Synthesis of 3-O-(2-acetamido-2-deoxy-3-O-β-d-galactopyranosyl-β-d-galactopyranosyl)-1,2-di-O-tetradecyl-sn-glycerol

Stereo- and regio-selective synthesis of 3-O-(2-acetamido-2-deoxy-3-O-β-d- galactopyranosyl-β-d-galactopyranosyl)-1,2-di-O-tetradecyl-sn-glycerol by use of 1,2-di-O-tetradecyl-3-O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-d-galactopyranosyl)-sn-glycerol as a key intermediate is described.     

Synthese der pentasaccharid-kette des forssman-antigens

The pentasaccharide chain of the Forssman antigen, O-(2-acetamido-2-deoxy-α-d-galactopyranosyl)-(1→3)-O-(2-acetamido-2-deoxy-β-d-galactopyranosyl)-(1→3)-O-α-d- galactopyranosyl-(1→4)-O-β-d-galactopyranosyl-(1→4)-d-glucopyranose (46) was synthesized by a block synthesis in which an α-d-glycoside linkage between two d-galactose residues was formed. The trisaccharide O-(6-O-acetyl-2-azido-3,4-di-O-benzoyl-2-deoxy-α-d-galactopyranosyl)- (1→3)-O-(6-O-acetyl-4-O-benzyl-2-deoxy-2-phthalimido-β-d-galactopyranosyl)-(1→3)-6-O-acetyl-2,4-di-O-benzyl- α-d-galactopyranosyl bromide (40) (this was obtained through acetolysis of O-(6-O-acetyl-2-azido-3,4-di-O-benzoyl-2-deoxy-α-d-galactopyranosyl)- (1→3)-O-(6-O-acetyl-4-O-benzyl-2-deoxy-2-phthalimido-β-d-galactopyranosyl)-(1→3)-1,6-anhydro-2,4-di-O-benzyl-β-d- galactopyranose to the acetyl derivative, followed by reaction with titanium tetrabromide under anhydrous conditions) was condensed with benzyl-4-O-(6-O-benzoyl-2,3-di-O-benzyl-β-d-galactopyranosyl)-2,3,6- tri-O-benzyl-β-d-glucopyranoside were in the presence of silver carbonate and perchlorate. The resulting pentasaccharide was deprotected to give 46.     

Characterization of new glycolipid biosurfactants,tri-acylated mannosylerythritol lipids,produced by <Emphasis Type="Italic">Pseudozyma</Emphasis> yeasts

Mannosylerythritol lipids (MELs) are glycolipid biosurfactants produced by Pseudozyma yeasts. They show not only the excellent interfacial properties but also versatile biochemical actions. In the course of MEL production from soybean oil by P. antarctica and P. rugulosa, some new extracellular glycolipids (more hydrophobic than the previously reported di-acylated MELs) were found in the culture medium. The most hydrophobic one was identified as 1-O-alka(e)noyl-4-O-[(4′,6′-di-O-acetyl-2′,3′-di-O-alka(e)noyl)-β-d-mannopyranosyl]-d-erythritol, namely tri-acylated MEL. Others were tri-acylated MELs bearing only one acetyl group. The tri-acylated MEL could be prepared by the lipase-catalyzed esterification of a di-acylated MEL with oleic acid implying that the new glycolipids are synthesized from di-acylated MELs in the culture medium containing the residual fatty acids.     

Novel Glycerolipids and Glycolipids from the Surface Lipids of Nicotiana benthamiana

