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

Article abstract of DOI:10.1016/j.jphotochem.2014.05.003

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

Full text of DOI:10.1016/j.jphotochem.2014.05.003

Journal of Photochemistry and Photobiology A: Chemistry 288 (2014) 1–12
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology A:
Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
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
a b s t r a c t
Article history:
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 oxy-
gen 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.
Received 18 March 2014
Received in revised form 5 May 2014
Accepted 8 May 2014
Available online 17 May 2014
Keywords:
Cationic phthalocyanines
pH
© 2014 Elsevier B.V. All rights reserved.
Aggregate
Photodynamic activity
Amide
Tautomeric transformation
1. Introduction
terminal hydrophilic groups can be introduced at the peripheral
or axial positions [15].
Being a class of synthetic macrocyclic compounds with intrigu-
ing physical, chemical, and biological properties, phthalocyanines
(Pcs) have received considerable attention and have found appli-
cations 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 photosen-
sitizers for photodynamic therapy (PDT) owing to their desirable
features, such as high molecular absorption coefficients at pho-
totherapeutic 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 photochemi-
cal and photobiological activities which finally limit their potential
applications in PDT [13,14]. To generate hydrophilic and nonag-
gregated Pcs, water-soluble moieties and bulky fragments with
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 exam-
ple, 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 methyl-
ated. However, if some other functional groups of such cationic
Pcs can be affected by pH, their aggregation degree and photo-
dynamic 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 imido-
hydrine 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
∗
Corresponding author. Tel.: +86 025 85891761; fax: +86 025 85891761.
E-mail address: shwei@njnu.edu.cn (S. Wei).
http://dx.doi.org/10.1016/j.jphotochem.2014.05.003
1010-6030/© 2014 Elsevier B.V. All rights reserved.

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 imidohy-
drine may affect the intermolecular hydrogen bond and molecular
arrangement of cationic Pc, which finally affect its aggregate ten-
dency. 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) methy-
lamino)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 abil-
ity and efficiency of CT-DNA photodegradation were studied and
compared. The results showed that the base additive can signifi-
cantly 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 flu-
orescence spectrophotometer. The pH values were measured on
a FE20/EL20 pH meter, Mettler Toledo. Infrared spectra were mea-
sured in KBr pellets on IR-Spectrometer Nicolet Nexus 670. 1H NMR
and 13C 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 determi-
nations and photo-stability were done using a 665 nm LED.
2. Experimental
2.3. Synthesis
2.1. Materials
ZnPc1 was prepared by a five-step procedure (Figs. 1 and 2).
First, l-alanine was reacted with (chloromethanetriyl) triben-
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
zene (TrCl) to obtain the N-Trityl alanine
1 [28,29]. Then,
compound
1 was reacted with 4-aminophenol using N-(3-
Dimethylaminopropyl)-Nꢀ-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
3
to obtain compound 2. After then, the phthalonitrile 3 was pre-
pared 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 intermedi-
ate. The ZnPc3 was prepared by removing the N-Trityl protecting
group with CF3COOH. Finally, ZnPc1 was obtained by methylation
of ZnPc3 in CH3OH.
The ZnPc2 was prepared according to ZnPc1, but with a little
modification (Figs. 1 and 2). First 4-(aminomethyl) phenol and 4-
nitrophthalonitrile were reacting using K2CO3 as the base catalyst
to obtain phthalonitrile4. Then, compound 4 was reacted with com-
pound 1 to give the Pc precursor 5. After then, zinc acetate and 5
were employed to give N-Trityl ZnPc. By using CF3COOH to remove
protecting groups, ZnPc4 was obtained. Finally, according to the
method described for ZnPc1, ZnPc2 was obtained by methylation
of ZnPc4 in CH3OH.
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 tem-
perature, and then excess CH3OH was added. After evaporation
of the solvent, the white solid was redissolved in CH2Cl2 (20 mL),
and washed with distilled water (3× 15 mL) and saturated brine
(2× 15 mL). The organic layer was dried over anhydrous Na2SO4.
The crude product was purified by silica gel column chromatogra-
phy 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]). 1H 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). 13C 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)
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/CH3CN (5:1) was added Me3SiCl (1.08 mL, 8.42 mmol). The
solution was refluxed for 5 h and then cooled to room temperature.
A mixture of 1 (0.60 g, 1.81 mmol), N-(3-Dimethylamino-
propyl)-Nꢀ-ethylcarbodiimide hydrochloride (EDC·HCl, 0.42 g,
2.17 mmol), 1-Hydroxybenzotriazole (HOBT, 0.29 g, 2.17 mmol)
R
R
N
CF3COOH (1)
NaOH (2)
N
N
N
N
Zn(OAc)2, DBU
n-pentanol
3
5
N
N
Zn
N
R
R
H
N
O
C
CH3
O
C
CH3
H
N
R3
O
C
H
NH2
R4
O
C
H
NH2
ZnPc3
ZnPc4
CH3OH CH3I, K2CO3
R
R
N
N
N
N
N
N
N
Zn
N
R
R
H
N
O
CH3
O
C
CH3
H
N
I-
O
C
C
H
N
O
C
H
N
I-
R2
R1
ZnPc2
ZnPc1
Fig. 2. Synthetic route for the preparation of Pcs ZnPc1, ZnPc2, ZnPc3 and ZnPc4.

