Gold-promoted cascade reactions in biological media

0 Portada TFG_2023_24.pdf TFG [Subtítulo del documento] [FECHA] [NOMBRE DE LA COMPAÑÍA] [Dirección de la compañía] Ximena Sánchez Iglesias Gold-promoted cascade reactions in biological media José Luis Mascareñas Cid, titor e docente do Departamento de Química Orgánica, e María Tomás Gamasa, cotitora e docente do Departamento de Química Orgánica , autorizan a presentación do Traballo de Fin de Grao da alumna Ximena Sánchez Iglesias na convocatoria de xullo do curso 2023-2024, o cal foi realizado baixo a súa dirección no Centro Singular de Investigación en Química Biolóxica e Materiales Moleculares da Universidade de Santiago de Compostela (CiQUS). E para que así conste asinamos o presente informe en Santiago de Compostela o 30 de xuño de 2024. TOMAS GAMASA MARIA - 09417809E Firmado digitalmente por TOMAS GAMASA MARIA - 09417809E Fecha: 2024.06.30 00:35:40 +02'00' MASCAREÑAS CID JOSE LUIS - 34934771W Firmado digitalmente por MASCAREÑAS CID JOSE LUIS - 34934771W Fecha: 2024.06.30 08:09:42 +02'00' INDEX ABBREVIATIONS .................................................................................................................................... 1 ABSTRACT ............................................................................................................................................... 2 1. INTRODUCTION ............................................................................................................................. 4 1.1 Bioorthogonal chemistry ......................................................................................................... 4 1.2 Organometallic catalysis in biological media ........................................................................ 6 1.2.1 Ruthenium; roots of bioorthogonal organometallic chemistry ................................................... 7 1.2.2 Palladium ................................................................................................................................... 8 1.3 Gold reactivity in biological environments ............................................................................ 8 1.3.1 Historical review.................................................................................................................... 8 1.3.2 Cycloisomerization of alkynes and cascade reactions .......................................................... 11 1.3.3 Synthesis and relevance of carbazoles ................................................................................. 12 2. OBJECTIVES AND WORK PLAN .............................................................................................. 14 3. RESULTS AND DISCUSSION ...................................................................................................... 15 3.1 Preliminary assays for a gold-cascade synthesis of carbazoles .......................................... 15 3.2 In vitro optimization ............................................................................................................... 16 3.2.1 Screening of other reactions conditions ............................................................................... 16 3.2.2 Reaction in biorelevant media ............................................................................................. 21 3.2.3 Compatibility with specific biomolecules ............................................................................ 22 3.3 Fluorescence studies ............................................................................................................... 24 3.3.1 UV study............................................................................................................................... 24 3.3.2 Fluorescence study .............................................................................................................. 25 3.4 In cellulo experiments ............................................................................................................ 26 3.5 Experimental procedures ...................................................................................................... 28 3.5.1 General information for in vitro experiments ...................................................................... 28 3.5.2 Synthesis of 1-iodo-2-(phenylethynyl)benzene ..................................................................... 29 3.5.3 Synthesis of 2-{[2-(phenylethynyl)phenyl]ethynyl}aniline 1 ............................................... 29 3.5.4 Synthesis of 6-phenyl-11H-benzo[a]carbazole 2 ................................................................. 30 3.5.5 Representative procedure for reactions at 200 mM scale using iPrAuNTf2 as complex and MeCN as solvent (section 3.2.1) ........................................................................................................ 31 3.5.6 Representative procedure for the study of the dilution (section 3.2.1)* .............................. 32 3.5.7 Representative procedure for reactions at 1 mM scale using stock solutions in DMSO* (section 3.2.1) .................................................................................................................................... 32 3.5.8 Internal standard method for yield quantification ............................................................... 33 3.5.9 General information for in cellulo experiments .................................................................. 34 3.5.10 Representative procedure for in cellulo reactions ............................................................... 34 CONCLUSIONS ...................................................................................................................................... 35 REFERENCES ........................................................................................................................................ 38 ANNEX: CHARACTERIZATIONS...................................................................................................... 