Transition-Metal-Free CO-Releasing BODIPY Derivatives Activatable by Visible to NIR Light as Promising Bioactive Molecules

Article abstract of DOI:10.1021/jacs.5b10800

Carbon monoxide-releasing molecules (CORMs) are chemical agents used to administer CO as an endogenous, biologically active molecule. A precise spatial and temporal control over the CO release is the major requirement for their applications. Here, we report the synthesis and properties of a new generation of transition-metal-free carbon monoxide-releasing molecules based on BODIPY chromophores (COR-BDPs) activatable by visible-to-NIR (up to 730 nm) light. We demonstrate their performance for both in vitro and in vivo experimental settings, and we propose the mechanism of the CO release based on steady-state and transient spectroscopy experiments and quantum chemical calculations.

Full text of DOI:10.1021/jacs.5b10800

Article
pubs.acs.org/JACS
Transition-Metal-Free CO-Releasing BODIPY Derivatives Activatable
by Visible to NIR Light as Promising Bioactive Molecules
‡
†
‡,§
Eduardo Palao,†,∥ Tomas Slanina,†,∥ Lucie Muchova, Tomas Solomek, Libor Vítek, and Petr Klan*,†
̌
́
̌
́
́
̌
́
†Department of Chemistry and RECETOX, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic
‡Institute of Medical Biochemistry and Laboratory Diagnostics, 1st Faculty of Medicine, and §4th Department of Internal Medicine,
1st Faculty of Medicine, Charles University in Prague, Na Bojisti 3, 121 08 Praha 2, Czech Republic
̌
S
* Supporting Information
ABSTRACT: Carbon monoxide-releasing molecules (CORMs) are
chemical agents used to administer CO as an endogenous, biologically
active molecule. A precise spatial and temporal control over the CO
release is the major requirement for their applications. Here, we report
the synthesis and properties of a new generation of transition-metal-
free carbon monoxide-releasing molecules based on BODIPY
chromophores (COR-BDPs) activatable by visible-to-NIR (up to
730 nm) light. We demonstrate their performance for both in vitro
and in vivo experimental settings, and we propose the mechanism of
the CO release based on steady-state and transient spectroscopy
experiments and quantum chemical calculations.
light. CO release upon irradiation with 980 nm from a Mn-
based nanocarrier via upconversion has also been recently
shown.24 Nevertheless, the design of new photoCORMs, which
are chemically stable, nontoxic, and release CO efficiently upon
visible light exposure, is still a major challenge.
INTRODUCTION
■
Carbon monoxide is known for its lethal effects in mammals
because it binds to hemoglobin more strongly than oxygen.1
However, it is now evident that CO is an important cell-
signaling molecule with substantial therapeutic potential
protecting from vascular, inflammatory, or even cancer
diseases.2−6
PhotoCORMs based on organic compounds offer a great
promise to address this challenge. Several examples of organic
molecules, such as cyclopropenones,25−28 1,3-cyclobutane-
diones,29 or 1,2-dioxolane-3,5-diones,30 liberate CO upon
biologically adverse UV or near-UV (<420 nm) light. To our
knowledge, only two purely organic photoCORMs can be
activated by visible light. Liao and co-workers have developed a
system based on a cyclic aromatic α-diketone chromophore
that liberates CO upon irradiation with light below ∼500 nm.31
The molar absorption coefficients of such chromophores are
however relatively low (only ∼1000 dm3 mol−1 cm−1 at 458
nm32). Some of us have introduced a fluorescein analogue, 6-
hydroxy-3-oxo-3H-xanthene-9-carboxylic acid (1; Scheme 1a),
as a photoCORM that liberates CO along with the formation of
3,6-dihydroxy-9H-xanthen-9-one (2) upon irradiation below
520 nm in aqueous solutions at pH ≈ 7.33 Although the CO-
release quantum efficiency is low (Φ ≈ 7 × 10−4), the uncaging
cross-section18 evaluated as the product of Φ and the decadic
molar absorption coefficient at the wavelength of irradiation
(λirr), Φε, is large (on the order of 1−10). Unfortunately, the
synthesis of this compound is very difficult,33 and we could not
find an alternative pathway to produce it in sufficient quantities
for further mechanistic studies that would allow us to modify
Carbon monoxide-releasing molecules (CORMs) have been
developed to deliver CO into the cell.3,7−11 The most
important criteria for designing CORMs are chemical stability
when stored as well as aqueous solubility, temporal control over
the CO release, and low toxicity in their in vivo applications.
