A new method for the synthesis of 2′-O-benzyladenosine using mitsunobu reaction

Article abstract of DOI:10.1081/NCN-120019506

A new method to introduce a benzyl group onto the 2′-OH of purine ribonucleoside is described. Thus, 6-chloropurine 3′-O-benzoylriboside and its 5′-O-trityl congener were condensed with benzyl alcohol using the Mitsunobu reaction to give the 2′-O-benzyl derivative. The yields were varied from 4.6 to 62.9% depending on the solvent used. The product was converted to adenosine, indicating that the stereochemistry at C-2′ is retained.

Full text of DOI:10.1081/NCN-120019506

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A New Method for the Synthesis of 2′-O-
Benzyladenosine Using Mitsunobu Reaction
a
a
a
a b
Shigetada Kozai , Tomoyo Fuzikawa , Keisuke Harumoto & Tokumi Maruyama
a
Faculty of Pharmaceutical Sciences , Tokushima Bunri University , Yamashiro-cho,
Tokushima, Japan
b
Faculty of Pharmaceutical Sciences , Tokushima Bunri University , Yamashiro-cho,
Tokushima, 7708514, Japan
Published online: 07 Feb 2007.
To cite this article: Shigetada Kozai , Tomoyo Fuzikawa , Keisuke Harumoto & Tokumi Maruyama (2003) A New Method for the
Synthesis of 2′-O-Benzyladenosine Using Mitsunobu Reaction, Nucleosides, Nucleotides and Nucleic Acids, 22:2, 145-151, DOI:
10.1081/NCN-120019506
To link to this article: http://dx.doi.org/10.1081/NCN-120019506
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, No. 2, pp. 145–151, 2003
A New Method for the Synthesis of
0
2
-O-Benzyladenosine Using
Mitsunobu Reaction
Shigetada Kozai, Tomoyo Fuzikawa, Keisuke Harumoto,
*
and Tokumi Maruyama
Faculty of Pharmaceutical Sciences, Tokushima Bunri University,
Yamashiro-cho, Tokushima, Japan
ABSTRACT
0
A new method to introduce a benzyl group onto the 2 -OH of purine ribonucleo-
0
0
side is described. Thus, 6-chloropurine 3 -O-benzoylriboside and its 5 -O-trityl
congener were condensed with benzyl alcohol using the Mitsunobu reaction to
0
give the 2 -O-benzyl derivative. The yields were varied from 4.6 to 62.9% depend-
ing on the solvent used. The product was converted to adenosine, indicating that
0
the stereochemistry at C-2 is retained.
Antisense oligonucleotides have emerged as antiviral agents and for other ther-
0
[
1,2]
apeutic purposes.
ribonucleoside have been intensely investigated due to the importance of these
Therefore, several methods for the synthesis of the 2 -O-alkyl
0
monomers in this field. However, introduction of an alkyl group at the 2 -hydroxyl
0
group has been a challenging issue for nucleoside chemists since the 3 - and
*Correspondence: Tokumi Maruyama, Faculty of Pharmaceutical Sciences, Tokushima Bunri
University, Yamashiro-cho, Tokushima 7708514, Japan.
1
45
DOI: 10.1081/NCN-120019506
Copyright # 2003 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

