Polyethylene glycol-functionalized benzylidene cyclopentanone dyes for two-photon excited photodynamic therapy

Article abstract of DOI:10.1039/c0ob01278e

A series of polyethylene glycol-functionalized benzylidene cyclopentanone dyes with varying lipid/water partition coefficients were synthesized in high yields by a simple process. Detailed characterization and systematic studies of these molecules, including linear and nonlinear photophysical properties, reactive oxygen yields, and in vitro photodynamic therapy (PDT) activities, were conducted. Four of these dyes exhibited good solubility in PBS (>2 mg ml^-1, which is sufficient for clinical venous injection), high reactive oxygen yields, large two-photon absorption and low dark toxicity, under the therapy dosage. Among them, two dyes could be absorbed efficiently by human rectal cancer 1116 cells, and presented strong two-photon excited PDT activity in in vitro cell experiments.

Full text of DOI:10.1039/c0ob01278e

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Polyethylene glycol-functionalized benzylidene cyclopentanone dyes for
two-photon excited photodynamic therapy†
Yuxia Zhao,a Weijia Wang,a,c Feipeng Wu,*a Yang Zhou,b Naiyan Huang,b Ying Gu,b Qianli Zoua,c and
Wei Yanga,c
Received 31st December 2010, Accepted 10th March 2011
DOI: 10.1039/c0ob01278e
A series of polyethylene glycol-functionalized benzylidene cyclopentanone dyes with varying
lipid/water partition coefficients were synthesized in high yields by a simple process. Detailed
characterization and systematic studies of these molecules, including linear and nonlinear
photophysical properties, reactive oxygen yields, and in vitro photodynamic therapy (PDT) activities,
were conducted. Four of these dyes exhibited good solubility in PBS (>2 mg ml-1, which is sufficient for
clinical venous injection), high reactive oxygen yields, large two-photon absorption and low dark
toxicity, under the therapy dosage. Among them, two dyes could be absorbed efficiently by human rectal
cancer 1116 cells, and presented strong two-photon excited PDT activity in in vitro cell experiments.
Therefore, the spatial selectivity and accuracy of the treatment
Introduction
could be further enhanced.6
Photodynamic therapy (PDT), a promising noninvasive treatment
Conventional clinical PDT drugs were used as photosensitizers
in early TPE-PDT studies, such as Photofrin and Verteprofin.6,7
However, their two-photon absorption cross-section (s) values
are too small (for example, the s value of Photofrin is 7.4
GM6 at 850 nm and the s value of Verteprofin is 51 GM7 at
900 nm, 1 GM = 10-50 cm4 s molecule-1 photon-1) to be suitable
clinically because their dynamic power ranges for PDT (the ratio
of the threshold for photothermal or photomechanical damage
vs. the lowest light intensities required for a clear therapeutic
effect) are not large enough. For exploring the full potential
of TPE-PDT, recently various design strategies have been em-
ployed to synthesize water-soluble photosensitizers with large two-
photon absorption (TPA) cross-section and evident photodynamic
activity.8–13 Among them, porphyrin dimer derivatives8 have been
progressed to in vivo testing and proved the TPE-PDT effect
by selectively closing blood vessels. Moreover, other valuable
approaches such as encapsulation of TPA photosensitizer into
water-dispersed nanoparticles or encapsulation of photosensitizer
into amphiphilic TPA-chromophore-containing block copolymers
have been reported to successfully induce a TPE phototoxic
effect in in vitro cell testing.14,15 These exciting results have
attracted lots of attention for TPE-PDT, however, for realizing
its practical application, much more systematic work is still
required.
for cancer and nonmalignant tumors, has been used in clinical
treatment for a long period. Its mechanism is using light to
activate a tumor-localized photosensitizer, selectively to destroy
tumor tissue via the in situ generation of highly cytotoxic reactive
oxygen species (ROS), for example singlet oxygen (1O2) or super-
∑
oxide anion radical (O2 ).1 Compared to other cancer treatment
-
modalities, such as surgery, radio- and chemo-therapy, PDT has
advantages of high selectivity to malignant target vs. normal cells,
and low damage.2 However, due to the limited tissue penetration,
its applications are mainly confined to superficial tissues of an
organ, such as in esophageal cancer and skin disease.3 Two-photon
excited PDT (TPE-PDT) has attracted much attention in recent
years for its significant benefit of deep tissue penetration due to
the fact that two photons of infrared light rather than one photon
of visible light are absorbed by photosensitizer,4,5 which helps to
resolve the penetration problem mentioned above, in present PDT.
