17 e- rhenium dicarbonyl CO-releasing molecules on a cobalamin scaffold for biological application

Article abstract of DOI:10.1039/c1dt10649j

Cyanocobalamin (B₁₂) offers a biocompatible scaffold for CO-releasing 17-electron dicarbonyl complexes based on the cis-trans-[Re ^II(CO)₂Br₂]⁰ core. A Co-CN-Re conjugate is produced in a short time and high yield from the reaction of [Et₄N]₂[Re^IIBr₄(CO)₂] (ReCORM-1) with B₁₂. The B₁₂-Re^II(CO) ₂ derivatives show a number of features which make them pharmaceutically acceptable CO-releasing molecules (CORMs). These cobalamin conjugates are characterized by an improved stability in aqueous aerobic media over the metal complex alone, and afford effective therapeutic protection against ischemia-reperfusion injury in cultured cardiomyocytes. The non-toxicity (at μM concentrations) of the resulting metal fragment after CO release is attributed to the oxidation of the metal and formation in solution of the ReO₄^- anion, which is among the least toxic of all of the rare inorganic compounds. Theoretical and experimental studies aimed at elucidating the aqueous chemistry of ReCORM-1 are also described.

Full text of DOI:10.1039/c1dt10649j

Dalton
Transactions
An international journal of inorganic chemistry
www.rsc.org/dalton
Volume 41 | Number 2 | 14 January 2012 | Pages 309–680
ISSN 1477-9226
COVER ARTICLE
Zobi et al.
17 e rhenium dicarbonyl CO-releasing molecules on
a cobalamin scaffold for biological application

Dynamic Article Links
Dalton
Transactions
Cite this: Dalton Trans., 2012, 41, 370
www.rsc.org/dalton
PAPER
17 e- rhenium dicarbonyl CO-releasing molecules on a cobalamin scaffold for
biological application†
Fabio Zobi,*a Olivier Blacque,a Robert A. Jacobs,b Marcus C. Schaubc and Anna Yu. Bogdanovab
Received 13th April 2011, Accepted 22nd July 2011
DOI: 10.1039/c1dt10649j
Cyanocobalamin (B12) offers a biocompatible scaffold for CO-releasing 17-electron dicarbonyl
complexes based on the cis-trans-[ReII(CO)2Br2]0 core. A Co–C N–Re conjugate is produced in a short
time and high yield from the reaction of [Et4N]2[ReIIBr4(CO)2] (ReCORM-1) with B12. The
B12-ReII(CO)2 derivatives show a number of features which make them pharmaceutically acceptable
CO-releasing molecules (CORMs). These cobalamin conjugates are characterized by an improved
stability in aqueous aerobic media over the metal complex alone, and afford effective therapeutic
protection against ischemia-reperfusion injury in cultured cardiomyocytes. The non-toxicity (at mM
concentrations) of the resulting metal fragment after CO release is attributed to the oxidation of the
-
metal and formation in solution of the ReO4 anion, which is among the least toxic of all of the rare
inorganic compounds. Theoretical and experimental studies aimed at elucidating the aqueous
chemistry of ReCORM-1 are also described.
Despite its current evaluation in novel therapies, the use of
Introduction
gaseous CO poses several problems related to safe handling
In the last decade, the use of carbon monoxide (CO) as a
and delivery to specific target sites in a controlled and mea-
cytoprotective and homeostatic molecule has received increasing
surable fashion. The challenges associated with clinical applica-
attention in medicine due to its documented beneficial therapeutic
tion of the gas by inhalation have sparked the design of CO-
effects.1,2 There are three main areas where CO is evaluated
as a clinically valuable medical agent: 1) inflammation, 2)
releasing molecules (CORMs) as an alternative approach to the
administration of CO (e.g. orally or by injection). Transition
cardiovascular diseases and 3) organ preservation and trans-
metal carbonyl complexes have been predominantly evaluated
plantation. The anti-inflammatory properties of CO have been
as CORMs. There are numerous examples available in the
literature,13–25 of which three molecules have been most extensively
investigated in biology and medicine: the lipid-soluble tricar-
bonyldichlororuthenium(II) dimer ([Ru(CO)3Cl2]2, CO-RM2),26–28
the water soluble tricarbonylglycinatoruthenium(II) monomer
([Ru(CO)3Cl(glycinato)], CO-RM3)29–34 and the boron-containing
carboxylic acid Na2[H3BCO2] salt (CO-RMA1).35–38 Treatment of
several diseases with these CORMs has shown that the molecules
elicit the same type of therapeutic effects as CO gas.2
Design, identification and characterization of new CORMs
constitutes an active field of research not only because of the
aforementioned problems associated with the clinical application
of the gas, but also because in some cases the use of CORMs has
an advantage over CO inhalation. For instance, the bactericidal
effect of CO-RM3 can prevent sepsis-induced death in mice, while
CO gas does not seem to directly affect bacterial survival.32 As
recently pointed out,2 none of the characterized CO-releasing
carbonyl complexes displays acceptable properties as required for
a medical drug. Motterlini and Otterbein put forward a number
of requirements CORMs should meet.2 These include: 1) water
solubility and biocompatibility, 2) stability in aqueous aerobic
media, 3) slow decay of the M(CO)x fragment in the blood, 4) low
toxicity and rapid excretion of the metal scaffold after CO release.
