Phthalocyanine thio-pyridinium derivatives as antibacterial photosensitizers

Article abstract of DOI:10.1111/j.1751-1097.2012.01113.x

This study describes the synthesis of three new tetra- and octa-thio-pyridinium phthalocyanine derivatives. PSs 3a and 4a were prepared from the tetramerization of phthalonitriles 1 and 2, respectively, whereas PS 5 was prepared from the nucleophilic substitution of the 8 beta fluor atoms of hexadecafluorophthalocyaninatozinc(II) by mercaptopyridine, followed by cationization. The recombinant bioluminescent Escherichia coli strain was used to assess, in real time, the photoinactivation efficiency of these cationic phthalocyanines, under white and red light. The cellular localization and uptake were also determined to assess the potential of the new phthalocyanines as antibacterial agents. Derivative 3a was the most effective PS, causing a 5 logs reduction in bioluminescence after 30 min of irradiation under white or red lights. The photoinactivation efficiency of the phthalocyanine 4a was similar (5 logs reduction in bioluminescence) to that of 3a when irradiated with white light, but the efficiency of inactivation was reduced (2.1 logs reduction in bioluminescence) under red light. The tetra-substituted phthalocyanine 3a also generates high amounts of singlet oxygen, does not aggregate in PBS and is highly fluorescent, which makes it an effective PS and a promising fluorescent labeling. Three new cationic thio-pyridinium phthalocyanines (3a-5a) were prepared and their potential as photosensitizers (PSs) was determined against Escherichia coli. Derivative 3a was the most effective PS, causing a 5 logs reduction in bioluminescence under white or red lights.

Full text of DOI:10.1111/j.1751-1097.2012.01113.x

Photochemistry and Photobiology, 2012, 88: 537–547
Phthalocyanine Thio-Pyridinium Derivatives as Antibacterial
Photosensitizers†
Joana B. Pereira1,2, Eliana F. A. Carvalho1, Maria A. F. Faustino1, Rosa Fernandes3, Maria G. P. M. S.
1
Neves , Jose A. S. Cavaleiro , Newton C. M. Gomes , Angela Cunha , Adelaide Almeida* and
1
2
2
2
ˆ
´
Joao P. C. Tome*
1
˜
´
1Department of Chemistry and QOPNA, University of Aveiro, Aveiro, Portugal
2Department of Biology and CESAM, University of Aveiro, Aveiro, Portugal
3Faculty of Medicine, IBILI, University of Coimbra, Coimbra, Portugal
Received 3 December 2011, accepted 3 February 2012, DOI: 10.1111/j.1751-1097.2012.01113.x
ered an important and very promising one that in principle
would not lead to microbial resistance (3–6).
ABSTRACT
This study describes the synthesis of three new tetra- and octa-
thio-pyridinium phthalocyanine derivatives. PSs 3a and 4a were
prepared from the tetramerization of phthalonitriles 1 and 2,
respectively, whereas PS 5 was prepared from the nucleophilic
substitution of the 8 beta fluor atoms of hexadecafluorophtha-
locyaninatozinc(II) by mercaptopyridine, followed by cationiza-
tion. The recombinant bioluminescent Escherichia coli strain was
used to assess, in real time, the photoinactivation efficiency of
these cationic phthalocyanines, under white and red light. The
cellular localization and uptake were also determined to assess
the potential of the new phthalocyanines as antibacterial agents.
Derivative 3a was the most effective PS, causing a 5 logs
reduction in bioluminescence after 30 min of irradiation under
white or red lights. The photoinactivation efficiency of the
phthalocyanine 4a was similar (5 logs reduction in biolumines-
cence) to that of 3a when irradiated with white light, but the
efficiency of inactivation was reduced (2.1 logs reduction in
bioluminescence) under red light. The tetra-substituted phthalo-
cyanine 3a also generates high amounts of singlet oxygen, does
not aggregate in PBS and is highly fluorescent, which makes it
an effective PS and a promising fluorescent labeling.
Photodynamic therapy is based on the theory that a
photoactivable compound, a PS, can be preferentially localized
in certain cells ⁄ tissues and when activated by light of an
appropriate wavelength, generates singlet oxygen and ⁄ or free
radicals, which are cytotoxic to targeted cells (7). This
approach presents several advantages compared with tradi-
tional antimicrobials, namely: (1) a short time of inactivation;
(2) effectiveness independent of the antibiotic resistance
profile; (3) nondevelopment of photoresistance after multiple
treatments; (4) lack of mutagenicity; and (5) availability of
broad spectrum PSs with a wide range of microbial targets (8).
Several studies showed that phthalocyanines are efficient in
the photodynamic inactivation of both Gram-positive and
-negative bacteria (9–13).
Phthalocyanines, considered second generation PSs in PDT,
are macrocycles constituted by four isoindole units connected
by four nitrogen atoms, forming an internal 16-membered
ring, characterized with red wavelength absorption
(>670 nm), long triplet lifetime (ꢀ1 ms) and good quantum
yields of singlet oxygen generation (FDZnPc = 0.56 in DMF;
10,14,15). They can occur as metal-free phthalocyanines or
metallophthalocyanines if the hydrogen atoms of the central
cavity are replaced by a metal (16). These type of compounds
tend to aggregate easily, which can compromise their photo-
dynamic effect, but their water solubility can be enhanced
through the use of adequate substituents in peripheral posi-
tions of the macrocycle (17,18). Zinc phthalocyanines have
been studied as efficient drugs in microbial photodynamic
inactivation (11–13,19,20) as well as in cancer PDT (21–23). A
previous study showed that a tetra-substituted cationic Zn(II)
N-methylpyridyloxy phthalocyanine at 10 lM induced a 4.5 log
decrease of E. coli cell survival after a total light dose of
54 JÆcm)2 delivered at the fluence rate of 30 mWÆcm)2 of
visible light (12). An octa-cationic Zn(II) phthalocyanine with
four bis(N,N,N-trimethyl)amino-2-propyloxy groups, also
showed an efficient photosensitizing effect against E. coli after
irradiation with a diode laser operating at 200 mWÆcm)2 for
5 min (11). Another study involving cationic substituted Zn(II)
phthalocyanines, bearing anilinium groups in different posi-
tions, showed a Staphylococcus aureus survival decrease of
INTRODUCTION
The inappropriate prescription of antibiotics to treat infectious
diseases, with the consequent development of antibiotic
resistance among pathogenic bacteria was already recognized
by the scientific community as a major emerging health care
issue that needs urgent solutions (1). Bacterial replication is
very fast, hence a mutation that allows their survival in the
presence of a killing agent, such as an antibiotic drug, will
rapidly become predominant, leading to microbial resistance
(2). Therefore, alternatives to control microbial infections are
needed and photodynamic therapy (PDT) is already consid-
†This paper is part of the Symposium-in-Print on ‘‘Antimicrobial Photodynamic
Therapy and Photoinactivation.’’
