Synthesis, photophysical studies and 1O2 generation of carboxylate-terminated zinc phthalocyanine dendrimers

Article abstract of DOI:10.1016/j.jinorgbio.2014.02.007

Highly water-soluble dendrimers have been prepared consisting of a central zinc phthalocyanine moiety and dendritic wedges with terminal carboxylate groups. The biggest polyelectrolyte comprises 32 negative charges at the dendrimer surface. The photophysical studies reveal a strong correlation between the degree of dendritic environment, the extent of aggregation, and the ability to generate singlet oxygen in aqueous media. Compared to dendrimers having an axial derivatization the functionalization on the outer rim also significantly improves the phthalocyanine's ability to photosensitize singlet oxygen.

Full text of DOI:10.1016/j.jinorgbio.2014.02.007

JIB-09473; No of Pages 7
Journal of Inorganic Biochemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Synthesis, photophysical studies and 1O2 generation of carboxylate-terminated zinc
phthalocyanine dendrimers
Francesca Setaro a, Rubén Ruiz-González b, Santi Nonell b,1, Uwe Hahn a,c,1, Tomás Torres a,d,
⁎
a
Departamento de Química Orgánica (Modulo 01), Facultad de Ciencias, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain
Grup d'Enginyeria Molecular, Institut Químic de Sarriá, Universitat Ramon Llull, Barcelona, Spain
b
c
Laboratoire de Chimie des Matériaux Moléculaires, Université de Strasbourg et CNRS (UMR 7509), Ecole Européenne de Chimie, Polymères et Matériaux (ECPM),
25 rue Becquerel, 67087 Strasbourg Cedex 2, France
d
IMDEA-Nanociencia, c/Faraday, 9, Cantoblanco, 28049 Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Highly water-soluble dendrimers have been prepared consisting of a central zinc phthalocyanine moiety and
dendritic wedges with terminal carboxylate groups. The biggest polyelectrolyte comprises 32 negative charges
at the dendrimer surface. The photophysical studies reveal a strong correlation between the degree of dendritic
environment, the extent of aggregation, and the ability to generate singlet oxygen in aqueous media. Compared
to dendrimers having an axial derivatization the functionalization on the outer rim also significantly improves
the phthalocyanine's ability to photosensitize singlet oxygen.
Received 2 October 2013
Received in revised form 16 February 2014
Accepted 18 February 2014
Available online xxxx
Keywords:
Phthalocyanines
Dendrimers
© 2014 Elsevier Inc. All rights reserved.
Singlet oxygen generation
1. Introduction
unaffected. Several metallophthalocyanines disclosed their capability
to act as photosensitizing agents like e.g., zinc [29–36], silicon [37–41],
The structural motif of phthalocyanines has attracted significant
research efforts throughout the last couple of decades [1–6]. Whereas
this class of molecules has often been used as dyes and pigments in
the early days, this interest has shifted in recent decades towards their
utilization as building blocks for the construction of new molecular
materials for electronics and optoelectronics [1–6], and dye-sensitized
solar cells [7–14].
This notwithstanding, phthalocyanines have also demonstrated
their potential in the production of singlet molecular oxygen (1O2)
[15–17]. This reactive oxygen species is well known to play a crucial
role in applications such as photocatalysis [18–22] or photodynamic
therapy (PDT) [23–26]. Both applications rely on the interaction
between light in either the UV or visible region of the spectrum and a
photosensitizer (PS), but only the latter takes advantage of this effect
to selectively kill cancer cells [27,28]. The growing popularity of this
therapeutic method can be attributed to the high selectivity of
destructing diseased tissues and tumors through the localized genera-
tion of cytotoxic 1O2 while surrounding healthy cells remain mostly
or ruthenium as metal in the central cavity [42–45]. Some of these spec-
imens are dendritic structures in which the phthalocyanine macrocycle
is embedded inside the fractal scaffold [46,47]. One of the latest ap-
proaches has been described by Kataoka et al. [29,30]. It confers a prom-
ising example with a 100-fold photochemical enhancement of
transgene expression in vitro while at the same time showing reduced
photocytotoxicity. This ternary system consists of a core containing
DNA that is packaged with cationic peptides and surrounded at the
surface by a given number of anionic phthalocyanine dendrimers. This
sophisticated system was then also applied in animal experiments and
showed transgene expression only in the laser-irradiated region, thus
presenting the first example of photochemical-internalization-
mediated gene delivery in vivo. On the other hand, dendritic porphyrins
are not likely to be internalized by cancer cells, yet they can inflict lethal
photodynamic damage and kill them from the outside [48].
