Metalloregulation of triple helix formation by control of the loop conformation

Article abstract of DOI:10.1080/15257770802271813

The flexible polypyridine ligand, 2,2′:6′,2″-terpyridine (terpy), was built into the backbone of oligonucleotides to form DNA conjugates. The terpy unit functioned as a good loop when the conjugates formed the bimolecular triplexes with complementary oligopurine. The triplex structure was destabilized by the specific interaction with divalent transition metal ions (Cu2+, Zn2+, and Fe2+), in particular Cu2+ ions. This ion destabilized one of the triplexes by 4.2 kcalmol-1 or made the triplex formation constant less than 1/103 at 298 K. This result is attributed to the substantial turbulence of the terminal structure of the triplexes. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/15257770802271813

Nucleosides, Nucleotides and Nucleic Acids, 27:1084–1096, 2008
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770802271813
METALLOREGULATION OF TRIPLE HELIX FORMATION BY CONTROL
OF THE LOOP CONFORMATION
Toshihiro Ihara,1,2 Yoshinobu Sato,1 Hiroshi Shimada,1 and Akinori Jyo1
1Department of Applied Chemistry and Biochemistry, Graduate School of Science and
Technology, Kumamoto University, Kumamoto, Japan
2PRESTO, Japan Science and Technology Agency, Saitama, Japan
The flexible polypyridine ligand, 2,2ꢁ:6ꢁ,2ꢁꢁ-terpyridine (terpy), was built into the backbone of
2
oligonucleotides to form DNA conjugates. The terpy unit functioned as a good loop when the
conjugates formed the bimolecular triplexes with complementary oligopurine. The triplex structure
was destabilized by the specific interaction with divalent transition metal ions (Cu2+, Zn2+, and
Fe2+), in particular Cu2+ ions. This ion destabilized one of the triplexes by 4.2 kcalmol−1 or made
the triplex formation constant less than 1/103 at 298 K. This result is attributed to the substantial
turbulence of the terminal structure of the triplexes.
Keywords DNA triplex; terpyridine; metal ion; UV melting; amidite reagent
INTRODUCTION
Nucleic acids are the only materials that enable us to design and syn-
thesize, from scratch, the molecules that bind given nucleic acids according
to the rule of Watson-Crick base pairing.[1,2] In addition, the developing
techniques of in vitro evolution have extended the target molecules to non-
nucleosides.[3,4] Therefore, nucleic acids would be a versatile scaffold to
construct molecular devices for analyzing not only nucleic acids,[5–8,24] but
also other varieties of species in aqueous solution, such as metal ions,[9–13]
proteins,[14–17] and other physiologically active substances.[18–24] Recently,
researchers have been devoting their efforts to produce numbers of
Received 11 December 2007; accepted 22 May 2008.
The authors would like to thank Mr. Y. Isobe and Mr. J. Izumi for their assistance in measuring the
NMR. This work was partly supported by a Grant-in-Aid for Scientific Research from MEXT, Japan to
T. I.
Address correspondence to Toshihiro Ihara, Department of Applied Chemistry and Biochemistry,
Graduate School of Science and Technology, Kumamoto University, 2–39-1 Kurokami, Kumamoto 860-
8555, Japan. E-mail: toshi@chem.kumamoto-u.ac.jp
1084

Metalloregulation of DNA Triplex Structure
1085
molecular devices based on functional nucleic acids such as ribozymes,[19,25]
DNAzymes,[6–8,10–13] and aptamers.[5,9,14–23,25] Higher-order structures are
critically important for these molecules to function as they are expected.
The loop is a common secondary structure and an important element to
determine their three-dimensional structures.
DNA is chemically stable and easily modified with auxiliary chemical
groups carrying specific features.[26,27] If chemical groups that change their
conformation by certain stimuli are built into the backbone in the loop
moiety of the functional DNAs, we could regulate the functions indirectly by
controlling the conformational changes of the DNAs. The strategy is general
in the sense that it targets the conformation rather than each particular
function.
In this series of studies, we chose ligands such as bipy (2,2ꢁ-
bipyridine) and terpy (2,2ꢁ:6ꢁ,2ꢁꢁ-terpyridine) as the reversible conforma-
tional modulators.[28] When the appropriate metal ion is added as a
stimulus, pyridine units convergently coordinate to the metal ion to fix
the whole ligands’ structures. Thus, the length and flexibility of the loop
structure would be controlled by choosing appropriate metal ions to be
added (Figure 1). To date, it has been reported that the DNA hairpin
structure[29] and β-sheet structures of peptides[30] are controlled by specific
metal ions through their interaction with the substituted terpy and pyridine
unit, respectively, built into the backbone structure.
Here, we report the synthesis of the amidite reagent carrying a non-
substituted plain terpy unit. The unit was built into the middle of the
backbone of oligopyrimidines using an automated DNA synthesizer. The
OR
O
N
O
O
OR
N
N
N
N
N
O
OR'
linear state
bent state
OR'
FIGURE 1 Reversible loop modification. Conformation and rigidity of the loop terpyridine unit could
be controlled by complexation with metal ions.

