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The study of the provenance of dolomitic marble artefacts has become relevant since it was discovered that quarries of this marble other than that of Cape-Vathy located on the island of Thasos have been exploited since Antiquity. To improve our knowledge about the provenance of materials and the extent of their dispersion, multiple archaeometric studies were performed in the past including isotope analyses, petrography, cathodoluminescence, and elemental analyses. In the present work, solid-state nuclear magnetic resonance (NMR) spectroscopy has been added to this panel of techniques. NMR allows the characterization of the material at a molecular level by looking at different nuclei: carbon, magnesium, and calcium. Statistical analysis of the data collected on both quarry samples and archaeologic items was also implemented and clearly demonstrates the efficiency of a holistic approach for provenance elucidation. Finally, the first 25 Mg NMR tests have shown the potential of this technique to discriminate between dolomitic marbles of different provenance. The results are discussed in terms of their historical meaning and illustrate the exploitation of sources of dolomitic marbles other than the Greek Thasos source., Competing Interests: The authors declare no conflict of interest.

A series of mono-, di-, and tri-topic receptors in which H-bonding sites, complementary to those of barbituric acid (BA), are fused is used to induce the supramolecular assembly of n×m ladders containing 1, 2, or 3 triphenylenevinylene units appended with BA. The topological constraint enforced by the architectures induces through-space interactions between the electroactive moieties that are reflected in the electronic absorption and emission spectrum. The n=2, m=2 or m=3 architectures undergo two single electron oxidation events, indicative of the formation of the corresponding mono- and di-radical cation species with comproportionation constants of 340 and 70, respectively. Comparison of the electrochemical potentials suggests that the charges are delocalized over the electroactive units in the assembly., (© 2020 Wiley-VCH GmbH.)

Genome mining of the Gram-negative bacterium Pseudomonas fluorescens Pf0-1 showed that the strain possesses a silent NRPS-based biosynthetic gene cluster encoding a new lipopeptide; its activation required the repair of the global regulator system. In this paper, we describe the genomics-driven discovery and characterization of the associated secondary metabolite gacamide A, a lipodepsipeptide that forms a new family of Pseudomonas lipopeptides. The compound has a moderate, narrow-spectrum antibiotic activity and facilitates bacterial surface motility.

A molecular barbiturate messenger, which is reversibly released/captured by a photoswitchable artificial molecular receptor, is shown to act as an effector to control ring gliding on a distant hydrogen-bonding [2]rotaxane. Thus, light-driven chemical communication governing the operation of a remote molecular machine is demonstrated using an information-rich neutral molecule.

Polyphenols such as Epigallocatechin-3 gallate (EGCG) are currently bearer of hope to prevent or at least to slow down the deleterious effect of Tauopathies such as Alzheimer disease. One of the main effects of these neurodegenerative pathologies is the hyperphosphorylation and consequent aggregation of the Tau protein that leads to the irremediable neuronal cells death. In the present paper, we show how EGCG can play a crucial role to prevent Tau aggregation: (i) in binding Tau in its phosphorylation region with an affinity of the same order of magnitude than kinases (0.5 mM), hindering their access to the protein and (ii) in modifying the 3D-structure of Tau whose preferential conformation changes in the presence of EGCG. For this purpose, two peptides were synthesized, one of 20 residues long issued from the first Proline-rich region of Tau (171Ile-190Lys), the second of 50 residues long (171Ile-220Thr) corresponding to more than 50% of the Tau Proline rich domaine. The total attribution of all the 1H, 13C and 15N resonances of the two peptides has been achieved thanks to a "divide and conquer" strategy leading to their 3D structure preference and their affinity towards EGCG., Competing Interests: The authors declare to have no conflict of interests

Gaining insight into intermolecular interactions between multiple species is possible at an atomic level by looking at different parameters using different NMR techniques. In the specific case of the astringency sensation, in which at least three molecular species are involved, different NMR techniques combined with dynamic light scattering and molecular modeling contribute to decipher the role of each component in the interaction mode and to assess the thermodynamic parameters governing this complex interaction. The binding process between a saliva peptide, a polyphenol, and polysaccharides was monitored by following 1 H chemical shift variations, changes in NMR peak areas, and size of the formed complex. These NMR experiments deliver a complete picture of the association pathway, assessed by dynamic light scattering and molecular dynamics simulations: all of the data collected converge toward a comprehensive mode of interaction in which sugars indirectly play a role in astringency by sequestering part of the polyphenols, reducing their effective concentration to bind saliva proteins.

