Heteronuclear Multiple Quantum Research Articles

New bioactive C15 acetogenins from the red alga Laurencia obtusa

Background and Objective: With regard to the uniqueness of the red algae of the genus Laurencia as the source of C15-acetogenins, along with the diversity of biological applications; the acetogenin content of the Red Sea Laurencia obtusa was investigated. Materials and Methods: Fractionation and purification of the CH2Cl2/MeOH extract were carried out by applying several chromatographic techniques, including column and preparative thin-layer chromatography; followed by a series of 1H nuclear magnetic resonance measurements to give rise of some interesting notes. Toxicity to Artemia salina was evaluated. The apoptosis induced by these two compounds was demonstrated by DNA fragmentation assay and microscopic observation. Results: A new rare chloroallene-based C15 acetogenin, laurentusenin (1) along with a new furan ring containing C15 acetogenin, laurenfuresenin (2), were isolated from the red alga L. obtusa. Comparing 1D and 2D NMR, MS, ultraviolet and infrared radiation spectral data for the newly isolated compounds with the reported bromoallene containing acetogenins spectral data was played the crucial role for characterization of their chemical structures. 1 and 2 exhibited bare toxicity (LD50 >12 mM) in test organism, A. salina and induced apoptotic death confirmed by DNA fragmentation and microscopic investigations. Conclusion: The isolated metabolite 1 showed unusual substituted allene side chain, while 2 inserted furan ring as a new acetogenin nucleus. Both compounds may play a role in apoptosis induction and initiation and propagation of inflammatory responses. Abbreviations used: NMR: Nuclear magnetic resonance; MS: Mass spectrometry; UV: Ultraviolet spectroscopy; IR: infrared radiation; EIMS: Electron ionization mass spectra; TLC: Thin-layer chromatography; PPP: Platelet poor plasma; PRPDS: platelet-rich plasma derived serum. DEPT: Destortionless Enhancement by Polarization Transfer; NOESY: Nuclear Overhauser Effect Spectroscopy; HSQC: Heteronuclear Single Quantum Coherence; HMBC: Heteronuclear Multiple-quantum Correlation; 1H-1H COSY: Correlation Spectroscopy.

Reducing t1 noise through rapid scanning.

The so-called t1 noise, which arises due to random instabilities in the spectrometer hardware, remains the primary source of noise that limits the sensitivity of most 2D NMR experiments, particularly in the expanding group of solid-state NMR methods that utilize dipolar-recoupling. In this communication we revisit the relationship between the signal intensity and the t1 noise produced. It is shown that since the latter scales linearly with the signal strength, the use of a conventional relaxation delay of 1.3T1 may prove far from optimal. In cases where the fluctuations occur on a shorter timescale than the recycle delay, a considerably faster repetition rate should be used to maximize the time sensitivity in a 2D experiment than what is used to maximize the sensitivity in 1D. This is demonstrated with the acquisition of 1H{13C} Dipolar-mediated Heteronuclear Multiple-Quantum Correlation (D-HMQC) type spectra in which the sensitivity could be nearly doubled by choosing a very short relaxation delay corresponding to 0.2T1.

Observation of proximities between spin-1/2 and quadrupolar nuclei in solids: Improved robustness to chemical shielding using adiabatic symmetry-based recoupling

We introduce a novel heteronuclear dipolar recoupling based on the R21−1 symmetry, which uses the tanh/tan (tt) shaped pulse as a basic inversion element and is denoted R21−1(tt). Using first-order average Hamiltonian theory, we show that this sequence is non-γ-encoded and that it reintroduces the |m| = 1 spatial component of the Chemical Shift Anisotropy (CSA) of the irradiated isotope and its heteronuclear dipolar interactions. Using numerical simulations and one-dimensional (1D) 27Al-{31P} through-space D-HMQC (Dipolar Heteronuclear Multiple-Quantum Correlation) experiments on VPI-5, we compare the performances of this recoupling to those of other non-γ-encoded |m| = 1 heteronuclear recoupling schemes: REDOR (Rotational-Echo DOuble Resonance), SFAM (Simultaneous Frequency and Amplitude Modulation) and R42−1(tt). Such comparison indicates that the R21−1(tt) scheme is more robust to CSA, offset and radiofrequency field inhomogeneities than the other schemes. We take advantage of the high robustness of R21−1(tt) to CSA and offset to demonstrate the possibility to correlate the signals of 207Pb isotope with those of neighboring half-integer spin quadrupolar nuclei. Such approach is demonstrated experimentally by acquiring 11B-{207Pb} D-HMQC 2D spectra of Pb4O(BO3)2 crystalline powder.

