Synthesis, circular dichroism, DNA cleavage and singlet oxygen photogeneration of 4-amidinophenyl porphyrins

Article abstract of DOI:10.1142/S108842461100435X

5,10,15,20-tetrakis(4-amidinophenyl)porphyrin (Por 1), its Zn complex (Por 2) and its conjugate with a tetraphenylporphyrin to form a bisporphyrin (Por 3) were prepared. The monomeric Por 1 and Por 2 showed both intercalative and external binding with DNA whereas only external DNA binding was seen in the bisporphyrin, Por 3 by circular dichroism and UV-vis. The DNA photocleavage activities of these porphyrins followed the order: Por 1 ～ Por 2 > Por 3, which did not correlate with their measured ¹O₂ production rates. It suggests 4-amidinophenylporphyrins are promising new photodynamic therapeutic agents.

Full text of DOI:10.1142/S108842461100435X

Journal of Porphyrins and Phthalocyanines
J. Porphyrins Phthalocyanines 2012; 16: 85–92
DOI: 10.1142/S108842461100435X
Published at http://www.worldscinet.com/jpp/
Synthesis, circular dichroism, DNA cleavage and singlet
oxygen photogeneration of 4-amidinophenyl porphyrins
Kai Wanga,b,c, Chun T. Poonb, ChunY. Choib, Wai-Kwok Wong*b, Daniel W.J. Kwong*b,
Fa Q. Yuc, Heng Zhangc and ZaoY. Li*a
a College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, Hubei, P.R. China
b Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong, P.R. China
c Key Laboratory for Green Chemical Process of Ministry of Education, Wuhan Institute of Technology, Wuhan
430073, Hubei, P.R. China
Received 8 July 2011
Accepted 30 July 2011
ABSTRACT: 5,10,15,20-tetrakis(4-amidinophenyl)porphyrin (Por 1), its Zn complex (Por 2) and its
conjugate with a tetraphenylporphyrin to form a bisporphyrin (Por 3) were prepared. The monomeric
Por 1 and Por 2 showed both intercalative and external binding with DNA whereas only external DNA
binding was seen in the bisporphyrin, Por 3 by circular dichroism and UV-vis. The DNA photocleavage
activities of these porphyrins followed the order: Por 1 ~ Por 2 > Por 3, which did not correlate with their
measured 1O2 production rates. It suggests 4-amidinophenylporphyrins are promising new photodynamic
therapeutic agents.
KEYWORDS: amidinophenylporphyrin, singlet oxygen, DNA cleavage, binding mode.
signal indicates external binding whereas a negative
induced CD signal indicates intercalative binding with
INTRODUCTION
Photodynamic therapy (PDT), with its inherent
DNA [3, 4].
selectivity, has received increasing attention as an
emerging cancer treatment modality recently. In PDT, a
Cationic porphyrins, such as 5,10,15,20-tetrakis(4-
N-methylpyridinium)porphyrin (H2TMPyP), have been
non-toxic photosensitizer, which can be selectively taken
intensively studied as potential PDT agents for decades
up by tumor cells, generates reactive oxygen species,
due to their strong binding affinity towards DNA and their
such as singlet oxygen (1O2), upon photo-activation
water-solubility which make DNA-binding possible under
by visible light. The reactive oxygen species produced
physiologically relevant conditions [5, 6]. These crucial
causes oxidative damage to the tumor tissues, resulting
properties have been attributed to their cationic substituent
in cell death [1, 2].
which facilitates their binding interactions with the
Circular dichroism (CD) and UV-vis spectroscopy
anionic DNA in aqueous media [7, 8]. But DNA-binding
were applied to study the porphyrin-DNA interactions.
interactions can be achieved via complementary hydrogen
Previous studies showed that the sign of the induced
bonding with DNA bases as well, particularly at the minor
CD in the Soret region of the porphyrins can be used
groove region [9, 10]. In this work, a hydrogen bonding
as a signature of their binding modes with DNA under
low porphyrin loads (r < 0.1): a positive induced CD
functionality, i.e. the amidine group, was introduced onto
the tetraphenylporphyrin (TPP) by synthesis. Its Zn(II)
complex and its conjugate with another TPP via the
-O-(CH2)3-O- linker were prepared as well (Schemes
*Correspondence to: Zaoying Li, email: zyliwuc@whu.edu.
