Ligand-Directed Approach to Activity-Based Sensing: Developing Palladacycle Fluorescent Probes That Enable Endogenous Carbon Monoxide Detection

Article abstract of DOI:10.1021/jacs.0c06405

Carbon monoxide (CO) is an emerging gasotransmitter and reactive carbon species with broad anti-inflammatory, cytoprotective, and neurotransmitter functions along with therapeutic potential for the treatment of cardiovascular diseases. The study of CO chemistry in biology and medicine relative to other prominent gasotransmitters such as NO and H2S remains challenging, in large part due to limitations in available tools for the direct visualization of this transient and freely diffusing small molecule in complex living systems. Here we report a ligand-directed activity-based sensing (ABS) approach to CO detection through palladium-mediated carbonylation chemistry. Specifically, the design and synthesis of a series of ABS probes with systematic alterations in the palladium-ligand environment (e.g., sp3-S, sp3-N, sp2-N) establish structure-activity relationships for palladacycles to confer selective reactivity with CO under physiological conditions. These fundamental studies led to the development of an optimized probe, termed Carbon Monoxide Probe-3 Ester Pyridine (COP-3E-Py), which enables imaging of CO release in live cell and brain settings, including monitoring of endogenous CO production that triggers presynaptic dopamine release in fly brains. This work provides a unique tool for studying CO in living systems and establishes the utility of a synthetic methods approach to activity-based sensing using principles of organometallic chemistry.

Full text of DOI:10.1021/jacs.0c06405

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Article
Ligand-Directed Approach to Activity-Based Sensing: Developing
Palladacycle Fluorescent Probes That Enable Endogenous Carbon
Monoxide Detection
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Johannes Morstein, Denis Hofler, Kohei Ueno, Jonah W. Jurss, Ryan R. Walvoord, Kevin J. Bruemmer,
Samir P. Rezgui, Thomas F. Brewer, Minoru Saitoe, Brian W. Michel,*,# and Christopher J. Chang*
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ABSTRACT: Carbon monoxide (CO) is an emerging gasotrans-
mitter and reactive carbon species with broad anti-inflammatory,
cytoprotective, and neurotransmitter functions along with thera-
peutic potential for the treatment of cardiovascular diseases. The
study of CO chemistry in biology and medicine relative to other
prominent gasotransmitters such as NO and H2S remains
challenging, in large part due to limitations in available tools for
the direct visualization of this transient and freely diffusing small
molecule in complex living systems. Here we report a ligand-directed
activity-based sensing (ABS) approach to CO detection through
palladium-mediated carbonylation chemistry. Specifically, the design
and synthesis of a series of ABS probes with systematic alterations in
the palladium-ligand environment (e.g., sp3-S, sp3-N, sp2-N)
establish structure−activity relationships for palladacycles to confer selective reactivity with CO under physiological conditions.
These fundamental studies led to the development of an optimized probe, termed Carbon Monoxide Probe-3 Ester Pyridine (COP-
3E-Py), which enables imaging of CO release in live cell and brain settings, including monitoring of endogenous CO production that
triggers presynaptic dopamine release in fly brains. This work provides a unique tool for studying CO in living systems and
establishes the utility of a synthetic methods approach to activity-based sensing using principles of organometallic chemistry.
carbonylation chemistry for CO detection with high selectivity
over competing reactive oxygen, nitrogen, carbon, and sulfur
species. In parallel with our initial report, the He and Chen
laboratories described COSer,53 a first-generation fluorescent
protein sensor for CO. COP-1 has been independently applied
to monitor and study CO released by CORMs in biological
samples,54−57 and its organometallic trigger has been adapted by
others to develop various CO-responsive probes,58−61 including
fluorescent indicators for two-photon imaging,62 Raman
spectroscopy nanosensors,63 and CO-responsive nano-
particles.64 In addition, allyl ether-based fluorescent probes
have emerged as an alternative strategy for CO-detection,65−67
but the requirement for exogenous addition of free palladium
salts to biological samples makes this three-component system
more challenging to implement. These efforts have spawned
INTRODUCTION
■
Carbon monoxide (CO) is an essential gasotransmitter that
exhibits a diverse array of cytoprotective, anti-inflammatory, and
signaling effects in living systems.1−3 In addition to these roles in
basic biology, CO is being increasingly recognized for its
contributions to medicine as molecules with the capacity to
release CO in vivo, including Carbon Monoxide Releasing
Molecules (CORMs), are being evaluated as potential
therapeutics for a broad range of cardiovascular diseases.4−13
The biological and biomedical importance of CO motivates the
need to develop new methods to monitor dynamic changes in
CO fluxes with high selectivity and sensitivity in living systems
on a molecular level. In this context, fluorescent probes offer a
powerful set of chemical reagents for studying other transient
biologically active small molecules, including the gasotrans-
mitters NO14−16 and H2S17−27 as well as H2O2,28−43 presaging
the use of this approach to decipher the chemistry of CO in
biological settings.
Received: June 16, 2020
As part of a larger program in our laboratory on activity-based
sensing (ABS),44−48 we and others have become interested in
developing small-molecule probes for one-carbon species,49−51
and we reported the development of Carbon Monoxide Probe-1
(COP-1),52 which established the use of palladium-mediated
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broader strategies for exploiting the use of organometallic
reactivity in cells.68−77
Despite previous successes with COP-1, this first-generation
ABS probe exhibits shortcomings that limit its broader use in
biological settings, including a hydrophobic nature that
promotes punctate cellular staining, thereby effectively preclud-
ing visualization of substantial portions of the cell, as well as
relatively poor cellular retention, making washing steps (to
increase signal-to-noise responses) in imaging experiments more
challenging. We sought to address these issues by turning our
attention to studying the underlying fundamental organo-
metallic chemistry of CO detection by systematically modulat-
ing the metal−ligand environment of the reactive palladacycle
trigger. Specifically, using COP-1 as a starting point, tuning of
the covalently attached amine ligand with sp3-S, sp3-N, sp2-N
substitutions and the chloro-dimer-bridge were evaluated to
explore their influence on the reactivity of the CO probe. We
adapted the most promising reaction trigger identified in each
round of synthetic screening and explored further functionaliza-
tion of the BODIPY fluorophore to increase aqueous solubility,
cellular distribution, and retention properties. These founda-
tional structure−activity studies resulted in the development of
an optimized CO-probe, termed Carbon Monoxide Probe-3
Ester Pyridine (COP-3E-Py), which exhibits markedly increased
solubility in aqueous media and improved cellular retention. We
demonstrate the utility of COP-3E-Py for the detection of CO
release from CORMs in live cells. Included is the successful
visualization of endogenous release of CO during neural
stimulation in fly brain preparations using electrical and
chemical input, highlighting the potential of fundamental
organometallic chemistry for chemical biology applications.
Figure 1. Colocalization of COP-1 and COP-1′ with LysoTrackerRed
DND-99. (a−c) HEK293T cells were incubated with COP-1 (1 μM)
and LysoTracker (50 nM) for 30 min, resulting in the observed cellular
staining of (a) COP-1, (b) LysoTracker, and (c) overlay. (d−f)
HEK293T cells were incubated with COP-1′ (1 μM) and LysoTracker
(50 nM) for 30 min, resulting in the observed cellular staining of (d)
COP-1′, (e) LysoTracker, and (f) overlay. Scale bar represents 80 μm.
(g,h) Chemical structures of COP-1 and LysoTracker Red DND-99
showing shared alkylamine motifs.
RESULTS AND DISCUSSION
■
Imaging Studies Reveal Differences in Cellular Local-
ization for COP-1 and its Carbonylation COP-1′ Product.
