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

Article abstract of DOI:10.1007/s11696-019-00714-z

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.

Full text of DOI:10.1007/s11696-019-00714-z

Chemical Papers
https://doi.org/10.1007/s11696-019-00714-z
ORIGINAL PAPER
Prototypical cis‑ruthenium(II) complexes present diferential
fuorescent staining in walled‑cell models (yeasts)
Alexander Carreño1,2 · Dayán Páez‑Hernández1 · César Zúñiga1 · Angélica Ramírez‑Osorio2 · Jan Nevermann3 ·
María Macarena Rivera‑Zaldívar3 · Carolina Otero4 · Juan A. Fuentes3
Received: 14 December 2018 / Accepted: 6 February 2019
© Institute of Chemistry, Slovak Academy of Sciences 2019
Abstract
cis-Ru(deeb)23+ (R1; where deeb is 4,4′-diethanoate-2,2′-bipyridine) and cis-Ru(phen)23+ (R2; where phen is 1,10-phen-
anthroline) 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 demon-
strated 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 diferent walled-cell models (including bacteria and
yeasts). More importantly, we found that both R1 and R2 were able to fuorescently 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 diferential 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.
Keywords Ruthenium(II) complexes · Spin–orbit DFT · Cytotoxicity · Biomarkers · Yeasts
Electronic supplementary material The online version of this
Introduction
article (https://doi.org/10.1007/s11696-019-00714-z) contains
supplementary material, which is available to authorized users.
Research of ruthenium(II) nitrogenated complexes have
* Alexander Carreño
alexander.carreno@unab.cl
http://www.cans.cl
received interest as photosensitizers in dye solar cells
(DSSC) application due to their favorable photoelectro-
chemical properties and high stability in the oxidized state
(Bomben et al. 2010; Caramori et al. 2010; Nazeeruddin
et al. 2001; Oßwald et al. 2017; Sampaio et al. 2017; Zhong
et al. 2011). Development of ruthenium(II) complexes of
type cis-ruthenium(II) (N,N)23+, where N,N is a dinitrogen-
ated ligand such as 2,2′-bipyridine or 1,10-phenanthroline
derivatives, or monodentated such as halogens or ‒SCN,
among others, dates to the 1990s (Bignozzi et al. 1993; Bon-
hote et al. 1999; Juris et al. 1988). These complexes have
been described as useful in high efciency in the conversion
of high incident solar light into electricity, mainly due to
their wide absorption range from the visible light to the near
infrared (NIR) adsorption (Katsumata et al. 2017; Koyyada
et al. 2016; Sauvage et al. 1994). The absorption spectra
* Dayán Páez-Hernández
dayan.paez@unab.cl
http://www.cans.cl
* Juan A. Fuentes
jfuentes@unab.cl
1
Center of Applied Nanoscience (CANS), Universidad
Andres Bello, República 275, Santiago, Chile
2
Fondo Nacional de Desarrollo Científco y Tecnológico
(FONDECYT 11170637), Santiago, Chile
3
Laboratorio de Genética y Patogénesis Bacteriana, Facultad
de Ciencias de la Vida, Universidad Andrés Bello, República
330, Santiago, Chile
4
Escuela de Química y Farmacia, Facultad de Medicina,
Universidad Andres Bello, Santiago, Chile
Vol.:(0123456789)
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of ruthenium(II) dinitrogenated compounds can be modu-
lated according to the nature of their ligands, including the
HOMO and LUMO energy levels (Al-Noaimi and AlDamen
2012; Li et al. 2014; Sacksteder et al. 1990; Sangilipandi
et al. 2016; Song et al. 2017).
with suitable ancillary ligands allows an enough uptake
to permit their use as biomarkers in walled cells, includ-
ing bacteria and yeasts (Carreno et al. 2016b, 2017b, c).
