NMR spectroscopy: a versatile tool for the investigation of organic reaction mechanisms and metabolomics analyses

Olefin metathesis has become a powerful tool for the formation of carbon-carbon bonds and, therefore, for the synthesis of a number of molecules. This progress was recognized in 2005 with the award of NOBEL Prize in Chemistry to Yves Chauvin, Robert Grubbs and Richard Schrock for their work in this area. While the series of [2+2]cycloadditions and retro[2+2]cycloadditions that make up the pathways of ruthenium-catalysed metathesis reactions is wellestablished, the exploration of mechanistic aspects of alkene metathesis is going on. At first, we reported the tetramerization of (E)-2,4-dimethoxycinnamic acid ω-undecenyl ester with ethereal BF3. The reaction gave three stereoisomers 1a, 1b, and 1c, which were assigned as the chair, cone, and 1,2-alternate conformations, respectively. Undecenyl resorc[4]arene 1a, which featured the simplest pattern of substituent, was submitted to olefin metathesis using the second generation Grubbs complex as the catalyst. Depending on the reaction conditions, different products were isolated: a bicyclic alkene 2a, a linear dimer 3a, and a cross-linked homopolymer P1a. Moreover, we detected for the first time the formation of a ruthenium-carbene resorc[4]arene complex during the metathesis reaction of resorc[4]arene olefin 2a with the first generation Grubbs catalyst in CDCl3. We developed an NMR analytical protocol which proved capable of yielding both qualitative and quantitative information. In the first case, we were able to identify the complex 3a[Ru] as a key intermediate in the ROM-CM sequence of reactions, giving us a definitive proof of the previously hypothesized mechanism. As a further feedback of the pathway, we performed a quantitative analysis using benzene in the place of CDCl3, due to the poor stability of the catalyst in such a solvent. The reaction allowed the isolation of decomposition products of the ruthenium-carbene-resorc[4]arene complex 2a[Ru] such as compound 4a, which, due to the presence of still reactive alkene functions, proved to behave as propagating alkylidene species leading to further decomposition products.Metabolomics provides a direct measure of the state of the cell or biological system, where changes in the metabolome capture how the system responds to environmental or genetic stress. Specifically, a drug or an active chemical lead would be expected to perturb the metabolome of a cell or tissue upon treatment. The two leading analytical approaches to metabolomics are mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. In particular, NMR technique accounts for high reproducibility, quantitative determination of a wide dynamic range, and the capability to determine the structure of unknown metabolites. Cells of high-grade tumors, including medulloblastoma (MB) and glioblastoma (GBM), must similarly balance energy metabolism with the need to synthesize the macromolecules essential for tumor growth. Cells with large ATP requirements are likely to be disadvantaged by aerobic glycolysis because glycolysis generates less ATP per molecule of glucose than oxidative phosphorylation. Proliferating cells, however, may use aerobic glycolysis to satisfy the competing needs for both energy generation and the accumulation of biomass. Recently, it has been demonstrated that Sonic Hedgehog (SHH) pathway activation in granule cell progenitors (GCPs), responsible of MB development, induces transcription of hexokinase 2 (HK2) and pyruvate kinase M2 (PKM2), two key gatekeepers of glycolysis. The process is mediated by the canonical activation of the GLI transcription factors and causes a robust increase of extracellular lactate concentration. Glabrescione B (GlaB), an isoflavone naturally found in the seeds of Derris glabrescens (Leguminosae), turned out to be an efficient inhibitor of the growth of HH/GLI-dependent tumors and cancer stem cells in vitro and in vivo. Here, we reported the GlaB activity on both human MB DAOY and murine glioma GL261 cell models in vitro and in vivo. In order to evaluate how the GlaB-treatment affects cell metabolism as a consequence of GLI1 inhibition, untargeted NMR metabolomics analyses of cellular lysates and conditioned media were performed in both cell lines. For this purpose, a simple, fast, and reproducible sample preparation protocol was developed. To reduce bias in the interpretation of the experiments, it was decided to produce from five to seven biological replicates for each treated and untreated group. The 1D 1H NMR spectra were acquired to determine the metabolic fingerprints of the treated and untreated cancer cells. Notably, the NMR metabolomics approach revealed a typical endo-metabolic phenotype of the cells under investigation. Both the exo- and endo-metabolome of the DAOY and GL261 cell lines resulted to be completely changed after 24 h and 48 h of GlaB administration, respectively. The levels of most metabolites decreased after treatment, consistently with possible apoptosis phenomenon

NMR for sample quality assessment in metabolomics.

