Convenient synthesis of N2-isobutyryl-2′-O-methyl guanosine by efficient alkylation of O6-trimethylsilylethyl-3′,5′-di-tert-butylsilanediyl guanosine

Article abstract of DOI:10.1016/j.tet.2007.08.006

We present a novel route for the synthesis of N²-isobutyryl-2′-O-methyl guanosine, introducing 3′,5′-di-tert-butylsilyl and O⁶-trimethylsilylethyl groups as efficient protections during the 2′-O-methylation step with NaH/CH₃

Full text of DOI:10.1016/j.tet.2007.08.006

Tetrahedron 63 (2007) 11174–11178
Convenient synthesis of N2-isobutyryl-20-O-methy0l guanosine
by efficient alkylation of O6-trimethylsilylethyl-3 ,50-di-tert-
butylsilanediyl guanosine
*
Ivan Zlatev, Jean-Jacques Vasseur and Franc¸ois Morvan
´
Institut des Biomolecules Max Mousseron UMR 5247 CNRS-UM1-UM2, Universite Montpellier 2, CC1704, Place E. Bataillon,
´
34095 Montpellier Cedex 5, France
Received 21 May 2007; revised 1 August 2007; accepted 2 August 2007
Available online 8 August 2007
Abstract—We present a novel route for the synthesis of N2-isobutyryl-20-O-methyl guanosine, introducing 30,50-di-tert-butylsilyl and
O6-trimethylsilylethyl groups as efficient protections during the 20-O-methylation step with NaH/CH3I. These protections were then removed
simultaneously in a single step with TBAF. The eight-step synthesis is easy to perform, employing convenient commercially available
reagents; crude mixtures are of satisfying purity, so only three chromatography purifications were required. Title compound was obtained
in 25% overall yield from guanosine.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
However, use of hazardous diazomethane is the major incon-
venience of these methods. Even if it could be replaced by
less dangerous (CH3)3SiCHN215 this method is still not con-
venient. Use of classical NaH/MeI methylation conditions16
hampers the regioselectivity. So better results are obtained
when 30 and 50 hydroxyl groups are simultaneously protected
by a silyl containing cyclic protection.11 The regioselectivity
problem becomes even more complex when one considers
the nature of aglycone moiety. Indeed guanosine and uridine
are known to undergo base methylation due to the presence
of an acidic lactam type proton.16,17 In the present work, we
focused our attention on the synthesis of 20-O-methyl guano-
sine so further considerations will not refer to uridine. A
solution to avoid alkylation of N1 of guanine moiety is to
use 2-amino-6-chloropurine or 2,6-diaminopurine ana-
logues that must be transformed into guanine analogues after
the alkylation step.11,13,14,18 Unfortunately, these methods
are far more expensive than those starting from guanosine.
Recently a selective methylation of 30,50-O-protected guano-
sine was reported.19 However, although it provided good
yield and a good selectivity, this method is not convenient
since it requires a non-commercial disilyl cyclic protection
and the use of gaseous MeCl, difficult to handle. Otherwise,
if we consider guanosine as starting material, the way to
avoid N1 alkylation is to introduce a protecting group on
the O6 of guanosine. Hence, O6-nitrophenylethyl12,16 and
O6-nitrophenyl20 have been reported for successful guano-
sine alkylation while diphenylcarbamoyl protection gave
unsatisfying results.20 Moreover, Grotli et al.21,20 reported
O6-tert-butyldiphenylsilyl protection, which was used in
20-O-Methyl oligoribonucleotides (20-O-Me RNA) are an
attractive class of analogues for the antisense approach be-
cause of their physiological stability, resistance to nucleases
and high hybridization properties.1 Indeed, the thermal sta-
bility of their hybrids with complementary RNA is greater
than that of the RNA–DNA duplexes.2 More recently some
new applications in RNA splicing were reported3,4 and
20-O-Me RNA were also used for the synthesis of nuclease
resistant hammerhead ribozymes.5 Finally, due to the in-
creasing interest in RNA interference phenomena, the use
of 20-O-Me RNA was evaluated as a modification in
siRNA.6–10 In some cases fully modified 20-O-Me RNA
displayed enhanced enzymatic stability and similar or in-
creased in vitro potency compared to the unmodified
siRNA.6,8 These applications have made the synthetic re-
search area to develop the 20-O-methyl nucleosides of great
interest all along until today.
