Synthesis and triplex-forming ability of 2′,4′-BNAs bearing imidazoles as a nucleobase

Article abstract of DOI:10.1248/cpb.53.843

To expand the sequence of double-stranded DNA (dsDNA) targets in a triplex formation, 2′,4′-BNAs (2′-O,4′-C-methylene bridged nucleic acids) having imidazoles as a nucleobase were synthesized and incorporated into oligonucleotides. Triplex-forming ability

Full text of DOI:10.1248/cpb.53.843

July 2005
Notes
Chem. Pharm. Bull. 53(7) 843—846 (2005)
843
Synthesis and Triplex-Forming Ability of 2ꢀ,4ꢀ-BNAs Bearing Imidazoles
as a Nucleobase
Yoshiyuki HARI,a,1) Satoshi OBIKA,a,b Hiroyasu INOHARA,a Masahiro IKEJIRI,a Daisuke UNE,a and
,a
*
Takeshi IMANISHI
a Graduate School of Pharmaceutical Sciences, Osaka University; 1–6 Yamadaoka, Suita, Osaka 565–0871, Japan: and
b PRESTO, JST; 4–1–8 Honcho, Kawaguchi, Saitama 332–0012, Japan.
Received February 14, 2005; accepted April 26, 2005; published online May 10, 2005
To expand the sequence of double-stranded DNA (dsDNA) targets in a triplex formation, 2ꢀ,4ꢀ-BNAs (2ꢀ-
O,4ꢀ-C-methylene bridged nucleic acids) having imidazoles as a nucleobase were synthesized and incorporated
into oligonucleotides. Triplex-forming ability of the modified oligonucleotides was evaluated by using melting
temperature (Tm) measurements.
Key words methylene bridged nucleic acid (BNA); locked nucleic acid (LNA); triplex; imidazole; oligonucleotide
by using 120) as the starting material. Coupling reaction of 1
with 2-nitroimidazoles gave 2b, whereas the reaction of un-
substituted imidazole gave the desired compound 2a along
with an imidazolium salt 2aꢀ.21) Next, 2a and 2b were reacted
with K2CO3 in MeOH to give 3a and 3b. Reduction of 3a
and 3b smoothly proceeded to afford 2ꢀ,4ꢀ-BNA monomers
4a and 4b, respectively. Protection of an amino group in 4b
gave the corresponding N,N-dimethylformamidine derivative
4bꢀ. The 5ꢀ-hydroxy groups of 4a and 4bꢀ were then pro-
tected with a dimethoxytrityl (DMTr) group to afford 5a and
5b, respectively. The preparation of phosphoramidites 6a and
6b was carried out by phosphitilation of 5a and 5b, respec-
tively. Incorporation of the obtained amidites 6a and 6b into
oligonucleotides was successfully achieved by using a stan-
dard phosphoramidite method on an automated DNA synthe-
sizer. After purification by reverse-phase HPLC, composition
of the oligonucleotides was confirmed by MALDI-TOF-MS.
Triplex-forming oligonucleotide (TFO) is able to hybridize
with a double-stranded DNA (dsDNA) target in a sequence-
specific manner to form a triplex DNA, and it has attracted
great interest because of its applications to gene therapy and
diagnosis.2—4) There are two fashions in triplex formation.
One is a parallel motif triplex, where a TFO consisting of a
homopyrimidine sequence hybridizes with a dsDNA target in
parallel with a purine strand of the target duplex via Hoog-
steen hydrogen bond. The other is an antiparallel motif
triplex, in which the TFO and the purine strand of the duplex
are in an antiparallel orientation. In both triplex motifs, the
TFOs are able to hybridize only with a homopurine–homopy-
rimidine tract in the dsDNA target. This is a severe limitation
of the practical use of TFOs. To overcome this problem, ex-
tensive research on nucleic acid analogues has so far been
carried out.5—8) We have focused on the parallel type triplex,
and synthesized 2ꢀ-O,4ꢀ-C-methylene-bridged nucleic acid
(2ꢀ,4ꢀ-BNA5,9—13)/locked nucleic acid (LNA)14)) bearing un-
natural nucleobases to recognize pyrimidine–purine interrup-
tion in the target dsDNA.15—19) Recently, it was found that the
2ꢀ,4ꢀ-BNA bearing oxazole15,16) (OB: Fig. 1) or 2-pyridone17—
19)
as a nucleobase effectively recognized a CG interruption
in homopurine–homopyrimidine dsDNA.
