Second Generation Vibrational Reporter Protease Inhibitor

ABSTRACT The active site of the protease subtilisin was selectively and effectively probed with infrared spectroscopy using 4‐azidobenzenesulfonyl fluoride (ABSF), a covalent inhibitor containing an azide vibrational reporter sensitive to its local environment. Notably, mass spectrometry and x‐ray crystallography showed selective reactivity at the active site serine of subtilisin with ABSF, a significant improvement compared to the nitrile inhibitor, 4‐cyanobenzenesulfonyl fluoride (CBSF). However, the azide IR absorbance feature of the water‐soluble analogue sodium 4‐azidobenzenesulfonate (NaABSO) was found to be complex due to accidental Fermi resonance. Thus, several 15 N isotopologues of NaABSO were prepared to modulate this complexity. 15 N‐labeling of the middle nitrogen or the middle and terminal nitrogen atoms of the azide group significantly decreased the complexity of the IR feature in the azide asymmetric stretch region. Both of these azide reporter isotopologues were found to be sensitive to solvents mimicking the hydrophobic and hydrophilic environments found in proteins. The position of the azide asymmetric stretch frequency of the middle 15 N‐labeled ABSF isotopologue was then used to show that the active site of subtilisin is a hydrophilic/hydrated environment.