Recently, we described a series of ruthenium phthalocyanine
dendrimers with oligoethylene end groups which provided a straight-
forward means to switch their ability to produce 1O2 on and off between
non-polar and polar environments [42] (Chart 1). In continuation of our
research on phthalocyanine-derived PSs for photodynamic applications,
we report herewith on the photophysical characterization of a series of
zero to second generation zinc phthalocyanine dendrimers appended at
the surface through carboxylate moieties, whose potential to generate
1O2 was evaluated for pre-screening purposes. In contrast to the work
⁎
Corresponding author at: Departamento de Química Orgánica (Modulo 01), Facultad
de Ciencias, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain. Fax:
+34 91497 3966.
E-mail address: tomas.torres@uam.es (T. Torres).
Fax: +34 91497 3966.
1
http://dx.doi.org/10.1016/j.jinorgbio.2014.02.007
0162-0134/© 2014 Elsevier Inc. All rights reserved.
Please cite this article as: F. Setaro, et al., Synthesis, photophysical studies and 1O2 generation of carboxylate-terminated zinc phthalocyanine
dendrimers, J. Inorg. Biochem. (2014), http://dx.doi.org/10.1016/j.jinorgbio.2014.02.007

2
F. Setaro et al. / Journal of Inorganic Biochemistry xxx (2014) xxx–xxx
Chart 1. Structures of amphiphilic zinc phthalocyanine dendrimers ZnPc1–3 with up to 32 terminal carboxylate groups.
of Kataoka et al. [29,30,49], the primary photophysical properties and
the ability to generate 1O2 for the complete series of dendrimers
demonstrate a positive effect of the dendrimer shell.
2:1) to yield 5 (474 mg, 79%) as a white solid. 1H NMR (300 MHz,
CDCl3): δ = 8.68 (s, 1H, Har), 8.27 (s, 2H, Har), 7.74 (d, J = 9 Hz, 1H,
Har), 7.36 (d, J = 3 Hz, 1H, Har), 7.27 (dd, J = 9 Hz, J = 3 Hz, 1H, Har),
5.23 (s, 2H, CH2), 4.43 (q, J = 7 Hz, 4H, CH2), 1.43 (t, J = 7 Hz, 6H,
CH3) ppm. 13C NMR (75 MHz, CDCl3): δ = 165.2, 161.2, 135.4, 135.1,
132.4, 131.6, 130.7, 119.8, 119.5, 117.5, 115.3, 114.8, 108.1, 69.8, 61.6,
2. Materials and methods
2.1. Chemicals and materials
14.1 ppm. MALDI-TOF HRMS (matrix DCTB + PEGNa300 +
PEGNa400 + NaI): calc. for: C21H18N2NaO5 [M + Na+]: m/z =
401.1108, found 401.1118.
All reagents were used as purchased from commercial sources with-
out further purification. Solvents were dried using standard techniques
prior to use. Dendritic alcohols 2–4 have been prepared according to a
procedure as previously described in the literature [50]. All reactions
were performed in standard glassware under an inert argon atmo-
sphere. Reactions were monitored by thin-layer chromatography
(TLC) using TLC plates precoated with silica gel 60F254 (Merck). Column
chromatography was carried out on a Merck silica gel 60, 40–63 μm
(230–400 mesh). Gel permeation chromatography (GPC) was per-
formed using Biorad, Biobeads SX-1 (200–400 mesh). 1H and 13C NMR
spectra were recorded using Bruker AC 300 (300 MHz) instruments
(Hpc: phthalocyanine protons, Har: aromatic protons); the solvent signal
was used for internal calibration. UV–visible (UV–vis) spectra were re-
corded with a JASCO V-660 spectrophotometer. MALDI-TOF high reso-
lution mass spectra (HRMS) were recorded with a Bruker Reflex III
spectrometer.
2.2.2. Synthesis of first generation phthalonitrile (6)
To a solution of 1 (54 mg, 312 μmol) and dendritic benzyl alcohol 3
(170 mg, 279 μmol) in dry DMF (7 mL) was added anhydrous K2CO3
(188 mg, 1.36 mmol) in three portions. After addition was complete,
the reaction mixture was heated at 60 °C for 24 h under argon atmo-
sphere. After cooling to room temperature, the crude mixture was
poured into water and the aqueous phase extracted several times with
CH2Cl2. The combined organic phase was dried with Na2SO4, filtered
and the solvent evaporated to dryness. The resulting viscous oil was
purified by column chromatography (CH2Cl2/EtOAc 5:1) to yield 6
(140 mg, 68%) as a colorless viscous oil. 1H NMR (300 MHz, CDCl3):
δ = 8.63 (s, 2H, Har), 8.28 (s, 4H, Har), 7.69 (d, J = 9 Hz, 1H, Har),
7.29 (d, J = 3 Hz, 1H, Har), 7.21 (dd, J = 9 Hz, J = 3 Hz, 1H, Har),
6.63 (m, 3H, Har), 5.13 (s, 4H, CH2), 5.11 (s, 2H, CH2), 4.42 (q, J = 7 Hz,
8H, CH2), 1.42 (t, J = 7 Hz, 12H, CH3) ppm. 13C NMR (75 MHz, CDCl3):
δ = 165.5, 161.5, 160.0, 137.4, 137.2, 131.4, 130.2, 119.6, 117.4, 115.5,
115.1, 107.7, 106.4, 102.0, 70.6, 69.1, 61.5, 14.3 ppm. MALDI-TOF
HRMS (matrix ditranol + PEG600 + NaI): calc. for: C41H38N2NaO11
[M + Na+]: m/z = 757.2368, found 757.2404.
