Meso-functionalized aminoporphyrins as efficient agents for photo-antibacterial surfaces

Article abstract of DOI:10.1142/S1088424610002719

Anionic, neutral and cationic amino porphyrins were synthesized as precursors of photodynamic antimicrobial agents with an aim to functionalize cotton surface through 1,3,5-triazine link. Structures of porphyrin-triazine derivatives were characterized by

Full text of DOI:10.1142/S1088424610002719

Journal of Porphyrins and Phthalocyanines
J. Porphyrins Phthalocyanines 2010; 14: 925–931
DOI: 10.1142/S1088424610002719
Published at http://www.worldscinet.com/jpp/
Meso-functionalized aminoporphyrins as efficient agents for
photo-antibacterial surfaces
Cyril Ringot, Naima Saad, Robert Granet, Philippe Bressollier, Vincent Sol*
and Pierre Krausz
Université de Limoges, Laboratoire de Chimie des Substances Naturelles, EA 1069, 123 avenue Albert Thomas,
87060 Limoges, France
Received 10 June 2010
Accepted 14 September 2010
ABSTRACT: Anionic, neutral and cationic amino porphyrins were synthesized as precursors of photo-
dynamic antimicrobial agents with an aim to functionalize cotton surface through 1,3,5-triazine link.
Structures of porphyrin-triazine derivatives were characterized by 1H NMR, MS and UV-vis confirming
thefeasibilityofthisnovelconcept. Porphyriniccottonfabricshavebeendevelopedfromthesederivatives,
and tested in vitro against Staphylococcus aureus. These novel photodynamic surfaces showed strong
and varied antimicrobial activity.
KEYWORDS: cyanuric chloride, amino porphyrin, cotton fabric, antibacterial surface, photodynamic
therapy.
synthesized and numerous studies concerning structure-
INTRODUCTION
activity relationship have been described [4, 5].
Photosensitizing properties of porphyrinic macro-
Currently, the field of antimicrobial research is turn-
cycles and derivatives have been known since the early
ing toward the development of antimicrobial surfaces for
20th century. Meyer-Betz described in 1913 the com-
applications in the biomedical, medicine, food-process-
bined action of hematoporphyrin and visible light on can-
ing and textile industries. In the present case, the strategy
cer cells and introduced the concept of PhotoDynamic
consists in incorporating biocidal agents onto synthetic
Therapy (PDT) [1]. PDT is based upon the selective
surface structures like nylon, PET [6] or natural surface-
accumulation of photoreactive compounds (photosensi-
like cellulose. For example, antibacterial cellulosic sur-
tizers) in tumor tissue and on the production of singlet
faces or fabrics have been developed by grafting different
oxygen by irradiation of the sensitizer-enriched tumor
agents like quaternary ammonium salts, N-halamines,
with visible light. Formation of cytotoxic singlet oxy-
chitosan or antibiotics [7]. Only few studies have used
gen directly in tumor cells causes cell death and often
light-activated antimicrobial agents like photosensitizers
total tumor necrosis. Since these observations, scientists
to make photoactive surface [8] and results have shown
have wondered if these photosensitizing properties could
that photosensitizers can keep their antimicrobial proper-
kill bacterial cells. Nitzan and Malik [2] pioneered the
ties when grafted to polymers. Recently, we have shown
photodestruction of bacteria like Staphylococcus aureus
and Escherichia coli and established the first mechanism
elaboration of photobactericidal cotton fabrics with por-
phyrinic moieties using “Click-Chemistry” reaction as
of bacteria photokilling by porphyrinic compounds [3].
covalent binding protocol [9].
