Heteronuclear Multiple Bond Correlation Experiments Research Articles

Leveraging the HMBC to Facilitate Metabolite Identification.

The accuracy and ease of metabolite assignments from a complex mixture are expected to be facilitated by employing a multispectral approach. The two-dimensional (2D) 1H-13C heteronuclear single quantum coherence (HSQC) and 2D 1H-1H-total correlation spectroscopy (TOCSY) are the experiments commonly used for metabolite assignments. The 2D 1H-13C HSQC-TOCSY and 2D 1H-13C heteronuclear multiple-bond correlation (HMBC) are routinely used by natural products chemists but have seen minimal usage in metabolomics despite the unique information, the nearly complete 1H-1H and 1H-13C and spin systems provided by these experiments that may improve the accuracy and reliability of metabolite assignments. The use of a 13C-labeled feedstock such as glucose is a routine practice in metabolomics to improve sensitivity and to emphasize the detection of specific metabolites but causes severe artifacts and an increase in spectral complexity in the HMBC experiment. To address this issue, the standard HMBC pulse sequence was modified to include carbon decoupling. Nonuniform sampling was also employed for rapid data collection. A dataset of reference 2D 1H-13C HMBC spectra was collected for 94 common metabolites. 13C-13C spin connectivity was then obtained by generating a covariance pseudo-spectrum from the carbon-decoupled HMBC and the 1H-13C HSQC-TOCSY spectra. The resulting 13C-13C pseudo-spectrum provides a connectivity map of the entire carbon backbone that uniquely describes each metabolite and would enable automated metabolite identification.

Density functional theory calculations of δ(13 C) and δ(1 H) chemical shifts and 3 J(13 COO1 H) coupling constants as structural and analytical tools in hydroperoxides: Prospects and limitations of 1 H13 C heteronuclear multiple bond correlationexperiments.

Density functional theory (DFT) calculations of δ(13 C) and δ(1 H) chemical shifts and 3 J(13 COO1 H) coupling constants of three model hydroperoxides of the naturally occurring cis-11-OOH and trans-9-OOH isomers of oleate and 9-cis, 11-trans-16-OOH endo hydroperoxide of methyl linolenate are reported. The computational δ(OOH) for various functionals and basis sets were found to be nearly identical for the cis/trans geometric isomers. The chemical shifts of the methine CHOOH protons and carbons, on the contrary, are highly diagnostic for the identification of cis/trans geometric isomerism. The chemical shifts of the olefinic protons and carbons strongly depend on the orientation of the hydroperoxide unit relative to the double bond and, thus, of importance in conformational analysis. The results are in very good agreement with the available experimental data. For the various diastereomeric pairs of the model endo-hydroperoxide, the strongly deshielded OOH resonances, due to the presence of an intramolecular hydrogen bond between the hydroperoxide proton and an oxygen of the endo-peroxide ring, along with the δ(CHOOH), are highly diagnostic for identification and structure elucidation of complex erythro- and threo- diastereomeric pairs of endo-hydroperoxides; the computational results are in very good agreement with the available experimental data. The 3 J(13 COO1 H) coupling constants were found to be < Hz for the cis-trans geometric models and < Hz for the endo-hydroperoxide and, thus, unimportant in stereochemical analysis. Sharp resonances of the hydroperoxide protons, with Δν1/2 < 3 Hz, are required for the successful implementation of the 1 H13 C heteronuclear multiple bond correlation (HMBC) technique.

