Synthesis of galactofuranose disaccharides of biological significance

Methyl beta-D-galactofuranoside was readily obtained by tin(IV) chloride-catalyzed glycosylation of penta-O-benzoyl-alpha,beta-D-galactofuranose, followed by debenzoylation with sodium methoxide. Glycosylation of 1 with 2,3,5-tri-O-benzoyl-D-galactono-1,4-lactone or with the 6-O-trityl-lactone derivative 5 gave the benzoylated beta-D-galactofuranosyl-(1----6)-D-galactono-1,4-lactone 6 in excellent yield. The structure of disaccharide 6 was confirmed by borohydride reduction to the glycosyl-alditol 7. A byproduct of the condensation reaction of 1 with 4 or 5 was identified as the benzoylated (1----1)-beta,beta'-D-galactofuranosyl disaccharide 8. Compound 8 was readily prepared (88% yield) by controlled addition of water to 1, in the presence of stannic chloride. O-Debenzoylation of 8 afforded crystalline beta'-D-galactofuranosyl-(1----1)-beta-D-galactofuranoside. The glycosyl-lactone 6 constitutes a key intermediate for the synthesis of a disaccharide derivative having both units in the furanoid form. Thus, diisoamylborane reduction of the lactone function of 6 led to the disaccharide derivative 10, from which the methyl glycoside 12 was prepared. O-Debenzoylation of 12 gave the corresponding methyl beta-D-galactofuranosyl-(1----6)-beta-D-galactofuranoside. The free disaccharide beta-D-Galf-(1----6)-D-Galp and its acetylated derivative were also synthesized from 10.     

Cross metathesis for the synthesis of novel C-sialosides

Cross metathesis of both anomers of C-allyl sialoside (3alpha/3beta) with styrene catalyzed by the second generation Grubbs or Hoveyda-Grubbs catalysts gave the corresponding aryl derivatives (4alpha/4beta) in virtually quantitative yields. The products were hydrogenated to model compounds 5alpha/5beta. Similarly, reaction of the alpha-anomer 3alpha with galactose derivative 8 gave the olefin-linked disaccharide mimetic 9. Following hydrogenation and deprotection, the ethylene-bridged Neu5Acalpha(2-->6)Gal analogue 11 could be obtained.     

Mechanism for enterohepatic injury caused by circulatory disturbance of hepatic vessels in the rat

We reported previously that a transient occlusion followed by reperfusion of the portal vein and the hepatic artery of the rat significantly decreased the transhepatic transport of a cholephilic compound, and that this decrease was prevented by pretreating animals with poly(styrene co-maleic acid butyl ester)-conjugated superoxide dismutase (SM-SOD). To elucidate the mechanism for oxidative injury of the liver and the site for the generation of superoxide radicals, the effect of a portosystemic bypass on the liver function was examined in the rat whose hepatic vessels were temporarily occluded. A portosystemic bypass inhibited the reperfusion-induced decrease in hepatic transport of bromosulfophthalein as effectively as did SM-SOD. Kinetic analysis using 125I-labeled albumin revealed that the permeability of the small intestine markedly increased after a transient occlusion. The increase in intestinal permeability was also inhibited either by SM-SOD or by the portosystemic bypass. Xanthine oxidase activity in portal plasma markedly increased during occlusion and reperfusion, while it remained within normal ranges in the bypassed group. Thus, superoxide radical, and/or its metabolite(s), might play a critical role in increasing the intestinal permeability and in the pathogenesis of reperfusion-induced liver injury.     

The immobilization of enzymes and cells of Bacillus stearothermophilus onto poly(maleic anhydride/styrene)-Co-polyethylene and poly(maleic anhydride/vinyl acetate)-Co-polyethylene

The graft copolymer, poly(maleic anhydride/styrene)-co-polyethylene was prepared. The copolymer immobilized bovine serum albumin (BSA), but the amount coupled appeared to be effected by the amount of styrene in the graft copolymer, temperature, and pH of the coupling medium. Competition existed between hydrolysis of the grafted anhydride groups and the protein. A graft copolymer with 66% add-on immobilized 4.5 mg/glucose oxidase/g copolymer, 4.6 mg alkaline phosphates/g copolymer and 0.2 mg cell of Bacillus stearothermophilus/g copolymer. A number of copolymers containing poly(maleic anhydride/vinyl acetate)-co-polyethylene were prepared to cover a range of grafting levels. These immobilized larger quantities of BSA, alkaline phosphatase, and cells of B. stearothermophilus than did the styrene graft copolymer. The copolymer was also hydrolyzed to release the hydroxyl group from the poly(vinyl acetate) component of the grafted chains. Using p-benzoquinone as the "activating agent," the copolymer coupled to BSA and to acid phosphatase. Using p-toluene-sulfonyl chloride, the copolymer was very effective in immobilizing trypsin.     

