NMR and LC-MSn characterisation of two minor saponins from Ilex paraguariensis.

Two minor saponins obtained from the methanolic extract of the leaves of Ilex paraguariensis have been characterised by 13C-NMR, 1H-NMR, API-MS and chemical hydrolysis as oleanolic acid-3-O-(beta-D-glucopyranosyl-(1-->3)-alpha-L-arabinopyranosyl)-(28-->1)- beta-D-glucopyranosyl ester (guaiacin B) and oleanolic acid-3-O-(beta-D-glucopyranosyl-(1-->3)-(alpha-L-rhamnopyranosyl- (1-->2))-alpha-L-arabinopyranosyl)-(28-->1)-beta-D-glucopyranosyl ester (nudicaucin C). Both are isomeric forms of the known matesaponins 1 (MSP 1) and 2 (MSP 2) and differ only by the nature of the aglycone: they have oleanolic acid instead of ursolic acid, as found in the matesaponins. These minor saponins have not been fully separated from their major isomers MSP 1 and 2 and were characterised by in-mixture NMR analysis, LC-MS and LC-MSn experiments.

NMR chemical shifts of the rhodopsin chromophore in the dark state and in bathorhodopsin: a hybrid QM/MM molecular dynamics study.

We investigate nuclear magnetic resonance (NMR) parameters of the rhodopsin chromophore in the dark state of the protein and in the early photointermediate bathorhodopsin via first-principles molecular dynamics simulations and NMR chemical shift calculations in a hybrid quantum/classical (QM/MM) framework. NMR parameters are particularly sensitive to structural properties and to the chemical environment, which allows us to address different questions about the retinal chromophore in situ. Our calculations show that both the 13C and the 1H NMR chemical shifts are rather insensitive to the protonation state of Glu181, an ionizable amino acid side chain located in the vicinity of the isomerizing 11-cis bond. Thus, other techniques should be better suited to establish its protonation state. The calculated chemical shifts for bathorhodopsin further support our previously published theoretical structure, which is in very good agreement with more recent X-ray data.

The power of NMR.: in two and three dimensions

Soon after its discovery in the 1950s, NMR had become an indispensable tool fr chemists. In the 1970s and 1980s, the power of the technique was extended from one dimension to two and even three dimensions, opening up exciting applkications in both chemistry and biochemistry. the success of one dimensional. high-resolution NMR stems from the unique insights that it can provide about molecular structure. The chemical shift of a nucleus gives invaluable information abut the chemical environment in which that nucleus is located, Coupling interactions between hydorgen nuclei, as revealed by characteristic splitting patterns inthe 1H-NMR spectrum, provide informaton about the loaction of one group of hydorgen atoms relative to others inthe molecule. And the nuclearf Overhauser effect (nOe) can shed light on molecular stereochemistry.

Application of on-line C30 RP-HPLC-NMR for the analysis of flavonoids from leaf extract of Maytenus aquifolium

The application of on-line C30-reversed-phase high-pressure liquid chromatography-nuclear magnetic resonance spectroscopy is described for the analysis of tetraglycosylated flavonoids in aqueous and hydroalcoholic extracts of the leaves of Maytenus aquifolium (Celastraceae). Triacontyl stationary phases showed adequate separation for on-line 1H-NMR measurements at 600 MHz and allowed the characterisation of these flavonoids by detection of both aromatic and anomeric proton signals. Copyright (C) 2000 John Wiley and Sons, Ltd.

Determination of saturates, aromatics and ethanol in Brazilian commercial gasoline with low olefin content using 1 H NMR spectroscopy

In this work, a Hydrogen Nuclear Magnetic Resonance (1H NMR) method has been developed to determine aromatics and ethanol in Brazilian commercial gasoline with low olefin content. The proposed method involves subdividing an 1H NMR spectrum into regions, each of which is assumed to be associated with a specific type of structural group (OH, CH, CH2 and CH3). The method is based on the assignment of overlapping regions of 1H NMR spectra due to the signals of naphthene (N), iso and normal paraffins (P) and ethanol (E). Each 1H NMR spectrum was divided into 8 regions and the integration was correlated to the percentage of the substances to be determined. The results of the analysis by 1H NMR were compared with analysis of GC-FID obtained with the PONA system. The proposed technique of 1H NMR was shown to be an appropriate method for this sample type.

