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Comptes Rendus
Chimie
Andrey Kustov, Philipp Morshnev, Natal’ya Kukushkina, Mikhail
Krestyaninov, Nataliya Smirnova, Dmitry Berezin, Galina Kokurina
and Dmitry Belykh
The effect of molecular structure of chlorin photosensitizers on
photo-bleaching of 1,3-diphenylisobenzofuran—the possible evidence
of iodine reactive species formation
Volume 25 (2022), p. 97-102
<https://doi.org/10.5802/crchim.158>
© Académie des sciences, Paris and the authors, 2022.
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Comptes Rendus
Chimie
2022, 25, p. 97-102
https://doi.org/10.5802/crchim.158
Preliminary communication / Communication préliminaire
The effect of molecular structure of chlorin
photosensitizers on photo-bleaching of
1,3-diphenylisobenzofuran—the possible evidence
of iodine reactive species formation
Andrey Kustov a, b, PhilippMorshnev a, b, Natal’ya Kukushkina a, b,
Mikhail Krestyaninov a, Nataliya Smirnovaa, Dmitry Berezin ∗, b, Galina Kokurinab
and Dmitry Belykh c
a G.A. Krestov Institute of Solution Chemistry, Russian Academy of Sciences,
Academicheskaya str., 1, Ivanovo, 153045, Russia
b Ivanovo State University of Chemistry and Technology, Research Institute of
Macroheterocycles, Sheremetevsky av., 7, Ivanovo, 153000, Russia
c Institute of Chemistry of Komi Science Centre of the Ural Branch of Russian
Academy of Sciences, Pervomaiskaya str., 48, Syktyvkar, 167000, Russia
E-mails: kustov@isuct.ru (A. Kustov), morshnevphilipp@gmail.com (P. Morshnev),
nataliakukushkina05.1998@mail.ru (N. Kukushkina), mak1111@bk.ru
(M. Krestyaninov), smir141973@mail.ru (N. Smirnova), berezin@isuct.ru (D. Berezin),
kustov26@yandex.ru (G. Kokurina), belykh-dv@mail.ru (D. Belykh)
Abstract. In this short communication, we focus on the ability of chlorin e6 photosensitizers (PSs)
with one, two or three cationic groups to generate singlet oxygen species (1O2) in an apolar model
medium, viz. 1-octanol (OctOH). The quantum yield of 1O2 (Φ∆) is determined indirectly by the
chemical method for seven synthesized PSs using 1,3-diphenylisobenzofuran (DPBF) as a singlet
oxygen trap. The Φ∆ values of four PSs with one or two cationic groups occupying opposite positions
in the macrocycle are equal to 0.6, which is nearly identical to the values obtained by the time-
resolved fluorescence spectroscopy method. In contrast, three PSs containing a 3(1),3(2)-bis-(N,N,N-
trimethylaminomethylvinyl) iodide fragment with two adjacent cationic groups have an abnormally
high quantum yield reaching 0.8. It is apparent that this phenomenon results from faster trap oxidation
induced by radical reactions leading to molecular iodine formation.
Keywords. Antimicrobial photodynamic therapy, Cationic chlorin photosensitizer, Singlet oxygen
formation, 1,3-diphenylisobenzofuran, Reactive iodine species.
Manuscript received 10th August 2021, revised 20th November 2021 and 14th January 2022, accepted
15th February 2022.
∗Corresponding author.
ISSN (electronic) : 1878-1543 https://comptes-rendus.academie-sciences.fr/chimie/
98 Andrey Kustov et al.
1. Introduction
Antimicrobial photodynamic therapy (APDT) with
natural or synthetic photosensitizers seems to be a
very promising alternative or an important supple-
ment to classical antibiotic therapy of many localized
infections [1–5]. This bright technique consists of
three crucial components: a photosensitizer, molec-
ular oxygen and visible light taken together and leads
to the formation of reactive oxygen species (ROS)
such as singlet oxygen 1O2 (Type II photochemical
mechanism) or various radical forms (HO•, O•−2 )
and H2O2 (Type I photochemical mechanism) [6–8].
These ROS inflict a fatal damage towards bacterial
cells and provide the excellent killing effect, if the PS
molecule is able to penetrate a bacterial cell [1–3].
However, neutral and anionic PSs, which are cur-
rently in use for treating various tumors, have low
affinity towards the outer lipopolysaccharide mem-
brane of Gram-negative bacteria and can efficiently
kill these pathogens under irradiation [2,3]. In con-
trast, cationic PSs containing several charged groups
or bound to an appropriate carrier provide efficient
inactivation of Gram-negative pathogens [1–3,9,10].
