Photophysical and bactericidal properties of pyridinium and imidazolium porphyrins for photodynamic antimicrobial chemotherapy

Article abstract of DOI:10.3390/molecules26041122

Despite advances achieved over the last decade, infections caused by multi-drug-resistant bacterial strains are increasingly becoming important societal issues that need to be addressed. New approaches have already been developed in order to overcome this problem. Photodynamic antimicrobial chemotherapy (PACT) could provide an alternative to fight infectious bacteria. Many studies have highlighted the value of cationic photosensitizers in order to improve this approach. This study reports the synthesis and the characterization of cationic porphyrins derived from methylimidazolium and phenylimidazolium porphyrins, along with a comparison of their photophysical properties with the well-known N-methylpyridyl (pyridinium) porphyrin family. PACT tests conducted with the tetracationic porphyrins of these three families showed that these new photosensitizers may offer a good alternative to the classical pyridinium porphyrins, especially against S.aureus and E.coli. In addition, they pave the way to new cationic photosensitizers by the means of derivatization through amide bond formation.

Full text of DOI:10.3390/molecules26041122

molecules
Article
Photophysical and Bactericidal Properties of Pyridinium
and Imidazolium Porphyrins for Photodynamic
Antimicrobial Chemotherapy
Florent Le Guern 1
,2
, Tan-Sothéa Ouk 2 , Issabayev Yerzhan 3 , Yesmurzayeva Nurlykyz , Philippe Arnoux
3
3
,
Céline Frochot 3 , Stéphanie Leroy-Lhez 2 and Vincent Sol *
2,
1
Institut Lavoisier de Versailles, Université Paris-Saclay, UVSQ, CNRS, 78035 Versailles, France;
florent.le-guern@uvsq.fr
Laboratoire PEIRENE, Université de Limoges, EA 7500, 123 Avenue Albert Thomas,
2
3
8
7060 Limoges CEDEX, France; tan-sothea.ouk@unilim.fr (T.-S.O.); stephanie.lhez@unilim.fr (S.L.-L.)
Laboratoire Réactions et Génie des Procédés (LRGP), Université de Lorraine, UMR 7274 CNRS, ENSIC,
rue Grandville, 54000 Nancy, France; issabaev@gmail.com (I.Y.); yesmurzayeva.nurly@gmail.com (Y.N.);
1
philippe.arnoux@univ-lorraine.fr (P.A.); celine.frochot@univ-lorraine.fr (C.F.)
Correspondence: vincent.sol@unilim.fr
*
Abstract: Despite advances achieved over the last decade, infections caused by multi-drug-resistant
bacterial strains are increasingly becoming important societal issues that need to be addressed. New
approaches have already been developed in order to overcome this problem. Photodynamic antimi-
crobial chemotherapy (PACT) could provide an alternative to fight infectious bacteria. Many studies
have highlighted the value of cationic photosensitizers in order to improve this approach. This study
reports the synthesis and the characterization of cationic porphyrins derived from methylimidazolium
and phenylimidazolium porphyrins, along with a comparison of their photophysical properties with
the well-known N-methylpyridyl (pyridinium) porphyrin family. PACT tests conducted with the
tetracationic porphyrins of these three families showed that these new photosensitizers may offer
a good alternative to the classical pyridinium porphyrins, especially against S.aureus and E.coli. In
addition, they pave the way to new cationic photosensitizers by the means of derivatization through
amide bond formation.
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Citation: Le Guern, F.; Ouk, T.-S.;
Yerzhan, I.; Nurlykyz, Y.; Arnoux, P.;
Frochot, C.; Leroy-Lhez, S.; Sol, V.
Photophysical and Bactericidal
Properties of Pyridinium and
Imidazolium Porphyrins for
Photodynamic Antimicrobial
Chemotherapy. Molecules 2021, 26,
1
122. https://doi.org/10.3390/
molecules26041122
Keywords: photodynamic; therapy; antimicrobial; cationic; porphyrin; properties; photophysical
Academic Editor: Łukasz Sobotta
Received: 15 January 2021
Accepted: 17 February 2021
Published: 20 February 2021
1
. Introduction
Since the introduction of penicillin in the 1940s, the era of antibiotics has been consid-
ered as miraculous, given that these treatments had significantly decreased the number
of deaths resulting from bacterial infections [ ]. Despite the well-known rise in antibiotic-
resistant bacteria, these drugs have been wrongly used all around the world, due in
particular to faulty communications and common beliefs [ ]. In addition to antibioresis-
tance, leading to the emergence of superbugs or multidrug-resistant (MDR) strains, the
pace of new antibiotic discoveries has gradually slowed down in the recent years [ ]. The
issues due to microbial resistance are currently recognized worldwide by health organi-
zations [ ]. Consequently, alternative approaches have been investigated to reduce the
1
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
2
3,4
5
impact of microbial resistance.
Around the world, laboratories have focused their attention on a recent and promising
alternative to antibiotics, namely photodynamic antimicrobial chemotherapy (PACT). This
Copyright: © 2021 by the authors.
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/
technique relies on the light activation of photosensitizers (PS) in the presence of molecular
3
oxygen ( O ). Under appropriate light irradiation, PS activation leads to the production
2
1
of singlet oxygen ( O ) by energy transfer, or oxygen radicals (such as superoxide anion
2
•
−
•
•
O
2
, hydroxyl radical HO , hydroperoxyl radical HOO ) by electron transfer. These
4
.0/).
reactive oxygen species (ROS) are able to rapidly react with a large panel of molecules
Molecules 2021, 26, 1122. https://doi.org/10.3390/molecules26041122
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 1122
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(proteins, lipids, nucleic acids, metabolites) and cause cellular damages, which ultimately
lead to cell death. The number and diversity of the cellular targets greatly limit the
rise in bacterial strains that resist this treatment [ ]. Owing to their very short life
span, ROS do not end up in the waste system unlike common sanitizers, and are thus
environmentally friendly [ ]. Although PACT exhibits many advantages for bacterial
photoinactivation, its lack of specificity is a major drawback, as it may cause damages
to the host tissues as well [ 11]. PACT was first tried out against methicillin-resistant
6,7
6,8
9–
S. aureus (MRSA), with promising results [12]. Unfortunately, Gram-negative bacteria
have shown a poor sensitivity to the PS initially studied, which has been attributed the
protection provided by their outer membrane [13,14]. Nevertheless, it has been shown
that cationic PS exhibited a good affinity for bacteria by interacting through electrostatic
interactions, with the highly negatively charged cell wall components such as lipoteichoic
acid (LTA) and lipopolysaccharide (LPS) in Gram-positive and Gram-negative bacteria,
respectively [15–17]. Accordingly, cationic porphyrins displayed photoinduced activity
against a broad spectrum of bacteria [17
families of cationic PSs [21–23].
–20], and this trend was also confirmed with several
As members of the tetrapyrrolic PS, these compounds can be classified according to
the functions substituted at the meso positions: A porphyrins are related to symmetric and
4
uniform compounds, A B to asymmetric compounds with one meso position substituted by
3
a different function, and A B to porphyrins equally substituted by two different functions
2
2
(
cis or trans isomers) [24]. Unfortunately, a lack of data comparing the photophysical and
photobactericidal properties of cationic porphyrins may be problematic at the beginning
of a study. Except for aryl porphyrins carrying ammonium derivatives [25 27], these
cationic PS are mostly obtained by using N-methylated pyridyl (also called pyridinium)
groups [16 18 28 29]. Pyridinium porphyrins, as well as aryl porphyrin-carrying ammo-
–
–
, ,
nium derivatives, are well-known in PACT as these PS have shown very good activities
against several strains. As their efficacy is partially due to their quaternary ammonium
functions [17], porphyrins carrying imidazole groups (or N-methyl derivatives) should
also show interesting properties. Moreover, even if imidazole (metallo)porphyrins have
been studied for their conductivity properties [30
their photocytotoxicity has rarely been spotlighted [34
A B structures [36 38]. In the same way, phenylimidazole porphyrins have also been
–
32] or their antioxidant potencies [33],
,
35], especially in PACT as A or
4
–
3
presented in few reports, but never as A porphyrins [39]. We report herein the synthesis,
4
and photochemical and photophysical characterization of six different families of pyri-
dinium and imidazolium porphyrins along with a comparison of their efficiency against
Gram-positive and Gram- with a negative bacterial strains special underscoring on A3B
porphyrins. In connection with our research studies in PACT [40
synthetic routes to obtain porphyrin derivatives, which have been designed to selectively
target bacteria [45 46]. Through these studies, it occurred to us that amine or carboxylic acid
functions are very important to build amide bonds between PS and the targeting moieties
such as peptides). Thus, these functions have been distributed among the families of
porphyrins synthetized in this study (Table 1).
–44], we have devised
,
(

Molecules 2021, 26, 1122
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Table 1. Structures of neutral and cationic synthetized porphyrins.
Neutral Porphyrins
B
Cationic Porphyrins
Methylated A
Compound a
n
A
Compound a
4
-
1a [47,48]
2a [47,48]
2b [51,52]
2d [44]
4a [32]
4b
3
3
4
3
3
1b [49,50]
1d [53]
3a [54,55]
3b
-
3c
4c
4
3
-
5a [30]
6a [30,56]
5b
6b
5
e (isomer
6e (isomer
mixture)
2
mixture) [57]
a
Related compounds obtained after deprotection of carboxylic acid or amine functions, followed by previous studies using similar compounds.

Molecules 2021, 26, 1122
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2. Results and Discussion
2.1. Synthesis and Characterization
The porphyrins described in this study were synthesized using the Little [58] or
Adler–Longo [59] protocols. These porphyrins belong to six families named after their meso-
substituted groups: : Pyridyl, : N-methylpyridyl (or pyridinium), : Phenylimidazole,
: Phenylimidazolium, : Methylimidazole, and : Methylimidazolium. In these groups,
A porphyrins were first obtained (1a to 6a). Then, in each main family, A B porphyrins
1
2
3
4
5
6
4
3
were synthesized to observe the consequence of different functions on their photophysical
properties. Generally, these functions are introduced into the porphyrin structure by mixing
a functionalized benzaldehyde with other aldehydes during the synthesis, following the
Little protocol. In order to offer a global and efficient procedure for all porphyrins, only
protected functionalized aldehydes have been used in these syntheses. These protections
ease the purification steps, which are known to present difficulties. Indeed, the purification
of porphyrins carrying a free carboxylic acid function mainly depends on the brand of the
silica gel. Different functionalized benzaldehydes have been chosen to provide reactive
groups after deprotection: Methyl 4-formylbenzoate for a carboxylic acid function (b,
electron-withdrawing group (EWG)), ethyl 4-formylphenoxyacetate for a carboxylic acid
function on a hydroxyl-based porphyrin (c, EDG), and 4-acetamidobenzaldehyde for a
primary amine function (d, electron-donor group (EDG)) (Figure 1).
Figure 1. Deprotection of carboxylic acid and amine functions.
4
-acetamidobenzaldehyde was chosen to obtain porphyrins carrying an amine func-
tion but, according to other studies, these compounds can also be obtained by using
-nitrobenzaldehyde [60 61]. N-Methylated A B porphyrins carrying one carboxylic acid
4
,
3
function were directly prepared from the respective A B porphyrins (2b from 1b, for exam-
3
ple), as the carboxylic group only reacts with iodomethane under its carboxylate form. To
obtain the N-methylated A B porphyrin 2d carrying one primary amine, N-methylation
3
had to been done before deprotection to avoid any methylation of the primary amine.
Owing to the water-solubility of this porphyrin and the impossibility of washing out excess
salts, HCl was replaced by TFA for the deprotection step as TFA can be totally removed by
successive evaporation and freeze-drying steps. The different deprotection steps have been
performed with satisfying yields. Finally, A B porphyrins were also prepared to support
2
2
prospective works using two coupling agents (5e and 6e). Unfortunately, the separation
of trans and cis isomers could not be achieved using common purification procedures.
Therefore, all data concerning 5e and 6e are obtained by using an isomer mixture.
The porphyrin syntheses did present some yield fluctuations, depending on both the
family and the functionalized aldehyde (Table 2). The synthesis of pyridyl porphyrins
1
is well-known from the literature, whereas phenylimidazole
porphyrins are less common in PACT. Unfortunately, the final yield of 5a synthesis is
lower than that reported in the literature (generally about 10–12%) because of an interaction
3 and methylimidazole
5

