Synthesis of oxime-based CO-releasing molecules, CORMs and their immobilization on maghemite nanoparticles for magnetic-field induced CO release

Article abstract of DOI:10.1039/c5dt04888e

Oxime-based CO-releasing molecules (oximeCORMs) were immobilized with a catechol-modified backbone on maghemite iron oxide nanoparticles (IONPs) to give oximeCORM@IONP. The CO release from the free and immobilized oximeCORMs was measured using the standard myoglobin assay. The oximeCORM-nanoparticles were coated with dextran for improved water solubility and confined into an alginate shell for protection and separation from the surrounding myoglobin assay to allow for CO release studies by UV/Vis absorption without interference from highly-absorptive oximeCORM@IONP. Half-lifes of the oxime-based polymer-confined alginate@dextran@oximeCORM@IONPs were estimated at 20 °C to 814 ± 23 min, at 37 °C to 346 ± 83 min and at 50 °C to 73 ± 1 min. The alginate@dextran@oximeCORM@IONP composite showed a further decrease of the half-life of CO release to 153 ± 27 min at 37 °C through local magnetic heating of the susceptible iron oxide nanoparticles with application of an external alternating magnetic field (31.7 kA m^-1, 247 kHz, 39.9 mTesla). The activation energy for the CO release from molecular dicarbonylchlorido(imidazole-2-carbaldehydeoxime)(alkoxycarbonyl)ruthenium(ii) complexes is determined to be ～100 kJ mol^-1 for five different imidazole-oxime derivatives.

Full text of DOI:10.1039/c5dt04888e

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Synthesis of oxime-based CO-releasing molecules,
CORMs and their immobilization on maghemite
nanoparticles for magnetic-field induced CO
release†
Cite this: DOI: 10.1039/c5dt04888e
Hajo Meyer,a Markus Brenner,a Simon-P. Höfert,a Tim-O. Knedel,a Peter C. Kunz,a
Annette M. Schmidt,b Alexandra Hamacher,c Matthias U. Kassackc and
Christoph Janiak*a
Oxime-based CO-releasing molecules (oximeCORMs) were immobilized with a catechol-modified back-
bone on maghemite iron oxide nanoparticles (IONPs) to give oximeCORM@IONP. The CO release from
the free and immobilized oximeCORMs was measured using the standard myoglobin assay. The oxime-
CORM-nanoparticles were coated with dextran for improved water solubility and confined into an alginate
shell for protection and separation from the surrounding myoglobin assay to allow for CO release studies
by UV/Vis absorption without interference from highly-absorptive oximeCORM@IONP. Half-lifes of the
oxime-based polymer-confined alginate@dextran@oximeCORM@IONPs were estimated at 20 °C to 814
23 min, at 37 °C to 346 83 min and at 50 °C to 73 1 min. The alginate@dextran@oximeCORM@IONP
composite showed a further decrease of the half-life of CO release to 153 27 min at 37 °C through local
magnetic heating of the susceptible iron oxide nanoparticles with application of an external alternating
magnetic field (31.7 kA m−1, 247 kHz, 39.9 mTesla). The activation energy for the CO release from mole-
cular dicarbonylchlorido(imidazole-2-carbaldehydeoxime)(alkoxycarbonyl)ruthenium(II) complexes is
determined to be ∼100 kJ mol−1 for five different imidazole-oxime derivatives.
Received 15th December 2015,
Accepted 20th March 2016
DOI: 10.1039/c5dt04888e
www.rsc.org/dalton
order to regulate the circadian rhythms, memory and
hemodynamics.8–10 Due to its regulatory and cytoprotective
Introduction
The toxic physiological effects of carbon monoxide (CO) were effects CO has already been extensively investigated for
first described by John Haldane in 1927.1 CO is generally medical applications.9 Yet, when CO is administered to a
known as a highly toxic gas which hinders the oxygen trans- human organism as a gas, it is difficult to accumulate at
port in the blood system, by strong coordination to the oxygen a specific site (e.g. tumor or burned skin). Gaseous CO as a
binding site in hemoglobin. Even breathing of small quantities therapeutic agent features a half-life of 3 to 7 hours in the
(about 100 ppm in air) of this color- and odorless gas can be body and was well tolerated at 3 mg per kg for one hour. CO
potentially harmful to the human organism.2,3 On the other binds reversibly to cellular targets and the only way for CO to
hand, carbon monoxide is classified as a biochemical messen- exit mammal bodies in a therapeutic approach was by lung
ger molecule like hydrogen sulfide and nitrogen oxide.4–6 exhalation.11,12 In fact the gas also reaches healthy tissue and
Human organisms express heme oxygenase enzymes,7 which the dose needs to be relatively high in order to achieve an
are capable of producing carbon monoxide continuously in effect on the desired tissue. In addition, the quantity needs to
be carefully controlled and special administering equipment is
necessary.
aInstitut für Anorganische Chemie und Strukturchemie, Universität Düsseldorf,
CO releasing molecules (CORMs), primarily metal carbonyl
40204 Düsseldorf, Germany. E-mail: janiak@uni-duesseldorf.de
derivatives were invented for specific targeting and controlled
release of carbon monoxide. Metal carbonyl derivatives are
investigated also as antibacterial compounds and for their
anticancer therapeutic potential.13 However, the first CORMs
were hardly water-soluble and released CO a way too fast for
precise administering (half the gas load in less than one
minute under physiological conditions).14 Motterlini and co-
bInstitut für Physikalische Chemie, Universität zu Köln, Luxemburger Str. 116,
D-50939 Köln, Germany. E-mail: annette.schmidt@uni-koeln.de
cInstitut für Pharmazeutische und Medizinische Chemie, Universität Düsseldorf,
40204 Düsseldorf, Germany. E-mail: matthias.kassack@hhu.de
†Electronic supplementary information (ESI) available: Synthesis for oxime-
based CORMs, NMR, IR and ESI-mass spectra, myoglobin assay, leaching experi-
ments, DLS experiments, TEM measurements, toxicity tests, and activation
energy. See DOI: 10.1039/c5dt04888e
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workers3,15 introduced in 2003 the water-soluble complex perties of these large pores to allow polymers to enter the
[Ru(CO)3(glycinate)] (CORM-3) which released CO under tumor interstitium and to remain and accumulate there
physiological conditions16 and showed vasodilatative, anti- because the polymers are not removed by the lymphatic
inflammatory, renoprotective and anti-apoptosis effects in system.36
preclinical studies.17 Since the last decade, Ru-based CORMs have
become a very important field of research with several studies of tissue with the ability to release CO is an additional concept of
CORM-2 and the water-soluble CORM-3 (Scheme 1).9,14,16
developing polymer–CORM conjugates. In such conjugates the
The accumulation of functionalized polymers in tumor
The half-life of CO release from CORM-3 via a pseudo-first- use of a slow CO releaser from which the CO release can be
order reaction is 1 min at 37 °C in PBS buffer,18 2.3 min at triggered or activated by magnetic heating through alternating
30 °C in PBS buffer19 and 3.6 min in human plasma,20 appar- magnetic field should be of interest.
ently due to CO-abstraction by heme proteins.21 Recent studies
Different CORMs with ruthenium carbonyls are known and
presented CORMs which liberate CO upon external stimuli like the number is still increasing.37 Oresmaa et al. described a
change in the pH,22,23 increasing temperature, UV simple synthetic route to oxime-based CORMs for which they
irradiation24,25 and oxidative or enzymatic degradation.26,27 reported a longer half-life for CO release than for CORM-3 38
The immobilization of CORMs in28 and on29,30 other materials (Scheme 1).
opens new methodologies in medicinal chemistry.31,32
However, different possible medical applications of CORMs trolled CO release by covalently attaching the CO-releasing
will require CORMs with different release kinetics. molecule (a CORM-3 analogue) to the surface of iron oxide
In our previous work, we presented a new concept of con-
Drugs which are conjugated to polymers are restricted to nanoparticles which are susceptible to local heating through
the vascular system and can be transported directly to the an external alternating current magnetic field.39 The increased
target area.
