Formación de dímeros pirimidínicos en el estado excitado triplete: Ciclobutanos frente a fotoproductos (6,4)

[EN] UV radiation is able to induce changes in the chemical composition of DNA that have been closely related to skin cancer. Among the most relevant lesions, damage to pyrimidine bases leading to bipyrimidine units (cyclobutane dimers and (6,4) photoproducts) are found. The overall thesis objective is to clarify some of the DNA photoreaction mechanisms. First of all, the influence of C5 substitution on the photophysical properties of 2-thiopyrimidines (2-thiouracil (TU), 5-tert-butyl-2-thiouracil (BTU) and 2-thiothymine (TT)) was determined. The UV spectra displayed showed a maximum around 275 nm and a shoulder at ca. 290 nm. The three 2-thiopyrimidines exhibited a strong phosphorescence emission ET of ca. 307, 304 and 294 kJ/mol for TU, BTU and TT, respectively. Transient absorption spectra displayed after excitation at 308 nm gave rise to a broad band ranging from 500 nm to 700 nm. The triplet lifetime were 70 ns, 1.1 microseconds and 2.3 microseconds, for TU, BTU and TT, respectively. Theoretical calculations at the B3LYP/aug-ccpVDZ/PCM level were also done. Results were agree well with the experimental range of excited state energies and support the pp* nature of the lowest triplet states. Secondly, the influence of steric hindrance on the formation of bipyrimidine lesions was analyzed by introducing a bulky substituent at C5 position of uracil. Thus, the reactivity of 5-tert-butiluracil methyl ester (1c) was compared to its thymine analogue (2c) after BP, xanthone and acetone photosensitization. Benzophenone photoreaction led exclusively to oxetanes while acetone gave rise to uracil-5-tert-butiluracil heterodimers (1e-1 and 1e-2), an acetonyl derivative (1e-3) and to a dehydrogenation photoproduct (1e-4). In the case of xanthone only one oxetane (1f) was observed. However, parallel irradiations performed with 2c, revealed the formation of cyclobutane dimers in all cases. Then, the photochemistry of thymine from its upper np* triplet excited states, through the Norrish-Yang photocyclation was explored. This required design-ing a dyad that consisted of a 5-tert-butiluracil moiety covalently linked by an aliphatic amide to BP chromophore. Benzophenone was chosen as a photo-sensitizer. The multiphotonic excitation of the dyad was performed using a laser beam at high power (40 mJ/pulse) and then the reaction mixture was analyzed by UPLC-MS/MS. The results showed that the Norrish- Yang photo-cyclation was produced by the formation of expected pyrimidone compound. The chemical yield was 0.003%. The last chapter of this thesis addresses the possibility that the photoproduct (6,4) once formed in DNA, could act as an endogenous photosensitizer. This lesion is capable of absorbing light in the UVB-UVA region because of the presence in its structure of the 5-methyl-2-pyrimidone moiety (Pyo). It was irradiated in the presence of DNA to analyze the photoinduced damage in the biomacromolecule. The experiments established that Pyo acts as a photosensi-tizer in DNA. Indeed, photophysical studies conducted with Pyo confirmed that it can participate in energy transfer and oxidative processes Since the photochemical properties of the whole lesion may differ from the isolated chromophore Pyo, the 6,4 PP potential to act as a photosensitizer was also considered. Irradiation of 6,4 PP in the presence of DNA revealed that in fact, it can act as a photosensitizer too, although their photophysical properties are not entirely coincident. Thus, this chapter has served to establish that (6,4) photoproduct can act as a Trojan horse and extend the active fraction of UV radiation, causing the formation of pyrimidine dimers and oxidative damage, making it potentially more dangerous than estimated so far.[ES] La radiación UV está asociada a la formación de lesiones en el ADN que son el origen del cáncer piel. Las más relevantes son los DCBs y 6,4 PPs. El objetivo general de esta tesis doctoral es esclarecer algunos de los mecanismos de la fotorreacción del ADN. En primer lugar se estableció la influencia de la sustitución en C5 sobre las propiedades fotofísicas de 2-tiopirimidinas (2-tiouracilo (TU), 2-tiotimina (TT) y 5-tert-butiluracilo (BTU)). Los espectros de UV presentaron un máximo sobre 275 nm y un hombro sobre 290 nm. Las tres 2-tiopirimidinas exhibieron una intensa de fosforescencia, de cuyos espectros se determinaron las ET: 307, 304 y 294 kJ/mol para TU, BTU y TT, respectivamente. Los espectros de absorción transitoria obtenidos presentaron una banda desde 500 nm a 700 nm, que se asignó a la absorción triplete-triplete. Los tiempos de vida de triplete fueron de 70 ns, 1.1 microsegundos y 2.3 microsegundos para TU, BTU y TT, respectivamente. Además se realizaron cálculos teóricos B3LYP/aug-cc-pVDC/PCM. Los resultados obtenidos concordaron con los experimentales, respaldando la naturaleza pp* de los estados triplete excitados más bajos. En segundo lugar, se analizó la influencia del impedimento estérico en posición C5 sobre la formación de lesiones bipirimidínicas. Para ello se comparó la reactividad del 5-tert-butiluracilacetato de metilo (1c) con la de su análogo de timina (2c) tras fotosensibilizar con benzofenona, acetona y xantona. La reacción con benzofenona (BP) dio lugar exclusivamente a oxetanos. En el caso de usar acetona como sensibilizador se obtuvieron dímeros mixtos uracilo-5-tert-butiluracilo (1e-1 y 1e-2), el derivado acetonilo (1e-3) y un fotoproducto fruto de la deshidrogenación (1e-4). En el caso de la xantona solo se observó la formación de un único oxetano (1f). Sin embargo, las irradiaciones paralelas llevadas a cabo