Confocal and fluorescence lifetime imaging sheds light on the fate of a pyrene-tagged carbon monoxide-releasing Fischer carbene chromium complex

Article abstract of DOI:10.1039/c4dt03312d

The synthesis of a new pyrene-containing Fischer carbene complex is described. The complex has a broad absorbance spectrum between 300 and 400 nm and, on excitation at 345 nm in CH₂Cl₂ solution, emission is observed at 395 and 415 nm. Emission is also observed in PBS buffer, but in this case the resulting spectra are much broader. Confocal and fluorescence lifetime imaging indicate that emission occurs on treating HeLa cells with the complex and co-localisation studies demonstrate that this is from the mitochondria and lipid-rich regions of the cell. This journal is

Full text of DOI:10.1039/c4dt03312d

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Confocal and fluorescence lifetime imaging
sheds light on the fate of a pyrene-tagged
carbon monoxide-releasing Fischer carbene
chromium complex†
Cite this: Dalton Trans., 2015, 44,
4957
a
b
c
b
Rory L. Arrowsmith, Anthony J. Atkin, Stanley W. Botchway,* Ian J. S. Fairlamb,*
b
d
a
b,d
Jason M. Lynam,* James W. B. Moir, Sofia I. Pascu,* Jonathan S. Ward and
b
Wei-Qiang Zhang
The synthesis of a new pyrene-containing Fischer carbene complex is described. The complex has a
broad absorbance spectrum between 300 and 400 nm and, on excitation at 345 nm in CH Cl solution,
2
2
Received 27th October 2014,
Accepted 3rd December 2014
emission is observed at 395 and 415 nm. Emission is also observed in PBS buffer, but in this case the
resulting spectra are much broader. Confocal and fluorescence lifetime imaging indicate that emission
occurs on treating HeLa cells with the complex and co-localisation studies demonstrate that this is from
the mitochondria and lipid-rich regions of the cell.
DOI: 10.1039/c4dt03312d
www.rsc.org/dalton
5
6
5e
may release CO with a thermal, photochemical, hydrolytic,
Introduction
7
or enzyme trigger. In a number of important studies, it has
Carbon monoxide has a well-defined physiological role and been demonstrated that CO-RMs mimic the therapeutic ability
1
3
offers great potential in the therapeutic arena. It is produced of CO gas. Indeed, CO-RMs have been shown to be effective
8
endogenously during the catabolism of heme, a process which anti-bacterial agents, more so than CO gas alone. Given that
is catalysed by the heme-oxygenase series of enzymes (HO-1 one of the envisaged advantages of CO-RMs is their ability to
2
and HO-2) and CO acts as a gasotransmitter. Remarkably, it selectively localise, it is somewhat surprising that there have
has been shown that the administration of CO gas may have a only been a handful of studies focused on determining the cel-
beneficial effect: preventing organ rejection; as well as aiding lular fate of a CO-RM. For example, a study using Raman
3
the treatment of inflammation, hypertension, cerebral spectromicroscopy has shown that [Mn(tpm)(CO) ]Cl (tpm =
3
malaria, tumour growth and liver failure. CO also shows inter- tris(1-pyrazolyl)methane) localises at the nuclear membrane
9
esting effects against certain types of bacteria. Carbon monox- and the nucleolus of HT29 cancer cells, whereas IR spectromi-
ide-releasing molecules (CO-RMs) have been employed as croscopy has been used to study the localisation of a Mn-based
1
0
potential CO-prodrugs, which circumvent the requirement for CO-RM with a vitamin B12 ligand in 3T3 fibroblasts. In
3
non-selective administration of CO gas. The fundamental addition, confocal fluorescence microscopy has been used to
concept is that a CO-RM will be able to deliver a well-defined determine the fate of fac-[Re(bpy)(CO)
therapeutic quantity of CO in a selective fashion. bipyridine, thp tris(hydroxymethyl)phosphine)
There have been a number of different structural types PCC-1 human prostatic carcinoma cells.
3
(thp)]OTf (bpy = 2,2′-
=
in
1
1
4
investigated as potential CO-RMs such as boronate esters. The
In a recent study we have investigated the ability of a series
1
2
vast majority of CO-RMs, however, are metal carbonyls that of Group 6 metal carbonyl complexes to release CO. In the
family of Fischer carbene complexes [M(CO) (vCRR′)] (M = Cr,
5
Mo, W; R = alkyl, aryl, alkynyl; R′ = amino, alkoxy, thio) it was
demonstrated that both the nature of the metal and the substi-
a
Department of Chemistry, University of Bath, Claverton Down, Bath, BA2 7AY, UK.