Most of the methods for the preparation of 20-O-methyl
nucleosides were recently reviewed (Ref. 11 and references
cited in). Direct alkylation of non-protected ribose moiety
using diazomethane seems to be the shortest and the most
popular way of synthesis and a better regioselectivity can
be obtained when the alkylation is tin directed.12–14
Keywords: Alkylation; 20-O-Methyl guanosine; O6-Silyl protection.
* Corresponding author. Tel.: +33 467 144 961; fax: +33 467 042 029;
e-mail: morvan@univ-montp2.fr
0040–4020/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2007.08.006

I. Zlatev et al. / Tetrahedron 63 (2007) 11174–11178
11175
combination with silyl 30,50-TIPDS bridge. However, the
O6-silyl protecting group was partly unstable during chro-
matography on silica gel.20 While working on the
development of some dinucleotide 20-O-alkylated guanosine
analogues, we developed the idea to use the stable O6-trime-
thylsilylethyl (TSE) protection. This protection was recently
used in our group as a transient oligonucleotide protec-
tion.22,23
purification. The conformationally restricted sugar due to
the cyclic silanediyl protection was confirmed by NMR anal-
ysis sho0w0 ing a marked chemical shift difference between the
0
0
5 and 5 protons, and the anomeric H1 signal as a sharp sin-
glet.24 Subsequent O6 alkylation was performed in two steps
including sulfonate activation on the O6 lactim (enol) form
by tri-isopropylsulfonyl chloride activated by DMAP fol-
lowed by a nucleophilic displacement of the latter by
DABCO and 2-trimethylsilylethanol activated by DBU.28
Crude O6-TSE compound 3 was then deacetylated by
0.2 N NaOH, and the reaction was quenched with DOWEX
50W-X8 pyridinium form resin. After silica gel chromato-
graphy, the 20-hydroxyl derivative 4 was obtained with
a 40% overall yield from guanosine. The O6-TSE protection
was found to be stable during silica gel chromatography and
minimal hydrolysis of the DTBS group was observed during
the saponification step. Methylation reaction was performed
in anhydrous conditions using iodomethane and NaH in
DMF at 0 ꢀC to afford desired compound 5 after work-up
and silica gel chromatography with 70% yield (note that
this chromatography is optional). It was then quantitatively
protected on the N2 position with isobutyryl chloride pro-
viding 6. Finally the removal of silyl groups afforded
the N2-isobutyryl-20-O-methyl guanosine 7 useful for
further oligonucleotide construction (i.e., 50-DMTr and
30-phosphoramidite).
We describe herein an easy and efficient route to synthesize
N2-isobutyryl-20-O-methyl guanosine using stable 30,50-O
and O6-silyl protecting groups and a classical NaH/MeI
alkylation procedure. This synthesis requires a minimum
of purification steps.
Starting from inexpensive guanosine, di-tert-butylsilanediyl
(DTBS)24 was introduced first as a 30,50 cyclic protection
(Scheme 1). It was chosen considering its greater stability
to strong basic conditions compared to the Markiewicz
TIPDS protection25 and its lower cost.26 Indeed the TIPDS
protection is theorized to be fragile due to the inductive
effect of the oxygen atom interconnecting the two silicon
groups.27 The DTBS protection was introduced by its bis-
triflate derivative onto dried guanosine in dry DMF at
0 ꢀC. In a one-pot reaction, acetyl group was then selectively
introduced on the 20-hydroxyl using acetic anhydride. It is
well known that the amino function of guanine (in fact a gua-
nidine function) has a low nucleophilicity, hence it could not
react with acetic anhydride. It is worth noticing that the NH2
group on position 2 must be kept free to avoid any undesired
N2-alkylation. Indeed, its acylation would yield an N2 acid
