Photodynamic self–disinfecting surface using pyridinium phthalocyanine

Article abstract of DOI:10.1016/j.dyepig.2017.08.021

We have synthesized novel phthalocyanine with four pyridyl substituents connected to α-phthalo-positions via direct C-C bond. The Zn complex and tetracationic derivatives of phthalocyanine were also synthesized and the dyes were impregnated into filter pa

Full text of DOI:10.1016/j.dyepig.2017.08.021

Accepted Manuscript
Photodynamic self–disinfecting surface using pyridinium phthalocyanine
Lijo George, Alexander Müller, Beate Röder, Ville Santala, Alexander Efimov
PII:
S0143-7208(17)31111-7
10.1016/j.dyepig.2017.08.021
DYPI 6183
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 12 May 2017
Revised Date: 16 June 2017
Accepted Date: 14 August 2017
Please cite this article as: George L, Müller A, Röder B, Santala V, Efimov A, Photodynamic self–
disinfecting surface using pyridinium phthalocyanine, Dyes and Pigments (2017), doi: 10.1016/
j.dyepig.2017.08.021.
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ACCEPTED MANUSCRIPT
Photodynamic self –disinfecting surface using
pyridinium phthalocyanine
Lijo George, a Alexander Müller, b Beate Röder, b Ville Santala, a Alexander Efimov*a
a
Laboratory of Chemistry and Bioengineering, Tampere University of Technology, P. O. Box 541,
33101 Tampere, Finland
b
AG Photobiophysik, Humboldt-Universität zu Berlin, MNF, Institut für Physik, Newtonstrasse 15,
12489 Berlin, Raum 1'519
*Corresponding author
Tel.:+358405613419; Email: alexandre.efimov@tut.fi

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ABSTRACT
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We have synthesized novel phthalocyanine with four pyridyl substituents connected to α-phthalo-
positions via direct C-C bond. The Zn complex and tetracationic derivatives of phthalocyanine were
also synthesized and the dyes were impregnated into filter paper to prepare photoactive antimicrobial
surface. The photodynamic antimicrobial efficacy of the dyed paper samples was evaluated by a simple
and fast setup using bioluminescent microbes. Escherichia coli and Acinetobacter baylyi ADP1 strains
carrying bacterial luciferase genes were used in the screening experiment. The most efficient
compound, tetracationic zinc derivative 8, was investigated further. The compound was highly water
soluble, had high molar absorptivity and exhibited good adhesion to the filter paper without leaching
into the solution. The singlet oxygen quantum yield of tetracationic zinc derivative 8 in water was
found out to be 30 ± 20 %. According to the cell viability assay test performed on E. coli wild type in
solution, the molecule had similar or better photo toxicity as the reference photosensitizer, tetrakis (1-
methyl-pyridinium-4-yl)porphyrin (TMPyP). Antimicrobial efficacy of the dye 8 on photoactive
surface was studied by live cell assessment through colony forming unit (CFU) counting. The colored
surface demonstrated 3 log reduction in CFU against E. coli and A. baylyi ADP1 just after 1 h of
illumination with the white light of low intensity.
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KEYWORDS: Antimicrobial • Self-disinfecting surface •Pyridinium phthalocyanine • Singlet oxygen
• Photodynamic antimicrobial chemotherapy
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1.
INTRODUCTION
One of the major challenges of the 21st century is how to prevent the spread of life threatening
epidemics. Nosocomial or Healthcare associated infections (HAI) account for the major source of
transmission of infectious disease. Contaminated surfaces play a significant role in the spread of

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microbes. The contamination leads to the formation of biofilms, which facilitate microbial
proliferation.[1–6] Together with the emergence of drug resistant bacteria, the risk is multiplied several
times.[7–13] The so called “ESKAPE”- pathogens (Enterococcus faecium, Staphylococcus aureus,
Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter strains)
survive almost any individual antibiotic treatment.[14] Hence, in order to control the transmission of
the pathogenic microorganism, new approaches are required.
One such approach is photodynamic antimicrobial chemotherapy (PACT), which has been found to
be effective against drug resistant bacteria and biofilms. [15–19] The term “photodynamic reaction”
was introduced by Hv Tappeiner for inactivation of microbes by dyes in the presence of light; he also
demonstrated the involvement of oxygen in the process.[20,21] Inactivation is achieved via the
oxidative action of singlet oxygen produced by an organic dye upon light irradiation. Since the singlet
oxygen can diffuse in liquids and air, as well as through cell wall, [22,23] its applications are extended
in the preparation of photoactive self-disinfecting surfaces such as coatings, films, polymers, paints for
controlling microbial contamination [24–33], and to water sanitation.[34] Whether or not an organic
dye is suitable for the purpose, depends first of all on the quantum yield of singlet oxygen generation,
on extinction coefficient, photo- and thermal stability and appropriate wavelength absorptions.
Additional properties, such as dark toxicity, redox potentials of excited states, lipophilicity and
ionization degree must also be taken into consideration while selecting the photosensitizer. [35–39]
Many organic dyes such as methylene blue, toluidine blue O, acridine, rose bengal, and various
macrocyclic structures are capable of generating singlet oxygen.[23,40,41] In particular porphyrins,
such as haematoporphyrin and protoporphyrin and their various derivatives are excellent
photosensitizers for treatment of microbes and malignant cells.[42,43] Complexes of phthalocyanines
and napthalocyanines also exhibit considerable photobiological activity against tumors.[44] The level
of photo toxicity is strongly related to the size, charge, and hydrophobicity balance of the dye

