T1 relaxation measurement with solvent suppression

Three solvent-suppression pulse sequences of varying complexity were incorporated into the standard inversion recovery pulse program and experimentally evaluated. The least complex suppression sequence involves a composite 90 degrees pulse. A more complex sequence utilizes an excitation sculpting sequence requiring pulsed field gradients, and the most complex sequence incorporates an excitation sculpting sequence with selective rf pulses and gradient pulses. The quality of the spectral data and the accuracy of T(1) measurements of the investigated suppression schemes were evaluated using three aqueous samples with increasing proton content in the water solvent, i.e. by volume 100% D(2)O, 80/20% D(2)O/H(2)O, and 100% H(2)O. For lines removed from the water resonance the T(1) values were generally very consistent between all pulse sequences tested. For lines less than about 200 Hz from the water signal the T(1) measurements become less reliable but are still possible for most of the tested pulse programs.     

Orientations of the principal components of electric field gradients and internal motions in dihydrogen ligands from the 2H T1 NMR relaxation data in solution

The deuterium spin-lattice relaxation times in (D2) ligands of W, Ru and Os complexes are reviewed and analyzed in terms of the fast internal (D2) motions: free rotation, librations and 180 degrees jumps. The analysis was performed using quadrupolar coupling constant (DQCC) parameters taken from the solid-state 2H NMR spectra and density function theory calculations. It is shown that the calculated DQCC values can be corrected for further use in interpretations of deuterium relaxation times for Ru and Os dihydrogen complexes. The resulting data led to a criterion for using the relaxation data to distinguish fast-spinning dihydrogen ligands. It is shown that the principal components of electric field gradient tensors at D in the dihydrogen ligands are oriented closer to M-D directions.     

Advanced solvent signal suppression for the acquisition of 1D and 2D NMR spectra of Scotch Whisky

A simple and robust solvent suppression technique that enables acquisition of high‐quality 1D ¹H nuclear magnetic resonance (NMR) spectra of alcoholic beverages on cryoprobe instruments was developed and applied to acquire NMR spectra of Scotch Whisky. The method uses 3 channels to suppress signals of water and ethanol, including those of ¹³C satellites of ethanol. It is executed in automation allowing high throughput investigations of alcoholic beverages. On the basis of the well‐established 1D nuclear Overhauser spectroscopy (NOESY) solvent suppression technique, this method suppresses the solvent at the beginning of the pulse sequence, producing pure phase signals minimally affected by the relaxation. The developed solvent suppression procedure was integrated into several homocorrelated and heterocorrelated 2D NMR experiments, including 2D correlation spectroscopy (COSY), 2D total correlation spectroscopy (TOCSY), 2D band‐selective TOCSY, 2D J‐resolved spectroscopy, 2D ¹H, ¹³C heteronuclear single‐quantum correlation spectroscopy (HSQC), 2D ¹H, ¹³C HSQC‐TOCSY, and 2D ¹H, ¹³C heteronuclear multiple‐bond correlation spectroscopy (HMBC). A 1D chemical‐shift‐selective TOCSY experiments was also modified. The wealth of information obtained by these experiments will assist in NMR structure elucidation of Scotch Whisky congeners and generally the composition of alcoholic beverages at the molecular level.     

Water and oil signal assignment in low-moisture mozzarella as determined by time-domain NMR T2 relaxometry

A time-domain ¹H nuclear magnetic resonance relaxometry method was elaborated for the rapid microstructural characterization of mozzarella cheese. For this purpose, there is a strong need to know how the experimentally determined T₂ relaxation time distribution can be related to specific constituents in mozzarella. In this study, a detailed investigation is offered for fresh and aged low-moisture mozzarella cheese, often applied as a pizza cheese, by application of both a conventional Carr–Purcell–Meiboom–Gill (CPMG) sequence and a free-induction decay CPMG (FID-CPMG) sequence. The relaxation behavior was further elucidated by addition of deuterium oxide and by mild heat treatment of samples. The relaxation times of water protons in mozzarella were found to range from a few microseconds to some tens of milliseconds (in aged mozzarella) or to about hundred milliseconds (in fresh mozzarella). The upper limit of the T₂ distribution can even be extended to the seconds range upon releasing water protons from the mozzarella matrix using a mild heat treatment or upon addition of deuterated water. Both stimuli also provided evidence for the absorption of water into the cheese matrix. The potential release and uptake of water demonstrated that mozzarella acts as a very dynamic system during production and storage. The detected differences in the behavior of the water fraction between fresh and aged low-moisture mozzarella might be utilized to study the influence of either production and/or storage conditions on the cheese ripening process.     

