Synthesis of a spacer-linked derivative of heptopyranosyl(α1-3)heptopyranose expressed in lipooligosaccharide and lipopolysaccharide.

Glycosylation of penta-O-acetyl heptopyranosyl trichloroacetimidate with the 3-OH acceptor, methyl 2-O-benzyl-4,6-O-benzylidene-7,8-dideoxy-α-D-manno-oct-7-enopyranoside, gave the desired α1-3-linked disaccharide in a 94% yield. The oct-enopyranoside moiety of the disaccharide was converted to the heptoside by oxidative cleavage with osmium tetroxide/NaIO(4) and subsequent reduction with NaBH(4). The resulting α1-3-linked heptose disaccharide was converted to a tricholoroacetaimidate derivative containing a benzoyl group at C-2.

Western blot analysis of neisserial lipooligosaccharide at the lower femtomole level with normal human sera.

Lipooligosaccharide (LOS) is a major immunogenic component of pathogenic Neisseria species such as Neisseria meningitidis and N. gonorrhoeae. Recent immunochemical studies have found that normal human sera (NHS) contain bactericidal anti-LOS antibodies that bind to the oligosaccharide (OS) moiety of neisserial LOS.

The oligosaccharide of gonococcal lipooligosaccharide contains several epitopes that are recognized by human antibodies.

Recently, we isolated human IgG from normal human sera (NHS) using lipooligosaccharide (LOS) from gonococcal strain JW31R as an affinity ligand. We provided evidence that the oligosaccharide (OS) moiety of LOS was immunogenic in humans and that NHS contains functional antibodies that bind to the branched OS. The present study aimed to identify bactericidal antibodies that bind to partial core OS structures or their adjacent sites expressed in the 3,4-branched and 2,3:3,4-dibranched neisserial LOSs.

Anomeric O-acylation of Kdo using alkyl and aryl isocyanates.

To develop a convenient method for the preparation of an alpha-Kdo derivative carrying a functional spacer at the reducing end, we examined anomeric O-acylation using Kdo and halogenated alkyl/aryl isocyanates as nucleophile and electrophiles, respectively. Reaction of a Kdo derivative with 2-chloroethyl isocyanate in the presence of DMAP gave an alpha-spiro product (82%) and an alpha-Kdo derivative of a dimeric isocyanate adduct (10%). Similar reaction with 4-(chloromethyl)phenyl isocyanate gave only the corresponding alpha-spiro product (81%).

Normal human sera contain bactericidal IgG that binds to the oligosaccharide epitope expressed within lipooligosaccharides of Neisseria gonorrhoeae.

Although more than several investigators reported the presence of antibodies in normal human sera (NHS) that bind to lipooligosaccharide (LOS) of Neisseria gonorrhoeae, the specificities of those antibodies were not fully characterized. To identify anti-LOS antibodies in NHS, we used LOS from a serum-sensitive strain, JW31R, as an affinity ligand and purified IgG from NHS that bound to JW31R LOS. The affinity purified IgG (AP-IgG) binds to the oligosaccharide (OS) moiety of both the ligand LOS and its truncated form, 15253 LOS.

Mass spectrometric strategy for the characterization of lipooligosaccharides from Neisseria gonorrhoeae 302 using FTICR.

The lipooligosaccharides (LOS) of Neisseria gonorrhoeae 302 were profiled using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). Using techniques developed in this laboratory, the topology and some of the linkages of the LOS were determined. Mass spectrometric analysis in the negative ion mode yielded a glycoform of the composition: Hex3 Hep2 Hxn1 PEA1 KDO2 DPLA.

Synthesis of alpha-lactosyl-(1-->3)-L-glycero-alpha-D-manno-heptopyranoside, a partial oligosaccharide structure expressed within the lipooligosaccharide produced by Neisseria gonorrhoeae strain 15253.

The glycosyl donor, hepta-O-benzyl-beta-lactosyl trichloroacetimidate (4) was prepared by treating hepta-O-benzyl-lactose with trichloroacetonitrile in the presence of potassium carbonate. The acceptor, methyl 2-O-benzyl-4,6-O-benzylidene-7,8-dideoxy-alpha-D-manno-oct-7-enopyranoside (8) was synthesized by hydrolysis of a 3,4-butane diacetal of methyl L-glycero-alpha-D-manno-oct-enopyranoside and subsequent benzylidenation. Glycosidation of the donor 4 with the acceptor 8 in 1,4-dioxane using Me(3)SiOTf as a promoter for 1 h at room temperature gave methyl (2,3,4,6-tetra-O-benzyl-beta-D-galactopyranosyl)-(1-->4)-(2,3,6-tri-O-benzyl-alpha-D-glucopyranosyl)-(1-->3)-2-O-benzyl-4,6-O-benzylidene-7,8-dideoxy-alpha-D-manno-oct-7-enopyranoside (9) as a major product (59%).