Miloš Hricovíni

Conformation of heparin pentasaccharide bound to antithrombin III.

The interaction, in aqueous solution, of the synthetic pentasaccharide AGA*IA(M) (GlcN,6-SO(3)alpha 1-4GlcA beta 1-4GlcN,3,6-SO(3)alpha 1-4IdoA,2-SO(3)alpha 1-4GlcN,6-SO(3)alpha OMe; where GlcN,6-SO(3) is 2-deoxy-2-sulphamino-alpha-D-glucopyranosyl 6-sulphate, IdoA is l-iduronic acid and IdoA2-SO(3) is L-iduronic acid 2-sulphate), which exactly reproduces the structure of the specific binding sequence of heparin and heparan sulphate for antithrombin III, has been studied by NMR. In the presence of antithrombin there were marked changes in the chemical shifts and nuclear Overhauser effects (NOEs), compared with the free state. On the basis of the optimized geometry of the pentasaccharide the transferred NOEs were interpreted with full relaxation and conformational exchange matrix analysis. An analysis of the three-dimensional structures of the pentasaccharide in the free state, and in the complex, revealed the binding to be accompanied by dihedral angle variation at the A-G and I-A(M) (where G, I, A and A(M) are beta-d-glucuronic acid, 2-O-sulphated alpha-L-iduronic acid, N,6-O-sulphated alpha-D-glucosamine and the alpha-methyl-glycoside of A respectively) glycosidic linkages. Evidence is also provided that the protein drives the conformation of the 2-O-sulphated iduronic acid residue towards the skewed (2)S(0) form.

Structural features of a water-soluble l-arabino-d-xylan from rye bran

A water-soluble L-arabino-D-xylan, obtained from the chlorite holocellulose of de-lipidated, de-starched, and de-pectinated rye bran by ammoniacal extraction, was composed of L-Ara and D-Xyl in the molar ratio 7.8:10 and had Mw 36,500 and Mn 26,950. The backbone of the polysaccharide was shown to comprise (1----4)-linked beta-D-Xylp residues, with approximately 41% unsubstituted, approximately 33% 2- or 3-substituted, and approximately 26% disubstituted. Single alpha-L-Araf groups were attached to the xylan core; only a small proportion was 2-, 3-, or 5-linked.

Residual dipolar coupling investigation of a heparin tetrasaccharide confirms the limited effect of flexibility of the iduronic acid on the molecular shape of heparin

The solution conformation of a fully sulfated heparin-derived tetrasaccharide, I, was studied in the presence of a 4-fold excess of Ca2+. Proton–proton and proton–carbon residual dipolar couplings (RDCs) were measured in a neutral aligning medium. The order parameters of two rigid hexosamine rings of I were determined separately using singular value decomposition and ab initio structures of disaccharide fragments of I. The order parameters were very similar implying that a common order tensor can be used to analyze the structure of I. Using one order tensor, RDCs of both hexosamine rings were used as restraints in molecular dynamics simulations. RDCs of the inner iduronic acid were calculated for every point of the molecular dynamics trajectory. The fitting of the calculated RDCs of the two forms of the iduronic acid to the experimental values yielded a population of 1C4 and 2So conformers of iduronic acid that agreed well with the analysis based on proton–proton scalar coupling constants. The glycosidic linkage torsion angles in RDC-restrained molecular dynamics (MD) structures of I are consistent with the interglycosidic three-bond proton–carbon coupling constants. These structures also show that the shape of heparin is not affected dramatically by the conformational flexibility of the iduronic acid ring. This is in line with conclusions of previous studies based on MD simulations and the analysis of 1H-1H NOEs. Our work therefore demonstrates the effectiveness of RDCs in the conformational analysis of glycosaminoglycans.

Conformational analysis of heparin epoxide in aqueous solution. An NMR relaxation study

1H and 13C NMR relaxation measurements at various magnetic fields have been used to characterize the nature of overall and internal motions in heparin epoxide in aqueous solution. A two-dimensional homonuclear NOESY experiment showed a considerable number of cross-relaxing protons in the molecule. The inter-proton distances calculated from NOE data were compared with those obtained by molecular mechanics calculations. Several discrepancies between the experimental and the theoretical inter-proton distances as well as the variations in 13C spin-lattice relaxation times, measured at two magnetic fields, indicated that the polysaccharide tumbles anisotropically in solution. The rates of overall and internal motions as well as the order parameters have been calculated using a model-free spectral density function. The numerical values indicate that the correlation times which characterize overall molecular motion are outside the extreme narrowing limit (tau parallel = 8 x 10(-10) s and tau perpendicular = 4.2 x 10(-8) s) and that internal motion correlation time is on a picosecond timescale.

NMR study of virenose and dihydrohydroxystreptose isolated from Coxiella burnetii phase I lipopolysaccharide

A lipopolysaccharide (LPS) isolated from Coxiella burnettii in virulent phase I contains in its O-polysaccharide chain two unusual sugars, virenose (6-deoxy-3-C-methylgulose) and dihydrohydroxystreptose [3-C-(hydroxymethyl)lyxose]. The sugars were isolated from LPS I, after acid hydrolysis and removal of lipid A, by a combination of HPLC and preparative paper chromatography. Their enantiomeric forms and ring conformations were established from optical rotation and NMR data. Two-dimensional COSY, HSQC, and HMBC as well as one- and two-dimensional NOEs were used to assign all proton and carbon signals in both monosaccharides. Virenose was found to be the D-gulo enantiomer with the 4C1 ring conformation and dihydrohydroxystreptose was shown to be the L-lyxo enantiomer also with the 4C1 conformation. The latter sugar was reported [S. Schramek, J. Radziejewska-Lebrecht, and H. Mayer, Eur. J. Biochem., 148 (1985) 455-461] to be present in LPS I in a furanose form, and it appears that a furanose to pyranose tautomerization took place in the course of the isolation procedure.

