Synthesis, characterization, photophysical and DNA photocleavage properties of amphiphilic porphyrins

Article abstract of DOI:10.1142/S1088424613501101

A series of amphiphilic porphyrins appended with pyridinium cation and/or polyethylene glycol have been synthesized and fully characterized by ¹H NMR, IR and MALDI-TOF-MS. Their photophysical properties were determined and the singlet oxygen (<

Full text of DOI:10.1142/S1088424613501101

Journal of Porphyrins and Phthalocyanines
J. Porphyrins Phthalocyanines 2014; 18: 221–230
DOI: 10.1142/S1088424613501101
Published at http://www.worldscinet.com/jpp/
Synthesis, characterization, photophysical and DNA
photocleavage properties of amphiphilic porphyrins
Jie Zhang*a◊, Mingyu Tengb and Dan Lic
a School of Pharmaceutical and Chemical Engineering, Taizhou University, Taizhou 318000, P. R. China
b Faculty of Chemistry and Chemical Engineering, Yunnan Normal University, Kunming 650500, P. R. China
c Department of Scientific Research, Taizhou University, Linhai 317000, P. R. China
Received 26 June 2013
Accepted 30 September 2013
ABSTRACT: A series of amphiphilic porphyrins appended with pyridinium cation and/or polyethylene
1
glycol have been synthesized and fully characterized by H NMR, IR and MALDI-TOF-MS. Their
photophysical properties were determined and the singlet oxygen (1O2) quantum yields of these
compounds are in the range of 0.30–0.61 in CHCl3 and 0.048–0.18 in Tris buffer. The measured two-
photon absorption (TPA) cross-sections s(2) of these porphyrin derivatives are between 110 and 240
GM. These amphiphilic porphyrins show some photocleavage activities (10%–21%) towards the anionic
DNA observed at 100 mM.
KEYWORDS: singlet oxygen, two-photon absorption, DNA photocleavage, amphiphilic porphyrins.
[2a, 2c, 3]. There are also other important requirements
INTRODUCTION
such as low dark toxicity and good pharmacokinetics.
Photodynamic therapy (PDT) has recently emerged
Recently, a new approach to photogenerate singlet
as an inherently selective cancer treatment modality
rendered by precisely targeted photodamage of the
oxygen (1O2) via two photon excitation of a sensitizer
has been reported [4]. Two photon absorption (TPA) is
cancerous tissues. In PDT, non-toxic photosensitizer
an optical nonlinear process that involving simultaneous
preferentially accumulates inside the tumor cells and
generates cytotoxic reactive oxygen species (e.g. singlet
oxygen) only when photo-activated via absorption at
specific wavelengths [1].
absorption of two photons, in which the energy of a
photon is normally unable to induce excitation. It can be
accomplished by laser light with l ≥ 800 nm via TPA,
therefore allowing much better depth penetration through
The majority of photosensitizers are tetrapyrroles
tissues. Porphyrin-based materials show a strong linear
or porphyrinoids, and these drugs cause cell death by
absorption at about 400–500 nm. Thus, its TPA should be
transferring energy to oxygen, producing the highly
around 800–1000 nm [5]. It is widely accepted that the
1
reactive singlet excited state of molecular oxygen, O2
optimum excitation wavelength for PDT agents should
[2]. An efficient PDT photosensitizer requires a favorable
be within this range to increase the penetration of tissue
combination of the following factors: (1) light absorption
and minimize the absorption from water. Therefore,
at wavelengths which penetrate effectively into biological
porphyrin-based materials are the prior option in TPA-
tissue (700–950 nm), (2) a high quantum yield for singlet
PDT. However, the problem is that the TPA cross-section
oxygen generation (FD), (3) efficient uptake into the
of commercial available porphyrin-base materials is very
diseased tissues, and (4) localisation in regions of the
low. Therefore, many efforts have been focused on the
cell which are vulnerable to singlet oxygen damage
development of new porphyrin-based materials with high
TPA cross-section s(2) values [6].
◊SPP full member in good standing
In this study, we aim to develop porphyrin-based
photosensitizers that possess the above mentioned
properties optimized for PDT applications and excite
*Correspondence to: Jie Zhang, email: zhang_jie@tzc.edu.cn,
tel: +86 576-8513-7265
Copyright © 2014 World Scientific Publishing Company

222
J. ZHANG ET AL.
via TPA. To accomplish this goal, an amphiphilic
porphyrin with an appropriate hydrophobic/hydrophilic
balance was designed with the H2TPP (Scheme 1) or
H2TPEGPP (Scheme 2) and its cationic pyridinium group,
respectively, as the hydrophobic and the hydrophilic
ends. Herein, we report the synthesis, characterization,
photophysical and DNA photocleavage activities of
these amphiphilic porphyrin derivatives with pyridinium
cations and polyethylene glycol.
of 276 GW cm-2 from an optical parametric amplifier
operating at a repetition rate of 1 kHz generated from
a Ti:sapphire regenerative amplifier system [8]. The
laser beam was split into two parts by a beam splitter.