Furthermore, the quadratic dependence of two-photon excitation
on the laser intensity restricts excitation to the focus of a laser
beam, which allows the generation of cytotoxic ROS to be confined
to a small volume, reducing collateral damage to normal tissues.
aTechnical Institute of Physics and Chemistry, Chinese Academy of Sciences,
Beijing, 100190, P. R. China. E-mail: fpwu@mail.ipc.ac.cn
In our previous work, benzylidene cyclopentanone dyes were
found to have large s values (400–3300 GM around 800 nm,
which were comparatively high based on their simple molecular
structures), and high photosensitizing efficiencies.16–18 Unfortu-
nately, these dyes are all hydrophobic. In this work, we introduced
a number of low molecular weight polyethylene glycol (PEG)
bDepartment of Laser Medicine, Chinese PLA General Hospital, Beijing,
100853, P. R. China
cGraduate University of Chinese Academy of Science, Beijing, 100049, P. R.
China
† Electronic supplementary information (ESI) available: 1H and 13C NMR
spectra of all target dyes. See DOI: 10.1039/c0ob01278e
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Table 1 Solubility in PBS and lipid/water (octanol/water) partition
coefficient (PC) of dyes
Compound
Solubility in PBS/mg mL-1
log PC
A1
A2
A3
A4
B1
B2
B3
B4
—
>8
>7
>7
2.57
>8
2.99
3.04
2.32
0.0194
0.0035
4.6
—
2.8
2.6
5.3
Results and discussion
Synthetic strategy
The new series of dyes presented in this study were ob-
tained by a simple two-step synthesis process illustrated in
Scheme 1. The prototype dye is 2,5-bis[4-(diethylamino)benzyli-
dene]cyclopentanone (BDEA), which is insoluble in aqueous
media. Therefore, we introduce amphiphilic ethylene glycol moi-
eties, which are known for conferring cell permeability and
tumor targeting characteristics on photosensitizers,19–21 into the
parent structure. Intermediate products d1–d4 were prepared
by substitution reaction between 4-[bis(2-hydroxyethyl)amino]-
benzaldehyde or 4-[(2-hydroxyethyl)methylamino]benzaldehyde
and 1-(p-tolysulfonyl)-3,6-dioxoheptane (c1) or 1-(p-tolysulfonyl)-
3,6,9-trioxodecane (c2) in high yields (72–79%). Target dyes
A1–B4 were synthesized by base-catalyzed condensation of
aromatic aldehydes d1–d4 with cyclopentanone or 2-[4-
(diethylamino)benzylidene]cyclopentanone (DEA) and purified
by column chromatography on silica in good yields (61%–74%).
Solubility and lipid-water partition coefficient (PC)
As shown by the data in Table 1, four dyes (A4, B2, B3 and B4)
exhibit good water solubility in the range of 2.6–5.3 mg ml-1 in
PBS (pH = 7.4), while dyes A2 and A3 are only slightly soluble, and
dyes A1 and B1 are almost insoluble. This shows that increasing the
number of PEG chains or extending their length leads to increase
of solubility in PBS.
In PDT, photosensitizers are injected into a vein and trans-
ported to target tumor tissues through the blood circulating
system, and enter tumor cells and intercellular regions through
the lipid membranes of blood vessels and cells, which requires the
photosensitizers to be both water- and lipid-soluble. Thus a mod-
erate lipid/water PC is important for a PDT photosensitizer.22,23
The results show that the lipid/water (octanol/water) PC values
of dyes A1–A3 and B1 are very large (log PC >7), with almost
all dye in the organic phase, while A4 and B2–B4 have moderate
lipid/water PC values. For ensuring sufficient cellular uptake of
dyes, an appropriate PC value is required, i.e. a fair degree of water
solubility, as shown by dyes A4 and B2–B4.
Scheme 1 Synthesis of dyes A1–B4.
chains into the benzylidene cyclopentanone core (Scheme 1) to
improve their solubility in biological media and to optimize
their lipid/water partition coefficients (PC) through adjusting
the length and number of PEG chains covalently linked to the
dyes. After systematic studies of their water-solubility, linear and
non-linear photophysical properties, and PDT activities, two dyes
successfully demonstrated their PDT and TPE-PDT application
potential in in vitro cell testing.
One- and two-photon photophysical properties
The UV-Vis absorption spectra as well as one-photon excited
fluorescence emission spectra excited at 480 nm of all dyes in n-
octanol are almost exactly the same, as shown in Fig. 1A and Table
2, which proves that the terminal PEG chains have almost no effect
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Table 2 One- and two-photon photophysical properties of new dyesa
b
c
Compound
Solvent
lmaxabs /nm
104emax/M-1 cm-1
lmaxfl/nm
Uf
UD
UD
smax/GM
A1
A2
n-Octanol
n-Octanol
PBS
n-Octanol
n-Octanol
PBS
n-Octanol
n-Octanol
PBS
n-Octanol
PBS
n-Octanol
PBS
n-Octanol
486
486
505
486
488
507
486
487
508
487
511
486
509
490
6.3
6.2
4.7
6.1
6.2
4.8
6.2
6.3
5.0
6.1
4.5
6.0
5.1
6.8
578
578
636
577
578
636
578
578
637
578
636
578
636
578
0.30
0.28
0.028
0.27
0.29
0.023
0.28
0.29
0.019
0.28
0.026
0.29
0.02
0.21
0.22
0.20
—
0.19
0.18
—
0.21
0.20
—
0.21
—
0.24
0.27
—
0.26
0.24
—
0.23
0.30
—
0.22
—
869
814
—
861
986
—
783
781
—
917
—
990
A3
A4
B1
B2
B3
B4
0.18
—
0.23
0.26
—
0.28
—
1052
BDEA
a
abs
fl
lmax is one-photon absorption band-maximum; emax is molar absorption coefficient at lmaxabs ; lmax is fluorescence band maximum; Uf is fluorescence
quantum yield; UD is singlet oxygen quantum yield. b Measured by photochemical trap method, c Measured by 1O2 phosphorescence intensity; smax is the
maximum TPA cross section obtained within 720–880 nm.
