Synthesis of β-D-Fructopyranosyl-(2→6)-D-glucopyranose from D-Glucose and D-Fructose by a Thermal Treatment

The synthesis is reported of β-D-fructopyranosyl-(2→6)-D-glucopyranose that had previously been isolated from a fermented plant extract as a new saccharide. A disaccharide was predominately formed from an equal amount of D-glucose and D-fructose under melting conditions at 140 °C for 60 to 90 min. This saccharide was isolated from the reaction mixture by carbon-Celite column chromatography and preparative HPLC, and was confirmed to be β-D-fructopyranosyl-(2→6)-D-glucopyranose by TOF-MS and NMR analyses.     

Synthesis of β-(1→6)-linked N-acetyl-D-glucosamine oligosaccharide substrates and their hydrolysis by Dispersin B

Dispersin B (DspB) from Aggregatibacter actinomycetemcomitans is a β-hexosaminidase exhibiting biofilm detachment activity. A series of β-(1→6)-linked N-acetyl-D-glucosamine thiophenyl glycosides with degree of polymerisation (DP) of 2, 3, 4 and 5 were synthesized, and substrate specificity of DspB was studied on the obtained oligosaccharides. For oligomer synthesis a 1+2, 2+2, 1+4 coupling strategy was applied, using bromo-sugars as glycosyl donors. The formation of 1,2-trans interglycosidic bond has been ensured by 2-phtalimido protecting group; chloroacetyl group was installed to mask temporarily the 6-hydroxyl and acetate esters were applied as permanent protecting groups. Enzymatic studies revealed that DP of the GlcNAc oligomers strongly affected the hydrolysis rate, and the hydrolytic activity of DspB on the tetramer and pentamer have been found to be approximately 10-fold higher than that of the dimer. This fact indicates that four units are required for a strong binding at the active centre of DspB. The role of aromatic amino acids W237, Y187 and Y278 in substrate specificity and catalysis was also examined using mutant enzymes.     

Evidence for a C-2→C-1 intramolecular hydrogen-transfer during the acid-catalyzed isomerization of D-glucose to D-fructose ag]

In mechanistic studies by isotope-exchange tecniques of the conversion of D-fructose and D-glucose into 2-(hydroxyacetyl)furan, it was shown that both sugars are converted in acidified, tritiated water into the furan containing essentially no carbon-bound tritium. As the hydroxymethyl carbon atom of the furan corresponds to C-1 of the hexose, this result suggests that one of the hydrogen atoms in this group, when it is produced from D-glucose, must arise intramolecularly. This hypothesis was verified by synthesizing D-glucose-2-³H and converting it into the furan in acidified water. The 2-(hydroxyacetyl)furan obtained was labeled exclusively on the hydroxymethyl carbon atom, thus showing that intramolecular hydrogen-transfer occurs, during the conversion, from C-2 of D-glucose to the carbon atom corresponding to C-1. The specific activities of the product and reactant permitted calculation of the tritium isotope-effect (k_h/k_t4.4) for the reaction. The precise step for the transfer from C-2 of the aldose to the carbon atom corresponding to C-1 was found to be during the isomerization of D-glucose to D-fructose, as evidenced by the conversion of D-glucose-2-³H into D-fructose-1-³H in acidified water.     

The structure of a β-(1→6)-d-glucan from yeast cell walls

By selective enzymolysis, or chemical fractionation, a minor polysaccharide component has been isolated from yeast (Saccharomyces cerevisiae) glucan. This minor component has a degree of polymerization of about 130-140, a highly branched structure, and a high proportion of beta-(1-->6)-glucosidic linkages. The molecules also contain a smaller proportion of beta-(1-->3)-glucosidic linkages that serve mainly as interchain linkages, but some may also be inter-residue linkages.     