Another important way to improve the ability of PSs
to kill these microbes is to add non-toxic inorganic
salts such as azides, iodides or thiocyanides to a
PS formulation [4,5]. In particular, the addition of
various KI amounts to a 10 µM “Photofrin” (water-
soluble hematoporphyrin) solution provides the
concentration-dependent killing curve for five Gram-
negative bacteria [4]. Similar results were reported for
several cationic porphyrins but not for their neutral
derivatives [5]. This “extra killing effect” is recognized
to be dependent of the PS structure [4,5] and results
from several parallel processes initiated by the sin-
glet oxygen reaction with an iodide ion in water and
peroxyiodide formation [4,5]. The latter is unstable
and may suffer further decomposition via two differ-
ent pathways that depend on the degree of PS bind-
ing to a microbial cell [5]. The first of them involves
the formation of free iodine (I2/I−3 ) and hydrogen
peroxide. The second pathway leads to the formation
of reactive iodine species (I•−2 ) with a short diffusion
distance. In principle, both processes provide the
efficient killing of Gram-negative bacteria but in a
different way (see [4,5] and references therein).
With these results in mind, we studied photo-
bleaching of the well-established 1O2 trap, viz.
1,3-diphenylisobenzofuran, using several chlorin
PSs with one, two or three iodide ions in OctOH as
an appropriate lipid-like environment model [11].
Then, the respective photo-bleaching rates were
used to compute theΦ∆ values and to compare them
with the quantities obtained by direct spectroscopic
measurements [9].
2. Experimental section
2.1. Reagents
1-Octanol (Panreac, >98%) was dried with 4 Å molec-
ular sieves and distilled under reduced pressure at
353 K. Karl Fisher titration showed that the final
water content in OctOH was less than 0.02 wt%.
1,3-diphenylisobenzofuran (DPBF) (Sigma-Aldich,
>99%) and crystal iodine (Reachem, >99%) were
used as supplied.
PSs shown in Figure 1 were synthesized, puri-
fied and identified in our laboratory with 1H NMR,
MS- and UV-Vis spectra. 1H NMR spectra were
registered with the Bruker Avance II spectrome-
ter (300 MHz) or Bruker Avance III spectrometer
(500 MHz). CDCl3 or DMSO d6 were used as ap-
propriate solvents and TMS was applied as the in-
ternal standard for the 1H NMR measurements.
MS-spectra were obtained with the Thermo finni-
gan LCQ Flut (ESI) instrument and/or MALDI FAB
MS-spectrometer Shimadzu AXIMA Confidence with
α-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-
dihydroxybenzoic acid (DHB) as a matrix. UV-Vis
spectra were obtained at 293 K with the Drawell
D8 spectrophotometer in a highly diluted pigment
solution (∼5–8 µmol).
A detailed description of the synthetic and identi-
fication procedures of compounds 1, 2, 5–8 was given
earlier [9,10,12]. For newly synthesized compounds
3, 4 this information can be found in the Supplemen-
tary material file.
2.2. Determination of singlet oxygen quantum
yield
The singlet oxygen quantum yield was determined by
the indirect chemical method which was very simi-
lar to the technique described elsewhere [13,14]. In
brief, two quartz cuvettes with a 1 cm optical way
contained 3 ml of a PS + DPBF or St + DPBF solution
C. R. Chimie — 2022, 25, 97-102
Andrey Kustov et al. 99
Figure 1. Molecular structures of the PSs studied: (a) 13(1)-N-(2-N′N′N′-trimethylammonioethyl
iodide)amide chlorin e6 15(2),17(3)-dimethyl ester 1, (b) 3(2)-(N,N,N-trimethylaminomethyl io-
dide) chlorin e6 13(1)-N-methylamide 15(2),17(3)-dimethyl ester 2; (c) chlorin e6 13(1)-(4′-N,N-
dimethylpiperazinyl iodide)amide 15(2),17(3)-dimethyl ester 3; (d) 3(2)-(N,N,N-trimethylaminomethyl
iodide) chlorin e6 13(1)-N′-(2-N′′,N′′,N′′-trimethylammonioethyl iodide)amide 15(2),17(3)-dimethyl
ester 4; (e) 3(1),3(2)-bis-(N,N,N-trimethylaminomethyl iodide) chlorin e6 13(1)-N-methylamide
15(2),17(3)-dimethyl ester 5; (f ) 3(1),3(2)-bis-(N,N,N-trimethylaminomethyl iodide) chlorin e6 13(1)-N-
(2-myristoxyethyl)amide 15(2),17(3)-dimethyl ester 6; (g) 3(1),3(2)-bis-(N,N,N-trimethylaminomethyl io-
dide) chlorin e6 13(1)-N′-(2-N′′,N′′,N′′-trimethylammonioethyl iodide)amide 15(2),17(3)-dimethyl ester
7; (h) pheophorbide a 17(3)-methyl ester (methylpheophorbide a) 8.