During the last 30 years new photosensitizers have been
With the same strategy and in relation with our research
programs on photodynamic antimicrobial chemotherapy
(PACT) using natural polymeric surfaces, we report in
this paper the use of 2,4,6-trichloro-1,3,5-triazine with an
SPP full and student member in good standing
aim to graft porphyrin on cotton fabrics using covalent
bond. We describe the synthesis and characterization of
*Correspondence to: Vincent Sol, email: vincent.sol@unilim.fr,
tel: +33(0)-5-5545-7490; fax: +33(0)-5-5545-7202
Copyright © 2010 World Scientific Publishing Company

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C. RIngOt et al.
neutral, anionic and cationic amino porphyrins precur-
sors and their 1,3,5-triazinylporphyrin derivatives. Pho-
tosensitizing surfaces elaborated from these derivatives
were tested in vitro against Staphylococcus aureus, as a
pathogenic agent responsible for numerous nosocomial
infections.
iodomethane, followed by reduction with SnCl2/HCl led
to the final cationic compound 5. Methylation and reduc-
tion resulted in good yield (overall 85%). All compounds
and intermediates were characterized by H NMR, UV-
visible and mass spectroscopies.
1
Porphyrin-triazine link
In the aim to elaborate new antibacterial cotton fab-
rics, we have chosen to use the 1,3,5-triazine derivative
cyanuric chloride (2,4,6-trichloro-1,3,5-triazine). This
compound has been used as an initiator for the solution
phase synthesis of dendrimers [13], macrocycles [14],
and combinatorial libraries [15], taking advantage of
temperature-dependent stepwise substitution of its three
chlorine atoms by different nucleophiles. Furthermore,
near quantitative yields are routine for these reactions,
which could allow a one-pot protocol. Moreover, this
reactant has been frequently used in textile industries, is
inexpensive and possesses high reactivity. For these rea-
sons, this triazine derivative was used as grafting agent
between porphyrins and cellulose fabrics. Initially, we
devoted our attention to the reaction between 4-amino-
phenylporphyrin derivatives with triazine (Scheme 2) to
verify the grafting of one photosensitizer [16]. Moreover,
reaction between 1,3,5-triazine derivatives and tetrapy-
rrolic macrocycles like porphyrins and phthalocyanines
has been already reported [17].
Each 4-aminophenylporphyrin derivative was linked
to triazine at 0 °C, then addition of piperidine (for neu-
tral and cationic compound 2 and 5) or sulfanilate salts
(for anionic compound 3) motif allowed us to isolate,
purify and characterize triazine derivatives bearing one
photosensitizer and also confirmed the feasibility of this
method (Scheme 2). The first step between each amin-
oporphyrins and cyanuric chloride was carried out in
the presence of a base at 0 °C. After only 15 min, TLC
analysis showed the complete disappearance of the initial
RESULTS AND DISCUSSION
Synthesis of porphyrinic precursors
In the aim to graft porphyrins on triazine ring with
excellent yield, we substituted chlorine atoms of cya-
nuric chloride with porphyrin derivatives bearing
nucleophilic groups. We chose to prepare 4-amino-
phenylporphyrin derivatives 2, 3 and 5 as nucleophilic
agents (Scheme 1).
As shown in Scheme 1, 5-(4-nitrophenyl)-10,15,20-
triphenylporphyrin 1 was prepared according to classi-
cal porphyrin synthesis methods [10], by condensation of
4-nitrobenzaldehyde, benzaldehyde and pyrrole in propi-
onic acid. Compounds 2 and 3 were synthesized accord-
ing to the literature [11] with reasonable yields: reduction
of the nitro group by SnCl2/HCl resulted in 80% yield
of amino porphyrin 2 and hydrosoluble product 3 was
obtained by sulfonation of 2 in the presence of concen-
trated sulfuric acid (86% yield). Cationic aminoporphyrin
derivative 5 was synthesized by Lindsey’s method [12], a
2+2 Mc Donald type condensation: 5,15-bis(mesityl)-10-
(4-nitrophenyl)-20-(4-pyridyl)porphyrin 4 was obtained
after condensation of meso-(mesityl)-dipyrromethane,
pyridine-4-carbaldehyde and 4-nitrobenzaldehyde (in
stoichiometric proportions) catalyzed by TFA (TFA/alde-
hyde molar ratio = 4). After oxidation of porphyrinogen
by p-chloranil (tetrachloro-p-benzoquinone) and chro-
matographic purification, good yield of trans unsymmet-
rical precursor 4 was obtained (18.2%). Methylation with
Scheme 1. Synthetic route of neutral (2), anionic (3) and cationic (5) aminoporphyrin derivatives
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Scheme 2. Triazinyl porphyrinic derivatives
amino product and the formation of the derivative (2a, 3a
or 5a). These intermediates were very reactive, so they
were neither isolated nor characterized. They were used
in situ to react with an excess of piperidine (2a and 5a)
or sulfanilate salts (3a) at reflux for 24 h. Good yields
of products 6–8 were obtained (78, 90 and 52% respec-
tively). Analysis of these substituted triazine porphyrins
Table 1. UV-vis absorption data (a: CHCl3, b: MeOH)
Porphyrins
Absorption λmax, nm
2a
3b
5a
6a
7b
8a
421
416
424
420
415
423
517
514
523
516
519
521
554
552
572
552
557
567
591
590
595
591
586
592
648
648
656
647
641
654
1
by H NMR and mass spectrometry allowed us to con-
firm the presence of triazine link with each porphyrin.
