Ph-dependent photoinduced interconversion of furocoumaric and furocoumarinic acids

Article abstract of DOI:10.3390/molecules26092800

Photo-controlled or photo-regulated molecules, especially biologically active and operating in physiological conditions, are in steady demand. Herein, furocoumaric and furocoumarinic acids being (Z/E)-isomers relative to each other were obtained in two stages starting from psoralen: the alkaline solvolysis of psoralen led to furocoumaric acid, which was further Z → E photoisomerized (365 nm) to furocoumarinic acid. The kinetics of Z → E photoisomerization was monitored by HPLC and UV-vis spectrophotometry. Photophysical characteristics in the aqueous phase for both acids, as well as the reversibility of (Z/E) photoisomerization process, were also assessed. Furocoumarinic acid was found to be visibly fluorescent at pH 2.0–12.0, with the maxima of fluorescence emission spectra being pH-dependent. The reverse E → Z photoisomerization predicted by quantum chemistry calculations as energetically favorable for the monoanionic form of furocoumarinic acid was proved in the experiment while being complicated by pyrone ring closure back to psoralen in acidic and neutral conditions. The preparative synthesis of furocoumarinic acid outlined in this work is particularly valuable in view of a wide range of pharmacological effects previously predicted for this compound.

Full text of DOI:10.3390/molecules26092800

molecules
Article
pH-Dependent Photoinduced Interconversion of Furocoumaric
and Furocoumarinic Acids
Vladislav V. Skarga 1,2, Anton A. Matrosov 1, Artemiy I. Nichugovskiy 2 , Vadim V. Negrebetsky 1,
Mikhail A. Maslov 2 , Ivan A. Boldyrev 1,3 and Mikhail V. Malakhov 1,
*
1
Institute of Translational Medicine, Pirogov Russian National Research Medical University,
1 Ostrovityanov Str., 117997 Moscow, Russia; skargavlad@gmail.com (V.V.S.); fmpfan@rambler.ru (A.A.M.);
nmr_rsmu@yahoo.com (V.V.N.); ivan@lipids.ibch.ru (I.A.B.)
Lomonosov Institute of Fine Chemical Technologies, MIREA—Russian Technological University,
86 Vernadsky Ave., 119571 Moscow, Russia; ashpwnz77@gmail.com (A.I.N.); mamaslov@mail.ru (M.A.M.)
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,
16/10 Miklukho-Maklaya Str., 117997 Moscow, Russia
2
3
*
Correspondence: malakhov.mikhail@gmail.com; Tel.: +7-916-815-5258
Abstract: Photo-controlled or photo-regulated molecules, especially biologically active and operating
in physiological conditions, are in steady demand. Herein, furocoumaric and furocoumarinic acids
being (Z/E)-isomers relative to each other were obtained in two stages starting from psoralen: the al-
kaline solvolysis of psoralen led to furocoumaric acid, which was further Z
→
E photoisomerized
(365 nm) to furocoumarinic acid. The kinetics of Z E photoisomerization was monitored by HPLC
→
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
and UV-vis spectrophotometry. Photophysical characteristics in the aqueous phase for both acids,
as well as the reversibility of (Z/E) photoisomerization process, were also assessed. Furocoumarinic
acid was found to be visibly fluorescent at pH 2.0–12.0, with the maxima of fluorescence emission
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Skarga, V.V.; Matrosov,
A.A.; Nichugovskiy, A.I.;
spectra being pH-dependent. The reverse E
→
Z photoisomerization predicted by quantum chem-
Negrebetsky, V.V.; Maslov, M.A.;
Boldyrev, I.A.; Malakhov, M.V.
pH-Dependent Photoinduced
Interconversion of Furocoumaric and
Furocoumarinic Acids. Molecules
2021, 26, 2800. https://doi.org/
10.3390/molecules26092800
istry calculations as energetically favorable for the monoanionic form of furocoumarinic acid was
proved in the experiment while being complicated by pyrone ring closure back to psoralen in acidic
and neutral conditions. The preparative synthesis of furocoumarinic acid outlined in this work is
particularly valuable in view of a wide range of pharmacological effects previously predicted for
this compound.
Keywords: furocoumaric acid; furocoumarinic acid; (Z
photoisomerization; UV irradiation; pH dependence; fluorescence; photoswitch; quantum chemistry
calculations; psoralens; furocoumarins; photoproducts
→
E)-photoisomerization; (E
→
Z)-
Academic Editor: Maria João Matos
Received: 31 March 2021
Accepted: 7 May 2021
Published: 10 May 2021
1. Introduction
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Psoralens are plant-derived or synthetic linear furanocoumarins, which are com-
monly used as medicinal UVA-photosensitizers [1,2]. Current psoralen-based photother-
apies (PUVA-therapy and photopheresis) are versatile, cost-efficient, and safe protocols
widely used for the treatment of various T-cell mediated and hyperproliferative skin
disorders [3–6]. These therapies are based on antiproliferative, immunosuppressive, and
apoptotic effects toward keratinocytes and immune cells, thus being considered as pho-
tochemotherapy and photoimmunotherapy [
antibacterial effects of psoralens were also reported [10
cycle, apoptosis, and differentiation [13].
Photolysis of psoralens in vitro provides the complex mixtures of biologically active
photoproducts comprehensively reviewed by S. Caffieri [14]. Photoproducts of psoralens
were found to induce phototoxic and dark membranolytic effects and apoptosis, as well as
4
,
6
–
9
]. Anti-tumor, anti-inflammatory, and
Copyright:
© 2021 by the authors.
–12], as well as their effects on cell
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
affect cell proliferation and differentiation [13
psoralen photooxidation were able to induce the immunotherapeutic effect in vivo [20–22]
–20]. More importantly, photoproducts of
Molecules 2021, 26, 2800. https://doi.org/10.3390/molecules26092800
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 2800
2 of 14
and selectively induce apoptosis in vitro in the transformed T-cell lines (Jurkat cell line) [17],
suggesting the crucial role of these photoproducts in the realization of the immunothera-
peutic effects of psoralen-based phototherapies.
Furocoumaric acid [(Z)-3-(6-hydroxybenzofuran-5-yl)acrylic acid; Z-FCA] is an α-
pyrone ring-opening (photo)product of psoralen, while furocoumarinic acid [(E)-3-(6-
hydroxybenzofuran-5-yl)acrylic acid; E-FCA] is E-isomer of Z-FCA (Scheme 1). Recently,
a wide range of pharmacological effects was predicted for E-FCA by means of chemoinfor-
matic analysis [23]. These effects include a number of protective (i.e., neuro-, vaso-, and
radioprotective) effects, antimutagenic, anticarcinogenic, antioxidant, and anti-inflammatory
activities, and even a prospective dermatologic (in particular, antipsoriatic) therapeutic activity.
Scheme 1. Structure of the subject compounds.
Structurally similar acids and their O-methylated analogs were previously reported
for 8-methoxypsoralen (8-MOP) and 5-methoxypsoralen [24–27]. These compounds have
been construed as water-soluble precursors (“pre-psoralens”), yielding the corresponding
psoralens in situ in the course of photocyclization reaction. It was found that this reaction
proceeds at physiological pH and temperature through the stage of E→ Z photoisomeriza-
tion of the acid followed by the lactonization process [25–27].
Reversibly photoisomerizable (Z/E)-FCAs might be considered as a cinnamate light-
activated molecular switch (photoswitch). Photoswitches and molecular motors based
on photo-switchable molecules have found application in a number of areas, including
biomolecular and analytical chemistry, as well as material sciences [28
ical control of material functions using cinnamate photoswitches was reported for hy-
dro/nanogels, micelle solutions, and polymer networks [31 34]. o-Hydroxycinnamate
–30]. Photochem-
–
phototriggers were used for image-guided photo-regulated uncaging of biologically rele-
vant and cosmeceutical agents [35,36].
Taking into consideration the prospective pharmacological activities of (E)-FCA and
the practical relevance of its photophysical properties, this work was aimed to prepare
(Z/E)-FCAs using previously described protocol [24–27] and to assess their photophysical
behavior in the aqueous phase and reversibility of (Z/E) photoisomerization process.
2. Results and Discussion
2.1. Psoralen Pyrone Ring-Opening
α
-Pyrone ring-opening under alkaline conditions are well-known reactions [37,38],
and derivatives of 8-MOP and their O-methylated analogs were obtained earlier using this
approach [24–27].
Psoralen pyrone ring-opening after treatment with 1 M aq. NaOH was accompanied
by significant changes in the UV-vis absorption spectra (Figure 1). An absorption spectrum
of psoralen in PBS (pH 7.4, 1% of ethanol) was characterized with two absorption maxima
at 245 and 296 nm and no absorption above 400 nm. After the psoralen solution was
alkalized (pH 13.1), the maximum at 296 nm disappeared, and a new broad absorption
band with a maximum at 343 nm was formed. A weak absorption in the visible region
(up to 430 nm) also appeared. Earlier, similar spectral changes were observed for 8-MOP
ring-opening products in the alkaline water/ethanol mixture (95/5, pH 12) [24]. These
similarities with 8-MOP suggest that strong alkalizing of psoralen solution also results in
pyrone ring-opening yielding Z-FCA dianion.

