Synthesis of a novel bridged nucleoside bearing a fused-azetidine ring, 3′-amino-3′,4′-BNA monomer

Article abstract of DOI:10.1016/S0040-4039(03)01279-6

A novel bridged nucleic acid monomer, 3′-amino-3′-deoxy-5-methyl-3′-N,4′-C- methyleneuridine, was successfully synthesized via a useful and convenient azetidine ring formation under Staudinger's conditions. A ¹H NMR experiment and a PM3 calcula

Full text of DOI:10.1016/S0040-4039(03)01279-6

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 5267–5270
Synthesis of a novel bridged nucleoside bearing a fused-azetidine
ring, 3%-amino-3%,4%-BNA monomer
Satoshi Obika, Jyun-ichi Andoh, Mayumi Onoda, Osamu Nakagawa, Akiko Hiroto,
Tomomi Sugimoto and Takeshi Imanishi*
Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan
Received 28 March 2003; revised 12 May 2003; accepted 16 May 2003
Abstract—A novel bridged nucleic acid monomer, 3%-amino-3%-deoxy-5-methyl-3%-N,4%-C-methyleneuridine, was successfully syn-
1
thesized via a useful and convenient azetidine ring formation under Staudinger’s conditions. A H NMR experiment and a PM3
calculation revealed that the sugar moiety of the novel bridged nucleic acid monomer, 3%-amino-3%,4%-BNA, was restricted to S-type
conformation. © 2003 Elsevier Science Ltd. All rights reserved.
The synthesis of novel nucleoside analogues is of great
interest because of their application as a key intermedi-
ate for an antisense and/or an antigene molecule to
regulate target gene expression,1 as well as their direct
use for an anti-tumor or an antiviral compound. In
recent studies for developing an ideal and a practical
antisense molecule, the oligonucleotide analogues hav-
ing ‘non-genetic’ 2%,5%-phosphodiester linkages were
found to be a good candidate as an antisense molecule
due to their RNA selective hybridization property and
enzymatic stability.2 Furthermore, one of the 2%,5%-
linked oligonucleotides, 2-5A (2%,5%-oligoadenylate 5%-
triphosphate), is well known to activate a RNase L to
prevent viral infection;3 therefore, intensive efforts
towards the development of various 2-5A analogues
have been demonstrated to date.4
well known to have high binding affinity with ssRNA,
ssDNA and dsDNA.5 The sugar moiety of 3%-amino-3%-
deoxynucleoside prefers N-type puckering due to weak
gauche effect between the 3%-nitrogen and the 4%-oxygen.
This preference in the sugar puckering mode is thought
to be one major reason for the superior binding ability
of oligonucleotides N3%“P5% phosphoramidate.6
Recently, we achieved the synthesis and development of
a novel class of nucleic acid analogues, b
6 ridged n6 ucleic
a6
cid (BNA),7 such as 2%-O,4%-C-methylene BNA (2%,4%-
BNA)8–12 and 3%-O,4%-C-methylene BNA (3%,4%-BNA),13,14
as shown in Figure 1. The sugar conformation of
3%,4%-BNA was found to be restricted in S-type pucker-
ing mode, while that of 2%,4%-BNA is locked in N-type.
In addition, the 3%,4%-BNA oligonucleotides, including a
2%,5%-phosphodiester linkage and an oxetane-fused ribo-
furanose ring, showed favorable features as an anti-
sense molecule.15
3%-Amino-3%-deoxynucleoside is an essential component
of oligonucleotides N3%“P5% phosphoramidate that are
Figure 1. Structures of bridged nucleic acids (BNAs).
* Corresponding author. Tel.: +81-6-6879-8200; fax: +81-6-6879-8204; e-mail: imanishi@phs.osaka-u.ac.jp
0040-4039/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0040-4039(03)01279-6

