Synthesis and duplex-forming property of oligonucleotides bearing a novel polyamine-modified intercalator at the terminal or the internal position

Article abstract of DOI:10.1080/15257770600685982

Novel oligonucleotides bearing a polyamine-intercalator conjugate modified at the terminal or the internal position were reported. These modified oligonucleotides showed duplex-stabilization effect, and the thermodynamic analysis and the salt concentratio

Full text of DOI:10.1080/15257770600685982

Nucleosides, Nucleotides, and Nucleic Acids, 25:601–612, 2006
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770600685982
SYNTHESIS AND DUPLEX-FORMING PROPERTY OF
OLIGONUCLEOTIDES BEARING A NOVEL POLYAMINE-MODIFIED
INTERCALATOR AT THE TERMINAL OR THE INTERNAL POSITION
Tomohisa Moriguchi, Hirokazu Sekiguchi, Makoto Tachibana,
and Kazuo Shinozuka
Department of Chemistry, Faculty of Engineering,
2
Gunma University, Kiryu, Gunma, Japan
Novel oligonucleotides bearing a polyamine-intercalator conjugate modified at the terminal or the
2
internal position were reported. These modified oligonucleotides showed duplex-stabilization effect,
and the thermodynamic analysis and the salt concentration dependency of the duplex stability revealed
that the polyamine moiety also acted as the duplex stabilizer by neutralization of the phosphate negative
charge.
Keywords Anthraquinone; Antisense DNA; Intercalator
INTRODUCTION
Oligonucleotides capable of forming a stable duplex with its comple-
mentary mRNA have been attracting great interest since they would prevent
the expression of certain gene functions responsible for disease through the
antisense-mechanism.[1,2] Although the antisense effect would depend on
various factors, such as the nuclease-resistant property and cell permeability
of the antisense oligomer, the most basic feature to bring about the effect
is, naturally, the stability of the complementary duplex formed between the
antisense oligomer and the target mRNA.[3,4]Thus, the duplex-stabilizing
ability of the antisense oligomer toward its complement is the vital factor to
achieve efficient gene-silencing in the antisense methodology.
One common method to increase the duplex-stabilizing ability of an-
tisense oligomer is to covalently introduce an intercalative moiety to the
oligomer.[5,6] For example, several modified oligonucleotides bearing an
Received 10 January 2006; accepted 14 February 2006.
This work was supported by the Gunma University Foundation for Science and Technology, and
Novartis Foundation (Japan) for the Promotion of Science.
This article is dedicated to Professor Eiko Ohtsuka on the occasion of her 70th birthday.
Address correspondence to Kazuo Shinozuka, Faculty of Engineering, Gunma University, 1-5-1
Tenjincho, Kiryu, Gunma, 376-8515, Japan. E-mail: sinozuka@chem.gunma-u.ac.jp
601

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anthraquinone moiety have been synthesized to show improved duplex-
stabilizing ability (higher Tm values) compared to their unmodified
counterparts.[7] Meanwhile, it is also well known that the incorporation
of some cationic moieties, such as polyamine moieties, to oligonucleotide
brings about enforcement of the ability.[8−14]These facts suggest that the si-
multaneous introduction of both an intercalative moiety and a polyamine
moiety to oligonucleotide would bring about substantial enhancement of
its duplex-stabilizing ability. However, reports dealing with the combination
of both an intercalative moiety and a polyamine moiety on the duplex sta-
bility are quite rare. This prompted us to synthesize novel anthraquinone-
polyamine conjugates. The conjugates were successfully incorporated into
either the C-5 position of certain pyrimidine nucleoside locating at the mid-
dle portion of the sequence or the 5ꢁ-OH terminus of oligonucleotide. Here,
we would like to report the synthesis of the modified oligodeoxynucleotides
bearing the conjugates and their duplex-stabilizing ability including the re-
sults of thermodynamic analysis of the duplex.
The 5ꢁ-terminal modified oligonucleotide was prepared by the succes-
sive solid-phase DNA synthesis strategy, and we needed to prepare the
phosphoramidite of the anthraquinone-polyamine derivatives (Scheme 1).
SCHEME 1 a. H2N(CH2)6OH, xylene-pyridine (3:1, v/v), reflux, 11 h, 67%. b. (NH2CH2CH2)3, pyri-
dine, 100◦C, 3 h, 83%. c. CF3C(O)OEt, Et3N, MeOH, r.t., 5 h, 98%. d. NCCH2CH2OPClN(i-Pr)2,
(i-Pr)2NEt, CH2Cl2, r.t., 30 min, 35%. e. NCCH2CH2OPClN(i-Pr)2, (i-Pr)2NEt, CH2Cl2, r.t., 30 min,
55%.

