Antibacterial photoactivity and photosensitized oxidation of phenols with meso -tetra-(4-benzoate, 9-phenanthryl)-porphyrin and its metal complexes (Zn and Cu)

Article abstract of DOI:10.1080/00958972.2014.917633

We performed the synthesis and characterization of meso-tetra-(4-benzoate- 9-phenanthryl)-porphyrin and its Zn and Cu complexes. Synthesis of meso-tetra-(4-benzoate-9-phenanthryl)-porphyrin was carried out by dry gaseous HCl, meso-tetra-(4-carboxyphenyl)-porphyrin, and 9-phenantrol in tetrahydrofuran. The preparation of metal complexes was carried out using the method of the acetates. All porphyrins were characterized by FT-IR, NMR ( ¹H and ¹³C), MS, UV-visible, and fluorescence spectroscopy. Their photophysical properties: photostability, fluorescence quantum yields, energy transfer, and generation of singlet oxygen were determined and compared with the meso-tetraphenylporphyrin. Photochemical studies on their effectiveness as photosensitizers were performed through photo-oxidation of alcohols, phenol, and 2-naphthol. Higher efficiency of degradation was obtained with photostable TB9FPCu. The antibacterial photoactivity assay was tested against Escherichia coli and its viability was measured by chemiluminescence. The highest inactivation levels were obtained by ester TB9FP and Zn complex. The properties of the photosensitizer and its efficiency vary as a result of modifying its structure. The results obtained show that the efficiency of a photosensitizer depends on multiple factors. Thus, we can say that the copper complex is efficient in degradation of alcohol, while the metal-free porphyrin is better for antibacterial applications. 2014

Full text of DOI:10.1080/00958972.2014.917633

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Antibacterial photoactivity and
photosensitized oxidation of phenols
with meso-tetra-(4-benzoate, 9-
phenanthryl)-porphyrin and its metal
complexes (Zn and Cu)
ab
a
a
Olycen Oviedo , Tamara Zoltan , Franklin Vargas , Marcel
a
a
Inojosa & Julio C. Vivas
a
Photochemistry Laboratory, Chemical Center, Venezuelan
Institute for Scientific Research (IVIC), Caracas, Venezuela
b
Faculty of Science, Department of Chemistry, University of Los
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Andes, Mérida, Venezuela
Published online: 22 May 2014.
To cite this article: Olycen Oviedo, Tamara Zoltan, Franklin Vargas, Marcel Inojosa & Julio C. Vivas
2014) Antibacterial photoactivity and photosensitized oxidation of phenols with meso-tetra-(4-
(
benzoate, 9-phenanthryl)-porphyrin and its metal complexes (Zn and Cu), Journal of Coordination
Chemistry, 67:10, 1715-1730, DOI: 10.1080/00958972.2014.917633
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Journal of Coordination Chemistry, 2014
Vol. 67, No. 10, 1715–1730, http://dx.doi.org/10.1080/00958972.2014.917633
Antibacterial photoactivity and photosensitized oxidation of
phenols with meso-tetra-(4-benzoate, 9-phenanthryl)-
porphyrin and its metal complexes (Zn and Cu)
OLYCEN OVIEDO†‡, TAMARA ZOLTAN*†, FRANKLIN VARGAS†,
MARCEL INOJOSA† and JULIO C. VIVAS†
†Photochemistry Laboratory, Chemical Center, Venezuelan Institute for Scientific Research (IVIC),
Caracas, Venezuela
‡Faculty of Science, Department of Chemistry, University of Los Andes, Mérida, Venezuela
(Received 14 February 2014; accepted 27 March 2014)
We performed the synthesis and characterization of meso-tetra-(4-benzoate-9-phenanthryl)-porphyrin
and its Zn and Cu complexes. Synthesis of meso-tetra-(4-benzoate-9-phenanthryl)-porphyrin was
carried out by dry gaseous HCl, meso-tetra-(4-carboxyphenyl)-porphyrin, and 9-phenantrol in tetra-
hydrofuran. The preparation of metal complexes was carried out using the method of the acetates.
1
13
All porphyrins were characterized by FT-IR, NMR ( H and C), MS, UV–visible, and fluorescence
spectroscopy. Their photophysical properties: photostability, fluorescence quantum yields, energy
transfer, and generation of singlet oxygen were determined and compared with the meso-tetraphenyl
*
Corresponding author. Email: tzoltan@ivic.gob.ve
2014 Taylor & Francis
©