*Corresponding author emails: aalmeida@ua.pt (Adelaide Almeida);
´
jtome@ua.pt (Joao P. C. Tome)
˜
ꢀ 2012 Wiley Periodicals, Inc.
Photochemistry and Photobiology ꢀ 2012 The American Society of Photobiology 0031-8655/12
537

538 Joana B. Pereira et al.
Sigma–Aldrich. Solvents were purified or dried according to the
literature procedures (39).
Synthesis of phthalocyanines and precursors. 4-Thiopyridylphthalo-
nitrile 1. A dimethylformamide (DMF) (5 mL) solution of 4-nitropht-
halonitrile (1.00 g, 5.78 mmol) and 4-mercaptopyridine (1.62 g,
ꢀ4.6 log, without affecting fibroblasts, when a concentration
of 0.1 lM and a total light dose of 15 JÆcm)2 delivered at the
fluence rate of 50 mWÆcm)2 of red light was used (24).
Regarding cancer PDT, Zn(II) phthalocyanine presented an
efficient selectivity and photosensitization of MS-2 fibrosar-
coma (21) and its liposome-incorporated preparation also
affected drastically cell survival of tumorigenic non- and highly
metastic transformed rat fibroblasts (22). Plus, a liposomal
preparation of zinc phthalocyanine (CGP55847) has under-
gone clinical trials (Phase I ⁄ II, Switzerland) against squamous
cell carcinomas of the upper aerodigestive tract (23). Thiol-
substituted phthalocyanine complexes are known to absorb
light at higher wavelengths (>700 nm) and to present good
photochemical and spectroscopic properties (25,26).
Although Gram-positive bacteria are generally susceptible
to the most PS used in conventional PDT (27–29), a different
situation is reported for the Gram-negative bacteria, that in
general are insensitive to photodynamic effects in the presence
of neutral or anionic PS (30–32). The different susceptibility to
the photodynamic process between the two types of bacteria is
due to structural differences on their cell wall. Although
Gram-positive bacteria have a much thicker peptidoglycan
layer than Gram-negative bacteria, their wall presents a rather
high degree of porosity. On the hand, the cell wall of Gram-
negative bacteria has an additional membrane layer, external
to the peptidoglycan, that presents an asymmetric lipid
structure composed by strong negatively charged lipopolysac-
charides (30,33). The external membrane markedly decreases
the bacterial permeability of Gram-negative bacteria. This
constraint can be overcome by using cationic PS that can
penetrate in cells by the self-promoted uptake pathway,
through the interaction of the positive charges of the PS with
negatively charged sites of the outer membrane, resulting in an
increased permeability and consequently increase of suscepti-
bility to the photodynamic effect (18,29,34–36).
The aim of this study was to evaluate and compare the
photoinactivation efficiency of the new tetra and octa-thio-
pyridinium phthalocyanines 3a–5a against Gram-negative
bacteria. Similar tetra- and octa-substituted compounds were
already produced in previous studies, but in very low yields
(12%; 37,38). Herein, we describe a new and simple synthetic
approach to prepare, in high yields, tetra- and octa-thio-
pyridinium phthalocyanines to test their potential as PS for
Gram-negative bacteria. The photoinactivation efficiency of
these cationic phthalocyanine derivatives was evaluated, in real
time, using a bioluminescent E. coli strain as a model of Gram-
negative pathogenic bacteria. The light and PS dose applied in
this study were somewhat higher than the ones used in other
studies to produce effective target cell destruction to facilitate
the comparison among the different PSs.
1.44 mmol) was stirred at room temperature under
a nitrogen
atmosphere for 10 min. Dry potassium carbonate (214 mg, 1.5 mmol)
was added and the mixture was heated at 50ꢁC for 5 h. The residue was
resuspended in water (80 mL) and extracted with ethyl acetate (three
portions of 50 mL). The fraction containing the 4-thiopyridylphthalo-
nitrile 1 was purified by recrystallization from dichloromethane,
yielding 0.96 g (70%). mp: 146–148ꢁC. 1H NMR (CDCl3): d 7.35
(dd, J = 1.6 and 4.5, 2H, Py-o-H), 7.91 (dd, J = 1.8 and 8.2, 1H,
H-5), 8.15 (d, J = 8.2, 1H, H-6), 8.28 (d, J = 1.8, 1H, H-3), 8.54 (dd,
J = 1.6 and 4.5, 2H, Py-m-H). 13C NMR (CDCl3): d 113.9, 115.4,
115.8, 116.1, 124.0, 134.9, 136.1, 136.2, 139.9, 143.8 and 150.5.
4,5-Dithiopyridylphthalonitrile 2. Dry potassium carbonate (0.5 g,
3.6 mmol) was added to a solution of 4-mercaptopyridine (0.677 g,
6.09 mmol) and 4,5-dichlorophthalonitrile (0.5 g, 2.5 mmol) in DMF
(3 mL) in an ice bath and under a nitrogen atmosphere. The reaction
was left at room temperature and nine more portions of dry potassium
carbonate (50 mg each portion) were sequentially added every 10 min.
