Mechanisms of orthogonal photodecarbonylation reactions of 3-hydroxyflavone-based acid-base forms

Article abstract of DOI:10.1021/acs.joc.9b03248

Carbon monoxide is a naturally occurring gasotransmitter combining inherent toxicity with a remarkable therapeutic potential and arduous administration. Photoactivatable carbon monoxide-releasing molecules (photoCORMs) are chemical agents that allow for precise spatial and temporal control over the CO release. In this work, we present a comprehensive mechanistic study of the photochemical CO release from 3-hydroxy-2-phenyl-4H-chromen-4-one, a π-extended 3-hydroxyflavone photoCORM, in methanol using steady-state and transient absorption spectroscopies and quantum chemical calculations. The multiplicity of the productive excited states and the role of oxygen (O₂) in the CO production are emphasized, revealing a photoreaction dichotomy of the 3-hydroxyflavone acid and base forms. The utilization of three major orthogonal mechanistic pathways, all of which lead to the CO release, can fuel future endeavors to improve the CO release efficacy of 3-hydroxyflavone-based derivatives and refine their potential medical applications as photoCORMs.

Full text of DOI:10.1021/acs.joc.9b03248

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Mechanisms of Orthogonal Photodecarbonylation Reactions of
3‑Hydroxyflavone-Based Acid−Base Forms
̌
́
́
Marina Russo, Peter Stacko, Dana Nachtigallova, and Petr Klan*
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ABSTRACT: Carbon monoxide is a naturally occurring gaso-
transmitter combining inherent toxicity with a remarkable
therapeutic potential and arduous administration. Photoactivatable
carbon monoxide-releasing molecules (photoCORMs) are chem-
ical agents that allow for precise spatial and temporal control over
the CO release. In this work, we present a comprehensive
mechanistic study of the photochemical CO release from 3-
hydroxy-2-phenyl-4H-chromen-4-one, a π-extended 3-hydroxyfla-
vone photoCORM, in methanol using steady-state and transient
absorption spectroscopies and quantum chemical calculations. The
multiplicity of the productive excited states and the role of oxygen
(O2) in the CO production are emphasized, revealing a
photoreaction dichotomy of the 3-hydroxyflavone acid and base forms. The utilization of three major orthogonal mechanistic
pathways, all of which lead to the CO release, can fuel future endeavors to improve the CO release efficacy of 3-hydroxyflavone-
based derivatives and refine their potential medical applications as photoCORMs.
these challenges. A number of organic molecules, such as
cyclopropenones,14 1,3-cyclobutadiones,15 or 1,2-dioxolane-
3,4-diones,16 liberate CO upon irradiation with biologically
adverse UV light. Cyclic aromatic α-diketone-17 and xanthene-
based carboxylic acids18,19 were shown to undergo CO
liberation initiated with visible light. A meso-carboxy BODIPY
reported by our group recently is currently the only transition-
metal-free photoCORM operating at the edge of a photo-
therapeutic window.20
INTRODUCTION
■
Carbon monoxide (CO) is a small gaseous molecule known for
its toxicity, which stems from its higher affinity for hemoglobin
than oxygen resulting in the formation of carbonylhemoglobin
(COHb) and subsequent hypoxia.1 On the other hand, CO is a
naturally occurring cell-signaling molecule that exhibits strong
cytoprotective,2 cardioprotective,3 anti-inflammatory,4 and
anti-microbial effects5 at submicromolar concentrations (0.2
μM).6 CO was also shown to induce an anti-Warburg effect.7
Cancer cells and tumors exposed to CO are compelled to
rapidly fuel oxidative metabolism, imposing extreme oxidative
stress on cells, leading to growth inhibition, cellular exhaustion,
and eventually death.8 The synergistic effect between CO and
chemotherapeutics is especially attractive as demonstrated by
an increased sensitivity of cancer cells toward camptothecin
and doxorubicin upon exposure to CO, while simultaneously
protecting normal cell growth and viability.8
Despite its remarkable therapeutic potential, one of the
central challenges associated with CO therapy is controlling its
local biological concentrations. This necessity has spurred the
development of CO-releasing molecules (CORMs). Enzymati-
cally triggered processes9 or a solvent-mediated ligand
exchange6 usually result in a poor spatial and temporal control
over the release profile. On the contrary, prevalent CO-
releasing metal−carbonyl complexes10−12 retain a metal
backbone upon CO release, which can lead to unwanted
reactions with adjacent cells.13
Berreau and co-workers have introduced a new type of
photoCORM, 3-hydroxy-2-phenyl-4H-chromen-4-one (1, Fig-
ure 1), based on a 3-hydroxyflavone (flavonol) motif, which
abs
max
efficiently releases CO upon irradiation with visible light (λ
> 400 nm) under biologically compatible conditions.21 This
structure belongs to the family of flavonoids that are well-
known natural antioxidants due to their ability to react with
different reactive oxygen species.22−24 3-Hydroxyflavone
derivatives were reported to undergo an oxygen-mediated
enzymatic25 reaction to release CO in bacteria and fungi. CO
can also be produced from these compounds upon irradiation
with UV light.26,27
3-Hydroxyflavone as an archetypal molecule exists in both
acid (2A) and base (2B) forms (Figure 1), and their
Received: December 2, 2019
Recently, transition-metal-free light-activated CORMs (pho-
toCORMs) have emerged with the promise of circumventing
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RESULTS AND DISCUSSION
Steady-State Spectroscopy. 3-Hydroxy-2-phenyl-benzo-
■
[g]chromen-4-one 1 exists as an acid (1A) form or a base (1B)
form (Scheme 2) in protic solvents.40 The phenolic hydrogen
Scheme 2. Acid−Base Equilibrium of 1: Formation of
1
Excited Tautomer 1Z*
Figure 1. 3-Hydroxyflavone derivatives and the photoproducts
obtained by irradiation of 2.