1
716
O. Oviedo et al.
porphyrin. Photochemical studies on their effectiveness as photosensitizers were performed through
photo-oxidation of alcohols, phenol, and 2-naphthol. Higher efficiency of degradation was obtained
with photostable TB9FPCu. The antibacterial photoactivity assay was tested against Escherichia coli
and its viability was measured by chemiluminescence. The highest inactivation levels were obtained
by ester TB9FP and Zn complex. The properties of the photosensitizer and its efficiency vary as a
result of modifying its structure. The results obtained show that the efficiency of a photosensitizer
depends on multiple factors. Thus, we can say that the copper complex is efficient in degradation of
alcohol, while the metal-free porphyrin is better for antibacterial applications.
Keywords: Porphyrin; Photosensitizer; Quantum yield; Photo-oxidation; Antibacterial activity
1. Introduction
Porphyrins and metalloporphyrins have been the subject of numerous experimental and the-
oretical studies due to their remarkable photochemical, electrochemical, and biochemical
properties [1]. Synthesis and functionalization of porphyrins have long been of interest
because of the potential of porphyrin derivatives in diverse fields [2–11]. Substitution on
the periphery of porphyrin rings of suitable organic groups as well as coordination of metal
in the center of the porphyrin ring permits the design of new synthetic porphyrins. These
modifications play a key role in the chemical, electronic absorption, and emission spectra,
as well as redox properties [12]. A variety of organic reactions using multiple substitu-
ents have been employed to improve the periphery of porphyrins. However, few studies are
found for modification of porphyrins by esterification reactions [12]. Substitution at meso
positions of highly aromatic and bulky groups, such as naphthyl, phenanthryl, or pyrenyl
porphyrins, have been reported, providing an increase in the quantum yields of the forma-
tion of reactive oxygen species (ROS) [13, 14]. It has also been reported that substitutions
in the meso positions distort the core porphyrin ring, which alters its aromatic character
decreasing its efficiency as a photosensitizer [15]. Thus, the main objective of this work is
the synthesis of photosensitizers with highly resonant groups without loss of structural
integrity of the porphyrin ring.
Considering that the meso-tetra-(4-carboxyphenyl)-porphyrin (TCPP) is a rigid and sym-
metric porphyrin, highly resonant groups without compromising the central ring may be
included. Thus, in this study, we describe the esterification of the carboxylic groups of
TCPP with highly resonant groups (phenanthryl) and its metal complexes. Synthesis and
characterization of three new synthetic porphyrins, meso-tetra-(4-benzoate-9-phenanthryl)-
porphyrin (TB9FP, 1), and its Zn and Cu metal complexes (TB9FPZn, 2 and TB9FPCu, 3)
(
figure 1) were carried out as well as their photophysical characterizations.
The effect of peripheral substitution and metal coordination was studied through photo-
chemical characterization. The effect on the quantum yield of singlet oxygen and fluorescence
and photostability of modifications to the porphyrin were studied. In addition, 1–3 were stud-
ied both in the photosensitized oxidation of phenols and photoinduced antibacterial efficiency.
2
2
. Materials and methods
.1. General aspect
All analytical or HPLC grade solvents, meso-tetra-(4-carboxyphenyl)porphyrin (TCPP),
-phenanthrol, Zn(CH COO) , Cu(CH COO) , and 9,10-diphenylanthracene (DPA) were
9
3
2
3
2
purchased from Sigma-Aldrich and used without purification.

Porphyrin complexes of Zn and Cu
1717
Figure 1. Structures of porphyrins used in this study: meso-tetra-(4-benzoate-9-phenanthryl)-porphyrin: (TB9FP,
); zinc meso-tetra-(4-benzoate-9-phenanthryl)-porphyrin: (TB9FPZn, 2); and copper meso-tetra-(4-benzoate-9-
phenanthryl)-porphyrin: (TB9FPCu, 3).
1
1
13
H NMR and C NMR spectra (figure 2) were recorded with a Bruker Avance 500 and
00 MHz, respectively, in chloroform-d with tetramethylsilane (Me Si) as an internal stan-
3
4
dard. Chemical shifts (δ) are given in parts per million. Infrared (IR) spectra were per-
formed 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 spectrometer coupled to a HPLC Electrospray. Elemental analyses were performed in a
Fisons Instrument EA-1108. The samples were prepared by addition of the compound of
interest to chloroform. Absorption spectra were recorded on a Perkin Elmer Lambda-35
UV–vis spectrophotometer. Fluorescence spectra and the quantum yields were registered in
a Perkin Elmer LS-45.
13
1
Figure 2. Assignment of signals C NMR (a) and H NMR (b) for TB9FP (1).

