Synthesis, characterization and biological evaluation of cationic porphyrin-terpyridine derivatives

Article abstract of DOI:10.1039/c6ra25373c

A simple access to a new series of cationic porphyrin-terpyridine derivatives is described. The key step to obtain the required neutral precursors as major products involved a Kr?hnke type approach. The methodology allowed also the isolation of the respective benzoporphyrins and porphyrin-chalcone type derivatives, and in one case a new 2-(2,4-terpyridin-6-yl)-porphyrin. The quaternization of the pyridyl groups was performed in the presence of the adequate alkyl iodide affording the dicationic derivatives in excellent yields. All the new conjugates were fully characterised and it was found that the cationic isomers can be efficiently differentiated by ESI-MS, as their behaviour can be intensively studied by mass spectrometry. The new methylated cationic porphyrin-terpyridine derivatives demonstrate an ability to generate singlet oxygen and their efficacy to photoinactivate bioluminescent Gram-negative E. coli was evaluated. A reduction in the bioluminescence signal, up to 5.4 log, was obtained with the most efficient photosensitiser.

Full text of DOI:10.1039/c6ra25373c

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PAPER
Synthesis, characterization and biological
evaluation of cationic porphyrin–terpyridine
derivatives†
Cite this: RSC Adv., 2016, 6, 110674
abc
*
Nuno M. M. Moura,
Catarina I. V. Ramos,d Ines Linhares,e Sergio M. Santos,f
ˆ ´
M. Amparo F. Faustino, Adelaide Almeida,e Jose A. S. Cavaleiro,a
a
*
´
Francisco M. L. Amado,d Carlos Lodeirobc and M. Graça P. M. S. Neves
a
*
A simple access to a new series of cationic porphyrin–terpyridine derivatives is described. The key step to
obtain the required neutral precursors as major products involved a Krohnke type approach. The
¨
methodology allowed also the isolation of the respective benzoporphyrins and porphyrin–chalcone type
derivatives, and in one case a new 2-(2,4-terpyridin-6-yl)-porphyrin. The quaternization of the pyridyl
groups was performed in the presence of the adequate alkyl iodide affording the dicationic derivatives in
excellent yields. All the new conjugates were fully characterised and it was found that the cationic
isomers can be efficiently differentiated by ESI-MS, as their behaviour can be intensively studied by mass
spectrometry. The new methylated cationic porphyrin–terpyridine derivatives demonstrate an ability to
generate singlet oxygen and their efficacy to photoinactivate bioluminescent Gram-negative E. coli was
evaluated. A reduction in the bioluminescence signal, up to 5.4 log, was obtained with the most efficient
photosensitiser.
Received 17th October 2016
Accepted 7th November 2016
DOI: 10.1039/c6ra25373c
www.rsc.org/advances
inactivation of microorganisms (PDI)8–11 also known as anti-
microbial photodynamic therapy (aPDT)12,13 or antibacterial
PDT.14,15
Introduction
Porphyrins are receiving a lot of attention from the scientic
community due to their high success in different elds like
catalysis, development of advanced biomimetic models for
photosynthesis, drugs, new electronic materials and sensors.1
The richness of porphyrin applications is due to their synthetic
versatility, thermal stability, large p-electron systems and
photochemical and photophysical properties.2,3 In particular,
porphyrin-based macrocycles are being exploited with high
success in medicine, namely as photosensitizers (PS) in
photodynamic therapy of tumours (PDT)4–7 or in photodynamic
PDT and PDI are nowadays considered efficient alternatives
to other techniques in the treatment of several diseases, such as
tumours, age macular degeneration (AMD), skin disorders, and
also in the inactivation of microorganisms, respectively.4,16 The
photodynamic process has as main advantages the fact that
occur only in the illuminated region and the possibility to occur
resistance is unlikely due to the non-specic killing mecha-
nism.5,17–19 Both therapies combine three key elements – light,
oxygen and a photosensitiser (PS).20 The combination of these
non-toxic elements provide an efficient formation of high
reactive oxygen species (ROS), such as the hydroxyl (cOH) and
superoxide (O2cꢀ) radicals and non-radical species, such as
singlet oxygen (1O2) and hydrogen peroxide (H2O2).21 These
species are responsible for multi-targeted damage and
destruction of living tissues. In porphyrin-mediated photody-
namic inactivation (PDI), 1O2 is in general considered the main
damaging species.22–24
aOrganic Chemistry Laboratory, QOPNA, Department of Chemistry, University of
Aveiro, 3810-193 Aveiro, Portugal. E-mail: nmoura@ua.pt; gneves@ua.pt;
faustino@ua.pt
bBIOSCOPE Research Team, UCIBIO, REQUIMTE, Departamento de Quımica,
´
ˆ
Faculdade de Ciencias
e Tecnologia, Universidade Nova de Lisboa, 2829-516
Caparica, Portugal
cProteoMass Scientic Society, Madan Parque, Rua dos Inventores, 2825-182 Caparica,
In last years, many studies proved the efficiency of cationic
porphyrins as PS agents, in the microbial inactivation of viruses,
fungi, protozoa, Gram-negative and Gram-positive bacteria.25–30
The most promising studies are based on 5,10,15,20-tetrakis(1-
alkylpyridinium-4-yl)porphyrin and its analogues where the
charge is localized in the meso-substituents. Notwithstanding,
there are few results in literature31–34 of microorganism inacti-
vation using cationic meso-tetraarylporphyrins bearing charges
Portugal
dMass Spectrometry Laboratory, QOPNA, Department of Chemistry, University of
Aveiro, 3810-193 Aveiro, Portugal
eDepartment of Biology, CESAM, University of Aveiro, 3810-193 Aveiro, Portugal
fCICECO, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal
† Electronic supplementary information (ESI) available: Scheme S1: mechanistic
pathway for the synthesis of compound 4. Fig. S1–S66: NMR (1D and 2D) and
HRMS spectra. Fig. S67: relationship between bioluminescence and viable
counts of overnight cultures of recombinant E. coli. See DOI: 10.1039/c6ra25373c
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in the beta-pyrrolic positions; the results already obtained show
that this type of PSs is also very promising.
This fact, and our nding of an easy synthesis for porphyrin
1a (see Fig. 1) bearing a terpyridine unit in the beta pyrrolic
position,35 prompted us to expand the studies to the syntheses
of the analogues 1b and 1c in order to use them as templates for
further cationization. So, herein, we report the synthesis and
the characterization of a series of neutral and cationic porphy-
rins bearing terpyridine units in one of their beta-pyrrolic
positions. In the course of the characterization of these
compounds we found that isomer differentiation of the cationic
compounds could be easily achieved by ESI-MS (electrospray
mass spectrometry).
Scheme 1 Synthetic route to porphyrinic derivatives 1, 2 and 3.
Table 1 Products obtained from the reaction of 2-formyl-porphyrin
(TPP-CHO) with acetylpyridines
Additionally, their efficacy on the photoinactivation of
bioluminescent E. coli was evaluated. The bioluminescent
genetically transformed E. coli was selected as a model of Gram-
negative bacteria to monitor in real-time photodynamic inacti-
vation through bioluminescence measurement, thus avoiding
the laborious and time-consuming conventional method of
counting colony-forming units.
Entry Py
Time (h) 1 (%) 2 (%) 3 (%) 4 (%) 5 (%)
1a
2a
3
4
45
47
29
27
10
12
—
4
—
—
3a
4b
6
4
16
45
7
21
33
11
—
—
7
—
5a
6b
6
4
18
48
8
24
36
8
—
—
2
—
Results and discussion
Synthesis of neutral derivatives
a
Acetylpyridine (5 equiv.), NH4OAc (8 equiv.), La(OTf)3 (20 mol%),
b
toluene, reux. Acetylpyridine (12 equiv.), NH4OAc (14 equiv.),
La(OTf)3 (20 mol%), toluene, reux.
The synthetic strategy to obtain the new b-pyrrolic cationic
derivatives 6 and 7 (see Scheme 2) required the previous
preparation of the corresponding neutral derivatives accord-
ing with experimental work summarized in Scheme 1. The
access to the new neutral porphyrins 1b and 1c bearing the
terpyridine units in the b-pyrrolic positions involved the
In order to force the formation of the desired terpyridine 1a,
the reaction was repeated in the presence of 2-acetylpyridine
during 4 h. The results summarized in Table 1 (entry 2) show
that the expected derivatives 1a, 2a and 3a were isolated in
yields similar to our previously used conditions. However,
a careful analysis of TLC data allowed us to detect a minor and
more polar red product. Aer purication, the structure of this
product, obtained in 4% yield, was established based on spec-
troscopic data (see infra and also ESI†) as the new 2-(2,4-
diarylpyridin-6-yl)-5,10,15,20-tetraphenylporphyrin 4 (Fig. 2).