840 nm in the range of 780–990 GM (shown in Fig. 2), which is
comparable with the 1052 GM of BDEA (without PEG chains),
indicating that the terminal PEG chains have little effect on the
TPA characterization of the benzylidene cyclopentanone core.
Fig. 2 Two-photon excitation spectra of dyes in n-octanol. The exciting
source is a mode-locked Tsunami Ti : sapphire laser (720–880 nm, 80 MHz,
<130 fs).
Photosensitization activity
Fig. 1 Normalized absorption and one-photon excited fluorescence
emission spectra of water-soluble dyes in n-octanol (A), excited at 480 nm,
and in PBS (B), excited at 530 nm.
By irradiating oxygen-saturated solutions of A4 and B2–B4 with a
532 nm laser in the presence of DMPO used as superoxide radical
(O2 ∑) spin-trapping reagent or TEMP used as a singlet oxygen
-
on the electron transition of the benzylidene cyclopentanone core.
The absorption and one-photon excited fluorescence emission
spectra excited at 530 nm (Fig. 1B) of water-soluble A4 and B2–B4
in PBS (PH = 7.4) are also very similar.
The two-photon excitation (TPE) spectra of all dyes within the
range 720–880 nm were measured using the TPE fluorescence
technique with a mode-locked Tsunami Ti : sapphire femtosecond
laser (80 MHz, <130 fs). Their maxima s values (smax) appear at
(1O2) spin-trapping reagent, an electron paramagnetic resonance
(EPR) signal appeared (inset in Fig. 4) with a g factor and
N
H
hyperfine coupling constants (g = 2.0056, a = 12.48 G, ab
=
H
10.36 G, ag = 1.35 G) in good agreement with those for a
∑
-
DMPO–O2 adduct,24 or (inset in Fig. 3) with triplet peaks in
equal intensity (g = 2.0056, a = 16.3 G) due to the TEMP–1O2
N
∑
adduct.24 The results confirm that both O2 and O2 species can
-
1
be generated by A4 and B2–B4 under 532 nm illumination. In
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shown in Fig. 5 and 6. The UD of PEG-functionalized dyes are
comparable with the data of BDEA, which also confirms that the
terminal PEG chains have little effect on the photosensitization
activity of the benzylidene cyclopentanone core. For each dye, its
UD value from the photobleaching method is relatively lower than
that from the 1O2 luminescence method. The latter values should
be more reliable as they are taken from a direct spectroscopic
measurement method.
Fig. 3 EPR signal intensity of TEMP–1O2 adducts as a function of
irradiation time (irradiated at 532 nm) for oxygen-saturated DMSO and
toluene solutions of dyes (1 ¥ 10-4 M). Inset: The EPR spectrum of
TEMP–1O2 adduct.
Fig. 5 Photosensitized DMA-bleaching by measuring the decrease in
absorbance (DA) at 400 nm as a function of irradiation time in oxygen-sat-
urated n-octanol solutions of dyes. The concentrations of dyes are 2 ¥
10-5 M. The excitation wavelength is 532 nm.
∑
-
Fig. 4 EPR signal intensity of DMPO–O2 adducts as a function of
irradiation time (irradiated at 532 nm) for oxygen-saturated DMSO and
toluene solutions of dyes (1 ¥ 10-4 M). Inset: The EPR spectrum of
∑
-
DMPO–O2 adduct.
addition, the efficiency to generate reactive oxygen species (ROS)
depends significantly on the solution environment. For example,
∑
-
for B3 the polar solvent DMSO facilitates the production of O2
while the nonpolar solvent toluene facilitates the production of
1O2. Dependences of EPR signal intensity on irradiation time of
the four dyes are also shown in Fig. 3 and 4 and it is clear that the
four dyes have similar photosensitization activity. Among them,
B3 is relatively better than the other three.
