Improved synthetic approaches toward 2'-O-methyl-adenosine and guanosine and their N-acyl derivatives

Article abstract of DOI:10.1016/S0040-4020(00)00002-8

We developed several improved approaches toward 2'-O-methyl adenosine and guanosine and their N-acyl derivatives. (a) Transglycosylation of N⁴- acetyl-5', 3'-di-O-acetyl-2'-O-methyl cytidine with N⁶-Bz-adenine provided N⁶-benzoyl-5'3'-di-O-acetyl-2'-O-methyl adenosine in 50% yield. (b) Regioselective methylation of 2-amino-6-chloro purine riboside with MeI/NaH followed by hydrolysis provided 2'-O-Me-guanosine in high yield. The same 2'- O-Me-precursor was transformed into 2'-O-Me-adenosine in 58% yield. (c) Very efficient transformation of 2,6-diamino-purine riboside into N²-isobutyryl (isopropylphenoxyacetyl) 2'-O-Me-guanosine through methylation of 5',3'-O- TIPDSi derivative followed by selective N²-acylation, deamination and desylilation provided target compounds in 70% combined yield. (d) Mg²⁺ and Ag⁺ directed methylation of N¹-Bzl-guanosine proceeded in >80% yield with ratio of 2'-O-Me-3'-O-Me=9:1. The same methylation of adenosine with Ag⁺ and Sr²⁺ acetylacetonates provided 2'-O-Me-adenosine in 75-80% yield. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(00)00002-8

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 1047–1056
Improved Synthetic Approaches Toward 20-O-Methyl-Adenosine
and Guanosine and Their N-Acyl Derivatives
*
Leonid Beigelman, Peter Haeberli, David Sweedler and Alexander Karpeisky
Department of Organic Chemistry, Ribozyme Pharmaceuticals Inc., 2950 Wilderness Place, Boulder, CO 80301, USA
Received 21 September 1999; accepted 22 December 1999
Abstract—We developed several improved approaches toward 20-O-methyl adenosine and guanosine and their N-acyl derivatives. (a)
Transglycosylation of N4-acetyl-50, 30-di-O-acetyl-20-O-methyl cytidine with N6-Bz-adenine provided N6-benzoyl-5030-di-O-acetyl-20-O-
methyl adenosine in 50% yield. (b) Regioselective methylation of 2-amino-6-chloro purine riboside with MeI/NaH followed by hydrolysis
provided 20-O-Me-guanosine in high yield. The same 20-O-Me-precursor was transformed into 20-O-Me-adenosine in 58% yield. (c) Very
efficient transformation of 2,6-diamino-purine riboside into N2-isobutyryl (isopropylphenoxyacetyl) 20-O-Me-guanosine through methyl-
ation of 50,30-O-TIPDSi derivative followed by selective N2-acylation, deamination and desylilation provided target compounds in 70%
combined yield. (d) Mg2ϩ and Agϩ directed methylation of N1-Bzl-guanosine proceeded in Ͼ80% yield with ratio of 20-O-Me/30-O-
Meˆ9:1. The same methylation of adenosine with Agϩ and Sr2ϩ acetylacetonates provided 20-O-Me-adenosine in 75–80% yield.
᭧ 2000 Elsevier Science Ltd. All rights reserved.
Introduction
glycosylation step typically provides a mixture of a and b
anomers and is especially problematic for adenosine and
guanosine nucleosides.
20-O-Methylribonucleosides are widely distributed in RNA1
and since the late 1950s have been the focus of considerable
synthetic efforts. These synthetic efforts were further stimu-
lated by recent applications of 20-O-methyloligoribonucleo-
tides in studying pre-mRNA splicing2 and the structure of
splicesomes3 as well as in the preparation of nuclease-
resistant hammerhead ribozymes.4 20-O-Alkyl substituted
oligonucleotides are also emerging as a second generation
antisense constructs with improved hybridization properties
and favorable nuclease resistance and pharmacological
profiles.5 In light of this interest in oligonucleotide based
therapeutics, it is increasingly important to develop cost
effective synthesis of 20-O-alkyl nucleosides that serves as
a key raw material. Previously developed methods for the
preparation of 20-O-Me nucleosides can be divided into five
categories.
Direct methylation of ribonucleosides
The observation by Broom and Robins, in 1965, that diazo-
methane in DME directed methylation of adenosine prefer-
entially to the cis-diol system11 resulted in the first practical
synthesis of 20-O-Me-adenosine,11,14 20-O-Me-cytidine13
and 20-O-Me-uridine13 and the indirect synthesis of 20-O-
Me-guanosine12 through methylation of 2-amino-6-chloro
purine riboside. Shugar et al. described alkylation of
cytidine15 and adenosine16 by methyl(ethyl)sulfate resulting
in the preparation of all possible alkylated products. Direct
methylation of uridine and guanosine by NaH/MeI reagent
causes undesired base methylation, but provide satisfactory
yields of 20-O-Me-Adenosine.17 This procedure was utilized
for the preparation of all four 20-O-Me(Et) ribonucleoside
phosphoramidites.18 In the case of uridine and guanosine,
protection of N3 and O6 of the base prior to alkylation was
necessary. Methylation of 50-O-DMT-N4-t-butylphenoxy-
acetyl(or benzoyl)cytidines by Ag2O/MeI in the presence
of a trace amount of pyridine resulted in exclusive 20-O-
methylation in 65–95% yield.19 In general, the above
methodology usually requires extensive chromatographic
separation of the 20 and 30-O-Me isomers.
Synthesis from carbohydrate precursors
This approach was pioneered by Haines6 and Imbach.7
Recent improvements involved optimization of the large
scale synthesis of the 2-O-Me ribofuranose precursor,8
synthesis of an alternately protected 2-O-Me-synthon9 and
the utilization of d-glucose as a starting material.10 In
general, this approach requires the multistep synthesis of
carbohydrate precursors for glycosylation. The critical
Metal-directed methylation of the activated cis-diol
group
Keywords: ribozymes; oligonucleotides; 20-O-Me-nucleosides; methyl-
ation.
Diazomethane mediated methylation of the cis-diol group
* Corresponding author. E-mail: lnb@rpi.com
0040–4020/00/$ - see front matter ᭧ 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00002-8

1048
L. Beigelman et al. / Tetrahedron 56 (2000) 1047–1056
using SnCl2·2H2O activation is the most popular method of
synthesis of all four 20-O-Me ribonucleosides. These proce-
dures typically produce mixtures of 20 and 30-O-Me isomers
under mild conditions.20 Direct methylation of guanosine by
this method provided 20-O-Me-guanosine in only a 15%
yield. Therefore, a better method was developed using the
same technique, but with 2,6-diaminopurine riboside as a
starting material, providing 20-O-Me-guanosine in 40%
yield.21 Application of this method for methylation of
50-O-TBDPSi22, 50-O-Tr23 and 50-O-DMT-N-NPEOC24
protected ribonucleosides resulted in preferential 20-O-
methylation. Recently, TMSiCHN2 substituted the hazar-
dous reagent diazomethane25 for the methylation of base
protected guanosine.
Optimized large scale preparation of N4-ibu-20-O-Me-
cytidine using this method was also reported.31 Sproat and
co-workers concentrated on development of a convenient
route to guanosine derivatives utilizing 50,30-O-TIPDSi
protected purine ribonucleosides. The best synthesis of
20-O-Me guanosine was first achieved from 50,30-O-
TIPDSi-2,6-dicloropurine precursor using new methylation
system MeI/BDDDP.32 This procedure was later extended to
the direct methylation of 50,30-O-TIPDSi-O6-TBDPSi-Guano-
sine with MeI/BEMP.33 High cost of the BEMP reagent,
relative instability of 50,30-O-TIPDSi-O6-TBDPSi-Guanosine
intermediate and modest overall yield (50%) warrant further
improvements in the synthesis of this compound.
