Dynamics of hyaluronan oligosaccharides revealed by 15N relaxation

Complex carbohydrates are considered to be flexible biomolecules, yet few experimental techniques are available to characterize their dynamics. In this study, we investigate the potential of 15N relaxation to probe the dynamics of hyaluronan oligosaccharides by adapting approaches previously applied to proteins. Unlike the 13C nucleus, 15N provides considerably enhanced spectral resolution, allowing position-specific information to be measured even in the middle of oligomers as large as decasaccharides. Moreover, isotopic incorporation maintains the 1H-15N group as an isolated spin-pair, allowing relaxation experiments to be performed and interpreted at low concentrations. A methodology is described for calculating the Lipari and Szabo model-free parameters at specific positions in hyaluronan oligomers and is used to produce a dynamic representation for the hexasaccharide. In this model, the glycosidic linkages and acetamido rotamer were determined to deviate by 18 degrees and 24 degrees from their mean positions, respectively. This approach allows the dynamic structural characterization of hyaluronan and other nitrogen-containing carbohydrates. The resultant models provide crucial insights into the physical properties and biology of these flexible molecules, which are at present poorly understood.     

Matrix assisted laser desorption ionization-time of flight mass spectrometry analysis of hyaluronan oligosaccharides

A new method is presented for the identification of oligosaccharides obtained by enzymatic digestion of hyaluronan (HA) with bacterial hyaluronidase (E.C. 4.2.2.1, from Streptomyces hyalurolyticus) using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOFMS). Mixtures containing HA oligosaccharides of tetrasaccharide (4-mer)-34-mer were analyzed using this method. The carboxyl groups of the glucuronate residues in the prepared HA oligomers, were modified as the acidic form (COOH), sodium salts (COONa), organic ammonium salts, or methylesters before MALDI-TOFMS measurement. Among these samples, the methylester form of glucuronate residues in HA oligosaccharides, prepared by methylation using trimethylsilyl diazomethane, afforded high sensitivity for spectra. This simple modification method for carboxyl group methylation of acidic polysaccharides [Hirano et al., Carbohydr. Res., 340, (2005) 2297-2304] provides samples suitable for MALDI-TOF mass spectrometric analysis throughout a significantly enhanced range of masses.     

Temperature dependencies of amide 1H- and 15N-chemical shifts in hyaluronan oligosaccharides

Temperature coefficients (Deltadelta/DeltaT) of amide chemical shifts of N-acetylglucosamine residues have been measured in a range of oligosaccharides of the important vertebrate polysaccharide hyaluronan. Odd- and even-numbered oligosaccharides with glucuronic acid, Delta-4,5-unsaturated glucuronic acid and N-acetylglucosamine at the termini were investigated. All amide proton temperature coefficients were only slightly less negative (-6.9 to - 9.1 ppb/ degrees C) than those of amide protons in free exchange with water (approximately equal to -11 ppb/ degrees C), indicating an absence of persistent intramolecular hydrogen bonds. With the exception of amide groups in reducing-terminal N-acetylglucosamine rings, all amide proton environments have the same temperature coefficient (-6.9 ppb/ degrees C), irrespective of differences in amide group chemical shifts and (3)J(HH) coupling constants, i.e. they do not sense subtle differences in orientation of the amide group. Amide nitrogen temperature coefficients report the same phenomena but with greater sensitivity. These data provide a set of reference values for temperature coefficients measured in other carbohydrates with acetamido sugars.     

