Mass‐Spectrometric Studies of Providencia SR‐Form Lipopolysaccharides and Elucidation of the Biological Repeating Unit Structure of Providencia rustigianii O14‐Polysaccharide

Enterobacteria Providencia are opportunistic human pathogens causing multiple types of infections. Earlier we have studied the S‐ and R‐form lipopolysaccharides (LPSs) of Providencia strains of various O‐serogroups and established the structures of the O‐polysaccharides (O‐antigens) and core‐region oligosaccharides, respectively. Now we report on mass spectrometric studies of oligosaccharides consisting of the core moiety with one O‐polysaccharide repeating unit attached, which were derived from the SR‐form LPSs of Providencia strains. The site of attachment of the O‐polysaccharide to the core and the structure of the O‐polysaccharide biological repeating unit were elucidated in Providencia rustigianii O14 using NMR spectroscopy.     

The Full Structure of the Carbohydrate Chain of the Lipopolysaccharide of Providencia alcalifaciens O19

An oligosaccharide isolated from the lipopolysaccharide of Providencia alcalifaciens O19 was found to consist of a single O‐antigen repeating unit linked to the core. The full oligosaccharide structure was elucidated by 2D ¹H, ¹³C, and ³¹P NMR spectroscopy. It was shown that the inner core region has the same structure as in other Providencia strains studied, whereas the outer core is structurally diverse between Providencia strains. A pyruvic acid acetal was found in the isolated oligosaccharide and in the long‐chain O‐antigen, whose structure has been established earlier. The biological O‐unit structure was elucidated in the short‐chain lipopolysaccharide.     

Characterization of the Core Oligosaccharide and the O‐Antigen Biological Repeating Unit from Halomonas stevensii Lipopolysaccharide: The First Case of O‐Antigen Linked to the Inner Core

A novel core structure among bacterial lipopolysaccharides (LPS) that belong to the genus Halomonas has been characterized. H. stevensii is a moderately halophilic microorganism, as are the majority of the Halomonadaceae. It brought to light the pathogenic potential of this genus. On account of their role in immune system elicitation, elucidation of LPS structure is the mandatory starting point for a deeper understanding of the interaction mechanisms between host and pathogen. In this paper we report the structure of the complete saccharidic portion of the LPS from H. stevensii. In contrast to the finding that the O‐antigen is usually covalently linked to the outer core oligosaccharide, we could demonstrate that the O‐polysaccharide of H. stevensii is linked to the inner core of an LPS. By means of high‐performance anion‐exchange chromatography with pulsed amperometric detection we were able to isolate the core decasaccharide as well as a tridecasaccharide constituted by the core region plus one O‐repeating unit after alkaline degradation of the LPS. The structure was elucidated by one‐ and two‐dimensional NMR spectroscopy, ESI Fourier transform ion cyclotron resonance (FT‐ICR) mass spectrometry, and chemical analysis.     

Chemical Synthesis and Immunological Evaluation of Helicobacter pylori Serotype O6 Tridecasaccharide O‐Antigen Containing a dd‐Heptoglycan

The development of glycoconjugate vaccines against Helicobacter pylori is challenging. An exact epitope of the H. pylori lipo‐polysaccharide (LPS) O‐antigens that contain Lewis determinant oligosaccharides and unique dd ‐heptoglycans has not yet been identified. Reported here is the first total synthesis of H. pylori serotype O6 tridecasaccharide O‐antigen containing a terminal Le^y tetrasaccharide, a unique α‐(1→3)‐, α‐(1→6)‐, and α‐(1→2)‐linked heptoglycan, and a β‐d ‐galactose connector, by an [(2×1)+(3+8)] assembly sequence. Seven oligosaccharides covering different portions of the entire O‐antigen were prepared for immunological investigations with a particular focus on elucidation of the roles of the dd ‐heptoglycan and Le^y tetrasaccharide. Glycan microarray analysis of sera from rabbits immunized with isolated serotype O6 LPS revealed a humoral immune response to the α‐(1→3)‐linked heptoglycan, a key motif for designing glycoconjugate vaccines for H. pylori serotype O6.     

