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.     

Structural characterization of the core region from the lipopolysaccharide of the haloalkaliphilic bacterium Halomonas alkaliantarctica strain CRSS

Halophilic and halotolerant Gram-negative bacteria are microorganisms which thrive in high salt environments. LPS are the major components of their outer leaflet, nevertheless very little is known about the role of this molecules in the adaptation mechanisms of extremophiles. Recently we determined the O-chain repeating unit structure of the LPS from Halomonas alkaliantarctica strain CRSS, an haloalkaliphilic Gram-negative bacterium isolated from salt sediments of a saline lake in Cape Russell in Antarctic continent. The polysaccharide is constituted of the trisaccharidic repeating unit: →3)-β-l-Rhap-(1→4)-α-l-Rhap-(1→3)-α-l-Rhap-(1→. In this paper we report the complete core LPS structure from this bacterium. The LPS was hydrolyzed both under mild acid and strong alkaline conditions. The MALDI spectra showed the presence of two glycoforms. The most abundant was recovered after HPAEC purification of the alkaline hydrolyzed product and was characterized by means of 2D-NMR spectroscopy. A comparison of the MALDI-PSD spectra of the two glycoforms suggested that the branched heptose was not stoichiometrically substituted.     

An unambiguous assignment and structural analysis using solution NMR experiments of O-antigen from Escherichia coli ATCC23505 (Serotype O9)

Bacterial lipopolysaccharide from Escherichia coli O9 (O9 LPS) has various characteristic biological activities other than endotoxic activities. The biological activities exhibited depend on the structure of the O-antigen. The O-antigen region of O9 LPS is composed of the mannose homopolysaccharide (MHP). This structure was reported previously, but not all its proton and carbon signals were assigned. In the present study, we completely assign all proton and carbon signals of the O-antigen of O9 LPS using (1)H- and (13)C-NMR spectroscopy, including the DQF-COSY, TOCSY, NOESY, HSQC, H2BC, HSQC-TOCSY and HMBC methods.     

Structural reinvestigation of the core oligosaccharide of a mutant form of Aeromonas salmonicida lipopolysaccharide containing an O-4 phosphorylated and O-5 substituted Kdo reducing end group using electrospray QqTOF-MS/MS

The molecular structure of the mutant form of the lipopolysaccharide of Aeromonas salmonicida was determined to contain an O-4 phosphorylated and O-5 substituted Kdo reducing group, and is proposed as the following: [molecular structure: see text] It was established that during the cleavage of this LPS with 1% acetic acid, to release the core oligosaccharide from the Lipid A portion, we obtained a degraded core oligosaccharide which eliminated its phosphate group with extreme facility. The precise molecular structure of this dephosphorylated core was deduced by electrospray mass spectrometry and is proposed as the following:[molecular structure: see text] Low energy collision ESI-QqTOF-MS/MS analysis of the dephosphorylated core oligosaccharide confirmed the presence of the O-5 glycosylated 4,8- and 4,7-anhydro derivatives of the enolizable alpha-keto-acids. The CID tandem mass spectrometric analysis of the heterogeneous mixture of the permethylated core oligosaccharide established the unreported methylation reaction on the diastereomeric 4,8- and 4,7-anhydro alpha-keto-acids and the complete permethylation and addition reaction of the O-5 glycosylated open chain reducing end terminal D-arabino-3-en-2-ulonic acid. The stereo-specific fragmentation routes obtained during the tandem mass spectrometric analysis permitted the precise sequencing of this dephosphorylated rough core oligosaccharide of the mutant LPS of A. salmonicida.     

Elucidation of the Lipopolysaccharide Core Structures of Bacteria of the Genus Providencia

Enterobacteria of the genus Providencia are opportunistic pathogens causing diarrhea in travelers and children. Lipopolysaccharide (LPS) is the major surface antigen of Providencia and the major target of the immune response. O‐polysaccharide structures have been elucidated in several Providencia O‐serogroups, but little is known about the LPS core structure. We isolated core oligosaccharides from the R‐type LPS of a number of Providencia O‐serogroups and studied them by high‐resolution mass spectrometry, including capillary skimmer dissociation technique; three selected oligosaccharides were analyzed also by NMR spectroscopy. The conserved inner core and variable outer core regions were distinguished and the full core oligosaccharide structures established in Providencia O8, O35, and O49. Providencia LPSs were found to share some structural features with Proteus LPSs.     