Studies on the carbohydrate moiety of α1-acid glycoprotein (orosomucoid) by using alkaline hydrolysis and deamination by nitrous acid

Alkaline hydrolysis followed by deamination with nitrous acid was applied for the first time to a glycoprotein, human plasma alpha(1)-acid glycoprotein (orosomucoid). This procedure, which specifically cleaves the glycosaminidic bonds, yielded well-defined oligosaccharides. The trisaccharides, which were obtained from the native protein, consisted of a sialic acid derivative, galactose and 2,5-anhydromannose. The linkage between galactose and 2,5-anhydromannose is most probably a (1-->4)-glycosidic bond. A hitherto unknown linkage between N-acetylneuraminic acid and galactose was also established, namely a (2-->2)-linkage. The three linkages between sialic acid and galactose described in this paper appear to be about equally resistant to mild acid hydrolysis. The disaccharide that was derived from the desialized glycoprotein consisted of galactose and 2,5-anhydromannose. Evidence was obtained for the presence of a new terminal sialyl-->N-acetylglucosamine disaccharide accounting for approximately 1mol/mol of protein. The presence of this disaccharide may explain the relatively severe requirements for the complete acid hydrolysis of the sialyl residues. The present study indicates that alkaline hydrolysis followed by nitrous acid deamination in conjunction with gas-liquid chromatography will afford relatively rapid determination of the partial structure of the complex carbohydrate moiety of glycoproteins.     

Synthesis of the alpha-D-GlcpA-(1-->3)-alpha-L-Rhap-(1-->2)-L-Rha trisaccharide isolated from the cell wall hydrolyzate of the green alga, Chlorella vulgaris

The title trisaccharide was synthesized from 6-O-acetyl-2,3,4-tri-O-benzyl-alpha-D-glucopyranosyl chloride (10), ethyl 2,4-di-O-benzyl-1-thio- (5) and benzyl 3,4-di-O-benzyl-alpha-L-rhamnopyranoside (9). The disaccharide 11 obtained from compounds 5 and 10 was used as the glycosyl donor to glycosylate the rhamnopyranoside derivative 9 having free OH-2 using the NIS-AgOTf-mediated glycosylation methodology. Zemplén deacetylation of the trisaccharide 12 resulted in the 6"-OH derivative (13), which was selectively oxidized with CrO3 to the uronic acid derivative 14. The benzyl groups were removed by catalytic hydrogenolysis to furnish the target trisaccharide (1).     

Facile synthesis of Nalpha-protected-L-alpha,gamma-diaminobutyric acids mediated by polymer-supported hypervalent iodine reagent in water.

Hofmann rearrangement of Nalpha-Boc-L-Gln-OH mediated by a polymer-supported hypervalent iodine reagent poly[(4-diacetoxyiodo)styrene] (PSDIB) in water afforded Nalpha-Boc-L-alpha,gamma-diaminobutyric acid (Boc-Dab-OH, 1) in 87% yield. Nalpha-Z-derivative (Z-Dab-OH, 2) was prepared with PSDIB in 83% yield. Since the reaction of Nalpha-Fmoc-Gln-OH by this procedure did not proceed because of the insolubility of Fmoc-Gln-OH in aqueous media, we synthesized Fmoc-Dab(Boc)-OH (5) from 2 in 54% yield. Polymyxin B heptapeptide (PMBH) which contains four Dab residues was successfully synthesized in a solution-phase synthesis.     

