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. This donor was glycosylated with 2-(carbobenzoxyamino)-1-ethanol to give an α spacer-linked disaccharide derivative in a 90% yield. Zemplén deacylation of the derivative and subsequent hydrogenolysis gave a 2-aminoethyl glycoside of heptopyranosyl(α1-3)heptopyranose.

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. Although affinity-purified anti-LOS antibodies can be characterized using 10-100 ng of LOS samples (up to a few tens of pmoles), a more sensitive immunoblotting assay must be established in order to analyze NHS directly and characterize anti-LOS antibodies without affinity purification. We examined analytical PAGE/blot conditions using a 15-well mini gel. For the first time, Western blot detection of LOS at the lower femtomole level was accomplished by both chromogenic and chemiluminescent detection. A model LOS, 15253 LOS, was detected in a low femtomole range (62.5-500 pg, 16-125 femtomole) even with 10 pM of a monoclonal antibody (MAb) 2C7. Furthermore, detection of similar amounts (50-250 femtomole) of neisserial LOSs and Salmonella truncated lipopolysaccharides (LPSs) was also possible with 1:50 and with 1:100 diluted NHS. The results obtained here indicate that the binding of IgG in NHS to the LOS and LPS samples is probably due to their carbohydrate moieties. The detection level accomplished in this study should help not only to further characterize anti-LOS antibodies in blood and body fluids but also to analyze carbohydrate structures that are recognized by them.

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. Using 15253 LOS from serum-resistant gonococcal strain 15253 as an affinity ligand, we isolated IgG2 and found that this preparation contained at least three different species. (i) One IgG2 species recognized a cross-reactive epitope that is expressed on 3,4-branched and 2,3:3,4-dibranched neisserial LOSs. (ii) Another IgG2 species was specific for JW31R LOS from a pyocin-resistant gonococcal strain; this IgG-defined epitope was not shared with the aforementioned branched LOSs. (iii) The third IgG2 species bound to the "Salmonella minnesota" Rb and Re mutant lipopolysaccharides (LPSs); this IgG2 recognizes a KDOalpha2-4KDO residue at the reducing end of the carbohydrate moiety of each LPS. The IgG2 was also found to be functional and facilitated the killing of strain 15253. The current results show that neisserial LOS contains several epitopes within its OS moiety that are recognized by human antibodies.

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%). The NMR data show that the pyranose rings of both the alkyl and aryl spiro products adopt the 5C2 conformation. Thus, we accomplished alpha-selective anomeric O-acylation by coupling the Kdo derivative with alkyl and aryl isocyanates.

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. Lipid A could be essential for maximum expression of the carbohydrate epitope that resides on 15253 OS. We also found that AP-IgG is capable of killing a serum-sensitive strain JW31R. The present work provided direct evidence that NHS contain bactericidal antibodies specific for a site close to the inner core OS expressed on gonococcal LOS. The present results not only show that anti-LOS antibodies specific for the inner core OS could play a major role in our defense against gram-negative bacteria. But also they demonstrated that such core OS or a nearby site could be utilized as possible targets for vaccine development against microbial infections.

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. The composition was confirmed through exact mass measurements, which showed only a 2 ppm error between the exact mass and theoretical mass. Although the core structure has been postulated previously, the positioning of the three hexose moieties were in question for this particular strain of N. gonorrhoeae. Topology assignment was performed through collision-induced dissociation analysis of the O-deacylated glycoform in the negative ion mode followed by submission to the saccharide topology analysis tool (STAT) computer program, which confirmed the topology assignment. It was found that the three hexoses were added to the Hep[I] of the conserved core of N. gonorrhoeae in a linear fashion, while Hep[II] remains unbranched. Linkage position analysis was performed through application of a mild acid hydrolysis technique followed by collision-induced dissociation of the sodiated precursor ions, yielding a 1 --> 4 linkage between the terminating and penultimate hexoses.

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%). The oct-enopyranoside moiety of the trisaccharide 9 was converted to a heptopyranoside (80%) by oxidative cleavage with OsO(4)-NaIO(4) and subsequent reduction. Hydrogenolysis of the resulting trisaccharide and subsequent acetylation gave the peracetate of alpha-lactosyl-(1-->3)-Hep. Deacetylation of the peracetate afforded the title trisaccharide.