A O l c 5-h b s u p h c p i m n s o w c e m s c a e d c w W d 0 1 E S L
5-Hydroxy-2'-deoxycytidirte (5-OHdC) 2 is one of the major stable "OH radicals and one-electron mediated oxidation products of 2'-deoxycytidine 1. Diflkrent studies have ahovm that the level of 2 increases substantially when DNA is exposed to UV, ionizing radiation or oxidizing agents'. In order to study the structural and the biological role of 5-OHdC 2 in DNA, it is necessary to use oligonucleotides containing the oxidative lesion 2 at defied sites. Due to the relative instability of the modified nucleoside 2 and the necessity to protect the additional hydroxyl group in position 5 of the base, this lesion to our knowledge has not been yet incorporated in oligonucleotides by chemical approaches.
We report herein the fist aite-specfic incorporation of 2 into oligonucleotides using the phosphoramidite chemistry..Prior to the preparation of the phosphoramidite aynthon 8, the stability of 5-OHdC 2 was checked at room temperature under the three main conditions used during solid support synthesis : 300/0 aqueous ammonia, SOY.acetic acid and a commercial oxidizing solution of iodine3. No detectable degradation of 2 was observed after 24h of incubation. Furthermore, the N4,05-diprotected nucleoaide 6 was quantitatively converted into the parent compound 2 upon treatment with aqueous ammonia for 2h at room temperature. The kinetic and stability studies showed the compatibility of 2 with the "Pac phosphoramidite" chemistry.
The synthesis of the target phosphoramidite 8 was achieved in 6 .s@ as shown m Scheme 1. First, 5-OHdC 2 was prepared by bromination of 2'-deoxycytidine 1 in an aqueous medium followed by 2,4,6-coUidrne treatment as described in the literatures. Compound 2 was then converted rnto the N4,05-diprotected intermediate 6 via the TIPDS ether 3. This was accomplished by treating 2 with TIPDSCIZ in DMFGm the presence of imidazole and N& The reaction gives the 3',5'-O-TIPDS derivative 3 and the 5,5'-O-TtPDS