Then porphyrin-triazine derivatives 2a, 3a and 5a were
grafted onto cellulose fabrics (3.5 × 3.5 squares) accord-
ing to a routine protocol [18].
Photoinactivation of bacterial cells
The photodynamic activity of the porphyrin-triazine
derivatives (2a, 3a and 5a) grafted on cotton fabrics was
tested in vitro against Staphylococcus aureus (S2375),
as model of Gram positive bacteria. The experimental
results are reported in Table 2. First of all, it appears nec-
essary to justify the different controls. All untreated cot-
ton samples (in darkness or irradiated) and treated cotton
samples (in darkness), permit bacterial growth of 4 log
compared with reference (t = 0, CFU number initially
present on textile squares). These controls demonstrate
that either chemical modification of cotton alone or light
dose (9.5 J/cm²) alone have no influence on bacterial
growth. Furthermore, these three controls allowed us to
distinguish between bacteriostatic or bactericidal proper-
ties of the photosensitive fabrics.
Spectroscopic studies of porphyrinic derivatives
The absorption spectra of the amino anionic and cat-
ionic porphyrins were recorded in CHCl3 or MeOH and
compared to reference neutral compound 2. The spectra
showed typical Soret and Q-bands characteristic of free-
base porphyrin derivatives. The maximum absorption is
summarized in Table 1. With regard to anionic sulfonated
derivatives, porphyrin 3 and porphyrin-triazine 7, a blue
shift was observed (intensified effect in MeOH), which is
the consequence of aggregation or stacking phenomenon
H-type or H-aggregate (face to face); contrary to cat-
ionic pyridinium derivatives 5 and 8 (red shift) where the
stacking effect is of J-type or J-aggregate [19]. These dif-
ferences of stacking effects are due to the trans function-
alized property of cationic compounds. All shifts were
measured and compared with neutral compounds.
Experimental data show that all surfaces cause a pho-
tobactericidal effect at quantity as low as ~10 µmol of
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Table 2. Bacterial counts (log10 CFU) of Staphylococcus aureus; (A) neutral cotton fabric, (B) anionic cotton fabric
and (C) cationic cotton fabric
Cotton fabrics
t = 0
Control
Darkness
Light
Darkness
Light
2a grafted: A
3a grafted: B
5a grafted: C
6.60 ± 0.03
6.30 ± 0.02
6.43 ± 0.01
10.16 ± 0.04
10.27 ± 0.09
10.36 ± 0.09
10.44 ± 0.03
10.3 ± 0.03
10.47 ± 0.04
9.18 ± 0.15
10.26 ± 0.04
5.73 ± 0.06
5.40 ± 0.5
6.10 ± 0.05
0
active compound per fabric square sample. After 24 h
exposure to light at fluence dose of 9.5 J/cm2 (h = 10 cm,
T = 37 °C), percentages of surviving bacteria were 63%,
6.3% and 0% for anionic, neutral and cationic surfaces
respectively. Values of photoinactivation (obtained by
comparison treated light vs. t = 0) depend on the global
charge of the photosensitizer. These results suggest a
structure-activity relationship on photoinactivation of
bacteria cells. For cationic cotton fabric, toxicity in
the dark is probably due to the presence of quaternary
ammonium, known for disorganizing cell walls [20].
