Synthesis, properties and near-infrared imaging evaluation of glucose conjugated zinc phthalocyanine via Click reaction

Article abstract of DOI:10.1142/S1088424611004361

In order to develop a novel near infrared fluorescence agent, glucose conjugated zinc phthalocyanine, [2,9(10),16(17),23(24)-tetrakis((1-(β-D- glucopyranose-2-yl)-1H-1,2,3-triazol-4-yl)methoxyl)phthalocyaninato]zinc(II), was synthesized via Click reaction

Full text of DOI:10.1142/S1088424611004361

Journal of Porphyrins and Phthalocyanines
J. Porphyrins Phthalocyanines 2012; 16: 77–84
DOI: 10.1142/S1088424611004361
Published at http://www.worldscinet.com/jpp/
Synthesis, properties and near-infrared imaging evaluation
of glucose conjugated zinc phthalocyanine via Click reaction
Feng Lv†, Xujun He†, li Lu, Li Wu and Tianjun Liu*◊
Institute of Biomedical Engineering, Chinese Academy of Medical Sciences & Peking Union Medical College,
The Tianjin Key Laboratory of Biomaterial Research, Tianjin 300192, PR China
Received 8 July 2011
Accepted 28 July 2011
ABSTRACT: In order to develop a novel near infrared fluorescence agent, glucose conjugated
zinc phthalocyanine, [2,9(10),16(17),23(24)-tetrakis((1-(b-D-glucopyranose-2-yl)-1H-1,2,3-triazol-4-
yl)methoxyl)phthalocyaninato]zinc(II), was synthesized via Click reaction. Their chemical structures
were characterized by mass spectrometry, nuclear magnetic resonance spectrum. Their light stability and
fluorescence quantum yield were evaluated by UV-visible and fluorescent spectroscopic method. Optical
imaging in vivo was performed with this saccharide conjugated phthalocyanine as probe on liver tumor-
bearing nude mice. Near-infrared imaging effect, organ aggregation as well as distribution of probe
in vivo were evaluated by in vivo fluorescence imaging technique. Results show that glucose conjugated
zinc phthalocyanine has favorable water solubility, good optical stability and high emission ability in
near infrared region. Imaging results demonstrate that saccharide conjugated phthalocyanine has possess
obvious imaging effect in vivo, which implies its potential in cancer diagnosis as near infrared optical
probe.
KEYWORDS: near infrared fluorescence probe, zinc phthalocyanines, saccharide conjugated,
Click reaction.
near-infrared image sensitivity and penetrating depth in
biological tissues [5, 6]. So near-infrared fluorescence
INTRODUCTION
Molecular imaging in vivo can display the biological
imaging can be used in deep tissues and organs
processes at the cellular and molecular level, reveal the
for detection and imaging owning to its specific
mechanism of physiological and pathological processes,
advantages [7–9].
and provide effective methods of detecting and tracking
However, imaging events in vivo by fluorescence is
the diseases treatments. The applications of molecular
limited by the probes available. As optical probe several
imaging include the early clinical disease diagnosis,
properties are required including light transmission
qualitative and quantitative evaluation of curative
in depth, the luminescence quantum yield, good
effect and so on [1, 2]. Comparing with CT, PET, MRI
biocompatibility as well as targeting ability, so a great
imaging modality, optical imaging has many advantages,
scientific effort has been focused on the development
such as low cost, non-ionic low-energy radiation, high
of near-infrared fluorescence probe. Phthalocyanines
sensitivity, continuous real-time monitoring, non-invasive
fluorescent dyes have large visible to NIR absorption
or minimally invasive [3, 4]. Furthermore, the fact that
molar extinction constant, acceptable fluorescent
organisms have low scattering effect and background
quantum yields, and good biocompatibility [10, 11]. Up
interference in near-infrared region(NIR), increases
to now, several kinds of phthalocyanines compounds with
emission in NIR region have been developed [12, 13].
