Synthesis of novel C4-linked imidazole ribonucleoside phosphoramidites for probing general acid and base catalysis in ribozyme

Article abstract of DOI:10.1016/j.tet.2005.09.063

We describe the synthesis of novel C4-linked imidazole ribonucleoside phosphoramidite (PA) 1a by which the imidazole moiety is incorporated into VS ribozyme to study its role in general acid and base catalysis. Investigation of protecting groups for the i

Full text of DOI:10.1016/j.tet.2005.09.063

Tetrahedron 61 (2005) 11976–11985
Synthesis of novel C4-linked imidazole ribonucleoside
phosphoramidites for probing general acid and base catalysis
in ribozyme
a
a
a
Lisa Araki,a Shinya Harusawa,a, Maho Yamaguchi, Sumi Yonezawa, Natsumi Taniguchi,
*
David M. J. Lilley,b Zheng-yun Zhaob and Takushi Kuriharaa
aOsaka University of Pharmaceutical Sciences, 4-20-1, Nasahara, Takatsuki, Osaka 569-1094, Japan
bDepartment of Biochemistry, MSI/WTB Complex, University of Dundee, Dundee DD1 5EH, UK
Received 28 July 2005; accepted 13 September 2005
Available online 10 October 2005
Abstract—We describe the synthesis of novel C4-linked imidazole ribonucleoside phosphoramidite (PA) 1a by which the imidazole moiety
is incorporated into VS ribozyme to study its role in general acid and base catalysis. Investigation of protecting groups for the imidazole-N
first indicated that pivaloyloxymethyl (POM) was adequate as an N-protecting group for the imidazole nucleoside, which could be readily
removed under mild basic conditions. Further, the synthetic method was extended to synthesis of 20-deoxy- and 20-O-allyl nucleoside PAs 1b
and 1c.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
The VS ribozyme is the largest of a group of nucleolytic
ribozymes that include hammerhead, hairpin, and HDV
ribozymes, and catalyzes the site-specific cleavage of a
phosphodiester linkage to generate products containing 20,
30-cyclic phosphate and 50-OH termini.1 We recently
indicated that the A730 loop of VS ribozyme was an
important component of the active site in general acid and
base catalysis (Fig. 1).2 In particular, the adenine (A756) in
the A730 loop is a critical base in the cleavage,3 because
sequence variants at the position 756 are especially
impaired in the cleavage and ligation activity. In these
studies, we have used a trans-acting VS ribozyme, where a
three-piece ribozyme-substrate system was used, as
illustrated in Figure 1.
Been and co-workers previously reported a phenomenon
called ‘imidazole rescue’: cleavage activity of the uracil-
substituted mutant (aC76U) at the active site in HDV
antigenomic ribozyme could be restored by addition of
exogeneous imidazole.4 This is probably due to rare
circumstances in which the active site region of HDV is
quite open to the solvent. By contrast, in the case of A756G
Figure 1. Schematic of a substrate and VS ribozyme, with helices
Keywords: Imidazole; Phosphoramidite; Ribozyme; POM; RNA.
*
numbered and the cleavage position arrowed. The trans-acting VS
ribozyme was used in our studies where the substrate and ribozyme were
separated. The A730 loop is boxed.
Corresponding author. Tel.: C81 72 690 1087; fax: C81 72 690 1086;
e-mail: harusawa@gly.oups.ac.jp
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.09.063

L. Araki et al. / Tetrahedron 61 (2005) 11976–11985
11977
Figure 2. Incorporation of the imidazole into A756 of VS ribozyme using phosphoramidite 1a.
or A756 abasic variants of VS ribozyme, addition of
imidazole in the medium failed to restore the activity,
possibly due to the fact that the active site is buried in a cliff
between stem I and IV, therefore preventing free imidazole
entering the active site.3
We herein describe the chemical synthesis of the imidazole
ribonucleoside PA 1a,6 which is a crucial building block in
the construction of the imidazole-containing oligonucleo-
tides, starting from 4(5)-2,3,5-tri-O-benzyl-b-ribofuranosyl-
imidazole 2.7 The key feature of the synthesis is the use of
the pivaloyloxymethyl (POM) group as an efficient
N-protecting group of imidazole. In connection with this
study, 20-deoxy-30-O-PA 1b and 20-O-allylribonucleoside-
300-O-PA 1c were synthesized, since removal of the
2 -hydroxy group from the ribose of A756 led to a small
(10-fold slower) reduction in the rate of cleavage.3 (Fig. 3)
From these results, we have developed a new chemical
strategy for determining the role of acid–base catalysis in a
ribozyme function, in which C4-linked imidazole was
placed into A756 of VS ribozyme covalently as a
pseudonucleobase (Fig. 2).5 In this study, a novel C4-linked
imidazole ribonucleoside phosphoramide (PA) 1a was
subjected to a tBDMS approach of RNA automatic
synthesis to provide an imidazole-substituted VS ribozyme
(A756 Imz) in an average 99% coupling yield. The A756
Imz catalyzed the almost-complete cleavage of a substrate
stem-loop at the correct position. Although the rate is slow
(KobsZ0.01 minK1), it was comparable to that of the uracil-
substituted HDV ribozyme in the presence of exogeneous
imidazole.4 The result powerfully supported a direct role of
the nucleobase at position 756 in the chemistry of natural
VS ribozyme.5
2. Results and discussion
2.1. An adequate protecting group for imidazole-N in the
RNA synthesis
We reported an efficient and stereoselective synthesis of tri-
O-benzyl-b-ribofuranosylimidazole 2, 4(5)-b-D-ribofurano-
sylimidazole 3a, its Nim-ethoxycarbonyl compound 3b,
which were useful intermediates to supply related nucleo-
sides.7,8 First, 50-O-dimethoxytritylation of non-protected
or Nim-ethoxycarbonylated imidazole C-nucleosides (ICNs)
3a or 3b was attempted according to the ordinary PA
synthesis.9 Although the ethoxycarbonyl group of imida-
zole-N could be removed by aqueous ammonia,7b
dimethoxytritylation of 3a or 3b failed0to give the desired
compounds (Scheme 1). Instability of 5 -O-dimethoxytrityl
(DMT) products seems responsible for the failure, but the
result emphasized the need for a suitable protecting group of
imidazole-N.