Unlike other signaling molecules, such as NO or H2S, CO is a
stable and inert molecule that reacts only with transition
metals.1 Thus, with a few exceptions, such as boranocarbon-
ates12 or an interesting “click and release” prodrug system,13
metal−carbonyl complexes are used as CORMs. The most
common activation of CO release from these compounds in
aqueous media is a solvent-mediated ligand exchange that starts
immediately upon CORM administration,3,4,7−10 but CO can
also be liberated from CORMs via enzyme-triggered
processes.14−16
Light-triggered CO liberation from a photochemically active
CORM (photoCORM17) is an alternative activation strategy
that allows for a precise spatial and temporal control over the
CO release.18 Transition metal-based complexes offer a
relatively good flexibility and diversity in terms of their
chemical composition and release efficiencies;7,9,19 however,
only a few such photoCORMs, for example, polypyridyl
metallodendrimers,20 Mn-complexes of azaheteroaromatic
ligands,21 or azopyridine,22,23 absorb biologically benign visible
Received: June 26, 2015
© XXXX American Chemical Society
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1 should possess frontier molecular orbitals (MOs) with similar
nodal properties at the meso position.34
Scheme 1. (a) 6-Hydroxy-3-oxo-3H-xanthene-9-carboxylic
Acid (1) as a PhotoCORM; and (b) Comparison of the
Frontier MOs (Left, HOMO; Right, LUMO) of 6-Hydroxy-
Using Huckel molecular orbital (HMO) calculations, we
̈
3-oxo-3H-xanthene (Top) and BODIPY (Bottom; Figure 1)
screened the frontier molecular orbitals (MOs) of several well-
known chromophores with strong absorption in the visible
region, and we identified that the boron-dipyrromethene
(BODIPY) molecule has a similar antisymmetric highest
occupied MO (HOMO) as compared to that of 1 (Scheme
1b).35 HMO predicts an increase in electronic density on the
meso carbon atom upon the HOMO−LUMO excitation for
both systems. Therefore, we deduced that meso-carboxy
BODIPY derivatives (3, Figure 1) are promising photoCORM
candidates. Many BODIPY derivatives have already received
considerable attention in the past decade due to their
distinctive and easily tunable optical properties.36−38
a
Chromophores Using the Huckel MO Theory
̈
We first synthesized the model COR-BDP 3a to evaluate its
properties and ability to release CO. The preparation of a meso-
carboxy BODIPY derivative analogous to 3a has recently been
reported.39 The benzyl ester 4a (Figure 1) was prepared as a
synthetic precursor from benzyl chlorooxalate and 2-methyl-
pyrrole.40 This compound was then hydrogenated on Pd/C to
obtain 3a in 82% yield. Compound 3a is soluble in aqueous
solutions (>5 mM), and the pKa of its carboxylic group in an
aqueous solution, determined by potentiometric titration, was
found to be (3.0 0.2) (Figure S30). This value corresponds
well with the pKa’s of coumarine-3-carboxylic acid41 or
xanthene-9-carboxylic acid (1).33 Compound 3a therefore
exists as its conjugate base at physiological pH. Another
protonation step occurs at a lower pH with pKa′ = 1.69 0.02
that affects the absorption properties of the chromophore
(Figure S31). It is possible that a BF2 moiety is displaced at
such a low pH as observed in the case of other BODIPY
derivatives.42 The compound quickly decomposes at pH < 0.5.
It is stable in the crystalline state (>3 weeks at room
temperature) as well as in aqueous solutions (pH = 7.4, >2
weeks) in the dark. The absorption spectrum of an aqueous
solution of 3a (phosphate buffered saline, PBS; pH = 7.4; the
ionic strength, I = 0.15 mol dm−3) shows a major band with
λmax = 502 nm (Figure 2). The compound exhibits a bright
fluorescence (Figure 2) with a quantum yield Φf of 67%. The
corresponding excitation spectrum matches that of the
absorption (Figure S17).
a
The meso position in both chromophores is indicated by an arrow.
The BF2 group of BODIPY (in gray) was not explicitly considered in
the Huckel calculation.
̈
the chromophore properties and shift its absorption to longer
wavelengths. We therefore decided to find an alternative
chromophore that can be easily synthesized and exhibits
photoreactivity similar to that of 1.
Herein, we report the synthesis and properties of the meso-
carboxy BODIPY (COR-BDP) derivatives 3a,b (Figure 1) and
Figure 1. Structures of the BODIPY-based photoCORMs (COR-
BDPs) 3a−c and the synthetic precursors 4a,b.
demonstrate their ability to efficiently release CO. We tested
their bioavailability and potential toxicity in both in vitro and in
vivo experimental settings, and we propose the mechanism of
the CO release on the basis of steady-state and transient
spectroscopy experiments and DFT calculations.
RESULTS AND DISCUSSION
■
In our previous work, we hypothesized that an α-lactone is
formed in the meso position prior to the CO-release step upon
irradiation of 1.33 Such an intermediate should be formed by a
formal addition of the carboxylate oxygen to the meso position
in an excited singlet or triplet state, possibly involving a
photoinduced one-electron reduction step. Therefore, an
alternative chromophore that preserves the photoreactivity of
Figure 2. Absorption (solid lines) and normalized emission (dashed
lines) spectra of 3a (red) and 3b (blue) (c = 1 × 10−5 mol dm−3;
phosphate buffered saline pH = 7.4; I = 0.15 mol dm−3).