Synthesis, NMR, X-ray crystallography and DFT studies of some regioisomers possessing imidazole heterocycles

• Imidazopyridines and imidazopyrimidines are formed by fusing imidazole ring with pyridine and pyrimidine, respectively. • Some 2D-NMR techniques including 1 H- 15 N HMBC experiment were used for the structural elucidation of regioisomeric products. • Structure confirmations of 2c and 3c were also supported by their XRD crystallography. • The quantum chemical calculations of synthesized compounds were performed by choosing DFT. Imidazole-containing heterocycles: Imidazopyridines, imidazopyrimidines can exist in several tautomeric forms. Their regioselectivities were determined for N -alkylations with 4-chlorobenzyl bromide under basic conditions (K 2 CO 3 ) in DMF. We observed that, regioisomers were mainly formed as a mixture in this reaction and N -benzylation occurs at a higher ratio on six membered heterocycles. Their structural assignments were made with the use of two-dimensional 1 H– 1 H NOE (Nuclear Overhauser Effect Spectroscopy, NOESY) and 1 H- 15 N HMBC (Heteronuclear Multiple Bond Correlation) spectra. Further confirmation of the structure of 2c and 3c were obtained from X-ray crystallography. Experimental data was confirmed by Density Functional Theory (DFT) studies as well.

Synthesis and Cytotoxicity of Some Imidazo[4,5‐b]pyridine Derivatives and Their Regioselective N‐Alkylation

AbstractSome imidazopyridine‐carboxamides and carboximidamides were synthesized and their cytotoxic activities were tested against human leukemia cell lines (K562 and HL‐60), human colon cancer cell line (HCT‐116), human multiple myeloma cell lines (U266 and H929) and normal mouse fibroblast cell line (L929) by MTT (Tetrazolium salt colorimetric assay). Among them, imidazopyridine‐2‐phenylcarboxamide analogues 21 a–23, gave the lowest IC50 value with the range of 5.9–9.8 μM. Since the imidazopyridine ring exist in three tautomeric forms, N‐alkylation with benzyl bromide under basic conditions (K2CO3, in DMF) formed the mixture of three regioisomers. Their structural assignments (16 a, 20 a, 16 b, 20 b–22 b and 16 c) were made with the use of two‐dimensional 1H−1H NOE (Nuclear overhauser effect spectroscopy, NOESY) and 1H/13C‐15N HMBC (Heteronuclear Multiple Bond Correlation) experiments. We observed that, N‐benzylation occurs at a higher ratio on the pyridine moiety as N4‐regiosomer. In order to analyze the possible interactions of compounds 21 a–23, which were found to display good cytotoxic effect against the cancer cell lines tested were selected to dock into Aurora A kinase (AURKA) active site. The results reported that the compounds occupied the ATP pocket via forming interactions with Lys141, Lys162, Thr217, and Tyr 219 indicating the binding affinity to the AURKA.

Biological evaluation and molecular docking study of metabolites from Salvadora Persica L. Growing in Egypt

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is a resistant staph bacterium to several antibiotics causing several lives-threating diseases such as pneumonia and sepsis. Meswak, Salvadora persica, exhibited promising antimicrobial properties before. Objective: Exploring the anti-MRSA activity of S. persica L. metabolites and its mechanism of action on a molecular level. Materials and Methods: Structure elucidation of the isolated metabolites was carried out by spectroscopic data (one-dimensional and two-dimensional nuclear magnetic resonance). The biological activities of the isolated metabolites against MRSA were evaluated and the molecular mode of action against the dehydrosqualene synthase enzyme have been done. Results: Four compounds have been isolated and identifies to be; apigenin (1), luteolin (2), astragalin (3), and kaempferol-3-O-rhamnoside (4). Compounds 1–4 showed good anti-MRSA activities with IC50 values of 10.3, 11.5, 3.5, and 4.5 μg/mL, respectively. In consistent, astragalin and kaempferol-3 rhamnoside showed close high docking scores. Herein, we are reporting the molecular determinates of activity of these new scaffolds as anti-MRSA, which would be of great importance to developing new anti-MRSA candidates. Abbreviations Used: 1D: One-dimensional; 2D: Two-dimensional; CC: Column chromatography; COSY: Correlations spectroscopy; DMSO: Dimethyl sulfoxide; HMBC: Heteronuclear multiple-bond correlation experiment; HRESIMS: High-resolution electrospray ionization mass spectrometry; HSQC: Heteronuclear single-quantum correlation; IR: Infrared; MRSA: Methicillin-resistant Staphylococcus aureus; NMR: Nuclear magnetic resonance; RP: Reversed phase; TLC: Thin-layer chromatography; UV: Ultraviolet; VLC: Vacuum liquid chromatography.