Miscellaneous
The activation of cis-diol groups by derivatization with
Bu2SnO followed by methylation with MeI provided 20
and 30-O-Me derivatives in the case of uridine but not for
other nucleosides.26 The combination of dibutylstannylene
activation followed by methylation with diazomethane
allowed to obtain 20-O-Me adenosine are uridine, but failed
in the case of cytidine and guanosine.27 Several metal
acetylacetonates used together with trimethylsulfonium
hydroxide directed methylation primarily to 20 and 30-hydrox-
yls for uridine, adenosine are cytidine, but guanosine was
methylated at 20 and 30-hydroxyls and N1 of the base.28
Once again, the above methodology usually requires exten-
sive chromatographic separation of 20 and 30-O-Me isomers.
Since the direct methylation of uridine and guanosine with
NaH or Ag2O/MeI requires the protection of acidic N3 and
N1 atoms of the pyrimidine and purine bases, it was logical
to develop new approaches which introduce the precursors of
the methyl group without protection of acidic functions on
the base. Sekine and Hata34 reported the introduction of a 1,3-
benzodithiol-2-yl group into 50,30-O-TIPDSi-uridine without
protection of N3 nitrogen; the 20-O-methyl group was regen-
erated after desulfurization with Raney Ni. Methylthio-
methyl (MTM)35 or methylthiophenyl (MTPh)10 ethers
were introduced at 20-OH of 50,30-O-TIPDSi-uridine with-
out base protection. Desulfurization with Raney Ni (MTM)
or radical reduction (MTPh) regenerated the methyl group.
Methylation of 30, 50-O-TIPDSi-protected
ribonucleosides
Pyrimidine 2,20-anhydronucleosides are very attractive
starting materials for the synthesis of 20-O-methyl nucleo-
sides because of their ease of synthesis, scalability and
susceptibility to the 2,20-anhydro ring opening by many
nucleophiles. It was demonstrated recently that Mg2ϩ and
Ca2ϩ alkoxides can open 50-O-protected pyrimidine 2,20-
anhydronucleosides providing 20-O-alkyl ribonucleosides
in 50–90% yield.36 Opening of unprotected 2,20-anhydro
pyrimidine nucleosides by action of B(OMe)3 at high
temperature resulted in the preparation of 20-O-Me-uridine
(Ͼ90% yield).37 In addition, Mg (OMe)2 was also effective
in this reaction.38
Introduction of selective and simultaneous protection of 30-
and 50-hydroxyl groups in ribonucleosides through a 50,
30-O-triisopropyldisiloxane bridge, simplified direct selec-
tive alkylation of 20-hydroxyls in such compounds. It is
notable that mostly MeI/Ag2O reagent was used so far.
Convenient preparation of 20-O-Me-uridine and cytidine
through the common precursor 50,30-O-TIPDSi-4-(2-nitro-
phenyl)-2-pyrimidinone using MeI/Ag2O was described by
Nyilas and Chattopadhyaya.29 A comprehensive investiga-
tion of the methylation of 50,30-O-TIPDSi-protected ribo-
nucleosides by MeI/Ag2O reagent was described by Inoue
and Otsuka.30 50,30-O-TIPDSi N4-Bz-Cyd was methylated
directly in 70% yield. The uridine derivative required N3-Bz
protection and the adenosine derivative was prepared
through methylation of the 6-Cl-purine intermidiate.
Attempts to methylate 50,30-O-TIPDSi-N 2-ibu-Guo or its
N-protected derivative were not successful and guanosine
synthone was prepared by methylation with diazomethane.
Results and Discussion
Synthesis of 20-O-Me-adenosine through transgly-
cosylation (Scheme 1)
In 1982 Imbach et al.7 demonstrated the utilization of
2-O-methyl-1,3,5-tri-O-benzoyl-a-d-ribofuranose for the
Scheme 1. Synthesis of 20-O-Me-adenosine by transglycosylation: (i) Ac2O, pyridine/DMF; (ii) N6-benzoylaminopurine, BSA, TMStriflate, MeCN, 75ЊC, 16h.

L. Beigelman et al. / Tetrahedron 56 (2000) 1047–1056
1049
stereospecific synthesis of 20-O-Me pyrimidine-b-d-ribo-
nucleosides through glycosylation of silylate bases in the
presence of Lewis acids as catalysts. This procedure was
optimized for a large scale preparation of 20-O-methyl-
pyrimidine ribonucleosides by Ross et al.8 Even after
optimization, this procedure required methylation of 1,3,5-
tri-O-benzoyl-a-d-ribofuranose with a large excess of
diazomethane which is potentially explosive. To overcome
this problem, we decided to utilize transglycosylation of a
suitably protected 20-O-Me-pyrimidine ribonucleosides
that were obtained through B(OMe)3 mediated opening of
2,20-anhydronucleosides.
N 6-benzoyl-50,30-di-O-acetyl-20-O-methyl adenosine (3)
that can be isolated in 50% yield. Several other products
were also formed along with a-isomer of 3 that contributed
to the overall moderate yield. When N6-phenoxyacetyl-
aminopurine was used instead of N6-benzoyladenine under
the same conditions extensive decomposition of initially
formed adenosine derivative was observed and target
nucleoside was not isolated.
The described procedure for transglycosylation is a useful
alternative in the synthesis of 20-O-Me adenosine derivative
since the starting cytidine synthone can be easily prepared
37
on large scale by modified procedure based on B(OMe)3
The transglycosylation reaction proceed with high b-selec-
tivity if carbohydrate donor contains 20-O-acyl group
capable to stabilize postulated C-1 carboxonium ion for
exclusive b-attack by an incoming silylated base. The
stereochemical outcome of transglycosylation reactions
with a carbohydrate donor without participating group at
20-OH (such as 20-O-Me) usually resulted in a mixture of
a,b nucleosides as documented for the synthesis of 20-azido
purine ribonucleoside.39
mediated opening of 2,20-O-anhydro-Cyd followed by
acylation.
Synthesis of 20-O-Me-guanosine and adenosine from
2-amino-6-chloropurine riboside (Scheme 2)
High regioselectivity in the methylation of 2-amino-6-
choloropurine riboside by diazomethane was reported by
Robins in 1966.12 Despite exclusive methylation of the
20-OH, the desired 20-O-Me product was not isolated.
Subsequent transformations of this key intermediate
resulted in the preparation of 20-O-Me-guanosine in 15%
yield. Since diazomethane is not practical for large scale
preparations, it was reasonable to investigate other
methylation reagents.