After the last addition, the reaction was kept under stirring for 2 h
more. Distilled water was added and the precipitate formed was
filtered and purified by chromatography over a silica gel column using
a mixture of CH2Cl2 ⁄ MeOH (9:1) as eluent. The resulting product was
recrystallized from CH2Cl2, yielding 0.60 g of 2 (50%) mp: 238–240ꢁC.
1H NMR (CDCl3): d 7.25 (dd, J = 1.5 and 4.5, 4H, Py-o-H), 7.57 (s,
2H, H-3,6), 8.67 (dd, J = 1.5 and 4.5, 4H, Py-m-H). 13C NMR
(CDCl3): d114.2, 114.8, 125.4, 135.0, 141.7, 142.6, 151.2. MS (ESI-
TOF) m ⁄ z: 347 [M+H]+
.
ZnPcH12SPy4 3. A mixture of phthalonitrile 1 (400 mg, 1.69 mmol)
and zinc acetate (276 mg, 2.02 mmol) in dimethylaminoethanol
(DMAE, 1.5 mL) was placed under reflux (140ꢁC) for 15 h. After
cooling to room temperature, the reaction mixture was washed with
MeOH ⁄ H2O (9:1) and the residue was filtered and washed with
methanol. Metallophthalocyanine
3 was obtained in 82% yield
(351 mg) after vacuum drying. mp: >300ꢁC. 1H NMR (DMSO-
d6 + TFA): d 7.79–7.92 (m, 8H, Py-o-H), 8.29–8.45 (m, 4H, Pc-b-H),
8.65–8.70 (m, 8H, Py-m-H), 8.90–9.45 (m, 8H, Pc-a-H). UV–Vis
(DMSO) kmax (log e): 347 (4.98), 617 (4.55), 684 (5.37) nm. MS
(MALDI-TOF) m ⁄ z: 1013.07 [M+H]+
.
ZnPcH8SPy8 4. A mixture of phthalonitrile 2 (285 mg, 0.82 mmol)
and zinc acetate (138 mg, 1.01 mmol) in DMAE (1 mL) was placed
under reflux (140ꢁC) for 15 h. After cooling to room temperature, the
reaction mixture was washed with MeOH ⁄ H2O (9:1) and the residue
was filtered and washed with methanol. The product 4 was dried
under vacuum, yielding 254 mg (85%). mp: >300ꢁC. 1H NMR
(DMSO-d6 + TFA): d 8.02 (d, J = 6.0, 16H, Py-o-H), 8.70 (d,
J = 6.0, 16H, Py-m-H), 10.15 (s, 8H, Pc-a-H). UV–Vis (DMSO-d6)
kmax (log e): 371 (4.59), 632 (4.47), 663 (4.68), 702 (5.03) nm. HRMS
(MALDI-TOF) m ⁄ z: calcd for C72H40N16S8Zn ([M]+) 1448.0673,
found 1448.0643.
ZnPcF8SPy8 5. DEA (5.0 mL) was added to a DMF (50 mL)
solution of ZnPcF16 (200 mg, 0.23 mmol) and 4-mercaptopyridine
(205 mg, 1.85 mmol) and the reaction mixture was kept under stirring
at room temperature for 24 under N2 atmosphere. After this period,
the DMF was evaporated under vacuum and the resulting product was
washed with acetone. The desired derivative 5 was obtained in 89%
(328 mg) yield after crystallization from water ⁄ acetone. mp: >300ꢁC.
1H NMR (DMSO-d6): d 7.28 (br s, 16H, Py-o-H), 8.19 (br s, 16H,
Py-m-H). 19F NMR (DMSO-d6): d)126.7 and )127.2 (2s, 8F, Pc-a-F).
UV–Vis (DMSO) kmax (log e): 384 (4.82), 690 (5.04), 720 (5.09) nm.
HRMS (MALDI-TOF) m ⁄ z: calcd for C72H33F8N16S8Zn 1592.9998
([M+H]+), found 1592.9927.
MATERIALS AND METHODS
1
General methods. H, 13C and 19F solution NMR spectra were
Methylation of metallophthalocyanines 3–5. A large excess of methyl
iodide (4 mL) was added to a stirred solution (or suspension) of
metallophthalocyanines 3–5 (100 mg) in dry DMF (20 mL). The
reaction mixture was heated at 40ꢁC overnight in a sealed tube. The
reaction mixture was cooled in an ice bath and the cationic
phthalocyanines were precipitated with diethyl ether, filtered and
washed several times with diethyl ether. The solid was dissolved in
acetone ⁄ H2O (1:1) and reprecipitated with acetone. The products were
dried under reduced pressure and obtained in quantitative yields.
analyzed on a BrukerAvance-300 spectrometer at 300.13, 75.47 and
282.38 MHz, respectively. CDCl3 and DMSO-d6 were used as solvents
and tetramethylsilane (TMS) as internal reference; the chemical shifts
are expressed in d (ppm) and the coupling constants (J) in Hertz (Hz).
Mass spectra were recorded on a MALDI-TOF ⁄ TOF 4800 Applied
Biosystems. UV–Vis spectra were obtained on a Shimadzu UV-
2501PC spectrophotometer. Column chromatography was carried out
using silica gel (Merck; 35–70 mesh). All chemicals were supplied by

Photochemistry and Photobiology, 2012, 88 539
ZnPcH12(SPyMe)4 3a – mp: >300ꢁC. 1H NMR (DMSO-d6): d
4.21 (4s, 12H, CH3), 7.87–7.99 (m, 8H, Py-o-H), 8.35–8.51 (m, 4H,
Pc-b-H), 8.67–8.74 (m, 8H, Py-m-H), 9.30 (br s, 8H, Pc-a-H). UV–Vis
(DMSO): kmax (log e): 352 (4.60), 616 (4.39), 685 (5.20) nm. MS
fischeri, required to produce light. The plasmid pHK724 contains a
ColE1 replicon, an ampicillin resistance marker and luxR gene whose
gene product is a transcription regulatory protein. The plasmid
pHK555 contains a P15A replicon, a chloramphenicol resistance
marker and a functional luxICDABE operon. The luxR gene of
pHK555 is inactive because of the insertion of phage DNA. When
pHK724 is transformed into E. coli containing pHK555, the resultant
colonies grow on selective media and are bioluminescent.
