The mechanism of the acid-catalysed hydrolysis of polysaccharides

The kinetics of homogeneous and heterogeneous acid-catalyzed hydrolysis of polysaccharides has been studied. The hydrolysis of O-methylcellulose in solutions of hydrochloric, sulfuric, and perchloric acids, and the acetolysis of cellulose triacetate were found to follow the mechanism established earlier for glycosides. Degradation of O-ethylcellulose films in hydrochloric acid vapour occurred in the kinetic region.     

Biotransformation pathway and kinetics of the hydrolysis of the 3-O- and 20-O-multi-glucosides of PPD-type ginsenosides by ginsenosidase type I

Ginsenosidase type I from Aspergillus niger g.48 can hydrolyze the 3-O- and 20-O-multi-glycosides of PPD-type ginsenosides. The enzyme molecular weight is approximately 74 kDa. When hydrolyzing the glycosides of Rb1, Rb3, Rb2 and Rc, the structures of which only differ in their terminal 20-O-glycosides, ginsenosidase type I hydrolyzes both the 3-O- and 20-O-glycosides of Rb1 and Rb3 using two pathways, but the enzyme first hydrolyzes the 3-O-glucosides of Rb2 and Rc using one pathway. One pathway of Rb1 hydrolyzes the 20-O-Glc of Rb1 to Rd→F2→C-K; another pathway hydrolyzes the 3-O-Glc of Rb1 to Gyp17→Gyp75→C-K. Two hydrolysis pathways are used to hydrolyze the 20-O-Xyl and the 3-O-Glc of Rb3. According to the enzyme reaction parameters K_m, V_max and V₀ at a 10 mM substrate concentration, the enzyme hydrolysis velocity values decrease in the following order: the 20-O-Xyl of Rb3→Rd> the 20-O-Glc of Rb1→Rd> the 3-O-Glc of Rc> the 3-O-Glc of Rb2> the 3-O-Glc of Rd> the 3-O-Glc of Rb3→C-Mx1> the 3-O-Glc of Rb1→Gyp17> the 3-O-Glc of F2> the 3-O-Glc of 20(S)-Rg3.     

Screening of Arabidopsis thaliana stems for variation in cell wall polysaccharides

A high-throughput method is described by which Arabidopsis thaliana stems can be screened for variation in cell wall composition after hydrolysis with Driselase or trifluoroacetic acid (TFA). Driselase, a mixture of fungal enzymes, hydrolyses cellulose (to glucose) and all the major matrix polysaccharides (to monosaccharides and/or characteristic disaccharides); TFA hydrolyses the matrix polysaccharides, but not cellulose, to monosaccharides. Two different wild-type ecotypes, Columbia and Wassilewskija, showed only minor differences in wall carbohydrate composition. A small number of T-DNA-tagged populations that were screened contained individuals in which the proportion of cellulose, xyloglucan or xylan differed quantitatively from the wild-type. Differences from the wild-type were also observed in the susceptibility of the hemicelluloses to hydrolysis by Driselase, probably reflecting differences in wall architecture.     

Methylation analysis of carrageenans from the seaweed Iridaea undulosa

Two carrageenans from Iridaea undulosa, isolated by precipitation of the crude polysaccharide at O.70–1.05 M and 1.55–1.65 M KCl concentrations, were studied by methylation analysis. Acid hydrolysis of the methylated derivative of the less soluble carrageenan (molar ratio galactose: 3,6-anhydrogalactose: sulphate 1.00: 0.50: 1.20) yielded major amounts of 2,6-di-O-methylgalactose (51.3 mol %), 4,6-di-O-methylgalactose (25.6%) and 4-O-methylgalactose (51.3mol%), 4,6-di-O-methylgalactose (25.6%) and 4-O-methylgalactose (13.4%). Minor quantities of 3-O-methylgalactose (4.6%) and 6-O-methylgalactose (3.2%) were found together with traces of 2,3,6- and/or 2,4,6-tri-O-methylgalactose, 2-O-methylgalactose and galactose. Oxidative acid hydrolysis produced 3,6-anhydro-2-O-methylgalactonic acid and 3,6-anhydrogalactonic acid in a molar ratio 3.5-4.0:1.0. The methylated derivative of the more soluble carrageenan (molar ratio galactose:3,6-anhydrogalactose:sulphate 1.00:0.04:1.43) gave on acid hydrolysis, 2,3,4,6-tetra-O-methylgalactose (4.6%), 2,3,6-tri-O-methylgalactose (4.2%), 2,4,6-tri-O-methylgalactose (10.7%), 4,6-di-O-methylgalactose (24.1%), 3,6-di-0-methylgalactose (8.0%), 2,3-di-O- methylgalactose (3.4%), 2,4-di-O-methylgalactose (4.6%), 2,6-di-O-methylgalactose (4.2%), 3-O-methylgalactose (19.5%),4-O-methylgalactose (9.6%),6-O-methylgalactose(3.1%),galactose (3.4%)and traces of 2-O-methylgalactose.     