Synthesis of the (1→6)-linked thiodisaccharide of galactofuranose: Inhibitory activity against a β-galactofuranosidase

A new (1→6)-linked thiodisaccharide formed by two galactofuranosyl units has been synthesized. Methyl (methyl α,β-d-galactofuranosid)uronate was employed as the starting compound, which was per-O-silylated with TBSCl and reduced with LiAlH₄ to afford methyl 2,3,5-tri-O-tert-butyldimethylsilyl-β-d-galactofuranoside (2β) as a key precursor for the preparation of methyl per-O-tert-butyldimethylsilyl-6-thio-β-d-galactofuranoside (12). The free thiol group of 12 was glycosylated and the product O-deprotected to afford the target β-d-Galf-S-(1→6)-β-d-Galf-OMe (14). The conformations of this thiodisaccharide were preliminarily studied using combined theoretical calculations and NMR data. Furthermore, the glycomimetic 14 showed to be a competitive inhibitor of the β-galactofuranosidase from Penicillum fellutanum (K_i = 3.62 mM).     

Enzymic hydrolysis of barley and other β-glucans by a β-(1→4)-glucan hydrolase

1. A barley glucan with 68% of beta-(1-->4)-linkages and 32% of beta-(1-->3)-linkages was exhaustively hydrolysed with an Aspergillus niger beta-(1-->4)-glucan 4-glucanohydrolase (EC 3.2.1.4) (Clarke & Stone, 1965b). The hydrolysis products were separated and estimated. 2. The lower-molecular-weight products were identified as: glucose, 1.4%; cellobiose, 11.9%; 3(2)-O-beta-glucosylcellobiose, 45.0%; a tetrasaccharide(s), which was a substituted cellobiose, 16.4%. A series of unidentified higher-molecular-weight products (26.5%) were also found. 3. The identity of the products suggests that the A. niger beta-(1-->4)-glucan hydrolase hydrolyses beta-glucosidic linkages joining 4-O-substituted glucose residues. 4. When an enzyme fraction containing the beta-(1-->4)-glucan hydrolase and an exo-beta-(1-->3)-glucan hydrolase was used, the same products were found, but the higher-molecular-weight products were observed to have only a transient existence in the hydrolysate and were virtually absent after prolonged incubation. It is suggested that these oligosaccharides are resistant to attack by beta-(1-->4)-glucan hydrolase but are partially hydrolysed by the exo-beta-(1-->3)-glucan hydrolase and therefore possess one or more (1-->3)-linked glucose residues at their non-reducing end.     

Synthesis of Some New α- and β-(l→2) Linked Disaccharides Containing L-Quinovose (6-Deoxy L-Glucose)

Hepta-O-acetyl-2-0-β-l-quinovopyranosyl-α-d-glucose (VI) and hepta-O-acetyl-2-O-α-l-quinovopyranosyl-β-d-gIucose (VIII) were prepared by the coupling of 2,3,4-tri-O-acetyl-α-l-quinovopyranosyl bromide (IV) with l,3,4,6-tetra-O-acetyl-α-D-glucose (V) in the presence of mercuric cyanide and mercuric bromide in absolute acetonitrile.

Similarly, hepta-O-acetyW-O-α-l-quinovopyranosyl-α-d-galactose (X) and hepta-O-acetyl-2-O-β-L-quinovopyranosyl-α-d-galactose (XI) were prepared by the reaction of IV with 1,3,4,6-tetra-O-acetyl-α-d-galactose (IX).

Removal of the protecting groups of VI, VIII, X and XI afforded the corresponding disaccharides. On treatment with hydrogen bromide, VI, VIII, X and XI gave the corresponding acetobromo derivatives.     

Structure of a complex asparagine bound glycopeptide from horse pancreatic ribonuclease containing (2→3) and (2→6) linked sialic acid residues

The primary structure of the main glycopeptide obtained by pronase digestion of horse pancreatic ribonuclease has been investigated by 360 MHz ¹H-NMR spectroscopy and methylation analysis. The results demonstrate that this glycopeptide has the following structure:

This is the first time that the presence of both (2→3) and (2→6) linked sialic acid residues in a glycopeptide has been demonstrated.     