Ingenine F: A New Cytotoxic Tetrahydro Carboline Alkaloid from the Indonesian Marine Sponge Acanthostrongylophora ingens.

Background:Marine organisms are established to be a wealthy source of bioactive compounds with diverse chemical structures and bioactivities. Acanthostrongylophora ingens is known to be rich with pyrimidine b-carboline and manzamine-type alkaloids. The goal of the present work is to isolate and identify new alkaloids from A. ingens as well as to assess the cytotoxic potential of these metabolites towards various cancer cell lines.Methods:The crude MeOH extract of the sponge was separated by vacuum liquid chromatography (VLC), using n-hexane, EtOAc, and MeOH. The EtOAc fraction was chromatographed on VLC, SiO2, sephadex LH-20, and RP18 columns, affording four metabolites. Their structures were identified using infrared, ultraviolet, high-resolution mass spectrometry, and nuclear magnetic resonance spectroscopic techniques, as well as comparison with the published data.Results:A new 1,2,3,4-tetrahydro-β-carboline (THβCs) alkaloid, ingenine F (4) and three known compounds: Annomontine (1), acanthomine A (2), and 1-oxo-1,2,3,4-THβCs (3) were isolated and identified. Ingenine F (4) exhibited cytotoxic activity toward hormone-dependent breast carcinoma (MCF7), colon carcinoma (HCT116), and lung carcinoma (A549) cell lines with IC50 values of 2.82, 1.00, and 2.37 μM, respectively, compared to doxorubicin (IC50 0.012, 0.036, and 0.102 μM, respectively).Conclusion:It is the first report for the isolation of THβCs alkaloids from A. ingens. The THβCs alkaloid with N-methylbutyramide unit as found in ingenine F is very rarely encountered in nature. Ingenine F may provide new promising candidates for potential cytotoxic agent.SUMMARY Ingenine F, a new 1,2,3,4-THβCs derivative (4) and three known alkaloids (1-3) were isolated from A. ingens. Their structures were verified by various spectroscopic analyses. Compound 4 had potent cytotoxic effect toward MCF7, HCT116, and A549 cancer cell lines. Abbreviations used: 1D: One-dimensional; 2D: Two-dimensional; CC: Column chromatography; COSY: Correlations spectroscopy; DMSO: Dimethyl sulfoxide; HMBC: Heteronuclear multiple bond correlation experiment; HRESIMS: High resolution electrospray ionization mass spectrometry; HSQC: Heteronuclear single quantum correlation; IR: Infrared; LCQ: Liquid chromatography quadrupole; LTQ: Linear trap quadropole; NMR: Nuclear magnetic resonance; RP: Reversed phase; SiO2: Silica gel; TLC: Thin-layer chromatography; UV: Ultraviolet; VLC: Vacuum liquid chromatography.

The microstructure determination of ethylene‐styrene‐propylene terpolymers at triad level by high‐temperature two‐dimensional NMR spectra

ABSTRACTTo further extend temperature range of application and low temperature performance of the ethylene‐styrene copolymers, a series of poly(ethylene‐styrene‐propylene) samples with varying monomer compositions and relatively low glass‐transition temperatures (Tg = −28 – 22 °C) were synthesized by Me2Si(Me4Cp)(N‐t‐Bu)TiCl2/MMAO system. Since the 13C NMR spectra of the terpolymers were complex and some new resonances were present, 2D‐1H/13C heteronuclear single quantum coherence and heteronuclear multiple bond correlation experiments were conducted. A complete 13C NMR characterization of these terpolymers was performed qualitatively and quantitatively, including chemical shifts, triad sequence distributions, and monomer average sequence lengths. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2018, 56, 340–350

A tridentate CNO‐donor palladium(II) complex as efficient catalyst for direct C―H arylation: Application in preparation of imidazole‐based push–pull chromophores