photophysics and photochemistry have already been inves-
1
tigated in detail. The singlet excited state 2A* undergoes
rapid (<125 fs) excited-state intramolecular proton transfer
(ESIPT) resulting in the formation of a Stokes-shifted emitting
phototautomer (zwitterion) 12Z* (Scheme 1).28−31 Mat-
Scheme 1. Excited-State Intramolecular Proton Transfer
upon Irradiation of 2A
acidity of 2A in the electronic ground state in an ethanol/water
(1:1) solution was determined to be pKa = 9.60 (and pKa =
9.81, when extrapolated to pure water) by Rodembusch and
co-workers.41 In this work, we found the pKa of 1 in a water/
DMSO (1:1) mixture to be 9.25 0.03 (the value is corrected
for this solvent mixture;42 Figure 1a). The absorption
spectrum of 1A in this solvent shows two major bands at
abs
λ
= 344 and 401 nm (Figure 2a, black line), whereas that of
max
abs
1B exhibits a single band with λ = 472 nm (Figure 2a, red
max
line). Owing to the poor solubility of flavonol 1 in aqueous
media and especially the need to use highly concentrated
samples for transient spectroscopy measurements, further
experiments were carried out in methanol as a solvent. The
conjugate acid 1A is the sole species observed in a methanol
solution. Figure S7 depicts the transition of 1A to 1B upon the
addition of methanolic sodium methoxide, the spectra of which
suura32−34 and later Chou35,36 and their co-workers have
studied the mechanism of CO photoproduction in the
presence and absence of molecular oxygen. Several different
reaction pathways in solvents of different polarity have been
proposed to show that ground-state oxygen, as well as excited
(singlet) oxygen, can act as an oxidant in the production of
salicylic acid ester 3 and CO, whereas a lactone (4) along with
CO is formed under anaerobic conditions (Figure 1).
abs
abs
are essentially identical (1A, λ = 345 and 399 nm; 1B, λ
=
max
max
474 nm; Figure 2b) to those observed in water/DMSO.
The emission spectrum of the conjugate acid 1A in
methanol exhibits two distinct bands: a very weak fluorescence
em
max
em
max
band at λ = 472 nm and an intense band at λ = 595 nm
The π-extended 3-hydroxyflavone derivative 1 and its
derivatives, proposed and studied by Berreau and co-workers,
represent photoCORMs absorbing at longer wavelengths than
2 and releasing CO in high chemical and quantum
yields.20,21,31,37−39 In addition, incorporating logic gate features
such as thiol sensing and anti-cancer/anti-inflammatory effects
of CO photoreleased from this scaffold has been demon-
strated.31,37 The effects of solvent polarity or the presence of
oxygen, metal ions, and proteins20 has been studied to
rationalize the biological applications of 1 as a photoCORM.
The mechanism of CO liberation from 1 was assumed to be
similar to that of 2, but it has not been studied in detail yet.
In this paper, we report a kinetic study of the photo-
chemistry of both the acid and base forms of 1 in methanol
using steady-state, femtosecond pump−probe and nanosecond
laser flash spectroscopies and quantum chemical calculations.
We present a comprehensive mechanistic scheme to reveal the
multiplicity of the productive excited states and to emphasize
and specify the role of oxygen (O2) in the CO production.
(Table 1; Figure 2b). The former signal corresponds to the
“normal” excited-state 11A*, whereas the latter one is assigned
as a phototautomer (zwitterion) 11Z* on the basis of
spectroscopic studies with parent flavonol 2A (Scheme 1). A
Stokes shift of over 200 nm (Figure 2b) for this species is the
result of an efficient excited-state intramolecular proton
transfer (ESIPT), identified and studied for 3-hydroxyflavone
2A28,30,43 and also for its π-extended analogue 1.20 The
fluorescence quantum yields were determined for each
emission band to show that the emission from the photo-
tautomer is approximately 30 times more intense than that
1
from 1A*. The emission spectrum of 1B displays only a
single, less intense emission band,44,45 with λ at 637 nm
em
max
(Table 1, Figure 2b).
Photochemistry. The photochemistry of both 1A and 1B
(1.5 equiv of NaOCH3) in methanol solutions (irradiated at
λirr = 405 and 450 nm, respectively) was investigated, and the
course of the reaction was monitored using UV−vis absorption
spectroscopy (Figure 3). The disappearance quantum yield of
B
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Figure 2. (a) Spectroscopic determination of the pKa of 1 in water/
DMSO (1:1), depicting two distinct absorption bands of 1A (pH =
6.1, black bold line) and 1B (pH = 12.3, red bold line) with an
isosbestic point at 427 nm. Volume changes in the titration were
corrected. (b) Absorption (solid lines; left ordinate) and normalized
emission (dashed lines; right ordinate) spectra of 1A (black; in neat
solvent) and 1B (red; with 1.5 equiv of NaOCH3) in methanol.
Figure 3. Irradiation of (a) 1A at λirr = 405 nm (the absorption
spectra were taken every 5 s) and (b) 1B at λirr = 450 nm (the
absorption spectra were taken every 30 s) in aerated methanol. The
spectra prior to (red line) and after (blue line) irradiation are
highlighted.
headspace to monitor the photoproducts: 3-(benzyloxy)-2-
naphthoic acid 5, 3-phenylnaphtho[2,3-c]furan-1(3H)-one 6,
and CO (Table 2), which were the same species identified by
Berreau and co-workers before38 and were analogous to those
produced upon irradiation of flavonol 2.34,35 In this study, it
was essential to evaluate the photoreaction under the same
conditions as those used in time-resolved spectroscopy studies.
The maximum chemical yields of 5 and CO in irradiated
aerated methanol solutions of 1 were 91 and 80%, respectively,
which were the magnitudes comparable to those found for the
photochemistry of 1 in acetonitrile.38 In contrast, the yields of
lactone 6 and CO formation in degassed samples were
moderate (52−55%; Table 2). Lower chemical yields indicate
competing photoreactions, but no other specific side products
were identified by our analytical methods. The presence of
oxygen alters the reaction mechanism, as is also supported by
the labeling experiments of Berreau, providing evidence that
1A in an aerated solution was found to be Φr = (3.1 0.1) ×
10−2 (Table 2). It compares well to that reported for the
photochemistry of 1A in an acetonitrile solution, Φr = (0.7
0.02) × 10−2.38 Practically no absorption above 300 nm
remained after exhaustive photolysis. The photolysis of 1A in a
methanol solution degassed by three freeze−pump−thaw
cycles was more than 2 orders of magnitude less efficient
(Φr ∼ 1.2 × 10−4), demonstrating a profound influence of
oxygen on the reaction course (Figure S12). In addition, the
photodegradation of 1B (1.5 equiv of NaOCH3) in aerated
methanol at λirr = 450 nm was nearly 3 orders of magnitude
more efficient (Φr = (2.1 0.1) × 10−2) than that in a solution
degassed by three freeze−pump−thaw cycles (Φr ∼ 1.0 ×
10−5; Table 2).