Endophytic Pseudomonas poae strain RE*1-1-14 was originally isolated from internal root tissue of sugar beet plants and shown to suppress growth of the fungal pathogen Rhizoctonia solani both in vitro and in the field. To identify genes involved in its biocontrol activity, RE*1-1-14 random mutagenesis and sequencing led to the identification of a nonribosomal peptide synthetase (NRPS) gene cluster predicted to encode a lipopeptide (LP) with a 10-amino-acid peptide moiety. The two unlinked gene clusters consisted of three NRPS genes, designated poaA (cluster 1) and poaB and poaC (cluster 2), spanning approximately 33.7 kb. In silico analysis followed by chemical analyses revealed that the encoded LP, designated poaeamide, is a structurally new member of the orfamide family. Poaeamide inhibited mycelial growth of R. solani and different oomycetes, including Phytophthora capsici, P. infestans, and Pythium ultimum. The novel LP was shown to be essential for swarming motility of strain RE*1-1-14 and had an impact on root colonization of sugar beet seedlings The poaeamide-deficient mutant colonized the rhizosphere and upper plant cortex at higher densities and with more scattered colonization patterns than the wild type. Collectively, these results indicate that Pseudomonas poae RE*1-1-14 produces a structurally new LP that is relevant for its antagonistic activity against soilborne plant pathogens and for colonization of sugar beet roots.

Sensory properties of red wine tannins are bound to complex interactions between saliva proteins, membranes taste receptors of the oral cavity, and lipids or proteins from the human diet. Whereas astringency has been widely studied in terms of tannin-saliva protein colloidal complexes, little is known about interactions between tannins and lipids and their implications in the taste of wine. This study deals with tannin-lipid interactions, by mimicking both oral cavity membranes by micrometric size liposomes and lipid droplets in food by nanometric isotropic bicelles. Deuterium and phosphorus solid-state NMR demonstrated the membrane hydrophobic core disordering promoted by catechin (C), epicatechin (EC), and epigallocatechin gallate (EGCG), the latter appearing more efficient. C and EGCG destabilize isotropic bicelles and convert them into an inverted hexagonal phase. Tannins are shown to be located at the membrane interface and stabilize the lamellar phases. These newly found properties point out the importance of lipids in the complex interactions that happen in the mouth during organoleptic feeling when ingesting tannins.

Chromatographic separation using fluorescence as a detection mode revealed, besides a series of flavan-3-ols, the recurrent presence of an undefined compound in Bordeaux red wine. Its isolation and structure characterization by complementary means (high-resolution mass spectrometry, nuclear magnetic resonance, and chemical synthesis) has permitted us to identify it as the nitrogen-containing glycoconjugate 3-indolyl-(2R)-O-β-D-glucosyl-lactic acid. Its quantification was performed for different wines of different vine varieties and terroirs with the aim to assess whether this compound may be used as a terroir, variety, or wine process tag.

Three new aromatic acids, named lahorenoic acids A (1), B (2), and C (3), have been isolated along with the known compounds phenazine-1-carboxylic acid (4), 2-hydroxyphenazine-1-carboxylic acid (5), 2-hydroxyphenazine (6), 2,8-dihydroxyphenazine (7), cyclo-Pro-Tyr (8), cyclo-Pro-Val (9), cyclo-Pro-Met (10), and WLIP (11) and characterized from the biocontrol strain Pseudomonas aurantiaca PB-St2. The structures of these compounds were deduced by 1D and 2D NMR spectroscopic and mass spectral data interpretation. Compounds 2, 4, and 7 showed moderate antibacterial activity against mycobacteria and other Gram-positive bacteria, while 4 was also found to exhibit cytotoxic and antifungal properties.

While the definition of tannins has been historically associated with its propensity to bind proteins in a nonspecific way, it is now admitted that specific interaction also occurs. The case of the astringency perception is a good example to illustrate this phenomenon: astringency is commonly described as a tactile sensation induced by the precipitation of a complex composed of proline-rich proteins present in the human saliva and tannins present in beverages such as tea or red wines. In the present work, the interactions between a human saliva protein segment and three different procyanidins (B1, B3, and C2) were investigated at the atomic level by NMR and molecular dynamics. The data provided evidence for (i) an increase in affinity compared to shortest human saliva peptides, which is accounted for by protein "wraping around" the tannin, (ii) a specificity in the interaction below tannin critical micelle concentration (CMC) of ca. 10 mM, with an affinity scale such that C2 > B1 > B3, and (iii) a nonspecific binding above tannin CMC that conducts irremediably to the precipitation of the tannins/protein complex. Such physicochemical findings describe in accurate terms saliva protein-tannin interactions and provide support for a more subtle description by oenologists of wine astringency perception in the mouth.

