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Therapeutic application of many drugs is often hampered by poor or denied access to intracellular targets. A case in point is miltefosine (MT), an orally active antiparasitic drug, which becomes ineffective when parasites develop dysfunctional uptake systems. We report here the synthesis of a fluorescent BODIPY-embedding MT analogue with appropriate thiol functionalization allowing linkage to the cell-penetrating Tat(48-60) peptide through disulfide or thioether linkages. The resulting constructs are efficiently internalized into the otherwise MT-invulnerable R40 Leishmania strain, resulting in fast parasite killing, and hence successful avoidance of the resistance. In the disulfide-linked conjugate, an additional fluoro tag on the Tat moiety allows to monitor its reductive cleavage within the cytoplasm. Terminally differentiated cells such as peritoneal macrophages, impervious to MT unless infected by Leishmania, can uptake the drug in its Tat-conjugated form. The results afford proof-of-principle for using CPP vectors to avert drug resistance in parasites, and/or for tackling leishmaniasis by modulating macrophage uptake. © 2014 Springer-Verlag Wien.

The ether-phospholipid edelfosine, a prototype antitumor lipid (ATL), kills yeast cells and selectively kills several cancer cell types. To gain insight into its mechanism of action, we performed chemogenomic screens in the Saccharomyces cerevisiae gene-deletion strain collection, identifying edelfosine-resistant mutants. LEM3, AGP2, and DOC1 genes were required for drug uptake. Edelfosine displaced the essential proton pump Pma1p from rafts, inducing its internalization into the vacuole. Additional ATLs, including miltefosine and perifosine, also displaced Pma1p from rafts to the vacuole, suggesting that this process is a major hallmark of ATL cytotoxicity in yeast. Radioactive and synthetic fluorescent edelfosine analogues accumulated in yeast plasma membrane rafts and subsequently the endoplasmic reticulum. Although both edelfosine and Pma1p were initially located at membrane rafts, internalization of the drug toward endoplasmic reticulum and Pma1p to the vacuole followed different routes. Drug internalization was not dependent on endocytosis and was not critical for yeast cytotoxicity. However, mutants affecting endocytosis, vesicle sorting, or trafficking to the vacuole, including the retromer and ESCRT complexes, prevented Pma1p internalization and were edelfosineresistant. Our data suggest that edelfosine-induced cytotoxicity involves raft reorganization and retromer- and ESCRT-mediated vesicular transport and degradation of essential raft proteins leading to cell death. Cytotoxicity of ATLs is mainly dependent on the changes they induce in plasma membrane raft-located proteins that lead to their internalization and subsequent degradation. Edelfosine toxicity can be circumvented by inactivating genes that then result in the recycling of internalized cell-surface proteins back to the plasma membrane. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

Lipid rafts and mitochondria are promising targets in cancer therapy. The synthetic antitumor alkyl-lysophospholipid analog edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) has been reported to target lipid rafts. Here, we have found that edelfosine induced loss of mitochondrial membrane potential and apoptosis in human cervical carcinoma HeLa cells, both responses being abrogated by Bcl-xL overexpression. We synthesized a number of new fluorescent edelfosine analogs, which preserved the proapoptotic activity of the parent drug, and colocalized with mitochondria in HeLa cells. Edelfosine induced swelling in isolated mitochondria, indicating an increase in mitochondrial membrane permeability. This mitochondrial swelling was independent of reactive oxygen species generation. A structurally related inactive analog was unable to promote mitochondrial swelling, highlighting the importance of edelfosine molecular structure in its effect on mitochondria. Raft disruption inhibited mitochondrial localization of the drug in cells and edelfosine-induced swelling in isolated mitochondria. Edelfosine promoted a redistribution of lipid rafts from the plasma membrane to mitochondria, suggesting a raft-mediated link between plasma membrane and mitochondria. Our data suggest that direct interaction of edelfosine with mitochondria eventually leads to mitochondrial dysfunction and apoptosis. These observations unveil a new framework in cancer chemotherapy that involves a link between lipid rafts and mitochondria in the mechanism of action of an antitumor drug, thus opening new avenues for cancer treatment.

New hybrid dyes, analogs of the BODIPY laser dye PM567, with the chromophore group covalently bound to a POSS T8 group, have been prepared and their photophysical properties in ethyl acetate solution and in poly(methyl methacrylate) (PMMA) thin film solution have been studied. In PMMA solution and under comparative conditions, the dyes show higher thermostability than the parent dye PM567, the related compound POSS(iBu)8, and the corresponding unbound model dyes, because the first mass loss takes place at higher temperatures. In addition, their photostabilities in the same medium under irradiation with 365 nm light are much higher than those of the dye PM567 or an equimolar mixture of PM567 plus POSS(iBu)8 under the same conditions, indicating that the POSS moiety photostabilizes the bound BODIPY group. The different photostabilities could be a consequence of different reactivities of the hybrid dyes with self-generated singlet oxygen, because they show lower reaction rate constant values than PM567, although the observed tendency of these values does not correlate with that of the photodegradation quantum yield values. The new hybrid materials can be employed in applications where highly photostable and highly emissive dyes are required.