Characterization of the O-4 phosphorylated and O-5 substituted Kdo reducing end group and sequencing of the core oligosaccharide of Aeromonas salmonicida ssp salmonicida lipopolysaccharide using tandem mass spectrometry

The molecular structure of the wild strain of the lipopolysaccharide core of Aeromonas salmonicida, ssp salmonicida has been sequenced using tandem mass spectrometry. The core oligosaccharide was determined to contain an O-4 phosphorylated and O-5 substituted Kdo reducing group, and its structure is proposed as the follows: [structure: see text] After the core oligosaccharide of LPS was released from the lipid A portion by conventional treatment with 1% acetic acid, we demonstrated the existence of a homogeneous mixture composed mainly of the native core oligosaccharide containing the Kdo with its O-4 phosphate group intact, and a degraded core oligosaccharide mixture, which eliminated the O-4 phosphate group with extreme facility. The precise molecular structure and glycone sequence of the homogeneous mixture of phosphorylated and dephosphorylated core oligosaccharides was determined by electrospray ionization (ESI) mass spectrometry and tandem mass spectrometric analysis. CID-MS/MS of the homogeneous mixture of permethylated core oligosaccharides afforded a series of diagnostic product ions which confirmed the established sequence of the glycones to be determined. Matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry reconfirmed the molecular structure of the dephosphorylated homogeneous permethylated mixture of the core oligosaccharides containing the diastereomeric 4,8- and 4,7-anhydro-alpha-keto acids.     

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.     

Electron delocalization from the fullerene attachment to the diiron core within the active-site mimics of [FeFe]hydrogenase

Attachment of the redox-active C(60)(H)PPh(2) group modulates the electronic structure of the Fe(2) core in [(μ-bdt)Fe(2)(CO)(5)(C(60)(H)PPh(2))]. The neutral complex is characterized by X-ray crystallography, IR, NMR spectroscopy, and cyclic voltammetry. When it is reduced by one electron, the spectroscopic and density functional theory results indicate that the Fe(2) core is partially spin-populated. In the doubly reduced species, extensive electron communication occurs between the reduced fullerene unit and the Fe(2) centers as displayed in the spin-density plot. The results suggest that the [4Fe4S] cluster within the H cluster provides an essential role in terms of the electronic factor.     

Synthesis of a common tetrasaccharide motif of Haemophilus influenzae LPS inner core structures

A conserved tetrasaccharide structure, L-glycero-alpha-D-manno-heptopyranosyl-(1-->2)-(6-O-aminoethylphosphono-L-glycero-alpha-D-manno-heptopyranosyl)-(1-->3)-[beta-D-glucopyranosyl-(1-->4)]-L-glycero-alpha-D-manno-heptopyranose, from the LPS inner core of Haemophilus influenzae has been synthesised as its ethylamino glycosides to allow later conjugations. Starting from a previously synthesised suitably protected trisaccharide intermediate, the third heptose and subsequently the spacer were introduced using thioglycoside donor chemistry. The phosphoethanolamine was formed employing a Boc-protected phosphoamidite. Final deprotection and conjugation to biotin gave conjugates that will be used to study the specificity of MAbs raised against native LPS structures.     

A directly fused tetrameric porphyrin sheet and its anomalous electronic properties that arise from the planar cyclooctatetraene core

Oxidation of a directly meso-meso linked cyclic porphyrin tetramer 2 gave a porphyrin sheet 3. The symmetric square structure of 3 is indicated by its simple 1H NMR spectrum that exhibits only two signals for the porphyrin beta-protons. The absorption spectrum of 3 displays characteristic Soret-like broad bands and weak Q-bands, and its magnetic circular dichroism (MCD) spectrum exhibits a negative Faraday A term at the 762 nm band as a rare case, indicating the absorption as a transition from a nondegenerate level to a degenerate level. A slightly longer S1-state (1.1 ps) and smaller TPA cross section (2750 GM) than a tetrameric linear porphyrin tape also indicate its unique electronic properties. The porphyrin sheet 3 forms stable 1:2 complexes with guest molecules G1 and G2, whose 1H NMR spectra exhibit remarkable downfield shifts for the guest protons that are located just above the cyclooctatetraene (COT) core of 3, whereas the imidazolyl protons bound to the zinc(II) porphyrin local cores are observed at slightly upfield positions. These results have been qualitatively accounted for in terms of the presence of a strong paratropic ring current around the COT core that propagates through the whole pi-electronic network of 3, hence competing with and cancelling the weak diatropic ring currents of the local zinc(II) porphyrins. This explanation was supported by DFT calculation performed at the GIAO-B3LYP/6-31G level, which indicated large positive NICS values within the COT core and small NICS values within the local zinc(II) porphyrins.     

