Conformational and dynamical properties of disaccharides in water: a molecular dynamics study

Explicit-solvent molecular dynamics simulations (50 ns, 300 K) of the eight reducing glucose disaccharides (kojibiose, sophorose, nigerose, laminarabiose, maltose, cellobiose, isomaltose, and gentiobiose) have been carried out using the GROMOS 45A4 force field (including a recently reoptimized carbohydrate parameter set), to investigate and compare their conformational preferences, intramolecular hydrogen-bonding patterns, torsional dynamics, and configurational entropies. The calculated average values of the glycosidic torsional angles agree well with available experimental data, providing validation for the force field and simulation methodology employed in this study. These simulations show in particular that: 1) (1-->6)-linked disaccharides are characterized by an increased flexibility, the absence of any persistent intramolecular hydrogen bond and a significantly higher configurational entropy (compared to the other disaccharides); 2) cellobiose presents a highly persistent interresidue hydrogen bond and a significantly lower configurational entropy (compared to the other disaccharides); 3) persistent hydrogen bonds are observed for all disaccharides (except (1-->6)-linked) and typically involve a hydrogen donor in the reducing residue and an acceptor in the nonreducing one; 4) the probability distributions associated with the glycosidic dihedral angles and psi are essentially unimodal for all disaccharides, and full rotation around these angles occurs at most once or twice for (never for psi) on the 50-ns timescale; and 5) the timescales associated with torsional transitions (except around and psi) range from approximately 30 ps (rotation of hydroxyl groups) to the nanosecond range (rotation of the lactol and hydroxymethyl groups, and around the omega-glycosidic dihedral angle in (1-->6)-linked disaccharides).     

Aglycone specificity of Escherichia coli alpha-xylosidase investigated by transxylosylation

The specificity of the aglycone-binding site of Escherichia coli alpha-xylosidase (YicI), which belongs to glycoside hydrolase family 31, was characterized by examining the enzyme's transxylosylation-catalyzing property. Acceptor specificity and regioselectivity were investigated using various sugars as acceptor substrates and alpha-xylosyl fluoride as the donor substrate. Comparison of the rate of formation of the glycosyl-enzyme intermediate and the transfer product yield using various acceptor substrates showed that glucose is the best complementary acceptor at the aglycone-binding site. YicI preferred aldopyranosyl sugars with an equatorial 4-OH as the acceptor substrate, such as glucose, mannose, and allose, resulting in transfer products. This observation suggests that 4-OH in the acceptor sugar ring made an essential contribution to transxylosylation catalysis. Fructose was also acceptable in the aglycone-binding site, producing two regioisomer transfer products. The percentage yields of transxylosylation products from glucose, mannose, fructose, and allose were 57, 44, 27, and 21%, respectively. The disaccharide transfer products formed by YicI, alpha-D-Xylp-(1-->6)-D-Manp, alpha-D-Xylp-(1-->6)-D-Fruf, and alpha-d-Xylp-(1-->3)-D-Frup, are novel oligosaccharides that have not been reported previously. In the transxylosylation to cello-oligosaccharides, YicI transferred a xylosyl moiety exclusively to a nonreducing terminal glucose residue by alpha-1,6-xylosidic linkages. Of the transxylosylation products, alpha-d-Xylp-(1-->6)-D-Manp and alpha-d-Xylp-(1-->6)-D-Fruf inhibited intestinal alpha-glucosidases.     