Different roles of phosphorus in the nucleation of lithium aluminosilicate glasses

Glasses in the Li2O–Al2O3–SiO2 (LAS) system with different Al2O3Li2O ratio and with or without addition of P2O5 have been synthesized and characterized by thermal analysis. The glass structure and the phosphorus environments in the glasses and their evolutions with annealing have been studied by 31P Solid-State Nuclear Magnetic Resonance (NMR) and 27Al{31P} D-HMQC (Dipolar Hetero-nuclear Multiple-Quantum Coherences) NMR correlation technique. The crystallization behavior has been studied by X-ray diffraction, NMR and Raman spectroscopy. The results showed a different role for P2O5 depending on the Al2O3Li2O ratio. For low alumina content, P2O5 plays a nucleating role while it acts as crystallization inhibitor for high alumina content. These different roles are related to different local environments of the phosphorus in the as-cast glasses. Our study emphasizes the importance of POAl complexes in the crystallization of aluminosilicate glasses.

Heteronuclear Correlation Experiments Such as Heteronuclear Single-Quantum Correlation Spectroscopy (HSQC), Heteronuclear Multiple-Quantum Correlation Spectroscopy (HMQC) and Heteronuclear Multiple-Bond Correlation Spectroscopy (HMBC) Comparative Study On Malignant and Benign Human Endocrinology and Thyroid Cancer Cells and Tissues Under Synchrotron Radiation

Heteronuclear Correlation Experiments Such as Heteronuclear Single-Quantum Correlation Spectroscopy (HSQC), Heteronuclear Multiple-Quantum Correlation Spectroscopy (HMQC) and Heteronuclear Multiple-Bond Correlation Spectroscopy (HMBC) Comparative Study On Malignant and Benign Human Endocrinology and Thyroid Cancer Cells and Tissues Under Synchrotron Radiation

Isolation, elucidation, and molecular docking studies of active compounds from Phyllanthus niruri with angiotensin-converting enzyme inhibition

Background: Phyllanthus niruri, in Indonesia, is known as “Meniran” has a long history of use in ethnic or traditional medicine worldwide, mainly as an antihypertensive agent. Objective: The present study was designed to isolate and identify active compounds with angiotensin-converting enzyme (ACE) inhibition activity from P. niruri herb and confirm the mechanism of action, affinity, and domain specificity interactions of the isolated compounds. Materials and Methods: Some fractions of P. niruri methanolic extract were subjected to column chromatography and preparative thin-layer chromatography to get active compounds. Structural elucidation was determined via spectroscopic methods. ACE inhibition activity was measured using hippuryl-L-histidyl-L-leucine as a substrate in vitro assay. Furthermore, confirmation of the mechanism of action, affinity, and domain specificity interaction of the isolated compounds on ACE complex macromolecule (protein database id: 1O86) was performed by in silico molecular docking studies. Results: In this work, four active compounds were isolated from aerial part of P. niruri, including hypophyllantin (50% inhibition concentration [IC50] = 0.180 μg/mL), phyllantin (IC50 = 0.140 μg/mL), methyl gallate (IC50 = 0.015 μg/mL), and quercetin 3-O-β-D-glucopyranosyl-(1'''-6'')-α-rhamnoside (IC50 = 0.086 μg/mL). In silico molecular docking method emphasizes ligand-residue interactions, thereby predicting the inhibitory activity of these compounds. After docking to an ACE complex macromolecule, quercetin 3-O-β-D-glucopyranosyl-(1'''-6'')-α-rhamnoside obtained more interactions than lisinopril. Conclusion: The results were obtained from in silico and in vitro experiments and confirm the potential active compound is an ACE inhibitor and a new antihypertensive agent. Abbreviations Used: P. niruri: Phyllanthus niruri; ACE: Angiotensin-converting enzyme; HHL: Hippuryl-L-histidyl-L-leucine; HA: Hippuric acid; PDB: Protein database; IC50: 50% inhibition concentration; FH: N-hexane fraction; FE: Ethyl acetate fraction; TLC: Thin layer chromatography; UV-VIS: Ultraviolet-visible; NMR: nuclear magnetic resonance; FTIR: Fourier–Transform infrared; MS: Mass spectrometry; HMQC: Heteronuclear Multiple-Quantum Correlation; HMBC: Heteronuclear multiple bond correlation; TADOK: Tugas Akhir Mahasiswa Doktor.

