N-Nitrosamine-{cis-Re[CO]2}2+ cobalamin conjugates as mixed CO/NO-releasing molecules

Article abstract of DOI:10.1039/c5dt03402g

Mixed CO/NO-releasing molecules were prepared by conjugation of the 17-electron rhenium dicarbonyl cis-[Re(CO)₂Br₄]^2- complex to N-nitrosamine modified cyanocobalamin (B12) bio-vectors. The species were fully characterized by standard analytical techniques, including X-ray crystallography for cyanocobalamin-5′-O-pyrazine and (1) and its N-pyrazine nitrosylated derivative (1a). The N-nitrosamine B12 derivatives are able to liberate low NO doses in buffer solutions and appear to be "activated" towards NO release if in contact with cultured cells. Coordination of the cis-[Re(CO)₂Br₄]^2- complex on the axial cyanide of B12 allows for the combined loss of CO and NO from the conjugates. The mixed CO/NO-releasing molecules show cytoprotection in an ischemia-reperfusion model but no significant enhanced synergistic effects over the relative NORMs and CORMs building constituents.

Full text of DOI:10.1039/c5dt03402g

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ARTICLE
2
+
N-Nitrosamine-{cis-Re[CO] } Cobalamin Conjugates as
2
Mixed CO/NO-Releasing Molecules
Received 00th January 20xx,
Accepted 00th January 20xx
a
b
b
a
c
Giuseppe Santoro, Ruben Beltrami, Emmanuel Kottelat, Olivier Blacque, Anna Yu. Bogdanova
b
and Fabio Zobi*
DOI: 10.1039/x0xx00000x
Mixed CO/NO-releasing molecules were prepared by the conjugation of the 17-electron rhenium dicarbonyl cis-
www.rsc.org/
2
-
[
2 4
Re(CO) Br ] complex to N-nitrosoamine modified cyanocobalamin (B12) bio-vectors. Speceis were fully characterize by
standard analytical techniques, including X-ray crystallography for cyanocobalmin-5’-O-pyrazine and (1) and its N-pyrazine
nitrosilated derivative (1a). The N-nitrosamine B12 derivative are able to libarate low NO doses in buffer solutions and
2
-
2 4
appear to be “activated” towards NO release if in contact to cultured cells. Coordiation of the cis-[Re(CO) Br ] complex on
the axial cyanide of B12 allows for the combined loss of CO and NO from the conjugates. The mixed CO/NO-releasing
molecules show cytotoprotection in an ischemia-reperfusion model but no significant enhanced synergistic effects over
the relative NORMs and CORMs building constituents.
in severe pathologies such as oncotransformation,
7
hypertension, chemoreceptorion, cardiovascular diseases.
-9
Introduction
Restoration of NO/CO bioavailability upon exhaustion of their
pools in diseased tissues may be achieved by administration of
Nitric oxide (NO) and carbon monoxide (CO) are established
gasotransmitters involved in multiple signalling pathways in
cells and tissues. NO is formed enzymatically as a product of L-
1
0, 11
NO and CO releasing molecules.
Nitric oxide releasing
molecules (NORMs) have been used for more than 100 years
1
arginine cleavage by NO synthases (NOS) and upon non-
-
2-14
for therapeutic use.
More recently, CORMs (CO releasing
1
enzymatic reduction of NO
2
by deoxygenated haemoglobin
5-20
molecules) have been the subject of intense scrutiny.
Like
and myoglobin. CO is produced during the degradation of
heme by heme oxygenases (HOs). Crucial importance of NO
and CO for the living organisms was confirmed by the studies
of HOs and NOS knockout mice. Biochemical parallels between
the two gases are particularly striking in the circulation and
nervous system where both NO and CO may function as co-
their nitric oxide-releasing counterparts, CORMs are pro-drugs
that upon activation release therapeutic doses of carbon
monoxide and display beneficial effects in e.g. atherosclerosis,
vascular constriction, inflammation, cellular protection,
2
1
hyperoxia-induced injury, and other physiological situations.
1, 2
In light of the akin biological roles of NO and CO, synergistic
cytoprotective effects of two gases might be expected. Indeed
those were demonstrated in an acute hepatic and vascular
transmitters in the same neurons. Strong evidence supports
the notion that interactions of CO and NO may influence the
physiological responses to each of these gases at the enzymes
biosynthetic level via a positive feedback loop mechanism
whereby NO elevates the levels of heme oxygenase-1 mRNA
2
2, 23
injury models.
Intrigued by those reports, we
hypothesized that mixed CO/NO-releasing molecules could
provide an advantageous alternative to CORMs and NORMs, as
such entities should be able of delivering both gasotrasmitters
and thus in principle promote, or exploit, the positive feedback
loop mechanism now recognized for the two diatomic
molecules in higher organisms. To the best of our knowledge,
a chemical approach to these types of molecules has not yet
been described.
3
-5
6
and protein, and CO increases the steady state level of NO.
Uncoupling or dysregulation of NO and CO production results
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Scheme 1. Synthetic procedure to mixed CO/NO-releasing molecules. Conditions cyanocobalamin. NO-releasing molecules (NORMs 1a
are: i) DMSO, CDT, 12h, 60 C followed by ii) selected macrocycle; iii) AcOH,
NaNO , 2h, RT; iv) MeOH, [Et N] [Re(CO) Br ], N2, 1h, RT.
