**2.4 RPy-bonded dicationic P-porphyrins at** *meso* **position: (R'***m***)2P(RPyTpp)2+**

At first, 5,10,15-triphenyl-20-(4-pyridinyl)porphyrin (PyTpp) was prepared by reaction of pyrrole (1.55 mL), benzaldehyde (1.83 mL), and 4-formylpyridine (0.56 mL) in propanoic acid (100 mL) in an oil bath heated at 140°C for 1 h to give PyTpp (533 mg, 14%) [24]. PyTpp (101 mg) was reacted with phosphoryl chloride (POCl3, 2.0 mL) in pyridine (10 mL) in a pressure bottle heated at 180°C for 1 day to give dichloro[triphenyl(4-pyridinyl)porphyrinato]phosphorus chloride ([Cl2P(PyTpp)]Cl, 99.0 mg) in 81% yield. Substitution of the axial chloro ligand with a methoxo group was performed by refluxing [Cl2P(PyTpp)]Cl (82.7 mg) in MeOH (20 mL)-pyridine (0.25 mL) for 3 days until the Soret band shifted from 435 to 424 nm. Dimethoxo[5-(1-hexyl-4-pyridinio)-10,15,20-triphenyl-porphyrinato] phosphorus (V) dichloride ((Me)2P(HexPyTpp)2+) was prepared by reaction of [(MeO)2P(PyTpp)]Cl (62.0 mg) with 1-iodohexane (2 mL) in DMF (5 mL) in the presence of K2CO3 (19 mg) at 100°C for 2 h. (Me)2P(HexPyTpp)2+ was purified through anion exchange with chloride ions, as follows. An aqueous solution (10 mL) of AgBF4 (115 mg) was added to a MeCN-MeOH (1:1 v/v, 20 mL) solution of the porphyrins. After stirring for 24 h at room temperature, the solution was washed with water (100 mL) and an aqueous NaCl solution (100 mL) three times and subjected to precipitation with hexane (200 mL) [24].

**125**

*Photodynamic Inactivation of Escherichia coli with Cationic Porphyrin Sensitizers*

[Cl2P(PyTpp)]Cl (78–100 mg) was reacted with ethylene glycol derivatives (H(OCH2CH2)*m*OR', R' = Me, *n*-Bu, *n*-Hex, 5–7 mL) in MeCN (10 mL) in the presence of pyridine (0.75 mL) for 24 h to give bis(2-alkyloxyethoxo)- 5-(4-pyridinyl)-10,15,20-triphenylporphyrinatophosphorus (V) chloride ([(R'*m*)2P(PyTpp)]Cl) in 66–88%. Bis(2-methoxyethoxo)-5-(1-hexyl-4 pyridinyl)-10,15,20-triphenylporphyrinatophosphorus (V) bromide, chloride ((Me*1*)2P(HexPyTpp)2+) was prepared by reaction of [(Me*1*)2P(PyTpp)]Cl (51 mg)

with 1-iodohexane (2 mL) in DMF (5 mL) in the presence of K2CO3 (19 mg) in an oil bath heated at 100°C for 2 h. After anion-exchange, dichloride salt of (Me*1*)2P(HexPyTpp)2+ (27 mg, 78%) was obtained. Also, other *meso*-RPy-bonded dicationic P-porphyrins (61–90 mg) were reacted with MeI (1.2 mL) in DMF (7.5 mL) in the presence of K2CO3 (43 mg) by heating at 100°C for 24 h to give an *N*-methyl-substituted complex. After anion exchange, (Me1)2P(HexPyTpp)2+ (35 mg, 94%), (Bu*2*)2P(MePyTpp)2+ (13.7 mg, 32%), and (Hex*2*)2P(MePyTpp)2+

*E. coli* K-12 (IFO 3301) was cultured aerobically at 30°C for 8 h in a LB medium

suspended in physiological saline, resulting in a cell suspension of *E. coli*. The cell concentrations were determined using a calibration curve and turbidity quantified

PDI of *E. coli* was performed as follows. A phosphate buffer (0.1 M, pH 7.6) was prepared by dissolving Na2HPO4 (2.469 g) and NaH2PO4 (0.312 g) in 100 mL of water.

