Visible-light promoted bioorthogonal photocatalysis

FACULTADE DE QUÍMICA GRAO EN QUÍMICA VISIBLE-LIGHT PROMOTED BIOORTHOGONAL PHOTOCATALYSIS LARA TRONCOSO AFONSO 2020-2021 UNIVERSIDADE DE SANTIAGO DE COMPOSTELA 2 3 TRABALLO FIN DE GRAO Rama de coñecemento: Química Orgánica. Departamento, centro, institución ou empresa: Centro de Investigación en Química Biolóxica e Materiais Moleculares (CiQUS). Titor: José Luis Mascareñas Cid Cotitora: María Tomás Gamasa Autorización dos titores: D. José Luis Mascareñas Cid, Catedrático do Departamento de Química Orgánica da Universidade de Santiago de Compostela. Dra. María Tomás Gamasa, Investigadora JIN do Departamento de Química Orgánica da Universidade de Santiago de Compostela. Certifican: que a presente memoria adxunta, titulada ‘’Visible-light promoted bioorthogonal photocatalysis’’, que presenta Lara Troncoso Afonso, foi realizada baixo a súa dirección nos laboratorios do Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares (CiQUS). Considerando que a nomeada memoria constitúe o seu traballo fin de grado, autorizan a súa presentación na Universidade de Santiago de Compostela. Para que así conste, firman o presente informe en Santiago de Compostela a día 5 de Xullo de 2021. MASCAREÑAS CID JOSE LUIS - 34934771W Firmado digitalmente por MASCAREÑAS CID JOSE LUIS - 34934771W Fecha: 2021.07.05 10:21:47 +02'00' TOMAS GAMASA MARIA - 09417809E Firmado digitalmente por TOMAS GAMASA MARIA - 09417809E Fecha: 2021.07.05 10:33:56 +02'00' 4 AGRADECEMENTOS Desculpándome de antemán por si esquezo mencionar alguén, gustaríame agradecer a todas as persoas que me apoiaron ao longo da miña formación: En primeiro lugar, a Xulián, María e José Luis pola implicación, apoio e dedicación ao longo do desenvolvemento do presente traballo. Seguidamente, a todos os profesores que durante estes anos foron quen de transmitirme a súa curiosidade científica e afán pola química. Finalmente, á miña familia en xeral e, en particular, a meus pais polo apoio incondicional, á miña irmá Carlota por sacar o mellor de min e ás miñas avoas, Carmen e Pilar, por ser os meus máis valiosos exemplos de esforzo, perseverancia e traballo constante dende nena. 5 TABLE OF CONTENTS TABLE OF CONTENTS ................................................................................................. 5 ABSTRACT ................................................................................................................. 6 1. INTRODUCTION .................................................................................................. 8 1.1. Bioorthogonal chemistry ....................................................................................... 8 1.1.1. Bioorthogonal ligation reactions .......................................................................................... 9 1.1.2. Bioorthogonal cleavage reactions ...................................................................................... 11 1.1.3. Requirements for achieving bioorthogonality ................................................................... 12 1.2. Photocatalysis ..................................................................................................... 14 1.2.1. Photosensitization and photoredox catalysis .................................................................... 14 1.2.2. Photocatalysis in water ...................................................................................................... 16 1.2.3. Bioorthogonal photocatalysis ............................................................................................ 17 1.2.4. Photocatalysts: organic dyes and transition-metal complexes .......................................... 18 1.2.5. Substrates: aryldiazonium salts ......................................................................................... 20 2. OBJECTIVES AND PLAN ..................................................................................... 22 2.1. Objective ............................................................................................................. 22 3. RESULTS AND DISCUSSION ............................................................................... 23 3.1. Synthesis of phenanthrenes ................................................................................ 23 3.1.1. Optimization ...................................................................................................................... 25 3.1.2. Bioorthogonality ................................................................................................................ 29 3.2. Synthesis of benzotiophenes ............................................................................... 30 3.2.1. Optimization ...................................................................................................................... 33 3.3. Synthesis of coumarins ........................................................................................ 34 3.4. Reactions to be explored in cells ......................................................................... 36 CONCLUSIONS ......................................................................................................... 39 References ............................................................................................................... 41 6 ABSTRACT Biological chemistry deals with the study of the chemical processes that occur inside living beings, from a molecular perspective. Understanding the cellular and molecular mechanisms underlying biological functions is fundamental for the treatment of many diseases. Furthermore, being able to manipulate, monitor and transform cellular behaviour is key to the development of modern medicine. However, interfering with cell’s functioning is not a trivial task and it requires the development of tools to carry out designed transformations under the complex environment of biological habitats. The chemical transformation of exogenous or endogenous substances inside living beings requires bioorthogonality and selectivity. One way to perform biocompatible reactions could be based on the use of photochemically-induced transformations, promoted by visible light. Combining photochemistry and bioorthogonal methods leads to bioorthogonal photochemistry, a new field of research that is still in its infancy. During this TFG, some preliminary work in this area have been carried out. RESUMEN La química biológica consiste en el estudio de los procesos químicos que ocurren en el interior de los seres vivos desde el punto de vista molecular. Entender los mecanismos celulares y moleculares relativos a las funciones biológicas es fundamental para el tratamiento de muchas enfermedades. Es más, ser capaz de manipular, monitorizar y transformar el comportamiento celular es clave para el desarrollo de la medicina moderna. Sin embargo, interferir en las funciones celulares no es una tarea trivial y requiere el desarrollo de nuevas herramientas para llevar a cabo las transformaciones diseñadas