We initiated structure−activity studies with extensive character-
ization of COP-1 and the carboxylic acid product formed from
reaction of COP-1 and CO, termed COP-1′. This postcarbon-
ylation product is presumably formed following migratory
insertion of COP-1 with CO to provide a Pd-acyl intermediate
and subsequent reductive hydrolysis. Confocal microscopy
images of HEK293T cells loaded with COP-1 showed punctate
staining. This phenomenon was revealed to arise from lysosomal
localization through a costaining experiment with lysosome
marker LysoTracker Red DND-99. Interestingly, this experi-
ment showed strong colocalization for COP-1 with LysoTracker
Red DND-99 (PC: 0.792, M1:0.584) but not for the more polar
carboxylic acid product COP-1′ (PC: 0.600, M1:0.158) (Figure
1). We initially hypothesized that COP-1 accumulates in acidic
stores due to protonation of the basic directing group.78
However, while this alkylamine functionality is shared by COP-
1, COP-1′, and LysoTracker Red DND-99 (Figure 1h), the
presence of an additional polar carboxyl group on COP-1′
mitigates lysosomal localization. We further investigated the
reaction of COP-1 with H2S by subjecting COP-1 to excess
Na2S in a suspension of CH2Cl2 and water. Subsequent LCMS
analysis revealed that the product of this reaction is indeed the
protodemetalation product (Figure S1).
weight would result in increased hydrophilicity and CO
reactivity. To this end, we conducted a bridge-splitting reaction
with pyridine as the σ-donating ligand to synthesize the
monomeric probe COP-1-Py.79 This reaction was conducted
with a suspension of COP-1 in CH2Cl2, and upon addition of an
equimolar amount of pyridine (1:1 with Pd) at room
temperature, we observed an immediate increase in solubility
in CH2Cl2 through the formation of a homogeneous solution
(Figure 2a). The resulting COP-1-Py complex was isolated and
behaved similarly in vitro to samples prepared from the μ-chloro
dimer COP-1, reacting with CO to give the same COP-1′
product. Single crystals of COP-1, COP-1-Py, and the
carbonylation product COP-1′ were obtained from slow
evaporation of concentrated dichloromethane solutions and
characterized by single crystal X-ray crystallography to provide
molecular connectivity for all three compounds (Figure 2b). As
anticipated, the palladacycles of COP-1 and COP-1-Py are
perpendicular to the BODIPY plane and the palladium centers
adopt a square planar geometry. The crystal structure suggests
that COP-1 exists as a dimer with two μ-chloro bridges linking
the palladium(II) centers, whereas COP-1-Py exists as a
monomer with a single square-planar palladium center. A
hydrogen-bonded zwitterion is observed for the COP-1′
carbonylation product after CO-induced palladium release.
The acidic hydrogen of the ammonium group, located in the
electron density map, is 1.636 Å from the nearest oxygen of the
carboxylate moiety.
Synthesis, Structure, and CO Reactivity of a Mono-
meric Analogue of COP-1. With data on COP-1′ showing
that a more hydrophilic COP analogue could promote more
even and diffuse cellular staining, we targeted the synthesis of a
monomeric COP dye in hopes that the decreased molecular
Modulation of the Directing Group Ligands on the
COP Platform to Tune CO-Dependent Palladacycle
Carbonylation Reactivity. Since dimeric COP-1 and
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Figure 2. Synthesis and reactivity of the of the monomeric CO indicator COP-1-Py and crystal structures of COP-1, COP-1-Py, and COP-1′. (a)
Reagents and conditions: (i) CO, wet CH2Cl2, 31 °C, 14 h; (ii) pyridine, CH2Cl2, rt, 5 h, 79%. (b) X-ray crystal structures: hydrogen atoms have been
omitted for clarity, except for the H-bonding hydrogen in COP-1′. Thermal ellipsoids are shown at the 70% probability level.
monomeric COP-1-Py showed the same CO-dependent
carbonylation reactivity, we decided to move forward and
design monomeric palladacycles to tune CO reactivity by
systematic modulation of the directing group ligand on
palladium. In particular, we reasoned that the chelating
heteroatom ligand, which is the directing group for the
cyclopalladation reaction, does not exhibit fluxional behavior
and is therefore expected to potentially have the most profound
effect on CO reactivity. We therefore focused our efforts on
varying the chelating heteroatom ligand and explored a set of
sp3-S, sp3-N, and sp2-N donors. Indeed, these types of directing
ligands are known to facilitate the formation of palladacycles and
to undergo CO insertion in an abiological experimental setting.
We sought to explore varied σ-donating directing group ligands
to evaluate its influence on both reactivity and subcellular
localization. Finally, in addition to N-basic directing groups, we
sought to characterize the potential of a sp3-S-derived pallada-
cycle for CO-sensing applications. Previous mechanistic studies
in abiotic reaction environments suggest that these palladacycles
undergo more facile CO-insertion compared to sp3-N-derived
palladacycles.79,80 As such, we envisioned that this promotion of
CO-insertion could potentially allow for faster reaction kinetics
and improved selectivity of the probe scaffold over H2S, a
competing gasotransmitter and a substrate for an off-pathway
protodemetalation.
formation, and acetal hydrolysis to provide aldehyde 6.
Condensation with hydroxyl amine then afforded aldoxime 7.
2-Pyridinyl 9 was prepared from the bromo BODIPY 8 via a
Stille cross-coupling. All probes were prepared from their
precursors via directed palladation, appropriate anion meta-
thesis, and bridge-splitting with pyridine.
With this family of CO probes in hand, we evaluated their
comparative reactivity with CO and H2S in vitro as well as their
subcellular localization and staining pattern in live HEK293T
cells (Figure 3). Specifically, we evaluated the CO responses of
these reagents in buffered aqueous solution using [Ru(CO)3Cl-
(glycinate)] (CORM-3) as a CO source. COP-2-Py did not
exhibit a significant fluorescent turn-on response with either CO
or H2S in phosphate buffered saline, suggesting that sp3-S-
palladacycles do not undergo facile CO-insertion under
physiological conditions relative to sp3-N-palladacycle con-
geners. Moreover, COP-4-Py and COP-5-Py were found to be
less chemoselective for CO over H2S than COP-1. Notably, both
probes exhibit a stronger relative and absolute fluorescence
response with H2S compared to CO. Whereas COP-4-Py shows
a continual rise in fluorescence turn-on response with H2S (over
the time course of the experiments), COP-5-Py exhibits a rapidly
declining fluorescence turn-off response toward H2S after 15
min. Some possible speculations for this observation are
aggregation and/or precipitation of the expected nonpolar
product from protodemetalation. In contrast to CO, which gives
a carbonylated product upon activity-based sensing, H2S is
presumably undergoing protodemetalation due to its acidic
nature and potential ability to form a metal-sulfide precipitate.17
Of this series, COP-3-Py exhibits the strongest fluorescence
response toward CO with minimal reactivity with H2S, offering
superior specificity over the first-generation COP-1 reagent.
Taken together, these structure−activity relationships for a
family of monomeric COP dyes suggest that sp3-N-palladacycles
appear to be best-suited for the development of palladacycle
An sp3-S-derived CO-probe, termed COP-2-Py, and a
comparably less-basic sp3-N-derived morpholino congener to
COP-1, termed COP-3-Py, were obtained via similar synthetic
procedures as COP-1-Py (Scheme 1). In addition, an oxime-
palladacycle probe, COP-4-Py, and a pyridine-based dye, COP-
5-Py, were synthesized via newly developed synthetic routes
(Scheme 1). Specifically, compounds 2 and 3 were prepared in a
similar fashion to the COP-1 precursor, i.e. via a nucleophilic
substitution of the benzylic chloride 1. The oxime-containing
synthon 7 was prepared from 1,4-phthalaldehyde by monoacetal
protection (to differentiate the two aldehydes), BODIPY
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a
Scheme 1. Synthesis of COP-2-Py, COP-3-Py, COP-4-Py, and COP-5-Py
a
Reagents and conditions: (i) CH2Cl2, rt, 14 h; (ii) DIPEA, BF3·OEt2, CH2Cl2, rt, 3 h, 28% (2 steps); (iii) L2: NaSMe, EtOH, rt, 25 h, 45%; L3: KI,
K2CO3, CH3CN, morpholine, 80−100 °C, 60 min, 92%; (iv) L2: Pd(OAc)2, AcOH, 120 °C, 55 min; L3: Pd(OAc)2, C6H6, 50 °C, 14 h; (v) LiCl,
acetone, rt, 4 h; (vi) pyridine, CH2Cl2, rt, 5 h, 22% for L2 (3 steps); 13% for L3 (3 steps); (vii) ethylene glycol, p-toluenesulfonic acid, 130 °C, 4 h,
24%; (viii) 2,4-dimethylpyrrole, TFA, CH2Cl2,16 h, rt, then DIPEA, BF3·OEt2, MePh, 45 °C, 50 min, 30%; (ix) acetone, 10% HCl, reflux, 2 h, 78%;
(x) hydroxylamine hydrochloride, KOAc, EtOH, H2O, 70 °C, 3 h, 91%; (xi) di-μ-chlorobis[2-[(dimethylamino)methyl]phenyl-C,N]-
dipalladium(II), CHCl3/AcOH, 50 °C, 16 h; (xii) pyridine, CH2Cl2, rt, 16 h, 69% (2 steps); (xiii) CH2Cl2, rt, 14 h, then Et3N, BF3·OEt2, rt,
20 h, 50%; (xiv) 2-(tributylstannyl)pyridine, toluene, Pd(Ph3P)2Cl2, 110 °C, 48 h, 65%; (xv) Pd(OAc)2, PhH, 50 °C, 14 h then LiCl, acetone, rt, 4
h then pyridine, CH2Cl2, rt, 74%.
activity-based sensing probes for CO that achieve improved
selectivity over H2S.
analogue COP-3-Py still exhibits minor punctate staining in
HEK293T cells owing to its basic nitrogen donor. As such, we
subjected COP-3-Py to a second round of structure
optimization focusing on the fluorophore backbone.
