Synthesis and characterization of a new fluorogenic active-site titrant of serine proteases

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Abstract
The molecule 3'',6''-bis(4-guanidinobenzoyloxy)-5-[N''-(4-carboxyphenyl)thioureido]spiro[isobenzofuran-1-(3H),9''-[9H]xanthen]-3-one (FDE) was designed and synthesized as a fluorogenic active-site titrant for serine proteases. It is an analog of p-nitrophenyl p-guanidinobenzoate (NPGB) in which a fluorescein derivative is substituted for p-nitrophenol. FDE and NPGB exhibit similar kinetic characteristics in an active-site titration of [bovine pancreatic] trypsin in phosphate-buffered saline, pH 7.2. The rate of acylation with FDE is extremely fast (k2 = 1.05 s-1) and the rate of deacylation extremely slow (k3 = 1.66 .times. 10-5 s-1). The Ks is 3.06 .times. 10-6 M, and the Km(app) is 4.85 .times. 10-11 M. With 2 of the serine proteases involved in fibrinolysis, the rate of acylation with FDE is also fast, k2 = 0.112 s-1 for urokinase and 0.799 s-1 for [dog] plasmin, and the rate of deacylation is slow, k3 = 3.64 .times. 10-4 s-1 for urokinase and 6.27 .times. 10-6 s-1 for plasmin. The solubility limit of FDE in phosphate-buffered saline is 1.3 .times. 10-5 M, and the 1st-order rate constant for spontaneous hydrolysis is 5.1 .times. 10-6 s-1. The major difference between FDE and NPGB is the detectability of the product in an active-site titration. p-Nitrophenol can be detected at concentrations no lower than 10-6 M whereas fluorescein can be detected at concentrations as low as 10-12 M. FDE should be useful in quantitatively assaying serine proteases at very low concentrations.