SYNTHESIS OF 2',3',5'-TRIDEOXYURIDINE-5'-METHYLPHOSPHONIC ACID AND ITS DIPHOSPHATE DERIVATIVE - STUDY OF THE INTERACTION WITH HIV-1 REVERSE-TRANSCRIPTASE

2',3',5'-Trideoxyuridine-5'-methylphosphonate, [8], was synthesized fr om ddU. The 5',6' carbon-carbon bond was formed by condensing the 5'-a ldehyde of ddU and a Wittig reagent. The binding interaction of the di phosphate derivative [10] of the phosphonate [8] with HIV-1 reverse tr anscriptase (RT) was studied using methods based primarily on fluoresc ence spectroscopy. From the quenching of intrinsic tryptophan fluoresc ence of RT during its titration against [10], a dissociation constant of 24 mu M was calculated at 25 degrees C. In the presence of a DNA/DN A template/primer of defined sequence and RT, [10] and a fluorescent d erivative of ddTTP competed for binding to RT without incorporation of ddU-5'-methylphosphonate. in the presence of a 0.5 mM concentration o f [10], the RT activity measured with Poly(rA)/(dT)(15) and [H-3] dTTP was lowered to 65%. All our observations are consistent with suppress ion of the catalysis of bond formation between the OH at the primer 3' -end and alpha-P of [10] after formation of the complex between RT, te mplate/primer and [10].