2.2. Chemistry
2.2.1. Synthesis of generation zero phthalonitrile (5)
To a solution of 1 (305 mg, 1.76 mmol) and dendritic benzyl alcohol
2 (400 mg, 1.59 mmol) in dry DMF (16 mL) was added anhydrous
K2CO3 (975 mg, 7.05 mmol) in three portions. After addition was com-
plete, the reaction mixture was heated at 60 °C for 24 h under argon at-
mosphere. After cooling to room temperature, the crude mixture was
poured into water and the aqueous phase extracted several times with
CH2Cl2. The combined organic phase was dried with Na2SO4, filtered
and the solvent evaporated to dryness. The resulting viscous oil was pu-
rified by gradient column chromatography (CH2Cl2 to CH2Cl2/EtOAc
2.2.3. Synthesis of second generation phthalonitrile (7)
To a solution of 1 (14.2 mg, 82.0 μmol) and dendritic benzyl alcohol
3 (120 mg, 90.8 μmol) in dry DMF (7 mL) was added anhydrous K2CO3
(63 mg, 456 μmol) in three portions. After addition was complete, the
reaction mixture was heated at 60 °C for 24 h under argon atmosphere.
After cooling to room temperature, the crude mixture was poured into
water and the aqueous phase extracted several times with CH2Cl2. The
Please cite this article as: F. Setaro, et al., Synthesis, photophysical studies and 1O2 generation of carboxylate-terminated zinc phthalocyanine
dendrimers, J. Inorg. Biochem. (2014), http://dx.doi.org/10.1016/j.jinorgbio.2014.02.007

F. Setaro et al. / Journal of Inorganic Biochemistry xxx (2014) xxx–xxx
3
Scheme 1. Preparation of dendritic phthalonitriles 5–7 with terminal ethyl ester functions. Reagents and conditions: (i) dendritic alcohol 2–4, K2CO3, DMF, reflux, 1d (5: 79%, 6: 68%, 7:
86%).
combined organic phase was dried with Na2SO4, filtered and the solvent
evaporated to dryness. The resulting viscous oil was purified by gradient
column chromatography (CH2Cl2 to CH2Cl2/EtOAc 8:1) to yield 7
(102 mg, 86%) as a pale yellow viscous oil. 1H NMR (300 MHz, CDCl3):
δ = 8.63 (s, 4H, Har), 8.29 (s, 8H, Har), 7.68 (d, J = 9 Hz, 1H, Har), 7.30
(d, J = 3 Hz, 1H, Har), 7.22 (dd, J = 9 Hz, J = 3 Hz, 1H, Har), 6.69
(m, 3H, Har), 6.59 (m, 6H, Har), 5.13 (s, 8H, CH2), 5.11 (s, 4H, CH2),
5.00 (s, 2H, CH2), 4.42 (q, 16H, CH2), 1.39 (t, 24H, CH3) ppm. 13C
NMR (75 MHz, CDCl3): δ 165.5, 165.2, 160.2, 159.8, 139.3, 137.6,
137.4, 135.4, 132.5, 131.4, 130.2, 119.9, 119.6, 117.6, 117.5, 106.6,
106.4, 106.3, 101.6, 101.6, 70.6, 69.1, 69.9, 61.4, 14.1. MALDI-TOF
HRMS (matrix ditranol + NaI): calc. for: C81H78N2NaO23 [M + Na+]:
m/z =1469.4888, found 1469.4846.
2.2.5. Synthesis of generation zero ester-appended zinc phthalocyanine (8)
Ester-appended zinc phthalocyanine: Prepared from dendritic
phthalonitrile 5 (250 mg, 661 μmol), Zn(OAc)2 (48 mg, 262 μmol) and
a few drops of DBU in n-pentanol (8 mL). Purification by column chro-
matography (SiO2; EtOAc/Hexane 10:1) followed by GPC (eluent:
THF) yielded dendritic zinc phthalocyanine 8 (167 mg, 53%) as dark
green solid. NMR data indicate that transesterification of ethyl to pentyl
end groups occurred during the synthesis. 1H NMR (300 MHz, THF-d8):
δ = 8.76–8.98 (m, 4H, Hpc), 8.62 (m, 12H, Har), 8.30–8.56 (m, 4H, Hpc),
7.50–7.73 (m, 4H, Hpc), 5.66 (m, 8H, CH2), 4.44 (q, J = 7 Hz, 16H, CH2),
1.88 (m, 16H, CH2), 1.50 (m, 32H, CH2), 0.98 (m, 24H, CH3) ppm. UV–vis
(THF): λmax (log ε) = 675 (5.3), 610 (4.6), 350 (4.97), 284 (4.6). MALDI-
TOF (matrix DHB): calcd. for C108H120N8O20Zn [M]+: m/z = 1914.8,
found 1914.8.
