Synthesis of 2'-O-methoxyethylguanosine using a novel silicon-based protecting group.

Article abstract of DOI:10.1021/jo025970e

A short and efficient synthesis of 2'-O-methoxyethylguanosine (8) is described. Central to this strategy is the development of a novel silicon-based protecting group (MDPSCl(2), 2) used to protect the 3',5'-hydroxyl groups of the ribose. Silylation of guanosine with 2 proceeded with excellent regioselectivity and in 79% yield. Alkylation of the 2'-hydroxyl group of 6 proceeded with methoxyethyl bromide and NaHMDS and afforded compound 7 in 85% yield, without any noticeable cleavage of the silyl protecting group and without the need to protect the guanine base moiety. Finally, deprotection of 7 was achieved using TBAF and produced 8 in 97% yield.

Full text of DOI:10.1021/jo025970e

Syn th esis of 2′-O-Meth oxyeth ylgu a n osin e
Usin g a Novel Silicon -Ba sed P r otectin g
Gr ou p
to degradation by various nucleases and form hybrids of
high thermal stability with complimentary RNA, render-
ing them suitable for a second generation of antisense
constructs.6
,7
†
†
‡
Given the medicinal importance of 2′-O-MOE oligori-
bonucleotides, a considerable effort has been devoted
toward their synthesis using a combination of chemical
and enzymatic approaches. Although these strategies
have been successfully implemented to the production of
Ke Wen, Suetying Chow, Yogesh S. Sanghvi, and
Emmanuel A. Theodorakis*,†
Department of Chemistry and Biochemistry,
University of California, San Diego, 9500 Gilman Drive,
La J olla, California 92093-0358, and ISIS Pharmaceuticals,
Inc., Carlsbad Research Center, 2292 Faraday Avenue,
Carlsbad, California 92008
8
pyrimidine-containing 2′-O-MOE nucleosides, they are
much less efficient in the case of the purine analogues.
In the latter case, the problems derive from the inherent
reactivity of the purine bases, requiring the use of
appropriate protecting groups and the need for selective
etheodor@ucsd.edu
Received May 25, 2002
9
protection of the C3′ and C5′ hydroxyl groups. Efforts
to overcome these problems have led to attempts to
Abstr a ct: A short and efficient synthesis of 2′-O-methoxy-
ethylguanosine (8) is described. Central to this strategy is
the development of a novel silicon-based protecting group
simultaneously protect the 3′- and 5′-hydroxyl groups,
10
2
using tetraisopropyldichlorodisilyloxane (TIPDSCl , 1)
11
or TBSCl
2
.
However, both of these reagents proved to
(MDPSCl2, 2) used to protect the 3′,5′-hydroxyl groups of
be labile during the strongly basic conditions required
the ribose. Silylation of guanosine with 2 proceeded with
excellent regioselectivity and in 79% yield. Alkylation of the
for methoxyethylation of the adjacent 2′-hydroxyl group.
1
2
Sterically hindered organic bases, such as BEMP, have
also been tested, but their cost renders them unsuitable
for large-scale application of the derived nucleosides.
Recognizing the significance of the above problem and
the current limitations in the choice of protecting groups,
we sought to develop a novel silicon-based protecting
group for a 3′- and 5′-selective protection of the ribose
2
′-hydroxyl group of 6 proceeded with methoxyethyl bromide
and NaHMDS and afforded compound 7 in 85% yield,
without any noticeable cleavage of the silyl protecting group
and without the need to protect the guanine base moiety.
Finally, deprotection of 7 was achieved using TBAF and
produced 8 in 97% yield.
scaffold. We theorized that the fragility of TIPDSCl
2
Modified oligonucleotides are currently under evalua-
tion for their medicinal potential against a variety of
viral, infectious, or cancer-related diseases and have
marked the beginning of antisense strategies as effective
under basic conditions may be due to the inductive effect
of the oxygen atom that interconnects the two silicon
groups. Based on this hypothesis, we decided to synthe-
size and study compound 2, in which a methylene unit
is used to bridge the two silicon groups. Being isosteric
to 1, we expected that 2 would exhibit a similar reactivity
and selectivity profile with 1 but display an extended
stability under basic conditions.