Perfil metabólico por RMN de 1H e avaliação citotóxica do fungo endofítico Colletotrichum gloeosporioides isolado de Eugenia jambolana, em diferentes meios de cultivo

Pós-graduação em Química - IQ

Structure and dynamics of supramolecular assemblies studied by advanced solid-state NMR spectroscopy

Ziel der vorliegenden Arbeit ist die Aufklärung von Struktur und Dynamik komplexer supramolekularer Systeme mittels Festkörper NMR Spektroskopie. Die Untersuchung von pi-pi Wechselwirkungen, welche einen entscheidenden Einfluss auf die strukturellen und dynamischen Eigenschaften supra- molekularer Systeme haben, hilft dabei, die Selbst- organisationsprozesse dieser komplexen Materialien besser zu verstehen. Mit dipolaren 1H-1H and 1H-13C Wiedereinkopplungs NMR Methoden unter schnellem MAS können sowohl 1H chemische Verschiebungen als auch dipolare 1H-1H und 1H-13C Kopplungen untersucht werden, ohne dass eine Isotopenmarkierung erforderlich ist. So erhält man detaillierte Informationen über die Struktur und die Beweglichkeit einzelner Molekül- segmente. In Verbindung mit sogenannten nucleus independent chemical shift (NICS) maps (berechnet mit ab-initio Methoden) lassen sich Abstände von Protonen relativ zu pi-Elektronensystemen bestimmen und so Strukturvorschläge ableiten. Mit Hilfe von homo- und heteronuklearen dipolaren Rotationsseitenbandenmustern könnenaußerdem Ordnungs- parameter für verschiedene Molekülsegmente bestimmt werden. Die auf diese Weise gewonnenen Informationen über die strukturellen und dynamischen Eigenschaften supramolekularer Systeme tragen dazu bei, strukturbestimmende Molekül- einheiten und Hauptordnungsphänomene zu identifizieren sowie lokale Wechselwirkungen zu quantifizieren, um so den Vorgang der Selbstorganisation besser zu verstehen.

13C NMR of a single molecule magnet: analysis of pseudocontact shifts and residual dipolar couplings

Paramagnetic triple decker complexes of lanthanides are promising Single Molecule Magnets (SMMs), with many potential uses. Some of them show preferable relaxation behavior, which enables the recording of well resolved NMR spectra. These axially symmetric complexes are also strongly magnetically anisotropic, and this property can be described with the axial component of the magnetic susceptibility tensor, χa. For triple decker complexes with phthalocyanine based ligands, the Fermi˗contact contribution is small. Hence, together with the axial symmetry, the experimental chemical shifts in 1H and 13C NMR spectra can be modeled easily by considering pseudocontact and orbital shifts alone. This results in the determination of the χa value, which is also responsible for molecular alignment and consequently for the observation of residual dipolar couplings (RDCs). A detailed analysis of the experimental 1H-13C and 1H-1H couplings revealed that contributions from RDCs (positive and negative) and from dynamic frequency shifts (negative for all observed couplings) have to be considered. Whilst the pseudocontact shifts depend on the average positions of 1H and 13C nuclei relative to the lanthanide ions, the RDCs are related to the mobility of nuclei they correspond to. This phenomenon allows for the measurement of the internal mobility of the various groups in the SMMs.

