Regioselective Glycosylation Strategies for the Synthesis of Group Ia and Ib Streptococcus Related Glycans Enable Elucidating Unique Conformations of the Capsular Polysaccharides

Abstract

Group B Streptococcus serotypes Ia and Ib capsular polysaccharides are key targets for vaccine development. In spite of their immunospecifity these polysaccharides share high structural similarity. Both are composed of the same monosaccharide residues and differ only in the connection of the Neu5Acα2-3Gal side chain to the GlcNAc unit, which is a β1-4 linkage in serotype Ia and a β1-3 linkage in serotype Ib. The development of efficient regioselective routes for GlcNAcβ1-3[Glcβ1-4]Gal synthons is described, which give access to different group B Streptococcus (GBS) Ia and Ib repeating unit frameshifts. These glycans were used to probe the conformation and molecular dynamics of the two polysaccharides, highlighting the different presentation of the protruding Neu5Acα2-3Gal moieties on the polysaccharide backbones and a higher flexibility of Ib polymer relative to Ia, which can impact epitope exposure.

Keywords: carbohydrates; conformation analysis; glycosylation; regioselectivity; therapeutics.

Figure 1

A) Chemical structure of GBS CPS Ia and Ib. B) Chemical structure of the target fragments from GBS CPS Ia (1 and 2) and CPS Ib (3 and 4). Synthesis of an unsialylated form of CPS Ia repeating units 5 was also envisaged.

Scheme 1

A) Preparation of disaccharide intermediates 12–15 for the synthesis of GBS CPS Ia repeating unit. Promoters and conditions are described in Table 1. B) Preparation of disaccharide intermediates 21–25 for the synthesis of GBS PSIb repeating unit. Promoters and conditions are described in Table 2.

Scheme 2

Assembly of GBS CPS Ia repeating unit. Reagents and conditions: a) TMSOTf, CH₂Cl₂ dry, −10 °C, (β‐) 75 % from 12 a, 68 % (β‐) from 14 a; b) Me₃N⋅BH₃, BF₃ ⋅Et₂O, MeCN, 0 °C, 70 %; c) TMSOTf, CH₂Cl₂ dry, 0 °C, (β‐) 75 %; d) TfOH, NIS, CH₂Cl₂ dry, −40 °C, (β‐) 73 %; e) LiI, Py, 120 °C; H₂NCH₂CH₂NH₂, EtOH, 90 °C; Ac₂O, Py; MeONa, MeOH; H₂, Pd‐C, 40 % (over five steps); f) 3 m NaOH, THF, reflux; Ac₂O, MeOH; H₂, Pd‐C, 45 % (over three steps).

Scheme 3

Assembly of linear GBS PS Ia fragments 2. Reagents and conditions: a) TMSOTf, CH₂Cl₂ dry, −10 °C, (β‐) 68 %; b) Me₃N⋅BH₃, BF₃ ⋅Et₂O, MeCN, 0 °C, 65 %; c) TMSOTf, CH₂Cl₂ dry, 0 °C, (β‐) 65 %; d) LiI, Py, 120 °C; H₂NCH₂CH₂NH₂, EtOH, 90 °C; Ac₂O, Py; MeONa, MeOH; H₂, Pd‐C, 33 % (over five steps); e) TFACl, Cs₂CO₃, CH₂Cl₂, 61 %; f) TMSOTf, −20 °C, CH₂Cl₂, (β‐) 72 %; g) PdCl₂, MeOH; H₂NCH₂CH₂NH₂, EtOH, 90 °C; Ac₂O, Py; MeONa, MeOH; H₂, Pd‐C, 42 % (over five steps).

Scheme 4

Assembly of GBS PSIb pentasaccharide branched unit 3. Reagents and conditions: a) TMSOTf, CH₂Cl₂ dry, 0 °C, (β‐) 63 % for 41, (β‐) 70 % or 42; b) piperidine, CH₂Cl₂ dry, 92 %; c) TMSOTf, CH₂Cl₂ dry, 0 °C, (β‐) 80 %; d) TfOH, NIS, CH₂Cl₂ dry, −40 °C, (β‐) 65 %; e) HF/pyridine, 0 °C; 3 m NaOH, THF, reflux; Ac₂O/MeOH; H₂/Pd‐C, 40 %.

Scheme 5

Synthetic route to type Ib linear repeating unit. Reagents and conditions: a) TMSOTf, CH₂Cl₂ dry, 0 °C, (β‐) 55 %; b) piperidine, CH₂Cl₂, 90 %; c) TMSOTf, CH₂Cl₂ dry, 0 °C, (β‐) 66 %; d) TfOH, NIS, CH₂Cl₂ dry, −40 °C, (β‐) 40 %; e) HF/pyridine; 3 m NaOH, THF, reflux; Ac₂O/MeOH; H₂/Pd‐C, 40 %.

Figure 2

Superimposition of the major conformations for pentasaccharides 1 (lime) and 3 (grey), with the exo‐anti‐Φ geometry around the Neu5Acα2‐3Gal linkage.

Figure 3

Glycosidic linkage analysis for GBS Ia (A) and Ib (B) polysaccharides: Φ/Ψ plots for representative glycosidic bonds of a 10 repeating unit model along the 2.5 μs MD simulation.

Figure 4

¹H‐¹³C‐HSQC spectrum recorded for the pentasaccharide repeating unit of GBS Ia at 600 MHz, 298 K, D₂O (A and B) and for GBS CPS Ib at 800 MHz, 318 K, D₂O, showing the assignment of the ¹H and ¹³C NMR signals. As expected, the matching is excellent except for some signals of residue E.

Figure 5

The key regions of the NOESY spectra recorded for the GBS CPS Ia (A and C) and GBS CPS Ib (B and D) polysaccharides showing the essential inter‐residue cross peaks.

Figure 6

Model structures for the GBS Ia and Ib polysaccharides: A) Ia with the major exo‐anti‐Φ conformation around all the Neu5Acα2‐3Gal linkages B) Ib with exo‐anti‐Φ conformation around all Neu5Acα2‐3Gal linkages, and C) Ib with the major exo‐syn‐Φ conformation around all Neu5Acα2‐3Gal linkages.