1086
T. Ihara et al.
oligopyrimidine is designed to be folded in half at the terpy position when
it binds with the complementary oligopurine to form a triplex with a parallel
motif.[31] The formation of the bimolecular triplexes were studied with UV
melting experiments in the presence of several metal ions.
RESULTS AND DISCUSSION
Preparation of Terpy-Modified Oligopyrimidines
The 2,2ꢁ:6ꢁ,2ꢁꢁ-terpyridine-based amidite reagent was synthesized accord-
ing to Scheme 1. Generally, a diol is the key compound when one designs
the synthetic scheme of an amidite reagent. One of its hydroxyl groups is
tritylated by 4,4ꢁ-dimethoxytrityl chloride, and then the other is phosphity-
lated by chloro(diisopropylamino) (β-cyanoethoxy)phosphine to form the
desired structure of the amidite reagent, which could be directly used in an
automated DNA synthesizer. Here, the terpy diol (6) was synthesized as the
key compound.
H SO
2
4
O
O
O
THF
HO
HO
OH
+
RT
3
1
2
3
NaH
Br
Br
O
O
O
O
O
OH
OH
N
N
O
O
N
N
HCl, MeOH
Reflux
N
N
N
(CH OCH CH ) O
3
2
2 2
N
N
110 o
C
O
4
6
5
OCH
OCH
3
3
Cl
O
P
N
CN
O
O
O
OCH
3
O
O
O
OCH
3
8
DMTr-Cl
Pyridine
Et N
3
N
N
N
CH Cl , (iPr) NEt
2
2
2
N
N
CN
RT
RT
O
P
N
N
O
OH
7
9
SCHEME 1 Synthesis of the terpy amidite.
One of the hydroxyl groups of ethylene glycol (1) was protected by
3,4-dihydro-2H-pyrane (2) to form 2-tetrahydropyranethanol (3). The 3 and
6,6ꢁꢁ-dibromo-2,2ꢁ:6ꢁ,2ꢁꢁ-terpyridine (4) was coupled by the Williamson reac-
tion to form the protected diol 5. This was then deprotected quantitatively
in HCl-MeOH solution to afford the diol (6) as a white powder. Both of

Metalloregulation of DNA Triplex Structure
1087
FIGURE 2 Sequences of the oligonucleotides used in this study (a) and the configuration of bimolecu-
lar triplexes (b).
the hydroxyl groups in 6 are primary ones and are supposed to have the
same reactivity toward tritylation with 4,4ꢁ-dimethoxytrityl chloride. There-
fore, to obtain the monotritylated alcohol (7) in good yield, the reaction
conditions such as the feeding ratio of 6 and 4,4ꢁ-dimethoxytrityl chloride,
reaction time, and temperature are critical. The crude product was chro-
matographed on a silica gel column with dichloromethane-diethylamine
(100 : 1) to give 7 as a pale yellow powder with no decomposition during the
chromatography. Finally, the amidite (9) was prepared by phosphitylation
of 7 with chloro(diisopropy1amino)-(β-cyanoethoxy)phosphine (8). Three
DNA conjugates carrying a (dT)nterpy(dT)n loop (n = 0, 1, or 2 for tp,
TtpT, or T2tpT2, respectively) were synthesized using this amidite, 9, with
the automated DNA synthesizer (Figure 2). All oligonucleotides were puri-
fied with the conventional two-step procedure using RP-HPLC (reversed-
phase HPLC, linear gradient: 0.1 M triethylammonium acetate (TEAA),
pH7.0/acetonitrile) and identified with MALDI-TOF mass spectrometry.[27]
Metal-Ion Effect on the Stabilities of Bimolecular Triplexes
The effect of coexisting metal ions (Cu2+, Fe2+, and Zn2+) on the bi-
molecular triple helix formation was studied using UV melting experiments.

