Leonardo Scaglioni

Taste-guided identification of high potency TRPA1 agonists from Perilla frutescens

Perilla frutescens is a food plant widely used in Asian cuisine. This plant was investigated for its interesting taste and somatosensory properties. Perillaldehyde and perillaketone are among the components of the aromatic extracts from P. Frutescens. These compounds were shown here to activate the cloned TRPA1 channel when expressed in an heterologous cell system and are therefore suggested to be responsible for the chemesthetic properties of this plant.

Synthesis and cytotoxic activity of a new series of topoisomerase I inhibitors.

A series of structurally simple analogues of natural topopyrone C were synthesized and tested for cytotoxic and topoisomerase I inhibitory activities. The removal of the hydroxyl groups at the 5 and 9 positions resulted in an increased cytotoxic potency and ability to stabilize topoisomerase-mediated cleavage. In addition, the results suggest that some structural features, such as the pyrone ring and a polar group in position 11, are fundamental for topoisomerase I inhibitory effect. These structural requirements are also consistent with the cytotoxic activity.

Alkalizing Reactions Streamline Cellular Metabolism in Acidogenic Microorganisms

An understanding of the integrated relationships among the principal cellular functions that govern the bioenergetic reactions of an organism is necessary to determine how cells remain viable and optimise their fitness in the environment. Urease is a complex enzyme that catalyzes the hydrolysis of urea to ammonia and carbonic acid. While the induction of urease activity by several microorganisms has been predominantly considered a stress-response that is initiated to generate a nitrogen source in response to a low environmental pH, here we demonstrate a new role of urease in the optimisation of cellular bioenergetics. We show that urea hydrolysis increases the catabolic efficiency of Streptococcus thermophilus, a lactic acid bacterium that is widely used in the industrial manufacture of dairy products. By modulating the intracellular pH and thereby increasing the activity of β-galactosidase, glycolytic enzymes and lactate dehydrogenase, urease increases the overall change in enthalpy generated by the bioenergetic reactions. A cooperative altruistic behaviour of urease-positive microorganisms on the urease-negative microorganisms within the same environment was also observed. The physiological role of a single enzymatic activity demonstrates a novel and unexpected view of the non-transcriptional regulatory mechanisms that govern the bioenergetics of a bacterial cell, highlighting a new role for cytosol-alkalizing biochemical pathways in acidogenic microorganisms.

Taste-Active Compounds in a Traditional Italian Food: ‘Lampascioni’

Nature is a rich source of taste‐active compounds, in particular of plant origin, many of which have unusual tastes. Many of these are found in traditional food, where spontaneous plants are used as ingredients. Some taste‐active compounds were identified in the bulbs of Muscari comosum, a spontaneous plant belonging to the family of the Liliaceae, very common in the Mediterranean area, and used in traditional gastronomy (called ‘lampascioni’ in South Italy). The bulbs were extracted with a series of solvents of different polarity. The different fractions were submitted to a preliminary sensory evaluation, and the most interesting ones, characterized by a strong bitter taste and some chemestetic properties, were submitted to further purification and structural analysis. From the ethereal extract, several 3‐benzyl‐4‐chromanones and one stilbene derivative were isolated. Pure compounds were examined for their taste activity by means of sensory evaluation, and proved to be responsible for the characteristic taste of this food. Some of these compounds have been synthesized de novo to confirm their structure.

Alkaloids of Uncaria elliptica

Abstract Ajmalicine, formosanine, isomitraphylline and mitraphylline have been isolated from the bark of Uncaria elliptica . Differences in alkaloid content within the species have been observed.

Phytochemicals from Ruta graveolens Activate TAS2R Bitter Taste Receptors and TRP Channels Involved in Gustation and Nociception

Ruta graveolens (rue) is a spontaneous plant in the Mediterranean area with a strong aroma and a very intense bitter taste, used in gastronomy and in folk medicine. From the leaves, stems and fruits of rue, we isolated rutin, rutamarin, three furanocoumarins, two quinolinic alkaloids, a dicoumarin and two long chain ketones. Bitter taste and chemesthetic properties have been evaluated by in vitro assays with twenty receptors of the TAS2R family and four TRP ion channels involved in gustation and nociception. Among the alkaloids, skimmianine was active as a specific agonist of T2R14, whereas kokusaginin did not activate any of the tested receptors. The furanocoumarins activates TAS2R10, 14, and 49 with different degrees of selectivity, as well as the TRPA1 somatosensory ion channel. Rutamarin is an agonist of TRPM5 and TRPV1 and a strong antagonist of TRPM8 ion channels.

