Abiotic reactions promoted by the cell machinery PART A

GRAO EN QUÍMICA UNIVERSIDADE DE SANTIAGO DE COMPOSTELA Facultade de Química TRABALLO FIN DE GRAO Curso 2022/23 Alumno/a: <Título> Abiotic reactions promoted by the cell machinery PART A David Montoto Pintos 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 do alumno David Montoto Pintos na convocatoria de xullo do curso 2022-2023, o cal foi realizado baixo a súa dirección no Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares da Universidade de Santiago de Compostela (CiQUS). E para que así conste asinamos o presente informe en Santiago de Compostela o 3 de xullo de 2023. TOMAS GAMASA MARIA - 09417809E Firmado digitalmente por TOMAS GAMASA MARIA - 09417809E Fecha: 2023.07.06 15:29:30 +02'00' MASCAREÑ AS CID JOSE LUIS - 34934771W Firmado digitalmente por MASCAREÑAS CID JOSE LUIS - 34934771W Fecha: 2023.07.06 16:19:06 +02'00' David Montoto Pintos - Degree in Chemistry Final Project Page 3 of 49 Abbreviations ADP adenosine diphosphate ATRP atom transfer radical polymerization BNAH N-benzyl-1,4- dihydronicotinamide CuAAC copper-promoted azide-alkyne cycloaddition d doublet DMEM Dulbecco’s modified Eagle’s medium DMSO dimethyl sulfoxide E. coli Escherichia coli e.g. exempli gratia EET extracellular electron transfer ETC electron transport chain eq. equivalents EWG electron withdrawing group EY eosin Y FBS fetal bovine serum g relative centrifugal force GSH L-glutathione GSSG glutathione disulfide 1H-NMR proton NMR HS heat shock IED-DA inverse electron-demand Diels-Alder reaction IR infrared L. lactis Lactococcus lactis LB Lysogeny broth m multiplet MW molecular weight NaAsc sodium ascorbate NAD+ nicotinamide adenine dinucleotide (oxidized form) NADH nicotinamide adenine dinucleotide (reduced form) NAD(P)H nicotinamide adenine dinucleotide or dinucleotide phosphate (reduced form) NMR nuclear magnetic resonance PBS phosphate-buffered saline ppm parts per million RAFT reversible additionfragmentation chain transfer ROS reactive oxygen species r.t. room temperature (approximately 21 ºC) Ru(bpy)3 tris(bipyridine)ruthenium(II) hexafluorophosphate ! density s singulet S. cerevisiae Saccharomyces cerevisiae SET single electron transfer SPAAC strain-promoted azide-alkyne cycloaddition TEMPO (2,2,6,6-tetramethylpiperidin- 1-yl)oxyl THF tetrahydrofuran UV ultraviolet v/v volume/volume Abiotic reactions promoted by the cell machinery Page 4 of 49 Table of contents Abstract ............................................................................................................................. 6 1. Introduction ................................................................................................................... 8 1.1. Bioorthogonal chemistry ....................................................................................... 8 1.2. Biocompatible chemistry ....................................................................................... 9 1.2.1. Definition and applications of biocompatible chemistry .............................. 9 1.2.2. Reactions promoted by cellular redox processes ........................................ 10 1.2.3. Reactions promoted by abiotic catalysts .................................................... 12 1.2.4. Future projection of abiotic biocompatible chemistry ................................ 12 1.3. Chemistry of arenediazonium salts ..................................................................... 13 1.3.1. General reactivity of arenediazonium salts ................................................ 13 1.3.2. The Meerwein arylation ............................................................................. 14 2. Objectives and work plan ............................................................................................ 14 2.1. Objectives ............................................................................................................ 14 2.2. Work plan ............................................................................................................ 15 3. Results ......................................................................................................................... 15 3.1. (Non-)Photocatalyzed reactions in cells: a frustrating finding. ........................... 15 3.2. Evaluation of the photocatalyzed reaction .......................................................... 16 3.3. In vitro experiments ............................................................................................. 18 3.3.1. Screening of different biological reductants ............................................... 18 3.3.2. Optimization of conditions and study of the NADH-promoted reaction ... 20 3.3.3. Evaluation of alternative acceptors ............................................................ 25 3.3.4. Reactions with alkynes ............................................................................... 25 3.4. Experiments in cells ............................................................................................ 26 3.4.1. ATP viability assay ..................................................................................... 26 3.4.2. Evaluation of the reaction in HeLa cells .................................................... 27 3.4.3. Cell fractionation experiments ................................................................... 28 3.4.4. Experiments in E. coli ................................................................................ 30 3.5. Experimental procedures ..................................................................................... 31 3.5.1. General information for in vitro experiments ............................................. 31 3.5.2. Synthesis of p-methoxyphenyldiazonium tetrafluoroborate (1) ................. 32 3.5.3. Synthesis of 2-(4-methoxyphenyl)naphthalene-1,4-dione (2) .................... 32 3.5.4. Synthesis of 2,3-bis(4-methoxyphenyl)naphthalene-1,4-dione (3) ............ 33 David Montoto Pintos - Degree in Chemistry Final Project Page 5 of 49 3.5.5. Representative procedure for irradiation experiments in subsection 3.2 ... 33 3.5.6. Representative procedure for reactions in subsection 3.3 .......................... 34 3.5.7. Representative procedure for reactions in subsection 3.3.4 ....................... 34 3.5.8. Internal standard method for yield quantification ...................................... 34 3.5.9. General information for cell culture experiments ...................................... 36 3.5.10. Luminescence viability assay ..................................................................... 36 3.5.11. Experiment in HeLa cells ........................................................................... 36 3.5.12. Cell fragmentation assay ............................................................................ 