Cellular uptake of a polypyridyl ruthenium complex revealed using a fluorescent rhodamine-modified ruthenium complex

Article abstract of DOI:10.1002/jccs.201200172

A fluorescent polypyridyl ruthenium complex was successfully prepared using an amide bond linkage to link two rhodamine moieties through bipyridine groups. Although photo-induced electron transfer (PET) quenched the fluorescent intensity, the quantum yield of the rhodamine-modified Ru(II) complex was 0.17 in water, sufficient for observing the fluorophore behaviour in biological systems. The rhodamine-modified Ru(II) complex was found to inhibit the bacterial growth of E. coli. In vitro fluorescence images of human hepatoma cells (SK-Hep1) showed that a fluorescent polypyridyl ruthenium complex not only supported the above observation but also preferably accumulated in the cytoplasmic region inside the cell. These observations suggest that in addition to strong Ru-DNA interactions, Ru-protein interactions in the cytoplasmic regions are strong and are therefore important to the development of metallopharmaceuticals.

Full text of DOI:10.1002/jccs.201200172

JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
Communication
Cellular Uptake of a Polypyridyl Ruthenium Complex Revealed Using a Fluorescent
Rhodamine-modified Ruthenium Complex
Yu-Ru Jhou,a Chien-Hui Chiou,a Lin-Kia Ni,a Li-Chou Chen,a Yau-Hung Chenb and
Chien-Chung Chenga,*
aDepartment of Applied Chemistry, National Chia-Yi University, Chia-Yi City, Taiwan 60004, R.O.C.
bDepartment of Chemistry, Tamkang University, Tamsui, Taipei, Taiwan, R.O.C.
(Received: Mar. 28, 2012; Accepted: May 28, 2012; Published Online: ??; DOI: 10.1002/jccs.201200172)
A fluorescent polypyridyl ruthenium complex was successfully prepared using an amide bond linkage to
link two rhodamine moieties through bipyridine groups. Although photo-induced electron transfer (PET)
quenched the fluorescent intensity, the quantum yield of the rhodamine-modified Ru(II) complex was
0.17 in water, sufficient for observing the fluorophore behaviour in biological systems. The rhodamine-
modified Ru(II) complex was found to inhibit the bacterial growth of E. coli. In vitro fluorescence images
of human hepatoma cells (SK-Hep1) showed that a fluorescent polypyridyl ruthenium complex not only
supported the above observation but also preferably accumulated in the cytoplasmic region inside the cell.
These observations suggest that in addition to strong Ru–DNA interactions, Ru-protein interactions in the
cytoplasmic regions are strong and are therefore important to the development of metallopharmaceuticals.
Keywords: Ruthenium; Rhodamine; Polypyridine; Uptake; Fluorescence.
The development of imaging sensors for metal ions
can facilitate investigations into the biological behavior of
metal ions.1,2 Fluorophores associated with metal ions,
such as Zn(II),3,4 Ca(II),5,6 or Cu(I)/(II),7-9 have been used
to study the role of these metals in brain function and mus-
cle action. Many synthetic metal complexes containing
metal ions, such as platinum, bismuths, and gold, are not re-
quired for daily life but are important in the treatment and/
or diagnosis of diseases.10 A better understanding of their
interaction modes and pathways may guide the develop-
ment of improved therapeutics. Recently, ruthenium com-
plexes have been considered as alternatives to platinum
drugs in antitumor therapy.10-12 The synthetic polypyridyl
chlororuthenium complexes constitute one class of poten-
tial antitumor reagents. Unlike Ru(III) ionic species gener-
ated from [Ru(Im)(DMSO)Cl4]– (Im: imidazole),13-15 poly-
pyridyl chlororuthenium complexes are believed to exert
antitumor activity as an integral metal complex.11 In addi-
tion to Ru(II)–DNA interactions,13,16 the cytotoxicity of
polypyridyl ruthenium complexes has been studied in
murine and human tumour cells in vitro, and the bio-reac-
tivity of the complexes has been evaluated in zebrafish em-
bryos, revealing that the complex targets proliferating fin
mesenchymal cells.17 The transport of polypyridyl ruthe-
nium complexes through E. coli cell membranes was found
to be related to stress-regulating proteins (the outer mem-
brane proteins, OmpF), in a study that employed pro-
teomics techniques in conjunction with ICP-OES mass
spectrometry.18 A systematic study of the inductive proper-
ties of polypyridyl ruthenium complexes showed that these
complexes significantly influence the total cellular protein
distribution, which can affect several metabolic pathways
in E. coli. The main biological processes associated with
the down-regulated proteins were related to carbohydrates,
including transport, the tricarboxylic acid (TCA) cycle,
glycolysis, and gluconeogenesis.19 This work suggests that
Ru–protein interactions in the cytoplasm contribute signifi-
cantly to the modulation of cell growth, in addition to the
effects of Ru–DNA interactions in the cell nuclei. Visual-
ization of ruthenium complex localization inside cells
would assist in differentiating the effects of interactions
with genomic DNA or cytoplasmic protein.
