Methyl Resonances Research Articles

Synthesis of ethyl 4-(1,2,3-thiadiazol-4-yl)-5-(bromomethyl)furan-2-carboxylate and its reactions with nucleophiles

By cyclization of carboethoxyhydrazone of ethyl 4-acetyl-5-methylfuran-2-carboxylate under the conditions of Hurd–Mori reaction ethyl 4-(1,2,3-thiadiazol-4-yl)-5-methylfuran-2-carboxylate was synthesized. The ester obtained was brominated with N-bromosuccinimide at the methyl group in the furan ring. This bromide reacts with various N-, S-, O-, and P-nucleophiles to form the corresponding substitution products. Furylthiadiazole fragment remains stable in the course of these transformations.

A Semiautomated Assignment Protocol for Methyl Group Side Chains in Large Proteins.

The developments of biosynthetic specific labeling strategies for side-chain methyl groups have allowed structural and dynamic characterization of very large proteins and protein complexes. However, the assignment of the methyl-group resonances remains an Achilles' heel for NMR, as the experiments designed to correlate side chains to the protein backbone become rather insensitive with the increase of the transverse relaxation rates. In this chapter, we outline a semiempirical approach to assign the resonances of methyl-group side chains in large proteins. This method requires a crystal structure or an NMR ensemble of conformers as an input, together with NMR data sets such as nuclear Overhauser effects (NOEs) and paramagnetic relaxation enhancements (PREs), to be implemented in a computational protocol that provides a probabilistic assignment of methyl-group resonances. As an example, we report the protocol used in our laboratory to assign the side chains of the 42-kDa catalytic subunit of the cAMP-dependent protein kinase A. Although we emphasize the labeling of isoleucine, leucine, and valine residues, this method is applicable to other methyl group side chains such as those of alanine, methionine, and threonine, as well as reductively methylated cysteine side chains.

Visibility of lipid resonances in HR-MAS spectra of brain biopsies subject to spinning rate variation

Lipid resonances from mobile lipids can be observed by 1H NMR spectroscopy in multiple tissues and have also been associated with malignancy. In order to use lipid resonances as a marker for disease, a reference standard from a healthy tissue has to be established taking the influence of variable factors like the spinning rate into account. The purpose of our study was to investigate the effect of spinning rate variation on the HR-MAS pattern of lipid resonances in non-neoplastic brain biopsies from different regions and visualize polar and non-polar lipids by fluorescence microscopy using Nile Red staining. 1H HR-MAS NMR spectroscopy demonstrated higher lipid peak intensities in normal sheep brain pure white matter biopsies compared to mixed white and gray matter biopsies and pure gray matter biopsies. High spinning rates increased the visibility particularly of the methyl resonances at 1.3 and the methylene resonance at 0.89ppm in white matter biopsies stronger compared to thalamus and brainstem biopsies, and gray matter biopsies. The absence of lipid droplets and presence of a large number of myelin sheaths observed in white matter by Nile Red fluorescence microscopy suggest that the observed lipid resonances originate from the macromolecular pool of lipid protons of the myelin sheath's plasma membranes. When using lipid contents as a marker for disease, the variable behavior of lipid resonances in different neuroanatomical regions of the brain and at variable spinning rates should be considered. The findings may open up interesting possibilities for investigating lipids in myelin sheaths.

The use of a selective saturation pulse to suppress t1 noise in two-dimensional 1H fast magic angle spinning solid-state NMR spectroscopy