2.2.4. General procedure for the preparation of ester-appended zinc
phthalocyanines (8–10)
2.2.6. Synthesis of first generation ester-appended zinc phthalocyanine (9)
Ester-appended zinc phthalocyanine: Prepared from dendritic
phthalonitrile 6 (197 mg, 268 μmol), Zn(OAc)2 (20 mg, 109 μmol) and
a few drops of DBU in n-pentanol (4 mL). Purification by column chro-
matography (SiO2; EtOAc/Hexane 10:1) followed by GPC (eluent:
THF) yielded dendritic zinc phthalocyanine 9 (97 mg, 39%) as dark
green viscous oil. NMR data indicate that transesterification of ethyl to
pentyl end groups occurred during the synthesis. 1H NMR (300 MHz,
THF-d8 + 1% (CD3)2SO): δ = 8.55 (s, 8H, Har), 8.41 (br m, 4H, Hpc),
A mixture of the corresponding dendritic phthalonitrile (4 eq) and
Zn(OAc)2 (1.2 eq) in n-pentanol was heated at 90 °C. At this tempera-
ture a few drops of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) were
added and the mixture heated at reflux overnight under argon atmo-
sphere. After cooling to room temperature, the solvent was removed
under reduced pressure to give a greenish oil, which was purified by
column chromatography and size exclusion chromatography.
Scheme 2. Preparation of zinc phthalocyanine dendrimers 8–10 with terminal ethyl ester functions and subsequent saponification under the formation of polyelectrolyte dendrimers
ZnPc1–3. Reagents and conditions: (i) Zn(AcO)2, DBU, n-pentanol, 90 °C to reflux, 16–20h (8: 53%, 9: 39%, 10: 31%); (ii) NaOH, H2O/MeOH (v/v: 1:4), 40 °C, 4 h (ZnPc1: 81%, ZnPc2:
83%, ZnPc3: 83%).
Please cite this article as: F. Setaro, et al., Synthesis, photophysical studies and 1O2 generation of carboxylate-terminated zinc phthalocyanine
dendrimers, J. Inorg. Biochem. (2014), http://dx.doi.org/10.1016/j.jinorgbio.2014.02.007

4
F. Setaro et al. / Journal of Inorganic Biochemistry xxx (2014) xxx–xxx
8.33 (s, 16H, Har), 7.71 (m, 4H, Hpc), 7.38 (m, 4H, Hpc), 6.82 (s, 8H, Har),
6.76 (s, 4H, Har), 5.25 (s, 8H, CH2), 5.23 (s, 4H, CH2), 4.33 (t, 32H, J =
7 Hz, CH2), 1.78 (m, 32H, CH2), 1.42 (m, 64 H, CH2), 0.93 (m, 48H,
CH3) ppm. UV–vis (THF): λmax (log ε) = 677 (4.4), 611 (3.7), 350 (4.2).
MALDI-TOF HRMS (matrix ditranol): calcd. for: C212H248N8O44Zn [M+]:
m/z = 3677.6764, found 3677.6900.
CH2) ppm. UV–vis (H2O): λmax (log ε) = 686 (4.0), 638 (4.0), 642
(4.4), 275 (4.6).
2.3. Photophysical measurements
Absorption spectra were recorded on a double beam Cary 6000i spec-
trophotometer (Varian, Palo Alto, CA). Time-resolved near-infrared phos-
phorescence measurements were carried out using a customized setup
composed by PicoQuant Fluotime 200 system and its software for the
data analysis. Direct 1O2 phosphorescence detection was achieved by
means of a diode-pumped pulsed Nd:YAG laser (FTSS355-Q, Crystal
Laser, Berlin, Germany) working at a 10 kHz repetition rate for sample ex-
citation at 355 nm (5 mW, 0.5 μJ per pulse). A 1064 nm rugate notch filter
(Edmund Optics, U.K.) was placed at the exit port of the laser to remove
any residual component of its fundamental emission in the near-IR
region. The luminescence exiting from the 90° side of the sample was
filtered by two long-pass filters of 355 and 532 nm (Edmund Optics,
York, U.K.) and two narrow bandpass filters at 1275 nm (NB-1270-010,
Spectrogon, Sweden; bk-1270-70-B, bk Interferenzoptik, Germany) to
remove any scattered laser radiation. A near-IR sensitive photomultiplier
tube assembly (H9170-45, Hamamatsu Photonics, Hamamatsu City,
Japan) was used as the detector. Photon counting was achieved with a
multichannel scaler (PicoQuant's Nanoharp 250).