One was monitored by a photodiode (D1) as the incident
intensity reference, I0, and the other was detected as the
transmitted intensity by another photodiode (D2). After
passing through a lens with f = 20 cm, the laser beam was
focused and passed through a quartz cell. The position of
the sample cell, z, was moved along the direction of the
laser beam (z axis) by a computer-controlled translatable
table so that the local power density within the sample cell
could be changed under the constant incident intensity
laser power level. Finally, the transmitted intensity from
the sample cell was detected by the photodiode D2. The
photodiode D2 was interfaced to a computer for signal
acquisition and averaging. Each transmitted intensity
datum represents the average of over 100 measurements.
Assuming a Gaussian beam profile, the nonlinear
absorption coefficient, b, can be obtained by curve-fitting
totheobservedopen-aperturetraces,T(z), withEquation1
[9], where a0 is the linear absorption coefficient, l is
the sample length (the 1 mm quartz cell) and z0 is the
diffraction length of the incident beam. After obtaining
the nonlinear absorption coefficient, b, the TPA cross-
section, s(2), of the sample molecule (in units of 1 GM =
10–50 cm4.s.photon-1) can be determined by using
Equation 2, where NA is Avogadro’s constant, d is the
concentration of the sample compound in solution, h is
Planck’s constant and v is the frequency of the incident
laser beam.
EXPERIMENTAL
Materials and methods
The chemicals were purchased from Sigma-Aldrich
Company and used as received. Silica gel 60 (0.04–
0.063 mm) for column chromatography was purchased
from Merck. NMR spectra were recorded on a Bruker
Ultrashield 400 Plus NMR spectrometer. High-resolution
matrix-assisted laser desorption/ionization time-of-flight
(MALDI-TOF) mass spectra were recorded with a
Bruker Autoflex MALDI-TOF mass spectrometer. All
solution spectroscopic measurements were prepared in
spectrophotometric grade CHCl3 and DMSO (Aldrich)
and degassed with freeze-pump-thaw cycles (three
times). Electronic absorption spectra in the UV-vis
region were recorded with a Varian Cary 100 UV-vis
spectrophotometer. The IR spectra (KBr pellets) were
recorded with a Nicolet Magna 550 FTIR spectrometer.
Steady-state visible fluorescence, PL excitation spectra,
singlet oxygen and near-IR (NIR) emission spectra were
recorded on a Photon Technology International (PTI)
Alphascanspectrofluorimeterandvisibledecayspectraon
a pico-N2 laser system (PTI Time Master) with lexc = 337
nm. Quantum yields of visible emissions were computed
according to the literature method using H2TPP as the
reference standard [7]. The absorbance of the solutions
for fluorescence quantum yield measurements was less
than 0.02 at the excitation wavelength, so that attenuation
of the excitation beam in the sample was small. It was
computed by using the expression Fs = Fr(Br/Bs)(ns/
nr)2(Ds/Dr), where the subscripts r and s denote reference
standard and the sample solution, respectively, and n,
D, and F are the refractive index of the solvents, the
integrated intensity, and the luminescence quantum yield,
respectively. The quantity B was calculated by using
B = 1–10-A, where A is the absorbance at the excitation
wavelength. All measurements were performed at
ambient temperature (20 2 °C) under atmospheric
pressure.
-a0l
bI (1 - e
)
2
0
(1)
T(z) = 1-
2a [1 + z/z )]
0
o
1000bhv
2
(2)
s
=
N d
A
DNA photocleavage assay (plasmid DNA
relaxation assay)
The DNA photocleavage activities of the test samples
were measured using the plasmid DNA relaxation
assay. Briefly, the plasmid DNA (pBluescript), enriched
with the covalently-closed circular conformer, and the
one-phor-all plus buffer (10 mM Tris-acetate, 10 mM
magnesium acetate, 50 mM potassium acetate, pH 7.5)
was vortexed. Aliquots of the DNA were pipetted into
different Eppendorf tubes. Various amounts of autoclaved
water (control sample) or test samples were added into
the Eppendorf tubes to give a final volume 20 L in each
sample tube. The sample mixtures were then photo-
irradiated either at 400–500 nm for 30 or 45 min using
a transilluminator (Vilber Lourmat) containing 4 × 15 W
light tubes (Aqua Lux) with maximum emission at 455 nm
or at > 605 nm using a 50 W halogen lamp equipped
with an optical filter which cutoff all wavelengths shorter
Two-photon absorption measurements
The two-photon-absorption spectra (i.e. Z-scan traces)
were measured at 800 nm by the open-aperture Z-scan
method using 100 fs laser pulses with a peak power
Copyright © 2014 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2014; 18: 222–230

SYNTHESIS, CHARACTERIZATION, PHOTOPHYSICAL AND DNA PHOTOCLEAVAGE PROPERTIES
223
than 605 nm. After photo-irradiation, 3 mL of the 6X
4.24 (t, J = 6.4 Hz, 2H), 3.51 (t, J = 6.4 Hz, 2H),
32.02–2.00 (m, 4H), 1.68–1.66 (m, 4H) and -2.77 (s,
2H). HRMS (MALDI-TOF, positive mode, CHCl3): m/z
794.2469 (calcd. for [M]+ 794.2449). IR (KBr): n, cm-1
3314m, 3052w, 2927m, 1596m, 1466s, 1245s, 1175s,
965s, 780vs and 701s. UV-vis (CHCl3, 20 °C): lmax, nm
(log e, M-1.cm-1) 418 (5.63), 516 (4.18), 551 (3.81), 591
(3.54) and 647 (3.57).
