Dimensional NMR Spectroscopy Research Articles

Cytotoxic ent-Kaurane Diterpenoids from Rabdosia Rubescens.

One new (1) and 11 reported ent-kaurane diterpenoids (2-12) were received from the ethanol extract of the air-dried aerial parts of Rabdosia rubescens collected in Jiyuan. Their structures were determined in accordance with high resolution electrospray ionization mass spectroscopy, one dimensional (1D) and two-dimensional (2D) NMR spectroscopy and the data published in the literature. The cytotoxic activity of these isolated compounds was assessed against SMMC-7721, A-549, H-1299 and SW-480 cancer cell lines. Compounds 2-6 revealed significant cytotoxic activity on lung cancer cell lines A549 with IC50 values from 6.2 to 28.1 μM. Analysis of structure-activity relationship of these tested compounds indicated the carbonyl at C-15 and hydroxy at C-1 together could be crucial groups for inhibiting lung cancer cell lines A549 proliferation.

Chemical Constituents from the Leaves of Pachyrhizus erosus Collected in Vietnam.

A phytochemical investigation of methanol extract from leaves of Pachyrhizus erosus (L.) Urban, a leguminous shrub distributed in Vietnam and other tropical and subtropical countries led to the isolation of a new prenylated chalcone, erosusone (1) and a new megastigmane glycoside epimer, 3-episedumoside F1 (9), together with thirteen known compounds including flavonoids (2-6), a 3-benzoxepine lactone (7), a pyridine-4,5-diol derivative (8), megastigmanes and megastigmane glycosides (10-15). Their structures were elucidated by means of high resolution-electrospray ionization (HR-ESI)-MS, one dimensional (1D) and two-dimensional NMR (2D-NMR) spectroscopy as well as comparison with the data reported in the literature. The cytotoxic effects on LU-1 (lung carcinoma), HepG2 (hepatocellular carcinoma), and MCF7 (breast carcinoma) cell lines were assessed. Prenylated chalcones 1-2 and isoflavone 3 exhibited cytotoxicity against all tested cell lines with IC50 values ranging from 22.04 to 45.03 µM.

Induction of New Lactam Derivatives From the Endophytic Fungus Aplosporella javeedii Through an OSMAC Approach.

Fermentation of the endophytic fungus Aplosporella javeedii on solid rice medium in presence of either 3.5% NaNO3 or 3.5% monosodium glutamate caused a significant change of the fungal metabolite pattern compared to fungal controls grown only on rice. Chemical investigation of the former fungal extracts yielded 11 new lactam derivatives, aplosporellins A–K (2–12), in addition to the known compound, pramanicin A (1). All of these compounds were not detected when the fungus was grown on rice medium without these activators thereby indicating the power of this OSMAC approach. The structures of the new compounds were elucidated by one- and two- dimensional NMR spectroscopy, DFT-NMR calculations and by mass spectrometry as well as by comparison with the literature whereas the absolute configuration of the lactam core was determined by TDDFT-ECD and OR calculations. Pramanicin A (1) showed strong cytotoxicity against human lymphoma (Ramos) and leukemia (Jurkat J16) cells with IC50 values of 4.7 and 4.4 μM, respectively. Mechanistic studies indicated that 1 activates caspase-3 and induces apoptotic cell death.

Ebenamariosides A-D: Triterpene Glucosides and Megastigmanes from the Leaves of Diospyros maritima.

The 1-BuOH-soluble fraction of the methanol (MeOH) extract of Diospyros maritima was separated by chromatographic techniques to give three new oleanane-type and one new ursane-type triterpene glucoside, named ebenamariosides A-D (1-4); two megastigmanes were also isolated. The structures of triterpene glucosides was elucidated with extensive investigation by one and two dimensional NMR spectroscopy and the structures were confirmed by partial enzymatic hydrolyses to give the corresponding mono-glucosides and aglycones. The structures of the megastigmanes, including their absolute stereochemistries, were elucidated by spectroscopic evidence and by the modified Mosher's method. Two megastigmanes were chemically correlated and their absolute structures were unambiguously determined. The cytotoxicity of the triterpene glucosides and their degradation products were assayed. They did not show any significant activity.