Figure 3. Novel C4-linked imidazole ribonucleoside PAs.

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L. Araki et al. / Tetrahedron 61 (2005) 11976–11985
into the imidazole-N of 2 via the corresponding chlorides to
give 5a (74%), 5b (89%), and 5c (94%) as single isomers
(Table 1). The location of the protecting groups was
tentatively assigned to be t-nitrogen because N-acylation of
the imidazole ring occurs regioselectively on the less-
hindered t-nitrogen.15 The substituents on the imidazole-N
could be readily removed to give back 2 by aqueous
ammonia–MeOH (1/3, v/v) at rt after 3 h in 73–92% yields.
Scheme 1. Attempted direct tritylation of 3a and 3b.
Next, the protected imidazoles 5a–c were evaluated under
catalytic debenzylation as shown in Table 2. Hydro-
genolysis of 5a (An) or 5b (Troc) over Pd–C cleaved their
Nim-substituted groups as well as benzyl groups to give a
non-protected imidazole C-nucleoside 3a (R1, R2, R3ZH,
quant) (Table 2, entries 1 and 2). However, POM-protected
imidazole 5c interestingly maintained the POM group under
the reduction conditions to give partial debenzylated
products 6 and 7 (Table 2, entry 3). Further, treatment of
5c with Pd(OH)2–C/cyclohexene in refluxing ethanol
produced N-POM–ICN 8 in quantitative yield. Accordingly,
the POM group satisfied the factors (2) and (3) required for
the protecting group of imidazole-N.
Bergstrom et al.10 previously reported the synthesis of 20-
deoxy-b-ribofuranosylimidazole with p-nitrophenylethyl
(PNPE)11 as a protecting group at imidazole-N. They
described that PNPE could be removed by treatment with
DBU, but they did not give any experimental details. We
thus examined whether the PNPE group at the imidazole-N
could be easily removed by DBU in the ribo-situation. As a
nitro group could be reduced under debenzylation-condition
such as catalytic hydrogenation, we assumed a protecting
group, p-trifluoromethylphenylethyl group, which could be
stable under such reductive conditions. Reaction of
tribenzyl compound 2 with PNPE bromide afforded
PNPE-protected ICN 4a (86%) as an 8:1 isomeric mixture
at the Nim position (Scheme 2). Treatment of 4a with DBU
(5 equiv) in acetonitrile at room temperature (rt) did not
remove the PNPE group, but prolonged refluxing (20 h) of
the reaction mixture gave 2 in only 22% yield. These results
indicated that the PNPE group was not appropriate for
protection of imidazole-N in the RNA synthesis. p-Tri-
fluoromethylphenylethyl-protected ICN 4b (36%) was
similarly obtained from 2, but reaction of 4b with DBU
was ineffective.
Zaramella and co-workers recently reported a convenient
method for positioning of the imidazole-protecting group, in
which 1H–15N heteronuclear multiple bond correlation
(HMBC) in the NMR method was used for several histidine
derivatives.16 We then applied the method to 8 to determine
the location of the POM group at the t-nitrogen: a
1H(C10)/15N(p) cross-peak could clearly be seen in
1H–15N HMBC signals [d (ppm) 15N (t) 176.1, 15N (p)
247.1, and 1H (C10) 4.66] (Scheme 3), since the signal of the
substituted imidazole-N always appeared at lower chemical
shift (d) than that of the unsubstituted one.
We envisaged the indispensable factors as a protecting
group for imidazole-N in RNA synthesis by the tBDMS
approach: (1) The factor, which might contribute to the
The utility of the POM group may be pointed out in RNA
Scheme 2. Attempted imidazole N-protection and deprotection.
stability of the synthetic intermediates in the whole process
via the building block 1a from 2. (2) The factor, which
should be compatible with the deprotection-condition [28%
aqueous ammonia–ethanol (3/1, v/v)] at the end of the solid-
phase RNA synthesis by the tBDMS–phosphoramidite
approach. (3) The factor, which0 coul0d be tolerated under
the debenzylation-condition of 2 ,30,5 -tri-O-benzyl-N-pro-
tected ICNs 5.7b (4) Further, the factor which might increase
the lipophilicity of imidazole, which facilitated the
extraction and isolation of each synthetic intermediate.
We investigated the protecting group from the viewpoint of
the factors (2) and (3), and focused our attention on
p-anisoyl (An),12 2,2,2-trichloroethoxycarbonyl (Troc),13
and the POM groups14 as candidates. They were introduced
Table 1. Preparation of imidazole N-protected nucleosides
Entry Reaction condition (equiv, h)
5 (%) 5 to 2
(%)
1
2
3
p-CH3OC6H4COCl (1.1), iPr2NEt, pyridine, 1.5 5a, 74 73
Cl3CCH2OCOCl (1.1), DMAP, benzene, 4
(CH3)3CCO2CH2Cl (1.5), NaH, THF, 3
5b, 89 89
5c, 94 92

L. Araki et al. / Tetrahedron 61 (2005) 11976–11985
Table 2. Catalytic debenzylation of N-protected imidazoles
11979
Entry
Reaction conditions
Product
Yield (%)
1
2
3
5a (An), H2/10% Pd–C (3 kg/cm2), 16 h
5b (Troc), H2/5% Pd–C (1 kg/cm2), 6 h
5c (POM), H2/10% Pd–C (3 kg/cm2), 16 h
3a, R1, R2, R3ZH
Quant
Quant
22
33
Quant
3a, R1, R2, R3ZH
6, R1ZPOM, R2, R3ZBn
7, R1ZPOM, R2ZH, R3ZBn or R1ZPOM, R2ZBn, R3ZH
8, R1ZPOM, R2, R3ZH
4
5c (POM), 20% Pd(OH)2–C, cyclohexene,
reflux, 3 h
˚
Scheme 3. Preparation of imidazole PA 1a. Reagents and conditions: (a) DMTCI, Et3N, cat. DMAP, pyridine; (b) TBDMSOTf, pryidine, K40 8C, MS 4 A; (c)
(iPr2N)2POCH2CH2CN, 4,5-DCl, ClCH2CH2Cl, 40 8C, 15 h.
