Prototypical cis-ruthenium(II) complexes present differential fluorescent staining in walled-cell models (yeasts)

  • Alexander CarreñoEmail author
  • Dayán Páez-HernándezEmail author
  • César Zúñiga
  • Angélica Ramírez-Osorio
  • Jan Nevermann
  • María Macarena Rivera-Zaldívar
  • Carolina Otero
  • Juan A. FuentesEmail author
Original Paper


cis-Ru(deeb) 3 2+ (R1; where deeb is 4,4′-diethanoate-2,2′-bipyridine) and cis-Ru(phen) 3 2+ (R2; where phen is 1,10-phenanthroline) were synthesized. Although the presence of the cell wall (a structure that is present in yeasts and bacteria,) was previously described as a natural barrier that hampers the uptake of d6-based luminescent complexes, we previously demonstrated that rhenium(I) tricarbonyl complexes were useful to stain both yeasts and bacteria. Even though several studies of classical ruthenium(II) complexes can be found, none of those studies aimed to determine the potential of these compounds as biomarkers for walled cells, testing only cell lines that lack this permeability barrier. Walled cells exhibit a relatively rigid structure, mainly constituted by carbohydrates and proteins, and surround the plasma membrane. In this manuscript, we observed that both R1 and R2 exhibited very low cytotoxicity in different walled-cell models (including bacteria and yeasts). More importantly, we found that both R1 and R2 were able to fluorescently stain Candida albicans (yeast), with a simple and fast procedure, without the need of additional permeabilizer molecules and antibodies. Interestingly, R1 remained retained in a discrete central structure consistent with the cell nucleus, whereas R2 seemed to be accumulated in the cell wall. These results show that these two complexes can be used as biomarkers for walled cells as differential staining, supporting the fact that, as well as with rhenium(I) complexes, biomarkers properties can be modulated by changing the substituents in ruthenium(II)-derivative luminescent stains, even for walled cells.


Ruthenium(II) complexes Spin–orbit DFT Cytotoxicity Biomarkers Yeasts 



We thank FONDECYT 11170637, 1181638, and 1180017. We thank Dr, Ramiro Arratia-Pérez (Center of Applied Nanoscience, Universidad Andres Bello) for the Computational Resources Facilities and Dr. Ivonne Chávez M (Departamento de Química Inorgánica, Pontificia Universidad Católica de Chile) for instrumental facilities.

Compliance with ethical standards

Conflicts of interest

The authors declare no conflicts of interest.


This manuscript is dedicated to Professor Dr. Ramiro Arratia-Pérez (UNAB, director of Center of Applied Nanoscience, Chile), for his relevant contributions to the relativistic quantum chemistry in Chile, where he founded the first Molecular Relativistic School in Latin America, and in the world.

Supplementary material

11696_2019_714_MOESM1_ESM.docx (421 kb)
Supplementary material 1 (DOCX 421 kb)