Scopoletin – an anti-hyperglycemic Coumarin from the fruit of <em>Averrhoa carambola</em> L. (Star fruit)

Purpose: Averrhoa carambola L. has a long history of its use in traditional medicine for the treatment of various diseases such as diabetes, intermittent fevers, intestinal worms, scabies and inflammatory conditions. The fresh fruit of A. carambola is very popular in Sri Lanka for its glucose-lowering effect and is taken as good remedy for diabetes. The present study was carried out to isolate and characterize anti-hyperglycemic principles of the fruit of A. carambola to obtain additional scientific evidence for its usage in traditional medicine in the management of diabetes. Methods: The dried and powdered fruits of A. carambola were successively extracted with n-hexane and CH2Cl2 using soxhlet apparatus. The CH2Cl2 extract was subjected to silica gel column chromatography, eluting in a stepwise gradient with hexane and ethyl acetate (EtOAc) mixtures. The fraction eluted with hexane:EtOAc (60:40) yielded a mixture which with preparative thin-layer chromatography afforded a pure compound designated as JW-AC-3. The structure of JW-AC-3 was elucidated on the basis of its Ultra- violet spectroscopy (UV), Infra-Red spectroscopy (IR), Mass Spectroscopy (MS), and Nuclear Magnetic Resonance spectroscopy (NMR) including Distortionless Enhancement Polarization Transfer (DEPT), Homonuclear Shift-Corrilation Spectroscopy (COSY), Nuclear Overhauser Spectroscopy (NOESY), Hetero Multiple Bond Connectivities (HMBC) and Heteronuclear Multiple-Quantum Coherence (HMQC) experiments and direct comparison with reported data. Results: JW-AC-3 was identified as scopoletin by direct comparison of its spectral data with reported data of scopoletin. To our knowledge, scopoletin has not been reported from A. carambola. Scopoletin has been shown to possess significant anti-hyperglycemic activity by previous workers. The results of this study clearly indicate that the majority of the anti-hyperglycemic activity of the fruits of A. carambola could be attributed to scopoletin. The results also provide for the use of scopoletin as a bio-marker in the standardization and quality control of products derived from the fruits of A. carambola . Conclusion: The results of this study demonstrate potential use of scopoletin in the development of drugs of natural origin in the treatment of diabetes. The results could also be used in the rationalization of ethnomedical use of the fruits of A. carambola .

71Ga-77Se connectivities and proximities in gallium selenide crystal and glass probed by solid-state NMR

We introduce two-dimensional (2D) 71Ga-77Se through-bond and through-space correlation experiments. Such correlations are achieved using (i) the J-mediated Refocused Insensitive Nuclei Enhanced by Polarization Transfer (J-RINEPT) method with 71Ga excitation and 77Se Carr-Purcell-Meiboon-Gill (CPMG) detection, as well as (ii) the J- or dipolar-mediated Hetero-nuclear Multiple-Quantum Correlation (J- or D-HMQC) schemes with 71Ga excitation and quadrupolar CPMG (QCPMG) detection. These methods are applied to the crystalline β-Ga2Se3 and the 0.2Ga2Se3-0.8GeSe2 glass. Such glass leads to a homogeneous and reproducible glass–ceramic, which is a good alternative to single-crystalline Ge and polycrystalline ZnSe materials for making lenses transparent in the IR range for thermal imaging applications. We show that 2D 71Ga-77Se correlation experiments allow resolving the 77Se signals of molecular units, which are not resolved in the 1D 77Se CPMG spectrum. Additionally, the build-up curves of the J-RINEPT and the J-HMQC experiments allow the estimate of the 71Ga-77Se J-couplings via one and three-bonds in the three-dimensional network of β-Ga2Se3. Furthermore, these build-up curves show that the one-bond 1J71Ga-77Se couplings in the 0.2Ga2Se3-0.8GeSe2 glass are similar to those measured for β-Ga2Se3. We also report 2D 71Ga Satellite Transition Magic-Angle Spinning (STMAS) spectrum of β-Ga2Se3 using QCPMG detection at high magnetic field and high Magic-Angle Spinning frequency using large radio frequency field. Such spectrum allows separating the signal of β-Ga2Se3 and that of an impurity.