Thus, we wondered whether Ru(II) complexes, substituted
with diferent dinitrogenated ligands (i.e. 2,2′-bipyridine or
1,10-phenanthroline), are suitable as biomarkers in walled
cell such as bacteria (Gram-negative bacteria: Salmonella
enterica; Gram-positive bacteria: Staphylococcus aureus
and Bacillus cereus) and yeasts (Candida albicans and
Cryptococcus spp.). For that aim, we synthesized two pro-
totypical cis-Ruthenium(II)(N,N)23+ complexes (Wang et al.
2016; Zhou et al. 2005): cis-Ru(deeb)23+ (R1; where deeb is
4,4′-diethanoate-2,2′-bipyridine) and cis-Ru(phen)23+ (R2;
where phen is 1,10-phenanthroline) (Scheme 1). In this con-
text, the focus of the present study was not the full chemical
characterization of either R1 or R2, but the determination
of their potential properties as biomarkers for walled-cell
models. In addition, to better understand the emission of
these compounds, we performed a relativistic computational
protocol to explore not only the nature and assignment of
the transition but also the possible mechanism. It is neces-
sary to consider the relativistic efects and, particularly, the
role of the spin–orbit coupling because it determines the
amount of diferent spin-free multiplicities involved in the
emission process (Carreno et al. 2019; Rojas-Poblete et al.
2018). In addition, we performed assays to determine the
potential cytotoxicity of both R1 and R2 in diferent cell
models, including bacteria (walled prokaryotic cells) and
yeasts (walled eukaryotic cells).
Normally, ruthenium(II) polypyridyl complexes have
been described as possible anticancer agents (Poynton
et al. 2017), including Ru(II) cyclometallated complexes
(Biancalana et al. 2017). Interestingly, intracellular locali-
zation of Ru(II)-based complexes has been monitored by
confocal microscopy, showing that these complexes are
normally retained in the cell nucleus in non-walled cells
(e.g., epithelial cells). These fndings are consistent with
the previously described DNA-disrupting activity of some
Ru(II) complexes (Li et al. 2015; Schatzschneider 2018).
The use of confocal microscopy, which allows obtaining
images through the Z axis, has also remarked an alterna-
tive use of Ru(II) complexes as cellular biomarkers (Byrne
et al. 2016). Nevertheless, Ru(II)-based biomarkers have
been exclusively tested in non-walled eukaryotic cells
(e.g, baby hamster kidney cells BHK, human embryonic
kidney cells HEK-293, and liver carcinoma HEp-G2) (Li
et al. 2015), where their potential use as biomarkers have
not been assessed in walled cells such as bacteria (prokary-
otic) and yeasts (fungi, eukaryotic). Both bacteria and fungi
(yeasts) exhibit a rigid structure in their envelop, called cell
wall (Caveney et al. 2018; Sanz et al. 2017). Although these
structures are not chemically related in these two biologi-
cal groups, in both cases, the presence of a cell wall could
hamper the uptake of foreign molecules, including bio-
markers. In this context, bacteria and fungi are considered
as “walled cells”, since these cell types harbor a cell wall,
whereas epithelial cells are “non-walled cells” because they
lack this structure. In fact, it has been stated that, at least for
Re(I) tricarbonyl complexes, the presence of the cell wall
can be considered as a barrier that impairs the uptake of
biomarkers (Amoroso et al. 2007). However, we reported
that the use of monocationic Re(I) tricarbonyl complexes
Regarding the potential as biomarkers in walled cells,
we found that both R1 and R2 efciently stained yeasts,
including Candida albicans and Cryptococcus spp. with a
simple and fast procedure, without the need of additional
permeabilizer molecules and antibodies. Interestingly, R1
and R2 exhibited diferential staining patterns, where R1
specifcally was retained in a central structure that could
be interpreted as the nucleus, and R2 stained a periph-
eric structure that could correspond to the cell wall. In
Scheme 1 Chemical
structure of the ligands
deeb (4,4′-diethanoate-
2,2′-bipyridine), and phen
(1,10-phenanthroline); and
the cis-ruthenium(II)(N,N)32+
complexes R1 (cis-Ru(deeb)32+
)
and R2 (cis-Ru(phen)32+
)
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summary, we showed that cis-Ruthenium(II)(N,N)23+ com-
plexes present properties as suitable biomarkers for walled
cells in a simple and fast procedure, without the need of
additional permeabilized molecules and antibodies.
