A reaction-based fluorescent probe for selective imaging of carbon monoxide in living cells using a palladium-mediated carbonylation

Article abstract of DOI:10.1021/ja307017b

Carbon monoxide is a member of the gasotransmitter family, which also includes NO and H₂S, and has been implicated in a variety of pathological and physiological conditions. Whereas exogenous therapeutic additions of CO to tissues and whole ani

Full text of DOI:10.1021/ja307017b

Communication
pubs.acs.org/JACS
A Reaction-Based Fluorescent Probe for Selective Imaging of Carbon
Monoxide in Living Cells Using a Palladium-Mediated Carbonylation
†
†,§
,†,‡
Brian W. Michel, Alexander R. Lippert, and Christopher J. Chang*
†
‡
Department of Chemistry and Howard Hughes Medical Institute, University of California, Berkeley, California 94720, United States
S Supporting Information
*
14−18
including NO,19−22 H S,
23−29
30−40
settings,
and H O ,
but
2
2
2
ABSTRACT: Carbon monoxide is a member of the
there have been no reports of analogous indicators for CO.
Herein we present the design, synthesis, and biological
evaluation of a new type of chemical reagent for selective CO
detection in living cells based on palladium-mediated carbon-
ylation chemistry. CO Probe 1 (COP-1) represents a unique
first-generation chemical tool that features a robust turn-on
response to CO with selectivity over reactive nitrogen, oxygen,
and sulfur species and can be used to detect this
gasotransmitter in aqueous buffer and in live-cell specimens.
Our overall strategy for imaging CO in live biological systems
relies on exploiting selective CO-induced reaction chemistry for
its detection. Recent work from our laboratory on reaction-
23
gasotransmitter family, which also includes NO and H S,
2
and has been implicated in a variety of pathological and
physiological conditions. Whereas exogenous therapeutic
additions of CO to tissues and whole animals have been
well-studied, the real-time spatial and temporal tracking of
CO at the cellular level remains an open challenge. Here
we report a new type of turn-on fluorescent probe for
selective CO detection based on palladium-mediated
carbonylation reactivity. CO Probe 1 (COP-1) is capable
of detecting CO both in aqueous buffer and in live cells
with high selectivity over a range of biologically relevant
reactive small molecules, providing a potentially powerful
approach for interrogating its chemistry in biological
systems.
30−38
based fluorescent probes for imaging of H O2
and H2S
2
have taken advantage of the respective nucleophilic oxidative
and reductive abilities of these small molecules. In contrast, CO
is not a particularly nucleophilic or electrophilic species and is
better known for its inorganic coordination chemistry and
subsequent organometallic reactivity. Therefore, we envisioned
metal-mediated carbonylation chemistry as a potential means to
design a reaction-based fluorescent CO probe, as covalent
incorporation of CO into a dye scaffold can significantly alter its
electronic characteristics. In particular, we turned our attention
to palladium because of the established reactivity of this metal
arbon monoxide is best known as a toxic gas inhaled from
common sources such as smoke and car exhaust, but
C
emerging studies have shown that this reactive small molecule
is also continuously produced in the body via the breakdown of
1
−3
heme by heme oxygenase enzymes.
Similar to the other
major gasotransmitter molecules NO and H S, CO is proposed
2
41−47
to play significant roles in modulating responses to both
in catalytic carbonylation reactions
as well as recent
4
−7
chemical and physical stresses.
In one example, exogenous
reports demonstrating the compatibility of organometallic
and endogenous CO can provide protection against tissue
damage during myocardial ischemia/reperfusion, and to this
reactions with cellular systems, including Ahn’s indicators for
8
48
palladium/platinum 4 and Meggers’ ruthenium-induced allyl
9
end, CO-releasing molecules (CORMs) based on transition-
metal carbonyl complexes have been developed as potential
therapeutics that allow for more targeted release of CO in
carbamate cleavage, where cell viability was maintained.
Additionally, Bradley and co-workers elegantly illustrated the
use of palladium-functionalized microspheres to elicit both
4
,9,10
50
comparison with direct gas inhalation.
deallylation and cross-coupling reactions in live cells. On the
Despite the important signal/stress dichotomy of CO, many
aspects of its chemistry in biological systems remain elusive, in
part because of the lack of ways to track this transient small
molecule selectively within intact, living biological specimens.