1 and 2). The efficiency of cellular uptake of the drug is
cn, tel: +86 27-87219084, fax: +86 27-68754067; Wai-Kwok
very important to play a therapeutic role, which depends
on hydrophilic and lipophilic nature of molecular structure
[11]. Since the previously prepared bisporphyrin with
Wong, email: wkwong@hkbu.edu.hk and Daniel W.J. Kwong,
email: dkwong@hkbu.edu.hk, tel: +852 3411-7063, fax: +852
3411-7348
Copyright © 2012 World Scientific Publishing Company

86
K. WANG ET AL.
CN
CN
N
N
N
N
NH
N
N
Zn(OAC)2
Zn
CN
CN
NC
NC
HN
(a) LiN(SiMe3)2/THF
(b) H2O
CN
CN
H2N
NH
NH2
NH
HN
N
N
N
M
N
H2N
Por1, M = 2H
Por2, M = Zn
HN
NH2
Scheme 1
CN
CN
NH
N
N
NH
NH
N
N
N
NH
N
N
K2CO3/DMF
+
OH
O
O
O
NC
NC
Br
N
HN
HN
HN
HN
CN
CN
(a) LiN(SiMe3
(b) H2O
)2/THF
Br(CH2
)3Br
K2CO3/DMF
K
2CO3/DMF
H2N
NH
CN
HN
NH
N
N
NH
N
N
O
O
NH
N
N
NH
N
N
HN
HN
+
H2
N
O
Br
NC
HO
HN
HN
NH2
Por3
HN
CN
Scheme 2
hydrophilic (TMPyP) and lipophilic (TPP) structure in
our lab had a good cellular uptake [12], amidine group as
the hydrophilic part replaced TMPyP in the bisporphyrin
structure design. Furthermore, the amidine substituent, in
addition to its hydrogen-bonding capability, can also form
amidinium-carboxylate salt bridge with biomolecules
bearing a pendant carboxylate/carboxylic acid. Such
intermolecular amidinium-carboxylate bridges have
recently been shown to facilitate photo-excited energy
transfer with rates much faster than those predicted by
Copyright © 2012 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2012; 16: 86–92

DNA CLEAVAGE AND SINGLET OXYGEN PHOTOGENERATION OF 4-AMIDINOPHENYL PORPHYRINS
87
the Forster mechanism [13]. Herein we reported their
binding modes with DNA as studied by circular dichroism
spectroscopy and UV-vis. Their DNA photocleavage
1
activities and their relative O2 production rates were also
investigated.
RESULTS AND DISCUSSION
Binding mode of Por 1, Por 2 and Por 3 with DNA
The absorption titration spectra of these 4-amidi-
nophenylporphyrins with calf thymus DNA (ct-DNA)
was given in Fig. 1. Without DNA, lmax of the Soret
bands of Por 1, Por 2 and Por 3 were 414, 423 and 422
nm, respectively. In the presence of low concentrations
of ct-DNA (i.e., r = [porphyrin]/[DNA] > 0.6), the Soret
bands of Por 1, Por 2 and Por 3 exhibited substantial
hypochromicity (75.9%, 76.1% and 77.1%, respectively)
but no significant spectral shift, which suggests an
external DNA binding mode with modest effect on the
p–p* Soret absorption. Further addition of ct-DNA to
Por 1, Por 2 and Por 3 (i.e., r < 0.26) resulted in
a red shift of 11, 4 and 10 nm, respectively. The
substantial bathochromic shifts and hypochromicity
observed suggest binding interactions between the
4-amidinophenylporphyrins and ct-DNA [14, 15]. An
apparent binding constant, Kapp, can be calculated from
these data using Eq. (1) as follows [16].


[DNA]total
1
1
=
[DNA]total +


(| eapp -e f |)
(| e -e )
{Kapp (| eb -e f |)}
|


b
f
(1)
where eapp, ef and eb correspond to Aobsd/[porphyrin], the
molar absorptivity of the free porphyrin, and the molar
absorptivity of the DNA-bound porphyrin, respectively.
By plotting [DNA]total/(|eapp - ef |) vs. [DNA]total, Kapp of Por
1, Por 2 and Por 3 were determined to be (1.53 ± 0.69) ×
106 M-1, (7.25 ± 6.02) × 105 M-1 and (5.81 ± 1.14) × 105
M-1, respectively.
