Novel Disaccharides Containing 3-Deoxy-D-Manno-2-Octulosonic Acid (KDO) and Lipid a Subunit Analogs

Abstract

3-Deoxy-D-manno-2-octulosonic acid (KDO)₁ is a prominent constituent of bacterial lipopolysaccharides (LPS),₂ and recently it has been demonstrated _3,4 that the KDO part in the core region of LPS is α-ketosidically linked to 0-6′ of the o-glucosamine-disaccharide backbone of lipid A. Although the portion, including KDO, has been suggested_5,6 to be necessary for expression of antitumor and interleukin 1 (IL 1)-induction activities of LPS, the disticnt biological roles of KDO in LPS are still obscure.     

Mechanistic divergence of two closely related aldol-like enzyme-catalysed reactions

The analysis of the interaction of threose 4-phosphate and 2-deoxyerythrose 4-phosphate with 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) reveals previously unrecognised mechanistic differences between the DAH7PS-catalysed reaction and that catalysed by the closely related enzyme, 3-deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS).     

Probing the Subunit-Subunit Interaction of the Tetramer of E. coli KDO8P Synthase by Electrospray Ionization Mass Spectrometry

Escherichia coli 3-Deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase catalyzes the condensation reaction between D-arabinose 5-phosphate (A5P) and phosphoenolpyruvate (PEP) to form KDO8P and inorganic phosphate (Pi). This enzyme exists as a tetramer in solution, which is important for catalysis. Two different states of the enzyme were obtained: i) PEP-bound and ii) PEP-unbound. The effect of the substrates and products on the overall structure of KDO8P synthase in both PEP-bound and unbound states was examined using electrospray ionization mass spectrometry. The analysis of our data showed that the complexes of the PEP-unbound enzyme with PEP (or Pi) favored the formation of monomers, while the complexes with A5P (or KDO8P) mainly favored dimers. The PEP-bound enzyme was found to exist in the monomer and dimer with a small amount of the tetramer, whereas the PEP-unbound form primarily exists in the monomer and dimer, and no tetramer was observed, suggesting that the bound PEP have a role in stabilization of the tetrameric structure. Taken together, the results imply that the addition of the substrates or products to the unbound enzyme may alter the subunit-subunit interactions and/or conformational change of the protein at the active site, and this study also demonstrates that the electrospray ionization mass spectrometric method may be a powerful tool in probing the subunit-subunit interactions and/or conformational change of multi-subunit protein upon binding to ligand.     

Two Related Porous Thioindates with T3 Clusters, (H2DAH)3In10S18·6H2O and (H2DAH)3In10S18

Two high porous thioindates with 1,6‐diaminohexane (DAH), DAH‐InS‐ 1 and DAH‐InS‐ 2 , were prepared by solvothermal reaction. Though the same T₃ supertetrahedral frameworks were reported, the cations are different. DAH is a non‐cyclic diamine and also an economical reagent for synthesis. Because of the structural difference of the structural directing agents, the dimensions of crystal cells of DAH‐InS‐ 1 and DAH‐InS‐ 2 are elongated and the total volumes for the cation and solvent are 74.6 and 72.1 % (Platon) larger relative to those reported previously. The two compounds were obtained in one reaction system by controlling the reaction time. Their structures are briefly discussed and the correlativity of crystal shape with the parameters of microstructure is discussed.     

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.     

Preparation of novel pyranosyl fluorides of 3-deoxy-D-manno-2-octulosonic acid (KDO) feasible for synthesis of KDO α-glycosides

α-Ketopyranosyl fluorides, 2 and 3, of 4,5:7,8-di-O-isopropylidene KDO methyl and benzyl esters were prepared and shown to act as effective glycosyl donors. X-Ray structure analysis of 2 and ¹H NMR study established the boat (B_3,6) conformation of the di-O-isopropylidene derivatives of KDO and enabled the assignment of their anomeric configurations.     