Molecules 2021, 26, 1122
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of the compound with silica gel used for chromatography. Thus, alumina is usually advised
for such compounds [34]. 3a was the easiest porphyrin to purify because of the particularly
high final yield. Whereas the formation of A B porphyrins from
1
and
have been obtained with very good yields, which are
three times higher than the average. Carboxylic porphyrins ( and ) have been recovered
5 leads to similar
3
yields, asymmetric porphyrins from
3
b
c
with similar results, but the use of 4-acetamidobenzaldehyde has induced a significant
drop in yield. Furthermore, methyl 4-formylbenzoate seemed to be very reactive as the
presence of all possible porphyrins was found in the crudes (B , AB , A B ) despite the
4
3
2 2
advised reactant proportions for A B synthesis (3 eq of A, 1 eq for B).
3
Table 2. Average yields of porphyrin syntheses.
Type
Porphyrin
Average Yield (%)
1
3a
5
a
10
15
2
A4
a
1
1
b
d
5
2
A3B
3b
13
10
3c
5
b
5
5
A2B2
5e
The synthesized porphyrins exhibited varied solubility properties. Generally, phenylim-
idazole porphyrins were less soluble than all other porphyrins, even if a high purity of
3
porphyrin usually involves some solubility issues that can be resolved by adding minute
amounts of methanol into usual apolar solvents. N-Methyl porphyrins are supposed to
be water-soluble but a primary dissolution into a DMSO solution is strongly advised to
avoid any incomplete dissolution. Due to the solubility issues, the analysis of porphyrins
may appear difficult in classical solvent. To obtain NMR analysis of
1, 3, and 5, a mixture
with CDCl and CD OD has been used that may induce some peak shifting and multi-
3
3
plication depending on the device used. Fortunately, all porphyrins could be identified
1
by MS (ESI +) and H NMR (Supplementary Materials Figures S1 to S18). Due to the
asymmetric methylimidazole group in
Thus, NMR spectra of porphyrins have shown some peak multiplication, especially for
the signals attributed to N-methyl protons. The total N-methylation of the imidazole group,
in porphyrin family , led to symmetric signals that could indicate an even distribution of
5, different stereoisomers were actually obtained.
5
6
the positive charge within the imidazole ring.
The carboxylic acid reactivity of the different water-soluble porphyrins was also
investigated (Figure 2). N-Methylation of 1b (leading to 2b) induced a drop in the reactivity
of the carboxylic function. Indeed, 2b has shown a poor reactivity in different conditions
(buffers or DMSO), in spite of the use of different catalysts (carbodiimide or uronium salts).
Thus, in order to obtain a conjugate based on 2a, the coupling should be done before the
N-methylation step, which implies that the new moiety should not react with iodomethane
afterward. Otherwise, the carboxylic acid of 1b should be activated by coupling an N-
hydroxysuccinimide group before N-methylation [49]. On the other hand, other porphyrins
(
4b, 4c, 6b, 6e) were able to react with these catalysts, leading then to the formation of an
amide function in the presence of a primary amine.

Molecules 2021, 26, 1122
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Figure 2. Investigation of carboxylic acid reactivities of cationic porphyrins using coupling agents.
2.2. Photophysical Properties
The photophysical properties of the different porphyrins have been investigated in
ethanol (Figures 3 and 4 and Tables 3 and 4). In order to correlate the following biological
trials, the photophysical properties of cationic porphyrins had also been investigated
through further studies in aqueous solution (D O).
2
− −1
1
Table 3. Maximum absorbance wavelengths (
porphyrins in ethanol solution.
λ, nm) and molar absorptivities (log ε, L mol cm ) of the cationic
−
1
−1
λmax (nm) (log ε; L mol cm )
Compd
Soret
14
3.92)
15
5.10)
16
4.82)
15
5.16)
26
5.40)
28
5.16)
17
3.71)
17
5.14)
18
5.23)
Qy(1,0)
Qy(0,0)
Qx(1,0)
Qx(0,0)
Compd
4a
Soret
Qy(1,0)
Qy(0,0)
Qx(1,0)
Qx(0,0)
4
509
(2.56)
546
(2.00)
588
(2.00)
645
(1.75)
416
(5.62)
513
(4.24)
547
(3.86)
590
(3.71)
645
(3.45)
1
a
(
(
(
(
(
(
(
(
(
4
512
(3.86)
544
(3.51)
589
(3.44)
645
(3.15)
417
(5.67)
513
(4.30)
547
(3.94)
590
(3.78)
646
(3.57)
1
1
b
d
4b
4c
5a
5b
5e
6a
6b
6e
4
519
(3.93)
555
(3.78)
594
(3.67)
655
(3.54)
418
(5.57)
514
(4.20)
549
(3.89)
591
(3.69)
649
(3.55)
4
524
(4.44)
556
(4.26)
594
(4.19)
646
(3.87)
416
(5.44)
509
(4.24)
543
(3.48)
585
(3.75)
658
(3.63)
2
a
4
517
(4.26)
554
(3.91)
592
(3.82)
649
(3.43)
415
(5.28)
510
(4.04)
543
(3.36)
586
(3.54)
655
(3.20)
2
2
b
d
4
520
(4.10)
566
(3.93)
593
(3.92)
657
(3.54)
415
(5.45)
511
(4.12)
545
(3.52)
586
(3.62)
650
(3.01)
4
516
(2.22)
558
(2.09)
592
(1.97)
650
(1.91)
415
(4.58)
510
(3.41)
545
(3.25)
582
(3.32)
637
(3.08)
3
a
4
515
(3.80)
549
(3.55)
592
(3.36)
648
(3.28)
416
(5.42)
510
(4.23)
542
(3.66)
583
(3.86)
634
(3.69)
3
b
4
516
(4.07)
555
(3.92)
593
(3.78)
652
(3.74)
417
(5.59)
514
(4.35)
544
(3.68)
583
(3.97)
632
(3.21)
3
c

Molecules 2021, 26, 1122
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Figure 3. Absorption spectra of the different porphyrins in ethanol: (A) 1 and 2, (B) 3 and 4, (C) 5 and 6.

Molecules 2021, 26, 1122
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Figure 4. Fluorescence spectra of the different porphyrins in ethanol: (
A) 1 and 2, (B) 3 and 4, (C) 5 and 6. In deuterium
oxide: (D) 2, 4, and 6 (λexc = 414 nm). Structures of quenched porphyrins are also represented (only in D O for 4b).
2

Molecules 2021, 26, 1122
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Despite a few discrepancies due to the poor solubility of pyridinium porphyrins in
ethanol, absorption spectra of
1 and 2 are fairly similar (Figure 3). Indeed, the main dis-
tinctions are due to aggregation phenomena that can be observed through the broadening
of the Soret band [62]. On the opposite, florescence emission is greatly influenced by the
kind of substituent (Figure 4A). Surprisingly, fluorescence spectra of 1a and 2a are similar
in ethanol, which indicate that the different substituents (pyridyl or N-methylpyridinium)
have no significant influence on the excited state properties of these compounds in this
solvent. Lifetimes are also consistent with the usual data of porphyrins (ca. 10 ns). How-
ever, the Q (0,0) band of porphyrins 1a and 2a is less intense than the Q (0,1) band (ratio
Q(0,0)/Q(0,1) < 1), which differs from the usual fluorescence emission spectra of por-
phyrins (Table 4). According to Vergeldt and co-workers [63], this discrepancy is due to the
electron-accepting properties of pyridyl groups that have a direct influence on the Q (0,0)
bands. Thus, the presence of a carboxylic acid group, instead of a pyridyl or pyridinium
group in 1b and 2b, respectively, leads to a ratio Q (0,0)/Q (0,1) > 1. Moreover, a red-shift
in the fluorescence emission of 2b is observed, which is coherent with its absorption spec-
trum. In D O, in addition to a lower fluorescence quantum yield, the ratio Q (0,0) / Q
2
(
0,1) dropped below 1 for 2b, and the emission spectrum of this compound then became
very similar to the well-known structureless broad band found in the spectrum of 2a in
D O [63]. As the fluorescence emission properties of 2a and 2b are similar in D O, it is
2
2
likely that, in this solvent, the carboxylic acid group and the pyridinium groups share equal
electron-accepting properties. These involvements in fluorescence and the shorter lifetimes
may also indicate a new nonradiative deactivation path. On the other hand, the aniline
group of 1d and 2d causes a drastic quenching of fluorescence in both solvents. Indeed,
primary amines (because of their electron lone-pair) are well-known to favor intramolecular
electron-transfers (IET), which cause emission quenching [64]. The lifetimes (shorter and
bi-exponential for 1d and not even determined for 2d) are consistent with this behavior.
A drop in pH (by addition of minute amounts of acetic acid, data not shown) induces a
partial restoration of 2d fluorescence emission.
Porphyrins
emission spectra are also similar to a classic porphyrin pattern showing a strong Q (0,0)
band (Figure 4B). Families and display close photophysical properties, similar to those
of tetraphenyl porphyrin (TPP) [65]. However, fluorescence quantum yields of porphyrins
are lower than those of other porphyrins in ethanol. In D O, unlike family , the ratio
(Table 4). Whereas the increase in
3 and 4 have similar absorption profiles. Moreover, their fluorescence
3
4
4
2
2
Q (0,0)/Q (0,1) of family 4 is still higher than that of 1
solvent polarity leads to an enhancement in fluorescence emission (twice the yield recorded
in ethanol) for 4a and 4c, it also causes emission quenching of 4b. Based on the results
obtained with 2b, this is probably due to the enhanced electron-withdrawing properties of
the carboxylic acid group in these conditions. Moreover, the core of porphyrins
4 seems
to have a lower pKa than other porphyrins (green coloration of pH = 2 solutions), which
could indicate that the phenylimidazole groups behave as EDG. Thus, in D O, in the case
2
of 4b, a photoinduced intramolecular charge-transfer (ICT) may occur, as this compound
is actually behaving as a donor-acceptor (phenylimidazolium groups and carboxylic acid
groups, respectively). ICT is well-known to lead to red-shifted and lower fluorescence
emissions. In order to verify this hypothesis, an additional porphyrin carrying a primary
amine, from this family (4d), has been tested and no quenching was observed (Figure S19).
As the replacement of the EWG by an EDG involved the recovery of the emissions, our
hypothesis seems to be consistent with the data.
Even if
have been spotted. First, the Q
functions. Indeed, for family , a slight blue shift (∆λ = 3 nm) of the Q
5
and
6
seemed to display similar absorption spectra, some discrepancies
(0,0) band is very sensitive to the number of carboxylic
(0,0) band, as well
x
5
x
as a hypochromic effect, and a full-width at half-maximum (fwhm) decrease were observed
with 5b, in comparison with 5a; the same remarks hold for 5e, following the increasing
number of carboxylic acid groups on the bay region of the porphyrins (Table 4). Regarding
x
porphyrins 6a and 6b, Q (0,0) is also red-shifted (∆λ = 3 nm), but the hypochromic effect