A new phase modulated binomial‐like selective‐inversion sequence for solvent signal suppression in NMR

A new 8 ‐pulse P hase M odulated binomial‐like selective inversion pulse sequence, dubbed ‘8PM’, was developed by optimizing the nutation and phase angles of the constituent radio‐frequency pulses so that the inversion profile resembled a target profile. Suppression profiles were obtained for both the 8PM and W5 based excitation sculpting sequences with equal inter‐pulse delays. Significant distortions were observed in both profiles because of the offset effect of the radio frequency pulses. These distortions were successfully reduced by adjusting the inter‐pulse delays. With adjusted inter‐pulse delays, the 8PM and W5 based excitation sculpting sequences were tested on an aqueous lysozyme solution. The 8 PM based sequence provided higher suppression selectivity than the W5 based sequence. Two‐dimensional nuclear Overhauser effect spectroscopy experiments were also performed on the lysozyme sample with 8PM and W5 based water signal suppression. The 8PM based suppression provided a spectrum with significantly increased (~ doubled) cross‐peak intensity around the suppressed water resonance compared to the W5 based suppression. Copyright © 2016 John Wiley & Sons, Ltd.     

Standardization of chemical shifts of TMS and solvent signals in NMR solvents

The standard for chemical shift is dilute tetramethylsilane (TMS) in CDCl3, but many measurements are made relative to TMS in other solvents, the proton resonance of the solvent peak or relative to the lock frequency. Here, the chemical shifts of TMS and the proton and deuterium chemical shifts of the solvent signals of several solvents are measured over a wide temperature range. This allows for the use of TMS or the solvent and lock signal as a secondary reference for other NMR signals, as compared with dilute TMS in CDCl3 at a chosen temperature; 25 degrees C is chosen here. An accuracy of 0.02 ppm is achievable for dilute solutions, provided that the interaction with the solvent is not very strong. The proton chemical shift of residual water is also reported where appropriate.     

Gradient echo single scan inversion recovery: application to proton and fluorine relaxation studies

Single scan longitudinal relaxation measurement experiments enable rapid estimation of the spin‐lattice relaxation time (T₁) as the time series of spin relaxation is encoded spatially in the sample at different slices resulting in an order of magnitude saving in time. We consider here a single scan inversion recovery pulse sequence that incorporates a gradient echo sequence. The proposed pulse sequence provides spectra with significantly enhanced signal to noise ratio leading to an accurate estimation of T₁ values. The method is applicable for measuring a range of T₁ values, thus indicating the possibility of routine use of the method for several systems. A comparative study of different single scan methods currently available is presented, and the advantage of the proposed sequence is highlighted. The possibility of the use of the method for the study of cross‐correlation effects for the case of fluorine in a single shot is also demonstrated. Copyright © 2015 John Wiley & Sons, Ltd.     

Characterization of the complex formation of 1,6‐anhydro‐β‐maltotriose with potassium using 1H and 39K NMR spectroscopy

The ¹H and ³⁹K longitudinal relaxation times (T₁) and ¹H diffusion coefficients were measured to investigate the complex formation of 1,6‐anhydro‐β‐maltotriose and potassium ions. Although the ¹H‐T₁ values of H3′, H5′, H1″ and H4″ decreased in the presence of potassium ions, ¹H chemical shifts and ¹H diffusion coefficients did not show significant changes. The long‐range coupling constants of ³J_C?H around the glycosyl bonds did not show significant changes either. In the measurements of ³⁹K spectra, the ³⁹K signal obviously broadened and the ³⁹K‐T₁ values decreased in the presence of 1,6‐anhydro‐β‐maltotriose, indicating the complex formation of 1,6‐anhydro‐β‐maltotriose and potassium ions. These results indicate that the conformation and molecular volume were unaffected in the complex formation. Copyright © 2009 John Wiley & Sons, Ltd.     

