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Current methods for the preparation of functional small-molecule fluorophores generally require labor-intensive, multi-step synthetic routes for all the major chromophore groups. In spite of recent significant contributions from numerous laboratories, the paucity of rapid, straightforward and wide-scope synthetic strategies in this field is limiting the development of advanced probes for bioimaging, sensing and therapeutic applications. We describe herein a general and robust methodology for the one-step fluorescent labeling of a wide variety of molecules having C-, N-, P-, O-, S-, or halide-nucleophilic centers, using stable and readily available acetoxymethyl-BODIPYs as reagents in the presence of an acid catalyst. This modular methodology allows a very facile preparation of mono- and di-functional probes incorporating a broad assortment of biomolecules, enzyme cofactors, natural products, and other chromophores, as well as chemical functionalities for a wide range of applications including bioorthogonal conjugation, polymerization, and supramolecular chemistry, among others. The photophysical properties and preliminary applications of the new probes in live-cell imaging were also studied. The described strategy enables the high-throughput engineering of novel BODIPY dyes with diverse functionalities for basic and applied science with potential for innovative technological applications.

[ES] Compuestos para etiquetado fluorescente. La presente invención se refiere a unos compuestos de estructura BODIPY con buenas propiedades para el etiquetado fluorescente específico de C-nucleófilos, así como su procedimiento de obtención que permite de manera eficiente y económica la preparación de una gran diversidad de compuestos de forma directa, rápida, y con buenos rendimientos de reacción. La aplicación de estos compuestos incluye, aunque no exclusivamente, su uso como marcadores biológicos para la tinción selectiva de células y microrganismos vivos, como sondas fluorescentes en biosensores en bioimagen de organismos vivos o los captadores de energía en células solares, o como emisores en láseres de colorante o como emisores en sistemas OLED., [EN] Fluorescent labeling compounds. The present invention refers to compounds with a BODIPY structure with good properties for the specific fluorescent labeling of C-nucleophiles, as well as its obtaining procedure that allows efficiently and economically the preparation of a great diversity of compounds directly, quickly. , and with good reaction yields. The application of these compounds includes, although not exclusively, their use as biological markers for the selective staining of living cells and microorganisms, as fluorescent probes in biosensors in bioimaging of living organisms or energy harvesters in solar cells, or as emitters in lasers. dye or as emitters in OLED systems.

[EN] The present invention relates to compounds of BODIPY structure having good properties for the specific fluorescent labelling of C-nucleophiles and to the process of obtainment thereof, which enables the efficient and economic preparation of a great diversity of compounds in a direct, fast way and with good reaction yields. The application of these compounds includes, but is not limited to, their use as biomarkers for selective staining of living cells and microorganisms, as fluorescent probes in biosensors in bioimaging of living organisms or energy scavengers in solar cells, or as emitters in dye lasers or as emitters in OLED systems., [ES] La presente invención se refiere a unos compuestos de estructura BODIPY con buenas propiedades para el etiquetado fluorescente específico de C-nucleófilos, así como su procedimiento de obtención que permite de manera eficiente y económica la preparación de una gran diversidad de compuestos de forma directa, rápida, y con buenos rendimientos de reacción. La aplicación de estos compuestos incluye, aunque no exclusivamente, su uso como marcadores biológicos para la tinción selectiva de células y microrganismos vivos, como sondas fluorescentes en biosensores en bioimagen de organismos vivos o los captadores de energía en células solares, o como emisores en láseres de colorante o como emisores en sistemas OLED., [FR] La présente invention concerne des composés de structure BODIPY ayant de bonnes propriétés pour l'étiquetage fluorescent spécifique de C-nucléophiles, ainsi que leur procédé d'obtention permettant de manière efficace et économique la préparation d'une grande variété de composés de manière directe, rapide et avec de bons rendements de réaction. L'application de ces composés comprend, même de manière non exclusive, leur utilisation comme marqueurs biologiques pour la coloration sélective de cellules et de micro-organismes vivants, comme sondes fluorescentes dans des biocapteurs en bio-imagerie d'organismes vivants ou capteurs d'énergie dans des cellules solaires ou comme émetteurs dans des lasers à colorant ou comme émetteurs dans des systèmes OLED.

