DNA binding and photocleavage specificities of a group of tricationic metalloporphyrins

Article abstract of DOI:10.1016/j.saa.2009.12.065

The interactions of 5,10,15-tris(1-methylpyridinium-4-yl)-20-(4-hydroxyphenyl)porphyrinatozinc(II) Zn[TMPyHP]³⁺ (2) along with Cu[TMPyHP]³⁺ (3), Co[TMPyHP]⁴⁺ (4), Mn[TMPyHP]⁴⁺ (5) and the free base porphyrin H₂[TMPyHP]³⁺ (1) with duplex DNA have been studied by using a combination of absorption, fluorescence titration, surface-enhanced Raman spectroscopy (SERS), induced circular dichroism (ICD) spectroscopy, thermal DNA denaturation, viscosity measurements as well as gel electrophoresis experiment. Their binding modes and intrinsic binding constants (K_b) to calf DNA (CT DNA) were comparatively studied and were found significantly influenced by different metals coordinated with the porphyrin plane. Except 3, which has four-coordination structure at the metal, all the metal derivatives showed non-intercalative DNA-binding mode and lower K_b than the free base porphyrin 1, most probably due to the steric hindrance results from the axial ligands of the inserted metals which are five or six-coordination structures. Meanwhile, the insertion of metals into cationic porphyrin greatly removed the self-aggregation of the metal-free porphyrins, and thus fully enhanced the singlet oxygen (¹O₂) productivities in the DNA photocleavage experiments. Therefore, these metalloporphyrins have comparable DNA cleavage ability with the free base porphyrin.

Full text of DOI:10.1016/j.saa.2009.12.065

Spectrochimica Acta Part A 75 (2010) 1108–1114
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
DNA binding and photocleavage specificities of a group of
tricationic metalloporphyrins
Ping Zhaoa,b, Jin-Wang Huanga,∗, Wen-Jie Meib, Juan Heb, Liang-Nian Jia
a MOE Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry and Chemical Engineering, Sun Yat-Sen University, No. 135, Xin Gang Xi Road,
Guangzhou 510275, PR China
b School of Chemistry and Chemical Engineering, Guangdong Pharmaceutical University, Guangzhou 510275, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
The interactions of 5,10,15-tris(1-methylpyridinium-4-yl)-20-(4-hydroxyphenyl)porphyrinatozinc(II)
Zn[TMPyHP]3+ (2) along with Cu[TMPyHP]3+ (3), Co[TMPyHP]4+ (4), Mn[TMPyHP]4+ (5) and the free base
porphyrin H2[TMPyHP]3+ (1) with duplex DNA have been studied by using a combination of absorp-
tion, fluorescence titration, surface-enhanced Raman spectroscopy (SERS), induced circular dichroism
(ICD) spectroscopy, thermal DNA denaturation, viscosity measurements as well as gel electrophoresis
experiment. Their binding modes and intrinsic binding constants (Kb) to calf DNA (CT DNA) were com-
paratively studied and were found significantly influenced by different metals coordinated with the
porphyrin plane. Except 3, which has four-coordination structure at the metal, all the metal derivatives
showed non-intercalative DNA-binding mode and lower Kb than the free base porphyrin 1, most prob-
ably due to the steric hindrance results from the axial ligands of the inserted metals which are five or
six-coordination structures. Meanwhile, the insertion of metals into cationic porphyrin greatly removed
the self-aggregation of the metal-free porphyrins, and thus fully enhanced the singlet oxygen (1O2) pro-
ductivities in the DNA photocleavage experiments. Therefore, these metalloporphyrins have comparable
DNA cleavage ability with the free base porphyrin.
Received 15 September 2009
Received in revised form
21 December 2009
Accepted 22 December 2009
Keywords:
Metalloporphyrin
DNA binding
Steric hindrance
DNA photocleavage
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
Co(III), Ni(II) and Mn(III) with tetra-kis(1-methylpyridinium-4-
yl)porphyrin (H2TMPyP)4+ have been intensely investigated in the
The design of small complexes that bind and react with DNA
becomes important as we begin to delineate the expression of
genetic information on a molecular level. A complete understand-
ing of how to target DNA sites with specificity will lead not only
to novel chemotherapeutics but also to a greatly expanded ability
for chemists to probe DNA and to develop highly sensitive diagnos-
tic agents [1–5]. Transition-metal complexes are being used at the
forefront of many of these efforts. Stable, inert, and water-soluble
complexes containing spectroscopically active metal centers are
extremely valuable in the researches of biological systems. More
recently, such metal complexes have been applied to probe both
structural and functional aspects of nucleic acid chemistry [6–8].