Structural Studies on the Lipopolysaccharide Core of Bacteria of the Genus Citrobacter: Two Different Core Structures in Citrobacter O14 Serogroup

Core oligosaccharides were released by mild acid hydrolysis of the lipopolysaccharides of 25 strains of bacteria Citrobacter representing 19 O-serogroups. Screening by sugar analysis and high-resolution ESI MS revealed structural heterogeneity of the core within each strain and core structure variations between strains. The core oligosaccharides from Citrobacter werkmanii O14 strains PCM 1548 and 1549 were further purified by anion-exchange chromatography and their full structures were elucidated by methylation analysis and two-dimensional NMR spectroscopy; similar inner core regions and significantly different outer core pentasaccharide regions were established. D-Galacturonic acid 3-(2-aminoethyl)phosphate was found to be a characteristic component of the Citrobacter core.     

Kdo Glycosylations and Their Application in Oligosaccharide Synthesis**

Higher carbon saccharide 3-deoxy-d -manno-oct-2-ulosonic acid (Kdo) is a structural unit of bacterial lipopolysaccharides (LPSs) and capsular polysaccharides (CPSs). Kdo is present in the inner core region of LPSs, and this region is structurally conserved. Being non-mammalian in origin, Kdos are effectively recognized by the native and adaptive immune systems. Therefore, the synthesis of new Kdo derivatives and neoglycoconjugates is highly important for the development of vaccines. This review highlights recent accomplishments related to α-glycosylations, β-glycosylations and C-glycosylations of Kdos and their application to the stereoselective synthesis of inner core oligosaccharides.     

Structural Study and Conformational Behavior of the Two Different Lipopolysaccharide O‐Antigens Produced by the Cystic Fibrosis Pathogen Burkholderia multivorans

Lipopolysaccharides (LPSs) are virulence factors expressed by Gram‐negative bacteria; they are among those mainly responsible for bacterial virulence. In this work we define the primary structure and the conformational features of the O‐chain from the LPS produced by the highly virulent clinical isolate Burkholderia multivorans strain C1576, an opportunistic human pathogen isolated in a cystic fibrosis center and causative of an outbreak with lethal outcome. We demonstrate that the LPS from this clinical isolate consists of two O‐polysaccharide chains present in different amounts and made up of repeating units, both containing deoxy sugar. Additionally, conformational studies have been performed to establish and compare the spatial arrangements of the two polysaccharides and differences in their shape have been highlighted. The comprehension of the structural and conformational features of the two repeating units may help to explain their biological significance, the molecular shape of the bacterial external surface, and the comprehension at the molecular level of the recognition mechanisms of the antibodies.     

Synthesis,characterization, and curing kinetics of novel ladder‐like polysilsesquioxanes containing side‐chain maleimide groups

Two ladder‐like polysilsesquioxanes (LPS) containing side‐chain maleimide groups have been synthesized successfully by reacting N‐(4‐hydroxyphenyl)maleimide (HPM) with LPS containing 100 mol % of chloropropyl groups (Ladder A ) and 50 mol % of each methyl and chloropropyl group (Ladder B ). HPM was synthesized by reacting maleic anhydride with 4‐aminophenol, and the resulting amic acid was imidized using p‐toluenesulfonic acid as a catalyst (Scheme 1 ). The LPSs were characterized by Fourier transform infrared (FTIR), ¹H nuclear magnetic resonance (NMR), proton‐decoupled ¹³C NMR, ²⁹Si NMR, wide‐angle X‐ray diffraction (WAXD), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). Characterization indicated that these polymers had ordered ladder‐like structures with possible defects. These polymers were soluble in common solvents at ambient temperature, which suggested that they were not crosslinked. Both the polymers and the HPM were cured, and their kinetics were followed by dynamic DSC. The Ozawa and Kissinger methods were used to calculate activation energies for curing. Curing increased the temperature at which both 5% weight loss and maximum rate of weight loss were observed. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 4036–4046, 2004     