γ-Independent through-space hetero-nuclear correlation between spin-1/2 and quadrupolar nuclei in solids

We introduce novel sequences using indirect detection to correlate quadrupolar nuclei and spin-1/2 isotopes, other than 1H and 19F. These sequences use γ-encoded symmetry-based RNnν schemes that reintroduce the space component |m| = 1 of the heteronuclear dipolar coupling. These schemes can be applied to the indirectly detected spin in Dipolar-mediated Heteronuclear Multiple-Quantum Correlation (D-HMQC) sequence or to the detected isotope in a novel sequence, named Dipolar-mediated Heteronuclear Universal-Quantum Correlation (D-HUQC). We show that the signal of these sequences using γ-encoded recoupling does not depend on the γ Euler angle relating the inter-nuclear vector between the coupled spins to the MAS rotor-fixed frame. Therefore, the transfer efficiency of these sequences is in principle higher than that of D-HMQC methods using non-γ-encoded recoupling. Furthermore, numerical simulations show that the heteronuclear correlation experiments employing γ-encoded recoupling are more robust to Chemical Shift Anisotropy (CSA) of the irradiated spin and MAS frequency fluctuations. These results are confirmed by 13C-{15N} heteronuclear correlation on glycine and 31P-27Al ones on VPI-5 and Na7(AlP2O7)4PO4. These experiments indicate that R1635 recoupling produces the highest signal-to-noise ratio in heteronuclear correlation 2D experiments when the detected spin-1/2 nuclei are subject to large CSA.

Fast Magic-Angle Spinning Three-Dimensional NMR Experiment for Simultaneously Probing H-H and N-H Proximities in Solids.

A fast magic-angle spinning (MAS, 70 kHz) solid-state NMR experiment is presented that combines 1H Double-Quantum (DQ) and 14N-1H HMQC (Heteronuclear Multiple-Quantum Coherence) pulse-sequence elements, so as to simultaneously probe H-H and N-H proximities in molecular solids. The proposed experiment can be employed in both two-dimensional (2D) and three-dimensional (3D) versions: first, a 2D 14N HMQC-filtered 1H-DQ experiment provides specific DQ-SQ correlation peaks for proton pairs that are in close proximities to the nitrogen sites, thereby achieving spectral filtration. Second, a proton-detected three-dimensional (3D) 1H(DQ)-14N(SQ)-1H(SQ) experiment correlates 1H(DQ)-1H(SQ) chemical shifts with 14N shifts such that longer range N···H-H correlations are observed between protons and nitrogen atoms with internuclear NH distances exceeding 3 Å. Both 2D and 3D versions of the proposed experiment are demonstrated for an amino acid hydrochloride salt, l-histidine·HCl·H2O, and a DNA nucleoside, guanosine·2H2O. In the latter case, the achieved spectral filtration ensures that DQ cross peaks are only observed for guanine NH and CH8 1H resonances and not ribose and water 1H resonances, thus providing insight into the changes in the solid-state structure of this hydrate that occur over time; significant changes are observed in the NH and NH21H chemical shifts as compared to the freshly recrystallized sample previously studied by Reddy et al., Cryst. Growth Des. 2015, 15, 5945.