1
718
.2. Synthesis of the porphyrin derivatives
.2.1. Meso-tetra-(4-benzoate,9-phenanthryl)-porphyrin (TB9FP, 1). TB9FP (1) was
O. Oviedo et al.
2
2
synthesized (scheme 1) by adapting the procedure used by Vargas et al. [16]. The esterifica-
tion was carried out by passing a stream of dry gaseous HCl through a solution of
meso-tetra-(4-carboxyphenyl)porphyrin (TCPP, 201.3 mg, 0.255 mM) and 9-phenanthrol
(
497.0 mg, 2.56 mM) in THF (50 mL) without heating for five days. The reaction progress
was followed by thin-layer chromatography and the crude product was separated and
purified by column chromatography (CHCl /MeOH, 2 : 8). The purple crystals were washed
3
with methanol. Yield: 41% (156.6 mg).
−
1
FT-IR (KBr, thin film) νmax (cm ): 3435 (N–H), 3138–2853 (=C–H), 1744 (C=O), 1400
C–N), 1384 (C–O–C), 1116 (C–O–C), 1102 (N–H), 802 (N–H), 617 (=C–H). MS: m/z
(
+
+
1
293.80 (M + H) Phenantrol (cal: 1292.41); 1496.53 (M) . Analysis, C H N O calcu-
52 31 2 4
1
lated %C 69.065, %H 5.190, %N 1.870; found: %C 69.032, %H 5.236, %N 1.770. H
NMR (500 MHz, CDCl , δ, J = Hz): −2.808 (s, 4H, imino protons), 8.824 (s, 8H, pyrrols
3
protons), 8.652 (d, J = 7.5, 4H, H3), 8.581 (d, J = 7.5, 4H, H4), 8.450 (d, J = 7.5, 4H, H5),
8
.361 (d, J = 7.5, 4H, H6), 8.305 (d, J = 7.5, 4H, H7), 7.753 (d, J = 7.5, 4H, H8), 7.681 (t,
J = 7.5, 4H, H9), 7.622 (t, J = 7.5, 4H, H10), 7.535 (t, J = 7.5, 4H, H11), 7.496 (t, J = 7.5,
1
3
4
1
1
1
H, H12), 6.972 (s, 4H, H13). C NMR (200 MHz, CDCl ), δ = ppm: 166.688 (C=O, C1),
3
52.684 (C–N, C2), 146.721 (C–O, C3), 134.566 (C4), 132.910 (C5), 131.309 (C6),
29.879 (C7), 129.583 (C8), 128.003 (C9), 127.287 (C10), 127.160 (C11), 126.911 (C12),
26.610 (C13), 126.472 (C14), 126.400 (C15), 124.270 (C16), 122.566 (C17), 122.517
(
C18), 122.499 (C19), 119.416 (C20), 102.649 (C21). UV–vis (CHCl ), λ
(nm): 420,
max
3
5
16, 550, 590, 648.
2
.2.2. Zn-meso-tetra-(4-benzoate,9-phenanthryl)-porphyrin (TB9FPZn, 2) and Cu-meso-
tetra-(4-benzoate,9-phenanthryl)-porphyrin (TB9FPCu, 3). Metallization of the TB9FP
1) was performed similarly for both products [17]: the dissolved ester (12.0 mg,
(
Scheme 1. Synthesis of meso-tetra-(4-benzoate-9-phenanthryl)-porphyrin: (TB9FP, 1).