Here, we have selected imidazoles as unnatural nucle-
obases for recognition of pyrimidine–purine interruption.
Synthesis of the 2ꢀ,4ꢀ-BNA monomers bearing imidazoles
(IB and aIB: Fig. 1) and triplex-forming ability of the oligonu-
cleotide derivatives are described.
Results and Discussion
Synthesis of 2ꢀ,4ꢀ-BNA Monomers and Their TFO De-
rivatives As shown in Chart 1, 2ꢀ,4ꢀ-BNA amidite units
bearing imidazole and 2-aminoimidazole were synthesized
Reagents: a) 1-trimethylsilylimidazole, trimethylsilyl trifluoromethanesulfonate, 40%
(2a) and 24% (2aꢀ), or N,O-bis(trimethylsilyl)acetamide, 2-nitro-1-imidazole, tri-
methylsilyl trifluoromethanesulfonate, 93% (2b); b) K2CO3, MeOH, 94% (3a) and 87%
(3b); c) 20% Pd(OH)2–C, cyclohexene, EtOH, 78% (4a) and 90% (4b); d)
Me2NCH(OMe)2, MeOH, 93% (4bꢀ); e) DMTrCl, pyridine, 47% (5a) and 87% (5b); f)
(iPr2N)2POCH2CH2CN, diisopropylammonium tetrazolide, THF–MeCN, 42% (6a) and
87% (6b).
Fig. 1. Structures of the 2ꢀ,4ꢀ-BNAs Bearing Oxazole (OB), Imidazole (IB)
and 2-Aminoimidazole (aIB) as Unnatural Nucleobases and the 2ꢀ,4ꢀ-BNA
without a Nucleobase (HB)
Chart 1
∗ To whom correspondence should be addressed. e-mail: imanishi@phs.osaka-u.ac.jp
© 2005 Pharmaceutical Society of Japan

844
Vol. 53, No. 7
Fig. 2. Sequences of the TFOs and the dsDNA Targets Used in This Study
C means 5-methylcytidine.
Table 1. Tm Values (°C) for Dissociation of the Triplexes Comprising
TFOs 7—13 and dsDNA Targets at pH 7.0a)
dsDNA target (YZ)
TFO
CG
GC
TA
AT
Fig. 3. Plausible Structures of IB·CG, OB·CG, aIB·GC and aIB·TA Base
Triads
7 (IB)
8 (aIB)
9 (OB)b)
10 (HB)c)
11 (T)
21
17
29
24
25
25
20
23
33
22
20
20
43
23
25
19
27
20
17
16
27
23
19
23
16
44
19
16
oligonucleotide derivatives revealed that the 2ꢀ,4ꢀ-BNA with
unsubstituted imidazole (IB) had only low affinity with CG
interruption in the homopurine·homopyrimidine dsDNA tar-
get, while OB, which has an oxazole moiety as a nucleobase,
successfully interacted with the CG interruption.15) This re-
sult suggests importance of the oxygen atom in OB for recog-
nition of the CG interrption. On the other hand, IB showed
moderate binding affinity with TA interruption, and the other
2ꢀ,4ꢀ-BNA analogue aIB, which has a 2-aminoimidazole moi-
ety, formed a relatively stable aIB·GC triad. These results
would be useful for further development of highly effective
TFOs.
12 (C)
13 (G)
a) Conditions: 7 mM sodium phosphate buffer (pH 7.0) containing 140 mM KCl and
10 mM MgCl2. The concentration of triplexes was 1.5 mM. b) Ref. 15. c) Ref. 18. C
means 5-methylcytidine.
The sequences of the TFOs used in this study are shown in
Fig. 2.