Organic dyes based on F-BODIPY derivatives and carnitine, method of producing same and their use for specific fluorescent labeling or tagging of mitochondria in live cells

Organic dyes based on F-BODIPY derivatives and carnitine, method of producing same and their use for specific fluorescent labeling or tagging of mitochondria in live cells.

5-methoxy tryptophan (5-MTP) is an anti-fibrotic metabolite made by fibroblasts and epithelial cells, present in a micromolar concentrations in human blood, and is associated with the progression of fibrotic kidney disease, but the mechanism is unclear. Here, we show by microscopy and functional assays that 5-MTP influences mitochondria in human peripheral blood monocyte-derived macrophages. As a result, the mitochondrial membranes are more rigid, more branched, and are protected against oxidation. The macrophages also change their metabolism by reducing mitochondrial import of acyl-carnitines, intermediates of fatty acid metabolism, driving glucose import. Moreover, 5-MTP increases the endocytosis of collagen by macrophages, and experiments with inhibition of glucose uptake showed that this is a direct result of their altered metabolism. However, 5-MTP does not affect the macrophages following pathogenic stimulation, due to 5-MTP degradation by induced expression of indole-amine oxygenase-1 (IDO-1). Thus, 5-MTP is a fibrosis-protective metabolite that, in absence of pathogenic stimulation, promotes collagen uptake by anti-inflammatory macrophages by altering the physicochemical properties of their mitochondrial membranes.

[EN] The upsurge of antimicrobial resistance (AMR) is one of the greatest and most urgent global risks to human and animal health today, which may severely impact future human development. This work is a part of a long-term project with the radical vision of developing a fundamentally new strategy against microbial infections that effectively kills pathogens without eliciting resistance. To this end, our research group is developing an innovative approach for fighting pathogenic bacteria using a sustainable, bio-based nanodevice with a novel mode of action, based on cellulose nanocrystals (CNC) as one of its key components. In this work, we have prepared mono- and bi-functional CNCs having a photosensitizer (Rose Bengal) and one of two types of molecules (D-mannose or a lipophilic quaternary ammonium salt) designed to promote the binding of bacteria to the cellulose surface to enhance the antibacterial photodynamic activity of the photosensitizer., [ES] El aumento de la resistencia a los antimicrobianos es uno de los mayores y más urgentes riesgos globales para la salud humana y animal en la actualidad, que puede afectar gravemente al desarrollo humano futuro. Este trabajo es parte de un proyecto a largo plazo con la visión radical de desarrollar una estrategia fundamentalmente nueva contra las infecciones microbianas que mate de manera efectiva a los patógenos sin generar resistencia. Con este objetivo, nuestro grupo de investigación está desarrollando una estrategia innovadora para combatir las bacterias patógenas utilizando un nanodispositivo sostenible y de base biológica con un modo de acción novedoso, que se basa en los nanocristales de celulosa (CNC) como uno de sus componentes clave. En este trabajo, hemos preparado CNCs mono- y bifuncionalizados con un fotosensibilizador (Rosa de Bengala) y uno de dos tipos de moléculas (D-manosa o una sal de amonio cuaternario lipofílica) diseñadas para promover la unión de la bacteria a la superficie de la celulosa para potenciar la actividad fotodinámica antibacteriana del fotosensibilizador.