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. 1H NMR
0.6
0.5
0.4
0.3
0.2
0.1
0.0
ZnPc1
ZnPc2
(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). 13C 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 C28H26N2O2:
C, 79.59; H, 6.20; N, 6.63. Found: C, 79.30; H, 6.21; N, 6.77.
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 anhy-
drous K2CO3 (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. 1H 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). 13C NMR (100 MHz, DMSO-
d6): 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,
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 CH2Cl2 (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 CH2Cl2 (3× 15 mL), and the
combined organic portion was dried over anhydrous Na2SO4. 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):
0.5
A
0.5
0.4
Redistilled water
A
acid
0.4
base
0.3
aggregate
0.3
monomer
0.2
0.1
0.0
0.2
0.1
0.0
300
400
500
600
700
800
300
400
500
600
700
800
Wavelength(nm)
Wavelength(nm)
0.5
0.165
0.48
B
B
0.4
0.3
0.2
0.1
0.0
Redistilled water
0.160
0.155
0.150
0.145
0.140
0.135
0.46
0.44
0.42
0.40
0.38
acid
base
monomer (700 nm)
aggregate (641 nm)
0
5
10
15
20
25
300
400
500
600
700
800
Time(min)
Wavelength(nm)
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).
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
5
115.87, 108.20, 71.29, 53.41, 22.45. Anal. Calcd. For C36H28N4O2:
C, 78.81; H, 5.14; N, 10.21. Found: C, 79.02; H, 5.20; N, 10.11.
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.
1H NMR (400 MHz, CDCl3): ı (ppm) 7.72 (d, 1H, 8.8 Hz, HNC O),
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). 13C 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 C36H28N4O2: C, 78.81; H, 5.14; N, 10.21. Found: C,
78.40; H, 5.34; N, 10.12.
2.3.4. 4-(4-(aminomethyl) phenoxy)phthalonitrile (4, Fig. 1)
Under nitrogen atmosphere, a mixture of 4-(aminomethyl) phe-
nol (1.00 g, 8.12 mmol), 4-Nitrophthalonitrile (1.41 g, 8.12 mmol)
and finely ground anhydrous K2CO3 (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. 1H NMR (400 MHz, DMSO-
d6): 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). 13C 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 C15H11N3O: C, 72.28; H, 4.45;
N, 16.86. Found: C, 72.10; H, 4.65; N, 16.80.
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 CH3OH to afford crude green solid.
After filtration, the crude product was purified by silica gel column
chromatography using CH3OH/CH2Cl2 (1:40, v/v) as the eluent.
After evaporating the eluent in vacuo, the blue solid was redis-
solved in CH2Cl2 (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 CH2Cl2 till the filtrate was col-
orless. 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
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 CH2Cl2 (15 mL) was stirred at room tem-
perature for 5 h. The mixture was washed with distilled water (3×
10 mL) and saturated brine (2× 10 mL). After drying by Na2SO4, the
crude product was purified by silica gel column chromatography
0.5
0.60
NaOH:ZnPc1
A
NaOH:ZnPc2
C
0.4
0.3
0.2
0.1
0.0
0:1
1:1
2:1
3:1
4:1
5:1
6:1
7:1
8:1
0:1
0.45
1:1
2:1
3:1
0.30
4:1
5:1
6:1
0.15
7:1
8:1
0.00
300
400
500
600
700
800
300
400
500
600
700
800
Wavelength(nm)
Wavelength(nm)
0.43
0.42
0.41
0.40
0.39
0.38
0.37
0.36
0.35
0.45
0.40
0.35
0.30
0.25
0.20
0.15
monomer
aggregate
D
B
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
Base(equiv)
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. 1H NMR (400 MHz, DMSO-d6
with a drop of CF3COOD): 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).
13C NMR (100 MHz, DMSO-d6): ı (ppm) 167.72, 162.06, 158.89,