A selective saturation pulse at fast magic angle spinning (MAS) frequencies (60+kHz) suppresses t1 noise in the indirect dimension of two-dimensional 1H MAS NMR spectra. The method is applied to a synthetic nucleoside with an intense methyl 1H signal due to triisopropylsilyl (TIPS) protecting groups. Enhanced performance in terms of suppressing the methyl signal while minimising the loss of signal intensity of nearby resonances of interest relies on reducing spin diffusion – this is quantified by comparing two-dimensional 1H NOESY-like spin diffusion spectra recorded at 30–70kHz MAS. For a saturation pulse centred at the methyl resonance, the effect of changing the nutation frequency at different MAS frequencies as well as the effect of changing the pulse duration is investigated. By applying a pulse of duration 30ms and nutation frequency 725Hz at 70kHz MAS, a good compromise of significant suppression of the methyl resonance combined with the signal intensity of resonances greater than 5ppm away from the methyl resonance being largely unaffected is achieved. The effectiveness of using a selective saturation pulse is demonstrated for both homonuclear 1H–1H double quantum (DQ)/single quantum (SQ) MAS and 14N–1H heteronuclear multiple quantum coherence (HMQC) two-dimensional solid-state NMR experiments.

Crystal structure of rac-(3aR,4S,5aR,6S,9R,10aS,10bR)-3a,5a,9-tri-methyl-tetra-deca-hydro-6,9-ep-oxy-cyclo-hepta-[e]inden-4-ol monohydrate.

The title hydrate, C17H28O2·H2O, was synthesized in order to determine the relative configuration of the tetra­cyclic framework. The fused 5,6,7-tricarbocyclic core exhibits an entire cis-annulation, featuring a 1,4-cis-relation of the angular methyl groups in the six-membered ring. The oxa bridge of the ep­oxy­cyclo­heptane moiety is oriented towards the concave face of the boat-shaped mol­ecule, whereas the angular methyl groups are directed towards the convex face. The asymmetric unit of the crystal contains two nearly identical formula units, which are related via a pseudo-centre of symmetry. The structure could be solved in the space groups I-4 and I41/a. The refinement in the acentric space group, however, gave significantly better results and these are used in this paper. O—H⋯O hydrogen bonds are observed between the organic mol­ecules, between the organic mol­ecules and the water mol­ecules, and between the water mol­ecules, forming a chain along the c-axis direction.

OP41 - Methylation at positon 7’ but not 5’ and the isoprenoid chain are required for vitamin E to induce P-glycoprotein protein expression and activity in the human colon carcinoma cell line LS180

The eight congeners of vitamin E differ in their methylation pattern and either possess an alkyl (tocopherols, T) or an isoprenoid (tocotrienols, T3) side chain. The different chemical structures influence the biological activity of the vitamin, like the binding to the nuclear pregnane X receptor (PXR). P-glycoprotein (P-gp), a PXR target gene, is an efflux transporter expressed at various barriers in the body including small intestine, bile ducts and the blood-brain-barrier. We therefore investigated the impact of methylation of the chromanol ring (position 5’ and 7’ = α; position 7’ = γ) and the structure of the side chain on the expression and activity of P-gp. LS180 cells, which have a low basal P-gp expression, are a good model to study the induction of P-gp protein expression and activity. LS180 cells were either treated with the positive control rifampicin (25 µmol/L), a known P-gp inducer, the appropriate solvent control, or the respective vitamin E congener for 48 hours. αT was investigated in the range of 10–100 µmol/L, γT 10-50 µmol/L, αT3 5-50 µmol/L and γT 5-25 µmol/L due to differences in cell toxicity. Protein expression was determined by Western blot analysis and P-gp activity via the efflux of the fluorescent substrate rhodamine 123 in the presence or absence of the P-gp specific inhibitor elacridar. αT, γT and αT3 neither influenced the protein expression nor the activity of P-gp. γT3 induced P-glycoprotein activity and protein expression which is in accordance with the literature. To induce P-glycoprotein, both methylation at position 7’ but not 5’ of the chromanol ring as well as an isoprenoid side chain are required as none of the two structural features individually showed an effect.