Molecules 2021, 26, 2800
3 of 14
(A)
(B)
Figure 1. Psoralen pyrone ring-opening. (A) Reaction scheme. (
B) UV-vis absorption spectra of
0.1 mM psoralen solution in PBS (pH 7.4, 1% of ethanol) and after alkalizing to pH 13.1.
Preparative isolation and characterization of Z-FCA were not performed since these
processes are complicated due to the well-studied process of pyrone ring back-closure in
neutral and acidic conditions previously noted for 8-MOP [25–27].
2.2. Z→ E Photoisomerization
According to earlier findings, UVA irradiation of the dianionic 8-MOP ring-opening
product in alkaline conditions results in its Z
solutions, as well as the absence of oxygen, are needed to avoid dimerization and/or
photooxidation processes [24 27]. Z E photoisomerization of psoralen ring-opening
→
E photoisomerization, and highly diluted
–
→
product (Z-FCA dianion) at pH 13.1 was performed using increasing fluences of UVA
irradiation (365 nm, 0–252 kJ m−2) and monitored using reverse-phase HPLC and UV-vis
absorption spectroscopy.
In order to make the samples suitable for HPLC analysis, the obtained alkaline UVA-
irradiated solutions were acidified to pH 2 with 1 M aq. HCl prior to analysis. HPLC
analysis of the acidified non-irradiated sample provides a picture of a single peak with
the retention time (Rt) of 14.3 min, which corresponds to Rt of psoralen (Figure S1). Thus,
the acidification of the initial non-irradiated sample leads to complete Z-FCA pyrone ring
back-closure to psoralen similarly to 8-MOP [25
may serve as a quantitative measure of Z-FCA remained after UVA irradiation.
Fluence-dependent HPLC changes during the Z E photoisomerization process
are summarized in Figure 2. At the minimal UVA fluence (4.2 kJ m−2), HPLC analysis
–27], and the HPLC peak area for psoralen
→
showed about a half-decrease in Z-FCA in solution, and the second peak with Rt = 13.1 min
which may be attributed to fully protonated E-FCA was also detected (Figure S1). Further
increase in UVA fluences (up to 50.4 kJ m−2) resulted in the maximal consumption of
Z-FCA (of ~95%), and the peak of E-FCA reached the plateau. At higher UVA fluences
(50.4–252 kJ m−2), no valuable changes for both Z-FCA and E-FCA amounts were found
on chromatograms. In addition, no other peaks comparable with those of Z-FCA and
E-FCA were found even at the highest UVA fluence (252 kJ m−2), which corresponded to
5 min of irradiation using a high radiant flux UVA source (Figure S1). This suggests that
,
no other sufficient photoinduced or post-irradiation processes occur during or after Z
E photoisomerization and this approach seems applicable for the preparative synthesis
of E-FCA.
→

