A Fluorescent‐Labeled Phosphono Bisbenzguanidine As an Activity‐Based Probe for Matriptase

Abstract

Activity-based probes are compounds that exclusively form covalent bonds with active enzymes. They can be utilized to profile enzyme activities in vivo, to identify target enzymes and to characterize their function. The design of a new activity-based probe for matriptase, a member of the type II transmembrane serine proteases, is based on linker-connected bis-benzguanidines. An amino acid, introduced as linker, bears the coumarin fluorophore. Moreover, an incorporated phosphonate allows for a covalent interaction with the active-site serine. The resulting irreversible mode of action was demonstrated, leading to enzyme inactivation and, simultaneously, to a fluorescence labeling of matriptase. The ten-step synthetic approach to a coumarin-labeled bis-benzguanidine and its evaluation as activity-based probe for matriptase based on in-gel fluorescence and fluorescence HPLC is reported. HPLC fluorescence detection as a new application for activity-based probes for proteases is demonstrated herein for the first time.

Recent Developments in Peptidyl Diaryl Phoshonates as Inhibitors and Activity-Based Probes for Serine Proteases

“…The probe allowed for direct matriptase-2 detection in a complex protein mixture separated by gel electrophoresis. In continuation, a similar coumarin-based probe of matriptase was developed by the corresponding design and synthesis approach (Figure 1, compound a ) [44]. To label the inhibitor, 6,7-dimethoxycoumarin-3-carboxylic was coupled with ω-amino group of the P2 lysine residue.…”

Design of an Activity-Based Probe for Human Neutrophil Elastase: Implementation of the Lossen Rearrangement To Induce Förster Resonance Energy Transfers

“…Coumarins with donor groups at the position 7, such as coumarin 343, represent a widely used Stokes shifts, as well as chemical and enzymatic stability are their favored properties. 25,34 We aimed at synthesizing a small series of linker-connected N-(mesyloxy)phthalimides (Scheme 2). The nitro-substituted N-hydroxyphthalimide 1 was chosen as the starting compound whose nitro group was reduced using Pd/C to afford compound 2.…”

A Short Peptide Inhibitor as an Activity-Based Probe for Matriptase-2

“…Here we present a short biotinylated peptide probe with a chloromethyl ketone warhead (biotin-RQRR-CMK) as an irreversible inhibitor and activity-based probe for MT-2 ( Figure 1 ). This probe was originally developed for the related enzyme matriptase [ 30 , 31 ], which exhibits a similar substrate specificity [ 32 ]. Selectivity for MT-2 over matriptase has rarely been observed within series of synthetic inhibitors [ 9 , 18 , 23 , 24 ], but has been achieved with certain SFTI-1 analogues [ 21 ] and, in particular, peptidic ketones containing unnatural amino acids, such as l - allo -threonine at P2 position [ 20 ].…”