1
,2
therapeutic approaches. Some of the common chemical
modifications3 include alkylations of the 2′-hydroxyl
4
group of the ribose unit, giving rise to 2′-O-methyl or
5
2
′-O-methoxyethyl (MOE) nucleotides. Among them, the
MOE nucleotides were found to exhibit high resistance
*
To whom correspondence should be addressed. Tel: 858-822-0456.
Fax: 858-822-0386.
†
University of California.
ISIS Pharmaceuticals, Inc.
‡
(1) For selected recent reviews in this area, see: Sohail, M. Drug
Discuss. Today 2001, 6, 1260-1261. Dean, N. M. Curr. Opin. Biotech-
nol. 2001, 12, 622-625. Crooke, S. T. Oncogene 2000, 19, 6651-6659.
Green, D. W.; Roh, H.; Pippin, J .; Drebin, J . A. J . Am. Coll. Surg. 2000,
1
91, 93-105. Koller, E.; Gaarde, W. A.; Monia, B. P. Trends Pharmacol.
Sci. 2000, 21, 142-148. Sanghvi, Y. S. In Comprehensive Natural
Products Chemistry; Barton, D. H. R., Nakanishi, K., Eds.-in-Chief;
Vol. 7: DNA and Aspects of Molecular Biology; Kool, E. T., Ed.;
Academic Press: New York, 1999; pp 258-311.
The synthesis of compound 2 is shown in Scheme 1.
Commercially available disilane 3 was converted to
tetraalkylated silane 4 upon treatment with the ap-
(
2) For a recent monograph on this topic, see: Antisense Drug
Technology Principles, Strategies and Applications; Crooke, S. T., Ed.;
Marcel Dekker: New York, 2001.
(
3) Herdewijin, P. Antisense Nucl. Acid Drug Dev. 2000, 10, 297-
(6) Dean, M. N.; Butler, M.; Monia, B. P.; Manoharan, M. In ref 2,
pp 319-338. Altmann, K.-H.; Martin, P.; Dean, N. M.; Monia, B. P.
Nucleosides Nucleotides 1997, 16, 917-926.
(7) The first generation of antisense nucleosides refers to modifica-
tions of the phosphodiester backbone by replacing the equatorial
oxygen with a sulfur.
3
10. An, H.; Wang, T.; Maier, M. A.; Manoharan, M.; Ross, B. S.; Cook,
P. D. J . Org. Chem. 2001, 66, 2789-2801. Andrade, M.; Scozzari, A.
S.; Cole, D. L.; Ravikumar, V. T. Nucleosides Nucleotides 1997, 16,
1
617-1620.
4) Beigelman, L.; Haeberli, P.; Sweedler, D.; Karpeisky, A. Tetra-
(
hedron 2000, 56, 1047-1056. Ross, B. S.; Springer, R. H.; Tortorice,
Z.; Dimock, S. Nucleosides Nucleotides 1997, 16, 1641-1643. Chan-
teloup, L.; Thuong, N. T. Tetrahedron Lett. 1994, 35, 877-880. Roy,
S. K.; Tang, J .-Y. Org. Process Res. Devel. 2000, 4, 170-171. Von Matt,
P.; Lochmann, T.; Kesselring, R.; Altmann, K.-H. Tetrahedron Lett.
(8) Legorburu, U.; Reese, C. B.; Song, Q. Tetrahedron 1999, 55,
5635-5640. Altmann, K.-H.; Bevierre, M.-O.; Mesmaeker, A. D.; Moser,
H. E. Bioorg. Med. Chem. Lett. 1995, 5, 431-436. Cook, P. D.; Springer,
R. H.; Sprankle, K. G.; Ross, B. S. U.S. Patent No. 5,861,493, 1999.
(9) Grotli, M.; Douglas, M.; Beijer, B.; Garcia, R. G.; Eritja, R.;
Sproat, B. J . Chem. Soc., Perkin Trans. 1 1997, 2779-2786. Gundlach,
C. W., IV; Ryder, T. R.; Glick, G. D. Tetrahedron Lett. 1997, 38, 4039-
4042.
1
999, 40, 1873-1876. Beigelman, L.; Sweedler, D.; Haeberli, P.;
Karpeisky, A. U.S. Patent No. 5,962,575, 1999.