One of the most versatile metal complexes for nucleic acid
research is the metalloporphyrins. A number of metalloporphyrins
have been used in various fields of DNA study such as DNA breaks
[9,10], probes of DNA structure [11], enhancement of restric-
tion enzyme activity [12] and photoactive insecticides [13]. Most
especially, the cationic metalloporphyrin derivatives of Cu(II),
past [14–18]. Nevertheless, so far no studies have focused on the
role unsymmetrical cationic metalloporphyrins played in the DNA
research.
We recently reported the synthesis and the DNA binding and
photocleavage properties of a series of metal-free tricationic pyrid-
ium porphyrins with different peripheral active groups [19–22]. It
was found that, among them, only the tricationic pyridium por-
phyrin appending a hydroxyphenyl group, H2[TMPyHP]3+, could
intercalate into DNA base pairs, cleave DNA efficiently and hence
may be a candidate for PDT instead of (H2TMPyP)4+. In this paper,
we synthesized the zinc(II) (2), copper(II) (3), cobalt(III) (4), man-
ganese(III) (5) (Fig. 1) complexes of H2[TMPyHP]3+ (1), and studied
their binding properties with CT DNA. Meanwhile, the photo-
cleavage of pBR322 plasmid DNA by these porphyrins were also
comparatively investigated.
2. Experimental
2.1. Materials and chemicals
∗
The metalloporphyrin derivatives were prepared by mixing the
free base porphyrin 1 with corresponding acetate salts in the dark
Corresponding author. Tel.: +86 20 84113317; fax: +86 20 84112245.
E-mail address: ceshjw@163.com (J.-W. Huang).
1386-1425/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2009.12.065

P. Zhao et al. / Spectrochimica Acta Part A 75 (2010) 1108–1114
1109
Fig. 1. The molecular structures of porphyrins 1–5.
[21–23]. The formation of the metalloporphyrins was confirmed by
the measurement of UV–vis absorption spectra.
spectrofluorophotometer at room temperature. SERS spectra were
carried out on an inVia Laser Micro-Raman Spectrometer of Ren-
ishaw, with a power of 20 mW at the samples. ICD Spectra were
recorded on a JASCO-J810 spectrometer. Thermal denaturation
studies were carried out with a PerkinElmer-Lambda-850 spec-
trophotometer equipped with a Peltier temperature-controlling
programmer ( 0.1 ◦C). The melting curves were obtained by mea-
suring the absorbance at 260 nm for solutions of CT DNA (100 ␮M)
in the absence and presence of the porphyrin compound (10 ␮M) as
a function of the temperature. The temperature was scanned from
The spectroscopic results obtained from 5,10,15-tris(1-
methylpyridinium-4-yl)-20-(4-hydroxyphenyl)porphyrinatozinc
(2): 1H NMR (300 MHz, DMSO): chemical shift ı: 9.48(d, J = 5.94 Hz,
6H, 2, 6-pyridinium), 9.11(s, 4H, ␤-pyrrole), 9.07(s, 4H, ␤-pyrrole),
9.00(d, J = 5.51 Hz, 6H, 3,5-pyridinium), 8.02(d, J = 8.13 Hz, 2H, 2,6-
phenyl), 7.26(d, J = 8.06 Hz, 2H, 3,5-phenyl), 4.73(s, 9H, N+–Me),
3.01(s, 1H, OH). ES-MS [EtOH, m/z]: 247 ([M]3+). UV–vis (10 ␮M in
Tris buffer), ꢀmax(nm) (log ε): 425(4.9), 563(3.4), 583(3.3).
The spectroscopic results obtained from 5,10,15-tris(1-me-
thylpyridinium-4-yl)-20-(4-hydroxyphenyl)porphyrinatocopper
(3): ES-MS [EtOH, m/z]: 246 ([M]3+). UV–vis (10 ␮M in Tris buffer),
ꢀmax(nm) (log ε): 426(4.8), 560(3.2), 584(3.1).