,
2a and
, scheme 1) were first obtained by N,N-carbonylditriazole
CDT) amide coupling of the desired macrocycle on the ribose
’-OH functionality, followed by NaNO nitrosation to the
°
3
a
2
4
2
2
4
(
5
2
To test our hypothesis we prepared mixed CO/NO-releasing
molecules from the reaction of the 17-electron organometallic
corresponding N-nitrosamines. Overall, this procedure affords
reasonable yields, but the efficiency of the reaction is rather
poor, particularly in case of hexacyclen. Specifically, we found
2
-
CORM cis-[Re(CO) Br ]
2
complex with N-nitrosamine
4
functionalized cyanocobalamin (B12) bio-vectors. The design
of the molecules was based on previous findings published by
5
our group. Indeed in the last few years we showed that d
carbonyl Re complexes possess features which make them
2
4, 25
attractive as CORMs
and if appended to B12 are able to aid
that the critical synthetic step is the first amide coupling with
typical isolated yield of ca. 70, 40 and 15% for pyrazine,
tetraazacyclodecane and hexacyclen respectively. Conversely,
nitrosation affords complete conversion of the precursors to
the differentiation of embryonic stem cells into energetically-
efficient cardiomyocytes which may contribute to functional
2
6, 27
repair in progressive degenerative cardiac diseases.
In this
contribution we report the synthesis and characterization of
the above mentioned mixed CO/NO-releasing molecules and
the cytoprotective effects of the same tested on cultured 3T3
fibroblasts under conditions of ischemia-reperfusion
mimicking settings. While all molecules show cytoprotection in
our model, no significant enhanced synergistic effects over the
relative NORMs and CORMs building constituents was
observed.
species 1a, 2a and 3a. Mixed CO/NO-releasing molecules (1b,
2
b
and 3b in scheme 1) were finally isolated following
2
-
conjugation of the 17-electron cis-[Re(CO)
2
Br
4
]
anion to the
2
. Coordination
6
axial cyano group of derivatives 1a, 2a and 3a
of the organometallic species was confirmed by the
hypsochromic shift of the
2
these metal conjugates)
ν
CN stretching frequency (typical of
8-31
and the presence characteristic
2b and 3b. Inductively
νCO bands in infrared spectrum of 1b
,
coupled plasma/optical emission spectrometry further
confirmed the expected percentage concentration of rhenium
in the species, calculated as the fraction of the weight of the
samples.
Results and discussion
Synthesis and characterization of compounds.
With the long-term aim of applying the molecules in vivo, we
selected as
a biocompatible scaffold for our chemistry,
2
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2
-
All species were characterized by standard analytical The CO releasing profile of the cis-[Re(CO)
techniques, including X-ray crystallography for and 1a (see previously reported and herein confirmed for 1b
Fig. 1 and 2 respectively and ESI for crystallographic details). (see ESI for a typical spectrum).
Compound crystallized in the P2 space group (typical for properties of molecules 1a 2a and 3a were also evaluated via
2
Br
4
]
species were
2b and 3b
The nitric oxide releasing
1
,
2
6, 38
1
1
2
1
2
1
,
3
9
B12 species) and showed no significant structural differences the well-known myoglobin assay. The assay consists in
when compared to B12. Due to low diffraction of the crystals, monitoring via UV-visible spectroscopy the conversion of
2
and to the presence of 11 disordered H O solvent molecules deoxy-myoglobin (deoxy-Mb) with a maximum absorption
the R1 value for this species is relatively high if compared to peak of the Q band at 560 nm, to carboxy-myoglobin (Mb-CO)
other B12-based solid-state structures (10.4 % vs 7-9% on or nitrosyl-myoglobin (Mb-NO) which show two absorption
4
0
average, see experimental section for further details). maxima in the 540-548 and 575-580 nm for the same band.
Compound 1a crystallized as a dimer in the P2 space group, a The reaction between deoxy-Mb and NO was initiated by
rare example of a vitamin B12 monomer whose solid state adding a small aliquot of a freshly prepared concentrated
1
3
2-35
structure is not found in P2 2 2 (Fig. 2).
1 1 1
The pyrazine rings aqueous solution of either 1a
, 2a and 3a to a Mb solution in
in both molecules of the unit cell were found to be disordered, phosphate buffer (0.1 M, pH 7.4) previously treated with
4
1
probably due to different conformations imposed by the high sodium dithionite (final concentrations: 12
ꢀ
M deoxy-Mb
Interestingly, the NO bond lengths and 50µM for NORM’s). Incubation of the deoxy-Mb solution
are different in the two molecules. These are 1.096 (13) and with 1a caused little spectroscopic change over a 5 h
.217 (14) for atoms labelled N17-O16 and N34-O32 monitoring period, indicating that the molecule is poor NO
3
6, 37
N-NO rotational barrier.