The singlet oxygen quantum yields (UD) of all dyes were
determined by a photochemical bleaching method24 using 9.10-
dimethylanthracene (DMA) as 1O2 acceptor and 1O2 phosphores-
cence emission at 1270 nm13 using Rose Bengal in n-octanol (UD =
1) as reference, respectively. The results are listed in Table 2 and
Fig. 6 Emission of singlet oxygen at 1270 nm generated by irradiating
dyes in n-octanol with Rose Bengal (RB) as reference. The concentrations
of dyes are 2 ¥ 10-4 M. The excitation wavelength is 532 nm.
Intracellular distribution
Confocal fluorescence microscopy was used to monitor the uptake
and intracellular distribution of the water-soluble dyes. Solutions
of all the dyes in PBS were diluted into cell culture medium and
incubated with human rectal cancer 1116 (HRC-1116) cells for
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4 h. The fluorescence images of cells incubated with the dyes are
shown in Fig. 7. It is clear that all dyes can taken up by HRC-1116
cells. However, their uptake ratios are different. B2 and B3 in cells
show much stronger fluorescence compared to A4 and B4, which
illustrates that B2 and B3 are absorbed more than A4 and B4 by
cells. This result coincides with the relatively better lipid-solubility
of B2 and B3.
Fig. 7 Confocal fluorescence images obtained following incubations of
HRC-1116 cells with 2 mg mL-1 dyes for 4 h. The excitation wavelength
was 488 nm.
Fig. 9 Viability of HRC-1116 cells (1 ¥ 104 cells per well) with (light)
or without (dark) irradiation in the presence of dyes (10 mg mL-1). The
excitation wavelength is 457 nm, 20 mW cm-2, 1000 s light dose (20 J
cm-2). The error bars denote standard deviation from three replicates.
Fig. 8 shows the intracellular localization of the dyes in HRC-
1116 cells after 4 h incubation, together with the localization
of Mito-Tracker Green. The presence of B2 and B3 are evident
from their red fluorescence (third column), while Mito-Tracker
Green is detected by its intense green fluorescence (second
column). The colocalization of dye and Mito-Tracker results in
a yellow fluorescence (final column). It is clear that both B2
and B3 accumulate mainly in the mitochondria. No visible red
fluorescence is observed in the co-staining results of A4 and B4
with Mito-Tracker Green, which should be due to their weak
uptake.
better phototoxicity compared to PSD-007, illustrating they are
promising candidates for further study in PDT applications.
The TPE-PDT experiments were also conducted on HRC-1116
cells. After a cancer cell incubated with B3 is selectively irradiated
for 5 min under focused 800 nm laser pulses (80 MHz, 80 fs) with
an average power of 16 mW, a drastic hydropic degradation of
the cell membrane is clearly observed (Fig. 10), while the non-
irradiated cells at ambient sites remain intact. The same result is
confirmed by nine replicates. Furthermore, it is very exciting to
find that even under short irradiation time for 30 s and removal
of light, hydropic degradation of cell can also be observed 10 min
later (Fig. 11), which means that very short irradiation time is
sufficient to induce cell death with TPE-PDT. Under the same
condition, the degradation speed of cells incubated with B2 is a
little slower than that of cells incubated with B3, which may be
due to their different uptake ratio by cells; this factor needs to be
studied in more detail in the future. Parallel experiment without
photosensitizer shows no PDT effect. The results prove the B2 and
B3 have great potential in TPE-PDT.
Fig. 8 Confocal fluorescence images obtained following incubations of
HRC-1116 cells with 2 mg mL-1 dyes for 4 h and Mito-tracker green for
30 min. The excitation wavelength is 488 nm.
One- and two-photon PDT activity
For evaluating the biological PDT activity of the four water-
soluble dyes (A4, B2, B3 and B4) in vitro, PSD-007 is used as
reference, which is a clinical available dye and its active ingredient
is hematoporpyhrin monomethyl ether.25 The viability of HRC-
1116 cells was estimated based on measurements of MTT assay.
It is shown (Fig. 9) that the four dyes all exhibit low dark toxicity
(percentage of cell survival >90%), indicating that these dyes have
good safety under the therapy dosage in the dark. However, only
B2 and B3 show high phototoxicity. The possible reason is due
to their optimized amphiphilicities with two PEG chains leading
to higher uptake by cells compared to A4 and B4 with four
PEG chains. It is notable that B2 and B3 exhibit equal or even
Fig. 10 Photographs of human rectal cancer 1116 cells incubated with
2 mg mL-1 B3 before (A) and after (B) 5 min irradiation with 80 fs pulses
at 800 nm (output power 16 mW).