Abstract The EU Framework 7 project SPIDIA was the occasion for development of NMR approaches to evaluate the impact of different pre-analytical treatments on the quality of biological samples dedicated to metabolomics. Systematic simulation of different pre-analytical procedures was performed on urine and blood serum and plasma. Here we review the key aspects of these studies that have led to the development of CEN technical specifications, to be translated into ISO/IS in the course of the EU Horizon 2020 project SPIDIA4P. Inspired by the SPIDIA results, follow-up research was performed, extending the analysis to different sample types and to the different effects of long-term storage. The latter activity was in conjunction with the local European da Vinci Biobank. These results (which partially contributed to the ANNEX of CEN/TS 16945"MOLECULAR IN VITRO DIAGNOSTIC EXAMINATIONS - SPECIFICATIONS FOR PRE-EXAMINATION PROCESSES FOR METABOLOMICS IN URINE, VENOUS BLOOD SERUM AND PLASMA") are presented in detail

Inhibition of Hedgehog-dependent tumors and cancer stem cells by a newly identified naturally occurring chemotype

Hedgehog (Hh) inhibitors have emerged as valid tools in the treatment of a wide range of cancers. Indeed, aberrant activation of the Hh pathway occurring either by ligand-dependent or -independent mechanisms is a key driver in tumorigenesis. The smoothened (Smo) receptor is one of the main upstream transducers of the Hh signaling and is a validated target for the development of anticancer compounds, as underlined by the FDA-approved Smo antagonist Vismodegib (GDC-0449/Erivedge) for the treatment of basal cell carcinoma. However, Smo mutations that confer constitutive activity and drug resistance have emerged during treatment with Vismodegib. For this reason, the development of new effective Hh inhibitors represents a major challenge for cancer therapy. Natural products have always represented a unique source of lead structures in drug discovery, and in recent years have been used to modulate the Hh pathway at multiple levels. Here, starting from an in house library of natural compounds and their derivatives, we discovered novel chemotypes of Hh inhibitors by mean of virtual screening against the crystallographic structure of Smo. Hh functional based assay identified the chalcone derivative 12 as the most effective Hh inhibitor within the test set. The chalcone 12 binds the Smo receptor and promotes the displacement of Bodipy-Cyclopamine in both Smo WT and drug-resistant Smo mutant. Our molecule stands as a promising Smo antagonist able to specifically impair the growth of Hh-dependent tumor cells in vitro and in vivo and medulloblastoma stem-like cells and potentially overcome the associated drug resistance

A Smo/Gli multitarget hedgehog pathway inhibitor impairs tumor growth

Pharmacological Hedgehog (Hh) pathway inhibition has emerged as a valuable anticancer strategy. A number of small molecules able to block the pathway at the upstream receptor Smoothened (Smo) or the downstream effector glioma-associated oncogene 1 (Gli1) has been designed and developed. In a recent study, we exploited the high versatility of the natural isoflavone scaffold for targeting the Hh signaling pathway at multiple levels showing that the simultaneous targeting of Smo and Gli1 provided synergistic Hh pathway inhibition stronger than single administration. This approach seems to effectively overcome the drug resistance, particularly at the level of Smo. Here, we combined the pharmacophores targeting Smo and Gli1 into a single and individual isoflavone, compound 22, which inhibits the Hh pathway at both upstream and downstream level. We demonstrate that this multitarget agent suppresses medulloblastoma growth in vitro and in vivo through antagonism of Smo and Gli1, which is a novel mechanism of action in Hh inhibition

A unique high-diversity natural product collection as a reservoir of new therapeutic leads

