A novel class of DNA analogs bearing 5'-C-phosphonothymidine units: synthesis and physicochemical and biochemical properties

S(C) and R(C) diastereomers of 5'-C-(O,O-diethyl)-phosphonylthymidine ((R)T and (S)T) were used for the synthesis of the dimers T(R)T and T(S)T, respectively. These dimers were incorporated at selected sites in oligonucleotide constructs. Melting temperature (Tm) experiments demonstrated that relative to the unmodified oligodeoxyribonucleotide, the presence of the (R)T moiety reduced the thermal stability of the duplexes by approximately 5.0 degrees C per modification, whereas their (S)T counterparts only slightly destabilized the duplex structure (deltaTm < or = 1 degree C/modification). The stability of the triple-helical complexes containing one, two, or three modified thymidines is slightly higher than that of the parent complex. Nuclease resistance studies performed with snake venom phosphodiesterase, calf spleen phosphodiesterase, and 3'-exonuclease from human plasma showed that cleavage of the oligonucleotides at the site of the modification was completely suppressed regardless of the stereochemistry of the 5'-C-chiral center. The influence of the (R)T and (S)T modification in the recognition sequence of HindIII, EcoRI, and HpaI restriction endonucleases was also investigated. Although the catalytic activity of HindIII was not affected by the presence of the 5'-C-ethoxyphosphonyl modification, the activities of the two remaining restriction enzymes were partially suppressed depending on the site of modification or the stereochemistry of the modification or both ((R)T vs. (S)T).