A passive accumulation of macromolecular temperature of the nanoparticles, due to the external alternat-
systems in tumor tissues due to the so-called enhanced per- ing magnetic field, decreased the half-life from 13 2 min to
meability and retention (EPR) effect was first described and
investigated by Maeda et al.33,34
7
2 min in a buffered myoglobin-assay solution kept at 25 °C.
Here we target the covalent immobilization of relatively
The growth of tumors (>2 mm) is ensured by fast growing stable (long half-life) oxime-based CORMs on the surface of
blood vessels to supply nutrients and dioxygen. The blood iron oxide nanoparticles. In addition a dextran coating is
vessels are destabilized due to vascular endothelial growth applied for better water solubility and as a barrier against
factors (VEGFs) into the extracellular space and have higher heme proteins.
permeability than normal grown blood vessels.35 The resulting
At the same time the strong absorptivity of the black IONP
blood vessels lack tight junctions that are normally present dispersion hampers CO release studies with the standard myo-
between adjacent vascular endothelial cells. This deficiency globin assay in the same dispersion. The CO release by a myo-
leaves holes on an order of magnitude greater than normal globin assay is followed by UV/Vis spectrometry. Therefore, we
vascular pores (healthy tissue has pores of 5 to 8 nm, tumor used an alginate confinement of “water-soluble” dextran@
tissue can have pores of about 50 nm and inter-endothelial oximeCORM@IONP to achieve a spatial separation from the
junctions of about 500 nm). The EPR effect refers to the pro- surrounding myoglobin assay.
Results and discussion
Synthesis
The covalent immobilization of the oxime-based CORMs on an
iron oxide nanoparticle (IONP) surface is depicted in
Scheme 2.
Starting with the commercially available 2-chloro-3′,4′-di-
hydroxyacetophenone 3 (Scheme 3), we first tried unsuccess-
fully a simple condensation reaction with 1H-imidazole-
2-carbaldehyde 4 to try to obtain the condensation product
Scheme 1 Scheme of the synthesized oxime-based CORMs from
Oresmaa et al. with their half-lifes. The half-lifes were determined at
ambient (room) temperature in a phosphate buffered saline solution
(pH = 6.8).38
Scheme 2 Covalent immobilization of oxime-based CORM on an iron
oxide nanoparticle (IONP) surface through a catecholate group.
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Scheme 4 Synthesis of CORMs with the diol-protected catechol oxime
8 as methoxy- (10) and ethoxy- (11) ruthenium complexes (yields in
parentheses).
Scheme 3 Synthetic route (with yields) for the coupling of the catechol
group with 1H-imidazol-2-carbaldehyde (4) to 1-(2-(3,4-dihydroxy-
phenyl)-2-oxoethyl)-1H-imidazole-2-carbaldehyde (5) and the acetate
protected (8) and unprotected oxime (9). (a) Acetonitrile, sodium car-
bonate, 60 °C, 5 d; (b) dry toluene, dry acetone, phosphorus pentoxide,
75 °C, 4 h; (c) acetonitrile, sodium carbonate, 60 °C, 7 d; (d) acetic acid,
hydrochloric acid, 100 °C, 2 h; (e) sodium carbonate, hydroxylammo-
nium chloride, 70 °C, 24 h.
1-(2-(3,4-dihydroxyphenyl)-2-oxoethyl)-1H-imidazole-2-carbaldehyde
5 in a single step. The single-step route failed so that the diol
was first protected by an acetal group to form product 6 in
good yields. The condensation of 6 with 4 to give 1-(2-(3,4-di-
hydroxyphenyl)-2-oxoethyl)-1H-imidazole-2-carbaldehyde 7 in
acetonitrile with an equimolar ratio of a base proved success-
ful. Acid cleavage of the acetal protecting group afforded the
desired product 1-(2-(3,4-dihydroxyphenyl)-2-oxoethyl)-1H-
imidazole-2-carbaldehyde 5 in high yields. Introducing the
oxime function into compounds 7 and 5 was done similarly to
ref. 38 (Scheme 3). The reaction times were extended up to
24 h to obtain good yields for (E)-1-(2-(2,2-dimethylbenzo[d]-
[1,3]dioxol-5-yl)-2-oxoethyl)-1H-imidazol-2-carbaldehyde-oxime
8 (75%) and 1-(2-(3,4-dihydroxyphenyl)-2-oxoethyl)-1H-imid-
azole-2-carbaldehyde-oxime 9 (47%).
The synthesis of the diol-protected ruthenium carbonyl
oximes 10 and 11 was performed as described in the litera-
ture38 with [Ru(CO)3Cl2]2 (12) and an extended reaction time of
5 d (Scheme 4). To obtain the methoxy-compound 10 the reac-
tion was carried out in methanol and for the ethoxy-compound
11 in ethanol. CORMs 10 and 11 precipitated from the solution
as the modified backbone leads to lower solubility in polar sol-
vents than for the parent compounds a and b (Scheme 1).
For immobilization on an iron oxide nanoparticle surface
the dihydroxyphenyl-oxime 9 was reacted with [Ru(CO)3Cl2]2
(12) to give the ruthenium carbonyl oximes 13 and 14 as
shown in Scheme 5.
Scheme 5 Synthesis of CORMs with the (unprotected) catechol oxime
9 as methoxy- (13) and ethoxy- (14) ruthenium complexes (yields in
parentheses).
Electron spray ionization mass spectrometric analysis of
10–14 in acetonitrile solution showed ligand lability (see
Fig. S3, ESI†). For compounds 10 and 11 or 13 and 14, respect-
ively, identical mass spectra were obtained and the isotopic
pattern showed a ruthenium complex from which the methoxy
(or ethoxy) group (OR−), a CO unit and/or a chloride ligand
had been lost. In addition, a proton was abstracted, pre-
sumably from the N–OH group, which together with CO and
OR− amounts to the liberation of formic acid ester or with OR−
only to the liberation of the alcohol ROH. Instead, acetonitrile
was coordinated under ESI-MS conditions. Hence, the domi-
nant fragments (for 102Ru) of m/z = 499.1 (10, 11) or 459.2 (13,
14) correspond to [M − OR− − CO − Cl− − H+ + CH3CN]+ and
m/z = 527.0 (10, 11) or 487.0 (13, 14) to [M − OR− − Cl− − H+ +
CH3CN]+ (R = Me, Et). The lability of carbonyl complexes in
ESI mass spectrometric analysis is known.40
IR spectroscopic measurements of 10–14 show the expected
strong carbonyl absorptions (Fig. S1 in the ESI†) which agree
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Table 1 Carbonyl stretching frequencies of oxime ruthenium CORMsa
The IR spectrum of the composite material 17 (Fig. S4 in
the ESI†) shows the OH-vibrations of the dextran at
3600–3000 cm−1 and the carbonyl region exhibits three strong
Compound
νCO [cm−1
]
νCO [cm−1
]
Ref.d
absorptions at 2064, 1995 and 1961 cm−1
.