E-mail: S.Pascu@bath.ac.uk
b
tuents on the carbene ligand affect the rate of release. For
example, complexes based on chromium release CO rapidly;
those based on molybdenum are slower; whereas the tungsten
complexes are extremely slow. The nature of the π-donor sub-
stituent on the carbene also had a profound effect on the rate
of CO-release with good donor such as amino-groups release
Department of Chemistry, University of York, Heslington, York, YO10 5DD, UK.
E-mail: ian.fairlamb@york.ac.uk, jason.lynam@york.ac.uk
c
Rutherford Appleton Laboratory, STFC, Harwell Oxford, Didcot, Oxfordshire OX11
OQX, UK. E-mail: stan.botchway@stfc.ac.uk
d
Department of Biology, University of York, Heslington, York, YO10 5DD, UK
Electronic supplementary information (ESI) available: Further details of
†
microscopy studies, methodology for DFT calculations, Alamar Blue and LDH
assays. See DOI: 10.1039/c4dt03312d
3
4
CO at a slow rate (t1/2 ca. 10 –10 s), whereas those containing
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a sulfur substituent released CO more rapidly (t1/2 ca. 300 s).
Given this level of control on the rate of CO-release and given
that, in principle, up to a total of five molecules of CO may be
liberated from each metal, we decided to explorer this series of
compounds further. In particular, the incorporation of an
emissive pyrene tag into the structure of the Fischer carbene
could enable us to determine the site of cellular localisation
for [Cr(CO) (vCRR′)] complexes.
5
Results and discussion
The synthesis of the pyrene-substituted carbene complex, 4,
was undertaken using a modification of Fischer and Maasböl’s
original
method
to
obtain
5
[Cr(CO) (vCPh{OMe})]
1
3
n
(Scheme 1). Lithiation of 1-bromopyrene, 1, with BuLi at
6
−78 °C followed by addition of Cr(CO) leads to the formation
of the acyl anion 3. This which was characterised in situ by IR
−
1
spectroscopy (bands at 1908 and 2039 cm in CH Cl solu-
2
2
14
tion). Methylation with [Me
of 4 which could be isolated as orange crystals. The identity of
was confirmed by NMR and IR spectroscopic analysis. For
3 4
O]BF resulted in the formation
4
1
3
1
example, in the C{ H} NMR spectrum a resonance was
observed at δ 358.9 for the carbene carbon. Resonances at
2
16.4 (CO ligands cis to the carbene) and 225.3 (CO ligand
trans to the carbene), along with infrared bands at 2063 and
948 cm− in the IR spectrum confirmed the presence of the
1
1
Cr(CO) group.
5
2 2
The UV-vis spectrum of 4 recorded in CH Cl solution
exhibited two peaks in the visible region 374 nm (ϵ = 2.20 ×
4
3
−1
−1
4
3
−1
−1
1
(
0 dm mol cm ) and 337 nm (ϵ = 2.17 × 10 dm mol cm )
Fig. 1a). The former having a shoulder and being somewhat
similar to the lowest energy band in [Cr(CO) (vCPh{OMe})]
Fig. 1b) whereas the peak at 334 nm occurred at a similar
energy to those observed in the hydroxyl-substituted pyrene
(CH OH (Fig. 1c). TD-DFT calculations (see ESI† for
5
(
C
16
H
9
2 4
)
details) indicate that for [Cr(CO) (vCPh{OMe})] there are
5
three transitions at λ > 300 nm, the most intense (and highest
energy in this region) is predicted to be at 372 nm. These
Fig. 1 UV-vis spectra of (a) 4, (b) [Cr(vCPh{OCH
CH OH recorded in CH Cl solution.
3 5 9
})(CO) ], (c) C16H -
(
2
)
4
2
2
correspond to MLCT transitions from filled metal-based orbi-
tals to the LUMO, which is located on the carbene ligand. In
the case of 4, the calculations indicate a total of seven tran-
sitions between 316 and 466 nm, consistent with the presence
of the extra bands in this region in the experimental spectrum
(
Fig. 1a) when compared to [Cr(CO) (vCPh{OMe})] (Fig. 1b).
5
Scheme 1 Preparation of pyrene-substituted carbene complex 4 using
a three step synthetic sequence.