con 2c, condujeron a la formación de dímeros ciclobutánicos en todos los casos. A continuación se exploró la fotoquímica de la timina desde su estado excitado superior triplete np* a través de la fotorreacción de Norrish-Yang. La estrategia a seguir consistió en poblar dicho estado por transferencia de energía desde un estado excitado triplete superior de un fotosensibilizador adecuado. Para ello se diseñó una diada (1a) formada por el compuesto 5-tert-butiluracilo enlazado mediante una amida alifática al cromóforo benzofenona (BP). Se llevó a cabo la excitación multifónica de 1a con un haz de láser a una potencia elevada (40 mJ/pulso) y la mezcla de reacción se analizó mediante UPLC-MS/MS. Los resultados evidenciaron la formación de la pirimidona esperada tras tener lugar la reacción de Norrish-Yang con un rendimiento químico de formación de fotoproducto del 0.003 %. El último capítulo de la tesis aborda la posibilidad de que el fotoproducto (6,4) una vez formado en el ADN, actúe a su vez como fotosensibilizador endógeno, debido a que esta lesión es capaz de absorber la luz en la región UVB-UVA debido a la presencia en su estructura de 5-metil-2-pirimidona (Pyo). Para ello se sintetizó Pyo y se irradió en presencia de ADN. El análisis de los daños foto-inducidos en la biomacromolécula permitió establecer que Pyo puede ser un fotosensibilizador del ADN. Además, los estudios fotofísicos llevados a cabo con Pyo confirmaron que puede participar tanto en procesos de TE como en procesos oxidativos Este capítulo ha servido para establecer que el propio daño 6,4 PP puede actuar como caballo de Troya y extender la fracción activa de la radiación UV, provocando la formación de dímeros pirimidínicos y daños oxidativos, convirtiéndolo en un daño potencialmente más peligroso de lo estimado hasta la fecha.[CA] La radiació UV està associada amb la formació de lesions en l'ADN que son l'origen del càncer de pell. Les més rellevants DCBs i 6,4 PPs. L'objectiu general d'aquesta tesi doctoral va ser aclarir alguns dels mecanismes de la fotorreacció de l'ADN. Primer es va establir la influència de la substitució en C5 sobre les propietats fotofísiques de 2-tiopirimidines (2-tiouracil (TU), 2-tiotimina (TT) i 2-tert-butiluracil (BTU)). Els espectres UV registrats presentaren un màxim sobre 275 nm. Les tres 2-tiopirimidines exhibiren una intensa fosforescència, a partir dels espectres de la qual es van determinar les energies dels seus estats excitats triplets: 307, 304 y 294 kJ/mol per TU, BTU i TT, respectivament. Els espectres d'absorció transitòria obtinguts per excitació làser (308 nm) presentaren una banda des de 500 nm a 700 nm. Els temps de vida de triplet van ser 70 ns, 1.1 microsegons i 2.3 microsegons per a TU, BTU i TT, respectivament. A més, es van realitzar càlculs teòrics B3LYP/aug-cc-pVDC/PCM. Els resultats obtinguts van concordar amb els experimentals, recolzant la natura pp* dels estats triplet excitats més baixos. En segon lloc, es va analitzar la influència de l'impediment estèric en posició C5 sobre la formació de lesions bipirimidíniques. Així es va comparar la reactivitat del 5-tert-butiluracilacetat de metil (1c) amb el seu anàleg de timina (2c) al fotosensibilitzar amb benzofenona (BP), acetona i xantona. La reacció amb BP originà exclusivament oxetans. En el cas d'usar acetona com a fotosensibilització es van obtenir dímers mixtes uracil-5-tert-butiluracil (1e-1 i 1e-2), el derivat acetonil (1e-3) i un fotoproducte fruit d' una deshidrogenació (1e-4). En el cas de la xantona, sols es va observar a formació d'un únic oxetà (1f). No obstant, les irradiacions paral·leles dutes a terme amb 2c van conduir a la formació de dímers ciclobutànics en tots els casos. A continuació, es va explorar la fotoquímica de la timina des del seu estat excitat superior triplet np* a través de la fotorreacció de Norrish-Yang. L' estratègia a seguir va consistir a poblar el mencionat estat per transferència d'energia des d'un estat excitat triplet superior d'un fotosensibilitzador adequat. Per dur-ho a terme, es va dissenyar una diada formada pel compost 5-tert-butiluracil, susceptible de sofrir un procés de Norrish-Yang, enllaçat per una amida alifàtica al cromòfor BP. Així, es va dur a terme una excitació multifotònica de la diada amb un fluix de làser a una potencia elevada (40 mJ/pols) i posteriorment la barreja de reacció es va analitzar amb un UPLC-MS/MS. Els resultats evidenciaren la formació de la pirimidona esperada al tenir lloc la reacció de Norrish-Yang amb un rendiment químic de formació de fotoproucte del 0.003 %. En l'últim capítol de la tesi s'aborda la possibilitat de que el fotoproducte (6,4) una vegada format a l'ADN, actue a la vegada com a fotosensibilitzador endogen, ja que aquesta lesió té la capacitat d'absorbir en la regió UVB-UVA per la presència en la seua estructura de 5-metil-2-pirimidona (Pyo). Així, es va sintetitzar Pyo i es va irradiar en presència d'ADN. L'anàlisi de les lesions fotoin-duïdes en la biomacromolécula van permetre establir que Pyo pot ser un foto-sensibilitzador d' ADN. A més, els estudis fotofísics fets amb Pyo van confirmar que pot ser partícep tant en processos de transferència d'energia com en pro-cessos oxidatius. Aquest capitol ha servit per establir que el propi dany 6,4 PP pot actuar com a cavall de Troia i estendre la fracció activa de la radiació UV, provocant la for-mació de dímers pirimidínics i danys oxidatius, convertint-lo en un dany potencialment més perillós d'allò estimat fins la data.Vendrell Criado, V. (2015). Formación de dímeros pirimidínicos en el estado excitado triplete: Ciclobutanos frente a fotoproductos (6,4) [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/5861

