Carbon Resonances Research Articles

Nitrogen-detected CAN and CON experiments as alternative experiments for main chain NMR resonance assignments

Heteronuclear direct-detection experiments, which utilize the slower relaxation properties of low gamma nuclei, such as (13)C have recently been proposed for sequence-specific assignment and structural analyses of large, unstructured, and/or paramagnetic proteins. Here we present two novel (15)N direct-detection experiments. The CAN experiment sequentially connects amide (15)N resonances using (13)C(alpha) chemical shift matching, and the CON experiment connects the preceding (13)C' nuclei. When starting from the same carbon polarization, the intensities of nitrogen signals detected in the CAN or CON experiments would be expected four times lower than those of carbon resonances observed in the corresponding (13)C-detecting experiment, NCA-DIPAP or NCO-IPAP (Bermel et al. 2006b; Takeuchi et al. 2008). However, the disadvantage due to the lower gamma is counteracted by the slower (15)N transverse relaxation during detection, the possibility for more efficient decoupling in both dimensions, and relaxation optimized properties of the pulse sequences. As a result, the median S/N in the (15)N observe CAN experiment is 16% higher than in the (13)C observe NCA-DIPAP experiment. In addition, significantly higher sensitivity was observed for those residues that are hard to detect in the NCA-DIPAP experiment, such as Gly, Ser and residues with high-field C(alpha) resonances. Both CAN and CON experiments are able to detect Pro resonances that would not be observed in conventional proton-detected experiments. In addition, those experiments are free from problems of incomplete deuterium-to-proton back exchange in amide positions of perdeuterated proteins expressed in D(2)O. Thus, these features and the superior resolution of (15)N-detected experiments provide an attractive alternative for main chain assignments. The experiments are demonstrated with the small model protein GB1 at conditions simulating a 150 kDa protein, and the 52 kDa glutathione S-transferase dimer, GST.

ChemInform Abstract: Chromones and Flavanones from Artemisia campestris subsp. maritima.

Abstract From the acetone extract of Artemisia campestris subsp. maritima six flavanones, two chromones and the coumarin scopoletin were isolated. 5-Hydroxy-7-methoxychromone and 5,7-dimethoxychromone are new compounds, while the flavanone eriodictyol-7,3′-dimethyl ether is reported for the first time in this species. The structures were elucidated by 1D and 2D NMR techniques. The unequivocal assignments of carbon resonances, mainly made by using 1D selective INEPT and 2D HETCOR experiments, allowed us to correct some 1H and 13C resonances of the isolated flavanones and also to differentiate between the flavanone isomers sakuranetin⧹isosakuranetin and eriodictyol-7,3′-dimethyl ether⧹eriodictyol-7,4′-dimethyl ether.

ChemInform Abstract: Synthesis and Solution Structure Analysis of a Bispyrenyl Bishydroxamate Calix[4]arene-Based Receptor, a Fluorescent Chemosensor for Cu2+ and Ni2+ Metal Ions.

A bispyrenyl calix[4]arene-based receptor, 1, incorporating two hydroxamic acid functionalities has been designed for the optical detection of Cu2+ and Ni2+ metal ions in solution. The synthetic route is based on the utilization of pyrene-labeled, O-protected hydroxylamines that represent versatile building blocks for the generation of fluorescent siderophore-based chelators. Compound 1 is shown to exist in CDCl3 solution at room temperature as a mixture of mainly two conformers, namely, the cone and partial-cone species (partial-cone/cone ratio ca. 57/43). The full assignment of the proton and carbon resonances of the calix[4]arene core for both conformers is described. Ligand 1 exhibited in solution a dual fluorescence emission spectrum composed of monomer and excimer contributions, the latter resulting from intramolecular interactions between pyrene nuclei in the excited state. In methanol/water (80/20 v/v), the fluorescence intensity was shown to be sensitive to proton concentration and to the presenc...

