Solution NMR techniques for large molecular and supramolecular structures

Transverse relaxation-optimized spectroscopy (TROSY) or generation of heteronuclear multiple quantum coherences during the frequency labeling period and TROSY during the acquisition period have been combined either with cross-correlated relaxation-induced polarization transfer (CRIPT) or cross-correlated relaxation-enhanced polarization transfer (CRINEPT) to obtain two-dimensional (2D) solution NMR correlation spectra of (15)N,(2)H-labeled homo-oligomeric macromolecules with molecular weights from 110 to 800 kDa. With the experimental conditions used, the line widths of the TROSY-components of the (1)H- and (15)N-signals were of the order of 60 Hz at 400 kDa, whereas, for structures of size 800 kDa, the line widths were about 75 Hz for (15)N and 110 Hz for (1)H. This paper describes the experimental schemes used and details of their setup for individual measurements. The performance of NMR experiments with large structures depends critically on the choice of the polarization transfer times, the relaxation delays between subsequent recordings, and the water-handling routines. Optimal transfer times for 2D [(15)N,(1)H]-CRIPT-TROSY experiments in H(2)O solutions were found to be 6 ms for a molecular weight of approximately 200 kDa, 2.8 ms for 400 kDa, and 1.4 ms for 800 kDa. These data validate theoretical predictions of inverse proportionality between optimal transfer time and size of the structure. The proton longitudinal relaxation times in H(2)O solution were found to be of the order of 0.8 s for structure sizes around 200 kDa, 0.4 s at 400 kDa, and 0.3 s at 800 kDa, which enabled the use of recycle times below 1 s. Since improper water handling results in severe signal loss, the water resonance was kept along the z-axis during the entire duration of the experiments by adjusting each water flip-back pulse individually.     

Synthesis of novel antifungal phthalides produced by a wheat rhizosphere fungus

Two antifungal phthalides produced by a wheat rhizosphere fungus have been synthesized using the Alder-Rickert reaction to construct their common isobenzofuranone core structure. The absolute configuration of one of the two phthalides has been determined to be S by synthesizing its (S)- and (R)-enantiomer and comparing their optical rotations with that of the natural product.     

Purity determination of a new antifungal drug candidate using quantitative 1H NMR spectroscopy: Method validation and comparison of calibration approaches

Quantitative nuclear magnetic resonance (qNMR) is an analytical technique that offers numerous advantages in pharmaceutical applications including minimum sample preparation and rapid data collection times with no need for response factor corrections, being a powerful tool for assaying drug content in both drug discovery and early drug development. In the present work, we have applied qNMR, using both the internal standard and the electronic reference to access in vivo concentrations 2 calibration methods, to assess the purity of RI76, a novel antifungal drug candidate. NMR acquisition and processing parameters were optimized in order to obtain spectra with intense, well-resolved signals of completely relaxed nuclei. The analytical method was validated following current guidelines, demonstrating selectivity, linearity, accuracy, precision, and robustness. The calibration approaches were statistically compared, and no significant difference was observed when comparing the obtained results and their dispersion in terms of relative standard deviation. The proposed qNMR method may, therefore, be used for both qualitative and quantitative assessments of RI76 in early drug development and for characterization of this compound.     

Analysis of macromolecular structures by pulsed NMR

The T₂ spin-spin relaxation curves obtained by pulsed NMR techniques can readily be used to study important features of macromolecular systems quite distinct from their chemical structure. Such features refer to more physical properties such as molecular size, flexibility and mobility, the influence of solvent and temperature on this motion (which is related to viscosity), crystalline fraction and the rate of crystallization, polymerisation and other chemical reactions where there is a considerable change in dimensions etc. It can also serve to determine the degree of crosslinking, where this forms a partial or complete network. However it appears to indicate the presence of a network even when no permanent network is revealed by alternative and well-established techniques such as solubility and swelling which require much longer times. This difference is ascribed to the presence of some intermolecular binding somewhat akin to permanent crosslinks, but of a very shortlived dynamic nature, and this is referred to as due to entanglements between adjacent macromolecules. The T₂ measurements reveal their presence if the life-time of these entanglements is comparable or longer than the period ofmeasurement by pulsed NMR.The usual formulae used to determine network formation by permanent crosslinks can be applied to such systems with entanglements or with entanglements plus crosslinks, so that the elastic properties can be determined by NMR T₂ measurements. Over a long time only the permanent crosslinks will provide elastic recovery but for sufficiently short times the entanglements provide an additional restoring force and this may be taken to be the cause of the rheological property referred to as creep and viscosity. Since the entanglements but not the permanent crosslinks depend on temperature, many of these physical properties and their variation with temperature can be related directly to the effect of these entanglements as determined by these T₂ measurements and derived from pulsed NMR.Another feature which emerges from these investigations is their dependence on solvent where present. The total variation can be ascribed to molecular dimensions and the free volume available for their motion (and hence their freedom to become disentangled). This free volume is influenced by temperature and concentration of solvent where present.The meaning of these T₂ responses have been deduced from the changes in pulsed NMR responses to a series of macromolecular systems whose properties have been modified to known extents by known radiation doses. The interpretation of the T₂ relaxation patterns obtained from other macromolecular structures now becomes possible. We can therefore hope to see this technique used not only for polymers but especially for biological systems where considerable changes of molecular behaviour such as conformation and motion can result from very minute chemical modifications. Such sensitive features might be for example molecular entanglements and concentration, radiation or chemically-induced crosslinking or degradation (scission), disruption of a regular refolding sequence etc. This T₂ technique is particularly suitable for following such changes.     