The induced CD spectra in the Soret region of Por 1,
Por 2 and Por 3 (at r([porphyrin]/[DNA]) = 0.05) were
given in Fig. 2. From this figure, Por 1 showed two
positive signals at 421 and 443 nm, and one negative
signal at 431 nm. Por 2 showed a strong negative signal
at 427 nm and a strong positive signal at 450 nm. These
features suggest that both Por 1 and Por 2 could bind to
ct-DNA via both external bounds and intercalation. Por 3
showed a very weak and broad positive signal centered at
440 nm, indicating that it bond to ct-DNA rather weakly
with outside binding mode in the high DNA concentration.
Fig. 1. Absorption titration spectra of the amidinophenyl-
porphyrins (4.0 mM), Por 1 (a), Por 2 (b) and Por 3 (c), with
increasing concentrations of ct-DNA (r = [porphyrin]/[DNA base
pairs] = `, 7.14, 3.57, 1.80, 0.91, 0.62, 0.26, 0.20, 0.17, 0.11) in
buffered solution (pH = 7.4, 5 mM tris-HCl, 50 mM NaCl)
The gel images are given in Fig. 3. For Por 1 and its
Zn(II) complex (Por 2), their photocleavage activities
were comparable and were ca. 4-fold lower than that of
H2TMPyP. At 10 mM, their DNA photocleavage activities
DNA photocleavage ability
DNA photocleavage activities of Por 1, Por 2 and Por 3
were measured using plasmid DNA relaxation assay [17].
Copyright © 2012 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2012; 16: 87–92

88
K. WANG ET AL.
its DNA-binding property, we measured the
1
relative O2 production rates of Por 1, Por 2,
1
Por 3 and H2TMPyP based on the O2-induced
bleaching of 1,3-diphenylisobenzofuran (DPBF,
lmax = 418 nm) [21, 22]. According to the
relationship between A/A0 absorbed by DPBF
and illumination time, one may indirectly obtain
1
the O2 yield of those porphyrins compared with
H2TMPyP. All three 4-amidinophenylporphyrins
1
produced O2 more efficiently than H2TMPyP in
the following order: Por 2 > Por 1 ~ Por 3 >
H2TMPyP (Fig. 4).
Incontrast, Zn-porphyrin2showedagreateryield
1
of O2, as other Zn(II)-porphyrin derivatives, and
this is attributed to its correspondingly high triplet
quantum yield, FT [23]. The lack of correlation
between the observed DNA photocleavage activities
of these porphyrins and their relative 1O2 production
rates suggests that their DNA-binding properties
must be a more important factor. Presumably, the
intercalative binding exhibited by H2TMPyP, Por
1 and Por 2 with DNA was crucial to their higher
DNA photocleavage activities than Por 3 which binds to
DNA only externally.
Fig. 2. Induced CD spectra of Por 1, Por 2 and Por 3 in the tris-HCl
buffer solution with the [porphyrin] = 10 mM and [ct-DNA] = 200 mM,
r = 0.05
were 69% and 74%, respectively, which was ca. 4-fold
higher than that of Por 3 (19%).
For Por 3, its maximum photocleavage activity
of ca. 36% was seen at 75 mM. The significant DNA
photocleavage activities were likely the result of a
binding interaction with DNA and a more effectively
EXPERIMENTAL
1
oxidative attack from close range with O2 [18–20]. It
General
also suggests that Por 1, Por 2 and Por 3 with hydrogen-
bonding capability have relatively weaker electrostatic
interaction with DNA compared with H2TMPyP.
