Site-specific insertion of the (5R*) and (5S*) diastereoisomers of 1-[2-deoxy-beta-D-erythro-pentofuranosyl]-5-hydroxyhydantoin into oligodeoxyribonucleotides

The insertion of the (5R(*)) and (5S(*)) diastereoisomers of 1-[2-deoxy- be ta -D-erythro-pentofuranosyl] -5-hydroxyhydantoin (1)-a major oxidation pro duct of 2'-deoxycytidine upon exposure to OH radicals, excited photosensiti zers, or ozone - into oligonucleotides is reported. It was achieved by mean s of phosphoramidite chemistry, using the solid-phase synthesis approach. T he synthesis of the phosphoramidite synthon 7 required 6 steps from 3 '-O-( tert-butyldimethylsilyl)-2 '-deoxycytidine and involved protection of the s econdary hydroxy group (5-OH) of the modified base by the nonstandard levul inyl group. The modified phosphoramidite synthon 7 was efficiently incorpor ated into several oligonucleotides (3-mer, 14mer, 22-mer) by solid-support assembling. The presence and the integrity of the (5R(*)) and (5S(*)) diast ereoisomers of 1-[2deoxy-beta -D-eryihro-pentofuranosyl] -5-hydroxyhydantoi n in the synthetic oligomers was confirmed by electrospray ionization mass spectrometry, together with HPLC and MALDITOF mass-spectrometric analyses o f enzymatic digestions. The use of exonucleases (calf spleen phosphodiester ase and bovine intestinal mucosa phosphodiesterase) clearly showed that enz ymatic hydrolysis of the phosphodiester bonds between the (5R(*)) and (5S(* )) diastereoisomers of 1-[2-deoxy-beta -D-erythro-pentofuranosyl]-5-hydroxy hydantoin and normal 2'-deoxyribonucleosides is prevented, while endonuclea se (nuclease P-1) is able to cleave the lesion residue from the oligonucleo tides.