Current methods for the preparation of functional small-molecule fluorophores generally require labor-intensive, multi-step synthetic routes for all the major chromophoric groups. In spite of recent significant contributions from numerous laboratories, the paucity of rapid, straightforward and wide-scope synthetic strategies in this field is limiting the development of advanced probes for bioimaging, sensing and therapeutic applications. We describe herein a general and robust methodology for the one-step fluorescent labeling of a wide variety of molecules having C-, N-, P-, O-, S-, or halide-nucleophilic centers, using stable and readily available acetoxymethyl-BODIPYs as reagents in the presence of an acid catalyst. This modular methodology allows a very facile preparation of mono- and di-functional probes incorporating a broad assortment of biomolecules, enzyme cofactors, natural products, and other chromophores, as well as chemical functionalities for a wide range of applications including bioorthogonal conjugation, polymerization, and supramolecular chemistry, among others. The photophysical properties and preliminary applications of the new probes in live-cell imaging were also studied. The described strategy enables the high-throughput engineering of novel BODIPY dyes with diverse functionalities for basic and applied science with potential for innovative technological applications.

Current methods for the preparation of functional small-molecule fluorophores generally require labor-intensive, multi-step synthetic routes for all the major chromophore groups. In spite of recent significant contributions from numerous laboratories, the paucity of rapid, straightforward and wide-scope synthetic strategies in this field is limiting the development of advanced probes for bioimaging, sensing and therapeutic applications. We describe herein a general and robust methodology for the one-step fluorescent labeling of a wide variety of molecules having C-, N-, P-, O-, S-, or halide-nucleophilic centers, using stable and readily available acetoxymethyl-BODIPYs as reagents in the presence of an acid catalyst. This modular methodology allows a very facile preparation of mono- and di-functional probes incorporating a broad assortment of biomolecules, enzyme cofactors, natural products, and other chromophores, as well as chemical functionalities for a wide range of applications including bioorthogonal conjugation, polymerization, and supramolecular chemistry, among others. The photophysical properties and preliminary applications of the new probes in live-cell imaging were also studied. The described strategy could enable the high-throughput engineering of novel BODIPY dyes with diverse functionalities for basic and applied research with potential for innovative technological applications.

Compuestos y sus usos como sondas fluorescentes. Colorantes orgánicos basados en derivados de F-BODIPY y carnitina, su procedimiento de obtención y su aplicación para el etiquetado o marcaje fluorescente especifico de mitocondrias en células vivas.

[EN] Organic dyes based on F -BODIPY derivatives and camitine, method for producing same and their use for the specific fluorescent labelling or tagging of mitochondria in living cells., [ES] Colorantes orgánicos basados en derivados de F-BODIPY y carnitina, su procedimiento de obtención y su aplicación para el etiquetado o mareaje fluorescente específico de mitocondrias en células vivas.

The first fluorescent probes that are actively channeled into the mitochondrial matrix by a specific mitochondrial membrane transporter in living cells have been developed. The new functional probes (BCT) have a minimalist structural design based on the highly efficient and photostable BODIPY chromophore and carnitine as a biotargeting element. Both units are orthogonally bonded through the common boron atom, thus avoiding the use of complex polyatomic connectors. In contrast to known mitochondria-specific dyes, BCTs selectively label these organelles regardless of their transmembrane potential and in an enantioselective way. The obtained experimental evidence supports carnitine-acylcarnitine translocase (CACT) as the key transporter protein for BCTs, which behave therefore as acylcarnitine biomimetics. This simple structural design can be readily extended to other structurally diverse starting FBODIPYs to obtain BCTs with varied emission wavelengths along the visible and NIR spectral regions and with multifunctional capabilities. BCTs are the first fluorescent derivatives of carnitine to be used in cell microscopy and stand as promising research tools to explore the role of the carnitine shuttle system in cancer and metabolic diseases. Extension of this approach to other small-molecule mitochondrial transporters is envisaged.

Cellulose nanocrystals (CNCs) display a set of unique properties such as surface charge, nanometric size, and giant dipole moment that have not been considered so far to tame the luminescent properties of organic dyes upon soft and hard photoexcitation. Herein, the first comprehensive analysis on the photonic behavior of laser dyes adsorbed onto CNCs through physically dynamic driving forces is reported. By systematically varying the solvent, the dye molecular structure, and dye/CNC ratios, it is shown that the dye absorption and emission signatures are drastically modified when CNCs are incorporated into the solutions. The photonic characterization, together with molecular dynamic simulations, allows to unravel the labeling interaction processes and unambiguously assigns both the physicochemical properties of the surrounding media and the giant permanent dipole moment of CNCs as the most important factors taming the photophysical and lasing properties of dyes adsorbed on it. Accordingly, a CNC¿induced Stark effect accounts for the absorption and emission spectral changes recorded upon CNC incorporation. Herein, the widely expanding applications of CNCs toward the design of advanced photoactive nanomaterials based on these renewable building blocks are opened up.

