(C18 Plus SepPak) using anhydrous acetonitrile to elute. 50 mg of N-hydroxysuccinimide (NHS) was added to the purified product and cooled to 0 ºC in an ice bath for 5 minutes followed by the addition of (diacetoxyiodo)benzene then stirred at 0 ºC for 15 minutes. The reaction was warmed to room temperature for 5 minutes prior to semi-preparative RP-HPLC purification using a Phenomenex C18(2) 100Å, 250x10 mm, 5 µ column, 35/65 acetonitrile/water with 0.1% TFA solvent system at a flow rate of 7 mL/min and UV detection at 254 nm. [18F]SFB was collected and concentrated via SPE and eluted in ACN. Radiochemical yields ranged from decay-corrected from fluoride. Section 2- IC50 and mode of inhibition determinations: General method for IC50 value determination: Working solutions of the substrate acid, (PABGgG) and inhibitor were prepared in Tris buffer (50 mM, pH 7.4). Working solutions of purified PSMA were diluted in Tris buffer (50 mM, pH 7.4 containing 1% Triton X-100) to provide 15-20% conversion of substrate to product in the absence of inhibitor. A typical incubation mixture (final volume of 250 was μL) prepared by the addition of either 25 of an inhibitor solution or 25 TRIS buffer (50 mM, μL μL pH 7.4) to 175 TRIS buffer (50 mM, pH 7.4). PABGgG (25 10 was added to the μL μL, μM) above solution. The enzymatic reaction was initiated by the addition of 25 of the PSMA μL working solution. In all cases, the final concentration of PABGgG was 1 while the enzyme μM was incubated with five serially diluted inhibitor concentrations providing a range of inhibition from 10% to 90%. The reaction was allowed to proceed for 15 min with constant shaking at 37 oC and was terminated by the addition of 25 methanolic TFA (2% v/v trifluoroacetic acid in μL
(C18 Plus SepPak) using anhydrous acetonitrile to elute. 50 mg of N-hydroxysuccinimide (NHS) was added to the purified product and cooled to 0 ºC in an ice bath for 5 minutes followed by the addition of (diacetoxyiodo)benzene then stirred at 0 ºC for 15 minutes. The reaction was warmed to room temperature for 5 minutes prior to semi-preparative RP-HPLC purification using a Phenomenex C18(2) 100Å, 250x10 mm, 5 µ column, 35/65 acetonitrile/water with 0.1% TFA solvent system at a flow rate of 7 mL/min and UV detection at 254 nm. [18F]SFB was collected and concentrated via SPE and eluted in ACN. Radiochemical yields ranged from decay-corrected from fluoride. Section 2- IC50 and mode of inhibition determinations: General method for IC50 value determination: Working solutions of the substrate acid, (PABGgG) and inhibitor were prepared in Tris buffer (50 mM, pH 7.4). Working solutions of purified PSMA were diluted in Tris buffer (50 mM, pH 7.4 containing 1% Triton X-100) to provide 15-20% conversion of substrate to product in the absence of inhibitor. A typical incubation mixture (final volume of 250 μL) was prepared by the addition of either 25 μL of an inhibitor solution or 25 μL TRIS buffer (50 mM, pH 7.4) to 175 μL TRIS buffer (50 mM, pH 7.4). PABGgG (25 μL, 10 μM) was added to the above solution. The enzymatic reaction was initiated by the addition of 25 μL of the PSMA working solution. In all cases, the final concentration of PABGgG was 1 μM while the enzyme was incubated with five serially diluted inhibitor concentrations providing a range of inhibition from 10% to 90%. The reaction was allowed to proceed for 15 min with constant shaking at 37 oC and was terminated by the addition of 25 μL methanolic TFA (2% v/v trifluoroacetic acid in
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