Two diastereoisomeric 2,3:5,6-di-O-ethylidene-β-D-alloses and related derivatives

Direct condensation of β-D-allose with acetaldehyde in the presence of sulfuric acid formed two of eight possible 2,3:5,6-di-O-ethylidene-D-alloses in overall yields of 84–96%. Conditions of the reaction were varied to favor formation of either isomer. The presence of a furanose ring in both isomers was established by converting the diastereoisomers into 1,4-di-O-acetyl-D-allitol analogs. P.m.r. analysis of the reducing isomers, their 3-deuterio analogs, their 1-O-acetyl derivatives, and the 1,5,6-triacetate of a common hydrolysis product, 2,3-O-ethylidene-D-allose, established the anomeric configuration of D-allose as β-, and the C-2′ atom in the 2,3-O-ethylidene ring as R and as either R or S in the 5,6-O-ethylidene ring.     

Evaluation of Processing Technology for Triarrhena sacchariflora (Maxim.) Nakai for Ethanol Production

The effects of dilute H₂SO₄ concentration, forage:sulfuric acid ratio, digestion time, and digestion temperature were evaluated to determine effects on ethanol yield of Triarrhena sacchariflora (Maxim.) Nakai. Twenty single factor experiments were conducted to evaluate H₂SO₄ concentration (0.5, 1.0, 1.5, 2.0, and 2.5%, w/w), forage:sulfuric acid ratio (1∶6, 1∶8, 1∶10, 1∶12, and 1∶14, g/ml), digestion time (15, 30, 45, 60, and 90, min), digestion temperature (80, 100, 110, 120, and 125 °C) for 3 replicates of the 5 levels of each factor. Based on results of the single factor experiments, an incomplete factorial was designed to evaluate ethanol yield from the best combinations of single factors. Finally, the best combination was tested by enzymatic hydrolysis and fermentation experiment in selected combinations according to pretreatment results. Percentage cellulose, hemicellulose, and lignin contents of forage residue after pretreatment, and glucose and xylose concentrations of the filtrate were analyzed prior to enzymatic hydrolysis, and percentage crystallinity was observed in untreated grass and pretreated residue. In addition, the solid residues were then hydrolysed and fermented by cellulase and yeast, the concentrations of glucose and ethanol being monitored for 96 h. Results showed that the order of the effect of main effect factors was as follows: digestion temperature > dilute H₂SO₄ concentration > digestion time > forage:sulfuric acid ratio. The best process parameters evaluated were sulfuric acid concentration of 1.5%, forage:sulfuric acid ratio of 1∶6, digestion time of 15 min, and digestion temperature of 120°C. With this combination of factors, 80% of the cellulose was hydrolysed in 96 h, and 78% converted to ethanol. The findings identified that hemicelluloses were the key deconstruction barrier for pretreatment of Triarrhena sacchariflora (Maxim.) Nakai for ethanol production. The results of this research provide evidence of appropriate combinations of processing factors for production of ethanol from Triarrhena sacchariflora (Maxim.) Nakai.     

The elongation factor Tu·GTPase reaction: Effect of 2′(3′)-O-aminoacyl oligoribonucleotides

The activity of synthetic (2′(3′)-O-aminoacyl trinucleotides, C-C-A-Phe, C-C-U-Phe, C-U-A-Phe, U-C-A-Phe and C-A-A-Phe, in promoting the EF-Tu·70 S ribosome-catalyzed GTP hydrolysis was investigated. It was found that the activity decreases in the order C-C-A-Phe > C-U-A-Phe > U-C-A-Phe > C-A-A-Phe ⪢ C-C-U-Phe. Thus, the substitution in ‘natural’ C-C-A sequence with other nucleobases weakens binding of 2′(3′)-O-aminoacyl trinucleotides to EF-Tu, with the substitution at the 3′-position having the most profound effect. Since the 2′(3′)-O-aminoacyl oligonucleotides mimic the effect of the aa-tRNA 3′-terminus on EF-Tu·GTPase, it follows that EF-Tu probably directly recognizes structure of nucleobases in the aa-tRNA 3′-terminus, with the 3′-terminal adenine playing the most important role.     