Synthesis and Characterization of COP-3E-Py Bearing
a More Hydrophilic BODIPY Core. The foregoing structure−
activity studies led to the identification of a sp3-N morpholino
directing ligand on COP-3-Py as an improved activity-based
sensing trigger for CO detection. To further optimize this
scaffold, we next decided to functionalize the BODIPY core with
Evaluation of the staining patterns and localizations of all
monomeric COP dyes showed observably less punctate staining
(Figure 3e−h) relative to COP-1 (Figure 1). In addition to
making probes less hydrophobic upon reduction in size from
dimer to monomer, these results give some indication that less
basic directing group ligands can decrease probe localization in
acidic stores, presumably by lowering protonation under
physiological conditions. However, the sp3-N morpholino
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Figure 3. Evaluation of COP-2-Py, COP-3-Py, COP-4-Py, and COP-5-Py reactivity in vitro and in cellulo. (a−d) Fluorescent responses of the
respective COP reagent (1 μM) to CO and H2S. Bars represent integrated fluorescence intensities between 520 and 620 nm normalized to the
respective probe in PBS at 0 min. Analytes were incubated at 50 μM concentration in pH 7.4 PBS at 37 °C and the bars correspond to the time points 0,
5, 15, 30, 45, and 60 min after addition of the respective analyte. Legend: (1) control; (2) CO; (3) H2S. CORM-3 was used as a CO source. (e−h)
Cellular staining of the respective probes (1 μM) on a HEK293T cell monolayer after 60 min of incubation show basal localization of the dyes. Images
were acquired with individualized microscope settings and normalized for staining comparison. Scale bar represents 80 μm.
a
Scheme 2. Synthesis of COP-3E-Py and COP-3E-Py′
a
Reagents and conditions: (i) H2, Pd/C, acetone, rt, 12 h; (ii) TFA, 40 °C, 10 min, 48% (2 steps); (iii) p-(chloromethyl)benzoyl chloride, CH2Cl2,
rt, 14 h; (iv) DIPEA, BF3·OEt2, CH2Cl2, rt, 3 h, 22% (2 steps); (v) KI, K2CO3, morpholine, CH3CN, 60 °C, 3 h, 94%; (vi) Pd(OAc)2, C6H6, 50
°C, 14 h; (vii) LiCl, acetone, rt, 4 h; (viii) Pyridine, CH2Cl2, rt, 5 h, 70% (3 steps); (ix) CO, wet CH2Cl2, 31 °C, 14 h, 92%.
E
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ester chains to increase aqueous solubility and hydrophilicity
and improve cellular retention by exploiting native intracellular
esterase activity.81 The probe Carbon Monoxide Probe-3 Ester
Pyridine (COP-3E-Py) was designed and synthesized according
to Scheme 2. Initial deprotection of a commercially available
pyrrole gave the more electron-rich pyrrole 10. This
intermediate was unstable due to rapid oxidation and was
therefore used directly to form BODIPY 11 via condensation
with 4-chlorobenzoyl chloride and complexation with BF3.
Nucleophilic substitution of chlorine with morpholine, directed
palladium metalation, ligand exchange, and palladacycle-bridge-
splitting gave access to the final COP-3E-Py probe. The
carbonylated product COP-3E-Py′ was synthesized by reacting
COP-3E-Py under a CO atmosphere in a solvent mixture of
CH2Cl2 and water. In contrast to protodemetalation, the direct
observation of carbonylated products formed in the presence of
CO in vitro illustrate the unique nature of our ABS strategy.
In aqueous PBS solution, we observed an 11-fold fluorescence
turn-on for COP-3E-Py (1 μM) upon addition of CORM-3 (50
μM) as a CO source within 60 min. Both the COP-3E-Py
indicator (λmax = 521 nm, λem = 535 nm, Φfl = 0.12, ε521 = 31 000
Figure 5. Live-cell imaging comparison between COP-1 and COP-3E-
Py establishing improved diffuse staining of the more hydrophilic COP-
3E-Py congener compared to lysosomal localization of COP-1. (a,b,d,e)
CO-detection in live HEK293T cells using COP-3E-Py and COP-1.
HEK293T cells were incubated with 1 μM COP 1 (a,b) or COP-3E-Py
(d,e) for 120 min total, with negative control vehicle (a,d) or CORM-3
(50 μM) added for the positive control for the final 60 min (b,e). (c,f)
Representative images taken at 63× for staining pattern comparison.
Scale bar represents 80 μm (a,b,d,e) or 20 μm (c,f).
M−1 cm−1) and its carbonylated product COP-3E-Py′ (λmax
=
521 nm, λem = 537 nm, Φfl = 0.51, ε521 = 43 000 M−1 cm−1)
exhibit a red shift compared to the parent COP-1 (λmax = 499
nm, λem = 503 nm). Other potentially competing analytes,
including reactive nitrogen, oxygen, and sulfur species, were
tested at the same concentration (50 μM) for reactivity with
COP-3E-Py (Figure 4). Only minimal responses were observed
and ER-Tracker Red E34250 in the presence of CO indicate
some colocalization with the ER (PC: 0.913, M1: 0.682, Figure
6).9 Notably, the observation that this CO detection reagent can
Figure 4. Turn-on fluorescence response to CO and analyte selectivity
of COP-3E-Py in buffered aqueous solution. (a) Turn-on fluorescence
response of COP-3E-Py (1 μM) to CORM-3 (50 μM) in pH 7.4 PBS at
37 °C (λex = 515 nm, emission collected from 520−630 nm). Time
points taken at 0, 5, 15, 30, 45, and 60 min after addition of CORM-3.
(b) Fluorescence responses of COP-3E-Py (1 μM) to CO and other
biologically relevant reactive small molecules. Bars represent integrated
fluorescence intensity between 520 and 620 nm normalized to COP-
3E-Py in PBS at 0 min. Analytes were incubated at 50 μM concentration
in pH 7.4 PBS at 37 °C and the bars correspond to the time points 0 (or
1 for (8)), 5, 15, 30, 45, and 60 min after addition of the respective
analyte. Legend: (1) control; (2) CORM-3; (3) H2O2; (4) NaOCl; (5)
TBHP; (6) KO2; (7) ONOO−; (8) H2S.
with other biologically relevant small molecules in this assay,
showing the high specificity for activity-based sensing of CO by
the COP-3E-Py palladacycle.
Figure 6. Live-cell imaging comparison between COP-3E-Py and
COP-1 reagents showing superior retention of the COP-3E-Py dye
after sample washing. Panels (a-c) show subcellular localization of
COP-3E-Py. HEK293T cells were stained with COP-3E-Py (a, 1 μM)
and ER-Tracker Red E34250 (b, 1 μM). (d−g) Confocal microscopy
images of HEK293T cells with COP-3E-Py (d,e) or COP-1 (f,g) were
taken before and after a buffer exchange was conducted. The images
after buffer-exchange were recorded after an equilibration period of 10
min. (h) Mean fluorescence intensities of images acquired 10 min after
buffer exchange as fractions of the initial fluorescence before buffer
exchange. Error bars denote SEM (n = 3). **P < 0.002. Scale bars
represent 40 μm (for a−g).
We next evaluated the ability of COP-3E-Py to image CO in
biological samples and compared its performance to COP-1.
Administration of CORM-3 to live HEK293T cells in the
presence of COP-1 or COP-3E-Py yielded a robust fluorescent
turn-on response in both cases (Figure 5). However, in contrast
to COP-1, which accumulated in lysosomes, COP-3E-Py gave a
more diffuse, homogeneous staining pattern in live cells.
Moreover, dual-dye imaging experiments with COP-3E-Py
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Figure 7. Endogenous CO release monitored with COP-3E-Py. (a) Schematic of the fly brain experiment. (b) Representative images of COP-3E-Py
fluorescence observed at the α3/α′3 compartments of the mushroom body vertical lobes of the fly brain preparation. Scale bar represents 25 μm. (c−f)
Trace and quantification of turn-on response after stimulation of the antennal lobe and NMDA stimulation using COP-3E-Py or COP-1 respectively.
*p < 0.05; **p < 0.01; Bonferroni post hoc test.
monitor changes in CO fluxes associated with the ER is desirable
owing to the ER-membrane localization of heme oxygenase-1
(HO-1) which a major endogenous source for production of
CO.82 As such, the increased hydrophilicity of COP-3E-Py
relative to COP-1 affords improved detection of CO through
broader distribution of the probe throughout the cell.
in which simultaneous activation of acetylcholine and glutamate
receptors has taken place. Fly brains were isolated and the
mushroom body vertical lobes were imaged with COP-3E-Py,
with a washing step after probe incubation (Figure 7). Two
different types of stimulation were employed to evoke dopamine
release and neurotransmission in this biological model: (1)
electrical stimulation of the antennal lobe (acetylcholine input)
and (2) bath application with NMDA (N-methyl-D-aspartate;
glutamate input). A clear increase in COP-3E-Py fluorescence
was observed, confirming that this probe could monitor
endogenous CO release mediated in this fly brain region.