A combined photophysical and computational study on the binding of mycophenolate mofetil and its major metabolite to transport proteins

[EN] Binding of the inmunodrepresive agent mycophenolate mofetil (MMP) and its pharmacologically active metabolite mycophenolic acid (MPA) to human serum albumin (HSA) and ¿1-acid glycoprotein (HAAG) has been investigated by an integrated approach involving selective excitation of the drug fluorophore, following their UV-A triggered fluorescence and docking studies. The formation of the protein/ligand complexes was evidenced by a dramatic enhancement of the fluorescence intensity and a hypsochromic shift of the emission band. In HSA, competitive studies using oleic acid as site I probe revealed site I as the main binding site of the ligands. Binding constants revealed that the affinity of the active metabolite by HSA is four-fold higher than its proactive form. Moreover, the affinity of MMP by HSA is three-fold higher than by HAAG. Docking studies revealed significant molecular binding differences in the binding of MMP and MPA to sub-domain IIA of HSA (site 1). For MPA, the aromatic moiety would be in close contact to Trp214 with the flexible chain pointing to the other end of the sub-domain; on the contrary, for MMP, the carboxylate group of the chain would be fixed nearby Trp214 through electrostatic interactions with residues Arg218 and Arg222.Financial support from the Spanish Ministry of Economy and Competiveness (CTQ2013-47872-C2-1-P, CTQ2016-78875-P, SAF2016-75638-R), the Xunta de Galicia (Centro singular de investigacion de Galicia accreditation 2016-2019, ED431G/09), the European Union (European Regional Development Fund-ERDF) and the Generalitat Valenciana (PROMETEO/2017/075) is gratefully acknowledgedVendrell-Criado, V.; González-Bello, C.; Miranda Alonso, MÁ.; Jiménez Molero, MC. (2018). A combined photophysical and computational study on the binding of mycophenolate mofetil and its major metabolite to transport proteins. Spectrochimica Acta Part A Molecular and Biomolecular Spectroscopy. 199:308-314. https://doi.org/10.1016/j.saa.2018.03.064S30831419

Photophysical properties of 5-substituted 2-thiopyrimidines

The aim of the present work is to determine the influence of C5 substitution on the photophysical properties of 2-thiopyrimidines (2-TPyr). For this purpose, 2-thiouracil, 5-t-butyl-2-thiouracil and 2-thiothymine (TU, BTU and TT, respectively) have been selected as target thionucleobases for the experimental studies and, in parallel, for DFT theoretical calculations. The UV spectra displayed by TU, BTU and TT in EtOH were very similar to each other. They showed a maximum around 275 nm and a shoulder at ca. 290 nm. The three 2-TPyr exhibited a strong phosphorescence emission; from the recorded spectra, triplet excited state energies of ca. 307, 304 and 294 kJ mol(-1) were determined for TU, BTU and TT, respectively. Laser excitation at 308 nm gave rise to a broad transient absorption band from 500 nm to 700 nm, which was in principle assigned to triplet-triplet absorption. This assignment was confirmed by energy transfer experiments using biphenyl (E-T = 274 kJ mol(-1)) as an acceptor. The triplet lifetimes were 70 ns, 1.1 mu s and 2.3 mu s, for TU, BTU and TT, respectively. The obtained photophysical data, both in phosphorescence and transient absorption measurements, point to significantly different properties of the TT triplet excited state in spite of the structural similarities. Theoretical calculations at the B3LYP/aug-cc-pVDZ/PCM level agree well with the experimental range of excited state energies and support the pi pi(star) nature of the lowest triplet states.Financial support by the Spanish Government (CTQ2009-13699, CTQ2012-32621, RyC-2007-00476 to V. L.-V., and contracts JAE-Predoc 2011-00740 and JAE-Doc 2010-06204 to V. V.-C. and J. A. S. respectively) and the computing facilities provided by the Theoretical Organic Chemistry group at the Universitat de Valencia (http://utopia.uv.es) are acknowledged.Vendrell Criado, V.; Sáez Cases, JA.; Lhiaubet, VL.; Cuquerella Alabort, MC.; Miranda Alonso, MÁ. (2013). Photophysical properties of 5-substituted 2-thiopyrimidines. Photochemical & Photobiological Sciences Photochemical and Photobiological Sciences. 12(8):1460-1465. doi:10.1039/c3pp50058fS14601465128Kumar, R. (1997). Synthesis and studies on the effect of 2-thiouridine and 4-thiouridine on sugar conformation and RNA duplex stability. Nucleic Acids Research, 25(6), 1272-1280. doi:10.1093/nar/25.6.1272Sintim, H. O., & Kool, E. T. (2006). Enhanced Base Pairing and Replication Efficiency of Thiothymidines, Expanded-size Variants of Thymidine. Journal of the American Chemical Society, 128(2), 396-397. doi:10.1021/ja0562447Favre, A., & Fourrey, J.-L. (1995). Structural Probing of Small Endonucleolytic Ribozymes in Solution Using Thio-Substituted Nucleobases as Intrinsic Photolabels. Accounts of Chemical Research, 28(9), 375-382. doi:10.1021/ar00057a003Cooper, D. S. (2005). Antithyroid Drugs. New England Journal of Medicine, 352(9), 905-917. doi:10.1056/nejmra042972Reader, S. C. J., Carroll, B., Robertson, W. R., & Lambert, A. (1987). Assessment of the biopotency of anti-thyroid drugs using porcine thyroid cells. Biochemical Pharmacology, 36(11), 1825-1828. doi:10.1016/0006-2952(87)90245-0Massey, A., Xu, Y.-Z., & Karran, P. (2001). Photoactivation of DNA thiobases as a potential novel therapeutic option. Current Biology, 11(14), 1142-1146. doi:10.1016/s0960-9822(01)00272-xKuramochi, H., Kobayashi, T., Suzuki, T., & Ichimura, T. (2010). Excited-State Dynamics of 6-Aza-2-thiothymine and 2-Thiothymine: Highly Efficient Intersystem Crossing and Singlet Oxygen Photosensitization. The Journal of Physical Chemistry B, 114(26), 8782-8789. doi:10.1021/jp102067tHarada, Y., Okabe, C., Kobayashi, T., Suzuki, T., Ichimura, T., Nishi, N., & Xu, Y.-Z. (2009). Ultrafast Intersystem Crossing of 4-Thiothymidine in Aqueous Solution. The Journal of Physical Chemistry Letters, 1(2), 480-484. doi:10.1021/jz900276xFavre, A., Saintomé, C., Fourrey, J.-L., Clivio, P., & Laugâa, P. (1998). Thionucleobases as intrinsic photoaffinity probes of nucleic acid structure and nucleic acid-protein interactions. Journal of Photochemistry and Photobiology B: Biology, 42(2), 109-124. doi:10.1016/s1011-1344(97)00116-4Coleman, R. S., & Siedlecki, J. M. (1992). Synthesis of a 4-thio-2’-deoxyuridine containing oligonucleotide. Development of the thiocarbonyl group as a linker element. Journal of the American Chemical Society, 114(23), 9229-9230. doi:10.1021/ja00049a089Hafner, M., Landthaler, M., Burger, L., Khorshid, M., Hausser, J., Berninger, P., … Tuschl, T. (2010). Transcriptome-wide Identification of RNA-Binding Protein and MicroRNA Target Sites by PAR-CLIP. Cell, 141(1), 129-141. doi:10.1016/j.cell.2010.03.009Basnak, I., Balkan, A., Coe, P. L., & Walker, R. T. (1994). The Synthesis of Some 5-Substituted and 5,6-Disubstituted 2′-Deoxyuridines. Nucleosides and Nucleotides, 13(1-3), 177-196. doi:10.1080/15257779408013234Becke, A. D. (1988). 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Photobehavior of the antipsychotic drug cyamemazine in a supramolecular gel protective environment