Conclusions
We implemented a successful strategy to explore a series of novel
PEG-functionalized benzylidene cyclopentanone dyes for TPE-
PDT application. The results showed that all of them had large
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with CH2Cl2 (3 ¥ 100 mL). The organic phase was dried over
anhydrous MgSO4 and rotary evaporated to give a viscous liquid,
which was purified by silica gel column chromatography, elution
of the column with ethyl acetate and petroleum ether mixture
(1 : 1) gave a yellow viscous liquid d1 (2.92 g, yield 72%). 1H NMR
(400 MHz, CDCl3): d (ppm) 3.35 (s, 6H), 3.53–3.65 (m, 24H),
6.73 (d, J = 9 Hz, 2H), 7.67 (d, J = 9 Hz, 2H), 9.70 (s, 1H).
Fig. 11 Photographs of human rectal cancer 1116 cells incubated with
2 mg mL-1 B3 before (A) and after (B) 30 s of irradiation, and (C) 10 min
after irradiation.
d2–d4. A procedure identical to that described above for d1
was utilized with different hydroxy-substituted benzaldehyde and
tosylated glycol as reactants, correspondingly. d2 (yield 75%): 1H
NMR (400 MHz, CDCl3): d (ppm) 3.07 (s, 3H), 3.34 (s, 3H), 3.51–
3.65 (m, 12H), 6.71 (d, J = 9 Hz, 2H), 7.69 (d, J = 9 Hz, 2H), 9.70
TPA within 700–900 nm and high ROS yields. Among them,
four dyes exhibited good solubility in PBS (>2 mg ml-1, which is
sufficient to clinical venous injection) and low dark toxicity under
the therapy dosage. Finally, two dyes showed efficient uptake by
HRC-1116 cells and were proved to have large potential in TPE-
PDT application by in vitro cell experiments. Owing to their simple
molecular structures and synthesis, we believe they will be good
candidates for TPE-PDT applications in the future.
1
(s, 1H). d3 (yield 79%): H NMR (400 MHz, CDCl3): d (ppm)
3.36 (s, 6H), 3.52–3.63 (m, 32H), 6.71 (d, J = 9 Hz, 2H), 7.66 (d,
J = 9 Hz, 2H), 9.68 (s, 1H). d4 (yield 72%): 1H NMR (400 MHz,
CDCl3): d (ppm) 3.09 (s, 3H), 3.36 (s, 3H), 3.52–3.68 (m, 16H),
6.73 (d, J = 8.8 Hz, 2H), 7.68 (d, J = 8.8 Hz, 2H), 9.70 (s, 1H).
2-[4-(Diethylamino)benzylidene]cyclopentanone (DEA)18. Cy-
clopentanone (8.41 g, 0.1 mol) and 4-(diethylamino)benzaldehyde
(5.32 g, 0.03 mol) were dissolved in a mixed solution of ethanol
(30 mL) and H2O (10 mL) together with 0.07 g NaOH as catalyst.
The reaction was performed in an ice-bath and N2 atmosphere for 2
h and an additional 1 h at room temperature, using TLC to monitor
the reaction progress. The obtained products were a mixture of
mono- and di-benzylidene cyclopentanones. The desired mono-
product was purified by silica gel column chromatography. Elution
of the column with ethyl acetate and petroleum ether mixture
(1 : 3) gave a red brown solid DEA (3.12 g, yield 42%). 1H NMR
(400 MHz, CDCl3): d (ppm) 1.19 (t, J = 7.2 Hz, 6H), 2.04 (m, 2H),
2.34 (t, 2H), 2.95 (t, 2H), 3.40 (q, J = 7.2 Hz, 4H), 6.69 (d, J = 8.5
Hz, 2H), 7.51 (d, J = 8.5 Hz, 3H).
Experimental
5,5-Dimethyl-1-pyrroline-N-oxide (DMPO), 2,2,6,6-tetramethyl-
4-piperidone (TEMP) and 9,10-dimethylanthracene (DMA)
were purchased from Sigma Aldrich Chemical Company.
Phosphate buffered saline (PBS) solution (pH = 7.4), Dul-
becco’s modified Eagle’s medium (DMEM), penicillin, strep-
tomycin, fetal bovine serum (FBS) and Mito-tracker green
were purchased from Beyotime Institute of Biotechnology. 4-
[N-methyl-N-(2-hycroxyethyl)amino]benzaldehyde and other ma-
terials were purchased from Beijing Chemical Co. Ltd. 4-
[N,N-bis(2-hydroxyethyl)amino]benzaldehyde, 1-(p-tolysulfonyl)-
3,6-dioxoheptane (c1) and 1-(p-tolysulfonyl)-3,6,9- trioxodecane
(c2) were synthesized according to the literature.26,27 Solvents used
for spectroscopy experiments were spectrophotometric grade.
UV-vis spectra were recorded on a Hitachi U-3900 spectropho-
tometer. Steady-state fluorescence was performed using a Hitachi
F-4500 spectrometer. Fluorescence quantum yields (Uf) were
measured in dilute solutions (6 ¥ 10-7 mol L-1) using Rhodamine B
in methanol as a standard (Uf = 0.7).28 The 1O2 phosphorescence
emission at 1270 nm was measured by a near infrared spectrometer
(NIR-512L-1.7T1) with a 532 nm Millennia laser as light source.
