The First Enantioselective Organocatalytic Synthesis of 3-Spiro-α-Alkylidene-γ-Butyrolactone Oxindoles.

A new and flexible methodology catalyzed by bifunctional chiral thioureas has been developed to react ß-nitro oxindoles 1 with aldehydes. This approach allowed us to achieve the first enantioselective organocatalytic synthesis of 3-spiro-α-alkylidene-γ-butyrolactone oxindoles 3. We examined the scope of the two starting materials and, varying the structure of the ß-nitro oxindole 1, intriguing new products, derived from unexpected transformations, have been stereoselectively obtained. The aim of this study was to merge two potentially bioactive structural motifs: the spirooxindole substructure and the α-alkylidene-γ-butyrolactone moiety. A preliminary NMR study on the ability to reversibly trap 2-aminoethanethiol gave us promising results.

Synthesis of 1,2-Dioxetanes as Thermochemiluminescent Labels for Ultrasensitive Bioassays: Rational Prediction of Olefin Photooxygenation Outcome by Using a Chemometric Approach.

Great interest in new thermochemiluminescent (TCL) molecules, for example, in bioanalytical assays, has prompted the design and synthesis of a small library of more than 30 olefins to be subjected to photooxygenation, with the aim of obtaining new 1,2-dioxetane-based TCL labels with optimized properties. Fluorine atoms on the acridan system remarkably stabilize 1,2-dioxetanes when they are located in the 3- and/or 6-position (4 h and 4 i). On the other hand, 2,7-difluorinated acridan dioxetane (4 j) showed a significantly enhanced fluorescence quantum yield with respect to the unsubstituted dioxetane (4 a). Some of the synthesized olefins did not undergo singlet oxygen addition and a rationale was sought to ease the photooxygenation step, leading to the TCL dioxetanes. A chemometric approach has been adopted to exploit principal component analysis and linear discriminant analysis of the structural and electronic molecular descriptors obtained by DFT optimizations of olefins 3. This approach allows the steric and electronic parameters that govern dioxetane formation to be revealed.

Highly stereoselective [4+2] and [3+2] spiroannulations of 2-(2-oxoindolin-3-ylidene)acetic esters catalyzed by bifunctional thioureas.

A new Michael-Michael cascade reaction between 2-(2-oxoindolin-3-ylidene)acetic esters 1 and nitroenoates 2, catalyzed by bifunctional thioureas, is investigated. The combination of the two Michael reactions results in a novel and facile [4+2] or [3+2] spiroannulation process, which is characterized by the following features: 1) two carbon-carbon bonds and four stereocenters, including a quaternary spiro carbon, are formed under mild conditions; 2) an unprecedented and stereochemically defined substitution pattern on the spirocarbocyclic unit is obtained; 3) the double-bond configuration of the donor-acceptor nitroenoate 2 determines the absolute configuration of the spiro center, whereas the remaining stereocenters are formed under control of the catalyst. The effect on the final stereochemical outcome of structural variations of each starting material, catalyst, and experimental conditions is analyzed in detail. In particular, the use of specifically designed chiral nitroenoates enables diverse polyfunctional spirocyclohexane derivatives containing six consecutive stereogenic centers to be constructed. To our knowledge, this is the first asymmetric organocatalytic strategy enabling both five- and six-membered ß-nitro spirocarbocyclic oxindoles.

Organically modified silica nanoparticles doped with new acridine-1,2-dioxetane analogues as thermochemiluminescence reagentless labels for ultrasensitive immunoassays.

