# Cationic Porphyrin Induced a Telomeric DNA to  G-Quadruplex Form in Water

Authors: Takashi Murashima; Daichi Sakiyama; Daisuke Miyoshi; Masanori Kuriyama; Takashi Yamada; Toshifumi Miyazawa; Naoki Sugimoto
Journal: Bioinorganic Chemistry and Applications
 (2008)

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## Abstract

The formation of the DNA G-quadruplex is induced by the addition of a novel porphyrin carrying four cationic tethers. Circular dichroism spectroscopy reveals that the porphyrin binds toTetrahymena telomeric repeat to form G-quadruplex under stabilizing-cation-deficient and no buffer conditions.


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## Body

## 1. Introduction

Guanine-rich tandem repeats can be folded to form quadruplex structures through Hoogsteen hydrogen bonding [1].
These guanine-rich segments are found in biologically important regions such as
telomeres [2],  c-myc gene promoter
regions [3], immunoglobulin switch regions [4], and fragile X-syndrome triplet
repeats [5]. Not only because their biological importance but also because the
potential applications in supramolecular chemistry and nanotechnology, a
G-quadruplex attracted much attention in the field of medicinal chemistry,
pharmaceutical biology, and material sciences. For example, telomere sequences
have been used for the construction of nanomolecular machines [6–8] and DNA logic
gates [9–11].Because the G-quadruplex polymorphism
and the duplex-quadruplex conversion are affected by environmental factors, we
[12–14] and others
[15, 16] have investigated the structure and thermodynamic stability of DNAs
under molecular crowding conditions as a mimic of the cellular conditions.
Another important factor for the stabilization and polymorphism of the
G-quadruplex structure is the presence of certain metal cations, effectively K+ and Na+ [1, 17, 18].  Most of
the previous studies about the structure and stability of G-quadruplex were
conducted in cation-containing solutions, and to our knowledge, only a few
reports have been published using salt-deficient conditions in buffer solution
[19–28]. Since DNA
secondary structures such as  quadruplex
exist only within a narrow range of biophysical conditions or their mimic
conditions, chemical probe that traps quadruplex structure under
simple conditions is desired. Here, we report that small organic molecule
induced a telomeric DNA to G-quadruplex formation under noncrowding, and no
buffer conditions in the absence of added cation.


## 2. Experimental

## 2.1. Materials and Methods

Most
of the reagents and solvents were purchased from Wako Pure Chemical Industries,
Ltd., Tokyo Kasei Kogyo Co., Japan, and Sigma-Aldrich Co., Mo, USA, and used without further
purification.1H NMR spectra were recorded on a Varian UNITY 300
spectrometer at 299.94 MHz by using CDCl3 or CD3OD as a
solvent and tetramethylsilane as an internal standard, and J values are given in Hz. Mass spectra were measured with a PerSeptive
Biosystems Voyager DE-Pro. Circular dichroism (CD) spectra of DNA quadruplexes
were obtained by using a Jasco J-820 spectropolarimeter.


## 2.2. Synthesis of a Novel Cationic Porphyrin

As
a G-quadruplex-inducing agent, a novel cationic porphyrin5 was prepared according to Scheme 1. The molecule 5 was designed to bind to G-quadruplex by
a stacking interaction between porphyrin ring and G-quartet plane, together
with electrostatic interaction between ammonium cations and DNA backbone. In
spite of a postulated low affinity for G-quadruplex that is due to the bulky
substituents, porphyrin 5 is
expected to grab at the G-quadruplex with the corporation of π-stacking and electrostatic
interactions. All the reactions were conducted in the dark.Scheme 1
Synthesis of cation tethered porphyrin5.


## 2.3. Preparation of 5,10,15,20-Tetrakis(3’-Hydroxyphenyl)Porphyrin 1

To
a solution of 3-hydroxybenzaldehyde (3.66 g, 30 mmol) in propionic acid (70 mL),
was added pyrrole (2.40 g, 36 mmol) at 110°C under nitrogen atmosphere and
stirred for 1 hour. Then, the solution was stirred over night at room temperature
under air to afford oxidation of the precursor to porphyrin. The solvent was
removed and the residue was purified by silica gel column chromatography using
chloroform/methanol = 7/1 as an eluent to obtain compound 1 as a purple crystal (1.06 g, 21% yield).