(1→2) and (1→6)-linked β-d-galactofuranan of microalga Myrmecia biatorellae, symbiotic partner of Lobaria linita

A structural study of the cell wall polysaccharides of Myrmecia biatorellae, the symbiotic algal partner of the lichenized fungus Lobaria linita was carried out. It produced a rhamnogalactofuranan, with a (1→6)-β-d-galactofuranose in the main-chain, substituted at O-2 by single units of β-d-Galf, α-l-Rhap or by side chains of 2-O-linked β-d-Galf units. The structure of the polysaccharide was established by chemical and NMR spectroscopic analysis, and is new among natural polysaccharides. Moreover, in a preliminary study, this polysaccharide increased the lethality of mice submitted to polymicrobial sepsis induced by cecal ligation and puncture, probably due to the presence of galactofuranose, which have been shown to be highy immunogenic in mammals.     

Efficient release of thermostable β-galactosidase by glycine addition and thermal treatment of Escherichia coli cells

Efficient release of thermostable β-galactosidase from a recombinant Escherichia coli by the addition of glycine to the culture broth and subsequent thermal treatment was investigated. The enzyme release rate was strongly dependent on glycine concentration. The enzyme release rate was almost proportional to glycine concentrations up to 2% in phosphate buffer; however, inactivation of the enzyme was not observed following incubation for up to 3 h at 70°C even in the presence of 10% glycine. In a preliminary experiment, severe thermal inactivation was observed in the presence of polyethylene glycol (PEG), but glycine was able to suppress the inactivation. Thermal treatment of the cell suspension was effective for the improvement of the enzyme release rate. In the absence of glycine, the enzyme release rate was low at 37 and 45°C, even though the initial release rate was high at 0.5 h and 60°C. The combination of thermal treatment and addition of glycine to the cell suspension significantly improved the initial enzyme release rate and the amount of enzyme released to the extracellular fraction at 37 and 45°C was as high as that at 60°C during a 2-h incubation.     

Synthesis of oligosaccharides by reversal of a fungal β-glucanase

Summary A crude commercial preparation of -glucanase fromPenicillium emersonii was used to synthesise glucose-containing oligosaccharides by condensation reactions in high concentrations of glucose at elevated temperature. Gentiobiose, laminaribiose, cellobiose, isomaltose and trehalose were identified as products. Heterooligosaccharides were produced by enzyme in some mixtures of glucose and an acceptor sugar. High performance ion-exchange chromatography was used to analyse synthetic products.     

Properties of a β-(1→4)-glucan hydrolase from Aspergillus niger

1. A beta-(1-->4)-glucan hydrolase prepared from Aspergillus niger, as described by Clarke & Stone (1965a), showed a pH optimum in the range 4.5-6 and K(m) 0.25% when acting on a cellulose dextrin sulphate substrate. 2. The hydrolase rapidly decreased the specific viscosity of carboxymethylcellulose with a small increase in the production of reducing sugars. The identity of the products of hydrolysis of cellotetraose, cellopentaose and their reduced analogues indicate a preferential cleavage of non-terminal glucosidic linkages. The enzyme may be described as beta-(1-->4)-glucan 4-glucanohydrolase (EC 3.2.1.4). 3. In addition to carboxymethylcellulose, cellulose dextrins, cellopentaose and cellotetraose the enzyme fraction hydrolysed lichenin, oat and barley glucans, ivory-nut mannan and a glucomannan from Konjak flour. No hydrolysis of wheat-straw beta-(1-->4)-xylan, Lupinus albus beta-(1-->4)-galactan, pneumococcal type III polysaccharide, chitin, hyaluronic acid, laminarin, pachydextrins, carboxymethylpachyman or beta-(1-->3)-oligoglucosides was detected. 4. The hydrolase showed no transglycosylase activity from cellodextrin or cellopentaose substrates to glucose or methanol acceptors. 5. The hydrolysis of cellodextrins was inhibited completely by 1.0mm-Hg(2+), 0.7mm-phenylmercuric nitrate and 1.0mm-iodine.     