Attempts to improve compound 4 yield by increasing the
reaction time were not successful. The formation of the new
derivative 4 can be explained in a similar manner as the one
proposed for derivative 1a,35 using as template the porphyrin–
chalcone type derivative 3a (see Scheme S1 of ESI†). However,
instead of a Michael addition involving the 2-acetylpyridine that
led to compound 1a, an aldol condensation, followed by reac-
tion with NH3 occurs in the case of compound 4.
¨
Krohnke type approach described previously for the synthesis
of porphyrin 1a.35 In this methodology the terpyridine moiety
was built directly from the easy accessible 2-formyl-5,10,15,20-
porphyrin (TPP-CHO)36 by reaction with 2-acetylpyridine (5
equiv.) in the presence of ammonium acetate (8 equiv.) and
La(OTf)3 (20 mol%).35 The procedure led to compound 1a in
good yield (45%) affording as minor compounds the benzo-
porphyrin 2a (29%) and the porphyrin–chalcone 3a (10%)
(Table 1, entry 1).
Based on that synthetic strategy we envisaged, just by
changing the 2-acetylpyridine by the adequate 3- and 4-ace-
tylpyridine the syntheses of the new neutral terpyridine deriv-
atives 1b and 1c.
Fig. 1 Porphyrin–terpyridine derivatives 1a–c used as template in this
study.
Fig. 2 Structures of compounds 4 and 5b, 5c.
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Our rst experiments with 3- and 4-acetylpyridine, were pyrrolic proton H-3 and an AB system at ca. d 7.5 ppm due to the
performed also in reuxing toluene, in the presence of 5 equiv. resonance of the two protons from the a,b-unsaturated ketone.
of these ketones, ammonium acetate and using La(OTf)3 as The trans conguration of the chalcone moiety was clearly
catalyst as it was described for 2-acetylpyridine. However, under conrmed by the coupling constant (J ¼ 15.7 Hz). The 13C NMR
these conditions and even aer 6 h of reaction, besides the large spectra show a distinctive signal at ca. d 191.0 ppm corre-
decomposition observed at the bottom of TLC, the main prod- sponding to the carbonyl carbon. Again, the mass spectra of
ucts isolated and structurally characterized (see below) aer compounds 3b and 3c present a peak, at m/z 746.3, that corre-
work up and chromatographic purication were the porphyrin– sponds to the [M + H]+ ion and conrms the proposed struc-
chalcone derivatives 3b (33%) and 3c (36%) instead of the tures (Fig. S25–S36†).
desired 2-(terpyridin-4-yl)porphyrin derivatives 1b (16%) and 1c
The 1H NMR patterns of the derivatives synthesized from 3-
(18%), (Table 1, respectively entries 3 and 5). The low yields and 4-acetylpyridine are remarkably different. While the rst
obtained for these porphyrin–terpyridines and also for the ones show four signals in the range of approximately d 9.0 ppm
benzoporphyrins 2b (7%) and 2c (8%), also obtained from the to d 7.4 ppm, the derivatives from 4-acetylpyridine present two
same reactions, suggested that 3- and 4-acetylpyridines are less signals at ca. d 8.7 and d 7.7 (8.0 ppm for 1c) ppm indicating
reactive than the 2-acetylpyridine.
highly symmetric structures. The 2D COSY experiments allowed
For the reactions with 3- and 4-acetylpyridines, small the unequivocal attribution of the resonances of the protons
amounts of new derivatives, identied as compounds 5b (2%) from the pyridine moieties.
and 5c (7%) (Fig. 2), were also formed. The oxidative process
In all cases the presence of the two protons in the inner core
involved in formation of terpyridine units is probably respon- of the porphyrinic macrocycle was conrmed by a singlet at ca. –
sible by the appearance of these reduced porphyrin–chalcone d 2.6 ppm.
derivatives.
The 1H NMR of derivative 4 has a similar prole to the NMR
In a further attempt to obtain the desired porphyrin derivatives obtained for its isomer 1a, but it is indicative of a much less
1b and 1c in higher yields, we decided to perform the reactions in symmetric structure. This asymmetry is particularly noticeable
the presence of a higher number of equivalents of the acetylpyr- in the signals generated by the resonances of the three pyridyl
idines (12 equiv.) and consequently of ammonium acetate and groups of the terpyridine moiety, namely in the central pyridyl
La(OTf)3 (Table 1, entries 4 and 6). Under these conditions and where the resonances of the two protons generated two signals
aer 4 h of reaction, we were able to isolate derivatives 1b and 1c (d 8.75 and d 8.09 ppm) instead of only one singlet (ca. d 7.5
as the main reactional products and in comfortable yields (45% ppm) as in the case of derivative 1a. The mass spectrum of the
and 48% respectively). Under these conditions the second most compound 4 presents a peak at m/z 846.3 corresponding to the
abundant products were the benzoporphyrin derivatives 2b, 2c (21 [M + H]+ ion of the proposed structure (see ESI, Fig. S37–S42†).
1
and 24%) followed by the porphyrin–chalcone type derivatives 3b,
3c (11 and 8%, respectively).
The H NMR of compounds 5 shows two multiplets at ca.
d 3.4 ppm and d 3.3 ppm due to the resonance of the aliphatic
protons from the reduced chain of the propanone moiety. The
mass spectra of these compound are also in accordance with
proposed structures, presenting a peak at m/z 748.3, corre-
Structural characterization
The structural elucidation of all compounds involved the use 1D sponding to the [M + H]+ ion (Fig. S43–S48 of ESI†).
(1H and 13C spectra) and 2D [(1H,1H) COSY, NOESY, (1H,13C)
HSQC and (1H,13C) HMBC] NMR techniques, high resolution
mass spectrometry (HRMS-ESI) and UV-Vis spectroscopy.
Synthesis of cationic derivatives
In particular, the 1H NMR of the new porphyrin–terpyridine The quaternisation of the pyridyl groups of the porphyrin–ter-
derivatives 1b and 1c are consistent with b-substituted pyridine derivatives 1 was performed in the presence of methyl
porphyrins showing the resonance of seven beta pyrrolic iodide or ethyl iodide under conventional conditions as it is
protons as multiplet at ca. d 8.9 ppm. The resonances of the two outlined in Scheme 2. A typical reaction was performed in N,N0-
protons from the central pyridyl show a characteristic singlet at dimethylformamide (DMF) for 24 h at 40 ꢁC in the presence of
ca. d 7.8 ppm. The meso-phenyl protons appear as multiplets the respective 2-(2,6-diarylpyridin-4-yl)-porphyrin 1a–c and
between ca. d 7.1 and d 8.3 ppm. The obtained ESI† mass spectra using an excess of methyl or ethyl iodide. Aer that period,
present a peak at m/z 846.3, corresponding to the molecular ion when methyl iodide was used, the reaction control by TLC
[M + H]+, in agreement with the proposed structures for 1b and conrmed the total conversion of all starting porphyrins 1 to
1c (ESI, Fig. S1–S12†).
more polar compounds that were identied as the corre-
In the case of compounds 2a and 2c the 1H NMR spectra sponding dicationic products 6. All the derivatives were ob-
show the resonances of only six b-pyrrolic protons, consistent tained pure, in excellent yields (87–97%), directly from
with a benzoporphyrin derivative. The mass spectra obtained crystallization in CH2Cl2/hexane.
for both compounds show a peak at m/z 743.2 corresponding to
When ethyl iodide was used only the derivatives 7b (96%)
and 7c (95%) were isolated, also in excellent yields and directly
the [M + 2H]+c ion (see Fig. S13–S24 of ESI†).
1
Considering the H NMR spectra of compounds 3b and 3c from crystallization in CH2Cl2/hexane. All attempts to ethylate
the most important features are the presence of a characteristic the pyridine units of compound 1a were not successful and only
singlet at ca. d 9.1 ppm corresponding to the resonance of the b- the starting porphyrin was recovered.
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For the methylated isomers, the compounds 6, the expected
doubly charged M2+ ions, m/z 437.8, were observed with high
abundances. Triply charged species corresponding to [M + H]3+
ions, m/z 292.2, were also identied in the case of compounds
6b and 6c, the abundance being higher for the former. As can be
observed in Fig. 3 this species is absent in the mass spectra of
isomer 6a (ortho) allowing its differentiation from the other two
isomers.
The absence of the triply charged species [M + H]3+ for
compound 6a points to the occurrence of the protonation
preferentially in the terpyridine moiety, since if it did occur in
the core of the macrocycle, the formation of the [M + H]3+ ions
should also be observed for isomer 6a. This behaviour was not
unexpected because, as mentioned once, the sterical hindrance
is higher in the case of the alkylated ortho isomers.
Scheme 2 Alkylation of porphyrin–terpyridine derivatives 1a–c.
Monocharged ions, m/z 1002.3, corresponding to the
formation of adducts with the iodide counter ion, [M2+ + Iꢀ]+,
were observed for the meta isomer, compound 6b. This char-
acteristic feature allows the differentiation of this isomer and
points to a preferential formation of its [M + Iꢀ]+ adduct. In
order to understand this behavior, theoretical studies were
performed for the terpyridine moieties. As it can be seen in
Fig. 4 the stereochemistry of the meta isomer allows a closer
approach of the iodide anion to the positive charges of the
nitrogen atoms of the pyridinium cations, resulting in an
increase of the coulombic attractive forces and consequently in
a preferential formation of the [M + Iꢀ]+ adduct.
