Synthesis and characterization of an O(6)-2'-deoxyguanosine-alkyl-O(6)-2'-deoxyguanosine interstrand cross-link in a 5'-GNC motif and repair by human O(6)-alkylguanine-DNA alkyltransferase.

O(6)-2'-Deoxyguanosine-alkyl-O(6)-2'-deoxyguanosine interstrand DNA cross-links (ICLs) with a four and seven methylene linkage in a 5'-GNC- motif have been synthesized and their repair by human O6-alkylguanine-DNA alkyltransferase (hAGT) investigated. Duplexes containing 11 base-pairs with the ICLs in the center were assembled by automated DNA solid-phase synthesis using a cross-linked 2'-deoxyguanosine dimer phosphoramidite, prepared via a seven step synthesis which employed the Mitsunobu reaction to introduce the alkyl lesion at the O(6) atom of guanine. Introduction of the four and seven carbon ICLs resulted in no change in duplex stability based on UV thermal denaturation experiments compared to a non-cross-linked control. Circular dichroism spectra of these ICL duplexes exhibited features of a B-form duplex, similar to the control, suggesting that these lesions induce little overall change in structure. The efficiency of repair by hAGT was examined and it was shown that hAGT repairs both ICL containing duplexes, with the heptyl ICL repaired more efficiently relative to the butyl cross-link. These results were reproducible with various hAGT mutants including one that contains a novel V148L mutation. The ICL duplexes displayed similar binding affinities to a C145S hAGT mutant compared to the unmodified duplex with the seven carbon containing ICLs displaying slightly higher binding. Experiments with CHO cells to investigate the sensitivity of these cells to busulfan and hepsulfam demonstrate that hAGT reduces the cytotoxicity of hepsulfam suggesting that the O(6)-2'-deoxyguanosine-alkyl-O(6)-2'-deoxyguanosine interstrand DNA cross-link may account for at least part of the cytotoxicity of this agent.

Synthesis of building blocks and oligonucleotides with {G}O6-alkyl-O6{G} cross-links.

This unit describes two methods for preparing oligonucleotides containing an O(6)-2'-deoxyguanosine-alkyl-O(6)-2'-deoxyguanosine interstrand cross-link by a solid-phase synthesis approach. Depending on the desired orientation of the cross-link in the DNA duplex, either a bis- or a mono-phosphoramidite synthesis strategy can be employed. Both procedures require the preparation of a protected 2'-deoxyguanosine-containing dimer where the two nucleosides are attached at the O(6)-atoms by an alkyl linker. This linker is introduced as a protected diol using two successive Mitsunobu reactions to produce a cross-linked amidite that is incorporated into an oligonucleotide via solid-phase synthesis. The use of a protected diol lends versatility to this method, as cross-links of variable chain length may be prepared. The bis-phosphoramidite approach is a direct method to preparing the cross-linked duplex, whereas the mono-phosphoramidite strategy requires additional manipulation of the solid support to prepare cross-linked oligonucleotides. Once all synthetic steps are completed, these oligonucleotides can then be removed from the solid support and deprotected, and then purified via ion-exchange HPLC to produce sufficient quantities of substrates that can be used in DNA repair studies.