Synthetic oligosaccharides as tools to demonstrate cross-reactivity between polysaccharide antigens

Abstract

Escherichia coli O148 is a nonencapsulated enterotoxigenic (ETEC) Gram negative bacterium that can cause diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome in humans. The surface-exposed O-specific polysaccharide (O-SP) of the lipopolysaccharide of this bacterium is considered both a virulence factor and a protective antigen. It is built up of the linear tetrasaccharide repeating unit [3)-α-L-Rhap-(1→2)-α-D-Glcp-(1→3)-α-D-GlcNAcp-(1→3)-α-L-Rhap-(1→] differing from that of the O-SP of Shigella dysenteriae type 1 (SD) only in that the latter contains a D-Galp residue in place of the glucose moiety of the former. The close similarity of the O-SPs of these bacteria indicated a possible cross-reactivity. To answer this question we synthesized several oligosaccharide fragments of E. coli O148 O-SP, up to a dodecasaccharide, as well as their bovine serum albumin or recombinant diphtheria toxin conjugates. Immunization of mice with these conjugates induced anti-O-SP-specific serum IgG antibody responses. The antisera reacted equally well with the LPSs of both bacteria, indicating cross-reactivity between the SD and E. coli O148 O-SPs that was further supported by Western-blot and dot-blot analyses, as well as by inhibition of binding between the antisera and the O-SPs of both bacteria.

Figure 1

Spacer-equipped oligosaccharide fragments of the O-SP of E. coli O148 synthesized in this study

Figure 2

Retrosynthetic strategy toward the targeted oligosaccharides

Figure 3

Tetra- 41, octa- (42), and dodecasaccharide (14) methoxycarbonylpentyl glycosides

Figure 4a

¹H-coupled 2D HSQC spectrum of octasaccharide 42.

Figure 4b

¹H-coupled 2D HSQC spectrum of dodecasaccharide 14.

Figure 5

Western immunoblot of S. dysenteriae type 1 and E. coli O148 LPS with sera induced by whole bacteria or synthetic oligosaccharides bound to the recombinant Diphteria Toxin (DT). The terminal sugars are as follow: 10mer: GlcNAc; 11mer: Gal; 12 and 13mers: Rha. EC: E. coli O148, SD: S. dysenteriae type 1 1. S. dysenteriae type 1 LPS, 2. E. coli O148 LPS

Figure 5

Western immunoblot of S. dysenteriae type 1 and E. coli O148 LPS with sera induced by whole bacteria or synthetic oligosaccharides bound to the recombinant Diphteria Toxin (DT). The terminal sugars are as follow: 10mer: GlcNAc; 11mer: Gal; 12 and 13mers: Rha. EC: E. coli O148, SD: S. dysenteriae type 1 1. S. dysenteriae type 1 LPS, 2. E. coli O148 LPS

Scheme 1a

Synthesis of glucosyl trichloroacetimidate 11 ^aReagents and conditions: (a) NaOMe, MeOH, quant.; (b) BnBr, NaH, DMF, 95%; (c) AcOSi(CH₃)₃ (excess), reflux, 2 h, 93%; (d) PhSSi(CH₃)₃, BF₃, Et₂O, CH₂Cl₂, 0 °C, 90 min, 84%; (e) MeONa, MeOH, 4 h, 97%; (f) MBnCl, NaH, DMF, 91%; (g) (CF₃CO₂)₂Hg, CH₂Cl₂, H₂O, 97%; (h) CCl₃CN, Cs₂CO₃, CH₂Cl₂, quantitative.

Scheme 2a

Synthesis of the trisaccharide trichloroacetimidate 28. ^aReagents and conditions: (a) 1.6 equivalents of 11, TMSOTf, −40 °C → 0 °C, 2h, 90%; (b) H₂ (200 psi), Pd/C, Et₃N, EtOAc, EtOH, Ac₂O; (c) Ce(NH₄)₂(NO₃)₆, CH₃CN, H₂O, yields: 57% for 10, 11% for 25; (d) Ac₂O, C 5H₅N, DMAP, CH₂Cl₂, quantitative; (e) TFA, CH₂Cl₂, 80%; (f) CCl₃CN, DBU, CH₂Cl₂, 95%.

Scheme 3a

Synthesis of the tetrasaccharide trichloroacetimidate 8. ^aReagents and conditions: (a) 4.5 equivalents of 9, TMSOTf, CH₂Cl₂, 0 °C → 23 °C, 3 h, 91%, (b) TFA, CH₂Cl₂, 23 °C, 3 h, 65%; (c) CCl₃CN, DBU, CH₂Cl₂, 0 °C → 23 °C, 1 h, 78%.

Scheme 4a

Synthesis of the spacer-linked tetrasaccharide acceptor 32 ^aReagents and conditions: (a) 1.4 equivalents of 31, TMSOTf, CH₂Cl₂, 0 °C, 1 h, 81%; (b) CS(NH₄)₂, C₅H₅N, DMF, 23 °C, 12 h, 83%.

Scheme 5a

Synthesis of the higher-membered oligosaccharides 34–40 Reagents and conditions: (a) 2.3 equivalents of 33, TMSOTf, CH₂Cl₂, 0 °C, 45 min, 91%; (b) 2.3 equivalents of 28, TMSOTf, CH₂Cl₂, 0 °C, 40 min, 60%; (c) 2.1 equivalents of 8, TMSOTf, CH₂Cl₂, 0 °C → 23 °C, 1 h, 88%; (d) CS(NH₄)₂, C₅H₅N, DMF, 23 °C, 16 h, 77%; (e) 5.2 equivalents of 33, TMSOTf, CH₂Cl₂, 0 °C → 23 °C, 1 h, 88%; (f) 3.5 equivalents of 28, TMSOTf, CH₂Cl₂, 0 °C → 23 °C, 1 h, 86%; (g) 1.9 equivalents of 8, TMSOTf, CH₂Cl₂, 0 °C → 23 °C, 1 h, 64%.

Scheme 5a

Synthesis of the higher-membered oligosaccharides 34–40 Reagents and conditions: (a) 2.3 equivalents of 33, TMSOTf, CH₂Cl₂, 0 °C, 45 min, 91%; (b) 2.3 equivalents of 28, TMSOTf, CH₂Cl₂, 0 °C, 40 min, 60%; (c) 2.1 equivalents of 8, TMSOTf, CH₂Cl₂, 0 °C → 23 °C, 1 h, 88%; (d) CS(NH₄)₂, C₅H₅N, DMF, 23 °C, 16 h, 77%; (e) 5.2 equivalents of 33, TMSOTf, CH₂Cl₂, 0 °C → 23 °C, 1 h, 88%; (f) 3.5 equivalents of 28, TMSOTf, CH₂Cl₂, 0 °C → 23 °C, 1 h, 86%; (g) 1.9 equivalents of 8, TMSOTf, CH₂Cl₂, 0 °C → 23 °C, 1 h, 64%.

Scheme 6

Removal of the protecting groups and attachment of the linker moiety Pg = protecting group

Scheme 7

Synthesis of neoglycoproteins by oxime-conjugation between carbohydrates and proteins.