O‐Specific Polysaccharide of Vibrio cholerae O139: Improved Synthesis and Conjugation to BSA by Squaric Acid Chemistry

Abstract

The sequence α‐Colp‐(1→2)‐4,6‐P‐β‐d‐Galp‐(1→3)‐[α‐Colp‐(1→4)]‐β‐d‐GlcpNAc‐(1→4)‐α‐d‐GalpA‐(1→3)‐β‐d‐QuipNAc constitutes the complete O‐specific polysaccharide (O‐antigen, O‐SP) of Vibrio cholerae O139. It was chemically synthesized in a linker‐equipped, conjugation‐ready form (7) and conjugated to a model protein carrier, bovine serum albumin. The preparation involved the synthesis of a tetrasaccharide intermediate sequence β‐d‐Galp‐(1→3)‐β‐d‐GlcpNAc‐(1→4)‐α‐d‐GalpA‐(1→3)‐β‐d‐QuipNAc→linker by coupling of two disaccharide intermediates followed by a stepwise, two single‐site colitosylations. The present synthesis is an alternative to the academic, small‐scale synthesis developed earlier where the final hexasaccharide assembly was achieved by simultaneous, two‐sites colitosylation. The approach reported herein comprises a few more synthetic steps but is experimentally less demanding, minimizes separation difficulties and is more convenient when tens of mgs of the final product is required. Syntheses of non‐phosphorylated and methyl ester analogues of 7 are also described.

Defining Polysaccharide-Specific Antibody Targets against Vibrio cholerae O139 in Humans following O139 Cholera and following Vaccination with a Commercial Bivalent Oral Cholera Vaccine, and Evaluation of Conjugate Vaccines Targeting O139

“…Complex structure and polymolecularity make such substances unattached to the core virtually inaccessible in pure form either by regulated chemical synthesis or by isolation from bacteria. Availability of specimens of synthetic OSP of Vibrio cholerae O139, its fragments ( 25 – 30 ), and homogeneous native OSP-core ( 18 ) provided us with an unprecedented opportunity to subject these LPS-related antigens to the immunological studies presented here.…”

“…We generated three preparations of native V. cholerae O139 OSP-core fragment as previously described ( 18 ): (i) an initial preparation was generated by delipidation of LPS followed by purification by Bio-Gel P30 size exclusion chromatography (SEC), to afford material we termed “crude” O139 OSPc (preparation 1 [ Table 1 ]); (ii) the crude preparation underwent additional purification, to substantial but not complete purity, by semipreparative high-performance liquid chromatography (HPLC), to obtain an “intermediate” preparation (preparation 2); and (iii) the intermediate preparation underwent further purification by analytical HPLC to obtain “pure” OSP-core (hexasaccharide-core, preparation 3), which was homogeneous by NMR spectroscopy, analytical HPLC, and electrospray ionization-mass spectrometry (ESI-MS), as previously described ( 18 ). We also synthesized the complete V. cholerae O139 OSP (hexasaccharide), as well as a series of derivatives and fragments of the OSP from disaccharide to hexasaccharide (conjugates 4 to 18 [ Table 1 ]) ( 25 – 30 ). These include compounds lacking one or both colitose units or the phosphate residue or having a methyl galacturonate instead of a galacturonic acid residue.…”

Zwitterionic Phosphodiester-Substituted Neoglycoconjugates as Ligands for Antibodies and Acute Phase Proteins

“…Hydrogenation reactions in methanol or ethanol solutions, however, resulted in the simultaneous formation of reductive amination products, which could not be separated from 22 . This side reaction has been noted in the literature . Finally, the hydrogenation was carried out in a THF/water mixed solvent containing 1% acetic acid.…”

Semi- and fully synthetic carbohydrate vaccines against pathogenic bacteria: recent developments

“…The first chemical synthesis of the complete protective ready-for-conjugation antigen of V. cholerae O139 [ 80 ] was improved from a small-scale chemical synthesis into a less demanding experimental method. A linker-equipped synthesis of the O-specific polysaccharide of V. cholerae O139 allowed for conjugation to a carrier protein, BSA ( Figure 11 ) [ 84 ]. Glycoclusters displaying synthetic oligosaccharides of the O-specific polysaccharide of V. cholerae O1 ST Inaba were conjugated to BSA.…”