Oligonucleotides Synthesis and Duplex-Forming Property
603
The anthraquinone-polyamine conjugate (3) was synthesized from 1,5-
dichloroanthraquinone (1). In brief, 1,5-dichloroanthraquinone was reacted
with 6-amino-1-hexanol in a mixture of pyridine/toluene to give the mono-
reacted compound (2) as the main product. 2 was reacted with tris(2-
aminoethyl)amine in pyridine to give the anthraquinone-polyamine conju-
gate (3). After protection of the primary amine moieties with trifluoroacetyl
function by the reaction of trifluoroacetic acid ethyl ester, compound 3 was
converted to the corresponding phosphoramidite derivative (5) in the usual
manner. It should be noted that the phosphoramidite 5 is somewhat unsta-
ble under storage; therefore, it was used immediately after the purification
with silica-gel column chromatography. For the synthesis of ODN-2, non-
polyamine conjugated anthraquinone derivative[15] (6) was also converted
to the corresponding phosphoramidite derivative (7) in the usual manner.
The incorporation of 5 and 7 into oligonucleotide were carried out the same
as the incorporation of normal nucleoside phosphoramidites. However, ex-
tended coupling period (360 s) was needed to achieve the satisfactory cou-
pling yield estimated by the usual trityl-color assay.
Another modified oligonucleotide, namely the anthraquinone-
polyamine conjugate incorporated at the middle position of oligonu-
cleotide, was prepared by the post-synthetic procedure according to our
previous report (Scheme 2). The reaction of 1,5-dichloroanthraquinone
with hexamethylenediamine in xylene gave 5-(6-aminohexylamino)-1-
chloroanthraquinone (8) as the main product. After protection of the
primary amine moiety with a monomethoxytrityl group (MMTr), 9 was
reacted with tris(2-aminoethyl)amine in toluene to give the anthraquinone-
polyamine conjugate (10). The primary amine moieties of 10 were protected
with the trifluoroacetyl function as described above and the resultant 11
was treated with trichloroacetic acid to remove the MMTr group to afford
compound 12.
The incorporation of 11 into the C-5 position of the pyrimidine base was
hardly achievable, and was carried out by treatment with a 0.15 M solution of
compound 12 in dimethylacetamide after standard DNA synthesis. However,
no satisfactory result was given in spite of the investigation of various reac-
tion conditions. Therefore, the modified nucleoside, namely the conjugate
introduced at the 5-position of the deoxyuridine derivative, was prepared
in advance, and the phosphoramidite of this modified nucleoside derivative
was applied to the DNA synthesizer.
The previously prepared compound 12 was allowed to react with the
5-cyanomethoxycarbonylmethyl-2ꢁ-deoxyuridine derivative (13) to give the
successfully introduced product (14) in 72% yield. The phosphitylation of
14 by the standard method gave the phosphoramidite derivative 15; however,
purification of 15 by silica gel column chromatography was difficult due
to the rapid decomposition in the purification step. Therefore, the given