Regioselective synthesis and structural elucidation of 1,4-disubstituted 1,2,3-triazole derivatives using 1D and 2D NMR spectral techniques

Regioselective synthesis of 2-[1-(2-oxo-2-phenylethyl)-1H-[1,2,3]triazol-4-ylmethoxy]-benzaldehyde derivatives was achieved by [3 + 2] cycloaddition reaction of 2-(prop)-2-ynyloxy-benzaldehyde derivatives with phenacyl azide. The regiochemistry and the spectral assignments of the synthesized triazole derivatives were studied using both 1D and 2D NMR spectral techniques in solution.     

Reduction of conformational mobility and aggregation in W60G β2‐microglobulin: assessment by 15N NMR relaxation

The amyloid pathology associated with long‐term haemodialysis is due to the deposition of β₂‐microglobulin, the non‐polymorphic light chain of class I major histocompatibility complex, that accumulates at bone joints into amyloid fibrils. Several lines of evidence show the relevance of the tryptophan residue at position 60 for the fibrillogenic transition of the protein. A comparative ¹⁵N NMR relaxation analysis is presented for wild‐type human β₂‐microglobulin and W60G β₂‐microglobulin, i.e. the mutant with a glycyne replacing the natural tryptophan residue at position 60. The experimental data, collected at 11.4 T and 310 K, were analyzed by means of the reduced spectral density approach. Molecular dynamics (MD) simulations and corresponding thermodynamic integration, together with hydrodynamic calculations were performed to support data interpretation. The analysis results for the mutant protein are consistent with a reduced aggregation with respect to the wild‐type counterpart, as a consequence of an increased conformational rigidity probed by either NMR relaxation and MD simulations. Although dynamics in solution is other than fibrillar competence, the assessed properties of the mutant protein can be related with its reduced ability of forming fibrils when seeded in 20% trifluoroethanol. Copyright © 2013 John Wiley & Sons, Ltd.     

Structure elucidation of the intermediate in triethylborane-mediated radical addition of oxime ethers with 2D- and 3D-DOSY NMR

The structures of the components in the triethylborane-mediated radical addition reaction of oxime ether were investigated by 1H- and 3D-DOSY NMR methods. It has been impossible to physically separate the unstable intermediates; therefore, the structures were thus far unidentified. It has been possible to elucidate the structures of these unstable intermediates using Diffusion-Ordered Spectroscopy (DOSY) methods for the reaction in an NMR tube. The DOSY methods resolved the spectra of each starting compound, intermediate and product having different diffusion coefficients. The structure of the intermediate was shown to be due to the bonding of diethylborane to the nitrogen atom of the alkoxyamino group.     

1H, 13C and 15N NMR spectroscopic characterization of unexpected degradation products of the nifedipine analogue bis(2‐cyanoethyl) 2,6‐dimethyl‐4‐(2‐nitrophenyl)‐1,4‐dihydro‐3,5‐pyridinedicarboxylate

In the course of saponification experiments with bis(2‐cyanoethyl) 2,6‐dimethyl‐4‐(2‐nitrophenyl)‐1,4‐dihydro‐3,5‐pyridinedicarboxylate ( 1 ), an analogue of the calcium channel blocker nifedipine, three unexpected degradation products were isolated. The compounds were identified as 3‐(2‐acetamido‐1‐carboxy‐1‐propenyl)‐1‐hydroxy‐2‐indolecarboxylic acid ( 3 ), 9‐hydroxy‐1,3‐dimethyl‐β‐carboline‐4‐carboxylic acid ( 4 ) and 6‐hydroxy‐2,4‐dimethyl‐5‐oxo‐5,6‐dihydrobenzo[c][2,7]naphthyridine‐1‐carboxylic acid ( 6 ). The structures of these compounds were deduced from one‐ and two‐dimensional ¹H, ¹³C and natural abundance ¹⁵N NMR experiments (¹H,¹H‐COSY, gs‐HSQC, gs‐HMBC, ¹⁵N gs‐HMBC), and corroborated by comparison of their NMR data with the respective data for structurally similar compounds. Copyright © 2002 John Wiley & Sons, Ltd.     