Indeed, the primary methods for interrogating the biological
effects of CO to date involve detecting a gross anatomical
change of some observable parameter, such as infarct size in
studies of the effects of CO on ischemia/reperfusion or offline
basis of these considerations, we designed and synthesized the
cyclopalladated species COP-1, anticipating that the presence
of palladium would quench the fluorescence of the
borondipyrromethene difluoride (BODIPY) core via heavy-
atom electronic effects and that upon binding of CO, a
carbonylation reaction would concomitantly release reduced
Pd(0) and a more fluorescent species. To this end, we prepared
COP-1 by alkylation of benzyl chloride 2 with dimethylamine
1
1,12
and subsequent cyclometalation with Pd(OAc) , which was
extracellular measurements using myoglobin
or dirhodium-
2
13
then converted to the chloride dimer (Scheme 1).
supported particles for colorimetric readouts. We reasoned
that the development of a CO-responsive small-molecule
fluorescent probe would meet a critical need for new
technologies to monitor this reactive small molecule in
biological systems with spatial and temporal information.
This approach has proved useful for studying the contributions
of a variety of small signal/stress molecules in biological
With COP-1 in hand, we tested its fluorescence properties
and CO reactivity in aqueous solution buffered to physiological
pH. For the present in vitro analysis, we utilized the water-
Received: July 17, 2012
©
XXXX American Chemical Society
A
dx.doi.org/10.1021/ja307017b | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
a
Scheme 1. Synthesis and Reactivity of COP-1
a
Reagents and conditions: (a) CH Cl , reflux, 2 h, then solvent swap
2
2
(
∼10:1 toluene:CH Cl ), Et N, BF ·Et O, 50 °C, 1 h. (b) Me NH,
2
2
3
3
2
2
K CO , KI, CH CN, microwave, 80 to 100 °C, 1 h. (c) Pd(OAc) ,
2
3
3
2
benzene, 50 °C, 14 h, then solvent swap (acetone), LiCl. (d) Wet
CH Cl , CO, 31 °C, 14 h.
Figure 1. COP-1 shows a robust and selective turn-on response to CO
in buffered aqueous solution. (a) Turn-on fluorescence response of 1
μM COP-1 to 50 μM CORM-3 in pH 7.4 DPBS at 37 °C (λex = 475
nm, emission collected at 490−630 nm). Time points are represented
by spectra taken 0, 5, 15, 30, 45, and (red) 60 min after the addition of
CORM-3. (b) Fluorescence responses of 1 μM COP-1 to CO and
biologically relevant reactive oxygen, nitrogen, and sulfur species. Bars
represent normalized integrated fluorescence intensity responses
between 490 and 630 nm with λex = 475 nm for the respective
analytes (50 μM) at t = 0, 5, 15, 30, 45, and (red) 60 min. Data were
acquired in pH 7.4 DPBS buffer at 37 °C. Legend: (1) control; (2)
2
2
soluble complex [Ru(CO) Cl(glycinate)] (CORM-3) as an
3
5
1,52
easy-to-handle CO source.
As expected, COP-1 was weakly
fluorescent in Dulbecco’s phosphate-buffered saline (DPBS)
buffered to pH 7.4 (λem = 503 nm, Φ = 0.01; see Figure S2 in
the Supporting Information). Addition of 50 μM CORM-3 to a
solution of COP-1 at 37 °C triggered a robust fluorescence
turn-on response due to CO-induced formation of the
carbonylation product 4, which was synthesized and charac-
•−
CORM-3; (3) H O ; (4) tBuOOH; (5) NaOCl; (6) O ; (7) NO;
2
2
2
−
terized independently for verification (λ
= 499 nm, ε =
(8) ONOO ; (9) H S.