Molecules 2021, 26, 2800
4 of 14
(A)
(B)
Figure 2. Fluence-dependent HPLC changes during the Z
action scheme. (B) Z-FCA and E-FCA peak areas as monitored by HPLC.
→
E photoisomerization process. (A) Re-
Absorption changes during the Z E photoisomerization process in alkaline solutions
→
were also fluence-dependent (Figure 3A). After minimal UVA irradiation (4.2 kJ m−2), the
Z-FCA dianion absorption maxima at 245 and 343 nm were shifted to 249 and 360 nm,
respectively. Upon gradual increase in UVA fluences up to 50.4 kJ m−2, the absorption at
249 nm was also increased with a small shift of maximum to 250 nm, while the red shift of
the rising 360 nm peak was more pronounced (to 376 nm). At higher fluences, no valuable
changes of absorption or peak positions were found. The presence of isobestic points at
240, 314, and 350 nm further implies the Z-FCA dianion conversion to E-FCA dianion.
Figure 3. UV-vis absorption changes upon Z→ E photoisomerization process. Conditions: (
A) spectral changes immediately
after irradiation of the alkaline solution of psoralen (0.1 mM, 1% of ethanol, pH 13.1) with increasing fluences of UVA
irradiation (365 nm, 0–252 kJ m−2). The insert shows the photometric changes at 410 nm; (B) spectral changes after strong
acidification of the irradiated samples to pH 2. The insert shows the photometric changes at 380 nm.
Spectral changes after acidification of the irradiated samples to pH 2 were less promi-
nent (Figure 3B). The absorption spectrum of acidified non-irradiated solution coincided
with the psoralen absorption spectrum (Figure 1), closely corresponding to HPLC analy-
sis data (Figure S1). At low fluences (4.2–12.6 kJ m−2), the acidified irradiated solutions
consist of the newly formed product (presumably E-FCA) and psoralen, the latter being
the product of the pyrone ring back-closure of non-isomerized Z-FCA. Thus, upon an
increase in UVA fluences, the absorption spectrum shifted gradually to the spectrum of

Molecules 2021, 26, 2800
5 of 14
E-FCA through the isobestic points at 304 and 332 nm. At higher fluences (equal or above
25.2 kJ m−2), the quantities of psoralen were too small to contribute significantly to the
absorption of solutions, and the absorption spectra might be attributed to the spectrum of
fully protonated E-FCA with characteristic maxima at 245, 290, and 342 nm.
Fluence-dependent photometric changes both in alkaline and acidified irradiated solu-
tions (Figure 3A,B, inserts) are in close agreement with HPLC analysis data (Figure 2). Thus,
Z
→
E photoisomerization process may be monitored by routine photometric data acquisition.
The above-described Z E photoisomerization approach with photometric Z
→
→
E
conversion monitoring was scaled up for preparative synthesis of E-FCA (see Section 3.4).
It was found that UVA-irradiation of 0.6 L sample for 9 min was aligned with maximal
absorbance changes at 410 nm (Figure S2). According to HPLC analysis, this time point
matches with 93% conversion of Z-FCA to E-FCA. The isolated and purified E-FCA was
characterized using NMR and MS methods. The recorded 1H and 13C-NMR spectra of
E-FCA are presented in Figures S3 and S4. The coupling constant values for
protons (both of about 16 Hz) imply the E character of this bond. Similar observations were
made earlier for the methoxy derivative of E-FCA prepared from 8-MOP [24 27]. HRMS
α,β vinyl
–
spectra recorded both in positive and negative modes are presented in Figures S5 and S6,
and the values obtained for [M+H]+ and [M−H]– correspond to calculated values.
2.3. pH-Dependent Absorption and Fluorescence Characteristics of (Z/E)-FCAs
Both Z-FCA and E-FCA have acidic dissociable protons at the carboxyl and phenolic
groups. Thus, depending on pH, these acids may present in fully protonated, monoanionic,
and dianionic forms, as well as mixtures thereof.
However, a process of pyrone ring back-closure to psoralen in neutral and acidic
conditions previously noted for methoxy derivative of Z-FCA [25–27] and confirmed for
Z-FCA in the present work (see Section 2.2. above) governs the presence of Z-FCA in protic
solvents as a dianionic form at the alkaline pH range only.
Theoretical pH-dependent relative distribution of fully protonated and ionized forms
of E-FCA was calculated using the Marvin pKa plugin (Figure S7). The acid-base dissoci-
ation constants for E-FCA in aqueous solutions were predicted as being pKa1 = 3.61 (for
the carboxyl proton dissociation) and pKa2 = 8.43 (for the phenolic proton dissociation).
In addition, it was calculated that a fully protonated form of E-FCA is predominant at
pH < 2.0, with monoanionic and dianionic forms being predominant around pH 6.0 and at
pH > 10, respectively.
UV-vis absorption changes upon pH titration of the aqueous E-FCA solution in the
range of pH 2.0–12.0 are presented in Figure 4. The absorption spectrum of E-FCA at
pH 2.0 was characterized with three absorption maxima at 245, 290, and 342 nm. Upon
increase in pH values, all the maxima showed the hypsochromic shifted to 243, 280,
and 330 nm, respectively, with the minor changes in absorbance values (Figure 4A). The
most vivid changes at pH 6.5~7.0 can be attributed to the complete dissociation of the
carboxylic protons. Further increase in pH values (to alkaline region) was accompanied
by the dramatic changes of the absorption spectra, which resulted from the dissociation
of the phenolic protons. At the pH > 8.0, the absorption spectra underwent the vivid
transformations (Figure 4B): the maximum at 243 nm was shifted to 250 nm, and two other
maxima at 280 and 330 nm were transformed to a shoulder at ~270 nm and a new broad
maximum at 376 nm (i.e., with a bathochromic shift of 46 nm) with a prominent absorption
in the visible region. According to the obtained absorption spectra, the predicted pKa1 and
pKa2 values should be corrected to about 4.5 and 9.2, respectively, especially considering
the fact that the close values were previously reported for the methoxy derivative of Z-
FCA [24]. In addition, the absence of significant changes in absorbances at pH < 3.0 may be
indicative of the pH range with the predominance of fully protonated E-FCA.

