Synthesis of Oligonucleotides Containing the (4R) and (4S) Diastereoisomers of 4,8-Dihydro-4-hydroxy-8-oxo-2′-deoxyguanosine

The insertion of the (4R) and (4S) diastereoisomers of 4,8-dihydro-4-hydroxy-8-oxo-2′-deoxyguanosine, the two main singlet oxygen oxidation products of 2′-deoxyguanosine, (4R)-4-OH-8-oxodGuo (3a) and (4S)-4-OH-8-oxodGuo (3b), into oligonucleotides has been achieved by means of phosphoramidite chemistry using the solid-phase synthesis approach. The synthesis of the phosphoramidite synthons 8a and 8b required 6 steps from 2′-deoxyguanosine and involved the simultaneous protection of the additional tertiary hydroxy (4-OH) and N2-amino groups of the modified base by acetylation. The modified phosphoramidites 8a, b were efficiently incorporated into several oligonucleotides (3-mer, 9-mer, 14-mer, and 22-mer). The presence and the integrity of the 3a and 3b in the synthetic oligomers was confirmed by electrospray ionization mass spectrometry, together with HPLC and MALDI-TOF mass-spectrometric analyses of enzymatic digestions. The use of various enzymes, including endonucleases (nuclease P1) and exonucleases (snake venom phosphodiesterase and calf spleen phosphodiesterase), clearly shows that the enzymatic hydrolysis of phosphodiester bonds between 3a,b and normal 2′-deoxyribonucleosides is inhibited.