## 2.4. Alkylation of Phenolic Hydroxyl Groups of 1

To
a solution of1 (0.34 g, 0.5 mmol)
in DMF (10 mL) were
added potassium carbonate (5.5 g, 40 mmol) and ethyl α-bromoacetate (3.4 g, 20 mmol).
The mixture was stirred at room temperature for 24 hours. The reaction was
quenched with water (100 mL) and the mixture was extracted with chloroform (3×30 mL). The combined organic phase was washed with water (3×30 mL) and brine
(1×30 mL), then dried over Na2SO4. The solvent was
removed and the residue was purified by silica gel column chromatography using
chloroform/ethyl acetate = 10/1 mixed solvent as an eluent to give compound 2 as a purple crystal (0.42 g, 82%
yield); 1H NMR (CDCl3, 300 MHz) δ -2.84 (s, 2H, NH), 1.25 (t, 12H, J =
7.2, CH3), 4.26 (q, 8H, J = 7.2, CH2CH3), 4.80 (s, 8H, OCH2), 7.34 (dd, 4H, J = 2.9, 8.6, Ar-H), 7.64 (t, 4H, J = 8.1, Ar-H), 7.75 (s, 4H, Ar-H),
7.83 (d, 4H, J = 7.2, Ar-H), 8.84 (s, 8H, pyrrole-β-H); m/z (MALDI-TOF) 1023
(M+ + H, 100%).


## 2.5. Hydroxylation of2

To
a solution of2 (0.31 g, 0.3 mmol)
in DMF (250 mL) was added 2N NaOHaq (50 mL). The solution was stirred over
night at 50°C. The solution was concentrated to a total volume of 150 mL, and
the pH of the solution was adjusted to 2.0 with 1N HCl to give a green
precipitate. The precipitate was washed with chloroform to give compound 3. This compound was used for the next
reaction without further purification.


## 2.6. Amidation of3 with Monoprotected Ethylenediamine

To
a solution of3 (45 mg, 0.05 mmol)
in DMF/DMSO = 3/1 (4 mL) were added N-tert-butoxycarbonylaminoethylamine (40 mg, 0.25 mmol), 1-hydroxybenzotriazole (HOBt) (41 mg, 0.3 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDC) (58 mg, 0.3 mmol). After the mixture was stirred over night
at room temperature, the reaction was quenched with water (50 mL). The
resulting mixture was extracted with dichloromethane (3×50 mL) and the
combined organic layer was washed with saturated NaHCO3aq (3×30 mL) and brine (1×30 mL), then dried over Na2SO4. The
solvent was removed and the residue was purified by silica gel column
chromatography using dichloromethane/ methanol = 15/1 as an eluent to give
compound 4 as a purple crystal
(0.059 g, 79% two steps overall yield); 1H NMR (CDCl3,
300 MHz) δ -2.83 (s, 2H, pyrrole-NH), 1.31 (s, 36H, tert-Bu),
3.31 (q, 8H, J = 5.3, CH2), 3.48 (q, 8H, J = 5.6, CH2), 4.69 (s, 8H, OCH2),
4.93 (brs, 4H, NH), 7.34 (dd, 4H, J = 2.1, 8.1, Ar-H), 7.40 (brs, 4H, NH),
7.65 (t, 4H, J = 8.1, Ar-H), 7.81 (s, 4H, Ar-H), 7.87 (d, 4H, J = 7.8,
Ar-H), 8.83 (s, 8H, pyrrole-β-H).


## 2.7. Preparation of Cationic Porphyrin5

A
solution of4 (29 mg, 0.02 mmol) in
trifluoroacetic acid (5 mL) was stirred at room temperature for 2 hours. After
the solvent was evaporated off, diethyl ether (50 mL) was added to give a green
precipitate. To the solution of the precipitate in water was added excess
NaOHaq. and the resulting solid was dissolved in chloroform. 1 M HCl was added
to this solution to give green precipitate of porphyrin tetra hydrochloric acid
salt 5 (22 mg, 92% yield); 1H
NMR (CD3OD, 300 MHz) δ  3.20 (t, 8H, J = 5.3, CH2),
3.70 (t, 8H, J = 5.6, CH2), 4.94 (s, 8H, OCH2), 7.69 (dd, 4H, J = 2.6, 8.3, Ar-H), 7.99 (t, 4H, J = 8.1,
Ar-H), 8.20 (s, 4H, Ar-H), 8.23 (brs, 4H, Ar-H), 8.85 (s, 8H, pyrrole-β-H); m/z (MALDI-TOF) 1079
(M+ + H, 100%).


