Chemoenzymatic synthesis of homogeneous chondroitin polymers and its derivatives.

Chondroitin sulfate is a linear glycosaminoglycan widely distributed as an important extracellular matrix component of mammalian cells. It participates in numerous pathological processes, however, illustration of its diverse biological roles is hampered by the unavailability of structurally defined chondroitin polymers and their derivatives. Herein, we report a novel homogeneous chondroitin polymers synthetic strategy which combines stepwise oligosaccharides synthesis with one-pot homogeneous chondroitin chain polymerization. Exogenous trisaccharide was proved to be the necessary acceptor for PmCS-catalyzed homogeneous chondroitin polymers synthetic reactions. The strategy exhibited a well-controlled relationship between the final sugar chain length and the molar ratios of reaction substrates that could synthesize homogenous chondroitin polymers with unprecedented narrow molecular weight distribution. More importantly, the strategy was further expanded to synthesis of unnatural zwitterionic and N-sulfonated chondroitin polymers by incorporation of sugar nucleotide derivatives into the synthetic approach.

Structural comparison, antioxidant and anti-inflammatory properties of fucosylated chondroitin sulfate of three edible sea cucumbers.

Three fucosylated chondroitin sulfate (fCS) were obtained from edible sea cucumbers Apostichopus japonicus, Stichopus chloronotus and Acaudina molpadioidea collected from China. fCS from Stichopus chloronotus was firstly reported. The detailed structures of fCSs, particularly the fucose branches, were investigated and compared. 1H and 13C NMR of the polysaccharide identified three sulfation patterns of fucose branches: 4-O-, 2,4-di-O, and 3,4-di-O-sulfation variously existed in different fCSs. The backbone structure was confirmed by the monosaccharide composition and two-dimensional NMR. Antioxidant properties of fCSs were evaluated by the scavenging abilities on 1,1-diphenyl-2-picrylhydrazyl, nitric oxide radicals and inhibition of lipid peroxidation. The results showed that their activities could be affected by the sulfation patterns of the fucose branches, and O-4 sulfation is particularly important for its activities. The anti-inflammatory assays of fCS-Am showed significant reduction of the carrageenan induced edema in a dose depended manner, which could be used as a potential antiallergic agent.

Effects of cytotoxic cis- and trans-diammine monochlorido platinum(II) complexes on selenium-dependent redox enzymes and DNA.

Here we present the preparation of 14 pairs of cis- and trans-diammine monochlorido platinum(II) complexes, coordinated to heterocycles (i.e., imidazole, 2-methylimidazole and pyrazole) and linked to various acylhydrazones, which were designed as potential inhibitors of the selenium-dependent enzymes glutathione peroxidase 1 (GPx-1) and thioredoxin reductase 1 (TrxR-1). However, no inhibition of bovine GPx-1 and only weak inhibition of murine TrxR-1 was observed in in vitro assays. Nonetheless, the cis configured diammine monochlorido Pt(II) complexes exhibited cytotoxic and apoptotic properties on various human cancer cell lines, whereas the trans configured complexes generally showed weaker potency with a few exceptions. On the other hand, the trans complexes were generally more likely to lack cross-resistance to cisplatin than the cis analogues. Platinum was found bound to the nuclear DNA of cancer cells treated with representative Pt complexes, suggesting that DNA might be a possible target. Thus, detailed in vitro binding experiments with DNA were conducted. Interactions of the compounds with calf thymus DNA were investigated, including Pt binding kinetics, circular dichroism (CD) spectral changes, changes in DNA melting temperatures, unwinding of supercoiled plasmids and ethidium bromide displacement in DNA. The CD results indicate that the most active cis configured pyrazole-derived complex causes unique structural changes in the DNA compared to the other complexes as well as to those caused by cisplatin, suggesting a denaturation of the DNA structure. This may be important for the antiproliferative activity of this compound in the cancer cells.

Chondroitin-based nanoplexes as peptide delivery systems--Investigations into the self-assembly process, solid-state and extended release characteristics.