C. R. Chimie — 2022, 25, 97-102
100 Andrey Kustov et al.
Table 1. Singlet oxygen quantum yield for methylpheophorbide a (compound 8) and seven chlorin
photosensitizers (compounds 1–7) in liquid 1-OctOH
PS
1 2 3 4 5 6 7 8
0.63±0.05a 0.63±0.02 0.64±0.04 0.60±0.03 0.77±0.02 0.79±0.02 0.77±0.05 0.49±0.05
0.65±0.07b - - - 0.60±0.06b 0.53±0.05b 0.47±0.05b
aUncertainties represent a twice standard deviation. For the determination of theΦ∆ value for compound
1 we have used compound 8 as the appropriate standard with the quantum yield equals 0.47 [9].
bValues obtained from the direct measurements of 1O2 luminescence intensity at 1270 nm [9].
with the absorbance of 0.2–0.3 at the Q-band and 0.9–
1.2 at 444 nm. The cuvettes were irradiated with a red
light diode panel (BIC, Minsk) [9,10]. This panel emit-
ting between 590 and 720 nm with the maximum at
662 nm provided the light spot of 10 × 10 cm. The
power density of red light was chosen to be equal
to 3 mW·cm−2. At this irradiance a fluence (light
dose) of 0.18 J·cm−2 was delivered every minute. The
monocationic chlorin (see compound 1 in Figure 1)
with the known Φ∆ value equaling to 0.67 [9] was
used as an appropriate standard.
DPBF degradation in both cuvettes was monitored
spectrophotometrically each minute, and each irra-
diation session contained usually six-eight measure-
ments. Then, the rate-of-degradation constants kd
were evaluated in terms of the first order exponen-
tial decay. All the photo-bleaching experiments were
repeated from six to eight times. The Φ∆ values were
derived from the following equation:
Φ∆PS = kdPS
kdSt
I PSt
I PPS
Φ∆St, (1)
where the PS and St symbols refer to the photosen-
sitizer and the standard, respectively. The I P values
were computed numerically by:
I P =
∫ λ2
λ1
I0(λ)(1−10−A)dλ, (2)
where λ1 and λ2 are the initial and the final wave-
lengths of the region, in which the panel and PS spec-
tra overlap, I0 (λ) is the LED panel intensity (between
590 and 720 nm) as a function of λ and A is the solu-
tion optical density. The photo-bleaching correction
term was omitted because the PS concentration was
constant over all the irradiation period.
3. Results and discussion
The PS molecules shown in Figure 1 in their ground
state, i.e. singlet electronic state (S0), contain two
electrons with the opposite spins located in the most
favorable molecular orbital [6]. Absorbance of red
light at 660 nm leads to the transfer of the macrocycle
to an excited singlet state (S1). The excited PS mol-
ecule is capable of intersystem crossing and transi-
tion to the long-lived biradical triplet state (T1) with
an inverted spin of one electron [4,6]. Then, the PS
molecule in this triplet state may transfer its energy
to molecular oxygen, 3O2, which leads to the forma-
tion of a reactive singlet oxygen species, 1O2. This
so-called Type II process is characteristic of apolar
media, while in H-bonded solvents, especially water,
some fraction of radical forms is detected [15].
We have recently shown [9] that in many chlorin-
type PSs without metal ions in their coordination
sphere, the Φ∆ values range from 0.47 to 0.67 regard-
less of the macrocycle structure. Hence, the quan-
tum yield of singlet oxygen of a chlorin photosen-
sitizer is within 0.55 ± 0.1 in any weakly polar sol-
vent. This finding is in agreement with chemical in-
tuition and the available literature values [16–19]. Ta-
ble 1 shows that for compounds 1–4 and 8 the corre-
sponding quantum yields agree with this estimation.
It is interesting to note that the correspondingΦ∆ val-
ues are larger for chlorin PSs (see compounds 1–4)
compared to phorbin-type pigments (compound 8).
Moreover, for compounds 1, 8 the indirectly obtained
quantum yields are seen to be in very good agree-
ment with the previously reported values [9].
However, compounds 5–7 demonstrate signifi-
cantly higher quantum yields than what could be ex-
pected from direct measurements (see Table 1). This
difference results from the higher rate of DPBF
C. R. Chimie — 2022, 25, 97-102
Andrey Kustov et al. 101
oxidation that leads to larger kd values (see (1)).