## 3. Results and Discussion

Tetrahymena telomeric sequence d(T2G4)4 was chosen as a
model molecule and the cationic porphyrin 5 was used as a G-quadruplex inducing agent. Besides the effect of monovalent
cation, the difference between intrastrand and interstrand G-quadruplexes was
also examined to compare sequences d(T2G4)4 and
d(TG4T2G4T).The structure of d(T2G4)4 in the presence and absence of cationic porphyrin 5 was verified by circular dichroism spectroscopy in a buffer
containing 100 mM KCl and
50 mM Tris-HCl (see Figure 1(a)), in 100 mM KCl solution (see Figure 1(b))),
and in water (see Figure 1(c)). CD spectrum of (T2G4)4 without porphyrin 5 had shoulder
around 295 nm (see Figures 1(a)  and 1(b)) or positive peak around 295 nm (as
shown in Figure 1(c)). These CD spectra indicate a hybrid G-quadruplex
structure of (T2G4)4 without porphyrin 5, whose CD spectrum has positive
intensities both at 260 and 295 nm. On the other hand, the positive peak or
shoulder around 295 nm disappeared by the addition of the porphyrin. This
indicates that a structural transition of (T2G4)4 from a hybrid to a parallel G-quadruplex was induced by the porphyrin.
Therefore, these results lead us to conclude that porphyrin 5 induces a parallel G-quadruplex
structure of (T2G4)4 under various conditions.
Bisignate signals of porphyrin were strongly induced in a buffer solution,
suggesting that the porphyrin was stacked on the G-quadruplex surface. Relatively,
weak signals for the porphyrin were observed in solutions without Tris-HCl. The
self-aggregation of porphyrin in such solutions seems to be the reason for
these weak interactions.CD spectra of d(T2G4)4 (25 μM) with (red) or without (black) porphyrin 5 (75 μM) in buffer containing 100 mM KCl and 50 mM Tris-HCl (pH 7.5) (a), in 100 mM KCl (b), and in water (c) at 0°C.
(a)(b)(c)We
investigated the CD spectral changes of d(T2G4)4 by the addition of porphyrin 5 in
the absence of added cation (see Figure 1(c)). All DNA samples with or without
porphyrin were annealed by heating to 90°C followed by slow cooling to 0°C
over 8 hours. Both spectra shown in Figure 1(c) were measured using a d(T2G4)4 solution in Milli-Q water with no addition of external cation. In the absence
of cationic porphyrin 5, d(T2G4)4 did not form a G-quadruplex. However, when the sample was prepared in the
presence of porphyrin 5, the CD
spectrum exhibited both a positive signal around 260 nm and a negative signal
around 240 nm, indicating  the formation
of the d(T2G4)4 G-quadruplex. A weak induced
CD signal for the porphyrin 5 was
also observed between 420 and 460 nm. The induced CD signal is additional
evidence for the interaction between d(T2G4)4 and 5, since its appearance in the CD
spectra is indicative of the change in the chirality of the proximal chemical
environment of 5.The
structure of a half-length ofTetrahymena telomere sequence, d(TG4T2G4T), was studied by
CD spectroscopy. CD spectra of 50 μM of d(TG4T2G4T)
(G-quadruplex concentration: 25 μM)
in a 100 mM KCl solution had positive and negative peaks near 260 and 240  nm,
respectively, both in the absence and presence of porphyrin 5, indicating parallel G-quadruplex (see
Figure 2(a)). These spectral features were similar to those of d(T2G4)4.
On the other hand, in the absence of added cation, CD spectrum of d(TG4T2G4T)
was completely different from that of d(T2G4)4 without porphyrin 5; while in the
presence of 5, the locations of positive and
negative CD signals of d(TG4T2G4T) and d(T2G4)4 were almost identical (as in Figure 2(b)). These results indicate that, while
these two sequences have different structure in water, both are induced to form
parallel G-quadruplex by the addition of cationic porphyrin 5.CD spectra of d(TG4T2G4T) (25 μM) with (red) or without (black) porphyrin 5
(75 μM) in buffer containing 100 mM KCl and 50 mM Tris-HCl (pH 7.5) (a) and in water (b) at 0°C.
(a)(b)In
conclusion, the novel porphyrin carrying four cationic tethers5 induced the formation of parallel
G-quadruplex with Tetrahymena telomeric sequence. The effect of G-quadruplex induction was strong enough and
made both sequences d(T2G4)4 and d(TG4T2G4T)
folded into G-quadruplex without the addition of cation and buffer. The formation of the G-quadruplex under in vitro environment is mandatory for
recent applications of G-quadruplex to nano-materials. Thus, the simple
conditions for the formation of G-quadruplex are desired. The system described
here must be useful for the development of nanobiomaterials. In more general,
it will be important to control the structure of biomolecules with small
organic compounds for nanobiotechnology.



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*Source: 294756-2008-03-02.xml*