Porphyrin complexes of Zn and Cu
1719
0
.00802 mM) in chloroform with the corresponding metal acetate [Cu(CH COO) 8.25
3
2
mg, 0.0413 mM; Zn(CH COO) 9.10 mg, 0.0414 mM] was taken under constant stirring
3
2
at room temperature. The metal coordination was followed by UV–visible spectroscopy
and was stopped when there was no change in the absorption spectra in the Q band
regions. The reaction mixture, in each case, was concentrated until dry. The solids
obtained were washed with methanol to remove acetates. Yield of TB9FPZn (2) and
TB9FPCu (3) was 89.74% and 72.83%, respectively.
−
1
2
2
.2.2.1. TB9FPZn (2). FT-IR (KBr, thin film) νmax (cm ): 3119.47 (=C–H), 2923.69–
853.14 (=C–H), 1731.33 (C=O), 1636.65 (C=C), 1384.54 (C–O–C), 1108.24 (C–O–C),
1
1102 (N–H), 617.91 (=C–H). UV–vis (CHCl ), λ
(nm): 421, 548, 631. The NMR ( H
and C) spectra showed no significant changes in the observed signals except for the disap-
pearance of the signal at −2 ppm as a result of the metallization.
3
max
1
3
−
1
2
2
(
.2.2.2. TB9FPCu (3). FT-IR (KBr, thin film) νmax (cm ): 3120.25 (=C–H), 2920.57–
850.79 (=C–H alquenos), 1717.34 (C=O), 1607.12 (C=C), 1384.48 (vta C–O–C), 1100.97
1
13
C–O–C). UV–vis (CHCl ), λmax (nm): 417, 539, 675. The NMR ( H and C) spectra
3
showed no significant changes in the observed signals except for the disappearance of the
signal at −2 ppm as a result of the metallization.
2.3. Irradiation
All irradiations were carried out using a Rayonet photochemical reactor, consisting of a
−
2
series of fluorescent lamps UV-A (320–400 nm, 3.3 mW cm ) and UV-B (280–320 nm,
−
2
3
.3 mW cm ) along the cylindrical wall of the reactor surrounding the cell. The reactor has
a fan to keep the sample at room temperature reducing thermal excitation of the solution.
2
2
.4. Photochemical characterization
.4.1. Photobleaching studies. Solutions of porphyrins 1–3 in CHCl (1 × 10 M L )
−
5
−1
3
were irradiated at room temperature in a Rayonet photochemical reactor at a distance of 10
cm between the lamp surface and the solution. The light emission was maximum for the
−
2
−1
photochemical reactor in UV-A 320–400 nm (3.3 mW cm , 45.575 Lux seg ) and in UV-
−
2
B (280–320 nm, 3.3 mW cm ), as measured with a UVX Digital Radiometer at 25 °C
under oxygen. The course of the reaction was followed by UV–vis spectrophotometry using
a Perkin Elmer Lambda-35 instrument. The absorbance spectra were recorded every 30 s
for 10 min to observe the possible photobleaching by reduction of the photosensitizer con-
centration. The photostability was expressed as A /A (A = absorbance of the Soret band at
t
0
t
a given time of irradiation, A = absorbance of the Soret band before irradiation).
0
2
.4.2. Energy transfer. The efficiency of energy transfer from aromatic substituents
towards the porphyrin ring was determined. Porphyrin derivatives 1–3 in CHCl
3
−
5
−1
(
1 × 10 M L ) were excited at the wavelength of the single substituent (9-phenantrol,
λexc: 242 nm) obtaining the signal emission of the substituent 9-phenantrol (λ : 739 nm)
em
inside the synthesized porphyrin derivative. Variations in the fluorescence spectra allow
determining the energy transferred from the substituents to the porphyrin.

1
720
O. Oviedo et al.
2.4.3. Fluorescence quantum yields. The relative fluorescence quantum yields for 1–3
were determined at room temperature by the comparative method using meso-tetraphenyl-
−
5
−1
porphyrin (TPP) as standard (1 × 10 M L in CHCl ; Ф 0.11 [18]). Fluorescence
3
F
quantum yields (Ф ) were determined using equation (1) [19],
F
FAStdg2
UF ¼ UF(Std)
(1)
FStdAg2
Std
where F and FStd are the areas under the fluorescence curves of 1–3 and the standard,
respectively, A and AStd are the absorbances of the sample and standard at the excitation
wavelengths, η and ηStd are the refractive indices of solvents used, respectively, for the sam-
ple and the standard.
2
.4.4. Singlet oxygen generation. Singlet oxygen was measured by the DPA bleaching
method [20]. The singlet oxygen with DPA reaction occurs with 100% chemical quenching
no competing physical quenching), and its disappearance kinetics can be followed by
(
UV–vis spectrophotometry by gradually decreasing absorbance at 374 nm with respect to
irradiation time (scheme 2) [20]. In a typical experiment, a DPA solution in chloroform
−
5
−1
−5
−1
(
1.5 mL, 1 × 10 M L ) and porphyrin solution (1.5 mL, 1 × 10 M L ) in chloroform
were placed in a quartz cuvette with a 1 cm optical path and irradiated with visible light
>420 nm) under oxygen and gentle magnetic stirring for different periods of time up to
(
2
0 min (2, 4, 6, 8, 10, 15, and 20 min).
Quantum yields of singlet oxygen photogeneration were determined in air (no oxygen
bubbled) using the relative method with TPP as reference and DPA as a chemical quencher
of singlet oxygen, from equation (2):
W
WStd
U
¼ U (Std)
(2)
4
4
where Φ (Std) are the singlet oxygen quantum yields for the standard, TPP in chloroform
Δ
Std
being (0.50) [4], W and W are the DPA photobleaching rates in the presence of the
porphyrin complex and the standard, respectively [21].
2
.5. Photosensitized oxidation of aromatic alcohols
The photodegradation assays were performed by the reaction of phenol and 2-naphthol with
singlet oxygen generated by photosensitization to obtain 1,4-benzoquinone and 1,2-naph-
thoquinone, respectively (scheme 3) [22]. Photodegradation of phenol was carried out using
Scheme 2. Singlet oxygen oxidation of DPA to its endoperoxide.