1088
T. Ihara et al.
FIGURE 3 Normalized UV melting curves of the bimolecular triplexes. The solutions containing the
triplexes (pH 5.0) were heated at a rate of 0.5 deg min−1 in the absence (without dot) and presence of
Cu2+ (with dot). Solid curves: melting of tp/target triplex; broken curves: melting of T3/target control
triplex.
All the triple helixes melted cooperatively at once. The UV melting curves
for the T3/target and tp/target in the presence and absence of Cu2+ are
shown in Figure 3. The maximum temperatures of the first derivative of
the curves, Tmax, are summarized in Table 1 and used as indicators of the
triplex stabilities. The triplex of oligopyrimidines of the conjugates carry-
ing a (dT)nterpy(dT)n loop (tp/target, TtpT/target, and T2tpT2/target)
provided higher Tmax than that of the control (dT)3 loop (T3/target).
The (dT)nterpy(dT)n loop seems to function as an excellent loop when
forming the bimolecular triplexes, as compared with (dT)3, probably due
to the stacking interaction with the terminal base triplet (CG.C+) and their
appropriate length and flexibility.
Although the divalent transition metal ions scarcely change the Tmax
of the control triplex (T3/target), they significantly decreased the Tmax
of the triplexes containing a terpy loop (tp/target, TtpT/target, and
T2tpT2/target). The order of the influence on the thermal stabilities of
the triplexes was Cu2+ > Zn2+ > Fe2+. This order coincides with the order
of magnitude of stability constants between the terpy ligand and these metal
ions.[32] Therefore, the metal ions seem to destabilize the triplex structures
by their complexation with the terpy unit. The effect was already significant

Metalloregulation of DNA Triplex Structure
1089
TABLE 1 The Tmax of bimolecular triplex formation in the presence and absence of three transition
metal ionsa
T
max/◦C
ꢀTmax/◦Cb
[Cu2+]/µM
[Zn2+]/µM
[Fe2+]/µM
Pyrimidine
strands
No metal
54.3
1
5
10
54.7
1
5
10
1
5
10
T3
tp
54.2 54.9
−0.1 0.6
54.0
54.2
54.9
53.8
−0.5
—
54.6
0.3
—
55.3
1.0
56.4
0.4
−0.3 −0.1 0.6
58.1
—
—
44.0
—
—
52.0
−14.1
47.5
−6.1
−1.7
54.5
−3.7
58.2
TtpT
58.2
59.9
49.3 48.3
53.3
52.8
51.8
55.7
55.5
−2.7
—
−8.9 −9.9
−10.7
−4.9 −5.4 −6.4 −2.5
T2tpT2
—
—
51.2
—
—
56.3
—
−8.7
−3.6
−1.7
aTmax is defined as the temperature that provides the maximum of the 1st derivative ((dA/dT)max
)
of the melting curve.
bThe values of the lower row in each cell indicated in italics are the differentials with Tmax of “no
metal”.
—
: not measured.
with the equimolar ratio of metal ions to the terpy units, it then became
somewhat more remarkable when 5 and 10 equivalents of the ions were
added. The metal ions would bind selectively to the terpy loop of the
conjugates and change the conformation and flexibility of the loop. The
thermodynamic parameters of triplex formation were estimated by fitting
the melting curves shown in Figure 3 to the theoretical equation, which is
derived based on the two state model.[33] The parameters are summarized
in Table 2. Ten equivalents (10 µM) of Cu2+ decreased the free energy of
the triplex formation by 4.2 kcalmol−1 at 298 K, which corresponds to a
decrease of the binding constant to less than 1/103. The distance between
˚
the two phosphates at both ends of the tp loop is ca. 21.6 A in all of its
trans conformations, which is almost the same with the diameter of DNA
triplex. Therefore, the tp loop would cap one of the ends of the triplex by
bridging both pyrimidine strands that was proportional to its length. That
is, three pyridine rings would stack well on the terminal CG.C+ to form a
stable hairpin-turn structure in the absence of the metal ions. When it binds
TABLE 2 Thermodynamic parameters of the tp/target triplex formation
ꢀH◦/kcal
ꢀS◦/cal
ꢀG◦298/kcal
mol−1
mol−1K−1
mol−1
K
298/ M−1
tp/target
−84.1
−70.0
−226
−192
−16.8
−12.6
2.05 × 1012
1.85 × 109
tp/target + 10 Cu2+