Due to the insoluble and immobilized property of the
photosensitizers, mechanistic interpretations of these
experiments must take into account the generation of a
reactive species, such as singlet oxygen, on the mate-
rial surface [8], followed by its diffusion and eventual
interaction with the target cell. Midden and co-workers
[21] have already shown that such photoinhibition is
due to the type II photochemical process implying sin-
glet oxygen (1O2) that ultimately damages the cell enve-
lope since the photosensitizer does not penetrate the
bacterial cell.
Photosensitizer synthesis
5-(4-nitrophenyl)-10,15,20-triphenyl porphyrin (1).
To a solution of 4-nitrobenzaldehyde (302 mg, 2 mmol,
1 equiv.) and benzaldehyde (613 µL, 6 mmol, 3 equiv.)
in propionic acid (60 mL) under magnetic stirring at
120 °C for 1 h, freshly distilled pyrrole (555 µL, 8 mmol,
4 equiv.) was added dropwise. The mixture was kept
under magnetic stirring at 120 °C for 1 h. After removing
of solvent, the crude product was purified by chromatog-
raphy on silica gel (CHCl3/petroleum ether: 6/4), to give
96 mg of 1, purple solid (7.2% yield). TLC (CHCl3/petro-
leum ether: 6/4): Rf = 0.62. UV-vis (CHCl3): λmax, nm
(ε × 10-3) 420 (241), 516 (12.2), 552 (7.1), 591 (4.4), 647
(3.1). 1H NMR (CDCl3): δ, ppm 8.89 (d, 2H, J = 4.8 Hz,
Hb-pyrrolic), 8.86 (br s, 4H, Hb-pyrrolic), 8.73 (d, 2H, J = 4.8 Hz,
Hb-pyrrolic), 8.62 (d, 2H, J = 8.5 Hz, H3,5-aryl), 8.39 (d, 2H,
J = 8.5 Hz, H2,6-aryl), 8.21 (d, 6H, J = 6.8 Hz, H2,6-phenyl),
7.76 (d, 9H, J = 7.3 Hz, H3,4,5-phenyl), -2.78 (br s, 2H, NHint.).
MS (MALDI): m/z for C44H29N5O2, calcd. 659.23, found
660.07 [M + H]+.
5-(4-aminophenyl)-10,15,20-triphenyl porphyrin
(2). Porphyrin 1 (80 mg, 121 µmol) was dissolved in
20 mL of chloroform and a solution (20 mL) of SnCl2
(3 equiv./NO2) in concentrated HCl was added. Acetic
acid (20 mL) was added to form homogeneous solution
and the resulting mixture was heated at 70–80 °C, under
stirring overnight. Reaction was stopped by neutraliza-
tion with 2 M NaOH (150 mL). Mixture solution was
washed with water (2 × 100 mL) then dried on MgSO4,
and filtered; after purification by chromatography on sil-
ica gel (CHCl3), compound 2 (purple solid) was obtained
(61 mg, 80%). TLC (CHCl3): Rf = 0.50. UV-vis (CHCl3):
From these preliminary results, we can conclude that
all tested surfaces exhibit good Gram positive bacteria
photoinactivation and seem to be promising for antibac-
terial application.
EXPERIMENTAL
All solvents and reagents were purchased from
Aldrich, Prolabo or Acros. Analytical thin layer chroma-
tography (TLC) was performed on Merck 60F254. Col-
umn chromatography was carried out with Merck silica
gel (60 ACC; 15–40 µm).
λ
max, nm (ε × 10-3) 421 (369), 517 (14.0), 554 (7.6), 591
1
(4.4), 648 (4.0). H NMR (CDCl3): δ, ppm 8.94 (d, 2H,
J = 4.7 Hz, Hb-pyrrolic), 8.83 (br s, 6H, Hb-pyrrolic), 8.21 (br
d, 6H, J = 7.6 Hz, H2,6-phenyl), 7.99 (d, 2H, J = 8.2 Hz,
H2,6-aryl), 7.74 (br d, 9H, J = 7.4 Hz, H3,4,5-phenyl), 7.04 (d,
2H, J = 8.2 Hz, H3,5-aryl), 3.99 (s, 2H, NH2), -2.75 (br
s, 2H, NHint.). MS (MALDI): m/z for C44H31N5, calcd.