UV Melting Experiments Triplex-forming ability of the
TFOs 7—13 was evaluated by UV melting experiments
under neutral pH conditions (7 mM sodium phosphate buffer
(pH 7.0) containing 140 mM KCl and 10 mM MgCl2). The re-
sults are summarized in Table 1. The TFO 7 containing IB
showed low affinity with the dsDNA containing CG interrup-
tion (Tmꢁ21 °C), while OB in TFO 9 successfully recognized
the CG interruption (Tmꢁ29 °C).15) This reflects the signifi-
cant importance of an oxygen atom in the oxazole moiety of
OB for recognition of a CG base pair. One possibility is that
the oxygen atom directly makes a hydrogen bond with the 4-
amino group of C (Fig. 3), though we previously proposed
the interaction between the nitrogen atom of the oxazole and
Experimental
General All melting points were measured on a Yanagimoto micro
melting points apparatus and are uncorrected. Optical rotations were
recorded on a JASCO DIP-370 instrument. IR spectra were recorded on a
JASCO FT/IR-200 spectrometer. 1H-NMR spectra were recorded on a JEOL
EX-270 (270 MHz) and 31P-NMR spectrum was recorded on a Varian VXR-
200 (31P, 81.0 MHz). Mass spectra of nucleoside analogues were recorded on
a JEOL JMS-600 or JMS-700 mass spectrometer. For column chromatogra-
phy, Fuji Silysia BW-300 (200—400 mesh) and BW-127ZH (100—270
mesh) were used. MALDI-TOF-Mass spectra were recorded on an Applied
Biosystems Voeyger®-DE.
1-[2-O-Acetyl-3,5-di-O-benzyl-4-(p-toluenesulfonyloxymethyl)-b-D-ri-
the 4-amino group of C.15) Although no appropriate hydrogen bofuranosyl]imidazole (2a) Under a N2 atmosphere, 1-trimethylsilylimi-
dazole (0.73 ml, 5.00 mmol) and trimethylsilyl trifluoromethanesulfonate
bonding scheme between the TA base pair and an oxazole or
(1.00 ml, 5.53 mmol) were added to a solution of 120) (1.00 g, 1.67 mmol) in
imidazole moiety can be proposed, both IB and OB showed
anhydrous 1,2-dichloroethane (20 ml) at room temperature and the mixture
was stirred at room temperature for 24 h. After addition of a saturated aque-
ous NaHCO3, the mixture was extracted with AcOEt. Usual work-up and
moderate affinity with a TA base pair. The Tm values of the
triplexes having IB·TA and OB·TA triads were 25 °C and
purification by flash column chromatography [CHCl3/Et2O (5/1)] afforded
2a (402 mg, 40%) as a colorless oil along with 2aꢀ (512 mg, 24%) as a col-
orless prism. 2a: [a]D28 ꢂ30.7° (cꢁ1.00, CHCl3). IR nmax (KBr): 1748, 1363,
1229, 1180 cmꢂ1. 1H-NMR (CDCl3) d: 2.01 (3H, s), 2.42 (3H, s), 3.54, 3.56
(2H, AB, Jꢁ10 Hz), 4.16, 4.22 (2H, AB, Jꢁ11 Hz), 4.46—4.48 (3H, m),
4.48, 4.54 (2H, AB, Jꢁ12 Hz), 5.28 (1H, dd, Jꢁ6, 6 Hz), 5.61 (1H, d,
Jꢁ6 Hz), 6.99—7.00 (2H, m), 7.19—7.41 (12H, m), 7.57 (1H, s), 7.72 (2H,
d, Jꢁ8 Hz). Mass (EI): m/z 606 (Mꢃ, 2.7), 91 (100). Anal. Calcd for
C32H34N2O8S·1/3H2O: C, 62.73; H, 5.70; N, 4.57; S, 5.23. Found: C, 62.75;
H, 5.74; N, 4.31; S, 5.03. 2aꢀ: mp 59—61 °C. [a]D26 ꢂ4.0° (cꢁ0.86, CHCl3).