The spectroscopic results obtained from 5,10,15-tris(1-me-
thylpyridinium-4-yl)-20-(4-hydroxyphenyl)porphyrinatocobalt
(4). ES-MS [EtOH, m/z]: 245 ([M]3+). UV–vis (10 ␮M in Tris buffer),
ꢀmax(nm) (log ε): 425(5.0), 563(3.4).
The spectroscopic results obtained from 5,10,15-tris(1-methy-
lpyridinium-4-yl)-20-(4-hydroxyphenyl)porphyrinatomanganese
(5). ES-MS [EtOH, m/z]: 243 ([M]3+). UV–vis (10 ␮M in Tris buffer),
ꢀmax(nm) (log ε): 425(4.8), 561(3.3), 585(3.1).
30 to 90 ◦C at a speed of 1 ◦C min−1. The melting temperature (Tm
)
was taken as the mid-point of the hyperchromic transition.
In the SERS experiment, Ag colloids were prepared by reducing
AgNO3 with EDTA according to the reported method [25]. The Ag
colloid/compound (or Ag colloid/DNA) SERS-active systems were
prepared by mixing equal volume of the porphyrin (or DNA) solu-
tion with the Ag colloid in buffer A to obtain the desired porphyrin
or DNA concentrations. In the porphyrin/DNA complex experi-
ments, the solution of DNA was mixed with the porphyrin solution
at a DNA/porphyrin ratio of 30:1, then an equal volume of the mixed
solution was fully mixed with the Ag colloid, and the spectrum was
immediately measured at room temperature. The final concentra-
tions of porphyrins and DNA in all of the SERS-active systems were
5 and 150 ␮M, respectively.
For the gel electrophoresis experiment, pBR322 supercoiled
plasmid DNA (0.1 ␮g) was treated with the porphyrin compound
in buffer C (50 mM Tris–HCl, 18 mM NaCl, pH 7.2), and the solution
was then irradiated at room temperature with a high-pressure mer-
cury lamp/monochromator assembly. The samples were analyzed
by electrophoresis for 1 h at 120 V in Tris–acetate buffer containing
1% agarose gel. The gel was stained with 1 ␮g ml−1 3,8-diamino-5-
ethyl-6-phenylphenanthridinium bromide (EB) and photographed
under UV light.
Buffer
A (5 mM Tris–HCl, 50 mM NaCl, pH 7.2, Tris = Tris
(hydroxymethyl)aminomethane) solution was used in all the
experiments except for the thermal denaturation studies in which
buffer B (1.5 mM Na2HPO4, 0.5 mM NaH2PO4, 0.25 mM Na2H2EDTA
(H4EDTA = N,Nꢀ-ethane-1,2-diylbis[N-(carboxymethyl)glycine]),
pH 7.0) was used. CT DNA and pBR322 supercoiled plasmid
DNA were obtained from the Sigma Company. A solution of CT
DNA in the buffer A gave a ratio of UV absorbance at 260 and
280 nm of 1.85:1, indicating that the DNA was sufficiently free of
protein. The DNA concentration per nucleotide was determined
by absorption spectroscopy using the molar absorption coefficient
(6600 M−1 cm−1) at 260 nm [24]. Unless otherwise stated, reagents
were commercially available and of analytical grade.
3. Results and discussion
2.2. Measurements
3.1. DNA-binding studies
Electrospraymass spectra (ES-MS)wererecorded onaLCQ DECA
XP liquid chromatography–mass spectrometry (Thremo, USA).
1H NMR spectra were recorded on a Mercury-Plus 300 Nuclear
Magnetic Resonance Spectrometer (Varian, USA). UV–vis spectra
were recorded on a PerkinElmer-Lambda-850 spectrophotome-
ter. Fluorescence spectra were recorded on a PerkinElmer L55
3.1.1. Absorption titrations
One of the most common techniques in DNA-binding studies of
porphyrins is electronic absorption spectroscopy. The magnitude
of spectral perturbation is an evidence for DNA-binding [9,14,15].
As is well known, intercalation of porphyrin into DNA base pairs is
characterized by a bathochromism (>8 nm) and hypochromism (up

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P. Zhao et al. / Spectrochimica Acta Part A 75 (2010) 1108–1114
Fig. 3. Emission spectra for
2 in the absence and presence of CT DNA, in
buffer A. Arrows show the intensity change upon increasing DNA concentrations.