[EN] In this work, a molecular hydrogel made of gelator (S)-4-((3-methyl-1-(nonylamino)-1-oxobutan-2-yl)amino)-4-oxobutanoic acid (SVN) has been employed as soft container to modify the photochemical and photophysical behavior of the antipsychotic drug cyamemazine (CMZ). The interaction of CMZ with the gel network has been evidenced by fluorescence spectroscopy through a hypsochromic shift of the emission band (from lambda(max) = 521 nm in solution to lambda(max) = 511 nm in the gel) and an increase of the fluorescence lifetime (5.6 ns in PBS vs. 7.2 ns in the gel). In the laser flash photolysis experiments on CMZ/SVN systems, the CMZ triplet excited state ((3)CMZ*), monitored at lambda = 320 nm, has been more efficiently generated and became much longer-lived than in solution (2.7 mu s vs. 0.7 mu s); besides, photochemical ionization leading to the radical cation CMZ(+center dot) was disfavored. In the steady-state experiments, photooxidation of CMZ to afford the N,S-dioxide derivative CMZ-SONO has been retarded in the gel, which provides a more lipophilic and constrained microenvironment. Both the photophysical properties and the photoreactivity are in agreement with CMZ located in a less polar domain when entrapped in the supramolecular gel, as result of the interaction of the drug with the fibers of the supramolecular SVN gel.Financial support from the Spanish Government (CTQ2016-78875-P and CTQ2015-71004-R), the Generalitat Valenciana (PROMETEO/2017/075) and the European Union is gratefully acknowledged.Vendrell-Criado, V.; Angulo-Pachón, CA.; Miravet, JF.; Galindo, F.; Miranda Alonso, MÁ.; Jiménez Molero, MC. (2020). Photobehavior of the antipsychotic drug cyamemazine in a supramolecular gel protective environment. Journal of Photochemistry and Photobiology B Biology. 202:1-4. https://doi.org/10.1016/j.jphotobiol.2019.111686S14202Bourin, M., Dailly, E., & Hascöet, M. (2006). Preclinical and Clinical Pharmacology of Cyamemazine: Anxiolytic Effects and Prevention of Alcohol and Benzodiazepine Withdrawal Syndrome. CNS Drug Reviews, 10(3), 219-229. doi:10.1111/j.1527-3458.2004.tb00023.xConilleau, V., Dompmartin, A., Michel, M., Verneuil, L., & Leroy, D. (2000). Photoscratch testing in systemic drug-induced photosensitivity. Photodermatology, Photoimmunology and Photomedicine, 16(2), 62-66. doi:10.1034/j.1600-0781.2000.d01-5.xMorlière, P., Bosca, F., Miranda, M. A., Castell, J. V., & Santus, R. (2004). Primary Photochemical Processes of the Phototoxic Neuroleptic Cyamemazine: A Study by Laser Flash Photolysis and Steady-state Irradiation¶. Photochemistry and Photobiology, 80(3), 535. doi:10.1562/0031-8655(2004)0802.0.co;2Morlière, P., Haigle, J., Aissani, K., Filipe, P., Silva, J. N., & Santus, R. (2004). An Insight into the Mechanisms of the Phototoxic Response Induced by Cyamemazine in Cultured Fibroblasts and Keratinocytes¶. Photochemistry and Photobiology, 79(2), 163. doi:10.1562/0031-8655(2004)0792.0.co;2Weiss, R. G. (2014). The Past, Present, and Future of Molecular Gels. What Is the Status of the Field, and Where Is It Going? Journal of the American Chemical Society, 136(21), 7519-7530. doi:10.1021/ja503363vDraper, E. R., & Adams, D. J. (2017). Low-Molecular-Weight Gels: The State of the Art. Chem, 3(3), 390-410. doi:10.1016/j.chempr.2017.07.012Lan, Y., Corradini, M. G., Weiss, R. G., Raghavan, S. R., & Rogers, M. A. (2015). To gel or not to gel: correlating molecular gelation with solvent parameters. Chemical Society Reviews, 44(17), 6035-6058. doi:10.1039/c5cs00136fSegarra-Maset, M. D., Nebot, V. J., Miravet, J. F., & Escuder, B. (2013). Control of molecular gelation by chemical stimuli. Chem. Soc. Rev., 42(17), 7086-7098. doi:10.1039/c2cs35436eJones, C. D., & Steed, J. W. (2016). Gels with sense: supramolecular materials that respond to heat, light and sound. Chemical Society Reviews, 45(23), 6546-6596. doi:10.1039/c6cs00435kHirst, A. R., Escuder, B., Miravet, J. F., & Smith, D. K. (2008). High-Tech Applications of Self-Assembling Supramolecular Nanostructured Gel-Phase Materials: From Regenerative Medicine to Electronic Devices. Angewandte Chemie International Edition, 47(42), 8002-8018. doi:10.1002/anie.200800022Mayr, J., Saldías, C., & Díaz Díaz, D. (2018). Release of small bioactive molecules from physical gels. Chemical Society Reviews, 47(4), 1484-1515. doi:10.1039/c7cs00515fMaiti, B., Abramov, A., Pérez-Ruiz, R., & Díaz Díaz, D. (2019). The Prospect of Photochemical Reactions in Confined Gel Media. Accounts of Chemical Research, 52(7), 1865-1876. doi:10.1021/acs.accounts.9b00097Escuder, B., Rodríguez-Llansola, F., & Miravet, J. F. (2010). Supramolecular gels as active media for organic reactions and catalysis. New Journal of Chemistry, 34(6), 1044. doi:10.1039/b9nj00764dMiravet, J. F., & Escuder, B. (2005). Reactive Organogels:  Self-Assembled Support for Functional Materials. Organic Letters, 7(22), 4791-4794. doi:10.1021/ol0514045Guler, M. O., & Stupp, S. I. (2007). A Self-Assembled Nanofiber Catalyst for Ester Hydrolysis. Journal of the American Chemical Society, 129(40), 12082-12083. doi:10.1021/ja075044nRodríguez-Llansola, F., Escuder, B., & Miravet, J. F. (2009). Switchable Perfomance of an l-Proline-Derived Basic Catalyst Controlled by Supramolecular Gelation. Journal of the American Chemical Society, 131(32), 11478-11484. doi:10.1021/ja902589fGalindo, F., Isabel Burguete, M., Gavara, R., & Luis, S. V. (2006). Fluorescence quenching in organogel as a reaction medium. Journal of Photochemistry and Photobiology A: Chemistry, 178(1), 57-61. doi:10.1016/j.jphotochem.2005.06.021Burguete, M. I., Izquierdo, M. A., Galindo, F., & Luis, S. V. (2008). Time resolved fluorescence of naproxen in organogel medium. Chemical Physics Letters, 460(4-6), 503-506. doi:10.1016/j.cplett.2008.06.045Díaz Díaz, D., Kühbeck, D., & Koopmans, R. J. (2011). Stimuli-responsive gels as reaction vessels and reusable catalysts. Chem. Soc. Rev., 40(1), 427-448. doi:10.1039/c005401cPérez-Ruiz, R., & Díaz Díaz, D. (2015). Photophysical and photochemical processes in 3D self-assembled gels as confined microenvironments. Soft Matter, 11(26), 5180-5187. doi:10.1039/c5sm00877hArnau del Valle, C., Felip-León, C., Angulo-Pachón, C. A., Mikhailov, M., Sokolov, M. N., Miravet, J. F., & Galindo, F. (2019). Photoactive Hexanuclear Molybdenum Nanoclusters Embedded in Molecular Organogels. Inorganic Chemistry, 58(14), 8900-8905. doi:10.1021/acs.inorgchem.9b00916Dawn, A., Fujita, N., Haraguchi, S., Sada, K., & Shinkai, S. (2009). An organogel system can control the stereochemical course of anthracene photodimerization. Chemical Communications, (16), 2100. doi:10.1039/b820565eShumburo, A., & Biewer, M. C. (2002). Stabilization of an Organic Photochromic Material by Incorporation in an Organogel. Chemistry of Materials, 14(9), 3745-3750. doi:10.1021/cm020421aBhat, S., & Maitra, U. (2007). Hydrogels as Reaction Vessels: Acenaphthylene Dimerization in Hydrogels Derived from Bile Acid Analogues. Molecules, 12(9), 2181-2189. doi:10.3390/12092181Bachl, J., Hohenleutner, A., Dhar, B. B., Cativiela, C., Maitra, U., König, B., & Díaz, D. D. (2013). Organophotocatalysis in nanostructured soft gel materials as tunable reaction vessels: comparison with homogeneous and micellar solutions. Journal of Materials Chemistry A, 1(14), 4577. doi:10.1039/c3ta01109gTorres-Martínez, A., Angulo-Pachón, C. A., Galindo, F., & Miravet, J. F. (2019). In between molecules and self-assembled fibrillar networks: highly stable nanogel particles from a low molecular weight hydrogelator. Soft Matter, 15(17), 3565-3572. doi:10.1039/c9sm00252aVendrell-Criado, V., González-Bello, C., Miranda, M. A., & Jiménez, M. C. (2018). A combined photophysical and computational study on the binding of mycophenolate mofetil and its major metabolite to transport proteins. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 199, 308-314. doi:10.1016/j.saa.2018.03.064Vayá, I., Andreu, I., Jiménez, M. C., & Miranda, M. A. (2014). Photooxygenation mechanisms in naproxen–amino acid linked systems. Photochem. Photobiol. Sci., 13(2), 224-230. doi:10.1039/c3pp50252