Developing fluorescent hyaluronan analogs for hyaluronan studies

Two kinds of fluorescent hyaluronan (HA) analogs, one serving as normal imaging agent and the other used as a biosensitive contrast agent, were developed for the investigation of HA uptake and degradation. Our approach of developing HA imaging agents depends on labeling HA with varying molar percentages of a near-infrared (NIR) dye. At low labeling ratios, the hyaluronan uptake can be directly imaged while at high labeling ratios, the fluorescent signal is quenched and signal generation occurs only after degradation. It is found that the conjugate containing 1%-2% NIR dye can be used as a normal optical imaging agent, while bioactivable imaging agents are formed at 6% to 17% dye loading. It was determined that the conjugation of dye to HA with different loading percentages does not impact HA biodegradation by hyaluronidase (Hyal). The feasibility of using these two NIR fluorescent hyaluronan analogs for HA investigation was evaluated in vivo with optical imaging. The data demonstrates that the 1% dye loaded fluorescent HA can be used to monitor the behavior of HA and its fragments, whereas bioactivatable HA imaging agent (17% dye in HA) is more suitable for detecting HA fragments.     

NMR structural study of hydrogels based on partially deacetylated hyaluronan

Hydrogels have been prepared using two partially deacetylated hyaluronan (deHA) samples having a molar ratio of free amino groups to total initial N-acetamido groups equal to 23 and 33%, respectively. Taking advantage of such primary amino functionalities, polymeric networks could be easily obtained in aqueous media by means of two different crosslinking procedures, namely: a) Ugi multicomponent reactions and, more simply, b) glutaraldehyde reticulation. In this paper, we wish to report on the synthesis of deHA based hydrogels and, in particular, on their structural elucidation as obtained by NMR studies.

Structure of HA (R = COCH₃) and de-HA (R = H) repeat units.     

Therapeutic use of hyaluronan

Hyaluronan used today as a therapeutic agent in human and animal medicine must be in a highly purified form, free of immunologically active protein molecules, from endotoxin and from inflammatory molecules originating from the tissues or bacterial cultures—the source of hyaluronan. All hyaluronans, whether in liquid or in covalently crosslinked gel form, must have certain elastoviscous properties to be usable for current therapeutic applications. Elastoviscous properties are especially important in the ophthalmic viscosurgical use. The metabolism of hyaluronan is very fast in most tissues, except in the vitreus. The ability of injected hyaluronan with a short half-life time in the joint to accomplish long-lasting analgesia represents a challenge for the design of the various products. The history of the therapeutic use of hyaluronan and its present status is described, with emphasis on its use in ophthalmic surgery and for a long-lasting analgesia in arthritic joints in humans and animals. The use of hyaluronan gels is described in viscoaugmentation as injected into the dermal layer of the skin. Hyaluronan gels were also used as viscosupplements injected into sphincter muscles to improve their function in aging or disease. The use of hyaluronan and its gel for drug delivery was suggested several decades ago, and is also mentioned.     

Theoretical study of ibuprofen phototoxicity

The photochemical properties and degradation of the common nonsteroid anti-inflammatory drug ibuprofen is studied by means of hybrid density functional theory. Computed energies and properties of various species show that the deprotonated form dominates at physiological pH, and that the species will not be able to decarboxylate from a singlet excited state. Instead, decarboxylation will occur, with very high efficiency, provided the deprotonated compound can undergo intersystem crossing from an excited singlet to its excited triplet state. In the triplet state, the C-C bond connecting the carboxyl group is elongated, and the CO2 moiety detaches with a free energy barrier of less than 0.5 kcal/mol. Depending on the local environment, the decarboxylated product can then either be quenched through intersystem crossing (involving the possible formation of singlet oxygen) and protonation, or serve as an efficient source for superoxide anions and the formation of a peroxyl radical that will initiate lipid peroxidation.     

Theoretical study of single-bubble sonochemistry

Numerical simulations of bubble oscillations in liquid water irradiated by an ultrasonic wave are performed under the experimental condition for single-bubble sonochemistry reported by Didenko and Suslick [Nature (London) 418, 394 (2002)]. The calculated number of OH radicals dissolving into the surrounding liquid from the interior of the bubble agrees sufficiently with the experimental data. OH radicals created inside a bubble at the end of the bubble collapse gradually dissolve into the surrounding liquid during the contraction phase of an ultrasonic wave although about 30% of the total amount of OH radicals that dissolve into the liquid in one acoustic cycle dissolve in 0.1 micros at around the end of the collapse. The calculated results have indicated that the oxidant produced by a bubble is not only OH radical but also O atom and H2O2. It is suggested that an appreciable amount of O atom is produced by bubbles inside a standing-wave-type sonochemical reactor filled with water in which oxygen is dissolved as in the case of air.     