2.2.7. Synthesis of second generation ester-appended zinc phthalocyanine
(10)
Ester-appended zinc phthalocyanine: Prepared from dendritic
phthalonitrile 7 (119 mg, 82.2 μmol), Zn(AcO)2 (6 mg, 32.7 μmol) and
a few drops of DBU in n-pentanol (2 mL). Purification by column
chromatography (SiO2; EtOAc/CH2Cl2 10:1) followed by GPC (eluent:
THF) yielded dendritic zinc phthalocyanine 10 (45 mg, 31%) as dark
green viscous oil. NMR data indicate that transesterification of ethyl to
pentyl end groups occurred during the synthesis. 1H NMR (300 MHz,
THF-d8 + 1% (CD3)2SO): δ = 9.26 (m, 4H, Hpc), 9.03 (m, 4H, Hpc),
8.51 (br s, 16H, Har), 8.31 (br s, 32H, Har), 7.83 (m, 4H, Hpc), 7.09 (m,
8H, Har), 6.73–6.93 (br m, 28H, Har), 5.63 (br s, 8H, CH2), 5.26 (m,
48H, CH2), 4.29 (br s, 64H, CH2), 1.73 (br s, 64H, CH2), 1.36 (br s,
128H, CH2), 0.88 (s, 96H, CH3) ppm. UV–vis (THF): λmax (log ε) =
678 (4.94), 612 (4.12), 350 (4.7), 275 (4.92). MALDI-TOF (matrix
DCTB): calcd. for: C324H312N8O92Zn [M+]: m/z = 7201.4, found 7201.4.
Transient absorption experiments in the UV–visible (UV–vis) region
were carried out using a home-built nanosecond laser flash photolysis
system. In this instrument, the 3rd harmonic (355 nm) of a Continuum
Surelite I-10 Nd:YAG laser (10 Hz, 5 ns pulse width, 1–10 mJ per pulse)
was directed onto the sample. Changes in the sample absorbance were
detected by a Hamamatsu R928 photomultiplier in order to monitor the
intensity variations of an analyzing beam produced by a 75 W short arc
Xe lamp (USHIO) and spectral discrimination was provided by a PTI 101
monochromator. The signal was fed to a Lecroy Wavesurfer 454 oscillo-
scope for digitizing and averaging (3–10 shots, typically) and finally
transferred through a GPIB interface (National Instruments) to a PC
computer for data storage and analysis. The TTL sync output of the
2.2.8. General procedure for the hydrolysis of ester-appended zinc
phthalocyanine dendrimers
The corresponding ester-terminated zinc phthalocyanine 8–10 was
dissolved in a small amount of THF and the solution added slowly to a
saturated solution NaOH in a mixture of water/methanol (v/v: 1:4).
The solution was then heated at 40 °C until total consumption of the
starting material (ZnPc1–3: 4 h). The organic solvents were evaporated
and the remaining aqueous solution was subjected to dialysis. Finally,
the solid was treated with CH2Cl2, acetone, and ethyl acetate and
dried to give the final product.
2.2.9. Synthesis of generation zero zinc phthalocyanine polyelectrolyte
dendrimer (ZnPc1)
Prepared from dendritic zinc phthalocyanine
8 (60 mg,
38.0 μmol) in THF (3 mL) and saturated solution of NaOH in water/
methanol (v/v 1:4; 25 mL). Carboxylate-terminated zinc phthalocy-
anine dendrimer ZnPc1 was obtained after dialysis as a dark green
powder (47 mg, 81%). 1H NMR (300 MHz, D2O + 1% (CD3)2SO):
δ = 9.26 (br m, 4H, Hpc), 8.84 (m, 4H, Hpc), 8.61 (br, 12H, Har),
7.97 (m, 4H, Hpc), 5.45 (s, 8H, CH2) ppm. UV–vis (H2O): λmax
(log ε) = 684 (4.4), 639 (4.2), 344 (4.33). MALDI-TOF (matrix
DHB): calc. for: C68H40N8O20Zn [M+]: m/z 1354.2, found 1354.1.
2.2.10. Synthesis of first generation zinc phthalocyanine polyelectrolyte
dendrimer (ZnPc2)
Prepared from dendritic zinc phthalocyanine 9 (20 mg, 6.66 μmol)
in THF (1 mL) and saturated solution of NaOH in water/methanol (v/v
1:4; 15 mL). Carboxylate-terminated zinc phthalocyanine dendrimer
ZnPc2 was obtained after dialysis as a dark green powder (16 mg,
83%). 1H NMR (300 MHz, D2O + (CD3)2SO): δ = 8.17 (s, 8H, Har), 7.90
(s, 16H, Har), 6.68 (s, 16H, CH2), 6.63 (s, 8H, CH2) ppm. UV–vis (H2O):
λmax (log ε) = 680 (3.9), 638 (3.97), 336 (4.1).
