Novel porphyrin-daunomycin hybrids: Synthesis and preferential binding to G-quadruplexes over i-motif

Article abstract of DOI:10.1016/j.saa.2014.08.123

Encouraged by the enormous importance attributed to the structure and function of human telomeric DNA, herein we focused our attention on the interaction of a serious of newly prepared porphyrin-daunomycin (Por-DNR) hybrids with the guanine-rich single-strand oligomer (G4) and the complementary cytosine-rich strand (i-motif). Various spectral methods such as absorption and fluorescence titration, surface-enhanced Raman and circular dichroism spectrum were integrated in the experiment and it was found that these Por-DNR hybrids could serve as prominent molecules to recognize G4 and i-motif. What is more, interesting results were obtained that the hybrids with longer flexible links are more favorable in binding with both G4 and i-motif than the hybrid with shorter linkage. These Por-DNR hybrids may help to develop new ideas in the research of human telomeric DNA with small molecules.

Full text of DOI:10.1016/j.saa.2014.08.123

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 137 (2015) 227–235
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Novel porphyrin–daunomycin hybrids: Synthesis and preferential
binding to G-quadruplexes over i-motif
a
b,
a
a
a
Ping Zhao a, , Shu-fang Jin , Jia-Zheng Lu , Jun-liang Lv , Gong-qing Wu , Pan-Pan Chen ,
⇑
⇑
Cai-Lian Tan a, Dian-Wen Chen a
a School of Chemistry and Chemical Engineering, Guangdong Pharmaceutical University, No. 13, Changmingshui Road, Zhongshan 528458, PR China
b School of Pharmacy, Guangdong Pharmaceutical University, No. 280, Waihuandong Road, Education Mega Centre, Guangzhou 510006, PR China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
The newly synthesized porphyrin–daunomycin hybrids could serve as prominent molecules to recognize
guanine-rich (G4) and cytosine-rich (i-motif) DNA strands, with bis-intercalation to G4 (a) and non-spe-
cific binding to i-motif DNA (b).
ꢀ
ꢀ
Cationic porphyrin was firstly linked
to a clinic drug, daunomycin.
The porphyrin–daunomycin hybrids
bind preferentially to G4 over i-motif
DNA.
ꢀ
Long-linked hybrids are more
favorable in binding with G4 or
i-motif structures.
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 May 2014
Received in revised form 18 August 2014
Accepted 24 August 2014
Available online 4 September 2014
Encouraged by the enormous importance attributed to the structure and function of human telomeric DNA,
herein we focused our attention on the interaction of a serious of newly prepared porphyrin–
daunomycin (Por–DNR) hybrids with the guanine-rich single-strand oligomer (G4) and the complemen-
tary cytosine-rich strand (i-motif). Various spectral methods such as absorption and fluorescence
titration, surface-enhanced Raman and circular dichroism spectrum were integrated in the experiment
and it was found that these Por–DNR hybrids could serve as prominent molecules to recognize G4 and
i-motif. What is more, interesting results were obtained that the hybrids with longer flexible links are more
favorable in binding with both G4 and i-motif than the hybrid with shorter linkage. These Por–DNR hybrids
may help to develop new ideas in the research of human telomeric DNA with small molecules.
Ó 2014 Elsevier B.V. All rights reserved.
Keywords:
Porphyrin–daunomycin hybrids
G-quadruplexes
i-Motif
Introduction
Telomeres are specific nucleoprotein structures and the telo-
⇑
Corresponding authors. Tel./fax: +86 760 88207939 (P. Zhao). Tel./fax: +86 20
39352021 (J.-Z. Lu).
meric DNA has a unique mode of replication based on a special
reverse transcriptase enzyme called telomerase. By catalysing the
synthesis of telomeric DNA, telomerase could maintain telomeric
E-mail addresses: zhaoping666@163.com (P. Zhao), lujia6812@163.com
(J.-Z. Lu).
http://dx.doi.org/10.1016/j.saa.2014.08.123
1386-1425/Ó 2014 Elsevier B.V. All rights reserved.

228
P. Zhao et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 137 (2015) 227–235
DNA integrity and prevent critical telomere shortening with suc-
cessive cell division so that cells cannot reach crisis points of
senescence and apoptosis. Thus, telomerase play a critical role in
cellular aging and cancer since they are essential for chromosomal
stability and genome integrity [1,2]. Telomerase is up-regulated in
80–85% of tumor cells in comparison to normal somatic cells and is
a major factor in cancer cell immortalisation. This unique activity
of telomerase makes the enzyme an ideal probe for tumor diagno-
sis and a target for cancer chemotherapy. Therefore, currently
there is considerable interest in finding molecules that can inhibit
telomerase and potentially act as anti-cancer drugs [3–5].
