Molecular Self-Assembly of an Unusual Dinuclear Ruthenium(III) Complex Based on the Nucleobase Guanine

The study of crystal structures based on complexes containing purine nucleobases is a significant research subject, mainly regarding the diagnosis and treatment of some diseases and the investigation of genetic mutations and biochemical structures in life sciences. We have obtained and characterized a new dinuclear ruthenium(III) complex based on guanine with the formula [{Ru(μ-Cl)(μ-gua)}2Cl4]·2H2O (1) (gua = guanine). 1 was characterized by means of Fourier transform infrared spectroscopy (FT–IR), scanning electron microscopy and energy dispersive X-ray analysis (SEM–EDX), single-crystal X-ray diffraction (XRD), Hirshfeld surface analysis and cyclic voltammetry (CV). The study of its electrochemical properties allowed us to investigate the presence of guanine molecules when linked to the ruthenium(III) ion in 1. The well-resolved voltammetric response together with the reliability and stability achieved through 1 could provide a step forward to developing new ruthenium-based platforms, devices and modified electrodes adequate to study this purine nucleobase.


Introduction
Guanine is one of the main nitrogenous bases found in the nucleotides of the nucleic acids (DNA and RNA) present in the cells of living organisms and viruses. Besides being involved in the storage and the expression of genetic information, it participates in many other cellular biochemical processes and structures [1][2][3]. Given that this purine nucleobase can be present in the fluids of human beings, their levels have been proposed as direct and indirect biomarkers for a variety of diseases and pathologies, such as Alzheimer's disease [4], epilepsy [5] and human immunodeficiency virus (HIV) infection [6,7]. Hence, sensitive and selective methods for determining both this and other purine nucleobases are being investigated in several research areas [8,9].
1 is the first dinuclear ruthenium(III) compound based on guanine which has been reported so far. Scheme 1. Molecular structure of guanine.

Reagents and Instruments
Guanine (99.90%) was purchased from Acros Organics and the ruthenium precursor K2[RuCl5(H2O)] (99.95%) was acquired from Alfa Aesar. The elemental analyses (C, H, N) and X-ray microanalysis were performed through Central Service for the Support to Experimental Research (SCSIE) at the University of Valencia. Images and results of scanning electron microscopy (SEM-EDX) were obtained through a Hitachi S-4800 field emission scanning electron microscope, with 20 kV and 9.0 mm of accelerating voltage and working distance, respectively. Each infrared spectrum (FT-IR) was performed on KBr pellets and was obtained through a PerkinElmer Spectrum 65 FT-IR spectrometer in the 4000-500 range (cm −1 ) with 25 scans and a spectral resolution of 4 cm −1 . The study of the electrochemical properties was carried out on 1 by means of an Autolab/PGSTAT 204 scanning potentiostat at a scan rate working in the 10-250 range (mVs −1 ) and with Metrohm electrodes. CV curves were obtained with a 0.1 M (NBu4)[PF6] solution, as supporting electrolyte, and a 10 −3 M solution of 1 in dry N,N'-dimethylformamide (DMF). A glassy carbon disk of 0.32 cm 2 was used as working electrode, polished with 1.0 µm diamond powder, which was sonicated and then washed with absolute ethanol and acetone, and air-dried. The AgCl/Ag reference electrode was separated from the test solution through a salt bridge with the solvent and supporting electrolyte. The platinum electrode was auxiliary. The study was performed in a standard electrochemical cell at 20 °C with flowing argon. The studied potential range was from −1.5 to +1.5 V vs. AgCl/Ag. Ferrocene (Fc) was added as an internal standard at the end of the measurements. (1) K2[RuCl5(H2O)] (11.2 mg, 0.03 mmol) and guanine (6.80 mg, 0.03 mmol) were reacted through a solvothermal synthesis in HCl (4 mL, 3.0 M) at 90 °C for three days, then a 12 h cooling process took place to room temperature. Dark brown parallelepipeds of 1 were obtained. Yield: ca. 53%. Anal. Calcd. for C10H10Cl6N10O2Ru2 (1)