The Longin SNARE VAMP7/TI-VAMP Adopts a Closed Conformation

SNARE protein complexes are key mediators of exocytosis by juxtaposing opposing membranes, leading to membrane fusion. SNAREs generally consist of one or two core domains that can form a four-helix bundle with other SNARE core domains. Some SNAREs, such as syntaxin target-SNAREs and longin vesicular-SNAREs, have independent, folded N-terminal domains that can interact with their respective SNARE core domains and thereby affect the kinetics of SNARE complex formation. This autoinhibition mechanism is believed to regulate the role of the longin VAMP7/TI-VAMP in neuronal morphogenesis. Here we use nuclear magnetic resonance spectroscopy to study the longin-SNARE core domain interaction for VAMP7. Using complete backbone resonance assignments, chemical shift perturbations analysis, and hydrogen/deuterium exchange experiments, we conclusively show that VAMP7 adopts a preferentially closed conformation in solution. Taken together, the closed conformation of longins is conserved, in contrast to the syntaxin family of SNAREs for which mixtures of open and closed states have been observed. This may indicate different regulatory mechanisms for SNARE complexes containing syntaxins and longins, respectively.

Selective formation of glycosidic linkages of N-unsubstituted 4-hydroxyquinolin-2-(1 H)-ones

A comparative study for selective glucosylation of N-unsubstituted 4-hydroxyquinolin-2(1 H)-ones into 4-(tetra- O-acetyl-β- d-glucopyranosyloxy)quinolin-2(1 H)-ones is reported. Four glycosyl donors including tetra- O-acetyl-α- d-glucopyranosyl bromide, β- d-glucose pentaacetate, glucose tetraacetate and tetra- O-acetyl-α- d-glucopyranosyl trichloroacetimidate were tested, along with different promoters and reaction conditions. The best results were obtained with tetra- O-acetyl-α- d-glucopyranosyl bromide with Cs 2CO 3 in CH 3CN. In some cases the 4-O-glucosylation of the quinolinone ring was accompanied by 2-O-glucosylation yielding the corresponding 2,4-bis(tetra- O-acetyl-β- d-glucopyranosyloxy)quinoline. Next, 4-(tetra- O-acetyl-β- d-glucopyranosyloxy)quinolin-2(1 H)-ones were deacetylated into 4-(β- d-glucopyranosyloxy)quinolin-2(1 H)-ones with Et 3N in MeOH. In some instances the deacetylation was accompanied by the sugar–aglycone bond cleavage. Structure elucidation, complete assignment of proton and carbon resonances as well as assignment of anomeric configuration for all the products under investigation were performed by 1D and 2D NMR spectroscopy.

Hyperpolarized 13C magnetic resonance metabolic imaging: application to brain tumors

In order to compare in vivo metabolism between malignant gliomas and normal brain, (13)C magnetic resonance (MR) spectroscopic imaging data were acquired from rats with human glioblastoma xenografts (U-251 MG and U-87 MG) and normal rats, following injection of hyperpolarized [1-(13)C]-pyruvate. The median signal-to-noise ratio (SNR) of lactate, pyruvate, and total observed carbon-13 resonances, as well as their relative ratios, were calculated from voxels containing Gadolinium-enhanced tissue in T(1) postcontrast images for rats with tumors and from normal brain tissue for control rats. [1-(13)C]-labeled pyruvate and its metabolic product, [1-(13)C]-lactate, demonstrated significantly higher SNR in the tumor compared with normal brain tissue. Statistical tests showed significant differences in all parameters (P < .0004) between the malignant glioma tissue and normal brain. The SNR of lactate, pyruvate, and total carbon was observed to be different between the U-251 MG and U-87 MG models, which is consistent with inherent differences in the molecular characteristics of these tumors. These results suggest that hyperpolarized MR metabolic imaging may be valuable for assessing prognosis and monitoring response to therapy for patients with brain tumors.