146
Kozai et al.
0
[3]
-hydoroxyl groups should be protected prior to alkylation. An attempted
5
direct reaction of adenosine with phenyldiazomethane gave 2 -O-benzyladenosine
0
[
4]
0
in only 27% yield. Also, Moffatt et al. reported the preparation of the 2 - and
0
0 0
3 -O-monoalkylated ribonucleosides using 2 ,3 -O-(dibutylstannylene)-nucleosides
as an intermediate. An efficient protecting group for the specific reaction onto
the 2 -hydroxyl group was established by 3 ,5 -O-(tetra-isopropyldisiloxane-1,3-diyl)
(TIPS) ribonucleosides. In a large scale, however, it is difficult to use this protecting
group because of the cost for reagent. Recently, direct 2 -O-alkylation of the
protected cytidine was achieved with alkyl halide in the presence of 1.5–2.5 equiva-
[5]
0
0
0
[6]
0
[7]
lents of silver oxide. A drawback to this method is the use of silver which is thought
to be a causative substance of pollution, and there has been no report regarding
purine nucleosides.
The Mitsunobu reaction is a universal method to condense the acid and alcohol
[
8]
accompanied with inversion of the configuration of the alcoholic hydroxyl group.
One exceptional case is a sterically hindered sugar, 1,2:5,6-di-O-isopropylidene-D-
[9]
gluco-furanose, in which resistance to S 2 displacement was reported. Also,
N
[
8]
intramolecular ethereal bond formation was reported in the trans-diol sugar.
0
Wentwarth and Janda reported the displacement of arabinoside to 2 -O-benzylated
10]
riboside using the Mitsunobu reaction with benzyl alcohol. This method could be
an alternative approach to obtain the 2 -O-alkylated ribonucleoside (Chart 1).
However, it takes several steps to prepare arabinoside.
[
0
This background prompted us to develop a method to introduce an alkyl group
onto the purine ribonucleoside. In this report the Mitsunobu reaction of the 6-chloro-
0
purine 3 -O-benzylriboside with benzyl alcohol is described.
0 0
6-Chloropurine 3 -O-benzoylriboside (1a) and its 5 -O-trityl congener (1b) were
prepared by the method as described in an earlier report. Then, compound 1b was
[
11]
0
0
[12]
subjected to the reaction with benzyl alcohol (4 eq.) in the presence of N,N,N ,N -
0
tetramethylazodicarboxamide [TMAD], 1,1 -azobis-(N,N-dimethylformamide)
and triphenylphosphine (TPP) in a solvent. The reaction was monitored by high-per-
formance liquid chromatography (HPLC), and the results are presented in Table 1.
In an aprotic polar solvent such as N,N-dimethylformamide (DMF), the peak of the
0
2
-O-benzyl congener (2b) appeared in low yield. Also, the reaction in tetrahydro-
furan (THF) gave 2b in 14% yield. In spite of this result, a similar reaction performed
in 1,4-dioxane gave 2b in 45% yield. This discrepancy is interesting since the effects of
both solvents are thought to be similar. The best result was obtained when the
reaction was carried out in benzene, in which conversion was estimated to be
6
2.5% yield (Table 1). After work-up of the solution, 2b was obtained in 57% yield.
Chart 1.

0
Synthesis of 2 -O-Benzyladenosine
147
Table 1. The Mitsunobu reaction of 1b with benzyl alcohol (10 eq.) in solvent.
Mitsunobu reagent
Reagent
Solvent
1b remain (%) 2b yields (%)
4
4
4
4
4
4
4
eq TMAD
eq TMAD
eq TMAD
eq TMAD
eq TMAD
eq DIAD
eq TMAD
4 eq TPP
4 eq TPP
4 eq TPP
4 eq TPP
4 eq TPP
4 eq TPP
THF
DMF
1,4-dioxane
Benzene
0.6
0.3
2.9
0.5
1.3
43.7
1.0
14.4
4.6
45.1
62.5
59.7
17.7
45.0
Benzene : hexane (1 : 2)
1,4-dioxane
1,4-dioxane
TPP, (NH
4
)
2
SO
4
An attempt to change TMAD to diisopropyl azodicarboxylate (DIAD) decreased
the yield of 2b to 17.7% in the 1,4-dioxane solvent system. To evaluate the role of
0
0
the 5 -O-protecting group, 3 -O-benzoylriboside 1a was subjected to a similar reac-
0
tion and the results are presented in Table 2. It appeared that the 5 -O-protection
did not benefit the condensation and, as a whole, the yields were even improved
as demonstrated in the case of 1,4-dioxane. The reaction mechanism could be
explained as follows. It is well known that 2 -OH is more acidic than 5 -OH. There-
fore, the initial reaction of 1 with N-phosphonium salt gives an intermediary benzyl-
0
0
0
oxyphosphonium ion, which receives a nucleophilic displacement of the 2 -alkoxide
0
anion of 1a,b. The other route that form an intermediary 2 -O-phosphinio-riboside is
considered to be unfavourable since the b-site of this intermediate is tight, therefore,
less accessible to nucleophilic displacement of benzyloxy anion. Compound 2b was
0
[13]
treated with NH in MeOH to afford 2 -O-benzyladenosine (3), which showed
0
nuclear Overhauser effect (NOE) between H2 and H3 in the two-dimensional
3
0
NOE (NOESY) spectrum as shown in Fig. 1. Thus, the configuration of 3 was iden-
0
tified as a 2 (R) -riboside structure. A part of the product was converted to the ade-
nosine 4, for which the measured data were identical with those of the authentic
sample. To substantiate the efficacy of this method, compound 1b was condensed
0
with 3,4,5-trimethoxybenzyl alcohol to give 2 -O-(3,4,5-trimethoxybenzyl) derivative
in 56% yield. However, an attempt to condense with 2,4-dimethoxybenzyl alcohol
5
Table 2. The Mitsunobu reaction of 1a with benzyl alcohol (10 eq.) in solvent.
Mitsunobu reagent Reagent
Solvent
1a remain (%) 2a yields (%)
4
4
4
4
4
4
4
4
4
eq TMAD
eq TMAD
eq TMAD
eq TMAD
eq TMAD
eq TMAD
eq TMAD
eq TMAD
eq DIAD
4 eq TPP
4 eq TPP
4 eq TPP
4 eq TPP
4 eq TPP
THF
DMF
1,4-dioxane
5.2
7.5
4.7
0
25.5
0.4
61.0
61.6
60.9
62.9
55.8
62.6
26.6
CHCl
Benzene
3
0
4 eq TPP Benzene : 1,4-dioxane (1 : 1)
1.2
3.6
3.9
54.7
4 eq TPP
4 eq TBP
4 eq TPP
Benzene : hexane (1 : 2)
1,4-dioxane
1,4-dioxane

