Activity-based probes are small molecules that bind specifically and irreversibly to active proteases. They consist of a reactive functional group (“electrophilic warhead”) that binds to the active site of the target enzyme, a tag that enables enzyme detection, and a linker that is a peptide based sequence that connects the warhead with the tag and provides the selectivity towards an enzyme of interest.
Diazoketone (Nα-benzyloxycarbonyl-Nβ-tert-butoxycarbonyl-3,7-diamino-1-diazo-2-oxoheptane) was synthesized from Z-Lys(Boc)-OH following a procedure described in the literature. Acyloxymethyl ketone (AOMK) (Nα-benzyloxycarbonyl-Nβ-tert-butoxycarbonyl-3,7-diamino-2-oxoheptyl 2,6-dimethylbenzoate) was then synthesized from the diazoketone using different methods.
AOMK was first synthesized via direct substitution reaction between the diazoketone and 2,6-dimethylbenzoic acid in a fluorinated solvent (TFE). Synthesis of AOMK through the p-tosylate derivative was unsuccessful as the derivative decomposed. AOMK was then synthesized through a chloromethyl ketone according to a modified literature procedure.
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