Molecules 2021, 26, 1122
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is less pronounced. Nonetheless, the intense hypochromic effect and enlargement of the Q
0,0) band were observed for 6e. For this compound, moreover, a split of this band was
x
(
recorded. In the case of 5e, a split can almost be distinguished. The symmetrization of the
porphyrin leads to this effect, in accordance with the four-orbital model of Gouterman [66].
Secondly, a shoulder at 444 nm was recorded in the absorption spectrum of 6a (Figure 3).
This is probably due to a J -aggregation that has already been described for some cationic
2
porphyrins [67]. Whereas the weak shifts in fluorescence emissions of
with their absorbance, a usual profile with a stronger Q(0,0) band was obtained with 5a
6a, and 6b in ethanol (Figure 4C). When comparing with , imidazole groups seem
to have a weaker electron-withdrawing effect, probably because of the steric hindrance
and thus absence of planarity and lack of conjugation), in a manner identical to that
5 and 6 are consistent
,
5b
,
1
2
(
of the ortho-pyridinium groups [63]. Once again, the carboxylic acid group has a weak
involvement on the fluorescence emission in ethanol, whereas the decrease in the ratio Q
(
0,0)/Q (0,1) of 6b in D O indicates a stronger electron-withdrawing effect of this group in
2
a polar solvent. On the other hand, 5e and 6e have a weak Q (0,0) band, which is consistent
with the low epsilon value recorded from their respective Qx (0,0) and Qy (0,0) bands.
Table 4. Photophysical properties of the porphyrins in ethanol (λexc = 414 nm).
Ethanol
D O
2
a
b
e
a
b
g
λ
λ
τ
τ∆
(µs)
λ
λ
τ
F
(ns)
τ∆
(µs)
f1
f2
c
d
F
f1
f2
c
f
Compd
λ /λ
Φ
Φ∆
λ /λ
Φ
F
Φ∆
f1 f2
F
f1 f2
(
nm)
645
646
(nm)
(ns)
10.0
10.1
(nm)
(nm)
1
a
712
0.75
1.06
0.10
0.12
0.74
0.77
15
−
−
−
−
−
−
−
−
−
−
−
−
−
−
1
b
713
17
3
.2
57%)
.5
1
d
663
716
1.29
0.02
0.05
16
−
−
−
−
−
−
−
(
(
8
43%)
10.0
8.9
2
a
645
656
654
652
650
653
713
716
719
718
717
719
0.75
1.27
0.97
1.89
1.7
0.09
0.12
0
0.65
0.82
0
16
16
nd
16
17
16
657
655
nd
−
−
−
709
709
nd
−
−
−
0.63
0.59
nd
−
−
−
0.07
0.07
0.01
−
−
−
0.90
0.93
nd
−
−
−
6.1
4.8
4.6
−
−
−
69
66
nd
−
−
−
2
2
b
d
nd
3
a
0.13
0.09
0.08
0.66
0.57
0.49
9.5
3
b
10.1
9.8
3
c
2.05
−
−
−
−
−
−
−
4
a
648
649
651
648
650
649
716
716
718
714
714
711
1.3
1.43
1.7
0.06
0.07
0.07
0.14
0.15
0.10
0.51
0.54
0.53
0.74
0.78
0.81
10
9.4
9.6
8.7
12.3
9.7
16
15
16
15
16
15
644
660
648
−
−
−
704
719
707
−
−
−
1.33
1.45
1.53
−
−
−
0.14
0.01
0.14
−
−
−
0.83
nd
0.62
−
−
−
11.2
10.8
10.1
−
−
−
65
nd
68
−
−
−
4
b
4
c
5
a
1.26
1.25
0.62
5
b
5
e
−
−
−
−
−
−
−
6
a
639
638
639
709
705
704
1.75
1.73
0.39
0.13
0.13
0.11
0.63
0.58
0.78
12.7
10.4
11.9
17
15
15
635
636
638
702
702
698
2.66
1.53
0.39
0.15
0.13
0.09
0.60
0.83
0.86
17.4
16.1
14.9
60
67
69
6
b
6
e
a
Q (0,0). b Q (0,1). c TPP as standard (ΦF (toluene) = 0.11) [68]. d TPP–COOH as standard (Φ∆ (EtOH) = 0.54) [69]. e Literature value~14.5
µs
in EtOH [70]. f TMPyP as standard (Φ∆ (D2O) = 0.90) [68]. Literature value~67 µs in D2O [71,72].
g

Molecules 2021, 26, 1122
11 of 21
2.3. Singlet Oxygen Production
Overall singlet oxygen quantum yields of
1
and
2
are significantly high in either
ethanol or D O (0.65 < Φ∆ < 0.93) (Table 4). The presence of one carboxylic acid group
2
seems to slightly increase the different yields, even if 2a has a lower yield in ethanol,
probably because of its aggregation. However, 1d and 2d were characterized by a very low
singlet oxygen production (not even measurable), because of, once again, the quenching of
emissions induced by the IET. Nevertheless, after the addition of a few drops of acetic acid
(data not shown), a characteristic singlet oxygen emission was observed for 2d. According
to the literature, the presence of bacteria may also induce a restoration of the production
of singlet oxygen, as this PS has shown a photobactericidal activity [73]. As previously
mentioned, families
3 and 4 have shown results similar to those of TPP. Indeed, the singlet
oxygen production yield of these porphyrins in ethanol is ca. 0.55 (0.49 < Φ∆ < 0.66), which
is very similar to the TPP–COOH value (0.54). However, in D O, porphyrins 4a and 4c have
2
shown a significant singlet oxygen production increase (Φ∆ = 0.83 and 0.62, respectively).
Efficient singlet oxygen productions have also been observed with porphyrins
5
and
6
(
0.58 < Φ∆ < 0.86) in ethanol. In D O, these values are, once again, slightly higher. In
2
both families
5 and 6, in ethanol as in D O, the singlet oxygen production quantum
2
yield increases with the number of carboxylic groups and with the symmetrization of the
porphyrin (Φ = 0.86 for 6e in D O).
∆
2
Based on this study, different phenomena have been observed in the various families
of porphyrins. Whereas intramolecular transfers (especially in aqueous solutions) have
been detected in pyridyl and phenylimidazole-substituted porphyrins and their N-methyl
derivatives, methylimidazole groups induce fewer effects on the porphyrin core. More-
over, the symmetrization of imidazole porphyrins has a positive influence on their singlet
oxygen production. Thus, studies based on asymmetric cationic porphyrins should in-
clude a judicious selection of the kind of porphyrin in order to avoid any quenching in
aqueous solution (especially if they are supposed to serve as controls). The presence of
one carboxylic acid group in 1, 2, 5, and
singlet oxygen, whereas a carboxylic acid group should be avoided in
methylimidazolium porphyrins seem to be promising candidates for the studies that plan
6
seems to slightly increase the production of
4. On the other hand,
6
to use two coupled moieties in trans positions, as 6e has shown a significant singlet oxygen
production in aqueous solution.
2.4. Bacterial Photoinactivation
We compared the bacterial photoinactivation efficiency of each porphyrin family
against three bacterial species known to be involved in nosocomial infections (Staphylococcus
aureus, Escherichia coli, and Pseudomonas aeruginosa). This antibacterial study focused on
N-methylporphyrins. Indeed, pyridinium porphyrin 2a is already renowned as a better
PACT photosensitizer than 1a [29], which may suggest a similar difference between other
porphyrins and their respective N-methyl form. As the functionalized porphyrins (2b, 2c,
4
b
,
4c
,
6b
,
6e) have been devised in view of their further optimized derivatization and use
4a, and 6a were used in these biological assays. Moreover,
in PACT, only A porphyrins 2a
,
4
the impact due to the different cationic groups can be better compared in the absence of
any interfering chemical group.
The photoinactivation capacity of compounds 2a
Table 5). Inactive in the dark, these porphyrins led to bacterial inhibition of all strains
after light irradiation. Despite the narrow range of the minimum bactericidal concentra-
tions (MBCs), porphyrin 4a was more efficient against S. aureus (1.5 M). Assays against
E. coli revealed discrepancies between the different PS. Whereas 4a was characterized by
a low MBC (2.5 M), significantly higher concentrations of 2a and 6a were required to
photoinactivate this strain (>18.0 M). Concerning P. aeruginosa, all MBCs tightly clustered
around 22.0 M. In comparison with porphyrin 2a (already known as an efficient PS [74]),
showed the highest MBCs against all strains. Based on the presumptive relation between
PS efficacy and their positive charge, this result is surprising as this porphyrin bears the
, 4a, and 6a has been confirmed
(
µ
µ
µ
µ
6a