We first investigated the transglycosylation of 50,30-di-O-
acetyl-20-O-Me uridine obtained by acylation of 20-O-Me-
uridine. The transglycosylation of 50,30-di-O-acetyl-20-O-
Me uridine with 3 equiv. of N 6-benzoylaminopurine and
3 equiv. TMStriflate in CH3CN at 75ЊC for 16 h resulted
in an inseparable mixture of a and b isomers of N6-
benzoyl-50,30-di-O-acetyl-20-O-methyl adenosine in a 60%
yield and a 1:1 ratio (a/b). Since the nature of the aglycone
can also influence the product distribution in trans-
glycosylation reactions,40 we decided to try N 4-acetyl-
50,30-di-O-acetyl-20-O-methyl cytidine (2) as a carbo-
hydrate donor. Utilization of this donor under the same
conditions as for the 20-O-Me uridine derivative
resulted in predominant formation of the b anomer of
Methylation of 2-amino-6-chloropurine riboside with a
small excess of NaH/MeI reagent in DMF at Ϫ20ЊC resulted
in 20-O-Me nucleoside (5) in a 65% isolated yield together
with the formation of 20,30 bis-O-Methyl derivative in 15%
yield; no 30-methylation was observed under these condi-
tions. Several procedures were tried for the transformation
of intermediate (5) into 20-O-Me-guanosine (9), the best
result was obtained then intermediate (5) was treated with
Scheme 2. Synthesis of 20-O-methyl-adenosine and guanosine from 2-amino-6-chloropurine riboside: (i) NaH/MeI/DMF; (ii) Ac2O,DMAP,Et3N/CH3CN; (iii)
DABCO/H2O; (iv) isoamyl nitrile/THF; (v) NH3/MeOH.

1050
L. Beigelman et al. / Tetrahedron 56 (2000) 1047–1056
1,4-diazabicyclo[2.2.2]octane (1 equiv.) and water at 90ЊC
for 45 min with subsequent hydrolysis by 2 M NaOH at pH
12.41 The target 20-O-Me-guanosine was obtained in 65%
yield.
In 2,6-diamino-b-d-ribofuranosylpurine the guanosine
C6-N1 acidic amide function is replaced by weekly basic
amidine function. Therefore, one would expect that such
replacement should reduce the extent of methylation at N 1
under basic conditions (i.e. NaH/MeI). In order to increase
regioselectivity we decided to use 50,30-O-tetraisopropyl-
disiloxane-1,3-diyl protection, which was reported to be
relatively stable under NaH/MeI methylation conditions.9
The 50,30-O-tetraisopropyldisiloxane-1,3-diyl protection
was introduced by a standard procedure; utilization of ethyl-
acetate–water extraction allowed the isolation of pure
protected derivative (11) in 90% yield in a crystalline
form due to its low solubility in the above system.
The high regioselectivity in the methylation of 2-amino-6-
chloropurine riboside allows one to obtain gram quantities
of 20-O-Me-synthone (5) which can serve as a key inter-
mediate in the preparation of not only 20-O-Me-guanosine
but also 20-O-Me adenosine. This latter transformation
was achieved through radical deamination42,43 of 30,50-di-
O-acetyl-20-O-methyl-6-Cl-2-aminopurine riboside (6)
which provided 30,50-di-O-acetyl-20-O-methyl-6-chloro-
purine riboside 7 in 72% yield from 5. Subsequent amina-
tion of 7 with methanolic ammonia at 125ЊC for 4 h
provided 20-O-Me-adenosine (8) in 80% yield.
We investigated several methylation procedures for the
NaH/MeI system, varying the amount and type of solvent
and equivalents of NaH and MeI. The best results were
obtained when methylation was performed at 0ЊC in DMF
with 1.5 equiv. of NaH and 3 equiv. of MeI. If the reaction
was performed at higher temperature or in a more concen-
trated solution, extensive hypermethylation occurred.
Utilization of a lower amount of MeI required longer
reaction time and opening of the cyclic silyl group was
observed with simultaneous methylation of both 2 and
30-hydroxyls. The 20-O-Me derivative (12) can be isolated
in 90% yield without column chromatography by crystal-
lization from ethanol–water.
The convergent synthesis of both 20-O-Me-Ado and Guo
from the same precursor is attractive for scale up since the
starting 2-amino-6-chloropurine riboside is available
from guanosine in three steps without chromatographic
separations in 75% yield.44
Synthesis of N2-acyl(isobutyryl or isopropylphenoxy-
acetyl)-20-O-methylguanosine from 2,6-diamino-
b-d-ribofuranosylpurine (Scheme 3)
It was reported that the diazomethane methylation of 2,6-
diamino-b-d-ribofuranosylpurine in the presence of
SnCl2·2H2O provided a 1:1 mixture of 20 and 30-O-methyl-
ated derivatives in a quantitative yield. These compounds
can be separated on a Dowex 1 (OH)Ϫ column and deami-
nated to the corresponding guanosine derivatives with
adenosine deaminase.21 Literature analysis also indicated
that direct methylation of guanosine (NaH/MeI or diazo-
methane) usually resulted in a preferential methylation at
N 1 or/and N 7 position of the base.17,18
In order to synthesize N2-acyl-20-O-methylguanosine deriva-
tives (14) and (15) we decided to explore selective acylation
of the 2-amino group of intermediate (12) followed by
chemical deamination of the 6-amino group. Two factors
are critical for the success of this approach: the degree of
selectivity in acylation of the N-2 amino group vs. the
6-amino group in the intermediate (12); and the stability
of N-2-protection to acidic conditions of deamination.45
We found that when acylation of diaminopurine (12) was
Scheme 3. Synthesis of N2-acyl derivatives of 20-O-methyl-guanosine from 2,6-diaminopurine riboside: (i) TIPDSiCl/pyridine; (ii) MeI, NaH/DMF 0ЊC; (iii)
iPrPacCl/pyridine; (iv) HOAc/NaNO2/H2O; (v) TEA·3HF.

L. Beigelman et al. / Tetrahedron 56 (2000) 1047–1056
1051
Scheme 4. N1-Benzyl guanosine route: (i) N,N-dimethylformamide dibenzyl acetal, 80ЊC, 18h; 2N NaOH; (ii) Metal acetylacetonate, Me3SOH, 80ЊC, 2h.; (iii)
Naϩ naphthalene.
performed at Ϫ10ЊC with 1.1 equiv. of acyl chloride, the
exclusive acylation of 2-amino group in (12) occurred: for
example, the N 2-isobutyryl intermediate (13) was isolated
in 97% yield. The structure of (13) was confirmed by NMR
data and its deamination to N 2-isobutylryl-20-O-methyl-
guanosine (14) by NaNO2/CH3COOH followed by desilyl-
ation with TEA·3HF. We also observed that during
deamination with NaNO2/CH3COOH the 30,50-O-cyclic
silyl group partially opened, presumably at the 50 end.
Therefore, this intermediate was not isolated but desilylated
directly with TEA·3HF. It is worth noting that the N2-iso-
butyryl group was completely stable during deamination
and that the presence of the hydrophobic silyl group in
intermediate allowed for easy purification by extraction
from an excess of inorganic salts. This allowed subsequent
desilylation without isolation in direct preparation of (14)
from (13).
We also applied this procedure to the synthesis of N 2-
isopropylphenoxyacetyl-20-O-methylguanosine on 50 g
scale. The N2-isopropylphenoxyacetyl protecting group
was sufficiently stable for acidic diazotization conditions,
although minor loss of this group was observed resulting
in 83% overall yield from (12). To the best of our
knowledge this is the most efficient synthesis of N2-acyl
derivatives of 20-O-Me-Guanosine (13) and (14) (70%
overall yield from 2,6-diaminopurine riboside).
