A regular thymine tetrad and a peculiar supramolecular assembly in the first crystal structure of an all-LNA G-quadruplex.

Locked nucleic acids (LNAs) are formed by bicyclic ribonucleotides where the O2' and C4' atoms are linked through a methylene bridge and the sugar is blocked in a 3'-endo conformation. They represent a promising tool for therapeutic and diagnostic applications and are characterized by higher thermal stability and nuclease resistance with respect to their natural counterparts. However, structural descriptions of LNA-containing quadruplexes are rather limited, since few NMR models have been reported in the literature. Here, the first crystallographically derived model of an all-LNA-substituted quadruplex-forming sequence 5'-TGGGT-3' is presented refined at 1.7 Šresolution. This high-resolution crystallographic analysis reveals a regular parallel G-quadruplex arrangement terminating in a well defined thymine tetrad at the 3'-end. The detailed picture of the hydration pattern reveals LNA-specific features in the solvent distribution. Interestingly, two closely packed quadruplexes are present in the asymmetric unit. They face one another with their 3'-ends giving rise to a compact higher-order structure. This new assembly suggests a possible way in which sequential quadruplexes can be disposed in the crowded cell environment. Furthermore, as the formation of ordered structures by molecular self-assembly is an effective strategy to obtain nanostructures, this study could open the way to the design of a new class of LNA-based building blocks for nanotechnology.

Thermodynamic signature of secondary nano-emulsion formation by isothermal titration calorimetry.

The stabilization of oil in water nano-emulsions by means of a polymer coating is extremely important; it prolongs the shelf life of the product and makes it suitable for a variety of applications ranging from nutraceutics to cosmetics and pharmaceutics. To date, an effective methodology to assess the best formulations in terms of thermodynamic stability has yet to be designed. Here, we perform a complete physicochemical characterization based on isothermal titration calorimetry (ITC) compared to conventional dynamic light scattering (DLS) to identify polymer concentration domains that are thermodynamically stable and to define the degree of stability through thermodynamic functions depending upon any relevant parameter affecting the stability itself, such as type of polymer coating, droplet distance, etc. For instance, the method was proven by measuring the energetics in the case of two different biopolymers, chitosan and poly-L-lysine, and for different concentrations of the emulsion coated with poly-L-lysine.

Increasing the X-ray diffraction power of protein crystals by dehydration: the case of bovine serum albumin and a survey of literature data.

Serum albumin is one of the most widely studied proteins. It is the most abundant protein in plasma with a typical concentration of 5 g/100 mL and the principal transporter of fatty acids in plasma. While the crystal structures of human serum albumin (HSA) free and in complex with fatty acids, hemin, and local anesthetics have been characterized, no crystallographic models are available on bovine serum albumin (BSA), presumably because of the poor diffraction power of existing hexagonal BSA crystals. Here, the crystallization and diffraction data of a new BSA crystal form, obtained by the hanging drop method using MPEG 5K as precipitating agent, are presented. The crystals belong to space group C2, with unit-cell parameters a = 216.45 Å, b = 44.72 Å, c = 140.18 Å, ß = 114.5°. Dehydration was found to increase the diffraction limit of BSA crystals from ~8 Å to 3.2 Å, probably by improving the packing of protein molecules in the crystal lattice. These results, together with a survey of more than 60 successful cases of protein crystal dehydration, confirm that it can be a useful procedure to be used in initial screening as a method of improving the diffraction limits of existing crystals.

Tritoniopsins A-D, cladiellane-based diterpenes from the South China Sea nudibranch Tritoniopsis elegans and its prey Cladiella krempfi.

Four diterpenes, tritoniopsins A-D (1-4), have been isolated from the South China Sea nudibranch Tritoniopsis elegans and its prey, the soft coral Cladiella krempfi. They display an unprecedented pyran ring in the cladiellane framework, thus representing a novel cladiellane-based diterpene family. Their structures have been mainly characterized by NMR and mass spectrometric techniques, whereas the relative configuration of compound 1 was secured by X-ray analysis. Antiproliferative assays on tumor and nontumor cell lines have been carried out for the main metabolite, tritoniopsin B (2).

Design and synthesis of spirotryprostatin-inspired diketopiperazine systems from prolyl spirooxoindolethiazolidine derivatives.

Based on the spirotryprostatin-A structure, we designed, synthesized, and evaluated different series of compounds belonging to the diketopiperazine structural class as potential cell cycle modulators and cytotoxic agents. Starting from the spirooxoindolthiazolidine scaffold, amide coupling with Pro derivatives and intramolecular cyclization reactions are suitable synthetic methods to generate chemically diverse diketopiperazine system, such as hexahydropyrrolo[1,2-a][1,3]thiazolo[3,2-d]pyrazine-5,10-dione (structure I), hexahydropyrrolo[1,2-a] [1,3]thiazolo[3,4-d]pyrazine-5,10-dione (structure II) and spiroindol-2-one[3,3']hexahydro-5,10H-pyrrolo[1,2-a][1,3]thiazolo[3,4-d]pyrazine-5,10-dione (structure III). Some of these compounds, especially those who belong to the series I and II, showed interesting cytotoxic activity.