1aa,c
2064
2067
2056–2059
2075
2065
2077
2064
2061
2071, 2015
1990
1994
1980–1985
1989
1988
Fig. S2-1a
Fig. S2-1a
38
Fig. S2-1b
Fig. S2-1b
Fig. S2-2
Fig. S2-3
Fig. S24
Fig. S24
2aa,c
The amount of ruthenium in the dextran@oxime-
CORM@IONP (17) composite was determined by atomic
absorption spectroscopy (AAS) to an average of 0.60 wt% ruthe-
nium and a molar Ru : Fe ratio of 1 : 25 (see Experimental
section or the ESI† for details). This is the same molar Ru : Fe
ratio as in our previous report on a CORM-3 analog on IONP
(Ru : Fe 1 : 2539).
Further, composite 17 was incorporated into calcium algi-
nate spheres. A mixture of 17 with sodium alginate was added
dropwise to a calcium chloride solution. Calcium cross-linked
alginate spheres were rapidly formed which almost quantitat-
ively incorporate the dextran@oximeCORM@IONP substrate
as is evident from the clear, non-colored surrounding solution
(Fig. S26 in the ESI†). The IR spectrum of the composite
material 18 (Fig. S28 in the ESI†) shows two strong absorptions
at 2061 and 1991 cm−1, which are indicators for the immobi-
lized carbonyls on the nanoparticle surface. Yet, the still
necessary use of water to anchor 14 on IONPs to give 16 and in
the dextran and alginate coating of 16 to 17 and further to 18
represents a drawback and already induces partial decompo-
sition with a loss of CO (see below and Table S1 in the ESI†).
Future work has to address replacing water by other solvents.
1ab,c, 2ab,c
10a
11a
14a
2012
17a
1995, 1961e
1991
18a
18a after CO release
1992
a KBr disks. b FT-IR in DMSO. c Formula in Scheme 1. d This work with
reference to the figure showing the IR spectrum, unless indicated
otherwise. e We trace the third CO band to partial CO release during
CORM deposition on IONP and inclusion in dextran in water
(cf. Scheme 6).
well with the ones reported by Oresmaa et al.38 for a and b in
the regions 2056–2059 cm−1 and 1980–1985 cm−1 (Table 1).
Maghemite nanoparticles (15) were synthesized by the
known method from Ewijk et al.41 A catechol based anchor
group is common for immobilizing molecules on an IONP
surface.42 The unprotected catechol–oxime ruthenium
complex 14 was immobilized on the surface of maghemite
nanoparticles. Compound 14 was dissolved in water under
basic conditions and stirred with maghemite nanoparticles.
To prevent agglomeration of the nanoparticles the combined
solutions of both compounds were kept below pH 8. After pre-
cipitation with acetone and washing with ethanol, the oxime-
CORM@IONP composite nanoparticles 16 were not able to
build a stable dispersion in MOPS-buffer or water but sedi-
mented within a few seconds (see the ESI† for DLS and TEM
analysis of 16). To increase the solubility of the oxime-
CORM@IONP nanoparticles in water, they were coated by
using dextran polymers to give dextran@oximeCORM@IONP
(17). Water-soluble dextran (M = 500 000 g mol−1) consists of
glucose monomers with α-1,4- and α-1,6-connectivity and was
added during the reaction of 14 with the iron oxide
nanoparticles.
CO release
The half-lifes t1/2 of the CO release from compounds 1, 2, 10,
11 and 18 were measured using the myoglobin assay to eluci-
date differences in the CO release kinetics at 20, 37 and 50 °C
(Fig. 1a).
A 3-(N-morpholino)propanesulfonic acid buffer
Fig. 1 Time-dependent visible absorption spectra (left) and the plot
of the intensity changes of selected wavelengths (right) of alginate@
dextran@oximeCORM@IONP (18) with the myoglobin assay at 37 °C (a)
without the influence of an external alternating magnetic (AC) field to
t1/2 = 346 83 min, and (b) with alternating field t1/2 = 155 27 min.
Arrows in the absorption spectra mark the selected wavelengths with
the direction of their time-dependent change.
Scheme 6 Synthesis of the composite material alginate@dextran@
oximeCORM@IONP (18) by immobilization of the oximeCORM 14 on the
maghemite nanoparticle surface 15.
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(pH = 7.4, 0.1 M) was used by us instead of the phosphate alginate-separation method allows for working with the widely
buffer which Oresmaa et al. described to prevent further accepted myoglobin assay.
agglomeration of the IONP-containing samples and to make the
results comparable to our previously presented system.39 All coated iron oxide nanoparticles presents a membrane, which
measurements were done three times to ensure reproducibility. allows for diffusion of gaseous CO but prevents the interaction
The calcium alginate shell around the “soluble” dextran-
The half-lifes of the CO release are listed in Table 2 (for with the outside protein and sodium dithionite of the myo-
kinetic data see Fig. S5–S25 in the ESI†). Oresmaa et al. globin assay.42
described a slightly faster CO release from the methoxy-1a
Previous CO-release studies from CORM-2 and CORM-3
(t1/2 60 min) than from the ethoxy-2a oximeCORM showed that the rate depended on myoglobin and was accele-
=
(cf. Scheme 1) (t1/2 = 70 min) at ambient temperature, albeit rated by an increasing concentration of the reducing agent,
not further specified temperature.38 From our thermostated sodium dithionite.21 The loss of carbon monoxide was shown
measurement at 20 °C, 37 °C and 50 °C, the methoxy- (1a) and when CORM-3 was preincubated with sodium dithionite in the
ethoxy- (2a) derivatives have the same half-life within the absence of myoglobin. After reduction of the myoglobin and
experimental error. We note that the oximeCORM compounds removal of the sodium dithionite in the assay, no liberation of
1a and 2a show a slower CO release than CORM-3 at 37 °C CO from CORM-3 was seen.19 The interactions of CORM-3 with
(t1/2 ≤ 1 min) in the presence of the myoglobin assay.
proteins result in the loss of a chloride ion, glycinate, and one
The diol-protected catechol oximeCORMs 10, 11 and the CO ligand because of rapid formation of stable protein–Ru
unprotected catechol oximeCORM 14 also show similar half- (CO2) adducts.21
lifes within the experimental error with a slightly faster release
than from 1a and 2a at 20 °C, which might correlate to the the alkoxycarbonyl group plays an important role in CO
modified backbone. release. Whether the role is merely to destabilize carbonyl
The dark “soluble” dextran-coated iron oxide nanoparticles ligands or act as a source of CO remains to be studied”.38
17 do not lend themselves to follow the CO release with the Furthermore, the alginate spheres containing the iron oxide
For the oxime-based CORMs (1, 2) Oresmaa suggested “that
myoglobin assay due to their strong UV/Vis absorption.42 nanoparticles were magnetic and could be retained at the
Dextran coating leads to a stable dispersion with no sedimen- bottom of the cuvette with a magnet to prevent them from
tation anymore. Thus, the high background absorption from moving into the light path during UV/Vis measurement
the dark color from the iron oxide nanoparticles masks the (Fig. S22 in the ESI†). Spheres of the composite material 18
absorption changes of the myoglobin assay. The confinement were transferred within 30 min after their synthesis into the
of dextran@oximeCORM@IONP in calcium alginate spheres cuvette under nitrogen to avoid contamination with dioxygen.
allowed for minimization of the spectral interference by separ- MOPS-buffer and myoglobin solution (containing Na2S2O3)
ation within the myoglobin assay.42 We briefly note that there were added and the CO release measurements were started
are other methods available for CO release studies from immediately by collecting time-dependent UV/Vis spectra at
CORMs. Electrochemical sensing,43 fluorescent probes,44,45 20, 37 and 50 °C. The half-lifes were determined from the
gas-phase IR spectroscopy,43 or gas chromatography46,47 have kinetic curves of the absorption changes with time (see the
been reported in the literature. Yet, the inexpensive and easy ESI†) and are listed in Table 2.