Association of procyanidins with enzymes has drawn attention over the past few years. This work aimed to bring insights on interaction of the protease trypsin with the procyanidin dimer (B3). This interaction was characterized by fluorescence quenching, saturation transfer difference (STD) NMR, molecular modeling, and through an enzymatic inhibition assay. Further studies were conducted regarding the influence of pectin on the binding process. A general overview of the binding process may be outlined as follows: a) at low procyanidin concentrations (below the critical micellar concentration-(CMC)) a specific interaction probably driven by hydrogen bonds between the protein backbone and the procyanidin occurs and is associated with the reduction of both enzyme activity and fluorescence; b) at high procyanidin concentration (above the CMC) the interaction becomes nonspecific. This variation in both nature and extent of the interaction with the variation of procyanidin concentration shows how tannin self-association may affect the interaction between tannins and proteins. It was also shown that the mechanism through which pectin affects the interaction between procyanidin B3 and trypsin is of a competitive type.

Tannins represent a key element in red wine flavors, so researchers have made a lot of effort to try to understand the role of their structure in wine taste in recent decades. We report some new routes to achieve a true structure-taste relationship for the major tannins found in wine, which are procyanidins in their monomeric or oligomeric state. All these routes use synthetic standards. New advances in their synthesis and their analyses using chromatographic methods, NMR spectroscopy, and mass spectrometry to obtain more precise information about their chemical structure, including their stereochemistry and their precise degree of polymerization and galloylation, are described.

Astringency is a sensation in the mouth used in judging the quality of red wine. The rough, dry, and puckering sensation called astringency is the result of an interaction between tannins and saliva proteins, mainly proline-rich proteins (PRP), which leads to the formation and precipitation of a complex. A dry and rough sensation is then perceived in the mouth. To get an insight into astringency at the molecular level we investigated: (i) An efficient and iterative method for 4-8 procyanidin synthesis, which gives rise to all possible 4-8 procyanidins up to the tetramer with total control of degree of oligomerization and stereochemistry. (ii) The 3D-structural preferences, which take into account their internal movements, using 2D NMR and molecular modeling. (iii) The self-association process in water or hydroalcoholic solutions using diffusion NMR spectroscopy that gives the active proportion of tannins able to fix proteins. (iv) A comprehensive description of the PRP-procyanidin complex formation to get information about stoichiometry, binding site localization, and affinity constants for different procyanidins. The data collected suggest that the interactions are controlled by both procyanidin conformational and colloidal state preferences. All these results provide new insights into the molecular interpretation of tannin astringency., (© Georg Thieme Verlag KG Stuttgart · New York.)

In organoleptic science, the association of tannins to saliva proteins leads to the poorly understood phenomenon of astringency. To decipher this interaction at molecular and colloidal levels, the binding of 4 procyanidin dimers (B1-4) and 1 trimer (C2) to a human saliva proline-rich peptide, IB7(14), was studied. Interactions have been characterized by measuring dissociation constants, sizes of complexes, number, and nature of binding sites using NMR (chemical shift variations, diffusion-ordered spectroscopy, and saturation transfer diffusion). The binding sites were identified using molecular mechanics, and the hydrophilic/hydrophobic nature of the interactions was resolved by calculating the molecular lipophilicity potential within the complexes. The following comprehensive scheme can be proposed: 1) below the tannin critical micelle concentration (CMC), interaction is specific, and the procyanidin anchorage always occurs on the same three IB7(14) sites. The tannin 3-dimensional structure plays a key role in the binding force and in the tannin's ability to act as a bidentate ligand: tannins adopting an extended conformation exhibit higher affinity toward protein and initiate the formation of a network. 2) Above the CMC, after the first specific hydrophilic interaction has taken place, a random hydrophobic stacking occurs between tannins and proteins. The whole process is discussed in the general frame of wine tannins eliciting astringency.