A simple method for the assay of colistin in human plasma, using pre-column derivatization with 9-fluorenylmethyl chloroformate in solid-phase extraction cartridges and reversed-phase high-performance liquid chromatography

A simple, selective and sensitive high-performance liquid chromatographic (HPLC) method is described for the determination of colistin in human plasma. Derivatization with 9-fluorenylmethyl chloroformate was performed in the same solid-phase extraction C₁₈ cartridge used for sample pre-treatment, followed by reversed-phase HPLC with fluorimetric detection. Quantification was achieved using the ratio of the summed peak areas of colistin A and B derivatives to that of the derivative of netilmicin (internal standard). Linear calibration curves were obtained within the concentrations of colistin sulfate from 0.10 to 4.0 mg/l in plasma. Accuracy was within 10% and reproducibility (RSD) was less than 10%.     

Synthetic mucin fragments: methyl 3-O-(2-acetamido-2-deoxy-beta-D-galactopyranosyl-beta-D- 3-O-(2-acetamido-2-deoxy-3-O-beta-D-galactopyranosyl- beta-D-glucopyranosyl)-beta-D-galactoyranoside. pyranosyl)-beta-D-galactopyranoside

Methyl 2,4,6-tri-O-benzyl-beta-D-galactopyranoside (5) was obtained crystalline by way of its 3-O-allyl derivative, which was in turn obtained by ring-opening of a presumed 3,4-O-stannylene derivative of methyl beta-D-galactopyranoside, followed by benzylation. Condensation of 5 with 2-methyl-(2-acetamido-3,4,6-tri-O-acetyl-1,2-dideoxy-beta-D-glucopyra no)-[2,1-d]-2-oxazoline in 1,2-dichloroethane in the presence of p-toluenesulfonic acid afforded the disaccharide derivative methyl 3-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glucopyranosyl)-2, 4,6-tri-O-benzyl-beta-D-galactopyranoside (6) Deacetylation of 6 in methanolic sodium methoxide afforded the disaccharide derivative 7, which was acetalated with alpha, alpha-dimethoxytoluene to afford the 4',6'-O-benzylidene acetal (10). Catalytic hydrogenolysis of the benzyl groups of 7 afforded the title disaccharide 8. Glycosylation of 10 with 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl bromide in 1:1 benzene-nitromethane in the presence of mercuric cyanide gave the fully protected trisaccharide derivative 12. Systematic removal of the protecting groups of 12 then furnished the title trisaccharide 14. The structures of 5, 8, and 14 were all confirmed by 13C-n.m.r. spectroscopy. The 13C-n.m.r. chemical shifts for methyl alpha- and beta-D-galactopyranoside, and also those of their 3-O-allyl derivatives, are recorded, for the sake of comparison, in conjunction with those of compound 5.     

Linear copolymeric poly(thia-alkanedioates) by lipase-catalyzed esterification and transesterification of 3,3'-thiodipropionic acid and its dimethyl ester with alpha,omega-alkanediols

Linear copolymeric polyesters (polyoxoesters) containing thioether functions [poly(3,3'-thiodipropionic acid-co-alpha,omega-alkanediols)] were formed in good yield by esterification of an equimolar mixture of 3,3'-thiodipropionic acid (4-thiaheptane-1,7-dioic acid) and 1,6-hexanediol (weight average molecular mass, M(W) >600 Da: approximately 81% after 6 h) or 1,12-dodecanediol (M(W) > 900 Da: approximately 90% after 6 h) catalyzed by immobilized lipase B from Candida antarctica (Novozym 435) for up to 336 h in moderate vacuo without a solvent or drying reagent in the reaction mixture. Poly (3,3'-thiodipropionic acid-co-1,6-hexanediol) and poly (3,3'-thiodipropionic acid-co-1,12-dodecanediol) were extracted from the reaction mixtures using tetrahydrofurane and precipitated from tetrahydrofurane-iso-hexane (1:1, v/v) at approximately 0 degrees C. The precipitate of poly(3,3'-thiodipropionic acid-co-1,6-hexanediol) showed a maximum molecular weight of 6 x 10(5) Da corresponding to a M(W) of approximately 24,200 Da and a degree of polymerization of up to 2,150 monomer units. The precipitated poly(3,3'-thiodipropionic acid-co-1,12-dodecanediol) showed a maximum molecular weight of 8 x 10(5) Da corresponding to a M(W) of approximately 27,200 Da and a maximum degree of polymerization of up to 2,200 monomer units. The chemical structures of both polyesters containing thioether functions were confirmed by chemical derivatization and NMR spectrometry. The chemical structures of various low-molecular weight reaction intermediates of the esterification of 3,3'-thiodipropionic acid with 1,6-hexanediol were elucidated by GC-MS.     