Solid-state NMR investigation of phosphonic acid based proton conducting materials

In this work, new promising proton conducting fuel cell membrane materials were characterized in terms of their structure and dynamic properties using solid-state nuclear magnetic resonance (NMR) spectroscopy and X-ray diffraction. Structurally different, phosphonic acid (PA) containing materials were systematically evaluated for possible high-temperature operation (e.g. at T>100°C). Notably, 1H, 2H and 31P magic angle spinning (MAS) NMR provided insight into local connectivities and dynamics of the hydrogen bonded network, while packing arrangements were identified by means of heteronuclear dipolar recoupling techniques.rnThe first part of this work introduced rather crystalline, low molecular weight ionomers for proton conducting membranes, where six different geometries such as line, triangle, screw, tetrahedron, square and hexagon, were investigated. The hexagon was identified as the most promising geometry with high-temperature bulk proton conductivities in the range of 10-3 Scm-1 at a relative humidity of 50%. However, 2H NMR and TGA-MS data suggest that the bulk proton transport is mainly due to the presence of crystal water. Single crystal X-ray data revealed that in the tetrahedron phosphonic acids form tetrameric clusters isolating the mobile protons while the phosphonic acids in the hexagon form zigzag-type pathways through the sample.rnThe second part of this work demonstrates how acid-base pairing and the choice of appropriate spacers may influence proton conduction. Different ratios of statistical copolymers of poly (vinylphosphonic acid) and poly (4-vinylpyridine) were measured to derive information about the local structure and chemical changes. Though anhydrous proton conductivities of all statistical copolymers are rather poor, the conductivity increases to 10-2 S cm-1 when exposing the sample to relative humidity of 80%. In contrast to PVPA, anhydride formation of phosphonic acids in the copolymer is not reversible even when exposing the sample to a relative humidity of 100%.rnIn addition, the influence of both spacers and degree of backbone crystallinity on bulk proton conductivity was investigated. Unlike in systems such as poly benzimidazole (PBI), spacers were inserted between the protogenic groups along the backbone. It was found that dilution of the protogenic groups decreases the conductivity, but compared to PVPA, similar apparent activation energies for local motions were obtained from both variable temperature 1H NMR and impedance spectroscopy data. These observations suggest the formation of phosphonic acid clusters with high degrees of local proton motion, where only a fraction of motions contribute to the observable bulk proton conductivity. Additionally, it was shown that gradual changes of the spacer length lead to different morphologies.rnIn summary, applying advanced solid-state NMR and X-ray analysis, structural and dynamic phenomena in proton conducting materials were identified on a molecular level. The results were discussed with respect to different proton conduction mechanisms and may contribute to a more rational design or improvement of proton conducting membranes.rn

Solid-state NMR on columnar supramolecular structures

The present work deals with the characterisation of three columnar self-assembled systems, that is, benzene-1,3,5-tricarboxamides, a peripherally thioalkyl-substituted phthalocyanine, and several oligo-(p-phenylenevinylene)s. In order to probe the supramolecular organisation solid-state NMR has been used as the main technique, supported by X-ray measurements, theoretical methods, and thermal analysis. rnrnBenzene-1,3,5-tricarboxamides (BTAs) turned out to be well suited model compounds to study various fundamental supramolecular interactions, such as π-π-interactions, hydrogen bonding, as well as dynamic and steric effects of attached side chains. Six BTAs have been investigated in total, five with a CO-centred amide group bearing different side chains and one with an inverted N-centred amide group. The physical properties of these BTAs have been investigated as a function of temperature. The results indicated that in case of the CO-centred BTAs the stability of the columnar mesophase depends strongly on the nature of the side chains. Further experiments revealed a coplanar orientation of adjacent BTA molecules in the columnar assembly of CO-centred BTAs, whereas the N-centred BTA, showed a deviating not fully coplanar arrangement. These differences were ascribed to distinct hydrogen bonding schemes, involving a parallel alignment of hydrogen bonds in case of CO-centred BTAs and an antiparallel alignment in case of the N-centred counterpart.rnrn The fundamental insights of the supramolecular organisation of BTAs could be partially adapted to an octa-substituted phthalocyanine with thiododecyl moieties. Solid-state NMR in combination with chemical shift calculations determined a tilted herringbone arrangement of phthalocyanine rings in the crystalline phase as well as in the mesophase. Moreover, 1H NMR measurements in the mesophase of this compound suggested an axial rotation of molecules, which is inhibited in the crystalline phase.rnrnAs a third task, the supramolecular assembly of oligo-(p-phenylenevinylene)s of varying length and with different polar head groups have been investigated by a combined X-ray and solid-state NMR study. The results revealed a columnar structure formation of these compounds, being promoted by phase separation of alkyl side chains and aromatic rigid rods. In this system solid-state NMR yielded meaningful insight into the isotropisation process of butoxy and 2-S-methylbutoxy substituted oligo-(p-phenylenevinylene) rods.rn