In this case, the dominant contributions to the transitions are
4958 | Dalton Trans., 2015, 44, 4957–4962
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from filled orbitals based on both the metal and the pyrene
fragment to unoccupied orbitals with significant localisation
on the pyrene and carbene carbon.
The effect of the incorporation of the chromium carbene on
the emission spectra of the pyrene was then investigated. Exci-
2 2
tation of a CH Cl solution of 4 resulted in emission at 395
and 415 nm (Fig. 2a). Emission was also observed in PBS solu-
tion (Fig. 2b), although the bands were somewhat broader and
the intensity reduced. Only weak emission was observed from
3 5
[Cr(vC{OCH }Ph)(CO) ] under identical conditions, indicating
the incorporation of the pyrene groups was vital to this effect.
The ability of 4 to release CO was estimated via an assay
1
5
based on the capture of liberated CO by deoxy-myoglobin.
This process results in the formation of carboxymyoglobin,
Mb–CO, and the change was monitored spectrophotometri-
cally. In this case a correction was applied to the data to
account for the absorbance of 4 in the region of the proteins
Fig. 3 CO-release profiles for assays performed on 60 μM, 40 μM and
20 μM solutions of 4.
1
5
Q-bands: the resulting data are shown in Fig. 3. CO-release is
typically quantified by the half-life, t1/2, (defined as the which provides a mechanism-independent measure of CO-
amount of time required for an X μM solution of CO-RM to release. For complex 4 the t1/2 values were 2773 s, 2765 s and
generate a solution of Mb–CO with concentration X/2 μM) 1764 s for solutions of concentration of 60 μM, 40 μM and
20 μM respectively, which is somewhat slower than [Cr(vC-
{
OCH }Ph)(CO) ] (t 332 s for a 60 μM solution) and more
3
5
1/2
1
2
similar to the amino-substituted carbenes [Cr(vC{NR
2
}R )-
1
2
(
CO) ]. The stability of 4 in EtOH solution was assessed by
5
UV-vis spectroscopy and demonstrated that over a period of
ca. 2 hours, the bands due to the complex depleted in inten-
sity, mirroring the behaviour shown in the CO-release assay
(
see ESI†). However, it did not prove to be possible to assess the
correlation between the CO-release behaviour with the stability
of 4 in a 9 : 1 H O : EtOH mixture as a significant baseline drift
2
was observed.
Having established that 4 was a viable CO-releasing mole-
cule which was emissive in both organic media and aqueous
solution, we elected to investigate if the localisation of this
complex with cancer cells could be observed by confocal
microscopy. Compound 4 and 1-bromopyrene, 1, (used as a
ligand model) were incubated with HeLa cells as described in
the Experimental and imaged using laser scanning confocal
microscopy and fluorescence lifetime imaging microscopy
(
FLIM). The free ligand precursor showed negligible uptake,
whereas addition of the chromium complex 4 resulted in
notable fluorescence emission when excited at 405 nm, with
insignificant emission observed when exciting at 488 nm and
543 nm (see ESI†). It was therefore appropriate to carry out
colocalisation experiments with dyes such as Mitotracker
Red® and Nile Red, which displayed strong corroboration with
complex 4 (Fig. 4 and 5), indicating that this compound loca-
lises both in mitochondria and lipid-rich regions of the cell.
Furthermore, blebbing (which sometimes accompanies cell
1
6
death)
was observed upon continuous illumination for
10 min with lasers of 488 nm (see ESI†). Currently there is no
technique that provides a full understanding of complex stabi-
lity in cells, however based upon our previous studies, the
FLIM is capable of providing information on the chemical
2 2
Fig. 2 Emission spectra of 4 recorded in (a) CH Cl solution at 25 °C,
excitation wavelength 345 nm. (b) PBS solution at 25 °C, excitation
wavelength 345 nm.
1
7
environment of a molecule, hence speciation. FLIM experi-
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Fig. 4 Laser scanning confocal microscopy of complex 4 incubated with HeLa cells for 15 minutes at 50 µM (2% ethanol), co-stained with Mito-
tracker Red® at 200 nM (a) DIC micrograph, (b) green channel with λex = 405 nm, (c) red channel with λex = 543 nm and (d) is an overlay of the pre-
vious three images. Where green represents fluorescence emission from compound 4, red is emission from Mitotracker Red® and yellow represents
colocalisation of both dyes.
