Effects of pH on aggregation and photodynamic activities of cationic zinc phthalocyanines substituted with amides

Journal of Photochemistry and Photobiology A: Chemistry 288 (2014) 1–12

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Effects of pH on aggregation and photodynamic activities of cationic zinc phthalocyanines substituted with amides Ao Wang, Xianglan Chen, Liu Zhang, Guangyi Zhang, Lin Zhou, Shan Lu, Jiahong Zhou, Shaohua Wei ∗ School of Chemistry and Materials Science, Jiangsu Key Laboratory of Biofunctional Materials, Key Laboratory of Applied Photochemistry, Nanjing Normal University, Nanjing 210046, PR China

a r t i c l e

i n f o

Article history: Received 18 March 2014 Received in revised form 5 May 2014 Accepted 8 May 2014 Available online 17 May 2014 Keywords: Cationic phthalocyanines pH Aggregate Photodynamic activity Amide Tautomeric transformation

a b s t r a c t This work reports on the effects of pH (especially base addition) on the properties of two cationic phthalocyanines substituted with amide groups. The absorption spectra, photo-stability, singlet oxygen generation ability and photodegradation of CT DNA of the two cationic phthalocyanines in different pH were studied and compared. Results indicated that the base additive can promote a shift in the amide/imidohydrine balance toward imidohydrine, which resulted in the reduction of aggregate and enhancement of photodynamic activities. This base induced tautomeric transformation could be a novel way to generate low aggregated and high photodynamic efficiency cationic Pcs. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Being a class of synthetic macrocyclic compounds with intriguing physical, chemical, and biological properties, phthalocyanines (Pcs) have received considerable attention and have found applications in various fields including in medicine, materials science and nanotechnology [1–6]. Among these various applications, Pcs have received particular interest as a new generation of photosensitizers for photodynamic therapy (PDT) owing to their desirable features, such as high molecular absorption coefficients at phototherapeutic window (ε ≈ 105 l mol−1 cm−1 at 600–900 nm), high chemical stability, high efficiency in singlet oxygen generation and low dark toxicity [7–10]. Although Pcs possess so many desirable features, the poor water solubility hamper their delivery in vivo. Besides, Pcs tend to form aggregate in water due to their extended ␲-systems usually give rise to strong ␲-stacking [11,12]. Normally, the aggregation phenomena of Pcs would reduce their photochemical and photobiological activities which finally limit their potential applications in PDT [13,14]. To generate hydrophilic and nonaggregated Pcs, water-soluble moieties and bulky fragments with

∗ Corresponding author. Tel.: +86 025 85891761; fax: +86 025 85891761. E-mail address: [email protected] (S. Wei). http://dx.doi.org/10.1016/j.jphotochem.2014.05.003 1010-6030/© 2014 Elsevier B.V. All rights reserved.

terminal hydrophilic groups can be introduced at the peripheral or axial positions [15]. Cationic Pcs are well known for their enhanced water-solubility and cellular uptake efficiency [16–18]. However, for some cationic Pcs, the aggregation phenomenon in aqueous systems still existed [19–22]. It is well known that the pH environment can change the solubility and aggregation tendency of the photosensitizers, thereby affecting their photodynamic activities [23,24]. For example, Ng and co-workers have reported a series of Pcs-polyamine conjugates as pH-responsive photosensitizers for photodynamic therapy [25,26]. However, to our knowledge, no reports indicated that pH can affect the aggregate degree of cationic Pcs. The cationic Pcs were always considered to be less affected by pH because their pH- sensitive amine groups have been methylated. However, if some other functional groups of such cationic Pcs can be affected by pH, their aggregation degree and photodynamic activities could also be regulated. It is well known that the tautomeric transformation between amide and imidohydrine always exist in water. While, because of the instability of imidohydrine in thermodynamics and dynamics, it cannot exist under normal conditions. Yet, the base addition may promote a shift in the amide/imidohydrine balance toward imidohydrine-dominate. Furthermore, if the imidohydrin could conjugate with the extend ␲-system of Pc, it will exist stably in water. So, we suppose that if a cationic Pc was amide substituted, its existing form can be changed

2

A. Wang et al. / Journal of Photochemistry and Photobiology A: Chemistry 288 (2014) 1–12

by adjusting the pH to alkaline. The transfer from amide to imidohydrine may affect the intermolecular hydrogen bond and molecular arrangement of cationic Pc, which finally affect its aggregate tendency. To prove such hypothesis, in this work, we synthesized two cationic Pcs substituted with amides ZnPc1 (Quaternized 2(3), 9(10), 16(17), 23(24)-tetra-(4-(N-(2-amino) propanamide) amino) phenoxy) phthalocyaninato-zinc (II)) and ZnPc2 (Quaternized 2(3), 9(10), 16(17), 23(24)-tetra-(4-(N-(2-amino) propanamide) methylamino)phenoxy) phthalocyaninato-zinc (II)). The effects of pH (especially base addition) on the properties of the two cationic Pcs such as aggregation, photostability, singlet oxygen generation ability and efficiency of CT-DNA photodegradation were studied and compared. The results showed that the base additive can significantly reduce aggregation and enhance photodynamic activities of ZnPc1, while had little but negative effects on ZnPc2.

otherwise stated. Disodium salt of 9, 10-anthracenedipropionic acid (ADPA) and CT-DNA were purchased from Sigma–Aldrich. 2.2. Equipment and characteristics The Pcs were prepared as stock solutions and diluted to the final concentrations with water for all the experiments. All data of ZnPc1 were measured until it reached its dynamic stability. Absorption spectra were recorded on spectrophotometer Cary 5000, Varian. Fluorescence spectra were recorded on Perkin Elmer LS 50B fluorescence spectrophotometer. The pH values were measured on a FE20/EL20 pH meter, Mettler Toledo. Infrared spectra were measured in KBr pellets on IR-Spectrometer Nicolet Nexus 670. 1 H NMR and 13 C NMR spectra were recorded on a Bruker Advance 400 MHz NMR spectrometer. Elemental analyses were taken with Vario MICRO, Elementar. Photo-irradiation for singlet oxygen determinations and photo-stability were done using a 665 nm LED.