automatic synthesis: (1) In the capping step, the unreacted
50-hydroxy group is acetylated with acetic anhydride to
prevent the growing oligonucleotide chain with a nucleoside
deletion,9 whereas activated Nim-carbonyl groups (as in
5a,b) may be susceptible to potential exchange-reactions at
this stage, leading to complexities. (2) Base-protecting
groups are conventionally removed in the final step of RNA
synthesis by an ammonia and ethanol mixture [28%
aqueous NH3–EtOH (3/1, v/v)] at 60 8C for 16 h. As the
POM group can be removed under faster and milder
conditions, it is particularly attractive to sensitive RNA such
as Cy5 labelled RNA where deprotection is recommended at
rt to minimize the destruction of cyanine dye. Obviously,
the very mild conditions require other bases to be protected
with more labile groups such as PAC or TAC for RNA
monomers A, G, and C. Hence, the N-POM group is the
most suitable and practical protecting group for the
imidazole RNA.
2.2. Synthesis of imidazole ribonucleoside PA 1a
50-O-DMT-20-O-tert-butyldimethylsilyl(TBDMS)-30-O-
cyanoethyldiisopropylphosphoramidite 1a was synthesized
in three steps from the N-POM-imidazole 8 (Scheme 3).
After DM-tritylation of 8, 50-O-DMT-ribonucleoside 9
(80%) was isolated immediately through a basic silica gel
bed, because the DMT group of 9 was removed by neutral or
standard silica gel chromatography. In this note, the basic
silica gel was used for the purification and isolation of the
labile compounds containing the DMT group. Although the
silylation reaction of the 20-hydroxy group can be controlled
to some extent by using AgNO3 as an additive,17 silylation
of 9 with TBDMSCl did not proceed in the presence of
AgNO3 and pyridine in THF. On the other hand, treatment
of 9 with TBDMSOTf in pyridine led to an inseparable 1:1
mixture 10ab (52%) of 20-O-TBDMS and 30-O-TBDMS
isomers, together with a 20,30-bis-O-substituted derivative

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L. Araki et al. / Tetrahedron 61 (2005) 11976–11985
dicyanoimidazole (DCI) as an activator and heating at 40 8C
for 15 h in 1,2-dichloroethane improved the reaction to
provide 1a and 12 in the range of 49–31 and 25% yields,
respectively. Since the final phosphoramidite 1a was
decomposed on a standard TLC plate (Merck 60 F254), the
purification of the crude product was not straightforward.
However, further efforts clarified that the phosphoramidite
1a could be purified by a basic preparative TLC plate.
The stereochemical assignment of phosphoramidites 1a and
12 was indicated by the observation of an NOESY between
the C10 and Si-Me protons of 1a, as illustrated in Figure 4,
while NOE enhancement between C10 and NCHMe2
protons in 12 was observed. It was further supported
Figure 4. The NOESY experiments of phosphoramidities 1a and 12.
Arrows indicate interactions between sets of two protons.
11 (17%). Unfortunately, since the TBDMS group readily
migrated between the 20- and the 30-hydroxy function, the
desired 20-protected ribonucleoside 10a was not separated
as a stable compound.18 Thus, the mixture 10ab was
subjected to phosphitylation. Treatment of 10ab with
2-cyanoethyl N,N,N0,N0-tetraisopropylphosphodiamidite in
the presence of diisopropylammonium tetrazolide (DIPT) in
dichloromethane at rt (90 h) generated 30- and 20-phosphor-
amidites 1a and 12 in low yields. Alternatively, use of 4,5-
31
by observation of a P–1H (C30) coupling constant
1
[3JP–H(C3 )Z13.4 Hz] with the aid of the 31P-decoupled H
0
NMR spectrum of 1a [31P NMR: d 148.9 ppm (CD3CN) as a
singlet-like peak for two P-diastereoisomers]. Although MS
measurement of PAs has been problematic owing to their
labile properties, we have very recently found that the
accurate molecular weight (MW) of PAs may be generally
Scheme 4. Preparation of imidazole PA 1b. Reagents and conditions: (a) NaH, POMCl, rt; (b) 28% NH3–MeOH (1/4, v/v), rt, 3 h; (c) 20% Pd(OH)2–C,
cyclohexene, EtOH, reflux, 3 h; (d) DMTCl$Et3N, DMAP (cat.), py, rt; (e) (iPr2N)2POCH2CH2CN, DIPT, 40 8C, 46 h.
Scheme 5. Preparation of imidazole 20-allyl ribonucleoside PA 1c. Reagents and condition: (a) Table 2, entry 4; (b) TIPDSCl2, 0 8C then py, rt, 3 h; (c) allyl
ethyl carbonate, Pd2(dba)3, Ph3P, reflux, 15 h; (d) HF, TMEDA, 0 8C then rt, 2.5 h; (e) DMTCl, Et3N, DMAP (cat.), py, rt, 19 h; (f) (iPr2N)2POCH2CH2CN, 4,
5-DCl, rt, 24 h.

L. Araki et al. / Tetrahedron 61 (2005) 11976–11985
11981
determined by using a suitable matrix system, triethanol-
amine (TEOA)–NaCl, on liquid secondary ion (LSI) MS
equipped with a double-focusing mass spectrometer. The
present method successfully revealed the molecular-related
ion [MCNa]C of 1a at m/z 953.4625, leading to the
composition formula (C50H71N4O9PSi).19 Purified imida-
zole phosphoramidite 1a was stored for several months at
Further work on application of the imidazole oligonucleo-
tides is under way and will be published in due course.
4. Experimental
4.1. General
1
K20 8C to check its long-term stability, but 31P and H
NMR measurements of the stored compound did not show
any substantial decomposition.
The melting points were determined on a hot-stage
apparatus and are uncorrected. IR spectra were recorded
on a Shimadzu IR-435 spectrometer. 1H and 13C NMR
spectra were taken with tetramethylsilane as an internal
standard on a Varian Gemini-200, Varian Mercury-300, and
Varian UNITY INOVA-500 spectrometers. Reactions with
air- and moisture-sensitive compounds were carried out
under an argon atmosphere. Unless otherwise noted, all
extracts were dried over Na2SO4, and the solvent was
removed in a rotary evaporator under reduced pressure.