bajo el complejo ambiente de los sistemas biológicos. Las transformaciones químicas de sustancias exógenas o endógenas en el interior de los seres vivos requiere 7 bioortogonalidad y selectividad. Un modo de llevarlas a cabo podría basarse en el uso de transformaciones inducidas fotoquímicamente, promovidas por la luz visible. La combinación de la fotoquímica y los métodos bioortogonales abre paso a la química fotobioortogonal, un novedoso campo de investigación que se encuentra todavía en su infancia. Durante este TFG, se ha llevado a cabo trabajo preliminar en este área. RESUMO A química biolóxica consiste no estudo dos procesos químicos que acontecen no interior dos seres vivos para entender os seres vivos dende o punto de vista molecular. Entender os mecanismos celulares e moleculares relativos as funcións biolóxicas é fundamental para o tratamento de moitas enfermidades. É máis, ser capaz de manipular, monitorizar e transformar o comportamento celular é clave para o desenvolvemento da medicina moderna. Non obstante, interferir nas funcións celulares non é unha tarefa trivial e require o desenvolvemento de novas ferramentas para levar a cabo as transformacións deseñadas baixo o complexo ambiente dos sistemas biolóxicos. As transformacións químicas de substancias exóxenas e endóxenas no interior dos seres vivos require bioortogonalidade e selectividade. Un modo de levalas a cabo podería basearse no uso de transformacións inducidas fotoquímicamente, promovidas pola luz visible. A combinación da fotoquímica e os métodos bioortogonales abre paso a química fotobioortogonal, un novidoso campo de investigación que se encontra aínda na súa infancia. Durante este TFG, levouse a cabo traballo preliminar nesta área. 8 1. INTRODUCTION 1.1. Bioorthogonal chemistry Bioorthogonal chemistry refers to a series of quick, nonnative abiotic chemical reactions that occur in complex biological environments. Moreover, for these transformations to be considered bioorthogonal, they need to be carried out with good selectivity in the presence of biological components. (1; 2) The development of this type of biocompatible chemical reactions is not trivial. While the first examples of bioorthogonal reactions reported were restricted to the modification of nucleophilic amino acids (e.g., cysteines), today many other options are available, including catalytic methods based on transition metal reagents. (3) In nature, many transformations in complex physiological conditions and inside cells are mediated by enzymes and metalloenzymes. These natural catalysts are implied in all the vital functions of living organisms. Chemists have been trying for years to obtain artificial versions of enzymes to catalyze nonnative reactions in a bioorthogonal manner. Research on this area has led to the design of different artificial metalloenzymes with relatively good success. (4; 5) One alternative to carry out bioorthogonal reactions in live settings is based on the use of discrete transition metal catalysts. These artificial catalysts can promote a great variety of reactions in biological systems and also in cells, as for example, gold-promoted hydroarylations or ruthenium isomerizations, as demonstrated by our group. (6; 7) There are other metal-free bioorthogonal reactions such as Staudinger ligations, inverse electron-demand Diels Alder reactions or cycloadditions promoted by light. In general, the complexity of physiological media, with different concentration of salts, biomolecules and ions, makes the development of bioorthogonal methods really challenging. There are mainly two types of transformations: bioorthogonal ligation reactions (figure 1) and bioorthogonal cleavage reactions (figure 4). 9 1.1.1. Bioorthogonal ligation reactions This type of strategies were the first so far developed. Staudinger azidetriphenylphosphine ligation, inverse electron-demand Diels-Alder reactions and copper catalyzed azide-alkyne cycloadditions were the pioneering bioorthogonal ligations and their applications were focused on the labelling biomolecules, and on the synthesis of sophisticated pharmaceuticals. (8) Nevertheless, these reactions were only the starting point of a large number of bioorthogonal processes that have been developed over the last years. Figure 1 Scheme for bioorthogonal ligation processes. (8) In general, metal complexes are really versatile agents for promoting chemical transformations as they allow to change not only the main metal but also the ligands coordinated to the central cation. In other words, they can be designed with specific physical and chemical characteristics and therefore, they can be adapted to different reaction media by changing its solubility or to different substrates by modulating the electron-donor character of the complex. This flexibility led chemists to consider the utility of this complexes to perform bioorthogonal ligations. Cross-coupling of biomolecules catalyzed by palladium (e.g. Suzuki-Miyaura (9), Sonogashira reactions (10)…) or cross-metathesis reactions catalyzed by ruthenium (11) constitute some examples. Albeit the more famous is the copper-catalyzed cycloaddition between alkynes and azides. Over the years, strategies to avoid the use of copper in bioorthogonal reactions have been developed. This is due to its toxicity and instability in the presence of O2 and H2O2. Bioorthogonal ligation Biomolecule Functional groups Probe 16 this treatment have led to three generations of photosensitizers, from simple natural occurring porphyrins to sophisticated aggregation-induced emission luminogens (AIEgens) and metal-organic frameworks (MOFs), going through the use of heavy-metal based sensitizers. (20) 1.2.2. Photocatalysis in water Although the combination of photocatalysis and aqueous media can be considered a powerful tool to perform sustainable organic transformations, this is a field in its total infancy. There are some photoinduced reactions that have been performed in water such as arylation of pyridines with aryldiazonium salts (21) (figure 10a), synthesis of trifluoromethylated dihydroisoquinolines (22) (figure 10b) or amide bond formation (23) (figure 10c). However, many transformations still require organic solvents, as some reactants are either poorly soluble in water or likely prone to suffer from hydrolysis. (18) Figure 10 Examples of photoinduced transformations performed in water. Substitution of traditional-based organic solvents by water reduces difficult-to-treat wastes and risks related to heat and gas release from the reaction. (18) Moreover, water is an abundant natural liquid, innocuous and it is ubiquitously present in all living organisms. N R1 N2BF4 R2 [Ru(bpy)3]Cl2·6H2O (2.5 mol%) 25 oC, H2O, Ar, 80 h 45 W bulb N R1 R2 (a) R1 N OMe +CF3SO2Na [Ru(bpy)3]Cl2 (2 mol%) TBHP (1.5 equiv.) 