1
respectively in Fig. 2. This dimer may persist in solution as releaser. However, when 2a or 3a was used in the same assay,
revealed by the splitting of the pyrazine resonances evidenced the deoxy-Mb spectrum evolved the expected spectral
1
by H-NMR spectroscopy (Fig. 2, ESI). In general, in comparison changes associated with NO release (Fig. 4). Under the
to the free vitamin, the most significant feature observed in experimental conditions Mb-NO concentration reached
1
a
the H-NMR spectra of these species, is a progressive shift to maximum within 5h of incubation. After this time, the two Mb-
lower magnetic field of the ribose R4, R5a and R5b protons as NO absorption maxima at 548 and 583 nm began to steadily
a consequence of amide
decrease while peaks at 505 and 630 nm increased in intensity.
The changes are associated with the conversion of Mb-NO to
metmyoglobin (met-Mb), due to slow oxygen dissolution in the
reaction medium.
Fig. 1. X-ray structure of compound 1 with thermal ellipsoids drawn at 30%
probability. Hydrogen atoms are omitted for clarity. Crystallographic details are
summarized in ESI.
coupling of the heterocycles and their subsequent nitrosation.
Furthermore, resonances of the heterocycles appear as broad
signals which can be attributed to their fluxionality and
possibly to tautomerization and interconversion of the
anchoring nitrogen between its amide and imidic acid forms.
1
To illustrate these features Fig. 3 compares the H-NMR
spectra of B12,
2 and 3. There both the shifts of the ribose
protons and the broad heterocycles resonances are clearly
seen.
CO and NO releasing properties of 1a-3a and 1b-3b.
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In the presence of dioxygen, nitric oxide can catalyze the
Fig. 2. X-ray structure (top) of compound 1a with thermal ellipsoids drawn at oxidation to Mb to met-Mb through the formation of a
0% probability. Hydrogen atoms are omitted for clarity. Crystallographic details
3
42-44
peroxynitrite derivative (referred to as met-Mb(OONO)).
1
are summarized in ESI. H-NMR (5.25 - 3.8 ppm, D
species 1 and C) species 1a. Peaks labeled Pz# refer to resonances of pyrazine,
2
O, 298 K of A) vitamin B12, B)
This species is also generally assumed to be the first
intermediate of reaction between oxy-myoglobin (oxy-Mb)
and NO. In order to understand if 2a-, 3a-derived NO could
also elicit the spectroscopic changes linked to oxy-Mb to met-
Mb conversion, a fresh solution of oxy-Mb was prepared
according to the method of Bauer and Pacyna and incubated
while R4, R5a and R5b are those of the ribose sugar (see text further details).
4
5
with the molecules. As Fig. 4 shows 3a-derived NO provided
a clean conversion between the two Mb species. In the
presence of 3a, the two absorption maxima of oxy-Mb at 542
and 579 nm steadily decreased over a 24 h period while the
met-Mb peaks at 505 and 630 nm increased. A total of five
isosbestic points at 350, 411, 478, 524 and 580 nm
accompanied the conversion.
Table 1. Amount of nitric oxide released by 1a-3a and 1b-3b (100 ꢀM solutions) in PBS
buffer and 3T3 cell culture after 30 h of incubation.
PBS Total (ꢀM)
3.7 ± 0.4
3.2 ± 0.6
7.1 ± 1.1
3.9 ± 0.3
Cell Culture Total (ꢀM)
4.1 ± 0.9
1
1a
Fig. 3.
species
H
-
NMR (5.25 - 3.8 ppm, D
Signals enclosed in segmented boxes are those of 1,4,8,11-
tacd and hexacyclen hacd respectively. Peaks
2
O, 298 K) of A) vitamin B12, B) species 2 and C)
2
a
6.9 ± 0.5
14.9 ± 2.0
4.4 ± 0.8
3.
tetraazacyclotetradecane
(
)
(
)
3a
1b
labeled R4, R5a and R5b refer to resonances of the ribose sugar.
2
3
b
b
3.6 ± 0.7
8.7 ± 0.9
7.7 ± 0.4
19.0 ± 1.8
Fig. 5. Reductive gas-phase chemiluminescence detection of NO released by 100
B
ꢀ
M solutions: of 1a
(
cell culture and of 1b
■
(
), 2a (●) and 3a
(
▲
) in:
A
, PBS buffer (pH 7.4) and
, PBS buffer (pH 7.4) and
, 3T3
, 3T3
■), 2b (●) and 3b
(▲
) in:
C
D
cell culture.
Fig. 4. Spectrum changes of a deoxy-Mb (top) and an oxy-Mb (bottom) solution
(
both 12 ꢀM) incubated with a 50 ꢀM solution of 3a over a 24 h period (1h
intervals). Arrows indicate the relative spectroscopic variations. Insets detail The total amount of NO released by 1a-3a and 1b-3b was
changes of the Q band. In the top figure insert, the initial growth of Mb-NO (over
determined by reductive gas-phase chemiluminescence
detection in PBS buffer and in cell culture medium overlaying
ca. 5h) is followed by its disappearance and conversion to met-Mb (over
remaining 19h). See text for further details.
the monolayer of 3T3 fibroblasts (data relative to 100
4
ꢀM
The method entails the
6, 47
solutions, see table 1 and Fig. 5).
-
oxidation of all NOx species in solution to NO
3
followed by its
4
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-
Fig. 6. Effects of active (blue) and inactive (orange) B12 species 1a
3b on the viability of fibroblasts in cell cultures under in an ischemia-
reperfusion model. Non-treated control is shown in grey. Data are means of 8
The ensuing reaction is then followed by light emission experiments ± SEM. B12 species concentration is 30 μM; incubation time 48 h
see ESI for details).* and ** stand for p<0.05 and p< 0.01 compared to the non-
, 2a, 3a and 1b,
step-wise reduction to NO
2
and NO which is finally carried in a
in a reaction chamber.