  1. Al-Noaimi M, AlDamen MA (2012) Ruthenium complexes incorporating azoimine and α-diamine based ligands: synthesis, crystal structure, electrochemistry and DFT calculation. Inorg Chim Acta 387:45–51. CrossRefGoogle Scholar
  2. Amoroso AJ et al (2007) Rhenium fac tricarbonyl bisimine complexes: biologically useful fluorochromes for cell imaging applications. Chem Commun. Google Scholar
  3. Amoroso AJ et al (2008) 3-Chloromethylpyridyl bipyridine fac-tricarbonyl rhenium: a thiol-reactive luminophore for fluorescence microscopy accumulates in mitochondria. New J Chem 32:1097–1102. CrossRefGoogle Scholar
  4. Ardo S, Sun Y, Staniszewski A, Castellano FN, Meyer GJ (2010) Stark effects after excited-state interfacial electron transfer at sensitized TiO(2) nanocrystallites. J Am Chem Soc 132:6696–6709. CrossRefGoogle Scholar
  5. Averkiev BB, Dreger ZA, Chaudhuri S (2014) Density functional theory calculations of pressure effects on the structure and vibrations of 1,1-diamino-2,2-dinitroethene (FOX-7). J Phys Chem A 118:10002–10010. CrossRefGoogle Scholar
  6. Biancalana L, Pampaloni G, Marchetti F (2017) Arene ruthenium(II) complexes with phosphorous ligands as possible anticancer agents. Chimia 71:573–579. CrossRefGoogle Scholar
  7. Bignozzi CA, Chiorboli C, Murtaza Z, Jones WE, Meyer TJ (1993) Photophysical and photochemical behavior of nitro complexes of ruthenium(Ii). Inorg Chem 32:1036–1038. CrossRefGoogle Scholar
  8. Bomben PG, Koivisto BD, Berlinguette CP (2010) Cyclometalated Ru complexes of type [Ru(II)(N–N)(2)(C–N)](z): physicochemical response to substituents installed on the anionic ligand. Inorg Chem 49:4960–4971. CrossRefGoogle Scholar
  9. Bonhote P, Moser JE, Humphry-Baker R, Vlachopoulos N, Zakeeruddin SM, Walder L, Gratzel M (1999) Long-lived photoinduced charge separation and redox-type photochromism on mesoporous oxide films sensitized by molecular dyads. J Am Chem Soc 121:1324–1336. CrossRefGoogle Scholar
  10. Byrne A, Burke CS, Keyes TE (2016) Precision targeted ruthenium(ii) luminophores; highly effective probes for cell imaging by stimulated emission depletion (STED) microscopy. Chem Sci 7:6551–6562. CrossRefGoogle Scholar
  11. Caramori S, Cristino V, Argazzi R, Meda L, Bignozzi CA (2010) Photoelectrochemical behavior of sensitized TiO(2) photoanodes in an aqueous environment: application to hydrogen production. Inorg Chem 49:3320–3328. CrossRefGoogle Scholar
  12. Carreno A et al (2016a) Theoretical and experimental characterization of a novel pyridine benzimidazole: suitability for fluorescence staining in cells and antimicrobial properties. New J Chem 40:2362–2375. CrossRefGoogle Scholar
  13. Carreno A et al (2016b) Fluorescence probes for prokaryotic and eukaryotic cells using Re(CO)(3)(+) complexes with an electron withdrawing ancillary ligand. New J Chem 40:7687–7700. CrossRefGoogle Scholar
  14. Carreno A et al (2019) Cyclic voltammetry, relativistic DFT calculations and biological test of cytotoxicity in walled-cell models of two classical rhenium (I) tricarbonyl complexes with 5-amine-1,10-phenanthroline. Chem Phys Lett 715:231–238. CrossRefGoogle Scholar
  15. Carreño A, Aros AE, Otero C, Polanco R, Gacitúa M, Arratia-Pérez R, Fuentes JA (2017a) Substituted bidentate and ancillary ligands modulate the bioimaging properties of the classical Re(i) tricarbonyl core with yeasts and bacteria. New J Chem 41:2140–2147. CrossRefGoogle Scholar
  16. Carreño A, Gacitúa M, Molins E, Arratia-Pérez R (2017b) X-ray diffraction and relativistic DFT studies on the molecular biomarker fac-Re(CO)3(4,4′-dimethyl-2,2′-bpy)(E-2-((3-amino-pyridin-4-ylimino)-methyl)-4,6-di-tert-butylphenol)(PF6). Chem Pap 71:2011–2022. CrossRefGoogle Scholar
  17. Casida M (2009) Time-dependent density-functional theory for molecules and molecular solids. J Mol Struct (Thoechem) 914:3–18. CrossRefGoogle Scholar
  18. Caveney NA, Li FK, Strynadka NC (2018) Enzyme structures of the bacterial peptidoglycan and wall teichoic acid biogenesis pathways. Curr Opin Struct Biol 53:45–58. CrossRefGoogle Scholar
