Efficient incorporation of a copper hydroxypyridone base pair in DNA

Article abstract of DOI:10.1021/ja027175o

Recently, we reported the first artificial nucleoside for alternative DNA base pairing through metal complexation (J. Org. Chem. 1999, 64, 5002-5003). In this regard, we report here the synthesis of a hydroxypyridone-bearing nucleoside and the incorporati

Full text of DOI:10.1021/ja027175o

Published on Web 09/25/2002
Efficient Incorporation of a Copper Hydroxypyridone Base
Pair in DNA
Kentaro Tanaka,† Atsushi Tengeiji,† Tatsuhisa Kato,‡ Namiki Toyama,‡
Motoo Shiro,§ and Mitsuhiko Shionoya*,†
Contribution from the Department of Chemistry, Graduate School of Science,
The UniVersity of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan,
Institute for Molecular Science, Myodaiji, Okazaki 444-8585, Japan, and
Rigaku Corporation, 3-9-12 Matsubaracho, Akishima, Tokyo 196-8666, Japan
Received June 5, 2002
Abstract: Recently, we reported the first artificial nucleoside for alternative DNA base pairing through metal
complexation (J. Org. Chem. 1999, 64, 5002-5003). In this regard, we report here the synthesis of a
hydroxypyridone-bearing nucleoside and the incorporation of a neutral Cu2+-mediated base pair of
hydroxypyridone nucleobases (H-Cu-H) in a DNA duplex. When the hydroxypyridone bases are
incorporated into the middle of a 15 nucleotide duplex, the duplex displays high thermal stabilization in the
presence of equimolar Cu2+ ions in comparison with a duplex containing an A-T pair in place of the H-H
pair. Monitoring temperature dependence of UV-absorption changes verified that a Cu2+-mediated base
pair is stoichiometrically formed inside the duplex and dissociates upon thermal denaturation at elevated
temperature. In addition, EPR and CD studies suggested that the radical site of a Cu2+ center is formed
within the right-handed double-strand structure of the oligonucleotide. The present strategy could be
developed for controlled and periodic spacing of neutral metallobase pairs along the helix axis of DNA.
Introduction
base-pairing modes in which hydrogen-bonded base pairs
present in natural DNA are replaced by metal-mediated ones.
Efforts toward the expansion of the genetic alphabet have
been made extensively with respect to artificial gene control as
well as development of functionalized biopolymers.1,2 Recently,
we3 and others4,5 have reported a few examples of alternative
Along this strategy, we have designed a novel hydroxypyridone
nucleobase (H) as a planar bidentate ligand (Chart 1). Since
hydroxypyridone is known to form, with concomitant depro-
tonation, a stable, neutral complex with a divalent transition
metal ion such as Cu2+,6 the resulting square-planar complex
should replace hydrogen-bonded natural base pairs. We report
here the synthesis of hydroxypyridone-bearing nucleoside 1,
efficient incorporation of 1 into oligonucleotides, 2 and 3, and
the influence of a copper-mediated pair of the chelator-type
nucleobases on the thermal stability of the DNA duplex.
* To whom correspondence should be addressed. Phone: +81-3-5841-
8061. Fax: +81-3-5841-8061. E-mail: shionoya@chem.s.u-tokyo.ac.jp
† The University of Tokyo.
‡ Institute for Molecular Science.
§ Rigaku Corporation.
(1) (a) Switzer, S.; Moroney, S. E.; Benner, S. A. J. Am. Chem. Soc. 1989,
111, 8322. (b) Piccirilli, J. A.; Krauch, T.; Moroney, S. E.; Benner, S. A.
Nature 1990, 343, 33. (c) Switzer, C. Y.; Moroney, S. E.; Benner, S. A.
Biochemistry 1993, 32, 10489. (d) Horlacher, J.; Hottiger, M.; Podust, V.
N.; Hu¨bscher, U.; Benner, S. A. Proc. Natl. Acad. Sci. U.S.A. 1995, 92,
6329. (e) Lutz, M. J.; Held, H. A.; Hottiger, M.; Hu¨bscher, U.; Benner, S.