[Por] = 10 ␮M; ꢀex = 449 nm.
Fig. 2. Absorption spectra of 2 in buffer A at 25 ◦C in the presence of increasing
amounts of CT DNA. [Por] = 10 ␮M. Arrows indicate the change in absorbance upon
increasing the DNA concentration.
the plot of [DNA]/(εa − εf) versus [DNA], Kb of the porphyrin com-
pounds were calculated and the results were listed in Table 1, too.
From Table 1, we can find that the values of Kb have strict rela-
tionship with respect to the coordinated metal in the porphyrin
core. The free base porphyrin 1 and the metalloporphyrin 3 with
Cu(II) in the centre have larger Kb than the porphyrins coordinated
with Zn(II), Co(III), Mn(III). From the spectrophotometric titrations
of the porphyrin solutions, 1 and 3 were proved to employ inter-
calative (which is more advantageous) modes when bind with DNA
while porphyrins 2, 4 and 5 via non-intercalative modes. Thus, we
suppose that the different Kb may mainly result from the distinct
DNA-binding modes.
to 40%) in the Soret band of UV–vis spectra; groove binding mode
shows no (or minor) and outside binding mode exhibits moder-
ate change in UV–vis spectra of porphyrins [9,14,15,19–22]. The
DNA titration absorption spectroscopy of porphyrin 2 is shown in
Fig. 2 and analogous spectra were obtained in the case of other
porphyrins (see supporting materials). All physical data about the
change of porphyrins’ absorption spectra in the presence of increas-
ing amounts of CT DNA are collected in Table 1. With increasing
CT DNA concentration, different extents of bathochromism and
hypochromism are observed in the Soret bands for all the metallo-
porphyrins, indicating different DNA-binding modes of them. The
large bathochromism and hypochromism of porphyrin 3 (9 nm and
45.9%) indicate that it may intercalate into DNA, which is similar to
the DNA-binding behavior of its free base porphyrin ligand 1. How-
ever, the moderate or minor changes in the absorbance spectra of
2, 4 and 5 suggest that these three metalloporphyrins may employ
non-intercalative binding modes.
3.1.2. Fluorescence titration
Fluorescence titration experiments in the presence of CT DNA
were performed. In our previous study, the fluorescence emission
of the free base porphyrin 1 decreased remarkably at the initial
stage of DNA titration and then increased significantly at higher
concentrations of DNA [19,20]. It could be ascribed to self-stacking
of the porphyrin molecules along the DNA surface, and is widely
accepted as a criterion for intercalation [17,19]. Large increases in
intensities of fluorescence emission are depicted for porphyrin 2 in
the presence of increasing DNA amount (Fig. 3), which implies that
Zn(II) porphyrin can be protected by the hydrophobic environment
inside the DNA helix efficiently. Since no obvious decease of fluo-
rescence emission for porphyrin 2 was found at any stage of DNA
titration, non-intercalative DNA-binding mode maybe employed
by the Zn(II) porphyrin.
Cu(II), Co(III) and Mn(III) porphyrins 3, 4 and 5 show negligi-
ble luminescence in buffer A and neglectable changes were found
with the addition of DNA. For complexes exhibiting a weak or no
emission intensity in the presence of DNA, competitive binding to
DNA with EB provides rich information regarding the nature of DNA
binding [26,27]. EB emits intense fluorescence in the presence of
DNA, due to its strong intercalation between the adjacent DNA base
pairs of DNA [28]. The emission spectra of DNA-bound EB solution
significantly quenched with increasing concentrations of the met-
alloporphyrins and the quenching plots were given in Fig. 4. From
Fig. 4, we can find that porphyrin 3 could quench the emission of EB
to a greater extent than porphyrin 4 and 5. It is widely accepted that
the emission decreases of DNA-bound EB solution results from the
extrusion of EB from DNA duplex structures. Generally, the extru-
sion of EB occurs by replacing of other intercalating compounds, or
In order to compare the affinities of the porphyrins bound to CT
DNA quantitatively, the intrinsic binding constants Kb were mea-
sured by monitoring the changes of absorbance in the Soret band
with increasing concentration of CT DNA using the following equa-
tion [15]:
[DNA]
(εa − εb)
[DNA]
(εa − εf)
1
=
+
Kb(εb − εf)
where [DNA] is the concentration of DNA in base pairs, εa, εf and
εb correspond to the apparent absorption coefficient Aobsd/[Por]
([Por], the concentration of porphyrin), the extinction coefficient
for the free porphyrin complex and the extinction coefficient for
the porphyrin complex in the fully bound form, respectively. From
Table 1
Physical data of porphyrins binding with CT DNA.