Formation of a quadruplex DNA structure, which is incompati-
ble with telomerase attachment to the 30 end, is a promising
approach to inhibit the enzyme. Human telomeric DNA consists
of multiple repeated 50-TTAGGG sequence on one strand and
repeated 50-CCCTAA sequence on the other one. However, the telo-
meric 30-terminal DNA is composed of a single-stranded G-rich
TTAGGG repeated overhang (approximately 100–200 bases) that
can form G-quadruplex (G4) DNA structures in vitro [6–10]. Simi-
larly, the complementary C-rich strand may also self-associate at
pH 5.5 (a value close to the pKa for free cytosine) into an interca-
lated structure called the i-motif based on intercalated CAC+ base
pairs [11,12] (Fig. 1). It is reported that quadruplex-binding and
quadruplex-promoting small molecules can in principle effectively
inhibit both the catalytic and capping functions of telomerase.
Many small aromatic ligands, including porphyrins, quinacridones,
anthraquinones, phenanthrolines, substituted triazines and
acridines, have been shown to bind and stabilize the quadruplex
structure of G-quadruplex DNA [13–15].
porphyrin (5,10,15,20-tetrakis(1-methyl-4-pyridyl)-21H,23H-por-
phine) (TMPyP4) has been intensively researched as a good G-
quadruplex-binding model because of its unique symmetric struc-
ture and high positive charge [16,17]. On the other hand, dauno-
mycin (DNR) is routinely used in the clinic for the treatment of
leukemia, ovarian and breast carcinoma. In the past years, numer-
ous anthracyclines derivatives have been synthesized with the aim
of finding related compounds showing better DNA binding, which
is closely related to higher therapeutic efficacy [18–20].
However, it is worth pointing out that the efforts on the
research of DNR interactions with G-quadruplex and i-motif DNA
are still inadequate in contrast to its considerable use as duplex
DNA binders. Herein, we attached the clinic DNR molecules to cat-
ionic porphyrin through different flexible carbochains and tested
their interactions with both G-quadruplex and i-motif DNA
through the combination of various spectral methods. Since both
the cationic porphyrin and DNR structures have positive charges
and large molecular planes, it is expected that the integration of
these two structures will be much favorable in DNA binding. Since
it is well known that the structure of G-quadruplex DNA is cat-
ionic-sensitive (antiparallel in the presence of Na+ and parallel in
K+ solution) while the i-motif is pH-sensitive (stable at a maximum
pH value at 5.5) [6–12,21,22], all the experiments in this study
were carried out in buffer A.
Experimental
Materials
Porphyrins with their large aromatic structure and the four
meso-positions available for substitution are interesting scaffold
for the targeting of G-quadruplex DNA. In particular, the free base
Daunorubicin hydrochloride (Sigma), 1,3-dibromopropane,
hexamethylene dibromide and 1,9-dibromononane (Sigma), silica
gel (Qingdao) and chloroform (Guangzhou) were commercially
Fig. 1. Structures of a G-quartet showing the anti-parallel G-quadruplex topology in the sodium solution (a), i-motif (b), Hoogstaeen base pairing (c), C+AC base pair (d) and of
human telomeric sequences AG3(T2AG3)3 and C3(TA2C3)3. Arrows denoted 50 ? 30 strand alignment for each DNA.

P. Zhao et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 137 (2015) 227–235
229
available and of analytical grade. MilliQ water was used to prepare
buffer solutions. Buffer A (10 mM Tris–HCl, 1 mM EDTA and
100 mM NaCl, pH 5.5) solution was used in spectral, SERS and EB
competing experiments and buffer B (10 mM Na2HPO4/NaH2PO4,
100 mM NaCl and 1 mM Na2EDTA, pH 5.5) was used for thermal
denaturation studies.
measured at room temperature. The final concentrations of hybrids
and DNA in all of the SERS-active systems were 5 and 150
respectively.
lM,
Synthesis of hybrids
Porphyrin
tris(4-pyridiniumyl)-porphyrin (BrPPTPyP). 5-(4-Hydroxyphenyl)-
10,15,20-tris(4-N-pyridiniumyl)porphyrin (HTPyP, formerly
derivates. 5-(1-Bromopropylhydroxylphenyl)-10,15,20-
DNA oligomers 50-AGGGTTAGGGTTAGGGTTAGGG-30 (22 AG)
and its corresponding complementary strand 50-CCCTAACCC-
TAACCCTAACCCT-30 (22 CT) were purchased from Shanghai Sangon
Biological Engineering Technology & Services Co., Ltd. (China) and
used without further purification. The formations of intramolecu-
lar G4 and i-motif were carried out as follows: the oligonucleotide
samples were dissolved in buffer A and heated to 90 °C for 5 min.
