Absolute Metabolite Quantification Using Pure Shift NMR: Toward Quantitative Metabolic Profiling of Aqueous Biological Samples.

Accurate quantification of metabolites by nuclear magnetic resonance (NMR) is of prime importance in the field of health sciences for understanding the metabolic pathways of the investigated system, to address the mechanisms of action of diseases, and improving their diagnosis, treatment, and prognosis. Unfortunately, the absolute quantitative analysis of complex samples is still limited by sensitivity and resolution issues that are intrinsic to this technique. Ultrahigh-resolution pure shift methods have especially shown to be suitable for interpreting mixtures of metabolites in biological samples. Here, we introduce a robust analytical protocol based on the use of a pure shift library of calibration reference spectra to fit the fingerprint of each metabolite of interest and determine its concentration. The approach based on the SAPPHIRE pulse sequence enhanced with a block for solvent suppression has been validated through the results of a series of model mixtures, exhibiting excellent trueness (slope values in the range of 0.93-1.02) and linearity (R2 > 0.996) in a total time (a few hours) that is fully compatible with metabolomics studies. Furthermore, we have successfully applied our method to determine the absolute metabolite concentrations in a lymphoma extracellular medium, which improves metabolomic protocols reported to date by providing a quantitative and highly resolved vision of metabolic processes at play.

NMR applications for identifying ß-TrCP protein-ligand interactions.

In the absence of crystallographic data, NMR has emerged as the best way to define protein-ligand interactions. Using NMR experiments based on magnetization transfer, one can sort bound from unbound molecules, estimate the dissociation constant, identify contacts implied in the binding, characterize the structure of the bound ligand and conduct ligand competition assays.

3-carboxy-4-phosphonocyclopentane amino acids: new metabotropic glutamate receptor ligands.

Two glutamic acid analogs (1 SR,3 RS,4 RS)- and (1 SR,3 SR,4 SR)-1-amino-4-phosphono cyclopentane-1,3-dicarboxylic acids (APCPD) have been synthesized. Pure E-(diethoxy-phosphoryl)-acrylic acid ethyl ester was obtained from ethyl propiolate, phenol and triethylphosphite. It was used as dienophile in a Diels-Alder reaction. Oxidation and cyclization afforded 3-(ethoxy-carbonyl)-4-(diethoxy-phosphoryl)-cyclopentanone. Bucherer-Bergs reaction and hydrolysis yielded APCPD-III and -IV which are inactive on mGlu1a receptor and antagonists on mGlu2 and mGlu8a receptors.

Sheep prion protein synthetic peptide spanning helix 1 and beta-strand 2 (residues 142-166) shows beta-hairpin structure in solution.

According to the "protein only" hypothesis, a conformational conversion of the non-pathogenic "cellular" prion isoform into a pathogenic "scrapie" isoform is the fundamental event in the onset of prion diseases. During this pathogenic conversion, helix H1 and two adjacent surface loops L2 and L3 of the normal prion protein are thought to undergo a conformational transition into an extended beta-like structure, which is prompted by interactions with the pre-existing beta-sheet. To get more insight into the interaction between the helix and one of the beta-strands in the partially unfolded prion protein, the solution structure of a synthetic linear peptide spanning helix H1 and beta-strand S2 (residues 142-166 in human numbering) was studied by circular dichroism and nuclear magnetic resonance spectroscopies. We found that, in contrast to many prion fragments studied earlier, this peptide (i) is highly soluble and does not aggregate up to a millimolar concentration range in aqueous medium and (ii) exhibits an intrinsic propensity to a beta-hairpin like conformation at neutral pH. This beta-propensity can be one of the internal driving forces of the molecular rearrangement responsible for the pathogenic conversion of the prion protein.

Purification and structure of the major product obtained by reaction of NADPH and NMNH with the myeloperoxidase/hydrogen peroxide/chloride system.

The first spectrophotometric study of the reaction of the myeloperoxidase/H2O2/Cl- system with NADPH and NMNH showed that the reaction products were not the corresponding oxidized nucleotides and that modifications would take place on the nicotinamide part of the molecule [Auchère, F. & Capeillère-Blandin, C. (1999) Biochem. J. 343, 603-613]. In this report, in order to obtain more precise information on the structural modifications and mechanism of the reaction, we focus on the purification and isolation of products derived from NADPH and NMNH by RP-HPLC. Electrospray ionization mass spectra indicated that the relative height of the peaks reflected that of the natural isotopic abundance of 35Cl and 37Cl, providing evidence that the products derived from NADPH and NMNH were monochlorinated. Moreover, calculated masses revealed the 1 : 1 addition of HOCl to the molecule. Various 1D and 2D NMR experiments provided data for the assignments of the chemical shifts of protons and carbons and the coupling constants of the protons of the chlorinated nucleotides. Further NOESY experiments allowed the characterization of the spatial structure of the chlorinated product and showed that trans HOCl addition occurred at the C5=C6 carbon double bond of the nicotinamide ring, leading to a chlorohydrin.

