H. Ozaki et al. / Bioorg. Med. Chem. Lett. 13(2003) 2441–2443
Table 2. Melting temperatures of the modified ODN/DNA
2443
convenient synthesis of the ODNs containing arabino-
nucleosides. The ODNs containing arabinofur-
anosylisocytosine or arabinofuranosyl-5-methylisocytosine
formed a stable duplex with complementary DNA con-
taining isoguanosine and also the substitution of amino
group to 2-aminoethylamino group at C-2 position led
to the destabilization of the duplex. Also, C5 substitu-
tion of arabinofuranosyluracil residue (ODN2c) did not
so much affect on duplex formation with a com-
plementary DNA. The study on the synthesis of a novel
modified ODN containing arabinonucleosides is in pro-
gress using our developed method.
ODN
X
cDNA1(dA)
cDNA2(disoG)
Tm/ꢁC ÁT/ꢁC(vs ODN2b) Tm/ꢁC ÁT/ꢁC(vs cDNA1)
N-ODN
T
61.6
4.0
ꢀ8.6
ꢀ8.3
ꢀ0.2
0
ꢀ0.1—
ꢀ8.0
ꢀ8.3
ꢀ9.7
ꢀ8.7
ꢀ13.1
ꢀ8.8
—
—
47.1
—
—
—
ODN1a 1a 49.0
ODN1b 1b 49.3
ODN2a 2a 57.4
ODN2b 2b 57.6
ODN2c 2c 57.5
ODN3a 3a 49.6
ODN3b 3b 49.3
ODN3c 3c 47.9
ODN4a 4a 48.9
ODN4b 4b 44.5
ODN4c 4c 48.8
ꢀ2.2
—
—
—
—
—
—
63.6
62.7
—
48.5
49.4
14.3
14.8
—
4.0
0.6
References and Notes
ODN: 50-d(CGC TTC TXC CTG CCA)-30, which X is a modified
nucleoside. cDNA1: 50-d(TGG CAG GAA GAA GCG)-30 cDNA2:
50-d(TGG CAG GisoGA GAA GAC)-30, which is isoG is deoxy-
isoguanosine.
Condition: Conc. of DNA, 2 mM; buffer, 150 mM sodium chloride/
10 mM sodium phosphate (pH 7.0).
1. (a) Damha, M. J.; Wilds, C. J.; Noronha, A.; Brukner, I.;
Borkow, G.; Arion, D.; Parniak, M. A. J. Am. Chem. Soc.
1998, 120, 12976. (b) Noronha, A. M.; Wilds, C. J.; Lok, C.-
N.; Viazovkina, K.; Arion, D.; Parniak, M. A.; Damha, M. J.
Biochemistry 2000, 39, 7050.
2. Denisov, A. Y.; Noronha, A. M.; Wild, C. J.; Trempe, J.-
F.; Pon, R. T.; Gehring, K.; Damha, M. J. Nucleic Acids Res.
2001, 29, 4284.
3. Min, K.-L.; Viazovkina, E.; Galarneau, A.; Parniak, M. A.;
Damha, M. J. Bioorg. Med. Chem. Lett. 2002, 12, 2651.
4. Legorburu, U.; Reese, C. B.; Song, Q. Tetrahedron 1999,
55, 5635.
5. Brown, D. M.; Parihar, D. B.; Todd, A.; Varadarajan, S. J.
Chem. Soc. 1958, 3028.
6. Doerr, I. L.; Fox, J. J. J. Org. Chem. 1967, 32, 1462.
7. Ross, B. S.; Springer, R. H.; Tortorici, Z.; Dimock, S.
Nucleosides and Nucleotides 1997, 16, 1641.
8. Ogilvie, K. K.; Iwacha, D. Can. J. Chem. 1969, 47, 495.
9. Arabinoaminooxazoline allowed to react with ethyl
a-(bromomethyl)-acrylate and the product was treated with
potassium t-butoxide. Ref.; Sawai, H.; Nakamura, A.; Haya-
shi, H.; Shinozuka, K. Nucleosides and Nucleotides 1994, 13,
1647.