Then the samples were gently cooled to room temperature, and
incubated at 4 °C overnight. Concentrations of these oligomers
were determined by measuring the absorbance at 260 nm after
melting. Single-strand extinction coefficients were calculated from
mononucleotide data using a nearest-neighbor approximation
with the molar extinction coefficients at 260 nm are 215,000 and
185,900 Mꢁ1 cmꢁ1 for 22 AG and 22 CT, respectively [23].
prepared [25]) was dissolved in a mixture of N,N-dimethylform-
amide (DMF) 6 mL. Then, Na2CO3 (100 mg) and 1,3-dibromopro-
pane (68.7 mg, 0.34 mmol) were added. The reaction mixture
was stirred at room temperature for 20–28 h. The progress of the
reaction was monitored by TLC. After the reaction was complete,
the reaction mixture was diluted with CHCl3 and poured into
water. The organic layer was separated and washed with water
until neutral pH, dried over anhydrous Na2SO4, and evaporated
under diminished pressure. The purple powder was dissolved in
methylene chloride and chromatographed on silica gel using meth-
ylene chloride and ethanol (35:1) as eluent, collecting the first
band from the column as the objective BrPPTPyP (178 mg,
0.153 mmol, 61.5% yield). ESI-MS m/z 753.2 ([MꢁH]ꢁ, calcd for
Instruments
C44H32BrN7O: 754.3).
5-(Bromohexylhydroxylphenyl)-10,15,20-tris(4-pyridiniumyl)-por-
Element analysis (C, H, and N) was carried out with a Perkin-
Elmer 240 Q elemental analyzer. 1H NMR spectra were recorded
on a Varian-300 spectrometer. All chemical shifts are given relative
to tetramethylsilane (TMS). Electrospray Ionization mass spectra
(ESI-MS) were recorded on a LCQ DECA XP system (Thremo,
USA). UV–Vis spectra were recorded on a Hitech UV-3900 spectro-
photometer. Fluorescence spectra were recorded on a Perkin-Elmer
L55 spectrofluorophotometer. Circular dichroism (CD) spectra
were recorded on a JASCO-J810 spectrometer. Surface-enhanced
Raman spectroscopy (SERS) spectra were carried out on a Laser
Micro-Raman Spectrometer of Renishaw in-via, with a power of
20 mW at the samples.
phyrin (BrHPTPyP) and 5-(1-bromodecylhydroxylphenyl)-10,15,20-
tris(4-pyridiniumyl)-porphyrin (BrDPTPyP) were similarly prepared,
replacing 1,3-dibromopropane by hexamethylene dibromide and
1,10-dibromononane, respectively.
BrHPTPyP. Yield: 63.2%. ESI-MS m/z 795.2 ([MꢁH]ꢁ, calcd for
C
47H38BrN7O: 796.2).
BrDPTPyP. Yield: 68.6%. ESI-MS m/z 851.3 ([MꢁH]ꢁ, calcd for
C
51H46BrN7O: 852.3).
Synthesis of Por–DNR hybrids. The synthetic scheme of Por–DNR
hybrids is shown in Scheme 1.
Synthesis of hybrid 1. Daunorubicin hydrochloride was firstly
dissolved in double distilled water and 5% NaOH solution was
added to adjust the pH to 10. The daunorubicin was then precipi-
tated and extracted by dichloromethane. The organic phase was
evaporated and the deprotonated daunorubicin was obtained after
drying under vacuum. Deprotonated daunorubicin (0.5 mmol,
0.272 g) and anhydrous K2CO3 (0.7 g) in dry DMF (20 mL) were
stirred for 2 h at room temperature. Then BrPPTPyP (0.5 mmol,
0.376 g) was added to the activated daunorubicin, and the mixture
was then stirred for 36 h at room temperature. The progress of the
reaction was monitored by TLC. The reaction mixture was then
diluted by CHCl3 and DMF was washed out by water. The extracted
crude material was purified by chromatography. Evaporation of
solvent afforded compound, 5-(1-bromopropyl)-10,15,20-tris(4-
Npyridiniumyl)porphyrin, (1a in Scheme 1) as a purple powder
(yield: 0.394 g, 65%). 1a was methylated in 5 mL of DMF with
methyl iodide (0.8 mL) for 3 h at room temperature. The solvent
and methyl iodide were removed under vacuum. Subsequently,
the residue was dissolved in a 1 M solution of hydrochloride in
methanol and then precipitated with ethyl ether as hydrochloride
salt, which was washed with ethyl ether until neutral pH and dried
under vacuum. Compound 1 was thus obtained quantitatively.