A. Nucleic Acids Res. 1996, 24, 1308.
Results and Discussion
A synthetic route for a hydroxypyridone-bearing nucleoside
1 is shown in Scheme 1. A key step was a Lewis acid-catalyzed
coupling reaction7 between fully protected deoxyribofuranose
68 and 2-methyl-3-(benzyloxy)-4-pyridone (7).9 The resulting
mixture of R- and â-anomers 8 (R:â ) 3:7) was used for the
following deprotection without separation. After removal of the
benzyl group, the desired â-N-nucleoside 9 was successfully
isolated by recrystallization from EtOH, and its anomeric
configuration was determined by X-ray crystal analysis (Figure
(2) (a) Kool, E. T. Biopolymers 1998, 48, 3. (b) Guckian, K. M.; Krugh, T.
R.; Kool, E. T. Nat. Struct. Biol. 1998, 11, 954. (c) Kool, E. T.; Morales,
J. C.; Guckian, K. M. Angew. Chem., Int. Ed. 2000, 39, 990. (d) McMinn,
D. L.; Ogawa, A. K.; Wu, Y.; Liu, J.; Schultz, P. G.; Romesberg, F. E. J.
Am. Chem. Soc. 1999, 121, 11585. (e) Ogawa, A. K.; Wu, Y.; McMinn,
D. L.; Liu, J.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc. 2000,
122, 3274. (f) Wu, Y.; Ogawa, A. K.; Berger, M.; McMinn, D. L.; Schultz,
P. G.; Romesberg, F. E. J. Am. Chem. Soc. 2000, 122, 7621. (g) Brotschi,
C.; Ha¨berli, A.; Leumann, C. J. Angew. Chem., Int. Ed. 2001, 40, 3012.
(3) (a) Tanaka, K.; Shionoya, M. J. Org. Chem. 1999, 64, 5002. (b) Cao, H.;
Tanaka, K.; Shionoya, M. Chem. Pharm. Bull. 2000, 48, 1745. (c) Shionoya,
M.; Tanaka, K. Bull. Chem. Soc. Jpn. 2000, 73, 1945. (d) Tanaka, K.;
Tasaka, M.; Cao, H.; Shionoya, M. Eur. J. Pharm. Sci. 2001, 13, 77. (e)
Tasaka, M.; Tanaka, K.; Shiro, M.; Shionoya, M. Supramol. Chem. 2001,
13, 671. (f) Tanaka, K.; Tasaka, M.; Cao, H.; Shionoya, M. Supramol.
Chem. 2002, 14, 255. (g) Tanaka, K.; Yamada, Y.; Shionoya, M. J. Am.
Chem. Soc. 2002, 124, 8802.
(6) (a) El-Jammal, A.; Howell, P. L.; Turner, M. A.; Li, N.; Templeton, D. M.
J. Med. Chem. 1994, 37, 461. Here the X-ray structure of a square-planar
Cu2+ complex in the trans form is reported. (b) Ahmed, S. I.; Burgess, J.;
Fawcett, J.; Parsons, S. A.; Russell, D. R.; Laurie, S. H. Polyhedron 2000,
19, 129. (c) Griffith, W. P.; Mostafa, S. I. Polyhedron 1992, 11, 2997.
(7) Mao, D. T.; Driscoll, J. S.; Marquez, V. S. J. Med. Chem. 1984, 27, 160.
(8) Gold, A.; Sangaiah, R. Nucleosides Nucleotides 1990, 9, 907.
(9) Harris, R. L. N. Aust. J. Chem. 1976, 29, 1329.
(4) (a) Meggers, E.; Holland, P. L.; Tolman, W. B.; Romesberg, F. E.; Schultz,
P. G. J. Am. Chem. Soc. 2000, 122, 10714. (b) Atwell, S.; Meggers, E.;
Spraggon, G.; Schultz, P. G. J. Am. Chem. Soc. 2001, 123, 12364.
(5) (a) Weizman, H.; Tor, Y. Chem. Commun. 2001, 453. (b) Weizman, H.;
Tor, Y. J. Am. Chem Soc. 2001, 123, 3375.