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.     

The structure of bael (Aegle marmelos) gum

Purified, bael-gum polysaccharide containsd-galactose (71%),l-arabinose (12.5%),l-rhamnose (6.5%), andd-galacturonic acid (7%). Hydrolysis of one mole of the fully methylated polysaccharide gave: (a) from the neutral part, 2,3,4-tri-O-methyl-l-rhamnose (2 moles), 2,3,5-tri-O-methyl-l-arabinose (4 moles), 2,3,4,6-tetra-O-methyl-d-galactose (8 moles), 3,4-di-O-methyl-l-rhamnose (2 moles), 2,5-di-O-methyl-l-arabinose (1 mole), 2,4,6-tri-O-methyl-d-galactose (10 moles), 2,3-di-O-methyl-l-arabinose (1 mole), 2,4-di-O-methyl-d-galactose (14 moles), and 2-O-methyl-d-galactose (2 moles); and (b) from the acidic part, 2,3,4-tri-O-methyl-d-galacturonic acid (1 mole), 2,4,6-tri-O-methyl-3-O-(2,3,4-tri-O-methyl-d-galactopyranosyluronic acid)-d-galactose (2.6 moles), and 2,4,6-tri-O-methyl-3-O-[2,4,6-tri-O-methyl-3-O-(2,3,4-tri-O-methyl-d-galactopyranosyluronic acid)-d-galactopyranosyl]-d-galactose (1 mole). Mild hydrolysis of the whole gum yielded oligosaccharides from which 3-O-β-d-galactopyranosyl-l-arabinose, 5-O-β-d-galactopyranosyl-l-arabinose, 3-O-β-d-galactopyranosyl-d-galactose, and 6-O-β-d-galactopyranosyl-d-galactose could be isolated and characterized. The results of methylation, periodate oxidation, Smith degradation, Barry degradation, and graded hydrolysis studies were employed for the elucidation of the structure of the whole gum.     

Structural studies of 4-O-acetyl-α-N-acetylneuraminyl-(2→3)-lactose, the main oligosaccharide in echidna milk

The main oligosaccharide (50%) in the milk of the Australian echidna (Tachyglossus aculeatus) has been identified unequivocally as 4-O-acetyl-α-N-acetylneur-amínyl-(2→3)-lactose. The 4-O-acetyl substituent of the sialic acid residue was characterised by g.l.c.-m.s. of the isolated (after mild, acid hydrolysis) and trimethyl-silylated/esterified sialic acid, and by m.s. (after derivatisation) and 500-MHz, ¹H-n.m.r. spectroscopy of the intact oligosaccharide. Information about the glycosidic bonds was obtained by methylation analysis and 500-MHz, ¹H-n.m.r. spectroscopy. This animal species is the third one known to produce 4-O-acetylated sialic acid.     

Cellulose whiskers extracted from mulberry: A novel biomass production

A kind of cellulose whiskers were extracted from the branch-barks of mulberry (Morus alba L.) by an alkali treatment at 130 °C and subsequently a sulfuric acid hydrolysis. AFM image showed that the diameter of obtained whiskers was ranged from 20 to 40 nm. The chemical compositions analysis, FT-IR, XRD results indicated that the hemicellulose and lignin were removed extensively in the cellulose whiskers, with a crystallinity of 73.4%. The TGA curves implied a two-stage thermal decomposition behavior of cellulose whisker due to the introduction of sulfated groups into the crystals in the sulfuric acid hydrolysis process. The obtained whiskers may have the potential applications in the fields of composites as a reinforcing phase, as well as in pharmaceutical and optical industries as additives.     

