Structural characterization of oligosaccharides isolated from the pectic polysaccharide rhamnogalacturonan II

Rhamnogalacturonan II (RG-II) is a structurally complex pectic (d-galactosyl-uronic acid-rich) polysaccharide that is present in the primary (growing) cell-walls of higher plants. RG-II is composed of ∼60 glycosyl residues. The isolation and structural characterization of 23 oligosaccharide fragments of the residue of RG-II that remained after removal of hepta- and di-saccharides by partial hydrolysis with acid are reported. In order to obtain the oligosaccharide fragments characterized herein, the carboxyl groups of RG-II were dideuterio-reduced, and the carboxyl-reduced polysaccharide was per-O-methylated. The per-O-methylated polysaccharide was fragmented by partial hydrolysis with acid, producing partially O-methylated oligosaccharides. These derivatized oligosaccharides were reduced, to afford a mixture of partially O-methylated oligoglycosyl-alditols, which was then per-O-methylated. The structures of the resulting per-O-methylated oligoglycosylalditols were determined by chemical-ionization mass spectrometry, electron-impact mass spectrometry, fast-atom-bombardment mass spectrometry, ¹H-n.m.r. spectroscopy, and analysis of corresponding, partially O-acetylated, partially O-methylated alditols. Seventeen of the oligosaccharides isolated from RG-II were parts of a single heptasaccharide, namely.     

Structure of the extracellular polysaccharide produced by the bacterium Alcaligenes (ATCC 31555) species

The extracellular anionic polysaccharide produced by the bacterium Alcaligenes (ATCC 31555) contains l-mannose, l-rhamnose, d-glucose, and d-glucuronic acid in the molar ratios 1.0:4.5:3.1:2.3. Analysis of the methylated and methylated, carboxyl-reduced polysaccharide indicated terminal non-reducing rhamnose and mannose, (1→4)-linked rhamnose, (1→3)- and (1→3,1→4)-linked glucose, and (1→4)-linked glucuronic acid to be present in the ratios 1.0:0.8:2.1:2.2:2.0:2.2. Partial acid hydrolysis and base-catalysed β-elimination gave a series of oligosaccharides that were isolated as their alkylated alditol derivatives by reverse-phase h.p.l.c. and characterised by f.a.b.-m.s., e.i.-m.s., and ¹H-n.m.r. spectroscopy. The repeating unit 1, excluding O-acyl groups, is proposed.

     

An electron-impact,mass-spectrometric fragment-ion that identifies 3-linked d-glucopyranosyl residues in per-O-alkylated,linear β-d-glucopyrano-oligosaccharide-alditols

A previously unreported fragment-ion, designated J₀, was observed in the electron-impact, mass spectra of per-O-alkylated, linear di- and tri-β-d-glucopyranosylalditols containing 3-linked d-glucopyranosyl residues. The J₀ fragment-ion was absent from, or present in very low abundance in, the spectra of per-O-alkylated, linear di- and tri-β-d-glucopyranosylalditols composed of only 2-, 4-, or 6-linked residues. The presence of the J₀ fragment-ion and the absence of the J₁ fragment-ion were found indicative of the presence of 3-linked d-glucopyranosyl residues, and may be indicative of the presence of all 3-linked-glycosyl residues in per-O-alkylated oligosaccharide-alditols. A possible mechanism for the formation of the J₀ fragment-ion is proposed.     

Depolymerization of pectin with diazomethane in the presence of a small proportion of phosphate buffer

Treatment of pectin with diazomethane in diethyl ether in the presence of a small proportion of phosphate buffer resulted in considerable depolymerization of the polysaccharide chain and concomitant methylation of the hydroxyl groups. A mixture of the depolymerized products was fractionated by gel-filtration on a Toyopearl HW-40S column to give di- and tri-saccharide fractions, which were subsequently permethylated with diazomethane-boron trifluoride etherate. The resulting permethylated di- and tri-saccharide fractions were separated into two di- and four tri-sacchraides by reversed phase l.c., followed by adsorption l.c. The ¹H-n.m.r. spectra of the isolated oligosacchraides were studied by homonuclear, spin-decoupling experiments, COSY-45 two-dimensional homonuclear correlation experiments, two-dimensional J-resolved methods, and two-dimensional ¹³C¹H correlation experiments. The structures (1 and 2) for the two disaccharides, and those (3 and 4) for two of the four trisaccharides were determined. The structures 5 and 6 for the remaining trisaccharides were tentatively deduced from their ¹H-n.m.r. spectral data.     

