http://www.unc.edu/courses/2009spring/envr/740/001 overhead 18Begin 03/31/09The next structure is the N6-dAdo adduct of the (-)-trans-anti-BPDE in a synthetic oligonucleotide containing the N-Ras sequence from codons 60 – 62 and the CAA (Gln) triplet atcodon 61 is modified at position 2 by the BPDE adduct. The choice of the N-Ras codon and position of the adduct was made based on the known transforming mutation at codon 61. It is important to point out that the adduct studied is with one of the less active metabolites. In this study, the association of the BPDE adduct with the mutation, however is not as direct as in the case of the p53 hot spots in the DNA binding domain. Here, the model is based primarily on the assumption that, since mutations in codon 61 are found in tumors from animals exposed to BPDE, BPDE must be responsible for the initial lesion. The base sequence in codon 61 is CAA, and since pyrimidines are much less reactive than purines, one of the As would be the target. In addition, a promutagenic adduct at position 3 might give a redundant codon (CAG, if A → G transition), the authors assumed that the adduct would be at position 2. Regarding the choice of the less active (-)-anti enantiomer, the authors did not indicate why they did not attempt to modelthe adduct with (+)-anti enantiomer, or whether they were unsuccessful in obtaining a structure with the (+)-trans-anti enantiomer. They simply comment that they are reporting the structure of the (-)-trans-anti compound. The authors do indicate that the most active of the metabolites is the(+)-trans-anti BPDE but do not address the obvious question of why they did not attempt to characterize the (+)-trans-anti adduct. It is surprising to me that the reviewers did not point this out and require the authors at least to comment on it. No structure of the (+)-trans-anti- adduct has been deposited in the Protein Data Bank since publication of the review.[OH; (-)-trans-anti-BPDE: N6-dAdo adduct structure]The view looking down the helix axis shows that the planar PAH structure intercalates between the bases, so the adduct behaves very differently from the (-)-trans-anti N2-dGuo adduct (groove binder). The aromatic moiety lies over the 5'-face of the modified dAdo, and the partner dThyd ispushed slightly out of the way, but not totally out of an H-bonding position with respect to the modified dAdo. Despite this distortion, there is no major distortion of the helix.1There is one structure of double stranded DNA with dAdo modified by a (+)-cis-anti BPDE adduct, which is shown on the next slide.[OH22; adduct of (+)-cis-anti BPDE with N6 of dAdo]The (+)-cis-anti adduct flanked by dCyds and adopts a conformation with the aromatic moiety intercalated to the 5'-side of the modified dAdo. As in the case of the (-)-trans-anti BPDE, pairing remains intact, although the complementary bases are buckled in this case, and there is a bulge in the DNA helix.To summarize what we might have learned from the BPDE adducts: comparison of the dAdo with the dGuo structures points up the fact that it is not possible, at the moment, to draw any conclusions regarding the significance of the different structures for biological activity. However,the NMR structures indicate that sequence context influences conformation around a lesion, which would in turn be expected to influence the mutagenic potential. This is consistent with the existence of mutagenic hotspots in vivo and may in part explain this phenomenon. Having examined the series of adduct structures with BPDE, a pertinent question to ask is whether there are any correlations between diolepoxide stereochemistry, DNA adduct conformation and relative biological activity for other PAH besides BP. This is area that requiresmore work before definitive answers are possible. The racemic anti-diolepoxides – racemic is the term applied when a 1:1 mixture of the () enantiomers has been used – the racemic anti-diolepoxides have been determined to be the most active synthetic derivatives of benzanthracene and the structurally related potent carcinogen 7,12-DMBA. Similarly, for both 5-methylchryseneand the symmetric unmethylated parent, chrysene – the structure is shown at the bottom of the next slide – the (±)-anti derivatives are the most active. In the case of 5-methylchrysene, where the bay regions - “bay region” is the term applied to the 3-sided indentation on the PAH periphery – where the bay regions are inequivalent as a result of the methyl substituent, the diolepoxide on the hindered bay region is the more active - and also, perhaps surprisingly, the predominant metabolite.2Another question that has been explored is the relative activities of methylated PAH compared tounsubstituted parent PAH- for example 7,12-DMBA compared to benzanthracene; 5-methylchrysene compared to chrysene. When the methyl group projects into the bay region, biological activity increases. Crystal structures have demonstrated that steric hindrance caused by intrusion of the methyl group into a 3-sided or 4-sided peripheral indentation forces the PAH to become slightly non-planar. (The 4-sided indentation is called a “fjord”.)While this sterically induced non-planarity seems to correlate with increased activity, there is at this moment no physico-chemical explanation for this correlation. The authors of the CRT review have also determined the structure of the N2-dGuo adduct of the (-)-trans-anti diolepoxide of 5-methylchrysene in which the diolepoxide is on the hindered indentation. The top panel of the next slide shows that,[OH23; stereo view of (-)-trans-anti-5-MeC adduct with dGuo]like the (-)-trans-anti analogue BPDE, the chrysene adduct lies in the minor groove, with the distal end of the PAH oriented towards the 3′-end of the modified strand. The effect of the hydrophobic methyl group is to try to insert into the helix to avoid contact with the polar solvent,with the result that the aromatic residue is oriented more edge-on within the minor groove than the aromatic pyrenyl residue of the (-)-trans-anti adduct of BP. However, no structures of other 5-MeC diolepoxide stereoisomers have been reported, so it is impossible at this point to make any further generalizations regarding adduct orientations of groove binders, although it is tempting to predict that when the structure of the (+)-trans-anti adduct of 5-MeC is determined, it will be a groove-binder with the distal end oriented