Cyanine-Flavonol Hybrids for Near-Infrared Light-Activated Delivery of Carbon Monoxide

Article abstract of DOI:10.1002/chem.202003272

Carbon monoxide (CO) is an endogenous signaling molecule that controls a number of physiological processes. To circumvent the inherent toxicity of CO, light-activated CO-releasing molecules (photoCORMs) have emerged as an alternative for its administration. However, their wider application requires photoactivation using biologically benign visible and near-infrared (NIR) light. In this work, a strategy to access such photoCORMs by fusing two CO-releasing flavonol moieties with a NIR-absorbing cyanine dye is presented. These hybrids liberate two molecules of CO in high chemical yields upon activation with NIR light up to 820 nm and exhibit excellent uncaging cross-sections, which surpass the state-of-the-art by two orders of magnitude. Furthermore, the biocompatibility and applicability of the system in vitro and in vivo are demonstrated, and a mechanism of CO release is proposed. It is hoped that this strategy will stimulate the discovery of new classes of photoCORMs and accelerate the translation of CO-based phototherapy into practice.

Full text of DOI:10.1002/chem.202003272

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Medicinal Chemistry |Hot Paper|
Cyanine-Flavonol Hybrids for Near-Infrared Light-Activated
Delivery of Carbon Monoxide
[a]
[a]
[b]
[a]
[b]
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ˇ
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Lenka Stackovꢀ, Marina Russo, Lucie Muchovꢀ, Vojtech Orel, Libor Vꢁtek,
[a]
[a]
Peter Stacko,* and Petr Klꢀn*
Abstract: Carbon monoxide (CO) is an endogenous signal-
ing molecule that controls a number of physiological pro-
cesses. To circumvent the inherent toxicity of CO, light-acti-
vated CO-releasing molecules (photoCORMs) have emerged
as an alternative for its administration. However, their wider
application requires photoactivation using biologically
benign visible and near-infrared (NIR) light. In this work, a
strategy to access such photoCORMs by fusing two CO-re-
leasing flavonol moieties with a NIR-absorbing cyanine dye
is presented. These hybrids liberate two molecules of CO in
high chemical yields upon activation with NIR light up to
820 nm and exhibit excellent uncaging cross-sections, which
surpass the state-of-the-art by two orders of magnitude. Fur-
thermore, the biocompatibility and applicability of the
system in vitro and in vivo are demonstrated, and a mecha-
nism of CO release is proposed. It is hoped that this strategy
will stimulate the discovery of new classes of photoCORMs
and accelerate the translation of CO-based phototherapy
into practice.
Introduction
matory, and anti-microbial effects at submicromolar concentra-
tions.[8] Taking advantage of an anti-Warburg effect,[9] cancer
cells and tumors exposed to CO are forced to switch to oxida-
tive metabolism, leading eventually to growth inhibition, cellu-
lar exhaustion, and death.[10] Moreover, the sensitivity of cancer
cells towards chemotherapeutics is significantly increased (up
to 1000-fold) upon exposure to CO while simultaneously pro-
tecting normal cell growth and viability. CO also exhibits
potent multifactorial inhibitory and anti-angiogenic effects on
cancer proliferation.[11,12]
Use of light as a control stimulus offers unparalleled advantag-
es in terms of availability, adjustability, high spatial and tempo-
ral precision, high orthogonality towards biochemical systems,
and the minimization of waste products. The irradiation wave-
lengths applicable in living organisms are restricted by the ad-
verse effects of UV- and visible-light absorption and optical
scattering by endogenous chromophores, which limits the
depth of tissue penetration.[1,2] Successful application of photo-
dynamic therapy (PDT) lends credibility to the use of light in
the near-infrared (NIR) range of 650–900 nm, known as the
phototherapeutic window, in clinical applications.[3]
However, owing to its inherent toxicity and arduous admin-
istration of CO, many CO-releasing molecules (CORMs), primari-
ly based on transition metal-carbonyl complexes, have been
developed.[4,13,14] Generally, these CORMs rely on enzymes as
the release triggers or on a solvent-mediated ligand exchange
reaction in aqueous media, methods that exhibit poor spatial
and temporal control over the release profile.[15,16] Furthermore,
