NMR Evidence for Complexing of Na+ in Muscle, Kidney, and Brain, and by Actomyosin. The Relation of Cellular Complexing of Na+ to Water Structure and to Transport Kinetics

The nuclear magnetic resonance (NMR) spectrum of Na⁺ is suitable for qualitative and quantitative analysis of Na⁺ in tissues. The width of the NMR spectrum is dependent upon the environment surrounding the individual Na⁺ ion. NMR spectra of fresh muscle compared with spectra of the same samples after ashing show that approximately 70% of total muscle Na⁺ gives no detectable NMR spectrum. This is probably due to complexation of Na⁺ with macromolecules, which causes the NMR spectrum to be broadened beyond detection. A similar effect has been observed when Na⁺ interacts with ion exchange resin. NMR also indicates that about 60% of Na⁺ of kidney and brain is complexed. Destruction of cell structure of muscle by homogenization little alters the per cent complexing of Na⁺. NMR studies show that Na⁺ is complexed by actomyosin, which may be the molecular site of complexation of some Na⁺ in muscle. The same studies indicate that the solubility of Na⁺ in the interstitial water of actomyosin gel is markedly reduced compared with its solubility in liquid water, which suggests that the water in the gel is organized into an icelike state by the nearby actomyosin molecules. If a major fraction of intracellular Na⁺ exists in a complexed state, then major revisions in most theoretical treatments of equilibria, diffusion, and transport of cellular Na⁺ become appropriate.     

Connectivity of proton and carbon spectra of the blue copper protein,plastocyanin, established by two-dimensional nuclear magnetic resonance

NMR studies of plastocyanin have centered on the ligands to the copper atom at the active site, particularly histidines-37 and -87. Heteronuclear (¹³C, ¹H) J-connectivity spectroscopy has enabled cross assignment of ¹H and ¹³C NMR resonances from the two copper-ligated histidines. In addition to providing assignments of the ¹³C resonances, the two-dimensional Fourier transform NMR results require the reversal of the original ¹H NMR assignments to the ring protons of histidine-37. The line widths of the ring protons of histidine-87 are field-dependent leading to determination of the reduced lifetime of the proton on the N_δ atom (about 400 μs).     

Oriciopsin,a new ring-D cleaved tetranortriterpenoid,and flindersiamine from Oriciopsis glaberrima

A novel ring-D cleaved tetranortriterpenoid, oriciopsin and flindersiamine have been isolated from the whole plant extracts of Oriciopsis glaberrima. The structure of the new limonoid was determined from its ¹H NMR and ¹³C NMR spectra and by mass spectrometry.     

Complete H and C NMR chemical shift assignments of mono-, di-, and trisaccharides as basis for NMR chemical shift predictions of polysaccharides using the computer program casper

The computer program casper uses ¹H and ¹³C NMR chemical shift data of mono- to trisaccharides for the prediction of chemical shifts of oligo- and polysaccharides. In order to improve the quality of these predictions the ¹H and ¹³C, as well as ³¹P when applicable, NMR chemical shifts of 30 mono-, di-, and trisaccharides were assigned. The reducing sugars gave two distinct sets of NMR resonances due to the α- and β-anomeric forms. In total 35 ¹H and ¹³C NMR chemical shift data sets were obtained from the oligosaccharides. One- and two-dimensional NMR experiments were used for the chemical shift assignments and special techniques were employed in some cases such as 2D ¹H,¹³C-HSQC Hadamard Transform methodology which was acquired approximately 45 times faster than a regular t₁ incremented ¹H,¹³C-HSQC experiment and a 1D ¹H,¹H-CSSF-TOCSY experiment which was able to distinguish spin-systems in which the target protons were only 3.3 Hz apart. The ¹H NMR chemical shifts were subsequently refined using total line-shape analysis with the PERCH NMR software. The acquired NMR data were then utilized in the casper program (http://www.casper.organ.su.se/casper/) for NMR chemical shift predictions of the O-antigen polysaccharides from Klebsiella O5, Shigella flexneri serotype X, and Salmonella arizonae O62. The data were compared to experimental data of the polysaccharides from the two former strains and the lipopolysaccharide of the latter strain showing excellent agreement between predicted and experimental ¹H and ¹³C NMR chemical shifts.     

