A Photo‐Crosslinkable Biotin Derivative of the Phosphoantigen ( E )‐4‐Hydroxy‐3‐Methylbut‐2‐Enyl Diphosphate (HMBPP) Activates Vγ9Vδ2 T Cells and Binds to the HMBPP Site of BTN3A1

Vγ9Vδ2 T cells play an important role in the cross talk of the innate and adaptive immune system. For their activation by phosphoantigens (PAgs), both cell surface receptors, the eponymous Vγ9Vδ2 T cell antigen receptors (Vγ9Vδ2 TCRs) on Vγ9Vδ2 T cells and butyrophilin 3A1 (BTN3A1) on the phosphoantigen‐“presenting” cell, are mandatory. To find yet undetected but further contributing proteins, a biotinylated, photo‐crosslinkable benzophenone probe BioBP‐HMBPP ( 2 ) was synthesized from a known allyl alcohol in nine steps and overall 16 % yield. 2 is based on the picomolar PAg ( E )‐4‐hydroxy‐3‐methylbut‐2‐enyl diphosphate (HMBPP, 1 ). Laser irradiation of 2 at 308 nm initiated the photo‐crosslinking reaction with proteins. When the B30.2 domain of BTN3A1, which contains a positively charged PAg‐binding pocket, was exposed to increasing amounts of HMBPP ( 1 ), labeling by BioBP‐HMBPP ( 2 ) was reduced significantly. Because BSA labeling was not impaired, 2 clearly binds to the same site as natural ligand 1 . Thus, BioBP‐HMBPP ( 2 ) is a suitable tool to identify co‐ligands or receptors involved in PAg‐mediated T cell activation.