corroborated in a large number of animal models including cere-
bral malaria inflammation,3 rheumatoid arthritis,4 autoimmune
neuroinflammation,5 sickle cell disease,6,7 diabetes8,9 and acute
hepatitis.10,11 The protective effects of CO as a vasodilator have
been successfully evaluated for several cardiovascular diseases
including pulmonary arterial hypertension, for which there is no
cure.12 Carbon monoxide proved effective in prolonging organ
graft survival, particularly in heart and kidney transplants for
which CO inhalation (3 mg per kg for 1 h) has entered Phase II
clinical trials.2
aInstitute of Inorganic Chemistry, University of Zu¨rich, Winterthurerstrasse
190, CH-8057, Zu¨rich, Switzerland. E-mail: fzobi@aci.uzh.ch; Fax: (+41)
044 635 6802; Tel: (+41) 044 635 4623
bInstitute of Veterinary Physiology, University of Zu¨rich, Winterthur-
erstrasse 260, CH-8057, Zu¨rich, Switzerland
cInstitute of Pharmacology and Toxicology, University of Zu¨rich, Win-
terthurerstrasse 190, CH-8057, Zu¨rich, Switzerland
† Electronic supplementary information (ESI) available: Spectroscopic
details (HPLC, MS, IR, UV-Vis, 1H-NMR) of B12-ReCORM-2 and 4
and of molecules 3 and 5. Theoretical details and UV-Vis absorption
spectra. Effect of CORMs’ supplementation on the oxygen levels in the cell-
free culture medium and on NRCs respiration. CCDC reference numbers
817626 and 817627 (compounds 3 and 5). For ESI and crystallographic
data in CIF or other electronic format see DOI: 10.1039/c1dt10649j
370 | Dalton Trans., 2012, 41, 370–378
This journal is
The Royal Society of Chemistry 2012
©

Within this context, we have recently described a family of
ReII-based CORMs.39,40 These uncommon monomeric 17-electron
dicarbonyl species are unique in the field as the rate of CO-
release (and thus the decay of the Re(CO)2 fragment) can be
conveniently modulated by pH and by ligand variation on the
basic cis-trans-[Re(CO)2Br2]0 core. The molecules were further
shown to protect cultured cardiomyocytes against ischemia-
reperfusion injury by increasing cell survival by up to 83%.39
However, most complexes showed poor water solubility. In order
to meet the above mentioned requirement for a pharmaceutically
acceptable CORM, we have begun to study the reactivity of the
[Et4N]2[ReIIBr4(CO)2] (ReCORM-1) salt with biomolecules. In this
contribution we present cyanocobalamin (B12) derivatives of 17-
electron rhenium dicarbonyl species which, as a second generation
of ReII-based CORMs, show several improved features over the
original complexes. The derivatives herein described: a) are fully
water soluble and biocompatible, b) show improved stability in
aqueous aerobic media over the metal complex alone, c) are
non-toxic towards cultured cardiomyocytes, d) protect these cells
against ischemia-reperfusion injury and e) after CO release, in
water under aerobic conditions, the rhenium complex is oxidized
to ReO4- which is among the least toxic of all of the rare inorganic
compounds.41
the [Et4N]2[ReIIBr4(CO)2] (ReCORM-1) salt in methanol. HPLC-
MS traces recorded during the reaction revealed the formation of
a single product in quantitative yield with a mass of m/z = 1758.4
and an isotope pattern corresponding exactly to the theoretical
mass of B12-ReCORM-2 after loss of a solvent molecule (see
ESI†). The absence of a negative MS signal corresponding to
an adduct with three coordinated bromides and the recovery
of nearly two equivalents of [Et4N]Br after workup are all in
agreement with the proposed formulation of B12-ReCORM-2.
The IR spectrum of B12-ReCORM-2 showed a single nCN band at
2184 cm-1, which is shifted by 50 cm-1 to higher frequency than the
corresponding stretching vibration of B12. Similar shifts to higher
energy have been reported for other B12-metal complex adducts
and are characteristic for bridging cyanides.42–44 Alongside the nCN
band at 2184 cm-1 the IR spectrum of B12-ReCORM-2 shows
two nCO stretching frequencies at 1989 and 1839 cm-1 which are
characteristic of cis-trans-[ReII(CO)2Br2L2] complexes (ESI†).39,40
The anion of ReCORM-1 rapidly coordinates to the cyano
group of B12. Under our conditions the reaction is complete in
ca. 90 min. In comparison similar adducts of cyanocobalamin
with fac-[ReI(CO)3]+ complexes and cisplatin are formed at higher
temperatures after prolonged reaction times (up to 16 h).42–44 If the
reaction is allowed to proceed for 12 h the major B12-Re adduct
was identified as a B12 complex of fac-[ReI(CO)3Br(H2O)]+. This
product (5 in Scheme 1) was isolated and it was possible to grow
X-ray quality crystals (Fig. 1).45 Compound 5 crystallized in the
P212121 space group. The Re atom (ca. 50% occupancy) sits on
Finally, as it is our intention to further develop these potential
drugs, we have devised for the species a general nomenclature
of the type: X-MCORM-#; where X = carrier biomolecule (here
B12); M = transition metal (here Re); CORM explicitly denotes
the function; and # = associated numeric value to differentiate the
molecules. This nomenclature is used throughout the text.
˚
top of the cyano ligand with a Re–N distance of 2.24 A, ca.
˚
0.09 A longer than the same distance found in the other two
known B12-ReI(CO)3 adducts.42 The C–N–Re angle has a value
of 160◦ being significantly more acute than those found in Re
and Pt analogues.42–44 The cobalamin bond lengths and angles are
similar to those of native B12. Due to the inherent instability of
B12-ReCORM-2 (vide infra) it was not possible to obtain single
X-ray quality crystals of the compound. However, in order to
obtain insights into the structure of this adduct, calculations at the
density functional level of theory were performed. The calculated
structure of B12-ReCORM-2 is shown in Fig. 1. Bond lengths
and angles around the Re coordination sphere were found to be
in good agreement with those of the cis-[ReII(CO)2Br3(NCCH3)]-
anion previously published.40 The DFT calculations also correctly
predicted the bending of the two coordinated trans bromides away
from the CO ligands always observed in the solid state structures
of the family of cis-trans-[ReII(CO)2Br2L2] complexes.
Results and discussion
Synthesis and characterization
The B12-ReII(CO)2 adduct (B12-ReCORM-2, see Scheme 1) was
prepared in good yield from the reaction of cyanocobalamin and
The UV-visible spectrum of B12-ReCORM-2 is virtually iden-
tical to that of native B12 except for a pronounced absorption
at 406 nm ascribed to the presence of the coordinated ReII
complex (Fig. 2).39,40 In water the intensity of this signal de-
creased over time. Theoretical calculations of a model cis-trans-
[ReII(CO)2Br2(H2O)(N C–H)] complex revealed that in water the
observed changes are consistent with the substitution of the bound
trans bromide ions by water molecules and the formation of a
cis-[ReII(CO)2(H2O)3(N C–H)]2+ species (ESI†). The presence in
solution of the B12-cis-[ReII(CO)2(H2O)3]2+ adduct (2a in Scheme
2, vide infra) was also confirmed via HPLC-MS analysis.
Scheme 1 Synthesis of B12-adduct 3 and B12-ReII(CO)2 conjugates
B12-ReCORM-2 and B12-ReCORM-4. Reagents and conditions: i)
[Et4N]2[ReIIBr4(CO)2] (ReCORM-1), methanol/water, 1.5 h, 40–50 ◦C;
ii) CDI, DMSO, 12 h, 60 ◦C followed by 4-picolylamine, 0.5 h, RT; iii)
ReCORM-1, methanol/water, 12 h, 40–50 ◦C.
In order to assess the versatility of B12-ReCORM-2 for ap-
plications in biology, knowledge about the water stability of
the compound is crucial. Immediately after dissolution in water
(at 25 ◦C, aerobic conditions), the HPLC-MS trace reveals the
This journal is
The Royal Society of Chemistry 2012
Dalton Trans., 2012, 41, 370–378 | 371
©

Scheme 2 The postulated sequence of events leading to CO-release from
B12-ReCORM-2.
B12-ReCORM-2 exchanged the coordinated bromides for water
molecules. It is however difficult to pinpoint the right sequence
of events which lead to the formation of a mono carbonyl species
after release of carbon monoxide from the cis-[ReII(CO)2] core.
Analysis of the kinetics of CO release is described in the following
paragraphs. Here it should be noted that solvation of B12-CORM-2
and CO release might be concurrent events. However, the absence
of MS signals corresponding to a Re mono carbonyl species
containing bromide ions is in support of the sequence illustrated
in Scheme 2.
