An efficient preparation of protected ribonucleosides for phosphoramidite RNA synthesis

Article abstract of DOI:10.1016/S0040-4039(02)00181-8

An efficient synthesis of protected ribonucleosides useful for phosphoramidite RNA synthesis is described. Di-t-butylsilylene group was employed for simultaneous protection of 3′- and 5′-hydroxyl functions of nucleoside. Subsequent silylation of free 2′-O

Full text of DOI:10.1016/S0040-4039(02)00181-8

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 1983–1985
An efficient preparation of protected ribonucleosides for
phosphoramidite RNA synthesis
Vladimir Serebryany and Leonid Beigelman*
Department of Organic Chemistry, Ribozyme Pharmaceuticals Inc., 2945 Wilderness Place, Boulder, CO 80301, USA
Received 7 January 2002; accepted 24 January 2002
Abstract—An efficient synthesis of protected ribonucleosides useful for phosphoramidite RNA synthesis is described. Di-t-butylsi-
lylene group was employed for simultaneous protection of 3%- and 5%-hydroxyl functions of nucleoside. Subsequent silylation of
free 2%-OH group followed by introduction of suitable protection on the base moiety, removal of cyclic silyl protection and
tritylation of 5%-OH gave target compounds in 60–66% overall yield. © 2002 Elsevier Science Ltd. All rights reserved.
Increasing demand for synthetic RNA-based therapeu-
tics stimulated a search for efficient and practical routes
toward protected ribonucleosides utilized in solid-phase
RNA synthesis. Two approaches are currently used for
the preparation of 5%-O-DMT-N-acyl-2%-O-TBDMS
pound to a mixture of 2%- and 3%-isomers, this step is
very time and raw material consuming. Jones’s method-
ology represents significant improvement but still
requires chromatographic separation of 2(3%) phospho-
nate intermediates. In addition, high 2%-regioselectivity
(8–9:1) in the phosphonylation–silylation reaction is
1
precursors: (a) the original synthesis that starts with
3
preparation of N-acyl-protected nucleosides using
observed only for purine ribonucleosides.
2
‘
transient protection’ as the first step, followed by
protection of 5%-hydroxy group as 4,4%-dimethoxytrityl
ether and silylation of 2%-OH; (b) an elegant synthesis
by Jones and co-workers that relies on regioselective
phosphonylation–silylation of 5%-O-DMT-N-acyl-pro-
tected ribonucleosides followed by removal of the 3%-
We challenged ourselves with the task of developing
general methodology for the preparation of 5%-O-DMT-
N-acyl-2%-O-TBDMS precursors that would address the
above shortcomings and would be amenable to scale-
up. We argued that this objective can be achieved if one
identifies suitable protecting group for simultaneous
protection of 3%- and 5%-hydroxyl groups of nucleoside
that would be stable during introduction of the 2%-O-
TBDMS group and can then be removed selectively
and without undesirable 2%“3%silyl migration.
phosphonate
group
without
migration
of
3
2%-O-TBDMS-protection.
The main drawback of the first methodology is the lack
of silylation regioselectivity that results in formation of
the mixture of 2%-O-, 3%-O- and 2%,3%-O-bis silyl ethers.
It was shown that the use of silver nitrate or silver
Initially, we turned our attention to tetra-
5
perchlorate in the silylation reaction can increase the
isopropyldisiloxane protection (TIPDS) that is widely
4
2%-O-regioselectivity.
However, in the case of
used for simultaneous protection of 3%- and 5%-hydroxyls
of ribonucleosides. Unfortunately, in our hands 2%-O-
tert-butyldimethylsilyl ether did not withstand the con-
ditions commonly used for removal of the TIPDS
group (n-Bu NF/THF or Et N·3HF/THF). As an alter-
guanosine derivatives, approximately 1:1 mixture of 2%-
and 3%-silyl ethers is obtained even if silver nitrate or
perchlorate additives are used. To the best of our
knowledge, only column chromatography allows sepa-
ration of pure 2%-O-silyl protected nucleosides.