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 C68H56N16O8Zn: 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. 1H NMR (400 MHz, DMSO-d6 with
a drop of CF3COOD): 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). 13C 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
C72H64N16O8Zn: 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)
2.3.8. ZnPc1 (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
CH3OH (50 mL) to afford a green solid. The crude product was
purified by silica gel column chromatography using CH3OH/CH2Cl2
(1:40, v/v) as the eluent. The obtained product was not the final
product. It was dissolved in CH2Cl2 (10 mL), and excess trifluo-
roacetic acid (TFA) (2 mL) was added. The solution was stirred at
room temperature for 3 h. After filtration, the cake was washed with
CH2Cl2. Thereafter, the crude product was dissolved in water and
precipitated by adjusted pH to 9–10. After filtration, the product
A mixture of ZnPc3 (0.3 g, 0.23 mmol) and K2CO3 (0.26 g,
1.86 mmol) in CH3OH (10 mL) was stirred at reflux. Excess CH3I
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 CH3OH/CH2Cl2
(1:1, v/v) and filtered to remove the residual K2CO3. 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. 1H
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). 13C NMR (100 MHz,
0.60
0.5
A
C
0.4
0.45
0 min
0.3
0.30
0.15
0.00
0.2
5 min
0.1
0.0
550
600
650
700
750
800
550
600
650
700
750
800
Wavelength(nm)
Wavelength(nm)
0.50
0.70
B
ZnPc2
4eq base +ZnPc2
8eq base+ZnPc2
4eq acid+ZnPc2
D
0.65
0.60
0.55
0.50
0.45
0.40
0.35
0.30
0.25
4eq acid+ZnPc1
4eq base+ZnPc1-a
8eq base+ZnPc1-a
4eq base+ZnPc1-m
8eq base+ZnPc1-m
ZnPc1
0.49
0.48
0.47
0.46
0.45
0.44
0.43
0
1
2
3
4
5
6
0
1
2
3
4
5
6
Time(min)
Irradiation 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
pH
4eq acid
Nothing
1eq base
2eq base
3eq base
4eq base
8eq base
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
The solutions of ZnPc1 (C = 5 × 10−6 M).
The solutions of ZnPc2 (C = 5 × 10−6 M).
b
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
C80H84I4N16O8Zn: 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 C84H92I4N16O8Zn: 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)
2.4. Singlet oxygen generation detection
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 K2CO3 (0.13 g, 0.88 mmol) with excess CH3I in
CH3OH (0.20 g, 90.1%). M.P. >200 ◦C. IR (KBr, cm−1): 3430, 2905,
1671 (C O), 1603, 1468, 1231, 1088, 944. 1H 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). 13C NMR (100 MHz,
The ability of 1O2 generation of the Pcs in solutions at differ-
ent pH was determined using ADPA as the probe, which has been
described before [30]. The rate of singlet oxygen generation is cal-
culated by the following equation [31]:
ꢀ
ꢁ
[ADPA]t
[ADPA]0
ln
= −kt
0.0
1.0
A
0min
-0.2
-0.4
-0.6
-0.8
-1.0
0.8
B
0.6
4eq base
4eq acid
4eq acid+ZnPc1
ZnPc1
2eq base+ZnPc1
4eq base+ZnPc1
8eq base+ZnPc1
5min
0.4
0.2
0.0
300
350
400
450
0
1
2
3
4
5
Wavelength(nm)
Time(min)
0.00
-0.05
-0.10
-0.15
-0.20
-0.25
-0.30
-0.35
C
ZnPc2
4eq acid+ZnPc2
2eq base+ZnPc2
4eq base+ZnPc2
8eq base+ZnPc2
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.
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.
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 pho-
totherapeutic 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 per-
formed 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.
3.3. 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
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
350
A
B
0min
0min
300
300
250
250
200
200
4min
150
4min
150
100
50
100
50
0
0
500
550
600
650
700
750
800
500
550
600
650
700
750
800