Yield: 0.384 g, 94%. (Found: C, 67.38; H, 6.13; N, 8.32%. Calc. for
Methods
Absorption, fluorescence, CD spectrum
For absorption and fluorescence spectra, the aliquot DNA pre-
pared in buffer A solution was added stepwise to the sample cell
containing the Por–DNR hybrids. After equilibration for 10 min
absorption or fluorescence spectrum were recorded. The titration
processes were repeated until there was no change in the spectra
for at least three titrations indicating the binding saturation had
been achieved.
For CD spectra, Por–DNR hybrids mixed with G4 or i-motif
DNAs at a ratio of [DNR]/[DNA] = 5 in the buffer A. The mixture
was incubated at 4 °C overnight. Each measurement was the aver-
age of three repeated scans recorded with a 0.1 mm quartz cell
with reaction volume of 1 mL at 25 °C. A background CD spectrum
of corresponding buffer solution was subtracted from the average
scan for each sample. CD spectrum of the compound alone
(5 lM) was also measured in the buffer solution. Final analysis of
the data was carried out using Origin 7.5 (Origin Lab Corp.).
SERS experiment
Ag colloids were prepared by reducing AgNO3 with EDTA
according to the reported method [24]. The Ag colloid/hybrid (or
Ag colloid/DNA) SERS active systems were prepared by mixing
equal volume of the hybrids (or DNA) solution with the Ag colloid
in buffer A buffer to obtain the desired hybrid or DNA concentra-
tions. In the hybrid/DNA complex experiments, the solution of
DNA was mixed with the Por–DNR hybrid solution at a DNA/hybrid
ratio of 30:1, then an equal volume of the mixed solution was fully
mixed with the Ag colloid, and the spectrum was immediately
C75H73I3O11N8.4H2O: C, 67.51; H, 6.07; N, 8.40%). ESI-MS m/z
319.3 (M4+, calcd for C75H73O11N8: 1262.4). 1H NMR (300 MHz,
DMSO): chemical shift d 9.37 (d, J = 5.9 Hz, 6H, 2, 6-pyridinium),
9.07 (d, J = 6.6 Hz, 6H, 3,5-pyridinium), 8.90 (s, 8H, b-pyrrole),
8.16 (s, 2H, Por-2, 6-phenyl), 8.06 (s, 1H, DNR-phenyl), 7.83 (s,
1H, DNR-phenyl), 7.45 (s, 2H, Por-3,5-phenyl), 7.38 (s, 1H, DNR-
phenyl), 6.1 (s, 4H, OH), 5.92 (s, 2H, NH2), 5.2 (s, H, DNR-cyclo-
H), 4.79 (s, 1H, DNR-cyclo-H), 4.73 (s, 9H, N+ACH3), 4.21 (s, 1H,
DNR-cyclo-H), 4.11 (s, 2H, ACH2AO), 3.95 (s, 3H, AOACH3), 3.91

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P. Zhao et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 137 (2015) 227–235
Scheme 1. The synthetic scheme of cationic Por–DNR hybrids.
(s, 1H, DNR-cyclo-H), 3.2 (s, 2H, CH2AN), 3.11 (s, 1H, DNR-cyclo-H),
2.97 (d, J = 5.6 Hz, 2H, DNR-cyclo-H), 2.54 (s, 2H, ACH2A), 2.41 (s,
3H, O@CACH3), 2.35 (d, J = 4.8 Hz, 2H, DNR-cyclo-H), 1.79 (s, 2H,
DNR-cyclo-H), 1.06 (s, 3H, CH3), ꢁ3.10 (s, 2H, NH pyrrole).
(M4+, calcd for C82H86O11N8: 1360.3).1H NMR (300 MHz, DMSO):
chemical shift d: 9.37 (d, J = 5.9 Hz, 6H, 2,6-pyridinium), 9.04 (d,
J = 6.6 Hz, 6H, 3,5-pyridinium), 8.91 (s, 8H, b-pyrrole), 8.13 (s, 2H,
Por-2, 6-phenyl), 8.06 (s, 1H, DNR-phenyl), 7.83 (s, 1H, DNR-phe-
nyl), 7.4 (s, 2H, Por-3,5-phenyl), 7.35 (s, 1H, DNR-phenyl), 6.1 (s,
4H, OH), 6.01 (s, 2H, NH2), 5.17 (s, H, DNR-cyclo-H), 4.8 (s, 1H,
DNR-cyclo-H), 4.73 (s, 9H, N+ACH3), 4.2 (s, 1H, DNR-cyclo-H),
4.02 (s, 2H, ACH2AO), 3.94 (s, 3H, s, 3H, AOACH3), 3.88 (s, 1H,
DNR-cyclo-H), 3.11 (s, 1H, DNR-cyclo-H), 3.02 (s, 2H, CH2AN),
2.97 (d, J = 5.6 Hz, 2H, DNR-cyclo-H), 2.4 (s, 3H, O@CACH3), 2.34
(d, J = 4.8 Hz, 2H, DNR-cyclo-H), 1.83 (d, J = 4.8 Hz, 4H,
A(CH2)2A), 1.76 (s, 2H, DNR-cyclo-H), 1.54 (d, J = 4.8 Hz, 4H,
A(CH2)2A), 1.26 (d, J = 5.6 Hz, 8H, A(CH2)4A), 1.04 (s, 3H, DNR-
cyclo-CH3), ꢁ2.94 (s, 2H, NH pyrrole).