3 W blue LED, H2O air, r.t., 24 h R1 N OMe O CF3 O (b) (c) R1 O SH N H R3 R2 CdSNPs, 30 W CFL H2O r.t., 3 h R1 O N R3 R2 17 Therefore, using water as a solvent is not only ecological but also provides for developing biocompatible transformations. In fact, the development of photocatalytic synthetic strategies in water media is fundamental to discover light-induced bioorthogonal strategies. 1.2.3. Bioorthogonal photocatalysis The maintenance of all the vital functions needed for an organism to stay alive requires a perfect functioning of the cellular enzymatic machinery. These natural biocatalysts are implied in cellular metabolism, respiration and reproduction enabling living beings to growth and stay healthy. In fact, alterations in enzymatic functions often trigger diseases and pathologic processes. In this sense, absorption of light is a well-known natural method that living organisms use to activate their biocatalysts and this fact has inspired the development of artificial visible light induced catalysis inside cells. (20) The combination of photocatalysis and bioorthogonal chemistry has led to the birth of a new field, bioorthogonal photochemistry, with great potential to face problems and foster progress in biomedical engineering, nanomaterials, nanomedicine, drug discovery and human health. (3; 20) Curiously, although visible light is highly compatible with living systems, there are few applications of photochemical reactions in biology. Some of those reports include: fotoredox dimerization of tyrosines (figure 11a), (24) bioorthogonal photocatalyzed oxidation of a prodrug (figure 11b), (2) or photocatalyzed reduction of azides with bioimaging purposes (figure 11c). (25) The reason behind this scarcity might be related to the notion of incompatibility between radical chemistry and biological environments as well as to the need of using cell-damaging UV-light to promote photochemical reactions. 18 Figure 11 Some examples of bioorthogonal photocatalysis. a) Example of protein dimerization through ligation of tyrosines. b) Releasing of Pt IV prodrug; c) Releasing of a fluorophore for bioimaging. The idea behind this project is to explore the viability of achieving visible light initiated photocatalyzed processes in biological media. 1.2.4. Photocatalysts: organic dyes and transition-metal complexes In visible-light-induced redox transformations it is crucial to choose an adequate catalyst with a high absorptivity coefficient in the visible region of the spectra and a long lifetime excited state as well as a suitable redox potential. Organic dyes are chromophores with a high absorption coefficient in the visible region. Their capacity to undergo electron and energy transfer processes after being excited have extended their use as catalysts. In addition, they avoid the use of toxicmetallic complexes, and they are usually cheaper. Protein1 OH Protein2 OH [Ru(bpy)3]2+ hv Protein1 OH Protein2 OH Pt Cl NH3 NH3 Cl Riboflavin hv Pt Cl NH3 NH3 Cl OO O O 2 O O O O O O O O (a) (b) N NH O Cl Cl HO [Ru(bpy)3]Cl2, NaAsc hv N NH O Cl Cl O OO N3 (c) Fluorophore PtIV prodrug 19 However, transition-metal complexes show unique redox properties and several examples have been described for ruthenium (II)/iridium (III) polypyridyl complexes, cobalt and palladium species. Especially remarkable are ruthenium polypyridyl complexes and, in particular, [Ru(bpy)3](PF6)2 satisfies the above conditions: the wavelength for maximum absorbance is 452 nm (visible-near UV region) allowing the reaction to be performed in biological media. Figure 12 a) Eosin Y as an example of organic dye. b) [Ru(bpy)3]2+ as an example of transition-metal complex. After absorption of a photon, electron transfer from t2g ruthenium orbital to 𝜋∗ ligand orbital takes place, resulting in a long lifetime excited state (1100 ns) that triggers electron or energy transfer processes. As a result, excited-state catalyst can lead to radical formation from different substrates and gives a new approach to radical chemistry, traditionally dependent on powerful radical initiators (AIBN, Bu3SnH…) and high temperatures. Finally, taking into account the biocompatible excitation wavelength and solubility in water of [Ru(bpy)3](PF6)2, combination of both constitutes an environmentally friendly method to access free radical intermediates. In this sense, diazonium salts are suitable oxidative quenchers in photoredox chemistry and there are several examples in literature supporting their use as nice precursors of radicals obtained by electron transfer processes triggered by these catalysts. (26; 27) O Br Br O Br Br HO NN Ru N NN N 2+ Eosin Y O O [Ru(bpy)3]2+ a) b) 20 1.2.5. Substrates: aryldiazonium salts Diazonium salts, in particular those derived from anilines, are inexpensive and versatile compounds appropriated for loads of synthetic routes in organic chemistry. Aryldiazonium salts are suitable precursors for transformations in water as they are commonly soluble and highly reactive. Mainly, aryldiazonium salts can easily undergo two types of reactions: nitrogen-removal or nitrogen-retention processes. (28) On one hand, the driving force of the first type of chemistry is the release of N2 from the aryldiazonium substrate. This ability has turned them into highly suitable precursors for Meerwein-type alkene functionalization or Sandmeyer-type reactions for C-S, C-P and C-B bond formation. On the other hand, concerning the nitrogen-retention processes, aryldiazonium salts act as electrophiles in N-N bond forming transformations or as radical acceptors in radical addition reactions. (28) On these grounds, this project is focused on N2 releasing reactions that result in aryl radical formation in presence of a redox-active transition-metal complex after irradiation with light. Even though many diazonium salts can be directly photolyzed, UV photons are needed. Therefore, photoredox catalysts are used to sensitize these organic molecules through electron or energy transfer processes. Under these conditions, aryldiazonium salts are able to take up an electron from the catalyst and the energy required for the redox reaction to proceed is provided by light. The reaction takes place at room temperature and, as nitrogen is released, it does not interfere with the reaction mixture. Moreover, these transformations undergo through radical species and the chemoselectivity is high. Overall, these salts are extremely useful for synthetic methodologies as they play a fundamental role in the transformation and functionalization of the aromatic carbon they are attached to, leading to different types of bond formation (C-H, C-C-, C-S, C-P…) and lots of different aromatic derivatives. 