43 1 ABBREVIATIONS allyl prop-2-en-1-yl NADH nicotinamide adenine dinucleotide (reduced) BSA bovin serum albumin NMR nuclear magnetic resonance 13C-NMR carbon NMR NTF2 bis(trifluoromethylsulfonyl)amide CuAAC copper-catalyzed azide-alkyne cycloaddition OSCs organic solar cells d doublet OTf trifluoromethanesulfonate dba dibenzylideneacetone DEPT-135 distortionless enhancement by polarization transfer PBS phosphate-buffered saline δ chemical shift PhSH phenylthiol DMEM Dulbecco’s Modified Eagle Medium ppm parts per million DMSO dimethyl sulfoxide PTA 1,3,5-triaza-7-phospaadamantane e.g. exempli gratia r.t. room temperature eq. equivalents Rf retention factor Et3N triethylamine ROS reactive oxygen species EtOAc ethylacetate s singlet GSH L-glutathione SM starting material 1H-NMR proton NMR SPAAC strain-promoted azide-alkyne cycloaddition HPLC-MS high performance liquid chromatography – mass spectrometry TMB 1,3,5-trimethoxybenzene HSA human serum albumin THF tetrahydrofuran IED-DA inverse electron-demand Diels-Alder reaction TLC thin layer chromatography J coupling constant t triplet JohnPhos 2-(di-tertbutylphosphino)biphenyl tr retention time λ wavelength UV ultraviolet m multiplet vol volume MeCN acetonitrile w/o without 2 ABSTRACT In recent years, the possibility of introducing abiotic reactions inside living systems in a biocompatible way has aroused great interest, thanks to the multiple applications in chemical biology and biomedicine that could lead to the development of new therapies and treatments. Within these reactions, the use of transition metals as catalytic structures can open the door to new reactivities to be carried out within biological systems. Among them, gold, a precious metal long considered highly inert, has begun to gain importance in the field of Bioorthogonal Chemistry. In this Final Degree Project, it is proposed the development of a gold-promoted double cyclization through a cascade mechanism within biological media. Thus, the study involves the optimization of the transformation towards biological conditions, that is, high dilution, water and air compatibility, as well as the tolerance to the presence of different biomolecules and complex media. We have obtained promising results suggesting the viability of these cascade processes in biological environments and even in live mammalian cells (A549). This strategy constitutes an important new approach to build up high complexity molecules from simple compounds in a straightforward, elegant and bioorthogonal manner in living cells. RESUMEN En los últimos años, la posibilidad de introducir reacciones abióticas dentro de sistemas vivos de manera biocompatible ha despertado un gran interés, gracias a las múltiples aplicaciones en química biológica y biomedicina que podrían conducir al desarrollo de nuevas terapias y tratamientos. Dentro de estas reacciones, el uso de metales de transición como estructuras catalíticas permite abrir la puerta a nuevas reactividades que llevar a cabo dentro de sistemas biológicos. Entre ellos, el oro, un metal precioso que durante mucho tiempo ha sido considerado inerte, ha comenzado a ganar relevancia en el campo de la Química Bioortogonal. En este Trabajo de Fin de Grado se propone el estudio de una reacción de doble ciclación promovida por oro a través de un mecanismo en cascada en el interior de medios biológicos. Así, se ha optimizado la transformación para alcanzar condiciones biológicas; esto es, altas diluciones y compatibilidad con el aire y el agua, así como tolerancia a la presencia de diferentes biomoléculas y medios complejos. Los resultados obtenidos son prometedores, sugiriendo la viabilidad de estos procesos en cascada, no solo en medios biológicos, sino también en el interior de células de mamífero (A549). Esta estrategia constituye un importante nuevo enfoque para la construcción de moléculas de alta complejidad a partir de compuestos sencillos de una manera simple, elegante y bioortoogonal dentro de células vivas. 3 RESUMO Nos últimos anos, a posibilidade de introducir reaccións abióticas dentro de sistemas vivos de maneira biocompatible despertou un gran interese, grazas ás múltiples aplicacións en química biolóxica e biomedicina que poderían conducir ao desenvolvemento de novas terapias e tratamentos. Dentro destas reaccións, o uso de metais de transición como estruturas catalíticas permite desenvolver novas reactividades dentro de sistemas biolóxicos. Entre eles, o ouro, un metal precioso que durante moito tempo foi considerado inerte, comezou a gañar relevancia no campo da Química Bioortogonal. Neste Traballo de Fin de Grao proponse o estudo dunha reacción de dobre ciclación promovida por ouro a través dun mecanismo en cascada no interior de medios biolóxicos. Así, optimizouse a transformación para alcanzar condicións biolóxicas; isto é, altas dilucións e compatibilidade co aire e auga, así como tolerancia á presencia de diferentes biomoléculas e medios complexos. Os resultado obtidos son prometedores, suxerindo a viabilidade destes procesos en cascada, non solo en medios biolóxicos, senón tamén no interior de células de mamífero (A549). Esta estratexia constitúe un importante novo enfoque para á construción de moléculas de alta complexidade a partir de compostos sinxelos dunha maneira simple, elegante e bioortoogonal dentro de células vivas. 4 1. INTRODUCTION Nature has historically served as a source of inspiration for science. The complex network of chemical transformations that makes life possible constitutes the basis of Chemical Biology, which applies chemical knowledge to the exploration of living systems, the development of techniques for the synthesis and modification of biomolecules, as well as the design of new chemical processes based on biology. 