(MALDI-TOF) m ⁄ z: 1027.10 [M-3CH3]+
.
ZnPcH8SPyMe8 4a – mp: >300ꢁC. 1H NMR (DMSO-d6): d 4.23
(s, 24H, CH3), 8.10 (d, J = 7.1, 16H, Py-o-H), 8.72 (d, J = 7.1, 16H,
Py-m-H), 10.15 (s, 8H, Pc-a-H). 13C NMR (DMSO-d6): d 46.9, 122.9,
133.1, 133.5, 141.2, 144.5, 153.4, 160.8. UV–Vis (DMSO) kmax (log e):
383 (4.55), 630 (4.38), 702 (5.03) nm. MS (MALDI-TOF) m ⁄ z: 1463.0
Before each photoinactivation assay, E. coli was aseptically spread-
plated on TSA with the antibiotics ampicillin (100 mgÆmL)1) and
chloramphenicol (25 mgÆmL)1) and grown for 1 day at 26ꢁC. One
isolated colony was aseptically inoculated on Luria Broth (LB; Merck)
with both the antibiotics and grown overnight at 26ꢁC under stirring
(130 rpm). An aliquot (240 lL) of this culture was subcultured in LB
(30 mL) with antibiotics and grown overnight under stirring (130 rpm)
at 26ꢁC.
[M-7CH3]+
.
ZnPcF8SPyMe8 5a – mp: >300ꢁC. 1H NMR (DMSO-d6): d 4.22
(s, 24H, CH3), 8.25 (d, J = 7.0, 16H, Py-o-H), 8.77 (d, J = 7.0, 16H,
Py-m-H). 19F NMR (DMSO-d6): d)127.24 (s, 8F, Pc-a-F). UV–Vis
(DMSO) kmax (log e): 407 (4.46), 647 (4.29), 722 (4.84) nm. MS
(MALDI-TOF) m ⁄ z: 1608.9 [M-7CH3]+
.
Phthalocyanine solubility studies. The solubility of cationic phthalo-
cyanines 3a–5a in DMSO and PBS was assessed by UV–Visible
spectroscopy. Concentrations, between 0.625 and 25 lmolÆL)1, ob-
tained by the addition of aliquots of each phthalocyanine stock
solution, were analyzed. The intensity of the Q-band versus phthalo-
cyanine concentration was plotted in a graphic for linear regression to
determine if these concentrations follow the Beer–Lambert law.
Photostability studies. The photobleaching rates of compounds 3a–
5a were determined by irradiating 2 mL of a diluted solution of each
phthalocyanine in PBS (Abs ꢁ1) under the same conditions used in the
biological assays (150 mWÆcm)2). During their radiation the solutions
were magnetically stirred and kept at room temperature. The concen-
tration of the phthalocyanine derivative was quantified by visible
absorption spectroscopy at regular time intervals. UV–Visible spec-
troscopy assessed the intensity of the Q-band at different intervals of
time and the photostability was expressed as It ⁄ I0 (%; It = intensity of
the band at given time of irradiation, I0 = intensity of the band before
irradiation). Similar assays were performed in the dark to account for
the effect of aggregation as a source of light-independent decay.
Singlet oxygen generation. The ability of the PS to generate singlet
oxygen was qualitatively evaluated following the photooxidation of
3-diphenylisobenzofuran (DPBF), a singlet oxygen quencher (40).
Stock solutions of each cationic phthalocyanine at 0.1 mmolÆL)1 in
DMF and a stock solution of DPBF at 10 mmolÆL)1 in DMF ⁄ H2O
(9:1) were prepared. The reaction mixtures of 50 lmolÆL)1 of DPBF
and 0.5 lmolÆL)1 of each phthalocyanine derivative in DMF ⁄ H2O
(9:1) were irradiated, in a glass cuvette at room temperature and under
gentle magnetic stirring, with white light filtered through a cut-off filter
for wavelengths <550 nm, at a fluence rate of 9.0 mWÆcm)2. The
absorption decay of DPBF at 415 nm was measured at irradiation
intervals of 1 up to 10 min. The percentage of the DPBF absorption
decay, proportional to the production of 1O2, was assessed by the
difference between the initial absorbance and the absorbance of DPBF
after a given period of irradiation.
Cellular uptake of phthalocyanines. A bacterial suspension (107 to
108 cellsÆmL)1
) was incubated for 10 min in the dark at room
temperature in the presence of the same PS concentration used in
the inactivation studies (20 lmolÆL)1). The unbound PS was removed
out of the suspension by centrifugation at 13 000 g for 10 min
(Eppendorf Microcentrifuge 5414). For the digestion, the pellets were
resuspended in 1 mL of a digestion solution containing 0.5 mL of 2%
aqueous SDS (Merck) and incubated at room temperature for at least
24 h. The concentration of the phthalocyanine derivatives in the
digested extracts was analyzed by fluorimetry with a Fluoromax 3
(Horiba JovinYvon). The samples were excited at 425 nm and the
fluorescence emission of the PS was monitored in the 440–900 nm
range. The measured fluorescence intensity allowed the determination
of the corresponding concentration by interpolation with a calibration
plot built with known concentrations of each PS using the digestion
solution as solvent. Parallel aliquots of the bacteria incubated in the
presence of the PS were serially diluted and spread-plated in TSA for
the determination of the concentration of viable E. coli (CFUÆmL)1).
The adsorption value (PS CFU)1) was calculated according to the
literature (43). For each PS three independent assays were carried out.
Cellular localization of phthalocyanines. Bacterial cells (ꢀ108 CFU Æ
mL)1) were incubated with each PS, as described in cellular uptake.