37 3.5.13. Experiments in E. coli ................................................................................ 37 3.5.14. Quantification by HPLC-MS ...................................................................... 38 4. Conclusions ................................................................................................................. 38 References ....................................................................................................................... 40 Annex: NMR spectra ....................................................................................................... 49 Abiotic reactions promoted by the cell machinery Page 6 of 49 Abstract The development of new-to-nature, non-enzymatic biocompatible reactions that interact with the metabolism of living systems represents an emerging field at the interface of chemical synthesis and synthetic biology. These reactions could be used to access novel synthetic routes, and they have potential applications in biomedicine and biology. In this Degree Final Project, we report the discovery of a Meerwein arylation reaction between diazonium salts and naphthoquinone derivatives promoted by living cells. The process has been carried out in mammalian cells (HeLa and A549) and E. coli, and it has been found to proceed in isolated mitochondria and mitochondria lysates. Low-weight biological reductants such as NADH, glutathione and ascorbate promote the reaction in abiotic media. Finally, preliminary studies to expand the scope of this transformation to other diazonium salts or other radical acceptor partners have been undertaken, opening the door to interesting structures like benzothiophenes and phenanthrenes. Resumen El desarrollo de reacciones biocompatibles no enzimáticas nuevas en la naturaleza que interactúan con el metabolismo de los sistemas vivos representa un campo emergente en la interfase entre la síntesis química y la biología sintética. Estas reacciones podrían utilizarse para acceder a nuevas rutas sintéticas, y tienen aplicaciones potenciales en biomedicina y biología. En este Trabajo de Fin de Grado, se describe el descubrimiento de una reacción de arilación de Meerwein entre sales de diazonio y derivados de la naftoquinona inducida por células vivas. El proceso se ha llevado a cabo en células de mamífero (HeLa y A549) y E. coli, y se ha observado que tiene lugar en mitocondrias aisladas y lisados de mitocondrias. Los reductores biológicos de bajo peso molecular como el NADH, el glutatión y el ascorbato favorecen la reacción en medios abióticos. Finalmente, se han llevado a cabo estudios preliminares para ampliar el alcance de esta transformación a otras sales de diazonio u otros sustratos aceptores de radicales, abriendo la puerta a estructuras interesantes como los benzotiofenos y los fenantrenos. Resumo O desenvolvemento de reaccións biocompatibles non enzimáticas novas na natureza que interactúan co metabolismo dos sistemas vivos representa un campo David Montoto Pintos - Degree in Chemistry Final Project Page 7 of 49 emerxente na interfase entre a síntese química e a bioloxía sintética. Estas reaccións poderían utilizarse para acceder a novas vías sintéticas, e teñen aplicacións potenciais en biomedicina e bioloxía. Neste Traballo de Fin de Grao, descríbese o descubrimento dunha reacción de arilación de Meerwein entre sales de diazonio e derivados da naftoquinona inducida por células vivas. O proceso levouse a cabo en células de mamífero (HeLa e A549) e E. coli, e observouse que ten lugar en mitocondrias illadas e lisados de mitocondrias. Os redutores biolóxicos de baixo peso molecular coma o NADH, o glutatión e o ascorbato favorecen a reacción en medios abióticos. Finalmente, realizáronse estudos preliminares para ampliar o alcance desta transformación a outras sales de diazonio ou outros substratos aceptores de radicais, abrindo a porta a estruturas interesantes coma os benzotiofenos e os fenantrenos. Abiotic reactions promoted by the cell machinery Page 8 of 49 1. Introduction 1.1. Bioorthogonal chemistry The concept of bioorthogonal chemistry was introduced at the beginning of the 21st century by Carolyn R. Bertozzi, and refers to chemical reactions that do not affect the biochemical processes of living systems.1 This represents a great challenge, as biological systems are extremely complex: they are composed of myriads of small biomolecules with nucleophilic, electrophilic, and redox properties, as well as inorganic ions at different concentrations, and catalytically-active macromolecules –enzymes and enzymatic complexes, many of them with metal-based cofactors–. Biological milieus have also very specific pH and temperature conditions, and they are heavily compartmentalized. The first bioorthogonal reactions were initially developed as probing methods for monitoring biological processes,2 but other applications have emerged since then, such as intracellular drug activation for targeted therapy, and in situ generation of imaging probes for diagnosis.3 The introduction of the concept of bioorthogonality opened the door to a new field fresh for improvement, and its importance was most recently highlighted with the awarding of the 2022 Nobel Prize in Chemistry to Bertozzi –shared with Sharpless and Meldal–, with emphasis on the conceptual nature of her contributions.4 Bioorthogonal reactions are usually required to possess the following characteristics:5–7 – Chemoselectivity: the reactants do not present side reactions with components of the biological system –that means they are biologically inert– and react only with their corresponding reaction partners. – Biocompatibility: the reaction must take place at biological conditions of aqueous medium, pH and temperature. Also, the reactants and reagents should be non-toxic at the required concentrations and stable in biological media. – Fast kinetics: a high reaction rate helps avoiding undesirable side reactions, and it is also necessary for the reaction to successfully take place when the reactants are present at very low concentrations (typical conditions to prevent toxicity). The first bioorthogonal reaction to be reported was the Staudinger ligation between an azide and a triphenylphosphine.8 Subsequently, metal catalysts have been successfully employed both for bioorthogonal reactions that involve either bond formation (copper-catalyzed azide-alkyne cycloaddition (CuAAC),9 palladium-catalyzed cross-coupling reactions,10,11 ruthenium-catalyzed cross-metathesis reactions)12 or David Montoto Pintos - Degree in Chemistry Final Project Page 9 of 49 cleavage reactions (ruthenium-13,14 or palladium-catalyzed deallylation,15 palladium-catalyzed depropargylation).16 Other metal-free bioorthogonal reactions have also been developed, like strain-promoted cycloadditions between strained cycloalkynes and 1,3-dipoles (SPAAC),17 and the inverse