Unless otherwise stated, Fuji Silysia FL-60D silica gel, Fuji
Silysia BW-127ZH silica gel, and Merck 60 F254 were used
for flash column chromatography, column chromatography
and thin-layer chromatography (TLC), respectively. As for
the basic (N–H) silica gel, Chromatorex NH-DM 1020 (Fuji
Silisia Chemical Ltd) was used. The accurate molecular
weight measurements of nucleoside PAs 1a, 1b, and 1c were
determined by MS spectrometry using a novel matrix
system, triethanolamine (TEOA)–NaCl, on LSIMS
equipped with a double-focusing MS spectrometer.19
2.3. Synthesis of 20-deoxy- and 20-O-allyl nucleoside PAs
1b and 1c
The synthetic method using POM protection of imidazole-N
was extended to the preparation of 20-deoxy- and 20-O-allyl
imidazole C-nucleoside PAs 1b and 1c (Schemes 4 and 5). It
is noted that 20-O-allyl nucleosides are potent antisense
molecules because0of0 their high nuclease resistance.20 The
imidazole-N of 3 ,5 -di-O-benzyl-20-deoxy-D-ribonucleo-
sides 137b was analogously protected by the POM group
to give 30,50-di-O-benzyl-N-protected ICN 14 (90%).
Subseq0uent debenzylation followed by DM-tritylation
gave 5 -O-DMT-20-deoxyribonucleoside 16 (51%)0. Phos-
phitylation of 16 produced the fully protected 2 -deoxy
nucleoside PA 1b (32–14%). The moderate yield of 1b was
due to isolation problems caused by column chromato-
graphy. The feasibility of 1b as a protecting group of the
Nim-POM group on 20-deoxy-D-ribonucleosides was
examined by restoration of N-protected imidazole 14 into
unsubstituted imidazole 13 in aqueous ammonia–MeOH.
4.1.1. 4(5)-(2,3,5-Tri-O-benzyl-b-D-ribofuranosyl)-1-[2-
(4-nitrophenyl)ethyl]imidazole (4a). To a solution of 2
(81 mg, 0.17 mmol) in 4-methyl-2-pentanone (0.5 ml) was
added a solution of p-nitrophenethyl bromide (56 mg,
0.24 mmol) in 4-methyl-2-pentanone (0.5 ml) followed by
potassium carbonate (50 mg, 0.36 mmol). The resulting
mixture was refluxed for 5 h and then evaporated. The
residue was dissolved with EtOAc and the resulting solution
was washed with water, brine. The organic layers were
dried, and evaporated. The crude oil was purified by flash
column chromatography [80% EtOAc in hexane] to give 4a
(90 mg, 86%) as a colorless oil; IR (film, cmK1) 1600, 1510,
For the synthesis of 20-O-allyl imidazole C-nucleoside PAs
1c, 30,50-O-TIPDS-protection (TIPDS Z1,1,3,3-tetra-
isopropyldisiloxanediyl) was carried out to give 17 (67%),
starting from tribenzylated POM–ICN 5c (Scheme 5), as the
regioselective formation for 20-O-allyl ribonucleosides had
been reported.21a Palladium-catalyzed allylation21a,b of the
20-hydroxy group of 17 produced a 200-O-allyl compound 18
(38%). Desilylation of 18 gave a 30, 5 -dihydroxy compound
19 (74%). Further, DM-tritylation (quant) followed by
phosphitylation provided the desired 20-O-allyl nucleoside
PA 1c (60–27%).
1
1450, 1345 (NO2); H NMR (CDCl3) d 2.95 (t, 6/4H, JZ
7.2 Hz), 3.09 (t, 2/4H, JZ7.2 Hz), 3.50 (dd, 1H, JZ9.5,
3.8 Hz), 3.77 (dd, 1H, JZ9.5, 3.8 Hz), 3.98 (t, 2H, JZ
3.6 Hz), 4.02–4.72 (m, 8H), 4.96 (d, 1/4H, JZ6.5 Hz), 5.10
(d, 3/4H, JZ3.8 Hz), 6.86 (s, 1H), 7.08 (d, 2H, JZ8.6 Hz),
7.15 (s, 1H), 7.25–7.35 (m, 15H), 8.08 (d, 2H, JZ8.6 Hz);
HRMS(EIMS) calcd for C37H37N3O6 [(M)C]: 619.2680,
found 619.2681.
3. Conclusion
A novel C4-linked ICN PA 1a was designed and
synthesized starting from tribenzylribofuranosylimidazole
2. The imidazole PA enabled incorporation of the imidazole
moiety into VS ribozyme in order to elucidate its roles in the
general acid and base catalysis of ribozyme. In the synthetic
study, POM was first introduced as a protecting group of
imidazole-N and it could be readily removed under mild
basic conditions. Further, the imidazole nucleoside 1a
places the base at the natural C1-atom, while other modified
nucleosides with imidazole attached to the nucleobase have
been reported.22 As the imidazole base may be incorporated
at any site in an RNA sequence using 1a, the present
approach would be applied to other situations in which
nucleobase participation is suspected. 20-Deoxy and 20-O-
allyl analogues 1b and 1c could be applicable to insert the
imidazole into modified DNA and RNA oligonucleotides.
4.1.2. 4(5)-(2,3,5-Tri-O-benzyl-b-D-ribofuranosyl)-1-[2-
(4-trifluoromethylphenyl)ethyl]imidazole (4b). The
same procedure (4a) was used for the preparation of 4b
(44 mg, 36%, a colorless oil) from 2 (90 mg, 0.19 mmol);
1H NMR (CDCl3) d 2.92 (t, 6/4H, JZ6.8 Hz), 3.05 (t, 2/4H,
JZ7.2 Hz), 3.62 (dd, 1H, JZ10.3, 3.5 Hz), 3.76 (dd, 1H,
JZ10.3, 3.5 Hz), 3.97 (t, 2H, JZ6.8 Hz), 4.02–4.68 (m,
9H), 4.98 (d, 1/4H, JZ6.8 Hz), 5.10 (d, 3/4H, JZ3.5 Hz),
6.85 (s, 1H), 7.07 (d, 2H, JZ7.2 Hz), 7.12–7.39 (m, 16H),
7.48 (d, 2H, JZ7.2 Hz).