Characterization of two cell-wall polysaccharides from Fusicoccum amygdali

1. The nature of two polysaccharides (s(0) (20) values 6S and 2S respectively in 1m-sodium hydroxide), comprising a fragment (fraction BB, [alpha](D) +236 degrees in 1m-sodium hydroxide), previously isolated from cell walls of Fusicoccum amygdali, has been investigated. 2. Both the major (2S) and minor (6S) components were affected by incubation with alpha-amylase. The 6S polysaccharide was also attacked by exo-beta-(1-->3)-glucanase, which is evidence that it contained both alpha-(1-->4)- and beta-(1-->3)-glucopyranose linkages. By fractionation of the products of alpha-amylase-treated fraction BB it was possible to obtain a water-insoluble polysaccharide, fraction P ([alpha](D) +290 degrees in 1m-sodium hydroxide, 67% of fraction BB) and a water-soluble polysaccharide, fraction Q ([alpha](D) +16 degrees in 1m-sodium hydroxide, 11% of fraction BB), both of which sedimented as single boundaries with s(0) (20) values (in 1m-sodium hydroxide) of 1.7S and 4.6S respectively. 3. Evidence from periodate oxidation, methylation analysis, i.r. spectroscopy and partial acid hydrolysis showed that fraction P consisted of linear chains of alpha-(1-->3)-glucopyranose units with blocks of one or two alpha-(1-->4)-glucopyranose units interspersed at intervals along the main chain. The 2S polysaccharide, from which fraction P is derived, evidently also contains longer blocks of alpha-(1-->4)-glucopyranose units, that are susceptible to alpha-amylase action. 4. Fraction Q consisted of glucose (88%) with small amounts of galactose, mannose and rhamnose. Evidence from digestion with exo- and endo-beta-(1-->3)-glucanases, periodate oxidation and methylation analysis suggests that fraction Q consists of a branched galactomannorhamnan core, to which is attached a beta-(1-->3)-, beta-(1-->6)-glucan. In the cell wall, chains of alpha-(1-->4)-linked glucopyranose units are linked to fraction Q to form the 6S component of fraction BB.     

Synthesis of [C]Cellobiose with Clostridium thermocellum Cellobiose Phosphorylase

Cellobiose labeled either at the reducing end (4-O-beta-d-glucopyranosyl-d-[U-C]glucopyranose) or at the nonreducing end (4-O-beta-d-[U-C]glucopyranosyl-d -glucopyranose) was synthesized with Clostridium thermocellum cellobiose phosphorylase at greater than 98% purity. The radioactive cellobioses were identical to authentic cellobiose in melting point, optical rotation, isotopic dilution, and chromatographic properties.     

Enzymic alpha-galactosylation of a cyclic glucotetrasaccharide derived from alternan

Alternanase catalyzes the hydrolysis of alternan, an alpha-(1-->3)-alpha-(1-->6)-D-glucan produced by Leuconostoc mesenteroides, resulting in the formation of a cyclic tetramer cyclo -->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->(2) (cGlc(4)). Two alpha-galactosidases, one from coffee bean and the other produced by a fungus, currently described as Thermomyces lanuginosus, were found to catalyze an efficient 6-O-alpha-D-galactopyranosylation of cGlc(4). The attachment of a nonreducing alpha-D-galactopyranosyl residue to the cGlc(4) molecule opens new possibilities for future applications of the cyclic tetramer, since the D-galactopyranosyl residue can be easily modified by D-galactose oxidase to introduce a reactive aldehyde group. The results also extend our knowledge about the synthetic potential of T. lanuginosus alpha-galactosidase.     

Reaction of some D-glucobioses with 2,2-dimethoxypropane

Laminarabiose, cellobiose, and gentiobiose were acetonated with 2,2-dimethoxy-propane under various conditions. Two isopropylidene acetals in which the reducing D-glucose residue had the furanoid form were obtained from laminarabiose, and two, in which the reducing D-glucose residue formed the acyclic dimethyl acetal, from cellobiose. Gentiobiose gave both types of isopropylidene compound.     

