UNIVERSIDAD COMPLUTENSE DE MADRID FACULTAD DE CIENCIAS QUÍMICAS TESIS DOCTORAL Design of diruthenium metalloproteins as structural models: synthetic aspects, structural characterization and properties Diseño de metaloproteínas de dirrutenio como modelos estructurales: aspectos sintéticos, caracterización estructural y propiedades MEMORIA PARA OPTAR AL GRADO DE DOCTOR PRESENTADA POR Aarón Terán More DIRECTORES Santiago Herrero Domínguez Ana Edilia Sánchez Peláez © Aarón Terán More, 2023 UNIVERSIDAD COMPLUTENSE DE MADRID FACULTAD DE CIENCIAS QUÍMICAS TESIS DOCTORAL DESIGN OF DIRUTHENIUM METALLOPROTEINS AS STRUCTURAL MODELS: SYNTHETIC ASPECTS, STRUCTURAL CHARACTERIZATION, AND PROPERTIES DISEÑO DE METALOPROTEÍNAS DE DIRRUTENIO COMO MODELOS ESTRUCTURALES: ASPECTOS SINTÉTICOS, CARACTERIZACIÓN ESTRUCTURAL Y PROPIEDADES MEMORIA PARA OPTAR AL GRADO DE DOCTOR PRESENTADA POR Aarón Terán More DIRECTORES Dr. Santiago Herrero Domínguez Dra. Ana Edilia Sánchez Peláez UNIVERSIDAD COMPLUTENSE DE MADRID FACULTAD DE CIENCIAS QUÍMICAS DOCTORADO EN QUÍMICA AVANZADA TESIS DOCTORAL DESIGN OF DIRUTHENIUM METALLOPROTEINS AS STRUCTURAL MODELS: SYNTHETIC ASPECTS, STRUCTURAL CHARACTERIZATION, AND PROPERTIES DISEÑO DE METALOPROTEÍNAS DE DIRRUTENIO COMO MODELOS ESTRUCTURALES: ASPECTOS SINTÉTICOS, CARACTERIZACIÓN ESTRUCTURAL Y PROPIEDADES MEMORIA PARA OPTAR EL GRADO DE DOCTOR PRESENTADA POR: Aarón Terán More DIRECCIÓN: Dr. Santiago Herrero Domínguez Dra. Ana Edilia Sánchez Peláez Madrid, 2023 2 If you surrender to the air, you can ride it. -Toni Morrison Agradecimientos (Acknowledgements) El camino de mi tesis ha estado lleno de sorpresas. Una montaña rusa constante donde muchas veces no veía el final. Sin embargo, después de casi cuatro años, me siento más seguro que nunca diciendo que he elegido el camino correcto. En primer lugar, quiero agradecer a mis directores de tesis, Ana y Santi. Sin duda, todo lo que he logrado ha sido gracias a la confianza incondicional que habéis depositado en mí cada día. Ha sido un placer poder trabajar a vuestro lado, crecer como científico y como persona y entender lo bonito que es la química. Creedme que me llevo conmigo lecciones que no siempre se explican en una clase. Cuando pienso en qué me hizo querer investigar, siempre recuerdo a mis profesores de la carrera. Gracias a ellos, se despertó en mí una inquietud por querer entender lo que sucede a mi alrededor. Cómo no recordar las grandes clases de Marina, Marifeli, José Calbet, José Antonio o Emilio. Si no fuera por ellos, estoy convencido de que no hubiera seguido por este camino. Alguien que también me inspiró durante estos años fue mi mentor Javier Carvajal. Disfruté cada una de las reuniones que tuvimos y realmente me enseñaste a ver todas las posibilidades que me rodean. Tenemos que conocer nuestro potencial, saber marcar límites y hasta dónde nos gustaría llegar. Aunque en esta tesis sólo vaya mi nombre, no podría haber terminado si no fuera por el apoyo de los grandes amigos que tengo. Por un lado, quiero mencionar a mis compañeros de la Residencia de Estudiantes (Águeda, Aitor, Adrián, Bea, Carlos, Cristina, Jesús, Pedro y Julián), con quién pude convivir durante dos maravillosos años y aprender un poquito de todos ellos. También quiero agradecer a mis amigos de la estancia. Quién me iba a decir a mí que tres meses en Nápoles iban a dar para tanto. Grazie a tutti. Gracias a la profesora Giarita Ferraro y al profesor Antonello Merlino, que me acogieron desde el primer día en el laboratorio como si fuera uno más, aunque no consiguieron que bebiera un espresso cada mañana. Cómo no acordarme en este momento de mis amigos de la carrera (Alessandra, Andrés, Linneth, Ignacio, Paloma y Sara) o de mis amigos de la facultad (Aida, Almu, Elena, Patri, Laura y Sergio). Gracias por seguir ahí y por vuestro apoyo durante todo este tiempo. Y, no quiero olvidarme de mi grupo de investigación MatMoPol y de los profesores del departamento de Química Inorgánica, de quienes he 6 aprendido tantísimo, tanto dentro como fuera del laboratorio. También me gustaría agradecer a todo el personal de la Facultad de Ciencias Químicas, desde secretaría, dirección, mantenimiento y limpieza (mención especial para Gema). Habéis hecho que todo el tiempo que he pasado aquí sienta este lugar como mi casa. Y, por último, quiero agradecer a mi familia, a mis padres (Juan y Margarita), a mis hermanos (Adrián y Ariana) y a Jorge. De verdad, no sabéis lo que significa para mi todo lo que me habéis demostrado durante este tiempo. No puedo sentirme más feliz de poder teneros a mi lado. Y, aunque ya no esté conmigo, sé que mi abuela Sabina estaría muy orgullosa de verme llegar hasta aquí. Gracias a ti y a todo lo que me enseñaste, sé que puedo conseguir cualquier cosa. Table of contents List of abbreviations .......................................................................................................... 1 Abstract ............................................................................................................................. 5 Resumen ......................................................................................................................... 11 Chapter I. Overview ........................................................................................................ 23 Chapter II. Aims and objectives ....................................................................................... 47 Chapter III. Ultrasound-assisted synthesis of water-soluble monosubstituted diruthenium compounds ..................................................................................................................... 53 Chapter IV. Effect of equatorial ligand substitution on the reactivity with proteins of paddlewheel diruthenium complexes: Structural Studies .............................................. 62 Chapter V. Charge effect in protein metalation reactions by diruthenium complexes .. 71 Chapter VI. Steric hindrance and charge influence on the cytotoxic activity and protein binging properties of diruthenium complexes ................................................................ 83 Chapter VII. Applications ................................................................................................. 98 Chapter VIII. Discussion ................................................................................................. 102 Conclusions ................................................................................................................... 116 Conclusiones ................................................................................................................. 122 References .................................................................................................................... 128 Annexes ......................................................................................................................... 146 1 List of abbreviations 2,4-D 2,4-Dichlorophenoxyacetate AD Alzheimer's disease ADME Absorption, distribution, metabolism, and elimination Asn Asparagine Asp Aspartic acid Aspi Aspirinate Arg Arginine Aβ Amyloid-β CD Circular dichroism CV Cyclic voltammetry Cys Cysteine DAniF- N,N´-bis(p-methoxy)phenylformamidinate DPhF- N,N´-diphenylformamidinate D-p-CNPhF- N,N´-bis(4-cyanophenyl)formamidinate D-p-FPhF- N,N′-bis(4-fluorophenyl)formamidinate D-o/m/p-TolF- N,N´-bis(2/3/4-tolyl)formamidinate DNA Deoxyribonucleic acid DFT Density Functional Theory DMSO Dimethyl sulfoxide EB776 Deprotonated form of (2-phenylindol-3-yl)glyoxyl-L-phenylalanine-L- leucine EB106 Deprotonated form of (2-phenylindol-3-yl)glyoxyl-L-leucine-L- phenylalanine ESI Electrospray ionization FDA Food and Drug Administration GLA γ-Linolenic acid Gly Glycine Hade Adenine Haden Adenosine HcAMP Adenosine 3´,5´-cyclic monophosphate 2 Hcyti Cytidine Hcyto Cytosine HEWL Hen egg white lysozyme His Histidine HSA Human serum albumin IAA Indole-3-acetate Ibp Ibuprofenate Ind Indomethacinate IRES Internal ribosome entry sites Ket Ketoprofenate Leu Leucine Lys Lysine MS Mass spectrometry MTT 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide MWS Microwave-assisted solvothermal synthesis NAA 1-Naphthaleneacetate NacCMP Cytidine 2´,3´-cyclic monophosphate sodium salt NCB Non-covalent bonding NMR Nuclear magnetic resonance Npx Naproxenate NSAIDs Nonsteroidal anti-inflammatory drugs PDB Protein Data Bank p-TolA- N-4-tolylamidate RNA Ribonucleic acid Sec Selenocysteine SHAPE Selective 2′ hydroxyl acylation analysed by primer extension SPLNs Solid polymer-lipid nanoparticles (S)-BPTPI (S)-3-(benzene-fused-phthalimido)-2-piperidonate (S)-NTTL (S)-2-(1,3-dioxo-1H,3H-benzo[de]isoquinolin-2-yl)-3-methyl-butyrate (S)-PTAD (S)-adamantan-1-yl-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-acetate (S)-PTTL N-phthaloyl-(S)-tert-leucinate 3 (S)-TCPTAD (S)-adamantan-1-yl-(4,5,6,7-tetrachloro-1,3-dioxo-1,3-dihydro- isoindol-2-yl)-acetate (S)-TCPTTL N-tetrachlorophthaloyl-(S)-tert-leucinate (S)-TPPTTL N-tetraphenylphthaloyl-(S)-tert-leucinate THF Tetrahydrofuran TPLNs Terpolymer-lipid nanoparticles Tyr Tyrosine USS Ultrasound-assisted synthesis UV Ultraviolet Vis Visible XRD X-ray diffraction ZFS Zero field splitting 4 5 Abstract bstract A 6 7 Bimetallic complexes with multiple metal–metal bonds have drawn intense interest across various fields due to their unique properties. Particularly, in the bioinorganic chemistry area, paddlewheel diruthenium compounds are increasingly receiving attention due to the unique versatility of the diruthenium core that cannot be achieved with monometallic compounds. Diruthenium complexes have been widely studied as promising anticancer metallodrugs, testing their cytotoxic properties against a great variety of cancer cell lines. In recent studies, a synergetic effect between the drug and the diruthenium core is searched. But the mechanism of action of these compounds is still unknown. Nevertheless, evidences suggest that the pharmacological features of these complexes rely on the specific interactions with proteins, and their selectivity could be related with steric hindrance or charge effects. This dissertation aims to provide the structural basis to better understanding the molecular behaviour of diruthenium compounds and their biological activity. Therefore, charge and steric hindrance parameters have been evaluated in the interaction of diruthenium compounds with proteins. The protein hen egg white lysozyme (HEWL) has been selected as structural model. Deciphering the affinity of diruthenium compounds for the different amino acids present in a protein allows to assess the potential of diruthenium compounds to interact with a protein structure and to form metalloproteins. This information can also be employed to predit the behaviour of diruthenium species with other biomolecules. To achieve this objective, it is necessary to work with compounds that are soluble and stable in aqueous media to favour their interaction with biomolecules without decomposition. However, the number of diruthenium compounds fulfilling these properties is quite scarce. For this reason, we will focus on the use of equatorial ligands (formamidinate and/or carbonate ligands) to provide stability to the diruthenium core and to control the charge and steric hindrance parameters. We have synthesised four types of water-soluble diruthenium compounds: the well- known K3[Ru2(CO3)4] and the novel derivatives [Ru2Cl(L-L)(O2CCH3)3], K2[Ru2(L-L)(CO3)3], and [Ru2Cl(L-L)2(O2CCH3)2] (L-L = formamidinate ligand). They differ in the charge of the 8 species generated in solution and/or number of bulky groups around the Ru2 5+ core. [Ru2Cl(L-L)(O2CCH3)3] and [Ru2Cl(L-L)2(O2CCH3)2] form cationic species in solution of coordinating solvents because they can replace the axial chloride ligand. K3[Ru2(CO3)4] and K2[Ru2(L-L)(CO3)3] compounds contain anion complexes. [Ru2Cl(L-L)(O2CCH3)3] and K2[Ru2(L-L)(CO3)3] derivatives contain one formamidinate ligand (monosubstituted complexes), while [Ru2Cl(L-L)2(O2CCH3)2] contains two formamidinate ligands (disubstituted compounds). Therefore, the introduction of one or two formamidinate ligands increases the steric hindrance around the ruthenium atoms, while the exchange between acetate and carbonate ligands allows obtaining cationic or anionic species in solution. To prepare [Ru2Cl(L-L)(O2CCH3)3] compounds, a selective substitution of a single acetate ligand of the starting compound [Ru2Cl(O2CCH3)4] has been carried out. For this purpose, a new methodology based on the use of ultrasound-assisted synthesis has been employed. In the case of K2[Ru2(L-L)(CO3)3], the substitution of the acetate by carbonate ligands has been carried out starting from [Ru2Cl(L-L)(O2CCH3)3] complexes under mild- conditions. Finally, a fine control of the synthesis conditions was necessary to achieve the substitution of two acetate ligands from [Ru2Cl(O2CCH3)4] due to their high reactivity to obtain [Ru2Cl(L-L)2(O2CCH3)2] compounds. All the complexes were characterised by a multi-technique approach to identify its structure and physico-chemical properties. We have evaluated the protein binding properties of diruthenium complexes as function of their charge and steric hindrance in solution and in the solid-state through different biophysical methods and single-crystal X-ray diffraction. For these studies, we have selected the model protein hen egg white lysozyme (HEWL), which has been widely used in the study of interactions of metal compounds with protein systems. The number of artificial metalloproteins with metal‒metal bonded binuclear compounds is very scarce. Before the work here presented, only one example of diruthenium metalloproteins had been described. In this dissertation, eight diruthenium compounds have been studied with HEWL, and up to fourteen novel diruthenium metalloproteins have been isolated under different conditions. The information that has been drawn from this study is related to the stability, selectivity, and coordination modes of these complexes in the presence of the model protein HEWL. 9 Our data demonstrates that the coordination of one or two formamidinate ligands to the bimetallic core increases the stability of the Ru2 5+ core in solution. Moreover, a remarkable coordination versatility of these compounds has been observed. Covalent or non-covalent bonding, axial or equatorial coordination, and cis or trans configuration of the protein residues respect to the bulky ligands have been described. The bonding mode seems to depend on the medium, the charge of the complexes and the bulky groups around the bimetallic core. The affinity of these complexes for binding proteins is quite high since they can lose (or release) part of their ligands to the medium to bind the side chain of different protein residues, maintaining the integrity of the multiple metal‒metal bond. Our results suggest that a proper choice of the diruthenium equatorial ligands can lead to compounds with improved biological properties and different reactivity with proteins. Indeed, the potential use of the complexes [Ru2Cl(DPhF)2(O2CCH3)2], [Ru2Cl(DPhF)(O2CCH3)3], and K2[Ru2(DPhF)(CO3)3] as anticancer drugs has been evaluated on a cell-based model. Preliminary screening for biological application was done by studying their cytotoxicity against different cancer cell lines. Results reveal that the complex [Ru2Cl(DPhF)2(O2CCH3)2] is a promising candidate with high selective activity against HeLa cells. A comparative study of the applicability of these species was carried out after understanding how charge and steric hindrance affect to the properties of diruthenium compounds in a biological system. Two compounds, K3[Ru2(CO3)4] and [Ru2Cl(D-p- FPhF)(O2CCH3)3], were selected with the aim to find out if 1) they were able to inhibit amyloid-β aggregation; and 2) how the different charge and steric hindrance of these complexes affected to this property. Results indicate that only the latter has the potential to inhibit aggregation and this is closely related to both the charge of the compound and the presence of aromatic ligands. This doctoral thesis proposes to develop fundamental research to broaden the knowledge about the behaviour of diruthenium species using new reaction media and thus help to reach their maximum potential. This research provides the basis of fundamental knowledge for the reaction between diruthenium species and proteins, 10 which can be applied in a variety of fields, such as the design of artificial diruthenium- containing metalloenzymes and diruthenium-based metallodrugs. 11 Resumen esumen R 12 13 Los complejos bimetálicos que presentan un enlace metal‒metal múltiple han suscitado un gran interés en distintos campos de investigación debido a sus propiedades únicas. En particular, en el área de la química bioinorgánica, los compuestos de dirrutenio con estructura de rueda de paletas están recibiendo cada vez más atención, debido a la versatilidad excepcional del centro de bimetálico, que no es posible en compuestos mononucleares. Los complejos de dirrutenio se han estudiado ampliamente como metalofármacos anticancerígenos, probando sus propiedades citotóxicas en una gran variedad de células malignas. Recientemente, se ha visto que se produce un efecto sinérgico entre el fármaco y el centro de dirrutenio. Aún se desconoce el mecanismo de acción de estos compuestos. No obstante, las evidencias sugieren que las características farmacológicas de este tipo de sistemas dependen de las interacciones específicas con proteínas y su selectividad podría estar relacionada con la carga de los complejos o con efectos de impedimento estérico alrededor del núcleo inorgánico. El objetivo de esta tesis es sentar las bases estructurales para comprender mejor el comportamiento molecular de los compuestos de dirrutenio y su actividad biológica. Para ello, se han evaluado la influencia de la variación de los parámetros de carga e impedimento estérico en la interacción de estos compuestos con proteínas. Se ha seleccionado la proteína lisozima de la clara de huevo de gallina (del inglés hen egg white lysozyme, HEWL) como modelo estructural. Conocer la afinidad de los compuestos de dirrutenio por los distintos aminoácidos presentes en una estructura proteica permite evaluar el potencial de estas especies para interaccionar con diferentes proteínas y, así, formar metaloproteínas artificiales. Esta información también puede emplearse para predecir el comportamiento de las especies de dirrutenio con otras biomoléculas. Para lograr este objetivo, es necesario trabajar con compuestos que sean solubles y estables en medios acuosos con la finalidad de favorecer su interacción con biomoléculas, pero sin descomponerse en el medio. Sin embargo, el número de compuestos de dirrutenio que cumplen estas dos propiedades es bastante escaso. Por ello, el trabajo se centra en el uso de ligandos ecuatoriales (ligandos formamidinato y/o carbonato) que den estabilidad y solubilidad al núcleo de dirrutenio, pero que también permitan evaluar a su vez los parámetros de carga e impedimento estérico. 14 Se han sintetizado cuatro tipos de compuestos de dirrutenio solubles en agua: el ya conocido K3[Ru2(CO3)4] y los nuevos derivados [Ru2Cl(L-L)(O2CCH3)3], K2[Ru2(L-L)(CO3)3] y [Ru2Cl(L-L)2(O2CCH3)2] (L-L = ligando formamidinato). Estos complejos difieren en la carga de las especies generadas en solución y/o en el número de grupos voluminosos alrededor del núcleo de Ru2 5+. Los compuestos [Ru2Cl(L-L)(O2CCH3)3] y [Ru2Cl(L-L)2(O2CCH3)2] forman especies catiónicas en disoluciones de disolventes coordinantes porque pueden sustituir al ligando cloruro de la posición axial. Los compuestos K3[Ru2(CO3)4] y K2[Ru2(L-L)(CO3)3] se comportan en disolución como complejos aniónicos. Por otra parte, los derivados [Ru2Cl(L-L)(O2CCH3)3] y K2[Ru2(L-L)(CO3)3] tienen solo un ligando formamidinato (complejos monosustituidos), mientras que [Ru2Cl(L-L)2(O2CCH3)2] contiene dos ligandos formamidinato (compuestos disustituidos). La introducción de uno o dos ligandos formamidinato en el complejo supone un aumento del impedimento estérico alrededor de los átomos de rutenio, mientras que el intercambio entre ligandos acetato y carbonato permite obtener especies catiónicas o aniónicas en disolución. Para preparar los compuestos de tipo [Ru2Cl(L-L)(O2CCH3)3], se ha llevado a cabo la sustitución selectiva de un único ligando acetato del compuesto de partida [Ru2Cl(O2CCH3)4]. Para ello, se ha empleado una nueva metodología basada en el uso de síntesis asistida por ultrasonidos. En el caso de los complejos K2[Ru2(L-L)(CO3)3], la sustitución de los ligandos acetato por los ligandos carbonato se ha llevado a cabo partiendo de los complejos de tipo [Ru2Cl(L-L)(O2CCH3)3] en condiciones suaves. Finalmente, fue necesario un control preciso de las condiciones de síntesis para conseguir la sustitución de dos ligandos acetato a partir de [Ru2Cl(O2CCH3)4], debido a la alta reactividad de los compuestos disustituidos [Ru2Cl(L-L)2(O2CCH3)2]. Todos los complejos se han caracterizado mediante las técnicas adecuadas para identificar su estructura y propiedades fisicoquímicas. Se han evaluado las propiedades de unión a proteínas de los complejos de dirrutenio arriba propuestos en función de su carga e impedimento estérico, en disolución y en estado sólido, mediante diferentes métodos biofísicos y difracción de rayos X de monocristal. Para estos estudios, se ha seleccionado la proteína HEWL, que ha sido 15 ampliamente utilizada en el estudio de las interacciones de compuestos metálicos con sistemas proteicos. El número de metaloproteínas artificiales con compuestos binucleares con enlace metal– metal es muy reducido. Antes de este trabajo, sólo se había descrito un ejemplo de metaloproteínas de dirrutenio. En esta tesis se han estudiado hasta ocho compuestos de dirrutenio distintos con la proteína HEWL, y se han aislado hasta catorce nuevas metaloproteínas de dirrutenio en diferentes condiciones. La información que se ha extraído de este estudio está relacionada con la estabilidad, selectividad y modos de coordinación de estos complejos en presencia de HEWL. Los datos recogidos en esta tesis demuestran que la coordinación de uno o dos ligandos formamidinato al centro bimetálico aumenta la estabilidad del núcleo Ru2 5+ en disolución. Además, se ha observado una notable versatilidad de coordinación de estos compuestos. Se han descrito enlaces covalentes o no covalentes, coordinación axial o ecuatorial, y configuración cis o trans de los residuos proteicos respecto a los ligandos voluminosos. El modo de enlace parece depender del medio, la carga de los complejos y los grupos voluminosos alrededor del núcleo bimetálico. La afinidad de estos complejos para unirse a proteínas es bastante elevada, ya que pueden perder (o liberar al medio) parte de sus ligandos para unirse a la cadena lateral de diferentes residuos proteicos, manteniendo la integridad del enlace metal–metal múltiple. Los resultados sugieren que una elección adecuada de los ligandos ecuatoriales del complejo de dirrutenio puede dar lugar a especies con mejores propiedades biológicas y diferente reactividad con las proteínas. De hecho, se ha evaluado el uso potencial de los complejos [Ru2Cl(DPhF)2(O2CCH3)2], [Ru2Cl(DPhF)(O2CCH3)3] y K2[Ru2(DPhF)(CO3)3] como fármacos anticancerígenos en líneas celulares de cáncer. Los resultados revelan que el complejo [Ru2Cl(DPhF)2(O2CCH3)2] es un candidato prometedor que presenta una elevada selectividad por las células malignas de HeLa. Se llevó a cabo un estudio comparativo de la aplicabilidad de estas especies tras comprender cómo afectan la carga y el impedimento estérico a las propiedades de los compuestos de dirrutenio en un sistema biológico. Para ello, se seleccionaron dos 16 compuestos, K3[Ru2(CO3)4] y [Ru2Cl(D-p-FPhF)(O2CCH3)3], con el objetivo de averiguar si: 1) eran capaces de inhibir la agregación del polipéptido β-amiloide; y 2) cómo afectaban a esta propiedad la distinta carga e impedimento estérico de estos complejos. Los resultados indican que el complejo [Ru2Cl(D-p-FPhF)(O2CCH3)3] tiene la capacidad de inhibir la agregación de β-amiloide y esto está estrechamente relacionado tanto con la carga del compuesto como con la presencia de ligandos aromáticos. La investigación desarrollada en esta tesis permite comprender el comportamiento de las especies de dirrutenio en un medio biológico. En concreto, la investigación desarrollada proporciona la base del conocimiento fundamental para la comprensión de la reacción entre proteínas y compuestos de dirrutenio, lo que permite su aplicación en diversos campos, como el diseño de metaloenzimas artificiales o metalofármacos que contengan compuestos de dirrutenio. 17 As result of this Thesis, the following research articles have been published: Chapter III Terán, A., Cortijo, M., Gutiérrez, Á., Sánchez-Peláez, Ana E., Herrero, S. and Jiménez- Aparicio, R. Ultrasound-assisted synthesis of water-soluble monosubstituted diruthenium compounds. Ultrason. Sonochem. (2021). 80, 105828. https://doi.org/10.1016/j.ultsonch.2021.105828 All the experiments included in this article were performed at Complutense University of Madrid under the supervision, orientation and guidance of Dr. Santiago Herrero and Dr. Ana E. Sánchez-Peláez, the thesis supervisors and corresponding authors of the paper. The contributions to the paper are as follows: • Aarón Terán performed the synthesis and full characterization of all diruthenium complexes described in this article. He proposed and developed the methodology to obtain this type of complexes. In addition, he carried out the investigation, formal data analysis, discussion, and the writing of the original draft. • Dr. Miguel Cortijo performed the formal analysis of magnetic measurements for all diruthenium complexes. • Dr. Ángel Gutiérrez performed the resolution of the crystal structure of compounds [Ru2Cl(D-p-CNPhF)(O2CCH3)3] and [Ru2Cl(p-TolA)(O2CCH3)3]. He was also in charge of the crystallographic training of the PhD candidate. • Dr. Ana E. Sánchez-Peláez, Dr. Reyes Jiménez-Aparicio, and Dr. Santiago Herrero were in charge on the visualization, supervision, discussion, and writing-review and editing. Dr. Santiago Herrero was also in charge on the project administration and funding acquisition. Chapter IV Terán, A., Ferraro, G., Sánchez-Peláez, Ana E., Herrero, S. and Merlino, A. Effect of Equatorial Ligand Substitution on the Reactivity with Proteins of Paddlewheel Diruthenium Complexes: Structural Studies. Inorg. Chem. (2023). 62, 670-674. https://doi.org/10.1021/acs.inorgchem.2c04103 The experiments included in this article were performed part at Complutense University of Madrid with supervision, orientation and guidance of Dr. Ana E. Sánchez-Peláez and https://doi.org/10.1016/j.ultsonch.2021.105828 https://doi.org/10.1021/acs.inorgchem.2c04103 18 Dr. Santiago Herrero (corresponding author of the paper) and another part in collaboration with Dr. Antonello Merlino (the co-corresponding author of the article) at the University of Naples Federico II (Naples, Italy). The contributions to the paper are as follows: • Aarón Terán performed the synthesis and characterization of the diruthenium complex [Ru2Cl(D-p-FPhF)(O2CCH3)3] in Complutense University of Madrid. As part of a three-month predoctoral stay in the group of Dr. Antonello Merlino at the University of Naples Federico II, he was able to learn and carry out crystallisation assays, X-ray diffraction data collection in the Elettra synchrotron facility at Trieste (Italy), and the crystal structure resolution of the Ru2:HEWL adducts. In addition, he carried out the investigation, validation, formal analysis, discussion, and the writing of the original draft. • Dr. Giarita Ferraro performed the crystallisation assays, X-ray diffraction data collection in the Elettra synchrotron facility at Trieste (Italy), the crystal structure resolution supervision and the solution measurements related with the Ru2:HEWL adducts. In addition, she carried out the formal analysis, discussion, and writing- review and editing. • Dr. Ana E. Sánchez-Peláez, Dr. Santiago Herrero, and Dr. Antonello Merlino were in charge on the visualization, supervision, and writing-review and editing. Dr. Santiago Herrero was also in charge on the project administration and funding acquisition. Chapter V Terán, A., Ferraro, G., Sánchez-Peláez, Ana E., Herrero, S. and Merlino, A. Charge effect in protein metalation reactions by diruthenium complexes. Inorg. Chem. Front. (2023). 10, 5016-5025. https://doi.org/10.1039/D3QI01192E (This article is part of the themed collection: 2023 Inorganic Chemistry Frontiers HOT articles). The experiments included in this article were performed part at Complutense University of Madrid with supervision, orientation and guidance of Dr. Ana E. Sánchez-Peláez and Dr. Santiago Herrero (corresponding author of the paper) and another part in collaboration with Dr. Antonello Merlino (the co-corresponding author of the article) at https://doi.org/10.1039/D3QI01192E 19 the University of Naples Federico II (Naples, Italy). The contributions to the paper are as follows: • Aarón Terán performed the synthesis and characterization of all diruthenium complexes in Complutense University of Madrid. As part of a three-month predoctoral stay in the group of Dr. Antonello Merlino at the University of Naples Federico II, he was able to learn and carry out crystallisation assays, X-ray diffraction data collection in the Elettra synchrotron facility at Trieste (Italy), and the crystal structure resolution of the Ru2:HEWL adducts. In addition, he carried out the investigation, validation, formal analysis, discussion, and the writing of the original draft. • Dr. Giarita Ferraro performed crystallisation assays, X-ray diffraction data collection in the Elettra synchrotron facility at Trieste (Italy), the crystal structure resolution supervision and the solution measurements related with the Ru2:HEWL adducts. In addition, she carried out the formal analysis, discussion, and writing- review and editing. • Dr. Ana E. Sánchez-Peláez, Dr. Santiago Herrero, and Dr. Antonello Merlino were in charge on the visualization, supervision, and writing-review and editing. Dr. Santiago Herrero was also in charge on the project administration and funding acquisition. Chapter VI Terán, A., Ferraro, G., Imbimbo, P., Sánchez-Peláez, Ana E., Monti, D. M., Herrero, S. and Merlino, A. Steric hindrance and charge influence on the cytotoxic activity and protein binding properties of diruthenium complexes. Int. J. Biol. Macromol. (2023). 253. 126666. https://doi.org/10.1016/j.ijbiomac.2023.126666 The experiments included in this article were performed part at Complutense University of Madrid with supervision, orientation and guidance of Dr. Ana E. Sánchez-Peláez and Dr. Santiago Herrero (corresponding author of the paper) and another part in collaboration with Dr. Antonello Merlino (the co-corresponding author of the article) and Dr. Daria Maria Monti at the University of Naples Federico II (Naples, Italy). The contributions to the paper are as follows: https://doi.org/10.1016/j.ijbiomac.2023.126666 20 • Aarón Terán performed the synthesis and characterization of all diruthenium complexes and part of solution measurements (UV-Vis spectroscopy) of Ru2:HEWL adducts in Complutense University of Madrid. As part of a three-month predoctoral stay in the group of Dr. Antonello Merlino at the University of Naples Federico II, he was able to learn and carry out crystallisation assays, X-ray diffraction data collection in the Elettra synchrotron facility at Trieste (Italy), and the crystal structure resolution of the Ru2:HEWL adducts. In addition, he carried out the investigation, validation, formal analysis, and the writing of the original draft. • Dr. Giarita Ferraro performed crystallisation assays, X-ray diffraction data collection in the Elettra synchrotron facility at Trieste (Italy), the crystal structure resolution supervision and the solution measurements (circular dichroism and fluorescence spectroscopy) related with the Ru2:HEWL adducts. In addition, she carried out the formal analysis, discussion, and writing-review and editing. • Dr. Paola Imbimbo performed all the biological essays and the discussion and writing-review and editing. • Dr. Daria Maria Monti was in charge on the supervision, discussion and writing- review and editing of the biological experiments. • Dr. Ana E. Sánchez-Peláez, Dr. Santiago Herrero, and Dr. Antonello Merlino were in charge on the visualization, supervision, and writing-review and editing. Dr. Santiago Herrero was also in charge on the project administration and funding acquisition. Chapter VII As an evidence of the applicability of the knowledge obtained from this doctoral thesis, two articles were published in collaboration with other research groups. . In both cases, the contribution of the PhD candidate was in the synthesis and characterisation of the diruthenium compounds, as well as in the writing and revision of both manuscripts: 1. Inchausti, A., Terán, A., Manchado-Parra, A., Marcos-Galán, A., Perles, J., Cortijo, M., González-Prieto, R., Herrero, S. and Jiménez-Aparicio, R. New insights into progressive ligand replacement from [Ru2Cl(O2CCH3)4]: synthetic strategies and 21 variation in redox potentials and paramagnetic shifts. Dalton Trans. (2022). 51, 9708-9719. https://doi.org/10.1039/d2dt00909a 2. La Manna, S., Di Natale, C., Panzetta, V., Leone, M., Mercurio, Flavia A., Cipollone, I., Monti, M., Netti, P. A., Ferraro, G., Terán, A., Sánchez-Peláez, Ana E., Herrero, S., Merlino, A. and Marasco, D. A diruthenium complex as a potent inhibitor of Amyloid-β aggregation: synergism of mechanisms of action. (2023). Submitted. https://doi.org/10.1039/d2dt00909a 22 The content of this Thesis is organized in eight chapters, with a general abstract and conclusions, and a section of annexes containing supplementary material associated with each publication: Chapter I is a general introduction about the current state of diruthenium chemistry and artificial metalloproteins. Chapter II summarise our previous hypotheses and the main objectives of this dissertation. Chapter III is focused on the synthesis and characterization of water-soluble, and air- and water-stable monosubstituted diruthenium compounds with general formula [Ru2Cl(L- L)(O2CCH3)3] (L-L = DPhF-, D-p-CNPhF-, DAniF-, D-o/m/p-TolF-, and p-TolA-). Chapter IV describes the reactivity of compound [Ru2Cl(D-p-FPhF)(O2CCH3)3] with the model protein HEWL under four different conditions as a first approach to design artificial diruthenium metalloproteins. Chapter V explains the different binding modes between anionic and cationic diruthenium species with the model protein HEWL. Here, the synthesis and characterization of the anionic compounds K2[Ru2(L-L)(CO3)3] (L-L = D-p-FPhF- or DAniF-) is discussed. In Chapter VI, a comparison of coordination modes between compounds [Ru2Cl(DPhF)(O2CCH3)3], K2[Ru2(DPhF)(CO3)3] and [Ru2Cl(DPhF)2(O2CCH3)2] and HEWL is carried out. Here, the synthesis and characterization of K2[Ru2(DPhF)(CO3)3] and the first disubstituted water-soluble compound, [Ru2Cl(DPhF)2(O2CCH3)2], are described. In addition, the first cytotoxic studies of these species are described against HeLa cells. The Chapter VII show the applicability of methodology and knowledge gained from this dissertation. Chapter VIII contains the integrative discussion of the results included in Chapters III-VII. 23 Chapter I. Overview hapter I C Overview 24 25 Metal‒metal bonded complexes are an important research field in inorganic, physical, and theoretical chemistries. The interplay of two or more metal centres, homo- or heterometallics, enables physicochemical properties that are impossible to achieve using monometallic species. The direct electronic communication between metal ions allows tuning the reactivity (activity and selectivity) at the coordination sites, the control of multielectron redox processes, and the modulation of other intrinsic properties such as photoluminescence, stability, solubility, magnetism, and structural conformation.1–3 The field of bimetallic compounds has grown up steadily with a wide range of metallic elements able to form metal‒metal (M‒M) bonds. The preparation of bimetallic complexes has largely been dominated by transition metals whose s, p and d valence orbitals provide a colourful palette of metal‒metal bonding compounds.4–8 The first unambiguous recognition of a M−M bond (single bond) by X-ray diffraction was for [M2(CO)10] (M = Mn and Re) compounds.9 Later, multiple metal‒metal bond chemistry was developed following the discovery of [Re3Cl12]3- (double bond)10–12 and [Re2Cl8]2- (quadruple bond)13 compounds. In the last years, seminal works have revealed a formal bond order of five for a Cr2 2+ complex,14,15 and a bond order of six has been proposed for Mo2 2+ and W2 2+ in gas phase.16 In addition, examples for s-block, p-block and even f-block elements have also been described with interesting catalytic and magnetic properties.8,17–20 Over the years it has been seen that metal‒metal bonds are not just a synthetic matter for inorganic chemists, but they are present in a variety of biological structures. There are few examples that have been experimentally characterised that suggest that metal– metal bonds are formed during the activity of multinuclear metallocofactors such as hydrogenase enzymes which serve as active sites for redox transformations.21–24 Many of these cofactors contain metals that are electronically coupled through bridging ligands such as thiolates, carboxylates, sulphides, or hydroxides. The formation of metal–metal bonds can be viewed as a mechanism by which the cofactor thermodynamically compensates for the loss of a bridging hydride ligand, stabilizing vacant states in the course of catalysis.25,26 These biological systems have served as an inspiration to different groups to obtain synthetic analogues in order to probe the synergistic effect of multiple metals in diverse biological processes.27–30 26 1. Paddlewheel diruthenium compounds (Ru2 5+) One of the most intensively developed groups of metal−metal bonded compounds are paddlewheel-type binuclear complexes. The first example of a binuclear complex with paddlewheel structure was the [Cr2(O2CCH3)4] compound, described in 1844 but structurally characterised in 1970.31 Since then, various kinds of paddlewheel binuclear complexes have been synthesised and studied thoroughly because they exhibit intriguing electrochemical, magnetic, and optical properties derived from the metal–metal interactions.32–35 In 1966, Stephenson and Wilkinson described the first reported synthesis of diruthenium compounds formulated as [Ru2Cl(O2CR)4] (R = -CH3, -CH2CH3 or -CH2CH2CH3), by refluxing RuCl3·3H2O in the appropriate carboxylic/acid anhydride mixture.36 Later, Cotton and co- workers described the first structure of a diruthenium compound via single crystal X-ray diffraction. This analysis showed a paddlewheel-type structure (Figure 1) composed by two ruthenium atoms in different oxidation states: Ru(II) and Ru(III). The short Ru‒Ru bond distance was consistent with a multiple metal‒metal bond.37 The metal‒metal bond order is 2.5 which favours the electronic delocalization between the ruthenium atoms. Therefore, these species must be better considered compounds with average valence (Ru2 5+) than mixed-valence complexes. Carboxylate ligands were coordinated in a bidentate fashion at the equatorial positions and the chloride ligands were at the axial positions bridging adjacent ruthenium dimers. Figure 1. Schematic representation of diruthenium compound structures. Reactive positions are marked with an asterisk (*). Since then, a great variety of diruthenium complexes have been synthesised. Most of the works related with diruthenium chemistry have been focused on the Ru2 5+ species but well-documented homovalent compounds containing Ru2 4+ and Ru2 6+ cores have also 27 been reported.38–47 Although paddlewheel-type structure is the most common arrangement in diruthenium compounds, there are examples of compounds with open- paddlewheel type structure (Figure 1).48 These species are characterised by being reactive species. In the solid-state, diruthenium compounds can show different type of structures. The most common examples usually are discrete species, polymeric chains (linear or zig-zag) or anion-cation pairs (Figure 2). However, the use of ligands with multiple coordination positions or other coordination compounds can lead to more complex structures (2D or 3D networks).49–59 Figure 2. Most common structural types of Ru2 5+ compounds: discrete species (a-d), polymeric chains, linear (e) or zig-zag (f), and anion-cation pairs (g). 