Porphyrin
UV titration
ICDsignal
ꢁTm (◦C)
Kb
ꢁꢀ (nm)
H%
1
2
3
4
5
10
3
9
5
6
47.5
29.8
45.9
30.8
36.8
−
9.1
3.4
7.8
4.6
5.0
4.9 × 105
1.8 × 105
4.1 × 105
2.4 × 105
3.3 × 105
+
−
+
+
−

P. Zhao et al. / Spectrochimica Acta Part A 75 (2010) 1108–1114
1111
Fig. 4. Fluorescence quenching plots of DNA-bound EB by 3 ( ), 4 ( ) and 5 (
in buffer A. [DNA] = 100 ␮M, [EB] = 16.0 ␮M, and ꢀex = 537 nm.
)
it can occur indirectly by changing the DNA conformation as a result
of binding of non-intercalating compounds [26–28]. The quenching
extent of direct EB replacement is always larger than the indirect
DNA change. Thus, it is suggested that the Cu(II) porphyrin 3 inter-
calate into DNA duplexes while the Co(III) and Mn(III) porphyrins
4 and 5 may change the conformation of DNA when bound with it.
3.1.3. SERS investigation
SERS is a powerful tool for the study of the interactions of drugs,
especially fluorescence molecules, with biomacromolecules at very
low concentrations. The metal colloids can be adapted for the appli-
cation in the study of biological objects, because they do not modify
to a great extent the structures of biological molecules adsorbed
on their surface [29]. To further prove the interactions of these
metalloporphyrins with CT DNA, SERS spectra were measured.
The SERS spectra of the porphyrins in the absence and presence
of CT DNA are shown in Fig. 5. Table 2 summarized the main SERS
bands of the porphyrins and their assignments. Fig. 5 also exhibited
the SERS spectrum of free CT DNA. It is found that no substantial
SERS signal is given by free CT DNA, since the Ag colloids have neg-
ative charges on their surface, which repulses the adsorption of
negative-charged DNA molecules [30].
Fig. 5. SERS spectra (250–3000 cm−1) of CT DNA and porphyrins in the absence
(1–5) and presence (1ꢀ–5ꢀ) of CT DNA. (1), (2), (3), (4), (5) for free porphyrins 1–5,
respectively; (1ꢀ), (2ꢀ), (3ꢀ), (4ꢀ), (5ꢀ) for DNA complexes with 1–5, respectively.
[DNA]/[Por] = 30:1.
marker band of Por ring doubtlessly proves the vanishment of
porphyrin molecules surrounding Ag colloids after the interaction
between metalloporphyrins and DNA [31–33]. The vanishment of
bands centered at 411, 814, 965, 1001, 1098, 1141 nm also indi-
cates the porphyrins are protected by DNA from adsorbing on Ag
particles.
Moreover, the spectral changes confirm that the cationic por-
phyrins efficiently bind to DNA even in Ag sols. In this medium,
the preference of cationic porphyrins for polyanionic DNA prevails
over negatively charged Ag nanoparticles. This also reveals the high
binding affinities between the cationic drugs and DNA [34].
Curves 1–5 and 1ꢀ–5ꢀ in Fig. 5 show the SERS spectra of the por-
phyrins in the absence and presence of DNA, respectively. In the
presence of DNA, most bands of the porphyrin compounds dis-
appear, indicating that the interactions of porphyrins with DNA
can largely reduce the amount of porphyrins adsorbed on Ag col-
loids [31]. Especially, the disappearance of the band at 330 cm−1
which is mainly assigned to the bending vibration and is the
Table 2
Raman frequencies and assignments for porphyrins.a.
