Synthesis and Characterization of a New Fluorogenic Active-Site Titrant of Serine Proteases

Abstract

The molecule 3’,6'-bis(4-guanidinobenzoyloxy)- 5-[N'-(4-carboxyphenyl)thioureido] spiro [isobenzofuran-1-(3H),9'-[9H]xanthen]-3-one, abbreviated FDE, was designed and synthesized as a fluorogenic active-site titrant for serine proteases. It is an analogue 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 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 (k2 = 1.66 X 10-5 s-1). The Ks is 3.06 X 10-6 M, and the Km(app) is 4.85 X 10µM. With two 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 plasmin, and the rate of deacylation is slow, k3 = 3.64 X 10-4 s-1 for urokinase and 6.27 X 10-6 s-1 for plasmin. The solubility limit of FDE in phosphate-buffered saline is 1.3 X 10-5 M, and the first-order rate constant for spontaneous hydrolysis is 5.1 X 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 10-0 M whereas fluorescein can be detected at concentrations as low as 10-12 M. Thus, FDE should be useful in quantitatively assaying serine proteases at very low concentrations. © 1981, American Chemical Society. All rights reserved.