Experimental
All starting materials and solvents were purchased from
Merck and Aldrich and used with no further purifcation.
The purity of each compound (deeb, R1 and R2) was
checked by 1H NMR recorded on a Bruker AVANCE 400
spectrometer at 400 MHz at 25 °C. Samples were dissolved
in deuterated DMSO (for R1 and R2), or chloroform (for
deeb). In all the cases, we used tetramethylsilane as an inter-
nal reference.
Quantum chemistry calculations
All calculations reported here were carried out in the
framework of the density functional theory (DFT) using
the Amsterdam Density Functional computational package
(ADF) via the zeroth-order regular approximation (ZORA)
Hamiltonian, including spin–orbit (SO) relativistic cor-
rections and Slater Type Orbitals (STO) basis sets with
triple–ζ accuracy plus double polarization function (TZ2P)
for all the atoms (Muñoz-Castro et al. 2017). The molecu-
lar geometry of the ground states was fully optimized via
GGA-BP86 functional (Averkiev et al. 2014; Micciarelli
et al. 2017). In all cases, frequency analysis was performed
after each geometry optimization, where we obtained only
positive frequencies verifying the local minima. Time-
dependent DFT (TDDFT) was used to determine the ori-
gin of the emission of the complexes and to assign the
transitions (Yang et al. 2006; Zarate et al. 2013). Since
calculations use the physical meaning of phosphorescence,
a previously known excitation wavelength is not necessary
to obtain the theoretical emission (Macleod-Carey et al.
2018). Solvation efects were considered via the conduc-
tor-like screening model (COSMO) using acetonitrile as
solvent (Sinnecker et al. 2006; Sistla and Khanna 2011).
More detailed calculations were performed by the self-
consistent two-component spin–orbit coupling TDDFT
(SOC-TDDFT) within the ZORA Hamiltonian (Fantacci
et al. 2005; Grotjahn et al. 2017). ADF allowed us to
better estimate the emission wavelength. In all cases, at
least fve spin-mixed excitations were calculated in the
full SOC-TDDFT calculations (Franco de Carvalho et al.
2017; Grotjahn et al. 2017; Páez-Hernández et al. 2012;
Younker and Dobbs 2013). It is important to remark that,
in a non-relativistic framework, spin functions can be clas-
sifed depending on the spin-projection quantum number
(ms) as α = |1/2,1/2 > and β = |1/2,− 1/2 > functions. When
spin–orbit coupling is included in the calculations, the spin
is no longer a good quantum number, and α and β func-
tions mix between them. In this scenario, all these efects
are treated at scalar relativistic time-dependent density
functional theory with ZORA Hamiltonian (SR-TDDFT/
ZORA) (Weerawardene and Aikens 2018; Zalis et al.
2015) and spin–orbit time-dependent density functional
theory with ZORA Hamiltonian (SO-TDDFT/ZORA) (Van
Kuiken et al. 2013).
Antimicrobial activity
Both R1 and R2 complexes were evaluated for their in vitro
growth inhibitory activity against the clinical yeasts Candida
albicans and Cryptococcus spp., obtained from the Hospi-
tal Clínico of the Universidad de Chile, Santiago, Chile;
the Gram-negative pathogen Salmonella enterica serovar
Typhimurium ATCC14028s; the Gram-positive pathogen
Staphylococcus aureus; and the sporulated Gram-positive
Bacillus cereus obtained from the Hospital Clínico of the
Universidad de Chile, Santiago, Chile. Minimum inhibi-
tory concentration (MIC) was obtained by broth dilution as
described (Cuenca-Estrella et al. 2003). The MIC is defned
as the lowest concentration of the tested compounds at which
no growth of the strain was observed after the incubation
(Cuenca-Estrella et al. 2003). Yeasts were previously cul-
tured in Sabouraud agar (Bacto peptone, 10 g/L; glucose,
40 g/L; agar, 15 g/L; pH 5.6) at 28 °C. Bacteria were pre-
viously cultured in Luria–Bertani broth (Bacto peptone,
10 g/L; NaCl, 5.0 g/L; peptone, 10 g/L; yeast extract 10 g/L).