1H and 13C NMR spectra were obtained on a Bruker DPX 400
spectrometer. The high-resolution mass spectrometry (HRMS)
analyses were carried out on a Bruker apex_IV_FT mass spec-
trometer. Elemental analyses were performed on a Flash EA1112
elemental analyzer. The EPR signals were recorded on a Bruker
ESP-300E spectrometer. All measurements were carried out at
room temperature.
Target dye A1. DEA (2.43 g, 10 mmol) and d2 (2.81 g,
10 mmol) were dissolved in ethanol (20 mL) together with 0.06 g
NaOH as catalyst. The mixed solution was refluxed in N2 atmo-
sphere for 8 h, then cooled to room temperature, followed by rotary
evaporation to obtain the crude product. The desired dye A1 was
obtained by purification using silica gel column chromatography.
Elution of the column with methanol–chloroform (1 : 50) gave
deep red viscous liquid A1 (3.27 g, yield 65%). lmax (n-octanol)/nm
(log e) = 486 (4.80). 1H NMR (400 MHz, CDCl3): d (ppm) 1.21 (t,
J = 7.2 Hz, 6H), 3.05 (s, 3H), 3.07 (s, 4H), 3.37 (s, 3H), 3.43 (q,
J = 7.2 Hz, 4H), 3.52–3.67 (m, 12H), 6.69 (d, J = 8.5 Hz, 4H), 7.51
(d, J = 8.5 Hz, 6H). 13C NMR (400 MHz, CDCl3): d (ppm) 12.8,
26.8, 39.2, 44.6, 52.2, 59.2, 68.7, 70.8, 70.9, 71.0, 72.1, 111.3, 111.7,
123.6, 124.4, 132.8, 133.1, 133.4, 133.7, 133.8, 148.5, 149.7, 196.2.
HR-MS (ESI): m/z calc. for C31H43N2O4 [M + H]+ 507.32173;
found 507.32192. Anal. Calc. for C31H42N2O4: C, 73.49; H, 8.36;
N, 5.53. Found: C, 73.44; H, 8.39; N, 5.61%.
A2–B4 were obtained by the procedure described above. All
the prepared dyes are deep red viscous liquid. The yield, NMR
and HR-MS data are listed as follows: A2 (yield 72%): lmax (n-
octanol)/nm (log e) = 486 (4.79). 1H NMR (400 MHz, CDCl3): d
(ppm) 3.05 (s, 6H), 3.08 (s, 4H), 3.37 (s, 6H), 3.51–3.67 (m, 24H),
6.73 (d, J = 8.6 Hz, 4H), 7.51 (d, J = 8.6 Hz, 6H). 13C NMR
(400 MHz, CDCl3): d (ppm) 26.8, 39.0, 52.0, 59.0, 68.5, 70.6, 70.7,
70.8, 71.9, 111.1, 111.7, 124.1, 132.7, 133.4, 133.5, 149.6, 196.0.
HR-MS (ESI): m/z calc. for C35H51N2O7 [M + H]+ 611.36908;
4-[N,N-Bis(3,6,9-trioxodecyl)amino]benzaldehyde
To stirred solution of 4-[N,N-bis(2-hydroxyethyl)amino]-
(d1)29.
a
benzaldehyde (2.09 g, 10 mmol) in freshly distilled THF (30 mL),
KOH (2.7 g, 48 mmol) was added and the reaction mixture
was refluxed for 1 h. Then, a solution of 1-(p-tolysulfonyl)-3,6-
dioxoheptane (6.31 g, 23 mmol) in freshly distilled THF (20 mL)
was added dropwise over a period of 1 h. After the addition,
the reaction mixture was refluxed for 24 h. Excess solvent was
neutralized by 2 M H2SO4 and evaporated under reduced pressure
and the obtained residue was dissolved in water and extracted
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found 611.36926. Anal. Calc. for C35H50N2O7: C, 68.83; H, 8.25;
N, 4.59. Found: C, 68.91; H, 8.41; N, 4.79%. A3 (yield 69%): lmax
(n-octanol)/nm (log e) = 486 (4.67). 1H NMR (400 MHz, CDCl3):
d (ppm) 1.19 (t, J = 7.2 Hz, 6H), 3.05 (s, 4H), 3.37 (s, 6H), 3.43
(q, J = 7.2 Hz, 4H), 3.53–3.65 (m, 24H), 6.74 (d, J = 8.6 Hz,
4H), 7.52 (d, J = 8.6 Hz, 6H). 13C NMR (400 MHz, CDCl3):
d (ppm) 12.7, 26.7, 44.5, 51.0, 59.1, 68.5, 70.6, 70.7, 70.8, 72.0,
111.3, 111.7, 123.4, 124.3, 132.7, 132.9, 133.0, 133.2, 133.7, 133.8,
148.4, 148.5, 196.0. HR-MS (ESI): m/z calc. for C37H55N2O7 [M +
H]+ 639.40038; found 639.40072. Anal. Calc. for C37H54N2O7: C,
69.56; H, 8.52; N, 4.39. Found: C, 69.38; H, 8.68; N, 4.54%. A4
solubility. For experiments of determining the partition coefficient
(PC), photosensitizers were diluted to a concentration of 20 mM
in 2 mL PBS (PH = 7.4), and then 2 mL n-octanol was added. The
mixture of the two solvents was vigorously stirred and sonicated
for 2 min, then centrifuged at 4000 rpm for 10 min to separate
the phases. The photosensitizer concentrations in n-octanol phase
and PBS phase were determined by UV spectroscopy. Then
the final lipid-water PC values were calculated by the ratio of
photosensitizer concentration in n-octanol to that in PBS solution.