sample dye solution (which contained 20% glycerol,
0.25% bromophenol blue and 0.25% xylene cyanol FF)
was added to each Eppendorf tube and mixed well by
centrifugation. The sample mixtures were loaded onto
a 0.8% (v/v) agarose gel (dimension: 13 cm × 10 cm),
with 1X Tris-borate-EDTA buffer (89 mM Tris-borate,
2.5 mM EDTA, pH 8) used as supporting electrolyte,
and electrophoresized at 1.3 V.cm-1 for 3 h using mini
gel set (CBS Scientific Co., Model MGU-502T). After
electrophoresis, the gel was stained with 0.5 g/mL
ethidium bromide solution for 10 min and then destained
with deionized water for 3 min. The resulting gel image
was viewed under 365 nm light and captured digitally
using a gel documentation system (BioRad).
TPP-OC6-Py-OH. 4-hydroxyl pyridine (120 mg,
1.26 mmol) was added to a suspension of TPP-OC6Br
(100 mg, 0.13 mmol) and anhydrous K2CO3 in dry DMF
(10 mL). The mixture was stirred at room temperature,
and the progress of the reaction was monitored by TLC.
After 24 h, the mixture was evaporated to dryness under
vacuum. The residue was redissolved in a CHCl3 and
chromatographed on a silica gel column using CHCl3/
CH3OH as eluent. The third band gave the product TPP-
OC6-Py-OH. Yield 73 mg (71. 8%). 1H NMR (400 MHz;
CDCl3; Me4Si): dH, ppm 8.88–8.87 (m, 8H), 8.85–8.21
(m, 6H), 8.11 (d, J = 8.4 Hz, 2H), 7.78–7.75 (m, 9H),
7.29–7.24 (m, 4H), 6.43 (d, J = 6.4, 2H), 4.22 (t, J = 6.4
Hz, 2H), 3.78 (t, J = 7.2 Hz, 2H), 1.97–1.92 (m, 2H),
1.89–1.82 (m, 2H), 1.66–1.62 (m, 2H), 1.50–1.44 (m,
2H) and -2.78 (s, 2H). HRMS (MALDI-TOF, positive
mode, CHCl3): m/z 808.3604 (calcd. for [M]+ 808.9855).
IR (KBr): n, cm-1 3430m, 3313m, 2934m, 1636m, 1578s,
1468s, 1243s, 1182s, 965s, 799s and 747vs. UV-vis
(CHCl3, 20 °C): lmax, nm (log e, M-1.cm-1) 418 (5.66),
515 (4.27), 551 (4.00), 590 (3.84) and 647 (3.85).
ZnTPP-OC6Br. The complex was prepared by
refluxing TPP-OC6Br (570 mg, 0.72 mmol) with an
excess amount of zinc(II) acetate in CHCl3/methanol for
5 h; it was then purified by column chromatography on
silica gel with CHCl3 as eluent. Yield 520 mg, 84.6%.1H
NMR (CDCl3): dH, ppm 8.99–8.94 (m, 8H), 8.23–8.21
(m, 6H), 8.10 (d, J = 8.4, 2H), 7.77–7.71 (m, 9H), 7.24–
7.23 (m, 2H), 4.21 (m, 2H), 3.49 (t, J = 6.4, 2H), 2.00–
1.97 (m, 4H) and 1.65–1.63 (m, 4H). HRMS (MALDI-
TOF, positive mode, CHCl3): m/z 856.1600 (calcd. for
[M]+ 856.1577). IR (KBr): n, cm-1 3434m, 3049m, 2929s,
1596s, 1486s, 1248vs, 1174s, 1003vs, 798vs and 701s.
UV-vis (CHCl3, 20 °C): lmax, nm (log e, M-1.cm-1) 419
(5.78), 547 (4.28) and 585 (3.11).
Preparations of porphyrin derivatives
with pyridinium cations and polyethylene glycol
p-OH-TPP. A solution of benzaldehyde (18.29 mL,
180 mmol) and p-hydroxybenzaldehyde (7.32 g, 60 mmol)
in propionic acid (700 mL) was heated to 130 °C. Then
freshly distilled pyrrole (16.65 mL, 240 mmol) in pro-
pionic acid (50 mL) was added slowly to the solution over
a period of 0.5 h.The reaction mixture was heated to reflux
for another 0.5 h and then cooled to rt. Then the volume
of the reaction mixture was reduced to about half of its
original volume under reduced pressure, and methanol
(300 mL) was added to the concentrated solution. The
resultant solution was kept in a refrigerator overnight to
give dark purple solid, which was filtered, redissolved in
a minimum amount of CHCl3 and chromatographed on a
silica gel column with CH2Cl2 as eluent. The second band
gave the desired p-OH-TPP. Yield 1.8 g (4.8%). 1H NMR
(400 MHz; CDCl3; Me4Si): dH, ppm 8.86–8.84 (m, 8H),
8.23–8.20 (m, 6H), 8.06 (d, J = 8.4 Hz, 2H), 7.77–7.74
(m, 9H), 7.17 (d, J = 8.8 Hz, 2H), 5.07 (s, 1H) and -2.78
(s, 2H). HRMS (MALDI-TOF, +ve mode, CHCl3): m/z
630.2455 (calcd. for [M]+ 630.2414). IR (KBr): n, cm-1
3502w, 3314w, 3053w, 1662m, 1594m, 1472s, 1170m,
965s, 798s and 699s.