21 However, these compounds can be unstable. In fact, the stability of diazonium salts moves in a wide range from those that are explosive to those that are difficult to decompose. As a result, these salts can be really hard to isolate. Nevertheless, some of them are isolable, such as aryl diazonium tetrafluoroborate, tosylate, disulfonimide or carboxylate (29) and they are commonly storaged under special conditions, such as low temperatures (below 0 oC) or avoiding contact with light. The stability of these diazonium salts is subjected to the type and position of the substituents on the aromatic ring. The decomposition temperature of these chemicals can be determined by using differential scanning calorimetry (DSC) and in general, not many trends are observed. (29) 22 2. OBJECTIVES AND PLAN 2.1. Objective As commented, even though photocatalytic methods are a valuable tool in synthesis, there are little examples of bioorthogonal photocatalytic strategies. The reason behind that might be the formation of highly reactive radical species that restrict the applications. Nevertheless, there are several examples of reactions in aqueous media. The objective of this project is the design and evaluation of different photochemical transformations in aqueous media, and the viability of performing them in a bioorthogonal manner. For this purpose, intermolecular processes that encompass both C-C and C-heteroatom bond formation are chosen for the obtention of interesting structures, such as phenanthrenes, benzotiophenes or coumarins. Moreover, to prove bioorthogonality and compatibility with biological media, the reactions will be tested in more complex media such as DMEM, BSA solution, lysates and finally in presence of HeLa cells. Finally, preliminary experiments might be carried out in presence of living cells, consisting in the synthesis of a highly fluorescent product, an AIE fluorophore by means of a bioorthogonal photocatalytic reaction. 23 3. RESULTS AND DISCUSSION 3.1. Synthesis of phenanthrenes In 2012, Zhou and co-workers reported the synthesis of monoand di-substituted phenanthrenes by Eosin-Y catalyzed visible light induced [4+2] benzannulation. (26) In the present work, the reaction between biaryldiazonium tetrafluoroborate salt 3 and methyl propiolate 4 displayed in figure 13 was chosen as a model reaction. Figure 13 Reaction conditions for phenanthrene formation. While the authors described the use of Eosin-Y in acetonitrile, preliminary results of the group led us to introduce two initial changes. On the one hand, [Ru(bpy)3](PF6)2 was used instead of Eosin-Y. As mentioned in section 1.2.4., the use of [Ru(bpy)3](PF6)2 is preferred because of the solubility in aqueous media as well as biocompatible excitation wavelength and long-life excited state. On the other hand, deoxygenated PBS (saline phosphate buffer pH = 7.4) was selected as reaction media, which works as a first approximation to cellular media. In addition, to avoid degradation of the reactants, inert atmosphere was used. Taking into account these aspects, for the experiments biaryldiazonium salt 3 (1 eq.) and [Ru(bpy)3](PF6)2 were loaded inside a sealed vial under inert atmosphere. Then, PBS was added, followed by methyl propiolate 4 (5 eq.). The vial was introduced inside the photoreactor (for technical characteristics see annex IV) and was irradiated using blue LED lamp (figure 14). Once the reaction was completed, the crude was subjected to adequate work-up and the final product was isolated by flash column chromatography for characterization purposes (for further details see annex II: 2.1.1. and annex III: 2.1.1). 3 4 5 OMe OOMe O N2+BF4- [Ru(bpy)3](PF6)2 deox. PBS Ar atm., r.t. 24 Figure 14 Set-up for photocatalyzed reactions. The reaction mechanism proposed by the authors for phenanthrene formation is displayed in figure 15, taking into account the use of [Ru(bpy)3](PF6)2 instead of Eosin-Y. As showed in the reaction scheme, reaction starts with excitation of ruthenium complex ( 𝜆 = 452'𝑛𝑚 ), followed by single electron transference to biaryldiazonium salt, resulting in biphenolic radical formation and releasing of N2. This radical is added to the alkyne and cyclization affords phenanthrene structure. Finally, electron transference back to ruthenium complex, regenerates the catalyst and resulting carbocation recovers aromaticity after deprotonation. Figure 15 Reaction mechanism proposed by the authors adjusted to [Ru(bpy)3](PF6)2. (26) N2 +BF4 - N2 + BF4 - OMe O OMe O OMe O H OMe O H OMe O[Ru(bpy)3](PF6)2 [Ru(bpy)3](PF6)2 [Ru(bpy)3](PF6)2 hv = 452 nm SET BF4 HBF4 25 The reaction was optimized for the photocatalyzed synthesis of methyl 9phenanthrene carboxylate 5. In this case, for yield determination (annex IV), nitromethane in CDCl3 was used as an internal standard. In concrete, the relation between the signal for nitromethane ( 𝛿' = 4.32 ppm) and that for the metoxi group ( 𝛿' = 4.05 ppm) was calculated to get the yield as shown in figure 16. Moreover, two additional signals (8.9 ppm and 8.5 ppm) that ideally integrate by 1H could be used too, therefore yields were estimated as an average of the three values. Figure 16 Example of yield's estimation for phenanthrene formation. 3.1.1. Optimization First experiments were carried out with a concentration of biaryldiazonium salt 3 of 330 mM, and the reaction was evaluated after different irradiation times: 1, 5, 15, 30 min and 2, 6 and 24 h. The irradiation time refers to the time the samples are exposed to the LED lamp. In the following experiments, irradiation time is almost equal to the reaction time as samples were introduced inside the photoreactor just after addition of the reactants following the procedure described in annex II (2.1.1.). 0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.0 f1 (ppm) 1.82 1.00 0.59 1.22 0.61 OMe O 3 %"#$%& =1.82/3 + 0.59 + 0.61 334100 =60.2% 32 As it was needed to introduce a characteristic signal in the NMR spectra of the product, it was considered the idea of introducing a metoxi group in one of the reagents, as it would give an aliphatic and isolated signal. In concrete, it was important to introduce it in the limiting reagent (e.g. aryldiazonium tetrafluoroborate salt), otherwise it would remain after completion of the reaction, preventing the yield to be determined properly. Therefore, the next step consisted in trying to perform the reaction using aryldiazonium salt 6b, synthesized from p-anisidine (following protocols 1.1.2. and 1.2.2. described in annex II). As it is displayed in figure 21, all the other reaction conditions (alkyne, catalyst, irradiation time, temperature, concentration…) were maintained. Figure 21 Scheme of the reaction using aryldiazonium salt 6b and alkyne 7a. Gratifyingly, product 6b was obtained by following the protocol described in annex II (2.2.1.) , isolated by flash column chromatography in 37% yield and characterized by 1H-NMR (annex III: 2.2.2.). As for phenanthrenes, in order to determine the yield (annex IV), nitromethane in CDCl3 was used as an internal standard. In concrete, the relation between the signal for nitromethane ( 𝛿' = 4.32 ppm) and that for the metoxi group ( 𝛿' = 3.89 ppm) was calculated to get the yield as shown in figure 22. 5 mol% Eosin Y DMSO LED 530 nm atm. Ar, 14 h, 20 oC S 7a N2BF4 S + 6b MeO MeO 8b 33 Figure 22 Example of yield's estimation for benzotiophene formation. 