A new type of self-assembled polyelectrolyte complex nanocarrier composed of chondroitin (CHON) and protamine (PROT) was designed and the ability of the carriers to bind salmon calcitonin (sCT) was examined. The response of sCT-loaded CHON/PROT NPs to a change in the properties of the liquid medium, e.g. its pH, composition or ionic strength was studied and in vitro peptide release was assessed. The biocompatibility of the NPs was evaluated in Caco-2 cells. CHON/PROT NPs were successfully obtained with properties that were dependent on the concentration of the polyelectrolytes and their mixing ratio. X-ray diffraction determined the amorphous nature of the negatively charged NPs, while those with the positive surface potential were semi-crystalline. sCT was efficiently associated with the nanocarriers (98-100%) and a notably high drug loading (13-38%) was achieved. The particles had negative zeta potential values and were homogenously dispersed with sizes between 60 and 250 nm. CHON/PROT NPs released less than 10% of the total loaded peptide in the first hour of the in vitro release studies. The enthalpy of the decomposition exotherm correlated with the amount of sCT remaining in NPs after the release experiments. The composition of medium and its ionic strength was found to have a considerable influence on the release of sCT from CHON/PROT NPs. Complexation to CHON markedly reduced the toxic effects exerted by PROT and the NPs were compatible and well tolerated by Caco-2 cells.

TGF-ß3 immobilized PLGA-gelatin/chondroitin sulfate/hyaluronic acid hybrid scaffold for cartilage regeneration.

Although most in vitro studies indicate that transforming growth factor ß3 (TGF-ß3) immobilized scaffold is suitable for cartilage tissue engineering, in vivo studies of implanting immobilized scaffold for chondral defect repair are still lacking. This study is to evaluate the potentials of TGF-ß3 immobilized poly-(lactic-co-glycolic acid)-gelatin/chondroitin sulfate/hyaluronic acid (PLGA-GCH) hybrid scaffold for cartilage regeneration. The scaffold was fabricated by incorporating GCH micro-sponges into PLGA frameworks and then crosslinked with TGF-ß3 to mimic natural cartilaginous extra cellular matrix (ECM). In vitro study demonstrated that MSCs proliferated vigorously and produced abundant ECM on scaffold. The immunohistochemistry staining and alcian blue staining confirmed the cartilaginous ECM production. The chondrogenic differentiation of MSCs on scaffold was proved by the expression of collagen II gene in mRNA and protein level. Then MSCs/TGF-ß3 immobilized scaffolds were implanted in rabbits for chondral defects repair. After eight weeks, histological observation showed that differentiated MSCs were located in lacunae within the metachromatic staining matrix and exhibited typical chondrocyte morphology. Histological grading scores also indicated the congruent cartilage was regenerated. In conclusion, the TGF-ß3 immobilized PLGA-GCH hybrid scaffold has great potential in constructing the tissue-engineered cartilage.

Diammine dicarboxylic acid platinum enhances cytotoxicity in platinum-resistant ovarian cancer cells through induction of apoptosis and S-phase cell arrest.

PURPOSE: Polysaccharides such as chondroitin play a potent role in tumor growth, tissue repair and angiogenesis. These properties make chondroitin a good candidate for novel drug delivery systems. Diammine dicarboxylic acid platinum (DDAP), a novel polymeric platinum compound, was developed by conjugating the platinum analogue to aspartate-chondroitin for drug delivery to tumor cells. DDAP improves platinum solubility which may reduce systemic toxicity and be more efficacious than cisplatin in killing tumor cells. METHODS: We tested and compared the cytotoxic effects of DDAP and CDDP on the platinum-sensitive 2008 and A2780 ovarian cancer cell lines and their platinum-resistant sublines 2008.C13 and A2780cis; we also investigated DDAP's mechanism of action. RESULTS: In the platinum-sensitive cell lines, the cytotoxic effects of DDAP and CDDP were comparable. However, in the platinum-resistant sublines, significantly greater cell-growth inhibition was induced by DDAP than by CDDP, especially at lower doses. DDAP also induced more apoptosis than CDDP did in the 2008.C13 subline, which was partially mediated by the caspase 3-dependent pathway. In addition, lower (but not higher) doses of DDAP arrested 90% of S-phase 2008.C13 cells, which might be associated with up-regulation of p21 and maintenance of low cyclin A expression. Furthermore, greater cellular uptake of DDAP was seen in platinum-resistant than in platinum-sensitive ovarian cancer cells. CONCLUSIONS: Low-dose DDAP enhances drug delivery to platinum-resistant ovarian cancer cells and substantially inhibits their growth by inducting apoptosis and arresting cells in the S-phase, suggesting that DDAP may overcome platinum resistance in ovarian cancer.