It is interesting to note that, besides the described species,
a monocharged ion, at m/z 860.4, is also present for the ortho
isomer. This ion is also diagnostic for the differentiation of this
isomer and probably results from a reduction process involving
the formation of the hypervalent radical ions [M2+ + eꢀ]+ ¼ M+c.
Aromatic hypervalent pyridinium radicals, contrary to aliphatic
ammonium hypervalent radicals, which are transient species,39
are relatively stable due to charge delocalisation throughout the
macrocycle. However, they fragment by loss of alkyl radicals
forming neutral pyridyl functionalities, whenever this frag-
mentation is energetically favourable (Scheme 3). The forma-
tion and fragmentation of hypervalent ions by loss of alkyl
The proximity of the nitrogen atoms in this terpyridine
moiety is probably compromising the nucleophilic attack on the
more bulky ethyl iodide and it is why we were not able to isolate
any N-alkylated derivative in this reaction.
It is worth to mention that all the attempts to alkylate the
third pyridyl unit by increasing the reaction time or by using
dimethyl sulphate as alkylating agent were not successful. This
fact may be explained by the reduced basicity of the central
pyridine unit aer cationization of the other two pyridine units.
The structure of the alkylated derivatives 6a–c and 7b, c were
conrmed by recording their 1H-NMR, 13C-NMR and mass
spectra. In particular, the 1H NMR spectra present
a pronounced deviation for low elds of the signals in aromatic
region, namely, the peaks generated by the resonances of the
protons from the pyridine rings. The resonances of the protons
from the methyl groups (6a–c) generated a distinctively singlet
at ca. d 4.5 ppm, while the resonances of protons from the ethyl
chains (7b, c) generated two typically signals, a quartet at ca.
d 4.8 ppm and a triplet at ca. d 1.7 ppm. The characteristic signal
due to resonances of the inner NH protons appears, as expected,
at high eld at ca. d ꢀ2.7 ppm (see ESI, Fig. S49–S66†).
Characterization of the cationic derivatives by electrospray
mass spectrometry
In the course of the characterization of the obtained cationic
compounds we found that we could easily differentiate these
isomers by ESI-MS. These ndings led us to investigate, in
further detail, the gas-phase behavior of the neutral compounds
1 and of their corresponding methylated and ethylated coun-
terparts, the cationic compounds 6 and 7, respectively.
In the mass spectra of the neutral isomeric compounds 1,
the expected [M + H]+ ions, m/z 846.4, were observed with high
relative abundance. Higher abundant [M + 2H]2+ ions, m/z
423.7, were also observed. Formation of [M + 2H]2+ ions was
already observed by us for neutral porphyrin–chalcone struc-
tures37 and for neutral imidazole porphyrins and metal-
loporphyrins.38 However, in the case of the imidazole-
porphyrins and metalloporphyrins studied, the [M + 2H]2+
ions showed lower abundance, when compared with the cor-
responding monoprotonated species.
Fig. 3 Mass spectra of (A) compound 6a; (B) compound 6b; (C)
compound 6c.
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Photophysical properties and biological evaluation
Singlet oxygen generation. In order to evaluate the potenti-
ality of new materials for the photodynamic inactivation of
bacteria, their ability to generate singlet oxygen should be
previously estimated. In these studies, the ability to produce 1O2
by the cationic porphyrins 6a–c was qualitatively estimated
using 1,3-diphenylisobenzofuran (DPiBF) as 1O2 indicator.42
The yellow DPiBF (with a maximum wavelength at 415 nm)
Fig. 4 Lowest energy conformations for the iodide adducts of ter-
pyridine moieties of compounds 6a, 6b and 6c; color code: blue –
nitrogen; grey – carbon; white – hydrogen; purple – iodine.
1
reacts with O2 generated by the combined action of light, PS
and dissolved oxygen in a [4 + 2] cycloaddition affording a col-
ourless o-dibenzoylbenzene. The decay of DPiBF absorption
band at 415 nm allows to evaluate the ability of each porphyrin
to generate 1O2. As reference was used 5,10,15,20-tetrakis(1-
methylpyridinium-4-yl)porphyrin (Tetra-Py+-Me) that was
proved to be an excellent singlet oxygen generator and efficient
in the photodynamic inactivation of microorganisms.43
The results obtained with the cationic porphyrins (6a–c) are
Scheme 3 Formation of the [M ꢀ CH3]+ ions from a methyl loss represented in Fig. 6 and conrm that all the studied porphy-
following a reduction process with formation of the hypervalent cation
rins are able to cause a decay of DPiBF absorption within 10 min
M+c.
when irradiated with light at an irradiance of 4.0 mW cmꢀ2
.
Compounds 6a and 6c showed a distinctively aptitude to
generate singlet oxygen when compared with 6b; the efficiency
of this derivative to generate O2 generation is around 25% of
radicals, in positive ion mode ESI-MS, was previously reported
by us for other cationic porphyrins.40,41
1
the reference Tetra-Py+-Me, while the others varied between ꢂ70
and ꢂ85%. These data demonstrate that the localisation of the
positive charges could be a determinant factor for the 1O2
generation. The results found allow to state that compounds 6
exhibit potentiality to be used as PS in the photodynamic
inactivation of microorganisms.
Bioluminescence versus colony forming units of an over-
night culture. The correlation between the colony-forming units
(CFU) and the bioluminescence signal of E. coli was evaluated
and a high correlation (R ¼ 0.9846) between viable counts and
the bioluminescence signal of overnight cultures was observed
(Fig. S67, ESI†). Thus, the bioluminescence results reect the
viable bacterial abundance, allowing to obtain results in real
time in a cost effective way. Consequently, the effect of bacterial
photoinactivation by the porphyrin derivatives was assessed
using the bioluminescent method.44
The above mentioned processes leading to the formation of
the [M ꢀ CH3]+ ion are favoured in the case of ortho isomer due
to the higher sterical hindrance induced by the proximity of the
methyl substituents.
In the case of the ethylated isomers 7, the ortho isomer could
not be made probably due to the increased sterical hindrance
caused by the longer alkyl chain. The gas phase behavior of the
ethylated meta and para isomers is similar to the obtained for
the corresponding methylated meta and para isomers, as it can
be seen in Fig. 5.
ESI-MS proved to be a useful technique for the differentia-
tion of the alkylated isomers through the formation of different
diagnostic ions. The absence of the [M + H]3+ ion and the
formation of the monocharged [M ꢀ CH3]+ ion allowed the
differentiation of the ortho isomer 6a from the others two
isomers. On the other hand, formation of the [M + Iꢀ]+ adducts
is a feature that is specic of the meta alkylated isomers, 6b and
7b, enabling their differentiation from the para isomers, 6c and
7c.
Fig. 6 Time dependent photodecomposition of DPiBF (50 mM) pho-
tosensitized by derivatives 6a–c and Tetra-Py+-Me in DMF/H2O (9 : 1)
upon irradiation with red light LEDs (654 nm ꢃ 20 nm) with an irra-
diance of 4.0 mW cmꢀ2 with and without porphyrin–terpyridine
derivatives (0.5 mM).
Fig. 5 Mass spectra of (A) compound 7b; (B) compound 7c.
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photoinactivation of bioluminescent E. coli. The results of the
experiments carried out under PAR white light (380–700 nm)
conditions (4.0 mW cmꢀ2) showed that the cationic porphyrins
under study are able to cause a decrease in the bioluminescent
signal emitted by E. coli. In the three independent experiments
(Fig. 7) the compound 6a was the most effective derivative
causing a reduction of ca. 2.4 log on bioluminescence signal
aer 270 min of irradiation. At same light conditions,
compounds 6b and 6c were less effective causing a reduction of
ca. 1.4 log and ca. 2.0 log on bacterial bioluminescence,
respectively (Fig. 7). The inactivation prole was similar in the
three independent assays, even with different bacteria densities
(Fig. 7).
The better efficiency of 6a and 6c when compared with 6b is
probably associated with their much higher ability to produce
Fig. 8 Bioluminescence monitoring of E. coli treated with 6a at 5, 10,
20 and 40 mM after 30, 90, 120, 180 and 270 min of irradiation with
white light at an irradiance of 4.0 mW cmꢀ2. The values are expressed
as the means of three independent experiments; error bars indicate
1
1O2. However, the production of O2 even for 6b compound is
considered to be enough to inactivate the bacteria when
compared with other PS from previous studies.45 So other the standard deviation; DC dark control; LC light control.
factors are also contributing for this inactivation prole such as
structural features which can affect the adequate conformation
to interact with the bacterial targets. In fact, the terpyridine
units in the b-pyrrolic position differ in the position of the
nitrogen linked to central pyridyl. Probably the charge distri-
bution in 6a and 6c turn these molecules more accessible to
interact with the external structure of the bacteria when
compared with the derivative 6b. A possible explanation can be
due to the closer approach of the iodide anion to the positive
charges of the nitrogen atoms of the pyridinium cations in
derivative 6b as it was veried by mass spectrometry studies.
