A phthalocyanine-peptide conjugate with high in vitro photodynamic activity and enhanced in vivo tumor-retention property

Article abstract of DOI:10.1002/chem.201103516

A novel zinc(II) phthalocyanine conjugated with a short peptide with a nuclear localization sequence, Gly-Gly-Pro-Lys-Lys-Lys-Arg-Lys-Val, was synthesized by click chemistry and a standard Fmoc solid-phase peptide synthesis protocol. The conjugate was pur

Full text of DOI:10.1002/chem.201103516

FULL PAPER
DOI: 10.1002/chem.201103516
A Phthalocyanine–Peptide Conjugate with High In Vitro Photodynamic
Activity and Enhanced In Vivo Tumor-Retention Property
Mei-Rong Ke,[a] Sin-Lui Yeung,[b] Wing-Ping Fong,[b] Dennis K. P. Ng,*[a] and
Pui-Chi Lo*[a]
Abstract: A novel zinc(II) phthalocya-
nine conjugated with a short peptide
with a nuclear localization sequence,
Gly-Gly-Pro-Lys-Lys-Lys-Arg-Lys-Val,
was synthesized by click chemistry and
a standard Fmoc solid-phase peptide
synthesis protocol. The conjugate was
purified by HPLC and characterized
with UV/Vis and high-resolution mass
spectroscopic methods. Both this com-
pound and its non-peptide-conjugated
analogue are essentially non-aggregat-
ed in N,N-dimethylformamide and can
generate singlet oxygen effectively with
quantum yields (FD) of 0.84 and 0.81,
respectively, relative to unsubstituted
zinc(II) phthalocyanine (FD =0.56).
Conjugation of the peptide sequence,
however, can enhance the cellular
uptake, efficiency in generating intra-
cellular reactive oxygen species, and
photocytotoxicity of the phthalocya-
nine-based photosensitizer against
HT29 human colorectal carcinoma
cells. The IC50 value of the conjugate is
as low as 0.21 mm. In addition, the con-
jugate shows an enhanced tumor-reten-
tion property in tumor-bearing nude
mice. After 72 h post-injection, the dye
concentration in the tumor was signifi-
cantly higher than that in other organs.
The results suggest that this phthalo-
cyanine–peptide conjugate is a highly
promising photosensitizer for photody-
namic therapy.
Keywords:
peptides
·
photo-
dynamic therapy · photosensitizers ·
phthalocyanines · singlet oxygen
Introduction
proach is through conjugation to monoclonal antibodies
(mAbs) that are specific for tumor-associated antigens.[3]
However, the large size of mAbs hinders tissue penetration
and lowers cellular uptake when used in vivo. The attach-
ment of photosensitizers to mAbs may also reduce the anti-
genic specificity of the mAbs. As a result, smaller antibody
fragments, such as single-chain Fv fragments (scFv) have re-
ceived considerable attention as alternative carriers.[4] Con-
jugation of photosensitizers with albumins and low-density
lipoproteins has also been studied, but only moderate target
specificity has been achieved so far.[2b] An alternative strat-
egy involves encapsulation of photosensitizers in colloidal
carriers, such as liposomes,[5] polymeric micelles,[6] and silica-
based nanoparticles.[7] These nanomedicines can achieve
a long circulation time and allow significant accumulation in
tumors through the enhanced permeability and retention
(EPR) effect. In addition, some activatable photodynamic
molecular beacons have recently been developed.[8] Their
photodynamic action can be triggered by specific tumor-as-
sociated stimuli. This represents another promising ap-
proach to achieve the targeting effect.
Among the various vectors for active drug targeting, syn-
thetic peptides are of particular interest.[9] Peptides with ap-
propriate sequences can specifically bind to different surface
markers of cancer cells and tumor vasculatures.[10] Peptide-
based drugs also generally have a number of advantages,
such as ease of preparation and modification, non-immuno-
genicity, high tissue permeability, and rapid clearance from
the body. As a result, synthetic peptides have been widely
used to deliver various agents, such as chemotherapeutic
Photodynamic therapy (PDT) is a promising modality for
cancer treatment.[1] It utilizes the combined action of a pho-
tosensitizer, light, and molecular oxygen to generate reactive
oxygen species (ROS), particularly singlet oxygen, to eradi-
cate cancer cells. Compared with the traditional therapeutic
methods, such as chemotherapy, radiotherapy, and surgery,
PDT has several potential advantages.[1b] Generally, it is
non-invasive and has tolerance of repeated doses, few side
effects, and high specificity that can be achieved through
precise delivery of light. The therapeutic outcome depends
on several interdependent factors, particularly the selectivity
of the photosensitizer toward cancer cells and their efficien-
cy in generating ROS. Targeted delivery of photosensitizers
remains as major challenge in PDT, which has stimulated
much research effort over the past decade.[2]
Various approaches have been explored to improve the
tumor-targeting property of photosensitizers. One logical ap-
[a] M.-R. Ke, Prof. D. K. P. Ng, Prof. P.-C. Lo
Department of Chemistry, The Chinese University of Hong Kong
Shatin, N.T., Hong Kong (P.R. China)
Fax : (+852)2603-5057
E-mail: dkpn@cuhk.edu.hk
pclo@cuhk.edu.hk
[b] S.-L. Yeung, Prof. W.-P. Fong
School of Life Sciences, The Chinese University of Hong Kong
Shatin, N.T., Hong Kong (P.R. China)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201103516.