Compounds
2 and 3 were similarly prepared, replacing
BrPPTPyP by BrHPTPyP and BrDPTPyP, respectively.
2, yield: 89%. (Found: C, 69.13; H, 6.22; N, 8.23%. Calc. for C78-
H78I3O4N8ꢂ3H2O: C, 69.03; H, 6.19; N, 8.26%). ESI-MS m/z 330.3
(M4+, calcd for C78H78O4N8: 1304.2). 1H NMR (300 MHz, DMSO):
chemical shift d: 9.38 (d, J = 5.9 Hz, 6H, 2, 6-pyridinium), 9.05 (d,
J = 6.6 Hz, 6H, 3,5-pyridinium), 8.92 (s, 8H, b-pyrrole), 8.14 (s, 2H,
Por-2, 6-phenyl), 8.04 (s, 1H, DNR-phenyl), 7.83 (s, 1H, DNR-phe-
nyl), 7.4 (s, 2H, Por-3,5-phenyl), 7.37 (s, 1H, DNR-phenyl), 6.1 (s,
4H, OH), 5.92 (s, 2H, NH2), 5.19 (s, H, DNR-cyclo-H), 4.8 (s, 1H,
DNR-cyclo-H), 4.73 (s, 9H, N+ACH3), 4.21 (s, 1H, DNR-cyclo-H),
3.99 (s, 2H, ACH2AO), 3.95 (s, 3H, AOACH3), 3.91 (s, 1H, DNR-
cyclo-H), 3.2 (s, 2H, CH2AN), 3.11 (s, 1H, DNR-cyclo-H), 2.97 (d,
J = 5.6 Hz, 2H, DNR-cyclo-H), 2.41 (s, 3H, O@CACH3), 2.34 (d,
J = 4.8 Hz, 2H, DNR-cyclo-H), 1.83 (d, J = 4.8 Hz, 4H, A(CH2)2A),
1.76 (s, 2H, DNR-cyclo-H), 1.26 (d, J = 4.8 Hz, 4H, A(CH2)2A), 1.04
(s, 3H, DNR-cyclo-CH3), ꢁ2.95 (s, 2H, NH pyrrole).
Results and discussion
Synthesis
In our previous work, we have successfully obtained porphyrin–
anthraquinone hybrids by attaching flexible carbochains to the
anthraquinone molecules firstly and then linked to the porphyrin
3, yield: 87% (Found: C, 68.44; H, 6.65; N, 7.78%. Calc. for C82H86-
I3O11N8.4.5H2O: C, 68.38; H, 6.61; N, 7.78%). ESI-MS m/z 344.1

P. Zhao et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 137 (2015) 227–235
231
Fig. 2. Absorption spectra of 2 in buffer A at 25 °C in the presence of increasing amounts of 22 AG (a) or 22 CT (b). [DNR] = 10 lM. Arrows indicate the change in absorbance
upon increasing the DNA concentration.
moiety [25]. However, this experience failed in synthesizing Por–
DNR hybrids because the DNR molecule has more active groups
than anthraquinone. Without the complex and troublesome group
protection process, the active OH and NH2 groups in DNR mole-
cules will form rings when react with brominated carbon chains.
Thus, we have to attach the flexible chains to the porphyrin part
firstly rather than employ the synthetic scheme similar to porphy-
rin–anthraquinone hybrids. This method has the advantages of
high productivity and effortlessness, with no group protection pro-
cess needed.
475 nm (hybrid’s maximum excitation wavelength), the hybrids
have similar emission spectra, ranging from 520 nm to 670 nm.
In Fig. 3, fluorescence emission spectrum of 1 was selected to show
the spectral change with increasing G4-DNA and i-motif and we
can find that the fluorescence intensities were remarkably
enhanced. Hybrids 2 and 3 have similar results and all these
hybrids have smaller fluorescence change under i-motif than that
under G4-DNA. Since the hydrophobic environment provided by
DNA can protect the hybrid from quenching by water molecules,
the emission increase is widely admitted as an indication of the
interaction between compounds and DNA [28]. Thus, the different
emission increase also suggests that the hybrids interact with G4-
DNA more strongly than that with i-motif.