9
12494
J. AM. CHEM. SOC. 2002, 124, 12494-12498
10.1021/ja027175o CCC: $22.00 © 2002 American Chemical Society

Copper Hydroxypyridone Base Pairs in DNA
A R T I C L E S
Chart 1
a natural-type oligoduplex (4‚5), d(5′-CACATTAATGTTGTA-
3′)‚d(3′-GTGTAATTACAACAT-5′), in which the H-H base
pair is replaced by an A-T base pair, melted at 44.2 °C (Figure
3b). Thus, in the absence of Cu2+ ions, the H-H base pair
behave as a mispair to destabilize the duplex, although the pair
could possibly be hydrogen-bonded (Figure 1). In contrast,
addition of equimolar Cu2+ ions (2 µM) to the duplex 2‚3 led
to base pairing with higher thermal stability. Thermal denatur-
ation of 2‚3 resulted in a biphasic melting curve with a
melting point of 50.1 °C (Figure 3c), which is higher than that
of 4‚5 in the absence of Cu2+ ions. Thus, the Cu2+-assisted base
pair in 2‚3 stabilized the duplex by about 13 °C,11 whereas the
natural duplex 4‚5 was hardly affected by addition of Cu2+ ions
(Figure 3d).
Figure 4 shows the temperature dependence of the UV
absorption for the duplex 2‚3 in the presence of equimolar Cu2+
ions ([2‚3] ) [Cu2+] ) 2 µM). As is evidenced in Figure 4a, a
peak at 314 nm that appeared at low temperature indicates
that Cu2+ ions interact preferentially with the hydroxypyri-
done moiety and that the resulting Cu2+-mediated base pair
(H-Cu-H) is stoichiometrically formed inside the duplex.12
As the temperature was raised from 10 to 85 °C, this peak at
314 nm characteristic for the Cu2+ complex gradually disap-
peared, where absorbance at 260 nm simultaneously increased
with two isosbestic points as shown in Figure 4a. Absorption
changes (∆A/∆Amax from 10 to 85 °C) at 260 nm (broken line
in Figure 4b) are in fair agreement with those at 314 nm (solid
line in Figure 4b). This result suggests that, at elevated
temperature after melting, the hydroxypyridone bases no longer
bind to Cu2+ ions.
1).10 After removal of the acetyl groups, the 5′ and phenolic
hydroxy groups of 1 were selectively protected in order by 4,4′-
dimethoxytrityl and pivaloyl groups, respectively. Nucleoside
11 thus obtained was finally converted to phosphoramidite 12.
Incorporation into oligodeoxynucleotides and deprotection of
the fully protected products were carried out with standard
protocols using an automated DNA synthesizer.
We have chosen a Cu2+ ion that forms a 1:2 square-planar
complex with deprotonated hydroxypyridone ligands.6a UV-
absorption change upon complexation was employed as a
quantitative structural probe to verify the presence of bound
Cu2+ ions. Figure 2 shows UV-absorption changes of 1 when
the concentration of Cu2+ ions is increased. With an increase
in the Cu2+ concentration, the absorbance at 282 nm gradually
decreased while a new peak at 303 nm appeared with two
isosbestic points in the range of [Cu2+]/[1] from 0.0 to 0.5. This
simultaneous change indicates the deprotonation of phenolic
hydroxy groups of hydroxypyridone ligands upon Cu2+ com-
plexation. Absorption data at 282 and 303 nm are plotted as a
function of the ratio of Cu2+ to 1 (Figure 2, inset), clearly
showing the complex formation between Cu2+ and hydroxy-
pyridone ligand 1 in a 1:2 ratio. The final product was confirmed
by electrospray ionization time-of-flight mass spectrometry
(found, m/z 566.10, calcd for [M + Na]+, m/z 566.09, where
M represents a 1:2 complex, Cu2+‚2(1 - H+)).
The circular dichroism (CD) spectrum of the duplex 2‚3
without Cu2+ ion has a characteristic feature of typical right-
handed B-DNA (Figure 5).13 The metalloduplex showed almost
the same spectrum except an additional positive Cotton effect
was observed at 314 nm (Figure 5, solid line). This result
indicates that a Cu2+-hydroxypyridone complex incorporated
hardly affects the B-DNA helix sense.