Condensation reactions of malvidin-3-glucoside with two representative oak wood furanic aldehydes (furfural and methylfurfural) were conducted in wine-like model solutions. Methylfurfural led to the formation of malvidin-3-glucoside-methylfurfural (603 m/z), whereas furfural led to the formation of malvidin-3-glucoside-furfural (589 m/z). The latter was structurally characterized by 1D and 2D NMR, allowing an elucidation of the formation mechanism of these anthocyanin-furanic aldehyde adducts in the absence of flavanols.

The mechanism of the ring-opening polymerization (ROP) of lactide catalyzed by two partner hydrogen-bonding organocatalysts was explored. New amidoindoles 4 a,c, thioamidoindoles 4 b,d, amidobenzimidazoles 5 a,c, and thioamidobenzimidazoles 5 b,c were synthesized and used as activators of the monomer. In the solid state and in solution, compounds 4 and 5 showed a propensity for self-association, which was evaluated. (Thio)Amides 4 and 5 do catalyze the ROP of lactide in the presence of a cocatalyst, tertiary amine 3 a or 3 b, which activates the growing polymer chain through hydrogen-bonding. Reactions were conducted in 2-24 h at 20 degrees C; conversion yields ranged between 22 and 100 %. A detailed study of the intermolecular interactions undertaken between the participating species showed that, as expected, simultaneous weak hydrogen bonds do exist to activate the reagents. Moreover, interactions have been revealed between the partner catalysts 4/5+3. ROP catalyzed by these partner activators is thus governed by multiple dynamic equilibria. The latter should be judiciously adjusted to fine-tune the catalytic properties of (thio)amides and organocatalysts, more generally.

Together with leucoanthocyanidin reductase, anthocyanidin reductase (ANR) is one of the two enzymes of the flavonoid-biosynthesis pathway that produces the flavan-3-ol monomers required for the formation of proanthocyanidins or condensed tannins. It has been shown to catalyse the double reduction of anthocyanidins to form 2R,3R-flavan-3-ols, which can be further transformed to the 2S,3R isomers by non-enzymatic epimerization. ANR from grape (Vitis vinifera) was expressed in Escherichia coli and purified. Unexpectedly, RP-HPLC, LC-MS and NMR experiments clearly established that the enzyme produces a 50:50 mixture of 2,3-cis and 2,3-trans flavan-3-ols which have been identified by chiral chromatography to be 2S,3S- and 2S,3R-flavan-3-ols, i.e. the naturally rare (+)-epicatechin and (-)-catechin, when cyanidin is used as the substrate of the reaction. The first three-dimensional structure of ANR is described at a resolution of 2.2 A and explains the inactivity of the enzyme in the presence of high salt concentrations.

The colloidal behavior of eight synthetic procyanidins (three monomers, four dimers, and a trimer) has been investigated in water or in a winelike medium using DOSY NMR spectroscopy and molecular dynamics simulations. Different behavior was observed for monomers and oligomers. Monomers self-associate with a high affinity constant (37-53 M(-1)) to form micelles at low cmc (critical micelle concentration) values (1-5 g.L(-1)). These micelles undergo a time-dependent coalescence process to form hazes and precipitates. As for dimers and the trimer, self-association also occurs but with a lower affinity (approximately 6 M(-1)) and at higher cmc values (10-20 g.L(-1)) to form small micelles (<5 nm) that remain stable throughout the experiment. The presence of 10% ethanol does not significantly affect the self-association constant for monomers and oligomers but increases their cmc values by approximately 50% and decreases the micelle size by a factor 2. However, the presence of 20 mM NaCl appears to negate the effect of ethanol. This study helps to clarify the role of procyanidin monomers versus oligomers in wine turbidity and demonstrates that procyanidin oligomers are fully available to interact with saliva proteins.

Water-soluble benzoate-terminated dendrimers of four generations (from G0 with 9 branches to G3 with 243 branches) were synthesized and fully characterized. They form water-soluble assemblies by ion-pairing interactions with three cations of medicinal interest (acetylcoline, benzyltriethylammonium, and dopamine), which were characterized and investigated by 1H NMR spectroscopy, whereas such interactions do not provoke any significant shift of 1H NMR signals with the monomeric benzoate anion. The calculated association constants confirm that the dendritic carboxylate termini reversibly form ion pairs and aggregates. Diffusion coefficients and hydrodynamic diameters of the dendrimers, as well as changes thereof on interaction with the cations, were evaluated by DOSY experiments. The lack of increase of dendrimer size on addition of the cations and the upfield shifts of the 1H NMR signals of the cation indicate encapsulation within the hydrophobic dendrimer interiors together with probable backfolding of the benzoate termini.