1.1 Bioorthogonal chemistry Bioorthogonal chemistry, defined as reactions that do not disrupt the intrinsic biochemical processes that take place inside living systems,1 was first described by Carolyn R. Bertozzi in 2003. These chemical reactions, characterized by a good biocompatibility and high selectivity, must tolerate physiological conditions of pH and temperature.2 Nevertheless, developing new bioorthogonal transformations in complex living systems is really challenging, due to the networks of interconnected metabolites, ions and biopolymers responsible for the array of cellular processes that happen within cells, that create an intricate media to conduct non-natural reactions.3 The rise of bioorthogonal transformations in recent years has been possible due to their impact in multiple fields. It can be implemented in medicinal chemistry (e.g. biomedical imaging or biotherapeutics), materials science (e.g. polymer synthesis or energy storage materials), basic research (e.g. protein modification and synthesis) and biotechnology.4,5 This opens the possibility of evolving efficient methods to perform genetic code expansion, drug labeling identification and delivery, metabolic engineering and different types of bioconjugation.5 It was this significant impact what earned Bertozzi (ft. Meldal and Sharpless) the Nobel Prize in 2022, “for the development of click chemistry and bioorthogonal chemistry”. However, compared to traditional organic synthesis, where concentration of reactants, solvents and temperature can be modulated, air and moisture can be eliminated and catalyst can be added, if necessary, bioorthogonal reactions face an uncontrollable environment.4 Thus, to successfully carry out a chemical reaction under physiological conditions, some requirements should be followed: o Chemoselectivity. Reactions must be selective for a certain functional group and inert to the vast compendium of chemical functionalities found in vivo,1 avoiding competing side reactions engaging endogenous biological reactants.4 o Biocompatibility. Living environments imply aqueous media and an elevated control of pH and temperature, to avoid performing toxic reactions that could interfere in the current function of the medium.5 5 o Reaction rate. It is important to optimize the kinetics of the reactions with the objective of reducing the concentrations used in vivo, considering that a great part of these reactions follow second-order rates, dependent on concentration of the two reactants.1 Analyzing these characteristics, such as selectivity and functional tolerance, it is noted that bioorthogonal chemistry is, although conceptually different, quite close to click chemistry in its attributes, being fairly common for a bioorthogonal reaction to meet the requirements of the latter.4 Studying its history, the beginnings of bioorthogonal chemistry can be linked to protein bioconjugation in the pursue of a selective method for monitoring biochemical processes within cells.1 In this research, the first approaches to bioorthogonal reactions were condensations between aldehydes/ketones and hydrazides, hydrazine or aminooxy compounds, where selectivity issues were notable. Thus, the first success in the search for bioorthogonality was achieved with azides, which demonstrated to be inert substrates under biological environments. They debuted in the Staudinger ligation,6 a transformation of azides into amides by reaction with arylphosphine derivatives (Scheme 1).4 Scheme 1. Staudinger ligation. From then on, an important set of bioorthogonal strategies where developed, classified in two families of processes: bioorthogonal bond-forming reactions and bioorthogonal bond-cleavage reactions. Scheme 2. Representative: (a) bioorthogonal bond-forming reaction, (b) bioorthogonal bond-cleavage reaction. Within the first type of transformations, graphically represented in Scheme 2a, it is worth highlighting the well-known copper-catalyzed alkyne azide cycloaddition (CuAAC),7,8 reported by Sharpless and Meldal (Scheme 3). 6 Scheme 3. Copper-catalyzed alkyne azide cycloaddition. Essentially, the reaction consists of a [3+2] addition between terminal alkynes and azides, to regioselectively afford 1,4-disubstituted-1,2,3-triazoles. It shows high selectivity, great kinetics and bioorthogonality, though it must be said that its use, mostly related to the modification of cell surface glycans and proteins,9 is limited by the toxicity of copper and ascorbate (used to obtain CuI from a CuII source), able to generate ROS (reactive oxygen species).10 However, many alternatives have been described to avoid these issues, both in the modification of azides11 and the ligands to protect the copper complex.12,13 In the following years, more biofriendly alternatives to the CuAAC reaction have been developed, such as the metal free cycloaddition with strained cycloalkynes (SPAAC),14 or the inverse electron-demand Diels-Alder15 (IED-DA).2,4 The expansion of this field has also allowed to develop new procedures, such as palladium-catalyzed cross-couplings and ruthenium-catalyzed cross-metathesis reactions, among other examples.2 The second group of bioorthogonal transformations involves bond-breaking reactions (Scheme 2b). Typical synthetic methods to cleave chemical bonds use harsh conditions based on acidicbasic, nucleophilic or redox reagents not always compatible with physiological conditions. An alternative approach takes advantage of the great reactivity of the organometallic chemistry. Towards this idea, many bond breaking bioorthogonal reactions promoted by transition metal catalysts have already been described and incorporated to the armory of bioorthogonal chemistry.2 Along the years, the field of biorthogonal organometallic chemistry has acquired a synthetic aim with numerous examples of intracellular bond-forming processes in live cells. 