Molecules 2021, 26, 2800
6 of 14
Figure 4. UV-vis absorption changes upon pH titration of the aqueous E-FCA solution (12.5 µM, 0.5% of ethanol). Conditions:
(A) absorbance changes upon neutral to acidic titration of E-FCA with 1 M aq. HCl; (B) absorbance changes upon neutral-
to-alkaline titration of E-FCA with 1 M aq. NaOH.
Fluorescence changes upon pH titration of E-FCA were also pH-dependent (Figure 5)
and visible to the naked eye (Figure 5A). Two wavelengths, i.e., 376 and 420 nm, were
chosen for excitation of the aqueous E-FCA solution in the range of pH 2.0–12.0: the first
one is the “universal” excitation wavelength for all three forms of E-FCA, while the second
one is specific for the dianionic form of E-FCA.
The fluorescence emission spectra of E-FCA at pH 2.0 and pH 3.0 had equal intensities
and a peak at 536 nm (Figure 5B). Increasing the pH value to 4.0–6.0 resulted in a hyp-
sochromic shift of the emission maximum to ~470 nm with the transformation of the peak at
536 nm to a shoulder. Further increase in pH value in the range of 6.0–7.4 was accompanied
by a slight increase in fluorescence intensity at ~470 nm and the appearance of another
peak at ~540 nm. The fluorescence intensity of the latter notably increased in the range of
6.0–7.4, and finally, this peak became predominant at pH 7.4 with the emission maximum at
543 nm. All these transformations of the emission spectra at the acidic-to-neutral pH range
might indicate the complete dissociation of the carboxylic protons of E-FCA, while the new
rising peak at pH 6.0–7.4 can be attributed to the initial stages of dianion formation. These
features are in suitable agreement with absorption changes upon pH titration presented in
Figure 4A. Moreover, the strong contribution of dianionic form in the fluorescence intensity
at pH 6.0–7.4 suggests that this form is more fluorescent than the others.
The fluorescence intensity measurements at the neutral-to-alkaline pH range finalized
the peak value for the dianionic form of E-FCA (Figure 5C,D). Under excitation at 420 nm,
the fluorescence emission maximum for the dianionic form of E-FCA was found to be equal
to 555 nm (Figure 5C). Meanwhile, at pH values in the range of 7.4–9.0, the contribution of
other forms is still visible, especially under excitation at 376 nm where all three forms of
E-FCA absorb (Figure 5D). These features are also in suitable agreement with absorption
changes upon pH titration presented in Figure 4B. Furthermore, the vivid amplification of
fluorescence intensity for the dianionic form of E-FCA needs to be noted.

Molecules 2021, 26, 2800
7 of 14
Figure 5. Fluorescence changes upon pH titration of the aqueous E-FCA solution (12.5
images of the aqueous E-FCA solution at various pH values under UV LED light (365 nm) (
upon neutral to acidic titration of E-FCA with 1 M aq. HCl under excitation at 376 nm ( ); fluorescence changes upon
neutral-to-alkaline titration of E-FCA with 1 M aq. NaOH under excitation at 420 nm (C) or 376 nm (D).
µM, 0.5% of ethanol). Fluorescence
A
); fluorescence changes
B
The fluorescence quantum yields (
to quinine sulfate in 0.5 M H2SO4 ( = 54.6%) used as a standard (Figure S8). The calculated
data confirmed our prediction, which was made in the course of spectral studies: the
of dianionic form of E-FCA at pH 12.0 was 7-fold higher than that of the fully protonated
form (2.1% vs. 0.3%, respectively). Moreover, the values calculated for E-FCA at pH 6.0
and 7.4 (1.6% vs. 2.0%, respectively) were found to be remarkably close to the value of
Φ) of E-FCA at various pH were measured relative
Φ
Φ
Φ
Φ
the dianionic form of E-FCA. Combined with fluorescence spectra presented in Figure 5B,
these data argue for the complex character of E-FCA solutions at neutral pH region, which
presumably contains the monoanionic form of E-FCA with admixtures of fully protonated
and dianionic forms, the latter strongly contributing to fluorescence intensity.
pH-Dependent photophysical characteristics of (Z/E)-FCAs described above were
summarized below in Table 1, being added with absorption data for E-FCA in ethanol
(Figure S9) and photophysical data for methoxy derivative of E-FCA [24].
The photophysical stability of E-FCA in different pH regions was confirmed by flu-
orescence measurements during cyclic changing of pH values between pH 2.0 and 12.0.
Monitoring of fluorescence emission under excitation at 376 and 420 nm at these pH
values revealed cyclic recovery of emission intensities without any significant changes
(Figure S10).

Molecules 2021, 26, 2800
8 of 14
Table 1. Photophysical characteristics of (Z/E)-FCAs and relative compounds.
Compound
Parameter 1
Absorption
Emission
pH 2.0 2
pH 6.0 2
pH 7.4 2
pH 12.0 2
Ethanol
245 (15200)
343 (4500)
Z-FCA
3
-
245 (20700)
290 (10900)
342 (5500)
243 (25200)
280 (9700)
330 (5600)
243 (25100)
280 (9700)
330 (5500)
250 (21200)
376 (6700)
245 (25600)
286 (11000)
342 (6600)
Absorption
Emission
E-FCA
536 (0.3)
470 (1.6)
543 (2.0)
365 (1000) 4b
540 (0.4) 4d
555 (2.1)
256 (27000) 4c
378 (7000) 4c
468 (7.0)
250 (30000)
289 (14500)
336 (5200)
294 (16000) 4a
Absorption
Methoxy E-FCA 4
Emission
530 (3.0)
1
Absorption = peak maxima in nm (molar extinction coefficient in (M cm)−1); Emission = peak maximum in nm (fluorescence quantum
2
yield in %). Conditions: pH 2.0 and pH 6.0 = aqueous solutions acidified with 1 M aq. HCl to the indicated pH values; pH 7.4 = solutions
in phosphate buffered saline (PBS); pH 12.0 = aqueous solution basified with 1 M aq. NaOH to the indicated pH value. Concentrations:
3
absorption measurements—0.1 mM, 1% of ethanol; fluorescence measurements—12.5
µ
M, 0.5% of ethanol. Non-fluorescent, upon UVA
irradiation readily isomerize to E-FCA (see Section 2.2. above). 4 Data presented are from [24]. Conditions: 4a = peak value in water/ethanol
mixture (95/5) acidified to pH 1.0 with 1 N aq. HCl; 4b = shoulder value in water/ethanol mixture (95/5) at pH 7.0; 4c = peak values in
water/ethanol mixture (95/5) basified to pH 12.0 with 0.01 N aq. NaOH; 4d = peak value in water/ethanol mixture (95/5) at pH 7.0.
2.4. Computational Studies
Quantum chemistry calculations were used to obtain optimized geometries and corre-
sponding energy gaps for the protonated, monoanionic, and dianionic forms of E-FCA and
Z-FCA (Figure 6). Molecular orbitals for all forms of (Z/E)-FCAs are shown in Figure S11.
Figure 6. Optimized geometries for the dianionic, monoanionic, and protonated forms of (Z/E)-FCAs (with charges of −2,
−1, and 0, respectively). Bars represent energy gaps (eV) between corresponding forms of E-FCA and Z-FCA.
The calculated energies for dianionic and protonated forms of Z-FCA were found to
be higher than the energies of corresponding forms of E-FCA. Thus, Z → E isomerization
is energetically favorable for these forms. In contrast, the energy of monoanionic E-
FCA is lower than the energy of monoanionic Z-FCA, and the reverse process of E
isomerization becomes energetically favorable.
→
Z
The values of dihedrals, angles, and bond lengths calculated for (Z/E)-FCAs are
presented in Table 2. It can be noticed that the spatial structure of E-FCA does not change
sufficiently during the loss or addition of a proton (Figure 6). The molecule is almost planar:

