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

Abstract

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.

The Role of the Phosphorus Atom in Drug Design

“…Therefore, multiplication of Vγ9Vδ2 T cells is stimulated by the presence of these organophosphorus compounds that bind to the signaling protein butyrophilin 3A1. The most effective natural agonist for this protein is ( E )‐4‐hydroxy‐3‐methyl‐but‐2‐enyl diphosphate (HMBPP, 139 ), which is a metabolite of the 1‐deoxy‐ d ‐xylulose 5‐phosphate (DOXP) pathway found in plants and Apicomplexan parasites . Isopentenyl diphosphate ( 64 ) and dimethylallyl diphosphate ( 63 ) behave as weak phosphoantigens.…”

“…This compound, which contains a biotin molecule at one end and HMBPP at the other, bound through its oxygen atom, also has two linkers and a molecule of benzophenone at its center . The benzophenone moiety was the actual probe to interact with proteins . The title compound 154 could be efficiently synthesized in nine steps from the previously described allylic alcohol 155 .…”

“…Compound 154 was able to be bound to bovine serum albumin upon laser irradiation at 308 nm. This probe was 3000‐fold less efficient than 139 , but at the same site, suggesting that 154 might be a powerful tool to explore proteins associated with the activation of Vγ9Vδ2 T cells . The structures of compounds 139 – 155 are illustrated in Figure .…”

“…The most effective natural agonist for this protein is (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate( HMBPP, 139), which is am etabolite of the 1-deoxy-d-xylulose 5-phosphate (DOXP)p athway found in plants and Apicomplexan parasites. [129] Isopentenyl diphosphate (64)a nd dimethylallyl diphosphate (63)b ehavea sw eak phosphoantigens.I nf act, risedronate( 58,s ee Figure 9) and zoledronate (61,s ee Figure 9) activateV g9Vd2Tcellsv ia accumulation of isopentenyl diphosphate as ac onsequence of blocking farnesyl diphosphate synthase.A ctivation of Vg9Vd2Tcells mayb ea ppliedt od ifferent parasitic and bacterial diseases including malaria, toxoplasmo- ChemMedChem 2019, 14,190 -216 www.chemmedchem.org sis, andtuberculosis. Awide varietyoftumor cells could be targeted by this behavior as well.…”

“…To identifyc o-ligands or receptors involved in phosphoantigen-mediatedT -cell stimulation, ap hoto-cross-linkable biotin derivative (154)w as synthesized.T his compound, which containsabiotin molecule at one end and HMBPP at the other,b ound throughi ts oxygen atom, also has two linkers and am olecule of benzophenone at its center. [129] The benzophenonem oiety was the actual probe to interactw ith proteins. [129] The title compound 154 could be efficiently synthesized in nine steps from the previously described allylic alcohol 155.…”