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molecule.[16,39] It was found that cationic photosensitizers have higher activity than anionic or neutral
ones against both Gram-negative and Gram-positive bacteria.[45–48] Such difference can be explained
by the fact that even though photosensitizers are not required to penetrate into the cells, the electrostatic
interaction between cationic dye and poly-anionic lipopolysaccharide layer of the cell wall structure of
the bacteria may result in its destabilization and thus facilitates the subsequent photosensitization.
[33,49,50] On the other hand, testing the photosensitizers is a long and time-consuming procedure. To
the best of our knowledge, there is no fast and simple procedure for screening of potential photoactive
molecules; this fact considerable restricts their development.
Inspired by the above observations, we synthesized novel tetrakis(α-phthalo-pyridyl) substituted
phthalocyanine, its zinc complex and their tetracationic salts, which can serve as a potent
photosensitizer for antimicrobial treatment. In our present work, a photoactive self-disinfecting surface
was prepared by immobilizing the photosensitizer on to the filter paper by a simple technique. The
surface hence prepared was found to be stable without any leaching into water. We also propose a
simple and fast method to evaluate the antimicrobial efficacy of the surface using bioluminescent
microbes. In addition, photo inactivation of the surface was also confirmed by conventional CFU
counting method using two different microbes. However, we have also tested the phototoxic action of
the dye in solutions.
2.
MATERIALS AND METHODS
2.1. General Methods.
Reagents and solvents were purchased from TCI Europe, Sigma Aldrich Co. or from VWR and were
used without further purifications unless otherwise mentioned. Purification of the products was carried
out either by column chromatography on Silica gel 60 or Silica gel 100 (Merck) or on preparative TLC
plates (Merck) coated with neutral aluminum oxide 60 F254. NMR spectra were recorded using Varian

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Mercury 300 MHz spectrometer using TMS as internal standard. HRMS measurements were done with
Waters LCT Premier XE ESI-TOF bench top mass spectrometer. Lock-mass correction (leucine
enkephaline as a reference compound), centering and calibration were applied to the raw data to obtain
accurate mass. UV-Vis absorption spectra were recorded using Shimazu spectrophotometer.
2.2. Synthesis.
The compounds 3-hydroxyphthalonitrile 1, 2,3-dicyanophenyl trifluoromethanesulfonate 2 and 4-
(5,5-dimethyl-1,3,2-dioxaborinan-2-yl) pyridine 3 were synthesized according to the literature
procedure.[51,52] The synthetic route for compounds 2-8 were described in scheme 1.
2.2.1. Synthesis of 3-(pyridin-4-yl) phthalonitrile 4
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Pyridine boronate ester 3 (120 mg, 0.628 mmol), triflate phthalonitrile 2 (174 mg, 0.628 mmol),
PdCl2(dppf)•DCM (26 mg, 0.0314 mmol), K3PO4 (400 mg, 1.884 mmol) was dissolved in a mixture of
7.5 ml water and 7.5 ml of toluene, and was heated at vigorous stirring at 90 °C for 2 h. The reaction
mixture was extracted with CHCl3 and washed with brine, and dried over anhydrous Na2SO4. The
solvent was evaporated under reduced pressure to yield 105 mg of a crude product. The pure product 4
was isolated by re-precipitating from CHCl3/ hexane. Yield 75 mg, 60 %.
+
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MS (ESI-TOF): [M+H]+ calcd for C13H7N3 , 206.0718; found, 206.0719. H NMR (300 MHz, CDCl3,
TMS): δ = 8.83 (d, J = 5.86 Hz, 2 H), 7.93-7.77 (m, 3 H), 7.50 (d, J = 5.86 Hz, 2 H); 13C NMR (75
MHz, CDCl3, 25 °C, TMS): δ = 150.65, 144.35, 143.77, 133.72, 133.26, 123.03, 117.81, 115.24,
114.54 ppm.
2.2.2. Synthesis of 1, 8, 15, 22-tetra(pyridin-4-yl)-29H,31H-phthalocyanine 5
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Freshly cut lithium shots (57 mg, 8.212 mmol) were dissolved in n-butanol (5.7 ml) at 90 °C under
argon atmosphere. The reaction mixture was allowed to cool to room temperature and pyridine
phthalonitrile 2 (80 mg, 0.3898 mmol) was added to the above solution under argon atmosphere. The