the metal backbone left upon CO release from these metal-car-
bonyl complexes can lead to uncontrolled reactions with adja-
cent cells, resulting in their damage and a major barrier to in
vivo CORM applications.[6] Transition-metal-free light-triggered
CORMs (photoCORMs) have recently appeared with the prom-
ise of circumventing these challenges. Many organic mole-
cules, such as cyclopropenones,[17–19] 1,3-cyclobutadiones,[20] or
1,2-dioxolane-3,4-diones,[21] liberate CO upon irradiation with
biologically adverse UV light. Cyclic aromatic a-diketones[22]
Carbon monoxide (CO) has recently emerged as a promising
target for photodelivery, possessing remarkable therapeutic
potential.[4–7] It is a naturally occurring cell-signaling molecule
exhibiting strong cytoprotective, cardioprotective, anti-inflam-
ˇ
ˇ
[a] Dr. L. Stackovꢀ, M. Russo, V. Orel, Dr. P. Stacko, Prof. P. Klꢀn
Department of Chemistry and RECETOX, Faculty of Science
Masaryk University, Kamenice 5, 62500 Brno (Czech Republic)
E-mail: stacko@mail.muni.cz
klan@sci.muni.cz
[b] Dr. L. Muchovꢀ, Prof. L. Vꢁtek
Institute of Medical Biochemistry and Laboratory Diagnostics
General Faculty Hospital and 1st Faculty of Medicine
Charles University, Na Bojisti 3, 12108 Praha 2 (Czech Republic)
ˇ
(lmax =468 nm) and a xanthene-based carboxylic acid[23] (lmax
=
Supporting information and the ORCID identification number(s) for the
author(s) of this article can be found under:
https://doi.org/10.1002/chem.202003272.
488 nm) have been designed to undergo photochemical decar-
bonylation with visible light. Recently, a class of p-extended
flavonols 1 based on a naturally occurring flavone scaffold has
been shown to efficiently release CO upon irradiation at 400–
540 nm (Figure 1a, left),[24–27] and the effects of liberated CO on
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original work is properly cited.
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biology, and medicine.[31–34] For instance, the most prominent
cyanine dye, indocyanine green, is FDA approved and currently
enrolled in hundreds of clinical trials despite being discovered
over 60 years ago.[35] The symmetric nature of cyanines also
allows the installation of two flavonol moieties in the molecule,
facilitating a potential release of two equivalents of CO (Fig-
ure 1c). In addition, such a system exhibits high customization
potential for further applications owing to the presence of sev-
eral versatile structural elements. The N-substituents of the in-
dolenine heterocycles can be used to tune its solubility in
aqueous media or for bioconjugation of enzyme ligands and
antibodies,[36] whereas the C4’ position of the heptamethine
chain can be used to tune absorption spectra[37] or photostabil-
ity,[38] or to attach targeting residues.[39,40] The phenyl substitu-
ent of the flavonol moiety is introduced at a late stage of the
synthesis, which allows for the installation of additional sub-
stituents. In this work, we present a strategy to access potent
NIR-absorbing photoCORMs by designing flavonol-cyanine hy-
brids 5a and b (Figure 1c), and we describe their photochemi-
cal behavior and demonstrate their biological applicability in in
vivo experiments.
We synthesized hybrid 5a and its analog 5b featuring trime-
thylammonium substituents to facilitate its solubility in aque-
ous media (Figure 1c). We reasoned that the flavonol core
must be constructed prior to assembling the cyanine scaffold,
which is susceptible to strongly basic and oxidative conditions
required in the flavonol preparation. The synthesis of 5a–b
was started by the construction of an indolenine core 7 from 6
and 3-methyl-2-butanone in 97% yield (Scheme 1). The me-
thoxy group of 7 was removed with BBr3, and the resulting
phenolic group was acylated with acetyl chloride. Subsequent
Fries rearrangement of 9 with AlCl3 at 1908C provided the de-
sired methylketone 10 in 80% yield in a regioselective fashion,
presumably because of steric hindrance imposed by the dime-
thylmethylene substituent. Algar–Flynn–Oyamada reaction of
10 with benzaldehyde or 4-(dimethylamino)benzaldehyde in
the presence of H2O2 provided flavonols 11 a–b in 22% and
38% yields, respectively. Methylation with TfOMe or MeI afford-
ed the building blocks 12a–b, which were subsequently con-
densed with commercially available enamine 13 (see the Sup-
porting Information) to access the final cyanine-flavonol hy-
brids 5a–b in 55% and 51% yields, respectively.