Conversion of 4a into 2. A mixture of 4a (45 mg,
0.07 mmol) and DBU (56 mg, 0.37 mmol) in acetonitrile

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L. Araki et al. / Tetrahedron 61 (2005) 11976–11985
(2 ml) was refluxed for 20 h. After cooling, acetic acid
(28 mg) was then added. The reaction mixture was
evaporated to give a residue, which was subsequently
dissolved in CH2Cl2. The organic layer was washed with
water, dried over anhydrous MgSO4, and evaporated to give
a residue. Chromatography using EtOAc in hexane
(60–100%) gave a mixture (colorless oil, 37 mg), showing
4.26 (m, 1H), 4.44–4.67 (m, 6H), 5.06 (d, 1H, JZ2.9 Hz),
5.64 (dd, 1H, JZ19.5, 7.3 Hz), 7.03 (s, 1H), 7.21–7.38 (m,
15H), 7.61 (s, 1H).
Conversion of 5c into 2. A mixture of 5c (20 mg,
0.04 mmol) in methanol (1.5 ml) and 28% aqueous NH3
(0.5 ml) was stirred 3 h at rt. After evaporation, the resulting
residue was purified by flash column chromatography using
AcOEt to give 2 (15 mg, 92%) as a colorless oil. By the
same procedure, 5a and 5b were converted into 2 in 74 and
89% yields, respectively.
1
ca. 1:3 ratio of 2 (22%) and 4a (65%) from H NMR.
4.1.3. 4-(2,3,5-Tri-O-benzyl-b-D-ribofuranosyl)-1-(4-
methoxybenzoyl)imidazole (5a). To a mixture of 2
(101 mg, 0.22 mmol) and diisopropylethylamine (56 mg,
0.43 mmol) in pyridine (1 ml) was added p-anisoyl chloride
(74 mg, 0.43 mmol) in pyridine (1 ml). The resulting mixtue
was stirred for 1.5 h and then evaporated to give a residue,
which was subsequently diluted with EtOAc. The organic
layer was washed with 1.5 N HCl followed by brine. The
organic layers were dried, and evaporated. The resulting
crude oil was purified by flash column chromatography on
silica gel using 50% EtOAc in hexane to give compound 5a
Catalytic debenzylation of 5a (Table 2, entry 1). A solution
of 5a (52 mg, 0.09 mmol) in EtOH (8 ml) was hydrogenated
over 10% Pd on carbon23 (35 mg) at 3.0 kg/cm2 for 16 h.
After filtration through Celite, the filtrate was evaporated to
give a residue, which was purified by column chromato-
graphy to give 3a7b (17 mg, quant).
Catalytic debenzylation of 5b (Table 2, entry 2). By the
same procedure as above (Table 2, entry 1), 5b (30 mg,
0.05 mmol) in EtOH (5 ml) was hydrogenated over 5% Pd
on carbon23 (20 mg) at 1.0 kg/cm2 for 6 h to give 3a7b
(10 mg, quant).
1
(96 mg, 74%) as a colorless oil; H NMR (CDCl3) d 3.61
(dd, 1H, JZ10.3, 4.8 Hz), 3.70 (dd, 1H, JZ10.3, 4.0 Hz),
3.88 (s, 3H), 4.06 (t, 1H, JZ5.6 Hz), 4.25 (t, 1H, JZ
4.8 Hz), 4.33 (dt, 1H, JZ5.6, 4.0 Hz), 4.47–4.69 (m, 6H),
5.11 (d, 3/4H, JZ4.0 Hz), 6.98 (d, 2H, JZ9.1 Hz), 7.21–
7.35 (m, 15H), 7.48 (s, 1H), 7.76 (d, 2H, JZ9.1 Hz), 8.09
(d, 2H, JZ1.4 Hz); HRMS(EIMS) calcd for C37H36N2O6
[(M)C]: 604.2571, found 604.2571.
Catalytic debenzylation of 5c (Table 2, entry 3). By the
same procedure as above (Table 2, entry 1), 5c (36 mg,
0.06 mmol) in EtOH (6 ml) was hydrogenated over 5% Pd
on carbon23 (25 mg) at 3.0 kg/cm2 for 16 h to give 6 (7 mg,
22%) and 7 (8 mg, 33%).7b
4.1.4. 2,2,2-Trichloroethyl 4-(2,3,5-tri-O-benzyl-b-D-
ribofuranosyl)imidazole-1-carboxylate (5b). A mixture
of 2 (57 mg, 0.12 mmol), trichloroethoxycarbonyl chloride
(29 mg, 0.13 mmol), pyridine (12 mg, 0.16 mmol), and a
catalytic amount of 4-DMAP (1 mg) in benzene (3.5 ml)
was stirred for 3 h. After H2O (0.5 ml) was added to the
mixture, the solvent was evaporated to give a residue, which
was subsequently dissolved with EtOAc. The organic layer
was washed with water, brine, dried, and evaporated to give
a crude oil. It was purified by flash column chromatography
using 20% EtOAc in hexane to give 5b (69 mg, 89%) as a
4.1.6. [4-(b-D-Ribofuranosyl)imidazolyl]methyl 2,2-
dimethylpropiolate (8) (Table 2, entry 4). A mixture of
5c (206 mg, 0.35 mmol), 20% Pd(OH)2–C23 (124 mg), and
cyclohexene (1.1 ml, 10.56 mmol) in EtOH (10 ml) was
refluxed for 3 h. After filtration through Celite, the filtrate
was evaporated to give a residue, which was purified by
column chromatography [MeOH–EtOAc (1/20)] to give 8
(111 mg, quant) as a colorless oil; 1H NMR (CD3OD) d 1.18
(s, 9H), 3.52–4.19 (m, 5H), 4.66 (d, 1H, JZ5.4 Hz), 6.02 (s,
2H), 7.45 (s, 1H), 8.36 (s, 1H). LSIMS m/z: 315 [(MCH)C].