629.26, found 630.11 [M + H]+.
1H NMR spectroscopy was performed with a Bruker
DPX 400 spectrometer. Chemical shifts are reported as
δ (ppm), downfield from internal TMS and are listed
according to the standard numbering of meso-arylpor-
phyrins. UV-vis spectra were recorded on a Perkin
Elmer Lambda 25 double-beam spectrophotometer using
10-mm quartz cells. MALDI-TOF mass spectra were
recorded with a Voyager Elite (Framingham MA, USA)
time-of-flight mass spectrometer equipped with a 337 nm
nitrogen laser, VSL 337ND (Université Pierre et Marie
Curie, Paris).
5-(4-aminophenyl)-10,15,20-tri(4-sulfonatophenyl)
porphyrin trisodium (3). Porphyrin 2 (75 mg, 119 µmol)
was dissolved in 5 mL of 95% H2SO4 and heated at 70 °C,
under stirring overnight. Water (100 mL) was added and
the mixture was neutralized by saturated Na2CO3. After
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water removal, sodium salts (containing hydrosoluble
product 3) were precipitated by an excess of methanol.
This operation was carried out twice. Salts were filtered
and dissolved in minimum water. After 4 days of dialysis
(cut-off: 1000 Da), pure product 3 (brown-green solid)
was lyophilized and obtained with 86% yield (95.6 mg).
TLC (CHCl3/MeOH/H2O: 60/45/12): Rf = 0.34. UV-
vis (MeOH): λmax, nm (ε × 10-3) 416 (354), 514 (16.6),
purple solid, were obtained (90%). TLC (CHCl3/MeOH:
8/2): Rf = 0.43. UV-vis (CHCl3): λmax, nm (ε × 10-3) 424
(76), 523 (7.1), 572 (7.2), 595 (5.4), 656 (4.7). 1H NMR
(CDCl3): δ, ppm 9.67 (s, broad, 2H, H2,6-pyridyl), 8.84 (d,
J = 4.7 Hz, 2H, Hb-pyrrolic), 8.73 (d, J = 4.4 Hz, 2H, H3,5-pyridyl),
8.68 (br s, 4H, Hb-pyrrolic), 8.61 (d, J = 4.7 Hz, 2H, Hb-pyrrolic),
7.91 (d, J = 8.2 Hz, 2H, H2,6-aryl), 7.18 (s, 4H, Hmesityl),
6.99 (d, J = 8.2 Hz, 2H, H3,5-aryl), 5.03 (s, 3H, Nmethyl), 2.54
(s, 6H, CH3,p-mesityl), 1.73 (s, 12H, CH3,o-mesityl), -2.58 (br
s, 2H, NHint.). MS (MALDI): m/z for C50H46N6O, calcd.
746.37, found 729.20 [M - OH]+.
1
552 (10.1), 590 (5.8), 648 (4.8). H NMR (DMSO): δ,
ppm 8.98 (d, 2H, J = 4.5 Hz, Hb-pyrrolic), 8.83 (br s, 6H,
Hb-pyrrolic), 8.20 (d, 6H, J = 8.0 Hz, H3,5-phenyl), 8.07 (d, 6H,
J = 8.0 Hz, H2,6-phenyl), 7.89 (d, 2H, J = 8.2 Hz, H2,6-aryl),
7.02 (d, 2H, J = 8.2 Hz, H3,5-aryl), 5.61 (br s, 2H, NH2),
-2.86 (br s, 2H, NHint.). MS (MALDI): m/z for C44H28N5-
S3O9Na3, calcd. 935.07, found 936.09 [M + H]+, 1871.26
[M2 + H]+.
