Point-of-Care Laboratory Data Collection During Critical Care Transport.

OBJECTIVE: Critical care transport involves a high level of intensive clinical care in a resource-limited environment. These patients require multiple assessments guiding specialty treatments, including blood product administration, intravenous electrolyte replacement, ventilator management, and extracorporeal membrane oxygenation. This study aims to measure the usage of point-of-care (POC) laboratory data during critical care transport. METHODS: Data were collected via electronic medical record review over 1 year of use in a hospital-based critical care rotor wing, fixed wing, and ground critical care transport team in the Southeastern United States. RESULTS: One hundred twenty POC tests were performed during 1,075 critical care transports over the 1-year period (8.9%). Patient transportations involved 35 extracorporeal membrane oxygenation, 21 medical, 17 cardiac, 13 neonatal, 11 respiratory failure, 8 gastrointestinal bleeding, 6 neurologic, 5 pediatrics, 3 trauma, and 1 organ donor. Seventy-eight POC laboratory tests (65%) required intervention, including ventilator changes (39.7%), electrolyte replacement (35.8%), blood products (7.6%), and other (12.8%). The remaining 42 (35%) POC laboratory tests confirmed no intervention was necessary (n = 35) and that ongoing treatments were effective (n = 7). CONCLUSION: POC laboratory testing performed during critical care transport guides providers in performing essential emergent interventions in a timelier manner that may benefit critically ill patients.

Synthesis and characterization of heparosan-granulocyte-colony stimulating factor conjugates: a natural sugar-based drug delivery system to treat neutropenia.

Many injectable drugs require delivery strategies for enhancing their pharmacokinetics due to rapid loss via renal filtration if possess low molecular weight (<60-70 kDa) and/or clearance by the body's components (e.g., proteases, antibodies, high-efficiency receptors) in their native form. FDA-approved polyethylene glycol (PEG) is a vehicle for improving therapeutics, but artificial polymers have potential biocompatibility and immunogenicity liabilities. Here, we utilized a natural vertebrate carbohydrate, heparosan (HEP), the biosynthetic precursor of heparan sulfate and heparin, to enhance performance of a biologic drug. The HEP polysaccharide was stable with a long half-life (~8 days for 99-kDa chain) in the nonhuman primate bloodstream, but was efficiently degraded to very short oligosaccharides when internalized by cells, and then excreted into urine and feces. Several HEP-modified human granulocyte-colony stimulating factor (G-CSF) conjugates were synthesized with defined quasi-monodisperse HEP polysaccharide chains. Single dosing of 55- or 99-kDa HEP-G-CSF in rats increased blood neutrophil levels comparable to PEG-G-CSF conjugates. Repeated dosing of HEP-G-CSF or HEP alone for 2 weeks did not cause HEP-specific toxic effects in rats. HEP did not possess the anticoagulant behavior of its daughter, heparin, based on testing in rats or clinical diagnostic assays with human plasma. Neither anti-HEP IgG nor IgM antibodies were detected in a long-term (9 doses over 7 months) immunogenicity study of the HEP-drug conjugate with rats. These proof-of-concept experiments with HEP-G-CSF indicate that it is a valid drug candidate for neutropenia and suggest the potential of this HEP-based platform as a safe alternative delivery vehicle for other therapeutics.

Heparosan-coated liposomes for drug delivery.