Nitrous acid deamination of methylated amino-oligosaccharide glycosides

G.l.c.-mass spectrometry has been used to characterize the products of N-deacetylation-nitrous acid deamination of per-O-methylated derivatives (8–11) of methyl 2-acetamido-2-deoxy-3-O-β-D-galactopyranosyl-α-D-glucopyranoside(1), methyl (2) and benzyl (3) 2-acetamido-2-deoxy-4-O-β-D-galactopyranosyl-β-D-glucopyranosides, and methyl 2-acetamido-2-deoxy-6-O-β-D-galactopyranosyl-α-D-glucopyranoside (4). 2,5-Anhydrohexoses have been converted into alditol trideuteriomethyl ethers, alditol acetates, and aldononitriles. The importance of side reactions that lead to the formation of 2-deoxy-2-C-formylpentofuranosides is discussed.     

Quantitative methods for determining the points of O-(carboxymethyl) substitution in O-(carboxymethyl)-guar

Two methods are described for locating the O-(carboxymethyl) groups in O-(carboxymethyl)guar. In Method I, O-(carboxymethyl)guar was depolymerized by methanolysis, the O-(carboxymethyl) groups were reduced, and the mixture of methyl glycosides and O-(2-hydroxyethyl)-substituted methyl glycosides was converted into a mixture of per-O-acetylated alditols and partially O-(2-acetoxyethyl)ated, partially O-acetylated alditols. Analysis of these alditols by gas-liquid chromatography-mass spectrometry allowed the positions of substitution of the O-(carboxymethyl) groups on the galactosyl groups and mannosyl residues to be determined. However, this method did not distinguish between O-(carboxymethyl) substitution on 4-linked and 4,6-linked mannosyl residues. This limitation was overcome by the more-detailed analysis provided by Method II, in which O-(carboxymethyl)guar was carboxyl-reduced, the product methylated, the glycosyl residues hydrolyzed, the sugars reduced, and the alditols acetylated to yield a mixture of partially O-acetylated, partially O-methylated alditols and partially O-acetylated, partially O-(2-methoxyethyl)ated, partially O-methylated alditols. These derivatives, when separated and quantitated by g.l.c., and identified by g.l.c.-m.s., gave a quantitative measure of every type of carboxymethyl substitution in guar.     

Regioselective synthesis of novel 3-alkoxy (phenyl) thiophosphorylamido-2-(per-O-acetylglycosyl-1′-imino)thiazolidine-4-one derivatives from O-alkyl N-glycosyl(thiosemicarbazido)phosphonothioates

A series of 3-alkoxy(phenyl)thiophosphorylamido-2-(per-O-acetylglycosyl-1′-imino)thiazolidine-4-one derivatives were prepared by the reaction of 1-alkoxy(phenyl)thiophosphoryl-4-(per-O-acetylglycosyl) thiosemicarbazides with ethyl bromoacetate. ¹H/¹³C HMBC measurements corroborated by X-ray crystallographic results revealed the exclusive regioselectivity of these ring closures toward the N-2 position of the thiosemicarbazide moiety. The bioactivity data of 3a-k suggest that the thiazolidine-4-one ring is critical for the herbicidal and fungicidal activities.     

Enhancement of protonated molecular ions and ammonium·molecular ion complexes in direct-liquid-introduction-mass spectrometry of carbohydrates

Addition of ammonium acetate to the mobile phase in direct-liquid-introduction mass spectrometry enhances the abundance of the protonated molecular ion or ammonium·molecular ion complex for compounds of biological interest. The efficacy of the method was investigated by comparing mass spectra obtained, with and without ammonium acetate, for a variety of underivatized, per-O-acetylated, and per-O-alkylated carbohydrates, and for several underivatized peptides. The mass spectra of the per-O-alkylated carbohydrates obtained by direct-liquid-introduction mass spectrometry with ammonium acetate were also compared to those obtained by thermospray mass spectrometry.     