X-ray Diffraction Data Collection and Structure Refinement
Data collection from a single crystal of 1 (with dimensions of 0.46 × 0.13 × 0.09 mm 3 ) was performed on a Bruker D8 Venture diffractometer with graphite-monochromated Mo-Kα radiation (λ = 0.71073 Å). Table 1 summarizes the crystal parameters and refinement results for 1. The structure of 1 was solved by standard direct methods and then completed by Fourier recycling through the SHELXTL program. The model was refined

Reagents and Instruments
Guanine (99.90%) was purchased from Acros Organics and the ruthenium precursor K 2 [RuCl 5 (H 2 O)] (99.95%) was acquired from Alfa Aesar. The elemental analyses (C, H, N) and X-ray microanalysis were performed through Central Service for the Support to Experimental Research (SCSIE) at the University of Valencia. Images and results of scanning electron microscopy (SEM-EDX) were obtained through a Hitachi S-4800 field emission scanning electron microscope, with 20 kV and 9.0 mm of accelerating voltage and working distance, respectively. Each infrared spectrum (FT-IR) was performed on KBr pellets and was obtained through a PerkinElmer Spectrum 65 FT-IR spectrometer in the 4000-500 range (cm −1 ) with 25 scans and a spectral resolution of 4 cm −1 . The study of the electrochemical properties was carried out on 1 by means of an Autolab/PGSTAT 204 scanning potentiostat at a scan rate working in the 10-250 range (mVs −1 ) and with Metrohm electrodes. CV curves were obtained with a 0.1 M (NBu 4 )[PF 6 ] solution, as supporting electrolyte, and a 10 −3 M solution of 1 in dry N,N'-dimethylformamide (DMF). A glassy carbon disk of 0.32 cm 2 was used as working electrode, polished with 1.0 µm diamond powder, which was sonicated and then washed with absolute ethanol and acetone, and air-dried. The AgCl/Ag reference electrode was separated from the test solution through a salt bridge with the solvent and supporting electrolyte. The platinum electrode was auxiliary. The study was performed in a standard electrochemical cell at 20 • C with flowing argon. The studied potential range was from −1.5 to +1.5 V vs. AgCl/Ag. Ferrocene (Fc) was added as an internal standard at the end of the measurements.

X-ray Diffraction Data Collection and Structure Refinement
Data collection from a single crystal of 1 (with dimensions of 0.46 × 0.13 × 0.09 mm 3 ) was performed on a Bruker D8 Venture diffractometer with graphite-monochromated Mo-K α radiation (λ = 0.71073 Å). Table 1 summarizes the crystal parameters and refinement results for 1. The structure of 1 was solved by standard direct methods and then completed by Fourier recycling through the SHELXTL program. The model was refined with version 2018/1 of SHELXL against F 2 on all data by full-matrix least-squares [29]. The non-hydrogen atoms were anisotropically refined, and the H atoms of the guanine molecules were set in calculated positions and refined isotropically. The H atoms of the water molecules in 1 were neither detected nor included in the model, this fact being due to the thermal disorder observed on the water molecules, which increases the R 1 and wR 2 parameters in this system ( Table 1). The graphic manipulations were performed with the DIAMOND program [30] of CCDC 2081706. Table 1.
Summary of the crystal data and structure refinement parameters for 1 (see Supplementary Material).

Synthetic Procedure
The ruthenium(III) precursor K 2 [RuCl 5 (H 2 O)] was made to react with guanine in hydrochloric acid (3.0 M) solutions, thus we prepared a new purine-based ruthenium(III) complex of the formula [{Ru(µ-Cl)(µ-gua)} 2 Cl 4 ]·2H 2 O (1) (Figure 1). The synthetic procedure was carried out by means of heating this mixture at 90 • C in a solvothermal reaction and, in order to crystallize, the process continued for a further 12 h cooling step to room temperature. In this way, dark brown crystals of 1 were obtained with a satisfactory yield (53%). It is worth mentioning that compound 1 was also obtained by replacing hydrochloric acid with acetic acid, but the yield was too low (less than 10%). These results support the fact that this is an adequate synthesis to prepare purine-based dinuclear ruthenium(III) complexes.