A series of imidazolium chlorides for the formation of tridentate CNO‐donor palladium(II) complexes featuring N‐heterocyclic carbene moieties have been developed from cheap and readily available starting materials with high yields. Their palladium complexes were prepared by reactions between the ligand precursors and PdCl2 using K2CO3 as base in pyridine with reasonable yields. These air‐stable metal complexes were characterized using 1H NMR and 13C{1H} NMR spectroscopy and elemental analyses. Heteronuclear multiple bond correlation experiments were performed to identify key NMR signals of these compounds. The structures of two of the complexes were also established by single‐crystal X‐ray diffraction analysis. One of these complexes was successfully applied in the direct C―H functionalization reactions between heterocyclic compounds and aryl bromides, producing excellent yields of coupled products. The coupling reactions were scalable, allowing grams of coupled products to be obtained with a mere 2 mol% of Pd loading. The catalyst system developed allowed the large‐scale preparation of several push–pull chromophores straightforwardly. Photophysical properties based on UV–visible and fluorescence spectroscopy for these chromophores were investigated. Deep blue photoluminescence with moderate quantum efficiency and twisted intramolecular charge transfer excited state were observed for these chromophores. Density functional theory (DFT) and time‐dependent DFT calculations were performed to support the experimental results.

Antimicrobial constituents from endophytic fungus Fusarium sp.

ObjectiveTo evaluate the antimicrobial potential of fraction of the fungus Fusarium sp. and study the tentative identification of their active constituents. MethodsSix compounds were purified from an fraction of endophytic fungus Fusarium sp. using column chromatography and their structures have been confirmed based on 1H and 13C nuclear magnetic resonance spectra, distortionless enhancement by polarization transfer, 2D COSY, heteronuclear multiple quantum correlation and heteronuclear multiple bond correlation experiments. The six isolated compounds were screened for antimicrobial activity using the agar well diffusion method. ResultsPhytochemical investigation of endophytic fungus Fusarium sp. lead to the isolation and identification of the following compounds viz., colletorin B, colletochlorin B, LL-Z1272β (llicicolin B), 4,5-dihydroascochlorin, ascochlorin, and 4,5-dihydrodechloroascochlorin. Colletorin B and colletochlorin B displayed moderate herbicidal, antifungal and antibacterial activities towards Chlorella fusca, Ustilago violacea, Fusarium oxysporum, and Bacillus megaterium. On the other hand LL-Z1272β (llicicolin B) showed moderate antifungal activity towards Ustilago violacea and Fusarium oxysporum while 4,5-dihydroascochlorin showed strong antibacterial activity towards Bacillus megaterium. Furthermore, 4,5-dihydrodechloroascochlorin showed very strong antifungal activity towards Eurotium repens. ConclusionsAntimicrobial activities demonstrated by five of the six isolated compounds clearly demonstrate that these fungi extracts and active compounds present a great potential for the food, cosmetic and pharmaceutical industries.

29Si–1H IMPACT HMBC: a suitable tool for analyzing silylated derivatives

A modified version of the IMPACT heteronuclear multiple bond correlation (HMBC) has allowed the characterization of an organosilane and a tetrasilylated yttrium complex. With the help of this sequence, an average gain in sensitivity close to 2 has been obtained compared with the standard HMBC experiment for disilanes as well as for yttrium complexes containing silylated ligands. This modified version of this long-range correlation experiment opens the way for following kinetics in the range of a fraction of a minute and to study by NMR low concentrated samples and low abundant nuclei.