Although the yield in this step can be increased by
subsequent equilibration of undesirable 3%-silyl com-
4
3
native, the di-tert-butylsilylene group, first introduced
6
7
by Trost and Corey, and later employed by Furusawa
8
et al. for simultaneous protection of 3%- and 5%-hydroxy-
ls of nucleosides was investigated. This group appeared
to be more attractive since it could be cleaved with
fluoride ion under very mild conditions.
6,8,9
*
0
Corresponding author. E-mail: lnb@rpi.com
040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)00181-8

1
984
V. Serebryany, L. Beigelman / Tetrahedron Letters 43 (2002) 1983–1985
The reaction of nucleosides 1a–c with 1.1 equiv. of
di-tert-butylsilyl bis(trifluoromethanesulfonate) in
DMF at 0°C proceeded smoothly and in high yield to
afford 3%,5%-O-protected intermediates 2a–c. Without
isolation from the reaction mixture, compounds 2a–c
were silylated affording derivatives 3a–c as crystalline
solids in 80–87% yield.
In conclusion, we have developed a general and
efficient methodology for the preparation of 5%-O-
DMT-N-acyl-2%-O-TBDMS-protected nucleoside pre-
cursors for phosphoramidite RNA synthesis. The
utilization of this methodology for the synthesis of
2%-O-alkylribonucleosides, as well as 2%-O-tri-
10
12
isopropylsyliloxymetyl protected precursors for phos-
phoramidite RNA synthesis will be reported shortly.
Because all sugar hydroxyls of 3a–c are now protected,
base amino functions can be easily derivatized with
2
suitable protecting group. Acylation of N -position of
Acknowledgements
guanosine derivative 3a with 2 equiv. of isobutyryl
chloride in a dichloromethane–pyridine mixture pro-
vided, after short methylamine treatment, crystalline 4a
We thank Dr. A. Karpeisky and Dr. J. Matulic-Adamic
for helpful discussions during this work and Dr. N.
Usman for continuous support.
6
in nearly quantitative yield. N -Benzoyladenosine
derivative 4b was prepared using benzoyl chloride and a
similar sequence of reactions in 77% yield after crystal-
lization from acetonitrile. Crude cytidine derivative 3c
was acetylated using 1.5 equiv. of acetic anhydride to
furnish 4c in 76% yield (overall from cytidine) after
crystallization from ethylacetate.
References
1. Ogilvie, K. K.; Sadana, K. L.; Thompson, E. A.; Quil-
liam, M. A.; Westmore, J. B. Tetrahedron Lett. 1974, 15,
2861–2864.
2. Ti, G. S.; Gaffney, B. L.; Jones, R. A. J. Am. Chem. Soc.
1982, 104, 1316–1319.
Removal of di-tert-butylsilylene protection using HF–
Py in dichloromethane at 0°C furnished compounds
a–c in a high yield. The purity of compounds 5a–c
5
after aqueous work up was high enough to proceed to
the next step without additional purification. Reaction
with dimethoxytrityl chloride in pyridine gave com-
3. Song, Q.; Wang, W.; Fisher, A.; Zhang, X.; Gaffney, B.
L.; Jones, R. A. Tetrahedron Lett. 1999, 40, 4153–4156.
4. Hakimelahi, G. H.; Proba, Z. A.; Ogilvie, K. K. Can. J.
Chem. 1982, 60, 1106–1113.
5. Markiewicz, W. T. J. Chem. Res. (S) 1979, 24–25.
6. Trost, B. M.; Caldwell, C. G. Tetrahedron Lett. 1981, 22,
4999–5002.
7. Corey, E. J.; Hopkins, P. B. Tetrahedron Lett. 1982, 23,
4871–4874.
8. Furusawa, K.; Ueno, K.; Katsura, T. Chem. Lett. 1990,
97–100.