Doped organically modified silica nanoparticles (ORMOSIL NPs) with luminescent molecules represent a potent approach to signal amplification in biomolecule labeling. Herein, we report the synthesis of new ORMOSIL NPs incorporating thermochemiluminescent (TCL) 1,2-dioxetane derivatives to prepare TCL labels for ultrasensitive immunoassay, displaying a detectability comparable to those offered by other conventional luminescence-based systems. Amino-functionalized ORMOSIL NPs were synthesized for inclusion of acridine-containing 1,2-dioxetane derivatives with a fluorescence energy acceptor. The doped ORMOSIL NPs were further functionalized with biotin for binding to streptavidin-labeled species to be used as universal detection reagents for immunoassays. A quantitative non-competitive immunoassay for streptavidin has been developed by immobilizing anti-streptavidin antibody to capture streptavidin, then the antibody-bound streptavidin was detected by the biotinylated TCL ORMOSIL NPs. The analytical performance was similar to that obtained by chemiluminescent (CL) detection using horseradish peroxidase (HRP) as label, being the limits of detection 2.5-3.8 and 0.8 ng mL(-1) for TCL and CL detection, respectively. In addition, since the TCL emission is simply initiated by thermolysis of the label, chemical reagents were not required, thus allowing reagentless detection with a simplification of the analytical protocols. A compact mini dark box device based on the use of a cooled charge-coupled device (CCD) and a miniaturized heater has been developed and used to quantify the light emission after heat decomposition of the label at a temperature of 90-120 °C. These characteristics make TCL-doped ORMOSIL NPs ideal universal nanoprobes for ultrasensitive bioassays such as immuno- and DNA-based assay.

Quantitative chemical imaging of the intracellular spatial distribution of fundamental elements and light metals in single cells.

We report a method that allows a complete quantitative characterization of whole single cells, assessing the total amount of carbon, nitrogen, oxygen, sodium, and magnesium and providing submicrometer maps of element molar concentration, cell density, mass, and volume. This approach allows quantifying elements down to 10(6) atoms/µm(3). This result was obtained by applying a multimodal fusion approach that combines synchrotron radiation microscopy techniques with off-line atomic force microscopy. The method proposed permits us to find the element concentration in addition to the mass fraction and provides a deeper and more complete knowledge of cell composition. We performed measurements on LoVo human colon cancer cells sensitive (LoVo-S) and resistant (LoVo-R) to doxorubicin. The comparison of LoVo-S and LoVo-R revealed different patterns in the maps of Mg concentration with higher values within the nucleus in LoVo-R and in the perinuclear region in LoVo-S cells. This feature was not so evident for the other elements, suggesting that Mg compartmentalization could be a significant trait of the drug-resistant cells.

A novel fluorescent chemosensor allows the assessment of intracellular total magnesium in small samples.

The present study investigated the analytical capabilities of a new fluorescent chemosensor, named DCHQ5, a phenyl derivative belonging to the family of diaza-crown-hydroxyquinolines, for the quantitative assessment of total intracellular Mg content. The results obtained were compared to the analytical performances of DCHQ1 - the parent probe of the series which so far was the only suitable species for the quantitative assessment of the intracellular total magnesium and showed comparable results to atomic absorption spectroscopy. Different protocols were tested in several cell lines both by flow cytometry and by steady state fluorescence spectroscopy assays. The results obtained support the possibility to use DCHQ5 to accurately quantify the intracellular total Mg in much smaller samples than DCHQ1, also displaying an increased stable intracellular staining. These features, combined with the high fluorescence enhancement upon cation binding, and the possibility to be excited both in the UV and visible region, make DCHQ5 a valuable and versatile analytical tool for Mg assessment in biological samples.

Development of Lab-Scale Continuous Stirred-Tank Reactor as Flow Process Tool for Oxidation Reactions Using Molecular Oxygen.

The use of sustainable oxidants is of great interest to the chemical industry, considering the importance of oxidation reactions for the manufacturing of chemicals and society's growing awareness of its environmental impact. Molecular oxygen (O2), with an almost optimal atom efficiency in oxidation reactions, presents one of the most attractive alternatives to common reagents that are not only toxic in most cases but produce stoichiometric amounts of waste that must be treated. However, fire and explosion safety concerns, especially when used in combination with organic solvents, restrict its easy use. Here, we use state-of-the-art 3D printing and experimental feedback to develop a miniature continuous stirred-tank reactor (mini-CSTR) that enables efficient use of O2 as an oxidant in organic chemistry. Outstanding heat dissipation properties, achieved through integrated jacket cooling and a high surface-to-volume ratio, allow for a safe operation of the exothermic oxidation of 2-ethylhexanal, surpassing previously reported product selectivity. Moving well beyond the proof-of-concept stage, we characterize and illustrate the reactor's potential in the gas-liquid-solid triphasic synthesis of an endoperoxide precursor of antileishmanial agents. The custom-designed magnetic overhead stirring unit provides improved stirring efficiency, facilitating the handling of suspensions and, in combination with the borosilicate gas dispersion plate, leading to an optimized gas-liquid interface. These results underscore the immense potential that lies within the use of mini-CSTR in sustainable chemistry.