Potassium and Mixed Lithium/Potassium Complexes of Deprotonated 1,2‐Bis(neopentylamino)benzene

AbstractDeprotonation of 1,2‐bis(neopentylamino)benzene (1) with an excess of KH in tetrahydrofuran (thf) yields the singly metalated derivative [(thf)K{C6H4‐1‐(NH‐CH2tBu)‐2‐(N‐CH2tBu)}] (2a). Performance of the metalation reaction in tetrahydropyran (thp) leads to formation of [(thp)2K{C6H4‐1‐(NH‐CH2tBu)‐2‐(N‐CH2tBu)}] (2b). The second amino group of 2a can be lithiated by butyllithium yielding heterobimetallic [(thf)2LiK{C6H4‐1,2‐(N‐CH2tBu)2}] (3). The structures demonstrate that ethers as well as aromatic π systems represent suitable bases for the rather soft potassium ions. Intermolecular potassium π interactions lead to the formation of strands in the crystalline state. For complex 2a, a dynamic exchange process of the N‐bound hydrogen atom with an energy barrier of 45.6 kJ·mol–1 is observed in [D8]THF solution. Heterobimetallic 3 also shows a temperature‐dependent dynamic behavior that gives rise to splitting of the methylene and aromatic resonances suggesting overlying metal exchange and aggregation equilibria.

Methyl resonances of ubiquitin S65E in complex with R0RBR domain (141-465) of parkin

Methyl resonances of ubiquitin S65E in complex with R0RBR domain (141-465) of parkin

Heterospin complex showing spin transition at room temperature

New nitronyl nitroxides LMe and LMe-CP containing a 4-methylpyridin-3-yl substituent were synthesized. It was found that the interaction of Сu(hfac)2 with LMe and LMe-CP gave binuclear [Cu(hfac)2LMe]2 and [Cu(hfac)2LMe-CP]2·Solv (Solv=n-C6H14, n-C10H22, n-C16H34) and chain polymer {[[Cu(hfac)2]2LMe2][Cu(hfac)2]}∞ heterospin complexes. An important structural peculiarity of LMe and LMe-CP is a large dihedral angle between the planes of the O–N−CN→O paramagnetic fragment and the pyridine ring: 55.2 and 56.1°, respectively. The presence of a methyl group in the pyridine ring of the nitroxide molecule in {[[Cu(hfac)2]2LMe2][Cu(hfac)2]}∞ proved favorable for spin transition at nearly room temperature.

In vivo proton magnetic resonance spectroscopy of liver metabolites in non-alcoholic fatty liver disease in rats: T2 relaxation times in methylene protons

The aim of this study was to evaluate the transverse relaxation time of methylene resonance as compared to other lipid resonances. The examinations were performed using a 3.0 T scanner with a point-resolved spectroscopy (PRESS) sequence. Lipid relaxation time in a lipid phantom filled with canola oil was estimated with a repetition time (TR) of 6000ms and echo time (TE) of 40–550ms. For in vivo proton magnetic resonance spectroscopy (1H-MRS), eight male Sprague–Dawley rats were given free access to a normal-chow (NC) and another eight male Sprague–Dawley rats were given free access to a high-fat (HF) diet. Both groups drank water ad libitum. T2 measurements in the rats’ livers were conducted at a fixed TR of 6000ms and TE of 40–220ms. Exponential curve fitting quality was calculated through the coefficients of determination (R2). Chemical analyses of the phantom and livers were not performed, but T2 decay curves were acquired. The T2 relaxation time of methylene resonance was estimated as follows: NC rats, 37.1±4.3ms; HF rats, 31.4±1.8ms (p<0.05). The extrapolated M0 values were higher in HF rats than in NC rats (p<0.005). This study of 1H MRS led to sufficient spectral resolution and signal-to-noise ratio differences to characterize the T2 relaxation times of methylene resonance. 1H MRS relaxation times may be useful for quantitative characterization of various liver diseases, including fatty liver disease.

Conformational and structural studies of N-methylacetohydroxamic acid and of its mono- and bis-chelated uranium(VI) complexes