5) Martin, P. Helv. Chim. Acta 1995, 78, 486-504.
(
1
0.1021/jo025970e CCC: $22.00 © 2002 American Chemical Society
Published on Web 09/27/2002
J . Org. Chem. 2002, 67, 7887-7889
7887

SCHEME 1a
TABLE 1. Effect of Ba se d u r in g Alk yla tion of 6
base (3.0 equiv) T (°C) TBAI (equiv) results compd ratio 5/6/7
NaH
NaH
t-BuOK
BEMP
CsOH
25
0
25
25
0
0.1
N/A
0.1
3:7:0
2:8:0
3:7:0
0:0:others
9:1:0
a
LiHMDS
KHMDS
NaHMDS
NaHMDS
-20
-20
-10
-20
0.3
0.3
no reaction
1:8:0
b
1:8:1:others
0.3
0:1:9c
a
N alkylation(90%) together with dialkylation(10%) were ob-
a
served. b 2′-MOE-alkylated product (10%) and some (<10%) cleaved
material were formed. A ratio of 1:9 of starting material/desired
Reagents and conditions: (a) 4.4 equiv of i-PrMgCl (2.0 M in
c
THF), 0.01 equiv of CuCl2, THF, 65 °C, 5 h, 78%; (b) 1.0 equiv of
CCl4, 0.02 equiv of PdCl2, 60 °C, 2 h, 87%.
product was observed.
SCHEME 2a
somewhat slower, presumably due to the decreased
reactivity of the silyl chloride functionality. Nonetheless,
the regioselectivity of the reaction was excellent and
comparable to that observed for 1. This is attributed to
the steric effects of the isopropyl groups in 1 and 2 that
propel the silicon chloride to react initially with the
primary 5′-hydroxyl group and subsequently with the
neighboring 3′-hydroxyl unit.10 It is also worth mention-
ing that compound 6 can be easily crystallized, and its
structure was further confirmed via a single-crystal X-ray
analysis.16
The conversion of 6 to compound 7 was examined
under a variety of alkylation conditions summarized in
Table 1. Use of sodium hydride, potassium tert-butoxide,
and CsOH as base resulted in partial desilylation when
the reaction was performed at 0-25 °C. In contrast to
the above conditions, use of BEMP as the base did not
1
result in desilylation but yielded N -alkylated product.
Promising results were obtained when NaHMDS was
examined as the base, leading predominantly to the
desired alkylated product 7. This reaction was further
improved by adding a catalytic amount of TBAI and
performing the alkylation at -20 °C. Under these opti-
mized conditions, purified compound 7 was isolated in
a
Reagents and conditions: (a) 1.15 equiv of 2, 5.0 equiv of imid,
DMF, 0-25 °C, 5 h, 79%; (b) 3.0 equiv of NaHMDS, 0.3 equiv of
TBAI, 3.0 equiv of MeOCH2CH2Br, DMF, -20 °C, 3 h, 85%; (c)
1
.0 equiv of TBAF (1.0 M in THF), THF, 35 °C, 5 h, 97%.
8
5% yield. Interestingly, use of NaHMDS under the
above conditions did not produce any N-alkylated adduct,
thus eliminating the need to protect the nucleobase
during alkylation. The excellent regioselectivity of alky-
lation under these conditions may be explained if we
consider that HMDS reacts transiently with the guanine
propriate Grignard reagent in the presence of catalytic
CuCl . Chlorination of silane 4 was achieved in refluxing
carbon tetrachloride using catalytic PdCl
filtration of the residual PdCl under inert atmosphere,
2
1
3
2
.
Following
2
compound 2 was isolated after vacuum distillation (112-
6
17
nucleus to produce the corresponding O -TMS ether.
Although this group is known to be unstable during
1
14 °C at 0.45 mmHg) in 68% combined yield.