We have previously shown that the [Et4N]2[ReIIBr4(CO)2] salt
(ReCORM-1) reacts with pyridine and imidazole type ligands
yielding stable complexes.39,40 In order to further improve the
stablility of the B12-ReII(CO)2 adduct, B12 was derivatized with a
pendent pyridine arm at the 5¢-OH position of the ribose according
to a modified procedure recently described by Grissom et al. (3 in
Scheme 1).46 The X-ray structure of 3 is shown in Fig. 1 confirming
the presence of a pyridine unit on the sugar moiety.47 The adduct
crystallized in the P212121 space group. Bond lengths and angles
are not significantly different than those of native B12.
The reaction of 3 with ReCORM-1, under similar conditions
to those described for the formation of B12-ReCORM-2, gave a
single product in quantitative yield (B12-ReCORM-4 in Scheme
1). The isolated product showed a mass of m/z = 1893.4 with
an isotope pattern corresponding to the theoretical mass of B12-
ReCORM-4 after loss of a solvent molecule. The IR spectrum of
B12-ReCORM-4 showed a single nCN band at 2184 cm-1 and nCO
stretching frequencies at 1989 and 1839 cm-1 at the same energy
to those of B12-ReCORM-2, indicating the formation of the same
Co–C N–Re conjugate. Under conditions of limiting amounts
of 3 (i.e. with 5 fold excess of ReCORM-1) we found evidence of
a bis-Re adduct of 3 but by allowing the reaction to proceed for
several hours, a mixture of products comprising derivatives of the
fac-[ReI(CO)3]+ core was obtained (ESI†). In water B12-ReCORM-
4 showed similar behaviour to that described for B12-ReCORM-2.
The optimized structure of B12-ReCORM-4 is shown in Fig. 1.
The calculated structure parameters are similar to those found in
B12-ReCORM-2.
Fig.
1
DFT calculated structures of B12-ReCORM-2 and
B12-ReCORM-4 and ORTEP plots of X-ray structures of 3 and 5.
Ellipsoids are drawn at 50% probability, hydrogen atoms are omitted
for clarity. Selected bond lengths (A) and angles ( ) for 5 are: Re–Br1
2.6482(19); Re–O18 2.072(12); Re–N7 2.221(7); Re–C67 1.926(16);
Br1–Re–N7 89.32(18), O18–Re–N7 79.3(4); C67–Re–N7 170.5(6).
Crystallographic details are summarized in the ESI.†
◦
˚
Fig. 2 Changes in the UV-visible spectrum of an unbuffered aqueous
solution of B12-ReCORM-2. Spectra were recorded at fixed intervals of
5 min at 25 ◦C.
CO releasing properties of B12-ReCORM-2 and B12-ReCORM-4
The carbon monoxide releasing properties of B12-ReCORM-2
and B12-ReCORM-4 were evaluated by the myoglobin assay (Fig.
3).30,31 In these experiments, an aliquot of a freshly prepared
concentrated solution of the cobalamin adducts was added to
a buffered solution of horse skeletal myoglobin (Mb), freshly
reduced with excess sodium dithionite under N2. The conversion
of Mb to carbon monoxide myoglobin (MbCO) was followed over
presence of a single peak ascribed to B12-ReCORM-2. After
30 min six distinct peaks were observed. These were identified
by HPLC-MS as a mixture of B12-ReCORM-2, 2a, 2b free B12,
aquocobalamin, ReO4 and a Re complex whose coordination
sphere could not be unambiguously assigned (Scheme 2, ESI†).
It is clear that after dissolution in water the ReII(CO)2 species in
-
372 | Dalton Trans., 2012, 41, 370–378
This journal is
The Royal Society of Chemistry 2012
©

slowly disappeared and after ca. 30 min a single major peak was
detected at a frequency of 1887 cm-1 (ESI†).
The liquid IR evidence in phosphate buffer clearly points to
a reduction of the cis-[ReII(CO)2]2+ core and formation of a
complex comprising a cis-[ReI(CO)2]+ moiety. This reduction is
evidenced by the shift of carbonyl stretching frequencies of ca.
100 cm-1 to lower frequency when compared to the spectrum of
ReCORM-1 in methanol (nCO frequencies at 2000 and 1856 cm-1,
see reference 40). After 30 min the presence of a single C
O
stretching frequency indicates a monocarbonyl complex, pointing
at the release of 1 equivalent of CO. The reduction of ReCORM-
1 in water was further confirmed by MS data (ESI†). The
MS spectrum of an aqueous solution of ReCORM-1, recorded
soon after sample preparation (ESI†), showed two clear peaks
assigned to a [ReII(CO)2Br3]- ion (m/z = 481.7) and to a reduced
[ReI(CO)2Br2(H2O)(CH3OH)]- species (m/z = 454.0). After 12 h
the same sample showed signals at m/z = 250.2 and 428.9 assigned
Fig. 3 A typical spectrum of conversion of deoxy-myoglobin (Mb)
to carbonmonoxy myoglobin (MbCO) by cobalamin derivatives
B12-ReCORM-2 and B12-ReCORM-4. Here reproduced are the changes of
the spectrum of a 30 mM Mb solution after the addition of B12-ReCORM-2
(30 mM, 25 ◦C, 0.1 M phosphate buffer pH 7.4, spectra intervals = 5 min).
to ReO4 and the monocarbonyl [ReI(CO)Br2(H2O)3]- complex
-
respectively (ESI†).
Rapid titration of an aqueous solution of ReCORM-1 (0.1 M
◦
KNO3, 25 C, completed within 15 min) indicated Br- as the
possible reducing agent. Base titration of an aqueous solution
of ReCORM-1 showed three distinct acid–base equilibria (ESI†).
The corresponding pKa values are 3.8, 6.0 and 8.9. The first two
steps are tentatively assigned to the formation of the mononuclear
deprotonation products cis-[ReII(CO)2Br(H2O)2(OH)] and cis-
[ReI(CO)2Br(H2O)2(OH)]- (1b and 1d respectively in Scheme 3).
The pKa value at 8.9, on the other hand, is attributed to the
presence in solution of hypobromite (BrO-), in agreement with
the value reported for the oxidation of bromide-containing water
and hypobromite formation.48
time by measuring the changes in the absorption spectra of the Q
band region of this protein at 25 ◦C and at pH 7.4 after addition of
B12-ReCORM-2 and B12-ReCORM-4. The maximum absorption
peak of Mb at 551 nm was rapidly converted over time to the
spectrum of MbCO, with two maximum absorption peaks at 540
and 578, respectively. A typical spectrum is shown in Fig. 3.
Both compounds elicited the spectral changes associated with
CO release. The amount of MbCO formed over time after addition
of the cobalamin adducts to the Mb solution was calculated
according to the known extinction coefficients.30,31 The rate of
CO loss from B12-ReCORM-2 and B12-ReCORM-4 was found to
be the same within experimental error with a half life (t1/2) of ca.