Biopolymer Skeleton Produced by Rhizobium radiobacter: Stoichiometric Alternation of Glycosidic and Amidic Bonds in the Lipopolysaccharide O‐Antigen

The lipopolysaccharide (LPS) O‐antigen structure of the plant pathogen Rhizobium radiobacter strain TT9 and its possible role in a plant‐microbe interaction was investigated. The analyses disclosed the presence of two O‐antigens, named Poly1 and Poly2. The repetitive unit of Poly2 constitutes a 4‐α‐l ‐rhamnose linked to a 3‐α‐d ‐fucose residue. Surprisingly, Poly1 turned out to be a novel type of biopolymer in which the repeating unit is formed by a monosaccharide and an amino‐acid derivative, so that the polymer has alternating glycosidic and amidic bonds joining the two units: 4‐amino‐4‐deoxy‐3‐O‐methyl‐d ‐fucose and (2′R,3′R,4′S)‐N‐methyl‐3′,4′‐dihydroxy‐3′‐methyl‐5′‐oxoproline). Differently from the O‐antigens of LPSs from other pathogenic Gram‐negative bacteria, these two O‐antigens do not activate the oxidative burst, an early innate immune response in the model plant Arabidopsis thaliana, explaining at least in part the ability of this R. radiobacter strain to avoid host defenses during a plant infection process.     

Diversity of non‐stoichiometric substitutions on the lipopolysaccharide of E. coli C demonstrated by electrospray ionization single quadrupole mass spectrometry

The lipopolysaccharide (LPS) of enterobacteria frequently contains various numbers of charged non‐stoichiometric substituents such as phosphate (P) and ethanolamine (EtN) groups and a third residue of 3‐deoxy‐D ‐manno‐2‐octulosonic acid (KDO) on the R‐core polysaccharide backbone. These substituents can modify the biological activities of LPS including varying the stability of the outer membrane, tolerance to cationic antibiotics, pathogenicity, and sensitivity to enterobacteria bacteriophages. These diverse substituents can be clearly detected in degraded samples of LPS from E. coli C using electrospray ionization single quadrupole mass spectrometry (ESI‐Q‐MS) from a 0.1 mg/mL solution in a 50:50 mixture of methanol and 10 mM ammonium acetate (pH 6.8). The O‐deacylated derivative showed multiple peaks of [M–3H]^3? ions which corresponded to species having up to eight phosphates, two ethanolamines, and an additional KDO on the backbone of Hex₅ Hep₃ KDO₂ GlcN₂ C14:0(3‐OH)₂. The major components of the O,N‐deacylated derivative were the species associated with four and five phosphates on Hex₅ Hep₃ KDO₂ GlcN₂. The polysaccharide portion of LPS also revealed species which corresponded to Hex₅ Hep₃ KDO associated with two to four phosphates and an ethanolamine. The present method was proved to be useful to investigate the structural diversity of enterobacterial LPS. Copyright © 2009 John Wiley & Sons, Ltd.     

Synthesis of the Branched Trisaccharide L‐Glycero‐α‐D‐manno‐heptopyranosyl‐(1 → 3)‐ [β‐D‐glucopyranosyl‐(1 → 4)]‐L‐glycero‐α‐D‐manno‐heptopyranose,Protected to Allow Flexible Access to Neisseria and Haemophilus LPS Inner Core Structures