1H, 13C and 31P MAS NMR studies of lyophilized brain tumors

1H, 13C and 31P magic angle spinning magnetic resonance spectra (MAS NMR) of lyophilized brain tissue specimens were recorded. Among the 35 cases of brain tumors there were 24 glioblastomas, seven meningiomas and a few other types. 1H NMR measurements were performed with a MAS speed of 33 kHz. The intense CH 3, CH 2 and CH peaks in the 1H spectrum result from fatty acid residues of phospholipids, which are “mobile enough” besides the anhydrous environment. 13C CPMAS spectra revealed the resonances of creatine and guanidine carbons; the high intensity signals arise from carbonyl groups and methylene carbons of lipids. In particular we found a fraction of mobile lipids, characterized by narrow resonances and long T 1 ρ H Overlapped resonances of phospholipids head groups contributed to the peak at 4–7 ppm in the 31P MAS NMR spectra. Our results indicate that 1H and 13C MAS NMR are able to characterize tumor types: differentiate glioblastomas from meningiomas and shed light on tumor biochemical characteristics. However, water soluble metabolites are not observed and macromolecules yield broad overlapped resonances. Generally, lyophilization significantly decreases discriminative potential of NMR analysis.

13C NMR spectroscopy for determining the acylglycerol positional composition of lampante olive oils. Chemical shift assignments and their dependence on sample concentration

Two Lampante olive oils with free acidity expressed as oleic acid of 25.9 and 8.4 g per 100 g were used to carry out the 13C NMR study for the assignment of all resonances of free fatty acid fraction and of the acylglycerol fraction comprising 1(3)-mono- (1(3)-MAG), 1,3-di- (1,3-DAG), 1,2-di- (1,2-DAG) and triacylglycerols (TAG). The glycerol carbon resonances were found to be resolved according to the esterification degree of the glycerol molecule but no data were available on the length and the unsaturation pattern of the acyl chains. It was the spectral region where the C-1 carboxy carbons resonate that enabled the determination of free fatty acids and of different acylglycerol species. Moreover, within each acylglycerol class, the acyl chain distribution between the 1,3- and 2- glycerol positions was also calculated where the saturated, vaccenate, oleate and linoleate chains were detected as baseline resolved resonances in 1(3)-MAG, 1,3-DAG, 1,2-DAG and TAG. The variability of chemical shifts to sample concentration changes was also evidenced in all the frequency ranges being studied where the methylene C-2 and C-3 carbons underwent the most dramatic changes in their resonance patterns which prevented any resonance assignment.

Some reactions of an η3-tetracyanobutadienyl-ruthenium complex

In the eta(3)-butadienyl complex Ru{eta(3)-C(CN)(2)CPhC=C(CN)(2)}(PPh(3))Cp 1, which is formed from Ru(C[triple bond]CPh)(PPh(3))(2)Cp and tcne, a CN group reacts with MeO(-) to give the methoxy-amide Ru{NH=C(OMe)C(CN)=CCPh=C(CN)(2)}(PPh(3))Cp 2, in which the NH has displaced the C=C from the Ru centre with formation of a RuC(3)N ring. "Click addition" of azide to a CN group in 1 gives the oligomeric tetrazolato complex Ru{N(3)N[Na(OEt(2))]=CC(CN)=CCPh=C(CN)(2)}(PPh(3))Cp 3, also containing a RuC(3)N ring. Salt-elimination reactions of 3 with MeOTf, FeCl(dppe)Cp, RuCl(dppe)Cp* and trans-PtCl(2){P(tol)(3)}(2) result in selective substitution at one nitrogen atom of the RuC(3)N ring. Geometries of 1 and the anion in 3 were computed by DFT methods. Preferences for CN groups attacked in the nucleophilic and cycloaddition reactions of 1 are supported by NBO calculations. Alkylation of 1 in reactions with 1,2-dimethoxyethane gave two isomers of Ru{N(3)[CH(CH(2)OMe)(OMe)]N=CC(CN)=CCPh=C(CN)(2)}(PPh(3))Cp 8 and 9, differing in the sites of attachment of the alkyl group, likely by radical processes. The molecular structures of eight complexes are reported, including a re-determination of 1. Computed NMR chemical shifts are used to reassign the butadienyl carbon resonances in the (13)C NMR spectrum of 1.