Infrared Spectroscopy
The infrared (IR) spectra of 1 and that of the free guanine ligand are given in Figure 2. The IR spectrum of guanine has been previously studied [31,32], so that it has been added in this work only for comparison. In general, the vibrational bands mainly associated with N-H symmetric (ν s ) and asymmetric (ν as ) stretching for the free guanine molecule were more intense and complex than those of 1 in the ca. 3800-2000 cm −1 region (Figure 2), which would be due to a more ordered hydrogen-bonding network in the crystalline solid for the free nucleobase [31,32]. In the case of the IR spectrum of 1, the values associated to the vibrational ν as (NH 2 ) (3325 cm −1 ) and ν s (NH 2 ) (3114 and 3062 cm −1 ) bands were very similar to those obtained for the free guanine [31,32] (Figure 2). In the 1800-500 cm −1 region, the most interesting features were the two strong vibrational bands associated to the stretching ν(C=N) and ν(C=C) and bending δ(NH 2 ), mainly scissoring, which were found at 1695 and 1672 cm −1 for the guanine molecule [31,32]. In the IR spectrum of 1, these two vibrational bands were shifted to 1708 and 1635 cm −1 , respectively, with this fact indicating the coordination of the Ru(III) metal ions to guanine molecules in compound 1 (Figure 2).

Infrared Spectroscopy
The infrared (IR) spectra of 1 and that of the free guanine ligand are given in Figure  2. The IR spectrum of guanine has been previously studied [31,32], so that it has been added in this work only for comparison. In general, the vibrational bands mainly associated with N-H symmetric (νs) and asymmetric (νas) stretching for the free guanine molecule were more intense and complex than those of 1 in the ca. 3800-2000 cm −1 region (Figure 2), which would be due to a more ordered hydrogen-bonding network in the crystalline solid for the free nucleobase [31,32]. In the case of the IR spectrum of 1, the values associated to the vibrational νas(NH2) (3325 cm −1 ) and νs(NH2) (3114 and 3062 cm −1 ) bands were very similar to those obtained for the free guanine [31,32] (Figure 2). In the 1800-500 cm −1 region, the most interesting features were the two strong vibrational bands associated to the stretching ν(C=N) and ν(C=C) and bending δ(NH2), mainly scissoring, which were found at 1695 and 1672 cm −1 for the guanine molecule [31,32]. In the IR spectrum of 1, these two vibrational bands were shifted to 1708 and 1635 cm −1 , respectively, with this fact indicating the coordination of the Ru(III) metal ions to guanine molecules in compound 1 (Figure 2).

Description of the Crystal Structure
The crystal structure of 1 was obtained by single-crystal X-ray diffraction. A CSD survey revealed that 1 displays the first reported crystal structure based on a dinuclear Ru(III) compound containing guanine. Compound 1 crystallizes in the monoclinic system with space group C2/c ( Table 1). The crystal structure of 1 is made up of neutral [{Ru(µ-Cl)(µ-gua)}2Cl4] units and H2O molecules. The asymmetric unit of 1 consists of half a [{Ru(µ-Cl)(µ-gua)}2Cl4] complex and one H2O molecule (Figure 1).
In this dinuclear complex, each Ru(III) ion is linked to four chloride ions and two nitrogen atoms (these are N3 and N9) from two guanine molecules in an almost regular Oh geometry. The two Ru(III) ions are connected to each other through two guanine molecules and two chloro-bridges (Figure 1). A very short intramolecular Ru···Ru distance [Ru (1)

Description of the Crystal Structure
The crystal structure of 1 was obtained by single-crystal X-ray diffraction. A CSD survey revealed that 1 displays the first reported crystal structure based on a dinuclear Ru(III) compound containing guanine. Compound 1 crystallizes in the monoclinic system with space group C2/c ( Table 1) In this dinuclear complex, each Ru(III) ion is linked to four chloride ions and two nitrogen atoms (these are N3 and N9) from two guanine molecules in an almost regular Oh geometry. The two Ru(III) ions are connected to each other through two guanine molecules and two chloro-bridges (Figure 1). A very short intramolecular Ru···Ru distance [Ru (1)