Vinyl Carbanions. Part 36.. Synthesis of 3-deoxy-D-manno-2-octulosonic acid (KDO) and derivatives

The β-C-lithiated acrylamide 3A has been proven to be an ideal pyruvate β-carbanion equivalent useful in a highly diastereoselective KDO synthesis. The starting material 3 was prepared from pyruvate diethyl acetal in four convenient steps. Direct lithiation with 2 equiv. of LDA generated the dilithiated species 3A quantitatively. Reaction with 2,3:4,5-di-O-isopropylidene-D -arabinose ( 11 ) was highly D -manno-selective. The product 12 was obtained readily from the reaction mixture via crystallization. Ring closure to the butenolide 13 , subsequent PhS-group removal with Bu₃SnH and pyridinium bromide, and hydrogenolytic debenzylation afforded the known butenolide 19 ; this KDO precursor gives KDO in two convenient steps. Butenolide 19 was also transformed via two high-yielding steps into the 4,5:7,8-di-O-cyclohexylidene-KDO derivative 22 , a valuable starting material for KDO α-glycoside syntheses.     

A New Synthesis of 3-Deoxy-D-Manno-Octulosonic Acid (KDO) From D-Mannose Via Condensation of Dioxene

Abstract

The title compound has long been known as an essential component of lipopolysaccharides (LPS) and capsular polysaccharides which exist in the outer membrane of Gram-negative bacteria. KDO has attracted additional attention as a consequence of some remarkable discoveries; i) mutants unable to produce KDO are non-viable.¹ ii) KDO is not present in mammalian cells,² iii) the 2-deoxy analog of β-KDO represents a new class of synthetic antimicrobial agent.^3,4 Although syntheses of KDO have been reported by many groups,^5–8 a more efficient synthetic method endowed with the potential for synthesis of not only complex glycoconjugates but also biologically significant analogs is required.     

An in-depth study on ring-closing metathesis of carbohydrate-derived alpha-alkoxyacrylates: efficient syntheses of DAH, KDO, and 2-deoxy-beta-KDO

Novel, efficient synthetic pathways to DAH, KDO, and 2-deoxy-beta-KDO are described. Ring-closing metathesis (RCM) of highly functionalized alpha-alkoxyacrylate fragments resulted in a series of synthetically versatile oxygen heterocyclic intermediates. Further functionalization of the resulting enol ether double bond and subsequent deprotection provided the natural products in high overall yields, starting from commercially available protected sugars.     

A new synthesis of 3-deoxy-d-manno-2-octulosonic acid (kdo) from d-mannose

A new efficient chemical synthesis of KDO (1) with the natural configuration was achieved via the condensation of a protected D-mannitol triflate (3) with methyl glyoxylate dithioacetal (2) as a key step.     

Structural studies on Helicobacter pylori 3‐deoxy‐D‐manno‐2‐octulosonate‐8‐phosphate synthase using electrospray ionization mass spectrometry: a tetrameric complex composed of dimeric dimers

Helicobacter pylori 3‐deoxy‐D ‐manno‐2‐octulosonate‐8‐phosphate (KDO8P) synthase catalyzes the conversion of D ‐arabinose‐5‐phosphate (A5P) and phosphoenolpyruvate (PEP) to produce KDO8P and inorganic phosphate. Since this protein is absent in mammals, it might therefore be an attractive target for the development of new antibiotics. Unlike E. coli KDO8P synthase (class I), the H. pylori counterpart is a class II enzyme, where it requires a divalent transition metal ion for catalysis. Although the metal ions have been shown to be important for catalysis, their role in the structure is not understood. Using electrospray ionization mass spectrometry (ESI‐MS), the role of the metal ions in H. pylori KDO8P synthase has been investigated. This protein is found to be a tetramer in the gas phase but dissociates into the dimer with increasing declustering potential (DP2) suggesting an existence of a ‘structurally specific’ tetramer. An examination of mass spectra revealed that the tetrameric state of the Cd²⁺‐reconstituted enzyme is less stable than those of the Zn²⁺‐, Co²⁺‐ and Cu²⁺‐enzymes. The stoichiometry of metal binding to the protein depends on the nature of the metal ion. Taken together, our data suggest that divalent metal ions play an important role in the quaternary structure of the protein and the tetrameric state may be primarily responsible for catalysis. This study demonstrates the first structural characterization and stoichiometry of metal binding in class II KDO8P synthase using electrospray ionization quadrupole time‐of‐flight mass spectrometry under nondenaturing conditions. Copyright © 2009 John Wiley & Sons, Ltd.     