Festkörper-NMR an polymeren Kompositmaterialien

In dieser Arbeit wurden polymere Kompositmaterialien mit Hilfe von Festkörper-NMR-Techniken untersucht, um den Einfluß von Polymer-Festkörper-Kontakten auf molekulare Materialeigenschaften zu betrachten. Dabei wurden sowohl Analysen am Polymer als auch am Füllmaterial durchgeführt.rnrnIm ersten Teil der Arbeit wurde die Dynamik von Poly(ethylmethacrylat) (PEMA) in sphärischen Bürstenpartikeln gemessen. Diese Bürsten bestanden aus einem Poly(silsesquioxan)-Kern und verpfropften PEMA-Ketten, die über ATRP (atom transfer radical polymerization) an verschiedenen Kettensequenzen mit 13C an der Carboxylgruppe markiert wurden. Statische 13C-NMR-Messungen konnten zeigen, dass die Dynamik dieser Sequenzen unabhängig vom Abstand zur Oberfläche verlangsamt ist, was auf eine eingeschränkte Reptation zurückgeführt wurde.rnrnDer zweite Teil der Arbeit beschäftigt sich mit den molekularen Unterschieden von Silika-Naturkautschuk-Kompositen, die über mechanisches Mischen bzw. über eine Sol-Gel-Reaktion hergestellt wurden. Durch kinetische 1H-NMR-Messungen wurde der Umsatz der Sol-Gel-Reaktion bestimmt. Mittels heteronuklearen 29Si{1H}-NMR-Korrelationsexperimenten wurde ein direkter räumlicher Kontakt zwischen dem Inneren der Partikel und dem Polymer nachgewiesen. Dies belegt experimentell, dass im Kompositmaterial die Polymerketten in den durch Sol-Gel-Reaktion hergestellten Silikapartikeln eingeschlossen sind.

Time-dependent interactions of the two porphyrinic compounds chlorin e6 and mono-L-aspartyl-chlorin e6 with phospholipid vesicles probed by NMR spectroscopy

The distribution processes of chlorin e6 (CE) and monoaspartyl-chlorin e6 (MACE) between the outer and inner phospholipid monolayers of 1,2-dioleoyl-phosphatidylcholine (DOPC) vesicles were monitored by 1H NMR spectroscopy through analysis of chemical shifts and line widths of the DOPC vesicle resonances. Chlorin adsorption to the outer vesicle monolayer induced changes in the DOPC 1H NMR spectrum. Most pronounced was a split of the N-methyl choline resonance, allowing for separate analysis of inner and outer vesicle layers. Transbilayer distribution of the chlorin compounds was indicated by time-dependent characteristic spectral changes of the DOPC resonances. Kinetic parameters for the flip-flop processes, that is, half-lives and rate constants, were obtained from the experimental data points. In comparison to CE, MACE transbilayer movement was significantly reduced, with MACE remaining more or less attached to the outer membrane layer. The distribution coefficients for CE and MACE between the vesicular and aqueous phase were determined. Both CE and MACE exhibited a high affinity for the vesicular phase. For CE, a positive correlation was found between transfer rate and increasing molar ratio CE/DOPC. Enhanced membrane rigidity induced by increasing amounts of cholesterol into the model membrane was accompanied by a decrease of CE flip-flop rates across the membrane. The present study shows that the movement of porphyrins across membranes can efficiently be investigated by 1H NMR spectroscopy and that small changes in porphyrin structure can have large effects on membrane kinetics.