群多普利溶液构象的NMR研究

采用1D和2D NMR技术对血管紧张素转化酶抑制剂群多普利在CDCl₃溶液中存在的两种构象进行了结构解析, 并结合分子动力学和密度泛函理论方法对其进行了结构的几何优化和能量计算. 结果表明, 群多普利因分子中酰胺键的旋转而形成反式构象A和顺式构象B, 两种构象的能量差为6.35 kJ/mol, 且顺式构象为该药物的优势构象.     

Synthesis of a novel bicyclic scaffold inspired by the antifungal natural product sordarin

A simplified bicyclic scaffold inspired by the antifungal natural product sordarin was designed and synthesized which maintains the carboxylic acid/aldehyde (or nitrile) pharmacophore. Docking studies with the target for sordarin, the fungal protein eukaryotic elongation factor 2 (eEF2), suggested that the novel scaffolds may bind productively. A densely functionalized chiral cyclopentadiene was constructed in 8 steps and utilized in a Diels-Alder reaction with acrylonitrile. The resulting [2.2.1] cycloheptene was transformed into a scaffold possessing vicinal carboxylic acid and nitrile groups, with orientations predicted to provide high affinity for eEF2. The synthetic approach disclosed here sets the stage for a renewed medicinal chemistry campaign against eEF2.     

TAT-liposomes: a novel intracellular drug carrier

TAT peptide was attached to the surface of plain and PEGylated liposomes. These TAT peptide-modified liposomes have been shown to translocate into a variety of normal and cancer cells if a non-hindered interaction between the cell surface and liposome-attached TAT peptide was made possible. TAT peptide-liposomes translocated into cells remain intact within first few hours as proved by a co-localization of fluorescent markers entrapped inside liposomes and incorporated into the liposomal membrane. After 2 hours liposomes had slowly migrating towards cell nuclei. Liposomes had completely disintegrated with their inner marker released by approximately 9 hours. TAT peptide-liposomes were made slightly cationic by adding up to 10 mol %. of a cationic lipid (DOTAP). These slightly cationic liposomes were non-toxic towards cells, formed firm complexes with DNA (plasmid encoding for the formation of the Green Fluorescent Protein), and efficiently transfected a variety of cells. TAT peptide-liposomes can be considered as promising carriers for the non-endocytotic intracellular delivery of drugs and DNA.     

Identification of complex structures of paintings on canvas by NMR: Correlation between NMR profile and stratigraphy

Paintings on canvas are complex structures created by superimposing layers of different composition. Investigations on the structure of these artworks can provide essential information on their state of conservation, pictorial technique, possible overpaintings, and in planning a proper conservation plan. Standard methods of investigation consist in sampling a limited number of fragments for stratigraphic analyses. Despite the recognized validity of these methods, they are affected by evident limitations. Nuclear magnetic resonance (NMR) profiling, often named NMR stratigraphy, is an NMR relaxometry technique applied by single-sided portable devices developed to overcome the disadvantages of microinvasive stratigraphic analyses. The potential of this approach on artworks, including wall paintings and a few examples of painted canvas, is described in the literature. In this study, NMR profiles of painting on canvas were examined by analyzing transverse relaxation time data by T₂ quasi-continuous distributions and the results compared with standard stratigraphic cross-sections analysis. Combining signal intensity and T₂ quasi-continuous distributions, the identification of textile, preparatory, and paint layers was enhanced. The diction “NMR stratigraphy” for these inhomogeneous layered artworks is also discussed. Indeed, unlike the stratigraphic cross-sections, NMR profiles provide information on a volume (flat slice), rather than on a surface, and the collected signal can derive from nonuniform and partially overlapping layers. This study paves the way for extensive investigations on relaxation time quasi-continuous distributions in various binder/pigment mixtures in order to improve the reliability of NMR profile as an innovative, non–invasive, and nondestructive method for analyzing paintings on canvas.     