1.2 Organometallic catalysis in biological media Metabolism is sustained by myriads of enzymes that work to conduct properly each reaction that takes place within living systems. Many of these biocatalysts have metal ions embedded in their active sites, taking part as redox centers or Lewis acids, such as Fe, Mn, Zn or Mg.16 It is interesting to notice that natural metalloenzymes do not use second or third row transition metal catalysts, being limited their reactivity mainly to redox processes or acid-base transformations. Whereas the use of these metalloenzymes in synthetic chemistry together with the development of artificial ones has received a lot of attention, the exploration of artificial transition metal catalysis inside live cells have been unconsidered for a long time. 13 Scheme 12. Mechanism proposed by Hirano et al. for cascade cyclization with carbazole formation. It can be observed that this procedure is favored by the ortho substitution of the arylalkyne and the nucleophilic group, forcing proximity of both reactants.47 The mechanism is supported by checking the reaction of a plausible intermediate (similar to intermediate III lacking the gold atom), which when treated under the standard reaction conditions affords the same carbazole.57 14 2. OBJECTIVES AND WORK PLAN The present work, in the framework of Final Degree Project, aims to broaden the scope of intracellular bioorthogonal reactions utilizing gold catalysts. We proposed the development of new biocompatible gold-promoted cascade reactions in biological or even cellular environments. Specifically, we set out these goals: (a) Synthesis of bioactive skeletons such as carbazoles, by the intramolecular gold-promoted double cyclization of designed substrates through a cascade process. (b) Optimize the reaction towards biocompatible conditions. (c) Confirm the tolerance of the reaction to different biological media and biomolecules. (d) Translate the reaction into live mammalian cells. In the consecution of these objectives, the following plan was proposed: MONTH TASK November Bibliographical research and familiarization with the lab December Synthesis and characterization of reactant 1 January Synthesis and characterization of product 2 February Screening of gold catalysts March Optimization of in vitro conditions April Refinement of the final conditions May Biological media screening and biocompatibility study with biomolecules Memory writing June Experiments in live mammalian cells Memory writing 15 3. RESULTS AND DISCUSSION Introducing a new chemical transformation into a living system requires to follow certain steps to ensure the viability of the process, that include the study of the reaction and the optimization of the conditions to guarantee its compatibility to aqueous and complex biological media. Finally, the viability of the reaction in living cells can be explored. The present work was developed under the direct supervision of last year PhD student Cinzia D’Avino. 3.1 Preliminary assays for a gold-cascade synthesis of carbazoles Aiming to study new gold-promoted bioorthogonal cascade reactions, we selected the one described in organic solvents by Hirano et al (Scheme 11).48 The substrate 1 was prepared through a two-step synthesis, starting from commercial reagents. A Sonogashira coupling between o-diiodobenzene and phenylacetylene using bis(triphenylphospine)palladium(II) and copper(I) iodide, in butylamine, provided 1-iodo-2- (phenylethynyl)benzene in 39% yield. This compound was subjected to a second Sonogashira coupling with 2-ethynylaniline, this time in THF and Et3N, obtaining 2-((2phenylethynyl)phenyl)ethynyl)aniline (1) in 75% yield. The overall yield of this synthesis was 29%. Scheme 13. Synthetic route of 2-((2-phenylethynyl)phenyl)ethynyl)aniline 1. Once obtained compound 1, the gold-promoted cyclization was attempted following Hirano’s organic reaction conditions. Thus, this aniline derivative was treated with chloro(triphenylphosphine)gold(I), using AgOTf as chloride scavenger in MeCN and stirred for 4.5 hours. The product 2 was formed in 50% yield, although in a lower efficiency to the reported by Hirano (81%, in only 1.5 hours). 16 Scheme 14. Synthesis of 6-phenyl-11H-benzo[a]carbazole 2 under Hirano’s conditions. To translate organic reactions to biological media it is mandatory to perform the transformations at physiological temperature, that is, 37 ºC. Thus, we explored the reaction at a lower temperature. When the reaction was carried out at room temperature (25 ºC) and time was rise to 7 hours, a 69% yield was obtained, similar to the 70% reported in bibliography. In this case, the yield of 2 was determined by 1H-NMR using 1,3,5-trimethoxybenzene (TMB) as internal standard. Importantly, it was also confirmed that the reaction is unviable without the catalyst. Scheme 15. Cyclization performed at r.t. with and without catalyst. [Au] = Ph3PAuCl. 3.2 In vitro optimization 3.2.1 Screening of other reactions conditions The first study undertaken was the selection of the gold catalyst using different gold complexes. 17 0% 96% 0% 99% AuCl AuCl3 85% 75% 23% 55% HAuCl4·3H2O w/o 66% 0% Scheme 16. Catalyst screening for the synthesis of carbazole 2.a aConditions: 200 mM of 1 (0.12 mmol), 20 mol% Au catalyst, in MeCN (0.59 mL), overnight, under N2 atmosphere at room temperature. bYields were determined by 1H-NMR using TMB as internal standard. Important information can be extracted from this study. First, the reaction with PPh3AuCl without silver salt led to the recovery of compound 1, confirming the need of the chloride scavenger. This was also observed with iPrAuNTf2 and iPrAuCl (96% and 0%, respectively). Moreover, gold(I) complexes showed a better performance compared to the gold(III) salts. In this screening, two catalysts, JohnPhos and iPrAuNTf2, show excellent yields, above 90%. Since the first complex presents low cell-uptake and high cytotoxicity (as previously found by the group), the studies continued with iPrAuNTf2. Using this complex, it was found that high yields can be obtained only after 10 min of reaction (Table 1, entry 3). 18 Table 1. Time screening for the synthesis of carbazole 2.a Entry Time Yield (%)b 1 7 hours 97 2 1 hour 98 3 10 min 99 aConditions: 200 mM of 1 (0.12 mmol), 20 mol% iPrAuTf2, in MeCN (0.59 mL), under N2 atmosphere at room temperature. bYields were determined by 1H-NMR using TMB as internal standard. In the next set of experiments, the performance of the reaction in different solvents together with the water compatibility was studied. The results, shown in Table 2, demonstrated that an organic media was not necessary, obtaining comparable yields when an aqueous milieu was used (entry 3). In this case, the reactions were stirred for 1 h to ensure the total consumption of substrate under the new reaction media. However, it should be pointed out the insolubility of the substrate and product in H2O, using the term on water to refer to the heterogeneous character of the reaction. Table 2. Solvent screening for the synthesis of carbazole 2.a Entry Solvent Yield (%)b 1 MeCN 98 2 DMSO 100 3 H2O 93 aConditions: 200 mM 1 (0.12 mmol), 20 mol% iPrAuTf2, in different solvents (0.59 mL), one hour and under N2 atmosphere at r.t. bYields were determined by 1H-NMR using TMB as internal standard. Once the water compatibility was confirmed, the reaction was tested in an atmosphere open to air, essential to the development of future in cellulo experiments. Simultaneously, time was overlooked again, due to the latter changes. The results are summarized in Table 3. 