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.     

Very-high-field n.m.r. studies of bovine lung heparan sulphate tetrasaccharides produced by nitrous acid deaminative cleavage. Determination of saccharide sequence, uronate composition and degrees of sulphation.

Tetrasaccharides with the general structure UA-GlcNAc-GlcUA-aManOH (where UA represents uronate, GlcNAc N-acetylglucosamine, GlcUA glucuronate and aManOH anhydromannitol) were prepared from low-sulphated heparan sulphates of bovine lung origin by complete nitrous acid deaminative cleavage followed by reduction and fractionated by gel filtration. Ion-exchange chromatography of the tetrasaccharides yielded three major fractions in approximate yields of 37%, 45% and 14%. These were shown to be non-, mono- and di-sulphated respectively. Complete structural characterization of the tetrasaccharide fractions by quantitative high-field n.m.r. spectroscopy showed that each fraction contained only two discrete species and led to the following observations. (1) All of the uronate residues in the tetrasaccharides (and in larger oligosaccharides) are unsulphated, and hence sulphated iduronate [IdUA(2SO3)] must occur exclusively within -GlcNSO3-IdUA(2SO3)-GlcNSO3- sequences (where GlcNSO3 represents N-sulpho-glucosamine) in the parent polymers. (2) The GlcNAc residues in the tetrasaccharides are more highly C-6-O-sulphated than are the aManOH residues, and furthermore sulphation on the aManOH appears to occur only where the GlcNAc is also sulphated. (3) Where the GlcNAc is C-6-O-sulphated, iduronate is the major non-reducing terminal residue, whereas glucuronate predominates in this position if the GlcNAc is unsulphated. The quantitative data obtained are used to determine the degree of C-6-O-sulphation of glucosamine residues in specific sequences within the parent heparan sulphates.     

Periodate oxidation and alkaline degradation of heparin-related glycans

Heparin, heparan sulphate, and various derivatives thereof have been oxidised with periodate at pH 3.0 and 4° and at pH 7.0 and 37°. Whereas oxidation under the latter conditions destroys all of the nonsulphated uronic acids, treatment with periodate at low pH and temperature causes selective oxidation of uronic acid residues. The reactivity of uronic acid residues depends on the nature of neighbouring 2-amino-2-deoxyglucose residues. d-Glucuronic acid residues are susceptible to oxidation when flanked by N-acetylated amino sugars, but resistant when adjacent residues are either unsubstituted or N-sulphated. L-Iduronic acid residues in their natural environment (2-deoxy-2-sulphoamino-d-glucose) are resistant to oxidation, whereas removal of N-sulphate groups renders a portion of these residues periodate-sensitive. Oxidised uronic acid residues in heparin-related glycans may be cleaved by alkali, producing a series of oligosaccharide fragments. Thus, periodate oxidation-alkaline elimination provides an additional method for the controlled degradation of heparin.     

Characterization of oligosaccharides from the chondroitin sulfates. (1)H-NMR and (13)C-NMR studies of reduced disaccharides and tetrasaccharides.

Chondroitin sulfates were fragmented using the enzymes chondroitin sulfate ABC endolyase and chondroitin ACII lyase; both disaccharide and tetrasaccharide fragments were isolated after reduction to the corresponding 2-deoxy-2-N-acetylamino-D-galactitol (GalNAc-ol) form. These have the structures: Delta UA(beta 1--3)GalNAc4S-ol, Delta UA(beta 1--3)GalNAc6S-ol, Delta UA2S(beta 1--3)GalNAc6S-ol, Delta UA(beta 1--3)GalNAc4S(beta 1--4)L-IdoA(alpha 1--3)GalNAc4S-ol, Delta UA(beta 1--3)GalNAc4S(beta 1--4)GlcA(beta 1--3)GalNAc4S-ol, Delta UA(beta 1--3)GalNAc6S(beta 1--4)GlcA(beta 1--3)GalNAc4S-ol, Delta UA(beta 1--3)GalNAc6S(beta 1--4)GlcA(beta 1--3)GalNAc6S-ol, Delta UA2S(beta 1--3)GalNAc6S(beta 1--4)GlcA(beta 1--3)GalNAc4S-ol and Delta UA2S(beta 1--3)GalNAc6S(beta 1--4)GlcA(beta 1--3)GalNAc6S-ol, where Delta UA represents a 4,5-unsaturated hexuronic acid (4-deoxy-alpha-Lthreo-hex-4-enepyranosyluronic acid) and 6S/4S/2S represent O-ester sulfate groups at C6/C4/C2 sites. Complete (1)H-NMR and (13)C-NMR data are derived for these species, which may help to alleviate some of the significant difficulties resulting from signal complexity that are currently hindering the characterization and assignment of major and minor structural components within chondroitin sulfate and dermatan sulfate polymers.     