Tetrahydropyrimidinones/thiones stabilized by trifluoromethyl-containing β-diketones

A library of new hydropyrimidinone/thione compounds was synthesized via the classical Biginelli reaction using hydrated cerium(III) chloride as the catalyst. The presence of a trifluoromethyl or methyl group in the diketone starting material has been established to selectively control the outcome of the Biginelli reaction where one of the two possible pyrimidinone/thione compounds is formed. The results showed that the electronic effects of substituents of the diketone directly affect the product formation. The synthesized compounds were fully characterized using 1H, 13C, and two dimensional NMR (2D NMR) spectroscopy, single crystal X-ray diffractometry, FT-IR, and ESI-HDMS techniques. We also report on the uncommon one-bond correlations which were observed in the HMBC spectra and the interesting long-range heteronuclear coupling of fluorine to hydrogen and carbon.

TWO NOVEL FLAVONE C-GLYCOSIDES ISOLATED FROM AFROCARPUS GRACILIOR: POM ANALYSES AND IN VITRO CYTOTOXIC ACTIVITY AGANIST HEPATOCELLULAR CARCINOMA

Objective: Cancer is considered as one of the top reasons of death and the number of cases increasing gradually. Cancer is severe clinical difficulty to the health caution system. This study explored two novel polyphenols of Afrocarpus gracilior Pilger growing in Egypt and evaluated their cytotoxic activity.
 Methods: Methanolic (80%) extract of the leaves of A. gracilior was subjected to column chromatography; the chemical structures of the isolated compounds were established by advanced spectral techniques: UV, 1H, 13C NMR, two dimensional NMR (2D NMR) and electron spray ionization mass spectroscopy (ESI-MS). Compounds 1 and 2 were studied for their cytotoxic activity against hepatocellular carcinoma (Hep-G2) using sulforhodamine B (SRB) assay. Furthermore the pharmacokinetics profiles of these molecules were accessed by employing Petra/Osiris/Molinspiration (POM) analyses.
 Results: Two novel C-flavonoid glycosides were isolated [1: Apigenin 8-C-β-D-glucopyranosyl-(1```→4``)-O-β-D-glucopyranoside] and [2: 7-O methyl-luteolin 8-C-β-glucopyranosyl-(1```→4``)-O-β-D-glucopyranoside]. They exhibited significant cytotoxic activity (IC50 = 9.02 and 15.61 µg/ml, respectively) against Hep-G2 cells. The POM analyses revealed that the activity of these two compounds depends on the presence of glucosyl and alkyl groups at the internal and terminal atmosphere of the compounds.
 Conclusion: These findings demonstrated that the leaves of A. gracilior contain a series of bioactive polyphenolic compounds with significant cytotoxic properties against hepatocellular carcinoma and may be used as alternative anticancer agents for doxorubicin. On the basis of POM calculations, it will be interesting to develop some alternative flavones because the deglucosylated derivatives have a better drug score than parent molecules. This preliminary study will be extended to other strains of cancer.

A new antibacterial dibenzofuran-type phloroglucinol from myrtus communis linn

Our continuation on the bio-assay guided isolation from Myrtus communis Linn. led to the discovery of a new dibezofuran type phloroglucinol 1,1'-(1,3,7,9-tetrahydroxydibenzo[b,d]furan-2,8-diyl)bis(ethan-1-one) 1. The structure was established through detailed spectroscopic studies including one and two dimensional NMR spectroscopy and electrospray ionization high resolution mass spectrometer (ESI-HRMS). The crude acetone extract from M. communis (AMA), dichloromethane fraction (DCM), and the isolated pure compound 1 were tested against pathogenic bacteria. Compound 1 displayed higher antibacterial activity against the Gram-positive and Gram-negative Staphlocococus aureus and Escherichia coli respectively as compared to the crude extract and fractions.

