Nuclear Overhauser Enhancement Experiments Research Articles

Anions as Dynamic Probes for Ionic Liquid Mixtures.

Ionic liquid (IL) mixtures have been proposed as a viable alternative to rationally fine-tune the physicochemical properties of ILs for a variety of applications. The understanding of the effects of mixing ILs on the properties of the mixtures is however only in the very early stages. Two series of ionic liquid mixtures, based on the 1-ethyl-3-methylimidazolium and 1-dodecyl-3-methylimidazolium cations, and having a common anion (tetrafluoroborate or bis(trifluoromethylsulfonyl)imide), have been prepared and deeply characterized via multiple NMR techniques. Diffusion and relaxation methods combined with 2D ion–ion correlation (nuclear Overhauser enhancement) experiments have been used for a better understanding of the interplay between dynamics and structure of IL mixtures. A crucial role of the anion in driving the mixture’s behavior emerged, making them important “dynamic probes” for gaining information of the polar and nonpolar regions of ionic liquids and their mixtures.

I-PINE web server: an integrative probabilistic NMR assignment system for proteins.

Various methods for understanding the structural and dynamic properties of proteins rely on the analysis of their NMR chemical shifts. These methods require the initial assignment of NMR signals to particular atoms in the sequence of the protein, a step that can be very time-consuming. The probabilistic interaction network of evidence (PINE) algorithm for automated assignment of backbone and side chain chemical shifts utilizes a Bayesian probabilistic network model that analyzes sequence data and peak lists from multiple NMR experiments. PINE, which is one of the most popular and reliable automated chemical shift assignment algorithms, has been available to the protein NMR community for longer than a decade. We announce here a new web server version of PINE, called Integrative PINE (I-PINE), which supports more types of NMR experiments than PINE (including three-dimensional nuclear Overhauser enhancement and four-dimensional J-coupling experiments) along with more comprehensive visualization of chemical shift based analysis of protein structure and dynamics. The I-PINE server is freely accessible at http://i-pine.nmrfam.wisc.edu . Help pages and tutorial including browser capability are available at: http://i-pine.nmrfam.wisc.edu/instruction.html . Sample data that can be used for testing the web server are available at: http://i-pine.nmrfam.wisc.edu/examples.html .

Solid State NMR Investigations of Lipid Bilayers in Interaction with Amphiphilic Triblock Copolymers

Pluronics are triblock copolymers consisting of hydrophilic poly(ethylene oxide) end blocks and a hydrophobic poly(propylene oxide) middle block. They are widely used in pharmacological industry. Depending on the lengths of the different blocks, Pluronics can influence membrane properties in a variety of ways, e.g., increase or decrease membrane permeability. This variable influence could be explained by different ways of incorporation1, i.e., spanning the membrane or just penetrating it as hairpin.Our work focuses on Pluronics as well as its variations, obtained by replacing the PEO-blocks by the more bulky poly(glycerol monomethacrylate) (PGMA) blocks. The latter have a higher propensity to act as hydrogen bond donor and acceptor, potentially increasing the interaction with the lipid head group.2 Using solid state MAS NMR, we investigate structure and dynamics of model membranes consisting of phosphatidylcholines with variable bilayer thickness and saturation, in mixture with copolymers of variable block length. 1H-1H Nuclear Overhauser Enhancement (2D-NOESY) experiments were applied to confirm the insertion of the copolymer into the bilayer. Motionally averaged dipolar couplings yield information on molecular dynamics.For the lipids, heteronuclear 13C-1H dipolar coupling constants were determined by the R-PDLF experiment, obtaining similar results for all samples and the pure lipids, confirming a weak influence of the polymers on lipid dynamics. Homonuclear 1H-1H residual couplings were measured with the Back-to-Back (BaBa) double-quantum recoupling pulse sequence3, which is suitable for measuring the very small coupling constants of the polymers. In this way, we were able to quantify the relative amount of inserted vs. non-inserted polymer and conclude on its conformation.[1] H. Rabbel et al., Macromolecules, 48, 4724, 2015.[2] C. Schwieger et al., Matter,10, 6147, 2014.[3] K. Saalwachter et al., J. Magn. Reson., 212, 204, 2011.