electron-demand Diels-Alder (IED-DA) reaction.18 1.2. Biocompatible chemistry 1.2.1. Definition and applications of biocompatible chemistry Biocompatible chemistry refers to non-enzymatic chemical reactions that can occur under mild conditions and are compatible with living systems.19 Depending on the authors, the same reactions and experimental settings have been described either as bioorthogonal or biocompatible, but biocompatible reactions do not necessarily fit all the requirements of bioorthogonal chemistry. Abiotic biocompatible reactions that interface with the metabolism would offer new possibilities for interrogating or manipulating biological systems and contributing to synthetic biology. These reactions could have biomedical applications, such as in new therapies and diagnostic imaging, and provide valuable tools for basic research in biology. Abiotic biocompatible chemistry also offers the possibility of interfacing synthetic chemistry and synthetic biology, potentially increasing the number of reactions obtainable through biological systems with the far more ample repertoire of classical organic synthesis.20,21 A first report on the biocompatible manipulation of cellular metabolism by non-enzymatic chemistry was reported by Neuberg and Hirsch in 1919. They significantly increased the production of glycerol from glucose by fermentation in Saccharomyces cerevisiae in presence of sodium bisulfite (glycerol is a precursor of nitroglycerine, and Neuberg discoveries allegedly benefited the German war effort during the First World War). Bisulfite anion acts as a protecting group for acetaldehyde, blocking the last step of ethanol fermentation and preventing the reoxidation of NADH formed in glycolysis. This promotes the biosynthesis of glycerol via a branching pathway of glycolysis, with concurrent oxidation of NADH to NAD+ (Scheme 1).19 Abiotic reactions promoted by the cell machinery Page 16 of 49 Scheme 5. (a) Initial attempts to develop bioorthogonal photocatalysis in live cells. (b) This work: developing new biocompatible transformations promoted by the live cell machinery. 3.2. Evaluation of the photocatalyzed reaction The initial efforts were focused on investigating this Meerwein arylation between p-methoxyaryldiazonium tetrafluoroborate (1) and 2-(4-methoxyphenyl)naphthalene- 1,4-dione (2) (Scheme 5a). First attempts were carried out by dissolving compounds 1 (1 eq., 10 mM), 2 (3 eq.) and Ru(bpy)3 (0.05 eq.) in deoxygenated DMSO (1 mL) under inert atmosphere and irradiating with blue light (450 nm, 60 mW/cm2). All components were apparently soluble in the choice solvent. A ratio 3:1 of naphthoquinone / diazonium salt had been previously demonstrated to be necessary to achieve high yields. Adequate controls were performed in the absence of photocatalyst and illumination. Yield of 3 was determined by 1H-NMR with dibromomethane as internal standard, unless otherwise noted. Results are summarized in Table 2. The arylated naphthoquinone 3 was obtained in 85% yield after 2 h under irradiation in presence of Ru(bpy)3 (entry 1). Bubbling due to the extrusion of nitrogen was immediately observed, and the solution turned from orange to dark red in 10 min. In the absence of blue irradiation, trace amounts of the product were observed, either under the lab’s ambient illumination (entries 2 and 5) or in total darkness (entries 3 and 6). Surprisingly, a yield of 63% (isolated yield of 38%) was obtained under blue illumination in the absence of catalyst (entry 4). O O OMe + N2 OMe BF4-O O OMe OMe Blue light, DMSO, 2 h, r.t. 123 O O OMe + N2 OMe BF4-O O OMe OMe DMEM, DMSO, r.t. 123 HeLa cells Ru(bpy)3 a) b) David Montoto Pintos - Degree in Chemistry Final Project Page 17 of 49 Table 2. Evaluation of the photocatalyzed reaction.a Entry Catalyst Illumination Time/h Yield of 3/% 1 Ru(bpy)3 Blue 2 85 2 Ru(bpy)3 Ambient 2 Traces 3 Ru(bpy)3 Darkness 2 Traces 4 - Blue 2 63 (38 isolated) 5 - Ambient 2 Traces 6 - Darkness 2 Traces (a) Reaction conditions (unless otherwise noted): 1 (1 eq., 10 mM), 2 (3 eq.), Ru(bpy)3 (0.05 eq.), DMSO (entries 1–3 and 5–6: 1 mL; entry 4: 20 mL), r.t., inert atmosphere. Yields determined (unless otherwise noted) by 1H-NMR using dibromomethane as internal standard. Traces defined as less than 7% yield. In 2022, Nagar and Dhar44 reported the photocatalyzed arylation of naphthoquinones using eosin Y (EY) as photocatalyst under green irradiation. Surprisingly, they did not observe any conversion in the absence of catalyst. These authors proposed a mechanism for their reaction under EY catalysis and green light, and an adapted mechanism is here proposed for the Ru(bpy)3-catalyzed reaction (Scheme 6). After excitation of the photocatalyst, the reaction would begin with a single electron transfer (SET) from the excited Ru(II) complex to 1, which generates an aryl radical by nitrogen extrusion, and Ru(III). The aryl radical is trapped by 2 to give a new radical species. The catalytic cycle closes with a new SET from this species to Ru(III) to regenerate Ru(II) and give a carbocation, which deprotonates to yield product 3.44 Scheme 6. Proposed mechanism for the photocatalyzed Meerwein arylation using Ru(bpy)3 as catalyst, based on the mechanism proposed for the eosin Y-catalyzed reaction.44 N2BF4 SET Ru(II)* N2 + + BF4- O O MeO O O MeO O O MeO O O MeO Blue light -H+ MeO MeO MeO H H Ru(III) Ru(II) Abiotic reactions promoted by the cell machinery Page 18 of 49 Assuming that the blue light-promoted reaction also proceeds via an aryl radical in the absence of catalyst, it seems unlikely that either the BF4- counterion or the solvent participate directly in the electron transfer process. In 2021, Witzel et al.45 had reported the arylation of furan by tetrafluoroborate arenediazonium salts in methanol under blue illumination. The transformation did not take place in DMSO. That was explained by the absence of light absorption of arenediazonium salts and DMSO in that range of the spectrum, while methanol can absorb blue light. In the case of arylation of naphthoquinone derivatives, the aryl radical may be generated by interaction with the naphthoquinone 2, which would act as the photocatalyst. Wang et al.46 reported a similar case in which boron dipyrromethene dyes self-promoted their arylation with diazonium salts when excited with visible light. Nevertheless, more research is needed to clarify this point, but, although the possibility of a new catalyst-free photoinduced arylation of naphtoquinones is interesting, it falls out of the scope of the present work. 3.3. In vitro experiments 3.3.1. Screening of different biological reductants Living cells contain several reducing biomolecules of low molecular weight that serve as coenzymes in multitude of redox enzymatic reactions (both prosthetic groups permanently bounded to proteins, and free coenzymes