Conjugation of oligosaccharides by reductive amination to amine modified chondroitin oligomer and gamma-cyclodextrin.

Carbohydrates present on cell surfaces participate in numerous biological recognition phenomena including cell-cell interactions, cancer metastasis and pathogen invasion. Therefore, synthetic carbohydrates have a potential to act as pharmaceutical substances for treatment of various pathological phenomena by inhibiting specifically the interaction between cell surface carbohydrates and their protein receptors (lectins). However, the inherently low affinity of carbohydrate-protein interactions has often been an obstacle for successful generation of carbohydrate based pharmaceuticals. Multivalent glycoconjugates, i.e. structures carrying several copies of the active carbohydrate sequence in a carrier molecule, have been constructed to overcome this problem. Here we present two novel types of multivalent carbohydrate conjugates based on chondroitin oligomer and cyclodextrin carriers. These carriers were modified to express primary amino groups, and oligosaccharides were then bound to carrier molecules by reductive amination. Multivalent conjugates were produced using the human milk type oligosaccharides LNDFH I (Lewis-b hexasaccharide), LNnT, and GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc.

Cartilage regeneration using mesenchymal stem cells and a PLGA-gelatin/chondroitin/hyaluronate hybrid scaffold.

The study was to produce a novel hybrid poly-(lactic-co-glycolic acid) (PLGA)-gelatin/chondroitin/hyaluronate (PLGA-GCH) scaffold and evaluate its potentials in cartilage repair. The porous PLGA-GCH scaffold was developed to mimic the natural extra cellular matrix of cartilage. The differentiated mesenchymal stem cells (MSCs) seeded on PLGA-GCH or PLGA scaffold were incubated in vitro and showed that, compared to PLGA scaffold, the PLGA-GCH scaffold significantly augmented the proliferation of MSCs and GAG synthesis. Then autologous differentiated MSCs/PLGA-GCH was implanted to repair full-thickness cartilage defect in rabbit, while MSCs/PLGA for the contra lateral cartilage defect (n=30). Fifteen additional rabbits without treatment for defects were used as control. Histology observation showed the MSCs/PLGA-GCH repair group had better chondrocyte morphology, integration, continuous subchondral bone, and much thicker newly formed cartilage compared with MSCs/PLGA repair group 12 and 24 weeks postoperatively. There was a significant difference in histological grading score between these two groups, which both showed much better repair than control. The present study implied that the hybrid PLGA-GCH scaffold might serve as a new way to keep the differentiation of MSCs for enhancing cartilage repair.

Enzymatic synthesis of chondroitin and its derivatives catalyzed by hyaluronidase.

The enzymatic polymerization to provide synthetic chondroitin and its derivatives is reported here, the first example of such in vitro synthesis to date. N-Acetylchondrosine (GlcAbeta(1-->3)GalNAc) oxazoline (1a) and its derivatives (1b-1f) were designed and synthesized as novel transition state analogue substrate monomers for catalysis by hyaluronidase. Hyaluronidase is a hydrolysis enzyme of chondroitin that also catalyzes the formation of repeated glycosidic bonds in in vitro synthesis, rather than in the catabolic direction. Monomers of 2-methyl (1a), 2-ethyl (1b), and 2-vinyl (1f) oxazoline derivatives were polymerized using this enzyme, via ring-opening polyaddition with total control of regioselectivity and stereochemistry. These reactions provided the corresponding synthetic chondroitin (natural type; N-acetyl, 2a) and the derivatives (unnatural type) with N-propionyl (2b) and N-acryloyl (2f) functional groups at the C2 position of all the galactosamine units, in good yields. Monomers of 2-n-propyl (1c) and 2-isopropyl (1d) oxazoline derivatives were polymerized to produce 2c and 2d in low yield. The 2-phenyl oxazoline derivative (1e) did not afford any enzyme-catalyzed products. M(n) values of 2a and 2b reached 4800 and 4000, respectively. The M(n) value of 2a corresponds to that of the naturally occurring chondroitin. Thus, hyaluronidase catalysis allows the in vitro production of not only natural type but also the formation of unnatural type chondroitins.