The thermodynamics and kinetics of the cis/trans isomerism of N-methylacetohydroxamic acid (NMAH) and its conjugated base (NMA−) have been reinvestigated in aqueous media by 1H NMR spectroscopy. Hindered rotation around the central C–N bond due to electronic delocalization becomes slow enough on the NMR time scale to observe both rotamers in equilibrium in D2O at room temperature. By properly assigning the methyl group resonances, evidence for the prevalence of the E over the Z form is unambiguously provided [K300=[E]/[Z]=2.86(2) and 9.63(5) for NMAH and NMA−, respectively], closing thereby a long-lasting dispute about the most stable conformer. To that end, calculations of the chemical shifts by density functional theory (DFT), which accurately reproduced the experimental data, turned out to be a much more reliable method than the direct computation of the relative energy for each conformer. The Z⇌E interconversion dynamics was probed at 300K in D2O by 2D exchange-correlated spectroscopy (EXSY), affording the associated rate constants [kZE=9.0(2) s−1 and kEZ=3.14(5) s−1 for NMAH, kZE=0.96(3) s−1 and kEZ=0.10(2)s−1 for NMA−] and activation barriers at 300K [ΔG≠ZE=68.0kJmol−1 and ΔG≠EZ=70.6kJmol−1 for NMAH, ΔG≠ZE=73.6kJmol−1 and ΔG≠EZ=79.2kJmol−1 for NMA−]. For the first time, mono- and bis-chelated uranium(VI) complexes of NMA− have been isolated. Crystals of [UO2(NMA)(NO3)(H2O)2] and [UO2(NMA)2(H2O)] have been characterized by X-ray diffractometry, infrared and Raman spectroscopies.

Synthesis of (11)C-Labeled Thiamine and Fursultiamine for in Vivo Molecular Imaging of Vitamin B1 and Its Prodrug Using Positron Emission Tomography.

To enable in vivo analysis of the kinetics of vitamin B1 (thiamine) and its derivatives by positron emission tomography (PET), (11)C-labeled thiamine ([(11)C]-1) has been synthesized. This was carried out via a rapid, multistep synthesis consisting of Pd(0)-mediated C-[(11)C]methylation of a thiazole ring for 3 min and benzylation with 5-(bromomethyl)pyrimidine for 7 min. The [(11)C]-1 was also converted to (11)C-labeled fursultiamine ([(11)C]-2), a prodrug of vitamin B1, by disulfide formation with S-tetrahydrofurfurylthiosulfuric acid sodium salt. Characterization of [(11)C]-1 and [(11)C]-2 showed them to be suitable for use as PET probes for in vivo pharmacokinetic and medical studies. The total durations of the preparations of [(11)C]-1 and [(11)C]-2 were shorter than 60 and 70 min, respectively. The [(11)C]CH3I-based decay-corrected radiochemical yields of [(11)C]-1 and [(11)C]-2 were 9-16% and 4-10%, respectively. The radioactivities of the final injectable solutions of [(11)C]-1 and [(11)C]-2 were 400-700 and 100-250 MBq, respectively. The radiochemical purity of both [(11)C]-1 and [(11)C]-2 was 99%, and the chemical purities of [(11)C]-1 and [(11)C]-2 were 99% and 97-99%, respectively. In vivo PET imaging of normal rats was illustrated by the distribution of [(11)C]-1 and [(11)C]-2 following intravenous injection.

SU-E-I-64: Transverse Relaxation Time in Methylene Protons of Non-Alcoholic Fatty Liver Disease Rats

Purpose: The aim of this study was to evaluate transverse relaxation time of methylene resonance compared to other lipid resonances. Methods: The examinations were performed using a 3.0 T scanner with a point — resolved spectroscopy (PRESS) sequence. Lipid relaxation time in a lipid phantom filled with canola oil was estimated considering repetition time (TR) as 6000 msec and echo time (TE) as 40 — 550 msec. For in vivo proton magnetic resonance spectroscopy ({sup 1}H — MRS), eight male Sprague — Dawley rats were given free access to a normal - chow (NC) and eight other male Sprague-Dawley rats were given free access to a high — fat (HF) diet. Both groups drank water ad libitum. T{sub 2} measurements in the rats’ livers were conducted at a fixed TR of 6000 msec and TE of 40 – 220 msec. Exponential curve fitting quality was calculated through the coefficients of determination (R{sup 2}). Results: A chemical analysis of phantom and liver was not performed but a T{sub 2} decay curve was acquired. The T{sub 2} relaxation time of methylene resonance was estimated as follows: NC rats, 37.07 ± 4.32 msec; HF rats, 31.43 ± 1.81 msec (p < 0.05). Themore » extrapolated M0 values were higher in HF rats than in NC rats (p < 0.005). Conclusion: This study of {sup 1}H-MRS led to sufficient spectral resolution and signal — to — noise ratio differences to characterize all observable resonances for yielding T{sub 2} relaxation times of methylene resonance. {sup 1}H — MRS relaxation times may be useful for quantitative characterization of various liver diseases, including fatty liver disease. This study was supported by grant (2012-007883 and 2014R1A2A1A10050270) from the Mid-career Researcher Program through the NRF funded by Ministry of Science. In addition, this study was supported by the Industrial R&D of MOTIE/KEIT (10048997, Development of the core technology for integrated therapy devices based on real-time MRI-guided tumor tracking)« less