The samples of 1 in methanol solutions were irradiated to
complete conversion and were analyzed by HPLC and GC-
Table 1. Photophysical Properties of 1A and 1B in Methanol
a
a
b
b
b
compound
λambasx/nm
ε/(M−1 cm−1
)
λemmax/nm
ΦF
τF/ns
1A
344, 401
472
9720
472 (11A*)
595 (11Z*)
637
0.024 0.004 (11A*)
0.72 0.06 (11Z*)
0.16 0.01
5.1 1.6
3.2 0.9
1B
10800
c
c
0.15
2.2
a
b
c
c = 1 × 10−4 M. c = 1 × 10−5 M. From ref 45.
C
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Table 2. Photochemistry of 1 to Give Photoproducts 5 and 6
a
b,
c
b
,c
b,
c
d
e
compound
Φr/10−2
yield of 5/%
yield of 6/%
yield of CO/%
ΦΣ
ΦΔ
b
b
b
1A
3.1 0.1
91
8
80
52
5
3
ineff
0.14 0.01
c
c
c
0.012
3.1
55
30
5
e
b
b
b
c
1B
2.1 0.1
55
5
55
30
3
4
0.26
0.07 0.01
c
c
0.00
4
f
0.28
a
b
The disappearance quantum yield of 1 (c = 1 × 10−4 M) upon direct irradiation. Formation chemical yields of the corresponding photoproducts
c
d
in aerated solutions. Formation chemical yields of the corresponding photoproducts in degassed solutions. The quantum yield of 1 disappearance
e
f
in the presence of 1O2 generated by excited rose bengal; ineff = inefficient. The quantum yield of singlet-oxygen production from excited 1. The
disappearance quantum yield measured in the presence of a large excess of a singlet-oxygen trap (furfuryl alcohol; 0.01 M).
a
Table 3. Photochemistry of 1A and 1B in Methanol
b
c
d
e,f
compound
τS/ps
11 (11Z*)
k
ISC/s−1
ΦISC
τT/μs
e
f
1A
(3.94 0.43) × 109
0.23 0.02
n.d.
0.1421 0.0003
1.630 0.008
f
e
1B
(3.47 0.21) × 109
n.d.
0.0052 0.0004
a
b
c
d
Data obtained by TR spectroscopy measurements. Singlet-state lifetime obtained by femtosecond spectroscopy. The rate constant of ISC. ISC
quantum yield.51 Triplet-state lifetime obtained by nanosecond spectroscopy in aerated methanol solutions. Triplet-state lifetime obtained by
e
f
nanosecond spectroscopy in degassed methanol solutions.
1
the incorporated oxygen atoms in the product 5 originate from
molecular oxygen.38 The same photoproducts as those in the
case of 1A irradiation, including CO, were detected upon
irradiation of both aerated and degassed methanol samples of
1B, albeit the chemical yields were generally lower (Table 2).
Such results were anticipated because Chou and co-workers
identified the corresponding photoproducts upon irradiation of
3-hydroxyflavone 2B.35 Again, no other photoproducts were
found in this case, and because the maximum CO yields match
those of the related side products, we imply that the mass loss
is related to different competing photoprocesses.
parent flavonol 2B was shown to react with O2 in a basic aq
solution with a similar rate constant of kΣ = 2.2 × 108 M−1 s−1,
whereas its conjugate acid 2A was found to be only weakly
reactive (kΣ = 2.5 × 105 M−1 s−1).35 In the presence of a large
excess of FFA (c = 1 × 10−3 M), the Φr for 1A remained
unchanged, but it decreased by a factor of 7 for 1B (Φr = from
2.1 × 10−2 to 2.8 × 10−3; Table 2), which implies that the
starting material was degraded by self-sensitized photo-
oxygenation (see later). The maximum chemical yields of
both 5 and CO produced from 1B in the presence of 1O2 were
found to be ∼60%, thus comparable to those obtained by
direct irradiation of 1B in aerated methanol.
As we demonstrated above, the role of oxygen (O2) in the
kinetics of CO production from pure acid and base forms of 1
is significant. Although oxygen can act as a triplet-state
quencher, it can also be a reagent (oxidant) in its ground (3O2)
Production of Singlet Oxygen. Singlet-oxygen produc-
tion from the excited forms 1A and 1B is direct evidence for
the involvement of their triplet excited states. Such a result
would also be important for the CO production itself because
1O2 hereby formed could subsequently react with the ground-
state 1B (and eventually inefficiently with 1A), as demon-
strated in the previous paragraph. The quantum yield of
1
and excited (singlet oxygen, O2) states. Therefore, we aimed
our research to discriminate the individual effects of O2 on the
reaction.
Reaction with Singlet Oxygen. We used rose bengal
1
(RB) as an auxiliary O2 generator to learn whether the acid
3
singlet-oxygen production (ΦΔ) by triplet excited 1A* was
and base forms of 1 in the ground state can react with singlet
oxygen. Irradiation of RB (λirr = 572 nm) in the presence of the
acid 1A in methanol resulted in a very inefficient (and
incomplete) degradation even in a considerably prolonged
experiment (16 h; Figure S22). On the contrary, the base 1B
disappeared within minutes under the same conditions (Figure
S23). The quantum yield of 1B disappearance, ΦΣ = 0.26,
determined using furfuryl alcohol (FFA) as a singlet-oxygen
trap.48 DPBF could not be used because of its significant
spectral overlap with 1A. Thus, a mixture of 1A and FFA in
aerated methanol was irradiated to complete conversion, and at
the same time, 1A was used as an actinometer because its Φr
1
was determined independently (Table 2). The O2 photo-
1
production was found to be relatively efficient, ΦΔ = 0.14
0.01. Because 1A was found essentially unreactive toward
singlet oxygen, the self-sensitized photooxygenation pathway
was excluded, and we concluded that O2 produced by 1A*
was not involved in the CO release from 1A upon irradiation.