A comprehensive discussion of hmbc pulse sequences, part 1: The classical HMBC

AbstractSince its introduction in 1986, the heteronuclear multiple bond correlation (HMBC) experiment has modernized nuclear magnetic resonance spectroscopy for the structural studies of small to medium molecules. The experiment provides information about which protons are near through bonds (but not directly bonded to) different carbons or to another heteronucleus. This experiment (in conjunction with the heteronuclear single‐quantum coherence experiment) provides significant amount of information about molecular structure, as the long‐range proton–carbon or proton–nitrogen correlations can include quaternary carbons or nitrogens, in addition to protonated carbons or nitrogens. Impressive examples for structure elucidation of organic molecules, peptides, or natural products have been published in the last 20 years. After a short introduction into the basic theory, this article will provide a comprehensive and pedagogical description of the classical HMBC pulse sequences, with the aid of product operator formalism. Building from basic principles, low‐pass filtering for rejecting unwanted 1 JCH correlations, and the role of pulsed‐field gradients for coherence transfer pathway selection are explained in detail. This article is intended to be used as a basic coverage for students and researchers seeking a simple theoretical understanding of the fundamental tools of the HMBC experiment. © 2012 Wiley Periodicals, Inc. Concepts Magn Reson Part A 40A: 101–127, 2012.

Alkaloids and flavonoids of Argemone mexicana

A new protopine alkaloid, protomexicine and a new isoflavonoid, mexitin, together with 8-methoxydihydrosanguinarine, 13-oxoprotopine, rutin and quercetrin have been isolated from the aerial part of the methanolic extract of Argemone mexicana. The structures of these compounds have been established by various spectral data. The structure of protomexicine was further confirmed by heteronuclear multiple bond correlation experiment. Protomexicine and mexitin are new compounds, and other compounds are first reported from A. mexicana and genus Argemone.

Naphthotriazole derivatives: Synthesis and fluorescence properties

Eight fluorescent compounds containing a naphthotriazole moiety substituted at position 2 by a (vinylsulfonyl)aryl group or its precursors, containing either a hydroxyl or sulphonic acid groups or N-methylglycine, were prepared and characterized. The products were recovered in moderate yield after column chromatography or recrystallization and identified using 1H and 13C NMR; double resonance, heteronuclear multiple quantum coherence and heteronuclear multiple bond correlation experiments were carried out for complete assignment of proton and carbon signals. Absorption and emission spectra were obtained, in acetonitrile and fluorescence quantum yield determined. All compounds offer promise as fluorescent probes owing to their high fluorescence quantum yield.

Structural determination of the O-antigenic polysaccharide from the verocytotoxin-producing Escherichia coli O176

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O176 has been determined. Component analysis together with 1H and 13C NMR spectroscopy was employed to elucidate the structure. Inter-residue correlations were determined by 1H, 1H NOESY and 1H, 13C heteronuclear multiple-bond correlation experiments. The PS is composed of tetrasaccharide repeating units with the following structure: [Display omitted] Cross-peaks of low intensity from α-linked mannopyranosyl residues were present in the 1H, 1H TOCSY NMR spectra and further analysis of these showed that they originate from the terminal part of the polysaccharide. Consequently, the biological repeating unit has a 3-substituted N-acetyl- d-galactosamine residue at its reducing end. The repeating unit of the E. coli O176 O-antigen is similar to those from E. coli O17 and O77, thereby explaining the reported cross-reactivities between the strains, and identical to that of Salmonella cerro (O:6, 14, 18).

Laccase-Mediated Michael Addition of 15N-Sulfapyridine to a Model Humic Constituent

Chemical incorporation of sulfonamide antimicrobials into natural organic matter may represent an important process influencing the fate of these synthetic, primarily agents in soil and sediment environments. We previously demonstrated that a fungal peroxidase mediates covalent coupling of sulfonamide antimicrobials to model humic constituents; reactions with the 2,6-dimethoxyphenol syringic acid produced Schiff bases (Bialk et al. Environ. Sci. TechnoL 2005, 39, 4436-4473). Here, we show that fungal laccase-mediated reaction of sulfapyridine with the orthodihydroxyphenol protocatechuic acid yields a Michael adduct. We synthesized 15N-enriched sulfapyridine to facilitate determination of the covalent linkage(s) formed between sulfapyridine and protocatechuic acid by NMR spectroscopy. 1H-(15)N heteronuclear multiple bond correlation experiments and tandem mass spectrometry demonstrated that the sulfapyridine anilinic nitrogen engaged in a Michael addition reaction to oxidized protocatechuic acid to form an anilinoquinone. Michael adducts are more stable than the previously reported imine linkages between sulfonamides and 2,6-dimethoxyphenols. Michael addition to quinone-like structures in soil organic matter is expected to diminish the mobility and biological activity of sulfonamide antimicrobials.