Synthetic Studies on Nephritogenic Glycosides. Synthesis of β-Glc-(1→6)-α-Glc-(l→6)-α-Glc-(1→Asn)

Gibberellins (GAs) A₉, A₁₅, A₁₉, A₂₀, A₂₉, A₃₅, A₄₄, A₅₀ and A₆₁ were identified by capillary gas chromatography/selected ion monitoring (GC/SIM) in immature seeds of loquat (Eriobotrya japonica Lindl). Furthermore, five unknown GA-like compounds with apparent parent ions of m/z 418, 504 or 506 (as methyl ester trimethylsilyl ether derivatives) were found by GC/mass spectrometry (GC/MS) in the biologically active fractions. The m/z 418 and 504 compounds may have been C-11β hydroxylated GA₉ and dehydro-GA₃₅, respectively. The bioassay and GC/MS results suggest that the major GAs were GA₅₀ and the five unknown GA-like compounds. In the immature seeds, at least two GA metabolic pathways may thus exist, one being the non-hydroxylation pathway of GA₁₅→GA₂₄→GA₉, and the other, the early C-13 hydroxylation pathway of GA₄₄→GA₁₉→GA₂₀→GA₂₉. A late C-11β hydroxylation pathway is also possible.     

Synthesis and Biological Evaluation of β-D-Pentofuranonucleoside Derivatives of 2-Azidoadenine and 6-Azidopurines

Abstract

β-D-pentofuranonucleoside derivatives of 2-azidoadenine and 6-azidopurines have been synthesized. The azido-tetrazolo tautomerism observed on such nucleoside analogues has been studied. The compounds were tested for their activity against HIV and HBV but they did not show significant antiviral effect.     

Structural and Enzymatic Characterization of Os3BGlu6, a Rice β-Glucosidase Hydrolyzing Hydrophobic Glycosides and (1→3)- and (1→2)-Linked Disaccharides