Synthesis and protein binding properties of T-antigen containing GlycoPAMAM dendrimers.

Allyl O-(beta-D-galactopyranosyl)-(1-3)-2-acetamido-2-deoxy-alpha-D-galactopyranoside (8) was prepared in excellent yield from the corresponding galactosyl bromide (6, 7) and allyl 2-acetamido-4,6-benzylidene-2-deoxy-alpha-D-galactopyranoside (5) using Hg(CN)2 as a promoter. Compound 5 was obtained from N-acetylglucosamine 1 following sequential protecting group strategy and C-4 epimerization as a key step. Carboxylic acid functionalized T-antigen derivative 15, obtained by radical addition of 3-mercaptopropionic acid to allyl disaccharide 10, was conjugated to PAMAM dendritic cores 13-16 by an efficient amide coupling strategy using TBTU. GlycoPAMAM dendrimers having T-antigen residues with 4, 8, 16 and 32 valencies (17-20) were obtained in 73 to 99% yields. Their protein binding properties were demonstrated using peanut lectin from Arachis hypogaea and a mouse monoclonal IgG antibody. The higher valency conjugates generated stronger binding interactions indicating a cluster effect. The inhibitory potential of these glycoPAMAM conjugates toward antibody-coating antigen interactions was enhanced up to 3800 times over that of the monomeric T-antigen residue (10).     

Enzymatic synthesis and characterization of oligosaccharides structurally related to the repeating unit of Pullulan

A trisaccharide (Glcalpha1-4Glcalpha1-6Glc) and a tetrasaccharide (Glcalpha1-4Glcalpha1-4Glcalpha1-6Glc) the structures of which are related to that of repeating unit of pullulan have been obtained, exploiting the transglycolytic activity of Aspergillus niger cyclodextrin glucanotransferase. Both products were obtained in one-pot reaction using as a donor the alpha-cyclodextrin and as an acceptor the disaccharide isomaltose. The regioselectivity of the reaction was 85% for the tetrasaccharide and 80% for the trisaccharide. The yield of reaction resulted to be 42% for the synthesis of trisaccharide and 25% for that of tetrasaccharide. Purification of products was performed by size exclusion chromatography and by semipreparative reverse phase HPLC after reversible derivatization with 2-aminopyridine. Structural characterization was performed by capillary electrophoresis, ion-spray mass spectrometry, and by 13C-NMR spectroscopy. A comparison of these results with those obtained by using alpha-D-glucosidase, which had been effective for the synthesis of the disaccharide isomaltose, is reported.     

The alkali-labile linkage between keratan sulphate and protein

Keratan sulphate was isolated from adult intervertebral disc in 90% yield by sequential digestion of the whole tissue with papain, Pronase and Proteus vulgaris chondroitin sulphate lyase. Treatment of this preparation with alkali cleaved a glycosidic bond between N-acetylgalactosamine and threonine and produced, by an alkali-catalysed ;peeling' reaction, an unsaturated derivative of N-acetylgalactosamine which reacted as a chromogen in the Morgan-Elson reaction, but remained covalently bonded to the keratan sulphate chain. This derivative was reduced and labelled by alkaline NaB(3)H(4). The substituent at position 3 of N-acetylgalactosamine in the keratan sulphate-protein linkage was identified as a disaccharide, N-acetylneuraminylgalactose, which was isolated from the reaction mixture after alkali treatment.     

Changes in the antioxidant properties of substituted phenol polydisulfides during interaction with human serum albumin

Gallic acid polydisulfide and poly(2-aminodisulfide-4-nitrophenol) in aqueous solutions were shown to form polycomplexes with human serum albumin. This process was accompanied by considerable changes in the spectrum of protein circular dichroism recorded in distilled water in the far UV range at 20 degrees C. Complex formation between human serum albumin and polydisulfides was followed by a marked decrease in the content of alpha-helices and increase in the count of antiparallel beta-structures in the protein. Stable complexes containing 1.5, 2.8, and 7.7 poly(2-aminodisulfide-4-nitrophenol) molecules per human serum albumin molecule were formed in bicarbonate buffer (pH 9.0). In these complexes, the secondary protein structure underwent changes similar to those in polycomplexes of human serum albumin and polydisulfides. Gallic acid polydisulfide and poly(2-aminodisulfide-4-nitrophenol) inhibited the catalase-induced degradation of 50 mM H2O2. Complexes of human serum albumin and poly(2-aminodisulfide-4-nitrophenol) increased the catalytic activity and operational stability of catalase 1.5 and 4-7-fold, respectively. This was characterized by the effective reaction rate constant (kin, s-1). Our results indicate that complexes of human serum albumin and substituted phenol polydisulfides act as potent protectors and activators of catalase during enzymatic degradation of H2O2 at high concentrations.     