After the incubation period, samples were centrifuged (12 000 g,
6 min), and bacterial cells were washed twice with 1 mL of PBS to
remove unbound PS. Cells were fixed with 4% paraformaldehyde in
PBS for 30 min at room temperature. Cells were washed twice with
1 mL of PBS and permeabilized for 10 min in 500 lL of 0.1% Triton
X-100 (Merck) in PBS, pH 7.4 at 50ꢁC. The cells were washed twice
with 1 mL of PBS, stained with the membrane marker FM1-43
(25 lmolÆL)1, Molecular Probes; Invitrogen) during 15 min at room
temperature in the dark, washed twice with 1 mL of PBS. A 10 lL of
glycerol were added to the pellet. Images of PS and FM1-43
fluorescence were acquired with a confocal microscope (Zeiss LSM
710). The preparation was excited at 488 nm and light emitted above
493 nm was collected for analysis of FM1-43. For analysis of the PS,
each preparation was excited at 633 nm and emitted light was collected
above 650 nm.
Correlation between bioluminescence and colony-forming units. To
assess the correlation between the bioluminescent signal (in relative
light units, RLU) of E. coli and the colony-forming units (CFU)
number, two independent assays were carried out in dark conditions.
A bioluminescent E. coli overnight culture (ꢀ107 CFUÆmL)1) was
serially diluted (10)1 to 10)7) in PBS. The nondiluted and diluted
aliquots were read on a luminometer (Turner Designs – 20 ⁄ 20) and
pour plated in TSA medium. After 24 h of incubation at 37ꢁC, the
number of colonies was counted in the most convenient dilution series.
Two independent assays were conducted.
Photoinactivation procedure. Bacterial cultures grown overnight
were diluted 10-fold in PBS to a final concentration of ꢀ106 colony-
forming units per milliliter (CFU mL)1). This bacterial suspension was
equally distributed in 100 mL sterilized and acid-washed glass beakers.
Appropriate quantities of the three stock solution of the phthalocy-
anines derivatives 3a–5a under study (500 lmolÆL)1 DMSO) were
added to achieve final concentrations of 20 lmolÆL)1 (test sample) in a
total volume of 10 mL per beaker. The samples were protected from
light with aluminium foil and incubated for 10 min under 100 rpm
stirring, at 25–30ꢁC, to promote PS binding to E. coli cells. Light and
dark controls were included in the experiments. The light control was
Fluorescence quantum yield. The fluorescence quantum yields (FF)
of the phthalocyanine derivatives in DMF were measured in 1 · 1 cm
quartz optical cells under normal air conditions on a spectrofluo-
rimeter Fluoromax 3 (Horiba JovinYvon). The fluorescence quantum
yields (FF) of the phthalocyanine derivatives were calculated by
comparison of the area below the corrected emission spectrum (600–
800 nm) with that of phthalocyaninatozinc (II; ZnPc). ZnPc was used
as fluorescence standard (kexc = 425 nm) with FF = 0.28 in DMF
(12). In all cases, the absorbance of the sample and reference
solutions was kept at 0.02 at 425 nm, the excitation wavelength.
Fluorescence quantum yield was calculated according equation
(Eq. 1):
AUCsampleð1 ꢂ 10ꢂAbs
Þ
ref
UsFample ¼ UrFef
ð1Þ
AUCrefð1 ꢂ 10ꢂAbs
Þ
sample
where AUC is the integrated area under the fluorescence curves of each
phthalocyanine and the standard and Abs is the absorbance of the
samples and the standard at the excitation wavelength, respectively.
Bacterial culture. The bioluminescent E. coli strain used in this
study was obtained in a previous study (41) and stored at )80ꢁC in
10% glycerol. The E. coli Top10 (Invitrogen) was cloned with two
plasmids as described by Maniatis et al. (42). These plasmids contain
the lux operon from the bioluminescent marine bacterium Vibrio

540 Joana B. Pereira et al.
irradiated without phthalocyanine. The dark control contained
20 lmolÆL)1 of phthalocyanine, but was protected from light with
aluminium foil. Two independent assays were conducted for each
condition.
chromic shift of ꢀ55 nm when compared with the same
compounds in PBS. The broadening of the Q-bands and the
appearance of two new bands in the Q-band regions of the
metallophthalocyanine complexes in DMSO is suggestive of
aggregation phenomena (44,45).
Irradiation conditions. Following the preincubation period, the
samples were irradiated with white light (400–800 nm) or red light
(620–750 nm) delivered by an illumination system (LC-122 LumaCare,
London) equipped with a halogen ⁄ quartz 250 W lamp coupled to two
different interchangeable optic fiber probes (400–800 nm and 620–
Phthalocyanine solubility
750 nm). The lights were delivered at a fluence rate of 150 mWÆcm)2
,
The phthalocyanines solubility in DMSO and PBS were mea-
sured by UV–Visible spectroscopy in concentrations between
0.625 and 25 lmol Æ L)1 to determine if the phthalocyanines, at
this concentration range, follow the Beer–Lambert law. The
graphics obtained from the plotting of the Q-band intensity
versus phthalocyanine concentration in DMSO (Fig. 2) show a
nonlinear regression for all the cationic phthalocyanines under
study confirming that aggregation processes are occurring. A
different situation occurs in PBS for compounds 3a and 4a at
concentrations below 25 lmol Æ L)1 where the Beer–Lambert
law is followed for both derivatives (Fig. 3). However, the
behavior of phthalocyanine 5a did not improve in PBS main-
taining its high tendency to aggregate such as in DMSO.
measured with an energy meter Coherent FieldMaxII-Top combined
with a Coherent PowerSensPS19Q energy sensor. All samples were
irradiated for 30 min under 100 rpm stirring, in a water bath at 25ꢁC.