Porphyrin-triazine link
5-[4-((4,6-(bis)piperidyl)-1,3,5-triazinyl)-amino-
phenyl]-10,15,20-triphenyl porphyrin (6). Porphyrin 2
(35 mg, 56 µmol, 1 equiv.) in THF (5 mL) was cooled
at0°C,thencyanuricchloride(10.3mg,56µmol,1equiv.)
and N,N-diisopropyl ethylamine, DIPEA (12 µL,
67 µmol, 1.2 equiv.) were added. The mixture was kept
under magnetic stirring at room temperature. After 15
min, TLC analysis (petroleum ether/ethyl acetate: 2/1)
showed a complete disappearance of porphyrin 2 and
the formation of derivative 2a (Rf = 0.7). Product 2a was
not isolated, but it was further reacted with an excess of
piperidine (28 µL, 280 mmol, 5 equiv.) and 2.4 equiv. of
DIPEA were added then the reaction was heated at reflux
under stirring for 24 h. THF was removed under vacuum
and compound 6 was obtained after purification by pre-
parative TLC, petroleum ether/ethyl acetate: 8/2, (purple
solid, 39 mg, 78%). TLC (petroleum ether/ethyl acetate:
8/2): Rf = 0.46. UV-vis (CHCl3): λmax, nm (ε × 10-3) 420
5,15-bis(mesityl)-10-(4-nitrophenyl)-20-(4-pyridyl)
porphyrin (4). Meso-(mesityl)dipyrromethane (600 mg,
2.27 mmol, 2 equiv.) solubilized in anhydrous CH2Cl2
(227 mL), 172 mg of 4-nitrobenzaldehyde (1.135 mmol,
1 equiv.) and 108 µL of pyridine-4-carbaldehyde (1.135
mmol, 1 equiv.) were added. The mixture was bubbled
under argon for 15 min then 315 µL of trifluoroacetic
acid (4.1 mmol, 3.6 equiv.) was introduced dropwise.
The reaction was stirred under argon for 45 min. 560 mg
of p-chloranil (2.27 mmol, 2 equiv.) were added and
the reaction was kept under stirring for 90 min at room
temperature. After purification by chromatography on
silica gel (CHCl3), 155 mg of porphyrin 4 were collected
(purple solid, 18.2%). TLC (CHCl3/EtOH: 98/2): Rf =
0.45. UV-vis (CHCl3): λmax, nm (ε × 10-3) 419 (363), 515
(18.6), 550 (7.6), 590 (5.6), 648 (3.9). 1H NMR (CDCl3):
δ, ppm 8.96 (d, J = 5.1 Hz, 2H, H2,6-pyridyl), 8.68 (br s, 6H,
Hb-pyrrolic), 8.63 (d, J = 4.6 Hz, 2H, Hb-pyrrolic), 8.55 (d, J =
8.4 Hz, 2H, H3,5-aryl), 8.33 (d, J = 8.4 Hz, 2H, H2,6-aryl), 8.11
(d, J = 5.1 Hz, 2H, H3,5-pyridyl), 7.18 (s, 4H, Hmesityl), 2.56 (s,
6H, CH3,p-mesityl), 1.76 (s, 12H, CH3,o-mesityl), -2.73 (br s, 2H,
NHint.). MS (MALDI): m/z for C49H40N6O2, calcd. 744.32,
found 745.33 [M + H]+.
5-(4-aminophenyl)-10,20-bis(mesityl)-15-(4-N-
methylpyridinium)porphyrin (5). To a solution of 4
(76 mg, 0.1 mmol, 1 equiv.) in anhydrous DMF (10 mL),
an excess of iodomethane (62 µL, 1 mmol, 10 equiv.) was
added under argon atmosphere. The mixture was kept
under magnetic stirring at room temperature for 24 h.
