Synthesis and performance of acyloxy-diene-fe(CO)3 complexes with variable chain lengths as enzyme-triggered carbon monoxide-releasing molecules

Article abstract of DOI:10.1021/om301233h

Novel η⁴-acyloxy-cyclohexadiene-Fe(CO)₃ complexes (with variable length of the acyloxy chain) were synthesized as potential enzyme-triggered carbon monoxide (CO)-releasing molecules (ET-CORMs). The molecular structure of two complexes was additionally confirmed by X-ray crystallography. The enzyme-triggered CO-releasing activity of the compounds was assessed under physiological conditions (37 C, 0.1 M phosphate buffer, pH = 7.4) by headspace gas chromatography (GC) and additionally by means of a myoglobin assay (UV). The relative rate of CO release and the amount of liberated CO were found to depend on the length of the acyloxy chain and its position at the diene unit (outer or inner position). Some of the new ET-CORMs exhibited very good biological activity as assessed in different cellular assays (cytotoxicity, protective effect against hypothermia-associated cell damage, and inhibition of TNF-α-mediated VCAM-1 expression).

Full text of DOI:10.1021/om301233h

Article
pubs.acs.org/Organometallics
Synthesis and Performance of Acyloxy-diene-Fe(CO)3 Complexes
with Variable Chain Lengths as Enzyme-Triggered Carbon Monoxide-
Releasing Molecules
Svetlana Botov,† Eleni Stamellou,‡ Steffen Romanski,† Miguel Guttentag,§ Roger Alberto,§
Jorg-Martin Neudorfl,† Benito Yard,*,‡ and Hans-Gunther Schmalz*,†
̈
̈
̈
†Department fur Chemie, Universitat zu Koln, Greinstrasse 4, D-50939 Koln, Germany
̈
̈
̈
̈
‡Medizinische Klinik, Universitatsmedizin Mannheim, 68167 Mannheim, Germany
§Institut fur Anorganische Chemie, Universitat Zurich, CH-8057 Zurich, Switzerland
̈
̈
̈
̈
̈
S
* Supporting Information
ABSTRACT: Novel η4-acyloxy-cyclohexadiene-Fe(CO)3
complexes (with variable length of the acyloxy chain) were
synthesized as potential enzyme-triggered carbon monoxide
(CO)-releasing molecules (ET-CORMs). The molecular
structure of two complexes was additionally confirmed by X-
ray crystallography. The enzyme-triggered CO-releasing
activity of the compounds was assessed under physiological
conditions (37 °C, 0.1 M phosphate buffer, pH = 7.4) by
headspace gas chromatography (GC) and additionally by
means of a myoglobin assay (UV). The relative rate of CO
release and the amount of liberated CO were found to depend on the length of the acyloxy chain and its position at the diene
unit (outer or inner position). Some of the new ET-CORMs exhibited very good biological activity as assessed in different
cellular assays (cytotoxicity, protective effect against hypothermia-associated cell damage, and inhibition of TNF-α-mediated
VCAM-1 expression).
equivalents of CO upon enzymatic cleavage of the ester function
under slightly oxidizing conditions.18
1. INTRODUCTION
Despite the high toxicity1 of carbon monoxide (CO) gas, this
very small natural product plays an essential physiological role as
a signaling molecule in humans and other mammals. It is
endogenously produced during oxidative heme degradation by
heme oxygenase (HO)2,3 and exhibits a variety of beneficial
biological activities, such as cytoprotective, anti-inflammatory,
vasodilatory, and antiproliferative effects.4,5 The obvious bio-
logical potential of CO has induced intense research activities
aiming at its use as a therapeutic agent.5,6 In this context, CO-
releasing molecules (CORMs) have been developed as attractive
tools to liberate and deliver CO to cellular systems in a controlled
fashion.7,8 After the first CORMs had been introduced (by
Motterlini and co-workers)9,10 a broad range of transition metal
carbonyl complexes were shown to release CO under certain
conditions, and their biological effects were the subject of many
investigations.11−13 Because CORMs of the first generation act in
a rather uncontrolled fashion, it is a major challenge to develop
novel CORMs that liberate their CO load only after activation of
a trigger such as pH change,14 ligand exchange in the cellular
medium,15 or photoactivation.16
Figure 1. Regioisomeric acyloxydiene-Fe(CO)3 complexes of types 1
and 2.
The proposed mechanism of action (Scheme 1) is based on
the observation that dienol-Fe(CO)3 complexes of type 3 are
highly labile and decompose under mildly oxidative conditions
(possibly after formation of a 16-VE intermediate of type 4).
Overall, the enzymatic hydrolysis of the dienol ester complexes of
type 3 should thus result in the liberation of up to three
molecules of CO besides the enone ligand (5) and an Fe3+ ion.
In our previous studies18 we have synthesized ET-CORMs of
type 1 and 2 (with R = Me, t-Bu, n-C15H31) and assessed their
CO-releasing properties (Figure 1). Additionally, we could show
that diene-Fe(CO)3 complexes with monomethylated phosphor-
As a new and conceptually different concept, we recently
introduced enzyme-triggered CORMs (ET-CORMs).17 In this
context, we showed that acyloxy-cyclohexadiene-Fe(CO)3
complexes of type 1 and 2 (Figure 1) release up to three
Received: December 20, 2012
© XXXX American Chemical Society
A
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hexadienolate with TMSCl. Complexation of 6 by thermal
treatment with Fe2(CO)9 in diethyl ether afforded complex rac-
8. This compound could not be purified because the TMS group
was cleaved off during chromatography on SiO2.18 In contrast,
the corresponding triisopropylsilyloxydiene complex rac-9,
prepared by trapping the dienolate with TIPS-OTf and
subsequent complexation, proved to be sufficiently stable to be
isolated and purified by chromatography.18 However, because of
the high price of TIPS-OTf (as compared to TMSCl), we
routinely used the TMS-protected intermediate rac-8 as a
precursor complex for the synthesis of different ET-CORMs. For
this purpose, the crude complexation product (rac-8) was
dissolved in DMF and deprotected by addition of TBAF (under
inert gas conditions). The in situ-formed dienol complex was
then directly O-acylated with an acid chloride in the presence of
Scheme 1. Proposed Mechanism of Action of Enzyme-
Triggered CO-Releasing Molecules (ET-CORMs)
yloxy substituents represent water-soluble, phosphatase-acti-
vated ET-CORMs.19 For the detection of CO we used both the
myoglobin-based assay established by Motterlini et al.13 and gas
chromatographic measurements. In vitro studies showed that the
esterase-triggered CO release from the pivalates (R = t-Bu) and
palmitates (R = C15H31) was slower than from the acetates (R =
Me). In general, complexes with the ester function at the outer
position of the diene ligand (type 2) released CO much faster
than those with the ester function at the inner position (type 1).