Fig. 5 Laser scanning confocal microscopy of complex 4 incubated in HeLa cells for 15 minutes at 50 µM (2% ethanol), co-stained with Nile Red at
00 ng mL− (a) DIC micrograph, (b) green channel with λex = 405 nm, (c) red channel with λex = 543 nm and (d) is an overlay of the previous three
1
1
images. Where green represents fluorescence emission from compound 4, red is emission from Nile Red and yellow represents colocalisation of
both dyes.
ments were therefore carried out to better understand how imaging measurements have allowed for the visualisation of
complex 4 interacts with cells. First, excited state lifetime this process.
experiments in solution, were carried out in DMSO using time-
correlated single photon counting (TCSPC). The ligand precur-
sor, 1, required fitting to two exponential decay components,
with a short first component, τ of 0.6 ns and a much longer
1
Conclusions
2
second component τ
4
2
of 2.6 ns (χ = 1.00). In contrast complex The pyrene-substituted carbene chromium complex 4 is an
2
possessed a single component, which was 4.3 ns (χ = 1.25). effective CO-releasing molecule which is emissive in both
Interestingly, in HeLa cells complex 4 localised in both punc- organic and aqueous media. Microscopy studies have demon-
tate and cytoplasmic regions and displayed two components, a strated that the addition of 4 to HeLa cells also results in emis-
short component of 0.3 ns ± 0.2 ns and a longer component of sive behaviour, which is not observed in the case of 1.
6
.5 ns ± 2.8 ns. Furthermore, in the punctuated regions the Although these data do not unambiguously demonstrate that 4
longer component lifetime was significantly higher and was in is intact in cells, the observation of emission from the pure
excess of 10 ns. The proligand did not enter cells, therefore complex in water which is similar to that observed in CH Cl
2
2
this indicates that either dissociation occurred after entering (in which the complex is stable) and the lack of emission in
the cell or that the presence of the ligand was evident (by the the microscopy measurement from 1-bromopyrene appears to
lifetime component). Furthermore, the increase of the excited indicate that this behaviour is related to the complex. Localis-
state for the second component is probably an indication of ation occurs within both lipid droplets and mitochondria, the
the stability of the complex. The cytotoxicity of 4 over a latter being especially important, as cytochrome c-oxidase (the
1
0–100 μM concentration range was evaluated by Alamar Blue terminal oxidant in the respiratory chain) is a known target of
1
8
and LDH assays against RAW264.7 Murine Macrophages (see CO. Further, the application of FLIM and TSCPC has allowed
ESI†). The resulting data demonstrated a small loss of cell via- us to better understand the fate of the complex in live cells.
bility at concentrations of 10 μM of 4 over a 4 hour (Alamar
Blue) and 15 min (LDH) time period, whereas concentrations
of 100 μM resulted in a ca. 90% loss of viability in both cases.
Therefore, it appears that 4 is cytotoxic to both the HeLa and
Experimental
macrophages at high concentration (as is the case for many All manipulations were accomplished using standard Schlenk
organic compounds). The mechanism for the toxicity is line and Glove Box apparatus. The THF was distilled from
unknown, although the results we have obtained through the sodium benzophenone/ketyl prior to use. All other solvents
4960 | Dalton Trans., 2015, 44, 4957–4962
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were AR grade and used without further purification. Com- free EMEM was added to the cells followed by 20 µL from a
plexes [Cr(vC{OCH }R)(CO) ] (R = CH , C H ) were prepared 2.5 mM ethanol solution of the compound (giving a final con-
3
5
3
6
5
1
9
according to literature procedures.
pyrene, 1, and Cr(CO)
was sublimed prior to use. Myoglobin, light mineral oil and being returned to serum free EMEM prior to imaging.
[Me
3
4
O]BF , 1-bromo- centration of 50 µM, 2% ethanol). This was incubated for
6
were purchased from Aldrich the latter 15 minutes and washed 3 times with 1 mL of HBSS before
PBS were purchased from Sigma and sodium dithionite from
Alfa Aeasar. Ultra violet-visible spectra were recorded using a ESI.†
JASCO V-560 instrument. IR spectra were acquired on a
Details on RAW 264.7 murine macrophage culture is in the
Colocalisation staining was performed as follows. Cells
Mattson Research Series FTIR spectrometer using CsCl solu- were washed 5 times with 1 mL Hank’s Balanced Salt Solution
tion cells. NMR spectra were recorded on a Bruker AMX 300 (HBSS) and subsequently 990 µL serum free EMEM was added
1
13
Spectrometer (Operating frequencies H 300.13 MHz;
7
C
to the cells followed by 10 µL of the colocalisation dye in
1
3
6.98 MHz). For C NMR data, “cis” and “trans” carbonyl reso- DMSO. Nile Red (Invitrogen) Mitotracker Red® were prepared
nances refer to orientation related to the carbene substituent. as final concentrations of 100 ng mL− and 200 nM respect-
Mass spectra were obtained on a Bruker microTOF instrument. ively and incubation times of 10 minutes and 30 minutes
Emission spectra were obtained on Toshiba F-4500 FL Spectro- correspondingly. After washing a further 3 times, cells were
photometer. Assessment of CO-release by the myoglobin assay returned to serum free EMEM and complex 4 or its ligand pre-
1
1
5
was performed as detailed previously. Confocal microscopy cursor were added as described above.