28 In 1979, Norman and co-workers, through a detailed theoretical analysis of a Ru2 5+ tetracarboxylate complex model, assigned the electronic configuration of Ru2 5+ compounds as σ2π4δ2(π*δ*)3 (S = 3/2) resulting in a quadruplet ground state (Figure 3).60 The quasi degeneration of the π* and δ* levels cause in most of the cases the presence of three unpaired electrons per dimer. Figure 3. Qualitative molecular orbital diagram of concerning orbitals in the metal-metal bond of Ru2 5+ compounds. The 11 electrons in the Ru2 5+ core have been generally represented as Q8(π*δ*)3 in which Q8 denotes the eight electrons in the underlying σ2π4δ2 core. However, the type of equatorial bridging ligands as well as the type of axial ligands is of great importance because they affect the structural characteristics and electronic structures of these compounds, which in turn have great influence on their magnetic properties. Based on this, three possible electronic configurations may arise depending on the relative energy of π* and δ* orbitals: two a priori indistinguishable quartet states, Q8π*2δ*1/Q8δ*1π*2, and two doublets, Q8π*3 and Q8δ*2π*1. 29 1.1. Properties of Ru2 5+ compounds 1.1.1. Magnetic properties Magnetism in diruthenium chemistry has been a topic of considerable interest for its importance from a theoretical61 and applied perspective such as, for example, in molecular wires,62–64 as magnetic65 or optical66 materials or as sensors.67 The different nature ligands coordinated to the binuclear core (axial or equatorial) are involved in the valence electronic configuration of diruthenium complexes, in their different geometries (linear or zigzag polymer chains)68–70, packing forces,71 torsion angles72,73 and dimensionality (molecular,48,74–76 cationic-anionic units,77,78 1D,79 2D,80,81 or 3D55,58,82). All these factors have been found to give rise to a wide variety of variable temperature magnetic behaviours83 for Ru2 5+ complexes. The most common magnetic behaviour is high-spin (three unpaired electrons) with zero field splitting (ZFS).4,55,68– 70,72,79,82 However, other cases have been reported such as low-spin (one unpaired electron)74 or several intermediate-spin systems48,71 such as quantum mechanical spin admixed,84 Boltzmann distribution between π*3 and (π*δ*)3 homoelectronic configurations75 and physical spin mixtures.73,76 1.1.2. Electrochemical properties The study of electrochemical properties of diruthenium compounds has aroused interest since their discovery due to the huge versatility in the electrochemical behaviour of Ru2 n+ compounds. The electrochemistry of metal‒metal bonded diruthenium complexes has been described as a function of the electrochemical solvent, the oxidation state (Ru2 4+, Ru2 5+ or Ru2 6+) and the type and number of ligands coordinated to the binuclear core.85 The early electrochemical studies only involved compounds with formula [Ru2Cl(O2CR)4] (R = alkyl or aryl group).86 With these species, the research was limited to the use of aqueous media due to poor solubility in organic solvents of the tetracarboxylate compounds. The introduction of long alkyl chains or the replacing of O,O’-donor ligands for O,N- or N,N’-donor bridging ligands enabled electrochemical studies in non-aqueous solvents (i.e., DMSO, CH2Cl2, CHCl3, CH3CN, THF, among others) which allowed to observe the formation of different Ru2 n+ species.85 The majority of redox processes described to 30 date for Ru2 5+ compounds involve reversible redox reactions in a wide potential window which permits to study multiple oxidation and reduction reactions for a given compound. Table 1 summarize the various oxidation states reported for Ru2 5+ complexes containing O,O´-, O,N- and N,N´-donor bridging ligands. Table 1. Electrochemical electron transfer reaction for diruthenium paddlewheel complexes with different ligands. Type of ligand Reduction Oxidation O,O´-donor Ru2 5+ + e‒ ⇄ Ru2 4+ Ru2 5+ ‒ e‒ ⇄ Ru2 6+ O,N-donor Ru2 5+ + e‒ ⇄ Ru2 4+ Ru2 5+ ‒ e‒ ⇄ Ru2 6+ N,N´-donor Ru2 5+ + e‒ ⇄ Ru2 4+ Ru2 4+ + e‒ ⇄ Ru2 3+ Ru2 3+ + e‒ ⇄ Ru2 2+ Ru2 5+ ‒ e‒ ⇄ Ru2 6+ Ru2 6+ ‒ e‒ ⇄ Ru2 7+ Ru2 7+ ‒ e‒ ⇄ Ru2 8+ 1.2. Synthetic aspects The general starting material to obtain new complexes in diruthenium chemistry is the [Ru2Cl(O2CCH3)4] compound. This derivative shows diverse ligand exchange possibilities which has permitted the replacement of the acetate ligands, partially (mono-, di- or tri- substituted species) or totally (tetra-substituted species) (Figure 4). Depending on the degree of substitution and the type of equatorial ligands coordinated to Ru2 5+ core, axial positions may be or may be not occupied. Figure 4. [Ru2Cl(O2CCH3)4] as model starting material for ligand exchange reactions to obtain partially (mono-, di- or tri-substituted) or fully substituted (tetra-substituted) compounds. 31 The precise control of the synthetic conditions (temperature, solvent, atmosphere, or reagents ratio) is essential to obtain diruthenium derivatives. The most common synthetic activation method described in the literature is conventional heating (refluxing solvent) under inert atmosphere (N2 or Ar). However, different articles have demonstrated that the use of inert atmosphere it is not indispensable to obtain Ru2 5+ compounds.58,69,70,72,87,88 The most common equatorial ligands (Figure 5) employed in diruthenium chemistry are bridging ligands (L-L) such as O,O´-donor (e.g., carboxylates,68,86 carbonates,44,56,57,81,82,89– 94 sulfates,95 phosphates95 or phosphonates62,96,97), O,N-donor (e.g., amidates,72 oxypyridinates98 or 2-piperidonates99) or N,N´-donor (e.g., amidinates,100 2- anilinopyridinates101,102 or guanidinates103), among others. Axial positions usually are coordinated by σ- and/or π-donating electron rich ligands such as halides,69,87 aryls,104 or alkynyls63,102,105,106 but different examples with small molecules such as H2O, NO3 -, CN-, CO, N3 -, NCS-, pyridine derivatives, and OP(CH3)3 have also been described.74,101,104,107,108 Figure 5. Common bridging equatorial ligands (L-L) in diruthenium chemistry. The most synthesised compounds are tetrasubstituted species, [Ru2Cl(L-L)4]. Usually, these complexes are obtained by the direct reaction in refluxing solvents or in melted ligand precursors.109–113 In the last years, the application of microwave-assisted solvothermal synthesis (MWS) to achieve tetrasubstituted species has been gained attention.114 The use of microwaves as molecular activator allows to obtain better yields and more environmentally friendly reaction conditions than the ones reported by other conventional methods. This technique leads to an easy and fast substitution of acetate groups from the starting material [Ru2Cl(O2CCH3)4] by different bridging donor ligands. 32 The number of intermediate substitution species, [Ru2Cl(L-L)x(O2CCH3)4-x] (x = 1, 2, 3) is more limited (Figure 4),48,110,111,115–120 and their properties differ significantly from the tetrasubstituted diruthenium compounds in terms of solubility, geometry, electrochemical properties and reactivity. The lack of abundant literature regarding the synthesis of intermediate species is due to the synthetic challenge which often result the isolation of these products. Their preparation is based on a very precise temperature and time control,48,110,119 the use of bulky ligands that limit the reaction rate109,111,118 or the use of chromatographic techniques for the separation of the different substitution intermediates.101,116,118,120 It has been shown that the substitution of O,O´-donor ligands by N,N´- and N,O-donor ligands gives rise to species with higher stability, but at the expense of decreasing the water solubility of the starting compounds. There are a few examples of water-soluble monosubstituted diruthenium species that were obtained through a reflux synthetic method with low yields. The results remark the relevance on the preparation of series of water-soluble compounds because it is key to develop complexes with high cytotoxicity and selectivity toward certain cancer cell lines.101 1.3. Applications of diruthenium compounds 1.3.1. Paddlewheel diruthenium compounds (Ru2 5+) in catalysis Diruthenium complexes (chiral or achiral) have been used as homogeneous or heterogeneous catalysts in different reactions. The chemistry behind this process derived from the different equatorial bridging ligands coordinated to the diruthenium core which offers a great richness in the physicochemical properties of these species.121 Diruthenium complexes have showed significant catalytic properties, particularly in redox processes, either as a catalyst for reduction or, more commonly, for oxidation reactions. This dual behaviour is ascribed to the mixed valence nature of the binuclear core, containing formally two ruthenium atoms in +2 and +3 oxidation states. The catalytic activity and selectivity of these complexes are usually compared with dirhodium catalysts. The higher oxidation states in diruthenium compounds with respect to dirhodium 33 counterparts (Rh2 4+), suggest that diruthenium species could exhibit much higher Lewis acidity, especially in their cationic form, resulting in different catalytic activity. In 1970, it was described the first example of catalytic activity for the diruthenium compound [Ru2Cl(O2CCH3)4]. In the presence of triphenylphosphine, this complex is an active catalyst for the homogeneous hydrogenation of alkenes, alkynes, and other unsaturated substances.122 To the best of our knowledge, this is the unique reduction reaction involving a diruthenium complex. Since then, different organic transformations with diruthenium compounds have been studied. The most frequent application of Ru2 5+ complexes are oxidative transformations. Within this category we can highlight the oxidation of alcohols to aldehydes, ketones, acids or lactones,96,117,123 the oxygenation of organic sulphides to sulfoxides or sulfones,124–128 the oxidation transformation of secondary amines to imines,129 the oxidative cleavage of olefins,130 and the oxidative inter- or intramolecular amination of C–H bonds.6,98 There are examples where diruthenium complexes are involved in the catalysis of skeletal reorganization of 1,6-enynes, leading to the production of 1-vinylcyclopentene derivatives,131 the photochemical production of hydrogen from water132 or the electrochemical conversion of ammonia to dinitrogen in a direct ammonia fuel cell.133 The potential of diruthenium catalysts as a platform for asymmetric catalysis has been explored. Chiral tetrasubstituted diruthenium complexes bearing amidate or carboxylate ligands (Figure 6) have been synthesised and characterised.99,134,135 They show remarkable catalytic performance and robustness in hetero-Diels-Alder reactions, asymmetric intramolecular C−H amination reactions and asymmetric cyclopropanation reactions of olefins with high enantioselectivity.99 In addition, catalytic asymmetric nitrene-transfer reactions with enol silyl ethers134 and enantioselective cyclopropanation with donor/acceptor carbenes derived from aryldiazoacetates have been studied.135 The substrate scope of the diruthenium catalyst was superior to that of analogous chiral dirhodium catalysts. The evaluation of other first-row transition-metal congener, Cu2 4+ and Co2 4+, showed little to no asymmetric catalysis induction. These results corroborate the potential of diruthenium compounds as promising chiral catalysts. 34 Figure 6. Structures of chiral ligands (S)-PTAD ((S)-adamantan-1-yl-(1,3-dioxo-1,3- dihydro-isoindol-2-yl)-acetate), (S)-TCPTAD ((S)-adamantan-1-yl-(4,5,6,7-tetrachloro-1,3- dioxo-1,3-dihydro-isoindol-2-yl)-acetate), (S)-PTTL (N-phthaloyl-(S)-tert-leucinate), (S)- TPPTTL (N-tetraphenylphthaloyl-(S)-tert-leucinate), (S)-NTTL ((S)-2-(1,3-dioxo-1H,3H- benzo[de]isoquinolin-2-yl)-3-dimethyl-butyrate), (S)-TCPTTL (N-tetrachlorophthaloyl-(S)- tert-leucinate), and (S)-BPTPI ((S)-3-(benzene-fused-phthalimido)-2-piperidonate). In 2017, the catalytic activity of the sole diruthenium artificial metalloenzyme was studied.136 The metalloprotein [HEWL/Ru2(O2CCH3)2(OH2)2] (Figure 7) shows catalytic activity in the green aerobic oxidation of hydroxylamines to nitrones. The same activity was reported for the complex [Ru2Cl(O2CCH3)4], but the protein derivative suppresses the formation of by-products and improve the regioselectivity of the process. This was the first proof of concept of the catalytic activity of diruthenium metalloproteins. Figure 7. Overall structure of [HEWL/Ru2(O2CCH3)2] metalloprotein (PDB: 4OOO).137 35 1.3.2. Diruthenium compounds as drug carrier: biological activity, molecular targets, and mechanisms of action The serendipitous discovery of the anticancer activity of cisplatin, cis-[PtCl2(NH3)2], and its approval by the Food and Drug Administration (FDA) for clinical use as chemotherapy drug138,139, set a milestone in the employment of metal ions in biomedicine fields for biosensing, disease diagnosis and treatment.140–144 Metal-based drugs are interesting alternatives treatments to organic molecules. They offer novel chemistry, including different types of ligand substitution, metal- and ligand- based redox processes, and catalytic properties. These therapeutics are often prodrugs which undergo activation en route to or at the target site. This can involve dissociation or displacement mechanism of one or more labile ligands, chelate ring-opening, or changes in the oxidation state of the metal atom and/or ligand(s). Alternatively, an external stimulus (e.g., light, radiation, sound or heat) can selectively activate metallodrugs at the target site.145 In this regard, paddlewheel Ru2 5+ complexes have been used as molecular building blocks to develop new metallodrugs. Diverse complexes have been synthesised varying the axial or equatorial ligands.146–148 The cytotoxic properties of these compounds have been studied mainly in diruthenium tetracarboxylate complexes. The first compounds studied were [Ru2Cl(O2CCH3)4] and [Ru2Cl(O2CCH2CH3)4], which showed slight antineoplastic activity against P388 leukemia cell lines.149 The introduction of solubilising groups in the carboxylate ligands, as in the complex [Ru2(O2C-m-C6H4SO3)4]3-, improved the cytotoxic properties against CoLo 320DM cancer cells.150 The lower water-solubility of the first diruthenium compounds may have affected their antioncogenic properties. Further efforts to increase the biological activity of these complexes gave rise to replace these carboxylates by other biologically active carboxylate ligands. For example, diruthenium derivatives containing nonsteroidal anti-inflammatory drugs (NSAIDs) such as ibuprofenate (Ibp), aspirinate (Aspi), naproxenate (Npx), ketoprofenate (Ket) and indomethacinate (Ind) (Figure 8) have displayed a wide range of pharmacological properties for these derivatives. NSAIDs are coordinated through their carboxylate group 36 to the equatorial positions of the diruthenium core to give rise to [Ru2(NSAID)4]+ complexes. Figure 8. Chemical structure of nonsteroidal anti-inflammatory drugs (NSAIDs) after deprotonation: aspirinate (Aspi), ibuprofenate (Ibp), indomethacinate (Ind), ketoprofenate (Ket), naproxenate (Npx). These species have clearly higher cytotoxic effects than the free NSAID molecules. In addition, their properties differ greatly from those of their parent drugs. This difference is related to the stability of diruthenium-NSAID complex in biological media. Table 2 summarises the Ru2 5+-NSAIDs compounds tested in different biological assays. Table 2. Ru2 5+-NSAIDs compounds and their biological targets. Compound Biological targets Reference [Ru2Cl(Ibp)4] Inhibition of development of carrageenin-induced edema in rats 151 [Ru2Cl(Ibp)4] Hep2 human larynx (non-activity), T24/83 human bladder tumour (non-activity) and C6 rat glioma cell (best results for naproxen and ibuprofen metallodrugs) 152 [Ru2Cl(Aspi)4] [Ru2(Npx)4(H2O)2](PF6) [Ru2(Ind)4(H2O)2](PF6) [Ru2Cl(Ibp)4] C6 rat glioma 153 [Ru2Cl(Ket)4] Human colon carcinoma cells HT-29 and Caco-2 154 [Ru2Cl(Ibp)4] [Ru2(Npx)4(H2O)2](PF6) [Ru2Cl(Ibp)4] Human glioma cell with p53 wild-type and p53 mutant cells 155 [Ru2Cl(Ibp)4] U87MG and A172 human glioma cell (best results for [Ru2Cl(Ibp)4]) 156 [Ru2(Ibp)4(CF3SO3)] g[Ru2(Ibp)4(EtOH)2](PF6) 37 The coordination of γ-linolenic acid (GLA, Figure 9) after deprotonation to the Ru2 5+ core give rise to obtain the [Ru2Cl(GLA)4] compound. This derivative inhibits in vitro and in vivo C6 rat glioma cell proliferation. This complex causes alterations in the tumour morphology, related to effective intracellular absorption and collagen fiber-binding in the extracellular matrix.157 Figure 9. Chemical structure of γ-linolenic acid (GLA). Two different indolylglyoxylyl dipeptide isomers, EB106 and EB776 (Figure 10), were coordinated to the diruthenium core to obtain [Ru2Cl(EB776)4] and [Ru2Cl(EB106)4] complexes (EB776 is the deprotonated form of (2-phenylindol-3-yl)glyoxyl-L- phenylalanine-L-leucine and EB106 is the deprotonated form of (2-phenylindol-3- yl)glyoxyl-L-leucine-L-phenylalanine). The results showed no antiproliferative activity for [Ru2Cl(EB106)4] and more activity against glioblastoma model U87MG for [Ru2Cl(EB776)4] compound. This difference is related with the steric effect of ligands around the diruthenium core.158,159 Figure 10. Chemical structure of indolylglyoxylyl dipeptides EB106 and EB776 after deprotonation. But the low solubility of these diruthenium metallodrugs is still their major limitation for clinical employment. Generally, for cellular experiments, diruthenium compounds must be solubilised in organic solvents such as dimethyl sulfoxide (DMSO) or ethanol.158–162 In last years, to overcome the low water-solubility problem and to facilitate the internalization in cells, encapsulation methods have been tested with [Ru2Cl(Ibp)4] and 38 [Ru2Cl(Npx)4] complexes using injectable solid polymer-lipid nanoparticles (SPLNs)160 and terpolymer-lipid nanoparticles (TPLNs).161 The Ru2 5+-SPLNs showed enhanced cytotoxicity in relation with the corresponding free metallodrugs in triple negative breast (EMT6 and MDA-MB-231) and prostate (DU145) cancer cells in vitro while Ru2 5+-TPLN nanoformulations improve the medicinal efficacy against glioblastoma cancer cells. The comparison between the activities of the free-drugs, free-metallodrugs and their encapsulated forms points out that the encapsulation improves their anticancer activity and that the bimetallic unit is not a mere drug carrier but plays a key role in the cytotoxic activity. It seems that there is a synergistic effect between the drug and the diruthenium core and that the pharmacokinetic properties of the metallodrugs are enhanced due to the encapsulation process. Besides applications in cancer research, compounds based on [Ru2Cl(DPhF)3(L-L)] and [Ru2Cl(DAniF)3(L-L)] motifs (DPhF- = N,N´-diphenylformamidinate and DAniF- = N,N´-bis(p- methoxy)phenylformamidinate) have been developed to carry bioactive carboxylate ligands (L-L) called auxins: indole-3-acetate (IAA), 2,4-dichlorophenoxyacetate (2,4-D), and 1-naphthaleneacetate (NAA) (Figure 11).163,164 Figure 11. Schematic representation of [Ru2Cl(DPhF)3(L-L)] and [Ru2Cl(DAniF)3(L-L)] compounds. Auxins molecules are phytohormones involved in different processes during plant growth and development and regulating cell proliferation and differentiation in various tissues. Biological assays in Arabidopsis thaliana transgenic plants have demonstrated that these compounds are stable at physiological pH, but the release of the auxins is faster at slightly 39 acidic media (pH = 6.5). The study of the auxins release, studied using a fluorimetric quantitative assay, allows determining the influence of different formamidinate ancillary ligands and the nature of the outgoing auxin ligand in the release process. These results reinforce the idea of employing diruthenium molecules as molecular carriers. The mechanism of action of diruthenium metallodrugs is still unknown, but accumulated evidence suggests that the biological function is highly related with their interactions with biomolecules,148,157 which may be associated to metallodrug transformations (reduction- oxidation and/or ligand substitution reactions). Different research groups have studied experimental and theoretical models to discern the coordination capacity of Ru2 5+ compounds with biomolecules. To study potential interactions of diruthenium complexes with DNA, three diruthenium complexes of the form [Ru2(O2CCH3)4L2](PF6), with the biologically relevant axial ligands (L = imidazole, 7-azaindole and caffeine, Figure 12), were synthesised and characterised by single-crystal X-ray diffraction.165 Different hydrogen-bonding interactions involving the axial ligands were described for these compounds: a) Intramolecular interactions with the diruthenium tetraacetate core. b) Intermolecular interactions with counterions. c) Intermolecular interactions with any molecules of solvation that may be present. d) Intermolecular interactions with adjacent diruthenium units. Authors suggest that these would be the most likely interactions that could be seen when these compounds bind the nucleotide bases of DNA. Figure 12. Chemical structure of imidazole, 7-azaindole and caffeine ligands. Black dashed lines show the coordination position to the axial positions of the diruthenium core. Pink dashed lines show atoms involved in hydrogen bonding. 40 The interactions of the open-paddlewheel compound [Ru2Cl2(DPhF)3] with RNA using two viral internal ribosome entry site (IRES) elements were analysed by SHAPE technique. This method allows to analyse the secondary structure of long RNA molecules in solution. The results support the use of [Ru2Cl2(DPhF)3] as RNA probing compound because it preferentially attacks residues located at or close to the junctions of the RNA structure unlike other chemical reagents (e.g., NMIA, IA, 1M7, and 1M6) which join to bulges and loops in the structure.166 The coordination capacity of [Ru2Cl2(DPhF)3] to different nitrogenous bases, nucleosides and nucleotides (Figure 13) such as cytosine (Hcyto), cytidine (Hcyti), cytidine 2´,3´-cyclic monophosphate sodium salt (NacCMP), adenine (Hade), adenosine (Haden) and adenosine 3´,5´-cyclic monophosphate (HcAMP) has been studied to clarify the bonding mode of this compound to RNA.167 The single-crystal X-ray diffraction results for [Ru2Cl(DPhF)3(cyto)] and [Ru2Cl(DPhF)3(ade)] compounds revealed a preferential binding as N,N´-bridging ligands of the nitrogenous bases to the equatorial positions of the diruthenium core. The axial positions are still available for additional interactions with other residues that could explain the preference of [Ru2Cl2(DPhF)3] towards RNA junctions. Figure 13. Chemical structure of cytosine (Hcyto), cytidine (Hcyti), cytidine 2´,3´-cyclic monophosphate sodium salt (NacCMP), adenine (Hade), adenosine (Haden) and adenosine 3´,5´-cyclic monophosphate (HcAMP). Black dashed lines mark the coordination positions to the equatorial positions of the diruthenium core. Proteins are relevant biomolecules as targets for metallodrugs. The side chains of different amino acids possess appropriate donor atoms such as nitrogen, oxygen, or sulphur to interact with a metallodrug (Figure 14). 41 Figure 14. Structure of the 21 amino acids needed to make all the proteins found in the human body. The starting compound [Ru2Cl(O2CCH3)4] has been studied as a model complex for thermodynamic and kinetic reaction studies with amino acids, biological relevant reducing agents, and nucleophilic protein sites. The dissolution of [Ru2Cl(O2CCH3)4] in water leads mainly to [Ru2(O2CCH3)4(OH2)2]+ complex. This species can undergo axial ligand substitution reactions by amino acid ligands (His, Cys, Trp, and Gly).168 Calculations showed that these reactions are thermodynamically favourable and are enthalpy driven. Results showed that the [Ru2(O2CCH3)4]+ unit is retained in the presence of these amino acids. When reducing agents such as ascorbic acid or glutathione are in the medium, an axial coordination of these nucleophiles takes place.169 This reaction is favoured for the [Ru2(O2CCH3)4(OH2)2]+ complex, because of the stronger nucleophile nature of the chloride axial ligand in [Ru2Cl(O2CCH3)4] compound. The coordination of reducing agents is followed by an intramolecular electron transfer process, reduction of the diruthenium 42 core from Ru2 5+ to Ru2 4+ and oxidation of the reducing agents. The mechanisms for these reactions were proposed as a dissociative interchange mechanism. The reaction between [Ru2Cl(O2CCH3)4] and the model protein hen egg white lysozyme (HEWL) was investigated through ESI-MS, UV-Vis spectroscopy, and X-ray diffraction analysis.137 The complex binds the protein through the equatorial positions, exchanging one of the acetate ligands in favour of the side chain of Asp101 or Asp119 residues (Figure 15). A second acetate ligand is replaced by two water molecules and the axial positions are also occupied by water molecules. Figure 15. Details of the binding sites of [HEWL/Ru2(O2CCH3)2] metalloprotein at the side chain of Asp101 (a) and Asp119 (b). 2Fo − Fc electron density maps are contoured at 1.0 σ (blue). Axial water molecules are omitted for clarity (PDB code: 4OOO).137 It has been described that HSA plays a key role in the pharmacological properties and efficacy of mononuclear ruthenium drugs. Human serum albumin (HSA) is the most abundant serum protein, which transport bioactive molecules from blood stream to specific targets. Therefore, three diruthenium complexes, [Ru2Cl(O2CCH3)4], [Ru2Cl(Ibp)4] (Ibp = ibuprofenate), and [Ru2Cl(Ket)4] (Ket = ketoprofenate), were tested with HSA as a potential carrier in the blood plasma.170 These complexes seem to interact with the protein through electrostatic forces and induce conformational changes on his secondary structure. Authors described that these complexes form adducts with HSA via non- covalent interactions. The molecular affinity of [Ru2(O2CCH3)4(OH2)2]+, [Ru2Cl(O2CCH3)4(OH2)], and [Ru2Cl(O2CCH3)4(OH)]- for the binding to selected nucleophile molecules (Arg, Cys, His, 43 Lys, Sec) was determined by Density Functional Theory (DFT) approaches.171,172 The authors proposed that the axial ligands are promptly substituted by suitable binding sites with high exergonicity, thus anchoring the diruthenium core to the surface of the protein. Acidic environments favour reactions involving the exchange of axial water and selectively targeting Arg-, Cys-, and Sec-. The presence of an axial hydroxide, following the water coordination, yields the chloride substitution possible, but only at high pH that favours the hydroxo over the aquo form. These studies suggest that the chemoselectivity of diruthenium compounds in a biological context is related with steric and charge effects around the binuclear core. All these studies evidence that diruthenium metallodrugs are very valuable systems due to their structural features and pharmacological properties. This doctoral thesis intends to continue on the path of understanding and further developing the potential of these compounds. 2. Artificial metalloproteins Metalloproteins perform diverse functions in nature that are essential to life, including electron transfer,173 transition metal ion transport/storage,174 gas sensing/transport175 and catalytic transformations of complex molecules.176 This impressive range of functions is performed with the limited toolbox of earth-abundant and bioavailable metals and biosynthetically accessible ligands.177,178 The design of artificial metalloproteins is a powerful approach for understanding the possible metal sites of inorganic compounds in biology through reproduction of coordination environments within a peptidic platform. The engineering fundamental requisites for constructing artificial metalloproteins are based on the field of bioinorganic coordination chemistry. To succeed in this area, it is necessary to understand the thermodynamic balance between the protein stability and metal ion coordination preferences. Two main approaches are used to develop artificial metalloproteins: protein redesign or de novo protein design. Protein redesign encompasses a variety of approaches including redesign using non-metalloprotein scaffolds as a starting point and using existing metal sites and redesigning them into new metal sites with altered properties. On the other 44 hand, de novo metalloprotein design, requires both design of a unique protein structure and the incorporation of the metal site(s).179,180 This dissertation is focused on protein redesign. Designing metalloproteins from native proteins provides many more scaffold choices than de novo design. In addition, native proteins can be easier to crystallize, making three- dimensional characterization possible.181 The insertion of metal compounds into a protein system can be accomplished by: a) Formation of a direct dative covalent bond with an amino acid ligand. b) Covalent attachment to the protein by a bioconjugate moiety. c) Noncovalent anchoring in the protein environment. The need to understand metal-based structure–function relationships is a timeless endeavour, not only in the study of metalloproteins but also to study emerging bio- inspired, semi-synthetic and de novo protein systems, as well as metallodrugs and for the development of catalytic metallodrugs.182 Different model proteins, such as ubiquitin, lysozyme, cytochrome C, proteinase K and bovine pancreatic ribonuclease, have been used to understand how protein metalation takes place. The binding responses of different metal compounds to model proteins have been studied by a diverse spectrum of strategies with the aim of profiling interactions between them. In this context, two atomic resolution techniques stand out, nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography. On one hand, NMR spectroscopy is very useful for atomic-resolution structural biology of flexible biomolecules and enables their characterization in aqueous solution at ambient temperatures. In contrast, X-ray crystallography requires a well-ordered crystal of the biomolecule in which each unit cell has the same static arrangement of atoms. However, X-ray crystallography can resolve atomic-resolution structures of small to very large biomolecules and biomolecular complexes, whereas NMR spectroscopy has historically been limited by the biomolecule size. This thesis is based on the use of protein X-ray crystallography. This technique is a well-stablished biophysical method which provides 45 atomic resolution structures, crucial to understand the molecular mechanisms and the specific biomolecular targets for the analysed metal-based compounds.183,184 The key step in crystallographic analysis is the preparation of high-quality protein crystals. Several techniques are used to obtain high-quality protein crystals including the application of magnetic fields, microgravity, solution flow, gel-based growth media, and the ceiling crystallisation method.185 The hanging-drop vapour diffusion has been chosen in this thesis as crystal growth technique (Figure 16). Figure 16. Schematic representation of protein crystallisation using the hanging drop vapour diffusion technique. In the hanging-drop method, the droplet is suspended on a siliconized coverslip that is used to seal the well in which the reservoir solution is placed. This method relies on the principle of vapour diffusion between a droplet containing the protein and the precipitant. Initially, the protein concentration in the droplet is low, but as soon as the osmolarity of droplet and the reservoir solution are equal, nucleation and crystal growth take place.186 In this research, the selected model protein is hen egg white lysozyme (HEWL). Lysozyme was the first enzyme structure to be solved by X-ray crystallography and has subsequently become a widely used model protein in a variety of contexts.187,188 It is a small (129 residues, 14.3 kDa) and stable protein that in its hydrogenated form can be acquired at low cost. Lysozyme has been used as a model protein to conduct amyloid research, crystallography, and nanomaterial research.189,190 This protein has also served as model for probing the structural interactions of different medicinal metal complexes with proteins because its relative propensity to crystallise under a wide range of crystallisation conditions which allows structural studies using X-ray diffraction.191–195 46 This protein could be a good alternative for understanding how the charge and steric hindrance of diruthenium compounds affect to their protein binding properties and further to examine the mechanism underlying the interaction(s) between diruthenium species and biomolecules. 47 Chapter II. Aims and objectives hapter II C Aims and objectives 48 49 The general purpose of this thesis is focused on understanding the molecular basis of diruthenium compounds (Ru2 5+) in a biological system, in particular, the interaction of those compounds with proteins. The following milestones are required to achieve that goal: • The synthesis of diruthenium derivatives with different characteristics for their interaction with the model protein hen egg white lysozyme (HEWL). • The preparation of Ru2-protein adducts or artificial diruthenium metalloproteins to study the binding response modulation of diruthenium compounds into a protein structure. The process to reach the target of this thesis is divided into two main sections: • Synthesis and characterization of water-soluble and air- and water-stable diruthenium complexes, changing their charge and number of bulky groups around the diruthenium core. • Study of the influence of charge and steric effects on the protein binding properties of the proposed diruthenium compounds. Previous studies about the interaction of diruthenium compounds with biomolecules had remarked that water solubility is a key to obtain high reactive species. The number of diruthenium species that are water-soluble was limited, and those that were soluble in water exhibited low stability. Therefore, it was necessary to focus on which species could be soluble in water and stable at the same time. For this purpose, the following concepts were put forward: • Formamidinate ligands (L-L) are widely applied to obtain diruthenium complexes because they can act as bridging equatorial ligands by the virtue of the two nitrogen atoms present in its structure. Formamidinates are a desirable ligand choice in the pursuit of designing robust functional materials. • Diruthenium compounds [Ru2Cl(L-L)x(O2CCH3)4-x] containing three or four formamidinates (x = 3, 4) have been found to be insoluble in water. However, the water solubility of compounds with one or two L-L ligands (x = 1, 2) was not known. The hypothesis was that the synthesis of compounds bearing one or even 50 two formamidinate ligands can allow to obtain complexes with high stability in solution and may be still soluble in aqueous solutions like [Ru2Cl(O2CCH3)4]. In this work, a family of formamidine ligand-precursors with different substitution patterns are synthesised to obtain formamidinate complexes with higher steric hindrance. The functional groups of formamidines (R) also influence the physicochemical properties of the Ru2 5+ core. In general, formamidine ligand-precursors can be synthesised following a well-established condensation reaction between the corresponding aniline and triethyl orthoformate, generating off-white microcrystalline solids (Scheme 1).196 Scheme 1. Synthesis of N,N´-diarylformamidine ligand-precursors. The planning synthesis of target diruthenium compounds is shown in Scheme 2: • The first step corresponds to the synthesis of monosubstituted compounds (x = 1) with general formula [Ru2Cl(L-L)(O2CCH3)3] (L-L = formamidinate ligand). However, the lack of a general and simple method to isolate those compounds with good yields made necessary at the beginning of the research to explore new synthetic pathways. This type of species usually generates cationic diruthenium complexes in coordinating solvents due to the easy substitution of the axial chloride ligand. • All studies on the interaction of diruthenium compounds with biomolecules used small cationic species. In addition to the steric effect, it is interesting to study the charge effect on the binding response of diruthenium complexes. For this reason, it was planned to study the substitution of the acetate by carbonate ligands in [Ru2Cl(O2CCH3)4] and the monosubstituted complexes to obtain K3[Ru2(CO3)4] and K2[Ru2(L-L)(CO3)3] compounds. The anionic diruthenium complexes with different charge and steric hindrance can allow the evaluation of the possible charge-dependent interactions of these species. Again, no heteroleptic diruthenium compounds with formamidinate and carbonate ligands were 51 available, so it was necessary to develop an accurate synthesis methodology for this type of compounds. • The water-soluble disubstituted diruthenium species (x = 2) with formula [Ru2Cl(L-L)2(O2CCH3)2] is also contemplated in this study. The substitution of two acetate by formamidinate ligands from the starting material [Ru2Cl(O2CCH3)4] generates molecules with higher steric hindrance around the diruthenium core, which may result in a different protein binding selectivity. As with monosubstituted complexes, the availability of disubstituted compounds was rather limited and it was necessary to search a new methodology to obtain them in high yield. Scheme 2. Target-oriented synthesis of diruthenium complexes [Ru2Cl(L-L)(O2CCH3)3], [Ru2Cl(L-L)2(O2CCH3)2], and K2[Ru2(L-L)(CO3)3]. L-L = formamidinate ligand. All compounds are characterised through a multi-technique approach including elemental analysis, mass spectrometry, Fourier-transform infrared spectroscopy, electrochemistry, X-ray crystallography, magnetic measurements, and UV-Vis 52 spectroscopy. The various techniques allow the determination of the composition, structure, stability and physicochemical properties. The study of these diruthenium complexes with different steric hindrance and charge in solution allows understanding the modulation of the binding properties of diruthenium compounds respect to biomolecules. Biophysical techniques such as UV-Vis absorption spectroscopy, fluorescence spectroscopy, and circular dichroism are used to study in solution the interaction and effect of diruthenium compounds on the protein structure. Crystallisation experiments are set up to determine the three-dimensional structure of the metal-protein adducts. Crystals of the proteins are grown by the hanging drop vapour diffusion method and the metal-protein adducts are obtained by a soaking procedure. The interactions between the model protein and diruthenium compounds are analysed by X-ray diffraction. 53 Chapter III. Ultrasound-assisted synthesis of water-soluble monosubstituted diruthenium compounds hapter III C Ultrasound-assisted synthesis of water-soluble monosubstituted diruthenium compounds 54 60 Abstract A novel synthetic route based on the use of ultrasound-assisted synthesis (USS) has been developed to find the best experimental conditions to prepare elusive monosubstituted diruthenium complexes. Seven monosubstituted diruthenium complexes, [Ru2Cl(DAniF)(O2CCH3)3] (1), [Ru2Cl(DPhF)(O2CCH3)3] (2), [Ru2Cl(D-p-CNPhF)(O2CCH3)3] (3), [Ru2Cl(D-o-TolF)(O2CCH3)3] (4), [Ru2Cl(D-m-TolF)(O2CCH3)3] (5), [Ru2Cl(D-p-TolF)(O2CCH3)3] (6) and [Ru2Cl(p- TolA)(O2CCH3)3] (7) (DAniF- = N,N´-bis(4-anisyl)formamidinate; DPhF- = N,N´- diphenylformamidinate; D-p-CNPhF- = N,N´-bis(4-cyanophenyl)formamidinate; D-o/m/p- TolF- = N,N´-bis(2/3/4-tolyl)formamidinate; p-TolA- = N-4-tolylamidate), have been synthesised and characterised. Two different types of ligands, formamidinate (1-6) and amidate (7), have been used to check the generality of this procedure for the obtention of monosubstituted complexes. Five new compounds (2-6) have been obtained, the synthesis of a previously described compound (1) has been improved, and an unprecedented monoamidate complex has been achieved (7). This method is simpler and greener than the tedious procedures previously described in the literature. The substitution of one acetate ligand by a higher Lewis base retaining the metal‒metal bond has permitted to obtain water-soluble and stable complexes with good yields in a short period of time. The redox properties of all compounds were analysed using cyclic voltammetry. The substitution of one acetate bridging ligand (O,O´-donor) by one formamidinate (N,N´- donor) or amidate (N,O-donor) bridging ligand increases the electron density on the diruthenium core and also favour the reversibility of the redox process Ru2 5+/Ru2 4+. The crystal structures of compounds 3 and 7 have been solved by single crystal X-ray diffraction. These compounds show the typical paddlewheel structure with three acetate ligands and one formamidinate (3) or amidate (7) bridging ligand at the equatorial positions. The axial positions are occupied by the chloride ligand giving rise to one- dimensional polymer structures that were previously unknown for monosubstituted compounds. 