The t positioning of POM group on the imidazole ring was
determined by means of a 1H–15N HMBC experiment
1
colorless oil; H NMR (CDCl3) d 3.60 (dd, 1H, JZ10.9,
4.3 Hz), 3.70 (dd, 1H, JZ10.9, 3.9 Hz), 4.05 (t, 1H, JZ
5.8 Hz), 4.21 (t, 1H, JZ4.8 Hz), 4.32 (dt, 1H, JZ5.8,
4.3 Hz), 4.45–4.65 (m, 6H), 4.91 (d, 1H, JZ11.6 Hz), 5.00
(d, 1H, JZ11.6 Hz), 5.07 (d, 1H, JZ4.8 Hz), 7.18–7.36 (m,
15H), 7.44 (s, 1H), 8.12 (s, 1H).
1
[conditions: 25 8C, 499.7 MHz for H and 50.7 MHz for
15N; d (ppm) relative to external DMF (103.2 ppm)];
1H–15N HMBC (CD3OD) d 176.1 [15N (t)], 247.1 [15N
(p)], and the cross peak coordinate 247.1/4.66 [1H(C10)].16
4.1.5. [4-(2,3,5-Tri-O-benzyl-b-D-ribofuranosyl)imidazo-
lyl]methyl 2,2-dimethylpropiolate (5c). Under stirring,
60% NaH (72 mg, 1.80 mmol) in mineral oil was added to
THF (7 ml) to give a suspension. A solution of 2 (562 mg,
1.20 mmol) in THF (3 ml) was added to the suspension, and
the resulting mixture was stirred at rt for 3 h. Then, a
solution of chloromethyl pivaloate (271 mg, 1.80 mmol) in
THF (8 ml) was added. After 1 h, H2O (0.5 ml) was added
and the whole was evaporated to give a residue, which was
subsequently dissolved with EtOAc. The organic layer was
washed with water, brine, dried, and evaporated. The crude
product was purified by flash column chromatography on
silica gel using 50% EtOAc in hexane to give compound 5c
4.1.7.
methyl 2,2-dimethylpropiolate (9). Compound
[4-(5-O-DMT-D-b-ribofuranosyl)imidazolyl]
8
(111 mg, 0.35 mmol) was coevaporated with pyridine
(2 ml) three times and redissolved in pyridine (2 ml)
again. DMTCl (188 mg, 0.51 mmol), Et3N (0.07 ml,
0.51 mmol), and DMAP (1 mg, 0.01 mmol) were added to
the pyridine solution of 8. After the mixture was stirred
overnight, methanol (1 ml) was added to the reaction
mixture. The solvent was removed to give a residue,
which was purified through NH-silica gel bed with
chloroform to give compound 9 (174 mg, 80%) as white
amorphous product; 1H NMR (CD3OD) d 1.09 (s, 9H),
3.15–3.38 (overlapped with CD3OD), 3.76 (s, 6H), 3.99–
4.13 (m, 3H), 4.79 (d, 1H, JZ5.3 Hz), 5.73–5.92 (m, 2H),
1
(654 mg, 94%) as a colorless oil; H NMR (CDCl3) d 1.15
(s, 9H), 3.63 (dd, 1H, JZ7.3, 3.1 Hz), 3.71 (dd, 1H, JZ7.3,
3.1 Hz), 4.03 (t, 1H, JZ3.1 Hz), 4.20 (t, 1H, JZ2.9 Hz),

L. Araki et al. / Tetrahedron 61 (2005) 11976–11985
11983
6.82 (d, 4H, JZ8.3 Hz), 7.12–7.49 (m, 10H), 7.81 (s, 1H);
LSIMS m/z 617 [(MC1)C].
(CDCl3) d K4.8, K4.7, 18.2, 20.4, 24.5, 24.6, 24.6, 24.7,
25.9, 26.9, 27.0, 29.7, 38.7, 42.8, 42.9, 55.2, 58.6, 58.7,
64.3, 67.7, 73.9, 76.5, 78.5, 82.7, 86.2, 113.0, 113.1, 117.7,
118.4, 126.6, 127.7, 128.4, 130.3, 130.3, 136.1, 136.1,
138.0, 141.7, 145.0, 158.4, 177.6; 31P NMR (202 MHz,
CD3CN) d 148.9; HRMS(LSIMS)19 calcd for C50H71N4O9-
SiPCNa [(MCNa)C] 953.4621, found 953.4625.
Compound 12. A white powder; Rf Z0.75 (50% EtOAc/
4.1.8. [4-(5-O-DMT-2(3)-O-TBDMS-b-D-ribofuranosyl)
imidazolyl]methyl 2,2-dimethylpropiolate (10ab). Com-
pound 9 (314 mg, 0.51 mmol) was coevaporated with
pyridine (2 ml) three times and dissolved in pyridine
(5 ml) again. TBDMSOTf (0.13 ml, 0.56 mmol) was
added to a solution of 9 at K40 8C. The reaction mixture
was stirred for 5 min and then evaporated. The crude
product was chromatographied by NH-silica gel chroma-
tography using gradient solvent system [20–30% EtOAc in
hexane] to give compound 11 (33 mg, 8%) and 10ab
(250 mg, 67%) in that order. Compound 10ab. A pale
1
hexane); H NMR (500 MHz, CD3CN) d K0.10 (s, 3H),
0.02 (s, 3H), 0.77 (s, 9H), 1.07 (s, 9H), 1.08 (d, 3H, JZ
5.0 Hz), 1.16 (d, 9H, JZ8.3 Hz), 2.52–2.53 (m, 2H), 3.00
(dd, 1H, JZ11.0, 7.4 Hz), 3.30 (dd, 1H, JZ7.4, 3.7 Hz),
3.55–3.65 (m, 2H), 3.75 (s, 6H), 3.70–3.85 (m, 2H), 3.95–
4.05 (m, 1H), 4.30–4.35 (m, 2H), 4.92 (s, 1H), 5.75 (s, 2H),
6.84 (d, 4H, JZ8.0 Hz), 7.14 (s, 1H), 7.2–7.5 (br m, 9H),
7.61 (s, 1H); 31P NMR (202 MHz, CD3CN) d 149.1, 150.7;
HRMS (LSIMS)19 calcd for C50H71N4O9SiPCNa [(MC
Na)C] 953.4621, found 953.4618.