Synthesis and structure elucidation of 25,26,27,28-tetramethylcalix[4]arene tetraketone using 1D and 2D NMR spectroscopies

The 1H and 13C NMR spectra of the new calix[4]arene-based tetraketone were completely assigned by one- and two-dimensional homo- and heteronuclear experiments ( 1H– 1H COSY, 1H– 13C HMQC, and HMBC) at 400 and 100 MHz, respectively, at 25 °C standard pulse sequence. 1H and 13C spectra were measured at room temperature for 25,26,27,28-tetramethylcalix[4]arene tetraketone ( 1). 13C{ 1H}, DEPT and NMR techniques were used to distinguish the methyl, methylene and methine carbon resonance signals of calix[4]arene 1. Correlation of 1D ( 1H, 13C{ 1H}, DEPT) and 2D (HMQC and HMBC) NMR data was used to completely assign various overlapping and broad signals of calix[4]arene 1. Heteronuclear multibond correlation (HMBC) studies were used to completely assign various carbon resonances. Our results show that the conformation of calix[4]arene 1 in the solution is very similar to the cone conformation reported in the literature.

Analysis of possible use of neutron filtered beam technique at the reactor for the carbon resonance parameters determination in the 150 keV energy region

Analysis of possible use of neutron filtered beam technique at the reactor for the carbon resonance parameters determination in the 150 keV energy region

Study and characterization of crystalline hydrate/polymorph forms of 5,11-dihydro-11-ethyl-5-methyl-8-(2-(1-oxido-4-quinolinyl)ethyl-6H-dipyrido(3,2-B:2′,3′-E)(1,4)diazepin-6-one by solid-state NMR and solution NMR

A novel inhibitor of reverse transcriptase was studied by solid-state NMR. Three phases of the compound were examined which included the dihydrate and two anhydrous polymorphs (Form I and Form III). By correlating 1H and 13C solution NMR with the solid-state 13C NMR CP/MAS and CPPI spectral editing experiments, comparative 13C assignments were made for each phase. Polymorphs of Form I and Form III and the dihydrate were easily distinguished based upon chemical shift patterns of the carbon resonances. The 1H spin-lattice relaxation times were also measured for each phase which provided information on the mobility and relative crystallinity. The 13C ssNMR spectrum of Form I showed the presence of a minor component identified as the dihydrate. Weight/percent quantitation of major and minor components in Form I was obtained from integrated intensities of a 50:50 mixture containing weighed amounts of Form I and the pure dihydrate. Comparison of the ssNMR and X-ray powder diffraction techniques is discussed.

One- and two-dimensional NMR studies of methyl acrylate/vinyl acetate/ N-vinyl carbazole terpolymers

Terpolymers of methyl acrylate/vinyl acetate/ N-vinyl carbazole (M/A/C) with different compositions were synthesized by solution polymerization using AIBN as an initiator. Composition of terpolymers was determined from quantitative 13C{ 1H} NMR spectrum. Two-dimensional heteronuclear single quantum correlation (HSQC) and total correlated spectroscopy (TOCSY) were used to assign the methylene and methine carbon resonances by analyzing two and three bond order couplings. Various resonance signals were assigned to different compositional and configurational sequences with the help of one- and two-dimensional NMR spectra. Three and four bond order coupling between carbonyl carbon and other neighboring protons have been investigated with the help of 2D heteronuclear multiple bond correlation (HMBC) spectra. The complex and overlapped 1H NMR spectrum of terpolymer was analyzed completely with the help of 2D HSQC and TOCSY spectra.