Heterogeneous Solution NMR Signal Amplification by Reversible Exchange

A novel variant of an iridium‐based organometallic catalyst was synthesized and used to enhance the NMR signals of pyridine in a heterogeneous phase by immobilization on polymer microbead solid supports. Upon administration of parahydrogen (pH₂) gas to a methanol mixture containing the HET‐SABRE catalyst particles and the pyridine, up to fivefold enhancements were observed in the ¹H NMR spectra after sample transfer to high field (9.4 T). Importantly, enhancements were not due to any residual catalyst molecules in solution, thus supporting the true heterogeneity of the SABRE process. Further significant improvements may be expected by systematic optimization of experimental parameters. Moreover, the heterogeneous catalyst is easy to separate and recycle, thus opening a door to future potential applications varying from spectroscopic studies of catalysis, to imaging metabolites in the body without concern of contamination from expensive and potentially toxic metal catalysts or accompanying organic molecules.     

Coordination Chemistry of Gold Catalysts in Solution: A Detailed NMR Study

Coordination chemistry of gold catalysts bearing eight different ligands [L=PPh₃, JohnPhos (L2), Xphos (L3), DTBP, IMes, IPr, dppf, S‐tolBINAP (L8)] has been studied by NMR spectroscopy in solution at room temperature. Cationic or neutral mononuclear complexes LAuX (L=L2, L3, IMes, IPr; X=charged or neutral ligand) underwent simple ligand exchange without giving any higher coordinate complexes. For L2AuX the following ligand strength series was determined: MeOH?hex‐3‐yne <MeCN≈OTf^??Me₂S<2,6‐lutidine<4‐picoline<CF₃CO₂^?≈DMAP<TMTU<PPh₃<OH^?≈Cl^?. Some heteroligand complexes DTBPAuX exist in solution in equilibrium with the corresponding symmetrical species. Binuclear complexes dppf(AuOTf)₂ and S‐tolBINAP(AuOTf)₂ showed different behavior in exchange reactions with ligands depending on the ligand strength. Thus, PPh₃ causes abstraction of one gold atom to give mononuclear complexes LLAuPPh₃⁺ and (Ph₃P)_nAu⁺, but other N and S ligands give ordinary dicationic species LL(AuNu)₂²⁺. In reactions with different bases, LAu⁺ provided new oxonium ions whose chemistry was also studied: (DTBPAu)₃O⁺, (L2Au)₂OH⁺, (L2Au)₃O⁺, (L3Au)₂OH⁺, and (IMesAu)₂OH⁺. Ultimately, formation of gold hydroxide LAuOH (L=L2, L3, IMes) was studied. Ligand‐ or base‐assisted interconversions between (L2Au)₂OH⁺, (L2Au)₃O⁺, and L2AuOH are described. Reactions of dppf(AuOTf)₂ and S‐tolBINAP(AuOTf)₂ with bases provided more interesting oxonium ions, whose molecular composition was found to be [dppf(Au)₂]₃O₂²⁺, L8(Au)₂OH⁺, and [L8(Au)₂]₃O₂²⁺, but their exact structure was not established. Several reactions between different oxonium species were conducted to observe mixed heteroligand oxonium species. Reaction of L2AuNCMe⁺ with S^2? was studied; several new complexes with sulfide are described. For many reversible reactions the corresponding equilibrium constants were determined.     

