A novel bridged nucleoside bearing a conformationally switchable sugar moiety in response to redox changes

Article abstract of DOI:10.1039/c0cc02484h

A novel nucleoside analog with a disulfide bridge structure at the sugar moiety, which shows redox-responsive reversibility of the sugar conformation due to formation and scission of the disulfide bond, was designed and synthesized.

Full text of DOI:10.1039/c0cc02484h

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A novel bridged nucleoside bearing a conformationally switchable sugar
moiety in response to redox changesw
Takeshi Baba,z Tetsuya Kodama,z Kazuto Mori, Takeshi Imanishi and Satoshi Obika*
Received 10th July 2010, Accepted 6th September 2010
DOI: 10.1039/c0cc02484h
A novel nucleoside analog with a disulfide bridge structure at the
sugar moiety, which shows redox-responsive reversibility of the
sugar conformation due to formation and scission of the disulfide
bond, was designed and synthesized.
2. Since it is well known that sulfur nucleophiles react with
2,20-anhydronucleosides at the C20-position,15 2,20-anhydro-
nucleoside 2 was subjected to potassium thioacetate at 120 1C
to obtain bis(thioacetate) 4. However, nucleoside 2 was
converted to the 20-thio-LNA derivative16 3 via monothio-
acetylated compound 20.
It is known that sugar moieties of nucleosides, nucleotides and
nucleic acids exist in an equilibrium between S-type and N-type
conformations.1 These sugar conformations are recognized
selectively by biomolecules, such as adenosine deaminase,
DNA kinase, polymerase, RNase H and ATP receptors, to
perform biological events based on the selected sugar confor-
mation on the nucleoside, nucleotide or nucleic acid.2–5 Restricting
the nucleosugar conformations is thus an effective way to
discover compounds having high affinity for biomolecules,
and to inhibit enzymes6,7 and/or enhance binding affinity for
complementary strands.8,9 Therefore, an intense effort has
been made to develop a wide variety of conformationally
restricted nucleosides, nucleotides and oligonucleotides.
Controlling the conformational change of small molecules
by external stimuli is expected to be applied to nano-machines
and nano-devices such as molecular switches.10 In nucleic acid
chemistry, photo-,11 pH-,12 and redox-responsive13 nucleic acids
have been reported; however, there are few examples of
conformationally-switchable nucleosugar moieties to control
the affinity for biomolecules and/or complementary strands.
Here, we designed a novel bridged nucleic acid (BNA)
monomer bearing a disulfide bridged structure (disulfide-type
BNA, Fig. 1). The disulfide bridge is expected to lock the sugar
into the N-type conformation under oxidative conditions.
On the other hand, cleavage of the bridge under reductive
conditions should release the sugar conformational restric-
tions. In other words, the nucleosugar restriction of this
disulfide-type BNA monomer could be controlled by changing
the redox condition, making it applicable to a redox switch for
various biomolecules.14
Therefore, another synthetic pathway (Scheme 2) was
used to prepare the BNA monomer 8. Hydroxyl groups of
nucleoside 1 were triflated and the following alkali-mediated
hydrolysis gave arabino-type nucleoside 5. Subsequently,
C20-hydroxyl group was triflated to give ditriflate 6.17 As
expected, the thioacetylation of 6 proceeded easily at room
temperature and furnished desired 4 in 40% yield from nucleo-
side 1. Subsequently, the acetyl groups of 4 were removed with
aqueous ammonia solution under dilute conditions to afford
disulfide-bridged nucleoside 7. Finally, 7 was treated with
boron trichloride to yield the desired BNA monomer 8.
Next, structural changes of the BNA monomer under
reductive and oxidative conditions were examined using
1H-NMR spectroscopy (Fig. 2) and mass spectrometry (ESIw).
Dithiothreitol (DTT) and hydrogen peroxide (H2O2) were
used as redox reagents. In the absence of DTT, the 10-H
resonance of deuterated monomer 8-D was observed at
6.58 ppm as a singlet (Fig. 2a). After adding DTT, the singlet
resonance decreased significantly and the 10-H resonance of
the deuterated monomer 9-D was observed at 5.95 ppm as a
doublet (J = 10 Hz) (Fig. 2b). Furthermore, after adding
H2O2, the doublet decreased and the singlet reappeared at
6.58 ppm (Fig. 2c). This observation suggested that the disulfide
bridge of 8 cleaved and reformed reversibly in response to the
reducing agent DTT and the oxidizing agent H2O2.18 We also
confirmed the change in the structure by electrospray ionization
mass spectrometry (ESIw).19 In the case of 8 in H2O–MeOH
solution, m/z 319 was observed as [M + H]+. In the presence of
excess DTT, m/z 321, 641 and 663 appeared, which were
identical to [M + H]+, [2M + H]+ and [2M + Na]+ for 9,
respectively. These results suggest that disulfide-type BNA 8
could function as a molecular switch by forming and cleaving
the disulfide bridge reversibly in response to redox changes.
Next, redox reactions of BNA monomer 8 using DTT
and H2O2 were repeated (Fig. 3), showing that the reaction
To synthesize the disulfide-type BNA monomer, we examined
the effective introduction of sulfur functional groups into
nucleosides (Scheme 1). At first, the known nucleoside
