SUGARS IN DNA BINDERS--STRUCTURE, FUNCTION, AND DESIGN

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Oligosaccharides are components of many antitumor antibiotics that bind to DNA. However, very little is known about the role of the oligosaccharides in any DNA binders. The long-term goal of the proposed research is to understand how the oligosaccharides in three specific antitumor antibiotics - calicheamicin gamma1, chromomycin A3, and ciclamycin 0 - are involved in DNA binding. The ultimate goal is to use this understanding to design simpler carbohydrate-based DNA binders with therapeutic activity. The specific aims of the proposed research are: I. Understanding the role of the oligosaccharide-aryl tail of calicheamicin gamma1 a) To synthesize analogues in which key structural features of the oligosaccharide-aryl tail (e.g., the N-O bond, the thiobenzoate ring) have been altered; b) To assess the effects of the structural changes on DNA binding and specificity using footprinting and affinity cleavage; c) to develop a structural model for the calicheamicin-DNA complex using NMR; the point is to relate the solution conformation of the oligosaccharide to the bound conformation and identify contacts to the DNA. II. Understanding the role of sugars in chromomycin A3 a) To determine which sugars of the C-D-E trisaccharide are minimally required for formation of a dimer metal complex in solution; to this end, UV/VIS, CD, and NMR spectroscopy will be used to characterize the metal complexes formed by degradation products of CRA3; b) To determine how dimer stability in solution relates to DNA binding affinity; to this end, the DNA binding affinity of the degradation products of CRA3 will be evaluated using UV/VIS spectroscopy; c) To synthesize a simplified analogue of CRA3 containing only the chromophore and C-D-E trisaccharide and evaluate its ability to dimerize and bind to DNA. III. Understanding ciclamycin 0 a) To synthesize ciclamycin 0; b) To determine whether ciclamycin 0 binds to DNA using 1-D NMR spectroscopy to monitor DNA imino protons upon addition of drug. If binding is indicated, the role of the trisaccharide in DNA binding will be investigated.