Synthesis of 2-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-D-mannose,and its interaction with D-mannose-specific lectins

Condensation of 3,4:5,6-di-O-isopropylidene-D-mannose dimethyl acetal with 2-methyl-(3,4,6-tri-O-acetyl- 1,2-dideoxy-α-D-glucopyrano)-[2′, 1′:4,5]-2-oxazoline in the presence of a catalytic amount of p-toluenesulfonic acid afforded crystalline 2-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-3,4:5,6-di-O-isopropylidene-D-mannose dimethyl acetal (3) in 25% yield. Catalytic deacetylation of 3 with sodium methoxide, followed by hydrolysis with dilute sulfuric acid, gave 2-O-(2-acetamido-2-deoxy-α-D-glucopyranosyl)-D-mannose (4). Treatment of 3 with boiling 0.5% methanolic hydrogen chloride under reflux gave methyl 2-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-α-D-mannopyranoside (5) and methyl 2-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-α-D-mannofuranoside (6). The inhibitory activities of 4, 5, and 6 against the hemagglutinating and mitogenic activities of Lens culinaris and Pisum sativum lectins and concanavalin A were assayed. From the results of these hapten inhibition studies, subtle differences of specificity between these D-mannose-specific lectins were confirmed.     

Removal of enzymatic and fermentation inhibitory compounds from biomass slurries for enhanced biorefinery process efficiencies

Within the biorefinery paradigm, many non-monomeric sugar compounds have been shown to be inhibitory to enzymes and microbial organisms that are used for hydrolysis and fermentation. Here, two novel separation technologies, polyelectrolyte polymer adsorption and resin-wafer electrodeionization (RW-EDI), have been evaluated to detoxify a dilute acid pretreated biomass slurry. Results showed that detoxification of a dilute acid pretreated ponderosa pine slurry by sequential polyelectrolyte and RW-EDI treatments was very promising, with significant removal of acetic acid, 5-hydroxymethyl furfural, and furfural (up to 77%, 60%, and 74% removed, respectively) along with >97% removal of sulfuric acid. Removal of these compounds increased the cellulose conversion to 94% and elevated the hydrolysis rate to 0.69 g glucose/L/h. When using Saccharomyces cerevisiae D₅A for fermentation of detoxified slurry, the process achieved 99% of the maximum theoretical ethanol yield and an ethanol production rate nearly five-times faster than untreated slurry.     

Effect of Castanospermine and Related Polyhydroxyalkaloids on Purified Myrosinase from Lepidium sativum Seedlings

Myrosinase (β-thioglucoside glucohydrolase, EC 3.2.3.1) was purified to apparent homogeneity from light-grown cress (Lepidium sativum L.) seedlings. This enzyme, which catalyzes hydrolysis of the glucosinolate sinigrin (K_m, 115 micromolar) at an optimum pH of 5.5 in sodium citrate buffer, had a native molecular weight of 130 ± 5 kilodaltons and an isoelectric point of 4.7 to 4.9. SDS-PAGE revealed two polypeptides with molecular weights of 62 and 65 kilodaltons. Both subunits contained carbohydrate as shown by periodic acid-Schiff staining. The purified enzyme hydrolyzed p-nitrophenyl-β-d-glucoside (K_m, 2.0 millimolar) at an optimum pH of 6.5 in phosphate buffer. The indolizidine alkaloid castanospermine, a known inhibitor of O-glycosidases, competitively inhibited the hydrolyses of sinigrin (thioglucosidase activity) and p-nitrophenyl-β-d-glucoside (O-glucosidase activity) with K_i values of 5 and 6 micromolar, respectively. In contrast, the related polyhydroxyalkaloids swainsonine and deoxynojirimycin were without effect upon these hydrolyses.     

Manufacture of cellulose nanocrystals by cation exchange resin-catalyzed hydrolysis of cellulose

Cellulose nanocrystals (CNC) were prepared from microcrystalline cellulose (MCC) by hydrolysis with cation exchange resin (NKC-9) or 64% sulfuric acid. The cation exchange resin hydrolysis parameters were optimized by using the Box–Behnken design and response surface methodology. An optimum yield (50.04%) was achieved at a ratio of resin to MCC (w/w) of 10, a temperature of 48 °C and a reaction time of 189 min. Electron microscopy (EM) showed that the diameter of CNCs was about 10–40 nm, and the length was 100–400 nm. Regular short rod-like CNCs were obtained by sulfuric acid hydrolysis, while long and thin crystals of cellulose were obtained with the cation exchange resin. X-ray diffraction (XRD) showed that, compared with MCC, the crystallinity of H₂SO₄-CNC and resin-CNC increased from 72.25% to 77.29% and 84.26%, respectively. The research shows that cation exchange resin-catalyzed hydrolysis of cellulose could be an excellent method for manufacturing of CNC in an environmental-friendly way.     