Bioluminescence monitoring. In all experiments, aliquots (50 lL)1
)
of treated and control samples were collected at time 0 and after 2.5, 5,
10, 15, 20, 25 and 30 min of irradiation for bioluminescence measure-
ment in a luminometer (TD-20 ⁄ 20 Luminometer; Turner Designs, Inc.).
Statistical analysis. Statistical analysis was performed with SPSS
package (SPSS 15.0 for Windows, SPSS Inc.). Normal distributions
were assessed by the Kolmogorov–Smirnov test. The significance of
both irradiation time and type of PS on bacterial inactivation was
assessed by two-way univariate analysis of variance (ANOVA) model
with the Bonferroni post hoc test. A value of P < 0.05 was considered
significant.
RESULTS
Photostability, singlet oxygen generation and fluorescence
quantum yield
Synthesis of phthalocyanines and precursors
The synthetic routes used to prepare the novel cationic
phthalocyanine derivates 3a–5a are summarized in Scheme 1
and involved the previous preparation of the adequate neutral
precursors 3–5. The first two, 3 and 4 were prepared
respectively from the tetramerization of phthalonitriles 1 and
2, whereas 5 was prepared from the nucleophilic substitution
of the eight beta fluor atoms of the synthetic available
hexadecafluorophthalocyaninatozinc(II) by 4-mercaptopyri-
dine. In both methodologies, the neutral precursors were
isolated in yields higher than 80%.
The methylation of these metallophthalocyanines, carried
out in the presence of a large excess of methyl iodide in DMF
at 40ꢁC afforded, after 16 h, followed by precipitation with
diethyl ether, the cationic derivatives 3a–5a quantitatively. The
UV–Vis spectra of these phthalocyanines show the typical
Soret and Q-bands, of beta substituted phthalocyanines
(Fig. 1). In DMSO, the Q-bands of 3a–5a present a batho-
The photostability studies showed that the three cationic
compounds 3a–5a when irradiated with white light or red light
in PBS, under the same conditions used in the biological assays
(30 min at a fluence rate of 150 mWÆcm)2), do not suffer
pronounced changes in the residual absorbance (Table 1 and
Fig. S1–S6), indicating that the new derivatives are photosta-
ble in the conditions used.
The results of the photooxidation of DPBF in the presence
of the cationic phthalocyanines show that they are able to
generate singlet oxygen, causing photodegradation of DPBF
when irradiated with light at a fluence rate of 9 mW cm)2
(Table 1 and Fig. S7). However, the decay caused by tetra-
substituted phthalocyanine 3a was much higher (90% of
DPBF decay after 5 min of irradiation) than those caused by
the octa-substituted compounds (4a, 5a), indicating a higher
singlet oxygen rate production by this PS.
R3
R3
CN
CN
R1,R2
R3
R1,R2
R3
N
i)
R2
R3
H
R1
H
3
4
N
Zn
N
S
SPy
3
1
N
N
N
iv)
iv)
H
H
SPyMe
SPy
3a
4
H
N
N
N
N
S
S
CN
CN
R3
N
R3
R1
ii)
SPy
SPyMe
SPy
SPyMe
4a
5
H
R1,R2
2
R3
R3
R2
SPy
F
F
F
F
3-5
F
iv)
F
F
5a SPyMe
SPyMe
F
F
N
N
S
S
SPy =
F
F
N
N
iii)
N
Zn
N
N
5
SPyMe =
N CH3
.I-
N
N
F
F
F
F
F
i) ZnCl2, DMAE, 140 ºC, 82%; ii) ZnCl2, DMAE, 140 ºC, 85%;
F
F
ZnPcF16
iii) HSPy, DEA, DMF, r. t., 89%; iv) CH3I, DMF, 40 ºC, Quantitative yield
Scheme 1.

Photochemistry and Photobiology, 2012, 88 541
Figure 1. Normalized UV–Vis absorption spectra of phthalocyanines 3a–5a in DMSO and PBS.
All new cationic derivatives were able to show fluorescence
emission after excitation with visible light. In Table 1 are
summarized the fluorescence quantum yields obtained for the
derivatives 3a–5a in DMF. According to the results obtained,
compound 3a (0.43) showed higher fluorescence quantum yield
followed by compounds 4a (0.25) and 5a (0.16).
affected by light alone (light control) nor by the direct effect of
any of the tested PS (dark controls; Fig. 7). Significant
differences between the independent assays conducted for
each phthalocyanine were not found (ANOVA, P > 0.05).
Comparing the bioluminescence values obtained in the
experiments carried out under the white light (Fig. 7a), a clear
difference in the photoinactivation patterns of the three
phthalocyanines was observed. Compounds 3a and 4a were
more efficient than 5a (P < 0.05, ANOVA). The first two
caused a 5 log (99.999% of reduction) decrease of biolumi-
nescence after 30 min of irradiation, whereas the last one
caused only 2.1 log reduction (ꢀ99.33% of reduction). The
experiments carried out under red light also showed different
patterns of inactivation with the three PSs (Fig. 7b). The
efficiency of photodynamic inactivation of 3a was not very
different from that obtained with white light (>5 log decrease
of bioluminescence) after 30 min of irradiation. However, the
photodynamic inactivation with red light in the presence of
compound 4a was lower than that observed under white light
(3.5 log decrease after 30 min of irradiation). In these
conditions, compound 5a was even less effective than under
white light, causing 1 log decrease in bacterial biolumines-
cence.
Cellular uptake of phthalocyanines
The values of cationic phthalocyanines uptake by the E. coli,
obtained after 10 min of incubation in the dark at a concen-
tration of 20 lmolÆL)1, and after two washings are summa-
rized in Fig. 4. Compound 5a showed the highest amount of
phthalocyanine retention in E. coli cells, with an average value
of 9.99 · 1021 molecules CFU-1. The tetra-substituted phtha-
locyanine 3a and the octa-substituted 4a presented a similar
uptake, with 5.24 · 1021 and 5.11 · 1021 molecules CFU)1
,
respectively. The amount of phthalocyanine taken up by the
bacterial cells decreased after each washing, and this decrease
was more evident for 5a (Fig. 4).