which are transitorily attached to enzymes), and also as antioxidants that remove harmful ROS from the cell. Aryldiazonium salts are known oxidants, and it has been reported that some biological reductants are able to reduce aryldiazonium compounds to aryl radicals.47 It was thus hypothesized that low-weight reducing biomolecules could be directly responsible for promoting this Meerwein arylation between 1 and 2. Three biomolecules were selected to test their ability to promote the reaction in abiotic conditions: glutathione (GSH), nicotinamide-adenine dinucleotide (NADH) and ascorbate. GSH is the main antioxidant present in cells.48 It is a tripeptide with structure γ-L-glutamyl-L-cysteinyl-glycine (Figure 1a). Its oxidation proceeds by disulfide bond formation between the thiol groups of two molecules of GSH to give glutathione disulfide. The usual concentration of GSH in the cytoplasm ranges between 0.1–10 mM,49 although in most cells the concentration is 1–2 mM.48 However, GSH has been found in elevated concentrations in several types of cancer.29 GSH concentration David Montoto Pintos - Degree in Chemistry Final Project Page 19 of 49 and GSSG/GSH ratio are considered important indicators of the cytosolic redox state,49 and concentrations of GSSG are usually very low, of 5–10% of the total.48 Figure 1. Structures of biological reductants: (a) L-glutathione; (b) NADH; (c) ascorbate. NADH (Figure 1b) is the main redox coenzyme of the cell. It is formed by two 5’-phosphate nucleosides: adenosine 5’-phosphate and another 5’-phosphate nucleoside containing nicotinamide at the 1’ carbon. The nucleosides are joined by an ester bond between the phosphates. NADH is oxidized by net donation of a hydride (H-) to give NAD+. Cellular concentration of NAD+ averages 365 μM,50,51 with a NAD+/NADH ratio of around 10:1,52 but these values vary greatly between organelles: for the cytoplasm, the values of 50 μM53 and a 600–700:1 ratio54,55 have been reported for free (non protein-bounded) NAD+ and NADH, and the corresponding values for mitochondria are 246 μM56 and 8:1.54 Moreover, NADH has been reported to generate aryl radicals from aryldiazonium salts in abiotic conditions.47 Ascorbate (Figure 1c), also known as vitamin C, serves as a coenzyme and an antioxidant. Its concentration in mammalian cells is in general 1–5 μM, although specific cell types can reach 10 mM or more.57 Ascorbate has also been reported to generate aryl radicals by reduction of arenediazonium compounds in abiotic media.47 The ability of these three selected biological reductants to promote this Meerwein reaction was then explored. NADH disodium salt and sodium ascorbate (NaAsc) were used as sources for NADH and ascorbate, respectively. Compounds 1 (1 eq., 10 mM), 2 (3 eq.) and the biological reductant (1 eq.) were dissolved in 1 mL of DMSO or DMSO:H2O (1:1) under inert atmosphere (Table 3). All reductants were apparently soluble in DMSO. However, naphtoquinone 2 was insoluble in this mixture of DMSO:H2O. Yield of 3 was determined by 1H-NMR with dibromomethane as internal standard. All three reductants were found to promote the reaction, both in absence and presence of water. The decreased yields in the latter case were attributed to the poor solubility of compound 2 in water. The highest yields were obtained for NADH in both O O N H H N NH3 OSH O O O N N N N NH2 O OHOH HH HH O P O- O O N O OHOH HH HH O P O- O O NH2 H H O HO HO OH O HO H a) b) c) Abiotic reactions promoted by the cell machinery Page 20 of 49 conditions (77% in DMSO, entry 3, and 36% in DMSO:H2O, entry 4). For this reason, NADH was selected for the study of the in vitro reaction, and new experiments were conducted to optimize conditions for the NADH-promoted reaction. Table 3. Screening of biological reductants.a Entry Additive Water/% Yield/% 1 GSH 0 53 2 GSH 50 12 3 NADH 0 77 4 NADH 50 36 5 NaAsc 0 33 6 NaAsc 50 28 (a) Reaction conditions (unless otherwise noted): 1 (1 eq., 10 mM), 2 (3 eq.), additive (1 eq.), DMSO or DMSO:H2O (1 mL), 2 h, r.t., inert atmosphere, ambient light. Yields determined by 1H-NMR using dibromomethane as internal standard. 3.3.2. Optimization of conditions and study of the NADH-promoted reaction The reversible oxidation of NADH (Scheme 7a) takes place with the transference of two electrons and a proton, but it is still debated whether the electron transference is concerted or stepwise (Scheme 7b).58 These two mechanisms can be also thought of as extremes of a continuous spectrum,59 and the observation of one or the other would depend in each case of the rate constants for each elementary step.60 Nonetheless, the radical NAD• has been identified in enzymatic60 and non-enzymatic reactions.61 Scheme 7. (a) Redox NAD+/NADH pair.62 (b) Stepwise mechanism for NADH reduction.60 O O OMe + N2 OMe BF4-O O OMe OMe DMSO, 2 h, r.t. inert atmosphere 123 Biological reductant N Ribose-ADP NH2 O +H++2e- N Ribose-ADP NH2 O H H NADH -e- NADH•+ -H+ NAD• -e- NAD+ a) b) David Montoto Pintos - Degree in Chemistry Final Project Page 21 of 49 Regarding the reduction of arenediazonium compounds, Yasui et al.59 suggested an outer sphere SET mechanism for dediazoniation promoted by BNAH (a NADH analogue). Substoichiometric amounts of BNAH would initiate a free radical chain which is inhibited by oxygen, and also by stoichiometric or higher than stoichiometric quantities of BNAH. Reszka and Chignell47 hypothesized that generated NAD• would in turn react with another diazonium cation, as it is a powerful oxidizing agent (Scheme 8). Scheme 8. NADH-promoted homolytic dediazoniation by outer sphere SET.47 On the other hand, Tatunashvil et al.63 have reported that Hantzsch ester, which is a stronger hydride donor than NADH,64 induces fragmentation of arenediazonium compounds to give aryl radicals via a hydride transfer mechanism. They observed that the reaction only took place in presence of oxygen, and proposed a mechanism in which oxygen induced the fragmentation of an arenediimide intermediate (Scheme 9). Scheme 9. Formation of aryl radicals from arenediazonium cations by hydride transfer from Hantzsch ester followed by oxygen-mediated fragmentation of intermediate diimide.63 To get more information about the reaction, different experiments were performed changing the concentration of NADH. Besides, to study the role of oxygen in the process, the influence of using deoxygenated solvents as well as nitrogen or open-air atmosphere was explored (Table 4). The yield of 3 increased using higher quantities of NADH (entries 1–6) until reaching a maximum at 1.0 eq. (entries 5–6). Lower yields of product 3 were formed with superstoichiometric amounts of the reducing agent (entries 8–9). This could be explained by a «discoordination» between the aryl radical generation and the radical attack to the double bond of 2. In the presence of more than