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drugs,[11] radiopharmaceuticals,[12] antisense oligonucleo-
tides,[13] and theranostic nanoparticles.[14] A number of pep-
tide-conjugated photosensitizers,[15] including chlorins,[16]
porphyrins,[17] pyropheophorbide a,[18] 5-aminolaevulinic
acid,[19] and phthalocyanines[20] have been synthesized and
evaluated for their potential in targeted PDT. Improved
properties have been observed for some of these conjugates
as a result of peptide conjugation. As part of our continuing
effort in development of efficient and selective photosensi-
tizers for PDT,[21] we report herein a novel peptide-conjugat-
ed zinc(II) phthalocyanine that not only exhibits high in
vitro photodynamic activity, but also shows improved in
vivo tumor-retention property.
by HPLC analysis (Figure S1 in the Supporting Informa-
tion). The Fmoc protecting group on NLS-resin was re-
moved with piperidine (20%) in DMF to give 11 (Boc=
tert-butoxycarbonyl, Pbf=2,2,4,6,7-pentamethyldihydroben-
zofurane-5-sulfonyl), then the N terminus of the peptide was
coupled with the carboxyl group of 10 by using 1-hydroxy-
benzotriazole (HOBt) and O-(benzotriazol-1-yl)-N,N,N’,N’-
tetramethyluronium hexafluorophosphate (HBTU) as the
activating agents. Finally, all the remaining protecting
groups on the peptide and the resin were removed by a mix-
ture of trifluoroacetic acid (TFA, 95%), triisopropylsilane
(TIS, 2.5%), and dichloromethane (2.5%). Conjugate 12
was purified by repeated precipitation by using dimethylsulf-
oxide (DMSO) and diethyl ether. It was found to be pure
by HPLC analysis (Figure S2 in the Supporting Information)
and was characterized with UV/Vis spectroscopy and high-
resolution electrospray ionization (ESI) mass spectrometry.
All the other new compounds were fully characterized
with various spectroscopic methods and elemental analysis.
NMR spectra of phthalocyanines 7, 9, and 10 were recorded
in CDCl3 with a trace amount of [D5]pyridine to reduce
their aggregation. All of them showed a typical pattern for
di-a-substituted phthalocyanines in the aromatic region.[22]
Figure S3 (in the Supporting Information) shows the
1H NMR spectrum of 10 as an example. The multiplet at d=
9.35–9.39 and doublet at d=9.27 (integrated as 4:2) can be
assigned to the 6a protons of phthalocyanine (Pc-Ha), and
the multiplet at d=8.08–8.11 and singlet at d=7.41 (inte-
grated as 6:2) are due to the 8b phthalocyanine ring protons
(Pc-Hb). The triazole proton resonates as a singlet at d=
7.69. The signals for the remaining methylene and methyl
protons can also be easily seen and assigned.
Results and Discussion
Molecular design and synthesis: In this conjugate, 1,4-bis(tri-
ethylene glycol)-substituted zinc(II) phthalocyanine was em-
ployed as the photosensitizing unit.[22] Having two hydro-
philic triethylene glycol chains at the a positions, this p sy-
stem can absorb at a longer-wavelength position [at ca. 690
vs. 670 nm for unsubstituted analogue (ZnPc)], which allows
deeper penetration of light. These substituents can also
lower the aggregation tendency and enhance the amphiphi-
licity of the macrocycle, which can promote singlet oxygen
generation and cellular uptake. All these characteristics are
desirable for PDT application. For the peptide unit, we em-
ployed the short sequence Gly-Gly-Pro-Lys-Lys-Lys-Arg-
Lys-Val, which is a nuclear localization signal (NLS) pep-
tide.[23] It was expected that this multicationic peptide could
exhibit a high level of cell permeability and target vulnera-
ble intracellular sites, such as mitochondria and nuclei.[9]
Scheme 1 shows the synthetic route used to prepare the
phthalocyanine–peptide conjugate. Treatment of 2,3-dicya-
nohydroquinone (1) with 1 equiv triethylene glycol methyl
ether tosylate (2)[24] in the presence of K2CO3 in N,N-dime-
thylformamide (DMF) led to monosubstitution and gave
phthalonitrile 3. Further substitution with alkynyl tosylate
4[25] afforded the unsymmetrical phthalonitrile 5. This com-
pound then underwent lithium-promoted mixed cyclization
with excess of phthalonitrile (6) in the presence of Zn-
Electronic absorption and photophysical properties: The
UV/Vis spectra of 10 and 12 were measured in DMF (Fig-
ure S4 in the Supporting Information) and the data are
listed in Table 1. Both compounds gave typical absorption
Table 1. Electronic absorption and photophysical data for 10 and 12 in
DMF.
[b]
[c]
Compound labs [nm] (loge)
lem [nm][a] FF
FD
10
12
336 (4.70), 619 (4.55), 687 (5.30) 698
340 (4.78), 622 (4.56), 691 (5.30) 700
0.08 0.81
0.07 0.84
ACHTUNGTRENNUNG(OAc)2·2H2O in n-pentanol to afford the “3+1” unsymmet-
rical phthalocyanine 7 in 57% yield. Due to the presence of
two triethylene glycol chains at the a positions, this com-
pound was soluble in common organic solvents and could be
purified readily by column chromatography followed by size
exclusion chromatography. This alkynyl phthalocyanine was
then treated with azido ester 8[26] under typical click reaction
conditions to give 9, which was hydrolyzed with NaOH in
MeOH/H2O/tetrahydrofuran (THF) to yield the carboxy
phthalocyanine 10. This compound served as the non-pep-
tide-conjugated analogue for comparison in the following
studies. The NLS-resin was prepared manually by using a 9-
fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide syn-
thesis (SPPS) protocol.[27] The peptide Fmoc-Gly-Gly-Pro-
Lys-Lys-Lys-Arg-Lys-Val was found to be essentially pure
[a] Excited at 620 nm; [b] relative to ZnPc in DMF as the reference
(FF =0.28); [c] relative to ZnPc as the reference (FD =0.56 in DMF).
