Kdo Disaccharide Research Articles

Exploring the cross-reactivity of S25-2: complex with a 5,6-dehydro-Kdo disaccharide.

The near-germline antibody S25-2 exhibits a remarkable cross-reactivity for oligosaccharides containing the bacterial lipopolysaccharide carbohydrate 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo). The recent synthesis of a variety of Kdo analogues permits a detailed structural analysis of the importance of specific interactions in antigen recognition by S25-2. The Kdo disaccharide analogue Kdo-(2→4)-5,6-dehydro-Kdo lacks a 5-OH group on the second Kdo residue and has been cocrystallized with S25-2. The structure reveals that the modification of the Kdo residue at position 5 results in a rearrangement of intramolecular hydrogen bonds in the antigen that allows it to assume a novel conformation in the antibody-combining site. The cross-reactive binding of S25-2 to this synthetic ligand highlights the adaptability of this antibody to non-natural synthetic analogues.

Molecular Basis of Lipopolysaccharide Heterogeneity in Escherichia coli: ENVELOPE STRESS-RESPONSIVE REGULATORS CONTROL THE INCORPORATION OF GLYCOFORMS WITH A THIRD 3-DEOXY-α-d-MANNO-OCT-2-ULOSONIC ACID AND RHAMNOSE

Mass spectrometric analyses of lipopolysaccharide (LPS) from isogenic Escherichia coli strains with nonpolar mutations in the waa locus or overexpression of their cognate genes revealed that waaZ and waaS are the structural genes required for the incorporation of the third 3-deoxy-α-D-manno-oct-2-ulosonic acid (Kdo) linked to Kdo disaccharide and rhamnose, respectively. The incorporation of rhamnose requires prior sequential incorporation of the Kdo trisaccharide. The minimal in vivo lipid A-anchored core structure Kdo(2)Hep(2)Hex(2)P(1) in the LPS from ΔwaaO (lacking α-1,3-glucosyltransferase) could incorporate Kdo(3)Rha, without the overexpression of the waaZ and waaS genes. Examination of LPS heterogeneity revealed overlapping control by RpoE σ factor, two-component systems (BasS/R and PhoB/R), and ppGpp. Deletion of RpoE-specific anti-σ factor rseA led to near-exclusive incorporation of glycoforms with the third Kdo linked to Kdo disaccharide. This was accompanied by concomitant incorporation of rhamnose, linked to either the terminal third Kdo or to the second Kdo, depending upon the presence or absence of phosphoethanolamine on the second Kdo with truncation of the outer core. This truncation in ΔrseA was ascribed to decreased levels of WaaR glycosyltransferase, which was restored to wild-type levels, including overall LPS composition, upon the introduction of rybB sRNA deletion. Thus, ΔwaaR contained LPS primarily with Kdo(3) without any requirement for lipid A modifications. Accumulation of a glycoform with Kdo(3) and 4-amino-4-deoxy-l-arabinose in lipid A in ΔrseA required ppGpp, being abolished in a Δ(ppGpp(0) rseA). Furthermore, Δ(waaZ lpxLMP) synthesizing tetraacylated lipid A exhibited synthetic lethality at 21-23°C pointing to the significance of the incorporation of the third Kdo.

Synthesis of Neoglycoconjugates Containing 4‐Amino‐4‐deoxy‐L‐arabinose Epitopes Corresponding to the Inner Core of Burkholderia and Proteus Lipopolysaccharides