Cellular assays also revealed that the complexes greatly differed in
their cytotoxicity and their ability to suppress NO production
(due to iNOS inhibition).17,18 To refine the emerging structure−
activity relationship patterns, the target of the present study was
to synthesize, characterize, and investigate a set of new ET-
CORMs of types 1 and 2 with different lengths of the aliphatic
ester side chain R (Figure 1).
Hunig’s base (DIPEA). This way, the complexes rac-10b, rac-
̈
10c, rac-10d, and rac-10e were obtained in high yields (after
chromatographic purification) and with good reproducibility
(Table 1). For the synthesis of complexes of type rac-13,
Table 1. Complexes of Type rac-10 and rac-13 Synthesized
According to Scheme 2
entry
R
product
yield (%)
1
CH3
rac-10a
rac-10b
rac-10c
rac-10d
rac-10e
rac-10f
rac-13a
rac-13b
rac-13c
rac-13d
rac-13e
rac-13f
7217
2
C3H7
81
3
C5H11
C7H15
C11H23
C15H31
CH3
81
4
70
5
83
6
9118
7917
91
7
8
C3H7
2. RESULTS AND DISCUSSION
9
C5H11
C7H15
C11H23
C15H31
84
10
11
12
81
2.1. Synthesis. The synthesis of acyloxydiene-Fe(CO)3
complexes of type rac-10 and rac-13 with variable ester side
chains is displayed in Scheme 2. Following the general procedure
developed in this laboratory,18 we first prepared the silyloxy-
substituted complexes (rac-8, rac-9, and rac-12, respectively)
from which the target acyloxy derivatives were obtained in a
divergent manner. The silyloxydiene 6 was prepared by
deprotonation of 2-cyclohexen-1-one (5) under kinetic control
(LDA, THF, −78 °C) and trapping the resulting 2,5-cyclo-
75
9718
cyclohexenone (5) was converted into the thermodynamically
more stable dienolate using LiHMDS in the presence of TPPA
(as an HMPA substitute) followed by O-silylation (TIPS-OTf).
The resulting silyloxydiene 11 was then again reacted with
Fe2(CO)9 to afford rac-12, from which the complexes rac-13b,
rac-13c, rac-13d, and rac-13e were then obtained by desilylation
(TBAF) and O-acylation in excellent overall yields (Scheme 2,
Table 1).
Scheme 2. Synthesis of Complexes of Type rac-10 and rac-13
a
(compare Table 1)
2.2. Structural Characterization. The various acyloxy-
cyclohexadiene-Fe(CO)3 complexes (Scheme 2) were fully
characterized by the common spectroscopic methods (IR,
NMR, MS). In addition, the molecular structures of compounds
rac-13b and rac-13c were determined by X-ray crystallography
(Figure 2).
As expected, the Fe(CO)3-complexed diene units are virtually
planar (torsion angle <2°) with the Fe atom coordinated to all
four diene carbon atoms with Fe−C bond lengths between 2.04
and 2.13 Å. The Fe(CO)3 tripod adopts a conformation with one
CO ligand eclipsing the formal C−C single bond of the diene.
Selected structural parameters are given in the Supporting
Information.
2.3. Detection of CO Release. In our previous studies we
had identified pig liver esterase (PLE) and lipase from Candida
rugosa (LCR) as particularly suitable esterases for investigating
the hydrolysis-triggered CO-releasing properties of ET-CORMs
of type rac-10 and rac-13.18 Therefore, the CO liberation was
a
(a) LDA, THF, −78 °C, then TMSCl or TIPS-OTf; (b) Fe2(CO)9,
Et2O, 40 °C, 16 h; (c) TBAF, DIPEA, DMF, rt, then RCOCl; (d)
LiHMDS, TPPA, THF, −78 °C. TMSCl: trimethylsilyl chloride;
TIPS-OTf: triisopropylsilyl trifluoromethanesulfonate; TBAF: tetra-n-
butylammonium fluoride; DIPEA: diisopropylethylamine; LiHMDS:
lithium hexamethyldisilazide; TPPA: trispyrrolidinophosphoric acid
triamide.
B
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Table 2. Detection and Quantification of the CO Release from
Various Acyloxydiene-Fe(CO)3 Complexes Triggered by
Suitable Enzymes As Determined by Means of Headspace GC
a
b
c
d
e
substrate
enzyme
released CO (equiv)
time (min)
t0.5 (min)
rac-10a
rac-10b
rac-10c
rac-10d
rac-10e
rac-10f
rac-13a
rac-13b
rac-13c
rac-13d
rac-13e
rac-13f
LCR
LCR
LCR
LCR
LCR
LCR
PLE
PLE
PLE
PLE
PLE
PLE
2.40
1.07
1.67
1.78
1.28
0.58
2.40
2.08
2.69
2.82
2.30
1.33
10 020 n.f.
9256 n.f.
9178 n.f.
9178 n.f.
7770 n.f.
7200 n.f.
3415
128
5332
3482
3142
3785
6145
12
Figure 2. Structure of rac-13b and rac-13c in the crystalline state (gray:
C; white: H; red: O; yellow: Fe).
4763
30
monitored in the presence of these enzymes using two different
techniques: (1) the myoglobin (Mb) assay detecting changes of
the UV absorption spectra associated with the conversion of
deoxymyoglobin (deoxy-Mb) to carbon monoxymyoglobin
(MbCO)13 and (2) headspace gas chromatography (GC),
which also allows to quantify the amount of released CO and
provides more reliable information on the (relative) rates. In
contrast to the Mb assay, which is performed under reductive
conditions (dithionite) and does not tolerate much oxygen
because of reoxidation of the CO-binding Fe(2+) form of Mb to
the nonbinding Fe(3+) form, the GC-based CO release
measurements can also be performed at higher concentrations
of oxygen, which is important in the case of fast reacting ET-
CORMs. Moreover, the GC-based CO detection method also
allowed to monitor the consumption of oxygen associated with
the enzyme-triggered CO release from the acyloxydiene-
Fe(CO)3 complexes studied (Figure 3).20
6130 n.f.