Synthesis of 2,5-dideoxy-2-C-methyl-d-arabinose derivatives from methyl 2,3-anhydro-5-deoxy-α-d-ribofuranoside

Treatment of methyl 2,3-anhydro-5-deoxy-α-d-ribofuranoside with lithium dimethyl cuprate gave methyl 2,5-dideoxy-2-C-methyl-α-d-arabinofuranoside (54% yield) and methyl 3,5-dideoxy-3-C-methyl-α-d-xylofuranoside (10%). The former was converted into its 3-O-acetyl and 3-O-benzyl derivatives, which, upon acid hydrolysis, afforded 3-O-acetyl- and 3-O-benzyl-2,5-dideoxy-2-C-methyl-d-arabinofuranose in 60–75% overall yield. Treatment of the 3-O-benzyl compound with ethanethiol in the presence of trifluoromethanesulfonic acid afforded 3-O-benzyl-2,5-dideoxy-2-C-methyl-d-arabinose diethyl dithioacetal (20%) and ethyl 3-O-benzyl-2,5-dideoxy-2-C-methyl-1-thio-α-d-arabinoside (73%). The former, which was also available from the latter by equilibration in acidic ethanethiol, was acetylated at O-4 and the product converted into the corresponding dimethyl acetal (85% overall yield). This compound was, after debenzylation, hydrolyzed with acid, to provide 4-O-acetyl-2,5-dideoxy-2-C-methyl-d-arabinose in 70% overall yield.     

Studies on cellulolytic enzymes I: The use of 3,4-dinitrophenyl glycosides as substrates

The syntheses of 3,4-dinitrophenyl β-d-glucoside, β-cellobioside, β-cellotrioside, and β-cellotetraoside and their use to monitor the purification of two enzymes from a crude commercial cellulase preparation from Trichoderma viride are described. The enzymes isolated are an endo-β-1,4-d-glucan glucanohydrolase (EI) of molecular weight ca. 12 000 which catalysed the release of 3,4-dinitrophenol from 3,4-dinitrophenol-β-cellotetraoside, and an enzyme of molecular weight about 76 000 which catalysed the hydrolysis of 3,4-dinitrophenyl β-d-glucoside (EII) and is probably a cellobiase or exo-β-1,4-d-glucan glucohydrolase. Kinetic parameters are reported for the hydrolyses of 3,4-dinitrophenyl β-cellobioside, β-cellotrioside, and β-cellotetraoside catalysed by enzyme EI. In the presence of cellotriose, cellotetraose, or cellopentaose 3,4-dinitrophenyl β-d-glucoside underwent induced hydrolyses by EI. Similar but faster induced hydrolyses were shown by 3,4-dinitrophenyl β-d-xyloside and 3,4-dinitrophenyl β-d-6-deoxyglucoside; 3,4-dinitrophenyl 6-chloro-6-deoxy-β-d-glucoside and 3,4-dinitrophenyl 6-O-methyl-β-d-glucoside underwent slower induced hydrolyses than the glucoside. p-Nitrophenyl β-d-glucoside also underwent an induced hydrolysis in the presence of cellopentaose and the enzyme EI, but p-nitrophenyl 2-deoxy-β-d-glucoside did not. These results are discussed and compared with the results obtained previously on induced hydrolyses found with lysozyme. Kinetic parameters are reported for the hydrolysis of 3,4-dinitrophenyl and p-nitrophenyl β-d-glucosides catalysed by the enzyme EII. 3,4-Dinitrophenyl 6-deoxy-β-d-glucoside, β-d-xyloside, 6-chloro-6-deoxy-β-d-glucoside, 6-O-methyl-β-d-glucoside and p-nitrophenyl-β-d-galactopyranoside and 2-deoxy-β-d-glucopyranoside were hydrolysed 10² to 10³ times slower by EII than the corresponding glucosides, but 3,4-dinitrophenyl 2-acetamido-2-deoxy-β-d-glucoside was only hydrolysed about 25 times slower than 3,4-dinitrophenyl β-d-glucoside. The significance of these results is discussed. EII catalysed the release of 3,4-dinitrophenol from 3,4-dinitrophenyl β-cellobioside, β-cellobioside, and β-cellotetraoside, but these reactions showed induction periods which are consistent with stepwise removal of glucose residues from the oligosaccharide chains before release of the phenol.     