Wampetin,a furocoumarin from Clausena wampi

A new furocoumarin wampetin has been isolated from Clausena wampi (syn. Clausena lansium). The structure was established from ¹H NMR, ¹³C NMR, MS and chemical data.     

Bacterial synthesis,purification, and solubilization of membrane protein KCNE3, a regulator of voltage-gated potassium channels

An efficient method is described for production of membrane protein KCNE3 and its isotope labeled derivatives (¹⁵N-, ¹⁵N-/¹³C-) in amounts sufficient for structural-functional investigations. The purified protein preparation within different detergent micelles was characterized using dynamic light scattering, CD spectroscopy, and NMR spectroscopy. It is shown that within DPC/LDAO micelles the protein is in monomeric form and acquires mainly α-helical conformation. The existence of cross-peaks for all glycines of the ¹⁵N-HSQC NMR spectra as well as relatively small line widths (∼20 Hz) confirm the high quality of the preparation and the possibility of obtaining structural-dynamic information on KCNE3 by high resolution heteronuclear NMR spectroscopy.     

4D Non-uniformly sampled C,C-NOESY experiment for sequential assignment of 13C,15N-labeled RNAs

¹³C spin diluted protein samples can be produced using [1-¹³C] and [2-¹³C]-glucose (Glc) carbon sources in the bacterial growth medium. The ¹³C spin dilution results in favorable ¹³C spectral resolution and polarization transfer behavior. We recently reported the combined use of [1-¹³C]- and [2-¹³C]-Glc labeling to facilitate the structural analysis of insoluble and non-crystalline biological systems by solid-state NMR (ssNMR), including sequential assignment, detection of long-range contacts and structure determination of macromolecular assemblies. In solution NMR the beneficial properties of sparsely labeled samples using [2-¹³C]-glycerol (¹³C labeled Cα sites on a ¹²C diluted background) have recently been exploited to provide a bi-directional assignment method (Takeuchi et al. in J Biomol NMR 49(1):17–26, 2011 ). Inspired by this approach and our own recent results using [2-¹³C]-Glc as carbon sources for the simplification of ssNMR spectra, we present a strategy for a bi-directional sequential assignment of solid-state NMR resonances and additionally the detection of long-range contacts using the combination of ¹³C spin dilution and 3D NMR spectroscopy. We illustrate our results with the sequential assignment and the collection of distance restraints on an insoluble and non-crystalline supramolecular assembly, the Salmonella typhimurium type III secretion system needle.     

Garuganin III,a macrocyclic biphenyl ether from Garuga pinnata

The structure of garuganin III, a novel macrocyclic biphenyl ether isolated from Garuga pinnata, was established by spectroscopic analyses, including ¹H NMR and ¹³C NMR decoupling experiments and chemical transformations     

43Ca and 67Zn NMR study of Ca2+, Zn2+-thermolysin complexes

⁴³Ca NMR spectroscopy of Ca²⁺-thermolysin complexes reveals that the structure and/or exchange rate of Ca²⁺ bound to the regulative-site of the enzyme are not essentially changed by adding Zn²⁺ or an inhibitor, L-leucine hydroxamate, both of which may be bound to the active-site of the enzyme. It is shown that the chemical exchange mechanism dominates the ⁴³Ca NMR of Ca²⁺ bound to the enzyme on the basis of temperature-dependences of the NMR. In contrast with the ⁴³Ca NMR findings, first application of ⁶⁷Zn NMR to the Zn²⁺-thermolysin complexes offers convincing evidences that the structure and/or exchange rate of Zn²⁺ bound to the active-site of the enzyme are remarkably changed by adding Ca²⁺ or the inhibitor, L-leucine hydroxamate.     

