Synthesis and Metabolism of BTN3A1 Ligands: Studies on Modifications of the Allylic Alcohol

Abstract

(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.

Figure 1

Relevant phosphoantigens. Endogenous phosphoantigens (1a and 2a) and their monophosphate analogs (1b and 2b). The most potent natural ligand for BTN3A1, HMBPP (3). Phosphonate analogs C-HMBP (4a) and C-HMBPP (4b). Bromohydrin diphosphate (5) and 3-formyl-1-butyl diphosphate (6).

Scheme 1. Synthesis of C-DMAP and Its Phosphonamidate Derivative

Scheme 2. Synthesis of C-HMBP C5 Aldehydes of Phosphonamidates and Their Previously Reported Alcohols

Scheme 3. Synthesis of C-HMBP C5 Modified Aldoxime

Scheme 4. Synthesis of POM₂-C-HMBP Aldehyde by Oxidation with the Dess–Martin Periodinane

Scheme 5. Synthesis of C-HMBP Aldehydes of Mixed Aryl Acyloxyalkyl Phosphonate Esters

Scheme 6. Attempted Synthesis of the Aldehyde Salt 30

Figure 2

Conversion of selected aldehyde (or aldoxime) prodrugs to their free acid alcohol forms in K562 cells. Treatment for 1 h with the indicated compound results in a two-step biological release of the phosphoantigen payload from the prodrug groups to generate the phosphono monoacid and free-acid forms. Simultaneously, the allylic aldehyde is reduced to the alcohol form (n = 2).