Electron paramagnetic resonance (EPR) was used as a
quantitative structural probe to verify the presence of bound
Cu2+ ions. The X-band EPR spectrum at 1.5 K of a frozen
aqueous solution of the duplex 2‚3 with equimolar Cu2+ ions
is compared with that of a 1:2 Cu2+-complex with mononucleo-
side 1 (Figure 6). Both spectra are comparable with those of
Cu2+-porphyrins14 and are due to a doublet (S ) 1/2) radical of
a Cu2+ center in the square-planar ligand field. A significant
difference was the line sharpening of the spectrum of the Cu2+
complex with the duplex 2‚3. This line sharpening could reflect
the isolation of the radical site from hydrated water into the
hydrophobic environment. This fact suggests that the radical
site of a Cu2+ center is formed within the right-handed double-
strand structure of oligonucleotide.
To examine the influence of Cu2+ ions on the thermal stability
of an H-H base pair in DNA, the pair was introduced in the
middle of a 15-nucleotide DNA duplex (2‚3), d(5′-CACAT-
TAHTGTTGTA-3′)‚d(3′-GTGTAATHACAACAT-5′), and the
melting temperature in the absence or presence of Cu2+ ions
was determined by UV-monitored thermal denaturation ([du-
plex] ) 2 µM). In the absence of Cu2+ ions, the duplex 2‚3
showed a melting temperature of 37.0 °C (Figure 3a), whereas
(10) Crystal data for 9: C15H19NO7, fw ) 325.32, trigonal, space group P32, a
) 7.7066(3) Å, c ) 44.782(1) Å, V ) 2303.9(1) Å3, Z ) 6, µ ) 1.12
cm-1, Dcald ) 1.407 g cm-3, F(000) ) 1032.00. Reflection data were
collected on a Rigaku RAXIS-RAPID imaging plate diffractometer with
Mo KR radiation (λ ) 0.71073 Å) at 103 K. Of the 27 442 reflections
which were collected, 5511 were unique (Rint ) 0.029); equivalent
reflections were merged. Convergence to the final R values of R1 ) 0.057,
Rw ) 0.095, and R1 ) 0.039 (I > 2σ(I)) for 9 was achieved by using 5502
unique reflections and 422 parameters (on F2), and R (Rw) ) 0.057 (0.095)
for all the data. The goodness-of-fit on F2 is 1.16. The maximum and
minimum peaks on the final diffraction Fourier map corresponded to 0.33
and -0.42 e-/Å3, respectively.
(11) Additional Cu2+ ions further increased the stability only slightly.
(12) It should be noted that under this condition a Cu2+-mediated base pair is
formed only when incorporated into DNA oligomers. For example, the
binding constant determined voltammetrically for a 2:1 complex between
deprotonated 1,2-dimethyl-3-hydroxy-4-pyridone (pKa ) 9.86 ( 0.02) and
Cu2+ is log â2 ) 21.7 ( 0.8; see ref 6a. The Cu2+ complexation inside the
DNA should be reinforced cooperatively by the presence of surrounding
hydrogen-bonded and stacked base pairs in the hydrophobic environ-
ment.
(13) Saenger, W. Principles of Nucleic Acid Structure; Springer-Verlag: New
York, 1984.
(14) Wolberg, A.; Manassen, J. J. Am. Chem. Soc. 1970, 92, 2982.
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Tanaka et al.
Scheme 1. Synthetic Route for Nucleoside 1 and Its Phosphoramidite 12a
a Reagents and conditions: (a) (i) hexamethyldisilazane, (NH4)2SO4, reflux; (ii) 1,3,5-tri-O-acetyl-2-deoxy-D-ribofuranose (6), trimethylsilyl trifluo-
romethanesulfonate, CH3CN, rt, 70%; (b) H2, Pd/C, AcOEt, rt, 30% (for â-anomer); (c) saturated aqueous NH4OH, MeOH, rt, quant; (d) DMTr-Cl, pyridine,
rt, 77%; (e) pivalic anhydride, iPr2EtN, THF, rt, 61%; (f) 2-cyanoethyl N,N-diisopropylchlorophosphoramidite, iPr2EtN, CH2Cl2, rt, 61%.
Figure 1. ORTEP drawing for a hydrogen-bonded, paired structure of â-N-
Figure 3. Melting curves of the duplexes 2‚3 (a, c) and 4‚5 (b, d). [2‚3]
nucleoside 9 with 50% thermal ellipsoids.
) [4‚5] ) 2.0 µM in 10 mM Na phosphate buffer, 50 mM NaCl (pH 7.0).