Novel Fluorescent Labeled Octasilsesquioxanes Nanohybrids as Potential Materials for Latent Fingerprinting Detection

The recent demand for fluorescent labeled materials (FLMs) in forensic security concepts such as latent fingerprints (LFs) that encodes information for anti-counterfeiting and encryption of confidential data makes necessary the development of building new and innovative materials. Here, novel FLMs based on Polyhedral Oligomeric Silsesquioxanes (POSS) functionalized with fluorophores via “click” reactions have been successfully synthesized and fully characterized. A comprehensive study of their photophysical properties has displayed large Stokes’s shift together with good photostability in all cases, fulfilling the fundamental requisites for any legible LF detection on various surfaces. The excellent performance of the hetero-bifunctional FLM in the visualization of LF is emphasized by their legibility, selectivity, sensitivity and temporal photostability. In this study, development mechanisms have been proposed and the overall concept constitute a novel approach for vis-à-vis forensic investigations to trace an individual’s identity.Alexander von Humboldt FoundationGeneralitat ValencianaUniversität RegensburgUniversidad de La LagunaMinisterio de Ciencia, Innovación y Universidade

Blocking cyclobutane pyrimidine dimer formation by steric hindrance

The efficiency of thymine (Thy) and uracil (Ura) to form cyclobutane pyrimidine dimers (CPDs) in solution, upon UV irradiation differs by one order of magnitude. This could to be partially related to the steric hindrance induced by the methyl at C5 in thymine. The aim of the present work is to establish the influence of a bulky moiety at this position on the photoreactivity of pyrimidines. With this purpose, photosensitization with benzophenone and acetone of a 5-tert-butyl uracil derivative (1) and the equivalent Thy (2) has been compared. Introduction of the tert-butyl group completely blocks CPD formation. Moreover, the mechanistic insight obtained by laser flash photolysis is in accordance with the observed photoreactivity.Financial support by the Spanish Government (CTQ2012-32621, CTQ2015-70164-P and contract JAE-Predoc 2011-00740 to V. V.-C.) and the Generalitat Valenciana (PROMETEOII/2013/005) is gratefully acknowledged.Vendrell Criado, V.; Lhiaubet, VL.; Yamaji, M.; Cuquerella Alabort, MC.; Miranda Alonso, MÁ. (2016). Blocking cyclobutane pyrimidine dimer formation by steric hindrance. Organic and Biomolecular Chemistry. 14(17):4110-4115. https://doi.org/10.1039/c6ob00382fS41104115141

Highly efficient latent fingerprint detection by eight-dansyl-functionalized octasilsesquioxane nanohybrids

The largely demand in continued security issues makes necessary the development of novel materials with outstanding properties to improve the current detection techniques. In this context, latent fingerprint (LF) by fluorescent labeled materials (FLM) is one of the most attractive personnel identification methodologies. Here, two FLM based on polyhedral oligomeric silsesquioxane (POSS) nanohybrids labeled with dansyl chromophores have been synthesized and fully characterized. Their photophysical properties have confirmed that these materials clearly possess the prime qualifications as suitable LF sensing platforms. In fact, they adequately detect LFs on glassy surface with excellence legibility.Alexander von Humboldt FoundationGeneralitat ValencianaUniversit¨at RegensburgMinisterio de Ciencia, Innovación y Universidade

Investigation of metabolite-protein interactions by transient absorption spectroscopy and in silico methods