The complete structure of the trifoliin a lectin-binding capsular polysaccharide of Rhizobium trifolii 843

The complete structure of the acidic, extracellular, capsular polysaccharide of Rhizobium trifolii 843 has been elucidated by a combination of chemical, enzymic, and spectroscopic methods, confirming an earlier proposed sugar sequence and assigning the locations of the acyl substituents. The polysaccharide was depolymerized by a lyase into octasaccharide units which were uniform in carbohydrate composition and linkage. These units also contained a uniform distribution of acetyl and pyruvic acetal [O-(1-carboxyethylidene)] groups, and half of them were further acylated with d-3-hydroxybutanoyl groups. A much smaller proportion (<5%) of the oligomers was further acylated by a second d-3-hydroxy-butanoyl group. The locations of the substituents were determined chemically and by J-correlated, ¹H-n.m.r. spectroscopy, proton nuclear Overhauser effect (n.O.e.)_ measurements, doubie-resonance ¹H-n.m.r. spectroscopy, and ¹³C-n.m.r. spectroscopy. The composition and structure of the carbohydrate chain were determined by methylation analysis using g.l.c.-m.s. fast-atom-bombardment mass spectrometry, and n.m.r. studies on the reduced, deacylated oligomer. Structural studies were supplemented by n.m.r. analyses on the original polymer. The oligosaccharides were found to be branched octasaccharides with four sugar residues in each branch, and the carbohydrate sequence agreed well with that expected from earlier work. In the abbreviated sequence and structure (1a), the sugar residues are labelled “a” through “h”. The main chain (a–d) is composed of a 4-deoxy-α-l-threo-hex-4-enopyranosyluronic acid group (a) that is linked to O-4 of a 3-O-acetyl-d-glucosyluronic acid residue (b) which is β-linked to O-4 of a d-glucosyl residue (c). Residue c is β-linked to O-4 of the branching d-linked to O-4 of a d-glucosyl residue (d). The side chain consists of a substituted d-galactosyl group (h) which is β-linked to O-3 of residue 9 of a β-(1→4)-linked d-glucose trisaccharide (fragment e–f–g). The reducing end of the resulting tetrasaccharide (e–f–g–h) is β-linked to O-6 of the branching d-glucose residue (d). In the native polymer, this branching residue is α-linked to O-4 of the modified d-glucuronic acid residue (a) which is the unsaturated sugar in the oligomer. A small proportion of the O-2 atoms of the acetylated d-glucosyluronic acid residues is acetylated because of ester migration. The two terminal sugars (g and h) of the branch chain bear 4,6-O-(1-carboxyethylidene) groups. The d-galactosyl groups of half of the oligomers are acylated by d-3-hydroxybutanoyl groups at O-3. About 5% of the oligomers bear a second d-3-hydroxybutanoyl group at O-2 of the d-galactosyl group (h).     

Improved preparations of some per-O-acetylated aldohexopyranosyl cyanides

3,4,6-Tri-O-acetyl-1,2-O-[1-(exo-, endo-cyano)ethylidene]-α-d-galacto- (1a/b), -α-d-gluco- (2a/b), and -β-d-manno-pyranose (3a/b) were stereoselectively isomerized to the corresponding per-O-acetylated 1,2-trans-aldohexopyranosyl cyanides in 75, 16, and 62% yield, respectively, by treatment with boron trifluoride etherate in dry nitromethane. The corresponding per-O-acetylated 1,2-cis-aldohexopyranosyl cyanides were obtained concurrently in respective yields of 1.9, 0.9, and 4.8%. The per-O-acetylaldohexopyranosyl cyanide products were found stable to the reaction conditions and were readily isolated following completion of the rearrangement. It had previously been proved that reaction of 2,3,4,6-tetra-O-acetyl-α-d-manno- and -gluco-pyranosyl bromide with mercuric cyanide in nitromethane generates, in the ratio of ∼1:1, the desired 1,2-trans-glycosyl cyanides and the corresponding 1,2-O-(1-cyanoethylidene) isomers (3a/b and 2a/b, respectively). Treatment of these reaction-mixtures with boron trifluoride etherate in nitromethane effected the rearrangement of 3a/b and 2a/b, thereby facilitating the isolation, and increasing the overall yields, of the per-O-acetylated 1,2-trans-d-manno and -gluco-pyranosyl cyanides (58 and 30% total yield, respectively) relative to the earlier procedures. The boron trifluoride etherate-mediated reaction of per-O-acetyl-α- and -β-d-galacto, -α- and -β-d-gluco-, -α-d-manno-, and -2-deoxy-2-phthalimido-β-d-gluco-pyranoses with trimethylsilyl cyanide in nitromethane was also investigated. This reaction provides a “one-flask” synthesis of the corresponding per-O-acetylated 1,2-trans-aldohexopyranosyl cyanides in which 1,2-O-(1-cyanoethylidene) derivatives are isomerized in situ. Finally, improved preparations of the (not readily accessible) per-O-acetylated 1,2-cis-d-manno- and -gluco-pyranosyl cyanides are described. Thus, 2,3,4,6-tetra-O-acetyl-α- and -β-d-mannopyranosyl cyanide (48 and 16% total yield, respectively) and -α- and -β-d-glucopyranosyl cyanide (12 and 39% total yield, respectively) were synthesized by fusion of the corresponding -α-d-glycosyl bromides with mercuric cyanide.     