27 °C, respectively, while that of the triplex containing G·TA
triad was 27 °C.22) This moderate affinity of IB and OB with
the TA base pair might involve shape recognition of the TA
base pair by five-membered heteroaromatics. Introduction of
a 2-amino group into the imidazole ring of IB decreased the
affinity with the TA base pair (Tmꢁ19 °C), while the triplex
containing aIB·GC triad was found to be stable (Tmꢁ33 °C).
It was supposed that the 2-amino group of aIB interacted with
the 6-carbonyl oxygen of G, while the interaction between
the 2-amino group of aIB and the 4-carbonyl oxygen of T was
obstructed by steric hindrance of the 5-methyl group of T
(Fig. 3).
IR nmax (KBr): 1751, 1364, 1268, 1097 cmꢂ1 1H-NMR (CDCl3) d: 2.07
.
(6H, s), 2.43 (6H, s), 3.40, 3.80 (4H, AB, Jꢁ11 Hz), 4.03 (2H, d, Jꢁ11 Hz),
4.42 (2H, d, Jꢁ12 Hz), 4.33—4.56 (8H, m), 4.69 (2H, d, Jꢁ6 Hz), 5.51 (2H,
dd, Jꢁ2, 6 Hz), 5.72 (2H, d, Jꢁ2 Hz), 7.19—7.37 (24H, m), 7.43 (2H, d,
Jꢁ1 Hz), 7.71 (4H, d, Jꢁ8 Hz), 9.29 (1H, s). Mass (FAB): m/z 1145 (Mꢃ),
149 (CF3SO3ꢂ). Anal. Calcd for C61H65N2O16S2·CF3SO3: C, 57.49; H, 5.06;
N, 2.16. Found: C, 57.25; H, 5.04; N, 2.19.
In conclusion, we have synthesized 2ꢀ,4ꢀ-BNA monomers
bearing imidazole and 2-aminoimidazole as an unnatural nu-
cleobase. UV melting experiments of the corresponding
1-[2-O-Acetyl-3,5-di-O-benzyl-4-(p-toluenesulfonyloxymethyl)-b-D-ri-

July 2005
845
bofuranosyl]-2-nitroimidazole (2b) Under a N2 atmosphere, 2-nitroimi- C, 50.65; H, 6.46; N, 19.69. Found: C, 50.89; H, 6.42; N, 19.39.
dazole (277 mg, 2.45 mmol) and N,O-bis(trimethylsilyl)acetamide (0.71 ml,
2.87 mmol) were added to a solution of 1 (1.22 g, 2.04 mmol) in anhydrous
1,2-dichloroethane (20 ml) at room temperature and the mixture was re-
1-[5-O-(4,4ꢀ-Dimethoxytrityl)-2-O,4-C-methylene-b-D-ribofuranosyl]-
imidazole (5a) Under a N2 atmosphere, DMTrCl (83 mg, 0.24 mmol) was
added to a solution of 4a (40 mg, 0.19 mmol) in anhydrous pyridine (1 ml) at
fluxed for 1 h. After the mixture cooled, trimethylsilyl trifluoromethanesul- room temperature and stirred for 3.5 h. The reaction was quenched by addi-
fonate (0.15 ml, 0.83 mmol) was added to the mixture and the mixture was tion of a saturated aqueous NaHCO3. The mixture was extracted with
refluxed for 5 h. After addition of a saturated aqueous NaHCO3, the mixture AcOEt. Usual work-up and purification by silica gel column chromatogra-
was extracted with AcOEt. Usual work-up and purification by flash column phy [CHCl3/AcOEt/Et3N (80/4/1)] afforded 5a (46 mg, 47%) as a white
chromatography [n-hexane/AcOEt (12/5)] afforded 2b (1.24 g, 93%) as a
powder. mp 92—94 °C (n-hexane/AcOEt). [a]D24 ꢂ20.9° (cꢁ0.66, CHCl3).
white powder. mp 38—41 °C. [a]D22 ꢃ2.1° (cꢁ1.10, CHCl3). IR nmax (KBr): IR nmax (KBr): 3135, 2938, 1615, 1508, 1251, 1040 cmꢂ1
.