UV/Vis absorption spectra of cyanine-flavonol hybrids 5a–b
in methanol display an intense absorption band typical for the
heptamethine cyanine dyes with lmax =791 and 793 nm, re-
spectively (Figure 2a and Table 1).[37] The compounds also ex-
hibit emission at lem =815 and 819 nm, respectively, with the
intensity comparable to that of indocyanine green (ICG). Com-
pound 5b in methanol or DMSO at spectroscopic concentra-
tions was found stable at 48C for days.
Irradiation of 5a–b (cꢀ4ꢃ10À6 m) in methanol at 770
(ꢀ60 mWcmÀ2) or 820 nm (ꢀ14 mWcmÀ2) with commercial
LEDs was initially (ꢀ50 min) accompanied by a small hypso-
chromic shift (ꢀ3 nm) of the major absorption bands and a
decrease of their intensities, and concurrent disappearance of
the absorption bands at 343 and 422 nm (Figure 2b and Fig-
ure S52 in the Supporting Information). Because the substitu-
Figure 1. (a) Flavonol-based 1 (left) and BODIPY-based 2 (right) photo-
CORMs. (b) The concept of fusing a flavonol photoCORM moiety and a hep-
tamethine cyanine dye into the conjugated system proposed in this work.
Structural elements suitable for further modifications are depicted. (c) Photo-
chemical release of CO from cyanine-flavonol hybrids 5a–b prepared and
studied herein. Trimethylammonium groups installed in 5b facilitate solubili-
ty in aqueous media. Counter anions are omitted for clarity.
the cells were unequivocally demonstrated.[28] Our recent
mechanistic investigation of this system revealed the existence
of three orthogonal pathways for the CO photorelease.[29]
meso-Carboxy BODIPY derivative 2 is currently the only organic
photoCORM operating at the edge of a phototherapeutic
window (lmax =652 nm), but it suffers from a low uncaging
cross-section Femax (Figure 1a, right).[30]
Results and Discussion
After elucidating the mechanism of photochemical CO release
from flavonol 1,[29] we envisioned that fusing its analog 3 with
an established chromophore could serve as a general strategy
for designing new classes of photoCORMs, especially those ab-
sorbing in the NIR region (Figure 1b). Heptamethine cyanine
(Cy7) chromophore 4 was chosen for its strong absorption in
the center of the phototherapeutic window, synthetic versatili-
ty, and its well-established role in contemporary chemistry,
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Scheme 1. Synthesis of the cyanine-flavonol hybrids 5a–b. Reaction conditions: (i) 3-methyl-2-butanone, AcOH, 1108C, 97%; (ii) BBr3, CH2Cl2, 08C to rt, 95%;
(iii) AcCl, Et3N, CH2Cl2, rt, 59%; (iv) AlCl3, neat, 1908C, 80%; (v) NaOH, 30% H2O2, MeOH, 08C. 11 a: 22%. 11 b: 38%; (vi) 12a: MeOTf, CH2Cl2, 86% or 12b:
1) MeI, MeCN, 1008C; 2) AgOTf, MeOH 81%; (vii) enamine 13, AcONa, EtOH, reflux. 5a: 55%. 5b: 51%.