Chondroitin O-methyl ester: an unusual substrate for chondroitin AC lyase.

Chondroitin O-methyl ester was depolymerized by chondroitin AC lyase (EC 4.2.2.5) from Flavobacterium heparinum. The major product isolated from the depolymerization reaction was found to be methyl alpha-L-threo-hex-4-enopyranosyluronate-(1-->4)-2-acetamido-2-deoxy-alpha,beta-D-galactopyranoside.

Fibroblasts induce heparin synthesis in chondroitin sulfate E containing human bone marrow-derived mast cells.

Human bone marrow-derived mast cells (hBMMCs), differentiated in vitro in suspension culture and under the influence of human peripheral blood mononuclear cells conditioned medium (hCM), were tested for their response to recombinant human interleukin-3 (rhIL-3) and for their behavior in different microenvironments. The hBMMCs were incubated in the presence of rhIL-3 and the changes in their proliferation rate were determined. Recombinant hIL-3 induced a more than sixfold increase in 3H-thymidine uptake into the hBMMC DNA in a dose-dependent manner. Human CM used as a control for proliferation response induced a more than eightfold maximal proliferation rate increase. Rabbit anti-rhIL-3 completely inhibited hBMMC 3H-thymidine uptake induced by rhIL-3 and decreased the hCM-induced proliferation by approximately 50%. These hBMMCs were cocultured with four different mytomicin C-treated cell monolayers and assayed for phenotypic changes. After only 2 days in coculture with either embryonic mouse skin-derived fibroblasts (MESFs) or human skin-derived fibroblasts (HSFs), a marked increase in granule number and density was noted on staining with toluidine blue. Mast cells that initially stained alcian blue+/safranin- at day 0 of coculture became alcian blue+/safranin+ during the coculture period. Human BMMC proteoglycan synthesis shifted from approximately 85% chondroitin sulfate E to approximately 60% heparin within 14 to 19 days of coculture with the MESF monolayer and to approximately 50% heparin within 19 days of coculture with the HSF monolayer. None of the above-mentioned changes were noted in cocultures of hBMMCs with 3T3 cell line fibroblast monolayers or in cocultures with bovine vascular endothelium (BVE) cell monolayers. These results demonstrate microenvironmental effects exerted by the MESF and HSF monolayers on IL-3-dependent hBMMCs similar to those reported in the conversion of murine mast cell phenotype.

Presence and function of chondroitin-4-sulfate on recombinant human soluble thrombomodulin.

We constructed a human soluble thrombomodulin (sTM) expression vector using the RSV promoter. Recombinant sTM (rsTM) was expressed in CHO cells and was recovered from culture medium by ion exchange chromatography. Two active fractions, designated as rsTM alpha (low salt elution) and rsTM beta (high salt elution), were detected and further purified by immunoaffinity chromatography. Purified rsTM beta contained bound chondroitin-4-sulfate as judged by HPLC detection of the chondroitinase ABC and AC I digestion product, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose. The apparent Kd values for thrombin of alpha and beta were 7.4 and 1.4 nM respectively. RsTM beta was more effective at inhibition of thrombin clotting activity and had antithrombin III-dependent anticoagulant activity which was not possessed by rsTM alpha. Both anticoagulant activities were lost after chondroitinase treatment of rsTM beta.

Connective-tissue macromolecules in Golgi chicken tendon organs and at their interface with muscle fibers and adjoining tendinous structures.