spectra for non-aggregated phthalocyanines and showed an
intense and sharp Q band at 687 nm (10) or 691 nm (12),
which strictly followed the Lambert–Beer law. Upon excita-
tion at 620 nm, these compounds showed a weak fluores-
cence emission at 698–700 nm with a fluorescence quantum
yield (FF) of 0.07–0.08 relative to ZnPc (FF =0.28).[28]
The singlet oxygen quantum yields (FD) of these com-
pounds were also determined in DMF by a steady-state
method by using 1,3-diphenylisobenzofuran (DPBF) as the
scavenger. The concentration of the quencher was moni-
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A Phthalocyanine–Peptide Conjugate for PDT
FULL PAPER
Scheme 1. Synthesis of phthalocyanine–peptide conjugate 12.
tored spectroscopically at 415 nm during irradiation, from
which the values of FD could be determined. As shown in
Figure S5 (in the Supporting Information), both 10 and 12
are highly efficient singlet oxygen generators and the effi-
ciency is significantly higher than that of ZnPc. Their FD
values reach 0.81 (10) to 0.84 (12) compared to ZnPc (FD =
0.56; Table 1).[29] The high singlet oxygen quantum yields
and low fluorescence quantum yields of these compounds
are in accord with the results found for other 1,4-disubstitut-
ed zinc(II) phthalocyanines.[22]
In vitro photodynamic activities: The in vitro photodynamic
activities of compounds 10 and 12 formulated with Tween-
80 were investigated against HT29 human colorectal carci-
noma cells. Figure 1 shows their dose-dependent survival
curves both in the absence and presence of light. It is clear
that both compounds are non-cytotoxic in the dark, but ex-
Figure 1. Comparison of the cytotoxic effects of 10 (squares) and 12 (tri-
angles) on HT29 cells in the absence (closed symbols) and presence
(open symbols) of light (l>610 nm, 40 mWcmÀ2, 48 JcmÀ2). Data are ex-
pressed as mean value Æ standard error of the mean (SEM) of three in-
dependent experiments, each performed in quadruplicate.
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D. K. P. Ng, P.-C. Lo et al.
hibit high cytotoxicity upon illumination. The peptide conju-
gate 12, which has an IC50 value of 0.21 mm, shows a higher
potency than 10 (IC50 =0.39 mm). The photocytotoxicity is
also significantly higher than that of the classical photosensi-
tizer, porfimer sodium, which has an IC50 value of
4.5 mgmLÀ1 under the same experimental conditions (vs.
0.4 mgmLÀ1 for 12).
To account for the different photodynamic activities of
these two compounds, their aggregation behavior in DMEM
cell culture medium was first examined by absorption and
fluorescence spectroscopic methods. As shown in Figure 2,
the Q band of conjugate 12 is slightly broadened and less in-
tense, and its fluorescence emission is weaker compared
with that of 10. The results suggest that 12 is slightly more
aggregated than 10 in the culture medium probably due to
the intermolecular interactions of the peptide sequence. The
higher aggregation tendency of 12 seems to be in contrast
with its higher photocytotoxicity.
Figure 3. a) Bright-field (upper row) and intracellular fluorescence (lower
row) images of HT29 cells after incubation with: i) 10, or ii) 12 (5 mm) for
2 h. b) Comparison of the intracellular fluorescence intensity of 10 and
12. Data are expressed as mean value Æ standard deviation (number of
cells=50). c) Percentage cellular uptake of 10 and 12 determined by an
extraction method. Data are expressed as mean value Æ standard devia-
tion of three independent experiments.
determined and is depicted in Figure 3b. It can be seen that
compound 10 exhibits strong fluorescence in the cytoplasm,
whereas the intracellular fluorescence of the peptide conju-
gate 12 is very weak. The intracellular fluorescence intensity
of 12 is about eightfold lower than that of 10.
Figure 2. a) UV/Vis absorption, and b) fluorescence spectra (excited at
620 nm) of 10 (c) and 12 (a) both at 2 mm, formulated with Tween-
80 (0.05% by volume) in DMEM.
To take into account that these two compounds could
have different fluorescence quantum yields inside the cells,
we also employed an extraction method to quantify the cel-
lular uptake. After incubation with the phthalocyanines,
DMF was used to lyze the cells and extract the dyes. The
dye concentrations inside the cells were quantified by meas-
uring their Q band absorbance. As shown in Figure 3c, the
To further address this issue, the cellular uptake of these
compounds was examined by fluorescence microscopy and
absorption spectroscopy. Figure 3a shows the bright-field
and fluorescence microscopic images of HT29 cells after in-
cubation with 10 or 12 (5 mm) for 2 h. The average relative
fluorescence intensity per cell of these compounds was also
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A Phthalocyanine–Peptide Conjugate for PDT
FULL PAPER
cellular uptake of 12 was about fivefold higher than that of
10. Hence, even though conjugate 12 shows weaker intracel-
lular fluorescence, it in fact has higher cellular uptake,
which is clearly due to the peptide sequence. The weak fluo-
rescence could be a result of aggregation, quenching by the
amino groups on the peptide chain, and/or enhanced inter-
system crossing to the triplet state.
To shed light on this issue, the intracellular production of
ROS by these compounds was also studied by using 2’,7’-di-
chlorodihydrofluorescein diacetate (DCFDA) as the
quencher.[30] As shown in Figure 4, both 10 and 12 cannot
generate ROS in the dark, but upon illumination, they can
sensitize the formation of ROS. The peptide conjugate 12 is
particularly efficient, which can be partly attributed to its
higher cellular uptake. The results also suggest that the
weak intracellular fluorescence of 12 is mainly due to en-
hanced intersystem crossing, which facilitates the population
of triplet state and formation of ROS. Hence, the higher
Figure 5. Fluorescence images of HT29 cells after incubation with: a) 10,
or b) 12 (10 mm) in the culture medium for 2 h.
For the latter, fluorescence was not observed in the nucleus
despite the presence of an NLS peptide. Similar results have
been observed for some NLS-conjugated porphyrin-based
photosensitizers.[17a,d–f] It seems that the hydrophobic macro-
cyclic core of these conjugates counteracts the directing
effect of the peptide.