2b,
He flow and allowed to interact with O
3
(
detected by a photomultiplier (see experimental for further
details). For species 1a 3a in PBS, 3a released the highest total
detectable amount of NO, ca. 7 M, while 1a and 2a released
treated control.
-
ꢀ
about half the same amount. Similar results were found in cell
culture. In this case, however, the total amount of NO released
by 2a and 3a was twice that determined in PBS (ca. 7 and 15
was associated with prevention from de-attachment of 3T3
cells from the bottom of the petri-dish and reduction of stress
fibers formation. No other significant morphological changes
could be observed in 3T3 cells treated with our compounds
(Fig. 7). The lack of additional cytoprotective effects of NO
when applied along with CO suggests that both gases may
share similar pathways of cytoprotection in our experimental
ꢀM respectively) while it was constant for 1a. Thus, it appears
that 2a and 3a are activated by cultured 3T3 fibroblast towards
NO liberation, with relative total concentrations of released
3
6, 37
NO comparable to other N-nitrosamine derivatives.
CO/NO molecules 1b 3b released comparable amounts of NO,
with 3b giving the highest total measured concentration of the
gas (ca. 20 M, table 1).
Mixed
-
4
9
settings.
ꢀ
Cytoprotective properties of 1a-3a and 1b-3b.
The cytoprotective properties of mixed CO/NO-releasing
molecules 1b
reperfusion model on the 3T3 fibroblast cell culture and
compared to NORMs 1a 2a and 3a and the CO-releasing
, 2b and 3b were tested in an ischemia-
,
2
6
molecule B12-ReCORM-2 (Fig. 6). The latter afforded the
pure CORM-only model for direct comparison to the species
herein described. Compounds were administered at
reperfusion at a 30 μM concentration and responses of cells to
metabolic depletion were measured after 48 h as reflected in
the percentage of dead cells in the monolayer. Identical sets of
experiments were performed with two sets of samples, one of
which was treated with fresh-dissolved compounds (blue bars
in Fig. 6) and the other with compounds, which were in
aqueous solution for more than 50 h to release all the gaseous
molecules (orange bars in Fig. 6). B12-ReCORM-2 was used to
assess the possible additivity of the NO and CO in protection
from ischemia-reperfusion injury. As follows from the Fig. 6,
the number of dead cells reduced when cells were treated
with any of gas-emitting compounds compared to that in the
non-treated control (grey bar). The protective effect was lost
when compounds were administered after they released the
gasses (orange bars). The presence of NO did not result in any
additional protection than that provided by B12-ReCORM-2
4
8
alone. In agreement with our previous findings, protection
accomplished by the combined CO/NO-releasing compounds
Fig. 7. A: Representative histochemical staining of (A) untreated 3T3 fibroblasts
control) and (B) the cells treated with 30 μM 3b. Stained in blue (DAPI) are the
nuclei, actin filaments (phalloidin) are shown in red.
(
Conclusions
In summary, we have presented in this contribution the
synthesis and characterization of the first examples of mixed
CO/NO-releasing molecules based on N-nitrosamine-{cis-
2
+
Re[CO]
2
}
cobalamin conjugates. The derivatives were shown
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to be able to release both gases, albeit NO in relative small Analytical HPLC Methods.
doses. When testes in an ischemia-reperfusion model on 3T3
Method 1. Hitachi LaChrom L-7000 instrument. Column:
Marcherey-Nagel, EC250/3 Nucleosil 100-5 C18. Flow rare 0.5
mL/min. Absorbance monitored at 250nm. Solutions: A: 0.1%
trifluoroacetic acid in water; B: methanol. Chromatographic
method: 0-5 min: isocratic flow of 75% A – 25%B (A = TFA 0.1%
in water; B = Methanol); 5-40min: linear gradient to 100%
methanol. Method 2. Hitachi LaChrom L-7000 instrument.
Column: Nucleodur 100-5 C18. Flow rare 0.5 mL/min.
fibroblast cell cultures, all species afforded cytoprotection but
we found no significant enhanced synergistic effects over the
relative NORMs and CORMs alone. Despite the lack of
enhanced cytoprotection exhibited by the mixed CO/NO-
releasing molecules in the specific model we selected, we
believe this new class of compounds may be possibly
considered as alternative therapeutic agents along classical
NORMs and CORMs.
Absorbance monitored at 250nm. Solutions:
A:0.1%
trifluoroacetic acid in water; B: methanol. Chromatographic
method: 0-5 min: isocratic flow of 90% A – 10%B; 5-35min:
Acknowledgements
linear gradient to 100% B; 35-40min: isocratic flow of 100%B
.
Financial support from the SNSF grant PP00P2_144700 is
gratefully acknowledged.
Preparative HPLC method.
Preparative HPLC purifications were run either on a Varian Pro
Star or an Hitachi LaChrom L-7000 instrument using a
Nucleodur C18 Gravity column. Gradient used: A: 0.1%
trifluoroacetic acid in water; B: methanol.; flow rate = 30
ml/min; 0–10 min 75% A, 10–110 min 25 – 100% B.