6130 n.f.
6130
36
36
261
110
2100
a
The data for substrates rac-10a, rac-10f, rac-13a, and rac-13f were
b
taken from ref 18. In the GC-based investigations low-purity PLE
with 15 U/mg was used (in contrast to the Mb assay, for which PLE of
higher purity (130 U/mg) was required, as the less pure PLE (15 U/
c
mg) reacted with Mb). The quantification was complicated by the
consumption of O2, which somewhat varied for the individual
substrates. The average consumption of O2 was considered in the
calibration, but deviations in the range of 10% cannot be excluded.
Cases where the CO release still proceeded when the detection was
stopped are indicated by “n.f.” (not finished). t0.5 indicates the time
d
e
required for the release of 0.5 molar equivalents of CO.
CO even after prolonged reaction times), the relative CO-
releasing rates of the other complexes did not correlate
with the chain lengths. For instance, the hexanoate (rac-
10c) was slightly faster than the octanoate (rac-10d) and
the laurate (rac-10e), while the butyrate (rac-10b) proved
to react much more slowly.
• Although the kinetics of the two types of complexes (rac-
10/rac-13) cannot be directly compared, because different
enzymes were used and the enzymatic cleavage leads to
different intermediates for the two series, it can clearly be
seen that complexes of type 2 (i.e., rac-13) (Figure 4, B)
generally reacted much faster. This is most likely due to a
faster enzymatic cleavage, because in the case of the
dienylphosphate-based CORMs19 the compound sub-
stituted at the inner position of the diene unit showed a
faster CO release. Nevertheless, the different reactivity of
the resulting dienol-Fe(CO)3 intermediates in the two
series might also be important.21 As a characteristic
pattern, all complexes of type 2 exhibited a nonlinear time
dependency of CO release. With the exception of the
longer chain esters rac-13e and rac-13f these complexes
almost instantly released about two equivalents of CO
before the reactions continued to proceed much more
slowly with only minor differences in the relative rates.
The acetate (rac-13a) reacted slightly more slowly as
compared to the octanoate (rac-13d) and the hexanoate
(rac-13c).
Figure 3. LCR-induced CO release from rac-10c as a function of the
time associated with the consumption of oxygen as monitored by
headspace GC.
The results of the CO release study performed with complexes
rac-10a−f and rac-13a−f are summarized in Table 2. In addition,
the relative kinetics of the complexes of type 1 (graph A) and of
type 2 (graph B) are depicted in Figure 4. These data reveal the
following trends:
These observations might be rationalized in the following
fashion:
• All complexes of type 1 (i.e., rac-10) (Figure 4, A) showed
a more or less linear time dependency of the amount of
CO released, with the exception of the acetate rac-10a,
which almost instantly released the first equivalent of CO.
The rate of CO release depended significantly on the
length of the aliphatic ester chain. While the palmitate rac-
10f reacted the slowest (liberating only small amounts of
• With the exception of the acetate rac-10a the enzymatic
cleavage of complexes of type 1 (rac-10) occurs with a
similar (comparably slow) rate to the subsequent
decomposition connected to CO liberation.
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• Compounds of type 2 (rac-13) are rapidly cleaved by the
enzyme (PLE) under the chosen conditions, with the
exception of the long chain compounds rac-13e and rac-
13f, which react a bit more slowly. The resulting dienol
complex intermediate (rac-3) then almost instantaneously
decomposes under release of two equivalents of CO. The
third equivalent of CO is then liberated much more slowly
(if at all), which may indicate a stabilized intermediate still
containing one CO unit.
While future investigations will have to prove (or disprove)
these assumptions, the different CO-releasing properties of the
complexes are connected to pronounce differences in their
biological activities as described in the following section.
2.4. Biological Activity. The vascular endothelium is
critically involved in the regulation of inflammation, including
the coordination of leukocyte trafficking to specific tissues.22
Upon exposure of endothelial cells (ECs) to inflammatory
mediators such as TNF-α, ECs modify their phenotype and up-
regulate an array of proinflammatory genes, including the
adhesion molecules VCAM-1, ICAM-1, and E-selectin.23−25
Because CO is able to down-regulate VCAM-1 expression and to
protect ECs against hypothermia-mediated cell damage, the
biological activity of ET-CORMs of both series (rac-10 and rac-
13) was assessed with respect to their cytotoxicity, their
efficiency to protect human umbilical vein endothelial cells
(HUVECs) against hypothermia-associated cell damage, and
their ability to inhibit TNF-α-mediated VCAM-1 expression.
Cytotoxicity was assessed by means of MTT assay. In both series
of compounds (i.e., rac-10 and rac-13, Scheme 2) the cytotoxicity
decreased with the elongation of the ester side chain (Figure 5).
Also, the position of the acyloxy substituent at the complexed
diene moiety clearly influenced the cytotoxicity. In fact, with the
exception of rac-13e, complexes of type 2, with the ester
positioned at the outer position (rac-13b−d), proved to be more
toxic than their isomers (rac-10a−e) bearing the ester function at
the inner position (Figure 5, measuring at 100 μM of accordant
ET-CORMs). Apparently, both the rate of ester hydrolysis and
the reactivity of the resulting (oxidation-sensitive) intermediates
control the toxicity and other biological effects. Larger (more
lipophilic) or bulkier ester moieties exhibit the slowest rate of
hydrolysis, which results in a lower toxicity. This may also explain
Figure 4. Monitoring the enzyme-triggered CO release by headspace
GC (A) from ET-CORMS of type 1 (rac-10) using LCR; (B) from ET-
CORMs of type 2 (rac-13) using PLE.
Figure 5. Cytotoxicity was assessed by means of MTT. HUVECs were grown in 96-well plates until confluence and subsequently stimulated for 24 h
with concentrations at 100 μM. Untreated cells (medium) served as control. (A) Cell toxicity of ET-CORMs of type 1: rac-10b, rac-10c, rac-10d, and
rac-10e. (B) Cell toxicity of ET-CORMs of type 2: rac-13b, rac-13c, rac-13d, and rac-13e. The results of a representative experiment are depicted.
Dose−response experiments have been preformed and are presented in Figure S31 (Supporting Information).
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Figure 6. Inhibition of hypothermic damage assessed by lactate dehydrogenase release (LDH). HUVECs were cold stored at 4 °C for 24 h in the
presence of different ET-CORMs (100 μM). Untreated cells (medium) served as a control.
