G-Quadruplex DNA Selective Binders
FULL PAPER
For the quadruplex experiments, samples were dissolved in the same
buffer as for the duplex experiments. Spectra were recorded under the
same conditions for the duplex experiments except samples were pre-
129.307, 130.649, 134.576, 144.494, 149.653, 153.082 ppm; MS (ESI,
2
+
CH
275.56986, found 275.56913 with Pt.
Synthesis of [(PIN)Pt(en)][PF (3): Complex 3 was prepared in the
same manner as complex 2 except (PIN)PtCl (69.8 mL, 0.098 mmol) and
ethylenediamine (60.1 mg, 0.98 mmol) was used. Yield: 65% H NMR
500 MHz, [D ]DMSO): d=9.455 (dd, 2H), 9.082 (d, 2H), 9.080 (m,
3
CN, trace of H
2
O and formic acid): m/z (%): calculated [Mꢀ2PF
6
]
1
95
II
pared as a 1:1 ratio of quadruplex in tetrad to Pt at a concentration of
A
H
R
U
G
6 2
]
3
2
4.8 mm. Samples were heated to 958C for 10 min and then cooled over
–3 h at room temperature. CD spectra were collected before and after
2
1
heating at room temperature.
(
6
Competitive dialysis: A dialysis solution containing a 10 mm solution of
the Pt complex (80 mL), 200x the total amount of nucleic acid to be
1H), 8.324 (dd, 2H), 8.211 (d, 1H), 8.157 (d, 1H), 8.123 (d, 1H), 717.796
(t, 1H), 7.692 (m 2H), 6.936 (br s, 4H), 2.804 ppm (br s, 4H); C NMR
II
13
tested, in a 1 mm NaH
prepared. Nucleic acids to be dialysed against the Pt complexes were
prepared to be 75 mm in either base pair or base quartet in buffer
2
PO
4
/Na
2
HPO
4
and 2 mm NaCl buffer (pH 7.0) was
(300 MHz, [D
126.606, 127.359, 128.348, 128.658, 130.102, 130.879, 133.526, 134.667,
144.378, 149.604, 152.997 ppm; MS (ESI, CH CN, trace of H O and
formic acid): m/z (%): calculated [Mꢀ2PF 300.577685, found
300.57691 with Pt.
6
]DMSO): d=47.251, 125.332, 125.775, 126.333, 126.455,
II
3
2
2
+
(
200 mL). The dialysis units were positioned slightly above the dialysis so-
6
]
1
95
lution within the beaker and were covered with parafilm and aluminum
foil. The setup was then allowed to equilibrate for 24 h with stirring at
room temperature. After equilibration, the dialysis solution (180 mL) was
added to a microcentrifuge tube along with a 10% (w/v) SDS solution
Molecular modeling of the quadruplex/Pt complex system: The initial
quadruplex structure was constructed from the reported NMR structure
of
a
quadruplex
DNA
made
of
four
DNA
(5’-
(
20 mL) to give a final concentration of 1% SDS. The SDS treated solu-
D(*TP*TP*GP*GP*GP*GP*T)-3’) units (PDB code: 139D). Two layers
of thymines were added on the 5’-end using InsightII as a graphical inter-
face to more accurately represent the experimental system. Subsequently,
II
tions and the dialysis buffer containing the Pt complex were then ana-
lyzed by UV/Vis spectroscopy after 10 min to determine the amount of
intercalator present inside and outside of the dialysis bag.
II
the Pt complexes were constructed and optimized through B3LYP den-
Synthesis of naphthylphenanthroimidazole (PIN) ligand: The naphthyl
sity functional theory computations at the B3LYP/LACV3P* level of
II
derivatized ligand was synthesized using the procedure as outlined by
theory. Specific Pt force field parameters were developed from single-
[
35]
II
Steck and Day. Column conditions were modified, so the mixture was
first eluted with a hexanes/ethyl acetate mixture (70:30) to remove start-
ing material and the product was then eluted with a dichloromethane/
methanol mixture (97:3). The product was then evaporated to yield an
point DFT computations of various distorted structures of the Pt com-
plex at the B3LYP/LACV3P**+ level of theory. LACV3P**+ is a
triple-z basis set with polarization and diffuse functions on all atoms
except hydrogens. The standard triple-zeta 6–31G basis set is used for all
light elements and the Los Alamos non-relativistic effective core poten-
oily residue. A small amount of CHCl
product as pale yellow solid. Yield: 29% H NMR (500 MHz,
]DMSO): d=9.111 (d, 1H), 9.047(br. d, 2H), 8.958 (br. s, 2H), 8.125
m, 2H), 8.069 (d, 1H), 7.863 (m, 2H), 7.737 (t, 1H), 7.681 (t, 1H),
3
was added to precipitate the
II
1
tial was used for Pt . All DFT computations were performed using the
a
Jaguar7.0 program package. The developed force field parameters were
incorporated into the Generalized Amber Force Field (GAFF). The plat-
inum complex atomic partial charges were derived from the atomic elec-
trostatic potential.
