The influences of the number of the ammonium groups and their arrangement manner on the photophysical properties of the quaternized zinc phthalocyanines

Article abstract of DOI:10.1016/j.inoche.2015.01.009

The influences of the number of the ammonium groups and their arrangement manner on the photophysical properties of the quaternized zinc phthalocyanines were studied. The aggregation tendency, singlet oxygen generation ability and the fluorescence intensi

Full text of DOI:10.1016/j.inoche.2015.01.009

Inorganic Chemistry Communications 53 (2015) 15–19
Contents lists available at ScienceDirect
Inorganic Chemistry Communications
journal homepage: www.elsevier.com/locate/inoche
The influences of the number of the ammonium groups and their
arrangement manner on the photophysical properties of the quaternized
zinc phthalocyanines
⁎
⁎
Liu Zhang, Ao Wang, Shan Lu, Lin Zhou , Jiahong Zhou, Yun Lin, Shaohua Wei
School of Chemistry and Materials Science, Jiangsu Collaborative Innovation Center of Biomedical Functional Materials, Jiangsu Key Laboratory of Biomedical Materials, Key Laboratory of Applied
Photochemistry, Nanjing Normal University, Nanjing 210023, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The influences of the number of the ammonium groups and their arrangement manner on the photophysical
properties of the quaternized zinc phthalocyanines were studied. The aggregation tendency, singlet oxygen gen-
eration ability and the fluorescence intensity properties of the quaternized zinc phthalocyanines were studied
and compared. Results indicated that more quaternized ammonium groups and the straight chain arrangement
manner for the quaternized zinc phthalocyanines can decrease aggregation degree and increase singlet oxygen
generation and fluorescence intensity.
Received 27 October 2014
Received in revised form 15 December 2014
Accepted 2 January 2015
Available online 6 January 2015
Keywords:
Photodynamic therapy
Phthalocyanine
© 2015 Elsevier B.V. All rights reserved.
Quaternized nitrogen number
Molecular configuration
Singlet oxygen generation
Fluorescence intensity
Photodynamic therapy (PDT) is a minimal invasive treatment and
shows negligible toxicity when compared with the traditional cancer
treatments [1]. The general procedure of PDT begins with the injection
of photosensitizer (Ps) to human body followed by illumination with
appropriate wavelength light [2,3]. In the presence of oxygen, Ps can
transfer absorbed photon energy to neighboring oxygen molecules
and produce reactive oxygen species (ROSs), including singlet oxygen
(1O2), superoxide anion (O−2 ) and hydroxyl radical [4]. It's generally ac-
cepted that 1O2 is the most effective cytotoxic ROS in PDT process
among the current Pss [5,6].
Meanwhile, in the PDT process, upon light activation, Ps can emit
fluorescence due to the relaxation of the excited-state Ps back to the
ground state [7]. This emitted fluorescence can be used for molecular
imaging (MI) which has the potential to provide real time parameters
about the tumor environment [8]. Thus, organic fusion of PDT and MI
enables the treatment and monitoring simultaneously and is meaning-
ful to cancer treatment [9]. So, the ideal Ps should be an agent that has
1O2 generation for PDT and high fluorescence intensity for MI [10,11]
and finding Ps with these ideal properties has attracted great attentions
in the field of PDT.
ZnPcs often show high fluorescence intensity which is mainly because
of the inhibition of intramolecular photoinduced electron transfer
(PET) process [13–16]. However, the influences of the number of the
ammonium groups and their arrangement manner on the aggregation
tendency, 1O2 generation ability and fluorescence intensity have never
been studied and reported to our knowledge. These properties are
vital as they greatly determined the application potentials of the phtha-
locyanines (Pcs) for photo-induced PDT and MI.
Therefore, in this paper, we synthesized and researched four
quaternized ZnPcs with different quaternized ammonium groups and
configurations (straight or branched chain arrangement manner)
(Fig. 1), and compared their aggregation tendency, 1O2 generation
ability and fluorescence intensity.
ZnPcs substituted with methylamino and 1,2-diethylamino were
synthesized and converted to their quaternized derivatives (ZnPc1,
ZnPc2) according to the literature [17,18]. The structures of the com-
pounds were characterized by a combination of IR, 1H NMR, elemental
analysis and UV–Vis spectrum.
