O-2-(4-Formylphenoxy)ethylnitrilotris(methylenephosphonic acid)
(3)
phases were then removed and the supports were washed with
water and treated with a mixture of saturated aq. ammonia and
ethanol (3 : 1, v/v) at 55 ◦C for 8 h, after which the solution
phases were collected and evaporated to dryness. The residues
were purified by denaturing polyacrylamide gel electrophore-
sis (20% acrylamide) and RP HPLC. MALDI-TOF-MS: m/z
DNA20LNTP: 6753.3 (calcd 6754.5); DNA20RNTP: 6663.4 (calcd
6657.4); DNA20LEDTP: 6762.8 (calcd 6759.4); DNA20REDTP:
6798.5 (calcd 6794.5); DNA20LNTP2: 7092.2 (calcd 7096.8);
DNA20RNTP2: 7124.0 (calcd 7131.8); DNA20LEDTP2: 7367.9
(calcd 7370.2); DNA20REDTP2: 7396.1 (calcd 7405.2).
Nitrilotris(methylenephosphonic acid) (16.7 mmol, 5.0 g) was
suspended in a mixture of acetic anhydride (20 mL) and DMF
(20 mL). The resulting mixture was stirred under reflux for 3 h,
after which it was evaporated to dryness. The residue (12.2 mmol,
3.0 g) and 4-(2-hydroxyethoxy)benzaldehyde (12.0 mmol, 2.0 g)
were suspended in anhydrous DMSO (20 mL) and the resulting
◦
mixture was stirred under nitrogen atmosphere at 90 C for 3 h.
Water (10 mL) was added and the stirring at 90 ◦C continued for
1 h, after which the reaction mixture was evaporated to dryness.
The residue was dissolved in water (100 mL) and washed with ethyl
acetate (100 mL). The aqueous layer was evaporated to dryness
and the residue purified by semipreparative HPLC. Overall yield
starting from nitrilotris(methylenephosphonic acid) was 31%. 1H
NMR (dH)(500 MHz, D2O) 3.68 (d, 4H, J = 11 Hz), 3.79 (d, 2H,
J = 11 Hz), 4.33–4.36 (m, 4H), 7.18 (d, 2H, J = 7 Hz), 7.93
(d, 2H, J = 7 Hz), 9.77 (s, 1H). 31P NMR (dP)(202 MHz, D2O)
8.42 (2P), 10.68 (1P). ESI−-MS: m/z 445.85 [M − H]−.
Conclusions
Synthesis of oligonucleotide conjugates bearing one or two ter-
minal nitrilotris(methylenephosphonic acid) (NTP) or ethylene-
diaminetetrakis(methylenephosphonic acid) (EDTP) ligands has
been achieved using on-support oximation of aminooxy-
functionalized oligonucleotides with 2-(4-formylphenoxy)ethyl
esters of NTP and EDTP. Single-stranded gap sites formed
by hybridizing a DNA substrate with two of these conjugates
are efficiently hydrolyzed under conditions where other single-
stranded regions of the substrate remain intact. In other words, the
system described allows site-selective hydrolysis of single-stranded
DNA targets of arbitrary length using short (e.g. 20mer) additive
oligonucleotide conjugates.
O-2-(4-Formylphenoxy)ethylethyl-
enediaminetetrakis(methylenephosphonic acid) (4)
Ethylenediaminetetrakis(methylenephosphonic acid) (11.5 mmol,
5.0 g) was suspended in a mixture of acetic anhydride (20 mL)
and DMF (20 mL). The resulting mixture was stirred under reflux
for 3 h, after which it was evaporated to dryness. The residue
(8.8 mmol, 5.0 g), 4-(2-hydroxyethoxy)benzaldehyde (48.0 mmol,
8.0 g) and a catalytic amount of DMAP were suspended in anhy-
drous DMSO (15 mL) and the resulting mixture was stirred under
nitrogen atmosphere at 100 ◦C for 2 h. Water (10 mL) was added
and the stirring at 100 ◦C continued for 2 h, after which the reaction
mixture was evaporated to dryness. The residue was dissolved in
water (150 mL) and washed with ethyl acetate (2 × 100 mL).