An enzymatically produced novel cyclic tetrasaccharide, cyclo-{-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->} (cyclic maltosyl-(1-->6)-maltose), from starch

A bacterial strain M6, isolated from soil and identified as Arthrobacter globiformis, produced a novel nonreducing oligosaccharide. The nonreducing oligosaccharide was produced from starch using a culture supernatant of the strain as enzyme preparation. The oligosaccharide was purified as a crystal preparation after alkaline treatment and deionization of the reaction mixture. The structure of the oligosaccharide was determined by methylation analysis, mass spectrometry, and (1)H and (13)C NMR spectroscopy, and it was demonstrated that the oligosaccharide had a cyclic structure consisting of four glucose residues joined by alternate alpha-(1-->4)- and alpha-(1-->6)-linkages. The cyclic tetrasaccharide, cyclo-{-->6)-alpha-D-Glcp(1-->4)-alpha-D-Glcp(1-->6)-alpha-D-Glcp(1-->4)-alpha-D-Glcp(1-->}, was found to be a novel oligosaccharide, and was tentatively called cyclic maltosyl-maltose (CMM). CMM was not hydrolyzed by various amylases, such as alpha-amylase, beta-amylase, glucoamylase, isoamylase, pullulanase, maltogenic alpha-amylase, and alpha-glucosidase, but hydrolyzed by isomalto-dextranase to give rise to isomaltose. This is the first report of the cyclic tetrasaccharide, which has alternate alpha-(1-->4)- and alpha-(1-->6)-glucosidic linkages.     

Automated docking of alpha-(1-->4)- and alpha-(1-->6)-linked glucosyl trisaccharides and maltopentaose into the soybean beta-amylase active site

The Lamarckian genetic algorithm of AutoDock 3.0 was used to dock alpha-maltotriose, methyl alpha-panoside, methyl alpha-isopanoside, methyl alpha-isomaltotrioside, methyl alpha-(6(1)-alpha-glucopyranosyl)-maltoside, and alpha-maltopentaose into the closed and, except for alpha-maltopentaose, into the open conformation of the soybean beta-amylase active site. In the closed conformation, the hinged flap at the mouth of the active site closes over the substrate. The nonreducing end of alpha-maltotriose docks preferentially to subsites -2 or +1, the latter yielding nonproductive binding. Some ligands dock into less optimal conformations with the nonreducing end at subsite -1. The reducing-end glucosyl residue of nonproductively-bound alpha-maltotriose is close to residue Gln194, which likely contributes to binding to subsite +3. In the open conformation, the substrate hydrogen-bonds with several residues of the open flap. When the flap closes, the substrate productively docks if the nonreducing end is near subsites -2 or -1. Trisaccharides with alpha-(1-->6) bonds do not successfully dock except for methyl alpha-isopanoside, whose first and second glucosyl rings dock exceptionally well into subsites -2 and -1. The alpha-(1-->6) bond between the second and third glucosyl units causes the latter to be improperly positioned into subsite +1; the fact that isopanose is not a substrate of beta-amylase indicates that binding to this subsite is critical for hydrolysis.     

Modes of action of acarbose hydrolysis and transglycosylation catalyzed by a thermostable maltogenic amylase, the gene for which was cloned from a Thermus strain

A maltogenic amylase gene was cloned in Escherichia coli from a gram-negative thermophilic bacterium, Thermus strain IM6501. The gene encoded an enzyme (ThMA) with a molecular mass of 68 kDa which was expressed by the expression vector p6xHis119. The optimal temperature of ThMA was 60 degrees C, which was higher than those of other maltogenic amylases reported so far. Thermal inactivation kinetic analysis of ThMA indicated that it was stabilized in the presence of 10 mM EDTA. ThMA harbored both hydrolysis and transglycosylation activities. It hydrolyzed beta-cyclodextrin and starch mainly to maltose and pullulan to panose. ThMA not only hydrolyzed acarbose, an amylase inhibitor, to glucose and pseudotrisaccharide (PTS) but also transferred PTS to 17 sugar acceptors, including glucose, fructose, maltose, cellobiose, etc. Structural analysis of acarbose transfer products by using methylation, thin-layer chromatography, high-performance ion chromatography, and nuclear magnetic resonance indicated that PTS was transferred primarily to the C-6 of the acceptors and at lower degrees to the C-3 and/or C-4. The transglycosylation of sugar to methyl-alpha-D-glucopyranoside by forming an alpha-(1,3)-glycosidic linkage was demonstrated for the first time by using acarbose and ThMA. Kinetic analysis of the acarbose transfer products showed that the C-4 transfer product formed most rapidly but readily hydrolyzed, while the C-6 transfer product was stable and accumulated in the reaction mixture as the main product.     