A fully automatic reference deconvolution strategy to increase the accuracy of in vivo lipid signal quantification.

Lipid signals measured by (1)H MR spectroscopy cannot be adequately quantified by common fitting routines like VARPRO or AMARES, if lipid spectra are distorted by irregular spatial and temporal inhomogeneities of the static magnetic field during readout. A fully automatic reference deconvolution algorithm is presented that eliminates these distortions before application of fitting routines. The measured signal of the dominant methyl resonance is isolated with aid of a spectral estimator (estimation of parameters via rotational invariance techniques) and used as reference signal for estimation of distortions. A Wiener filter is applied to deconvolve those distortions in the lipid spectrum. Performance of the algorithm is assessed for different bandwidths and shapes of distortions, using artificially distorted as well as measured data. Application of the fully automatic reference deconvolution algorithm on simulated spectra yields a distinct increase in quantification accuracy. Deconvolved in vivo spectra of subcutaneous fat indicate reduced spectral overlap after application of the proposed strategy. The proposed method is helpful for in vivo magnetic resonance spectroscopy of adipose tissue to correct for effects of field inhomogeneities within the voxel and for inevitable eddy current effects. Quantification accuracy is improved by eliminating distortions before application of fitting routines.

Assignment of methyl NMR resonances of a 52 kDa protein with residue-specific 4D correlation maps

Methyl groups have become key probes for structural and functional studies by nuclear magnetic resonance. However, their NMR signals cluster in a small spectral region and assigning their resonances can be a tedious process. Here, we present a method that facilitates assignment of methyl resonances from assigned amide groups. Calculating the covariance between sensitive methyl and amide 3D spectra, each providing correlations to C(α) and C(β) separately, produces 4D correlation maps directly correlating methyl groups to amide groups. Optimal correlation maps are obtained by extracting residue-specific regions, applying derivative to the dimensions subject to covariance, and multiplying 4D maps stemming from different 3D spectra. The latter procedure rescues weak signals that may be missed in traditional assignment procedures. Using these covariance correlation maps, nearly all assigned isoleucine, leucine, and valine amide resonances of a 52 kDa nonribosomal peptide synthetase cyclization domain were paired with their corresponding methyl groups.

O-tert-Butyltyrosine, an NMR tag for high-molecular-weight systems and measurements of submicromolar ligand binding affinities.

O-tert-Butyltyrosine (Tby) is an unnatural amino acid that can be site-specifically incorporated into proteins using established orthogonal aminoacyl-tRNA synthetase/tRNA systems. Here we show that the tert-butyl group presents an outstanding NMR tag that can readily be observed in one-dimensional (1)H NMR spectra without any isotope labeling. Owing to rapid bond rotations and the chemical equivalence of the protons of a solvent-exposed tert-butyl group from Tby, the singlet resonance from the tert-butyl group generates an easily detectable narrow signal in a spectral region with limited overlap with other methyl resonances. The potential of the tert-butyl (1)H NMR signal in protein research is illustrated by the observation and assignment of two resonances in the Bacillus stearothermophilus DnaB hexamer (320 kDa), demonstrating that this protein preferentially assumes a 3-fold rather than 6-fold symmetry in solution, and by the quantitative measurement of the submicromolar dissociation constant Kd (0.2 μM) of the complex between glutamate and the Escherichia coli aspartate/glutamate binding protein (DEBP, 32 kDa). The outstanding signal height of the (1)H NMR signal of the Tby tert-butyl group allows Kd measurements using less concentrated protein solutions than usual, providing access to Kd values 1 order of magnitude lower than established NMR methods that employ direct protein detection for Kd measurements.