The CO release of the composite material 18 was also
measured at 37 °C (surrounding solution temperature) under
the influence of an external alternating magnetic (AC) field
Table 2 Half-lifes of the CO release from oximeCORMs determined
using the myoglobin assay at different temperatures
t1/2 [min]
Compound
20 °C
∼25 °C
37 °C
21
50 °C
1a
220 23
2
1
5
6
1
1aa
60
70
2a
226
9
18
1
2aa
10
11
14
18
172 23
155 13
18
18
16
346 83
153 27
3
1
1
4
4
3
1
1
1
1
207
6
814 23
73
18 in AC fieldb
a The work of Oresmaa et al.38 does not specify a temperature. Thus,
ambient or room temperature can be assumed which can be –
depending on season, climate and weather – somewhere between
15 °C to 30 °C. The half-life values from Oresmaa et al. must be traced
to a temperature higher than 20 °C or to buffer influences. b With
Fig. 2 Half-lifes for temperature-dependent CO release from alginate@
dextran@oximeCORM@IONP (18) without and with applied alternating
(AC) magnetic field (at 37 °C only).
applied alternating magnetic field (31.7 kA m−1
39.9 mTesla).
,
247 kHz,
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(31.7 kA m−1, 247 kHz, 39.9 mTesla) to determine the accele- a half-life of 890 70 min at 20 °C, 172 27 min at 37 °C and
rated CO release due to magnetic heating of the iron oxide par- 45 7 min at 50 °C with an AC field to 155 18 min at 20 °C,
ticles (Fig. 2). The estimated half-life at 37 °C with an AC field 65 5 min at 37 °C and 30 3 min at 50 °C.42 Thus, the accele-
is 153 27 min (ESI, Fig. S27†). The magnetic heating of the ration of CO release or lowering of the CORM half-life by the
maghemite nanoparticles leads to an accelerated CO release by AC field will be even larger at 20 °C.
a factor 2.3 through the increase in the surface temperature of
the IONP, which affects the attached CORM (Fig. 3a).
IR spectroscopic measurements showed shifted carbonyl
absorptions after the CO release of the composite material
We determined the AC effect only at the physiologically rele- alginate@dextran@oximeCORM@IONP (18) from 2061 and
vant temperature of 37 °C. We have shown recently with a 1991 cm−1 to 2071, 2015 and 1992 cm−1 (ESI, Fig. S28†). The
CORM-3@IONP analog that the CO-release is accelerated from complete disappearance of the carbonyl absorption bands
could not be observed after the CO release studies with the
myoglobin assay. Therefore, the release of both CO-ligands
from oximeCORMs does not take place. For CORM-3 only one
mol of CO can be released from [Ru(CO)3Cl(O2CCH2NH2)] in
PBS buffered solution with myoglobin. After the release of one
CO from CORM-3 an inactive form, termed iCORM is formed.
The inactive iCORM-form of CORM-3 is not able to release
further CO after the loss of one CO.16
The absolute amount of released CO was determined with
the Lambert–Beer law and gave the formation of 0.2(1) mol
carbon monoxide per mol of Ru from alginate@dextran@
oximeCORM@IONP (18), 0.9(1) mol for 10, 0.8(1) mol for 11
and 0.9(1) mol for 14 in the myoglobin assay (see eqn (1) in
the ESI†). The low CO amount per Ru in 18 results apparently
from the CO loss during the synthesis. It is known from recent
work that CORM-2 ([Ru(CO)3Cl2]2) will lose 1.8 CO equivalents
as CO2 in water within 24 h.37 In order to anchor 14 on maghe-
mite nanoparticles (15) and coat with dextran to yield dextra-
n@oximeCORM@IONP (17), the mixture had to be stirred in
water for 3 h. Inclusion of 17 in the alginate spheres, giving 18
Fig. 3 (a) Correlation of ln(t1/2) versus the temperature for 18 for AC on
and off to estimate the IONP surface temperature upon magnetic
added another ∼35 min of water contact before the CO release
measurements. Attempts to replace water by methanol in the
heating, thereby assuming a direct correlation between their half-life
and temperature. The initial temperature position of the red dot (AC on)
is the set solution temperature. The AC field then increases the tempera-
formation of 17 led to agglomeration of the IONPs.
ture on the IONP surface as traced by the blue arrows. (b–d) Arrhenius
In our previous work on a CORM-3@IONP analog we rea-
plots for 18 (b), 11 (c) and 14 (d) of ln k = ln(ln(2)/t1/2) versus 1/TKelvin
lized that ln(t1/2) shows a linear correlation with temperature
and that the ln(t1/2) values at 37 °C (AC off) and 20 °C (solution
to determine the activation energy from the slope (−EA/R,
R =
8.314 J mol−1 K−1) of the graph.
Table 3 Cytotoxicity test of ruthenium CORMs and cisplatin against the human ovarian cancer cell line A2780, the human tongue cancer cell line
Cal27 and the non-cancer cell line HEK293. The inhibition of cell growth is expressed as % of untreated controla
Cell line
A2780
Cal27
100
HEK293
100
Concentration/µmol L−1
Compound
100
10
10
10
Cisplatin
CORM-2
CORM-3
14b
93.2
0
5.20
12.7
—
0
5.73
3.07
95.2
20.8
22.0
29.3
—
0
2.25
2.65
94.3
13.9
18.0
33.9
—
9.23
7.04
0
Concentration/µg mL−1
100
10
100
0
10
0
100
10
0
Dextran@oxime-CORM@IONP (17)
0
0
0
a Growth inhibition values are the average of experiments performed in triplicate. b See Scheme 5 for the formula of the ethoxy-oximeCORM 14.
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temperature, AC on) were very close, with ln(172) = 5.15 and ln myoglobin assay. An alternating current magnetic field (31.7
(155), respectively = 5.04. From this, we deduced that the AC kA m−1, 247 kHz, 39.9 mTesla) leads to a decrease of the half-
field at 20 °C generates an IONP surface temperature of about life at 37 °C from 346 83 (without AC field) to 153 27 (with
37 °C.42 The surface temperature of the IONP through the AC AC field).
field was determined through a plot of ln(t1/2) versus the temp-
erature (Fig. 3a). Under the assumption that the half-life for
the same material is only a function of temperature, then the
ln(t1/2) value reached under the AC magnetic field would corre-
late with an IONP surface temperature of 43 °C in the sur-
Experimental section
rounding solution temperature of 37 °C (Fig. 3a).