The application of disilane 2 to the synthesis of 2′-O-
MOE-guanosine is summarized in Scheme 2. Treatment
of guanosine (5) with a slight excess of disilane 2 in the
isolation,9
nucleobase, hence allowing alkylation exclusively at the
′-hydroxyl group.18 It is also worth mentioning that
,12
it could lead to a temporarily protected
2
presence of imidazole produced the desired 3′,5′-protected
nucleoside 6 in 79% yield.1
4,15
guanosine protected with disilane 1 underwent complete
deprotection under the above conditions, supporting our
hypothesis that the fragility of 1 derives from the
As compared to similar
protection with disilane 1, the reaction with 2 was
(
10) Markiewicz, W. T. J . Chem. Res., Synop. 1979, 24-25. Beijer,
B.; Grotli, M.; Douglas, M. E.; Sproat, B. S. Nucleosides Nucleotides
994, 13, 1905-1927.
11) Wada, T.; Tobe, M.; Nagayama, T.; Furusawa, K.; Sekine, M.
Tetrahedron Lett. 1995, 36, 1683-1684.
12) Grotli, M.; Douglas, M.; Beijer, B.; Eritja, R.; Sproat, B. Bioorg.
Med. Chem. Lett. 1997, 7, 425-428.
13) Ferreri, C.; Costantino, C.; Romeo, R.; Chatgilialoglu, C.
Tetrahedron Lett. 1999, 40, 1197-1200.
14) The effects of different bases (pyridine, imidazole, triethylamine,
(15) Under similar experimental conditions the selective 3′,5′ protec-
tion of other nucleic acids with 2 proceeded in 79-92% yield. See the
Supporting Information for more details.
(16) For the X-ray structure of 6, see the Supporting Information.
To the best of our knowledge, this is the first crystal stucture of any
3′,5′-silylated nucleoside.
1
(
(
(
(17) Our efforts to identify by spectroscopic means the formation of
-TMS ether prior to aqueous extraction met with failure. However,
O
6
(
a small peak corresponding at the molecular weight of the O -TMS
6
and lutidine) and temperature (0-70 °C) for the protection of 5 were
investigated. Among them, imidazole was found to produce the best
results.
protected alkylated nucleoside (MW ) 653) was detected by LCMS
analysis of the crude reaction mixture. We thank the reviewer for this
suggestion.
7
888 J . Org. Chem., Vol. 67, No. 22, 2002

inductive effect of the oxygen atom. Moreover, exposure
of alkylated adduct 7 to excess NaHMDS even at 25 °C
did not yield any deprotected material, indicating that
compound 7 is stable to any base-induced desilylation.
The TBAF-induced desilylation of 7 was slower than
the one performed in the identical substrate protected
with 1. Nonetheless, heating a solution of 7 in THF at
Com p ou n d 6. Guanosine (5) (1.0 g, 3.5 mmol) and imidazole
1.13 g, 17 mmol) were dried by coevaporation with pyridine (2
(
mL), dissolved in 20 mL of dry DMF, and treated with 2 (1.27
g, 4.1 mmol) added dropwise at 0 °C. The temperature was
gradually increased to 25 °C. After 5 h of reaction, TLC showed
no further change. The reaction mixture was poured into ice-
water, and the precipitated white solid was filtered to afford
compound 6 (1.45 g, 2.8 mmol, 79% yield). An analytical sample
was obtained by crystallization from MeOH. 6: R
0% methanol in dichloromethane); mp ) 270-271 °C dec; [R]
15 (c ) 0.3, CH Cl
NMR (400 MHz, DMSO-d
f
) 0.3 (silica,
3
5 °C in the presence of 1 equiv of TBAF produced the
2
1
5
1
-
D
desired product 8 in 97% yield. This deprotection was also
achieved in the presence of 0.6 equivalents of TBAF in
wet THF at 50 °C. In the latter case, however, the
reaction was considerably slower (24 h) and produced 8
in 90% yield.19
In summary, we describe herein an efficient procedure
for the synthesis of 2′-MOE-guanosine (8). Essential to
our strategy was the development of a novel silicon-based
2
2
); IR (film) νmax 1352, 1530, 1683, 3357; H
6
) δ 10.61 (s, 1 H), 7.76 (s, 1 H), 6.48
(
s, 2 H), 5.67 (s, 1 H), 5.47 (d, J ) 4.4 Hz, 1 H), 4.25 (dd, J )
