Synthesis and characterization of oligonucleotides containing an O6 -deoxyguanosine-alkyl-O6 -deoxyguanosine interstrand cross-link

摘要

Studies have shown that resistance to bifunctional alkylating agents used in the treatment of chronic myelogenous leukemia can result in diminished effectiveness of these therapeutics. To further understand this process we have developed clinically relevant models of cross-linked DNA duplexes to study the structure and repair of lesions induced by the bifunctional alkylating agents: busulfan and hepsulfam. To emulate the known deoxyguanosine- N 7 -alkyl- N 7 -deoxyguanosine lesion formed by hepsulfam and the unidentified lesion(s) formed by busulfan, we have designed three synthetic approaches leading to the synthesis of six stable deoxyguanosine dimers. General mono, bi and tripartite syntheses were developed for the preparation of 1,1 and l,3-deoxyguanosine- O 6 -alkyl- O 6 -deoxyguanosine cross-links employing 'fast-deprotecting' phenoxyacetyl protection at the N 2 position for facile removal. The O 6 -alkyl couplings are performed via a Mitsunobu reaction between a nucleoside and a mono-protected diol. The phosphoramidites of synthesized dimers were incorporated into one helical turn and 'sticky-end' DNA duplexes via solid-phase synthesis employing a DNA synthesizer. All duplexes were of defined structure and sequence for the purpose of further conformational analysis and enzymatic repair studies. Sequence composition was confirmed via enzymatic digestion and purity by subsequent HPLC analysis. A variety of methods including ultraviolet transition thermal denaturation (T m ), circular dichroism (CD) and gel electrophoretic studies were employed to characterize the cross-linked duplexes. CD and T m studies suggest little deformation from native B-form conformation for the hepsulfam mimics. Sufficient quantities of the mono and bipartate heptyl cross-linked duplexes were obtained for various structural studies with required quantities for biological investigations obtained for the monopartate synthesis.