A new water-soluble polyanionic dendrimer containing 81 benzoate termini (diameter: 11+/-1 nm from DOSY NMR spectroscopy) has been synthesized; it interacts with acetylcholine cations in water-soluble assemblies in which each carboxylate terminus reversibly forms contact ion pairs and aggregates at the tether termini, as shown by 1H NMR spectroscopy.

The spontaneous emulsification of alcoholic solutions of trans-anethole (t-A) in water is investigated using EXSY and DOSY NMR techniques. The system investigated (5-10 mM t-A in 5% EtOH/H2O solution) is exceptional in providing sharp, clearly resolved signals for both t-A that is dissolved in the aqueous phase (free t-A) and t-A that is incorporated in aggregates (3-6 nm diameter) thus allowing both fractions to be probed simultaneously. This feature is utilized to explore the initial events that occur during the spontaneous emulsification process. Upon mixing, the majority of the t-A (ca. 75%) undergoes nucleation to form small aggregates (ca. 10 nm diameter), while 15% (corresponding to [t-A] = 7.5 x 10(-4) M) is dissolved in the aqueous phase. The kinetic rates governing the exchange process between aggregated and free t-A are found to be time-dependent and slow on the NMR time scale (k = 0.8-2 s(-1)). DOSY experiments indicate that the initially formed small aggregates undergo rapid coalescence to form larger droplets. Ostwald ripening of these droplets at the expense of the remaining small aggregates is responsible for the subsequent, slower time-evolution of the system.

The structure and conformation of two native procyanidin trimers in water have been determined using 2D NMR and molecular mechanics. The results show the existence of four rotameric forms, one of which is predominant (60 to 80%). These four rotamers are shown to be in slow to intermediate exchange on the NMR timescale. Both trimers, whose structures vary owing to a different substitution of one carbon atom, adopt conformations in which stacking between different phenolic rings is favored., (Copyright (c) 2006 John Wiley & Sons, Ltd.)

Alpha-amylase, a major human salivary protein, and IB8c, a representative of the proline-rich proteins, were obtained by isolation from saliva and by solid-phase synthesis, respectively. The interactions between these proteins and condensed tannins isolated from grape seeds were studied at different protein and tannin concentrations by measuring their aggregation. Pectic polysaccharides were isolated from wine, and their effect on protein tannin aggregation was assessed. The results presented in this study showed that the most acidic fractions of arabinogalactan proteins have the ability to inhibit the formation of aggregates between the grape seed tannins and the two different salivary proteins. Rhamnogalacturonan II has the same ability toward alpha-amylase but not IB8c under the conditions of the present study. Polysaccharides show effects at concentrations at which they are present in wine, which could mean an influence in wine astringency. The interaction between condensed tannins and alpha-amylase is differently affected by ionic strength when compared with IB8c.

The three-dimensional structures of 5 procyanidin dimers have been determined in a hydro-alcoholic medium and in water using 2D NMR and molecular mechanics. They are made from monomers of catechin (CAT) and epicatechin (EPI)-B1: EPI-CAT, B2: EPI-EPI, B3: CAT-CAT, B4: CAT-EPI and B2g: EPI-EPI-3-O-gallate. These tannins exist in two conformations that are in slow exchange in the NMR timescale (s), one is compact and the other extended. The compact form is found to dominate (76-98%) when the dimer is made of at least one CAT monomer (B1, B3, B4). Both forms are found in even proportions only in the case of procyanidin B2. The latter tannin can be converted into a dominant compact form when the lower EPI unit is galloylated. The finding of a predominant compact form for procyanidin dimers is discussed in relation with tannin-saliva protein interactions that are of importance for the wine-tasting/making processes., (Copyright (c) 2006 John Wiley & Sons, Ltd.)

