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.

A novel linker containing biotin, alkyne and benzophenone groups (1) 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 1 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 1. The biotin was utilized via biotin-avidin binding to identify the cross-linked complex. To evaluate the effectiveness of 1, it was applied in a model system using an allene oxide synthase (AOS) from the model moss Physcomitrella patens (PpAOS1) and an AOS inhibitor that contained azide group (3). The cross-linked complex consisting of PpAOS1, 1 and 3 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.