DNA-binding properties
Absorption titrations
Fig. 2 exemplifies the absorption spectral changes of hybrid 2
upon G4-DNA and i-motif addition. With the increasing of G4-
DNA (22 AG) and i-motif (22 CT) concentration, the absorption
spectrum of the cationic Por–DNR hybrid 2 was significantly dis-
turbed with hypochromism and bathochromism. This is an intui-
tional evidence of the compound–DNA binding behavior [26,27].
Similar spectral change was observed for hybrids 1 and 3.
Table 1 summarizes the detailed titration data on G4-DNA and
i-motif of all these three Por–DNR hybrids. From Table 1, all Por–
DNR hybrids have larger hypochromism and red shift when bind-
ing with G4-DNA than with i-motif, suggesting that these hybrids
have more intense interactions with the former. Meanwhile,
hybrid 1 with shorter flexible linkages has smaller absorption
change than 2 and 3, from which we could consider that the Por
and DNR moieties in hybrid 1 may suffer from steric hindrance
and thus could not bind with the DNA structures flexibly. However,
the longer bridging chain lengths in 2 and 3 decrease the steric hin-
drance between Por and DNR and the two moieties could employ
more favorable binding modes.
CD spectra
The CD spectral method, which is a very useful in distinguishing
the DNA secondary structures, was employed to study the interac-
tion of these hybrids with G4 and i-motif DNA. As shown in Fig. 4,
none of these hybrids displays any CD spectral signal in the ultra-
violet region. In the presence of Na+, the CD spectrum of 22 AG
shows a strong positive band at around 290 nm and a negative
band at around 265 nm, which indicates the formation of an
anti-parallel G-quadruplex structure [21]. Meanwhile, the CD spec-
tra of C-rich strand alone showed a large positive band with a peak
at 287 nm and a negative band centered at 255 nm, which is char-
acteristic of the i-motif structure [22].
As to G4-DNA, in the presence of the hybrids 1–3, the intensities
of both positive and negative bands are increased significantly
(Fig. 4a), which suggests that an anti-parallel structure is enhanced
and stabilized by compounds [26]. However, excepted for slight
red shifts in the positive peak and negative bands, no substantial
changes were observed for i-motif in the presence of hybrids
(Fig. 4b), implying that the hybrids affected the structure of i-motif
to a certain extent [14,29,30]. The distinct CD spectral change
between G4 and i-motif DNA indicates that the former may employ
a more efficient binding with the hybrids than the latter. This
Fluorescence titration studies
Fluorescence titration experiments of the hybrids in the pres-
ence of G4-DNA and i-motif were performed. When excited with
Table 1
Physical data of Por–DNR hybrids binding with G4 and i-motif DNAs.
Hybrid
UV–Vis spectra
G4
D
Tm (°C)
Kb (ꢃ106 Mꢁ1
)
i-Motif
G4
i-Motif
G4
i-Motif
D
k (nm)
H%a
D
k (nm)
H%
1
2
3
10
17
16
31.47
53.04
51.35
5
8
7
22.31
27.32
32.78
4.1
14.2
9.3
5.8
6.7
4.2
8.3
9.2
8.5
4.4
5.8
5.1
a
H% refers to the extent of hypochromism for absorption intensity.

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P. Zhao et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 137 (2015) 227–235
Fig. 3. Fluorescence emission spectra of 1 in buffer A upon increasing concentrations of 22 AG (a) or 22 CT (b). kex = 475 nm. [DNR] = 10 lM. Arrows indicate the change in
absorbance upon increasing the DNA concentration.
Fig. 4. CD spectra of free Por–DNR hybrids 1 (. . .), 2 (ꢂ –ꢂ –) and 3 (– – –), 22 AG (a) and 22 CT (b) in the absence (—) and in the presence of 1 ( ), 2 ( ) and 3 ( ) in buffer A.
[DNR]/[DNA] = 5. Arrows indicate the change in CD spectra upon increasing the concentration of the compounds.
result is in high consistent with the absorption and fluorescence
titration experiments above.
centered at 330, 411, 814, 965, 1001, 1098, 1141 nm which are
attributed to the porphyrin ring, indicating that the porphyrin
structures of Ag-hybrid complexes were undetectable [25,31–36].