A Rapid Microwave Induced Synthesis of Isonicotinamide Derivatives and their Antifungal Activity

Ten novel isonicotinamide derivatives were prepared by quaternization reactions of isonicotinamide with methyl iodide and nine differently substituted 2 bromoacetophenones under rapid microwave irradiation of 10 minutes. The microwave preparations were significantly faster and with yields higher up to 8 times, than the preparations by conventional method. The structures of synthesized molecules were determined by one- and two dimensional NMR and IR spectroscopy, mass spectrometry and elemental analysis. Antifungal activity of all compounds was tested in two different concentrations (10 and 100 µg mL–1) against Fusarium oxysporum, Fusarium culmorum, Macrophomina phaseolina and Sclerotinia sclerotiorum in vitro. From the antifungal assay it can be seen that the most prepared compounds have moderate to weak activity against M. phaseolina and F. culmorum. A very high inhibitory rate was observed against S. sclerotiorum, 62–87.5 % in concentration of 10 µg mL–1 and 83.7–93.2 % in concentration of 100 µg mL–1.

Substituted l-tryptophan-l-phenyllactic acid conjugates produced by an endophytic fungus Aspergillus aculeatus using an OSMAC approach.

The endophytic fungus Aspergillus aculeatus isolated from leaves of the papaya plant Carica papaya was fermented on solid rice medium, yielding a new l-tryptophan-l-phenyllactic acid conjugate (1) and thirteen known compounds (11, 14–25). In addition, an OSMAC approach was employed by adding eight different sodium or ammonium salts to the rice medium. Addition of 3.5% NaNO3 caused a significant change of the metabolite pattern of the fungus as indicated by HPLC analysis. Subsequent isolation yielded several new substituted l-tryptophan-l-phenyllactic acid conjugates (1–10) in addition to three known compounds (11–13), among which compounds 2–10, 12–13 were not detected in the rice control culture. All structures were unambiguously elucidated by one and two dimensional NMR spectroscopy and by mass spectrometry. The absolute configuration of the new compounds was determined by Marfey's reaction and X-ray single crystal diffraction. Compounds 19–22 showed cytotoxicity against the L5178Y mouse lymphoma cell line with IC50 values of 3.4, 1.4, 7.3 and 23.7 μM, respectively.

Bio-guided Isolation, Purification and Chemical Characterization of Epigallocatechin; Epicatechin, Stigmasterol, Phytosterol from of Ethyl Acetate Stem Bark Fraction of Spondias mombin (Linn.)

Spondias mombin (Linn.) is a widely cultivated edible plant used in folkloric medicine for the treatment of severe infection and health disorders. This research work was carried out to isolation, purification and chemical characterization the bioactive constituents of the ethyl acetate stem bark fraction of Spondias mombin (Linn.), a medicinally important plant of the Anacardiaceae family. This study revealed the presence of flavonoid and steroids, which have been found to be important hormone regulators which possess antimicrobial, anti-inflammatory, antioxidant properties. The chemical investigation resulted in the isolation of (C15H14O6.) 5, 7, 3', 4'-pentahydroxy flavanol (Epicatechin), (C15H14O7.) Epigallocatechin (C29H48O.), Stigmasterol phytosterol. It is here reported isolated from Spondias mombin for the first time, this makes the Spondias mombin very important medicinal plant in Nigeria and west Africa. EGC and EC arts as a strong inhibitor of HIV replication in cultured peripheral blood cells and inhibition of HIV-1 reverse transcriptase in vitro. EGC binds directly to CD4 molecules with consequent inhibition of Gp 120 binding and inactivate viruses in-vitro by deformation of phospholipids. Stigmasterol phytosterol have been shown to lower/reduce blood cholesterol and this lowering may reduce the risk of coronary heart disease. The structure was elucidated using two dimensional NMR spectroscopy, NMR (1H,13C) spectroscopy in combination with Infra-red (IR) and Mass spectrometer (MS) spectra data.

