Influence of charge and metal coordination of meso-substituted porphyrins on bacterial photoinactivation

Article abstract of DOI:10.1016/j.saa.2014.07.053

The photodynamic effect of meso-substituted porphyrins with different charges and metal ions: meso-tetraphenylporphyrin tetrasulfonate 1, its nickel 2 and zinc complexes 3; meso-tetranaphthylporphyrin tetrasulfonate 4, and its zinc complex Zn 5; and tetra piridyl ethylacetate porphirins 6 and their nickel 7 and zinc 8 complexes, were synthesized and studied their antimicrobial activity against Escherichia coli. Fluorescence quantum yields (Φ_F) were measured in water using reference TPPS₄, obtaining higher values for complexes 3 and 4. The singlet oxygen Φ_Δ were measured using histidine as trapping singlet oxygen and Rose Bengal as a reference standard. Complexes 1, 2 and 6 have the highest quantum yields of singlet oxygen formation, showing no relation with the peripheral charges and efficiency as Type II photosensitizers. Meanwhile complexes 3, 8 and 4 were the most efficient in producing radical species, determined with their reaction with NADH. The photoinduced antibacterial activity of complex was investigated at different concentrations of the photosensitizers with an irradiation time of 30 min. The higher antibacterial activities were obtained for the complexes 1-3 that are those with greater production of ROS and minor structural deformations. Complexes 7 and 8 had moderate activity, while 4-6 a low activity. Thus, in this work demonstrates that the production of ROS and structural deformations due to peripheral substituents and metal coordination, influence the activity of the complexes studied. Therefore, is important to perform comprehensive study physics and structurally when predicting or explain such activity.

Full text of DOI:10.1016/j.saa.2014.07.053

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 135 (2015) 747–756
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Influence of charge and metal coordination of meso-substituted
porphyrins on bacterial photoinactivation
Tamara Zoltan a, , Franklin Vargas , Verónica López , Valery Chávez , Carlos Rivas , Álvaro H. Ramírez
⇑
a
a
b
a
c
a
Laboratorio de Fotoquímica, Centro de Química, Instituto Venezolano de Investigaciones Científicas I.V.I.C., Apartado 20632, Caracas 1020-A, Venezuela
Laboratorio de Bioquímica Celular, Centro de Microbiología y Biología Celular, Instituto Venezolano de Investigaciones Científicas I.V.I.C., Apartado 20632, Caracas 1020-A, Venezuela
Laboratorio de Biología de virus, Centro de Microbiología y Biología Celular, Instituto Venezolano de Investigaciones Científicas I.V.I.C., Apartado 20632, Caracas 1020-A, Venezuela
b
c
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
4
A: The series of TNPS have higher structural deformity, low production of ROS and reduced antibacterial
activity. B: The series of TPPS have a low structural deformity, high production of ROS and the greater
4
antibacterial activity.
ꢀ
ꢀ
ꢀ
ꢀ
Were synthesized and characterized 8
meso substituted porphyrins with
different substituents.
Effect of charges and metal
coordination in their photochemical
characteristics was studied.
We performed a detailed study of
photoinduced antibacterial
structure–activity.
It was obtained a relation among the
structural integrity, ROS production
and antibacterial activity.
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 May 2014
Received in revised form 7 July 2014
Accepted 17 July 2014
Available online 1 August 2014
The photodynamic effect of meso-substituted porphyrins with different charges and metal ions: meso-tet-
raphenylporphyrin tetrasulfonate 1, its nickel 2 and zinc complexes 3; meso-tetranaphthylporphyrin tet-
rasulfonate 4, and its zinc complex Zn 5; and tetra piridyl ethylacetate porphirins 6 and their nickel 7 and
zinc 8 complexes, were synthesized and studied their antimicrobial activity against Escherichia coli. Fluo-
rescence quantum yields (
U
F
) were measured in water using reference TPPS
4
, obtaining higher values for
complexes 3 and 4. The singlet oxygen
U
D
were measured using histidine as trapping singlet oxygen and
Keywords:
Rose Bengal as a reference standard. Complexes 1, 2 and 6 have the highest quantum yields of singlet
oxygen formation, showing no relation with the peripheral charges and efficiency as Type II photosensi-
tizers. Meanwhile complexes 3, 8 and 4 were the most efficient in producing radical species, determined
with their reaction with NADH. The photoinduced antibacterial activity of complex was investigated at
different concentrations of the photosensitizers with an irradiation time of 30 min. The higher antibacte-
rial activities were obtained for the complexes 1–3 that are those with greater production of ROS and
minor structural deformations. Complexes 7 and 8 had moderate activity, while 4–6 a low activity. Thus,
in this work demonstrates that the production of ROS and structural deformations due to peripheral sub-
stituents and metal coordination, influence the activity of the complexes studied. Therefore, is important
to perform comprehensive study physics and structurally when predicting or explain such activity.
Ó 2014 Elsevier B.V. All rights reserved.
Photoinactivation
Water-soluble metallic porphyrins
Reactive oxygen species
Escherichia coli
Metal and charge effect
⇑
Corresponding author.
E-mail address: tzoltan@ivic.gob.ve (T. Zoltan).
http://dx.doi.org/10.1016/j.saa.2014.07.053
386-1425/Ó 2014 Elsevier B.V. All rights reserved.
1

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T. Zoltan et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 135 (2015) 747–756
Introduction
Benzaldehyde, a-naftaldehyde, chloro ethyl acetate, pyrrole,
propionic acid, tetra-pyridyl porphyrin, chloroform, histidine, Rose
Bengal, horseradish peroxidase (HRP), nicotinamide adenine dinu-
cleotide phosphate reduced form (NADPH), and p-nitrosodimethyl
aniline were purchased from Sigma–Aldrich (St. Louis, MO, USA).