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S. Obika et al. / Tetrahedron Letters 44 (2003) 5267–5270
Staudinger reaction of 1,3-azido alcohol is a very useful
entry for azetidines.19 After benzyloxycarbonylation or
trifluoroacetylation of the azetidine 6 (93–96%, 6“7),
acetolysis was performed to give the diacetate 8 in
82–83% yields. Coupling reactions of 8 with silylated
thymine effectively proceeded to give the desired b-
anomer of 9 (76–85%), exclusively. Deprotection of the
2%- and 5%-hydroxy groups in 9a gave the nucleoside
analogues 1a in 86% yields (two steps). A similar reac-
tion of 9b proceeded only in 28% yield (two steps),
probably due to instability of the trifluoroacetyl group
in 9b under the alkaline conditions. The treatment of 1b
with conc. ammonia gave the unsubstituted azetidine
derivative 1c in 87% yield. Thus, we achieved the
synthesis of the novel azetidine-fused nucleoside ana-
logue 1.
On the other hand, 3%-amino-3%,4%-BNA monomer 1,
which has an azetidine-fused furanose ring, is one of
the isosteres of the 3%,4%-BNA monomer, and is poten-
tially capable of introducing some functional groups at
the 3%-nitrogen atom, such as a fluorescent or a chemi-
luminescent probe, an intercalator and DNA scissors.16
Furthermore, the 3%-amino-3%,4%-BNA monomer 1 is
applicable to the oligonucleotides N3%“P5% phospho-
ramidate with S-type sugar conformation, when 1 is
introduced into oligonucleotides via the 3%-amino and
5%-hydroxy groups. The effect of S-type sugar confor-
mation on hybridizing ability of the oligonucleotides
N3%“P5% phosphoramidate is of great interest. Thus,
the 3%-amino-3%,4%-BNA monomer 1 would be an atrac-
tive and potential synthon for a novel class of antisense
and antigene molecules.17 Here, we would like to
describe the synthesis and conformational analysis of a
novel nucleoside analogue 1.
Next, conformational analysis of 3%-amino-3%,4%-BNA
monomer 1 was performed. On 1H NMR measure-
ments of 1a and 1c, relatively large J1%2% values (7.6 Hz
for 1a and 6.0 Hz for 1c) were observed,20 which mean
that the azetidine derivative 1 was conformationally
restricted in S-form (S%=95% for 1a and 72% for
1c),21 in analogy with the parent oxetane derivative
(S%=91–96% for 3%,4%-BNA monomers).13,14 In addi-
tion, the conformational energy of 1c was analyzed by
means of the PM3 semi-empirical calculation.22 The
result demonstrated in Figure 2 clearly shows that the
C1%-exo puckering mode (conformation A, pseudorota-
tional phase angle P=ca. 130°) is the most favorable
form. The heat of formation of conformer A is ca. 4.6
kcal/mol lower than that of another conformer B (C1%-
endo form, P=ca. 300°), and relatively large energy
The synthesis of 1 was successfully achieved by using
3-azido derivative 210 as the starting material (Scheme
1). An azido group in 2 was reduced by the usual
hydrogenolysis, and the resulting amino alcohol 3 was
treated with p-toluenesulfonyl chloride to give 4 in 92%
yield (two steps). Reaction of 4 with sodium hydride
effectively caused an azetidine ring formation to afford
5 (90%), and reductive detosylation of 5 with sodium in
amyl alcohol gave 6 in 66% yield. On the other hand,
the azetidine derivative 6 was also obtained in 99%
yield directly from azido alcohol 2 via the aza-ylide
intermediate
under
Staudinger’s
conditions
(triphenylphosphine, o-xylene, D).18 To our best knowl-
edge, this is the first example clearly exhibiting that a
Scheme 1. Reagents and conditions: (a) H2 (1 atm), 10% Pd–C, AcOEt, rt. (b) TsCl, DMAP, Et3N, CH2Cl2, rt, (92%, two steps).
(c) NaH, THF, rt, 90%. (d) Na, n-AmOH, 120–130°C, 66%. (e) Ph3P, o-xylene, reflux, 99%. (f) CbzCl or (CF3CO)2O, DMAP,
CH2Cl2, 0°C, 96% for 7a, 93% for 7b. (g) Ac2O, AcOH, H2SO4, rt, 83% for 8a, 82% for 8b. (h) 2TMS·T, TMSOTf, ClCH2CH2Cl,
rt, 76% for 9a, 85% for 9b. (i) K2CO3, MeOH, 0°C, then n-Bu4NF, THF, rt, 86% for 1a, 28% for 1b. (j) NH3 aq., p-dioxane, rt,
87%.