5-[p-(3-bromopropoxy)phenyl]-10,15,20-tris-
phenylporphyrin, 5-(p-hydroxyphenyl)- 10,15,20- trisph-
enylporphyrin and meso-tetrakis (N-methylpyridinium-
4-yl)porphyrin (H2TMPyP) were prepared according
to literature procedures [24, 25]. Other chemicals were
obtained from Sigma-Aldrich Company and used without
further purification. Silica gel 60 (0.04–0.063 mm) for
Photogeneration of 1O2
Since the DNA photocleavage activity of
photosensitizer depends on its O2 yield as well as
a
1
Fig. 3. Agarose gel electrophoresis images of DNA photocleavage assay of (a) Por 1, (b) Por 2, (c) Por 3 and (d) H2TMPyP at
different concentrations. Photo-irradiation was conducted using a transilluminator at 455 nm for 45 min
Copyright © 2012 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2012; 16: 88–92

DNA CLEAVAGE AND SINGLET OXYGEN PHOTOGENERATION OF 4-AMIDINOPHENYL PORPHYRINS
89
evaporated to dryness. After adding chloroform
(300 mL) to the residue and mixing for 3 h, it was
filtered, and chloroform layer was washed with
water (300 mL × 3), which was dried by anhydrous
sodium sulfate. After it had been filtered and
concentrated, the residue was purified by silica gel
column chromatography using dichloromethane as
eluent. The first fraction was 5,10,15,20-tetrakis(p-
cyanophenyl)porphyrin. Yield 220 mg (1.5%), mp
> 300 °C. MS (FAB): m/z 714.6 (calcd. for [M]+
714.2); the second fraction was the title blue-violet
product.Yield 665 mg (4.7%) after recrystallization
from chloroform/methanol, mp > 300 °C. IR (KBr):
n, cm-1 3310.9, 2227.6, 1604.3. UV-vis (CHCl3):
l
max, nm (log e) 422 (5.62), 517 (4.24), 553 (3.90),
591 (3.73), 648 (3.68). MS (FAB): m/z 706.3 (calcd.
for [M + H]+ 705.2).
Fig. 4. Normalized absorbance of DPBF (50 mM in DMSO) at
418 nm as a function of photo-irradiation time in the absence
(control) and presence of 1 mM of H2TMPyP, Por 1, Por 2 and Por 3
5,10,15,20-tetrakis(p-cyanophenyl)-Zn-
porphyrin(ZnTCNPP). The complex was
prepared by refluxing H2TCNPP (100 mg, 0.14 mmol)
with an excess amount of zinc acetate in CHCl3/methanol
for 5 h and purified by column chromatography on silica
gel using chloroform as eluent. Yield 103 mg (95%), mp
> 300 °C. 1H NMR (400 MHz; CDCl3; Me4Si): dH, ppm
8.09–8.11 (8H, d, J = 8.0 Hz, porPhHm), 8.33–8.35 (8H,
d, J = 8.0 Hz, porPhHo), 8.90 (8H, s, pyrrole H). IR (KBr):
v, cm-1 2228, 1603, 996 (ZnII, OSMB). UV-vis (CHCl3):
column chromatography was obtained from Merck. Calf
thymus DNA (ct-DNA) was obtained from Pharmacia
Biotech Company. The concentration of ct-DNA in base-
pairs was determined spectrophotometrically using the
extinction coefficient e260 = 6600 M-1.cm-1 [26].
NMR spectra were recorded on a Varian INOVA 400
NMR spectrometer. High-resolution matrix-assisted laser
l
max, nm (log e) 427 (5.76), 557 (4.35), 597 (3.74). HRMS
(MALDI-TOF, in chloroform): m/z 776.1421 (calcd. for
[M]+ 776.1409, Dm = 1.4288 ppm).
desorption/ionization-time of flight (MALDI-TOF) mass
spectra were recorded on an Autoflex Bruker MALDI-
TOF mass spectrometer. FAB-mass spectra were obtained
on a Finnigan TSQ710 mass spectrometer. Electronic
absorption spectra in the UV-vis region were recorded
with a Varian Cary 100 UV-vis spectrophotometer.
Elemental analyses were performed on an instrument of
VarioEL III. The IR spectra (KBr pellets) were recorded
on a Nicolet Magna-FTIR 550 spectrometer. CD spectra
were recorded using a JASCO J-720 spectropolarimeter.
The CD spectral and UV-vis measurements were
performed in 5 mM Tris-HCl buffer (pH 7.4) containing
50 mM NaCl.