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mixture was stirred at 90 °C for 18 h. The product was extracted with CHCl3 and washed with water
several times until the pH of the aqueous layer was neutral. The organic layer was evaporated under
reduced pressure to get a crude residue. The residue was washed with acetonitrile and purified by
column chromatography (neutral alumina, 1% EtOH in CHCl3) to yield free base phthalocyanine 5 (40
mg, 50 %).
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MS (ESI-TOF): [M+H]+ calcd for C52H30N12+, 823.2795; found, 823.2832. H NMR (300 MHz,
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CDCl3, TMS): δ = 9.01-8.95 (m, 5 H), 8.86-8.84 (m, 2 H), 8.60-8.59 (m, 6 H), 8.55-8.53 (m, 1 H),
8.35-8.33 (m, 1 H), 8.28-8.26 (m, 2 H), 8.11-8.08 (m, 1 H), 8.02-7.96 (m, 3 H), 7.86-7.80 (m, 8 H),
7.67-7.61 (m, 5 H), 7.57-7.51 (m, 7 H), 7.44-7.42 (m, 2 H), 1.65 (br, 1 H), 2.21 (br, 1 H). UV/Vis
(CHCl3): λ (ɛ) = 715 (100000), 680 nm (87037), 650 nm (31481), 615 nm (20370 Lmol-1cm-1).
2.2.3. Synthesis [1,8,15,22-tetra(pyridin-4-yl)-29H,31H-phthalocyaninato(2-)-
κ4N29,N30,N31,N32]zinc 6
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Tetrapyridinyl phthalocyanine free base 5 (12 mg, 0.0146 mmol) was dissolved in CHCl3 (1.5 ml)
and ZnOAc•2H2O (12 mg, 0.0547 mmol) in 120 µl H2O was added into it. The reaction mixture was
stirred at 60 °C for 2 h. The product was extracted with CHCl3 (20 ml) and washed with water (25 ml x
3), dried over anhydrous Na2SO4 and evaporated under reduced pressure to yield crude residue. The
product was purified with column chromatography. (Neutral alumina, 10 % EtOH in CHCl3) and later
washed with diethyl ether and acetonitrile to yield a pure compound 6 (11 mg, 85 %).
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MS (ESI-TOF): [M+H]+ calcd for C52H28N12Zn+, 885.1929; found, 885.1970. H NMR (300 MHz,
DMSO D6, TMS): δ = 9.52-9.50 (m, 1 H), 9.10-9.06 (m, 2 H), 8.62 (br, 8 H), 8.45 (br, 2 H), 8.31 (br, 2
H), 8.16-8.14 (m, 1 H), 7.78-7.58 (m, 19 H). UV/Vis (CHCl3): λ (ɛ) = 695 (113077), 634 nm (24615
Lmol-1cm-1).

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2.2.4. Synthesis of [1,8,15,22-tetra(pyridin-4-yl)-29H,31H-phthalocyaninato(2-)-
κ4N29,N30,N31,N32]zinc 6 (direct method)
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A mixture of pyridine phthalonitrile 4 (77 mg, 0.3752 mmol) and anhydrous zinc acetate (84.67 mg,
0.4615 mmol) in dimethylaminoethanol (DMAE, 810 µl) was heated at reflux at 140 °C for 12 h. The
reaction mixture was cooled to room temperature and the product was precipitated by adding a mixture
of MeOH/ H2O (9:1). The green solid was filtered and washed with methanol to yield the product 6 (80
mg, 96 %).
MS (ESI-TOF): [M+H]+ calcd for C52H28N12Zn+, 885.1929; found, 885.1904
2.2.5. Synthesis of 4,4',4'',4'''-(29H,31H-phthalocyanine-1,8,15,22-tetrayl)tetrakis(1-
methylpyridinium) tetraiodide 7
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Phthalocyanine 5 (15 mg, 0.0182 mmol) was dissolved in DMF (3 ml) and methyl iodide (1 ml,
16.0642 mmol) was added into solution. The reaction mixture was stirred at 45 °C for 18 h. The
reaction mixture was cooled in an ice bath and product was precipitated with diethyl ether (15 ml). The
solid was filtered, washed with diethyl ether several times and later with acetone/diethyl ether to yield
pure product 7 (14.8 mg, 58.32 %).
MS (ESI-TOF): [M-4I]4+ calcd for C56H38N124+, 220.5914; found, 220.5889; [M-3I]3+ calcd for
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C56H39IN123+, 336.4233; found, 336.4204. H NMR (300 MHz, DMSO D6, TMS): δ = 9.69-9.55 (m, 5
H), 9.14-9.07 (m, 8 H), 8.77-8.69 (m, 6 H), 8.46-8.32 (m, 9 H), 4.88-4.61 (m, 12 H). UV/Vis (DMF): λ
(ɛ) = 702 (141250), 635 nm (28750 Lmol-1cm-1).
2.2.6. Synthesis of {4,4',4'',4'''-(29H,31H-phthalocyanine-1,8,15,22-tetrayl-
κ4N29,N30,N31,N32)tetrakis[1-methylpyridiniumato(2-)]}zinc(4+) tetraiodide 8