19 Table 3. Time and atmosphere screening for the synthesis of carbazole 2.a Entry Time Yield (%)b 1 10 min 96 2 30 min 94 3 1 hour 89 4 24 hours 89 aConditions: 200 mM 1 (0.12 mmol), 20 mol% iPrAuTf2, in H2O (0.59 mL) under open atmosphere, r.t. bYields were determined by 1H-NMR using TMB as internal standard. These results again revealed the fast kinetics of the reaction, as well as the tolerance to oxygen and humidity. Consequently, from now on, all the experiments are performed open to air, unless otherwise noted. The next question raised is whether the catalyst loading and starting material concentration could be reduced, since in cellulo experiments are highly sensitive to elevated amounts of reagents, producing stress, behavior alteration or even cytotoxicity. Firstly, the reaction performed under optimized conditions, that is, at 200 mM scale of substrate, in water, r.t., 10 min, but decreasing the gold loading to 10 mol%, led to 91% yield (Table 4, entry 1). Subsequently, the influence of the concentration of substrate was studied. As shown in the table, the molarity could be reduced up to 40 times, to 5 mM of starting material, keeping the yields above 80% (entries 3, 5, 7 and 9). The reaction also took place at 1 mM, with an acceptable 65% (entry 11). Importantly, no product was detected in the absence of the gold complex (entries 2, 4, 6, 8 and 10). Table 4. Screening of catalyst loading and concentration of 1 for the synthesis of carbazole 2.a 20 Entry [1] (mM) mmol SM Vol (mL) Catalyst Yield (%)b 1 200 0.12 0.59 Yes 91 2 200 0.12 0.59 - 0 3 100 0.034 0.34 Yes 88 4 100 0.034 0.34 - 0 5 50 0.034 0.68 Yes 83 6 50 0.034 0.68 - 0 7 10 0.034 3.4 Yes 87 8 10 0.034 3.4 - 0 9 5 0.034 6.8 Yes 87 10 5 0.034 6.8 - 0 11 1 0.034 34.1 Yes 65 12 1 0.034 34.1 - 0 aConditions: Different concentration of 1 (0.12 mmol at 200 mM and 0.034 mmol in the following), 10 mol% iPrAuTf2, in H2O (0.59 mL in 200 mM to 34.1 mL in 1 mM) under open atmosphere, r.t. bYields were determined by 1H-NMR using TMB as internal standard. During this experimentation, it became evident the necessity of reducing the reaction volume to be able to finally transfer the reaction into living systems. For this, the operational protocol was changed, requiring the preparation of stock solutions from both the substrate and the catalyst. To achieve it considering the lack of solubility of the reactants in water, it was proposed the addition of DMSO. Thus, stock solutions of compound 1 and the gold catalyst in DMSO were prepared, and then added to the water used as reaction media, in a Schlenk tube, in a proportion 9 to 1 (H2O/DMSO). Again, a time screening was developed in the aim of a wider understanding of the transformation taking place. As indicated in Table 5, the yields after 10 min were remarkable. Table 5. Time screening for the synthesis of carbazole 2 at 1 mM scale.a 21 Entry Time Yield (%)b 1 10 min 74 2 30 min 71 3 1 hour 77 aConditions: 1 mM of 1 (0.0058 mmol), 10 mol% iPrAuTf2, in 9/1 H2O/DMSO (5.8 mL) under open atmosphere, r.t. bYields were determined by 1H-NMR using TMB as internal standard. Bearing in mind all this, it was determined that the final optimized conditions in aqueous media are those indicated in Scheme 17. Scheme 17. Final optimized conditions for the synthesis of carbazole 2 in aqueous media. 3.2.2 Reaction in biorelevant media When a bioorthogonal research is being conducted, the analysis of the reaction within biological milieu is an interesting in vitro study, since the increasing complexity of the media can enlighten about the viability of the process. The following mediums were tested: • PBS (phosphate buffered saline solution, pH 7.4). Solution of inorganic salts (NaCl, KCl, Na2HPO4 and KH2PO4). It is commonly used for washes, dilutions and to maintain the integrity of tissues and cells in biological and biochemical investigations. • DMEM (Dulbecco’s Modified Eagle Medium). Cell culture medium designed for eukaryotic cells’ growth, containing nutrients and different growth factors. In its composition there are diverse amino acids, vitamins, inorganic salts and other components like phenol red, sodium pyruvate and dextrose. • Cellular lysates. Extraction of cellular content resuspended in PBS. Concentration given in mg protein/mL PBS. 22 Firstly, the reaction was performed in the presence of PBS, observing the formation of the product in an excellent yield (99%). In DMEM the reaction was also effective, although the yield decreased to 69%. Remarkable, the reaction is also possible in presence of cellular lysates, leading to a 59% yield of the product. [Au] w/o [Au] w/o [Au] w/o PBS DMEM LYSATES 0 50 100 Yield (%) Scheme 18. Bar graphic showing the results of the reaction in different biological media, with and without [Au]. Conditions: 1 mM of 1 (0.0034 mmol), 10 mol% iPrAuTf2, in 9/1 media/DMSO (3.4 mL) under open atmosphere, r.t. Yields were determined by 1H-NMR using TMB as internal standard. PBS 1x: 136.9 mM of NaCl, 2.68 mM of KCl, 10.14 mM of Na2HPO4 and 1.76 mM of KH2PO4. Lysates: 2.05 mg protein/mL of sample. These results allow to conclude that the reaction is highly orthogonal, tolerating the presence of different type of salts and molecules. Considering the outcome of the experimentation, the next studies were carried out in PBS, where the effect of the studied biomolecules can be easily spotted. 