3.2.1. Optimization In order to turn reaction conditions into more biocompatible, several changes were introduced, starting with the reaction media. In concrete, first attempts were focussed on checking the compatibility of the reaction with aqueous media (e.g. deoxygenated PBS). With this purpose, the reaction was performed in PBS, keeping all the other reaction conditions (catalyst, irradiation time, temperature, concentration…). Table 5 Yield obtained for the synthesis of benzotiophene 6b as a function of reaction media and photocatalyst.a S MeO 6b %"#$%& =1.12 3,-100 = 37.3% 0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0 f1 (ppm) 1.12 1.00 Entry Reaction media Photocatalyst Yield % 1 DMSO Eosin Y 34 2 PBS Eosin Y 45 3 PBS [Ru(bpy)3](PF6)2 26 N2+BF4SMe + 5 mol% photocatalyst reaction media LED Ar atm., 14 h, 20 oC S 6b 7a 8b 250 mM MeO MeO 34 a Reaction conditions: 6a (0.25 mmol), 7a (1.25 mmol), photocatalyst (0.0125 mmol) in the different reaction media (1.0 mL), LED (452 nm), Ar atm. and r.t. Gratifyingly, the yield is even improved when using PBS as reaction media (Table 5). Encouraged by the results, the following step, consisted in using different catalysts to compare their behaviour for the synthesis of benzotiophenes. In concrete, the reaction using either Eosin Y or [Ru(bpy)3](PF6)2 was carried out and the yields were determined, as shown in Table 5. As better results were obtained using Eosin Y in PBS, next step implied the comparison between different catalyst loadings, keeping all reaction conditions used before. The yield afforded by the different catalytic loadings is shown in Table 6. Table 6 Yields obtained for benzotiophene formation as a function of catalyst loading. a aReaction conditions: 6a (0.25 mmol), 7a (1.25 mmol), photocatalyst (0.0125 mmol) in the different reaction media (1.0 mL), LED (452 nm), Ar atm. and r.t. From the obtained results, it can be deduced that 5% catalyst loading affords the highest yield. However, the yield obtained is still low and further experiments must be performed. 3.3. Synthesis of coumarins In this case, the starting point is the photoinduced synthesis of 3-difluoroacetylated coumarins described by Baoming and coworkers in 2015. The authors reported as the highest-yielded the transformation of phenyl alkynoate derivatives and ethyl-2-bromo2,2-difluoroacetate into the desired coumarin after photoexcitation of [fac-Ir(ppy)3] in DMF and in presence of K2CO3 as a base. (31) Entry Catalyst loading % Yield % 1 5% 45 2 7.5% 42 3 10% 40 35 Figure 23 Reaction conditions reported by Baoming et al. (31) However, inspired on the previous experiences using diazonium salts as source of radicals, it was hypothesized that these versatile substrates could be used to participate in the formation of coumarins. The use of different photocatalysts was considered as well. Therefore, the mechanism shown in figure 24 is adjusted to the use of any aryldiazonium salt and any photocatalyst (commonly Eosin Y or [Ru(bpy)3]2+). The plausible reaction mechanism is similar to that for phenanthrenes and benzotiophenes, but in this case, intermolecular C(sp2)-C(sp2) bond formation is followed by intramolecular cyclization of the alkyne structure to afford the skeleton of coumarins. Figure 24 Reaction mechanism proposed by the authors and adjusted to any aryldiazonium salt and photocatalyst (PC). (31) Particularly, p-metoxi aryldiazonium tetrafluoroborate salt 10 was used in order to enable the monitorization of the reaction by NMR-spectroscopy with internal standard O O R1 +BrCF2COOEt 2 mol% fac-Ir(ppy)3 K2CO3 DMF 5W blue LED O CF2COOEt O R1 N2 + BF4 - hv SET HCO3 H2CO3 O O R1 O O R1 N2 +BF4 - R2 R2 R O O R1 R2 PC PC PC O O R1 R2 O O R1R2 36 as this metoxi substituent would hopefully give an isolated signal in the aliphatic region of the spectra. Moreover, first experiments were focused on using 9 synthesized from phenol as described in annex II (1.3.1). The reason behind is the fluorescence product 11 might show that would allow to follow the reaction inside cells in future experiments. As this transformation has not been previously described, different catalysts and reaction media were scanned, as well as the presence or absence of base. In general, the reaction conditions are summarized in figure 25. Figure 25 Resumed reaction conditions for coumarin formation. Unfortunately, none of the combinations enabled the identification of the product. Even though the reaction was followed by TLC (thin layer chromatography) and consumption of the starting reactants was observed, the crude consisted in a complex mixture of products from which the desired product could not have been isolated and characterized yet. 3.4. Reactions to be explored in cells The final purpose of the project was to perform light-induced photocatalytic reactions inside living cells. This requires the production of a fluorescent compound in order to follow the reaction in real time by using fluorescence microscopy. However, neither phenanthrene nor benzotiophene derivatives prepared before were fluorescent. Therefore, a new transformation, which gives fluorescent compound 14 was proposed. O O Ph +5 mol% photocatalyst K2CO3, deox. solvent, irradiation atm. Ar, 24 h, t.a. O O Ph N2BF4 OMe 910 11 OMe NN 37 Figure 26 Scheme of reaction for the synthesis of AIE fluorophore. As preliminary examples on the viability of this transformation had been developed in the group, it was checked if it would be possible to perform the process in the presence of living cells. In concrete, HeLa cells were treated with the photocatalyst [Ru] during 30 minutes before being washed with DMEM to remove extracellular [Ru]. Then, they were incubated with substrates 12 and 13 for 30 minutes before irradiation with blue light ( 𝜆' = 460 nm) for 10 minutes (for detailed procedure see annex II 2.4.). Additionally, two control experiments were carried out consisting in incubation for 30 minutes of 50 µM [Ru] and 100 µM of substrates 12 and 13. Finally, the following micrographies shown in figure 27 were taken. Figure 27 Bioorthogonal photocatalytic reaction inside HeLa cells. Fluorescence micrographies (brightfield) after incubation with: (a) substrates 12 and 13; (b) [Ru]; (c) [Ru], washing step, substrates 12 and 13; (d) same as (c) but 10 min of irradiation at 𝜆 = 460 nm. Blue channel: Iexc = 385 nm Iem = 410-480 nm. From the comparison of the fourth, it can be concluded that there is not an observable intracellular fluorescence in the case of control experiments (a) and (b). In panel (c), without irradiation, no intracellular staining is observed, while there is an increase of blue fluorescence for experiments (d) corresponding with the intracellular formation of the product after irradiation. N2BF4 OMe 13 Ph Ph Ph + Ph Ph Ph OMe [Ru(bpy)3](PF6)2 12 14 A) B) C) D) 38 The results are very promising but very preliminar since this part of the project has been recently initiated and it must be exhaustively studied. 