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Tetrapyridyl phthalocyanine zinc 6 (20 mg, 0.0225 mmol) was dissolved in DMF (3 ml), and methyl
iodide (1 ml, 16.0642 mmol) was added to solution. The reaction mixture was stirred at 45 °C for 18 h.
The reaction mixture was cooled in an ice bath and product was precipitated by adding diethyl ether
(15 ml). The solid was filtered and washed several times with diethyl ether and later with mixture of
acetone/ H2O (1:1) to yield the product 8 (15 mg, 45.71 %).
MS (ESI-TOF): [M-4I]4+ calcd for C56H36N12Zn4+, 236.0698; found, 236.0692; [M-3I]3+ calcd for
C56H37IN12Zn3+, 357.0612; found, 357.0617. 1H NMR (300 MHz, DMSO D6, TMS): δ = 9.77-9.70 (m,
1 H), 9.95-9.54 (m, 5 H), 9.21-9.04 (m, 8 H), 8.76 (br, 3 H), 8.62 (br, 2 H), 8.46-8.35 (m, 6 H), 8.25-
8.19 (m, 1 H), 4.85-4.59 (m, 12 H). UV/Vis (DMF): λ (ɛ) = 692 (128500), 625 nm (22000 Lmol-1cm-1).
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Scheme 1. Synthetic route for the preparation of pyridine substituted phthalocyanine.
2.3. Singlet oxygen measurement.
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Singlet oxygen kinetics were monitored in aqueous solution via time-correlated multi-photon counting
(TCMPC) at 1270 ± 15 nm, the characteristic singlet oxygen luminescence wavelength. For sample

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excitation, a LMD-405D diode laser (Omikron-Laserage, Rodgau-Dudenhofen, Germany) was used:
excitation wavelength 405 nm, pulse width 120 ns, channel width 20 ns, average power 1.2 W, duration
of measurement 60 s. A TCMPC-1270 Singlet Oxygen Luminescence Detection System by SHB
Analytics (Berlin, Germany) was used for luminescence signal detection. Singlet oxygen quantum
yields were determined indirectly from fitting the luminescence signal and using TMPyP, optical
density adjusted for the excitation wavelength, as reference.[41] Fits of the data were conducted
following the standard bi-exponential model for singlet oxygen kinetics and an additional mono-
exponential phosphorescence term for TMPyP.[53] The goodness of the fit is indicated by the reduced
χ2 -test.
2.4. Antimicrobial tests.
2.4.1. Screening test on dyed paper.
The efficiency of dyes was screened by conducting antimicrobial test with bioluminescent bacterial
strains Escherichia coli (XL1-Blue, Stratagene, USA) pBAV1C-T5-lux and Acinetobacter baylyi
ADP1 (DSM 24193) carrying plasmid pBAV1C-T5-lux, The plasmid was constructed by replacing gfp
with lux in pBAV1C-T5-GFP. [54] The lux operon was cloned from the pBAV1K-T5-lux plasmid,
kind gift from Ichiro Matsumura (Addgene plasmid # 55800)[55] using standard BioBrick cloning.
Whatman 1 filter papers (area 12.25 cm2) were soaked in solutions of dyes 5-8 (0.9 mg dye in 200 µl
solvent) and allowed to dry out. After drying, three discs 0.5 cm in diameter were cut from each dyed
paper and pasted on the LA agar gel plate (15 g/l agar, 10 g/l tryptone, 5 g/l yeast extract, 5 g/l NaCl) in
a 3×4 grid. As shown in Figure 1, each column contains 4 disks with dyes 5-8, while the rows contain
disks of the same dye. A dark screen with a square hole was placed over the agar plate in such a way
that, one column of the four dyes was under the dark area while the two other columns would be
accessible for light. Blank control samples were prepared by cutting neat uncolored filter papers of the