2. Experimental 2.3. Synthesis 2.1. Materials All of the necessary organic solvents were of analytical grade and used after purified according to the reported procedures [27]. The water was triply distilled and the pH of it was 6.81 at the room temperature. The necessary chemicals were obtained from the commercial suppliers and used without further purification unless

ZnPc1 was prepared by a five-step procedure (Figs. 1 and 2). First, l-alanine was reacted with (chloromethanetriyl) tribenzene (TrCl) to obtain the N-Trityl alanine 1 [28,29]. Then, compound 1 was reacted with 4-aminophenol using N-(3Dimethylaminopropyl)-N -ethylcarbodiimide hydrochloride (EDC· HCl) and 1-Hydroxybenzotriazole (HOBT) as the bridging agent

Fig. 1. Synthetic route for the preparation of phthalonitriles 3 and 5.

A. Wang et al. / Journal of Photochemistry and Photobiology A: Chemistry 288 (2014) 1–12

to obtain compound 2. After then, the phthalonitrile 3 was prepared by the reaction of 4-nitrophthalonitrile with compound 2. In the presence of 1, 8-diazabicyclo [5,4, 0]-undec-7-ene (DBU), phthalonitrile 3 reacted with zinc acetate to give a ZnPc intermediate. The ZnPc3 was prepared by removing the N-Trityl protecting group with CF3 COOH. Finally, ZnPc1 was obtained by methylation of ZnPc3 in CH3 OH. The ZnPc2 was prepared according to ZnPc1, but with a little modification (Figs. 1 and 2). First 4-(aminomethyl) phenol and 4nitrophthalonitrile were reacting using K2 CO3 as the base catalyst to obtain phthalonitrile 4. Then, compound 4 was reacted with compound 1 to give the Pc precursor 5. After then, zinc acetate and 5 were employed to give N-Trityl ZnPc. By using CF3 COOH to remove protecting groups, ZnPc4 was obtained. Finally, according to the method described for ZnPc1, ZnPc2 was obtained by methylation of ZnPc4 in CH3 OH.

3

Addition of triethylamine (2.35 mL, 16.83 mmol) was followed by a solution of (chloromethanetriyl) tribenzene (1.56 g, 5.61 mmol) in CHCl3 (15 mL). The solution was stirred for 2 h at room temperature, and then excess CH3 OH was added. After evaporation of the solvent, the white solid was redissolved in CH2 Cl2 (20 mL), and washed with distilled water (3× 15 mL) and saturated brine (2× 15 mL). The organic layer was dried over anhydrous Na2 SO4 . The crude product was purified by silica gel column chromatography using petroleum ether/ethyl acetate (2:1, v/v) as the eluent to afford 1 as a white solid (1.42 g, 76.3%). M.P. = 156 ◦ C (156–157 ◦ C in the literature [28,29]). 1 H NMR (400 MHz, DMSO-d6 ): ı (ppm) 12.56 (s, 1H, COOH) 7.43 (t, 6H, 4.4 Hz, ArH), 7.26–7.30 (m, 6H, ArH), 7.17–7.21 (m, 3H, ArH), 3.07–3.12 (m, 1H, CH), 1.09 (d, 3H, 6.8 Hz, CH3 ). 13 C NMR (100 MHz, CDCl3 ): ı (ppm) 179.58, 145.39, 128.71, 128.04, 126.81, 71.56, 52.40, 20.96. 2.3.2. N-(4-hydroxyphenyl)-2-(tritylamino) propanamide (2, Fig. 1) A mixture of 1 (0.60 g, 1.81 mmol), N-(3-Dimethylaminopropyl)-N -ethylcarbodiimide hydrochloride (EDC·HCl, 0.42 g, 2.17 mmol), 1-Hydroxybenzotriazole (HOBT, 0.29 g, 2.17 mmol)

2.3.1. 2-(Tritylamino) propanoic acid (1, Fig. 1, [28,29]) To a stirred suspension of l-alanine (0.5 g, 5.61 mmol) in 18 mL of CHCl3 /CH3 CN (5:1) was added Me3 SiCl (1.08 mL, 8.42 mmol). The solution was refluxed for 5 h and then cooled to room temperature.

R

R N 3

Zn(OAc)2, DBU

5

n-pentanol

CF3COOH (1) NaOH (2)

N N

N N

Zn N

N N

R

R

R3

O

O H N C

H N

CH3 C H

NH2

R4

O

ZnPc3

O

CH3

C

C H

NH2

ZnPc4 CH3OH

CH3I, K2CO3 R

R N N N

N N

Zn N

N N

R

R

R1

O

O H N C ZnPc1

H N

CH3 C H

N

I-

R2

O

O

CH3

C

C H

ZnPc2

Fig. 2. Synthetic route for the preparation of Pcs ZnPc1, ZnPc2, ZnPc3 and ZnPc4.

N

I-

4

A. Wang et al. / Journal of Photochemistry and Photobiology A: Chemistry 288 (2014) 1–12

3300 (OH), 1654 (C O), 1519 (NH), 1443, 1223, 851, 698. 1 H NMR (400 MHz, DMSO-d6 ): ı (ppm) 9.07 (s, 1H, NH), 9.06 (s, 1H, OH), 7.44 (d, 6H, 7.2 Hz, ArH), 7.25 (t, 6H, 7.6 Hz, ArH), 7.12 (t, 3H, 7.4 Hz, ArH), 6.98–7.01 (m, 2H, ArH), 6.56–6.59 (m, 2H, ArH), 3.12 (d, 1H, 8.8 Hz, CH), 1.15–1.19 (m, 3H, CH3 ). 13 C NMR (100 MHz, DMSO-d6 ): ı (ppm) 174.11, 153.24, 145.17, 129.54, 128.75, 128.20, 127.14, 122.04, 115.52, 71.94, 54.36, 21.62. Anal. Calcd. For C28 H26 N2 O2 : C, 79.59; H, 6.20; N, 6.63. Found: C, 79.30; H, 6.21; N, 6.77.