1090
T. Ihara et al.
FIGURE 4 One of the possible structures of the bimolecular triplexes carrying terpy loop, tp/target,
in the absence (left) and presence (right) of metal ions. White and black strands shows tp and target,
respectively. The structures were optimized with an AMBER∗ force field using Macromodel (ver. 9.5,
Schro¨dinger Inc.).
the metal ions, the pyridine rings flip over and direct three nitrogen atoms
convergently to make space to accommodate the ions. This would make the
terpy structure shorter and more rigid; the distance between the α-oxygens
˚
would be changed from ca. 12.2 to 7.4 A by forming the complex. The
possible structures of the tp triplex before and after complexation, which
are optimized by AMBER∗, are shown in Figure 4. While the free terpy stacks
well on adjacent bases before complexation, an apparent perturbation of
the stacking is found in the structure after complexation. The substantial
destabilization of the triplexes by Cu2+ would be attributed to such a
turbulence of the terminal structure of the triplex. The destabilization effect
by the transition metal ions seemed to decrease with elongation of the
loop length. This is because the strain or distortion induced by the metal
complexation would be alleviated more by longer flexible loops.
CONCLUSIONS
The terpy structure was built into the backbone of oligopyrimidines
to form the conjugates, tp, TtpT, and T2tpT2. The terpy unit functioned

Metalloregulation of DNA Triplex Structure
1091
as an excellent loop when the conjugates formed the triplexes with com-
plementary oligopurine, target. The triplex structure was destabilized by
the specific interaction with divalent transition metal ions. Among them, the
effect of Cu2+ was most significant especially for tp; Cu2+ destabilized the
tp/target triplex by 4.2 kcalmol−1 or made the binding constant less than
1/103 at 298 K. This technique could be applied for the controlled release
of DNA or RNA drugs. The loop would also be applicable as a modulator of
functional nucleic acids such as ribozymes, DNAzymes, and aptamers.
EXPERIMENTAL
General
400 MHz 1H and 100 MHz 13C NMR spectra were measured on a EX400
spectrometer (JEOL, Japan) at 298 K. 31P NMR spectrum was measured on
a Unity Inova 400 (Brucker, Germany). Chemical shifts (δ) are expressed in
1
ppm relative to SiMe4 for H and 13C, and phosphate for 31P-NMR. Multi-
plicities are indicated as the following: s (singlet); d (doublet); dd (double
doublets); dt (double triplets); t (triplet); m (multiplet). 1H NMR coupling
constants (J ) are reported in Hz. IR spectra were collected on an FTIR
Spectrum one spectrometer (Perkin Elmer, USA). Elemental analysis was
performed on a CHN Corder MT-6 (Yanaco, Japan). Matrix-assisted laser
desorption ionization mass spectrometry measurements (MALDI-TOF/MS)
were carried out using a Voyager RP mass spectrometer (PerSeptive, USA).
Electrospray ionization-time of flight mass spectrometry (ESI-TOF/MS)
measurements of terpy amidite was carried out using a solution of acetoni-
trile saturated with lithium chloride with a LCT spectrometer (Micromass,
UK) in positive ion mode.[34]
Synthesis of Terpy Amidite (9)
Protected diol (3): Ethylene glycol (1) (0.45 mL, 8.1 mmol) and 3,4-
dihydro-2H-pyrane (2) (0.88 mL, 9.7 mmol) were dissolved in 5 mL of
THF under an atmosphere of argon. Sulfuric acid (0.012 mL) was added to
the mixture and the mixture was stirred for 2 hours at room temperature.
The mixture was diluted with 10 mL of ethyl acetate and washed with 5
mL of brine and saturated sodium hydrogen carbonate. The organic phase
was separated, dried with anhydrous MgSO4, and concentrated to dryness
under reduced pressure. The residue was subjected to silica gel column
chromatography (hexane : ethyl acetate = 4 : 1) and the fraction of Rf 0.25
was collected. The fraction was concentrated to dryness in vacuo to afford a
light yellow liquid.