Characterisation of oligosaccharides from the chondroitin/dermatan sulphates: H and C NMR studies of oligosaccharides generated by nitrous acid depolymerisation

The polymers chondroitin sulphate and dermatan sulphate have been fragmented by an anhydrous hydrazine/nitrous acid procedure. The resulting disaccharides from the polymer repeat sequences were reduced with NaBH₄ and purified by ion exchange chromatography. Whereas enzymatic depolymerisation leads to the loss of the distinction between glucuronic and iduronic acids of CS and DS in the resultant disaccharides, nitrous acid depolymerisation retains these structures. Complete ¹H and ¹³C NMR data have been derived for the major components which were shown to have the structures: GlcA-(β1→3)-anTal6S-ol (I) and l-IdoA-(α1→3)-anTal4S-ol (II), where anTal-ol represents (2,5)anhydro-d-talitol and 6S/4S represent O-ester sulphate groups at C-6/C-4 sites.     

The structure of the keratan sulphate chains attached to fibromodulin from human articular cartilage

The small keratan sulphate proteoglycan, fibromodulin, has been isolated from pooled human articular cartilage. The main chain repeat region and the chain caps from the attached N-linked keratan sulphate chains have been fragmented by keratanase II digestion, and the oligosaccharides generated have been reduced and isolated. Their structures and abundance have been determined by high pH anion-exchange chromatography. These regions of the keratan sulphate from human articular cartilage fibromodulin have been found to have the following general structure: Significantly, both α(2-6)- and α(2-3)-linked N-acetyl-neuraminic acid have been found in the capping oligosaccharides. Fucose, which is α(1-3)-linked as a branch to N-acetylglucosamine, has also been found along the length of the repeat region and in the capping region. The chains, which have been found to be very highly sulphated, are short; the length of the repeat region and chain caps is ca. nine disaccharides. These data demonstrate that the structure of the N-linked keratan sulphate chains of human articular cartilage fibromodulin is similar, in general, to articular cartilage derived O-linked keratan sulphate chains. Further, the general structure of the keratan sulphate chains attached to human articular cartilage fibromodulin has been found to be generally similar to that of both bovine and equine articular cartilage fibromodulin. Abbreviations: KS, keratan sulphate; IEC, ion-exchange chromatography; ELISA, enzyme linked immunosorbent assay; Gal, β-D-galactose; Fuc, α-L-Fucose; GlcNAc, N-acetylglucosamine (2-acetamido-β-D-glucose); GlcNAc-ol, N-acetylglucosaminitol (2-acetamido-D-glucitol); NeuAc, N-acetyl-neuraminic acid; 6S/(6S), O-ester sulphate group on C6 present/sometimes present; NMR -nuclear magnetic resonance; HPAE, high pH anion-exchange; PED, pulsed electrochemical detection; HPLC, high performance liquid chromatography This revised version was published online in November 2006 with corrections to the Cover Date.     

Very-high-field n.m.r. studies of bovine lung heparan sulphate oligosaccharides produced by nitrous acid deaminative cleavage. 13C-n.m.r. study of methylene resonances: degree and positions of C-6 sulphation.