In vivo studies: To evaluate the biodistribution of 10 and 12
in vivo, nude mice bearing a HT29 tumor were treated with
an intravenous dose of these phthalocyanines (1 nmolgÀ1).
Their fluorescence images, which reflect the quantity of the
dyes, were monitored continuously up to 48 h (10) or 72 h
(12; Figure 6). Shortly after injection, both of the dyes
spread throughout the whole body. The metabolic clearance
of 10 appeared to be faster than that of 12. Interestingly, the
peptide conjugate 12 showed promising tumor-retention
property. After 24–30 h post-injection, bright fluorescence
was observed virtually only in the tumor. The fluorescence
diminished gradually after about 2 days. To quantify the
fluorescence intensities in different organs, the mice were
sacrificed after 48 h (10) or 72 h (12). The tumor and other
organs, including skin, liver, kidney, stomach, and spleen
were then harvested, and their relative fluorescence intensi-
Figure 4. ROS production induced by 10 (squares) and 12 (triangles) in
HT29 cells in the absence (closed symbols) and presence (open symbols)
of light (l>610 nm, 40 mWcmÀ2, 48 JcmÀ2). Data are expressed as mean
value Æ SEM of three independent experiments, each performed in
quadruplicate.
photocytotoxicity of 12 is be-
lieved to be a result of its
higher cellular uptake and
higher efficiency in generating
ROS inside the cells.
The subcellular localization
of these two compounds was
also analyzed. Figure 5 shows
the fluorescence microscopic
images of HT29 cells after incu-
bation with a higher concentra-
tion of 10 or 12 (10 mm) for 2 h.
Although both compounds give
substantial intracellular fluores-
cence, a closer examination re-
veals that 10 is distributed
throughout
the
cytoplasm,
whereas conjugate 12 tends to
Figure 6. Fluorescence images of tumor-bearing nude mice before and after intravenous injection of: a) 10, or
localize in the cell membrane. b) 12.
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D. K. P. Ng, P.-C. Lo et al.
ties (per unit area) were measured. It can be seen in
Figure 7 that conjugate 12 has much better tumor selectivity
than the non-conjugated analogue 10. It is clear that the
Conclusion
We have prepared and characterized a carefully designed
zinc(II) phthalocyanine conjugated with an NLS peptide.
The compound exhibits high in vitro photodynamic activity
with an IC50 value of 0.21 mm for HT29 cells. The peptide se-
quence can enhance the cellular uptake of the photosensitiz-
er and promote the generation of intracellular ROS. In addi-
tion, it imparts a tumor-retention property to the dye. These
properties make this conjugate a highly promising photosen-
sitizer for targeted PDT.
Experimental Section
Materials and methods: All the reactions were performed under an at-
mosphere of nitrogen. DMF was dried over barium oxide and distilled
under reduced pressure. Dichloromethane and n-pentanol were distilled
from calcium hydride and sodium, respectively. Chromatographic purifi-
cations were performed on silica gel columns (Macherey–Nagel, 70-230
mesh) with the indicated eluents. Size-exclusion chromatography was car-
ried out on Bio-Rad Bio-Beads S-X1 beads (200–400 mesh) by using
THF as the eluent. All other solvents and reagents were of reagent grade
and used as received. The Sieber amide resin and all the Fmoc-protected
amino acids were purchased from Chem-Implex International Inc., and
GL Biochem Ltd., respectively. Compounds 2,[24] 4,[25] and 8[26] were pre-
pared as described.
1H and 13C{1H} NMR spectra were recorded on a Bruker Avance III 400
spectrometer (1H, 400; 13C, 100.6 MHz) in deuterated solvents. Spectra
were referenced internally by using the residual solvent [1H: d=7.26
(CDCl3), 2.05 ([D6]acetone)] or solvent [13C: d=77.2 (CDCl3), 206.3
([D6]acetone)] resonances relative to SiMe4. ESI mass spectra were re-
corded on a Thermo Finnigan MAT 95XL mass spectrometer. Elemental
analyses were performed by the Shanghai Institute of Organic Chemistry,
Chinese Academy of Sciences, China.
UV/Vis and steady-state fluorescence spectra were taken on a Cary 5G
UV/Vis/NIR spectrophotometer and a Hitachi F-7000 spectrofluorome-
ter, respectively. The fluorescence quantum yields (FF) of the samples
Figure 7. Relative fluorescence intensities (per unit area) of the tumor
and different organs in nude mice treated with: a) 10, or b) 12. Five mice
were used for each compound and the data are expressed as mean value
Æ standard deviation.
were determined by the equation: FF(sample) =(Fsample/Fref)(Aref
/
[33]
Asample)(n2sample/n2ref)FF(ref)
,
where F, A, and n are the measured fluores-
cence (area under the emission peak), the absorbance at the excitation
position (620 nm), and the refractive index of the solvent, respectively.
ZnPc in DMF was used as the reference (FF(ref) =0.28).[28] To minimize
reabsorption of radiation by the ground state species, the emission spec-
tra were obtained in very dilute solutions of which the absorbance at
620 nm was about 0.03. The singlet oxygen quantum yields (FD) were
measured in DMF by the method of chemical quenching of DPBF by
using ZnPc as the reference (FD =0.56).[29]
peptide plays an important role in enhancing the tumor-re-
tention property of 12. Although a significant number of
peptide-conjugated photosensitizers have been reported,
their in vivo tumor-localization properties have been little
studied. Systems that show enhanced accumulation in
tumors are usually conjugated with a tumor-targeting ligand,
such as the cyclic RGD peptide[17b] or folate.[18] Tung et al.
have reported a chlorin e6-containing polylysine grafted
with monomethoxy polyethylene glycol, which also accumu-
lates in tumors over time.[31] The result has been attributed
to the EPR effect, which in fact is very common for poly-
meric drugs.[32] While the detailed mechanism for the high
tumor-retention property of 12 remains elusive at this stage,
it is believed that the EPR effect induced by the peptide is
an important factor.