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same size and placing them in the dark and illuminated areas of LA agar plate. Background
luminescence arising from the setup was recorded (Xenogen IVIS Lumina, Caliper Life Sciences,
USA). Microbial strains were inoculated in 5 ml of LB medium (10 g/l tryptone, 5 g/l yeast extract, 5
g/l NaCl) containing 0.5% glucose and 25 µg/ml of chloramphenicol and incubated at 30 °C 300 rpm.
After overnight cultivation, 100 µl of the culture was diluted with 5 ml of LB medium containing 0.5%
glucose and 25 µg/ml of chloramphenicol and incubated for 3 h at 30 °C 300 rpm. Microbial solution
thus prepared, was pipetted over the paper discs (5 µl per disk). To check the influence of filter paper
on the bioluminescence of bacteria the microbial solution was pipetted as well straight on the agar in
the dark region of the plate. Luminescence of the plate with microbes deposited was recorded by IVIS
and the plate was subjected to illumination. The whole plate was placed inside solar simulator
(Luzchem, Canada) and the light intensity was adjusted to 18 mW cm-2 by lifting the plate up/down.
Two filters [KG3 band pass with 315–750 nm transmittance and YG-17 filter with transmittance > 485
nm] were placed over the square hole to remove the infrared and UV radiation. After 1 hour of
illumination, the luminescence was measured again, and the antimicrobial efficacy of dyes was
compared.
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Figure 1. Schematic diagram of the setup for screening dyes.

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2.4.2. Cell viability assay.
For relative cell viability tests a resazurin assay was used.[56] Two different sample sets with
E. coli wild type (ATCC 25922) cell suspension in PBS (approx. 4×108 cfu/ml) were incubated for 2
hours under standard ambient temperature in the dark and under low white-light illumination
conditions (fluence rate of 8 ± 2 mW cm-2). First set: 200 µl cell suspension on M9-minimal-agar
substrate in 24-well-plates. Second set: 1 ml cell suspension without any agar in 24-well-plates. A
photosensitizer concentration of 5 µM was used. After addition of the resazurin reactant (for each well
900 µl of 0.05 g/l Resazurin sodium salt, Sigma-Aldrich, Germany) all samples were incubated for
another 4 hours in the dark under gentle stirring. The relative viability was then determined from
resorufin fluorescence using a VICTOR3 plate reader, PerkinElmer Inc., USA. Per sample, three wells
were used measuring each well nine times.
2.4.3. Determination of optimal dye loading on filter paper.
The filter papers with different dye loading of were prepared in the following way. First four
solutions of phthalocyanine 8 (1 mg, 0.5 mg, 0.25 mg and 0.1 mg) in 200 µl milli Q water was prepared
in 4 different vials. Whatman 1 filter paper of size 3.5 cm x 3.5 cm (12.25 cm2) soaked in the solution
containing 1 mg dye gives 0.081 mg/cm2 dye loading. The filter paper of same size soaked in 0.5 mg
dye solution gives loading of 0.04 mg/cm2. Similarly, 0.25 mg dye solution and 0.1 mg dye solution
gives dye loading of 0.02 mg/cm2 and 0.008 mg/cm2 respectively. A filter paper of same size without
dye was kept as a control.
Antimicrobial efficiency of the filter papers was confirmed by live cell assessment through CFU
counts using microbe Acinetobacter baylyi ADP1 (ATCC 33305). Microbial strain was inoculated in a
solution of 5 ml of LB medium and supplemented with 1 % glucose at 30 °C and 300 rpm overnight.
The overnight-cultivated solution (100 µl) was diluted with 5 ml of LB medium and 1 % glucose and

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shaken (300 rpm) at 30 °C for 3 h. The optical density of culture was measured at 600 nm. The
microbial solution was centrifuged for 5 minutes at 6500 rpm and the LB medium was decanted out
from the vial. The residual microbes were suspended in 5 ml of PBS (phosphate-buffered saline)
buffer. Circular discs (cut from filter papers of different dye loading and control paper) were placed in
the wells of a microplate and microbial solution (25 µl) was pipetted over the disks. The microplate
was illuminated in the solar simulator for 1 hour. UV and IR radiations were cut off using a
combination of KG3 band pass filter (315-750 nm transmittance) and YG-17 filter (transmittance > 485
nm) and the overall light intensity kept at 18 mW cm-2. After 1 h of illumination, the microbes were
extracted from wells with 975 µl of LB medium and serial dilutions (up to two times) were made from
each extract. The dilutions were then plated on LA agar plates and incubated at 30 °C overnight. The
number of colonies grown on the agar plate were counted and CFUs per milliliter were calculated from
it and the filter paper with optimal dye loading was determined from it.
2.4.4. Determination of antimicrobial efficacy of dyed-filter paper.
The E. coli MG1655 (E. coli Genetic Resources at Yale) and Acinetobacter baylyi ADP1 (ATCC
33305) strains were used in determining antimicrobial efficacy. The cultivations and resuspensions
were carried out as described above. Two sets of paper discs (original and duplicate), with
phthalocyanine 8 and an uncoloured blank control, were placed in the wells of a microplate and
microbial solution (25 µl) was pipetted over the disks. The microplate was illuminated in the solar
simulator for 1 hour. UV and IR radiations were cut off using a combination of KG3 band pass filter
(315-750 nm transmittance) and YG-17 filter (transmittance > 485 nm) and the overall light intensity
kept at 18 mW cm-2. Dark control samples were prepared by depositing microbial medium over dyed
and uncoloured disks and keeping the microplate in dark for 1 h at room temperature inside the laminar
hood. After 1 h of illumination or incubation, the microbes were extracted from wells with 975 µl of