High field dynamic nuclear polarization NMR with surfactant sheltered biradicals.

We illustrate the ability to place a water-insoluble biradical, bTbk, into a glycerol/water matrix with the assistance of a surfactant, sodium octyl sulfate (SOS). This surfactant approach enables a previously water insoluble biradical, bTbk, with favorable electron–electron dipolar coupling to be used for dynamic nuclear polarization (DNP) nuclear magnetic resonance (NMR) experiments in frozen, glassy, aqueous media. Nuclear Overhauser enhancement (NOE) and paramagnetic relaxation enhancement (PRE) experiments are conducted to determine the distribution of urea and several biradicals within the SOS macromolecular assembly. We also demonstrate that SOS assemblies are an effective approach by which mixed biradicals are created through an assembly process.

Synthesis, Structures, and Properties of Thieno[3,2-b]thiophene and Dithiophene Bridged Isoindigo Derivatives and Their Organic Field-effect Transistors Performance

Organic semiconductors (ITTI and IDTI) characterized of thieno[3,2-b]thiophene and dithiophene bridged isoindigo derivatives were designed and successfully synthesized. 1H–13C HMBC, 1H–1H nuclear Overhauser enhancement spectroscopy (NOESY) spectra, and 1H nuclear Overhauser enhancement (NOE) experiments were employed to determine the configurations of the synthesized molecules, indicating they belong to stable Z,Z configurations. Organic field-effect transistors based on ITTI and IDTI through a solution processed method have good air stabilities and exhibit hole mobilities of 0.045 and 0.075 cm2 V–1 s–1 after high-temperature annealing in air, respectively.

ChemInform Abstract: C-Nucleosides. Part 18. Stereoselective Annulation of 6-Acetoxy-6-(2,3, 5-tri-O-benzoyl-β-D-ribofuranosyl)pyran-3(2H,6H)-one with Ethylene Glycol to Pyrano(2,3-b)-1,4-dioxine Glycosides

The synthesis of (4aS,8aS)- and (4aR,8aR)-4a-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-2,3,8,8a-tetrahydropyrano[2,3-b]-1,4-dioxin-7(4aH,6H)-one 9a and 9b is described. Treatment of the 6-acetoxypyranulose glycoside 2 with ethylene glycol in the presence of toluene-p-sulfonic acid in acetone for 1 h afforded the (6S)- and (6R)-6-(2-hydroxyethoxy)pyranulose glycosides 3a and 3b in 14 and 40% yield, respectively. The stereochemistry of compounds 3a and 3b was assigned as 6S and 6R by comparison of their CD spectra. When compounds 3a and 3b were treated with toluene-p-sulfonic acid at room temperature for 5 h, they reacted to give bicyclic compounds 9a and 9b in 65% yield, respectively. The stereochemistry of the bicyclic compounds 9a and 9b was established by nuclear Overhauser enhancement experiments.

Determination of the Absolute Configurations of (+)-N-((3S)-3- {[(4-methylphenyl)sulfonyl]amino}-1-oxaspiro[4.5]deca-6,9- dien-2,8-dion-7-yl) Acetamide and Benzamide

We recently reported the asymmetric synthesis of the two title compounds without the configurational assignments of the newly formed chiral spirocarbons. We now wish to report that both compounds have a (R)-configuration at the spirocarbon based on 1D and 2D nuclear Overhauser enhancement (nOe) experiments.

Novel triterpenoid from Citrus aurantium L. possesses chemopreventive properties against human colon cancer cells