A
All chemicals were purchased from Acros®, Aldrich®, VWR®
determination of the activation energy EA for the CO release and Fluka® and used as received. [Ru(CO)3Cl2]2 (12) was syn-
from the alginate composite material 18 with an Arrhenius plot thesized according to literature procedures.48 Doubly-de-
of ln(ln(2)/t1/2) versus 1/T gives a value of 62(17) kJ mol−1 ionized (d.d.) water was obtained by Millipore Synergy with a
(AC off) (Fig. 3b). This activation energy (with its error margins) conductivity of 0.05 μS cm−1
.
compares well to the value of 78 kJ mol−1 for the CO release
1H-NMR spectra were recorded with a 300 or 600 MHz
from a CORM-3 analog embedded in a similar alginate@ Bruker FT-NMR Spectrometer Avance III. For measurements
dextran@CORM@IONP composite.42 Furthermore, we deter- with CDCl3, tetramethylsilane (TMS) was used as an internal
mined the activation energy for the CO release from 1a to 100(3) reference. Measurements in other solvents were referenced to
kJ mol−1, from 2a to 96(12) kJ mol−1, from 10 to 99(1) kJ mol−1
,
the solvent signal (DMSO-d6 2.50 ppm, acetone-d6 2.04 ppm).
from 11 to 96(1) kJ mol−1 and from 14 to 111(2) kJ mol−1 (see All chemical shifts δ are given in ppm. Spectra were recorded
ESI, Fig. S39†). The low correlation for 18 is due to its hetero- at 300 K. H and 13C NMR spectra are displayed in Fig. S1a–i
1
geneous nature, whereas the CO release from compounds 1a, in the ESI† and illustrate the purity of the compounds.
2a, 10, 11 and 14 occurred in homogeneous solutions.
Mass spectra were obtained with a HR-MS UHR-QTOF
Toxicity tests of dextran@oximeCORM@IONP (17) against maXis 4G (Bruker Daltonics) or with an ESI Ion-Trap-API-
the three cell lines A2780, Cal27 and HEK293 showed no Massenspektrometer Finnigan LCQ Deca (Thermo Quest).
growth inhibition up to a concentration of 17 of 100 µg mL−1 Ruthenium-containing fragments show the Ru-96, -98, -99,
(see ESI, Fig. S35–S38†). The three reference compounds -100, -101, -102, and -104 isotope distribution patterns. The
CORM-2, CORM-3, and 14 also showed no or low cytotoxic most intense peak of the isotope distribution is noted in the
effects with 0–9.23% growth inhibition, respectively, at mass spectrometric assignments (based on 102Ru, mass
10 µmol L−1. A higher concentration of 100 µmol L−1 showed 101.904 with 31.6% abundancy).
moderate growth inhibition of 14 up to 33.9%. For compari-
IR spectra were recorded on a Bruker Tensor 37 IR spectro-
son, 100 µmol L−1 cisplatin inhibited cell growth by >93% meter as KBr discs or using an attenuated total reflectance
(Table 3).
(ATR) technique. Care was taken to keep the amount of the
nanoparticle composites as low as possible by using only
1–2 mg composite material/30–50 mg KBr because of the
strong absorption of the dark nanoparticles. In order to see
the relevant carbonyl bands the amount of substance needed
Conclusions
We have presented the formation of an oxime-based CORM co- to be higher than for pure compounds because of the low
valently anchored on iron oxide nanoparticles and “solubi- ruthenium carbonyl percentage in dextran@oxime-
lized” by a dextran coating. For this, we implemented a CORM@IONP (17) and alginate@dextran@oximeCORM@
synthesis for the immobilization and backbone modification IONP (18). Attempted IR spectroscopic measurements of
of oxime-based CORMs on iron oxide nanoparticles with a materials containing the black-colored nanoparticles in ATR
catechol anchor group. A dextran polymer coating with a mode gave only very small signal intensities. Only compounds
molar mass of 500 000 g mol−1 was applied to achieve good without nanoparticles were measured by ATR. The integration
water “solubility” or rather a stable suspension of the modified time was set to 50 scans per wavenumber to obtain good
iron oxide nanoparticles. For the measurement of the CO quality spectra. The IR spectra are given in the ESI.†
release with the myoglobin assay, the dark-colored CORM-
Dynamic light scattering measurements were carried out
functionalized iron oxide nanoparticles had to be separated to with a Malvern Zetasizer Nanoseries. For determination of
allow for reliable UV/Vis absorption measurements. This the nanoparticles size distribution, a dispersion (“solution”)
in situ separation was achieved by incorporating the “soluble” with a minimum concentration of 1 mg mL−1 in doubly de-
dextran@oximeCORM@IONP into a calcium alginate sphere. ionized water was prepared. Measurements at different con-
The composite material alginate@dextran@oximeCORM@ centrations of the dispersion yielded the same hydrodynamic
IONP shows an extended half-life for CO release at 37 °C of diameter.
346 83 min compared to the molecular oximeCORMs with
Alternating current (AC) field generator measurements were
the diol-protected catechol backbone 10 of 18 ∼3 min and performed with a Hüttinger HF generator AXIO T5, equipped
the unprotected catechol compound 14 of 16 ∼1 min in the with a water-cooled copper induction coil. The system was
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operated at 247 kHz, with a magnetic field amplitude of 31.7 dimer [Ru(CO)3Cl2]2 (12) in 1.5 mL of methanol and 66 mg
kA m−1 and 39.9 mTesla.
(0.60 mmol) of imidazole-2-carbaldehydeoxime (synthesized
UV/Vis-absorption spectra were collected on a Specord according to ref. 50) in another 1.5 mL of methanol were com-
S 600 UV/Vis-spectrometer of Analytik Jena Co. This work was bined and stirred for 5 d at 30 °C. The solution became slightly
carried out under nitrogen or argon and with thermostated red and a colorless solid was formed. The solid was separated
cuvettes.
by centrifugation and washed with cold methanol (3 × 2 mL).
Atomic absorption spectroscopy (AAS) measurements were Yield: 43 mg (31%, lit.: 41%38). IR (KBr): ˜ν [cm−1] = 2068, 1990.
performed with a Perkin Elmer AAS AAnalyst200 by MicroLab 1H NMR (300 MHz, CD3OD): δ [ppm] = 8.43 (s, 1H, C–CHvN),
Kolbe, Höhenweg 17, D-45470 Mülheim. 7.42 (s, 1H, imi-CHvCH), 7.37 (s, 1H, imi-CHvCH), 3.36 (s,
Transmission electron microscopy (TEM) images were 3H, CH3) (matching NMR in ref. 38).
obtained with a Zeiss 902 TEM at the Center for Advanced
Synthesis of dicarbonylchlorido(imidazol-2-carbaldehydeox-
Imaging (CAi) in the University of Düsseldorf. For the sample ime)(ethoxycarbonyl)ruthenium(II) (2a). 100 mg (0.20 mmol)
preparation about 1 mg of the particles was dispersed by soni- of tricarbonyldichloridoruthenium(II)-dimer (12) were dis-
cation and fast up-and-down pipetting using a 1 mL syringe in solved in 1.5 mL of ethanol. 66 mg (0.60 mmol) of imidazole-
deionized water (2 mL). This dispersion was diluted 1 : 10 with 2-carbaldehydeoxime (synthesized according to ref. 50) was
deionized water to yield a very low concentration of the nano- dissolved in another 1.5 mL of ethanol and subsequently
particles. One drop of this dispersion was placed on a holey mixed together. The vessel was closed and stirred for 5 d at
thin film of amorphous carbon on a copper grid. After a 30 °C. The solution became slightly red and a colorless solid
contact time of 15 min, the grid was floated on a water surface was formed. The solid was centrifuged and washed with cold
and held vertically to rinse of the solvent.
ethanol (3 × 2 mL). Yield: 68 mg (45%, lit.: 53%38). IR (KBr):
1
Column chromatography was performed using a Reveleris ˜ν [cm−1] = 2067, 1994. H NMR (300 MHz, CD3OD): δ [ppm] =
chromatograph from Grace.
