Wanda Sicinska

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.

Solvent effects on nitrogen NMR shieldings in azines

Abstract The ranges of solvent effects on the nitrogen shielding are reported for pyridine, 1,2-1,3-, and 1,4-diazine, and 1,3,5-triazine. These ranges are both significant and variable from about 48 ppm for 1,2-diazine to about 1 I ppm for the triazine. The former is the largest solvent-induced range so far observed in nitrogen NMR. Contributions to the nitrogen shielding ranges from hydrogen bonding and solvent polarity effects are estimated. The former effect usually provides a dominant contribution and can be associated with the strength of the hydrogen bond formed from the solvent to the solute. The contribution to the change in nitrogen shielding from a change in solvent polarity is also significant but usually less than that due to hydrogen bonding. Both effects operate to produce changes in the nitrogen shielding in the same direction. The polarity effects are unusually large in the case of 1,2-diazine and this is corroborated by INDO/S-SOS shielding calculations using the solvaton model.

13C Chemical shifts of some azaindolizines versus electron charge distribution

Abstract Azaindolizines, which contain all possible combinations of nitrogen atoms within the five-membered ring moiety, are used as models for the investigation of a relationship between electron charge distribution and 13C shifts. A linear correlation is observed between the shifts and total rather than π-charge densities as calculated by the INDO-MO method. The average excitation energy (AEE) approximation in the theory of nuclear screening is shown to hold separately for the CH moieties and the carbon atoms at the ring junction in indolizines. An empirical correlation with charge densities is obtained from the AEE method, as a result of the compensation of effects within the local paramagnetic term and the prevailing contribution to the latter of the effective nuclear charge.13C shifts afford a reasonable measure of the total net charges at the carbon atoms of indolizines. The INDO calculations indicate that the π-charges follow the pattern suggested by simple resonance structures but the overall charge density depends heavily on σ-core polarization effects.

Model of three-dimensional structure of vitamin D receptor and its binding mechanism with 1alpha,25-dihydroxyvitamin D(3).

Comparative modeling of the vitamin D receptor three‐dimensional structure and computational docking of 1α,25‐dihydroxyvitamin D3 into the putative binding pocket of the two deletion mutant receptors: (207–423) and (120–422, Δ [164–207]) are reported and evaluated in the context of extensive mutagenic analysis and crystal structure of holo hVDR deletion protein published recently. The obtained molecular model agrees well with the experimentally determined structure. Six different conformers of 1α,25‐dihydroxyvitamin D3 were used to study flexible docking to the receptor. On the basis of values of conformational energy of various complexes and their consistency with functional activity, it appears that 1α,25‐dihydroxyvitamin D3 binds the receptor in its 6‐s‐trans form. The two lowest energy complexes obtained from docking the hormone into the deletion protein (207–423) differ in conformation of ring A and orientation of the ligand molecule in the VDR pocket. 1α,25‐Dihydroxyvitamin D3 possessing the A‐ring conformation with axially oriented 1α‐hydroxy group binds receptor with its 25‐hydroxy substituent oriented toward the center of the receptor cavity, whereas ligand possessing equatorial conformation of 1α‐hydroxy enters the pocket with A ring directed inward. The latter conformation and orientation of the ligand is consistent with the crystal structure of hVDR deletion mutant (118–425, Δ [165–215]). The lattice model of rVDR (120–422, Δ [164–207]) shows excellent agreement with the crystal structure of the hVDR mutant. The complex obtained from docking the hormone into the receptor has lower energy than complexes for which homology modeling was used. Thus, a simple model of vitamin D receptor with the first two helices deleted can be potentially useful for designing a general structure of ligand, whereas the advanced lattice model is suitable for examining binding sites in the pocket. Proteins 2001;44:188–199.

NMR assignments of tryptophan residue in apo and holo LBD-rVDR.

Binding sites in the full‐length, ligand‐binding domain of rat vitamin D receptor (LBD‐rVDR) for an active hormone derived from vitamin D (1α,25‐dihydroxyvitamin D3) and three of its C‐2 substituted analogs were compared by nuclear magnetic resonance (NMR) spectroscopy. Specific residue labeled with [UL]‐15N2 Trp allowed assignment of the side‐chain Hϵ1 and Nε1 resonances of the single tryptophan residue at position 282 in LBD‐rVDR. Comparison of 1H[15N] Heteronuclear Single Quantum Correlation (HSQC) spectra of apo and holo LBD‐rVDR revealed that the position of the Trp282 Hε1 and Nε1 signals are sensitive to the presence of the ligand in the receptor cavity. Binding of the ligands to LBD‐rVDR results in a shift of both Trp Hε1 and Nε1 resonances to lower frequencies. The results indicate that the interaction between the ligands and Trp282 is not responsible for differences in calcemic activity observed in vitamin D analogs. Proteins 2005.