10. Sawai, H.; Nakamura, A.; Sekiguchi, S.; Yumoto, K.;
Endoh, M.; Ozaki, H. J. Chem. Soc., Chem. Commun. 1994,
1997.
cDNA1 were lower than araU derivatives-containing
ODNs (ODN2a–c) by >8 ꢁC. This may be because
these anhydroU derivatives (1a–c) and arabinofur-
anosylisocytosine derivatives (3a–c and 4a–c) don’t have
an imino proton at N3 position, which is required for
the Watson–Crick base-pairing with adenine residue on
the complementary DNA. Also, 2,20-anhydrouridine
derivatives may have rigid conformation due to a 2,20-
ether bond. Isocytosine is known to form a base-pair
with isoguanine.15 To confirm the formation of base
pair between arabinofuranosylisocytosine derivatives
and isoganine, a complementary DNA bearing deoxy-
isoguanosine (cDNA2) was synthesized and melting
experiments of ODN/cDNA2 were carried out. The
results for several ODN were listed in Table 2. Tm of
ODN1b containing 2,20-anhydrothymidine with cDNA2
is lower than that with cDNA1 by ꢀ2.2 ꢁC (ÁT vs
cDNA1). This is due to no amino group at C2. On the
other hand, Tm’s of ODN containing arabinofur-
anosylisocytosine derivatives (ODN3b, ODN3c,
ODN4b, and ODN4c) with cDNA2 is higher than that
with cDNA1. Especially, the duplex of ODN3b con-
11. ESI-MS data. 2a: m/z calcd for C9H12N2NaO6+
(M+Na)+267.06, found 266.9. 2b: m/z calcd for
C10H15N2O+6 (M+H)+ 259.09, found 259.1. 2c: m/z calcd for
C11H15N2O+8 (M+H)+ 303.08, found 303.4. 3a: m/z calcd for
C9H14N3O+5 (M+H)+ 244.09, found 244.1. 3b: m/z calcd for
C10H16N3O+5 (M+H)+ 258.11, found 257.9. 3c: m/z calcd for
C11H17N4O+6 (M+H)+ 301.11, found 301.3. 4a: m/z calcd for
C11H19N4O+5 (M+H)+ 287.14, found 287.0. 4b: m/z calcd for
C12H21N4O+5 (M+H)+ 301.15, found 301.2. 4c: m/z calcd for
C15H27N6O+6 (M+H)+ 387.2, found 387.3.
taining
ODN3c
arabinofuranosyl-5-methylisocytosine
containing arabinofuranosyl-5-carboxy-
or
methylisocytosine with cDNA2 showed higher Tm which
was comparative to that of normal DNA duplex N-
ODN/cDNA1 (Tm 61.6 ꢁC). These results suggest that
arabinofuranosylisocytosine derivatives formed a base
pair with isoguanine residue in cDNA2. Also, a sub-
stitution to ethylenediamine at N2 amino group
(ODN4b and ODN4c) caused a destabilization of
duplex due to the steric hindrance by the substituent
compared with ODN3b and ODN3c, respectively.
12. Atkins, T.; Smith, M. In Oligonucleotides Synthesis— A
Practical Approach; Gait, M. J., Ed.; IRL Press: Oxford, 1984;
pp 35–81.
13. For example, 1a in addition of four natural deoxynucleo-
sides was detected on HPLC when ODN4a, which was identi-
fied by ESI-MS, was degraded by nucleases (snake venom
phosphodiesterase, nuclease P1, and alkaline phophatase).
14. (a) Hirata, M. Chem. Pham. Bull. 1968, 16, 437. (b) Delia,
T. J.; Beranek, J. J. Carb. Nucleosides Nucleotides 1977, 4, 349.
15. Switzer, C. Y.; Moroney, S. E.; Benner, S. A. Biochemistry
1993, 32, 10489.
In this paper, we indicated that ODN containing araU,
araT, or ara(isoC) derivatives were easily prepared by a
post-synthetic modification. This method will provide a