Disaccharides that contain 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) and d-glycero-d-talo-oct-2-ulosonic acid (Ko) substituted at the 8-position by 4-amino-4-deoxy-β-l-arabinopyranosyl (Ara4N) residues have been prepared. Coupling an N-phenyltrifluoroacetimidate-4-azido-4-deoxy-l-arabinosylglycosyl donor to acetyl-protected allyl glycosides of Kdo and Ko afforded anomeric mixtures of disaccharide products in 74 and 90 % yield, respectively, which were separated by chromatography. Further extension of an intermediate Ara4N-(1→8)-Kdo disaccharide acceptor, which capitalized on a regioselective glycosylation with a Kdo bromide donor under Helferich conditions, afforded the branched trisaccharide α-Kdo-(2→4)[β-l-Ara4N-(1→8)]-α-Kdo derivative. Deprotection of the protected di- and trisaccharide allyl glycosides was accomplished by TiCl4-promoted benzyl ether cleavage followed by the removal of ester groups and reduction of the azido group with thiol or Staudinger reagents, respectively. The reaction of the anomeric allyl group with 1,3-propanedithiol under radical conditions afforded the thioether-bridged spacer glycosides, which were efficiently coupled to maleimide-activated bovine serum albumin. The neoglycoconjugates serve as immunoreagents with specificity for inner core epitopes of Burkholderia and Proteus lipopolysaccharides.

Topography of Lipid A Assembly and Modification in the Inner Membrane of Francisella

Francisella tularensis is Gram‐negative bacteria that cause tularemia in mammals. The outer leaflet of its outer membrane is formed by lipid A, the hydrophobic membrane anchor of lipopolysaccharide (LPS). However, about two thirds of the lipid A molecules in Francisella do not have a core oligosaccharide. We have discovered that LPS and free lipid A are not fully separated from each other during purification. We took advantage of a mutation in a mannosyl transferase (LpcC), which adds the proximal mannose residue to Kdo2‐lipid A in the first step of outer core assembly. The LpcC mutant produces a truncated LPS with Kdo residues as the only remaining core sugars. The Kdo modified lipid A molecules lack of both the 1 and 4′‐phosphate groups, whereas free lipid A only lacks the 4′‐phosphate residue. This difference is attributed to the selectivity of the 1‐phosphatase LpxE, which located on the outer surface of the inner membrane and is dependent upon the presence of the Kdo disaccharide for activity. Likewise, LpcC, which faces the cytoplasm, is selective in vivo and in vitro for acceptor substrates with two Kdo units instead of one. Our findings have implications for the order and topography of lipid A modification reactions in the Francisella inner membrane. Supported by NIH grant GM‐51796 to C.R.H.R.

3-Deoxy-d-manno-octulosonic Acid (Kdo) Hydrolase Identified in Francisella tularensis, Helicobacter pylori, and Legionella pneumophila

3-Deoxy-D-manno-octulosonic acid (Kdo) is an eight-carbon sugar ubiquitous in Gram-negative bacterial lipopolysaccharides (LPS). Although its biosynthesis is well described, no protein has yet been identified as a Kdo hydrolase. However, Kdo hydrolase enzymatic activity has been detected in membranes of Helicobacter pylori and Francisella tularensis and may be responsible for the removal of side-chain Kdo from the LPS core saccharides. We now report the identification of genes encoding a Kdo hydrolase in F. tularensis Schu S4 and live vaccine strain strains, in H. pylori 26695 strain and in Legionella pneumophila Philadelphia 1 strain. We have renamed the genes kdhA for keto-deoxyoctulosonate hydrolase A. Deletion of kdhA abolished Kdo hydrolase activity in membranes of F. tularensis live vaccine strain. The F. tularensis kdhA mutant synthesized a core oligosaccharide containing a Kdo disaccharide with one of the Kdo residues being a terminal side chain. This side-chain Kdo monosaccharide was absent in the wild-type core oligosaccharide. Expression in Escherichia coli of recombinant KdhA from F. tularensis, H. pylori, and L. pneumophila resulted in a reduction of membrane-associated side-chain Kdo. The identification of this previously faceless enzyme will accelerate study of the biosynthetic basis and biologic impact for postbiosynthetic LPS structural modification.