SPP full member in good standing
However, their low water solubility and no cell specificity
were some obstacles for biomedical applications. The
combination of carbohydrate moieties with macrocycles
can not only improve the substrate solubility and
*Correspondence to: Tianjun Liu, email: liutianjun@hotmail.com,
nanobme@163.com, tel/fax: +86 22-87893236
†These authors contributed equally to this work
Copyright © 2012 World Scientific Publishing Company

78
F. LV ET AL.
biocompatibility [14], but also increase tumor targeting
function of imaging probes or Photodynamic Therapy
(PDT) sensitizer [15, 16]. In NIR optical imaging field,
glucose conjugated phthalocyanine as NIR fluorescence
probe has not been reported before. In this paper,
near-infrared fluorescent probe was designed to meet
molecular probes function and improve imaging effects
by combining phthalocyanine with saccharide.
recrystallization from 95% ethanol, the compound (7)
was obtained as a white solid in 89% yield. Reaction
of 4-propyne oxide phthalonitrile (4) and 1,2,3,5-tetra-
O-acetyl-b-D-glycopyranosyl azide (7) in the presence
of anhydrous CuSO4 and sodium ascorbate in dichloro-
methane/methanol at room temperature for 48 h gave
compound (8) 4-((1-(1,2,3-trihydroxy-4-(hydroxymethyl)-
tetrahydro-2H-pyran-2-yl)-1H-1,2,3-triazol-4-yl)methoxy)
phthalonitrile in 60%yield. Compound (8) was deprotected
in dry MeOH and NaOMe to give compound (9)
4-((1-(3,4,5-trihydroxy-6-(hydroxymethyl)-tetrahydro-2H-
pyran-2-yl)-1H-1,2,3-triazol-4-yl)methoxy)phthalonitrile.
Without further purification, the deprotected 9 reacted
in a mixture of DMAE and n-butanol, zinc chloride
under N2 for 24 h at 100 °C. After recrystallization from
water and acetone, glucose conjugated phthalocyanine
[2,9(10),16(17),23(24)-tetrakis((1-(b-D-glucopyranose-
2-yl)-1H-1,2,3-triazol-4-yl)methoxyl)phthalocyaninato]
zinc(II) (1) was obtained as a green solid in 62% yield.
The structures of products were characterized by
NMR spectroscopy and MS. 1H NMR (DMSO-d6,
300 MHz) shows typical signals of phthalcyanine and
glucose. Peak at 8.437 ppm (s, 12H) is assigned as
aromatic proton, broad peak at 7.07 ppm as hydroxyl
protons (s, 16OH), peak from 5.562 to 5.191 (s, 8H)
assigned as triazole and related sugar protons. High
resolution MS recorded by Agilent 6510 confirmed
the target compound. The optical characters of target
compound were evaluated by UV-vis and fluorescence
spetra. The absorption and fluorescence spectra of
glucose conjugated zinc phthalocyanines in DMSO and
in water were shown in Fig. 1. The spectrum in DMSO
shows no intermolecular aggregation. Characteristic
sharp bands of the zinc phthalocyanines are seen at 618
nm and at 682 nm. However, the absorption spectrum
in water differs remarkably from that in DMSO. In
water, the intensity of absorption peak is much lower
and broader than in DMSO, which can be attributed to
cofacial aggregation of phthalocyanines in water [25,
26]. Consistent with the aggregation difference in DMSO
and water, the emission ability in these two solvents also
show large difference [27, 28]. Glucose conjugated zinc
phthalocyanine in water has weak fluorescence signal due
to its aggregation, while strong fluorescence at 690 nm
with excitation wavelength at 618 nm (Ff = 0.482) was
detected in DMSO. However the following experiment
in vivo demonstrated that the low fluorescent ability of
glucose conjugated phthalocyanines in water does not
limit its application as fluorescence probe in vivo.