Tendon organs from leg and forearm muscles of white leghorn chickens were examined with a library of monoclonal antibodies to determine the composition of their connective-tissue framework and the types of connective-tissue macromolecules that occur at the sites where muscle fibers attach to the receptors. The capsules of the tendon organs were positive for connective-tissue macromolecules typical of basal lamina (collagen type IV, laminin, and heparin sulfate proteoglycan) and for tenascin, collagen types III and VI, and fibronectin. Connective-tissue bundles in the lumen of a receptor reacted primarily with antibodies against collagen type I and 4-chondroitin sulfate. The narrow partitions that divide each lumen into compartments stained for collagen type III. Toward its tendinous end, a receptor made few contacts with muscle fibers. Instead, the capsule and the collagenous bundles blended gradually with the intermuscular portions of tendons. At the muscular end, the connections were more complex. Muscle fibers that attached in series to tendon organs split to produce basal lamina-covered, finger-like extensions, which were separated from each other by fissures. Tongues of connective tissue containing tenascin, collagen types I and VI, and fibronectin extended into the fissures. Distally the tongues were continuous with the tenascin in the capsule and just internal to the capsule, fibronectin and basal lamina macromolecules in the capsule, and collagen type I in the collagenous bundles. The uninterrupted presence of these macromolecules around terminating muscle fibers and in the capsule and/or the intraluminal collagen bundles suggests that muscle fibers that attach in series at the muscular end exert a force during muscular contraction on the intraluminal collagen bundles and on the receptor capsule.

Influence of collagen lattice on the metabolism of small proteoglycan II by cultured fibroblasts.

Small dermatan sulphate proteoglycan II from cultured human skin fibroblasts interacts with type I collagen in vitro and in vivo. When fibroblasts are maintained in a type I collagen lattice the proteoglycan remains exclusively within the lattice, and its association with fibrils can be demonstrated immunocytochemically. On the basis of [35S]sulphate incorporation, small proteoglycan II comprises about 80% of total proteoglycans secreted by cells in monolayer culture. In a collagen lattice, fibroblasts down-regulate its synthesis to the level of large chondroitin sulphate/dermatan sulphate and of heparan sulphate proteoglycans, the synthesis of which remains unaffected. Compared with the product from monolayer cultures, small proteoglycan II from collagen gels contained a longer polysaccharide chain which is characterized by a larger proportion of disulphated and a smaller proportion of monosulphated glucuronic acid-containing disaccharides. The half-life varied between 60 and 110 h. It is suggested that the compositional differences between the proteoglycan from monolayer cultures and from cells in a collagen lattice are related to the slower intracellular trafficking of the proteoglycan under the latter culture conditions.

Synthesis of chondroitin sulfate D and heparin proteoglycans in murine lymph node-derived mast cells. The dependence on fibroblasts.

Proteoglycans synthesized in cultured mast cells derived from horse serum-immunized lymph node cells were analyzed. Treatment of the 35S-proteoglycans extracted from these cells with either chondroitinase ABC or AC resulted in 95% +/- 7% and 84% +/- 7%, respectively (mean +/- S.E., n = 3), of the radioactivity associated with disaccharides eluting in the included volume of PD-10. The 35S-proteoglycans were not hydrolyzed by nitrous acid elimination treatment. The chondroitinase ABC-generated disaccharides were analyzed by aminocyano high performance liquid chromatography. 35S-Disaccharides eluted in a major peak at a retention time of 8.1 min, corresponding to the disaccharide of chondroitin 4-sulfate proteoglycan (delta Di-4S), and a second peak at 12 min, corresponding to the disaccharide of chondroitin sulfate D proteoglycan (delta Di-diSD). Further treatment with chondro-4-sulfatase did not affect the retention time of the disaccharide corresponding to delta Di-diSD whereas this peak disappeared after the digested proteoglycan was treated either by chondro-6-sulfatase or by both sulfatases. Therefore, this disaccharide was identified as chondroitin sulfate D. Quantification of the radiolabeled disaccharides showed that delta Di-diSD contributed 20% +/- 2% (n = 3) of the total sulfated disaccharides of the chondroitin sulfate of these cultured cells. The role of fibroblasts in inducing the shift of chondroitin sulfate D into heparin proteoglycan in these mast cells was also investigated by using three types of monolayers: mouse embryonic skin fibroblasts (MESF), rat embryonic skin fibroblasts (RESF), and 3T3 fibroblasts. 35S-Proteoglycans that were extracted from the lymph node-derived mast cells cultured for 30 days on MESF and on 3T3 fibroblast monolayers were 93% +/- 4% and 30% +/- 7% (n = 3) susceptible to nitrous acid elimination, respectively. No degradation by nitrous acid was observed in 35S-proteoglycans extracted from cells cultured on RESF monolayer. Since the MESF was found to be the most potent monolayer in the induction of heparin synthesis, the kinetics of changes in the synthesis of proteoglycan types were determined in lymph node-derived mast cells cultured on MESF for up to 30 days. It was found that the synthesis of chondroitin sulfate gradually declined whereas that of heparin starting between 4 and 7 days after plating gradually increased. From the 17th day on, only the synthesis of heparin was detected.