1H-NMR (ace-
1
1752, 1537, 1478, 1363, 1225, 1181, 1096 cmꢂ1. H-NMR (CDCl3) d: 2.11 tone-d6) d: 3.47, 3.57 (2H, AB, Jꢁ11 Hz), 3.79 (6H, s), 3.94, 3.99 (2H, AB,
(3H, s), 2.43 (3H, s), 3.42, 3.91 (2H, AB, Jꢁ11 Hz), 4.12 (1H, d, Jꢁ12 Hz),
4.24—4.47 (4H, m), 4.44 (1H, d, Jꢁ6 Hz), 4.54 (1H, d, Jꢁ12 Hz), 5.40 (1H,
Jꢁ8 Hz), 4.31 (1H, s), 4.44 (1H, s), 5.83 (1H, s), 6.87—6.92 (4H, m), 6.98
(1H, s), 7.23—7.53 (10H, m), 7.80 (1H, s). Mass (EI): m/z 514 (Mꢃ, 6.2),
dd, Jꢁ2, 6 Hz), 6.10 (1H, d, Jꢁ2 Hz), 6.84 (1H, d, Jꢁ1 Hz), 7.17—7.34 303 (100). Anal. Calcd for C30H30N2O6·1/3H2O: C, 69.22; H, 5.94; N, 5.38.
(12H, m), 7.73 (1H, d, Jꢁ1 Hz), 7.79 (2H, d, Jꢁ8 Hz). Mass (EI): m/z 605
Found: C, 69.39; H, 5.88; N, 5.21.
1-[5-O-(4,4ꢀ-Dimethoxytrityl)-2-O,4-C-methylene-b-D-ribofuranosyl]-
(MꢃꢂNO2, 0.1), 560 (MꢃꢂBn, 0.1), 91 (100). Anal. Calcd for
C32H33N3O10S: C, 58.98; H, 5.10; N, 6.45; S, 4.92. Found: C, 58.72; H, 5.19; 2-[N-(dimethylaminomethylidene)amino]imidazole (5b) Compound 5b
N, 6.06; S, 4.86.
was obtained from 4bꢀ (68 mg, 0.24 mmol) using the same procedure em-
ployed for the preparation of 5a (a pale yellow powder, 122 mg, 87%). mp
106—110 °C. [a]D22 ꢂ4.9° (cꢁ0.92, CHCl3). IR nmax (KBr): 1631, 1510,
1-(3,5-Di-O-benzyl-2-O,4-C-methylene-b-D-ribofuranosyl)-imidazole
(3a) K2CO3 (481 mg, 3.48 mmol) was added to a solution of 2a (707 mg,
1.16 mmol) in MeOH (15 ml) at room temperature and the mixture was
stirred for 16 h. After removal of the solvent, water was added to the residue
and the mixture was extracted with AcOEt. Usual work-up and purification
by flash column chromatography [CHCl3/AcOEt (3/1)] afforded 3a (432 mg,
1
1251, 1038 cmꢂ1. H-NMR (acetone-d6) d: 3.02 (3H, s), 3.11 (3H, s), 3.45,
3.54 (2H, AB, Jꢁ11 Hz), 3.78 (6H, s), 3.88, 3.96 (2H, AB, Jꢁ8 Hz), 4.22
(1H, s), 4.45 (1H, s), 5.81 (1H, s), 6.62 (1H, d, Jꢁ2 Hz), 6.87—6.90 (4H,
m), 7.03 (1H, d, Jꢁ2 Hz), 7.23—7.41 (7H, m), 7.52—7.55 (2H, m), 8.40
(1H, s). Mass (EI): m/z 584 (Mꢃ, 43.3), 303 (100). Anal. Calcd for
94%) as a colorless oil. [a]D26 ꢂ7.5° (cꢁ0.68, CHCl3). IR nmax (KBr): 2945,
1492, 1216, 1025 cmꢂ1. H-NMR (CDCl3) d: 3.81 (2H, s), 3.95, 4.08 (2H, C33H36N4O6·3/2CH3OH: C, 65.49; H, 6.69; N, 8.85. Found: C, 65.74; H,
1
AB, Jꢁ8 Hz), 4.13 (1H, s), 4.21 (1H, s), 4.56 (2H, s), 4.62 (2H, s), 5.73 (1H, 6.39; N, 8.76.
s), 6.91 (1H, s), 7.08 (1H, s), 7.23—7.35 (10H, m), 7.60 (1H, s). Mass (EI):
m/z 392 (Mꢃ, 9.4), 91 (100). Anal. Calcd for C23H24N2O4·1/3H2O: C, 69.33;
H, 6.24; N, 7.03. Found: C, 69.10; H, 6.14; N, 6.95.