2.2.11. Synthesis of second generation zinc phthalocyanine polyelectrolyte
dendrimer (ZnPc3)
Prepared from dendritic zinc phthalocyanine 10 (25 mg,
4.27 μmol) in THF (1.5 mL) and saturated solution of NaOH in
water/methanol (v/v 1:4; 20 mL). Carboxylate-terminated zinc phtha-
locyanine dendrimer ZnPc3 was obtained after dialysis as a dark green
powder (20 mg, 83%). 1H NMR (300 MHz, D2O + (CD3)2SO): δ = 8.15
(s, 16H, Har), 7.90 (s, 32H, Har), 6.62 (s, 24H, Har), 6.63 (br, 48H,
Fig. 1. Normalized absorption spectra of anionic phthalocyanines ZnPc1 (red solid line),
ZnPc2 (green dotted line) and ZnPc3 (blue dashed line) in DMSO (a) or D2O (b). The
bulk concentration is ca. 10 μM for all compounds. (For interpretation of the references
to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: F. Setaro, et al., Synthesis, photophysical studies and 1O2 generation of carboxylate-terminated zinc phthalocyanine
dendrimers, J. Inorg. Biochem. (2014), http://dx.doi.org/10.1016/j.jinorgbio.2014.02.007

F. Setaro et al. / Journal of Inorganic Biochemistry xxx (2014) xxx–xxx
5
O2
Table 1
oxygen concentration afforded kq as the slope of the linear fit. The
concentration of oxygen in the solutions was changed by gentle bub-
bling with solvent-saturated argon or oxygen for at least 30 min. The
Summary of the photophysical characterization of the anionic zinc dendrimer
phthalocyanines in D2O.
O2
b
2
2
O f
Compound
Q-band λmax/nma
ΦΔ
τΔ/μsc
kqO /M−1 s−1d
τT/μse
FT
fraction of triplets quenched by oxygen, FT , was calculated from the
kq and kT0 values following Eq. (3):
O2
ZnPc1
ZnPc2
ZnPc3
675 (682)
682 (685)
679 (684)
0.02
0.06
0.15
55.1
59.8
62.8
5.8 × 108
6.2 × 108
6.1 × 08
38
59
171
0.82
0.88
0.95
kOq ꢀ ½O2ꢁ
2
FTO
¼
:
ð3Þ
2
a
DMSO as solvent in parentheses.
k0T þ kOq 2 ꢀ ½O2ꢁ
b
c
d
e
f
Singlet oxygen quantum yield in air-saturated solutions.
Singlet oxygen lifetime in air-saturated solutions.
Rate constant for triplet quenching by oxygen.
Triplet lifetime in argon-saturated solutions.
3. Results and discussion
Fraction of phthalocyanine triplet excited states deactivated by oxygen in air-
O2
saturated solutions (FT ).
3.1. Synthesis and characterization
laser was used to trigger the oscilloscope. The energy of the laser pulse
was varied by controlling the Q-switch delay and measured with a py-
roelectric energy meter (RJP 735 and RJ 7610) from Laser Precision
Corp. The system was controlled by the in house-developed LKS soft-
ware (LabView, National Instruments).
The singlet oxygen quantum yields (ΦΔ) were determined by compar-
ing the intensity of the 1270 nm signals to those of optically-matched
solutions of a reference PS. 5,10,15,20-Tetrakis(4-sulfonatophenyl)
porphine (TPPS, ΦΔ, D2O = 0.64) was used as reference [51]. Measure-
ments were carried out in 1 × 1 cm quartz fluorescence cuvettes at
room temperature. The concentration of the phthalocyanines was in the
range 1–10 μM. Time-resolved phosphorescence signals (St) were then
fitted by Eq. (1) using the PicoQuant FluoFit 4.0 data analysis software,
thereby extracting amplitude (A) and lifetime values (τΔ and τT). Since
A is an instrumental quantity proportional to the quantum yield, ΦΔ
values were calculated from the amplitudes according to Eq. (2):
Dendritic molecules have been an inspiration to chemists since its
discovery in the late 1970s. The structural characteristics brought
along with such macromolecular architectures remain appealing not
least due to the possibility to tune and control the overall properties
by pure chemical means. Likewise, the generation-dependant behavior
offers the incentive to study the often peculiar properties of a given core
moiety that is surrounded by an increasing branched shell. In this nexus,
hydrophilic dendrimers can be easily obtained through the implemen-
tation of polar groups, which assist to overcome the intrinsically strong
lipophilic character of phthalocyanine macrocycles [52]. This can be
accomplished upon decoration of the dendritic scaffolds by multiple
charges at the surface with for instance modified Fréchet-type
polyarylether dendrons, or aliphatic Newkome-type dendrons. Similarly,
introducing polar entities such as oligoethylene glycol chains confers a
certain hydrophilicity [42,53–56]. Whereas the former approach relying
on multiple charges allows to obtain highly water-soluble scaffolds, the
latter comprising neutral polar end groups offers the possibility to exam-
ine the material properties in both common organic solvents as well as
aqueous media [42,53–56].