B
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Compound 3a underwent a complete decomposition upon
irradiation at ∼500 nm (Figure 3; top) with quantum yields of
Our GC/RGA analysis of CO released into the headspace of
septum-sealed vials containing an irradiated sample evidenced
that the decomposition of 3a was accompanied by the
formation of CO. Its total maximum chemical yield reached
87% upon complete conversion in the absence of oxygen
(Table 1); that is, the quantum yield of decomposition
corresponds reasonably to that of CO release. The maximum
yield decreased to ∼45% in aerated solutions, which was
probably caused by bleaching of the starting material by the
generated singlet oxygen (see later in the text). Only UV-light
absorbing photoproducts were formed in the solution upon
exhaustive irradiation. This is a great advantage as the
photoproducts do not interfere with the photochemical process
as internal filters.18 HRMS analysis showed that the major
products were 2-methylpyrrol and 2H-pyrrole-4-carbaldehyde
(Figure S16) in accord with the reports that describe
photochemical degradation of other BODIPY derivatives.44
For comparison, the ester 4a was irradiated under the same
condition as 3a. This compound was photochemically stable,
and no CO release was detected. The carboxylic functional
group is therefore essential for the reaction.
Encouraged with the results, we decided to design COR-
BDP with a π-extended chromophore to shift the absorption to
the phototherapeutic (or tissue-transparent) window (the
region of 650−950 nm),45 desired for biological and medical
applications. There is currently no transition-metal-free caged
compound that can be directly activated by light in this
wavelength region. Only a few chemical systems releasing a
chemical species upon irradiation with wavelengths above 450
but below ∼600 nm are known,18 for example, the (6-hydroxy-
3-oxo-3H-xanthen-9-yl)methyl33 or pyronin46 derivatives in-
troduced by our laboratory, 4-aryloxy BODIPY derivatives
proposed by Urano and co-workers,47 and the meso-
methylhydroxy BODIPY scaffold designed simultaneously by
the groups of Weinstain48 and Winter.49
We synthesized COR-BDP 3b by condensation of 4a with
the corresponding PEG-substituted benzaldehyde in the
presence of piperidine in glacial acetic acid and subsequent
hydrogenation on Pd/C in 56% yield. The 3,5-distyryl groups
in 3b affect the absorption properties of the BODIPY
chromophore by extending the π-conjugation, whereas PEG
chains enhance its aqueous solubility. The major absorption
band of this compound (652 nm, tails to ∼750 nm; Figure 2) is
bathochromically shifted by ∼150 nm as compared to that of
3a. Excitation of 3b at either of the main absorption bands (λmax
= 368 and 652 nm) resulted in the same fluorescence spectrum
(Figure 2), that is, in the same emitting excited state. The
presence of two styryl groups in 3b is probably responsible for
its lower fluorescence quantum yield (Φf = 12%) as compared
to that of 3a due to enhanced radiationless decay of the singlet
excited state.50,51
Figure 3. Irradiation of 3a at 500 nm (top) and 3b at 625 nm
(bottom) in aerated phosphate buffer solutions (pH = 7.4). The
spectra prior to (red line) and after (blue line) the irradiation are
highlighted.
(2.7 0.4) × 10−4 and (1.1 0.1) × 10−4 in degassed and
aerated PBS solutions (pH = 7.4), respectively (Table 1).43 The
product of Φεmax on the order of 10 is comparable to that of
some of the common caged compounds, especially those
absorbing near 400 nm.18 The quantum yields were
independent of the concentration of 3a (Figures S27−S29).
Table 1. Photochemistry of 3a and 3b
a
b
c
d
e
compd
Φ
decomp/%
yield/%
εmax
Φεmax
3a (deg)
3a (aer)
3b (deg)
3b (aer)
(2.7 0.4) × 10−2
(1.1 0.1) × 10−2
(1.2 0.4) × 10−3
(1.4 0.4) × 10−3
87
44
91
42
49 500
52 000
13
5
Two protonation steps with pKa′ = (4.71 0.01) and pKa =
(3.0
0.2), respectively, have been identified; thus the
0.6
0.7
conjugate base of 3b is present at physiological pH (Figure
S32). Irradiation of 3b in an aqueous solution (PBS, pH = 7.4)
released CO not only upon excitation at the major absorption
band maxima (368 and 652 nm) but also at the absorption tail
in the near-infrared region (732 nm). To our knowledge, COR-
BDP 3b is therefore the first caged compound that efficiently
releases a molecule upon direct irradiation with near-infrared
photons. Although the product of Φε decreases at longer
wavelengths (see Table 1), it is still suitable for biological
applications because the light penetrates deeply into the tissue.
a
In degassed (deg) or aerated (aer) phosphate buffer solutions (I = 0.1
M, pH = 7.4). 133 was used as an actinometer for 3a and 3b;
ferrioxalate actinometer was also used for 3b. Compounds 3a and 3b
were irradiated at λirr = 505 and 365 nm (the second major absorption
b
c
band), respectively. The total maximum chemical yields of released
CO, monitored by a GC/RGA headspace analysis, were obtained upon
d
exhaustive irradiation. The molar absorption coefficient, εmax/(mol−1
e
dm−3 cm−1). The uncaging cross-section at λmax
.