3-Aminophthalhydrazide (Luminol) and hydrogen peroxide, 30
wt.% solution in water, were purchased from Aldrich (Milwaukee,
USA). Phosphate buffered saline solution (PBS) pH 7.4 (0.01 mol
Due to the wide variety of pathogens and how fast their
evolutionary changes to become resistant, the field of antimicro-
bial chemotherapy is an area of study in constant development.
Photodynamic therapy (PDT) has been successfully applied in
treating cancer as well as in non-cancerous condition [1,2].
Furthermore, there has also been an increasing interest in the
application of PDT in the treatment of infectious diseases [3–5].
PDT involves the use of non-toxic dyes, which act as photoactive
drugs called photosensitizers (PS) with the visible light of the
proper wavelength to excite the PS. The light raises the PS to an
excited state, which then interacts with oxygen, leading to the for-
mation of reactive oxygen species (ROS) such as singlet oxygen,
resulting in cytotoxicity and direct cell kill [6].
New developments in PDT applications are several approaches,
which innovate in the design of PS is very wide. The chemical
structure of a photosensitizer, charge and hydrophobicity influ-
ences their efficiency of ROS productions and determines how it
interacts with itself and with its environment. Theoretically,
photosensitizers can be properly designed a function of its
chemical structure, but more realistically their effectiveness is
determined as a function of the targets to be confronted [7].
The design of porphyrins as PS with microbial activity PDT has
been extensively studied mainly focused on develop cationic com-
plexes, due to possibility of interaction with the cell membrane
ꢁ1
ꢁ1
l
phosphate buffer and 0.135 mol l NaCl) was prepared daily
before each experiment.
1H NMR and 13C NMR spectra were recorded with a Brucker
Avance 500 and 300 MHz respectively, in chloroform-d with tetra-
4
methylsilane (Me Si) as an internal standard. Chemical shifts (d)
are given in parts per million. Infrared spectroscopy (IR) spectra
were performed using a Nicolet Magna 560 FT-IR spectrometer.
ElectroSpray Ionization mass spectra and MS/MS spectra were
obtained with a Thermo-Finnigan TSQ Quantum Ultra AM spec-
trometer coupled to a HPLC Electrospray (ESI). Elemental analyses
were performed in a Fisions Instrument EA-1108. The samples
were prepared by addition of the compound of interest to water.
The absorption spectra were recorded on a Perkin Elmer Lambda-
35 UV–Vis spectrophotometer. The fluorescence spectra and the
quantum yields were registered in a Perkin Elmer LS-45.
Synthesis of the porphyrins derivatives
[
8,9]. The development of neutral or anionic porphyrins, regardless
Tetraphenyl porphyrins (TPP)
of their efficiency to produce reactive oxygen species, does not
receive great attention because of its low interaction on the
membrane [10,11]. Many of those studies report that the latter
porphyrins have negligible activity against Gram-negative bacteria
such as Escherichia coli [12]. It is important to note that the exper-
imental method, bacterial washes are performed system before
irradiation, thereby eliminating the PS not attached to the bacteria
The synthesis of TPP (Scheme 1) was carried out following the
experimental procedure reported by Alder and coworkers [15]
with some modifications: benzaldehyde (0.67 mol) and pyrrole
(0.65 mol) were added simultaneously to refluxing propionic acid
(200 ml) and the mixture was refluxed for 1 h before being allowed
to cool and stand at room temperature overnight. The product was
filtered off and washed with water and methanol to give purple
ꢁ1
[
13,14]. Taking into account that in large-scale applications, such
crystals. Yield 20% (20.5 g). F.T-I.R (KBr, thin film)
mmax (cm ):
as in wastewater treatment, these washings not performed, the
efficiency of ROS production in the entire system plays an impor-
tant role in bacterial photoinactivation. Thus, evaluating new
photosensitizers on bacterial cultures without pre-irradiation
washes, would allow obtaining information about the system
under real operating conditions. In this sense, in this work the syn-
thesis and photochemical characterization 8 hydrophilic porphy-
rins were performed. The aim of this paper is to examine their
efficiency as PS in cultures of E. coli (no previous washes), and
the impact of structural changes in this efficiency.
3309 (NAH); 3047 (C@C); 2361 (C@N); 1593, 1466 (aromatic
ꢁ5
ꢁ1
C@C); 698 (CAH). UV–vis (1.0 ꢂ 10
mol l
(nm): 419, 515, 550, 590, 674; H NMR (500 MHz, CDCl
d = ppm: 8.87 (s, 8H, H-pyrrolic), 8.21–8.28 (d, J = 5.0 Hz, 8H, o-
3
in CHCl ), kmax
1
3
)
13
phenyl), 7.72–7.85 (m, 12H, H-phenyl), ꢁ2.73 (s, NH). C NMR
(125 MHz, CHCl ) d = ppm: 142.21 (2C-phenyl), 134.6 (CH-o-phe-
3
nyl), 131.15 (CH-pyrrolic), 127.74 (2C-pyrrolic), 126.72 (2CH-phe-
nyl), 120.18 (2C).