Raman shift (cm−1
)
Assignmentsb
3.1.4. ICD studies
The ICD spectrum experiment may be one of the most direct
means in examining the binding modes of the porphyrin com-
pounds to DNA. None of these compounds as well as DNA by
themselves displays any CD spectra signal in the visible region,
but ICD spectra were observed in the Soret band of these com-
pounds in the presence of DNA due to the interaction between the
transition moments of the achiral porphyrin and chirally arranged
DNA base transitions. The sign of the ICD spectrum of DNA in the
Soret region can be used as a sensitive signature for the binding
modes of porphyrins to DNA: a positively ICD band is indicative
of external (minor groove) binding, a negatively ICD band is pro-
duced upon intercalation and a conservative bisignal ICD band is
the characteristic of outside binding [14,15].
330
411
814
965
1001
1098
1191
1214
1242
1337
1141
1554
ı(por)
ı(por) + ꢂ(Ag–N)
pyr ꢂ(N+–CH3), ıs(por)
ꢂ(C␣–C␤
ꢂ(Cm–C␣
ıs(C␤–H)
)
)
ı(pyr), ꢂ(N+–CH3)
ı(pyr), ꢂ(C␣–N)
ꢂ(Cm–pyr)
ꢂs(N–C␣
ꢂ(C␣–N)
ꢂ(C␤–C␤
)
)
a
Band assignments are according to Refs. [29–34].
b
Abbreviations: ꢂ, stretching mode; ı, bending mode; s, symmetric mode; pyr,
N-methylpyridinium; por, porphyrin core.

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P. Zhao et al. / Spectrochimica Acta Part A 75 (2010) 1108–1114
Fig. 6. ICD spectra of porphyrins in the absence (dot line) and presence (solid line)
of CT DNA in buffer A. [Por] = 10 ␮M; [Por]/[DNA] = 0.1.
Fig. 7. Melting curves of CT DNA at 260 nm in the absence ( ) and the presence of
1 ( ), 2 ( ), 3 ( ), 4 ( ) and 5 ( ), in buffer B. [Por] = 10 ␮M; [DNA] = 100 ␮M.
Fig. 6 and Table 1 give the results of ICD study which was also
carried out to investigate the binding modes of the cationic por-
phyrins. As shown in Fig. 6, free base porphyrin 1 shows strong
negative peak centered at ca. 445 nm upon binding to CT DNA,
indicating that it is an excellent DNA intercalator. Similar negative
signal was observed in the case of its copper derivative 3, suggest-
ing that the coordination of Cu(II) in the porphyrin core does not
influence the intercalation of porphyrin plane into the DNA bases.
As to the Zn(II)−, Mn(III)− complexes of 1, positive CD spectra in
the range of 410–430 nm were observed, which is consistent with
groove binding mode. As to Co(II) complexes 4, in the presence of
DNA, both positive and negative ICD spectra appeared, indicating
an outside binding mode. The result of ICD experiment unambigu-
ously proves the binding modes we proposed in the absorption and
fluorescence experiments.
From the experimental results, we can find that Cu(II) com-
plexes of free base porphyrin 1 can still intercalate into DNA bases
while Zn(II)−, Co(III), Mn(III) complexes can only employ outside
or groove binding modes when interact with DNA. This may result
from that, similar to the case of (H2TMPyP)4+, when inserts in the
porphyrin core of 1, Cu(II) has four-coordination structure and no
extra ligands on the axial position of porphyrin plane; Zn(II) has
five-coordination structure with a water molecule as the axial lig-
and; Co(III), Mn(III) have six-coordination structure with a water
molecule and a Cl atom as axial ligands [7,14,15,17]. The axial lig-
ands of these metalloporphyrins increase the steric hindrance of
the porphyrin molecules and thus, only Cu(II) derivative, porphyrin
3, is sterically appropriate to bind with DNA in intercalative mode
while the metalloporphyrins 2, 4 and 5 are not.
of DNA in the presence and absence of porphyrins, respectively.
The large increases of Tm in the presence of 1 and 3 (ꢁTm = 9.1
and 7.8 ◦C, respectively) indicate that these two porphyrins interact
with DNA in a typical intercalative mode and have strong DNA-
binding affinities. The small increases of Tm in the presence of 2,
4 and 5 (ꢁTm = 3.4, 4.6 and 5.0 ◦C, relatively) give us the informa-
tion that these metalloporphyrins do not intercalate into DNA and
have relatively weaker DNA-binding affinity than the free base por-
phyrin and its Cu(II) complex. These results are in good agreement
with the experiments above.
