Polyene sphingolipids with latent fluorescence: new tools to study the biophysical properties of cellular membranes

One of the ultimate goals in biomedicine is to understand the relationship between structure, function, and dynamics of biomolecules in living cells. The biophysical tools designed to gain more insight into metabolism, trafficking and interaction of the sphingolipids (SLs), require the use of high molar concentrations of fluorescence lipid analogues, labelled with bulky chromophores, such BODIPY or NBD, resulting in altered biophysical properties of the cell membrane. Recently, lipids tagged with conjugated linear polyene moieties, which are strongly absorbing chromophores that may attain modest but useful fluorescence yields, have been reported to behave in vivo like their endogenous counterparts. Nevertheless, when these labelled lipids are part of the N-acyl or the O-acyl chains of the SLs, the fluorophore can be released by hydrolysis of the amide or the ester bonds, and the resulting sphingoid base is no longer traceable. Taking into account the above considerations, the main objective of this thesis is to synthesize novel intrinsically-fluorescent sphingosine (Sph) and ceramide (Cer) probes, labelled with five conjugated double bonds in the sphingoid base backbond, in order to analyse membrane microdomains that are temporarily enriched in certain lipids. Additionally, the fluorescence of these probes can be modulated with total spatiotemporal control by the presence of a suitable radical quencher that can be removed by the action of a specific enzyme. Initially, as a proof of concept, three probes derived from γ-aminobutyric acid (GABA), based on a conjugated pentaene system with a radical scavenger (n-DOXYL free radical group) at different positions, were synthesised. These probes are structural analogues of palmitoyl-Cer lacking the hydroxyl group at C1 of the sphingoid chain. In addition, the secondary hydroxyl group at C3 has been replaced with an ester group. Despite the overall effect is a lower polarity of the headgroup, GABA-pentaene probes were still useful tools to evaluate the biophysical properties of these dual fluorophore-quencher systems in model membranes. With the GABA-pentaene probes in hand, different biophysical studies were developed in lipid vesicles. Fluorescence studies, differential scanning calorimetry and electron paramagnetic resonance measurements exhibited the ability of these probes to detect membrane lipids in gel phase, being relevant in view of the novel evidences pointing at the existence of gel microdomains in cell membranes. In addition, these Cer analogues may be particularly useful probes for the observation of highly-ordered bilayers by confocal microscopy, since its emission was higher in gel than in fluid domains, and in liquid-ordered than in liquid-disordered areas. Finally, different synthetic protocols for the construction of conformationally constrained pentaene-Sph and -Cer analogues were designed. The combination of Horner-Wadsworth-Emmons (HWE) and Wittig olefinations gave access to triene and pentane alkyne intermediates. In turn, these alkynes were found to be versatile synthons for the preparation of SLs analogues. By means of nucleophilic alkynylations, polyenyne-Sph analogues were obtained, whereas a hydrozirconation approach allowed the synthesis of the first pentaene palmitoyl-Cer analogue. Additionally, in light of the apparent instability of the conjugated pentaene system under acidic conditions, an alternative unreported serinal derivative building block was synthesised, which enabled a selective deprotection under mild basic conditions. Overall, synthetic approaches to polyene sphingosine analogues have been achieved. Remarkably, the pentaene moiety has been introduced in the sphingoid base backbond for the first time, providing new tools to directly observe their distribution in lipid bilayers. In addition, biophysical studies revealed the suitability of polyene moieties for fluorescence detection, by means of the simplified models GABA-pentaene probes.Actualmente, uno de los retos de la biomedicina es profundizar más en el metabolismo, señalización e interacciones de los esfingolípidos (SLs), mediante empleo de técnicas biofísicas que no alteren las propiedades de las membranas celulares. El estudio y la visualización de los SLs, generalmente, va asociado al uso de concentraciones molares muy elevadas de sondas marcadas con grupos fluorescentes, cuyos cromóforos presentan estructuras rígidas, de considerable tamaño y volumen (NBD, BODIPY), generando un escenario artificial que dista de las condiciones fisiológicas. Con objeto de reducir el impacto que produce el volumen de los grupos cromóforos en la estructura y empaquetamiento de los lípidos de membrana, se han utilizado sistemas de dobles enlaces conjugados, fluorescentes, los cuales se comportan in vivo de forma similar a sus análogos naturales. Sin embargo, hasta el momento, no se ha diseñado ninguna sonda que contenga el sistema pentaénico incorporado en la estructura de la base esfingoide, el cual permitiría observar la distribución de especies tipo esfingosina (Sph), por visualización directa. Con objeto de analizar los microdominios de membrana que son temporalmente enriquecidos en ciertos lípidos, en la presente tesis doctoral se han diseñado diversos protocolos sintéticos para la obtención de sondas análogas a la Sph y a la ceramida (Cer). Las nuevas sondas obtenidas, pentaenino-Sph y palmitoil pentaeno-Cer, han sido marcadas, por primera vez, en la base esfingoide con una estructura de cinco dobles enlaces conjugados, confiriendo un sistema intrínsecamente fluorescente. Además, el diseño de los pentaeno-SLs permite la introducción en la cadena N-acilo de un agente radical atenuador interno (n-DOXYL), el cual latentizaría la fluorescencia con total control espaciotemporal. Por acción de una enzima específica, se liberaría el atenuador, recuperando la fluorescencia del sistema pentaénico, lo que permitiría la visualización directa de los SLs a concentraciones más cercanas a las fisiológicas. Asimismo, como prueba de concepto, se han sintetizado tres sondas derivadas del ácido γ- aminobutírico (GABA), como modelos simplificados de la palmitoil-Cer natural, los cuales contienen un sistema pentaénico con un atenuador radicalario en la misma molécula. Los ensayos biofísicos llevados a cabo en membranas modelo con estas sondas alternativas, nos han proporcionado información interesante de las propiedades biofísicas que presenta el sistema dual pentaeno-atenuador radicalario en un entorno lipídico