proton 0and hence alkylation would proceed on the both N2
and O2 atoms. After work-up, the resulting crude mixture
was pure enough and used in the next step without further
Elimination conditions of silyl groups were investigated in
order to find a simple procedure leading directly to the de-
sired final compound 7. On one hand, treatment with the
HF–pyridine complex in pyridine at room temperature gave
a complete cleavage of the cyclic protection within 15 min
while TSE group was only partially removed after 4 h. So, af-
ter aqueous work-up the mixture was treated with 0.1 N
iPr
iPr
O
SiMe3
S
iPr
O
O
O
N
O
N
N
N
N
N
N
N
NH
NH2
N
N
O
NH2
NH2
O
N
O
O
O
O
iv
i, ii
iii
Si
t-Bu
Si
t-Bu
Si
Guanosine
t-Bu
t-Bu
O
OAc
t-Bu
O
OAc
t-Bu
O
OAc
1
2
3
v
SiMe3
NH2
SiMe3
NHR
O
N
O
N
O
N
N
N
N
N
N
N
N
N
NH
NHiBu
viii
vi
O
HO
O
Si
t-Bu
O
O
O
Si
t-Bu
t-Bu
t-Bu
O
OH
O
OMe
OH OMe
7
5 R = H
4
vii
6 R = iBu
Scheme 1. Reagents and conditions: (i) t-BuSi(OTf)2, DMF, 0 ꢀC to rt, 1 h; (ii) Ac2O, DMAP, pyridine, rt, overnight; (iii) 2,4,6-tri-isopropylsulfonyl chloride,
˚
DMAP, NEt3, CH2Cl2, rt, 2 h, 95%; (iv) DABCO, 2-trimethylsilylethanol, DBU, dioxane, 3 A molecular sieve, rt, overnight; (v) 0.2 N NaOH in THF/MeOH/
+
ꢀ
ꢀ
˚
H2O—5:3:1 (v/v/v), 0 C, 5 min—then DOWEX 50W-X8-pyrH , 42%; (vi) NaH—60% in mineral oil, MeI, DMF, 3 A molecular sieve, 0 C, 1 h, 70%; (vii)
isobutyryl chloride, pyridine, rt, 30 min, quant; (viii) 1 M TBAF in THF, rt, 1 h, 90%.

11176
I. Zlatev et al. / Tetrahedron 63 (2007) 11174–11178
AcOH in methanol for 20 h providing nucleoside 7. On the
other hand, a more efficient treatment with 1 M TBAF in
THF provided the expected N2-isobutyryl-20-O-methyl gua-
nosine 7 within 1 h at room temperature. For each procedure,
pure 7 was obtained in 90% yield after chromatography.
Signal assignments were based on 2D homo-1H/1H or heter-
onuclear-1H/13C correlations and D2O exchange.
2.1.1. 20-O-Acetyl-30,50-O-di-tert-butylsilanediyl guano-
sine 1. To a suspension of guanosine hydrate (1.4 g,
5 mmol, 1 equiv), dried by azeotropic distillation with anhy-
drous pyridine, in anhydrous DMF (10 mL) at 0 ꢀC was
added dropwise di-tert-butylsilyl-bistriflate (1.8 mL,
5.5 mmol, 1.1 equiv). Solution was stirred for an hour at
room temperature and then DMAP (122 mg, 1 mmol,
0.2 equiv), 8 mL of anhydrous pyridine and 1.2 mL of acetic
anhydride (12.5 mmol, 2.5 equiv) were added. Mixture was
stirred overnight at room temperature. Reaction was
quenched with 20 mL of methanol. Solvents were removed
under reduced pressure, and residue dissolved in ethyl
acetate and washed three times with saturated bicarbonate
solution. Organic layers were combined, dried over
Na2SO4 and evaporated to dryness. Product 1 was then
used in the next step without further purification. Rf:
0.69—CH2Cl2/MeOH—9:1 (v/v). 1H NMR: (DMSO-d6,
200 MHz) d 10.72 (1H, s, br, H1); 7.98 (1H, s, H8);
We have described a new multi-step synthetic method
affording N2-isobutyryl-20-O-methyl guanosine 7. For that
purpose, we used two stable silyl protecting groups on the
30,50-hydroxyl (DTBS) and on the O6 position (TSE), then
a 20-O-methylation was easily performed using classic
NaH/iodomethane conditions. After N2 protection, the both
silyl groups were efficiently and simultaneously removed
with TBAF affording the title compound. The reaction
scheme is easy to perform, crude mixtures are of satisfying
purity affording minimum chromatography purification
steps and the overall yield starting from guanosine was
25% in eight steps. The three reactions 20-methylation,
N2-protection and O6,30,50-deprotection were performed
without intermediate chromatography. Thus N2-isobutyryl-
20-O-methyl guanosine 7 was obtained after only three
chromatographies.
