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. 2022 Jan 27;13(2):164-170.
doi: 10.1021/acsmedchemlett.1c00408. eCollection 2022 Feb 10.

Synthesis and Metabolism of BTN3A1 Ligands: Studies on Diene Modifications to the Phosphoantigen Scaffold

Nyema M Harmon  1 , Michael M Poe  2 , Xueting Huang  2 , Rohit Singh  2 , Benjamin J Foust  1 , Chia-Hung Christine Hsiao  2 , David F Wiemer  1   3 , Andrew J Wiemer  2   4
Affiliations

Synthesis and Metabolism of BTN3A1 Ligands: Studies on Diene Modifications to the Phosphoantigen Scaffold

Nyema M Harmon et al. ACS Med Chem Lett. .

Abstract

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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Conflict of interest statement

The authors declare the following competing financial interest(s): A.J.W. and D.F.W. own shares in Terpenoid Therapeutics, Inc. The current work did not involve the company. The other authors have no financial conflicts of interest.

Figures

Figure 1
Figure 1
Activation of Vγ9Vδ2 T cells by HMBPP and its diene analogs. (A) HMBPP and analogs bind to the intracellular domain of BTN3A1 in the antigen presenting cell (APC). BTN2A1 and 3A1 interact to form the HMBPP receptor. Ligand-induced conformational changes to the receptor modify the extracellular domain of 2A1 to allow detection by the Vγ9Vδ2 TCR within its Vγ9 protein. (B) Structure of HMBPP (1). (C) Prior analogs used as controls (2, 3) and the diene scaffold (4, this work) investigated in this study.
Scheme 1
Scheme 1. Synthesis of Compounds 8 and 9
Reagents and conditions: (a) diisopropylamine, n-BuLi, −78 °C, then 3-methyl-2-butenal, 2 h, 64%; (b) H2SO4, tetrahydrofuran (THF), reflux, 0.5 h, 10%; (c) NaI, POM-Cl, CH3CN, reflux, 16 h, 41% of 8, 18% of 9.
Scheme 2
Scheme 2. Synthesis of 13
Reagents and conditions: (a) TEMPO, bis(acetoxy)iodobenzene, CH3CN; (b) TMMBP, NaH, THF; (c) POM-Cl, NaI, CH3CN, 14% of diene 13 over three steps.
Scheme 3
Scheme 3. Synthesis of 16
Reagents and conditions: (a) TEMBP, KH, THF; (b) TMSBr then NaOH.
Scheme 4
Scheme 4. Synthesis of Dienes 20 and 21
Reagents and conditions: (a) TEMBP, NaH, THF; (b) TMSBr, CH2Cl2 then (COCl)2, N,N-dimethylformamide (DMF); (c) phenol, Et3N, CH2Cl2, 35%; (d) phenol, Et3N, CH2Cl2 then Ala-OCH3, Et3N, CH2Cl2, 39%.
Scheme 5
Scheme 5. Synthesis of Diene 24
Reagents and conditions: (a) (COCl)2, DMF, CH2Cl2; (b) phenol, THF, Et3N; (c) TMSBr then Ag2CO3, POM-Cl, reflux, 42% overall.
Scheme 6
Scheme 6. Synthesis of 25
Reagents and conditions: (a) CH2Cl2, TMSBr, collidine, 0 °C to room temperature (RT), then NaOH, 91%.
Figure 2
Figure 2
Metabolism of dienes 8, 9, 13, 21, and 24. K562 cells were treated for 1 h with 100 μM of the indicated compound. The displayed metabolites were detected, including the ionized form of 25. The retention times, m/z, and integrated peak intensities are reported.
Figure 3
Figure 3
Diene salt forms compete with probe binding to BTN3A1. Compounds 1, 2, 16, and 25 were tested for binding to BTN3A1 full intracellular domain (BFI). * p < 0.05, ANOVA with Tukey’s post hoc test, relative to probe only. # p < 0.05, relative to probe plus protein control.
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