EPR experiments. Experimental details are as follows, mi-
crowave bridge: X-band with 100 Hz field modulation; sweep
width: 100 G; modulation amplitude: 1.0 G; modulation fre-
quency, 100 kHz; receiver gain: 1 ¥ 105; microwave power: 5 mW.
Samples were purged with oxygen for 15 min in the dark, and
then injected into the specially made quartz capillaries for EPR
analyses. According to the experimental requirement, samples
were illuminated directly in the cavity of the EPR spectrometer
with a Nd:YAG laser (532 nm, 5–6 ns of pulse width, repetition
frequency: 10 Hz, 10 mJ/pulse energy). The kinetics of spin adduct
generation were studied by recording peak heights of EPR spectra
every 40 s. DMPO and TEMP were used as superoxide radical and
singlet oxygen spin-trapping reagent, respectively. The solution
components were as follows: 100 mL dye (1 ¥ 10-4 M) plus 3 mL
pure TEMP or 20 mL 0.2 M DMPO. The solvents were DMSO
and toluene, respectively.
1
(yield 73%): lmax (n-octanol)/nm (log e) = 488 (4.79). H NMR
(400 MHz, CDCl3): d (ppm) 3.06 (s, 4H), 3.38 (s, 12H), 3.52–3.61
(m, 48H), 6.74 (d, J = 8.5 Hz, 4H), 7.49 (d, J = 8.5 Hz, 6H). 13
C
NMR (400 MHz, CDCl3): d (ppm) 26.7, 51.0, 59.2, 68.5, 70.7,
70.8, 70.9, 72.1, 111.7, 124.3, 132.8, 133.5, 133.6, 148.7, 196.1.
HR-MS (ESI): m/z calc. for C47H75N2O13 [M + H]+ 875.52636;
found 875.52309. Anal. Calc. for C47H74N2O13: C, 64.51; H, 8.52;
N, 3.20. Found: C, 63.92; H, 8.24; N, 3.05%. B1 (yield 72%): lmax
(n-octanol)/nm (log e) = 486 (4.79). 1H NMR (400 MHz, CDCl3):
d (ppm) 1.21 (t, J = 7.2 Hz, 6H), 3.05 (s, 3H), 3.07 (s, 4H), 3.37
(s, 3H), 3.41 (q, J = 7.2 Hz, 4H), 3.52–3.67 (m, 16H), 6.71 (d, J =
8.4 Hz, 4H), 7.51 (d, J = 8.4 Hz, 6H). 13C NMR (400 MHz, CDCl3):
d (ppm) 12.8, 26.8, 39.2, 44.6, 52.2, 59.2, 68.7, 70.8, 70.9, 71.0,
72.1, 111.3, 111.7, 123.6, 124.4, 132.8, 133.1, 133.4, 133.7, 133.8,
148.5, 149.7, 196.2. HR-MS (ESI): m/z calc. for C33H47N2O5 [M +
H]+ 551.34795; found 551.34846. Anal. Calc. for C33H46N2O5: C,
71.97; H, 8.42; N, 5.09. Found: C, 71.65; H, 8.26; N, 4.94%. B2
Singlet oxygen quantum yield. In the photochemical method,
9.10-dimethylanthracene (DMA) was used as 1O2 acceptor. A
532 nm diode laser was used as the light source. Dyes and DMA
were dissolved in n-octanol and saturated with oxygen in the
dark. The concentrations of all the photosensitizers were adjusted
to possess the same absorbance at 532 nm. Experiments were
performed by monitoring the bleaching of the absorption band of
DMA at 400 nm as a function of the irradiation time. The slope of
the curves obtained was an indication of the efficiency to generate
singlet oxygen (the larger the slope, the better the efficiency).