TPP-OC6Br. 1,6-dibromohexane (1.23 mL, 8.0 mmol)
was added to a suspension of p-OH-TPP (500 mg,
0.80 mmol) and anhydrous K2CO3 in dry DMF (20 mL).
The mixture was stirred at rt, and the progress of the
reaction was monitored by TLC. After 24 h, CH2Cl2
(20 mL) was added to the reaction mixture, which was
then washed with water (6 × 20 mL) until all of the DMF
and K2CO3 were removed. The organic phase was dried
with Na2SO4, filtered and the solvents were evaporated to
dryness under vacuum. The residue was redissolved in a
CHCl3/hexane mixture (1:1, v/v) and chromatographed
on a silica gel column using CHCl3/hexane (5:1, v/v) as
eluent. The first band gave the product TPP-OC6Br. Yield
ZnTPP-OC6-Py-OH. 4-hydroxyl pyridine (240 mg,
2.53 mmol) was added to a suspension of ZnTPP-OC6Br
(200 mg, 0.23 mmol) and anhydrous K2CO3 in dry DMF
(10 mL). The mixture was stirred at room temp, and the
progress of the reaction was monitored by TLC. After
24 h, the mixture was evaporated to dryness in vacuo. The
residue was redissolved in a CHCl3 and chromatographed
on a silica gel column using CHCl3/CH3OH as eluent.
The third band gave the product TPP-OC6-Py-OH. Yield
1
120 mg (59.1%). H NMR (400 MHz; CDCl3; Me4Si):
dH, ppm 8.84 (m, 8H), 8.83–8.82 (m, 6H), 7.99 (d, J =
8.4 Hz, 2H), 7.72–7.65 (m, 9H), 7.08 (d, J = 8.4 Hz, 2H),
6.28 (d, J = 7.6 Hz, 2H), 4.04 (d, J = 6.8, 2H), 3.91 (t, J =
6.4, 2H), 2.91 (t, J = 6.8, 2H), 1.56 (m, 2H), 1.19–1.14
1
544 mg (86.3%). H NMR (400 MHz; CDCl3; Me4Si):
dH, ppm 8.89–8.88 (m, 8H), 8.22–8.21 (m, 6H), 8.11 (d,
J = 8.8 Hz, 2H), 7.78–7.73 (m, 9H), 7.28–7.26 (m, 2H),
Copyright © 2014 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2014; 18: 223–230

224
J. ZHANG ET AL.
(m, 4H) and 0.83–0.81 (m, 2H). HRMS (MALDI-TOF,
positive mode, CHCl3): m/z 870.2807 (calcd. for [M]+
870.2780). IR (KBr): n, cm-1 3434m, 2926m, 1635vs,
1560vs, 1486s, 1247s, 1186s, 992s, 797s and 751s.
UV-vis (CHCl3, 20 °C): lmax, nm (log e, M-1.cm-1) 420
(5.73), 549 (4.24) and 589 (3.23).
3.75–3.74 (m, 10H), 3.64–3.62 (m, 6H), 3.43 (s, 9H),
3.32–3.29 (m, 2H) and -2.78 (s, 2H). HRMS (MALDI-
TOF, positive mode, CHCl3): m/z 1403.5291 (calcd. for
[M + H]+ 1403.5234). IR (KBr): n, cm-1 3318m, 2876m,
1607s, 1508s, 1472s, 1247vs, 1176s, 967s, 804s and
740m. UV-vis (CHCl3, 20 °C): lmax, nm (log e, M-1.cm-1)
421 (5.35), 519 (3.11), 555 (1.86) and 651 (2.37).
p-OHTPEGPP. A solution of 4-[tri(ethylene glycol)
monomethyl ether] benzaldehyde (13 g, 0.048 mol) and
p-hydroxybenzaldehyde (1.97 g, 0.016 mol) in propionic
acid (300 mL) was heated to 130 °C. Then freshly
distilled pyrrole (4.47 mL, 0.064 mol) in propionic acid
(50 mL) was added slowly to the solution over a period
of 0.5 h. The reaction mixture was heated to reflux for
another 0.5 h and then cooled to rt. Then the volume of
the reaction mixture was reduced to about 20 mL under
reduced pressure, and ethanol (300 mL) was added to
the concentrated solution. The resultant solution was
kept in a refrigerator overnight to give purple solid,
which was filtered, redissolved in a minimum amount
of CHCl3 and chromatographed on a silica gel column
with CH2Cl2/CH3OH as eluent. The first band gave the
5,10,15,20-tetraPEGphenylporphyrin. The second band
gave the desired p-OHTPEGPP, Yield 600 mg (3.4%).