Cellular localization of phthalocyanines
The confocal immunofluorescence microscopy with FM1-43 as
a membrane cell marker showed that all the compounds, even
5a with a much higher uptake than 3a and 4a, have a similar
behavior, with a uniform redistribution within the cell (Fig. 5).
DISCUSSION
The possibility of designing an enormous variety of structur-
ally different phthalocyanines with high absorption in the red
region of the electromagnetic spectrum places this class of
second generation PSs among the most promising for the
inactivation of pathogenic microorganisms in the clinic area.
This fact supports the effort of several synthetic groups to
obtain new derivatives based on phthalocyanines with im-
proved features to be used as PS (30).
Bioluminescence versus CFU of an overnight culture
The bioluminescence results reflect the bacterial abundance of
the bioluminescent E. coli strain (Fig. 6). A significant linear
correlation (R2 = 0.980) was observed between biolumines-
cence units and colony counts.
In this study an easy and efficient synthetic approach to
obtain three new cationic phthalocyanines based on nucleo-
philic substitutions of commercial available precursors by
4-mercatopyridine is described. This selection was based on the
knowledge that pyridine units after being quaternarized by
Photoinactivation of bioluminescent E. coli
The results of the photoinactivation experiments show that the
viability of the recombinant bioluminescent E. coli was not

542 Joana B. Pereira et al.

Photochemistry and Photobiology, 2012, 88 543
Table 1. Photostability, fluorescence quantum yield and relative
photooxidation of DPBF by singlet oxygen generated by cationic
phthalocyanine derivatives.
Considering the biological results this study shows that:
(1) the three new cationic thio-pyridinium phthalocyanines
with different physico-chemical properties have different
photoinactivation efficiencies against a Gram-negative bacte-
rium; (2) Phthalocyanines 3a and 4a, have high potential to
be used as antimicrobial PSs under irradiation with white
light (5 log reduction in E. coli bioluminescence); and (3)
phthalocyanine 3a is the most promising of the three PS
under red light (5.5 log reduction in bioluminescence). The
best performance of the tetra- and of the octa-substituted
phthalocyanines 3a and 4a, when compared with the octa-
substituted one 5a, in the photoinactivation of Gram-negative
bacteria can be explained by the tendency of 5a to aggregate
Photostability (%)
Compounds White light* Red light† DPBF decay‡ (%)
§
F
3a
4a
5a
90
99
97
90
100
99
90
13
11
0.43
0.25
0.16
*Upon 30 min of irradiation in PBS with white light (400–800 nm) at a
fluence rate of 150 mW cm)2. †Upon 30 min of irradiation in PBS with
red light (620–750 nm) at a fluence rate of 150 mW cm)2. ‡Upon 5 min
of irradiation in DMF ⁄ H2O (9:1) with white light filtered through a
1
1
with consequent low O2 production. The production of O2
by phthalocyanine 3a is approximately nine times higher than
the octa-substituted 4a and 5a, probably due to the lower
substitution of the Pc core by thiol groups. It is well known
that 1O2 can be quenched by thiols (46). According to the
cutoff filter for wavelengths <550 nm, at
a fluence rate of
§
9.0 mW cm)2. Reference ZnPc in DMF.
alkylation can confer an efficient antimicrobial photosensi-
tizing activity (27). The neutral derivatives 3 and 4 were
obtained respectively from the nucleophilic substitution of the
4-nitrophthalonitrile and 4,5-dichlorophthalonitrile with the
selected pyridine derivative followed by the tetramerization
step. Terivative 5 was obtained from the direct substitution,
with the same nucleophile, of eight fluorine atoms from the
commercially available hexadecafluorophthalocyaninatozinc
(II). The neutral derivatives were obtained in yields higher
than 80% and the methylation process allowed us to isolate the
required cationic PS 3a–5a quantitatively. This new series of
derivatives with four (derivative 3a) and eight charges (4a and
8a) allows us to increase the number of cationic phthalocya-
nines with potentiality to be used as PS (11,12,24,37,38). The
yields obtained in this study were higher than the ones referred
to in literature for analogue derivatives (37,38; >80% versus
12%), and the compounds 4a and 5a were isolated as single
compounds. Although cationic phthalocyanines (as a mixture
of regioisomers) namely with eight charges were already
reported in literature (11,24) and their efficiency as antibac-
terial PSs was evaluated, the charge was conferred by different
groups from the ones studied here: trimethylammonium or
anilinium versus pyridinium groups.
1
literature, O2 is the main ROS through which the PS exerts
their photodynamic action (47–50). Although the overall
production of 1O2 by 4a is also reduced, the photoinactiva-
tion results show that it is still enough to photoinactivate
efficiently the bacteria under white light. The low tendency of
4a to aggregate can justify the different profile of the two-
eight-positive charge PS.
The presence of one or more positively charged groups
plays an essential role in driving the PS toward sites that are
critical for the stability of cell organization and ⁄ or the cell
functions (34,51,52). In fact, several studies demonstrate a high
rate of bacterial inactivation with tri- and tetracationic
porphyrinic PS compared with di- and monocationic mole-
cules (31,52). However, other studies report on contradicting
results (31,53) and it was even suggested that a high number of
positive charges could decrease the PS efficiency (Jori, pers.
comm.). These results are according to previously reported
high PDI activity for tetra-cationic pyridinium zinc(II) phtha-
locyanine (PPc), a tetra-cationic methylpyridyloxy zinc(II)
phthalocyanine and octa-cationic zinc(II) phthalocyanine
bearing four bis(N,N,N-trimethyl)amino-2-propyloxy groups
against E. coli (11,13,19).
Figure 4. Adsorption of phthalocyanines 3a–5a to E. coli in the presence of 20 lmolÆL)1 of each PS, after incubation 10 min in the dark. Error bars
represent the standard deviation.