Molecules 2021, 26, 2800
9 of 14
0
0
0
0
0
◦
◦
the 5-3 -2 -1 and 6-5-3 -2 dihedral values are close to 180 and 0 , respectively. The length
of the 30-20 (double) and 5-30 (single) bonds are also conventional for conjugated systems.
Table 2. The values of dihedrals, angles, and bond lengths calculated for (Z/E)-FCAs.
◦
◦
Dihedrals ( )
Angles ( )
Bond Lengths (Å)
Compound
(Charge)
DH
5-3 -2 -1
DH
6-5-3 -2
A
5-3 -2
A
3 -2 -1
0
0
0
0
A 4-5-3
B 5-3
B 3 -2
0
0
0
0
0
0
0
0
0
0
E-FCA (0)
E-FCA (-1)
E-FCA (-2)
Z-FCA (0)
Z-FCA (-1)
Z-FCA (-2)
179.9
176.3
176.6
5.2
9.4
2.3
0.2
2.0
2.5
43.2
26.8
175.7
117.3
118.0
116.8
117.8
151.1
124.1
130.3
131.3
131.7
131.3
138.9
136.9
123.2
122.2
122.6
129.2
136.0
136.0
1.457
1.464
1.464
1.470
1.463
1.463
1.345
1.340
1.344
1.338
1.352
1.355
In contrast, the geometries of protonated and both ionized forms of Z-FCA are notably
different (Figure 6). The protonated and monoanionic forms of Z-FCA are not planar, as the
5-30-20-10 and especially the 6-5-30-20 dihedral values are far from 0◦ and 180◦, respectively.
In the case of protonated form, a deviation from planarity is bigger, and the 6-5-30-20
dihedral value is about 43◦. Moreover, the large degree of deviation from planarity also
affects the double bond conjugation in the protonated Z-FCA. The 30-20 (double) bond
shortens to 1.338 Å, while the 5-30 (single) bond elongates to 1.470 Å (i.e., the degree of
bond conjugation decreases).
0
0
0
The dianionic form of Z-FCA appeared to be planar, since calculated the 5-3 -2 -1 and
the 6-5-30-20 dihedral values are close to 0◦ and 180◦, respectively.
Furthermore, a sufficient deviation of bond angles from conventional values (of 120◦
for sp2 hybridization) was revealed for all forms of Z-FCA, as well as the formation of the
internal hydrogen bond in the monoanionic form of Z-FCA.
2.5. E → Z Photoisomerization
According to computational studies, the E
→
Z photoisomerization process is ener-
getically favorable for the monoanionic form of E-FCA. With the proviso that this form of
E-FCA is predominant at neutral pH region, the E Z photoisomerization process was
monitored by HPLC analysis of E-FCA solutions UVA-irradiated at pH 6.5 (Figure 7).
→
(A)
(B)
Figure 7. HPLC-controlled fluence-dependent E
→
Z conversion in E-FCA solutions (0.1 mM, 1%
ethanol) UVA-irradiated at pH 6.5. (A) Reaction scheme. (B) Normalized conversion of E-FCA to
Z-FCA as monitored by HPLC (raw HPLC data are presented in Figure S12).

Molecules 2021, 26, 2800
10 of 14
It was found that non-irradiated E-FCA solutions contained ~1.7% of Z-FCA as
an impurity after synthesis. Upon UVA irradiation, gradual fluence-dependent E
Z conversion was observed. Similar to E Z photoisomerization, HPLC-controlled
formation of psoralen during Z-FCA pyrone ring back-closure was used as a measure of E
→
→
→
Z conversion. At fluences above 151 kJ m−2, the process of E
→
Z photoisomerization
was slowed down. Moreover, at higher fluences (>252 kJ m−2), psoralen content reached
the plateau (or even slightly decreased), while E-FCA content still continuously diminished.
This effect might indicate a simultaneous formation of Z-FCA accompanied by partial
photolysis of psoralen newly formed in the course of E → Z conversion.
3. Materials and Methods
3.1. Materials
Psoralen ( 98% purity) was purchased from ChemicalPoint (Deisenhofen, Germany).
≥
Other reagents and solvents were purchased from commercial suppliers (Sigma-Aldrich,
St. Louis, MO, USA; Acros Organics, Geel, Belguim; Fisher Scientific, Loughborough, UK;
J.T. Baker, Deventer, Netherlands) and used without additional purification. All the organic
solvents were of HPLC grade.
Ultra-pure water (18.2 M
Ω cm) produced on an OMNI-Analytic water purification
system (Xiamen RSJ Scientific Instruments Co., Xiamen, China) was used for the prepa-
ration of all aqueous solutions, as well as for spectral and HPLC analyses. Phosphate
buffered saline (PBS, pH 7.4
Solon, OH, USA) was prepared by dissolving the PBS tablets in ultra-pure water. Stock
±
0,1, Biotechnology grade, VWR Life Science AMRESCO,
solutions of psoralen and E-FCA were prepared in distilled ethanol and stored in the dark.
3.2. UVA Irradiation Procedures
UVA irradiation procedures were performed on air at ambient temperature (23 ± 2 ◦C)
with continuous stirring using a magnetic stirrer.
For research studies, the samples were UVA-irradiated in a 5 mL optic glass cuvette
(1.0 cm path length) using a high radiant flux UV LED array OTLH-0480-UV (365 nm,
full width half maximum = 14.5 nm, Opto Technology Inc.; Wheeling, IL, USA). The UVA
fluence rate on the front wall of the cuvette (840 W m−2) was measured using Waldmann
UV-meter (H. Waldmann GmbH & Co., Villingen-Schwenningen, Germany).
For preparative purposes, a large-volume (0.6 L) sample was UVA-irradiated in a 1 L
flat-bottomed glass flask using BIO-LINK BLX-365 irradiation system (365 nm, 5
UVA tubes, Vilber Lourmat, Collégien, France).
×
8 W
3.3. Analytical Procedures
All analytical procedures were performed on air at ambient temperature (23 ± 2 ◦C).
UV-vis absorption spectra were recorded in a 1.0 cm path length quartz cuvette
(Hellma Analytics, Müllheim, Germany) using a Shimadzu UV-1601PC spectrophotometer.
The corrected fluorescence emission spectra were recorded in a 1.0 cm path length
quartz cuvette (Hellma Analytics, Müllheim, Germany) using a Hitachi F-7000 spectrofluo-
rometer. Both excitation and emission slits were set at 5 nm. To minimize the reabsorption
effect, fluorescence measurements were performed in samples with absorbances not ex-
ceeded 0.1 at and above the excitation wavelength. The fluorescence quantum yields (Φ)
of E-FCA were measured relative to quinine sulfate in 0.5 M H2SO4 (
Φ = 54.6%) as a
standard [39].
Analytical reverse-phase HPLC analyses were performed using an Agilent 1200 DAD-
HPLC system (Agilent Technologies, Santa Clara, CA, USA) in gradient elution mode under
conditions indicated in the Electronic Supplementary Information file. Data acquisition and
processing were performed using a ChemStation® software system (Agilent Technologies,
Waldbronn, Germany).

