New photo-CORMs: Deeply-coloured biocompatible rhenium complexes for the controlled release of carbon monoxide

Article abstract of DOI:10.1016/j.ica.2015.05.035

Air- and water-stable rhenium carbonyl complexes are investigated as CO-carriers for studying the light-dependent release of small amounts of carbon monoxide under physiological conditions. The reported very low quantum yields are in a suitable range for

Full text of DOI:10.1016/j.ica.2015.05.035

Accepted Manuscript
New Photo-CORMs: Deeply-coloured biocompatible Rhenium Complexes for
the Controlled Release of Carbon Monoxide
Elham Kianfar, Christine Schäfer, Mohammad Reza Lornejad-Schäfer,
Engelbert Portenkirchner, Günther Knör
PII:
S0020-1693(15)00320-5
http://dx.doi.org/10.1016/j.ica.2015.05.035
ICA 16588
DOI:
Reference:
Inorganica Chimica Acta
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2 March 2015
26 May 2015
28 May 2015
Please cite this article as: E. Kianfar, C. Schäfer, M.R. Lornejad-Schäfer, E. Portenkirchner, G. Knör, New Photo-
CORMs: Deeply-coloured biocompatible Rhenium Complexes for the Controlled Release of Carbon Monoxide,
Inorganica Chimica Acta (2015), doi: http://dx.doi.org/10.1016/j.ica.2015.05.035
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New Photo-CORMs: Deeply-coloured biocompatible
Rhenium Complexes for the Controlled Release of Carbon
Monoxide
Elham Kianfar,a Christine Schäfer,b Mohammad Reza Lornejad-Schäfer,b Engelbert Portenkirchner c
and Günther Knör *a
a Institute of Inorganic Chemistry, Johannes Kepler University Linz (JKU), 4040 Linz, Austria
b BioMed-zet Life Science GmbH, 4020 Linz, Austria
c Institute of Physical Chemistry, University of Innsbruck, 6020 Innsbruck, Austria
* Corresponding Author, Tel: +43 732 2468 5100, E-mail: guenther.knoer@jku.at
A R T I C L E I N F O
A B S T R A C T
Article History
Received
Accepted
Air- and water-stable rhenium carbonyl complexes are
investigated as CO-carriers for studying the light-
dependent release of small amounts of carbon
monoxide under physiological conditions. The reported
very low quantum yields are in a suitable range for
studying potential applications as photochemical CO-
releasing molecules (Photo-CORMs) without rapidly
reaching cytotoxic levels of carbon monoxide.
Preliminary results on bio-compatibility and selective
uptake of the compounds into human colon carcinoma
cells (Caco-2 cell line) are described.
Available online
Keywords
Photochemistry • Carbon Monoxide • Heme
Oxygenase Models • Rhenium Complexes • Cellular
Uptake • Biocompatibility
1. Introduction
Carbon monoxide usually is considered to be highly toxic
to humans, due to its fatal function as a respiratory inhibitor.
Nevertheless, small quantities of this gaseous molecule are
essential for a healthy life. As a result of enzymatic heme
oxidation, a significant amount of gaseous CO is generated
naturally in the human body where it acts as a small
molecular messenger and plays an important role in
regulating different cell functions. CO gas is known to dilate
blood vessels and to induce beneficial anti-inflammatory,
neuroprotective and anti-apoptotic effects.1 Synthetic CO-
releasing molecules (CORMs) are therefore widely used to
explore the therapeutic potential of future carbon monoxide
based drugs including applications in wound healing, organ
transplantation and cardiovascular protection.
Fig. 1. Structures of the investigated rhenium(I)-complexes.
2. Results and discussion
The intensely red-brown coloured organometallic
compounds 1-3 (Fig. 1) of the type fac-Re(1,2-
diimine)(CO)3X with X = Cl selected for the present study
have recently been synthesized and structurally characterized
in our group.4 In the solid state and in solution under ambient
conditions, these chloro-tricarbonyl rhenium complexes
display an excellent stability with no indication of
decomposition or loss of CO. When exposed to light,
however, the compounds are gradually transformed in a
photochemical reaction sequence leading to permanent
spectral changes shown here for the neutral complex 2 (Fig.
2). Irradiated solutions of 1 and 3 display a very similar
behaviour (ESI†). Closer analysis of the light-induced
spectral variations indicates a loss of the isosbestic points in
the visible spectral region, which is consistent with the
occurrence of a stepwise conversion.
An interesting approach to control the release of small
amounts of carbon monoxide under physiological conditions
is to develop biocompatible precursor compounds that can be
photochemically activated with visible light as an external
signal (Photo-CORMs).2,3 Here we describe the investigation
of air- and water-stable rhenium carbonyl complexes that can
be applied as non-cytotoxic CO-carriers for triggering the
release of carbon monoxide under physiological conditions.
Quantum yields for photochemical CO-release and
preliminary studies on the incorporation of the metal
complexes into cells of the human colon adeno-carcinoma
cell line Caco-2 are reported.

