The NMR solution structures of benzo(a)pyrene modified deoxyadenosine in DNA
Abstract
Two benzo (a) pyrene epoxide adducts to the N$\sp6$ amino of deoxyadenosine in duplex DNA were studied. The first modified oligonucleotide, d(GGTC- (BaP) A-CGAG), in which ($-$)-(7S,8R)-dihydroxy-(9R,10S)-epoxy-benzo (a) pyrene (BaP) was covalently bonded to the exocyclic N$\sp6$ amino of deoxyadenosine through trans addition at C10 (R chirality at C10) of the epoxide was incorporated into a DNA 9-mer duplex, d(G$\rm\sb1G\sb2T\sb3C\sb4$- (BaP) A$\rm\sb5$-C$\sb6G\sb7A\sb8G\sb9)\cdot$ d(C$\rm\sb{10}T\sb{11}C\sb{12}G\sb{13}G\sb{14}G\sb{1 5}A\sb{16}C\sb{17}C\sb{18}$) containing a dG mismatch opposite the modified dA (trans ($-$)-DE2(10R)-dA-adduct). The second modified undecamer duplex contained (7R,8S)-dihydroxy-(9R, 10S)-epoxy-benzo (a) pyrene which was covalently bonded through trans addition to the N$\sp6$ amino of deoxyadenosine (dA* 6) with R chirality at C10 of the benzo (a) pyrene (trans ($-$)-DE1 (10R)-dA-adduct). The undecamer duplex d(C$\rm\sb1G\sb2G\sb3T\sb4C\sb5A\sp{\*}\sb6C\sb7G\sb8A\sb9$ G$\rm\sb{10}G\sb{11})\cdot d(C\sb{12}C\sb{13}T\sb{14}C\sb{15}$ G$\rm\sb{16}T\sb{17}G\sb{18}A\sb{19}C\sb{20}C\sb{21}G\sb{22})$ has a dT opposite the modified dA. The proton assignments for the solution structures of the adducts were made using 2D TOCSY and NOESY NMR specua. The complete hybrid relaxation matrix program, MORASS2.0 was used to generate NOESY two-spin flatwell distance constraints for iterative refinement using distance restrained molecular dynamics calculations with AMBER4.0. The iteratively refined structure of the trans ($-$)-DE2(10R)-dA-adduct showed the benzo (a) pyrene (BaP) intercalated from the major groove immediately below the dC(4)-dG(15) base pair. The modified dA was in an anti configuration with the dG of the GA mismatch turned out into the major groove. The refined structure of the trans ($-$)-DE1 (10R)-dA-adduct showed the benzo (a) pyrene intercalated from the major groove between the dA*6-dT17 and dC5-dG18 base pairs. The modified dA*6 was in the normal anti configuration and formed Watson-Crick base pairing to dT17 opposite. Automated procedures for the synthesis of RNA and dithio modified oligonucleotides on Pharmacia's Gene Assembler Plus were developed. The automated synthesis used solid phase DNA oligonucleotide synthetic procedures with modified coupling times and oxidation cycles. The thiophosphoramidites were synthesized in a one pot reaction.
Degree
Ph.D.
Advisors
Gorenstein, Purdue University.
Subject Area
Organic chemistry
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