Synthesis of 1-(2′-O-methyl-β-D-ribofuranosyl)-5-nitroindole and its phosphoramidite derivative

Article abstract of DOI:10.1016/j.tetlet.2004.05.116

The synthesis of 1-(2′-O-methyl-β-D-ribofuranosyl)-5- nitroindole, a new nucleoside containing the universal base 5-nitroindole, and its phosphoramidite derivative for incorporation into oligonucleotides is described.

Full text of DOI:10.1016/j.tetlet.2004.05.116

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 5629–5632
Synthesis of 1-(20-O-methyl-b-
D
-ribofuranosyl)-5-nitroindole
and its phosphoramidite derivative
Gilles Gaubert and Jesper Wengel*
Nucleic Acid Center, Department of Chemistry, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark
Received 1 April 2004; revised 17 May 2004; accepted 24 May 2004
Available online 11 June 2004
Abstract—The synthesis of 1-(20-O-methyl-b-
D-ribofuranosyl)-5-nitroindole, a new nucleoside containing the universal base
5-nitroindole, and its phosphoramidite derivative for incorporation into oligonucleotides is described.
Ó 2004 Elsevier Ltd. All rights reserved.
The naturally occurring base hypoxanthine, as its ribo-
nucleoside or deoxyribonucleoside I has been used for
many years as a universal base analogue, that is an ‘inert
nucleotide’ forming isoenergetic base pairs witheachof
the natural DNA bases (DNA nucleotide monomers).1;2
This has led to applications in primers3;4 and in probes
for hybridization.1;5;6 However, nucleotide I is not
indiscriminate in its base pairing properties and a wide
range of melting temperatures are found when it is
paired opposite the natural bases in duplexes,5;7 and
multiple incorporations of I often induce a large de-
crease in the stabilities of the duplexes formed between
these modified oligonucleotides and DNA comple-
ments.7 These drawbacks have encouraged many efforts
towards the discovery of new universal bases.8 Among
these, the 5-nitroindole 20-deoxyribonucleoside II was
found to be superior, giving more favourable duplex
stabilities and behaving indiscriminately towards each of
the four natural bases in duplex melting experiments
(Fig. 1).9
O
O2N
O2N
N
N
HN
OH
N
N
N
O
HO
HO
HO
O
O
OH
OH
OMe
I
II
III
Figure 1.
venience of not needing a protective group for the 20-
hydroxy group, we preferred the 20-O-methyl-RNA
derivative over the corresponding RNA derivative. To
our knowledge, only one publication from a Russian
group has dealt with the preparation of the ribo-ana-
logue of III.13 This synthetic sequence included glyco-
sylation of 2,3-dihydro-5-nitroindole with a protected
sugar, protection of the hydroxy groups, oxidation of
the heterocyclic base, separation of the two anomers and
finally complete deprotection. This process appeared
rather long and tedious to us and furthermore furnished
the RNA, and not the 20-O-methyl-RNA, derivative.
This letter reports the synthesis of the novel 20-O-
methyl-5-nitroindole ribofuranoside III and its phos-
phoramidite derivative suitable for incorporation into
oligonucleotides.
20-O-Methyl-RNA10;11 oligonucleotides are among the
preferred modifications for antisense oligonucleotides.
Their structure mimics the structure of RNA,12 and we
became interested in synthesizing the novel 5-nitroindole
20-O-methylribonucleoside III to evaluate the properties
of the 5-nitroindole base surrogate in an RNA-type
oligonucleotide. For synthetic reasons, that is the con-
Bromination of tetra-O-acetyl-b-D-ribofuranose 1 using
trimethylsilyl bromide provided the bromo sugar
derivative 2 as a mixture of the two anomers. The
sodium salt of 5-nitroindole was formed by the action of
sodium hydride on 5-nitroindole in acetonitrile, and was
Keywords: 5-Nitroindole ribonucleoside; Phosphoramidite synthesis;
Universal hybridization; Universal base; 20-O-Me-RNA Monomer.
* Corresponding author. Tel.: +45-65502510; fax: +45-66158780;
e-mail: jwe@chem.sdu.dk
0040-4039/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.05.116