1
yellow amorphous product; H NMR (CDCl3) d 0.00 (s,
3H), 0.06 (s, 3H), 0.88 (s, 9H), 1.16 (s, 9H), 2.70 (m, 1H),
3.16 (dd, 1/3H, JZ11.0, 4.3 Hz), 3.25 (dd, 2/3H, JZ11.0,
4.3 Hz), 3.37 (dd, 1/3H, JZ11.0, 3.1 Hz), 3.38 (dd, 2/3H,
JZ11.0, 3.1 Hz), 3.78 (s, 6H), 4.07 (m, 2/3H), 4.24 (m,
2/3H), 4.46 (t, 1H, JZ4.3 Hz), 4.76 (d, 2/3H, JZ5.1 Hz),
4.82 (d, 1/3H, JZ5.1 Hz), 5.73 (q, 2H, JZ10.2 Hz), 6.80 (d,
4H, JZ10.2 Hz), 7.08 (s, 2/3H), 7.12 (s, 1/3H), 7.16–7.52
(m, 9H), 7.62 (s, 1H); HRMS(EIMS) calcd for
C41H54N2O8Si [(M)C] 730.3647, found 730.3649.
Compound 11. A pale yellow amorphous; 1H NMR
(CDCl3) d K0.10 (s, 3H), K0.08 (s, 3H) K0.04 (s, 6H),
0.79 (s, 9H), 0.82 (s, 9H), 1.10 (s, 9H), 3.18 (dd, 1H, JZ
10.3, 4.5 Hz), 3.37 (dd, 1H, JZ10.3, 4.5 Hz), 3.76 (s, 6H),
4.04 (t, 1H, JZ4.3 Hz), 4.09 (m, 1H), 4.22 (t, 1H, JZ
4.3 Hz), 4.84 (d, 1H, JZ4.3 Hz), 5.61 (d, 1H, JZ10.8 Hz),
5.72 (d, 1H, JZ10.8 Hz), 6.80 (d, 4H, JZ9.0 Hz), 7.08 (s,
1H), 7.14–7.51 (m, 9H), 7.59 (s, 1H).
4.1.10. [4-(3,5-Di-O-benzyl-2-deoxy-b-D-ribofuranosyl)
imidazolyl]methyl 2,2-dimethylpropiolate (14). By the
same procedure as used for the preparation of 5c, 2-deoxy
compound 137b (1480 mg, 4.06 mmol) was converted to 14
(1750 mg, 90%) as a colorless oil; 1H NMR (CDCl3) d 1.14
(s, 9H), 2.20–2.38 (m, 2H), 3.53 (dd, 1H, JZ7.0, 5.8 Hz),
3.64 (dd, 1H, JZ7.0, 4.7 Hz), 4.08–4.26 (m, 2H), 4.55 (d,
4H, JZ6.25 Hz), 5.13 (dd, 1H, JZ5.8, 4.7 Hz), 7.00 (s,
1H), 5.75 (s, 2H), 7.20–7.40 (m, 11H), 7.60 (s, 1H);
HRMS(EIMS) calcd for C28H34N2O5 [(M)C] 478.2466,
found 478.2464.
Conversion of 14 into 13. To a solution of 14 (114 mg,
0.24 mmol) in methanol (10 ml) 28% aqueous NH3 (2.5 ml)
was added and then the whole was stirred for 3 h at rt. After
evaporation, the resulting residue was purified by flash
column chromatography (70–100% AcOEt) to give 13
(87 mg, quant) as a colorless oil.
4.1.9. {4-[5-O-DMT-2-O-TBDMS-3-O-(2-CE-N,N-diiso-
propylphosphoramidite)-b-D-ribofuranosyl]imidazolyl}-
methyl 2,2-dimethylpropiolate (1a) and {4-[5-O-
DMT-3-O-TBDMS-2-O-(2-CE-N,N-diisopropylphos-
phoramidite)-b-D-ribofuranosyl]imidazolyl}methyl 2,2-
dimethylpropiolate (12). Compound 10ab (93 mg,
0.13 mmol) was coevaporated with dichloroethane (2 ml)
three times and dissolved in dichloroethane (1.5 ml) again.
To the solution was added 4,5-DCI (19 mg, 0.16 mmol) and
2-cyanoethyl N,N,N0,N0-tetraisopropylphosphodiamidite
(0.09 ml, 0.26 mmol). The resulting mixture was stirred at
40 8C for 15 h and then evaporated. The residual oil was
chromatographed (25% EtOAc in hexane) by NH-silica gel
chromatography to give partially purified 12 (25%) and 1a
(49%) in that order. The desired PA 1a was further carefully
purified on a preparative NH-TLC with 50% EtOAc in
hexane to give 1a (37 mg, 31%), while PA 12 remained
without further purification owing to its instability.