Reversed-phase analytical HPLC experiments were performed on
a Apollo-C18 column (5 mm, 4.6 mmꢁ250 mm) or on a XBridge-C18
column (5 mm, 10 mmꢁ250 mm) by using a Shimadzu CBM-20A control-
ler with a SPD-M20A diode array detector. The conditions were set as
follows: solvent A=0.1% TFA in distilled water; solvent B=0.1% TFA
in acetonitrile. Gradient for the Fmoc-protected peptide sequence: 80%
A+20% B in the first 5 min, then changed to 0% A+100% B in
35 min, further to 80% A+20% B in 5 min, and finally maintained at
this condition for
a further 5 min. The flow-rate was fixed at
0.6 mLminÀ1. Gradient for the phthalocyanine–peptide conjugate 12:
95% A+5% B in the first 5 min, then changed to 0% A+100% B in
35 min, kept at this condition for 5 min, and finally changed to 95% A+
5% B in 5 min. The flow-rate was fixed at 3.0 mLminÀ1
.
Phthalonitrile 3:
A mixture of 2,3-dicyanohydroquinone (1; 2.40 g,
15.0 mmol) and K2CO3 (4.14 g, 30.0 mmol) in DMF (20 mL) was heated
to 908C. A solution of triethylene glycol monomethyl ether tosylate (2;
4.77 g, 15.0 mmol) in DMF (15 mL) was added dropwise to the above
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A Phthalocyanine–Peptide Conjugate for PDT
FULL PAPER
mixture while being stirred. The mixture was stirred at 908C for 8 h, then
the volatiles were removed in vacuo. The residue was mixed with water
(40 mL) and acidified with HCl solution (2m, 40 mL), then the mixture
was extracted with ethyl acetate (50 mLꢁ3). The combined organic layer
was washed with saturated aqueous NaCl solution, dried over anhydrous
Na2SO4, and then evaporated to dryness under reduced pressure. The res-
idue was purified by silica gel column chromatography by using CH2Cl2/
CH3OH (40:1, v/v) as the eluent to give a white solid (1.75 g, 38%).
1H NMR ([D6]acetone): d=7.53 (d, J=9.6 Hz, 1H, ArH), 7.38 (d, J=
9.6 Hz, 1H, ArH), 4.32 (virtual t, J=4.8 Hz, 2H, CH2), 3.85 (virtual t, J=
4.8 Hz, 2H, CH2), 3.65–3.67 (m, 2H, CH2), 3.55–3.60 (m, 4H, CH2), 3.44–
3.47 (m, 2H, CH2), 3.27 ppm (s, 3H, CH3); 13C{1H} NMR ([D6]acetone):
d=155.9, 155.3, 123.5, 121.5, 114.2 (two overlapping signals), 104.2, 102.4,
72.5, 71.4, 71.1, 70.9, 70.7, 70.0, 58.6 ppm; MS (ESI): m/z: 329 (100%,
[M+Na]+); HRMS (ESI): m/z calcd for C15H18N2NaO5 [M+Na]+:
329.1108; found: 329.1113; elemental analysis calcd (%) for C15H18N2O5:
C 58.82, H 5.92, N 9.15; found: C 58.94, H 5.99, N 9.02.
hydrous Na2SO4 and evaporated to dryness under reduced pressure. The
residue was purified by silica gel column chromatography by using
CHCl3/CH3OH (30:1, v/v) as the eluent to afford a dark-green solid
(70 mg, 56%). 1H NMR (CDCl3 with a trace amount of [D5]pyridine):
d=9.37–9.41 (m, 4H, Pc-Ha), 9.30 (d, J=6.8 Hz, 2H, Pc-Ha), 8.09–8.13
(m, 6H, Pc-Hb), 7.63 (s, 1H, triazole-H), 7.45 (s, 2H, Pc-Hb), 5.00 (s, 2H,
CH2), 4.92 (t, J=5.2 Hz, 4H, CH2), 4.62 (s, 2H, CH2), 4.49–4.52 (m, 4H,
CH2), 4.12–4.15 (m, 4H, CH2), 4.10 (q, J=7.2 Hz, 2H, CH2), 3.82–3.87
(m, 4H, CH2), 3.67–3.70 (m, 4H, CH2), 3.63–3.65 (m, 2H, CH2), 3.51–
3.53 (m, 2H, CH2), 3.33 (s, 3H, CH3), 1.14 ppm (t, J=7.2 Hz, 3H, CH3);
13C{1H} NMR (CDCl3 with a trace amount of [D5]pyridine): d=166.2,
153.7, 153.6, 153.5, 153.4, 152.4, 152.3, 150.2, 150.1, 145.5, 138.9, 138.5,
138.4, 129.0, 128.9, 127.1, 124.2, 122.7, 122.5, 114.7, 72.0, 71.2, 70.9, 70.7,
70.6, 69.8, 69.1, 69.0, 64.6, 62.3, 59.1, 50.8, 14.0 ppm (some of the signals
are overlapped); MS (ESI): an isotopic cluster peaking at m/z 1054
(100%, [M+H]+); HRMS (ESI): m/z calcd for C52H52N11O10Zn [M+H]+
1054.3185; found: 1054.3195; elemental analysis calcd (%) for
C52H53N11O11Zn (9·H2O): C 58.18, H 4.98, N 14.35; found: C 58.58, H
5.07, N 14.19.