20 min. In comparison ReCORM-1 under the same conditions
shows a t1/2 for CO release of 6 min.39 This difference further
corroborates the notion that simple ligand substitution of the cis-
trans-[ReII(CO)2Br2]0 core is able to modulate the kinetics of the
thermal dissociation of carbon monoxide and thus the decay of
the Re(CO)2 fragment. When the experiments were performed
under conditions of a limiting amount of B12-ReCORM-2 or B12-
ReCORM-4, taking into account the molar extinction coefficient
of MbCO, it was found that approximately 1 mol of CO was
released per mole of the corresponding complex within 2 h.
Scheme 3 A possible mechanism underlying the CO-release from
-
ReCORM-1 and its subsequent oxidation to the ReO4 anion.
Taken together, these results suggest the following mechanism
for the CO release of ReCORM-1 (Scheme 3). When dissolved
in water three bromides in ReCORM-1 are rapidly exchanged
for solvent molecules. Theoretical calculations of the progressive
aquation of ReCORM-1 are in full support of this assignment
(ESI†). Of the calculated UV-Vis spectra of the different aquo
complexes the only one closely matching the experimental spec-
trum (two absorption maxima at 335 and 389 nm) is the one
assigned to species 1a in Scheme 3 (ESI†). Substitution of three
Br- by three water molecules is postulated to increase the ReI
→ ReII redox couple of 1a to a value sufficient to drive the
oxidation of Br- to BrO-. The value for the ReI → ReII redox
couple of ReCORM-1 is reported at ca. +0.1V (vs. NHE).40
According to the electrochemical parameters described by Lever,49
substitution of three Br- by three water molecules should increase
a metal M(n-1)/M(n) redox potential by +0.78 V. This value is
Aqueous chemistry of ReCORM-1
The aqueous chemistry of ReCORM-1 was studied in detail in
order to delineate a possible mechanism of CO release from
the compounds herein described. Due to the rapid CO loss,
the paramagnetic nature of ReCORM-1 and the lack of ligands
bearing active NMR probes the main techniques employed in this
study were IR spectroscopy and MS. The salt of ReCORM-1 is
soluble in water and shows two absorption maxima at 335 and
389 nm (ESI†). When a 1 mM solution of ReCORM-1 was
prepared in distilled water the pH dropped rapidly to 3. The liquid
IR spectrum of ReCORM-1 in phosphate buffer (0.1 M, pH =
7.4) showed three distinct signals within only a few minutes after
dissolution. Two signals were observed at 1884 and 1782 cm-1
together with a small peak at 1925 cm-1 (ESI†). These signals
This journal is
The Royal Society of Chemistry 2012
Dalton Trans., 2012, 41, 370–378 | 373
©

already sufficient to drive the oxidation of bromide in water to
hypobromite whose value for the redox reaction is reported as
+0.76 V (vs. NHE).48
Following reduction of the solvated ReII complex the corre-
sponding ReI species is formed (1c in Scheme 3) which may
undergo further acid–base equilibrium (pKa 6.0) to generate the
cis-[ReI(CO)2Br(H2O)2(OH)]- complex (1d). One of the reduced
cis-[ReI(CO)2] species is then postulated to be the CO-releasing
entity liberating 1 equivalent of carbon monoxide within 30 min
under physiological conditions. Finally in water under aerobic
conditions, the monocarbonyl ReI complex (clearly detected by
-
liquid IR and MS, ESI†) is oxidized to ReO4 .
Cytoprotective effects of B12-ReCORMs
Fig.
4
Cytoprotective effects of conjugates B12-ReCORM-2 and
B12CO-RM-4 (30 mM) against “ischemia-reperfusion” injury (I/R). Cell
damage in neonatal rat ventricular cardiomyocytes (NRCs) after 16 h
of ischemia (hypoxia, aglycemia, and acidosis). CORMs or the aliquot
of solvent (DMSO) were added to the cell culture medium at the
onset of the reperfusion period of 9 h at 30 mM concentration. Bars
indicate the % of PI-positive (dead) cells. ** denotes p < 0.01 when I/R
B12-ReCORM-2-treated cells are compared to I/R control. # denotes p <
0.05 when normoxic cells are compared with I/R control.
B12-ReCORM-2 and 4 were tested for their cytotoxic and their
cytoprotective effects using the neonatal rat cardiomyocyte (NRC)
cell-based model of ischemia-reperfusion injury (I/R) as previ-
ously described.39 Cytotoxicity of some ReII-based CORMs ob-
served earlier was most likely associated with their accumulation in
the cells, followed by formation of cytotoxic intermediates such as
4-methylpyridine which may interfere with the cell metabolism.39
The membrane-impermeable ReII-CORMs studied so far were
non-toxic in the micromolar concentration range. To test for the
possible uptake of B12-ReCORMs by cells, incubation of NRCs
with 30 mM of B12-ReCORM-2 and 4 was performed and cell
culture medium samples were collected over 180 min of incubation.
Atomic absorption spectroscopy (AAS) measurements showed
that the rhenium concentration in the medium supplemented with
B12-ReCORM-2 and B12-ReCORM-4 did not change over time.
This observation implies that complexes (or the dissociated Re
fragment) did not enter the cells through the cell surface membrane
during a 3 h incubation period (data not shown). The fraction of
dead cells tended to decrease in the presence of CORMs, but,
due to the high variability, the differences were not statistically
significant (2.7 1.7% in control vs. 1.1 0.3 and 1.0 0.2 in the
presence of B12-ReCORM-2 and B12-ReCORM-4 respectively).
Exposure of the NRCs to the conditions mimicking ischemia-
reperfusion (I/R) resulted in a 5-fold increase in the number
of dead cells (control in Fig. 4). Administration of 30 mM
B12-ReCORM-2 at the “onset of reperfusion” nearly prevented
cell mortality (cell death was reduce by ca. 80% as compared
to control) whereas 30 mM B12-ReCORM-4 reduced cell death
by ca. 50%. Thus, B12-ReCORM-2 proved to be more efficient
in preventing I/R-induced cell death than B12-ReCORM-4.
Therefore B12-ReCORM-2 was chosen for further investigation
and compared to [Et4N]2[ReIIBr4(CO)2] (ReCORM-1) and the
previously reported cis-trans-[ReII(CO)2Br2(Im)2] complex (where
Im = imidazole, ReCORM-6) that also exhibited substantial cy-
toprotective effects.39 All three compounds release CO in aqueous
solution, albeit at different rates, and with the exception of the
lipophilic ReCORM-6 show good water solubility. Under normal
physiological conditions the compounds showed no cytotoxicity
towards NCRs up to a tested concentration of 120 mM.
significant effect on oxygen consumption by NRCs within the
concentration range relevant for cytoprotection within 30 min of
observation (ESI†). The lack of effect neither depended on the
rate of CO release nor on the compound solubility. Interestingly,
addition of ReCORM-1, and to a lesser extent of B12-ReCORM-2,
resulted in deoxygenation of the cell-free medium within minutes
after administration. This CORM-induced deoxygenation was
dose-dependent (ESI†) and most likely reflected the reaction
describing the formation of ReO4- in Scheme 3. The deoxygenation
efficiency followed the kinetics of CO release (and thus the decay
of the Re(CO)2 fragment) and was maximal for the fast-releasing
ReCORM-1 compound (Fig. 5A and ESI†).