Tetranaphthylporphirins (TNP)
In this paper the synthesis of the following water-soluble por-
phyrins is presented: meso-tetraphenylporphyrin (TPP) tetrasulfo-
The synthesis of TNP (Scheme 2) was carried out following the
experimental procedure reported by Regimol and coworkers [16]
with some modifications: benzaldehyde (0.10 mol) and pyrrole
(0.14 mol) were added simultaneously to refluxing propionic acid
(100 ml). The reaction mixture was left at reflux for a ten days,
after which it was allowed to cool to room temperature, and then
it was kept at 5 °C overnight. The product was filtered off and
washed with water and methanol and the solid obtained was puri-
fied by preparative chromatography on silica plates, using chloro-
form as mobile phase, yielding purple crystals. Yield of 19.8%
nate (S
TPPZnS
onate (TNPS
4
): (TPPS
, 3) as well as the meso-tetranaphthylporphyrin tetrasulf-
, 4) its Zn complexes (TNPZnS , 5) and tetra piridyl
4 4
, 1), its nickel (TPPNiS , 2) and zinc complexes
(
4
4
4
ethylacetate porphirins (TPyEtAcP, 6) and their Ni and Zn com-
plexes (TPyEtAcPNi, 7; TPyEtAcPZn, 8). Studied photophysical
properties of absorption, fluorescence emission and the photo-
chemical characteristic of singlet oxygen generation and oxygen
free radicals by synthetized porphyrins are compared in depen-
dence on the chemical structure. Finally, a comparison of the pho-
tochemical properties of the synthesized compounds and its
efficiency in the photoinduced antibacterial activity is performed.
ꢁ1
(5.44 g). F.T-I.R (KBr, thin film)
mmax (cm ): 3432 (NAH), 2357
(C@N), 3048 (C@C), 1568, 1499 (aromatic C@C), 1021 (CAH).
ꢁ
5
ꢁ1
), kmax (nm): 423. 1H NMR
3
UV–vis (1.0 ꢂ 10 mol l in CHCl
500 MHz, CDCl
(
3
) d = ppm: 8.47 (d, 8H, J = 4.5 Hz, H-pyrrolic),
.28–7.73 (m, 28H, H-naphthalene), ꢁ2.76 (s, 2H, NH). MS (APCI)
8
+
Materials and methods
38 4
m/z: 815.22 (M + H) , (cal: M:C60H N = 814.97).
Chemicals
Tetrapiridyl ethylacetate porphirins
TPyEtAcP, 6): The synthesis of water-soluble porphyrin TPyE-
(
All analytical or HPLC grade solvents were obtained from Merck
Darmstadt, Germany) and used without further purification.
tAcP (Scheme 3) was performed according to changes in the meth-
odology reported by Berezin and coworkers [17]: tetrapiridyl
(

T. Zoltan et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 135 (2015) 747–756
749
Scheme 1. Synthesis of tetraphenyl porphyrins (TPP).
Scheme 2. Synthesis of tetranaphthylporphirins (TNP).
Scheme 3. Synthesis of tetrapiridyl ethylacetate porphirins (TPyEtAcP).
1
3
porphirin (0.16 mmol) in 30 mL de CHCl
3
was placed under reflux.
J = 5.50 Hz, CH
3
-Ethyl), ꢁ3.02 (s, NH). C NMR (125 MHz, DMSO-
+
After the reflux was stable an excess of ethyl chloroacetate (6 mL)
was added drop wise. The mixture was left at reflux for 48 h; after
that which it was evaporated to dryness, and the product obtained
d
6
) d = ppm: 14.5 (CH -Ethyl), 60.57 (CH -N ), 62.99 (CH Ethyl),
3 2 2
115.81 (2C-pyrrolic), 129.52 (2CH-pyrrolic), 124.6 (2C piridyl),
+
145.4 (C-N -Piridyl), 148.86 (C-meso-porphyrin), 158.24 (C-meso-
was washed with CHCl
3
to remove starting materials, thus getting
piridyl), 167.06 (C-acetate). MS = m/z: 791.23 (M-2EtAc)+ (cal: M
dark pink crystals. Yield of 69.6% (0.125 g). F.T-I.R (KBr, thin film)
38 8 4
– 2EtAc (C48H N O + H) = 791.31).
ꢁ
1
m
1
max (cm ): 3318, 3090, 1632, 1561, 1502, 1459, 1365, 1285,
ꢁ5
ꢁ1
093, 1000, 716, 657. UV–vis (1.0 ꢂ 10 mol l in H
2
O, pH 7.2),
General procedure of the sulfonation reaction
The synthesis of TPPS , 1 (or TNPS , 4) was carried out following
1
6
kmax (nm): 422, 519, 557, 584, 641. H NMR (500 MHz, DMSO-d )
4
4
d = ppm: 9.66–9.63 (d, 8H, J = 5.0 Hz, H-piridyl), 9.13–9.10 (d, 8H,
J = 5.0 Hz, H-piridyl), 8.29 (s, 4H, H-acetate), 6.13 (s, 2H, H-pyrro-
lic), 4.45–4.41 (c, 8H, J = 5.50 Hz, CH -Ethyl), 1.42–1.40 (t, 12,
2
the general procedure with some modifications, described by Sri-
vastava and coworkers [18] (Scheme 4). Meso-tetraphenylporphine
(
1.6 mmol, 1 g) was mixed with 20 ml of concentrated sulfuric

7
50
T. Zoltan et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 135 (2015) 747–756
Scheme 4. General procedure of the sulfonation reaction.
acid. The mixture was heated at 100 °C, controlling the tempera-
ture for 24 h. Afterward, the solution at room temperature, was fil-
tered through a glass filter funnel and the filtrate was diluted
carefully to 500 mL with water. The dilute green solution was
heated and calcium oxide (CaO) was added slowly with stirring
until the solution changed to a permanent purple color. Calcium
sulfate was filtered off and washed with a minimum quantity of
hot water, which was then combined with the filtrate. Crushed
Dry Ice was added to the filtrate and was filtered. The filtrate
was concentrated to a small volume (about 100 mL) and the pH
of the final warm solution was regulated in 9 with sodium carbon-
ate solution. Calcium carbonate was removed by filtration and
washed with water, which was then combined with the filtrate.
The resulting solution was carried to dryness and the solid dis-
solved in MeOH, filtered to remove excess carbonate formed. The
resulting solution was evaporated and recrystallized with acetone
to get purple crystals highly soluble in water of tetrasodium meso-
sulfonatophenyl porphyrin (TPPZnS
4
3), yield 71.4% (0.764 g). UV–
O, pH 7.2), (kmax nm). 421, 556, 595.