[EN] Transient absorption spectroscopy in combination with in silico methods has been employed to study the interactions between human serum albumin (HSA) and the anti-psychotic agent chlorpromazine (CPZ) as well as its two demethylated metabolites (MCPZ and DCPZ). Thus, solutions containing CPZ, MCPZ or DCPZ and HSA (molar ligand:protein ratios between 1:0 and 1:3) were submitted to laser flash photolysis and the Delta A(max) value at lambda = 470 nm, corresponding to the triplet excited state, was monitored. In all cases, the protein-bound ligand exhibited higher Delta Amax values measured after the laser pulse and were also considerably longer-lived than the non-complexed forms. This is in agreement with an enhanced hydrophilicity of the metabolites, due to the replacement of methyl groups with H that led to a lower extent of protein binding. For the three compounds, laser flash photolysis displacement experiments using warfarin or ibuprofen indicated Sudlow site I as the main binding site. Docking and molecular dynamics simulation studies revealed that the binding mode of the two demethylated ligands with HSA would be remarkable different from CPZ, specially for DCPZ, which appears to come from the different ability of their terminal ammonium groups to stablish hydrogen bonding interactions with the negatively charged residues within the protein pocket (Glu153, Glu292) as well as to allocate the methyl groups in an apolar environment. DCPZ would be rotated 180 degrees in relation to CPZ locating the aromatic ring away from the Sudlow site I of HSA. (C) 2019 Elsevier B.V. All rights reserved.Financial support from Ministerio de Economia, Industria y Competitividad (CTQ2016-78875-P, SAF2016-75638-R, BES-2011-043706), Generalitat Valenciana (Prometeo 2017/075), Xunta de Galicia [Centro Singular de Investigacion de Galicia accreditation 2016-2019 (ED431G/09, ED431B 2018/04) and post-doctoral fellowship to E. L.] and European Union (European Regional Development Fund-ERDF) is gratefully acknowledged. I. A. holds a "Miguel Servet" contract (CP1116/00052) funded by the Carlos III Health Institute. We are grateful to the Centro de Supercomputacion de Galicia (CESGA) for computational facilities.Limones Herrero, D.; Palumbo, F.; Vendrell Criado, V.; Andreu Ros, MI.; Lence, E.; González-Bello, C.; Miranda Alonso, MÁ.... (2020). Investigation of metabolite-protein interactions by transient absorption spectroscopy and in silico methods. Spectrochimica Acta Part A Molecular and Biomolecular Spectroscopy. 226:1-8. https://doi.org/10.1016/j.saa.2019.117652S18226Yang, G. X., Li, X., & Snyder, M. (2012). Investigating metabolite–protein interactions: An overview of available techniques. Methods, 57(4), 459-466. doi:10.1016/j.ymeth.2012.06.013S. Hage, D., Anguizola, J., Barnaby, O., Jackson, A., J. Yoo, M., Papastavros, E., … Tong, Z. (2011). Characterization of Drug Interactions with Serum Proteins by Using High-Performance Affinity Chromatography. Current Drug Metabolism, 12(4), 313-328. doi:10.2174/138920011795202938Matsuda, R., Bi, C., Anguizola, J., Sobansky, M., Rodriguez, E., Vargas Badilla, J., … Hage, D. S. (2014). Studies of metabolite–protein interactions: A review. Journal of Chromatography B, 966, 48-58. doi:10.1016/j.jchromb.2013.11.043López-Muñoz, F., Alamo, C., cuenca, E., Shen, W., Clervoy, P., & Rubio, G. (2005). History of the Discovery and Clinical Introduction of Chlorpromazine. Annals of Clinical Psychiatry, 17(3), 113-135. doi:10.1080/10401230591002002Beckett, A. H., Beaven, M. A., & Robinson, A. E. (1963). Metabolism of chlorpromazine in humans. Biochemical Pharmacology, 12(8), 779-794. doi:10.1016/0006-2952(63)90108-4Chetty, M., Moodley, S. V., & Miller, R. (1994). Important Metabolites to Measure in Pharmacodynamic Studies of Chlorpromazine. Therapeutic Drug Monitoring, 16(1), 30-36. doi:10.1097/00007691-199402000-00004Hubbard, J. W., Midha, K. K., Hawes, E. M., McKAY, G., Marder, S. R., Aravagiri, M., & Korchinski, E. D. (1993). Metabolism of Phenothiazine and Butyrophenone Antipsychotic Drugs. British Journal of Psychiatry, 163(S22), 19-24. doi:10.1192/s0007125000292556García, C., Oyola, R., Piñero, L. E., Arce, R., Silva, J., & Sánchez, V. (2005). Substitution and Solvent Effects on the Photophysical Properties of Several Series of 10-Alkylated Phenothiazine Derivatives. The Journal of Physical Chemistry A, 109(15), 3360-3371. doi:10.1021/jp044530jNavaratnam, S., Parsons, B. J., Phillips, G. O., & Davies, A. K. (1978). Laser flash photolysis study of the photoionisation of chlorpromazine and promazine in solution. Journal of the Chemical Society, Faraday Transactions 1: Physical Chemistry in Condensed Phases, 74(0), 1811. doi:10.1039/f19787401811Palumbo, F., Garcia-Lainez, G., Limones-Herrero, D., Coloma, M. D., Escobar, J., Jiménez, M. C., … Andreu, I. (2016). Enhanced photo(geno)toxicity of demethylated chlorpromazine metabolites. Toxicology and Applied Pharmacology, 313, 131-137. doi:10.1016/j.taap.2016.10.024Garcia, C., Smith, G. A., McGimpsey, W. G., Kochevar, I. E., & Redmond, R. W. (1995). Mechanism and Solvent Dependence for Photoionization of Promazine and Chlorpromazine. Journal of the American Chemical Society, 117(44), 10871-10878. doi:10.1021/ja00149a010Nath, S., & Sapre, A. V. (2001). Photoinduced electron transfer from chloropromazine and promethazine to chloroalkanes accompanied by cleavage of C–Cl bond. Chemical Physics Letters, 344(1-2), 138-146. doi:10.1016/s0009-2614(01)00685-6Joshi, R., Ghanty, T. K., & Mukherjee, T. (2008). Reactions and structural investigation of chlorpromazine radical cation. Journal of Molecular Structure, 888(1-3), 401-408. doi:10.1016/j.molstruc.2008.01.025He, X. M., & Carter, D. C. (1992). Atomic structure and chemistry of human serum albumin. Nature, 358(6383), 209-215. doi:10.1038/358209a0Sharples, D. (1974). The binding of chlorpromazine to human serum albumin. Journal of Pharmacy and Pharmacology, 26(8), 640-641. doi:10.1111/j.2042-7158.1974.tb10679.xVerbeeck, R. K., Cardinal, J.