Theoretical study of free-radical migrations

Free-radical migrations have been investigated by ab initio molecular orbital theory to account for the facts that 1,2-migrations are observed for Cl-atoms, aryl or vinyl groups, whereas for H atoms only 1,5- and 1,6-migrations have been seen in contrast to the easy 1,2-hydride shifts in cations. The optimum geometry and energy models of the transition states of 1,2- and 1,5-migrations, and of their corresponding initial states have been determined using the Gaussian 70 STO-3G RHF method. The calculated activation energies ΔE are in agreement with experimental observations. The main features of the 1,2-migration reactions are (i) ΔE is more important for elements of the first and second row of the periodic classification than for those belonging to the third row; (ii) protonation strongly reduces ΔE. For both 1,2- and 1,5-migrations a correlation exists between ΔE and the energetic change of the frontier orbital, Δ?_somo: the reactions are under frontier orbital control.     

Synthesis of two hyaluronan trisaccharides

[formula: see text] The synthesis of two hyaluronan trisaccharides, methyl O-(beta-D-glucopyranosyluronic acid)-(1,3)-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-(1,4)-O-beta-D- glucopyranosiduronic acid and methyl O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-(1,4)-O-beta- D-glycopyranosyluronic acid)-(1,3)-O-(2-acetamido-2-deoxy-beta-D-glucopyranoside, are described. Construction of the target molecules was achieved though a combination of the phenyl sulfoxide and trichloroacetimidate glycosylation methodologies. This is the first report on the synthesis of the beta-methyl derivatives, which represent the smallest fragments that incorporate all the structural features of polymeric hyaluronan.     

Theoretical study of I2O

We present high-level density functional calculations (DFT) on the unknown I₂O molecule. The results suggest that the compound may be sufficiently stable for detection and synthesis. Our results also suggest that the DFT method is a reliable and computationally cheap alternative to G2, for estimating thermodynamic properties. The trends in relative stabilities within the HOX and X₂O series are discussed (X=halogen). © 1998 John Wiley & Sons, Inc. Heteroatom Chem 9:383–385, 1998     

Theoretical study of formamide decomposition pathways

The chemical transformations of formamide (NH(2)CHO), a molecule of prebiotic interest as a precursor for biomolecules, are investigated using methods of electronic structure computations and Rice-Rampserger-Kassel-Marcus (RRKM) theory. Specifically, quantum chemical calculations applying the coupled-cluster theory CCSD(T), whose energies are extrapolated to the complete basis set limit (CBS), are carried out to construct the [CH(3)NO] potential energy surface. RRKM theory is then used to systematically examine decomposition channels leading to the formation of small molecules including CO, NH(3), H(2)O, HCN, HNC, H(2), HNCO, and HOCN. The energy barriers for the decarboxylation, dehydrogenation, and dehydration processes are found to be in the range of 73-78 kcal/mol. H(2) loss is predicted to be a one-step process although a two-step process is competitive. CO elimination is found to prefer a two-step pathway involving the carbene isomer NH(2)CHO (aminohydroxymethylene) as an intermediate. This CO-elimination channel is also favored over the one-step H(2) loss, in agreement with experiment. The H(2)O loss is a multistep process passing through a formimic acid conformer, which subsequently undergoes a rate-limiting dehydration. The dehydration appears to be particularly favored in the low-temperature regime. The new feature identifies aminohydroxymethylene as a transient but crucial intermediate in the decarboxylation of formamide.     