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T. Moriguchi et al.
SCHEME 2 a. H2N(CH2)6NH2, xylene, 110◦C, 2 h, 63%. b. MMTrCl, CH2Cl2, r.t., 1 h, 98%. c.
(H2NCH2CH2)3N, toluene, reflux, 2 h, 77%. d. CF3C(O)OEt, Et3N, CH2Cl2, r.t., 5 h, 98%. e. 10%
TCA/CH2Cl2, 1 h, 89%. f. 13, triazole, DMA, 40◦C, 24 h, 72%. g. NCCH2CH2OPClN(i-Pr)2, (i-Pr)2NEt,
CH2Cl2, r.t., 87%.
phosphoramidite derivative was applied to the DNA synthesizer after the
simple purification, reprecipitation by hexane.
The incorporation into DNA was carried out following the modified
procedure on DNA synthesizer, the extended coupling period (360 s) and
the twice coupling reaction. The modified DNA was given after the stan-
dard deprotection procedure, and the structure of the synthesized modi-
fied DNA was confirmed by the ESI-mass,[16] and the introduction of the

Oligonucleotides Synthesis and Duplex-Forming Property
605
TABLE 1 Melting Temperature of the Modified DNA
ODN-N ODN-1 ODN-2
ODN-3
ODN-4
Tm (◦C)
63.3
—
68.4
+5.1
68.3
+5.0
67.7
+4.4
68.5
+5.2
ꢀTm (◦C)
anthraquinone moiety was also confirmed by the comparison of the ratio
of the absorption maximum of the 260 nm and 554 nm, the absorption
maximum of anthraquinone, with the calculated value. The structure and
sequence of the synthesized oligonucleotides are shown in Figure 1. ODN-1
and -2 represent the oligonucleotides bearing non-trisamine conjugated an-
thraquinone derivatives. ODN-3 and 4 represent the oligonucleotides bear-
ing the anthraquinone-trisamine conjugates. Moreover, ODN-1 and -3 are
introduced the intercalative moiety at the 5ꢁ-terminus of oligonucleotides,
whereas ODN-2 and -4 are introduced the intercalative moiety at the central
position of the oligonucleotides.
Next, to evaluate the stabilizing effect of the anthraquinone-polyamine
conjugates toward the duplex DNA, the thermal property and the thermody-
namic property were discussed. At first, the thermal property was evaluated
by the comparison of the melting temperature (Tm) of the duplex of the
modified DNA and the corresponding complementary DNA. The thermal
stability of the duplexes containing modified oligonucleotides was measured
under the following physiological conditions; each oligonucleotide was dis-
solved in 10 mM phosphate buffer containing 100 mM NaCl, and the oligonu-
cleotide concentration was 2.5 µM. Table 1 shows that all the modified ODN
stabilized the duplex formed with the complementary DNA, and the mag-
nitude of the stabilization effects was almost the same as that of the non-
polyamine conjugates. Moreover, no difference in thermal stability of the
FIGURE 1 Sequence and structure of the oligonucleotides in this work.

606
T. Moriguchi et al.
TABLE 2 Thermodynamic Parameter of the Modified DNA
ODN-N
ODN-1
ODN-2
ODN-3
ODN-4
ꢀH (kcal/mol)
ꢀS (cal/mol · K)
ꢀG (kcal/mol)
−85.9
−227.1
−18.7
−101.4
−274.2
−19.6
−108.8
−290.0
−22.3
−133.6
−363.2
−25.3
−126.3
−341.6
−24.4
duplexes between the positions of the incorporation of the anthraquinone
moiety was observed. This enhanced stabilization was due to the introduc-
tion of the anthraquinone moiety into the oligonucleotide; however, in this
thermal profile experiment, no effect of the polyamine moiety was observed.
Next, in order to discuss the effect of the polyamine moiety on the duplex
stability, the thermodynamic analysis was explored (Table 2). The thermody-
namic parameter was calculated from the slope of the 1/Tm vs. ln(Ct/4) after
the Tm experiment at various oligonucleotide concentrations (0.63–10 µM).
All data was approximated to the first functional lines by high correlation
coefficient (Figure 2). The ꢀH values were reduced about 30 kcal/mol with
the introduction of the anthraquinone moiety, and this indicated the an-
thraquinone moiety stabilized the duplex DNA. Moreover, the additive in-
crease of the ꢀH values by the introduction of the polyamine moiety was also
observed. However, the loss of the ꢀS values was also observed. The reason
for the ꢀS loss seemed to be the enhancement of the structural rigidity by
the introduction of the polyamine moiety.
Totally, the introduction of an anthraquinone moiety and a
polyamine moiety seems to promote the stabilization of the duplex DNA
thermodynamically.
In order to estimate the effect of the polyamine moiety, the salt concen-
tration dependency against duplex stability was discussed. The relationship
between 1/Tm and ln(Na+) of all duplexes is shown in Figure 3. The thermal
FIGURE 2 Plots of 1/Tm versus ln(Ct /4). Closed squares, closed circles, closed triangles, open circles,
and open triangles represent ODN-N, -1, -2, -3, and -4, respectively.