1H NMR (400 MHz; CDCl3; Me4Si): dH, ppm 8.83–8.77
(m, 8H), 8.03–7.94 (m, 8H), 7.19–7.11 (m, 8H), 5.70 (s,
1H), 4.34–4.26 (m, 6H), 4.02–3.98 (m, 6H), 3.85–3.72
(m, 18H), 3.61–3.60 (m, 6H), 3.41 (s, 9H) and -2.81 (s,
2H). HRMS (MALDI-TOF, positive mode, CHCl3): m/z
1117.5109 (calcd. for [M + H]+ 1117.5168). IR (KBr): n,
cm-1 3320m, 2923m, 1607s, 1508s, 1472s, 1247s, 1176s,
967s, 805s and 741vs. UV-vis (CHCl3, 20 °C): lmax, nm
(log e, M-1.cm-1) 421 (5.35), 519 (3.50), 553 (3.19) and
651 (3.15).
TPEGPP-PEG-Py-OH. 4-hydroxylpyridine (21 mg,
2.2 mmol) was added to a suspension of TPEGPP-PEG-I
(32 mg, 0.022 mmol) and anhydrous K2CO3 in dry DMF
(3 mL). The mixture was stirred at 60 °C, and the progress
of the reaction was monitored by TLC. After 24 h, the
mixture was evaporated to dryness in vacuo. The residue
was redissolved in a CHCl3 and chromatographed on a
silica gel column using CHCl3/CH3OH as eluent. The
third band gave the product TPEGPP-PEG-Py-OH. Yield
17 mg (54.8%). 1H NMR (400 MHz; CDCl3; Me4Si): dH,
ppm 8.90 (s, 8H), 8.10 (d, J = 8.4 Hz, 8H), 7.33–7.26 (m,
10H), 6.37 (d, J = 7.6, 2H), 4.43–4.40 (m, 8H), 4.06–
4.04 (m, 8H), 3.88–3.79 (m, 16H), 3.74 (m, 10H), 3.63–
3.62 (m, 10H), 3.42 (s, 9H) and -2.72 (s, 2H). HRMS
(MALDI-TOF, positive mode, CHCl3): m/z 1370.6454
(calcd. for [M]+ 1370.6488). IR (KBr): n, cm-1 3316m,
2922m, 1639s, 1503s, 1454s, 1246vs, 1176s, 966s, 803s
and 741m. UV-vis (CHCl3, 20 °C): lmax, nm (log e, M-1.
cm-1) 421 (5.28), 518 (3.55), 555 (3.33), 591 (2.56) and
650 (2.88).
RESULT AND DISCUSSION
Synthesis of amphiphilic porphyrins
TPEGPP. Yield 505 mg (2.5%). 1H NMR (400 MHz;
CDCl3; Me4Si): dH, ppm 8.85 (s, 8H), 8.10 (d, J = 8.8
Hz, 8H), 7.30–7.25 (m, 8H), 4.43–4.40 (m, 8H), 4.06–
4.03 (m, 8H), 3.89–3.87 (m, 8H), 3.80–3.78 (m, 8H),
3.75–3.73 (m, 8H), 3.63–3.61 (m, 8H), 3.42 (s, 12H)
and -2.77 (s, 2H). HRMS (MALDI-TOF, positive mode,
CHCl3): m/z 1263.6148 (calcd. for [M + H]+ 1263.6112).
IR (KBr): n, cm-1 3320m, 2925s, 1606s, 1502s, 1455s,
1246s, 1177s, 967s, 803s and 741vs. UV-vis (CHCl3,
20 °C): lmax, nm (log e, M-1.cm-1) 421 (5.51), 519 (3.78),
555 (3.54), 594 (1.27) and 652 (3.33).
TPEGPP-PEG-I. 1-iodo-2-(2-(2-(2-iodoethoxy)-
ethoxy)ethoxy)ethane (444 mg, 1.074 mmol) was added
to a suspension of p-OHTPEGPP (60 mg, 0.0537 mmol)
and anhydrous K2CO3 in DMF (5 mL). The mixture was
stirred at room temp, and the progress of the reaction was
monitored byTLC.After 24 h, the mixture was evaporated
to dryness in vacuo. The residue was redissolved in
CHCl3 and chromatographed on a silica gel column
using CHCl3/CH3OH as eluent. The first band gave the
product TPEGPP-PEG-I. Yield 50 mg (66.7%). 1H NMR
(400 MHz; CDCl3; Me4Si): dH, ppm 8.85 (s, 8H), 8.11
(d, J = 8.4 Hz, 8H), 7.30–7.29 (m, 8H), 4.44 (m, 8H),
4.07 (m, 8H), 3.89–3.88 (m, 8H), 3.81–3.79 (m, 10H),
The synthetic routes for amphiphilic porphyrins
TPP-OC6-Py-OH and ZnTPP-OC6-Py-OH are shown in
Scheme 1. Carbon chain was attached to p-OHTPPH2 by
nucleophilic substitution reaction between phenol group
of the porphyrin and bromo-group of 1,6-dibromohexane
in the presence of a base (K2CO3) using DMF as the
solvent. The product TPP-OC6Br could be easily purified
by column chromatography using a silica gel column
and the yield was about 86%. TPP-OC6Br was then
further reacted with 4-hydroxyl pyridine in DMF to
give TPP-OC6-Py-OH in about 72% yield. Reaction of
TPP-OC6Br with an excess amount of zinc(II) acetate in
CHCl3/methanol give the product ZnTPP-OC6Br in about
85% yield. ZnTPP-OC6Br was then further reacted with
4-hydroxyl pyridine in DMF to give ZnTPP-OC6-Py-OH
in about 59% yield. The advantage of this synthetic route
is that the carbon length can be adjusted.