En esta Memoria se estudia el comportamiento láser de colorantes con emisión en el rojo, utilizando tanto colorantes comerciales como nuevos colorantes sintetizados en nuestros laboratorios. El objetivo último es lograr láseres de colorante en estado sólido con emisión eficiente, estable y sintonizable más allá de los 600 nm, así como entender las relaciones composición-estructura-propiedades-aplicaciones de estos sistemas que permitan rediseñar y simplificar las rutas de síntesis de materiales con propiedades optimizadas para aplicaciones específicas. Los resultados alcanzados son consecuencia de un esfuerzo multidisciplinar en el que han intervenido grupos de investigación de los Institutos de Química Orgánica General, Ciencia y Tecnología de Polímeros y Química Física “Rocasolano”, todos del CSIC, y de la Universidad del País Vasco.

En esta Memoria se estudia el comportamiento láser de colorantes con emisión en el rojo, utilizando tanto colorantes comerciales como nuevos colorantes sintetizados en nuestros laboratorios. El objetivo último es lograr láseres de colorante en estado sólido con emisión eficiente, estable y sintonizable más allá de los 600 nm, así como entender las relaciones composición-estructura-propiedades-aplicaciones de estos sistemas que permitan rediseñar y simplificar las rutas de síntesis de materiales con propiedades optimizadas para aplicaciones específicas. Los resultados alcanzados son consecuencia de un esfuerzo multidisciplinar en el que han intervenido grupos de investigación de los Institutos de Química Orgánica General, Ciencia y Tecnología de Polímeros y Química Física “Rocasolano”, todos del CSIC, y de la Universidad del País Vasco.

Para llevar a cabo la incorporación de grupos fluorescentes a compuestos lipídicos con actividad bio-farmacológica se eligieron tres tipos de estructuras relacionadas químicamente: alquilfosfocolinas, éter-lípidos y ácidos grasos de cadena lineal. La elección de estas familias concretas de lípidos estaba condicionada por la existencia en nuestro entorno de equipos de investigadores trabajando activamente en problemas mecanísticos relacionados con su bioactividad y que, además, disponían de los métodos de fluorescencia apropiados para la explotación de los posibles compuestos fluorescentes obtenidos a lo largo de este trabajo.

Di- y trifenilen-borodipirrometenos y su utilización como colorantes láser en disolución y en estado sólido. Colorantes láser di- y trifenilen-borodipirrometenos, basados en la unión covalente de dos cromóforos de distinta naturaleza, cuales son, por un lado el anillo tricíclico de un complejo dipirrometeno.BF2, al cual se le ha unido en su posición 8 un grupo de dos o tres anillos fenilénicos, como medio activo para la generación de radiación láser, que una vez mecanizados y pulidos, con el fin de obtener la forma geométrica deseada, preferentemente cilíndrica de 1 cm de altura y diámetros variables, con o sin una cara lateral plana de dimensión variable, son adaptables a cualquier cavidad láser para su utilización en optoelectrónica.

Para llevar a cabo la incorporación de grupos fluorescentes a compuestos lipídicos con actividad bio-farmacológica se eligieron tres tipos de estructuras relacionadas químicamente: alquilfosfocolinas, éter-lípidos y ácidos grasos de cadena lineal. La elección de estas familias concretas de lípidos estaba condicionada por la existencia en nuestro entorno de equipos de investigadores trabajando activamente en problemas mecanísticos relacionados con su bioactividad y que, además, disponían de los métodos de fluorescencia apropiados para la explotación de los posibles compuestos fluorescentes obtenidos a lo largo de este trabajo.