9. Tang, X.-Q.; Liao, X.; Piccirilli, J. A. J. Org. Chem. 1999,
64, 747–754.
10. In the case of cytidine the addition of trifluoromethane-
sulfonic acid (1 equiv.) was necessary to improve the
selectivity of the reaction.
11. The typical procedure is as follows: Guanosine (11.33 g,
40 mmol) was suspended in DMF (80 mL) and di-tert-
butylsilyl ditriflate (14.3 mL, 44 mmol) was added drop-
1
1
pounds 6a–c; no significant 2%“3%-silyl migration was
observed under the reaction conditions. Guanosine and
cytidine derivatives 6a and 6c were isolated by crystal-
lization. For the isolation of adenosine derivative 6b,
silica gel chromatography on a short column was neces-
sary. The overall yield of target compounds 6a–c from
the starting nucleosides 1a–c was 60–66%. This is com-
parable to the yields obtained using the procedure
3
reported by Jones. Another advantage of this method-
ology is that all key intermediates as well as the final
compounds 6a and 6c are crystalline, thus, eliminating
chromatographic purification steps. In the synthesis of
6
b only one simple chromatography step is necessary.
When the above procedure (without acylation step) was
applied to the preparation of 5%-O-DMT-2%-O-TBDMS
11
uridine the target compound was obtained in 71%.

V. Serebryany, L. Beigelman / Tetrahedron Letters 43 (2002) 1983–1985
1985
wise during 15 min under stirring at 0°C. The resulting
solution of 2a was stirred at 0°C for 30 min and treated
with imidazole (13.6 g, 200 mmol). The mixture was
stirred for 5 min at 0°C and then at room temperature for
crystallized from dichloromethane (20 mL) and ether (200
mL) to provide 6a as a white fine powder. Yield 24.16 g
1
(78.4%). Mp 141–142°C. H NMR (400 MHz, DMSO-
d6), l: 12.16 (1H, s, NH), 11.70 (1H, s, NH), 8.19 (1H, s,
H8), 7.30 (13H, m, Ph), 5.96 (1H, d, J1%,2%=5.6 Hz, H1%),
2
5 min. tert-Butyldimethylchlorosilane (7.24 g, 48 mmol)
was added and the reaction was allowed to proceed at
0°C for 2 h. The precipitate of 3a was separated by
5.15 (1H, d, JOH,3%=5.2 Hz, 3%-OH), 4.69 (1H, dd, J2%,1%
=
6
5.6 Hz, J2%,3%=5.2 Hz, H2%), 4.22 (1H, dd, J3%,2%=5.2 Hz,
J3%,OH=5.2 Hz, H3%), 4.15 (1H, m, H4%), 3.80 (6H, s,
OMe), 3.35 (2H, m, H5%), 2.83 (1H, m, iBu-CH), 1.19
(3H, s, iBu), 1.17 (3H, s, iBu), 0.83 (9H, s, t-Bu), 0.06
(3H, s, Me), 0.05 (3H, s, Me).