148
Kozai et al.
6
Figure 1. NOESY correlation of compound 3 in DMSO-d .
was unsuccessful, suggesting the methoxy group at the ortho-position disturbs the
approach of the anion species (Chart 2).
0
In conclusion, a new method to introduce a benzyl group at 2 -OH of purine
ribonucleoside using the Mitsunobu reaction was developed. The acidity of the
0
2
-OH group of 1 is increased by electron-withdrawing groups, 6-chloropurine and
Chart 2.

0
Synthesis of 2 -O-Benzyladenosine
149
0
0
-O-benzyol groups. These groups also evolve steric hindrance in 2 -OH; therefore
3
the stereochemistry of sugars is retained. This method could be an alternative
[
9]
0
way to obtain the 2 -O-benzylnucleosides.
EXPERIMENTAL
Melting points (mp) were determined using a Yanagimoto micro-melting point
apparatus (hot stage type) and are uncorrected. UV spectra were recorded with a
Shimadzu UV-190 digital spectrometer. Low-resolution mass spectra were obtained on
a Shimadzu-LKB 9000B mass spectrometer in the direct-inlet mode. High-resolution
mass spectra were obtained on a JMS AX-500 spectrometer in the direct-inlet mode.
1
H-NMR spectra were recorded on either Varian UNITY 200 (200 MHz) or Varian
UNITY600(600MHz)inCDCl (ordimethyl sulfoxide(DMSO)-d )withtetramethyl-
3
6
silane as an internal standard. Merck Art 5554 plates precoated with silica gel 60 con-
taining fluorescent indicator F254 were used for thin-layer chromatography and silica
gel 60 (Merck 7734, 60–200 mesh) was employed for column chromatography.
High-Performance Liquid Chromatography
The apparatus for high performance liquid chromatography (HPLC) were
CCPD pump (Toso Co.) and SPD-M10A photo diode array UV-VIS detector
(
Shimadzu Co.). The HPLC conditions were as follows: the columns, connection with
Cosmosil Guard Column 5C18-MS (4.6 ꢀ 10 mm, Nacalai Tesque INC.) and Cosmo-
sil Packed Column 5C18-MS (4.6 ꢀ 150 mm, Nacalai Tesque Inc.); eluent, 10 mM
phosphoric acid-MeOH (2 : 3) for 2a, and (1 : 4) for 2b; flow rate, 1 mL=min; column
ꢁ
temperature, 50 C. The weight of compounds were calculated by the absolute cali-
bration method from the peak areas, and the yields were converted to percentages.