3.2.3 Compatibility with specific biomolecules As previously stated, the complex network of biomolecules that exist within cells is an aspect to take into consideration when an abiotic reaction is intended to be introduced in cellular environments. Therefore, the performance of the reaction in the presence of some representative biomolecules was next investigated. 29 They were followed by a gradual change of 5 min to H2O/MeCN (5:95). The latest conditions were kept 1 min. Then, settings were returned to the initial ones (45:55) for 2 min. The chromatogram was recorded using a UV detection at λ = 300 nm. 3.5.2 Synthesis of 1-iodo-2-(phenylethynyl)benzene Procedure adapted from Hirano et al.48 Bis(triphenylphosphine)palladium(II) dichloride (182.5 mg, 0.26 mmol, 2.5 mol%) and copper(I) iodide (49.3 mg, 0.26 mmol) were dissolved in butylamine (20 mL) in a purged round flask under nitrogen atmosphere. Phenylacetylene (1.2 mL, 11.2 mmol, 1.06 eq) and 1,2diiodobenzene (1.4 mL, 10.6 mmol, 1 eq) were added to the stirred solution, again under N2 flow. The mixture was stirred for 24 hours at 60 ºC under argon, showing the TLC (silica gel, nHexane/EtOAc 20:1) complete disappearance of the starting material. The mixture was cooled to room temperature, quenched by addition of saturated aqueous NH4Cl and washed three times with brine. The organic layer was dried over Na2SO4, filtrated and concentrated under vacuum. The residue was purified by column chromatography on silica gel with n-hexane/ethyl acetate 20:1 to afford 1-iodo-2-(phenylethynyl)benzene (1.15 mg, 36%) as a pale yellow oil. 1H-NMR (300 MHz, d-chloroform): δ 7.88 (d, 3J = 8.0 Hz, 1H), 7.63-7.58 (m, 2H), 7.53 (d, 3J = 7.7 Hz, 1H), 7.40-7.29 (m, 4H), 7.01 (t, 3J = 7.7 Hz, 1H); 13C-NMR (75 MHz, d-chloroform): δ 138.9 (CHAr), 132.6 (CHAr), 131.8 (2xCHAr), 130.0 (CAr), 129.5 (CHAr), 128.8 (CHAr), 128.5 (2xCHAr), 128.0 (CHAr), 123.1 (CAr), 101.3 (CAr), 93.2 (Calk), 91.8 (Calk). Rf: 0.47 (hexane/ethyl acetate 20:1). Data in accordance with the literature.58 3.5.3 Synthesis of 2-{[2-(phenylethynyl)phenyl]ethynyl}aniline 1 Procedure extracted from Hirano et al.48 30 A mixture of 1-iodo-2-(phenylethynyl)benzene (1.15 mg, 3.77 mmol, 1.0 eq), copper(I) iodide (35.7 mg, 0.050 eq) and bis(triphenylphosphine)palladium(II) dichloride (131.6 mg, 5 mol%) were purged in a round flask and subjected to nitrogen atmosphere. Once they were dissolved with Et3N (10.2 mL), a solution of ethynylaniline (470.0 mg, 4.012 mmol) in THF (3.9 mL) was added to the mixture, that was stirred at 80 ºC for 5.0 h under argon with reflux. The mixture was cooled to room temperature, quenched by addition of saturated aqueous NH4Cl and extracted three times with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtrated and concentrated under vacuum. The product 2-((2- (phenylethynyl)phenyl)ethynyl)aniline (1) was purified by column chromatography on silica gel with hexane/EtOAc 4:1 as a pale yellow solid (843.5 mg, 76%). 1H-NMR (300 MHz, d-chloroform): δ 7.63-7.53 (m, 4H), 7.42-7.30 (m, 6H), 7.14 (t, 3J = 7.7 Hz, 1H), 6.70 (t, 3J =8.9 Hz, 2H), 4.43 (s, 2H); 13C-NMR (75 MHz, d-chloroform): δ 148.4 (CAr), 132.3 (CHAr), 132.1 (CHAr), 132.0 (2xCHAr), 131.6 (CHAr), 130.1 (CHAr), 128.7 (CHAr), 128.5 (2xCHAr), 128.3 (CHAr), 127.9 (CHAr), 126.0 (CAr), 125.2 (CAr), 123.2 (CAr), 117.8 (CHAr), 114.2 (CHAr), 107.8 (CAr), 93.9 (Calk), 93.2 (Calk), 90.6 (Calk), 89.0 (Calk). Rf: 0.36 (hexane/AcOEt 8:2); 0.37 (hexane/toluene 2:1). C22H15N [M+]: 294.23. tr: 10.5 min. Data in accordance with the literature.48 3.5.4 Synthesis of 6-phenyl-11H-benzo[a]carbazole 2 Procedure adapted from Hirano et al.48 A mixture of chloro(triphenylphosphine)gold(I) (2.9 mg, 0.0059 mmol, 5 mol%) and silver trifluoromethanesulfonate (1.5 mg, 0.0059 mmol, 0.05 eq) were dissolved in acetonitrile (0.59 mL) in a purged Schlenk tube under argon atmosphere. Once 2-((2- (phenylethynyl)ethynyl)aniline (35.2 mg, 0.12 mmol, 1.0 eq) was added, the mixture was stirred at 80 ºC until TLC (silica gel, n-hexane/EtAOc 9:3) showed complete conversion of the starting material (4.5 h). After cooling the Schlenk to room temperature, the mixture was diluted with ethyl acetate, washed three times with saturated aqueous NH4Cl and brine, dried over Na2SO4, filtered and concentrated under vacuum. 31 The product 2 was purified by column chromatography (silica gel) with n-hexane/toluene (2:1), obtaining 6-phenyl-11H-benzo[a]carbazole as pale orange solid (17.6 mg, 50%*). 1-H-NMR (300 MHz, d-chloroform): δ 8.91 (s, 1H), 8.17 (d, 3J= 7.8 Hz, 1H), 8.01 (d, 3J= 7.9 Hz), 1H), 7.70 (d, 3J = 7.4 Hz, 2H), 7.64-7.51 (m, 7H), 7.45 (d, 3J = 8.1 Hz, 1H), 7.37 (t, 3J = 7.6 Hz, 1H), 7.05 (t, 3J = 7.6 Hz, 1H). 13C-NMR (75 MHz, d-chloroform): δ 141.4 (CAr), 138.9 (CAr), 136.8 (CAr), 135.4 (CAr), 132.3 (CAr), 129.5 (2xCHAr), 129.1 (CHAr), 128.5 (2xCHAr), 127.7 (CHAr), 125.8 (CHAr), 125.6 (CHAr), 124.8 (CHAr), 124.1 (CAr), 122.3 (CHAr), 121.1 (CHAr), 120.5 (CHAr), 120.4 (CAr), 119.8 (CHAr), 117.0 (CAr), 111.0 (CHAr). Rf: 0.42 (hexane/ethyl acetate 9:3); 0.29 (hexane/toluene 2:1). C22H15N [M+]: 294.24. tr: 9.9 min. Data in accordance with the literature.59 * Yield slightly overrated due to remaining toluene solvent. The further purification for the isolation of the product was performed via HPLC-preparative (60-95% of MeCN in water for 32 min). 3.5.5 Representative procedure for reactions at 200 mM scale using iPrAuNTf2 as complex and MeCN as solvent (section 3.2.1) Aniline 1 (35.2 mg, 0.12 mmol, 1.0 eq) and iPrAuNTf2 (20.8 mg, 0.024 mmol, 20 mol%) were added to a Schlenk tube and subjected to a purge by three vacuum-nitrogen cycles.* Then, MeCN (0.59 mL) was added to the mixture, which was magnetically stirred at room temperature overnight. Over time, the solution turned yellow. After approximately 16 hours, the mixture was diluted with ethyl acetate, washed with saturated aqueous NH4Cl and brine, dried over Na2SO4, filtered and concentrated under vacuum. A yield of 96% was determined by 1H-NMR with TMB as internal standard. The starting material conversation was complete. * When the reactions are performed open to air, both compound 1 and catalyst are added to a Schlenk tube in open atmosphere. 