0
6.50 (2H, s, br, NH2); 5.90 (1H, s, H1 ); 5.65 (1H, d, H2 ,
0
3
3
0
0
0
0
0
JH2 –H3 ¼5.5 Hz); 4.55 (1H, dd, H3 , JH2 –H3 ¼5.5 Hz);
3
0
0
0
0
4.40 (1H, dd, H5 , JH4 –H5 ¼4.3 Hz); 4.05 (2H, m, H4 ,
00
2. Experimental
2.1. General
H5 ); 2.15 (3H, s, CH3acetyl); 1.05–1.10 (18H, 2s, CH3DTBS).
FAB+ (GT) m/z 466 (M+H)+; 315 (Su)+; 152 (BH2)+; FABꢁ
(GT) m/z 464 (MꢁH)ꢁ; 150 (B)ꢁ.
TLC was performed on Merck silica coated plates 60F254
(art. 5554). Compounds were revealed on UV light
(254 nm) and after spraying with 10% sulfuric acid/ethanol
solution. Column chromatography was performed on silica
gel Merck 60 (art. 9385). Reverse-phase liquid chromato-
graphy was performed using silica C18 Merck LiChroprepÒ
RP-18 (art. 9303), ISCO TRIS Pump and UA-6 detector
(254 nm). Ion exchange reactions were performed using
DOWEX 50W-X8 H+ resin (Aldrich); different ion forms
were obtained after washing with corresponding 2 M aque-
ous solutions and then water until neutrality.
2.1.2. O6-[(2,4,6-Tri-isopropyl)-benzenesulfonyl]-20-O-
acetyl-30,50-O-di-tert-butylsilanediyl guanosine 2. To a
solution of crude 1 (5 mmol, 1 equiv), dried by azeotropic
distillation with anhydrous acetonitrile, in 30 mL of anhy-
drous dichloromethane were added DMAP (122 mg,
1 mmol, 0.2 equiv), triethylamine (3 mL, 20 mmol, 4 equiv)
and 2,4,6-tri-isopropylbenzenesulfonyl chloride (5 g,
16 mmol, 3 equiv). After 2 h of stirring at room temperature
the mixture was diluted with 100 mL of ethyl acetate,
washed three times with saturated bicarbonate solution and
three times with brine. The organic layers were combined,
dried over Na2SO4 and evaporated to dryness. Resulting
oil was quickly purified by column chromatography (iso-
cratic cyclohexane/AcOEt—7:3 (v/v)) and pure compound
2 obtained as a yellow foam (3.5 g, 95% from guanosine).
Rf: 0.33—cyclohexane/AcOEt—7:3 (v/v). 1H NMR:
All moist-sensitive reactions were carried out under anhy-
drous conditions using dry glassware, anhydrous solvents
and argon atmosphere. Acetonitrile was distilled from
CaH2 after one night stirring at room temperature and 2 h
˚
stirring under reflux, and then stored over 3 A molecular
(CDCl3, 200 MHz) d 7.70 (1H, s, H8); 7.22 (2H, s, Harom);
0
3
0
0
0
sieves. Pyridine and triethylamine were distilled from
CaH2 after one night stirring at room temperature and 2 h
stirring under reflux. Dichloromethane was distilled from
P2O5 after stirring 2 h under reflux. Dioxane was dried
5.85 (1H, s, H1 ); 5.75 (1H, d, H2 , JH2 –H3 ¼5.3 Hz); 4.90
3
3
0
0
0
0
0
(1H, dd, H3 , JH2 –H3 ¼5.3 Hz, JH3 –H4 ¼9.1 Hz); 4.45
i
3
0
(1H, m, H5 ); 4.30 (2H, septet, CHo– Prarom, J¼6.7 Hz);
i
0
00
4.05 (2H, m, H4 and H5 ); 2.91 (1H, septet, CHp– Prarom
,
over Al2O3, and then stored over 3 A molecular sieves. All
other commercially available anhydrous solvents were
3J¼6.9 Hz); 2.20 (3H, s, CH3acetyl); 1.20–1.25 (18H, 2d,
CH3–iPrarom, 3J¼6.7 Hz); 1.05 (18H, 2 s, CH3DTBS). FAB+
(GT) m/z 732 (M+H)+; 418 (BH2)+; 315 (Su)+.
˚
˚
used as received. Molecular sieves (3 A) were dried in
a 300 ꢀC oven during 3 h prior to use.
