Abstract

The work reported in this article has evaluated the relative molecular activity of the 5′-triphosphate of a novel β-l-nucleoside with an unsaturated ribose residue, β-l-2′,3′-dideoxy-2′,3′-didehydro-5-fluorocytidine (β-l-Fd4CTP), with that of β-l-2′,3′-dideoxy-5-fluorocytidine (β-l-FddCTP) and 2′,3′-dideoxycytidine (ddCTP), on DNA strand elongation by human immunodeficiency virus-1 reverse transcriptase (HIV RT) and human DNA polymerases α (pol α), β (pol β), γ (pol γ), and ε (pol ε). The concentrations of β-l-Fd4CTP that inhibited the yield of products by 50% were 0.20 μm, 1.8 μm, and 4.0 μm for HIV RT, pol γ, and pol β, respectively. The β-l-Fd4CTP at a concentration as high as 40 μm had no inhibitory effect on pol ε, but could inhibit pol α by 10–20% at 20 μm. TheK_m and relativeV_max values of β-l-Fd4CTP, β-l-FddCTP, and ddCTP for incorporation into the standing start point of 5′-[³²P]-oligonucleotide primer annealed with M13mp19 phage DNA by HIV RT and human DNA polymerases were evaluated. The efficiency of incorporation (V_max/K_m) of β-l-Fd4CTP by HIV RT was about 4-fold and 12-fold higher than that of ddCTP and β-l-FddCTP, respectively. In contrast, theV_max/K_mratio of β-l-Fd4CTP for pol γ was 7-fold lower than that of ddCTP, but 4-fold higher than that of β-l-FddCTP. Pol α could use β-l-Fd4CTP as a substrate, but only at a high concentration (>20 μm). Incorporation of β-l-Fd4CTP by pol ε could not be detected. A hypothesis about the preferable recognition of the 2′,3′-dideoxy-2′,3′-didehydro- structure of β-l-Fd4CTP to that of the 2′,3′-dideoxy-structure of β-l-FddCTP by HIV RT is discussed.