Convenient syntheses of deoxypyranose sugars from glucuronolactone

One of the characteristic reactions of glucuronic acid derivatives is the base-catalysed elimination of a 4-(substituted) hydroxy group to generate a Δ_4,5 pyranose. Following hydrogenation, proceeding mainly from the α-face provided the anomeric configuration is β, the initial C(5)-configuration is restored. This sequence affords access to a number of 4-deoxypyranoses: thus 4-deoxyglucoses are readily available by reduction at C(6). Conversion to a glycal, then cis-dihydroxylation at C(2)/C(3) leads to the d-lyxo configuration (found in neosidomycin). Finally a less obvious relationship to the KDO series is revealed, again by dihydroxylation.     

Quantum mechanical calculation of aqueuous uranium complexes: carbonate, phosphate, organic and biomolecular species

Background  

Quantum mechanical calculations were performed on a variety of uranium species representing U(VI), U(V), U(IV), U-carbonates, U-phosphates, U-oxalates, U-catecholates, U-phosphodiesters, U-phosphorylated N-acetyl-glucosamine (NAG), and U-2-Keto-3-doxyoctanoate (KDO) with explicit solvation by H₂O molecules. These models represent major U species in natural waters and complexes on bacterial surfaces. The model results are compared to observed EXAFS, IR, Raman and NMR spectra.     

Comparison of the Efficiency of the One and Two-Step Process for the Production of BSCCO (2212) Powders by Means of Analytical Techniques

 The efficiency of the production of the high temperature superconducting powders Bi₂Sr₂CaCu₂O_8+x (BSCCO 2212) using the solid state reaction in one or two step processes under different thermal treatment was compared by means of different modern analytical techniques. Through the same techniques the optimization of the production of the production of Bi-2212 powder produced by the two step process, was achieved. X-ray diffraction analysis (XRD) and Raman spectroscopy were used to characterize the products for their stoichiometry and phase-purity. The grain size of the powders was observed by scanning electron microscopy (SEM), while their superconducting properties were tested by electrical DC-resistivity and magnetic susceptibility measurements using a superconducting quantum interference device (SQUID). It resulted that the two step process gives a high quality BSCCO 2212 superconducting powder with T _c = 85 K, in a shorter time and with a greater recovery rate than the one step process. Received May 3, 1999. Revision April 27, 2000.     

Synthesis of the Core Oligosaccharides of Lipooligosaccharides from Campylobacter jejuni: A Putative Cause of Guillain–Barré Syndrome

The chemical synthesis of the highly branched core oligosaccharides of lipooligosaccharides (LOSs) found in Campylobacter jejuni, which causes Guillain–Barré syndrome by a preceding infection, is described. The target LOS mimics, consisting of eight or nine monosaccharides, were classified into three groups as key building blocks: ganglioside-core tetra-/pentasaccharides (GM1-/GD1a-like), l -glycero-d -manno-heptose-containing trisaccharides, and 3-deoxy-d -manno-2-octulosonic acid (KDO) residues. These synthetic fragments were obtained from commercially available monosaccharides. Less obtainable l -glycero-d -manno-heptose and KDO residues, as key components of the LOSs, were synthesized from p-methoxyphenyl d -mannoside and di-O-isopropylidene-protected d -mannose, respectively. The synthesis of α-KDO glycoside, as one of the most difficult stereocontrolled glycosidic constructions, was achieved by treating a 2,3-ene derivative of KDO with phenylselenyl trifluoromethanesulfonate as a suitable α-directing reagent. All synthetic blocks were constructed through a convergent synthetic route, which resulted in the first synthesis of structurally challenging LOS core glycans containing ganglioside GM1 and GD1a-core sequences.     

Concise synthesis of 3-deoxy-d-manno-oct-2-ulosonic acid (KDO) as a protected form based on a new transformation of α,β-unsaturated ester to α-oxocarboxylic acid ester via diol cyclic sulfite

A concise synthesis of KDO (1) as the suitably protected form (2) from 2,3:5,6-di-O-isopropylidene-α-d-mannofuranose (3) was achieved in five steps (overall 65% yield). The key step is the efficient transformation of readily available α,β-unsaturated ester to α-oxocarboxylic acid ester. The newly β-elimination of the corresponding diol cyclic sulfite and the in situ trap (DBU/TMSCl) into enol silyl ether was developed to give the tautomeric equivalent of α-oxocarboxylic acid ester. The deprotection of acid labile TMS ether provided the desired product.     