Solvent polarity and hydrogen-bonding effects on the nitrogen NMR shieldings of N-nitrosamines and DFT calculations of the shieldings of C-, N-, and O-nitroso systems

High-precision nitrogen NMR shieldings, bulk susceptibility corrected, are reported for dimethyl-N-nitrosamine (I) and diethyl-N-nitrosamine (II) in a variety of solvents which represent a wide range of solvent properties from the point of view of polarity as well as hydrogen bond donor and acceptor strength. The observed range of solvent-induced nitrogen shielding variations of (I) and (II) is significant for the amino-type nitrogens, up to about 16 ppm, and originates essentially from the deshielding effect of the increasing polarity of solvent. On the other side, the nitroso nitrogen shieldings reveal an even stronger response to solvent effects, within about 20 ppm, but in this case the increasing polarity and hydrogen bond donor strength of solvent produce enhanced shielding. DFT quantum-mechanical calculations using the GIAO/B3PW91/6-311++G** approach and geometry optimizations employing the same basis set and hybrid density functionals show an excellent correlation with the experimental data on C-, N-, and O-nitroso moieties and reproduce not only major changes but also most of the subtle variations in the experimental nitrogen shieldings of the nitroso systems as a whole. A combination of the calculations involving the corresponding N and O-protonated species and the trends observed in the solvent-induced nitrogen shielding variations shows clearly that the prime acceptor site for hydrogen bonding is the nitroso oxygen atom.

Solvent-Induced Effects on the Nitrogen NMR Shieldings of Some Nitrosobenzene Systems

High‐precision 14N NMR measurements of solvent‐induced shielding variations are reported for some nitrosobenzene systems. These variations are shown to result from a combination of three major factors, solvent to solute hydrogen bonding where the solute nitrogen lone pair electrons are involved, solvent polarity and interactions between the electron‐deficient benzene ring of the nitrosobenzenes and basic centres in the solvent molecules. The last of these three factors produces nitrogen deshielding of the nitroso group, and in the present work this interaction was found to be the largest of its type so far observed. Consequently, this implies that, in nitroso aromatic compounds, the benzene ring shows a remarkable deficit of electronic charge. The former two factors produce an increase in nitroso nitrogen shielding, thus indicating a strong electron‐withdrawing effect of the nitroso group which is consistent with previous observations. INDO/S parameterized molecular orbital calculations of solute nitrogen shieldings, incorporating the Solvaton model of non‐specific solute–solvent interactions, predict that the nitrogen shielding will increase as the polarity of the medium increases. This is in very good agreement with the observation that the nitroso nitrogen shielding analysis yields a large and positive value for the s term which describes the influence of solvent polarity/polarizability on the shielding variation as a function of solvent. For the 0.2 M solutions studied at 35°C, a significant amount of the dimeric isodioxy form is only observed for o‐nitrosotoluene.

Solvent Effects on Nitrogen NMR Shieldings of 1,2,4-Triazine

Abstract Nitrogen NMR shieldings of 1,2,4-triazine are shown, as an example, to be capable of providing deep insight into solvent-induced, site-oriented electric charge redistributions and solvent-solute hydrogen bonding effects in an unsymmetrical molecule. A sharp contrast is observed in the solvent effects on the nitrogen atoms in positions 1 and 2 with respect to that at position 4. The former pair of atoms exhibits a remarkable affinity to hydrogenbond donor solvents. Their electron densities appear to be significantly dependent upon the solvent polarity. In contrast N4 appears to be relatively uninfluenced by solvent effects.

A NMR study of solute-solvent interactions as a function of the nitrogen shielding of pyridine N-oxide

Abstract The range of solvent effects on the nitrogen shielding of pyridine N-oxide is found to be comparable to that of pyridine, amounting to about 30 ppm. The largest contribution to this range arises from hydrogen bonding from the solvents to the oxygen atom of pyridine N-oxide. For the first time it is demonstrated that hydrogen bonding to a neighboring atom can have a comparable effect on nitrogen shielding as would direct hydrogen bonding to the nitrogen atom. Medium polarity effects on the nitrogen shielding of pyridine and its N-oxide are of the same sign and order of magnitude. This is corroborated by INDO/S-SOS shielding calculations using the solvaton model. These medium polarity effects amount to a nitrogen shielding variation not exceeding 6 ppm while those due to hydrogen bonding exceed 20 ppm.

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