Practical, Catalytic, Asymmetric Synthesis of β-Lactones via a ...

Purohit et al.
Fluorogenic Assay for Detection of Enzyme Inhibition.
Expression of the recombinant thioesterase domain of FAS was
performed as described previously, 9 and large-scale expression was
performed by Invitrogen Corp. (Madison, WI). The synthetic
fluorogenic substrate, 4-methylumbelliferyl heptanoate (4-MUH),
was purchased from Sigma (St. Louis, MO). The reaction mixture
consisted of 45 µL of 500 nM FAS TE in buffer A (100 mM Tris-
HCl, 50 mM NaCl at pH 7.4) which was preincubated with 2.5 µL
of stock solutions of test β-lactones dissolved in DMSO at final
concentrations of 0.32-100 µM at37°C for 30 min. The reaction
was initiated by addition of 5 µL of 1.25 mM 4-MUH in 1:1 DMSO:
buffer A. The resulting fluorescence from liberated 4-methylumbelliferone
was measured every 5 min at 350/450 nm for 40-60
min. Results are the average of triplicate time points in which the
typical standard deviation was