filtration, washed with cold methanol and dried under
reduced pressure to give 18.81 g of 3a (87.4%), mp
3
75–380°C (dec.). Isobutyryl chloride (10.4 mL, 100
mmol) was added dropwise to a stirred suspension of 3a
26.89 g, 50 mmol) in dichloromethane (100 mL) and
1
(
6b: Overall yield 62% from 1b. H NMR (400 MHz,
pyridine (30 mL) at 0°C. The reaction was allowed to
proceed for 3 h at room temperature, then diluted with
methanol (40 mL) and cooled in an ice bath. A solution
of methylamine in ethanol (8 M, 25 mL, 200 mmol) was
added slowly to the reaction mixture. After 30 min the
reaction mixture was concentrated to give a slurry that
was diluted with methanol (100 mL) and left for 2 h at
DMSO-d ), l: 11.28 (1H, s, NH), 8.74 (1H, s, H8), 8.67
(1H, s, H2), 8.11 (2H, d, J=7.6 Hz, Ph), 7.35 (12H, m,
Ph), 6.93 (4H, m, Ph), 6.15 (1H, d, J1%,2%=4.8 Hz, H1%),
6
5.27 (1H, d, JOH,3%=5.6 Hz, 3%-OH), 4.97 (1H, dd, J2%,1%
4.8 Hz, J2%,3%=5.2 Hz, H2%), 4.35 (1H, dd, J3%,2%=5.2 Hz,
3%,OH=5.6 Hz, H3%), 4.21 (1H, m, H4%), 3.79 (6H, s,
=
J
OMe), 3.35 (2H, m, H5%), 0.84 (9H, s, t-Bu), 0.05 (3H, s,
0
°C. Precipitate was filtered off, washed with cold
methanol and dried to give 29.26 g (96.2%) of 4a, mp
18–223°C (dec.). Hydrogen fluoride–pyridine (Aldrich, 4
Me), 0.05 (3H, s, Me).
6c: Overall yield 60% from 1c. Mp 222–223°C (EtOAc–
1
2
hexanes). H NMR (400 MHz, DMSO-d
6
), l: 10.94 (1H,
mL, 154 mmol) was carefully diluted with pyridine (25
mL) under cooling. The resulting solution was added
slowly to a stirred at 0°C suspension of 4a (24.36 g, 40
mmol) in dichloromethane (200 mL) and the reaction was
allowed to proceed for 2 h at 0°C. The reaction mixture
was washed with water followed by saturated sodium
bicarbonate solution. The organic layer was dried over
magnesium sulfate and evaporated to give crude 5a as a
semi-crystalline material. The latter was dissolved in pyri-
dine (80 mL) and dimethoxytrityl chloride (14.91 g, 44
mmol) was added at 0°C. The reaction mixture was kept
at 0°C overnight, quenched with anhydrous methanol
s, NH), 8.37 (1H, d, J6,5=7.2 Hz, H6), 7.40 (9H, m, Ph),
7.07 (1H, d, J5,6=7.2 Hz, H5), 6.97 (4H, m, Ph), 5.80
(1H, d, J1%,2%=1.6 Hz, H1%), 5.19 (1H, d, JOH,3%=6.4 Hz,
3%-OH), 4.18 (3H, m, H2%, H3%, H4%), 3.82 (6H, s, OMe),
3.41 (2H, m, H5%), 2.17 (3H, s, Ac), 0.95 (9H, s, t-Bu),
0.19 (3H, s, Me), 0.15 (3H, s, Me).
5%-O-DMT-2%-O-TBDMS-U: Overall yield 71% from
uridine. NMR (DMSO-d ), l: 11.45 (1H, s, NH), 7.82
6
(1H, d, J6,5=8.2 Hz, H6), 7.44–7.33 (9H, m, Ph), 6.98
(4H, m, Ph), 5.83 (1H, d, J1%,2%=4.4 Hz, H1%), 5.37 (1H, d,
J
5,6=8.2 Hz, H5), 5.20 (1H, d, JOH,3%=6.4 Hz, 3%-OH),
4.28 (1H, dd, J2%,1%=4.4 Hz, J2%,3%=4.8 Hz, H2%), 4.15 (1H,
m, H3%), 4.07 (1H, m, H4%), 3.82 (6H, s, OCH ), 3.38 (2H,
(0.5 mL) and concentrated. The residue was partitioned
3
between dichloromethane and water. The organic layer
was washed with saturated sodium bicarbonate solution
and dried over magnesium sulfate. The solvent was
removed under reduced pressure and the residue was
m, H5%), 0.93 (9H, s, t-Bu), 0.14 (3H, s, Me), 0.13 (3H, s,
Me).
12. Wu, X.; Pitsch, S. Nucleic Acids Res. 1998, 26, 4315–
4323.