Organocatalytic conjugate addition of nitroalkanes to 3-ylidene oxindoles: a stereocontrolled diversity oriented route to oxindole derivatives.

An efficient and highly enantioselective Michael addition of nitroalkanes to 3-ylidene oxindoles is described, mediated by thiourea-based bifunctional organocatalysts. The stereochemistry at C(α) and C(ß) centers is perfectly controlled, and the intermediate C-3 enolate is trapped with a second Michael acceptor. The developed one-pot three-component consecutive reactions generate up to four contiguous stereocenters, including the C-3 all-carbon quaternary center, in a perfectly defined configuration. The conversion of the ß-nitro oxindole into the corresponding ß-amino derivative discloses synthetically useful transformations, exploitable to generate pharmaceutically attractive molecular targets.

Preparation and characterization of thermochemiluminescent acridine-containing 1,2-dioxetanes as promising ultrasensitive labels in bioanalysis.

Thermochemiluminescence is the luminescence process in which a thermodynamically unstable molecule decomposes with light emission when heated above a threshold temperature. We recently reported the thermochemiluminescence properties of an acridine-containing 1,2-dioxetane, which emits at relatively low temperatures (i.e., below 100 °C). Herein, we explored the effect of the introduction of methyl substituents in the acridine system. The methyl group did not determine an excessive destabilization of 1,2-dioxetane ring nor significantly affect the general physical properties of the molecule. Monosubstituted methyl derivatives and a series of derivatives bearing several combinations of two, three, and four methyl groups were prepared. The rate of formation of 1,2-dioxetane derivatives 1b-k strongly depended on the methyl substitution pattern. All members of this library of mono-, di-, tri-, and tetramethyl-substituted derivatives were characterized in terms of photophysical and thermochemiluminescence properties. The introduction of methyl groups into the acridine ring caused a marked decrease in the activation energy of the thermochemiluminescent reaction. Tri- and tetramethyl-substituted acridones had the highest fluorescence quantum yields, in the range 0.48-0.52, and the corresponding 1,2-dioxetanes 1h and 1j showed in thermochemiluminescence imaging experiments limit of detection values more than ten times lower with respect to the unsubstituted derivative.

Dioxetane-doped silica nanoparticles as ultrasensitive reagentless thermochemiluminescent labels for bioanalytics.

Thermochemiluminescence (TCL; the light emission originating by the thermally triggered decomposition of a molecule) was proposed in the late 1980s as a detection technique for immunoassays. However, after little pioneering work, this technique was abandoned because of the high temperatures required and the poor detectability in comparison to other labels. Here we describe for the first time a thermochemiluminescent acridine-based 1,2-dioxetane with a remarkably low (i.e., below 100 °C) emission-triggering temperature, which made it possible to obtain light emission even in an aqueous environment, as well as amino-functionalized silica nanoparticles loaded with this compound and the fluorescent energy acceptor dipyridamole. Thanks to the signal amplification due to the large number of 1,2-dioxetane molecules in each nanoparticle (about 10(4)) and the increased emission efficiency due to energy transfer to the fluorescent acceptor, the doped nanoparticles could be revealed with a detectability close to that of chemiluminescent enzyme labels (the limit of detection of doped nanoparticles by TCL imaging was 1 × 10(-16) mol mm(-2), thus approaching the value of 5 × 10(-17) mol mm(-2) obtained for the enzyme label horseradish peroxidase with chemiluminescence detection). They could thus be used as highly detectable labels in the development of sensitive TCL-based immunoassays and nucleic acid hybridization assays, in which the detection step does not require any additional chemical reagent. We believe that these doped silica nanoparticles could pave the way for the revival of TCL detection in bioanalytics, taking advantage of the reagentless detection and the high signal/noise ratio in comparison with conventional luminescence detection techniques.

Microwave assisted synthesis of a small library of substituted N,N'-bis((8-hydroxy-7-quinolinyl)methyl)-1,10-diaza-18-crown-6 ethers.