Effects of intracameral injection of viscoelastic solutions on corneal endothelium in dogs.

Contact in vivo wide-field specular microscopy was performed on right eyes of 20 healthy dogs after sodium hyaluronate (1%, n = 5), sodium chondroitin sulfate (4%) and sodium hyaluronate (3%, n = 5), hydroxypropyl methylcellulose (2%, n = 5), or balanced salt solution (control, n = 5) was injected into the anterior chamber. Using computerized morphometric analysis and pachymetry, changes in endothelial cell density, cell morphologic features, and corneal thickness from baseline values were evaluated at postinjection hour (PIH) 72 and PIH 168. Changes were not seen in endothelial cell density or cell morphologic features in any treated eye. The mean corneal thickness of all treated eyes at PIH 72 increased 6%, significantly greater than that of the nontreated eyes (P = 0.03). Mean corneal thickness of treated and nontreated eyes was similar at baseline and PIH 168 in all treatment groups.

A method for the sequence analysis of dermatan sulphate.

We are attempting to develop methods for the sequencing of glycosaminoglycans from their reducing end. Here we describe a procedure for the analysis of dermatan sulphate from pig skin. The glycosaminoglycan is released from its parent proteoglycan by exhaustive proteolysis by using both endo- and exo-peptidases. The amino group of the residual serine residue is conjugated with a p-hydroxyphenyl group, which in turn is iodinated with 125I (the Bolton-Hunter reagent, BHR). The ion-exchange-purified end-labelled dermatan sulphate is then degraded partially or completely by various enzymic or chemical means to yield fragments extending from the labelled serine residue to the point of cleavage. The various products are separated by gradient PAGE, detected by autoradiography and quantified by videodensitometry. Complete digestion with chondroitin ABC lyase affords the labelled fragment delta HexA-GalNAc(-SO4)-GlcA-Gal-Gal-Xyl-Ser(-BHR). The structure was confirmed by sequential degradation from the non-reducing end by chondroitin AC lyase, HgCl2, and beta-galactosidase. Periodate oxidation cleaves most of the Xyl even without treatment with alkaline phosphatase, showing that Xyl is not substituted with phosphate. Results from partial and selective periodate oxidation indicate that most of the non-sulphated IdoA residues are located towards the non-reducing end. Partial or complete digestions with testicular hyaluronidase (in the presence of an excess of beta-glucuronidase) or chondroitin AC lyase identify the positions of GlcA residues. The results confirm that HexA next to Gal is always GlcA. Moreover, GlcA is common in the first three disaccharide repeats. Results with testicular hyaluronidase indicate that the distribution of clustered GlcA-GalNAc repeats is periodic and peaks at positions 1-3, 8-9 and around 25. Although there must be chains that contain IdoA in nearly all of the available positions, regions that have not been fully processed during biosynthesis are markedly non-random.

Syntheses of the methyl glycosides of the repeating units of chondroitin 4- and 6-sulfate.