equimolar quantities of NADH, the aryl radical will probably form faster, which can in turn favor competitive reactions. Deoxygenation of the solvents gave higher yields when substoichiometric quantities of NADH were used (entries 1–4), but almost no difference was observed for 1.0 eq. of NADH (entries 5–6). When the reaction was carried out under open ArN2 + + NADH Ar• + N2 + NAD• + H+ ArN2 + + NAD•Ar• + N2 + NAD+ N H H H O RO O OR Ar N N DMSO N O RO O OR H +N N Ar HO2Ar + N2 + OO H Abiotic reactions promoted by the cell machinery Page 22 of 49 atmosphere, a slightly reduced yield was observed (entry 7). These findings support the outer sphere SET mechanism, as oxygen is not necessary for the reaction to proceed. Inhibition by oxygen is probably due to trapping of intermediate radicals. Addition of TEMPO, a radical trapping agent, also lowered the yield (entries 10–12), which corroborates the participation of radicals in the process. Table 4. Optimization of reaction conditions with NADH.a Entry NADH/eq. Deoxygenation Atmosphere Yield/% 1 0.1 Yes N2 56 2 0.1 No N2 43 3 0.8 Yes N2 58 4 0.8 No N2 67 5 1.0 Yes N2 72 6 1.0 No N2 77 7 1.0 No Open 65 8 1.5 Yes N2 65 9 2.0 Yes N2 39 10 0.1 Yes N2 19b 11 1.0 No N2 58b 12 1.0 No N2 27c (a) Reaction conditions (unless otherwise noted): 1 (1 eq., 10 mM), 2 (3 eq.), DMSO (1 mL), 2 h, r.t., inert atmosphere, ambient light. (b) 1 eq. of TEMPO. (c) 10 eq. of TEMPO. Yields determined by 1H-NMR using dibromomethane as internal standard. The reaction time was next investigated. A substantial yield of 56% was achieved with substoichiometric amounts of NADH after 2 h (0.1 eq, Table 4, entry 1), and similar results were obtained with reduced reaction times of 1 h or 30 min (Table 5, entries 1–2). Indeed, the HPLC-MS analysis of reaction crude for a 1 h reaction with 1.0 eq. of NADH showed that NADH was completely consumed, as only NAD+ (m/z of 664) was detected (Table 6). This suggests that, in abiotic conditions, NADH acts as radical initiator for a radical chain process. David Montoto Pintos - Degree in Chemistry Final Project Page 23 of 49 Table 5. Yields at different times with NADH.a Entry NADH/eq. Time/min Yield/% 1 0.1 30 54b 2 0.1 60 53b 3 1.0 1 29 4 1.0 5 64 5 1.0 10 67 (a) Reaction conditions (unless otherwise noted): 1 (1 eq., 10 mM), 2 (3 eq.), NADH, DMSO (1 mL), r.t., inert atmosphere, ambient light. (b) Deoxygenated solvent. Yields determined by 1H-NMR using dibromomethane as internal standard. To further test this hypothesis, an experiment was devised in which a second equivalent of diazonium salt 1 was added after 1 h to the reaction with 0.1 eq. of NADH. An increment in the yield from 0.56 eq. to 0.76 eq. was observed, proving that radical intermediates remained in the reaction milieu and were able to promote the fragmentation of the additional quantity of 1. The reaction was also evaluated for 1.0 eq. of NADH at different reaction times, showing the fast kinetics of the process. After 10 minutes, there were almost no changes in the yield obtained (Table 5, entries 3–5). The reaction was also attempted with naphthoquinone 2 as limiting reactant (10 mM of 2, 3.0 eq. of 1, 0.1 eq. of NADH, deoxygenated solvent), obtaining a decreased yield of 32%. This is explained because aryl radicals are highly reactive species, and an excess of the acceptor alkene 2 is necessary for the reaction to adequately compete with side reactions. Additional experiments were conducted to determine the water sensitivity of the reaction. As previously mentioned, 50% of water decreased the reaction yield from 77% to 36% (Table 3, entries 3–4). However, the same yield of 36% was obtained when water content was reduced to 10% in the same conditions. The reaction was also attempted in other organic solvents (acetonitrile and THF), but only trace amounts of product were observed. This was attributed to the poor solubility of NADH in both solvents. Therefore, after the exhaustive optimization process, the optimized conditions were selected as 10 mM of 1, 3 eq. of 2, 1 eq. of NADH, DMSO, 2 h, r.t. and inert atmosphere. Abiotic reactions promoted by the cell machinery Page 24 of 49 To further evaluate the mechanism, identification of reaction subproducts was attempted. In order to monitor the process by 1H-NMR, the optimized reaction was carried out in d6-DMSO, but a very complex spectra was obtained, and no new species could be positively identified. Table 6. Identification of subproducts by HPLC-MS Species Detected masses/m/z Conditions NADH 666 (M+1) a,b NAD+ 664 (M+1), 123 (nicotinamide+1) c–f 4 770 (M+1), 542 (M−nicotinamide−PhOCH3), 229 (nicotinamide+PhOCH3+1), 108 (PhOCH3+1) d,f 5 R = CH3 171 (M+1), 154 (M−CH3) c,e R = CD3 174 (M+1), 157 (M−CD3) d,f 6 243 (M+1) c–f Conditions: 1 mL, 1 h, r.t., inert atmosphere, ambient light. (a) NADH (10 mM), DMSO. (b) NADH (10 mM), d6-DMSO. (c) 1 and NADH (both 10 mM), DMSO. (d) 1 and NADH (both 10 mM), d6-DMSO. (e) 1 (10 mM), 2 (30 mM) and NADH (10 mM), DMSO. (f) 1 (10 mM), 2 (30 mM) and NADH (10 mM), d6-DMSO. In contrast, HPLC-MS allowed for identification of several subproducts (Table 6). The detection of aryl methyl sulfoxide (5) in all reactions is consistent with reports of Ru(bpy)3-and-light-catalyzed methylsulfoxidation of arenediazonium salts.65 Methylsulfoxidation could thus represent an important side reaction in the selected conditions. Additionally, in those transformations carried out in deuterated solvent, a compound with mass that could agree with that of the product of arylation of NAD+ in position 4 of the nicotinamide (4) could be detected. A possible mechanism for this N N N N NH2 O OHOH HH HH O P HO O O N O OHOH HH HH O P HO O O NH2 OCH3 H3CO S R O OCH3 N N H3CO David Montoto Pintos - Degree in Chemistry Final Project Page 25 of 49 transformation would be the radical coupling of NAD• and the aryl radical derived from the diazonium salt. This would contribute to explaining the decrease in yield when more than 1 eq. of NADH is used, since NADH would be capturing the aryl radicals. 3.3.3. Evaluation of alternative acceptors Three 1,4-naphthoquinones which were available at the lab were evaluated in vitro to widen the scope of the transformation (Scheme 10). Products were not isolated, as this was a first preliminary test, and the yield was determined by 1H-NMR comparing with the reported spectra. Reactions with naphthoquinones proceeded with acceptable yields, but lower than for derivative 2. R = CH3, 7, 66% R = Br, 8, 58% R = OH, 9, 46% Scheme 10. NADH mediated arylation reactions of three different 1,4-naphthoquinones. Reaction conditions: 1 (1 eq., 10 mM), starting naphtoquinone (3 eq.), NADH (1 eq.), DMSO (1 mL), r.t ., inert atmosphere, ambient light. Reaction time was 4 h for 7 and 2 h for 8 and 9. Yields determined by 1H-NMR using dibromomethane as internal standard. Peaks were identified by comparison with reported spectra. 