After precipitation with diethyl ether and filtration, the
corresponding cationic nitro porphyrin was obtained with
94% yield (87 mg). Then, the procedure was the same as
for synthesis of product 2. To a solution of cationic nitro
porphyrin (71 mg, 80 µmol, 1 equiv.) in CHCl3 (10 mL),
a solution of SnCl2 (54 mg, 240 µmol, 3 equiv.) in 10 mL
37% HCl was added. Acetic acid (10 mL) was added to
form homogeneous solution and resulting mixture was
heated at 70–80 °C, overnight under stirring. Reaction was
stopped by neutralization with 1 M NaOH (100 mL). The
mixture solution was washed with water (2 × 100 mL),
dried on MgSO4 and filtered. 54 mg of porphyrin 5,
1
(723), 516 (18.4), 552 (11.8), 591 (6.1), 647 (4.6). H
NMR (CDCl3): δ, ppm 8.95 (d, 2H, J = 4.7 Hz, Hb-pyrrolic),
8.84 (d, 6H, J = 5.1 Hz, Hb-pyrrolic), 8.21 (d, 6H, J = 7.0 Hz,
H2,6-phenyl), 8.14 (d, 2H, J = 8.4 Hz, H3,5-aryl), 8.0 (d, 2H, J =
8.4 Hz, H2,6-aryl), 7.76 (m, 9H, H3,4,5-phenyl), 7.07 (s, broad,
1H, NH), 3.94 (s, 8H, Ha-piperidyl), 1.66 (s, 12H, Hb,g-piperidyl),
-2.75 (br s, 2H, NHint.). MS (MALDI): m/z for C57H50N10,
calcd. 874.42, found 875.14 [M + H]+.
5-[4-((4,6-(bis)sulfanilatyl)-1,3,5-triazinyl)-amino-
phenyl]-10,15,20-tri(4-sulfonatophenyl)porphyrin
(7). To a solution of porphyrin 3 (30 mg, 32 µmol) in
ultra pure water (10 mL) cooled at 0 °C were added THF
(1 mL), a solution of cyanuric chloride (7.1 mg, 38 µmol,
1.2 equiv.) and a saturated solution of Na2CO3 (2 mL).
After 30 min, the reaction was complete, as witnessed by
TLC analysis (CHCl3/MeOH/H2O: 6/4/1), showing the
formation of derivative 3a (Rf = 0.25). Product 3a was
not isolated, but it reacted with an excess of sulfanilic
acid (55 mg, 10 equiv.), prepared with 5 mL of saturated
solution of Na2CO3. The resulting mixture was stirred at
reflux for 24 h and the solution was dialyzed (cut-off:
1000 Da) for 48 h. Compound 7 (40.4 mg, 90%), red
solid, was obtained after lyophilization. TLC (CHCl3/
MeOH/H2O: 6/4/1): Rf = 0.19. UV-vis (MeOH): λmax, nm
(ε × 10-3) 415 (254), 519 (10.4), 557 (7.0), 586 (4.8), 641
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(3.4). 1H NMR (DMSO): δ, ppm 8.93 (d, 2H, J = 3.9 Hz,
Hb-pyrrolic), 8.86 (br s, 6H, Hb-pyrrolic), 8.20 (d, 12H, J = 7.6
Hz, Hphenyl), 8.07 (d, 8H, J = 7.9 Hz, Hsulfanilic), 7.85-7.60
(m, 4H, Haryl), -2.89 (br s, 2H, NHint.). MS (MALDI): m/z
for C59H37N10Na5O15S5, calcd. 1400.05, found 1401.09
[M + H]+.
with 1 mL of bacterial inoculum at cell density of approxi-
mately 106 colonies forming units/mL (CFU/mL), depos-
ited in a sterile Petri dish and then incubated at 37 °C
for 24 h under white light irradiation (fluence dose = 9.5
J/cm2, h = 10 cm) in wet atmosphere. Then, each sample
was removed and transferred into 20 mL of extraction
solution (Triton X-100, 0.5% v/v). After 15 min of gentle
stirring at room temperature, serial dilutions of this sus-
pension were prepared. One hundred µL of each dilution
were plated on Tryptic Soy agar plates. Colonies were
counted after 24 h incubation at 37 °C. Results were
expressed as the number of CFU per textile square.