Zinc meso-tetrasulfonatonaphthyl porphyrin (TNPZnS 5) with a
in H O, pH
ꢁ
5
ꢁ1
vis (1.0 ꢂ 10 mol l in H
2
4
ꢁ5
ꢁ1
yield 85.73% (0.578 g). UV–vis (1.0 ꢂ 10
mol l
2
7.2), kmax (nm): 426, 467, 557, 594. Nickel meso-tetrapiridyl ethyl
acetate porphyrin (TPyEtAcPNi 7), yield 54.6% (0.288 g). UV–vis
ꢁ
5
ꢁ1
(1.0 ꢂ 10
mol l
in H
2
O, pH 7.2), kmax (nm): 418, 532, 563.
ꢁ
MS = m/z: 674.09 (M-4EtAc + 2H) , (calc M-4EtAc + 2H: (C40
H
24
N
8
Ni) =
674.15). Zinc meso-tetrapiridyl ethylacetate porphyrin (TPyEtAcPZn 8)
ꢁ5
ꢁ1
yields 54.6% (0.288 g). UV–vis (1.0 ꢂ 10
mol l
2
in H O, pH
+
7.2), kmax (nm): 437, 564 608. MS = m/z: 681.05 (M + H) , (calc:
1
13
24 8
M + H: (C40H N Zn) = 681.15). The NMR ( H and C) and I.R.
spectra showed no significant differences with those obtained in
the compounds without metal.
Irradiation
tetra (p-sulfophenyl) porphine, TPPS
4
(or tetrasodium meso-tetra
All processes of irradiation were carried out using an illumina-
tor LuzChem LZC 4V Photoreactor equipped with 14 lamps with
(
p-sulfonaphthyl) porphine, TNPS
TPPS 1, yield 57.8% (0.946 g). F.T-I.R (KBr, thin film)
cm ): 3360, 3320, 3010, 1940, 1630, 1548, 1390, 1300, 1110,
4
).
ꢁ2
4
m
max
emission in the UV-A (320–400 nm, 3.3 mW cm ) and in the vis-
ible, keeping a distance of 10 cm between the lamp surface and the
solution flask, varying the time periods of exposure at 37 °C under
ꢁ1
(
1
H
(
(
ꢁ1
ꢁ5
ꢁ1
060, 995, 739, 700, 638 cm . UV–vis (1.0 ꢂ 10
O, pH 7.2), kmax (nm): 413, 515, 633, 579 553. H NMR
500 MHz, DMSO-d ) d = ppm: 8.81 (s, 6H, H-pirrolyc), 8.11–8.21
d, 8H, J = 10 Hz H-m-phenyl), 7.8–8.05 (d, 8H, J = 10 Hz H-o-phe-
mol l
in
1
2
2
continuous shaking. The radiation dose was 4.5 J/cm as measured
6
with a model of UVX Digital Radiometer after 1 h of continued
illumination.
1
3
nyl), ꢁ2.97 (s, NH). C NMR (125 MHz, DMSO-d
6
) d = ppm: 148.1
2C-phenyl), 141.7 (CH-o-phenyl), 134.1 (C@N-pyrrolic), 131.7
2C-pyrrolic), 124.6 (CH-m-phenyl), 120.1 (2C). TNPS 4, yield
O, pH 7.2), kmax
(
(
8
(
Fluorescence quantum yields
4
ꢁ5
ꢁ1
5.73% (0.578 g). UV–vis (1.0 ꢂ 10 mol l in H
2
The relative quantum yields of fluorescence for porphyrins
complexes (1–8) were determined at room temperature by the
comparative method using Rose Bengal as standard (at a concen-
nm): 419, 633, 578, 556.
ꢁ
6
ꢁ1
tration of 1 ꢂ 10 mol l in ethanol; fluorescence quantum yield,
General procedure of the metallation reaction
The reaction was carried out as described by Herrman and
coworkers [19] with modifications (Scheme 5): 50 mg of TPPS
TNPS or TPyEtAc, was added to 100 ml of refluxing distilled and
ꢁ1
0
.11) or else with that of quinine bisulfate in 0.05 mol l
2 4
H SO
(
fluorescence quantum yield, 0.55) [20]. Fluorescence quantum
4
,
yields (U
F
) were determined using the Eq. (1) [21]:
4
deionized water, containing 1 g of metal oxide (ZnO or NiO). The
mixture was refluxed for 24 h monitoring the progress of the
reaction until the fusion the four bands Q in two. After that, the
2
FAStd
g
U
F
¼
U
F
ðStdÞ F
ð1Þ
2
Std
Std
Ag
mixture was filtrated on a 0.22
to dryness under reduced pressure dried at 120 °C under vacuum,
obtained the metalloporphyrins. Nickel meso-tetrasulfonatophenyl
l
Millipore filter and evaporated
where F and FStd are, respectively, the areas under the fluorescence
curves of the samples (1–8) and the standard. A and AStd are the
respective absorbance of the sample and of the standard at the exci-
ꢁ5
porphyrin (TPPNiS
4
2), yield 42.3% (0.482 g). UV–vis (1.0 ꢂ 10
tation wavelengths.
g and gStd are the refractive indices of solvents
ꢁ1
mol l in H
2
O, pH 7.2), (kmax nm). 414, 515, 553. Zinc meso-tetra-
used, for the sample and the standard respectively.

T. Zoltan et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 135 (2015) 747–756
751
Scheme 5. General procedure of the metallation reaction.