Synthesis of diverse spiroisoxazolidinohydantoins by totally regio- and diasteroselective 1,3-dipolar cycloadditions

5-Carboxymethylene hydantoins have been synthesized in high yield and under very mild conditions (20 °C, dichloromethane) through a highly (often totally) regio- and diastereoselective domino process involving readily accessible carbodiimides and acetylenedicarboxylic acid monoesters. Such compounds could be considered very intriguing intermediates for the preparation of spirohydantoins and other hydantoin derivatives. For instance, they react smoothly with either acyclic and cyclic nitrones to form spiroisoxazolidinohydantoins in high yield and with total control of the regio- and diastereoselectivity. Starting from an enantiomerically pure chiral pyrroline nitrone, the reaction was also enantiospecific. Both processes have been studied in detail and the diastereoselectivities seen have been rationalized upon examination of the alternative transition states and by density functional theory calculations of the cycloaddition step.

Interaction between double helix DNA fragments and a new topopyrone acting as human topoisomerase I poison

A water soluble derivative (2) of topopyrones was selected for NMR studies directed to elucidate the mode of binding with specific oligonucleotides. Topopyrone 2 can intercalate into the CG base pairs, but the residence time into the double helix is very short and a fast chemical exchange averaging occurs at room temperature between the free and bound species. The equilibria involved become slow below room temperature, thus allowing to measure a mean lifetime of the complex of ca. 7 ms at 15 degrees C. Structural models of the complex with d(CGTACG)(2) were developed on the basis of DOSY, 2D NOESY and (31)P NMR experiments. Topopyrone 2 presents a strong tendency to self-associate. In the presence of oligonucleotide a certain number of ligand molecules are found to externally stack to the double-helix, in addition to a small fraction of the same ligand intercalated. The external binding to the ionic surface of the phosphoribose chains may thus represents the first step of the intercalation process.

1H-NMR study and structure determination of 4,4- and 4,6-dimers from electrochemical reduction of NADP+

The products arising from one-electron electrochemical reduction of the coenzyme nicotinamide adenine dinucleotide phosphate (NADP+) have been studied by HPLC chromatography and 1H-NMR spectroscopy. HPLC and NMR analyses have shown seven dimeric species, the most abundant of which (40%) has been isolated and has resulted to be an NADP 4,4-linked dimer. The other two diastereoisomeric 4,4-dimers present for the 25% and 10%, respectively, have been detected in the crude reaction mixture, but have not been isolated. The 4,4-tetrahydrobipyridine structure and the stereochemistry at the ring-ring junction for these three isomers have been determined on the basis of their NMR parameters. Preparative HPLC chromatography also led to two fractions enriched in another four dimers, present in the crude mixture, which turned out to have a 4,6-tetrahydrobipyridine structure. All the chemical shifts and the H,H coupling constants of the 4,4- and 4,6-tetrahydrobipyridine systems have been obtained for the seven compounds. For the most abundant among the 4,4-dimers the NMR analysis also gave the coupling constant values of the ribose-diphosphate chain.

Molecular recognition in naphthoquinone derivatives — G-quadruplex complexes by NMR

BACKGROUND G-quadruplexes have become important drug-design targets for the treatment of various human disorders such as cancer, diabetes and cardiovascular diseases. Recently, G-quadruplex structures have been visualized in the DNA of human cells and appeared to be dynamically sensitive to the cell cycle and stabilized by small molecule ligands. A small library of isoxazolo naphthoquinones (1a-h), which exhibited a strong antiproliferative activity on different cancer cell lines, was studied as potential ligands of G-quadruplex DNA. METHODS The DNA binding properties of a series of the selected compounds have been analyzed by fluorescence assays. NMR/modeling studies were performed to describe the complexes between G-quadruplex DNA sequences and two selected compounds 1a and 1b. RESULTS 1a and 1b in the presence of G-quadruplexes, d(T(2)AG(3)T)(4), d(TAG(3)T(2)A)(4) and d(T(2)G(3)T(2))(4), showed good ability of intercalation and the formation of complexes with 2:1 stoichiometry. 1a showed an important interaction with the sequence Pu22 belonging to the promoter of oncogenes c-myc. CONCLUSIONS The ligands directly interact with the external G-tetrads of the G-quadruplexes, without alterations in the structure of the G-quadruplex core. The role of the adenine moieties over the G-tetrads in the stabilization of the complexes was discussed. GENERAL SIGNIFICANCE The results obtained suggested that the strong antiproliferative activity of isoxazolo naphthoquinones is not due to the Hsp90 inhibition, but mainly to the interaction at the level of telomeres and/or at the level of gene promoter. These findings can be used as a basis for the rational drug design of new anticancer agents.