5-[p-(3-bromopropoxy)phenyl]-10,15,20-tris(4-
cyanophenyl)porphyrin. In the presence of anhydrous
potassium carbonate (1.0 g), 5-(p-hydroxyphenyl)-
10,15,20-tris(4-cyanophenyl)porphyrin (100 mg, 0.14
mmol) and 1,3-dibromopropane (0.57 g, 2.83 mmol) in
dry DMF (10 mL) was heated to 65 °C for 4 h with stirring
under nitrogen. After cooling to room temperature, the
reaction mixture was poured into water saturated with
sodium chloride (30 mL) and then filtered. The precipitate
was purified by silica gel column chromatography using
chloroform as eluent. The first fraction was the title blue-
violet product.Yield 82 mg (70.8%) after recrystallization
from chloroform/methanol. mp > 300 °C. 1H NMR (400
MHz; CDCl3; Me4Si): dH, ppm -2.88 (2H, s, pyrrole ring
NH), 2.50–2.52 (2H, m, -CH2- CH2- CH2-), 3.77–3.80
(2H, t, J = 6.4 Hz, CH2Br), 4.39–4.42 (2H, t, J = 5.6 Hz,
OCH2), 7.29–7.31 (2H, m, O-PhHo), 8.06–8.10 (8H, m,
porPhHm), 8.31–8.33 (6H, d, J = 7.2 Hz, porPhHo), 8.73–
8.76 (8H, m, pyrrole H). UV-vis (CHCl3): lmax, nm (log e)
420 (5.65), 517 (4.22), 553 (3.85), 591 (3.70), 647 (3.66).
MS (FAB): m/z 827.3 (calcd. for [M]+ 826.7).
Synthesis routes and procedures of the 4-amidino-
phenylporphyrins
The synthetic routes for the preparation of porphyrins
with tris- and tetra-amidino groups are outlined in
Schemes 1 and 2. The detailed procedures for the
preparation of all the involved compounds, together with
their characterization data, are given in the following
sections.
5-(p-hydroxyphenyl)-10,15,20-tris(4-cyanophenyl)-
porphyrin and 5,10,15,20-tetrakis(p-cyanophenyl)-
porphyrin (H2TCNPP). Pyrrole (5.36 g, 0.08 mmol) was
added dropwisely to a solution of 4-cyanobenzaldehyde
(2.62 g, 0.06 mmol) and 4-hydroxybenzaldehyde
(2.44 g, 0.02 mmol) in refluxing propionic acid
(200 mL). The mixture was refluxed for 1 h and then
Free CN-bisporphyrin. In the presence of anhydrous
potassium carbonate (1.0 g), 5-(p-hydroxyphenyl)-
10,15,20-tris(4-cyanophenyl)porphyrin (100.0 mg, 0.14
mmol) and 5-[p-(3-bromopropoxy)phenyl]-10,15,20-
trisphenylporphyrin (106.5 mg, 0.14 mmol) [or 5-[p-
(3-bromopropoxy)phenyl]-10,15,20-tris(4-cyanophenyl)-
Copyright © 2012 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2012; 16: 89–92

90
K. WANG ET AL.
porphyrin (100 mg, 0.12 mmol) and 5-(p-hydroxyphenyl)-
10,15,20-trisphenylporphyrin (76 mg, 0.12 mmol)] in dry
DMF (15 mL) was heated to 65 °C for 5 h with stirring
under nitrogen. After cooling to room temperature, the
reaction mixture was poured into water saturated with
sodium chloride (50 mL) and then filtered. The precipitate
was purified by silica gel column chromatography
using chloroform as eluent. The second fraction was
the title blue-violet product. Yield 135 mg (70.4%)
after recrystallization from chloroform/methanol, mp >
4-amidinophenylbisporphyrin(Por3).Aslurryoffree
CN-bisporphyrin (50 mg, 0.036 mmol) and LiN(SiMe3)2
(0.16 mmol/mL in THF solution, 0.32 mmol) in dry THF
(15 mL) was sonicated at room temperature for 5 h. The
solvent was removed in vacuum. The residue obtained
was washed by water. Yield 50 mg (97%), mp > 300 °C.
Anal. calcd. for C94H70N14O2: C, 78.96; H, 4.90; N,
13.72. Found: C, 78.82; H, 4.92; N, 13.63. 1H NMR (400
MHz; CDCl3; Me4Si): dH, ppm -2.76 (4H, s, pyrrole ring
NH), 2.57–2.67 (2H, m, -CH2- CH2- CH2-), 4.50–4.63
(4H, m, CH2), 7.37–7.40 (4H, m, O-PhHo), 7.61–7.77
(15H, m, porPhHm and portrisPhHp), 8.09–8.22 (16H,
m, porPhHo), 8.83–8.85 (16H, m, pyrrole H). IR (KBr):
n, cm-1 3318, 3036, 2918, 1674, 1598, 1482. UV-vis
(DMSO): lmax, nm (log e) 421 (5.77), 516 (4.44), 552
(4.14), 591 (3.88), 647 (3.89). HRMS (MALDI-TOF, in
methanol): m/z 1428.5971 (calcd. for [M]+ 1428.5909,
Dm = 4.2923 ppm).