Molecules 2021, 26, 1122
12 of 21
same positive charge, as 2a and 4a; this positive charge is distributed on substituents of
smaller size that then present a higher positive charge density. Thus, this lower activity
is probably due to the lower production of singlet oxygen of 6a. Porphyrin 4a has shown
promising results against S. aureus and, interestingly, against E. coli. Considering MBCs,
the efficiency of 4a can be estimated as 7 times better than that of 2a against E. coli. The
photophysical properties and, especially, the respective singlet oxygen productions of 2a
and 4a do not offer enough discrepancy to justify these results. Thus, a specific interaction
between 4a and E. coli could be the reason of this higher efficiency.
Table 5. Minimum bactericidal concentrations (MBCs) of the cationic porphyrins against S. aureus,
2
2
E. coli, and P. aeruginosa after 20 h of white light irradiation (4.83 mW/cm , totaling 348 J/cm fluence)
◦
at 37 C. Each MBC is the concentration that inactivated at least 99.9% of bacterial cells, in comparison
with the untreated control. Each experiment was performed six times.
MBC (µM)
2a
4a
6a
S. aureus
E. coli
P. aeruginosa
5.0
18.0
20.0
1.5
2.5
22.0
6.2
40.0
25.0
An additional experiment was performed to confirm these previous results (Figure 5).
Bacteria were harvested and put in contact with the different compounds in PBS. After
◦
3
0 min of incubation at 37 C in the dark, bacteria were washed twice, resuspended in
PBS, and then irradiated as before. The steepest decline in survival rate was obtained with
2
S. aureus irradiated after the contact with porphyrin 4a. Indeed, a fluence of 14 J/cm
induced a reduction of 2 log in the survival rate; compounds 2a and 6a were signficantly less
efficient. This result could be due to a better affinity of 4a for the cell envelope of S. aureus.
2
Interestingly, a fluence of 35 J/cm with this same compound induced a reduction of 3 log
in the survival rate of E. coli in accordance with MBC values (Table 5). For P. aeruginosa, a
2
fluence of 35 J/cm was also necessary to induce a reduction of 3 log in the survival rate,
but in presence of a higher concentration of the cationic porphyrins during the incubation
(2 µM for E. coli against 20 µM for P. aeruginosa). This set of assays showed that porphyin 4a
has a better affinity for bacteria, especially for S. aureus and E. coli, than others porphyrins.
This effect allows this compound to induce a better photocidal activity against these strains,
which is coherent with the first set of biological assays.
3. Materials and Methods
3.1. General Methods
All organic materials and microbial nutrients were purchased from commercial sup-
pliers (Acros Organics, Alfa Aesar, Grosseron, Sigma-Aldrich, TCI). All reactants for the
synthesis of porphyrins were obtained from Sigma-Aldrich and directly used, except ethyl
4
-formylphenoxyacetate, which was prepared from 4-hydroxybenzaldehyde. Solvents
used for the UV-Vis and fluorescence measurements were of spectroscopic grade and were
stored in a dark place. Analytical thin-layer chromatography (TLC) was performed on
Merck 60F254 silica gel. Column chromatography was carried out with silica gel (60 ACC,
1
5–40
on a RediSep Rf C18 column (43 g) mounted on an Interchim puriFlash
provided by Interchim, Montluçon, France) with acetonitrile (HPLC grade +0.1% TFA)
and distilled water (+0.1%TFA) as eluents. Continuous monitoring of effluent absorbance
µM, Merck, Darmstadt, Germany). Reverse-phase chromatography was performed
®
®
™
430 apparatus
(
1
13
allowed the detection of cationic porphyrins (
λ
= 418 nm). H and C nuclear magnetic
resonance ( H-NMR and C-NMR) spectra were recorded in deuterated solvents, with
Bruker DPX 400 and 500 spectrometers. Chemical shifts are reported as (parts per million),
1
13
δ
downfield from internal TMS. Multiplicities are reported as follows: s = singlet, d = doublet,
m = multiplet. Electron spray ionization mass spectra (ESI-MS) were recorded on a Sciex 4000
®
Q-TRAP , monitored by Sciex Analyst 1.6.2 (Applied Biosystems, Loughborough, UK).

Molecules 2021, 26, 1122
13 of 21
Figure 5. Time course of cell survival during photodynamic inactivation of S. aureus (
A
), E. coli
◦
(B
), and P. aeruginosa (
C
). Bacteria were incubated for 30 min in the dark at 37 C in solutions of
porphyrins 2a 4a, and 2a (2
with PBS, bacteria were irradiated by white LEDs (4.83 mW/cm ). Survival rates were expressed as
percent of the initial counts and plotted as a function of cumulative light fluence (J/cm ).
,
µ
M for S. aureus and E. coli, 20
µ
M for P. aeruginosa). After 2 washings
2
2
3.2. Chemical Synthesis
3.2.1. Preparation of Ethyl 4-Formylphenoxyacetate
4
-hydroxybenzaldehyde (1.00 g, 8.20 mmol, 1 eq), ethyl bromoacetate (0.90 mL,
8
.20 mmol, 1 eq), and a large excess of K CO (3 g) were stirred in a solution of DMF
2
3
◦
(100 mL) for 3 h at 95 C. The reaction was monitored by TLC (hexane/ethyl acetate (70/30,
v/v)). After removal of the solvent in vacuo, the product was extracted in CHCl , washed
3
with distilled water, and dried over mgSO . The solvent was removed and the protected
4
aldehyde was obtained as a yellowish powder (1.53 g, yield 90%).
3.2.2. General Procedure for the Synthesis of Porphyrins
Freshly distilled pyrrole (6.66 mL, 96.0 mmol, 4 eq) was added dropwise to a refluxed
solution of propionic acid (120 mL) containing aromatic aldehyde (4-pyridylcarboxaldehyde,
-imidazolebenzaldehyde, or N-methyl-4-imidazolecarboxaldehyde, 4 eq for A por-
4
4
phyrins, 3 eq for A B porphyrins, 2 eq for A B porphyrins) and functionalized aldehyde
3
2 2
(
methyl 4-formylbenzoate, 4-acetamidobenzaldehyde, or ethyl 4-formylphenoxyacetate, 1
eq for A B porphyrins, 2 eq for A B porphyrins). The mixture was stirred under reflux
3
2 2

Molecules 2021, 26, 1122
14 of 21
for 3 h. After removal of propionic acid in vacuo, the crude solid was dissolved in CHCl3,
filtered through a small pad of silica gel, and washed with CHCl /EtOH (90/10, v/v).
3
After removal of the solvent under vacuum, porphyrins were purified by silica gel column
chromatography (CHCl /EtOH (98/2, v/v)) to afford A porphyrin (yield 10% for 1a, yield
3
4
1
5% for 3a, yield 2% for 5a), A B porphyrin (yield 2–5% for
1
, yield 10–13% for
3, yield 5%
3
for 5), or A B porphyrin (yield 5% for 5) as light purple powders.
2
2
1
1
a (tetrakis(4-pyridyl)porphyrin): H NMR (500 MHz, CDCl3):
δ (ppm) = 9.06 (d,
J = 6.0 Hz, 8H, Ho-py), 8.87 (s, 8H, Hpyrr), 8.16 (d, J = 6.0 Hz, 8H, Hm-py), -2.92 (s, 2H,
2
+
NH); MS (ESI+) [C H N ]: calcd [M + 2H] 310.11, found 310.31.
40
26
8
1
3
a (tetrakis(4-imidazol-phenyl)porphyrin): H NMR (500 MHz, CDCl 9/1 CD OD):
3 3
δ
(ppm) = 8.92 (s, 8H, Hpyrr), 8.36 (d, J = 8.3 Hz, 8H, Ho-phe), 8.26 (s, 4H, Him-2), 7.86 (d,
J = 8.3 Hz, 8H, Hm-phe), 7.67 (s, 4H, Him-5), 7.34 (s, 4H, Him-4); MS (ESI+) [C H N ]:
5
6
38 12
calcd [M + 2H]2 440.16, found 440.04.
+
1
5
a (tetrakis(N-methylimidazol-2-yl)porphyrin): H NMR (500 MHz, CDCl 9/1
3
CD OD):
δ
(ppm) = 8.88 (m, 8H, Hpyrr), 7.67 (d, J = 1.3 Hz, 2H, Him-5), 7.65 (d, J = 1.3 Hz,
3
2
H, Him-4), 7.51 (d, J = 1.1 Hz, 2H, Him-5), 7.50 (d, J = 1.1 Hz, 2H, Him-4), 3.45 (m, 12H,
2+
N-CH3); MS (ESI+) [C H N ]: calcd [M + 2H] 316.13, found 316.26.
36
30 12
3
.2.3. General Procedure for the Deprotection of Carboxylic Acid
Esters of A B porphyrins (200 Mol) were saponified in a refluxing solution of KOH
2 mL, 2 mol/L) and EtOH (for methyl ester) or MeOH (for ethyl ester) (50 mL) for 24 h.
µ
3
(
After removal of the solvent, an excess of HCl was added until the green coloration of the
solution appeared. The crude product was extracted in CHCl /TEA (99/1, v/v), washed
3
with distilled water, and dried over mgSO . The residue was purified by silica gel column
4
chromatography (CHCl /EtOH (80/20, v/v)) to obtain A B porphyrins with deprotected
3
3
carboxylic acid functions, as purple solids.
1
b (5-(4-carboxyphenyl)-10,15,20-tri(4-pyridyl)-21H,23H-porphyrin): 178
µ
Mol, yield
9%. H NMR (500 MHz, CDCl 9/1 CD OD): δ (ppm) = 9.40 (d, J = 6.5 Hz, 6H, Ho-py),
3 3
1
8
8
8
.97 (m, 6H, Hpyrr), 8.93 (t, J = 6.5 Hz, 6H, Hm-py), 8.87 (d, J = 4.7 Hz, 2H, Hpyrr-phe),
.58 (d, J = 8.0 Hz, 2H, Ho-phe), 8.42 (d, J = 8.0 Hz, 2H, Hm-phe); MS (ESI+) [C H N O ]:
42
27
7
2
2+
calcd [M + 2H] 331.61, found 331.64.
3
b (5-(4-carboxyphenyl)-10,15,20-tri(4-imidazol-phenyl)-21H,23H-porphyrin): 182
µ
Mol,
1
yield 91%. H NMR (500 MHz, CD Cl 9/1 CD OD):
δ
(ppm) = 8.83 (s, 8H, Hpyrr), 8.35
2
2
3
(d, J = 8.2 Hz, 2H, Ho-pheCOOH), 8.23 (m, 11 H, Hm-pheCOOH + Ho-phe + Him-2), 7.77 (d,
J = 8.3 Hz, 6H, Hm-phe), 7.62 (s, 3H, Him-5), 7.24 (s, 3H, Him-4); MS (ESI+) [C H N O ]:
54
36 10
2
calcd [M + 2H]2 429.15, found 429.03.
+
3
c (5-(4-phenyoxyacetic)-10,15,20-tri(4-imidazol-phenyl)-21H,23H-porphyrin): 184
µ
Mol,
1
yield 92%. H NMR (500 MHz, CDCl 9/1 CD OD):
δ
(ppm) = 8.88 (m, 8H, Hpyrr), 8.31
3
3
(
(
d, J = 8.1 Hz, 6H, Ho-phe), 8.19 (s, 3H, Him-2), 8.14 (d, J = 8.5 Hz, 2H, Ho-phe
d, J = 8.1 Hz, 6H, Hm-phe), 7.59 (s, 3H, Him-5), 7.38 (s, 3H, Him-4), 7.31 (d, J = 8.5 Hz,
o
), 7.78
2
[
H, Hm-phe
o
), 4.77 (s, 2H, O-CH -),
−
2.75 (s, 2H, NH); MS (ESI+) [C H N O ]: calcd
55 38 10 3
2
M +2H ]2 444.15, found 444.17.
+
5
b (5-(4-carboxyphenyl)-10,15,20-tri(N-methylimidazol-2-yl)-21H,23H-porphyrin):
1
1
72
µMol, yield 86%. H NMR (500 MHz, CDCl 9/1 CD OD):
δ
(ppm) = 8.88 (m, 8H,
3
3
Hpyrr), 8.42 (m, 2H, Ho-pheCOOH), 8.19 (m, 2H, Hm-pheCOOH), 7.65 (m, 3H, Him-5), 7.51
2
+
(
m, 3H, Him-4), 3.45 (m, 9H, N–CH ); MS (ESI+) [C H N O ]: calcd [M +2H] 336.13,
3 39 30 10 2
found 336.15.
5
e (di(4-carboxyphenyl)- di(N-methylimidazol-2-yl)-21H,23H-porphyrin): 174
µ
Mol,
1
yield 87%. H NMR (500 MHz, CDCl ):
δ (ppm) = 8.85 (m, 8H, Hpyrr), 8.43 (m, 4H, Ho-
3
pheCOOH), 8.19 (m, 4H, Hm-pheCOOH), 7.67 (s, 2H, Him-5), 7.47 (s, 2H, Him-4), 3.44 (m,
2
+
6
H, N–CH3),
−
2.78 (m, 2H, NH); MS (ESI+) [C H N O ]: calcd [M + 2H] 356.12,
42 30 8 4
found 356.16.