39 CONCLUSIONS The experimental work of this project consisted in the study of different photoinduced reactions in aqueous media and in presence of biologically relevant solvents. The intermolecular processes explored encompass both C-C and C-heteroatom bond formation for the obtention of interesting structures, such as phenanthrenes, benzotiophenes or coumarins. The synthesis of phenanthrenes was studied, optimizing reaction conditions using ruthenium complex in PBS by using different techniques such as slow addition methods. Moreover, the reaction was successfully performed in different biological media, such as DMEM, BSA solution, cell lysates and even in presence of HeLa cells. Regarding the synthesis of benzotiophenes, the compatibility of the system with more complex media (e.g., PBS) was explored comparing the activity of different catalysts as preliminary studies needed to achieve bioorthogonality. However, in relation to the synthesis of coumarins, the desired product could not be isolated. Finally, preliminary experiments carried out in living cells for the synthesis of an AIE fluorophore showed that the transformation can occur. CONCLUSIONES El trabajo experimental en este proyecto consistió en el estudio de diferentes reacciones fotoinducidas en medio acuoso y en la presencia de disolventes biológicos. Los procesos intermoleculares estudiados incluyen la formación de enlaces C-C y Cheteroátomo para la obtención de estructuras interesantes como los fenantrenos, benzotiofenos y cumarinas. La síntesis de fenantrenos se ha estudiado, optimizando las condiciones de reacción empleando el complejo de rutenio en PBS y utilizando diferentes técnicas como los métodos de adición lenta. Así mismo, la reacción se llevó a cabo en diferentes medios biológicos, como DEMEM, solución de BSA, lisados celulares e incluso en presencia de 40 células HeLa. Respecto a la síntesis de benzotiofenos, se ha explorado la compatibilidad del sistema con medios más complejos (por ejemplo PBS), comparando la actividad de diferentes catalizadores y cargas catalíticas, como estudios preliminares necesarios para alcanzar la bioortogonalidad del proceso. Sin embargo, en relación a la síntesis de cumarinas, el producto deseado no se ha podido aislar. Finalmente, los experimentos preliminares llevados a cabo en células vivas para la síntesis de un AIE fluoróforo muestran que la transformación se puede llevar a cabo. CONCLUSIÓNS O traballo experimental neste proxecto consistiu no estudo de diferentes reaccións fotoinducidas en medio acuoso e na presencia de disolventes biolóxicos. Os procesos intermoleculares estudiados inclúen a formación de enlaces C-C e C-heteroátomo para a obtención de estruturas interesantes como fenantrenos, benzotiofenos e cumarinas. A síntese de fenantrenos someteuse a estudo, optimizando as condicións de reacción, empregando o complexo de rutenio en PBS e utilizando diferentes técnicas como os métodos de adición lenta. Asimesmo, a reacción se levouse a cabo en diferentes medios biolóxicos, como DEMEM, solución de BSA, lisados celulares e incluso en presencia de células HeLa. Respecto da síntese de benzotiofenos, explorouse a compatibilidade do sistema con medios más complexos (por exemplo PBS), comparando a actividade de diferentes catalizadores e cargas catalíticas, como estudios preliminares necesarios para alcanzar a bioortogonalidade do proceso. Non obstante, en relación a síntese de cumarinas, o produto desexado non puido illarse. Finalmente, os experimentos preliminares levados a cabo en células vivas para a síntese dun AIE fluoróforo mostran que a transformación pode levarse a cabo. 41 References 1. Scinto, S. L.; Bilodeau, D. A.; Hincapie, R.; Lee, W.; Nguyen, S. S.; Xu, M.; Ende, C. W.; Finn, M. G.; Lang, K.; Lin, Q.; Pezaki, J. P.; Prescher, J. A.; Robillard, M. S. and Fox, J. M. (2021). Bioorthogonal chemistry. Nat. Rev. Methods Primers, 1 (30). 2. Alonso-de Castro, S.; Ruggiero, E.; Ruiz-de-Angulo, A.; Rezabal, E.; Mareque-Rivas, J. C.; Lopez, X.; López-Gallego, F. and Salassa, L. (2017). Riboflavin as a bioorthogonal photocatalyst for the activation of a PtIV prodrug. Chem. Sci., 8 (6), 4619-4625. 3. Wang, J.; Wang, X.; Fan, X. and Chen, P. R. (2021). Unleashing the power of bond cleavage chemistry in living systems. ACS Cent. Sci., 7 (6), 929-943. 4. Vornholt, T.; Christoffel, F.; Pellizzoni, M. M.; Panke, S.; Ward, T. R. and Jeschek, M. (2021). Systematic engineering of artificial metalloenzymes for new-to-nature reactions. Science Advances, 7, eabe4208. 5. Davis, H. J. and Ward, T. R. (2019). Artificial metalloenzymes: Challenges and opportunities. ACS Cent. Sci., 5, 1120-1136. 6. Vidal, C.; Tomás-Gamasa, M.; Destito, P.; López, F. and Mascareñas, J. L. (2018). Concurrent and orthogonal gold (I) and ruthenium (II) catalysis inside living cells. Nat. Commun., 9 (1913). 7. Vidal, C.; Tomás-Gamasa, M.; Gutiérrez-González, A. and Mascareñas, J. L. (2019). Ruthenium-catalyzed redox isomerizations inside living cells. J. Am. Chem. Soc., 141 (13), 5125-5129. 8. Fu, H. and Li, Y. (2020). Bioorthogonal ligations and cleavages in chemical biology. ChemistryOpen, 9 (8), 835-853. ANNEX II 4 Figure 2 Reaction scheme for 4-metoxi-2-(metylthio)aniline 2. A mixture of p-anisidine (2.00 g, 16.3 mmol, 1 eq.) and KSCN (3.20 g, 32.6 mmol, 2 eq.) is dissolved in glacial acetic acid (27.5 mL) and cooled down until 10 °C. Then, a solution of Br2 (1.6 mL, 32.6 mmol, 2 eq.) in glacial acetic acid (2.5 mL) is added dropwise and the resulting mixture is stirred for 3 h at 10 °C and for additionally 45 min at room temperature. The precipitate is filtered, redissolved in warm water and basified with saturated NaOH solution until pH = 8. Benzothiazole is obtained after filtration as a nude precipitate in 42% yield (1.20 g, 6.8 mmol). Finally, benzothiazole (1.20 g, 6.8 mmol, 1 eq.) is dissolved in 25% aqueous KOH and refluxed for 17 h. Then, the solution is cooled down to room temperature, previously to the addition, in only one portion, of iodomethane (440.0 µL, 6.8 mmol, 1 eq.) and stirred for an additional hour at room temperature. The crude is extracted with diethyl ether and isolated by flash column chromatography on silica gel (hexane / ethyl acetate 7:3) as a green oil in 52% yield. Rf = 0.320 (hexane / ethyl acetate 7:3). Yield = 52% (947.6 g, 3.5 mmol). 1H-NMR (300 MHz, CDCl3): δ (ppm) 6.93 (d, J = 2.6 Hz, 1H), 6.80 – 6.65 (m, 2H), 3.75 (s, 3H), 2.39 (s, 3H). 1.2. Synthesis of aryldiazonium salts: 1.2.1. Procedure A NH2 MeO MeO S N NH2 NH2 SMe MeO CH3COOH glacial 1. KSCN 2. Br2, 10 oC, 3 h 3. Stirring, r.t., 45 min 25% aq. KOH MeI 2 ANNEX II 5 Aryldiazonium tetrafluoroborate derivatives are synthesized according to the procedure described by Youn et al. and spectroscopic data are in agreement with the reported values. (3) Figure 3 Reaction scheme for the synthesis of aryldiazonium salts. These compounds are obtained by nitrosation of aniline derivative using tert-butyl nitrate in acidic media. With this purpose, aniline (1 eq.) and tetrafluoroboronic acid (2 eq.) are dissolved in ethanol and the mixture is cooled down to 0 °C. Tert-butyl nitrate (2 eq.) is added dropwise and the resulting solution is stirred during 1 h at room temperature to afford a solid product after precipitation with diethyl ether. 