1
300 °C. H NMR (400 MHz; CDCl3; Me4Si): dH, ppm
-2.86 (2H, s, trisphenyl pyrrole ring NH), -2.79 (2H, s,
tris(cyanophenyl) pyrrole ring NH), 2.63–2.66 (2H, m,
-CH2- CH2- CH2-), 4.60–4.64 (4H, m, CH2), 7.37–7.42
(4H, m, O-PhHo), 7.64–7.77 (9H, m, portrisPhHm,p),
8.31–8.33 (22H, m, porPhHo and trisCNPhHo), 8.68–8.99
(16H, m, pyrrole H). IR (KBr): v, cm-1 3311, 2227, 1604.
UV-vis (CHCl3): lmax, nm (log e) 418 (5.53), 517 (4.18),
553 (3.88), 592 (3.65), 647 (3.62). MS (FAB): m/z 1376.6
(calcd. for [M + H]+ 1375.5).
Absorption titration of Por 1, Por 2 and Por 3
with DNA
5,10,15,20-tetrakis(4-amidinophenyl)porphyrin
(Por 1). A slurry of H2TCNPP (150 mg, 0.21 mmol)
and LiN(SiMe3)2 (0.16 mmol/mL in THF solution,
0.32 mmol) in dry THF (20 mL) was sonicated at room
temperature for 5 h [27]. The solvent was removed under
vacuum. The residue was then washed with distilled
water and chloroform, purified by recrystallization
from methanol and acetonitrile. The resulting title
compound was characterized by NMR, UV-vis, MS and
elemental analysis. Yield 164 mg (91%), mp > 300 °C.
Anal. calcd. for C48H38N12: C, 73.53; H, 4.85; N, 21.45.
The sample solutions were prepared by mixing 2 mM
4-amidinophenylporphyrin stock solution (in DMSO)
and 0.169 mM ct-DNA stock solution in appropriate
ratios and then diluted with Tris-HCl buffer solution
to obtain the final desired concentration ratios (r =
[porphyrin]/[ct-DNA]). UV-vis absorption spectra of these
porphyrin-DNA sample mixtures were recorded from
350–500 nm.
Circular dichroism (CD) measurement of Por 1, Por 2
and Por 3 with DNA
1
Found: C, 73.60; H, 4.79; N, 21.50. H NMR (400
MHz; DMSO-d6; Me4Si): dH, ppm -2.95 (2H, s, pyrrole
ring NH), 8.24 (8H, s, porPhHm), 8.26 (8H, s, porPhHo),
8.87 (8H, s, pyrrole H). IR (KBr): n, cm-1 3318, 3201,
1667, 1607, 1507, 1440, 966, 863, 799, 502. UV-vis
(DMSO): lmax, nm (log e) 421 (5.43), 515 (4.07), 551
(3.72), 589 (3.51), 645 (3.44). HRMS (MALDI-TOF,
in methanol): m/z 783.3456 (calcd. for [M]+ 783.3420,
Dm = 1.1489 ppm).
Appropriate amount of calf thymus DNA was added
to a Tris-HCl buffered 10 mM porphyrin solution to
obtain a porphyrin-DNA mixture with a concentration
ratio r ([porphyrin]/[ct-DNA]) = 0.05.After an incubation
period of 45 min, the sample mixture was scanned in the
Soret region of porphyrin (i.e., 400–500 nm). Baseline
correction was obtained by scanning the same buffer
solution without the porphyrin. The spectral bandwidth
and the cell length for the CD measurements were 1 nm
and 1 cm, respectively.
Zn(II)-5,10,15,20-tetrakis(4-amidinophenyl)-
porphyrin (Por 2). A slurry of ZnTCNPP (50 mg, 0.06
mmol) and LiN(SiMe3)2 (0.16 mmol/mL in THF solution,
0.32 mmol) in dry THF (20 mL) was sonicated at room
temperature for 5 h. The solvent was removed in vacuum.