Potential cancer preventive constituents of sour orange ( Citrus aurantium L.) were isolated and identified from EtOAc extract of sour orange. Crude EtOAc extract was purified using silica gel column chromatography to isolate two putative bioactive compounds. The purity of the isolated compounds was analyzed by TLC and HPLC. The structures of the two compounds were identified by one-dimensional ( 1H, 13C) and two-dimensional ( 1H–H and 1H– 13C) NMR experiments as isolimonic acid and a novel compound named as ichanexic acid. Stereochemical assignment of the protons for both the compounds was made using one-dimensional nuclear Overhauser enhancement (nOe) experiments. The identified compounds were tested for the inhibition of human colon cancer cells (HT-29) proliferation, apoptosis, and on non-cancerous (COS-1 fibroblast) cells. Cell proliferation, arrest of cell growth, and induction of apoptosis were determined by MTT assay, flow cytometry, and nuclear staining methods, respectively. The MTT assay indicated that both the compounds exhibited differential inhibition at various concentrations. Significant arrest of cell growth by isolimonoic acid was noticed within 24 h of treatment on the HT-29 colon cancer cells at a concentration as low as 5.0 μM ( P = 0.005) and by ichanexic acid at 10.0 μM ( P = 0.011). None of the compounds exerted any apparent cytostatic effects on the non-cancerous COS-1 fibroblast cells. Both the compounds exerted nearly 4- to 5-fold increase in the counts of G2/M stage cells at 5 μ M indicating a potential role in the cell cycle arrest as well as possible lead structures for the development of cancer chemopreventive and therapeutic agents. To the best of our knowledge, this is the first report on isolation, identification of isolimonic acid in its native form, and compound 2 was found to a novel and identified as ichanexic acid.

Solution Structure of a Post-transition State Analog of the Phosphotransfer Reaction between the A and B Cytoplasmic Domains of the Mannitol Transporter IIMannitol of the Escherichia coli Phosphotransferase System

The solution structure of the post-transition state complex between the isolated cytoplasmic A (IIAMtl) and phosphorylated B (phospho-IIBMtl) domains of the mannitol transporter of the Escherichia coli phosphotransferase system has been solved by NMR. The active site His-554 of IIAMtl was mutated to glutamine to block phosphoryl transfer activity, and the active site Cys-384 of IIBMtl (residues of IIBMtl are denoted in italic type) was substituted by serine to permit the formation of a stable phosphorylated form of IIBMtl. The two complementary interaction surfaces are predominantly hydrophobic, and two methionines on IIBMtl, Met-388 and Met-393, serve as anchors by interacting with two deep pockets on the surface of IIAMtl. With the exception of a salt bridge between the conserved Arg-538 of IIAMtl and the phosphoryl group of phospho-IIBMtl, electrostatic interactions between the two proteins are limited to the outer edges of the interface, are few in number, and appear to be weak. This accounts for the low affinity of the complex (Kd approximately 3.7 mm), which is optimally tuned to the intact biological system in which the A and B domains are expressed as a single polypeptide connected by a flexible 21-residue linker. The phosphoryl transition state can readily be modeled with no change in protein-protein orientation and minimal perturbations in both the backbone immediately adjacent to His-554 and Cys-384 and the side chains in close proximity to the phosphoryl group. Comparison with the previously solved structure of the IIAMtl-HPr complex reveals how IIAMtl uses the same interaction surface to recognize two structurally unrelated proteins and explains the much higher affinity of IIAMtl for HPr than IIBMtl.

Complete 1H and 13C NMR assignments of aurasperone A and fonsecinone A, two bis‐naphthopyrones produced by Aspergillus aculeatus

Complete assignments of 1H and 13C NMR chemical shifts of the polyketides aurasperone A and fonsecinone A were made by means of nuclear Overhauser enhancement and heteronuclear NMR correlation experiments. These compounds were isolated for the first time from Aspergillus aculeatus, an endophytic fungus obtained from leaves of Melia azedarach(Meliaceae).

1H and 13C NMR conformational study of 2‐(benzotriazol‐1‐yl)‐substituted tetrahydrofurans

AbstractAssignments of the proton and carbon NMR signals for 2‐(benzotriazol‐1‐yl)tetrahydrofurans 1–6 are based on 1H–1H COSY, NOESY, 1H–13C heteronuclear HETCOR, 1H–13C heteronuclear long‐range HETCOR‐LR and nuclear Overhauser enhancement experiments, and supported by selective spin decoupling experiments. The electronic and steric effects of the 2‐benzotriazolyl substituent on the 1H and 13C NMR chemical shifts are evaluated and discussed. Conformational isomers cis and trans are distinguished by the magnitude of the J(H−2,H−3) coupling constant, by one‐bond 1J(C−2, H−2) coupling constants and by 13C chemical shifts of the C‐2 anomeric carbon. The benzotriazol‐1‐yl group prefers the pseudo‐axial orientation in each of 1–6. The stereochemistry and conformation of 6 are rigorously demonstrated by an x‐ray structure. Copyright © 2003 John Wiley & Sons, Ltd.