The synthesis of the zinc phthalocyanine dendrimers presented
herein started with the preparation of dendritic wedges 2–4, which
have been obtained following the procedure of Fréchet et al. [50]. The
different alcohols of generation zero to two have then been subjected
to ipso-substitution reactions with 4-nitrophthalonitrile (1) in the
presence of potassium carbonate as base to provide the branched
ortho-dinitriles in good to high yields of 68 to 86%. With these
dendritically modified phthalonitriles 5–7 in hand, the corresponding
cyclotetramerizations have been conducted in the presence of zinc ace-
tate as metal template (Scheme 1). The target dendrimers 8–10 have
been obtained as intense green products in reasonably good yields of
31 to 53% after purification by gradient column chromatography and
size exclusion chromatography. However, it has to be noted that the
often observed transesterification occurred during the synthesis giving
rise to the formation of the pentylester-terminated dendrimers rather
than the ethyl-appended derivatives. The transesterification products
have been obtained quantitatively as can be deduced from the 1H NMR
integrals. In addition, no signals have been observed in the MALDI-TOF
mass spectra for partially transesterified products. As the last step of the
synthetic route, the terminal ester groups were saponified under the
use of sodium hydroxide in a mixture of water and methanol (v/v: 1:4)
and furnished the final dendrimers ZnPc1–3 after dialysis in high yields
(ZnPc1: 81%, ZnPc2: 83%, ZnPc3: 83%) (Scheme 2).
ꢀ
ꢁ
τΔ
τΔ−τT
−t=τT
Δ
St ¼ A ꢀ
ꢀ
e−t=τ −e
ð1Þ
ð2Þ
AðsampleÞ
AðrefÞ
ΦΔðsampleÞ ¼ ΦΔðrefÞ ꢀ
:
The rate constant for oxygen quenching of the triplet state of ZnPcs
O2
1–3 (kq ) was determined by measuring the lifetime of the triplet–
triplet transient-absorption signals as a function of oxygen concen-
tration in solution. The samples were excited at 355 nm and the tran-
sients were observed at 635 nm. A plot of the reciprocal lifetime vs
All phthalonitriles (5–7) and phthalocyanines with ester end groups
(8–10) have been characterized by common techniques including NMR,
MALDI-TOF mass spectrometry and UV–vis spectroscopy. Due to the
multiple charges prevailing in ZnPc1–3, the characterization of these
polyelectrolytes was more difficult. Using the NMR technique, the corre-
sponding resonances in the proton spectra have been obtained as broad
signals even after accumulation over an elongated period of time.
MALDI-TOF mass spectrometry was attempted under the use of the re-
spective uncharged acid species. However, these attempts proved only
Fig. 2. Time-resolved near-IR signals of 1O2 photosensitized by ZnPc1 (red), ZnPc2 (green),
ZnPc3 (blue) and reference TPPS (gray) in D2O upon excitation at 355 nm. The changes in
signal intensity are greater in this order. (For interpretation of the references to color in
this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: F. Setaro, et al., Synthesis, photophysical studies and 1O2 generation of carboxylate-terminated zinc phthalocyanine
dendrimers, J. Inorg. Biochem. (2014), http://dx.doi.org/10.1016/j.jinorgbio.2014.02.007

6
F. Setaro et al. / Journal of Inorganic Biochemistry xxx (2014) xxx–xxx
Fig. 3. Transient absorption decays of ZnPc1 (red, top line in panels a and b), ZnPc2 (green, middle line in panels a and b) and ZnPc3 (blue, bottom line in panels a and b) excited at 355 nm
and observed at 635 nm in argon-saturated solutions (a), air-saturated solutions (b) or oxygen-saturated solutions (c) in D2O. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article.)
successful for the generation zero derivative and no suitable signal has
been observed neither for the first nor for the second generation
dendrimer.