150
Kozai et al.
(2a). To a
9-(3-O-Benzoyl-2-O-benzyl-b-D-ribofuranosyl)-6-chloropurine
mixture of 6-chloro-9-(3-O-benzoyl-b-D-ribofuranosyl) purine (1a, 390 mg, 1 mmol)
and benzyl alcohol (1.04 mL, 10 mmol) in 1,4-dioxane (25 mL) were added triphenyl-
phosphine (1.05 g, 4 mmol) and TMAD (668 mg, 4 mmol). The solution was stirred
ꢁ
at 50 C overnight, then concentrated to a small volume. The residual solution
was chromatographed over a column of silica gel G (2.2 ꢀ 34 cm) using 25–50%
AcOEt in hexane (1.4 l) to give 12a as white crystals (231 mg, 48%), mp 167.5–
ꢁ
1
69.5 C. Anal Calcd for C H ClN O : C, 59.94; H, 4.40; N, 11.65. Found:
5
2
4
21
4
þ
C, 59.97; H, 4.49; N, 11.60. MS m=z: 450, 452 (M -CH O). UV l
nm: 265. H-NMR (600 MHz, CDCl ) d: 8.55 (1H, s, H8), 8.15–8.16 (2H, m, two
(MeOH)
max
2
1
3
of Ph), 8.10 (1H, s, H2), 7.63–7.66 (1H, m, one of Ph), 7.51–7.54 (2H, m, two of
0
Ph), 6.84–6.94 (5H, m, Ph), 5.91 (1H, d, J ¼ 8.2 Hz, H1 ), 5.90 (1H, d, J ¼ 5.2 Hz,
0
0
0
0
H3 ), 5.55 (1H, br s, 5 OH), 4.89–4.91 (1H, m, H2 ), 4.55–4.57 (2H, m, H4 and
0
one of CH ), 4.29 (1H, d, J ¼ 12.1 Hz, one of CH ), 3.97–4.00 (1H, m, H5 a),
2
2
0
3
.86–3.88 (1H, m, H5 b).
9-(3-O-Benzoyl-2-O-benzyl-5-O-trityl-b-D-ribofuranosyl)-6-chloropurine
(2b).
To
(
were added triphenylphosphine (1.05 mg, 4 mmol) and TMAD (688 mg, 4 mmol).
a mixture of 6-chloro-9-(3-O-benzoyl-5-O-trityl-b-D-ribofuranosyl)purine
1b, 633 mg, 1 mmol) and benzyl alcohol (1.04 mL, 10 mmol) in 1,4-dioxane (25 mL)
ꢁ
The solution was stirred at 50 C overnight, then concentrated to a small volume.
The residual solution was chromatographed over a column of silica gel G
(
5
2.5 ꢀ 45 cm) using 20–25% AcOEt in hexane (1.4 l) to give a caramel (411 mg,
þ
1
7%). MS m=z: 479, 481 (M -Tr). H-NMR (600 MHz, CDCl ) d: 8.10, 8.51 (each
3
1
C H CO), 7.48–7.52 (2H, m, two of C H CO), 7.22–7.44 (ca 15H, m, Tr), 6.95–
H, s, H2, H8), 8.11–8.13 (2H, m, two of C H CO), 7.62–7.65 (1H, m, one of
6
5
6
5
6
5
0
0
7
5
4
.11 (5H, m, Ph), 6.14 (1H, d, J ¼ 6.6 Hz, H1 ), 5.75 (1H, dd, J ¼ 3.0, 5.2 Hz, H3 ),
0
.04 (1H, dd, J ¼ 5.5, 6.6 Hz, H2 ), 4.63 (1H, d, J ¼ 12.4 Hz, one of CH C H ),
2
6
5
0
.54–4.56 (1H, m, H4 ), 4.45 (1H, d, J ¼ 12.4 Hz, one of CH C H ), 3.56 (1H, dd,
2
6
5
0
0
J ¼ 3.8, 10.7 Hz, H5 a), 3.49 (1H, dd, J ¼ 4.1, 10.4 Hz, H5 b).
0
2
10 mL) saturated with ammonia at 0 C was heated in a steel bomb at 120 C over-
-O-Benzyladenosine (3). A solution of 12a (250 mg, 0.52 mmol) in MeOH
ꢁ ꢁ
(
night, then ice-cooled. The solution was concentrated to a small volume and chromato-
graphed over a column of silica gel G (2.7 ꢀ 33 cm) using a 0–33% EtOH in CHCl .
3
Evaporation of the fraction and crystallization from CCl and CHCl gave white
3
4
ꢁ
[4]
ꢁ
crystals (173 mg, 93%). mp 150–151.5 C. (lit.
C H N O 0.7 H O: C, 55.19; H, 5.56; N, 18.93. Found: C, 55.09; H, 5.65; N,
147–150 C). Anal Calcd for
1
7
19
5
4
2
þ
1
1
8.94. MS m=z: 327 (M -CH O). UV l
(MeOH) nm: 261. H-NMR (600 MHz,
max
2
DMSO-d ) d: 8.33 (1H, s, H8), 8.09 (1H, s, H2), 7.35 (2H, br s, NH ), 7.15–7.22
6
2
0
0
(
5
5H, m, Ph), 6.07 (1H, d, J ¼ 6.3 Hz, H1 ), 5.46 (1H, dd, J ¼ 4.4, 7.4 Hz, 5 OH),
0
.35 (1H, d, J ¼ 5.2 Hz, 3 OH), 4.57 (2H, dd, J ¼ 12.1, 12.5 Hz, CH ), 4.55 (1H,
2
0
0
dd, J ¼ 4.9, 6.3 Hz, H2 ), 4.37 (1H, dt, J ¼ 3.0, 4.9 Hz, H3 ), 4.03–4.04 (1H, m,
0
0
0
H4 ), 3.67–3.69 (1H, m, H5 a), 3.55–3.59 (1H, m, H5 b).
Adenosine (4). A suspension of 13 (60 mg, 0.17 mmol) and palladium on
activated carbon (10%, 60 mg) in EtOH (20 mL) was stirred vigorously under H2