Porphyrin complexes of Zn and Cu
1721
Scheme 3. Sensitized photo-oxidation reactions for phenol and 2-naphthol.
a molar ratio of 1 : 20 (porphyrins : alcohol) in chloroform. A mixture of alcohol and photo-
sensitizer were irradiated with a Rayonet photochemical reactor, keeping 10 cm from lamp
2
surface to sample solution, with an emission maximum in UV-A 320–400 nm (3.3 mW cm ,
−
1
4
5.575 Lux seg ) for 45 min. The reaction was followed by the appearance of the corre-
sponding band, 247 nm for 1,4-benzoquinone [22] and 241 nm for 1,2-naphthoquinone [23]
in a Perkin Elmer Lambda-35 UV–vis spectrophotometer. Photodegradation efficiency of
the sensitizers used was quantified by production yields of benzoquinone and naphthoqui-
none, based on the ideal performance assuming 100% of conversion.
2
.6. Antibacterial activity
Antibacterial assays were carried out using Escherichia coli (ATCC 8739) and their prolif-
eration and viability were obtained by chemiluminescence using BacTiter- Glo Microbial
Cell (Promega, USA). Compounds 1–3 were prepared in H O : DMSO : Tween-80 : NaCl
2
−
6
−6
−1
(
95.16 : 4.0 : 0.04 : 0.8) in a concentration range of 1.0 × 10 to 5.0 × 10 M L . We took
into account that different bacteria have diverse amounts of ATP per cell, and values
reported of ATP level in cells vary considerably. Factors that affect the ATP content of cells,
such as growth phase, culture medium, and presence of metabolic inhibitors, can influence
the relationship between cell concentration and luminescence. The antibacterial photoactivi-
ties were carried out under irradiation with an illuminator LuzChem LZC 4 V Photoreactor
−
2
−1
using 14 lamps with emission in UV-A (320–400 nm, 3.3 mW cm , 45.575 Lux seg ),
keeping 10 cm between the lamp surface and the solution flask, varying the time periods of
exposure at 37 °C under continuous shaking.

1
722
O. Oviedo et al.
The BacTiter-Glo Microbial Cell Viability Assay is a homogeneous method to determine
the number of viable bacterial cells in a culture based on quantification of ATP present.
ATP is an indicator of metabolically active cells. The homogeneous assay procedure
involves adding a single reagent (BacTiter-Glo Reagent) directly to the bacterial cells in LB
broth and measuring the luminescence. The luminescent signal is relative to the amount of
ATP present, which is directly proportional to the number of living cells in the culture.
The recorded luminescence signals (Luminescence (RLU.): relative 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 background.
A direct relationship (linear correlation) exists between luminescence measured with the
BacTiter-Glo Microbial Cell Viability Assay and the number of cells in culture over five
orders of magnitude. Values represent the mean ± SD of four replicates for each cell num-
ber. The luminescent signal from 50 E. coli cells is greater than three standard deviations
above the background signal resulting from serum-supplemented medium without cells.
2
There is a linear relationship (r = 0.99) between the luminescent signal and the number of
cells from 0 to 50,000 cells per well.
2
.7. Statistical treatment of results
In each test, at least three independent experiments were performed unless stated otherwise.
The figures show either single representative results or means (± SD where appropriate).
3
3
. Results and discussion
.1. Primary photophysical properties of 1–3
The fundamental step of any photochemical process is to absorb a photon by a photoexcit-
able molecule. In this way, absorption spectra describe the regions of the UV–visible, where
these compounds absorb and hence are excited to higher energy states providing important
data for characterization of the photosensitizer. Spectrophotometric studies of 1–3 were car-
ried out using known concentrations in chloroform.
Absorption spectra of 1–3 show the typical porphyrins Soret band near 400 nm and four
Q bands in the visible region. A band near 250 nm is associated with electronic transitions
of phenanthryl. Absorption maxima and absorption coefficients of the sensitizers are pre-
sented in table 1, along with wavelengths of excitation and emission for each.
A small red shift was observed in the Soret band of 1 regarding TCPP, which could be
attributed to a slight loss of planarity of the macrocycle due to the peripheral substituents
[
15]. The UV–vis spectra of metal complexes (table 1) all show the characteristic Soret
band maxima and the corresponding displacement with respect to free base. This displace-
6
10
ment is an indication of metal complexes with metals of configuration d –d [1]. Both
metal complexes show a red shift, characteristic of structural deformation as a result of
metal coordination on the porphyrin ring [24]. The number of Q bands is reduced to two, in
line with the expected D4 h symmetry.
By comparing UV–vis spectra (table 1) of the porphyrin complex of zinc (2) with copper
porphyrin complex (3), a blue shift of the spectral bands of the copper complex is shown. As
described by Gouterman [25], these blue shifts of the Soret and Q bands are attributed to
strong effects of conjugation between Cu(II) orbitals and the π electrons of the tetrapyrrole