Abstract

An efficient synthesis of the protected branched trisaccharide (2′S,3′S)‐(7‐O‐benzyl‐6‐O‐chloroacetyl‐3,4‐O‐(2′,3′‐dimethoxybutane‐2′,3′‐diyl)‐2‐O‐p‐methoxybenzyl‐L‐glycero‐α‐D‐manno‐heptopyranosyl)‐(1 → 3)‐[(2,3,4,6‐tetra‐O‐benzoyl‐β‐D‐glucopyranosyl)‐(1 → 4)]‐7‐O‐acetyl‐1,6‐anhydro‐2‐O‐benzyl‐L‐glycero‐β‐D‐manno‐heptopyranose, which is a key intermediate in the synthesis of inner core structures of Haemophilus and Neisseria LPSs, is described. The heptoses were formed by Grignard reactions using a benzyloxymethyl chloride or a commercial vinyl reagent. The anhydro bridge was formed by treatment of a 6‐OH methyl α‐heptoside precursor with FeCl₃. The protecting group pattern allows modifications at the 2‐, 3‐, 4‐, and 6‐positions of the second heptose moiety and also, after acetolysis of the anhydro bridge, elongation at the reducing end, all known alterations found in the bacterial LPSs.     

Chemical Synthesis and Immunological Evaluation of Helicobacter pylori Serotype O6 Tridecasaccharide O-Antigen Containing a dd-Heptoglycan

The development of glycoconjugate vaccines against Helicobacter pylori is challenging. An exact epitope of the H. pylori lipo-polysaccharide (LPS) O-antigens that contain Lewis determinant oligosaccharides and unique dd -heptoglycans has not yet been identified. Reported here is the first total synthesis of H. pylori serotype O6 tridecasaccharide O-antigen containing a terminal Le^y tetrasaccharide, a unique α-(1→3)-, α-(1→6)-, and α-(1→2)-linked heptoglycan, and a β-d -galactose connector, by an [(2×1)+(3+8)] assembly sequence. Seven oligosaccharides covering different portions of the entire O-antigen were prepared for immunological investigations with a particular focus on elucidation of the roles of the dd -heptoglycan and Le^y tetrasaccharide. Glycan microarray analysis of sera from rabbits immunized with isolated serotype O6 LPS revealed a humoral immune response to the α-(1→3)-linked heptoglycan, a key motif for designing glycoconjugate vaccines for H. pylori serotype O6.     

Efficient synthesis of β‐(1,6)‐linked oligosaccharides through microwave‐assisted glycosylation

A microwave‐assisted glycosylation method was developed for efficient synthesis of oligosaccharides. Di‐functional AB monomers, 2,3,4‐tri‐O‐acetyl‐α‐d ‐galactopyranosyl bromide ( 3a ) and 2,3,4‐tri‐O‐acetyl‐α‐d ‐glucopyranosyl bromide ( 3b ) were designed and synthesized as weakly reactive monomers to avoid unwanted glycosylation or degradation during preparation and storage. The glycosylations of these monomers gave low conversions and low molecular weight oligosaccharides at rt, reflux, and under low microwave energy irradiation. However, the glycosylation became very effective when high microwave energy was applied, giving 100% conversion and producing oligosaccharides with M_n = 4.76 kDa for 3a and M_n = 4.05 kDa for 3b. The acetylated oligosaccharides were further subjected to deprotection for structural analysis, which indicated the oligosaccharides contain predominantly linear β‐(1,6)‐glycosyl linkages. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2013, 51, 3693–3699     

Characterization of intact lipopolysaccharides from the Haemophilus influenzae strain RM 118 using electrophoresis-assisted open-tubular liquid chromatography-mass spectrometry