1092
T. Ihara et al.
1
Yield 0.59 g (50%); yellow liquid; H-NMR (CDCl3) δ 4.58 (1H, m, O-
CH-O), 3.90–4.00, 3.65–3.80, 3.50–3.60 (1H, 4H, 1H, m, O-CH2), 3.02 (1H,
s, OH), 1.72–1.90, 1.50–1.63 (2H, 4H, m, C-CH2-C).
Protected terpy diol (5):[35] Sodium hydride (60 mg, 1.5 mmol) was
suspended in 3 mL dry diglyme (bis(2-methoxyethyl) ether) under an
atmosphere of argon. Tetrahydropyran ethanol (3) (0.21 mL, 1.5 mmol)
was added slowly to the solution in an ice bath. After the termination of
gas generation, dibromoterpyridine (4) (0.1 g, 0.26 mmol) was added into
the solution and stirred at 110◦C for 12 hours. Analysis by RP-TLC indicated
the presence of a new component (Rf 0.2, acetonitrile : water = 6 : 1)
and the disappearance of 8. After standing to cool, the resulting white
precipitate was filtered. The filtrate was diluted with ethyl acetate and
washed twice with water. The organic phase was separated, dried with
anhydrous MgSO4, and concentrated to dryness under reduced pressure.
Recrystallization from 1-butanol afforded 5 as white crystals.
1
Yield 92 mg (69%); white crystals, m.p. 107–108◦C; H NMR (CDCl3)
δ 8.30 (2H, d, J = 7.81, Ar-5,5ꢁꢁH), 8.14 (2H, d, J = 7.81, Ar-3,3ꢁꢁH), 7.82
(1H, t, J = 7.71, Ar-4ꢁH), 7.66 (2H, t, J = 7.81, Ar-4,4ꢁꢁH), 6.76 (2H, d, J =
8.30, Ar-3ꢁ,5ꢁH), 4.60, 4.06, 3.83, 3.45 (14H, m, OCH2, THP-2,2ꢁ,6,6ꢁH) 1.62
(12H, m, THP-3,4,5,3ꢁ,4ꢁ,5ꢁH); 13C NMR (CDCl3) δ 180.8 (C), 172.8 (C),
171.7 (C), 157.1 (CH), 155.3 (CH), 138.4 (CH), 131.6 (CH), 129.2 (CH),
116.6 (CH) and 83.5 (CH2) 82.6 (CH2) 79.9 (CH2) 48.3 (CH2) 43.1 (CH2)
37.1 (CH2); IR (KBr) ν 3448, 2945, 1742, 1601, 1572, 1470, 1380, 1322, 1337,
1265, 1235, 1201, 1184, 1143, 1128, 1076, 1937, 1024, 991, 944, 930, 907,
889, 874, 815, 794, 763, 747, 632 cm−1; Anal. Calcd for C29H35N3O6: C 66.78,
H 6.76, N 8.06%, Found: C 66.75, H 6.65, N 8.25%.
Terpy diol (6): Protected terpy diol (0.24 g, 0.47 mmol) was dissolved
in 10 mL HCl-CH3OH (1 : 4) and the solution was refluxed for 1 hour.
After standing to cool, the solution was basified by potassium carbonate.
The resulting white precipitate was collected and washed with water. The
product was used for the next step synthesis without further purification,
because an NMR study showed that the purity was more than 99%.
Yield 0.16 g (95%); white crystals, m.p. 203.8–204.0◦C; 1H NMR (DMSO-
d6) δ 8.37 (2H, d, J = 7.81, Ar-5,5ꢁꢁH), 8.19 (2H, d, J = 7.32, Ar-3,3ꢁꢁH), 8.09
(1H, t, J = 7.81, Ar-4ꢁ), 7.90 (2H, t, J = 7.81, Ar-4,4ꢁꢁ), 6.92 (2H, d, J = 8.30,
Ar-5ꢁ,3ꢁH), 4.90 (2H, t, J = 5.37, OH), 4,46 (4H, t, J = 5.37, Ar-OCH2),
3.81 (4H, t, J = 5.37, CH2-OH); 13C NMR (DMSO) δ 162.9 (C) 154.5
(C), 152.6 (C), 140.2 (CH), 138.4 (CH), 120.5 (CH), 113.6 (CH), 111.4
(CH), 67.3 (CH2), 59.4 (CH2); IR (KBr) ν 3327, 2953, 1603, 1572, 1471,
1438, 1380, 1339, 1323, 1272, 1074, 1047, 991, 943, 888, 792, 735, 634 cm−1
;
Anal. Calcd for C19H19N3O4: C 64.58, H 5.42, N 11.89%, Found: C 64.54,
H 5.49, N 11.80%; MALDI-TOF/MS (CHCA matrix) Calcd: 354.14, Found:
352.76.