The determination of ΦΔ for 1B was problematic because of its
demonstrated its high reactivity toward O2. Using furfuryl
alcohol (FFA; kΣ = 1.03 × 108 M−1 s−1) and 1,3-
diphenylisobenzofuran (DPBF; kΣ = 1.1 × 109 M−1 s−1)46 as
singlet-oxygen traps, we determined the rates of the reaction of
1
3
1
both 1A and 1B with O2 to be 4.3 × 105 and 4.7 × 108 M−1
s−1, respectively.47 For comparison, the conjugate base of
D
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high reactivity toward 1O2 and thus a rapidly changing reaction
composition (the absorbance of the irradiated solution).
Therefore, a large excess of DPBF and short irradiation
times were utilized to mitigate this issue. A solution of 1B was
irradiated at the tail of its absorption band at 507 nm, and the
nm within 100 fs after the excitation (Figure 5). It was assigned
to the singlet excited tautomer 1Z*, generated rapidly from
1
1O2 production quantum yield of ΦΔ = 0.07
0.01 was
obtained (Table 2).
Time-Resolved Spectroscopy. Time-resolved (TR) spec-
troscopy was used to identify short-lived reaction intermediates
produced from both 1A and 1B and to determine rate
constants of the elementary reaction steps.
Nanosecond time-resolved spectroscopy of 1A in aerated
methanol (c ∼ 10−4 M; λexc = 355 nm; 1A has a strong
absorption band at ∼400 nm) revealed a transient signal with
λmax = 440 nm. The lifetime of this species was strongly
dependent on the concentration of oxygen. The kinetic traces
were fitted with a first-order rate law (the oxygen
concentration in an aerated methanol solution at 20 °C is
almost 30 times higher, ∼2.2 × 10−3 M,49 than that of 1A) to
provide the lifetime of 142 ns in aerated methanol (Table 3;
Figure S28). The lifetimes in oxygen-saturated and degassed
methanol were determined to be 56 ns and 1.63 μs,
respectively. The triplet-state lifetime of parent flavonol 2A
measured in methylpentane of τT = 7.8 μs has been determined
before.50 We first attempted to assign the signal to either triplet
3
3
1
excited 1Z* or 1A*. In the latter case, 1Z*, formed from
11A* via ESIPT (see above), would have to undergo a reverse
proton transfer during intersystem crossing to give 31A*.
Furthermore, a fluorescence signal up to 20 ns after the
excitation pulse (λmax = 600 nm, Figure 4) was still detectable
in these experiments, which is consistent with the relatively
long fluorescence lifetime measured by a steady-state
technique (τF = 5.1 ns; Table 1).
Figure 5. Femtosecond transient spectroscopy of 1A (c ∼ 1.0 × 10−3
M in methanol; excitation by a 387 nm laser pulse): (a) spectra
obtained from 100 fs to 19 ps after the pulse; (b) spectra obtained
from 5 to 500 ps after the pulse (initial state = red line; end state =
blue line).
11A* (see above) via an analogous process observed for the
excited tautomer of parent 3-hydroxyflavone 2.29 The
corresponding decay kinetic data were fitted with a first-
order rate law to provide the 11Z* lifetime of 11 ps (Table 3).
In the case of parent 3-hydroxyflavone 2, Harris and co-
workers observed a fast ESIPT (240 fs) in nonpolar solvents,
whereas the transfer was assumed to be much faster in
methanol (<125 fs; faster than their instrument response).29
1
The intersystem crossing rate constant of 1Z* to give triplet
3
excited state 1Z* (λmax at 440 nm), the species assigned by a
ns TR spectroscopy, was kISC = (3.94 0.43) × 109 s−1 (Table
Figure 4. Transient absorption spectra of 1A in aerated methanol (c =
8 × 10−5 M) taken after a 355 nm flash at different delay times (black
= 10 ns, red = 20 ns, blue = 100 ns, magenta = 1 μs).
3).
The femtosecond spectroscopy of 1B in methanol (λexc
=
475 nm) provided quite different results from those of 1A
because the compound cannot undergo ESIPT. The first signal
with λmax = 543 nm was assigned to the excited singlet state
11B*. Its depletion with concomitant formation of a new band
with λmax = 549 nm was attributed to intersystem crossing and
Nanosecond time-resolved spectroscopy of 1B in both
aerated and degassed methanol (λexc = 355 and 532 nm; 1B
has a strong absorption band at ∼472 nm) did not show any
signal of transient species (Figures S32, S33, and S34). We
3
3
assumed that the 1B* lifetime is too short to be determined
the new transient to 1B* (Figure 6). The kinetic traces were
by our nanosecond equipment; therefore, a femtosecond
pump-and-probe technique was used for its evaluation.
Femtosecond TR absorption experiments with a 1A
methanol solution (λexc = 387 nm) provided transient-
absorption spectra, which possessed a signal with λmax at 531
fitted by a first-order rate law to give the rate constant of (3.47
0.21) × 109 s−1, representing the rate constant of the triplet-
state formation.
DFT Calculations for 1A and 1Z. We calculated the
absorption maxima and excited-state energies for species 1A
E
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́
Figure 7. Jabłonski diagrams of (a) 1A and (b) 1B photochemistry in
aerated (aer) methanol solutions. The adiabatic-state energies were
calculated.
