Protein Expr Purif. and parasitic infections show that they are important drug targets. Here, we present the development and software of a fluorescence polarization binding assay to identify small molecule inhibitors of flavin monooxygenases. Since in all of these enzymes FH1 (BRD-K4477) NADPH is definitely a common substrate, we designed an ADP-based fluorescently-labeled ligand, which has affinity to several monooxygenases. It was demonstrated that substrates and products displace the ADP-chromophore, indicating that the chromophore binds in the active site of both SidA and KMO. A display of a small molecular library was performed and an inhibitor of SidA was recognized. Furthermore, we display that this assay has a Z element of 0.77 0.01 and displays good heat and dimethyl sulfoxide (DMSO) tolerance. More importantly, we display that this assay can be generally applied PRKAA to additional flavin monooxygenases, such as FMO and mycobacterium SidA was performed as previously explained [5, 15]. Manifestation and purification of enzyme, MbtG) was performed as previously explained [5, 15]. The synthetic flavin monooxygenase gene from . Kynurenine 3-monooxygenase from was a nice gift from Dr. Graham Moran, University or college of Wisconsin, Milwaukee . Synthesis of ADP chromophores AMP triethylammonium salt 1 Dowex 50WX8-200 (H+) resin (5 g) in Et3N (7 mL) and H2O (43 mL) was stirred at space heat (rt) for 5 h. After filtration, the resin was washed with H2O and dried to give Dowex 50WX8-200 (Et3NH+) resin. This resin (2 g) was added to a solution of adenosine monophosphate (AMP) (673 mg, 1.72 mmol) in H2O (10 mL), and the suspension was stirred at rt over night before filtration and concentration to give triethylammonium salt 1 (800 mg, 99%). 1H NMR (400 MHz, D2O) 8.53 (s, 1H), 8.23 (s, 1H), 6.11 (d, = 6.1 Hz, 1H), 4.77 C 4.74 (m, 1H), 4.48 (dd, = 5.1, 3.4 Hz, 1H), 4.38-4.34 (m, 1H), 4.04 (dd, = 4.7, 3.0 Hz, 2H), 3.18 (q, = 7.3 Hz, 6H), 1.25 (t, = 7.3 Hz, 9H) (Number S1). ADP-linker conjugate 3 Dimethylpyridine (114 L, 0.9 mmol), Et3N (63 L, 0.45 mmol) and trifluoroacetic anhydride (1mL, 1.4 M in acetonitrile) was added dropwise at 0 C to a suspension of AMP triethylammonium salt 1 (100 mg, 0.22 mmol) in acetonitrile (3 mL). The producing red brown answer was stirred for 15 min before becoming FH1 (BRD-K4477) concentrated and redissolved in acetonitrile (3 mL). After successive addition of molecular sieves (4 ?, 100 mg), Et3N (153 L, 1.1 mmol), and methylimidazole (96 L, 1.2 mmol) at 0 C, a solution of phosphate 2 (70 mg, 0.18 mmol) in acetonitrile (1 mL) was added dropwise to the suspension at 0 C, and the suspension was stirred at 0 C for 1 h and at rt for 3 h. The suspension was then filtered and washed with H2O. The filtrate, was concentrated and purified by silica gel adobe flash FH1 (BRD-K4477) chromatography (CHCl3: MeOH: 1M NH4OAc = 5:4:1) to give ADP conjugate 3 (70 mg, 0.11 mmol, 50%). 1H NMR (400 MHz, D2O) 8.49 (s, 1H), 8.18 (s, 1H), 6.09 (d, = 5.6 Hz, 1H), 4.73 (t, = 5.4 Hz, 1H), 4.50 (t, = 4.2 Hz, 1H), 4.36 (s, 1H), 4.20 (s, 2H), 3.80 (d, = 6.3 Hz, 2H), 3.11 (t, = 7.1 Hz, 2H), 1.45 C 1.37 (m, 2H), 1.36 C 1.27 (m, 2H), 1.14 C 1.02 (m, 4H). HRMS (MALDI-TOF): calcd. for C18H26F3N6O11P2 (M-H)-: 621.1087, found 621.1071 (Number S2). ADP conjugated amine 4 ADP-linker conjugate 3 (35 mg, 0.056 mmol) was dissolved in 3 M NH4OH (5 mL) and the resulting solution was stirred at rt for 2 h. After becoming concentrated = 6.2 Hz, 1H), 4.72 C 4.69 (m, 1H), 4.54 C 4.49 (m, 1H), 4.39 C 4.34 (m, 1H), 4.21 C 4.17 (m, 2H), 3.84 C 3.78 (m, 2H), 2.89 (t, = 7.5 Hz, 2H), 1.58 C 1.37 (m, 4H), 1.22-1.66 (m, 4H). HRMS (MALDI-TOF): calcd. for C16H27N6O10P2 (M-H)-: 525.1264, found.