Sapofectosid – Ensuring non-toxic and effective DNA and RNA delivery

Different methods are being deployed for non-viral DNA/RNA delivery. However non-viral formulations for DNA/RNA-delivery are often accompanied by severe toxicity and thus low efficiency. Particular costly cell culture media are required as well. Here we introduce sapofection as a valuable enhancing method for non-viral DNA/RNA delivery. Sapofection is based on the application of DNA/RNA nanoplexes and sapofectosid, a plant derived natural transfection reagent. Sapofectosid was produced from plant raw material by chromatographic methods and characterized by tandem mass spectrometry and intensive one and two dimensional NMR-spectroscopy. Sapofectosid did enhance the transfection efficiency of different DNA- and RNA-nanoplexes formulated with liposomes, polyethylenimine (PEI) or targeted and non-targeted oligo-lysine peptides. All nanoplexes were characterized physicochemically and the influence of sapofectosid on the nanoplex integrity was determined by DNA complexation assays. The nanoplexes and sapofectosid were administered to a variety of cancer cell lines and the transfection efficiency was investigated by flow cytometry and confocal microscopy. Dependent on the cell line the transfection efficiencies varied from 6 to 76%. The saponin- and receptor-mediated endocytosis of nanoplexes was investigated by flow cytometry. As demonstrated by impedance based live cell imaging sapofection was non-toxic. The findings show the great potential of sapofection to be used as an effective and non-toxic transfection enhancing method.

Molecular Level-based Analysis of Organosolv Wastewater

Background: Identification of monomeric and oligomeric organic compounds in Organosolv process wastewater is of growing importance in the context of isolating high value chemicals and producing biofuels. There were many studies devoted to identification of such compounds in precipitated Organosolv lignin, all of them using demanding, mostly hyphenated techniques such as HPLC-MS/MS and two dimensional NMR spectroscopy. However, the resulting Organosolv wastewater has not been studied as intensively in this respect, even though the application of these techniques to identify the components of dried/lyophilized Organosolv wastewater would be straightforward. Objective: The objective was to identify lignin-based breakdown products in Organosolv wastewater resulting from pulping of beech with aqueous ethanol solutions. Method: Low resolution GC/MS analysis in combination with derivatization reactions using nonlabeled and isotopically labeled derivatization agents and retention index information (where available) were used. Results: The application of isotopically labeled derivatization agents such as BSTFA-d9 (N,Obis( trimethylsilyl)trifluoracetamide-d9) turned out especially useful in structural identification work. In addition to monomers such as abundant syringaldehyde and vanillin, a multitude of dimeric products of the guaiacyl(syringyl) glycerol-β-guaiacyl(syringyl) ether-type, phenylcoumaran type and lignan-type could be detected, the most abundant surrogate being syringaresinol. Syringyl-type surrogates were more abundant compared to guaiacyl-type surrogates. Formation pathways of the monomeric breakdown products such as syringaldehyde and vanillin were proposed on the basis of the solvolytic cleavage of β-O-4 aryl ether linkages. Conclusion: Solvolytic cleavage of β(α)-O-4, β-5 and β-β linkages within the macromolecular lignin network under Organosolv process conditions results in dimeric products. Further activities should be focused on the use of high resolution MS and the synthesis of authentic standards. Keywords: Aryl ether bonds, derivatization, GC/MS analysis, lignin, NMR spectroscopy, organosolv wastewater.

Stereoselective green synthesis and molecular structures of highly functionalized spirooxindole-pyrrolidine hybrids – A combined experimental and theoretical investigation

Highly functionalized spirooxindole-pyrrolidine hybrids have been synthesized stereoselectively through a [3 + 2] cycloaddition strategy in an ionic liquid, 1-butyl-3-methylimidazolium bromide ([bmim]Br). The structure of these spiro heterocyclic hybrids was elucidated using one and two dimensional NMR spectroscopy, single crystal X-ray crystallographic studies and Density Functional Theory (DFT) calculations. The calculated geometric parameters are in good agreement with the experimental data obtained from the X-ray structures. The Natural Bond Orbital (NBO) calculations on these molecules confirm the electron rich carbonyl oxygen and electron deficient NH groups. The 1H and 13C NMR chemical shifts calculated using GIAO method are in good agreement with the experimental data. The DFT computed polarizability values also suggest the possible NLO activity of these molecules.

New α-Pyrone derivatives from the endophytic fungus Embellisia sp

Two new α- Pyrone derivatives (1,2) have been isolated from the endophytic fungus Embellisia sp. The isolated compounds were chemically identified as 5-(3-S-hydroxybutyl)-4-methoxy-6-methyl-2H-pyran-2-one (1) and 4-(4-methoxy-6-methyl-2-oxo-2H-pyran-5-yl)butanoic acid (2). The chemical structures of 1 and 2 were determined based on one and two dimensional NMR spectroscopy and high resolution mass spectrometry. Absolute configuration of compound 1 was determined using modified mosher ester reaction. In vitro antileishmanial, antifungal, antibacterial and antimalarial activities of 1 and 2 were examined but they didn’t show promising activities.

