Characterization of the extracellular,water-insoluble α-D-glucans of oral streptococci by methylation analysis,and by enzymic synthesis and degradation

Methylation analysis of water-insoluble α-D-glucans synthesized from sucrose by culture filtrates from several strains of Streptococcus spp. has proved that all of the glucans were highly branched and that the chains contained (1→6)- and (1→3)-linked D-glucose residues not involved in branch points. Hydrolysis of the glucans with a specific endo-(1→3)-α-D-glucanase demonstrated that the majority of the (1→3)-linked glucose residues were arranged in sequences. D-Glucose was the major product of the hydrolysis, and a small proportion of nigerose was also released. The use of a specific endo-(1→6)-α-D-glucanase similarly indicated that the glucans also contained sequences of (1→6)-linked α-D-glucose residues, and that those chains were branched. Two D-glucosyltransferases (GTF-S and GTF-I), which reacted with sucrose to synthesize a soluble glucan and a water-insoluble glucan, respectively, were separated from culture filtrates of S. mutans OMZ176. The soluble glucan was characterized as a branched (1→6)-α-D-glucan, whereas the insoluble one was a relatively linear (1→3)-α-D-glucan. The hypothesis is advanced that the glucosyltransferases can transfer glucan sequences by means of acceptor reactions similar to those proposed by Robyt for dextransucrase, leading to the synthesis of a highly branched glucan containing both types of chain. The resulting structure is consistent with the evidence obtained from methylation analysis and enzymic degradations, and explains the synergy displayed when the two D-glucosyltransferases interact with sucrose. Variations in one basic structure can account for the characteristics of water-insoluble glucans from S. sanguis and S. salivarius, and for the strain-dependent diversity of S. mutans glucans.     

Construction of Chimeric Glucansucrases for Analyzing Substrate-binding Regions That Affect the Structure of Glucan Products

A gene that encodes dextransucrase S (dsrS) from Leuconostoc mesenteroides NRRL B-512F encodes a glucansucrase dextransucrase S (DSRS) which mainly produces water-soluble glucan (dextran), while the dsrT5 gene derived from dsrT of the B-512F strain encodes an enzyme dextransucrase T5 (DSRT5), which mainly produces water-insoluble glucan. Tyr340-Asn510 of DSRS and Tyr307-Asn477 of DSRT5 (Site 1), Lys696-Gly768 of DSRS and Lys668-Gly740 of DSRT5 (Site 2), and Asn917-Lys1131 of DSRS and Asn904-Lys1118 of DSRT5 (Site 3) were exchanged and six different chimeric enzymes were constructed. Water-soluble glucan produced by recombinant DSRS was composed of 64% 6-linked glucopyranoside (Glcp), 9% 3,6-linked Glcp, and 13% 4-linked Glcp. Water-insoluble glucan produced by recombinant DSRT5 was composed of 47% 6-linked Glcp and 43% 3-linked Glcp. All of the chimeric enzymes produced glucans different from the ones produced by their parental enzymes. Some of the glucans produced by chimeric enzymes were extremely changed. The Site 1 chimeric enzyme of DSRS (STS1) produced water-soluble glucan composed mostly of 6-linked Glcp. That of DSRT5 (TST1) produced water-insoluble glucan composed mostly of 4-linked Glcp. The Site 3 chimeric enzyme of DSRS (STS3) produced mainly water-insoluble glucan, DSRT5 (TST3) produced mainly water-soluble glucans, and all of the glucan fractions consisted of 3-Glcp, 4-Glcp, and 6-Glcp. The amounts of the three linkages in the water-soluble glucan produced by TST3 were about 1:1:1. Site 1 was assumed to be important for making or avoiding making α-1,4 linkages, while Site 3 was assumed to be important for determining the kinds of glucosyl linkages made.     