Liposomal encapsulation is a useful drug delivery strategy for small molecules, especially chemotherapeutic agents such as doxorubicin. Doxil® is a doxorubicin-containing liposome ("dox-liposome") that passively targets drug to tumors while reducing side effects caused by free drug permeating and poisoning healthy tissues. Polyethylene glycol (PEG) is the hydrophilic coating of Doxil® that protects the formulation from triggering the mononuclear phagocyte system (MPS). Evading the MPS prolongs dox-liposome circulation time thus increasing drug deposition at the tumor site. However, multiple doses of Doxil® sometimes activate an anti-PEG immune response that enhances liposome clearance from circulation and causes hypersensitivity, further limiting its effectiveness against disease. These side effects constrain the utility of PEG-coated liposomes in certain populations, justifying the need for investigation into alternative coatings that could improve drug delivery for better patient quality of life and outcome. We hypothesized that heparosan (HEP; [-4-GlcA-ß1-4-GlcNAc-α1-]n) may serve as a PEG alternative for coating liposomes. HEP is a natural precursor to heparin biosynthesis in mammals. Also, bacteria expressing an HEP extracellular capsule during infection escape detection and are recognized as "self," not a foreign threat. By analogy, coating drug-carrying liposomes with HEP should camouflage the delivery vehicle from the MPS, extending circulation time and potentially avoiding immune-mediated clearance. In this study, we characterize the postmodification insertion of HEP-lipids into liposomes by dynamic light scattering and coarse-grain computer modeling, test HEP-lipid immunogenicity in rats, and compare the efficacy of drug delivered by HEP-coated liposomes to PEG-coated liposomes in a human breast cancer xenograft mouse model.

A refined model for the TSG-6 link module in complex with hyaluronan: use of defined oligosaccharides to probe structure and function.

Tumor necrosis factor-stimulated gene-6 (TSG-6) is an inflammation-associated hyaluronan (HA)-binding protein that contributes to remodeling of HA-rich extracellular matrices during inflammatory processes and ovulation. The HA-binding domain of TSG-6 consists solely of a Link module, making it a prototypical member of the superfamily of proteins that interacts with this high molecular weight polysaccharide composed of repeating disaccharides of D-glucuronic acid and N-acetyl-D-glucosamine (GlcNAc). Previously we modeled a complex of the TSG-6 Link module in association with an HA octasaccharide based on the structure of the domain in its HA-bound conformation. Here we have generated a refined model for a HA/Link module complex using novel restraints identified from NMR spectroscopy of the protein in the presence of 10 distinct HA oligosaccharides (from 4- to 8-mers); the model was then tested using unique sugar reagents, i.e. chondroitin/HA hybrid oligomers and an octasaccharide in which a single sugar ring was (13)C-labeled. The HA chain was found to make more extensive contacts with the TSG-6 surface than thought previously, such that a D-glucuronic acid ring makes stacking and ionic interactions with a histidine and lysine, respectively. Importantly, this causes the HA to bend around two faces of the Link module (resembling the way that HA binds to CD44), potentially providing a mechanism for how TSG-6 can reorganize HA during inflammation. However, the HA-binding site defined here may not play a role in TSG-6-mediated transfer of heavy chains from inter-α-inhibitor onto HA, a process known to be essential for ovulation.

Proteoglycans and their heterogeneous glycosaminoglycans at the atomic scale.

Proteoglycan spatiotemporal organization underpins extracellular matrix biology, but atomic scale glimpses of this microarchitecture are obscured by glycosaminoglycan size and complexity. To overcome this, multimicrosecond aqueous simulations of chondroitin and dermatan sulfates were abstracted into a prior coarse-grained model, which was extended to heterogeneous glycosaminoglycans and small leucine-rich proteoglycans. Exploration of relationships between sequence and shape led to hypotheses that proteoglycan size is dependent on glycosaminoglycan unit composition but independent of sequence permutation. Uronic acid conformational equilibria were modulated by adjacent hexosamine sulfonation and iduronic acid increased glycosaminoglycan chain volume and rigidity, while glucuronic acid imparted chain plasticity. Consequently, block copolymeric glycosaminoglycans contained microarchitectures capable of multivalent binding to growth factors and collagen, with potential for interactional synergy at greater chain number. The described atomic scale views of proteoglycans and heterogeneous glycosaminoglycans provide structural routes to understanding their fundamental signaling and mechanical biological roles and development of new biomaterials.

Microsecond kinetics in model single- and double-stranded amylose polymers.