Replacement of Lipopolysaccharide with Free Lipid A Molecules in Escherichia coli Mutants Lacking All Core Sugars

Escherichia coli mutants deficient in 2-keto-3-deoxy-D-manno-octulosonic acid (Kdo) biosynthesis are conditionally lethal, but their phenotypes are bypassed by certain suppressor mutations or by overexpression of MsbA, the inner membrane flippase for core-lipid A. These strains grow on broth with the tetraacylated precursor lipid IV(A) replacing lipopolysaccharide [Meredith, T. C., et al. (2006) ACS Chem. Biol. 1, 33-42]. Deletion of kdtA, which encodes the Kdo transferase, is possible under these conditions. We now show that lipid IV(A) reaches the outer surface of the outer membrane in these strains, as judged by its accessibility to the lipase PagL. On the assumption that MsbA is optimized to transport penta- or hexaacylated lipid A, we overexpressed the lauroyl- or the myristoyltransferase of lipid A biosynthesis, encoded by lpxL and lpxM, respectively, and demonstrated that kdtA deletion mutants were also viable in this setting. Although E. coli LpxL is stimulated by the presence of the Kdo disaccharide in its acceptor substrate, LpxL does slowly acylate lipid IV(A). Overexpression of LpxL from a plasmid suppressed the lethality of kdtA deletions on nutrient broth at 30 or 37 degrees C without the need for MsbA overproduction. These strains accumulated penta- and hexaacylated free lipid A containing a secondary laurate chain or a laurate and a myristate chain, respectively. Deletion of kdtA in strains overexpressing LpxM accumulated pentaacylated lipid A with a secondary myristate moiety. None of the strains lacking kdtA grew in the presence of bile salts at any temperature or on nutrient broth at 42 degrees C. Our findings show that the main function of Kdo is to provide the right substrates for the acyltransferases LpxL and LpxM, resulting in the synthesis of penta- and hexaacylated lipid A, which is optimal for the MsbA flippase.

The role of CDR H3 in antibody recognition of a synthetic analog of a lipopolysaccharide antigen

In order to explore the structural basis for adaptability in near germline monoclonal antibodies (mAb), we have examined the specificity of the promiscuous mAb S67-27 to both naturally derived carbohydrate antigens and a variety of synthetic nonnatural antigens based on the bacterial lipopolysaccharide component 3-deoxy-alpha-D-manno-oct-2-ulosonic acid (Kdo). One such analog, a 7-O-methyl (7-O-Me) Kdo disaccharide, was found to bind to the antibody with at least 30-fold higher affinity than any other antigen tested. The structure of S67-27 in complex with this analog and three other naturally occurring Kdo antigens revealed that the enhanced affinity of the mAb for the synthetic analog was accomplished by the strategic positioning of CDR H3 away from a conserved Kdo binding pocket that allowed the formation of new antibody-antigen contacts. Furthermore, the comparison of this structure with the structures of related mAbs revealed how the position and structure of CDR H3 influence the specificity or promiscuity of near-germline carbohydrate-recognizing antibodies by altering the architecture of the combining site.

Synthesis and antigenic properties of C-7-modified Kdo mono- and disaccharide ligands and Kdo disaccharide interresidue lactones

In order to define binding interactions of Kdo-specific monoclonal antibodies directed against the chlamydial α-(2→8)-linked Kdo disaccharide epitope on a molecular level, modifications at the 7-position of the proximal and distal Kdo unit were investigated. The synthesis of 7- O-methyl and 7-azido-7-deoxy-7- epi-Kdo monosaccharide derivatives was achieved via an 8- O-TBS protected derivative, whereas methylation of O-7 at the proximal Kdo unit of the α-(2→8)-linked Kdo disaccharide was conveniently accomplished via a 4,5; 4′,5′; 7′,8′-tri- O-carbonyl-protected disaccharide intermediate. Attempted epimerization at C-5 of the inner unit of a α-(2→4)-linked Kdo disaccharide, however, resulted in formation of the corresponding 5,6-dehydro derivative, which was fully deprotected. Treatment of unprotected α-(2→8)- as well as α-(2→4)-linked Kdo disaccharides in neat acetic acid furnished the corresponding interresidue lactone derivatives. The lactones displayed limited stability under neutral conditions and were hydrolyzed at pH 7 within 3 days. Access to the lactones, however, provides a means for selective derivatization of the carboxylic group located at the distal Kdo residue, which was demonstrated by methanolysis of the lactone to afford the monomethyl ester of the α-(2→8)-linked Kdo disaccharide. ELISA inhibition experiments of the ligands with two Kdo-specific monoclonal antibodies showed slightly reduced reactivity for the binding of the α-(2→8) Kdo-specific antibody S25-2, whereas the 7- O-methyl disaccharide antigen displayed high binding affinity toward the Kdo monosaccharide-specific antibody S67-27.