61 Ultrasonics Sonochemistry 80 (2021) 105828 Available online 12 November 2021 1350-4177/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Ultrasound-assisted synthesis of water-soluble monosubstituted diruthenium compounds Aarón Terán , Miguel Cortijo , Ángel Gutiérrez , Ana E. Sánchez-Peláez *, Santiago Herrero *, Reyes Jiménez-Aparicio Universidad Complutense de Madrid, Facultad de Ciencias Químicas, Departamento de Química Inorgánica, Avda. Complutense s/n, 28040 Madrid, Spain A R T I C L E I N F O Dedicated to the memory of Prof. Carlos A. Murillo. Keywords: Sonochemistry Substitution reaction Diruthenium compounds Water-soluble compounds A B S T R A C T The elusive monosubstituted diruthenium complexes [Ru2Cl(DAniF)(O2CMe)3] (1), [Ru2Cl(DPhF)(O2CMe)3] (2), [Ru2Cl(D-p-CNPhF)(O2CMe)3] (3), [Ru2Cl(D-o-TolF)(O2CMe)3] (4), [Ru2Cl(D-m-TolF)(O2CMe)3] (5), [Ru2Cl(D- p-TolF)(O2CMe)3] (6) and [Ru2Cl(p-TolA)(O2CMe)3] (7) have been synthesized using for the first time ultrasound-assisted synthesis to carry out a substitution reaction in metal–metal bonded dinuclear compounds (DAniF− = N,N′-bis(4-anisyl)formamidinate; DPhF− = N,N′-diphenylformamidinate; D-p-CNPhF− = N,N′-bis(4- cyanophenyl)formamidinate; D-o/m/p-TolF− = N,N′-bis(2/3/4-tolyl)formamidinate; p-TolA− = N-4-tolylami date). This is a simpler and greener method than the tedious procedures described in the literature, and it has permitted to obtain water-soluble complexes with good yields in a short period of time. A synthetic study has been implemented to find the best experimental conditions to prepare compounds 1–7. Two different types of ligands, formamidinate and amidate, have been used to check the generality of the method for the preparation of monosubstituted complexes. Five new compounds (2–6) have been obtained using a formamidinate ligand, the synthesis of the previously described compound 1 has been improved, and an unprecedented monoamidate complex has been achieved (7). The crystal structures of compounds 3 and 7 have been solved by single crystal X- ray diffraction. These compounds show the typical paddlewheel structure with three acetate ligands and one formamidinate (3) or amidate (7) bridging ligand at the equatorial positions. The axial positions are occupied by the chloride ligand giving rise to one-dimensional polymer structures that were previously unknown for mon osubstituted compounds. 1. Introduction Since mid-1960’s when Stephenson and Wilkinson [1] described the synthesis of the first mixed-valent [Ru2Cl(O2CR)4] (R = alkyl) com pound and Cotton and col [2] the first crystal structure, a big number of Ru2 5+ paddlewheel diruthenium complexes with Ru-Ru bond order of 2.5 have been synthesized and characterized [3–8]. These compounds have attracted interest for their practical applications in catalysts [9–16], biomedicine [17–20], biotechnology [21–26] or by their po tential in material science due to their unique magnetic [27–36] and redox properties [37]. The precise control of synthetic procedures is essential for the obtention of new diruthenium derivates. The most common synthetic routes are conventional methods, like the direct reaction in refluxing solvents or in melted ligand precursors. In the last decade, our research group has investigated the application of microwave-assisted synthesis (MWS) as a greener molecular activation method to prepare dir uthenium compounds [38–45]. The MWS has demonstrated to be very useful to substitute the four acetate ligands in [Ru2Cl(O2CMe)4] by other bridging ligands. However, this method focuses mainly on obtaining fully substituted species, [Ru2Cl(L-L)4] (L-L = O,O-donor, N,Ń-donor or N,O-donor bridging ligands), and it does not generally lead to the in termediate substitution species, [Ru2Cl(L-L)x(O2CMe)4-x] (x = 1, 2, 3). The properties of monosubstituted complexes, [Ru2Cl(L-L) (O2CMe)3], are quite unknown because they have been especially elusive with any synthetic procedure. With N,Ń-donor ligands there are two examples of monoformamidinatodiruthenium(II,III) complexes [7,8], one 2-amino-4,6-dimethylpyridinate derivative [46], one 2- amino-4,6-dimethylpyrimidinate compound [47] and several mono anilinopyridinatodiruthenium(II,III) complexes [48,49]. Their obten tion, based on a conventional synthesis method, is facilitated using a steric hindrance ligand or requires chromatographic column * Corresponding authors. E-mail addresses: aesanche@ucm.es (A.E. Sánchez-Peláez), sherrero@ucm.es (S. Herrero). Contents lists available at ScienceDirect Ultrasonics Sonochemistry journal homepage: www.elsevier.com/locate/ultson https://doi.org/10.1016/j.ultsonch.2021.105828 Received 29 September 2021; Received in revised form 29 October 2021; Accepted 11 November 2021 mailto:aesanche@ucm.es mailto:sherrero@ucm.es www.sciencedirect.com/science/journal/13504177 https://www.elsevier.com/locate/ultson https://doi.org/10.1016/j.ultsonch.2021.105828 https://doi.org/10.1016/j.ultsonch.2021.105828 https://doi.org/10.1016/j.ultsonch.2021.105828 http://crossmark.crossref.org/dialog/?doi=10.1016/j.ultsonch.2021.105828&domain=pdf http://creativecommons.org/licenses/by-nc-nd/4.0/ Ultrasonics Sonochemistry 80 (2021) 105828 2 purification. To date, no synthetic procedures have been described to carry out monosubstitution processes using O,O- or N,O-donor bridging ligands and only a few cases of bisubstituted species using carboxylate ligands with high steric hindrance have been published [50,51]. In most cases, only the tetrasubstituted species were described. In the search of a more direct and greener synthetic method for the preparation of monosubstituted compounds, sonochemistry arises as a promising alternative [52]. In fact, ultrasound-assisted synthesis (USS) is becoming one of the most popular techniques for the preparation of organic [53] or inorganic materials [54]. In addition, USS is associated with faster reaction rates, better yields and higher purity of products, better selectivity, lower energy consumption and less use of hazardous materials participating in the chemical processes [55]. This technique has been recently applied to carry out a self-assembly reaction of a heterometallic diruthenium(II,III) carbonate, Na[Ni(H2O)4Ru2(CO3)4]⋅ 3H2O, improving their synthesis conditions and magnetic properties [56]. Inspired by this approach, we now report a novel and general route for the preparation of monosubstituted Ru2 5+ derivatives using ultrasound-assisted synthesis. To generalize this technique, six different formamidinates and one amidate ligands have been tested. Thus, the complexes [Ru2Cl(DAniF)(O2CMe)3] (1), [Ru2Cl(DPhF)(O2CMe)3] (2), [Ru2Cl(D-p-CNPhF)(O2CMe)3] (3), [Ru2Cl(D-o-TolF)(O2CMe)3] (4), [Ru2Cl(D-m-TolF)(O2CMe)3] (5), [Ru2Cl(D-p-TolF)(O2CMe)3] (6) and [Ru2Cl(p-TolA)(O2CMe)3] (7) have been synthesized (DAniF− = N,N′- bis(4-anisyl)formamidinate; DPhF− = N,N′-diphenylformamidinate; D- p-CNPhF− = N,N′-bis(4-cyanophenyl)formamidinate; D-o/m/p-TolF− = N,N′-bis(2/3/4-tolyl)formamidinate; p-TolA− = N-4-tolylamidate). The complexes have been characterized by elemental analyses, IR and UV–Vis spectroscopy, ESI-MS, cyclic voltammetry, and magnetization measurements. Single-crystal X-ray diffraction studies for compounds 3 and 7 have also been carried out. 2. Experimental The Scheme 1 is a summary of the chemistry displayed in the present work. Compounds 1–7 have been prepared according to the following procedure: To a round-bottomed flask containing [Ru2Cl(O2CMe)4] (0.4264 g, 0.9 mmol) and the corresponding N,N’-́diarylformamidine (1–6) or amide (7) (0.9 mmol) was added Et3N (300 µL) and ethanol (50 mL). The mixture was sonicated during 150 min (for 1, 5 and 6), 180 min (for 2 and 3), 210 min (for 4), and 300 min (for 7) at 80 kHz, room temperature and atmospheric pressure. The mixture was filtered through Celite® in order to remove the unreacted [Ru2Cl(O2CMe)4] and the solvent was evaporated under vacuum. The solid was washed with ethyl ether (50 mL) for compounds 1, 3–7 or toluene (10 mL) for com pound 2, solved in distilled water (150 mL), and the solution was filtered through Celite®. The aqueous solution was placed in a separatory fun nel, treated with brine (5 mL) and extracted with dichloromethane (2 × 50 mL). To the organic solution MgSO4 was added. The solution was filtered off and concentrated to dryness. The solid was washed with a 10:6 mixture of hexane and acetone only in the case of compound 7 (10 mL). Then, the product was dried under vacuum. [Ru2Cl(DAniF)(O2CMe)3] (1). Anal. Calcd for C21H24ClN2O8R u2⋅H2O (688.03 g/mol), C, 36.66; H, 3.81; N, 4.07; found C, 36.96; H, 4.10; N, 4.06; ESI-MS (CH2Cl2) m/z calcd for C21H24N2O8Ru2 [M− Cl]+ 636.0, found 635.9; Yield: 0.4066 g (65%). [Ru2Cl(DPhF)(O2CMe)3] (2). Anal. Calcd for C19H20ClN2O6R u2⋅H2O (627.99 g/mol), C, 36.34; H, 3.53; N, 4.46; found C, 36.61; H, 3.82; N, 4.60; ESI-MS (CH2Cl2) m/z calcd for C19H20N2O6Ru2 [M− Cl]+ 575.0, found 574.9; Yield: 0.4863 g (86%). [Ru2Cl(D-p-CNPhF)(O2CMe)3] (3). Anal. Calcd for C21H18ClN4O6Ru2⋅H2O (678.00 g/mol), C, 37.20; H, 2.97; N, 8.26; found C, 37.45; H, 3.23; N, 8.12; ESI-MS (CH2Cl2) m/z calcd for C21H18N4O6Ru2 [M− Cl]+ 624.9, found 624.9; Yield: 0.4511 g (74%). [Ru2Cl(D-o-TolF)(O2CMe)3] (4). Anal. Calcd for C21H24ClN2O6R u2⋅H2O⋅0.5CH2Cl2 (698.51 g/mol), C, 36.97; H, 3.90; N, 4.01; found C, 36.99; H, 4.16; N, 4.03; ESI-MS (CH2Cl2) m/z calcd for C21H24N2O6Ru2 [M− Cl]+ 603.9, found 603.9; Yield: 0.3951 g (63%). [Ru2Cl(D-m-TolF)(O2CMe)3] (5). Anal. Calcd for C21H24ClN2O6Ru2 (638.02 g/mol), C, 39.53; H, 3.79; N, 4.39; found C, 39.70; H, 4.14; N, 4.48; ESI-MS (CH2Cl2) m/z calcd for C21H24N2O6Ru2 [M− Cl]+ 603.9, found 603.9; Yield: 0.4713 g (80%). [Ru2Cl(D-p-TolF)(O2CMe)3] (6). Anal. Calcd for C21H24ClN2O6R u2⋅H2O (656.04 g/mol), C, 38.45; H, 3.99; N, 4.27; found C, 38.41; H, 4.03; N, 4.27; ESI-MS (CH2Cl2) m/z calcd for C21H24N2O6Ru2 [M− Cl]+ 603.9, found 603.9; Yield: 0.4976 g (84%). [Ru2Cl(p-TolA)(O2CMe)3] (7). Anal. Calcd for C14H17ClNO7Ru2 (548.88 g/mol), C, 30.64; H, 3.12; N, 2.55; found C, 30.75; H, 3.40; N, 2.76; ESI-MS (CH2Cl2) m/z calcd for C14H17NO7Ru2 [M− Cl]+ 514.0, found 514.0; Yield: 0.3219 (65%). Scheme 1. Synthesis of monosubstituted diruthenium compounds. A. Terán et al. Ultrasonics Sonochemistry 80 (2021) 105828 3 3. Results and discussion 3.1. Synthetic method According to the literature, all the methods described to obtain in termediate substitution species, using formamidinate ligands, have been carried out through conventional synthesis where the activation energy was heat. Table 1 summarizes the experimental conditions for the two monoformamidinatodiruthenium(II,III) compounds previously reported and the conditions used in the present work for complexes [Ru2Cl (DAniF)(O2CMe)3] (1), [Ru2Cl(DPhF)(O2CMe)3] (2), [Ru2Cl(D-p- CNPhF)(O2CMe)3] (3), [Ru2Cl(D-o-TolF)(O2CMe)3] (4) [Ru2Cl(D-m- TolF)(O2CMe)3] (5) [Ru2Cl(D-p-TolF)(O2CMe)3] (6) and [Ru2Cl(p-TolA) (O2CMe)3] (7). To optimize the synthesis conditions of compounds 1–7, a study of the most crucial variables involved in the process has been carried out: a) Formamidine nature. Compounds 1–6 differ in the nature (donor/ acceptor properties) of the R substituent on the phenyl ring of the formamidinates. Depending on the substituent nature the reaction rate varies. Formamidines with electron donating group (R = OMe, Me) show higher reaction rates while the formamidine with an electron withdrawing substituent (R = CN) requires longer reaction times. An exceptional case occurs in the synthesis of compound 4 (R = Me). Although the methyl group should make the formamidine more reactive, its position at ortho causes a great steric hindrance and a longer reaction time is needed for the substitution process. The amidine, as expected, is less reactive than the formamidines [39,40]. Therefore, compound 7 needs more time. b) Atmosphere. In general, the compounds with metal–metal bonds are quite air-sensitive. Several authors have described different synthetic pathways for Ru2 5+ species under inert atmosphere (N2 or Ar) [3,7,57] in order to protect the metal–metal bond, which adds dif ficulty to the procedure. In this work, all reactions were carried out in air and without dry solvents, maintaining the stability and prop erties of the binuclear core as demonstrated the physicochemical analyses. This had already been demonstrated by our research group through different articles involving conventional synthesis [30,58] and microwave-assisted synthesis [25,38–45], and it is also appli cable to ultrasound-assisted synthesis. c) Reagents. Although the use of LiCl is necessary for the synthesis of some intermediate species, [Ru2Cl(L-L)x(O2CMe)4-x] (x = 2 and 3) [7,59], to improve the yield, in this case the synthesis with or without LiCl, gives the same results, so it has been omitted. d) Solvent. The best solvent to carry out the sonochemical substitution of Ru2 5+ derivatives is ethanol in which the yield of the reaction is high. With other common organic solvents such as tetrahydrofuran (THF), dichloromethane (DCM), acetone, or toluene the substitution reaction does not occur. This inefficient sonochemical process can be explained by the physical properties of these organic solvents (Table 2) which produce low ultrasonic power (Up). Different at tempts were also made to execute the synthesis in aqueous medium where the starting reagent [Ru2Cl(O2CMe)4] is soluble. However, the low solubility of the formamidines avoided the substitution reaction. e) Time and temperature. The reaction times described in this work are the minimum to consume the most [Ru2Cl(O2CMe)4] starting ma terial and the corresponding formamidine. Long sonication times generate an increase of solvent temperature (T > 45 ◦C) which may lead to increase the formation of by-products. The synthesis of [Ru2Cl(DAniF)(O2CMe)3] (1) complex had been described in the literature and involves inert atmosphere (N2), THF as solvent, a 1:1.5 M ratio [Ru2Cl(O2CMe)4]:HDAniF, temperature lower than 45 ◦C and 12 h of reaction time. A purification process of the final product through filtration and chromatographic column is also neces sary. The application of USS has significatively enhanced the obtention of compound 1, with a reduction of temperature and reaction time, without the need of inert atmosphere or chromatographic column dur ing purification and achieving more than double yield. With the appli cation of USS to the obtention of monosubstituted compounds, it becomes possible to reach some of the twelve principles of green chemistry, such as atom economy, the avoid of high temperatures and pressures or the design of energy-efficient processes. This work describes for the first time that USS can be applied to carry out ligand substitution reactions in metal–metal bonded binuclear compounds. 3.2. Structural characterization and properties The isolated compounds 1–7 are stable in both solution (in water, dichloromethane, tetrahydrofuran, dimethyl sulfoxide, acetone, meth anol, and ethanol) and in the solid state under ambient conditions. FTIR spectra of the compounds confirmed the presence of repre sentative functional groups due to the coordination of formamidinate or amidate ligands to the diruthenium core (Fig. S1, ESI). Compounds 1–7 spectra contain the aromatic and non-aromatic C–H stretches around 3040 cm− 1 and 2930 cm− 1, respectively, and sharp peaks at ≈1640 and 1600 cm− 1 (ν(C=C)Ar), ≈1490 cm− 1 (νa(O-C-O)), ≈1350 cm− 1 (νs (O-C- O)), ≈1070, 1020, 950 cm− 1 (δip(C=C–H)Ar) and ≈690 cm− 1 (δoop(C=C–H)Ar). The spectrum of compound 7 containing an amidate group shows a band at ≈3340 cm− 1 due to N–H stretching vibration and a strong band in the 1430–1400 cm− 1 region associated with a combi nation of the ν(C=O), ν(C-N) and ν(C–C) vibration. For compounds with formamidinate ligands (1–6), strong bands at ≈1435, 1310 and 1225 cm− 1 related to ν(C-N) are observed. The spectra of derivatives 1 and 3 show representative peaks corresponding to the para substituent at 1025 cm− 1 (ν(C-OCH3)), and 2217 cm− 1 (ν C≡N), respectively. The compounds show the typical pattern of diruthenium isotopic distribution in ESI-MS. The maximum m/z peaks in all mass spectra are ascribed to the [M− Cl]+ and [M+CH3CO]+ fragments that agree with the simulated MS patterns (Fig. S2, ESI). When ionization is performed by MALDI-MS, only the fragment [M− Cl]+ appears in the spectrum, suggesting the absence of by-products. These results corroborate the integrity of paddlewheel structure maintaining three acetates and one formamidine or amidate bridging ligand bonded to the diruthenium core. This technique has also allowed us to monitor the substitution reaction for compounds 1–7. It was recorded a spectrum of crude Table 1 Comparison of the experimental conditions to obtain monosubstituted com plexes between conventional methods and sonochemical assisted synthesis. Compound Solvent T (◦C) t (h) Yield (%) Ref [Ru2Cl(DXyl2,6F) (O2CMe)3] Toluene ≈110 36 32 [7,8] [Ru2Cl(DAniF) (O2CMe)3] (1) THF ≈45 12 28 [8] 1–6 Ethanol RT 2.5–3.5 63–86 This work 7 Ethanol RT 5 h 65 This work Table 2 Ultrasonic power (Up) and physical properties of tested solvents [60]. Solvent Vapor pressure (kPa) Viscosity (mPa⋅s) Surface tension (Dyn/cm) Up (W) Acetone 24.53 0.33 23.3 1.98 Toluene 2.9 0.59 28.5 2.87 THF 17.3 0.55 28 2.14 DCM 46.99 0.44 28.1 1.38 Ethanol 5.9 1.08 22.3 3.47 A. Terán et al. Ultrasonics Sonochemistry 80 (2021) 105828 4 reaction at 1.5 h for all the complexes (Fig. S3, ESI). There are two peaks for compounds 1–3, 5 and 6 that can be assigned to the corresponding mono- and bis-substituted species losing the axial chloride ligand. The main peak corresponds to the monosubstituted species, [M− Cl]+, being the other one almost residual. After purification, this by-product is eliminated. For compound 4, the bisubstituted species does not appear probably because its formation is hindered by the steric effect of the methyl group at ortho position of the aromatic ring in the formamidinate ligand. For compound 7 there is also no species with a higher substitu tion degree, but this can be associated with the lower reactivity of the amides with respect to the formamidines. This lower reactivity has already been observed in the tetrasubstitution reactions carried out by microwave-assisted solvothermal synthesis. The reactions with for mamidines require around 1–2 h [39], while 16 h are necessary when amides are used instead [40,43,44]. Complexes 1–7 show intense colors both in the solid state and in solution. The electronic absorption spectra in the vis-NIR region were recorded in dichloromethane solution (Fig. S4, ESI). Compounds 1–6 display the typical profile of other high-spin (S = 3/2) partial substituted diruthenium complexes reported in the literature [6]. In compound 7, with an amidate ligand, the absorption spectrum is very similar to the monosubstituted compounds with a formamidinate ligand. In general, it can be observed a band around 370 nm that can be tentatively assigned to a π(Cl) → π*(Ru2) transition, a second band (which in some com pounds appears with a shoulder) in the 450–650 nm region that can be attributed to π(RuO/N,Ru2) → π*(Ru2) transitions, and a less intense band in the 900–1050 nm region that can be attributed to a δ(Ru2) → δ* (Ru2) transition (Table S1, ESI). Magnetization measurements at variable temperature were carried out for compounds 1–7. The χMT values at room temperature of 1–7 are in the 2.23–1.84 cm3⋅K⋅mol− 1 range (Table 3). These values are close to the expected spin-only value, 1.87 cm3⋅K⋅mol− 1 (g = 2), for a quartet state arising from a σ2δ2π4(δ*π*)3 electronic configuration [3,6,61]. On cooling, χMT value decreases continuously (Figs. S5–S11, ESI), which is typically observed in this type of complexes and ascribed to a strong zero-field splitting (D) and a weak intermolecular antiferromagnetic coupling [3,6]. For this reason, the χM and the χMT data were fitted using the Cukiernik model [62–64] taking into account a weak intermolecular antiferromagnetic coupling (zJ) and a temperature-independent para magnetism (TIP) term in addition to the D and g parameters (see equa tions S1-S5, ESI). The values obtained from the fits are shown in Table 3 and are similar to those previously reported for another mono formamidinatodiruthenium(II,III) complex [8]. According to the Cambridge Structural Database [65] (CSD 2020), no crystal structure of monosubstituted diruthenium compounds with amidate ligands has been reported and there are only two crystal structures with a formamidinate ligand (HDXyl2,6F) [8]. In the present work, the crystal structures of compounds 3 and 7 have been solved by single crystal X-ray diffraction (see Tables S2 and S3, ESI). Both com pounds, 3⋅CH2Cl2 and 7⋅2CH2Cl2, crystallize with dichloromethane molecules in the monoclinic system, space group P21/n for compound 3 and P21/c for compound 7. These compounds show the typical paddlewheel structure (Fig. 1). The coordination environment for both ruthenium atoms is octahedral. Each diruthenium unit has retained three acetate ligands along with one formamidinate (3) or amidate (7) bridging ligand at the equatorial po sitions, while the axial positions are occupied by bridging chloride li gands. The Ru-Ru bond distances of 2.306 Å (3) and 2.280 Å (7) are in the range described for related Ru2 5+ derivatives [6]. The chloride is aligned with the diruthenium core with a Ru2-Ru1-Cl1 angle of 175.38◦ (3) and 176.61◦ (7). The presence of a different ligand (amidate or formamidinate) in the structure results in two types of acetates, being the Ru-O bond distance of the acetate trans to the formamidinate (3) or amidate (7) longer than the acetates trans to each other, as it has been also observed for other mono-substituted compounds described in the literature [8,46–48]. This fact can be attributed to the better donor character of the N-donor ligand relative to the corresponding carbox ylate ligand. In both compounds the chloride ligand bridges two diruthenium units, giving rise to a polymeric structure. For complex 3, the Ru1-Cl1- Ru2 angle is 174.02◦ giving an almost linear polymeric chain whereas for compound 7 this angle is 124.25◦, giving rise to a clear zigzag chain (Fig. 2). In both compounds, the Ru1-Cl1 and Cl1-Ru2 distances are similar (2.625 and 2.637 Å for 3; 2.534 and 2.548 Å for 7), indicating the formation of an almost symmetrical bridge between the diruthenium units. These bond distances are longer for compound 3 than those usu ally observed for analogous compounds [8], suggesting weaker interactions. In compound 3 (Fig. 2a), the steric hindrance of the formamidinate substituents imposes a rotation of 140◦ between the formamidinate li gands of consecutive dimeric units, resulting in a staggered conforma tion along the chains. The dichloromethane solvent molecule allows weak interchain (H22A⋅⋅⋅N3) and intrachain (H22A⋅⋅⋅O2 and H22B⋅⋅⋅O3) interactions that keep the different chains in contact. For compound 7 (Fig. 2b), the adjacent diruthenium units are rotated 180◦ along the zig-zag chains. The only observed interchain interaction is a weak π-π overlap between the aromatic rings of neighboring amidate units with an average interplanar distance of 3.38 Å. As in the previous compound, the dichloromethane solvent molecules participate in weak intrachain interactions (H15A⋅⋅⋅Cl1, H15B⋅⋅⋅O3, H16B⋅⋅⋅Cl1 and H16B⋅⋅⋅O5). The redox properties of compounds 1–7 were analyzed using cyclic voltammetry (CV). The results are plotted in Fig. S12 and the electro chemical data are summarized in Table 4. All the complexes show only one-electron redox couple within the cathodic limit of water. This is assigned to the Ru2 5+/Ru2 4+ redox couple according to other diruthenium complexes with similar ligands in aqueous and nonaqueous solvents [48,49,58,66–72]. The redox prop erties of these compounds vary depending on the nature of the ligand. The substitution of one acetate bridging ligand (O,Ó-donor) for one formamidinate (N,Ń-donor) or amidate (N,O-donor) bridging ligand increases the electron density on the diruthenium core which results in more negative redox potentials relative to the starting [Ru2Cl(O2CMe)4] (E1/2 = 0.04 V [48]). For complexes 1–6, the presence of electron donating substituents in the aromatic ring (compounds 1, 4–6) gener ates more negative potentials, while the presence of an electron- Table 3 Magnetic parameters obtained from the magnetic data of 1–7. Compound χMT at 300 K (cm3⋅K⋅mol− 1) g D (cm− 1) zJ (cm− 1) TIP (cm3⋅mol− 1) σ2** 1 2.19 2.00* 68 − 0.28 1.14 × 10− 3 2.41 × 10− 4 2 2.10 2.00* 68 − 0.32 8.75 × 10− 4 2.69 × 10− 4 3 2.23 2.19 67 − 0.85 1.33 × 10− 4 3.58 × 10− 4 4 1.84 2.00* 75 − 0.25 1.83 × 10− 12 3.62 × 10− 4 5 2.09 2.09 53 − 0.41 7.45 × 10− 14 2.95 × 10− 4 6 2.00 2.00 63 − 0.58 6.43 × 10− 4 3.04 × 10− 4 7 2.18 2.11 76 − 1.28 6.28 × 10− 4 4.37 × 10− 4 *A g value of 2 was fixed in these fittings. Otherwise, values of 1.97, 1.98 and 1.95 were obtained for 1, 2 and 4, respectively. ** σ2 = ∑( χM⋅Tcalcd − χM⋅Texp )2 / ∑( χM⋅Texp )2 . A. Terán et al. Ultrasonics Sonochemistry 80 (2021) 105828 5 withdrawing substituent (compound 3) causes a positive cathodic shift. The substituent position on the aromatic ring (ortho, meta or para) seems to be more relevant that the electron donating character of functional group (i.e., E1/2 = -0.35 V for compound 1 (p-OMe) and 6 (p-Me)). Moreover, it seems that the influence of substituent in ortho position is greater than in para position (i.e., E1/2 = -0.37 V for compound 4 (o-Me), and being almost negligible when the electron donating substituent is located in meta position (E1/2 = -0.32 V for compound 5) compared to the unsubstituted formamidinate derivative (E1/2 = -0.33 for compound 2). The amidate ligand presents a lower donating character than the formamidinate, a fact reflected in the low negative reduction potential observed for the corresponding derivative, 7. 4. Conclusions This work presents an alternative method for the synthesis of met al–metal bonded compounds based on the sonochemical activation (compounds 1–7). This procedure permits an effective replacement of only one acetate group. It works not only with different types of for mamidines but also with amides which suggests that this is a general method of preparation for monosustituted complexes with N,N- or N,O- donor ligands. Additionally, this method of synthesis considerably reduces reaction times, uses a greener solvent (ethanol) and saves energy with respect to other methods described for dimetallic compounds. The aqueous media solubility of these complexes may be very important for their potential in fields such as biomedicine or biotechnology. Author contributions A.T. carried out the experiments and wrote the original draft. M.C. described the magnetic behavior. A.G. solved the crystal structures. A.E. S.-P., S.H. and R.J.-A., realized the supervision of the work and the writing-review. S.H. was also in charge of the project administration. Fig. 1. Asymmetric unit representation of compound 3⋅CH2Cl2 (a) and compound 7⋅2CH2Cl2 (b). Hydrogen atoms have been omitted for clarity. Fig. 2. Crystal packing of compound 3⋅CH2Cl2 (a) and compound 7⋅2CH2Cl2 (d) with the representative interactions marked in blue. Some hydrogen atoms have been omitted for clarity. Table 4 Electrochemical data (V, vs. Ag/AgCl) from CV of complexes 1–7. Compound E1/2 ΔE 1 − 0.35 0.15 2 − 0.33 0.12 3 − 0.18 0.15 4 − 0.37 0.12 5 − 0.32 0.19 6 − 0.35 0.10 7 − 0.16 0.16 A. Terán et al. Ultrasonics Sonochemistry 80 (2021) 105828 6 CRediT authorship contribution statement Aarón Terán: Methodology, Investigation, Writing – original draft, Visualization. Miguel Cortijo: Formal analysis. Ángel Gutiérrez: Formal analysis. Ana E. Sánchez-Peláez: Supervision, Writing – review & editing. Santiago Herrero: Supervision, Writing – review & editing, Conceptualization, Project administration, Funding acquisition. Reyes Jiménez-Aparicio: Supervision, Writing – review & editing. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements The Government of Comunidad de Madrid is gratefully acknowl edged for financial support (Project S2017/BMD-3770-CM). A.T. ac knowledges to Universidad Complutense for a Predoctoral Grant (CT63/ 19-CT64/19) and to the Spanish Ministry of Science and Innovation for a Postgraduate Fellow at Residencia de Estudiantes (2020-2021). We also thank Elena García-Chamocho for her help with cyclic voltammetry measurements. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.ultsonch.2021.105828. References [1] T.A. Stephenson, G. Wilkinson, New ruthenium carboxylate complexes, J. Inorg. Nucl. Chem. 28 (10) (1966) 2285–2291, https://doi.org/10.1016/0022-1902(66) 80118-5. [2] M.J. Bennett, K.G. Caulton, F.A. Cotton, Structure of tetra-n-butyratodiruthenium chloride, a compound with a strong metal-metal bond, Inorg. Chem. 8 (1) (1969) 1–6, https://doi.org/10.1021/ic50071a001. [3] F.A. Cotton, C.A. Murillo, R.A. 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Terán et al. https://doi.org/10.1016/j.ccr.2020.213706 https://doi.org/10.1039/C003411H https://doi.org/10.1039/C0GC00800A https://doi.org/10.1039/C0GC00800A https://doi.org/10.1039/C2DT30939D https://doi.org/10.1039/C2DT30939D https://doi.org/10.1016/j.ica.2014.07.063 https://doi.org/10.1016/j.ica.2014.07.063 http://refhub.elsevier.com/S1350-4177(21)00370-9/h0210 http://refhub.elsevier.com/S1350-4177(21)00370-9/h0210 http://refhub.elsevier.com/S1350-4177(21)00370-9/h0210 http://refhub.elsevier.com/S1350-4177(21)00370-9/h0210 https://doi.org/10.3390/cryst7070192 https://doi.org/10.3390/cryst7070192 https://doi.org/10.1039/C3DT52727A https://doi.org/10.3390/inorganics2030524 https://doi.org/10.1021/ic971428a https://doi.org/10.1016/S0020-1693(02)00712-0 https://doi.org/10.1016/S0020-1693(02)00712-0 https://doi.org/10.1021/acs.inorgchem.9b02838 https://doi.org/10.1021/ic8017369 https://doi.org/10.1021/ic8017369 https://doi.org/10.1007/s10876-010-0310-1 https://doi.org/10.1039/C6DT00569A https://doi.org/10.1039/C9GC02534K https://doi.org/10.1016/j.ultsonch.2016.10.010 https://doi.org/10.1016/j.ultsonch.2020.105384 https://doi.org/10.1016/j.ultsonch.2019.104722 https://doi.org/10.1016/j.ultsonch.2019.104722 https://doi.org/10.1039/C9CC07876B https://doi.org/10.1021/om050068t https://doi.org/10.1021/ic052174t https://doi.org/10.1016/j.inoche.2003.09.016 https://doi.org/10.1016/j.inoche.2003.09.016 https://doi.org/10.1016/j.ultsonch.2016.03.014 https://doi.org/10.1016/j.ultsonch.2016.03.014 https://doi.org/10.1021/ja00512a025 https://doi.org/10.1021/ja00512a025 https://doi.org/10.1021/ic971366o https://doi.org/10.1021/ic00188a019 https://doi.org/10.1021/ic00188a019 http://refhub.elsevier.com/S1350-4177(21)00370-9/h0325 http://refhub.elsevier.com/S1350-4177(21)00370-9/h0325 https://doi.org/10.1021/ic00196a011 https://doi.org/10.1021/ic00196a011 https://doi.org/10.1021/ic9602658 https://doi.org/10.1021/ic9602658 https://doi.org/10.1021/ic020590x https://doi.org/10.1021/ic034722d https://doi.org/10.1021/ic060267k https://doi.org/10.1021/om100263c https://doi.org/10.1021/om100263c https://doi.org/10.1021/ic5007605 62 Chapter IV. Effect of equatorial ligand substitution on the reactivity with proteins of paddlewheel diruthenium complexes: Structural Studies hapter IV C Effect of equatorial ligand substitution on the reactivity with proteins of paddlewheel diruthenium complexes: structural studies 63 69 Abstract Prior to this article there was only one precedent on the study of the interaction of diruthenium compounds with proteins. The [Ru2Cl(O2CCH3)4] complex was reported to react with the model protein hen egg white lysozyme (HEWL) forming two adducts bound to Asp101 and Asp119 side chains upon releasing of one acetate ligand. Here, it is studied the effect of the substitution of equatorial ligands in diruthenium compounds on their reactivity and selectivity with the model protein HEWL. We selected the complex [Ru2Cl(D-p-FPhF)(O2CCH3)3] (D-p-FPhF- = N,N′-bis(4- fluorophenyl)formamidinate) to interact with HEWL under four different conditions. To ascertain the stability of [Ru2Cl(D-p-FPhF)(O2CCH3)3] compound in water and in the conditions used to perform the protein binding studies, UV-Vis absorption spectroscopy, fluorescence, and circular dichroism were used. The introduction of a formamidinate ligand (D-p-FPhF-), increase the stability of this species in solution in the conditions studied, unlike [Ru2Cl(O2CCH3)4] or other Rh2 4+ species which show breakage of the metal- metal bond or aggregation processes under similar conditions. Four diruthenium metalloproteins were formed and studied by X-ray diffraction. Our data demonstrate that this compound also binds the protein forming adducts bound to Asp side chains (101 and 119) upon releasing of an acetate ligand. The other ligands can be retained and the D-p-FPhF ligand can be cis or trans to the Asp side chain probably due to steric hindrance. In addition, Lys or Arg side chains or even main chain carbonyl groups can coordinate to diruthenium core at the axial site. Our data help to understand the reactivity of paddlewheel diruthenium complexes with proteins, providing useful information for the design of new artificial diruthenium- containing metalloenzymes. 70 Effect of Equatorial Ligand Substitution on the Reactivity with Proteins of Paddlewheel Diruthenium Complexes: Structural Studies Aarón Terán, Giarita Ferraro, Ana E. Sánchez-Peláez, Santiago Herrero,* and Antonello Merlino* Cite This: Inorg. Chem. 2023, 62, 670−674 Read Online ACCESS Metrics & More Article Recommendations *sı Supporting Information ABSTRACT: The paddlewheel [Ru2Cl(O2CCH3)4] complex was previously reported to react with the model protein hen egg white lysozyme (HEWL), forming adducts with two diruthenium moieties bound to Asp101 and Asp119 side chains upon the release of one acetate. To study the effect of the equatorial ligands on the reactivity with proteins of diruthenium compounds, X-ray structures of the adducts formed when HEWL reacts with [Ru2Cl(D-p-FPhF)(O2CCH3)3] [D-p-FPhF = N,N′-bis(4- fluorophenyl)formamidinate] under different conditions were solved. [Ru2Cl(D-p-FPhF)(O2CCH3)3] is bonded through their equatorial positions to the Asp side chains. Protein binding occurs cis or trans to D-p-FPhF. Lys or Arg side chains or even main- chain carbonyl groups can coordinate to the diruthenium core at the axial site. Data help to understand the reactivity of paddlewheel diruthenium complexes with proteins, providing useful information for the design of new artificial diruthenium-containing metalloenzymes with potential applications in the fields of catalysis, biomedicine, and biotechnology. Most of the diruthenium complexes with the general formula [Ru2X(L−L)4] contain a strong metal−metal interaction (bond order of 2.5), four bridging equatorial ligands (L−L) arranged in a lantern-like fashion around the dimetallic center, and donor ligands (X) at the axial positions.1−3 These molecules have attracted considerable interest for their application in catalysis4−10 or biomedicine11 and for their peculiar magnetic and redox properties.12−14 The prototype of the diruthenium compounds family, [Ru2Cl(O2CCH3)4], has interesting pharmaceutical properties and has been used as a precursor to prepare promising anticancer compounds.11,15−17 For example, diruthenium compounds containing nonsteroidal antiinflammatory drugs18−20 or γ-linolenic acid21,22 as ligands have been found to be active against glioma tumor models in vitro and in vivo and, in particular, against human glioblastoma cell lines, while the [Ru2Cl(EB776)4] complex [where EB776 is the deproto- nated form of (2-phenylindol-3-yl)glyoxyl-L-phenylalanine-L- leucine] was found to be active against a glioblastoma cell line.23 The diruthenium ibuprofenate complex also shows antiinflammatory properties with reduced gastric ulceration in vivo compared to the copper ibuprofenate complex.24 In earlier work, the interaction of [Ru2Cl(O2CCH3)4] with the model protein hen egg white lysozyme (HEWL) was studied to give the first key insights into the biological targets and mode of action of the diruthenium metallodrugs: the diruthenium center binds the protein, retaining the Ru−Ru bond and replacing one acetate ligand by an Asp side chain; a second acetate is then replaced by two water (H2O) molecules.25 Recently, it has been suggested that the use of bulky equatorial substituents on the diruthenium core may constitute an approach to increase the selectivity of diruthenium complexes toward anticancer targets.26,27 Many diruthenium complexes of the type [Ru2Cl(L−L)4] (L−L = O,N donors,28,29 N,N′-donors30,31 or other O,O′-donors3,32) have been synthesized. However, intermediate substitution species [Ru2Cl(L−L)x(O2CCH3)4−x] (x = 1−3) are quite scarce.14,33−35 These molecules show variations in the stability, solubility, redox potential, and paramagnetic behavior compared to [Ru2Cl(O2CCH3)4] and to their fully substituted species. Here, we investigate interaction of the monosubstituted diruthenium compound [Ru2Cl(D-p-FPhF)(O2CCH3)3], where D-p-FPhF = N,N′-bis(4-fluorophenyl)formamidinate (Figure 1), with HEWL under four different experimental conditions. [Ru2Cl(L−L)(O2CCH3)3] compounds are soluble in H2O and are expected to be more stable than [Ru2Cl- (O2CCH3)4] according to the previous results.34 X-ray structures of adducts formed upon reaction of the protein with [Ru2Cl(D-p-FPhF)(O2CCH3)3] are reported. The results are compared with those obtained when proteins react with [Ru2Cl(O2CCH3)4] 25 and various dirhodium compounds.36−40 The stability of [Ru2Cl(D-p-FPhF)- (O2CCH3)3] was first studied in solution by UV−vis absorption spectroscopy (Figure 2). The spectrum in dichloro- methane showed only one transition band around 480 nm.34 However, in H2O, the electronic spectrum shows bands in the UV (243 and 338 nm) and visible (∼420 and 520 nm) regions. A recent study by Kadish and co-workers41 with similar compounds showed the sensitivity of the axial positions to Received: November 21, 2022 Published: January 4, 2023 Communicationpubs.acs.org/IC © 2023 The Authors. Published by American Chemical Society 670 https://doi.org/10.1021/acs.inorgchem.2c04103 Inorg. Chem. 2023, 62, 670−674 D ow nl oa de d vi a U N IV C O M PL U T E N SE D E M A D R ID o n Ja nu ar y 30 , 2 02 3 at 1 3: 07 :4 5 (U T C ). Se e ht tp s: //p ub s. ac s. or g/ sh ar in gg ui de lin es f or o pt io ns o n ho w to le gi tim at el y sh ar e pu bl is he d ar tic le s. https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Aaro%CC%81n+Tera%CC%81n"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Giarita+Ferraro"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Ana+E.+Sa%CC%81nchez-Pela%CC%81ez"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Santiago+Herrero"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Antonello+Merlino"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdf https://pubs.acs.org/action/showCitFormats?doi=10.1021/acs.inorgchem.2c04103&ref=pdf https://pubs.acs.org/doi/10.1021/acs.inorgchem.2c04103?ref=pdf https://pubs.acs.org/doi/10.1021/acs.inorgchem.2c04103?goto=articleMetrics&ref=pdf https://pubs.acs.org/doi/10.1021/acs.inorgchem.2c04103?goto=recommendations&?ref=pdf https://pubs.acs.org/doi/10.1021/acs.inorgchem.2c04103?goto=supporting-info&ref=pdf https://pubs.acs.org/toc/inocaj/62/2?ref=pdf https://pubs.acs.org/toc/inocaj/62/2?ref=pdf https://pubs.acs.org/toc/inocaj/62/2?ref=pdf https://pubs.acs.org/toc/inocaj/62/2?ref=pdf pubs.acs.org/IC?ref=pdf https://pubs.acs.org?ref=pdf https://pubs.acs.org?ref=pdf https://doi.org/10.1021/acs.inorgchem.2c04103?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as https://pubs.acs.org/IC?ref=pdf https://pubs.acs.org/IC?ref=pdf https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ https://acsopenscience.org/open-access/licensing-options/ donor ligands [H2O or dimethyl sulfoxide (DMSO)] and suggested coordination to the axial positions. The higher-energy UV band is usually assigned to an axial ligand-to-metal charge transfer.42 The peak at 338 nm can be assigned to ligand-to-metal transitions [π(N) → σ*/π*/ δ*(Ru2)], while the peaks in the visible region can be assigned to an allowed ligand-to-metal charge transfer [π(N), π(axial) → π*(Ru2)]. 