and FLIM measurements were performed on a modified Nikon
TE2000-U at the Rutherford Appleton Laboratory.
Synthesis of [Cr(CO) (vC{OMe}C H )] 4
5
16
9
Acknowledgements
1
-Bromopyrene (0.33 g, 1.17 mmol) was dissolved in THF
This work was funded in part by the EPSRC (grant EP/
E005152/1), Leverhume Trust (F/00 2244/AA), BBRSC (student-
ship to JSW) and Royal Society (University Research Fellow-
ships to I.J.S.F. and S.I.P). We are also grateful to STFC for
funding to S.I.P. and S.W.B. The University of Bath, Cancer
Research at Bath and Alumni Funds are also thanks for finan-
cial support and for a studentship to R.L.A.
n
(
(
10 ml) and the solution cooled to −78 °C. A solution of Li Bu
1 ml, 1.2 M diluted in 10 ml THF) was added dropwise. After
3
6
0 min, Cr(CO) (0.303 g, 1.38 mmol) was added and the solu-
tion stirred for 20 min and warmed to 0 °C and stirred for a
further 30 min, during which time the solution changed
colour from yellow to orange/brown. Following addition of
[
Me O]BF (0.191 g, 1.29 mmol) the solution was stirred for at
3 4
0
C for 20 min before warming to room temperature and stir-
ring for a further 30 min, during which time a colour change
to dark red occurred. The reaction was then quenched with
Notes and references
0
3
.2 M NaHCO before extracting with hexane. After removal of
the solve the resulting residue was purified by chromatography
1 R. Motterlini, P. Sawle, J. Hammad, B. E. Mann,
T. R. Johnson, C. J. Green and R. Foresti, Pharmacol. Res.,
2013, 68, 108.
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C. Green and R. Motterlini, J. Inorg. Biochem., 2003, 96, 40;
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on silica gel using a 4 : 1 (v/v) mixture of hexane–CH Cl as
2
2
eluent. Complex 4 was isolated as a dark red solid. Yield
1
1
3
12 mg, 25%. H NMR (300 MHz, CDCl
3
) δ (ppm): 4.18 (br,
1
3
1
H, OMe), 7.60–8.25 (m, 9H, pyrene), C{ H} NMR (100 MHz,
) δ 66.8 (OMe), 119.8–147.9 (pyrene), 216.4 (cis-CO),
25.3 (trans-CO), 358.9 (CrvC); IR (CH
CDCl
2
3
−1
2 2
Cl , cm ): 2063 s,
1
C
C
948 vs; HRMS (ESI): m/z 437.0125 (437.0123 calculated for
−
23
H
H
13CrO
12CrO
6
[M + H] ). Elemental analysis expected for
C 63.31% H 2.77%, found C 63.89% H 2.85%.
3 F. Zobi, Future Med. Chem., 2013, 5, 175.
23
6
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Cell culture of HeLa cells was carried out in T75 flasks with
Eagle’s Minimum Essential Media (EMEM) containing 10%
fetal calf serum (FCS), this was supplemented with final con-
centrations of 1% L-glutamine (200 mM), 0.5% penicillin/strep-
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−
1
−1
tomycin (10 000 IU mL /10 000 mg mL ). After the EMEM
containing FCS was aspirated phosphate buffered saline (PBS)
was used (3 × 5 mL) to wash the cells, which was subsequently
aspirated. To this a 2.5% trypsin solution was added to detach
the cells from the flask, which was incubated at 37 °C for
5
–10 minutes, after which 5 mL EMEM containing FCS was
added. Next, the cells were seeded in glass bottomed dishes
for 12 hours. Cells were washed 5 times with 1 mL Hank’s
Balanced Salt Solution (HBSS) and subsequently 980 µL serum
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4962 | Dalton Trans., 2015, 44, 4957–4962
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