Glycoside hydrolase family 1 (GH1) β-glucosidases play roles in many processes in plants, such as chemical defense, alkaloid metabolism, hydrolysis of cell wall-derived oligosaccharides, phytohormone regulation, and lignification. However, the functions of most of the 34 GH1 gene products in rice (Oryza sativa) are unknown. Os3BGlu6, a rice β-glucosidase representing a previously uncharacterized phylogenetic cluster of GH1, was produced in recombinant Escherichia coli. Os3BGlu6 hydrolyzed p-nitrophenyl (pNP)-β-d-fucoside (k_cat/K_m = 67 mm⁻¹ s⁻¹), pNP-β-d-glucoside (k_cat/K_m = 6.2 mm⁻¹ s⁻¹), and pNP-β-d-galactoside (k_cat/K_m = 1.6 mm⁻¹s⁻¹) efficiently but had little activity toward other pNP glycosides. It also had high activity toward n-octyl-β-d-glucoside and β-(1→3)- and β-(1→2)-linked disaccharides and was able to hydrolyze apigenin β-glucoside and several other natural glycosides. Crystal structures of Os3BGlu6 and its complexes with a covalent intermediate, 2-deoxy-2-fluoroglucoside, and a nonhydrolyzable substrate analog, n-octyl-β-d-thioglucopyranoside, were solved at 1.83, 1.81, and 1.80 Å resolution, respectively. The position of the covalently trapped 2-F-glucosyl residue in the enzyme was similar to that in a 2-F-glucosyl intermediate complex of Os3BGlu7 (rice BGlu1). The side chain of methionine-251 in the mouth of the active site appeared to block the binding of extended β-(1→4)-linked oligosaccharides and interact with the hydrophobic aglycone of n-octyl-β-d-thioglucopyranoside. This correlates with the preference of Os3BGlu6 for short oligosaccharides and hydrophobic glycosides.β-Glucosidases (EC 3.2.1.21) have a wide range of functions in plants, including acting in cell wall remodeling, lignification, chemical defense, plant-microbe interactions, phytohormone activation, activation of metabolic intermediates, and release of volatiles from their glycosides (Esen, 1993). They fulfill these roles by hydrolyzing the glycosidic bond at the nonreducing terminal glucosyl residue of a glycoside or an oligosaccharide, thereby releasing Glc and an aglycone or a shortened carbohydrate. The aglycone released from the glycoside may be a monolignol, a toxic compound, or a compound that further reacts to release a toxic component, an active phytohormone, a reactive metabolic intermediate, or a volatile scent compound (Brzobohatý et al., 1993; Dharmawardhama et al., 1995; Reuveni et al., 1999; Lee et al., 2006; Barleben et al., 2007; Morant et al., 2008). Indeed, the wide range of glucosides of undocumented functions found in plants suggests that many β-glucosidase functions may remain to be discovered.Plant β-glucosidases fall into related families that have been classified as glycosyl hydrolase (GH) families GH1, GH3, and GH5 (Henrissat, 1991; Coutinho and Henrissat, 1998, 1999). Of these, GH1 has been most thoroughly documented and shown to comprise a gene family encoding 40 putative functional GHs in Arabidopsis (Arabidopsis thaliana) and 34 in rice (Oryza sativa) in addition to a few pseudogenes (Xu et al., 2004; Opassiri et al., 2006). In addition to β-glucosidases, plant GH1 members include β-mannosidases (Mo and Bewley, 2002), β-thioglucosidases (Burmeister et al., 1997), and disaccharidases such as primeverosidase (Mizutani et al., 2002) as well as hydroxyisourate hydrolase, which hydrolyzes the internal bond in a purine ring rather than a glycosidic bond (Raychaudhuri and Tipton, 2002). The specificity for the glycone in GH1 enzymes varies. Some enzymes are quite specific for β-d-glucosides or β-d-mannosides, while many accept either β-d-glucosides or β-d-fucosides, and some also hydrolyze β-d-galactosides, β-d-xylosides, and α-l-arabinoside (Esen, 1993). However, most GH1 enzymes are thought to hydrolyze glucosides in the plant, and it is the aglycone specificity that determines the functions of most GH1 enzymes.Aglycone specificity of GH1 β-glucosidases ranges from rather broad to absolutely specific for one substrate and is not obvious from sequence similarity. For instance, maize (Zea mays) ZmGlu1 β-glucosidase hydrolyzes a range of glycosides, including its natural substrate, 2-O-β-d-glucopyranosyl-4-dihydroxy-1,4-benzoxazin-3-one (DIMBOAGlc), but not dhurrin, whereas sorghum (Sorghum bicolor) Dhr1, which is 72% identical to ZmGlu1, only hydrolyzes its natural cyanogenic substrate dhurrin (Verdoucq et al., 2003). Similarly, despite sharing over 80% amino acid sequence identity, the legume isoflavonoid β-glucosidases dalcochinase from Dalbergia cochinchinensis and Dnbglu2 from Dalbergia nigrescens hydrolyze each other''s natural substrate very poorly (Chuankhayan et al., 2007). Thus, small differences in the amino acid sequence surrounding the active site may be expected to account for significant differences in substrate specificity.GH1 is classified in GH clan A, which consists of GH families whose members have a (β/α)₈-barrel structure with the catalytic acid/base on strand 4 of the β-barrel and the catalytic nucleophile on strand 7 (Henrissat et al., 1995; Jenkins et al., 1995). As such, all GH1 enzymes have similar overall structures, but it has been noted that four variable loops at the C-terminal end of the β-barrel strands, designated