Saccharide conjugated macrocyclic compounds usually
were achieved using coupling methods such as esterification,
amidation or etherification [17, 18]. Recently, saccharide
decorated macrocyclic compounds including porphyrin or
phthalocyanine were synthesized by Click reaction as a
novel method instead of traditional synthesis method [19,
20]. Click chemistry has been exploited for the generation
of neoglycoconjugates and been applied in a wide variety of
researchareas,includingmaterialscience,polymerchemistry,
and pharmaceutical sciences because of the simplicity of
this reaction and the easy workup procedure for the resulting
products [21]. The characteristics of tolerance of typical
biological conditions, tolerance of most functional groups
makes click reaction particularly ideal for bioconjugations
[22, 23]. In carbohydrate chemistry, propargyl glycosides
have thus been widely utilized with alkyl azides. Based
on this approach, we synthesized glucose conjugated
phthalocyanine [2,9(10),16(17),23(24)-tetrakis((1-(b-D-
glucopyranose-2-yl)-1H-1,2,3-triazol-4-yl)
methoxyl)
phthalocyaninato]zinc(II). The fluorescent imaging
in vivo with glucose conjugated phthalocyanine as probe
was reported with liver tumor-bearing athymic nude mice
as animal model.
RESULTS AND DISCUSSION
Glucose conjugated phthalocyanine [2,9(10),16(17),
23(24)-tetrakis((1-(b-D-glucopyranose-2-yl)-1H-1,2,3-
triazol-4-yl)methoxyl)phthalocyaninato]zinc(II) (1) was
achieved according to Ref. 24 by condensation of
its precursor 4-((1-(3,4,5-trihydroxy-6-(hydroxymethyl)-
tetra-hydro-2H-pyran-2-yl)-1H-1,2,3-triazol-4-yl-
methoxy)phthalonitrile(9), whichwasachievedinaroutine
three step route from 4-propyne oxide phthalonitrile(4)
and 1,2,3,5-tetra-O-acetyl-b-D-glycopyranosyl azide (7)
through Click reaction (Scheme 1). 4-propyne oxide
phthalonitrile (4) was achieved from reaction of
4-chlorophthalonitrile (2) and propiolic alcohol (3) in
DMF for 24 h at 60 °C in the presence of potassium
carbonate. After recrystallization from methanol, the
compound (4) was obtained as a yellow-white solid in
65% yield. Hydrogen bromide in acetic acid reacted
with 1,2,3,4,6-penta-O-acetyl-b-D-glucopyranose (5)
in CH2Cl2 at 0 °C to give 2,3,4,6-tetra-O-acetyl-b-
D-glycopyranosyl bromide (6) in 80% yield. Then
compound (6) reacted with NaN3 in water at 70 °C
overnight with benzyltriethylammonium chloride as
phase transfer catalyst to give corresponding 1,2,3,
5-tetra-O-acetyl-b-D-glycopyranosyl azide (7). After
Photo-bleaching experiment demonstrated that
glucose conjugated phthalocyanine had relative high
photostability. After continuous irradiation by 0.1 w/
cm2/s laser with light emerge density of 60 J/cm2 at
690 nm, its absorbance spectrum was recorded every 10
minirradiation.Infirst10min,itsopticaldensitydecreased
less than 10%, 20 min vs. 20%, 40 min to 50%, after
continuous irradiation for 70 min, it decreased 70% or so.
Copyright © 2012 World Scientific Publishing Company
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SYNTHESIS, PROPERTIES AND NEAR-INFRARED IMAGING EVALUATION OF GLUCOSE
79
CI
CN
CN
CN
CN
(i)
O
OH
4
2
3
OAc
OAc
6
OAc
(iii)
(ii)
O
O
O
AcO
AcO
AcO
AcO
AcO
AcO
OAc
Br
N3
OAc
OAc
OAc
7
5
OAc
OAc
CN
CN
N
(iv)
N
N
O
O
O
AcO
AcO
AcO
+
O
CN
CN
N3
AcO
OAc
OAc
4
8
7
OH
N
(v)
N
N
O
HO
O
CN
CN
HO
OH
9
OH
H
HO
O
H
N
HO
N
OH
N
H
H
H
HO
OH
H
OH
O
H
H
HO
O
(vi)
N
N
1
N
N
O
N
N
N
Zn
N
O
N
N
N
N
N
N
O
OH
H
OH
H
H
O
HO
H
O
H
OH
HO
N
H
H
N
OH
H
OH
N
OH
Scheme 1. Synthetic route of glucose conjugated zinc phthalocyanines. (i) DMF, K2CO3, 60 °C; (ii) CH2Cl2, 33% HBr/AcOH, 0 °C;
(iii) CHCl3, NaN3, PTC, 70 °C; (iv) CuSO4, Na ascorbate, CH2Cl2/MeOH (4:1), rt; (v) MeOH, NaOMe, rt; (vi) DMAE, n-butanol,
ZnCl2, N2, 100 °C
As a NIR probe, stability of probe in physiological
conditions such as salt or serum was important. The
emission of probe was measured in different physiological
conditions. It can be seen that emission fluorescence
was greatly reduced in serum or salt solution owning to
molecule aggregation. After incubation in DMSO, FCS
or physiological salt solution at 37 °C for 1 h, the total
fluorescence emission of probe showed a slightly increase
in first 20 min and then maintained at a stable level for
40 min, which indicated that the probe was pretty stable
in serum or salt solution. Considering that optical image
recording costs just several minutes, the photo stability of
this compound was good enough as probe.
