The overall goal of this Project is to develop versatile and efficient synthesis of oligonucleotides containing adducts of the diol epoxides of polycyclic aromatic hydrocarbons (PAHs) in which the site of attachment of th PAH moiety to the DNA and stereochemistry are rigorously defined. The general approach involves a reversal of the normal electrophile-nucleophile relationship of carcinogen and DNA; aminotriols derived from the diol epoxides are condensed with halopurine analogs of deoxyadenosine and deoxyguanosine. A post-oligomerization strategy is employed; the condensations are carried out after the halopurine deoxynucleosides have been incorporated into oligodeoxynucleotides. The method permits structurally defined adducts to be placed in any DNA sequence.
Four specific aims will be addressed. (1) Enantioselective syntheses of a number of the important PAH diol epoxides will be developed in the new grant period so that the individual enantiomers can be used as the starting point for preparation of adducted oligonucleotides rather than the racemic diol epoxides presently being used. (2) The post-oligomerization synthesis of oligonucleotides having PAH diol epoxide adducts on adenine N6 will be improved by altering the DNA synthesizer chemistry so that the halopurine-containing oligonucleotides can be deprotected and released from the solid support prior to the adduction reaction. This enhancement in the methodology will facilitate synthesis of adducted oligomers with fjord region aminotriols. (3) The post-oligomerization approach with sterically encumbered bay region aminotriols of PAHs gives only marginal yields for oligonucleotides having adducts on the N2 position of guanine although less sterically demanding amines give excellent results. Better leaving groups will be used on the purine ring which should cause this procedure to become the method of choice for synthesis of guanine-adducted oligonucleotides. (4) The final goal involves the possibility that poorly characterized, transient adducts, e.g., N1 adducts on adenine and O6 adducts on guanine, may arise in significant quantities from reactions of PAH diol epoxides with duplexed DNA and are sufficiently stable to contribute to the genotoxicity of PAHs. Attempts will be made to characterize these intermediates.
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