3.3.4. Reactions with alkynes Several photocatalytic reactions between arenediazonium salts and alkynes have been reported. Some of these transformations have been previously studied in the research group as candidate bioorthogonal reactions, so three adequate aryldiazonium salts (10– 12) were already synthetized and were generously shared. Hari et al.66 demonstrated that benzothiophenes can be synthetized from alkynes and 1,2-methylthioarenediazonium salts employing green light an EY as photocatalyst. The reaction was thus attempted using diazonium salt 10 and phenylacetylene in presence of NADH. An initial yield of 24% was determined for 13, but an increase from 3 to 10 eq. of the alkyne gave a yield of 43% (Scheme 11a). Xiao et al.67 also reported synthesis of phenanthrenes by cascade radical reactions initiated by EY and visible light. In this case, treatment of diazonium salts 11 and 12 and O O R OMe O O R 1 eq. NADH DMSO, r.t., innert atmosphere + N2 OMe BF4- Abiotic reactions promoted by the cell machinery Page 32 of 49 Analytical HPLC was performed on an HPLC-MS Thermo Ultimate 3000 coupled to a Bruker AmaZon SL mass spectrometer, using electrospray ionization (ESI) and a flow rate of 0.35 mL/min at room temperature. 3.5.2. Synthesis of p-methoxyphenyldiazonium tetrafluoroborate (1) Some diazonium salts are stable and can be isolated, as is the case of compound 1. The synthesis was performed by the classical diazotization procedure.75 p-Anisidine (1.0 g, 8.12 mmol, 1 eq.) was suspended in 2.4 mL of water in a 25 mL round-bottom flask. Tetrafluoroboric acid (2.1 mL, 16.24 mmol, 2 eq.) was added to give a black solution. The reaction was cooled to 0 ºC in a water-ice bath. A cold solution of sodium nitrite (1.12 g, 16.24 mmol, 2 eq.) in 2.4 mL of water was added dropwise. The mixture turned dark green, and the formation of a grey solid was observed. After 1 h 15 min, the solid was isolated by filtration as a grey powdery solid, washed with cold diethyl ether, redissolved in acetone, and recrystallized in diethyl ether to give pure 1 as a grey solid (1.2 g, 66%). 1H-NMR (300 MHz, d6-DMSO) δ 8.61 (d, J = 9.0 Hz, 2H), 7.48 (d, J = 9.0 Hz, 2H), 4.04 (s, 3H). Data in accordance with the literature.76 3.5.3. Synthesis of 2-(4-methoxyphenyl)naphthalene-1,4-dione (2) Procedure was adapted from literature.77 Tetrakis(triphenylphosphine)palladium(0) (28.9 mg, 0.025 mmol, 0.025 eq.) was dissolved in 4.0 mL of anhydrous THF in a purged Schlenk tube under nitrogen atmosphere. 2-Bromonaphthalene-1,4-dione (237.5 mg, 1.0 mmol, 1 eq.), cesium carbonate (488.7 mg, 1.5 mmol, 1.5 eq.) and 4-methoxyphenylboronic acid (227.9 mg, 1.5 mmol, 1.5 eq.) were successively added to the stirred solution under N2 flow. Then, 0.6 mL of water were added. The mixture was heated to 65 ºC until TLC (silica gel, n-hexane/ethyl acetate 9:1) showed complete consumption of the starting material (20 h). After cooling to ambient temperature, the mixture was diluted with water (15 mL) and MeO NH2NaNO2, HBF4 H2O, 0 ºC, 1 h 15 min MeO N2BF4 1 O O O O OMe + Br BOHHO OMe Pd(PPh3)4, Cs2CO3 THF, H2O, 65 ºC, 24 h 2 David Montoto Pintos - Degree in Chemistry Final Project Page 33 of 49 extracted three times with ethyl acetate. The combined organic layers were washed with brine, dried over MgSO4, and concentrated under vacuum. The product was purified by flash chromatography on silica gel (n-hexane/ethyl acetate, gradient from 0% to 20%) as an orange solid (238.4 mg, 90%), which turned red after dissolving in dichloromethane and concentrating under vacuum. 1H-NMR (300 MHz, d-chloroform) δ 8.21–8.08 (m, 2H), 7.79–7.78 (m, 2H), 7.59 (d, J = 8.5 Hz, 2H), 7.08–6.96 (m, 3H), 3.87 (s, 3H). Rf = 0.33 (n-hexane/ethyl acetate 9:1). Data in accordance with the literature.44 3.5.4. Synthesis of 2,3-bis(4-methoxyphenyl)naphthalene-1,4-dione (3) Compound 3 was synthesized for characterization following standard blue-light-promoted Meerwein arylation between compounds 1 and 2. Diazonium salt 1 (44.4 mg, 0.20 mmol, 1 eq.) and naphthoquinone 2 (158.6 mg, 0.6 mmol, 3 eq.) were introduced under nitrogen flow in a purged 50 mL Schlenk tube. The tube was then purged with three vacuum-nitrogen cycles. 20 mL of anhydrous DMSO was added to yield an orange solution. The solution was irradiated with a blue LED lamp (456 nm, 60 mW/cm2, distance 10 cm). Bubbling was observed, and the solution turned dark red in 10 min. After 2 h, the solution was diluted with water (100 mL) and extracted three times with diethyl ether. The combined organic layers were washed with brine, dried over MgSO4, and concentrated under vacuum. The product was purified by flash chromatography on silica gel (n-hexane/ethyl acetate, gradient from 0% to 20%) as a red solid (28.4 mg, 38%). 1H-NMR (300 MHz, d-chloroform) δ 8.17 (d, J = 4.0 Hz, 2H), 7.76 (d, J = 4.1 Hz, 2H), 7.03 (d, J = 8.3 Hz, 4H), 6.78 (d, J = 8.5 Hz, 4H), 3.78 (s, 6H). Rf = 0.22 (n-hexane/ethyl acetate 9:1). Data in accordance with the literature.44 3.5.5. Representative procedure for irradiation experiments in subsection 3.2 Diazonium salt 1 (2.2 mg, 0.01 mmol, 1 eq.), naphthoquinone 2 (7.9 mg, 0.03 mmol, 3 eq.) and Ru(bpy)3 (0.4 mg, 0.5 nmol, 0.05 eq.) were introduced under nitrogen flow in a purged 10 mL Schlenk tube. The tube was then purged with three vacuum-nitrogen cycles. 1 mL of anhydrous DMSO was added to yield an orange O O OMe + N2BF4 OMe O O OMe OMe Blue light, DMSO, 2 h, r.t. 123 Abiotic reactions promoted by the cell machinery Page 34 of 49 solution. The solution was magnetically stirred and irradiated with a blue LED lamp (456 nm, 60 mW/cm2, distance: 10 cm). Bubbling was observed, and the solution turned dark red in 10 min. After 2 h, the solution was diluted with water (5 mL) and extracted three times with diethyl ether. The combined organic layers were washed with brine, dried over MgSO4, and concentrated under vacuum. A yield of 85% was determined by 1H-NMR using dibromomethane as internal standard. 3.5.6. Representative procedure for reactions in subsection 3.3 Diazonium salt 1 (2.2 mg, 0.01 mmol, 1 eq.), naphthoquinone 2 (7.9 mg, 0.03 mmol, 3 eq.) and NADH disodium salt (7.1 mg, 0.01 mmol, 1 eq.) were introduced under nitrogen flow in a purged 10 mL Schlenk tube. The tube was then purged with three vacuum-nitrogen cycles. 1 mL of anhydrous DMSO was added to yield an orange solution. The solution was magnetically stirred. Bubbling was observed, and the solution turned dark red in 10 min. After 2 h, the solution was diluted with water (5 mL) and extracted three times with diethyl ether. The combined organic layers were washed with brine, dried over MgSO4, and concentrated under vacuum. A yield of 77% was determined by 1H-NMR using dibromomethane as internal standard. 