8.43 (s, 1H, C–CHvN), 7.43 (s, 1H, imi-CHvCH), 7.37 (s, 1H,
imi-CHvCH), 3.62 (q, 3J = 7.1 Hz, 2H, CH2), 1.20 (t, 3J =
7.1 Hz, 3H, CH3) (matching NMR in ref. 38).
Toxicity tests
Cisplatin was purchased from Sigma-Aldrich, and 3-(4,5-di-
Synthesis of 1-(2-(3,4-dihydroxyphenyl)-2-oxoethyl)-1H-imi-
methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dazole-2-carbaldehyde (5). A mixture of 2.0 g (7.0 mmol) of 7
from Serva (Germany). All other reagents were supplied by PAN in 200 mL of conc. acetic acid, 66 mL of d.d. water and 26 mL
Biotech (Germany).
of 6 mol L−1 hydrochloric acid was stirred for 2 h at 100 °C.
Cell lines and cell culture. The human epithelial ovarian After this the solvent was removed under vacuum, the residue
cancer cell line A2780 was obtained from the European Collec- was washed with 80 mL of d.d. water and the solid was
tion of Cell Cultures (ECACC, UK). The human tongue cell line removed by centrifugation. The aqueous phase was neutralized
Cal27 and the human embryonic kidney cell line HEK293 were with sodium hydroxide, the precipitate was collected by cen-
obtained from the German Collection of Microorganisms and trifugation and dried under vacuum. Yield: 1.47 g (85%).
1
Cell Cultures (DSMZ, Germany). All cell lines were grown at ESI-MS: m/z = 265.2 [M + H + H2O]+, 247.2 [M + H]+. H NMR
37 °C under a humidified atmosphere containing 5% CO2 in (300 MHz, DMSO-d6): δ [ppm] = 9.64 (d, 5J = 0.8 Hz, 1H,
3
RPMI 1640 (A2780) or DMEM (Cal27, HEK293) containing 10% C–CHvO), 7.56 (t, 3/5J = 0.8, 1H, imi-CHvCH), 7.35 (dd, J =
4
3
fetal calf serum, 120 IU mL−1 penicillin, and 120 µg mL−1 8.4 Hz, J = 2.2 Hz, 1H, Caryl–H), 7.30 (d, J = 0.8 Hz, 1H, imi-
streptomycin. The cells were grown to 80% confluency before CHvCH), 7.25 (d, 4J = 2.1 Hz, 1H, Caryl–H), 6.65 (d, 3J = 8.3 Hz,
using them for the appropriate assays.
1H, C
aryl–H), 5.88 (s, 2H, C(O)–CH2–NN). 13C-NMR (see
MTT cell viability assay. The rate of cell survival under the Fig. S1a†) (75 MHz, DMSO-d6): δ [ppm] = 189.95 (CvO),
action of test compounds was evaluated by an improved MTT 181.85 (CHvO), 153.52 (aryl-COH), 146.15 (aryl-COH), 143.51
assay as previously described.49 In brief, A2780, Cal27 or (N–CvN), 130.82 (aryl-C–CO), 128.72 (Caryl–H), 124.99 (imi-
HEK293 were seeded at a density of 5000 (A2780) or 2000 CHvCH), 121.52 (imi-CHvCH), 115.35 (Caryl–H), 114.32
(Cal27, HEK293) cells per well in 96-well plates (Corning, (Caryl–H), 53.39 (CH2).
Germany). After 24 h, the cells were exposed to increased con-
Synthesis of 2-chloro-1-(2,2-dimethylbenzo[d][1,3]dioxol-5-yl)
centrations of test compounds. Incubation was ended after ethanone (6). A mixture of 6.0 g (32.6 mmol) of 2-chloro-2,4-
72 h and the cell survival was determined by addition of MTT dihydroxy-acetophenone with 0.95 g phosphorus pentoxide in
solution (5 mg mL−1 in phosphate buffered saline). The forma- 120 mL of dry toluene was stirred under nitrogen at 75 °C and,
zan precipitate was dissolved in DMSO (VWR, Germany). every 20 min a portion of 0.55 mL acetone and every 30 min
Absorbance was measured at 544 nm and 690 nm using a 0.85 g phosphorus pentoxide were added over a period of 4 h.
FLUOstar microplate reader (BMG LabTech, Germany).
After adding 24 mL of aqueous 20% sodium hydroxide, the
aqueous phase was removed by decantation and discarded.
The organic phase was washed with d.d. water (3 × 60 mL).
Synthesis
Synthesis of dicarbonylchlorido(imidazole-2-carbaldehyde- The toluene solvent was removed and the solid was dried
oxime)(methoxycarbonyl)ruthenium(II) (1a). Solutions
of under vacuum. Yield: 6.6 g (87%). 1H NMR (see Fig. S1b†)
100 mg (0.20 mmol) of tricarbonyldichloridoruthenium(II)- (300 MHz, acetone-d6): δ [ppm] = 7.64 (dd, J = 8.2 Hz, J = 1.8
3
4
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Hz, 1H, Caryl–H), 7.37 (dd, 4J = 1.8 Hz, 5J = 0.4 Hz, 1H, Caryl–H), (Fig. S1g†) (125 MHz, DMSO-d6): δ [ppm] = 190.62 (CvO),
6.90 (dd, 3J = 8.2 Hz, 5J = 0.4 Hz, 1H, Caryl–H), 4.91 (s, 2H, 151.22 (aryl-COH), 145.37 (aryl-COH), 141.44 (CHvNOH),
CH2–Cl), 1.71 (s, 6H, CH3).
140.41 (s, 1C, N–CvN), 128.34 (aryl-C–CO), 126.30 (Caryl–H),
Synthesis of 1-(2-(2,2-dimethylbenzo[d][1,3]dioxol-5-yl)-2- 124.77 (imi-CHvCH), 121.14 (imi-CHvCH), 115.20 (Caryl–H),
oxoethyl)-1H-imidazole-2-carbaldehyde (7). 5.0 g (22.1 mmol) 114.69 (Caryl–H), 53.33 (CH2).
of 6, 2.34 g (22.1 mmol) Na2CO3 and 4.24 g (44.2 mmol) imid-
Synthesis of [Ru(η2-8)(methoxycarbonyl)(CO)2Cl] (10).