Kdo-(2 → 8)-Kdo-(2 → 4)-Kdo but not Kdo-(2 → 4)-Kdo-(2 → 4)-Kdo is an acceptor for transfer of L-glycero-α-D-manno-heptose by Escherichia coli heptosyltransferase I (WaaC)

Early steps in the biosynthesis of lipopolysaccharide (LPS) involve the transfer of 3-deoxy-alpha-D-manno-oct-2-ulopyranosonic acid (Kdo) to lipid A. Whereas Kdo transferases (WaaA) of Escherichia coli generate a (2 --> 4)-linked Kdo disaccharide, Chlamydiae contain tri- or tetra-functional WaaA generating oligosaccharides with (2 --> 8)- and (2 --> 4)-linkages between Kdo. It has been suggested that the transfer of L-glycero-alpha-D-manno-heptose (Hep) to Kdo by an E. coli WaaC may not be possible in the presence of (2 --> 8)-linked Kdo. E. coli double-mutants deficient in heptosyltransferases I (waaC) and II (waaF) and expressing waaA of Chlamydiae instead of their own, make Chlamydia-type Kdo oligosaccharides which are attached to an E. coli lipid A. Using such strains expressing waaA of Chlamydophila pneumoniae, Chlamydophila psittaci, or Chlamydia trachomatis, we have studied the effect of E. coli waaC gene expression on LPS structure. Structural analyses revealed the formation of two novel oligosaccharides Hep-(1 --> 5)[Kdo-(2 --> 4)]-Kdo and Hep-(1 --> 5)[Kdo-(2 --> 8)-Kdo-(2 --> 4)]-Kdo showing that Hep is transferred in the presence of (2 --> 8)-linked Kdo. Surprisingly, the transfer of Hep onto Kdo-(2 --> 4)-Kdo-(2 --> 4)-Kdo did not occur, despite the fact that Hep-(1 --> 5)[Kdo-(2 --> 4)-Kdo-(2 --> 4)]-Kdo is found in nature as a partial structure of E. coli LPS. The premature end of the biosynthesis and incorporation of Hep into the LPS indicated that WaaC had access to the substrate before Kdo transfer was completed. We have observed differences between WaaA of C. trachomatis, C. pneumoniae and C. psittaci which indicate mechanistic differences between these Kdo transferases.

Dodecaprenyl Phosphate-Galacturonic Acid as a Donor Substrate for Lipopolysaccharide Core Glycosylation in Rhizobium leguminosarum

The lipid A and inner core regions of Rhizobium leguminosarum lipopolysaccharide contain four galacturonic acid (GalA) residues. Two are attached to the outer unit of the 3-deoxy-D-manno-octulosonic acid (Kdo) disaccharide, one to the mannose residue, and one to the 4'-position of lipid A. The enzymes RgtA and RgtB, described in the accompanying article, catalyze GalA transfer to the Kdo residue, whereas RgtC is responsible for modification of the core mannose unit. Heterologous expression of RgtA in Sinorhizhobium meliloti 1021, a strain that normally lacks GalA modifications on its Kdo disaccharide, resulted in detectable GalA transferase activity in isolated membrane preparations, suggesting that the appropriate GalA donor substrate is available in S. meliloti membranes. In contrast, heterologous expression of RgtA in Escherichia coli yielded inactive membranes. However, RgtA activity was detectable in the E. coli system when total lipids from R. leguminosarum 3841 or S. meliloti 1021 were added. We have now purified and characterized dodecaprenyl (C60) phosphate-GalA as a minor novel lipid of R. leguminosarum 3841 and S. meliloti. This substance is stable to mild base hydrolysis and was purified by DEAE-cellulose column chromatography. Its structure was established by a combination of electrospray ionization mass spectrometry and gas-liquid chromatography. Purified dodecaprenyl phosphate-GalA supports the efficient transfer of GalA to Kdo2-1-dephospho-lipid IV(A) by membranes of E. coli cells expressing RgtA, RgtB, and RgtC. The identification of a polyisoprene phosphate-GalA donor substrate suggests that the active site of RgtA faces the periplasmic side of the inner membrane. This work represents the first definitive characterization of a lipid-linked GalA derivative with the proposed structure dodecaprenyl phosphate-beta-D-GalA.