Enzymatic synthesis of a 2-O-alpha-D-glucopyranosyl cyclic tetrasaccharide by kojibiose phosphorylase

The glucosyl transfer reaction of kojibiose phosphorylase (KPase) from Thermoanaerobacter brockii ATCC35047 was examined using cyclo-{-->6)-alpha-d-Glcp-(1-->3)-alpha-d-Glcp-(1-->6)-alpha-d-Glcp-(1-->3)-alpha-d-Glcp-(1-->} (CTS) as an acceptor. KPase produced four transfer products, saccharides 1-4. The structure of a major product, saccharide 4, was 2-O-alpha-d-glucopyranosyl-CTS, cyclo-{-->6)-alpha-d-Glcp-(1-->3)-alpha-d-Glcp-(1-->6)-[alpha-d-Glcp-(1-->2)]-alpha-d-Glcp-(1-->3)-alpha-d-Glcp-(1-->}. The other transfer products, saccharides 1-3, were 2-O-alpha-kojibiosyl-, 2-O-alpha-kojitriosyl-, and 2-O-alpha-kojitetraosyl-CTS, respectively. These results showed that KPase transferred a glucose residue to the C-2 position at the ring glucose residue of CTS. This enzyme also catalyzed the chain-extending reaction of the side chain of 2-O-alpha-d-glycopyranosyl-CTS.     

Enzymic transfer of α-L-arabinopyranosyl residues to exogenous 1,4-linked β-D-galacto-oligosaccharides using solubilized mung bean (Vigna radiata) hypocotyl microsomes and UDP-β-L-arabinopyranose

A single alpha-L-arabinopyranosyl (alpha-L-Arap) residue was shown, by a combination of chemical and spectroscopic methods, to be transferred to O-4 of the nonreducing terminal galactosyl (Gal) residue of 2-aminobenzamide (2AB)-labeled galacto-oligosaccharides when these oligosaccharides were reacted with UDP-ss-L-arabinopyranose (UDP-ss-L-Arap) in the presence of a Triton X-100-soluble extract of microsomal membranes isolated from mung bean (Vigna radiata, L. Wilezek) hypocotyls. Maximum-(1-->4)-arabinopyranosyltransferase activity was obtained at pH 6.0-6.5 and 20 degrees C in the presence of 25 mM Mn2+. The enzyme had an apparent K m of 45 microM for the 2AB-labeled galactoheptasaccharide and 330 microM for UDP-ss-L-Arap. A series of 2AB-labeled galacto-oligosaccharides with a degree of polymerization (DP) between 6 and 10 that contained a single alpha-L-Arap residue linked to the former nonreducing terminal Gal residue were generated when the 2AB-labeled galactohexasaccharide (Gal6-2AB) was reacted with UDP- ss-L-Ara p in the presence of UDP-beta-D-Galp and the solubilized microsomal fraction. The mono-arabinosylated galacto-oligosaccharides are not acceptor substrates for the galactosyltransferase activities known to be present in mung bean microsomes. These results show that mung bean hypocotyl microsomes contain an enzyme that catalyzes the transfer of Arap to the nonreducing Gal residue of galacto-oligosaccharides and suggest that the presence of a alpha-L-Arap residue on the former terminal Gal residue prevents galactosylation of galacto-oligosaccharides.     