N,N'-bis-((8-hydroxy-7-quinolinyl)methyl)-1,10-diaza-18-crown-6 ether 1a and its analogue 1c are known as fluorescent sensors of magnesium in living cells. With the aim to investigate the effects of the substitution pattern on the photophysical properties of ligands 1 and their metal complexes, we developed an efficient microwaves enhanced one-pot Mannich reaction to double-armed diaza-crown ligands 1 carrying a variety of substituents. This new protocol is characterized by shorter reaction times, enhanced yields, and improved product purities with respect to the use of conventional conductive heating.

Evaluation of the Pharmacophoric Role of the O-O Bond in Synthetic Antileishmanial Compounds: Comparison between 1,2-Dioxanes and Tetrahydropyrans.

Leishmaniases are neglected diseases that can be treated with a limited drug arsenal; the development of new molecules is therefore a priority. Recent evidence indicates that endoperoxides, including artemisinin and its derivatives, possess antileishmanial activity. Here, 1,2-dioxanes were synthesized with their corresponding tetrahydropyrans lacking the peroxide bridge, to ascertain if this group is a key pharmacophoric requirement for the antileishmanial bioactivity. Newly synthesized compounds were examined in vitro, and their mechanism of action was preliminarily investigated. Three endoperoxides and their corresponding tetrahydropyrans effectively inhibited the growth of Leishmania donovani promastigotes and amastigotes, and iron did not play a significant role in their activation. Further, reactive oxygen species were produced in both endoperoxide- and tetrahydropyran-treated promastigotes. In conclusion, the peroxide group proved not to be crucial for the antileishmanial bioactivity of endoperoxides, under the tested conditions. Our findings reveal the potential of both 1,2-dioxanes and tetrahydropyrans as lead compounds for novel therapies against Leishmania.

Mathematical form factor studies on the effect of water on airborne particles morphology using a bi-dimensional TEM image processing.

Mathematical morphology is a tool for extracting image components that are useful for representation and description. The technique consists of a set-theoretic method of image analysis providing a quantitative description of geometrical structures. A simple application of mathematical morphology to a bi-dimensional processing of TEM images of airborne particles allows us to distinguish between particles grown and/or transported in atmosphere under dry conditions or in rainy days by a simple comparison of the corresponding image form factors. The form factors range in the 0.385-0.031 interval in the case of particles sampled in rainy days, and in the 0.103-0.006 interval in the case of non-rainy conditions. The same classification criterion was applied to filters collected under dry conditions and plunged in water. The results demonstrate that a morphological change may be artificially induced to the particle structure. The artificially wet particles, indeed, display an apparent contraction of their structures evidenced by a two-fold increase of the average values of their form factors. The last experiment roughly simulates the impact of particles on membranes of the respiratory tract.

A modular approach to catalyst hydrophobicity for an asymmetric aldol reaction in a biphasic aqueous environment.

Catalyst 5, an ion pair consisting of a hydrophilic cation and a lipophilic anion, fulfils the solubility requirements needed to couple efficiency (enantioselectivities and anti-diastereoselectivities up to > or = 99%) and catalyst recyclability in asymmetric aldol reactions under aqueous biphasic conditions.

Thermochemiluminescent semiconducting polymer dots as sensitive nanoprobes for reagentless immunoassay.

Thermochemiluminescence (TCL) is a potentially simple and sensitive detection principle, as the light emission is simply elicited by thermally-triggered decomposition of a molecule to produce a singlet excited-state product. Here we report about TCL semiconductive polymer dots (TCL-Pdots) obtained by doping fluorescent cyano-polyphenylene vinylene (CN-PPV) Pdots with an acridine 1,2-dioxetane derivative. The TCL-Pdots showed remarkable stability over time and minimum leaching of the thermo-responsive species. Furthermore, detectability of TCL-Pdots was improved by taking advantage of both the high number of 1,2-dioxetanes entrapped in each nanoparticle (about 20 molecules per Pdot) and the 5-fold enhancement of TCL emission due to energy transfer from 1,2-dioxetane to the polymer matrix, which itself acted as an energy acceptor. Indeed, upon heating the TCL-Pdots to 110 °C, 1,2-dioxetane decomposes generating an acridanone product in its electronically excited state. The latter transfers its energy to the surrounding CN-PPV chains via the Förster mechanism (φFRET about 80%), resulting in intense yellow light emission (550 nm wavelength). We next conjugated streptavidin onto the surface of these TCL-Pdots and demonstrated their suitability for use in biological studies. In particular, we used TCL-Pdots as labels in a model non-competitive immunoassay for IgG detection, which showed a LOD of 13 nM IgG and a dynamic range extending up to 230 nM. By combining the biocompatibility, brightness and tunability of Pdot fluorescence emission with the thermally-triggered reagentless light generation from TCL 1,2-dioxetanes, a broad panel of ultrabright TCL nanosystems could be designed for a variety of bioscience applications, even in multiplexed formats.