La emisión de fluorescencia en fase condensada es un proceso de origen electrónico, lo que ha retrasado históricamente su comprensión, a pesar de que las primeras observaciones del fenómeno se realizaron en Sevilla ya en el siglo XVI.1 Hoy en día se sabe que las molécula excitadas electrónicamente por absorción fotónica permanecen nanosegundos en dicho estado excitado, un tiempo que es ~ 106 veces mayor que el requerido para la excitación. En la mayoría de los casos, la molécula pierde este exceso de energía electrónica a través de procesos no radiantes. Sin embargo, para un número reducido de compuestos la probabilidad de la emisión es mayor que la de los procesos no radiantes y, por tanto, se produce emisión de fluorescencia. Como era de esperar, se ha dedicado un gran esfuerzo investigador a lo largo de casi un siglo para determinar los factores electrónicos y estructurales que optimizan las propiedades emisivas de una molécula; sin embargo, todavía hoy el diseño de nuevos grupos fluorescentes (fluoróforos) se lleva a cabo de forma semi-empírica. En contraste, el desarrollo de las técnicas instrumentales para la excitación y detección de fluorescencia ha progresado considerablemente.

New tailormade BODIPY dyes have been synthesized by a simple protocol to reach wavelength finely tunable laser action from 540 to 625 nm while maintaining highly efficient and photostable laser emission. The new dyes are analogues of the commercial dye PM597 with the eight position free (PTH8) or substituted by the groups acetoxymethyl (PTAlk) or p-acetoxymethylphenyl (PTAr). The photophysical properties strongly depend on the geometrical distortion from planarity of the indacene core generated by the presence of the bulky 2,6-di-tert-butyl groups and the eight substituent. In both liquid and polymeric solid solutions, lasing efficiencies of up to 63 and 48%, respectively, were observed under transversal pumping at 532 nm with high photostabilities. In the case of PTAlk incorporated into silicon-containing solid organic matrices, the laser emission remained at 92% of its initial intensity value after 100 000 pumping pulses in the same position of the sample at 30 Hz repetition rate. The laser action of the new dyes enhances that of the parent dye PM597 and outperforms the lasing behavior of dyes considered to be benchmarks over the green-yellow to red spectral region.

We report the synthesis, photophysical properties and evaluation of laserdye of a newBODIPYdye with a 3-styryl substituent, PMS, and with the rest of the substituents as in the commercial dyePM567. PMS shows an emission band at 584 nm in methanol, i.e. displaced ca. 50 nm to longer wavelengths with regard to the green-emission band of PM567, as well as a high-fluorescence quantum yield (0.82) and also a high-molar absorption coefficient (105 M−1 cm−1) in the same solvent. The laser action of the newdye has been analyzed under transversal pumping at 532 nm, 5.5 mJ pulse−1 and up to 10 Hz repetition rate, in both liquid phase and incorporated into solid polymeric matrices of methyl methacrylate copolymerized with crosslinking or fluorinated monomers. Lasing emission at 602–610 nm, with maximum efficiencies of 18%, were reached in these media. In solid-fluorinated matrices, good lasing photostabilities were established, with 30% of the initial laser output remaining after 100,000 pump pulses at 10 Hz

Two general synthetic methods are described, by which the highly fluorescent and photostable BODIPY group can be inserted in and aligned with the alkyl backbone of linear lipids. These methods have been used to prepare strongly emitting analogues of the leishmanicidal drug miltefosine, in which the antiparasite activity in vitro of the original drug is preserved

Di- and triphenylene boron dipyrromethene laser colorants, based on the covalent binding of two chromophores of different types, which comprise the tricyclical ring of a dipyrromethene.BF 2 complex, to which a group of two or three phenylenic rings have bonded, as the active medium for generation of laser radiation, which after machining and polishing, with a view to obtaining the required geometric shape, preferably cylindrical of 1 cm in height and variable diameters, with or without a plane side face of variable sizes, are adaptable to any laser cavity for use thereof in optoelectronics., Colorantes láser di- y trifenilen-borodipirrometenos, basados en la unión covalente de dos cromóforos de distinta naturaleza, cuales son, por un lado el anillo tricílico de un complejo dipirrometeno, BF 2 al cual se le ha unido en su posición 8 un grupo de dos o tres anillos fenilénicos, como medio activo para la generación de radiación láser, que una vez mecanizados y pulidos, con el fin de obtener la forma geométrica deseada, preferentemente cilíndrica de 1 cm de altura y diámetros variables, con o sin una cara lateral plana de dimensión variable, son adaptables a cualquier cavidad láser para su utilización en optoelectrónica.

Bichromatic laser emission from dipyrromethene-based solid-state dye lasers is reported. The dependence of this dual emission on different factors and its origin and causes are discussed in the light of different models proposed in the literature. Our experimental results indicate that the long-wavelength emission can be explained in terms of reabsorption/reemission effects and inhomogeneous broadening of the S0-S1 transition. The short-wavelength emission corresponds to the usual S0-S1 transition and dominates at low dye concentration.