Metabolic differences between cold stored and machine perfused porcine kidneys: A 1H NMR based study

Hypothermic machine perfusion (HMP) and static cold storage (SCS) are the two methods used to preserve deceased donor kidneys prior to transplant. This study seeks to characterise the metabolic profile of HMP and SCS porcine kidneys in a cardiac death donor model.Twenty kidneys were cold flushed and stored for two hours following retrieval. Paired kidneys then underwent 24 h of HMP or SCS or served as time zero controls. Metabolite quantification in both storage fluid and kidney tissue was performed using one dimensional 1H NMR spectroscopy. For each metabolite, the net gain for each storage modality was determined by comparing the total amount in each closed system (i.e. total amount in storage fluid and kidney combined) compared with controls.26 metabolites were included for analysis. Total system metabolite quantities following HMP or SCS were greater for 14 compared with controls (all p < 0.05). In addition to metabolic differences with control kidneys, the net metabolic gain during HMP was greater than SCS for 8 metabolites (all p < 0.05). These included metabolites related to central metabolism (lactate, glutamate, aspartate, fumarate and acetate).The metabolic environments of both perfusion fluid and the kidney tissue are strikingly different between SCS and HMP systems in this animal model. The total amount of central metabolites such as lactate and glutamate observed in the HMP kidney system suggests a greater degree of de novo metabolic activity than in the SCS system. Maintenance of central metabolic pathways may contribute to the clinical benefits of HMP.

NMR structure of dual site binding of mitoxantrone dimer to opposite grooves of parallel stranded G-quadruplex [d-(TTGGGGT)

The formation of complex between anti-cancer drug mitoxantrone (MTX) and tetra-molecular parallel G-quadruplex DNA [d-(TTGGGGT)]4 has been studied by solution state one and two dimensional NMR spectroscopy. Mitoxantrone forms a head-to-tail dimer and binds at two opposite grooves of the G-quadruplex. The Job's method of continuous variation and thermal melting studies independently ascertain binding stoichiometry of 4:1 in mitoxantrone:DNA complex. The existence of only four guanine NH peaks corresponding to the four G-quartets during the course of titration shows that C4 symmetry of G-quadruplex is intact upon binding of mitoxantrone. The specific inter molecular short distance contacts between protons of two mitoxantrone molecules of dimer, that is, ring A protons with ring C and side chain methylene protons, confirms the formation of mitoxantrone head-to-tail dimer. The observed 38 Nuclear Overhauser Enhancement (NOE) cross peaks between MTX and G-quadruplex DNA indicate formation of a well-defined complex. The three dimensional structure of 4:1 mitoxantrone:[d-(TTGGGGT)]4 complex computed by using experimental distance restraints followed by restrained Molecular Dynamics (rMD) simulations envisages the critical knowledge of specific molecular interactions within ligand-G-quadruplex complex. The findings are of direct interest in development of anti-cancer therapeutic drug based on G-quadruplex stabilization, resulting in telomerase inhibition.

Enhanced biosynthetically directed fractional carbon-13 enrichment of proteins for backbone NMR assignments