Organofunctionalized kenyaite for dye removal from aqueous solution

Crystalline layered sodium kenyaite was exchanged to proton kenyaite when reacted with hydrochloric acid solution, providing a new surface with available silanol groups that are able to couple with N-3-trimethoxysilylpropylethylenediamine silylating agent, after prior expansion of the basal distance with the polar organic solvent dimethyl sulfoxide. The resulting organofunctionalized nanomaterial (2N-Ken) was characterized by elemental analysis, infrared spectroscopy, X-ray diffraction, carbon and silicon nuclear magnetic resonances in the solid state, surface analysis, porosity, thermogravimetry, and electron scanning microscopy. The quantity of silylating agent incorporated into the nanospace, calculated from the nitrogen elemental analysis, was determined as 0.48 mmol g −1, after expanding of the acidic precursor basal distance from 1.62 to 1.99 nm. The presence of a covalent silicon–carbon bond of the organosilyl moiety on the inorganic layered structure was confirmed through nuclear magnetic resonance. This new nanomaterial has the ability to extract the Sumifix Brilliant Orange 3R textile dye from aqueous solution, using a batchwise process. The effects of stirring time, adsorbent dosage, and pH on the adsorption capacity demonstrated that 4 h is enough to reach equilibrium at 298 ± 1 K under pH 4.0. Based on error function values ( F error) the data were best fitted to fractional-order and chemisorption kinetic models when compared to pseudo-first-order and pseudo-second-order kinetic models. The equilibrium data were better fitted to the Sips isotherm model.

Vanadocalixarenes on Silica: Requirements for Permanent Anchoring and Electronic Communication

Grafted V(V)−calixarene complexes that are 1,3-disubstituted at the lower rim have been synthesized using an extraordinarily mild procedure, using the V(III)−calixarene precursor p-But−calix[4]−(OMe)2(O)2V−Cl (1c), which is subsequently grafted onto silica and, afterward, oxidized to yield material 2c. The calixarene acts as a sterically bulky chelating ligand and enforces isolated pseudo-octahedral vanadium centers on silica, as ascertained by 51V NMR spectroscopy and analysis of the UV/vis spectrum of materials after calixarene removal via calcination. 13C CP/MAS NMR spectroscopy of 2c reveals lack of a downfield-shifted ipso carbon resonance, which is strongly indicative of a high d-orbital electron density for the V metal centers in 2c, a result that is expected on the basis of a proposed simple geometric model that is consistent with single-crystal X-ray crystallographic data of early transition metal metallocalixarenes. This result motivates future programming of grafted metal centers with chemical ...

Microstructure determination of poly(acrylonitrile-co-methyl methacrylate-co-methyl acrylate) terpolymers by 2D HMBC

A compositional and configurational sequence analysis from two-dimensional heteronuclear multiple bond correlation (HMBC) spectra of acrylonitrile/methyl methacrylate/methyl acrylate (A/B/M) terpolymer is being reported here. The methylene and methine carbon resonances assigned from 2D HSQC spectra were established by analyzing two and three bond order couplings with methyl, methylene and methine protons. α-Methyl carbon resonance of B-unit and methine carbon resonances of A- and M-unit were assigned up to triad compositional and configurational sequences while methylene carbon resonances were assigned up to tetrad compositional and configurational sequences. The assignments of carbonyl carbon resonances based on analysis of three bond couplings with methyl and methylene protons are reported. Quaternary carbon resonance of B-unit was assigned completely with the help of two bond order couplings with methyl and methylene protons. Both carbonyl and quaternary carbon resonances were found to be sensitive up to triad compositional and configurational sequences. Complete spectral assignment of quaternary and carbonyl carbon resonances showing the critical contribution of 2D HMBC spectra is indirect analysis of carbon resonances.

Stereochemical elucidation of complex α‐methyl and β‐methylene carbon resonances in poly(2‐hydroxy ethyl methacrylate‐co‐methacrylonitrile) by 2D NMR

AbstractCopolymers of 2‐hydroxy ethyl methacrylate and methacrylonitrile (H/M) of different composition were synthesized by free radical bulk polymerization using azobisisobutyronitrile (AIBN) as an initiator under nitrogen atmosphere. The copolymers composition were calculated from 1H and quantitative 13C{1H}NMR spectra. The complete spectral assignment of complex and overlapped α‐methyl and β‐methylene carbon regions in 13C{1H} NMR spectrum in term of compositional and configurational sequences of H/M copolymers were done with the help of two‐dimensional heteronuclear single quantum coherence (HSQC) and total correlation spectroscopy (TOCSY). © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2009