Structural and immunochemical analysis of the lipopolysaccharide from Acinetobacter lwoffii F78 located outside Chlamydiaceae with a Chlamydia-specific lipopolysaccharide epitope

Chemical analyses, NMR spectroscopy, and mass spectrometry were used to elucidate the structure of the rough lipopolysaccharide (LPS) isolated from Acinetobacter lwoffii F78. As a prominent feature, the core region of this LPS contained the disaccharide alpha-Kdo-(2-->8)-alpha-Kdo (Kdo=3-deoxy-d-D-manno-oct-2-ulopyranosonic acid), which so far has been identified only in chlamydial LPS. In serological investigations, the anti-chlamydial LPS monoclonal antibody S25-2, which is specific for the epitope alpha-Kdo-(2-->8)-alpha-Kdo, reacted with A. lwoffii F78 LPS. Thus, an LPS was identified outside Chlamydiaceae that contains a Chlamydia-specific LPS epitope in its core region.     

The complete structure and pro-inflammatory activity of the lipooligosaccharide of the highly epidemic and virulent gram-negative bacterium Burkholderia cenocepacia ET-12 (strain J2315)

Members of genus Burkholderia include opportunistic Gram-negative bacteria that are responsible for serious infections in immunocompromised and cystic fibrosis (CF) patients. The Burkholderia cepacia complex is a group of microorganisms composed of at least nine closely related genomovars. Among these, B. cenocepacia is widely recognized to cause epidemics associated with excessive mortality. Species that belong to this strain are problematic CF pathogens because of their high resistance to antibiotics, which makes respiratory infections difficult to treat and impossible to eradicate. Infection by these bacteria is associated with higher mortality in CF and poor outcomes following lung transplantation. One virulence factor contributing to this is the pro-inflammatory lipopolysaccharide (LPS) molecules. Thus, the knowledge of the lipopolysaccharide structure is an essential prerequisite to the understanding of the molecular mechanisms involved in the inflammatory process. Such data are instrumental in aiding the design of antimicrobial compounds and for developing therapeutic strategies against the inflammatory cascade. In particular, defining the structure of the LPS from B. cenocepacia ET-12 clone LMG 16656 (also known as J2315) is extremely important given the recent completion of the sequencing project at the Sanger Centre using this specific strain. In this paper we address this issue by defining the pro-inflammatory activity of the pure lipopolysaccharide, and by describing its full primary structure. The activity of the lipopolysaccharide was tested as a stimulant in human myelomonocytic U937 cells. The structural analysis was carried out by compositional analysis, mass spectrometry and 2D NMR spectroscopy on the intact lipooligosacchride (LOS) and its fragments, which were obtained by selective chemical degradations.     

Synthesis of the core structure of the lipoteichoic acid of Streptococcus pneumoniae

Streptococcus pneumoniae LTA is a highly complex glycophospholipid that consists of nine carbohydrate residues: three glucose, two galactosamine and two 2‐acetamino‐4‐amino‐2,4,6‐trideoxygalactose (AATDgal) residues that are each differently linked, one ribitol and one diacylated glycerol (DAG) residue. Suitable building blocks for the glucose and the AATDgal residues were designed and their synthesis is described in this paper. These building blocks permitted the successful synthesis of the core structure Glcβ(1‐3)AATDgalβ(1‐3)Glcα(1‐O)DAG in a suitably protected form for further chain extension ( 1 b , 1 c ) and as unprotected glycolipid ( 1 a ) that was employed in biological studies. These studies revealed that 1 a as well as 1 lead to interleukin‐8 release, however not via TLR2 or TLR4 as receptor.     

Octasubstituted metal-free phthalocyanine as core of phosphorus dendrimers: a probe for the properties of the internal structure