32 3.5.6 Representative procedure for the study of the dilution (section 3.2.1)* Aniline 1 (10.0 mg, 0.034 mmol, 1.0 eq) and iPrAuNTf2 (3.0 mg, 0.0035 mmol, 10 mol%) were stirred in a round flask with water (0.34 mL) at room temperature under open atmosphere for 10 min. The mixture, a cloudy suspension that turns yellow over time, was diluted with ethyl acetate, washed with saturated aqueous NH4Cl and brine, dried over Na2SO4, filtered and concentrated under vacuum. A yield of 88% was determined by 1H-NMR with TMB as internal standard. The starting material conversation was complete. * Alterations: concentration of 1 was gradually reduced and, therefore, volume of water was increased. 3.5.7 Representative procedure for reactions at 1 mM scale using stock solutions in DMSO* (section 3.2.1) A stock solution of iPrAuNTf2 (2.0 mM) in DMSO and another one of SM 1 (20 mM), again in DMSO, were previously prepared to add to water (5.2 mL) in a Schlenk tube. Both solutions were pipetted with a volume of 290 μL, first the substrate followed by the catalyst. Due to the insolubility of the starting material, a suspension was formed. It was stirred at room temperature for 10 minutes. The mixture was extracted with ethyl acetate three times, dried over Na2SO4 and then filtered with Florisil® (60-100 mesh) to eliminate the gold catalyst. Then, the solution was concentrated under vacuum. A yield of 74% was determined by 1H-NMR with TMB as internal standard. The starting material conversation was complete. * Times were screened. For the study of the biocompatibility, a third solution of 1.0 eq of a biomolecule was prepared in PBS (0.1 mL) and added before compound 1 and catalyst to the reaction media (3.0 mL). 33 3.5.8 Internal standard method for yield quantification Nuclear magnetic resonance (NMR) spectroscopy allows elucidation of small and macro molecules, as well as quantification of signals thanks to the proportionality between the resonance line and the number of resonant nuclei.60 This enables the utilization of an internal standard to analyze reaction crudes, allowing the easy obtention of the transformation yield by comparing a well-known peak of the internal standard with one of the product whose integration have been previously identified. This way, an ideal internal standard would be one highly purified, stable, chemically inert and soluble in the NMR solvent used.61 In this research it was used 1,3,5-trimethoxybenzene (TMB), where its aromatic peak (δ= 6.09 ppm) can be used to determine the yield of the reaction. The comparation was made based on the mean of three signals of the product: two aromatic H from the carbazole moiety (δ = 8.17 ppm, d, 1H; δ = 8.01 ppm, d, 1H) and the H from the NH group (δ = 8.91 ppm, s, 1H). The known amount of TMB added was calculated in base of a 1:1 relation between the investigated signals, assuming a100% yield. To simplify the calculations, a factor of 1/3 was employed. This adjustment accounts for the fact that the utilized peak of TMB integrates for three protons (3H), while the corresponding peaks from the product integrate for one proton (1H). Thus, for the experiments performed in Table 5 (Entry 1): 𝑀𝑎𝑠𝑠𝑇𝑀𝐵 = 0.006 𝑚𝑚𝑜𝑙 𝑝𝑟𝑜𝑑𝑢𝑐𝑡 · 1 𝑚𝑚𝑜𝑙 𝑇𝑀𝐵 1 𝑚𝑚𝑜𝑙 𝑝𝑟𝑜𝑑𝑢𝑐𝑡 · 1 𝐻 3 𝐻 · 168.19 𝑚𝑔 𝑇𝑀𝐵 1 𝑚𝑚𝑜𝑙 𝑇𝑀𝐵 = 0.34 𝑚𝑔 𝑇𝑀𝐵 Scheme 25. Example of yield determination by internal standard of 1,3,5-trimethoxybenzene. Spectrum corresponding to procedure described in Table 5. The determined amount of internal standard would be added to the crude with the previous preparation of stock solutions. 34 3.5.9 General information for in cellulo experiments All steps were performed on a sterile clean bench Tesltar AV-100 at room temperature. Solutions stored in a fridge were pre-warmed in a water bath (37ºC) before use. A549 cell cultures were incubated in DMEM (Dulbecco’s modified Eagle’s medium) acquired from Sigma-Aldrich, which was enriched with penicillin (100 units/mL), streptomycin (100 units/mL) and glutamine 5 mM, all from Invitrogen. Cells were seeded in well plates at the specified concentration two days prior to treatment. Proliferating cultures were maintained in an incubator at 37 ºC, 5% CO2 and 95% humidity. 3.5.10 Representative procedure for in cellulo reactions To perform the different experiments, 1.5 million cells per plate (100 nm) were seeded two days before treatment. In the beginning of the experimentation, the growing media was retired prior to the addition of the solutions previously prepared in DMEM. Treatment of the cells was initiated with the first one containing the aniline 1 (100 or 50 μM, final volume of 3 mL), for 30 minutes, using freshly prepared stock solution (10 mM in DMSO). Once the time is over, the media was aspired and cells were washed twice with PBS (3 mL). Then, the second DMEM solution was added, containing iPrAuNTf2 (50 and 25 μM, final volume of 3 mL), from again freshly prepared stock solution (10 mM in DMSO). After 3 hours, the media was collected and kept for posterior analysis, as well as the 2 new washes with PBS (3 mL). All these fractions were lyophilized and filtered by HPLC filter before being subjected to HPLC-MS. Finally, cells were extracted with MeCN (1 mL) three times. The portions were concentrated under vacuum, agitated by vortex and subjected to sonication for 5 min. Then, they were filtered with HPLC filters and analyzed by HPLC-MS to determine the product 2 formation. 