1H NMR of glycosaminoglycans and hyaluronic acid oligosaccharides in aqueous solution: The amide proton environment (original) (raw)
Related papers
13 C-NMR Studies of Hyaluronan: Conformational Sensitivity to Varied Environments †
Macromolecules, 1996
Hyaluronan (HA) samples ranging in size from small oligosaccharides to high molecular weight polymer have been studied by 13 C-NMR spectroscopy. In neutral aqueous solutions, the chemical shifts of carbons directly involved in the -1,3 glucuronidic linkage are found to be sensitive to (1) residue linkage position in short chains, (2) oligomer degree of polymerization, (3) solvent ionic strength, and (4) monovalent vs divalent counterions. The carbons of the -1,4-glucosaminidic linkage show less sensitivity to the above conditions. Thus conformational versatility for HA in aqueous solution is correlated with a chemical shift change primarily in carbons of the -1,3 linkage. We have also compared the 13 C spectrum of HA in neutral aqueous salt solutions to spectra observed in dimethyl sulfoxide (DMSO) solution (ordered 2-or 4-fold HA form) or the solid state (Na + counterion, tetragonal 4-fold helical HA form). The solid state spectrum is similar to that found in DMSO but differs substantially from the aqueous solution spectrum. The differences are attributed to (1) rotation of the acetamido group, with concomitant change in hydrogen bonding and average conformation at the -1,4 linkage, and (2) loss of hydrogen bonds in aqueous solution and consequent change in average conformation at the -1,3 linkage.
Glycobiology, 2004
The glycosaminoglycan hyaluronan is a vital structural component of extracellular matrices with diverse biological functions, a molecular understanding of which requires a detailed description of secondary and tertiary solution structures. Various models of these structures have been proposed on the basis of 1 H and 13 C natural-abundance nuclear magnetic resonance (NMR) experiments, but resonance overlap limits further progress with these techniques. We have therefore produced 15 N-and 13 Cisotopically-labeled hyaluronan oligosaccharides and applied triple-resonance and 3D experiments to overcome this restriction. Spectra recorded on oligosaccharides (of lengths 4, 6, 8, 10, and 12 sugar rings), reveal that the 15 N nucleus allows resolution of the amide groups in a decamer at high magnetic field, whereas 13 C natural-abundance NMR can only resolve internal groups up to hexamers. Complete 15 N sequencespecific assignments of these oligosaccharides indicate that the chemical shift dispersion can be explained by end-effects, which are seen even in the middle of octamers. Tripleresonance and 15 N-edited 3D experiments, among the first of their kind in oligosaccharides, have been used to achieve resolution of ring 1 H and 13 C nuclei where not possible previously. The subtle chemical shift perturbations resolved suggest that different conformations and dynamics occur at the ends, which may contribute to the range of biological activities displayed by varying lengths of hyaluronan. 15 N-NMR in carbohydrates has not received much attention before, however, this study demonstrates it has clear advantages for achieving resolution and assessing dynamic motion. These conclusions are likely to be applicable to the study of the structure and dynamics of other nitrogencontaining carbohydrates.
Macromolecules, 2001
The conformational dynamics of low molecular weight hyaluronan (HA) polymer and oligosaccharides have been assayed by 13 C NMR T1 relaxation time measurements. For polymeric HA in aqueous solution, the average relaxation time for ring carbons was not significantly affected by changing the ionic strength or by changing counterion type. The average relaxation time of HA was found to be similar to that for chondroitin 4-sulfate or chondroitin 6-sulfate in aqueous solution. As has been observed previously for other polysaccharides, the segmental motions of HA chains studied at room temperature in aqueous solution appear to be dominated by viscous damping of the chain motions. Within a chain structure, there are variations in observed T1 that correspond to differences in relative mobility. The hydroxymethyl substituent group shows a difference in relaxation rate relative to the ring to which it is attached, depending on the ring configuration (glucose vs galactose). These differences correlate with relative rotational isomerization rates measured by ultrasonic relaxation. In short HA chains, the nonreducing and reducing terminal residues show much greater mobility than penultimate residues, and these are in turn more mobile than interior residues. In contrast, the more rigid hydrogen-bonded conformation of an HA tetrasaccharide in dimethyl sulfoxide solution shows less position-dependent variation in T 1. These data are in accord with the dynamic nature of conformation-stabilizing hydrogen bonds for HA chains in aqueous solution.
Hydrogen-bonded conformation of hyaluronate oligosaccharide fragments in aqueous solution
Febs Lett, 1983
The hydrogen bonding in hyaluronate oligosaccharide fragments was studied in aqueous solution using hydrogen-tritium exchange techniques. The data reveal an acetamido hydrogen exchange rate that is 5-6-fold slower than that seen in model compounds. The magnitude of the slowing is interpreted as reflecting the participation of an acetamido hydrogen in a relatively labile intramolecular hydrogen bond.