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This premise was confirmed by an in vivo experiment with a
nude SKH1 mouse strain (Table 2). Hairless mice were used to
Table 2. CO Release from 3b upon Irradiation in Vivo
d
e
f
blood
COHb
liver
CO
kidney
group
w
/%
c
/pmol
c
/pmol
CO
a
control
0.36 0.06
0.57 0.04
0.78 0.18
5.6 1.2
7.1 1.1
11.3 2.7
7.1 1.7
7.0 0.8
10.1 0.8
b
g
dark
c
gh
,
gh
,
gh
,
irradiated
a
A control group of 6 animals (no 3b application). Another group of
12 animals received an intraperitoneal injection of 3b in saline (50
b
μmol per kg of body weight), from which 6 mice were kept in the
c
dark and 6 mice were irradiated with white light focused to the
d
blood
abdominal area.
w
: the relative amounts of COHb in the total
COHb
e
f
kidney
liver
CO
amount of Hb in blood (in %); c and c
: the amounts of CO in
CO
pmol per 1 mg of a fresh liver and kidney tissue, respectively. The
g
h
statistical significance: P-value ≤0.05 vs “control group”; P-value
Figure 4. CO production from 3a. 3a (500 μM, PBS solution) was
irradiated at 510 nm (11.6 mW cm−2). The CO released to the vial
headspace was measured by GC/RGA in 2−4 h intervals and
expressed as the amount of CO (in ppm) released from 3a. When
irradiation (on) was interrupted (off), the vial was kept in the dark at 4
°C.
≤0.05 vs “dark group”; n = 6 in each mice group.
facilitate light penetration through the cutaneous barrier. One
group of mice that was not treated with 3b served as a control
group. Two other groups received intraperitoneal application of
3b (50 μmol per kg of body weight). One group was left in the
dark while the other was irradiated with white light focused to
the abdominal area of mice for 4 h. Irradiation resulted in a
substantial increase in both the carbonylhemoglobin (COHb)
concentration in blood and the CO content in hepatic and
kidney tissues, when compared to both nontreated and
nonirradiated control groups. It should be noted that, although
nonirradiated mice treated with 3b were carefully kept in the
dark, inadvertent light exposure of biological material (blood,
liver), still containing unreacted 3b during sampling, resulted in
increased levels of COHb. Nevertheless, statistically unequiv-
ocal increase in the CO levels in both blood and tissues of
irradiated mice as compared to both control groups (by factors
of 1.5−2) clearly demonstrates that CO was photoreleased in
vivo. Heme oxygenase induction, a possible source of CO
overproduction, was excluded by a direct determination of
heme oxygenase activity (Figure S49). In addition, an in vitro
sample of blood containing 3b was irradiated with white light,
and the production of CO was detected by GC (Table S2).
The CO release from 3b is accompanied by the concomitant
formation of a yet-unidentified primary photoproduct that
remains in the solution even after a prolonged photolysis
(<10% conversion after 24 h) at wavelengths close to its
absorption maximum (λmax = 578 nm; Figure 3b). On the basis
of the absorption spectrum, this compound still probably
contains a BODIPY chromophore, but it does not represent an
internal filter when the irradiation is conducted at wavelengths
longer than the λmax of 3b. The decomposition quantum yields,
the yields of CO release upon exhaustive irradiation, and the
uncaging cross sections, Φεmax, of 3b when aerated or degassed
samples were used are summarized in Table 1.
The influence of oxygen on the photolysis quantum yield of
3a (Φdecomp is lower by a factor of 2 in an aerated sample; Table
1) suggested that its triplet state might be involved in the CO
release. However, intersystem crossing (isc) is not efficient in
3a as evidenced by its high fluorescence quantum yield (Φf =
67%), which explains rather low decomposition quantum yields
shown in Table 1. We thus synthesized a diiodo derivative 3c
(Figure 5; the synthesis and its photochemical properties are
Figure 5. A diiodo BODIPY derivative 3c.
described in the Supporting Information) to enhance the isc
rate via a heavy-atom effect as demonstrated on other BODIPY
derivatives. For example, the isc quantum yield can reach 0.66
in a hexabromo-substituted BODIPY.53 The absorption
properties of 3c are similar to those of 3a (Figures 2 and
S23); however, its fluorescence quantum yield dropped to Φf =
2% as expected. Unfortunately, the total chemical yield of
released CO from 3c was also very low (∼3%). We concluded
that the triplet excited 3c undergoes a photoreaction that does
not lead to the CO production (see the Supporting
Information) either due to an alternative reaction pathway
(i.e., a C−I bond fission) or due to bleaching by the generated
singlet oxygen.
Nevertheless, laser flash spectroscopy of the compound 3c
helped us to assign the transient absorption signals of triplet 3a
that are of low intensity due to its inefficient isc. The transient
triplet−triplet absorption spectrum of 3c possesses three
absorption maxima at 325, 415, and 660 nm (Figures S36−
S38), which exhibit the same decay kinetics. Triplet-state
absorption bands in the region of 410−450 nm have already
been reported for analogous brominated BODIPY derivatives.53
The lifetimes of the triplet 3c under different conditions are
shown in Table S1. The triplet state is markedly quenched by
A controlled, steady, and long-lasting CO production is
crucial for biological applications. Our GC/RGA analysis
revealed a steady CO release for 100 and 26 h using 3a and
3b, respectively. The release rate depends on the intensity of
the incident light. The formation of CO ceased when the
irradiation was interrupted, allowing for complete control over
the CO release (Figure 4).