azole-2-carboxaldehyde (4) were dissolved in 200 mL of aceto- 270 mg (0.9 mmol) of 8 and 170 mg (0.3 mmol) of
nitrile and stirred for 10 d at 60 °C under a dry atmosphere. [Ru(CO)3Cl2]2 (12) were separately dissolved in 2.5 mL of
The solid was removed by centrifugation and the supernatant methanol, combined and stirred for 5 d at 30 °C in a closed
solution was poured into 1 L of d.d. water. The formed solid vessel. The solid was sedimented by centrifugation at 5 °C,
was separated by centrifugation and dried under vacuum. The washed with cold methanol (3 × 5 mL) and dried under
crude product was dissolved in 60 mL of ethyl acetate and vacuum. Yield: 249 mg (50%). IR (ATR): ˜ν [cm−1] = 2074; 1985,
purified using a Reveleris Chromatograph from Grace (4 runs IR (KBr): ˜ν [cm−1] = 2075; 1989. ESI-MS: m/z = 527.0 [M −
with 120 g silica column, ethyl acetate 40 mL min−1). Yield: OMe− − Cl− − H+ + CH3CN]+, 499.1 [M − CO/OMe− − Cl− − H+
2.5 g (40%). 1H NMR (Fig. S1c†) (300 MHz, acetone-d6): δ + CH3CN]+. 1H NMR (Fig. S1h†) (300 MHz, acetone-d6): δ
5
3
[ppm] = 9.67 (d, J = 0.9 Hz, 1H, C–CHvO), 7.71 (dd, J = 8.2 [ppm] = 8.75 (“d” = 2s for different cis- and trans ligands, 1H,
Hz, 4J = 1.8 Hz, 1H, Caryl–H), 7.48 (t, 3/5J = 0.9 Hz, 1H, imi- C–CHvN), 7.72 (dd, 3J = 8.2, 1.8 Hz, 1H, Caryl–H), 7.51–7.46
CHvCH), 7.40 (d, 4J = 1.8 Hz, 1H, Caryl–H), 7.30 (d, 3J = 0.9 Hz, (m, 2H, imi-CHvCH), 7.42 (d, 4J = 1.8 Hz, 1H, Caryl–H), 6.97
3
1H, imi-CHvCH), 6.94 (d, 3J = 8.1 Hz, 1H, Caryl–H), 5.99 (s, (d, J = 8.2 Hz, 1H, Caryl–H), 6.05 (s br, 2H, C(O)–CH2–N), 3.75
2H, C(O)–CH2–N), 1.72 (s, 6H, CH3). 13C NMR (Fig. S1d†) (s, 3H, COOCH3), 1.72 (s, 6H, (CH3)2).
(75 MHz, acetone-d6): δ [ppm] = 190.56 (CvO), 182.61
Synthesis of [Ru(η2-8)(ethoxycarbonyl)(CO)2Cl] (11). 250 mg
(CHvO), 153.11 (aryl-COH), 148.99 (aryl-COH), 144.84 (N– (0.83 mmol) of 8 and 160 mg (0.313 mmol) of [Ru(CO)3Cl2]2
CvN), 131.82 (aryl-C–CO), 129.79 (Caryl–H), 129.02 (imi- (12) were separately dissolved in 2.5 mL of ethanol, combined
CHvCH), 124.87 (imi-CHvCH), 120.67 (C–(CH3)2), 108.89 and stirred for 5 d at 30 °C in a closed vessel. The solid was
(Caryl–H), 108.04 (Caryl–H), 54.33 (CH2), 25.89 (CH3).
sedimented by centrifugation at 5 °C, washed with cold
Synthesis of (E)-1-(2-(2,2-dimethylbenzo[d][1,3]dioxol-5-yl)-2- ethanol (3 × 5 mL) and dried under vacuum. Yield: 221 mg
oxoethyl)-1H-imidazol-2-carbaldehyde-oxime (8). A mixture of (47%). IR (ATR): ˜ν [cm−1] = 2059; 1986, IR (KBr): ˜ν [cm−1] =
200 mg (0.70 mmol) of 7 with 74 mg (0.7 mmol) of sodium car- 2065, 1988. ESI-MS (Fig. S3a†): m/z = 527.0 [M − OEt− − Cl−
−
bonate and 94 mg (1.4 mmol) of hydroxyl ammonium chlor- H+ + CH3CN]+, 499.1 [M − CO/OEt− − Cl− − H+ + CH3CN]+. H
ide, [NH3OH]Cl in 4 mL of d.d. water was stirred for 24 h at NMR (Fig. S1i†) (300 MHz, acetone-d6): δ [ppm] = 8.73 (“d” =
70 °C. The obtained solid was separated by centrifugation at 2s for different cis- and trans ligands, 1H, C–CHvN), 7.72 (dd,
5 °C, washed with ethyl acetate (3 × 5 mL) and dried under 3J = 8.2 Hz, 4J = 1.8 Hz, 1H, Caryl–H), 7.51–7.47 (m, 2H, imi-
vacuum. Yield: 158 mg (75%). ESI-MS: m/z = 302.1 [M + H]+. 1H CHvCH), 7.42 (d, 4J = 1.8 Hz, 1H, Caryl–H), 6.97 (d, 3J = 8.2 Hz,
NMR (Fig. S1e†) (300 MHz, DMSO-d6): δ [ppm] = 11.22 (s, 1H, 1H, Caryl–H), 6.06 (s br, 2H, C(O)–CH2–N), 4.26 (t, 3J = 8 Hz,
1
3
4
NO–H), 7.97 (s, 1H, C–CHvN), 7.65 (dd, J = 8.2 Hz, J = 2.4 2H, –OCH2–), 1.72 (s, 6H, C(CH3)2), 1.28 (t, 3J = 7 Hz, 3H,
4
3
Hz, 1H, Caryl–H), 7.44 (d, J = 1.7 Hz, 1H, Caryl–H), 7.24 (d, J = –CH3).
0.9 Hz, 1H, imi-CHvCH), 7.04 (d, 3J = 1 Hz, 1H, imi-CHvCH),
Synthesis of [Ru(η2-9)(methoxycarbonyl)(CO)2Cl] (13). 50 mg
7.02 (d, 3J = 8.35 Hz, 1H, Caryl–H), 5.86 (s, 2H, C(O)–CH2–N), (0.19 mmol) of 9 and 48 mg (96 µmol) of [Ru(CO)3Cl2]2 (12)
1.70 (s, 6H, CH3).
were separately dissolved in 1 mL of methanol, combined and
Synthesis of 1-(2-(3,4-dihydroxyphenyl)-2-oxoethyl)-1H-imid- stirred for 5 d at 30 °C in a closed vessel. The solvent was
azole-2-carbaldehyde-oxime (9). 300 mg (1.22 mmol) of 5, removed under vacuum and the solid was analysed as a crude
95 mg (1.22 mmol) of Na2CO3 and 169 mg (2.44 mmol) of product. Compound 13, which was detected by mass spectro-
hydroxyl ammonium chloride were dissolved in 10 mL of d.d. metric analysis, did not precipitate in a concentrated or cold
water and stirred for 24 h at 70 °C. The solid was separated by solution and we were not able to purify the crude product.
centrifugation at 5 °C, washed with d.d. water (3 × 5 mL) and Therefore, compound 14 which could be precipitated from the
dried under vacuum. Yield: 151 mg (47%). IR (KBr): ˜ν [cm−1] = solution as a colorless product and purified with cold ethanol
1692, 1472. High-resolution, HR-ESI MS: m/z = 262.0822, calc. was used for the CO release experiments. Yield: 88.5 mg (crude
for [M + H]+ m/z = 262.0823, matching with the exact mass for product). ESI-MS: m/z = 487.0 [M − OMe− − Cl− − H+
+
the indicated fragment; the absence of other significant peaks CH3CN]+, 459.2 [M − CO/OMe− − Cl− − H+ + CH3CN]+.