41 These signals do not experience any change after 24 h. UV−vis spectra of [Ru2Cl(D-p-FPhF)(O2CCH3)3] were also collected in the conditions that are used to grow HEWL crystals in the absence (Figures S1A−D) and presence of HEWL (protein to diruthenium compound molar ratio of 1:3; Figures S2A−D). In almost all of the conditions used for the crystallization experiments, ignorable variations in the spectral profiles of the compound are observed, while minimal variations are found in the presence of HEWL (Figures S1A−D and S2A−D). These findings suggest that the diruthenium compound is more stable than [Ru2Cl- (O2CCH3)4] in different aqueous solutions25 and could bind the protein. Circular dichroism spectra of the protein in the absence and presence of diruthenium are superimposable, with negligible variations of the molar ellipticity (Figure S3). These data suggest that HEWL retains its secondary structure and is presumably well-folded in the presence of [Ru2Cl(D-p- FPhF)(O2CCH3)3]. Fluorescence data confirm that the diruthenium compound binds the protein; indeed, a quenching of the intrinsic fluorescence of HEWL is observed when the metal compound concentration is increased (Figure S4A−F). The quenching is not accompanied by a change of the maximum emission wavelength, thus suggesting that metal compound binding occurs without the overall protein structure being altered. Crystals of the adducts formed upon incubation of the protein with the metal compound were obtained by a soaking procedure under four different experimental conditions (see the Supporting Information for further details). The resolution of the structures ranges from 1.07 to 1.81 Å. Data collections and refinement statistics are reported in Table S1. The structures of the protein in the four adducts are very similar to Figure 1. Asymmetric unit of [Ru2Cl(D-p-FPhF)(O2CCH3)3]n· 2nCH2Cl2. Solvent molecules and H atoms have been omitted for clarity. Figure 2. Time course UV−vis spectra of 50 μM [Ru2Cl(D-p- FPhF)(O2CCH3)3] in Milli-Q water. No appreciable spectral changes were observed within 24 h. Figure 3. Overall structures of the adducts formed in the reaction of [Ru2Cl(D-p-FPhF)(O2CCH3)3] with HEWL under different experimental conditions: (A) structure 1 (20% ethylene glycol, 0.1 M sodium acetate buffer at pH 4.0, and 0.6 M sodium nitrate); (B) structure 2 (2.0 M sodium formate and 0.1 M HEPES buffer at pH 7.5); (C) structure 3 (0.8 M succinic acid at pH 7.0); (D) structure 4 (1.1 M sodium chloride and 0.1 M sodium acetate buffer at pH 4.0). Coordinates and structure factors are deposited in the PDB with codes 8BPH (structure 1), 8BPU (structure 2), 8BPJ (structure 3), and 8BQM (structure 4). Ru atoms are in green. Inorganic Chemistry pubs.acs.org/IC Communication https://doi.org/10.1021/acs.inorgchem.2c04103 Inorg. Chem. 2023, 62, 670−674 671 https://pubs.acs.org/doi/suppl/10.1021/acs.inorgchem.2c04103/suppl_file/ic2c04103_si_001.pdf https://pubs.acs.org/doi/suppl/10.1021/acs.inorgchem.2c04103/suppl_file/ic2c04103_si_001.pdf https://pubs.acs.org/doi/suppl/10.1021/acs.inorgchem.2c04103/suppl_file/ic2c04103_si_001.pdf https://pubs.acs.org/doi/suppl/10.1021/acs.inorgchem.2c04103/suppl_file/ic2c04103_si_001.pdf https://pubs.acs.org/doi/suppl/10.1021/acs.inorgchem.2c04103/suppl_file/ic2c04103_si_001.pdf https://pubs.acs.org/doi/suppl/10.1021/acs.inorgchem.2c04103/suppl_file/ic2c04103_si_001.pdf https://pubs.acs.org/doi/suppl/10.1021/acs.inorgchem.2c04103/suppl_file/ic2c04103_si_001.pdf https://pubs.acs.org/doi/suppl/10.1021/acs.inorgchem.2c04103/suppl_file/ic2c04103_si_001.pdf https://pubs.acs.org/doi/10.1021/acs.inorgchem.2c04103?fig=fig1&ref=pdf https://pubs.acs.org/doi/10.1021/acs.inorgchem.2c04103?fig=fig1&ref=pdf https://pubs.acs.org/doi/10.1021/acs.inorgchem.2c04103?fig=fig1&ref=pdf https://pubs.acs.org/doi/10.1021/acs.inorgchem.2c04103?fig=fig1&ref=pdf https://pubs.acs.org/doi/10.1021/acs.inorgchem.2c04103?fig=fig2&ref=pdf https://pubs.acs.org/doi/10.1021/acs.inorgchem.2c04103?fig=fig2&ref=pdf https://pubs.acs.org/doi/10.1021/acs.inorgchem.2c04103?fig=fig2&ref=pdf https://pubs.acs.org/doi/10.1021/acs.inorgchem.2c04103?fig=fig2&ref=pdf https://pubs.acs.org/doi/10.1021/acs.inorgchem.2c04103?fig=fig3&ref=pdf https://pubs.acs.org/doi/10.1021/acs.inorgchem.2c04103?fig=fig3&ref=pdf https://pubs.acs.org/doi/10.1021/acs.inorgchem.2c04103?fig=fig3&ref=pdf https://pubs.acs.org/doi/10.1021/acs.inorgchem.2c04103?fig=fig3&ref=pdf pubs.acs.org/IC?ref=pdf https://doi.org/10.1021/acs.inorgchem.2c04103?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as each other and are not significantly affected by interaction with the metal compound (Figure 3). The root-mean-square deviation of Cα atoms from the structure of the metal-free protein (PDB code 193L)43 is within the range 0.17−0.21 Å. In structure 1 (Figure 3A), diruthenium centers are found to be close to the side chains of Asp101 or Asp119 (Figures 4A,B). The diruthenium-containing fragment bound close to Asp119 is well-defined (Figure 4B), while the D-p-FPhF ligand of the diruthenium bound to Asp101 is less ordered (Figure 4A). The two diruthenium-containing fragments are alternative to each other with 0.50 and 0.40 occupancies, respectively. The Asp119 side chain changes its conformation to coordinate the diruthenium center at the equatorial site (Figure S5), while Asp101 is already in the right position in the metal-free protein to link the dimetallic center. Close to the Asp119 side chain, the diruthenium unit binds two acetate ligands along with one L−L bridging ligand and the carboxylate group of Asp at the equatorial position. Close to the Asp101 side chain, acetate ligands could be replaced by H2O molecules. Because of the crystallization conditions (low pH and high concentration of sodium nitrate), we cannot exclude that, in structure 1, acetate ligands were replaced by nitrate ions. The axial positions of the diruthenium center bound to Asp119 are occupied by H2O molecules, while at the Asp101 binding site, axial ligands were not added to the model because of low electron density. Notably, close to Asp119, the diruthenium-containing frag- ment binds the Asp side chain cis to the D-p-FPhF ligand, while close to Asp101, the side chain is trans to the L−L ligand. As was already observed, bisubstituted species usually have cis configuration. The trans disposition has only been obtained with bulky equatorial ligands.14 In structure 2 (Figure 3B), similar results were obtained (Figure 4C−E): the diruthenium center binds Asp101, with the side chain trans to the L−L ligand (occupancy = 0.70; Figure 4C) and to Asp119 (occupancy = 0.55; Figure 4D). Interestingly, in this structure, the diruthenium bound to Asp101 with the side chain trans to the D-p-FPhF ligand (Figure 4C) is better defined than that bound to Asp119 (Figure 4D). In the former diruthenium binding site, inspection of the electron density maps suggests that two formate ions have replaced acetate ligands (Figure 4C). Axially coordinated H2O molecules can also be confidently modeled. In the latter, the electron density is disordered and diruthenium ligands have been interpreted as H2O molecules (Figure 4D). An additional diruthenium binding site with low occupancy (0.20) is found to be close to the side chain of Asp18 (Figure 4E). Here, only two Ru atoms and a few H2O molecules as ligands have been modeled. The Ru atoms are at ∼3 Å from the atoms of the Asn19 side chain. Additional binding sites for the diruthenium core were found also in structure 3 (Figure 3C). In this structure, a diruthenium center is axially coordinated to the carbonyl of Asp101 (Figure 4F) and to the Lys33 side chain (Figure 4G). Unfortunately, in both of these additional diruthenium binding sites, metal ligands cannot be confidently modeled because of the low occupancy of the metal (0.25 and 0.20, respectively) and conformational disorder. The carbonyl group probably competes with H2O molecules in solution. Nevertheless, the axial coordination of dimetallic compounds to both a residue side chain and a carbonyl oxygen in the solid state was already observed in the adducts formed upon the reaction of dirhodium compounds with proteins.36−40,44 In structure 4 (Figure 3D), binding of the dimetallic center occurs at the level of the side chains of Asp101 (Figure 4H), Asp119 (Figure 4I), and Arg125 (Figure 4J). However, in this structure, especially close to the side chain of Asp101 (Figure 4H), interpretation of the map is complicated by conforma- tional disorder, by the presence of multiple conformations of the diruthenium center, and by the presence of a high concentration of chloride ions, which could replace the diruthenium ligands.45 Interestingly, the diruthenium center close to the side chain of Arg125 is axially coordinated (Figure 4J). Overall, these data indicate that Ru−Ru bonds remain stable upon reaction with HEWL regardless of the experimental conditions used. Interestingly, the structures of the adducts of HEWL with dirhodium tetraacetate and derivatives under the same conditions show breakage of the Rh−Rh bond.36,38,39 In conclusion, here we have studied the reactivity of [Ru2Cl(D-p-FPhF)(O2CCH3)3] with HEWL under different experimental conditions. Our data unambiguously demonstrate that the compound binds the protein, forming adducts with dimetallic moieties bound to the Asp side chains upon the release of an acetate ligand. In the adduct, excluding the acetate replaced by the Asp side chain, the other ligands can be retained and the D-p-FPhF ligand can be cis or trans to the Asp side chain probably due to steric hindrance. These data confirm that diruthenium compounds react with proteins, Figure 4. Diruthenium binding sites in the adducts formed upon the reaction of HEWL with [Ru2Cl(D-p-FPhF)(O2CCH3)3] in structures 1 (panels A and B), 2 (panels C−E), 3 (panels F and G), and 4 (panels H−J). The electron density maps are very well-defined and unambiguously indicate that the compound retains the diruthenium center and L−L ligands upon protein binding. Axial H2O molecules are omitted for the sake of clarity. 2Fo − Fc electron density maps are contoured at 1.0 σ (salmon). Inorganic Chemistry pubs.acs.org/IC Communication https://doi.org/10.1021/acs.inorgchem.2c04103 Inorg. Chem. 2023, 62, 670−674 672 https://pubs.acs.org/doi/suppl/10.1021/acs.inorgchem.2c04103/suppl_file/ic2c04103_si_001.pdf https://pubs.acs.org/doi/10.1021/acs.inorgchem.2c04103?fig=fig4&ref=pdf https://pubs.acs.org/doi/10.1021/acs.inorgchem.2c04103?fig=fig4&ref=pdf https://pubs.acs.org/doi/10.1021/acs.inorgchem.2c04103?fig=fig4&ref=pdf https://pubs.acs.org/doi/10.1021/acs.inorgchem.2c04103?fig=fig4&ref=pdf pubs.acs.org/IC?ref=pdf https://doi.org/10.1021/acs.inorgchem.2c04103?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as forming adducts with Asp side chains at equatorial sites24 and keeping their paddlewheel structure. The results also indicate that monosubstituted diruthenium compounds present a different reactivity with proteins compared to diruthenium tetraacetate25 and to paddlewheel dirhodium com- pounds.36−40,44 Our data also suggest the possibility of an axial bond of the diruthenium core to the side chains of Lys or Arg and to backbone carbonyl groups. It is possible that the binding of the Asp residues at the equatorial position is a late event in the reaction of paddlewheel complexes with proteins and that the coordination of protein nucleophile sites at the axial position of the bimetallic scaffolds can be an early event in the dimetallic compound/protein recognition process. The binding to the axial site not only anticipates the later acetate detachment but also could exert a structural destabilization that facilitates its eventual occurrence, as indicated by computational studies.26 The combination of axial and equatorial coordinative binding has been postulated as a way to establish specific interactions between [Ru2Cl2(formamidinate)3(DMSO)] and ribonucleic acid.46 ■ ASSOCIATED CONTENT *sı Supporting Information The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.inorgchem.2c04103. Materials, solution studies, crystallization and X-ray structure solution and refinement, Figures S1−S5, and Table S1 (PDF) ■ AUTHOR INFORMATION Corresponding Authors Antonello Merlino − Department of Chemical Sciences, University of Naples Federico II, Naples 80126, Italy; orcid.org/0000-0002-1045-7720; Email: antonello.merlino@unina.it Santiago Herrero − Departamento de Química Inorgánica, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, Madrid E-28040, Spain; orcid.org/0000-0002- 9901-1142; Email: sherrero@ucm.es Authors Aarón Terán − Departamento de Química Inorgánica, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, Madrid E-28040, Spain Giarita Ferraro − Department of Chemical Sciences, University of Naples Federico II, Naples 80126, Italy Ana E. Sánchez-Peláez − Departamento de Química Inorgánica, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, Madrid E-28040, Spain Complete contact information is available at: https://pubs.acs.org/10.1021/acs.inorgchem.2c04103 Notes The authors declare no competing financial interest. ■ ACKNOWLEDGMENTS The authors thank the Elettra staff for technical assistance. Comunidad de Madrid is gratefully acknowledged for financial support (Project S2017/BMD-3770-CM). A.T. acknowledges the Universidad Complutense for a Predoctoral Grant (CT63/ 19-CT64/19) and Research Stay Grant (EB25/22) and the Spanish Ministry of Science and Innovation for a Postgraduate Fellowship at Residencia de Estudiantes (2021−2022). ■ REFERENCES (1) Cotton, F. A.; Murillo, C. A.; Walton, R. A. Multiple Bonds between Metal Atoms, 3rd ed.; Springer: New York, 2005. (2) Bennett, M. J.; Caulton, K. G.; Cotton, F. A. Structure of tetra-n- butyratodiruthenium chloride, a compound with a strong metal-metal bond. 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Charge effect in protein metalation reactions by diruthenium complexes hapter V C Charge effect in protein metalation reactions by diruthenium complexes 72 73 Abstract Charge effects on diruthenium complexes interaction with proteins is a key factor to take into account since these biomolecules do not show a uniform surface charge distribution. To compare the reactivity of ionic diruthenium compounds (cationic vs anionic) with the model protein hen egg white lysozyme (HEWL), the well-known anionic complex [Ru2(CO3)4]3-, two new anionic species, [Ru2(D-p-FPhF)(CO3)3]2- and [Ru2(DAniF)(CO3)3]2-, and their analogues, [Ru2Cl(D-p-FPhF)(O2CCH3)3] and [Ru2Cl(DAniF)(O2CCH3)3] that generate cationic species in solution (D-p-FPhF- = N,N′-bis(4- fluorophenyl)formamidinate; DAniF- = N,N′-bis(4-methoxyphenyl)formamidinate) were prepared. The interaction of these compounds with HEWL was investigated using UV-Vis absorption spectroscopy, intrinsic fluorescence, circular dichroism, and X-ray crystallography. The binding properties of paddlewheel diruthenium compounds significantly depend on the nature of their bridging equatorial ligands. The charge of the complexes permits to modulate the covalent and non-covalent bonding. Multivalent anionic diruthenium compounds remain interacting with the surface while monoanionic, neutral or cationic species can covalently bind the protein. The molecular structures of the adducts differ in the number of metal binding sites, in the binding mode and in the type of fragments that are bound to the protein. The new data suggest that the conditions used for the protein metalation can also significantly affect the structures of the reaction products, i.e. the final metal/protein adducts. Overall, these results may help designing diruthenium/protein adducts with the diruthenium species covalently or non-covalently bound to the protein surface by a proper choice of the diruthenium equatorial ligands. This study can have great implications for further studies in the tailoring of artificial diruthenium-containing metalloenzymes. 74 INORGANIC CHEMISTRY FRONTIERS RESEARCH ARTICLE Cite this: Inorg. Chem. Front., 2023, 10, 5016 Received 26th June 2023, Accepted 11th July 2023 DOI: 10.1039/d3qi01192e rsc.li/frontiers-inorganic Charge effect in protein metalation reactions by diruthenium complexes† Aarón Terán, a Giarita Ferraro,b Ana E. Sánchez-Peláez, a Santiago Herrero *a and Antonello Merlino *b The properties of paddlewheel diruthenium compounds significantly depend on the nature of the brid- ging equatorial ligands. The charge of these complexes is a factor to take into account when studying their interaction with proteins. To compare the reactivity of diruthenium compounds with the model protein hen egg white lysozyme (HEWL), we have prepared the well-known anionic complex [Ru2(CO3)4] 3−, two new anionic species, [Ru2(D-p-FPhF)(CO3)3] 2− and [Ru2(DAniF)(CO3)3] 2−, and their ana- logues [Ru2Cl(D-p-FPhF)(O2CCH3)3] and [Ru2Cl(DAniF)(O2CCH3)3] that generate cationic species in solu- tion (D-p-FPhF− = N,N’-bis(4-fluorophenyl)formamidinate and DAniF− = N,N’-bis(4-methoxyphenyl)for- mamidinate). The interaction of these compounds with HEWL was investigated by UV-vis absorption spectroscopy, circular dichroism, intrinsic fluorescence and X-ray crystallography. The molecular struc- tures of the adducts differ in the number of metal binding sites, in the binding mode and in the type of fragments that are bound to the protein. The charge of the diruthenium complexes in aqueous solutions strongly influences their protein binding properties. High-negative charge complexes are non-covalently bound. However, the replacement of carbonate ligands changes the negative charge of these complexes and favours covalent binding. These results have great implications for further studies in the tailoring of artificial diruthenium-containing metalloenzymes. Introduction Diruthenium(II,III) paddlewheel complexes contain a Ru–Ru core bridged by four equatorial ligands, which in the first com- pounds, synthesized by Wilkinson and Cotton, were carboxylates.1,2 In the solid state, they can show polymeric structures with axial ligands bridging the diruthenium units to form linear or zig-zag chains, discrete molecular species or cation–anion complexes.3,4 Besides carboxylates, a variety of N,O- and N,N′-bridging bidentate ligands have been employed to obtain diverse diruthenium motifs.5–9 The synthesis and reactivity of these complexes have been intensively studied in the last few years due to their singular electrochemical10,11 and magnetic properties.12–16 Diruthenium(II,III) paddlewheel complexes have also been used as scaffolds to prepare anticancer agents17–25 and to produce stable metalloenzymes with fascinating catalytic properties.26,27 In this frame, it has been shown that [Ru2(O2CCH3)4] + forms a metalloenzyme with the model protein hen egg white lysozyme (HEWL)26 which is able to cat- alyse the aerobic oxidation of hydroxylamines to nitrones imparting complete chemoselectivity to the reaction, in con- trast to the metal complex alone.27 In our continuous effort to study the reactivity of metal compounds with physiologically relevant molecules such as proteins, we have recently described the interaction of [Ru2(D- p-FPhF)(O2CCH3)3] + (D-p-FPhF− = N,N′-bis(4-fluorophenyl)for- mamidinate) with HEWL under different conditions in order to compare the effect of bulky equatorial ligands.28 The intro- duction of a formamidinate ligand increases the stability of the diruthenium core in solution. It has been shown that under all circumstances the formamidinate ligand is retained. In addition, this N,N′-bridging ligand can adopt a cis or a trans configuration with respect to the bridging coordinating residue side chains. The diruthenium core not only can bind at the equatorial positions of the side chain of Asp101 and 119, but also can bind to the Lys or Arg side chains or even the oxygen atom of a main chain carbonyl group at the axial sites. The diruthenium(II,III) compounds employed until now to study their interaction with proteins form monocationic species in solution. The charge of the diruthenium complexes †Electronic supplementary information (ESI) available. See DOI: https://doi.org/ 10.1039/d3qi01192e aMatMoPol Research Group, Department of Inorganic Chemistry, Faculty of Chemical Sciences, Complutense University of Madrid, Avda. Complutense s/n, 28040 Madrid, Spain. E-mail: sherrero@ucm.es bDepartment of Chemical Sciences, University of Naples Federico II, Complesso Universitario di Monte Sant’Angelo, via Cinthia, 21, 80126 Naples, Italy. E-mail: antonello.merlino@unina.it 5016 | Inorg. Chem. Front., 2023, 10, 5016–5025 This journal is © the Partner Organisations 2023 O pe n A cc es s A rt ic le . P ub lis he d on 1 1 Ju ly 2 02 3. D ow nl oa de d on 1 0/ 29 /2 02 3 6: 57 :5 1 PM . T hi s ar tic le is li ce ns ed u nd er a C re at iv e C om m on s A ttr ib ut io n 3. 0 U np or te d L ic en ce . View Article Online View Journal | View Issue http://rsc.li/frontiers-inorganic http://orcid.org/0000-0001-6126-6230 http://orcid.org/0000-0002-2258-1921 http://orcid.org/0000-0002-9901-1142 http://orcid.org/0000-0002-1045-7720 https://doi.org/10.1039/d3qi01192e https://doi.org/10.1039/d3qi01192e https://doi.org/10.1039/d3qi01192e http://crossmark.crossref.org/dialog/?doi=10.1039/d3qi01192e&domain=pdf&date_stamp=2023-08-19 http://creativecommons.org/licenses/by/3.0/ http://creativecommons.org/licenses/by/3.0/ https://doi.org/10.1039/d3qi01192e https://pubs.rsc.org/en/journals/journal/QI https://pubs.rsc.org/en/journals/journal/QI?issueid=QI010017 is expected to affect the type of adducts that will be formed upon reaction of the compounds with proteins.29 In this regard, anionic species may be the door towards a new family of diruthenium metalloproteins. However, the number of known anionic diruthenium complexes is rather limited and most of them contain two labile anionic ligands at the axial positions.30–34 Another possibility is the use of high-charge anionic bridging ligands. In fact, our group and others have been interested in the use of the anionic unit [Ru2(CO3)4] 3− to develop heterometallic building blocks with different topolo- gies and interesting magnetic properties.35–43 Here we report the synthesis and characterization of K3[Ru2(CO3)4], K2[Ru2(DAniF)(CO3)3], K2[Ru2(D-p-FPhF)(CO3)3], and [Ru2Cl(DAniF)(O2CCH3)3] (DAniF− = N,N′-bis(4-methoxy- phenyl)formamidinate; Fig. 1) to prepare new diruthenium metalloproteins. We show crystallographic evidence of different binding modes depending on the cationic/anionic nature of the complexes in aqueous solutions ([Ru2(CO3)4] 3−, [Ru2(D-p-FPhF)(CO3)3] 2−, [Ru2(DAniF)(CO3)3] 2−, and [Ru2(DAniF)(O2CCH3)3] +). Data have been compared with those reported for [Ru2(O2CCH3)4] + and [Ru2(D-p-FPhF)(O2CCH3)3] + complexes.26,28 Results and discussion Synthesis and characterization of K2[Ru2(D-p-FPhF)(CO3)3] and K2[Ru2(DAniF)(CO3)3] The preparation of K3[Ru2(CO3)4]·4H2O and [Ru2Cl(DAniF) (O2CCH3)3]·H2O derivatives is already described in the literature.44,45 Here, the synthesis of two new heteroleptic anionic complexes, K2[Ru2(D-p-FPhF)(CO3)3]·3H2O·EtOH and K2[Ru2(DAniF)(CO3)3]·3H2O, from the corresponding [Ru2Cl(L– L)(O2CCH3)3] is reported (Scheme S1, ESI†). The replacement of acetate by carbonate bridging ligands proceeds successfully overnight under mild conditions in 95–99% yields. All the dir- uthenium complexes are air stable and water-soluble and can be handled without special caution. The new complexes were determined by elemental analysis, ATR-FT-IR (attenuated total reflection Fourier transform infrared) spectroscopy, UV-Vis spectroscopy, electrospray ionization mass spectrometry and cyclic voltammetry. The ATR-FTIR spectra of the compounds (Fig. S1, ESI†) are similar to each other and to their analogs with acetate ligands. A wide band in the 3500–3400 cm−1 range and a band centred around 1640 cm−1 suggest the presence of water in both com- plexes, in accordance with elemental analysis. Carbonate vibrational modes corresponding to COO stretching (asym- metric + symmetric) and bending can be observed around 1450 and 694 cm−1, respectively. Some of the formamidinato ligand vibration modes are observed at 1529 cm−1 (ν(CvCarom)) and 1296 and 1210 cm−1 (ν(C–N)).46 These results confirmed the presence of representative functional groups coordinated to the diruthenium core. Electrospray ionization mass spectrometry was used to verify the stoichiometry and elemental composition of the dir- uthenium complexes. The mass spectra of K2[Ru2(D-p-FPhF) (CO3)3]·3H2O·EtOH and K2[Ru2(DAniF)(CO3)3]·3H2O support the full exchange of all acetate molecules by carbonate ligands retaining the formamidinate ligand. The dominant peaks correspond to the intact complex with the loss of solvent mole- cules (S = H2O and/or EtOH), [M − S + H+]+, or with the ionized forms by loss of K+ counterions and solvent molecules, [M − K+ − S + 2H+]+ and [M − 2K+ − S + 3H+]+. All the ion peaks show reasonable agreement between the experimental and simulated isotopic distribution and support the assigned stoichiometries and formulations of the new diruthenium derivatives (Fig. S2 and S3, respectively, ESI†). Cyclic voltammetry (CV) was used to evaluate the effect of ligand substitution on the electrochemical responses of dir- uthenium complexes in aqueous solutions. CV measurements were carried out in 0.10 M KCl solution at 0.1 V s−1 (Fig. S4, ESI†); the data were compared with those obtained for [Ru2Cl (DAniF)(O2CCH3)3] 45 (reversible one-electron reduction, Ru2 5+/ 4+) and [Ru2Cl(D-p-FPhF)(O2CCH3)3] 47 (quasi-reversible one- electron reduction, Ru2 5+/4+). The properties of [Ru2Cl (O2CCH3)4] 9 (quasi-reversible one-electron reduction, Ru2 5+/4+) and K3[Ru2(CO3)4] 44 (quasi-reversible one-electron oxidation, Ru2 5+/6+) were also measured because they had been previously reported under different conditions. Table 1 presents the half- wave peak potentials (E1/2) and anodic to cathodic peak poten- tial separation (ΔE) for [Ru2Cl(O2CCH3)4], K3[Ru2(CO3)4], [Ru2Cl(L–L)(O2CCH3)3] and K2[Ru2(L–L)(CO3)3] (L–L = DAniF− Fig. 1 Molecular structure of diruthenium derivatives: (a) K3[Ru2(CO3)4], (b) K2[Ru2(DAniF)(CO3)3] and K2[Ru2(D-p-FPhF)(CO3)3], and (c) [Ru2Cl (DAniF)(O2CCH3)3] and [Ru2Cl(D-p-FPhF)(O2CCH3)3]. Table 1 Electrochemical data (V vs. Ag/AgCl) from cyclic voltammetry measurements of diruthenium complexes in 0.10 M KCl aqueous solutions Compound E1/2 ΔE [Ru2Cl(O2CCH3)4] −0.10 0.27 K3[Ru2(CO3)4] 0.68 0.21 [Ru2Cl(D-p-FPhF)(O2CCH3)3] −0.32 0.28 K2[Ru2(D-p-FPhF)(CO3)3] 0.75 0.08 [Ru2Cl(DAniF)(O2CCH3)3] −0.35 0.15 K2[Ru2(DAniF)(CO3)3] 0.68 0.08 Inorganic Chemistry Frontiers Research Article This journal is © the Partner Organisations 2023 Inorg. Chem. Front., 2023, 10, 5016–5025 | 5017 O pe n A cc es s A rt ic le . P ub lis he d on 1 1 Ju ly 2 02 3. D ow nl oa de d on 1 0/ 29 /2 02 3 6: 57 :5 1 PM . T hi s ar tic le is li ce ns ed u nd er a C re at iv e C om m on s A ttr ib ut io n 3. 0 U np or te d L ic en ce . View Article Online http://creativecommons.org/licenses/by/3.0/ http://creativecommons.org/licenses/by/3.0/ https://doi.org/10.1039/d3qi01192e or D-p-FPhF−) derivatives. Formamidinate10 (L–L) and carbon- ate44 ligands increase the electron density on the diruthenium core and seem to favour a Ru2 5+/6+ reversible oxidation process. Besides, the presence of electron-donating substituents in the aromatic rings (–OCH3) produces lower oxidation potentials, while the presence of an electron-withdrawing substituent (–F) causes a positive cathodic shift. Stability of [Ru2(CO3)4] 3−, [Ru2(D-p-FPhF)(CO3)3] 2−, [Ru2(DAniF)(CO3)3] 2− and [Ru2(DAniF)(O2CCH3)3] + in aqueous solutions To ascertain the stability of [Ru2(CO3)4] 3−, [Ru2(D-p-FPhF) (CO3)3] 2−, [Ru2(DAniF)(CO3)3] 2− and [Ru2(DAniF)(O2CCH3)3] + in aqueous solutions and under the conditions used to study their binding to HEWL, UV-Vis absorption spectra were col- lected as a function of time. The monitoring of the UV-Vis bands of the four compounds in pure water and in different buffers is reported (Fig. S5 and S6–S9 panels A and C, ESI†). In the visible region (400–700 nm), absorptions bands are assigned to allowed ligand-to-metal charge transfers [π(N/O), π(axial) → π*(Ru2)], while the UV bands are usually related to an axial ligand-to- metal charge transfer and ligand-to-metal transitions [π(N) → σ*/π*/δ*(Ru2)].9,28 No appreciable spectral changes were observed within 24 h. However, under the conditions used for the crystallization experiments, some changes were found after a week. In all cases, the absorption profiles show regularity in the shape and position of the bands and are very similar to other monosubstituted diruthenium compounds. Slight vari- ations due to a possible substitution of the O,O′-donor equa- torial ligands (acetates or carbonates) for other O,O′-donor ligands present under the corresponding conditions (e.g., nitrates, acetates, or formates) are noticed. This is related to the high concentration of these ions in the buffer medium which may lead to different partial substitution reactions.48 Spectra were also collected as a function of time in the pres- ence of HEWL under the same experimental conditions. Under almost all of the conditions used for the crystallization experiments, no significant differences between the spectral profiles of the compounds in the absence and in the presence of the protein are evidenced (Fig. S6–S9 panels B and D, ESI†). Binding to HEWL: fluorescence and circular dichroism studies To further study the HEWL binding ability of [Ru2(CO3)4] 3−, [Ru2(D-p-FPhF)(CO3)3] 2−, [Ru2(DAniF)(CO3)3] 2− and [Ru2(DAniF)(O2CCH3)3] +, intrinsic fluorescence and circular dichroism (CD) measurements were collected. First, the fluo- rescence quenching measurements were registered (Fig. 2A–D and Fig. S10–S13, ESI†). Basically, HEWL fluorescence is caused by tryptophan, tyrosine and phenylalanine residues (n → π* transition). As a result of the binding, an enhancement or a decrease of the fluorescence should occur. Titration experiments carried out by adding [Ru2(CO3)4] 3−, [Ru2(D-p- FPhF)(CO3)3] 2−, [Ru2(DAniF)(CO3)3] 2− and [Ru2(DAniF) (O2CCH3)3] + to a HEWL solution show a reduction of the fluo- rescence intensity of the protein, thus indicating that all these metal compounds could bind to the protein under the investi- gated experimental conditions. Circular dichroism (CD) spectroscopy provides information on the protein secondary structure content and can be used to investigate the structure and dynamics of interactions of pro- teins with foreign moieties. Far-UV CD spectra of HEWL at 25 °C in the absence and in the presence of the four com- pounds at different HEWL : Ru2 molar ratios in 10 mM Hepes buffer pH 7.5 and 10 mM sodium acetate buffer pH 4.0 were collected and are reported in Fig. 2E–H and Fig. S14, ESI,† respectively. The spectrum of metal-free HEWL presents a double minimum at 208 and 222 nm, as expected for a protein with a large content of α-helix structural elements. Surprisingly, we noticed that the addition of some Ru2 5+ com- pounds caused an increase in the negative ellipticity without any significant shift of the bands. Previous results with the [Ru2(D-p-FPhF)(O2CCH3)3] + complex28 showed that the incu- bation of this compound with the protein did not change the CD spectrum. In this case, its anionic analogue, [Ru2(D-p- FPhF)(CO3)3] 2−, did not significantly change the CD signal. However, with compounds [Ru2(CO3)4] 3−, [Ru2(DAniF)(CO3)3] 2− and [Ru2(DAniF)(O2CCH3)3] +, an increase in the negative ellip- ticity signal is observed. This can be associated with a kosmo- tropic-like property of these diruthenium derivatives. A kosmo- trope strengthens the hydrogen bonds of water molecules and stabilizes the intramolecular interactions in the structure of a biomolecule.49 The increase of self-association processes within the HEWL chain, in the presence of diruthenium com- plexes, can create a more prevalent secondary structure which would increase the CD signal.50,51 In all cases, the global stabi- lization of the protein secondary structure produced upon interaction with the diruthenium species is not associated with a change in their thermal denaturation, since the protein in the presence of the diruthenium complexes has almost the same denaturation temperature as in their absence (Table S1, ESI†). The character of the changes suggests that the diruthe- nium-induced variations do not generate a different confor- mation but more likely affect the compactness/dynamics of the polypeptide chain. This phenomenon seems to be quite sig- nificant, especially for the DAniF derivatives, where the pres- ence of the methoxy group is expected to generate a higher number of interactions with the surrounding water molecules. Binding to HEWL: crystallographic studies X-ray crystallography was used to study the structure of the adducts formed upon the reaction of HEWL with [Ru2(CO3)4] 3−, [Ru2(D-p-FPhF)(CO3)3] 2−, [Ru2(DAniF)(CO3)3] 2−, and [Ru2(DAniF)(O2CCH3)3] +. The structures of the adducts were prepared by a soaking method using protein crystals grown under two different experimental conditions: 20% ethyl- ene glycol, 0.6 M sodium nitrate, and 0.1 M sodium acetate pH 4.0 (condition A) and 2 M sodium formate and 0.1 M Hepes (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid), pH 7.5 (condition B). The crystals of the adducts were freezed after 14 days of soaking. They diffracted X-ray at a resolution within the range of 1.46–1.03 Å. Data collection and refinement statistics Research Article Inorganic Chemistry Frontiers 5018 | Inorg. Chem. Front., 2023, 10, 5016–5025 This journal is © the Partner Organisations 2023 O pe n A cc es s A rt ic le . P ub lis he d on 1 1 Ju ly 2 02 3. D ow nl oa de d on 1 0/ 29 /2 02 3 6: 57 :5 1 PM . T hi s ar tic le is li ce ns ed u nd er a C re at iv e C om m on s A ttr ib ut io n 3. 0 U np or te d L ic en ce . View Article Online http://creativecommons.org/licenses/by/3.0/ http://creativecommons.org/licenses/by/3.0/ https://doi.org/10.1039/d3qi01192e are reported in Table S2, ESI.† The structures refine to Rfactor and Rfree values within the range of 0.179–0.205 and 0.202–0.245, respectively. Analysis of the Fourier difference electron density map indicated that attempts to obtain crystals of the adduct of [Ru2(CO3)4] 3− with HEWL under condition B failed. The structures are deposited in the PDB (https://www. rcsb.org) as entries 8PFU (HEWL with [Ru2(CO3)4] 3− under con- dition A), 8PFT (HEWL with [Ru2(D-p-FPhF)(CO3)3] 3− under condition A), 8PFX (HEWL with [Ru2(D-p-FPhF)(CO3)3] 3− under condition B), 8PFW (HEWL with [Ru2(DAniF)(CO3)3] 2− under condition A), 8PFY (HEWL with [Ru2(DAniF)(CO3)3] 2− under condition B) and 8PFV (HEWL with [Ru2(DAniF)(O2CCH3)3] + under condition A). Structure of the adduct formed upon the reaction of HEWL with [Ru2(CO3)4] 3− under condition A The overall structure of the adduct that HEWL forms with [Ru2(CO3)4] 3− under condition A (Fig. 3), which was refined at a resolution of 1.18 Å, is nearly identical to that of the metal- Fig. 2 (A–D) Fluorescence quenching spectra of HEWL (2 μM) in 10 mM Hepes buffer pH 7.5 by [Ru2(CO3)4] 3− (A), [Ru2(D-p-FPhF)(CO3)3] 2− (B), [Ru2(DAniF)(CO3)3] 2− (C), and [Ru2(DAniF)(O2CCH3)3] + (D) (upon increasing the concentrations from 0 to 20 μM). Spectra exciting the protein at 295 nm are reported in the ESI.† At the concentration used for these experiments the metal compounds do not significantly absorb. (E–H) Far-UV CD spectra of HEWL (7.0 μM concentration) incubated for 24 h in the presence of [Ru2(CO3)4] 3− (E), [Ru2(D-p-FPhF)(CO3)3] 2−(F), [Ru2(DAniF)(CO3)3] 2− (G), and [Ru2(DAniF)(O2CCH3)3] + (H) in 10 mM Hepes buffer pH 7.5 in different HEWL : Ru2 molar ratios. Fig. 3 Diruthenium binding sites in the structure of the adduct formed upon the reaction of HEWL with [Ru2(CO3)4] 3− after 14 days of soaking under condition A. 2Fo − Fc electron density maps are contoured at the 1.0σ (grey) level. Inorganic Chemistry Frontiers Research Article This journal is © the Partner Organisations 2023 Inorg. Chem. Front., 2023, 10, 5016–5025 | 5019 O pe n A cc es s A rt ic le . P ub lis he d on 1 1 Ju ly 2 02 3. D ow nl oa de d on 1 0/ 29 /2 02 3 6: 57 :5 1 PM . T hi s ar tic le is li ce ns ed u nd er a C re at iv e C om m on s A ttr ib ut io n 3. 0 U np or te d L ic en ce . View Article Online https://www.rcsb.org https://www.rcsb.org https://www.rcsb.org http://creativecommons.org/licenses/by/3.0/ http://creativecommons.org/licenses/by/3.0/ https://doi.org/10.1039/d3qi01192e free protein: the root mean square deviation of the carbon alpha atoms (rmsd) between the structure of the adduct and that of the HEWL used as a starting model (PDB code: 193L52) is as low as 0.12 Å. The main differences are located at the level of the diruthenium binding sites. In the structure of the adduct, diruthenium centres are found close to the interface between two symmetry-related molecules (occupancy = 0.20) and at the side chains of Asn103 (occupancy = 0.2 and the dis- tance from the nearest protein atom ≈ 2.1 Å) and Asp101 (occupancy = 0.20 and the distance Ru-OD ≈ 2.5 Å). In all these sites, diruthenium ligands cannot be defined. B-factors of Ru atoms are within the range of 63.6–28.6 Å2. Our data suggest that under the investigated experimental conditions, diruthenium tetracarbonate loses at least a part of the carbon- ate ligands and that a diruthenium containing fragment, prob- ably coordinated to solvent molecules and to a part of carbon- ate ligands originally present in the [Ru2(CO3)4] 3− structure, can react with the protein. An additional diruthenium centre was found at the C-terminal tail (distance Ru-OX ≈ 2.2 Å), non- covalently bound to the protein. Here the ligands are not well defined, but one carbonate could be bound to the metals. At this site, Ru atoms have an occupancy of 0.40 and B-factors of 63.6 and 42.8 Å2. Structures of the adducts formed upon the reaction of HEWL with the [Ru2(D-p-FPhF)(CO3)3] 2− complex Superimposition of the structures of the adducts formed upon the reaction of HEWL with [Ru2(D-p-FPhF)(CO3)3] 2− under con- ditions A and B gave a rmsd value of 0.15 Å, suggesting simi- larity of the overall protein conformation in the adducts obtained under the two conditions (Fig. 4A and 5A). Similar results were obtained upon superimposition of these struc- tures with that of the metal-free protein (rmsd = 0.16 Å for con- ditions A and B). The structure obtained under condition A was resolved at 1.30 Å resolution, while that under condition B was resolved at 1.03 Å resolution. In the crystal structure of the adduct formed upon the reac- tion of HEWL with [Ru2(D-p-FPhF)(CO3)3] 2− under condition A (Fig. 4A), resolved at 1.42 Å resolution, electron density for two diruthenium containing fragments (occupancy = 0.35) was observed (Fig. 4B and C). The side chain of Asp101 is co- ordinated to the diruthenium core together with D-p-FPhF, a carbonate ion, and two equatorial and two axial water mole- cules, [Ru2(Asp101)(D-p-FPhF)(CO3)(OH2)4] + (Fig. 4B). D-p-FPhF is trans to the side chain of the Asp (Fig. 4B). In our model this diruthenium center was interpreted as an alternative to that found close to Asp119. The second diruthenium fragment is the same found close to Asp101, i.e., a [Ru2(Asp119)(D-p-FPhF) (CO3)(OH2)4] + moiety. This fragment has the D-p-FPhF cis to the side chain of Asp119 (Fig. 4C). These findings confirm that anionic monosubstituted diruthenium paddlewheel complexes can bind to protein residue side chains both in cis and trans Fig. 4 Diruthenium binding sites in the structure of the adduct formed upon the reaction of HEWL with [Ru2(D-p-FPhF)(CO3)3] 2− after 14 days of soaking under condition A (panel A). Binding sites have been observed close to the side chains of Asp101 (panel B) and Asp119 (panel C). 2Fo − Fc electron density maps are contoured at the 1.0 σ (grey) level. Fig. 5 Covalent and non-covalent (NCB) diruthenium binding sites in the structure of the adduct formed upon the reaction of HEWL with [Ru2(D-p-FPhF)(CO3)3] 2− after 14 days of soaking under condition B (panel A). Binding sites have been observed close to the side chain of Asp101 (panel B). 2Fo − Fc electron density maps are contoured at the 1.0 σ (grey) level. Axial H2O molecules are omitted for the sake of clarity. Research Article Inorganic Chemistry Frontiers 5020 | Inorg. Chem. Front., 2023, 10, 5016–5025 This journal is © the Partner Organisations 2023 O pe n A cc es s A rt ic le . P ub lis he d on 1 1 Ju ly 2 02 3. D ow nl oa de d on 1 0/ 29 /2 02 3 6: 57 :5 1 PM . T hi s ar tic le is li ce ns ed u nd er a C re at iv e C om m on s A ttr ib ut io n 3. 