Amylose, a component of starch with increasing biotechnological significance, is a linear glucose polysaccharide that self-organizes into single- and double-helical assemblies. Starch granule packing, gelation and inclusion-complex formation result from finely balanced macromolecular kinetics that have eluded precise experimental quantification. Here, graphics processing unit (GPU) accelerated multi-microsecond aqueous simulations are employed to explore conformational kinetics in model single- and double-stranded amylose. The all-atom dynamics concur with prior X-ray and NMR data while surprising and previously overlooked microsecond helix-coil, glycosidic linkage and pyranose ring exchange are hypothesized. In a dodecasaccharide, single-helical collapse was correlated with linkages and rings transitioning from their expected syn and (4)C1 chair conformers. The associated microsecond exchange rates were dependent on proximity to the termini and chain length (comparing hexa- and trisaccharides), while kinetic features of dodecasaccharide linkage and ring flexing are proposed to be a good model for polymers. Similar length double-helices were stable on microsecond timescales but the parallel configuration was sturdier than the antiparallel equivalent. In both, tertiary organization restricted local chain dynamics, implying that simulations of single amylose strands cannot be extrapolated to dimers. Unbiased multi-microsecond simulations of amylose are proposed as a valuable route to probing macromolecular kinetics in water, assessing the impact of chemical modifications on helical stability and accelerating the development of new biotechnologies.

Does microsecond sugar ring flexing encode 3D-shape and bioactivity in the heparanome?

The biological information encoded in carbohydrate sequences dwarfs that of proteins and nucleic acids. Deciphering structure-function relationships in heparin and heparan sulfate (the heparanome) is further compounded by extreme sequence diversity, experimental difficulties, and the computational cost of rigorous modeling. Here we perform unbiased microsecond dynamics simulations of 11 heparanome oligosaccharides (55 microseconds total) to investigate the effect of sequence on 3D-structure and to underpin a coarse-grained model that is consistent with long-time scale experimentally validated atomic motions in water. Pyranose ring flexing (puckering) in 2-O-sulfo-α-l-iduronic acid, which underlies heparin-mediated anticoagulation, was modulated by polymerization (chain position and adjacent residues), which is supported by previous experiments. Furthermore, in coarse-grained simulations, inclusion of puckering was essential to predict macroscopic hydrodynamic properties of heparan sulfate chains containing hundreds of monosaccharaides. Our structural findings and model enable rational molecular design, and we propose that, in the heparanome, puckering, polymer 3D-shape, and bioactivity are inextricably linked.

Quantification of free ligand conformational preferences by NMR and their relationship to the bioactive conformation.

Accurate unbound solution 3D-structures of ligands provide unique opportunities for medicinal chemistry and, in particular, a context to understand binding thermodynamics and kinetics. Previous methods of deriving these 3D-structures have had neither the accuracy nor resolution needed for drug design and have not yet realized their potential. Here, we describe and apply a NMR methodology to the aminoglycoside streptomycin that can accurately quantify accessible 3D-space and rank the occupancy of observed conformers to a resolution that enables medicinal chemistry understanding and design. Importantly, it is based upon conventional small molecule NMR techniques and can be performed in physiologically-relevant solvents. The methodology uses multiple datasets, an order of magnitude more experimental data than previous NMR approaches and a dynamic model during refinement, is independent of computational chemistry and avoids the problem of virtual conformations. The refined set of solution 3D-shapes for streptomycin can be grouped into two major families, of which the most populated is almost identical to the 30S ribosomal subunit bioactive shape. We therefore propose that accurate unbound ligand solution conformations may, in some cases, provide a subsidiary route to bioactive shape without crystallography. This experimental technique opens up new opportunities for drug design and more so when complemented with protein co-crystal structures, SAR data and pharmacophore modeling.

Assigning kinetic 3D-signatures to glycocodes.