Oligonucleotides Synthesis and Duplex-Forming Property
607
FIGURE 3 Plots of Tm−1 versus ln(Na+). Closed squares, closed circles, closed triangles, open circles, and
open triangles represent ODN-N, -1, -2, -3, and -4, respectively.
stability of no polyamine-tethering ODNs (ODN-N, -1, and -2) decreased ac-
cording to the decrease of the NaCl concentration. However, the polyamine-
tethering ODNs (ODN-3 and -4) showed the independency of the duplex
stability against low salt concentration. Therefore, the polyamine moiety sta-
bilizes the duplex DNA by the neutralization of the negative charge of the
phosphate backbone.
In conclusion, we have synthesized a novel modified DNA bearing the
anthraquinone-polyamine conjugate attached at the 5ꢁ-terminus or internal
position of the DNA. The modified DNA stabilized the DNA duplex forming
with the complementary DNA, and the thermodynamic analysis indicated
that the intercalative moiety and the cationic moiety act as stabilizers for the
duplex formation.
EXPERIMENTAL
General Procedures
CH2Cl2, MeCN and triethylamine were freshly distilled from CaH2, after
being refluxed several hours. Pyridine was distilled from CaH2, after being
refluxed several hours, and stored over KOH pellet. 1H NMR was obtained
at 300 MHz with a JEOL-AL-300 spectrometer with tetramethylsilane (TMS)
as an internal standard in CDCl3. 31P NMR was obtained at 121 MHz with a
JEOL-AL-300 spectrometer with 85% H3PO4 as an external standard in all
solvents. UV spectra were recorded with a Shimadzu UV-2450 spectropho-
tometer. Reverse-phase HPLC was performed on a JASCO PU-2089 gradient
pump system with a Wakosil 5C18(10 ml × 250 mm). ESI-TOF mass spec-
trometry was obtained with a Perkin Elmer Sciex API-100.