Plants represent a rich source of structurally diverse secondary metabolites, which can be exploited in the development of new clinically important compounds. Indeed, due to their biodiversity, medicinal plants represent the largest library of compounds that has ever existed. To date less than 1% of this vast biodiversity has been exploited in drug discovery, due to several factors, including the lack of an appropriate multidisciplinary perspective. Here we review the successful application of computer-aided methods in screening a unique and high-diversity in house collection library composed of around 1000 individual natural products, isolated mainly from indigenous plants collected in biodiversity-rich countries, especially of the tropics and subtropics, and enlarged with their semi-synthetic and synthetic derivatives, as well as plant material extracts, up to around 2000 components. During the last ten years, the in house library has provided several lead compounds that have been developed, and in some cases patented, as anticancer and antimicrobial agents. The main classes of the library are described, including (but not limited to) alkaloids, terpenoids, Diels–Alder-type adducts, isoflavones, chalcones, and cannabinoids. The main focus is on the chemical characteristics and biological activity of these identified compounds, with particular attention being given to those currently under patent or in the preclinical phase. We also assess the use of computer-aided methods in screening this unique and diverse in house collection of natural products that, over the last ten years, has provided some lead compounds that have been developed, and in some cases patented, as anticancer and antimicrobial agents. Finally, this review highlights the potential use of plant food extracts as a source of nutraceuticals and functional foods. The multidisciplinary approach described herein may further motivate research groups involved in natural product chemistry to potentially benefit from a limitless source of novel bioactive compounds

Anodic microbial community analysis of microbial fuel cells based on enriched inoculum from freshwater sediment.

Abstract: The characterization of anodic microbial communities is of great importance in the study of microbial fuel cells (MFCs). These kinds of devices mainly require a high abundance of anode respiring bacteria (ARB) in the anode chamber for optimal performance. This study evaluated the effect of different enrichments of environmental freshwater sediment samples used as inocula on microbial community structures in MFCs. Two enrichment media were compared: ferric citrate (FeC) enrichment, with the purpose of increasing the ARB percentage, and general enrichment (Gen). The microbial community dynamics were evaluated by polymerase chain reaction followed by denaturing gradient gel electrophoresis (PCR-DGGE) and real time polymerase chain reaction (qPCR). The enrichment effect was visible on the microbial community composition both during precultures and in anode MFCs. Both enrichment approaches affected microbial communities. Shannon diversity as well as β-Proteobacteria and γ-Proteobacteria percentages decreased during the enrichment steps, especially for FeC (p < 0.01). Our data suggest that FeC enrichment excessively reduced the diversity of the anode community, rather than promoting the proliferation of ARB, causing a condition that did not produce advantages in terms of system performance. Graphical abstract: [Figure not available: see fulltext.]

A Multimethodological Characterization of Cannabis sativa L. Inflorescences from Seven Dioecious Cultivars Grown in Italy: The Effect of Different Harvesting Stages

The chemical profile of the female inflorescence extracts from seven Cannabis sativa L. dioecious cultivars (Carmagnola, Fibranova, Eletta Campana, Antal, Tiborszallasi, Kompolti, and Tisza) was monitored at three harvesting stages (4, 14, and 30 September), reaching from the be ginning of flowering to end of flowering/beginning of seed formation, using untargeted nuclear magnetic resonance (NMR) and targeted (ultra-high-performance liquid chromatography (UHPLC) and spectrophotometry) analyses. The tetrahydrocannabinol content was always below the legal limits (<0.6%) in all the analyzed samples. The NMR metabolite profile (sugars, organic acids, amino acids, and minor compounds) subjected to principal components analysis (PCA) showed a strong variability according to the harvesting stages: samples harvested in stage I were characterized by a high content of sucrose and myo-inositol, whereas the ones harvested in stage II showed high levels of succinic acid, alanine, valine, isoleucine, phenylalanine, and threonine. Samples harvested in stage III were characterized by high levels of glucose, fructose, choline, trigonelline, malic acid, formic acid, and some amino acids. The ratio between chlorophylls and carotenoids content indicated that all plants grew up exposed to the sun, the Eletta Campana cultivar having the highest pigment amount. Tiborszallasi cultivar showed the highest polyphenol content. The highest antioxidant activity was generally observed in stage II. All these results suggested that the Cannabis sativa L. inflorescences of each analyzed dioecious hemp cultivar presented a peculiar chemical profile affected by the harvesting stage. This information could be useful for producers and industries to harvest inflorescences in the appropriate stage to obtain samples with a peculiar chemical profile suitable for proper applications.15s