Porphyrin complexes of Zn and Cu
1723
−
5
−1
Table 1. Absorption spectra of porphyrins 1–3 in chloroform (1 × 10 M L ).
4
−1
−1
Porphyrin
TCPP*
Soret band, nm (ε × 10 L M cm
)
Q Bands (nm)
λ
exc (nm)
λ
em (nm)
415.36 (105.9)
513.50
415.37
650.00
5
5
6
35.77
48.50
47.03
TB9FP (1)
420.84 (6.30)
516.00
417.90
650.38
5
5
6
50.96
90.90
48.57
TB9FPZn (2)
TB9FPCu (3)
421.89 (3.27)
417.54 (1.89)
548.89
30.99
539.05
75.82
419.50
422.00
597.90
656.29
6
6
−
7
−1
*
1 × 10 M L in MeOH.
ring. These effects of conjugation cause a decrease in the energy of orbitals a1u (π) and a2u
π) relative to the orbitals e (π*) and consequently an increase in the energy available for
(
g
the electronic transitions [25].
Table 1 shows that in all cases, the fluorescence excitation spectrum coincides with the
Soret band in the absorption spectrum, confirming that the excitation is associated directly
with the energy absorption process. Furthermore, the wavelength of the fluorescent emission
for 1 compared with TCPP indicates that inclusion of the aromatic groups has no effect on
radiant deactivation processes. Likewise, if the metal coordination has an effect, a blue shift
for Cu and red for Zn are observed.
3.1.1. Photobleaching studies. Energies and lifetimes of the excited states of the porphy-
rin complexes are dependent on the structure of the metal coordination complex and the
nature of the solvent [26]. Substitutions in the macrocycle and metal ion at the center of the
molecule give the greatest effects on stability of excited molecules. The photostability of
the complexes is important to ensure the structural integrity of the complex when exposed
to visible radiation and oxygen. In order to establish the rate of photodegradation of the
compounds, we performed photostability studies under the same conditions as those used
for the photooxidations and biological assay. Spectrophotometric studies carried out for esti-
mating the photostability of 1–3 are shown in figure 3.
Figure 3 shows that the tendency of stability against UVA irradiation is TB9FPCu >
TB9FPZn. This tendency is consistent with those reported by Fleischer [27] that describe
the tendency of stability is directly associated with distance from the metal–nitrogen bond
in metalloporphyrins, greater distance, less stability. The high stability observed in the cop-
per complex can be explained due to the Cu–N bond of 1.98 Å, while the Zn–N bond dis-
tance is 2.05 Å, explaining the low stability against UVA irradiation. Thus, for TB9FPZn,
UVA radiation is sufficiently strong to compromise its structural integrity, whereas for
TB9FPCu integrity is not affected by radiation.
To explain the behavior of the Soret band for the free base (1) shown in figure 3, it is
necessary to observe their behavior throughout the electromagnetic spectrum (figure 4). As
irradiation time increases, the band at ~450 nm increases with simultaneous decrease in the
Soret band at 420 nm. In addition, in the region between 500 and 700 nm, there is a
decrease of the intensity of the four Q bands simultaneously with increase of two new

1
724
O. Oviedo et al.
Figure 3. Porphyrin derivatives photobleaching at different irradiation times. The porphyrin bleaching was moni-
tored by measuring the decrease of absorbance at the maximum of the Soret band: 421 nm TB9FP 1 (*, 422 nm
TB9FPZn 2) (* and 417 nm TB9FPCu 3 (▲)).
Figure 4. Spectral changes of TB9FP (1) upon UV-A irradiation at different times.
bands at ~548 and 647 nm. The changes observed in spectra of the free base can be attrib-
+
2 (solv)
uted to protonation of nitrogen of pyrrole as the solvent effect [H P
H4P
+ H
↔
2
(solv)
+
2 (solv)
]. Based on the proton donating ability of chloroform and similarity in metallo-
porphyrins, degeneration of the electronic levels is obtained with an increased molecular
symmetry from D2 h to D4 h [28, 29] and a variation in the absorption spectrum between
5
00 and 700 nm.

