Bay-region diol epoxide (DE) metabolites of carcinogenic polycyclic aromatic hydrocarbons are believed to initiate cell transformation by covalent modification of DNA. Two diastereomeric DEs, each of which exists as a pair of enantiomers, are formed metabolically in mammals: DE-1, in which the benzylic hydroxyl group and epoxide oxygen are cis, and DE-2, in which these substituents are trans. The primary targets in DNA for these DEs are the exocyclic N-2 and N-6 amino groups of deoxyguanosine (dG) and deoxyadenosine (dA), respectively. The goal of our research is to elucidate the relationships between the DNA adducts formed from specific DEs, their physical structures and their biochemical processing both with purified enzymes and in intact cells. Efforts during the past year have focused on the development of efficient synthetic methodology for the construction of protected DE adducts of dG and dA as their 3'-O-phosphoramidite derivatives for use in solid-phase DNA synthesis. We are now in a position to synthesize oligonucleotides of any desired sequence containing adducts derived from either cis or trans opening of DE-1 or DE-2 by the exocyclic amino groups of dA or dG. These oligonucleotides are currently the subject of conformational analysis by 2D NMR as well as biochemical and molecular biological studies to assess the effects of different adducts on DNA replication and repair. Several years ago, we reported the synthesis of phosphoramidites derived from the adducts formed by trans opening of DEs by the exocyclic amino groups of dA and dG. The critical step in the construction of these phosphoramidites involves bond formation between a suitably protected, fluorinated purine nucleoside and the amino triol derived from trans ring opening of the DE by ammonia. The adducts derived from cis opening of DEs are less accessible synthetically by this general approach, since efficient syntheses of the cis opened amino triols are unavailable. We have now developed an extremely facile synthesis of cis opened adducts of benzo[a]pyrene 7,8-diol 9,10-epoxide (BaP DE-2) from the corresponding 7,8-dihydrodiol. Reaction of BaP 7,8-dihydrodiol with dA as its di-O-tert-butyldimethylsilyl derivative under conditions of the Sharpless aminohydroxylation reaction (tert-butyl hypochlorite, hydroquinidine 1,4-phthalazinediyl diether, potassium osmate dihydrate) gave the desired cis-opened dA adduct in 85% yield in a single step, as compared with six steps for the alternative route via the cis opened amino triol. Addition occurs exclusively on one face of the hydrocarbon to give the cis opened DE-2 adduct. Efforts are currently in progress to extend the scope of this reaction to other hydrocarbons and other nucleoside derivatives. However, we have to date not found conditions under which analogous N-2 dG derivatives can be obtained by this route. Availability of methods for the construction of DE adducted oligonucleotides of defined adduct stereochemistry and nucleotide sequence makes possible the systematic study of effects of these structural features on mutations that result from DNA replication past the adducts. A set of eight 16-mer oligonucleotides containing dA adducts derived from trans opening of each enantiomer of BaP DE-1 and DE-2 in two different sequence contexts and another set of eight 16-mers containing the analogous dG adducts in two other sequence contexts were ligated into single-stranded DNA from bacteriophage M13mp7L2 and allowed to undergo replication after transfection into E. coli. The predominant substitution mutations observed were A to T and G to T transversions. Adduct structure and sequence context were both found to influence the distribution of specific mutations. The sequence had a marked effect on the frequency of mutations induced by all the dA adducts, such that an adduct (A*) in the partial sequence TTTA*GAG resulted in a 3- to 10-fold greater frequency of substitution mutations overall than an adduct in the partial sequence CAGA*TTT. The effect of sequence on the frequency of substitution mutations was less pronounced for the dG adducts, but single-base deletions were highly sensitive to sequence, occurring with high frequency on replication of the partial sequence GGGG*TTC but not at all with TTCG*ATT. Experiments currently in progress are designed to test the effect of all possible nearest-neighbor contexts (16 triplet sequences for each adduct) on the frequency and distribution of mutations induced by both cis and trans opened DE-2 adducts. Previous mutation studies of this type in other laboratories have been limited to a few oligonucleotides. Our new synthetic methodology allows the number of different oligonucleotides investigated in a single study to be increased by at least an order of magnitude.
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