2.1.3. O6-[(2-Trimethylsilyl)-ethyl]-20-O-acetyl-30,50-O-
di-tert-butylsilanediyl guanosine 3. To a solution of 2
(3.5 g, 4.8 mmol, 1 equiv) in anhydrous dioxane (80 mL)
were added DABCO (1.2 g, 10.6 mmol, 2 equiv) and 3 g
FAB-MS and FAB-HRMS spectra were recorded on a JEOL
SX102 mass spectrometer using a mixture of glycerol/thio-
glycerol [50:50, v/v (GT)] as a matrix. Sugar (Su) and base
(B) fragments are given if observed. 1H and 13C NMR spec-
˚
of dry 3 A molecular sieves. After 30 min of stirring at
€
tra are recorded at room temperature on a Bruker spectrom-
room temperature were added 2-(trimethylsilyl)ethanol
(4 mL, 26.6 mmol, 5 equiv) and DBU (2 mL, 13.8 mmol,
2.5 equiv). Mixture was stirred overnight under argon, fil-
tered off and filtrate was then diluted with 250 mL of ethyl
acetate and washed three times with brine. Organic layers
eter at 400 or 200 MHz (1H) and 100 MHz (13C). Chemical
shifts are given in parts per million referenced to the solvent
residual peak (CDCl3—7.27 and 77.0 ppm; DMSO-d6—
2.49 and 39.5 ppm). Coupling constants are given in hertz.

I. Zlatev et al. / Tetrahedron 63 (2007) 11174–11178
11177
were combined, dried over Na2SO4 and evaporated to
dryness. Product 3 was used in next step without further
purification.
100 MHz) d 161.5 (C6); 159.3 (C2); 152.9 (C4); 137.6
0
0
0
(C8); 116.4 (C5); 89.2 (C1 ); 82.1 (C2 ); 77.2 (C3 ); 74.4
(C4 ); 67.6 (C5 ); 65.0 (O–CH2TSE); 59.3 (20-O–CH3);
27.4; 26.9 (CH3DTBS); 22.8; 20.3 (CDTBS); 17.6 (CH2–
TMSTSE); ꢁ1.4 (CH3–SiTSE). FAB+ (GT) m/z 538
(M+H)+; 252 (BH2)+. HRMS:FAB+ (GT) m/z calcd for
(M+H)+: 538.2881, found: 538.2878.
0
0
Rf: 0.44—CH2Cl2/AcOEt—9:1 (v/v). 1H NMR: (CDCl3,
0
200 MHz) d 7.65 (1H, s, H8); 5.85 (1H, s, H1 ); 5.80 (1H,
3
0
0
0
0
d, H2 , JH2 –H3 ¼5.3 Hz); 4.95 (1H, m, H3 ); 4.60 (2H, m,
0
0
00
O–CH2TSE); 4.45 (1H, pd, H5 ); 4.10 (2H, m, H4 and H5 );
2.20 (3H, s, CH3acetyl); 1.20 (2H, m, TMS–CH2TSE); 1.05–
1.10 (18H, 2s, CH3DTBS); 0.10 (9H, s, Si–CH3TSE). FAB+
(GT) m/z 566 (M+H)+; 315 (Su)+; 252 (BH2)+; FABꢁ (GT)
m/z 464 (MꢁHꢁTSE)ꢁ; 250 (B)ꢁ.
2.1.6. N2-Isobutyryl-O6-[(2-trimethylsilyl)-ethyl]-20-O-
methyl-30,50-O-di-tert-butylsilanediyl guanosine 6. To
a solution of 5 (0.21 g, 0.39 mmol, 1 equiv) dried by azeo-
tropic distillation with anhydrous pyridine, in 5 mL of anhy-
drous pyridine at 0 ꢀC was added dropwise isobutyryl
chloride (82 mL, 0.78 mmol, 2 equiv). After 1 h of stirring
at room temperature the reaction was quenched by adding
10 mL of methanol. Solvents were then removed under re-
duced pressure, resulting residue dissolved in 20 mL of ethyl
acetate and washed with saturated bicarbonate solution.
Organic layers were combined, dried over Na2SO4 and evap-
orated to dryness. Product 6 was used in next step without
further purification.