An inner membrane enzyme in Salmonella and Escherichia coli that transfers 4-amino-4-deoxy-L-arabinose to lipid A: induction on polymyxin-resistant mutants and role of a novel lipid-linked donor.

Attachment of the cationic sugar 4-amino-4-deoxy-l-arabinose (l-Ara4N) to lipid A is required for the maintenance of polymyxin resistance in Escherichia coli and Salmonella typhimurium. The enzymes that synthesize l-Ara4N and transfer it to lipid A have not been identified. We now report an inner membrane enzyme, expressed in polymyxin-resistant mutants, that adds one or two l-Ara4N moieties to lipid A or its immediate precursors. No soluble factors are required. A gene located near minute 51 on the S. typhimurium and E. coli chromosomes (previously termed orf5, pmrK, or yfbI) encodes the l-Ara4N transferase. The enzyme, renamed ArnT, consists of 548 amino acid residues in S. typhimurium with 12 possible membrane-spanning regions. ArnT displays distant similarity to yeast protein mannosyltransferases. ArnT adds two l-Ara4N units to lipid A precursors containing a Kdo disaccharide. However, as shown by mass spectrometry and NMR spectroscopy, it transfers only a single l-Ara4N residue to the 1-phosphate moiety of lipid IV(A), a precursor lacking Kdo. Proteins with full-length sequence similarity to ArnT are present in genomes of other bacteria thought to synthesize l-Ara4N-modified lipid A, including Pseudomonas aeruginosa and Yersinia pestis. As shown in the following article (Trent, M. S., Ribeiro, A. A., Doerrler, W. T., Lin, S., Cotter, R. J., and Raetz, C. R. H. (2001) J. Biol. Chem. 276, 43132-43144), ArnT utilizes the novel lipid undecaprenyl phosphate-alpha-l-Ara4N as its sugar donor, suggesting that l-Ara4N transfer to lipid A occurs on the periplasmic side of the inner membrane.

Synthesis of neoglycoproteins containing Kdo epitopes specific for Chlamydophila psittaci lipopolysaccharide.

The oligosaccharides alpha-Kdop-(2-->8)-alpha-Kdop-(2-->6)-beta-D- GlcpNAc-(1-->OAll) 4, alpha-Kdop-(2-->4)-alpha- Kdop-(2-->4)-alpha-Kdop-(2-->6)-beta-D-GlcpNAc-(1-->OAll+ ++) 10, and the branched Kdo tetrasaccharide alpha- Kdop-(2-->4)-[alpha-Kdop-(2-->8)]-alpha-Kdop-(2-->4)-a lpha-Kdop-(2-->OAll) 21 have been prepared using en bloc transfer of Kdo oligosaccharide bromide donors to protected mono- or disaccharide acceptors. Radical addition of cysteamine to the anomeric allyl glycosides afforded good yields of the corresponding 3-(2-aminoethylthio)propyl glycosides 5, 11 and 22. The spacer ligands were activated with thiophosgene and reacted with bovine serum albumin to give the neoglycoconjugates 6, 12 and 23 which were used to prepare solid-phase antigens in enzyme immuno-assays for the characterization of monoclonal antibodies against chlamydial LPS. The data showed that the (2-->8)-linked Kdo disaccharide and the (2-->8)-(2-->4)-linked Kdo trisaccharide portion of the neoglycoconjugate 23 were not available for binding of antibodies which recognize these structures as di- and trisaccharide, respectively.