Singlet oxygen generation
phosphate buffer 0.01 M, pH 7.4) was irradiated with visible light
>420 nm) under oxygen and gentle magnetic stirring for different
(
Singlet oxygen was measured by photosensitized degradation
periods of time up to 30 min (5, 10, 15, 20, 25 and 30) with the
respective controls been protected from light. The concentration
of L-histidine was determined by a colorimetric reaction using
ꢁ5
ꢁ1
of
[
L-histidine with synthetic porphyrins (1–8, 4 ꢂ 10 mol l
)
22,23]. In a typical experiment, a mixture (equal volumes and con-
centrations) of porphyrins complex (1–8) and
L-histidine (in
phosphate buffer, sulfanilic acid, sodium nitrite, sodium carbonate
and ethanol as reagents. The optical density was read on a spectro-
photometer at 440 nm against a blank reagent (a modified Pauly
reaction), and by bleaching of p-nitrosodimethylaniline [19,24].
Table 1
Another ‘‘trap’’ method has been successfully used to detect the
ꢁ
5
ꢁ1
1
Absorption spectra of Porphyrins 1–8, in water (1 ꢂ 10 mol l ).
generated
O
in a variety of samples. This method is based on
2
3
ꢁ1 ꢁ1
ꢁ1
following the consumption of a chemical trap (Furfuryl alcohol,
FFA) that reacts with singlet oxygen. The consumption of FFA
Porphyrins
Soret band (nm) (
e
ꢂ 10 (mol l
)
cm
)
Q bands (nm)
515.53
TPPS
4
(1)
413.32 (129)
was followed by HPLC using a 90:10 H
composition. The wavelength detection used for monitoring FFA
consumption was at 222 nm. Rose Bengal a well known 1
sensitizer, was used as a standard for comparison with synthetic
2 3
0/CH CN mobile phase
5
5
6
52.40
79.58
33.24
O
2
TPPS
TPPS
4
Ni (2)
Zn (3)
413.91 (51)
421.38 (174)
419.22 (107)
515.53
52.87
1
5
porphyrins for O
[24,25].
2
formation, under same conditions of photolysis
4
556.51
95.34
5
TNPS
4
(4)
516.65
Quantum yields of singlet oxygen
5
5
6
56.05
78.06
32.82
Quantum yields of singlet oxygen photogeneration were deter-
mined in air (no oxygen bubbled) using the relative method with
Rose Bengal as reference and histidine as a chemical quencher of
singlet oxygen, from Eq. (2):
TNPS
4
Zn (5)
426.23 (126)
422.94 (125)
556.96
94.79
5
TPyEtAcP (6)
519.18
5
5
6
57.97
84.73
41.03
W
U
D
¼
U
D
ðStdÞ
ð2Þ
Std
W
TPyEtAcPNi (7) 418.08 (77)
532.50
62.80
5
Std
where UD are the singlet oxygen quantum yields for the standard,
Rose Bengal in water (0.79) [26]. W and W are the p-nitrosodime-
thylaniline photobleaching rates in the presence of the porphyrins
complexes and the standard, respectively [27].
Std
TPyEtAcPZn
8)
436.96 (153)
563.97
608.47
(

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T. Zoltan et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 135 (2015) 747–756
ꢁ1
prepared daily in 2 mol l of NaOH and diluted with PBS). The por-
phyrins-induced CL at different concentrations was dispensed after
irradiation in presence of NADPH. The generated CL at 37 °C was
measured continuously for 10 min in
a Luminoskan Ascent
luminometer (ThermoLabsystems, Finland) in a 96-well Thermo
Labsystems Microtiter plate [28–30].
Antibacterial photoactivity
Antibacterial assay was carried out using E. coli (ATCCÒ 8739TM
[
31]) and their proliferation and viability were obtained by chemi-
luminescence using BacTiter-Glo Microbial Cell (Promega, USA).
The compounds used 1–8 were prepared in H O, in a concentration
2
ꢁ4
ꢁ6
ꢁ1
range of 1.0 ꢂ 10 to 5.0 ꢂ 10 mol l . Taking into account that
different bacteria have different amounts of ATP per cell, and val-
ues reported for the ATP level in cells vary considerably. Factors
that affect the ATP content of cells such as growth phase, culture
medium, and the presence of metabolic inhibitors, can influence
the relationship between cell concentration and luminescence.
The antibacterial photoactivity was carried out under irradiation
with an illuminator LuzChem LZC 4V Photoreactor using 14 lamps
with emission in UV-A (320–400 nm with a fluence rate of 3.3 mW
cm , 45.575 Lux seg and light dose of 5.94 J cm ), keeping a
distance of 10 cm between the lamp surface and the solution flask,
varying the time periods of exposure at 37 °C under continuous
shaking.
Fig. 1. Absorption spectra of: meso-tetraphenylporphyrin tetrasulfonate
TPPS , 1), meso-tetranaphthylporphyrin tetrasulfonate ( TNPS , 4), meso-
tetrapiridyl ethylacetate porphirin ( TPyEtAcP, 6).
(
4
4
Detection of others reactive oxygen species
Chemiluminescence (CL) was generated in cell-free systems;
ꢁ2
ꢁ1
ꢁ2
ꢁ
1
2
H O
2
-induced CL (as a blank): H
2 2
O (3.5 mmol l ) was added to
ꢁ1
phosphate buffered saline solution (PBS, 0.01 mol l phosphate
buffer and 0.135 mol l NaCl, pH 7.2) and luminol (250 lmol l ,
ꢁ1
ꢁ1
Fig. 2. Absorption spectra of: (a) meso-tetraphenylporphyrin tetrasulfonate (
tetraphenylporphyrin tetrasulfonate ( TPPZnS , 3); (b) meso-tetranaphthylporphyrin tetrasulfonate (
TNPZnS , 5) and (c) meso-tetrapiridyl ethylacetate porphirin ( TPyEtAcP, 6) Nickel meso-tetrapiridyl ethylacetate porphirin, (
ethylacetate porphirin ( TPyEtAcPZn, 8).