Preparation of some (1→6)-linked disaccharides,and their derivatives suitable for protein modification

Synthetic methods for the preparation of per-O-acetylated, (1→6)-linked disaccharides containing either a d-galactose or a d-glucose residue at the reducing end are described. In these methods, 1,2,3,4-tetra-O-acetyl-6-O-trityl-β-d-glucopyranose was first converted into 1,2,3,4-tetra-O-acetyl-β-d-glucopyranose (1) by rapid treatment with 90% trifluoroacetic acid, followed by rapid isolation designed to minimize O-acyl migration. Disaccharides were formed by glycosylation of 1 or 1,2:3,4-di-O-isopropylidene-d-galactopyranose with per-O-acetylglycosyl halides. Isopropylidene groups in the resulting disaccharide, if present, were removed, and the disaccharide was per-O-acetylated. Per-O-acetylated β-Gal-(1→6)-Glc and β-GlcNAc-(1→6)-Gal, and a mixture of per-O-acetylated α-Gal-(1→6)-Gal and β-Gal-(1→6)-Gal (in the ratio of 3:7) were thus obtained. The per-O-acetylated Gal-(1→6)-Gal disaccharides were converted, by a reaction sequence previously reported, into (2,2-dimethoxyethyl)aminocarbonylmethyl 1-thio-β-d-glycosides, which could then be coupled to proteins via reductive alkylation. For the anomeric mixture of per-O-acetylated Gal-(1→6)-Gal, conversion into the corresponding 1-thioglycoside permitted resolution of the isomers by chromatography on silica gel. When disaccharides, as borate complexes, were chromatographed on a column of a strong, anion-exchange resin, all of the (1→6)-linked disaccharides of neutral sugars tested (including melibiose) were eluted later than analogous disaccharides having other linkages, and also later than any neutral monosaccharides.     

Identification of ketoses by use of their peracetylated oxime derivatives: A G.L.C.-M.S. approach

A method based on peracetylated oxime (PAKO) derivatives has been developed for rapid g.l.c.-m.s. survey of ketoses. This derivatization procedure (and the chromatographic analysis of these derivatives) is identical to one previously employed to identify aldoses by means of peracetylated aldononitrile (PAAN) derivatives. The production of chemically different derivatives from the aldoses and ketoses by the same derivatization procedure greatly simplifies the chromatographic separation of the derivatives of the ketoses from those of the aldoses, and also results in distinctively different, mass-spectral fragmentation-pathways for the two sets of derivatives. Both the electron-impact (e.i.) and ammonia chemical-ionization (c.i.) mass spectra of PAKO derivatives have been examined. Extensive differences between the fragmentation-pathways of the PAAN and the PAKO derivatives have been observed both by e.i.m.s. and ammonia c.i.m.s. The g.l.c.-m.s. of these PAKO derivatives, in conjunction with various, isotopic variants of the derivatization process, can yield extensive structural information with regard to the starting saccharides associated with the known, or unknown, g.l.c. peaks. The g.l.c. and mass-spectral properties of highly O-methylated PAKO derivatives of d-fructose are compared, and contrasted, to those of the PAKO derivatives of non-O-methylated saccharides. The chromatographic properties of derivatives of oligosaccharides that result from the PAAN-PAKO derivatization procedure have also been studied.     

Evidence that the acidic polysaccharide secreted by Agrobacterium radiobacter (ATCC 53271) has a seventeen glycosyl-residue repeating unit.