Neither 3a,b nor their photoproducts displayed toxicity52 in
our in vitro experiments on hepatoblastoma HepG2 and
neuroblastoma SH-SY5Y cell lines up to concentrations of 100
μmol L−1 (Figure S39).
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oxygen; the lifetime of (34.5 0.62) μs in a degassed aqueous
buffer solution at pH = 7.4 dropped to (0.50 0.02) μs in an
oxygen-saturated sample. The triplet state of 3a possesses
absorption maxima at 430 and 640 nm with a lifetime of ∼(5
1) μs in an aerated solution (Figure S35).
Scheme 2. Proposed Mechanism of COR-BDP
Phototransformation To Release CO
Final evidence that a triplet state is involved in the CO
release was provided by irradiation of 3a in a degassed aqueous
solution containing CsCl (1 M aqueous solution; an analogous
NaCl-containing sample was used as a reference) as a heavy-
atom effect mediator54 (Figure S34). We monitored the major
absorption band of 3a and observed that the decomposition
quantum yield (Φdecomp) increased by a factor of 1.6 in the
presence of Cs+ ions due to the isc-rate enhancement, while the
fluorescence quantum yield decreased from 67% to 53%. The
rate of the CO release monitored by a GC/RGA headspace
analysis increased accordingly and matched that of 3a
decomposition.
This result motivated us to further explore the reaction
mechanism. When a solution of 3a was irradiated at pH = 2.5
where only 25% of 3a exists in the form of the conjugate base, a
decrease in Φdecomp by a factor of ∼4 was observed (Figure
S29). This suggests that the negative charge on the carboxyl
group is essential for the reaction. The proton dissociation
strongly influences the redox properties. For example,
carboxylic acids have redox potentials, which are more positive
than those of the corresponding carboxylates by ∼0.5 V.55 The
ground-state redox potential for 3a was determined by cyclic
voltammetry (−1.02 V vs SCE; the one-electron oxidation of
3a (as a conjugate base) occurs at +0.13 V vs SCE; Figure S33).
The former value corresponds well to the published data for
other BODIPY derivatives.56,57 The energy of the triplet excited
state of a BODIPY could be estimated from the phosphor-
escence spectra of the dibromo (λphos = 740 nm, 1.68 eV)58 or
diiodo derivatives (λphos = 795 nm, 1.56 eV).59 Using the latter
value, the triplet state redox potential equals +0.54 V vs SCE.
As a result, ΔGeT of an intramolecular electron transfer between
the carboxylate anion attached to an aromatic system and the
triplet excited BODIPY moiety equals approximately −0.41 eV
(−9.5 kcal mol−1). In contrast, electron transfer between the
protonated carboxylic acid and the BODIPY would be slightly
endothermic (∼ +0.09 eV; +2 kcal mol−1). Therefore, we
hypothesized that an intramolecular photoinduced electron
transfer (PET) takes place between the carboxylate and the
BODIPY system in the triplet excited state.
This PET would result in the formation of a triplet diradical
5 (Scheme 2) with one electron on the carboxylate moiety and
the other largely localized in the meso position of the BODIPY
core (see HMOs in Scheme 1b). Such a structure resembles the
oxyallyl diradical, the formation of which via photolysis of p-
hydroxyphenacyl derivatives in aqueous solution was recently
studied by some of us.60 Its decay from a triplet to an open-
shell singlet state is followed by the formation of a
cyclopropanone intermediate that can be considered as a
structural analogue of the anticipated α-lactone 6 (Scheme 2).
It has already been reported that liberation of CO from α-
lactones can occur in the dark.61,62 In our previous study on 6-
hydroxy-3-oxo-3H-xanthene-9-carboxylic acid (1),33 the density
functional theory (DFT) calculations failed to provide an
evidence for the existence of the related α-lactone. We
therefore decided to examine whether 6 may be formed prior
to the CO release (Scheme 2). In this work, we attempted to
find a potential energy minimum that would correspond to the
structure of 6 on the singlet ground-state potential energy
surface. By screening a series of well-performing functionals
(see the Supporting Information for a detailed technical
description), we found that the heterolysis of the α-lactone
ring represents a difficult case to DFT methods. Thorough test
calculations with a model smaller than but structurally similar to
6 (Supporting Information), which allowed the use of accurate
perturbation and coupled-cluster methods, showed that the
M06HF functional, which predicted that an energy minimum
for 6 does exist, performs best of all of the DFT methods. We
therefore conclude that the formation of 6 is feasible.
On the basis of our experimental and computational
evidence, we propose the mechanism of CO release from
COR-BDPs 3 (Scheme 2). Upon excitation, a strongly
fluorescent, lowest excited singlet state of 3 undergoes relatively
inefficient intersystem crossing to the triplet state where an
exergonic PET from the carboxylate to the BODIPY
chromophore takes place, resulting in the formation of an
oxyallyl-type triplet diradical 5. The subsequent intersystem
crossing leads to the formation of α-lactone 6 on the singlet
ground-state potential energy surface. Compound 6 then
undergoes a nonphotochemical fragmentation to release CO.