Applications of isothermal titration calorimetry in biophysical studies of G-quadruplexes.

G-quadruplexes are higher-order nucleic acids structures formed by G-rich sequences that are stabilized by tetrads of hydrogen-bonded guanine bases. Recently, there has been growing interest in the study of G-quadruplexes because of their possible involvement in many biological processes. Isothermal titration calorimetry (ITC) has been proven to be a useful tool to study the energetic aspects of G-quadruplex interactions. Particularly, ITC has been applied many times to determine the thermodynamic properties of drug-quadruplex interactions to screening among various drugs and to address drug design. In the present review, we will focus on the ITC studies of G-quadruplex structures and their interaction with proteins and drugs and the most significant results will be discussed.

Duplex/quadruplex oligonucleotides: Role of the duplex domain in the stabilization of a new generation of highly effective anti-thrombin aptamers.

Recently, mixed duplex/quadruplex oligonucleotides have attracted great interest for use as biomedical aptamers. In the case of anti-thrombin aptamers, the addition of duplex-forming sequences to a G-quadruplex module identical or very similar to the best-known G-quadruplex of the Thrombin Binding Aptamer (HD1) results in new or improved biological properties, such as higher activity or different recognition properties with respect to HD1. Remarkably, this bimodular fold was hypothesized, based on its sequence, for the only anti-thrombin aptamer in advanced clinical trial, NU172. Whereas cation modulation of G-quadruplex conformation and stability is well characterized, only few data from similar analysis on duplex/quadruplex oligonucleotides exist. Here we have performed a characterization of structure and stability of four different duplex/quadruplex anti-thrombin aptamers, including NU172, in the presence of different cations and in physiological-mimicking conditions in comparison to HD1, by means of spectroscopic techniques (UV and circular dichroism) and differential scanning calorimetry. Our data show a strong reciprocal influence of each domain on the stability of the other and in particular suggest a stabilizing effect of the duplex region in the presence of solutions mimicking the physiological conditions, strengthening the idea that bimodular aptamers present better therapeutic potentialities than those containing a single G-quadruplex domain.

Relative stereochemistry of a diterpene from Salvia cinnabarina.

The relative stereochemistry of 3,4-secoisopimara-4(18),7,15-triene-3-oic acid, a diterpenoid with antispasmodic, hypotensive and antibacterial activities isolated from Salvia cinnabarina, was determined by an X-ray diffraction analysis of a single crystal of a suitable crystalline derivative.

Relative stability of quadruplexes containing different number of G-tetrads.

The aim of this work is to compare the physicochemical properties of three oligonucleotidic sequences, d(TGGGT), d(TGGGGT) and d(TGGGGGT), which assemble to form quadruplex structures with the same molecularity, but containing three, four, and five G-quartets, respectively. The addition of one or two G-tetrads greatly increases both the enthalpy and Tm values of the quadruplex dissociation.

Antifungal Activity of Some Constituents of Origanum vulgare L. Essential Oil Against Postharvest Disease of Peach Fruit.

Plant essential oils (EOs) can potentially replace synthetic fungicides in the management of postharvest fruit and vegetable diseases. The aim of this study was to evaluate in vitro and in vivo effectiveness of thymol, carvacrol, linalool, and trans-caryophyllene, single constituents of the EO of Origanum vulgare L. ssp. hirtum against Monilinia laxa, M. fructigena, and M. fructicola, which are important phytopathogens and causal agents of brown rot of pome and stone fruits in pre- and postharvest. Moreover, the possible phytotoxic activity of these constituents was assessed and their minimum inhibitory concentration (MIC) was determined. In vitro experiment indicated that thymol and carvacrol possess the highest antifungal activity. Results of in vivo trials confirmed the strong efficacy of thymol and carvacrol against brown rot of peach fruits. The thymol MIC resulted to be 0.16 µg/µL against M. laxa and M. fructigena and 0.12 µg/µL against M. fructicola, whereas for carvacrol they were 0.02 µg/µL against the first two Monilinia species and 0.03 µg/µL against the third. Results of this study indicated that thymol and carvacrol could be used after suitable formulation for controlling postharvest fruit diseases caused by the three studied Monilinia species.

Linkage of proton binding to the thermal dissociation of triple helix complex.