Two-dimensional exchange NMR spectroscopy has been employed to study the isomerization process of copper(I) complexes formed upon complexation of Cu+ with a racemic mixture of the atropoisomer diimine benzimidazole-pyridine ligands 1-3 and evaluate the configurational stability of the pseudotetrahedral complexes [Cu(1-3)2]PF6. Racemization of the heterochiral isomers RSLambda/RSDelta proceeds through an intramolecular ligand rearrangement on a time scale of about 1.9 s(-1) for 1, 4.4 s(-1) for 2, and 0.3 s(-1) for 3 in CD2Cl2 at room temperature. The intramolecular Lambda/Delta isomerizations in the homochiral diastereoisomers RRDelta/SSLambda and RRLambda/SSDelta of [Cu(1)2]PF6 proceed at room temperature on a time scale of about 0.6 s(-1) for the conversion of RRDelta/SSLambda into RRLambda/SSDelta and 13 s(-1) for the conversion of RRLambda/SSDelta into RRDelta/SSLambda. The kinetics of these intramolecular exchange processes were found to be sensitive to the stabilizing interligand pi-stacking interactions that develop within the [Cu(1-3)2]+ structure and to the bulkiness of the benzimidazole aryl substituents. The kinetics of racemization in the heterochiral RSLambda/RSDelta isomers of [Cu(3)2]PF6 with the bulky cumyl-derived ligand were 1 order of magnitude lower than in [Cu(2)2]PF6 with the tolyl-based ligand. Slower intermolecular ligand exchanges between all the isomers have also been shown to occur at ambient temperature in CD2Cl2 through complete ligand dissociation. Free energies at 298 K varying between 66.7 and 74.4 kJ.mol(-1) and entropies varying between -26.4 and 28.3 J.K(-1).mol(-1) were determined for the intramolecular Lambda/Delta isomerizations. For the intermolecular ligand exchanges free energies at 298 K varying between 55.6 and 62.5 kJ.mol(-1) and entropies varying between -97.9 and -74.5 J.K(-1).mol(-1) were measured.

An unexpected six-membered silacycle was obtained using ring-closing metathesis of diallylsilane 3. The ring size of the compound formed was derived using a 2D 13C-13C inadequate experiment. All proton and carbon-13 resonances were assigned from classical 2D NMR experiments and the proton coupling pattern was clarified. Molecular modeling calculation was then applied using NMR data as constraints, giving the 3D structure of the synthesized compound., (Copyright 2004 John Wiley & Sons, Ltd.)

The interactions between the B3 (catechin-4alpha,8-catechin) red wine tannin and the human salivary protein fragment IB7(14) (SPPGKPQGPPPQGG) were monitored by (1)H magic angle spinning NMR, circular dichroism, electrospray ionization mass spectrometry, and molecular modeling. It is found that the secondary structure of IB7(14) is made of a type II helix (collagen helix) and random coil. The central glycine 8 appears to act as a flexible rotula separating two helix II regions. Three tannin molecules tightly complex the peptide, without modifying its secondary structure, but seem to reduce its conformational dynamics. The binding dissociation constant is in the millimolar range. B3 tannins with a "tweezers" conformation bind to the hydrophilic side of the saliva peptide, suggesting that the principal driving forces toward association are governed by hydrogen bonding between the carbonyl functions of proline residues and both the phenol and catechol OH groups. These findings are further discussed in the frame of an astringency phenomenon.

The solid phase synthesis of a 59 amino acid human salivary protein IB7 has been accomplished using Fmoc strategy. Because the protein contains 25 proline, 13 glycine and 9 glutamine residues the coupling time, piperidine delivery and acetic anhydride reaction time were increased. Yield after HPLC purification was 35%. Circular dichroism studies revealed that about one third of IB7 residues adopted a type II helix secondary structure, as found in collagen helices. The rest of the sequence adopts a random coil secondary structure.

A new family of atropoisomeric bidentate ligands that have a dissymmetric benzimidazole-pyridine binding site has been synthesized. Aromatic rings, that is, naphthyl, tolyl and cumyl, were introduced in order to fine tune the complexation properties of the ligands. The tetrahedral copper(I) complexes L2Cu were prepared and the structure of the complex with the naphthyl-substituted ligand was established by X-ray diffraction. The behavior of the L2Cu complexes in solution was studied by 1H NMR spectroscopy. With the most crowded cumyl-derived ligand, ligand self-recognition based on chirality occured: 95% of the complex was present in solution as a racemate RRdelta/SSlambda, the heterochiral RSdelta/SRlambda isomers represented only 5 % of the mixture, and the RRlambda/SSdelta isomers were not detected. Owing to lower steric repulsions within the other L2Cu complexes (i.e., with the naphthyl- and tolyl-based ligands) the homorecognition is less pronounced, as diastereomeric excesses of 6 and 26% were measured, respectively.