Chemical analysis of new water-soluble (1→6)-, (1→4)-α, β-glucan and water-insoluble (1→3)-, (1→4)-β-glucan (Calocyban) from alkaline extract of an edible mushroom, Calocybe indica (Dudh Chattu)

Two different glucans (PS-I, water-soluble; and PS-II, water-insoluble) were isolated from the alkaline extract of fruit bodies of an edible mushroom Calocybe indica. On the basis of acid hydrolysis, methylation analysis, periodate oxidation, and NMR analysis ((1)H, (13)C, DEPT-135, TOCSY, DQF-COSY, NOESY, ROESY, HMQC, and HMBC), the structure of the repeating unit of these polysaccharides were established as: PS-I: →6)-β-D-Glcp-(1→6)-β-D-glcp-(1→6)-)-β-D-Glcp-(1→ α-D=Glcp (Water-soluble glucan). PS-II: →3)-β-D-Glcp-(1→3)-β-D-glcp-(1→3)-)-β-D-Glcp-(1→3)-β-D-Glcp-(1→ β-D-Glcp (Water-insoluble glucan, Calocyban).     

Construction of chimeric glucansucrases for analyzing substrate-binding regions that affect the structure of glucan products

A gene that encodes dextransucrase S (dsrS) from Leuconostoc mesenteroides NRRL B-512F encodes a glucansucrase dextransucrase S (DSRS) which mainly produces water-soluble glucan (dextran), while the dsrT5 gene derived from dsrT of the B-512F strain encodes an enzyme dextransucrase T5 (DSRT5), which mainly produces water-insoluble glucan. Tyr340-Asn510 of DSRS and Tyr307-Asn477 of DSRT5 (Site 1), Lys696-Gly768 of DSRS and Lys668-Gly740 of DSRT5 (Site 2), and Asn917-Lys1131 of DSRS and Asn904-Lys1118 of DSRT5 (Site 3) were exchanged and six different chimeric enzymes were constructed. Water-soluble glucan produced by recombinant DSRS was composed of 64% 6-linked glucopyranoside (Glcp), 9% 3,6-linked Glcp, and 13% 4-linked Glcp. Water-insoluble glucan produced by recombinant DSRT5 was composed of 47% 6-linked Glcp and 43% 3-linked Glcp. All of the chimeric enzymes produced glucans different from the ones produced by their parental enzymes. Some of the glucans produced by chimeric enzymes were extremely changed. The Site 1 chimeric enzyme of DSRS (STS1) produced water-soluble glucan composed mostly of 6-linked Glcp. That of DSRT5 (TST1) produced water-insoluble glucan composed mostly of 4-linked Glcp. The Site 3 chimeric enzyme of DSRS (STS3) produced mainly water-insoluble glucan, DSRT5 (TST3) produced mainly water-soluble glucans, and all of the glucan fractions consisted of 3-Glcp, 4-Glcp, and 6-Glcp. The amounts of the three linkages in the water-soluble glucan produced by TST3 were about 1:1:1. Site 1 was assumed to be important for making or avoiding making alpha-1,4 linkages, while Site 3 was assumed to be important for determining the kinds of glucosyl linkages made.     

Synthesis of 7-(tetrahydropyran-2-yl)- and 7-(4-acetoxytetrahydropyran-2-yl)-ε-rhodomycinone and 7-(tetrahydropyran-2-yl)-ε-pyrromycinone

The ¹³C.n.m.r spectra of water-soluble and -insoluble glucans synthesized by enzymes isolated from six strains of Streptococcus mutans are interpreted. The glucans are shown to be composed primarily of α(1→3)- and α-(1→6)-linked glucosyl residues, and the relative abundance of each linkage is estimated from peak areas. Treatment of water-insoluble glucans with dextranase is found to result in water-soluble and -insoluble products, the former enriched in α-(1→6)-linkages and the latter in α-(1→3)-linkages. The structural conclusions arrived at by ¹³C-n.m.r. spectroscopy are consistent with data from methylation analysis and ¹H-n.m.r. spectroscopy.     

The production of glucans via glucansucrases from Lactobacillus satsumensis isolated from a fermented beverage starter culture

Several starter cultures used in the production of fermented beverages were screened for lactic acid bacteria that produced water-insoluble polysaccharides from sucrose. The strain producing the greatest amount was identified as Lactobacillus satsumensis by its 16S RNA sequence and was deposited in the ARS culture collection as NRRL B-59839. This strain produced at least two α-d-glucans from sucrose. One was a water-soluble dextran, consisting of predominantly α-(1?→?6)-linked d-glucose units, and the other was a water-insoluble glucan containing both α-(1?→?6)-linked and α-(1?→?3)-linked d-glucose units. The culture fluid was found to contain glucansucrases responsible for the two glucans, and no significant level of fructansucrase was detected. Glucansucrase activity was not present in the culture fluid when the bacteria were grown on glucose, fructose, or raffinose as the carbon source. Although the water-soluble glucans produced by cell-free enzyme and by cell suspensions were essentially identical, the same was not true for the water-insoluble glucans. The water-insoluble glucan produced by cell-free culture fluid contained a higher proportion of α-(1?→?3)-linked d-glucose units than the water-insoluble glucan produced by cell suspensions.     