Quantitative detection of photoinduced CO-loss by the
complexes 1-3 was carried out with direct FTIR-
spectroscopic method described elsewhere in detail7 (Fig. 3).
a
Fig. 2. Absorption spectral changes during irradiation of the rhenium
carbonyl complex 2 in acetonitrile solution (29 K, 1 cm cell, c =
8.7 × 10-5 M, t = 0-2 h exposure to a 150 W Xe-
amp equipped with
8
8
Fig. 3. Headspace gas FTIR difference absorpption spectra of dark
control (dashed line) and light-exposed sampples of the rhenium
ll
a water filter).
carbonyl complex 2 in acetonitrile solution (2988 K, GC vial, c = 4.1
× 10-4 M, t = 0, 2 and 4 h exposure to a 150
with a water filter).
WW Xe-lamp equipped
The quantum yields for this UV-light depeendent photolysis
of the rhenium complexes are rather low (< 1%). Values of
φ(280 nm) = 2.9 × 10-3, 4.8 × 10-3 an
measured for compounds 1 and 2 in CH3C
the water-soluble derivative in
respectively. A quite similar reactivity wi
d
3.8 × 10-3 were
In a typical experiment, 2 mg (2.5 µmol) of 2 were
dissolved in 6 ml of dry CH3CN and the sample vial was
sealed with a gas-tight septum inside a gloovve box. From dark
NN solution and for
3
a
q
t
ueous solution,
th an irreversible
control and irradiated samples, headspace gas was drawn
with a syringe and injected into the FTIR measurement cell
to detect the amount of free carbon monoxxide. While no CO
was lost in the dark, a quantum yield of φ(CO) = 0.04 was
determined for CO-photorelease of the rhhenium complex 2.
Comparable values of φ(CO) = 0.03 and φ(CO) = 0.01 were
obtained with this method for complex 1 in CH3CN and
complex 3 in aqueous solution, respectivelyy (ESI†).
wavelength-dependent photolysis in CH3C
N
N
and a quantum
ppectral region has
yield maximum of φ = 3 × 10-3 in the UV-s
been reported before for the corresponding
with R = phenyl (Fig. 1).5 The quantu
r
r
henium complex
mm yield for this
decomposition reaction rapidly drops to zero with
monochromatic light in the visible sp
ee
ctral range. In
agreement with our earlier study and als
UV-photoreactivity reported for the closely related family of
o
Light-induced formation of free carbon monoxide with
Photo-CORMs such as 2 is therefore in thhe activity range of
160 nmol CO/h/mg under the conditionss reported here. It
can be estimated that in 4h of photoolysis about 50%
conversion of the Photo-CORM takes plaace, assuming that
only one carbon monoxide molecule per rhhenium center can
be ejected. Such a slow process may be partially due to a
competing back-reaction of the photolyzeed complex in the
presence of dissolved CO. In aqueous ssolution the water
fac-Mn(1,2-diimine)(CO)3X systems,3,6 the spectral changes
observed here (Fig. 2) are tentatively inte
r
preted as a UV-
light induced fac ꢀ mer isomerization of t
hh
e complexes 1-3
and a photosubstitution of the coordinateed chloro ligand
against a solvent molecule. Such a reactivity pattern was also
supported by positive ESI-mass spectrometry of the
photolyzed samples. Comparing the tren
yield data confirms our assumption that the presence of more
bulky aryl substituents on the nitrogen a oms of the 1,2-
diimine ligand can favour the release of t e rhenium bound
chloride ligand, which is also of considerable interest for
catalytic applications of these compounds 7
Note that the
UV-light induced reactivity described abo e, which is most
probably involving the direct population of a high-lying
repulsive metal-centered excited state of li
and field (1MC)
origin, does not lead to a permanent loss o
dd of the quantum
t
soluble complex
3 is permanently releasing carbon
h
monoxide with a rate of 40-50 nmoll CO/h/mg when
activated by light under the conditionns reported here.
Compared to many other compounds sugggested as CORMs
s
vv
.
for potential biomedical applications, thes
first glance seem to be not very promisingg. It should be kept
in mind, however, that the balance betw en any beneficial
ee low values at the
g
e
e
f
f
CO. This is also
therapeutic action of carbon monoxide and negative effects
of CO on oxygen transport and cellular resspiration has to be
very carefully controlled.1a In fact, the activity of natural
heme oxygenase (HO) enzymes respponsible for the
consistent with the IR-spectroscopic da
photolysis.5
tta obtained after
Upon polychromatic irradiation in solutiion, however, the
completely dark-stable complexes 1-3 were found to release
controlled release of carbon monoxide in
typically in the range of around 1 nmol C
still 50 times lower than the value off Photo-CORM 3
c
ells and tissues is
OO
/h/mg,8,9 which is
free carbon monoxide in significant
indicating a branching of the excited
a
mm
ounts. This is
sstate deactivation
pathways at lower energies, which can open additional
photoreactivity patterns involving the chr mophoric charge
transfer (CT) excited state manifold5,6 wh
ch is dominating
the absorption properties of the compou ds in the visible
spectral region. It is interesting to note, that such a an
additional photochemical reactivity arising from lower-lying
charge transfer excited states of otherwise eemissive metal-to-
ligand (MLCT) or ligand-to-ligand (LLCT) origin could also
explain the unusual non-luminescent behaviour reported
earlier for this kind of rhenium complexes.5
reported here. It should also be mentioned iin this context that
a convenient up- or down-regulation of t
o
i
nn
o
his inducible heme
oxygenase-like activity can be achieved
a
quantum yield by controlling the appliied light-intensity,
which is a well-known general advantagee of photochemical
enzyme model systems.10
In
a
preliminary study for poteential biomedical
applications of the new Photo-CORMs as photoactivable
metal-based drugs,11 we have started tto investigate the
properties of the novel water-soluble deerivative 3 under
physiological conditions. Different conccentrations of the