1.2.2. Procedure B p-Methoxyaryldiazonium tetrafluoroborate derivatives are synthesized according to the procedure described by Youn et al. and spectroscopic data are in agreement with the reported values. (3) Figure 4 Reaction scheme for the synthesis of para-metoxi aryldiazonium salts. The compounds are obtained by nitrosation of aniline derivative using sodium nitrate in acidic media. With this purpose, aniline (1 eq.) and tetrafluoroboronic acid (2 eq.) are dissolved in distilled water and the mixture is cooled down until 0 °C. Tert-butyl nitrate (2 eq.) is added dropwise and the resulting solution is stirred during 40 min at 05 °C temperature to afford a solid product after filtration. The solid is re-dissolved in acetone and precipitated with diethyl ether to obtain the final product. NH2 1) HBF4 2) t-BuONO, 0 oC EtOH 1 h, r.t. N2BF4 RR NH2 1) HBF4 2) NaNO2, 0 oC distilled H2O 40 min 0-5 oC N2BF4 MeO MeO RR ANNEX II 6 Table 1 Yields obtained for the synthesis of the different aryldiazonium tetrafluoroborate salt derivates. Compound Name R group Yield% Colour 3 Biaryldiazonium tetrafluoroborate salt Ph 70% Light yellow 6a 2-(Methylthio)benzenediazonium tetrafluoroborate salt SMe 96% Intense yellow 6b 4-Methoxy-2-(methylthio) benzenediazonium tetrafluoroborate salt SMe 97% Light green 10 4-Methoxybenzenediazonium tetrafluoroborate salt H 80% Purple 1H-NMR (300 MHz, DMSO): δ (ppm) 8.91 (d, 3J = 8.3 Hz, 1H), 8.35 (t, 3J = 7.1 Hz, 1H), 8.12 – 7.96 (m, 2H), 7.92-7.82 (m, 2H), 7.74 – 7.65 (m, 3H). 1H-NMR (300 MHz, DMSO): δ (ppm) 8.66 (d, 3J = 8.3 Hz, 1H), 8.15 (t, 3J = 7.9 Hz, 1H), 7.97 (d, 3J = 7.4 Hz, 1H), 7.71 (t, 3J = 7.9 Hz, 1H), 2.84 (s, 3H). 1H-NMR (300 MHz, DMSO): δ (ppm) 8.62 (d, 3J = 8.8 Hz, 2H), 7.49 (d, 3J = 7.4 Hz, 2H), 4.04 (s, 3H). N2 +BF4 - Ph 3 N2 +BF4 - SMe 6a N2 +BF4 - 10 MeO ANNEX II 7 1H-NMR (300 MHz, DMSO): δ (ppm) 8.58 (d, 3J = 9.3 Hz, 1H), 7.32 (d, 3J = 2.3 Hz, 1H), 7.27 (dd, 3J = 9.3, 2.3 Hz, 1H), 4.09 (s, 3H), 2.85 (s, 3H). 1.3. Synthesis of alkynoates: 1.3.1. Synthesis of 2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-yl 3phenylpropiolate ( 9 ) Alkynoate 9 is synthesized following the procedure described by Vidal et al. and spectroscopic data are in agreement with the reported values. (4) Figure 5 Reaction scheme for synthesis of compound 9. Phenyl propiolic acid (200.0 mg, 1.1 mmol, 1 eq.) is added to a heat gun dried round bottom flask equipped with stir bar under nitrogen and dissolved with dichloromethane (1.6 mL). The resulting solution is stirred at 0 °C in an ice/brine bath for 1 min. Then, DIC (N,N-dicyclohexylcarbodiimide, 250.0 µL, 1.6 mmol, 1.5 eq.) is added causing the precipitation of a white solid. The mixture is stirred for a minute before addition of solution of 8-hydroxyjulolidine (200.0 mg, 1.4 mmol, 1.3 eq.) in dichloromethane (2.5 mL) followed by addition of 4-(dimethylamino)pyridine (DMAP, 32.0 mg, 1.05 mmol, 0.25 eq.) in dichloromethane (250.0 µL) that turns solution into orange. Final mixture is stirred until consumption of 8-hydroxyjulolidine is observed by TLC. Finally, the reaction mixture is filtered through Kieselguhr, concentrated under vacuum and the final product is obtained after flash column chromatography (hexane / acetate 8:2) yielding product 9 as an orange solid. N2 +BF4 - 6b MeO SMe HO O Ph + 1. DIC 2. DMAP CH2Cl2, 0 ºC O O Ph N OH N 9 ANNEX II 8 Rf = 0.300 (hexane / ethyl acetate 8:2). Yield = 54% (175.0 mg, 0.6 mmol). 1H-NMR (300 MHz, CDCl3): δ (ppm) 7.63 (d, 3J = 8.3 Hz, 2H), 7.48 (t, 3J = 7.5 Hz, 1H), 7.40 (t, 3J = 7.7 Hz, 2H), 6.81 (d, 3J = 8.1 Hz, 1H), 6.34 (d, 3J = 7.9 Hz, 1H), 3.14 (q, 3J = 5.7 Hz, 4H), 2.75 (t, 4J = 6.5 Hz, 2H), 2.65 (t, 3J = 6.6 Hz, 2H), 1.97 (m, 4H). 2. SYNTHESIS OF PRODUCTS 2.1. Synthesis of phenanthrenes: The procedure is adapted from Zhou et al. and 1H NMR is in concordance with the data reported in the literature. (5) 2.1.1. Representative general procedure for experiments at 330 mM Figure 6 Scheme of reaction conditions for biaryldiazonium salt 3 concentration of 330 mM. In an 8.0 mL glass vial equipped with magnetic stirring bar, biaryldiazonium tetrafluoroborate salt 3 (53.60 mg, 0.2 mmol, 1 eq.) and [Ru(bpy)3(PF6)2] (1.71 mg, 0.002 mmol, 0.01 eq.) are introduced and degassed by vacuum-nitrogen cycles (x3) via a syringe needle. The vial is sealed under an argon atmosphere and the mixture is dissolved in deoxygenated PBS (0.6 mL). Then, methyl propiolate 4 (89.0 µL, 1 mmol, 5 eq.) is added via Hamilton syringe and the vial is irradiated with blue LED lamp (452 nm). After irradiation, the crude is diluted with water and the aqueous phase is extracted with ethyl acetate (3 x 30.0 mL). The combined organic layers are dried over MgSO4, filtered and concentrated under reduced pressure. The final residue is purified by flash chromatography on silica gel (hexane / ethyl acetate 9:1). During the optimization process, the crude is analysed by 1H NMR. The yield is determined by 1H NMR using nitromethane as internal standard. OMe OOMe O 330 mM 1 mol% [Ru(bpy)3](PF6)2 deox. PBS LED 452 nm Ar atm., r.t. 45 3 N2BF4 ANNEX II 9 Rf = 0.34 (hexane / ethyl acetate 9:1). 1H-NMR (300 MHz, CDCl3): δ (ppm) 8.98 – 8.87 (m, 1H), 8.78 – 8.65 (m, 2H), 8.48 (s, 1H), 7.97 (d, 3J = 7.9 Hz, 1H), 7.81 – 7.58 (m, 4H), 4.06 (s, 3H). LR-MS Calculated for [C16H13O2]+: 237.09; found: 236.80. 2.1.2. Representative general procedure for experiments at 10 mM In case of 10 mM experiments, similar protocol is used but a catalyst loading of 5 mol% of [Ru(bpy)3(PF6)2] is used instead of 1 mol%. Figure 7 Scheme of reaction conditions for biaryldiazonium salt 3 concentration of 10 mM. 2.1.3. General procedure for slow addition of diazonium salt 3 exemplified for experiments at 10 mM The stock solution is prepared by dissolving biaryldiazonium salt 3 (26.8 mg, 0.1 mmol) in PBS (400 µL) inside an Eppendorf (1mL). Then, 200 µL of the solution are added via syringe into the sealed vial, containing the reactants left, and using an automatic pump machine with addition rate of 0.04 mL/min in order to ensure the continuous and slow addition as well as the reproducibility of the process. 