3.1.6. Viscosity measurement
In the absence of X-ray structural data, viscosity measurement
is regarded as the least ambiguous and the most critical test of a
DNA-binding model in solution and provides strong arguments for
intercalative DNA-binding mode. Intercalative mode is expected
to lengthen the DNA helix as the base pairs are pushed apart to
accommodate the bound ligand, leading to an increase in the DNA
viscosity. In contrast, a partial, non-classical intercalation of ligand
could bend (or kink) the DNA helix and reduce its effective length
and, concomitantly, its viscosity. When outside binding occurs, the
viscosity of DNA would not change basically [36].
Fig. 8 shows the effects of porphyrins on the viscosity of rod like
DNA, which is very sensitive to the binding modes of the ligands.
On increasing the amounts of 1 and 3, the relative viscosity of DNA
increases steadily. However, the increases of porphyrins 2, 4 and 5
have no obvious effect on the relative viscosity of DNA. The viscosity
results show that the free base porphyrin 1 and its Cu(II) derivative
intercalate between the base pairs of DNA, while the its Zn(II)−,
Co(III)−, Mn(III) complexes bind to DNA in outside or groove bind-
ing mode. The distinct results of viscosity experiment convincingly
prove the conclusion above.
3.1.5. Thermal denaturation studies
The melting temperature (Tm) of DNA is sensitive to its dou-
ble helix stability and the binding of compounds to DNA alters the
Tm depending on the strength of interactions [14,15]. Therefore,
it can be used as an indicator of binding properties of porphyrins
to DNA and their binding strength. Tm will considerably increase
when intercalation binding mode occurs [14,35].
The melting curves of CT DNA in the absence and presence
of porphyrins are presented in Fig. 7. The Tm of CT DNA is
(61.1 0.2) ◦C in the absence of the porphyrins. When mixed with
the porphyrins at a concentration ratio [Por]/[DNA] of 1:10, the
observed melting temperatures of CT DNA increase to different
degrees. The differences, designed as ꢁTm, are collected in Table 1.
Here ꢁTm = Tm − Tm0 , Tm and Tm0 refer to the melting temperature
3.2. DNA photocleavage experiment
The cleavage reaction of porphyrins on plasmid DNA could be
monitored by agarose gel electrophoresis. When circular plasmid
DNA is subject to electrophoresis, relatively fast migration will be
observed for the intact supercoil form (Form I). If scission occurs on
one strand (nicking), the supercoil will relax to generate a slower
moving open circular form (Form II). If both strands are cleaved, a
linear form (Form III) that migrates between Forms I and II will be
generated [37].

P. Zhao et al. / Spectrochimica Acta Part A 75 (2010) 1108–1114
1113
Fig. 10. Decomposition of DPBF by porphyrins 1 ( ), 2 ( ), 3 (
), 4 ( ) and 5
Fig. 8. Plots of the relative viscosity change of CT DNA in the presence of 1 ( ), 2
), 3 ( ), 4 ( ) and 5 (
) in buffer A at (30 0.1) ◦C. [DNA] = 0.5 mM.
(
). Porphyrin (1 ␮M) and DPBF (100 ␮M) were irradiated in pyridine.
(
Table 3
The slopes (S) of the plots of bleached absorption of DPBF by photosensition of
porphyrins.
In our previous research, porphyrin 1 was proved to employ 1O2
mediated mechanism [19–21] in DNA cleavage, which is a common
DNA photocleavage mechanism of porphyrins [38,39]. In order to
establish the reactive species responsible for the DNA photocleav-
age of the metal derivatives of 1, we investigated the influence of
different potentially inhibiting agents. Porphyrin 2 was taken as
an example and the result is shown in Fig. 9. Study with NaN3, a
1O2 quencher [40], was carried out and the cleavage was signif-
icantly inhibited (lane 2). The photocleavage efficiency was also
greatly inhibited when irradiation reaction was done under an Ar
atmosphere (lane 3). On the other hand, the photocleavage ability
was enhanced by replacing the reaction media H2O by 70% D2O
which makes the life span of 1O2 longer (lane 4). These results sug-
gest that 1O2 is likely to be one of the reactive species responsible
for the cleavage reaction. Meanwhile, the cleavage of the plasmid
DNA was not inhibited in the presence of hydroxyl radical scav-
engers such as ethanol (lane 5), methanol (lane 6) and DMSO (lane
7) even at high concentration, suggesting that hydroxyl radical may
not be the cleaving agent. Similar cases have also been observed
for other metalloporphyrins under identical conditions (data not
shown), indicating that 1O2 is the reactive species responsible for
the cleavage reaction of all these metalloporphyrins.