Theoretical study of cooperativity in biotin

To give a deeper insight into the widely discussed catalytic mechanism of biotin, four representative model molecules and their aggregates hydrogen bonding (H-bonding) to water molecules were investigated by means of ab initio calculations and compared with molecular dynamics simulations. The roles of the ureido group, the sulfur atom, and the side chain of biotin are examined and discussed, respectively. Some significant H-bonding cooperativities are theoretically demonstrated in the ureido group of biotin. The pi-electron delocalization of the ureido group makes the system a good candidate for the H-bonding cooperativities, which in turn increases the covalent character of the corresponding H-bonds and facilitates the electrophilic substitution of the nitrogen atoms in the ureido group. The sulfur of biotin may participate in the delocalized pi-electron system of the ureido group via special sulfur-nitrogen bonding interactions, which reinforces the H-bonding cooperativities of the ureido group. The side chain of biotin not only reduced the accessibility of 3-NH due to steric hindrance but also enhanced the H-bonding cooperativities of the ureido group by folding over to hydrogen bond to more water molecules. The folded states are a probable way of activating 1-NH by strong cooperative effects. In addition, the H-bonding cooperativities may be a significant reason for the strong and specific binding between biotin and streptavidin.     

Theoretical study of acridane oxidation reactions

The interactions of acridanes with oxidants were modeled using combined methods of quantum chemistry (DFT B3LYP/6-311G(d,p)), molecular mechanics, MERA model, and MOPS algorithm. The results of simulation are compared with XRD data, oxidation potentials of acridanes, and the reduction potentials of the products of the oxidation reaction. It is shown that elimination of the hydride anion by the reactions of acridanes with oxidants is a consequence of a two-step process; the first step is the transfer of electron density from the HOMO of acridane to the LUMO of the oxidant; the second step is hydrogen elimination from the acridane molecule due to hydrogen bonding with the oxygen atom of the oxidant. The details of the mechanism were established by modeling the acridane-oxidant complexes using the MOPS algorithm including the continuum model of the solvent. The logarithms of the rate constants of the processes depend on the structure parameters of the model complexes with correlation coefficients of at least 0.93.     

Theoretical study of ScCO2+

The structure, binding energy, and vibrational frequencies have been determined for ScCO₂⁺. The inserted OSc⁺CO structure in the ¹A′ state is the most stable isomer and lies 43.2 kcal/mol below the ground-state Sc⁺+ CO₂ asymptote. The linear η¹-O Sc⁺(SINGLE BOND)OCO ³Δ state is bound by a charge-quadrupole interaction and has a binding energy of 13.9 kcal/mol. © 1997 John Wiley & Sons, Inc. Int J Quant Chem 63: 523–528, 1997     

Theoretical study of metallofullerenes M@C32

14 systems of metal atoms, embedded inside the C₃₂ cage, have been calculated and analyzed using the ab initio quantum chemistry Gamess program. We study their electronic structure, compare their stability. The metal atom with even electrons interjected inside the C₃₂ cage is more stable than the metal atom with odd electrons. Ti@C₃₂ system is the most stable in the 14 metallofullerenes.     

Theoretical study of hydrogen-bonded formaldehyde complexes

The hydrogen-bonded complexes involving formaldehyde and a series of proton donors of varying strengths, have been investigated at different levels of ab initio MO theory. The structures of the studied complexes were SCF optimized at the 6-31G basis set level. The binding energy was estimated employing basis set superposition correction, zero-point vibrations and MP2 correlation contribution at the different basis set: STO-3G; 6-31G; MP2/6-31G; 6-31G**; MP2/6-31G**; 6-311G(2d, 2p) and MP2/6-311G(2d, 2p). Linear relationships were found of the calculated binding energy with: the calculated shift in the carbonyl stretching frequency, the changes in carbonyl bond length and the optimum value of hydrogen-bond distance; furthermore the calculations confirm a parallel trend between the proton-donor ability and the strength of the hydrogen bond.