Identification and quantitative analysis of the phytoecdysteroids in Silene species (Caryophyllaceae) by high-performance liquid chromatography. Novel ecdysteroids from S. pseudotites.

Many species in the genus Silene (Caryophyllaceae) have previously been shown to contain ecdysteroids and this genus is recognised as a good source of novel ecdysteroid analogues. We have used ecdysteroid-specific radioimmunoassays and the microplate-based Drosophila melanogaster B(II) cell bioassay for ecdysteroid agonist and antagonist activities to identify further phytoecdysteroid-containing species in this genus. The main ecdysteroid components from 10 Silene species (S. antirrhina, S. chlorifolia, S. cretica, S. disticha, S. echinata, S. italica, S. portensis, S. pseudotites, S. radicosa, S. regia) were isolated and identified, mainly by normal-phase and reversed-phase high-performance liquid chromatography. The amount of each ecdysteroid was determined by comparing chromatogram peak areas with those for reference 20-hydroxyecdysone (20E) on reversed-phase HPLC. 20E is the most abundant ecdysteroid in each of the Silene extracts. Polypodine B, 2-deoxy-20-hydroxyecdysone and ecdysone are also common ecdysteroids in these Silene species, but the proportions of these ecdysteroids vary between the Silene species. HPLC proved to be a quick and effective way to screen Silene species, determine ecdysteroid profiles and, hence, identify extracts containing novel analogues. An extract of the aerial parts of S. pseudotites was found to contain several new ecdysteroids. These have been isolated and identified spectroscopically (by NMR and mass spectrometry) as 2-deoxyecdysone 22beta-D-glucoside, 2-deoxy-20,26-dihydroxyecdysone and 2-deoxypolypodine B 3beta-D-glucoside. Additionally, (5alpha-H)-2-deoxyintegristerone A (5alpha-2H 91%, 5alpha-1H 9%) was isolated as an artefact. This study contributes to the understanding of ecdysteroid distribution in Silene species and provides further information on the chemotaxonomic significance of ecdysteroids in Silene species.

Conformations in solution and bound to bacterial ribosomes of ketolides, HMR 3647 (telithromycin) and RU 72366: a new class of highly potent antibacterials.

The new class of antibiotics called ketolides is endowed with remarkable antibacterial activity against macrolide-resistant strains. Further modifications of the 3 keto-macrolactone backbone led to 11,12-hydrazonocarbamate ketolides with an imidazolyl pyridine chain: the file-leader of ketolide class, HMR 3647 (telithromycin), and its N-bis-demethyl-derivative, RU 72366. The potency of HMR 3647 is higher than that of RU 72366. Stereospecific 1H and 13C resonance assignments of HMR 3647 and RU 72366 have been determined and have allowed a detailed quantitative conformational analysis of the uncomplexed form of the molecules. The comparative conformation of HMR 3647 in solution and its N-bis-demethyl-derivative in D2O has been carried out using different heteronuclear correlation experiments in conjunction with nuclear Overhauser effect experiments and in particular long-range 3J(CH) coupling constants and using molecular dynamics (MD) methods. The study of ketolide ribosome interaction has been investigated using two-dimensional transferred nuclear Overhauser effect spectroscopy (TRNOESY). The database of ribosome-bound ketolide structures has been used to compare the structure(s) of ketolide in ribosome-ketolide complexes with the conformational preferences of free ketolides and to highlight the significant differences between HMR 3647 and RU 72366. A comparison of the conformations bound to ribosome was made with those of other previously studied ketolide (RU 004) and macrolides and would explain the remarkable potencies of HMR 3647 in inhibiting protein synthesis.

Lincomycin and clindamycin conformations. A fragment shared by macrolides, ketolides and lincosamides determined from TRNOE ribosome-bound conformations.