with 200 mL of glucose conjugated phthalocyanines at
2 × 10–4 M through the tail vein, another was injected with
equal amount of saline as control. The in vivo imaging at
12 h post injections were acquired respectively using a
Kodak In Vivo FX Professional Imaging System equipped
with fluorescent filter sets (excitation/emission, 625/700
nm) near optimal wavelength. As shown in Fig. 2, a
significant luminescence signal was observed in the body
after intravenous injection of probe for 12 h, whereas
no significant luminescence signal was observed in the
control group. Considerable fluorescence was detected
in liver and kidney at experiment group after injecting
near infrared fluorescence agent. These results indicate
that in vivo, glucose conjugated zinc phthalocyanines are
emissive and do not undergo substantial phase transitions
that drive phthalocyanines aggregation or facilitate
The NIR fluorescent imaging was conducted with
liver tumor-bearing athymic nude mice as animal model.
Two groups of model mice, one group was injected
Copyright © 2012 World Scientific Publishing Company
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80
F. LV ET AL.
(a)
Fig. 2. Optical imaging in vivo with glucose conjugated zinc
phthalocyanines as probe, mice bearing cancer as model
(injected with 200 mL of 2 × 10-4 M with an exposure time of
1min (filters: excitation 625 nm, emission 700 nm)
intermembranous fluorophore transfer to surrounding
biological structures.
To further investigate the probe distribution in various
organs, the animals were sacrificed immediately after
intravenous injection of probe at 12 h time point, and
the kidney, heart, liver, lung, spleen, muscle and tumor
were harvested and subjected to the imager. Fluorescence
could be detected clearly in the lung, kidney and liver, a
little in the heart and muscle (Fig. 3). Comparing with the
control group, the intensity of sample group was about
two-fold higher. This further confirmed that the probe
was accumulated in these organ after enough circulation
time. Because the lung, kidney and liver are the main
metabolism organs, the probe as external matter was
accumulated at relative large amount in these organs.
In order to investigate the influence of glucose substituted
zinc phthalocyanines to organ, histological analysis were
performed. Large amount of fluorescence agent aggregated
in the liver and kidney (Fig. 4), while little fluorescence
was found in the other organs, which was in accordance
with the result of NIR fluorescent imaging of organs. These
results further revealed the distribution of fluorescence
agent. Toxic effects are an important issue for probe.
Histological analysis showed that the glucose conjugated
phthalocyanines had no damage to the organ according to
the HE staining, which proved the safety of fluorescence
agent as the near infrared fluorescence probe in vivo.