Oligosaccharides with the general structure UA-(GlcNAc-GlcUA-)m-aManOH (m = 1-5) (where UA represents uronic acid, GlcNAc N-acetylglucosamine, GlcUA glucuronic acid and aManOH anhydromannitol) were prepared from low-sulphated heparan sulphates of bovine lung origin by nitrous acid deaminative cleavage followed by reduction. Analysis of the methylene signals in the 100 MHz 13C-n.m.r. spectrum of the tetrasaccharide (m = 1) shows that, whereas the extent of C-6 O-sulphation in the GlcNAc is approx. 65%, in the aManOH [formerly a GlcNSO3 (N-sulphoglucosamine) residue in the parent heparan sulphate] it is only approx. 10%. In the higher oligosaccharides (m = 2-5) the gross extent of C-6 O-sulphation of GlcNAc residues falls systematically with increasing oligosaccharide size, whereas that in the aManOH residues remains below 10%. There is also evidence that the C-6 O-sulphation of the GlcNAc residues is confined to the GlcNAc residue adjacent to the non-reducing terminal uronic acid residue. It is therefore tentatively proposed that the GlcNAc in the sequence -GlcNSO3-UA-GlcNAc- might be a favoured substrate for the 6-O-sulphotransferase. It is concluded that in the low-sulphated heparan sulphates GlcNSO3 residues that do not occur in (GlcNSO3-UA-)n blocks tend to have a significantly smaller extent of C-6 O-sulphation than do GlcNAc residues that occur in -GlcNSO3-UA-GlcNAc-GlcUA-GlcNSO3-sequences.     

Investigation of 13C relaxation and nuclear Overhauser enhancement parameters for heparinoid systems: comparison with data from dextrans

Data are presented for ¹³C spin-lattice (T₁) relaxation times and nuclear Overhauser enhancement (NOE) values in a range of heparinoid glycosaminoglycans. These paramaters are compared with T₁and NOE values for simple dextrans of $\text{M}{}_{\mathrm{<mtext>W</mtext>}}\text{= 19 900, 40 000}\text{and}\text{150 000}$. For the heparinoid molecules, significant and consistent differences in relaxation times are observed for different ring carbons, for which simple models do not provide an adequate explanation. No such variations are found for the dextrans. Both families of polysaccharides exhibit NOE values significantly reduced from the theoretical maximum. These changes are discussed in terms of molecular rotational correlation times (τ_c) which are similar in magnitude to the resonance frequencies used for n.m.r. measurements. It is concluded that for the n.m.r. investigation of polysaccharides at high field strengths (${}^{13}\text{C resonance frequencies > 25 MHz}$) considerable economy of time will be achieved by the use of INEPT experiments rather than conventional Overhauser signal enhancement via proton-noise decoupling.     

Both the D-(+) and L-(-) enantiomers of nicotine inhibit Abeta aggregation and cytotoxicity

The underlying cause of Alzheimer's disease is thought to be the aggregation of monomeric beta-amyloid (Abeta), through a series of toxic oligomers, which forms the mature amyloid fibrils that accumulate at the center of senile plaques. It has been reported that L-(-)-nicotine prevents Abeta aggregation and toxicity, and inhibits senile plaque formation. Previous NMR studies have suggested that this could be due to the specific binding of L-(-)-nicotine to histidine residues (His6, His13, and His14) in the peptide. Here, we have looked at the effects of both of the L-(-) and D-(+) optical enantiomers of nicotine on the aggregation and cytotoxicity of Abeta(1-40). Surprisingly, both enantiomers inhibited aggregation of the peptide and reduced the toxic effects of the peptide on cells. In NMR studies with Abeta(1-40), both enantiomers of nicotine were seen to interact with the three histidine residues. Overall, our data indicate that nicotine can delay Abeta fibril formation and maintain a population of less toxic Abeta species. This effect cannot be due to a highly specific binding interaction between nicotine and Abeta, as previously thought, but could be due instead to weaker, relatively nonspecific binding, or to the antioxidant or metal chelating properties of nicotine. D-(+)-nicotine, being biologically much less active than L-(-)-nicotine, might be a useful therapeutic agent.