Oxidative stress triggered by acute hyperoxygenation is a
hallmark of reperfusion injury. Based on the findings presented
in Fig. 5A, selected ReII-based-CORMs may be viewed as
oxygen scavengers which, when applied early on during reper-
fusion, may reduce the cellular oxidative damage. To test this
hypothesis intracellular reduced glutathione (GSH) levels were
determined in cells 10 min after the “onset of reperfusion” in
the presence or absence of 30 mM ReCORM-1, ReCORM-6,
and B12-ReCORM-2. As shown in Fig. 5B, presence of the
ReCORM-1 complex at reperfusion resulted in an increase in
the intracellular reduced glutathione (GSH) levels reflecting its
antioxidative action. This acute antioxidative effect of ReCORM-
1 most likely reflects the ability of the intermediates formed
-
upon CO release to react with oxygen ultimately yielding ReO4
(see Scheme 3).
Although the molecular mechanisms of the observed cyto-
protective effects of the above-mentioned ReII-based-CORMs
remain unknown, their cytoprotective action may, at least in
part, be attributed to the extracellular release of CO and to the
de-oxygenating effect described above. Finally, our observations
suggest that the protective effect of the ReII-based CORMs is not
related to a decrease in mitochondrial respiration and secondary
free radical production.50
Carbon monoxide is known to suppress respiration in different
cell types by inhibiting the mitochondrial electron transfer chain
and by interfering with oxygen binding.50 Therefore, the effect
of the three selected CORMs on oxygen consumption by NRCs
was investigated. None of the tested compounds displayed any
374 | Dalton Trans., 2012, 41, 370–378
This journal is
The Royal Society of Chemistry 2012
©

underlying the cytoprotective effects of these species and to the
synthesis of ReII(CO)2 conjugates of other naturally occurring
molecules.
Experimental section
Chemicals and solvents were purchased from standard sources.
All synthesis were performed under an inert N2 atmosphere
unless otherwise noted. ReCORM-1 was synthesized according
to literature.40 Elemental analyses (EA) were performed on a Leco
CHNS-932 elemental analyzer. IR spectra were recorded in a
PerkinElmer Spectrum BX FT-IR spectrometer. Crystallographic
data were collected at 183(2) K with Mo-Ka radiation (l =
˚
0.7107 A) that was monochromated with help of a graphite
monochromator on an Oxford Diffraction Xcalibur system with
a Ruby detector. Suitable crystals were covered with oil (Infineum
V8512), mounted on top of a glass fiber and immediately trans-
ferred to the diffractometer. The program suite CrysAlisPro was
used for data collection, semi-empirical absorption correction and
data reduction.51 Structures were solved with direct methods using
SIR9752 and were refined by full-matrix least-squares methods on
F2 with SHELXL-97.53 The structures were checked for higher
symmetry with help of the program Platon.54
Analytical HPLC method
Instrument: MERCK HITACHI LaChrom with a D-7000 inter-
face coupled with a Diode Array detector L-7455 and a pump L-
7100 system. Column: Macherey-Nagel, EC250/3 Nucleosil 100-
5 C18. Flow rate 0.5 mL min-1. Absorbance monitored at 250
nm. Solutions: A: 0.1% trifluoroacetic acid in water; B: methanol.
Chromatographic method: 0–5 min: isocratic flow of 75% A–25%
B; 5–30 min: linear gradient to 100% B; 30–35 min: isocratic flow
of 100% B; 35–40 min linear gradient to 75% A–25% B.
Fig. 5 Regulation of oxygen availability and the intracellular reduced
glutathione (GSH) content by ReII-based-CORMs. A: Reduction in oxygen
content in the incubation medium within 10 min after addition of 60 mM
of ReCORM-1, ReCORM-6, and B12-ReCORM-2; N = 6–8. Given are
means SEM. B: Intracellular reduced glutathione (GSH) content in
NRCs exposed to 16 h of ischemia and 10 min of reperfusion in the presence
or absence of ReII-based-CORMs. N = 4. Data are means SEM.
Conclusions
Computational details
Cyanocobalamin (B12) derivatives of CO-releasing 17-electron rhe-
nium dicarbonyl species were presented. As a second generation
of ReII-based CORMs, the B12 adducts were found to be fully
water soluble, biocompatible and showed an improved stability
in aqueous aerobic media over the original family of complexes
alone. In all cases formation of a Co–C N–Re conjugate was
observed from the reaction of the vitamin derivatives and the
[Et4N]2[ReIIBr4(CO)2] salt (ReCORM-1). Theoretical and exper-
imental studies suggested that aquation of the bound bromide
ions of the ReII-based CORMs is the first step in the mechanism
underlying CO release.
The B12-ReII(CO)2 derivatives are non-toxic towards cultured
cardiomyocytes and provided an effective therapeutic protection
against ischemia-reperfusion injury to the cells. Experimental data
suggest that the cytoprotective action of ReII-based CORMs may
be attributed to both the extracellular release of CO and to
antioxidative action of the molecules. None of the tested rhenium
adducts interfered with mitochondrial respiration. Chemical and
biochemical evidence indicate that the non-toxicity of the ReII-
based CORMs may be ascribed to the oxidation of the metal and
formation in solution of the ReO4- anion which is among the least
toxic of all of the rare inorganic compounds.41 Current efforts
are directed towards the elucidation of the molecular mechanisms
Geometry optimizations as well as frequency calculations for all
“small” molecules, were performed at the Density Functional
level of theory with the Gaussian03 program package55 using the
hybrid B3LYP functional56 in conjunction with the LanL2DZ
basis set.57–59 Pure basis functions (5d, 7f) were used in all
calculations. Geometries were fully optimized without symmetry
restrictions. The nature of the stationary points was checked by
computing vibrational frequencies in order to verify true minima.
The lowest 30 singlet excitation energies were computed by means
of time-dependent DFT (TD-DFT) methodology60–62 to simulate
the absorption electronic spectra. Solvent effects were taken into
account using the polarizable continuum model (PCM)63 with
water as solvent for the TD-DFT calculations. The molecular
structures of B12-ReCORM-2 and B12-ReCORM-4 were optimized
by the two-layer ONIOM method64,65 using the DFT method
B3LYP/LanL2DZ for the high layer and the molecular mechanics
method UFF for the low layer (see description of the layers in the
ESI†).66
Detection of CO release using the myoglobin assay
The release of CO from compounds B12-ReCORM-2 and B12-
ReCORM-4 was assessed spectrophotometrically by measuring
This journal is
The Royal Society of Chemistry 2012
Dalton Trans., 2012, 41, 370–378 | 375
©

the conversion of deoxymyoglobin (Mb) to carbonmonoxy myo-
globin (MbCO) as previously reported.30,31 A small aliquot of a
freshly prepared concentrated aqueous solution of the selected ReII
complex was added to 1 ml of the Mb solution in phosphate buffer
(0.1 M, pH 7.4, final concentrations: 30 mM for ReII complex and
Mb). Changes in the Mb spectra were recorded over time at 25 ◦C.