In summary, a novel water-soluble glucose conjugated
zinc phthalocyanines was synthesized. With mice
bearing liver cancer as animal model in vivo fluorescent
imaging was conducted. Near-infrared imaging effect,
distribution in organs as well as its histological analysis
were also assessed. The results proved that glucose
conjugated phthalocyanines has obvious imaging effect
(b)
(c)
Fig. 1. (a) UV and FL spectrum of glucose conjugated
phthalocyanines in different solvents (2 × 10-4 M; solid line
UV: spectrum in DMSO; dot line: FL spectrum in DMSO;
dash dot line: UV spectrum in H2O; short dot line: FL
spectrum in H2O). (b) Photobleaching experiment recorded
by UV-vis (light intensity was 0.1 w/cm2/s and light emerge
density was 60 J/cm2. The photobleaching measurements
was carried from 0 to 70 min). Inset: absorbance spectra
data after different irradiation time. (c) Optical stability of
glucose conjugated phthalocyanines incubation in DMSO,
FCS or physiological salt solution at 37 °C (5 × 10-5 M,
solid line: DMSO; dash line: DMSO:FCS = 1:1; dot line:
DMSO:physiological saline = 1:1)
Copyright © 2012 World Scientific Publishing Company
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SYNTHESIS, PROPERTIES AND NEAR-INFRARED IMAGING EVALUATION OF GLUCOSE
81
20 mmol) were stirred for 24 h in DMF (40 mL) at 60 °C in
the presence of potassium carbonate (8.3 g, 60 mmol). The
reaction was then followed to complete byTLC. The reaction
mixture was poured into ice-water to give a green-brown
precipitate, which was extracted with dichloromethane. The
organic extracts were dried over anhydrous MgSO4 and
concentrated under vacuum to give a yellow-brown solid.
After recrystallization from methanol, the title compound
was obtained as a yellow-white solid.Yield 1.2 g (65%), mp
112.3–114.1 °C. ESI-MS: m/z (C11H6N2O) 183.0826143
(calcd. for [M + H]+ 183.0553). IR (solid): n, cm-1 3288.3
(C≡H); 2232.9 (CN); 1593.1, 1494.3 (C=C phenyl).
1H NMR (CDCl3, 300 MHz): TM, ppm 7.762 (d, 1H, J = 8.7
Hz), 7.373 (d, 1H, J = 2.4 Hz), 7.314 (dd, 1H, J = 2.4, 8.7
Hz), 4.816 (d, 2H, J = 2.4 Hz), 2.636 (t, 1H, J = 2.4,2.7 Hz).
1,2,3,5-tetra-O-acetyl-b-D-glycopyranosyl azide (7).
To a stirring solution of 1,2,3,4,6-penta-O-acetyl-b-D-
glucopyranose (2.4 g, 6.1 mmol) in CH2Cl2 (20 mL) was
added HBr/HOAc (10 mL, 33%) solution at 0 °C. The
resulting solution was stirred for 7 h until 1,2,3,4,6-penta-
O-acetyl-b-D-glucopyranose completely disappeared.
The solution was neutralized with saturated NaHCO3
aqueous solution before being dried (MgSO4), filtered,
and evaporated under vacuum to give the crude glycosyl
bromide as a yellow oil. After recrystallization from
ethanol, the compound was obtained as a white solid
(2.0 g, 80%). 2,3,4,6-tetra-O-acetyl-b-D-glycopyranosyl
bromide (0.5 g, 1.2 mmol) was dissolved in CHCl3
(20 mL) and stirred with NaN3 (0.3 g, 4.6 mmol) in
water (20 mL) at 70 °C overnight in the presence of
benzyltriethylammonium chloride (0.1 g, 0.4 mmol). The
reaction mixture was diluted with water and extracted
with CHCl3. The combined organic layers were dried
(MgSO4), filtered, and concentrated under vacuum to give
an orange oil. After recrystallization from 95% ethanol, the
title compound 1,2,3,5-tetra-O-acetyl-b-D-glycopyranosyl
azide was obtained as a white solid. Yield 0.4 g (89%),
mp 129.7–130.5 °C. ESI-MS: m/z (C14H19N3O9) 396.1013
(calcd. for [M + Na]+ 396.1014). IR (solid): n, cm-1 2233.3
(N=N=N), 1737.8 (C=O). 1H NMR (CDCl3, 300 MHz):TM,
ppm 5.247 (t, 1H, J = 9.3, 9.6 Hz), 5.133 (t, 1H, J = 9.6,
9.9 Hz), 4.982 (t, 1H, J = 8.7, 9.6 Hz), 4.661 (d, 1H, J =
8.7Hz), 4.301 (dd, 1H, J = 4.8, 12.6 Hz), 4.184 (dd, 1H, J
= 2.1, 12.3 Hz), 3.821–3.764 (m, 1H), 2.165 (s, 3H, CH3),
2.099 (s, 3H, CH3), 2.074 (s, 3H, CH3), 2.027 (s, 3H, CH3).