Next, we conducted comparative cellular retention assays with
COP-1 and COP-3E-Py (Figure 6). Probes were loaded into
cells and imaged, and then washed by a buffer exchange and
imaged again. Under these conditions, the fraction of final
fluorescence intensity relative to initial fluorescence intensity of
COP-1 was 57%, with COP-3E-Py offering an improvement to
76% retention of fluorescence signal after washing (Figure 6).
The observed increase in cellular retention for COP-3E-Py
expands opportunities for using this reagent for CO detection in
biological settings that benefit from protocols with washing
steps. In order to assess the most useful time window for COP-
3E-Py probe applications in biological samples, we conducted a
cell viability experiment (Figure S3). After 3 h, a decrease in
viability was observed, suggesting that this probe is most useful
to monitor CO release on shorter time scales. These data
establish that systematic modification of the activity-based
sensing palladacycle trigger for selective tuning of CO reactivity
as well as increasing hydrophilicity of the BODIPY core for
improved cellular localization and retention leads to COP-3E-Py
as an advanced reagent for biological CO detection with
enhanced properties.
COP-3E-Py Enables Detection of Endogenous CO
Release in Fly Brain Models. To showcase the utility of
palladacycle COP-3E-Py for biological CO imaging applica-
tions, we sought to apply this reagent to observe endogenous
CO signaling events. To this end, we utilized COP-3E-Py to
monitor the postsynaptic release of CO in a fly brain model, as a
novel role for CO as a retrograde messenger in noncanonical
dopamine release was recently established.83 Thus, dopamine
release from dopaminergic neuron terminals on the mushroom
body is induced by CO generated in the mushroom body itself,
CONCLUDING REMARKS
■
In summary, we have presented a ligand-directed approach to
activity-based sensing of CO using organometallic reactivity. In-
depth evaluation of the first-generation palladacycle probe
COP-1 and systematic modulation of the ligand environment on
palladacycle reactivity under physiological conditions laid a
foundation for the development of next-generation molecular
imaging probes for biological CO. The data revealed that sp3-N-
palladacycles are superior to other common classes of
palladacycles investigated. Remarkably, sp3-N-palladacycles
undergo facile CO insertion and are also sufficiently stable
toward protodemetalation under physiological conditions as
shown by comparative reactivity studies with the competing
gasotransmitter H2S. Further functionalization of the core
BODIPY fluorophore with hydrophilic ester substituents gave
rise to the development of COP-3E-Py, an optimized scaffold
with improved properties for CO detection, including more
diffuse cellular staining and enhanced cellular retention. This
advanced CO probe enabled detection of biological CO in both
cell and fly brain models, as shown by monitoring CORM-3-
mediated CO release in live HEK293T cells and visualization of
endogenous CO release in fly brain preparations upon electrical
and NMDA stimulation. In addition to providing a unique
chemical tool for deciphering biological and biomedical
contributions of CO, this study provides a starting point for
G
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redissolved in minimal CH2Cl2, and hexanes were added. The resulting
precipitate was collected by vacuum filtration to provide COP-2 (39
mg, 0.037 mmol). This material was suspended in minimal CH2Cl2.
Pyridine (2.1 equiv relative to Pd) was added and the reaction mixture
was allowed to stir for 5 min. CH2Cl2 was removed in vacuo, and the
excess pyridine was removed by azeotroping with CH2Cl2 (×5). The
resulting orange solid was collected in minimal CH2Cl2 and hexanes
was added. The resulting precipitate was collected by vacuum filtration,
the broader use of organometallic principles in the design of
activity-based sensing probes for chemical biology applications.
EXPERIMENTAL SECTION
■
General Methods. Reactions utilizing air- or moisture-sensitive
reagents were performed in oven- or flame-dried glassware under an
atmosphere of dry N2. Reagents from commercial sources were used as
received without further purification. Reagents were purchased from
Sigma-Aldrich (St. Louis, MO). All solvents were of reagent grade.
COP-1,52 COP-1′,52 and CORM-384 were synthesized according to
literature procedures. Silica gel P60 (SiliCycle) was used for all column
chromatography purifications and SiliCycle 60 F254 silica gel
(precoated sheets, 0.25 mm thick) was used for thin layer
1
to yield COP-2-Py (15 mg, 22%) as an orange solid. H NMR (400
MHz, chloroform-d) 8.77 (d, J = 5.8 Hz, 2H), 7.81 (m, 1H), 7.37 (m,
2H), 7.18 (d, J = 7.8 Hz, 1H), 6.87 (d, J = 7.7 Hz, 1H), 6.14 (s, 1H),
5.93 (s, 2H), 4.38 (d, J = 14.2 Hz, 1H), 4.12 (d, J = 14.1 Hz, 1H), 2.74
(s, 3H), 2.49 (s, 6H), 1.41 (s, 3H), 1.38 (s, 3H). δ 155.3, 155.0, 153.4,
153.0, 152.4, 148.7, 143.2, 142.5, 142.2, 138.9, 133.5, 132.7, 131.4,
131.3, 125.6, 125.0, 124.3, 123.8, 121.1, 120.9, 48.6, 34.7, 25.3, 23.0,
20.7, 14.5. HRMS(ESI) calcd for C25H28BF2N4PdS [M − Cl − Py +
2MeCN]+: 571.1131; found: 571.1124.
Synthesis of 3. 1 (0.222 g, 0.596 mmol, 1.0 equiv), KI (0.198 g,
1.19 mmol, 2.0 equiv), and K2CO3 (0.165 mg, 1.19 mmol, 2.0 equiv)
were suspended in CH3CN (1.8 mL), and morpholine (260 μL, 2.98
mmol, 5.0 equiv) was added. The reaction mixture was heated via
microwave irradiation to 80 °C for 5 min, then to 100 °C for 60 min and
cooled to room temperature. The mixture was diluted with CH2Cl2 (60
mL), washed with water and brine, dried over Na2SO4, filtered, and
concentrated in vacuo. Purification with flash chromatography on silica
gel eluting with a 4:1 mixture of hexanes/CH2Cl2 and running a shallow
gradient from 0 to 5% EtOAc in this mixture yielded 3 (0.325 g, 0.768
mmol, 92%) as an orange solid. 1H NMR (600 MHz, CDCl3): δ 7.45
(d, J = 7.7 Hz, 2H), 7.22 (d, J = 7.8 Hz, 2H), 5.96 (s, 2H), 3.73 (t, J = 4.6
Hz, 4H), 3.59 (s, 2H), 2.54 (s, 6H), 2.46 (t, J = 4.5 Hz, 4H), 1.36 (s,
6H). 13C NMR (151 MHz, CDCl3): δ 155.3, 142.9, 141.6, 138.6, 133.8,
131.4, 129.8, 127.9, 121.1, 121.1, 66.9, 62.9, 53.5, 14.5, 14.3.
HRMS(ESI) calcd for C24H29BF2N3O+ [M + H]+: 424.2366; found:
424.2365.
Synthesis of COP-3-Py. Amine 3 (0.212 g, 0.501 mmol, 1.05
equiv), Pd(OAc)2 (0.107 g, 0.477 mmol, 1.00 equiv), and benzene (5
mL) were added to a 20 mL vial under an atmosphere of N2. The
mixture was heated to 50 °C in an oil bath under stirring for 16 h. The
solution was cooled to room temperature, concentrated in vacuo. The
mixture was then dissolved in CH2Cl2 and filtered over a fritted funnel
and concentrated in vacuo. A volume of 5 mL of a saturated LiCl
solution in acetone was added, and the reaction mixture was stirred for
16 h. The reaction mixture was filtered over a fritted funnel with
CH2Cl2 and methanol, concentrated in vacuo, and dissolved in CH2Cl2
(5 mL). Pyridine (81.0 μL, 1.00 mmol, 2.00 equiv) was added, and the
reaction mixture was stirred 16 h at room temperature. The reaction
mixture was concentrated in vacuo, suspended in CH2Cl2, and filtered
1
chromatography. H NMR and 13C NMR spectra were acquired at
25 °C with Bruker AVB-400, AVQ-400, or AV-300 at the College of
Chemistry NMR facility at UC Berkeley (UCB). Signals were internally
calibrated to solvent peaks. High resolution mass spectrometry (ESI-
MS) data were provided by the mass spectrometry department of UCB.