  19. Chaves GM, da Silva WP (2012) Superoxide dismutases and glutaredoxins have a distinct role in the response of Candida albicans to oxidative stress generated by the chemical compounds menadione and diamide. Mem I Oswaldo Cruz 107:998–1005. CrossRefGoogle Scholar
  20. Cuenca-Estrella M et al (2003) Multicenter evaluation of the reproducibility of the proposed antifungal susceptibility testing method for fermentative yeasts of the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antimicrobial Susceptibility Testing (AFST-EUCAST). Clin Microbiol Infect 9:467–474CrossRefGoogle Scholar
  21. de Menorval MA, Mir LM, Fernandez ML, Reigada R (2012) Effects of dimethyl sulfoxide in cholesterol-containing lipid membranes: a comparative study of experiments in silico and with cells. PLoS ONE 7:e41733. CrossRefGoogle Scholar
  22. Donnici CL et al (1998) Synthesis of the novel 4,4′- and 6,6′-dihydroxamic-2,2′-bipyridines and improved routes to 4,4′- and 6,6′- substituted 2,2′-bipyridines and mono-N-oxide-2,2′-bipyridine. J Braz Chem Soc 9:1. CrossRefGoogle Scholar
  23. Dreyse P, Loeb B, Soto-Arriaza M, Tordera D, Orti E, Serrano-Perez JJ, Bolink HJ (2013) Effect of free rotation in polypyridinic ligands of Ru(II) complexes applied in light-emitting electrochemical cells. Dalton Trans 42:15502–15513. CrossRefGoogle Scholar
  24. Fantacci S, De Angelis F, Sgamellotti A, Marrone A, Re N (2005) Photophysical properties of [Ru(phen)2(dppz)]2+ intercalated into DNA: an integrated Car-Parrinello and TDDFT study. J Am Chem Soc 127:14144–14145. CrossRefGoogle Scholar
  25. Farnum BH, Jou JJ, Meyer GJ (2012) Visible light generation of I-I bonds by Ru-tris(diimine) excited states. Proc Natl Acad Sci USA 109:15628–15633. CrossRefGoogle Scholar
  26. Favereau L et al (2016) A molecular tetrad that generates a high-energy charge-separated state by mimicking the photosynthetic Z-scheme. J Am Chem Soc 138:3752–3760. CrossRefGoogle Scholar
  27. Franco de Carvalho F, Pignedoli CA, Tavernelli I (2017) TDDFT-based spin-orbit couplings of 0D, 1D, and 2D carbon nanostructures: static and dynamical effects. J Phys Chem C 121:10140–10152. CrossRefGoogle Scholar
  28. Gajardo F, Barrera M, Vargas R, Crivelli I, Loeb B (2011) Influence of the nature of the absorption band on the potential performance of high molar extinction coefficient ruthenium(II) polypyridinic complexes as dyes for sensitized solar cells. Inorg Chem 50:5910–5924. CrossRefGoogle Scholar
  29. Gow NAR, Latge JP, Munro CA (2017) The fungal cell wall: structure, biosynthesis, and function. Microbiol Spectr. Google Scholar
  30. Grotjahn R, Maier TM, Michl J, Kaupp M (2017) Development of a TDDFT-based protocol with local hybrid functionals for the screening of potential singlet fission chromophores. J Chem Theory Comput 13:4984–4996. CrossRefGoogle Scholar
  31. Juris A, Balzani V, Barigelletti F, Campagna S, Belser P, von Zelewsky A (1988) Ru(II) polypyridine complexes: photophysics, photochemistry, eletrochemistry, and chemiluminescence. Coordin Chem Rev 84:85–277. CrossRefGoogle Scholar
  32. Katsumata K, Matsui H, Yamaguchi T, Tanabe N (2017) 6-(2-Quinolinyl)-2,2′-bipyridine ruthenium complexes for near-infrared sensitization in dye-sensitized solar cells. Inorg Chim Acta 463:118–125. CrossRefGoogle Scholar
  33. Kendall K, Roberts AD (2015) van der Waals forces influencing adhesion of cells Philosophical transactions of the Royal Society of London Series B. Biol Sci 370:20140078. CrossRefGoogle Scholar
  34. Koyyada G et al (2016) New terpyridine-based ruthenium complexes for dye sensitized solar cells applications. Inorg Chim Acta 442:158–166. CrossRefGoogle Scholar
  35. León-García MC, Ríos-Castro E, López-Romero E, Cuéllar-Cruz M (2017) Evaluation of cell wall damage by dimethyl sulfoxide in Candida species. Res Microbiol 168:732–739. CrossRefGoogle Scholar
  36. Li HF, Zhang LS, Lin H, Fan XL (2014) A DFT-D study on the electronic and photophysical properties of ruthenium(II) complex with a chelating sulfoxide group. Chem Phys Lett 604:10–14. CrossRefGoogle Scholar
