Intercation covalence

The non-covalently bound BPDEs to DNA formed initially appear to be intercalation complexes (1 6,52-55) Meehan et al. (1 6) report that the BPDE intercalates into DNA on a millisecond time scale while the BPDE alkylates DNA on a time scale of minutes. Most of the BPDE is hydrolyzed to tetrols (53-56). Geacintov et al. (5l ) have shown with linear dichroism spectral measurements that the disappearance of intercalated BPDE l(+) is directly proportional to the rate of appearance of covalent adducts. These results suggest that either there may be a competition between the physically non-covalently bound BPDE l(+) and an externally bound adduct or as suggested by the mechanism in the present paper, an intercalative covalent step followed by a relaxation of the DNA to yield an externally bound adduct. Their results for the BPDE i(-) exhibit both intercalative and externally bound adducts. The linear dichroism measurements do not distinguish between physically bound and covalent bound forms which are intercalative in nature. Hence the assumption that a superposition of internal and external sites occurs for this isomer. 

A theoretical analysis is presented for the binding of the four dia-stereoisomers of benzo[a]pyrene diol epoxides (BPDEs) to N2(g), N6(a), 06(G) and NU(c). Molecular models for binding and stereoselectivity involving intercalation, intercalative covalently and externally bound forms are presented. Molecular mechanics calculations provide the energetics which suggest possible structures for the formation of each of the principal DNA-BPDE complexes. Stereographic projections are used to illustrate the molecular structures and steric fits. The results of previous calculations on intercalation and adduct formation of BPDE l(+) in kinked DNA (37) are summarized and extended to include the four diastereoisomers l( ) and II( ). The theoretical model is consistent with the observed experimental data. 

Formation of the BPDE-DNA adduct requires a study of (l) Benzo ring conformations of BPDEs and adducts (2) The rehybridization of amino groups on the benzo for the C10-N bond formation (3) The receptor sites resulting from a conformational adjustment of DNA to accommodate an intercalated and finally an intercalative covalently bound BPDE, and the base sequence specificity in the formation of the receptor site (U) Classical intercalation and the orientation of CIO of the BPDEs toward the reactive N2(G), N6(a), 06(G) and... 

NU(C) base atoms (5) The stereoselectivity of the BPDEs during intercalative covalent binding in kinked DNA and (6) The possible reorientation of the complex to yield an externally bound adduct. The energetics for each of these processes will be presented to identify the important steps that influence the binding of specific isomers. It will be shown that the orientation of each diastereoisomer of BPDE about specific base atoms in kinked receptor sites in the duplex DNA during covalent bond formation is the determining factor in stereoselectivity. 

Table V. Definition of Binding Sites for Intercalation and Intercalative Covalent Binding3...

Figure 8. Intercalative covalent binding to N2(G). Trans BPDE-adducts are bound to the 5 side of G in the +G C,C G,BPT,C G,C G+ complex in DNA kinked to site KMa.

Although we postulate that this receptor site results in stereoselectivity, it may not be the final state. The orientation of the long axis of the pyrene moiety is approximately 80°-90° and this implies quasi-intercalation of site IQ (56-58). The kinked site proposed by Hogan et al. (50) and studied by Taylor and Miller (MO for l(+)-N2(G) binding in retrospect appears to represent different binding sites. The orientation of the pyrene moiety of a(BPDE) = 1+3° and the local DNA axis in the kink of y(DNA) = 29° (50) will be explained by external binding in the next section. The intercalative covalent binding in a kinked site is an intermediate step between intercalation and the final structure for the externally bound BPDE-N2(G) adduct, but it becomes the final structure for the quasi intercalated BPDE-N6(g), 06(g) and N +(c) adducts. 

Table XII. Stereoselectivity of BPDEs for Base Atoms by the Intercalative Covalent Binding Step in Kinked DNA and Predicted Final Binding Site3...

The energies reported in Table XIII for the externally bound forms are measured relative to that for the intercalative covalently bound form. Thus, the trans BPDE l(+)-N2(G) adduct is 10.1 kcal/ mole more stable and the trans BPDE II(-)-N6(a) adduct is 12.7 kcal/mole less stable in the externally bound form. Similarily, the trans BPDE Il(+)-N2(G) adduct is -13.8 kcal/mole more stable and the trans BPDE I(-)-N6(a) adduct is 7 5 kcal/mole less stable. Therefore, site IQ (intercalative covalent) which is favored by the i(-) isomer (5l) may be due to n6(a) and NH(c) adduct formation, specifically trans addition. 

Intercalative covalent binding stereoselectivity final predominant structure for trans addition to n6(a) and 06(a 5 of BPDE I(-) and Il(-). 

The main features of this proposed mechanism are (l) the stereoselectivity of the BPDEs by the DNA during intercalative covalent binding and (2) the final orientation of the bound pyrene which may be oriented internally (intercalative covalent) or externally (outside the helix). The stereoselectivity occurs during covalent bond formation and after intercalation. Relaxation of the DNA allows the adduct to adjust to its final orientation. If the experimental measurements are assumed to be made on the DNA-adducts after the final orientation has been achieved, then the following interpretations can be made. 

The l(+) and Il(+) isomers are stereoselected by N2(G), whereas the i(-) and II(—) isomers are stereoselected by the n6(a) and 06(G) during intercalative covalent steps with trans addition. The l( + )-and Il(+)-N2(G) adducts are rearranged to an externally bound form with the pyrene in the minor groove, but the I(-)-N6(a) and II(-)-06(g) adducts remain quasi intercalated. This is determined by the relative energy change between the two forms as we see from Table XIII. However, there is a superposition of the two types of sites, IQ and IIX (51 57,58), and BPDE i(-) DNA adducts exhibit both types of binding. By symmetry, the cis BPDE l(-)-N2(G) adduct is predicted to behave similarily to the trans l(+)-N2(G) adduct. It should be externally bound. 

From the susceptibility isotherms it may be expected that three kinds of information may be obtained first, the oxidation state of the paramagnetic ion second, evidence of intercation covalence and third, the effectiveness of dispersion of the paramagnetic ions. There will now be presented specific applications of the method. 

If this were the only example of diminished mi netic moments, then such an explanation in terms of intercation covalency would be indefensible. But the effect actually occurs with most of the transition group oxides. Even in supported chromia the measured moment is about 10 per cent too small, while in molybdenum dioxide the expected moment of 2.8 Bohr magnetons is actually found to be zero. Other examples of the effect will be mentioned later. 

Supported iron is thus seen to resemble supported chromia and (+3) manganese, with the important difference that intercation covalency is a major factor in the structure of this system. 

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