One approach to visualizing the in vitro localization
of ruthenium complexes involves introducing a fluorescent
group to a ruthenium complex. [Ru(terpy)(dppz)Cl]+ (terpy:
terpyridine, dppz: dipyrido[3,2-a:2¢,3¢-c]phenazine) and
its analogues have been developed as potential candidate
fluorescent ruthenium complexes.20 The electronic state
absorption properties and DNA affinity of [Ru(terpy)-
(dppz)Cl]+ have been well characterized; however, the
binding affinity of this complex may be heavily influenced
by the high affinity of dppz toward DNA via p–p interac-
* Corresponding author. Tel: +886-5-2766084; Fax: +886-5-2717901; E-mail: cccheng@mail.ncyu.edu.tw
J. Chin. Chem. Soc. 2012, 59, 000-000
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Communication
Jhou et al.
tions of the aromatic rings.21
fluxing compound 4 with Ru(terpy)Cl3 in an ethanolic so-
lution in the presence of LiCl and triethylamine to give a
deep brown-red precipitate in a yield of 24%.
An alternative visualization approach involves the
linking of fluorophores containing a modified bipyridine at
the 4,4¢-position to generate a fluorescent ruthenium com-
plex,22 as shown in Figure 1. This paper describes the prep-
aration of a fluorescent ruthenium complex into which a
rhodamine-modified bipyridine moiety has been intro-
duced to link two rhodamine moieties through an amide
bond. The rhodamine moiety was selected for introduction
into the framework of the ruthenium complexes due to its
reasonable solubility in water, relative chemical inertness
in biological systems, and acceptable quantum yield for
fluorescence visualization. The ruthenium complex was
characterized by NMR and mass spectroscopy. Visualiza-
tion of the fluorescent ruthenium complex localization was
accomplished by fluorescence imaging of human hepatoma
cells (SK-Hep1). The large rhodamine-modified Ru(II)
complex appeared to be transportable into SK-Hep1cells,
and it accumulated mainly in the cytoplasm.
Synthesis of a rhodamine-modified ruthe-
nium complex via amide bond linkage
Scheme I
The rhodamine-modified ruthenium complex was
characterized by solution NMR and MALDI mass spec-
troscopy. Because the ruthenium center was divalent and
diamagnetic, compound 1 displayed distinct peaks in the
NMR spectrum. The chemical shifts of compound 1 were
affected by the ring current effects of the terpyridine
ligand, so the chemical shifts of the pyridine moieties in the
bipyridine derivative differed from one another. Com-
pound 1 displayed asymmetric proton shifts in the bipyr-
idine ligand, which complicated the assignment of the pro-
ton NMR spectrum. To deconvolute the chemical struc-
Compound 1 was prepared by synthesizing the rho-
damine-modified bipyridine, 4, followed by a reaction with
Ru(terpy)Cl3 in EtOH for 18 hr, as shown in Scheme I. The
preparation of a rhodamine-modified bipyridine was
achieved by the following procedure. The rhodamine de-
rivative, 3, was prepared with an N-terminus by coupling a
carboxylic group to an ethylenediamine moiety in the pres-
ence of 1,1-carbonyldiimidazole (CDI) under anhydrous
conditions with a yield of 66%.23 4,4¢-Dicarboxylate bi-
pyridine, 2, was reacted with thionyl chloride under reflux
conditions for 4 hr to form the diacyl chloride derivative for
use in the next step. Compound 4 was produced by the slow
addition of the diacyl chloride derivative in a CHCl3 solu-
tion to a CHCl3 solution of the rhodamine derivative, 3, at
room temperature. The reaction was then refluxed for an
additional 5 hr to obtain a deep purple-red solid in a yield of
50%. The fluorescent ruthenium complex, 1, was prepared
according to the previously reported procedure24 by re-
1
tures, compounds 1 and 4 were analysed by H-COSY
NMR techniques. The assignment of compound 1 is illus-
trated in Figure 2. One of the 7-positions of the bipyridine
ligand showed a downfield shift from 8.76 ppm in the
rhodamine-modified bipyridine to 10.12 ppm in the ruthe-
nium complex. The peak at 1.36 ppm suggested the pres-
ence of ring current effects due to the terpyridine in com-
pound 1. The formation of a ruthenium complex was con-
firmed by analysis of the MALDI mass spectra, which re-
vealed a molecular ion peak, [M]+, with a mass-charge ratio
(m/z) of 1546.4.