The amount of MbCO formed was determined by measuring the
absorbance at 540 nm (extinction coefficient = 15.4 M-1 cm-1). The
MbCO concentration was plotted over time and directly related to
the equivalents of CO released from the compounds. The half-life
of CO release from B12-ReCORM-2 and B12-ReCORM-4 was then
estimated from the graphs. Control experiments were run under
identical conditions but without addition of the metal complexes.
All manipulations were performed under a pure N2 atmosphere in
a wet box.
X-ray diffraction were grown by slow diffusion of acetone into an
aqueous solution of the compound.
Synthesis of B12-ReCORM-4
As described for B12-ReCORM-2 but starting from 3 instead of
cyanocobalamin. Compound B12-ReCORM-4 was obtained as a
red microcrystalline powder. Yield: 95%. HPLC analysis showed
a single peak with a retention time of 25.3 min. Analytical data
for B12-ReCORM-4: ESI-MS analysis (positive mode) gave peaks
at m/z = 1893.4 [M+H+]+ and 946.8 [M+H+]2+. I.R. (solid state,
KBr, cm-1): nC 2184, nC 1989, 1839.
N
O
In vitro model of “ischemia-reperfusion” stress of cardiomyocyte
Animal keeping, breeding and experimentation were reviewed,
approved, and carried out in accordance with the Swiss ani-
mal protection laws and institutional guidelines. Neonatal rat
cardiomyocytes (NRCs) were isolated from ventricular tissue of
Wistar rat pups at postnatal day 3 by collagenase digestion
followed by separation on Percoll density gradient as described
elsewhere. The cells were maintained in culture for 4 days and
then used for experiments. Conditions of ischemia-reperfusion
were mimicked as follows. The “ischemic incubation medium” was
glucose- and serum-free with its pH adjusted to 6.0. Immediately
after replacing the normal cell culture medium by ischemic
medium the NRCs were placed into the hypoxic incubators in
which oxygen concentration was reduced to 1% for 16 h. Thereafter
the medium was replaced by the one at pH 7.4 containing
5 mM glucose, 10% fetal calf serum (FCS). Reoxygenation and
restoration of pH, glucose and FCS supplementation imitated
“reperfusion” phase. Rhenium-based CORMs were added from
the 30 mM stock solution prepared in DMSO to the cell culture
medium immediately after the onset of “reperfusion” to a final
concentration of 30 mM. Equal amounts of solvent were added to
the control samples. The cells were placed into an incubator in the
atmosphere of 95% air and 5% CO2 for a further 9 h and then the
percentage of dead cells was determined using a combined stating
with Hoechst 33342 (0.1 mg ml-1) and propidium iodide, PI (0.5 mg
ml-1). The resulting nuclear fluorescence was monitored using an
inverted fluorescent microscope Axiovert 200 M equipped with
DAPI (409 nm excitation/450 nm emission) and Cy3 (550 nm
excitation/570 nm emission) filters. The total number of cells per
field (Hoechst 33342-positive nuclei) and the number of dead cells
(PI-positive nuclei) were counted for 5 fields per 4.5 cm2 Petri-
dish. Experiments were repeated 6–8 times with cells obtained in
3 independent isolations.
Synthesis of B12-ReCORM-2
Cyanocobalamin (10 mg, 7.4 mmol) and ReCORM-1 (10 mg,
12 mmol) were added as solids to a round bottom flask. Methanol
(6 mL) was added, stirring began and then the flask was lowered
into an oil bath preheated to 50 ◦C. The temperature was then low-
ered to 40 ◦C within 1 h. After 1.5 h HPLC analysis showed a single
B12 derivative. Heating was stopped and the solvent removed under
reduced pressure. The resulting red powder was washed several
times with CH2Cl2 (ca. 1.7 eq. of [Et4N]Br was thus recovered) and
then acetone until washings were clear. Compound B12-ReCORM-
2 was thus obtained as a red microcrystalline powder. Yield:
12.8 mg, 98%. HPLC analysis showed a single peak with a
retention time of 21.9 min. Analytical data for B12-ReCORM-
2: ESI-MS analysis (positive mode) gave peaks at m/z = 1758.4
[M+H+]+ and 879.8 [M+H+]2+. I.R. (solid state, KBr, cm-1): nC
N
2184, nC 1989, 1839.
O
Synthesis of 3
Cyanocobalamin (400 mg, 0.3 mmol) was dissolved in 10 mL of
anhydrous DMSO and carbonyldiimidazole (CDI, 1 g, 6.2 mmol)
was added. The solution was stirred and heated to 60 ◦C for 12 h.
The solution was allowed to cool to room temperature (RT) and
then slowly added to a rapidly stirring mixture of 300 mL 1 : 1
diethyl ether : chloroform. The red precipitate was collected by
vacuum filtration, washed with 50 mL of acetone and vacuum
dried. Yield: 395 mg. This product was not purified but used
directly for the next reaction. 100 mg of the activated vitamin
was dissolved in 5 mL of anhydrous DMSO and 4-picolylamine
(100 ml, 1 mmol) was added. The solution was stirred at RT for
30 min and then slowly added to a rapidly stirring mixture of
150 mL 1 : 1 diethyl ether : chloroform. The red precipitate was
collected by vacuum filtration, washed with 30 mL of acetone and
vacuum dried. Yield: 109 mg. This precipitate consists of a mixture
of products. Compound 3 was then purified by HPLC (retention
time 15.5 min). Analytical data for 3: yield 45 mg. ESI-MS analysis
(positive mode) gave a single peak at m/z = 745.6 corresponding to
[M+H+]2+. 1H NMR, 500 MHz (D2O, d (ppm), aromatic region):
8.28 (s, 2H, py), 7.36 (s, 2H, py), 7.27 (s, 1H, B7), 7.12 (s, 1H, B2),
6.55 (s, 1H, B4), 6.33 (s, 1H, R1), 6.11 (s, 1H, C10). See also ESI.†
I.R. (solid state, KBr, cm-1): nC N 2134. Single crystals suitable for
Effect of selected ReII-based CORMs on oxygen consumption by
NRCs and oxygen content on the cell-free culture medium
The effect of B12-ReCORM-2, compounds ReCORM-1 and
ReCORM-6 on the oxygen consumption by the NRCs was
examined in cell culture medium at 37 ◦C using a high-resolution
Oroboros Oxygraph-2k (Oroboros, Innsbruck, Austria). The Oxy-
graph is a two-chamber titration-injection respirometer with an
oxygen detection limit of up to 0.5 pmol sec-1 mL-1. Standardized
instrumental and chemical calibrations were performed to correct
for back-diffusion of oxygen into the chamber from the various
components, leak from the exterior, oxygen consumption by
376 | Dalton Trans., 2012, 41, 370–378
This journal is
The Royal Society of Chemistry 2012
©

the chemical medium, and sensor oxygen consumption. Oxygen
flux was resolved by software allowing nonlinear changes in
the negative time derivative of the oxygen concentration signal
(Oxygraph 2k, Oroboros, Innsbruck, Austria). Following 4 days
of culture the cells were trypsinised, resuspended in the cell culture
medium, and transferred into their respective respiratory chamber
(~1 Mio of cells per chamber), which were then tightly sealed.