cells mL<sup>−</sup><sup>1</sup>

studied sensitizers (25–100 μM, 0.1 mL), and the phosphate buffer (0.1 M, pH 7.6, 8.9 mL) were introduced into L-type glass tubes, resulting in a buffer solution (10 mL)

dark conditions, the L-type glass tubes were set on a reciprocal shaker and shaken at 160 rpm at room temperature for 2 h [24]. And then the L-type glass tubes were irradiated using a fluorescent lamp (Panasonic FL-15ECW, Japan; wave length = 400–

temperature. A portion of the reaction mixture (0.1 mL) was taken up to 2 h at 20-min intervals and plated on LB plates. The LB plates were incubated for 30 h at 30°C.

The amount of the living cells (*B*) was defined as the average number of *E. coli* colonies that appeared after an incubation period of 30 h in three replicate plates. The *B* values for the PDI sensitizers were recorded at each irradiation time.

Incorporation of porphyrin sensitizers inside cells can be examined by fluorescence microscopy images of *E. coli* on a confocal laser scanning microscope (CLSM) under laser excitation at 543 nm. The aqueous solution containing the porphyrin sensitizers and *E. coli* was incubated for 3 h at 25°C. The concentrated solution was sandwiched between a cover slip and an agar pad on a bottom cover slip to maintain

cells mL<sup>−</sup><sup>1</sup>

723 nm; the maximum intensity: 545 nm; 10.5 W cm<sup>−</sup><sup>2</sup>

its position within the same focal plane [36].

the harvested cells were washed with physiological saline (NaCl, 7 g L<sup>−</sup><sup>1</sup>

by the absorbance measured at 600 nm on an UV–Vis spectrometer [24].

). After centrifugation of the cultured broth at 12,000 rpm for 10 min,

), yeast extract (5 g L<sup>−</sup><sup>1</sup>

, 1.0 mL), an aqueous solution of the

) on a reciprocal shaker at room

) and the studied sensitizers (0.25–1.0 μM). Under

), and NaCl

) and then

*DOI: http://dx.doi.org/10.5772/intechopen.82645*

(28.0 mg, 45%) were formed [24].

(10 g L<sup>−</sup><sup>1</sup>

**2.6 PDI of** *E. coli*

**2.5 Preparation of** *E. coli* **suspension**

The suspension of *E. coli* cells (1 × 105

containing *E. coli* (1 × 104

**2.7 Fluorescence imaging**

(pH 6.5) consisting of bactotryptone (10 g L<sup>−</sup><sup>1</sup>

*Photodynamic Inactivation of Escherichia coli with Cationic Porphyrin Sensitizers DOI: http://dx.doi.org/10.5772/intechopen.82645*

[Cl2P(PyTpp)]Cl (78–100 mg) was reacted with ethylene glycol derivatives (H(OCH2CH2)*m*OR', R' = Me, *n*-Bu, *n*-Hex, 5–7 mL) in MeCN (10 mL) in the presence of pyridine (0.75 mL) for 24 h to give bis(2-alkyloxyethoxo)- 5-(4-pyridinyl)-10,15,20-triphenylporphyrinatophosphorus (V) chloride ([(R'*m*)2P(PyTpp)]Cl) in 66–88%. Bis(2-methoxyethoxo)-5-(1-hexyl-4 pyridinyl)-10,15,20-triphenylporphyrinatophosphorus (V) bromide, chloride ((Me*1*)2P(HexPyTpp)2+) was prepared by reaction of [(Me*1*)2P(PyTpp)]Cl (51 mg) with 1-iodohexane (2 mL) in DMF (5 mL) in the presence of K2CO3 (19 mg) in an oil bath heated at 100°C for 2 h. After anion-exchange, dichloride salt of (Me*1*)2P(HexPyTpp)2+ (27 mg, 78%) was obtained. Also, other *meso*-RPy-bonded dicationic P-porphyrins (61–90 mg) were reacted with MeI (1.2 mL) in DMF (7.5 mL) in the presence of K2CO3 (43 mg) by heating at 100°C for 24 h to give an *N*-methyl-substituted complex. After anion exchange, (Me1)2P(HexPyTpp)2+ (35 mg, 94%), (Bu*2*)2P(MePyTpp)2+ (13.7 mg, 32%), and (Hex*2*)2P(MePyTpp)2+ (28.0 mg, 45%) were formed [24].