4-((1-(1,2,3-trihydroxy-4-(hydroxymethyl)-tetra-
hydro-2H-pyran-2-yl)-1H-1,2,3-triazol-4-yl)methoxy)-
phthalonitrile (8). A mixture of 4-propyne oxide phtha-
lonitrile (4) (500 mg, 2.7 mmol), 1,2,3,5- tetra-O-acetyl-
b-D-glycopyranosyl azide (7) (1100 mg, 2.9 mmol),
anhydrous CuSO4 (45 mg, 0.28 mmol), and sodium
ascorbate (110 mg, 0.56 mmol) in dichloromethane/
methanol = 4:1 (15 mL) was stirred at room temperature
for 48 h. After being filtered under vacuum, washed
with water, extracted with dichloromethane, the
organic extracts were dried over anhydrous MgSO4 and
concentrated under vacuum. The residue was purified
(a)
(b)
Fig. 3. (a) Distribution and fluorescent intensity of dissected
organs. (a) Ex vivo imaging with an exposure time of 1 min
(filters: excitation 625 nm, emission 700 nm). (b) Distribution and
fluorescent intensity of dissected organs. B: Quantitative analysis
and exhibits its potential as near infrared optical probe in
the diagnosis of cancer in future.
EXPERIMENTAL
General methods and materials
The 1H NMR spectra were recorded onVarian Mercury
at 300 MHz using CDCl3 or DMSO-d6 as solvent and
TMS as internal reference. MS were recorded on Varin
FT-ICR-MS and Agient 6510 Q-Tof instrument. The
UV-vis and fluorescence spectra were recorded on a
ThermoFisher scientific Varioskan TM Flash multimode
microplate spectra photometer. All purchased materials
were used without further purification.
Synthesis
4-propyne oxide phthalonitrile (4). 4-chlorophtha-
lonitrile (1.638 g, 10 mmol) and propiolic alcohol (1.2 g,
Copyright © 2012 World Scientific Publishing Company
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F. LV ET AL.
Fig. 4. Fluorescent image of liver (a) and kidney (b) tissue slice recorded by confocal laser microscopy
Copyright © 2012 World Scientific Publishing Company
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SYNTHESIS, PROPERTIES AND NEAR-INFRARED IMAGING EVALUATION OF GLUCOSE
83
by column chromatography (silica gel: ethyl acetate-
and Use of Laboratory Animals, Peking Union Medical
College, China. Animals can access free to food and
water. Tumor-bearing mice were prepared by injecting a
suspension of 1 × 106 liver tumor cells in physiological
saline (100 mL) into the subcutaneous left flank. Tumors
develop within a periods of 1 week. Athymic nude mice
were randomly assigned to perform as follows: glucose
conjugated zinc phthalocyanines, control group (n = 6
for each group). In experimental group, near infrared
fluorescence agent was injected into the tail vein of
the liver tumor-bearing mice at 200 mL of 2 × 10-4 M.
Images were taken using a Kodak Image Station in vivo
FX (filters: excitation 625 nm, emission 700 nm) with
an exposure time of 1 min. At the end of the imaging,
anesthetized mice were sacrificed and images of organs
were made to evaluate the distribution of near infrared
fluorescence agent. Fluorescence images of organ were
analyzed using the Kodah Image Analysis Software.
After imaging, organ tissues were immediately immersed
into 4% formaldehyde in phosphate-buffered saline of
pH 7.4 at 4 °C for 24 h. After fixation, the samples were
embedded in paraffin and sectioned to 5-mm-thick slices.
