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.