Experimental
Chemicals and solvents were purchased from standard
sources. Chemicals were of reagent grade and used as
purchased without further purification. Solvents were of
reagent grade or distilled under anaerobic conditions. Water
was doubly distilled before use. Reactions performed in a
glove box work were performed with solvents which were
dried and deoxygenated by stirring and refluxing under a
nitrogen atmosphere over the appropriate drying agent and,
subsequently, distilled off. Degassed (not dried) solvents were
prepared by freeze-thaw cycles or by sonication under
vacuum. All synthetic work was carried out under a nitrogen
atmosphere using standard Schleck techniques. Experiments
involving myoglobin and reactions were the CDT coupling was
required were prepared in a glove box (Siemens MBraun
Crystal Structure Determination.
X-ray analyses were performed at 183(2) K on an Agilent
5
Technologies Xcalibur diffractometer with a Ruby detector
0
using a single wavelength Enhance X-ray source with Mo Kα
radiation (λ = 0.71073 Å) from a micro-focus X-ray source and
an Oxford Instruments Cryojet XL cooler. Suitable crystals were
covered with oil (Infineum V8512, formerly known as Paratone
N). Pre-experiment, data collection, data reduction and
absorption correction were performed with the program suite
5
1
52
CrysAlisPro. Using Olex2, the structures were solved with
5
the ShelXS-97 structure solution program and refined with
3
2
Labmaster DP) with N as working gas and automatic oxygen
5
4
the SHELXL-2014/6 program package by full-matrix least-
squares minimization on F2. Isolated solvent molecules could
be identified around the main species but could not be
included in the refinement cycles due to serious disorders.
and moisture purifier. Reaction controls were conducted via
HPLC (default gradient as described in method 1, vide infra).
Elemental analyses (EA) were performed on a Leco CHNS-932
elemental analyzer. For EA measurements, samples of the
vitamin B12 species were purified via HPLC, lyophilized and
subjected to high vacuum for 7 days. Often deviations from
expected values were recorded. In these cases addition of
5
5
With the use of the PLATON SQUEEZE tool, their contribution
was calculated as 432 electrons within the cell of and
1
attributed to 44 water molecules (11 molecules per
asymmetric unit with Z = 4) and 522 electrons within the cell of
2 5
vanadium pentoxide (V O ) improved results. UV-visible
1
a
attributed to 2 acetone molecules and 46 water molecule (1
and 23 solvent molecules per asymmetric unit with Z = 2,
respectively). CCDC-1054820 (for ) and CCDC-1054821 (for
) contain the supplementary crystallographic data for this
paper. These data can be obtained free of charge from The
Cambridge Crystallographic Data Centre via
spectra were performed using a Cary 50 spectrometer, with 1
3
cm quartz cells. Cuvettes were adapted with silicon septa lids
1
which ensured that samples were kept under an inert
atmosphere. ESI-MS measurements were performed on a
Bruker Esquire HCT (ESI) instrument. The solvent flow rate was
1
a
5
μl/min, a nebulizer pressure of 15 psi and a dry gas flow rate
www.ccdc.cam.ac.uk/data_request/cif.
of 5 L/min at a dry gas temperature of 300 °C were used. NMR
spectra were recorded on Bruker AV2-400, AV2-500 and
1
Bruker DRX-500 spectrometers ( H at 400 MHz and 500 MHz
Synthesis of compounds 1-3.
respectively). Chemical shifts are reported relative to residual In a round-bottom flask cyanocobalamin (100 mg, 0.074 mmol)
solvent resonance. Inductively coupled plasma/optical was dissolved in 1-5 ml of dry DMSO. A total of 25 mg of N.N-
emission spectrometry (ICP/OES) measurements were carbonylditriazole (CDT, 0.15 mmol) was added and the
performed on a Perkin Elmer Optima 7300 V HF ICP-OES resulting mixture was heater to 60°C for 12h with constant
Spectrometer.
stirring. The solution was allowed to cool to RT and then a
total of 1 mmol of the heterocyclic compound of interest (i.e.
6
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pyrazine = pz, 1,4,8,11-tetraazacyclotetradecane = tacd and preparative HPLC. CAUTION
:
during the procedure an
hexacyclen= hacd for 2 and 3 respectively) was added. The overpressure builds inside the reaction vessel. All precautions
1
,
mixture was stirred for 12h at RT and then slowly added to a should be taken to avoid possible explosions.
rapidly stirring mixture of 150 mL 1:1 diethyl ether:chloroform. Analytical data for 1a: yield 13.7 mg, 71%. HPLC (method 2):
The red precipitate was collected by vacuum filtration, washed retention time 20.4 min. ESI-MS analysis (positive mode) m/z =
+
1
with 30 mL of acetone and vacuum dried. The resulting red 1519.0 [M+Na ]. H NMR (400 MHz, D
powder was then purified by preparative HPLC. Note: For the resonances) 0.63 (s, 3H), 1.37 (s, 3H), 1.42 (d, 3H), 1.57 (s, 3H),
2
O) δ/ppm = a) (B12
synthesis of
DMSO.