CONCLUSIONS
■
We report a straightforward synthesis of a new generation of
transition-metal-free CORMs based on BODIPY chromo-
phores (COR-BDPs) that are activatable by visible-to-NIR
(up to 730 nm) light. Such wavelengths are highly advanta-
geous due to a better penetration of the light into biological
tissues. We show the ability of COR-BDPs to efficiently release
CO in a fully controllable way, and demonstrate their
performance in both in vitro and in vivo experimental settings.
The fluorescent nature of these chromophores could also be
desirable for simultaneous in vivo imaging. We propose a
mechanism of the CO release based on steady-state and
transient absorption spectroscopy experiments and quantum
chemical calculations. The BODIPY molecular scaffold allows
for fine-tuning of the physicochemical properties of COR-BDPs
(such as optical properties or aqueous solubility) using simple
structural modification and promises a rapid development of
improved CORM molecules. Further biological studies to
prove the therapeutic potential of COR-BDPs are under
investigation in our laboratories.
EXPERIMENTAL SECTION
Synthesis of 4a. Benzyl chlorooxalate (556 mg, 2.8 mmol) was
added to a solution of 2-methylpyrrole (500 mg, 6.16 mmol) in
■
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dichloromethane at 0 °C, and the mixture was stirred at 0 °C for 4 h.
Triethylamine (1.96 mL, 14 mmol) was added, followed by an
addition of BF3·Et2O (1.68 mL, 14 mmol). The solution was warmed
to room temperature and stirred for 2 h. The reaction was quenched
with aq HCl (10%), and the crude product was extracted with ethyl
acetate. The organic layers were washed with water, dried with MgSO4,
filtered, and the solvents were evaporated to dryness. The compounds
were purified by flash chromatography on silica gel using hexane/ethyl
acetate (9:1) to give the pure compound 4a. Yield: 110 mg (11%).
1575, 1302, 1132, 1007, 944 cm−1. HRMS (ESI): calcd for
(C40H47BF2N2O10) 764.3292, found 764.3291 (Figure S14).
General Procedure for Irradiation in UV Cuvettes. A solution
of a compound in the given solvent (3 mL) in a matched 1.0 cm quartz
PTFE screw-cap cuvette equipped with a stir bar was stirred and
irradiated with a light source of 32 LEDs emitting at the selected
wavelength: λmax = 365, 503, 525, 590, or 625 nm (the bandwidths at
half height = 30 nm). Light pulses (425.6 Hz repetition rate, ≤150 fs
pulse length, and energy of ∼7.5 0.3 mW) from a Ti:Saphire laser
coupled to a noncollinear optical parametric amplifier (NOPA) with
the wavelength set to 505 nm (bandwidth at half height of ∼15 nm)
were used to irradiate the samples. A 400 W broad-band halogen lamp
with a filter (transmittance spectrum is shown in Figure S23) was used
to irradiate the samples at 732 nm. The progress of reactions was
monitored by UV−vis spectrometry using a diode-array spectropho-
tometer in a kinetic mode.
Quantum Yield Determination. The decomposition quantum
yields of 3a and 3b were determined using a solution of xanthene-9-
carboxylic acid (1, c = 1.0 × 10−5 M) in aq phosphate buffered saline
(PBS, I = 0.1 M, pH = 7.4; Φdisapp. = (6.8 3.0) × 10−4)33 as an
actinometer and light pulses at 505 7 nm (see above). The quantum
yield of photodegradation of 3b was determined at 365 nm with
ferrioxalate actinometer63 according to the published procedure.64 The
data were processed using a single value decomposition (SVD)
software.
1
Purple solid. Mp: >300 °C (decomp.). H NMR (500 MHz, CDCl3):
δ (ppm) 7.48−7.40 (m, 5H, 5CH), 7.19 (d, J = 3.8 Hz, 2H, 2CH),
6.30 (d, J = 3.9 Hz, 2H, 2CH), 5.45 (s, 2H, CH2), 2.64 (s, 6H, 2CH3)
(Figure S1). 13C NMR (125 MHz, CDCl3): δ (ppm) 163.7 (C), 160.7
(C), 134.7 (C), 133.5 (C), 130.8 (CH), 128.8 (CH), 128.6 (CH),
128.0 (C), 120.7 (CH), 120.6 (CH), 68.2 (CH2), 15.2 (CH3) (Figure
S2). FTIR (neat): 3065, 1725, 1567, 1458, 1374, 1163, 1101 cm−1.
HRMS (APCI): calcd for (C19H17BF2N2O2) 353.1460, found
353.1459 (Figure S11).
Synthesis of 4b. 4-{2-[2-(2-Methoxyethoxy)ethoxy)ethoxy}-
benzaldehyde (66 mg, 0.22 mmol), glacial acetic acid (0.08 mL, 1.8
mmol), piperidine (0.12 mL, 1.8 mmol), and a small amount of
molecular sieves were added to a solution of 4a (40 mg, 0.11 mmol)
dissolved in anhydrous benzene (10 mL) in a round-bottomed flask
equipped with a Dean−Stark head under argon atmosphere. The
mixture was refluxed for 1 h. The solvent was then removed under
reduced pressure, and the residue was diluted with ethyl acetate and
washed with water. The organic layer was dried with MgSO4, filtered,
and concentrated under reduced pressure. The crude mixture was
purified by flash chromatography on silica gel using hexane/
dichloromethane (2:8) to give 4b. Yield: 66 mg (70%). Green solid.