We used 31P-NMR spectroscopy to investigate the response of living C6 glioma cells to stimulation by a beta-adrenergic agonist, isoproterenol. In the presence of 3-isobutyl-1-methylxanthine, stimulation induced an accumulation of cAMP, making possible the NMR detection of the second messenger in living cells grown on microcarrier beads and perfused in the NMR tube. The cAMP signal rose to a maximum level within 20-25 min of stimulation; thereafter it decreased to the detection threshold within 60 min. At the same time, 40% increases of phosphomonoester and diphosphodiester signals were observed, whereas no significant change in phosphocreatine and nucleotide signals was detected. The kinetics of changes of the cellular content in phosphorylated metabolites were analyzed after recording 31P-NMR spectra of cell perchloric acid extracts as a function of time of stimulation. cAMP accumulation in stimulated cells was evidenced by a near linear increase of its NMR signal as a function of incubation time (from 0 to 60 min). Concomitantly with the production of cAMP, the data showed 30% decreases of phosphocreatine and ATP levels within 60 min of stimulation, and an unexpected redistribution of pyrimidine and purine nucleoside triphosphates. At the same time, levels of phosphomonoesters (phosphorylcholine and phosphorylethanolamine) and phosphodiesters (glycerophosphorylcholine and glycerophosphorylethanolamine) rose (50% increase). 13C-NMR spectra of cell perchloric acid extracts prepared after isoproterenol stimulation of cells incubated in the presence of [1-13C]glucose indicated a higher glucose content in stimulated cells, whereas the resonance of ribose C1 was diminished. Moreover, the resonances of C1 of ethanolamine and choline (and their derivatives) were increased in spectra of stimulated cells, whereas that of C3 of serine was decreased. In addition, the 13C-NMR data indicated that neither the pattern of glutamate carbon enrichment nor the glutamate/glutamine ratio was modified in stimulated cells. On the other hand, the heteronuclear coupling pattern of the lactate (methyl group) resonance in 1H-NMR spectra of cell incubation media indicated that no change occurred in the carbon flux through the pentose-phosphate shunt under stimulation. The results of this multinuclear NMR approach are discussed in terms of metabolic responses of C6 cells to beta-adrenergic stimulation and cAMP overproduction.

Rat astroglial cells in primary culture (95% enrichment) and C6 glioma cells were adapted to grow on microcarrier beads. In vivo 31P NMR spectra were collected from cell-covered beads perfused in the NMR tube. The NMR-visible phosphorylated metabolite contents of both cell types were determined using saturation factors calculated from the values of longitudinal relaxation times determined for C6 cells using progressive saturation experiments. On the other hand, the amounts of phosphorylated metabolites in cells were determined from proton decoupled 31P NMR spectra of cell perchloric acid extracts. The results indicate that the NTP and Pi contents of the normal and tumoral cells were similar, whereas the PCr level was higher in C6 cells and the NDP and phosphomonoester levels higher in astrocytes. The comparison of 1H NMR spectra of cell perchloric acid extracts evidenced larger inositol and alanine contents in C6 cells, whereas larger taurine and choline (and choline derivatives) contents were found in astrocytes. The Glu/Gln ratio was very different, 3.5 and 1 in C6 cells and astrocytes, respectively. In both cases, the more intense resonance in the 1H NMR spectrum was assigned to glycine. Based on the comparison of the metabolite content of a tumoral and a normal cell of glial origin, this work emphasizes the usefulness of a multinuclear NMR study in characterizing intrinsic differences between normal and tumoral cells.

A new process for embedding cells in agarose is described. Beads were obtained by extruding an ultralow gelling temperature agarose solution in a capillary containing a hydrophobic medium flowthrough. The toxicity of the procedure has been evaluated by monitoring the energy status of agarose-embedded C(6) glioma cells with (31)P nuclear magnetic resonance (NMR). Suspension and microbead cultures of hybridoma cell line were compared. In suspension culture the number of cells and the antibody concentrations increased for 5 days before the stationary phase began, when the cultures were stopped. In agarose bead cultures, the gel provided an enormous support surface area (50 m(2)/ mL of gel). It was possible to seed 20-fold more cells. The gel pressure modified the proliferative process and antibody pattern secretion. In particular, the antibodies could be harvested for two weeks.