3
, best results are obtained by using 1 ml of dry 1.58 (s, 3H), 1.61 (s, 3H), 2.04 (s, 3H), 2.44 (s, 6H), 2.71 (s, 3H),
2.74 (s, 3H), 1.14-4.46 (m’s, 32H), 4.50 (m, 2H), 5.06 (m, 1H),
Analytical data for
1: yield 89 mg, 76%. HPLC (method 1): 6.23 (s, 1H), 6.47 (d, 1H), 6.69 (s, 1H), 7.28 (s, 1H), 7.43 (s, 1H);
retention time 14.9 min. ESI-MS analysis (positive mode) m/z = b) (pz resonances) broad signals 4.58 (m, 2H), 4.11 (m, 2H),
+
1
-1
1
467.1 [M ]. H NMR (400 MHz, D
resonances) 0.36 (s, 3H), 1.09 (s, 3H), 1.16 (d, 3H), 1.29 (s, 3H),
.31 (s, 3H), 1.34 (s, 3H), 1.76 (s, 3H), 2.15 (d, 6H), 2.45 (s, 3H), (4882), 521 (6510), 545 (6533). Elemental Anal. Calcd. for
.48 (s, 3H), 1.00-4.26 (m’s, 31H), 4.52 (d, 1H), 4.87 – 4.76 (m, 16P (1496.49), C 54.58%, H 6.40%, N 15.91%.
2
O) δ/ppm = a) (B12 4.00 (broad, 2H), 3.83 (broad, 2H). I.r. (solid state, KBr, cm ):
-1
-1
ν
C≡N 2134. UV-Vis (nm, MeOH, ε: cm M ): 360 (20334), 397
1
2
3
1
2
68
C H93boN17O
H), 5.98 (s, 1H), 6.23 (d, 1H), 6.41 (s, 1H), 6.99 (s, 1H), 7.15 (s, Found: C 53.83%, H 6.64%, N 15.52%. Single crystals suitable
H); b) (pz resonances) broad overlapped signals 3.71-3.23 (m, for X-ray diffraction analysis were grown by slow diffusion of
-1
0H). I.r. (solid state, KBr, cm ):
15P (1467.49), C 55.65%, H 6.59%, N Analytical data for 2a: yield 15.4 mg, 77%. HPLC (method 2):
5.27%. Found: C 54.68%, H 6.76%, N 14.77%. Single crystals retention time 21.7 min. ESI-MS analysis (positive mode) m/z =
νC≡N 2134. Elemental Anal. acetone into an aqueous solution of 1a.
Calcd. for C68H96CoN16O
1
+
-1
suitable for X-ray diffraction analysis were grown by slow 1691.3 [M+Na ]. I.r. (solid state, KBr, cm ):
ν
C≡N 2134. UV-Vis
: cm M ): 361 (19050), 399 (3779), 519 (5635),
: yield 50 mg, 46%. HPLC (method 1): 549 (6230). Elemental Anal. Calcd. for C74
retention time 13.3 min. ESI-MS analysis (positive mode) m/z = (1668.68), C 53.26%, H 6.46%, N 17.63%. Found: C 51.04%, H
-1
-1
diffusion of acetone into an aqueous solution of
Analytical data for
1.
(nm, MeOH, ε
2
18
H107CoN21O P
+
1
1
582.4 [M+H ]. H NMR (500 MHz, MeOD) δ/ppm = a) (B12 6.66%, N 17.05%.
resonances) 0.42 (s, 3H), 1.17 (s, 3H), 1.23 (d, 3H), 1.34 (s, 3H), Analytical data for 3a: yield 16 mg, 75%. HPLC (method 2):
1
2
4
.36 (s, 3H), 1.43 (s, 3H), 1.86 (s, 3H), 2.26 (d, 6H), 2.51 (s, 3H), retention time 14.6 min. ESI-MS analysis (positive mode) m/z =
+
-1
.56 (s, 3H), 1.00-2.63 (m’s, 24H), 3.64 (m, 4H), 4.11 (d, 1H), 1806.8 [M+Na ]. I.r. (solid state, KBr, cm ):
ν
C≡N 2134. UV-Vis
-1
-1
.55 – 4.20 (m, 6H), 6.05 (s, 1H), 6.32 (d, 1H), 6.57 (s, 1H), 7.07 (nm, MeOH,
ε: cm M ): 361 (20976), 400 (3238), 518 (5991),
(
20
s, 1H), 7.22 (s, 1H); b) (tacd resonances) broad overlapped 548 (6721). Elemental Anal. Calcd. for C76H111boN25O P
-1
signals 3.45-3.10 (m’s, 20H). I.r. (solid state, KBr, cm ):
ν
C≡N (1784.76), C 51.14%, H 6.27%, N 19.62%. Found: C 48.95%, H
15P (1581.68), C 6.52%, N 18.93%.