Each test was repeated three times, and was conducted
along with necessary controls: after bacterial impregna-
tion, photosensitive textiles were processed immediately
(t = 0), another one was incubated 24 h at 37 °C in the
dark; unmodified cotton was used in the same conditions
(24 h at 37 °C in the dark and under white light irradia-
tion). Moreover, Triton X-100 used was tested in order to
determine its influence on bacterial viability; the extract-
ing agent Triton X-100 (solution at 0.5% v/v) had no
deleterious effect on S. aureus.
5-[4-((4,6-(bis)piperidyl)-1,3,5-triazinyl)-amino-
phenyl]-10,20-bis(mesityl)-15-(4-N-methylpyridin-
ium)porphyrin (8). Porphyrin 5 (43 mg, 58 µmol,
1 equiv.) in THF (10 mL) was cooled at 0 °C, then cya-
nuric chloride (11 mg, 58 µmol, 1 equiv.) and DIPEA
(12 µL, 70 µmol, 1.2 equiv.) were added. The mixture
was stirred for 30 min then was left to reach room tem-
perature. TLC analysis (CHCl3/MeOH: 9/1) showed a
complete disappearance of porphyrin 5 and the forma-
tion of derivative 5a. An excess of piperidine (57 µL,
580 mmol, 10 equiv.) and 2.4 equiv. of DIPEA were
added and the reaction was heated at reflux under stir-
ring for 24 h. THF was removed and compound 8 was
obtained after purification by preparative TLC, CHCl3/
MeOH: 9/1 (purple solid, 30 mg, 52%). TLC (CHCl3/
MeOH: 9/1): Rf = 0.34. UV-vis (CHCl3): λmax, nm (ε ×
10-3) 423 (146), 521 (12.7), 567 (11), 592 (8.2), 654 (6.6).
1H NMR (CDCl3): δ, ppm 9.73 (s, broad, 2H, H2,6-pyridyl),
8.85 (d, J = 4.6 Hz, 2H, Hb-pyrrolic), 8.73 (s, 2H, H3,5-pyridyl),
8.67 (br s, 4H, Hb-pyrrolic), 8.62 (d, J = 4.6 Hz, 2H, Hb-pyr-
rolic), 8.05 (d, J = 8.2 Hz, 2H, H3,5-aryl), 7.93 (d, J = 8.2 Hz,
2H, H2,6-aryl), 7.20 (s, 4H, Hmesityl), 5.05 (s, 3H, N-methyl),
3.76 (s, 8H, Ha-piperidyl), 2.54 (s, 6H, CH3,p-mesityl), 1.73
(s, 12H, CH3,o-mesityl), 1.57 (s, 12H, Hb,g-piperidyl), -2.60 (br
s, 2H, NHint.). MS (MALDI): m/z for C63H65N11O, calcd.
991.54, found 974.67 [M - OH]+.
CONCLUSION
In summary, we have obtained neutral, anionic and
cationic aminoporphyrins with an overall satisfying
yield. Moreover cyanuric chloride has been efficient in
grafting porphyrins onto cotton fabrics. The photoin-
activation preliminary results confirmed the interest in
studying these surfaces as potential photoactive materials
for antimicrobial applications. Antimicrobial evaluation
against other bacterial strains (like Gram negative bacte-
ria) and antifungal activity against Candida albicans are
in progress in our laboratory.
Grafting of cotton fabrics
Experimental conditions are similar to porphyrin-
triazine link characterization. From 10 mg of amin-
oporphyrins 2, 3 or 5, porphyrin derivatives 2a, 3a and
5a were obtained. Then, cotton fabrics (3.5 × 3.5 cm
squares), previously soaked in 0.5 M NaOH for 24 h,
were introduced. After 24 h of grafting reaction, modi-
fied surfaces were washed with DMF under reflux dur-
ing 24 h then dried at 100 °C for 1 h. Grafting yields
(determined by UV-vis titration) have been evalu-
ated to 57, 73 and 54% for aminoporphyrins 2, 3 and
5 respectively.
Acknowledgements
We thank Dr. Sandra Alves for MS analyses, Dr.
Michel Guilloton for help in editing this manuscript
and the ‘Conseil Régional du Limousin’ for financial
support.
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