Fig. 1. Design of a rhodamine derivative of a fluores-
cent Ru complex.
Fig. 2. 1H-NMR spectrum of compound 1.
2
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J. Chin. Chem. Soc. 2012, 59, 000-000

JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
Cellular Uptake a Fluorescent Ruthenium Complex
Table 1. UV-vis spectroscopic comparison of the ligands and the corresponding ruthenium complex, showing the absorption
wavelength, absorption coefficients, emission wavelength, and quantum yield
N
O
O
HN
O
NH
N+
Cl-
N
N
Cl-
Cl-
O
O
N
N
N
N
N Ru
H
N
Cl-
O
N
N
O
N
O
N
Cl
NH
NH
N+
O
HN
O
Structure
O
O
NH
HN
OH
O
O
N
N
N
Compound
Rhodamine B
553 (107000)
4
1
lmax (nm)(e M-1cm-1)
557 (145551)[a]
557 (15380)
548 (317085)/(MeOH)
575 (536)
547 (1509919)/(MeOH)
574 (536)
lem (lex) (nm)
F
572 (500)
0.48 (water);
0.80 (MeOH)
0.35 (water)
0.17 (water)
[a] H2O, pH 2
The electronic spectra of the fluorescent ruthenium
complex were measured in support of the physical charac-
terization studies of compound 1. The electronic absorp-
tion spectrum of compound 1 showed an absorption band at
a wavelength of 547 nm in methanol, which was compara-
ble to the absorption at 548 nm for compound 4, and 553
nm for rhodamine B. These results suggested that the elec-
tronic structure of the dye was not significantly affected by
the bipyridine linkage, as shown in Table 1. The emission
spectrum of compound 1 revealed an emission band at 574
nm in water upon excitation at 536 nm. The emission spec-
tra of the ligand, compound 4, and rhodamine 6B were sim-
ilar. However, the emission intensity of compound 1 in wa-
ter was dramatically reduced by a factor of 2 relative to the
emission intensity of rhodamine-modified bipyridine. Flu-
orescence can be easily quenched in aqueous solutions or
transition metal ions containing d-orbital electrons; e.g.,
Ni(II), Cu(II), and Ru(II) ions.25 Therefore, the fluores-
cence intensity of compound 1 was inevitably quenched to
a certain degree by the presence of ruthenium ions. The
quantum yield of compound 1 was reduced to 0.17 in wa-
ter; however, this quantum yield was sufficient for visual-
ization studies in biological systems.
for [Ru(terpy)(dmbpy)Cl]+ (dmbpy: 4,4¢-dimethylbipyri-
dine) to 23–36 Å for compound 1. The transport properties
of compound 1 were evaluated in the context of BL21 cell
growth inhibition, as reported previously. Compound 1
was found to have an inhibition concentration (IC50) of 60
mM, comparable to that measured for the [Ru(terpy)-
(dmbpy)Cl]+ complex under similar conditions. The outer
membrane protein (ompF) involved in [Ru(terpy)(dmbpy)-
Cl]+ transport in E. coli is also involved in stress-regulation
processes.18 The X-ray structure of ompF revealed a tri-
meric protein structure in which each monomer was ap-
proximately cylindrical with a radius of 30 Å.26 This sug-
gests that it was reasonable for compound 1, with a large
volume of rhodamine moieties, to penetrate E. coli cells
through the stress-regulation pathway. The concentration
of compound 1 in the cell media could not be increased
without producing a red precipitate. Compound 1 appar-
ently has limited solubility in water, possibly due to the
hydrophobicity of the aromatic rings.