  37. Li X, Gorle AK, Ainsworth TD, Heimann K, Woodward CE, Collins JG, Keene FR (2015) RNA and DNA binding of inert oligonuclear ruthenium(II) complexes in live eukaryotic cells. Dalton Trans 44:3594–3603. CrossRefGoogle Scholar
  38. Macleod-Carey D, Caramori GF, Guajardo-Maturana R, Paez-Hernandez D, Muñoz-Castro A, Arratia-Perez R (2018) Advances in bonding and properties of inorganic systems from relativistic calculations in Latin. Am Int J Quant Chem. Google Scholar
  39. Micciarelli M, Curchod BFE, Bonella S, Altucci C, Valadan M, Rothlisberger U, Tavernelli I (2017) Characterization of the photochemical properties of 5-benzyluracil via time-dependent density functional theory. J Phys Chem A 121:3909–3917. CrossRefGoogle Scholar
  40. Muñoz-Castro A, Páez-Hernández D, Arratia-Pérez R (2017) Spin-orbit effect into isomerization barrier of small gold Clusters. Oh ↔ D2h Fluxionality of the Au6 2+ cluster Investigated by relativistic methods. Chem Phys Lett 683:404–407. CrossRefGoogle Scholar
  41. Nazeeruddin MK et al (2001) Engineering of efficient panchromatic sensitizers for nanocrystalline TiO2-based solar cells. J Am Chem Soc 123:1613–1624. CrossRefGoogle Scholar
  42. Nir S, Andersen M (1977) Van der Waals interactions between cell surfaces. J Membr Biol 31:1–18CrossRefGoogle Scholar
  43. Oßwald S, Breimaier S, Linseis M, Winter RF (2017) Polyelectrochromic vinyl ruthenium-modified tritylium dyes. Organometallics 36:1993–2003. CrossRefGoogle Scholar
  44. Páez-Hernández D, Murillo-López JA, Arratia-Pérez R (2012) Optical and magnetic properties of the complex bis(dicyclooctatetraenyl)diuranium. A Theoretical View Organometallics 31:6297–6304. CrossRefGoogle Scholar
  45. Poynton FE, Bright SA, Blasco S, Williams DC, Kelly JM, Gunnlaugsson T (2017) The development of ruthenium(ii) polypyridyl complexes and conjugates for in vitro cellular and in vivo applications. Chem Soc Rev 46:7706–7756. CrossRefGoogle Scholar
  46. Rojas-Poblete M, Carreño A, Gacitúa M, Páez-Hernández D, Rabanal-Leon WA, Arratia-Pérez R (2018) Electrochemical behaviors and relativistic DFT calculations to understand the terminal ligand influence on the [Re63-Q)8X6]4− clusters. New J Chem 42:5471–5478. CrossRefGoogle Scholar
  47. Rowe RK, Ho PS (2017) Relationships between hydrogen bonds and halogen bonds in biological systems. Acta Crystallogr B Struct Sci Cryst Eng Mater 73:255–264. CrossRefGoogle Scholar
  48. Sacksteder L, Zipp AP, Brown EA, Streich J, Demas JN, Degraff BA (1990) Luminescence studies of pyridine alpha-diimine rhenium(I) tricarbonyl complexes. Inorg Chem 29:4335–4340. CrossRefGoogle Scholar
  49. Sampaio RN, Muller AV, Polo AS, Meyer GJ (2017) Correlation between charge recombination and lateral hole-hopping kinetics in a series of cis-Ru(phen’)(dcb)(NCS)2 dye-sensitized solar cells ACS. Appl Mater Interfaces 9:33446–33454. CrossRefGoogle Scholar
  50. Sangilipandi S, Sutradhar D, Bhattacharjee K, Kaminsky W, Joshi SR, Chandra AK, Mohan Rao K (2016) Synthesis, structure, antibacterial studies and DFT calculations of arene ruthenium, Cp∗Rh, Cp∗Ir and tricarbonylrhenium metal complexes containing 2-chloro-3-(3-(2-pyridyl)pyrazolyl)quinoxaline ligand. Inorg Chim Acta 441:95–108. CrossRefGoogle Scholar
  51. Sanz AB, Garcia R, Rodriguez-Pena JM, Arroyo J (2017) The CWI Pathway: regulation of the transcriptional adaptive response to cell wall stress in yeast. J Fungi (Basel). Google Scholar
  52. Sauvage JP et al (1994) Ruthenium(II) and osmium(II) bis(terpyridine) complexes in covalently-linked multicomponent systems—synthesis, electrochemical-behavior, absorption-spectra, and photochemical and photophysical properties. Chem Rev 94:993–1019. CrossRefGoogle Scholar
  53. Schatzschneider U (2018) Metallointercalators and metalloinsertors: structural requirements for DNA recognition and anticancer activity. Met Ions Life Sci. Google Scholar
  54. Sinnecker S, Rajendran A, Klamt A, Diedenhofen M, Neese F (2006) Calculation of solvent shifts on electronic g-tensors with the conductor-like screening model (COSMO) and its self-consistent generalization to real solvents (direct COSMO-RS). J Phys Chem A 110:2235–2245. CrossRefGoogle Scholar