rhenium carbonyl complex up to 1000 µg/ml were applied in A reliable quantitative measure for potential dark
a cell culture medium (Fig. 4). It turned out that complex 3 cytotoxicity and cytolysis mediated by the Photo-CORM 3 in
displays excellent solubility in the test medium (DMEM vitro using the lactate dehydrogenase (LDH) assay17 could
without fetal bovine serum and phenol red).
not be obtained, because the deeply-coloured rhenium
carbonyl complex (Fig. 1) interfered with LDH-detection
usually carried out at
a wavelength of 490 nm in
commercially available enzymatic assays (data not shown).
Therefore, in order to determine the potential toxic effect of
the carbonyl complex 3 in Caco-2 cells, we tested poly ADP-
ribose polymerase (PARP) cleavage18 as an apoptosis marker
(ESI†). It turned out that neither the cell morphology nor
Fig. 4. Solubility of the rhenium carbonyl complex 3 at increasing
concentrations in DMEM cell culture medium with 4.5 g/liter
glucose, 4 mM glutamine, 100 units/ml penicillin, 100 mg/ml PARP cleavage was affected in the dark control or
streptomycin, 1% nonessential amino acids.
illuminated cells in the presence and absence of Photo-
CORM 3, and also the protein expression of the reference
marker GAPDH was relatively unchanged (Fig. 6).
Therefore, we assume that apoptosis effects by Photo-
CORM 3, as well as light-induced apoptosis (light control),
and apoptosis potentially induced by the experimental setting
used (dark control) did not a play role. Photo-CORM 3 will
therefore be very useful for further in vitro studies to explore
new biomedical applications because it is not leading to cell
death under these conditions.
We also investigated cell morphology effects and cellular
uptake of the rhenium carbonyl complex 3 with the human
cell line Caco-2 in culture (ESI†). Caco-2 cells are derived
from human colorectal adenocarcinoma and form
monolayers (like human intestinal epithelium) under
conventional culture conditions.12 They have been widely
used as a potent in-vitro model to predict drug absorption in
humans.13,14 Cell morphology did not change markedly after
24 h treatment with the rhenium carbonyl complex 3 which
was dissolved in the test medium (Fig. 5a, 5b). Therefore, we
suppose no significant cytotoxicity up to 24 h. However,
regions of intense cellular uptake of the red-brown coloured
complex were located within the cell monolayer (Fig. 5c),
and therefore also the dose-dependent uptake of 3 into
differentiated Caco-2 cells was spectrophotometrically
analyzed after cell lysis (ESI†). These observations are
currently investigated more closely by us.
Fig. 6. Effect of carbonyl complex 3 on the apoptosis marker “PARP
cleavage” using western blot analysis. D: dark control, L: light
exposed samples in the presence (+) or absence (-) of 3.
3. Conclusion
In summary, we have reported here the characterization of
three deeply coloured rhenium(I) carbonyl complexes
suitable for the photochemically controlled release of small
amounts of carbon monoxide in solution. Among these
compounds,
a
sulfonated fac-Re(1,2-diimine)(CO)3Cl
derivative (3) is described, which displays several highly
desirable properties necessary for potential biomedical
applications as a Photo-CORM including excellent aqueous
solubility, air- and water-stability and a reasonable com-
patibility with physiological conditions. First very promising
observations on apparently negligible dark-cytotoxicity and
selective cellular uptake are reported. Moreover, the
observed quantum yields for light-triggered CO release are
in an advantageous range, which should allow to
photochemically mimick some of the beneficial protective
effects of an up-regulated inducible heme oxygenase (HO-1)
function on demand without risking to reach otherwise toxic
levels of carbon monoxide under physiological conditions,
which might be a serious problem with many other CORMs
reported in the literature.1
Fig. 5. Cell morphology monitored with a light microscope after
differentiated Caco-2 cells were treated with compound 3 for 24
hours. a) control (0 µg/ml); b) 1000 µg/ml carbonyl complex 3; c)
1000 µg/ml carbonyl complex 3 after removal of the culture medium
and washing step.
A selective absorption and/or accumulation of the rhenium
carbonyl complex 3 in a distinct subpopulation of intestinal
cells, for instance colon cancer stem cells, would be of great
interest for new biomedical applications against colorectal
cancer. Colorectal cancer is one of the leading causes of
cancer deaths and metastatic colorectal cancer is badly
curable with currently available systemic therapeutic
options.15 It should be kept in mind that the Caco-2 cell line
consists of several subpopulations. For instance, this cell line
has previously been demonstrated to sustain a subpopulation
of cancer stem cells with tumor initiating capacity
characterized by the expression of the CD133 and CD44
surface markers.16
Acknowledgments
Financial support of this work by the Austrian Science
Fund (FWF project P25038: “Functional Light-responsive