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LB medium (10 g/l tryptone, 5 g/l yeast extract, 5 g/l NaCl) and serial dilutions (up to 10-6) were made
from each extract. The dilutions were then plated on LA agar plates and incubated at 30 °C overnight.
The number of colonies grown on the agar plate was counted and CFUs per milliliter were calculated to
determine the antimicrobial efficacy.
3.
RESULTS AND DISCUSSION
3.1. Synthesis.
Photodyanamic antimicrobial activities of pyridine substituted phthalocyanines are already
known.[57,58] However most of these compounds describe substitution in beta-phthalo position via
oxy or thio-bridge. In the present work, we give a first example of direct C-C link between the α-
phthalo position and pyridine unit. Novel pyridine-containing phthalocyanine and its zinc complex
were synthesized according to Scheme 1. The triflate phthalonitrile 2 was prepared from 3- hydroxy
phthalonitrile 1 which in turn synthesized from commercially available 3-nitro phthalonitrile by
following the literature procedures reported elsewhere.[51]-[52] Pyridine phthalonitrile 4 was prepared
with 80 % yield by coupling pyridine boronate ester 3 and triflate phthalonitrile 2. It should be
mentioned that the coupling reaction between commercially available pyridine boronic acid and triflate
phthalonitrile 2 did not produce reasonable yield of pyridine phthalonitrile. Therefore, pyridine boronic
acid was converted into boronate ester 3 by reacting with neopentyl glycol. When the reaction was
accomplished in the presence of molecular sieves, the ester 3 precipitated in 1,4- dioxane at room
temperature and its separation from the molecular sieves was difficult. Therefore, we used the
azeotropic distillation in the synthesis and obtained boronic ester 3 with high yield (ca. 80 %). Free
base phthalocyanine 5 was prepared by the tetramerization of pyridine phthalonitrile 4 with 50 % yield.
The zinc complex 6 was synthesized by reacting free base phththalocyanine 5 with zinc acetate in a
mixture of chloroform and methanol with a yield of around 85 %. Nontheless, direct synthesis of zinc
complex 6 produced better overall yield than converting free base phthalocyanine 5.[58]

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The free base 5 and phthalocyanines zinc complex 6 were converted into cationic salts 7 and 8
respectively by methylation with iodomethane in DMF. High-resolution MS spectrometry was used to
identify the molecules. The 0.25 Da separation between the peaks in the MS signals confirmed the tetra
cationic charge of the molecule. However, since the substance was a mixture of regioisomers, NMR
spectra were rather broad and difficult to interpret (see SI).
UV-visible absorption spectra were measured in chloroform (for 5 and 6) and DMF (7 and 8) and
shown in figure 2. The Q band peak for free base phthalocyanine 5 was split into two when compared
to corresponding zinc phthalocyanine 6.[59] However, broadening of the peaks after methylation for
the free base cationic phthalocyanine 7 indicates the aggregation.
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Figure 2. UV-visible absorption spectra of phthalocyanines.
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Overall, we synthesized a novel phthalocyanine with pyridyl substitutuentsat α phthalo positions
through direct C-C linkage. Cationic tetra salts were found to be soluble in water, ethanol, could be
easily precipitated into a solid, and gave a clear mass-spectrum, which suggests good degree of
1
quaternization. Integrals of H-NMR signals support a good purity of the obtained salt, though the
signals are broad indeed.
3.2. Singlet oxygen quantum yield.

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Quantitative measurement of singlet oxygen for cationic zinc phthalocyanine 8 was done in water
(shown in figure 3). The singlet oxygen quantum yield of phthalocyanine 8 was calculated to be 30 ±
20 % by comparing the phosphorescence signal intensity at 1270 nm of with that of TMPyP as
reference with a quantum yield of 74 %. This value was reasonably good since water was known to
quench the singlet oxygen. [60] However, cationic pyridine free base phthalocyanine 7 did not produce
any signal for singlet oxygen in water. Most probable reason may be the aggregation of the molecule in
water as previously explained in the discussion of UV- visible absorption spectra.
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Figure 3. Time resolved singlet oxygen measurement of TMPyP and cationic zinc phthalocyanine 8.
The optical density of TMPyP is adjusted for the excitation wavelength at 405 nm. Pearson residuals
illustrate the goodness of the fit (reduced chi-squared below 1,01 for both fits). No distinct signal for
cationic free base phthalocyanine 7 could be observed.
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3.3. Screening of dyes’ efficiency.
In our search for surfaces with photodynamic antimicrobial effect, we decided to identify the most
efficient dye from the set of synthesized phthalocyanines, by comparing its antimicrobial capability on
a solid support. In particular, the dyes 7 and 8 were highly soluble in water and readily adsorbed to the
filter paper to form stable and permanent color. The leaching of the dyes to the medium was tested in
by sonicating a piece of the filter paper impregnated with cationic phthalocyanines (7 and 8) in 3 ml of
milli Q water for 30 minutes. The UV visible absorption measurement of the resultant water sample
did not show any indication of the dye in the water nor the color of the filter paper faded out after 30
minutes of sonication. Moreover, even an overnight incubation of filter paper in milli Q water at room