Additionally, three control experiments: 1, no irradiation; 2,
no photosensitizer; and 3, aerate with N2 to remove O2, were
performed, none of these experiments resulted in any absorbance
changes in the reaction mixture, which proved that the generation
of singlet oxygen was an integrative action with three components:
photosensitizer, light and oxygen, none is dispensable. In the 1O2
phosphorescence method, samples in n-octanol were prepared
with a concentration of 2 ¥ 10-4 M. A 532 nm diode laser was
used as the light source. A near infrared fiber spectrometer (NIR-
512L-1.7T1) was used as monitor. Rose Bengal (RB) in n-octanol
was used as a reference standard with UD = 1.
1
(yield 72%): lmax (n-octanol)/nm (log e) = 487 (4.68). H NMR
(400 MHz, CDCl3): d (ppm) 3.05 (s, 6H), 3.07 (s, 4H), 3.36 (s,
6H), 3.51–3.67 (m, 32H), 6.72 (d, J = 8.5 Hz, 4H), 7.49 (d, J = 8.5
Hz, 6H). 13C NMR (400 MHz, CDCl3): d (ppm) 26.8, 39.0, 52.0,
59.0, 68.5, 70.6, 70.7, 70.8, 71.9, 111.1, 111.7, 124.1, 132.7, 133.4,
133.5, 149.6, 196.0. HR-MS (ESI): m/z calc. for C39H59N2O9 [M +
H]+ 699.42151; found 699.42167. Anal. Calc. for C39H58N2O9: C,
67.02; H, 8.36; N, 4.01. Found: C, 66.73; H, 8.28; N, 4.06%. B3
1
(yield 74%): lmax (n-octanol)/nm (log e) = 487 (4.80). H NMR
(400 MHz, CDCl3): d (ppm) 1.20 (t, J = 7.3 Hz, 6H), 3.05 (s,
4H), 3.37 (s, 6H), 3.41 (q, J = 7.2 Hz, 4H), 3.53–3.65 (m, 32H),
6.71 (d, J = 8.5 Hz, 4H), 7.50 (d, J = 8.5 Hz, 6H). 13C NMR
(400 MHz, CDCl3): d (ppm) 12.7, 26.7, 44.5, 51.0, 59.1, 68.5, 70.6,
70.7, 70.8, 72.0, 111.3, 111.7, 123.4, 124.3, 132.7, 132.9, 133.0,
133.2, 133.7, 133.8, 148.4, 148.5, 196.0. HR-MS (ESI): m/z calc.
for C41H63N2O9 [M + H]+ 727.45281; found 727.45418. Anal. Calc.
for C41H62N2O9: C, 67.74; H, 8.60; N, 3.85. Found: C, 67.29; H,
8.43; N, 3.74%. B4 (yield 61%): lmax (n-octanol)/nm (log e) = 486
(4.70). 1H NMR (400 MHz, CDCl3): d (ppm) 3.06 (s, 4H), 3.38 (s,
12H), 3.52–3.61 (m, 64H), 6.74 (d, J = 8.7 Hz, 4H), 7.49 (d, J =
8.7 Hz, 6H). 13C NMR (400 MHz, CDCl3): d (ppm) 26.7, 51.0,
59.2, 68.5, 70.7, 70.8, 70.9, 72.1, 111.7, 124.3, 132.8, 133.5, 133.6,
148.7, 196.1. HR-MS (ESI): m/z calc. for C55H91N2O17 [M + H]+
1051.63122; found 1051.63098. Anal. Calc. for C55H90N2O17: C,
62.84; H, 8.63; N, 2.66. Found: C, 62.33; H, 8.51; N, 2.72%.
TPA characterization. TPA cross-section (s) values of com-
pounds in n-octanol (2 ¥ 10-4 M) were determined using the
two-photon excited fluorescence (TPEF) technique following the
experimental protocol described in detail by Xu and Webb.30 The
excitation light sources were a mode-locked Tsunami Ti : sapphire
laser (720–880 nm, 80 MHz, <130 fs). Two-photon excited
fluorescence spectra were recorded in a direction perpendicular to
the laser beam using a fiber spectrometer (Ocean Optics USB2000
CCD) as detector. Rhodamine B in methanol solution (10-4 M)
was used as reference. To avoid any contribution from other
photophysical or photochemical processes, the intensity of input
Solubility and lipid-water partition coefficient. To determine
the maximum solubility in PBS, absorption spectra for photo-
sensitizer dissolved in PBS solution in a series of concentrations
were measured. The top value at absorption band maximum in
the linear range of the plot was recognized to be the maximum
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pulses were adjusted to a appropriate regime to ensure a quadratic
dependence of the fluorescence intensity vs. excitation pulse energy
(shown in Fig. S1, ESI†).
Notes and references
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DMEM supplemented with 100 unit/ml penicillin, 100 mg ml-1
streptomycin and 10% FBS at 37 ◦C in a humidified atmosphere
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This work was supported by the National Natural Science
Foundation of China (No. 60978057). The authors are grateful to
Dr Hongyan Zhang for her expertise assistance with the confocal
fluorescence microscopy.
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The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 4168–4175 | 4175
©