Two important lincosamide antibiotics, lincomycin and clindamycin were studied in the complex state with the bacterial ribosome after a conformational analysis by 1H and 13C NMR spectroscopy and molecular modelling of the unbound molecules. Lincosamide-ribosome interactions were investigated using two-dimensional transferred nuclear Overhauser effect spectroscopy (TRNOESY), resulting in a bound structure compatible with the experimental NMR data. The results compared with the conformational analysis of the substrates in solution indicate that specific conformations are preferred in the bound state. Clindamycin, the more bioactive antibiotic studied, displayed a stronger NMR response than lincomycin showing that in lincosamide-ribosome interactions, a low affinity binding level is associated to the tight binding one and is related to biological activity. This study shows that conformation plays an essential role for the low affinity binding site. Superimposition of lincosamide, macrolide and ketolide bound structures exhibited conformational similarities in a particular fragment which is in agreement with a hypothesis of partial overlapping lincosamide and macrolide binding sites.

Microbial hydroxylation/functionalization of terpenoid synthons derived from communic acids.

Incubation of a communic acid-derived synthon with Cunninghamella elegans quantitatively affords 1 beta, 3 beta- and 7 beta- monohydroxylated derivatives.

Conformational analysis of ketolide, conformations of RU 004 in solution and bound to bacterial ribosomes.

A new structurally distinct class of 14-membered-ring macrolides is characterized by a keto-function instead of the cladinose sugar, well-known for its fragility even in weakly acidic media. This new class called ketolides is endowed with remarkable antibacterial activity against macrolide-resistant strains. A complete assignment of the 1H and 13C NMR spectra of RU 004 in deuteriochloroform, methanol-d4 and D2O has been made using different two-dimensional (2D) chemical-shift correlation methods. The study of ketolide-ribosome interaction has been investigated using 2D transferred nuclear Overhauser effect spectroscopy (TRNOESY). A comparison of the conformations in solution and bound to ribosomes was made with those of previous macrolides. This study can highlight some of the significant differences between RU 004 and other antibiotics.

Solution conformation of methylated macrolide antibiotics roxithromycin and erythromycin using NMR and molecular modelling. Ribosome-bound conformation determined by TRNOE and formation of cytochrome P450-metabolite complex.

Conformational study of methylated derivatives of macrolide antibiotics roxithromycin (6-OMe-roxithromycin and 6,11-OMe-roxithromycin) has been achieved by NMR in solution and molecular dynamics (MD) simulations and compared to 6-OMe-erythromycin (clarithromycin). A complete conformational study by NMR has been led by determination of homonuclear coupling constants and NOEs. Heteronuclear 1H-13C coupling constants were also measured to investigate the orientation of the sugar moieties with respect to the erythronolide. MD simulations were performed using the crystallographic coordinates as the starting conformation. For each compound, experimental results were compared to calculated conformations in order to identify eventual conformational equilibrium in solution. It is shown that the effect of the methylation is opposite for roxithromycin compared to erythromycin especially on motional properties as the roxithromycin derivatives gain in mobility while the erythromycin derivatives behaves as a more restrained molecule. The study of macrolide-ribosome interactions has been investigated using transferred NOESY 1H NMR experiments and the conformations weakly bound to bacterial ribosomes were determined. Biological interactions of these compounds with membranar liver protein cytochrome P450 was also discussed with regard to their structural properties.

Transferred nuclear Overhauser effect study of macrolide-ribosome interactions: correlation between antibiotic activities and bound conformations.

The study of macrolide-ribosome interactions has been investigated using two-dimensional transferred nuclear Overhauser effect spectroscopy (TRNOESY). A new medically important macrolide antibiotic, roxithromycin, with the replacement of the 9-keto group in erythromycin by a 9-oxime chain, was studied in the complex state with the bacterial ribosome. Analysis of transferred nuclear Overhauser effect (TRNOE) experiment resulted in a set of constraints for all protons pairs. These constraints were used in structure determination procedures based on molecular modelling to obtain a bound structure compatible with the experimental NMR data. The results compared with the conformational analysis of the substrate in solution indicate that only one specific conformation is preferred in the bound state while in the free state the sugar ring moities were relatively disordered. The bioactive macrolide antibiotics studied roxithromycin and erythromycin which displayed a strong NMR response, are metabolized in RU39001 and erythralosamine respectively which do not retain antimicrobial activity. The inactive major metabolites were used to define if TRNOEs observation may be characteristic of a biological activity. These control experiments gave essentially blank TRNOESY spectra. This study shows that Mg2+ does not play a direct role for the low affinity binding site studied by TRNOE what is in agreement with an hypothesis of two distinct binding levels, with a low affinity binding level necessary for the tight binding one.