Logic Structure Determination (LSD) as a Computer Assisted Structure Elucidation (Case) for Molecular Structure Determination of Cytotoxic Cembranoids from Soft Coral

The tropical Indonesian soft corals are a valuable resources that produce pharmacological cytotoxic cembranoids. However, manual structure determination into these compounds requires adequate knowledge of organic chemistry. This study presents the application of Logic Structure Determination (LSD), as a freeware Computer Assisted Structure Elucidation (CASE) for automatic molecular structure determination of cembranoid compounds from soft corals species. 12 Nuclear Magnetic Resonance (NMR) dataset of cytotoxic cembranoids were used to evaluate the accuracy of LSD in generating the possible structures. Results of this study shows LSD generated numerous possible molecular structures, as the data input file derived only from 2D-NMR HMQC (Heteronuclear Multiple-Quantum Correlation), COSY (Homonuclear Correlation Spectroscopy), and HMBC (Heteronuclear Multiple Bond Correlation). The accuracy significantly enhanced (only 2-4 possible cembranoid structures from each NMR dataset) with addition of H2BC (Heteronuclear 2 Bond Correlation) experimental data. This may indicates that VLRC (Very Long-Range Correlations) affects significantly to LSD capability. Furthermore, LSD with direct 2 bond NMR experimental data is a reliable CASE technique for identification of cembranoid compounds. In general, this freeware-CASE is potential to be applied to other type of small molecule compounds and may serves as a potential solution for elucidation bottleneck step in Indonesian natural products studies.

The Heteronuclear Multiple-Quantum Correlation Experiment: Perspective from Classical Vectors, Nonclassical Vectors, and Product Operators

It is usually accepted that most 2D-NMR experiments cannot be approached using classical models. Instructors argue that Product Operators (PO) or density matrix formalisms are the only alternative to get insights into complex spin evolution for experiments involving Multiple-Quantum Coherence, such as the Heteronuclear Multiple-Quantum Correlation (HMQC) technique. Nevertheless, in recent years, several contributions have been published to provide vectorial descriptions for the HMQC taking PO formalism as the starting point. In this work we provide a graphical representation of the HMQC experiment, taking the basic elements of Bloch’s vector model as building blocks. This description bears an intuitive and comfortable understanding of spin evolution during the pulse sequence, for those who are novice in 2D-NMR. Finally, this classical vectorial depiction is tested against the PO formalism and nonclassical vectors, conveying the didactic advantage of shedding light on a single phenomenon from different perspectives. This comparative approach could be useful to introduce PO and nonclassical vectors for advanced upper-division undergraduate and graduate education.

Ursolic Acid, a Natural Pentacylcic Triterpene from Ochrosia elliptica and Its Role in The Management of Certain Neglected Tropical Diseases.