544 Joana B. Pereira et al.
Figure 5. Confocal fluorescence microscopy images of E. coli, double stained with the PS (3a, 4a and 5a) and with the cell membrane marker,
FM1-43. Right panels show the superimposed images from PS (red) and FM1-43 (green). The last row shows representative bacteria amplified from
the merged images.
In this study, under white light, the tetra- (3a) and octa- (4a)
show similar photoinactivation effect suggesting that the high
number of positive charges does not affect PS efficiency.
All cationic compounds show higher photoinactivation
efficiency under white light (400–800 nm) comparatively to
red light (620–750 nm), most probably due to the higher
overlap of the Q-bands with the emission spectrum of white
light (see Fig. S8). The light wavelength necessary to induce
microorganism photoinactivation depends on the electronic
absorption spectrum of the PS, and the emission spectrum of
the light source must cover all the PS absorption spectrum or
at least some of the absorption bands (54). Under red light
irradiation, the photoinactivation efficiency of PS 4a was
significantly lower (P < 0.05) than the one observed for 3a. In
this case, however, the overlap of the 3a Q-band with the red
light emission is lower than the corresponding 4a Q-band
overlap (see Fig. S8), the lower molar extinction coefficients of
4a Q-band in that region can explain the much lower

Photochemistry and Photobiology, 2012, 88 545
emission after a 30 min of irradiation (total light dose
270 JÆcm)2) with white light and 5.5 log under red light.
Phthalocyanines with high amounts of thio-pyridinium groups
showed a significant reduction in singlet oxygen generation;
however, Pc 4a under white light showed similar photoinac-
tivation efficiency than the tetra-cationic Pc 3a. Confocal
immunofluorescence microscopy showed that phthalocyanines
are uniformly distributed in the cell wall and within the cells.
Under the studied conditions, compound 5a did not show
photoinactivation activity; however, the direct synthesis of Pc
5a, from the commercial perfluorinated ZnPcF16, can still
justify the use of this template to prepare novel cationic PSs,
eventually more active, if a different disposition ⁄ number from
the combination used here, or if other cationic groups, rather
than the pyridinium ones, were considered.
Figure 6. Linear correlation between the bioluminescence signal and
colony counts of overnight cultures of recombinant bioluminescent
E. coli serially diluted in PBS. Bacterial counts are expressed
in CFU mL)1 and bioluminescence in relative light units (RLU). Each
value represents average
SD of two independent experiments.
Acknowledgements—The authors would like to thank University of
Aveiro, FCT (Portugal) and FEDER for funding the Organic
Chemistry research unit QOPNA (project PEst-C ⁄ QUI ⁄ UI0062 ⁄
2011), the associated laboratory CESAM, the Portuguese National
production of 1O2 due to less photons to be absorbed. The
high photodynamic efficiency of compound 3a under red light
is of special interest considering potential clinical applications,
because it penetrates deeper into human tissues than lower
wavelengths (e.g. blue light).
NMR Network and the grant PTDC ⁄ QUI ⁄ 65228 ⁄ 2006 to J. Tome.
´
SUPPORTING INFORMATION
Compound 5a unexpectedly displayed the highest values of
cellular adsorption. The compounds 3a and 4a showed similar
uptake, although lower than 5a. This suggests that the high
uptake observed for 5a is more likely an artifact related to
aggregation in PBS. On the other hand, it has been shown that
antibacterial photoinactivation is generally not dependent on
surface-bound PS, but on the permeabilization of the cell
membrane by reactive species produced by unbound PS
molecules (55). Localization of the compounds may point
out the sites of direct photodamage. Confocal microscopy
images show that after 10 min incubation all compounds have
an identical cellular localization.
Additional Supporting Information may be found in the online
version of this article:
Figure S1. Photostability of 3a upon 30 min of irradiation
in PBS with white light (400–800 nm) at a fluence rate of
150 mW cm)2
Figure S2. Photostability of 3a upon 30 min of irradiation
.
in PBS with red light (620–750 nm) at a fluence rate of
.
150 mW cm)2
Figure S3. Photostability of 4a upon 30 min of irradiation
in PBS with white light (400–800 nm) at a fluence rate of
.
150 mW cm)2
Figure S4. Photostability of 4a upon 30 min of irradiation
in PBS with red light (620–750 nm) at a fluence rate of
.
150 mW cm)2
Figure S5. Photostability of 5a upon 30 min of irradiation
CONCLUSION
Phthalocyanines 3a and 4a were prepared from pyridylph-
thalonitriles in very good yields. Phthalocyanine 5a was also
obtained, in good yields, via nucleophilic substitution of the
fluor atoms of the hexadecafluorophthalocyaninatozinc(II) by
4-mercaptopyridine, followed by cationization.
The tetra-cationic phthalocyanine 3a generates high
amounts of singlet oxygen, which makes it an effective PS
against E. coli reaching a 5 log reduction in bioluminescence
in PBS with white light (400–800 nm) at a fluence rate of
.
150 mW cm)2
Figure S6. Photostability of 5a upon 30 min of irradiation
in PBS with red light (620–750 nm) at a fluence rate of
150 mW cm)2
.
Figure S7. Photooxidation of DPBF in the presence of the
photosensitizers under study irradiated in DMF ⁄ H2O (9:1)
Figure 7. Photoinactivation bioluminescent E. coli in the presence of 20 lM of each PS under white light (a) or red light (b) at 150 mWÆcm)2. Each
value represents the average SD of two independent experiments.

546 Joana B. Pereira et al.
cationic water-soluble zinc phthalocyanine to photoinactivate
both Gram-negative and Gram-positive bacteria. J. Photochem.
Photobiol., B 32, 159–164.
with white light filtered through a cutoff filter for wavelengths
<550 nm, at a fluence rate of 9.0 mW cm)2
Figure S8. Normalized UV–Vis spectra of 3a–5a in PBS and
white and red light source emission.
Please note: Wiley-Blackwell is not responsible for the
content or functionality of any supporting information supplied
by the authors. Any queries (other than missing material) should
be directed to the corresponding author for the article.
.
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