3.5.7. Representative procedure for reactions in subsection 3.3.4 Diazonium salt 10 (5.4 mg, 0.02 mmol, 1 eq.) and NADH disodium salt (14.2 mg, 0.02 mmol, 1 eq.) were introduced under nitrogen flow in a purged 10 mL Schlenk tube. The tube was then purged with three vacuum-nitrogen cycles. Phenylacetylene (22.0 μL, 0.20 mmol, 10 eq.) was dissolved in 1 mL of anhydrous DMSO and added to the Schlenk to yield a yellow solution. The solution was magnetically stirred. Bubbling was observed, and the solution turned orange in 10 min. After 2 h, the solution was diluted with water (5 mL) and extracted three times with diethyl ether. The combined organic layers were washed with brine, dried over MgSO4, and concentrated under vacuum. A yield of 43% was determined by 1H-NMR using dibromomethane as internal standard. 3.5.8. Internal standard method for yield quantification NMR has been established as a state-of-the-art method for determination of reaction yields from non-purified crudes, provided that the product presents one or more well-resolved signals. As the area under an NMR peak is usually proportional to the number of nuclei, calibrating with an adequate internal standard allows for precise quantification.78 David Montoto Pintos - Degree in Chemistry Final Project Page 35 of 49 Initially, nitromethane was selected as internal standard for this project, but integration of signals for excess of compound 2 when following the studied reaction yielded inconsistent and usually excessive values. This was attributed to the high volatility of nitromethane. Dibromomethane in CDCl3 (δ=4.95 ppm, s) was finally selected as internal standard due to the proximity of its signal to that of the methoxy groups of product 3. For validation of dibromomethane concentration, 1,3,5-trimethoxybenzene (δ=3.8 ppm, s) was used as secondary internal standard, but its overlapping with a signal of product 3 prevented its use for direct yield determination on the reaction crudes. For determining the yield of product 3, the signals for the methoxy moiety (δ=3.78 ppm, s, 3H) and two aromatic H from the naphthoquinone moiety (δ=6.78 ppm, d, 2H) were used. When the yields calculated from the two signals differed in more than 7%, only the lower value was considered. The necessary volume of dibromomethane was added so that for a yield of 100% the relation between the internal standard signal and the methoxy group signal would be 1:1 (Equation 1). !"#$%&!"#!$!=%"#&()"*)+ × 6 . %"#)"*)+ ⁄ 2. %"#)"*)123%.% ⁄ ×45!"#!$! 6$3789!"#!$!× :!"#!$! = 0.010)%%"# × 3 × 173.835)%B)%%"#&' 0.99 × 2.477)%B)EF&' = 2.13)EF Equation 1. Figure 4. Example of yield determination by internal standard of dibromomethane (spectrum corresponding to procedure described in subsection 3.5.6). For the compounds in subsections 3.3.3 and 3.3.4, the following peaks were employed for quantification based on literature reports: 7: 3.86 (s, 3H),44 2.12 (s, 3H). 8: O O OMe OMe 3 Abiotic reactions promoted by the cell machinery Page 36 of 49 3.88 (s, 3H).44 9: 3.86 (s, 3H).79 13: 3.89 (s, 3H).66 14: 4.05 (s, 3H).67 15: 4.05 (s, 3H), 4.03 (s, 3H), 8.48 (s, 1H).67 3.5.9. General information for cell culture experiments HeLa and A549 cell cultures were incubated in DMEM (Dulbecco’s modified Eagle’s medium) from Sigma-Aldrich supplemented with 10% v/v fetal bovine serum (FSB), 1% penicillin/streptomycin and 1% L-glutamine from Thermo Fisher. Cultures were incubated at 37 ºC, 5% CO2 and 95% humidity. DMEM was purchased from Sigma-Aldrich. The other components were from Thermo Fisher. E. coli cultures were incubated in LB (lysogeny broth) composed of 10 g of Bacto Triptone from Gybco, 5 g of yeast extract from AppliChem and 10 g of NaCl from Sigma-Aldrich. Sterile PBS and DMSO of biological grade from Sigma-Aldrich were employed as solvents. Luminescence was measured with an Infinite M Plex Tecan plate reader. Work was performed on a HR1200-IIA2 biological safety cabinet. 3.5.10. Luminescence viability assay HeLa cells were seeded in a black wall 96-well plate with a confluence of 15 000 cells/well (1 day assay) or 1 000 cells/well (2 days assay, with starvation) and were incubated for 1 day in DMEM supplemented with 10% FBS (1 day assay), or 2 days changing the medium on the night of the second day with DMEM supplemented with 1% FBS (2 days assay). After that, the cells were treated with 100 μM of a suspension of compounds 1, 2 or 3, or 1 and 2 in DMEM with 0.5% v/v DMSO. After 1 h, the medium was changed for 100 μL of DMEM without phenol red and 100 μM of the commercial reagent from the CellTiter-Glo luminescent cell viability assay, and the luminescence signal was measured on a plate reader. 3.5.11. Experiment in HeLa cells HeLa cells were incubated on four 100 mm Petri dish with a confluence of 6x106 cells/dish with 3 mL of a suspension of compounds 1 and 2 (both 100 μM) in DMEM with 0.5% v/v of DMSO. After 1 h, the culture medium was collected and kept for posterior analysis. Cells were washed two times with 1 mL of PBS, which were collected separately for analysis. Cells were treated with 1 mL of Triton X-100 for lysing. All four fraction were lyophilized and dissolved in 1 mL of a 100 μM solution of coumarin in acetonitrile by vortex agitation and sonication for 15 min at 45 ºC. The mixture was separated by centrifugation at 7 800 g for 10 min, and the supernatant was collected, filtered by HPLC filter, and analyzed by HPLC-MS. David Montoto Pintos - Degree in Chemistry Final Project Page 37 of 49 3.5.12. Cell fragmentation assay A549 cells were seeded in four 100 mm Petri dishes with a confluence of 700 000 cells/plate. Cells were washed with PBS, harvested with a scrapper, and resuspended in 1 mL of PBS. 2x1 mL were separated into two 2 mL Eppendorf tubes ((a) for whole mitochondria experiments, and (b) for mitochondrial lysate experiments), and 250 μL were separated to another tube for control with whole cells (c). The mitochondria isolation kit from cultured cells from Thermo Fisher (option A of the instructions) was employed to obtain a mitochondria-enriched fraction from tubes (a) and (b). 