SK-Hep1 cells were used in fluorescence imaging ex-
periments to visualize the localization of compound 1 in-
side cells. SK-Hep1 cells were cultured in LDF medium
containing 10 ppm compound 1 for 24 hr at 37 °C. Red flu-
orescence due to compound 1 was observed primarily in
the cytoplasm, as shown in Figure 3. A control reaction in-
volving staining with rhodamine 6B yielded with nonspe-
cific location in SK-Hep 1 cells. Under similar conditions,
The large volume of compound 1, which included a
ruthenium complex and two rhodamine moieties, could po-
tentially prevent transport into cells. The diameter of the
ruthenium complex was estimated to increase from 13 Å
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Communication
Jhou et al.
cytoplasm. The possibility that compound 1 was too large
in size to transport into the nucleus could not be excluded.
Fluorescence imaging suggested that the Ru–protein inter-
actions in the cytoplasm were important for bacterial
growth inhibition; Ru–DNA interactions in the nuclei may
also play a role in this regard. This work provides a new
fluorescence visualization tool to assist in the development
of ruthenium-based metallopharmaceuticals.
ACKNOWLEDGEMENT
The authors thank the Academia Sinica in Taiwan for
kindly providing the mass spectroscopy facilities. We
gratefully acknowledge the financial support of the Na-
tional Science Council of the Republic of China.
Fig. 3. Fluorescent imaging of SK-Hep1 cells. (A)
Staining with compound 1 (red fluorescence);
(B) staining with DAPI (blue fluorescence) to
visualize the nuclei; (C) expression levels of
P21 detected with mouse against human P21
(1:1000; Santa Cruz) as a primary antibody,
and with Alexa Fluor 488 rabbit anti-mouse-
IgG (1:200; Molecular Probes) as the second-
ary antibody (green fluorescence); and (D)
merged signal (orange fluorescence), indicat-
ing overlap between the signals corresponding
to compound 1 (red) and P21 (green).
REFERENCES
1. Domaille, D. W.; Que, E. L.; Chang, C. J. Nat. Chem. Biol.
2008, 4, 168.
2. Terai, T.; Nagano, T. Curr. Opin. Chem. Biol. 2008, 12,
515-21.
3. Kimura, E.; Aoki, S. BioMetals 2001, 14, 191.
4. Tomat, E.; Lippard, S. J. Curr. Opin. Chem. Biol. 2010, 14,
225.
5. Tsien, R. Y. Biochemistry 1980, 19, 2396.
6. Mac, M.; Uchacz, T.; Danel, A.; Danel, K.; Kolek, P.; Kulig,
E. J. Fluorescence 2011, 21, 375.
SK-Hep1 cells were counter-stained with 4¢,6-diamidino-
2-phenylindole (DAPI, excitation wavelength at 358 nm/
emission wavelength at 461 nm), which yields a blue fluo-
rescence and binds strongly to A-T rich regions in DNA, to
visualize the nuclei of the SK-Hep1cells, as shown in Fig-
ure 3B. A comparison of the fluorescence images sug-
gested that the red fluorescence of compound 1 was not sig-
nificantly accumulated in the nuclei.27,28 Compound 1-
treated SK-Hep1 cells were stained with a green fluores-
cent protein-labelled mouse anti-human P21 antibody, as
shown in Figure 3C, to visualize the expression of P21. The
yellow fluorescence of the antibody overlaid with the fluo-
rescence of compound 1, suggesting that compound 1-
treated SK-Hep1cells induced P21 expression and resulted
in apoptosis.29 As a result, compound 1 not only was inter-
nalized by the cells, but it predominantly accumulated in
the cytoplasm region.
7. Neupane, L. N.; Thirupathi, P.; Jang, S.; Jang, M. J.; Kim, J.
H.; Lee, K.-H. Talanta 2011, 85, 1566.
8. Liu, Z.-C.; Yang, Z.-Y.; Li, T.-R.; Wang, B.-D.; Li, Y.; Qin,
D.-D.; Wang, M.-F.; Yan, M.-H. Dalton Trans. 2011, 40,
9370.