Further dilutions of microorganisms (0.5 McFarland) were
performed with Bacto Tryptic Soy broth (pancreatic digest
casein 17.0 g/L, papaic digest of soybean 3.0 g/L, dextrose
2.5 g/L, sodium chloride 5.0 g/L, dipotassium phosphate
2.5 g/L). Stock solutions of the tested compounds were pre-
pared in dimethyl sulfoxide (DMSO). The inoculated wells
were then incubated at 28 °C (yeasts) or 37 °C (bacteria) for
24, 48 and 72 h. The MIC values of the tested compounds
were obtained as mM. All the experiments were performed
in biological triplicate, each in technical triplicate.
Fluorescence microscopy studies
Bacteria (Salmonella enterica serovar Typhimurium
ATCC14028s, Staphylococcus aureus, and Bacillus cereus)
were grown 24 h at 37 °C in Luria–Bertani plates (Bacto
peptone, 10 g/L; NaCl, 5.0 g/L; peptone, 10 g/L; yeast
extract 10 g/L; agar 15 g/L). Yeasts (Candida albicans and
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Cryptococcus spp.) were grown 48 h at 28 °C in Sabouraud
agar. For staining, either bacteria or yeasts (approximately
109 cfu/ml) were washed twice and resuspended in one
volume of phosphate bufer solution (PBS). The microor-
ganisms were mixed with either R1 or R2 (2 mM as fnal
concentration, 40% v/v or 5.6 M DMSO), incubated 90 min
at 37 °C and subsequently washed three times with PBS
to remove the excess of unbound complexes. It has been
stated that the DMSO is a relatively innocuous solvent for
yeasts, at least at 6 M (Chaves and da Silva 2012; León-
García et al. 2017; Westwater et al. 2005). Finally, micro-
organisms were resuspended in one volume of PBS prior to
being immobilized with 1% agarose in PBS and observed at
1000×(100×objective, 10×ocular, NA 1.4, with immer-
sion oil 1.510) with confocal microscopy (Leica MicroSys-
tems). Since yeasts present autofuorescence, we set detec-
tion threshold (fuorescence emission was obtained by laser
excitation at 405 nm with 20% power intensity) using yeasts
treated with DMSO alone (Carreno et al. 2016a). Emission
was collected with a long-pass emission flter in the range of
450–727 nm. To avoid photobleaching, samples were pho-
tographed before 5 min of exposure under the microscope.
Synthesis of cis‑ruthenium(II) (phen)3(PF6)2 (R2)
The procedure was similar to that described for R1. The
orange-light precipitate was fltered, washed with ethanol
and diethyl ether. Yield 63%. 1H NMR (400 MHz, DMSOd6,
2.50 ppm); 8.77 (d, 2H), 8.39 (s, 2H), 8.09 (d, 2H), 7.77 (dd,
2H) (Wang et al. 2016).
Results and discussion
Experimental and theoretical calculations
The 4,4′-diethanoate-2,2′-bipyridine (deeb) ligand was syn-
thesized from the 4,4′-dicarboxylic-2,2′-bipyridine acid, as
previously reported (Donnici et al. 1998). R1 and R2 were
synthesized by a traditional route involving the reaction of
the corresponding dinitrogenated ligand (deeb or phen)
with ruthenium(III) at refux under inert atmosphere in the
dark, to obtain the more stable cis-ruthenium(II) complexes
(Dreyse et al. 2013; Gajardo et al. 2011). The complexes
−
were precipitated by adding PF6 and recrystallized from
ethanol/diethyl ether (2:1) with a good yield. Both structure
and purity of complexes were corroborated by 1H NMR (see
Figures S1, S2 and S3 in the ESI†) (Tsai and Chang 2017;
Wang et al. 2016; Zhou et al. 2005).