Figure 2. (a) UV/Vis absorption (solid) and emission (dotted) spectra of the hybrids 5a (red) and 5b (blue). (b) Irradiation of 5b (cꢀ3.8ꢃ10À6 m) at 820 nm in
aerated methanol followed by UV/Vis spectroscopy at 10 min intervals (from blue to red lines). The inset depicts a kinetic trace of the absorption at 793 nm
superimposed with the liberation of CO in time (black). (c) Total chemical yields of CO produced in the dark or upon exhaustive irradiation of 5a (red) or 5b
(blue) in methanol or PBS (pH 7.4, 10 mm, I=100 mm) at 770 or 820 nm, respectively. The error bars represent the standard deviation of the mean from four
independent samples. (d) Time-dependent CO release from 5b (cꢀ4ꢃ10À6 m); blue line: irradiated at 770 nm in methanol; red line: irradiated at 820 nm in
methanol; black line: irradiated at 820 nm in PBS. The CO released to the headspace was determined by GC and is expressed as the total chemical yield.
tion of the aromatic cores of the cyanine heterocycles affects
the cyanine absorption maxima relatively insignificantly owing
to its weak interaction with the HOMO and LUMO of the cya-
nine chromophore, we hypothesized that this process corre-
sponds to the photochemical cleavage of the terminal flavonol
rings (see below). Upon extended irradiation, the disappear-
ance of the major absorption bands at approximately 790 nm
and the appearance of very weak bands at about 420 nm were
observed, which is attributed to the subsequent photooxida-
tion of the cyanine heptamethine chain with singlet oxygen
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gassed methanol prepared under hypoxic conditions (
ꢀ3 ppm of O2 in the atmosphere) and irradiated at 770 nm
produced CO less efficiently (ꢀ30%, ꢀ108%, and ꢀ128%
after 1, 24, and 36 h, respectively). This demonstrates the
pivotal role of oxygen in the photodecarbonylation process
but also indicates that the system can operate under the
hypoxic conditions often found in tumors, albeit with lower
efficiency (Figure S62 in the Supporting Information). Per-
forming the photolyzes in minimum essential medium
(MEM) or the presence of bovine serum albumin (BSA,
20 mgmLÀ1) did not influence the chemical yield of the re-
leased CO. Irradiation of 5b in aerated methanol at 420 nm
produced CO in approximately 160% chemical yield.
Table 1. Photophysical and photochemical properties of cyanine-flavonol
hybrids 5a and 5b.
[a]
[e]
labs lem emax
FD
Yield
[%][c]
FCO
[%][d]
FCOemax
[mÀ1 cmÀ1
[nm] [nm] [mÀ1 cmÀ1] [%][b]
]
5a 791 815 254000
5b 793 819 251000
0.34Æ0.01 125Æ15 (2.0Æ0.3)ꢃ10À2 51Æ8
0.62Æ0.02 131Æ6 (3.0Æ0.5)ꢃ10À2 75Æ12
All measurements were performed in methanol. [a] The molar absorption co-
efficient, emax. [b] The singlet oxygen production quantum yield was deter-
mined by using diphenylisobenzofurane (DPBF). Compounds 5a and 5b
were irradiated with 770 nm LEDs and ICG as a 1O2 generator (FD =0.0077)
and used as a reference. [c] The total chemical yield of released CO, moni-
tored by GC headspace, obtained upon exhaustive irradiation at 770 nm.
[d] Absolute quantum yields of CO release at lmax determined by using a
calibrated Si-photodiode. [e] The CO uncaging cross-section at lmax : FCOemax
.
Subsequently, the efficiency of the NIR-light-triggered CO
photorelease was assessed. Owing to the lack of suitable ac-
tinometers operating in the NIR region, we instead opted to
use a calibrated Si-photodiode to measure the photon flux.
Coupling this with GC headspace analysis allowed us to direct-
ly determine the absolute quantum yields of CO release (FCO)
to be (2.0Æ0.3)ꢃ10À4 and (3.0Æ0.5)ꢃ10À4 for 5a and 5b in
aerated methanol, respectively (Table 1). The excellent uncag-
ing cross-section FCOemax of 5a–b on the order of 50–
75mÀ1 cmÀ1 is comparable to that of the parent flavonol 1
(ꢀ84mÀ1 cmÀ1)[26] when operating at wavelengths batho-
chromically shifted by nearly 400 nm. The only transition-
metal-free photoCORM 2 activated by near-infrared light re-
ported to date possesses Femax of 0.6.[30] Compared with this
value, the present system constitutes up to 110-fold increase
in the efficiency of the photochemical CO release with a simul-
taneous shift of the absorption maximum by approximately
140 nm to the NIR region. The strong absorption in the photo-
therapeutic window, high chemical yields of CO, and excellent
uncaging cross-section FCOemax showcase the practical utility
and applicability of the hybrids 5a–b in biological settings.