1-[3-O-[2-Cyanoethoxy(diisopropylamino)phosphino]-5-O-(4,4ꢀ-
dimethoxytrityl)-2-O,4-C-methylene-b-D-ribofuranosyl]imidazole (6a)
Under a N2 atmosphere, 2-cyanoethyl-N,N,Nꢀ,Nꢀ-tetraisopropylphosphorodi-
1-(3,5-Di-O-benzyl-2-O,4-C-methylene-b-D-ribofuranosyl)-2-nitroimi-
dazole (3b) Compound 3b was obtained from 2b (1.09 g, 1.67 mmol)
amidite (44 ml, 0.14 mmol) was added to
a solution of 5a (60 mg,
0.12 mmol) and diisopropylammonium tetrazolide (14 mg, 81.7 mmol) in an-
using the same procedure employed for the preparation of 3a (pale yellow hydrous MeCN/THF (3/1, 2 ml) at room temperature and stirred for 5.5 h.
needles, 639 mg, 87%). mp 126—127 °C. [a]D23 ꢃ73.2° (cꢁ1.25, CHCl3). The solvent was concentrated under reduced pressure. The residue was puri-
IR nmax (KBr): 1536, 1473, 1362, 1052 cmꢂ1 1H-NMR (CDCl3) d: 3.80, fied by flash silica gel column chromatography [n-hexane/AcOEt/Et3N
.
3.84 (2H, AB, Jꢁ11 Hz), 3.87, 4.06 (2H, AB, Jꢁ8 Hz), 4.10 (1H, s), 4.40 (50/50/1)] and precipitated from n-hexane/AcOEt to give 6a (35 mg, 42%)
(1H, s), 4.49, 4.55 (2H, AB, Jꢁ12 Hz), 4.65, 4.67 (2H, AB, Jꢁ12 Hz), 6.24
(1H, s), 7.12 (1H, d, Jꢁ1 Hz), 7.18—7.40 (10H, m), 7.63 (1H, d, Jꢁ1 Hz).
Mass (EI): m/z 391 (MꢃꢂNO2, 0.2), 346 (MꢃꢂBn, 5.0), 91 (100). Anal.
as a colorless oil. 31P-NMR (acetone-d6) d: 149.45, 149.66.
1-[3-O-[2-Cyanoethoxy(diisopropylamino)phosphino]-5-O-(4,4ꢀ-
dimethoxytrityl)-2-O,4-C-methylene-b-D-ribofuranosyl]-2-[N-(dimethyl-
Calcd for C23H23N3O6: C, 63.15; H, 5.30; N, 9.61. Found: C, 63.08; H, 5.37; aminomethylidene)amino]imidazole (6b) Compound 6b was obtained
N, 9.43. from 5b (55 mg, 94.1 mmol) using the same procedure employed for the
1-(2-O,4-C-Methylene-b-D-ribofuranosyl)imidazole (4a) Twenty per- preparation of 6a (a white powder, 64 mg, 87%). mp 66—69 °C. 31P-NMR
cent Pd(OH)2–C (54 mg) and cyclohexene (0.74 ml, 7.31 mmol) were added
(CDCl3) d: 148.66, 148.88.