Materials
Both R1 and R2 have been previously characterized,
including theoretical studies. Nevertheless, the optical prop-
erties of these complexes, a very relevant aspect considering
their potential use as biomarkers, have not been previously
addressed in depth. In this context, we performed calcula-
tions using SOC-TDDFT level of theory, including relativ-
istic efects to describe electronic transitions in terms of the
molecular spinors. For both complexes, the emission has a
LMCT character and can be assigned as π*→d transition.
For R1, the emission band is centered at 633 nm and, for
R2, at 688 nm. These values agree with the experimental
reports for these systems (Ardo et al. 2010; Farnum et al.
2012; Favereau et al. 2016). We also calculated the emission
lifetime, which was 1.4×10−5 s and 2.2×10−5 s for R1 and
R2, respectively, suggesting that the emission mechanism is
phosphorescence in both cases. The nature of states involved
in the emission corroborates this assertion because we found
that, in both complexes, the spin-free triplet state contributes
with 95% to the emissive state. In this case, it is important
to underline that the normal selection rules, which forbid
the spin fip transitions, need a reformulation when the
spin–orbit interaction is considered. The molecular spinors
have a contribution of both kinds of spin functions, |α>and
|β >, and due to that the spin fip transitions are allowed
in these cases. In our case, we consider both situations,
SR-TDDFT/ZORA and SO-TDDFT/ZORA levels (Casida
2009), of calculations to describe the emission spectra of
The synthetic procedure to obtain the ligand, R1 and R2
complexes, was reported in literature (Donnici et al. 1998).
Synthesis of 4,4′‑diethanoate‑2,2′‑bipyridine (deeb)
This compound was prepared by reaction of 4,4′-dicarboxy-
2,2′-bipyridine in a mixture of sulfuric acid and absolute eth-
anol according a published procedure (Donnici et al. 1998).
Yield 60%. 1H NMR (400 MHz, chloroform-d, 7.24 ppm):
8.92 (d, 2H); 8.86 (d, 2H); 7.91 (dd, 2H); 4.45 (quartet,
–CH2–), 1.44 (t, –CH3).
Synthesis of cis‑ruthenium(II) (deeb)3(PF6)2 (R1)
We dissolved RuCl3 × H2O (0.22 mmol), and hydroqui-
none (0.65 mmol) in 25 ml of ethanol. Then, add deeb
(0.71 mmol) and refux with stirring in the dark, and under
nitrogen atmosphere for 24 h. The mixture was cooled to
room temperature and add 0.1 g of NH4PF6 and stirring for
24 h. The precipitate was fltered, washed with petroleum
ether, and vacuum-dried, to obtain an orange powder (Zhou
1
et al. 2005). Yield 50%. H NMR (400 MHz, DMSO-d6,
2.50 ppm); 9.33 (s, 2H), 7.96 (d, 2H), 7.84 (d, 2H), 4.43
(quartet, –CH2–), 1.34 (t, –CH3).
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both R1 and R2 (data not shown). Figure 1 shows molecu-
lar orbitals involved in the emission of R1. The peripheral
substituents were demitted during the optimization to avoid
convergence problems due to their rotation.
Nevertheless, and as stated above, Ru(II)-based biomarkers
have been exclusively tested in non-walled eukaryotic cells
(Li et al. 2015). Considering that the presence of the cell
wall plausibly could impair the uptake of foreign complexes,
as previously suggested for monocationic Re(I) tricarbonyl
complexes (Amoroso et al. 2007, 2008), we tested both R1
and R2 as biomarkers with the same cell models described
above. Regarding bacteria, we found that both R1 and R2
were unable to reveal the presence of cells under the tested
conditions (data not shown). By contrast, we observed that
both R1 and R2 were suitable to stain yeasts, including both
Candida albicans (Fig. 2) and Cryptococcus spp. (see Fig-
ure S5 in the ESI†), although these complexes seem to be
more suitable for Candida albicans. The diference found
between bacteria and fungi could be explained by a def-
cient uptake, due to the presence of a bacterial cell wall
chemically diferent from the fungal cell wall (Gow et al.