The water-soluble derivative 5b exhibited cytotoxic effects
in in vitro studies on the HepG2 hepatoblastoma cell line after
a 24 h exposure from concentrations as high as 100 mm (Fig-
ure S72 in the Supporting Information). We further probed the
cytotoxic effects of the photoproducts by exhaustive irradia-
tion of 5b in the absence of HepG2 cells and subsequent ex-
posure of HepG2 cells to the mixture of photoproducts for
24 h (Figure S73 in the Supporting Information). This approach
enabled us to assess the effects of photoproducts independ-
produced by sensitization of the oxygen present in the solu-
tion (Figure S63 in the Supporting Information).[41] Self-sensi-
tized photooxygenation is known to be a primary photo-
bleaching pathway of cyanines and has recently been utilized
for NIR uncaging.[42,43] These two distinct photochemical pro-
cesses were also clearly distinguished for 5b from the kinetic
trace detected at 793 nm (Figure 2b, inset). The phototransfor-
mation efficiency of 5b in degassed methanol was severely
suppressed; only an approximately 20% decrease in absorb-
ance at 793 nm was observed after irradiation for 16 h (Figur-
es S54 and S55 in the Supporting Information).
In the next step, we assessed whether the photolysis of 5a–
b with NIR light is accompanied by the anticipated formation
of CO (Figure 2c). Septum-sealed vials containing 5a–b (cꢀ4–
10ꢃ10À6 m) in methanol or phosphate-buffered saline (PBS,
pH 7.4, 10 mm, I=100 mm) were irradiated or kept in the dark,
and the headspace above the solution was analyzed by using
gas chromatography. Indeed, exhaustive irradiation of 5a–b in
methanol at 820 nm resulted in the formation of CO in (125Æ
15)% and (131Æ6)% chemical yields, respectively, whereas the
samples kept in the dark liberated only negligible (<2%)
amounts of CO, demonstrating that the release is of photo-
chemical origin. The photolysis of water-soluble 5b in PBS
(pH 7.4, 10 mm, I=100 mm) at 770 or 820 nm generated CO in
(102Æ9)% and (110Æ6)% chemical yields, respectively, where-
as no CO was detected in the samples kept in the dark. Irradia-
tion of the parent cyanine 4 for 16 h did not lead to the pro-
duction of CO (<2%). These results show that 5a–b can re-
lease in principle two molecules of CO from both flavonol moi-
eties, corresponding to approximately 65% and 55% yields of
CO per flavonol unit in methanol and PBS, respectively. The
chemical yield values obtained in both methanol and aqueous
solutions are slightly lower than those observed for the parent
flavonol 1.[29] The majority of CO was released within 1–2 h of
irradiation (Figure 2d) and is related to the first photochemical
process detected by UV/Vis spectroscopy (Figure S57 in the
Supporting Information). Under the given conditions, the ther-
apeutic levels of CO (1 mm,[44] corresponding to ꢀ25% chemi-
cal yield) were reached after about 15 min of irradiation and
the total dose of light (ꢀ6.3 WcmÀ2) is comparable to doses
routinely used in PDT.[45] On the contrary, solutions of 5b in de-
1
ently from those of CO or O2. The photoproducts were found
to be non-toxic up to concentrations as high as 200 mm. Irradi-
ation of 5b at 780 nm for 1 h incubated together with the
presence of HepG2 cells (Figure S74 in the Supporting Informa-
tion) had no significant effect on cell viability up to the con-
centration of 100 mm as measured 24 h post-irradiation, indi-
cating the low toxicity of the photoproducts. Encouraged by
these findings, we decided to evaluate the performance of 5b
in in vivo experiments on nude SKH1 mice. Hairless mice were
used to facilitate light penetration through the cutaneous bar-
rier. One group of mice was not treated with 5b, but received
vehicle only, and served as a control group. Another group re-
ceived an intraperitoneal injection of 5b (50 mmolkgÀ1 of body
weight) in saline (10 mLgÀ1, with 5% DMSO), and the mice
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