0.6

ZnPc1 ZnPc2

Absorbance

0.5 0.4 0.3 0.2 0.1 0.0 300

400

500

600

700

800

Wavelength(nm) Fig. 3. Absorption spectra of ZnPc1 and ZnPc2 in water (C = 5 × 10−6 M).

and triethylamine (1 mL, 7.17 mmol) in CH2 Cl2 (15 mL) was stirred at room temperature for 1 h. Then 4-aminophenol (0.20 g, 1.81 mmol) in DMF (2 mL) was added dropwise. After stirring overnight, the solvent was evaporated. The ice water (20 mL) was poured into residue, and then, HCl was added until the pH reached 7. The mixture was extracted with CH2 Cl2 (3× 15 mL), and the combined organic portion was dried over anhydrous Na2 SO4 . The crude product was purified by silica gel column chromatography using petroleum ether/ethyl acetate (1:1, v/v) as the eluent to afford 2 as a white solid (0.65 g, 85%). M.P. = 104 ◦ C. IR (KBr, cm−1 ):

2.3.3. N-(4-(3, 4-dicyanophenoxy) phenyl)-2-(tritylamino) propanamide (3, Fig. 1) Under nitrogen atmosphere, a mixture of 2 (0.50 g, 1.18 mmol), 4-Nitrophthalonitrile (0.21 g, 1.18 mmol) and finely ground anhydrous K2 CO3 (0.33 g, 2.37 mmol) in DMF (10 mL) was heated at 40 ◦ C for 4 h. The mixture was cooled and then poured into water (100 mL). After filtration, the cake was washed with distilled water (3× 30 mL). The crude product was purified by silica gel column chromatography using petroleum ether/ethyl acetate (2:1, v/v) as the eluent to afford 3 as a white solid (0.59 g, 90.9%). M.P. = 110 ◦ C. IR (KBr, cm−1 ): 3330, 2235 (CN), 1680 (C O), 1586, 1500 (NH), 1256, 1205, 859, 707. 1 H NMR (400 MHz, DMSO-d6 ): ␦ (ppm) 9.44 (s, 1H, O CNH), 8.08 (d, 1H, 8.8 Hz, ArH), 7.69 (d, 1H, 2.4 Hz, ArH), 7.46 (t, 6H, 4.2 Hz, ArH), 7.31–7.35 (m, 3H, ArH), 7.13–7.29 (m, 6H, ArH), 7.12 (t, 2H, 7.2 Hz, ArH), 7.02–7.05 (m, 2H, ArH), 3.14 (d, 1H, 8.8 Hz, CH), 1.23 (d, 3H, 7.2 Hz, CH3 ). 13 C NMR (100 MHz, DMSOd6 ): ı (ppm) 174.01, 162.04, 148.88, 146.55, 137.18, 136.74, 128.95, 128.20, 126.71, 122.59, 121.85, 121.29, 120.96, 117.08, 116.39,

0.5

A

0.5

0.4

A

Redistilled water acid base

Absorbance

0.4 Absorbance

aggregate 0.3

monomer 0.2

0.3

0.2

0.1 0.1

0.0

0.0 300

400

500

600

700

300

800

500

600

700

800

700

800

Wavelength(nm)

Wavelength(nm)

0.5 0.48

0.165

B

0.4

0.160

B

Redistilled water acid base

0.44

aggregate (641 nm)

0.150

0.42 0.145 0.140 0.135 0

5

10

15

20

Absorbance

monomer (700 nm)

Absorbance

0.46

0.155

Absorbance

400

0.3

0.2

0.40

0.1

0.38

0.0

25

Time(min) Fig. 4. (A) UV–vis spectra changes of ZnPc1 in water over times (0–25 min). (B) The absorption change of monomer (700 nm) and aggregate (641 nm) of ZnPc1 over times (0–25 min).

300

400

500

600

Wavelength(nm) Fig. 5. Change in absorption spectrum of (A) ZnPc1 and (B) ZnPc2 upon adding base and acid (>20 ␮M) in water.

A. Wang et al. / Journal of Photochemistry and Photobiology A: Chemistry 288 (2014) 1–12

115.87, 108.20, 71.29, 53.41, 22.45. Anal. Calcd. For C36 H28 N4 O2 : C, 78.81; H, 5.14; N, 10.21. Found: C, 79.02; H, 5.20; N, 10.11. 2.3.4. 4-(4-(aminomethyl) phenoxy)phthalonitrile (4, Fig. 1) Under nitrogen atmosphere, a mixture of 4-(aminomethyl) phenol (1.00 g, 8.12 mmol), 4-Nitrophthalonitrile (1.41 g, 8.12 mmol) and finely ground anhydrous K2 CO3 (2.24 g, 16.24 mmol) in DMF (10 mL) was heated at 40 ◦ C for 5 h. The mixture was cooled and poured into ice water. After filtration, the cake was washed with water and then purified by silica gel column chromatography using methanol/ethyl acetate (1:3, v/v) as the eluent to give 4 as a sallow solid (1.20 g, 59.4%). M.P. =82 ◦ C. IR (KBr, cm−1 ): 3378, 3320, 3050, 2235 (CN), 1595, 1480, 1250, 1200, 710. 1 H NMR (400 MHz, DMSOd6 ): ı (ppm) 8.04 (d, 1H, 8.8 Hz, Ar), 7.74 (s, 1H, Ar), 7.46 (d, 2H, 8.0 Hz, Ar), 7.34 (t, 1H, 4.2 Hz, Ar), 7.14 (d, 2H, 8.0 Hz, Ar), 3.76 (s, 2H, CH2 ). 13 C NMR (100 MHz, DMSO-d6 ): ı (ppm) 161.86, 152.45, 142.01, 136.74, 129.69, 122.81, 122.05, 120.59, 117.10, 116.39, 115.87, 108.32, 45.29. Anal. Calcd. For C15 H11 N3 O: C, 72.28; H, 4.45; N, 16.86. Found: C, 72.10; H, 4.65; N, 16.80. 2.3.5. 4-(4-(Aminomethyl) phenoxy)phthalonitrile (5, Fig. 1) A mixture of 1 (0.73 g, 2.2 mmol), 4 (0.55 g, 2.2 mmol), EDC·HCl (0.51 g, 2.64 mmol), HOBT (0.36 g, 2.64 mmol) and triethylamine (0.62 mL, 4.4 mmol) in CH2 Cl2 (15 mL) was stirred at room temperature for 5 h. The mixture was washed with distilled water (3× 10 mL) and saturated brine (2× 10 mL). After drying by Na2 SO4 , the crude product was purified by silica gel column chromatography