A new fluorescent probe has been developed by slightly modifying the structure of a BODIPY-carnitine probe previously described by our group. [1] The introduction of Br atoms at the available positions of the chromophore (2 and 6) had the desired effect of causing the probe to generate significant amounts of singlet oxygen when submitted to green light irradiation, while retaining some of the initial probe¿s fluorescence. In vitro fluorescence microscopy studies have shown staining of mitochondria as well as other parts of the cell at concentrations as low as 5 µM. Furthermore, apoptosis of approximately 80% of the irradiated cells previously treated with moderate concentrations of the new probe could be confirmed by flow cytometry only two hours after irradiation, whereas no significant degree of apoptosis was observed in the control experiment without irradiation. These findings support our hypothesis, suggesting that the new probe does not provoke cell death while kept in darkness and that the apoptosis observed in irradiated cells can be attributed to phototoxicity caused by the release of vast amounts of reactive oxygen species during irradiation. Both its fluorescence and its ability to selectively destroy cells by irradiating the desired region with green light make this new probe a promising candidate for applications in bioimaging and photodynamic therapy.

We have developed a general post-synthetic strategy for the direct one-step incorporation of hydroxy acids and diacids at the boron atom of F-BODIPY dyes. The tethering reaction uses easy-to-handle reagents, has broad functional group compatibility and proceeds under mild conditions without requiring any pre-functionalization of the starting F-BODIPY to afford the corresponding O-BODIPY derivative in good yield. The orthogonal geometrical arrangement of the hydroxyl acid or diacid moiety with respect to the mean plane of the BODIPY chromophore hampers intermolecular aggregation processes that quench fluorescence, while the covalent attachment to the boron atom has a minimal effect on the absorption properties of the chromophore. We will describe the application of this strategy to the one-step tagging of BODIPYs for the custom modification of their solubility properties and to the preparation of fluorescent probes for mitochondria targeting via a functional membrane transporter. In the first application, fully water-soluble and hydrolytically stable BODIPYs were prepared in a single step by incorporating a hydroxy acid solubility module having either neutral (tetra- and octa-ethylene glycol chains) or switterionic (sulfobetaine) hydrophilic tags. The resulting BODIPYs are valuable live cell imaging probes. In the second application, we have designed the first fluorescent probes that are actively channelled into the mitochondrial matrix by the carnitine system in living cells. Our functional probes have a minimalist structural design based on the BODIPY chromophore and carnitine as a biotargeting element. The new fluorescent probes selectively label mitochondria regardless of their membrane potential and in an enantioselective way.

We describe a general and practical strategy for the direct one-step incorporation of a tunable solubility module at the boron atom of F-BODIPY dyes. The tethering reaction uses easy-to-handle reagents, has broad functional group compatibility and proceeds under mild conditions without requiring any pre-functionalization of the starting F-BODIPY to yield the solubility-tagged O-BODIPY derivative in 51–86% yield. The module can be introduced at the end of the synthetic route without perturbing the fluorophore scaffold, thus minimizing difficulties in product isolation. Its orthogonal geometrical arrangement with respect to the plane of the BODIPY chromophore hampers intermolecular aggregation processes that quench fluorescence, while the covalent attachment to the boron atom has a minimal effect on the absorption properties. Fully water-soluble and hydrolytically stable BODIPYs were prepared by incorporating either neutral (tetra- and octaethylene glycol chains) or zwitterionic (sulfobetaine) hydrophilic tags in the module. The new dyes are valuable live cell imaging probes that ameliorate the undesired partitioning into lipophilic compartments that is often observed for standard BODIPYs. This strategy can be readily adapted to the general and highly practical post-synthetic introduction of new functionalities into F-BODIPY dyes, including phase-tagging, by appropriately choosing the nature of the chemical tags attached to the module.