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temperature did not induce leaching of the dye into water. Leaching was observed only after acidifying
the extraction water down to pH 2. In this case, phthalocyanine tetra salts were obviously extracted into
water, however not completely.
As we had four dyes to test, we needed a simple and rapid way of evaluation of the phototoxic effect.
One possibility to identify the efficient photosensitizer was to use bioluminescent bacteria as reporter
cells. The bioluminescence-based screening method has been applied in multiple studies[61] as it
allows a growth-independent and sensitive monitoring of toxic effects of different agents on the cells.
The intensity of bioluminescence arising from the bacteria on a surface is directly related to the
metabolic status of the bioluminescent bacteria.[58] In other words, when an efficient dye effectively
inactivates bioluminescent microbes, we expect a sharp decrease in the signal intensity. The
bioluminescence arising from surface of filter paper soaked with bioluminescent E. coli cell solution
was recorded before and after illumination as shown in Figure 4a. As expected, surfaces with cationic
derivatives 7 and 8 were more effective against E. coli. Moreover, cationic zinc derivative 8 had shown
higher efficacy than free base phthalocyanine 7. It should be noted that the bioluminescense from the
paper with dye 8 incubated in the dark region of the plate was also reduced. However, this decrease
may not necessarily arise from the dark toxicity of the substance, but rather can be attributed to the
photo toxicity induced by the stray light, since the incubation was done in the same plate.

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Figure 4. (a) Bioluminescent images of E. coli (carrying pBAV1C-T5-lux plasmid) on surface, before
and after illumination (clock wise direction: background image, before illumination, after illumination).
(b) Graph showing the antibacterial activity of the phthalocyanine phthalocyanine after illumination for
1 h with light of intensity 18 mW cm-2 and wavelength 485-750 nm. The data and graphs for back
ground luminescence and before illumination were shown in supplementary information.
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Similar test was conducted using filter paper soaked with more resistant bacteria - bioluminescent
Acinetobacter baylyi ADP1 carrying plasmid pBAV1C-T5-lux. In this case, the phthalocyanines (6, 7
and 8) showed much activity (Figure 5). However, the cationic zinc derivative was far more efficient in
inactivation of microbes. This time also the signal from the filter paper incubated in the dark region
was absent probably due to the stray light exposure. These experiments concluded that tetra cationic
derivates of pyridine phthalocyanines (7 and 8) are more efficient dyes compared to the neutral ones.
The extra cationic charges of the molecules might had played an important role in binding the gram
negative bacteria towards the surface of the filter paper there by ensuring a better photodynamic
inactivation.

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Figure 5. (a) Bioluminescent images of Acinetobacter baylyi ADP1 (carrying plasmid pBAV1C-T5-
lux) on surface, before and after illumination (clock wise direction: background image, before
illumination, after illumination). (b) Graph showing the antibacterial activity of the phthalocyanine
after illumination for 1 h with light of intensity 18 mW cm-2 and wavelength 485-750 nm. The data and
graphs for back ground luminescence and before illumination were shown in supplementary
information.
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3.4. Cell viability assay.
In order to understand the antimicrobial efficacy of the photosensitizers directly in a medium
without substrate support, the cell viability assay of both cationic derivatives (7 and 8) were tested in
M9- agar medium and PBS suspension. The results concluded that cationic zinc phthalocyanine 8 was
highly phototoxic towards E. coli wild type upon 2 h of illumination. The antimicrobial efficacy of the
compound was found to be superior to that of the reference photosensitizer tetrakis(methylpyridinum
iodide) porphyrin TMPyP. However, the free base cationic phthalocyanine 7 had comparable
phototoxicity to that of reference photosensitizer. The results are presented in Figure 6.