Synthesis of 20-O-Me-guanosine and 20-O-Me-adenosine
via metal-directed methylation (Scheme 4)
It was demonstrated that metal acetylacetonates direct the
methylation of ribonucleosides with trimethylsulfonium
hydroxide mostly to 20 and 30 hydroxyls in the case of
uridine, cytidine and adenosine.28 Application of this pro-
cedure to guanosine resulted in the isolation of six
compounds including products with methylation in both
the base and ribose moieties. When N 1-methyl-guanosine
was subjected to the same methylation conditions 1,2 and
1,30 di-N,O methyl guanosines were isolated in 82% yield.28
The high yield on all steps of transformation from (12) to
(14) prompted us to combine all reactions into ‘one pot’
procedure without isolation of intermediate (13). Selective
acylation of (12) with isobutyryl chloride followed by
deamination with NaNO2/CH3COOH and desilylation with
TEA·3HF resulted in the preparation of N 2-isobutyryl-20-O-
methylguanosine (14) in 88% yield.
We decided to investigate metal-directed methylation of
N 1-protected guanosine with trimethylsulfonium hydroxide
Scheme 5. Metal directed methylation of Adenosine: (i) Metal acetylacetonate, Me3SOH, 80ЊC, 2h.

1052
L. Beigelman et al. / Tetrahedron 56 (2000) 1047–1056
to optimize the 20:30 ratio of methylated products with
subsequent separation and deblocking in order to obtain
20-O-Me guanosine.
Experimental
NMR spectra were recorded on a Varian Gemini 400 spec-
trometer operating at 400.075 MHz for proton and
161.947 MHz for phosphorus. Chemical shifts in ppm
refer to TMS and H3PO4, respectively. Analytical thin-
layer chromatography (TLC) was performed with Whatman
The protection of N 1 in guanosine was achieved using
benzylation with N,N-dimethylformamide dibenzyl acetal.
We observed, however, that the complete cleavage of
20,30-orthoamide required more drastic condition than
reported (2 N NaOH vs MeOH/NH3)46. The desired
compound (16) was isolated in 80% yield after crystal-
lization.
˚
MK6F silica gel 60 A F254 plates and column chromato-
graphy using Merck 0.040–0.063 mm Silica gel 60.
N4-Acetyl-20-O-methyl cytidine (1). To an oven baked
stainless steel bomb (300 mL), equipped with magnetic
stirrer and purged with argon, 50 mL anhydrous methanol
was added followed by the addition of commercially avail-
able 1 g 2,20-Anhydro-1-(b-d-arabinofuranosyl)cytosine-
acetate (Aldrich) (3.5 mmol). To the resulting slurry,
8 mL trimethylborate (70 mmol) was added in the presence
of boron trifluoride-methanol (50%) (1.5 mL, 8.84 mmol).
The bomb was sealed and then heated in an oil bath at
130ЊC for 38–48 h. Upon cooling, the resulting clear,
slightly colored reaction mixture was evaporated in vacuo
to afford an off white foam. After drying in vacuo, the crude
foam was dissolved in anhydrous DMF (50 mL) in the
presence of acetic anhydride (0.36 mL, 3.85 mmol) which
was added drop-wise to the reaction mixture. The resulting
clear, light yellow solution was stirred overnight at room
temperature. The reaction mixture was evaporated in vacuo.
Crystallization of the crude product from methanol–
ethyacetate gave a pure compound 1 (0.94 g, 90%). Mp
220–223ЊC 1H NMR DMSO-d6: d 10.89 (exch. s, 1H
NH), 8.46 (d, J6,5ˆ7.4 Hz, 1H, H6), 7.18 (d, J5,6ˆ7.4 Hz,
We investigated several metal acetylacetonates in the
methylation reaction. Whereas Cu2ϩ acetylacetonate
mediated methylation provided a 1:1 ratio of 20- and 30-O-
methylated products, Mg2ϩ and Agϩ directed methylation
improved the ratio to 9:1. With Agϩ the overall conversion
was higher than with Mg2ϩ, resulting in a 70% isolated yield
of 20-O-Me-N1-Bzl-guanosine. The separation of 20-O-and
30-O-Me-N1-Bzl-guanosine derivative was achieved on a
preparative scale using
a
Waters Delta-Pak ODS
50×300 mm HPLC column. Removal of N 1-Bzl protection
with Naϩ naphthalene provided 20-O-Me-guanosine in 90%
yield.
Encouraged by results obtained with N1-Bzl-guanosine, we
investigated several different acetylacetonate in direct
methylation of adenosine (Scheme 5).
Whereas Fe and Cu acetylacetonates provided 1:1 and
2:1 ration of 20- and 30-O-Me isomers, we discovered
that Agϩ and St2ϩ both shifted the equilibrium toward
20-O-Me isomer, providing 4:1 and 8:1 ratio, respec-
tively. This allows isolation of 20-O-Me adenosine in
75–80% yield. It is also helpful for the process scale-
up that St2ϩ acethylacetonate is eight times cheaper than
Agϩ derivative.
0
0
0
1H, H5), 5.83 (d, J1 ,2 ˆ2.5 Hz, 1H, H1 ), 5.18 (exch. t,
0
0
00
JOH,5 ˆ4.6 Hz, JOH,5 ˆ4.9 Hz, 1H, 5 OH), 5.08 (exch. d,
0
0
0
0
0
0
JOH,3 ˆ6.7 Hz, 1H, 3 OH), 4.04 (t, J3 ,2 ˆ4.9 Hz, J3 ,4
ˆ
6.8 Hz, 1H, H30), 3.88 (m, 1H, 0 H40), 3.75 (dd,
0
0
0
00
00
0
J5 ,4 ˆ2.3 Hz, J
ˆ12.2 Hz, 1H, H5 ), 3.59 (dd, J5 ,4
ˆ
5 ,5
00
00
0
2.5 Hz, J5 ,5 ˆ12.2 Hz, 1H, H5 ), 3.45 (s, 3H, OCH3),
2.10 (s, 3H, CH3).
N6-Benzoyl-50,30-di-O-acetyl-20-O-methyl adenosine (3).
To a solution of N 4-acetyl-20-O-methyl cytidine(1) (1.87 g,
6.25 mmol) stirring at room temperature under argon in
DMF–pyridine (20 mL, 20 mL) was added acetic anhydride
(1.76 mL, 18.75 mmol). The reaction mixture was stirred at
room temperature for 1 h then quenched with EtOH (2 mL).
The reaction mixture was evaporated to dryness in vacuo
and partitioned between dichloromethane (50 mL) and satu-
rated NaHCO3 (20 mL). The aqueous layer was extracted
with additional dichloromethane (50 mL) and the combined
organics dried over Na2SO4. The filtrate was evaporated in
vacuo to afford a white foam.
Conclusion
In this communication we disclose several improved
synthetic methods toward 20-O-methyl adenosine (guano-
sine) and their N-acyl derivatives. We demostrated that:
(1) transglycosylation of N4-acetyl-50,30-di-O-acetyl-20-O-
methyl cytidine with N6-Bz-adenine provided N6-benzoyl-
50,30-di-O-acetyl-20-O-methyl adenosine in 50% yield. (2)
Regioselective methylation of 2-amino-6-chloropurine ribo-
side with MeI/NaH followed by hydrolysis resulted in 20-O-
Me-guanosine in high yield. The same 20-O-Me-precursor
can be transformed into 20-O-Me-adenosine in 58% yield.
(3) Very efficient transformation of 2,6-diamino-purine
riboside into N 2-isobutyryl (isopropylphenoxyacetyl) 20-
O-Me-guanosine can be achieved through methylation
of 50,30-O-TIPDSi derivative followed by selective N 2-
acylation, deamination and desilylation providing target
compounds in 70% combined yield. (4) Mg2ϩ and Agϩ
directed methylation of N1-Bzl-guanosine with trimethyl-
sulfonium hydroxide proceeded in Ͼ80% yield with ratio
of 20-O-Me/30-O-Meˆ9:1. The same methylation of adeno-
sine with Agϩ and Sr2 acetylacetonates provided 20-O-Me-
adenosine in 75–80% yield.