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G. Gaubert, J. Wengel / Tetrahedron Letters 45 (2004) 5629–5632
AcO
AcO
AcO
OAc
O
O
b
a
Br
OAc
OAc
OAc
O
O
2
1
NO2
AcO
N
O
O
O
O
OAc
OAc
OAc
4
3
HO
AcO
NO2
NO2
O
O
c
O
O
N
N
O
O
OH
OAc
5
6
Scheme 1. Reagents and conditions: (a) TMSBr, CHCl3, 0 °C; (b) sodium salt of 5-nitroindole, CH3CN; (c) NH3, MeOH, 78% from 1, mixture of
diastereoisomers.
TBDMSO
TBDMSO
TBDMSO
O
O
O
a
b
OH
D-Ribose
Cl
N
O
O
O
O
N
8
7
NO2
NO2
HO
O
O
c
d
O
O
OH
OH
10 + α-anomer
9 + α-anomer
NO2
f
NO2
N
O
O
N
O
i-Pr2Si
i-Pr2Si
e
O
O
O
OH
Si
O
OMe
Si
i-Pr2
i-Pr2
12
11
NO2
R1O
N
O
g
OR2
OMe
13 R1 = H, R2 = H
h
i
14 R1 = DMT; R2 = H
15 R1 = DMT; R2 = P(N(i-Pr)2)(OCH2CH2CN)
Scheme 2. Reagents and conditions: (a) Ref. 17; (b) CCl4, P(NMe2)3, THF, )80 °C; (c) sodium salt of 5-nitroindole, CH3CN; (d) TFA/H2O 9/1, 35%
from 7; (e) TIPDSCl2, pyridine, 60%; (f) BDDDP, MeI, CH3CN, 75%; (g) NEt3/3HF, THF, 95%; (h) DMTCl, pyridine, 80%; (i) ClP(N(i-
Pr)2)(OCH2CH2CN), DIPEA, CH2Cl2, 68%.

