(E)-4-Hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) and its phosphonate analogs are potent phosphoantigens. HMBPP contains an (E)-allylic alcohol which interacts with the molecular target BTN3A1 giving an antigenic signal to activate Vγ9Vδ2 T cells. As probes of BTN3A1 function, we prepared prodrug derivatives of the HMBPP analog C-HMBP that lack the (E)-allylic alcohol or have modified it to an aldehyde or aldoxime and evaluated their biological activity. Removal of the alcohol completely abrogates phosphoantigenicity in these compounds while the aldoxime modification decreases potency relative to the (E)-allylic alcohol form. However, homoprenyl derivatives oxidized to an aldehyde stimulate Vγ9Vδ2 T cells at nanomolar concentrations. Selection of phosphonate protecting groups (i.e., prodrug forms) impacts the potency of phosphoantigen aldehydes, with mixed aryl acyloxyalkyl forms exhibiting superior activity relative to aryl amidate forms. The activity correlates with the cellular reduction of the aldehyde to the alcohol form. Thus, the functionality on this ligand framework can be altered concurrently with phosphonate protection to promote cellular transformation to highly potent phosphoantigens.

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Phosphoantigens (pAgs) are small organophosphorus compounds such as (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) that trigger an immune response. These molecules bind to butyrophilin 3A1 (part of the HMBPP receptor) and activate Vγ9Vδ2 T cells. To explore the structure–activity relationships underlying this process, we evaluated a series of novel diene analogs of HMBPP. Here we report that prodrug forms of [(1E)-4-methylpenta-1,3-dien-1-yl] phosphonic acid that lack the allylic alcohol of HMBPP but instead contained a diene scaffold exhibit mid-nanomolar potency for the activation of Vγ9Vδ2 T cells. The compounds also trigger the production of T-cell interferon γ upon exposure to loaded K562 cells. Although both the allylic alcohol and the diene scaffold boost pAg activity, the combination of the two decreases the activity and results in glutathione conjugation. Together, these data show that the diene scaffold results in intermediate pAgs that may have implications for the mechanisms regulating the HMBPP receptor.

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