Direct visualization of enzyme inhibitors using a portion mixing inhibitor library containing a quenched fluorogenic peptide substrate. Part 1. Inhibitors for subtilisin Carlsberg

Abstract

A PEGA-resin was derivatized with a 2 : 1 mixture of 4-hydroxymethylbenzoic acid and Fmoc-Lys(Boc)-OH and the fluorogenic substrate Ac-Y(NO₂)FQPLAVK(ABz)-PEGA was assembled using the active ester approach. Following esterification of the 4-hydroxymethylbenzoic acid with Fmoc-Val-OH a library X₁X₂X₃-x₄-X₅X₆V was assembled by the portion mixing method. The library was subjected to extensive hydrolysis by subtilisin Carlsberg and the rate of hydrolysis was highly dependent on the potential inhibitor contained in each bead. The most persistently dark beads were collected and sequenced to yield repeatedly highly lipophilic peptides. Some of these were synthesized and found to be strong inhibitors of subtilisin Carlsberg in solution.