A stirred solution of N6-benzoylaminopurine (Lancaster)
(1.23 g, 5.16 mmol) in anhydrous acetonitrile (50 mL)
under an argon atmosphere was treated with BSA
(3.82 mL, 15.48 mmol) at reflux for 3 h. Upon cooling, a
solution of N 4-acetyl-50,30-di-O-acetyl-20-O-methyl cyti-
dine (2) (see above) (0.66 g, 1.72 mmol) in 20 mL
anhydrous acetonitrile was added to the reaction mixture
followed by TMStriflate (1.03 mL, 5.16 mmol). The reac-
tion mixture was then heated to 75ЊC for 16 h under positive
pressure of argon. Upon cooling, an additional 1.03 mL
(5.1 mmol) of TMStriflate was added, and the reaction

L. Beigelman et al. / Tetrahedron 56 (2000) 1047–1056
1053
mixture heated to 75ЊC for an additional 20 h. Once cool,
the reaction mixture was diluted with two volumes of
dichloromethane and washed with saturated NaHCO3. The
organic layer was then dried over Na2SO4 and evaporated in
vacuo. Flash chromatography employing a gradient of
10–80% ethyl acetate–hexanes afforded (3) as a white
foam; 0.403 g, 50% yield. Anal. Calcd for C22H23N5O7
(469.45): C, 56.29; H, 4.94; N, 14.92. Found: C, 56.10; H,
(297.27): C. 44.44; H, 5.09; N 23.56. Found: C, 44.34; H,
5.03; N, 23.36. The product was identical to authentic
sample by HPLC, UV, 1H NMR spectroscopy.
6-Chloro-9-[30,50-di-O-acetyl-20-O-methyl-b-d-ribofura-
nosyl] purine (7). To the solution of compound 5 (1.25 g,
3.96 mmol), 4-dimethylaminopyridine (39 mg, 0.32 mmol)
and triethylamine (0.37 mL, 2.64 mmol) in dry acetonitrile
was added acetic anhydride (0.9 mL, 9.5 mmol) and the
reaction mixture was left at room temperature for 40 min.
It was then quenched with MeOH (10 mL) and evaporated
to dryness. The residue was dissolved in methylene chloride
and washed with 1% aqueous acetic acid, saturated aqueous
sodium bicarbonate and brine. The organic layer was dried
over sodium sulfate and evaporated to dryness yielding
diacetate 6. The residue was additionally dried in vacuo
for 3 h, dissolved in dry THF and degassed with dry
argon. To the above boiling solution under positive pressure
of argon, isoamylnitrite (10 equiv.) was added dropwise.
After 2 h the solvent was removed in vacuo and the residue
was dissolved in methylene chloride, washed with saturated
aqueous NaHCO3 and brine. After evaporation of organic
phase the residue was purified by flash chromatography on
silica gel. Elution with hexanes–ethyl acetate (1:1) mixture
provided 1.1 g (72%) of compound 7 as yellow oil. Anal.
Calcd for C15H17N4O6Cl (384.78): C, 46.82; H, 4.45; N,
14.56. Found: C, 46.76; H, 4,56; N, 14.43. 1H NMR
(CDCl3): d d 2.13 (3H, s, 30-OAc or 50-OAc); 2.18 (3H, s,
30-OAc or 50-OAc); 3.44 (3H, s, 20-OCH3); 4.43 (3H, m,
1
5.15; N, 14.69; H NMR (CDCl3): d 8.88 (br s, 1H, NH),
8.81 (s, 1H, H8), 8.31 (s, 1H, H2), 8.11–7.53 (m, 5H,
0
0
0
0
0
benzoyl), 6.18 (d, J1 ,2 ˆ4.8 Hz, 1H, H1 ), 5.41 (t, J3 ,2
ˆ
0
0
0
0
0
0
4.8 Hz, J3 ,4 ˆ4.8 Hz, 1H, H3 ), 4.75 (t, J2 ,1 ˆ4.8 Hz,
0
0
0
0
J2 ,3 ˆ4.8 Hz, 1H, H2 ), 4.50–4.34 (m, 3H, H4 , H5 ,
H500), 3.44 (s, 3H, OCH3), 2.19 (s, 3H, OAc), 2.14 (s, 3H,
OAc).
2-Amino-6-chloro-9-[20-O-methyl-b-d-ribofuranosyl]
purine (5). Sodium hydride (0.44 g, 18.2 mmol) was added
to the cooled (Ϫ20ЊC) solution of 2-amino-6-chloropurine
riboside4 4 (5 g, 16.6 mmol) in dry dimethylformamide
(100 mL) under stirring. After 1 h the solution of CH3I
(1.24 mL, 19.9 mmol) in dry dichloromethane (10 mL)
was added dropwise to the reaction mixture during 1 h.
The resulting yellow solution was stirred at Ϫ20ЊC for
additional 2 h until TLC (methylene chloride–methanol
9:1) showed complete dissappearance of the starting
material. Reaction mixture was quenched with methanol
(20 mL) warmed to the room temperature and evaporated
to dryness in vacuo. The residue was dissolved in water
(200 mL) and extracted with methylene chloride
(3×200 mL), organic layer was back extracted with water
(100 mL). Combined aqueous phase was evaporated to
dryness and the residue was purified by flash chromato-
graphy on silica using gradient of MeOH (7–10%) in
methylene chloride to give 3.4 g (65%) of the compound
5. Flash chromatography purification can be substituted by
several crystallization from acetonitrile, mp 216–217ЊC
Anal. Calcd for C11H14N5O4Cl (331.10): C, 41.85; H,
40-H, 50-CH2); 4.69 (1H, 20-H, J2 ,3 ˆ5.04 Hz); 5.36 (1H, t,
0
0
0
30-H, J3 ,4 ˆ4.28 Hz); 6.13 (1H, d, 1 -H, J1 ,2 ˆ4.88 Hz);
0
0
0
0
8.31 (1H, s, 8-H); 8.77 (1H, s, 2-H).
20-O-Methyl adenosine (8). Solution of compound 7
(0.45 g, 1.17 mmol) in saturated methanolic ammonia
(20 mL) was autoclaved at 125ЊC for 4 h. The solvent was
removed in vacuo and the remaining residue was purified by
flash chromatography on silica gel. Elution with methylene
chloride–methanol (9:1) mixture provided 0.25 g (80%) of
20-O-methyl adenosine 8 as a white solid. The analytical
sample was recrystallized from abs. EtOH. mp 203–
204ЊC. Lit.20 mp 202–203.5ЊC (EtOH). Anal. Calcd for
C11H15N5O4 (384.78): C, 47.00; H, 5.30; N, 24.90. Found:
C, 47.03; H, 5.28; N, 24.85. The product was identical to
authentic sample by HPLC, UV, 1H NMR-spectroscopy.