Synthesis of COP-1-Py. A round-bottom flask was charged with
COP-1 (78.3 mg, 0.15 mmol, 1 equiv). CH2Cl2 (15 mL) and pyridine
(14.5 μL, 0.18 mmol, 1.2 equiv) were added and the solution was stirred
for 4 h. Hexanes were added, and the precipitate was collected with a
Buchner funnel to yield COP-1-Py (80.9 mg, 0.15 mmol, 98%) as an
̈
orange solid. 1H NMR (400 MHz, CDCl3): δ 8.77 (d, J = 5.4 Hz, 2H),
7.78 (t, J = 7.9 Hz, 1H), 7.36−7.29 (m, 2H), 7.09 (d, J = 7.5 Hz, 1H),
6.88 (d, J = 7.3 Hz, 1H), 5.93 (s, 2H), 5.92 (s, 1H), 4.06 (s, 2H), 2.94 (s,
6H), 2.50 (s, 6H), 1.42 (s, 6H).
Synthesis of 1. 4-(Chloromethyl)benzoyl chloride (5.00 g, 26.5
mmol, 1.0 equiv) was dissolved in dry CH2Cl2 (350 mL) and the
solution was sparged with N2 for 40 min. 2,4-Dimethylpyrrole (6.80
mL, 66.1 mmol, 2.5 equiv) was added, and the mixture was stirred for 16
h at rt. Dry diisopropylethylamine (23.0 mL, 132 mmol, 5.0 equiv) was
added within 1 min, and stirring was continued at room temperature for
15 min. BF3·OEt2 was added dropwise to the flask, and the mixture was
allowed to stir at room temperature for 15 min. The flask was then
equipped with a water-cooled condenser and heated to 50 °C for 50
min. The mixture was then allowed to cool to room temperature and
concentrated in vacuo. The resulting residue was dissolved in CH2Cl2,
washed with water, dried over Na2SO4, filtered, and concentrated in
vacuo. Purification with flash chromatography on silica gel eluting with
CH2Cl2:hexanes (50:50 → 60:40 → 75:25) to provide 1 (2.74 g, 7.35
mmol, 28%) as an orange solid. 1H NMR (400 MHz, CDCl3) spectrum
showed accordance with the literature.52 1H NMR (400 MHz, CDCl3):
δ 7.52 (d, J = 8.1 Hz, 2H), 7.26 (d, J = 8.1 Hz, 1H), 5.99 (s, 2H), 4.66 (s,
2H), 2.56 (s, 6H), 1.38 (s, 6H).
Synthesis of 2. 1 (558 mg, 1.50 mmol, 1.0 equiv) and NaSMe (210
mg, 3.00 mmol, 2.0 equiv) were dissolved in EtOH under an N2
atmosphere. The mixture was stirred for 25 h at rt. It was diluted with
CH2Cl2, washed with H2O, dried over Na2SO4, filtered, and
concentrated in vacuo. Purification with flash column chromatography
with CH2Cl2:hexanes (60:40 → 100:0) yielded 2 (259 mg, 0.68 mmol,
45%) as a red solid. NMR: 1H NMR (600 MHz, CDCl3) δ 7.42 (d, J =
7.5 Hz, 2H), 7.22 (d, J = 7.6 Hz, 2H), 5.97 (s, 2H), 3.74 (s, 2H), 2.54 (s,
6H), 1.96 (s, 3H), 1.38 (s, 6H). 13C NMR (151 MHz, CDCl3) δ 155.54,
143.10, 141.59, 139.30, 133.73, 131.54, 129.81, 128.18, 121.32, 38.01,
14.47. HRMS(ESI) calcd for C21H23BF2N2S [M]: 384.1638; found:
384.1633.
Synthesis of COP-2-Py. A round-bottom flask equipped with a
water condenser was charged with 2 (50.0 mg, 0.130 mmol, 1.05 equiv)
and Pd(OAc)2 (27.8 mg, 0.124 mmol, 1 equiv) under a nitrogen
atmosphere. AcOH (2 mL) was added. The mixture was stirred in an oil
bath at 120 °C for 55 min and then allowed to cool to room
temperature. CH2Cl2 (10 mL) was added, and the solution was washed
with H2O (15 mL × 2). The organic layer was collected and
concentrated in vacuo. The resulting crude solid was dissolved in a
saturated solution of LiCl in acetone (3 mL) and was allowed to stir for
30 min at room temperature. Acetone was removed in vacuo, and the
residue was dissolved in CH2Cl2. The mixture was passed through a
plug of Celite and concentrated, yielding an orange solid. The solid was
over a Buchner funnel. Addition of hexanes led to the precipitation of a
̈
solid which was collected. This solid was again precipitated with
CH2Cl2 and hexanes. The generated solids were again collected. The
solids were dissolved in CH2Cl2 and filtered over a Buchner funnel. The
̈
mother liquor was concentrated in vacuo to provide COP3-Py as an
orange solid (0.043 g, 0.063 mmol, 13%).52 1H NMR (400 MHz,
CDCl3): δ 8.90−8.79 (m, 2H), 8.78−8.70 (m, 2H), 7.77 (dddt, J = 7.5,
5.7, 3.7, 1.8 Hz, 2H), 7.33 (tdd, J = 10.5, 5.7, 1.4 Hz, 4H), 7.13 (d, J =
7.6 Hz, 1H), 6.89 (dd, J = 7.5, 1.6 Hz, 1H), 5.92 (s, 2H), 4.43 (s, 2H),
4.10 (ddt, J = 9.8, 7.3, 3.2 Hz, 4H), 3.99−3.85 (m, 2H), 2.84 (dt, J =
13.6, 4.3 Hz, 2H), 2.49 (s, 6H), 1.39 (s, 6H). 13C NMR (101 MHz,
CD3OD) δ 155.1, 153.3, 153.1, 150.1, 146.5, 142.7, 142.4, 138.6, 138.5,
132.1, 131.3, 130.7, 125.4, 124.9, 124.2, 122.5, 120.9, 68.1, 62.4, 58.8,
14.5, 14.4, 14.3. HRMS(ESI) calcd for C34H38BF2N5OPd+ [M + H]+:
687.2167; found: 687.2167.
Synthesis of 4. A round-bottom flask was charged with
terephthalaldehyde (25.5 g, 190 mmol, 1.00 equiv), ethylene glycol
(11.2 mL, 200 mmol, 1.05 equiv), toluene (300 mL), and p-
toluenesulfonic acid (1.81 g, 9.5 mmol, 0.05 equiv). A Dean−Stark
trap was attached, and the reaction mixture was refluxed at 130 °C on an
oil bath for 4 h. The oil bath was removed and the reaction mixture was
concentrated under reduced pressure. The crude product was purified
via flash chromatography on silica gel eluting with 20% CH2Cl2, 80%
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hexanes → 20% CH2Cl2, 12% ethyl acetate, 68% hexanes → 20%
CH2Cl2, 16% ethyl acetate, 64% hexanes. The product mixture was
refined with a second round of flash chromatography on silica gel
eluting with 100% CH2Cl2 providing the desired product (8.20 g, 46.0
mmol, 24%) as a colorless liquid. Not all product containing fractions
were further purified from the unprotected and diprotected mixture. An
acquired 1H NMR (600 MHz, CDCl3) spectrum showed accordance to
the literature.85 1H NMR (400 MHz, CDCl3): δ 10.04 (s, 1H), 7.91 (d,
J = 8.2 Hz, 2H), 7.66 (d, J = 8.1 Hz, 2H), 5.88 (s, 1H), 4.24−3.95 (m,
4H).
Synthesis of 5. A two-necked round-bottom flask was charged with
dry CH2Cl2 (500 mL) which was sparged with N2 for 15 min. Aldehyde
4 (2.07 g, 11.6 mmol, 1.0 equiv), 2,4-dimethylpyrrole (2.51 mL, 24.4
mmol, 2.1 equiv), and 1 drop of trifluoroacetic acid were added. The
reaction mixture was stirred 16 h at room temperature. A slurry of 2,3-
dichloro-5,6-dicyano-1,4-benzoquinone (2.63 g, 11.6 mmol, 1.0 equiv)
in dry CH2Cl2 (150 mL) was added to the reaction mixture. Stirring was
then continued for 60 min. The reaction mixture was concentrated in
vacuo until approximately 100 mL of solvent was left. Dry toluene (300
mL) and dry diisopropylethylamine (10.1 mL, 58.0 mmol, 5.0 equiv)
were added. BF3·OEt2 (7.16 mL, 58.0 mmol, 5.0 equiv) was added, and
the mixture was heated up to 50 °C in an oil bath for 45 min. The oil
bath was removed, and the reaction mixture was concentrated in vacuo.
The crude solid was dissolved in CH2Cl2, washed three times with H2O,
dried over Na2SO4, filtered over Celite, and concentrated in vacuo.