Simultaneous observation of NMR spin noise and maser spontaneous emission

Spin noise emission, absorbed noise, and spontaneous maser emission are distinguished in the hexanol molecule by changing spin noise dynamics along with coupling the spin system to a probe circuit. Inverted magnetization leads to spontaneous maser emission of methyl and methylene resonances permitting separation from broad overlapped peaks. The maser emission resonance shows random intensity the scattering range of which depends on the length of the gradient following magnetization inversion. Combination of thermal noise and spin noise can be altered by varying magnetization orientation. This results in radiation damping which alters how fluctuation signals are coupled with the probe circuit.

Synthesis of 2- and 3-acetylfuran carbethoxyhydrazones and investigation of their reaction with thionyl chloride

By the reaction of 2- and 3-acetylfurans with ethoxycarbonyl hydrazine in presence of acetic acid a series of carboethoxyhydrazones was synthesized. On the basis of the 1H NMR spectra it was shown that in the compounds obtained furyl substituent and ethoxycarbonyl group might be located both in Z- and in E-position with respect to the C=N bond. The isomer ratio for various types of substitution in the furan ring was established. It was shown that the introduction of two methyl groups in the positions adjacent to the hydrazone group leads to thermal lability of the corresponding hydrazone. 2-Acetyl-, 5-methyl-2-acetyl-, and 3-acetylfuran carbethoxyhydrazones under the action of thionyl chloride undergo cyclization to 4-furyl-1,2,3-thiadiazoles. In the case of 3-methyl-5-acetyl- and 2-methyl-3-acetylfuran a mixture of corresponding thiadiazoles and the products of chlorination of carbethoxyhyrazone fragment was formed. It was shown that the introduction of methyl group in the furan ring leads to thermal lability of furylthiadiazoles.

Antioxidant activities of tocopherols/tocotrienols and lipophilic antioxidant capacity of wheat, vegetable oils, milk and milk cream by using photochemiluminescence

The purpose of this study was to measure the antioxidant activity (AOA) of tocopherols and tocotrienols by using photochemiluminescence (PCL). This method enables to detect total lipophilic antioxidants. The AOA of all vitamin E isomers depended on number and position of methyl groups in the chroman ring. Correlation between the AOA and the redox potential and the biological activity of the tocochromanols was observed. The second aim was to analyse different kinds of wheat, vegetable oils, milk and milk cream on their antioxidant capacity (AOC) by using PCL and α-TEAC. The contents of vitamin E and carotenoids were analysed by HPLC. Correlations between the sum of carotenoids and vitamin E and the AOC were detected. Based on high vitamin E contents, the oils had the highest and in contrast, the product macaroni showed the lowest AOC. A concentration-dependent effect was observed in both assays, PCL and α-TEAC.

Mechanistic Elucidation of the 2‐Norbornyl to 1,3‐Dimethylcyclopentenyl Cation Isomerization

AbstractExtremely complicated isomerization mechanisms for the isomerization of the 2‐norbornyl to 1,3‐dimethylcyclopentenyl cation observed in the gas phase have been elucidated through Born–Oppenheimer molecular dynamics simulations and ab initio computations. These quantum chemical explorations revealed that there are two viable complex rearrangement pathways. The longer pathway involves consecutive ring‐openings with the formation of acyclic allylic cation intermediates. Although these are avoided in the shorter pathway, both routes are feasible, because the energy barriers of their first steps differ by only 1.0 kcal/mol. Both pathways eventually converge to the same methylcyclohexenyl cation, which undergoes ring contraction and methyl and hydride shifts to yield the C7H11+ global minimum, namely the 1,3‐dimethylcyclopentenyl cation.