DFT Calculations for 1B. We also calculated the vertical
excitation energies of three lowest excited states of 1B (Table
S2), which gives the transition energies at 2.127 eV (583 nm),
2.852 eV (435 nm), and 3.236 eV (383 nm). Contrary to the
results for 1A, these calculations gave larger errors when
compared to the experimental data; in particular, the
absorption energy of the lowest excited state was lower by
∼0.5 eV. Such an underestimation of the absorption energies
of an anionic form has also been observed for 3-
hydroxyflavone.53 The excited-state 11B* relaxes to its
minimum at 1.878 eV above the ground-state minimum
(Table S3), and the predicted emission energy from this state
at 717 nm compares reasonably well to the experimental value
of 637 nm (Table 1 and Table S3). These data are in accord
with those obtained for 3-hydroxyflavone 2.52 The correspond-
Figure 6. Femtosecond transient spectroscopy of 1B (c ∼ 1.0 × 10−3
M in methanol; excitation by a 475 nm laser pulse: (a) spectra
obtained from 1 to 20 ps after the pulse; (b) spectra obtained from 3
ps to 3.1 ns (initial state = red line; end state = blue line).
and 1Z at the DFT level employing a B3LYP functional. The
ground-state energy of 1A was found to be lower than that of
1Z by 0.52 eV (11.98 kcal mol−1), which has also been
reported for 3-hydroxyflavone 2A (Figure 7).52 The calculated
excitation energies (Table S2) of first two excited states were
2.985 and 3.532 eV (f = 0.21 and 0.28 for S1 and S2 states,
respectively), which is in very good agreement with the
observed maxima of ∼3.1 and 3.6 eV (401 and 344 nm,
respectively; Table 1). The calculations of the S1 excited-state
3
ing triplet-state 1B* was found lower in energy than that of
11B* by 0.706 eV.
Photorelease Mechanism. Our experimental results
showed that the triplet excited state formed from 1A must
exclusively be responsible for the formation of CO and
photoproducts. Upon irradiation, 1A in a methanol solution is
1
1
minima for both 1A* and 1Z* resulted in stable structures
(Table S3); the latter conformer is more stable by 0.26 eV
(Tables S3 and S5). The calculated emission wavelengths of
1
excited to its singlet excited state 1A*, which is converted to
excited phototautomer 11Z* in less than 100 fs. This species is
highly fluorescent; the total emission quantum yield from both
excited tautomers is ∼0.74 (ΦF(11Z*) = 0.72; Table 1). The
subsequent fast 31Z* formation (kISC = (3.9 0.4) × 109 s−1)
is efficient (ΦISC = 0.23; Table 3). The photoreaction quantum
yield (Φr) of 0.03 found for aerated solutions is related to the
formation of CO and naphthoic acid derivative 5 as the
exclusive photoproducts obtained from 31Z*. This species also
sensitizes oxygen to form 1O2 with ΦΔ = 0.14 (Table 2) under
the given experimental conditions (the initial O2 concentration
in methanol is ∼2.2 × 10−3 M49). The remaining triplet
undergoes intersystem crossing to give the ground-state
tautomer, which slowly regenerates36 the starting material
1A. The Φr value did not change in the presence of a large
1
1
481 and 670 nm for 1A* and 1Z*, respectively, are in good
harmony with the experimental values of 472 and 595 nm
(Table 1), and they support the assignment of the ESIPT
1
process responsible for the formation of 1Z*. In addition, we
evaluated the relative triplet energies of species 31A* and 31Z*,
showing that the latter zwitterionic state is lower in energy by
0.48 eV (Table S4). This allowed us to support our
conclusions that the observed triplet excited state is the
3
tautomer 1Z*. The vertical T1 → Tn (n = 2−6) transition
energies were calculated to compare them with those found by
our transient spectroscopy. The T1 → T3/T4/T5/T6 values for
31Z* of 529, 526, 490, and 465 nm (Table S4), respectively,
could be assigned to the bands in the region of 450−550 nm
(Figure 4).
1
excess of FFA as a O2 trap (Table 2); thus singlet oxygen,
F
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3
produced by 1Z* sensitization, cannot be responsible for the
rearrangement to give lactone 6 as the only identified side
product (Scheme 3b). This reaction most probably proceeds
via an intermediate 8, proposed by Yokoe58 and computation-
ally predicted by Kubinyi57 and their co-workers in the case of
3-hydroxyflavone 2 (Scheme S1). Because the oxygen
sensitization pathway is not available and the reaction quantum
yield is insignificant, 31Z* must efficiently regenerate the
starting material or be destroyed via an even less efficient
chemical process. This reaction pathway represents an
unimportant side process in the overall mechanism, and it is
not observed in aerated solutions.
3
formation of the photoproducts. The reaction of 1Z* with
ground-state oxygen (3O2) is a spin-allowed process; in
1
contrast, the ground-state (normal form) 1A reacts with O2
very slowly and inefficiently. The only detected photoproducts,
5 and CO, are formed in very high chemical yields; no
detectable amounts of photoproduct 6 were found in aerated
solutions. The mechanism of the final photooxygenation step
has been a subject of investigations with parent flavonol
2.32−35,38,53−56 Chou and co-workers proposed that the
reaction of triplet phototautomer 32Z* with ground-state
oxygen takes place via the formation of an endoperoxide
intermediate.35,53−55 Recent DFT calculations by Kubinyi and
co-workers gave evidence that the addition of O2 on 2Z*
gives an endoperoxide and not a hydroperoxide as an
intermediate.57 Inspired by these studies, we show an
analogous endoperoxide intermediate 7, formed upon the
The photochemistry of 1B is fundamentally different from
that of 1A because it cannot undergo phototautomerization.