1 H Nuclear Magnetic Resonance Study of Oxazolidinone Binding to Bacterial Ribosomes

The oxazolidinones are a novel class of antibiotics that inhibit initiation of protein synthesis in bacteria. In order to investigate their novel mechanism of action, the interactions of several oxazolidinones with bacterial 70S ribosomes, 50S subunits, and 30S subunits have been characterized by (1)H nuclear magnetic resonance (NMR) line-broadening analyses and transferred nuclear Overhauser enhancement (TRNOE) experiments. PNU-177553 and PNU-100592 (eperezolid) and their corresponding enantiomers, PNU-184414 and PNU-107112, were studied. The dissociation constants were determined to be 94 +/- 44 microM and 195 +/- 40 microM for PNU-177553 and eperezolid, respectively. There was a approximately 4-fold decrease in affinity for their corresponding enantiomers. The NMR-derived dissociation constants are consistent with their antibacterial activity. PNU-177553 and eperezolid were found to bind only to the 50S subunit, with similar affinity as to the 70S ribosome, and to have no affinity for the 30S subunit. Specific binding of PNU-177553 was further confirmed in TRNOE experiments in which positive NOEs observed for the small molecule alone were changed to negative NOEs in the presence of bacterial 70S ribosomes. The observed NOEs indicated that PNU-177553 did not adopt a significantly different conformation when bound to the 70S ribosome, compared to the extended conformation that exists when free in solution. Since this is likeliest the case for each of the four compounds included in this study, the A ring C5 side chain may be positioned in the proper orientation for antibacterial activity in PNU-177553 and eperezolid but not in their inactive enantiomers.

NMR Studies of the Antibody-Bound Conformation of a Carbohydrate-Mimetic Peptide,

Transferred nuclear Overhauser enhancement (TRNOE) experiments have been performed at 800 MHz to investigate the bound conformation of the hexapeptide DRPVPY, a functional molecular mimic of the group A Streptococcus cell-wall polysaccharide. The hexapeptide binds to the monoclonal antibody SA-3, mimicking the branched trisaccharide repeating unit, L-Rha-alpha-(1 --> 2)-(D-GlcNAc-beta-(1 --> 3))-alpha-L-Rha (Rha, rhamnose; GlcNAc, N-acetylglucosamine). The peptide adopts a tight turn conformation with close contacts between the side chains of valine and tyrosine. Relaxation network editing experiments (QUIET-NOESY) were used to confirm the validity of the observed contacts and to evaluate the presence of spin diffusion pathways. Saturation transfer difference (STD-NMR) experiments with selective saturation of protein resonances revealed enhancements of many of the peptide resonances due to close contacts between the peptide and the protein within the antibody combining site.

An 1H and 13C NMR conformational study of 2-(benzotriazol-1-yl)-substituted tetrahydropyrans

Proton and carbon NMR signals for 2-(benzotriazol-1-yl)tetrahydropyrans are assigned on the basis of spin decoupling, H,H-COSY, HETCOR, and nuclear Overhauser enhancement experiments. Conformational preferences are deduced. It was found that 2-(benzotriazol-1-yl) substituents: (i) prefer the equatorial orientation; and (ii) display a rotameric preference to minimize the electronic repulsion and (or) steric interaction.Key words: anomeric, NMR, tetrahydropyrans.

Protein-bound conformation of a specific inhibitor against Candida albicans myristoyl-CoA:protein N-myristoyltransferase in the ternary complex with CaNmt and myristoyl-CoA by transferred NOE measurements.