Hirshfeld Surface Analysis
The intermolecular interactions of the neutral [{Ru(µ-Cl)(µ-gua)} 2 Cl 4 ] complex were further studied through the CrystalExplorer program [33,34]. This program calculated the surfaces which allowed us the qualitative and quantitative investigation as well as the visualization of the main intermolecular contacts in 1 by mapping the distances from the surface to the nearest atom outside (d e ) and inside (d i ) this surface. Besides, a normalized contact distance called d norm was also taken into account to overcome some limitations generated by the atom size [33,34]. The Hirshfeld surfaces for complex 1 are given in Figure 5, the shorter contacts being shown with red color [34]. The intermolecular H···O contacts generated among the N-H and carbonyl groups of adjacent guanine molecules are approximately 16% of the complete fingerprint plot ( Figure 5). The Cl···H contacts involving Cl − anions and N-H groups of neighboring dinuclear [{Ru(µ-Cl)(µ-gua)} 2 Cl 4 ] units are the main interactions observed on the Hirshfeld surface, which covers ca. 24% ( Figure 5). Finally, further O···H contacts involving solvent water molecules and N-H groups of the guanine molecules are close to the 6% of the fingerprint plot ( Figure 5).

Hirshfeld Surface Analysis
The intermolecular interactions of the neutral [{Ru(µ-Cl)(µ-gua)}2Cl4] compl further studied through the CrystalExplorer program [33,34]. This program ca the surfaces which allowed us the qualitative and quantitative investigation as we visualization of the main intermolecular contacts in 1 by mapping the distances f surface to the nearest atom outside (de) and inside (di) this surface. Besides, a nor contact distance called dnorm was also taken into account to overcome some lim generated by the atom size [33,34]. The Hirshfeld surfaces for complex 1 are g Figure 5, the shorter contacts being shown with red color [34]. The intermolecul contacts generated among the N-H and carbonyl groups of adjacent guanine m are approximately 16% of the complete fingerprint plot ( Figure 5). The Cl···H c involving Cl − anions and N-H groups of neighboring dinuclear [{Ru(µ-Cl)(µ-gu

Scanning Electron Microscopy-Energy Dispersive X-ray Analysis
Compound 1 was studied by means of scanning electron microscopy and e dispersive X-ray analysis (SEM-EDX), these analyses being carried out as prev performed for other ruthenium systems [35,36]. The results of the microanalysis Ru/Cl molar ratio of 1:3 for 1. A recorded image of 1 is given in Figure 6. Crystals o shown as crystallized parallelepipeds in Figure 6.

Scanning Electron Microscopy-Energy Dispersive X-ray Analysis
Compound 1 was studied by means of scanning electron microscopy and energy dispersive X-ray analysis (SEM-EDX), these analyses being carried out as previously performed for other ruthenium systems [35,36]. The results of the microanalysis gave a Ru/Cl molar ratio of 1:3 for 1. A recorded image of 1 is given in Figure 6. Crystals of 1 are shown as crystallized parallelepipeds in Figure 6.