The results of control samples show that the viability of the
recombinant bioluminescent E. coli was not affected either by
irradiation (light control) or by any of the compounds tested
(dark control). The reduction of cell viability observed aer
irradiation of the treated samples is clearly due to the
photosensitisation effect of the cationic porphyrins as it was
demonstrated by the three independent experiments of photo-
inactivation performed (Fig. 7).
In order to evaluate the effect of PS concentration on the
E. coli photoinactivation efficiency, the most effective cationic
porphyrin 6a was irradiated using the same light conditions
(4.0 mW cmꢀ2) at different nal concentrations of 5, 10, 20 and
40 mM (Fig. 8). The photoinactivation efficiency of compound 6a
increases with the PS concentration but it seems to reach
a plateau aer 270 min of irradiation.
Knowing that the efficiency of the photoinactivation process
can be signicantly affected by the light irradiance44,46 we
decided to evaluate the efficacy of compound 6a at 20 mM but at
a higher light irradiance (150 mW cmꢀ2). The results depicted
in Fig. 9 show that the increase of light irradiance from 4.0 to
Fig. 7 Bioluminescence monitoring of E. coli treated with derivatives Fig. 9 Percentual of bioluminescence monitoring of E. coli treated
6a–c at 20 mM after 30, 90, 120, 180 and 270 min of irradiation with with 6a at 20 mM during 120 min of irradiation with white light at an
PAR white light at an irradiance of 4.0 mW cmꢀ2 in three independent irradiance of 150 mW cmꢀ2. The values are expressed as the means of
experiments with different bacteria density; DC dark control; LC light three independent experiments; error bars indicate the standard
control.
deviation.
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150 mW cmꢀ2 caused a signicant effect on the efficiency of 6a HSQC (1H/13C) and HMBC (delay for long-range J C/H couplings
to photoinactivate E. coli. were optimized for 7 Hz) experiments. Mass spectra were
At this irradiance of 150 mW cmꢀ2 and just aer 30 min (270 recorded using MALDI TOF/TOF 4800 Analyzer, Applied Bio-
J cmꢀ2), it was observed a reduction on the bioluminescence systems MDS Sciex, with CHCl3 as solvent and without matrix.
signal of 2.9 log, a value higher than the 2.4 log reduction ob- Mass spectra HRMS were recorded on a LTQ Orbitrap XL mass
tained for the same compound when exposed to white light spectrometer (Thermo Fischer Scientic, Bremen, Germany)
(PAR white light) at an irradiance of 4.0 mW cmꢀ2 during using CHCl3 as solvent. The UV-Vis spectra were recorded on an
270 min (64.8 J cmꢀ2). The porphyrin 6a reached reductions of UV-2501PC Shimadzu spectrophotometer using DMF as
the bioluminescence signal of 4.0 log aer 60 min of irradiation solvent. Preparative thin-layer chromatography was carried out
and of 5.4 log aer 210 min. According to the American Society on 20 ꢄ 20 cm glass plates coated with silica gel (0.5 mm thick).
for Microbiology, any new approach must achieve a reduction of Column chromatography was carried out using silica gel
at least 3 log10 CFU (killing efficiency of 99.9% or more) to be (Merck, 35–70 mesh). Analytical TLC was carried out on pre-
termed antimicrobial or antibacterial, which indicate that 6a coated sheets with silica gel (Merck 60, 0.2 mm thick).
compound is an effective PS.47,48 Control samples did not show
All the chemicals were used as supplied. Solvents were
signicant variation in the emission of bioluminescence, indi- puried or dried according to the literature procedures.49
cating that light alone (light control) or 6a at the studied
concentration of 20 mM in the dark (dark control) do not affect
bioluminescent E. coli viability.
Synthesis
Synthesis of the porphyrin precursor TPP-CHO. The 2-
formyl-5,10,15,20-tetraphenylporphyrin TPP-CHO was prepared
Conclusions
from 5,10,15,20-tetraphenylporphyrinatocopper(II), N,N0-dime-
New neutral porphyrin–terpyridine derivatives were efficiently thylformamide (DMF) and phosphorus oxychloride (POCl3),
prepared by reaction of 2-formyl-5,10,15,20-porphyrin with the according to literature procedure.36
appropriate acetylpyridine in the presence of ammonium
Synthesis of the beta functionalized meso-tetraarylporphyr-
acetate catalysed by La(OTf)3. Secondary compounds were also ins 1–5: general procedure. To a solution of the appropriate
isolated, the respective benzoporphyrins and porphyrin chal- acetylpyridine (5.0 or 12.0 equiv.) in dry toluene (1 mL) was
cones type derivatives. The studied conditions allowed also to added ammonium acetate (8.0 or 14 equiv.) and the mixture was
isolate for the rst time a 2-(2,4-terpyridin-6-yl)-porphyrin stirred for 30 min at room temperature. Aer this time, 2-
derivative. Two of the pyridine moieties in the terpyridine formyl-5,10,15,20-tetraphenylporphyrin
1 and La(OTf)3 (20
derivatives 1 were efficiently quaternised with methyl or ethyl mol%) were added to the mixture and it was heated at reux in
iodide leading, in general, to the formation of the respective the range of 3–6 h. Aer cooling, the reaction mixture was
dicationic derivatives.
washed with water and extracted with dichloromethane. The
A detailed characterization on gas phase of the neutral and organic phase was dried (Na2SO4) and the solvent was evapo-
cationic porphyrin–terpyridine derivatives using electrospray rated under reduced pressure. The crude mixture was
mass spectrometry demonstrated that ESI-MS is a useful and submitted to column chromatography (silica gel) using CH2Cl2
efficient technique to differentiate the alkylated isomers due to as eluent. The fractions obtained were full characterized by
the formation of different diagnostic ions.
The ability of the methylated terpyridine derivatives 6 to and yields are summarized in Table 1.
generate singlet oxygen was evaluated and conrmed their The characterization of compounds 1a, 2a and 3a were per-
NMR, mass and UV-Vis techniques. The reactional conditions
1
potential to be used as PSs. The photoinactivation of biolumi- formed by UV-vis, H NMR and mass spectrometry and all the
nescent E. coli in the presence of these photosensitisers showed experimental data are in agreement with the described litera-
that the PS concentration, oxygen singlet efficiency, the distri- ture data.35
bution of the charge in the porphyrin core, light irradiance are
important parameters to be taken into consideration.
2-[3,20:6,300-Terpyridin-4-yl]-5,10,15,20-tetraphenylporphyrin,
1b. 1H NMR (500 MHz, CDCl3): d 9.31 (2H, d, J ¼ 1.7 Hz, H-200),
8.91 and 8.89 (2H, AB, J ¼ 4.8 Hz, H-b), 8.85–8.83 (4H, m, H-b),
8.80 (1H, d, J ¼ 4.7 Hz, H-b), 8.71 (2H, dd, J ¼ 1.7 and 4.8 Hz, H-
600), 8.45 (2H, dt, J ¼ 1.7 and 8.1 Hz, H-400), 8.28–8.23 (6H, m, H-o-
Ph), 8.02–8.01 (2H, m, H-o-Ph), 7.81–7.75 (9H, m, H-m,p-Ph),
Experimental section
General remarks
1H and 13C solution NMR spectra were recorded on Bruker 7.73 (2H, s, H-30 and H-50), 7.47 (2H, ddd, J ¼ 0.6, 4.8 and 8.1 Hz,
Avance 300 (300.13 and 75.47 MHz, respectively), 500 (500.13 H-500), 7.17–7.14 (3H, m, H-m,p-Ph), ꢀ2.61 (2H, s, NH) ppm. 13C
and 125.76 MHz, respectively) and 700 (700.13 MHz) spec- NMR (125 MHz, CDCl3): d 153.4 (C-300), 149.9 (C-600), 149.5, 148.4
trometers. CDCl3, CD3OD and DMSO-d6 were used as solvent (C-200), 142.2, 142.0, 141.7, 140.6, 138.9, 135.7, 134.7, 134.6 (C-
and tetramethylsilane (TMS) as internal reference; the chemical 400), 134.5, 132.9–130.0 (C-b), 128.1, 128.0, 127.9, 126.9, 126.8,
shis are expressed in d (ppm) and the coupling constants (J) in 126.2, 123.6 (C-500), 121.26 (C-30 and C-50), 120.70, 120.66, 120.4,
Hertz (Hz).
120.3 ppm. UV-Vis (DMF): lmax (log 3) 420 (5.43), 516 (4.21), 552
Unequivocal 1H assignments were made using 2D COSY (3.80), 595 (3.63), 648 (3.55) nm. HRMS-ESI(+): m/z calcd for
(1H/1H), while 13C assignments were made on the basis of 2D
C
59H40N7 846.33397 [M + H]+; 846.33460 found.