Reactions of reversion occuring with theTrichoderma reesei CL-847 cellulase system

Summary Poplar wood chips were pretreated by steam explosion and the cellulosic residue was hydrolysed with the cellulase complex ofTrichoderma reesei CL 847. The hydrolysate contains the (1–6) disaccharide gentiobiose. Incubation of cellobiose with the same cellulase system led to the synthesis of Glc-Glc dimers which were characterized and quantified as gentiobiose, cellobiose, laminarabiose, sophorose and trehalose, whose origin was clarified by using carbon-13 glucose selectively labelled on C1. We propose a mechanism to explain these reversion reactions.     

Kinetics and mathematical model of hydrolysis and transglycosylation catalysed by cellobiase

The kinetics of hydrolysis and transglycosylation reactions catalysed by cellobiase (β-d-glucoside glucohydrolase, EC 3.2.1.21) from Aspergillus foetidus in the cellobiose-d-glucose reaction system have been studied. The formation of transglycosylation products was observed at cellobiose concentrations $\text{>10}{}^{\mathrm{?2}}\text{m}$, whereas at lower substrate concentrations the only reaction product was d-glucose. In the cellobiase-catalysed transglycosylation a (1→6)-β-linkage was formed after the transfer of a d-glucose residue to acceptor molecule. The basic transglycosylation products were isocellotriose and gentiobiose. A small amount of oligosaccharides with a higher degree of polymerization was also formed. The maximum content of transglycosylation products amounted to 25–30% of the total saccharide content in the system at the initial cellobiose concentration (0.1–0.3 m). The processes in the reaction system were inhibited by the substrate and product (d-glucose). A general scheme for cellobiose hydrolysis has been proposed and validated, allowing for the inhibition and transglycosylation effects. Based on this scheme, a mathematical model for cellobiose hydrolysis has been suggested to describe the kinetics of substrate consumption and product (d-glucose) accumulation, as well as the kinetics of formation and consumption of transglycosylation products throughout the course of enzymatic reaction with various initial amounts of cellobiose, starting from low concentrations up to 0.2–0.3 m (7–11% bv weight).     

Enzymatic characterization and substrate specificity of thermostable beta-glycosidase from hyperthermophilic archaea, Sulfolobus shibatae, expressed in E. coli

Enzymatic properties and substrate specificity of recombinant beta-glycosidases from a hyperthermophilic archaeon, Sulfolobus shibatae (rSSG), were analyzed. rSSG showed its optimum temperature and pH at 95 degrees C and pH 5.0, respectively. Thermal inactivation of rSSG showed that its half-life of enzymatic activity at 75 degrees C was 15 h whereas it drastically decreased to 3.9 min at 95 degrees C. The addition of 10 mM of MnCl2 enhanced the hydrolysis activity of rSSG up to 23% whereas most metal ions did not show any considerable effect. Dithiothreitol (DTT) and 2-mercaptoethanol exhibited significant influence on the increase of the hydrolysis activity of rSSG. rSSG apparently preferred laminaribiose (beta1-->3Glc), followed by sophorose (beta1-->2Glc), gentiobiose (beta1-->6Glc), and cellobiose (beta1--4Glc). Various intermolecular transfer products were formed by rSSG in the lactose reaction, indicating that rSSG prefers lactose as a good acceptor as well as a donor. The strong intermolecular transglycosylation activity of rSSG can be applied in making functional oligosaccharides.     