5-deoxystansioside,an iridoid glucoside from Tecoma stans

In addition to plantarenaloside and stansioside, a new iridoid glucoside with a formyl group at C-4 has been isolated from Tecoma stans. The new glucoside was shown to be 5-deoxystansioside by ¹³C NMR and ¹H NMR spectroscopy.     

Deuteron NMR spectroscopy of copper(II) complexes in solution. 1. (5,7,7,12,14,14,-hexamethyl-1,4,8,1 1-tetraazacyclotetradeca-4,11-diene)-copper(II) and its derivatives in nonaqueous solvent

¹H and ²H NMR spectra of the title copper(II) complexes and its derivatives have been measured. In contrast with their ¹H NMR spectra, ²H NMR spectra gave well resolved sharp signals, and demonstrated that two diastereomers attributable to two asymmetric ligand nitrogens are readily resolved. The remarkable linewidth-narrowing was found in the peripheral methyl groups, which make ²H NMR spectra very useful even for copper(II) complexes with a long electron spin relaxation time. By using ²H NMR spectra, meso-racemate equilibrium was pursued and examined in aqueous and acetonitrile solutions.     

Spin-Echo Nuclear Magnetic Resonance Evidence for Complexing of Sodium Ions in Muscle, Brain, and Kidney

Na⁺ in muscle, brain, and kidney is shown by spin-echo nuclear magnetic resonance (NMR) to consist of two fractions with different NMR parameters. The slow fraction of Na⁺ in these tissues has NMR relaxation times T₁ and T₂ of 10-15 × 10^-3 sec, which is approximately 4-5 times shorter than for Na⁺ in aqueous NaCl solution. The slow fraction may represent Na⁺ dissolved in structured tissue water. The fast fraction of tissue Na⁺, which is shown to represent approximately 65% of the total tissue Na⁺ concentration, has T₂ less than 1 × 10^-3 sec, which resembles the values of T₂ observed for Na⁺ complexed by synthetic ion-exchange resins. One is drawn to the conclusion that approximately 65% of total Na⁺ in muscle, brain, and kidney is complexed by tissue macromolecules.     

Uvariastrol,a novel cycloartane triterpene from the stem bark of Uvariastrum zenkeri

Uvariastrol, a cycloartane triterpene with a novel tetrahydrofuran/furanone side-chain, has been isolated from the stem bark of Uvariastrum zenkeri and characterized using spectral characteristics, notably high-field ¹H NMR and ¹³C NMR spectroscopy.     

Solanopubamine,a steroidal alkaloid from Solanum pubescens

Aerial parts of Solanum pubescens yielded a new steroidal alkaloid, solanopubamine, the structure of which was elucidated as 3β-amino-5α,22αH,25βH-solanidan-23β-ol by ¹³C NMR, ¹H NMR, IR, mass spectral analysis and chemical degradation methods.     

Innovations in Generation and Analysis of 2D [C,H] COSY NMR Spectra for Metabolic Flux Analysis Purposes

2D [¹³C,¹H] COSY NMR is used by the metabolic engineering community for determining ¹³C–¹³C connectivities in intracellular compounds that contain information regarding the steady-state fluxes in cellular metabolism. This paper proposes innovations in the generation and analysis of these specific NMR spectra. These include a computer tool that allows accurate determination of the relative peak areas and their complete covariance matrices even in very complex spectra. Additionally, a method is introduced for correcting the results for isotopic non-steady-state conditions. The proposed methods were applied to measured 2D [¹³C,¹H] COSY NMR spectra. Peak intensities in a one-dimensional section of the spectrum are frequently not representative for relative peak volumes in the two-dimensional spectrum. It is shown that for some spectra a significant amount of additional information can be gained from long-range ¹³C–¹³C scalar couplings in 2D [¹³C,¹H] COSY NMR spectra. Finally, the NMR resolution enhancement by dissolving amino acid derivatives in a nonpolar solvent is demonstrated.     

