Our laboratory has focused on the development of synthetic methods which make possible the relatively facile synthesis of PAH-adducted nucleosides, as well as site-specifically adducted oligonucleotides in any desired sequence context on a multi-milligram scale, suitable for NMR and X-ray crystallographic studies as well as for biochemical investigations. The approach taken is to prepare the appropriate adducted nucleosides in suitably protected form and to convert them to phosphoramidites for use in solid phase oligonucleotide synthesis. This year we have reported highly stereoselective methods for the synthesis of deoxyadenosine (dAdo) and deoxyguanosine (dGuo) adducts and their phosphoramidites derived from the prototypical carcinogenic PAH, benzo(a)pyrene (BaP). One such method involves the use of fluorinated alcohols as solvents for the ring opening of BaP diol epoxides by the exocyclic amino groups of protected purine nucleosides (1). By varying the molar ratios of trifluoroethanol (TFE), perfluoro-tert-butanol or hexafluoro-2-propanol (HFP) to diol epoxide, large changes in the stereoselectivity for cis vs. trans addition of the exocyclic 2-amino group of protected dGuo at the benzylic C10 position of BaP could be achieved. By use of this approach in combination with a newly developed and highly efficient HPLC separation method, the four possible phosphoramidites (cis/trans, R/S) of the BaP N2-dGuo adducts could be prepared on scales up to a gram for use in oligonucleotide synthesis. We have also employed a novel, highly regioselective substitution reaction (2) of the C10 acetoxy group of tetraol tetraacetates (acetylated hydrolysis products of the BaP diol epoxides) in TFE or HFP by protected dGuo to give the C10 adducts. This reaction, which proceeds in high yield, provides a new strategy for the preparation of PAH-adducted oligonucleotide building blocks. The fluorinated alcohol approach was also used to prepare dGuo adducts at C3 of cyclopenta(cd)pyrene 3,4-oxide from the corresponding 3,4-diol diacetates in 75-85% yield and with excellent regioselectivity (3).? ? Utilizing our synthetic methodology, the major BaP diol epoxide adduct in DNA, BaP-N2-deoxyguanosine, was placed at a template-primer junction in a synthetic oligonucleotide. Crystal structures (4) of this modified DNA in complex with the Y-family DNA polymerase Dpo4 revealed three possible consequences of interaction of the bulky PAH adduct with a polymerase enzyme: replication blockage, extension past a mismatched lesion, and a -1 frameshift mutation. In the productive structures, the bulky adduct was flipped/looped out of the DNA helix into a structural gap between the little finger and core domains of the enzyme. These observations, in combination with replication and mutagenesis data, suggest a model in which this gap provides a binding site that stabilizes the extrahelical nucleotide and permits lesion bypass by generation of base substitutions and -1 frameshift mutations.
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