Activity-based probes form covalent bonds with active enzymes and can be utilized to profile enzyme activities in vivo, to identify target enzymes and to characterize their function. They have a reactive head group that covalently binds to the target, a tag that allows detection (e.g. a fluorophore) and a linker to connect both.
Matriptase-2 is a transmembrane, multi-domain serine protease with primary substrate specificity for arginine in P1 position, which plays a key role in the human iron homeostasis. Our design of activity-based probes for matriptase-2 is based on linker-connected bis-benzguanidines. The two benzguanidine units interact as arginine mimetics with the S1 and the upper part of the S3/S4 pocket, respectively, and direct the inhibitor to the active site of the target enzyme. An amino acid was introduced as a linker, which 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 an enzyme inactivation and, simultaneously, to a fluorescence labeling of matriptase-2.
Herein, we present the preparation of coumarin-functionalized amino acids and the subsequent linear synthetic approach to coumarin-labeled bis-benzguanidines as activity-based probes for matriptase-2. Spectral properties and kinetic parameters for the reaction with matriptase-2 are reported.
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