Synthesis of 6-PEtN-α-D-GalpNAc-(1–>6)-β-D-Galp-(1–>4)-β-D-GlcpNAc-(1–>3)-β-D-Galp-(1–>4)-β-D-Glcp, a Haemophilus influenzae lipopolysacharide structure, and biotin and protein conjugates thereof

Abstract

Summary Background: In bacteria with truncated lipopolysaccharide structures, i.e., lacking the O-antigen polysaccharide part, core structures are exposed to the immune system upon infection and thus their use as carbohydrate surface antigens in glycoconjugate vaccines can be considered and investigated. One such suggested structure from Haemophilus influenzae LPS is the phosphorylated pentasaccharide 6-PEtN-α-D-GalpNAc-(1→6)-β-D-Galp-(1→4)-β-D-GlcpNAc-(1→3)-β-D-Galp-(1→4)-β-D-Glcp. Results: Starting from a spacer-containing lactose derivative a suitably protected lacto-N-neotetraose tetrasaccharide structure was constructed through subsequential couplings with two thioglycoside donors, a glucosamine residue followed by a galactose derivative, using NIS/AgOTf as promoter. Removal of a silyl protecting group at the primary position of the non-reducing end residue afforded an acceptor to which the terminal α-galactosamine moiety was introduced using a 2-azido bromo sugar and halide assisted coupling conditions. Global deprotection afforded the non-phosphorylated target pentasaccharide, whereas removal of a silyl group from the primary position of the non-reducing end residue produced a free hydroxy group which was phosphorylated using H-phosphonate chemistry to yield the phosphoethanolamine-containing protected pentasaccharide. Partial deprotection afforded the phosphorylated target pentasaccharide with a free spacer amino group but with a protected phosphoethanolamino group. Conjugation of the spacer amino group to biotin or dimethyl squarate followed by deprotection of the phosphoethanolamino group and, in the case of the squarate derivative, further reaction with a protein then afforded the title conjugates. Conclusion: An effective synthesis of a biologically interesting pentasaccharide structure has been accomplished. The target pentasaccharide, an α-GalNAc substituted lacto-N-neotetraose structure, comprises a phosphoethanolamine motif and a spacer aglycon. Through the spacer, biotin and protein conjugates of the title compound have been constructed to allow further use in biological experiments.

Synthesis of type 1 Lewis b hexasaccharide antigen structures featuring flexible incorporation of l-[U-13C6]-fucose for NMR binding studies

“…The corresponding unlabelled L-Fuc 5 35 , the D-GlcNH2 7 36 ,the D-Gal 8 37 thioglycoside donors, and the D-Lac acceptor 6 38 (Scheme 1) were prepared according to literature procedures. The assembly of the tetrasaccharide backbone 15 started with the NIS/AgOTf-mediated glycosylation at room temperature with D-Lac acceptor 6 and D-GlcNH2 donor 7 to give the corresponding 1,2-trans linked trisaccharide 14 38 The 1 H NMR spectrum of di-13 C-labelled hexasaccharide 2 shows the expected additional 13 C-1 H couplings corresponding to the labelled fucosyl residues (Figure 2A) which collapsed in the 13 C-1 H decoupled experiments, reassembling the corresponding spectrum of the unlabelled hexasaccharide 3. A standard 13 C NMR experiment displays only the 13 C enriched fucosyl residues, clearly showing the 13 C- 13 C couplings, and makes it a…”

Haemophilus parainfluenzae expresses diverse lipopolysaccharide O-antigens using ABC transporter and Wzy polymerase-dependent mechanisms

“…We found capsule synthesis genes in only one of our 18 H. parainfluenzae isolates (strain 18; unpublished data) and the sugar-specific genes were not H. influenzae -like, so capsule-based H. influenzae vaccines are unlikely to target H. parainfluenzae . Regarding potential NTHi vaccines, those based on cell-surface proteins may target both species, whilst those based on the LPS HMG unit ( Sundgren et al, 2010 ) would not impact directly on H. parainfluenzae as the O-unit structures produced by this species contain different sugar and linkage combinations to the HMG.…”

Synthesis of a Lewis b hexasaccharide thioglycoside donor and its use towards an extended mucin core Tn heptasaccharide structure and a photoreactive biotinylated serine linked hexasaccharide

“…Due to earlier findings, in which the removal of the phthalimido group in a fully assembled hexasaccharide was problematic and low yielding, 13 we decided to remove this protecting group and introduce the acetamide moiety prior to further glycosylation steps, despite being aware of problematic reports on the use of acetamido-containing acceptors. [29][30][31] The acetyl groups in trisaccharide 10 were removed using Zemplén conditions and the phthalimido group cleaved using hydrazine hydrate in ethanol. The amino group was then chemoselectively acetylated with acetic anhydride in methanol followed by benzoylation of the hydroxyl groups to give compound 11 in a yield of 83% over 4 steps.…”