13C-nmr spectroscopic investigation of two yeast cell wall β-D-glucans

By means of alkaline extraction of the cell walsl of the yeasts Saccharomyces cerevisiae and Canadida albicans, water-insoluble glucans were obtained. Methylation analysis and ¹³C-nmr investigation in dimethyl sulfoxide solution revealed the similar chemical structure of these glucans, being composed of β(1 → 3) glycosidically linked D -glucopyranosyl units with a small amount of β(1 → 6) linkages. More detailed study in dilute alkaline solutions and in the gel state at neutral pH, however, showed that an ordered helical conformation of the glucan chain is less stable in the case of the S. cerevisiae glucan in comparison with that of C. albicans. Measurements of the shift of the absorption maximum of the glucan complexes with Congo Red also demonstrated such difference. The S. cerevisiae glucan was also inable to form a gel at neutral pH. The difference in stability of helical conformation of the glucans is explained on the basis of the methylation analysis, so that the S. cerevisiae glucan possesses longer side chains, which hinder its adoption of a stable helical conformation.     

Structure of the water-insoluble α-d-glucan of streptococcus salivarius HHT

Water-insoluble, non-adherent α-d-glucans have been obtained from Streptococcus salivarius HHT under two sets of conditions: from a growing culture, or synthesized enzymically by using a glucosyltransferase. In the former case, the glucan ([α]_d + 197°) was shown by methylation analysis to have a slightly branched structure containing a relatively high proportion (80 %) of (1→3)-α-d-glucosidic linkages, together with small proportions of (1→6)- and (1→4)-α-d-glucosidic linkages. The enzymically synthesized glucan had a much less-branched structure, containing 88 % of (1→3)-α-d-glucosidic linkages. Both glucans, on Smith degradation (sequential periodate oxidation, borohydride reduction, and mild acid hydrolysis), gave linear, (1→3)-α-d-glucosidic polysaccharides (yields, 82-90%) that constitute the backbone chains. The presence of small proportions of glycerol, erythritol, 1-O-α-d-glucosyl-d-glycerol, and also 2-O-α-d-glucosyl-d-erythritol in the products of Smith degradation suggests that the short side-chains are attached to the backbone chain by (1→4)-, (1→6)-, and (1→3)-α-d-glucosidic linkages     

Structures and Antitumor Activities of the Polysaccharides Isolated from Fruiting Body and the Growing Culture of Mycelium of Ganoderma lucidum

Several β-D-glucans, appertaining to the same molecular species but having different degrees of branching, were isolated from water and alkali extracts of the fruiting body of Ganoderma lucidum (Reishi). The purified glucans that were mostly water-insoluble had a backbone of (1 →3)-linked D-glucose residues, attached mainly with single D-glucosyl units at 0-6 and also with a few short (l→4)-linked glucosyl units at 0-2 positions. However, their degrees of branching appeared to differ in the range of d.b. 1/3 ~ 1/23, depending on the extracted glucan fractions. In addition to the ^-glucans, the fruiting body contained water-soluble heteropolysaccharides, comprising D-glucose, D-galactose, D-mannose, L-(or D)-arabinose, D-xylose, and L-fucose.

A branched (1 →3)-β-D-glucan was also isolated from the culture filtrate of G. lucidum grown in a glucose-yeast extract medium. The extracellular β-D-glucan was less soluble in water after purification, but soluble in dilute alkali. This glucan has essentially the same structure as that of hot-water extracted polysaccharide from the fruiting body. The repeating unit of the glucan contains a backbone chain of (1 →3)-linked D-glucose residues, five out of sixteen D-glucose residues being substituted at 0-6 positions with single D-glucosyl units and one D-glucose residue at 0-2 positions probably with a cellobiose unit.