Diastereomeric difference of inclusion modes between (-)-epicatechin gallate, (-)-epigallocatechin gallate and (+)-gallocatechin gallate, with beta-cyclodextrin in aqueous solvent

Inclusion complexes of (-)-epicatechin gallate (ECg) as well as (+)-gallocatechin gallate (GCg) and beta-cyclodextrin (beta-CD) in an aqueous solution were investigated using several NMR techniques and a computational method. ECg and EGCg formed a 1:1 complex with beta-CD, in which the A ring and a portion of the C ring were included from the wide secondary hydroxyl group side of the beta-CD cavity, and the B and B' rings were left outside the cavity. GCg formed a 1:2 complex with beta-CD, in which the A and B rings of GCg were included by two molecules of beta-CD. The difference between the two modes of inclusion of the 1:1 complex of ECg, EGCg.beta-CD and the 1:2 complex of GCg.beta-CD might have resulted from the size of the space between the B and B' rings in aqueous solution. As a result of nuclear Overhauser effect (NOE) experiments, GCg was considered to have a large enough space between the B and B' rings to include the B ring in the beta-CD cavity; on the other hand, ECg and EGCg have no such large space.     

One‐step synthesis of 6‐acetamido‐3‐(N‐(2‐(dimethylamino) ethyl) sulfamoyl) naphthalene‐1‐yl 7‐acetamido‐4‐hydroxynaphthalene‐2‐sulfonate and its characterization with 1D and 2D NMR techniques

A one‐step method was reported for the synthesis of 6‐acetamido‐3‐(N‐(2‐(dimethylamino) ethyl) sulfamoyl) naphthalene‐1‐yl 7‐acetamido‐4‐hydroxynaphthalene‐2‐sulfonate by treating 7‐acetamido‐4‐hydroxy‐2‐naphthalenesulfonyl chloride with equal moles of N, N‐dimethylethylenediamine in acetonitrile in the presence of K₂CO₃. The chemical structure of the obtained compounds was characterized by MS, FTIR, ¹H NMR, ¹³C NMR, gCOSY, TOCSY, gHSQC, and gHMBC. The chemical shift differences of ¹H and ¹³C being δ 0.04 and 0.2, respectively, were unambiguously differentiated. Copyright © 2013 John Wiley & Sons, Ltd.     

NMR spectra and structures of oridonin derivatives complexes with β‐cyclodextrin

Complexations between three oridonin derivatives and β‐cyclodextrin (βCD) were studied by nuclear magnetic resonance (NMR) method. Job's plots for complexes were depicted by ¹H NMR spectra chemical shifts, which proved the 1:1 stoichiometry inclusion complex formation between each derivative and βCD. Two‐dimensional rotating frame overhauser effect spectroscopy (2D ROESY) support the above conclusion and also proved that ring A of each oridonin derivative deeply enters into hydrophobic cavity from the wider rim and the other parts are outside the cavity. Apparent formation constants (K_a) of complexes between three oridonin derivatives and two CDs are calculated according to Scott's equation. Copyright © 2011 John Wiley & Sons, Ltd.     

Improvements in lipid suppression for 1H NMR-based metabolomics: Applications to solution-state and HR-MAS NMR in natural and in vivo samples