608
T. Moriguchi et al.
1-Chloro-5-(6-hydroxyhexylamino)anthraquinone (2). 1,5-Dichloroanthra-
quinone (800 mg, 2.89 mmol) and 6-aminohexanol (778 mg, 6.63 mmol)
were dissolved in xylene-pyridine (8 ml, 3:1 (v/v)), and the mixture was
refluxed for 11 h. After cooling to room temperature, the reaction mixture
was diluted with CH2Cl2 (50 ml), washed twice with 0.1 M KOH and once
with brine, and the organic layer was dried over Na2SO4, filtered, and
concentrated to dryness. The residue was purified by silica gel column
chromatography (10% MeOH/CH2Cl2) to give the desired compound 2
1
(696 mg, 67%) as a dark red solid. H NMR (CDCl3): 8.10–8.18 (1H, d,
ArH), 7.38–7.63 (4H, m, ArH), 6.85–6.92 (1H, m, ArH), 3.55–3.65 (2H, t,
CH2 -OH), 3.15–3.25 (2H, q, CH2 -NH) 1.30–1.75 (8H, m, (CH2)4).
1-[2-[N,N-Bis-[(2-aminoethyl)amino]ethylamino]-5-(6-hydroxyhexylamino)-
anthraquinone (3). Compound 2 (450 mg, 1.25 mmol) was dissolved in dry
pyridine (15 ml), and to this solution was added tris(2-aminoethyl)amine
(1.0 ml, 6.68 mmol), and stirred at 100◦C for 3 h. After cooling to room
temperature, the reaction mixture was diluted with CH2CH2 and washed
twice with H2O, and the organic layer was dried over Na2SO4, filtered,
and concentrated to dryness. The residue was purified by silica gel column
chromatography (CH2Cl2:MeOH:NH3aq = 4:1:0.2 (v/v/v)) to give the
desired compound 3 (483 mg, 83%) as a dark red solid. 1H NMR (CDCl3):
7.40–7.56 (4H, m, ArH), 6.84–6.91 (2H, m, ArH), 3.55–3.65 (2H, t,
CH2 -OH), 3.30–3.36 (2H, q, TAEA), 3.20–3.24 (2H, q, CH2-NH), 2.75–2.83
(6H, m, TAEA), 2.55–2.60 (4H, m, TAEA), 1.30–1.75 (8H, m, (CH2)4).
1-[2-[N,N-Bis-[(2-trifluoroacetamidoethyl)amino]ethylamino]-5-(6-hydroxyhexyl-
amino)anthraquinone (4). Compound 3 (229 mg, 0.49 mmol) was dissolved
in MeOH (7 ml), and to this solution were added ethyl trifluoroacetate
(0.30 ml, 2.52 mmol) and triethylamine (0.35 ml, 2.52 mmol). After being
stirred at room temperature for 5 h, the reaction mixture was concentrated
to dryness. The residue was purified by silica gel column chromatography
(10% MeOH/CH2Cl2) to give the desired compound 4 (315 mg, 98%) as a
1
dark red solid. H NMR (CDCl3): 7.50–7.66 (4H, m, ArH), 6.98–7.05 (1H,
m, ArH), 6.89–6.93 (1H, m, ArH), 3.64–3.71 (2H, t, CH2-OH), 3.42–3.49
(4H, m, TAEA), 3.29–3.34 (2H, m, CH2-NH,TAEA), 2.85–2.92 (2H, t,
TAEA), 2.74–2.79 (4H, m, TAEA), 1.22–1.84 (8H, m, (CH2)4).
2-Cyanoethyl 6-[1-[2-[N,N-Bis-[(2-trifluoroacetamidoethyl)amino]ethylamino]
anthraquinone-5-yl]aminohexyl N,N-Diisopropylphosphoramidite (5). Compound
4 (224 mg, 0.34 mmol) and N,N-diisopropylethylamine (0.20 ml, 1.18 mmol)
were dissolved in dry CH2Cl2 (6 ml), and to this solution was added 2-
cyanoethyl N,N-diisopropyl phosphorochloridite (0.20 ml, 0.90 mmol)
at 0◦C. After being stirred at room temperature for 30 min, the reaction
mixture was diluted with CH2Cl2, washed twice with 5% NaHCO3aq and
once with brine, and the organic layer was dried over Na2SO4, filtered,
and concentrated to dryness. The residue was purified by silica gel column
chromatography (CH2Cl2:ethyl acetate: triethylamine = 45:45:10, (v/v/v))