The hot-water extractable fruiting body glucan and the extracellular glucan of the culture of growing mycelium showed relatively high growth-inhibition activities against Sarcoma 180 solid tumor in mice, when administered by. successive intraperitoneal injections. When the moderately branched glucans were modified to D-glucan-polyols by periodate oxidation and borohydride reduction, they exhibited higher antitumor activities, confirming the previous conclusion that the attachment of polyol groups to the (1 →3)-lmked backbone significantly enhances its host-mediated antitumor effect.     

Determination of the structure of dextran by 13C-nuclear magnetic resonance spectroscopy

The ¹³C-n.m.r. spectra have been recorded for a series of dextrans whose structures, in terms of degree and type of branching, had previously been determined by methylation analysis. The spectra established that all observable linkages in these dextrans are α-linked. Correlation of the spectra with methylation data indicated that the 75–85-p.p.m. spectral region is diagnostic for establishing the presence of α-D-(1→2)-, α-D-(1→3)-, or α-D-(1→4)-linkages. Each chemical shift has been found to be temperature-dependent (Δδ/ΔT) when referenced to either the deuterium lock or an external standard (tetramethylsilane). All carbohydrate Δδ/ΔT values are positive, and range from 0.01 to 0.03 p.p.m./°C. These values are considerably larger than analogous Δδ/ΔT values previously observed for smaller molecules. Larger than average Δδ/ΔT values are associated with the non-anomeric, sugar-linking carbon atoms.     

Purification and substrate specificity of an endo-dextranase of Streptococcus mutans K1-R

An extracellular endo-dextranase has been isolated from Streptococcus mutans K1-R. Incubation of cell-free culture fluid with sucrose permitted the removal of a large proportion of the extracellular d-glucosyltransferases by irreversible adsorption onto the insoluble glucans that these enzymes synthesize from sucrose. The remaining d-glucosyltransferases were separated from dextranase by precipitation with ammonium sulphate, chromatography on hydroxylapatite and DEAE-cellulose, followed by filtration on Ultrogel. The major products of action of the purified dextranase on (1→6)-α-d-glucans were isomaltotriose (IM₃), isomaltotetraose (IM₄), and isomaltopentaose (IM₅). Further hydrolysis of IM₄ and IM₅ occurred after prolonged incubation with excess of enzyme, to give d-glucose, IM₂, and IM₃. The relative rate of hydrolysis of isomaltose saccharides fell sharply with decreasing chainlength from IM₁₂ to IM₅. The hydrolysis of dextrans containing 96% or more of (1→6)-α-d-glucosidic linkages, expressed as apparent conversion into IM₃, was virtually complete, and substrates such as Streptococcus sanguis glucan, containing sequences of (1→6)-α-d-glucosidic linkages, were also effectively hydrolyzed. Dextranase activity towards the soluble glucan of Streptococcus mutans was limited, and there was no action on the insoluble glucan synthesized by S. mutans sucrose 3-d-glucosyltransferase.     

Fractionation of the beta-Linked Glucans of Bradyrhizobium japonicum and Their Response to Osmotic Potential

Bradyrhizobium japonicum USDA 110 synthesized both extracellular and periplasmic polysaccharides when grown on mannitol minimal medium. The extracellular polysaccharides were separated into a high-molecular-weight acidic capsular extracellular polysaccharide fraction (90% of total hexose) and three lower-molecular-weight glucan fractions by liquid chromatography. Periplasmic glucans, extracted from washed cells with 1% trichloroacetic acid, gave a similar pattern on liquid chromatography. Linkage analysis of the major periplasmic glucan fractions demonstrated mainly 6-linked glucose (63 to 68%), along with some 3,6- (8 to 18%), 3- (9 to 11%), and terminal (7 to 8%) linkages. The glucose residues were beta-linked as shown by H-nuclear magnetic resonance analysis. Glucan synthesis by B. japonicum cells grown on mannitol medium with 0 to 350 mM fructose as osmolyte was measured. Fructose at 150 mM or higher inhibited synthesis of periplasmic and extracellular 3- and 6-linked glucans but had no effect on the synthesis of capsular acidic extracellular polysaccharides.     