G. Gaubert, J. Wengel / Tetrahedron Letters 45 (2004) 5629–5632
5631
then reacted with 2 following the same procedure as
Acknowledgements
reported by Loakes and Brown9 for the synthesis of the
corresponding 20-deoxyribo-analogue. Interestingly, this
procedure did not yield the expected ribofuranose 4, but
instead compound 5 was obtained as a mixture of two
diastereoisomers. This result can be explained by intra-
molecular attack of the adjacent 2-O-acetyl group on the
anomeric position to give intermediate 3, followed by
nucleophilic attack by the 5-nitroindolyl anion on the
carbon atom of the O2-carbonyl moiety and not the C1-
carbon as would be expected. Similar products have
been reported in the literature by reacting other het-
erocyclic bases witha protected O2-benzoylated pro-
tected sugar.14;15 The fully deacetylated compound 6 was
obtained by treatment of 5 withsaturated methanolic
ammonia (78% from 1, mixture of diastereoisomers)
(Scheme 1). The structure of 6 was confirmed by high
The Danish National Research Foundation and Exiqon
A/S is thanked for financial support.
References and notes
1. Othsuka, E.; Matsuki, S.; Ikehara, M.; Takahashi, Y.;
Matsubara, K. J. Biol. Chem. 1985, 260, 2605–2608.
2. Takahashi, Y.; Kato, K.; Hayashizaki, Y.; Wakabayashi,
T.; Ohtsuka, E.; Matsuki, S.; Ikehara, M.; Matsubara, K.
Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 1931–1935.
3. Liu, H.; Nichols, R. Biotechniques 1994, 16, 24–26.
4. Kamaya, H.; Sakaguchi, T.; Murata, N.; Fujimuro, M.;
Miura, H.; Ishikawa, K.; Shimizu, M.; Inoue, H.;
Nishimura, S.; Matsukage, A.; Masutani, C.; Hanaoka,
F.; Ohtsuka, E. Chem. Pharm. Bull. 1992, 40, 2792–2795.
5. Martin, F. H.; Castro, M. M.; Aboul-ela, F.; Tinoco, I.
Nucleic Acids Res. 1985, 13, 8927–8938.
1
resolution mass spectroscopy, H NMR, 13C NMR and
elementary analysis.16
6. Van Aerschot, A.; Peeters, B.; Van Derhaegue, H.
Nucleosides Nucleotides 1987, 6, 437–439.
7. Kawase, Y.; Iwai, S.; Inoue, H.; Miura, K.; Ohtsuka, E.
Nucleic Acids Res. 1986, 14, 7727–7736.
In order to avoid the side reactions induced by the
presence of the participating 2-O-acetyl group of the
sugar moiety, the protected derivative 717 was selected as
starting material. After chlorination of ribofuranose 7
using CCl4 and P(NMe2)3 at low temperature,18 deriv-
ative 8 was reacted withthe sodium salt of 5-nitroindole
under the same conditions as applied above furnishing
the protected 5-nitroindole ribofuranoside 9 and the
corresponding a-anomer (inseparable by silica gel col-
umn chromatography) in a yield of 35% (from 7; ꢀ2:3
8. Loakes, D. Nucleic Acids Res. 2001, 29, 2437–2447.
9. Loakes, D.; Brown, D. M. Nucleic Acids Res. 1994, 22,
4039–4043.
10. Inoue, H.; Hayase, Y.; Imura, A.; Iwai, S.; Miura, K.;
Ohtsuka, E. Nucleic Acids Res. 1987, 15, 6131–6148.
11. Lesnik, E. A.; Guinosso, C. J.; Kawasaki, A. M.; Sasmor,
H.; Zounes, M.; Cummins, L. L.; Ecker, D. J.; Cook,
P. D.; Freier, S. M. Biochemistry 1993, 32, 7832–7838.
12. Blommers, M. J. J.; Pieles, U.; De Mesmaeker, A. Nucleic
Acids Res. 1994, 22, 4187–4194.
13. Mukhanov, V. I.; Miniker, T. D.; Preobrazhenskaya,
M. N. Zh. Org. Khim. 1977, 13, 214–218.
14. Ramasamy, K.; Robins, R. K.; Revankar, G. R.
J. Heterocycl. Chem. 1987, 24, 863–868.
15. Cristalli, G.; Franchetti, P.; Grifantini, M.; Nocentini, G.;
Vittori, S. J. Med. Chem. 1989, 32, 1463–1466.
16. Compound 6. Elemental analysis calcd for C15H16N2O7:
C, 53.57, H, 4.79, N, 8.33; found C, 53.58, H, 4.87, N,
8.08. HR-MS (MALDI) [M+Na]þ calcd: 359.0849; found
359.0848.
1
mixture of anomers according to H NMR). This mix-
ture was treated withaqueous TFA to cleave the pro-
tecting groups giving, in quantitative yield, a mixture of
fully deprotected 1-(b-D-ribofuranosyl)-5-nitroindole 10
and the corresponding a-anomer.19 In order to prepare
for O20-methylation, the 30- and 50-hydroxy groups of
nucleoside 10 (and its a-anomer) were protected by
reaction with1,3-dichloro-1,1,3,3-tetraisopropyldisilox-
ane (TIPDSCl2) in pyridine to give the desired b-anomer
11 in 60% yield after column chromatographic separa-
tion from a mixture of the two anomers. Attempts to
O20-methylate nucleoside 11 using NaH/MeI, Ag2O/MeI
or DBU/MeI failed. However, the use of BDDDP/MeI20
17. Kane, P. D.; Mann, J. J. Chem. Soc., Perkin Trans. 1 1984,
657–660.
1
18. Wilcox, C. S.; Otoski, R. M. Tetrahedron Lett. 1986, 27,
1011–1014.
19. The signals in the 1H NMR spectrum originating from one
of the anomers were identical to data reported earlier for
compound 10.12
provided the desired nucleoside in 75% yield. H NOE
experiments confirmed the b-
nucleoside 12.21 Cleavage of the silyl protective group
afforded 1-(20-O-methyl-b-
-ribofuranosyl)-5-nitroin-
D-ribo configuration of
D
dole 13 in 95% yield.22 In order to obtain the desired
building block for automated oligonucleotide synthesis,
nucleoside 13 was selectively protected at the primary
hydroxy group using 4,40-dimethoxytrityl chloride
(DMTCl) in pyridine to give nucleoside 14 (80% yield),
which was phosphitylated using the standard method to
give the desired phosphoramidite derivative 15 in 68%
yield (Scheme 2).23
20. Gotfredsen, C. H.; Jacobsen, J. P.; Wengel, J. Bioorg.
Med. Chem. 1996, 4, 1217–1225.
1
21. Compound 12: H NMR (CDCl3, 300 MHz) d 8.59–6.69
(5H, m, base), 6.01 (1H, s, H-10), 4.56–4.52 (1H, m, H-30),
4.29–4.04 (3H, m, H-40, H-50, H-500), 3.70 (1H, d, H-20,
J ¼ 4:6 Hz), 3.66 (3H, s, OMe), 1.57–0.99 (28H, m,
TIPDS). 13C NMR (CDCl3, 75 MHz) d 142.1, 137.5,
128.6, 127.5, 118.2, 117.5, 109.3, 104.7, 89.7, 84.3, 81.1,
70.2, 59.8, 17.5, 17.4, 17.3, 17.1, 17.0, 16.9, 13.4, 13.0, 12.6.
HRMS (MALDI) [M+Na]þ calcd: 573.2428; found:
573.2420. Key results of NOE experiments: irradiation
of H-30 gives 7.3% enhancement of the H-20 signal and
3.1% of the H-6 signal. Irradiation of H-6 gives 2.9%
enhancement of the H-30 signal and 1.4% of the H-20
signal.
In conclusion, we have developed a viable route to the
novel 20-O-methyl-5-nitroindole ribofuranoside and its
phosphoramidite derivative suitable for automated oli-
gonucleotide synthesis. Incorporation of this RNA-type
5-nitroindole nucleoside into oligonucleotides and
evaluation of its potential as a universal base will be
reported in due course.
22. Compound 13: 1H NMR (CD3OD, 300 MHz) d 8.66–6.88
(5H, m, base), 6.24 (1H, d, H-10, J ¼ 6:3 Hz), 4.56–4.53

5632
G. Gaubert, J. Wengel / Tetrahedron Letters 45 (2004) 5629–5632
HRMS (MALDI) [M+Na]þ: calcd: 331.0901; found:
(1H, m, H-30), 4.25–4.21 (2H, m, H-20, H-40), 3.99–3.87
(2H, m, H-50, H-500), 3.48 (3H, s, OMe). 13C NMR
(CD3OD, 75 MHz) d 143.0, 140.2, 129.6, 129.3, 118.5,
118.1, 111.1, 106.1, 88.8, 86.9, 85.1, 70.5, 62.7, 58.7.
331.0905.
23. Compound 15: 31P NMR (DMSO-d6, 121.5 MHz) d 151.4,
150.9.