The synthesis of a new family of phosphorus dendrimers built from an octasubstituted metal-free phthalocyanine core is described up to generation 5. This core is used as a sensor and a probe for analyzing the properties of the internal structure and the influence of each structural part (core, branches, surface) upon the whole structure. UV-visible spectra show both a hyperchromic and bathochromic effect on the Q-bands with increasing generation, indicating that the chromophore is more isolated, and that the dendritic shell mimics a highly polar solvent. There is no evidence for aggregation, except for generation 0, showing again the isolation of the core. However, the dendritic shell is permeable to aqueous acids and bases, as demonstrated by the reversible splitting of the Q-band in an acidic medium (neutral form of the phthalocyanine) and the single Q-band in a basic medium (dianionic form), even for generation 4. The fluorescence quantum yield for the neutral form increases with increasing generation. The dianionic form of generation 0 is poorly fluorescent, whereas generations 3 and 4 (G3 and G4) exhibit better fluorescence. The cores of G3 and G4 are highly sensitive optical sensors for H3O+ and OH-. These experiments are carried out in THF/water mixtures, and the influence of water on the structure has been checked. The hydrodynamic radius of generation 4 is measured by NMR diffusion (pulse gradient spin-echo) experiments. R(H) varies from 35.4 A at 4 mol % of water to 32.5 A at 64 mol % of water in THF, indicating the hydrophobic nature of these dendrimers.     

Thiol- and thioether-based bifunctional chelates for the {M(CO)3}+ core (M = Tc, Re)

By analogy to the recently described single amino acid chelate (SAAC) technology for complexation of the {M(CO)3}+ core (M = Tc, Re), a series of tridentate ligands containing thiolate and thioether groups, as well as amino and pyridyl nitrogen donors, have been prepared: (NC5H4CH2)2NCH2CH2SEt (L1); (NC5H4CH2)2NCH2CH2SH (L2); NC5H4CH2N(CH2CH2SH)2 (L3); (NC5H4CH2)N(CH2CH2SH)(CH2CO2R) [R = H (L4); R = -C2H5 (L5). The {Re(CO)3}+ core complexes of L1-L5 were prepared by the reaction of [Re(CO)3(H2O)3]Br or [NEt4]2[Re(CO)3Br3] with the appropriate ligand in methanol and characterized by infrared spectroscopy, 1H and 13C NMR spectroscopy, mass spectrometry, and in the case of [Re(CO)3(L2)] (Re-2) and [Re(CO)3(L1)Re(CO)3Br2] (Re-1a) by X-ray crystallography. The structure of Re-2 consists of discrete neutral monomers with a fac-Re(CO)3 coordination unit and the remaining coordination sites occupied by the amine, pyridyl, and thiolate donors of L2, leaving a pendant pyridyl arm. In contrast, the structure of Re-1a consists of discrete binuclear units, constructed from a {Re(CO)3(L1)}+ subunit linked to a {Re(CO)3Br2}- group through the sulfur donor of the pendant thioether arm. The series of complexes establishes that thiolate donors are effective ligands for the {M(CO)3}+ core and that a qualitative ordering of the coordination preferences of the core may be proposed: pyridyl nitrogen approximately thiolate > carboxylate > thioether sulfur > thiophene sulfur. The ligands L1 and L2 react cleanly with [99mTc(CO)3(H2O)3]+ in H2O/DMSO to give [99mTc(CO)3(L1)]+ (99m)Tc-1) and [99mTc(CO)3(L2)] (99mTc-2), respectively, in ca. 90% yield after HPLC purification. The Tc analogues 99mTc-1 and 99mTc-2 were subjected to ligand challenges by incubating each in the presence of 1000-fold excesses of both cysteine and histidine. The radiochromatograms showed greater than 95% recovery of the complexes.     

Structure-dependent modulation of a pathogen response in plants by synthetic O-antigen polysaccharides

Many phytopathogenic bacteria display lipopolysaccharides (LPS) with the O-chain repeating unit [alpha-l-Rha-(1-->3)-alpha-l-Rha-(1-->3)-alpha-l-Rha-(1-->2)](n)(). This trisaccharide unit was synthesized and oligomerized to obtain hexa- and nonasaccharides. The deprotected rhamnans were effective in suppressing the hypersensitive response (HR) and in inducing PR-1 gene expression in Arabidopsis thaliana. Conformational analysis of the oligorhamnans by NMR spectroscopy and molecular dynamics calculations revealed that a coiled structure develops with increasing chain length of the oligosaccharide. This is associated with increasing efficacy in HR suppression and PR-1 gene expression. We therefore infer that the coiled structure of phytopathogenic bacteria is a plant-recognizable pathogen-associated molecular pattern (PAMP).     