The surface lipids of Nicotiana benthamiana contained novel glycerolipids and several varieties of glycolipids. As glycerolipids, the triacylglycerol, 1,3-diacylglycerol, and 1,2-diacylglycerol types of glycerolipids were isolated and identified. Each lipid contained acetyl, 16–methylheptadecanoyl, and 18–methylnonadecanoyl moieties. The acetylated position of each lipid was determined by 2D-NMR, using the HMBC technique. The structures were 1,3-di-O-acetyl-2-O-acylglycerol, 1-O-acetyl-3-O-acylglycerol, and 1-O-acetyl-2-O-acylglycerol. As glycolipids, one glucose ester and four types of sucrose esters were isolated and identified. These glycolipids contained acetic acid and such branched short-chain fatty acids as 5-methylhexanoic, 4-methylhexanoic, 6-methylheptanoic, and 5-methylheptanoic acids. The structure of the glucose ester was 3,4-di-O-acyl-α-D-glucopyranose. The structures of the sucrose esters were 6-O-acetyl-4-O-acyl-α-D-glucopyranosyl-(3-O-acyl)-β-D-fructofuranoside, 4-O-acyl-α-D-glucopyranosyl-(3-O-acyl)-β-D-fructofuranoside, 3,4-di-O-acyl-α-D-glucopyranosyl-β-D-fructofuranoside, and 6-O-acetyl-α-D-glucopyranosyl-β-D-fructofuranoside.     

A synthesis of psi-cytidine

2-O-Benzoyl-3,6-di-O-benzyl-4-O-(chloroacetyl)-, 4-O-acetyl-2-O-benzoyl-3,6-di-O-benzyl-, and 2-O-benzoyl-3,4,6-tri-O-benzyl-α-d-galactopyranosyl chloride were converted into the corresponding 2,2,2-trifluoroethanesulfonates, and these were treated with allyl 2-O-benzoyl-3,6-di-O-benzyl-α-d-galactopyranoside, to give allyl 2-O-benzoyl-4-O-[2-O-benzoyl-3,6-di-O-benzyl-4-O-(chloroacetyl)-β-d-galactopyranosyl]-3,6-di-O-benzyl- α-d-galactopyranoside (26; 41% yield), allyl 4-O-(4-O-acetyl-2-O-benzoyl-3,6-di-O-benzyl-β-d-galactopyranosyl)-2-O-benzoyl-3,6-di-O-benzyl- α-d-galactopyranoside (27; 62% yield), and allyl 2-O-benzoyl-4-O-(2-O-benzoyl-3,4,6-tri-O-benzyl-β-d-galactopyranosyl)-3,6-di-O-benzyl-α-d-galactopyranoside (28; 65% yield). All disaccharides were free from their α anomers. Disaccharides 26 and 27 were found to be base-sensitive, and were de-esterified by KCN in aqueous ethanol, and debenzylated with H₂-Pd. Attempts to produce (1→4)-β-d-galactopyranosides from the coupling of a number of fully esterified d-galactopyranosyl sulfonates to allyl 2,3,6-tri-O-benzoyl-α-d-galactopyranoside were unsuccessful.     

Synthesis and characterization of propyl O-β-d-galactopyranosyl-(1→4)-O-β-d-galactopyranosyl-(1→4)-α-d-galactopyranoside

Allyl 4-O-(4-O-acetyl-2-O-benzoyl-3,6-di-O-benzyl-β-d-galactopyranosyl)-2-O-benzoyl-3,6-di-O-benzyl-α-d- galactopyranoside was O-deallylated to give the 1-hydroxy derivative, and this was converted into the corresponding 1-O-(N-phenylcarbamoyl) derivative, treatment of which with dry HCl produced the α-d-galactopyranosyl chloride. This was converted into the corresponding 2,2,2-trifluoroethanesulfonate, which was coupled to allyl 2-O-benzoyl-3,6-di-O-benzyl-α-d-galactopyranoside, to give crystalline allyl 4-O-[4-O-(4-O-acetyl-2-O-benzoyl-3,6-di-O-benzyl-β-d-galactopyranosyl)-2-O-benzoyl-3,6-di- O-benzyl-β-d-galactopyranosyl]-2-O-benzoyl-3,6-di-O-benzyl-α-d-galactopyranoside (15) in 85% yield, no trace of the α anomer being found. The trisaccharide derivative 15 was de-esterified with 2% KCN in 95% ethanol, and the product O-debenzylated with H₂-Pd, to give the unprotected trisaccharide. Alternative sequences are discussed.     

Abundance and Borrelia burgdorferi-infection Prevalence of Nymphal Ixodes scapularis Ticks along Forest–Field Edges

More than 19,000 human cases of Lyme disease (LD) are reported each year in the United States. Lyme disease cases occur when humans are exposed to the bacterium Borrelia burgdorferi through the bite of an infected ixodid tick. The probability of human exposure to infected ticks results from a combination of human behaviors and entomological risk. Human behaviors include use of tick habitats, use of protective clothing, and grooming for tick removal. Entomological risks include the density of ticks in a habitat and the proportion of these that are infected with B. burgdorferi. Recent studies have suggested that humans are at higher risk of exposure to B. burgdorferi near edges between forests and herbaceous communities, including lawns and old fields, but whether this increased risk is a function of human behaviors, entomological risk, or both, is unknown. We assessed entomological risk across forest–old field edges in Dutchess County, NY. Densities of ticks and of infected ticks were considerably higher within forests than at forest–field edges, and were lowest within fields. Thus, edges between forests and fields do not pose a higher entomological risk than do the forests themselves, although risk at the edge is higher than in herbaceous habitat. Landscapes with abundant edges between forested and herbaceous habitat, and roughly even proportions of both, might attract both heavy human use and pose moderately high entomological risk, and thus could be targeted for mitigation. We suggest that determining appropriate methods for reducing human exposure to LD requires differentiating entomological risk from human behaviors.     

Synthesis ofNeoglycoproteins containing the 3,6-di-O-methyl-β-d-glucopyranosyl epitope and their use in serodiagnosis of leprosy

A stratagem for the synthesis ofneoglycoproteins suitable for the selective serodiagnosis of leprosy is described in which synthetic 3,6-di-O-methyl--d-glucopyranose, the epitope of phenolic glycolipid I fromMycobacterium leprae, was used. Condensation of 8-methoxycarbonyloctanol with the acetobromo derivative of 3,6-di-O-methylglucose gave 8-methoxycarbonyloctyl 2,4-di-O-acetyl-3,6-di-O-methyl--d-glucopyranoside in 65% yield, and with absolute stereospecificity for the anomer. The deacylated product was converted to the crystalline hydrazide and coupled to bovine gamma globulin, bovine serum albumin and poly-d-lysinevia intermediate acyl azide formation to produce the 8-carbonyloctyl 3,6-di-O-methyl--d-glucopyranosyl polypeptides. Theneoglycoproteins were highly sensitive in ELISA and emulated the specificity of the native glycolipid in analysis of sera from patients throughout the spectrum of leprosy and from different geographical regions. The 8-carbonyloctyl 3,6-di-O-methyl--d-glucopyranoside-bovine serum albumin was also synthesized and shown to have about one-half the activity of the -linkedneoglycoprotein. A different synthetic approach produced the 8-carbonyloctyl 4-O-(3,6-di-O-methyl--d-glucopyranosyl)--l-rhamnopyranoside-bovine serum albumin which was also highly sensitive and specific for the serodiagnosis of leprosy. The presence of the second sugar unit, similar to that in the native glycolipid but for the absence ofO-methyl groups, seemed to provide a probe with greater felicity for the serological detection of tuberculoid leprosy.Thus, the results indicate that highly sensitive and specific antigen probes for the serodiagnosis of leprosy can be constructed based only on the terminal one or two sugars of phenolic glycolipid I, and the synthetic approach leads to the formation of haptens with absolute stereospecificity.Nomenclature BGG bovine gamma globulin - PGL-I phenolic glycolipid I - PDL poly-d-lysine - PBS phophate-buffered saline - 3,6-Me₂-Glc-Link-BSA 8-carbonyloctyl 3,6-di-O-methyl-glucopyranoside-bovine senalbumin - 3,6-Me₂-Glc-Rha-Link-BSA 8-carbonyloctyl 4-O-(3,6-di-O-methyl--d-glucopyranosyl)--l-rhan pyranoside-BSA     

Glycolipids Isolated from Spirulina maxima: Structure and Fatty Acid Composition

Several glycolipids were isolated from Spirulina maxima, an edible blue-green algae, by systematic fractionation with different solvents. Structural investigation by using methylation, GC-MS, and enzymic techniques indicated that the major glycolipids are O-β-d-galactosyl-(1→l′)-2′, 3′-di-O-acyl-d-glycerol, O-α-d-galactosyl-(l-→6)-O-β-d-galactosyl-(1→l′)-2′,3′-di-O-acyl-d-glycerol and 6-sulfo-O-α-quinovosyl-(l→l′)-2′, 3′-di-O-acyl-d-glycerol. Main fatty acid components of these glycolipids were identified as palmitic acid and linoleic or linolenic acid. Based on-these fatty acid compositions, Spirulina glycolipids were compared with those in higher plants.     

Simple Method to Determine the Absolute Configuration of the Glycerol Moiety in Glycosyl Glycerols Based on ORD and CD

A general method to determine the absolute configuration of the glycerol moiety in glycopyranosyl glycerols is presented, which involves per-O-benzylation and acid hydrolysis of the glycosyl glycerol to give optically active 1,2- or 2,3-di-O-benzylated sn-glycerol (III). ORD and CD measurements of III and its benzoylated derivatives gave intensive optical rotations or Cotton effects to determine the absolute configuration at C2.     

Regioselective synthesis of a glycomimetic trisaccharide of Sialyl Lewis (sLe)

Sialyl Lewis (sLe^x) is the smallest naturally occurring carbohydrate ligand that binds to E-Selectin on the activated endothelium. We report here the total synthesis of acetic acid-sLe^x analog (12), for testing as a therapeutic agent. Methoxyethyl 4-O-(3,4-O-isopropylidene-β-d-galactopyranosyl)-β-d-glucopyranoside (3) was prepared starting from the methoxyethyl-β-d-lactoside (2), which was selectively benzoylated to give the methoxyethyl 2,6-di-O-benzoyl-4-O-(2,6-di-O-benzoyl-3,4-O-isopropylidene-β-d-galactopyranosyl)-β-d-glucopyranoside (4). Glycosylation of acceptor 4 with methyl 2,3,4-tri-O-benzyl-1-thio-β-l-fucopyranoside (5) in the presence of cupric bromide and tetrabutylammonium bromide afforded the corresponding methoxyethyl 2,6-di-O-benzyl-3-O-(2,3,4-tri-O-benzyl-α-l-fucopyranosyl)-4-O-(2,6-di-O-benzyl-3,4-O-isopropylidene-β-d-galactopyranosyl)-β-d-glucopyranoside (6). Selective removal of the 4″,6″-O-isopropylidene group from 6 gave the deprotected trisaccharide 7. The regioselective esterification of O-3″ of trisaccharide 8 (obtained from the dibutylstannylene derivative of 7) with benzyl-2-bromoacetate and tetrabutylammonium bromide afforded the 3″-O-carbobenzyloxymethyl trisaccharide derivative 9, which on saponification and hydrogenolysis with palladium-charcoal afforded the target trisaccharide 12 glycomimetic of Sialyl Lewis (sLe^x) trisaccharide omitting the sialic acid moiety.     

Synthesis of a novel pentasaccharide core component from the lipooligosaccharide of Moraxella catarrhalis

The novel pentasaccharide [p-(trifluoroacetamido)phenyl]ethyl 3-O-β-d-glucopyranosyl-4-O-β-d-glucopyranosyl-6-O-[2-O-(α-d-glucopyranosyl)-β-d-glucopyranosyl]-α-d-glucopyranoside (1), which includes a linker moiety to enable facile coupling to an antigenic protein, was synthesised as a component of a potential vaccine candidate against the Gram-negative bacterium Moraxella catarrhalis. This microorganism is one of three principal causative agents of otitis media in children. The pentasaccharide represents a common cross-serotype (A, B and C) structure from the lipooligosaccharides of Moraxella catarrhalis.     

Production and structure elucidation of di- and oligosaccharide lipids (biosurfactants) from Tsukamurella sp. nov.

The bacterium Tsukamurella sp. nov., isolated from soil, was found to produce novel glycolipids when grown on sunflower oil as the sole carbon source. The glycolipids were isolated by chromatography on silica columns and their structures elucidated using a combination of multidimensional NMR and MS techniques. The three main components are 2,3-di-O-acyl-α-d-glucopyranosyl-(1-1)-α-d-glucopyranose, 2,3-di-O-acyl-β-d-glucopyranosyl-(1-2)-4,6-di-O-acyl-α-d-glucopyranosyl-(1-1)-α-d-glucopyranose and 2,3-di-O-acyl-β-d-glucopyranosyl-(1-2)-β-d-galactopyranosyl-(1-6)-4,6-di-O-acyl-α-d-glucopyranosyl-(1-1)-α-d-glucopyranosl which are linked to fatty acids varying in chain length from C₄ to C₁₈. The glycolipids are mainly extracellular but are also found attached to the cell walls. During the cultivation the composition of the glycolipids changed from disaccharide- to tri- and tetrasaccharide lipids. The glycolipids show good surface-active behaviour and have antimicrobial properties. Received: 22 May 1998 / Received revision: 24 August 1998 / Accepted: 26 August 1998     

Unusual properties of glycuronans [poly(glycosyluronic) compounds]

2-Methyl-[3,6-di-O-acetyl-2-deoxy-4-O-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)-α-d-glucopyrano]-[2,1-d]-2-oxazoline (4) was prepared from 2-acetamido-3,6-di-O-acetyl-2-deoxy-4-O-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)-α-d- glucopyranosyl chloride. Condensation of 3,4:5,6-di-O-isopropylidene-d-mannose dimethyl acetal with 4 in the presence of a catalytic amount of p-toluenesulfonic acid afforded O-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)-(1 → 4)-O-(2-acetamido-3,6-di-O-acetyl-2-deoxy-β-d-glucopyranosyl)-(1 → 2)-3,4:5,6-di-O-isopropylidene-d-mannose dimethyl acetal (6) in 8.6% yield. Catalytic deacetylation of 6 with sodium methoxide, followed by hydrolysis with dilute sulfuric acid, gave O-β-d-galactopyranosyl-(1 → 4)-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-(1 → 2)-d-mannose (7). The inhibitory activities of 7 and related sugars against the hemagglutinating activities of various lectins were assayed, and 7 was found to be a good inhibitor against Phaseolus vulgaris hemagglutinin.     

Lyme disease and current aspects of immunization

Lyme disease is a tick-borne multisystem disease that affects primarily the skin, nervous system, heart and joints. At least three species of Borrelia burgdorferi sensu lato, namely Borrelia burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii, can cause the disease. This review will focus mainly on the pathophysiology of Lyme arthritis, the long-term outcome of Lyme disease, and the recently licensed vaccine against Lyme disease.     

A new steroidal saponin from Allium ampeloprasum var. porrum with antiinflammatory and gastroprotective effects

A new steroidal saponin was isolated from the bulbs of Allium ampeloprasum var. porrum. On the basis of chemical conversions and detailed analyses of ¹H and ¹³C NMR spectra including 2D NMR spectroscopic techniques, its structure was established as 3-[(O-β-d-glucopyranosyl-(1 → 3)-β-d-glucopyranosyl-(1 → 2)-O-[O-β-d-glucopyranosyl-(1 → 3)]-O-β-d-glucopyranosyl-(1 → 4)-β-d-galactopyranosyl)oxy]-2,6-dihydroxy-(2α,3β,5α,6β,25R)-spirostane. Results of the present study indicated that the steroidal saponin showed haemolytic effects in the in vitro assays and demonstrated antiinflammatory activity and gastroprotective property using in vivo models.     

Detection of glycoproteins in Borrelia burgdorferi

The presence of carbohydrates on proteins of Borrelia burgdorferi, the causative agent of Lyme disease, was investigated by using a digoxigenin labeling method together with Schiff staining and N-glycosidase F assay. The two major outer surface exposed proteins of 31 kDa and 34 kDa showed to be glycosylated and gel filtration high pressure liquid chromatography (HPLC) of proteins of B. burgdorferi metabolically labeled with ¹⁴C-N-acetylglucosamine revealed the incorporation of the carbohydrate into the glycosyl residue of these proteins.Abbreviations N-glycosidase F peptide-N-glycosidase F (EC 3.5.1.52) - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - WB Western blotting - HPLC high pressure liquid chromatography - SDS sodium dodecyl sulphate - mAb monoclonal antibody - MIAF mouse immune ascitic fluid - SS Schiff staining - Osp Outer surface protein     

Synthesis of Glycoglycerolipids: 3-O-Mannooligosyl-l,2-di-O-tetradecyl-sn-glycerol

Synthetic routes for the following mannooligosylglycerolipids of biological interest were developed by using regioselectively protected monosaccharide synthons and l,2-di-O-alkyl-sn-glycerol; 3-O-(2-O-α-D-mannopyranosyl-α-D-mannopyranosyl)-l,2-di-O-tetradecyl-sn-glycerol; 3-O-[2-O-(2-O-α-D-mannopyranosyl-α-D-mannopyranosyl)-α-D-mannopyranosyl]-l,2-di-O-tetradecyl-sn-glycerol; 3-O-(6-O-α-D-mannopyranosyl-α-D-mannopyranosyl)-l,2-di-O-tetradecyl-sn-glycerol; and 3-O-(3,6-di-O-α-D-mannopyranosyl-α-D-mannopyranosyl)-1,2-di-α-tetradecyl-sn-glycerol.     

Synthesis of Neu5Ac- and Neu5Gc-α-(2→6′)-lactosamine 3-aminopropyl glycosides

In order to prepare 3-aminopropyl glycosides of Neu5Ac-α-(2→6′)-lactosamine trisaccharide 1, and its N-glycolyl containing analogue Neu5Gc-α-(2→6′)-lactosamine 2, a series of lactosamine acceptors with two, three, and four free OH groups in the galactose residue was studied in glycosylations with a conventional sialyl donor phenyl [methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio- -glycero-α- and β- -galacto-2-nonulopyranosid]onates (3) and a new donor phenyl [methyl 4,7,8,9-tetra-O-acetyl-5-(N-tert-butoxycarbonylacetamido)-3,5-dideoxy-2-thio- -glycero-α- and β- -galacto-2-nonulopyranosid]onates (4), respectively. The lactosamine 4′,6′-diol acceptor was found to be the most efficient in glycosylation with both 3 and 4, while imide-type donor 4 gave slightly higher yields with all acceptors, and isolation of the reaction products was more convenient. In the trisaccharides, obtained by glycosylation with donor 4, the 5-(N-tert-butoxycarbonylacetamido) moiety in the neuraminic acid could be efficiently transformed into the desired N-glycolyl fragment, indicating that such protected oligosaccharide derivatives are valuable precursors of sialo-oligosaccharides containing N-modified analogues of Neu5Ac.     

H9724, a Monoclonal Antibody to Borrelia burgdorferi's Flagellin, Binds to Heat Shock Protein 60 (HSP60) Within Live Neuroblastoma Cells: A Potential Role for HSP60 in Peptide Hormone Signaling and in an Autoimmune Pathogenesis of the Neuropathy of Lyme Disease

Although Borrelia burgdorferi, the causative agent of Lyme disease, is found at the site of many disease manifestations, local infection may not explain all its features. B. burgdorferi's flagellin cross-reacts with a component of human peripheral nerve axon, previously identified as heat shock protein 60 (HSP60). The cross-reacting epitopes are bound by a monoclonal antibody to B. burgdorferi's flagellin, H9724. Addition of H9724 to neuroblastoma cell cultures blocks in vitro spontaneous and peptide growth-factor–stimulated neuritogenesis. Withdrawal of H9724 allows return to normal growth and differentiation. Using electron microscopy, immunoprecipitation and immunoblotting, and FACS analysis we sought to identify the site of binding of H9724, with the starting hypotheses that the binding was intracellular and not identical to the binding site of II-13, a monoclonal anti-HSP60 antibody. The current studies show that H9724 binds to an intracellular target in cultured cells with negligible, if any, surface binding. We previously showed that sera from patients with neurological manifestations of Lyme disease bound to human axons in a pattern identical to H9724's binding; these same sera also bind to an intracellular neuroblastoma cell target. II-13 binds to a different HSP60 epitope than H9724; II-13 does not modify cellular function in vitro. As predicted, II-13 bound to mitochondria, in a pattern of cellular binding very different from H9724, which bound in a scattered cytoplasmic, nonorganelle-related pattern. H9724's effect is the first evidence that HSP60 may play a role in peptide-hormone–receptor function and demonstrates the modulatory potential of a monoclonal antibody on living cells.