TPPS
4
, 1), Nickel meso-tetraphenylporphyrin tetrasulfonate (
TNPS , 4), Zinc meso-tetranaphthylporphyrin tetrasulfonate
TPyEtAcPNi, 7), Zinc meso-tetrapiridyl
4
TPPNiS , 2), Zinc meso-
4
4
(
4

T. Zoltan et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 135 (2015) 747–756
753
Table 2
Relative fluorescence quantum yields (
U
F
) of synthesized compounds 1–8 in water.
em (nm)
Porphyrins
kexc (nm)
k
U
F
TPPS
TPPS
TPPS
TNPS
TNPS
TPyEtAcP (6)
TPyEtAcPNi (7)
TPyEtAcPZn (8)
4
4
4
(1)
Ni (2)
Zn (3)
(4)
Zn (5)
430.58
433.96
436.08
432.11
444.96
424.66
450.61
434.13
640.75
640.86
605.11
639.87
603.81
657.70
649.23
631.48
0.12a
0.070
0.103
0.07
0.06
0.02
4
4
0.003
0.044
a
Reported [41].
Fig. 4. Generation of peroxidic species determined by chemiluminescence gener-
ates for complexs 1–8. R.L.U. = relative light units. Data are the mean and SEM, (n =
4, p < 0.05 vs. control; analysis of variance).
The BacTiter-Glo. Microbial Cell Viability Assay is a homoge-
neous method to determine the number of viable bacterial cells
in culture based in quantification of ATP present; as it well known
ATP is an indicator of metabolically active cells. The homogeneous
assay procedure involves the addition of a single reagent (BacTiter-
Glo Reagent) directly on bacterial cells in LB Broth medium
followed by the measurement of luminescence. The luminescent
signal is proportional to the amount of ATP present, which in turn
is directly proportional to the number of viable cells in culture.
The recorded luminescence signals (Luminescence (R.L.U: rela-
tive light units) represent the mean of three replicates for each
measurement. The signal-to-noise ratio was calculated:
S:N = mean of signal mean of background/standard deviation of
the background.
Fig. 3. Effect of 30 min irradiation of the compounds 1 to 8 on the bleaching of
p-nitrosodimethylaniline in the presence of the histidine at 440 nm. .O.D represents
the difference in optical density of irradiated samples of compounds 1 to 8.
D
A direct correlation (linear correlation) exists between lumines-
cence measured with the BacTiter-Glo Microbial Cell Viability
Assay and the number of cells in culture over five orders of magni-
tude. Values represent the mean ± S.D. of four replicates for each
cell number. The luminescent signal from 50 E. coli cells is greater
than three standard deviations above the background signal result-
ing from serum-supplemented medium without cells. There is a
Table 3
D
Quantum yields (U ) and quantification of singlet oxygen by porphyrins 1–8 using
the methodology of the degradation of histidine [22].
1
mol 1
O /mol porphyrins
2
Porphyrins
U
D
[ O
2
] (mol/L)
ꢁ6
ꢁ5
ꢁ6
ꢁ7
ꢁ7
ꢁ5
ꢁ6
ꢁ7
TPPS
TPPS
TPPS
TNPS
TNPS
TPyEtAcP (6)
TPyEtAcPNi (7)
TPyEtAcPZn (8)
4
4
4
(1)
Ni (2)
Zn (3)
(4)
Zn (5)
0.620
0.918
0.142
0.053
0.065
0.768
0.167
0.033
9.04 ꢂ 10
1.34 ꢂ 10
2.07 ꢂ 10
7.80 ꢂ 10
9.48 ꢂ 10
1.12 ꢂ 10
2.43 ꢂ 10
4.77 ꢂ 10
120.59
178.48
27.65
10.40
12.65
149.31
32.45
6.36
2
linear relationship (r = 0.99) between the luminescent signal and
the number of cells from 0 to 50,000 cells per well.
4
4
Statistical treatment of results
At least three independent experiments were performed for
each compound except where indicated otherwise. The quantifica-
tion of results is expressed as a mean ± S.D. standard deviation
(
S.D.) is obtained from 3 to 4 observations. The level of significance
accepted was p 6 0.05.
Table 4
Production of oxygen free radicals of porphyrins 1–8 as a function of the quantifi-
cation of H on the chemiluminescence assay.
2 2
O
Results and discussions
ꢁ
1
Porphyrins
[H
ꢂ 10
2
O
2
] (mol l ± DS)
2 2
(mol H O /mol
porphyrins) ± S.D.
ꢁ
4
Synthesis and characterization of porphyrins
TPPS
TPPS
TPPS
TNPS
TNPS
4
4
4
(1)
Ni (2)
Zn (3)
(4)
Zn (5)
1.04 ± 0.08
1.26 ± 0.08
330 ± 20
2.3 ± 0.1
0.42 ± 0.03
1.4 ± 0.1
3 ± 1
3.8 ± 0.2
999 ± 63
6.9 ± 0.4
1.2 ± 0.1
4.1 ± 0.5
0.80 ± 0.05
To synthesize of TPP and TNP were used based methodologies
as reported by Alder et al. employing benzaldehyde and 1-naph-
thaldehyde. Yields obtained for meso-substituted porphyrins were
approximately 20%, which agrees well with the expected values.
The complexes metals free (1, 4 and 6) were obtained with high
yields (58–85%). Free complexes were metallized with the respec-
tive oxides (ZnO and NiO) in heterogeneous system using water as
a solvent. All the porphyrins complexes (1–8) were purified by
4
4
TPyEtAcP (6)
TPyEtAcPNi
0.27 ± 0.02
(
7)
TPyEtAcPZn
8)
62 ± 1
186 ± 4
(

7
54
T. Zoltan et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 135 (2015) 747–756
column chromatography and pure compounds were obtained, ver-
ified by H NMR and MS spectra.
concentration and are summarized in Table 2. The fluorescence
quantum yields were calculated using TPPS as reference 0.12 [41].