Molecules 2021, 26, 1122
15 of 21
3.2.4. General Procedure for the N-Methylation of Porphyrins
An excess of iodomethane (60 eq for A porphyrins, 45 eq for A B porphyrins, 30 eq
4
3
for A B porphyrins) was added to a solution of porphyrin (1 eq) dissolved in anhydrous
2
2
◦
DMF (25 mL). The mixture was stirred for 3 h at 140 C. DMF and excess iodomethane were
removed with a rotary vane pump under heating (70 C) to obtain N-methyl porphyrins in
◦
quantitative yields as light purple powders without further purification.
2
a (tetrakis(4-N-methylpyridyl)porphyrin): 1H NMR (500 MHz, DMSOd6):
δ (ppm) = 9.49
(
+
1
d, J = 6.0 Hz, 8H, Ho-py), 9.17(s, 8H, Hpyrr), 8.99 (d, J = 6.0 Hz, 8H, Hm-py), 4.74 (s, 12H
+
4+
water residual, N –CH ),
−
3.09 (s, 2H, NH); MS (ESI+) [C H I N ]: calcd [M + H]
3
44 38 4 8
69.83, found 169.81.
2
b (5-(4-carboxyphenyl)-10,15,20-tri(4-N-methylpyridyl)-21H,23H-porphyrin): 1H
NMR (500 MHz, DMSOd6):
δ (ppm) = 9.49 (d, J = 6.1 Hz, 6H, Ho-py), 9.15 (m, 4H, Hpyrr),
9
.07 (m, 4H, Hpyrr-phe), 9.01 (d, J = 6.2 Hz, 6H, Hm-py), 8.41 (m, 4H + chloroform residual,
+
2+
Hphe), 4.73 (s, 9H, N –CH ),
3
−
2.99 (s, 2H, NH); MS (ESI+) [C H I3N O ]: calcd [M]
45 36 7 2
3
53.14, found 352.65.
4
a (tetrakis(4-N-methylimidazol-phenyl)porphyrin): 1H NMR (500 MHz, DMSOd6):
δ
(ppm) = 10.13 (s, 4H, Him-2), 8.93 (s, 8H, Hpyrr), 8.65 (s, 4H, Him-4), 8.53 (d, J = 8.3 Hz,
+
8
H, Ho-phe), 8.27 (d, J = 8.3 Hz, 8H, Hm-phe), 8.14 (s, 4H, Him-5), 4.10 (s, 12H, N –CH ),
3
4+
−
2.88 (s, 2H, NH); MS (ESI+) [C H I N ]: calcd [M] 234.60, found 234.70.
60 50 4 12
4
b (5-(4-carboxyphenyl)-10,15,20-tri(4-N-methylimidazol-phenyl)-21H,23H-porphyrin):
(ppm) = 10.13 (s, 3H, Him-2), 8.93 (s, 6H, Hpyrr), 8.89 (s,
H, J = 4.2 Hz, Hpyrr-pheCOOH), 8.65 (s, 3H, Him-4), 8.54 (d, J = 8.2 Hz, 6H, Ho-phe), 8.42
1
H NMR (500 MHz, DMSOd6):
δ
2
(
6
[
d, J = 8.1 Hz, 2H, Ho-pheCOOH), 8.36 (d, J = 8.1 Hz, 2H, Hm-pheCOOH), 8.26 (d, J = 8.3 Hz,
+
H, Hm-phe), 8.14 (s, 3H, Him-4), 4.10 (s, 9H, N –CH ),
−
2.88 (s, 2H, NH); MS (ESI+)
3
3+
C H I N O ]: calcd [M] 300.47, found 300.41.
57 45 3 10 2
4
c (5-(4-phenyoxyacetic)-10,15,20-tri(4-N-methylimidazol-phenyl)-21H,23H-porphyrin):
1
H NMR (500 MHz, DMSO-d6): (ppm) = 10.13 (s, 3H, Him-2), 8.96 (d, J = 4.1 Hz, 2H,
δ
Hpyrr), 8.92 (s, 4H, Hpyrr), 8.88 (d, J = 4.1 Hz, 2H, Hpyrr), 8.65 (s, 3H, Him-4), 8.54 (d,
J = 8.3 Hz, 6H, Ho-phe) 8.26 (d, J = 8.3 Hz, 6H, Hm-phe), 8.15 (d, J = 8.6 Hz, 2H, Ho-phe
o
&
3
+
s, 3H, Him-5), 7.40 (d, J = 8.6 Hz, 2H, Hm-phe), 4.99 (s, 2H, O–CH ), 4.10 (s, 9H, N –CH ),
2
−
3+
−
2.87 (s, 2H, NH); MS (ESI+) [C H I N O ]: calcd [M + H] 310.79, found 310.75.
58 47 3 10 3
1
6a (tetrakis(N,N’-dimethylimidazol-2-yl)porphyrin): H NMR (500 MHz, DMSOd6):
δ
(ppm) = 9.40 (s, 8H, Hpyrr), 8.55 (s, 8H, Him), 3.75 (s, 24H, N–CH ), 3.23 (s, 2H, NH);
−
3
4+
MS (ESI+) [C H I N ]: calcd [M] 172.59, found 172.54.
40
42 4 12
6
b (5-(4-carboxyphenyl)-10,15,20-tri(N,N’-dimethylimidazol-2-yl)-21H,23H-porphyrin):
H NMR (500 MHz, DMSOd6): (ppm) = 9.23 (m, 8H, Hpyrr), 8.52 (s, 6H, Him), 8.47
d, J = 8.2 Hz, 2H, Ho-pheCOOH), 8.39 (d, J = 8.2 Hz, 2H, Hm-pheCOOH), 3.75 (m, 18H,
1
δ
(
3+
N–CH3),
−
3.03 (s, 2H, NH); MS (ESI+) [C H I N O ]: calcd [M] 238.44, found 238.50.
42 39 3 10 2
1
6e (di(4-carboxyphenyl) -di(N,N’-dimethylimidazol-2-yl)-21H,23H-porphyrin): H
NMR (500 MHz, DMSOd6):
δ (ppm) = 9.24–8.95 (m, 8H, Hpyrr), 8.51 (s, 4H, Him), 8.43 (d,
J = 8.1 Hz, 4H, Ho-pheCOOH), 8.38 (d, J = 8.1 Hz, 2H, Hm-pheCOOH), 3.75 (s, 12H, N-CH3),
2+
−
2.81 (s, 2H, NH); MS (ESI+) [C H I N O ]: calcd [M] 370.14, found 370.04.
44 36 2 8 4
3.2.5. Synthesis of 5-(4-aminophenyl)-10,15,20-tri(4-pyridyl)-21H,23H-porphyrin (1d)
5-(4-acetamidophenyl)-10,15,20-tri(4-pyridyl)-21H,23H-porphyrin (250 mg, 0.373 mmol)
was dissolved into a solution of HCl (5 M, 250 mL) under reflux for 3 h. After removal of
the solvent in vacuo, the product was extracted in CHCl /TEA (90/10, v/v), washed with
3
distilled water, and dried over mgSO . The solvent was removed to afford 1d as a purple
4
powder with a quantitative yield.
1
1
d: H NMR (500 MHz, CDCl3):
δ
(ppm) = 9.02 (d, J = 5.5 Hz, 6H, Ho-py), 9.02 (d,
J = 2.5 Hz, 2H, Hpyrr-phe), 8.81 (m, 6H, Hpyrr), 8.16 (d, J = 5.5 Hz, 6H, Hm-py), 7.98 (d,
J = 8.0 Hz, 2H, Ho-phe), 7.08 (d, J = 8.0 Hz, 2H, Hm-phe); MS (ESI+) [C H N ]: calcd [M +
41
28
8
2+
2
H] 317.12, found 316.93.

Molecules 2021, 26, 1122
16 of 21
3
.2.6. Synthesis of 5-(4-aminophenyl)-10,15,20-tri(4-N-methylpyridyl)-21H,23H-porphyrin (2d
)
5-(4-acetamidophenyl)-10,15,20-tri(4-pyridyl)-21H,23H-porphyrinwasdirectly N-methylated,
following the previous described procedure. Then, the crude was diluted in a solution
of H O/TFA (75/25, v/v) and stirred under reflux for 5 h. The aqueous solution was
2
removed and the product was freeze-dried to obtain 2d as a purple-brown powder with a
quantitative yield.
2
d: 1H NMR (500 MHz, DMSOd6):
δ (ppm) = 9.49 (d, J = 6.5 Hz, 6H, Ho-py), 9.11 (m,
8
2
H, Hpyrr), 9.01 (m, 2H, Hpyrr-phe), 9.00 (d, J = 6.5 Hz, 4H, Hm-py), 8.97 (d, J = 6.5 Hz,
H, Hm-py), 7.94 (d, J = 8.5 Hz, 2H, Ho-phe), 7.15 (d, J = 8.5 Hz, 2H, Hm-phe), 4.73 (s, 9H,
+
3+
N –CH ), −2.96 (s, 2H, NH); MS (ESI+) [C H I N ]: calcd [M] 225.77, found 225.74.
3
44 37 3
8
3.3. Spectroscopic Measurements
Absorption spectra were recorded on a UV-3600 UV-visible double-beam spectropho-
tometer (Shimadzu, Marne La Vallée, France). Fluorescence spectra were recorded on a
Fluorolog FL3-222 spectrofluorometer (Horiba Jobin Yvon, Longjumeau, France) equipped
◦
with a 450 W Xenon lamp, a thermostated cell compartment (25 C), a UV-visible R928 pho-
tomultiplier (Hamamatsu, Japan), and an InGaAs infrared detector (DSS-16A020L Electro-
Optical System Inc, Phoenixville, PA, USA). The excitation beam was diffracted by a double-
ruled-grating SPEX monochromator (1200 grooves/mm blazed at 330 nm). The emission
beam was diffracted by a double-ruled-grating SPEX monochromator (1200 grooves/mm
blazed at 500 nm). Singlet oxygen emission was detected through a double-ruled-grating
SPEX monochromator (600 grooves/mm blazed at 1 µM) and a long-wave pass (780 nm).
All spectra were recorded from solutions introduced in four-face quartz cuvettes. All the
emission spectra (fluorescence and singlet oxygen luminescence) have been displayed
with the same absorbance (less than 0.2) with lamp and photomultiplier correction. Flu-
orescence quantum yields (ΦF) were determined using a tetraphenyl porphyrin (TPP)
1
solution in toluene as the fluorescence standard (ΦF = 0.11). The quantum yield of O
2
1
production was determined by direct analysis of the O near-infrared luminescence at
2
1
270 nm. Whereas TPP–COOH was used as standard in ethanol (Φ∆ = 0.54), tetra(N-
methylpyridyl)porphyrin (2a) was chosen as the reference solution in D O due to its high
2
1
O quantum yield (Φ∆ = 0.90). Time-resolved experiments were performed using for
2
excitation a pulsed laser diode emitting at 407 nm (LDH-P-C-400M, FWHM < 70 ps, 1 MHz)
coupled with a PDL 800-D driver (both from PicoQuant GmbH, Berlin, Germany), and
for detection from an avalanche photodiode SPCM-AQR-15 (EG and G, Vaudreuil, QC,
Canada) coupled with a 550 nm long-wave pass filter as the detection system. Acquisi-
tion was performed by a PicoHarp 300 module with a PHR-800 4-channel router (both
PicoQuant GmbH, Berlin, Germany). Fluorescence decays were recorded using the sin-
gle photon counting method. Data were collected up to 1000 counts accumulated in the
maximum channel and analyzed using time-correlated single-photon counting (TCSPC)
software (PicoQuant GmbH, Berlin, Germany) based on iterative reconvolution using a
Levensberg–Marquandt algorithm, enabling the obtention of multi-exponential profiles
(mainly one or two exponentials in our cases). Singlet oxygen lifetime measurements
were performed on a TEMPRO-01 spectrophotometer (Horiba Jobin Yvon, Longjumeau,
France) composed of a SpectraLED-415 pulsed diode excitation source emitting at 415 nm,
a cell compartment, a Seya-Namioka-type emission monochromator (600–2000 nm), and a
H10330-45 near-infrared photomultiplier tube with a thermoelectric cooler (Hamamatsu,
Japan) as the detection system. The system was monitored by a FluoroHub-B single-photon
counting controller and the DataStation and DAS6 softwares (Horiba Jobin Yvon). Emis-
sion spectra were recorded on a Fluorolog FL3-22 spectrofluorometer (Horiba Jobin Yvon,
Longjumeau, France) equipped with a 450 W Xenon lamp, a xenon flash lamp, a ther-
◦
mostated cell compartment (25 C), and a R928 UV-visible photomultiplier (Hamamatsu,
Japan). The system was monitored by a FluoroHub-B single-photon counting controller.
The FluorEssence
™ software (Horiba Jobin Yvon) was used for recording emission spectra.