Routine staining was performed with hematoxylin-eosin.
petroleum ether, 1:1), to yield a white solid. Yield 899 mg
(60%), mp 161.5–162.8 °C. ESI-MS: m/z (C25H25N5O10)
578.2507 (calcd. for [M + Na]+ 578.1494). IR (solid):
1
n, cm-1 2233.3 (CN), 1737.8 (C=O). H NMR (CDCl3,
300 MHz):TM, ppm 7.907 (s, 1H), 7.751 (d, 1H, J = 8.7
Hz), 7.383 (d, 1H, J = 2.4 Hz), 7.359 (dd, 1H, J = 2.7,
8.7 Hz), 5.908 (d, 1H, J = 9.3 Hz), 5.476–5.206 (m, 5H),
4.359 (dd, 1H, J = 5.1, 12.6 Hz), 4.184 (dd, 1H, J = 2.1,
12.9 Hz), 4.058–4.007 (m, 1H), 2.178 (s, 3H, CH3), 2.090
(s, 3H, CH3), 2.079 (s, 3H, CH3), 2.038 (s, 3H, CH3).
[2,9(10),16(17),23(24)-tetrakis((1-(b-D-glucopy-
ranose-2-yl)-1H-1,2,3-triazol-4-yl)methoxyl)
phthalocyaninato]zinc(II)(I). 4-((1-(1,2,3-trihydroxy-
4-(hydroxymethyl)-tetrahydro-2H-pyran-2-yl)-1H-1,2,3-
triazol-4-yl)methoxy)phthalonitrile (8) (600 mg, 1.1
mmol) was suspended in dry MeOH (10 mL). NaOMe
(300 mL) was added and the solution was stirred for
4–5 h at room temperature. The ion exchanger was added
to neutralize the solution, then filtered off and the
solvent evaporated. Without further purification, to the
compound (9) 4-((1-(3,4,5-trihydroxy-6-(hydroxymethyl)-
tetrahydro-2H-pyran-2-yl)-1H-1,2,3-triazol-4-yl)
methoxy)phthalonitrile dissolved in a mixture of DMAE
(1 mL) and n-butanol (0.5 mL), zinc chloride (70 mg,
0.5 mmol) were added. The reaction mixture was stirred
under N2 for 24 h at 100 °C. After cooling, the solid
was reprecipitated by adding acetone and collected after
filtration. Yield 1.08 g (62%), mp > 300 °C. ESI-HRMS:
m/z (C68H68N20O24Zn) 1635.3891 (calcd. for [M + Na]+
1635.3899). 1H NMR (DMSO, 300 MHz): TM, ppm 8.437
(s, 12H), 7.076 (s, 16OH), 5.562 (d, 4H, J = 9 Hz), 5.439 (s,
4H), 5.301 (s, 4H), 5.191 (s, 8H), 3.797–3.016 (m, 28H).
Acknowledgements
This work is supported by the National Basic Research
Program of China (No. 2006CB705703) and the Ph.D.
Programs Foundation of Ministry of Education of China
(Nos. 20101106120052, 20070023020) and the Natural
Science Foundation of Tianjin (No. 09JCYBJC03500).
Supporting information
Experimental details for the synthesis, measurement
method, MS data and H NMR data are given in the
supplementary material. This material is available free of
charge via the Internet at http://www.worldscinet.com/
jpp/jpp.shtml.
Optical measurement
The optical characters of target compound were
evaluated by UV-vis and fluorescence spectrum. The
photobleaching measurements were carried out in
Multimode Microplate Spectraphotometer (VarioskanTM
Flash, ThermoFisher Scientific). Dye and buffer solutions
were prepared immediately prior to measuring. 200 mL
2 × 10-4 M in 96 wells was used as sample and sealed
by cover slips to avoid evaporation. The light intensity
was 0.1 w/cm2/s and light emerge density was 60 J/cm2.
The photobleaching measurements was carried from 0 to
70 min, every 10 min record one datum, with irradiation
wavelength at 690 nm. Optical stability of glucose
conjugated phthalocyanines incubation in DMSO, FCS
or physiological salt solution at 37 °C was measured
using 200 mL 5 × 10-5 M in 96 wells as sample.
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