1
4
The data of Table 2 clearly shows that include bulky groups
reduces fluorescence quantum yield of the anionic compounds 1
Primary photophysical properties of porphyrins 1–8
(
U
F
= 0.12) and 4 (U
F
= 0.07). Furthermore, it can be seen that the
= 0.02) provides an unfavorable effect on
cationic charge in 6 (U
F
Fundamental stage of any photochemical process is the absorp-
tion of a photon by a photo-excitable molecule. The absorbance
and emission spectra of the synthesized complexes provides
information for select the wavelength used and the processes of
deactivation of their excited states.
The absorption spectra of synthetized porphyrins in water (1–8)
show the typical Soret and Q bands of meso-substituted porphy-
rins. The absorption maxima and extinction coefficient of the in
water are presented in Table 1.
the fluorescence quantum yields. These results could be attributed
to the observed structural distortions and promotion of intersys-
tem crossing over the internal crossing [42]. Regarding the
decrease for metal complexes, it has been reported [43] that metal
coordination in porphyrins strongly affects the fluorescence. The
heavy metals may increase the radiationless decay rate for the
The absorption spectra of metal-free porphyrins 1, 4 and 6
(
Fig. 1) showed a Soret band around 420 nm arising from the tran-
⁄
sition of a1u
responding to the a2u
(p
) – e
g
(
p
), and Q bands between 515 and 640 nm cor-
⁄
(
p) – e
g
(p
) transition. A small red shift was
observed in the Soret band of 4 and 6 with respect to 1, that may
be attributed to a slight loss of planarity of the macrocycle due
to the peripheral substituents [32]. It has been reported that the
magnitude of the red shift is proportional to the extent of the dis-
tortion in the planarity of the molecule and depends on both the
shape and size of the substituents [33,34]. In this way this loss is
attributable to a steric effect of the substituent groups and repul-
sion effects associated with the positions of the substituents. These
effects can be clearly seen in Fig. S1 (supplementary material),
where the porphyrin structures were optimized using the Avoga-
dro software [35].
The UV–vis spectra of metal complexes (Fig. 2a–c) shows the
characteristic peak of the Soret band and a shift characteristic
regarding to the free base analogue of metal complexes with con-
6
10
figuration d –d [36]. For nickel complexes, a blue shift of the
Soret band was observed, indicating that no structural distortions
(
characterized by redshifts). This observation is consistent with
that due to the covalent radius of Ni (116 pm) that allows coordi-
nation without structural changes (‘‘hole’’ in the center of ring
2
00 pm) [37]. Therefore, the observed shift can be attributed to
its delocalized electron density decreasing metalloporphyrins,
p
increasing energy for the transition resulting in a blue shift of
the Soret bands [38]. Furthermore, for complexes of Zn, a displace-
ment of the Soret band towards red indicated structural deforma-
tion. The ionic radius of Zn (120 pm) may generate these
distortions characterized by these shifts. The optimized structures
[
35] of the metal complexes are shown in Fig. S2 (supplementary
material).
Fluorescence quantum yield
The wide variety of applications that have received the porphy-
rin derivatives is based on an ability of the complexes to generate
reactive oxygen species (ROS). A photosensitizer in its excited state
can interact with triplet oxygen in the ground state to generate sin-
glet oxygen [39]. This active molecule is responsible for many of
the photoinduced oxidative processes in the different applications
of porphyrins. Another important process that must be evaluated
in as regards to the energy transfer from the photoexcited sensi-
tizer is fluorescence emission. Information about the efficiency of
fluorescence emission as a route deactivation is utilized when por-
phyrin compounds are used as tumor or cellular marker [40].
Therefore, measuring the quantum yields was performed to evalu-
ate the potential use of these new porphyrin derivatives as
photosensitizers.
Fig. 5. Cell viability assay based on quantify ATP after irradiation (30 min) of E. coli
cultures at different concentration of complexes 1–8. All values are mean of three
independent assays. The error bars represent the standard deviation.
The values of fluorescence quantum yields (
F
U ) of the synthe-
sized compounds (1–8) were measured in water at a known

T. Zoltan et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 135 (2015) 747–756
755
intersystem crossing to the excited triplet state, resulting in a
decrease in the fluorescence quantum yield.
of hydrogen peroxide with luminol. Photogeneration of H
2
O
2
as a
function of light intensity (R.L.U: Relative Light Units) is shown
in Fig. 4. Quantifying the H (mol) produced by each complex
2 2
O
is shown in Table 4. No production of free radicals was observed
without irradiation (dark control).
Fig. 5 clearly shows that porphyrins with greater efficiency to
produce free radicals are 3, 8 and 4. It can be inferred then that com-
plexes 8 and 4 have as the only way of deactivation of triplet state
Singlet oxygen generation
1
Singlet oxygen ( O
2
) is one of the reactive oxygen species gen-
erated photochemically of great interest. To evaluate the photosen-
sitizing efficacy of the porphyrins synthetized, the singlet oxygen
production of radicals free (Type I photosensitizer), due to its low
quantum yields (
U
D
) were measured. Rose Bengal (
U
D
= 0.79 in
1
production of O
2
and fluorescence. Efficiency in producing free rad-
water) was used as the standard for determination of
U
D
(Table 3).
ꢁ5
icals is associated with great mean lifetimes of the triplet state char-
acteristic of photosensitizers with diamagnetic metals like Zn [45].