ZnPc3 and ZnPc4 were prepared according to the procedures shown
in Fig. 2: Firstly, a mixture of compounds 1, 2 and K2CO3 was dissolved
in DMF for reaction. After completion, the reaction mixture was poured
into ice water and the solid precipitation was purified by column chro-
matography to get compound 3. Then, through a two-step reaction, we
get compound 5 which has a good reactivity. Secondly, the solution of
compound 5 was added dropwise to the diethylenetriamine with stir-
ring at room temperature. After finished, the mixture was washed
Researches have demonstrated that quaternized zinc phthalocya-
nines (ZnPcs) are promising Pss due to their good water solubility, effi-
cient 1O2 generation and anticancer activity [12]. Besides, quaternized
⁎
Corresponding authors.
E-mail addresses: zhoulin@njnu.edu.cn (L. Zhou), shwei@njnu.edu.cn (S. Wei).
http://dx.doi.org/10.1016/j.inoche.2015.01.009
1387-7003/© 2015 Elsevier B.V. All rights reserved.

16
L. Zhang et al. / Inorganic Chemistry Communications 53 (2015) 15–19
Fig. 1. The molecular structures of the four quaternized ZnPcs.
with water and the organic extracts were dried with Na2SO4. Thirdly, di-
tert-butyl-di-carbonate ((Boc)2O) was added into the diluted organic
phase. After 4 h, the mixture was washed with saturated salt water
and purified by column chromatographic to get compounds 6 and 7.
Fourthly, a mixture of compound 6 (or 7), zinc acetate and DBU was dis-
solved in n-pentanol and heated to 140 °C with stirring for 24 h. After
cooling to room temperature, the solvent was removed and the raw
product was washed several times by ethyl ether and further purified
by column chromatography to get compound 8 (or 9). In the IR spec-
trum of 8 (or 9), the disappearance of the C`N stretching vibration
(2236 cm−1) and the intense absorption band of the amide bond
(3400 cm−1) suggested the formation of Pc. Their 1H NMR spectrums
which were tested in CDCl3 (or DMSO) also corresponded well with
the desired structures.
Fig. 2. The synthetic route of ZnPc3 and ZnPc4.

L. Zhang et al. / Inorganic Chemistry Communications 53 (2015) 15–19
17
0.6
0.5
0.4
0.3
0.2
0.1
0.0
ZnPcs was monitored using the molecular probe disodium ADMA
(9,10-anthracenediyl-bis(methylene)). In experiment, ZnPcs and
ADMA were mixed and irradiated with 665 nm LED light. The reaction
process was monitored by measuring the net loss of ADMA absorption
at 379 nm. The rate of singlet oxygen generation is calculated by the
following Eq. [22]:
ZnPc1
ZnPc2
ZnPc3
ZnPc4
lnð½ADMAꢀt=½ADMAꢀ0Þ ¼ −kt
[ADMA]t and [ADMA]0 are the concentrations of ADMA after and prior to
irradiation. Values of k are the rate of singlet oxygen generation and t is
the time of irradiation. As shown in Fig. 4a (ZnPc2 as an example), the
photo-oxidation of ADMA was induced by the ZnPc2 and the absorption
intensity of ADMA continued to decrease with the irradiation time in-
creasing. From Fig. 4b, the 1O2 generation rates of ZnPc1, ZnPc2, ZnPc3
and ZnPc4 were 1.18 × 10−2, 3.34 × 10−2, 8.55 × 10−2 and
6.51 × 10−2. Their difference in 1O2 generation rates may be explained
from two aspects. Firstly, with the number of ammonium groups in-
creased, their existence form is different in water which is shown in
Fig. 3. And research has confirmed that the monomeric form is more ef-
ficient in 1O2 generation [23]. Secondly, from ZnPc1 to ZnPc4, the in-
creasing number of I− counterion atoms may also contribute to the
1O2 generation since heavy atom effects have been proved to be benefi-
cial to 1O2 generation [24,25]. The heavy atom effects can be explained
from the mechanism of the 1O2 generation [26]. With the irradiation
of appropriate light, Ps was excited to 1Ps* but its lifetime is too short
to interact with the surrounding molecules. By intersystem crossing,
the 1Ps* can decay to the triplet state 3Ps* which lifetime is in the
300
400
500
600
700
800
Wavelength/nm
Fig. 3. The UV–Vis absorption spectra of four quaternized ZnPcs in water (C =
7 × 10−6 M).