3.6, 8.0 Hz, 1 H), 4.13 (t, J ) 4.4 Hz, 1 H), 4.01 (t, J ) 3.6 Hz,
1 H), 3.90 (d, J ) 8.4 Hz, 1H), 3.81-3.77 (m, 1H), 0.96-1.06 (m.
2
1
1
8H), 0.02 (s, 2H); 13C NMR (100 MHz, DMSO-d
50.8, 134.6, 117.1, 88.7, 81.0, 74.9, 70.5, 61.4, 18.5, 18.3, 18.3,
6
) δ 157.1, 154.1,
8.2, 18.1, 18.09, 18.0, 14.5, 14.4, 14.2, 14.1, -9.2; HRMS calcd
+
for C23
H
41
N
5
O
5
Si
2
(M + Na ) 546.2538, found 546.2522.
protecting group (MDPSCl
2
, 2), which, being isosteric to
Com p ou n d 7. To a solution of compound 6 (2.01 g, 3.8 mmol),
BrCH CH OCH (1.13 mL, 12.0 mmol), and TBAI (423 mg, 1.2
TIPDSCl (1), can selectively protect the 3′- and 5′-
2
2
2
3
hydroxyl groups of guanosine and withstands the basic
conditions required for alkylation of the 2′-hydroxyl
group. This maneuver allowed the synthesis of 8 from
guanosine (5) in three steps and 65% yield. An additional
advantage of silane 2 over 1 is that it produces com-
pounds that are highly crystalline and easily purified
without the need of chromatographic techniques. We are
currently exploring further applications of disilane 2 as
a new and versatile protecting group.
mmol) in 60 mL of DMF at -20 °C was added sodium bis-
(trimethylsilyl)amide (1.0 M in THF, 11.5 mL, 11.5 mmol), and
the mixture was stirred for 4 h under argon. After the reaction
was quenched with methanol, the THF was evaporated and the
residue was precipitated in ice to furnish compound 7 (1.89 g,
3
.3 mmol, 85% yield). 7:
R
f
) 0.4 (silica, 10% methanol in
-30.3 (c ) 0.6, CH Cl ); mp 229-231
1265, 1598, 1684, 2867, 2944, 3054;
) δ 10.67 (s, 1H), 7.74 (s, 1H), 6.48 (s,
H), 5.73 (s, 1H), 4.35 (dd, J ) 4.6, 9.2 Hz, 1H), 4.10 (d, J ) 4.8
Hz, 1H), 4.03 (d, J ) 11.4 Hz, 1H), 4.00-3.84 (m, 3H), 3.68-
25
dichloromethane); [R]
D
2
2
1
°C dec; IR (film) ν
max
H
NMR (400 MHz, DMSO-d
6
2
3
2
1
5
1
.84 (m, 2H), 3.40-3.52 (m, 1H), 3.45 (s, 3H), 0.96-1.10 (m,
Exp er im en ta l Section
8H), 0.00-0.10 (m, 2H); 13C NMR (100 MHz, CDCl
) δ 156.6,
3
Bis(d iisop r op ylsilyl)m eth a n e (4). A solution of bis(dichlo-
rosilyl)methane (3) (10.0 g, 0.05 mol) and CuCl (100 mg, 0.74
2
mmol) in 100 mL of THF was treated with isopropylmagnesium
chloride (2.0 M in THF, 103 mL, 0.206 mol) added dropwise at
53.8, 150.3, 134.1, 116.6, 86.6, 82.0, 80.5, 71.4, 70.4, 70.0, 60.6,
8.1, 18.1, 18.0, 17.9, 17.7, 17.6, 17.6, 17.5, 17.4, 13.9, 13.9, 13.8,
3.8, -9.4; HRMS calcd for C26
+
H
47
N
5
O
6
Si
2
(M + Na ) 604.2957,
found 604.2983.