1
4.47; N, 20.27. Found: C, 41.63; H, 4.35; N, 20.19. H
NMR (DMSO-d6 d 3.32 (3H, s, 20-OMe); 3.58 (1H,00dd,
50-H, J4 ,5 ˆ4.0 Hz, J5 ,5 ˆ12.0 Hz); 3.66 (1H, dd, 5 -H,
0
0
0
00
J4 ,5 ˆ4.0 Hz, J5 ,5 ˆ12.0 Hz); 3.95 (1H, dd, 40-H,
0
00
0
00
0
0
0
0
0
J3 ,4 ˆ3.6 Hz); 4.52 (1H, t, 2 -H, J2 ,3 ˆ4.0 Hz); 4.31 (1H,
m, 30-H); 5.07 (1H, bs, 30-OH exchangeable); 5.24 (1H, bs,
50-OH, exchangeable); 5.91 (1H, d, 10-H, J1 ,2 ˆ6.0 Hz);
0
0
6.96 (2H, bs, 2-NH2); 8.3 (1H, s, 8-H).
2,6-Diamino-9-[30,50-O-tetraisopropyldisiloxane-1,3-
diyl)-b-d-ribofuranosyl]purine (11). To an oven dried
500 mL three neck round bottom flask equipped with
mechanical stirrer, positive pressure argon and rubber
septum was added 2,6-diamino-9-(b-d-ribofuranosyl)-
purine21 (10) (10.0 g, 35.4 mmol), anhydrous DMF
(100 mL), and anhydrous pyridine (200 mL). The resulting
light brown suspension was cooled to 0ЊC in an ice–water
bath while stirring. TIPSDSiCl2 (42.48 mmol, 13.6 mL) was
added dropwise to the stirred reaction mixture via syringe
over a 20 min period, mantaining temperature at 0ЊC. The
reaction mixture was then warmed to rt, resulting in a homo-
genious solution. TLC indicated complete reaction after 3 h
at room temperature, at which time the reaction was
quenched by addition of ethanol (20 mL). The reaction
mixture was then evaporated in vacuo and the resulting
residue partitioned between ethyl acetate and saturated
20-O-Methyl guanosine (9). A mixture of 5 (2.05 g,
6.5 mmol), 1,4-diazabicyclo[2.2.2]octane (1 equiv.) and
water (30 mL) was heated to 90ЊC for 45 min. The solution
was then cooled to ambient temperature, adjusted to pH 12
with 2 M NaOH, and washed with methylene chloride
(3×60 mL). The aqueous phase was acidified to pH 6 with
6 M HCl and left at refrigerator (0ЊC) overnight. Precipitate
was filtered off, the mother liquor was evaporated to dryness
and residue was dissolved is water and applied on the short
column with RP-18 silica gel. Solid phase was washed with
water and remaining product was eluted with 5% aqueous
methanol. Appropriate fractions were combined, evaporated
to dryness and recrystallize from water to provide 1.25 g
(65%) of 20-O-methyl guanosine 9. Analytical sample was
recrystallized from methanol mp 234–236ЊC. Lit.21 mp
235–237ЊC (methanol). Anal. Calcd for C11H15N5O5

1054
L. Beigelman et al. / Tetrahedron 56 (2000) 1047–1056
0
0
0
0
0
aqueous NaHCO3 at which time (11) precipitated from the
organics layer. The aqueous layer was then extracted back
with ethyl acetate and the combined orgainics cooled to 0ЊC.
The precipitate was filtered and washed with ethyl acetate to
afford (11) as beige solid; 16.5 g, 89% yield. The analytical
sample was recrystallized from ethanol–water (2:1). Mp
174–176ЊC. Anal. Calcd for H22H40N6O5Si2 (524.77): C,
50.35; H, 7.68; N, 16.01. Found: C, 49.94; H, 7.79; N,
J2 ,3 ˆ5.2 Hz, 1H, H2 ), 4.15–3.91 (m, 3H, H4 , H5 ,
H500), 3.55 (s, 3H, OCH3), 2.87 (m, 1H, iBu-CH), 1.06–
0.96 (m, 34H, TIPDSi, iBu-(CH3)2).
N2-Isobutylryl-20-O-methylguanosine (14). A solution of
(12) (5.0 g, 9.28 mmol) in anhydrous pyridine (100 mL)
was cooled to Ϫ10ЊC in an ice–ethanol bath while stirred
under argon. Isobutyryl chloride (10.21 mmol, 1.07 mL)
was added dropwise to the stirred Ϫ10ЊC solution over a
period of 30 min. The reaction mixture was stirred at Ϫ10ЊC
for 2 h followed by 1 h at room temperature then quenched
with ethanol (20 mL). After evaporating the reaction
mixture to dryness in vacuo, the resulting residue was
partitioned between dichloromethane and saturated
NaHCO3. The aqueous layer was extracted back with
dichloromethane and the combined organics dried over
Na2SO4. Filtration and evaporation of the filtrate in vacuo
afforded a beige foam which was dissolved in glacial acetic
acid (80 mL). To the stirred acetic acid solution was added
water (40 mL) followed by NaNO2 (74.24 mmol, 5.12 g).
After 30 min another portion of NaNO2 (5.12 g,
74.24 mmol) was added and the reaction stirred at room
temperature for 48 h. The reaction mixture was diluted
with one volume of n-butanol and evaporated in vacuo to
50% of the original volume. Co-evaporation with n-butanol
(3×50 mL) was followed by partitioning the crude syrup
between ethyl acetate and saturated aqueous NaHCO3.
After extracting back the aqueous layer with ethyl acetate,
the combined organics were evaporated to dryness in vacuo.
The crude residue was then dissolved in anhydrous
dichloromethane (50 mL) and treated with a solution of
TEA·3HF (27.84 mmol, 4.54 mL) and TEA (8.17 mL), in
dichloromethane (20 mL). The reaction mixture was evapo-
rated to dryness in vacuo and subsequently dissolved in
additional dichloromethane (20 mL). Evaporation followed
by dilution was repeated three times, and the crude product
purified by flash chromatography. A gradient of 2–10%
ethanol in dichloromethane afforded (14) as light yellow
foam; 3.02 g, 88% yield. Anal. Calcd for C15H21N5O6
(367.66): C, 49.00; H, 5.77; N, 19.13. Found C, 49.05; H,
5,83; N, 19.20. 1H NMR (DMSO-d6): d 12.08 (s, exch., 1H,
NH), 11.63 (s, exch., 1H, NH), 8.29 (s, 1H, H8), 5.90 (d,
1
15.63; H NMR (DMSO-d6): d 7.77 (s, 1H, H8), 6.75 (s,
exch., 2H, N6-NH2), 5.74 (s, exch., 2H, N2-NH2), 5.71 (s,
0
1H, H10), 5.56 (d, JOH,2 ˆ5.0 Hz, 1H, 2 -OH), 4.43 (dd,
0
0
0
0
0
0
0
J3 ,2 ˆ4.5 Hz, J3 ,4 ˆ7.8 Hz, 1H, H3 ) 4.29 (m, J2 ,OH
ˆ
5.0 Hz), 4.06–3.08 (m, 3H, H40, H50, H500), 1.04 (m, 28H,
TIPDSi).
2,6-Diamino-9-[30,50-O-tetraisopropyldisiloxane-1,3-
diyl)-20-O-methyl-b-d-ribofuranosyl]purine (12). To an
oven baked 500 mL three neck round bottom flask equipped
with mechanical stirrer and positive pressure argon was
added (11) (15.6 g, 29.7 mmol) followed by anhydrous
DMF (300 mL) and methyl iodide (5.55 mL, 89.2 mmol).
The reaction mixture was cooled to 0ЊC in an ice–water bath
and 60% sodium hydride in oil (1.78 g, 4.46 mmol) added
slowly. A temperature of 0ЊC was maintained for 35 min at
which time the reaction was quenched with anhydrous
ethanol and diluted into two volumes of cold dichloro-
methane. The dilute reaction mixture was washed two
times with saturated NH4Cl, the aqueous layer was extracted
with dicloromethane, and the combined organics dried
over Na2SO4, filtered and evaporated to dryness in vacuo.