Phthalonitrile 5: A mixture of phthalonitrile 3 (1.30 g, 4.24 mmol), tosy-
late
4 (1.45 g, 4.24 mmol), and K2CO3 (1.17 g, 8.47 mmol) in DMF
(10 mL) was stirred at 908C for 24 h. The volatiles were removed under
reduced pressure. The residue was mixed with water (30 mL) and then
extracted with ethyl acetate (50 mLꢁ3). The combined organic layer was
dried over anhydrous Na2SO4 and then evaporated to dryness under re-
duced pressure. The residue was purified by silica gel column chromatog-
raphy by using CH2Cl2/CH3OH (50:1, v/v) as the eluent to give a pale
Carboxy phthalocyanine 10: A mixture of 9 (130 mg, 0.12 mmol) in a satu-
rated solution of NaOH in MeOH/H2O (5:1, v/v, 6 mL) was stirred at
608C for 12 h. The volatiles were removed in vacuo. The residue was
mixed with water (10 mL) and neutralized with HCl solution (1m) until
the formation of green precipitate. The precipitate was filtered off and
washed with water (5 mL), then dried in vacuo to afford a dark-green
1
yellow solid (1.80 g, 89%). H NMR (CDCl3): d=7.25 (s, 2H, ArH), 4.23
solid (90 mg, 71%). 1H NMR (CDCl3 with
a trace amount of
(virtual t, J=4.4 Hz, 4H, CH2), 4.18 (d, J=2.4 Hz, 2H, CH2), 3.88 (vir-
tual t, J=4.4 Hz, 4H, CH2), 3.72–3.74 (m, 4H, CH2), 3.62–3.70 (m, 10H,
CH2), 3.52–3.55 (m, 2H, CH2), 3.36 (s, 3H, CH3), 2.42 ppm (t, J=2.4 Hz,
[D5]pyridine): d=9.35–9.39 (m, 4H, Pc-Ha), 9.27 (d, J=6.4 Hz, 2H, Pc-
Ha), 8.08–8.11 (m, 6H, Pc-Hb), 7.69 (s, 1H, triazole-H), 7.41 (s, 2H, Pc-
Hb), 5.01 (s, 2H, CH2), 4.89 (virtual t, J=4.8 Hz, 4H, CH2), 4.65 (s, 2H,
CH2), 4.49 (t, J=5.2 Hz, 4H, CH2), 4.10–4.13 (m, 4H, CH2), 3.82–3.85
(m, 4H, CH2), 3.66–3.71 (m, 6H, CH2), 3.49–3.52 (m, 2H, CH2),
3.31 ppm (s, 3H, CH3); 13C{1H} NMR (CDCl3 with a trace amount of
[D5]pyridine): d=169.3, 153.4, 152.1, 150.0, 145.1, 138.7, 138.3, 128.8,
126.8, 124.2, 122.6, 122.4, 114.7, 71.9, 71.1, 70.8, 70.6, 70.5, 69.6, 69.0, 64.6,
58.9, 51.5 ppm (some of the signals are overlapped); MS (ESI): an isotop-
ic cluster peaking at m/z 1026 (100%, [M+H]+); HRMS (ESI): m/z
calcd for C50H48N11O10Zn [M+H]+ 1026.2872; found: 1026.2882; elemen-
tal analysis calcd (%) for C50H51N11O12Zn (10·2H2O): C 56.47, H 4.83, N
14.49; found: C 56.37, H 4.84, N 13.95.
1H, C CH); 13C{1H} NMR (CDCl3): d=155.4, 119.4, 113.1, 105.3, 79.7,
ꢀ
74.6, 71.9, 71.0, 70.6, 70.5, 70.4, 70.1 (two overlapping signals), 69.4 (two
overlapping signals), 69.1, 59.0, 58.4 ppm; MS (ESI): m/z: 499 (100%,
[M+Na]+); HRMS (ESI): m/z calcd for C24H32N2NaO8 [M+Na]+
499.2051; found: 499.2059; elemental analysis calcd (%) for C24H32N2O8:
C 60.49, H 6.77, N 5.88; found: C 60.60, H 6.97, N 5.77.
Alkynyl phthalocyanine 7:
A mixture of phthalonitrile 5 (100 mg,
0.21 mmol) and unsubstituted phthalonitrile (6; 0.27 g, 2.1 mmol) in n-
pentanol (8 mL) was heated to 1208C, then a piece of lithium chip
(102 mg, 14.7 mmol) was added. The mixture was stirred at 120–1308C
for 5 h, then Zn
G
Fmoc-Gly-Gly-Pro-LysACHTUNGTRNENU(G Boc)-LysACUTHNGTRNENU(GN Boc)-LysAHCTUNRTGEG(NNUN Boc)-ArgAHCTUNGTRNEUN(NG Pbf)-LysACHTUNGTRENNUNG(Boc)-Val-
was stirred at 120–1308C for a further 5 h. After being briefly cooled, the
volatiles were removed under reduced pressure. The residue was dis-
solved in CHCl3 (100 mL), and the solution was filtered to remove part
of the ZnPc formed. The filtrate was collected and evaporated to dryness
in vacuo. The residue was purified by silica gel column chromatography
by using CHCl3/CH3OH (30:1, v/v) as the eluent, followed by size-exclu-
sion chromatography by using THF as the eluent. The crude product was
further purified by silica gel column chromatography by using CHCl3/
CH3OH (30:1, v/v) as the eluent to give a dark-green solid (110 mg,
57%). 1H NMR (CDCl3 with a trace amount of [D5]pyridine): d=9.40–
9.43 (m, 4H, Pc-Ha), 9.33–9.35 (m, 2H, Pc-Ha), 8.10–8.14 (m, 6H, Pc-
Hb), 7.50 (s, 2H, Pc-Hb), 4.94 (t, J=5.2 Hz, 4H, CH2), 4.53 (t, J=5.2 Hz,
4H, CH2), 4.13–4.16 (m, 6H, CH2), 3.86 (t, J=5.2 Hz, 4H, CH2), 3.65–
3.73 (m, 6H, CH2), 3.51–3.53 (m, 2H, CH2), 3.33 (s, 3H, CH3), 2.37 ppm
resin (NLS-resin): This peptide resin (150 mg, 106.5 mmol) was synthe-
sized manually according to the Fmoc SPPS protocol with commercially
available N-a-Fmoc-protected amino acids. Sieber amide resin was used
as solid support, and HOBt and HBTU were used as the carboxyl group
activating agents. Excess (3 equiv) Fmoc-protected amino acids and acti-
vating agents were used for each coupling. After the final coupling, the
NLS-resin was washed with DMF and CH2Cl2, and then dried in vacuo.