Wavelength(nm)
Wavelength(nm)
0.6
0.5
0.4
0.3
0.2
0.1
0.0
4eq base+ZnPc1
ZnPc1
C
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
9
shows a distinct change after base addition. After the addition of
3.5. Effects of pH on singlet oxygen generation
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].
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 oxy-
gen 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 deter-
mined using a chemical method by the photo-oxidation of ADPA to
its endoperoxide derivative.
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).
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 gen-
eration ability was obtained when the mole rate of NaOH and
ZnPc1 was 4:1, which may be attributed to the lower aggrega-
tion 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 gener-
ation. 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 oxy-
gen 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
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 spec-
trophotometer, 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 irra-
diation 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, mean-
while without reducing its photo-stability. What’s more, upon the
addition of base or acid, both the two ZnPcs showed good photo-
stability.
I-
I-
N
N
I-
I-
N
N
O
O
NH
HN
HO
OH
N
N
O
O
O
O
O
O
N
N
N
N
N
N
OH-
H+
N
N
N
N
N
N
Zn
N
N
N
Zn
N
O
I-
O
I-
N
NH
HN
O
N
N
N
OH
O
I-
N
OH
I-
N
-
O
OH
O
H
N
H2O
OH-
R
O
R
O
N
R
O
N
I-
I-
I-
N
N
N
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−) addi-
tion would quench singlet oxygen. At this stage, additives (OH−)
is the predominant factor. Thus, the excess base addition would
decrease the singlet oxygen generation.
(Fig. 9C). However, in the absence of NaOH, only 24.65% binding
sites was destroyed (Fig. 9C).
3.7. Reason analysis and confirmation
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 sin-
glet 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).
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 con-
trast 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 trans-
formation 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
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 photosen-
sitizer. 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% bind-
ing sites was destroyed in the presence of 4 equivalent of NaOH
Fig. 11. 1H-NMR spectras of ZnPcs (carry out in DMSO-d6) in the absence (up) and presence (down) of NaOD (a drop of NaOD in D2O) (A) ZnPc1 (B) ZnPc2.

A. Wang et al. / Journal of Photochemistry and Photobiology A: Chemistry 288 (2014) 1–12
11
the hydrogen in water to give an imidohydrine Pc isomer of ZnPc1.
The formed carbon nitrogen double bond in the imidohydrin iso-
mer 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 sta-
bly. 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 conju-
gate 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 1H 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.
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In conclusion, we have synthesized and characterized two
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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
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aggregation of cationic Pc due to the intermolecular hydrogen
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This work was supported by the National Natural Science
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13KJA150003, 12KJB150015), the Scientific Research Foundation
of Nanjing Normal University (No. 2011103XG0249), the Prior-
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