Laura E. Lerner

A detailed 1H and 13C NMR study of a repeating disaccharide of hyaluronan: the effects of temperature and counterion type

For the first time, a detailed NMR study of the conformation of methyl 2-acetamido-2-deoxy-3-O-(beta-D-glucopyranosyluronic acid)-beta-D-glucopyranoside (disaccharide 1) in aqueous solution is reported. This disaccharide is a repeating unit of hyaluronan, a polysaccharide with widespread biological and pharmaceutical applications. Relatively small changes in temperature, over typical experimental conditions (0-37 degrees C), completely change the appearance of its one-dimensional 1H NMR spectrum at 500 MHz. To determine the underlying cause for this temperature sensitivity, we analyzed 1H and 13C chemical shifts, temperature coefficients (delta gamma/delta T), 1H-1H coupling constants, and interglycosidic 1H-13C coupling constants for 1 as a function of temperature. For comparison, we measured the temperature dependence of 1H chemical shifts and coupling constants for related monosaccharides: glucuronate (GlcUA or U) and N-acetylglucosamine (GlcNAc or N), and glucose (Glc). The temperature sensitivity of the 1H spectrum of 1 is caused by relatively larger values of delta delta/delta T for some ring protons, rather than a conformational change. The effect is mediated by strong coupling. To detect the presence of long-lived intramolecular hydrogen bonds in the disaccharide, we measured chemical shifts, delta delta/delta T, and coupling constants for hydroxyl protons of 1, GlcUA, and GlcNAc in 1:1 H2O-acetone-d6 at low temperature. We compared 1H NMR parameters for 1, GlcUA, and GlcNAc in water with published values measured in Me2SO-d6 and concluded that interactions with water predominated. We found no evidence for long-lived intramolecular hydrogen bonds occurring in 1 in aqueous solution.

Separation of hyaluronan oligosaccharides by the use of anion-exchange HPLC

There has been a recent surge of interest in hyaluronan (I-IA), a glycosaminoglycan. Hyaluronan is a linear polymer composed of the repeating disaccharide unit [(l + 3)-0-(2-acetamido-2-deoxy-&D-gIucopyranosyl)-(1 + 4&0-P-D-glucopyranuronosyl]. Originally thought to serve only a structural function in connective tissue, HA has now been found to play important roles in such diverse functions as cell-cell recognition’, limb, lung, and brain morphogenesis’,’ and as a potential regulator of tumor angiogenesis 3. It has also found biomedical applications in ophthalmic surgery and wound healing4. In the course of our Nh4R investigations of the solution conformation of hyaluronan as a function of chain length, we have developed a rapid, simple method for the preparation and analysis of monodisperse hyaluronan oligosaccharides. With this method we are able to separate HA oligomers of between 2 and 20 disaccharide units at a moderate pH so that chemical modification of the HA is unnecessary. Several methods for the separation of hyaluronan oligosaccharides already exist. Preparation of HA fragments has traditionally been accomplished by gel-permeation chromatography coupled with uranic acid analysis for peak identification5,6. Although this method is capable of separating HA oligomers up to 23 disaccharide units, it is tedious and time consuming, especially when used to prepare relatively large (> 10 mg) quantities of each fragment. HPLC methods including normalphase7, reversed-phase ion-pair’, and weak-anion exchange’ have also been used for the analysis and separation of HA oligosaccharides of varying length. Similar methods10-16 have been used to separate HA oligosaccharides from other glycosaminoglycans. However, the largest fragment that any of the published HPLC methods has been able to separate is six disaccharide units’. The development of a gel electrophoresis method allowed the analysis of oligosaccharides between 7 and 250 repeating disaccharide units 17*18 A capillary zone electrophoresis method has .

Solution dynamics of the 1,2,3,4,6-penta-O-acetyl-alpha-D-idopyranose ring.