The synthetic routes for the preparation of amphiphilic
porphyrin TPEGPP-PEG-Py-OH are shown in Scheme 2.
Reaction of polyethylene glycol (PEGOH) with meth-
anesulfonyl chloride gave the product SO2-PEG in good
yield. Polyethylene glycol substituted aldehyde was
synthesized by a nucleophilic reaction between 4-hydroxy-
benzaldehyde and SO2-PEG. Then, polyethylene glycol
Copyright © 2014 World Scientific Publishing Company
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SYNTHESIS, CHARACTERIZATION, PHOTOPHYSICAL AND DNA PHOTOCLEAVAGE PROPERTIES
225
CHO
CHO
OH
NH
N
N
NH
N
N
a
b
OH
O
+
+
Br
N
HN
HN
H
TPP-OC6Br
d
p-OH-TPP
c
N
N
N
N
Zn
O
Br
NH
N
N
O
N
HN
e
OH
ZnTPP-OC6Br
TPP-OC6-Py-OH
N
N
N
N
Zn
O
N
OH
ZnTPP-OC6-Py-OH
Scheme 1. Synthetic route for ZnTPP-OC6-PyOH. (a) propionic acid, reflux. (b) 1,6-dibromohexane, K2CO3, DMF, rt. (c) 4-hydroxyl
pyridine, DMF, rt. (d) zinc(II) acetate, CHCl3/methanol, reflux. (e) 4-hydroxyl pyridine, DMF, rt
O
O
O
O
CHO
O
NH
N
N
a
b
c
O
S
O
O
O
HO
O
O
O
OH
O
O
O
O
O
O
HN
O
O
O
SO2-PEG
PEG-aldehyde
O
O
p-OHTPEGPP
O
O
d
O
O
O
O
O
O
O
O
O
O
O
O
NH
N
N
NH
N
NH
N
N
N
e
O
O
O
O
O
O
O
O
O
+
O
O
O
O
O
O
HN
HN
HN
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
TPEGPP-PEG-Py-OH
TPEGPP-PEG-I
TPEGPP
I
N
HO
Scheme2.SyntheticroutesforTPEGPP-PEG-Py-OH.(a)sulfonylchloride,diisopropylethylamine,0°C,(b)3-hydroxybenzaldehyde,
K2CO3, THF, 70 °C, (c) 4-hydroxybenzaldehyde, pyrrole, propionic acid, reflux, (d) 1-iodo-2-(2-(2-(2-iodoethoxy) ethoxy) ethoxy)
ethane, K2CO3, DMF, rt, and (e) 4-Hydroxyl pyridine, DMF, 60 °C
Copyright © 2014 World Scientific Publishing Company
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226
J. ZHANG ET AL.
substituted aldehyde and p-hydroxybenzaldehyde
was refluxed in propionic acid to give the products
p-OHTPEGPP (3.4%) and TPEGPP (2.5%). PEG-I chain
was attached to p-OHTPEGPP and TPEGPP-PEG-I could
be easily purified by column chromatography with the
yield of 67%. TPEGPP-PEG-I was then further reacted
with 4-hydroxyl pyridine in DMF to give the target product
(TPEGPP-PEG-Py-OH) in about 55% yield.
Absorptions and emissions of amphiphilic
porphyrins
The UV-visible absorptions of these amphiphilic
porphyrins retain the characteristics features of H2TPP
and ZnTPP [10]. For example, TPP-OC6-Py-OH shows
a strong Soret band at 418 nm and four Q-bands at 515,
551, 590 and 647 nm. In contrast, Zn(II) complex ZnTPP-
OC6-Py-OH exhibits a similar Soret band at 420 nm
and two Q-bands at 549 and 589 nm. The absorption
spectra and data of these porphyrins are show in Fig. 1
and summarized in experimental part, respectively. The
fluorescence spectra of these amphiphilic porphyrins
also retain the characteristics features of H2TPP and
ZnTPP [10]. The metal-free products emitted at about
650 and 720 nm, whereas the zinc containing products
at about 600 and 650 nm. When monitoring the emission
wavelength at 650 nm, the excitation spectrum exhibits
an intense excitation band around 420 nm, which
corresponds to the ground-state absorption spectrum. In
any case, the visible emissions in the range of 597–727
nm of all of the porphyrins show quite fast decay time
(i.e. shorter than 1 ms). The fluorescence quantum yields
of these porphyrins are in the range of 0.029–0.13. In
Tris buffer, a decrease in quantum yields of fluorescence
emission is depicted for all the amphiphilic porphyrins
(Table 1). The fluorescence spectra these amphiphilic
porphyrins are shown in Fig. 2, and the photophysical
Fig. 2. Excitation and emission spectra of TPP-O-C6-Py-OH
(top) and ZnTPP-O-C6-Py-OH (middle) and TPEGPP-PEG-
Py-OH (bottom) in CHCl3 (2 × 10-6 M)
data are summarized in Table 1. The results show that the
addition of pyridinium and polyethylene glycol moiety
has only slight effect on both the absorption and emission
of the porphyrin moiety [11].