Reconciling glycocodes and their associated bioactivities, via 3D-structure, will rationalise burgeoning high-throughput functional glycomics data and underpin a new era of opportunity in chemical biology. A major impasse to achieving this goal is a detailed understanding of pyranose sugar ring 3D-conformation (or pucker) and the affiliated microsecond-timescale exchange kinetics. Here, we perform hardware-accelerated kinetically-rigorous equilibrium simulations of fundamental monosaccharides to produce the hypothesis that pyranoses have microsecond-timescale kinetic puckering signatures in water, classified as unstable (rare in the glycome), metastable (infrequently observed) and stable (prevalent). The predicted µs-metastability of ß-d-glucose explained hitherto irreconcilable experimental measurements. Twisted puckers seen in carbohydrate enzymes were present in the aqueous 3D-ensemble (suggesting preorganization) and pyranose-water interactions accounted for the relative stability of ß-d-galactose. Characteristic 3D-shapes for biologically- and commercially-important carbohydrates and new rules linking chemical modifications with pyranose µs-puckering kinetics are proposed. The observations advance structural-glycomics towards dynamic 3D-templates suitable for structure-based design.

Chemoenzymatic synthesis of sulfur-linked sugar polymers as heparanase inhibitors.

Complex carbohydrates (glycans) are major players in all organisms due to their structural, energy, and communication roles. This last essential role involves interacting and/or signaling through a plethora of glycan-binding proteins. The design and synthesis of glycans as potential drug candidates that selectively alter or perturb metabolic processes is challenging. Here we describe the first reported sulfur-linked polysaccharides with potentially altered conformational state(s) that are recalcitrant to digestion by heparanase, an enzyme important in human health and disease. An artificial sugar donor with a sulfhydryl functionality is synthesized and enzymatically incorporated into polysaccharide chains utilizing heparosan synthase. Used alone, this donor adds a single thio-sugar onto the termini of nascent chains. Surprisingly, in chain co-polymerization reactions with a second donor, this thiol-terminated heparosan also serves as an acceptor to form an unnatural thio-glycosidic bond ('S-link') between sugar residues in place of a natural 'O-linked' bond. S-linked heparan sulfate analogs are not cleaved by human heparanase. Furthermore, the analogs act as competitive inhibitors with > ~200-fold higher potency than expected; as a rationale, molecular dynamic simulations suggest that the S-link polymer conformations mimic aspects of the transition state. Our analogs form the basis for future cancer therapeutics and modulators of protein/sugar interactions.

Is N-acetyl-D-glucosamine a rigid 4C1 chair?

Understanding microsecond-timescale dynamics is crucial to establish three-dimensional (3D) structure-activity relationships in sugars but has been intractable to experiments and simulations. As a consequence, whether arguably the most important chemical scaffold in glycobiology, N-acetyl-d-glucosamine (GlcNAc), deviates from a rigid (4)C(1) chair is unknown. Here, conformer populations and exchange kinetics were quantified from the longest aqueous carbohydrate simulations to date (0.2 ms total) of GlcNAc, four derivatives from heparan sulfate and their methylglycosides. Unmodified GlcNAc took 3-5 µs to reach a conformational equilibrium, which comprised a metastable (4)C(1) chair that underwent (4)C(1) ↔ (1)C(4) transitions at a predicted forward rate of 0.8 µs(-1) with an average (1)C(4)-chair lifetime of 3 ns. These predictions agree with high-resolution crystallography and nuclear magnetic resonance but not with the hypothesis that GlcNAc is a rigid (4)C(1) chair, concluded from previous experimental analyses and non-aqueous modeling. The methylglycoside was calculated to have a slower forward rate (0.3 µs(-1)) and a more stable (4)C(1) conformer (0.2 kcal mol(-1)), suggesting that pivotal 3D intermediates (particularly (2)S(O), (1)S(5) and B(2,5)) increased in energy, and water was implicated as a major cause. Sulfonation (N-, 3-O and 6-O) significantly augmented this effect by blocking pseudorotation, but did not alter the rotational preferences of hydroyxl or hydroxymethyl groups. We therefore propose that GlcNAc undergoes puckering exchange that is dependent on polymerization and sulfo substituents. Our analyses, and 3D model of the equilibrium GlcNAc conformer in water, can be used as dictionary data and present new opportunities to rationally modify puckering and carbohydrate bioactivity, with diverse applications from improving crop yields to disease amelioration.

