Molecular specificity studies on modified nucleosides

Whi-Gun Chae, Purdue University

Abstract

I. Chemical modifications of DNA with methyl methanesulfonate (MeMS) and methylnitrosurea (MeNU) have been studied in vitro. We have developed a reverse-phase ion-phase ion-paired HPLC method for separating many modified deoxynucleosides. The structural specificity of HPLC is significantly enhanced by conjunction with other biochemical and physical methods. We have studied the specificity of enzymatic transformation of methyldeoxyribonucleosides by adenosine deaminase and purine nucleoside phosphorylase. All enzymic transformations were analyzed by HPLC using a photodiode array UV detector. The UV spectra and the retention time of the corresponding reference compound were used to verify the structural assignment of the product. In some cases, the enzymic reactions were also carried out in an NMR tube and directly monitored by $\sp1$H NMR. Simultaneous determination of many minor modified nucleosides was, however, not possible by HPLC. This problem has been overcome by tandem mass spectrometry (MS/MS). In conjunction with the resolving power of HPLC in the separation of isomers, desorption chemical ionization tandem mass spectrometry has been utilized in determination of the modified nucleosides at the picomole level using stable-isotope labeled compounds as internal references. II. A simple method for the production of modified dideoxyribonucleosides by either an enzymatic dideoxyribosyl transfer reaction or a simple chemical reaction was proposed. The reaction was monitored by HPLC and, in some cases, $\sp{19}$F NMR simultaneously. The product was isolated and purified by preparative HPLC and its structure was confirmed by $\sp1$H NMR (500 MHz) including 2D-NOE methods, and FAB-MS including high resolution mass measurement. Conformational analysis of dideoxyribose moieties was carried out from the observation of the coupling constant. The procedures were further applied to obtain new derivatives which have significant biological activity such as 2$\sp\prime$,5$\sp\prime$-dideoxy-6-thioguanosine and 2$\sp\prime$,5$\sp\prime$-dideoxy-1,$N\sp6$-ethenoadenosine. In some cases, facile chemical synthesis of 2$\sp\prime$,3$\sp\prime$-dideoxy-1,$N\sp6$-ethenoadenosine and 3$\sp\prime$-deoxy-1,$N\sp6$-ethenoadenosine were used as alternative methods. These modified nucleoside analogs have not been reported by others yet except for 2$\sp\prime$,5$\sp\prime$-dideoxy-5-fluorouridine.

Degree

Ph.D.

Advisors

Chang, Purdue University.

Subject Area

Analytical chemistry|Biochemistry

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