Alternansucrase acceptor reactions with methyl hexopyranosides

Alternansucrase (EC 2.4.1.140, sucrose: (1-->6), (1-->3)-alpha-D-glucan 6(3)-alpha-D-glucosyltransferase) is a D-glucansucrase that synthesizes an alternating alpha-(1-->3), (1-->6)-linked D-glucan from sucrose. It also synthesizes oligosaccharides via D-glucopyranosyl transfer to various acceptor sugars. We have studied the acceptor products arising from methyl glycosides as model compounds in order to better understand the specificity of alternansucrase acceptor reactions. The initial product arising from methyl beta-D-glucopyranoside was methyl beta-isomaltoside, which was subsequently glucosylated to yield methyl beta-isomaltotrioside and methyl alpha-D-glucopyranosyl-(1-->3)-alpha-D-glucopyranosyl-(1-->6)-beta-D-glucopyranoside. These products are analogous to those previously described from methyl alpha-D-glucopyranoside. The major initial acceptor product from methyl alpha-D-mannopyranoside was methyl alpha-D-glucopyranosyl-(1-->6)-alpha-D-mannopyranoside, but several minor products were also isolated and characterized, including a 3,6-di-O-substituted mannopyranoside. Methyl alpha-D-galactopyranoside yielded two initial products, methyl alpha-D-glucopyranosyl-(1-->3)-alpha-D-galactopyranoside and methyl alpha-D-glucopyranosyl-(1-->4)-alpha-D-galactopyranoside, in a 2.5:1 molar ratio. Methyl D-allopyranosides were glucosylated primarily at position 6, yielding methyl alpha-D-glucopyranosyl-(1-->6)-D-allopyranosides. The latter subsequently gave rise to methyl alpha-D-glucopyranosyl-(1-->6)-alpha-D-glucopyranosyl-(1-->6)-D-allopyranosides. In general, the methyl alpha-D-hexopyranosides were better acceptors than the corresponding beta-glycosides.     

Synthesis of beta-(1-->6)-branched beta-(1-->3) glucohexaose and its analogues containing an alpha-(1-->3) linked bond with antitumor activity

A beta-(1-->6)-branched beta-(1-->3)-glucohexaose, present in many biologically active polysaccharides from traditionally herbal medicines such as Ganoderma lucidum, Schizophyllum commune and Lentinus edodes, was synthesized as its lauryl glycoside 32, and its analogues 18, 20 and 33 containing an alpha-(1-->3) linked bond were synthesized. It is interesting to find that coupling of a 3,6-branched acylated trisaccharide trichloroacetimidate donor 9 with 3,6-branched acceptors 13 and 16 with 3'-OH gave the alpha-(1--> 3)-linked hexasaccharides 17 and 19, respectively, in spite of the presence of C-2 ester capable of neighboring group participation. However, coupling of 9 with 4-methoxyphenyl 4,6-O-benzylidene-beta-D-glucopyranoside (27) selectively gave beta-(1-->3)-linked tetrasaccharide 28. Simple chemical transformation of the tetrasaccharide 28 gave acylated tetrasaccharide trichloroacetimidate 29. Coupling of 29 with lauryl (1-->6)-linked disaccharide 26 with 3-OH gave beta-(1-->3)-linked hexasaccharide 30 as the major product. Bioassay showed that in combination with the chemotherapeutic agent cyclophospamide (CPA), the hexaose 18 at a dose of 0.5-1mg/kg substantially increased the inhibition of S(180) for CPA, but decreased the toxicity caused by CPA. Some of these oligosaccharides also inhibited U(14) noumenal tumor in mice effectively.     

The isolation and structure of the core oligosaccharide sequences of IgM.

Methods are presented for separating the three IgM heavy chain sialoglycopeptides associated with asparagines 170, 332, and 395. The core glycopeptide units containing the disaccharide fucosyl-N-acetylglucosamine were obtained through the use of an endo-beta-N-acetylglucosamindase from Diplococcus pneumoniae, following exoglycosidase treatment of the sialoglycopeptides. In addition to the core glycopeptides, high yields of a tetrasaccharide, (Man)3GlcNAc, were obtained. The fucose in the core disaccharide is glycosidically linked to the 6-O position of the N-acetylglucosamine residue in Asn-GlcNAc. This core unit is resistant to glycosyl asparaginase, but becomes susceptible to hydrolysis on removal of the fucosyl residue by a purified hen oviduct alpha-L-fucosidase. The core sequence of the immunoglobulin M sialoglycopeptides appears to be similar to that of most other asparagine-linked oligosaccharides in consisting of a basic unit composed of beta-D-Man-(1 leads to 4)beta-D-GlcNAc(1 leads to 4)beta-D-GlcNAc(1 leads to 4), but with L fucose linked alpha-(1 leads to 6) to the proximal GlcNAc. The two nonreducing terminal ends of (Man)3GlcNAc are linked to beta-D-Man by alpha-(1 leads to 3) and alpha(1 leads to 6) bonds, respectively.     