6.19%, H 7.01%, N 15.94%. Found: C 54.17%, H 7.26%, N Contrary to what observed for
2
5
1
134. Elemental Anal. Calcd. for C74H110CoN18O
1, nitrosation of 2 and 3 gave
5.08%.
derivatives (i.e. 2a and 3a) whose NMR spectra were severly
Analytical data for
3: yield 20 mg, 19%. HPLC (method 1): complicated not only by the different conformations of the
retention time 13.7 min. ESI-MS analysis (positive mode) m/z = pending tacd and hacd but also from an equilibrium between
+
639.9 [M+H ]. H NMR (500 MHz, D
1
1
2
O) δ/ppm = a) (B12 the base-on/off forms of the vitamin (spectra not shown).
resonances) 0.42 (s, 3H), 1.16 (s, 3H), 1.24 (d, 3H), 1.35 (s, 3H), Despite our efforts, we could not unambigously assign specific
1
2
3
1
1
.37 (s, 3H), 1.41 (s, 3H), 1.83 (s, 3H), 2.23 (s, 6H), 2.51 (s, 3H), sets of resonances.
.55 (s, 3H), 1.00-2.63 (m’s, 22H), 2.97 (dd, 1H), 3.16 (d, 1H),
.29 (dt, 2H), 3.45 (m, 1H), 3.58 (dd, 1H), 3.71 (t, 2H), 3.97 (d,
Synthesis of compounds 1b-3b.
The following general synthetic procedure was applied. In a
round-bottom flask with a side arm 10 mg of either 1a 2a or
3a and 10 mg of the [Et N] [Re(CO) Br ] salt were added as
H), 4.17 – 4.07 (m, 3H), 4.30 (dd, 1H), 6.05 (s, 1H), 6.32 (d,
H), 6.47 (s, 1H), 7.03 (s, 1H), 7.19 (s, 1H); b) (edac resonances)
,
broad overlapped signals 3.54-3.16 (m, 24H). I.r. (solid state,
-1
4
2
2
4
KBr, cm ): νC≡N 2134. Elemental Anal. Calcd. for
C H116CoN20O15P (1639.77), C 55.67%, H 7.13%, N 17.08%.
2
4
solids under nitrogen. A total of 6 ml of degassed methanol
were added giving a dark purple solution, stirring began and
then the flask was lowered into an oil bath preheated to 50 °C.
The temperature was kept constant for 1 h and then lowered
to 40 °C within 30 min. Heating was stopped and the solvent
76
Found: C 53.32%, H 7.30%, N 16.63%.
Synthesis of compounds 1a-3a.
The following general synthetic procedure was applied. In a removed under reduced pressure. The resulting dark red
round-bottom flask with a side arm 0.012 mmol of either powder was washed several times with CH Cl , acetone and
or were dissolved in 3 ml of acetic acid. A total of 100 mg of then again CH Cl until washings (containing a mixture of
NaNO (1.45 mmol) was added and the flask was sealed. NO unreacted [Et N] [Re(CO) Br ] and (Et N)Br) were clear.
gas began to evolve immediately. The mixture was stirred at Analytical data for 1b: HPLC: retention time 30.5 min. I.r.
room temperature for 2 hours and then the solution was dried (solid state, KBr, cm-1):
C≡O 1986, 1890; C≡N 2181. HR-ESI-
under reduced pressure. The red solid was then purified by MS analysis (positive mode) m/z Calcd.= 1900.4171 ; Found :
1,
3
2
2
5
2
2
2
4
2
2
4
4
ν
ν
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J. Name., 2013, 00, 1-3 | 7
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ARTICLE
Journal Name
1
900.4190. UV-Vis (nm, MeOH,
5487), 520 (6117), 545 (6158). Elemental Anal. Calcd. for
C71H99Br2CoN17O19PRe (1930.57), C 44.17%, H 5.17%, N
2.33%. Found: C 46.29%, H 6.37%, N 11.57%. ICP/OES
measurements of Re content by relative weight: calcd. 9.65;
found 9.28 0.12.
Analytical data for 2b: HPLC: retention time 32.7 min. I.r. (solid
state, KBr, cm-1):
C≡O 1990, 1841; C≡N 2183. HR-ESI-MS
analysis (positive mode) m/z Calcd.= 2071.5055 ; Found :
071.5178. UV-Vis (nm, MeOH, : cm-1 M-1): 361 (19041), 403
4550), 519 (5349), 550 (5957). Elemental Anal. Calcd. for
C77H111Br2CoN21O21PRe (2102.76), C 43.98%, H 5.32%, N
3.99%. Found: C 45.89%, H 5.57%, N 12.46%. ICP/OES
measurements of Re content by relative weight: calcd. 8.86;
found 8.36 0.24.
Analytical data for 3b: HPLC (method 2): retention time 28.7
min. I.r. (solid state, KBr, cm-1):
C≡O 1989, 1841; C≡N 2183.
HR-ESI-MS analysis (positive mode) m/z Calcd.= 2187.5390 ;
Found : 2187.5388. UV-Vis (nm, MeOH, : cm-1 M-1): 361
20556), 408 (4500), 517 (5556), 549 (6300). Elemental Anal.
Calcd. for C79H115Br2CoN25O23PRe (2218.83), C 42.76%, H
.22%, N 15.78%. Found: C 44.25%, H 5.38%, N 14.69%.
ICP/OES measurements of Re content by relative weight: calcd.
.39; found 8.08 0.29.
ε: cm-1 M-1): 360 (19626), 400
(
Cell culturing and experimentation conditions during metabolic
depletion.