Background:Leishmaniasis and African trypanosomiasis are recognized as the leading causes of mortality and morbidity with the greatest prevalence in the developing countries. They affect more than one billion of the poorest people on the globe.Objective:To find a cheap, affordable, safe, and efficacious antileshmanial and antitrypanosomal natural drug and to elucidate its probable mode of action.Materials and Methods:Phytochemical investigation of the non-polar fraction of the methanol extract of leaves of Ochrosia elliptica Labill. (Apocyanaceae) resulted in the isolation of ursolic acid, which was unambiguously determined based on HR-ESI-FTMS, extensive 1D and 2D NMR spectroscopy. It was further tested for its cytotoxicity, antimicrobial, antimalarial, antileishmanial, and trypanocidal potency. in-silico molecular modeling studies were conducted on six vital parasitic enzymes including farnesyl diphosphate synthase, N-myristoyl transferase, pteridine reductase 1, trypanothione reductase, methionyl-tRNA synthetase, and inosine–adenosine–guanosine nucleoside hydrolase to discover its potential mode of action as antitrypanosomal and antileishmanial agent.Results:Ursolic acid displayed considerable antitrypanosomal and antileishmanial activities with IC50 values ranging between 1.53 and 8.79 μg/mL. It showed superior antitrypanosomal activity as compared to the standard drug difluoromethylornithine (DFMO), with higher binding affinities towards trypanothione reductase and pteridine reductase 1. It displayed free binding energy of -30.73 and -50.08 kcal/mole towards the previously mentioned enzymes, respectively. In addition, ursolic acid exhibited considerable affinities to farnesyl diphosphate synthase, N-myristoyl transferase and methionyl-tRNA synthetase with free binding energies ranging from -42.54 to -63.93 kcal/mole.Conclusion:Ursolic acid offers a safe, effective and cheap antitrypanosomal and antileishmanial candidate acting on several key parasitic enzymes.SUMMARY The fresh leaves of Ochrosia elleptica Labill., family Apocyanaceae are a reliable source of ursolic acid.Ursolic acid displayed considerable antitrypanosomal and antileishmanial activities. It showed superior antitrypanosomal activity as compared to difluoromethylornithine (DFMO), potent antitrypanosomal reference drug.In silico molecular modeling studies revealed that the antileishmanial and antitrypanosomal activities of ursolic acid could be partially explained in view of its multiple inhibitory effects on vital parasitic enzymes with the highest potency exerted in the inhibition of pteridine reductase 1 and trypanothione reductase. Abbreviations used: AHT: African Human Trypanosomiasis, ATCC: American type cell culture, BuOH: n-butanol, DCM: dichloromethane, DFMO: difluoromethylornithine, EtOAc: ethyl acetate, FCS: fetal calf serum, HMBC: Heteronuclear Multiple Bond Correlation, HMQC: Heteronuclear Multiple-Quantum Correlation, HR-ESI-FTMS: High Resolution Electrospray ionozation Mass Spectrometry, MENA: Middle East and North Africa, MeOH: Methanol, MRSA: Methicillin-resistant Staphylococcus aureus, NTDs: Neglected tropical diseases, TLC: Thin layer chromatography, UA: Ursolic acid, UV: Ultra violet, WHO: World Health Organization.

Solid-state NMR indirect detection of nuclei experiencing large anisotropic interactions using spinning sideband-selective pulses

Under Magic-Angle Spinning (MAS), a long radio-frequency (rf) pulse applied on resonance achieves the selective excitation of the center-band of a wide NMR spectrum. We show herein that these rf pulses can be applied on the indirect channel of Hetero-nuclear Multiple-Quantum Correlation (HMQC) sequences, which facilitate the indirect detection via spin-1/2 isotopes of nuclei exhibiting wide spectra. Numerical simulations show that this indirect excitation method is applicable to spin-1/2 nuclei experiencing a large chemical shift anisotropy, as well as to spin-1 isotopes subject to a large quadrupole interaction, such as 14N. The performances of the long pulses are analyzed by the numerical simulations of scalar-mediated HMQC (J-HMQC) experiments indirectly detecting spin-1/2 or spin-1 nuclei, as well as by dipolar-mediated HMQC (D-HMQC) experiments achieving indirect detection of 14N nuclei via 1H in crystalline γ-glycine and N-acetyl-valine samples at a MAS frequency of 60kHz. We show on these solids that for the acquisition of D-HMQC spectra between 1H and 14N nuclei, the efficiency of selective moderate excitation with long-pulses at the 14N Larmor frequency, ν0(14N), is comparable to those with strong excitation pulses at ν0(14N) or 2ν0(14N) frequencies, given the rf field delivered by common solid-state NMR probes. Furthermore, the D-HMQC experiments also demonstrate that the use of long pulses does not produce significant spectral distortions along the 14N dimension. In summary, the use of center-band selective weak pulses is advantageous for HMQC experiments achieving the indirect detection of wide spectra since it (i) requires a moderate rf field, (ii) can be easily optimized, (iii) displays a high robustness to CSAs, offsets, rf-field inhomogeneities, and fluctuations in MAS frequency, and (iv) is little dependent on the quadrupolar coupling constant.

English

A pterocarpan, 2,3,4,8-tetramethoxy-6a,11a-dihydro-6H-benzo[4,5]furo[3,2-c]chromene-7,9-diol (compound 1) was isolated from the dichloromethane extract of the leaves of Abrus precatorius L. The structure of the compound was elucidated by detailed spectroscopic analysis such as 1H NMR, 13C NMR, Distortionless Enhancement by Polarisation Transfer (DEPT), Heteronuclear Multiple-Quantum Correlation (HMQC) and Heteronuclear Multiple-Bond Correlation (HMBC). The crude extracts displayed brine shrimp lethality and in vitro antimalarial activity. The methanolic crude extract demonstrated a A/B ratio of 9.7 signifying that it is more toxic against Plasmodium falciparum than brine shrimp. Key words: Fabaceae, Papilionaceae, Abrus precatorius, pterocarpan, brine shrimp lethality, antiplasmodial activity.