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2-[4,20:6,400-Terpyridin-4-yl]-5,10,15,20-tetraphenylporphyrin,
RSC Advances
526 (4.24), 566 (3.97), 605 (3.86), 666 (2.86) nm. HRMS-ESI(+): m/
z calcd for C52H36N5O [M + H]+ 746.29144; 746.29051 found.
2-[3-Oxo-3-(pyridin-4-yl)prop-1-en-1-yl]-5,10,15,20-
1c. 1H NMR (500 MHz, CDCl3): d 8.92 and 8.89 (2H, AB system, J
¼ 4.8 Hz, H-b), 8.85–8.83 (4H, m, H-b), 8.80–8.78 (5H, m, H-b, H-
200 and H-600), 8.27–8.22 (6H, m, H-o-Ph), 8.02 (4H, dd, J ¼ 1.6
and 4.6, H-300 and H-500), 8.00–7.97 (2H, m, H-o-Ph), 7.81 (2H, s,
H-30 and H-50), 7.80–7.74 (9H, m, H-m,p-Ph), 7.11–7.10 (3H, m,
H-m,p-Ph), ꢀ2.63 (2H, s, NH) ppm. 13C NMR (125 MHz, CDCl3):
d 153.3 (C-20), 150.7, 150.5 (C-200 and C-600), 149.8, 146.2 (C-400),
142.1, 142.0, 141.7, 140.5, 135.7 (C-300 and C-500), 134.6, 134.5,
133.1–130.0 (C-b), 128.1, 128.0, 127.9, 126.9, 126.8, 126.2, 122.4,
121.2 (C-30 and C-50), 120.8, 120.6, 120.5, 120.4 ppm. UV-Vis
(DMF): lmax (log 3) 421 (5.38), 516 (4.18), 552 (3.69), 592 (3.46),
651 (3.37) nm. HRMS-ESI(+): m/z calcd for C59H40N7 846.33397
[M + H]+; 846.33450 found.
tetraphenylporphyrin, 3c. 1H NMR (300 MHz, CDCl3): d 9.10
(1H, s, H-3), 8.86–8.77 (7H, m, H-b, H-200 and H-600), 8.73 (1H, d, J
¼ 4.9 Hz, H-b), 8.25–8.17 (6H, m, H-o-Ph), 8.06–8.03 (2H, m, H-o-
Ph), 7.84–7.72 (9H, m, H-m,p-Ph), 7.61–7.59 (2H, m, H-300 and H-
500), 7.55–7.53 (3H, m, H-m,p-Ph), 7.42 and 7.37 (2H, AB system, J
¼ 15.7 Hz, H-10 and H-20), ꢀ2.56 (2H, s, NH) ppm. 13C NMR (125
MHz, CDCl3): d 192.0 (C]O), 151.0, 150.6, 147.8, 147.2, 146.8,
146.5, 145.8, 144.9, 143.6, 142.1, 141.9, 141.6, 141.4, 139.2,
138.9, 134.6, 134.1, 132.7–130.1 (C-b), 129.0, 128.9, 128.8,
128.64, 128.57, 128.4, 128.1, 127.9, 127.1, 126.9, 126.8, 124.4,
122.1, 121.7, 1212, 120.9, 120.5, 120.4, 120.2 ppm. UV-Vis
22-(Pyridin-3-yl)-5,10,15,20-tetraphenylbenzo[b]porphyrin, 2b.
1H NMR (700 MHz, CDCl3): d 9.04 (1H, d, J ¼ 1.4 Hz, H-200), 8.93
(1H, d, J ¼ 4.9 Hz, H-b), 8.91 (1H, d, J ¼ 4.9 Hz, H-b), 8.85 (1H, d,
J ¼ 4.9, H-b), 8.84 (1H, d, J ¼ 4.9 Hz, H-b), 8.74 (2H, AB system, J
¼ 4.9 Hz, H-b), 8.64 (1H, dd, J ¼ 1.4 and 4.6 Hz, H-600), 8.24–8.21
(8H, m, H-o-Ph), 8.03 (1H, dt, J ¼ 1.4 and 7.9 Hz, H-400), 7.95–7.92
(2H, m, H-m,p-Ph), 7.86–7.75 (12H, m, H-m,p-Ph, H-10 and H-30),
7.35 (1H, ddd, J ¼ 0.6, 4.6 and 7.9 Hz, H-500), 7.29 (1H, d, J ¼
8.1 Hz, H-40), ꢀ2.65 (2H, s, NH) ppm. 13C NMR (125 MHz,
CDCl3): d 160.1 (C-300), 154.8, 151.7 (C-200), 149.7, 148.6, 147.9,
147.0, 143.1, 142.2, 142.0, 141.9, 140.2, 138.9, 138.0, 135.0,
134.8, 134.5, 134.1, 134.0, 133.9, 133.6, 132.6, 128.7, 128.3,
128.0, 127.9, 127.8, 127.7, 127.3, 127.0, 126.8, 126.8, 123.3,
121.3, 121.2, 117.8, 117.7, 116.7 ppm. UV-Vis (DMF): lmax (log 3)
425 (5.46), 517 (4.27), 593 (3.75) nm. HRMS-ESI(+): m/z calcd for
(DMF): lmax (log 3) 434 (4.89), 526 (3.97), 570 (3.62), 604 (3.52),
´
665 (1.95) nm. HRMS-ESI(+): m/z calcd for C52H36N5O [M + H]+
746.29144; 746.29031 found.
2-[2,20:4,200-Terpyridin-6-yl]-5,10,15,20-tetraphenylporphyrin, 4.
1H NMR (700 MHz, CDCl3): d 9.05 (1H, s, H-3), 8.87–8.82 (5H, m,
H-b), 8.76–8.73 (4H, m, H-b, H-600, H-6000 and H-30), 8.37 (1H, d, J
¼ 8.1 Hz, H-3000), 8.26 (2H, dd, J ¼ 1.6 and 7.6 Hz, H-o-Ph), 8.24–
8.20 (4H, m, H-o-Ph), 8.09 (1H, d, J ¼ 1.5 Hz, H-50), 7.98 (2H, d, J
¼ 6.0 Hz, H-o-Ph), 7.95 (1H, dt, J ¼ 1.0 and 7.7 Hz, H-300), 8.40
(1H, dt, J ¼ 1.8 and 7.8 Hz, H-400), 7.78–7.70 (10H, m, H-m,p-Ph
and H-4000), 7.34 (1H, ddd, J ¼ 1.1, 4.7 and 7.5 Hz, H-5000), 7.31
(1H, ddd, J ¼ 1.0, 4.9 and 7.7 Hz, H-500), 7.04–7.02 (1H, m, H-m-
Ph), 6.97 (2H, t, J ¼ 7.8 Hz, H-m-Ph), ꢀ2.58 (2H, s, NH) ppm. 13
C
NMR (75 MHz, CDCl3): d 157.9, 156.1, 155.7, 155.0, 149.9, 149.0
(C-600 and C-6000), 146.4, 142.34, 142.26, 141.9, 140.7, 136.8 (C-400),
136.6 (C-400), 135.7, 134.6 (C-3), 132.2–130.1 (C-b), 127.7, 127.2,
126.8, 126.64, 126.61, 125.8 (C-b), 123.5 (C-50), 121.8 (C-3000),
121.3, 121.2 (C-300), 120.4, 120.2, 120.0, 115.6 (C-30) ppm. UV-Vis
(DMF): lmax (log 3) 420 (5.46), 517 (4.18), 552 (3.72), 595 (3.62),
650 (3.52) nm. HRMS-ESI(+): m/z calcd for C59H40N7 846.33397
[M + H]+; 846.33411 found.
C
53H37N5 743.30490 [M + 2H]+c; 743.29159 found.
22-(Pyridin-4-yl)-5,10,15,20-tetraphenylbenzo[b]porphyrin, 2c.
1H NMR (700 MHz, CDCl3): d 8.94 (1H, d, J ¼ 4.9 Hz, H-b), 8.92
(1H, d, J ¼ 4.9 Hz, H-b), 8.86 (1H, d, J ¼ 4.9 Hz, H-b), 8.84 (1H, d,
J ¼ 4.9 Hz, H-b), 8.74 (2H, AB system, J ¼ 4.9 Hz, H-b), 8.67–8.66
(2H, m, H-200 and H-600), 8.25–8.21 (8H, m, H-o-Ph), 7.95–7.90
(2H, m, H-m,p-Ph), 7.86–7.53 (12H, m, H-m,p-Ph, H-10 and H-30),
7.68–7.66 (2H, m, H-300 and H-500), 7.31 (1H, d, J ¼ 8.2 Hz, H-40),
ꢀ2.67 (2H, s, NH) ppm. 13C NMR (125 MHz, CDCl3): d 160.0,
154.9, 151.4, 150.1, 146.4, 143.1, 142.2, 141.9, 141.8, 138.9,
138.1, 134.5, 134.1, 133.6, 132.6, 128.7, 128.4, 128.1, 127.9,
127.8, 127.3, 127.1, 126.84, 126.80, 121.4, 121.3, 117.9, 117.8,
117.1 ppm. UV-Vis (DMF): lmax (log 3) 426 (4.84), 517 (4.22), 592
(3.66) nm. HRMS-ESI(+): m/z calcd for C53H37N5 743.30490 [M +
2H]+c; 743.29054 found.