Molecules 2021, 26, 2800
11 of 14
1
Both H-NMR and 13C-NMR spectra were recorded on Bruker DPX 300 FT-NMR
spectrometer (at 300 and 75 MHz, respectively) using tetramethylsilane as an internal
standard and DMSO-d6 as a solvent.
The mass spectra were recorded both in positive and negative modes using an Orbitrap
Elite mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a
HESI probe (direct infusion rate = 5 µL min−1; spray voltage = 4 kV).
The pH values were measured using a Hanna HI 83,141 pH meter and corrected by
the addition of 1 M aq. HCl or 1 M aq. NaOH as appropriate.
3.4. Preparation and Characterization of E-FCA
Psoralen (112 mg, 0.602 mmol) was dissolved in 60 mL of EtOH and added to 540 mL
of 0.3 M aq. NaOH solution to provide 1 mM alkaline solution of Z-FCA containing 10%
of EtOH. This solution was transferred to a 1 L flat-bottomed glass flask (~2 cm layer)
and UVA-irradiated (365 nm) on air for 9 min with continuous stirring. The resulting
solution (0.6 L) was acidified to pH 3.0 by the addition of 1 M aq. HCl, then extracted with
ethyl acetate (3
×
80 mL), and the organic phases were washed with water (3
×
50 mL).
The combined organic extract was dried over Na2SO4, filtered, concentrated using rotary
vacuum evaporator, and purified by column flash-chromatography on silica gel (Kieselgel
60, 40–63 µm, Merck) eluting with ethyl acetate/petroleum ether (1:5 → 2:1, v/v) to yield
1
E-FCA (94 mg, 76.6%). H-NMR (300 MHz, DMSO-d6, ppm):
δ 6.48 (d, J = 16.05 Hz, 1H,
CH
COO–), 6.75 (dd, J = 2.23 Hz, 0.93 Hz, 1H, OCHC
(s, 1H, OCC COH), 7.72 (d, J = 2.22 Hz, 1H, OC CH), 7.78 (d, J = 16.07 Hz, 1H, C
13C-NMR (75 MHz, DMSO-d6, ppm):
H
), 7.10 (s, 1H, C
H
CCHCH), 7.71
H
H
H
CHCOO–).
δ
98.42, 107.00, 119.81, 119.94, 120.02, 123.22, 136.84,
145.12, 155.39, 156.30, 171.87. HRMS (HESI): m/z calc. for [M+H]+ and [M-H]–: 205.0501
and 203.0344; found: 205.0496 and 203.0359.
3.5. Theoretical Calculations
Calculations were performed for the dianionic, monoanionic, and protonated forms of
(Z/E)-FCAs (with charges of −2, −1, and 0, respectively). Geometries of (Z/E)-FCAs were
optimized using the Hartree Fock method with the def2-SVP basis set followed by B3LYP
−
geometry optimization with def2-TZVPP basis set, the latter also providing single point
energy. Calculations were carried out using the ORCA software [40].
4. Conclusions
pH-Dependent photoinduced interconversion of furocoumaric and furocoumarinic
acids was studied in this work. These acids were prepared using an approach previously
described for methoxy substituted psoralens [24
ring-opening in alkaline conditions to provide furocoumaric acid (Z-FCA) followed by
E photoisomerization (365 nm) yielding furocoumarinic acid (E-FCA). The Z
–27], i.e., by means of psoralen α-pyrone
Z
→
→
E
photoisomerization process was monitored by HPLC and spectrophotometry, and E-FCA
was obtained in preparatively useful yield. Assuming a wide range of pharmacological
effects previously predicted for E-FCA by means of chemoinformatic analysis [23], the
preparative synthesis of E-FCA outlined in this work is particularly valuable.
For both acids, the main photophysical characteristics were assessed. E-FCA was
found to be visibly fluorescent on the entire pH range studied (pH 2.0–12.0), with the
maxima of the emission spectra being pH-dependent. Hence, although not characterized
with significant fluorescence quantum yields, E-FCA might be considered as an acido-
/basochromic fluorophore in a similar manner to some other compounds previously
reported [41–43].
Based on quantum chemistry calculations, the reverse E
→
Z photoisomerization
process was proved to be energetically favorable for the monoanionic form of E-FCA.
Unfortunately, in acidic and neutral conditions, this process readily resulted in pyrone
ring closure back to psoralen. Thus, E-FCA still might be regarded as a photoisomerizable
water-soluble psoralen precursor while decategorized as an intrinsic photoswitch, which is

