ISOLATION AND IDENTIFICATION OF AZIDO-AMSA/DNA PHOTOADDUCTS

ERIC WILLIAM KOLODZIEJ, Purdue University

Abstract

Acridines have been used extensively as dyes and medicines for many years. Recently, the acridine drug AMSA (N- 4-(9-acridinyl)amino-3-methoxyphenyl) methanesulfonamide) has shown promising activity against a variety of tumors, especially against refractory childhood leukemias. The majority of research on the activity of the acridines indicates that they act by intercalation between the base pairs of DNA. Although much is known about the relationship between intercalating agents and mutagenesis, little is known about the exact mechanism of mutagenesis. The reason for the lack of knowledge is that intercalating agents, like the acridines, bind non-covalently to DNA making it difficult to determine their site specificity. In order to gain information on the site and base specificity of these agents, photoaffinity labelling azidoacridines, including 3-azido-AMSA (N- 4-(3-azido-9-acridinyl)amino-3-methoxyphenyl) methanesulfonamide), have been prepared. Photoaffinity labelling agents have been used previously to study the interactions of small molecules with macromolecules and receptors. Photoreactions of azido-AMSA with simple nucleophiles like diethylamine, mononucleosides, dinucleotides, blocked nucleosides, and DNA were studied in this project to examine the chemistry of the activated azide and to determine the site of interaction. The extent of base modification by the azide is extremely low. For this reason, mass spectrometry was used as the primary method of analysis. MS has been increasingly used for the elucidation of the molecular structure of biomolecules due to its advantages of increased sensitivity and specificity over other analytical techniques. Preliminary EI, CI, and FAB mass spectral studies have been carried out with acridines, nucleosides and nucleotides, and oligonucleotides to establish instrumental conditions, to optimize sample preparation procedures, and to determine key fragmentation patterns that would help in the identification of the adducts. FAB MS, MS/MS, and high resolution FAB MS of the photoadducts produced for the reactions of azido-AMSA with mononucleosides and blocked nucleosides have been done. This data suggests that the sites of interaction of the azide are exocyclic amino groups of guanosine, adenosine, and cytidine. Reactions with DNA in solution and in ice show that azidoacridine-DNA adducts are formed in such small amounts that adequate adduct characterization by mass spectrometry was only possible in 30-50 percent of the samples. For this reason, no base specificity could be detected by these methods.

Degree

Ph.D.

Subject Area

Pharmacology

COinS