The singlet excited state 11B* is only weakly fluorescent (ΦF =
0.16; τF = 3.4 ns), and it is rapidly ((3.47 0.21) × 109 s−1;
Table 1) converted to its triplet state (31B*). Unfortunately,
we were unable to determine ΦISC because its lifetime was too
short to be detected by our nanosecond LFP apparatus, but we
estimated an upper limit for ΦISC to be as high as 0.92 (thus, a
3
3
3
reaction of 1Z* with ground-state oxygen, in Scheme 3a; the
1
radiationless decay for 1B* is negligible). The triplet excited
Scheme 3. Major Reaction Pathways in the Photochemistry
of (a) 1A and (b) 1B under Aerobic (Red) and Anaerobic
(Blue) Conditions in Methanol
31B* sensitizes oxygen with ΦΔ = 0.07 (Table 2). The
irradiation of 1B in aerated methanol gives the same
photoproducts (5 and CO) as that of 1A with a similar
efficiency (Φr = 2.1 × 10−2) but lower maximum chemical
yields (55%). Thus, the triplet and probably singlet excited
states must undergo an efficient radiationless decay to
regenerate the starting material. The stark difference between
the photochemistry of 1A and 1B is represented by the
contrasting reactivity with singlet oxygen. 1B reacts more
1
efficiently with O2 (ΦΣ = 0.26; Table 2) than 1A by more
than 3 orders of magnitude, presumably because the addition
1
of electrophilic O2 onto the electron-rich anion core of the
ground state of 1B is favorable. The ΦΣ value of 2.0 × 10−2 for
1B calculated from ΦΔ, the bimolecular reaction rate of 1B
with 1O2 (kΣ), and the rate constant of 1O2 decay (kd) using a
steady-state approximation (see Supporting Information) is
comparable to the experimentally determined value of Φr =
(2.1 0.1) × 10−2 found for direct irradiation of 1B. At the
same time, we demonstrated that the chemical yield of CO was
unaffected by the type of reaction initiation (direct irradiation
versus RB sensitization). In an important experiment, the Φr
value considerably decreased in the presence of a large excess
1
of FFA as an O2 trap (Table 2). The Φr decrease from 2.1 ×
10−2 to 2.8 × 10−3 (Table 2) provides an inherent quantum
yield for the reaction of 1B with 1O2 under the given
conditions (1B and O2 concentrations in the sample), thus
(2.1 × 10−2) − (2.8 × 10−3) gives Φr of ∼1.8 × 10−2. The Φr
value of 2.8 × 10−3 must be related to the reaction of the triplet
31B* with ground-state oxygen (O2), representing the
efficiency as still 2 orders of magnitude larger than that of
the reaction under anaerobic conditions. Therefore, CO and
acid 5 are not formed exclusively by the reaction of ground-
state 1B with 1O2 generated by self-sensitization. The
mechanism of the photooxygenation of 3-hydroxyflavone 2B
has also been suggested to proceed via an endoperoxide
intermediate by Chou and co-workers (Supporting Informa-
tion),54 analogous to species 7 shown in Scheme 3b. The
previously studied mechanism of 2Z photooxygenation is
provided in Scheme S1. We conclude that the reaction of 31Z*
with 3O2 is a nearly exclusive pathway leading to these
3
photoproducts and that sensitization of oxygen by 1Z* is a
́
́
nonproductive process. Figure 7a shows a Jabłonski diagram
Jabłonski diagram for the 1B photochemistry is shown in
constructed from all our experimental and computational data.
Upon irradiation of 1A in a degassed methanol, the first fast
steps of the mechanism are identical to those of 1A in an
aerated solution. Upon the formation of triplet excited34 31Z*,
the compound undergoes a very inefficient (Φr = 1.2 × 10−4)
Figure 7b.
In the absence of oxygen, the formation of naphthoic acid
3
derivative 5 from 1B* is not observed; instead, CO and
lactone 6 are produced very inefficiently (Φr ∼ 10−5). These
photoproducts suggest a similar reaction pathway to that of
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31A*. This reaction is again insignificant in the overall
phototransformation.
Absorption spectra and the molar absorption coefficients were
obtained on a UV−vis spectrometer with matched 1.0 cm quartz cells.
Molar absorption coefficients were determined from the absorption
spectra (the average values were obtained from three independent
measurements with solutions of different concentrations). Fluores-
cence was measured on an automated luminescence spectrometer in
1.0 cm quartz fluorescence cuvettes at 23 1 °C. The corresponding
optical filters were used to avoid the second harmonic excitation/
emission bands induced by the grating. The samples of concentration
with the absorbance of ∼0.1 at the excitation wavelength were used.
Each sample was measured five times, and the spectra were averaged.
Emission and excitation spectra are normalized. Fluorescence
quantum yields were determined using an integration sphere as the
absolute values. For each sample, the quantum yield was measured
In the case of a mixture of 1A and 1B that forms at different
solution pHs, we have a collection of three major reaction
pathways involving both forms, all of which lead to CO
formation but with considerably different efficiencies and
oxygen demands, but only if both forms can be excited. The
common denominator for these processes is the triplet
multiplicity of the productive excited state. The presence of
two orthogonal reaction pathways for 1A and 1B thus implies
additional considerations for solutions where the concen-
trations of both species vary. The same photoproducts, 5 and
CO, will be produced by the reactions of triplet excited
1
five times and then it was averaged. H NMR spectra were recorded
3
3
3
intermediates 1Z* and 1B* with O2, whereas 1B will be
on 300 or 500 MHz spectrometers; 13C NMR spectra were obtained
1
1
substantially depleted by oxygenation with O2. Considering
on 125 or 75 MHz instruments in CDCl3, CD3OD, and D2O. H
just an acid−base equilibrium, the amount of CO generated
from 1B will increase with increasing acidity of the 3-
hydroxyflavone analogue or increasing pH of the solution. It
will also be strongly dependent on the emission spectrum of an
irradiation source due to considerably different absorption
spectra of both acid−base forms. Although other side reactions
may always compete (Table 2), they represent only a negligible
sink.
chemical shifts are reported in ppm relative to tetramethylsilane (δ =
0.00 ppm) using the residual solvent signal as an internal reference.
13C chemical shifts are reported in ppm with CDCl3 (δ = 77.67 ppm)
and CD3OD (δ = 49.30 ppm) as internal references. Deuterated
solvents were kept under a nitrogen atmosphere. The exact masses of
the synthesized compounds were obtained using a triple quadrupole
electrospray ionization mass spectrometer in a positive or negative
mode coupled with direct-inlet or liquid chromatography.
Spectrophotometric Determination of pKa. A freshly prepared
solution of 1A (c ∼ 8.5 × 10−5 M) in DMSO/H2O (1:1; 2.5 mL, I =
0.1 M) was transferred into a matched 1.0 cm quartz cuvette and its
UV−vis absorption spectrum was recorded. The solution was basified
by the addition of small aliquots of aq NaOH (typically 10 μL; 0.5
and 0.1 M), and the pH and UV−vis absorption spectra were
recorded after each addition. Volume changes in the titration were
corrected. The pKa value was determined by deconvolution using a
multivariate analysis.
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 stirring bar was
stirred and irradiated with a light source of 32 LEDs (λirr = 405 and
450 nm; Figure S37). The progress of the reactions was monitored at
the given time intervals by UV−vis spectrometry using a diode-array
spectrophotometer. In some experiments, light pulses (425.6 Hz
repetition rate, ≤150 fs pulse length, and energy of ∼7.5 0.3 mW)
from a Ti:sapphire laser coupled to a noncollinear optical parametric
amplifier (NOPA) with the wavelength set to 505 nm (fwhm ∼15
nm) were used to irradiate the samples.