The structural dynamics in the proton-conducting imidazolium oxalate

The 1H spin–lattice relaxation times and high-resolution solid-state nuclear magnetic resonance (NMR)under fast magic spinning were used to study the structural dynamics in the proton-conductingmaterial imidazolium oxalate. The measurements provide evidence for the ordered anddisordered domains within the studied material. The two components drastically differ in their1H spin–latticerelaxation times and 1H–13C cross-polarization magic-angle-spinning (CP/MAS) spectra. The coalescence phenomenonof the resonances of the basal carbons of the imidazole ring undergoing a reorientation isobserved only for mobile molecules in the disordered domains. Therefore, only thesemolecules can be responsible for proton conductivity allowing for the Grotthus mechanism.

Automated NMR Assignment of Protein Side Chain Resonances Using Automated Projection Spectroscopy (APSY)

This paper describes an automated method for sequence-specific NMR assignment of the aliphatic resonances of protein side chains in small- and medium-sized globular proteins in aqueous solution. The method requires the recording of a five-dimensional (5D) automated projection spectroscopy (APSY-) NMR experiment and the subsequent analysis of the APSY peak list with the algorithm ALASCA (Algorithm for local and linear assignment of side chains from APSY data). The 5D APSY-HC(CC-TOCSY)CONH experiment yields 5D chemical shift correlations of aliphatic side chain C-H moieties with the backbone atoms H(N), N, and C'. A simultaneous variation of the TOCSY mixing times and the projection angles in this APSY-type TOCSY experiment gives access to all aliphatic C-H moieties in the 20 proteinogenic amino acids. The correlation peak list resulting from the 5D APSY-HC(CC-TOCSY)CONH experiment together with the backbone assignment of the protein under study is the sole input for the algorithm ALASCA that assigns carbon and proton resonances of protein side chains. The algorithm is described, and it is shown that the aliphatic parts of 17 of the 20 common amino acid side chains are assigned unambiguously, whereas the remaining three amino acids are assigned with a certainty of above 95%. The overall feasibility of the approach is demonstrated with the globular 116-residue protein TM1290, for which reference assignments are known. For this protein, 97% of the expected side chain carbon atoms and 87% of the expected side chain protons were detected with the 5D APSY-HC(CC-TOCSY)CONH experiment in 24 h of spectrometer time, and all these resonances were correctly assigned by ALASCA. Based on the experience with TM1290, we expect that the approach presented in this work is routinely applicable to globular proteins with sizes up to at least 120 amino acids.

Determining Secondary Structure in Spider Dragline Silk by Carbon−Carbon Correlation Solid-State NMR Spectroscopy

Two-dimensional (2D) (13)C-(13)C NMR correlation spectra were collected on (13)C-enriched dragline silk fibers produced from Nephila clavipes spiders. The 2D NMR spectra were acquired under fast magic-angle spinning (MAS) and dipolar-assisted rotational resonance (DARR) recoupling to enhance magnetization transfer between (13)C spins. Spectra obtained with short (150 ms) recoupling periods were utilized to extract distinct chemical shifts for all carbon resonances of each labeled amino acid in the silk spectra, resulting in a complete resonance assignment. The NMR results presented here permit extraction of the precise chemical shift of the carbonyl environment for each (13)C-labeled amino acid in spider silk for the first time. Spectra collected with longer recoupling periods (1 s) were implemented to detect intermolecular magnetization exchange between neighboring amino acids. This information is used to ascribe NMR resonances to the specific repetitive amino acid motifs prevalent in spider silk proteins. These results indicate that glycine and alanine are both present in two distinct structural environments: a disordered 3(1)-helical conformation and an ordered beta-sheet structure. The former can be ascribed to the Gly-Gly-Ala motif while the latter is assigned to the poly(Ala) and poly(Gly-Ala) domains.