Comparison of various NMR methods for the indirect detection of nitrogen-14 nuclei via protons in solids

We present an experimental comparison of several through-space Hetero-nuclear Multiple-Quantum Correlation experiments, which allow the indirect observation of homo-nuclear single- (SQ) or double-quantum (DQ) 14N coherences via spy 1H nuclei. These 1H–{14N} D-HMQC sequences differ not only by the order of 14N coherences evolving during the indirect evolution, t1, but also by the radio-frequency (rf) scheme used to excite and reconvert these coherences under Magic-Angle Spinning (MAS). Here, the SQ coherences are created by the application of center-band frequency-selective pulses, i.e. long and low-power rectangular pulses at the 14N Larmor frequency, ν0(14N), whereas the DQ coherences are excited and reconverted using rf irradiation either at ν0(14N) or at the 14N overtone frequency, 2ν0(14N). The overtone excitation is achieved either by constant frequency rectangular pulses or by frequency-swept pulses, specifically Wide-band, Uniform-Rate, and Smooth-Truncation (WURST) pulse shapes. The present article compares the performances of four different 1H–{14N} D-HMQC sequences, including those with 14N rectangular pulses at ν0(14N) for the indirect detection of homo-nuclear (i) 14N SQ or (ii) DQ coherences, as well as their overtone variants using (iii) rectangular or (iv) WURST pulses. The compared properties include: (i) the sensitivity, (ii) the spectral resolution in the 14N dimension, (iii) the rf requirements (power and pulse length), as well as the robustness to (iv) rf offset and (v) MAS frequency instabilities. Such experimental comparisons are carried out for γ-glycine and l-histidine.HCl monohydrate, which contain 14N sites subject to moderate quadrupole interactions. We demonstrate that the optimum choice of the 1H–{14N} D-HMQC method depends on the experimental goal. When the sensitivity and/or the robustness to offset are the major concerns, the D-HMQC sequence allowing the indirect detection of 14N SQ coherences should be employed. Conversely, when the highest resolution and/or adjusted indirect spectral width are needed, overtone experiments are the method of choice. The overtone scheme using WURST pulses results in broader excitation bandwidths than that using rectangular pulses, at the expense of reduced sensitivity. Numerically exact simulations also show that the sensitivity of the overtone 1H–{14N} D-HMQC experiment increases for larger quadrupole interactions.

Improving the resolution in proton-detected through-space heteronuclear multiple quantum correlation NMR spectroscopy

Connectivities and proximities between protons and low-gamma nuclei can be probed in solid-state NMR spectroscopy using two-dimensional (2D) proton-detected heteronuclear correlation, through Heteronuclear Multiple Quantum Correlation (HMQC) pulse sequence. The indirect detection via protons dramatically enhances the sensitivity. However, the spectra are often broadened along the indirect F1 dimension by the decay of heteronuclear multiple-quantum coherences under the strong 1H–1H dipolar couplings. This work presents a systematic comparison of the performances of various decoupling schemes during the indirect t1 evolution period of dipolar-mediated HMQC (D-HMQC) experiment. We demonstrate that 1H–1H dipolar decoupling sequences during t1, such as symmetry-based schemes, phase-modulated Lee–Goldburg (PMLG) and Decoupling Using Mind-Boggling Optimization (DUMBO), provide better resolution than continuous wave 1H irradiation. We also report that high resolution requires the preservation of 1H isotropic chemical shifts during the decoupling sequences. When observing indirectly broad spectra presenting numerous spinning sidebands, the D-HMQC sequence must be fully rotor-synchronized owing to the rotor-synchronized indirect sampling and dipolar recoupling sequence employed. In this case, we propose a solution to reduce artefact sidebands caused by the modulation of window delays before and after the decoupling application during the t1 period. Moreover, we show that 1H–1H dipolar decoupling sequence using Smooth Amplitude Modulation (SAM) minimizes the t1-noise. The performances of the various decoupling schemes are assessed via numerical simulations and compared to 2D 1H–{13C} D-HMQC experiments on [U–13C]–L-histidine⋅HCl⋅H2O at various magnetic fields and Magic Angle spinning (MAS) frequencies. Great resolution and sensitivity enhancements resulting from decoupling during t1 period enable the detection of heteronuclear correlation between aliphatic protons and ammonium 14N sites in L-histidine⋅HCl⋅H2O.