3,4,6-Tri-O-acetyl-D-galactal was transformed into methyl 6-O-acetyl-2-azido-4-O-benzyl-2-deoxy-beta-D-galactopyranoside and its 4-O-acetyl-6-O-benzyl analogue, each of which was glycosylated with activated, O-acetylated derivatives of methyl D-glucopyranosyluronate. The resulting beta-(1----3)-linked disaccharide derivatives were each reductively N-acetylated, hydrogenolysed, O-sulfated, and saponified to afford the disodium salts of methyl 2-acetamido-2-deoxy-3-O-(beta-D-glucopyranosyluronic acid)-4-O-sulfo-beta-D-galactopyranoside and the 6-O-sulfo analogue. D-Galactal was also transformed into activated derivatives of 2-azido-3,6-di-O-benzyl-2-deoxy-D-galactopyranose and their 3,4-di-O-benzyl analogues with various substituents at O-4 and O-6. These glycosyl donors were condensed with 6-O-protected derivatives of methyl 2,3-di-O-benzyl-beta-D-glucopyranoside to give the beta-(1----4)-linked disaccharide derivatives, which were selectively deprotected, then oxidised at C-6 of the gluco unit, reductively N-acetylated, selectively deprotected, O-sulfated at C-4 or C-6 of the galacto unit, and hydrogenolysed to give the disodium salts of methyl 4-O-(2-acetamido-2-deoxy-4-O-sulfo-beta-D-galactopyranosyl)-beta-D- glucopyranosiduronic acid and the 6-O-sulfo analogue.

Partial characterization of dermatan sulfate by proton nuclear magnetic resonance spectroscopy.

The degree of galactosamine N-acetylation, iduronic acid composition, and total uronic acid/hexosamine ratios of the three dermatan sulfates of human skin, DS18, DS28, and DS35 (M. O. Longas et al. (1987) Carbohydr. Res. 159, 127-136), were determined by Fourier transform, proton nuclear magnetic resonance (FT 1H NMR) spectroscopy. Analysis of DS of varying ages was conducted at 400 MHz and 60 degrees C. Chemical shifts for H-1, H-2, H-4, and H-5 of L-IdUA were independent of those for the respective protons of D-GalNAc and D-GlcUA. The resonance intensities of H-1 and acetamido methyl protons of D-GalNac did not display the expected 1:3 ratios. Therefore, their integration values were employed to estimate the percentage N-acetylation (N-CH3/3 H-1) which was corroborated chemically. The L-IdUA content, relative to total uronic acid, was calculated from signal intensities of H-1 of L-IdUA and D-GlcUA and ascertained by quantitative chemical methods. Total uronic acid/hexosamine ratios were determined from both 1H NMR spectroscopy and chemical analyses. The data show the following N-acetylation (N-CH3/3 H-1) of galactosamine in DS:DS18, 61-72% between 17 and 60 years, unaffected by senescence; DS28, 78-86% with no age-related trend; DS35, 101% at 19 years. Furthermore, in all ages investigated, the percentage (wt/wt) L-IdUA relative to total uronic acid was 42-44% for DS18 and 37-40% for DS28. At age 19 years, DS35 had a 29% (wt/wt) L-IdUA. The total uronic acid/hexosamine ratios for DS18 and DS28 varied from 1.40:1.0 to 1.70:1.0 irrespective of age.

1H-n.m.r. investigation of naturally occurring and chemically oversulphated dermatan sulphates. Identification of minor monosaccharide residues.

The 1H-n.m.r. spectra of various dermatan sulphate preparations present, besides the major signals of the basic disaccharide unit, several other minor signals. We have assigned most of them by n.m.r., using two-dimensional proton-proton double-quantum-correlation and nuclear-Overhauser-effect spectroscopy experiments. This allowed us to identify 2-O-sulphated L-iduronic acid and D-glucuronic acid residues as well as 6-sulphated N-acetylgalactosamine (presumably 4-O-sulphated as well). 2-O-Sulphated iduronic acid was present to similar extents (6-10% of total uronic acids) in pig skin dermatan sulphate and pig intestine dermatan sulphate, whereas glucuronic acid represented 17% of the uronic acid of pig skin dermatan sulphate and was virtually absent (1%) from the other preparation. 6-O-Sulphated N-acetylgalactosamine was present in minor amounts in pig intestine dermatan sulphate only. The influence of sulphation of iduronic acid units on their conformation was assessed by using chemically oversulphated pig intestine dermatan sulphate. Introduction of sulphate groups in this unit in dermatan sulphate tends to shift the conformational equilibrium towards the 1C4 conformer.