Scanning Electron Microscopy-Energy Dispersive X-ray Analysis
Compound 1 was studied by means of scanning electron microscopy and energy dispersive X-ray analysis (SEM-EDX), these analyses being carried out as previously performed for other ruthenium systems [35,36]. The results of the microanalysis gave a Ru/Cl molar ratio of 1:3 for 1. A recorded image of 1 is given in Figure 6. Crystals of 1 are shown as crystallized parallelepipeds in Figure 6.   Figure 7.
Dinuclear ruthenium compounds containing metal-metal bonds have been studied for many years [37]. They show redox properties that are strongly dependent upon the solvent and the supporting electrolyte and are generally well known [38][39][40]. So, this type of Ru-Ru compound could be an acceptable reference in electrochemical studies [38][39][40]. In the CV curve of 1, three reduction processes can be observed (Figure 7). The first two reduction peaks were found between 0.0 V and −0.50 V, which would be associated with the formation of the mixed-valent Ru(II)-Ru(III) species (at −0.30 V) and also to that of the Ru(II)-Ru(II) system (at −0.50 V). These reduction potential values are close to those published for other dinuclear ruthenium complexes [38][39][40]. Nevertheless, much more interesting would be the third detected reduction peak at −0.89 V, which would be generated by the influence of the guanine molecule, see inset in Figure 7. It is worth mentioning that this value assigned to guanine is in agreement with those previously reported for this purine nucleobase in electrochemistry research works performed through modified electrodes based on polyaniline-MnO2 [41] and graphite-WS2 [42], which have shown high accuracy and promising redox activity toward purine nucleobases [41,42]. In order to analyze the repeatability of the CV curve, it was measured five times. Indeed, a relative standard deviation value of ca. 1.2% for the current response was obtained for 1. In any case, these results could establish a first step to develop new sensor devices suitable for the detection of purine nucleobases as guanine [25]. Nevertheless, this is an early stage of the research and, therefore, this type of diruthenium(III) systems must be further investigated. The comparison with other methods together with the study of the nature of the samples to be measured will be addressed subsequently in future works [25].

Conclusions
An unusual guanine-based dinuclear ruthenium(III) complex, of the formula [{Ru(µ-Cl)(µ-gua)}2Cl4]·2H2O (1) (gua = guanine), was prepared and characterized. Compound 1 is the first dinuclear ruthenium(III) compound based on guanine reported so far. Compound 1 was characterized by FT-IR, SEM-EDX, single-crystal X-ray diffraction (XRD), Hirshfeld surface analysis and cyclic voltammetry (CV). The study of its electrochemical properties revealed well-resolved, potentially useful, current peaks, which allowed us to investigate the guanine when linked to the ruthenium ion in 1. Dinuclear ruthenium compounds containing metal-metal bonds have been studied for many years [37]. They show redox properties that are strongly dependent upon the solvent and the supporting electrolyte and are generally well known [38][39][40]. So, this type of Ru-Ru compound could be an acceptable reference in electrochemical studies [38][39][40].
In the CV curve of 1, three reduction processes can be observed (Figure 7). The first two reduction peaks were found between 0.0 V and −0.50 V, which would be associated with the formation of the mixed-valent Ru(II)-Ru(III) species (at −0.30 V) and also to that of the Ru(II)-Ru(II) system (at −0.50 V). These reduction potential values are close to those published for other dinuclear ruthenium complexes [38][39][40]. Nevertheless, much more interesting would be the third detected reduction peak at −0.89 V, which would be generated by the influence of the guanine molecule, see inset in Figure 7. It is worth mentioning that this value assigned to guanine is in agreement with those previously reported for this purine nucleobase in electrochemistry research works performed through modified electrodes based on polyaniline-MnO 2 [41] and graphite-WS 2 [42], which have shown high accuracy and promising redox activity toward purine nucleobases [41,42]. In order to analyze the repeatability of the CV curve, it was measured five times. Indeed, a relative standard deviation value of ca. 1.2% for the current response was obtained for 1. In any case, these results could establish a first step to develop new sensor devices suitable for the detection of purine nucleobases as guanine [25]. Nevertheless, this is an early stage of the research and, therefore, this type of diruthenium(III) systems must be further investigated. The comparison with other methods together with the study of the nature of the samples to be measured will be addressed subsequently in future works [25].

Conclusions
An unusual guanine-based dinuclear ruthenium(III) complex, of the formula [{Ru(µ-Cl)(µ-gua)} 2 Cl 4 ]·2H 2 O (1) (gua = guanine), was prepared and characterized. Compound 1 is the first dinuclear ruthenium(III) compound based on guanine reported so far. Compound 1 was characterized by FT-IR, SEM-EDX, single-crystal X-ray diffraction (XRD), Hirshfeld surface analysis and cyclic voltammetry (CV). The study of its electrochemical properties revealed well-resolved, potentially useful, current peaks, which allowed us to investigate the guanine when linked to the ruthenium ion in 1.

Data Availability Statement:
The raw data that support the findings of this study are available from the corresponding author upon reasonable request.