A new water-soluble polysaccharide from the seeds of Cassia multijuga

A polysaccharide consisting of D-galactose, D-mannose, and D-xylose in the molecular ratios 5:1:2 has been isolated from the defatted seeds of Cassia multijuga. Methylation analysis yielded 2,3-di-O-methyl-D-galactose (2 mol), 2,3,6-tri-O-methyl-D-galactose (4 mol), 2,3,4,6-tetra-O-methyl-D-galactose (4 mol), 2,3-di-O-methyl-D-mannose (2 mol), 2-O-methyl-D-xylose (1 mol), 2,3-di-O-methyl-D-xylose (2 mol), and 2,3,4-tri-O-methyl-D-xylose (1 mol). Periodate oxidation indicated 32.4% of end-groups and methylation indicated 31.2%. Partial hydrolysis with acid gave 6-O-α-D-galactosyl-D-galactose, 6-O-α-D-galactosyl-D-mannose, 4-O-β-D-galactosyl-D-xylose, and 3-O-β-D-xylosyl-D-xylose, together with monosaccharides. The polysaccharide is highly branched, consisting of galactosyl, mannosyl, and xylosyl residues in the main chain, with (1→4)-β linkages, and galactosyl and xylosyl end-groups.     

Analytical and structural features of the gum exudate from Combretum hartmannianum schweinf.

The gum exudate from Combretum hartmannianum is water-soluble, forms very viscous solutions, and contains galactose (22%), arabinose (43%), mannose (10%), xylose (6%), rhamnose (4%), glucuronic acid (6%), 4-O-methylglucuronic acid (2%), and galacturonic acid (7%). The acidic components produced on hydrolysis of the gum were 6-O-(β-D-glucopyranosyluronic acid)-D-galactose, and two saccharides that had the same chromatographic mobility, and contained mannose and galacturonic acid, and galactose and 4-O-methylglucuronic acid, respectively. Methylation and methanolysis of the gum indicated the presence of terminal uronic acid, rhamnose, xylose, galactose, arabinofuranose, and arabinopyranose. Controlled, acid hydrolysis indicated the presence of (1→3)-linked arabinopyranose side-chains and (1→6)-linked galactose residues. C. hartmannianum gum, when subjected to two Smith-degradations, yielded Polysaccharides I and II, both of which contained galactose, arabinose, and mannose. Insufficient crude gum was available for a complete structural study, but the molecule was shown to contain long, sparsely branched chains of (1→6)-linked galactose residues, to which are attached (1→3)-linked arabinose and (1→3)-linked mannose side-chains.     

The linkage of 4-O-methyl-L-galactose in the sulphated polysaccharide of Aeodes ulvoidea

Methyl 2-acetamido-5,6-di-O-benzyl-2-deoxy-β-d-glucofuranoside (11) was obtained in six steps from the known methyl 3-O-allyl-2-benzamido-2-deoxy-5,6-O-isopropylidene-β-d-glucofuranoside. Mild acid hydrolysis, followed by benzylation gave the 5,6-dibenzyl ether. The benzamido group was exchanged for an acetamido group by strong alkaline hydrolysis, followed by N-acetylation, and the allyl group was isomerized into a 1-propenyl group that was hydrolyzed with mercuric chloride. Treatment of 11 with l-α-chloropropionic acid and with diazomethabe gave methyl 2-acetamido-5,6-di-O-benzyl-2-deoxy-3-O-[d-1-(methoxycarbonyl)ethyl]-β-d-glucofuranoside which formed on mercaptolysis the internal ester 16, further converted into 2-acetamido-4-O-acetyl-5,6-di-O-benzyl-2-deoxy-3-O-[d-1-(methoxycarbonyl)ethyl]-d-glucose diethyl dithioacetal (18) by alkaline treatment followed by esterification with diazomethane and acetylation. Attempts to remove the O-acetyl group of the corresponding dimethyl acetal 20 with sodium methoxide in mild conditions were not successful.