Metal-based new sulfonamides: Design,synthesis, antibacterial,antifungal, and cytotoxic properties

Cobalt(II), copper(II), nickel(II) and zinc(II) metal complexes with 5-chlorosalicyladehyde derived Schiff base sulfonamides have been synthesized and characterized. Structure and bonding nature of all the synthesized compounds have been deduced from physical, analytical, and spectral (IR, ¹H NMR, ¹³C NMR, Mass, electronic) data. An octahedral geometry has been proposed for all the metal complexes. The ligands and their metal complexes have been screened for their in vitro antibacterial, antifungal, and cytotoxic properties and results are reported.     

Cyanogenic glycosides and menisdaurin from Guazuma ulmifolia, Ostrya virgininana, Tiquilia plicata and Tiquilia canescens

The major cyanogenic glycoside of Guazuma ulmifolia (Sterculiaceae) is (2R)-taxiphyllin (>90%), which co-occurs with (2S)-dhurrin. Few individuals of this species, but occasional other members of the family, have been reported to be cyanogenic. To date, cyanogenic compounds have not been characterized from the Sterculiaceae. The cyanogenic glycosides of Ostrya virginiana (Betulaceae) are (2S)-dhurrin and (2R)-taxiphyllin in an approximate 2:1 ratio. This marks the first report of the identification of cyanogenic compounds from the Betulaceae. Based on NMR spectroscopic and TLC data, the major cyanogenic glucoside of Tiquilia plicata is dhurrin, whereas the major cyanide-releasing compound of Tiquilia canescens is the nitrile glucoside, menisdaurin. NMR and TLC data indicate that both compounds are present in each of these species. The spectrum was examined by CI-MS, ¹H and ¹³C NMR, COSY, 1D selective TOCSY, NOESY, and ¹J/^2,3J HETCOR experiments; all carbons and protons are assigned. The probable absolute configuration of (2R)-dhurrin is established by an X-ray crystal structure. The ¹H NMR spectrum of menisdaurin is more complex than might be anticipated, containing a planar conjugated system in which most elements are coupled to several other atoms in the molecule. The coupling of one vinyl proton to the protons on the opposite side of the ring involves a ⁶J- and a ^5/7J-coupling pathway. A biogenetic pathway for the origin of nitrile glucosides is proposed.     

Location, Structure, and Dynamics of the Synthetic Cannabinoid Ligand CP-55,940 in Lipid Bilayers

The widely used hydrophobic cannabinoid ligand CP-55,940 partitions with high efficiency into biomembranes. We studied the location, orientation, and dynamics of CP-55,940 in POPC bilayers by solid-state NMR. Chemical-shift perturbation of POPC protons from the aromatic ring-current effect, as well as ¹H NMR cross-relaxation rates, locate the hydroxyphenyl ring of the ligand near the lipid glycerol, carbonyls, and upper acyl-chain methylenes. Order parameters of the hydroxyphenyl ring determined by the ¹H-¹³C DIPSHIFT experiment indicate that the bond between the hydroxyphenyl and hydroxycyclohexyl rings is oriented perpendicular to the bilayer normal. ²H NMR order parameters of the nonyl tail are very low, indicating that the hydrophobic chain maintains a high level of conformational flexibility in the membrane. Lateral diffusion rates of CP-55,940 and POPC were measured by ¹H magic-angle spinning NMR with pulsed magnetic field gradients. The rate of CP-55,940 diffusion is comparable to the rate of lipid diffusion. The magnitude of cross-relaxation and diffusion rates suggests that associations between CP-55,940 and lipids are with lifetimes of a fraction of a microsecond. With its flexible hydrophobic tail, CP-55,940 may efficiently approach the binding site of the cannabinoid receptor from the lipid-water interface by lateral diffusion.     

Mukonal,a probable biogenetic intermediate of pyranocarbazole alkaloids from murraya koenigii

Mukonal, a carbazole alkaloid has been isolated from Murraya koenigii. The structure of the compound has been established as 2-hydroxy-3-formyl carbazole based on physical (UV, IR, ¹H NMR, ¹³C NMR and mass spectrometry) and chemical transformations.