[CuSO4] ) (a, b) 0 µM and (c, d) 2.0 µM.
multimetal arrays along the DNA helix axis is now underway
toward achieving molecular wires and magnets.
Experimental Section
Synthesis of Hydroxypyridone-Bearing Nucleosides and Nucle-
otides. General Methods. All reactions were carried out in oven-dried
glasswares under nitrogen atmosphere with commercial dehydrated
solvents (Wako). 1,3,5-Tri-O-acetyl-2-deoxy-D-ribofuranose and 2-methyl-
3-(benzyloxy)-4-pyridone were prepared according to previously
published procedures.8,9 All other reagents were purchased and were
used without further purification. Column chromatography was per-
formed using Wakogel C-300 silica gel (Wako).
1H NMR spectra were recorded on either a JEOL EX270 (270 MHz)
or a Bruker DRX500 (500 MHz) spectrometer. The spectra are
referenced to TMS in chloroform-d3. Chemical shifts (δ) are reported
in ppm; multiplicities are indicated by the following: s (singlet); d
(doublet); dd (doublet of doublets); ddd (doublet of doublet of doublets);
m (multiplet); br (broad). Coupling constants, J, are reported in Hz.
Electrospray ionization time-of-flight (ESI-TOF) mass spectra were
recorded on a Micromass LCT spectrometer.
Figure 2. UV absorption changes of 1 at various concentrations of CuSO4
at 25 °C. [1] ) 50 µM in 25 mM Na-Mops (pH 7.0). Inset: plot of
absorbance at 282 and 303 nm against the ratio of Cu2+ to 1.
Conclusion
Compound 9. 2-Methyl-3-(benzyloxy)-4-pyridone (7) (504 mg, 2.34
mmol) and a catalytic amount of (NH4)2SO4 were dissolved in
hexamethyldisilazane (HMDS, 5 mL), and the reaction mixture was
heated at reflux for 2 h before excess HMDS was distilled off. To the
resulting residue was added a solution of 1,3,5-tri-O-acetyl-2-deoxy-
D-ribofuranose (6) (669 mg, 2.57 mmol) in CH3CN (25 mL). Then
In this paper, we accomplished efficient incorporation of a
neutral Cu2+-mediated base pair of hydroxypyridone nucleo-
bases into a DNA duplex. The Cu2+-mediated base pair is
stoichiometrically formed inside the duplex and significantly
increases the thermal stability of the duplex. Nanoassembly of
9
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Copper Hydroxypyridone Base Pairs in DNA
A R T I C L E S
Figure 6. X-band EPR spectra of (a) Cu2+-mononucleoside, Cu‚2(1 -
H+), and (b) Cu2+-oligonucleotide, Cu‚(2‚3), obtained in a frozen aqueous
solution at 1.5 K. [Cu‚2(1 - H+)] ) [Cu‚(2‚3)] ) 100 µM in 10 mM
HEPES, 50 mM NaCl (pH 7.0). Both spectra are reproducible by the
simulation, as shown by the broken line. The parameters of the simulation
are anisotropic g values: Cu‚2(1 - H+), gx )gy ) 2.068, gz ) 2.262; Cu‚
(2‚3), gx ) gy ) 2.056, gz ) 2.262, and hyperfine coupling constants of
63CuII nuclear spin (I ) 3/2, 69.09%) and 65CuII (I ) 3/2, 30.91%);
Cu‚2(1 - H+), ax ) ay ) 1.6 mT, az ) 19.3 mT; Cu‚(2‚3), ax ) 2.8 mT,
ay ) 2.0 mT, az ) 21.4 mT, where ax, ay, and az are hyperfine coupling
constants of 63Cu and those of 65Cu are multiplied by 1.07.
Compound 8 (3.7 g, 8.9 mmol) was dissolved in AcOEt (100 mL),
and 10% Pd/C (500 mg, 0.47 mmol) was added to the reaction mixture.