The anticoagulant properties of heparin are thought to derive from the inhibition of thrombin and other coagulation-related proteases by the binding of heparin to cofactors such as antithrombin III and heparin cofactor II. The apparent minimum native heparin sequence which can bind to antithrombin III is a highly sulfated pentasaccharide which contains a 2-O-sulfo-α-L-idopyranosyluronic acid residue. The idopyranosyl residue has the unusual property of existing in the solution state as a mixture of ring conformers. Whereas most hexopyranose sugars exist as a single chair conformer (eg D-glucose exists overwhelmingly as a 4C1 chair), the idopyranosyl ring is known to rapidly exchange between at least two and often more distinct conformations, depending on type and number of substituents (hydroxyl, carboxyl, sulfate, etc.) and solvent conditions (solvent pH, salt concentration, temperature). It is believed that this flexibility of the idopyranosyl residue in heparin is related to its binding specificity.In the past, coupling constants and molecular dynamics have been used to estimate the relative populations of conformers in iduronate and related compounds. Here we report extensive NMR measurements, including line shape analysis, T1ρ measurements, T1 and NOE measurements and spectral density mapping, which have been used to study the dynamics of conformer interconversion in model compounds related to idose and glucose. The findings presented here indicate that 1,2,3,4,6-peneta-O-acetyl-α-D-idopyranose can be reasonably well described as existing in a two-state equilibrium consisting of the 4C1 and 0S2 conformers. 13C NMR line shape analysis yields a ΔH+ of 40 kJ mol-1 and a ΔS‡ of 31 J mol-1 K-1 for the 4C1→0S2 interconversion and a ΔH‡ of 31 kJ mol-1 and a ΔS‡ of 13 J mol-1K-1 for the 0S2→4C1 interconversion. This corresponds to exchange rates of 22 and 128 MHz, respectively, at room temperature.

A detailed 1H and 13C NMR study of a repeating disaccharide of hyaluronan: the effect of sodium and calcium ions.

Hyaluronan (HA), a polyanionic polysaccharide consisting of repeating glucuronate and N-acetylglucosamine residues, exists surrounded by ions in its physiological milieu. For example, the average concentration of sodium is 300 mM in bovine hyaline cartilage, or roughly twice that of typical extracellular fluid [1]. It has been shown that salts can modify strongly the properties of HA [2,3] and its constituent monomers [4,5]. For example, Van Damme et al. [2] reported that binding of HA to lysozyme is most efficient at pH 7.5 and 10-15 mM NaCI. Self-association of hyaluronate segments in aqueous solution requires a sodium concentration of 150 mM [3]. Several physical techniques (NMR [6-13], X-ray [14-17], CD [8,18], and IR [9,14]) have been employed to determine if salts interact with uronate residues non-specifically (via electrostatic interaction) [7] or through chelation by several ligands acting in concert. Most authors have postulated the existence of chelation sites. A review of studies conducted on HA in the solid and liquid states leads to the conclusion that the oxygens of the carboxyl groups on HA and of water molecules always take part in coordination of metal cations, while other oxygens around the sugar ring are sometimes involved as well. The type of

A comparison of the conformation of sucrose octasulfate, free and bound to acidic fibroblast growth factor

The conformation of sucrose octasulfate free in solution has been determined based on high-resolution NMR spectroscopy. Three-bond 1H-1H scalar coupling constants, laboratory and rotating frame NOEs, long-range 1H-13C scalar coupling constants, and chemical-shift temperature and ionic-strength dependence were used, aided by molecular mechanics calculations. By modification of a pulse sequence designed for measuring long-range 1H-13C coupling constants, it was possible to obtain an accurate value for the 3JC2f-H1g despite the confounding presence of 3JHH of similar value. Free sucrose octasulfate appears to assume a conformation significantly different from any of the eight conformations observed bound to acidic fibroblast growth factor, as determined in a previous X-ray crystallographic study [X. Zhu, B.T. Hsu, and D.C. Rees, Structure, 1 (1993) 27-34]. Strong electrostatic interactions between guest and host may be the dominant factor in deformation of sucrose octasulfate. The implications of this study for protein-carbohydrate interactions and the effects of the presence of sulfate groups on the flexibility of sucrose are also discussed.

Teaching high-resolution nuclear magnetic resonance to graduate students in biophysics

Teaching modern methods of high resolution, multi-dimensional nuclear magnetic resonance (NMR) spectroscopy in the limited time usually available is a challenge because of the breadth and complexity of the subject. Here we present two outlines for lectures on basic principles and biophysical applications of NMR, either in one lecture or in six to twelve lectures. We advocate emphasizing the versatility of NMR, and its numerous applications to biophysical questions. An annotated list of references is provided.