Investigating bacterial N-linked glycosylation: synthesis and glycosyl acceptor activity of the undecaprenyl pyrophosphate-linked bacillosamine

Abstract

The chemical synthesis and biological activity of undecaprenyl pyrophosphate bacillosamine (Und-PP-Bac), an obligatory intermediate in the asparagine-linked glycosylation pathway of Campylobacter jejuni, are reported. The key transformation involves the coupling of bacillosamine phosphate and undecaprenyl phosphate. The synthetic Und-PP-Bac can be used to investigate the activity of the enzyme PglA, which catalyzes the first glycosyl transfer in substrate biosynthesis for N-linked protein glycosylation in the pathogenic gram-negative bacterium. The availability of this synthetic substrate makes it possible to access polyprenyl-linked oligosaccharides, such as the GalNAc-alpha-1,3-bacillosamine-alpha-1-PP-Und intermediate, that will enable exploration of the remaining enzymes in the prokaryotic glycosylation pathway. Study of the bacterial glycosylation system will provide insight into the corresponding eukaryotic process, which is currently poorly understood.

Figure 1

Structure of the undecaprenyl-linked heptasaccharide.

Figure 2

(a) Reaction catalyzed by PglA. (b) Radioactive assay for PglA activity with Und-PP-Bac, 2. DPM (disintegrations per minute) denotes radioactive counts in the organic phase.

Figure 3

(a) Hydrolysis and 2-aminobenzamide labeling. (b) HPLC trace of 2AB-labeled GalNAc-Bac (fluorescence detection). * Denotes major product peak. (c) MALDI-MS of disaccharide (peak at 592.27 denotes sodium adduct of expected peak).

Scheme 1

Synthesis of Und-PP-Bac^{a a} Reagents and conditions: (a) benzoyl chloride, pyridine, –40 → 0 °C, 70%; (b) Tf₂O, CH₂Cl₂/pyridine, 0 °C → rt; (c) NaN₃, DMF, rt, 62% over two steps; (d) NaOMe, MeOH, rt, 80%; (e) TsCl, pyridine, 0 °C → rt, 75%; (f) NaI, MeCN, 80 °C, 70%; (g) H₂, Pd(OH)₂/C, DIPEA, MeOH, 32 °C; (h) Ac₂O, pyridine, rt, 65% over two steps; (i) NaOMe, MeOH, 90%; (j) BzCl, pyridine, 60%; (k) H₂, Pd(OH)₂/C, MeOH, 32 °C, 86%; (l) LiHMDS, −68 °C; then [(BnO)₂P(O)]₂O, −68 → 0 °C, 50%; (m) H₂, Pd/C, MeOH, 99%; (n) carbonyl diimidazole, DMF; then undecaprenyl phosphate, 50%; (o) NaOMe, MeOH, 99%.