Stimuli-sensitive core/shell template particles for immobilizing inorganic nanoparticles in the core

We synthesize and characterize stimuli-sensitive core/shell particles with functional group (or material) localized in the core. We previously reported two types of hybrid particles prepared by using the template particles which were synthesized by soap-free emulsion copolymerization with N-isopropylacrylamide and glycidyl methacrylate (GMA) as monomers but by different preparation methods. GMA has advantages in immobilizing materials having several functional groups such as thiol ones. In this study, to obtain the suitable template particles for immobilizing any inorganic nanoparticles in the core, we investigated the effect of feed ratio of the two monomers. Obtained template particles were modified by thiol compounds to introduce ionic groups. They were characterized by dynamic light scattering and scanning electron microscopy. After in situ synthesis of magnetic nanoparticles in the templates, the hybrid particles were characterized directly by transmission electron microscopy. Consequently, we could obtain the hybrid core/shell particles which contained a large amount of magnetic nanoparticles (∼33 wt%) in the core.     

Chemiluminescence change of polyphenol dendrimers with different core molecules

Second generation polyphenol dendrimers (PDs) with different core molecules were synthesized, and their chemiluminescence (CL) was measured by reacting the PDs with H2O2 under alkaline conditions. All of the PDs showed a strong CL, more than 120-fold greater than that of gallic acid. Various CL intensities of the PDs were obtained using different core molecules in the PDs. The distance between each dendron in the PD structure is crucial in the PD CL intensity.     

Dynamic nuclear polarization of amyloidogenic peptide nanocrystals: GNNQQNY, a core segment of the yeast prion protein Sup35p

Dynamic nuclear polarization (DNP) permits a approximately 10(2)-10(3) enhancement of the nuclear spin polarization and therefore increases sensitivity in nuclear magnetic resonance (NMR) experiments. Here, we demonstrate the efficient transfer of DNP-enhanced (1)H polarization from an aqueous, radical-containing solvent matrix into peptide crystals via (1)H-(1)H spin diffusion across the matrix-crystal interface. The samples consist of nanocrystals of the amyloid-forming peptide GNNQQNY(7-13), derived from the yeast prion protein Sup35p, dispersed in a glycerol-water matrix containing a biradical polarizing agent, TOTAPOL. These crystals have an average width of 100-200 nm, and their known crystal structure suggests that the size of the biradical precludes its penetration into the crystal lattice; therefore, intimate contact of the molecules in the nanocrystal core with the polarizing agent is unlikely. This is supported by the observed differences between the time-dependent growth of the enhanced polarization in the solvent versus the nanocrystals. Nevertheless, DNP-enhanced magic-angle spinning (MAS) spectra recorded at 5 T and 90 K exhibit an average signal enhancement epsilon approximately 120. This is slightly lower than the DNP enhancement of the solvent mixture surrounding the crystals (epsilon approximately 160), and we show that it is consistent with spin diffusion across the solvent-matrix interface. In particular, we correlate the expected DNP enhancement to several properties of the sample, such as crystal size, the nuclear T(1), and the average (1)H-(1)H spin diffusion constant. The enhanced (1)H polarization was subsequently transferred to (13)C and (15)N via cross-polarization, and allowed rapid acquisition of two-dimensional (13)C-(13)C correlation data.     

Luminescent core–shell nanoparticles with crosslinked aggregation-induced emission core structures: Emission both in solution and solid states

Luminescent core–shell nanoparticles (NPs) with crosslinked aggregation-induced emission (AIE) core structures, which exhibited excellent emission independent of the dispersion state of the NPs, have been developed by a facile one-pot method based on the self-assembly of an amphiphilic block copolymer poly(PEGMA)-b-poly(DB3VT). Core–shell micelles with a poly(DB3VT) core were formed from poly(PEGMA)-b-poly(DB3VT) in tetrahydrofuran (THF)/H₂O condition, and the crosslinked AIE-based structure was selectively incorporated into the core by the Suzuki coupling reaction between poly(DB3VT) blocks and tetraphenylethylene (TPE)-based coupling monomers at the same time. This method afforded a uniform NP with a crosslinked TPE-based AIE core structure. The obtained NP exhibited excellent emission both in diluted solution and solid states. This result indicated that the formed TPE-based AIE core structure was always aggregated regardless of NP dispersion owing to the crosslinking as we expected. The crosslinked TPE-based AIE core structure, which was related to the emission property, was readily tuned by the selection and combination of coupling monomers in the Suzuki coupling reaction. By incorporating electron-deficient units into the core, the emission color could be successfully tuned from yellow-green to orange and red while maintaining the emission property independent of the state of the NP dispersion. These results demonstrated that NPs with the crosslinked AIE core structures are a promising luminescent material design motif to realize emission independent on molecular dispersion.