Crystallization from ethanol–water 1:1 afforded 14.7 g of
(12), 92% yield, mp 156–158ЊC. Anal. Calcd for
C23H42N6O5Si2 (538.80): C, 51.27; H, 7.86; N, 15.60.
1
Found: C, 51.19; H, 7.86; N, 15.39; H NMR (CHCl3-d3):
d 7.75 (s, 1H, H8), 6.76 (s, exch., 2H, N6-NH2), 5.78 (s, 1H,
2
H10), 5.73 (s, exch., 2H, N -NH2) 4.58 (dd, J3 ,2 ˆ4.8 Hz,
0
0
0
0
0
0
0
0
J3 ,4 ˆ4.8 Hz, 1H, H3 ), 4.12 (d, J2 ,3 ˆ4.8 Hz, 1H, H2 ),
4.09–3.91 (m, 3H, H40, H50, H50 0), 3.54 (s, 3H, OCH3),
1.03 (m, 28H, TIPDSi).
2,6-Diamino-N2-isobutyryl-9-[3050-O-tetraisopropyldisil-
oxane-1,3-diyl)-20-O-methyl-b-d-ribofuranosyl]purine
(13). A solution of (12) (0.5 g, 0.93 mmol) in anhydrous
pyridine (20 mL) was cooled to Ϫ10ЊC in an ice–ethanol
bath while stirring under argon. Isobutyryl chloride
(0.11 mL, 1.02 mmol) was added dropwise to the stirred
(Ϫ10ЊC) solution over a period of 5 min. The reaction
mixture was stirred at Ϫ10ЊC for 2 h followed by 1 h at
room temperature then quenched with ethanol (2 mL).
After evaporating the reaction mixture to dryness in
vacuo, the resulting residue was partitioned between
dichloromethane and saturated aqueous NaHCO3. The
aqueous layer was extracted with dichloromethane
(50 mL) and the combined organic layer dried over
Na2SO4. Filtration and evaporation in vacuo afforded
beige foam which was purified by flash chromatography
using a gradient of 2–4% ethanol in dichloromethane and
afforded (13) as a white foam; 0.55 g, 97% yield. Anal.
Calcd for C27H48N6O6Si2 (609.25): C, 53.23; H, 7.94; N,
13.85. Found: C, 53.09; H, 8.01; N, 13.76; 1H NMR
(DMSO-d6): d 9.76 (s, exch., 1H, N2-NH), 8.04 (s, 1H,
0
0
0
0
0
J1 2 ˆ6.3 Hz, 1H, H1 ), 5.23 (d, JOH,3 ˆ4.9 Hz, 1H, 3 -OH),
00
00
5.07 (t, JOH,5 ˆ5.3 Hz, JOH,5 ˆ5.3 Hz, 1H, 5-OH), 4.30 (m,
0
0
0
0
0
0
0
J3 ,2 ˆ4.8 Hz, J3 4 ˆ3.3 Hz, 1H, H3 ), 4.22 (t, J2 ,1 ˆ6.3 Hz,
0
0
0
0
0
0
0
J2 ,3 ˆ4.8 Hz, 1H, H2 ), 3.93 (m, J4 ,3 ˆ3.3 Hz, J4 ,5
ˆ
0
0
0
00
3.9 Hz, J4 ,5 ˆ3.9 Hz, 1H, H4 ) 3.65–3.53 (m, 2H, H5 ,
H500), 3.33 (s, 3H, OCH3), 2.78 (m, 1H, iBu-CH), 1.12 (d,
6H, iBu-(CH3)2).
N2-Isopropylphenoxyacetyl-20-O-methylguanosine (15).
A solution of (12) (47.4 g, 88 mmol) in anhydrous pyridine
(500 mL) was cooled to Ϫ10ЊC in an ice–ethanol bath while
stirred under argon. Isopropylphenoxyacetyl chloride
(20.6 mL, 96.8 mmol) was added dropwise to the stirred
Ϫ10ЊC solution over a period of 5 min. The reaction
mixture was stirred at Ϫ10ЊC for 2 h followed by 1 h at
room temperature then quenched with ethanol (20 mL).
After evaporating the reaction mixture to dryness in
vacuo, the resulting residue was partitioned between
dichloromethane and saturated aqueous NaHCO3. The
aqueous layer was extracted back with dichloromethane
and the combined organics dried over Na2SO4. Filtration
H8), 7.20 (s, exch., 2H, N6-NH2) 5.88 (s, 1H, H10), 4.71
0
0
0
0
0
(dd, J3 ,2 ˆ5.2 Hz, J3 ,4 ˆ5.2 Hz, 1H, H3 ), 4.26 (d,

L. Beigelman et al. / Tetrahedron 56 (2000) 1047–1056
1055
and evaporation of the filtrate in vacuo afforded a beige
foam which was dissolved in glacial acetic acid
(1000 mL). To the stirred acetic acid solution was added
water (400 mL) followed by NaNO2 (742.4 mmol, 51.2 g).
Another portion of NaNO2 (742.4 mmol, 51.2 g) was added
after 30 min and the reaction stirred at room temperature for
48 h. The reaction mixture was diluted with one volume of
n-butanol and evaporated in vacuo to 50% of the original
volume. Co-evaporation with n-butanol (3×) was followed
by partitioning the crude syrup between ethyl acetate and
saturated aqueous NaHCO3. After extracting back the
aqueous layer with ethyl acetate, the combined organics
were evaporated to dryness in vacuo. The crude residue
was then dissolved in anhydrous dichloromethane
(500 mL) and treated with a solution of TEA·3HF
(278.4 mmol, 45.4 mL) and TEA (81.7 mL), in dichloro-
methane (200 mL). The reaction mixture was evaporated
to dryness in vacuo and subsequently dissolved in additional
dichloromethane (200 mL). Evaporation followed by dilu-
tion was repeated 3 times, and the crude product purified by
flash chromatography. A gradient of 2–10% ethanol in
dichloromethane afforded (5) as light yellow foam; 29.2 g,
85% yield. Anal. Calcd for C22H27N5O7 (473.79): C, 55.84;
HCl, and dried to a solid tar. The tar was dissolved in water
(500 mL) and filtered through a sintered glass funnel to
remove silver salts. The filtrate was evaporated to dryness
and a portion [10 g (26 mmol)] was purified on a Waters
Delta-Pak ODS 50×300 mm HPLC column in water. The
N1-Bz1-20-O-Me guanosine(17) eluted first (7 g, 70%).
Anal. Calcd for C18H21N5O5 (387.40): C, 55.81; H, 5.46 5;
1
N, 18.08. Found C, 55.67; H, 5.61; N, 17.96. H NMR
(DMSO-d6): d 8.02 (s, 1H, H8), 7.26 (m, 5H, Ph), 7.02
0
0
0
(bs, 2H, 2NH2), 5.82 (d, J1 2 ˆ6.4 Hz, 1H, H1 ), 5.23 (s,
0
0
2H, CH2-Bzl), 5.18 (d, JOH,3 ˆ4.8 Hz, 1H, 3 -OH), 5.04 (t,
0
0
00
0
0
JOH,5 ˆ5.2 Hz, JOH,5 ˆ5.6 Hz, 1H, 5 -OH), 4.28 (m, J3 ,2
ˆ
0
0
0
0
0
4.8 Hz, J3 4 ˆ3.2 Hz, 1H, H3 ), 4.20 (t, J2 ,1 ˆ6.4 Hz,
0
0
0
0
0
0
0
J2 ,3 ˆ4.8 Hz, 1H, H2 ), 3.90 (m, J4 ,3 ˆ3.6 Hz, J4 ,5
ˆ
4.0 Hz, 1H, H40), 3.65–3.53 (m, 2H, JOH,5 ˆ5.2 Hz,
0
0
00
0
0
J5 ,5 ˆ11.6 Hz, H5 , H5 ), 3.33 (s, 3H, OCH3).