Oxygen consumption in both chambers was monitored over time
with the oxygen flux expressed as pmol/(sec·Mio cells). After
basal oxygen consumption was established, either CORMs or
DMSO alone was introduced into the chamber and the change
in oxygen flux was measured as a function of time and CORM
concentrations. Similar measurements were repeated for the CO-
MRs added into the cell-free incubation medium to assess the
effect of CORM addition on the oxygen levels in the cell culture
medium. These experiments were performed as the some of the
reactive CORM intermediates were suggested to interact with
oxygen in the aqueous phase (see Scheme 3).
14 D. Crespy, K. Landfester, U. S. Schubert and A. Schiller, Chem.
Commun., 2010, 46, 6651–6662.
15 I. J. S. Fairlamb, A. K. Duhme-Klair, J. M. Lynam, B. E. Moulton,
C. T. O’Brien, P. Sawle, J. Hammad and R. Motterlini, Bioorg. Med.
Chem. Lett., 2006, 16, 995–998.
16 I. J. S. Fairlamb, J. M. Lynam, B. E. Moulton, I. E. Taylor, A. K.
Duhme-Klair, P. Sawle and R. Motterlini, Dalton Trans., 2007, 3603–
3605.
17 P. C. Kunz, W. Huber, A. Rojas, U. Schatzschneider and B. Spingler,
Eur. J. Inorg. Chem., 2009, 5358–5366.
18 B. E. Moulton, A. K. Duhme-Klair, I. J. S. Fairlamb, J. M. Lynam and
A. C. Whitwood, Organometallics, 2007, 26, 6354–6365.
19 J. Niesel, A. Pinto, H. W. P. N’Dongo, K. Merz, I. Ott, R. Gust and U.
Schatzschneider, Chem. Commun., 2008, 1798–1800.
20 H. Pfeiffer, A. Rojas, J. Niesel and U. Schatzschneider, Dalton Trans.,
2009, 4292–4298.
21 P. Sawle, J. Hammad, I. J. S. Fairlamb, B. Moulton, C. T. O’Brien,
J. M. Lynam, A. K. Duhme-Klair, R. Foresti and R. Motterlini, J.
Pharmacol. Exp. Ther., 2006, 318, 403–410.
22 U. Schatzschneider, Eur. J. Inorg. Chem., 2010, 1451–1467.
23 K. Splith, I. Neundorf, W. N. Hu, H. W. P. N’Dongo, V. Vasylyeva, K.
Merz and U. Schatzschneider, Dalton Trans., 2010, 39, 2536–2545.
24 W. Q. Zhang, A. J. Atkin, R. J. Thatcher, A. C. Whitwood, I. J. S.
Fairlamb and J. M. Lynam, Dalton Trans., 2009, 4351–4358.
25 W. Q. Zhang, A. C. Whitwood, I. J. S. Fairlamb and J. M. Lynam, Inorg.
Chem., 2010, 49, 8941–8952.
Acknowledgements
26 B. Chen, L. L. Guo, C. L. Fan, S. Bolisetty, R. Joseph, M. M. Wright,
A. Agarwal and J. F. George, Am. J. Pathol., 2009, 175, 422–429.
27 L. E. Otterbein, B. S. Zuckerbraun, M. Haga, F. Liu, R. P. Song, A.
Usheva, C. Stachulak, N. Bodyak, R. N. Smith, E. Csizmadia, S. Tyagi,
Y. Akamatsu, R. J. Flavell, T. R. Billiar, E. Tzeng, F. H. Bach, A. M.
K. Choi and M. P. Soares, Nat. Med., 2003, 9, 183–190.
28 M. Allanson and V. E. Reeve, Cancer Immunol. Immunother., 2007, 56,
1807–1815.
The Swiss National Science Foundation (Ambizione
PZ00P2_121989/1 and Grant# 310030_124970/1) is gratefully
acknowledged for financial support. We thank Prof. Roger
Alberto (Institute of Inorganic Chemistry) for helpful discussion
and Dr Nikolay Bogdanov (Institute of Veterinary Physiology) for
his assistance in the isolation of the neonatal rat cardiomyocytes.
29 M. J. Alcaraz, M. I. Guillen, M. L. Ferrandiz, J. Megias and R.
Motterlini, Curr. Pharm. Des., 2008, 14, 465–472.
30 J. E. Clark, P. Naughton, S. Shurey, C. J. Green, T. R. Johnson, B. E.
Mann, R. Foresti and R. Motterlini, Circ. Res., 2003, 93, E2–E8.
31 K. S. Davidge, G. Sanguinetti, C. H. Yee, A. G. Cox, C. W. McLeod, C.
E. Monk, B. E. Mann, R. Motterlini and R. K. Poole, J. Biol. Chem.,
2009, 284, 4516–4524.
32 M. Desmard, K. S. Davidge, O. Bouvet, D. Morin, D. Roux, R. Foresti,
J. D. Ricard, E. Denamur, R. K. Poole, P. Montravers, R. Motterlini
and J. Boczkowski, FASEB J., 2009, 23, 1023–1031.
References
1 B. E. Mann, Top. Organomet. Chem., 2010, 32, 247–285.
2 R. Motterlini and L. E. Otterbein, Nat. Rev. Drug Discovery, 2010, 9,
728–743.
3 A. Pamplona, A. Ferreira, J. Balla, V. Jeney, G. Balla, S. Epiphanio,
A. Chora, C. D. Rodrigues, I. P. Gregoire, M. Cunha-Rodrigues, S.
Portugal, M. P. Soares and M. M. Mota, Nat. Med., 2007, 13, 703–710.
4 M. L. Ferrandiz, N. Maicas, I. Garcia-Amandis, M. C. Terencio, R.
Motterlini, I. Devesa, L. A. B. Joosten, W. B. van den Berg and M. J.
Alcaraz, Ann. Rheum. Dis., 2008, 67, 1211–1217.
5 A. A. Chora, P. Fontoura, A. Cunha, T. F. Pais, S. Cardoso, P. P. Ho,
L. Y. Lee, R. A. Sobel, L. Steinman and M. P. Soares, J. Clin. Invest.,
2007, 117, 438–447.
6 J. D. Beckman, J. D. Belcher, J. V. Vineyard, C. S. Chen, J. Nguyen,
M. O. Nwaneri, M. G. O’Sullivan, E. Gulbahce, R. P. Hebbel and G.
M. Vercellotti, Am. J. Physiol. Heart Circ. Physiol., 2009, 297, H1243–
H1253.
7 S. K. Bains, R. Foresti, J. Howard, S. Atwal, C. J. Green and R.
Motterlini, Arterioscler. Thromb. Vasc. Biol., 2010, 30, 305–U331.
8 A. Nicolai, M. Li, D. H. Kim, S. J. Peterson, L. Vanella, V. Positano,
A. Gastaldelli, R. Rezzani, L. F. Rodella, G. Drummond, C. Kusmic,
A. L’Abbate, A. Kappas and N. G. Abraham, Hypertension, 2009, 53,
508–515.