0 U np or te d L ic en ce . View Article Online http://creativecommons.org/licenses/by/3.0/ http://creativecommons.org/licenses/by/3.0/ https://doi.org/10.1039/d3qi01192e configurations as their cationic analogs.28 In any case, since the occupancy is low, the coordination of an anion bridging ligand instead of two water molecules cannot be excluded (Fig. 4B). Under condition B, crystals of the adduct diffract at 1.03 Å resolution. Here, diruthenium containing fragments were observed close to the side chain of Asp101 (Fig. 5A) and close to a symmetry axis (Fig. 5B). A Hepes molecule was also added to the model (Fig. S15†). A [Ru2(Asp101)(D-p-FPhF)(CO3) (OH2)4] + moiety was formed similarly to that obtained under condition A. At this site, the diruthenium fragment presents an occupancy equal to 0.35. The B-factors of Ru atoms are 15.4 and 14.4 Å2. Two water molecules complete the octahedral coordination sphere of each Ru atom occupying the axial sites. An extra electron density neighbouring this diruthenium frag- ment was observed in the Fo − Fc and in the anomalous differ- ence electron-density maps. Here two Ru atoms were placed in the model, not coordinated to any residue side chains and with low occupancy (0.20) and B-factors of 15.5 and 14.5 Å2. Diruthenium centre ligands at this site are undefined. Another disordered Ru2-containing fragment, non-covalently bound to HEWL and with occupancy 0.20 (B-factors of 16.1 and 17.9 Å2), was found close to the symmetry axis. Here the ligands are not well defined. Structures of the adducts formed upon the reaction of HEWL with the [Ru2(DAniF)(CO3)3] 2− complex Crystals of the HEWL adducts with [Ru2(DAniF)(CO3)3] 2− obtained upon soaking of the metal compound within HEWL crystals grown under conditions A and B were also analysed. The structure of the adduct obtained under condition B (at a resolution of 1.19 Å) suggests a scarce binding of the com- pound to the protein. Only one minor peak of anomalous difference electron density map is observed in this structure, close to the Asp101 side chain (data not shown). In contrast, very clear electron density maps are observed in the structure of the adduct formed under condition A (at a resolution of 1.12 Å). Here, three different metal compound binding sites were found (Fig. 6A). The first diruthenium centre (occupancy = 0.50) is linked to the side chain of Asp119 cis to the DAniF ligand together with two equatorial carbonate ligands and two axial water mole- cules, [Ru2(Asp119)(DAniF)(CO3)2(OH2)2] − (Fig. 6C). The other two binding sites are found on the protein surface, where the diruthenium complexes are non-covalently bound to the protein (Fig. 6B and C). In both cases, the whole compound is modeled: a diruthenium compound that retains all its carbon- ate and DAniF ligands at the equatorial positions and with water molecules at the axial sites, [Ru2(DAniF)(CO3)3(OH2)2] 2−. The presence of carbonate ligands coordinated to the diruthe- nium center and a methoxy group attached to the aromatic rings of the formamidinate ligand (DAniF) allows one to estab- lish different interactions which stabilize the Ru2/HEWL adduct. One molecule (occupancy = 0.35) is close to Asp101 (Fig. 6D) and interacts with a NO3 − ion and with the side chains of Ser100, Lys97 and Gly102. Also, interactions with symmetry mates occur through the side chains of Arg5 and Gly4 and with the carbonate group of other diruthenium motif coordinated to Asp119. The second Ru2 complex (occupancy = 0.45) is close to Asp119 (Fig. 6E) and interacts with the main chain carbonyl group of Asp119 and with the carbonate group of the diruthenium complex bound to the side chain of Asp119. Here, the carbonate ligands interact with a symmetry- related molecule at the side chain of Gly117 and with the car- bonate group of a non-covalently bound diruthenium complex. The B-factors of Ru atoms in this structure are within the range of 12.8–9.9 Å2. Structures of the adducts formed upon the reaction of HEWL with the [Ru2(DAniF)(O2CCH3)3] + complex To compare the results obtained with [Ru2(DAniF)(CO3)3] 2− with a proper reference, the X-ray structure of the adducts of HEWL with [Ru2(DAniF)(O2CCH3)3] + formed under condition A was also resolved (Fig. 7A). The structure, which refines at 1.46 Å resolution, reveals the existence of a single binding site for [Ru2(DAniF)(O2CCH3)3] +, close to the side chain of Asp119 (occupancy = 0.35), with the Fig. 6 Overall structure of the adduct formed upon the reaction of HEWL with [Ru2(DAniF)(CO3)3] 2− under condition A (panel A) and details of the diruthenium covalent and non-covalent binding (NCB) sites (panels B–E). 2Fo − Fc electron density maps are contoured at the 1.0 σ (grey) level. Symmetry related molecules are colored grey. Axial H2O molecules are omitted for the sake of clarity. Inorganic Chemistry Frontiers Research Article This journal is © the Partner Organisations 2023 Inorg. Chem. Front., 2023, 10, 5016–5025 | 5021 O pe n A cc es s A rt ic le . P ub lis he d on 1 1 Ju ly 2 02 3. D ow nl oa de d on 1 0/ 29 /2 02 3 6: 57 :5 1 PM . T hi s ar tic le is li ce ns ed u nd er a C re at iv e C om m on s A ttr ib ut io n 3. 0 U np or te d L ic en ce . View Article Online http://creativecommons.org/licenses/by/3.0/ http://creativecommons.org/licenses/by/3.0/ https://doi.org/10.1039/d3qi01192e diruthenium centre bound to the side chain of the Asp residue cis to the DAniF ligand (Fig. 7B). Thus, in both the adducts of HEWL with [Ru2(DAniF)(CO3)3] 2− and [Ru2(DAniF)(O2CCH3)3] + binding to the Asp119 side chain, with the Asp bound cis to the formamidinate ligand, occurs. Thus, non-covalent binding was identified in the adduct of the protein with the anionic compound, while only the coordinative mode of binding was observed for [Ru2(DAniF)(O2CCH3)3] +. Structural comparison The structures of the adducts of two paddlewheel diruthenium compounds formed upon the reaction with HEWL have been resolved up to now, [Ru2(O2CCH3)4] + and [Ru2(D-p-FPhF) (O2CCH3)3] +.26,28 These data represent the only structural infor- mation on protein adducts of diruthenium compounds. [Ru2(O2CCH3)4] + binds to the side chains of Asp residues, losing one acetate ligand in favour of the Asp carboxylate and with the possibility to lose other acetate ligands in favour of water molecules. Upon reaction of the same protein with [Ru2(D-p-FPhF)(O2CCH3)3] +, diruthenium centres are bound to solvent-exposed protein residue side chains (Asp101, Asp119, Asn19, Lys33, and Arg125), retaining the formamidinate ligand, and with different reactivity depending on the experi- mental conditions. Comparing the behaviour of [Ru2(CO3)4] 3− and [Ru2(O2CCH3)4] +, it emerges that the different charge of the two complexes does produce significant differences in the reactivity with HEWL: both complexes bind to the Asp side chains, but the tetracarbonate compound can also bind to the Asn side chain and the C-terminal tail and can bind to the protein in a non-covalent fashion. In the case of [Ru2(D-p- FPhF)(CO3)3] 2− and [Ru2(DAniF)(CO3)3] 2−, two types of Ru2 fragments are described: those coordinated to Asp101 or Asp119 side chains and those that are non-covalently bound to the protein surface. The binding Asp residues are shared with their cationic analogues. However, there is a clear influence of the charge on the diruthenium compound binding to the protein, because as long as the complexes retain their high- negative charge (−2) they remain non-covalently bound to the protein. When the loss (or the replacement) of carbonate ligands occurs, i.e., when the complexes reduce their charge from −2 to −1 or they change from an anionic to a cationic form, they directly coordinate with the protein residue side chains, similarly to the cationic diruthenium compounds con- taining acetate ligands. Once the usual binding sites are occu- pied, additional molecules will remain on the surface interact- ing through charge or dipole interactions with adjacent groups, allowing stabilisation of the adduct structure. Thus, the non-covalent binding may be a step that precedes the coordination of the covalently bounded species to the protein, but it also can be the consequence of the full occupancy of the metal binding sites and of the charge of the compound. Notably, our data suggest that it is in principle possible to guide the diruthenium compound protein binding from non- covalent to covalent by substitution of carbonate ions by other ligands. Conclusions Structural studies on the products of the reaction of peptides and proteins with diruthenium paddlewheel complexes are important since they provide the molecular basis to explain the behaviour of artificial diruthenium-containing enzymes. These studies are also useful to define the interaction occur- ring in vivo when diruthenium paddlewheel complexes are used as metallodrugs. Here, we have reported six new crystal structures of the adducts formed upon the reaction of HEWL with four distinct diruthenium complexes: [Ru2(CO3)4] 3−, [Ru2(D-p-FPhF)(CO3)3] 2− [Ru2(DAniF)(CO3)3] 2−, and [Ru2(DAniF)(O2CCH3)3] +. Cationic diruthenium species react with HEWL by coordi- nating the side chains of Asp119 and Asp101 upon releasing one or two acetate ligands. In contrast, anionic diruthenium complexes may be non-covalently bound to the protein surface, unless carbonate ligands are lost or replaced. Crystallographic data indicate that the binding of these molecules does not alter the overall conformation of the protein, but it produces some changes around the dimetallic binding site. This work enlarges the repertoire of available structures of adducts formed upon the reaction of diruthe- nium compounds with proteins and provides new insights Fig. 7 Overall structure of the adduct formed upon the reaction of HEWL with [Ru2(DAniF)(O2CCH3)3] + under condition A (panel A) and details of the metal compound binding sites (panel B). 2Fo − Fc electron density maps are contoured at the 1.0 σ (grey) level. Axial H2O molecules are omitted for the sake of clarity. Research Article Inorganic Chemistry Frontiers 5022 | Inorg. Chem. Front., 2023, 10, 5016–5025 This journal is © the Partner Organisations 2023 O pe n A cc es s A rt ic le . P ub lis he d on 1 1 Ju ly 2 02 3. D ow nl oa de d on 1 0/ 29 /2 02 3 6: 57 :5 1 PM . T hi s ar tic le is li ce ns ed u nd er a C re at iv e C om m on s A ttr ib ut io n 3. 0 U np or te d L ic en ce . View Article Online http://creativecommons.org/licenses/by/3.0/ http://creativecommons.org/licenses/by/3.0/ https://doi.org/10.1039/d3qi01192e into the reactivity of these compounds with biological macro- molecules. Our results suggest that different diruthenium pad- dlewheel complexes can be used to prepare various artificial metalloenzymes with distinct properties. Data are significant because they support the idea that diruthenium complexes can be tuned, by changing the equatorial ligands, to direct their activity to specific cellular targets. Finally, our structures provide a very interesting system for molecular docking that can be used to predict metal compound binding sites on protein structures. Conflicts of interest Nothing to declare. Acknowledgements The authors thank Elettra staff for technical assistance. Comunidad de Madrid (Project S2017/BMD-3770-CM) and Universidad Complutense de Madrid (Program PR3/23) are gratefully acknowledged for the financial support. A. T. also acknowledges the Universidad Complutense for a Predoctoral Grant (CT63/19-CT64/19) and Research Stay Grant (EB25/22) and the Spanish Ministry of Science and Innovation for a Postgraduate Fellowship at Residencia de Estudiantes (2021–2022). References 1 T. A. Stephenson and G. Wilkinson, New ruthenium car- boxylate complexes, J. Inorg. Nucl. Chem., 1966, 28, 2285– 2291. 2 M. J. Bennett, K. G. Caulton and F. A. Cotton, Structure of tetra-n-butyratodiruthenium chloride, a compound with a strong metal-metal bond, Inorg. Chem., 1969, 8, 1–6. 3 M. A. S. Aquino, Recent developments in the synthesis and properties of diruthenium tetracarboxylates, Coord. Chem. Rev., 2004, 248, 1025–1045. 4 P. Delgado-Martínez, L. Moreno-Martínez, R. González- Prieto, S. Herrero, J. L. Priego and R. 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Acta, Part A, 2008, 70, 866–870. 52 M. C. Vaney, S. Maignan, M. Riès-Kautt and A. Ducruix, High-Resolution Structure (1.33 Å) of a HEW Lysozyme Tetragonal Crystal Grown in the APCF Apparatus. Data and Structural Comparison with a Crystal Grown under Microgravity from SpaceHab-01 Mission, Acta Crystallogr., Sect. D: Biol. Crystallogr., 1996, 52, 505–517. Inorganic Chemistry Frontiers Research Article This journal is © the Partner Organisations 2023 Inorg. Chem. Front., 2023, 10, 5016–5025 | 5025 O pe n A cc es s A rt ic le . P ub lis he d on 1 1 Ju ly 2 02 3. D ow nl oa de d on 1 0/ 29 /2 02 3 6: 57 :5 1 PM . T hi s ar tic le is li ce ns ed u nd er a C re at iv e C om m on s A ttr ib ut io n 3. 0 U np or te d L ic en ce . View Article Online https://doi.org/10.1039/D3QI00399J http://creativecommons.org/licenses/by/3.0/ http://creativecommons.org/licenses/by/3.0/ https://doi.org/10.1039/d3qi01192e 83 Chapter VI. Steric hindrance and charge influence on the cytotoxic activity and protein binging properties of diruthenium complexes hapter VI C Steric hindrance and charge influence on the cytotoxic activity and protein binding properties of diruthenium complexes 84 96 Abstract Paddlewheel diruthenium complexes have been used as molecular building blocks to develop new metal-based drugs. However, its therapeutic targets remain unidentified and their mechanism of action is still unknown. It has been proposed that charge and steric properties of the compounds determine their selectivity towards proteins. Here, these parameters thanks to the synthesis and characterization of the first water-soluble disubstituted diruthenium complex, [Ru2Cl(DPhF)2(O2CCH3)2] have been explored. Monosubstituted compounds [Ru2Cl(DPhF)(O2CCH3)3] and K2[Ru2(DPhF)(CO3)3] (DPhF- = N,N′-diphenylformamidinate) have been employed for comparison reasons. The antiproliferative properties of these complexes have been evaluated in a model based on eukaryotic cells. The new data indicate that there is a correlation between the number of formamidinate ligands and the anticancer activity of diruthenium derivatives against human epithelial carcinoma (HeLa) cells. Increased cytotoxicity may be related to increased steric hindrance and also Ru2 5+ core electronic density. However, the effect of increasing the lipophilicity of diruthenium species by introducing a second bidentate ligand N,N′-diphenylformamidinate must be also taken into account. The protein binding properties of these diruthenium compounds have been assessed using the model protein hen egg white lysozyme (HEWL). The results confirm that there is a relationship between the type of interaction (covalent or non-covalent) and the charge of those complexes. In addition, the crystallisation medium is found to be a key factor to obtain specific interactions through the equatorial or axial positions of the diruthenium core with protein residue side chains. In all cases, diruthenium species maintain the integrity of the M‒M bond and produce stable adducts. The molecular structures of these adducts provide new insights on the biological behaviour of diruthenium compounds. The proper choice of the diruthenium equatorial ligands can lead to complexes with improved biological properties and different reactivity with proteins. This work illustrates a systematic approach to shed light on the relevant properties of diruthenium compounds and provides a step forward to facilitate the design of metal-based metallodrugs and diruthenium metalloenzymes. 97 International Journal of Biological Macromolecules 253 (2023) 126666 Available online 1 September 2023 0141-8130/© 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). Steric hindrance and charge influence on the cytotoxic activity and protein binding properties of diruthenium complexes Aarón Terán a, Giarita Ferraro b, Paola Imbimbo b, Ana E. Sánchez-Peláez a, Daria Maria Monti b, Santiago Herrero a,*, Antonello Merlino b,* a MatMoPol Research Group, Department of Inorganic Chemistry, Faculty of Chemical Sciences, Complutense University of Madrid, Avda. Complutense s/n, 28040 Madrid, Spain b Department of Chemical Sciences, University of Naples Federico II, Complesso Universitario di Monte Sant’Angelo, Via Cinthia 21, 80126 Naples, Italy A R T I C L E I N F O Keywords: Diruthenium compounds Protein binding properties Cytotoxicity Crystal structure A B S T R A C T Paddlewheel diruthenium complexes are being used as metal-based drugs. It has been proposed that their charge and steric properties determine their selectivity towards proteins. Here, we explore these parameters using the first water-soluble diruthenium complex bearing two formamidinate ligands, [Ru2Cl(DPhF)2(O2CCH3)2], and two derivatives, [Ru2Cl(DPhF)(O2CCH3)3] and K2[Ru2(DPhF)(CO3)3] (DPhF− = N,N′-diphenylformamidinate), with one formamidinate. Their protein binding properties have been assessed employing hen egg white lysozyme (HEWL). The results confirm the relationship between the type of interaction (coordinate/non-coordinate bonds) and the charge of diruthenium complexes. The crystallization medium is also a key factor. In all cases, dir uthenium species maintain the M–M bond and produce stable adducts. The antiproliferative properties of these diruthenium complexes have been evaluated on an eukaryotic cell-based model. Our data show a correlation between the number of the formamidinate ligands and the anticancer activity of the diruthenium derivatives against human epithelial carcinoma cells. Increased cytotoxicity may be related to increased steric hindrance and Ru2 5+ core electronic density. However, the effect of increasing the lipophilicity of diruthenium species by introducing a second N,N′-diphenylformamidinate must be also considered. This work illustrates a systematic approach to shed light on the relevant properties of diruthenium compounds to design metal-based metallodrugs and diruthenium metalloenzymes. 1. Introduction The introduction of coordination complexes in medicine has become an emerging tool in drug discovery. In particular, paddlewheel Ru2 5+ complexes have been used as molecular building blocks to develop new metal-based drugs [1–4]. Clinical studies have been focused on diruthenium tetracarboxylate complexes. The first compounds studied were [Ru2Cl(O2CCH3)4] and [Ru2Cl(O2CCH2CH3)4], which showed a slight antineoplastic activity against P388 leukemia cell line [5]. The introduction of solubilising groups, as in complex [Ru2(O2C-m-C6H4SO3)4]3− , led to 5 times better cytotoxic properties against CoLo 320DM cancer cells [6]. Further efforts to improve the biological activity of these complexes gave rise to replace these carboxylates for other biologically active carboxylate ligands as strategy to design dual acting paddlewheel dir uthenium complexes. For example, diruthenium derivatives containing nonsteroidal anti-inflammatory drugs [7–11] or γ-linolenic acid [12,13] as ligands have been found to be active against glioma tumor models, while [Ru2(EB776)4]Cl (where EB776 is the deprotonated form of (2- phenylindol-3-yl)glyoxyl-L-phenylalanine-L-leucine) was found to be active against human glioblastoma multiforme U87MG [14]. However, the low solubility of diruthenium metallodrugs is still the major limi tation for their clinical employment. To overcome this problem and to facilitate the internalization in cells, encapsulation methods have been tested with various diruthenium species using injectable solid polymer- lipid nanoparticles (SPLNs) [15] and terpolymer-lipid nanoparticles (TPLNs) [16]. Those studies indicate that the dimetallic unit is not a mere drug carrier and, actually, plays a key role in the cytotoxic activity. Although the coordination of nucleotides [17] to the diruthenium core or the interaction with RNA [18] has been studied, the biological targets remain to date unknown. Nevertheless, the accumulated evi dences suggest that the pharmacological mechanism of action of these * Corresponding authors. E-mail addresses: sherrero@ucm.es (S. Herrero), antonello.merlino@unina.it (A. Merlino). Contents lists available at ScienceDirect International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac https://doi.org/10.1016/j.ijbiomac.2023.126666 Received 21 July 2023; Received in revised form 30 August 2023; Accepted 31 August 2023 mailto:sherrero@ucm.es mailto:antonello.merlino@unina.it www.sciencedirect.com/science/journal/01418130 https://www.elsevier.com/locate/ijbiomac https://doi.org/10.1016/j.ijbiomac.2023.126666 https://doi.org/10.1016/j.ijbiomac.2023.126666 https://doi.org/10.1016/j.ijbiomac.2023.126666 http://crossmark.crossref.org/dialog/?doi=10.1016/j.ijbiomac.2023.126666&domain=pdf http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ International Journal of Biological Macromolecules 253 (2023) 126666 2 metallodrugs involves the interaction with proteins [10,19–21]. The binding possibilities of different diruthenium compounds with the model protein hen egg white lysozyme (HEWL) have been studied. When [Ru2Cl(O2CCH3)4] reacts with HEWL the diruthenium center binds the protein retaining the Ru–Ru bond and losing one acetate ligand in favour of an Asp side chain [22]. DFT calculations have un veiled the scarce selectivity of this diruthenium complex in a biological context and suggested that anionic compounds or the use of bulky equatorial substituents could increase their protein binding selectivity [23,24]. For this reason, we decided to exchange an acetate by a bulky formamidinate ligand (L-L) in the structure of the diruthenium core to obtain [Ru2Cl(L-L)(O2CCH3)3] complexes [25]. These water-soluble complexes show an increase in their stability in aqueous solutions and a different behaviour compared to the tetracarboxylate derivative. The preferential binding sites are still Asp side chains through the equatorial positions, but also Lys or Arg side chains or even carbonyl groups of the main chain can coordinate the diruthenium core at the axial sites [26]. To study the charge effect on protein binding properties of diruthenium species, we synthesized the anionic complexes, [Ru2(CO3)4]3− and [Ru2(L-L)(CO3)3]2− (L-L = N,N′-bis(4-fluorophenyl)formamidinate, D-p- FPhF;− or N,N′-bis(4-methoxyphenyl)formamidinate, DAniF− ), to interact with HEWL. The molecular structures of these adducts differ in the number of metal binding sites, in the binding mode (coordinate or non-coordinate bonds) and in the type of diruthenium fragments that bind the protein. Our previous data suggest that complexes with high negative charges (regardless of their volumen) tend to remain outside the protein surface, and that only cationic, neutral and monovalent anionic species can be close enough to covalently bind the protein [27]. We describe below the influence of the charge (anionic vs cationic compounds) and, specially, the increase of bulky equatorial groups around the Ru2 core (one or two bidentate N,N′-diphenylformamidinate ligands) on the cytotoxic and protein binding properties of three water–soluble diruthenium complexes: [Ru2Cl(DPhF)(O2CCH3)3]⋅H2O, [Ru2Cl(DPhF)2(O2CCH3)2]⋅H2O, and K2[Ru2(DPhF)(CO3)3]⋅3H2O (DPhF− = N,N′-diphenylformamidinate). The structure of the complexes is shown in Fig. 1. 2. Material and methods 2.1. Synthesis of diruthenium derivatives All the reactions and manipulations were performed under air at mosphere. All reactants and solvents were obtained from commercial sources and used without further purification unless otherwise indi cated. Commercial HDPhF ligand (N,N′-diphenylformamidine) was recrystallized from a mixture of dichloromethane/hexane solution. The diruthenium starting material [Ru2Cl(O2CCH3)4] was prepared following procedures described in the literature [28]. The preparation of [Ru2Cl(DPhF)(O2CCH3)3] was previously re ported employing ultrasound-assisted synthesis (USS) [25]. [Ru2Cl (DPhF)2(O2CCH3)2] and K2[Ru2(DPhF)(CO3)3] derivatives were syn thetized following similar procedures for analogous compounds using conventional synthesis methods [27,29] (Scheme 1). [Ru2Cl(DPhF)2(O2CCH3)2]⋅H2O: Yield: 10 %. Elemental analysis found (calculated) for C30H30ClN4O5Ru2 (MW = 765.00 g/mol): C, 47.55 (47.15); H, 4.12 (3.96); N, 7.45 (7.33). FT-IR: 1634, 1595, 1525, 1481, 1442, 1318, 1217, 1073, 1022, 940, 780, 755, and 695 cm− 1. ESI (m/z): 711.8 [M – H2O – Cl]+, 789.7 [M – H2O + CH3CO]+. CV: E1/2 = − 0.45 V, ΔE = 0.15 V. K2[Ru2(DPhF)(CO3)3]⋅3H2O: Yield: 92 %. Elemental analysis found (calculated) for C16H17K2N2O12Ru2 (MW = 709.65 g/mol): C, 27.25 (27.08); H, 2.65 (2.41); N, 3.64 (3.95). FT-IR: 1655, 1518, 1480, 1447, 1303, 1270, 1221, 1041, 946, 815, 763, and 699 cm− 1. ESI (m/z): 617.6 [M – K+ − 3H2O + 2H+]+, 579.7 [M – 2 K+ − 3H2O + 3H+]+. CV: E1/2 = 0.57 V, ΔE = 0.09 V. 2.2. Characterization of diruthenium derivatives FTIR spectra (4000–500 cm− 1) were recorded with a Perkin-Elmer Spectrum 100 with a universal ATR accessory. Elemental analyses were performed at the Microanalytical Service of the Complutense University of Madrid. Mass spectrometry data (electrospray ionization) were recorded at the Mass Spectrometry Service of the Complutense University of Madrid, using an ion trap analyser HCT Ultra (Bruker Daltonics) mass spec trometer in water solution. Cyclic voltammetry (CV) measurements were conducted with a Metrohm Autolab PGSTAT204 potentiostat. The working electrode was Fig. 1. Structure of the paddlewheel compounds [Ru2Cl(DPhF)(O2CCH3)3] (a), [Ru2Cl(DPhF)2(O2CCH3)2] (b), and K2[Ru2(DPhF)(CO3)3] (c). A. Terán et al. International Journal of Biological Macromolecules 253 (2023) 126666 3 a glassy carbon electrode (GCE). A Pt wire served as the counter elec trode and Ag/AgCl was employed as the reference electrode. Electro chemical grade KCl at a concentration of 0.10 M was employed as the supporting electrolyte in voltammetric measurements. High pure N2 was used to deoxygenate the solution at least 10 min prior to each run and to maintain a nitrogen blanket. The ferrocenium/ferrocene couple was observed at 0.53 V (vs. Ag/AgCl) in 0.10 M tetrabutylammonium perchlorate (TBAP) dichloromethane solution. 2.3. Protein binding studies in solution UV–vis spectra of [Ru2Cl(DPhF)(O2CCH3)3], [Ru2Cl (DPhF)2(O2CCH3)2], and K2[Ru2(DPhF)(CO3)3] were recorded on a Cary 5G spectrophotometer using quartz cuvette of 1 cm path length, in water (500 μM diruthenium concentration) and in other two different exper imental conditions (50 μM diruthenium concentration), i.e. those used to grow HEWL crystals: 0.8 M succinic acid at pH 7.0 (condition A) and 2.0 M sodium formate and 0.1 M Hepes buffer at pH 7.5 (condition B) (Figs. S6-S9, ESI). Spectra were collected for 5 h continuously, and then after 24 h and 7 days, in the absence and in the presence of HEWL (protein to metal compound molar ratio 1:3). Each measurement was repeated twice. Fluorescence spectra of HEWL in the absence and in the presence of [Ru2Cl(DPhF)(O2CCH3)3], [Ru2Cl(DPhF)2(O2CCH3)2], and K2[Ru2(DPhF)(CO3)3] were collected using a HORIBA Fluoromax-4 spectrofluorometer equipped with a thermostat bath, upon excitation at 280 nm (to follow Tyr and Trp emission) and 295 nm (to follow Trp emission only) (Figs. S10-S12, ESI). A 2 mM Ru2 compound solution was used to titrate the protein (1.4 μM) to obtain different protein to metal molar ratios: 1:0.5, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:10. The titra tions were carried out in 10 mM succinic acid pH 7.0 and 10 mM Hepes buffer pH 7.5. Solutions were stirred and equilibrated for 5 min before recording the spectra. A 1 cm path length cuvette was used. Far UV-circular dichroism spectroscopy was used to record spectra of HEWL (7 μM) in the absence and in the presence of [Ru2Cl(DPhF) (O2CCH3)3], [Ru2Cl(DPhF)2(O2CCH3)2] and K2[Ru2(DPhF)(CO3)3] in the following protein to diruthenium molar ratios: 1:0.5, 1:1, 1:2, 1:3 (Figs. S13-S15, ESI). Spectra were recorded on a Jasco J-715 spec tropolarimeter equipped with a Peltier thermostatic cell holder (Model PTC-348WI) in the range of 195–250 nm, using a quartz cell with 0.1 cm path length. The protein was incubated with the metal compounds for 24 h in 10 mM succinic acid pH 7.0 and 10 mM Hepes buffer pH 7.5. Other experimental parameters were: 1.0 nm data pitch, 2.0 nm band width, 50 nm/min scanning speed, 2.0 s response time, and 25 ◦C. Each spectrum was obtained by averaging three scans. 2.4. Crystallization, X-ray diffraction data collection, structure solution and refinement of the adducts with HEWL HEWL was purchased from Sigma Chemical Co (Merck Life Science S. r.l., Milan, Italy) at the highest grade of purity. Crystals of the adducts formed upon reaction of HEWL with [Ru2Cl(DPhF)(O2CCH3)3], [Ru2Cl (DPhF)2(O2CCH3)2], and K2[Ru2(DPhF)(CO3)3] were obtained by soaking method using crystals of metal-free HEWL grown at 298 K with the hanging drop vapor diffusion method in 0.8 M succinic acid at pH 7.0 (condition A) or in 2.0 M sodium formate and 0.1 M Hepes buffer at pH 7.5 (condition B) (protein concentration of 13 mg⋅mL− 1). At those pH values the organic acids are almost completely deprotonated in aqueous solution [30]. Soaking was carried out for 14 days using satu rated solutions of the metal compounds freshly dissolved in the reser voir. The crystals were flash-cooled in liquid nitrogen and stored under cryogenic conditions until data collection. Diffraction data were collected on XRD2 beamline at Elettra syn chrotron in Trieste, Italy. Further details on the data collection param eters, structure solution and refinement are reported in the ESI. The crystals obtained for the adduct of HEWL–[Ru2Cl(DPhF)(O2CCH3)3] under condition B and HEWL–K2[Ru2(DPhF)(CO3)3] under condition A did not have enough quality to allow a good refinement. Atomic co ordinates and structure factors for the reported structures have been deposited in the Protein Data Bank as entries 8PH7 (HEWL–[Ru2Cl (DPhF)(O2CCH3)3], condition A), 8PH8 (HEWL–[Ru2Cl (DPhF)2(O2CCH3)2], condition A), 8PH5 (HEWL–[Ru2Cl (DPhF)2(O2CCH3)2], condition B) and 8PH6 (HEWL–K2[Ru2(DPhF) (CO3)3], condition B). Data collection and refinement statistics are re ported in Table S1 (see ESI). 2.5. Cell culture and cytotoxicity assay Immortalized murine fibroblasts (BALB/c-3T3) and human epithelial cervical cancer cells (HeLa) were obtained from ATCC. Immortalized human keratinocyte cells (HaCaT) were obtained from Innoprot (Biscay, Spain). All cell lines were cultured in Dulbecco’s Modified Eagle’s Me dium (Sigma-Aldrich, St Louis, Mo, USA), supplemented with 10 % foetal bovine serum (HyClone), 2 mM L-glutamine and antibiotics, all from Sigma-Aldrich, under a 5 % CO2 humidified atmosphere at 37 ◦C. For cytotoxic experiments, cells were seeded in 96-well plates at a density of 2 × 103 cells per well (HaCaT and HeLa cells), or 3 × 103 cells per well (BALB/c-3T3 cells). After 24 h, increasing concentrations (from 1 to 50 μM) of the three diruthenium complexes (dissolved in DMSO) were added to the cells. After 48 h incubation, cell viability was assessed by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro mide) assay, as previously described [31]. Cell survival was expressed as the percentage of viable cells in the presence of the drug compared to controls (i.e., untreated cells and cells supplemented with identical volumes of DMSO). Each sample was tested in three independent ana lyses, each carried out in triplicate. Results are reported as mean of re sults obtained from the three independent experiments (mean ± SD). Complexes containing N,N′-diphenylformamidinate and succinate or formate ligands have not been isolated. However, it is expected that the behaviour of the complexes with formamidinate and formate ligands is closer to those derivatives with acetate and formamidinate ligands. 3. Results and discussion 3.1. Physicochemical properties of diruthenium derivatives The FT-IR spectra of [Ru2Cl(DPhF)2(O2CCH3)2]⋅H2O and K2[Ru2(DPhF)(CO3)3]⋅3H2O (see Fig. S1, ESI) show the characteristic strong adsorptions of the ligands in the range of 4000–500 cm− 1. For both complexes, a wide band in the range 3400–3500 cm− 1 and a band Scheme 1. Reaction pathways to prepare [Ru2Cl(DPhF)(O2CCH3)3], [Ru2Cl(DPhF)2(O2CCH3)2], and K2[Ru2(DPhF)(CO3)3]. A. Terán et al. International Journal of Biological Macromolecules 253 (2023) 126666 4 centred at ~1645 cm− 1 suggest the presence of water in their structure, in accordance with elemental analysis. [Ru2Cl(DPhF)2(O2CCH3)2] show vibrational modes for the formamidinate ligand at 1525 cm− 1 (ν(C=Carom)), and 1217 cm− 1 (ν(C–N)). The carboxylate vibrational modes for stretching and bending appear at 1442 and 695 cm− 1, respectively [32]. In the case of K2[Ru2(DPhF)(CO3)3]⋅3H2O, carbonate vibrational modes corresponding to COO stretching and bending can be observed around 1447 and 699 cm− 1, respectively. Some of the for mamidinate ligand vibration modes are observed at 1518 cm− 1 (ν(C=Carom)), and 1270 and 1221 cm− 1 (ν(C–N)). The electrospray ionization mass spectra (ESI-MS) of [Ru2Cl (DPhF)2(O2CCH3)2] and K2[Ru2(DPhF)(CO3)3] compounds are shown in Figs. S2 and S3 (see ESI). We have compared the experimental molecular peaks with their calculated isotopic distribution. For [Ru2Cl (DPhF)2(O2CCH3)2], the main peaks reported with ESI-MS correspond to [M – H2O – Cl]+ and [M – H2O + CH3CO]+. The simplest ionization mechanism for mixed-valence diruthenium species is to lose the halide anion which is usually coordinated to the axial position. Our group and others have previously described the appearance of an acylium ion in their ESI spectra, related with a possible fragmentation of an acetate ligand during the experiment [29]. In the case of K2[Ru2(DPhF)(CO3)3], two dominant peaks appeared, which correspond to the ionized forms by the loss of K+ counterions and water molecules, [M – K+ − 3H2O + 2H+]+ and [M – 2 K+ − 3H2O + 3H+]+. Cyclic voltammetry (CV) was performed in 0.10 M KCl solution at 0.1 V/s with a three-electrode system to compare the electrochemical behaviour of [Ru2Cl(DPhF)2(O2CCH3)2] and K2[Ru2(DPhF)(CO3)3] with analogous compounds (Fig. S4 and S5, ESI). Both complexes show only one-electron redox couple within the limit of water. It has been described that the presence of electron-donating or electron- withdrawing groups in the aromatic rings influences the shift of the signals [33]. For K2[Ru2(DPhF)(CO3)3] (Fig. S4, ESI), a Ru2 5+/6+ reversible oxidation process occurs (E1/2 = 0.57 V, ΔE = 0.09 V). When compared to similar complexes [27] with p-OCH3 (E1/2 = 0.68 V, ΔE = 0.08 V) or p-F (E1/2 = 0.75 V, ΔE = 0.08 V) substituents on the N,N′- diphenylformamidinate ligand, the absence of substituents on the aro matic rings causes lower oxidation potential. CV measurement on [Ru2Cl(DPhF)2(O2CCH3)2] (Fig. S5, ESI) represents the first example of a CV experiment carried out in aqueous solution for a diruthenium com plex bearing two bidentate formamidinate ligands. Here a Ru2 5+/4+ pseudo-reversible reduction process occurs (E1/2 = − 0.45 V, ΔE = 0.15 V). Comparing these data with those obtained for [Ru2Cl(DPhF) (O2CCH3)3] under the same experimental conditions (E1/2 = − 0.33 V, ΔE = 0.12 V), we can see that the reduction potential shifts towards more negative values. It has been reported that as the number of for mamidinate ligands around the diruthenium core increases, the oxida tion process is facilitated [29,34]. 3.2. Stability of diruthenium complexes in solution To ascertain the stability of [Ru2Cl(DPhF)(O2CCH3)3], [Ru2Cl (DPhF)2(O2CCH3)2], and K2[Ru2(DPhF)(CO3)3] in water and in the conditions used to perform the protein binding studies and the biological tests, UV–vis absorption spectroscopy, fluorescence, and circular di chroism were used. The time course UV–vis absorption spectra of the three complexes in Milli-Q water (Fig. S6, ESI) displayed characteristic bands in the visible region (400–800 nm), assigned to allowed ligand-to-metal charge transfers [π(N/O), π(axial) → π*(Ru2)], and a UV band that it is usually related to a ligand-to-metal transitions [π(N) → σ*/π*/δ*(Ru2)]. No significant changes were observed after 2 weeks. UV–Vis spectra were also collected in the absence and in the presence of the model protein HEWL in the crystallization conditions A and B (Fig. S7-S9, ESI). In all the cases, ignorable variations in the spectral profiles were observed after one week. Results indicate that these diruthenium species show excellent stability to water exposure as their previous counterparts with other formamidinate ligands [26,27]. Fluorescence spectra of HEWL in the absence and in the presence of [Ru2Cl(DPhF)(O2CCH3)3], [Ru2Cl(DPhF)2(O2CCH3)2], and K2[Ru2(DPhF)(CO3)3] show a slight quenching of intrinsic fluorescence of the protein (Figs. S10-S12, ESI), thus suggesting that the three dir uthenium compounds are able to interact with the biological macro molecule under the investigated experimental conditions. The binding does not induce a variation of HEWL tertiary structure since there is no variation in the maximum emission wavelength. Superimposition of CD spectra of HEWL in the absence and in the presence of [Ru2Cl(DPhF)(O2CCH3)3], [Ru2Cl(DPhF)2(O2CCH3)2], and K2[Ru2(DPhF)(CO3)3] does not show significant differences, indicating that the binding of the diruthenium compounds to the protein does not alter HEWL secondary structure (Figs. S13-S15, ESI). 3.3. Effect of the ligand substitution on the binding properties of diruthenium complexes Previously, our groups have studied the influence of equatorial li gands on the protein binding response between diruthenium compounds and HEWL [22,26,27]. The anionic and cationic compounds K3[Ru2(CO3)4], [Ru2Cl(L-L)(O2CCH3)3] and K2[Ru2(L-L)(CO3)3] (L-L = D-p-FPhF− and DAniF− ), show different charge and/or higher steric ef fect around the Ru2 core than the starting material [Ru2Cl(O2CCH3)4]. However, we observed similar covalent binding sites (Asp101 and Asp119) in the adducts formed with HEWL. According to DFT calcula tions, an increase of the bulky groups around diruthenium should change the protein binding selectivity of these species [23,24]. There fore, we propose to study diruthenium complexes bearing two bidentate ligands to increase the steric effect around diruthenium core. De rivatives with the two formamidinates D-p-FPhF− or DAniF− showed very low water-solubility. In the search for different possibilities, it was found that with the complex [Ru2Cl(DPhF)2(O2CCH3)2] it was possible to increase the steric hindrance maintaining the water-solubility. For this reason, to have a comparative basis with the previous results, compounds [Ru2Cl(DPhF)(O2CCH3)3] and K2[Ru2(DPhF)(CO3)3] are also studied. The protein binding studies have been carried out in two conditions (A and B). A summary of the ligands and geometries of the diruthenium containing fragments found in the four new structures are described in Table S1. 