Porphyrin complexes of Zn and Cu
1725
Figure 5. Proposed structure of the J-aggregates of 1.
It has been reported that protonated form of the free base porphyrin in solution tends to
form aggregates of type H and/or J [28, 29]. A shift of the Soret band UV spectrum indi-
cates the presence of these aggregates. A bathochromic shift (blue shift) indicates side-by-
side aggregates (J-aggregates) while a hypsochromic shift (red shift) indicates one type or
face to face (H-aggregates). Given that the observed displacement in the Soret band is a red
shift, it can be inferred that J-aggregates are formed, without compromising the integrity of
the molecule. Figure 5 shows a graphical representation we have proposed for formation of
aggregates for 1.
3
.1.2. Energy transfer. The porphyrins synthesized show an intensive red emission at
5
97–656 nm (table 1). Complexes 1–3 were excited at the maximum absorption wavelength
of phenanthrene (251 nm), and the emission of porphyrin core was observed. The percent
yields of energy transferred from the substituents to the porphyrin ring were TB9FP (1)
8
4.81%, TB9FPZn (2) 90.62%, and TB9FPCu (3) 96.94%. These results show a significant
contribution to the energy of the fluorescence from phenanthryl groups in the compounds.
Thus, highly aromatic groups allow efficient transfer of the stored energy.
3
.2. Fluorescence quantum yield
The wide variety of applications of porphyrin derivatives is based on an ability of the com-
plexes to generate ROS. A photosensitizer in its excited state can interact with triplet oxy-
gen in the ground state to generate singlet oxygen [30], responsible for many of the
photoinduced oxidative processes in applications of porphyrins. Fluorescence emission must
be evaluated as a measure of energy transfer from the photoexcited sensitizer. Information
about the efficiency of fluorescence emission as a route to deactivation is utilized when por-
phyrin compounds are used as tumor or cellular markers [31]. Therefore, the quantum
yields were measured to evaluate the potential use of these new porphyrin derivatives as
photosensitizers.
The values of fluorescence quantum yields (Ф ) of 1–3 in chloroform are summarized in
F
table 2. The fluorescence quantum yields were calculated using TPP as reference 0.110 [18].

1
726
O. Oviedo et al.
From the values obtained for Ф , clearly there is a favorable effect on the fluorescence
F
quantum yield of TABP (Ф = 0.010) by including phenanthryl groups by esterification in 1
F
(
Ф = 0.078). In addition, the fluorescence quantum yield of the copper derivative (3, Ф =
F F
0
.063) is greater than that obtained for the zinc derivative (2, Ф = 0.047). Both metal
F
derivatives show a fluorescence quantum yield lower than 1. It has been reported [32] that
metal coordination in porphyrins strongly affects the fluorescence by increasing the radia-
tionless decay rate for intersystem crossing to the excited triplet state, resulting in a
decrease in the fluorescence quantum yield.
3
.3. Singlet oxygen generation
1
Singlet oxygen ( O ) is one of the ROS generated photochemically. To evaluate the photo-
2
sensitizing efficacy of the porphyrins, the singlet oxygen quantum yields (Φ ) were mea-
Δ
sured. TPP was used as the standard for determining Φ of the complexes, and the results
Δ
−
5
−1
are shown in table 3. Photogeneration of singlet oxygen of 1–3 (1 × 10 M L ) was stud-
ied and quantified indirectly using DPA as O trapping with an irradiation time of 20 min
1
2
(
figure 6).
Figure 6 shows that production of singlet oxygen is increased by esterification: TB9FP >
TBAP. Furthermore, both 2 and 3 show a singlet oxygen production lower than the free
base resulting in a tendency for the production of singlet oxygen: 1 > 2 > 3.
The efficiency of transfer of energy necessary for generation of singlet oxygen is depen-
dent on the rate (or lifetime) of deactivating mechanisms of photoexcited molecules. Copper
complex 3 is less efficient in the production of singlet oxygen. This phenomenon has been
reported previously for other porphyrin complexes with paramagnetic metals [33]. This
behavior is attributed to a non radiant relaxation given by the partially filled d orbitals of
the metal to the porphyrin in the excited triplet state not allowing energy transfer to molecu-
1
lar oxygen. Complexes 1 and 2 exhibit a substantial O quantum yield, which makes them
2
excellent potential PDT agents.
F
Table 2. Relative quantum yields (Ф ) of 1–3.
Porphyrins
λexc (nm)
λem (nm)
Ф
F
TABP
415.37
417.90
419.50
422.00
650.00
650.38
597.90
656.29
0.010
0.078
0.047
0.063
TB9FP (1)
TB9FPZn (2)
TB9FPCu (3)
Δ
Table 3. Quantum yields (Φ ) and quantification of singlet oxygen by 1–3 using
the methodology of the degradation of DPA.
1
1
−1
1
Porphyrins
Ф O
2
[ O
2
] M L
M O
2
/M Porphyrins
−
−
−
−
5
5
6
6
TABP
0.102
0.124
0.039
0.024
2.0 × 10
3.1 × 10
7.6 × 10
4.8 × 10
2.00
3.10
0.76
0.48
TB9FP (1)
TB9FPZn (2)
TB9FPCu (3)