CONCLUSIONS
■
Our experimental results revealed a reaction dichotomy of
acid−base forms of 3-hydroxy-2-phenyl-4H-chromen-4-one,
1A and 1B, manifested by three major orthogonal decarbon-
ylation pathways, which impose serious ramifications for the
future development of novel 3-hydroxyflavone-based photo-
CORMs. The reaction of the triplet excited tautomer of the
conjugate acid 1A, 31Z*, with 3O2 is a nearly exclusive reaction
pathway leading to the release of CO and photoproducts,
whereas 1A is nearly unreactive toward singlet oxygen,
3
produced from 1Z* sensitization as a side product. On the
contrary, the conjugate base 1B provides CO via an efficient
oxygenation reaction with singlet oxygen formed by sensitiza-
3
tion of 1B*; therefore, the major CO-releasing pathway is a
self-sensitized photooxygenation. CO can also be liberated by
3
3
very inefficient photorearrangements of both 1Z* and 1B*.
Because the triplet excited states have been established as
productive states, increasing the quantum yield of intersystem
crossing is desirable to improve the efficacy of the CO
Fluorescence Measurements. Fluorescence and excitation
spectra were measured using a fluorescence spectrometer in a 1.0
cm quartz fluorescence cuvette at 23
1 °C. The sample
1
concentrations were adjusted to keep the absorbance below 0.2 at
the corresponding excitation wavelength. Each sample was measured
five times, and the spectra were averaged. Emission and excitation
spectra were normalized and corrected by the photomultiplier
sensitivity function using correction files supplied by the manufac-
turer.
liberation. The intramolecular reaction rate of 1B with O2,
which is an order of magnitude below the diffusion limits,
brings another chance to significantly improve the CO release
quantum yield. Most importantly, 1B might also serve as an
attractive lead structure to achieve CO release using light in the
near-infrared region by harnessing its high affinity toward 1O2.
Schnermann has shown such an application in self-sensitized
photooxidation of near-infrared light-activated cyanine pho-
tocages and demonstrated the feasibility of this approach for
targeted in vivo delivery.59,60 The understanding of the
mechanism will guide future endeavors to improve the CO
release efficiency by 3-hydroxyflavone chromophores mod-
ifications and may help in their potential medical applications
as photoCORMs.
Decomposition Quantum Yield Determination. The decom-
position quantum yields of 1A in an aerated methanol solution were
determined using ferrioxalate (c = 1.0 × 10−5 M) in aqueous
phosphate buffered saline (PBS, I = 0.1 M, pH = 7.4) as an
actinometer, using a Xe lamp fitted with a monochromator (λirr = 400
2 nm). The subsequent quantum yields of decomposition were
determined relative to 1A using an LED source (λirr = 405 or 420 nm;
Figure S37).
Singlet-Oxygen Production Quantum Yields. A solution of
1,3-diphenylisobenzofuran (DPBF; c = 1 × 10−4) and either 1B (c = 5
× 10−5 M) or rose bengal (RB) as sensitizers (c = 5 × 10−6 M) in
methanol was prepared. The stirred solution (3.5 mL) in a quartz cell
(1 cm) was irradiated using LEDs at 507 nm, and the UV−vis spectra
were recorded periodically. The irradiation period was selected to
reach around 10% conversion of DPBF. The procedure was repeated
five times. The decomposition of DPBF monitored at 411 nm was
EXPERIMENTAL SECTION
■
Materials and Methods. Reagents and solvents of the highest
purity available were used as purchased, or they were purified/dried
using standard methods when necessary. The compounds 1, 5, and 6
were synthesized according to the published procedures.20,38,61,62
H
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fitted with a pseudo-first-order rate law, and the singlet-oxygen
formation quantum yield ΦΔ was calculated using RB as the reference
(ΦΔ = 0.7663).
the two beams. The pump−probe cross-correlation was <100 fs over
the entire spectrum. The measurements were performed at ambient
temperature (20 2 °C). The kinetic traces were fitted by single or
multiple exponential decay functions. In the case of the second-order
kinetics, the corresponding differential equations were solved using
the Levenberg−Marquard minimization algorithm.64
Quantum Chemical Calculations. The calculations of ground,
singlet (S1), and triplet (T1) excited state minima have been
performed with the clusters that consisted of 1A, 1Z, and 1B species
and one methanol molecule explicitly included in the quantum
mechanical description to account specifically for the proton transfer
in both ground and excited states. The additional solvent effects have
been modeled for by means of the polarizable continuum model
(PCM).65 The calculations were carried out at the DFT level
employing a B3LYP functional;66,67 a TD approach was used in the
calculations of the excited states.68,69 All calculations were performed
with a TZVP basis set.70,71 The same level of theory was also used for
calculations of all absorption spectra. The Gaussian 16 program
package was used for the calculations.72
A methanol solution containing furfuryl alcohol (c = 1.0 × 10−3 M)
and 1A (c = 1.0 × 10−4 M) was stirred and irradiated using LEDs at
405 nm until the 1A disappearance was complete. The amount of
FFA consumed by singlet oxygen was determined by HPLC using a
reported method.48 The quantum yield of the singlet-oxygen
production was calculated from Φr of 1A and a bimolecular rate
constant of the reaction of FFA with 1O2 in methanol (kr = 1.03 × 108
M−1 s−1).
Reaction of 1A and 1B with 1O2. Solutions containing 1,3-
diphenylisobenzofuran (DPBF; c = 2 × 10−4 M), 1A (c = 1 × 10−4 M)
or 1B (c = 1 × 10−4 M), and RB (c = 5 × 10−6 M) as a singlet-oxygen
sensitizer in methanol were prepared. A stirred solution (3.5 mL) in a
quartz cell (1.0 cm) was irradiated with an LED at 590 nm (Figure
S37), and UV−vis absorption spectra were recorded at the given
intervals. The rate constant of DPBF and of the 1A and 1B
decompositions were determined from the decay at the corresponding
absorption maxima (λmax). The bimolecular reaction rates of the
1
reaction of 1A or 1B with O2 were calculated using the known rate
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.joc.9b03248.