碱提水溶小皮伞多糖B_3的研究

用ＧＣ,ＩＲ,ＮＭＲ,ＧＣ－ＭＳ及高碘酸氧化,Ｓｍｉｔｈ降解,甲基化分析,部分酸水解等方法确定了一个新的碱提水溶小皮伞多糖的一级结构：分子主链α－Ｄ－（１→２）Ｍａｎ,支链α－（１→４）,α－（ａ→６）Ｇｌｃ,且均连在主链的０─６上。     

Vacuum-ultraviolet circular dichroism of acetylated glucans

We have measured the solution and film vacuum ultraviolet circular dichroism of a series of acetylated glucans containing α- and β-(1→3), (1→4), and (1→6) linkages. In addition to the 210-nm band studied previously, we observe the entire π-π* band near 190 nm; these bands are negative for all triacetates regardless of configuration and conformation. A band near 170 nm shows configurational sensitivity for (1→3)- and (1→6)-linked polysaccharides. The band is positive for both (1→4)-linked triacetates, but when cellulose triacetate is partially deacetylated, the 170-nm band becomes negative, thus making the correlation complete. The positive 170-nm band in cellulose triacetate films is more than an order of magnitude more intense than in any other case and, further, is accompanied by an equally large negative band near 153 nm, raising the possibility that the dichroism in the triacetate arises from strong excitonic interactions which are disrupted upon partial deacetylation.     

Fractionation of the β-Linked Glucans of Bradyrhizobium japonicum and Their Response to Osmotic Potential

Bradyrhizobium japonicum USDA 110 synthesized both extracellular and periplasmic polysaccharides when grown on mannitol minimal medium. The extracellular polysaccharides were separated into a high-molecular-weight acidic capsular extracellular polysaccharide fraction (90% of total hexose) and three lower-molecular-weight glucan fractions by liquid chromatography. Periplasmic glucans, extracted from washed cells with 1% trichloroacetic acid, gave a similar pattern on liquid chromatography. Linkage analysis of the major periplasmic glucan fractions demonstrated mainly 6-linked glucose (63 to 68%), along with some 3,6- (8 to 18%), 3- (9 to 11%), and terminal (7 to 8%) linkages. The glucose residues were β-linked as shown by ¹H-nuclear magnetic resonance analysis. Glucan synthesis by B. japonicum cells grown on mannitol medium with 0 to 350 mM fructose as osmolyte was measured. Fructose at 150 mM or higher inhibited synthesis of periplasmic and extracellular 3- and 6-linked glucans but had no effect on the synthesis of capsular acidic extracellular polysaccharides.     

Isolation and analysis of neutral glucans from Ecklonia radiata and Cystophora scalaris

Neutral glucans were isolated from the stipes and fronds of Eklonia radiata and Cystophora scalaris. Partial acid hydrolysis revealed the presence of gentiobiose and laminara-oligosaccharides. Methylation analysis, periodate oxidation, and enzyme studies indicated that the glucans contain β-(1→3) and β-(1→6) linkages. Methylation studies showed that branching in these glucans occurs via a 1,3,6-tri-O- substituted residue with a frequency of one branch point per seven glycosyl residues. In contrast to laminaran from Laminaria digitata, the intrachain (1→3)- and (1→6)- glucopyranoside occur in a molar ratio of 1:1. Enzymic hydrolysis confirmed the absence of long segments of (1→3)-linked residues in the glucans.     

A New Fungal α-D-glucan,elsinan, elaborated by Elsinoe leucospila

A new α-D-glucan, designated elsinan, has been isolated from the culture filtrate of Elsinoe leucospila grown in potato extract-sucrose medium. Acid hydrolysis of the methylated polysaccharide gave 2,3,6- and 2,4,6-tri-O-methyl-D-glucose, in the ratio of 2.5:1.0, together with small proportions of 2,3,4,6-tetra- (0.7%) and 2,4-di-O-methyl-D-glucose (0.5%), indicating that the glucan is an essentially linear polymer containing (1→4)- and (1→3)-α-D-glucosidic linkages. Periodate oxidation, followed by borohydride reduction and mild hydrolysis with acid (mild Smith degradation) yielded 2-O-α-D-glucosyl-D-erythritol and erythritol, in the molar ratio of 1.0:1.4, and a trace of glycerol. Partial acid hydrolysis, and also acetolysis, of elsinan gave nigerose, maltose, O-α-D-glucopyranosyl-(1→3)-O-α-D-glucopyranosyl (1→4)-D-glucopyranose, O-α-D-glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→3)-D-glucopyranose, maltotriose, and a small proportion of maltotetraose. It is concluded that elsinan is composed mainly of maltotriose residues joined by α-(1→3)-linkages, in the sequence →3)-α-D-Glcp-(1→4)-α-D-Glcp-(1→.The unique structural features of elsinan are discussed in comparison with other glucans.     