The suspended solution was stirred vigorously for 2 h under H2
atmosphere. After filtration and evaporation, the residue was recrystal-
lized from EtOH to afford the desired â-N-nucleoside 9 as colorless
plates (870 mg, 30%). 1H NMR (CDCl3): δ 7.68 (d, J ) 8.1 Hz, 1H),
6.45 (d, J ) 7.6 Hz, 1H), 6.01 (dd, J ) 5.7, 7.8 Hz, 1H), 5.27 (ddd, J
) 2.4, 2.4, 6.2 Hz, 1H), 4.22-4.42 (m, 3H), 2.51 (ddd, J ) 2.7, 5.9,
14.0 Hz, 1H), 2.45 (s, 3H), 2.33 (ddd, J ) 6.8, 7.0, 7.8 Hz, 1H), 2.11,
2.14 (s × 2, 6H).
Figure 4. (a) Temperature dependence from 10 to 85 °C of UV absorption
for the duplex 2‚3 in the presence of equimolar Cu2+ ions. [2‚3] ) [CuSO4]
) 2.0 µM in 10 mM Na phosphate buffer, 50 mM NaCl (pH 7.0). (b)
Comparison of absorption changes (∆A/∆Amax) at 260 and 314 nm (broken
and solid lines, respectively).
Compound 1. To a solution of nucleoside 9 (998 mg, 3.07 mmol)
in MeOH (40 mL) was added 28% aqueous NH4OH solution (10 mL),
and the mixture was stirred for 3 h at room temperature before
evaporation of the solvent. The residue was solidified in AcOEt, and
the colorless solid was collected (741 mg, 100%). Mp: 141.0-143.0
°C. 1H NMR (D2O): δ 7.95 (d, J ) 7.5 Hz, 1H), 6.50 (d, J ) 7.5 Hz,
1H), 6.21 (dd, J ) 6.3, 6.3 Hz, 1H), 4.41 (ddd, J ) 4.4, 4.4, 6.4 Hz,
1H), 4.05 (ddd, J ) 4.4, 4.4, 8.9 Hz, 1H), 3.80 (dd, J ) 3.5, 12.6 Hz,
1H), 3.71 (dd, J ) 5.3, 12.6 Hz, 1H), 2.49 (ddd, J ) 6.6, 6.6, 13.5 Hz,
1H), 2.40 (s, 3H), 2.32 (ddd, J ) 6.5, 6.5, 13.5 Hz, 1H). HRMS
(FAB): calcd for C11H16NO5 ([M + H]+), m/z 242.1028; found, m/z
242.1023.
Compound 10. To a solution of nucleoside 1 (290 mg, 1.20 mmol)
in anhydrous pyridine (2 mL) was added DMTr-Cl (570 mg, 1.68
mmol), and the reaction mixture was stirred for 2 h at room temperature.
After the reaction was quenched with MeOH, the mixture was poured
into ice-water (100 mL) and extracted with CHCl3. The organic layer
was dried over anhydrous MgSO4 and concentrated. The residue was
purified by silica gel column chromatography with CHCl3-MeOH (100:
1) to obtain 10 as a colorless foam (498 mg, 77%). 1H NMR (CDCl3):
δ 7.95 (d, J ) 5.7 Hz, 1H), 6.79-7.40 (m, 13H, including residual
CHCl3), 6.18 (d, J ) 7.3 Hz, 1H), 6.05 (br, 1H), 4.57 (br, 1H), 4.38
(br, 1H), 4.12 (br, 1H), 3.75 (s, 6H), 2.37 (s, 3H), 2.25-2.50 (br, 2H).
Compound 11. To a solution of nucleoside 10 (1.05 g, 1.93 mmol)
and iPr2EtN (404 µL, 2.32 mmol) in THF (7.7 mL) was added pivalic
anhydride (403 µL, 2.12 mmol), and the solution was stirred for 15 h
at room temperature. The reaction mixture was poured into CHCl3 (150
mL) and washed with brine. The organic layer was dried over MgSO4
Figure 5. CD spectra of the duplex 2‚3 in the absence (broken line) and
in the presence (solid line) of equimolar Cu2+ ions. [2‚3] ) 2.0 µM in 10
mM Na phosphate buffer, 50 mM NaCl (pH 7.0).
trimethylsilyl trifluoromethanesulfonate (465 µL, 2.57 mmol) was added
dropwise to the reaction mixture, and the resulting solution was stirred
for 24 h at room temperature. The reaction was quenched with saturated
aqueous NaHCO3 solution, and the solvent was evaporated. The residue
was taken up into CH2Cl2 and water, and the organic layer was washed
with saturated aqueous NaHCO3 solution and water, dried over
anhydrous Na2SO4, and then evaporated. Purification of the residue by
silica gel column chromatography with CHCl3-CH3OH (100:1) af-
forded 8 as a 3:7 mixure of R- and â-anomers (687 mg, 70%).
