compounds stepwise on an automated synthesizer. The feasibility of doing this was demonstrated first with thymidine O-methylphosphoramidites used in conjunction with the pimelate release resin. Hydrazinolysis left the methyl esters intact. Steps were taken to extend this approach to the synthesis of heterooligonucleotides, but stability problems that arose in the work with thymidine oligomers have cast doubt on the viability of the project.

Degree

Ph.D.

Advisors

Weith, Purdue University.

Subject Area

Pharmacology|Organic chemistry|Biochemistry

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