Oligonucleotides Synthesis and Duplex-Forming Property
609
to give the desired compound 5 (103 mg, 35%) as a dark red solid. 31P NMR
(CDCl3):147.83.
2-Cyanoethyl 6-(Anthraquinone-1-yl)aminohexyl N,N-Diisopropylphosphor-
amidite (7). 1-(6-hydroxyhexylamino)anthraquinone[15] (6) (120 mg,
0.37 mmol) and N,N-diisopropylethylamine (0.12 ml, 0.74 mmol) were
dissolved in dry CH2Cl2 (3 ml), and to this solution was added 2-cyanoethyl
N,N-diisopropyl phosphorochloridite (0.12 ml, 0.56 mmol) at 0◦C. After
being stirred at room temperature for 30 min, the reaction mixture was
diluted with CH2Cl2, washed twice with 5% NaHCO3aq and once with brine,
and the organic layer was dried over Na2SO4, filtered, and concentrated
to dryness. The residue was purified by silica gel column chromatography
(CH2Cl2:ethyl acetate:triethylamine = 45:45:10, (v/v/v)) to give the desired
compound 7 (107 mg, 55%) as a dark red solid. 31P NMR (CDCl3):148.01.
1-Chloro-5-(6-aminohexylamino)anthraquinone
(8). 1,5-Dichloroanthra-
quinone (1) (1.00 g, 3.61 mmol) and 1,6-hexamethylenediamine (965 mg,
8.30 mmol) were dissolved in xylene (7 ml), and this solution was stirred at
110◦C for 2 h. After cooling to room temperature, the reaction mixture was
diluted with CH2Cl2, washed twice with 0.1 M KOH and once with brine,
and the organic layer was dried over Na2SO4, filtered, and concentrated
to dryness. The residue was purified by silica gel column chromatography
(CH2Cl2: MeOH: NH3aq = 4:1:0.2 (v/v/v)) to give the desired compound
1
6 (814 mg, 63%) as a dark red solid. H NMR (CDCl3): 7.50–7.60 (1H, m,
ArH), 7.25–7.30 (4H, m, ArH), 6.94–6.98 (2H, dd, ArH), 3.30–3.30(2H, q,
CH2 -NH), 2.65–2.75 (2H, m, CH2 -NH), 1.30–1.75 (8H, m, (CH2)4).
1-Chloro-5-[6-[(monomethoxytrityl)amino]hexylamino]anthraquinone
(9).
Compound 8 (726 mg, 2.03 mmol) and monomethoxyltrityl chloride
(972 mg, 3.15 mmol) were dissolved in dry CH2Cl2 (10 ml), and this
solution was stirred at room temperature for 1 h. The reaction mixture
was diluted with CH2Cl2, washed three times with H2O, and the organic
layer was dried over Na2SO4, filtered, and concentrated to dryness. The
residue was purified by silica gel column chromatography (CH2Cl2: MeOH:
hexane: triethylamine = 90:3:6:1 (v/v/v/v)) to give the desired compound
1
9 (1.25 g, 98%) as a dark red solid. H NMR (CDCl3): 8.24–8.27 (1H, dd,
ArH), 7.16–7.70 (16H, m, ArH), 6.97–7.02 (1H, m, ArH), 6.77–6.82 (2H,
d, ArH), 3.76 (3H, s, OCH3), 3.25–3.32 (2H, q, CH2-NH), 2.12–2.17(4H, m,
CH2-NH), 1.43–1.73 (8H, m, (CH2)4).
1-[2-[N, N-Bis-(2-aminoethyl)amino]ethylamino]-5-[6-[(monometho xytrityl)-
amino]hexylamino]anthraquinone (10). Compound 9 (456 mg, 0.73 mmol)
and tris(2-aminoethyl)amine (1.0 ml, 6.7 mmol) were dissolved in toluene
(12 ml), and this solution was refluxed for 2 h. After cooling to room tem-
perature, the reaction mixture was diluted with CH2Cl2, washed three times
with H2O, and the organic layer was dried over Na2SO4, filtered, and con-
centrated to dryness. The residue was purified by silica gel column chro-
matography (CH2Cl2: MeOH: NH3aq = 4:1:0.2 (v/v/v)) to give the desired