Water- and alkali-soluble glucans from oak lichen

A water-soluble glucan, [α]²_D +217° (water), and an alkali-soluble glucan,

+152° (sodium hydroxide), have been isolated from the oak lichen Evernia prunastri (L.) Ach. On the basis of methylation analysis, periodate oxidation, and partial acid hydrolysis, the water-soluble polysaccharide has been shown to be a neutral, slightly branched glucan with a main chain composed of (1→3)- and (1→4)- linked glucopyranose residues in the ratio 1?:1. Branching occurs most probably at position 2 of (1→4)-linked glucopyranose residues. On the basis of optical rotation and i.r. spectral data, and enzymic hydrolysis, the α-D configuration has been assigned to the glycosidic linkages. Likewise, the alkali-soluble polysaccharide was shown to be a neutral, branched glucan with a main chain composed of (1→3)- and (1→4)-linked α-D-glucopyranose residues in the ratio 6:1. Each of the (1→4)-linked units was a branch point involving position 6. The presence of some β-D linkages is not excluded since hydrolysis with β-D-glucosidase occurred to a small extent.     

Solution Properties of Antitumor Sulfated Derivative of α-(1→3)-D-Glucan from Ganoderma lucidum

Four fractions of a water-insoluble α-(1→3)-D-glucan GL extracted from fruiting bodies of Ganoderma lucidum were dissolved in 0.25 M LiCl/DMSO, and then reacted with sulfur trioxide-pyridine complex at 80°C to synthesize a series of water-soluble sulfated derivatives S-GL. The degree of substitution of DS was measured by using IR infrared spectra, elemental analysis, and ¹³C NMR to be 1.2-1.6 in the non-selective sulfation. Weight-average molecular weight M_w and intrinsic viscosity [η] of the sulfated derivatives S-GL were measured by multi-angle laser light scattering and viscometry. The M_w value (2.4×10⁴) of sulfated glucan S-GL-1 was much lower than that (44.5×10⁴) of original α-(1→3)-D-glucan GL-1. The Mark-Houwink equation and average value of characteristic ratio C_∞ for the S-GL in 0.2 M NaCl aqueous solution at 25°C were found to be: [η]=1.32×10^-3M_w^1.06 (cm³ g^-1) and 16, respectively, in the M_w range from 1.1×10⁴ to 2.4×10⁴. It indicated that the sulfated derivatives of the α-(1→3)-D-glucan in the aqueous solution behave as an expanded chain, owing to intramolecular hydrogen bonding or interaction between charge groups. Interestingly, two sulfated derivatives synthesized from the α-(1→3)-D-glucan and curdlan, a β-(1→3)-D-glucan, all had significant higher antitumor activity against Ehrlich ascites carcinoma (EAC) than the originals. The effect of expanded chains of the sulfated glucan in the aqueous solution on the improvement of the antitumor activity could not be negligible.     

Acetolysis of disaccharides: Comparative kinetics and mechanism

The initial acetolysis rates of several disaccharides were compared using an assay procedure which involves adding portions of the reaction mixture to an alkaline sodium borohydride solution. After reduction, glycosidically-linked hexose was determined by the phenol-sulfuric acid method. For D-glucose disaccharides, β linkages were cleaved faster than α linkages, suggesting anchimeric assistance from the trans C-2 acetoxyl group. The acetolysis reaction rates for the various β-linked D-glucose disaccharides decreased in the order (1→6) ? (1→3) > (1→2) > (1»4). For the various α-linked disaccharides the order was (1→6) ? (1→4) > (1»3)> (1→2). The acetolysis rates for D-mannose disaccharides were in the order α-(1»6) ? α-(1→3) > β-(1»4) > α-(1»2). Turanose (3-O-α-D-glucopyranosyl-D-fructose) was cleaved at a much faster rate than either D-mannobiose or D-glucobiose with α-(1»2) or α-(1»3) linkages. A reaction mechanism is supported which features an acyclic intermediate, and, for certain -disaccharides, C-2 acetoxyl anchimeric assistance.