Porphyrin complexes of Zn and Cu
1727
Figure 6. Effect of 20 min irradiation of 1–3 and TBAP on the bleaching of DPA at 374 nm. ΔOD represents the
difference in optical density of irradiated sample of 1–3 and TBAP.
3.4. Photosensitized oxidation of aromatic alcohols
The photo-oxidative properties of 1–3 were investigated in the same conditions. Photodeg-
radation efficiencies of phenol (PN) and 2-naphthol (2-NP) were studied with 1, 2, and 3,
following changes in absorption spectra of both alcohols as a result of irradiation time. Ini-
tially, it was verified that NP or 2-NP will not degrade as a result of irradiation without por-
phyrins. The results obtained for degradation are shown in table 3.
These significant differences between the efficiencies in the degradation of the NP with
free porphyrin and metalloporphyrins indicate the importance of the metal coordination in
photodegradation mechanisms [34]. For the degradation of 2-PN, these differences are less
marked. It has been reported that the efficiency of a photosensitizer is related to its stability
and efficiency to produce ROS [35]. Thus, if we compare the stabilities of 2 and 3, we see
the high effectiveness for degradation of NP and 2PN by copper complex that can be
explained according of their great photochemical stability, reported for heterogeneous sys-
tems [36].
A further important observation is that the trends in both degradations are inverse to
quantum yields of singlet oxygen, which we can infer that these degradations involve other
ROS like radicals, which have been reported to degrade efficiently this type of alcohol [37].
The low activity exhibited by 1 may be explained for aggregate formation that reduces the
lifetime of the excited triplet state, making it inefficient on energy transfer and less efficient
as a photosensitizer [38].
3
.5. Antibacterial activity
Inactivation studies on E. coli (ATCC 8739) by 1, 2, and 3, performed under UV-A
−2
−1
(
320–400 nm, 3.3 mW cm , 45.575 Lux seg ), show that the complexes do not exhibit

1
728
O. Oviedo et al.
Figure 7. Cell viability assay (E. coli) in the presence of 1 (■), 2 ( ), and 3 (▲) at different concentrations. The
result is based on quantification of the ATP present measured by luminescence after 15 min irradiation at each
concentration.
1
Table 4. Yields of the phosensitized oxidation of phenol and 2-naphthol with O
irradiation.
2
, after an hour of
1
Porphyrins
Phenol degradation (%)
2-Naphthol degradation (%)
Ф O
2
TB9FP (1)
TB9FPZn (2)
TB9FPCu (3)
4.59
7.13
53.68
33.21
33.90
37.23
0.124
0.039
0.024
dark toxicity toward the bacteria at the tested concentrations (figure 7). Direct exposure
of these bacterial strains to light, without synthetized complexes (light control), did not
cause any cytotoxic effect.
Figure 7 shows comparative antibacterial action photoinduced only when 1–3 were irra-
diated for 15 min in the presence of E. coli at different concentrations. The compounds
exhibit a photoinduced dose-dependent activity, with a tendency 1 > 3 > 2, which does not
1
coincide with the production of O (table 4).
2
A T test for independent samples was carried out using the highest concentrations of por-
phyrin complexes, showing statistically significant differences at p < 0.05. The difference in
activity obtained between Zn complex and the free base confirms that the main mechanism
1
of bacterial photoinactivation is mediated by O [39]. The activity obtained with the cop-
2
per complex, despite its low quantum yield of singlet oxygen, can be explained by the pro-
duction of other ROS, which are involved in the photoinduced antibacterial activity [40].
4
. Conclusion
A porphyrin and metalloporphyrins TB9FP (1), TB9FPZn (2), and TB9FPCu (3) were
synthesized and characterized, with high yield under mild reaction conditions. The

Porphyrin complexes of Zn and Cu
1729
photochemical and photophysical characterization show the effect of substituent groups and
the porphyrin metal centers in characteristics of new photosensitizers. The variations
obtained in degradation of phenols, 3 > 2 > 1, are directly related to the photochemical sta-
bility of photosensitizers and production of other ROS. Furthermore, the antibacterial activ-
ity, 1 > 3 > 2, indicates that a synergy exists between the photochemical stability and singlet
oxygen production in such activity. Thus, it was demonstrated that the stabilities of the
photosensitizers and production of other ROS are responsible for efficiency of the synthe-
sized complexes. Finally, of the three complexes, TB9FPCu (3) has the best features to be
considered as an efficient photosensitizer.
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