Figure 4.

¹⁸O₂ incorporation studies. (A) Summary of proposed species observed in ¹⁸O₂ incorporation experiments. (B – D) ¹⁸O₂ incorporation with ORF36 and TDP-l-epi-vancosamine 10 incubated with ¹⁶O₂ (green) and ¹⁸O₂ (red). Masses shown under structures correspond to unlabeled species. HPLC/MS and MS/MS data were collected at a 15 minutes reaction time, when the hydroxylamine intermediate was most abundant. (B) TDP-l-epi-vancosamine 10 (t_R = 4.5 min). (C) hydroxylamine 13 (m/z = 560, t_R = 5.3 min). (D) Peaks at t_R = 7 – 9 min are proposed to correspond to the nitroso compound 14 (t_R = 8.8 min) and an additional oxidation product with m/z = 576 (t_R = 7.8 min). This new mass has a distinct retention time from the nitrososugar 14 and, based on MS/MS analysis, the additional mass in the m/z = 576 ion is constrained to the pyranose ring. One possible structure is the 4-keto-3-hydroxylamino sugar 12, which is detected as a hydrate 15 under ESI conditions. MS/MS analysis supports this hypothesis in that the labeled ion m/z = 578 readily fragments to m/z = 560.09 and this fragmentation pattern is identical to the reaction product of 9 shifted by 2 mass units.