The result was suggested that, when binding with 22 AG, the
porphyrin part of the hybrids may embed into the base pairs of
G4-DNA structures, and thus was well protected by DNA
molecules. Meanwhile, the band at 1545 cmꢁ1 which is the charac-
teristic band of anthraquinone plane significantly decreased [36],
SERS investigation
SERS is a powerful tool for the study of the interactions of com-
pounds, especially fluorescence molecules, with biomacromole-
cules at very low concentrations. The silver colloids can be
adapted for the application in the study of biological objects,
because they do not modify to a great extent the structures of
biological molecules adsorbed on their surface [24,31]. To further
clarify the binding modes of these compounds to G4 DNA and
i-motif, SERS spectra were measured.
The SERS spectra of free hybrid 1, the Por–DNR hybrids in the
presence of G4 and i-motif DNAs, are shown in Fig. 5. In the
absence of DNA, free hybrids 1–3 show very similar SERS spectra
because of their much similar molecular structures (see Fig. S1).
Table 2 summarized the main SERS bands of the hybrids and their
assignments. No substantial SERS signal is given by G4-DNA or i-
motif, since the Ag colloids have negative charges on their surface,
which repulses the adsorption of negative-charged DNA molecules
[31–33].
The SERS intensities for all these hybrids in the presence of 22
AG were significantly decreased. In terms of the short-range char-
acter of the Raman enhancement in a colloid system, the SERS
quenching is interpreted in term of a loss of accessibility for com-
pounds to the Ag colloids [25,31–36]. The vanishment of bands
Fig. 5. SERS spectra (300–3000 cmꢁ1) of 22 AG ( ), 22 CT ( ), free hybrid 1 (—),
22 AG bound 1 ( ), 2 ( ), 3 ( ) as well as 22 CT bound 1 ( ), 2 ( ), 3 ( ).
[DNA]/[DNR] = 30:1 in buffer A.

P. Zhao et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 137 (2015) 227–235
233
Table 2
On the other hand, in the presence of i-motif, most of the char-
acteristic porphyrin and DNR bands for these hybrids were still
remained although the intensities significantly decreased, suggest-
ing that the porphyrin and DNR moieties were still exists on the
surface of the hybrid–DNA complexes rather than embedded in
the i-motif molecules. The remained SERS signals indicate that
the studied hybrids may bind to the i-motif molecules in a groove
or end-pasting mode, which consists with the studies above.
Moreover, the chosen protocol for the SERS sample preparation
allows the G4-hybrid adduct to be formed before its addition to the
colloid solution (see Experimental). Consequently, the spectral
changes confirm that the Por–DNR hybrid sufficiently bind to G4
DNA or i-motif even in Ag sols. In this medium, the preference of
cationic hybrids for polyanionic G4-DNA or i-motif prevails over
negatively charged Ag nanoparticles [37]. This result proves the
tight binding affinities between the cationic hybrids and these
DNA structures.
Raman frequencies and assignments for Por–DNR hybrids.a
Raman shift
Assignmentsb
Raman shift
Assignmentsb
(cmꢁ1
)
(cmꢁ1
)
330
411
814
d(por)
1337
m
m
m
m
s(NAC )
s(CH2)
s(C14AN15
(AQ)
a
d(por) +
pyr
(N+ACH3)
ds(por)
m
(AgAN)
1443
1500
1554
1635
2580
m
)
965
1001
1098
1141
1191
1214
1242
m
m
(C ACb)
d(pyr) +
(CAH)
d(cyclohexane)
m
s(C@O)
a
(CmAC )
m
a
ds(CbAH)
(C AN)
2650
m
2730
m
m
m
m
(CAO12
s(CH2)
(CH3)
)
a
d(pyr),
d(pyr),
m
m
(N+ACH3) 2761
(C AN)
2854
2952
a
m
(CmApyr)
(CH2)
a
Band assignments are according to Refs. [25–34].
Abbreviations: m, stretching mode; d, bending mode; s, symmetric mode; pyr, N-
b
methylpyridinium; por, porphyrin core.
Competitive binding experiments with EB
For hybrids with multiple binding sites, traditional UV–Vis
titration method is not suitable to determine Kb between hybrids
and DNA [25,35]. Fluorescence spectrum was used to measure Kb
by competition between EB and the studied hybrids for binding
to DNA. This method measures the decrease of fluorescence of EB
bound to DNA in the presence of the hybrid of interest. It can be
used for all hybrids having a good affinity for DNA whatever their
binding modes may be because it only measures the ability of a
hybrid to compete with EB in binding with DNA [25,38,39].