Inorganic gold and polymeric poly(lactide-co-glycolide) nanoparticles as novel strategies to ameliorate the biological properties of antimicrobial peptides

Cationic antimicrobial peptides (AMPs) are an interesting class of gene-encoded molecules endowed with a broad-spectrum of anti-infective activity and immunomodulatory properties. They represent promising candidates for the development of new antibiotics, mainly due to their membrane-perturbing mechanism of action that very rarely induces microbial resistance. However, bringing AMPs into clinical field is hampered by some intrinsic limitations, encompassing low peptide bioavailability at the target site and high peptide susceptibility to proteolytic degradation. In this regard, nanotechnologies represent an innovative strategy to circumvent these issues. According to the literature, a large variety of nanoparticulate systems has been employed for drug-delivery, bioimaging, biosensors or as nanoantibiotics. The possibility of conjugating different types of molecules, including AMPs, to these systems, allows the production of nanoformulations able to enhance the biological profile of the compound, while reducing its cytotoxicity and prolonging its residence time. In this mini-review, inorganic gold nanoparticles (NPs) and biodegradable polymeric NPs made of poly(lactide-co-glycolide) are described with particular emphasis on examples of conjugation of AMPs to them, to highlight the great potential of such nanoformulations as alternative antimicrobials

DEVELOPMENT OF ArnT-MEDIATED COLISTIN RESISTANCE DITERPENE-BASED INHIBITORS

Colistin is a last-line antibiotic for the treatment of multidrug resistant Gram-negative bacterial infections.1 Recently, a natural ent-beyerene diterpene (ent-Beyer-15-en-18-O-oxalate) was identified as a promising inhibitor of the enzyme responsible for colistin resistance mediated by lipid A aminoarabinosylation in Gram-negative bacteria, namely, ArnT (undecaprenyl phosphate-alpha-4-amino-4-deoxy-l-arabinose arabinosyl transferase).2 To explore the structure-activity relationship (SAR), semi-synthetic analogs of hit were designed, synthesized and tested against colistin-resistant Pseudomonas aeruginosa strains, including clinical isolates (figure 1), in order to exploit the versatility of the diterpene scaffold. Microbiological assays coupled with molecular modeling demonstrated that an ent-beyerane scaffold bearing an oxalate like group at C-18/C-19, or a sugar residue at C-19 to resemble L-Ara4N is an essential requirement for a more efficient inhibition of bacterial growth likely resulting from a more efficient inhibition of ArnT activity. Importantly, the easy accessibility of ent-beyerane scaffold from Stevia rebaudiana secondary metabolites will provide a cost-effective key platform for the development of promising colistin resistance inhibitors

Olefin metathesis reaction as a locking tool for macrocycle and mechanomolecule construction

The present review deals with an updated visit to the olefin metathesis reaction as a powerful tool for the construction of sophisticated macromolecular architectures. Today, the reaction has proved to be one of the most popular processes, not only for the synthesis of novel cage and huge calixarene-based macrocycles, or more complex structures containing these privileged moieties, but also for the construction of complicated topologically intriguing molecules, such as catenanes, rotaxanes and knots. Notably, their use ranges from chemistry and biology, as catalysts and drug delivery systems, respectively, to materials research, as molecular motors and switches. The reaction effectiveness arises from the intrinsic specificity for the highly stable reactive end-groups (e.g. alkene moieties) and the feasibility to perform the reaction in relatively non-polar solvents, maximizing the supramolecular interactions often involved in the templation strategies. By moving from calixarene-based macrocycles to mechanically interlocked molecules, the review takes into account several synthetic strategies involving the olefin metathesis reaction highlighting its increasing utility and future potential in the synthesis of molecular machines