Pd(II)-mediated assembly of porphyrin channels in bilayer membranes.

A membrane-spanning bis(meso-3-pyridyl) porphyrin 1 has been synthesized, embedded in EYPC vesicles, and upon Pd(II) addition has been shown to form ionophores that allow the passage of anionic 5/6-carboxyfluorescein through membranes. The geometric matching of bis(meso-3-pyridyl) porphyrin 1 and trans-Pd(II) was designed to give a cyclic porphyrin trimer [PdCl(2)(1)](3). However, solution-phase studies showed that PdCl(2)(PhCN)(2) cross linked 1 into linear oligomers at porphyrin concentrations above 10 mM, although the formation of cyclic species was inferred from studies at concentrations below 2 µM. Fluorescence titrations showed that embedding porphyrin 1 in bilayers greatly reduced its affinity for Pd(II), but the combination of porphyrin 1 and Pd(II) gave an ionophoric species that increased the rate of 5/6-carboxyfluorescein (5/6-CF) transit through the phospholipid bilayer 12-fold. A maximum in the 5/6-CF release rate was observed at a Pd(II) concentration of 4 µM, and the application of a solution-phase binding model to the membrane phase showed that this peak in ionophoric activity corresponded to the greatest extent of porphyrin oligomerization. Further studies suggested these Pd(II)/porphyrin oligomers transported 5/6-CF via a channel mechanism.

A new route to carbohydrate secondary and tertiary structure using Raman spectroscopy and Raman optical activity.

The structural characterization of carbohydrate polymers is important for understanding their functions and behavior. However, mainstream structural biology tools are not applicable to many carbohydrate polymers, particularly at physiological concentrations. We report Raman and Raman optical activity spectra of hyaluronan polymer, the hyaluronan tetramer building block, and the two monosaccharide components glucuronic acid and N-acetylglucosamine and identify marker bands corresponding to primary and secondary structure in glycosaminoglycans. Furthermore, we show that the hyaluronan polymer does not adopt tertiary structure under near-physiological conditions, confirming a proposed model of hyaluronan structural organization.

Free energy landscapes of iduronic acid and related monosaccharides.

The pyranose ring of L-iduronic acid (IdoA), a major constituent of the anticoagulant heparin, is an equilibrium of multiple ring puckers that have evaded quantification by experiment or computation. In order to resolve this enigma, we have calculated the free energy landscape of IdoA and two related monosaccharides from extensive microsecond simulations. After establishing that the simulated puckers had reached equilibrium, hypotheses were confirmed that (a) IdoA (1)C(4)- and (4)C(1)-chair conformations exchange on the microsecond time scale, (b) C5 epimerization leads to a (4)C(1)-chair, and (c) IdoA 2-O-sulfation (IdoA2S) stabilizes the (1)C(4) conformer. The IdoA and IdoA2S (1)C(4) conformers were isoenergetic and computed to be 0.9 and 2.6 kcal mol(-1) lower in free energy than their respective (4)C(1)-chair conformations. The simulations also predicted that the IdoA (2)S(O)-skew-boat was less populated than previously thought. Novel chemical synthesis and ultra-high-field NMR supported these observations, but slight discrepancies in observed and predicted NMR vicinal couplings implied that the simulation overestimated the population of the IdoA (4)C(1)-chair with respect to (1)C(4)-chair due to small force field inaccuracies that only manifest in long simulations. These free-energy calculations drive improvements in computational methods and provide a novel route to carbohydrate mimetic biomaterials and pharmaceuticals.

Less is more when simulating unsulfated glycosaminoglycan 3D-structure: comparison of GLYCAM06/TIP3P, PM3-CARB1/TIP3P, and SCC-DFTB-D/TIP3P predictions with experiment.