Figure 7. HUVECs were treated for 24 h with 75 μM rac-10a, rac-10b, and rac-10c and with 100 μM rac-10d, rac-10e, and rac-13e in the presence of
TNF-α (50 ng/mL). Cells not stimulated (−) or stimulated with TNF-α alone served as control. Compounds rac-10a, rac-10b, rac-10c, rac-10d, and
rac-13e were found to inhibit VCAM-1 induction and induce HO-1 expression. Depicted is a representative Western blot. To confirm equal protein
loading, membranes were reprobed with a monoclonal anti-β-actin antibody. A concentration of 75 μM was used for those ET-CORMs that were found
to be toxic at 100 μM (as shown in previous experiments) when the cells were stimulated with TNF-α.
why rac-13e displayed a much lower toxicity in comparison to the
related ET-CORMs of type 2 (Figure 5).
with variable length of the ester chain. These compounds were
fully characterized by spectroscopic methods and, additionally,
by X-ray crystal structure analysis in two cases. The properties of
the complexes as enzyme-triggered CO-releasing molecules were
then assessed in vitro using either the myoglobin assay (UV) or
headspace gas chromatography for CO detection. In addition,
the biological activity (cytotoxicity, protection against hypo-
thermic damage, and inhibition of TNFα-mediated VCAM-1
expression) of the compounds was investigated using HUVEC
endothelial cells. This way, we could reveal a pronounced
influence of the position and the length of the ester substituents
on the biological properties. To explain the different behavior of
complexes of type 1 and 2, respectively, more detailed
mechanistic investigations will be required. However, our results
suggest that the biological (and pharmacological) properties of
this exciting new class of CORMs can be fine-tuned through the
choice of the ester moiety.
In our previous studies we demonstrated that CORM-3 was
able to protect endothelial cells against hypothermia-induced
damage.26 In a similar fashion, complexes of type 1 (rac-10a, rac-
10b, rac-10c, and rac-10d) were found to protect endothelial
cells against hypothermic damage (Figure 6). As a trend,
elongation of the ester chain (rac-10e) seems to decrease the
propensity of the complexes to protect endothelial cells. In
contrast, none of the type 2 complexes (rac-13a−e) exhibited
protective properties. While one may argue that the higher
toxicity of type 2 complexes may mask the protective effect of
released CO, it should be noticed that even the nontoxic
compound rac-13e did not protect HUVECs against cold
preservation injury.
We next investigated the ability of the ET-CORMs to inhibit
TNF-α-mediated VCAM-1 expression. With the exception of
rac-10e all compounds of type 1 (rac-10a, rac-10b, rac-10c, rac-
10d) were indeed able to inhibit TNF-α-mediated VCAM-1
expression. Among the type 2 compounds, only rac-13e was
tested, as all other members of this group were toxic also at lower
concentrations. Similar to the behavior of most of the type 1
compounds, rac-13e also inhibited TNF-α-mediated VCAM-1
expression. Inhibition of VCAM-1 expression was paralleled by
the induction of heme oxygenase (HO-1) (Figure 7).
EXPERIMENTAL SECTION
■
General Procedures. Unless otherwise stated all 1H and 13C NMR
spectra were recorded at room temperature in CDCl3 on Bruker
instruments (Avance DPX 300, Avance DRX 500). Chemical shifts (δ)
are reported in parts per million (ppm) from tetramethylsilane using the
residual solvent resonance as the internal standard (CDCl3: 7.24 ppm
for 1H NMR, 77.0 ppm for 13C NMR). Multiplicities are abbreviated as
follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br
= broad. Protons were assigned using 2D NMR techniques (H,H-
COSY, HSQC, and HMBC). UV/vis spectra were recorded in
phosphate buffer 0.1 M (pH = 7.4) on a Beckmann Coulter DU 800
(cell length 1 cm). IR spectra were recorded on a Perkin-Elmer Paragon
3. CONCLUSIONS
In summary, we have efficiently synthesized eight new (racemic)
acyloxy-cyclohexadiene-Fe(CO)3 complexes of types 1 and 2
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1000 FT-IR spectrometer in the ATR mode at room temperature.
Relative intensities of the signals are given as strong (s), medium (m),
weak (w), and broad (b). Direct inlet mass spectra (DIP-MS) were
recorded at the Analytical Service Unit, University of Cologne, on
Finnigan instruments (MAT Inocs 50 Galaxy system (for EI LR-MS)
and a MAT 900 (for HR-MS)). An Agilent Technologies model GC
6890N gas chromatograph coupled with an HP 5973N series mass
selective detector and an HP 7683 GC autosampler was employed for all
GC-MS analyses. Samples were separated on a 30 m × 0.25 mm HP-5
MS column. The column temperature was initially held at 50 °C for 2
min; then the temperature was raised to 300 °C at a rate of 25 °C per
min and held for 5 min. The total run-time was 17 min. Injector
temperature was maintained at 300 °C, and the injection volume was 1.0
μL in the split mode. Mass spectra were scanned from m/z 35 to 500.
Electron impact ionization energy was 70 eV. Gas chromatograms for
the CO detection were recorded using a Varian CP-3800 gas
chromatograph with helium as the carrier gas and a 3 m × 2 mm
packed molecular sieve 13X 80−100 column. The gases were detected
using a thermal conductivity detector (Varian) operated at 150 °C.
CHN analyses were measured on an Elementar Vario EL machine.
Analytical TLC was carried out using precoated silica gel plates (Merck
TLC plates silica gel 60F254). Flash column chromatography was
performed using silica gel (particle size 40−63 mM, Acros). All sensitive
reactions were carried out in flame-dried glassware under an argon
atmosphere. Chemicals were purchased from Merck, Sigma-Aldrich,
Fluka, Acros, Lancaster, or Strem and used without further purification.
Solvents were dried as follows: THF and Et2O were distilled from
sodium/benzophenone under an argon atmosphere. Cyclohexenone
was distilled and stored under argon. Diisopropylamine was refluxed
over KOH and stored on KOH. Lipase from Candida rugosa (>2 units/
mg; 90860) and esterase from porcine liver for Mb assay (PLE, >130
units/mg; 46058) or PLE for the GC measurements (>15 units/mg)
were purchased from Sigma-Aldrich.