S. Obika et al. / Tetrahedron Letters 44 (2003) 5267–5270
5269
Figure 2. PM3 calculations of 3%-amino-3%,4%-BNA monomer 1c.
barriers (P=ca. 20 and 230°) exist between the con-
former A and B, which come from the restrained struc-
ture of the azetidine-fused furanose ring.
Dev. 1995, 5, 3–11; (e) Prakash, T. P.; Jung, K.-E.;
Switzer, C. Chem. Commun. 1996, 1793–1794; (f) Shep-
pard, T. L.; Breslow, R. J. Am. Chem. Soc. 1996, 118,
9810–9811; (g) Kandimalla, E. R.; Manning, A.; Zhao,
Q.; Shaw, D. R.; Byrn, R. A.; Sasisekharan, V.; Agrawal,
S. Nucleic Acids Res. 1997, 25, 370–378.
Thus, we successfully synthesized a novel azetidine-
fused
nucleoside
analogue,
3%-amino-3%,4%-BNA
monomer 1, and also demonstrated that this compound
had predominantly S-form sugar puckering. We believe
that the nucleoside analogue 1 would be a potential
synthon for a novel antiviral agent, a highly functional
2%,5%-linked oligonucleotide, and an oligonucleotide
N3%“P5% phosphoramidate with a restricted S-type
sugar conformation and so on. Further studies on 1 are
now in progress.
3. Schro¨der, H. C.; Suhadolnik, R. J.; Pfleiderer, W.;
Charubala, R.; Mu¨ller, W. E. G. Int. J. Biochem. 1992,
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son, M. E.; Kong, N.; Guan, M.-X.; Henderson, E. E.;
Pfleiderer, W. Helv. Chim. Acta. 1997, 80, 1061–1072; (b)
Kvasyuk, E. I.; Kulak, T. I.; Tkachenko, U. V.; Sen-
tyureva, S. L.; Mikhaliopulo, I. A.; Suhadolnik, R. J.;
Henderson, E. E.; Horvath, S. E.; Guan, M.-X.;
Pfleiderer, W. Helv. Chim. Acta. 1998, 81, 1278–1284.
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7. Imanishi, T.; Obika, S. Chem. Commun. 2002, 1653–1659.
8. Obika, S.; Nanbu, D.; Hari, Y.; Morio, K.; In, Y.;
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Acknowledgements
Part of this work was supported by the Japan Securities
Scholarship Foundation (JSSF) Academic Research
Grant Program, Industrial Technology Research Grant
Program in 2000 from New Energy and Industrial
Technology Development Organization (NEDO) of
Japan, a Grant-in-Aid from the Japan Society for the
Promotion of Science, and a Grant-in-Aid from the
Ministry of Education, Science, and Culture, Japan.
9. Obika, S.; Nanbu, D.; Hari, Y.; Andoh, J.; Morio, K.;
Doi, T.; Imanishi, T. Tetrahedron Lett. 1998, 39, 5401–
5404.
10. Obika, S.; Andoh, J.; Sugimoto, T.; Miyashita, K.; Iman-
ishi, T. Tetrahedron Lett. 1999, 40, 6465–6468.
11. (a) Obika, S.; Hari, Y.; Morio, K.; Imanishi, T. Tetra-
hedron Lett. 2000, 41, 215–219; (b) Obika, S.; Hari, Y.;
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224; (c) Obika, S.; Hari, Y.; Sugimoto, T.; Sekiguchi, M.;
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