Molecules 2021, 26, 1122
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The DataStation and DAS6 softwares (Horiba Jobin Yvon) were used for phosphorescence
lifetime measurements.
3.4. Bacterial Cultures
Gram-positive (S. aureus CIP76.25) and Gram-negative (P. aeruginosa CIP76110 and
E. coli CIP54.8T) bacterial strains were obtained from Institut Pasteur (Paris, France). These
strains were cultured in liquid tryptic soy (pancreatic casein extract 17 g/L, soy flour papaic
digest 3 g/L, dextrose 2.5 g/L, NaCl 5 g/L, and K HPO 2.5 g/L) and incubated overnight
2
4
◦
at 37 C under aerobic conditions. Square plates were kept warm inside a SR1000 Thermosi
incubator. Bacterial concentrations were estimated by reading the turbidity at 600 nm in
a JENWAY 6320D spectrophotometer (Cole-Parmer Ltd., Roissy, France). Antimicrobial
experiments used a Heidolph Unkubator 1000 equipped with an Unimax 1010 orbital
platform shaker (Heidolph Instruments GmbH & CO. KG, Schwabach, Germany). An
Isotech Lightmeter 1335 light meter was used to measure the effective irradiation power
(
RS Components, CorbyNorthants, UK).
3.5. Bacterial Photoinactivation
A porphyrins were added to bacterial cultures during the log phase. However, 1a
,
4
3a, and 5a have poor solubility in DMSO and in aqueous solution. The bacterial assays
revealed that these compounds aggregated in the culture broth and precipitated during
incubation, which prevented any further observation (data not shown). Fresh solutions
of porphyrin 2a, 4a, and 6a in DMSO were mixed with tryptic soy culture medium. The
DMSO concentration never exceeded 1% (v/v). From these mixtures, 1 mL aliquots of serial
dilutions (100
Then, 1 mL of cultures containing 2
µ
M down to 156 nM) were transferred into two 24-well plates (BD Falcon).
5
×
10 CFU/mL were deposited in each well. The
2
24-well plates were irradiated with LED visible light. The fluence rate (4.83 mW/cm ) was
◦
measured with a light meter. Plates were incubated under shaking (200 rpm) at 37 C for
2
20 h (totaling 348 J/cm fluence). Controls consisting of 24-well plates were prepared in
the same conditions but kept in the dark. Six independent experiments were performed
with each strain. The bacterial count was performed after a 10-fold serial dilution of
each well. Each dilution was spread on tryptic soy agar plates using an automatic plater
®
◦
(
2
easySPIRAL , Interscience, Saint-Nom-la-Bretêche, France). After incubation at 37 C for
4 h, colonies were counted to determine the total CFU per mL (CFU/mL). The minimum
bactericidal concentration (MBC) corresponds to the concentration of the active compound
for which 99.9% of the bacteria have been killed (i.e., 3 log reduction compared to the
untreated control).
2
To observe the effects of light fluence (J/cm ) on the survival rate of each strain,
9
◦
bacteria (~10 CFU/mL) were incubated for 30 min at 37 C in the dark with 1 mL of the
different solutions (2 M for S. aureus and E. coli, 20 M for P. aeruginosa). Then, bacteria
µ
µ
were collected by centrifugation (10,000 rpm, 5 min) and washed twice with PBS 1
×
without Ca2 and mg ). Washed bacteria were resuspended in PBS, transferred into a
+
2+
(
2
9
6-well plate, and irradiated by white LEDs (4.83 mW/cm ). Survival rates, calculated in
relation to the initial count of each bacterial culture, were plotted as a function of cumulative
2
fluence (J/cm ). Four independent experiments were performed with each strain.
4. Conclusions
In order to photoinactivate MDR strains, previous studies have highlighted the effi-
ciency of cationic PS, including pyridinium porphyrins. In this work, different families of
cationic porphyrin have been investigated to support future studies. Even if pyridinium
A porphyrins are still very efficient, the functionalization of this family may induce some
4
reactivity issues (particularly in the case of the carboxylic acid function) or emissions
quenching (due to the presence of primary amine functions). Even if the participation of a
carboxylic acid function leads to emission quenching of these PS (as a consequence of an
ICT), phenylimidazolium porphyrins have shown several advantages, as easier synthesis,

Molecules 2021, 26, 1122
18 of 21
consistent photophysical properties (similar to those of TPP), along with a significantly
high photoinactivation activity against S. aureus and E. coli. Finally, the methylimidazolium
groups have not a strong electron-withdrawing effect on the porphyrin core, which allows
the designing of cationic PSs with different chemical functions without any emissions
quenching due to potential ICT/IET. Whereas 6a showed less efficiency against bacteria,
likely due to lower oxygen singlet production, porphyrin 6e has a significant singlet oxy-
gen production. Thus, further photobactericidal studies on porphyrins
compounds bearing two targeting groups.
6 should focus on
Supplementary Materials: The following are available online: Figures S1–S18. 1H NMR analyses,
Figure S19. Fluorescence spectrum of 4d.
Author Contributions: Conceptualization, T.-S.O. and V.S.; writing—original draft preparation,
F.L.G.; writing—review and editing, F.L.G., T.-S.O., P.A., C.F., S.L.-L. and V.S.; investigation, F.L.G.,
I.Y. and Y.N.; validation, T.-S.O., C.F. and S.L.-L.; project administration and supervision, V.S. All
authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Data Availability Statement: Data available on request to florent.le-guern@uvsq.fr or correspond-
ing authors.
Acknowledgments: P.A., C.F., S.L.-L. and V.S. are members of GDR 2067 “MARYRO.” We thank
“
Conseil Regional du Limousin” for his help (F.L.G.). The authors are indebted to Michel Guilloton for
help in manuscript editing, and Emilie Pinault and Yves Champavier (BISCEm—Limoges University)
for ESI-MS and NMR analyses, respectively.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are not available from the authors.
References
1
2
.
.
Prescott, L.M.; Willey, J.; Sherwood, L.; Woolverton, C.J. Microbiologie, 4th ed.; De Boeck: Louvain-la-Neuve, Belgique, 2013.
Cabral, C.; Ingram, J.; Lucas, P.J.; Redmond, N.M.; Kai, J.; Hay, A.D.; Horwood, J. Influence of Clinical Communication on Parents’
Antibiotic Expectations for Children with Respiratory Tract Infections. Ann. Fam. Med. 2016, 14, 141–147. [CrossRef] [PubMed]
Ventola, C.L. The Antibiotic Resistance Crisis. Pharm. Ther. 2015, 40, 277–283.
Wainwright, M.; Maisch, T.; Nonell, S.; Plaetzer, K.; Almeida, A.; Tegos, G.P.; Hamblin, M.R. Photoantimicrobials—Are We Afraid
of the Light? Lancet Infect. Dis. 2017, 17, e49–e55. [CrossRef]
3
4
.
.
5.
6.
7.
O’Neill, J. Tackling Drug-Resistant Infections Globally: Final Report and Recommendations. Review on Antimicrobial Resistance.
2
016. Available online: https://www.biomerieuxconnection.com/wp-content/uploads/2018/04/Tackling-Drug-Resistant-
Infections-Globally_-Final-Report-and-Recommendations.pdf (accessed on 18 February 2021).
Vatansever, F.; de Melo, W.C.M.A.; Avci, P.; Vecchio, D.; Sadasivam, M.; Gupta, A.; Chandran, R.; Karimi, M.; Parizotto, N.A.; Yin,
R.; et al. Antimicrobial Strategies Centered around Reactive Oxygen Species – Bactericidal Antibiotics, Photodynamic Therapy,
and Beyond. FEMS Microbiol. Rev. 2013, 37, 955–989. [CrossRef] [PubMed]
Tavares, A.; Carvalho, C.M.B.; Faustino, M.A.; Neves, M.G.P.M.S.; Tomé, J.P.C.; Tomé, A.C.; Cavaleiro, J.A.S.; Cunha, Â.; Gomes,
N.C.M.; Alves, E.; et al. Antimicrobial Photodynamic Therapy: Study of Bacterial Recovery Viability and Potential Development
of Resistance after Treatment. Mar. Drugs 2010, 8, 91–105. [CrossRef]
8
9
1
1
1
1
1
.
Dysart, J.S.; Patterson, M.S. Characterization of Photofrin Photobleaching for Singlet Oxygen Dose Estimation during Photody-
namic Therapy of MLL Cells in Vitro. Phys. Med. Biol. 2005, 50, 2597–2616. [CrossRef]
Dougherty, T.J.; Gomer, C.J.; Henderson, B.W.; Jori, G.; Kessel, D.; Korbelik, M.; Moan, J.; Peng, Q. Photodynamic Therapy. J. Natl.
Cancer Inst. 1998, 90, 889–905. [CrossRef]
.
0. Morton, C.A. Photodynamic Therapy in Acne: Can We Achieve Therapeutic Gain without Pain? Br. J. Dermatol. 2011, 165, 932–933.
CrossRef] [PubMed]
1. Alves, E.; Faustino, M.A.; Neves, M.G.; Cunha, A.; Tome, J.; Almeida, A. An Insight on Bacterial Cellular Targets of Photodynamic
Inactivation. Future Med. Chem. 2014, 6, 141–164. [CrossRef]
2. Fu, X.; Fang, Y.; Yao, M. Antimicrobial Photodynamic Therapy for Methicillin-Resistant Staphylococcus Aureus Infection. BioMed
Res. Int. 2013, 2013. [CrossRef] [PubMed]
3. Henderson, B.W.; Dougherty, T.J. How Does Photodynamic Therapy Work? Photochem. Photobiol. 1992, 55, 145–157. [CrossRef]
[
[PubMed]
4. Detty, M.R.; Gibson, S.L.; Wagner, S.J. Current Clinical and Preclinical Photosensitizers for Use in Photodynamic Therapy. J. Med.
Chem. 2004, 47, 3897–3915. [CrossRef] [PubMed]