Metalloregulation of DNA Triplex Structure
1093
Monotrityl terpy (7):[28] Terpy diol (6) (0.10 g, 0.28 mmol) was added
into 4 mL of anhydrous pyridine under an atmosphere of argon. To the sus-
pension, triethylamine (0.07 mL, 0.51 mmol) and 4,4ꢁ-dimethoxytrityl chlo-
ride (96 mg, 0.28 mmol) were added and then the suspension was stirred
for 2 hours at 25◦C. The reaction was quenched by addition of methanol
(1 mL). The reaction mixture was diluted with 10 mL of dichloromethane
and then washed twice with 5% aqueous NaOH (50 mL). The organic
phase was dried with anhydrous MgSO4 and concentrated in vacuo. The
residue was subjected to silica gel column chromatography. The column was
packed with the silica slurry suspended in the solution of dichloromethane-
diethylamine (100 : 5) and then washed with dichloromethane of three bed
volumes. Separation was carried out with hexane-ethyl acetate (3 : 2), and
the fraction of Rf 0.24 was collected. It was concentrated to dryness in vacuo
to give pale yellow crystals. The product was used for the next step synthesis
without further purification, because an NMR study showed that the purity
was more than 99%.
1
Yield 0.114 g (30%); yellow powder; H-NMR (CDCl3) δ 8.43 (1H, d,
J = 7.79, Ar-5ꢁꢁH), 8.28 (1H, d, J = 6.83, Ar-5H), 8.22 (2H, dd, J = 4.88,
5.87, Ar-3,3ꢁꢁH), 7.89 (1H, t, J = 7.83, Ar-4ꢁH), 7.76 (2H, dt, J = 6.35, 7.79,
Ar-4,4ꢁꢁH), 7.51–7.17 (9H, m, DMTr), 6.86–6.79 (6H, m, DMTr, Ar-3ꢁ,5ꢁH),
4.68 (2H, t, J = 4.87, Ar-OCH2), 4.63 (2H, t, J = 4.87, Ar-OCH2), 4.04 (2H,
t, J = 4.87, CH2-OH), 3.76 (6H, s, OCH3), 3.49 (2H, t, J = 4.87, CH2-O-
DMTr); 13C NMR (DMSO-d6) δ 163.3 (C), 148.4 (C), 155.4 (C), 154.7 (C),
153.6 (C), 153.3 (C), 145.0 (C), 139.8 (CH), 139.4 (CH), 137.7 (CH), 136.3
(CH), 130.1 (CH), 128.2 (CH), 127.7 (CH), 126.7 (CH), 120.9 (CH), 120.5
(CH), 114.4 (CH), 113.9 (CH), 113.0 (CH), 111.5 (CH), 85.9 (CH), 68.2
(CH2), 65.0 (CH2), 62.5 (CH2), 62.4 (CH2), 55.2 (CH).
Terpy amidite (9): Monotrityl terpy (7) (95 mg, 0.15 mmol) was dis-
solved in 3 mL of anhydrous dichloromethane under an atmosphere of
argon. To the solution, diisopropylethylamine (0.21 mL, 1.22 mmol) and
chloro(diisopropylamino)-(β-cyanoethoxy)phosphine (8) (0.068 mL, 0.30
mmol) was added and the solution was stirred for 1 hour at 25◦C. Analysis by
TLC (dichloromethane : ethyl acetate = 2 : 1, 1% triethylamine) indicated
the presence of a new component (Rf 0.90) and the disappearance of 7
(Rf 0.30). The reaction mixture was diluted with 5 mL of dichloromethane,
washed twice with 5% aqueous NaHCO3 (10 mL), dried with anhydrous
MgSO4, and concentrated in vacuo. The residue was purified by chromatog-
raphy on silica gel (dichloromethane : ethyl acetate : triethylamine = 100 :
50 : 1). The component of Rf 0.9 was collected and concentrated to dryness
in vacuo.
Yield 35 mg (24%); white powder; 1H-NMR (CDCl3) δ 8.29 (1H, d, J =
7.79, Ar-5ꢁꢁH), 8.24 (1H, d, J = 7.79, Ar-5H), 8.14 (2H, dd, Ar-3,3ꢁꢁH), 7.80
(1H, t, J = 7.83, Ar-4ꢁH), 7.66 (2H, dt, Ar-4,4ꢁꢁH), 7.43–7.11 (9H, m, DMTr),