Molecules 2021, 26, 2800
12 of 14
useful at physiological conditions. Instead, photoswitching between Z-FCA and E-FCA
accompanied with psoralen pyrone ring-opening/closure in neutral to basic conditions
could be noted. Considering the fact that the direction of photoisomerization is governed
by protonation/deprotonation processes, the combined basochromic and photochromic
behavior of Z/E-FCAs render them functional as a dual-responsive system [41,42].
Supplementary Materials: The following supporting data are available online: Figures S1–S12
(raw HPLC data; NMR and HRMS spectra; supporting absorption and fluorescence spectral data;
HOMO-LUMO molecular orbitals for (Z/E)-FCAs).
Author Contributions: Conceptualization, V.V.S., A.A.M., I.A.B. and M.V.M.; methodology, V.V.S.
and A.A.M.; investigation, V.V.S., A.A.M. and A.I.N.; computational studies, I.A.B.; resources, V.V.N.,
A.I.N. and M.A.M.; writing—original draft preparation, V.V.S., I.A.B. and M.V.M.; writing—review
and editing, V.V.N., M.A.M. and M.V.M.; funding acquisition, M.A.M., V.V.S. and I.A.B. All authors
have read and agreed to the published version of the manuscript.
Funding: This research was funded in part by the Russian Foundation for Basic Research, project
number 19-33-90277. Quantum chemistry calculations were funded by Russian Foundation for Basic
Research, project number 20-03-00770.
Data Availability Statement: The data presented in this study are available in this article.
Acknowledgments: The authors would like to thank Maria G. Zavialova for the assistance in
1
HRMS spectra acquisition. H-NMR and 13C-NMR spectra were recorded at the MIREA—Russian
Technological University Core Facilities Centre.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Not available.
References
1.
Bruni, R.; Barreca, D.; Protti, M.; Brighenti, V.; Righetti, L.; Anceschi, L.; Mercolini, L.; Benvenuti, S.; Gattuso, G.; Pellati, F.
Botanical Sources, Chemistry, Analysis, and Biological Activity of Furanocoumarins of Pharmaceutical Interest. Molecules 2019
24, 2163. [CrossRef]
,
2.
3.
Thakur, A.; Sharma, R.; Jaswal, V.S.; Nepovimova, E.; Chaudhary, A.; Kuca, K. Psoralen: A Biological Important Coumarin with
Emerging Applications. Mini-Rev. Med. Chem. 2020, 20, 1–7. [CrossRef]
Richard, E.G.; Hönigsmann, H. Phototherapy, psoriasis, and the age of biologics. Photodermatol. Photoimmunol. Photomed. 2013, 30,
3–7. [CrossRef]
4.
5.
Racz, E.; Prens, E.P. Phototherapy and Photochemotherapy for Psoriasis. Dermatol. Clin. 2015, 33, 79–89. [CrossRef]
Torres, A.E.; Lyons, A.B.; Hamzavi, I.H.; Lim, H.W. Role of phototherapy in the era of biologics. J. Am. Acad. Dermatol. 2021, 84,
479–485. [CrossRef] [PubMed]
6.
7.
8.
9.
Furuhashi, T.; Saito, C.; Torii, K.; Nishida, E.; Yamazaki, S.; Morita, A. Photo(chemo)therapy Reduces Circulating Th17 Cells and
Restores Circulating Regulatory T Cells in Psoriasis. PLoS ONE 2013, 8, e54895. [CrossRef]
Liang, Y.; Sarkar, M.K.; Tsoi, L.C.; Gudjonsson, J.E. Psoriasis: A mixed autoimmune and autoinflammatory disease. Curr. Opin.
Immunol. 2017, 49, 1–8. [CrossRef]
Nomura, T.; Honda, T.; Kabashima, K. Multipolarity of cytokine axes in the pathogenesis of atopic dermatitis in terms of age,
race, species, disease stage and biomarkers. Int. Immunol. 2018, 30, 419–428. [CrossRef]
Ho, A.W.; Kupper, T.S. T cells and the skin: From protective immunity to inflammatory skin disorders. Nat. Rev. Immunol. 2019
19, 490–502. [CrossRef]
,
10. Sturaro, G.; Cigolini, G.; Menilli, L.; Cola, F.; Di Liddo, R.; Tasso, A.; Conconi, M.T.; Miolo, G. Antiproliferative activity of
8-methoxypsoralen on DU145 prostate cancer cells under UVA and blue light. Photochem. Photobiol. Sci. 2017, 16, 1182–1193.
[CrossRef] [PubMed]
11. Rožman, K.; Alexander, E.M.; Ogorevc, E.; Bozovicˇar, K.; Sosicˇ, I.; Aldrich, C.C.; Gobec, S. Psoralen Derivatives as Inhibitors of
Mycobacterium tuberculosis Proteasome. Molecules 2020, 25, 1305. [CrossRef]
12. Ren, Y.; Song, X.; Tan, L.; Guo, C.; Wang, M.; Liu, H.; Cao, Z.; Li, Y.; Peng, C. A Review of the Pharmacological Properties of
Psoralen. Front. Pharmacol. 2020, 11, 571535. [CrossRef]
13. Viola, G.; Salvador, A.; Vedaldi, D.; Dall’Acqua, F.; Bianchi, N.; Zuccato, C.; Borgatti, M.; Lampronti, I.; Gambari, R. Differentiation
and Apoptosis in UVA-Irradiated Cells Treated with Furocoumarin Derivatives. Ann. N. Y. Acad. Sci. 2009, 1171, 334–344.
[CrossRef]
14. Caffieri, S. Furocoumarin photolysis: Chemical and biological aspects. Photochem. Photobiol. Sci. 2002, 1, 149–157. [CrossRef]

