A new approach for analysis of NMR parameters is proposed. The experimental data set includes scalar couplings, NOEs, and residual dipolar couplings. The method, which aims at construction of the conformational distribution function, is applied to R-cyclodextrin in isotropic solution and dissolved in a dilute liquid crystal. An attempt to analyze the experimental data using an average molecular conformation resulted in unacceptable errors. Our approach rests on the maximum entropy method (ME), which gives the flattest possible distribution, consistent with the experimental data. Very good agreement between experimental and calculated NMR parameters was observed. In fact, two conformational states were required in order to obtain a satisfactory agreement between calculated and experimental data. In addition, good agreement with Langevin dynamics computer simulations was obtained.Cyclodextrins (CDs) are cyclic R-(1f4)-linked carbohydrate oligomers constructed from glucose units. 1 The smallest homologue is R-CD, Figure 1, followed by βand γ-CDs, which comprise 6, 7, and 8 units of a glucopyranose monomer, respectively. Due to their unique structural features, these molecules are invaluable in a variety of industrial applications. Almost all applications involve the ability of the CDs to alter the physical, chemical, and biological properties of guest molecules through the formation of inclusion complexes. 1 It is therefore of utmost importance to determine details of the molecular structure of cyclodextrins in general and conformational transitions in particular. The new method, described here, aims at construction of conformational distribution functions for complex molecules in isotropic solution and in a dilute liquid crystal.In the present letter, we investigate the conformational properties of R-CD using NMR spectroscopy and computer simulations. The ultimate goal in the description of the molecular structure is the determination of conformational probability distributions, P(φ,ψ), where the two torsion angles, φ and ψ, are related to the glycosidic linkage, Figure 1. Using experimental NMR parameters such as J couplings, NOEs, and residual dipolar couplings (RDCs), we determine the torsion angle distribution function for R-CD. However, defining the structure of carbohydrates still poses problems since usually only a limited number of NMR observables are possible to obtain.

The molecular structure of alpha-L-Rhap-(1--> 2)-alpha-L-Rhap-OMe has been investigated using conformation sensitive NMR parameters: cross-relaxation rates, scalar 3J(CH) couplings and residual dipolar couplings obtained in a dilute liquid crystalline phase. The order matrices of the two sugar residues are different, which indicates that the molecule cannot exist in a single conformation. The conformational distribution function, P(phi, psi) related to the two glycosidic linkage torsion angles phi and psi was constructed using the APME method, valid in the low orientational order limit. The APME approach is based on the additive potential (AP) and maximum entropy (ME) models. The analyses of the trajectories generated in molecular dynamics and Langevin dynamics (LD) computer simulations gave support to the distribution functions constructed from the experimental NMR parameters. It is shown that at least two conformational regions are populated on the Ramachandran map and that these regions exhibit very different molecular order.

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The conformational dynamics of the human milk oligosaccharide lacto-N-fucopentaose (LNF-1), α-L-Fucp-(1 → 2)-β-D-Galp-(1 → 3)-β-D-GlcpNAc-(1 → 3)-β-D-Galp-(1 → 4)-D-Glcp, has been analyzed using NMR spectroscopy and molecular dynamics (MD) computer simulations. Employing the Hadamard (13)C-excitation technique and the J-HMBC experiment, (1)H,(13)C trans-glycosidic J coupling constants were obtained, and from one- and two-dimensional (1)H,(1)H T-ROESY experiments, proton-proton cross-relaxation rates were determined in isotropic D(2)O solution. In the lyotropic liquid-crystalline medium consisting of ditetradecylphosphatidylcholine, dihexylphosphatidylcholine, N-cetyl-N,N,N-trimethylammonium bromide, and D(2)O, (1)H, (1)H and one-bond (1)H, (13)C residual dipolar couplings (RDCs), as well as relative sign information on homonuclear RDCs, were determined for the pentasaccharide. Molecular dynamics simulations with explicit water were carried out from which the internal isomerization relaxation time constant, τ(N), was calculated for transitions at the ψ torsion angle of the β-(1 → 3) linkage to the lactosyl group in LNF-1. Compared to the global reorientation time, τ(M), of ∼0.6 ns determined experimentally in D(2)O solution, the time constant for the isomerization relaxation process, τ(N(scaled)), is about one-third as large. The NMR parameters derived from the isotropic solution show very good agreement with those calculated from the MD simulations. The only notable difference occurs at the reducing end, which should be more flexible than observed by the molecular simulation, a conclusion in complete agreement with previous (13)C NMR relaxation data. A hydrogen-bond analysis of the MD simulation revealed that inter-residue hydrogen bonds on the order of ∼30% were present across the glycosidic linkages to sugar ring oxygens. This finding highlights that intramolecular hydrogen bonds might be important in preserving well-defined structures in otherwise flexible molecules. An analysis including generalized order parameters obtained from nuclear spin relaxation experiments was performed and successfully shown to limit the conformational space accessible to the molecule when the number of experimental data are too scarce for a complete conformational analysis.

Background: Ultrafiltration (UF) is a conventional method for isolating the protein-unbound plasma fractions of therapeutic drugs. However, the ideal UF conditions for specific compounds remain largely unexplored. By comparing UF-derived unbound concentrations with the corresponding results obtained using a reference method, the authors sought to identify appropriate UF conditions for cefotaxime, cloxacillin, flucloxacillin, and piperacillin. Methods:In vitro microdialysis (MD) with a no-net-flux approach was used as a reference method for plasma protein separation, for which UF performance was assessed. Four levels of relative centrifugal force (2500-11,290g) and 2 levels of temperature (37 vs. 228C) during 10 minutes of UF centrifugation were evaluated. Ultrafiltrates and reference microdialysates were analyzed using liquid chromatography-tandem mass spectrometry to obtain unbound concentrations. After identifying the appropriate UF conditions in the spiked plasma samples, exploratory analyses of clinical samples (n = 10 per analyte) were performed.Results: Of the evaluated UF alternatives, the best overall agreement with the MD-derived reference concentrations was obtained with 11,290 g UF performed at 228C. For cloxacillin specifically, 378C UF yielded better agreement than 228C UF at 11,290 g. Clinical sample analyses indicated minimal differences between 228C and 378C at 11,290 g UF for cefotaxime and piperacillin. However, consistently lower levels of unbound cloxacillin (median: 223%, IQR: 219% to 224%) and flucloxacillin (median: 227%, IQR: 221 to 234%) were observed after UF at 228C versus 378C. Conclusions:For the evaluated UF device, 10 minutes of 11,290 g UF at 228C is appropriate for flucloxacillin, cefotaxime, and piper-acillin, and can arguably be justified for cloxacillin as well for laboratory practice purposes. Maintenance of 378C during highcentrifugal UF may lead to overestimation, particularly for unbound flucloxacillin.