The residue was then washed with distilled water and
chloroform and then purified by recrystallization from
methanol and acetonitrile. Yield 51 mg (94%), mp >
300 °C. Anal. calcd. for C48H36N12Zn: C, 68.14; H, 4.26;
DNA photocleavage assay
The DNA photocleavage activities of Por 1, Por 2, Por
3 and H2TMPyP were measured using the plasmid DNA
relaxation assay. Briefly, the plasmid DNA (pBluescript,
0.5 mg), enriched with the covalently-closed circular or
supercoiled conformer (Form I), 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
prophyrins (test sample) were added into the Eppendorf
tubes to give a final volume of 20 mL in each sample tube.
1
N, 19.86. Found: C, 68.07; H, 4.31; N, 19.79. H NMR
(400 MHz; methanol-d4; Me4Si): dH, ppm 8.10–8.12 (8H,
d, J = 8.0 Hz, porPhHm), 8.28–8.30 (8H, d, J = 8.0 Hz,
porPhHo), 8.82 (8H, s, pyrrole H). IR (KBr): n, cm-1 3372,
3326, 1633, 1606, 992 (ZnII, OSMB). UV-vis (DMSO):
l
max, nm (log e) 430 (5.55), 561 (4.16), 602 (3.83). HRMS
(MALDI-TOF, in methanol): m/z 845.2529 (calcd. for
[M]+ 845.2550, Dm = -2.4986 ppm).
Copyright © 2012 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2012; 16: 90–92

DNA CLEAVAGE AND SINGLET OXYGEN PHOTOGENERATION OF 4-AMIDINOPHENYL PORPHYRINS
91
The sample mixtures were then photo-irradiated at 400–450
is currently underway. Additionally, cellular uptake and
cytotoxicity tests are also in progress.
nm for 45 min using a transilluminator (Vilber Lourmat)
containing 4 × 15 W light tubes (Aqua Lux) with maximum
emission at 435 nm. After photo-irradiation, 2 mL of the 6x
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 (Gel dimension: 13 cm × 10 cm), with 1x TBE buffer
(89 mM tris-borate, 1 mM EDTA, pH 8) used as supporting
electrolyte, and electrophoresized at 1.3V.cm-1 for 3 h using
a mini gel set (CBS Scientific Co., Model No. MGU-502T).
After electrophoresis, the gel was stained with 0.5 mg/mL
ethidium bromide solution for 30 min and then destained
using deionized water for 10 min. The resulting gel image
was viewed under 365 nm and captured digitally using a gel
documentation system (BioRad).
Acknowledgements
The authors are grateful for the financial supports of
National Natural Science Foundation of China (Nos.
20902071, 20672082), Hong Kong Research Grants
Council (No. HKBU 202407) and Open Funds of Key
Laboratory for Green Chemical Process of Ministry of
Education in China (No. RGCT201003).
REFERENCES
1. Hasan T, Ortel B, Moor ACE and Pogue BW. In
Holland-Frei Cancer Medicine 6, Bast RC Jr, Kufe
DW, Pollock RE, Weichselbaum RR, Holland JF
and Frei E III. (Eds.) Decker and Hamilton: 2003;
pp 605–622.
2. Dolmans DEJGJ, Fukumura D and Jain RK. Nat.
Rev. Cancer 2003; 3: 380–387.
3. Pasternack RF. Chirality 2003; 15: 329–332.
4. Yellappa S, Seetharamappa J, Rogers LM, Chitta
R, Singhal RP and D’Souza F. Bioconjugate Chem.
2006; 17: 1418–1425.
The DNA photocleavage activities of Por 1, Por 2
and Por 3 were measured by monitoring the conversion
of supercoiled conformer (Form I) to the open-circular
conformer (Form II) upon nicking by a DNA-cleavage
agent.
Measurement of singlet oxygen production rate
5. Villaneuva A, Juarranz A, Diaz V, Gomez J and
Canete M. Anti-cancer Drug Des. 1992; 7: 297–303.
6. Villanueva A and Jori G. Cancer Lett. 1993; 73:
59–64.
7. Yamamoto T, Tjahjono DH, Yoshioka N and Inoue
H. Bull. Chem. Soc. Jpn. 2003; 76: 1947–1955.
8. McMillin DR, Shelton AH, Bejune SA, Fanwick
PE and Wall RK. Coord. Chem. Rev. 2005; 249:
1451–1459.
9. Pindur U, Jansen M and Lemster T. Curr. Med.
Chem. 2005; 12: 2805–2847.