derivative 19 was treated with trifluoromethanesulfonyl chloride
in the presence of triethylamine to afford 2,20-anhydronucleoside
Graduate School of Pharmaceutical Sciences, Osaka University,
1-6 Yamadaoka, Suita, Osaka, 565-0871, Japan.
E-mail: obika@phs.osaka-u.ac.jp; Fax: +81 6 6879 8204;
Tel: +81 6 6879 8200
w Electronic supplementary information (ESI) available: Full experi-
mental procedures, characterization data of all new compounds, redox
reactions using compound 8. CCDC 768234. For ESI and crystallo-
graphic data in CIF or other electronic format see DOI: 10.1039/
c0cc02484h
Fig. 1 Structure of disulfide-type BNA monomer and its change in
z Both authors contributed equally.
structure by reductant and oxidant.
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Scheme 1 Attempt to synthesize compound 4.
Fig. 2 Reversible redox reaction of BNA monomer 8 observed by
1H NMR spectroscopy: (a) absence of DTT; (b) after reaction with
DTT for 1 h; (c) after additional reaction with H2O2 for 1 h. Reaction
was performed in the mixed solvent of deuterated methanol (CD3OD)
and deuterated PBS (D2O was substituted for H2O, pH 7.4).
Scheme 2 Synthesis of BNA monomer 8.
successfully proceeded at least four times. No by-products such
as sulfoxide or sulfone were observed during these repetitions.
The switching of the molecular structure was also performed
with other redox agents: tris(2-carboxyethyl)phosphine
hydrochloride (TCEP-HCl), 2-mercaptoethanol, and sodium
tetrahydroborate as reductants, and iodine and 2,20-dithio-
dipyridine (2,20-DTDP) as oxidants (ESIw). The conforma-
tional switching was also observed in the presence of these
redox reagents. Under the presence of these redox reagents,
sugar conformations of monomer 8 or 9 switched almost
quantitatively between S-type and N-type, except for the case
where 2-mercaptoethanol was used as a reductant.
The structure of monomer 8 was confirmed by X-ray
crystallographic analysis (Fig. 4a) (see ESIw). Conformational
parameters (pseudorotation phase angle and torsion angles)
showed that the sugar conformation of 8 was fixed as the
N-type.20 Crystals of monomer 9 were not obtained because 9
was easily converted to 8 by air oxidation. Therefore, the
optimized structure of 9 was calculated.21 The structure
Fig. 3 Repetitive redox reaction of monomer 8. Reaction con-
ditions are shown in Fig. 2. The percentages of 8-D (K) and 9-D
(J) were obtained by the integration values from the 1H NMR
spectra.
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Fig. 4 (a) X-Ray crystal structure of BNA monomer 8 and
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The dihedral angle (H10–C10–C20–H20 = 163.51) obtained
from the optimized structure was in good agreement with
the coupling constant between 10-H and 20-H (J = 10 Hz)
observed in Fig. 2b.22 Furthermore, the dihedral angle
(H20–C20–C30–H30 = ꢀ39.61) was also in good agreement
with the observed coupling constant (J = 5 Hz). These results
clearly showed that the sugar moiety of 9 adopted the S-type
sugar conformation.
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In conclusion, we successfully synthesized a novel nucleo-
side with a disulfide bond forming a bridge between the 20 and
40 positions and showed that its sugar conformation was fixed
to the N-type by X-ray crystallographic analysis. We also
succeeded in switching the sugar conformation between the
N-type and S-type reversibly by changing the redox conditions.
An application study for antisense molecules, which change
its own properties in response to redox conditions, is now
ongoing, and will be reported elsewhere.
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14 A part of this work was presented at the 6th International
Symposium on Nucleic Acids Chemistry (Takayama, Japan);
T. Baba, T. Kodama, T. Imanishi and S. Obika, Nucleic Acids
Symp. Ser., 2009, 53, 107.
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17 M. Nishida, T. Baba, T. Kodama, A. Yahara, T. Imanishi and
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This work was supported by Sasagawa Science Foundation
Program (No. 21-334) of the Japan Science Society (JSS) and
Japan Society for the Promotion of Science (JSPS) KAKENHI
22655038 and 22651076.
18 We previously reported that the coupling constant between 10-H
and 20-H of light-responsive BNA changed from ca. 0 Hz to 8 Hz
as its bridge structure was cleaved in response to light stimulation.
See: K. Morihiro, T. Kodama, M. Nishida, T. Imanishi and
S. Obika, ChemBioChem, 2009, 10, 1784.
Notes and references
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19 Mass spectrum data of nucleosides 8, 9 and deuterated nucleosides
8-D, 9-D are available in ESIw.
20 The selected conformational parameters obtained by X-ray crystallo-
graphic analysis of 8 are shown in Table S1 and compared with those
of some 20,40-BNA analogs (Fig. S1) (see ESIw).
21 The calculation conditions for optimization and obtained
parameters of the optimized conformer are presented in ESIw.
22 (a) C. Altona and M. Sundaralingam, J. Am. Chem. Soc., 1972, 94,
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c
8060 Chem. Commun., 2010, 46, 8058–8060
This journal is The Royal Society of Chemistry 2010