The EB competitive binding experiments were carried out and
quenching plots are given in Fig. 6. The quenching plots of I0/I vs
[DNR]/[DNA] are in good agreement with the linear Stern–Volmer
equation. By taking Kb of 1.0 ꢃ 107 Mꢁ1 for EB with DNA, the Kb of
these hybrids with G4 DNA and i-motif were calculated in Table 1
[25,38,39]. The binding affinities of the Por–DNR hybrids with 22
AG are larger than those with 22 CT, indicating that the G-quadru-
plex is a more favorable target compared with i-motif DNA. Mean-
while, the binding affinities of the Por–DNR hybrids with longer
bridging links (2 and 3) are higher than that with shorter bridging
link (1), in the both cases of G4 and i-motif DNAs. We suppose that
both porphyrin and DNR moieties in the long-linked hybrids
intercalate into DNA bases and both can replace EB from DNA helix,
leading to relatively high binding affinities, whereas the short
linked hybrids could not bis-intercalate DNA because of the steric
hindrance.
Fig. 6. Fluorescence quenching plots of 22 AG-bound EB in buffer A by 1 ( ), 2 (
)
and 3 (j) in buffer A and 22 CT-bound EB by 1 ( ), 2 ( ) and 3 (h) in buffer A.
[DNA] = 100 M, [EB] = 16.0 M, kex = 537 nm.
l
l
indicating that the anthraquinone moieties in DNR are also well
protected by G4 DNA molecules. However, the remaining of bands
ranging from 2500 to 3000 nm, which can be mainly ascribed to
the sugar moieties in DNR and carbon links between porphyrin
and DNR parts in the molecule [34–36], suggests that the sugar
moieties and flexible carbon links may be still exposed in the
solvent. These significant changes in the SERS intensities of the
hybrids indicates that, the porphyrin and anthraquinone plane
intercalate into G4-DNA bases because of their excellent planarity,
while the sugar moieties and flexible carbon links bind on the sur-
face or groove of the DNA structures. Namely, a ‘‘sandwich’’-type
interaction mode was employed here.
Thermal denaturation studies
The melting curves of G4 DNA and i-motif in the absence and
presence of hybrids are presented in Fig. 7. When mixed with the
hybrids, the observed melting temperatures of G4-DNA and i-motif
Fig. 7. Melting curves of 22 AG (a) and 22 CT (b) in the absence (j) and presence of 1 ), 2 ( ) and 3 ( ), in buffer B. [DNR] = 10 lM, [DNA] = 100 lM.

234
P. Zhao et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 137 (2015) 227–235
Fig. 8. Job plot resulting from the method of continuous variation analysis for hybrids 1 ( ), 2 (j) and 3 ( ) with 22 AG (a) and 22 CT (b) in buffer A.
increase to different extents and the increases of Tm (
summarized in Table 1.
D
Tm) are
understanding of the biological implications of these structures
and for designing new compounds with enhanced activity and
minimized undesired toxicity. Further efforts on the biological
research such as cytotoxicity and cellular uptake are currently
underway.
From Table 1, we can find that, in the presence of 1, 2 and 3, the
values of Tm for 22 AG are relatively larger than those for 22 CT,
D
suggesting that the hybrids have stronger DNA binding affinities
with G4-DNA than with i-motif. [29,30]. This result is in consistent
with the SERS spectra, from which we could observe smaller signal
change of i-motif than G4-DNA. Meanwhile, the larger increases of
2 and 3 than 1 could be understood by the fact that the Por and
DNR moieties in 2 and 3 could bind more flexibly while in 1 they
suffer from steric hindrance. The results of thermal denaturation
further argue for the DNA binding modes we mentioned above.
Acknowledgements
This work was financially supported by the National Nature
Science Foundation of China (21101033) and Guangdong
Pharmaceutical University.
Appendix A. Supplementary material
Continuous variation analysis
Continuous variation analysis (Job plot) was employed to deter-
mine the binding stoichiometry of hybrids with G4 and i-motif
DNAs, as shown in Fig. 8. In the case of G4 DNA, the intersection
points were 0.50, 0.54, and 0.55 for hybrids 1, 2 and 3, respectively,
which corresponded to the stoichiometric ratio of 1:1 (one hybrid
molecule to per quadruplex), whereas the corresponding values
were 0.51, 0.53 and 0.53 for i-motif/hybrid, suggesting their bind-
ing stoichiometric ratios were also 1:1.
Ma et al. has reported a compound contained a pendant COOH
functional group which was found to have a strong preference for
binding to G-quadruplexes, which might be involved in H-bonding
interaction with the guanine in the external tetrad of G-quadruplex
DNA [40]. It is supposed that, besides the different binding modes,
the preferential selectivity of the Por–DNR hybrids to G4-DNA than
i-motif may also relate to the H-bonding interaction between the
guanine in the external tetrad of G-quadruplex DNA and the OH
groups in DNR moiety of the hybrids.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.saa.2014.08.123.
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