#### **2.5 Preparation of** *E. coli* **suspension**

*E. coli* K-12 (IFO 3301) was cultured aerobically at 30°C for 8 h in a LB medium (pH 6.5) consisting of bactotryptone (10 g L<sup>−</sup><sup>1</sup> ), yeast extract (5 g L<sup>−</sup><sup>1</sup> ), and NaCl (10 g L<sup>−</sup><sup>1</sup> ). After centrifugation of the cultured broth at 12,000 rpm for 10 min, the harvested cells were washed with physiological saline (NaCl, 7 g L<sup>−</sup><sup>1</sup> ) and then suspended in physiological saline, resulting in a cell suspension of *E. coli*. The cell concentrations were determined using a calibration curve and turbidity quantified by the absorbance measured at 600 nm on an UV–Vis spectrometer [24].

#### **2.6 PDI of** *E. coli*

*The Universe of Escherichia coli*

35%). The reaction of (Py3Sb(Tpp)+

shifted to 418 nm, respectively. Thus, bis[3-(4-pyridyl)propoxo]tetraphenyl-

90 mg) were obtained in 50% and 43% yields, respectively. (Py3)2Sb(Tpp)+

propoxo(methoxo)tetraphenylporphyrinatoantimony (V) bromide (Py3Sb(Tpp)<sup>+</sup>

(50 mg) was reacted with 1-bromohexane (0.5 mL) in MeCN (13 mL) at reflux temperature for about 24 h to give bis[3-(1-hexyl-4-pyridinio)-1-propoxo]-5,10,15,20 tetraphenylporphyrinatoantimony (V) tribromide ((HexPy3)2Sb(Tpp)3+, 20 mg,

in MeCN (13 mL) at reflux temperature for about 24 h gave α-(methoxo)-β-[3(1- -methyl-4-pyridinio)-1-propoxo]-5,10,15,20-tetraphenylporphyrinatoantimony (V) dibromide (MePy3Sb(Tpp)2+, 25 mg, 42%) and α-(methoxo)-β-[3 (1-hexyl-4-pyridinio)-1-propoxo]-5,10,15,20-tetraphenyl-porphyrinatoantimony (V)

, 83 mg) and 3-(4-pyridyl)

, 50 mg) with MeI and 1-bromohexane (0.5 mL

). The (Br5)2P(Tpp)+

(50 mg)

,

porphyrinatoantimony (V) bromide ((Py3)2Sb(Tpp)+

dibromide (HexPy3Sb(Tpp)2+, 20 mg, 25%), respectively [24].

porphyrinatophosphorus(V) chloride ((Br5)2P(Tpp)+

subjected to precipitation with hexane (200 mL) [24].

**2.3 Axially RPy-bonded tricationic P-porphyrins: (RPy5)2P(Tpp)3+**

Bis[5-(3-alkyl-1-pyridinio)-3-oxapentyloxo]tetraphenylporphyrinatophosphorus(V) dibromide, chloride ((RPy5)2P(Tpp)3+) was prepared from dihydroxo(tetraphenylporphyrinato)phosphorus chloride ([(HO)2P(Tpp)]Cl), which was prepared by hydrolysis of [Cl2P(Tpp)]Cl (300 mg) by refluxing in a mixed solvent of MeCN (160 mL) with pyridine (60 mL) and H2O (60 mL) [22]. Alkylation of [(HO)2P(Tpp)]Cl (80 mg) with di(2-bromoethyl) ether (1 mL) was performed in the presence of K2CO3 (19 mg) and 18-crown-6 ether (4.2 mg) in MeCN (5 mL) at 50°C to give bis(5-bromo-3-oxa-pentyloxo)tetraphenyl-

was reacted with 3-alkylpyridine (1.0 mL) in MeCN (10 mL) under heating at 100°C for 20–68 h for the preparations of (RPy5)2P(Tpp)3+ [22]. Similarly, bis[5-(4-ethyl-1-pyridinio)-3-oxapentyloxo]tetraphenylporphyrinatophosphor us(V) dibromide, chloride, (4EtPy5)2P(Tpp)3+ was prepared via the reaction of