Structural studies on the lipopolysaccharide from a rough strain of Ochrobactrum anthropi containing a 2,3-diamino-2,3-dideoxy- d-glucose disaccharide lipid A backbone

A degradation protocol using de- O-acylation and subsequent alkaline de- N-acylation was applied to the lipopolysaccharide of Ochrobactrum anthropi rough strain LMG 3301. Three main oligosaccharide bisphosphates containing core–lipid A backbone structures were obtained after fractionation by anion-exchange HPLC. Using 1 H and 13 C NMR spectroscopy, including two-dimensional COSY, TOCSY, and NOE spectroscopy (ROESY and NOESY), the following structures were established: where Kdo is 3-deoxy- d- manno-octulosonic acid, d-GlcN3N is 2,3-diamino-2,3-dideoxy- d-glucose and R is H or α- d-Gal pA or 4-deoxy- β- l- threo-hex-4-enopyranuronic acid, the latter sugar being derived from α- d-Gal pA by β-elimination of a substituent attached to O-4. This is the first report on the isolation from a lipopolysaccharide of an oligosaccharide containing GlcN3N in the lipid A backbone [ β- d-Glc pN3N4 P-(1→6)- α- d-Glc pN3N1 P]. Sugar and methylation analysis confirmed the presence of the GalA→Kdo disaccharide and non-stoichiometric substitution of GalA. It is suggested that Glc is the substituent at O-4 in GalA and that in the non-degraded lipopolysaccharide the amino group of GlcN is not acylated.

Synthesis of deoxy analogues of (2→8)-linked 3-deoxy-α-D-manno-oct-2-ulopyranosylonic acid (Kdo) disaccharides for binding studies with Chlamydia specific monoclonal antibodies

Deoxy analogues of the Chlamydia-specific, α-(2→8)-linked Kdo disaccharide epitope modified at the pyranose ring of the terminal Kdo unit have been prepared. Utilizing the 3,5-dideoxy-D-arabino-oct-2-ulosonate bromide donor [1] and the acceptor [2] under Helferich conditions, the 5-deoxy-α-Kdo-(2→8)-α-Kdo disaccharide [3] was obtained as the minor product together with unsaturated, α-(2→8)-glycosidically and (4→8)-ether-linked derivatives [5] and [7] as the major components. Deprotection afforded the disaccharide allyl glycosides [4], [6], and [8]. Further transformation of protected intermediates by hydrogenation followed by deblocking gave the propyl glycosides [12], [14] and [17]. The compounds may be used for binding studies with Chlamydia-specific and cross-reactive, Kdospecific monoclonal antibodies.

A Mono-functional 3-Deoxy-d-manno-octulosonic Acid (Kdo) Transferase and a Kdo Kinase in Extracts of Haemophilus influenzae

Lipopolysaccharide of Haemophilus influenzae contains a single 3-deoxy-D-manno-octulosonic acid (Kdo) residue, linked to the 6' position of lipid A. In Escherichia coli and related organisms, a Kdo disaccharide is attached to lipid A. In previous studies, we cloned the gene (kdtA) encoding the E. coli Kdo transferase and demonstrated that homogeneous preparations of KdtA polypeptide catalyzed the attachment of both Kdo groups to the precursor, lipid IVA. E. coli KdtA produced only traces of mono-glycosylated product. We now show that a single Kdo is transferred to lipid IVA in extracts of H. influenzae. The mono-functional Kdo transferase of H. influenzae is membrane-bound, and the reaction is dependent upon a CMP-Kdo-generating system, as in E. coli. The specific activity of Kdo transfer to lipid IVA is 0.5-1 nmol/min/mg in H. influenzae membranes. Utilizing solubilized H. influenzae membranes, milligram quantities of Kdo-lipid IVA were prepared for analysis. Matrix-assisted laser desorption/ionization mass spectrometry revealed a parent ion (M - H)- at m/z 1626.0, consistent with the addition of a single Kdo moiety. Like lipid IVA, Kdo-lipid IVA was an excellent substrate for the bi-functional Kdo transferase of E. coli. In membranes of H. influenzae, but not E. coli, Kdo-lipid IVA was further phosphorylated in the presence of ATP, yielding a mono-phosphorylated Kdo-lipid IVA with a parent ion (M - H)- at m/z 1703.9. The identification of the mono-functional H. influenzae Kdo transferase, which is encoded by a KdtA homologue that displays 50% identity to its E. coli counterpart, should facilitate the mechanistic dissection of more complex multi-functional Kdo transferases, like those of E. coli and Chlamydia trachomatis.