using ethyl acetate/petroleum ether (1:1, v/v) as the eluent to afford 5 as a pale yellow solid (1.12 g, 90.3%). M.P. = 94 ◦ C. IR (KBr, cm−1 ): 3404, 2237 (CN), 1654 (C O), 1595, 1485, 1248, 1205 715. 1 H NMR (400 MHz, CDCl ): ı (ppm) 7.72 (d, 1H, 8.8 Hz, HNC O), 3 7.32–7.38 (m, 6H, Ar), 7.04–7.31 (m, 14H, Ar), 7.01–7.03 (m, 2H, Ar), 4.13 (s, 2H, CH2 ), 3.39–3.42 (m, 1H, CH), 1.18 (d, 3H, 7.2 Hz, CH3 ). 13 C NMR (100 MHz, CDCl3 ): ı (ppm) 175.96, 161.75, 152.67, 145.48, 136.91, 135.41, 129.79, 128.79, 128.07, 126.98, 121.45, 121.39, 120.74, 117.68, 115.35, 114.93, 108.90, 71.89, 54.00, 21.78. Anal. Calcd. For C36 H28 N4 O2 : C, 78.81; H, 5.14; N, 10.21. Found: C, 78.40; H, 5.34; N, 10.12. 2.3.6. ZnPc3 (Fig. 2) A mixture of compound 3 (0.6 g, 1.09 mmol), Zn(OAc)2 (0.13 g, 0.68 mmol) and DBU (0.4 mL, 2.67 mmol) in n-pentanol (10 mL) was heated at 140 ◦ C for 24 h under nitrogen atmosphere. The solution was cooled and then poured into CH3 OH to afford crude green solid. After filtration, the crude product was purified by silica gel column chromatography using CH3 OH/CH2 Cl2 (1:40, v/v) as the eluent. After evaporating the eluent in vacuo, the blue solid was redissolved in CH2 Cl2 (10 mL). Under the condition of ice-water bath, excess trifluoroacetic acid (TFA) (0.5 mL) was added. The mixture was heated to room temperature and stirred for another 2 h. After filtration, the cake was washed with CH2 Cl2 till the filtrate was colorless. Thereafter, the dark blue product was dissolved in water and precipitated by adjusted pH to 9–10. The residue product collected by filtration was washed successively with water. The product was

0.5

0.60

NaOH:ZnPc1 0:1 1:1 2:1 3:1 4:1 5:1 6:1 7:1 8:1

0.3

0.2

0.1

NaOH:ZnPc2 0:1 1:1 2:1 3:1 4:1 5:1 6:1 7:1 8:1

C 0.45

Absorbance

A

0.4

Absorbance

5

0.30

0.15

0.0

0.00

300

400

500

600

700

800

300

400

500

Wavelength(nm)

600

700

800

Wavelength(nm) 0.43

0.45

monomer aggregate

B

0.40

D

0.42 0.41

Absorbance

Absorbance

0.35 0.30 0.25

0.40 0.39 0.38 0.37

0.20

0.36 0.35

0.15 0

1

2

3

4

5

Base(equiv)

6

7

8

9

0

1

2

3

4

5

6

7

8

9

Base(equiv)

Fig. 6. Spectra change of (A) ZnPc1 and (C) ZnPc2 upon continues addition of base; the change of Q-band of (B) ZnPc1 and (D) ZnPc2 versus the ratios of the base.

6

A. Wang et al. / Journal of Photochemistry and Photobiology A: Chemistry 288 (2014) 1–12

vacuum-dried for 12 h to afford ZnPc3 as a dark green solid (0.15 g, 42.5%). M.P. >200 ◦ C. IR (KBr, cm−1 ): 3404, 1650 (C O), 1519 (NH), 1390, 1330, 1224, 1104, 614. 1 H NMR (400 MHz, DMSO-d6 with a drop of CF3 COOD): ı (ppm) 8.83 (d, 4H, 6.8 Hz, NH), 8.38 (t, 8H, 13.2 Hz, ArH), 7.87–7.92 (m, 8H, PcH), 7.69 (d, 4H, 12 Hz, PcH), 7.49–7.57 (m, 8H, ArH), 4.13 (s, 4H, CH), 1.57 (s, 12H, CH3 ). 13 C NMR (100 MHz, DMSO-d ): ı (ppm) 167.72, 162.06, 158.89, 6 153.46, 152.87, 136.76, 133.21, 133.01, 130.04, 129.08, 121.17, 120.98, 118.56, 110.95, 110.65, 50.06, 49.48, 21.60, 17.66. Anal. Calcd. For C68 H56 N16 O8 Zn: C, 63.28; H, 4.37; N, 17.36. Found: C, 62.90; H, 4.66; N, 17.30.