1) Structures of the adducts formed upon reaction of HEWL with [Ru2Cl (DPhF)2(O2CCH3)2] and [Ru2Cl(DPhF)(O2CCH3)3] under condition A The overall structures of the adducts formed by HEWL with [Ru2Cl (DPhF)2(O2CCH3)2] and [Ru2Cl(DPhF)(O2CCH3)3] compounds obtained under condition A are reported in Fig. 2. The root mean square devia tion between the Cα atoms are 0.14 and 0.34 Å, respectively (see Table S1 for further details). A careful inspection of the 2Fo − Fc and Fo − Fc electron density (e.d. maps) and of the anomalous difference map revealed significant differences between the structures. The adduct with [Ru2Cl(DPhF)2(O2CCH3)2] (Fig. 3) was refined at ultrahigh resolution (1.29 Å). Two diruthenium centers were observed at level of the side chain of Asp101 (occupancy = 0.75) and Lys33 (occupancy = 0.40). The e.d. maps suggest a potential replacement of the acetate by succinate (suc) ligands in both binding sites and one axial water molecule coordinated to the Ru2 core. The Lys33 binding site (Fig. 3a) shows the diruthenium fragment cis- [Ru2(DPhF)2(suc)2(OH2)]− axially coordinated to the side chain. How ever, the Asp101 side chain (Fig. 3b) is coordinated as monodentate ligand at one axial position to the cis-[Ru2(DPhF)2(suc)2(OH2)]− frag ment. To the best of our knowledge this is the first time that this behaviour was observed for the diruthenium compounds [22,26,27]. This behaviour suggests that the cis-[Ru2(DPhF)2(suc)2(OH2)]− moiety is particularly stable under condition A and only the most labile A. Terán et al. International Journal of Biological Macromolecules 253 (2023) 126666 5 axial positions are available to interact with the protein. In the adduct formed with [Ru2Cl(DPhF)(O2CCH3)3], solved at 1.41 Å resolution, two diruthenium binding sites, close to the side chains of Lys33 (occupancy = 0.45) and Asp101 (occupancy = 0.40) are observed (Fig. 4). At the Lys33 binding site (Fig. 4a), the binding of the diruthenium centre also occurs through one axial position (axial binding). The dir uthenium fragment retains the formamidinate ligand, but two out of three acetate ligands have been replaced by two succinate bridges (0.8 M succinic acid at pH 7.0). This substitution probably facilitates the axial coordination of Lys33. The binding to the protein induces small structural modifications in the side chain of Trp62 that changes its conformation to form stacking interactions with the phenyl group of the N,Ń-diphenylformamidinate ligand. The electron density map at this site is rather well-defined and allows to model one water molecule at the remaining axial site. The substitution of acetate ligands by succinates before binding is expected. In fact, this is the common procedure to prepare diruthenium tetracarboxylates from [Ru2Cl(O2CCH3)4] [35]. The coordination of succinate ligands make the diruthenium complexes neutral or even anionic but also increases their steric hindrance which seems to facilitate the more accesible axial coordination. However, at the Asp101 binding site (Fig. 4b) unexpected results are obtained. The diruthenium centre seems to be coordinated to the side chain of Asp in a monodentate fashion but in a pseudo-equatorial binding upon release of one acetate ligand. This Ru2 motif shows one axial water molecule and retains one acetate ligand, while the other acetate (trans to Asp 101) is replaced by one succinate. The new ligand appears to be as monodentate, although it should be underlined that the electron density map for this ligand is not particularly well defined. This adduct seems to be a reaction intermediate in the ligand substitution/ binding to protein of the diruthenium core. 2) Structures of the adducts formed upon reaction of HEWL with [Ru2Cl (DPhF)2(O2CCH3)2] and K2[Ru2(DPhF)(CO3)3] under condition B The overall structures of the adducts formed by HEWL with [Ru2Cl (DPhF)2(O2CCH3)2] and K2[Ru2(DPhF)(CO3)3] complexes obtained under condition B are reported in Fig. 5. The root mean square devia tion between the Cα atoms are 0.15 and 0.17 Å, respectively (see Table S1 for further details). In the HEWL adduct with [Ru2Cl(DPhF)2(O2CCH3)2] (resolution = 1.29 Å) two binding sites were observed at level of the side chain of Asp101 (occupancy = 0.30) and Asp119 (occupancy = 0.30). In both metal binding sites an equatorial coordination occurs by the substitution of an acetate ligand (Fig. 6a and b). Besides, the e.d. maps suggest a Fig. 2. Overall structures of the adducts formed in the reaction of HEWL with a) [Ru2Cl(DPhF)(O2CCH3)3] and b) [Ru2Cl(DPhF)2(O2CCH3)2] under condi tion A. Atom colors for the complexes are green for C, blue for N, red for O, and dark green for Ru. Fig. 3. Close-up view of the diruthenium binding sites at Lys33 (a) and Asp101 (b) side chains in the adduct formed between HEWL and [Ru2Cl(DPhF)2(O2CCH3)2] under condition A. 2Fo − Fc electron density maps are contoured at 1.0 σ (salmon) level. Fig. 4. Close-up view of the diruthenium binding sites at Lys33 (a) and Asp101 (b) side chains in the adduct formed between HEWL and [Ru2Cl(DPhF) (O2CCH3)3] under condition A. 2Fo − Fc electron density maps are contoured at 1.0 σ (salmon) level. The axial water ligands have been omitted for clarity. A. Terán et al. International Journal of Biological Macromolecules 253 (2023) 126666 6 replacement of the second acetate ligand by a formate molecule and a water molecule at one axial position. The Asp119 side chain has to change its conformation to coordinate this diruthenium moiety. In the HEWL adduct with K2[Ru2(DPhF)(CO3)3], refined at 1.07 Å resolution, the e.d. maps reveal four binding sites (Fig. 7) at the side chain of Asp119 and Arg125 (occupancy = 0.30), and Asp101 (occu pancy = 0.30), and two non-covalent bonded (NCB) complexes (occu pancies = 0.30 and 0.35). We identifiy an alternate side-chain conformation for Arg125. The four diruthenium fragments were packed together with other three symmetry-related molecules (Fig. 7a). At the level of Asp119 and Arg125A (Fig. 7b) the diruthenium moiety is coordinated to both side chains through the equatorial posi tions releasing two carbonate ligands. The third carbonate ligand is probably substituted by a formate anion of the crystallization medium (2.0 M sodium formate). The carboxylate group of the side chain of Asp101 (Fig. 7c) is coordinated to the diruthenium core as a bridging equatorial ligand in cis configuration respect to the formamidinate ligand. With bulkier formamidinate ligands, the coordination to this residue was always in trans configuration [26,27]. This confirms the assumption that, unless there is a steric hindrance, the cis configuration is preferred. In addition, one carbonate ligand is replaced by a formate molecule and only one carbonate ligand remains. One of the axial positions is interacting with one alternate side chain of the Arg125B residue (distance = 2.6 Å) of other symmetry-related molecule (HEWL-2). Two NCB diruthenium complexes are observed in the structure of this adduct. These molecules are interacting through non-covalent in teractions with the surface of HEWL. One diruthenium unit is in the symmetry-axis (between HEWL-1 and HEWL-3) with three carbonate and one formamidinate ligands coordinated to the equatorial positions (Fig. 7d). The other Ru2 motif (Fig. 7e) show two carbonate, one for mamidinate and one formate ligands. In this case, one oxygen of a carbonate ligand seems to interact with one axial position of another symmetry-related diruthenium complexes in HEWL-4 (distance = 2.2 Å). This type of interaction is quite common for tetracarbonatodir uthenium compounds in the solid state [36–43]. 3.4. Structural comparison It was previously found that the exchange of one acetate by one formamidinate ligand in [Ru2Cl(O2CCH3)4] enhances the stability of the diruthenium species [22,26,27]. Here, we have studied for the first time the reactivity of a diruthenium complex containing two bidentate for mamidinate ligands, [Ru2Cl(DPhF)2(O2CCH3)2]. This complex is stable in aqueous medium, although less soluble than the diruthenium complex bearing only one bidentate formamidinate ligand. [Ru2Cl(DPhF) (O2CCH3)3] and K2[Ru2(DPhF)(CO3)3] have also been studied for com parison reasons. The preferential binding sites of diruthenium complexes are the side chains of the Asp101 and Asp119 through the equatorial positions. Generally, the coordination to Asp101 is in trans configuration respect to the formamidinate ligand, whereas to Asp119 is in cis configuration no Fig. 5. Overall structures of the adducts formed in the reaction of HEWL with a) [Ru2Cl(DPhF)2(O2CCH3)2] and b) K2[Ru2(DPhF)(CO3)3] under condition B. Atom colors for the complexes are green for C, blue for N, red for O, and dark green for Ru. Fig. 6. Close-up view of the diruthenium binding sites at Asp101 (a) and Asp119 (b) side chains in the adduct formed between HEWL and [Ru2Cl(DPhF)2(O2CCH3)2] under condition B. 2Fo − Fc electron density maps are contoured at 1.0 σ (salmon) level. A. Terán et al. International Journal of Biological Macromolecules 253 (2023) 126666 7 matter the crystallization media [26,27]. However, in this work with the DPhF− derivatives, a cis configuration can also be achieved in Asp101 (Figs. 3b and 7c). This may be due to a lower steric effect of DPhF− . We have described that multivalent anionic diruthenium compounds have the tendency to lose part of their ligands to be able to bind one protein residue [27]. Otherwise, they remain interacting with the sur face. Here, [Ru2(DPhF)(CO3)3]2− could bind simultaneously the side chains of Asp119 and Arg125 (Fig. 7b) through the equatorial positions releasing carbonate ligands, and thus stabilizing the paddlewheel structure. This is a unique case where the four equatorial ligands around the diruthenium core are different. When it is equatorial bound to the side chain of Asp101, its axial position can also interact with the side chain of Arg125 of other HEWL molecule (Fig. 7c). Furthermore, those molecules that remain non-covalently bound to the protein can interact via carbonate ligands with the axial position of another diruthenium unit (Fig. 7e), as it occurs for K3[Ru2(CO3)4]⋅4H2O in the solid state [37]. When succinate is present in the crystallization medium (condition A), axial coordination is preferentially achieved (Figs. 3 and 4). For [Ru2Cl(D-p-FPhF)(O2CCH3)3] (D-p-FPhF− = N,N′-bis(4-fluorophenyl) formamidinate), we reported an axial coordination employing the same medium [26], although the ligands around the diruthenium core could not be modelled. Our new results with [Ru2Cl(DPhF)(O2CCH3)3] and [Ru2Cl(DPhF)2(O2CCH3)2] endorse i) that axial coordination occurs preferably under condition A through the side chains of Asp101 and Lys33, ii) that the formamidinate ligand is retained and iii) that the acetate ligands tend to be replaced by succinate anions. The axial bond seems to be favoured by the greater steric hindrance of the diruthenium species and the higher coulombic repulsion with the protein due to the presence of two succinate ligands. When we changed the crystallization medium (condition B) for [Ru2Cl(DPhF)2(O2CCH3)2] (Fig. 6), the preferential equatorial binding to Asp101 and Asp119 was recovered. In this case, the presence of formate ions resulted in the substitution of the remaining acetate ligand according to the crystallographic data. This suggests that diruthenium complex bearing two bidentate for mamidinate ligands have a greater tendency to lose acetate ligands than those derivatives bearing only one formamidinate. 3.5. Cytotoxicity of [Ru2Cl(DPhF)(O2CCH3)3], [Ru2Cl (DPhF)2(O2CCH3)2], and K2[Ru2(DPhF)(CO3)3] on eukaryotic cell lines The potential use of the three diruthenium complexes as anticancer drugs was evaluated on a cell-based model. First, the effect on cell viability was evaluated on human cervical cancer cells (HeLa), incu bated with increasing concentration of complexes (1–50 μM) for 48 h. Cell viability was evaluated by the MTT assay (Fig. S16). IC50 values, i.e., the drug concentration able to reduce cell viability to 50 %, are reported in Table 1. Among the three complexes tested, [Ru2Cl (DPhF)2(O2CCH3)2] was found to be cytotoxic towards the cancer cell line (IC50 value 5 μM), whereas no noteworthy cytotoxicity was observed when cells were treated with [Ru2Cl(DPhF)(O2CCH3)3] or K2[Ru2(DPhF)(CO3)3], up to 50 μM. Thus, the effect of [Ru2Cl (DPhF)2(O2CCH3)2] was evaluated also on two non-cancer cell lines, immortalized human keratinocytes (HaCaT) and immortalized murine fibroblasts (BALB/c-3T3). Results in Table 1 indicate that [Ru2Cl(DPhF)2(O2CCH3)2] exerts a selective toxicity. Indeed, the Selectivity Index (SI), which is the ratio between the IC50 values of immortalized cells and cancer cells, was 3.6 for BALB/c-3T3/HeLa and 4.6 for HaCaT/HeLa. Previous results for the water-soluble complex [Ru2(O2C-m-C6H4SO3)4]3− revealed a very low cytotoxic effect against HeLa cells (ca. 600 μM) [6]. Then, the substi tution of two carboxylate bridging ligands by two formamidinates in creases the cytotoxicity of diruthenium species. In this respect it should be recalled that formamidinate ligands are cytotoxic [44], but they have to be liberated to exert their biological action. However, [Ru2Cl (DPhF)2(O2CCH3)2] is more stable than its monoformamidinate ana logues, and for all cases data indicate that the formamidinate ligand always remains attached to the diruthenium core. The increase in the Fig. 7. a) Surface representation of four symmetry-related HEWL molecules (HEWL-1/4) in the adduct formed between HEWL and K2[Ru2(DPhF)(CO3)3] under condition B. Close-up view of the diruthenium binding sites at Asp119 and Arg125 side chains (b), Asp101 side chain (c), and two NCB complexes (d and e). Black dashed lines in c) and e) indicate intermolecular interactions. 2Fo − Fc electron density maps are contoured at 1.0 σ (salmon) level. A. Terán et al. International Journal of Biological Macromolecules 253 (2023) 126666 8 cytotoxicity of this derivative could be related to the selectivity of these compounds due to steric hindrance as previously suggested by other authors [14,23,24]. In addition, the substitution of O,Ó-donor bridging ligands by N,Ń-donor bridging ligands permits to increase the electron density on ruthenium atoms, giving rise to a change in the electronic properties of these compounds. Therefore, the introduction of for mamidinate ligands makes the diruthenium species more difficult to be reduced and/or decomposed. This could help the species to reach their biological target(s). Moreover, the electronic density around the dir uthenium core is closely related to the coordinative capacity of the species and their binding selectivity. Then, the change on the cytotoxic properties of these diruthenium compounds may be related to their steric and/or electronic effects. Nevertheless, the increase of the dir uthenium species lipophilicity with the number of formamidinate li gands also have to be considered. All these factors may affect the antiproliferative action of the diruthenium-based anticancer drugs in a tumor microenvironment. 4. Conclusions The synthesis and characterization of three diruthenium paddle wheel complexes bearing N,N′-diphenylformamidinate ligands were reported: [Ru2Cl(DPhF)(O2CCH3)3], [Ru2Cl(DPhF)2(O2CCH3)2], and K2[Ru2(DPhF)(CO3)3]. All the compounds are water-soluble and mani fest good stability in all tested aqueous solutions. A preliminary screening for potential biological applications of these complexes was done by studying their cytotoxicity, through MTT assay against human cervical cancer cells, immortalized human keratinocytes and immor talized murine fibroblasts. Results reveal a promising biological activity for [Ru2Cl(DPhF)2(O2CCH3)2] with a Selectivity Index > 3.5, which can be related to the steric hindrance, lipophilicity and electronic properties of this compound. The protein binding properties of diruthenium complexes bearing one or two bidentate N,N′-diphenylformamidinate ligands with the model protein HEWL were also compared. Our study reinforces the idea of coordination versatility for diruthenium complexes that strongly de pends on their charge and the medium. Thus, coordinate or non- coordinate bonds, axial or equatorial coordination, and cis- or trans- configuration with respect to the bulky ligands can be obtained. This knowledge can help design compounds with specific characteristics and properties. The most common covalent binding sites found in the HEWL structure are the side chains of Asp101 and Asp119. Nevertheless, the diruthenium coordination sites can vary depending on the equatorial ligands and the medium. This generates different numbers of labile sites, which may have great relevance in the possible catalytic activity of diruthenium complexes. The tendency of diruthenium compounds to replace their acetate ligands can also be employed to design new water- soluble prodrugs by the introduction of bioactive ligands. Understand ing these interactions can help to develop new therapeutic molecules for cancer treatments. CRediT authorship contribution statement Aarón Terán: Methodology, Investigation, Formal analysis, Writing – original draft, Writing – review & editing. Giarita Ferraro: Method ology, Investigation, Writing – review & editing. Paola Imbimbo: Methodology, Investigation, Writing – review & editing. Ana E. Sánchez-Peláez: Supervision, Conceptualization, Writing – review & editing. Daria Maria Monti: Supervision, Conceptualization, Writing – review & editing. Santiago Herrero: Supervision, Conceptualization, Writing – review & editing. Antonello Merlino: Supervision, Concep tualization, Writing – review & editing. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data availability Data will be made available on request. Acknowledgements The authors thank Elettra staff for technical assistance. Comunidad de Madrid (Project S2017/BMD-3770-CM) and Complutense University of Madrid (GRFN32/23 and Program PR3/23) are gratefully acknowl edged for financial support. A.T. also acknowledges the Complutense University for a Predoctoral Grant (CT63/19-CT64/19) and Research Stay Grant (EB25/22) and the Spanish Ministry of Science and Innova tion for a Postgraduate Fellowship at Residencia de Estudiantes (2021–2022). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.ijbiomac.2023.126666. 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Applications hapter VII C Applications 99 100 The knowledge gained from this dissertation gave rise to the collaboration with other research groups to show the applicability of the water-soluble diruthenium compounds. In 2022, an article with Inchausti et al. was published119 where the synthetic methodology established in Chapter III was applied. This research was intended to study the complete series of [Ru2Cl(D-p-FPhF)x(O2CCH3)4−x] (x = 1–4; D-p-FPhF− = N,N’-bis(4- fluorophenyl)formamidinate) compounds, characterizing each compound by a multi- technique approach, including single crystal X-ray diffraction. Several synthetic attempts were made to obtain [Ru2Cl(D-p-FPhF)(O2CCH3)3]. It was tested a conventional synthesis method, through the reflux of the starting material [Ru2Cl(O2CCH3)4] with the formamidine ligand-precursor HD-p-FPhF in THF in the presence and absence of LiCl and Et3N and changing the reaction time. Nevertheless, this procedure was not appropriate because, in the absence of amines or LiCl, the yield of the reaction is very low (around 10%) and a lot of the starting material remained unreacted, while the addition of Et3N resulted in a mixture of compounds (x = 1–4). Therefore, ultrasound-assisted synthesis was tested. The use of this method reduced the reaction times and temperatures, requires neither chromatographic column purification nor an inert atmosphere during the synthesis, and improved the yield. In addition, the formation of mono-substituted diruthenium compounds was very selective. In this paper, the variation of physicochemical properties as a function of the number of formamidinate ligands was analysed. CV measurements showed an increasing tendency to oxidation of the compounds from x = 1 to 4, upon increasing the electronic donation from the equatorial ligands to the Ru2 5+ core. Besides, when the number of formamidinate ligands increases, the difference between the anodic and cathodic peaks decreases, indicating greater electrochemical reversibility, thus a lower barrier to electron transfer. The structural analysis of these paramagnetic compounds carried out by XRD and NMR showed a behaviour that has not been described before. In solid-state and in solution, it was observed that compounds [Ru2Cl(D-p-FPhF)(O2CCH3)3] and [Ru2Cl(D-p-FPhF)2(O2CCH3)2] tend to form oligomers unlike compounds [Ru2Cl(D-p-FPhF)3(O2CCH3)] and [Ru2Cl(D-p-FPhF)4] that remain as molecular species. 101 Very recently, La Manna et al. (submitted) have compared the ability of two diruthenium compounds, [Ru2Cl(D-p-FPhF)(O2CCH3)3] (compound 1) and K3[Ru2(O2CO)4] (compound 2), to act as inhibitors of amyloid-β (Aβ) aggregation of Aβ1-42 peptide and its peculiar fragments Aβ1-16 and Aβ21-40. These two diruthenium compounds were chosen to compare species with different charge and steric hindrance. Thus, we could verify all the findings of Chapters IV-VI and study the behaviour of these derivatives in a biological system. A wide analysis using different biophysical techniques was carried out to know the capacity of these compounds to inhibit the aggregation of Aβ peptides. Misfolding and extracellular aggregation of Aβ peptides are one of the recognized neuropathological hallmarks of Alzheimer's disease (AD). Therefore, it is important to know different ways to remedy their aggregation. It was found that differences in charge and steric hindrance were crucial in the inhibition of Aβ peptide aggregation. Only the compound [Ru2Cl(D-p-FPhF)(O2CCH3)3] could aid the interaction between the bimetallic unit and the polypeptide chain probably due to the different charge respect to K3[Ru2(O2CO)4]. In solution, [Ru2Cl(D-p-FPhF)(O2CCH3)3] forms cationic species, while K3[Ru2(O2CO)4] forms an anionic complex. We previously observed in Chapters V and VI that only cationic species can approach to the HEWL protein to bind it and form covalent bonds. Moreover, the aromatic groups of the formamidinate ligand of [Ru2Cl(D-p-FPhF)(O2CCH3)3] seems to favour the π- π interactions with other aromatic residues resulting in a complete inhibition of fibre formation and abolishes the cytotoxicity of Aβ fragments. In addition, the data show that [Ru2Cl(D-p-FPhF)(O2CCH3)3] can coordinate Histidine side chains, probably at the axial site. This dual and synergistic mechanism of action explains the greater effect exhibited by [Ru2Cl(D-p-FPhF)(O2CCH3)3] as inhibitor of Aβ-aggregation. Only compound 1 determines a deep rearrangement of the polypeptide chain of Aβ1-42 leading to oligomeric states quite different from that of amyloid alone to the point to completely suppress the cellular toxicity of the amyloid Aβ1- 42. With these results, we want to highlight the novel application of bimetallic Ru2-based drugs as inhibitors of Aβ aggregation, with important future impacts in the drug-discovery associated with Alzheimer’s disease. This result is also in agreement with predictions that steric and charge factors were crucial to achieve specific interactions between diruthenium compounds and biological systems. 102 Chapter VIII. Discussion hapter VIII C Discussion 103 104 The main objectives of this dissertation were the synthesis, characterization, and study of the protein binding properties of water-soluble, and air- and water-stable diruthenium complexes to obtain artificial diruthenium metalloproteins as structural models. The structural information on the protein-binding mode of these diruthenium coordination compounds permit a better understanding of their mode of action in a living system. It has been studied the solution properties and the crystal structure of different Ru2-protein adducts formed upon reaction of diruthenium derivatives with the model protein hen egg white lysozyme (HEWL) under different conditions. HEWL has been selected as structural model because it was employed in several structural studies of metal complexes, including one diruthenium compound, thus allowing a direct comparison. These objectives have been successfully achieved as described in Chapters III-VI. In addition, Chapter VII demonstrates that the methodology developed in this thesis is useful and shows how the compounds proposed here have potential biomedical applications based on their charge or steric hindrance. In this chapter, we will only discuss the results obtained in Chapters III-VI. Synthesis of diruthenium compounds A major part of this doctoral thesis has been focused on synthetic routes to obtain the compounds of interest (Scheme 3). Chapter III is focused on the synthesis of monosubstituted diruthenium compounds to obtain species with higher steric hindrance than [Ru2Cl(O2CCH3)4]. The [Ru2Cl(L-L)(O2CCH3)3] (L-L = formamidinate or amidate ligands) complexes were obtained by ultrasound-assisted synthesis (USS) by the substitution of one CH3CO2 - by one L-L ligand. In Chapters V and VI it is described the preparation of the anionic diruthenium complexes K3[Ru2(CO3)4], K2[Ru2(D-p- FPhF)(CO3)3], K2[Ru2(DAniF)(CO3)3], and K2[Ru2(DPhF)(CO3)3]. A conventional synthesis method was used at room temperature to prepare them. These species allowed to study the charge influence of the diruthenium complexes on their interaction with the model protein HEWL. Finally, in Chapter VI, the first example of a water-soluble disubstituted complex was reported: [Ru2Cl(DPhF)2(O2CCH3)2]. A reflux synthesis method is used to obtain this compound. 105 Scheme 3. Synthesis of [Ru2Cl(L-L)(O2CCH3)3], [Ru2Cl(L-L)2(O2CCH3)2] and K2[Ru2(L- L)(CO3)3] compounds (L-L = formamidinate or amidate ligands). Physicochemical properties of diruthenium compounds The properties of these complexes were studied by a multi-technique approach. Magnetization measurements were carried out for the complexes (Chapter III). Data reveal a high-spin electronic configuration with three unpaired electrons, σ2π4δ2(π*δ*)3 (S = 3/2) for the monosubstituted complexes. The solid-state structure of [Ru2Cl(p-TolA)(O2CCH3)3]n·2CH2Cl2 (p-TolA- = N-4- tolylamidate) and [Ru2Cl(D-p-CNPhF)(O2CCH3)3]n·CH2Cl2 (D-p-CNPhF- = N,N′-bis(4- cyanophenyl)formamidinate) were determined by X-ray diffraction analysis (Figure 17a and 17b, respectively). These complexes show the typical paddlewheel structure with one formamidinate or amidate ligand and three acetate ligands in the equatorial positions. In addition, in both complexes, the chloride ligand bridges two Ru2 5+ units, giving rise to a polymeric structure. [Ru2Cl(p-TolA)(O2CCH3)3]n·2CH2Cl2 forms a zig-zag chain, whereas Ru2Cl(D-p-CNPhF)(O2CCH3)3]n·CH2Cl2 forms an almost linear structure. All monosubstituted compounds described to date have shown a discrete molecule structure. The new results demonstrate that they can also form a 1D structure. 106 Figure 17. Crystal structure of a) [Ru2Cl(p-TolA)(O2CCH3)3]n·2CH2Cl2, b) [Ru2Cl(D-p- CNPhF)(O2CCH3)3]n·CH2Cl2 and c) [Ru2Cl(O2CCH3)4]n along the Ru‒Ru bond. Hydrogen atoms and solvent molecules are omitted for clarity. Color code: purple for Ru, green for Cl, red for O, blue for N, and black for C. In order to keep the water-solubility of the starting material only one acetate ligand was replaced. The water solubility of diruthenium species is associated with the dissociation of the axial chloride ligand due to the excellent solvating properties of H2O. Surprisingly, the solubility in aqueous solutions was higher for monoformamidinato complexes than for [Ru2Cl(O2CCH3)4] complex. This can be understood from the solid-state properties of these compounds. [Ru2Cl(O2CCH3)4] form linear or zig-zag polymeric chains (d(Ru‒Cl) = 2.566 Å for the linear complex197 and d(Ru‒Cl) = 2.572 Å for the zig-zag complex198). In [Ru2Cl(D-p-CNPhF)(O2CCH3)3], the formation of an almost linear polymeric compound has 107 also been seen (Figure 17c), but its Ru‒Cl bond distance is longer (Ru‒Cl = 2.635 Å). In solution, there is an equilibrium between neutral and cationic complexes due to the exchange between the chloride ligand and water molecules. A weaker Ru‒Cl bond in monoformamidinato complexes favours the formation of ionic species and make more difficult the polymerization of the neutral species. Diruthenium derivatives containing one formamidinate ligand were definitively adequate to try their interaction with the model protein HEWL because of their relatively high water-solubility and steric hindrance. Diformamidinato compounds showed lower water-solubility and only [Ru2Cl(DPhF)2(O2CCH3)2] was soluble enough to study its protein binding properties. Electrochemical studies in aqueous solution are reported for all complexes in Chapters III-VI. [Ru2Cl(L-L)(O2CCH3)3] complexes show only one-electron redox couple assigned to the Ru2 5+/Ru2 4+ redox couple (E1/2 = -0.37 to -0.16 V). The substitution of acetate by carbonate ligands in complexes K2[Ru2(L-L)(CO3)3] increases the electron density on the diruthenium core favouring a Ru2 5+/6+ reversible oxidation process (E1/2 = 0.57 to 0.75 V). The disubstituted complex [Ru2Cl(DPhF)2(O2CCH3)2] shows a Ru2 5+/4+ pseudo-reversible reduction (E1/2 = -0.45 V). As the number of formamidinate ligands around the diruthenium core increases, the oxidation process is facilitated. Sufficient stability is another important characteristic of the diruthenium compounds to be used in protein binding experiments. The L-L ligands studied are stronger Lewis bases than acetate ligands, so they can donate more electron density to the Ru2 5+ core and it was expected an increase of the stability of these species. The stability of the metal compounds in water and other aqueous media over time was confirmed by UV-Vis spectroscopy (Chapters IV-VI). The stability of diruthenium derivatives alone was verified for up to two weeks. Indeed, compared to other diruthenium compounds that only show carboxylate ligands in the equatorial positions and that tend to degrade or aggregate with time in aqueous solution, the introduction of formamidinate ligands allowed us to obtain quite robust species. When the same measurements were performed in the presence of the protein, it could be seen that the metal-protein adducts are also equally stable. Some of the bands are slightly shifted, which is related to a possible ligand substitution at the diruthenium core. 108 Binding properties of diruthenium compounds To investigate the protein binding possibilities of the above diruthenium complexes with the model protein HEWL, other solution measurements (fluorescence spectroscopy and circular dichroism) were performed (Chapters IV-VI). The diruthenium compounds studied in this dissertation do not exhibit fluorescence emission. However, the protein HEWL exhibits intrinsic fluorescence caused by the aromatic residues phenylalanine, tyrosine, and tryptophan. The incubation of the diruthenium compounds with HEWL results in a decrease in the intensity of the emission signal under different conditions. This led us to suppose that diruthenium compounds could modify the protein structure. To determine what type of changes in the secondary structure of the protein were occurring, circular dichroism measurements were performed. For most of the Ru2-HEWL adducts the same secondary structure of the free protein is maintained. However, for [Ru2(CO3)4]3-, [Ru2(DAniF)(CO3)3]2- and [Ru2(DAniF)(O2CCH3)3]+ compounds, an increase in the negative ellipticity signal is observed. This can be associated with a kosmotropic-like property of these derivatives. A kosmotrope strengthens the hydrogen bonds of water molecules and stabilizes the intramolecular interactions in the structure of a biomolecule. The increase of self-association processes within the HEWL chain, in the presence of diruthenium complexes, can create a more prevalent secondary structure which would increase the CD signal. The character of the changes suggests that the diruthenium- induced variations affect to the compactness/dynamics of the polypeptide chain. This phenomenon seems to be quite significant, especially for the DAniF derivatives, where the presence of the methoxy group is expected to generate a higher number of interactions with the surrounding water molecules. Upon the confirmation of the potential binding of diruthenium compounds to HEWL and the stability of these metal-protein adducts, crystallisation assays were performed. For this purpose, the hanging drop vapour diffusion method was used to grow single crystals of the native protein. Then, diruthenium compounds were added by a soaking technique. After a few weeks of incubation, the single crystals were preserved and frozen to carry out their structural determination. X-ray diffraction data collections were carried out on 109 Beamline XRD2 at Elettra synchrotron (Trieste, Italy), using a wavelength of 1.00 Å and a cold nitrogen stream of 100 K. X-ray diffraction studies of the diruthenium metalloproteins were described in the Chapters IV-VI (Figures 18-20). Up to fourteen adducts were obtained under different conditions. Prior to this research there was only one precedent on the interaction of diruthenium compounds with proteins. [Ru2Cl(O2CCH3)4] complex was reported to react with HEWL forming a metalloprotein with two binding sites (Asp101 and Asp119; Figure 18a and 18b) but only under one particular experimental condition due to the insufficient stability of [Ru2Cl(O2CCH3)4] in aqueous solutions.137 Since then, several attempts have been undertaken to understand how to modulate the response of diruthenium compounds in a biological context. In recent years, articles related with DFT studies have proposed that the chemoselectivity of diruthenium compounds in a biological context could be influenced by steric and charge effects.171,172 Both the effect of charge and steric hindrance on the recognition of diruthenium compounds with the model protein HEWL are studied in this dissertation. In Chapter IV the steric effect of cationic diruthenium species is boarded, in Chapter V the charge effect (anionic vs cationic complexes) is considered, and in Chapter VI the effect of further increase the steric hindrance around the Ru2 5+ core (mono- vs disubstituted complexes) is reported. Overall, the Ru‒Ru bonds remain stable regardless of experimental conditions and the formamidinate ligand is retained in all cases, despite the lability of the acetate/carbonate ligands. This is due to the stronger donor capacity of the formamidinate ligands (better Lewis bases), which favours the binding and increases their stability in solution. Diruthenium species show equatorial binding preference for the side chain of aspartate residues (Asp101 and Asp119). Two types of configurations have been found for the formamidinate ligand, cis- or trans- respect to the Asp101 and 119 side chains (Figures 18c-e, 19b-d and 20i). In addition, axial positions are involved in the interaction with the side chains of other residues such as lysine (Figures 18i and 20c), arginine (Figure 18l and 20i), aspartate (Figure 20d), and to backbone carbonyl groups (Figure 18h). 110 There is a clear influence of the charge on the diruthenium compound binding response respect to the surface of the model protein HEWL. Cationic complexes have always displayed covalent bonding. However, anionic complexes exhibited covalent or non- covalent bonding (NCB). As long as the complexes retain its high-negative charge, the species remain non-covalently bounded interacting with the surfaces of the protein (Figures 19a-c and 20j,k). In addition, non-covalent bonded species can interact via carbonate ligands with the axial position of another diruthenium species (Figure 20k), as usually occurs in the solid state for tetracarbonate derivatives. Due to the presence in the medium of other bidentate ionic species (e.g., acetates or formates) in high concentration, the loss or replacement of carbonate ligands seems to occur. The diruthenium complexes reduce their charge from -2 to -1 or change from an anionic to a cationic form, giving rise to a covalent bond similarly to the cationic analogues (Figures 19b,c and 20h,i). The further increase of the steric hindrance around Ru2 5+ core in Chapter VI for [Ru2Cl(DPhF)2(O2CCH3)2] complex showed the same binding sites for mono- and disubstituted complexes, the side chains of the Asp101 and Asp119. However, we observed that there is a relationship between the crystallisation medium and the binding mode. When succinato is used as crystallisation medium (condition A), axial coordination is preferentially achieved for [Ru2Cl(DPhF)(O2CCH3)3] and [Ru2Cl(DPhF)2(O2CCH3)2] complexes. The axial coordination occurs through the side chains of Asp101 and Lys33 (Figure 20a,b,d). Here, DPhF- ligand is retained and the acetate ligands seems to be replaced by succinate anions. Previously, we had seen this (Figures 18h and 18i), but the coordination environment of the diruthenium core could not be determined. When we changed the crystallisation condition (condition B) for [Ru2Cl(DPhF)2(O2CCH3)2] compound, the binding sites are the equatorial positions of the Ru2 5+ moiety by the side chains of Asp101 and Asp119 (Figures 20e and 20f). Here, the acetate ligands seem to be again replaced by formate ligands from the medium. 111 Figure 18. Diruthenium binding sites in the metalloproteins formed upon the reaction of HEWL with [Ru2Cl(O2CCH3)4] (a and b) and [Ru2Cl(D-p-FPhF)(O2CCH3)3] (c-l) under different conditions. Axial H2O molecules are omitted for clarity. 2Fo − Fc electron density maps are contoured at 1.0 σ. 112 Figure 19. Diruthenium metalloproteins formed upon the reaction of HEWL with a) K3[Ru2(CO3)4], b) K2[Ru2(D-p-FPhF)(CO3)3], c) K2[Ru2(DAniF)(CO3)3], and d) [Ru2Cl(DAniF)(O2CCH3)3] in condition A (20% ethylene glycol, 0.1 M sodium acetate at pH 4.5, and 0.6 M NaNO3) and/or condition B (2.0 M sodium formate and 0.1 M Hepes buffer pH 7.5). 113 Figure 20. Diruthenium binding sites of metalloproteins formed upon the reaction of HEWL with [Ru2Cl(DPhF)(O2CCH3)3] (a and b), [Ru2Cl(DPhF)2(O2CCH3)2] (c-f), and K2[Ru2(DPhF)(CO3)3] (g-k) in condition A (0.8 M succinic acid at pH 7.0) and/or condition B (2.0 M sodium formate, 0.1 M Hepes buffer pH 7.5). 2Fo − Fc electron density maps are contoured at 1.0 σ. Despite the fact that the same binding sites were observed for mono- and disubstituted complexes, cytotoxicity studies for [Ru2Cl(DPhF)(O2CCH3)3], [Ru2Cl(DPhF)2(O2CCH3)2] and K2[Ru2(DPhF)(CO3)3] compounds were carried out. MTT assay against immortalised human keratinocytes, immortalised murine fibroblasts and human cervical cancer cells reveal no cytotoxic effect of [Ru2Cl(DPhF)(O2CCH3)3] or K2[Ru2(DPhF)(CO3)3], while a 114 promising selective activity for [Ru2Cl(DPhF)2(O2CCH3)2] complex with a selectivity index > 3.5 was reported. This distinct activity has been related to the presence of two formamidinate ligands. Formamidines are known to show cytotoxic capacity.199 However, all the solution measurements we have performed indicate that the ligand is not released to the medium. Therefore, the higher cytotoxicity of [Ru2Cl(DPhF)2(O2CCH3)2] is related to the selectivity of this complex due to steric and/or electronic effects as previously suggested by other authors.158,171,172 The substitution of O,O´-donor bridging ligands by N,N´-donor bridging ligands permits to increase the electron density on the ruthenium atoms, giving rise to a change in the electronic properties of these compounds. The introduction of formamidinate ligands makes the diruthenium species more difficult to be reduced and/or decomposed and this could help the species to reach their biological target(s). Moreover, the electronic density around the diruthenium core is closely related to the coordinative capacity of the species and their binding selectivity. Nevertheless, the increase of the diruthenium species lipophilicity with the number of formamidinate ligands must also be considered. This can influence the ADME (absorption, distribution, metabolism, elimination) processes and affect to the activity, the toxicity, and the bioaccumulation of a potential drug.200 115 116 Conclusions onclusions C 117 118 The main objectives of this dissertation were successfully achieved. From the synthetic point of view, a series of water-soluble diruthenium complexes, [Ru2Cl(L-L)(O2CCH3)3], [Ru2Cl(L-L)2(O2CCH3)2], K3[Ru2(CO3)4] and K2[Ru2(L-L)(CO3)3] (L-L = DPhF-, D-p-CNPhF-, DAniF-, D-o/m/p-TolF-, and p-TolA-), were synthesised and characterised. An easy and selective synthetic method to obtain the hitherto elusive [Ru2Cl(L-L)(O2CCH3)3] family of compounds has been developed. This is the first time that metal–metal bonded compounds have been sonochemical activated to give rise an effective replacement of only one acetate ligand from [Ru2Cl(O2CCH3)4] starting material by N,N´- or N,O-donor ligands. This method considerably reduces reaction times, uses a greener solvent (ethanol) and saves energy with respect to other methods described for monosubstituted bimetallic compounds. Complexes K2[Ru2(L-L)(CO3)3] and [Ru2Cl(L-L)2(O2CCH3)2] were obtained efficiently using conventional methods. The physicochemical properties of all complexes have been studied by a multi-technique approach for coordination chemistry compounds. Magnetization measurements reveal a quartet state arising from a σ2δ2π4(δ*π*)3 electronic configuration for [Ru2Cl(L- L)(O2CCH3)3] complexes. Cyclic voltammetry studies in aqueous solution show only one- electron redox couple within the limit of water, Ru2 5+/4+ for [Ru2Cl(L-L)(O2CCH3)3] and [Ru2Cl(L-L)2(O2CCH3)2] derivatives and Ru2 5+/6+ for K2[Ru2(L-L)(CO3)3] compounds. Most importantly, those compounds resulted, as hypothesised, sufficiently stable and soluble in aqueous solutions to be used to prepare metalloproteins. From the point of view of obtaining model systems based on artificial diruthenium metalloproteins, all the objectives have also been achieved. We studied the stability and reactivity of K3[Ru2(CO3)4], [Ru2Cl(L-L)(O2CCH3)3], K2[Ru2(L-L)(CO3)3] (L-L = DPhF-, D-p- FPhF-, DAniF-) and [Ru2Cl(L-L)2(O2CCH3)2] (L-L = DPhF-) with the model protein hen egg white lysozyme (HEWL) through spectroscopic techniques (UV-Vis and fluorescence spectroscopies and circular dichroism) and X-ray diffraction under different experimental conditions. Although the diruthenium compounds are quite stable they are not inert and produce perturbations in the secondary structure of the protein in solution. Crystallographic data unambiguously indicate that diruthenium species bind the protein, 119 the overall conformation of the protein is not altered, the multiple Ru−Ru bond remain stable upon reaction with HEWL regardless of the experimental conditions used and the new diruthenium compounds show high coordinative versatility that depends on the nature of equatorial ligands. It has also been shown that cationic and anionic diruthenium species can react with HEWL through the equatorial positions with the side chains of Asp119 and Asp101 upon releasing one acetate/carbonate ligand and always retaining the formamidinate ligand. The coordination to side chains of the protein give rise to two types of configurations, cis- or trans- respect to the formamidinate ligand depending on steric hindrance. The replacement of equatorial carboxylates by carbonate ligands alters the reactivity of the diruthenium core. Anionic diruthenium compounds remain interacting with the surface of the protein, otherwise, they can lose part of their ligands to be able to bind one protein residue. Those molecules that remain non-covalently bound can interact via carbonate ligands with the axial position of another diruthenium unit. In all the complexes, equatorial ligands other than formamidinate ligand can be replaced in favour to other species such as formate or succinate anions from the crystallisation media. When succinate anions are present in the crystallisation medium, axial coordination is preferentially achieved probably due to charge/steric hindrance. The axial coordination occurs through the side chains of Asp101 and Lys33, the formamidinate ligand is retained and the other equatorial ligands tend to be replaced by succinate anions according to the crystallographic data. This is a very important finding that can allow to modulate the bonding position depending on the desired application. Finally, an objective that had not been initially considered, but which has also been achieved, is the applicability of the new diruthenium species as metallodrugs in biomedicine research. The cytotoxic properties of the [Ru2Cl(DPhF)2(O2CCH3)2], [Ru2Cl(DPhF)(O2CCH3)3], and K2[Ru2(DPhF)(CO3)3] compounds were evaluated against HeLa cells. The compound with the highest activity was [Ru2Cl(DPhF)2(O2CCH3)2]. The results indicate that there is a relationship between the steric effect and the anticancer activity of diruthenium derivatives. This result is in line with what was predicted by theoretical calculations. Nevertheless, the increase of the electronic density in the 120 diruthenium core makes these compounds more stable against reduction and can help them reaching the biological target. Also, the increase of the lipophilicity of the compounds with the number of formamidinate ligands can facilitate their cellular internalization. In addition, the use of two paddlewheel diruthenium complexes, [Ru2Cl(D-p-FPhF)(O2CCH3)3] and K3[Ru2(O2CO)4], have been tested as inhibitors of amyloid-β (Aβ) aggregation, which is closely related to Alzheimer's disease. The compound that showed the best result was [Ru2Cl(D-p-FPhF)(O2CCH3)3]. This is associated to the fact that the different charge in solution between these complexes (cationic vs anionic) favours the protein binding in the case of the cationic monosubstituted derivative. Moreover, the aromatic groups of the formamidinate ligand of [Ru2Cl(D-p- FPhF)(O2CCH3)3] seems to favour the π- π interactions with other aromatic residues resulting in a complete inhibition of fibre formation and abolishes the cytotoxicity of Aβ fragments. This result is also in agreement with predictions that steric and charge factors may be crucial to achieve specific interactions between diruthenium compounds and biological systems. In this study, 14 Ru2-HEWL metalloproteins have been analysed to better understand the molecular basis when diruthenium compounds interact with proteins. This work shows very interesting systems that can be easily tuned to interact with different protein binding sites. The results provide new insights into the reactivity of diruthenium compounds with biological macromolecules. They demonstrate that different diruthenium paddlewheel complexes can be used to prepare various artificial metalloenzymes with distinct properties. Data are also significant because they support the idea that diruthenium complexes can be modified, by changing the equatorial ligands, to direct their activity to specific biological targets. 