Quantification of 2-keto-3-deoxyoctonate in (lipo)polysaccharides by methanolytic release, trifluoroacetylation and capillary gas chromatography

Several conditions of acidic anhydrous methanolysis were examined to optimize the release and minimize the degradation of unphosphorylated 2-keto-3-deoxy-D-manno-octonic acid (KDO) from bacterial lipopolysaccharides and polysaccharides. The reaction was monitored by capillary gas chromatography after derivatization by trifluoroacetic anhydride. The best results were obtained by use of 2 M hydrochloric acid at 60 degrees C for 2 h. Under these conditions a single KDO component appeared, and KDO was quantitatively released from all model compounds except when glycosidically linked to hexosamines. For quantitative cleavage of this linkage a reaction time of 6 h was required at 60 degrees C, giving rise to 5-10% of secondary KDO products. The KDO detection limit was about 250 pmol (50 ng) and the molar response was the same as for glucose. The KDO derivative gave a mass spectrometric fragmentation pattern consistent with a pyranosidic methylketoside methyl ester structure. Differentiation of KDO linkage types could be obtained by determination of the rates of KDO release by mild methanolysis.     

Total synthesis of 3-deoxy-D-manno-2-octulosonic acid (KDO) and 2-deoxy-beta-KDO

[formula: see text] Total syntheses of KDO and 2-deoxy-beta-KDO are reported. The C2-symmetric dienediol 4 was desymmetrized by conversion to its corresponding 1,4-dioxanone 5. Ireland-Claisen rearrangement of 5 provided the 6-vinyldihydropyran-2-carboxylate template 6. Double-Sharpless asymmetric dihydroxylation gave the tetraol 7a, which was converted to KDO and 2-deoxy-beta-KDO using methods similar to those previously reported. This synthetic scheme provides a flexible route to KDO and KDO analogues.     

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.     

Transketolase and fructose-1,6-bis-phosphate aldolase, complementary tools for access to new ulosonic acid analogues

A new approach to the synthesis of ulosonic acids KDO and DAH is described. The key step is the C₅-C₆ bond formation catalysed by fructose-1,6-bisphosphate aldolase (for KDO) or transketolase (for DAH) using substituted acrylonitrile α-hydroxyaldehyde. All asymmetric carbon configurations are determined in an enzymatic step by the means of deshydrogenase or lipase. This strategy, using a non-metabolism pathway, allows access to novel precursors of KDO, DAH and analogues.     

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.     

Effects of Temperature and Energy on Stability of Oligomeric Enzyme Probed on Electrospray Ionization Mass Spectrometry

Escherichia coli 3-Deoxy-D-manno-octulosonate 8-phosphate（KDO8P） synthase catalyzed the condensation reaction between D-arabinose 5-phosphate（A5P） and phosphoenolpyruvate（PEP） to form KDO8P and inorganic phosphate（Pi）. The noncovalent tetrameric association ofKDO8P synthase was observed and dissociated in gas phase by means of electrospray ionization mass spectrometry under the very ＂soft＂ conditions. The results indicate that PEP-bound enzyme generated abundant tetrameric species as well as monomeric species at the ＂soft＂ conditions, whereas, the unbound enzyme favored the formation of a dimeric species. The mass spectra of the mixture of the enzyme with one of substrates, PEP, and A5P or one of products, KDO8P and Pi show that the complex of the unbound enzyme with PEP or Pi was prone to the formation of a monomeric species, whereas, that of the unbound enzyme with A5P or KDO8P was similar to the unbound enzyme. The intensity of the dimeric species increased with the increase of temperature at a collision voltage of 10 V. Taken together, the results presented here suggest that mass spectrometry will be a powerful tool to explore subtile conformational changes and/or subunit-subunit interactions of multiprotein assembly induced by ligand-binding and/or the changes of environmental conditions.     

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.     