Interactions of Polyvinylpyrrolidone with Chlorin e6-Based Photosensitizers Studied by NMR and Electronic Absorption Spectroscopy

Polyvinylpyrrolidone (PVP) can act as potential drug delivery vehicle for porphyrin-based photosensitizers in photodynamic therapy (PDT) to enhance their stability and prevent porphyrin self-association. In the present study the interactions of PVP (MW 10 kDa) were probed with five different derivatives of chlorin e6 (CE6) bearing either one of the amino acids serine, lysine, tyrosine or arginine, or monoamino-hexanoic acid as substituent. All derivatives of CE6 (xCE) formed aggregates of a similar structure in aqueous buffer in the millimolar range. In the presence of PVP monomerization of all xCE aggregates could be proved by 1H NMR spectroscopy. xCE-PVP complex formation was confirmed by 1H NMR T2 relaxation and diffusion ordered spectroscopy (DOSY). 1H1H-NOESY data suggested that the xCE uptake into the PVP polymer matrix is governed by hydrophobic interactions. UV–vis absorption and fluorescence emission bands of xCE in the micromolar range revealed characteristic PVP-induced bathochromic shifts. The presented data point out the potential of PVP as carrier system for amphiphilic derivatives of chlorin e6. The capacity of PVP to monomerize xCE aggregates may enhance their efficiency as possible photosensitizers in PDT.

NMR investigation of ferricytochrome c unfolding: Detection of an equilibrium unfolding intermediate and residual structure in the denatured state

Horse ferricytochrome c (cyt c) undergoes exchange of one of its axial heme ligands (Met-80) for one or more non-native ligands under denaturing conditions. We have used 1H NMR spectroscopy to detect two conformations of paramagnetic cyt c with non-native heme ligation through a range of urea concentrations. One non-native form is an equilibrium unfolding intermediate observed under partially denaturing conditions and is attributed to replacement of Met-80 with one or more Lys side chains. The second non-native form, in which the native Met ligand is replaced by a His, is observed under strongly denaturing conditions. Thermodynamic analysis of these data indicates a relatively small ΔG (17 kJ/mol) for the transition from native to the Lys-ligated intermediate and a significantly larger ΔG (47 kJ/mol) for the transition from native to the His-ligated species. Although CD and fluorescence data indicate that the equilibrium unfolding of cyt c is a two-state process, these NMR results implicate an intermediate with His-Lys ligation.

From traditional resource to global commodities: a comparison of Rhodiola species using NMR spectroscopy - metabolomics and HPTLC

The fast developing international trade of products based on traditional knowledge and their value chains has become an important aspect of the ethnopharmacological debate. The structure and diversity of value chains and their impact on the phytochemical composition of herbal medicinal products has been overlooked in the debate about quality problems in transnational trade. Different government policies and regulations governing trade in herbal medicinal products impact on such value chains. Medicinal Rhodiola species, including Rhodiola rosea L. and Rhodiola crenulata (Hook.f. & Thomson) H.Ohba, have been used widely in Europe and Asia as traditional herbal medicines with numerous claims for their therapeutic effects. Faced with resource depletion and environment destruction, R. rosea and R. crenulata are becoming endangered, making them more economically valuable to collectors and middlemen, and also increasing the risk of adulteration and low quality. We compare the phytochemical differences among Rhodiola raw materials available on the market to provide a practical method for Rhodiola authentication and the detection of potential adulterant compounds. Samples were collected from Europe and Asia and nuclear magnetic resonance spectroscopy coupled with multivariate analysis software and high performance thin layer chromatography techniques were used to analyse the samples. A method was developed to quantify the amount of adulterant species contained within mixtures. We compared the phytochemical composition of collected Rhodiola samples to authenticated samples. Rosavin and rosarin were mainly present in R. rosea whereas crenulatin was only present in R. crenulata. 30% of the Rhodiola samples purchased from the Chinese market were adulterated by other Rhodiola spp. Moreover, 7 % of the raw-material samples were not labelled satifactorily. The utilisation of both 1H-NMR and HPTLC methods provided an integrated analysis of the phytochemical differences and novel identification method for R. rosea and R. crenulata. Using 1H-NMR spectroscopy it was possible to quantify the presence of R. crenulata in admixtures with R. rosea. This quantitative technique could be used in the future to assess a variety of herbal drugs and products. This project also highlights the need to further study the links between producers and consumers in national and trans-national trade.