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A R T I C L E S
Tanaka et al.
and evaporated. The residue was purified by silica gel column
chromatography (CHCl3) and subsequent alumina column chromatog-
raphy (CHCl3) to afford 11 as a colorless foam (741 mg, 61%). H
and detritylated by OPC protocol. The yields and concentration of
solutions were determined by comparison of UV absorption at 260 nm
with nearest-neighbor parameters15 and molar extinction coefficient of
1 (ꢀ ) 5.34 × 103 at 260 nm).
1
NMR (CDCl3): δ 7.92 (d, J ) 7.9 Hz, 1H), 6.82-7.40 (m, 13H), 6.14
(d, J ) 7.9 Hz, 1H), 6.01 (dd, J ) 6.3, 6.3 Hz, 1H), 4.56 (br, 1H),
4.08 (br, 1H), 3.76 (s, 6H), 3.42 (br, 2H), 2.21 (s, 3H), 2.16-2.50 (br
× 2, 2H), 1.37 (s, 9H).
UV Absorption Measurements and UV-Melting Experiments. UV
absorption spectra were recorded on a Hitachi U-3500 spectrometer
equipped with a Peltier thermoelectric temperature control unit. Melting
curves (at 260 nm) were recorded for a consecutive heating (10-85
°C) heating-cooling-heating protocol with a linear gradient of 1.0
°C/min.
Compound 12. To a solution of 11 (342 mg, 545 µmol) and N,N-
diisopropylethylamine (238 µL, 1.36 mmol) in CHCl3 (10 mL) was
added dropwise 2-cyanoethyl N,N-diisopropylchlorophosphoramidite
(267 µL, 1.20 mmol). After 30 min, the reaction mixture was poured
into ice-water (30 mL) and extracted with CH2Cl2 (100 mL). The
organic layer was washed with water, dried over MgSO4, and then
evaporated. The residue was purified by silica gel column chromatog-
raphy (AcOEt containing 0.2%(v/v) pyridine) to afford a diastereomeric
Circular Dichroism Study. Experiments were performed with a
JASCO J-820 spectropolarimeter equipped with a Peltier thermoelectric
temperature control unit. A 1 cm path length quartz cuvette was used
for scans from 400 to 200 nm at 25 °C.
EPR Study. EPR spectra were recorded on a Bruker E500
spectrometer with an Oxford cryo-system.
1
mixture 12 as a colorless foam (275 mg, 61%). H NMR (CDCl3): δ
7.94 (m, 2H), 6.82-7.40 (m, 13H, including residual CHCl3), 6.15 (m,
1H), 6.01 (m, 1H), 4.69 (br, 1H), 4.17-4.22 (br, 1H), 3.35-4.90 (m,
12H), 2.47-2.62 (m, 2H), 2.31-2.50 (br × 2, 2H), 2.27 (s, 3H), 1.40
(s, 9H), 1.08-1.21 (m, 12H).
Oligonucleotide Synthesis. Oligonucleotides 2-5 were synthesized
on an ABI 394 DNA synthesizer using standard â-cyanoethyl phos-
phoramidite chemistry. Reagents and concentrations applied were the
same as those for syntheses of natural DNA oligomers. Syntheses were
performed on a 1 µmol scale trityl-on mode, according to the
manufacturer’s protocol. The only change made to the usual synthesis
cycle was the prolongation of the coupling time to 3 min. After the
oligomers were removed from the support and deprotected by treatment
with 25% NH3 (55 °C, 12 h), the crude oligonucleotides were purified
Acknowledgment. This work is supported by Grants-in-Aid
for Scientific Research (B), Nos. 13554024 and 14340206, and
by that on Priority Area, No. 13128202, to M.S. from the
Ministry of Education, Culture, Sports, Science, and Technology
of Japan.
Supporting Information Available: X-ray data (in CIF
format) for 9. This material is available free of charge via the
Internet at http://pubs.acs.org.
JA027175O
(15) Cantor, C. R.; Warshaw, M. M.; Shapiro, H. Biopolymers 1970, 9, 1059.
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