121 122 Conclusiones onclusiones C 123 124 Los principales objetivos de esta tesis se han alcanzado con éxito. Desde el punto de vista sintético, se han preparado y caracterizado distintos complejos de dirrutenio solubles y estables en agua, [Ru2Cl(L-L)(O2CCH3)3], [Ru2Cl(L-L)2(O2CCH3)2], K3[Ru2(CO3)4] y K2[Ru2(L-L)(CO3)3] (L-L = DPhF-, D-p-CNPhF-, D-p-FPhF-, DAniF-, D-o/m/p- TolF-, y p-TolA-). Se ha desarrollado un método sintético fácil y selectivo para obtener la familia de compuestos monosustituidos [Ru2Cl(L-L)(O2CCH3)3], hasta ahora muy poco representada. Además, es la primera vez que se activan sonoquímicamente compuestos de coordinación que presentan enlace metal-metal múltiple, para dar lugar a la sustitución eficaz de un único ligando acetato del material de partida [Ru2Cl(O2CCH3)4] por ligandos N,N'- o N,O-dadores. Este método reduce considerablemente los tiempos de reacción, utiliza un disolvente más ecológico (etanol) y ahorra energía con respecto a los escasos métodos descritos para compuestos similares. Por otro lado, los complejos K2[Ru2(L- L)(CO3)3] y [Ru2Cl(L-L)2(O2CCH3)2] se obtuvieron eficientemente utilizando métodos convencionales. Las propiedades fisicoquímicas de todos los complejos se han estudiado mediante distintas técnicas de caracterización para compuestos de coordinación. Las medidas de magnetización revelan un estado cuartete derivado de una configuración electrónica σ2δ2π4(δ*π*)3 para los complejos con fórmula [Ru2Cl(L-L)(O2CCH3)3]. Los estudios de voltamperometría cíclica en disolución acuosa muestran sólo un par redox dentro del límite del agua, Ru2 5+/4+ para los derivados [Ru2Cl(L-L)(O2CCH3)3] y [Ru2Cl(L-L)2(O2CCH3)2] y Ru2 5+/6+ para los compuestos K2[Ru2(L-L)(CO3)3]. Y, lo que es más importante, se confirma la hipótesis inicial de que los compuestos monoformamidinato son suficientemente estables y solubles en disoluciones acuosas, lo que permite su utilización para la síntesis de proteínas artificiales. Además, todos los compuestos obtenidos resultaron suficientemente estables y solubles en soluciones acuosas para ser utilizados en la preparación de metaloproteínas artificiales confirmando la hipótesis de trabajo. Desde el punto de vista de la obtención de sistemas modelo basados en metaloproteínas artificiales de dirrutenio, también se han alcanzado todos los objetivos. Se ha estudiado la estabilidad y reactividad de las especies K3[Ru2(CO3)4], [Ru2Cl(L-L)(O2CCH3)3], 125 K2[Ru2(L-L)(CO3)3] (L-L = DPhF-, D-p-FPhF-, DAniF-) y [Ru2Cl(L-L)2(O2CCH3)2] (L-L = DPhF- ) con la proteína modelo lisozima de la clara de huevo de gallina (HEWL) mediante técnicas espectroscópicas (espectroscopias UV-Vis y de fluorescencia y dicroísmo circular) y difracción de rayos X en diferentes condiciones experimentales. Aunque los compuestos de dirrutenio son bastante estables, no son inertes, ya que los resultados muestran que producen perturbaciones en la estructura secundaria de la proteína en disolución. Los datos cristalográficos indican inequívocamente que las especies de dirrutenio son capaces de unirse a la proteína HEWL, que la conformación global de la proteína no se altera, que el enlace Ru-Ru múltiple permanece estable al reaccionar con HEWL independientemente de las condiciones experimentales utilizadas y que los nuevos compuestos de dirrutenio muestran una gran versatilidad coordinativa que depende de la naturaleza de sus ligandos ecuatoriales. También se ha demostrado que las especies catiónicas y aniónicas de dirrutenio pueden reaccionar a través de sus posiciones axiales o ecuatoriales con HEWL, que pueden liberar los ligandos acetato/carbonato pero siempre mantienen el ligando formamidinato ecuatorial. Además, la coordinación de las cadenas laterales de la proteína da lugar a dos tipos de configuraciones, cis- o trans- respecto al ligando formamidinato, dependiendo del impedimento estérico alrededor del centro bimetálico. La sustitución de los ligandos carboxilato ecuatoriales por ligandos carbonato modifica la reactividad del centro de dirrutenio con la cadena polipeptídica. Los compuestos aniónicos de dirrutenio interactúan con la superficie de la proteína mediante interacciones electroestáticas, aunque también pueden perder parte de sus ligandos para poder unirse covalentemente a un residuo de la proteína. Las moléculas que se encuentran interaccionando de manera no covalente con la proteína pueden unirse mediante sus ligandos carbonato a la posición axial de otra unidad de dirrutenio. En general se ha visto que los ligandos ecuatoriales distintos del ligando formamidinato pueden sustituirse en favor de otras especies presentes en el medio, como los aniones formiato o succinato que se encontraban en el medio de cristalización. 126 Según los datos cristalográficos, cuando hay aniones succinato en el medio de cristalización, tiene lugar una coordinación axial de las cadenas laterales de la proteína al centro de dirrutenio, probablemente debido al impedimento de carga y/o estérico que generan los iones succinato al coordinarse al centro metálico. La coordinación axial se produce a través de las cadenas laterales de Asp101 y Lys33, el ligando formamidinato se mantiene y los otros ligandos ecuatoriales tienden a ser sustituidos por aniones succinato. Se trata de un hallazgo muy importante que puede permitir la modulación de la posición de coordinación en función de las condiciones del medio. Por último, un objetivo que no se había considerado inicialmente, pero que también se ha conseguido, es la aplicabilidad de las nuevas especies de dirrutenio como metalofármacos en la investigación biomédica. Concretamente, se han evaluado las propiedades citotóxicas de los compuestos [Ru2Cl(DPhF)2(O2CCH3)2], [Ru2Cl(DPhF)(O2CCH3)3] y K2[Ru2(DPhF)(CO3)3] frente a las células de cáncer de cuello uterino HeLa. El compuesto que presenta una mayor actividad es el complejo [Ru2Cl(DPhF)2(O2CCH3)2]. Los datos indican que existe una relación entre el efecto estérico de los ligandos y la actividad anticancerígena de los derivados del dirrutenio. Este resultado coincide con lo predicho por los cálculos teóricos. Además, el aumento de la densidad electrónica en el núcleo de dirrutenio hace que estos compuestos sean más estables frente a la reducción lo que puede ayudarles a alcanzar la diana biológica. Asimismo, el aumento de la lipofilicidad de los compuestos con el número de ligandos formamidinato puede facilitar su internalización celular. También se ha probado el uso de dos complejos de dirrutenio, [Ru2Cl(D-p- FPhF)(O2CCH3)3] y K3[Ru2(CO3)4], como inhibidores de la agregación de β-amiloide (Aβ), estrechamente relacionada con la enfermedad de Alzheimer. El compuesto que muestra mejores resultados es el complejo [Ru2Cl(D-p-FPhF)(O2CCH3)3]. Esto se asocia a la diferente carga en disolución entre estos complejos (catiónica vs aniónica) que favorece la unión a proteínas en el caso del derivado catiónico monosustituido. Además, los grupos aromáticos del ligando formamidinato en el complejo [Ru2Cl(D-p-FPhF)(O2CCH3)3] parecen favorecer las interacciones π- π con otros residuos aromáticos dando lugar a una inhibición completa de la formación de fibras y aboliendo la citotoxicidad de los fragmentos de Aβ. Este resultado también está de acuerdo con las predicciones de que 127 los factores estéricos y de carga son cruciales para lograr interacciones específicas entre los compuestos de dirrutenio y los sistemas biológicos. Este estudio incluye 14 nuevas metaloproteínas Ru2-HEWL. Su análisis detallado permite comprender mejor las bases moleculares de la interacción de los compuestos de dirrutenio con sistemas proteicos. Este trabajo muestra sistemas muy interesantes que presentan gran afinidad coordinativa por cadenas peptídicas y aporta nuevos conocimientos sobre la reactividad de los compuestos de dirrutenio con macromoléculas biológicas. Los resultados demuestran que pueden utilizarse diferentes complejos de dirrutenio con estructura de rueda de paletas para preparar diversas metaloenzimas artificiales con distintas propiedades. 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ESI+ mass spectra of compounds 1-7. ............................................................ 6 Figure S3. ESI+ mass spectra in ethanol solution of crude reaction mixture at 1.5 h of compounds 1-7. Each compound shows as molecular peak the [M-Cl]+ fragment and for compounds 1-3, 5, 6 also appears the corresponding bis-substituted derivative, [Ru2(L- L)2(O2CMe)2] + (L-L = formamidinate). ........................................................................... 7 Figure S4. Vis-NIR absorption spectra of compounds 1-7 in dichloromethane at 2×10-4 M. .......................................................................................................................................... 8 Table S1. Tentative assignment of the electronic spectra transitions for compounds 1-7. .......................................................................................................................................... 9 Figure S5. Temperature dependence of the M (green circles) and MT (red squares) for compound 1. Solid lines represent the best fit of the data using the model described in the text. ................................................................................................................................. 10 Figure S6. Temperature dependence of the M (green circles) and MT (red squares) for compound 2. Solid lines represent the best fit of the data using the model described in the text. ................................................................................................................................. 10 Figure S7. Temperature dependence of the M (green circles) and MT (red squares) for compound 3. Solid lines represent the best fit of the data using the model described in the text. ................................................................................................................................. 11 2 Figure S8. Temperature dependence of the M (green circles) and MT (red squares) for compound 4. Solid lines represent the best fit of the data using the model described in the text. ................................................................................................................................. 11 Figure S9. Temperature dependence of the M (green circles) and MT (red squares) for compound 5. Solid lines represent the best fit of the data using the model described in the text. ................................................................................................................................. 12 Figure S10. Temperature dependence of the M (green circles) and MT (red squares) for compound 6. Solid lines represent the best fit of the data using the model described in the text. ................................................................................................................................. 12 Figure S11. Temperature dependence of the M (green circles) and MT (red squares) for compound 7. Solid lines represent the best fit of the data using the model described in the text. ................................................................................................................................. 13 Equation S1 ................................................................................................................... 14 Equation S2 ................................................................................................................... 14 Equation S3 ................................................................................................................... 14 Equation S4 ................................................................................................................... 14 Equation S5 ................................................................................................................... 14 Table S2. Crystal data and structure refinement for compound 3 and 7........................ 15 Table S3. Selected bond lengths and angles, and torsion angles for compounds 3 and 7. ........................................................................................................................................ 16 Figure S12. Cyclic voltammograms of compounds 1-7 (vs. Ag/AgCl) in H2O containing KCl 0.1 M at 0.1 V/s. ..................................................................................................... 17 3 Experimental details The syntheses and manipulations were conducted under air atmosphere. The reactants and solvents were used as received from commercial sources and used without purification except for N,N´-diphenylformamidine (HDPhF) that was recrystallized in CH2Cl2. [Ru2Cl(O2CMe)4] was prepared according to the procedures reported in literature [1]. The formamidines HDAniF, HD-p-CNPhF, HD-o-TolF, HD-m-TolF and HD-p- TolF, were prepared according to a published general procedure using the corresponding primary amine (4-methoxyaniline, 4-cyanoaniline, 2-methylaniline, 3-methylaniline and 4-methylaniline, respectively) and triethyl orthoformate as reagents [2,3]. Ultrasound-assisted synthesis was carried out using an Elmasonic P 300 H (Elma- Hans Schmidbauer GmbH & Co, max. 1580 W and 80 kHz). Elemental analyses measurements were done by the Microanalytical Service of the Complutense University of Madrid using a LECO CHNS-932 analyzer. FT-IR (Fourier transform infrared) spectra were recorded in a Perkin Elmer Spectrum 100 with a universal ATR accessory. Mass spectra were performed on a Bruker HCT Ultra with electrospray ionization spectrometer or in a Bruker Ultraflex with MALDI-TOF/TOF spectrometer. Electronic spectra of the complexes were measured in dichloromethane solution in a Cary 5G spectrometer. Variable-temperature magnetic susceptibility measurements were performed on a Quantum Design MPMSXL SQUID magnetometer operating under a magnetic field of 0.5 T using 9.22, 16.97, 30.92, 15.07, 22.80, 37.58 and 6.20 mg for 1-7, respectively. The data were corrected considering the diamagnetic contribution of the samples and the sample holder. Single crystal X-ray diffraction experiments were carried out for compounds 3 and 7 in the X-ray Diffraction Service of the UCM. Crystals were obtained by very slow diffusion of hexane into a solution of the corresponding compound in dichloromethane. In both cases an adequate crystal was mounted on a D8 VENTURE diffractometer using Cu-Kα radiation (λ = 1.54178 Å). Data were collected at 100 K over a reciprocal space hemisphere using the Bruker APEX-II CCD diffractometer software. The cell parameters were determined and refined by least-squares fit of all the reflections collected and a semiempirical absorption correction was applied on the reduced data using the SADABS program [4]. The structures were solved by intrinsic phasing using the SHELXT solution program [5] and refined by full matrix least squares on F2 using the SHEXL refinement package [6] running in the Olex2 environment [7]. All non-hydrogen atoms were refined 4 anisotropically. The hydrogen atoms were included with fixed isotropic contributions at their calculated positions determined by molecular geometry. CCDC numbers for compounds 3 and 7 are 2098740 and 2098741, respectively. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_request@ccdc.cam.ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033. Cyclic voltammetry measurements were performed on a Metrohm Autolab PGSTAT204 potentiostat in KCl 0.1 M aqueous solution (N2-degassed), at a scan rate of 0.1 V/s and a three-electrode system: glassy-carbon as working electrode, Pt-wire as auxiliary electrode and Ag/AgCl as reference electrode. The ferrocenium/ferrocene couple was observed at 0.53 V (vs. Ag/AgCl) in tetrabutylammonium perchlorate (TBAP) 0.1 M dichloromethane solution. References: [1] R.W. Mitchell, A. Spencer, G. Wilkinson, Carboxylato-triphenylphosphine complexes of ruthenium, cationic triphenylphosphine complexes derived from them, and their behaviour as homogeneous hydrogenation catalysts for alkenes, J. Chem. Soc. Dalton Trans. (1973) 846–854. https://doi.org/10.1039/DT9730000846. [2] W. Bradley, I. Wright, 129. Metal derivatives of NN′-diarylamidines, J. Chem. Soc. Resumed. (1956) 640–648. https://doi.org/10.1039/JR9560000640. [3] C. Lin, J.D. Protasiewicz, E.T. Smith, T. Ren, Linear Free Energy Relationships in Dinuclear Compounds. 2. Inductive Redox Tuning via Remote Substituents in Quadruply Bonded Dimolybdenum Compounds, Inorg. Chem. 35 (1996) 6422– 6428. https://doi.org/10.1021/ic960555o. [4] SADABS, Bruker Nonius, Delft, The Netherlands, 2002. [5] G.M. Sheldrick, ıt SHELXT – Integrated space-group and crystal-structure determination, Acta Crystallogr. Sect. A. 71 (2015) 3–8. https://doi.org/10.1107/S2053273314026370. [6] G.M. Sheldrick, Crystal structure refinement with ıt SHELXL, Acta Crystallogr. Sect. C. 71 (2015) 3–8. https://doi.org/10.1107/S2053229614024218. [7] O.V. Dolomanov, L.J. Bourhis, R.J. Gildea, J.A.K. Howard, H. Puschmann, ıt OLEX2: a complete structure solution, refinement and analysis program, J. Appl. Crystallogr. 42 (2009) 339–341. https://doi.org/10.1107/S0021889808042726. 5 Figure S1. IR spectra of complexes 1-7. 6 Figure S2. ESI+ mass spectra of compounds 1-7. 7 Figure S3. ESI+ mass spectra in ethanol solution of crude reaction mixture at 1.5 h of compounds 1-7. Each compound shows as molecular peak the [M-Cl]+ fragment and for compounds 1-3, 5, 6 also appears the corresponding bis-substituted derivative, [Ru2(L- L)2(O2CMe)2] + (L-L = formamidinate). 8 Figure S4. Vis-NIR absorption spectra of compounds 1-7 in dichloromethane at 2×10-4 M. 9 Table S1. Tentative assignment of the electronic spectra transitions for compounds 1-7. Compound λ (nm) Absorbance (a.u) ε (M-1·cm-1) Assignment 1 370 0.4114 2743 π(Cl) → π*(Ru2) 515 0.3518 2345 π(RuO/N,Ru2) → π*(Ru2) 644 0.2141 1427 1070 0.0233 155 δ(Ru2) → δ*(Ru2) 2 364 0.4548 2274 π(Cl) → π*(Ru2) 519 0.7176 3588 π(RuO/N,Ru2) → π*(Ru2) 869 0.0230 115 δ(Ru2) → δ*(Ru2) 3 379 0.4692 2346 π(Cl) → π*(Ru2) 491 0.7844 3922 π(RuO/N,Ru2) → π*(Ru2) 896 0.0149 75 δ(Ru2) → δ*(Ru2) 4 358 0.5446 2723 π(Cl) → π*(Ru2) 483 1.2322 6161 π(RuO/N,Ru2) → π*(Ru2) 900 0.0253 126 δ(Ru2) → δ*(Ru2) 5 365 0.5339 2670 π(Cl) → π*(Ru2) 474 0.6381 3190 π(RuO/N,Ru2) → π*(Ru2) 527 0.7764 3882 900 0.0262 131 δ(Ru2) → δ*(Ru2) 6 372 0.5727 2863 π(Cl) → π*(Ru2) 477 0.6396 3198 π(RuO/N,Ru2) → π*(Ru2) 537 0.7284 3642 918 0.0323 162 δ(Ru2) → δ*(Ru2) 7 381 0.3330 1665 π(Cl) → π*(Ru2) 459 0.5825 2912 π(RuO/N,Ru2) → π*(Ru2) 1054 0.0243 121 δ(Ru2) → δ*(Ru2) 10 0 50 100 150 200 250 300 0,0 0,1 0,2 0,3 0,4 0,5 0,6 T / K 0,0 0,5 1,0 1,5 2,0 2,5  M T / c m 3 K m o l-1  M / e m u m o l-1 Figure S5. Temperature dependence of the M (green circles) and MT (red squares) for compound 1. Solid lines represent the best fit of the data using the model described in the text. 0 50 100 150 200 250 300 0,0 0,1 0,2 0,3 0,4 0,5 0,6 T / K 0,0 0,5 1,0 1,5 2,0  M T / c m 3 K m o l-1  M / e m u m o l-1 Figure S6. Temperature dependence of the M (green circles) and MT (red squares) for compound 2. Solid lines represent the best fit of the data using the model described in the text. 11 0 50 100 150 200 250 300 0,0 0,1 0,2 0,3 0,4 0,5 0,6 T / K 0,0 0,5 1,0 1,5 2,0  M T / c m 3 K m o l-1  M / e m u m o l-1 Figure S7. Temperature dependence of the M (green circles) and MT (red squares) for compound 3. Solid lines represent the best fit of the data using the model described in the text. 0 50 100 150 200 250 300 0,0 0,1 0,2 0,3 0,4 0,5 T / K 0,0 0,5 1,0 1,5 2,0  M T / c m 3 K m o l-1  M / e m u m o l-1 Figure S8. Temperature dependence of the M (green circles) and MT (red squares) for compound 4. Solid lines represent the best fit of the data using the model described in the text. 12 0 50 100 150 200 250 300 0,0 0,1 0,2 0,3 0,4 0,5 T / K 0,0 0,5 1,0 1,5 2,0  M T / c m 3 K m o l-1  M / e m u m o l-1 Figure S9. Temperature dependence of the M (green circles) and MT (red squares) for compound 5. Solid lines represent the best fit of the data using the model described in the text. 0 50 100 150 200 250 300 0,0 0,1 0,2 0,3 0,4 0,5 0,6 T / K 0,0 0,5 1,0 1,5 2,0  M T / c m 3 K m o l-1  M / e m u m o l-1 Figure S10. Temperature dependence of the M (green circles) and MT (red squares) for compound 6. Solid lines represent the best fit of the data using the model described in the text. 13 0 50 100 150 200 250 300 0,0 0,1 0,2 0,3 0,4 0,5 T / K 0,0 0,5 1,0 1,5 2,0 2,5  M T / c m 3 K m o l-1  M / e m u m o l-1 Figure S11. Temperature dependence of the M (green circles) and MT (red squares) for compound 7. Solid lines represent the best fit of the data using the model described in the text. 14 𝜒𝑀 = 𝜒ǁ + 2𝜒⊥ 3 Equation S1 𝜒ǁ = ( 𝑁𝑔2𝛽2 𝑘𝐵𝑇 ) [ 1 + 9 exp (−2𝐷 𝑘𝐵𝑇 ⁄ ) 4 + (1 + exp (−2𝐷 𝑘𝐵𝑇 ⁄ )) ] Equation S2 𝜒⊥ = ( 𝑁𝑔2𝛽2 𝑘𝐵𝑇 ) [ 4 + ( 3𝑘𝐵𝑇 𝐷 ) + (1 − exp (−2𝐷 𝑘𝐵𝑇 ⁄ )) 4 (1 + exp (−2𝐷 𝑘𝐵𝑇 ⁄ )) ] Equation S3 𝜒𝑀´ = 𝜒𝑀 + 𝑇𝐼𝑃 Equation S4 𝜒 = 𝜒𝑀´ 1 − ( 2𝑧𝐽 𝑁𝑔2𝛽2) 𝜒𝑀´ Equation S5 15 Table S2. Crystal data and structure refinement for compound 3 and 7. Compound 3 7 Empirical formula C22H20Cl3N4O6Ru2 C16H20Cl5NO7Ru2 Formula weight 744.91 717.72 Temperature/K 100 100.0 Crystal system monoclinic monoclinic Space group P21/n P21/c a/Å 8.4201(5) 8.0523(3) b/Å 22.9937(13) 13.1909(5) c/Å 13.7684(8) 24.6722(10) α/deg 90 90 β/deg 98.087(3) 92.638(2) γ/deg 90 90 Volume/Å3 2639.2(3) 2617.83(18) Z 4 4 Density (calculated)/g/cm3 1.875 1.821 Absorption coefficient/mm-1 12.462 14.356 F(000) 1468 1408 Crystal size/mm3 0.14 × 0.05 × 0.02 0.11 × 0.05 × 0.02 Radiation Cu-Kα (λ = 1.54178) Cu-Kα (λ = 1.54178) 2Θ range for data collection/deg 7.54 to 125.988 7.174 to 149.402 Index ranges -9 ≤ h ≤ 9, -26 ≤ k ≤ 26, -14 ≤ l ≤ 16 -10 ≤ h ≤ 10, -16 ≤ k ≤ 16, - 30 ≤ l ≤ 30 Reflections collected 47521 58372 Independent reflections 4265 [Rint = 0.1592, Rsigma = 0.0690] 5350 [Rint = 0.0834, Rsigma = 0.0376] Data/restraints/parameters 4265/297/337 5350/414/284 Goodness-of-fit on F2 1.032 1.037 Final R indexes [I>=2σ (I)] R1 = 0.0758, wR2 = 0.1880 R1 = 0.0420, wR2 = 0.1104 Final R indexes [all data] R1 = 0.1065, wR2 = 0.2170 R1 = 0.0492, wR2 = 0.1168 Largest diff. peak/hole/e·Å-3 2.34/-1.09 1.60/-1.24 16 Table S3. Selected bond lengths and angles, and torsion angles for compounds 3 and 7. Compound 3 Compound 7 Bond lengths (Å) Ru1-Ru2 2.307 (1) Ru1-Ru2 2.2805 (4) Ru1-Cl1 2.625 (3) Ru1-Cl1 2.548 (1) Cl1-Ru2 2.636 (3) Cl1-Ru2 2.564 (1) Ru1-N1 2.042 (9) Ru1-N1 2.015 (3) Ru2-N2 2.039 (8) Ru2-O1 2.013 (3) Ru1-O1 2.046 (7) Ru2-O3 2.035 (3) Ru2-O2 2.044 (7) Ru1-O4 2.039 (3) Ru1-O3 2.057 (7) Ru2-O5 2.052 (3) Ru2-O4 2.054 (7) Ru2-O7 2.029 (3) Ru1-O5 2.038 (8) Ru1-O6 2.039 (3) Ru2-O6 2.033 (8) Ru1-O2 2.031 (3) H22A-O2 2.434 (7) H15A-O3 2.536 (3) H22A-N3 2.616 (11) H15B-O6 2.717 (3) H22B-O3 2.498 (7) H16B-Cl1 2.764 (1) H16B-O5 2.420 (4) Bond angles (deg) and torsion angles (deg) Ru2-Ru1-Cl1 175.40 (7) Ru2-Ru1-Cl1 176.32 (3) Ru1-Cl1-Ru2 174.03 (11) Ru1-Cl1-Ru2 124.21 (4) N1-Ru1-Ru2-N2 -8.24 (5) O1-Ru2-Ru1-N1 1.61 (12) O1-Ru1-Ru2-O2 -4.78 (4) O2-Ru2-Ru1-O3 -1.22 (13) O3-Ru1-Ru2-O4 -5.8 (3) O4-Ru2-Ru1-O5 -1.51 (13) O5-Ru1-Ru2-O6 -5.3 (3) O6-Ru2-Ru1-O7 -0.76 (13) 17 Figure S12. Cyclic voltammograms of compounds 1-7 (vs. Ag/AgCl) in H2O containing KCl 0.1 M at 0.1 V/s. 148 1 Supporting Information Effect of equatorial ligand substitution on the reactivity with proteins of paddlewheel diruthenium complexes: structural studies Aarón Terán,a Giarita Ferraro,b Ana E. Sánchez-Peláez,a Santiago Herreroa* and Antonello Merlinob* aDepartamento de Química Inorgánica, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, E-28040 Madrid, Spain. bDepartment of Chemical Sciences, University of Naples Federico II, Complesso Universitario di Monte Sant’Angelo, via Cinthia, 21, 80126, Naples, Italy. *E-mails: antonello.merlino@unina.it or sherrero@ucm.es mailto:antonello.merlino@unina.it mailto:sherrero@ucm.es 2 Index Materials ...........................................................................................................................................................3 In solution studies.............................................................................................................................................3 Crystallization and X-ray structure solution and refinement ..........................................................................4 Figure S1. Time course UV–vis spectra of 50 μM [Ru2Cl(D-p-FPhF)(μ-O2CCH3)3] in: (A) 20% ethylene glycol, 0.1 M sodium acetate buffer at pH 4.0, 0.6 M sodium nitrate; (B) 2.0 M sodium formate, 0.1 M Hepes buffer pH 7.5; (C) 0.8 M succinic acid pH 7.0; (D) 1.1 M sodium chloride, 0.1 M sodium acetate buffer pH 4.0.........5 Figure S2. Time course UV–vis spectra of 50 μM [Ru2Cl(D-p-FPhF)(μ-O2CCH3)3] in the presence of HEWL (protein to diruthenium molar ratio= 1:3) in: (A) 20% ethylene glycol, 0.1 M sodium acetate buffer at pH 4.0, 0.6 M sodium nitrate; (B) 2.0 M sodium formate, 0.1 M Hepes buffer pH 7.5; (C) 0.8 M succinic acid pH 7.0; (D) 1.1 M sodium chloride, 0.1 M sodium acetate buffer pH 4.0. ..............................................................6 Figure S3. Far-UV CD spectra of HEWL (7.0 μM concentration) incubated for 1 h in the presence of [Ru2Cl(D- p-FPhF)(μ-O2CCH3)3] in 10 mM sodium acetate buffer pH 4.0 at protein to diruthenium molar ratio = 1:1 (violet) , 1:2 (red) and 1:3 (pink). CD of metal-free protein is in black. .............................................................7 Figure S4. Fluorescence emission spectra of HEWL in (A-B) 10 mM sodium acetate buffer pH 4.0, (C-D) 10 mM Hepes buffer pH 7.5 and (E-F) 10 mM succinic acid pH 7.0 upon titration with a solution of [Ru2Cl(D-p- FPhF)(μ-O2CCH3)3]. Spectra have been collected using λexc = 280 nm (panels A, C and E) and 295 nm (panels B, D and F). ........................................................................................................................................................8 Figure S5. Conformation of Asp119 side chain in the metal-free HEWL (panel A, violet) and in structure 2 (panel B, yellow). ...............................................................................................................................................9 Table S1. Data collection and refinement statistics. .......................................................................................10 3 Materials HEWL was purchased from Sigma Chemical Co (Merck Life Science S.r.l., Milan, Italy) at highest grade of purity and used without further purification. Synthesis of [Ru2Cl(D-p-FPhF)(O2CCH3)3] was carried out as done in a previous work1 using the following procedure. A round-bottom flask containing 0.142 g (0.30 mmol) of [Ru2Cl(O2CCH3)4] starting material, 0.070 g (0.30 mmol) of HD- p-FPhF ligand, 0.1 mL (0.72 mmol) of Et3N and 50 mL of ethanol was sonicated for 2 h at 80 kHz. The mixture was filtered through Celite® to remove unreacted [Ru2Cl(O2CCH3)4], and the solution was evaporated under vacuum. The solid was washed with diethyl ether (50 mL), extracted in distilled water (150 mL), and filtered through Celite®. The filtered solution was washed with 5 mL of brine and extracted with dichloromethane (2 × 50 mL). The organic solution was treated with MgSO4, filtered off, evaporated under vacuum giving rise to a red-wine solid. Yield: 150 mg (75%). Anal. found (calculated) for [Ru2Cl(N2F2C13H9)(O2C2H3)3(OH2)] (663.97 g⸱mol−1 ): C, 34.49 (34.37); H, 3.03 (3.04); N, 4.41 (4.22). MS (ESI+) m/z: 610 [M – Cl – H2O]+ (55%); 690 [M + CH3CO]+ (100%). IR signals: 3380w (νOH), 3056w (νCHar), 2936w (νCH), 1639m (δHOH), 1602w (νCCar), 1526w, 1497s, (νCN + νCCar), 1434s (νCOOa), 1411m (δCH), 1351m (νCOOs), 1311m 1201s (νCN), 1153s (νCF), 1095m (δCarCarH), 1014m (δCH), 944m (δCH), 865w (δNCH), 834s (δCarCarH), 797m (δCarCarH), and 687s (δCOO) cm−1. μeff (25 °C) = 4.0 μB. In solution studies UV-vis spectra of [Ru2Cl(D-p-FPhF)(O2CCH3)3] were recorded on a Jasco V-750 spectrophotometer using quartz cuvette of 1 cm path length, a diruthenium concentration of 50 μM in water and in other four different experimental conditions, i.e. those used to grow HEWL crystals: A) 20% ethylene glycol, 0.1 M sodium acetate buffer at pH 4.0, 0.6 M sodium nitrate; B) 2.0 M sodium formate, 0.1 M Hepes buffer pH 7.5; C) 0.8 M succinic acid pH 7.0; D) 1.1 M sodium chloride, 0.1 M sodium acetate buffer pH 4.0. Spectra were collected for 5 hours continuously, and then after 24 hours and 7 days, in the absence and in the presence of HEWL (protein to metal compound molar ratio 1:3). Other experimental parameters were: wavelength range 240 – 700 nm, data pitch 1.0 nm, scanning speed 400 nm/min, band width 2.0 nm. Each measurement was repeated twice. Far UV-CD spectra were recorded on a Jasco J-715 spectropolarimeter equipped with a Peltier thermostatic cell holder (Model PTC-348WI) in the range of 190-250 nm, using a protein concentration of 7 μM and a quartz cell with 0.1 cm path length. HEWL was incubated 1 h with an increasing amounts of [Ru2Cl(D-p-FPhF)(O2CCH3)3]. Protein to diruthenium molar ratios were: 1:1, 1:2 and 1:3. Spectra were collected in: 10 mM sodium acetate buffer at pH 4.0. Other experimental parameters were 1.0 nm data pitch, 2.0 nm bandwidth, 50 nm/min scanning speed, 2.0 s response time, and 25 °C. Each spectrum was obtained by averaging three scans. A HORIBA Fluoromax-4 spectrofluorometer equipped with a thermostat bath and a 1 cm path length cuvette was used to register fluorescence spectra of HEWL in the absence and in the presence of [Ru2Cl(D-p-FPhF)(O2CCH3)3] at 25°C. HEWL solutions at a concentration of 1.4 μM were titrated with a 2 mM Ru2 compound solution. The protein was excited at 280 nm (to follow Tyr and Trp emission) and 295 nm (to follow Trp emission only) over a range of wavelengths between 295 and 450 nm and between 310 and 450 nm, for excitation at 280 and 295 nm, respectively. Different protein to metal molar ratios were reached upon titration: 1:0.2, 1:0.5, 1:1, 1:2, 1:4, 1:6, 1:8, 1:10. Solutions were stirred and equilibrated for 5 minutes before recording the spectra. The titrations were carried out in three different buffers: 10 mM sodium acetate buffer pH 4.4, 10 mM Hepes buffer pH 7.5 and 10 mM succinic acid pH 7.0. When necessary, correction of the fluorescence spectra was employed to compensate the effects of the existing primary and/or secondary internal filter using the following equation: Fcorr = Fobs 10 (Aex + Aem) / 2 where Fcorr and Fobs are, respectively, the corrected and observed fluorescence intensities, while Aex and Aem are the absorbance values, respectively, at the excitation and emission wavelengths. Each measurement was repeated twice. 4 Crystallization and X-ray structure solution and refinement Crystals of HEWL were grown by the hanging drop vapor diffusion method mixing 1 μL of protein solution (concentration 13 mg/mL) with an equal volume of the reservoir solution containing the following conditions: A) 20% ethylene glycol, 0.1 M sodium acetate at pH 4.0, and 0.6 M sodium nitrate, B) 2.0 M sodium formate, 0.1 M Hepes buffer pH 7.5; C) 0.8 M succinic acid pH 7.0. D) 1.1 M sodium chloride, 0.1 M sodium acetate buffer pH 4.0. Crystals grew in few days. These crystals were then exposed to stabilizing solutions containing the mother liquors and a saturated solution of [Ru2Cl(D- p-FPhF)(O2CCH3)3] for a soaking time of two weeks. Crystals of the adducts of [Ru2Cl(D-p-FPhF)(O2CCH3)3] with HEWL obtained under different conditions diffract X-ray in the resolution range of 1.17-1.81 Å. X-ray diffraction data collections were carried out on Beamline XRD2 at Elettra synchrotron (Trieste, Italy) 2, using a wavelength of 1.00 Å and a cold nitrogen stream of 100 K. Before exposure to X- ray, crystals were cryoprotected using a solution of the reservoir with 25% glycerol. Data processing and scaling were performed using a Global Phasing autoPROC pipeline3. Data collection statistics are reported in Table S1. The structures were solved by molecular-replacement method using Phaser4 with PDB entry 193L5 as template. Refmac6 was used for the refinement and Coot7 for manual model building. The Ru atom positions were validated using anomalous difference electron density maps. Ligand positions were restraints to guide geometry optimization. Pymol (www.pymol.org) was used to generate molecular graphic figures. Coordinates and structure factors of the adducts were deposited in the Protein Data Bank under the accession codes 8BPH (structure 1), 8BPU (structure 2), 8BPJ (structure 3), 8BQM (structure 4). References 1. Inchausti, A.; Terán, A.; Manchado-Parra, A.; de Marcos Galán, A.; Perles, J.; Cortijo, M.; González-Prieto, R.; Herrero, S.; Jiménez-Aparicio, R. New insights into progressive ligand replacement from [Ru2Cl(O2CCH3)4]: synthetic strategies and variation in redox potentials and paramagnetic shifts. Dalton Trans. 2022, 51, 9708-9719. 2 Lausi, A.; Polentarutti, M.; Onesti, S.; Plaisier, J. R.; Busetto, E.; Bais, G.; Barba, L.; Cassetta, A.; Campi, G.; Lamba, D.; Pifferi, A.; Mande, S. C.; Sarma, D. D.; Sharma, S. M.; Paolucci, G. Status of the crystallography beamlines at Elettra. Eur. Phys. J. Plus, 2015, 130, 1–8. 3. Vonrhein C.; Flensburg C.; Keller P.; Sharff A.; Smart O.; Paciorek W.; Womack T.; Bricogne G. Data processing and analysis with the autoPROC toolbox. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2011, 67, 293-302. 4. McCoy A. J.; Grosse-Kunstleve R. W.; Adams P. D.; Winn M. D.; Storoni L. C.; Read R. J. Phaser crystallographic software. J. Appl. Crystallogr. 2007, 40, 658–674. 5. Vaney M. C.; Maignan S.; Riès-Kautt M.; Ducruix A. High-Resolution Structure (1.33 Å) of a HEW Lysozyme Tetragonal Crystal Grown in the APCF Apparatus. Data and Structural Comparison with a Crystal Grown under Microgravity from SpaceHab-01 Mission. Acta Crystallogr., Sect. D: Biol. Crystallogr. 1996, 52, 505-517. 6. Murshudov G.N.; Vagin A.A; Dodson E.J. Refinement of Macromolecular Structures by the Maximum-Likelihood method”. Acta Crystallogr., Sect. D: Biol. Crystallogr. 1997, 53, 240-255. 7. Emsley P.; Lohkamp B.; Scott W. G.; Cowtan K. Features and development of Coot. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 486–501. 5 A B C D Figure S1. Time course UV–vis spectra of 50 μM [Ru2Cl(D-p-FPhF)(O2CCH3)3] in: (A) 20% ethylene glycol, 0.1 M sodium acetate buffer at pH 4.0, 0.6 M sodium nitrate; (B) 2.0 M sodium formate, 0.1 M Hepes buffer pH 7.5; (C) 0.8 M succinic acid pH 7.0; (D) 1.1 M sodium chloride, 0.1 M sodium acetate buffer pH 4.0. 6 A B C D Figure S2. Time course UV–vis spectra of 50 μM [Ru2Cl(D-p-FPhF)(O2CCH3)3] in the presence of HEWL (protein to diruthenium molar ratio = 1:3) in: (A) 20% ethylene glycol, 0.1 M sodium acetate buffer at pH 4.0, 0.6 M sodium nitrate; (B) 2.0 M sodium formate, 0.1 M Hepes buffer pH 7.5; (C) 0.8 M succinic acid pH 7.0; (D) 1.1 M sodium chloride, 0.1 M sodium acetate buffer pH 4.0. 7 Figure S3. Far-UV CD spectra of HEWL (7.0 μM concentration) incubated for 1 h in the presence of [Ru2Cl(D-p- FPhF)(O2CCH3)3] in 10 mM sodium acetate buffer pH 4.0 at protein to diruthenium molar ratio = 1:1 (violet) , 1:2 (red) and 1:3 (pink). CD of metal-free protein is in black. 8 A B C D E F 9 Figure S4. Fluorescence emission spectra of HEWL in (A-B) 10 mM sodium acetate buffer pH 4.0, (C-D) 10 mM Hepes buffer pH 7.5 and (E-F) 10 mM succinic acid pH 7.0 upon titration with a solution of [Ru2Cl(D-p- FPhF)(O2CCH3)3]. Spectra have been collected using λexc = 280 nm (panels A, C and E) and 295 nm (panels B, D and F). 10 Figure S5. Conformation of Asp119 side chain in the metal-free HEWL (panel A, violet) and in structure 2 (panel B, yellow). 11 Table S1. Data collection and refinement statistics. Structure 1 Structure 2 Structure 3 Structure 4 PDB code 8BPH 8BPU 8BPJ 8BQM Crystallization conditions 20% ethylene glycol, 0.1 M sodium acetate buffer at pH 4.0, 0.6 M sodium nitrate 2.0 M sodium formate, 0.1 M Hepes buffer pH 7.5 0.8 M succinic acid pH 7.0 1.1 M sodium chloride, 0.1 M sodium acetate buffer pH 4.0 Soaking time Two weeks Two weeks Two weeks Two weeks Data collection Space group P43 21 2 P43 21 2 P43 21 2 P41 21 2 a (Å) 77.48 78.36 77.19 77.74 b (Å) 77.48 78.36 77.19 77.74 c (Å) 37.27 37.51 37.83 37.38 α/β/γ (°) 90.0/90.0/90.0 90.0/90.0/90.0 90.0/90.0/90.0 90.0/90.0/90.0 Molecules for asymmetric unit 1 1 1 1 Resolution range (Å) 54.79-1.38 (1.40-1.38) 55.41-1.81 (1.84-1.81) 38.60-1.07 (1.09-1.07) 54.970- 1.17 (1.19-1.17) Observations 244494 (11745) 228866 (10963) 949875 (16689) 852398 (19928) Unique reflections 23817(1153) 11121 (524) 49110 (1737) 39386 (1830) Completeness (%) 99.9 (100.0) 99.5 (98.5) 96.8 (69.7) 99.6 (95.0) Redundancy 10.3 (10.2) 206 (20.9) 19.3 (9.6) 21.6 (10.9) Rmerge (%) 0.047 (0.935) 0.191 (1.328) 0.042 (0.809) 0.040 (0.970) Average I/σ(I) 21.8 (2.3) 11.2 (2.5) 33.8 (2.8) 37.8 (2.3) CC1/2 0.999 (0.833) 0.993 (0.874) 1.000 (0.830) 1.000 (0.740) Anom. completeness (%) 100.0 (100.0) 99.5 (98.5) 96.6 (68.5) 99.7 (95.8) Anom. Multiplicity 5.5 (5.3) 11.3 (11.1) 10.3 (5.1) 11.5 (5.7) Refinement Resolution (Å) 1.38 1.81 1.07 1.17 N° reflections 22704 10488 46382 37067 N° reflections in working set 1638 749 2225 2603 Rfactor/Rfree 0.192/0.231 0.232/0.289 0.187/0.209 0.199/0.225 N° non-H atoms in the refin. 1257 1135 1308 1296 Ramachandran statistcs Most favoured 101 (95.28%) 121 (95.28%) 104 (96.30%) 97 (96.04%) Outliers 0 0 0 0 Rmsd bonds (Å) 0.013 0.008 0.016 0.015 Rmsd angles (°) 1.921 1.557 2.065 2.093 149 1 Supporting Information Charge effect in protein metalation reactions by diruthenium complexes Aarón Terán,a Giarita Ferraro,b Ana E. Sánchez-Peláez,a Santiago Herreroa* and Antonello Merlinob* aMatMoPol Reseach Group. Inorganic Chemistry Department, Faculty of Chemical Sciences, Complutense University of Madrid, Avda. Complutense s/n, E-28040 Madrid, Spain. bDepartment of Chemical Sciences, University of Naples Federico II, Complesso Universitario di Monte Sant’Angelo, via Cinthia, 21, 80126, Naples, Italy. *E-mail: antonello.merlino@unina.it; sherrero@ucm.es Electronic Supplementary Material (ESI) for Inorganic Chemistry Frontiers. This journal is © the Partner Organisations 2023 mailto:antonello.merlino@unina.it mailto:sherrero@ucm.es 2 Index Materials ........................................................................................................................................................... 4 Synthesis ........................................................................................................................................................... 4 Scheme S1. Synthesis of K2[Ru2(L-L)(CO3)3] compounds by the reaction of [Ru2Cl(L-L)(O2CCH3)3] with K2CO3 in H2O:EtOH (1:21.4) solution at room temperature (L-L = D-p-FPhF- or DAniF-). ................................................ 