Trifluoroacetic acid (TFA) is often applied in the amino deprotection
(i.e. Boc-, Trt-). In the experiment, compound 8 (or 9) was dissolved in a
mixture of CH2Cl2 and TFA (v/v = 1:1) to deprive the Boc-group. After
completion, the green solid was filtrated and the crude solid was dis-
solved into the dilute HCl aqueous solution. The dissolved Pcs can
reprecipitate by controlling the pH at ca. 9 with the addition of 5%
NaOH solution. This method was repeated three times to obtain pure
10 (or 11) and the spectrum of compound 10 (or 11) was tested in
DMSO with a trace amount of CF3COOD to reduce the aggregation. It's
worth noting that in their 1H NMR spectrums, the signals of the Boc-
moieties (δ = 1.41–1.51) on compound 8 (or 9) are absent, indicating
that all Boc-groups have been deprotected completely.
1.0
a
0s
0.8
Finally, compound 10 (or 11) was converted to its quaternized form.
A mixture of 10 (or 11) and methyl iodide was dissolved in methanol
with stirring at 40 °C for 24 h. After completed, the green solid was
got by filtration and the product was washed successively with ethyl
acetate, acetone, and chloroform to remove the impurities. After
vacuum-dried, the green powder ZnPc3 (or ZnPc4) was characterized
by 1H NMR and the peaks of ZnPc3 (or ZnPc4) are similar with the com-
pound 10 (or 11), except in δ = 3.21 ppm (δ = 3.18). A broad signal
around 3.21 ppm (or 3.18) was derived from methyl on the nitrogen,
corresponding well with the desired structure.
0.6
0.4
150s
0.2
0.0
300
UV–Vis spectrum is useful in verifying the structure and finding the
existing form of the Pcs [19]. The changes in the UV–Vis spectrums of
ZnPcs with different quaternized ammonium groups in water are
gathered and shown in Fig. 3. As shown in Fig. 3, all compounds exhibit
typical electronic spectra with B band at 300–400 nm and Q band at
350
400
450
Wavelength/nm
0.2
0.1
ZnPc1
b
⁎
ZnPc2
ZnPc3
ZnPc4
600–700 nm, correlating to π–π transitions. In addition, their existence
form is different in water. In UV–Vis spectrums of ZnPcs, monomer ex-
hibited absorption band at ~680 nm while aggregate exhibiting absorp-
tion band at 630–640 nm [20]. To better compare their existence form,
the radio of monomer and aggregate was roughly calculated from their
absorption wavelength at 680 and 640 nm [21]. The radio of monomer
and aggregate is ZnPc1 (0.46), ZnPc2 (0.75), ZnPc3 (1.07), and ZnPc4
(0.71). The increase of monomer proportion from ZnPc1 to ZnPc3 may
attribute to the increased steric hindrance and electrical charges. Since
ZnPc3 and ZnPc4 have the same electrical charges, their difference in
existence form may be caused by their arrangement manner and in
this case straight chain is conductive to disaggregation.
0.0
-0.1
-0.2
-0.3
-0.4
-0.5
The ability of 1O2 generation and fluorescence intensity is vitally im-
portant in the process of simultaneous PDT and MI. So to study the prop-
erties of Pcs, we should evaluate their ability of 1O2 generation and
fluorescence intensity. Taking the water environment of human body
into account, 1O2 generation and fluorescence intensity of these com-
pounds were measured in the water. The 1O2 generation ability of the
0
20
40
60
80
Time/s
100 120 140 160
Fig. 4. (a) UV–Vis spectrum for the determination of the 1O2 production rate of drugs use
ADMA as quencher in water (C = 7 × 10−6 M, ZnPc2 as an example); (b) first order plots
of ADMA absorbance versus time.

18
L. Zhang et al. / Inorganic Chemistry Communications 53 (2015) 15–19
800
700
600
500
400
300
200
100
0
ZnPc1
ZnPc2
ZnPc3
ZnPc4
650
700
750
800
Wavelength/nm
Fig. 5. The fluorescence emission spectra of four quaternized ZnPcs in water (Ex = 610 nm).
micro- to milli-second range, and this is long enough for energy transfer
and oxidative transfer. The effect of heavy atoms can affect the efficiency
of intersystem crossing from 1Ps* to 3Ps* and thus has a significant influ-
ence on the 1O2 generation. For ZnPc3 and ZnPc4, they have the same
charges, same I− counterion atoms and same ammonium groups, but
their 1O2 generation is different and this difference in 1O2 generation
may mainly caused by their existence form in water. As shown in
Fig. 3, ZnPc3 is mainly monomeric form, while ZnPc4 mainly aggrega-
tion, since research has confirmed that monomeric form is more effi-
cient in 1O2 generation [23].