2017; Yadav et al. 2018). In this case, bacterial cell wall
could be hampering the uptake of complexes. Alternatively,
it is also possible that the R1 and/or R2 are/is able to enter
bacteria, but they are unable to remain retained inside cells
after washing with phosphate bufer saline. Previously, simi-
lar results were reported with rhenium(I)-based complexes,
which were suitable as biomarkers only for yeasts, but not
for bacteria (Carreño et al. 2017a).
Cytotoxicity assays of R1 and R2
As a frst approach to determine the biological activity, we
determined the cytotoxicity of R1 and R2 in diferent cell
models. For this aim, we tested bacteria (prokaryotic walled
cells), including Gram-negative (Salmonella enterica sero-
var Typhimurium ATCC14028 s), Gram-positive (Staphy-
lococcus aureus), and sporulated Gram-positive (Bacillus
cereus) strains, as well as yeasts (eukaryotic walled cells),
including Candida albicans and the capsulated yeast, Cryp-
tococcus spp. We observed that, in all cases, both R1 and R2
exerted no cytotoxic efect under the tested conditions. The
only noticeable efect was observed with Cryptococcus spp.,
where both R1 and R2 presented a slight fungistatic efect
only at 24 h of incubation (see Figure S4 in the ESI†). The
fungistatic efect could be attributed to a fungal physiologi-
cal adaptation and/or to a decrease efective concentration
of compounds due to the presence of enzymes and other
biomolecules that might modify the Ru complexes. Never-
theless, this efect disappears after 48 h. These results show
that, both R1 and R2 exert low cytotoxicity, undistinguish-
able from the vehicle (DMSO) alone.
Our results showed that R1 presented a discrete distri-
bution inside cells, suggesting that this complex remained
retained in the nucleus. This result is consistent with the
previously reported data showing that Ru(II) complexes are
retained in the nucleus of non-walled cells (e.g,. epithelial
cells) (Li et al. 2015). Nevertheless, it is important to remark
that this is the frst report, to our knowledge, showing that
ruthenium-based compounds might be as useful as biomark-
ers for walled cells, opening new applications for this kind
of complexes. Interestingly, when R2 was tested, we also
observed a positive staining, but with a completely difer-
ent pattern. At a frst sight, we realized that R2 apparently
stained the whole cell, marking a sharp diference with the
Confocal microscopy
Previously, confocal microscopy has allowed determining
that dicationic Ru(II) polypyridyl complexes are retained in
the nucleus of non-walled eukaryotic cells (Li et al. 2015;
Schatzschneider 2018), presumably due to a possible inter-
action of the phosphate DNA groups with the positive charge
of Ru(II) complexes. In addition, the planar symmetry of this
kind of complexes could be also contributing to the inter-
action with DNA, as previously reported (Wu et al. 2018).
Fig. 1 Molecular orbitals
involved in the emission of R1
(similar to R2). The a1/2* repre-
sents the A irrep (group theory)
in the double-value group
representation. a1/2 corresponds
to the spin notation
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Fig. 2 Fluorescence confocal microscopy images showing Can-
dida albicans (yeast). The “Red channel” corresponded to excitation
of 405 nm and emission collected in a range of 450–727 nm. In all
cases, microorganisms were observed fresh, immobilized with 1%
agarose, using ×1000. DMSO alone was used to set the detection
threshold. The Z axis was divided into 10 confocal slices. Images
obtained for R2 show two confocal slices (2 and 4, respectively)
results obtained with R1 (Fig. 2, see Z = 2/10). However,
taking advantage of the confocal microscopy, a technique
that allows observing diferent focal felds through the Z
axis, we analyzed images in more detail. When we observed
the same images in a diferent slice, we realized that R2 was
retained in a structure that surrounds the cell (Fig. 2, see
Z=4/10). Considering the thickness of the stained structure,
we speculate that R2 could be useful to specifcally stain the
cell wall. In this case, we speculate that the diferential cell
localization of R1 and R2 can be attributed to the chemi-
cal nature of these two complexes. In R1, ruthenium(II) is
coordinated with three substituted 2,2′-bipyridine moie-
ties, whereas in R2, ruthenium(II) is coordinated with three
1,10-phenanthroline moieties (Scheme 1). Since 2,2′-bipy-
ridine moieties present ethyl ester, a polar substituent, and
1,10-phenanthroline are constituted by three fused aromatic
rings, without polar substituents, the possibility of interac-
tions via hydrogen bonds through carbonyls in R1 is higher
compared to R2. It has been reported that both DNA and
proteins, two biomolecules highly abundant in the nucleus,
are able to interact with exogenous molecules via hydrogen
bonds (Rowe and Ho 2017), explaining why R1 is appar-
ently retained in the cell compartment. On the other hand,
R2 does not present functional groups able to form hydro-
gen bonds, where only Van der Waals interactions are likely
possible. In the case of R2, it is possible that Van der Waals
interactions contributed to retain the complex in structures
found in the cells surface, such as the cell wall (Kendall and
Roberts 2015; Nir and Andersen 1977). Nevertheless, more
experimentation is needed to fully understand the molecular
mechanisms underlaying the diferential staining between
R1 and R2.