to a solution of 3a (57 mg, 0.15 mmol) in EtOH (2 ml) at room temperature
Synthesis and Purification of TFOs The modified oligonucleotides
and the mixture was refluxed for 1.5 h. After filtration of the solution, silica were synthesized on a 0.2 mmol scale on a Pharmacia Gene Assembler® Plus
(0.1 g) was added to the filtrate and the solution was concentrated under re-
duced pressure. The residue was purified by flash silica gel column chro-
matography [CHCl3/MeOH (10/1 to 5/1)] to give 4a (24 mg, 78%) as a col-
orless oil. [a]D22 ꢂ56.6° (cꢁ0.51, CH3OH). IR nmax (KBr): 3254, 3121,
or on an Applied Biosystems Expedite 8909 according to the standard phos-
phoramidite protocol. The oligonucleotide supported on CPG, which retains
a 5ꢀ-terminal DMTr group, was treated with concentrated ammonium hy-
droxide at 60 °C for 18 h, and the solvents were concentrated. After puri-
2961, 1494, 1226, 1051 cmꢂ1. 1H-NMR (CD3OD) d: 3.90 (2H, s), 3.83, 4.00 fication through NENSORBTM PREP, the oligonucleotide was purified
(2H, AB, Jꢁ8 Hz), 4.22 (1H, s), 4.27 (1H, s), 5.78 (1H, s), 7.02 (1H, s), 7.27 by reverse-phase HPLC (ChemcoPak® CHEMCOSORB 300-5C18,
(1H, s), 7.87 (1H, s). Mass (EI): m/z 212 (Mꢃ, 57.7), 69 (100). Anal. Calcd
4.6 mmꢄ250 mm) with a 11% MeCN in 0.1 M triethylammonium acetate
for C9H12N2O4·1/2H2O: C, 48.87; H, 5.92; N, 12.66. Found: C, 48.84; H, buffer (pH 7.0). MALDI-TOF-Mass data for TFO 7 [MꢂH]ꢂ: Found
5.77; N, 12.50.
4465.86, Calcd 4466.03; TFO 8 [MꢂH]ꢂ: Found 4480.85, Calcd 4481.04;
TFO 9 [MꢂH]ꢂ: Found 4467.24, Calcd 4467.01; TFO 10 [MꢂH]ꢂ: Found
4400.41, Calcd 4399.97.
2-Amino-1-(2-O,4-C-methylene-b-D-ribofuranosyl)imidazole (4b)
Compound 4b was obtained from 3b (200 mg, 0.46 mmol) using the same
procedure employed for the preparation of 4a (a colorless oil, 93 mg, 90%).
Tm Measurements UV melting experiments were carried out on a
[a]D28 ꢂ58.2° (cꢁ1.08, CH3OH). IR nmax (KBr): 3418, 1646, 1560 cmꢂ1. 1H- Beckmann DU-650 spectrophotometer equipped with Tm analysis accessory.
NMR (CD3OD) d: 3.81, 3.99 (2H, AB, Jꢁ8 Hz), 3.88 (2H, s), 4.28 (1H, s),
4.32 (1H, s), 5.55 (1H, s), 6.54 (1H, d, Jꢁ2 Hz), 6.77 (1H, d, Jꢁ2 Hz). Mass
(FAB): m/z 228 (MHꢃ). High-resolition Mass (FAB): 228.0981 (MHꢃ,
Calcd for C9H14N3O4: 228.0984).
The UV melting profiles were recorded in 7 mM sodium phosphate buffer
(pH 7.0) containing 140 mM KCl and 10 mM MgCl2 at a scan rate of
0.5 °C/min at 260 nm. The final concentration of each oligonucleotide was
1.5 mM. The Tm value was designated as the maximum of the first derivative
calculated from the UV melting profile.
2-[N-(Dimethylaminomethylidene)amino]-1-[2-O,4-C-methylene-b-D-
ribofuranosyl]imidazole (4bꢀ) 1,1-Dimethoxytrimethylamine (96 ml,
0.72 mmol) was added to a solution of 4b (66 mg, 0.29 mmol) in MeOH
(1 ml) at room temperature and the mixture was stirred for 11 h. After addi-
tion of silica (0.5 g), the solution was concentrated under reduced pressure.
The residue was purified by flash silica gel column chromatography
[CHCl3/MeOH (10/1 to 5/1)] to give 4bꢀ (76 mg, 93%) as a white powder.
mp 212—213 °C. [a]D28 ꢂ5.2° (cꢁ0.97, CH3OH). IR nmax (KBr): 3447,
References and Notes
1) Present address: Graduate School of Pharmaceutical Sciences, Nagoya
City University; 3–1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467–
8603, Japan.