NMR of a series of novel hydroxyflavothiones

Alkylated hydroxyflavothiones, namely flavothione, 5‐hydroxyflavothione, 5,7‐dihydroxyflavothione (chrysinthione), 7‐dodecyloxy‐5‐hydroxyflavothione, 7‐butyloxy‐5‐hydroxyflavothione, 2′,3,4′,7‐tetramethoxy‐5‐hydroxyflavothione, 3,3′,4′,7‐tetramethoxy‐5‐hydroxyflavothione, 7‐butyloxy‐4′,5‐dihydroxyflavothione and 7‐butyloxy‐4′,5‐hydroxyflavanonethione have been synthesized from the corresponding hydroxyflavones in two steps, alkylation of the non‐hydrogen‐bonded hydroxyl groups by bromoalkanes or dimethyl sulfate followed by conversion of the carbonyl group to a thione using Lawesson's Reagent under microwave irradiation and solvent‐free conditions. Part of the alkylated flavanone, 7‐butyloxy‐4′,5‐dihydroxyflavanone, was oxidized during the treatment with Lawesson's reagent to yield a second product 7‐butyloxy‐4′,5‐dihydroxyflavothione in addition to the target product butyloxy‐4′,5‐hydroxyflavanonethione. Deuterium isotope effects on 13C chemical shifts have been measured in hydroxyflavones, isoflavones, flavanones and the thio analogs. Formal four‐bond deuterium isotope effects on 13C chemical shifts, nΔC?S(OD) are very sensitive to variations in structures and substitution patterns. Density functional theory (DFT) calculations are carried out to obtain geometries. Correlations relating distances around the hydrogen bond system to the deuterium isotope effects on 13C chemical shifts are discussed. 13C chemical shifts are calculated by DFT methods. Effects of thiocarbonyl anisotropies are suggested. Copyright © 2009 John Wiley & Sons, Ltd.     

Solution structures of cyclosporin a and its complex with dysprosium(III) in SDS micelles: NMR and molecular dynamics studies

Cyclosporin A (CsA) is a cyclic naturally occurring peptide used to prevent graft rejection in organ transplantations. Its immunosuppressive activity is due to the formation of a complex with cyclophilin A (Cyp), in which the cis 9MeLeu-10MeLeu amide bond of CsA assumes a trans conformation. The mechanism of the conformational inversion has not been delineated, but it has been postulated that metal ions binding induces a conformational change that enables CsA to bind Cyp. In this work, we solved the structures of CsA in sodium dodecyl sulfate (SDS) micelles (which enhance its solubility and mimic the hydrophobic environment clinically used for drug delivery) and its complex with Dy(III) ion, whose coordination chemistry is frequently used to reproduce the effect of Ca(II). The paramagnetic properties of Dy(III) allowed us to build up a structure using proton relaxation enhancements, which remains stable in a MD simulation in the micelle environment.     

NMR Wool Tube: a novel method for NMR solution analysis of derivatized glass surfaces

Glass wool was placed within an NMR tube as a solid support for the covalent attachment of a molecule to allow for a simple one-dimensional 1H FT NMR solution analysis. This novel procedure avoids the use of expensive sample tubes or platforms, as required for magic angle or fast spinning, exotic pulse sequences, isotopic labeling or the use of a large number of scans to provide the ability to analyze the structure, mobility, ligand binding, and solvent interactions of the surface bound molecule.     

Response surface methodology in drug design: A case study on docking analysis of a potent antifungal fluconazole

Molecular docking is a valuable in silico technique for discovery/design of bioactive compounds. A current challenge within docking simulations is the incorporation of receptor flexibility. A useful strategy toward solving such problem would be the docking of a typical ligand into the multiple conformations of the target. In this study, a multifactor response surface model was constructed to estimate the AutoDock based binding free energy of fluconazole within multiple conformations of 14α-demethylase (CYP51) (cross docking) as a validated antifungal target. On the basis of developed models, individual and interactive effects of important experimental parameters on cross docking of fluconazole were elucidated. For this purpose, a set of high-resolution holo crystallographic structures from CYP51 of human pathogen Trypanosoma cruzi were retrieved to statistically model the binding mode and affinity of fluconazole. The changes of AutoDock binding free energy for the complexes of CYP51-fluconazole were elucidated with the simultaneous variations of six independent variables including grid size, grid spacing, number of genetic algorithm (GA) runs, maximum number of energy evaluations, torsion degrees for ligand and quaternion degrees for ligand. It was revealed that grid spacing (distance between adjacent grid points) and maximum number of energy evaluations were two significant model terms. It was also revealed that grid size, torsion degrees for ligand and quaternion degrees for ligand had insignificant effects on estimated binding energy while the effect of GA runs was non-significant. The interactive effect of “torsion degrees for ligand” with number of GA runs was found to be the significant factor. Other important interactive effects were the interaction of “number of GA runs” with “grid spacing” and “number of energy evaluations” with “grid size”. Furthermore; results of modeling studies within several CYP51 conformations exhibited that “number of GA runs” and “number of energy evaluations” were less sensitive to varied target conformations.     