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M9-agar
PBS suspension
2.5
2
1.5
1
1.5
1
0.5
0
0.5
0
TMPyP
dye 8
dye 7
ref.
TMPyP
dye 8
dye 7
ref.
phototox.
dark ctrl.
phototox.
dark ctrl.
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Figure 6. Relative cell viability was measured on M9-Agar (minimal medium for E. coli) and directly
in PBS suspension. The values are normalized with the reference (bacteria without any
photosensitizers) fluorescence. The photosensitizer concentration is 5 µM. Error bars result from
standard deviation and error propagation. Phototox.: illuminated samples, dark ctrl.: dark controls
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3.5. Live cell assessment through colony forming unit (CFU) counting.
All the above-mentioned tests pointed out that cationic zinc derivative 8 was best among the set of
phthalocyanines synthesized. Therefore, antimicrobial efficacy of the filter paper impregnated with
phthalocyanine 8 was determined by CFU counting. In order to control the growth of microbes during
the illumination experiment and on serial dilution, the LB medium was replaced with PBS before the
deposition on the filter paper. It must be mentioned that the paper impregnated with the photosensitizer
8 was highly toxic towards both E. coli and A. baylyi. If the dye loading was higher than 0.008 mg/cm2,
no any single bacterial colony could be found on LA plates plated with the microbial extracts from the
Table 1. Photoinactivation of A. baylyi under illumination.
Dye loading
Number of Colonies
Number of colonies
1st dilution
2nd dilution
0.08 mg/cm2
0.04 mg/cm2
0.02 mg/cm2
0.008 mg/cm2
Control
0
0
0
20
0
0
0
1
Too many to count
Too many to count
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illuminated filter papers even after overnight incubation. (Table:1)

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Hence, the filter paper with dye loading 0.008 mg/cm2 was found to be optimal for activity testing
and was used for further experiments. The optical densities of the microbial solutions measured before
deposition on the filter paper was 0.2 and 0.1 for E. coli and A. baylyi respectively. Thus, the higher
number of colonies of E. coli compared to A. baylyi grown after plating agree with the absorbance
values. We have found that photo inactivation against E. coli was as high as 2.7 log reduction in CFU,
whereas the phototoxic effect against A. baylyi demonstrated 3.4 log reduction in CFU (Figure 7).
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Figure 7. Antimicrobial efficacy of filter paper dyed with phthalocyanine 8 against E. coli and A.
baylyi after illumination for 1 h with light of intensity 18 mW cm-2 and wavelength 485-750 nm.
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These values are very well comparable with the best results reported in literature for the dyes
immobilized on similar surfaces. Ringot et al. prepared photoactive cotton fabrics by covalently
grafting anionic, neutral, and cationic amino porphyrins on cotton fabric via 1,3,5-triazine linker (dye
load 18 mg/g of substrate). When subjected to light irradiation of 0.16 mW/cm2 for 24 h (total light
dose 13.8 J/cm2), the cationic fabric exhibited 100 % photo inactivation against gram-positive bacteria
(Staphylococcus aureus) while did not show any activity against gram-negative bacteria (E.coli).[62]
Porphyrin- grafted filter paper through 1,3,5-triazine linker was prepared by Mbakidi et al. The

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substrate demonstrated antimicrobial activity of 4 and 2 log decrease in CFU against both S.aureus and
E.coli respectively under same illumination condition as mentioned above.[63] In these experiments the
dye load was 19 mg/g of substrate (0.03 µmol/mg, MW=672). Similarly Carpenter et al. was able to
achieve photo inactivation of 4 log CFU reduction against different types of bacterial strains using
porphyrin linked covalently to cellulose paper with the dye load ca. 8 mg/g (12.4 nmol/mg, MW=672),
and with the illumination intensity of 65 ± 5 mW/cm2 for 30 minutes (total light dose 117 J/cm2).[64]
In our case, similar activity was achieved with the white light dose 64.8 J/cm2 (1 h at 18 mW/cm2)and
the dye load of 1.2 mg/g of substrate (0.008 mg/cm2, paper density 68.8 g/m2, figure S16) prepared by
a simple method without any complex chemical modifications.
4.
CONCLUSIONS
Novel phthalocyanine with pyridine substitution at α- position, its zinc complexes and cationic
derivatives were synthesized in high yield. The dyes exhibited good dyeing ability on filter paper, good
stability against leaching, and good photostability. We have elaborated a fast and simple screening
setup for testing the photodynamic antimicrobial substances using bioluminescent bacteria E. coli and
A. baylyi. We applied the method to study the antimicrobial efficacy of self-disinfecting surfaces
prepared from the dyes. The tetracationic derivatives were found to be the most efficient. Cell viability
assay in M9 agar medium and PBS suspension clearly demonstrated the superior photo toxicity of
cationic zinc derivative of pyridine phthalocyanine 8. Finally, the antimicrobial activity using the filter
paper dyed with the photosensitizer 8 was studied by CFU counting method. We have achieved 2.7 log
CFU reduction against E. coli and 3.4 log CFU reduction against A. baylyi, respectively, which is
comparable with the best results reported to date.
Further study using different metal complexes of pyridine phthalocyanine and substrates for
immobilization of the photosensitizer are under progress.

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ACKNOWLEDGEMENTS
This research was funded by the Graduate School of Tampere University of Technology.
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APPENDIX A. Supporting information
Supporting information contains the spectra of the compounds synthesized (4-8), the graphs for
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background luminescence and before illumination for screening experiment using bioluminescent E.
coli and A. baylyi, and the data for CFU counting.
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