Synthesis of a spacer-linked derivative of heptopyranosyl(α1-3)heptopyranose expressed in lipooligosaccharide and lipopolysaccharide

Glycosylation of penta-O-acetyl heptopyranosyl trichloroacetimidate with the 3-OH acceptor, methyl 2-O-benzyl-4,6-O-benzylidene-7,8-dideoxy-α-D-manno-oct-7-enopyranoside, gave the desired α1-3-linked disaccharide in a 94% yield. The oct-enopyranoside moiety of the disaccharide was converted to the heptoside by oxidative cleavage with osmium tetroxide/NaIO(4) and subsequent reduction with NaBH(4). The resulting α1-3-linked heptose disaccharide was converted to a tricholoroacetaimidate derivative containing a benzoyl group at C-2. This donor was glycosylated with 2-(carbobenzoxyamino)-1-ethanol to give an α spacer-linked disaccharide derivative in a 90% yield. Zemplén deacylation of the derivative and subsequent hydrogenolysis gave a 2-aminoethyl glycoside of heptopyranosyl(α1-3)heptopyranose.     

Production of anti-(ADP-ribose) antibodies with the aid of a dinucleotide-pyrophosphatase-resistant hapten and their application for the detection of mono(ADP-ribosyl)ated polypeptides

Previous attempts to produce anti-(ADP-ribose) antibodies by immunization of rabbits with ADP-ribose conjugated to serum albumin had resulted in the production of 5'AMP-specific antibodies [Bredehorst et al. (1978) Eur. J. Biochem. 82, 105-113]. To obtain true anti-(ADP-ribose) antibodies an antigen was constructed that was resistant to enzymic degradation at the pyrophosphate group. The enzymically active beta-methylene derivative of NAD (NAD[CH2]) was synthesized from ADP containing a methylene bridge (CH2) instead of an oxygen in the diphosphate group. NAD[CH2] was converted to its N6-[(2-carboxyethyl)thiomethyl] derivative and hydrolyzed to the corresponding ADP[CH2]-ribose derivative which was then coupled to bovine serum albumin. The antibodies obtained with this antigen were specific for free or protein-bound ADP-ribose groups, except for a cross-reaction with FAD, AMP, ADP, ATP or poly(ADP-ribose) interfered with [3H]ADP-ribose tracer binding only at higher concentrations. No interference was observed with poly(A), RNA and DNA at 6000-fold excess. The antibodies were purified on a novel type of affinity matrix. This was formed from NAD and guanidinobutyrate by a cholera-toxin-catalyzed reaction and the product, ADP-ribosyl guanidinobutyrate, was bound to Affi Gel by carbodiimide-aided condensation. The purified antibodies allowed the detection of ADP-ribose conjugated to polypeptides in amounts lower than 1 pmol as demonstrated by immunoblotting of [14C]ADP-ribosylated elongation factor 2. They also could be used to observe in situ, by indirect immunofluorescence, the increased mono(ADP-ribosyl)ation of nuclear proteins in dimethyl-sulfate-treated cells, and to show that histone H2B was the principal histone acceptor of single ADP-ribose groups in alkylated 3T3 cells.     

Synthesis of 2-acrylamidoethylglycosides of 3-O-(beta-D- glucopyranosyluronic acid)-alpha-L-rhamnose and 3-O-(alpha-L- rhamnopyranosyl)-beta-D-glucopyranuronic acid and copolymeric artificial antigens based on them