Structural characterization of the O-antigenic polysaccharide of the lipopolysaccharide from Rhizobium etli strain CE3. A unique O-acetylated glycan of discrete size, containing 3-O-methyl-6-deoxy-L-talose and 2,3,4-tri-O-,methyl-l fucose

The O-antigenic polysaccharide of the Rhizobium etli CE3 lipopolysaccharide (LPS) was structurally characterized using chemical degradations (Smith degradation and beta-elimination of uronosyl residues) in combination with alkylation analysis, electrospray, and matrix-assisted laser desorption ionization-time of flight mass spectrometry, tandem mass spectrometry, and (1)H COSY and TOCSY nuclear magnetic resonance spectroscopy analyses of the native polysaccharide and the derived oligosaccharides. The polysaccharide was found to be a unique, relatively low molecular weight glycan having a fairly discrete size, with surprisingly little variation in the number of repeating units (degree of polymerization = 5). The polysaccharide is O-acetylated and contains a variety of O-methylated glycosyl residues, rendering the native glycan somewhat hydrophobic. The molecular mass of the major de-O-acetylated species, including the reducing end 3-deoxy-d-manno-2-octulosonic acid (Kdo) residue, is 3330 Da. The polysaccharide is comprised of a trisaccharide repeating unit having the structure -->4)-alpha-d-GlcpA-(1-->4)-[alpha-3-O-Me-6-deoxy-Talp-(1--> 3)]-alpha -l-Fucp-(1-->. The nonreducing end of the glycan is terminated with the capping sequence alpha-2,3, 4-tri-O-Me-Fucp-(1-->4)-alpha-d-GlcpA-(1-->, and the reducing end of the molecule consists of the non-repeating sequence -->3)-alpha-l-Fucp-(1-->3)-beta-d-Manp-(1-->3)-beta-QuiNA cp-(1-->4)-a lpha-Kdop-(2-->, where QuiNAc is N-acetylquinovosamine (2-N-acetamido-2,6-dideoxyglucose). The reducing end Kdo residue links the O-chain polysaccharide to the core region oligosaccharide, resulting in a unique location for a Kdo residue in LPS, removed four residues distally from the lipid A moiety. Structural heterogeneity in the O-chain arises mainly from the O-acetyl and O-methyl substitution. Methylation analysis using trideuteriomethyl iodide indicates that a portion of the 2,3,4-tri-O-methylfucosyl capping residues, typically 15%, are replaced with 2-O-methyl- and/or 2,3-di-O-methylfucosyl residues. In addition, approximately 25% of the 3,4-linked branching fucosyl residues and 10% of the 3-linked fucosyl residues are 2-O-methylated. A majority of the glucuronosyl residues are methyl-esterified at C-6. These unique structural features may be significant in the infection process.     

An Alkali-soluble Polysaccharide from the oak lichen Cetraria islandica (L.) Ach.

An alkali-soluble polysaccharide, [α]

+43° (M sodium hydroxide), containing d-glucose and d-glucuronic acid has been isolated from the oak lichen Cetraria islandica (L.) Ach. On the basis of methylation, periodate oxidation, and partial hydrolysis studies, the polysaccharide has been shown to contain (1→3)-linked glucopyranose and glucuronic acid residues and (1→4)- and/or (1→6)-linked gluco-pyranose residues as the main structural features of the basic chain. A preponderance of β linkages, indicated by the low optical rotation and the i.r. spectrum, was corro-borated by the formation of laminaribiose, cellobiose, and gentiobiose on partial hydrolysis.