A, B, C, and D, account for much of the differences in the active site architecture (Sanz-Aparicio et al., 1998). The similar structures with great diversity in substrate specificity make plant GH1 enzymes an ideal model system to investigate the structural basis of substrate specificity. To date, seven plant β-glucosidase structures have been reported, including three closely related chloroplastic enzymes from maize (Czjzek et al., 2000, 2001), sorghum (Verdoucq et al., 2004), and wheat (Triticum aestivum; Sue et al., 2006), the cytoplasmic strictosidine β-glucosidase from Rauvolfia serpentine (Barleben et al., 2007), and the secreted enzymes white clover (Trifolium repens) cyanogenic β-glucosidase (Barrett et al., 1995), white mustard (Sinapsis alba) myrosinase (thioglucosidase; Burmeister et al., 1997), and rice Os3BGlu7 (BGlu1; Chuenchor et al., 2008). These enzymes hydrolyze substrates with a range of structures, but they cannot account for the full range of β-glucosidase substrates available in plants, and determining the structural differences that bring about substrate specificity differences in even closely related GH1 enzymes has proven tricky (Verdoucq et al., 2003, 2004; Sue et al., 2006; Chuenchor et al., 2008).Amino acid sequence-based phylogenetic analysis of GH1 enzymes encoded by the rice genome showed that there are eight clusters containing both rice and Arabidopsis proteins that are more closely related to each other than they are to enzymes from the same plants outside the clusters (Fig. 1; Opassiri et al., 2006). In addition, there are a cluster of sixteen putative β-glucosidases and a cluster of myrosinases in Arabidopsis without any closely related rice counterparts. Comparison with characterized GH1 enzymes from other plants reveals other clusters of related enzymes not found in rice or Arabidopsis, including the chloroplastic enzymes, from which the maize, sorghum, and wheat structures are derived, and the cytoplasmic metabolic enzymes, from with the strictosidine hydrolase structure is derived (Fig. 1). Therefore, although the known structures provide good tools for molecular modeling of plant enzymes, most rice and Arabidopsis GH1 enzymes lack a close correspondence in sequence and functional evolution to these structures, suggesting that the variable loops that determine the active site may be different. It would be useful, therefore, to know the structures and substrate specificities of representative members of each of the eight clusters seen in rice and Arabidopsis. To begin to acquire this information, we have expressed Os3BGlu6, a member of cluster At/Os 1 in Figure 1, characterized its substrate specificity, and determined its structure alone and in complex with a glycosyl intermediate and a nonhydrolyzable substrate analog.Open in a separate windowFigure 1.Simplified phylogenetic tree of the amino acid sequences of eukaryotic GH1 proteins with known structures and those of rice and Arabidopsis GH1 gene products. The protein sequences of the eukaryotic proteins with known structures are marked with four-character PDB codes for one of their structures, including Trifolium repens cyanogenic β-glucosidase (1CBG; Barrett et al., 1995), Sinapsis alba myrosinase (1MYR; Burmeister et al., 1997), Zea mays ZmGlu1 β-glucosidase (1E1F; Czjzek et al., 2000), Sorghum bicolor Dhr1 dhurrinase (1V02; Verdoucq et al., 2004), Triticum aestivum β-glucosidase (2DGA; Sue et al., 2006), Rauvolfia serpentina strictosidine β-glucosidase (2JF6; Barleben et al., 2007), and Oryza sativa Os3BGlu7 (BGlu1) β-glucosidase (2RGL; Chuenchor et al., 2008) from plants, along with Brevicoryne brassicae myrosinase (1WCG; Husebye et al., 2005), Homo sapiens cytoplasmic (Klotho) β-glucosidase (2E9M; Hayashi et al., 2007), and Phanerochaete chrysosporium (2E3Z; Nijikken et al., 2007), while those encoded in the Arabidopsis and rice genomes are labeled with the systematic names given by Xu et al. (2004) and Opassiri et al. (2006), respectively. One or two example proteins from each plant are given for each of the eight clusters of genes shared by Arabidopsis (At) and rice (Os) and the Arabidopsis-specific clusters At I (β-glucosidases) and At II (myrosinases), with the number of Arabidopsis or rice enzymes in each cluster given in parentheses. These sequences were aligned with all of the Arabidopsis and rice sequences in ClustalX (Thompson et al., 1997), the alignment was manually edited, all but representative sequences were removed, and the tree was calculated by the neighbor-joining method, bootstrapped with 1,000 trials, and then drawn with TreeView (Page, 1996). The grass plastid β-glucosidases, which are not represented in Arabidopsis and rice, are shown in the group marked “Plastid.” Percentage bootstrap reproducibility values are shown on internal branches where they are greater than 60%. Except those marked by asterisks, all external branches represent groups with 100% bootstrap reproducibility. To avoid excess complexity, those groups of sequences marked with asterisks are not monophyletic and represent more branches within the designated cluster than are shown. For a complete phylogenetic analysis of Arabidopsis and rice GH1 proteins, see Opassiri et al. (2006).     