Carbohydrate Research, 1992
The 'H-and 'sC-NMR spectra of the ethyl and benzyl esters and the tetrabutylammonium and tetraethylammonium salts of hyaluronic acid I _)21-P-o-GlcpA-(1 + ~&P-D-GlcpNAc-(1-I+,) in Me,SO-d, have been assigned using 1D and ZD techniques. The chemical shifts of the resonance of GlcNAc C-3 suggest that the relative orientations of the monosaccharides at the (I + 31 linkage in the esters and salts are different. Small differences in the chemical shifts of the resonance GlcA C-4 suggest only a slight conformational variation around the (1 + 4) linkage. The 13C-NMR data also suggest similarities in conformation between the esters in Me,SO-d, and the salts in water. The chemical shifts of the 'H resonances for NH and OH groups and their temperature dependence for the esters and salts in Me,SO reveal markedly stronger inter-residue hydrogen bonds between the carboxyl and NH groups and between HO-4 of GlcA and O-5 of GlcNAc for the salts. The 3Jz,Nt, values indicate a slightly different orientation for the acetamido group. For solutions in Me,SO, the higher segmental flexibility of the esters is supported by the line widths, whereas the reduced viscosity for the tetrabutylammonium salt showed a sigmoidal concentration dependence and suggests association of chains which could contribute to the segmental rigidity. The linear concentration dependence for the benzyl ester suggests a higher overall flexibility without chain association.
Polymer Journal, 1978
We present the results of our theoretical conformational analysis of hyaluronic acid (HA) and sodium hyaluronate. Empirical potential energy functions for steric, electrostatic, hydrogen bonding, torsional, and solvation energies were used to evaluate the conformational energy with the CAMSEQ Software System. A trisaccharide model of the HA polymer was employed. Three chemical states were studied: HA-, charged HA in the absence of a counter-ion, neutral pH, and very low ionic strength; HAH, uncharged, protonated HA, low pH; HA-• Na +, charged HA with sodium ion present, neutral pH, and high ionic strength. Two sets of conformational energy data were collected. The first was generated by a series of sequential and random scans followed by gradient-search minimizations at each of the local minima. This was intended to give a general picture of the energy surface of each form of HA. The second data set consisted of 10,000 randomly generated conformations for each form. This was sufficient to account for the minima found in the first data set. The second set of energies was employed to estimate the partition function and corresponding chain dimensions for comparison with available hydrodynamic data. In addition, we compare and contrast our structural findings with the published crystal structures ofHA. KEY WORDS Hyaluronic Acid I Conformational Analysis I Glycosaminoglycans I * Sloan Research Fellow, to whom reprint requests should be sent. Early viscosity studies on HA isolated from vitreous humor, umbilical cord, and synovial fluid by Blix and Snellman 4 showed a molecular weight of 200,000 to 500,000 and particle lengths of 4800A to 10,000A. Several other molecular
Insight into the distribution of amino groups along the chain of chemically deacetylated hyaluronan
Carbohydrate Polymers, 2019
Deacetylated hyaluronan (daHA) containing reactive free amino groups is an important intermediate for further modification. Comparing direct and indirect NMR and HPLC to characterize the degree of HA deacetylation (DD), direct NMR approach using area ratio of anomeric CH and CH-NH 2 groups was the most precise one. To describe the substitution pattern, daHA was selectively cleaved by nitrous acid generated in situ or hyaluronan lyase from Streptococcus pneumoniae. The resulting oligomers were identified by LC-ESI-MS. The experimental distribution of these oligomers was compared with theoretically expected random oligomer distribution. Independently on the starting HA molecular weight and deacetylation conditions, the experimental data differed from the random distribution model and suggested that deacetylation of certain N-acetyl-D-glucosamine had reduced the probability of deacetylation at the neighbouring disaccharide. This phenomenon was explained by conformational changes of HA caused by intra-and intermolecular interactions between positively charged amino and negatively charged carboxylic groups.
Structural Chemistry, 2007
The Becke3LYP functional of DFT theory was used to investigate molecular structure and sodium affinity of the systems CH 3 CO 2 Na (1), CH 3-O-SO 3 Na (2), CH 3-NH-SO 3 Na (3), saccharide_1Na 2 (4), saccharide_2Na (5), saccharide_3Na 3 (6), saccharide_4Na 2 (7), and saccha-ride_5Na 2 (8), respectively, which are models of N-and Osulfate glycosaminoglycans. Interaction enthalpies, entropies and Gibbs energies of the sodium-coordinated systems in the gas phase were determined with the B3LYP/6-311+G(d,p) and B3LYP/6-31+G(d) methods. The computed Gibbs energies, DG o , of model systems 1-3 are negative and span a rather broad energy interval (from-500 to-1,500 kJ mol-1). Gibbs interaction energies for sodium acetate, sodium sulfate and sodium sulfamate functions of the five saccharides, systems 4-8 are always lower than those values found for the model compounds 1-3. The ionization of sodium salts of saccharides studied in gas phase is in most cases connected with considerable conformational rearrangement of the ionic species. This rearrangement causes an additional energetic stabilization of anionic species and is connected with the substantial release of entropy.
Glycobiology, 2010
The conformational features of hyaluronic acid, a key polysaccharide with important biological properties, have been determined through the combined used of nuclear magnetic resonance (NMR) spectroscopy and molecular modeling techniques. A decasaccharide fragment of sodium hyaluronate (HA) was submitted to 3.5 ns of molecular dynamics in explicit water environment form. The same decasaccharide was prepared by hyaluronidase digestion for the experimental study. The approach consisted in the measurements of NMR residual dipolar coupling (RDC) which were used to filter the molecular dynamics data by retaining those structures which were in agreement with the experimental observations. Further analysis of the new conformer ensemble (HA RDC ) and clustering the molecules with respect to their overall length led to seven representative structures, which were described in terms of their secondary motifs, namely the best fitting helix geometry. As a result, this protocol permitted the assessment that hyaluronic acid can adopt two different arrangements, which can be described by a threeor four-folded left-handed helix, with a higher occurrence of the first one.