NMR Solution Structure of Phospholamban

Phospholamban (PLN), an amphipathic intrinsic membrane protein of 52 amino acids, is the modulator of the Ca²⁺ pump of cardiac, slow‐twitch, and smooth‐muscle sarcoplasmic reticulum. In response to β‐adrenergic stimulation, it becomes phosphorylated at Ser¹⁶ and/or Thr¹⁷, and dissociates from the pump, which, in turn, achieves its full activity. Here we present the three‐dimensional structure of chemically synthesized, monomeric PLN in an organic solvent. Monomerization (PLN normally forms homopentamers) was obtained by replacing Cys⁴¹ with phenylalanine (Phe=F), a modification that did not affect biological activity. The structure was determined by high‐resolution NMR in CHCl₃/MeOH of the unphosphorylated state of [F⁴¹]PLN (C41F). Of the hydrophilic cytoplasmic parts IA (Met¹ to Pro²¹) and IB (Gln²² to Asn³⁰) and the membrane‐spanning hydrophobic domain II (Leu³¹ to Leu⁵²) of PLN, domain IA, which contains the two phosphorylation sites Ser¹⁶ and Thr¹⁷, and domain II have been suggested to be helical and connected through the less‐structured hinge‐region IB. In the structural study presented here, [F⁴¹]PLN is composed of two α‐helical regions connected by a β‐turn (type III). The residues of the β‐turn (type III) are Thr¹⁷, Ile¹⁸, Glu¹⁹, and Met²⁰, the first being one of the two phosphorylation sites (Ser¹⁶ and Thr¹⁷). The hinge region is located at the C‐terminal end of domain IA, and domain IB is part of a second helix. The two α‐helices comprising amino acids 4 – 16 and 21 – 49 are well‐defined (the root‐mean‐square deviations for the backbone atoms, calculated for a family of the structures, are 0.58 and 0.92 Å, resp.). Pro²¹ is at the beginning of the C‐terminal helix and in the trans conformation.     

Sequence-dependent bending of DNA induced by cisplatin: NMR structures of an A.T-rich 14-mer duplex

The NMR solution structure of the A.T rich DNA 14-mer duplex d(ATACATGGTACATA).d(TATGTACCATGTAT) is reported. This is compared with the NMR structure of the same duplex intrastrand cross-linked at the d(G*pG*) site by cis-(Pt(NH3)2?2+, derived from the anticancer drug cisplatin. The unmodified duplex has B-DNA geometry, but there is a large positive base-pair roll (roll angle 24 +/- 2 degrees) at the T9-A10 step on the 3' side of the central GG site. Platination of the DNA duplex causes the adjacent guanine bases to roll toward one another (roll angle 44 +/- 4 degrees), leading to an overall helix bend of 52 +/- 9 degrees. The platinum atom is displaced from the planes of the coordinated G7* and G8* by 0.8 A and 0.3 A, respectively. The minor groove opposite the platinum lesion is widened and flattened, with geometric parameters similar to those of A-form DNA. The unwinding of the helix at the platination site is 26 degrees. Platination causes the DNA duplex to bend toward the 3'-end (with respect to the G*G* strand), in contrast to G C-rich structures reported previously, which bend toward the 5'-end. This difference can be attributed to the predisposition of the A.T rich duplex toward bending in this region. Protein recognition of bent platinated G*G* lesions may therefore exhibit a strong dependence on the local DNA structure.     

Description of non-regular membrane structures: a novel phenomenological approach

A new phenomenological approach to the analysis of complex membrane structures and surfaces and the processing of corresponding experimental data obtained, for example, from the roughness study is presented. The methodology is based on a postulate about the crucial information contained in non-regularities of measured spatial dynamic variables, as well as on the acceptance of a new scaling equation. Accordingly, power spectra and structural functions of different orders are determined by non-regularities of different types resulting from dynamical spikes and jumps of the measured variables. It is also shown that equations for power spectra as well as for structural functions are the same at every spatial–temporal level of the system under consideration. It is demonstrated that multi-parametric invariant relationships characterize a new kind of self-similarity. Appropriate phenomenological parameters are introduced. It is shown that these parameters characterize self-similarity in the rate of loss of correlation links between non-regularities of one type as well as self-similarity in the dynamics of memory loss in the dynamic variable, as the spatial distance from any fixed point increases, for non-regularities of a second type. An algorithm is developed which makes it possible to obtain as many parameters as it is necessary for the characterization of the dynamic state of a system and changes of its state during evolution. Application of this approach to the analysis of surface roughness of a perfluorinated cation-exchange membrane coated with platinum layer is demonstrated.