Determination of the Photoproducts. 3a (500 mL, c ≈ 1 ×
10−5 M, aq solution) was irradiated in a large Petri dish (50 cm
diameter) with a halogen lamp (500 W, Pyrex filter) for 38 h to reach a
full conversion (the solution is photobleached). The resulting mixture
was lyophilized to give the photoproducts, which were analyzed by
HRMS (Figure S16).
1
Mp: >280 °C (decomp.). H NMR (500 MHz, CDCl3): δ (ppm)
7.55−7.49 (m, 6H, 6CH), 7.39−7.30 (m, 5H, 5CH), 7.25−7.22 (m,
4H, 4CH), 6.88 (d, J = 8.8 Hz, 4H, 4CH), 6.29 (d, J = 4.6 Hz, 2H,
2CH), 5.35 (s, 2H, CH2), 4.11 (t, J = 4.8 Hz, 4H, 2-OCH2), 3.82 (t, J
= 4.9 Hz, 4H, OCH2), 3.69−3.67 (m, 4H, OCH2), 3.63−3.58 (m, 8H,
OCH2), 3.49−3.47 (m, 4H, OCH2), 3.31 (s, 6H, OCH3) (Figure S3).
13C NMR (125 MHz, CDCl3): δ (ppm) 164.2 (C), 160.2 (C), 156.5
(C), 138.1 (CH), 136.0 (C), 135.0 (C), 129.6 (CH), 129.5 (CH),
129.4 (CH), 128.8 (CH), 128.7 (CH), 128.6 (CH), 122.6 (C), 117.4
(CH), 115.1 (CH), 72.0 (CH2), 70.9 (CH2), 70.7 (CH2), 70.6 (CH2),
69.7 (CH2), 67.6 (CH2), 59.0 (CH3) (Figure S4). FTIR (neat): 3085,
2920, 2851, 1717, 1593, 1349, 1158, 949 cm−1. HRMS (ESI): calcd for
(C47H53BF2N2O10) 854.3761, found 854.3756 (Figure S12).
Spectrophotometric Determination of pKa’s. A freshly
prepared solution of 3a−c (c = 2.5 × 10−5 M) in PBS (3 mL, I =
0.1 M, pH ≈ 7.4) was transferred into a matched 1.0 cm quartz
cuvette, and its UV−vis absorption spectrum was recorded. The
solution was acidified with HCl to pH ≈ 1 (10 μL of a 1 M solution)
and was titrated with small additions of aq NaOH (typically 10 μL;
0.1, 0.01, or 0.001 M; I = 0.1 M, adjusted by NaCl), and the pH and
UV−vis absorption spectra were monitored after each addition
(Figures S31, S32).
Potentiometric Determination of pKa’s. A freshly prepared
solution of 3a−c (c = 1 × 10−3 M) in distilled water (5 mL) was
stirred in a 25 mL beaker. The pH was measured potentiometrically,
and the solution was acidified by HCl to pH ≈ 1 (10 μL, 1 M
solution). The acidified solution was titrated with small additions of aq
NaOH (typically 10 μL; 0.1, 0.01, or 0.001 M; I = 0.1 M, adjusted by
NaCl), and the pH was measured after each addition. The volume of
the added aq NaOH corresponding to every change in pH was
recorded, and the titration curve was constructed (Figure S30).
Cyclic Voltammetry. Electrochemical studies were carried out
with compounds dissolved in acetonitrile containing 0.1 M tetra-n-
butylammonium hexafluorophosphate as a conducting salt using the
ferrocene/ferrocenium (Fc/Fc+) couple as an internal standard under
argon atmosphere (Figure S33). The measurements were carried out
in an optically transparent thin-layer electrochemical cell (0.1 mm
optical length). A Pt minigrid working electrode, Pt minigrid reference
electrode, and silver wire pseudoreference electrode were used. The
measured potentials were recalculated to potentials vs SCE according
to the published procedure.65
Synthesis of 3a. 4a (50 mg, 014 mmol) was dissolved in
dichloromethane (20 mL), and 10% Pd/C powder (5 mol %) was
added under argon atmosphere, followed by addition of methanol (20
mL). The mixture was saturated with hydrogen and stirred at 25 °C
for 2 h until no starting material remained. Pd/C was removed
through filtration, and the solvent was evaporated under reduced
pressure. The products were purified by flash chromatography using
dichloromethane/methanol (8:2) on silica gel to give 3a. Yield: 32 mg
1
(82%). Yellow-orange solid. Mp: >250 °C (decomp.). H NMR (500
MHz, D2O): δ (ppm) 7.13 (d, J = 4.1 Hz, 2H, 2CH), 6.43 (d, J = 4.1
Hz, 2H, 2CH), 2.53 (s, 6H, 2CH3) (Figure S5). 13C NMR (125 MHz,
D2O): δ (ppm) 170.5 (C), 158.6 (C), 139.0 (C), 131.2 (C), 129.7
(CH), 120.1 (CH), 14.2 (CH3) (Figure S6). FTIR (neat): 3321, 3065,
1725, 1567, 1261, 1101, 1007, 945 cm−1. HRMS (APCI): calcd for
(C12H11BF2N2O2) 262.0845, found 262.0844 (Figure S13).