610
T. Moriguchi et al.
compound 10 (408 mg, 77%) as a dark red solid. 1H NMR (CDCl3) δ 7.16–
7.54 (16H, m, ArH), 6.90–6.93 (2H, m, ArH), 6.78–6.81 (2H, m, ArH), 3.76
(3H, s, OCH3), 3.34–3.40 (2H, q, TAEA), 3.23–3.29 (2H, q, CH2 -NH), 2.78–
2.85 (6H, m, TAEA), 2.57–2.61 (4H, m, TAEA), 2.10–2.16 (2H, t, CH2 -NH),
1.40–1.72 (8H, m, (CH2)4).
1-[2-[N,N-Bis-(2-trifluoroacetamidoethyl)amino]ethylamino]-5-[6-(monometh-
oxytritylamino)hexylamino]anthraquinone (11). Compound 10 (408 mg,
0.55 mmol) was dissolved in dry CH2Cl2 (12 ml), and to this solution
were added ethyl trifluoroacetate (0.34 ml, 2.8 mmol) and triethylamine
(0.39 ml, 2.8 mmol). After being stirred at room temperature for 5 h, the
reaction mixture was concentrated to dryness. The residue was purified
by silica gel column chromatography (10% MeOH/CH2Cl2) to give the
desired compound 11 (501 mg, 98%) as a dark red solid. 1H NMR (CDCl3):
7.13–7.61 (16H, m, ArH), 6.98–7.01 (1H, m, ArH), 6.88–6.91 (1H, m,
ArH), 3.80 (3H, s, (OCH3)), 3.42–3.47 (4H, t, CH2 -NH), 3.27–3.36 (4H, m,
CH2 -NH,), 2.86–2.90 (2H, t, CH2 -NH), 2.74–2.77 (4H, t, CH2 -N), 2.10–2.16
(2H, t, CH2 -NH), 1.40–1.72 (8H, m, (CH2)4).
1-[2-[N,N-Bis-(2-trifluoroacetamidoethyl)amino]ethylamino]-5-(6-aminohexyl-
amino)anthraquinone (12). Compound 11 was treated with 10%
trichloroacetic acid in CH2Cl2 (10 ml) solution at room temperature
for 1 h. The reaction mixture was diluted with CH2Cl2, and washed twice
with 5% NaHCO3 and once with brine, and the organic layer was dried over
Na2SO4, filtered, and concentrated to dryness. The residue was purified
by silica gel column chromatography (CH2Cl2: MeOH: NH3aq = 4:1:0.2
(v/v/v)) to give the desired compound 12 (314 mg, 89%) as a dark
1
red solid. H NMR (CDCl3): 7.40–7.65 (4H, m, ArH) 6.88–6.91 (1H, m,
ArH), 6.78–6.84 (1H, m, ArH), 3.44–3.45 (4H, m, TAEA), 3.26–3.33 (4H,
m, CH2-NH, TAEA), 2.85–2.88 (2H, t, TAEA), 2.73–2.76 (6H, m, TAEA,
CH2-NH,), 1.41–1.74 (8H, m, (CH2)4).
1-[N-[6-[5-[2-[N,N-Bis-(2-trifluoroacetamidoethyl)amino]ethylamino]anthra-
quinone-5-yl]aminohexyl]carbamoylmethyl]-2ꢁ-deoxyuridine (14). Compound 12
(674 mg, 1.02 mmol), 1H-1,2,4-triazole (64 mg, 0.93 mmol) and 5-
cyanomethoxycarbonylmethyl-2ꢁ-deoxyuridine (13) (150 mg, 0.24 mmol)
were dissolved in N,N-dimethylacetamide (10 ml), and this solution was
stirred at 40◦C for 24 h. The reaction mixture was diluted with ethyl acetate,
and washed three times with H2O, and the organic layer was dried over
Na2SO4, filtered, and concentrated to dryness. The residue was purified
by silica gel column chromatography (10% 2-propanol in ethyl acetate
containing 2% triethylamine) to give the desired compound 14 (210 mg,
72%) as a dark red solid. 1H NMR (CDCl3): 7.82 (1H, s, H6), 7.40–7.65 (4H,
m, ArH), 7.18–6.92 (13H, m, DMTr), 6.88–6.91 (1H, m, ArH), 6.78–6.84
(1H, m, ArH), 6.45 (1H, t, H1ꢁ), 4.70–4.65 (1H, m, H3ꢁ), 4.15 (1H, m, H4ꢁ),
3.80(6H, s, DMTr-OCH3), 3.44–3.45 (4H, m, TAEA), 3.40 (2H, dd, H5ꢁ),