2-[3-Oxo-3-(pyridin-3-yl)propan-1-yl]-5,10,15,20-tetraphenyl-
porphyrin, 5b. 1H NMR (300 MHz, CDCl3): d 9.01 (1H, d, J ¼
1.6 Hz, H-200), 8.78 (2H, AB system, J ¼ 4.9 Hz, H-b), 8.74 (1H, d, J
¼ 4.9 Hz, H-b), 8.69–8.67 (3H, m, H-b), 8.56–8.53 (2H, m, H-
b and H-3), 8.14–8.04 (9H, m, H-o-Ph and H-400), 7.69–7.56 (m,
12H, H-m,p-Ph), 7.30 (1H, dd, J ¼ 4.8 and 8.0, Hz, H-500), 3.42–
3.36 (2H, m, H-20), 3.31–3.26 (2H, m, H-10), ꢀ2.84 (2H, s,
NH) ppm. 13C NMR (125 MHz, CDCl3): d 197.0, 164.9, 153.4,
149.6, 149.3, 146.8, 146.5, 146.2, 146.0, 145.9, 145.7, 145.1,
144.3, 142.6, 142.3, 142.0, 141.9, 139.8, 139.4, 139.1, 138.9,
138.8, 138.5, 135.3, 135.2, 134.6, 134.52, 134.46, 133.3, 132.2,
131.5, 130.4, 130.1, 129.98, 129.93, 128.7, 128.4, 128.3, 127.9,
127.8, 127.7, 127.6, 127.1, 126.8, 126.7, 126.6, 123.6, 123.4,
122.6, 122.5, 121.8, 121.2, 120.6, 120.2, 119.6, 40.4 (C-20), 39.6
(C-10) ppm. UV-Vis (DMF): lmax (log 3) 417 (5.49), 513 (4.19), 546
(3.69), 588 (3.63), 644 (3.49) nm. HRMS-ESI(+): m/z calcd for
2-[3-Oxo-3-(pyridin-3-yl)prop-1-en-1-yl]-5,10,15,20-tetraphenyl-
porphyrin, 3b. 1H NMR (300 MHz, CDCl3): d 9.09–9.07 (2H, m, H-
3 and H-200), 8.85–8.75 (7H, m, H-b and H-600), 8.26–8.24 (2H, m,
H-o-Ph), 8.21–8.19 (4H, m, H-o-Ph), 8.14 (1H, dt, J ¼ 1.9 and
7.9 Hz, H-400), 8.08–8.05 (2H, m, H-o-Ph), 7.84–7.72 (9H, m, H-
m,p-Ph), 7.59–7.57 (3H, m, H-m,p-Ph), 7.49–7.44 (3H, m, H-500,
H-10 and H-20), ꢀ2.56 (2H, s, NH) ppm. 13C NMR (125 MHz,
CDCl3): d 190.8 (C]O), 152.6, 149.8, 142.6, 142.1, 141.9, 141.7,
141.5, 136.1, 134.6, 134.55, 134.1, 133.9, 132.5–130.2 (C-b),
128.5, 128.1, 127.9, 127.1, 126.9, 129.8, 124.4, 123.5, 120.8,
120.5, 120.4, 120.3 ppm. UV-Vis (DMF): lmax (log 3) 437 (4.71),
C
52H38N5O 748.30709 [M + H]+; 748.30761 found.
2-[3-Oxo-3-(pyridin-4-yl)propan-1-yl]-5,10,15,20-tetraphenylpor-
phyrin, 5c. 1H NMR (300 MHz, CDCl3): d 8.84 (2H, AB system, J ¼
5.0 Hz, H-b), 8.81 (1H, d, J ¼ 4.9 Hz, H-b), 8.77–8.75 (4H, m, H-b,
H-200 and H-600), 8.62 (1H, d, J ¼ 4.9 Hz, H-b), 8.57 (1H, s, H-3),
8.22–8.19 (4H, m, H-o-Ph), 8.16–8.10 (4H, m, H-o-Ph), 7.76–
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766 (12H, m, H-m,p-Ph), 7.62 (2H, d, J ¼ 6.2 Hz, H-300 and H-500), H-300 and H-500), 8.95–8.80 (7H, m, H-b), 8.66 (2H, s, H-30and H-
3.44–3.39 (2H, m, H-20), 3.36–3.31 (2H, m, H-10), ꢀ2.77 (2H, s, 50), 8.27–8.05 (8H, m, H-o-Ph), 7.86–7.81 (9H, m, H-m,p-Ph),
NH) ppm. 13C NMR (125 MHz, CDCl3): d 197.6, 150.9, 150.7, 7.10–7.00 (3H, m, H-m,p-Ph), 4.45 (6H, s, N-CH3), ꢀ2.75 (2H, s,
146.8, 146.5, 146.3, 145.96, 145.9, 145.7, 145.1, 144.4, 142.8, NH) ppm. 13C NMR (125 MHz, DMSO-d6): d 162.3, 151.5, 149.4,
142.5, 142.3, 142.0, 141.9, 139.74, 139.69, 139.4, 139.1, 138.9, 146.1, 145.3, 141.2, 141.1, 140.8, 139.7, 135.8, 134.3, 134.2,
138.8, 138.2, 134.6, 134.5, 134.4, 133.3, 131.0, 130.4, 130.1, 132.8–129.8 (C-b), 127.2, 127.1, 126.2, 126.1, 124.5, 120.8, 120.5,
130.01, 129.96, 128.9, 128.7, 128.0, 128.3, 127.9, 127.83, 127.75, 120.3, 47.6 (CH3) ppm. UV-Vis (DMF): lmax (log 3) 423 (4.89), 518
´
127.68, 127.1, 126.8, 126.7, 126.6, 122.7, 122.5, 121.93, 121.85, (3.52), 554 (3.40), 597 (3.30), 649 (3.18) nm. HRMS-ESI(+): m/z
121.1, 121.0, 120.8, 120.7, 120.2, 119.6, 118.9, 40.5 (C-20), 39.8 for C61H45N7 calcd for 437.68684 [M2+]; 437.68495 found.
(C-10) ppm. UV-Vis (DMF): lmax (log 3) 417 (5.28), 513 (3.94), 547
Ethylation of porphyrin–pyridine derivatives 1b, c: general
(3.25), 586 (3.14), 646 (2.66) nm. HRMS-ESI(+): m/z calcd for procedure. To a solution of each porphyrin–pyridine derivatives
C
52H38N5O 748.30709 [M + H]+; 748.30705 found.
Methylation of porphyrin-pyridine derivatives 1a–c: general added an excess of ethyl iodide (60 equiv., 0.1 mL). The mixture
1 (20.0 mg, 2.7 ꢄ 10ꢀ5 mol) in DMF (1 mL) in a sealed tube was
ꢁ
procedure. To a solution of each porphyrin–pyridine derivatives was stirred for 24 h at 40 C. Aer cooling, the crude mixture
1a–c (20.0 mg, 2.7 ꢄ 10ꢀ5 mol) in DMF (1 mL) in a sealed tube was precipitated with diethyl ether, ltered, washed with diethyl
was added an excess of methyl iodide (60 equiv., 0.1 mL). The ether, dissolved in a mixture CH2Cl2/CH3OH (9 : 1) and the
ꢁ
mixture was stirred for 24 h at 40 C. Aer cooling, the crude solvent was evaporated under reduced pressure. Compounds 7b
mixture was precipitated with diethyl ether, ltered, washed and 7c were obtained without further purication by crystal-
with diethyl ether, dissolved in a mixture CH2Cl2/CH3OH (9 : 1) lisation from CH2Cl2/hexane in 96% and 95%, respectively. The
and the solvent was evaporated under reduced pressure. compounds obtained were fully characterised by NMR, mass
Compounds 6a–c were obtained without further purication by and UV-Vis techniques.
crystallisation from CH2Cl2/hexane in 87%, 96% and 97%,
2-[3,20:6,300-(10,100-Di-ethyl)terpyridinium-4-yl]-5,10,15,20-tetra-
respectively. The compounds obtained were fully characterised phenylporphyrin di-iodide, 7b. 1H NMR (300 MHz, DMSO-d6):
by NMR, mass and UV-Vis techniques.