Routes to carbon-13 enrichment of bacterially expressed proteins include achieving uniform or positionally selective (e.g. ILV-Me, or 13C′, etc.) enrichment. We consider the potential for biosynthetically directed fractional enrichment (e.g. carbon-13 incorporation in the protein less than 100%) for performing routine n-(D)dimensional NMR spectroscopy of proteins. First, we demonstrate an approach to fractional isotope addition where the initial growth media containing natural abundance glucose is replenished at induction with a small amount (e.g. 10%w/wu-13C-glucose) of enriched nutrient. The approach considered here is to add 10% (e.g. 200mg for a 2g/L culture) u-13C-glucose at the induction time (OD600=0.8), resulting in a protein with enhanced 13C incorporation that gives almost the same NMR signal levels as an exact 20% 13C sample. Second, whereas fractional enrichment is used for obtaining stereospecific methyl assignments, we find that 13C incorporation levels no greater than 20%w/w yield 13C and 13C–13C spin pair incorporation sufficient to conduct typical 3D-bioNMR backbone experiments on moderate instrumentation (600MHz, RT probe). Typical 3D-bioNMR experiments of a fractionally enriched protein yield expected backbone connectivities, and did not show amino acid biases in this work, with one exception. When adding 10% u-13C glucose to expression media at induction, there is poor preservation of 13Cα–13Cβ spin pairs in the amino acids ILV, leading to the absence of Cβ signals in HNCACB spectra for ILV, a potentially useful editing effect. Enhanced fractional carbon-13 enrichment provides lower-cost routes to high throughput protein NMR studies, and makes modern protein NMR more cost-accessible.

Spectral characterization and crystal structure of some 2,6-diarylthian-4-one hydrazone derivatives

A series of cis and trans 2,6-diarylthian-4-one hydrazone derivatives (11–16) have been synthesized and characterized by 1H, 13C and two dimensional NMR spectroscopy. For the 2r,6t-diphenylthian-4-one N-isonicotinoylhydrazone (14) X-ray diffraction have also been recorded. The coupling constants suggested that the cis-hydrazones (11–13), which have the phenyl groups in cis orientation, largely exist in chair conformations with equatorial orientation of the phenyl groups 11C. Analysis of the vicinal coupling constants of trans-hydrazones (14–16) suggests that boat forms 14B must make significant contributions to it and the relative population is 58%. Moreover, in solution chair conformations 14C and 14C′, may contribute to 14. The NOESY and X-ray diffraction of 14 gives definite evidence for the contribution of 14C.

Chemical structure of the cell wall-associated polysaccharide of Bifidobacterium animalis subsp. lactis LKM512.

We have demonstrated that Bifidobacterium animalis subsp. lactis LKM512 had some probiotic properties in vivo and in vitro. To further understand their mechanisms, the chemical structure of the extracellular polysaccharide that constructs the cell envelope was determined. The strain was anaerobically cultured in MRS broth at 37 °C for 20 h, then the bacterial cells were harvested by centrifugation and washed. The cell wall-associated polysaccharide (CPS) was prepared from the cell wall component digested by lysozyme. The results of anion exchange and gel filtration chromatography showed that the polysaccharide was negatively charged and had a high molecular mass. The CPS was found to compose of galactopyranosyl, galactofuranosyl, glucopyranosyl and rhamnopyranosyl residues in the molar ratio of 1:1:1:3 by using methylation analysis with GC-MS and HPLC profiling. From the results of the structural characterization by 1 dimensional and 2 dimensional NMR spectroscopy, the polysaccharide was established to be a hexasaccharide repeating unit with the following structure: LKM512.

Synthesis and structure elucidation of ethyliden-linked anthocyanin — Flavan-3-ol oligomers

The reaction of grape derived anthocyanins and catechin in the presence of acetaldehyde was examined in model wine solutions. The reaction profile was monitored by HPLC-DAD and HPLC-ESI-MSn which revealed the presence of 18 different higher oligomers in addition to the ethyliden-bridged dimers as the main products of the reaction. In addition to the ethyliden bridge between the monomeric subunits, a second bridge could be observed, which may be attributed to the reaction product of two vinyl-flavanol intermediates. The evolving dimers and higher oligomers were isolated by means of high-speed countercurrent chromatography and their structure was elucidated by two dimensional NMR spectroscopy. In addition to the ethyliden-catechin dimers of the anthocyanins malvidin-3-glucoside, petunidin-3-glucoside, delphinidin-3-glucoside, and cyanidin-3-glucoside, a trimer consisting of two catechin units and a central cyanidin-3-glucoside unit could be isolated and structurally characterized. An ethyliden bridged trimer containing an anthocyanin with ethyliden bridges at positions 6 and 8 is presented here for the first time. This structure confirms the possibility of branched oligomers and oligomers with more than two anthocyanin units that may account for color stabilization of red wine. The color stability of the isolated compounds was examined in model wine solution.