2.1.4. O6-[(2-Trimethylsilyl)-ethyl]-30,50-O-di-tert-butyl-
silanediyl guanosine 4. To a solution of crude 3 (4.8 mmol,
1 equiv) in THF/MeOH/H2O 5:3:1 (v/v/v) (180 mL) was
added at 0 ꢀC an aqueous 2 N sodium hydroxide solution
(12 mL, 24 mmol, 5 equiv). After 5 min of stirring at 0 ꢀC
the reaction mixture was neutralized by adding pyridinium
DOWEX 50W-X8 resin, and stirred for five more minutes.
The resin was then filtered off and rinsed with water and
THF. Filtrates were evaporated to dryness and the residue pu-
rified by column chromatography (gradient: cyclohexane with
AcOEt 30–50%). Pure 4 was obtained as colourless vitreous
solid (1.1 g, 42% from 2).
Rf: 0.36—cyclohexane/AcOEt—7:3 (v/v). 1H NMR: (CDCl3,
400 MHz) d 7.72 (1H, s, H8); 7.68 (1H, s, exch, NHibu); 5.81
2
0
(1H, s, H1 ); 4.53 (2H, 2dt, J¼9.8 Hz, O–CH2TSE); 4.38
3
0
0
(1H, pt, H3 , J¼4.8 Hz); 4.36 (1H, m, H5 ); 4.15 (1H, d,
Rf: 0.43—cyclohexane/AcOEt—5:5 (v/v). 1H NMR:
(DMSO-d6, 200 MHz) d 8.02 (1H, s, H8); 6.35 (2H, s, br,
H2 , JH2 –H3 ¼4.6 Hz); 4.06 (1H, m, H4 , 3J¼4.9 Hz); 3.92
3
0
0
0
0
2
3
00
0
00
0
00
(1H, dd, H5 , JH5 –H5 ¼9.2 Hz, JH4 –H5 ¼10.5 Hz); 3.57
(3H, s, 20-O–CH3); 2.85 (1H, septet, CHibu); 1.18 (6H, d,
CH3ibu); 1.12 (2H, m, TMS–CH2TSE); 1.00 (18H, 2s,
CH3DTBS); 0.05 (9H, s, Si–CH3TSE). 13C NMR: (CDCl3,
100 MHz) d 180.4 (COibu); 161.8 (C6); 161.2 (C2); 152.2
3
0
NH2); 5.80 (1H, s, H1 ); 5.75 (1H, d, br, OH2 , JH2 –OH2
0
0
0
¼
0
3.9 Hz); 4.50 (2H, m, H3 and H2 ); 4.40 (2H, m, O–
0
0
0
00
CH2TMSE); 4.30 (1H, m, H5 ); 4.00 (2H, m, H4 and H5 );
1.10 (2H, m, TMS–CH2TSE); 1.00–1.05 (18H, 2s,
CH3DTBS); 0.10 (9H, s, Si–CH3TSE). FAB+ (GT) m/z 524
(M+H)+; 252 (BH2)+; FABꢁ (GT) m/z 522 (MꢁH)ꢁ; 422
(MꢁHꢁTSE)ꢁ; 250 (B)ꢁ. HRMS:FAB+ (GT) m/z calcd
for (M+H)+: 524.2725, found: 524.2707.
0
0
(C4); 139.5 (C8); 118.8 (C5); 89.5 (C1 ); 82.1 (C2 ); 77.3
(C3 ); 74.6 (C4 ); 67.5 (C5 ); 65.9 (O–CH2TSE); 59.7 (20-O–
CH3); 35.5 (CHibu); 27.3; 27.3 (CH3DTBS); 19.3 (CH3ibu);
18.9; 18.3 (CDTBS); 17.5 (CH2-TMSTSE); ꢁ1.39 (CH3–
SiTSE). FAB+ (GT) m/z 608 (M+H)+; 508 (M+HꢁTSE)+;
FABꢁ (GT) m/z 606 (MꢁH)ꢁ.
0
0
0
2.1.5. O6-[(2-Trimethylsilyl)-ethyl]-20-O-methyl-30,50-O-
di-tert-butylsilanediyl guanosine 5. To a solution of 4
(0.19 g, 0.35 mmol, 1 equiv), dried by azeotropic distillation
with anhydrous acetonitrile, in 2.5 mL of anhydrous DMF at
2.1.7. N2-Isobutyryl-20-O-methyl guanosine 7. Method A:
To a solution of crude product 6 (0.34 mmol, 1 equiv) in
2 mL of anhydrous dichloromethane in a Teflon flask at
0 ꢀC was added 60 mL of HF$pyridine diluted in 0.37 mL
of anhydrous pyridine. The reaction mix was stirred for
1 h at 0 ꢀC, then diluted with 10 mL of ethyl acetate and
washed with 1 M TEAB solution. Aqueous layers contain-
ing fully desilylated nucleoside were kept away and the
organic layers, containing O6-TSE nucleoside were evapo-
rated to dryness and treated with 3 mL of 0.1 M aqueous ace-
tic acid in 3 mL of methanol for 20 h at room temperature.