To check the purity of the peptide, a portion of the NLS-resin was treat-
ed with a solution (0.2 mL) containing TFA (95%), TIS (2.5%), and
CH2Cl2 (2.5%) for 1 h to remove the Boc and Pbf protecting groups. The
resin was removed by filtration and the filtrate was precipitated by the
addition of diethyl ether. After centrifugation, the supernatant was re-
moved. The yellow solid was redissolved in DMSO (0.2 mL) and then
precipitated again by the addition of diethyl ether. This purification pro-
cedure was repeated twice and the product Fmoc-Gly-Gly-Pro-Lys-Lys-
Lys-Arg-Lys-Val was dried in vacuo before HPLC analysis. MS (ESI): an
isotopic cluster peaking at m/z: 1219 (100%, [M+H]+); HRMS (ESI):
m/z calcd for C59H96N17O11 [M+H]+ 1218.7470; found: 1218.7512. The
purity was found to be about 95%.
(t, J=2.4 Hz, 1H, C CH); 13C{1H} NMR (CDCl3 with a trace amount of
ꢀ
[D5]pyridine): d=153.7, 153.6, 153.5, 152.4, 150.2, 138.8, 138.4, 129.0,
128.9, 127.1, 122.7, 122.5, 114.7, 79.6, 74.6, 71.9, 71.1, 70.8, 70.6, 70.5, 70.4,
69.1, 69.0, 59.0, 58.4 ppm (some of the signals are overlapped); MS (ESI):
an isotopic cluster peaking at m/z 925 (100%, [M+H]+); HRMS (ESI):
m/z calcd for C48H45N8O8Zn [M+H]+ 925.2646; found: 925.2636; elemen-
tal analysis calcd (%) for C48H44N8O8Zn: C 62.24, H 4.79, N 12.10;
found: C 62.20, H 5.07, N 11.73.
Phthalocyanine–peptide conjugate 12: The Fmoc protecting group of gly-
cine was first removed from NLS-resin (15 mmol) with piperidine (20%)
in DMF, then the resin was washed with DMF and CH2Cl2. A mixture of
10 (38 mg, 37.0 mmol), HOBt hydrate (5.1 mg, 37.7 mmol), and HBTU
(14.3 mg, 37.7 mmol) in DMF (2 mL) was added to the peptide–resin in
the presence of N,N-diisopropylethylamine (20 mL). The mixture was
shaken under argon overnight at room temperature. The dark-green
resin was washed with DMF and CH2Cl2 and then dried in vacuo. The
resin was then treated with a solution (0.5 mL) containing TFA (95%),
Triazole phthalocyanine 9: A mixture of phthalocyanine
7 (110 mg,
0.12 mmol), azido ester (82 mg, 0.64 mmol), CuSO4·5H2O (16 mg,
8
64 mmol), and sodium ascorbate (25 mg, 0.13 mmol) in a 12:1:1 mixture
of CHCl3, EtOH, and water (3.5 mL) was stirred at room temperature
for 24 h. The mixture was mixed with water (15 mL) and then extracted
with CHCl3 (15 mLꢁ2). The combined organic layer was dried over an-
Chem. Eur. J. 2012, 18, 4225 – 4233
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
4231

D. K. P. Ng, P.-C. Lo et al.
TIS (2.5%), and CH2Cl2 (2.5%) for 1 h to remove all the protecting
groups on the peptide sequence and cleave the peptide from the resin.
The resin was removed by filtration and the filtrate was precipitated by
the addition of diethyl ether. After centrifugation, the supernatant was
removed. The green solid was redissolved in DMSO (0.5 mL) and then
precipitated by the addition of diethyl ether. This purification procedure
was repeated three times and the product was dried in vacuo before
HPLC analysis. MS (ESI): an isotopic cluster peaking at m/z 1003
(100%, [M+2H]2+); HRMS (ESI): m/z calcd for C94H132N28O18Zn
[M+2H]2+ 1002.9794; found: 1002.9787. The purity was found to be
about 100%.
(1.2 mL). The mixture was sonicated for 20 min and then centrifuged
again. The supernatants were transferred for UV/Vis spectroscopic meas-
urements. The absorbance at 689 nm (10) or 688 nm (12) was compared
with the respective calibration curves to give the uptake concentrations.
Cellular uptake (determined by confocal microscopy): HT29 cells (~3ꢁ
104) in the culture medium (2 mL) were seeded on a coverslip and incu-
bated overnight at 378C under 5% CO2. The medium was then removed.
The cells were incubated with a solution of 10 or 12 in the medium
(5 mm, 2 mL) for 2 h under the same conditions. The cells were then
rinsed with PBS and viewed with a Leica SP5 confocal microscope
equipped with a 633 nm helium neon laser. The compounds were excited
at 633 nm and monitored at 640–700 nm. The images were digitized and
analyzed by using the Leica Application Suite Advanced Fluorescence.
The intracellular fluorescence intensities (a total of 50 cells for each
sample) were also determined.
Fluorescence microscopic studies: HT29 cells (~3ꢁ104) in the culture
medium (2 mL) were seeded on a coverslip and incubated overnight at
378C under 5% CO2. The medium was then removed. The cells were in-
cubated with a solution of 10 or 12 in the medium (10 mm, 1.5 mL) for 2 h
under the same conditions. The cells were then rinsed with PBS and
viewed with an Olympus IX70 inverted microscope. The excitation light
source at 630 nm was provided by a multi-wavelength illuminator (Poly-
chrome IV, TILL Photonics). The emitted fluorescence at >660 nm was
collected, and digitized and analyzed by using MetaFluor V6.3 (Universal
Imaging).