was vacuum-dried to afford ZnPc4 as a dark green solid (0.17 g, 33.8%). M.P. >200 ◦ C. IR (KBr, cm−1 ): 3370, 3311, 1654, 1603, 1476, 1384, 1341, 1231, 1079, 944. 1 H NMR (400 MHz, DMSO-d6 with a drop of CF3 COOD): ı (ppm) 8.86 (s, 4H, NHC O), 8.45 (d, 4H, 9.6 Hz, PcH), 8.23 (d, 4H, 14.4 Hz, PcH), 7.67–7.73 (m, 4H, PcH), 7.44–7.58 (m, 16H, Ar), 4.51 (d, 8H, 4.8 Hz, CH2 ), 3.98 (t, 4H, 5.2 Hz, CH), 1.47 (t, 12H, 5.8 Hz, CH3 ). 13 C NMR (100 MHz, DMSO-d6 ): ı (ppm) 170.02, 165.02, 157.56, 153.24, 132.22, 130.43, 129.80, 121.24, 119.36, 118.02, 110.85, 49.96, 43.36, 17.02. Anal. Calcd. For C72 H64 N16 O8 Zn: C, 64.21; H, 4.79; N, 16.64. Found: C, 63.89; H, 4.88; N, 16.56.

2.3.7. ZnPc4 (Fig. 2) Under nitrogen atmosphere, a mixture of compound 5 (0.84 g, 1.49 mmol), Zn(OAc)2 (0.17 g, 0.93 mmol) and DBU (0.4 mL, 2.67 mmol) in n-pentanol (10 mL) was heated at 140 ◦ C for 24 h. After cooled to room temperature, the solution was poured into CH3 OH (50 mL) to afford a green solid. The crude product was purified by silica gel column chromatography using CH3 OH/CH2 Cl2 (1:40, v/v) as the eluent. The obtained product was not the final product. It was dissolved in CH2 Cl2 (10 mL), and excess trifluoroacetic acid (TFA) (2 mL) was added. The solution was stirred at room temperature for 3 h. After filtration, the cake was washed with CH2 Cl2 . Thereafter, the crude product was dissolved in water and precipitated by adjusted pH to 9–10. After filtration, the product

2.3.8. ZnPc1 (Fig. 2) A mixture of ZnPc3 (0.3 g, 0.23 mmol) and K2 CO3 (0.26 g, 1.86 mmol) in CH3 OH (10 mL) was stirred at reflux. Excess CH3 I was then added dropwise. After stirring for 12 h, the mixture was filtered while hot. The crude solid was washed thoroughly with ethanol. The product was redissloved in a mixture of CH3 OH/CH2 Cl2 (1:1, v/v) and filtered to remove the residual K2 CO3 . The final product was a dark green solid (0.39 g, 85.2%). M.P. > 200 ◦ C. IR (KBr, cm−1 ): 3450, 1630 (C O), 1502, 1223, 1079, 1028, 936. 1 H NMR (400 MHz, DMSO-d6 ): ı (ppm) 10.95 (s, 4H, NH), 8.87 (d, 4H, 7.6 Hz, ArH), 8.45 (d, 4H, 22 Hz, ArH), 7.89 (d, 8H, 15.6 Hz, PcH), 7.70 (t, 4H, 6.6 Hz, PcH), 7.53 (t, 8H, 11.2 Hz, ArH), 4.39 (s, 4H, CH2 ), 3.30 (br, 36H, CH3 ), 1.71 (s, 12H, CH3 ). 13 C NMR (100 MHz,

0.60

0.5

A

C

0.4

0.45

Absorbance

Absorbance

0 min 0.3

0.2

5 min

0.30

0.15

0.1

0.00

0.0 550

600

650

700

750

550

800

600

Wavelength(nm)

0.70

700

750

800

0.50

B

0.65

ZnPc2 4eq acid+ZnPc2 4eq base +ZnPc2 8eq base+ZnPc2

D

4eq acid+ZnPc1 4eq base+ZnPc1-m 4eq base+ZnPc1-a 8eq base+ZnPc1-m 8eq base+ZnPc1-a ZnPc1

0.60

0.49 0.48

Absorbance

0.55

Absorbance

650

Wavelength(nm)

0.50 0.45 0.40 0.35 0.30

0.47 0.46 0.45 0.44

0.25

0.43 0

1

2

3

4

Irradiation Time (min)

5

6

0

1

2

3

4

5

6

Time(min)

Fig. 7. Absorption change of ZnPc1 (A: ZnPc1 with 4eq NaOH aqueous as an example) and ZnPc2 (C: ZnPc2 with 4eq NaOH aqueous as an example) after irradiation with 665 LED light for 1–5 min; the change of Q-band of (B) ZnPc1 and (D) ZnPc2 versus irradiation time.

A. Wang et al. / Journal of Photochemistry and Photobiology A: Chemistry 288 (2014) 1–12

7

Table 1 pH values of the solutions of ZnPc1 and ZnPc2 upon adding of acid and base. Additives

4eq acid

Nothing

1eq base

2eq base

3eq base

4eq base

8eq base

pH

6.34a 6.43b

6.88a 6.85b

6.96a 6.92b

7.13a 7.10b

7.23a 7.23b

7.31a 7.38b

7.98a 8.06b

a b

The solutions of ZnPc1 (C = 5 × 10−6 M). The solutions of ZnPc2 (C = 5 × 10−6 M).

DMSO-d6 ): ı (ppm) 165.72, 158.92, 154.25, 153.90, 139.93, 134.32, 134.09, 133.30, 129.93, 124.30, 122.72, 120.56, 120.41, 116.17, 101.47, 70.49, 56.50, 52.01, 31.14, 18.98, 13.35. Anal. Calcd. For C80 H84 I4 N16 O8 Zn: C, 48.76; H, 4.30; N, 11.37. Found: C, 48.35; H, 4.35; N, 11.12.

DMSO-d6 ): ␦ (ppm) 167.20, 159.41, 157.72, 155.23, 135.56, 131.42, 129.22, 127.42, 124.80, 121.50, 119.91, 118.20, 103.42, 82.30, 51.24, 43.66, 10.35. Anal. Calcd. For C84 H92 I4 N16 O8 Zn: C, 49.78; H, 4.58; N, 11.06. Found: C, 49. 40; H, 4.84; N, 11.12.