0
Synthesis of 2 -O-Benzyladenosine
151
ꢁ
atmosphere at 50 C overnight. The catalyst was removed and the filtrate was concen-
trated to a small volume to give white crystals (45 mg, quantitative), which were
þ
identical in all respects with an authentic sample. MS m=z: 267 (M ). UV l
max
1
(
MeOH) nm:260.5. H-NMR (200 MHz, DMSO-d ) d: 8.41 (1H, s, H8), 8.20 (1H,
6
0
0
s, H2), 5.90 (1H, d, J ¼ 5.9 Hz, H1 ), 4.60 (1H, t, J ¼ 5.1 Hz, H2 ), 4.16 (1H, dd,
0
0
0
J ¼ 3.3, 7.0 Hz, H3 ), 3.99–4.00 (1H, m, H4 ), 3.53–3.72 (1H, m, H5 ).
-Chloro-9-[2-O-(3,4,5-trimethoxy)benzyl-3-O-benzoyl-5-O-trityl-b-D-ribofurano-
6
syl]purine (5). To a mixture of 6-chloro-9-(3-O-benzoyl-5-O-trityl-b-D-ribofurano-
syl) purine (1.89 g, 3 mmol) and 2,3,4-trimethoxybenzyl alcohol (4.8 mL, 30 mmol) in
benzene (75 mL) were added triphenylphosphine (3.15 g, 12 mmol) and TMAD
ꢁ
(
2.06 g, 12 mmol). The solution was stirred at 50 C overnight, then concentrated
to a small volume. The residual solution was chromatographed over a column of
þ
silica gel G to give a caramel (1.36 g, 56%). FAB-MS m=z: 835 (M þ Na - H).
1
UV l
(
(MeOH) nm: 265. H-NMR (600 MHz, CDCl )d: 8.61 (1H, s, H8), 8.25
max
3
0
1H, s, H2), 7.22–8.13 (ca 22H, m, Tr, Bz, Ph), 6.26 (1H, d, J ¼ 6.0 Hz, H1 ), 5.79
0
0
(
m, H4 , CH ), 3.87 (3H, s, OCH ), 3.85 (3H, s, OCH ), 3.77 (3H, s, OCH ), 3.52–
3
1H, dd, J ¼ 3.3, 5.2 Hz, H3 ), 5.15 (1H, dd, J ¼ 5.5, 6.0 Hz, H2 ), 4.45–4.59 (3H,
0
2
3
3
3
0
.59 (2H, m, H5 ).
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.
.
Baserga, R.; Denhardt, D.T. Antisense Strategies; Annals of the New York
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Temsamani, J.; Agarwal, S. Advances in Antiviral Drug Design; De Clerq, E.,
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Myles, A.; Pfleiderer, W. Chem. Ber. 1972, 105, 3327–3333.
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Perkin Trans. 1, 1997, 2779–2788.
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8.
9.
Hodge, R.P.; Sinha, N.D. Tetrahedron Lett. 1995, 36, 2933–2936.
Mitsunobu, O. Synthesis 1981, 1–28.
Grynkiewicz. Rocz. Chem. 1976, 50, 1499.
10. a) Chen, Y.-C.J.; Hansske, F.; Janda, K.D.; Robins, M.J. J. Org. Chem. 1991,
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Bull. 1999, 47, 966–970.
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1
2. Tsunoda, T.; Otsuka, J.; Yamamiya, Y.; Ito, S. Chem. Lett. 1994, 539–542.
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Received September 26, 2002
Accepted December 9, 2002