DNA binding constant experiments
We carried out the EB (ethidium bromide) competitive
binding with DNA experiments and the florescence
quenching plot are given in Fig. 3 [11b]. I0 and I are the
fluorescence intensities in the absence and presence of
the porphyrins, respectively. The I0/I vs. [Por]/[DNA]
plot is good agreement with the linear Stern–Volmer
equation with a slope of 0.69, 1.00, 2.40 and 5.09 for
TPP-OC6-Py-OH, ZnTPP-OC6-Py-OH, p-OHTPEGPP
and TPEGPP-PEG-Py-OH, respectively. We can also
Fig. 1. Absorption spectra of the products in CHCl3 (2 × 10-6 M)
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SYNTHESIS, CHARACTERIZATION, PHOTOPHYSICAL AND DNA PHOTOCLEAVAGE PROPERTIES
227
Table 1. Summary of fluorescence data in CHCl3 (2 × 10-6 M)
Compound
Excitation, nm
Emission, nm
Ffa
tfc
TPP-OC6Br
420, 516, 550, 591
420, 549
654, 720
597, 648
654, 719
597, 646
658, 723
657, 724
659, 723
658, 724
0.094
0.029
0.096 (0.019)b
0.037 (0.008)b
0.130
14.96 ns
1.99 ns
4.14 ns
2.13 ns
3.81 ns
10.07 ns
9.32 ns
10.61 ns
ZnTPP-OC6Br
TPP-OC6-Py-OH
ZnTPP-OC6-Py-OH
TPEGPP
420, 516, 551, 591
421, 549
422, 518, 555, 593
422, 523, 554, 594
422, 520, 556, 594
422, 520, 556, 595
p-OHTPEGPP
TPEGPP-PEG-I
TPEGPP-PEG-Py-OH
0.098 (0.012)b
0.096
0.098 (0.020)b
a The fluorescence quantum yields Ff were estimated in the benzene, using the integrated emission
intensity of 5,10,15,20-tetraphenyl porphyrin (H2TPP, Ff = 0.11 in benzene) as reference [7]
(under identical photoirradiation conditions (lexc = 420 nm, abs(lexc) = 0.02)). b The fluorescence
quantum yields Ff were estimated in the Tris buffer. c The fluorescene life is measured at excited
wavelength = 337 nm in benzene.
Fig. 3. Florescence quenching curves of DNA-bound EB by
amphiphilic porphyrins in Tris buffer (pH = 7.2, 0.05 M NaCl)
([DNA] = 100 mM, [EB] = 16.0 mM, lexc = 537 nm)
Fig. 4. Near-IR phosphorescence spectra of singlet oxygen
1O2 produced from the products in CHCl3 under identical
photoirradiation conditions (lexc = 420 nm, abs(lexc) = 0.02)
learn from Fig. 3 that 50% of EB molecules were
replaced from DNA-bound EB at a concentration ratio of
[Por]/[EB] = 9.7, 6.3, 2.9 and 1.3 for TPP-OC6-Py-OH,
ZnTPP-OC6-Py-OH, p-OHTPEGPP and TPEGPP-PEG-
Py-OH, respectively. The Kapp of EB in the experimental
condition is 1.0 × 107 M-1 [12], therefore, the Kapp of
TPP-OC6-Py-OH, ZnTPP-OC6-Py-OH, p-OHTPEGPP
and TPEGPP-PEG-Py-OH were 1.03 × 106 M-1 and
1.59 × 106 M-1, 3.49 × 106 M-1 and 7.69 × 106 M-1,
respectively [13].
Table 2. Summary of singlet oxygen quantum yields
Compound
FDa
FDb
TPP-OC6Br
0.61
0.53
0.46
0.36
0.36
0.33
0.33
0.30
ZnTPP-OC6Br
TPP-OC6-Py-OH
ZnTPP-OC6-Py-OH
TPEGPP
0.064
0.048
p-OHTPEGPP
TPEGPP-PEG-I
TPEGPP-PEG-Py-OH
0.18
Determination of singlet oxygen(1O2) quantum yield
0.094
The production of singlet oxygen (1O2) can be
measured by its phosphorescence at 1270 nm and its
quantum yield can be determined by comparing with
H2TPP (FD = 0.55). Figure 4 shows the phospho-
rescence spectra of singlet oxygen (1O2) generated
a The singlet oxygen quantum yields of these
compounds were measured in CHCl3 relative to
H2TPP (FD = 0.55). The experimental uncertainty
was ca. 20%. b Singlet oxygen quantum yields
estimated in Tris buffer.
