Bisubstrate analogs and inhibitors of farnesyl diphosphate synthase

Bisubstrate analog, 35-OPP, was synthesized and tested as a substrate for the enzyme catalyzed reaction of Farnesyl Diphosphate (FPP) Synthase. Analog 35-OPP was incubated with FPP synthase and samples were removed for analysis by reversed-phase C₁₈ HPLC. The HPLC traces showed evidence that a product was formed. The product of the enzymatic reaction was purified and 2-D NMR (NOESY, TOCSY, DQCOSY) revealed that the compound was a 7-membered cyclic allylic diphosphate 76-OPP . A tritim-labeled, bisubstrate analog 82-OPP was synthesized and this was used to determine the steady state kinetic constants K_m , and V_max. The catalytic efficiency (k_cat/K _m) for 82-OPP was calculated and found to be similar to the catalytic efficiencies (k_cat/K_m) for the normal substrates (IPP and DMAPP or GPP).; A new method was developed for synthesizing homoallylic diphosphates and phosphonophosphate inhibitors. Phosphonophosphate inhibitors were synthesized by treating dimethyl protected phosphonates with thiophenol, triethylamine, followed by dimethyl chlorophosphate. The phosphonophosphate was treated with trimethylsilyl. bromide to deprotect the methyl esters. The new method of synthesis eliminated the amount of tri-phosphate produced, which made the phosphonophosphate inhibitors easier to purify using cellulose chromatography The new method was used to synthesize isopentenyl diphosphate, and the phosphonophosphate, analogs of dimethylallyl and farnesyl diphosphates in moderate yields. This new methodology was also used in an attempt to synthesize bisubstrate analog inhibitors of FPP synthase.