Addition Reactions of Glycal Esters: Access to Glycosyl Donors of Kdo, d-glycero-d-talo- and d-glycero-d-galacto-2-Octulosonic Acid Residues

ABSTRACT Addition reactions of O-acetylated glycal esters of Kdo mono-, α-(2→8)- and α-(2→4)- linked Kdo disaccharide derivatives 1a - c with NIS in acetic acid afforded good yields of trans-diaxial as well as minor amounts of trans-diequatorial and cis-configured 2-O-acetyl-3-deoxy-3-iodo derivatives, which were efficiently reduced with Bu3SnH/AIBN to give the corresponding per-O-acetylated Kdo methyl ester derivatives. Similar reactions of 1a with NBS or NCS furnished the trans-diaxial 2-O-acetyl-3-bromo-3-deoxy- as well as 3-chloro-3-deoxy derivatives as the main products. Reaction of 1a with NBS in aqueous MeCN provided the 2,3-trans-bromohydrin derivative 11c, which upon treatment with DBU in MeCN gave the elimination product 11 and the α-2,3-anhydro derivative 12 as a suitable donor of glycosides with D-glycero-D-talo- or D-glycero-D-galacto configuration, respectively.

Crystal and molecular structure of allyl O-(sodium 3-deoxy-α- d- manno- 2-octulopyranosylonate)-(2 → 8)- O-(sodium 3-deoxy-α- d- manno- 2-octulopyranosidonate)-monohydrate

The α-(2 → 8)-linked Kdo disaccharide derivative allyl O-(sodium 3-deoxy-α- d- manno- 2-octulopyranosylonate)-(2 → 8)- O-(sodium 3-deoxy-α- d- manno-2-octulopyranosidonate)-monohydrate C 19H 28O 15Na 2·H 2O, M r = 542.32, is orthorhombic, P2 12 12 1 with a = 9.229(1), b = 12.036(1), c = 21.671(1) Å, and Z = 4. The structure was solved by direct methods and refined to R = 0.040 for 2677 observed reflections. The torsion angles about the (2 → 8)-glycosidic bond are stabilized by an intramolecular hydrogen bond between the carboxylate group at the anomeric carbon atom of the terminal Kdo residue and the hydroxyl group O-17 of the second Kdo moiety.

Synthesis of carboxyl-reduced analogues related to the Chlamydia-specific Kdo trisaccharide epitope

