Synthesis, Conformal Analysis, and Antibody Binding of Staphylococcus aureus Capsular Polysaccharide Type 5 Oligosaccharides

Abstract

Staphylococcus aureus is one of the most prominent pathogens responsible for life-threatening hospital acquired infections. Most clinical isolates belong to serotype 5 or 8, which express unique capsular polysaccharides (CP), composed of the rare N-acetyl-β-d-mannosaminuronic acid (β-d-ManNAcA), N-acetyl-α-l-fucosamine (α-l-FucNAc) and N-acetyl-β-d-fucosamine (β-d-FucNAc) that can be used for the development of conjugate vaccines. Different acetylation patterns of CP5 create microheterogeneous polymers, carrying partial zwitterionic character, which may be important for immunological activity. We here report on the assembly of a set of conjugation-ready CP5 oligosaccharides, ranging in length from trisaccharides to nonasaccharides. The developed protecting group strategy has allowed the incorporation of N-acetyl, -NH₃ ⁺ and O-acetyl groups. The reported syntheses offer solutions for the construction of the challenging cis-glycosidic linkages, the incorporation of many different functional groups and the installation of an appropriate linker for future conjugation purposes. Conformational analysis of the O-acetylated oligomers has revealed a distinctive linear conformation with the repeating units (RUs) being flipped ∼180° with respect to the flanking RUs. Binding studies with CP5-antibodies revealed the trisaccharide to be too short for relevant binding, while the hexa- and nonasaccharides exhibited strong binding. The l-FucNAc acetyl esters and d-FucNAc acetamides were shown to be crucial for binding.

Keywords: Conformational analysis; Oligosaccharides; Stereoselectivity; Synthetic vaccines.

Figure 1

Schematic representation of the repeating unit of CP5 and CP8.

Figure 2

Previously reported synthesis of CP5 trisaccharides and the set of CP5 oligosaccharides reported here.

Scheme 1

Retrosynthetic strategy for the assembly of the CP5‐oligosaccharides.

Scheme 2

a) Synthesis of trisaccharides 6–9. b) Deprotection of the trisaccharide to form target 1. Reaction conditions: a) a NIS, TMSOTf, DCM, –60 °C → –30 °C, 15:91%, α/β = 95:5; NIS, TBSOTf, DCM –78 °C → –0 °C, 16: 92%, α/β = 90:10; b NaOMe, MeOH, 17: 92%, 18: 79%; c NIS, TMSOTf, DCM, –60 °C → –10 °C, 6, 86%, α/β = 30:70, 7:91%, α/β = 21:79, 8:93%, α/β = 10:90, 9:86%, α/β = 10:90. b) d TBAF, AcOH, THF, 0 °C → rt, 81%, e ClC(═NPh)CF₃, K₂CO₃, acetone, 92%, f TBSOTf, DCM/MeCN, –50 °C, 73%, g i DDQ, DCM/H₂O, ii) Ac₂O, DMAP, pyridine, 89% over two steps, h zinc, AcOH, Ac₂O, THF, 50 °C, 18%, j Pd(OH)₂/C, AcOH, H₂, t‐BuOH/H₂O, 56%.

Scheme 3

Assembly of oligosaccharides 2–5. Reaction conditions:a) a i) TBAF, AcOH, THF, 0 °C → rt °C, ii) ClC(═NPh)CF₃, K₂CO₃, acetone, 96%, 25:87%, 26:79%, 27:83%, b TBSOTf, DCM/MeCN, –50 °C, 29:88%, β only, 31:79%, β only; c HCl in HFIP, TES, DCM, 0 °C, 30:76%, 32:75% d TBSOTf, DCM/MeCN, –78 °C, 33:70%, 34:80%, 36:75%; e HCl in HFIP, TES, DCM, 0 °C, 35:76%, 36:69%; f TBSOTf, DCM/MeCN, –78 °C, 38:79%, 39:65%. b) g for 2:i 33, DDQ, DCM/H₂O, ii) Ac₂O, DMAP, pyridine, 82% over two steps, iii) zinc, AcOH, Ac₂O, THF, 50 °C, 11%, iv) Pd(OH)₂/C, AcOH, H₂, t‐BuOH/H₂O, 47%; for 3:i 38, DDQ, DCM/H₂O, ii) Ac₂O, DMAP, pyridine, 74% over two steps, iii) zinc, AcOH, Ac₂O, THF, 50 °C, 16%, iv) Pd(OH)₂/C, AcOH, H₂, t‐BuOH/H₂O, 49%; h for 4:i 38, zinc, AcOH, Ac₂O, THF, 50 °C, ii) Pd(OH)₂/C, AcOH, H₂, t‐BuOH/H₂O, 60%; j i) 1 M aq. NaOH, MeOH, THF, (ii) 1,3‐propanedithiol, Et₃N, H₂O, pyridine, (iii) THF, H₂O, NaHCO₃, Ac₂O, (iv) Na, liq. NH₃, tBuOH, 3‐buten‐1‐ol, THF, –78 °C, (v) conc, NH₄OH, 60 °C, 38%.

Figure 3

Conformational analysis by NMR and MM calculations of trisaccharide 1, hexasaccharide 2, and nonasaccharide 3, showing the main conformations of the glycosidic linkages and the spatial orientation of the acetyl groups. a) Main conformation and 2D ROESY spectrum of trisaccharide 1. b) Main conformation, section of the two‐dimensional NOESY spectrum taken at the frequency of H1(A’), and 1D selective NOE spectra of hexasaccharide 2. c) Main conformation and 2D sections of the NOE correlations defining the main conformations around the glycosidic linkages of nonasaccharide 3.

Figure 4

a) Competitive SPR using the synthetic oligosaccharides and rat anti‐CP5 mAb showed that 1 was barely recognized by the antibody, while 2 and 3 bind the antibody well, leading to effective competition. b) IC50 values obtained through competitive SPR. **** p < 0.001, ** p, 0.01, ns = not significant.