2.3.9. ZnPc2 (Fig. 2) According to the procedure described for ZnPc1, compound ZnPc2 was prepared as a dark green solid by treating ZnPc4 (0.15 g, 0.11 mmol) and K2 CO3 (0.13 g, 0.88 mmol) with excess CH3 I in CH3 OH (0.20 g, 90.1%). M.P. >200 ◦ C. IR (KBr, cm−1 ): 3430, 2905, 1671 (C O), 1603, 1468, 1231, 1088, 944. 1 H NMR (400 MHz, DMSO-d6 ): ␦ (ppm) 9.16 (d, 4H, NHC O, 6.4 Hz), 8.99–9.05 (m, 4H, PcH), 8.57 (d, 4H, 12.4 Hz, PcH), 7.72–7.78 (m, 4H, PcH), 7.43–7.59 (m, 16H, Ar), 4.51 (t, 8H, 6.6 Hz, CH2 ), 4.19 (t, 4H, 6.2 Hz, CH), 3.22 (d, 36H, 4.0 Hz, CH3 ), 1.61 (t, 12H, 5.8 Hz, CH3 ). 13 C NMR (100 MHz,

2.4. Singlet oxygen generation detection

1.0

ln

[ADPA]0

= −kt

0.0

0min

-0.2

ln(At/A0)

0.6

5min

0.4 0.2 0.0 300

 [ADPA]t 

A

0.8

Absorbance

The ability of 1 O2 generation of the Pcs in solutions at different pH was determined using ADPA as the probe, which has been described before [30]. The rate of singlet oxygen generation is calculated by the following equation [31]:

B

-0.4

4eq base 4eq acid 4eq acid+ZnPc1 ZnPc1 2eq base+ZnPc1 4eq base+ZnPc1 8eq base+ZnPc1

-0.6 -0.8

350

400

-1.0

450

0

1

2

Wavelength(nm)

3

4

5

Time(min)

0.00

C

-0.05

ln(At/A0)

-0.10 -0.15

ZnPc2 4eq acid+ZnPc2 2eq base+ZnPc2 4eq base+ZnPc2 8eq base+ZnPc2

-0.20 -0.25 -0.30 -0.35 0

1

2

3

4

5

Time (min) Fig. 8. (A) Absorption changes for the determination of singlet oxygen generation ability of ZnPc1 and ZnPc2 using ADPA as the quencher (ZnPc1 without addition as an example, C = 5 × 10−6 M, irradiation with 665 nm LED); comparison of the rates of decay of ADPA as monitored by the decrease in the absorbance at 378 nm (B) ZnPc1 (C) ZnPc2.

8

A. Wang et al. / Journal of Photochemistry and Photobiology A: Chemistry 288 (2014) 1–12

In the equation, [ADPA]0 and [ADPA]t are the concentrations of ADPA after and prior irradiation, respectively. Values of k are the rate of singlet oxygen generation and t is the irradiation time. 2.5. Photodegradation of CT (Calf Thymus) DNA analysis To study the PDT properties of Pcs, the solution of CT DNA (20 ␮M) containing ethidium bromide (40 ␮M) was used as phototherapeutic target. After adding Pcs (5 ␮M) into the above mixture, the solution was irradiated with 665 nm LED, and the changes of the fluorescence spectrum at 610 nm were recorded every 1 min [32,33]. 3. Results and discussion 3.1. The pH values The pH values were measured on a pH meter. All data were performed in three times and the average of the results was used. The pH values of redistilled water used was 6.81. Table 1 shows the pH values of the solutions of ZnPc1 and ZnPc2 upon adding of base or acid in water. With the addition of acid and base, the pH values of ZnPc1 and ZnPc2 were changed as expected. 3.2. Ground state electronic absorption spectra The UV–vis spectra of ZnPc1 and ZnPc2 in water were shown in Fig. 3. Normally, the steric hindrance and electrostatic repulsion

350

3.3. Effects of pH on UV–vis spectra Fig. 5 shows the changes in the UV–vis spectrum of ZnPc1 (Fig. 5A) and ZnPc2 (Fig. 5B) in water upon adding base or acid. It can be seen that there are no significant changes in the absorbance of the two ZnPcs after acid addition. While there is only a little decrease in the absorption spectrum of ZnPc2, the spectra of ZnPc1

350

A

0min

300

250 200

Florescence

Florescence

300

forces caused by charged substituents of cationic Pcs might reduce aggregation of ZnPc1 and ZnPc2 [30]. However, the results were all different from what we had expected. Though the two ZnPcs were well soluble in water, their absorption spectra had typical shape for aggregate Pcs with a high energy B-band at about 341 nm and intense absorbance Q-band at 642 nm [34,35]. That may be result from the little steric hindrance of the substitution on the peripheral position and the free pending of charged substituents that may still allow ␲–␲ stacking of the hydrophobic cores [36,37]. Fig. 4 shows the changes in the UV–vis spectra of ZnPc1 in water over times. It can be seen that the absorbance at 641 nm which characterized as aggregate increases gradually over times, while absorption of monomer at 700 nm decreases at the same time (Fig. 4B). The whole process achieves a balance in about 25 min. This shows that in this condition, the monomer of ZnPc1 in water is dynamic unstable, and would transform into aggregate gradually in unaltered conditions. However, ZnPc2 has no obvious UV–vis spectra change over times. As ZnPc1 had a dynamic process, its related spectra were all measured after the system reaching the balance.

4min

150 100 50

250 200 150

4min

100 50

0 500

B 0min

0 550

600

650

700

750

800

500

550

600

Wavelength(nm)

650

700

750

800

Wavelength(nm)

Photodegradation Percentage

0.6

4eq base+ZnPc1 ZnPc1

C

0.5 0.4 0.3 0.2 0.1 0.0 0

1

2

3

4

Time(min) Fig. 9. Photodegradation of CT DNA by (A) ZnPc1 (B) ZnPc1 with 4eq NaOH; (C) photodegradation percentage of CT DNA by measuring the fluorescence decrease of EB.