9. Vinkenborg, J. L.; Koay, M. S.; Merkx, M. Curr. Opin.
Chem. Biol. 2010, 14, 231.
10. van Rijt, S. H.; Sadler, P. J. Drug Discovery Today 2009, 14,
1089.
11. Antonarakis, E. S.; Emadi, A. Cancer Chemother. Pharma-
col. 2010, 66, 1.
12. Clarke, M. J. Coordination Chem. Rev. 2003, 236, 209.
13. Brabec, V.; Nov<kov<, O. Drug Resistance Updates 2006, 9,
111.
14. Pluim, D.; van Waardenburg, R. C. M.; Beijnen, J. H.;
Schellens, J. H. M. Cancer Chemother. Pharmacol. 2004,
54, 71.
In summary, a fluorescent ruthenium complex was
successfully prepared by linking two rhodamine moieties
to the complex via an amide bond. Despite its large volume,
compound 1 was transported into cells, where it was found
to inhibit E. coli growth. Fluorescence images of SK-Hep1
cells incubated with compound 1 revealed that the poly-
pyridyl ruthenium complex preferably accumulated in the
15. Casini, A.; Mastrobuoni, G.; Terenghi, M.; Gabbiani, C.;
Monzani, E.; Moneti, G.; Casella, L.; Messori, L. J. Biol.
Inorg. Chem. 2007, 12, 1107.
16. Cheng, C. C.; Lee, W. L.; Su, J. G.; Liu, C. L. J. Chin. Chem.
Soc. 2000, 47, 213.
17. Wang, Y. H.; Cheng, C. C.; Lee, W. J.; Chiou, M. L.; Pai, C.
W.; Wen, C. C.; Chen, W. L.; Chen, Y. H. Chem-Biol. Inter-
4
www.jccs.wiley-vch.de
© 2012 The Chemical Society Located in Taipei & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
J. Chin. Chem. Soc. 2012, 59, 000-000

JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
Cellular Uptake a Fluorescent Ruthenium Complex
act. 2009, 182, 84.
2002, 21, 61.
18. Ho, M.-Y.; Chiou, M.-L.; Chang, R.-C.; Chen, Y.-H.; Cheng,
C.-C. J. Inorg. Biochem. 2010, 104, 614.
25. Posokhov, Y. O.; Kyrychenko, A.; Ladokhin, A. S. Anal.
Biochem. 2010, 407, 284.
19. Ho, M.-Y.; Chiou, M.-L.; Du, W.-S.; Chang, F. Y.; Chen,
Y.-H.; Weng, Y.-J.; Cheng, C.-C. J. Inorg. Biochem. 2011,
105, 902.
26. Cowan, S.; Garavito, R.; Jansonius, J.; Jenkins, J.; Karlsson,
R.; König, N.; Pai, E.; Pauptit, R.; Rizkallah, P.; Rosenbusch,
J.; Rummel, G.; Schirmer, T. Structrue 1995, 3, 1041.
27. Tian, X.; Gill, M. R.; Cantón, I.; Thomas, J. A.; Battaglia, G.
Chembiochem. 2011, 12, 548.
20. Kalsbeck, W.; Thorp, H. H. J. Am. Chem. Soc. 1993, 115,
7146.
21. Zeglis, B. M.; Pierre, V. C.; Barton, J. K. Chem. Commun.
2007, 4565.
28. Fernández-Moreira, V.; Thorp-Greenwood, F. L.; Coogan,
M. P. Chem. Commun. 2010, 46, 186.
22. Garelli, N.; Vierling, P. J. Org. Chem. 1992, 57, 3046.
23. Chen, Y.-H.; Chiou, C.-H.; Chen, W.-L.; Jhou, Y.-R.; Lee,
Y.-T.; Cheng, C.-C. J. Chin. Chem. Soc. 2010, 57, 1257.
24. Konno, H.; Ishii, Y.; Sakamoto, K.; Ishitani, O. Polyhedron
29. Peng, H. C.; Wang, Y. H.; Wen, C. C.; Wang, W. H.; Cheng,
C. C.; Chen, Y. H. Comp. Biochem. Physiol. C: Toxicol.
Pharmacol. 2010, 151, 480.
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