-A., Hill, A. G., & Midha, K. K. (1983). Binding of phenothiazine neuroleptics to plasma proteins. Biochemical Pharmacology, 32(17), 2565-2570. doi:10.1016/0006-2952(83)90019-9Silva, D., Cortez, C. M., & Louro, S. R. W. (2004). Quenching of the intrinsic fluorescence of bovine serum albumin by chlorpromazine and hemin. Brazilian Journal of Medical and Biological Research, 37(7), 963-968. doi:10.1590/s0100-879x2004000700004Lázaro, E., Lowe, P. J., Briand, X., & Faller, B. (2008). New Approach To Measure Protein Binding Based on a Parallel Artificial Membrane Assay and Human Serum Albumin. Journal of Medicinal Chemistry, 51(7), 2009-2017. doi:10.1021/jm7012826Kaddurah-Daouk, R., Kristal, B. S., & Weinshilboum, R. M. (2008). Metabolomics: A Global Biochemical Approach to Drug Response and Disease. Annual Review of Pharmacology and Toxicology, 48(1), 653-683. doi:10.1146/annurev.pharmtox.48.113006.094715Korkuć, P., & Walther, D. (2015). Physicochemical characteristics of structurally determined metabolite-protein and drug-protein binding events with respect to binding specificity. Frontiers in Molecular Biosciences, 2. doi:10.3389/fmolb.2015.00051Ohnmacht, C. M., Chen, S., Tong, Z., & Hage, D. S. (2006). Studies by biointeraction chromatography of binding by phenytoin metabolites to human serum albumin. Journal of Chromatography B, 836(1-2), 83-91. doi:10.1016/j.jchromb.2006.03.043Roelofs, K. G., Wang, J., Sintim, H. O., & Lee, V. T. (2011). Differential radial capillary action of ligand assay for high-throughput detection of protein-metabolite interactions. Proceedings of the National Academy of Sciences, 108(37), 15528-15533. doi:10.1073/pnas.1018949108Jimenez, M., & Miranda, M. (2015). Triplet Excited States as a Source of Relevant (Bio)Chemical Information. Current Topics in Medicinal Chemistry, 14(23), 2734-2742. doi:10.2174/1568026614666141216100907Jiménez, M. C., Miranda, M. A., & Vayá, I. (2005). Triplet Excited States as Chiral Reporters for the Binding of Drugs to Transport Proteins. Journal of the American Chemical Society, 127(29), 10134-10135. doi:10.1021/ja0514489Vayá, I., Bueno, C. J., Jiménez, M. C., & Miranda, M. A. (2006). Use of Triplet Excited States for the Study of Drug Binding to Human and Bovine Serum Albumins. ChemMedChem, 1(9), 1015-1020. doi:10.1002/cmdc.200600061Vayá, I., Jiménez, M. C., & Miranda, M. A. (2008). Transient Absorption Spectroscopy for Determining Multiple Site Occupancy in Drug−Protein Conjugates. A Comparison between Human and Bovine Serum Albumins Using Flurbiprofen Methyl Ester as a Probe. The Journal of Physical Chemistry B, 112(9), 2694-2699. doi:10.1021/jp076960qPérez-Ruiz, R., Bueno, C. J., Jiménez, M. C., & Miranda, M. A. (2010). In situ Transient Absorption Spectroscopy to Assess Competition between Serum Albumin and Alpha-1-Acid Glycoprotein for Drug Transport. The Journal of Physical Chemistry Letters, 1(5), 829-833. doi:10.1021/jz1000227Nuin, E., Jiménez, M. C., Sastre, G., Andreu, I., & Miranda, M. A. (2013). Drug–Drug Interactions within Protein Cavities Probed by Triplet–Triplet Energy Transfer. The Journal of Physical Chemistry Letters, 4(10), 1603-1607. doi:10.1021/jz400640sAlonso, R., Yamaji, M., Jiménez, M. C., & Miranda, M. A. (2010). Enhanced Photostability of the Anthracene Chromophore in Aqueous Medium upon Protein Encapsulation. The Journal of Physical Chemistry B, 114(34), 11363-11369. doi:10.1021/jp104900rAlonso, R., Jiménez, M. C., & Miranda, M. A. (2011). Stereodifferentiation in the Compartmentalized Photooxidation of a Protein-Bound Anthracene. Organic Letters, 13(15), 3860-3863. doi:10.1021/ol201209hKitamura, K., Fujitani, K., Takahashi, K., Tanaka, Y., Hirako, S., Kotani, C., … Takegami, S. (2000). Synthesis of [N-13CH3] drugs (chlorpromazine, triflupromazine and promazine). Journal of Labelled Compounds and Radiopharmaceuticals, 43(9), 865-872. doi:10.1002/1099-1344(200008)43:93.0.co;2-eGhuman, J., Zunszain, P. A., Petitpas, I., Bhattacharya, A. A., Otagiri, M., & Curry, S. (2005). Structural Basis of the Drug-binding Specificity of Human Serum Albumin. Journal of Molecular Biology, 353(1), 38-52. doi:10.1016/j.jmb.2005.07.075Pérez-Ruiz, R., Molins-Molina, O., Lence, E., González-Bello, C., Miranda, M. A., & Jiménez, M. C. (2018). Photogeneration of Quinone Methides as Latent Electrophiles for Lysine Targeting. The Journal of Organic Chemistry, 83(21), 13019-13029. doi:10.1021/acs.joc.8b01559Roe, D. R., & Cheatham, T. E. (2013). PTRAJ and CPPTRAJ: Software for Processing and Analysis of Molecular Dynamics Trajectory Data. Journal of Chemical Theory and Computation, 9(7), 3084-3095. doi:10.1021/ct400341