The effects of cytosine protonation on the thermodynamic properties of parallel pyrimidine motif DNA triplex were investigated and characterized by different techniques, such as circular dichroism (CD), ultraviolet spectroscopy (UV) and differential scanning calorimetry (DSC). A thermodynamic model was developed which, by linking the cytosine ionization equilibrium to the dissociation process of the triplex, is able to rationalize the experimental data and to reproduce the pH dependence of the free energy, enthalpy and entropy changes associated with the triplex formation. The results are useful to systematically introduce the effect of pH in a more general model able to predict the stability of DNA triplexes on the basis of the sequence alone.

Toward an antitumor form of bovine pancreatic ribonuclease: the crystal structure of three noncovalent dimeric mutants.

The cytotoxic action of bovine seminal ribonuclease (BS-RNase) depends on its noncovalent swapped dimeric form (NCD-BS), which presents a compact structure that allows the molecule to escape ribonuclease inhibitor (RI). A key role in the acquisition of this structure has been attributed to the concomitant presence of a proline in position 19 and a leucine in position 28. The introduction of Leu28, Cys31, and Cys32 and, in addition, of Pro19 in the sequence of bovine pancreatic ribonuclease (RNase A) has produced two dimeric variants LCC and PLCC, which do exhibit a cytotoxic activity, though at a much lower level than BS-RNase. The crystal structure analysis of the noncovalent swapped form (NCD) of LCC and PLCC, complexed with the substrate analogue 2 '-deoxycytidylyl(3 ',5 ')-2 '-deoxyguanosine, has revealed that, differently from NCD-BS, the dimers adopt an opened quaternary structure, with the two Leu residues fully exposed to the solvent, that does not hinder the binding of RI. Similar results have been obtained for a third mutant of the pancreatic enzyme, engineered with the hinge peptide sequence of the seminal enzyme (residues 16-22) and the two cysteines in position 31 and 32, but lacking the hydrophobic Leu residue in position 28. The comparison of these three structures with those previously reported for other ribonuclease swapped dimers strongly suggests that, in addition to Pro19 and Leu28, the presence of a glycine at the N-terminal end of the hinge peptide is also important to push the swapped form of RNase A dimer into the compact quaternary organization observed for NCD-BS.

3,6-Bis(dimethylamino)-10-(10-iododecyl)acridinium iodide.

In the title compound, C(27)H(39)IN(3)(+).I(-), the acridinium system shows the usual approximate mirror symmetry about the central C.N line, and the corresponding bond lengths and angles in the two halves agree within experimental error. The alkyl chain at the ring N atom is initially perpendicular to the ring plane and then bends sharply at the fourth C atom. Pairs of centrosymmetrically related cations overlap two of their rings and the dimethylamino groups are also partly involved in the overlap. Each I(-) ion is involved in short-range interactions with two cations. These interactions give rise to a 14-membered cyclic structure, which involves pairs of cations and anions across an inversion centre.

A new norkaurane-gamma-lactone from Parinari sprucei.

The title tetracyclic diterpenoid, 10,13,16,17-tetrahydroxy-9-methyl-15-oxo-20-norkaurane-18,10-carbolactone hemihydrate, C(20)H(28)O(6).0.5H(2)O, is a plant metabolite from Parinari sprucei, part of the Venezuelan Amazon flora. The asymmetric unit consists of two nearly identical molecules of the diterpenoid and one molecule of water. Some of the geometric parameters reflect steric strain in the molecule. The extended structure is characterized by hydrogen bonds and weaker hydrogen-mediated interactions, which involve all of the hydroxy groups and propagate in sheets that coincide with the (002) family of planes. The water molecule acts as a double hydrogen-bond donor and single acceptor and thus plays a critical role in the pattern of intermolecular interactions.

Solid-state photodimerization of steroid enones.

Androst-4-ene-3,17-dione (1) and 17alpha-methyltestosterone (2) are dimerized in the solid-state by UV radiation. These substances were selected by a search in the CSD among the steroid enones presenting in the crystalline state an intermolecular short contact between a hydrogen alpha to a carbonyl group and the oxygen of an enone system. Dimerization occurs by transfer of the hydrogen to the oxygen and connection between the two involved carbons. Androst-4-ene-3,17-dione (1) affords dimer 3 and trimer 4, both formed by connection of the C-16 of a molecule with the C-3 of a near one. Irradiation of 17alpha-methyltestosterone (2) gives the isomeric trienones 5 and 6. These compounds are reasonably formed by dehydration of unisolated intermediate products 7 and/or 8 obtained by coupling of two molecules through a linkage between the C-2 and the C-3' carbons. The formation mechanisms of the photoproducts are satisfactory explained on the basis of the molecular arrangement of the monomers in the crystal state. Modeling of the dimeric molecules was done using molecular mechanics calculations. A single-crystal X-ray of the dimer of androst-4-ene-3,17-dione confirms the structural interpretation of spectral data. The conformer found in the solid-state agrees well with the results of molecular mechanics calculations.