We have applied an electrophoresis-assisted open-tubular LC-MS method for analyzing intact lipopolysaccharides (LPSs) from Haemophilus influenzae strain RM118 (Rd). We were able to obtain structural information on both core oligosaccharides (OSs) and the lipid A moiety including the sialylation, glycylation, and the distribution of fatty acid residues on the disaccharide backbone of lipid A. The fragmentation patterns of sodiated and protonated LPS molecules were investigated for determining the location of sialic acid. It was found that the tandem mass spectra of sodiated ions provided unambiguous evidence of both sialylated lactose and sialylated lacto-N-neotetraose. In contrast, the fragment ions of protonated ions only offered the evidence for the existence of sialylated lacto-N-neotetraose. The lipid A of Gram-negative bacteria, as the principal endotoxic component of LPS, plays a major role in the pathogenesis of bacterial infections. We have previously characterized lipid A species after mild acid hydrolysis of LPS during which lipid A precipitates. In this study, intact LPS was directly introduced to a tandem mass spectrometer. In-source dissociation strategy was employed, followed by multiple-stage MS/MS on the ions originating from the lipid part to obtain structural information. This is the first time that the structure of lipid A of H. influenzae was characterized by MS/MS on intact LPS molecules without any prior chemical modifications. In the same way information on the OS can be obtained by MS/MS by focusing on ions originating from core OS.     

Chemical synthesis of Helicobacter pylori lipopolysaccharide partial structures and their selective proinflammatory responses

Helicobacter pylori is a common cause of gastroduodenal inflammatory diseases such as chronic gastritis and peptic ulcers and also an important factor in gastric carcinogenesis. Recent reports have demonstrated that bacterial inflammatory processes, such as stimulation with H. pylori lipopolysaccharide (LPS), initiate atherosclerosis. To establish the structures responsible for the inflammatory response of H. pylori LPS, we synthesized various kinds of lipid A structures (i.e., triacylated lipid A and Kdo‐lipid A compounds), with or without the ethanolamine group at the 1‐phosphate moiety, by a new divergent synthetic route. Stereoselective α‐glycosylation of Kdo N‐phenyltrifluoroacetimidate was achieved by use of microfluidic methods. None of the lipid A and Kdo‐lipid A compounds were a strong inducer of IL‐1β, IL‐6, or IL‐8, suggesting that H. pylori LPS is unable to induce acute inflammation. In fact, the lipid A and Kdo‐lipid A compounds showed antagonistic activity against cytokine induction by E. coli LPS, except for the lipid A compound with the ethanolamine group, which showed very weak agonistic activity. On the other hand, these H. pylori LPS partial structures showed potent IL‐18‐ and IL‐12‐inducing activities. IL‐18 has been shown to correlate with chronic inflammation, so H. pylori LPS might be implicated in the chronic inflammatory responses induced by H. pylori. These results also indicated that H. pylori LPS can modulate the immune response: NF‐κB activation through hTLR4/MD‐2 was suppressed, whereas production of IL‐18 and IL‐12 was promoted.     

Synthesis and mesomorphic properties of new chiral banana‐shaped liquid crystals with chiral 3‐(alkoxy)propoxy terminal groups

New chiral banana‐shaped liquid crystals with chiral 3‐(alkoxy)propoxy terminal groups (Pn‐O‐PIMB5*‐4O, n = 7, 8, 9 and 10) were synthesized and their mesomorphic properties and phase structures investigated by means of electro‐optic measurements, polarizing optical microscopy, differential scanning calorimetry, and second harmonic generation measurements. Most of these chiral bent‐core mesogens (n = 7–9) showed the antiferroelectric B2 phase, whereas P10‐O‐PIMB5*‐4O exhibited the B7 phase. Comparing with the previously reported homologue Pn‐O‐PIMB(n‐2)*, we conclude that the terminal chain structure, particularly the position of chiral centres, plays an important role in the emergence of particular phase structures.     

Studies on the Magnetic Ground State of a Spin Möbius Strip

Here we report the synthesis, structure and detailed characterisation of three n‐membered oxovanadium rings, Na_n[(V=O)_nNa_n(H₂O)_n(α, β, or γ‐CD)₂]?m H₂O (n=6, 7, or 8), prepared by the reactions of (V=O)SO₄?x H₂O with α, β, or γ‐cyclodextrins (CDs) and NaOH in water. Their alternating heterometallic vanadium/sodium cyclic core structures were sandwiched between two CD moieties such that O‐Na‐O groups separated the neighbouring vanadyl ions. Antiferromagnetic interactions between the S=1/2 vanadyl ions led to S=0 ground states for the even‐membered rings, but to two quasi‐degenerate S=1/2 states for the spin‐frustrated heptanuclear cluster.     