35 CONCLUSIONS In the present Final Degree Project, a new bioorthogonal reaction has been described based on the intramolecular double cycling promoted by gold of an aniline by means of a cascade mechanism. Its original parameters, based on organic synthesis, have been optimized, modifying catalysts and their loads, times, concentrations, solvents and atmosphere, finally determining that the reaction can be carried out in mild conditions. The perfected parameters allow the chemical transformation to be carried out in 9/1 H2O/DMSO, in 10 minutes, with a load of 10 mol% of iPrAuNTf2 as a catalyst, with a substrate concentration of 1 mM, at room temperature and in an open atmosphere. It has also been shown that the reaction is perfectly compatible with more complex media, obtaining good results in both PBS, DMEM and cell lysates. At the same time, the formation of carbazole from aniline has been found to be perfectly tolerant to the presence of biomolecules such as NADH, GSH, L-Cysteine or sodium ascorbate, which act with very little inhibition in in vitro tests. Finally, preliminary studies have been developed on the adaptation of the reaction to the interior of cellular media, using the A549 line for these in cellulo analyses, demonstrating its correct functioning and bioorthogonality. These results could open the way to the development of bioorthogonal reactions for the formation of structures of great molecular complexity through cascade mechanisms, acting in a simple and sustainable way. This could lead to the in situ formation of cyclic and heterocyclic structures of interest to Medicinal Chemistry, highlighting for example the antimicrobial and antitumor action of annulated derivatives of carbazoles. In this way, it is interesting to contemplate the future optimization of preliminary assays in cells, improving the conditions and carrying out quantitative studies that allow a better understanding of the reaction, as well as carrying it out in different cell lines to observe its effects. On the other hand, the future modification of the final structure of the cyclization is proposed with the aim of making it fluorescent, facilitating its in vivo study through fluorescence microscopy. CONCLUSIONES En el presente Trabajo de Fin de Grado, se ha descrito una nueva reacción bioortogonal basada en la doble ciclación intramolecular promovida por oro de una anilina mediante un mecanismo en cascada. Se han optimizado sus parámetros originales, fundamentados en la síntesis orgánica, modificando catalizadores y sus cargas, tiempos, concentraciones, disolventes y atmósfera, determinando finalmente que la reacción se puede realizar en condiciones suaves. 36 Los parámetros perfeccionados permiten llevar a cabo la transformación química en 9/1 H2O/DMSO, en 10 minutos, con una carga de 10 mol% de iPrAuNTf2 como catalizador, con una concentración de sustrato de 1 mM, a temperatura ambiente y en atmósfera abierta. También se ha demostrado que la reacción es perfectamente compatible con medios más complejos, obteniendo buenos resultados tanto en PBS, DMEM como en lisados celulares. Al mismo tiempo, la formación del carbazol a partir de la anilina ha resultado ser perfectamente tolerante a la presencia de biomoléculas como NADH, GSH, L-Cisteína o ascorbato sódico, las cuales actúan con una muy pequeña inhibición en ensayos in vitro. Por último, se han desarrollado estudios preliminares sobre la adaptación de la reacción al interior de medios celulares, utilizando la línea A549 para estos análisis in cellulo, demostrando su correcto funcionamiento y bioortogonalidad. Estos resultados podrían abrir el paso al desarrollo de reacciones bioortogonales de formación de estructuras de gran complejidad molecular a través de mecanismos en cascada, actuando de manera sencilla y sostenible. Esto podría dar lugar a la formación in situ de estructuras cíclicas y heterocíclicas de interés para la Química Médica, destacando por ejemplo la acción antimicrobiana y antitumoral de los derivados anillados de los carbazoles. De esta manera, es interesante contemplar la optimización futura de los ensayos preliminares en células, mejorando las condiciones y realizando estudios cuantitativos que permitan comprender mejor la reacción, así como llevarla a cabo en diferentes líneas celulares para observar sus efectos. Por otro lado, se propone la futura modificación de la estructura final de la ciclación con el objetivo de hacerla fluorescente, facilitando su estudio in vivo a través de microscopía de fluorescencia. CONCLUSIÓNS No presente Traballo de Fin de Grado, describiuse unha nova reacción bioortogonal baseada na dobre ciclación intramolecular promovida por ouro dunha anilina mediante un mecanismo en cascada. Optimizáronse os seus parámetros orixinais, fundamentados na síntese orgánica, modificando catalizadores e as súas cargas, tempos, concentración, disolventes e atmosfera, determinando finalmente que a reacción pódese realizar en condicións suaves. Os parámetros perfeccionados permiten levar a cabo a transformación química en 9/1 H2O/DMSO, en 10 minutos, cunha carga de 10 mol% de iPrAuNTF2 como catalizador, cunha concentración de substrato de 1 mM, a temperatura ambiente e atmosfera aberta. 37 Tamén demostrouse que a reacción é perfectamente compatible con medios máis complexos, obtendo bos resultados tanto en PBS, DMEM como en lisados celulares. Ao mesmo tempo, a formación do carbazol a partir da anilina resultou ser perfectamente tolerante á presencia de biomoléculas como NADH, GSH, L-Cisteína ou ascorbato sódico, os cales actúan cunha moi pequena inhibición en ensaios in vitro. Por último, desenvolvéronse estudos preliminares sobre a adaptación da reacción ao interior de medios celulares, utilizando a liña A549 para estes análises in cellulo, demostrando o seu correcto funcionamento e bioortogonalidade. Estes resultados poderían abrir o paso ao desenvolvemento de reaccións bioortogonais de formación de estruturas de gran complexidade molecular a través de mecanismos en cascada, actuando de maneira sinxela e sostible. Isto podería dar lugar á formación in situ de estruturas cíclicas e heterocíclicas de interese para a Química Médica, destacando por exemplo a acción antimicrobiana e antitumoral dos derivados anulados dos carbazoles. Desta maneira, é interesante contemplar a optimización futura dos ensaios preliminares en células, mellorando as condicións e realizando estudos cuantitativos que permitan comprender mellor a reacción, así como levala a cabo en diferentes liñas celulares para observar os seus efectos. Por outro lado, proponse a futura modificación da estrutura final da ciclación co obxectivo de facela fluorescente, facilitando o seu estudo in vitro a través de microscopio. 38 REFERENCES 1. Sletten, E. M.; Bertozzi, C. R. 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