A. Wang et al. / Journal of Photochemistry and Photobiology A: Chemistry 288 (2014) 1–12

shows a distinct change after base addition. After the addition of aqueous NaOH, the B-band at 341 nm shift to 354 nm, while sharp Q-band at 641 nm becomes a broad band at 693 nm. This shows the base additive disturb the aggregation and change the existing form of ZnPc1 in water [38]. In order to evaluate the effects of base addition more detailed, the spectra changes of ZnPc1 and ZnPc2 were further studied by a continuous variation method [39,40]. Fig. 6 shows the changes of ZnPc1 (Fig. 6A) and ZnPc2 (Fig. 6C) upon addition of aqueous NaOH in different ratios (from 0:1 to 8:1). As can be seen in Fig. 6B, when the mole ratio of aqueous NaOH and ZnPc1 is 4:1, the monomer and aggregate reaches a balance. The spectra would have little change upon continuing to add aqueous NaOH to the solution. However, the absorption intensity of ZnPc2 decreased as the base continuing addition (Fig. 6D). 3.4. Effects of pH on photo-stability Upon irradiation by light, most photosensitizers will degrade because of photobleaching. In this process, the intensity of the absorption will decrease [32]. Thus the Pcs used for PDT should not only have strong lethal effect on cancer cells but should also have good photo-stability [32]. By monitoring the decrease in the Q band absorption of the two Pcs before and after irradiation with 665 nm LED using UV–vis spectrophotometer, the photodegradation studies were carried out. In order to study the effects of pH on the photo-stability of the two Pcs, the photobleaching experiments were carried out. Upon irradiation by 665 nm LED light, the UV–vis spectra changes of ZnPc1 and ZnPc2 with different addition (aqueous NaOH or HCl) were recorded (Fig. 7). Though the ground state electronic absorption of ZnPc1 was changed distinctly upon the addition of base, its photo-stability was not obviously influenced. It can be seen that the base addition could reduce the aggregation of ZnPc1, meanwhile without reducing its photo-stability. What’s more, upon the addition of base or acid, both the two ZnPcs showed good photostability.

N O

I-

3.5. Effects of pH on singlet oxygen generation Singlet oxygen is one of the reactive oxygen species [41]. In PDT, the singlet oxygen generation ability is an important parameter to evaluate the application potential of a photosensitizer [42]. As the aggregation of ZnPc1 and ZnPc2 can be affected by the addition of base or acid, we believe that their singlet oxygen generation would also be influenced. We examined this aspect by comparing the singlet oxygen generation ability of ZnPc1 and ZnPc2 at different pH conditions. The singlet oxygen was determined using a chemical method by the photo-oxidation of ADPA to its endoperoxide derivative. Fig. 8A shows the ADPA was photo-oxidized by singlet oxygen and its absorbance at 378 nm decreased upon irradiation. From Fig. 8B, it can be seen that in the presence of base or acid but in the absence of photosensitizer, the decay of ADPA was negligible. Fig. 8B shows the results for ZnPc1. As shown in Fig. 8B, in the absence and presence of base or acid addition, ZnPc1 can induce the photobleaching of ADPA and the efficiency follows the order (4:1 base, k = 0.172) > (8:1 base, k = 0.127) > (2:1 base, k = 0.126) > (0:1 ZnPc1, k = 0.089) > (4:1 acid, k = 0.052). The best singlet oxygen generation ability was obtained when the mole rate of NaOH and ZnPc1 was 4:1, which may be attributed to the lower aggregation tendency of ZnPc1 in this condition. In accord with this trend, the addition of base reduces the aggregation of ZnPc1, therefore increases the singlet oxygen production. However, the excess base addition would have an opposite effect on the singlet oxygen generation. In our experiments, it seems the production of singlet oxygen is influenced by two principal factors: the existence form of ZnPcs and additives (OH− and H− ). The predominant factor is different in different stages. When the mole rate of NaOH and ZnPc1 is less than 4:1, though the OH− addition would quench the singlet oxygen [43,44], it reduces the aggregate of ZnPc1 and enhances singlet oxygen generation at the same time. At this stage, the existence form of ZnPc1 is the predominant factor. Therefore, the singlet oxygen generation ability of ZnPc1 increases. When the mole rate of NaOH and ZnPc1 is more than 4:1, the existence form of ZnPc1 would not

I- N

NH

N

I-

I- N

O

HN

HO

N

N

N

N

Zn N

N

OH-

N

N

O

O

N

N

H+

N

N

N N N

O

O

N

Zn N

N

N

NH

HN

O

N

O

O

I-

N

N

I- N

O

-

H O N

O OH N

I-

R O

N

OH

H2O

-

N

I-

OH

I- N

OH

R O

OH

O

O

I-

9

R O

N

N

I-

Fig. 10. Scheme representation of the mechanism of tautomeric transformation between amide and imidohydrine catalyzed by base.

10

A. Wang et al. / Journal of Photochemistry and Photobiology A: Chemistry 288 (2014) 1–12

change obviously. However, as always, the excess base (OH− ) addition would quench singlet oxygen. At this stage, additives (OH− ) is the predominant factor. Thus, the excess base addition would decrease the singlet oxygen generation. Though the acid addition had little effect on the absorption of ZnPc1, its singlet oxygen generation was reduced. Compared to ZnPc1, the effects of pH on the singlet oxygen generation of ZnPc2 was simple. All the addition would reduce the efficiency of singlet oxygen generation of ZnPc2. The efficiency followed the order (0:1 ZnPc2, k = 0.058) > (4:1 acid, k = 0.056) > (2:1 base, 0.054) > (4:1 base, 0.050) > (8:1 base, 0.046). 3.6. Effects of pH on photodegradation of CT DNA The ability of CT DNA photodegradation is another important parameter to evaluate the application potential of a photosensitizer. Fig. 9 shows the photodegradation of CT DNA by ZnPc1 in presence and absence of NaOH by measuring the fluorescence decrease of EB. Having better singlet oxygen generation ability, the ZnPc1 with base addition was believed to be better in the CT DNA degradation. As expected by us, when irradiation was carried out in the ZnPc1-EB-CT DNA solution with 665 nm LED, 47.12% binding sites was destroyed in the presence of 4 equivalent of NaOH

Fig. 11.