3‐O‐Methylquercetin Glucosides from Ophioglossum pedunculosum and Inhibition of Lipopolysaccharide‐Induced Nitric Oxide Production in RAW 264.7 Macrophages

The three new 3‐O‐methylquercetin glucosides 1 – 3 , together with three known congeners and 3‐O‐methylquercetin, were isolated from the fern Ophioglossum pedunculosum (quercetin=2‐(3,4‐dihydroxyphenyl)‐3,5,7‐trihydroxy‐4H‐1‐benzopyran‐4‐one). The new compounds were identified on the basis of spectroscopic analysis as 5′‐isoprenyl‐3‐O‐methylquercetin 4′,7‐di‐β‐D ‐glucopyranoside ( 1 ), 3‐O‐methylquercetin 4′‐β‐D ‐glucopyranoside 7‐[O‐β‐D ‐glucopyranosyl‐(1→2)‐β‐D ‐glucopyranoside] ( 2 ), and 3‐O‐methylquercetin 7‐[O‐β‐D ‐glucopyranosyl‐(1→2)‐β‐D ‐glucopyranoside] ( 3 ). The effect of the isolated compounds on lipopolysaccharide (LPS)‐induced NO production was evaluated. The inhibitory activity of 3‐O‐methylquercetin derivatives decreased markedly with the increasing number of glucosyl groups in the structures.     

New Dammarane Monodesmosides from the Acidic Deglycosylation of Notoginseng‐Leaf Saponins

Three new dammarane monodesmosides, named notoginsenosides Ft₁ ( 1 ), Ft₂ ( 2 ), and Ft₃ ( 3 ), together with three known ginsenosides, were obtained from a mild acidic hydrolysis of the saponins from notoginseng (Panax notoginseng (Burk .) F. H. Chen ) leaves. Their structures were elucidated to be (3β,12β,20R)‐12,20‐dihydroxydammar‐24‐en‐3‐yl O‐β‐D ‐xylopyranosyl‐(1 → 2)‐O‐β‐D ‐glucopyranosyl‐(1 → 2)‐β‐D ‐glucopyranoside ( 1 ), (3β,12β)‐12,20,25‐trihydroxydammaran‐3‐yl O‐β‐D ‐xylopyranosyl‐(1 → 2)‐O‐β‐D ‐glucopyranosyl‐(1 → 2)‐β‐D ‐glucopyranoside ( 2 ), and (3β,12β,24ξ)‐12,20,24‐trihydroxydammar‐25‐en‐3‐yl O‐β‐D ‐xylopyranosyl‐(1 → 2)‐O‐β‐D ‐glucopyranosyl‐(1 → 2)‐β‐D ‐glucopyranoside ( 3 ), by means of spectroscopic evidences. The known ginsenosides Rh₂ and Rg₃ 4 – 6 were obtained as the major products from this acidic deglycosylation.     

New Phenolic Constituents from the Stems of Spatholobus suberectus

Three new phenolic compounds, 5‐O‐(β‐apiosyl‐(1 → 2)‐O‐β‐xylopyranosyl)gentisic acid ( 1 ), 1‐O‐(β‐apiosyl‐(1 → 6)‐O‐β‐glucopyranosyl)‐3‐O‐methylphloroglucinol ( 2 ), and 15‐O‐(α‐rhamnopyranosyl)aloe‐emodin ( 3 ), together with the known compound aloe emodin ( 4 ), were isolated from the stems of Spatholobus suberectus. Their structures were characterized by chemical and spectroscopic methods. The absolute configurations of the sugar units were not determined.