It is important to remark that our staining protocol can
be performed without the need of additional permeabiliza-
tion steps. In this sense, DMSO has been known to enhance
cell membrane permeability of drugs or DNA, at least in
unwalled cells (i.e., epithelial cell line) (de Menorval et al.
2012). Nevertheless, we want to underline that, when we
tested other fuorescent, d6-metal complexes with this same
staining protocol, we were unable to obtain an efcient
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staining, indicating that the mere presence of DMSO does
not assure a successful result in this king of biomarkers
(Carreño et al. 2017a).
Altogether, these results show that Ru(II)-based com-
plexes might be useful to develop new biomarkers for walled
cells such as yeasts, similar to other Re(I)-based studies
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Conclusions
As determined by our relativistic DFT studies, both R1 and
R2 exhibit a similar emission of ligand–metal charge trans-
fer (LMCT) character. In addition, these complexes showed
very low cytotoxicity against diferent cell models, including
bacteria and yeasts. Regarding the biomarker potential, since
R1 and R2 presented diferent staining pattern in yeasts, we
concluded that changes in the substituents of the dinitrogen-
ated ligands can be used to modulate biomarker properties of
these kind of Ru(II) complexes, even for walled cells. These
results show that these two complexes could be used as bio-
markers for walled cells as luminescent staining. To our
knowledge, this is the frst report showing that ruthenium(II)
complexes can be used as biomarkers for walled cells.
Bomben PG, Koivisto BD, Berlinguette CP (2010) Cyclometalated
Ru complexes of type [Ru(II)(N–N)(2)(C–N)](z): physicochemi-
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Acknowledgements We thank FONDECYT 11170637, 1181638, and
1180017. We thank Dr, Ramiro Arratia-Pérez (Center of Applied Nano-
science, Universidad Andres Bello) for the Computational Resources
Facilities and Dr. Ivonne Chávez M (Departamento de Química
Inorgánica, Pontifcia Universidad Católica de Chile) for instrumental
facilities.
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. https
://doi.org/10.1039/c6nj00905k
Compliance with ethical standards
Carreno A et al (2019) Cyclic voltammetry, relativistic DFT calcula-
tions 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. https://doi.
org/10.1016/j.cplett.2018.11.043
Conflicts of interest The authors declare no conficts of interest.
Dedication This manuscript is dedicated to Professor Dr. Ramiro Arra-
tia-Pérez (UNAB, director of Center of Applied Nanoscience, Chile),
for his relevant contributions to the relativistic quantum chemistry in
Chile, where he founded the frst Molecular Relativistic School in Latin
America, and in the world.
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) tricar-
bonyl core with yeasts and bacteria. New J Chem 41:2140–2147.
https://doi.org/10.1039/c6nj03792e
Carreño A, Gacitúa M, Molins E, Arratia-Pérez R (2017b) X-ray dif-
fraction 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
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Casida M (2009) Time-dependent density-functional theory for mol-
ecules and molecular solids. J Mol Struct (Thoechem) 914:3–18.
https://doi.org/10.1016/j.theochem.2009.08.018
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