′-O-MOE G (8). To a solution of compound 7 (50 mg, 0.086
mmol) in THF at 25 °C was added TBAF (1 M in THF, 0.09 mL,
.09 mmol), and the mixture was stirred at 35 °C for 5 h. The
2
5 °C over a period of 2 h. After the addition, the mixture was
2
refluxed for 5 h. Water (300 mL) was then added, and the organic
phase was separated. The aqueous phase was extracted with
hexanes (2 × 50 mL), and the combined organic phases were
0
solvent was then evaporated under reduced pressure, and the
residue was filtered in a short pad of silica gel using 10%
methanol in dichloromethane to afford compound 8 (28.5 mg,
4
washed with water and brine and dried over MgSO . The organic
solvents were evaporated under reduced pressure, and the
residue was filtered through a 10 cm thick silica gel column
using hexane as the eluant to afford product 4 (8.9 g, 0.036 mol,
0
.082 mmol, 97% yield). 8: R
f
) 0.1 (silica, 10% methanol in
2
5
dichloromethane); [R]
D
-51 (c ) 1.4, CH Cl ); mp 247-248 °C
2
2
1
7
8% yield): bp 80-82 °C/0.45 mmHg; H NMR (300 MHz,
1
dec; IR (film) νmax 1265, 3055, 3223, 3338, 3443; H NMR (400
CDCl ) δ 3.61 (s, 2H), 0.99-1.10 (m, 28H), -0.35 (t, J ) 3.9 Hz,
3
MHz, DMSO-d
6
) δ 10.64 (s, 1H), 7.95 (s, 1H), 6.46 (s, 2H), 5.78
13
2
H); C NMR (75 MHz, CDCl
Bis(d iisop r op ylch lor osilyl)m eth a n e (2). To a mixture of
bis(diisopropylsilyl)methane (4) (5.0 g, 0.02 mol) and PdCl (72.6
mg, 0.41 mmol) was added dry CCl (1.98 mL, 0.021 mol) in one
portion. The reaction was kept under argon at 60 °C for 2 h.
The PdCl was filtered under argon, and the resulting mixture
3
) δ 19.0, 18.9, 11.9, -16.2.
(
1
3
d, J ) 6.5 Hz, 1H), 5.06 (d, J ) 5.0 Hz, 2H), 4.34 (t, J ) 5.5 Hz,
H), 4.24 (t, J ) 4.0 Hz, 1H), 3.88 (d, J ) 3.0 Hz, 1H), 3.66 (m,
13
2
H), 3.51-3.60 (m, 2H), 3.38-3.40 (m, 1H), 3.32 (s, 3H);
) δ 156.8, 153.8, 151.4, 135.5, 116.7,
5.8, 84.4, 81.3, 71.1, 69.0, 68.9, 61.3, 58.0.
C
4
NMR (125 MHz, DMSO-d
8
6
2
was distilled under reduced pressure to produce compound 2 (5.6
g, 0.02 mol, 87% yield) as a clear liquid. 2: bp 112-114 °C/0.45
Ack n ow led gm en t. Financial support from the Bio-
STAR (No. 00-10094) and ISIS Pharmaceuticals, Inc.,
is gratefully acknowledged. We thank Dr. P. Gantzel
1
mmHg; H NMR (400 MHz, C
1
-
6
D
6
) δ 1.20-1.10 (m, 4 H), 1.05-
) δ 17.6, 16.1,
.00 (m, 24 H), 0.28 (s, 2H); 13C (100 MHz, C
3.5.
6 6
D
(
UCSD, X-ray Facility) for his assistance with the
crystallographic analysis of 6.
(18) For relevant references on this topic, see: Zhang, Z.-H.; Li, T.-
S.; Yang, F.; Fu, C.-G. Synth. Commun. 1998, 28, 3105-3114. Langer,
S. H.; Connell, S.; Wender, I. J . Org. Chem. 1958, 23, 50-57. Sweeley,
C. C.; Bentley, R.; Makita, M.; Wells, W. W. J . Am. Chem. Soc. 1963,
Su p p or tin g In for m a tion Ava ila ble: A table for protec-
1
13
tion of different nucleic acids with 2, H and C NMR spectra
for compounds 2, 4, and 6-8, and X-ray data for compound 6.
This material is available free of charge via the Internet at
http://pubs.acs.org.
8
4
5, 2497-2506. Bruynes, C. A.; J urriens, T. K. J . Org. Chem. 1982,
7, 3966-3969.
(19) At present, the mechanism of the desilylation reaction using
subequivalent amounts of TBAF is not clear. Our data indicate that
in addition to fluoride anion, water is important for this deprotection
protocol.
J O025970E
J . Org. Chem, Vol. 67, No. 22, 2002 7889