Synthesis of 3b. 3b was synthesized according to the same
procedure as 3a from 4c (40 mg, 0.047 mmol). Yield: 19 mg (53%).
Blue solid. Mp: >255 °C (decomp.). 1H NMR (500 MHz, CD3OD): δ
(ppm) 7.62−7.57 (m, 6H, 6CH), 7.46 (d, J = 16.4 Hz, 2H, 2CH =
CH), 7.28 (d, J = 4.4 Hz, 2H, 2CH), 7.06−7.02 (m, 6H, 6CH), 4.24
(t, J = 4.5 Hz, 4H, OCH2), 3.89 (t, J = 4.6 Hz, 4H, OCH2), 3.74−3.72
(m, 4H, OCH2), 3.69−3.64 (m, 8H, OCH2), 3.56−3.54 (m, 4H,
OCH2), 3.37 (s, 6H, OCH3) (Figure S7). 13C NMR (125 MHz,
CD3OD): δ (ppm) 169.6 (C), 159.9 (C), 154.6 (C), 135.7 (CH),
133.8 (C), 129.7 (C), 128.5 (CH), 116.9 (CH), 115.3 (CH), 114.8
(CH), 71.5 (CH2), 70.4 (CH2), 70.2 (CH2), 70.0 (CH2), 69.4 (CH2),
67.4 (CH2), 57.7 (CH3) (Figure S8). FTIR (neat): 3331, 3075, 1721,
Determination of CO Concentrations. A solution of 3a−c
(0.25−2 mM) in PBS (20−150 μL, I = 0.1 M, pH ≈ 7.4) was added
into CO-free septum-sealed glass vials. After irradiation with white
light (433 mW cm−2; 10 cm from the light source) or 510 5 nm
(11.6 mW cm−2; Edmund Optics Barrington filter No. 65152), the CO
amount released into the vial headspace was determined by gas
chromatography with a reduction gas analyzer described previously.66
In Vitro Toxicity Determination. Human neuroblastoma
SHSY5Y and hepatoblastoma HepG2 cell lines were grown according
to the manufacturer’s protocol in a 96-well plate with solutions of 3a
and 3b or their photoproducts in the concentration range of 10−100
F
DOI: 10.1021/jacs.5b10800
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
μM for 4, 24, or 48 h. The viability of cell lines was determined by a 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay
(MTT). After 2 h incubation period with MTT, the absorbance of a
formazan-reduction product was measured at 545 nm with a standard
ELISA reader. Compounds 3a and 3b were found not to interfere with
the reduction of MTT.
AUTHOR INFORMATION
■
Corresponding Author
*klan@sci.muni.cz
Author Contributions
∥E.P. and T.S. contributed equally to this work.
In Vivo Experiments. Male nude SKH1 mice were allowed water
and standard granulated diet ad libitum. All studies in this work met
the criteria for the care and use of animals, and were approved by the
Animal Research Committee of the 1st Faculty of Medicine, Charles
University in Prague. Mice were anesthetized and then received an
intraperitoneal injection of saline (a control group) or a solution of 3b
in saline (50 μmol per 1 kg of body weight). An experimental group of
mice was irradiated with a lamp focused to the abdominal area for 4 h,
while a control group was kept in the dark. Subsequently, the animals
were sacrificed. Blood from the superior vena cava of each animal was
transferred to the test tubes containing aq solution of EDTA (2 μL;
0.5 M). The CO (as COHb) and total hemoglobin concentrations in
the sample of blood (1 μL) were determined using GC and a Drabkin
cyanmethemoglobin method described previously.67 Liver and kidneys
of each animal were then harvested, washed, put into an ice-cold
potassium phosphate buffer (c = 0.1 M, pH = 7.4) in a ratio of 1:4 (w/
w), and homogenized by sonication. The liver/kidneys homogenate
(40 μL) was added to CO-free septum-sealed vials containing 5 μL of
60% (v/w) sulfosalicylic acid. After incubation on ice (30 min), the
amount of the CO released into the vial headspace was determined by
GC/RGA as reported previously.68
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Support for this work was provided by the Czech Science
Foundation (GA13-25775S) (P.K.) and Czech Ministry of
Health (RVO-VFN64165/2015) (L.V.). The RECETOX
research infrastructure was supported by the projects of the
Czech Ministry of Education (LO1214 and LM2011028). We
̌
́
and Jakub Suk for their technical
also thank Marie Zadinova
assistance with in vivo experiments, Jana Vanıkova
performing MTT assays, Katerina Klanova for her help with
́
́
for
̌
́
́
TOC, and Jakob Wirz for fruitful discussions.
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* Supporting Information
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