Stereochemical and steric control of the relative spatial arrangement of the chromophoric units in multichromophoric systems offers an interesting strategy for raising unusual and appealing light-induced emission states. To explore and exploit this strategy, a series of conformationally restricted boron–dipyrromethene (BODIPY) dimers were designed by using tartaric acid as a symmetrical connector between the boron atoms of the dyes. The variety of stereoisomeric forms available for this bis(hydroxy acid) allows the relative spatial orientation of the chromophoric units in the dimer to be modified, which thus opens the door to modulation of the photophysical and chiroptical properties of the new bichromophoric systems. Chromophore alkylation introduces an additional level of control through distance-dependent steric interactions between the BODIPY units in the dimer, which also modulates their relative spatial disposition and properties.

Tunneling nanotubes (TnTs) are thin channels that temporally connect nearby cells allowing the cell-to-cell trafficking of biomolecules and organelles. The presence or absence of TnTs in human neoplasms and the mechanisms of TnT assembly remains largely unexplored. In this study, we have identified TnTs in tumor cells derived from squamous cell carcinomas (SCC) cultured under bi-dimensional and tri-dimensional conditions and also in human SCC tissues. Our study demonstrates that TnTs are not specific of epithelial or mesenchymal phenotypes and allow the trafficking of endosomal/ lysosomal vesicles, mitochondria, and autophagosomes between both types of cells. We have identified focal adhesion kinase (FAK) as a key molecule required for TnT assembly via a mechanism involving the MMP-2 metalloprotease. We have also found that the FAK inhibitor PF-562271, which is currently in clinical development for cancer treatment, impairs TnT formation. Finally, FAK-deficient cells transfer lysosomes/autophagosomes to FAK-proficient cells via TnTs which may represent a novel mechanism to adapt to the stress elicited by impaired FAK signaling. Collectively, our results strongly suggest a link between FAK, MMP-2, and TnT, and unveil new vulnerabilities that can be exploited to efficiently eradicate cancer cells.

We describe the design and synthesis of the first organic dyes enabling spontaneous formation of stable J-aggregates in common organic solvents without additives. The new dyes are O-BODIPYs with a B-spiranic 4,4-diacyloxyl substitution pattern. Key to the effectiveness of the J-aggregation process is the high conformational rigidity of the Bspiranic molecular design as well as the orthogonal disposition of the B-diacyloxyl substituent and the meso-aryl group with respect to the mean plane of the borodiazaindacene. Atomistic simulations, both in vacuum and in a solvent cage, support the dynamics of the J-aggregation process as well as its dependence on the alkylation pattern of the BODIPY chromophore. A detailed analysis of the photophysical and laser properties of the new dyes provides convincing evidence for the unambiguous assignment of these J-aggregates and their dependence on the environmental conditions.

The preparation of the first structurally well-defined Janus nanocube showing two chemically distinct opposed faces is described. The synthetic approach is based on a highly efficient and symmetrycontrolled CuAAC functionalization of an octa-azido cubic silsesquioxane with a conformationally constrained tetra-alkyne with an appropriate spatial orientation of the triple bonds.

Nonafluorobutanesulfonyl azide is a highly efficient reagent for the copper-catalyzed coupling of terminal alkynes to give symmetrical and unsymmetrical 1,3-diynes in good to excellent yields and with good functional group compatibility. The reaction is extremely fast (<10 min), even at low temperature (-78 °C), and requires substoichiometric amounts of a simple copper(I) or copper(II) salt (2-5 mol %) and an organic base (0.6 mol %). A possible mechanistic pathway is briefly discussed on the basis of model DFT theoretical calculations. The quantitative assessment of the safety of use and shelf stability of nonafluorobutanesulfonyl azide has confirmed that this reagent is a superior and safe alternative to other electrophilic azide reagents in use today.