Stereoselective synthesis of the alpha-glycoside of a KDO "C"-disaccharide

The reaction of tert-butyl (4,5,7, 8-tetra-O-acetyl-3-deoxy-alpha-D-manno-2-octulopyranosyl chloride)onate donor 7 with the 6-formylgalactopyranoside acceptor 4 in the presence of SmI(2) provided only the KDO alpha-C-disaccharide 8. The bulky tert-butyl ester in the donor was used to reverse the stereochemical outcome of C-glycosylation, stereoselectively forming the alpha-"C"-disaccharide of KDO.     

Modified method for the quantitative determination of certain deoxy sugars in lipopolysaccharides

An alkaline modification of the 2-thiobarbituric (2-TBA) method for the rapid determination of 2-keto-3-deoxy-D-mannooctonic acid (KDO) and of 3,6-dideoxyhexoses (3,6-DDHs) in hydrolysates of lipopolysaccharides is described. The modification is based on the performance of the periodate-2-TBA color reaction with the subsequent decomposition of the KDO chromophore by alkaline treatment and measurement of the optical densities of the solutions before and after this treatment. It has been found that the modification described is distinguished by high sensitivity, specificity, and reproducibility of the results. The standard deviation calculated from six determinations amounted to 2.84% for KDO and 3.93% for 3,6-DDHs. The method is simple to perform and needs only simple equipment and a small amount of reagents.     

Conversion of aquifex aeolicus 3-deoxy-d-manno-octulosonate 8-phosphate synthase, a metalloenzyme, into a nonmetalloenzyme

The Aquifex aeolicus 3-deoxy-d-manno-octulosonate 8-phosphate synthase (KDO8PS), a class II metalloenzyme, is converted into an active nonmetalloenzyme by a single amino acid mutation, namely, C11N. The result may provide insight into the evolutionary link between the two KDO8PS classes as well as the potential role of the metal and/or asparagine in the catalytic mechanism.     

Innate immune responses of synthetic lipid A derivatives of Neisseria meningitidis

Differences in the pattern and chemical nature of fatty acids of lipid A of Neisseria meningitides lipooligosaccharides (LOS) and Escherichia coli lipopolysaccharides (LPS) may account for differences in inflammatory properties. Furthermore, there are indications that dimeric 3-deoxy-D-manno-oct-2-ulosonic acid (KDO) moieties of LOS and LPS enhance biological activities. Heterogeneity in the structure of lipid A and possible contaminations with other inflammatory components have made it difficult to confirm these observations. To address these problems, a highly convergent approach for the synthesis of a lipid A derivative containing KDO has been developed, which relies on the ability to selectively remove or unmask in a sequential manner an isopropylidene acetal, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonate (Alloc), azide, and thexyldimethylsilyl (TDS) ether. The strategy was employed for the synthesis of N. meningitidis lipid A containing KDO (3). Mouse macrophages were exposed to the synthetic compound and its parent LOS, E. coli lipid A (2), and a hybrid derivative (4) that has the asymmetrical acylation pattern of E. coli lipid A, but the shorter lipids of meningococcal lipid A. The resulting supernatants were examined for tumor necrosis factor alpha (TNF-alpha) and interferon beta (IFN-beta) production. The lipid A derivative containing KDO was much more active than lipid A alone and just slightly less active than its parent LOS, indicating that one KDO moiety is sufficient for full activity of TNF-alpha and IFN-beta induction. The lipid A of N. meningitidis was a significantly more potent inducer of TNF-alpha and IFN-beta than E. coli lipid A, which is due to a number of shorter fatty acids. The compounds did not demonstrate a bias towards a MyD88- or TRIF-dependent response.     

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.     

Ring-closing metathesis of α-ester-substituted enol ethers: application to the shortest synthesis of KDO

Ring-closing metathesis reactions of α-ester-substituted enol ethers are described. In the case of unsubstituted terminal olefins, isomerization prior to cyclization was observed as an undesired side reaction, which could not be completely inhibited. Furthermore, this methodology was applied to a formal synthesis of KDO, which now represents the shortest synthetic pathway to KDO and its deoxy analogue. Interestingly, in this route olefin isomerization was not observed, presumably due to the increased steric environment of the double bond. Finally, an efficient two-step conversion to transform an alcohol into an α-alkoxy acrylate is also described.     

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.     

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.     