20-O-Methyl guanosine (9). The 0.6 M sodium naphthalene
solution was prepared from sodium (3.5 g, 0.152 M) and
naphthalene (21.2 g, 0.16 M) in dry THF (210 mL). This
solution (50 mmol, 90 mL) was added to N1-Bzl-20-O-
methyl guanosine (17) (2 g, 5.0 mm) and reaction mixture
was stirred overnight. The reaction was quenched with
10 mL of methanol and all solvent were removed in
vacuo. Water (100 mL) was added and resulted solution
was neutralized by 1 N HCl to pH 7. Naphthalene was
removed by extraction with toluene (3×100 mL) and
aqueous solution was purified on ODS Delta Pak column
to recover 20-O-Me guanosine nucleoside (9). The product
(1.3 g, 4.5 mmol, 90%) was identical to an authentic sample
1
H, 5.74; N, 14.84. Found C, 55.57; H, 5.78; N, 14.72. H
NMR (DMSO-d6): d 11.65 (s, exch., 2H, NH, NH), 8.30 (s,
1H, H8), 7.18–6.88 (dd, 4H, phenoxy), 5.91 (d,
0
0
0
0
0
J1 2 ˆ6.0 Hz, 1H, H1 ), 5.24 (d, JOH,3 ˆ4.8 Hz, 1H, 3 -
0
0
00
OH), 5.09 (t, JOH,5 ˆ5.6 Hz, JOH,5 ˆ5.2 Hz, 1H, 5 -OH),
0
0
0 0
4.82 (s, 2H, CH2), 4.31 (m, J3 ,2 ˆ4.8 Hz, J3 4 ˆ3.6 Hz,
1H, H30), 4.23 (t, J2 ,1 ˆ6.0 Hz, J2 ,3 ˆ4.8 Hz, 1H, H2 ),
0
0
0
0
0
1
0
0
0
0
0
00
3.93 (m, J4 ,3 ˆ3.6 Hz, J4 ,5 ˆ4.0 Hz, J4 ,5 ˆ3.9 Hz, 1H,
H40) 3.65–3.53 (m, 2H, H50, H500), 3.35 (s, 3H, OCH3),
2.84 (m, 1H, iPr-CH), 1.17 (d, 6H, iPr-(CH3)2).
by HPLC, UV–Vis and H NMR spectroscopy.
Trimethylsulfonium hydroxide (TMSH) solution in
methanol. A 0.2 M solution of trimethyl sulfonium iodide
(TMSI, 102 g, 0.5 mol), in 2.5 L of methanol and water
(9:1 v/v) was prepared by heating to 40ЊC and mixing
continuously for 30 min. The clear, colorless solution was
allowed to cool to room temperature. A glass chromato-
graphy column (Ace #50 thread; 75×300 mm) containing
Duolite 147 anion exchange resin (900 g) in the hydroxide
form was previously packed and used for the conversion of
TMSI to TMSH. The solution was pumped over the column
using a gear pump at 100 mL per minute. The resin was
washed with an additional 1 L of 90% methanol and the
total 3.5 L was reduced in volume to 500 mL using a rotary
evaporator with the bath set at 20ЊC. The solution was
checked for the presence of iodide ion using acidified silver
nitrate solution and found to be negative. The solution was
not characterized further and was stored in a teflon bottle
with a gas vent at 5ЊC.
N1-Benzyl guanosine (16). Guanosine hydrate (50 g,
177 mmol) was co-evaporated with dimethylformamide
(2×250 mL) and dissolved in dry DMF (400 mL). N,N-
dimethylformamide dibenzyl acetal was added (230 mL,
885 mmol) and the solution was heated with stirring to
80ЊC for 18 h. The excess of acetal was removed by
steam distillation on a rotary evaporator. The product was
recovered without chromatography by washing with
dichloromethane–hexanes (1:1 v/v) to yield 8 g of the
ortho-amide intermediate. The ortho-amide was cleaved
by treatment with 2 N NaOH (133 mL) at room temperature
for 4 h. The product was recrystallized from boiling water to
yield 54 g, 82% yield of pure (16). Mp 148–150ЊC (Lit.46
mp 149–150ЊC). Anal. Calcd for C17H19N5O5 (373.37): C,
54.69; H, 5.13; N, 21.43; Found: C, 54.75 H, 5.07; N, 21.29.
1H NMR (DMSO-d6): d 7.97 (s, 1H, H8), 7.29 (m, 5H, Bz),
0
0
0
7.02 (bs, 2H, 2NH2), 5.70 (d, J1 2 ˆ5.6 Hz, 1H, H1 ), 5.42
(bs, 1H, 20OH) 5.23 (s, 1H, CH2-Bz), 5.15 (bs, 1H, 30OH),
20-O-Methyl adenosine (8). To a solution of adenosine
(50 g, 187 mmol) in dimethylformamide (400 mL) silver
acetylacetonate (58 g, 28 mmol) and TMSH (280 mL of a
1 M) solution was added and the mixture was heated to 75ЊC
with stirring for 45 min. A sample of the mixture showed no
starting adenosine and two spots of which the predominant
one comigrated with an authentic sample of 20-OMe adeno-
sine. An HPLC assay showed a 9:1 ratio for 20 to 30-OMe
adenosine. The solvent was removed in vacuo. Residue
dissolved in 500 mL of water and neutralized by addition
250 mL of 1 N HCl to pH 7. The mixture was purified on
ODS 25×300 mm Delta Pak HPLC column using water as
5.00 (bs, 1H, 50-OH), 4.41 (t, J3 ,2 ˆ5.6 Hz, J3 4 ˆ4.0 Hz,
0
0
0 0
1H, H30), 4.08 (t, J2 ,1 ˆ6.3 Hz, J2 ,3 ˆ 4.8 Hz, 1H, H2 ),
0
0
0
0
0
0
0
0
0
0
3.85 (m, J4 ,3 ˆ3.5 Hz, J4 ,5 ˆ4.0 Hz, 1H, H4 ) 3.58–3.49
(m, 2H, H50, H500).
N1-Benzyl-20-O-methyl guanosine (17). To suspension of
16 (50 g, 134 mmol) and silver acetylacetonate (41 g
200 mmol) in dimethylformamide (400 mL) TMSH
(200 mL of 1 N solution in methanol) was added and reac-
tion mixture was heated to 70ЊC for 2 h. The solution was
cooled to ambient temperature, neutralized to pH 7 with 1 M

1056
L. Beigelman et al. / Tetrahedron 56 (2000) 1047–1056
21. Robins, M. J.; Hansske, F.; Bernier, S. E. Can. J. Chem. 1981,
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the eluant. Overall recovery was 42 g of (8) for a 75% yield
from adenosine. The product was identical to a authentic
sample by HPLC, UV, 1H NMR spectroscopy.
23. Ekborg, G.; Garegg, P. J. J. of Carbohydrates, Nucleosides &
Nucleotides 1980, 7, 57–60.
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