Cell lines and culture conditions: HT29 cells (ATCC, no. HTB-38) were
maintained in Dulbeccoꢂs modified Eagleꢂs medium (DMEM, Invitrogen,
no. 10313-021) supplemented with fetal calf serum (10%), penicillin/
streptomycin (100 unitsmLÀ1
, , respectively), l-glutamine
100 mgmLÀ1
(2 mm), and transferrin (10 mgmLÀ1). Cells (~3ꢁ104 per well) in the
media were inoculated in 96-well plates and incubated overnight at 378C
in a humidified 5% CO2 atmosphere.
Photocytotoxicity assay: Phthalocyanines 10 and 12 were first dissolved
in DMSO to give 1.6 mm solutions, which were diluted to 80 mm with an
aqueous solution of Tween-80 (Arcos, 0.5% by volume in these 80 mm
solutions). The solutions were filtered with a 0.22 mm filter, then diluted
with the culture medium to various concentrations. After being rinsed
with phosphate buffered saline (PBS) the cells were incubated with the
phthalocyanine solutions (100 mL) for 2 h at 378C under 5% CO2. The
cells were then rinsed again with PBS and the wells were refilled with
the culture medium (100 mL) before being illuminated at ambient tem-
perature. The light source consisted of a 300 W halogen lamp, a water
tank for cooling, and a color glass filter (Newport) cut-on 610 nm. The
fluence rate (l>610 nm) was 40 mWcmÀ2. Illumination of 20 min led to
In vivo imaging and ex vivo organ biodistribution: Female Balb/c nude
mice (20–25 g) were obtained from the Laboratory Animal Services
Centre at The Chinese University of Hong Kong. All animal experiments
were approved by the Animal Experimentation Ethics Committee of the
University. The mice were housed in pathogen-free conditions with
access to food and water. HT29 cells (~1ꢁ107 cells in 200 mL) were ino-
culated subcutaneously on the back of the mice. Once the tumors had
grown to 60–100 mm3, the mice were fed with low fluorescence diet
(TestDiet, no.: AIN-93M) for 4 days. Phthalocyanines 10 or 12 (25 nmol)
was first dissolved in DMSO (5 mL) and Tween-80 (5 mL). The solutions
were further diluted to appropriate concentrations (1 mmol of drug per
kg body weight) with distilled water (190 mL). These phthalocyanine solu-
tions (200 mL) were intravenously injected into the tail vein of the tumor-
bearing mice. In vivo fluorescence imaging was performed before and
after injection (at different time points) with an Odyssey infrared imag-
ing system (excitation wavelength=680 nm, emissionꢂ700 nm). The
images were digitized and analyzed by the Odyssey imaging system soft-
ware (Cat. no. 9201-500). After in vivo imaging studies, the animals were
euthanized at 48 or 72 h post-injection. Tumors and other organs were
harvested and their fluorescence intensities were measured. Five mice
were used for each compound.
a total fluence of 48 JcmÀ2
.
Cell viability was determined by means of the colorimetric MTT assay.[34]
After illumination, the cells were incubated at 378C under 5%
CO2overnight. MTT (Sigma) solution in PBS (3 mgmLÀ1, 50 mL) was
added to each well followed by incubation for 2 h under the same condi-
tions. A solution of sodium dodecyl sulfate (Sigma, 10% by weight,
50 mL) was then added to each well. The plate was incubated at 608C for
30 min, then isopropanol (80 mL) was added to each well. The plate was
agitated on a Bio-Rad microplate reader at ambient temperature for 10 s
before the absorbance at 540 nm for each well was taken. The average
absorbance of the blank wells, which did not contain cells, was subtracted
from the readings of the other wells. The cell viability was then deter-
mined by the following equation: %viability=[ꢀ(Ai/Acontrol ꢁ100)]/n,
where Ai is the absorbance of the i-th data (i=1, 2, …, n), Acontrol is the
average absorbance of the control wells in which the phthalocyanine was
absent, and n (=4) is the number of the data points.
ROS measurements: HT29 cells (~3ꢁ104) were placed in a 96-well plate
and incubated overnight at 378C in a humidified 5% CO2 atmosphere.
The cells were incubated with the phthalocyanine solutions for 2 h at
378C under 5% CO2. After being washed with PBS, the cells were incu-
bated with DCFDA solution in PBS (Molecular Probes, 10 mM, 100 mL)
at 378C for 30 min. The cells were then rinsed again with PBS and the
wells were refilled with PBS (100 mL) before photodynamic treatment.
Fluorescence measurements were made in a fluorescence plate reader
(TECAN Polarion) with a 485 nm excitation filter and a 535 nm emission
filter set at a gain of 60.
Acknowledgements
This work was supported by a Direct Grant for Research of The Chinese
University of Hong Kong (2009/10 and 2010/11).
Cellular uptake (determined by extraction): HT29 cells (ꢁ1ꢁ106) in
DMEM (2 mL) were seeded on a coverslip and incubated overnight at
378C under 5% CO2. The medium was removed and the cells were
rinsed with PBS (2 mL). The cells were incubated with a solution of 10
or 12 in the medium (1.0 mm with 0.06% DMF and 0.006% Cremophor
EL, 2 mL) for 2 h under the same conditions. The solution was then re-
moved and the cells were rinsed with PBS (2 mL) and then harvested by
trypsin (0.25%)/EDTA (Invitrogen, 500 mL) treatment. The trypsin was
then quenched with the medium (500 mL). The solution was transferred
to centrifuge tubes (1.5 mL) and centrifuged at 956 g for 3 min. The
pellet was then washed with PBS (1 mL) and the suspension was centri-
fuged again. After removing the PBS, the cells were lyzed with DMF
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A Phthalocyanine–Peptide Conjugate for PDT
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Received: November 8, 2011
Published online: February 29, 2012
Chem. Eur. J. 2012, 18, 4225 – 4233
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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4233