Synthesis of azacyclic 2-deoxy-KDO

Azacyclic analogues of 2-deoxy-KDO have been synthesized from KDO via reductive amination at C-2 and ring closure on C-6 with double inversion.     

Observation of a chemically labile,noncovalent enzyme intermediate in the reaction of metal‐dependent Aquifex pyrophilus KDO8PS by time‐resolved mass spectrometry

The direct detection of intermediates in enzymatic reactions can yield important mechanistic insights but may be difficult due to short intermediate lifetimes and chemical instability. Using a rapid‐mixing device coupled with electrospray ionization time‐of‐flight mass spectrometry, the noncovalent hemiketal intermediate in the reaction of metal‐dependent 3‐deoxy‐D‐manno‐octulosonate‐8‐phosphate (KDO8P) synthase from Aquifex pyrophilus was observed in the millisecond time range. Using single turnover conditions, the noncovalent complexes of enzyme with Cd²⁺:phosphoenolpyruvate, Cd²⁺:phosphate, Cd²⁺:KDO8P, and Cd²⁺:intermediate complexes were resolved. The intermediate complex is present during times ranging from 50–630 ms, indicating that the intermediate builds up at the ambient temperatures of the experiment. This represents the first direct detection of the intermediate with a native metal‐dependent KDO8PS, and further demonstrates that time‐resolved mass spectrometry is a useful tool in mechanistic studies of enzymatic reactions. Copyright © 2010 John Wiley & Sons, Ltd.     

Inhibition mode of a bisubstrate inhibitor of KDO8P synthase: a frequency-selective REDOR solid-state and solution NMR characterization

In this report the mode of inhibition of mechanism-based inhibitor (2, K(i) = 0.4 microM) of 3-deoxy-d-manno-2-octulosonate-8-phosphate synthase (KDO8PS), which was designed to mimic the combined key features of its natural substrates arabinose-5-phosphate (A5P) and phoshoenolpyruvate (PEP) into a single molecule, was investigated. Our earlier solid-state NMR observations identified the inhibitor to bind in a way that partly mimics A5P, while the phosphonate moiety of its PEP-mimicking part exhibits no interactions with enzyme residues. This result was apparently in disagreement with the competitive inhibition of 2 against PEP and with the later solved crystal structure of KDO8PS-2 binary complex identifying the interactions of its PEP-mimicking part with the enzyme residues that were not detected by solid-state NMR. To solve this discrepancy, further solid-state REDOR NMR and (31)P solution NMR experiments were applied to a variety of enzyme complexes with the substrates and inhibitor. In particular, a novel frequency-selective REDOR experiment was developed and applied. Integration of the solution and solid-state NMR data clearly demonstrates that under conditions of stoichiometric enzyme-ligand ratio at thermodynamic equilibrium (a) PEP binding is unperturbed by the presence of 2 and (b) both PEP and 2 can bind simultaneously to the synthase, i.e., form a ternary complex with PEP occupying its own subsite and 2 occupying A5P's subsite. The latter observation suggests that under the conditions used in our NMR measurements, the inhibition pattern of 2 against PEP should have a mixed type character. Furthermore, the NMR data directly demonstrate the distinction between the relative binding strength of the two moieties of 2: enzyme interactions with PEP-mimicking moiety are much weaker than those with the A5P moiety. This observation is in agreement with KDO8PS-2 crystal structure showing only remote contacts of the phosphonate due to large structural changes of binding site residues. It is concluded that these phosphonate-enzyme interactions evidenced by both (31)P solution NMR and X-ray are too weak to be preserved under the lyophilization of KDO8PS-2 binary complex and therefore are not evidenced by the solid-state REDOR spectra.     

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.     

Photodegradation of lipopolysaccharides and the inhibition of macrophage activation by anthraquinone-boronic acid hybrids

Target-selective photodegradation of 3-deoxy-D-manno-2-octulopyranosonic acid (KDO) was achieved without additives and under neutral conditions using a designed anthraquinone-boronic acid hybrid and long wavelength UV light irradiation. The hybrid can photodegrade lipopolysaccharides (LPS) and inhibit macrophage activation induced by LPS.