The extracellular anionic polysaccharide produced by the bacterium Agrobacterium radiobacter (ATCC 53271) contains D-galactose, D-glucose, and pyruvic acid in the molar ratio 2:15:2. Analysis of the methylated polysaccharide indicated the presence of terminal, non-reducing glucosyl, 3-, 4-, 6-, 2,4-, and 4,6-linked glucosyl residues, 3-linked 4,6-O-[(S)-1-carboxyethylidene]glucosyl residues, and 3-linked galactosyl residues. Partial acid hydrolysis of the methylated polysaccharide, followed by reduction with NaB2H4 and then O-ethylation, gave a mixture of alkylated oligoglycosyl alditols that were separated by reversed-phase h.p.l.c. and analyzed by 1H-n.m.r. spectroscopy, g.l.c.-m.s., and glycosyl-linkage composition analysis. Smith degradation of the polysaccharide gave three diglycosyl alditols that were separated by semi-preparative, high-pH anion-exchange chromatography, and were analyzed by 1H-n.m.r. spectroscopy, g.l.c.-m.s., and glycosyl-linkage composition analysis. The polymer obtained by NaBH4 reduction of the periodate-oxidized polysaccharide was methylated, and the noncyclic acetals were hydrolyzed with aq. 90% formic acid to generate a mixture of partially O-methylated mono- and di-glycosyl alditols. The partially O-methylated oligoglycosyl alditols were O-ethylated. The resulting alkylated oligoglycosyl alditols were separated by reverse-phase h.p.l.c. and then characterized by 1H-n.m.r. spectroscopy, g.l.c.-m.s., and glycosyl-linkage composition analysis. The results from the studies described here provide strong evidence that the acidic polysaccharide secreted by A. radiobacter (ATCC 53271) has a heptadecasaccharide repeating unit.     

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.     

Brassinolide-2,3-acetonide: A brassinolide-induced rice lamina joint inclination antagonist

A novel chemical tool compound that is an antagonist of brassinolide (BL, 1)-induced rice lamina joint inclination was developed. Although 2-O-, 3-O-, 22-O-, or 23-O-methylation of BL causes a critical decrease in biological activity,⁵ a crystal structure of the extracellular leucine-rich repeat (LRR) domain of BRASSINOSTEROID-INSENSITIVE I (BRI1) bound to BL3, 4 indicates that the loss of activity of the O-methylated BL may result from not only the low affinity to BRI1, but also from blocking the interaction with another BR signaling factor, a partner protein of BRI1 (e.g., BRI1-ASSOCIATED KINASE 1, BAK1). On the basis of this hypothesis we synthesized the BL 2,3-acetonide 2, the 22,23-acetonide 3, and the 2,3:22,23-diacetonide 4 to assess the possibility of 2-O- and 3-O- or/and 22-O- and 23-O-alkylated BL as an antagonist in BR signaling evoked by exogenously applied BL. The 2,3-acetonide 2 more strongly inhibited the lamina inclination caused by BL relative to the 22,23-acetonide 3, whereas the diacetonide 4 had no effect most likely due to its increased hydrophobicity. This suggested that the 2,3-hydroxyl groups of BL play a more significant role in the interaction with a BRI1 partner protein rather than BRI1 itself in rice lamina joint inclination. Taken together it was demonstrated that BL, the most potent agonist of BRI1, is transformed into an antagonist by functionalization of the 2,3-dihydroxyl groups as the acetonide. This finding opens the door to the potential development of a chemical tool that modulates protein–protein interactions in the BR signaling pathway to dissect the BR-dependent processes.     

Structural studies of an extracellular polysaccharide (S-198) elaborated by Alcaligenes ATCC 31853

The structure of an extracellular polysaccharide, S-198, elaborated by Alcaligenes ATCC 31853 has been investigated; methylation analysis, specific degradations, and ¹H-n.m.r. spectroscopy were the main methods used. It is suggested that the polysaccharide is composed of “repeating units” with the structure

A sugar residue in the chain may be either L-rhamnose or L-mannose and only ≈50% of the residues contain the branching α-L-rhamnopyranosyl group. The polysaccharide further contains O-acryl groups. It belongs to a group of polysaccharides, elaborated by Alcaligenes and Pseudomonas species, which all have the same linear backbone (except that some of them do not contain L-mannose) without branching or with branches that differ in their chemical structures and/or positions.     