1

(Fig. 9C). However, in the absence of NaOH, only 24.65% binding sites was destroyed (Fig. 9C). 3.7. Reason analysis and confirmation Cationic Pcs are traditionally known as less affected by acid or base because of the methylation of the pH sensitive amine groups [36,45]. However, the cationic Pc ZnPc1 shows some peculiar photochemical and photophysical properties which appear to be markedly base addition dependent. If we assume that the present results are due to the existence of cationic substituent, the contrast ZnPc2 should have similar phenomenon when NaOH was added in. In fact, the properties of ZnPc2 were little affected by the change of pH environment. Comparing the structure of ZnPc1 and ZnPc2, the only difference between them is that there is a methylene between the benzene ring and amide bond in ZnPc2. On the above account, we assume that there is a tautomeric transformation between amide and imidohydrine in ZnPc1. The scheme of this equilibrium is shown in Fig. 10. Due to the dual electron withdrawing effects of carbonyl group and cations, the hydrogen atom on the amide bond shows a certain degree of acidity. When OH− was added in, the hydrogen was removed, thus forming an imine intermediate. Then, the negative oxygen ions combine with

H-NMR spectras of ZnPcs (carry out in DMSO-d6 ) in the absence (up) and presence (down) of NaOD (a drop of NaOD in D2 O) (A) ZnPc1 (B) ZnPc2.

A. Wang et al. / Journal of Photochemistry and Photobiology A: Chemistry 288 (2014) 1–12

the hydrogen in water to give an imidohydrine Pc isomer of ZnPc1. The formed carbon nitrogen double bond in the imidohydrin isomer can conjugate with phthalocyanine macrocycle and benzene rings to give an even larger conjugated system. Just because of this, the imidohydrine Pc isomer can exist in water solutions stably. The intermolecular hydrogen bond and steric hindrance of the formed imidohydrine Pc isomer are changed, which result in the reduced aggregation. Though ZnPc2 also can be affected by the cations and carbonyl group, its imidohydrine isomer cannot conjugate with benzene rings because of obstruction of the methylene groups. Without conjugation, the isomer of ZnPc2 was unstable in water solution. Thus, ZnPc2 mainly existed in the form of amide. To verify our hypothesis, the 1 H NMR of ZnPc1 and ZnPc2 in the presence and absence of base were carried out in DMSO-d6 . As shown in Fig. 11A, the peaks of amide protons of ZnPc1 were clearly seen at ı 10.94. After adding a drop of aqueous NaOD (excessive), the peaks of amide protons disappeared. ZnPc2 was also studied in a similar manner under the same conditions. For ZnPc2, the peaks of amide protons existed both in the absence and presence (Fig. 11B) of NaOD. From the above results, we believed that the hypothesis was well confirmed. 4. Conclusion In conclusion, we have synthesized and characterized two cationic Pcs ZnPc1 and ZnPc2 which were substituted with amides. The effects of pH (especially base addition) on their properties were studied and compared. The results showed that the base additive can promote a shift in the amide/imidohydrine balance toward imidohydrine, and the formation of hyperconjugation makes it stably exist in water. The formed imidohydrine can greatly reduce aggregation of cationic Pc due to the intermolecular hydrogen bond and molecular arrangement; therefore improve its photodynamic activities without decreasing their photostability. This base induced tautomeric transformation could be a novel way to generate low aggregated cationic Pcs, improving their photosensitizing properties in aqueous media. Acknowledgments This work was supported by the National Natural Science Foundation of China (21201102), the Natural Science Foundation of Jiangsu Higher Education Institutions of China (No. 13KJA150003, 12KJB150015), the Scientific Research Foundation of Nanjing Normal University (No. 2011103XG0249), the Priority Academic Program Development of Jiangsu Higher Education Institutions(PAPD) and the Foundation of Jiangsu Collaborative Innovation Center of Biomedical Functional Materials. References [1] J. Jiang, Functional Phthalocyanine Molecular Materials, Springer, Berlin, 2010. [2] J.T.F. Lau, X.J. Jiang, D.K.P. Ng, P.C. Lo, A disulfide-linked conjugate of ferrocenyl chalcone and silicon(IV) phthalocyanine as an activatable photosensitiser, Chem. Commun. 49 (2013) 4274–4276. [3] I.M. Tynga, N.N. Houreld, H. Abrahamse, The primary subcellular localization of zinc phthalocyanine and its cellular impact on viability, proliferation and structure of breast cancer cells (MCF-7), J. Photochem. Photobiol. B 120 (2013) 171–176. [4] R.O. Ogbodu, T. Nyokong, Effects of number of ring substituents on the physicochemical properties of zinc aminophenoxy phthalocyanine-single walled carbon nanotube conjugate, J. Photochem. Photobiol. A 274 (2014) 83–90. [5] M.A. Zanjanchi, A. Ebrahimian, M. Arvand, Sulphonated cobalt phthalocyanineMCM-41: an active photocatalyst for degradation of 2, 4-dichlorophenol, J. Hazard. Mater. 175 (2010) 992–1000. [6] J.H. Shu, H.C. Wikle, B.A. Chin, Passive chemiresistor sensor based on iron (II) phthalocyanine thin films for monitoring of nitrogen dioxide, Sens. Actuators B 148 (2010) 498–503. [7] H. Ali, J.E. van Lier, Metal complexes as photo- and radiosensitizers, Chem. Rev. 99 (1999) 2379–2450.

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