Transferred nuclear Overhauser enhancement (trNOE) experiments have been performed to study the bioactive conformation(s) of Ro09-3472/000 derivatives in the ternary complex with Candida albicans myristoyl-CoA: protein N-myristoyltransferase (CaNmt) and myristoylCoA (MyrCoA). A critical step in the trNOE study is to identify 'true' trNOEs in the spectra. Nonspecific binding of ligands to target proteins and/or spin diffusion effects can give rise to 'false' trNOEs, which may lead to an incorrect conclusion if used to derive bound conformations. In this study for all ligands the observed trNOEs arose from specific binding interactions with the active site of CaNmt. This was shown by displacing the ligand with the known tightly binding active-site inhibitor 1 [Devadas, B., Zupec, M.E., Freeman, S.K., Brown, D.L., Nagarajan, S., Sikorski, J.A., McWherter, C.A., Getman, D. P. & Gordon, J.I. (1995) J. Med. Chem. 38, 1837-1840] and measuring the resonance linewidths in the NMR spectrum before and after addition of the competitive inhibitor. The compounds were also tested for nonspecific protein binding with bovine serum albumin (BSA) using the same Of the six compounds tested, Ro09-3700/001 (racemate) and its optically pure enantiomers, Ro09-4764/001(S) and Ro09-4765/001(R), showed both specific binding to CaNmt and no interaction with BSA. The NMR data of these molecules in the ternary complex with CaNmt/MyrCoA could thus be used for a detailed structural analysis. Thereby, the conformation of the bound ligand was obtained from a conformational search using the observed trNOEs as a selection filter. The NMR-determined conformations are in good agreement with the recently solved CaNmt-bound X-ray structures of two similar Ro09-3472/000 derivatives.

Study of the structure of Triton X-100 micelles in polyacrylamide aqueous solution by 2D heteronuclear Overhauser enhancement spectroscopy

Two-dimensional nuclear Overhauser enhancement (2D NOESY) and two-dimensional heteronuclear Overhauser enhancement (2D HOESY) experiments were performed on Triton X-100 (TX-100-polyacrylamide (PAAM) aqueous solution at concentrations of 104 gl(-1) TX-100 and 40 gl(-1) PAAM. The absence of the NOESY cross peaks between the protons of TX-100 and PAAM and their presence among TX-100 molecules significantly imply that interaction among the protons of TX-100 molecules is far stronger than that between the protons of TX-100 molecules and PAAM even at very high concentration. There are no HOESY cross peaks of protons of the system with either the carbonyl or the carboxylic carbon nuclei of PAAM. Therefore, there is no evidence for either hydrophobic (by 2D NOESY) or hydrophilic interaction (by 2D HOESY) between TX-100 and PAAM in aqueous solution. The appearance of cross correlation peaks between hydrophobic protons and hydrophilic carbon nuclei and Vice versa of the same pairs of groups in the 2D HOESY map gives strong evidence for the coiled and folded feature of the loosely packed hydrophilic chain of TX-100 upon micellization. This suggests that the conformation of the hydrophilic polyoxyethylene chain remains unchanged as the concentration of TX-100 increases even up to about 104 gl(-1). Copyright (C) 2001 John Wiley & Sons, Ltd.

Study of the structure of Triton X‐100 micelles in polyacrylamide aqueous solution by 2D heteronuclear Overhauser enhancement spectroscopy

Two-dimensional nuclear Overhauser enhancement (2D NOESY) and two-dimensional heteronuclear Overhauser enhancement (2D HOESY) experiments were performed on Triton X-100 (TX-100–polyacrylamide (PAAM) aqueous solution at concentrations of 104 g l−1 TX-100 and 40 g l−1 PAAM. The absence of the NOESY cross peaks between the protons of TX-100 and PAAM and their presence among TX-100 molecules significantly imply that interaction among the protons of TX-100 molecules is far stronger than that between the protons of TX-100 molecules and PAAM even at very high concentration. There are no HOESY cross peaks of protons of the system with either the carbonyl or the carboxylic carbon nuclei of PAAM. Therefore, there is no evidence for either hydrophobic (by 2D NOESY) or hydrophilic interaction (by 2D HOESY) between TX-100 and PAAM in aqueous solution. The appearance of cross correlation peaks between hydrophobic protons and hydrophilic carbon nuclei and vice versa of the same pairs of groups in the 2D HOESY map gives strong evidence for the coiled and folded feature of the loosely packed hydrophilic chain of TX-100 upon micellization. This suggests that the conformation of the hydrophilic polyoxyethylene chain remains unchanged as the concentration of TX-100 increases even up to about 104 g l−1. Copyright © 2001 John Wiley & Sons, Ltd.