The interaction of heme with plakortin and a synthetic endoperoxide analogue: new insights into the heme-activated antimalarial mechanism.

In the present work we performed a combined experimental and computational study on the interaction of the natural antimalarial endoperoxide plakortin and its synthetic analogue 4a with heme. Obtained results indicate that the studied compounds produce reactive carbon radical species after being reductively activated by heme. In particular, similarly to artemisinin, the formation of radicals prone to inter-molecular reactions should represent the key event responsible for Plasmodium death. To our knowledge this is the first experimental investigation on the reductive activation of simple antimalarial endoperoxides (1,2-dioxanes) by heme and results were compared to the ones previously obtained from the reaction with FeCl2. The obtained experimental data and the calculated molecular interaction models represent crucial tools for the rational optimization of our promising class of low-cost synthetic antimalarial endoperoxides.

An efficient high-yield synthesis of D-ribo-phytosphingosine.

[Structure: see text] [4R-[4alpha(S),5alpha]]-2,2-Dimethyl-4-(2-oxo-5-vinyl[1,3]dioxolan-4-yl)oxazolidine-3-carboxylic acid tert-butyl ester 5a, obtained in excellent yield and diastereoselectivity by the alpha-hydroxyallylation of the Garner aldehyde (4), is exploited in a novel high-yield synthesis of D-ribo-phytosphingosine (8), using microwave-enhanced cross metathesis as the key step in the chain elongation.

Particulate matter, gas-phase and particle-bound polycyclic aromatic hydrocarbons in an urban environment heavily impacted by vehicular traffic (Bologna, Italy).

A set of 8 polycyclic aromatic hydrocarbons (PAHs) has been analysed in a traffic-limited area in Bologna downtown, both in the gas-phase and in the particulate phase (PM10), and gas-to-particle partitioning has been investigated. From Sep 2002 to May 2003, 28 high volume PM10 samplings were carried out, and in 50 % of the samplings, PM10 concentrations exceeded the limit of 50 microg/m3 established by a 1999 EU directive. A precisely defined sampling strategy was adopted to limit artifacts (8 h sampling in the same time interval) in the 28 samplings carried out in different meteorological conditions. A linear log-log correlation was found between gas-particle partitioning coefficients KBp and the subcooled liquid vapour pressures pB0LB, with rP2P = 0.82 and slope = -0.59. This empirical correlation may be used to anticipate the total (gas + particle-bound) concentration of each PAHs in this urban site, once PM10 and the particle-bound concentration is measured. Parallel samplings of PM10 and of PM2.5 allowed us to ascertain that PM2.5 represents the gross contribution to PM10 and that most of the particle-bound PAHs reside on the finest fraction of particulate matter.

Further optimization of plakortin pharmacophore: structurally simple 4-oxymethyl-1,2-dioxanes with promising antimalarial activity.

For the optimization of the plakortin pharmacophore, we recently proposed a straightforward synthesis of 4-carbomethoxy-3-methoxy-1,2-dioxanes as potential antimalarial drug candidates. Herein we report the chemoselective reduction of the 4-carbomethoxy group which has allowed us to prepare in good yields twenty-four new endoperoxides carrying either the hydroxymethyl or the methoxymethyl group on C4 in various stereochemical arrangements with respect to the alkyl groups on C3 and C6 (the endoperoxide carbons). Some of these compounds showed promising in vitro antimalarial activities, both against chloroquine-resistant (CQ-R) and susceptible (CQ-S) strains of Plasmodium falciparum, with IC50 values in the range of 0.5-1.0 µM. Compound 8g showed activity against the CQ-R strain comparable to that of the structurally more demanding plakortin.