Fluorescence intensity is vitally important for MI. The fluorescence
emission spectra of these compounds were measured in water with
610 nm as the excitation wavelength. The fluorescence intensity of
ZnPcs is shown in Fig. 5. ZnPc3 shows the strongest fluorescence inten-
sity, then ZnPc2, and ZnPc1 is the worst, which is sorted by the number
of the quaternized ammonium groups. The fluorescence intensity of
ZnPc4 is quite weak when compared with ZnPc3 and this may be caused
by their aggregative existence form in water since research has already
confirmed that the fluorescence quantum yield of aggregates is quite
weak when compared with the monomeric form [23].
In conclusion, we have studied the relationships between the aggre-
gation tendency, 1O2 generation ability, fluorescence intensity and the
number of ammonium groups and their arrangement manner for
quaternized ZnPcs. The results showed that quaternized ZnPcs with
the more ammonium groups revealed the lower aggregation tendency,
higher 1O2 generation and fluorescence intensity. Besides, for ZnPcs
with the same ammonium groups, straight chain arrangement manner
is more excellent than branched chain. In view of the above researches
indicated that ZnPc3 had a great potential to be simultaneously used in
PDT and MI. And research about ZnPc3 and its related derivates will be
continued and report in due course.
Appendix A. Supplementary material
Supplementary information available: All the structures of com-
pounds were characterized by a combination of IR, 1H NMR, elemental
analysis and UV–Vis spectrum. And all the data of compounds were
processed in the document. Supplementary data to this article can be
found online at http://dx.doi.org/10.1016/j.inoche.2015.01.009.
References
[1] L.B. Josefsen, R.W. Boyle, Unique diagnostic and therapeutic roles of porphyrins and
phthalocyanines in photodynamic therapy, imaging and theranostics, Theranostics
9 (2012) 916–966.
[2] P. Huang, C. Xu, J. Lin, C. Wang, X. Wang, C. Zhang, X. Zhou, Light-triggered
theranostics based on photosensitizer-conjugated carbon dots for simultaneous
enhanced-fluorescence imaging and photodynamic therapy, Adv. Mater. 24
(2012) 5104–5110.
[3] Z.L. Chen, Y. Sun, P. Huang, X.X. Yang, X.P. Zhou, Studies on preparation of photosen-
sitizer loaded magnetic silica nanoparticles and their anti-tumor effects for targeting
photodynamic therapy, Nanoscale Res. Lett. (4) (2009) 400–408.
[4] J.P. Celli, B.Q. Spring, I. Rizvi, C.L. Evans, K.S. Samkoe, S. Verma, B.W. Pogue, T. Hasan,
Imaging and photodynamic therapy: mechanisms, monitoring, and optimization,
Chem. Rev. 110 (2010) 2795–2838.
[5] K. Ishii, Functional singlet oxygen generators based on phthalocyanines, Coord.
Chem. Rev. 256 (2012) 1556–1568.
[6] M. Gruner, V. Siozios, B. Hagenhoff, D. Breitenstein, C.A. Strassert, Structural and
photosensitizing features of phthalocyanine–zeolite hybrid nanomaterials,
Photochem. Photobiol. 89 (2013) 1406–1412.
[7] P. Rai, S. Mallidi, X. Zheng, R. Rahmanzadeh, Y. Mir, Development and applications of
photo-triggered theranostic agents, Adv. Drug Deliv. Rev. 62 (2010) 1094–1124.
[8] X. He, X. Wu, K. Wang, Methylene blue-encapsulated phosphonate-terminated silica
nanoparticles for simultaneous in vivo imaging and photodynamic therapy, Bioma-
terials 30 (2009) 5601–5609.
[9] Y. Cheng, J. Meyers, A. Broome, M. Kenney, J. Basilion, C. Burda, Deep penetration of a
PDT drug into tumors by noncovalent drug–gold nanoparticle conjugates, J. Am.
Chem. Soc. 133 (2011) 2583–2591.
[10] S. Wang, R. Gao, F. Zhou, M. Selke, Nanomaterials and singlet oxygen photosensi-
tizers: potential applications in photodynamic therapy, J. Mater. Chem. 14 (2004)
487–493.
[11] T.W.B. Liu, J. Chen, G. Zheng, Peptide-based molecular beacons for cancer imaging
and therapy, Amino Acids 41 (2011) 1123–1134.