1.592 mL of one of the cytosolic fractions were kept for the experiment in cytosolic fraction (d). The mitochondrial fractions were resuspended in 1.592 mL of PBS. Mitochondria on tube (b) were sonicated with 2 pulses of 5 s. All tubes were treated with 4 μL of a 40 μM suspension of compound 1 in DMSO and 4 μL of a 40 μM suspension of compound 2 in DMSO and incubated for 1 h at 37 ºC with agitation. Tubes (a) and (c) were centrifugated at 12 300 g for 10 min and 1 700 g for 5 min, respectively. The supernatant was then retired, and the pellets were washed with PBS and centrifugated again in the same conditions. Liquid fractions were lyophilized for all tubes, and pellets obtained from (a) and (c) were dissolved in methanol and concentrated under vacuum. Each fraction from each tube was then dissolved in 1 mL of acetonitrile and sonicated for 15 min at 45 ºC. The mixtures were centrifugated at 7 800g for 10 min, and the supernatant was collected, filtered with a HPLC filter, and analyzed by HPLC-MS. 3.5.13. Experiments in E. coli A 10 mL preculture of DH5-α E. coli was diluted in 100 mL of LB and incubated at 37 ºC with stirring until reaching an OD of 0.3. Six 10 mL aliquots of the culture were centrifugated at 3 000 g for 15 min. For normal treatment (a, b, c) the isolated pellet was resuspended in 1 mL of a solution 100 μM of compound 2 (a, c) or 100 μM of compounds 1 and 100 μM of 2 (b), in PBS with DMSO 0.5% v/v and incubated at 37 ºC for 2 h with stirring. The tubes were centrifugated at 3 000 g for 15 min, and the supernatant was collected. The pellets were washed with 1 mL of PBS, centrifugated at 3 000 g for 10 min, and the supernatant was again collected. 1 mL of a 100 μM solution of compound 1 in PBS with 0.5% v/v DMSO was added to tube (c) and incubated with stirring at 37 ºC for 1 h. Tube (c) was then centrifugated at 3 000 g for 10 min, and the supernatant was collected. For heat shock treatment (d, e, f), the first incubation was substituted by heat Abiotic reactions promoted by the cell machinery Page 38 of 49 shock (41 ºC for 20 s) followed by ice incubation for 30 min and stirred incubation at 37 ºC for 30 min. Liquid fractions were lyophilized. All fractions and pellets were dissolved in 1 mL of a 100 μM solution of coumarin in acetonitrile and sonicated for 15 min at 45 ºC. The mixtures were centrifugated at 7 800g for 10 min, and the supernatant was collected, filtered with a HPLC filter, and analyzed by HPLC-MS. 3.5.14. Quantification by HPLC-MS Concentration of 1 and 2 was determined from the areas under the absorbance peak at 270 nm in HPLC. Coumarin was used as internal standard. The calibration lines employed were y=0,09326332x+0,02085394 (R2=0,9984) for compound 2 and y=0,06245542x−0,000925 (R2=0,9997) for product 3, being y the quotient of the area under the peak for the compound and coumarin, and x the concentration of the compound in μM. The calibration curves were constructed and generously shared by member of the group. 4. Conclusions In this Degree Final Project, a new biocompatible reaction promoted intracellularly by mammal cells (HeLa and A549) based on a Meerwein arylation of a naphthoquinone is described. The reaction was also found to take place in presence of E. coli. The reaction is promoted in abiotic environments by low-weight biological reductants NADH, glutathione and ascorbate, and experimental results suggest that the NADH-promoted reaction begins with a single electron transfer from NADH to the diazonium salt to give a radical, and then proceeds by a radical chain mechanism. The reaction was found to occur in isolated mitochondria and in mitochondria lysate, although additional research is needed to clarify the mechanism by which the reaction is promoted in cells. Finally, preliminary studies in abiotic media showed that NADH can promote synthesis of benzothiophenes and phenanthrenes from diazonium salts and alkynes in abiotic media. These findings would enable the development of cell-promoted biocompatible reactions based on diazonium compounds with applications in biosynthesis and biomedicine. The study of these transformations in alternative cell types could be of great interest, as diazonium salts may be engineered to react selectively in highly reductive milieus, such as those present in some tumor microenvironments. This would allow for localized in situ synthesis of probes and drugs. David Montoto Pintos - Degree in Chemistry Final Project Page 39 of 49 Conclusiones En este Trabajo de Fin de Grado, se ha descrito una nueva reacción biocompatible promovida intracelularmente por células de mamífero (HeLa y A549) basada en la arilación de Meerwein de una naftoquinona. Se encontró que la reacción también tiene lugar en presencia de E. coli. En ambientes abióticos, promueven la reacción reductores biológicos de bajo peso molecular como el NADH, el glutatión y el ascorbato, y los resultados experimentales sugieren que la reacción promovida por el NADH comienza con una transferencia de un electrón del NADH a la sal de diazonio para dar un radical, y a continuación procede por un mecanismo radicalario en cadena. Se vio que la reacción transcurre en mitocondrias aisladas, aunque se requieren estudios adicionales para aclarar el mecanismo por el que las células promueven la reacción. Finalmente, se llevaron a cabo estudios preliminares que muestran que el NADH puede promover la síntesis de benzotiofenos y fenantrenos a partir de sales de diazonio y alquinos en medios abióticos. Estos resultados podrían permitir el desarrollo de reacciones biocompatibles promovidas por la célula basadas en compuestos de diazonio con aplicaciones en biosíntesis y biomedicina. El estudio de estas transformaciones en tipos celulares alternativos es de gran interés, ya que podrían diseñarse sales de diazonio que reaccionasen selectivamente en ambientes altamente reductores, como los que se encuentran en algunos microambientes tumorales. Ello permitiría la síntesis in situ localizada de sondas y fármacos. Conclusións Neste Traballo de Fin de Grao, describiuse unha nova reacción biocompatible promovida intracelularmente por células de mamífero (HeLa e A549) baseada nunha arilación de Meerwein dunha naftoquinona. Atopouse que a reacción tamén ten lugar en presencia de E. coli. En ambientes abióticos, promoven a reacción redutores biolóxicos de baixo peso molecular coma o NADH, o glutatión e o ascorbato, e os resultados experimentais suxiren que a reacción promovida polo NADH comeza cunha transferencia dun electrón do NADH ao sal de diazonio para dar un radical, e a continuación procede por un mecanismo radicalario en cadea. Atopouse que a reacción transcorre en mitocondrias illadas, aínda que se requiren estudos adicionais para aclarar o mecanismo polo que as células promoven a reacción. Finalmente, leváronse a cabo estudos Abiotic reactions promoted by the cell machinery Page 40 of 49 preliminares que mostran que o NADH pode promover a síntese de benzotiofenos e fenantrenos a partires de sales de diazonio e alquinos en medios abióticos. Estes resultados poderían permitir o desenvolvemento de reaccións biocompatibles promovidas pola célula baseadas en compostos de diazonio con aplicacións en biosíntese e biomedicina. O estudo destas transformación en tipos celulares alternativos é de gran interese, xa que poderían deseñarse sales de diazonio que reaccionasen selectivamente en ambientes altamente redutores, coma os que se atopan nalgúns microambientes tumorais. Elo permitiría a síntese in situ localizada de sondas e fármacos. References (1) Sletten, E. M.; Bertozzi, C. R. 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