Preparation and structure of an unusual dimeric furan from the acid decomposition of isomaltol

Isomaltol (1), an enolic nonenzymic browning-product, decomposes in dilute acid to form the new red-orange colored, symmetrical dimer, (E)-2-[1-(3-hydroxy-2-furanyl)ethylidene]-(2H)-furan-3-one (2). Compound 2 was obtained in 20.4% yield with toluenesulfonic acid (⩾3m) at 50°. The structure for 2 was assigned on the basis of spectral data (m.s., u.v., i.r., ¹³C- and ¹H-n.m.r.) and conversion into its mono-O-acetyl derivative (3).     

Deamination of 2-amino-2-deoxyhexitols and of their per-O-methylated derivatives with nitrous acid

The products of nitrous acid deamination of per-O-methylated 2-amino-2-deoxy-d-glucitol and $\text{2-}\text{amino}\text{-2-}\text{deoxy}\text{-3-O-β-}\text{d}\text{-galactopyranosyl-}\text{d}\text{-glucitol}$ and its per-O-methylated derivative have been characterized by g.l.c.—mass spectrometry after treatment with sodium borodeuteride and further substitution by acetylation, methylation, or (trideuteriomethyl)ation. The results confirm that the most important reaction pathway (1) involves a 1 → 2-hydride shift to give $\text{2-}\text{deoxy-}\text{d}\text{-arabino-}\text{hexoses}$, but that significant side-reactions include (2) solvolytic displacement at C-2, (3) a 3 → 2-hydride shift, to give $\text{2-}\text{deoxy-}\text{d}\text{-erythro-3-}\text{hexuloses}$, and (4) a C-4→C-2 migration to give $\text{2-}\text{deoxy}\text{-2-C-}\text{(hydroxymethyl)-}\text{d}\text{-ribose}$ and -d-arabinose. Reactions (3) and (4) result in elimination of the original 3-O-substituents, with the exposure of new reducing groups, from oligosaccharides terminated by 3-O-substituted 2-amino-2-deoxyhexitols.     

The action of alkali on di-O-(2-bromoethylidene)-d-mannitols: Formation of an unusually acid-stable acetal linkage

When cis,trans-1,2:5,6-di-O-(2-bromoethylidene)-d-mannitol and cis,cis-1,2:5,6-di-O-(2-bromoethylidene)-d-mannitol were treated with dilute, boiling sodium hydroxide, 5,6-O-(S)-(2-bromoethylidene)-3:1,2-O-[(R)-1-ethanyl-2-ylidene]-d-mannitol (3) and 3:1,2;4:5,6-di-O-[(R)-1-ethanyl-2-ylidene]-d-mannitol (10) were produced; the structures were established by a combination of chemical transformations, ¹H-n.m.r. spectroscopy, and mass spectrometry. The bicyclo ether-acetal linkage in 3 and 10 proved unusually resistant to hydrolysis by acid.     

Sucrochemistry : Part X. 1′,4,6′-tri-O-mesylsucrose penta-acetate: A comparison of the reactivity at the 4 and 1′ positions

The 1′,4,6′-trisulphonate 2, obtained by mesylation of sucrose 2,3,3′,4′,6-penta-acetate (1), undergoes nucleophilic substitution with sodium benzoate in hexamethylphosphoric triamide at positions 1′,4, and 6′ to give 1,6-di-O-benzoyl-β-D-fructofuranosyl 4-O-benzoyl-α-D-galactopyranoside penta-acetate (3), and selectively at positions 4 and 6′ to give 6-O-benzoyl-1-O-mesyl-β-D-fructofuranosyl 4-O-benzoyl-α-D-galactopyranoside penta-acetate (4). The products 3 and 4 were identified from their ¹H-n.m.r. spectra and by O-deacylation to give β-D-fructofuranosyl α-D-galactopyranoside (5) and its 1-methanesulphonate 6, respectively. Treatment of the trisulphonate 2 with sodium azide gave analogous products, namely, 1,6-diazido-1,6-dideoxy-β-D-fructofuranosyl 4-azido-4-deoxy-α-D-galactopyranoside penta-acetate (8) and 6-azido-6-deoxy-1-O-mesyl-β-D-fructofuranosyl 4-azido-4-deoxy-α-D-galactopyranoside penta-acetate (7).