Acknowledgment
[12] M. Durmus, H. Yaman, T. Nyokong, Water-soluble quaternized mercaptopyridine
substituted zinc phthalocyanines: synthesis, photophysical, photochemical and bo-
vine serum albumin binding properties, Dyes Pigments 91 (2011) 153–163.
[13] X.J. Jiang, S.L. Yeung, P.C. Lo, W.P. Fong, D.K.P. Ng, Phthalocyanine–polyamine conju-
gates as highly efficient photosensitizers for photodynamic therapy, J. Med. Chem.
54 (2011) 320–330.
[14] B. Valeur, I. Leray, Design principles of fluorescent molecular sensors for cation rec-
ognition, Coord. Chem. Rev. 205 (2000) 3–40.
[15] J. Marino, M.C.G. Vior, L.E. Dicelio, L. Roguin, J. Awruch, Photodynamic effects of
isosteric water-soluble phthalocyanines on human nasopharynx KB carcinoma
cells, Eur. J. Med. Chem. 45 (2010) 4129–4139.
This research was supported by the National Natural Science
Foundation of China (21201102), the Natural Science Foundation of
Jiangsu Higher Education Institutions of China (No. 13KJA150003 and
12KJB150015), the Scientific Research Foundation of Nanjing Normal
University (No. 2011103XG0249), the Priority Academic Program De-
velopment of Jiangsu Higher Education Institutions (PAPD), and the
Foundation of Jiangsu Collaborative Innovation Center of Biomedical
Functional Materials.

L. Zhang et al. / Inorganic Chemistry Communications 53 (2015) 15–19
19
[16] C.S. Prasanth, S.C. Karunakaran, A.K. Paul, V. Kussovski, V. Mantareva, D. Ramaiah, L.
Selvaraj, I. Angelov, L. Avramov, K. Nandakumar, N. Subhash, Antimicrobial photo-
dynamic efficiency of novel cationic porphyrins towards periodontal gram positive
and gram-negative pathogenic bacteria, Photochem. Photobiol. 90 (2014) 628–640.
[17] F. Dumoulin, M. Durmus, V. Ahsen, Synthetic pathways to water-soluble phthalocy-
anines and close analogs, Coord. Chem. Rev. 254 (2010) 2792–2847.
[18] A. Wang, Y.J. Ma, L. Zhou, Comparison of two strategies for conferring water solubil-
ity to a zinc phthalocyanine substituted with 1,2-diethylamino, Dyes Pigments 99
(2013) 348–356.
[19] M. Durmus, T. Nyokong, Synthesis and solvent effects on the electronic absorption
and fluorescence spectral properties of substituted zinc phthalocyanines, Polyhe-
dron 26 (2007) 2767–2776.
[20] M. Durmus, A. Erdogmus, A. Ogunsipe, T. Nyokong, The synthesis and
photophysicochemical behaviour of novel water-soluble cationic indium (III)
phthalocyanine, Dyes Pigments 82 (2009) 244–250.
[22] W. Tang, H. Xu, R. Kopelman, M. Philbert, Photodynamic characterization and
in vitro application of methylene blue-containing nanoparticle platform,
Photochem. Photobiol. 81 (2005) 242–249.
[23] X. Li, X. He, Dennis K.P. Ng, Influence of surfactants on the aggregation behavior of
water-soluble dendritic phthalocyanines, Macromolecules 33 (2000) 2119–2123.
[24] S. Kim, T.Y. Ohulchanskyy, D. Bharali, Y.H. Chen, Organically modified silica nanopar-
ticles with intraparticle heavy-atom effect on the encapsulated photosensitizer for
enhanced efficacy of photodynamic therapy, J. Phys. Chem. 29 (2009) 12641–12644.
[25] L. Zhou, S.H. Wei, X.F. Ge, J.H. Zhou, B.Y. Yu, J. Shen, External heavy-atomic construc-
tion of photosensitizer nanoparticles for enhanced in vitro photodynamic therapy of
cancer, J. Phys. Chem. 42 (2012) 12744–12749.
[26] L. Zhou, X.F. Ge, J.H. Liu, J.H. Zhou, Internal heavy atom effect of Au (III) and Pt (IV)
on hypocrellin A for enhanced in vitro photodynamic therapy of cancer, Bioorg.
Med. Chem. Lett. 23 (2013) 5317–5324.
[21] A. Wang, L. Zhou, Synthesis of novel octa-cationic and non-ionic 1,2-ethanediamine
substituted zinc (II) phthalocyanines and their in vitro anti-cancer activity compar-
ison, Eur. J. Med. Chem. 58 (2012) 12–21.