Two-photon chemistry from upper triplet states of thymine

Photolysis of the benzophenone chromophore by means of high energy laser pulses has been used as a tool to populate upper thymine-like triplet states via intramolecular sensitization. These species undergo characteristic n pi* photoreactivity, as revealed by the Norrish-Yang photocyclization of 5-tert-butyluracil.Financial support by the Spanish government (CTQ2009-13699, CTQ2012-32621, RyC-2007-00476 to V.L.-V. and JAE-Predoc 2011-00740 contract to V.V.-C.) and the Generalitat Valenciana (Prometeo program) is gratefully acknowledged. M.Y, is thankful for Grants-in-Aid for Scientific Research (KAKENHI) from JSPS (No. 23350059) and the "Element Innovation" Project by the Ministry of Education, Culture, Sports, Science, and Technology, Japan.Vendrell Criado, V.; Rodríguez Muñiz, GM.; Yamaji, M.; Lhiaubet, VL.; Cuquerella Alabort, MC.; Miranda Alonso, MÁ. (2013). Two-photon chemistry from upper triplet states of thymine. Journal of the American Chemical Society. 135(44):16714-16719. https://doi.org/10.1021/ja408997jS16714167191354

Two-Photon Chemistry from Upper Triplet States of Thymine

Photolysis of the benzophenone chromophore by means of high energy laser pulses has been used as a tool to populate upper thymine-like triplet states via intramolecular sensitization. These species undergo characteristic nπ* triplet photoreactivity, as revealed by the Norrish–Yang photocyclization of 5-<i>tert</i>-butyluracil