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228
J. ZHANG ET AL.
by the photo-irradiation of the samples in CHCl3. In
general, the singlet oxygen (1O2) quantum yields of
these porphyrins without polyethylene glycol moiety
are slightly greater than those with polyethylene glycol
moiety in CHCl3. However, a decrease in singlet
oxygen (1O2) quantum yields (0.048–0.18) for all the
amphiphilic porphyrins in Tris buffer (Table 2). Among
them, p-OHTPEGPP and TPEGPP-PEG-Py-OH showed
better result, which indicates nonpegylated porphyrins
much more tend to be aggregated in buffer solutions
[14]. The relative good singlet oxygen (1O2) quantum
yields indicate these amphiphilic porphyrins can be
applied in PDT application.
Fig. 5. Open-aperture Z-scan traces of products excited at
800 nm in DMSO (1 × 10-3 M)
Two photon absorption of these porphyrins
The TPA cross-sections s(2) of these porphyrins
were measured at 800 nm using 100 fs laser pulses
with the open-aperture Z-scan method. Figure 5
Table 3. Summary of TPA cross-section (s(2)) data in DMSO (1 × 10-3 M)
(2) [GM]
Compound
TPEGPP
s
(2) [GM]
shows the representative Z-scan traces of these
compounds, from which the absolute s(2) values
can be evaluated (Table 3). These porphyrins with
different substitutions exhibit slightly different
s(2) values between 110 and 240 GM. Although
the exceptionally large s(2) values more than
11000 GM on porphyrin derivatives has been
reported by Anderson et al. [5b], the s(2) values
of up to 240 GM are still high among previous
reported porphyrin derivatives to date. Therefore, these
porphyrins have the potential to produce singlet oxygen
(1O2) via two photon excitation, which is extremely
important in the photodynamic therapy (PDT).
Compound
s
TPP-OC6Br
110
171
132
170
198
189
240
214
ZnTPP-OC6Br
TPP-OC6-Py-OH
ZnTPP-OC6-Py-OH
p-OHTPEGPP
TPEGPP-PEG-I
TPEGPP-PEG-Py-OH
DNA photocleavage assay
The DNA photocleavage activities of these amphi-
philic porphyrins, namely, ZnTPP-OC6-Py-OH, TPP-
OC6-Py-OH, p-OHTPEGPP and TPEGPP-PEG-Py-OH,
Table 4. Agarose gel image of DNA photocleavage assay of amphiphilic porphyrins in 20% glycerol as a function
of its concentration. Lane 1: supercoiled DNA (Form I) alone (control); lane 2: 10 mM amphiphilic porphyrins;
lane 3: 20 mM amphiphilic porphyrins; lane 4: 100 mM amphiphilic porphyrins. Photo-irradiation conditions:
lirrad = 455 nm; duration 45 min
Conc., mM
0
10
20
100
Conc., mM
0
10
20
100
ZnTPP-OC6-Py-OH
TPP-OC6-Py-OH
Form II
2
4
96
2
6
94
4
12
88
10
2
3
97
1
4
96
2
4
96
2
Form I
98
0
98
0
Cleavage activity
Conc., mM
0
10
20
100
Conc., mM
0
10
20
100
p-OHTPEGPP
TPEGPP-PEG-Py-OH
Form II
2
98
0
4
96
2
8
92
6
23
77
21
2
98
0
4
96
2
4
96
2
4
96
2
Form I
Cleavage activity
Copyright © 2014 World Scientific Publishing Company
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SYNTHESIS, CHARACTERIZATION, PHOTOPHYSICAL AND DNA PHOTOCLEAVAGE PROPERTIES
229
were also measured. Among them, Only ZnTPP-OC6-
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Py-OH and p-OHTPEGPP showed some photocleavage
activities towards the anionic DNA, with 10% and 21%
cleavage activities observed at 100 mM, respectively
(Table 4). As for p-OHTPEGPP, its relatively significant
DNA photocleavage activities are likely the result of the
highest singlet oxygen (1O2) quantum yields and large
binding constant with DNA. This result underscores the
importance of good singlet oxygen (1O2) quantum yields
and a close range interaction with the target in effecting
the desired outcome [15].
Furthermore, photodynamic therapy (PDT) properties
of these products are in testing.
CONCLUSION
In conclusion, a series of amphiphilic porphyrins with
pyridinium cations and/or polyethylene glycol, namely,
ZnTPP-OC6-Py-OH, TPP-OC6-Py-OH, p-OHTPEGPP
and TPEGPP-PEG-Py-OH, were synthesized and fully
characterized by 1H NMR, IR and MALDI-TOF-MS. Their
photophysical properties were investigated in detail. These
porphyrin derivatives show good singlet oxygen (1O2)
quantum yield and exhibit large TPA cross-sections s(2)
values between 110 and 240 GM, which indicate they are
good candidate as photosensitizers in the PDT applications.
In addition, ZnTPP-OC6-Py-OH and p-OHTPEGPP show
moderate photocleavage activities (10–21%) towards the
anionic DNA observed at 100 mM.
Acknowledgements
This work was supported by the Zhejiang Provincial
Natural Science Foundation of China (Grant No.
LQ13B010001) and Scientific Research Foundation of
Taizhou University (Grant No. 2013PY28).
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