Metal Carbonyl Systems“) and the State of Upper Austria is
gratefully acknowledged.
14 C. Schäfer, K. R. Schröder, O. Höglinger, S.
Tollabimazraehno, M. R. Lornejad-Schäfer, Cell Physiol.
Biochem. 2013, 32, 431.
15 J. Ferlay, E. Steliarova-Foucher, J. Lortet-Tieulent, S.
Rosso, J. W. W. Coebergh, H. Comber, D. Forman, F. Bray,
Eur. J. Cancer 2013, 49, 1374.
Notes and references
†
Electronic Supplementary Information (ESI) available:
16 N. Haraguchi, M. Okhuma, H. Sakashita, S. S. Matsuzaki, F.
Tanaka, K. Mimori, Y. Kamohara, H. Inoue, M. Mori,
Ann. Surg. Oncol. 2008, 15, 2927.
Experimental details and further data. See DOI:
1
R. Motterlini, R. Foresti, Antioxid. Redox Signal. 2014, 20
1810; J. Chen, Rev. Neurosci. 2014, 25, 269; R. Motterlini,
L. E. Otterbein, Nat. Rev. Drug Discovery 2010, , 728; B.
,
17 C. Korzeniewski, D. M. Callewaert, J. Immunol. Meth. 1983,
64, 313.
9
18 S. Elmore, Toxicol Pathol. 2007, 35, 495.
E. Mann, Top. Organomet. Chem. 2010, 32, 247; R. Alberto,
R. Motterlini, Dalton Trans. 2007, 17, 1651; R. Motterlini, J.
E. Clark, R. Foresti, P. Sarathchandra, B. E. Mann, C. J.
Green, Circulation Res. 2002, 90, e17; Maines, M. D.,
Heme Oxygenase: Clinical Applications and Functions,
CRC, Boca Raton, FL, 1992.
2
3
S. García-Gallego, G. J. L. Bernardes, Angew. Chem. Int. Ed
2014, 53, 9712; R. D. Rimmer, A. E. Pierri, P. C. Ford,
Coord. Chem. Rev. 2012, 256 1509; G. Knör, U.
Monkowius, Adv. Inorg. Chem. 2011, 63 235; U.
Schatzschneider, Inorg. Chim. Acta, 2011, 374, 19.
.
,
,
V. Yempally, S. J. Kyran, R. K. Raju, W. Y. Fan, E. N.
Brothers, D. J. Darensbourg, A. A. Bengali, Inorg. Chem.
2014, 53, 4081; S. H. Heinemann, T. Hoshi, M.
Westerhausen, A. Schiller, Chem. Commun. 2014, 50, 3644;
S. J. Carrington, I. Chakraborty, P. K. Mascharak, Chem
Commun. 2013, 49, 11254; P. Peng, C. Wang, Z. Shi, V. K.
Johns, L. Ma, J. Oyer, A Copik, R. Igarashia, Y. Liao, Org.
Biomol. Chem. 2013, 11, 6671; J. S. Ward, J. M. Lynam, J.
W. B. Moir, D. E. Sanin, A. P. Mountford, A. P., I. J. S.
Fairlamb, Dalton Trans. 2012, 41, 10514.
4
5
E. Kianfar, U. Monkowius, E. Portenkirchner, G. Knör, Z.
Naturforsch. 2014, 69b, 691.
G. Knör, M. Leirer, A. Vogler, J. Inf. Recording 1998, 24, 69;
G. Knör, M. Leirer, T. Keyes, J. Vos, A. Vogler, Eur. J.
Inorg. Chem. 2000, 749.
6
I. R. Farrell, A. Vlček Jr., Coord. Chem. Rev. 2000, 208, 87;
F. Hartl, P. Rosa, L. Ricard, P. Le Floch, S. Záliš, Coord.
Chem. Rev. 2007, 251, 557.
7 E. Portenkirchner, K. Oppelt, C. Ulbricht, D. A. M. Egbe, H.
Neugebauer, G. Knör, N. S. Sariciftci, J. Organomet. Chem.
2012, 716, 19; E. Portenkirchner, E. Kianfar, N. S. Sariciftci,
G. Knör, ChemSusChem 2014,
7, 1347; M. Klein, U.
Neugebauer, A. Gheisari, A. Malassa, T. M. A. Jazzazi, F.
Froehlich, M. Westerhausen, M. Schmitt, J. Popp, J. Phys.
Chem. A, 2014, 118, 5381.
8
9
H. J. Vreman, D. K. Stevenson, Analyt. Biochem. 1988, 168,
31.
E. F. Chang, R. J. Wong, H. J. Vreman, T. Igarashi, E. Galo,
F. R. Sharp, D. K. Stevenson, L. J. Noble-Haeusslein, J.
Neurosci. 2003, 23, 3689.
10 G. Knör, Chem. Eur. J. 2009, 15, 568.
11 N. A. Smith, P. J. Sadler, J. Phil. Trans. R. Soc. A 2013, 371
,
20120519.
12 M. Pinto, S. Robine-Leon, M.-D. Appay, M. Kedinger, N.
Triadou, E. Dussaulx, B. Lacroix, P. Simon-Assmann, K.
Haffen, J. Fogh, A. Zweibaum, Biol. Cell 1983, 47, 323.
13 P. Artursson, J. Pharm. Sci. 1990, 79, 476.