2.1.4. Study of the reaction under biological relevant conditions In experiments carried out in complex biological media, standard conditions have been followed at 10 mM. The reaction is studied in different biological media: -Gibco DEMEM (Dubelcco’s Modified Eagle’s Medium) purchased from ThermoFisher Scientific. OMe OOMe O 10 mM 5 mol% [Ru(bpy)3](PF6)2 deox. PBS LED 452 nm Ar atm., r.t. 45 3 N2BF4 ANNEX II 10 -Bovine serum albumin (BSA) obtained from Sigma Aldrich and dissolved in PBS until getting a final concentration of 5 mg/mL. -HeLa cell lysates, obtained from 2 days cultured HeLa cells: after two washings with PBS, cells are scraped from the well, sonicated and diluted with PBS to reach the concentration of 1.03 cell lysates/mL. -Suspension of HeLa cells. The cells are cultured and suspended in PBS to achieve the concentration of 5 mg/mL. 2.2. Synthesis of benzotiophenes: 2.2.1. Representative general procedure for experiments at 250 mM in DMSO Benzotiophene derivatives are synthesized according to the procedure described by König et al. and spectroscopic data are in agreement with the reported values. (2) Figure 8 Scheme of reaction conditions for a concentration of aryldiazonium salt 6 equal to 250 mM. In an 8.0 mL glass vial equipped with a magnetic stirring bar, aryldiazonium tetrafluoroborate salt 6 (0.25 mmol, 1 eq.) and photocatalyst (0.0125 mmol, 0.05 eq.) are introduced and degassed by vacuum-nitrogen cycles (x3) via a syringe needle. The vial is sealed under an argon atmosphere and the mixture is dissolved in deoxygenated dimethylsulfoxide (1.0 mL). Then, phenylacetylene 7a (1.25 mmol, 5 eq.) is added and the vial is irradiated with green LED lamp (530 nm). After irradiation, the crude is diluted with diethyl ether, washed with water and the aqueous phase is extracted with diethyl ether (3 x 30.0 mL). The combined organic layers are dried over MgSO4, filtered and concentrated under reduced pressure. The final residue is purified by flash chromatography on silica gel. During the optimization process, the crude is analysed by 1H NMR. The yield is determined by 1H NMR using nitromethane as internal standard. N2+BF4SMe + 5 mol% photocatalyst DMSO LED 530 nm Ar atm., 14 h, 20 oC S 67a 8 RR ANNEX II 11 Rf = 0.51 (hexane). 1H-NMR (300 MHz, CDCl3): δ (ppm) 7.84 (d, 3J = 8.1 Hz, 1H), 7.78 (d, 3J = 7.2 Hz, 1H), 7.73 (d, 3J = 8.2 Hz, 2H), 7.55 (s, 1H), 7.49-7.40 (m, 2H), 7.40-7.28 (m, 3H). LR-MS Calculated for [C14H11S]+: 211.05; found: 210.81. Rf = 0.34 (hexane / ethyl acetate 9:1). 1H-NMR (300 MHz, CDCl3): δ (ppm) 7.73-7.62 (m, 3H), 7.47 (s, 1H), 7.46-7.37 (m, 2H), 7.37-7.27 (m, 2H), 6.98 (dd, 3J = 8.7, 2.4 Hz, 1H), 3.89 (s, 3H). LR-MS Calculated for [C15H13OS]+: 241.06; found: 240.80. When PBS is used as reaction media (1.0 mL), a different work-up must be performed. The crude is diluted with water and the aqueous phase is extracted with ethyl acetate (3 x 30.0 mL). The combined organic layers are dried over MgSO4, filtered and concentrated under reduced pressure to obtain the final product. Finally, for experiments with different catalytic loading, the amount of catalyst is changed while keeping all the other conditions. 2.3. Synthesis of coumarins: The procedure is adapted from Ji et al. (6) Figure 9 Scheme of reaction conditions for the synthesis of coumarin 11. S 8a S MeO 8b O O Ph +5 mol% photocatalyst K2CO3, deox. solvent, irradiation atm. Ar, 24 h, t.a. O O Ph N2BF4 OMe 910 11 OMe NN ANNEX II 12 In an 8.0 mL glass vial equipped with magnetic stirring bar, alkynoate 9 (0.25 mmol, 2.5 eq.), 4-methoxyaryldiazonium salt 10 (0.1 mmol, 1 eq.), photocatalyst (0.005 mmol, 0.05 eq.) and K2CO3 (0.1 mmol, 1 eq.) are introduced and degassed by vacuum-nitrogen cycles (x3) via a syringe needle. The vial is sealed under argon atmosphere and the mixture is dissolved in deoxygenated solvent (1.0 mL). Then, the vial is irradiated with suitable LED lamp during 24 h at room temperature. The reaction is followed by TLC (hexane / ethyl acetate 8:2) to completion of reactants. The crude of the reaction is subjected to suitable work-up to get the final product. 2.4. Preliminary reactions in cellular settings Figure 10 Scheme of reaction conditions for AIE fluorophore 14 formation. First, two days before treatment, HeLa cells are seeded on 12 well plates. Then, cells are incubated with the ruthenium complex [Ru(bpy)3](PF6)2 (50 µM) for 30 min in fresh DMEM. After 30 min, in order to remove extracellular ruthenium complex, two washing steps are needed (400 µM of DEMEM) . Resulting cells are incubated with substrates 12 and 13 (100 µM each) in fresh DMEM for 20 min to ensure intracellular reaction. In parallel, control experiments are performed by incubation of either [Ru] or substrates 12 and 13 in DEMEM for 30 and 20 min, respectively. Then, cells are irradiated at 460 nm for 10 min with the laser of the microscope before observation under the epifluorescence microscope (Iexc= 385 nm and Iem=450 nm). The digital pictures of the different samples were taken under identical conditions of gain and exposure under epifluorescence microscope. N2BF4 OMe 13 Ph Ph Ph + Ph Ph Ph OMe [Ru(bpy)3](PF6)2 12 14 ANNEX III 13 ANNEX III 1. SUBSTRATES: 1H-NMR SPECTRA. 1.1. 1H-NMR spectra for aniline derivatives 1 and 2 1.1.1. 1H-NMR spectra of 2-(methylthio)aniline 1 . 1.1.2. 1H-NMR spectra of 4-methoxy-2-(methylthio)aniline 2 . ANNEX IV 20 Figure 11 Example of yield's calculation for phenanthrene formation. On the other hand, for benzotiophenes formation, we simply calculate the relation between the signal for nitromethane ( 𝛿 = 4.32 ppm) and that for the metoxi group ( 𝛿 = 3.89 ppm) to get the yield as shown in figure 2. Figure 12 Example of yield's calculation for benzotiophene formation. 0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.0 f1 (ppm) 1.82 1.00 0.59 1.22 0.61 OMe O 3 %"#$%& =1.82/3 + 0.59 + 0.61 334100 =60.2% S MeO 6b %"#$%& =1.12 3,-100 = 37.3% 0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0 f1 (ppm) 1.12 1.00 ANNEX V 21 ANNEX V 1. INSTRUMENTATION 1.1. Photoreactor. One of the big drawbacks of photochemical methods is the difficulty related to standardization and reproducibility of the results. In order to get suitable and reproducible results is fundamental to control several parameters, mainly: the intensity of the radiation source, the uniformity of the optical pathway through the sample, the stirring intensity and the temperature. (7) The photoreactor used in this project is HepatoChem EvoluChemTM PhotoRedOx Box (HepatoChem Box). This instrument requires an external radiation source and samples are irradiated through a mirror system. This issue has a main advantage: as long as the lamp is available, any radiation (any 𝜆 ) can be used, but the intensity can only be controlled if it has its own control system. The refrigeration system consists on a fan that indirectly cools down the reactions vessels and it is located at the bottom of the photoreactor. Nevertheless, there is also a liquid cooled system available, but it was not used for this project. Concerning the stirring system, this is external too and it basically consist on a common magnetic stirring plate located just under the photoreactor to get a suitable agitation of the reaction samples. Figure 13 Set-up for photochemical methods. 22 REFERENCES 1. Basak, D.; Leusen, J.; Gupta, T.; Kögerler, P.; Bertolasi, V. and Ray, D. (2020) Unusually distorted pseudo-octahedral coordination environment around CoII from thioether shiff base ligands in dinuclear [CoLn] (Ln=La, Gd, Tb, Dy, Ho) complexes: synthesis, structure and understanding of magnetic behaviour. 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