■
constant of singlet oxygen quenching in methanol (kd = 9 × 104 s−1)
and the bimolecular reaction rate constant of DPBF with singlet
oxygen (kr = 1.2 × 109 M−1 s−1).46
sı
Determination of the Photoproducts. A solution of 1A or 1B
(3.5 mL, c ∼ 1 × 10−4 M, methanol) in a quartz cuvette was irradiated
with LEDs at 405 or 450 nm to complete conversion. The resulting
mixture was analyzed by HPLC (with a reversed-phase column and a
mixture of methanol and water (with 0.1% formic acid) with a
different gradient used as eluent; 1 mL min−1). The photoproducts as
analytical standards were prepared independently by known synthetic
routes (5,20,38 661) to unequivocally establish the structure of the
photoproducts.
Determination of CO Yield. A solution of 1A or 1B (500 μL, c ∼
1 × 10−4 M, in methanol) was irradiated with LEDs at 405 or 450 nm
in closed GC vials fitted with PTFE septa to complete conversion.
The released CO was analyzed and quantified by a GC-headspace
technique, which was calibrated using the photoreaction of cyclo-
propenone photoCORM14 (50−500 μL, c ∼ 5 × 10−4 M, in
methanol).
Transient Spectroscopy. The LFP nanosecond setup was
generally operated in a right-angle arrangement of the pump and
probe beams. Laser pulses of ≤170 or 700 ps duration at 355 nm
(20−180 mJ) were obtained from a Nd:YAG laser. The laser beam
was dispersed onto a 40 mm long and 10 mm wide modified
fluorescence cuvette held in a laying arrangement. An overpulsed Xe
arc lamp was used as the source of the probe light. Kinetic traces were
recorded using a photomultiplier. Transient absorption spectra were
obtained using an ICCD camera equipped with a spectrograph. The
samples were degassed by three freeze−pump−thaw cycles under
reduced pressure (0.04 Torr). Absorption spectra of the sample
solutions were measured regularly between laser flashes to test for
possible photodegradation of the solution components using a diode-
array spectrophotometer.64
Figures of NMR spectra, determination of pKa, UV−vis
absorption spectra, emission spectra, irradiation spectra,
HPLC spectra and calibrations, HPLC−HRMS analysis,
transient absorption spectra, kinetic decay, picosecond
LPF, heat maps, and normalized emission spectra,
equations of quantum-yield calculations, tables of ISC
quantum yields, calculated vertical excitation energies,
wavelengths, and oscillator strengths, calculated adia-
batic energies, and structures for compounds, and
schemes of photochemistry under aerobic and anaerobic
conditions (PDF)
AUTHOR INFORMATION
Corresponding Author
■
́
Petr Klan − Department of Chemistry and RECETOX, Masaryk
University, 625 00 Brno, Czech Republic; orcid.org/0000-
0001-6287-2742; Phone: +420-54949-4856; Email: klan@
sci.muni.cz; Fax: +420-54949-2443
Authors
Marina Russo − Department of Chemistry and RECETOX,
Masaryk University, 625 00 Brno, Czech Republic
̌
Peter Stacko − Department of Chemistry and RECETOX,
Masaryk University, 625 00 Brno, Czech Republic
́
Dana Nachtigallova − Institute of Organic Chemistry and
Biochemistry, 16610 Prague, Czech Republic; orcid.org/
Femtosecond transient absorption was measured with the pump−
supercontinuum probe technique using a Ti/Sa laser system (775 nm,
pulse energy 0.9 mJ, full width at half-maximum 150 fs, operating
frequency 426 Hz). Part of the beam was fed into a noncollinear
optical parametric amplifier (NOPA). The output at 387 nm (for 1A)
and 470 nm (for 1B) was frequency-doubled by a β-barium borate
(BBO) crystal to λ = 266 nm and, upon compression, elicited pump
pulses of 1 μJ energy and <150 fs pulse width. The probe beam was
generated by focusing the 775 nm beam in front of a CaF2 plate with a
2 mm path length that produced a supercontinuum spanning a
wavelength range of 270−690 nm. The pump and probe beams were
focused to a 0.2 mm spot on the sample that was flowing in an optical
cell with a thickness of 0.4 mm. The probe beam and a reference
signal obtained by passing the solution next to the pump beam were
spectrally dispersed and registered with two photodiode arrays (512
pixels). Transient absorption spectra were calculated from the ratio of
0000-0002-9588-8625
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.joc.9b03248
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Support for this work was provided by the Czech Science
Foundation (GA18-12477S) and also by the Czech Ministry of
Education, Youth and Sports (the RECETOX research
infrastructure: LM2018121 and the CETOCOEN EXCEL-
LENCE Teaming 2 project: 02.1.01/0.0/0.0/18_046/
̌
́
̌
0015975). We thank Lubos Jılek and Vojtech Orel (Masaryk
I
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(18) Antony, L. A. P.; Slanina, T.; Sebej, P.; Solomek, T.; Klan, P.
Fluorescein analogue xanthene-9-carboxylic acid: a transition-metal-
free CO releasing molecule activated by green light. Org. Lett. 2013,
15, 4552.
(19) Solomek, T.; Wirz, J.; Klan, P. Searching for Improved
Photoreleasing Abilities of Organic Molecules. Acc. Chem. Res. 2015,
48, 3064.
University) for their help with the experiments, Jakob Wirz
(University of Basel) for fruitful discussions, and Miroslava
́
Bittova (Masaryk University) for HRMS analyses. This work
was also a part of the research project RVO (61388963) of the
IOCB of the Czech Academy of Sciences (D.N.).
(20) Popova, M.; Soboleva, T.; Arif, A. M.; Berreau, L. M. Properties
of a flavonol-based photoCORM in aqueous buffered solutions:
influence of metal ions, surfactants and proteins on visible light-
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(21) Grubel, K.; Laughlin, B. J.; Maltais, T. R.; Smith, R. C.; Arif, A.
M.; Berreau, L. M. Photochemically-induced dioxygenase-type CO-
release reactivity of group 12 metal flavonolate complexes. Chem.
Commun. 2011, 47, 10431.
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