Synopsis for the Graphical Abstract
Bioorganometallic
Photochemistry
Air- and water-stable rhenium carbonyl
complexes are investigated as CO-
carriers for studying the light-dependent
release of small amounts of carbon
monoxide under physiological conditions.
The reported very low quantum yields
are in a suitable range for studying
potential applications as photochemical
CO-releasing molecules (Photo-CORMs)
without rapidly reaching cytotoxic levels
of carbon monoxide. Preliminary results
on bio-compatibility and selective uptake
of the compounds into human colon
carcinoma cells (Caco-2 cell line) are
described.
Elham Kianfar, Christine Schäfer,
Mohammad Reza Lornejad-Schäfer,
Engelbert Portenkirchner, Günther Knör *
New Photo-CORMs: Deeply-coloured
biocompatible Rhenium Complexes for
the Controlled Release of Carbon
Monoxide

New Photo-CORMs: Deeply-coloured Biocompatible Rhenium
Complexes for the Controlled Release of Carbon Monoxide
Elham Kianfar,a Christine Schäfer,b Mohammad Reza Lornejad-Schäfer,b Engelbert Portenkirchner c and Günther
Knör *a
Highlights :
•
•
•
•
Non-toxic dark-stable and water-soluble carbonyl carrier
Excellent biocompatibility and efficient cellular uptake
Light-triggered carbon monoxide release characterized
Photochemical modelling of heme oxygenase function
Keywords:
•
•
•
•
•
•
Photochemistry
Carbon Monoxide
Heme Oxygenase Models
Rhenium Complexes
Cellular Uptake
Biocompatibility