the dendrimer complexity [54,58,59]. Our series of phthalocyanines
behave in a similar fashion, as observed in Fig. 3 and Table 1, but the
effect must originate from the higher degree of disaggregation in the
more branched derivatives. Such effect is expected to also lead to an
increase in the ΦΔ value — as a result of the lower non-radiative deacti-
vation of its precursors, consistent with our observations. In addition,
3.2. Spectroscopic and photophysical characterization
O2
the fraction of triplets quenched by oxygen, FT , is very similar in all
Fig. 1 shows the normalized absorption spectra of all three phthalo-
cyanines either in deuterated water (D2O) or in DMSO. In this latter
media, compounds present a narrow shape and the maxima are found
in the less energetic but more intense band of the Q-region. When
changing to D2O, a remarkable change in the relative intensities of the
two bands of the Q-region appears, mainly due to different extents of
inter- or intramolecular aggregation effects. Comparison between
both solvents reveals that ZnPc3 is the only compound that maintains
the ratio between the two less energetic bands of the Q-region N1 in
aqueous media (least energetic band as dividend of the ratio). On the
contrary, for ZnPc1 and ZnPc2 this ratio is below 1, indicating that
these latter compounds are still largely aggregated. Moreover, for all
the cases a small but clear hypsochromic shift occurs and bands broad-
en, another archetypal aggregation feature. Altogether, the degree of
complexity of the dendrimer seems to be responsible for the extent of
aggregation, the more branched the easier to disaggregate in aqueous
media.
A detailed compilation of the spectroscopic and photophysical prop-
erties of the family of anionic zinc phthalocyanines ZnPc1–3 is summa-
rized in Table 1. Apart from the aforementioned spectral changes,
further differences are observed regarding their ability to sensitize the
production of 1O2. The photosensitizing properties of the phthalocya-
nines hinged on the degree of ramification, the more efficient the higher
the degree of dendrimerization.
As it can be seen in Table 1 and Fig. 2, the ΦΔ values increased 3-fold
and over 7-fold for ZnPc2 and ZnPc3, respectively, as compared to
parent zero generation ZnPc1 in D2O. A remarkable ΦΔ value of 0.15 is
achieved for ZnPc3, one order of magnitude higher than that of other
reported dendritic phthalocyanines [42]. On the other hand, the ester
form of ZnPc3 shows a ΦΔ value of 0.42 in tetrahydrofuran, where it is
predominantly in monomeric form.
Regarding the τΔ values, the compounds exhibited a small but not
negligible 1O2 quenching ability that correlated, once again, with the
level of complexity and degree of aggregation. Thus, ZnPc1 exhibited a
lifetime value of ca. 55 μs in solution, shorter than the reported value
of 65 μs in D2O [57], which is progressively more attained by ZnPc2
and ZnPc3.
compounds. This suggests that functionalization at the outer rim of
the phthalocyanine macrocycle is a valuable strategy for improving
the solubility of the phthalocyanines without shielding of the chromo-
phore from oxygen.
4. Conclusion
A new series of monodisperse generation zero to two zinc
phthalocyanine-centered dendrimers has been synthesized. Within
the last step of the synthetic route the terminal ethyl esters have been
saponified giving rise to a dramatic change in polarity under the forma-
tion of multiply charged polyelectrolyte species that are well-soluble in
aqueous media. Along with increasing dendritic shell surrounding the
photoactive zinc phthalocyanine centerpiece, a significant increase in
the generation of singlet oxygen has been observed even in air-
saturated solutions. Hence, such highly hydrophilic phthalocyanine
dendrimers may thus be advantageous for the purpose of producing
1O2 in biological media by photosensitization.
Abbreviations
1O2
A
singlet oxygen
amplitude
DBU
DCTB
1,8-diazabicyclo[5.4.0]undec-7-ene
trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene]
malononitrile
DHB
DMF
FT
2,5-dihydroxybenzoic acid
dimethylformamide
fraction of triplet states quenched by oxygen
gel permeation chromatography
high resolution mass spectrometry
rate constant for oxygen quenching
O2
GPC
HRMS
O2
kq
MALDI-TOF matrix-assisted laser desorption ionization
Nd:YAG neodymium-doped yttrium aluminum garnet
PDT
PEG
PS
photodynamic therapy
polyethylene glycol
photosensitizer
In contrast with apically-substituted dendritic phthalocyanines,
the kq values remained essentially constant among all compounds
(Table 1). It has been previously published that apical dendritic
wedges are able to partially shield the photoactive core from molecular
oxygen, resulting in an increase of the triplet lifetime upon increasing
St
time-resolved phosphorescence signals
tetrahydrofuran
thin-layer chromatography
5,10,15,20-tetrakis(4-sulfonatophenyl)porphine
transistor–transistor logic
O2
THF
TLC
TPPS
TTL
Please cite this article as: F. Setaro, et al., Synthesis, photophysical studies and 1O2 generation of carboxylate-terminated zinc phthalocyanine
dendrimers, J. Inorg. Biochem. (2014), http://dx.doi.org/10.1016/j.jinorgbio.2014.02.007

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ZnPc
ΦΔ
τ
zinc phthalocyanine
singlet oxygen quantum yields
lifetime values
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Please cite this article as: F. Setaro, et al., Synthesis, photophysical studies and 1O2 generation of carboxylate-terminated zinc phthalocyanine
dendrimers, J. Inorg. Biochem. (2014), http://dx.doi.org/10.1016/j.jinorgbio.2014.02.007