[
D
6
(
1
3
7
1
1
1
6
.633 ppm (t, 1H); C NMR (300 MHz, [D ]DMSO): d=123.556,
24.092, 124.403, 125.035, 125.371, 126.119, 126.522, 127.280, 127.445,
28.094, 128.496, 130.138, 130.229, 130.691, 133.733, 135.466, 135.731,
36.789, 143.162, 147.872, 150.770, 150.771, 151.047 ppm; MS (ESI, 90
Construction and refinement of the DNA/Pt complex system: Manual in-
tercalation of the platinum complex within the homologated quadruplex
was initially performed. Three potassium ions were added in the central
MeOH: 10 DMSO): m/z (%): calculated 346.12185, found 347.12873
[M+1]).
Synthesis of PIPPtCl
thesized by using the procedure as outlined by Steck and Day.
215 mg, 5.18 mmol) was first dissolved in DMSO (3 mL) and distilled
water (1 mL) and heated to near boiling. This hot solution was added to
hot solution of phenylphenanthroimidazole (153 g, 5.18 mmol) in
[
3]
(
channel between each of the G-tetrads, and the resulting system was
+
neutralized by addition of 27 K ions. These ions were added at the most
2
: The phenylphenanthroimidazole ligand was syn-
[
34]
negative locations as computed by the leap program and an additional 5
KCl molecules were added to simulate the ionic medium. After the addi-
2 4
K PtCl
(
+
tion of K ions, a periodic truncated octahedron box of water was added
corresponding to 10068 TIP3P water molecules. Stepwise relaxation was
then performed with the slow relaxation of the entire system commenc-
ing with the water molecules. In a subsequent step, the entire system was
fully minimized by using large constraints on the DNA heavy atoms and
iterated through the gradual decrease of constraints on these atoms. For
this purpose, the Sander program within the AMBER8.0 suite of pro-
grams was used with a combination of the parm99 force field for the G-
a
DMSO (5 mL). The reaction mixture was allowed to cool overnight and
then vacuum-filtered. The yellow dichloride product was washed with
water to remove excess K
ether to dry the product. Yield: 79% H NMR (300 MHz, [D
d=9.657 (d, 2H), 9.289 (d, 2H), 8.293 (d, 2H), 8.223 (t, 2H), 7.613 ppm
m, 3H); MS (ESI, CH CN and NaI): m/z (%): calculated 561.00868,
2 4
PtCl , a small amount of ethanol, and diethyl
1
6
]DMSO):
(
3
II
+
+
194
quadruplex and GAFF for the Pt complex. A combination of 5000 steps
found 582.99579 ([M +Na ]) with Pt.
Synthesis of PINPtCl : The same procedure was used as for the synthesis
of PIPPtCl , except (102 mg, 0.294 mmol) of the naphthylphenanthroimi-
dazole ligand and (122 mg, 0.294 mmol) of K PtCl were used. Yield:
]DMSO): d=9.613 (d, 2H), 9.294 (d, 2H),
.068 (d, 1H), 8.166 (4H), 8.105 (d, 1H), 7.778 (t, 1H), 7.683 ppm (m,
H); MS (ESI, CH CN and NaI): m/z (%): calculated 611.02433, found
of conjugate gradients minimization and 10000 steps of molecular dy-
namics at gradually increasing temperatures was used to relieve strain
within the system This minimization/MD simulation procedure was iter-
ated five times at 100 K, eight times at 200 K, and ten times at 300 K,
with the use of Berendsen temperature-coupling algorithm and the
SHAKE algorithm for all hydrogen atoms with 1 fs time steps. A cutoff
of 11 was used for the computation of the van der Waals and electro-
static interactions. As these systems are highly charged, the long-range
electrostatic interactions should be carefully computed. The Particle
Mesh Ewald summation term was enabled with the automatically defined
parameters. To maintain the integrity of the quadruplex, constraints were
added on the hydrogen bond network.
2
2
2
4
1
8
9
2
6
2% H NMR (500 MHz, [D
6
3
+
+
194
34.01243 ([M +Na ]) with Pt.
Synthesis of [(PIP)Pt(en)]
A
H
R
U
G
6
]
2
(2): Ethylenediamine (106 mL,
1
0
3
.57 mmol) was added to
.157 mmol) in ethanol (20 mL). The reaction mixture was refluxed for
h and a small amount of distilled water was added to the reaction mix-
a
suspension of (PIP)PtCl (88.5 mg,
2
ture until the solution turned a clear red color. The reaction mixture was
then gravity filtered to remove any unreacted material, and ammonium
hexafluorophosphate was added to precipitate the off-yellow product.
The product was filtered on a glass frit and washed with a small amount
1
of ethanol and diethyl ether. Yield: 62% H NMR (500 MHz,
Acknowledgements
[
D
6
]DMSO): d=9.224 (d, 2H), 9.055 (br s, 2H), 8.302, (br s, 2H), 8.247
(
4
d, 2H), 7.662 (br s, 2H), 7.616 (d, 1H), 6.920 (br s, 4H), 2.779 ppm (br s,
H); C NMR (500 MHz, [D ]DMSO): d=47.215, 126.497, 129.107,
6
This work was supported by NSERC, the Canada Foundation for Innova-
tion, Nanoquebec, Research Corporation, Center for Self-Assembled
1
3
Chem. Eur. J. 2008, 14, 1145 – 1154
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1153