Molecules 2021, 26, 2800
13 of 14
15. Marley, K.A.; Larson, R.A.; Davenport, R. Alternative Mechanisms of Psoralen Phototoxicity. ACS Symp. Ser. 1995, 616, 179–188.
[CrossRef]
16. Potapenko, A.; Malakhov, M.V.; Kyagova, A.A. Photobiophysics of furocoumarins. Biophysics 2004, 49, 307–324.
17. Caffieri, S.; Di Lisa, F.; Bolesani, F.; Facco, M.; Semenzato, G.C.; Dall’Acqua, F.; Canton, M. The mitochondrial effects of novel
apoptogenic molecules generated by psoralen photolysis as a crucial mechanism in PUVA therapy. Blood 2007, 109, 4988–4994.
[CrossRef] [PubMed]
18. Salvador, A.; Dall’Acqua, S.; Sardo, M.S.; Caffieri, S.; Vedaldi, D.; Dall’Acqua, F.; Borgatti, M.; Zuccato, C.; Bianchi, N.; Gambari,
R. Erythroid Induction of Chronic Myelogenous Leukemia K562 Cells Following Treatment with a Photoproduct Derived from
the UV-A Irradiation of 5-Methoxypsoralen. ChemMedChem 2010, 5, 1506–1512. [CrossRef]
19. Nevezhin, E.V.; Vlasova, N.V.; Pyatnitskiy, I.A.; Lysenko, E.P.; Malakhov, M.V. On the mechanism of erythrocyte hemolysis
induced by photooxidized psoralen. Biochemistry 2015, 80, 763–768. [CrossRef] [PubMed]
20. Kyagova, A.A.; Zhuravel, N.N.; Malakhov, M.V.; Lysenko, E.P.; Adam, W.; Saha-Möller, C.R.; Potapenko, A.Y. Suppression
of Delayed-type Hypersensitivity and Hemolysis Induced by Previously Photooxidized Psoralen: Effect of Fluence Rate and
Psoralen Concentration. Photochem. Photobiol. 1997, 65, 694–700. [CrossRef]
21. Potapenko, A.Y.; Kyagova, A.A.; Bezdetnaya, L.N.; Lysenko, E.P.; Chernyakhovskaya, I.Y.; Bekhalo, V.A.; Nagurskaya, E.V.;
Nesterenko, V.A.; Korotky, N.G.; Akhtyamov, S.N.; et al. Products of psoralen photooxidation possess immunomodulative and
antileukemic effects. Photochem. Photobiol. 1994, 60, 171–174. [CrossRef]
22. Kyagova, A.A.; Malakhov, M.V.; Potapenko, A. Immunosuppression caused by photochemo- and photodynamic therapy: Focus
on photosensitizer photoproducts. In Immunosuppression: New Research; Taylor, C.B., Ed.; Nova Science Publishers: Hauppauge,
NY, USA, 2009.
23. Skarga, V.V.; Zadorozhny, A.D.; Shilov, B.V.; Nevezhin, E.V.; Negrebetsky, V.V.; Maslov, M.A.; Lagunin, A.A.; Malakhov, M.V.
Prospective pharmacological effects of psoralen photooxidation products and their cycloadducts with aminothiols: Chemoinfor-
matic analysis. Bull. RSMU 2020, 5, 31–39. [CrossRef]
24. Décout, J.-L.; Lhomme, J. Photolysis of 8-methoxypsoralen in dilute deaerated aqueous and ethanolic solutions. Photochem.
Photobiophys. 1985, 10, 113–120.
25. Mohammad, T.; Morrison, H. In situ generation of psoralens by photolysis of water-soldble precursors. Photochem. Photobiol.
2008, 55, 631–638. [CrossRef]
26. Mohammad, T.; Morrison, H. Light-Mediated Cyclization Of (E)-3-(5-[6-Hydroxy-7-Methoxy] Benzofuranyl)Propenoic Acid,
A Water-Soluble Precursor Of 8-Methoxypsoralen. Photochem. Photobiol. 1994, 59, 248–251. [CrossRef]
27. Terrian, D.L.; Mohammad, T.; Morrison, H. Photocyclization of Ortho-Substituted Cinnamic Acids. J. Org. Chem. 1995, 60,
1981–1984. [CrossRef]
28. Feringa, B.L. The Art of Building Small: From Molecular Switches to Motors (Nobel Lecture). Angew. Chem. Int. Ed. 2017, 56,
11060–11078. [CrossRef]
29. García-López, V.; Liu, D.; Tour, J.M. Light-Activated Organic Molecular Motors and Their Applications. Chem. Rev. 2019, 120,
79–124. [CrossRef] [PubMed]
30. Baroncini, M.; Silvi, S.; Credi, A. Photo- and Redox-Driven Artificial Molecular Motors. Chem. Rev. 2019, 120, 200–268. [CrossRef]
31. Andreopoulos, F.M.; Beckman, E.J.; Russell, A.J. Photoswitchable PEG-CA Hydrogels and Factors That Affect Their Photo-
sensitivity. J. Polym. Sci. A Polym. Chem. 2000, 38, 1466–1476. [CrossRef]
32. Matsuhira, T.; Yamamoto, H.; Okamura, T.-A.; Ueyama, N. Manipulation of an intramolecular NHO hydrogen bond by
photoswitching between stable E/Z isomers of the cinnamate framework. Org. Biomol. Chem. 2008, 6, 1926–1933. [CrossRef]
[PubMed]
33. Sakai, H.; Taki, S.; Tsuchiya, K.; Matsumura, A.; Sakai, K.; Abe, M. Photochemical Control of Viscosity Using Sodium Cinnamate
as a Photoswitchable Molecule. Chem. Lett. 2012, 41, 247–248. [CrossRef]
34. Störmann, F.; Wischke, C.; Lendlein, A. Photo-Reversibility of Cinnamylidene Acetic Acid Derived Crosslinks in Poly(ε-
caprolactone) Networks. MRS Proc. 2015, 1718, 49–54. [CrossRef]
35. Paul, A.; Mengji, R.; Chandy, O.A.; Nandi, S.; Bera, M.; Jana, A.; Anoop, A.; Singh, N.D.P.; Singh, P.N. ESIPT-induced fluorescent
o-hydroxycinnamate: A self-monitoring phototrigger for prompt image-guided uncaging of alcohols. Org. Biomol. Chem. 2017
15, 8544–8552. [CrossRef] [PubMed]
,
36. Paul, A.; Bera, M.; Gupta, P.; Singh, N.D.P. o-Hydroxycinnamate for sequential photouncaging of two different functional groups
and its application in releasing cosmeceuticals. Org. Biomol. Chem. 2019, 17, 7689–7693. [CrossRef]
37. Garrett, E.R.; Lippold, B.C.; Mielck, J.B. Kinetics and Mechanisms of Lactonization of Coumarinic Acids and Hydrolysis of
Coumarins, I. J. Pharm. Sci. 1971, 60, 396–405. [CrossRef]
38. Lippold, B.C.; Garrett, E.R. Kinetics and Mechanisms of Lactonization of Coumarinic Acids and Hydrolysis of Coumarins II.
J. Pharm. Sci. 1971, 60, 1019–1027. [CrossRef]
39. Brouwer, A.M. Standards for photoluminescence quantum yield measurements in solution (IUPAC Technical Report). Pure Appl.
Chem. 2011, 83, 2213–2228. [CrossRef]
40. Neese, F. The ORCA program system. Wiley Interdiscip. Rev. Comput. Mol. Sci. 2012, 2, 73–78. [CrossRef]
41. Pu, S.-Z.; Sun, Q.; Fan, C.-B.; Wang, R.-J.; Liu, G. Recent advances in diarylethene-based multi-responsive molecular switches.
J. Mater. Chem. C 2016, 4, 3075–3093. [CrossRef]

Molecules 2021, 26, 2800
14 of 14
42. Liu, D.; Yu, B.; Su, X.; Wang, X.; Zhang, Y.-M.; Li, M.; Zhang, S.X.-A.; Yu, B. Photo-/Baso-Chromisms and the Application of a
Dual-Addressable Molecular Switch. Chem. Asian J. 2019, 14, 2838–2845. [CrossRef] [PubMed]
43. Medved’, M.; Hoorens, M.W.H.; Di Donato, M.; Laurent, A.D.; Fan, J.; Taddei, M.; Hilbers, M.; Feringa, B.L.; Buma, W.J.;
Szymanski, W. Tailoring the optical and dynamic properties of iminothioindoxyl photoswitches through acidochromism. Chem.
Sci. 2021, 12, 4588–4598. [CrossRef]