Purification via flash chromatography on silica gel eluting with 60%
CH2Cl2, 40% hexanes → 100% CH2Cl2 provided BODIPY 5 (1.39 g,
3.51 mmol, 30%) as an orange solid. 1H NMR (400 MHz, CDCl3): δ
7.61 (d, J = 7.9 Hz, 2H), 7.31 (d, J = 7.8 Hz, 2H), 5.97 (s, 2H), 5.85 (s,
1H), 4.24−4.13 (m, 2H), 4.13−4.01 (m, 2H), 2.55 (s, 6H), 1.36 (s,
6H). 13C NMR (101 MHz, CDCl3): δ 155.5, 143.1, 141.1, 138.8, 135.9,
131.3, 128.0, 127.4, 121.2,103.3, 65.4, 14.5, 14.5. HRMS(ESI) calcd for
C22H23BF2N2NaO+ [M + Na]+ 419.1713; found: 419.1715.
[(dimethylamino)methyl]phenyl-C,N]dipalladium(II) (0.090 g,
0.163 mmol, 1.0 equiv). The mixture was dissolved in a 1:1 solution
of CHCl3/acetic acid (20 mL). The reaction mixture was heated to 50
°C in an oil bath for 16 h under stirring. The oil bath was removed, and
the precipitates were collected on a Buchner funnel. The solids were
̈
transferred to a 20 mL vial and dissolved in 10 mL of CH2Cl2. Pyridine
(52 μL, 0.65 mmol, 2.0 equiv) was added, the reaction mixture was
stirred for 16 h at room temperature, filtered over Celite, and
concentrated to 5 mL, and hexanes (5 mL) were added, which led to
immediate precipitation. The precipitate was collected with a Buchner
̈
funnel to yield COP-4-Py (0.131 g, 0.223 mmol, 69%) as an orange
1
solid. H NMR (500 MHz, CDCl3): δ 10.20 (s, 1H), 8.78−8.70 (m,
2H), 7.91 (s, 1H), 7.87 (tt, J = 7.7, 1.7, 1H), 7.49−7.41 (m, 2H), 7.32
(d, J = 7.6, 1H), 6.98 (dd, J = 7.6, 1.6, 1H), 6.15 (d, J = 1.5, 1H), 5.94 (s,
2H), 2.49 (s, 6H), 1.43 (s, 6H). 13C NMR (126 MHz, CDCl3): δ 156.3,
155.4, 153.6, 152.5, 142.8, 142.4, 141.2, 139.0, 135.1, 130.9, 130.1,
126.3, 126.2, 124.7, 121.1, 14.5, 14.5. HRMS(ESI) calcd for
C25H25BClF2N4OPd+ [M + H]+ 587.0807; found: 587.0807.
Synthesis of 8. 4-Bromobenzoyl chloride (6.14 g, 26.3 mmol, 1
equiv) was dissolved in dry CH2Cl2 (65 mL) in an N2 atmosphere. 2,4-
Dimethylpyrrole (5.04 g, 53.0 mmol, 2 equiv) was added, and the
reaction mixture was stirred for 14 h at room temperature.
Triethylamine (11.0 mL, 78.3 mmol, 3 equiv) was added dropwise
and stirred for 15 min. BF3·OEt2 (17.0 mL, 137 mmol, 5.3 equiv) was
added dropwise, and the reaction mixture was stirred for 20 h. CH2Cl2
was added, and the organic layer was washed with water, dried over
Na2SO4, and concentrated in vacuo. The product was purified by flash
chromatography on silica gel, eluting with 50% CH2Cl2 in hexanes. 1H
NMR (400 MHz, CDCl3): δ 7.63 (d, J = 7.9 Hz, 2H), 7.14 (d, J = 8.0
Hz, 2H), 5.99 (s, 2H), 2.54 (s, 6H), 1.40 (s, 6H). 13C NMR (101 MHz,
CDCl3) δ 155.91, 142.95, 140.07, 133.94, 132.49, 131.20, 129.86,
123.31, 121.53, 14.69. HRMS(ESI) calcd for C19H18BBrF2N2 [M]:
402.0709; found: 402.0707.
Synthesis of 6. A 100 mL round-bottom flask equipped with a
water-cooled condenser was charged with BODIPY 5 (0.793 g, 2.00
mmol, 1.0 equiv), acetone (20 mL), and a 10% HCl (aq) solution (1.0
mL). The reaction mixture was put under N2 and refluxed on an oil bath
for 2 h. The oil bath was removed, and the reaction mixture was
concentrated in vacuo. The resulting residue was dissolved in CH2Cl2,
and the organic phase was washed three times with H2O, dried over
Na2SO4, filtered over Celite, and concentrated in vacuo. Purification via
flash chromatography on silica gel eluting with 60% CH2Cl2, 40%
hexanes → 100% CH2Cl2 provided aldehyde 6 (0.554 g, 1.57 mmol,
78%) as an orange solid.85 1H NMR (400 MHz, CDCl3): δ 10.10 (s,
1H), 8.02 (d, J = 7.8 Hz, 2H), 7.49 (d, J = 7.8 Hz, 2H), 5.99 (s, 2H),
2.55 (s, 6H), 1.34 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 191.5, 156.1,
142.7, 141.3, 139.6, 136.6, 130.7, 130.3, 129.0, 121.6, 14.6, 14.5.
HRMS(ESI) calcd for C20H20BF2N2O+ [M + H+] 353.1631; found:
353.1629.
Synthesis of 7. A round-bottom flask equipped with a water cooled
condenser was charged with hydroxylamine hydrochloride (0.160 g,
2.30 mmol, 3.0 equiv), potassium acetate (0.340 g, 3.45 mmol, 4.5
equiv), ethanol (10 mL), H2O (5 mL), and aldehyde 6 (0.280 g, 0.795
mmol, 1.0 equiv). The reaction mixture was put under N2 and heated up
to 70 °C in an oil bath for 3 h. The oil bath was removed and the
reaction mixture was concentrated in vacuo. The resulting residue was
dissolved in CH2Cl2 and concentrated in vacuo. CH2Cl2 was added and
the organic phase was washed one time with H2O, dried over Na2SO4,
filtered over Celite, and concentrated in vacuo. Purification via flash
chromatography on silica gel eluting with 100% CH2Cl2 → 5% ethyl
acetate, 95% CH2Cl2 provided aldoxime 7 (0.266 g, 0.724 mmol, 91%)
as an orange solid. 1H NMR (400 MHz, CDCl3): δ 8.21 (s, 1H), 7.72
(d, J = 8.2 Hz, 2H), 7.32 (d, J = 8.1 Hz, 2H), 5.99 (s, 2H), 2.56 (s, 6H),
1.40 (s, 6H). 13C NMR (101 MHz, acetone-d6): δ 156.2, 148.8, 143.8,
142.4, 136.5, 135.0, 131.9, 129.3, 128.2, 122.1, 122.1, 14.7, 14.6, 14.6,
14.6. HRMS(ESI) calcd for C20H21BF2N3O+ [M + H]+ 368.1740;
found: 368.1739.
Synthesis of 9. Compound 8 (770 mg, 1.91 mmol, 1 equiv) was
dissolved in toluene (25 mL), and 2-(tributylstannyl)pyridine (878 mg,
2.39 mmol, 1.25 equiv) was added under an N2 atmosphere.
Pd(Ph3P)2Cl2 (67.0 mg, 0.10 mmol, 0.05 equiv) was added. The
reaction mixture was heated to 110 °C for 48 h and cooled to rt, CH2Cl2
was added, and the mixture was filtered through Celite. It was
concentrated and purified with flash column chromatography in
CH2Cl2 → CH2Cl2 + 4% EtOAc to yield 9 as an orange solid (496 mg,
1.23 mmol, 65%). 1H NMR (400 MHz, CDCl3): δ 8.75 (d, J = 4.6 Hz,
1H), 8.18 (d, J = 8.1 Hz, 2H), 7.83 (t, J = 9.1 Hz, 2H), 7.41 (d, J = 8.1
Hz, 2H), 7.30 (t, J = 5.3 Hz, 1H), 6.01 (s, 2H), 2.59 (s, 6H), 1.46 (s,
6H). 13C NMR (101 MHz, CDCl3): δ 156.28, 155.59, 149.86, 143.20,
141.35, 139.98, 137.05, 135.67, 131.36, 128.55, 127.57, 122.72, 121.33,
+
120.65, 14.68. HRMS(ESI) calcd for C24H23BF2N3 [M + H]+:
402.1948; found: 402.1941.