Determination of Cell Toxicity in HUVECs. HUVECs were
cultured in 96-well plates until confluence. Afterward, the obtained cells
were cultured for 24 h in the presence of different concentrations (25,
50, 100 μM) of rac-10b, rac-10c, rac-10d, rac-10e, rac-13b, rac-13c, rac-
13d, and rac-13e. The toxicity was then determined by staining with (3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
according to the manufacturer’s instructions (Roche Diagnostics,
Mannheim, Germany). All measurements were performed in triplicate
in at least three different experiments.
Determination of Tolerance to Hypothermic Injury in
HUVECs. Tolerance to hypothermia-associated cell damage was
assessed by means of a lactate dehydrogenase (LDH) release assay.
HUVECs were seeded in 24-well plates and grown until confluence. The
medium was replaced by phenol-red-free medium, and the plates were
placed on ice for 24 h at 4 °C. ET-CORMs were added to the
preservation medium without prior pretreatment. Cell damage was then
assessed by determining LDH release in the supernatant according to
manufacturer’s instructions (Roche Diagnostics). All measurements
were performed in triplicate in at least three different experiments.
Western Blotting. HUVEC cell lysates were generated in lysis
buffer (10 mM Tris, 2% SDS, 0.5% β-mercaptoethanol) (all from Sigma-
Aldrich, St. Louis, MO, USA). Protein concentrations were measured
using Coomassie reagent (Pierce, Rockford, IL, USA). Samples (20 μg
of protein extract) were heated to 95 °C for 5 min, loaded, and separated
on 10% SDS-polyacrylamide gels followed by semidry blotting onto
PVDF membranes (Roche Diagnostics). The membranes were blocked
with blocking buffer (5% w/v nonfat dry milk in TBS/Tween 0.5%) and
incubated overnight at 4 °C, either with polyclonal anti-VCAM-1 (R&D
Systems, Wiesbaden, Germany) or anti-HO-1 (Enzo Life Sciences,
Lorrach Germany). Subsequently, the membranes were thoroughly
̈
washed with TBS/Tween and incubated with the appropriate
horseradish peroxidase conjugated secondary antibody (Jackson
ImmunoResearch, Baltimore, MD, USA). Proteins were visualized
using enhanced chemoluminescence technology, according to the
manufacturer’s instructions (Pierce). To confirm equal protein loading,
membranes were stripped and reprobed with monoclonal anti-β-actin
antibody (Abcam, Cambridge, UK).
Crystallographic Details. Measurements were made on a Nonius
Kappa-CCD diffractometer with Mo Kα radiation (λ = 0.71073), and
Denzo was used for data reduction. The structures were solved by direct
methods (Shelxs97). Shelxl97 was used for full-matrix least-squares
refinement on F2. All non-hydrogen atoms were refined anisotropically.
All hydrogen atoms were placed in geometrically idealized positions and
refined using a riding model. CCDC 927556−927557 contain the
supplementary crystallographic data for this paper and can be obtained
free of charge via www.ccdc.cam.ac.uk/data_request/cif.
Syntheses. Synthesis of Complexes of Type rac-10 and rac-13.
General Procedure for the Esterification Sequence. Procedure A:
Preparation of Dienyl-ester-Fe(CO)3 Complexes Starting from rac-8
or rac-9. To a solution of crude complexation product rac-8
(concentration of the theoretical amount of complex approximately
0.1 M) or complex rac-9 (1 equiv) in 40 mL of DMF was added a
solution of TBAF (1.2 M in THF, 5 mL, 6.0 mmol, 1.8 equiv). After 5
min (TLC control) DIPEA (1.0 mL, 6.0 mmol, 1.8 equiv) was added
dropwise, and the mixture was stirred for 15 min (TLC control). The
particular acyl chloride (3 equiv) was added, and the reaction mixture
was stirred for 30 min (TLC control). Afterward the mixture was diluted
with 30 mL of EtOAc/CyHex (1:1), washed with saturated aqueous
NaCl solution, and extracted with MtBE. The organic phase was dried
over MgSO4, and the solvent was evaporated. The crude product was
purified by column chromatography (silica gel, DCM/CyHex = 1:3).
Procedure B: Preparation of Dienyl-ester-Fe(CO)3 Complexes
Starting from rac-12. To a solution of complex rac-12 (1 equiv)
dissolved in 40 mL of DMF was added a solution of TBAF (1.2 M in
THF, 3.8 mL, 3.83 mmol, 3 equiv). After 5 min (TLC control) DIPEA
(0.6 mL, 3.83 mmol, 3 equiv) was added dropwise, and the mixture was
stirred for 15 min (TLC control). The particular acyl chloride (3 equiv)
was added, and the reaction mixture was stirred for 30 min (TLC
control). Afterward the mixture was diluted with 30 mL of EtOAc/
CyHex (1:1), washed with saturated aqueous NaCl solution, and
extracted with MtBE. The combined organic phases were dried over
MgSO4, and the solvent was evaporated under reduced pressure. The
crude product was purified by column chromatography (silica gel,
DCM/CyHex = 1:3).
General Procedure for CO Release Measurements in the
Presence of Oxygen (GC). All reactions were performed in 10 mL
headspace vials (BGB-Analytics, Cat. No. 200410-F). Prior to the
detection of the CO release a calibration was done. Calibrations were
done with the solvent mixture (phosphate buffer (1 mL, 0.1 M, pH =
7.4) and DMSO (0.2 mL)) that was used for the CO release
experiments. For the CO calibration, the consumption of oxygen had to
be included. For this purpose, the reaction vial was filled with the solvent
mixture and completely degassed with nitrogen. Afterward, 21% of the
gas volume was replaced by oxygen to generate the composition of air
(2.250 mL). To generate gas mixtures that are formed when a particular
amount of O2 is consumed in the course of the CO release, the volume
of added oxygen was decreased by the amount that is theoretically
needed for the release of the added CO (for a more detailed description
see Supporting Information). For the monitoring of the enzyme-
triggered CO release, the particular complex (36 μmol) was dissolved in
phosphate buffer (1.2 mL, 0.1 M, pH = 7.4). The particular enzyme (15
mg of PLE or 20 mg of LCR) was added, and the vial was closed with a
rubber vial cap. The reaction mixture was stirred at 37 °C. From time to
time samples (50 μL) were taken and the CO release was quantified.
The half-life times were determined directly from an exponential fit of
the CO release (first- or second-order). The same procedure was
repeated without enzyme for every complex, but there was virtually no
CO release without enzyme.