Synthesis and biological activity of O-(N-acetyl-β-muramoyl-l-alanyl-d-isoglutamine)-(1→6)-2-acylamino-2-deoxy-d-glucoses

Benzoylation of benzyl 2-acetamido-2-deoxy-4,6-O-isopropylidene-α-d-glucopyranoside, benzyl 2-deoxy-2-(dl-3-hydroxytetradecanoylamino)-4,6-O-isopropylidene-α-d-glucopyranoside, and benzyl 2-deoxy-4,6-O-isopropylidene-2-octadecanoylamino-β-d-glucopyranoside, with subsequent hydrolysis of the 4,6-O-isopropylidene group, gave the corresponding 3-O-benzoyl derivatives (4, 5, and 7). Hydrogenation of benzyl 2-acetamido-4,6-di-O-acetyl-2-deoxy-3-O-[d-1-(methoxycarbonyl)ethyl]-α-d-glucopyranoside, followed by chlorination, gave a product that was treated with mercuric actate to yield 2-acetamido-1,4,6-tri-O-acetyl-2-deoxy-3-O-[d-1-(methoxycarbonyl)ethyl]-β-d-glucopyranose (11). Treatment of 11 with ferric chloride afforded the oxazoline derivative, which was condensed with 4, 5, and 7 to give the (1→6)-β-linked disaccharide derivatives 13, 15, and 17. Hydrolysis of the methyl ester group in the compounds derived from 13, 15, and 17 by 4-O-acetylation gave the corresponding free acids, which were coupled with l-alanyl-d-isoglutamine benzyl ester, to yield the dipeptide derivatives 19–21 in excellent yields. Hydrolysis of 19–21, followed by hydrogenation, gave the respective O-(N-acetyl-β-muramoyl-l-alanyl-d-isoglutamine)-(1→6)-2-acylamino-2-deoxy-d-glucoses in good yields. The immunoadjuvant activity of these compounds was examined in guinea-pigs.     

The structure of a β-(1→3)-d-glucan from yeast cell walls

Yeast glucan as normally prepared by various treatments of yeast (Saccharomyces cerevisiae) cell walls to remove mannan and glycogen is still heterogeneous. The major component (about 85%) is a branched beta-(1-->3)-glucan of high molecular weight (about 240000) containing 3% of beta-(1-->6)-glucosidic interchain linkages. The minor component is a branched beta-(1-->6)-glucan. A comparison of our results with those of other workers suggests that different glucan preparations may differ in the degree of heterogeneity and that the major beta-(1-->3)-glucan component may vary considerably in degree of branching.     

β-Glucan hydrolases from Aspergillus niger. Isolation of a β-(1→4)-glucan hydrolase and some properties of the β-(1→3)-glucan-hydrolase components

1. The components of an enzyme preparation from Aspergillus niger, which hydrolysed substrates containing beta-(1-->3)- and beta-(1-->4)-glucosidic linkages, were separated by calcium phosphate and Dowex 1 column chromatography. 2. The hydrolytic activity of each fraction from both types of column towards laminaribiose, laminarin, carboxymethylpachyman, pachydextrins, salicin, cellobiose, cellopentaose and swollen cellulose was tested. 3. The activity towards the beta-(1-->3)-glucosidic substrates was found in three well-separated groups of fractions. The differences in action pattern of these groups is discussed. 4. Preparative-scale chromatography that enabled the separation of a beta-(1-->4)-glucan-glucanohydrolase component substantially free of activity towards beta-(1-->3)-glucosidic substrates is described. Residual beta-(1-->3)-glucan-hydrolase activity was removed by adsorption on to insoluble laminarin at pH3.5.