Synthesis of COP-5-Py. A round-bottom flask was charged with 9
(124 mg, 0.31 mmol, 1 equiv) and Pd(OAc)2 (69.9 mg, 0.31 mmol, 1
equiv). The flask was flushed with nitrogen to create an N2 atmosphere
and protected from light. Benzene (20 mL) was added, and the reaction
mixture was stirred for 14 h at 50 °C. CH2Cl2 was added, and the
mixture was filtered over Celite and concentrated in vacuo. A volume of
20 mL of a saturated solution of LiCl in acetone was added and stirred at
room temperature for 4 h. It was concentrated in vacuo, dissolved in
CH2Cl2, and filtered over Celite to remove excess LiCl. It was dissolved
in CH2Cl2(10 mL) and pyridine was added (25 uL, 1.2 equiv). COP-5-
Py (142 mg, 0.23 mmol, 74%) was obtained by precipitation in hexanes
as an orange solid. 1H NMR (500 MHz, CDCl3): δ 9.52 (d, J = 5.2 Hz,
1H), 8.85 (d, J = 5.0 Hz, 2H), 7.91−7.84 (m, 2H), 7.74 (d, J = 8.0 Hz,
1H), 7.60 (d, J = 7.8 Hz, 1H), 7.47−7.42 (m, 2H), 7.23 (t, J = 6.6 Hz,
1H), 7.02 (d, J = 8.9 Hz, 1H), 6.14 (s, 1H), 5.95 (s, 2H), 2.51 (s, 7H),
1.48 (s, 6H). 13C NMR (126 MHz, CDCl3): δ 165.01, 155.48, 155.12,
153.48, 152.82, 152.55, 146.55, 143.08, 141.92, 139.20, 138.88, 138.75,
136.06, 131.45, 131.19, 126.18, 125.13, 124.56, 123.75, 122.78, 121.25,
118.88, 14.76. HRMS(ESI) calcd for C31H28BF2N5Pd [M + MeCH/−
Cl]: 625.1441; found: 625.1435.
Synthesis of COP-4-Py. A two-necked round-bottom flask
equipped with a water-cooled condenser was charged with aldoxime
7 (0.119 g, 0.325 mmol, 1.0 equiv) and di-μ-chlorobis[2-
Synthesis of 10. A round-bottom flask was charged with methyl 5-
(benzyloxycarbonyl)-2,4-dimethylpyrrole-propionate (10.0 g, 31.8
I
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mmol, 1.0 equiv), palladium on carbon (0.20 g), and acetone (320 mL).
A hydrogen atmosphere was established by flushing the flask with
hydrogen gas and subsequent attachment of a hydrogen gas containing
balloon. Stirring was continued for 12 h at room temperature.
Palladium on carbon was filtered off over Celite. The reaction mixture
was concentrated in vacuo, trifluoroacetic acid (32 mL) was added, and
the reaction mixture was heated to 40 °C for 15 min. The oil bath was
removed and CHCl3 was added. The diluted reaction mixture was
washed with H2O and a saturated K2CO3 solution in H2O. The aqueous
phases were extracted with CHCl3, and the pooled organic phases were
dried over MgSO4 and concentrated in vacuo. Purification with flash
chromatography on silica gel with CH2Cl2 gave 10 (2.8 g, 15.4 mmol,
48%) as a yellow oil. 1H NMR (400 MHz, CDCl3): δ 8.16 (s, 1H), 6.49
(s, 1H), 3.81 (s, 3H), 2.90 (t, 2H), 2.63 (t, 2H), 2.31 (s, 3H), 2.21 (s,
3H).
Synthesis of 11. A round-bottom flask was charged with 4-
(chloromethyl)benzoyl chloride (1.46 g, 7.7 mmol, 1 equiv). 10 (2.8 g,
15.4 mmol, 2 equiv) was dissolved in dry CH2Cl2 (30 mL) and added
under an N2 atmosphere. The reaction mixture was stirred for 14 h at
room temperature. DIPEA (6.7 mL, 38.5 mmol, 5 equiv) was added
dropwise and stirred for 20 min. BF3·OEt2 (4.8 mL, 38.5 mmol, 5
equiv) was added dropwise, and the reaction mixture was stirred for 3 h.
CH2Cl2 was added, and the organic layer was washed with water, dried
over Na2SO4, and concentrated in vacuo. The product was purified by
flash chromatography on silica gel, eluting with 20% EtOAc, 80%
hexanes → 30% EtOAc, 70% hexanes. 11 (950 mg, 1.7 mmol, 22%) was
obtained as a red solid. 1H NMR (400 MHz, CDCl3): δ 7.51 (d, 2H),
7.26 (d, 2H), 4.66 (s, 2H), 3.64 (s, 6H), 2.62 (t, 4H), 2.53 (s, 6H), 2.34
(t, 4H), 1.29 (s, 6H). 13C NMR (101 MHz, CDCl3): δ 173.1, 154.4,
140.2, 139.4, 138.7, 135.6, 130.9, 129.4, 128.6, 51.8, 45.7, 34.3, 19.4,
12.7, 12.1. HRMS(ESI) calculated for [C28H32BClF2N2NaO4]+:
567.2004, found: 567.2005.
Synthesis of 12. A round-bottom flask was charged with 11 (950
mg, 1.7 mmol, 1 equiv), K2CO3 (470 mg, 3.4 mmol, 2 equiv) and KI
(564 mg, 3.4 mmol, 2 equiv). CH3CN (20 mL) and morpholine (741
μL, 8.5 mmol, 5 equiv) were added to the flask and the reaction mixture
was stirred at 60 °C for 3 h. CH2Cl2 was added and the organic layer was
extracted with water, dried over Na2SO4, and concentrated in vacuo.
The product was purified by flash chromatography on silica gel, eluting
with a 100% hexanes →100% EtOAc gradient. 12 (968 mg, 1.6 mmol,
94%) was obtained as a red solid. 1H NMR (400 MHz, CDCl3): δ 7.45
(d, 2H), 7.19 (d, 2H), 3.72 (t, 4H), 3.61 (s, 6H), 3.60 (s, 2H), 2.60 (t,
4H), 2.51 (s, 6H), 2.46 (s, 4H), 2.32 (t, 4H), 1.27 (s, 6H). 13C NMR
(101 MHz, CDCl3): δ 173.07, 154.05, 140.80, 139.36, 134.45, 130.93,
130.04, 129.18, 128.40, 128.13, 66.86, 63.01, 53.49, 51.70, 34.22, 29.72,
19.34, 12.62, 11.90. HRMS(ESI) calculated for [C32H41BF2N3O5]+:
596.3102, found: 596.3104.
Synthesis of COP-3E-Py. A round-bottom flask was charged with
12 (968 mg, 1.6 mmol, 1 equiv) and Pd(OAc)2 (347 mg, 1.5 mmol,
0.95 equiv). The flask was flushed with nitrogen to create an N2
atmosphere and protected from light. Benzene (40 mL) was added, and
the reaction mixture was stirred for 14 h at 50 °C. CH2Cl2 was added,
and it was filtered over Celite and concentrated in vacuo. A volume of
60 mL of a saturated solution of LiCl in acetone was added and stirred at
room temperature for 4 h. It was concentrated in vacuo, dissolved in
CH2Cl2, and filtered over Celite to remove excess LiCl. Hexanes were
Synthesis of COP-3E-Py′. A round-bottom flask was charged with
COP-3E-Py (60 mg, 0.073 mmol, 1 equiv). CH2Cl2 (20 mL) and water
(1 mL) were added and the flask was flushed with CO. A CO-filled
balloon was attached and the reaction mixture was stirred for 14 h at 31
°C while maintaining an atmosphere of CO. The reaction mixture was
cooled to room temperature, transferred to a separatory funnel, and
diluted with DCM (50 mL). The organic layer was washed with water,
dried over sodium sulfate, filtered, and concentrated in vacuo. The crude
product was purified by flash chromatography by elution with a EtOAc
→ 40% MeOH, 60% EtOAc gradient. COP-3E-Py′ (43 mg, 6.7 mmol,
92%) was isolated as a red solid. 1H NMR (400 MHz, CDCl3): δ 8.12
(d, 2H), 7.37 (d, 2H), 3.97 (s, 2H), 3.84 (s, 4H), 3.64 (s, 6H), 2.79 (s,
4H), 2.60 (t, 4H), 2.53 (s, 6H), 2.34 (t, 4H), 1.26 (s, 6H). 13C NMR
(101 MHz, CDCl3): δ 176.3, 173.2, 154.9, 138.9, 138.6, 137.1, 133.7,
132.4, 130.7, 129.7, 65.5, 61.9, 51.9, 51.6, 34.3, 29.8, 20.9, 19.4, 12.8,
12.4. HRMS(ESI) calculated for [C33H41BF2N3O7]+: 640.3000, found:
640.2995.