(RS)-Tricarbonyl-η4-(1-butyroyloxy-1,5-cyclohexadiene)iron (0)
(rac-10b). Following general procedure A (chromatography with silica
gel, DCM/CyHex = 1:3) complex rac-10b (0.81 g, 81%) was obtained as
Cells and Cell Culture. Human umbilical vein endothelial cells were
isolated from fresh umbilical cords as described previously.23 The cells
were grown in basal endothelial medium supplemented with 10% FBS
and essential growth factors until they formed a confluent monolayer.
1
a yellow oil. TLC: Rf (CyHex/EtOAc = 10:1): 0.67. H NMR (300
MHz, CDCl3): δ 5.50−5.48 (d, J = 6.4 Hz, 1H, H6), 3.34 (m, 1H, H2),
F
dx.doi.org/10.1021/om301233h | Organometallics XXXX, XXX, XXX−XXX

Organometallics
Article
2.84−2.82 (m, 1H, H5), 2.42−2.37 (t, J = 7.3 Hz, 2H, H8), 1.79−1.65
(m, 4H, H9, H3, H4), 1.55−1.52 (m, 2H, H3, H4), 1.00−0.95 (t, J = 7.3
Hz, 3H, H10). 13C NMR (75 MHz, CDCl3): δ 210.7 (Fe(CO)3), 173.0
(C7), 128.3 (C1), 79.7 (C6), 59.2 (C2), 52.0 (C5), 36.0 (C8), 24.5
(C3), 23.5 (C4), 18.2 (C9), 13.5 (C10). FT-IR (ATR) ν [cm−1]: 2960
(w, (Csp2-H)), 2926 (w, (Csp3-H)), 2873 (w, (Csp3-H)), 2040 (s,
(Fe(CO)3)), 1966 (s, (Fe(CO)3)), 1756 (s, (CO)). LR-MS (DIP-
MS, 70 eV): m/z (%) 278 (8, [M − CO]+), 250 (23, [M − 2CO]+), 220
(56, [M − 3O]+), 192 (13), 149 (48), 134 (51), 121 (19), 111 (24), 97
(53), 83 (49), 71 (67), 57 (87), 55 (100, [Fe]+). HR-MS (DIP-MS, 70
eV) ([M − CO]+): m/z 278.023 (calcd ([M − CO]+): m/z 278.024).
(RS)-Tricarbonyl-η4-(1-hexanoyloxy-1,5-cyclohexadiene)iron (0)
(rac-10c). Following general procedure A (chromatography with silica
gel, DCM/CyHex = 1:3) complex rac-10c (0.88 g, 81%) was obtained as
H3), 3.08−3.04 (m, 1H, H4), 2.29−2.24 (t, J = 7.4 Hz, 2H, H8), 2.17−
1.60 (m, 6H, H5, H6, H9), 0.95−090 (t, J = 7.4 Hz, 3H, H10). 13C NMR
(75 MHz, CDCl3): δ 211.3 (Fe(CO)3), 171.8 (C7), 103.3 (C1), 80.5/
80.3 (C2/C3), 60.3 (C4), 36.4 (C8), 26.7 (C6), 24.1 (C5), 18.3 (C9),
13.6 (C10). FT-IR (ATR): ν [cm−1] 2960 (w, (Csp3-H)), 2933 (w,
(Csp3-H)), 2866 (w, (Csp3-H)), 2040 (s, (Fe(CO)3)), 1956 (s,
(Fe(CO)3)), 1746 (s, (CO)). LR-MS (DIP-MS, 70 eV): m/z (%)
= 278 (8, [M − CO]+), 250 (34, [M − 2CO]+), 220 (100, [M −
3CO]+), 192 (16), 150 (35), 134 (51), 121 (21), 95 (7), 84 (15), 71
(12), 56 (49, [Fe]+). HR-MS (DIP-MS, 70 eV) ([M − 2CO]+): m/z
250.030 (calcd ([M − 2CO]+): m/z 250.029).
(RS)-Tricarbonyl-η4-(1-hexanoyloxy-1,3-cyclohexadiene)iron (0)
(rac-13c). Following general procedure B (chromatography with silica
gel, DCM/CyHex = 1:3) complex rac-13c (0.36 g, 84%) was obtained as
1
a yellow oil. TLC: Rf (CyHex/EtOAc = 10:1): 0.66. H NMR (300
1
MHz, CDCl3): δ 5.49−5.47 (d, J = 6.3 Hz, 1H, H6), 3.34 (m, 1H, H2),
2.84−2.82 (m, 1H, H5), 2.43−2.38 (t, J = 7.3 Hz, 2H, H8), 1.84−1.74
(m, 2H, H3), 1.68−1.63 (m, 2H, H9), 1.56−1.52 (m, 2H, H4), 1.31 (m,
4H, H10, H11), 0.88 (m, 3H, H12). 13C NMR (75 MHz, CDCl3): δ
210.8 (Fe(CO)3), 173.2 (C7), 128.4 (C1), 79.7 (C6), 59.2 (C2), 52.0
(C5), 34.2 (C8), 31.1 (C10/C11), 24.4 (C3), 23.5 (C9), 22.3 (C4),
13.9 (C12). FT-IR (ATR): ν [cm−1] 2926 (w, (Csp3-H)), 2853 (w,
(Csp3-H)), 2040 (s, (Fe(CO)3)), 1963 (s, (Fe(CO)3)), 1760 (m, (C
O)). LR-MS (DIP-MS, 70 eV): m/z (%) 306 (6, [M − CO]+), 278 (30,
[M − 2CO]+), 248 (100, [M − 3CO]+), 218 (11), 205 (22), 190 (10),
179 (23), 149 (44), 134 (56), 121 (25), 95 (16), 84 (19), 71 (15), 56
(78, [Fe]+). HR-MS (DIP-MS, 70 eV) ([M − 2CO]+): m/z 278.061
(calcd ([M − 2CO]+): m/z 278.061).