Molecules 2021, 26, 1122
19 of 21
1
5. Minnock, A.; Vernon, D.I.; Schofield, J.; Griffiths, J.; Parish, J.H.; Brown, S.B. Mechanism of Uptake of a Cationic Water-Soluble
Pyridinium Zinc Phthalocyanine across the Outer Membrane of Escherichia Coli. Antimicrob. Agents Chemother. 2000, 44, 522–527.
CrossRef]
[
1
6. Reddi, E.; Ceccon, M.; Valduga, G.; Jori, G.; Bommer, J.C.; Elisei, F.; Latterini, L.; Mazzucato, U. Photophysical Properties and
Antibacterial Activity of Meso-Substituted Cationic Porphyrins. Photochem. Photobiol. 2002, 75, 462–470. [CrossRef]
7. Alves, E.; Costa, L.; Carvalho, C.M.B.; Tomé, J.P.C.; Faustino, M.A.; Neves, M.G.P.M.S.; Tomé, A.C.; Cavaleiro, J.A.S.; Cunha, Â.;
Almeida, A. Charge Effect on the Photoinactivation of Gram-Negative and Gram-Positive Bacteria by Cationic Meso-Substituted
Porphyrins. BMC Microbiol. 2009, 9, 70. [CrossRef]
1
1
1
8. Saino, E.; Sbarra, M.S.; Arciola, C.R.; Scavone, M.; Bloise, N.; Nikolov, P.; Ricchelli, F.; Visai, L. Photodynamic Action of Tri-Meso
(N-Methyl-Pyridyl), Meso (N-Tetradecyl-Pyridyl) Porphine on Staphylococcus Epidermidis Biofilms Grown on Ti6Al4V Alloy.
Int. J. Artif. Organs 2010, 33, 636–645. [CrossRef] [PubMed]
9. Gyulkhandanyan, G.V.; Ghazaryan, R.K.; Paronyan, M.H.; Gyulkhandanyan, A.G.; Sheyranyan, M.A.; Dzhagarov, B.M.; Tuchina,
E.S.; Korchenova, M.A.; Tuchin, V.V. Photophysical Properties and Photodynamic Efficiency of Cationic Porphyrins. In Pro-
ceedings of the SPIE 8942, Dynamics and Fluctuations in Biomedical Photonics XI, San Francisco, CA, USA, 26 February 2014;
p. 894212.
2
2
2
0. Simões, C.; Gomes, M.C.; Neves, M.G.P.M.S.; Cunha, Â.; Tomé, J.P.C.; Tomé, A.C.; Cavaleiro, J.A.S.; Almeida, A.; Faustino,
M.A.F. Photodynamic Inactivation of Escherichia coli with Cationic Meso-Tetraarylporphyrins—The Charge Number and Charge
Distribution Effects. Catal. Today 2016, 266, 197–204. [CrossRef]
1. Schastak, S.; Ziganshyna, S.; Gitter, B.; Wiedemann, P.; Claudepierre, T. Efficient Photodynamic Therapy against Gram-Positive
and Gram-Negative Bacteria Using THPTS, a Cationic Photosensitizer Excited by Infrared Wavelength. PLOS ONE 2010, 5, e11674.
[CrossRef]
2. Simonetti, O.; Cirioni, O.; Orlando, F.; Alongi, C.; Lucarini, G.; Silvestri, C.; Zizzi, A.; Fantetti, L.; Roncucci, G.; Giacometti, A.;
et al. Effectiveness of Antimicrobial Photodynamic Therapy with a Single Treatment of RLP068/Cl in an Experimental Model of
Staphylococcus Aureus Wound Infection. Br. J. Dermatol. 2011, 164, 987–995. [CrossRef] [PubMed]
2
2
2
2
3. Caruso, E.; Banfi, S.; Barbieri, P.; Leva, B.; Orlandi, V.T. Synthesis and Antibacterial Activity of Novel Cationic BODIPY
Photosensitizers. J. Photochem. Photobiol. B 2012, 114, 44–51. [CrossRef]
4. Kadish, K.M.; Smith, K.M.; Guilard, R. The Porphyrin Handbook: Synthesis and Organic Chemistry; Academic Press: San Diego, CA, USA,
2000; ISBN 978-0-12-393201-3.
5. Maisch, T.; Bosl, C.; Szeimies, R.-M.; Lehn, N.; Abels, C. Photodynamic Effects of Novel XF Porphyrin Derivatives on Prokaryotic
and Eukaryotic Cells. Antimicrob. Agents Chemother. 2005, 49, 1542–1552. [CrossRef] [PubMed]
6. Prasanth, C.S.; Karunakaran, S.C.; Paul, A.K.; Kussovski, V.; Mantareva, V.; Ramaiah, D.; Selvaraj, L.; Angelov, I.; Avramov, L.;
Nandakumar, K.; et al. Antimicrobial Photodynamic Efficiency of Novel Cationic Porphyrins towards Periodontal Gram-Positive
and Gram-Negative Pathogenic Bacteria. Photochem. Photobiol. 2014, 90, 628–640. [CrossRef]
2
2
7. Caruso, E.; Malacarne, M.C.; Banfi, S.; Gariboldi, M.B.; Orlandi, V.T. Cationic Diarylporphyrins: In Vitro Versatile Anticancer and
Antibacterial Photosensitizers. J. Photochem. Photobiol. B Biol. 2019, 197, 111548. [CrossRef] [PubMed]
8. Sbarra, M.S.; Arciola, C.R.; Di Poto, A.; Saino, E.; Rohde, H.; Speziale, P.; Visai, L. The Photodynamic Effect of Tetra-Substituted
N-Methyl-Pyridyl-Porphine Combined with the Action of Vancomycin or Host Defense Mechanisms Disrupts Staphylococcus
Epidermidis Biofilms. Int. J. Artif. Organs 2009, 32, 574–583. [CrossRef]
2
9. Dosselli, R.; Tampieri, C.; Ruiz-González, R.; De Munari, S.; Ragàs, X.; Sánchez-García, D.; Agut, M.; Nonell, S.; Reddi, E.; Gobbo,
M. Synthesis, Characterization, and Photoinduced Antibacterial Activity of Porphyrin-Type Photosensitizers Conjugated to the
Antimicrobial Peptide Apidaecin 1b. J. Med. Chem. 2013, 56, 1052–1063. [CrossRef] [PubMed]
3
3
3
0. Machado, A.E.H.; Gomes, W.R.; Araújo, D.M.S.; Miglio, H.S.; Ueno, L.T.; Paula, R.D.; Cavaleiro, J.A.S.; Neto, N.M.B. Synthesis and
Spectroscopic Characterization of Two Tetrasubstituted Cationic Porphyrin Derivatives. Molecules 2011, 16, 5807–5821. [CrossRef]
1. Milgrom, L.R.; Dempsey, P.J.F.; Yahioglu, G. 5,10,15,20-Tetrakis(N-Protected-Imidazol-2-Yl) Porphyrins. Tetrahedron 1996, 52, 9877–9890.
[
CrossRef]
2. Kalita, D.; Morisue, M.; Kobuke, Y. Synthesis and Electrochemical Properties of Slipped-Cofacial Porphyrin Dimers of Ferrocene-
Functionalized Zn-Imidazolyl-Porphyrins as Potential Terminal Electron Donors in Photosynthetic Models. New J. Chem. 2006
0, 77–92. [CrossRef]
,
3
3
3
3
3. Kachadourian, R.; Johnson, C.A.; min, E.; Spasojevic, I.; Day, B.J. Flavin-Dependent Antioxidant Properties of a New Series of
0
Meso-N,N -Dialkyl-Imidazolium Substituted Manganese(III) Porphyrins. Biochem. Pharmacol. 2004, 67, 77–85. [CrossRef]
4. Tjahjono, D.H.; Akutsu, T.; Yoshioka, N.; Inoue, H. Cationic Porphyrins Bearing Diazolium Rings: Synthesis and Their Interaction
with Calf Thymus DNA. Biochim. Biophys. Acta BBA Gen. Subj. 1999, 1472, 333–343. [CrossRef]
5. Mroz, P.; Bhaumik, J.; Dogutan, D.K.; Aly, Z.; Kamal, Z.; Khalid, L.; Kee, H.L.; Bocian, D.F.; Holten, D.; Lindsey, J.S.; et al.
Imidazole Metalloporphyrins as Photosensitizers for Photodynamic Therapy: Role of Molecular Charge, Central Metal and
Hydroxyl Radical Production. Cancer Lett. 2009, 282, 63–76. [CrossRef] [PubMed]
3
6. Moura, N.M.M.; Esteves, M.; Vieira, C.; Rocha, G.M.S.R.O.; Faustino, M.A.F.; Almeida, A.; Cavaleiro, J.A.S.; Lodeiro, C.; Nevesa,
M.G.P.M.S. Novel
β-Functionalized Mono-Charged Porphyrinic Derivatives: Synthesis and Photoinactivation of Escherichia coli.
Dyes Pigments 2019, 160, 361–371. [CrossRef]