The synthesis of disaccharide repeating units, D-GlcA-(beta 1----3)-L-Rha (fragment A) and L-Rha-(alpha 1----3)-D-GlcA (fragment B), of the K54-antigenic polysaccharide from uropathogenic Escherichia coli 06:K54:H10 is described. Essential stages of the synthesis of fragment A involved the glycosylation of methyl 2,4-di-O-benzoyl-alpha-L-rhamnopyranoside followed by acetolysis of the methyl bioside obtained and further transformation into 2-(benzyloxycarbonylamino)ethyl glycoside; deprotection and, finally, conversion into 2-(acrylamido)ethyl glycoside. Selective opening of lactone ring in 2-azidoethyl 2,4-di-O-acetyl-beta-D-glucopyranoside-6,3-lactone was used for deprotection of 3-OH group in the synthesis of fragment B. Rhamnosylation of the glucuronic acid derivative thus obtained followed by transformation into 2-(acrylamido)ethyl glycoside and deprotection gave fragment B. Both fragments A and B were converted into artificial antigens of copolymer type.     

A novel glycoside hydrolase family 105: the structure of family 105 unsaturated rhamnogalacturonyl hydrolase complexed with a disaccharide in comparison with family 88 enzyme complexed with the disaccharide

YteR, a hypothetical protein with unknown functions, is derived from Bacillus subtilis strain 168 and has an overall structure similar to that of bacterial unsaturated glucuronyl hydrolase (UGL), although it exhibits little amino acid sequence identity with UGL. UGL releases unsaturated glucuronic acid from glycosaminoglycan treated with glycosaminoglycan lyases. The amino acid sequence of YteR shows a significant homology (26% identity) with the hypothetical protein YesR also from B. subtilis strain 168. To clarify the intrinsic functions of YteR and YesR, both proteins were overexpressed in Escherichia coli, purified, and characterized. Based on their gene arrangements in genome and enzyme properties, YteR and YesR were found to constitute a novel enzyme activity, "unsaturated rhamnogalacturonyl hydrolase," classified as new glycoside hydrolase family 105. This enzyme acts specifically on unsaturated rhamnogalacturonan (RG) obtained from RG type-I treated with RG lyases and releases an unsaturated galacturonic acid. The crystal structure of YteR complexed with unsaturated chondroitin disaccharide (UGL substrate) was obtained and compared to the structure of UGL complexed with the same disaccharide. The UGL substrate is sterically hindered with the active pocket of YteR. The protruding loop of YteR prevents the UGL substrate from being bound effectively. The most likely candidate catalytic residues for general acid/base are Asp143 in YteR and Asp135 in YesR. This is supported by three-dimensional structural and site-directed mutagenesis studies. These findings provide molecular insights into novel enzyme catalysis and sequential reaction mechanisms involved in RG-I depolymerization by bacteria.     

Design of polypeptide-functionalized polystyrene microspheres

In this contribution, the principle of spontaneous surface segregation has been applied for the preparation of polypeptide-functionalized polystyrene microspheres. For that purpose, an amphiphilic diblock copolymer was introduced in the mixture styrene/divinylbenzene and polymerized using AIBN as initiator. During the polymerization, cross-linked particles were obtained in which the diblock copolymer was encapsulated. The amphiphilic diblock copolymers used throughout this study contain a hydrophilic polypeptide segment, either poly(L-lysine) or poly(L-glutamic acid) and a hydrophobic polystyrene block. After 4 h of polymerization, rather monodisperse particles with sizes of approximately 3-4 microm were obtained. Upon annealing in hot water, the hydrophilic polypeptides migrate to the interface, hence, either positively charged or neutral particles were obtained when poly(L-lysine) is revealed at the surface and exposed to acidic or basic pH, respectively. On the opposite, negatively charged particles were achieved in basic pH water by using poly(L-glutamic acid) as additive. The surface chemical composition was modified by changing the environment of the particles. Thus, exposure in toluene provoked a surface rearrangement, and due to its affinity, the polystyrene block reorients toward the interface.     

N.m.r. studies of the disulphated disaccharide obtained by degradation of bovine lung heparin with nitrous acid

The disulphated disaccharide IdoA(2SO3)-anManOH(6SO3) was prepared from bovine lung heparin by treatment with nitrous acid followed by borohydride reduction. The 1H- (400 MHz) and 13C-n.m.r. (100 MHz) spectra of this disaccharide derivative have been assigned completely using homonuclear spin-decoupling experiments, 13C-1H correlations, and a COSY-45 two-dimensional homonuclear correlation experiment. The 3JH,H values show that the IdoA(2SO3) residue exists in a single conformation throughout the temperature range 20-90 degrees.