9 M. Di Pascoli, L. Rodella, D. Sacerdoti, M. Bolognesi, S. Turkseven
and N. G. Abraham, Biochem. Biophys. Res. Commun., 2006, 340, 935–
943.
33 E. Masini, A. Vannacci, P. Failli, R. Mastroianni, L. Giannini, M. C.
Vinci, C. Uliva, R. Motterlini and P. F. Mannaioni, FASEB J., 2008,
22, 3380–3388.
34 R. Motterlini, FEBS J., 2009, 276, 45–46.
35 R. Alberto and R. Motterlini, Dalton Trans., 2007, 1651–1660.
36 R. Motterlini, P. Sawle, S. Bains, J. Hammad, R. Alberto, R. Foresti
and C. J. Green, FASEB J., 2004, 18, 284–286.
37 T. S. Pitchumony, B. Spingler, R. Motterlini and R. Alberto, Chimia,
2008, 62, 277–279.
38 T. S. Pitchumony, B. Spingler, R. Motterlini and R. Alberto, Org.
Biomol. Chem., 2010, 8, 4849–4854.
39 F. Zobi, A. Degonda, M. C. Schaub and A. Y. Bogdanova, Inorg.
Chem., 2010, 49, 7313–7322.
40 F. Zobi, L. Kromer, B. Spingler and R. Alberto, Inorg. Chem., 2009,
48, 8965–8970.
41 T. J. Haley and Fd. Cartwrig, J. Pharm. Sci., 1968, 57, 321–323.
42 S. Kunze, T. Zobi, P. Kurz, B. Spingler and R. Alberto, Angew. Chem.,
Int. Ed., 2004, 43, 5025–5029.
43 S. Mundwiler, B. Spingler, P. Kurz, S. Kunze and R. Alberto, Chem.–
Eur. J., 2005, 11, 4089–4095.
44 P. Ruiz-Sanchez, S. Mundwiler, A. Medina-Molner, B. Spingler and R.
Alberto, J. Organomet. Chem., 2007, 692, 1358–1362.
45 5, Crystal data. 0.53(C63H86CoN14O14P) ·0.47(C66H88CoBrN14O18PRe)
10 B. S. Zuckerbraun, T. R. Billiar, S. L. Otterbein, P. K. M. Kim, F. Liu,
A. M. K. Choi, F. H. Bach and L. E. Otterbein, J. Exp. Med., 2003,
198, 1707–1716.
11 G. Sass, M. C. P. Soares, K. Yamashita, S. Seyfried, W. H. Zimmermann,
T. Eschenhagen, E. Kaczmarek, T. Ritter, H. D. Volk and G. Tiegs,
Hepatology, 2003, 38, 909–918.
12 B. S. Zuckerbraun, B. Y. Chin, B. Wegiel, T. R. Billiar, E. Czsimadia, J.
Rao, L. Shimoda, E. Ifedigbo, S. Kanno and L. E. Otterbein, J. Exp.
Med., 2006, 203, 2109–2119.
13 A. J. Atkin, S. Williams, P. Sawle, R. Motterlini, J. M. Lynam and I. J.
S. Fairlamb, Dalton Trans., 2009, 3653–3656.
·C3H6O·6.53(H2O), M = 1700.37, orthorhombic, a = 15.8806(2), b =
3
˚
˚
21.8255(3), c = 26.2738(4) A, U = 9106.6(2) A , T = 183 K, space group
P212121 (no.19), Z = 4, 79318 reflections measured, 20093 unique (Rint
0.0603) which were used in all calculations. The final wR(F2) was 0.1884
(all data).
=
46 M. Lee and C. B. Grissom, Org. Lett., 2009, 11, 2499–2502.
47 3, Crystal data. 2(C70H94CoN16O15P) ·5(C3H6O)·16(H2O), M = 3525.43,
˚
orthorhombic, a = 15.5066(4), b = 22.9558(7), c = 25.1741(7) A,
This journal is
The Royal Society of Chemistry 2012
Dalton Trans., 2012, 41, 370–378 | 377
©

3
˚
U = 8961.1(4) A , T = 183 K, space group P212121 (no.19), Z = 2,
71958 reflections measured, 16995 unique (Rint = 0.1164) which were
used in all calculations. The final wR(F2) was 0.1588 (all data).
48 U. Von Gunten and Y. Oliveras, Environ. Sci. Technol., 1998, 32, 63–70.
49 A. B. P. Lever, Inorg. Chem., 1990, 29, 1271–1285.
50 M. Bilban, A. Haschemi, B. Wegiel, B. Y. Chin, O. Wagner and L. E.
Otterbein, J. Mol. Med., 2008, 86, 267–279.
57 P. J. Hay and W. R. Wadt, J. Chem. Phys., 1985, 82, 270–283.
58 P. J. Hay and W. R. Wadt, J. Chem. Phys., 1985, 82, 299–310.
59 W. R. Wadt and P. J. Hay, J. Chem. Phys., 1985, 82, 284–298.
60 R. E. Stratmann, G. E. Scuseria and M. J. Frisch, J. Chem. Phys., 1998,
109, 8218–8224.
61 R. Bauernschmitt and R. Ahlrichs, Chem. Phys. Lett., 1996, 256, 454–
464.
51 Oxford Diffraction (2010), CrysAlis PRO, Oxford Diffraction Ltd,
Yarnton, England.
52 A. Altomare, M. C. Burla, M. Camalli, G. L. Cascarano, C. Giacov-
azzo, A. Guagliardi, A. G. G. Moliterni, G. Polidori and R. Spagna, J.
Appl. Crystallogr., 1999, 32, 115–119.
53 G. M. Sheldrick, Acta Crystallogr., Sect. A: Found. Crystallogr., 2008,
A64, 112–122.
54 A. L. Spek, J. Appl. Crystallogr., 2003, 36, 7–13.
55 S. S. Iyengar and M. J. Frisch, J. Chem. Phys., 2004, 121, 5061–
5070.
62 M. E. Casida, C. Jamorski, K. C. Casida and D. R. Salahub, J. Chem.
Phys., 1998, 108, 4439–4449.
63 J. Tomasi, B. Mennucci and R. Cammi, Chem. Rev., 2005, 105, 2999–
3093.
64 S. Dapprich, I. Komaromi, K. S. Byun, K. Morokuma and M. J. Frisch,
THEOCHEM, 1999, 462, 1–21.
65 T. Vreven, K. S. Byun, I. Komaromi, S. Dapprich, J. A. Montgomery,
K. Morokuma and M. J. Frisch, J. Chem. Theory Comput., 2006, 2,
815–826.
66 A. K. Rappe, C. J. Casewit, K. S. Colwell, W. A. Goddard and W. M.
Skiff, J. Am. Chem. Soc., 1992, 114, 10024–10035.
56 A. D. Becke, J. Chem. Phys., 1993, 98, 5648–5652.
378 | Dalton Trans., 2012, 41, 370–378
This journal is
The Royal Society of Chemistry 2012
©