Activity-based probes (ABPs) are active-site directed chemical probes that report on enzyme function in complex biological systems. The two essential elements of an ABP include i) a reactive group for covalent labeling of the enzyme active site, and ii) a reporter handle such as a fluorophore or biotin for visualization and/or enrichment of the labeled enzyme. The reporter handle may be replaced by a latent alkyne tag to allow downstream attachment to a fluorophore- or biotin-azide via the Huisgen Cu-Catalyzed Azide Alkyne Click (CuCAAC) reaction, post-covalent target engagement by the probe. We have developed novel alkyne tagged ABPs against a set of closely related proteases of therapeutic relevance. Fashioned off of the enzymes’ known inhibitor, these probes demonstrated similar inhibitory activity (kinact/KIapp 2500-4300 M-1s-1) as their parent compound (kinact/KIapp 3800 M-1s-1) in a biochemical assay using recombinant enzymes. In in-gel fluorescence profiling experiments, however, the said probes displayed less than satisfactory performance. Via a systematic investigation of the individual components of the experimental protocol, we found that the fidelity of enzyme visualization by in-gel fluorescence, in this case, was dictated primarily by the efficiency of the CuCAAC reaction between the target-engaged alkyne probe and the fluorophore (tetramethylrhodamine-TAMRA) azide. Specifically, we demonstrate that having a picolyl-azide group as the bioorthogonal handle on the TAMRA fluorophore enhances in-gel fluorescence yields dramatically (Figures 1 and 2) compared to corresponding alkyl azide. Herein, we wish to present this optimization effort and its utilization in subsequent biological follow up, including target engagement experiments in mucosal extracts.

Click chemistry optimization for activity-based investigation of enzyme function

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ACS Fall 2022

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