The disaccharides allyl O-(sodium 3-deoxy-α- d- manno-2-octulopyranosylonate)-(2 → 4)-3-deoxy-α- d- manno-2-octulopyranoside ( 8), allyl O-(3-deoxy-α- d- manno-2-octulopyranosyl)-(2 → 8)-(sodium 3-deoxy-α- d- manno-2-octulopyranosidonate) ( 24), and allyl O-(sodium 3-deoxy-α- d- manno-2-octulopyranosylonate)-(2 → 8)-3-deoxy-α- d- manno-2-octulopyranoside ( 35), and the trisaccharides allyl O-(sodium 3-deoxy-α- d- manno-2-octulopyranosylonate)-(2 → 8)-(sodium 3-deoxy-α- d- manno-2-octulopyranosylonate)-(2 → 4)-3-deoxy-α- d- manno-2-octulopyranoside ( 13) and allyl O-(3-deoxy-α- d- manno-2-octulopyranosyl)-(2 → 8)-(sodium 3-deoxy-α- d- manno-2-octulopyranosylonate)-(2 → 4)-(sodium 3-deoxy-α- d- manno-2-octulopyranosidonate) ( 30) were prepared. The ketosidic linkages were formed in good yields and high stereoselectivity by BF 3 · Et 2O-catalyzed reaction of the per- O-acetylated 3-deoxy-α- d- manno-2-octulopyranosyl fluoride derivative ( 16) with 8- O-SiBu tMe 2 derivatives 19 and 21. Coupling reactions using the Kdo monosaccharide bromide derivative 4 or the α-(2 → 8)-linked Kdo disaccharide bromide derivatives 9 and 26 were performed under Helferich conditions in MeCN or MeNO 2, respectively. The disaccharide halides were prepared in good overall yields starting from the readily available allyl β-glycoside of Kdo. The deprotected oligosaccharides correspond to the genus-specific lipopolysaccharide epitope of Chlamydia and part structures thereof, containing the carboxyl-reduced Kdo-residues at the distal and proximal position of the Kdo trisaccharide epitope, respectively.

Occurrence of antibodies against chlamydial lipopolysaccharide in human sera as measured by ELISA using an artificial glycoconjugate antigen.

An artificial glycoconjugate containing, as a ligand, the deacylated carbohydrate backbone of a recombinant Chlamydia-specific lipopolysaccharide was used as a solid-phase antigen in ELISA to measure antibodies against chlamydial LPS. The specificity and reproducibility of the assay was shown by using a panel of prototype monoclonal antibodies representing the spectrum of antibodies also occurring in patient sera. These mAbs recognized Chlamydia-specific epitopes [alpha 2-->8-linked disaccharide of 3-deoxy-D-manno-octulosonic acid (Kdo) or the trisaccharide alpha Kdo-(2-->8)-alpha Kdo-(2-->4)-alpha Kdo] or those shared between chlamydial and Re-type LPS (alpha Kdo, alpha 2-->4-linked Kdo disaccharide). The assay was used to measure IgG, IgA and IgM antibodies against chlamydial LPS in patients with genital or respiratory tract infections. In comparison to the results obtained with sera from blood donors, it became evident that both types of infection result in significant changes in the profile of LPS antibodies.

A synthetic analog of the 3-deoxy-D-manno-2-octulosonic acid disaccharide moiety of rough-type endotoxins does not bind to mouse peritoneal macrophages and human monocytes.

Strong evidence supports the concept that lipid A is the main biologically active region of endotoxins and is recognized by specific binding sites of different cell types. However, receptors for carbohydrates are also present on mononuclear phagocytes, and it has been suggested that one of these lectin-like proteins may be specific for the 3-deoxy-D-manno-2-octolosonic acid (Kdo) residues of endotoxins. To reexamine this hypothesis, we prepared a 125I-labeled conjugate consisting of a synthetic Kdo-2,4-Kdo disaccharide covalently linked to bovine serum albumin (125I-Kdo2-BSA). The Kdo disaccharide residues of this radiolabeled conjugate were fully accessible to a monoclonal antibody which reacts specifically with this epitope. However, 125I-Kdo2-BSA did not exhibit any detectable specific binding on thioglycolate-elicited mouse peritoneal macrophages or on human monocytes. Furthermore, the specific binding of biotin-labeled lipopolysaccharide derivatives to mouse macrophages and human monocytes was not inhibited by a soluble synthetic Kdo-2,4-Kdo-polyacrylamide copolymer or by a synthetic glycolipid consisting of an alpha-Kdo residue glycosidically linked to O-6 of allyl-4-O-phosphoryl-N-3-hydroxytetradecanoyl-beta-D-glucosaminide. These results indicate that binding sites specific for Kdo are not present (or not accessible) on the surface of mouse macrophages and human monocytes.