1094
T. Ihara et al.
6.77–6.71 (6H, m, DMTr, Ar-3ꢁ,5ꢁH), 4.59 (4H, m, Ar-OCH2), 4.04 (2H,
m, CH2-O-P), 3.78, 3.57 (4H, m, N-CH, P-OCH2), 3.69 (6H, s, Ar-OCH3),
3.42 (2H, t, J = 4.87, CH2-O-DMTr), 2.55 (2H, t, J = 4.87, CH2-CN), 1.18
(12H, m, (iPr)2); 13C NMR (CDCl3) δ 163.1, 158.4, 153.54, 145.1, 139.5,
139.4, 137.5, 136.3, 130.1, 128.3, 127.8, 126.7, 120.7, 120.6, 114.0, 113.8,
113.1, 111.4, 86.0, 65.5, 65.0, 62.4, 62.1, 60.4, 58.7, 58.5, 55.2, 53.4, 43.2,
43.1 29.7, 24.6, 20.4, 20.3; 31P-NMR (CDCl3) δ 147.16; ESI-TOF/MS for
C49H54N5O7P (mixed with saturated LiCl solution) Calcd: 862.39 [M+Li]+,
Found: 862.62.
Oligonucleotides: All oligonucleotides (ODNs) used in this study were
purchased from Hokkaido System Science Co., Ltd. (Japan), or synthesized
using Expedite 8900 (Applied Biosystems, USA) DNA synthesizer with
dimethoxytrityl phosphoramidite reagents purchased from Glen Research
(USA) or Proligo (Germany). All other reagents were purchased and used
without further purification. The synthesis followed the regular protocol
for the DNA synthesizer except for the cycle of terpy amidite (9), in which
prolonged coupling time of 10 minutes and capping time of 15 minutes
were used. The ODNs or ODN conjugates were purified from the crude
mixture by RP-HPLC (reversed phase-HPLC, LC-2000plus inert system,
Jasco) equipped with an ODS column (Wakopak-Wakosil-II 5C18 RS, 4.6
mm (ϕ) × 150 mm (w), Wako, Japan). MALDI-TOF/MS measurements
of ODNs and ODN conjugates were measured in negative ion mode
with 3-hydroxypicolinic acid as a matrix. The concentration of the ODN
solution was determined at 25◦C based on the molar extinction coefficients
calculated using those of nearest-neighbor dinucleoside monophosphates
at 260 nm obtained from the literature.[36]
MALDI-TOF/MS (3-HPA matrix) for tp Calcd: 6315.13, Found: 6316.25;
for TtpT Calcd: 6921.15, Found: 6921.24; for T2tpT2 Calcd: 7529.24,
Found: 7531.20
UV Melting[37]
Thermal denaturation experiments were carried out in sodium acetate
buffer solution (100 mM, pH 5.0) containing 10 mM NaCl and 10 mM
MgCl2 on a V-560 (Jasco, Japan) or UV-1650 (Shimadzu, Japan) UV/Vis
spectrophotometer equipped with a Peltier thermal controller. Concentra-
tions of each component of the triplexes (target, tp, TtpT, and T2tpT2)
were 1 µM. Prior to the beginning of each melt, the samples were degassed
at 85◦C for 5 minutes and then annealed with slow cooling to 0◦C. In the
denaturation experiments, the solutions were heated at a rate of 0.5 deg
min−1 after equilibration for 20 minutes at 0◦C.
The thermodynamic parameters of the triplex formation were estimated
by fitting the experimental curves to the theoretical one, which is derived

Metalloregulation of DNA Triplex Structure
1095
assuming the two-state model, using the Levenberg-Marquardt nonlinear
least-squares method.[33]
Molecular Modeling
The molecular models were constructed with Macromodel (ver. 9.5,
Schro¨dinger Inc., USA). The initial structure was built as a standard double
helix using the Maestro (Schro¨dinger, Inc.) fragment library. The simula-
tions were carried out with an AMBER∗ force field with GB/SA treatment of
solvation.
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