4 Characterization ................................................................................................................................................ 4 Crystallization, X-ray diffraction data collection, structure solution and refinement of the adducts with HEWL ................................................................................................................................................................. 6 Table S1. Estimated melting temperatures (Tm) of HEWL and HEWL in the presence of diruthenium compounds at 1:3 HEWL:Ru2 molar ratio under different conditions. ............................................................. 7 Table S2. Data collection and refinement statistics. ......................................................................................... 8 Figure S1. FT-IR spectra of K2[Ru2(D-p-FPhF)(CO3)3]·3H2O·EtOH (upper) and K2[Ru2(DAniF)(CO3)3]·3H2O (lower). .............................................................................................................................................................. 9 Figure S2. ESI mass peaks of K2[Ru2(D-p-FPhF)(CO3)3]·3H2O·EtOH acquired in water (Molecular weight = 791.70 g/mol). Experimental (upper) and simulated (lower) isotopic distribution. S = 3H2O + EtOH. ........... 10 Figure S3. ESI mass peaks of K2[Ru2(DAniF)(CO3)3]·3H2O acquired in water (Molecular weight = 769.70 g/mol). Experimental (upper) and simulated (lower) isotopic distribution. S = 3H2O. ................................................ 11 Figure S4. Cyclic voltammograms for [Ru2Cl(O2CCH3)4], K3[Ru2(CO3)4], [Ru2Cl(L-L)(O2CCH3)3] and K2[Ru2(L- L)(CO3)3] (L-L = DAniF- or D-p-FPhF-) derivatives. Experiments shown for 0.1 M KCl and scan rate = 100 mV/s. ......................................................................................................................................................................... 12 Figure S5. UV-vis spectra of K3[Ru2(CO3)4] (A), K2[Ru2(D-p-FPhF)(CO3)3] (B), K2[Ru2(DAniF)(CO3)3] (C), and [Ru2Cl(DAniF)(O2CCH3)3] (D) in pure water recorded as a function of time during the incubation for 24h. Metal compound concentration = 500 µM. ............................................................................................................... 13 Figure S6. Time course UV–vis spectra of 500 μM K3[Ru2(CO3)4] in 20% ethylene glycol, 0.1 M sodium acetate buffer at pH 4.0, 0.6 M sodium nitrate in the absence (A) and in the presence (B) of HEWL and in 2.0 M sodium formate, 0.1 M Hepes buffer pH 7.5 in the absence (C) and in the presence (D) of HEWL. ........................... 14 Figure S7. Time course UV–vis spectra of 500 μM K2[Ru2(D-p-FPhF)(CO3)3] in 20% ethylene glycol, 0.1 M sodium acetate buffer at pH 4.0, 0.6 M sodium nitrate in the absence (A) and in the presence (B) of HEWL and in 2.0 M sodium formate, 0.1 M Hepes buffer pH 7.5 in the absence (C) and in the presence (D) of HEWL. ......................................................................................................................................................................... 15 Figure S8. Time course UV–vis spectra of 500 μM [Ru2Cl(DAniF)(O2CCH3)3] in 20% ethylene glycol, 0.1 M sodium acetate buffer at pH 4.0, 0.6 M sodium nitrate in the absence (A) and in the presence (B) of HEWL and in 2.0 M sodium formate, 0.1 M Hepes buffer pH 7.5 in the absence (C) and in the presence (D) of HEWL. ......................................................................................................................................................................... 16 Figure S9. Time course UV–vis spectra of 500 μM K2[Ru2(DAniF)(CO3)3] in 20% ethylene glycol, 0.1 M sodium acetate buffer at pH 4.0, 0.6 M sodium nitrate in the absence (A) and in the presence (B) of HEWL and in 2.0 M sodium formate, 0.1 M Hepes buffer pH 7.5 in the absence (C) and in the presence (D) of HEWL. .......... 17 Figure S10. Fluorescence emission spectra of HEWL in (A-B) 10 mM sodium acetate buffer pH 4.0 and (C-D) 10 mM Hepes buffer pH 7.5 upon titration with a solution of K2[Ru2(CO3)4]. Spectra have been collected using λex = 280 nm (panels A and C) and 295 nm (panels B and D). ......................................................................... 18 Figure S11. Fluorescence emission spectra of HEWL in (A-B) 10 mM sodium acetate buffer pH 4.0 and (C-D) 10 mM Hepes buffer pH 7.5 upon titration with a solution of K2[Ru2(D-p-FPhF)(CO3)3]. Spectra have been collected using λex = 280 nm (panels A and C) and 295 nm (panels B and D). ................................................ 19 3 Figure S12. Fluorescence emission spectra of HEWL in (A-B) 10 mM sodium acetate buffer pH 4.0 and (C-D) 10 mM Hepes buffer pH 7.5 upon titration with a solution of [Ru2Cl(DAniF)(O2CCH3)3]. Spectra have been collected using λex = 280 nm (panels A and C) and 295 nm (panels B and D). ............................................... 20 Figure S13. Fluorescence emission spectra of HEWL in (A-B) 10 mM sodium acetate buffer pH 4.0 and (C-D) 10 mM Hepes buffer pH 7.5 upon titration with a solution of K2[Ru2(DAniF)(CO3)3]. Spectra have been collected using λex = 280 nm (panels A and C) and 295 nm (panels B and D). ................................................ 21 Figure S14. Far-UV CD spectra of HEWL (7.0 μM concentration) incubated for 24 h in the presence of K3[Ru2(CO3)4] (A), K2[Ru2(D-p-FPhF)(CO3)3] (B), K2[Ru2(DAniF)(CO3)3] (C), and [Ru2Cl(DAniF)(O2CCH3)3] (D) in 10 mM sodium acetate buffer pH 4.0 in different protein to diruthenium molar ratios. CD spectrum of metal- free protein is in black. .................................................................................................................................... 22 Figure S15. Hepes molecule found in the structure of the adduct formed in the reaction between HEWL and K2[Ru2(D-p-FPhF)(CO3)3] in the condition B. 2Fo-Fc electron density maps are contoured at 1.0 σ (grey) level. ......................................................................................................................................................................... 23 4 Materials All the reactions and manipulations were performed under air atmosphere. All reactants and solvents were obtained from commercial sources and used without further purification unless otherwise indicated. HEWL was purchased from Sigma Chemical Co (Merck Life Science S.r.l., Milan, Italy) at highest grade of purity. Synthesis The syntheses of [Ru2Cl(O2CCH3)4],1 K3[Ru2(CO3)4]·4H2O,2 [Ru2Cl(D-p-FPhF)(O2CCH3)3]3 and [Ru2Cl(DAniF)(O2CCH3)3]4 had been previously described. The HDAniF and HD-p-FPhF formamidines were prepared according to a published general procedure.5 [Ru2Cl(D-p-FPhF)(O2CCH3)3] and [Ru2Cl(DAniF)(O2CCH3)3] were used as starting material to prepare K2[Ru2(D-p-FPhF)(CO3)3] and K2[Ru2(DAniF)(CO3)3] compounds. Scheme S1. Synthesis of K2[Ru2(L-L)(CO3)3] compounds by the reaction of [Ru2Cl(L-L)(O2CCH3)3] with K2CO3 in H2O:EtOH (1:21.4) solution at room temperature (L-L = D-p-FPhF- or DAniF-). A mixture of a solution of the corresponding [Ru2Cl(L-L)(O2CCH3)3] (L-L = D-p-FPhF or DAniF) compound (0.1 mmol in 30 mL of EtOH) and a solution of K2CO3 (0.3 mmol in 1.4 mL of H2O) was vigorously stirred overnight. The dark-orange solid formed was filtred, washed with EtOH (5×10 mL) and dried under vacuum to give analytically pure materials. K2[Ru2(D-p-FPhF)(CO3)3]·3H2O·EtOH: Yield: 95%. Elemental analysis found (calculated) for C18H21F2K2N2O13Ru2 (MW = 791.70 g/mol): C, 27.11 (27.31); H, 2.57 (2.67); N, 3.76 (3.54). ATR-FT-IR: 1639, 1528, 1458, 1299, 1204, 1153, 1056, 947, 836, 780, 694 cm-1. ESI (m/z): 675.0 [M ‒ 3H2O ‒ EtOH + H+]+, 650.7 [M ‒ K+ ‒ 3H2O ‒ EtOH + 2H+]+, 615.7 [M ‒ 2K+ ‒ 3H2O ‒ EtOH + 3H+]+. K2[Ru2(DAniF)(CO3)3]·3H2O: Yield: 99%. Elemental analysis found (calculated) for C18H21K2N2O14Ru2 (MW = 769.70 g/mol): C, 27.86 (28.09); H, 3.19 (2.75); N, 3.45 (3.64). ATR-FT-IR: 1646, 1529, 1444, 1294, 1216, 1170, 1031, 945, 829, 768, 694 cm-1. ESI (m/z): 715.7 [M ‒ 3H2O + H+]+ and 671.7 [M ‒ K+ ‒ 3H2O + 2H+]+. Characterization FTIR spectra (4000–500 cm−1) were recorded with a Perkin-Elmer Spectrum 100 with a universal ATR accessory. Elemental analyses were performed at the Microanalytical Service of the Universidad 5 Complutense de Madrid. Mass spectrometry data (electrospray ionization) were recorded at the Mass Spectrometry Service of the Universidad Complutense de Madrid, using an ion trap analyser HCT Ultra (Bruker Daltonics) mass spectrometer in water solution. Cyclic voltammetry (CV) measurements were conducted with a Metrohm Autolab PGSTAT204 potentiostat. The working electrode was a glassy carbon electrode (GCE). A Pt wire served as the counter electrode and Ag/AgCl was employed as the reference electrode. Electrochemical grade KCl at a concentration of 0.10 M was employed as the supporting electrolyte in voltammetric measurements. High pure N2 was used to deoxygenate the solution at least 10 minutes prior to each run and to maintain a nitrogen blanket. The ferrocenium/ferrocene couple was observed at 0.53 V (vs. Ag/AgCl) in 0.1 M tetrabutylammonium perchlorate (TBAP) dichloromethane solution. UV-vis spectra of K3[Ru2(CO3)4], K2[Ru2(D-p-FPhF)(CO3)3], [Ru2Cl(DAniF)(O2CCH3)3] and K2[Ru2(DAniF)(CO3)3] were recorded on a Jasco V-750 spectrophotometer using quartz cuvette of 1 cm path length, a 50 μM diruthenium concentration in water and in other two different experimental conditions, i.e. those used to grow HEWL crystals: A) 20% ethylene glycol, 0.1 M sodium acetate buffer at pH 4.0, 0.6 M sodium nitrate; B) 2.0 M sodium formate, 0.1 M Hepes buffer pH 7.5. Spectra were collected for 5 hours continuously, and then after 24 hours and 7 days, in the absence and in the presence of HEWL (protein to metal compound molar ratio 1:3). Other experimental parameters were wavelength range 240–700 nm, data pitch 1.0 nm, scanning speed 400 nm/min, band width 2.0 nm. Each measurement was repeated twice. Far UV-CD spectra were recorded on a Jasco J-715 spectropolarimeter equipped with a Peltier thermostatic cell holder (Model PTC-348WI) in the range of 200-250 nm, using a protein concentration of 7 μM and a quartz cell with 0.1 cm path length. HEWL was incubated 24 h with increasing amounts of K3[Ru2(CO3)4], K2[Ru2(D-p-FPhF)(CO3)3], [Ru2Cl(DAniF)(O2CCH3)3] and K2[Ru2(DAniF)(CO3)3] to obtain the following protein to diruthenium molar ratios: 1:0.1, 1:1, 1:2, 1:3. Spectra were collected in: 10 mM sodium acetate buffer at pH 4.0 and 10 mM Hepes buffer at pH 7.5. Other experimental parameters were 1.0 nm data pitch, 2.0 nm bandwidth, 50 nm/min scanning speed, 2.0 s response time, and 25 °C. Each spectrum was obtained by averaging three scans. Thermal denaturation experiments were performed by following the CD signal at 222 nm as a function of temperature for HEWL and the adducts formed upon reaction with the diruthenium compounds (1 :3 protein : metal compound molar ratio) using 7 μM of protein at two different pH values, pH 7.5 (10 mM Hepes buffer) and pH 4.0 (10 mM sodium acetate buffer). A Peltier temperature controller was used to set up the temperature of the sample, with a slope of 1 °C per min. A HORIBA Fluoromax-4 spectrofluorometer equipped with a thermostat bath and a 1 cm path length cuvette was used to register fluorescence spectra of HEWL in the absence and in the presence of K3[Ru2(CO3)4], K2[Ru2(D-p-FPhF)(CO3)3], [Ru2Cl(DAniF)(O2CCH3)3] and K2[Ru2(DAniF)(CO3)3] at 25°C. HEWL solutions at a concentration of 1.4 μM were titrated with a 2 mM Ru2 compound solution. The protein was excited at 280 nm (to follow Tyr and Trp emission) and 295 nm (to follow Trp emission only) over a range of wavelengths between 295 and 450 nm and between 310 and 450 nm, for excitation at 280 and 295 nm, respectively. Different protein to metal molar ratios were reached upon titration: 1:0.2, 1:0.5, 1:1, 1:2, 1:4, 1:6, 1:8, 1:10. Solutions were stirred and equilibrated for 5 minutes before recording the spectra. The titrations were carried out in two different buffers: 10 mM sodium acetate buffer pH 4.0 and 10 mM Hepes buffer pH 7.5. When necessary, correction of the fluorescence spectra was employed to compensate the effects of the existing primary and/or secondary internal filter using the following equation: Fcorr = Fobs 10 (Aex + Aem) / 2 6 where Fcorr and Fobs are, respectively, the corrected and observed fluorescence intensities, while Aex and Aem are the absorbance values, respectively, at the excitation and emission wavelengths. Each measurement was repeated twice. Crystallization, X-ray diffraction data collection, structure solution and refinement of the adducts with HEWL Crystals of HEWL were grown by the hanging drop vapor diffusion method mixing 1 μL of protein solution (concentration 13 mg/mL) with an equal volume of the reservoir solution containing the following conditions: 20% ethylene glycol, 0.1 M sodium acetate at pH 4.0 (condition A), and 0.6 M sodium nitrate, B) 2.0 M sodium formate, 0.1 M Hepes buffer pH 7.5 (condition B). Crystals grew in few days. These crystals were then exposed to stabilizing solutions containing the mother liquors and a saturated solution of K3[Ru2(CO3)4], K2[Ru2(D-p-FPhF)(CO3)3], K2[Ru2(DAniF)(CO3)3] and [Ru2Cl(DAniF)(O2CCH3)3] for a soaking time of 14 days. Crystals of the adducts of the four compounds with HEWL were then soaked in the reservoir solution supplemented with 25% (v/v) glycerol for a few seconds, flash-cooled in liquid nitrogen and stored under cryogenic conditions until data collection. The crystals diffract X-ray in the resolution range of 1.46-1.03 Å. X-ray diffraction data collections were carried out on Beamline XRD2 at Elettra synchrotron (Trieste, Italy),6 using a wavelength of 1.00 Å and a cold nitrogen stream of 100 K. The total oscillation was 360°, with 1° per image, and the exposure time was 1 s per image. Data were processed and scaled using with the automated data-processing autoPROC pipeline. Data collection statistics are reported in Table S2. The structures were solved by molecular replacement using Phaser7 with the coordinates of metal- free HEWL deposited under the PDB accession codes 193L8 as template. REFMAC59 was used for the refinement and Coot10 for manual model editing. The Ru atom positions were identified using anomalous difference (ΔFano) and difference Fourier Fo−Fc electron density maps calculated using the Collaborative Computational Project Number 4 (CCP4) suite.11,12 Ligand positions were restraints to guide geometry optimization. Pymol (www.pymol.org) was used to generate molecular graphic figures. Refinement statistics are given in Table S2. Coordinates and structure factors of the adducts were validated using the Protein Data Bank validation server13 and deposited in the PDB (www.rcsb.org) as entries 8PFU (HEWL with [Ru2(CO3)4]3− in the condition A), 8PFT (HEWL with [Ru2(D-p-FPhF)(CO3)3]3- in the condition A), 8PFX (HEWL with [Ru2(D-p- FPhF)(CO3)3]3- in the condition B), 8PFW (HEWL with [Ru2(DAniF)(CO3)3]2- in the condition A), 8PFY (HEWL with [Ru2(DAniF)(CO3)3]2- in the condition B) and 8PFV (HEWL with [Ru2(DAniF)(O2CCH3)3]+ in the condition A). The results were visualized with PyMOL (Schrödinger).14 7 Table S1. Estimated melting temperatures (Tm) of HEWL and HEWL in the presence of diruthenium compounds at 1:3 HEWL:Ru2 molar ratio under different conditions. Entry Protein to metal compound molar ratio Tm (°C) 10 mM Hepes buffer pH 7.5 10 mM sodium acetate buffer pH 4.0 HEWL - 78 ± 1 80 ± 1 K3[Ru2(CO3)4]/HEWL 1:3 76 ± 1 79 ± 1 K2[Ru2(D-p-FPhF)(CO3)3] /HEWL 1:3 80 ± 1 80 ± 1 K2[Ru2(DAniF)(CO3)3] /HEWL 1:3 81 ± 1 82 ± 1 K2[Ru2(DAniF)(O2CCH3)3] /HEWL 1:3 77 ± 1 81 ± 1 8 Table S2. Data collection and refinement statistics. Compound HEWL + [Ru2(CO3)4]3- HEWL + [Ru2(D-p-FPhF)(CO3)3]2- HEWL + [Ru2(DAniF)(CO3)3]2- HEWL + [Ru2(DAniF)(O2CCH3)3]+ HEWL + [Ru2(D-p-FPhF)(CO3)3]2- HEWL + [Ru2(DAniF)(CO3)3]2- PDB code 8PFU 8PFT 8PFW 8PFV 8PFX 8PFY Crystallization conditions Condition A Condition B Soaking time 2 weeks 2 weeks Data collection Space group P41212 P41212 P41212 P41212 P41212 P41212 a (Å) 78.06 77.75 77.81 78.35 76.72 76.62 b (Å) 78.06 77.75 77.81 78.35 76.72 76.62 c (Å) 37.47 37.34 37.39 37.64 38.39 37.08 α/β/γ (°) 90.0/90.0/90.0 90.0/90.0/90.0 90.0/90.0/90.0 90.0/90.0/90.0 90.0/90.0/90.0 90.0/90.0/90.0 Molecules for asymmetric unit 1 1 1 1 1 1 Resolution range (Å) 55.20-1.18 (1.20-1.18) 54.98-1.42 (1.44-1.42) 55.02-1.12 (1.14-1.12) 55.40-1.46 (1.48-1.46) 38.36-1.03 (1.04-1.03) 54.18-1.19 (1.21-1.19) Observations 858346 (21017) 281127 (14230) 917971 (37961) 217102 (9627) 654453 (16800) 750551 (22007) Unique reflections 38622 (1683) 22171 (1093) 44515 (2171) 21101 (1035) 57486 (2835) 36175 (1773) Completeness (%) 99.2 (88.6) 99.8 (100.0) 99.9 (100.0) 99.9 (100.0) 100.0 (99.9) 100.0 (100.0) Redundancy 22.2 (12.5) 12.7 (13.0) 11.0 (9.1) 10.3 (9.3) 11.4 (5.9) 20.7 (12.4) Rmerge (%) 0.034 (1.003) 0.048 (1.273) 0.054 (1.324) 0.047 (0.997) 0.034 (0.753) 0.131 (1.106) Average I/σ(I) 42.5 (2.3) 24.0 (2.2) 27.2 (2.2) 23.7 (2.2) 33.7 (2.3) 14.4 (2.4) CC1/2 1.000 (0.750) 1.000 (0.833) 0.999 (0.807) 1.000 (0.762) 1.000 (0.858) 0.997 (0.843) Anom. completeness (%) 99.2 (88.6) 99.9 (100.0) 100.0 (100.0) 100.0 (100.0) 99.8 (98.3) 100.0 (100.0) Anom. Multiplicity 11.8 (6.5) 6.8 (6.8) 11.0 (9.1) 5.5 (4.9) 6.0 (3.1) 11.0 (6.4) Refinement Resolution (Å) 1.18 1.30 1.12 1.46 1.03 1.19 N° reflections 36163 24331 42224 19847 52328 34188 N° reflections in working set 2199 359 2946 1445 2721 2491 Rfactor/Rfree 0.179/0.202 0.205/0.245 0.203/0.239 0.181/0.211 0.193/0.214 0.195/0.212 N° non-H atoms in the refin. 1275 1208 1252 1171 1254 1128 Average B-factors (Å2) All atoms 16.8 26.2 17.8 23.6 12.3 15.9 Ru atoms 63.6/42.8 40.2/40.6 36.0/36.1 31.3/28.6 37.8/33.3 27.5/29.4 9.9/10.9 12.1/12.0 12.3/12.7 15.9/16.7 15.4/14.4 15.5/14.5 16.1/17.9 30.4 Ru occupancy 0.40/0.40 0.20/0.20 0.20/0.20 0.20/0.20 0.35/0.35 0.35/0.35 0.45/0.45 0.50/0.50 0.35/0.35 0.35/0.35 0.35/0.35 0.20/0.20 0.20/0.20 0.30 Ramachandran statistics Most favoured 96 (95.05%) 113 (95.76%) 107 (95.54%) 111 (93.28%) 108 (93.91%) 115 (95.04%) Outliers 0 0 0 0 0 0 Rmsd bonds (Å) 0.013 0.010 0.011 0.013 0.013 0.012 Rmsd angles (°) 1.827 2.525 3.691 1.996 1.933 1.827 †Rmerge = ΣhΣi |I(h,i)-|/ Σ hΣi I(h,i), where I(h,i) is the intensity of the ith measurement of reflection h and is the mean value of the intensity of reflection h Condition A: 20% ethylene glycol, 0.6 M NaNO3, 0.1 M sodium acetate pH 4.0 Condition B: 2 M sodium formate and 0.1 M Hepes, pH 7.5 9 Figure S1. FT-IR spectra of K2[Ru2(D-p-FPhF)(CO3)3]·3H2O·EtOH (upper) and K2[Ru2(DAniF)(CO3)3]·3H2O (lower). 10 Figure S2. ESI mass peaks of K2[Ru2(D-p-FPhF)(CO3)3]·3H2O·EtOH acquired in water (Molecular weight = 791.70 g/mol). Experimental (upper) and simulated (lower) isotopic distribution. S = 3H2O + EtOH. 11 Figure S3. ESI mass peaks of K2[Ru2(DAniF)(CO3)3]·3H2O acquired in water (Molecular weight = 769.70 g/mol). Experimental (upper) and simulated (lower) isotopic distribution. S = 3H2O. 12 Figure S4. Cyclic voltammograms for [Ru2Cl(O2CCH3)4], K3[Ru2(CO3)4], [Ru2Cl(L-L)(O2CCH3)3] and K2[Ru2(L- L)(CO3)3] (L-L = DAniF- or D-p-FPhF-) derivatives. Experiments shown for 0.1 M KCl and scan rate = 100 mV/s. 13 A B C D Figure S5. UV-vis spectra of K3[Ru2(CO3)4] (A), K2[Ru2(D-p-FPhF)(CO3)3] (B), K2[Ru2(DAniF)(CO3)3] (C), and [Ru2Cl(DAniF)(O2CCH3)3] (D) in pure water recorded as a function of time during the incubation for 24h. Metal compound concentration = 500 µM. 14 A B C D Figure S6. Time course UV–vis spectra of 500 μM K3[Ru2(CO3)4] in 20% ethylene glycol, 0.1 M sodium acetate buffer at pH 4.0, 0.6 M sodium nitrate in the absence (A) and in the presence (B) of HEWL and in 2.0 M sodium formate, 0.1 M Hepes buffer pH 7.5 in the absence (C) and in the presence (D) of HEWL. 15 A B C D Figure S7. Time course UV–vis spectra of 500 μM K2[Ru2(D-p-FPhF)(CO3)3] in 20% ethylene glycol, 0.1 M sodium acetate buffer at pH 4.0, 0.6 M sodium nitrate in the absence (A) and in the presence (B) of HEWL and in 2.0 M sodium formate, 0.1 M Hepes buffer pH 7.5 in the absence (C) and in the presence (D) of HEWL. 16 A B C D Figure S8. Time course UV–vis spectra of 500 μM [Ru2Cl(DAniF)(O2CCH3)3] in 20% ethylene glycol, 0.1 M sodium acetate buffer at pH 4.0, 0.6 M sodium nitrate in the absence (A) and in the presence (B) of HEWL and in 2.0 M sodium formate, 0.1 M Hepes buffer pH 7.5 in the absence (C) and in the presence (D) of HEWL. 17 A B C D Figure S9. Time course UV–vis spectra of 500 μM K2[Ru2(DAniF)(CO3)3] in 20% ethylene glycol, 0.1 M sodium acetate buffer at pH 4.0, 0.6 M sodium nitrate in the absence (A) and in the presence (B) of HEWL and in 2.0 M sodium formate, 0.1 M Hepes buffer pH 7.5 in the absence (C) and in the presence (D) of HEWL. 18 Figure S10. Fluorescence emission spectra of HEWL in (A-B) 10 mM sodium acetate buffer pH 4.0 and (C-D) 10 mM Hepes buffer pH 7.5 upon titration with a solution of K2[Ru2(CO3)4]. Spectra have been collected using λex = 280 nm (panels A and C) and 295 nm (panels B and D). 19 Figure S11. Fluorescence emission spectra of HEWL in (A-B) 10 mM sodium acetate buffer pH 4.0 and (C-D) 10 mM Hepes buffer pH 7.5 upon titration with a solution of K2[Ru2(D-p-FPhF)(CO3)3]. Spectra have been collected using λex = 280 nm (panels A and C) and 295 nm (panels B and D). 20 Figure S12. Fluorescence emission spectra of HEWL in (A-B) 10 mM sodium acetate buffer pH 4.0 and (C-D) 10 mM Hepes buffer pH 7.5 upon titration with a solution of [Ru2Cl(DAniF)(O2CCH3)3]. Spectra have been collected using λex = 280 nm (panels A and C) and 295 nm (panels B and D). 21 Figure S13. Fluorescence emission spectra of HEWL in (A-B) 10 mM sodium acetate buffer pH 4.0 and (C-D) 10 mM Hepes buffer pH 7.5 upon titration with a solution of K2[Ru2(DAniF)(CO3)3]. Spectra have been collected using λex = 280 nm (panels A and C) and 295 nm (panels B and D). 22 Figure S14. Far-UV CD spectra of HEWL (7.0 μM concentration) incubated for 24 h in the presence of K3[Ru2(CO3)4] (A), K2[Ru2(D-p-FPhF)(CO3)3] (B), K2[Ru2(DAniF)(CO3)3] (C), and [Ru2Cl(DAniF)(O2CCH3)3] (D) in 10 mM sodium acetate buffer pH 4.0 in different protein to diruthenium molar ratios. CD spectrum of metal-free protein is in black. 23 Figure S15. Hepes molecule found in the structure of the adduct formed in the reaction between HEWL and K2[Ru2(D-p-FPhF)(CO3)3] in the condition B. 2Fo-Fc electron density maps are contoured at 1.0 σ (grey) level. 24 REFERENCES 1 R. W. Mitchell, A. Spencer and G. Wilkinson, J. Chem. Soc., Dalton Trans., 1973, 846–854. 2 F. A. Cotton, L. Labella and M. Shang, Inorg. Chem., 1992, 31, 2385–2389. 3 A. Inchausti, A. Terán, A. Manchado-Parra, A. de Marcos-Galán, J. Perles, M. Cortijo, R. González- Prieto, S. Herrero and R. Jiménez-Aparicio, Dalton Trans., 2022, 51, 9708–9719. 4 A. Terán, M. Cortijo, Á. Gutiérrez, A. E. Sánchez-Peláez, S. Herrero and R. Jiménez-Aparicio, Ultrason. Sonochem., 2021, 80, 105828. 5 R. M. Roberts, J. Org. Chem., 1949, 14, 277–284. 6 A. Lausi, M. Polentarutti, S. Onesti, J. R. Plaisier, E. Busetto, G. Bais, L. Barba, A. Cassetta, G. Campi, D. Lamba, A. Pifferi, S. C. Mande, D. D. Sarma, S. M. Sharma and G. Paolucci, Eur. Phys. J. Plus, 2015, 130, 43. 7 A. J. McCoy, R. W. Grosse-Kunstleve, P. D. Adams, M. D. Winn, L. C. Storoni and R. J. Read, J. Appl. Crystallogr., 2007, 40, 658–674. 8 M. C. Vaney, S. Maignan, M. Riès-Kautt and A. Ducruix, Acta Crystallogr. D Biol. Crystallogr., 1996, 52, 505–517. 9 G. N. Murshudov, A. A. Vagin and E. J. Dodson, Acta Crystallogr. D Biol. Crystallogr., 1997, 53, 240–255. 10 P. Emsley, B. Lohkamp, W. G. Scott and K. Cowtan, Acta Crystallogr. D Biol. Crystallogr., 2010, 66, 486–501. 11 E. Potterton, P. Briggs, M. Turkenburg and E. Dodson, Acta Crystallogr. D Biol. Crystallogr., 2003, 59, 1131–1137. 12 M. D. Winn, C. C. Ballard, K. D. Cowtan, E. J. Dodson, P. Emsley, P. R. Evans, R. M. Keegan, E. B. Krissinel, A. G. W. Leslie, A. McCoy, S. J. McNicholas, G. N. Murshudov, N. S. Pannu, E. A. Potterton, H. R. Powell, R. J. Read, A. Vagin and K. S. Wilson, Acta Crystallogr. D Biol. Crystallogr., 2011, 67, 235–242. 13 H. Berman, K. Henrick and H. Nakamura, Nat. Struct. Mol. Biol., 2003, 10, 980–980. 14 The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC. 1 Supporting Information Steric hindrance and charge influence on the cytotoxic activity and protein binding properties of diruthenium complexes Aarón Terán,a Giarita Ferraro,b Paola Imbimbo,b Ana E. Sánchez-Peláez,a Daria Maria Monti,b Santiago Herrero,a* and Antonello Merlinob* aMatMoPol Research Group, Department of Inorganic Chemistry, Faculty of Chemical Sciences, Complutense University of Madrid, Avda. Complutense s/n, 28040, Madrid, Spain. bDepartment of Chemical Sciences, University of Naples Federico II, Complesso Universitario di Monte Sant’Angelo, via Cinthia, 21, 80126, Naples, Italy. *E-mail: sherrero@ucm.es; antonello.merlino@unina.it mailto:sherrero@ucm.es mailto:antonello.merlino@unina.it 2 Index Data collection, X-ray structure solution and refinement ................................................................................... 4 Figure S1. FT-IR spectra of K2[Ru2(DPhF)(CO3)3]·3H2O (upper) and [Ru2Cl(DPhF)2(O2CCH3)2]·H2O (lower). .......... 5 Figure S2. The only two peaks found in the ESI mass spectrum of [Ru2Cl(DPhF)2(O2CCH3)2]·H2O acquired in methanol. Experimental (red and blue bars) and simulated (black lines) isotopic distribution. ........................ 6 Figure S3. The two peaks observed in the ESI mass spectrum of K2[Ru2(DPhF)(CO3)3]·3H2O acquired in water. Experimental (orange and green bars) and simulated (black lines) isotopic distribution. ................................. 7 Figure S4. Cyclic voltammogram for K2[Ru2(DPhF)(CO3)3]·3H2O. Experiments shown for 0.1 M KCl and scan rate = 100 mV/s. ........................................................................................................................................................ 8 Figure S5. Cyclic voltammogram for [Ru2Cl(DPhF)2(O2CCH3)2]·H2O. Experiments shown for 0.1 M KCl and scan rate = 100 mV/s. ................................................................................................................................................ 9 Figure S6. Time course UV–vis spectra of a) [Ru2Cl(DPhF)(O2CCH3)3], b) K2[Ru2(DPhF)(CO3)3], and c) [Ru2Cl(DPhF)2(O2CCH3)2] in Milli-Q water at [C] = 500 μM. No appreciable spectral changes were observed within 2 weeks. ................................................................................................................................................ 10 Figure S7. Time course UV–vis spectra of 50 μM [Ru2Cl(DPhF)(O2CCH3)3] in 0.8 M succinic acid pH 7.0 (A and B) and 2.0 M sodium formate, 0.1 M Hepes buffer pH 7.5 (C and D) in the absence (A and C) and presence (B and D) of HEWL. ...................................................................................................................................................... 11 Figure S8. Time course UV–vis spectra of 50 μM [Ru2Cl(DPhF)2(O2CCH3)2] in 0.8 M succinic acid pH 7.0 (A and B) and 2.0 M sodium formate, 0.1 M Hepes buffer pH 7.5 (C and D) in the absence (A and C) and presence (B and D) of HEWL. ............................................................................................................................................... 12 Figure S9. Time course UV–vis spectra of 50 μM K2[Ru2(DPhF)(CO3)3] in 0.8 M succinic acid pH 7.0 (A and B) and 2.0 M sodium formate, 0.1 M Hepes buffer pH 7.5 (C and D) in the absence (A and C) and presence (B and D) of HEWL. .......................................................................................................................................................... 13 Figure S10. Fluorescence emission spectra of HEWL in (A-B) 10 mM succinic acid pH 7.0 and (C-D) 10 mM Hepes buffer pH 7.5 upon titration with a solution of [Ru2Cl(DPhF)(O2CCH3)3]. Spectra have been collected using λex = 280 nm (panels A, C) and λex =295 nm (panels B, D). ....................................................................................... 14 Figure S11. Fluorescence emission spectra of HEWL in (A-B) 10 mM succinic acid pH 7.0 and (C-D) 10 mM Hepes buffer pH 7.5 upon titration with a solution of [Ru2Cl(DPhF)2(O2CCH3)2]. Spectra have been collected using λex = 280 nm (panels A, C) and λex =295 nm (panels B, D). ..................................................................................... 15 Figure S12. Fluorescence emission spectra of HEWL in (A-B) 10 mM succinic acid pH 7.0 and (C-D) 10 mM Hepes buffer pH 7.5 upon titration with a solution of K2[Ru2(DPhF)(CO3)3]. Spectra have been collected using λex = 280 nm (panels A, C) and λex =295 nm (panels B, D). .............................................................................................. 16 Figure S13. Far-UV CD spectra of HEWL (7.0 μM concentration) incubated for 1 h in the presence of [Ru2Cl(DPhF)(O2CCH3)3] in (A) 10 mM succinic acid pH 7.0 and (B) 10 mM Hepes buffer pH 7.5 CD of metal-free protein is in black. ............................................................................................................................................ 17 Figure S14. Far-UV CD spectra of HEWL (7.0 μM concentration) incubated for 1 h in the presence of [[Ru2Cl(DPhF)2(O2CCH3)2] in (A) 10 mM succinic acid pH 7.0 and (B) 10 mM Hepes buffer pH 7.5 CD of metal- free protein is in black...................................................................................................................................... 18 Figure S15. Far-UV CD spectra of HEWL (7.0 μM concentration) incubated for 1 h in the presence of K2[Ru2(DPhF)(CO3)3] in (A) 10 mM succinic acid pH 7.0 and (B) 10 mM Hepes buffer pH 7.5 CD of metal-free protein is in black. ............................................................................................................................................ 19 3 Figure S16. Effect of diruthenium complexes on cell viability after 48 h incubation. (A) Dose-response curve of HeLa incubated in the presence of increasing concentration (0.1 – 50 µM) of [Ru2Cl(DPhF)(O2CCH3)3] (black circles), [Ru2Cl(DPhF)2(O2CCH3)2] (black squares), and K2[Ru2(DPhF)(CO3)3] (black triangles); (B) HaCaT cells (black circles) and BALB/c-3T3 cells (black squares) incubated with increasing concentration (0.1 – 50 µM) of [Ru2Cl(DPhF)2(O2CCH3)2]. Cell viability was assessed by the MTT assay and expressed. Values are given as means ± SD (n ≥ 3). ...................................................................................................................................................... 20 Table S1. Data collection and refinement statistics. ........................................................................................ 21 4 Data collection, X-ray structure solution and refinement Crystals of the adducts of [Ru2Cl(DPhF)(O2CCH3)3], [Ru2Cl(DPhF)2(O2CCH3)2], and K2[Ru2(DPhF)(CO3)3] with HEWL obtained under different conditions diffract X-ray in the resolution range of 1.07-1.41 Å. X-ray diffraction data collections were carried out on Beamline XRD2 at Elettra synchrotron (Trieste, Italy) [1], using a wavelength of 1.00 Å and a cold nitrogen stream of 100 K. The total oscillation was 360°, with 1° per image, and the exposure time was 1 s per image. Before exposure to X-ray, crystals were cryoprotected using a solution of the reservoir with 25% glycerol. The data were processed with the automated data-processing pipeline using Autoproc [2]. The structures were solved by molecular replacement with Phaser [3] in CCP4 using reported coordinates of the structures of metal-free HEWL (PDB code 193L) [4] from which solvent molecules had been removed, as search model. The structures were refined using Refmac5 [5] with manual editing in Coot [6]. Data collection and refinement statistics are reported in Table S1. Ru center positions have been identified analyzing Fourier difference and anomalous difference electron density maps. Metal and ligands occupancies were evaluated minimizing the positive and negative peaks on the metal center in the Fourier difference electron density maps. The quality of the final models was evaluated using the PDB validation server [7]. These data were made available to editors and peer reviewers during the review process. The results were visualized with PyMOL [8]. References [1] A. Lausi, M. Polentarutti, S. Onesti, J.R. Plaisier, E. Busetto, G. Bais, L. Barba, A. Cassetta, G. Campi, D. Lamba, A. Pifferi, S.C. Mande, D.D. Sarma, S.M. Sharma, G. Paolucci, Status of the crystallography beamlines at Elettra, Eur. Phys. J. Plus. 130 (2015) 43. https://doi.org/10.1140/epjp/i2015-15043-3. [2] C. Vonrhein, C. Flensburg, P. Keller, A. Sharff, O. Smart, W. Paciorek, T. Womack, G. Bricogne, Data processing and analysis with the ıt autoPROC toolbox, Acta Crystallographica Section D. 67 (2011) 293– 302. https://doi.org/10.1107/S0907444911007773. [3] A.J. McCoy, R.W. Grosse-Kunstleve, P.D. Adams, M.D. Winn, L.C. Storoni, R.J. Read, ıt Phaser crystallographic software, Journal of Applied Crystallography. 40 (2007) 658–674. https://doi.org/10.1107/S0021889807021206. [4] M.C. Vaney, S. Maignan, M. Riès-Kautt, A. Ducruix, High-Resolution Structure (1.33 Å) of a HEW Lysozyme Tetragonal Crystal Grown in the APCF Apparatus. Data and Structural Comparison with a Crystal Grown under Microgravity from SpaceHab-01 Mission, Acta Crystallogr. D. 52 (1996) 505–517. https://doi.org/10.1107/S090744499501674X. [5] G.N. Murshudov, A.A. Vagin, E.J. Dodson, Refinement of Macromolecular Structures by the Maximum- Likelihood Method, Acta Crystallographica Section D. 53 (1997) 240–255. https://doi.org/10.1107/S0907444996012255. [6] P. Emsley, B. Lohkamp, W.G. Scott, K. Cowtan, Features and development of ıt Coot, Acta Crystallographica Section D. 66 (2010) 486–501. https://doi.org/10.1107/S0907444910007493. [7] H. Berman, K. Henrick, H. Nakamura, Announcing the worldwide Protein Data Bank, Nature Structural & Molecular Biology. 10 (2003) 980–980. https://doi.org/10.1038/nsb1203-980. [8] The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC, (n.d.). 5 Figure S1. FT-IR spectra of K2[Ru2(DPhF)(CO3)3]·3H2O (upper) and [Ru2Cl(DPhF)2(O2CCH3)2]·H2O (lower). 6 Figure S2. The only two peaks found in the ESI mass spectrum of [Ru2Cl(DPhF)2(O2CCH3)2]·H2O acquired in methanol. Experimental (red and blue bars) and simulated (black lines) isotopic distribution. 7 Figure S3. The two peaks observed in the ESI mass spectrum of K2[Ru2(DPhF)(CO3)3]·3H2O acquired in water. Experimental (orange and green bars) and simulated (black lines) isotopic distribution. 8 Figure S4. Cyclic voltammogram for K2[Ru2(DPhF)(CO3)3]·3H2O. Experiments shown for 0.1 M KCl and scan rate = 100 mV/s. 9 Figure S5. Cyclic voltammogram for [Ru2Cl(DPhF)2(O2CCH3)2]·H2O. Experiments shown for 0.1 M KCl and scan rate = 100 mV/s. 10 Figure S6. Time course UV–vis spectra of a) [Ru2Cl(DPhF)(O2CCH3)3], b) K2[Ru2(DPhF)(CO3)3], and c) [Ru2Cl(DPhF)2(O2CCH3)2] in Milli-Q water at [C] = 500 μM. No appreciable spectral changes were observed within 2 weeks. 11 A B C D Figure S7. Time course UV–vis spectra of 50 μM [Ru2Cl(DPhF)(O2CCH3)3] in 0.8 M succinic acid pH 7.0 (A and B) and 2.0 M sodium formate, 0.1 M Hepes buffer pH 7.5 (C and D) in the absence (A and C) and presence (B and D) of HEWL. 12 A B C D Figure S8. Time course UV–vis spectra of 50 μM [Ru2Cl(DPhF)2(O2CCH3)2] in 0.8 M succinic acid pH 7.0 (A and B) and 2.0 M sodium formate, 0.1 M Hepes buffer pH 7.5 (C and D) in the absence (A and C) and presence (B and D) of HEWL. 13 A B C D Figure S9. Time course UV–vis spectra of 50 μM K2[Ru2(DPhF)(CO3)3] in 0.8 M succinic acid pH 7.0 (A and B) and 2.0 M sodium formate, 0.1 M Hepes buffer pH 7.5 (C and D) in the absence (A and C) and presence (B and D) of HEWL. 14 Figure S10. Fluorescence emission spectra of HEWL in (A-B) 10 mM succinic acid pH 7.0 and (C-D) 10 mM Hepes buffer pH 7.5 upon titration with a solution of [Ru2Cl(DPhF)(O2CCH3)3]. Spectra have been collected using λex = 280 nm (panels A, C) and λex =295 nm (panels B, D). A) [Ru2Cl(DPhF)(O2CCH3)3] under condition A B) [Ru2Cl(DPhF)(O2CCH3)3] under condition A C) [Ru2Cl(DPhF)(O2CCH3)3] under condition B D) [Ru2Cl(DPhF)(O2CCH3)3] under condition B 15 Figure S11. Fluorescence emission spectra of HEWL in (A-B) 10 mM succinic acid pH 7.0 and (C-D) 10 mM Hepes buffer pH 7.5 upon titration with a solution of [Ru2Cl(DPhF)2(O2CCH3)2]. Spectra have been collected using λex = 280 nm (panels A, C) and λex =295 nm (panels B, D). D) [Ru2Cl(DPhF)2(O2CCH3)2] under condition B C) [Ru2Cl(DPhF)2(O2CCH3)2] under condition B A) [Ru2Cl(DPhF)2(O2CCH3)2] under condition A B) [Ru2Cl(DPhF)2(O2CCH3)2] under condition A 16 Figure S12. Fluorescence emission spectra of HEWL in (A-B) 10 mM succinic acid pH 7.0 and (C-D) 10 mM Hepes buffer pH 7.5 upon titration with a solution of K2[Ru2(DPhF)(CO3)3]. Spectra have been collected using λex = 280 nm (panels A, C) and λex =295 nm (panels B, D). A) K2[Ru2(DPhF)(CO3)3] under condition A B) K2[Ru2(DPhF)(CO3)3] under condition A C) K2[Ru2(DPhF)(CO3)3] under condition B D) K2[Ru2(DPhF)(CO3)3] under condition B 17 Figure S13. Far-UV CD spectra of HEWL (7.0 μM concentration) incubated for 1 h in the presence of [Ru2Cl(DPhF)(O2CCH3)3] in (A) 10 mM succinic acid pH 7.0 and (B) 10 mM Hepes buffer pH 7.5 CD of metal-free protein is in black. A) [Ru2Cl(DPhF)(O2CCH3)3] under condition A B) [Ru2Cl(DPhF)(O2CCH3)3] under condition B 18 Figure S14. Far-UV CD spectra of HEWL (7.0 μM concentration) incubated for 1 h in the presence of [[Ru2Cl(DPhF)2(O2CCH3)2] in (A) 10 mM succinic acid pH 7.0 and (B) 10 mM Hepes buffer pH 7.5 CD of metal- free protein is in black. A) [Ru2Cl(DPhF)2(O2CCH3)2] under condition A B) [Ru2Cl(DPhF)2(O2CCH3)2] under condition B 19 Figure S15. Far-UV CD spectra of HEWL (7.0 μM concentration) incubated for 1 h in the presence of K2[Ru2(DPhF)(CO3)3] in (A) 10 mM succinic acid pH 7.0 and (B) 10 mM Hepes buffer pH 7.5 CD of metal-free protein is in black. A) K2[Ru2(DPhF)(CO3)3] under condition A B) K2[Ru2(DPhF)(CO3)3] under condition B 20 Figure S16. Effect of diruthenium complexes on cell viability after 48 h incubation. (A) Dose-response curve of HeLa incubated in the presence of increasing concentration (0.1 – 50 µM) of [Ru2Cl(DPhF)(O2CCH3)3] (black circles), [Ru2Cl(DPhF)2(O2CCH3)2] (black squares), and K2[Ru2(DPhF)(CO3)3] (black triangles); (B) HaCaT cells (black circles) and BALB/c-3T3 cells (black squares) incubated with increasing concentration (0.1 – 50 µM) of [Ru2Cl(DPhF)2(O2CCH3)2]. Cell viability was assessed by the MTT assay and expressed. Values are given as means ± SD (n ≥ 3). 21 Table S1. Data collection and refinement statistics. HEWL‒ [Ru2Cl(DPhF)2(O2CCH3)2] HEWL‒ K2[Ru2(DPhF)(CO3)3] HEWL‒ [Ru2Cl(DPhF)(O2CCH3)3] HEWL‒ [Ru2Cl(DPhF)2(O2CCH3)2] PDB code 8PH5 8PH6 8PH7 8PH8 Crystallization conditions 2.0 M sodium formate and 0.1 M Hepes buffer at pH 7.5 2.0 M sodium formate and 0.1 M Hepes buffer at pH 7.5 0.8 M succinic acid at pH 7.0 0.8 M succinic acid at pH 7.0 Soaking time 2 weeks 2 weeks 2 weeks 2 weeks Data collection Space group P42 12 P41 21 2 P42 12 P42 12 a (Å) 77.0 76.88 77.67 76.89 b (Å) 77.0 76.88 77.67 76.89 c (Å) 39.2 38.61 38.28 37.89 α/β/γ (°) 90.0/90.0/90.0 90.0/90.0/90.0 90.0/90.0/90.0 90.0/90.0/90.0 Molecules for asymmetric unit 1 1 1 1 Resolution range (Å) 39.20-1.29 (1.31-1.29) 54.36-1.07 (1.08-1.07) 54.92-1.41 (1.73-1.41) 54.37-1.29 (1.31-1.29) Observations 640879 (30686) 982587 (15986) 255213 (13505) 773576 (21894) Unique reflections 29909 (1489) 51851 (2477) 14789 (657) 37185 (1741) Completeness (%) 98.8 (99.5) 99.8 (97.0) 99.4 (98.4) 99.7 (95.6) Redundancy 21.4 (20.6) 19.0 (6.5) 19.3 (20.6) 20.8 (12.6) Rmerge (%) 0.094 (1.365) 0.038 (0.754) 0.161 (1.882) 0.074 (1.140) Average I/σ(I) 16.5 (2.4) 36.3 (2.3) 14.2 (2.0) 21.1 (2.2) CC1/2 0.999 (0.865) 1.000 (0.833) 0.999 (0.867) 0.999 (0.763) Anom. completeness (%) 98.8 (99.6) 99.6 (92.5) 99.5 (99.0) 99.7 (95.8) Anom. Multiplicity 11.5 (10.8) 10.0 (3.5) 10.6 (10.9) 11.0 (6.5) Refinement Resolution (Å) 1.29 1.07 1.41 1.29 N° reflections 28089 47360 14047 27741 N° reflections in working set 1882 2848 104 2035 Rfactor/Rfree 0.241/0.280 0.170/0.193 0.226/0.267 0.156/0.187 N° non-H atoms in the refin. 1253 1467 1217 1350 Ramachandran statistcs Most favoured 109 (96.46%) 83 (93.26%) 110 (94.83%) 107 (95.54%) Outliers 0 0 0 0 Rmsd bonds (Å) 0.013 0.012 0.009 0.013 Rmsd angles (°) 2.006 2.165 1.627 1.922 Portada Table of contents List of abbreviations Abstract Resumen Chapter I. Overview Chapter II. Aims and objectives Chapter III. Ultrasound-assisted synthesis of water-soluble monosubstituted diruthenium compounds Chapter IV. Effect of equatorial ligand substitution on the reactivity with proteins of paddlewheel diruthenium complexes: Structural Studies Chapter V. Charge effect in protein metalation reactions by diruthenium complexes Chapter VI. Steric hindrance and charge influence on the cytotoxic activity and protein binging properties of diruthenium complexes Chapter VII. Applications Chapter VIII. Discussion Conclusions Conclusiones References Annexes Button 1: