A simple one-pot method for the synthesis of partially protected mono- and disaccharide building blocks using an orthoesterification-benzylation-orthoester rearrangement approach

A simple one-pot method is reported for making partially protected glycosyl acceptors from O-methyl or S-alkyl/aryl glycosides of D-glucose, D-galactose, D-arabinose, L-rhamnose, L-fucose and lactose via orthoester formation, benzylation and selective hydrolysis.     

Synthesis of Lewis X trisaccharide analogues in which glucose and rhamnose replace N-acetylglucosamine and fucose,respectively

Two analogues of the Le(x) trisaccharide, alpha-L-Fucp-(1-->3)-[beta-D-Galp-(1-->4)]-D-Glcp were synthesized as allyl glycosides. In these derivatives either only the N-acetylglucosamine is replaced by glucose or both the N-acetylglucosamine and the fucosyl residue are replaced by glucose and rhamnose, respectively. Our synthetic scheme used armed beta-thiophenyl fuco- and rhamnoside glycosyl donors that were prepared anomerically pure from the corresponding alpha-glycosyl bromides. The protecting groups were chosen to allow access to the fully deprotected trisaccharides without reduction of the allyl glycosidic group. These analogues will be used as soluble antigens in binding experiments with anti-Le(x) antibodies and can also be conjugated to a carrier protein and used as immunogens. In the course of this synthetic work, we also describe the use of reversed-phase HPLC to purify key protected trisaccharide intermediates prior to their deprotection.     

The 13C-N.M.R. spectra of disaccharides of D-glucose, D-galactose, and L-rhamnose as models for immunological polysaccharides.

Complete assignments of the 13C-n.m.r. spectra of disaccharides having beta-glycosidic linkages are presented and discussed. The disaccharides of D-glucose, D-galactose, L-rhamnose, 2-acetamido-2-deoxy-D-glucose, and 2-acetamido-2-deoxy-D-galactose are model compounds for 13C-n.m.r. studies of immunological polysaccharides. Changing the nature of the reducing glucopyranose rings (D-glucose to L-rhamnose) has no important influence on the chemical shifts of the carbons of the non-reducing glucopyranose ring (D-glucose). The converse is also true: the chemical shifts of the carbons of the reducing glucopyranose ring (L-rhamnose) are not noticeably affected by a change of the non-reducing unit (D-glucose to D-galactose or 2-acetamido-2-deoxy-D-glucose).     

First total synthesis of alpha-(2-->3)/alpha-(2-->6)-disialyl lactotetraosyl ceramide and disialyl Lewis A ganglioside as cancer-associated carbohydrate antigens

The first total synthesis of alpha-(2-->3)/alpha-(2-->6)-disialyl lactotetraosyl (DSLc4) ceramide and alpha-(2-->3)/alpha-(2-->6)-disialyl Lewis A (DSLe(a)) ganglioside as cancer-associated antigens is described. The suitably protected lactotriose (Lc3) derivatives were successively glycosylated with sialic acid, sialyl-alpha-(2-->3)-D-galactose and/or L-fucose donors in a regio- and stereo-selective manner, to give the protected type I hexa- and hepta-saccharides, respectively, which were then converted to the target gangliosides by the introduction of ceramide and subsequent complete deprotection.     

Capsular polysaccharides and lipopolysaccharides from two Bacteroides fragilis reference strains: chemical and immunochemical characterization.

Fermentor growth of Bacteroides fragilis under controlled conditions in a complex medium containing 1% glucose and 10% fetal calf serum resulted in high yields of bacteria. After hot phenol-water extraction of the organisms, capsular polysaccharide was isolated from the aqueous phase and purified by Sephacryl S-300 chromatography in a buffer with 3% sodium deoxycholate. Lipopolysaccharide was isolated by phenol-chloroform-light petroleum ether extraction. The capsular polysaccharide from B. fragilis strain NCTC 9343 contained six sugars: L-fucose, D-galactose, D- and L-quinovosamine, D-glucosamine, and galacturonic acid. The capsule of strain ATCC 23745 also contained D-glucose, L-fucosamine, L-rhamnosamine, and a 3-amino-3,6-dideoxyhexose but lacked D-quinovosamine. The latter capsule also contained alanine (4%). The capsular polysaccharides were different immunochemically by ELISA inhibition. The lipopolysaccharide of both strains contained the same sugars (L-rhamnose, D-glucose, D-galactose, and D-glucosamine) and fatty acids (13-methyl-tetradecanoic and 3-hydroxy-hexadecanoic and 3-hydroxy-15 methyl-hexadecanoic as major constituents) and were identical by ELISA inhibition.     