Isolation and Structure Elucidation of Bioactive Compounds Chemical as Inhibitors of the Enzyme ?-Glucosidase Raru Bark Ethanol Extract (Vatica pauciflora Blume)

Inhibitory compound of ?-glucosidase bioactivity was isolated from ethanol extract of the stem bark of raru (Vatica pauciflora Blume) which, is a wild plant that grows in Tapanuli state in Indonesia.The ethanol extract of the stem bark of raru was partitioned and was chromatographed on columns with stationare fase silica gel 60 mesh F254 (0.063 mm-0.200 mm). The ethanol extract of isolates showed the enzyme ?-glucosidase inhibitor IC50 of 93.46. The next step is the determination of the chemical structure of the Ultraviolet–visible spectroscopy (UV Vis); FT-IR (Fourier Transform Infrared spectroscopy), NMR (Nuclear Magnetic Resonance) namely COSY, Heteronuclear Multiple-Quantum Correlation (HMQC); and Heteronuclear Multiple-Bond Correlation (HMBC). It found of compounds consist of two of methoxy, one of aromatic, and one of carbonil compounds. The isolat is a 3,4,9,Trihidroxy-2-(hydroxymethyl)-8-10-dimethoxy-2-3-4atetrahydropyrano(3,2-c)isochromen-6(10bH)-one acording to International Union of Pure and Applied Chemistry (IUPAC).

Site-specific NMR mapping and time-resolved monitoring of serine and threonine phosphorylation in reconstituted kinase reactions and mammalian cell extracts

We outline NMR protocols for site-specific mapping and time-resolved monitoring of protein phosphorylation reactions using purified kinases and mammalian cell extracts. These approaches are particularly amenable to intrinsically disordered proteins and unfolded, regulatory protein domains. We present examples for the ¹⁵N isotope-labeled N-terminal transactivation domain of human p53, which is either sequentially reacted with recombinant enzymes or directly added to mammalian cell extracts and phosphorylated by endogenous kinases. Phosphorylation reactions with purified enzymes are set up in minutes, whereas NMR samples in cell extracts are prepared within 1 h. Time-resolved NMR measurements are performed over minutes to hours depending on the activities of the probed kinases. Phosphorylation is quantitatively monitored with consecutive 2D ¹H-¹⁵N band-selective optimized-flip-angle short-transient (SOFAST)-heteronuclear multiple-quantum (HMQC) NMR experiments, which provide atomic-resolution insights into the phosphorylation levels of individual substrate residues and time-dependent changes thereof, thereby offering unique advantages over western blotting and mass spectrometry.

Measurement of the shortest hetero-nuclear distances in multiple-spin systems using constant-time correlation NMR methods

We demonstrate that the shortest hetero-nuclear distances in multiple-spin systems can be determined using Constant-Time Dipolar-mediated Hetero-nuclear Multiple-Quantum Correlation (CT-D-HMQC) two-dimensional NMR experiments. We show that this approach is applicable between two spin-1/2 isotopes, such as 13C and 15N, or between spin-1/2 and half-integer quadrupolar nuclei, such as 31P and 27Al. Furthermore, numerical simulations of spin dynamics and experiments on L-histidine·HCl and microporous aluminophosphate VPI-5 prove that the delay between zero-crossings of CT-D-HMQC trajectories is a reliable estimator of the largest dipolar couplings when the distances with the second neighbors are at least 60% larger than with the first neighbor, and there exists a known number of first neighbors at similar distances with an angle between the inter-nuclear vectors ranging from 30° to 150°.