d 9.91 (2H, s, H-200), 9.51 (2H, d, J ¼ 8.3 Hz, H-600), 9.26 (2H, d, J ¼
2-[2,20:6,200-(10,100-Dimethyl)terpyridinium-4-yl]-5,10,15,20-tetra- 6.1 Hz, H-400), 8.95–8.80 (7H, m, H-b), 8.45 (2H, s, H-30 and H-50),
phenylporphyrin di-iodide, 6a. 1H NMR (500 MHz, CDCl3/ 8.39 (2H, dd, J ¼ 6.1 and 8.3 Hz, H-500), 8.27–808 (8H, m, H-o-Ph),
CD3OD): d 9.33 (2H, d, J ¼ 5.9 Hz, H-600), 9.04 (1H, s, H-3), 8.90 7.88–7.83 (9H, m, H-m,p-Ph), 7.16–7.09 (3H, m, H-m,p-Ph), 4.82
(2H, AB system, J ¼ 4.9 Hz, H-b), 8.83 (1H, d, J ¼ 4.9 Hz, H-b), (4H, q, J ¼ 7.2 Hz, CH2CH3), 1.67 (6H, t, J ¼ 7.2 Hz, CH2CH3),
8.79 and 8.77 (2H, AB system, J ¼ 4.9 Hz, H-b), 8.75 (1H, d, J ¼ ꢀ2.74 (2H, s, NH). ppm. 13C NMR (125 MHz, DMSO-d6): d 151.9,
4.9 Hz, H-b), 8.63 (2H, t, J ¼ 7.9 Hz, H-400), 8.26–8.24 (4H, m, H-o- 149.5, 145.1, 141.2, 141.1, 140.8, 139.7, 135.9, 134.30, 134.26,
Ph and H-300) 8.21–8.17 (6H, m, H-o-Ph), 8.13 (2H, s, H-30 and H- 134.20, 133.2–130.2 (C-b), 128.3, 128.0, 127.2, 127.1, 126.3,
50), 7.78–7.71 (9H, m, H-m,p-Ph), 7.49–7.47 (2H, m, H-500), 7.31– 126.1, 125.0, 120.8, 120.5, 120.3, 56.0 (CH2CH3), 16.4 (CH2-
7.29 (3H, m, H-m,p-Ph), 4.58 (6H, s, N-CH3) ppm. 13C NMR (125 CH3) ppm. UV-Vis (DMF): lmax (log 3) 423 (4.72); 518 (3.81); 552
´
MHz, CDCl3/CD3OD): d 152.7, 151.1, 148.5, 148.3, 146.2, 141.6, (3.39); 595 (3.25); 651 (3.18) nm. HRMS-ESI(+): m/z for C63H49N7
141.4, 141.3, 137.1, 134.9, 134.8, 134.7, 133.9–133.3 (C-b), calcd for 451.70249 [M2+]; 451.70106 found.
131.84, 131.80, 130.6, 129.9, 129.3, 129.0, 128.9, 128.6, 128.3,
2-[4,20:6,400-(10,100-Di-ethyl)terpyridinium-4-yl]-5,10,15,20-tetra-
128.1, 127.2, 127.1, 127.0, 121.4, 120.9, 119.7, 50.2 ppm. UV-Vis phenylporphyrin di-iodide, 7c. 1H NMR (300 MHz, DMSO-d6):
(DMF): lmax (log 3) 422 (4.95), 518 (3.48), 553 (3.37), 595 (3.31), d 9.29 (4H, d, J ¼ 6.8 Hz, H-200 and H-600), 9.04 (H, d, J ¼ 6.8 Hz,
653 (3.19) nm. HRMS-ESI(+): m/z C61H45N7 calcd for 437.68684 H-300 and H-500), 8.95–8.80 (7H, m, H-b), 8.68 (2H, s, H-30 and H-
[M2+]; 437.68569 found.
50), 8.026–8.06 (8H, m, H-o-Ph), 7.88–7.83 (9H, m, H-m,p-Ph),
2-[3,20:6,300-(10,100-Dimethyl)terpyridinium-4-yl]-5,10,15,20-tetra- 7.11–7.04 (3H, m, H-m,p-Ph), 4.74 (4H, q, J ¼ 7.2 Hz, CH2CH3),
phenylporphyrin di-iodide, 6b. 1H NMR (300 MHz, DMSO-d6): 1.65 (6H, t, J ¼ 7.2 Hz, CH2CH3), ꢀ2.74 (2H, s, NH) ppm. 13C
d 9.89 (2H, s, H-200), 9.46 (2H, d, J ¼ 8.1 Hz, H-600), 9.13 (2H, d, J ¼ NMR (125 MHz, DMSO-d6): d 150.0, 149.1, 144.6, 143.1, 142.7,
6.0 Hz, H-400), 8.94–8.74 (7H, m, H-b), 8.42 (2H, s, H-30and H-50), 141.2, 141.1, 140.8, 139.7, 137.5, 135.9, 134.30, 134.28, 134.17,
8.36–802 (10H, m, H-o-Ph and H-500), 7.87–7.79 (9H, m, H-m,p- 133.3–130.3 (C-b), 128.5, 128.4, 128.3, 128.2, 128.1, 127.2, 127.1,
Ph), 7.18–7.07 (3H, m, H-m,p-Ph), 4.53 (6H, s, N-CH3), ꢀ2.74 126.1, 123.7, 120.9, 120.6, 120.33, 120.26, 57.0 (CH2CH3), 16.6
(2H, s, NH) ppm. 13C NMR (125 MHz, DMSO-d6): 162.3, 149.0, (CH2CH3) ppm. UV-Vis (DMF): lmax (log 3) 423 (4.88); 519 (3.63);
´
148.8, 145.6, 144.0, 142.2, 141.2, 141.1, 140.7, 139.6, 137.1, 554 (3.28); 596 (3.11); 652 (3.05) nm. HRMS-ESI(+): m/z for
137.0, 135.8, 134.3, 134.2, 134.1, 132.0, 128.3, 128.2, 128.1,
127.8, 127.7, 127.6, 127.2, 127.1, 126.7, 126.1, 125.6, 123.6,
123.5, 120.9, 120.50, 120.45, 120.3, 120.2, 44.2 ppm. UV-Vis
(DMF): lmax (log 3) 423 (4.89), 518 (3.50), 554 (3.04), 597 (3.22),
C
63H49N7 calcd for 451.70249 [M2+]; 451.70119 found.
Electrospray ionization mass studies
649 (3.07) nm. HRMS-ESI(+): m/z for C61H45N7 calcd for Electrospray ionization mass spectra were acquired with
437.68694 [M2+]; 437.68507 found.
a Micromass Q-Tof 2 (Micromass, Manchester, UK), operating
2-[4,20:6,400-(10,100-Dimethyl)terpyridinium-4-yl]-5,10,15,20-tetra- in the positive ion mode, equipped with a Z-spray source, an
phenylporphyrin di-iodide, 6c. 1H NMR (300 MHz, DMSO-d6): electrospray probe and a syringe pump. Source and desolvation
d 9.16 (4H, d, J ¼ 6.8 Hz, H-200 and H-600), 9.01 (4H, d, J ¼ 6.8 Hz, temperatures were 80 ꢁC and 150 ꢁC, respectively. Capillary
110682 | RSC Adv., 2016, 6, 110674–110685
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voltage was 3000 V. The spectra were acquired at a nominal
a
Power Meter Coherent FieldMaxII-Top combined with
resolution of 9000 and at cone voltages of 30 V. Nebulisation a Coherent PowerSens PS19Q energy sensor.
and collision gases were N2 and Ar, respectively. Porphyrin
Bacterial strains and growth conditions. Bioluminescent
solutions in methanol were introduced at a 10 mL minꢀ1 ow E. coli was grown on tryptic soy agar (TSA, Merck) supplemented
rate.
with 50 mg mLꢀ1 of ampicillin (Amp) and with 45 mg mLꢀ1 of
ꢁ
chloramphenicol (Cm), and stored at ꢀ80 C in 10% glycerol.
Before each assay, one isolated colony was aseptically trans-
ferred to 30 mL of triptic soy broth (TSB, Merck) medium
previously added with both antibiotics (150 mL Amp/100 mL TSB
Theoretical calculations
All calculations were performed with Gaussian09 using Density
Functional Theory with the B3LYP functional and the 6-
311G(d,p) basis set. Default optimization parameters were used
throughout the calculations. Vibrational analysis was per-
formed to assess the nature of the optimized structures and to
determine the zero-point energy corrections.
ꢁ
and 60 mL Cm/100 mL TSB) and was grown overnight at 25 C
under stirring (120 rpm). Aerwards, an aliquot was transferred
into fresh TSB under the same growth conditions to reach
stationary growth phase. An E. coli, an optical density at 600 nm
(OD600) of 1.6 ꢃ 0.1 corresponded to z108 colony forming units
(CFU) per mL.
Bioluminescence versus colony forming units. The correla-
tion between CFUs and the bioluminescent signal (in RLUs) of
bioluminescent E. coli strain was evaluated. Therefore, the
bacteria were grown under the aforementioned conditions.