Oligonucleotides Synthesis and Duplex-Forming Property
611
3.26–3.33 (4H, m, CH2-NH, TAEA), 2.85–2.88 (2H, t, TAEA), 2.73–2.76
(4H, m, TAEA), 2.65–2.75 (2H, m, CH2 -NH), 2.58–2.40 (4H, m, H2ꢁ and
-CH2-), 1.41–1.74 (8H, m, (CH2)4).
2-Cyanoethyl
1-[N-[6-[5-[2-[N,N-Bis-(2-trifluoroacetamidoethyl)amino]ethyl-
amino]anthraquinone-5-yl]aminohexyl]carbamoylmethyl]-2ꢁ -deoxyuridine-3ꢁ-yl
N,N-Diisopropylphosphoramidite (15). Compound 14 (166 mg, 0.14 mmol)
and N,N-diisopropylethylamine (69 µl, 0.41 mmol) were dissolved in dry
CH2Cl2 (6 ml), and to this solution was added 2-cyanoethyl N,N-diisopropyl
phosphorochloridite (75 µl, 0.34 mmol) at 0◦C. After being stirred at room
temperature for 30 min, the reaction mixture was diluted with CH2Cl2,
washed twice with 5% NaHCO3aq and once with brine, and the organic
layer was dried over Na2SO4, filtered, and concentrated to dryness. The
residue was dissolved in small amounts of CH2Cl2, reprecipitated into
hexane, and the resultant precipitate was collected by filtration to give the
desired compound 15 (167 mg, 87%) as a dark red solid. 31P NMR (CDCl3):
148.7, 149.0.
Oligonucleotides Synthesis
Oligonucleotides were synthesized on an Applied Biosystems 392A
DNA/RNA synthesizer on a 1 µmol scale, using the canonical phospho-
ramidites from Glen Research. A 0.1-M solution of anthraquinone deriva-
tives 5, 7, and 15 (for the synthesis of ODN-1, -3, and -4) was used,
and the coupling time was set up to be 360 s. For the synthesis of
ODN-2, 5-methoxycarbonylmethyl-2ꢁ-deoxyuridine was used at the modi-
fied nucleoside position, and after synthesis of oligonucleotide on the
DNA synthesizer, CPG-bound oligonucleotide was treated with 0.15 M 1-(6-
aminohexylamino)anthraquinone solution in N,N-dimethylacetamide for
24 h. The CPG was washed several times with CH2Cl2, and was further treated
with 25% NH3 at 55◦C for 24 h. All DMTr-on oligonucleotides were purified
by the reversed-phase HPLC, and were detritylated by the treatment with 10%
AcOH. All the purified oligonucleotides were given after EtOH precipitaion
and gel filtration.
Melting Temperature Analysis
A solution of the appropriate concentration of the oligonucleotides and
the complementary DNA was prepared in 10 mM sodium phosphate buffer
(pH 7.2) containing 100 mM NaCl. The Tm experiments were done on a
Shimadzu UV-2450 spectrophotometer and TMSPC system. The solution was
heated from 15 to 80◦C, and the heating rate was 0.1◦C/min. Each melting
curve was calculated by use of Igor Pro. Software (WaveMetrics, Inc.). For the
ion strength experiment, the concentration of each oligomer was performed
on 2.5 µM under the same buffer conditions.

612
T. Moriguchi et al.
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