**2.4 RPy-bonded dicationic P-porphyrins at** *meso* **position: (R'***m***)2P(RPyTpp)2+**

At first, 5,10,15-triphenyl-20-(4-pyridinyl)porphyrin (PyTpp) was prepared by reaction of pyrrole (1.55 mL), benzaldehyde (1.83 mL), and 4-formylpyridine (0.56 mL) in propanoic acid (100 mL) in an oil bath heated at 140°C for 1 h to give PyTpp (533 mg, 14%) [24]. PyTpp (101 mg) was reacted with phosphoryl chloride (POCl3, 2.0 mL) in pyridine (10 mL) in a pressure bottle heated at 180°C for 1 day to give dichloro[triphenyl(4-pyridinyl)porphyrinato]phosphorus chloride ([Cl2P(PyTpp)]Cl, 99.0 mg) in 81% yield. Substitution of the axial chloro ligand with a methoxo group was performed by refluxing [Cl2P(PyTpp)]Cl (82.7 mg) in MeOH (20 mL)-pyridine (0.25 mL) for 3 days until the Soret band shifted from 435 to 424 nm. Dimethoxo[5-(1-hexyl-4-pyridinio)-10,15,20-triphenyl-porphyrinato] phosphorus (V) dichloride ((Me)2P(HexPyTpp)2+) was prepared by reaction of [(MeO)2P(PyTpp)]Cl (62.0 mg) with 1-iodohexane (2 mL) in DMF (5 mL) in the presence of K2CO3 (19 mg) at 100°C for 2 h. (Me)2P(HexPyTpp)2+ was purified through anion exchange with chloride ions, as follows. An aqueous solution (10 mL) of AgBF4 (115 mg) was added to a MeCN-MeOH (1:1 v/v, 20 mL) solution of the porphyrins. After stirring for 24 h at room temperature, the solution was washed with water (100 mL) and an aqueous NaCl solution (100 mL) three times and

(63 mg) with 4-ethylpyridine (1.0 mL) in dry MeCN (10 mL) at

**124**

(Br5)2P(Tpp)+

100°C for 20 h.

PDI of *E. coli* was performed as follows. A phosphate buffer (0.1 M, pH 7.6) was prepared by dissolving Na2HPO4 (2.469 g) and NaH2PO4 (0.312 g) in 100 mL of water. The suspension of *E. coli* cells (1 × 105 cells mL<sup>−</sup><sup>1</sup> , 1.0 mL), an aqueous solution of the studied sensitizers (25–100 μM, 0.1 mL), and the phosphate buffer (0.1 M, pH 7.6, 8.9 mL) were introduced into L-type glass tubes, resulting in a buffer solution (10 mL) containing *E. coli* (1 × 104 cells mL<sup>−</sup><sup>1</sup> ) and the studied sensitizers (0.25–1.0 μM). Under dark conditions, the L-type glass tubes were set on a reciprocal shaker and shaken at 160 rpm at room temperature for 2 h [24]. And then the L-type glass tubes were irradiated using a fluorescent lamp (Panasonic FL-15ECW, Japan; wave length = 400– 723 nm; the maximum intensity: 545 nm; 10.5 W cm<sup>−</sup><sup>2</sup> ) on a reciprocal shaker at room temperature. A portion of the reaction mixture (0.1 mL) was taken up to 2 h at 20-min intervals and plated on LB plates. The LB plates were incubated for 30 h at 30°C.

The amount of the living cells (*B*) was defined as the average number of *E. coli* colonies that appeared after an incubation period of 30 h in three replicate plates. The *B* values for the PDI sensitizers were recorded at each irradiation time.

#### **2.7 Fluorescence imaging**

Incorporation of porphyrin sensitizers inside cells can be examined by fluorescence microscopy images of *E. coli* on a confocal laser scanning microscope (CLSM) under laser excitation at 543 nm. The aqueous solution containing the porphyrin sensitizers and *E. coli* was incubated for 3 h at 25°C. The concentrated solution was sandwiched between a cover slip and an agar pad on a bottom cover slip to maintain its position within the same focal plane [36].