(besides the isotope pattern) confirms the purity of compound
Synthesis of [Ru(η2-9)(ethoxycarbonyl)(CO)2Cl] (14). 50 mg
9 (see Fig. S4a†). 1H-NMR (Fig. S1f†) (600 MHz, DMSO-d6): (0.19 mmol) 9 and 32 mg 12 (63 µmol) were separately dis-
δ [ppm] = 11.21 (s, 1H, N–OH), 9.99 (s, 1H, C–OH), 9.45 (s, 1H, solved in 1 mL ethanol, combined and stirred for 5 d at 30 °C
C–OH), 7.96 (s, 1H, C–CHvN), 7.44 (dd, 3J = 8.27 Hz, 4J = in a closed vessel. The solid was sedimented by centrifugation
2.1 Hz, 1H, Caryl–H), 7.37 (d, 4J = 2.1 Hz, 1H, Caryl–H), 7.23 at 5 °C, washed with cold ethanol (3 × 5 mL) and dried under
(s br, 1H, imi-CHvCH), 7.03 (s br, 1H, imi-CHvCH), 6.87 (d, vacuum. Yield: 70 mg (70%). IR (KBr): ˜ν [cm−1] = 2064, 1995,
3J = 8.26, Caryl–H), 5.80 (s, 2H, C(O)–CH2–N). 13C-NMR 1678, 1478. ESI-MS (Fig. S3b†): m/z = 487.0 [M − OEt− − Cl−
−
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H+ + CH3CN]+, 459.1 [M − CO/OEt− − Cl− − H+ + CH3CN]+.
Myoglobin assay with molecular, soluble oxime-based
High-resolution, HR-ESI-MS: m/z = 486.9828 [M − OEt− − Cl− CORMs. Myoglobin from equine skeletal muscle (6.3 mg) was
− H+ + CH3CN]+, 458.9877 [M − CO/OEt− − Cl− − H+
+
dissolved in 5 mL 3-(N-morpholino)propanesulfonic acid
CH3CN]+, 419.9768 [M − CO/OEt− − Cl• + H]+, all matching buffer (pH 7.4; 0.1 mol L−1) and deoxygenated for 10 min with
with the calculated patterns for the indicated fragment masses nitrogen to yield a myoglobin solution (66 µmol L−1). Solid
(see Fig. S4b–d†). 1H NMR (500 MHz, acetone-d6): δ [ppm] = sodium dithionite was added (5 mg) to fully reduce the myo-
8.70 (s, 1H, C-CHvN), 7.60–7.38 (m, 4H, imi-CHvCH, 2Caryl
–
globin to the Fe(II) state and the solution was deoxygenated
H), 6.99 (d, 3J = 8.2, 1H, Caryl–H), 6.11–5.88 (m, 2H, C(O)–CH2– with nitrogen for an additional 10 min. To receive a 10 mmol
3
3
N), 3.57 (q, J = 6.5, 2H, –OCH2–), 1.12 (t, J = 6.7, 3H, –CH3). L−1 solution of the CORM, 1 mg of the compound was dis-
Calcd for C17H16ClN3O8Ru (526.85 g mol−1): C 38.76, H 3.06, solved in a corresponding amount of dimethylsulfoxide. A
N 7.98, found: C 38.72, H 3.09, N 7.47%.
thermostable quartz-cell was filled with 1.5 mL myoglobin
Immobilization of 14 on maghemite nanoparticles (16). A solution and 6 µL of the CORM-solution and sealed with a
suspension of 5 mg of 14 in 2 mL of d.d. water was mixed with Teflon plug to start the measurement immediately.
a few drops of 0.1 mol L−1 sodium hydroxide to form a clear
Myoglobin assay with alginate@dextran@oximeCOR-
solution. 30 mg of maghemite nanoparticles (15) were added M@IONP. A thermostable quartz-cell was filled with 1.3 mL
as an aqueous dispersion (3 mL, 10 mg mL−1) and the solution MOPS buffer solution, 15 spheres of alginate@dextran@oxime-
was stirred for 15 min. To prevent agglomeration of the nano- CORM@IONP (18), 200 µL of the above myoglobin/MOPS/
particles the combined solutions of both compounds were Na2S2O3 solution and sealed with a Teflon plug to start the
kept below pH 8. After neutralizing with hydrochloric acid the UV/Vis measurement immediately.
solution was combined with 100 mL acetone and the precipi-
CO release in alternating current (AC) field. Measurements
tated solid was removed by centrifugation. The solid was were performed with a Hüttinger HF generator AXIO T5,
washed with ethanol 3 × 5 mL and dried under vacuum. Yield: equipped with a water-cooled copper induction coil. The
27 mg, IR (KBr): ˜ν [cm−1] = 2038, 1916, 1633, 1487.
system was operated at 247 kHz, with a magnetic field ampli-
Immobilization of 14 on maghemite nanoparticles in tude of 31.7 kA m−1 and 39.9 mTesla. The thermostated
dextran (17). A suspension of 5 mg of 14 in 2 mL of d.d. water quartz-cell was placed inside the copper induction coil (see
was mixed with a few drops of 0.1 mol L−1 sodium hydroxide Fig. S40 in the ESI†). To keep the alginate spheres on the
to form a clear solution. 30 mg of maghemite nanoparticles bottom of the UV-cell, a small permanent magnet was placed
(15) were added as an aqueous dispersion (3 mL, 10 mg mL−1
)
under the quartz-cell. In order to keep the conditions reprodu-
and the solution was stirred for 15 min. Separately 60 mg cible, this permanent magnet was used during every measure-
dextran (M = 500 000 g mol−1) was dissolved in 5 mL of d.d. ment, even without alginate.
water, combined with the above solution and stirred for 3 h.
After neutralizing with hydrochloric acid, 100 mL of acetone
were added and the formed solid removed by centrifugation.
The solid was washed with ethanol 3 × 5 mL and the brown
Acknowledgements
solid, now called composite material dextran@oximeCORM@ We thank Dr Juri Barthel and the Ernst Ruska-Centre (ER-C)
IONP (17) was dried under vacuum and stored for several for Microscopy and Spectroscopy with Electrons, the Jülich
weeks. Yield: 69 mg, IR (KBr): ˜ν [cm−1] = 2064, 1995, 1961.
Research Centre and the RWTH Aachen University,
Embedding of 17 into alginate spheres (18). 10 mg of 17 52425 Jülich (Germany) for help and access to the HR-TEM
were dissolved in 0.5 mL MOPS buffer (pH 7.4). Mixing of facilities. Dr Peter Tommes is thanked for collecting the
0.5 mL of this solution with 0.5 mL alginate solution (50 mg HR-ESI mass spectra.
sodium alginate in 10 mL d.d. water) leads to a viscous solu-
tion which was added dropwise (by B-Braun, Sterican syringe
with a 0.60 × 80 mm BL/LB needle) into a calcium chloride
solution (2 g CaCl2·6H2O in 60 mL of d.d. water). The resulting
Notes and references
spheres were left to stand for 30 min in the calcium chloride
solution to form a fully cross-linked network of the alginate.
Then the formed spheres were washed three times each with
10 mL of MOPS buffer and used directly for kinetic CO release
experiments with the myoglobin assay.
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