a yellow oil. TLC: Rf (CyHex/EtOAc = 10:1): 0.62. H NMR (300
MHz, CDCl3): δ 5.35−5.34 (d, J = 4.3 Hz, 1H, H2), 5.10−5.06 (m, 1H,
H3), 3.07−3.05 (m, 1H, H4), 2.30−2.25 (t, J = 7.5, 16.1 Hz, 2H, H8),
2.17−1.60 (m, 6H, H5, H6, H9), 1.32−1.25 (m, 4H, H10, H11), 0.88−
0.84 (t, J = 6.7 Hz, 3H, H12). 13C NMR (75 MHz, CDCl3): δ 211.3
(Fe(CO)3), 172.0 (C7), 103.3 (C1), 80.5/80.3 (C2/C3), 60.2 (C4),
34.5 (C8), 31.2 (C10), 26.7 (C6), 24.5 (C9), 24.1 (C5), 22.3 (C11),
13.9 (C12). FT-IR (ATR): ν [cm−1] 2953 (m, (Csp3-H)), 2926 (m,
(Csp3-H)), 2853 (m, (Csp3-H)), 2040 (s, (Fe(CO)3)), 1950 (s,
(Fe(CO)3)), 1746 (s, (CO)). LR-MS (DIP-MS, 70 eV): m/z (%)
306 (5, [M − CO]+), 278 (32, [M − 2CO]+), 248 (100, [M − 3CO]+),
218 (7), 205 (20), 190 (9), 170 (21), 149 (51), 134 (55), 121 (29), 95
(11), 84 (19), 71 (12), 56 (65), [Fe]+). HR-MS (DIP-MS, 70 eV) ([M
− CO]+): m/z 306.053 (calcd ([M − CO]+): m/z 306.056).
(RS)-Tricarbonyl-η4-(1-octanoyloxy-1,5-cyclohexadiene)iron (0)
(rac-10d). Following general procedure A (chromatography with silica
gel, DCM/CyHex = 1:3) complex rac-10d (0.83 g, 70%) was obtained
as a yellow oil. TLC: Rf (CyHex/EtOAc = 10:1): 0.71. 1H NMR (300
MHz, CDCl3): δ 5.49−5.47 (d, J = 6.6 Hz, 1H, H6), 3.34 (m, 1H, H2),
2.84−2.82 (m, 1H, H5), 2.43−2.38 (t, J = 7.4 Hz, 2H, H8), 1.84−1.72
(m, 2H, H3), 1.67−1.63 (m, 2H, H9), 1.56−1.50 (m, 2H, H4), 1.29−
1.26 (m, 8H, H10, H11, H12, H13), 0.88−084 (t, J = 6.0 Hz, 3H, H14).
13C NMR (75 MHz, CDCl3): δ 210.8 (Fe(CO)3), 173.2 (C7), 128.4
(RS)-Tricarbonyl-η4-(1-octanoyloxy-1,3-cyclohexadiene)iron (0)
(rac-13d). Following general procedure B (chromatography with silica
gel, DCM/CyHex = 1:3) complex rac-13d (0.38 g, 81%) was obtained
as a yellow oil. TLC: Rf (CyHex/EtOAc = 10:1): 0.62. 1H NMR (300
MHz, CDCl3): δ 5.35−5.34 (d, J = 4.3 Hz, 1H, H2), 5.10−5.06 (m, 1H,
H3), 3.07−3.05 (m, 1H, H4), 2.30−2.25 (t, J = 7.5 Hz, 2H, H8), 2.17−
1.59 (m, 6H, H5, H6, H9), 1.30−1.25 (m, 8H, H10, H11, H12, H13),
0.87−0.83 (t, J = 6.6 Hz, 3H, H14). 13C NMR (75 MHz, CDCl3): δ
211.3 (Fe(CO)3), 172.0 (C7), 103.3 (C1), 80.5/80.3 (C2/C3), 60.2
(C4), 34.5 (C8), 31.6 (C12), 29.0 (C10), 28.9 (C11), 26.7 (C6), 24.9
(C9), 24.1 (C5), 22.6 (C13), 14.1 (C14). FT-IR (ATR): ν [cm−1] 2926
(s, (Csp3-H)), 2853 (m, (Csp3-H)), 2046 (s, (Fe(CO)3)), 1946 (s,
(Fe(CO)3)), 1750 (s, (CO)). LR-MS (DIP-MS, 70 eV): m/z (%) 334
(5, [M − CO]+), 306 (29, [M − 2CO]+), 276 (100, [M − 3CO]+), 246
(11), 234 (11), 218 (13), 205 (6), 180 (9), 170 (10), 156 (25), 149 (68),
134 (53), 124 (8), 121 (29), 110 (10), 95 (13), 84 (17), 77 (12), 56 (69,
[Fe]+). HR-MS (DIP-MS, 70 eV) ([M − CO]+): m/z 334.086 (calcd
([M − CO]+): m/z 334.087).
(C1), 79.7 (C6), 59.2 (C2), 52.0 (C5), 34.2 (C8), 31.6 (C10/C12),
28.9 (C11), 24.7 (C3), 24.6 (C9), 23.5 (C4), 22.6 (C13), 14.0 (C14).
FT-IR (ATR): ν [cm−1] 2920 (s, (Csp3-H)), 2846 (s, (Csp3-H)), 2040 (s,
(Fe(CO)3)), 1990 (s, (Fe(CO)3)), 1756 (s, (CO)). LR-MS (DIP-
MS, 70 eV): m/z (%) 334 (5, [M − CO]+), 306 (24, [M − 2CO]+), 276
(100, [M − 3CO]+), 246 (5), 234 (5), 218 (16), 179 (23), 170 (12), 156
(26), 149 (61), 134 (53), 121 (29), 110 (6), 97 (19), 84 (21), 56 (74,
[Fe]+). HR-MS (DIP-MS, 70 eV) ([M − CO]+): m/z 334.086 (calcd
([M − CO]+): m/z 334.087).