After completion of the reaction the mixture was neutralized
with 1 M TEAB and evaporated to dryness. The two frac-
tions containing deprotected 7 were put together and purified
by column chromatography (gradient: dichloromethane with
methanol 0–10%). Pure 7 was obtained after lyophilization
from water as a white spongy solid (0.11 g, 90%).
ꢀ
˚
0 C were added molecular sieves 3 A, iodomethane (65 mL,
1 mmol, 3 equiv) and sodium hydride 60% dispersion in
mineral oil (16 mg, 0.4 mmol, 1.1 equiv). The mixture was
stirred for 1 h at 0 ꢀC and then the same quantity of NaH
was added. Stirring was followed for 30 more minutes,
and then the mixture was hydrolyzed with 10 mL of absolute
ethanol, diluted with 50 mL of cold ethyl acetate and finally
washed with saturated ammonium chloride solution. The or-
ganic layers were combined, dried over Na2SO4 and evapo-
rated to dryness. Crude product was then purified by column
chromatography (gradient: cyclohexane with AcOEt 0–
30%) and pure compound 5 obtained as colourless vitreous
solid (0.13 g, 70%). This chromatography could be omitted.
Rf: 0.33—cyclohexane/AcOEt—7:3 (v/v). 1H NMR:
0
(CDCl3, 400 MHz) d 7.50 (1H, s, H8); 5.72 (1H, s, H1 );
3
0
4.59 (1H, dd, H3 , J¼4.7 Hz); 4.48 (2H, m, O–CH2TSE);
3
2
0
0
0
0
00
4.35 (1H, dd, H5 , JH5 –H4 ¼4.9 Hz, JH5 –H5 ¼9.0 Hz);
Method B: To a solution of crude 6 (0.48 mmol, 1 equiv) in
2.5 mL of anhydrous THF in a Teflon flask was added
5.2 mL (5.2 mmol, 10 equiv) of a 1 M TBAF solution in
THF. Mixture was stirred 1 h at room temperature and then
neutralized with 9 mL of 1 M TEAB and solvents were re-
moved under reduced pressure. The resulting brown residue
3
0
0
0
0
4.12 (1H, d, H2 , JH2 –H3 ¼4.7 Hz); 4.00 (1H, m, H4 ,
3
3
0
0
0
00
00
JH4 –H5 ¼4.9 Hz, JH4 –H5 ¼10.4 Hz); 3.91 (1H, dd, H5 ,
2
3
0
0
00
0
00
JH5 –H5 ¼9.1 Hz, JH4 –H5 ¼10.4 Hz); 3.55 (3H, s, 2 -O–
CH3); 1.12 (2H, m, TMS–CH2TSE); 1.00 (18H, 2s,
CH3DTBS); 0.05 (9H, s, Si–CH3TSE). 13C NMR: (CDCl3,

11178
I. Zlatev et al. / Tetrahedron 63 (2007) 11174–11178
7. Jackson, A. L.; Burchard, J.; Leake, D.; Reynolds, A.; Schelter,
J.; Guo, J.; Johnson, J. M.; Lim, L.; Karpilow, J.; Nichols, K.;
Marshall, W.; Khvorova, A.; Linsley, P. S. RNA 2006, 12,
1197–1205.
was purified by flash chromatography on reverse-phase
RP-18 (gradient: water with acetonitrile 0–50%) and the frac-
tions containing nucleoside 7 after evaporation were lyophi-
lized from water giving a white spongy solid (0.16 g, 90%).
8. Kraynack, B. A.; Baker, B. F. RNA 2006, 12, 163–176.
9. Prakash, T. P.; Allerson, C. R.; Dande, P.; Vickers, T. A.; Sioufi,
N.; Jarres, R.; Baker, B. F.; Swayze, E. E.; Griffey, R. H.; Bhat,
B. J. Med. Chem. 2005, 48, 4247–4253.
10. Yang, Z. Y.; Ebright, Y. W.; Yu, B.; Chen, X. M. Nucleic Acids
Res. 2006, 34, 667–675.