10. Neidle S. Nat. Prod. Rep. 2001; 18: 291–309.
11. Jiang FL, Poon CT, Wong WK, Koon HK, Mak NK,
Choi CY, Kwong DWJ and Liu Y. Chembiochem.
2008; 9: 1034–1039.
1,3-diphenylisobenzofuran (DPBF) was used as
a selective singlet oxygen (1O2) acceptor, which was
bleached upon reaction with O2. Five sample solutions
1
of DPBF in DMSO (50 mM) containing, respectively,
no porphyrin (control sample), H2TMPyP (1 mM), Por 1
(1 mM), Por 2 (1 mM) and Por 3 (1 mM) were prepared
in dark. Each sample container was covered with
aluminum foil with a yellow filter (with cutoff wavelength
< 500 nm) on one side. The samples were then exposed
to light (50 watt) through the filter. After irradiation,
visible spectra of the sample solutions were measured
spectrophotometrically. The normalized absorbances
of DPBF at 418 nm in these samples were reported as a
function of the photo-irradiation time. From this plot, the
1O2 production rates of Por 1, Por 2 and Por 3 relative to
that of H2TMPyP can be determined.
12. You Y, Gibson SL, Hilf R, Davies SR, Oseroff AR,
Roy I, Ohulchanskyy TY, Bergey EJ and Detty MR.
J. Med. Chem. 2003; 46: 3734–3747.
13. Otsuki J, Iwasaki K, NakanoY, Itou M, ArakiY and
Ito O. Chem. Eur. J. 2004; 10: 3461–3466.
14. Fiel RJ. J. Biomol. Struct. Dynam. 1989; 6:
1259–1274.
15. Pasternack RF, Gibbs EJ and Villafranca J.
Biochemistry 1983; 22: 2406–2414.
16. Mettath S, Munson BR and Pandey RK.
Bioconjugate Chem. 1999; 10: 94–102.
17. Nyarko E, Hanada N, Habib A and Tabata M. Inorg.
Chim. Acta. 2004; 357: 739–745.
18. Zheng YM, Wang K, Li T, Zhang XL and Li ZY.
Molecules 2011; 16: 3488–3498.
19. Wang K, Poon CT, Wong WK, Wong WY, Kwong
DWJ, Zhang H and Li ZY. Eur. J. Inorg. Chem.
2009; 922–928.
CONCLUSION
The monomeric Por 1 and Por 2 showed both
intercalative and external binding with DNA by the CD
spectra whereas only external DNA binding was seen
in the bisporphyrin, Por 3. The DNA photocleavage
activities of these porphyrins followed the order: Por 1
~ Por 2 > Por 3. All three 4-amidinophenylporphyrins
produced 1O2 more efficiently than H2TMPyP. It suggests
the porphyrins with strong hydrogen bonding capacity
are promising new photo-dynamic therapeutic agents. To
ascertain whether hydrogen bonding occurred between
the amidine group and the DNA bases in the observed
1
porphyrin-DNA binding interactions, H NMR study
using oligodeoxyribonucleotides as binding substrates
Copyright © 2012 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2012; 16: 91–92

92
K. WANG ET AL.
20. Ishikawa Y, Yamakawa N and Uno T. Bioorg. Med.
Chem. 2007; 15: 5230–5238.
24. Li ZY, Wang K, Zhao YM and Li HY. Chin. J. Org.
Chem. 2003; 23: 265–269. (in Chinese).
21. Oda K, Ogura S and Okura I. J. Photochem.
Photobiol. B 2000; 59: 20–25.
25. Zhu XJ and Thesis PhD. Hong Kong Baptist
University, Hong Kong, 2006.
22. Ishikawa Y, Yamakawa N and Uno T. Bioorg. Med.
Chem. 2002; 10: 1953–1960.
26. Reichmann ME, Rice SA, Thomas CA and Doty P.
J. Am. Chem. Soc. 1954; 76: 3047–3053.
23. Pineiro M, Carvalho AL, Pereira MM, d’AR
Gonsalves AM, Arnaut LG and Formosinho SJ.
Chem. Eur. J. 1998; 4: 2299–2307.
27. Zhu XJ, Wong WK, Jiang FL, Poon CT and Wong
WY. Tetrahedron Lett. 2008; 49: 2114–2118.
Copyright © 2012 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2012; 16: 92–92