Conformational properties of diastereomeric 5'-O-DMT-2'deoxythymidine 3'-O-(S-methyl-methanephosphonothiolate)s and -(se-methyl-methanephosphonoselenolate)s in solution as studied by NMR methods

Stereochemical characterization of diastereomerically pure 5'-O-DMT-2'deoxythymidine 3'-O-(S-methyl-methanephosphonothiolate)s and -(Se-methyl-methane-phosphono-selenolate)s by NMR methods are reported. (1)H-(1)H, (1)H-(31)P, and (13)C-(31)P coupling constants and nuclear overhauser enhancement (NOE) connectivities from transverse cross-relaxation experiments in rotating frame (T-ROESY) were measured to correlate the conformational properties of the isomers with the absolute configurations at the phosphorus obtained from X-ray studies of the relatives of S(p) configured isomers. Conformational differences between the stereoisomers were found to be restricted to the different orientation of the C3'-O3'-P bond. The NMR data reflected the preferred epsilon(-) conformation for the S(p) isomers, while in the R(p) isomers the conformational equilibrium was shifted toward the epsilon(t) domain. These results also indicated that for 5'-protected mononucleotides the absolute configuration at the phosphorus atom can be inferred from the NOE experiments and the trends observed in vicinal carbon-phosphorus coupling constants. Copyright 2000 Wiley-Liss, Inc.

Investigation of interpolymer complexation in swollen polyelectolyte networks using solid-state NMR spectroscopy

Copolymer networks of poly(methacrylic acid) (PMAA) and poly(ethylene glycol) (PEG) exhibit large changes in their swelling behavior over a narrow pH range due to the reversible formation/dissociation of interpolymer complexes between the polymer chains. Interpolymer complexation occurs in copolymer gels of PMAA and PEG due to hydrogen bonding between protonated acid groups and the ether groups of the PEG. Because of their nature, these gels have been identified for use as delivery vehicles for macromolecular drugs. In this work, solid-state, nuclear magnetic resonance nuclear Overhauser enhancement (NOE) experiments were performed to detect the molecular level complexation between PMAA and deuterated PEG in copolymer blends and crosslinked networks. For gels swollen in acidic media at room temperature or at 37°C, strong enhancements were detected in the 13 C resonance of the PEG carbons. The NOE was generated due to energy transfer between the rapidly rotating methyl group protons and the deuterated PEG carbons. The presence of the NOE was indicative of close packing of the polymer chains and was evidence of the presence of the intermacromolecular complexes. In basic solutions, no NOE was detected in the PEG, as the complexes were dissociated and the chains were separated in space.

Complexation of Voriconazole Stereoisomers with Neutral and Anionic Derivatised Cyclodextrins

A number of native, neutral derivatised and anionicderivatised cyclodextrins (CDs) were examined aschiral electrolyte additives in capillaryelectrophoresis (CE) to separate the fourstereoisomers of the new antifungal agent,voriconazole. A very large difference in interactionbetween each diastereoisomer and the CDs was observedin the CE study, where enantioselectivity was easilyobtained for one and extremely difficult to obtain forthe other. Nuclear magnetic resonance spectroscopy(1H-NMR) indicated a strong interaction betweenthe easily separated diastereoisomer and each of theCDs with enantiomeric shift nonequivalence values ofover 100 Hz obtained when using the anionicsulphobutylether-β-CD chiral solvating agent. Inaccordance with observations from the CE study, theopposite diastereoisomer indicated no shiftnonequivalence at all. The nature of the complexationbetween the easily separated diastereoisomer and theanionic sulphobutyletherβ-CD was also probedusing a two-dimensional nuclear Overhauser enhancementexperiment and a series of spin lattice relaxationtime measurements. It was found that theenantioselective interaction occurred through thepartial inclusion of a difluorophenyl group into theCD toroid which was also aided through a number ofadditional interactions between the drug molecule andthe sulphobutylether derivatives outside the CDtoroid.