2
max
1
−1
2
3 000 M− cm , λ = 507 nm, Φ = 0.44; see Figure S1).
em
Interestingly, this acid was the major product in aqueous
solution, in contrast to the dealkylative amide product typically
45
observed in organic solutions. Within 60 min of reaction
under these conditions, COP-1 produced a 10-fold increase in
fluorescence (Figure 1a). Moreover, we observed a dose-
dependent response of COP-1 to CORM-3 down to 1 μM
(
∼28 ppb CO) levels (Figure S3). Furthermore, the
fluorescence turn-on response for COP-1 was found to have
good selectivity over other biologically relevant reactive oxygen,
nitrogen, and sulfur species, including H O , tert-butyl
2
2
−
hydroperoxide (tBuOOH), hypochlorite (OCl ), superoxide
−
−
(
O ), NO, peroxynitrite (ONOO ), and H S, as exposure of
2
2
COP-1 to these molecules did not trigger fluorescence
responses to the same extent as exposure to CO (Figure 1b).
Finally, we evaluated the ability of COP-1 to visualize
changes in CO levels in live cells using confocal microscopy.
HEK293T cells were incubated with either CORM-3 (5 or 50
μM) or a vehicle control, and then the cells were treated with 1
μM COP-1 (Figure 2). A significant and dose-dependent
increase in intracellular fluorescence was observed in CORM-3-
treated cells (Figure 2b,c,e) over vehicle control samples
Figure 2. Confocal microscopy images of CO detection in live
HEK293T cells using COP-1. (a) HEK293T cells incubated with
COP-1 for 30 min at 37 °C. (b) HEK293T cells incubated with 5 μM
CORM-3 for 45 min at 37 °C and 1 μM COP-1 for the final 30 min.
(c) HEK293T cells incubated with 50 μM CORM-3 for 45 min at 37
°
C and 1 μM COP-1 for the final 30 min. (d) Bright-field image of the
cells in (c) overlaid with images of 1 μM Hoescht 33342-stained cells.
The scale bar represents 100 μM. (e) Mean fluorescence intensities of
representative images with (1) 1 μM COP-1, (2) 1 μM COP-1 and 5
μM CORM-3, and (3) 1 μM COP-1 and 50 μM CORM-3.
(
Figure 2a,e). In addition, we performed two independent
types of assays to show that the palladium-based probe and its
reactivity are nontoxic to the cellular specimens and compatible
with live-cell imaging over the course of these experiments.
First, we acquired bright-field images and overlaid them with
fluorescence images of the cells stained with Hoescht 33342
nuclear stain that clearly showed intact and viable nuclei
(Figure 2d). Second, as a further validation of cell viability, a
water-soluble tetrazolium salt (WST) cell proliferation assay
B
dx.doi.org/10.1021/ja307017b | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
was performed over a range of probe concentrations (500 nM
to 10 μM) with and without the addition of CORM-3 (100
μM). Cell viability remained constant over the range of probe
concentrations evaluated (Figure S4). We note that the
concentrations of CORM-3 employed are well within the
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shown not to alter cell viability, and the added CORM-3 in
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(
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In summary, we have presented a new approach to biological
CO detection through the synthesis and application of COP- 1,
a cyclopalladated probe that interacts with CO to trigger a
fluorogenic carbonylation reaction. COP-1 shows a robust turn-
on fluorescence response to CO that is selective over a variety
of reactive nitrogen, oxygen, and sulfur species and can be used
to image CO in living cells. Current efforts are focused on
designing indicators with improved sensitivity and greater turn-
on or ratiometric response, adapting this reaction-based
strategy to other imaging modalities, and using COP-1 and
next-generation chemical tools to study CO function in a
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ASSOCIATED CONTENT
Supporting Information
■
(
*
S
1
33, 10078−10080.
Experimental details, including synthesis and characterization,
selectivity assays, spectroscopic methods, cellular imaging
methods, and cell viability assays. This material is available
free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
chrischang@berkeley.edu
■
(
Present Address
Department of Chemistry, Southern Methodist University,
Dallas, Texas 75275-0314, United States.
§
(
(
4
(
Notes
The authors declare no competing financial interest.
Res. 2011, 44, 793−804.
ACKNOWLEDGMENTS
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■
This work was supported by Amgen, Astra Zeneca, Novartis,
and the Packard Foundation. C.J.C. is an Investigator of the
Howard Hughes Medical Institute. We thank Ann Fischer (UC
Berkeley Tissue Culture Facility) for expert technical assistance.
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