The direct and simple fluorination of representative organic laser dyes with emission covering the entire visible spectrum, from blue to red, including Coumarin 460, Pyrromethene 546, Rhodamine 6G and Perylene Red, enhances laser efficiencies by a factor up to 1.8 with respect to the corresponding non-fluorinated parent dyes. More importantly, fluorination also significantly enhances the photostability of the dyes, even under drastic laser pumping conditions.

Nuevos derivados clorados de BODIPY. La presente invención se refiere a unos nuevos colorantes con estructura de BODIPY caracterizados porque contienen al menos un átomo de cloro unido a los carbonos del sistema boradiazaindaceno, a su uso como colorantes láser y como marcadores fluorescentes y a un procedimiento de obtención de algunos de estos compuestos.

[EN] The invention relates to the use of novel dyes with a BODIPY structure, characterised in that they contain at least one chlorine atom bound to the carbons of the boradiazaindacene system, to the use thereof as laser dyes and fluorescent markers, and to a method for obtaining some of these compounds., [ES] La presente invención se refiere a unos nuevos colorantes con estructura de BODIPY caracterizado porque contienen al menos un átomo de cloro unido a los carbonos del sistema boradiazaindeceno, a su uso como colorantes láser y como marcadores fluorescentes y a un procedimiento de obtención de algunos de estos compuestos.

A general procedure for the assembly of hetero-bifunctional cubic silsesquioxanes with diverse functionality and a perfectly controlled distribution of functional groups on the inorganic framework has been developed. The method is based on a two-step sequence of mono- and hepta-functionalization through the ligand-accelerated copper(I)-catalyzed azide-alkyne cycloaddition of a readily available octaazido cubic silsesquioxane. The stoichiometry of the reactants and the law of binomial distribution essentially determine the selectivity of the key monofunctionalization reaction when a copper catalyst with strong donor ligands is used. The methodology has been applied to the preparation of a set of bifunctional nano-building-blocks with orthogonal reactivity for the controlled assembly of precisely defined hybrid nanomaterials and a fluorescent multivalent probe for application in targeted cell-imaging. The inorganic cage provides an improved photostability to the covalently attached dye as well as a convenient framework for the 3D multivalent display of the pendant epitopes. Thus, fluorescent bioprobes based on well-defined cubic silsesquioxanes offer interesting advantages over more conventional fully organic analogues and ill-defined hybrid nanoparticles and promise to become powerful tools for the study of cell biology and for biomedical applications. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Nonafluorobutanesulfonyl azide is an advantageous alternative to triflyl azide for the efficient synthesis of sulfonyl azides from sulfonamides in terms of its higher reactivity, lower cost, and non-hazardous nature. The reagent has proven its utility in a novel synthesis of N,N′-disulfonylamidines from available sulfonamides by a copper-catalyzed two-step, one-pot sequential process with added terminal alkynes. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A new procedure has been developed for the direct intermolecular C–H amination of simple hydrocarbons using shelf-stable nonafluorobutanesulfonyl azide in the presence of a dirhodium(II) tetracarboxylate catalyst undermild reaction conditions. Somemechanistic details are briefly discussed on the basis of control experiments.

A series of mono- to hexachlorinated BODIPY dyes have been prepared in good to excellent yields through the use of N-chlorosuccinimide as an inexpensive halogenating reagent. This library of chlorinated dyes allowed analysis in detail, from the experimental and theoretical points of view, of the dependency of the photophysical and optical properties of the dyes on the number and positions of the chlorine substituents on their BODIPY cores. Quantum mechanical calculations predict the regioselectivity of the halogenation reaction and explain why some positions are less prone to chlorination. The new chlorinated BODIPYs exhibit enhanced laser action with respect to their non-halogenated analogues, both in liquid solution and in the solid phase. In addition, chlorination is a facile and essentially costless protocol for overcoming important shortcomings exhibited by commercially available BODIPYs, which should favor their practical applications in optical and sensing fields. © 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.