Proton nuclear magnetic resonance (NMR) spectra of intact biological samples often show strong contributions from lipids, which overlap with signals of interest from small metabolites. Pioneering work by Diserens et al. demonstrated that the relative differences in diffusivity and relaxation of lipids versus small metabolites could be exploited to suppress lipid signals, in high-resolution magic angle spinning (HR-MAS) NMR spectroscopy. In solution-state NMR, suspended samples can exhibit very broad water signals, which are challenging to suppress. Here, improved water suppression is incorporated into the sequence, and the Carr-Purcell-Meiboom-Gill sequence (CPMG) train is replaced with a low-power adiabatic spinlock that reduces heating and spectral artefacts seen with longer CPMG filters. The result is a robust sequence that works well in both HR-MAS as well as static solution-state samples. Applications are also extended to include in vivo organisms. For solution-state NMR, samples containing significant amount of fats such as milk and hemp hearts seeds are used to demonstrate the technique. For HR-MAS, living earthworms (Eisenia fetida) and freshwater shrimp (Hyalella azteca) are used for in vivo applications. Lipid suppression techniques are essential for non-invasive NMR-based analysis of biological samples with a high-lipid content and adds to the suite of experiments advantageous for in vivo environmental metabolomics.     

Isotopic splitting patterns in the 13C NMR spectra of some partially deuterated 1‐aryl‐2‐(phenyldiazenyl)butane‐1,3‐dione and 4‐hydroxy‐3‐(phenyldiazenyl)‐2H‐chromen‐2‐one: evidence for elucidation of tautomeric forms

Nuclear magnetic resonance spectra of synthesized azo dyes derived from aniline derivatives in reaction with benzoylacetone and 4‐hydroxycoumarin were studied in both CDCl₃ and (CD₃)₂SO (two drops of D₂O were added into solutions of dyes). All dyes showed intramolecular hydrogen bonding. Dyes derived from o‐nitro aniline in the reaction with benzoylacetone, and 4‐hydroxycoumarin showed bifurcated intramolecular hydrogen bonds. The solvent‐substrate proton exchange of dyes derived from benzoylacetone and 4‐hydroxycoumarin was examined in the presence of two drops of D₂O. Among ten dye samples, two dyes derived from benzoylacetone did not show deuteration, three dyes showed partial deuteration and five dyes showed full deuteration under similar conditions. For the partially deuterated dyes the β‐isotope effect in ¹³C splitting was investigated and was used for the determination of the predominant tautomeric form. Copyright © 2016 John Wiley & Sons, Ltd.     

Solvent effect in 1H NMR spectra of 3'-hydroxy-4'-methoxy isoflavonoids from Astragalus membranaceus var. mongholicus

Four 3'-hydroxy-4'-methoxy-isoflavonoids: calycosin-7-O-beta-D-glucopyranoside (1), calycosin (2), pratensein-7-O-beta-D-glucopyranoside (3), and pratensein (4) were isolated from Astragalus membranaceus var. mongholicus, among which compound 4 was obtained from this plant for the first time. The solvent effect obscuring (1)H signal patterns of B ring of compounds 1-4 was reported to avoid mis-assignments. Previously reported NMR data of compounds 1 and 2 were corrected based on 1D and 2D NMR experiments.     

Complete 1H and 13C assignments of 8-C-beta-D-[2-O-(E)-p-coumaroyl]glucopyranosyl-2-(2-hydroxy)propyl-7-methoxy-5-methylchromone

1H and 13C NMR spectra of 8-C-beta-D-[2-O-(E)-p-coumaroyl] glucopyranosyl-2-(2-hydroxy)propyl-7-methoxy-5-methylchromone were completely assigned by 2D NMR observations. Especially the 1H assignments of the glucosyl and hydroxyl protons were achieved by utilizing HMQC, HMBC, 1H-1H COSY and DEPT techniques together with a heavy water exchange 1H NMR experiment.     

Structure determination of fexofenadine‐α‐cyclodextrin complex by quantitative 2D ROESY analysis and molecular mechanics studies

Complexation of fexofenadine with α‐cyclodextrin in aqueous medium was studied. The stoichiometry of the resulting inclusion complex was determined by ¹H NMR titration data. 2D ROESY data provided the evidence of formation of the complex by entry of the phenyl ring into the α‐cyclodextrin cavity probably from wider opening. Determination of relative peak intensities of intermolecular cross‐peaks for the most stable complexes obtained by molecular mechanics (MM2) studies and from 2D ROESY spectral data confirmed the presence of only one complex in solution that has been fully characterized. Copyright © 2012 John Wiley & Sons, Ltd.