  55. Sistla YS, Khanna A (2011) Validation and prediction of the temperature-dependent Henry’s constant for CO2–ionic liquid systems using the conductor-like screening model for realistic solvation (COSMO-RS). J Chem Eng Data 56:4045–4060. CrossRefGoogle Scholar
  56. Song HW et al (2017) Ultrafast relaxation dynamics of amine-substituted bipyridyl ruthenium(II) complexes. Chem Phys Lett 683:322–328. CrossRefGoogle Scholar
  57. Tsai KY, Chang IJ (2017) Oxidation of bromide to bromine by ruthenium(II) bipyridine-type complexes using the flash-quench technique. Inorg Chem 56:8497–8503. CrossRefGoogle Scholar
  58. Van Kuiken BE et al (2013) Simulating Ru L3-edge X-ray absorption spectroscopy with time-dependent density functional theory: model complexes and electron localization in mixed-valence metal dimers. J Phys Chem A 117:4444–4454. CrossRefGoogle Scholar
  59. Wang W, Zhang J, Wang H, Chen L, Bian Z (2016) Photocatalytic and electrocatalytic reduction of CO2 to methanol by the homogeneous pyridine-based systems. Appl Catal A 520:1–6. CrossRefGoogle Scholar
  60. Weerawardene KLDM, Aikens CM (2018) Origin of Photoluminescence of Ag25(SR)18—nanoparticles: ligand and doping effect. J Phys Chem C 122:2440–2447. CrossRefGoogle Scholar
  61. Westwater C, Balish E, Schofield DA (2005) Candida albicans-conditioned medium protects yeast cells from oxidative stress: a possible link between quorum sensing and oxidative stress resistance. Eukaryot Cell 4:1654–1661. CrossRefGoogle Scholar
  62. Wu Q et al (2018) Rigid dinuclear ruthenium-arene complexes showing strong DNA interactions. J Inorg Biochem 189:30–39. CrossRefGoogle Scholar
  63. Yadav AK, Espaillat A, Cava F (2018) Bacterial strategies to preserve cell wall integrity against environmental threats. Front Microbiol 9:2064. CrossRefGoogle Scholar
  64. Yang G, Guan W, Yan L, Su Z, Xu L, Wang EB (2006) Theoretical study on the electronic spectrum and the origin of remarkably large third-order nonlinear optical properties of organoimide derivatives of hexamolybdates. J Phys Chem B 110:23092–23098. CrossRefGoogle Scholar
  65. Younker JM, Dobbs KD (2013) Correlating experimental photophysical properties of iridium(III) complexes to spin-orbit coupled TDDFT predictions. J Phys Chem C 117:25714–25723. CrossRefGoogle Scholar
  66. Zalis S, Lam YC, Gray HB, Vlcek A (2015) Spin-orbit TDDFT electronic structure of diplatinum(II, II) complexes. Inorg Chem 54:3491–3500. CrossRefGoogle Scholar
  67. Zarate X, Schott E, Gomez T, Arratia-Perez R (2013) Theoretical study of sensitizer candidates for dye-sensitized solar cells: peripheral substituted dizinc pyrazinoporphyrazine-phthalocyanine complexes. J Phys Chem A 117:430–438. CrossRefGoogle Scholar
  68. Zhong YW, Wu SH, Burkhardt SE, Yao CJ, Abruna HD (2011) Mononuclear and dinuclear ruthenium complexes of 2,3-Di-2-pyridyl-5,6-diphenylpyrazine: synthesis and spectroscopic and electrochemical studies. Inorg Chem 50:517–524. CrossRefGoogle Scholar
  69. Zhou M, Robertson GP, Roovers J (2005) Comparative study of ruthenium(II) tris(bipyridine) derivatives for electrochemiluminescence application. Inorg Chem 44:8317–8325. CrossRefGoogle Scholar

Copyright information

© Institute of Chemistry, Slovak Academy of Sciences 2019

Authors and Affiliations

  • Alexander Carreño
    • 1
    • 2
    Email author
  • Dayán Páez-Hernández
    • 1
    Email author
  • César Zúñiga
    • 1
  • Angélica Ramírez-Osorio
    • 2
  • Jan Nevermann
    • 3
  • María Macarena Rivera-Zaldívar
    • 3
  • Carolina Otero
    • 4
  • Juan A. Fuentes
    • 3
    Email author
  1. 1.Center of Applied Nanoscience (CANS)Universidad Andres BelloSantiagoChile
  2. 2.Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT 11170637)SantiagoChile
  3. 3.Laboratorio de Genética y Patogénesis Bacteriana, Facultad de Ciencias de la VidaUniversidad Andrés BelloSantiagoChile
  4. 4.Escuela de Química y Farmacia, Facultad de MedicinaUniversidad Andres BelloSantiagoChile

Personalised recommendations