Kinetics of Zn/Zn(II) reactions in acidified solutions of potassium chloride

The title subject has been studied using stationary, single-pulse, and chronopotentiometric polarization measurements on the Zn/Zn(II) electrode and equilibrium measurements on the same and the Ag/AgCl electrode in 0.5–4 M chloride solutions at 25°C. The Zn/Zn(II) electrode reactions are found to occur in two consecutive charge-transfer steps with Zn(I) as intermediate. The ion-transfer step Zn/Zn(I) is too fast to exhibit its kinetics. The electron-transfer step Zn(I)/Zn(II) mostly occurs by the couple ZnCl₂(H₂O)_y/ZnCl₂(H₂O)_y, but species with one or no chloride ligand take over as the main electroactive ones at chloride (or salt) concentrations below 1 M. The value of y is not clearly revealed by the data. Some sluggishness in complex equilibration and some, double-layer effects are observed. A convenient scale for single-ion activities is described, used, and recommended.     

Optimisation of epsomite transformation into periclase using experimental design methodology

In this work, the experimental design methodology is applied to optimise MgO production from magnesium sulphate salt. This is a two‐step process through an intermediate product (Brucite: Mg(OH)₂). For the first step, a fractional factorial design and a centred composite one are used in order to establish appropriate experimental conditions for Mg(OH)₂ obtention. The most favourable conditions for the second step to obtain MgO were determined using only a fractional factorial design. The decomposition of the optimal precipitated Mg(OH)₂ was analysed by DTA/TGA and the crystallisation process was observed by XRD. The morphological properties of the MgO agglomerates were examined by SEM. Copyright © 2010 John Wiley & Sons, Ltd.     

Metal-organic deposition of YBa2Cu3Ox and Bi2Sr2Ca1Cu2Ox films on various substrates starting from different fluorine-free metallorganic compounds

YBa₂Cu₃O_x (Y-123) and Bi₂Sr₂Ca₁Cu₂O_x (Bi-2212) films on various substrates have been prepared by Metal-Organic Deposition starting from different metallorganic fluorine-free compounds and using a very simple instrumentation. The processing conditions include a rapid pyrolysis step in air and an annealing step in oxygen for Y-123 and in air for Bi-2212. The films obtained have been characterized by X-ray diffraction (XRD) and the formation of a superconducting phase of Y-123 or Bi-2212 was confirmed measuring the critical temperature (T _c) with Ac-susceptibility and resistive measurements. Microstructure and final cationic ratios have been studied by scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS).     

Improving prediction selectivity for on-line near-infrared monitoring of components in etchant solution by spectral range optimization

The components (H₃PO₄, HNO₃, CH₃COOH and water) in an etchant solution have been accurately measured in an on-line manner using near-infrared (NIR) spectroscopy by directly illuminating NIR radiation through a Teflon line. In particular, the spectral features according to the change of H₃PO₄ or HNO₃ concentrations were not mainly from NIR absorption themselves, but from the perturbation (or displacement) of water bands; therefore, the resulting spectral variations were quite similar to each other. Consequently partial least squares (PLS) prediction selectivity among the components should be the most critical issue for continuous on-line compositional monitoring by NIR spectroscopy. To improve selectivity of the calibration model, we have optimized the calibration models by finding selective spectral ranges with the use of moving window PLS. Using the optimized PLS models for each component, the resulting prediction accuracies were substantially improved. Furthermore, on-line prediction selectivity was evaluated by spiking individual pure components step by step and examining the resulting prediction trends. When optimized PLS models were used, each concentration was selectively and sensitively varied at each spike; meanwhile, when whole or non-optimized ranges were used for PLS, the prediction selectivity was greatly degraded. This study verifies that the selection of an optimal spectral range for PLS is the most important factor to make Teflon-based NIR measurements successful for on-line and real-time monitoring of etching solutions.