1
Two batches of NIH 3T3 Swiss Albino Mouse Fibroblast cells
(ECACC Catalogue Number 85022108) were cultured for 4 or 5
±
passages in Dulbecco’s Modified Eagle Medium (DMEM)
containing 5 mM glucose, 10% fetal calve serum (FCS), and
penicillin−streptomycin at pH 7.4. About one million cells were
ν
ν
2
plated per Petri dish of 4.5 cm area and kept proliferating to
2
ε
near confluence at 37 °C in the presence of 95% air and 5%
(
2
CO as described earlier (see e.g. references 17 and 28 in main
article). All cell culture materials were purchased from
Invitrogen (Invitrogen, Life Technologies, Paisley, UK).
1
The following protocol was used to mimic conditions of
ischemia-reperfusion. Ischemia was imitated by placing the
cells grown to 60-70% confluence and DMEM (GIBCO®
GlutaMAX™, Life Technologies, Paisley, UK) to the FCS-free
glucose-deprived medium (Invitrogen, Life Technologies,
Paisley, UK) and exposing them for 48 h to the atmosphere of
±
ν
ν
ε
(
1
2 2 2
% O , 94.8% N and 5% CO in the incubator HeraCell 240,
Heraeus. Thereafter the starvation medium was replaced by
the DMEM supplemented with 10% FCS and 5 mM glucose
without or with B12 species (final concentration 30 μM), which
were either fresh-dissolved or pre-dissolved for 54 h. This
simulated “reperfusion” period lasted for 12 h. Stock 30 mM
5
8
±
NO detection in PBS buffer and the cell culture medium in contact
with 3T3 cells.
solutions of B12 species 1a, 2a, 3a and 1b, 2b, 3b were
prepared in PBS.
NIH Stock solutions (1 mM) of 1a-3a, and 1b-3b were prepared
in sterile phosphate buffer (PBS, pH 7.4) and added either to
fresh PBS buffer, culture medium which had been previously in
contact with cells, or cultured cells to a final concentration of
Cell viability was then assessed by means of fluorescent
microscopy. To do so the cells were stained for 10 min with a
mixture of 1 μg/mL Hoechst-33342 and 10 μg/mL propidium
2+
2+
iodide (PI) (Invitrogen, Molecular Probes) in Ca /Mg -free
PBS (Gibco, Life Technologies, Paisley, UK). Hoechst-33342
100 μM. Aliquots (200 μL) of the resulting solutions were
sampled at 4, 9, 19 and 30h. For all collections, the samples
were filtered through a short C18 column after priming with
methanol (1.5 ml), and washing with distilled water (2 ml). The
samples were passed through the column followed by 1 ml of
PBS. Samples for each time point were prepared and handled
in triplicates. Nitrite and nitrate assessment was performed as
(
excitation and emission maxima of 360 and 450 nm,
respectively, Invitorgen, Molecular Probes) is membrane-
permeable and stains the nuclei of all cells by binding to
double-stranded nuclear DNA. Membrane-impermeable PI
(excitation and emission maxima of 555 and 620 nm,
respectively) interacts with DNA and RNA of cells with
ruptured a plasma membrane termed here as “dead”.
Fluorescent staining was recorded using the Axiovert 200-M
fluorescent microscope and analyzed using MCID image
analysis software. In each culture dish, cells were counted in 5
4
7, 48
previously described.
Nitrate was quantitatively reduced
to nitrite using Nitralyzer II kit (World Precision Instruments,
Inc.) in which copper-coated cadmium bids were used for
reduction. The samples (25, 50 or 100 μl) were injected into a
helium-gassed reducing Brown's solution containing 1.62 g KI,
2
2
predetermined areas of 0.5 mm , totalling 2.5 mm per dish,
three dishes tested for each condition. Experiments were
repeated four times (two 3T3 cell batches, two passages (P4
and P5) each. Data in each experiment were normalized to the
viability in non-treated control samples and expressed as the
means ± SEM. Statistical analysis was performed using
GraphPad Instat v.3, GraphPad Software Inc. Following the
normality test One-way Anova was used with the Dunnett-
post-test in which all data were compared to the viability of
cells in non-treated control.
0
.57 g I
warmed to 85
containing 0, 50, 100, 150 or 200 nM of NaNO
2
, 15 ml HPLC-grade water and 202 ml acetic acid
C. Calibration solutions were prepared
dissolved in
⁰
2
distilled water. Nitrite was quantitatively reduced to NO when
in Brown's solution and carried in a Helium flow to the
reaction chamber of CLD88 Nitric oxide analyzer (EcoMedics
3
Switzerland) in which NO was allowed to interact with O . The
ensuing reaction was then followed by light emission detected
by photomultiplier. Final results obtained from samples
dissolved in PBS were set as control while data obtained from
Additional batches of cells exposed to ischemia-reperfusion
protocol with or without the NO and CO releasing compounds
were prepared for staining with 4′,6-Diamidino-2-phenylindole
the addition of 1a
T3 cells were normalized to the data obtained from the fresh
culture medium alone.
, 2a and 3a to the medium in contact with
3
(DAPI) dihydrochloride (0.5 mg/ml) or phalloidin-rhodamine
8
| J. Name., 2012, 00, 1-3
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Journal Name
Sigma-Aldrich, 1:100 in PBS). Stainig was performed after 24
Ple Da sae l tdo onn To rt aa nd sj ua sct t mi o an r sg ins
View Article Online
DOI: 10.1039/C5DT03402G
ARTICLE
(
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