X-ray Crystallography. Single crystals were coated with Paratone-
N hydrocarbon oil and mounted on Kaptan loops. Temperature was
maintained at 100 K with an Oxford Cryostream 700 instrument during
data collection at the University of California, Berkeley, College of
Chemistry, X-ray Crystallography Facility. Samples were irradiated with
Mo Kα radiation with λ = 0.71073 Å using a Bruker APEX II QUAZAR
diffractometer equipped with a Microfocus Sealed Source (Incoatec
IμS) and APEX-II detector. The Bruker APEX2 ver. 2009.1 software
package was used to integrate raw data which were corrected for
Lorentz and polarization effects.86,87 A semiempirical absorption
correction (SADABS) was applied.44 Space groups were identified
based on systematic absences, E-statistics, and successive refinement of
the structures. The structures were solved using direct methods or the
Patterson method and refined by least-squares refinement on F2 and
standard difference Fourier techniques using SHELXL.88−90 Thermal
parameters for all non-hydrogen atoms were refined anisotropically,
and hydrogen atoms were included at ideal positions and refined
isotropically. The ammonium hydrogen in structure COP-1′ was
located in the electron density map.
Fluorescence Spectroscopy. Fluorescence spectra were recorded
on a Photon Technology International Quanta Master 4 L-format
scanning spectrofluorometer (Lawrenceville, NJ) equipped with an
LPS 220B 75 W xenon lamp and power supply, A 1010B lamp housing
with an integrated igniter, switchable 814 photon counting/analog
photomultiplier detection unit, and MD5020 motor driver. Samples for
emission measurements were contained in a quartz cuvette with a path
length of 1 cm and 1.5 mL cell volume (Starna, Atascadero, CA). CO-
probes were dissolved in DMSO (2 mM) and diluted to a final volume
of 1 μM in PBS or HEPES (Ca, Mg) buffer. The cuvette was placed in a
water bath at 37 °C, and after temperature equilibration a t = 0 a
spectrum was acquired. Subsequently 5 μL of a 10 mM solution of
CORM 3 (final concentration: 50 μM) in DMSO was added and the
cuvette was again placed in a water bath. Emission spectra were
recorded by quickly removing the cuvette from the water bath,
recording the spectrum and returning the cuvette to the bath. Spectra
were taken at t = 0, 5, 15, 30, 45, and 60 min. COP-3E-Py was excited at
515 nm and emission spectra were recorded from 520 to 620 nm in 1
nm intervals with an integration of 0.1. For selectivity assay the CO-
probes were treated with different compounds analogously to the
treatment with CORM-3: At 37 °C the tested species were added to a
final concentration of 50 μM to a 1 μM aqueous COP solution (PBS
(Ca, Mg) buffer). Emission spectra were recorded by quickly removing
the cuvette from the water bath, recording the spectrum, and returning
the cuvette to the bath. Spectra were taken at t = 0, 5, 15, 30, 45, and 60
min. For the selectivity assay, 10 mM solutions of H2O2, TBHP,
NaOCl, KO2, ONOO−, and H2S were prepared and 5 μL was added to
1 mL of a 1 μM solution of COP-3E-Py in PBS.
added, and a precipitate was collected with a Buchner funnel. The
̈
precipitate was dissolved in CH2Cl2. Pyridine (13 μL, 0.15 mmol, 2.2
equiv) was added, and the solution was stirred for 4 h. Hexanes were
added, and the precipitate was collected with a Buchner funnel to yield
̈
1
COP-3E-Py (90 mg, 0.11 mmol, 70%) as a red solid. H NMR (400
MHz, CDCl3): δ 8.81 (d, 1H), 8.72 (d, 1H), 7.75 (m, 1H), 7.31 (m,
2H), 7.11 (d, 2H), 6.86 (d, 2H), 5.84 (s, 1H), 4.43 (s, 2H) 4.08 (t, 4H),
3.91 (m, 2H) 3.63 (s, 6H), 2.84 (m, 2H), 2.59 (t, 4H), 2.46 (s, 6H),
2.31 (t, 4H), 1.30 (s, 6H). 13C NMR (101 MHz, CDCl3): δ 173.0,
153.7, 153.4, 153.3, 150.4, 146.6, 141.7, 139.2, 138.7, 138.6, 132.6,
131.0, 130.9, 129.0, 125.5, 125.1, 124.4, 122.7, 68.3, 62.6, 59.0, 51.8,
34.3, 19.4, 12.6, 12.1, 11.9. HRMS(ESI) calculated for
[C36H45BF2N5O5Pd]: 782.2517, found: 782.2513.
Cell Culture and Confocal Microscopy in Live Cells. Cells were
grown in the UC Berkeley Tissue Culturing Facility. HEK293T cells
were cultured in Dubelcco’s Eagle Medium (DMEM, Invitrogen)
supplemented with 10% fetal bovine serum (FBS, Hyclone) and
incubated at 37 °C in 5% CO2. One day before imaging, cells were
passaged and plated in phenol red-free medium on 8 well chamber
J
https://dx.doi.org/10.1021/jacs.0c06405
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
pubs.acs.org/JACS
Article
slides (Corning, Corning, NY) and grown to 70−80% (HEK293T)
confluency. 100 μL of 3 μM respective CO-Probe in DPBS was
prepared from a 0.4 mM COP in DMSO stock solution and added to
chambers filled with 200 μL of medium. For CO-response studies in
HEK293T cells, 100 μL medium was removed after 60 min of COP
incubation and 1.5 μL of 10 mM CORM-3 in DMSO was added (1.5
μL of DMSO for the controls), mixed and readded to the respective
chamber. Imaging was performed approximately 120 min after COP
addition. Confocal fluorescence microscopy was performed with a Zeiss
laser scanning microscope 710 equipped with a 40× water-immersion
and 63× oil-immersion objective lens, with Zen 2009 software (Carl
Zeiss). COP-1, and COP-1-Py to COP-5-Py were excited at 488 nm,
and COP-3E-Py was excited at 488 or 514 nm. The emission was
collected with a META detector. Hoechst 33342 and ER-Tracker Red
E34250 were used at 1 μM concentration and excited at 405 nm. Image
analysis was performed using FIJI (National Institute of Health).
Colocalization coefficients where calculated with the plugin JACoP.
Detection of CO in Fly Brain Models. Brains were prepared for
imaging as previously described.91 Briefly, brains were dissected in ice
cold 0 mM Ca2+ HL3 medium (in mM as follows: 70 NaCl, 115 sucrose,
5 KCl, 20 MgCl2, 10 NaHCO3, 5 Trehalose, 5 HEPES, pH 7.3, 359
mOsm) and placed in a recording chamber filled with normal, room
temperature HL3 medium (the same recipe as above, containing 1.8
mM CaCl2). COP-3E-Py was added to the buffer from a DMSO stock
solution and used at 3 μM final concentration. The antennal lobe (AL)
was stimulated (30 pulses, 100 Hz, 1.0 ms pulse duration) using glass
microelectrodes. For NMDA stimulation, 200 μM NMDA, diluted in
HL3 containing 4 mM Mg2+, was bath-applied to the recording
chamber. Fluorescent imaging was done with a confocal microscope
(A1R, Nikon) with a 20×x water-immersion lense (numerical aperture
0.5; Nikon). F0 was obtained by averaging the five sequential frames
before stimulus onset.
Jonah W. Jurss − Department of Chemistry, University of
California, Berkeley, California 94720, United States;
orcid.org/0000-0002-2780-3415
Ryan R. Walvoord − Department of Chemistry, University of
California, Berkeley, California 94720, United States
Kevin J. Bruemmer − Department of Chemistry, University of
California, Berkeley, California 94720, United States
Samir P. Rezgui − Department of Chemistry and Biochemistry,
University of Denver, Denver, Colorado 80210, United States
Thomas F. Brewer − Department of Chemistry, University of
California, Berkeley, California 94720, United States
Minoru Saitoe − Tokyo Metropolitan Institute of Medical Science,
Tokyo 1568506, Japan
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.0c06405
Author Contributions
#J.M. and B.W.M. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the NIH (ES 28096 and ES 4705 to C.J.C.) for
funding. J.M. was supported by a fellowship of the German
National Academic Foundation (Studienstiftung). D.H. was
supported by a fellowship of the Konrad−Adenauer Stiftung.
K.J.B. was supported by an NSF graduate fellowship. B.W.M.
was supported by an American Heart Association Fellowship.
B.W.M. and S.P.R. thank the NSF (CHE-1900482) for funding.
We also thank JSPS KAKENHI for funding (17K07122 to K.U.,
19H01013 to M.S., and Takeda Science Foundation to K.U.).
We thank Ann Fischer (UC Berkeley Tissue Culture Facility)
for expert technical assistance. We thank Allegra Aron, Lakshmi
Krishnamoorthy, Joseph A. Cotruvo, Jr., Zeming Wang, and
Sumin Lee for insightful discussion and experimental assistance.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.0c06405.
Experimental details, including characterization of com-
pounds (PDF)
REFERENCES
■
Crystallographic data for COP-1 (CIF)
Crystallographic data for COP-1-PY (CIF)
Crystallographic data for COP-1′ (CIF)
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AUTHOR INFORMATION
■
Corresponding Authors
Christopher J. Chang − Department of Chemistry, Molecular
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