The 3D-structure of extracellular matrix glycosaminoglycans is central to function, but is currently poorly understood. Resolving this will provide insight into their heterogeneous biological roles and help to realize their significant therapeutic potential. Glycosaminoglycan chemical isoforms are too numerous to study experimentally and simulation provides a tractable alternative. However, best practice for accurate calculation of glycosaminoglycan 3D-structure within biologically relevant nanosecond timescales is uncertain. Here, we evaluate the ability of three potentials to reproduce experimentally observed glycosaminoglycan monosaccharide puckering, disaccharide 3D-conformation, and characteristic solvent interactions. Temporal dynamics of unsulfated chondroitin, chondroitin-4-sulfate, and hyaluronan ß(1→3) disaccharides were simulated within TIP3P explicit solvent unrestrained for 20 ns using the GLYCAM06 force-field and two semi-empirical quantum mechanics methods, PM3-CARB1 and SCC-DFTB-D (both within a hybrid QM/MM formalism). Comparison of calculated and experimental properties (vicinal couplings, nuclear Overhauser enhancements, and glycosidic linkage geometries) showed that the carbohydrate-specific parameterization of PM3-CARB1 imparted quantifiable benefits on monosaccharide puckering and that the SCC-DFTB-D method (including an empirical correction for dispersion) best modeled the effects of hexosamine 4-sulfation. However, paradoxically, the most approximate approach (GLYCAM06/TIP3P) was the best at predicting monosaccharide puckering, 3D-conformation, and solvent interactions. Our data contribute to the debate and emerging consensus on the relative performance of these levels of theory for biological molecules.

Deconvolution of conformational exchange from Raman spectra of aqueous RNA nucleosides.

Ribonucleic acids (RNAs) are key to the central dogma of molecular biology. While Raman spectroscopy holds great potential for studying RNA conformational dynamics, current computational Raman prediction and assignment methods are limited in terms of system size and inclusion of conformational exchange. Here, a framework is presented that predicts Raman spectra using mixtures of sub-spectra corresponding to major conformers calculated using classical and ab initio molecular dynamics. Experimental optimization allowed purines and pyrimidines to be characterized as predominantly syn and anti, respectively, and ribose into exchange between equivalent south and north populations. These measurements are in excellent agreement with Raman spectroscopy of ribonucleosides, and previous experimental and computational results. This framework provides a measure of ribonucleoside solution populations and conformational exchange in RNA subunits. It complements other experimental techniques and could be extended to other molecules, such as proteins and carbohydrates, enabling biological insights and providing a new analytical tool.

Multiscale modeling of glycosaminoglycan structure and dynamics: current methods and challenges.

Glycosaminoglycans are long unbranched and complex polysaccharides that are an essential component of mammalian extracellular matrices. Characterization of their molecular structure, dynamics and interactions are essential to understand important biological phenomena in health and disease, and will lead to novel therapeutics and medical devices. However, this has proven to be a challenge experimentally and theoretical techniques are needed to develop new hypotheses, and interpret experiments. This review aims to examine the current theoretical (rather than experimental) methods used by researchers to investigate glycosaminoglycan structure, dynamics and interactions, from the monosaccharide to the macromolecular scale. It will consider techniques such as quantum mechanics, molecular mechanics, molecular dynamics, coarse graining and docking.

Hyaluronan: the local solution conformation determined by NMR and computer modeling is close to a contracted left-handed 4-fold helix.