1
2
3
4
5
S
0.25
0.33
0.29
0.35
0.32
the insertion of metal ions into the cationic porphyrin enhanced
the 1O2 productivities effectively. Since it has been reported that
the self-aggregation of cationic porphyrins is disadvantageous in
1O2 production [41,42], this enhancement seems to be derived
from the increased steric hindrance of metalloporpyrins than free
base porphyrin, which could relax the self-aggregation of porphyrin
1.
Fig. 11 shows the DNA photocleavage results of the porphyrins
under identical irradiation by high-pressure lamp. Without irra-
diation, no substantial cleavage of DNA was observed for all the
porphyrins (data not shown). Under comparative experimental
conditions, the cleavage abilities of these metalloporphyrins have
no obvious difference with the free base porphyrin 1. Extensive
studies have shown that the DNA photocleavage activity of por-
phyrins is always in parallel with the magnitude of their intrinsic
binding constants (Kb) and the DNA intercalators are often more
effective in DNA photocleavage than the non-intercalative binders
[40,43,44]. The result shown in Fig. 11 is somewhat inconsis-
tent with this experience, since it has been proved above that
the metalloporphyrins have lower Kb and more disadvantageous
DNA-binding modes than 1. Nevertheless, it is noteworthy that
the experience (the good agreement between DNA photocleav-
age and Kb) is based an assumption that the compounds have the
same 1O2 productivity. Here, the steric hindrance derived from the
insertion of metals into porphyrin acts as a “double-edged sword”:
bring smaller Kb but also higher 1O2 productivity for the metallo-
porphyrins than the metal-free one. Thus, the DNA photocleavage
abilities have no obvious between these metalloporphyrins and
their free base porphyrin. The results indicate that both the DNA-
Because 1O2 was clearly responsible for the DNA photocleav-
age by the porphyrins in our system, photosensitized productivity
of 1O2 was estimated quantitatively by measuring the decompo-
sition of 1,3-diphenylisobenzofuran (DPBF). DPBF directly reacts
with 1O2 and subsequently decomposes to 1,2-dibenzoylbenzene.
As shown in Fig. 10 for 1 and its metal complexes, absorbance
of DPBF (30 mM) at 415 nm linearly decreased in the presence
of each porphyrin as illumination time increased. The slopes of
the plots of bleached absorption of DPBF versus illumination time
are listed in Table 3. The values of the slopes for the metallopor-
phyrins are larger than the free base porphyrin, indicating that
Fig. 9. Photocleavage of pBR322 DNA in the presence of porphyrin 2 and different
inhibitors after irradiation by high-pressure mercury lamp for 20 min, in buffer C.
10 ␮L reaction mixtures contained 1.0 ␮g of plasmid DNA. [Por] = 1 ␮M. Lane 0: DNA
control; lane 1: in the presence of porphyrin, no inhibitor; lanes 2–6: in the presence
of porphyrin and inhibitor, (2) NaN3 (5 mM), (3) under an Ar atmosphere, (4) 70%
D2O, (5) ethanol (5 mM), (6) methanol (5 mM), and (7) DMSO (5 mM).
Fig. 11. Photoactivated cleavage of pBR322 DNA in the presence of compounds,
light after 20 min irradiation by high-pressure mercury lamp, in buffer C. Lane 0:
DNA controlled; lanes 1–5: in the presence of 1 ␮m porphyrins 1, 2, 3, 4 and 5,
respectively.

1114
P. Zhao et al. / Spectrochimica Acta Part A 75 (2010) 1108–1114
binding affinity and the 1O2 productivity are key factors for the DNA
photocleavage of porphyrins.
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Based on absorption, fluorescence, SERS, ICD spectroscopy, ther-
mal denaturation and viscosity measurements, the binding modes
of Cu(II)−, Zn(II), Co(III), Mn(III) tricationic metalloporphyrins to CT
DNA were investigated in comparison with the free base porphyrin,
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Acknowledgements
This work was financially supported by the three following units
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Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.saa.2009.12.065.
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