11. Beigelman, L.; Haeberli, P.; Sweedler, D.; Karpeisky, A.
Tetrahedron 2000, 56, 1047–1056.
12. Cramer, H.; Pfleiderer, W. Helv. Chim. Acta 1996, 79, 2114–
2136.
13. Khwaja, T. A.; Robins, R. K. J. Am. Chem. Soc. 1966, 88,
3640–3643.
14. Kore, A. R.; Parmar, G.; Reddy, S. Nucleosides Nucleotides,
Nucleic Acids 2006, 25, 307–314.
15. Leonard, T. E.; Bhan, P.; Miller, P. S. Nucleosides Nucleotides
1992, 11, 1201–1204.
16. Wagner, E.; Oberhauser, B.; Holzner, A.; Brunar, H.; Issakides,
G.; Schaffner, G.; Cotten, M.; Knollmuller, M.; Noe, C. R.
Nucleic Acids Res. 1991, 19, 5965–5971.
Rf: 0.44—CH2Cl2/MeOH—9:1 (v/v). 1H NMR: (DMSO-d6,
400 MHz) d 12.09 (1H, s, exch, NH1); 11.65 (1H, s, exch,
3
0
0
NHibu); 8.32 (1H, s, H8); 5.91 (1H, d, H1 , JH1 –H2
0
¼
3
0
0
0
6.1 Hz); 5.26 (1H, d, exch, OH3 , JOH3 –H3 ¼4.5 Hz); 5.09
3
0
0
0
0
(1H, t, exch, OH5 , JOH5 –H5 ¼5.1 Hz); 4.31 (1H, pd, H3 ,
3
3
0
0
0
0
0
JH3 –H4 ¼3.2 Hz); 4.23 (1H, pt, H2 , JH2 –H1 ¼5.6 Hz,
3
3
0
0
0
0
0
JH2 –H3 ¼4.8 Hz); 3.93 (1H, pd, H4 , JH4 –H3 ¼3.0 Hz);
0
0
3.50–3.65 (2H, m, H5 and H5 ); 3.30 (3H, s, 2 -O–CH3);
00
3
2.77 (1H, septet, CHibu, J¼6.9 Hz); 1.16 (6H, d, CH3ibu
,
3J¼7.2 Hz). 13C NMR: (DMSO-d6, 100 MHz) d 180.1
0
(COibu); 154.8 (C6); 148.2 (C2); 106.5 (C8); 86.1 (C4 );
0
0
0
0
0
84.4 (C1 ); 82.9 (C2 ); 68.6 (C3 ); 61.1 (C5 ); 57.5 (2 -O–
CH3); 34.7 (CHibu); 18.8 (CH3ibu). FAB+ (GT) m/z 735
(2M+H)+; 368 (M+H)+; 222 (BH2)+; 152 (BH2ꢁibu)+;
FABꢁ (GT) m/z 733 (2MꢁH)ꢁ; 366 (MꢁH)ꢁ; 220 (B)ꢁ.
HRMS:FAB+ (GT) m/z calcd for (M+H)+: 368.1570, found:
368.1558.
17. Yano, J.; Kan, L. S.; Tso, P. O. P. Biochim. Biophys. Acta 1980,
629, 178–183.
Acknowledgements
18. Linn, J. A.; Mclean, E. W.; Kelley, J. L. J. Chem. Soc., Chem.
Commun. 1994, 913–914.
This work was supported by grant from the ANRS. I.Z.
19. Chow, S.; Wen, K.; Sanghvi, Y. S.; Theodorakis, E. A. Bioorg.
Med. Chem. Lett. 2003, 13, 1631–1634.
ꢀ
thanks the ‘Ministere National de la Recherche et de la Tech-
nologie’ for the award of a research studentship.
20. Grotli, M.; Douglas, M.; Beijer, B.; Garcia, R. G.; Eritja, R.;
Sproat, B. J. Chem. Soc., Perkin Trans. 1 1997, 2779–2788.
21. Grotli, M.; Douglas, M.; Beijer, B.; Eritja, R.; Sproat, B.
Bioorg. Med. Chem. Lett. 1997, 7, 425–428.
References and notes
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