Conformations of (1→4)-linked α-d-galacturono-di- and -tri-saccharides in solution analysed by n.m.r. measurements and theoretical calculations

The conformations of the (1----4)-linked alpha-D-galacturono-di- (1) and -tri-saccharide (2) in aqueous solutions have been analysed by n.m.r. spectroscopy and MM2CARB calculations. The 3JC.H. and n.O.e. values did not change with temperature and were comparable for 1 and 2. Four energy regions were found on the relaxed (phi, psi) map for 1 computed by the MM2CARB method. Theoretical n.O.e. values, based on the geometry and the abundance of the most populated conformer, accorded with experimental values. The magnitudes of phi H and psi H for the glycosidic bond suggest that a right-handed three-fold helical arrangement can be formed by pectic acid oligosaccharides in solution.

Preparation of water-soluble/insoluble derivatives of hyaluronic acid by cross-linking with epichlorohydrin in aqueous NaOH/NH4OH solution

Hyaluronan (HA; 0.1 or 1 mmol) was cross-linked by using epichlorohydrin (E; 0.005-0.25 mol) in the presence of NaOH (0.005-0.25 mol) and without or with NH 4 OH (0.005 or 0.01 mol). The deacetylation of the N-acetyl-D-glucosamine units and a degradation of the product was confirmed in solution by NMR and the static light-scattering (LS) analysis. The products prepared with 0.005-0. mol of NaOH, at the presence of NH 4 OH, and with 0.005-0.05 mol of E were highly water soluble. But at 0.075 mol of E and 0. mol of NaOH/5 mol of water present, the prepared products were water-insoluble. For their analysis, the solid-state 13 C CP-MAS NMR spectroscopy was employed. By this method, the presence of both hydroxypropyl and hydroxypropylamine groups, partially overlapped with HA signals, were observed. The T 1ρ (H) relaxation experiments supported the assumption that the linkages between HA and hydroxypropylamine or hydroxypropyl groups were formed, because all the carbon signals resulted in close T 1ρ (H) values.

Nuclear overhauser effects and the flexibility of saccharides : methyl β-xylobioside

The behavior of methyl beta-xylobioside has been analysed by 1H-n.m.r. spectroscopy for solutions in water and methanol in the range - 15 degrees to 85 degrees. Experimental n.O.e. values did not change with temperature and solvent in contrast to the inter-glycosidic 3JC,H values. Experimental n.O.e. data accorded with the values calculated from time-averaged cross-relaxation terms. The tumbling times were determined from relaxation data and were in the sub-ns range.

Preparation of ion-exchangers by cross-linking of starch or polygalacturonic acid with 1,3-bis(3-chloro-2-hydroxypropyl)imidazolium hydrogen sulphate

Abstract The reaction conditions for cross-linking of starch (S) or polygalacturonic acid (PGA) with 1,3-bis(3-chloro-2-hydroxypropyl)imidazolium hydrogen sulphate (BCHIHS) in the presence of NaOH and water were studied in terms of the molar ratio of the reaction components, the yields of water-insoluble product, the nitrogen content and the BCHIHS conversion. The optimum for S-derivatives was at the molar ratio of S/BCHIHS/NaOH/H 2 O=0.01:0.02:0.04:0.333 with 348% yield on the amount of used S, 7.71% nitrogen content and 78% conversion of BCHIHS. Analogically on PGA the optimum was at PGA/BCHIHS/NaOH/H 2 O=0.005:0.005:0.1:0.2 with 125% yield, 7.99% nitrogen content and 63% conversion. The linkage of dihydroxypropylimidazolum (DHPI) groups was confirmed by NMR in solution on water-soluble products as well as by 13 C solid-state NMR and FTIR on insoluble products. Under the used conditions much more NaOH is consumed on PGA while the yields of water-insoluble materials are lower than on S. The calculated T 1 ρ (H) relaxation times gave similar values obtained previously on different polysaccharides cross-linked with different cross-linkers. The elemental composition of samples in different cycles confirmed the ionic interaction between the quaternary ammonium group and the PGA carboxyls.

Hydrogenation of 3(2H)-isoquinolinones and the stereochemistry of the products

Abstract A new route of preparing 1,4-dihydro-3(2 H )-isoquinolinones and 5,6,7,8-tetrahydrolsoquinolinones was developed by the hydrogenation of 3(2 H )-isoquinolinones. The 1,4-dihydro compounds 1 - 8 , were also prepared by synthesis, when the cis - trans ratio significantly differed from that of the products obtained by hydrogenation. Relative configurations and site of conformational equilibria were established by n.O.e. difference n.m.r. spectroscopy. Compounds 2 , 4 , 5 and 6 had practically one main conformation, whereas 1 , 3 , and 7 occurred as two boat conformers in equilibrium. Complete 1H and 13C signal assignments were achieved by different 2D homo- and heteronuclear correlation measurements.