Fresh overnight bacterial culture was serially diluted (10ꢀ1 to
10ꢀ5) in PBS. Non-diluted (10ꢁ) and diluted aliquots were pour-
plated on TSA medium (0.5 mL) and, simultaneously, were read
on a luminometer (0.5 mL) (TD-20/20 Luminometer, Turner
Designs, Inc., Madison, WI, USA) to determine the biolumi-
nescence signal.
Photosensitization procedure. Bacterial culture grown over-
night and tenfold diluted in phosphate buffered saline (PBS)
was equally distributed in sterilized and acid-washed beakers.
Aerwards, the appropriate volumes of each cationic porphyrin
(6a, 6b and 6c) were added to achieve a nal desired concen-
tration (total volume in the beakers was 15 mL per beaker). The
samples were protected from light with aluminium foil and
remained in the dark for 10 min to promote the porphyrin
binding to E. coli cells.
Light and dark controls were also carried out simultaneously
with the PDI procedure: the light control (LC) comprised
a bacterial suspension exposed to the same light protocol; and
the dark control (DC) comprised a bacterial suspension incu-
bated with PS at the maximum studied concentration but pro-
tected from light. Three independent experiments were
performed and, for each, two replicates were plated. Both
All geometry optimized structures show positive vibration
frequencies.
Singlet oxygen generation
Stock solution of each porphyrin derivative at 0.1 mM in DMF
and a stock solution of 1,3-diphenylisobenzofuran (DPiBF) at
10 mM in DMF were prepared. Aliquots of 2 mL of a solution of
each porphyrin (0.5 mmol dmꢀ3) and 1,3-diphenylisobenzofuran
(DPiBF, 50 mmol dmꢀ3) in DMF/H2O (9 : 1) were irradiated at an
irradiance of 4.0 mW cmꢀ2, in a glass cuvette, at room
temperature and under gentle magnetic stirring, with a home-
made red LED array in order to prevent the photodegradation of
DPiBF. The LED array is composed of a matrix of 5 ꢄ 5 LED that
makes a total of 25 light sources with an emission peak at
630 nm and a bandwidth at half maximum of ꢃ 20 nm. The
absorption decay of DPiBF at 415 nm was measured at irradi-
ation intervals up to 10 min. The production of singlet oxygen
was evaluated qualitatively through the DPiBF, a singlet oxygen
(1O2) quencher, due to the fact that DPiBF decays in a rst order
manner during continuous irradiation in a photosensitised
experiments. The irradiation of the PS in the presence of dis-
solved oxygen will result in the formation of 1O2, which is
trapped by DPiBF resulting in colourless o-dibenzoylbenzene,
1
aer the Diels–Alder like reaction with O2.50
Biologic evaluation studies
Photosensitizer stock solutions. Stock solutions of each controls were carried out during each experiment.
porphyrin derivative at 500 mM was prepared in DMSO and
Irradiation conditions. The samples were exposed to light
stored in the dark. Before each assay, the PS solution was under stirring and placed on a tray with clamps and the bottom
sonicated for 30 min at room temperature. of the tray was covered with water in order to maintain the
Light sources. The photodynamic effect of the cationic PS samples at constant temperature (25 ꢁC). For the PAR white
was evaluated by exposing the bacterial suspension in the light irradiation at an irradiance of 4.0 mW cmꢀ2 aliquots of 1.0
presence of PS to a set of uorescent PAR lamps or a halogen mL treated and control samples were collected at time 0 and
lamp. The rst light source is constituted by 13 uorescent aer 30, 90, 120, 180 and 270 min of light exposure and the
lamps OSRAM 21 of 18 W each one, PAR radiation (380–700 nm) bioluminescence signal was measured in the luminometer. For
at an irradiance of 4.0 mW cmꢀ2. The second light source used the white light at an irradiance of 150 mW cmꢀ2 aliquots of 1.0
is comprised by an illumination system (LumaCare®, USA, mL were collected from treated and control samples and the
model LC122) equipped with a halogen 250 W quartz type lamp bioluminescence signal was measured in the luminometer at
and coupled to an interchangeable bre optic probe (400–800 time 0 and aer each 15 min until a total of 210 min. Three
nm). This illumination system was used to irradiate the independent experiments were performed in two replicates and
microcosm's setup with white light (400–800 nm) at an irradi- the results were averaged.
ance of 150 mW cmꢀ2. All the irradiances were measured with
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The compounds 6a–c at concentration of 20 mM were irra- 12 R. Weijer, M. Broekgaarden, M. Kos, R. van Vught,
diated with white light at an irradiance of 4.0 mW cmꢀ2 while
E. A. J. Rauws, E. Breukink, T. M. van Gulik, G. Storm and
compound 6a was also tested at nal concentrations of 5, 10
M. Heger, J. Photochem. Photobiol., C, 2015, 23, 103.
and 40 mM. Compound 6a at 20 mM was irradiated with white 13 L. R. Pereira and F. C. da Silva, J. Med. Microb. Diagn., 2014, 3,
light at an irradiance of 150 mW cmꢀ2 with the Lumacare®
System.
158.
14 F. F. Sperandio, Y.-Y. Huang and M. R. Hamblin, Recent Pat.
Anti-Infect. Drug Discovery, 2013, 8, 108.
15 T. Maisch, R.-M. Szeimies, G. Jori and C. Abels, Photochem.
Photobiol. Sci., 2004, 3, 907.
Acknowledgements
Thanks are due to FCT/MEC for the nancial support to the 16 S. Perni, P. Prokopovich, J. Pratten, I. P. Parkin and
QOPNA research Unit (FCT UID/QUI/00062/2013), the CESAM
M. Wilson, Photochem. Photobiol. Sci., 2011, 10, 712.
(UID/AMB/50017/2013), CICECO-Aveiro Institute of Materials, 17 A. Tavares, C. M. B. Carvalho, M. A. Faustino,
´
´
POCI-01-0145-FEDER-007679 (FCT Ref. UID/CTM/50011/2013),
through national funds and when applicable co-nanced by
the FEDER, within the PT2020 Partnership Agreement and
M. G. P. M. S. Neves, J. P. C. Tome, A. C. Tome,
ˆ
J. A. S. Cavaleiro, A. Cunha, N. C. M. Gomes, E. Alves and
A. Almeida, Mar. Drugs, 2010, 8, 91.
´
´
“Compete” 2020, and also to the Portuguese NMR Network. 18 L. Costa, J. P. C. Tome, M. G. P. M. S. Neves, A. C. Tome,
ˆ
Scientic PROTEOMASS Association (Portugal) and the Asso-
J. A. S. Cavaleiro, M. A. F. Faustino, A. Cunha,
ciate Laboratory for Green Chemistry LAQV (FCT/MEC (UID/
N. C. M. Gomes and A. Almeida, Antiviral Res., 2011, 91, 278.
ˆ
QUI/50006/2013) and PT2020-POCI-01-0145-FEDER-007265) 19 E. Alves, M. A. F. Faustino, M. G. P. M. S. Neves, A. Cunha,
ˆ
´
and the Unidade de Ciencias Biomoleculares Aplicadas-
J. P. C. Tome and A. Almeida, Future Med. Chem., 2014, 6,
UCIBIO (FCT/MEC (UID/Multi/04378/2013) and PT2020-POCI-
141.
01-0145-FEDER-007728) for general funding. Nuno
Moura and Catarina I V Ramos thank FCT for their Post-Doc
M
M
20 R. J. Simmons, R. D. Griffith, L. A. Falto-Aizpurua and
K. Nouri, J. Eur. Acad. Dermatol. Venereol., 2015, 29, 1275.
scholarships SFRH/BPD/84216/2012 and SFRH/BPD/85902/ 21 M. Rajendran, Photodiagn. Photodyn. Ther., 2016, 13, 175.
ˆ
´
2012, respectively. Sergio M Santos thanks FCT for the FCT 22 E. Alves, A. C. Esteves, A. Correia, A. Cunha,
Investigator starting grant IF/00973/2014.The authors wish to
express their appreciation to Dr M Graça O Santana-Marques
M. A. F. Faustino, M. G. P. M. S. Neves and A. Almeida,
Photochem. Photobiol. Sci., 2015, 14, 1169.
(Department of Chemistry, University of Aveiro, Portugal) for 23 D. Mostafa and B. Tarakji, J. Clin. Med. Res., 2015, 7, 393.
all the helpful and valuable suggestions. Finally, we would like 24 A. Srivatsana, J. R. Missertb, S. K. Upadhyayc and
´
to thank MSc Barbara Coelho and graduation students Rafael
R. K. Pandey, J. Porphyrins Phthalocyanines, 2015, 19, 111.
´
Queiros Fernandes and Selesa Vanessa Shabudin for their help 25 M. P. Cormick, M. G. Alvarez, M. Rovera and E. N. Durantini,
in the synthesis of some of the compounds described.
Eur. J. Med. Chem., 2009, 44, 1592.
26 L. Costa, M. A. F. Faustino, J. P. C. Tome,
´
´
M. G. P. M. S. Neves, A. C. Tome, J. A. S. Cavaleiro,
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