31P-NMR spectroscopy has been used to study the energy metabolism and the NMR visibility of ATP and intracellular Pi of the C6 glioma cell line and rat astrocyte grown on microcarrier beads with the following results. 1. In vivo NMR spectra of C6 glioma cells and rat astrocytes indicate that these cells were able to maintain their level of ATP resonances during a long anoxic period (more than an hour). Both cell types were sensitive to ischemia which induced a loss of ATP resonances within 40 min. Glucose starvation induced by 40% decrease in ATP resonances correlated to a 50% increase in the intensity of the Pi signal. These changes corresponded to a new steady state which could be reversed by reperfusing the cells with a glucose-containing medium. 2. In contrast to in vivo data, 31P-NMR analyses of perchloric acid extracts of cells incubated in a glucose-free medium showed that their ATP and Pi contents were unchanged during starvation. The changes of NMR visibility of the metabolites in living C6 cells were correlated to modifications of their macroscopic longitudinal relaxation times, evolving from 0.30 +/- 0.08 s and 6.6 +/- 1.5 s in the presence of glucose to 0.68 +/- 0.26 s and 3.2 +/- 0.9 s in the absence of glucose for ATP and Pi, respectively. The changes of the NMR detectability of ATP and Pi indicate that changes in their microenvironment occur during glucose starvation, suggesting the existence of different pools of these metabolites within the cells. 3. Under various experimental conditions, i.e. anoxia, ischemia and glucose starvation, rat astrocytes in primary culture showed a very similar behavior to that of C6 cells, suggesting a similar adaptability to the nature of the energy supply for both the normal and the malignant cell.

Nuclear magnetic resonance (NMR) spectroscopy was used to study the metabolism of cells from the central nervous system both in vitro on perchloric acid extracts obtained either from cultured tumoral cells (C6 rat glioma) or rat astrocytes in primary culture, and in vivo within the human brain. Analysis of carbon 13 NMR spectra of perchloric acid extracts prepared from cultured cells in the presence of NMR [1-13C] glucose as substrate allowed determination of the glutamate and glutamine enrichments in both normal and tumoral cells. Preliminary results indicated large changes in the metabolism of these amino acids (and also of aspartate and alanine) in the C6 cell as compared to its normal counterpart. Localized proton NMR spectra of the human brain in vivo were obtained at 1.5 T, in order to evaluate the content of various metabolites, including glutamate, in peritumoral edema from a selected volume of 2 x 2 x 2 cm3. N-acetyl aspartate, glutamate, phosphocreatine, creatine, choline and inositol derivative resonances were observed in 15 min spectra. N-acetyl-aspartate was found to be at a lower level in contrast to glutamate which was detected at a higher level in the injured area as compared to the contralateral unaffected side.

When added to intact C6 glioma cells in the micromolar range of concentrations, ADP and ATP induce an inhibition of the isoproterenol-elicited cAMP responses. ATP is rapidly hydrolyzed by the ectonucleotidases present on these cells, with an apparent Km of 50 microM and a Vmax of 1.1 nmol/min/10(6) cells. cAMP responses are also inhibited by millimolar concentrations of either ATP in the presence of an ATP-regenerating system to prevent ADP accumulation or AMP-PCP. These observations show that, in C6 glioma cells, ADP is a more potent inhibitor of cAMP production than ATP, the latter acting indirectly, via its rapid hydrolysis to ADP. The additive inhibition of isoproterenol-elicited cAMP responses induced, on one hand, by the treatment of the cells with a phorbol ester and by addition of ADP to the cells, and, on the other hand, by the progressive disappearance of the effects of ADP and ATP when cells are treated with increasing concentrations of Pertussis toxin, demonstrate that ADP and ATP exert their action in C6 glioma cells via a P2 purinoceptor probably negatively coupled to adenylate cyclase and a G regulatory protein.

The activity of beta-adrenergic receptors at the plasma membrane level was investigated in viable, electropermeabilized C6 glioma cells. Electric field pulses were applied directly to the plated cells without any previous proteinase treatment. The affinity for isoproterenol and the density of the beta-adrenergic receptors, as judged from the number of [3H]CGP-12177 binding sites, were not affected by the electropermeabilization whereas the isoproterenol-stimulated cAMP accumulation was transiently impaired. This decrease in activity is due to an electropermeabilization-induced GTP leak. Normal activity could be obtained either by treating the cells by the electric field in a GTP-containing buffer, or by spontaneous recovery of the cells after the resealing of the plasma membrane, with a delay depending on the temperature. The activity of the receptors was not affected by the structural organization of the membrane associated to its electropermeabilization.