Figure 3: Formation and solution structures of uracil-based antigens that activate MAIT cells.

(a) Exclusive formation of 5-OP-RU 3c from 1 and 5c in DMSO-d₆, as monitored by ¹H NMR spectroscopy, with the characteristic signals of 1 and 5-OP-RU 3c as indicated. (b) Formation of 5-OP-RU 3c in DMSO (black) versus PBS buffer (red). In PBS buffer (red, 2.62 mM of 1, 7.86 mM of 5c, pH 7.4, 37 °C), 3c reached a maximum concentration corresponding to only 1.1% conversion of 1 at 5 min. In DMSO (black, 31.8 mM 1 and 34.5 mM 5c, room temperature), 5-OP-RU 3c reached 100% conversion after 2 days and then plateaued. (c) Stability of 5-OP-RU 3c in DMSO (black), PBS buffer (red) and after the DMSO solution was diluted into PBS buffer (blue). Data for PBS buffer were extracted from b (red) at the time the maximum 5-OP-RU 3c concentration was reached (that is, 5 min after mixing). (d–f) HMBC NMR spectra of 3a-c (DMSO-d₆), respectively. Square boxes indicate correlations for the aldehyde form of 3b, and circles for its hydrate form 3b′. Arrows and boxes indicate key ¹H–¹³C long-range correlations that unambiguously characterize the compounds. J refers to heteronuclear coupling through 1, 2, 3 or 4 bonds. (g) MAIT cell activation. Data represent mean±s.e.m. (n=3). The α-dicarbonyls 5a-c, lumazines 4a-b and the solvent DMSO-d₆ were inactive (up to 15 μM), while 4c and 4d had only very weak activity at the highest concentrations tested (EC₅₀>100 nM).