2) Guntaka R. V., Varma B. R., Weber K. T ., Int. J. Biochem. Cell Biol.,
35, 22—31 (2003).
3) Malvy C., Harel-Bellan A., Pritchard L. L., “Triple Helix Forming
Oligonucleotides,” Kluwer Academic Publishers, Boston, 1998.
4) Soyfer V. N., Potaman V. N., “Triple-Helical Nucleic Acids,” Springer-
Verlag, New York, 1995.
1
3222, 2932, 1638, 1523, 1393, 1041 cmꢂ1. H-NMR (CD3OD) d: 3.03 (3H,
s), 3.10 (3H, s), 3.81, 3.99 (2H, AB, Jꢁ8 Hz), 3.90 (2H, s), 4.21 (1H, s),
4.28 (1H, s), 5.78 (1H, s), 6.64 (1H, d, Jꢁ2 Hz), 6.99 (1H, d, Jꢁ2 Hz), 8.18
(1H, s). Mass (FAB): m/z 283 (MHꢃ). Anal. Calcd for C12H18N4O4·1/8H2O:

846
Vol. 53, No. 7
5) Obika S., Chem. Pharm. Bull., 52, 1399—1404 (2004) and references
cited therein.
15) Obika S., Hari Y., Morio K., Imanishi T., Tetrahedron Lett., 41, 221—
224 (2000).
6) Buchini S., Leumann C. J., Curr. Opin. Chem. Biol., 7, 717—726 16) Hari Y., Obika S., Sakaki M., Morio K., Yamagata Y., Imanishi T.,
(2003) and references cited therein.
Tetrahedron, 58, 3051—3063 (2002).
7) Robles J., Grandas A., Pedroso E., Luque F. J., Eritja R., Orozco M.,
Curr. Org. Chem., 6, 1333—1368 (2002) and references cited therein.
17) Obika S., Hari Y., Sekiguchi M., Imanishi T., Angew. Chem., Int. Ed.,
40, 2079—2081 (2001).
8) Sasaki S., Taniguchi Y., Takahashi R., Senko Y., Kodama K., Naga- 18) Obika S., Hari Y., Sekiguchi M., Imanishi T., Chem. Eur. J., 8, 4796—
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10) Imanishi T., Obika S., Chem. Commun., 2002, 1653—1659 (2002).
11) Obika S., Nanbu D., Hari Y., Morio K., In Y., Ishida T., Imanishi T.,
Tetrahedron Lett., 38, 8735—8738 (1997).
4802 (2002).
19) Torigoe H., Hari Y., Obika S., Imanishi T., Nucleosides Nucleotides
Nucleic Acids, 22, 1097—1099 (2003).
20) Koshkin A. A., Rajwanshi V. K., Wengel J., Tetrahedron Lett., 39,
4381—4384 (1998).
12) Obika S., Hari Y., Sugimoto T., Sekiguchi M., Imanishi T., Tetrahe- 21) It was demonstrated that the coupling reaction between a sugar deriva-
dron Lett., 41, 8923—8927 (2000).
13) Obika S., Uneda T., Sugimoto T., Nanbu D., Minami T., Doi T., Ima-
nishi T., Bioorg. Med. Chem., 9, 1001—1011 (2001).
tive and unsubstituted imidazole afforded the corresponding imida-
zolium dimer: Al Mourabit A., Beckmann M., Poupat C., Ahond A.,
Potier P., Tetrahedron: Asymmetry, 7, 3455—3464 (1996).
14) The 2ꢀ,4ꢀ-BNA was independently synthesized by the group of Wengel
immediately after our first report (ref. 11), and it is called a locked nu-
cleic acid (LNA). See: Singh S. K., Nielsen P., Koshkin A. A., Wengel
J., Chem. Commun., 1998, 455—456 (1998).
22) Among natural nucleobases A, G, T, and C, guanine nucleobase (G)
was found to most tightly interact with a TA base pair to form a rela-
tively stable G·TA triad in a parallel motif triplex: Griffin L. C., Der-
van P. B., Science, 245, 967—971 (1989).