Synthesis of Lewis X and three Lewis X trisaccharide analogues in which glucose and rhamnose replace N-acetylglucosamine and fucose, respectively

Three analogues of the Le(x) trisaccharide: alpha-L-Fucp-(1-->3)-[beta-D-Galp-(1-->4)]-D-GlcNAcp as well as the Le(x) trisaccharide itself were synthesized as methyl glycosides. In the analogues, either only the fucose residue is replaced by rhamnose or both the N-acetylglucosamine and the fucosyl residues are replaced by glucose and rhamnose, respectively. Our synthetic strategy relied on the use of lactoside and 2-azido lactoside derivatives as disaccharide acceptors, which were submitted to either fucosylation or rhamnosylation. Our results confirm that the reactivity of lactose in protection and glycosylation reactions is greatly affected by (1) the structure of the aglycone and (2) the presence of an azido substituent at C-2 of the glucose moiety. Thus, a methyl lactoside acceptor was easily glycosylated at O-3 with perbenzylated beta-thiophenyl fucoside and rhamnoside to give anomerically pure alpha-fucosylated and alpha-rhamnosylated trisaccharides, respectively. In contrast, the same reactions on a 2-azido methyl lactoside acceptor led to the formation of anomeric mixtures. While the alpha- and beta-fucosylated 2-azido trisaccharides could be separated by RP-HPLC, such separation was not possible for the rhamnosylated anomers. The desired rhamnosylated trisaccharide was finally obtained anomerically pure using an isopropylidene-protected rhamnosyl donor. The deprotection sequences also showed that the presence of a 2-azido substituent at C-2 of the glucose residue conferred stability to the vicinal fucosidic linkage at C-3. To test their relative affinity for anti-Le(x) Abs the Le(x) analogues will be used as competitive inhibitors against methyl Le(x). In addition, their conformational behavior will be studied by NMR spectroscopy and molecular modeling experiments.     

Structural studies on the specific type VII pneumococcal polysaccharide

The specific type VII pneumococcal polysaccharide was isolated from the crude capsular material by precipitative and chromatographic methods. It contained D-galactose, D-glucose, L-rhamnose, 2-acetamido-2-deoxy-D-glucose, and 2-acetamido-2-deoxy-D-galactose in the molar ratio of 3.5:2.3:3.0:2.1:1.0. Some of its structural features were revealed by methylation studies, time-lapse hydrolysis, periodate oxidation, and enzymic hydrolysis. The polysaccharide is branched at residues of D-galactose and 2-acetamido-2-deoxy-D-galactose. Non-reducing end groups consisted of D-galactopyranose and 2-acetamido-2-deoxy-D-glucopyranose residues, with the former predominating. Major components of the linear chains were (1→3)-linked L-rhamnose and (1→4)-linked D-glucose; the minor ones were (1→2)-linked L-rhamnose, (1→6)-linked D-galactose, and (1→6)-linked 2-acetamido-2-deoxy-D-glucopyranose. The (1→4)-linked D-glucose components may be present as cellobiose residues. The results are in accord with structural features deduced from the serological cross-reactivity of this polysaccharide.     

Homologous sugar transport proteins in Escherichia coli and their relatives in both prokaryotes and eukaryotes

Separate proteins for proton-linked transport of D-xylose, L-arabinose, D-galactose, L-rhamnose and L-fucose into Escherichia coli are being studied. By cloning and sequencing the appropriate genes, the amino acid sequences of proteins for D-xylose/H+ symport (XylE), L-arabinose/H+ symport (AraE), and part of the protein for D-galactose/H+ symport (GalP) have been determined. These are homologous, with at least 28% identical amino acid residues conserved in the aligned sequences, although their primary sequences are not similar to those of other E. coli transport proteins for lactose, melibiose, or D-glucose. However, they are equally homologous to the passive D-glucose transport proteins from yeast, rat brain, rat adipocytes, human erythrocytes, human liver, and a human hepatoma cell line. The substrate specificity of GalP from E. coli is similar to that of the mammalian glucose transporters. Furthermore, the activities of GalP, AraE and the mammalian glucose transporters are all inhibited by cytochalasin B and N-ethylmaleimide. Conserved residues in the aligned sequences of the bacterial and mammalian transporters are identified, and the possible roles of some in sugar binding, cation binding, cytochalasin binding, and reaction with N-ethylmaleimide are discussed. Each protein is independently predicted to form 12 hydrophobic, membrane-spanning alpha-helices with a central hydrophilic segment, also comprised of alpha-helix. This unifying structural model of the sugar transporters shares features with other ion-linked transport proteins for citrate or tetracycline.     

Human salivary fucosyltransferases: Evidence for two distinct α-3-L-fucosyltransferase activities one of which is associated with the lewis blood group Le gene

The occurrence of GDP-L-fucose:N-acetyl-β-D-glucosaminyl α-3-L-fucosyltransferase activity in human saliva was independent of Lewis blood group and ABH secretor status except insofar as the mean level of activity was higher in saliva from individuals with an $\text{Le}$ gene than in those whose red cells and saliva grouped as Le(a-b-). In contrast GDP-L-fucose:D-glucose α-3-L-fucosyltransferase activity was detectable in saliva from all Le(a+b-) and Le(a-b+) individuals but was absent from the salivas of Le(a-b-) donors. Isoelectric focusing experiments supported the inference that there are two distinct α-3-L-fucosyltransferase activities in saliva. Both enzymes appear to catalyse the transfer of L-fucose to the C-3 position of N-acetyl-β-D-glucosamine but only the transferase dependent upon the expression of the $\text{Le}$ gene has the capacity to transfer L-fucose to the C-3 position of D-glucose.     

Monomeric and Multimeric Blockers of Selectins: Comparison of <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> Activity

The potency of the oligosaccharides SiaLe(x), SiaLe(a), HSO(3)Le(x), and HSO(3)Le(a), their conjugates with polyacrylamide (PAA, 40 kD), and other monomeric and polymeric selectin inhibitors has been compared with that of the polysaccharide fucoidan. The following assay systems were used: 1) a 96-well assay based either on the use of recombinant E-, P-, and L-selectins or an analogous assay with natural P-selectin isolated from human platelets; 2) a platelet-based P-selectin cell assay; and 3) a rat model of peritoneal inflammation. IC(50) values for the neoglycoconjugate SiaLe(a)-PAA were 6, 40, and 85 microM for recombinant E-, P-, and L-selectins, respectively; all monomeric inhibitors were about two orders of magnitude weaker. PAA-conjugates, containing as a ligand tyrosine-O-sulfate (sTyr) in addition to one of the sialylated oligosaccharides, were the most potent synthetic blockers in vitro. Compared with fucoidan, the most potent known P- and L-selectin blocker, the bi-ligand glycoconjugate HSO(3)Le(a)-PAA-sTyr displayed similar inhibitory activity in vitro towards L-selectin and about ten times lower activity towards P-selectin. All of the tested synthetic polymers displayed a similar ability to inhibit neutrophil extravasation in the peritonitis model (in vivo) at 10 mg/kg. The data provide evidence that monomeric SiaLe(x) is considerably more effective as a selectin blocker in vivo than in vitro, whereas the opposite is true for fucoidan and the bi-ligand neoglycoconjugate HSO(3)Le(a)-PAA-sTyr.     

Acceptor specificity of cyclodextrin glycosyltransferase from Bacillus stearothermophilus and synthesis of alpha-D-glucosyl O-beta-D-galactosyl-(1----4)-beta-D-glucoside.

Bacillus stearothermophilus CGTase had a wider acceptor specificity than Bacillus macerans CGTase did and produced large amounts of transfer products of various acceptors such as D-galactose, D-mannose, D-fructose, D- and L-arabinose, D- and L-fucose, L-rhamnose, D-glucosamine, and lactose, which were inefficient acceptors for B. macerans CGTase. The main component of the smallest transfer products of lactose was assumed to be alpha-D-glucosyl O-beta-D-galactosyl-(1----4)-beta-D-glucoside.     

三齿草藤(Vicia bungei Ohwi)凝集素糖组分的高效液相色谱分析

本文报道经亲和层析纯化的三齿草藤凝集素(VBL)的糖含量和糖组分的测定结果。经酚-硫酸法测得VBL的总糖含量为4.7％。应用高效液相色谱法对一系列已知标准单糖的定性定量分析条件进行了探索,选用乙腈-水-甲醇=60:30:5体系作流动相,YWG-NH_2作固定相,在高效液相色谱仪中测出VBL含有核糖和鼠李糖,二者摩尔数之比为9.4:1。     

Induction and catabolite repression of L-rhamnose dehydrogenase in Pullularia pullulans.

The growth of Pullularia pullulans on L-rhamnose (6-deoxy-L-mannose) as the sole carbon source induces the synthesis of L-rhamnose dehydrogenase, a nicotinamide adenine dinucleotide-dependent enzyme that catalyzes the oxidation of the deoxy sugar to L-rhamnonolactone. The enzyme induction is inhibited by cycloheximide, suggesting de novo synthesis. The presence of d-glucose (0.2%) or D-galactose (0.2%) simultaneously with the inducer in the induction medium produced 50% repression of dehydrogenase synthesis, but no effect was detected with D-fructose and D-mannose at the same concentration. High levels of D-glucose (2%), under maximal catabolite repression conditions, produced a complete inhibition of enzyme synthesis.     

Partial characterization of Pseudomonas phage 2 receptor

The lipopolysacharide from Pseudomonas aeruginosa strain BI contains the receptors for phage 2 and strongly inactivates this phage in vitro (95-98% within 15 min). Several mono- and di-saccharides tested reduced phage 2 inactivation to 50% when present at the following concentrations: D-glucosamine, 0.25 M; maltose, 0.3M; lactose and cellobiose, 0.5 M; D-glucose, L-rhamnose, D-mannose, 2-deoxy-D-glucose, and sucrose, 1.0 M; D-galactose, D-xylose, and N-acetyl-D-glucosamine, 1.4 M; and melibiose. greater than 1.6 M. These results suggest the possibility that phage 2 receptors in lipopolysaccharide contain L-rhamnose, D-glucosamine, and (or) D-glucose, or a structurally related molecule. Either one of the latter two could be located at a terminal position alpha-linked to the adjacent residue, or located internally in the polysaccharide chain linked through its C-4 position.     

Neutral-sugar transport by rat liver lysosomes.

Transport of D-glucose was studied in Percoll-gradient-purified rat liver lysosomes. D-Glucose uptake had a Km of 22 mM and a t1/2 of approx. 30 s. D-Fucose, 2-deoxyglucose and methyl alpha-glucoside were the most effective competitors for uptake of D-glucose, although D-galactose, D-mannose, D-xylose and L-fucose also appeared to compete for uptake. L-Glucose was a poor competitor for uptake. No competition was observed with N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, D-glucuronic acid, N-acetylneuraminic acid, D-glucosamine or the amino acids L-glycine, L-lysine and L-proline. Uptake was unaffected by N-ethylmaleimide, dithiothreitol, KCl, NaCl, ATP/Mg or alteration of buffer pH. D-Glucose efflux from lysosomes was temperature-dependent, with a Q10 of 2.3, and was inhibited by cytochalasin B. Counter-transport could not be demonstrated. In contrast, L-fucose uptake had a Km of 65 mM and was largely unaffected by 5 M excess of neutral D-sugars. Both uptake and efflux of L-fucose were inhibited by cytochalasin B. It appears that lysosomes possess a facilitated transport system for D-glucose and perhaps other neutral D-sugars that is discrete from transport systems for acetylated and acidic sugars.     

Pentose metabolism in Mycobacterium smegmatis: specificity of induction of pentose isomerases.

The induction of D-xylose, D-ribose, L-arabinose, and D-lyxose isomerases by various sugars was studied to determine the configuration necessary for induction. D-Xylose isomerase was only induced by D-xylose, whereas D-ribose isomerase was induced by D-ribose, L-rhamnose, and L-lyxose. L-arabinose isomerase was induced by L-arabinose, D-galactose, L-arabitol, D-fucose, and dulcitol, whereas D-lyxose isomerase was induced by D-lyxose, D-mannose, D-ribose, dulcitol, and myoinositol. Some compounds such as dulcitol, D-galactose, and D- or L-fucose which do not support growth are still able to serve as inducers for various pentose isomerases.     

1H-nuclear magnetic resonance spectroscopy of reducing-residue anomeric protons of pertrifluoroacetylated carbohydrates

Eight monosaccharides (L-arabinoside, L-fucose, D-galactose, D-glucose, D-lyxose, D-mannose, L-rhamnose, and D-xylose), eight disaccharides (cellobiose, gentiobiose, isomaltose, lactose, maltose, nigerose, sophorose, and xylobiose), and three trisaccharides (isomaltotriose, maltotriose, and xylotriose) were derivatized with N-methylbis-(trifluoroacetamide) in pyridine solution to form trifluoroacetylated derivatives. These were analyzed by 1H-n.m.r. spectroscopy to determine the characteristics of the spectra and distributions of the reaction products. Peaks corresponding to reducing-residue anomeric protons were located significantly downfield of all others, and were in general 0.4 p.p.m. or more downfield of equivalent signals from the same carbohydrates when they were free or derivatized with other groups. Neither the location of anomeric proton peaks relative to each other nor the degree of spin-spin coupling between H-1 and H-2 varied greatly with type of derivatization. Spin-spin coupling, however, decreased for some beta-pyranose forms of xylobiose and the three trisaccharides. In all examples except some where H-2 was oriented equatorially to a pyranose ring, the proportion of the alpha-pyranose was either enhanced or not changed in concentration by trifluoroacetylation.     

L-rhamnose and L-fucose suppress cancer growth in mice

It is documented that deficient fucosylation may play an important role in the pathogenesis of cancer. Since the supplementation of L-fucose could restore fucosylation in both in vitro and in vivo conditions, our intent was to examine the effect of intraperitoneal administration of L-fucose and L-rhamnose (a similar deoxysaccharide) on tumour growth, mitotic activity and metastatic setting of a solid form of Ehrlich carcinoma as well as on the survival rate of tumour bearing mice. Both L-fucose and L-rhamnose exerted a significant suppressive effect on tumour growth (P<0.05). After 10 days of therapy, the greatest inhibition of tumour growth expressed as a percentage of controls was observed in L-rhamnose at a dose of 3 g/kg/day (by 62%) and L-fucose at a dose of 5 g/kg/day (by 47%). Moreover, the mitotic index decreased with increasing doses of L-fucose and L-rhamnose. Prolonged survival of tumour bearing mice was observed after 14 consecutive days of daily administering L-rhamnose. Its optimal dose was estimated to be 3.64 g/kg/day. L-Fucose, however, displayed only a slight effect on the survival of the mice. Our results suggest that L-fucose and especially L-rhamnose have anticancer potential. This study is the first to demonstrate the tumour-inhibitory effect of L-rhamnose.     

Chemical synthesis of disaccharides of the specific phenolic glycolipid antigens from Mycobacterium leprae and of related sugars

O-(3,6-Di-O-methyl-beta-D-glucopyranosyl)-(1----4)-2,3,-di-O-methyl-L -rhamnopyranose, which is the nonreducing disaccharide of the haptenic trisaccharide of the Mycobacterium leprae-specific, phenolic glycolipid I, O-(6-O-methyl-beta-D-glucopyranosyl)-(1----4)-2,3-di-O-methyl-L-rhamn opyranose, the nonreducing end of the specific, phenolic glycolipid III, and the nonhaptenic O-beta-(D-glucopyranosyl)-(1----4)-2,3-di-O-methyl-L-rhamnopyranose++ +, were synthesized in relatively good yield from 3-O-methyl-D-glucose, or D-glucose, and L-rhamnose via Koenigs-Knorr reactions. These disaccharides can be used as precursors in the synthesis of the trisaccharide unit of phenolic glycolipid I and of neoglycoconjugates suitable for the serodiagnosis of leprosy.     

Synthesis of the polysaccharide common antigen of Pseudomonas aeruginosa and its L-analog in the form of 6-aminohexyl glycosides

A synthesis of trisaccharide monomers built of D- or L-rhamnose residues is described. Their polycondensation in the presence of a 6-aminohexyl rhamnoside derivative afforded 6-aminohexyl glycosides of the natural common polysaccharide antigen of Pseudomonas aeruginosa and its L-analogue, respectively.