The polysaccharide hyaluronan (HA) is a ubiquitous component of the vertebrate extracellular matrix with diverse physiological roles from space-filling to acting as a scaffold for other macromolecules. The molecular interactions responsible for these solution properties have been the subject of much debate and, primarily due to the lack of residue-specific experimental data, no consensus model for the three-dimensional conformation nor dynamics of HA in solution has emerged. Here, the solution conformation of HA is investigated using molecular dynamics (MD) simulations and high-field nuclear magnetic resonance (NMR). In contrast to previous studies, MD simulations incorporated explicit water molecules and sodium ions, while NMR experiments utilized (15)N-enriched oligosaccharides to allow residue-specific information to be obtained. The resultant average conformation is predicted to be almost a contracted left-handed 4-fold helix; i.e. similar to that observed for sodium hyaluronate fibers by X-ray diffraction, but with the acetamido side-chain trans to H(2). The glycosidic linkages and acetamido side-chains are predicted to have standard deviation rotations of 13 degrees and 18 degrees around their mean conformations in free solution, respectively, and are not observed to be stabilized by strong intramolecular hydrogen bonds as X-ray fiber diffraction refinements describe for the solid-state. Rather, weak and transient hydrogen bonds that are in rapid interchange with solvent molecules are predicted. These predictions are quantitatively consistent with demanding residue-specific NMR data and correspond to an HA molecule that is rod-like as an oligosaccharide and behaves as a stiffened random coil at large molecular mass, in close agreement with previous hydrodynamic observations. This new description of the solution conformation of HA is consistent with all available experimental data and accounts for its viscoelastic space-filling properties. This representation can be used as a basis for modeling the association between HA and proteins, which will elucidate important aspects of extracellular matrix assembly.

Hyaluronan: the absence of amide-carboxylate hydrogen bonds and the chain conformation in aqueous solution are incompatible with stable secondary and tertiary structure models.

Contradictory descriptions for the aqueous solution conformation of the glycosaminoglycan hyaluronan (HA) exist in the literature. According to hydrodynamic and simulation data, HA molecules are stiffened by a rapidly interchanging network of transient hydrogen bonds at the local level and do not significantly associate at the global level. In marked contrast, models derived from NMR data suggest that the secondary structure involves persistent hydrogen bonds and that strong associations between chains can occur to form vast stable tertiary structures. These models require an extended 2-fold helical conformation of the HA chain and specific hydrogen bonds between amide and carboxylate groups. To test these descriptions, we have used 15N-labelled oligosaccharides and high-field NMR to measure pertinent properties of the acetamido group. The amide proton chemical shift perturbation and carboxylate group pK(a) value are inconsistent with a highly populated hydrogen bond between the amide and carboxylate groups. Amide proton temperature coefficients and chemical exchange rates confirm this conclusion. Comparison of oligomer properties with polymeric HA indicates that there is no discernible difference in amide proton environment between the centre of octasaccharides and the polymer, inconsistent with the formation of tertiary structures. A [1H-1H-15N] NOESY-HSQC (heteronuclear single-quantum correlation) spectrum recorded on an HA octasaccharide revealed that amide groups in the centre are in a trans orientation and that the average solution conformation is not an extended 2-fold helix. Therefore the two key aspects of the secondary and tertiary structure models are unlikely to be correct. Rather, these new NMR data agree with descriptions from hydrodynamic and simulations data.

Complete assignment of hyaluronan oligosaccharides up to hexasaccharides.

The glycosaminoglycan hyaluronan is involved in a diverse range of physiological and diseases processes and comprises repeated disaccharide units of N-acetyl-d-glucosamine (GlcNAc) and d-glucuronic acid (GlcA). A molecular description of the solution conformation of HA is required to account for this biology, which is best attained using nuclear magnetic resonance (NMR). NMR studies of the polymer, however, are frustrated by resonance overlap arising from the highly degenerate structure. In contrast, end-effects in oligosaccharides can produce some chemical shift dispersion, giving the possibility that their conformational properties can be measured and extrapolated to models of the polymer. We report the complete resolution and assignment of (1)H, (13)C and (15)N nuclei in hyaluronan oligosaccharides with seven different naturally occurring terminal rings. At 900MHz, all (1)H nuclei in the hexasaccharide GlcA-beta-(1-->3)-GlcNAc-beta-(1-->4)-GlcA-beta-(1-->3)-GlcNAc-beta-(1-->4)-GlcA-beta-(1-->3)-GlcNAc-OH were uniquely resolved and the two central rings were found to be a good model for the polymer environment. These assignments now allow resolved, unambiguous structural restraints to be acquired on this oligosaccharide and extrapolated to models for the solution conformation of the polymer.