(RS)-Tricarbonyl-η4-(1-lauroyloxy-1,5-cyclohexadiene)iron (0)
(rac-10e). Following general procedure A (chromatography with silica
gel, DCM/CyHex = 1:3) complex rac-10e (1.14 g, 83%) was obtained as
1
a yellow oil. TLC: Rf (CyHex/EtOAc = 10:1): 0.64. H NMR (300
(RS)-Tricarbonyl-η4-(1-lauroyloxy-1,3-cyclohexadiene)iron (0)
(rac-13e). Following general procedure B (chromatography with silica
gel, DCM/CyHex = 1:3) complex rac-13e (0.37 g, 75%) was obtained as
MHz, CDCl3): δ 5.49−5.47 (dd, J = 6.6 Hz, 1H, H6), 3.34 (m, 1H, H2),
2.84−2.82 (m, 1H, H5), 2.43−2.38 (t, J = 7.4 Hz, 2H, H8), 1.78−1.72
(m, 2H, H3), 1.67−1.60 (m, 2H, H9), 1.55−1.52 (m, 2H, H4), 1.28−
1.24 (m, 16H, H10, H11, H12, H13, H14, H15, H16, H17), 0.88−084
(t, J = 6.2 Hz, 3H, H18). 13C NMR (75 MHz, CDCl3): δ 210.8
(Fe(CO)3), 173.2 (C7), 128.4 (C1), 79.7 (C6), 59.2 (C2), 52.0 (C5),
34.2 (C8), 31.9 (C16), 29.6−29.0 (C10, C11, C12, C13, C14, C15),
24.7 (C3), 24.6 (C9), 23.5 (C4), 22.7 (C17), 14.1 (C18). FT-IR
(ATR): ν [cm−1] = 2920 (s, (Csp3-H)), 2846 (s, (Csp3-H)), 2040 (s,
(Fe(CO)3)), 1953 (s, (Fe(CO)3)), 1760 (s, (CO)). LR-MS (DIP-
MS, 70 eV): m/z (%) 390 (5, [M − CO]+), 362 (11, [M − 2CO]+), 334
(100, [M − 3CO]+), 254 (41), 239 (9), 226 (25), 212 (56), 198 (39),
184 (26), 170 (39), 149 (67), 134 (53), 121 (27), 110 (15), 97 (29), 84
(20), 71 (20), 56 (91, [Fe]+). HR-MS (DIP-MS, 70 eV) ([M − CO]+):
m/z 390.148 (calcd ([M − CO]+): m/z 390.149).
1
a yellow oil. TLC: Rf (CyHex/EtOAc = 10:1): 0.64. H NMR (300
MHz, CDCl3): δ 5.35−5.34 (d, J = 4.3 Hz, 1H, H2), 5.10−5.06 (m, 1H,
H3), 3.07−3.05 (m, 1H, H4), 2.30−2.25 (t, J = 7.5 Hz, 2H, H8), 2.17−
1.59 (m, 6H, H5, H6, H9), 1.23 (m, 16H, H10, H11, H12, H13, H14,
H15, H16, H17), 0.88−0.83 (t, J = 6.6 Hz, 3H, H18). 13C NMR (75
MHz, CDCl3): δ 211.3 (Fe(CO)3), 172.0 (C7), 103.3 (C1), 80.5/80.3
(C2/C3), 60.2 (C4), 34.5 (C8), 31.9 (C16), 29.6 (C10/C11), 29.4−
29.0 (C15, C12, C13, C14), 26.7 (C5), 24.9 (C6), 24.1 (C9), 22.7
(C17), 14.1 (C18). FT-IR (ATR): ν [cm−1] 2920 (s, (Csp3-H)), 2846 (s,
(Csp3-H)), 2040 (s, (Fe(CO)3)), 1956 (s, (Fe(CO)3)), 1743 (s, (C
O)). LR-MS (DIP-MS, 70 eV): m/z (%) 390 (3, [M − CO]+), 362 (11,
[M − 2CO]+), 334 (81, [M − 3CO]+), 254 (40), 239 (9), 226 (23), 212
(56), 198 (37), 184 (27), 170 (41), 151 (37), 149 (75), 121 (32), 110
(22), 97 (35), 83 (38), 71 (35), 69 (43), 57 (100), 56 (11, [Fe]+). HR-
MS (DIP-MS, 70 eV) ([M − CO]+): m/z 390.148 (calcd ([M − CO]+):
m/z 390.149).
(RS)-Tricarbonyl-η4-(1-butyroyloxy-1,3-cyclohexadiene)iron (0)
(rac-13b). Following general procedure B (chromatography with silica
gel, DCM/CyHex = 1:3) complex rac-13b (0.43 g, 91%) was obtained as
1
a yellow oil. TLC: Rf (CyHex/EtOAc = 10:1): 0.57. H NMR (300
MHz, CDCl3): δ 5.36−5.34 (d, J = 4.2 Hz, 1H, H2), 5.10−5.07 (m, 1H,
G
dx.doi.org/10.1021/om301233h | Organometallics XXXX, XXX, XXX−XXX

Organometallics
Article
(15) Desmard, M.; Foresti, R.; Morin, D.; Dagouassat, M.; Berdeaux,
A.; Denamur, E.; Crook, S. H.; Mann, B. E.; Scapens, D.; Montravers, P.;
Boczkowski, J.; Motterlini, R. Antioxid. Redox Signaling 2012, 16, 153−
163.
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthetic procedures and analytical data of all synthesized
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
(16) Schatzschneider, U. Inorg. Chim. Acta 2011, 374, 19−23.
(17) Romanski, S.; Kraus, B.; Schatzschneider, U.; Neudorfl, J.-M.;
̈
Amslinger, S.; Schmalz, H.-G. Angew. Chem., Int. Ed. 2011, 50, 2392−
2396.
AUTHOR INFORMATION
■
(18) Romanski, S.; Kraus, B.; Guttentag, M.; Schlundt, W.; Rucker, H.;
̈
Corresponding Author
*E-mail: schmalz@uni-koeln.de.
Notes
Adler, A.; Neudorfl, J.-M.; Alberto, R.; Amslinger, S.; Schmalz, H.-G.
̈
Dalton Trans. 2012, 41, 13862−13875.
(19) Romanski, S.; Rucker, H.; Stamellou, E.; Guttentag, M.; Neudorfl,
J.-M.; Alberto, R.; Amslinger, S.; Yard, B.; Schmalz, H.-G. Organo-
metallics 2012, 31, 5800−5809.
̈
̈
The authors declare no competing financial interest.
(20) There are also some other problems connected to the Mb assay.
For instance, it was found that the dithionite (necessary to keep the Mb
in the reduced form) influences the CO release from certain CORMs.
For details, see: McLean, S.; Mann, B. E.; Poole, R. K. Anal. Biochem.
2012, 427, 36−40.
(21) TLC analysis clearly revealed that the conversion of complexes of
type rac-13 was complete after 2 h (under the standard conditions used
for the GC monitoring of the CO release). The reaction of complexes of
type rac-10 was somewhat slower but definitely over after 24 h (while
the CO release still proceeded). Details of the decomposition
mechanism in the two compound series are the subject of a current
investigation in our lab.
ACKNOWLEDGMENTS
■
This work was supported by the Deutsche Forschungsgemein-
schaft (FOR 630) and the Fonds der Chemischen Industrie
(doctorate stipend to S.R.). We also thank the referees for
carefully reading the manuscript and for providing valuable
comments, which induced some very important corrections and
improvements.
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dx.doi.org/10.1021/om301233h | Organometallics XXXX, XXX, XXX−XXX