Tandem photoaffinity labeling of a target protein using a linker with biotin, alkyne and benzophenone groups and a bioactive small molecule with an azide group

Abstract

<div><p>A novel linker containing biotin, alkyne and benzophenone groups (<b>1</b>) was synthesized to identify target proteins using a small molecule probe. This small molecule probe contains an azide group (azide probe) that reacts with an alkyne in <b>1</b> via an azide–alkyne Huisgen cycloaddition. Cross-linking of benzophenone to the target protein formed a covalently bound complex consisting of the azide probe and the target protein via <b>1</b>. The biotin was utilized via biotin–avidin binding to identify the cross-linked complex. To evaluate the effectiveness of <b>1</b>, it was applied in a model system using an allene oxide synthase (AOS) from the model moss <i>Physcomitrella patens</i> (PpAOS1) and an AOS inhibitor that contained azide group (<b>3</b>)<b>.</b> The cross-linked complex consisting of PpAOS1, <b>1</b> and <b>3</b> was resolved via SDS–PAGE and visualized using a chemiluminescent system. The method that was developed in this study enables the effective identification of target proteins.</p></div