Inspection of the solute structures shown in Fig-
ure 1 indicates that all three PS molecules (see com-
pounds 5–7) contain two or even three iodine atoms
which may be responsible for the higher oxida-
tion rate of the trap. However, compound 4 also
contains two iodine atoms but has a “normal” Φ∆
value (see Table 1). Hence, the close location of
ionic groups in compounds 5–7 is much more im-
portant for the higher oxidation rate of the trap
than their relative number. In fact, two cationic
groups occupy opposite positions in compound 4,
while they are located in the 3(1),3(2)-bis-(N,N,N-
trimethylaminomethylvinyl) iodide fragment in
compounds 5–7. This close location seems to be
responsible for the higher oxidation rate of DPBF.
Another proof comes from our comparative anal-
ysis showing that the Φ∆ values for compounds 5
and 7 are almost identical, although the latter solute
contains an additional ionic group (see Figure 1).
Thus, the structure of the 3(1),3(2)-bis-(N,N,N-
trimethylaminomethylvinyl) iodide fragment with
two adjacent ionic groups is responsible for the in-
teraction between iodine and singlet oxygen [5]. The
intermediate of this reaction can further decom-
pose to free iodine (I2/I−3 ) and peroxyalcohol radicals
(ROO•) or short-lived reactive radicals I•−2 accord-
ing to the Scheme S1 shown in the Supplementary
material file [5].
In principal, both of these processes may lead
to the abnormal Φ∆ values due to faster oxidation
of the trap. However, it should be kept in mind
that the “extra killing effect” mentioned above takes
place in an aqueous medium, where each singlet
oxygen molecule interacts with well-hydrated iodide
ions [5]. However, all the cationic PSs shown in Fig-
ure 1 are weakly dissociated in apolar OctOH, and
1O2 molecules interact with the iodide ion bound to
the cationic group of PS.
To shed more light on the trap photobleaching
mechanisms in a lipid-like medium, we studied the
effect of ammonium iodide addition (∼0.2 mmol/kg)
to compound 5 and found that irradiation lead to
an increase in the solution absorbance at 365 nm
(see Figure S12 in the Supplementary material file).
According to the results mentioned above (see [4,5]
and references therein), this phenomenon can be
attributed to the formation of molecular iodine in
the liquid phase. To check this point, we dissolved an
appropriate amount of iodine and NH4I in OctOH
and found that the solution spectrum was very
similar to the spectrum of compound 5 + NH4I
shown in Figure S12. It is apparent that this proves
that singlet oxygen interacts with ammonium io-
dide, which leads to the formation of molecular io-
dine. We assume that a similar phenomenon oc-
curs in a solution of PSs with a 3(1),3(2)-bis-(N,N,N-
trimethylaminomethylvinyl) iodide fragment. This
induces faster oxidation of DPBF, which is responsi-
ble for the abnormalΦ∆ values.
4. Conclusions
In summary, our experimental efforts aimed to study
singlet oxygen generation by cationic chlorins indi-
cate that PSs with one or two cationic groups occu-
pying opposite positions in the macrocycle have nor-
mal Φ∆ values of 0.6, which is in good agreement
with the results of direct measurements. However,
PSs with two adjacent cationic groups have a larger
quantum yield that reaches 0.8. This phenomenon
results from faster trap oxidation due to radical reac-
tions that lead to the formation of molecular iodine.
CRediT authorship contribution statement
AVK: Conceptualization, Methodology, Investigation,
Writing—original draft, Review and editing, Fund-
ing acquisition. PKM: Investigation, Formal analy-
sis. NVK: Synthesis, Investigation. MAK: Methodol-
ogy, Investigation. NLS: Methodology, Investigation.
DBB: Conceptualization, Writing, Review and edit-
ing, Funding acquisition. GNK: Methodology, Formal
analysis. DVB: Investigation, Synthesis, Writing—
review and editing, Funding acquisition.
Conflicts of interest
Authors have no conflict of interest to declare.
Acknowledgment
This work was supported by the Russian Science
Foundation (project N 21-13-00398).
Supplementary data
Supporting information for this article is available on
the journal’s website under https://doi.org/10.5802/
crchim.158 or from the author.
C. R. Chimie — 2022, 25, 97-102
102 Andrey Kustov et al.
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C. R. Chimie — 2022, 25, 97-102


The effect of molecular structure of chlorin photosensitizers on photo-bleaching of 1,3-diphenylisobenzofuran—the possible evidence of iodine reactive species formation

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