Photogeneration of singlet oxygen of 1–8 porphyrins (4 ꢂ 10 mol
ꢁ1
1
l
) was studied and quantified indirectly using histidine as
O
2
trapping to an irradiation time of 30 min (Fig. 3).
Photoinduced antibacterial activity on E. Coli
Quantification of singlet oxygen generated by the complexes
(
1–8) was used calibration curves through the method previously
Fig. 5 shows a comparison of the photoinduced antibacterial
action when compounds 1–8, at different concentrations, were
irradiated during 30 min in the presence of E. coli. It is clearly seen
that all compounds show activity dose-dependent photoinduced of
the photosensitizer. It noted that the results obtained without radi-
ation did not show significant changes in cell viability.
reported [22]. The results obtained for quantification and quantum
yields are shown in Table 3.
Results of Table 3, the complexes with higher quantum yields of
singlet oxygen formation (Type II photosensitizer) are 1, 2 and 6,
with the TPPNiS (2) porphyrin with near unit efficiency. Demon-
4
strating that there is no linear relationship between the peripheral
charges and efficiency in producing of singlet oxygen.
In Fig. 5, a low level of complex 7 (5 l
mol lꢁ1) is observed
causes a bacterial growth, which can be associated with the Ni
presence. Recently has been studied the influence of different met-
als such as Cu, Ni and Ag on the growth of microorganisms as try-
ing to find new antibacterial alloys [46]. In this regard, it has been
shown that low concentrations of Ni increase the growth rate of
the bacterium E. coli, whereas at high concentrations, the effect is
opposite [47]. Considering the effect of Ni on the metabolism of
E. coli, and in the photoinactivation experiments the bacteria are
under conditions of minimal maintenance, it can be inferred that
low concentrations of Ni can be used to increase your metabolic
activity. This effect is counteracted by the increased concentration
obtaining the toxic effect of Ni.
The generation of singlet oxygen involves the transfer of energy
from the excited triplet state of the sensitizer to oxygen surround-
ing. In addition, the quantum yield of singlet oxygen is directly
associated with a high lifetime of the triplet state as well as a
low efficiency in the other deactivation mechanisms. In this regard,
the metal coordination effect has been reported decreases the
mean lifetime of the excited states increasing efficiency of other
deactivation mechanisms [44]. This effect is clear for porphyrins
4
, 5, 7 and 8 where the fluorescence and singlet oxygen quantum
yields are low (Tables 2 and 3). Moreover, for complexes 1, 2 and
shows a high and a low , indicating that there is an
6
U
D
U
F
increase in the efficiency of intersystem crossing [42].
In order to investigate the effect of peripheral charge, in each of
complexes (1–8) comparing the results of viability with a concen-
Chemiluminescence assay
ꢁ1
tration of 250
l
mol l was performed. Results of these studies are
shown in Fig. 6.
Efficiency in the photoinactivation of microorganisms mediated
by photosensitizers, is directly related to producing ROS. In this
sense, besides produce singlet oxygen determine efficiency to
generate free radicals is necessary.
In Fig. 6, complexes with higher antibacterial activity (lower via-
bility) are 1–3 consistent with high efficiency to produce ROS both
Type I and Type II. In this respect, the complexes 1 and 2 show a high
U
D
(Table 3) and a moderate production of free radicals (Table 4).
Meanwhile, the complex 3 presents a high efficiency in producing
free radicals and moderate (Tables 4 and 3 respectively).
Quantifying the generated hydrogen peroxide after 15 min irra-
diation of the compounds 1–8 with NADH concentrations
U
D
ꢁ
4
ꢁ1
1
.0 ꢂ 10 mol l of each complex were used. The determination
Furthermore, Figs. S1 and S2 show that the complexes 1–3 no signif-
icant structural deformations, thus reducing steric effects to interact
with the microorganism, facilitating an efficient attack of the ROS
produced [48]. In this way, we can infer that the complex 7 despite
their low yields of ROS, due to its planarity explain its moderate
antimicrobial activity. In this way, we can infer that the complex 7
despite their low yields of ROS due to its planarity, explain its mod-
erate antimicrobial activity, while 8 overcomes its deformation with
high production of free radicals (Table 4). Finally, the low cellular
activity of complexes 4–6 is associated both with the low efficiency
of ROS and its great structural deformity.
was performed using chemiluminescence produced by the reaction
The results obtained in this work show a direct relationship
between structure of porphyrins and antimicrobial activity. For a
greater understanding of this relationship, would be necessary to
conduct new studies focused on cellular mechanisms associated
to this activity.
Conclusions
In conclusion, this work provides information on photodynamic
activity of water-soluble porphyrins 1–8 with different charges on
the periphery of the tetrapyrrole macrocycle and metal
Fig. 6. Cell viability assay based on quantify ATP after irradiation (30 min) of E. coli
ꢁ
1
cultures at 250 mmol
L
of complexes 1–8. All values are mean of three
independent assays. The error bars represent the standard deviation.

7
56
T. Zoltan et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 135 (2015) 747–756
coordination. Photochemical characterization showed that the
mechanisms that the molecule deactivates excited state is influ-
enced both by the presence of metals as well as by the structure
of porphyrins. In this regard, the metal coordination decreases
fluorescence quantum yields while the charges showed no clear
trend. Determination of ROS showed that complex with Type II
photosensitizing characteristics were 1, 2 and 6 while the Type I
was 3, 8 and 4. In this way, it was observed that coordination with
diamagnetic metals favors the formation of free radicals, while
complex without metals tend to produce singlet oxygen. The
photoinduced antibacterial activity was closely related to the
efficiency of formation of ROS and low molecular deformation.
Complexes 1–3 showed the highest antibacterial activity. In this
way, this work demonstrated that the design of efficient photosen-
sitizers, an analysis must be made as to produce ROS as well as the
molecular interaction of the drug with the target.
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