The leishmanicidal mechanism of miltefosine (hexadecylphosphocholine, MT) is not clearly understood. Valuable insights into its mode of action could be obtained by fluorescence techniques, given suitably emitting analogues. In this regard, the synthesis and biological characterization of two fully competent MT fluorescent analogues is reported here: all-(E)-13-phenyltrideca-6,8,10,12- tetraenylphosphocholine (PTE-MT) and all-(E)-13-phenyltrideca-8,10,12-trien-6- ynylphosphocholine (PTRI-MT). Both compounds show large absorption coefficients and a modest, but usable, fluorescence yield. Their activities were very similar to that of MT and were recognized by the MT uptake system of Leishmania. Their localization in living L. donovani promastigotes by confocal microscopy show a homogeneous intracellular distribution of the fluorescence. The concentration of PTRI-MT within the parasites (ca. 1.7 mM) showed a 100-fold enrichment relative to its external concentration. These results are consistent with a multiple target leishmanicidal mechanism for MT and validate the application of these analogues for pharmacokinetic and diagnostic studies concerning the chemotherapy of leishmaniasis. © 2007 American Chemical Society.

In the search for more efficient and photostable solid-state dye lasers, newly synthesized analogs of the borondipyrromethene (BODIPY) dye PM567, bearing the polymerizable methacryloyloxypropyl group at position 2 (PMoMA) or at positions 2 and 6 (PDiMA), have been studied in the form of solid copolymers with methyl methacrylate (MMA). The parent dye PM567, as well as the model analogs bearing the acetoxypropyl group in the same positions, PMoAc and PDiAc, respectively, have been also studied both in liquid solvents and in solid poly(MMA) (PMMA) solution. Although in liquid solution PMoAc and PDiAc have the same photophysical properties as PM567, PDiAc exhibited a photostability up to 10 times higher than that of PM567 in ethanol under 310 nm-irradiation. The possible stabilization factors of PDiAc have been analyzed and discussed on the basis of the redox potentials, the ability for singlet molecular oxygen [O2(1Dg)] generation, the reactivity with O2(1Dg), and quantum mechanical calculations. Both PMoAc and PDiAc, pumped transversally at 532 nm, lased in liquid solution with a high (up to 58 %), near solvent-independent efficiency. This enhanced photostabilization has been also observed in solid polymeric and copolymeric media. While the solid solution of the model dye PDiAc in PMMA showed a lasing efficiency of 33 %, with a decrease in the laser output of ca. 50% after 60 000 pump pulses (10 Hz repetition rate) in the same position of the sample, the solid copolymer with the double bonded chromophore, COP(PDiMA-MMA), showed lasing efficiencies of up to 37 %, and no sign of degradation in the laser output after 100 000 similar pump pulses. Even under the more demanding repetition rate of 30 Hz, the laser emission from this material remained at 67% of its initial laser output after 400 000 pump pulses, which is the highest laser photostability achieved to date for solid-state lasers based on organic polymeric materials doped with laser dyes. This result indicates th

The asymmetrically substituted BODIPY dyes 9a and 9b have been synthesized through a key redox step involving the r-nitroso derivative of the starting pyrrol. Both dyes emit fluorescence with quantum yields of ca. 0.7, but only 8b behaves as a good laser dye, with an efficiency of 48% in ethanol solution.

Lipophilic fluorescent groups with a chain-like linear conformation emitting in the visible range and with high photostability are presently unavailable. These structures would be of great interest as labels for long-chain fatty acids and phospholipids lacking intrinsic fluorescent groups. With this aim in mind, we report the synthesis and the spectroscopic characterization of a series of emitting amphiphilic lipids that may approach that ideal fluorescent tag. Each lipid was constructed by attaching a linear, all-(E) conjugated pentaene, tetraenyne, -phenyltetraene, or -phenyltrienyne chromophore to a hydrophilic head-group through a polymethylene chain spacer. The key steps of the synthesis were the Pd0-mediated cross-coupling reaction between bromopolyenes and terminal acetylenic compounds, yielding tetraenynes or -phenyltrienynes, and the subsequent triple-bond partial reduction, producing the corresponding pentaenes or -phenyltetraenes in good overall yields. This method represents a further successful example of the so-called acetylenic approach to the indirect high-yield synthesis of polyene systems. In the case of -phenyltrienynes, a higher proportion of the all-(E) isomer was obtained using an alternative method based on the reaction of an -phenyldienylphosphonate with an -acetylenic aldehyde. Some of the resulting compounds exhibit spectral and photochemical properties that warrant their use as emitting lipophilic tags. Thus, the -phenyltetraene and -phenyltrienyne members of the series show intense absorption bands in the 320-370 nm range with fluorescence emission centered at 475 nm and quantum yields up to 0.25. These parameters are appropriate for the applications noted above.