Molecules 2021, 26, 1122
20 of 21
3
7. Moreira, X.; Santos, P.; Faustino, M.A.F.; Raposo, M.M.M.; Costa, S.P.G.; Moura, N.M.M.; Gomes, A.T.P.C.; Almeida, A.; Nevesa,
M.G.P.M.S. An Insight into the Synthesis of Cationic Porphyrin-Imidazole Derivatives and Their Photodynamic Inactivation
Efficiency against Escherichia Coli. Dyes Pigments 2020, 178, 108330. [CrossRef]
3
8. Vinagreiro, C.S.; Zangirolami, A.; Schaberle, F.A.; Nunes, S.C.C.; Blanco, K.C.; Inada, N.M.; da Silva, G.J.; Pais, A.A.C.C.; Bagnato,
V.S.; Arnaut, L.G.; et al. Antibacterial Photodynamic Inactivation of Antibiotic-Resistant Bacteria and Biofilms with Nanomolar
Photosensitizer Concentrations. ACS Infect. Dis. 2020, 6, 1517–1526. [CrossRef]
3
4
4
4
4
9. Seng, V.; Wang, X.; Ali, R.; Fang, W.; Charles-Pierre, F.; Bhagan, S.; Hyslop, A.G. Synthesis of Imidazole and Imidazolium
Porphyrins. J. Heterocycl. Chem. 2006, 43, 1077–1081. [CrossRef]
0. Sol, V.; Branland, P.; Granet, R.; Kaldapa, C.; Verneuil, B.; Krausz, P. Nitroglycosylated Meso-Arylporphyrins as Photoinhibitors
of Gram Positive Bacteria. Bioorg. Med. Chem. Lett. 1998, 8, 3007–3010. [CrossRef]
1. Sol, V.; Branland, P.; Chaleix, V.; Granet, R.; Guilloton, M.; Lamarche, F.; Verneuil, B.; Krausz, P. Amino Porphyrins as Photoin-
hibitors of Gram-Positive and -Negative Bacteria. Bioorg. Med. Chem. Lett. 2004, 14, 4207–4211. [CrossRef]
2. Ringot, C.; Naima, S.; Granet, R.; Bressollier, P.; Sol, V.; Krausz, P. Meso-Functionalized Aminoporphyrins as Efficient Agents for
Photo-Antibacterial Surfaces. J. Porphyr. Phthalocyanines 2010, 14, 925–931. [CrossRef]
3. Ringot, C.; Sol, V.; Barrière, M.; Saad, N.; Bressollier, P.; Granet, R.; Couleaud, P.; Frochot, C.; Krausz, P. Triazinyl Porphyrin-Based
Photoactive Cotton Fabrics: Preparation, Characterization, and Antibacterial Activity. Biomacromolecules 2011, 12, 1716–1723.
[CrossRef] [PubMed]
4
4
4
4. Mbakidi, J.-P.; Herke, K.; Alvès, S.; Chaleix, V.; Granet, R.; Krausz, P.; Leroy-Lhez, S.; Ouk, T.-S.; Sol, V. Synthesis and Photobiocidal
Properties of Cationic Porphyrin-Grafted Paper. Carbohydr. Polym. 2013, 91, 333–338. [CrossRef]
5. Le Guern, F.; Sol, V.; Ouk, C.; Arnoux, P.; Frochot, C.; Ouk, T.-S. Enhanced Photobactericidal and Targeting Properties of a Cationic
Porphyrin Following the Attachment of Polymyxin B. Bioconjug. Chem. 2017, 28, 2493–2506. [CrossRef]
6. Le Guern, F.; Ouk, T.-S.; Ouk, C.; Vanderesse, R.; Champavier, Y.; Pinault, E.; Sol, V. Lysine Analogue of Polymyxin B as a
Significant Opportunity for Photodynamic Antimicrobial Chemotherapy. ACS Med. Chem. Lett. 2018, 9, 11–16. [CrossRef]
[PubMed]
4
4
4
5
7. Kalyanasundaram, K.; Neumann-Spallart, M. Photophysical and Redox Properties of Water-Soluble Porphyrins in Aqueous
Media. J. Phys. Chem. 1982, 86, 5163–5169. [CrossRef]
8. Akins, D.L.; Zhu, H.-R.; Guo, C. Aggregation of Tetraaryl-Substituted Porphyrins in Homogeneous Solution. J. Phys. Chem. 1996
00, 5420–5425. [CrossRef]
,
1
9. Tomé, J.P.C.; Neves, M.G.P.M.S.; Tomé, A.C.; Cavaleiro, J.A.S.; Soncin, M.; Magaraggia, M.; Ferro, S.; Jori, G. Synthesis and
Antibacterial Activity of New Poly-S-Lysine−Porphyrin Conjugates. J. Med. Chem. 2004, 47, 6649–6652. [CrossRef] [PubMed]
0. Tomé, J.P.C.; Silva, E.M.P.; Pereira, A.M.V.M.; Alonso, C.M.A.; Faustino, M.A.F.; Neves, M.G.P.M.S.; Tomé, A.C.; Cavaleiro,
J.A.S.; Tavares, S.A.P.; Duarte, R.R.; et al. Synthesis of Neutral and Cationic Tripyridylporphyrin-d-Galactose Conjugates and the
Photoinactivation of HSV-1. Bioorg. Med. Chem. 2007, 15, 4705–4713. [CrossRef] [PubMed]
5
1. Dosselli, R.; Ruiz-González, R.; Moret, F.; Agnolon, V.; Compagnin, C.; Mognato, M.; Sella, V.; Agut, M.; Nonell, S.; Gobbo,
M.; et al. Synthesis, Spectroscopic, and Photophysical Characterization and Photosensitizing Activity toward Prokaryotic and
Eukaryotic Cells of Porphyrin-Magainin and -Buforin Conjugates. J. Med. Chem. 2014, 57, 1403–1415. [CrossRef] [PubMed]
2. Shi, D.-F.; Wheelhouse, R.T.; Sun, D.; Hurley, L.H. Quadruplex-Interactive Agents as Telomerase Inhibitors: Synthesis of
5
Porphyrins and Structure
PubMed]
−
Activity Relationship for the Inhibition of Telomerase. J. Med. Chem. 2001, 44, 4509–4523. [CrossRef]
[
5
5
5
3. Raffy, Q.; Ricoux, R.; Mahy, J.-P. Synthesis of a New Estradiol–Iron Metalloporphyrin Conjugate Used to Build up a New Hybrid
Biocatalyst for Selective Oxidations by the ‘Trojan Horse’ Strategy. Tetrahedron Lett. 2008, 49, 1865–1869. [CrossRef]
4. Jiang, W.; Yang, J.; Liu, Y.-Y.; Song, S.-Y.; Ma, J.-F. A Stable Porphyrin-Based Porous Mog Metal–Organic Framework as an Efficient
Solvent-Free Catalyst for C–C Bond Formation. Inorg. Chem. 2017, 56, 3036–3043. [CrossRef] [PubMed]
5. Jacobs, R.; Stranius, K.; Maligaspe, E.; Lemmetyinen, H.; Tkachenko, N.V.; Zandler, M.E.; D’Souza, F. Syntheses and Excitation
Transfer Studies of Near-Orthogonal Free-Base Porphyrin–Ruthenium Phthalocyanine Dyads and Pentad. Inorg. Chem. 2012
1, 3656–3665. [CrossRef]
,
5
5
6. Maximiano, R.V.; Piovesan, E.; Zílio, S.C.; Machado, A.E.H.; de Paula, R.; Cavaleiro, J.A.S.; Borissevitch, I.E.; Ito, A.S.; Gonçalves,
P.J.; Barbosa Neto, N.M. Excited-State Absorption Investigation of a Cationic Porphyrin Derivative. J. Photochem. Photobiol. Chem.
2
010, 214, 115–120. [CrossRef]
5
5
5
6
6
7. Fujimoto, J.; Manseki, K.; Miyaji, H. Dye-Sensitized Solar Cells Using Supramolecular Porphyrin Arrays Inspired by π-Stacking
Structures of Photosynthetic Light-Harvesting Complexes. Chem. Lett. 2013, 43, 207–209. [CrossRef]
8. Little, R.G.; Anton, J.A.; Loach, P.A.; Ibers, J.A. The Synthesis of Some Substituted Tetraarylporphyrins. J. Heterocycl. Chem. 1975
2, 343–349. [CrossRef]
,
1
9. Adler, A.D.; Longo, F.R.; Finarelli, J.D.; Goldmacher, J.; Assour, J.; Korsakoff, L. A Simplified Synthesis for Meso-
Tetraphenylporphine. J. Org. Chem. 1967, 32, 476. [CrossRef]
0. Meng, S.; Xu, Z.; Hong, G.; Zhao, L.; Zhao, Z.; Guo, J.; Ji, H.; Liu, T. Synthesis, Characterization and in Vitro Photodynamic
Antimicrobial Activity of Basic Amino Acid–Porphyrin Conjugates. Eur. J. Med. Chem. 2015, 92, 35–48. [CrossRef] [PubMed]
1. Kumar, D.; Chandra Shekar, K.P.; Mishra, B.; Kurihara, R.; Ogura, M.; Ito, T. Cationic Porphyrin–Quinoxaline Conjugate as a
Photochemically Triggered Novel Cytotoxic Agent. Bioorg. Med. Chem. Lett. 2013, 23, 3221–3224. [CrossRef] [PubMed]

Molecules 2021, 26, 1122
21 of 21
6
2. Sol, V.; Blais, J.C.; Carré, V.; Granet, R.; Guilloton, M.; Spiro, M.; Krausz, P. Synthesis, Spectroscopy, and Photocytotoxicity
of Glycosylated Amino Acid Porphyrin Derivatives as Promising Molecules for Cancer Phototherapy. J. Org. Chem. 1999
4, 4431–4444. [CrossRef]
,
6
6
6
6
3. Vergeldt, F.J.; Koehorst, R.B.; van Hoek, A.; Schaafsma, T.J. Intramolecular Interactions in the Ground and Excited States of
Tetrakis (N-Methylpyridyl) Porphyrins. J. Phys. Chem. 1995, 99, 4397–4405. [CrossRef]
4. Lakowicz, J.R. Principles of Fluorescence Spectroscopy; Springer Science & Business Media: Berlin, Germany, 2007; ISBN 978-0-387-
4
6312-4.
5. Dorough, G.D.; Miller, J.R.; Huennekens, F.M. Spectra of the Metallo-Derivatives of α, β, γ, δ-Tetraphenylporphine. J. Am. Chem.
Soc. 1951, 73, 4315–4320. [CrossRef]
6
6
6. Gouterman, M.; Wagnière, G.H.; Snyder, L.C. Spectra of Porphyrins. J. Mol. Spectrosc. 1963, 11, 108–127. [CrossRef]
7. Procházková, K.; Zelinger, Z.; Lang, K.; Kubát, P. Meso-Tetratolylporphyrins Substituted by Pyridinium Groups: Aggregation,
Photophysical Properties and Complexation with DNA. J. Phys. Org. Chem. 2004, 17, 890–897. [CrossRef]
6
6
7
7
8. Redmond, R.W.; Gamlin, J.N. A Compilation of Singlet Oxygen Yields from Biologically Relevant Molecules. Photochem. Photobiol.
1
999, 70, 391–475. [CrossRef]
9. Couleaud, P. Fonctionnalisation Par Des Peptides de Nanoparticules Hybrides Multifonctionnelles Pour de La Thérapie Photody-
namique Ciblant Neuropiline-1. Ph.D. Thesis, Université de Lorraine, Nancy, France, 2011.
0. Mathai, S.; Smith, T.A.; Ghiggino, K.P. Singlet Oxygen Quantum Yields of Potential Porphyrin-Based Photosensitisers for
Photodynamic Therapy. Photochem. Photobiol. Sci. 2007, 6, 995. [CrossRef]
1. Wilkinson, F.; Helman, W.P.; Ross, A.B. Rate Constants for the Decay and Reactions of the Lowest Electronically Excited Singlet
State of Molecular Oxygen in Solution. An Expanded and Revised Compilation. J. Phys. Chem. Ref. Data 1995, 24, 663–1021.
[CrossRef]
7
7
2. Ogilby, P.R.; Foote, C.S. Chemistry of Singlet Oxygen. 42. Effect of Solvent, Solvent Isotopic Substitution, and Temperature on the
Lifetime of Singlet Molecular Oxygen (1.DELTA.g). J. Am. Chem. Soc. 1983, 105, 3423–3430. [CrossRef]
3. Li, H.; Fedorova, O.S.; Grachev, A.N.; Trumble, W.R.; Bohach, G.A.; Czuchajowski, L. A Series of Meso-Tris(N-Methyl-
Pyridiniumyl)-(4-Alkylamidophenyl)Porphyrins: Synthesis, Interaction with DNA and Antibacterial Activity. Biochim. Biophys.
Acta 1997, 1354, 252–260. [CrossRef]
7
4. Maisch, T. Strategies to Optimize Photosensitizers for Photodynamic Inactivation of Bacteria. J. Photochem. Photobiol. B 2015
50, 2–10. [CrossRef]
,
1