Structural analysis of the O-antigen polysaccharide from Escherichia coli O152

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O152 has been determined. Component analysis together with 1H, 13C and 31P NMR spectroscopy were used to elucidate the structure. Inter-residue correlations were determined by 1H, 31P COSY, 1H, 1H NOESY and 1H, 13C heteronuclear multiple-bond correlation experiments. The PS is composed of pentasaccharide repeating units with the following structure: ▪ The structure is similar to that of the O-antigen polysaccharide from E. coli O173. The cross-reactivity between E. coli O152 and E. coli O3 may be explained by structural similarities in the branching region of their O-antigen polysaccharides.

Development of a Method for the Measurement of Long‐Range 13C—1H Coupling Constants from HMBC Spectra

AbstractFor Abstract see ChemInform Abstract in Full Text.

Development of a method for the measurement of long-range 13C– 1H coupling constants from HMBC spectra

This paper describes a number of improvements to a method, developed in this laboratory and described in J. Magn. Reson. 85 (1989) 111–113, which makes it possible to determine values of long-range 13C– 1H coupling constants from heteronuclear multiple bond correlation (HMBC) spectra. First, it is shown how pulsed-field gradients can be introduced into the HMBC experiment without perturbing the form of the cross-peak multiplets; a one-dimensional version of the experiment is also described which permits the rapid measurement of a small number of couplings. Second, the experiment is modified so that one-bond and long-range cross-peaks can be separated, and so that the one-bond cross-peaks have more reliable intensities. Finally, it is shown how these one-bond cross-peaks can be used to advantage in the fitting procedure.

Nitrite-induced nitration pathways of retinoic acid, 5,6-epoxyretinoic acid, and their esters under mildly acidic conditions: toward a reappraisal of retinoids as scavengers of reactive nitrogen species.

All trans retinoic acid (1), a cancer chemopreventive agent and a pluripotent morphogen, was found to react efficiently with nitrite ions in a biphasic system consisting of CH(2)Cl(2)/0.1 M phosphate buffer (pH 3) 1:1 v/v to give a complex mixture of nitration products. Repeated TLC fractionation of the reaction mixtures after methylation allowed isolation of the main products, which could be identified as the 12-nitro derivatives 3a,b and the decarboxylated 12,14-dinitro and 5,6-epoxy-14-nitro derivatives 4 and 5a by spectral analysis. Use of (15)NO(2)(-) followed by extensive 2D NMR analysis, including (1)H,(15)N heteronuclear multiple bond correlation experiments, allowed identification of nitronitrate derivatives as additional constituents of the mixture. Under similar conditions, 1 methyl ester gave mainly 3a,b. 5,6-Epoxyretinoic acid (2) reacted smoothly with acidic nitrite to give mainly 5a and its isomer 5b whereas its methyl ester afforded 14-nitro derivatives 9a,b as chief products. The observed patterns of reactivity along with mechanistic experiments would suggest that nitrite-induced nitration of 1 proceeds through complex reaction pathways set in motion by attack of NO(2) to the 12- and 14-positions. Separate experiments showed that 1 can inhibit nitrite-induced N-nitrosation of 2,3-diaminonaphthalene at pH values of 4 and 5.5, as well as decomposition of caffeic acid under similar conditions. Overall, these results provide the first detailed insight into the reaction behavior of a retinoid toward reactive nitrogen species and shed light on previously overlooked nitrite scavenging properties of 1 of potential relevance to the mechanism of its antiinflammatory, antimutagenic, and cancer chemopreventive action.

Use of Long-Range C-H Heteronuclear Multiple Bond Connectivity in the Assignment of the 13 C NMR Spectra of Complex Organic Molecules

Use of Long-Range C-H Heteronuclear Multiple Bond Connectivity in the Assignment of the 13 C NMR Spectra of Complex Organic Molecules