The aqueous layer was evaporated to dryness and the residue
purified by semipreparative HPLC. Overall yield starting from
ethylenediaminetetrakis(methylenephosphonic acid) was 26%. 1H
NMR (dH)(500 MHz, D2O) 3.11 (d, 2H, J = 10 Hz), 3.25 (d, 2H,
J = 10 Hz), 3.32 (m, 2H), 3.34 (d, 4H, J = 10 Hz), 3.50 (m, 2H),
4.29 (m, 2H), 4.35 (m, 2H), 7.19 (d, 2H, J = 9 Hz), 7.93 (d, 2H,
J = 9 Hz), 9.77 (s, 1H). 31P NMR (dP)(202 MHz, D2O) 11.14 (2P),
17.07 (1P), 19.62 (1P). ESI−-MS: m/z 583.05 [M − H]−.
Acknowledgements
This work was partially supported by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Science, Sports, Culture,
and Technology of Japan. Support by the Japan Society for
the Promotion of Science and by the Global COE Program for
Chemistry Innovation is also acknowledged.
References
1 M. Komiyama and J. Sumaoka, Curr. Opin. Chem. Biol., 1998, 2, 751;
N. H. Williams, B. Takasaki, M. Wall and J. Chin, Acc. Chem. Res.,
1999, 32, 485; A. Y. Chang and P. B. Dervan, J. Am. Chem. Soc., 2000,
122, 4856; A. Sreedhara and J. A. Cowan, JBIC, J. Biol. Inorg. Chem.,
2001, 6, 337; K. D. Copeland, M. P. Fitzsimons, R. P. Houser and J. K.
Barton, Biochemistry, 2002, 41, 343.
2 N. H. Williams and P. Wyman, Chem. Commun., 2001, 1268.
3 M. Komiyama, K. Matsumura, K. Yonezawa and Y. Matsumoto,
Chem. Express, 1993, 8, 85; M. Komiyama, T. Shiiba, T. Kodama,
N. Takeda, J. Sumaoka and M. Yashiro, Chem. Lett., 1994, 23, 1025;
B. K. Takasaki and J. Chin, J. Am. Chem. Soc., 1994, 116, 1121;
M. Komiyama, N. Takeda, Y. Takahashi, H. Uchida, T. Shiiba, T.
Kodama and M. Yashiro, J. Chem. Soc., Perkin Trans. 2, 1995, 269;
M. E. Branum, A. K. Tipton, S. Zhu and L. Que Jr., J. Am. Chem.
Soc., 2001, 123, 1898; M. Komiyama, N. Takeda and H. Shigekawa,
Chem. Commun., 1999, 1443; M. Komiyama, T. Shiiba, Y. Takahashi,
N. Takeda, K. Matsumura and T. Kodama, Supramol. Chem., 1994,
4, 31; M. Komiyama, J. Biochem., 1995, 118, 665; R. T. Kovacic, J.
T. Welch and S. J. Franklin, J. Am. Chem. Soc., 2003, 125, 6656; F.
H. Zelder, A. A. Mokhir and R. Kra¨mer, Inorg. Chem., 2003, 42,
8618.
4 W. Chen, Y. Kitamura, J.-M. Zhou, J. Sumaoka and M. Komiyama, J.
Am. Chem. Soc., 2004, 126, 10285.
5 A. N. Pudovik, Chemistry of Organophosphorus Compounds, MIR,
Moscow, 1983, p. 150.
6 H. Salo, P. Virta, H. Hakala, T. P. Prakash, A. M. Kawasaki,
M. Manoharan and H. Lo¨nnberg, Bioconjugate Chem., 1999, 10,
Preparation of the oligonucleotide multiphosphonate conjugates
DNA20LNTP, DNA20RNTP, DNA20LEDTP, DNA20REDTP,
DNA20LNTP2, DNA20RNTP2, DNA20LEDTP2 and
DNA20REDTP2
The oligodeoxyribonucleotides were assembled on a synthesizer
by the conventional phosphoramidite method, except that a
prolonged coupling time (600 s) was used for the protected
aminooxy building blocks 10, 11 and 12. The support-bound
oligonucleotides were treated with a mixture of hydrazine, pyridine
and acetic acid (1 : 32 : 8, v/v) for 30 minutes, after which
they were washed with pyridine, methanol and acetonitrile
and dried under vacuum. The supports were transferred to
microcentrifuge tubes, a solution of either 3 or 4 (20 lmol)
in water (200 lL) was added and the resulting heterogeneous
mixture was shaken at room temperature for 12 h. The solution
3586 | Org. Biomol. Chem., 2008, 6, 3580–3587
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