Compound 1a. A white foam; Rf Z0.69 (50% EtOAc/
4.1.11. [4-(5-O-DMT-2-deoxy-D-b-ribofuranosyl)imi-
dazolyl]methyl 2,2-dimethylpropiolate (16). By the
same procedures as used for the preparations of 8 and 9,
2-deoxy compound 14 (229 mg, 0.48 mmol) was converted
to a crude diol 15, which was subsequently tritylated to give
1
16 (146 mg, 51%) as a colorless oil; H NMR (CDCl3) d
1.16 (s, 9H), 2.10–2.44 (m, 2H), 3.12–3.26 (m, 1H), 3.32–
3.45 (m, 1H), 3.80 (s, 6H), 3.92–4.08 (m, 1H), 4.38–4.50
(m, 1H), 5.10–5.22 (m, 1H), 6.78 (s, 2H), 6.80 (d, 4H, JZ
8.0 Hz) 7.00 (s, 1H), 7.16–7.40 (m, 9H), 7.60 (s, 1H).
4.1.12. {4-[5-O-DMT-2-deoxy-3-O-(2-CE-N,N-diiso-
propylphosphoramidite)-b-D-ribo-furanosyl]imidazo-
lyl}methyl 2,2-dimethylpropiolate (1b). By the same
procedure for 1a, compound 16 (129 mg, 0.22 mmol) was
coevaporated with dichloroethane (2 ml) three times and
dissolved in dichloroethane (1.0 ml) again. To the
solution of 16 was a0dded DIPT (18 mg, 0.11 mmol) and
2-cyanoethyl N,N,N ,N0-tetraisopropylphosphodiamidite
(0.08 ml, 0.24 mmol). The resulting mixture was stirred at
40 8C for 46 h and then evaporated. The residual oil was
chromatographed by NH-silica gel chromatography to give
partially purified 1b (56 mg, 32%). PA 1b was further
purified on a preparative NH-TLC with 65% EtOAc in
1
hexane); H NMR (500 MHz, CDCl3) d K0.09 (s, 3H),
K0.03 (s, 3H), 0.82 (s, 9H), 1.00 (d, 6H, JZ7.0 Hz), 1.14
(s, 9H), 1.16 (d, 6H, JZ7.0 Hz), 2.55–2.68 (m, 2H), 3.16
(dd, 1H, JZ10.0, 4.0 Hz), 3.40 (dd, 1H, JZ10.0, 5.0 Hz),
3.52–3.60 (m, 2H), 3.78 (s, 6H), 3.82–3.88 (m, 1H), 3.90–
3.96 (m, 1H), 4.21 (t, 3/2H, JZ3.5 Hz), 4.49 (t, 1/2H, JZ
3.0 Hz), 4.49 (dd, 1H, JZ7.0, 4.0 Hz), 4.80 (d, 1H, JZ
7.0 Hz), 5.69 (d, 1H, JZ10.5 Hz), 5.75 (d, 1H, JZ10.5 Hz),
6.79 (dd, 4H, JZ9.0 Hz), 7.11 (d, 1H, JZ2.5 Hz), 7.16–
7.20 (m, 1H), 7.26–7.28 (m, 3H), 7.39 (dd, 3H, JZ6.0 Hz),
7.49–7.52 (m, 2H), 7.62 (d, 1H, JZ1.5 Hz); 13C NMR
1
hexane to give 1b (25 mg, 14%) as a colorless oil; 1b: H

11984
L. Araki et al. / Tetrahedron 61 (2005) 11976–11985
NMR (500 MHz, CDCl3) d 1.13 (s, 12H), 1.26 (s, 9H),
2.27–2.30 (m, 2H), 2.45 (dt, 2H, JZ6.5, 2.8 Hz), 3.23 (d,
2H, JZ5.0 Hz), 3.57–3.63 (m, 2H), 3.66–3.74 (m, 2H), 3.79
(s, 6H), 4.15–4.19 (m, 1H), 4.50–4.56 (m, 1H), 5.15 (dd, 1H,
JZ9.2, 5.8 Hz), 5.75 (d, 2H, JZ2.6 Hz), 6.81 (d, 4H, JZ
8.4 Hz), 7.04–7.06 (m, 1H), 7.17–7.22 (m, 1H), 7.23–7.29
(m, 3H), 7.30–7.36 (m, 4H), 7.43–7.47 (m, 2H), 7.61 (d, 1H,
JZ1.0 Hz); 13C NMR (CDCl3) d 20.2, 24.5, 24.7, 26.8,
29.7, 38.7, 43.3, 55.2, 59.4, 64.2, 67.7, 75.1, 76.5, 86.1,
97.5, 113.0, 116.6, 126.7, 127.7, 128.3, 130.2, 136.2, 138.0,
143.4, 158.4, 177.6; 31P NMR (202 MHz, CD3CN) d: 148.7;
HRMS (LSIMS)19 calcd for C44H57N4O8PCNa [(MC
Na)C] 823.3809, found 823.3801.
resulting mixture was stirred at 0 8C for 30 min and then at rt
for 2.5 h. The solvent was evaporated to give a residue,
which was subsequently purified by flash column chroma-
tography (5–15% MeOH in EtOAc) to obtain 19 (86 mg,
1
74%) as a colorless oil; H NMR (CDCl3) d 1.17 (s, 9H),
3.62 (dd, 1H, JZ12.6, 4.2 Hz), 3.77 (dd, 1H, JZ12.6,
3.1 Hz), 3.90–3.97 (m, 2H), 3.98 (ddt, 1H, JZ13.1, 5.3,
1.6 Hz), 4.08 (ddt, 1H, JZ13.1, 5.3, 1.6 Hz), 4.19 (dd, 1H,
JZ5.2, 4.5 Hz), 4.77 (d, 1H, JZ6.6 Hz), 5.07 (ddt, 1H, JZ
10.3, 1.6, 1.2 Hz), 5.16 (dq, 1H, JZ17.4, 1.6 Hz), 5.82 (ddt,
1H, JZ17.4, 10.3, 5.3 Hz), 5.94 (s, 2H), 7.32 (s, 1H), 7.84
(s, 1H); HRMS(EIMS) calcd for C17H27N2O6 [(MCH)C]
355.1867, found 355.1869.
4.1.13. {4-[3,5-O-(1,1,3,3-Tetraisopropyldisiloxane-1,3-
diyl)-b-D-ribofuranosyl]imidazolyl}methyl 2,2-dimethyl-
propiolate (17). A mixture of 5c (755 mg, 1.29 mmol), 20%
Pd(OH)2–C23 (453 mg), and cyclohexene (3.9 ml,
38.80 mmol) in EtOH (30 ml) was refluxed for 3 h. After
filtration through Celite, the filtrate was evaporated to give
triol 8 (437 mg), which was subsequently dissolved with dry
pyridine (18 ml). 1,3-Dichloro-1,1,3,3-tetraisopropyl-
disiloxane (0.4 ml) was added dropwise to the pyridine
solution of 8 at 0 8C. The resulting mixture was stirred for
1 h at the same temperature and then at rt for 3 h. After
evaporation, the crude product was purified by flash column
chromatography (30–50% EtOAc in hexanes) to obtain 17
(481 mg, 67%) as a colorless oil; 1H NMR (CDCl3) d 0.94–
1.12 (m, 28H), 1.18 (s, 9H), 3.00 (br s, 1H), 3.94–4.10 (m,
3H), 4.27 (dd, 1H, JZ12.0, 7.7 Hz), 4.46 (t, 1H, JZ
12.0 Hz), 4.77 (d, 1H, JZ7.7 Hz), 5.78 (s, 2H), 7.10 (s, 1H),
7.66 (s, 1H); HRMS(EIMS) calcd for C26H48N2O7Si2
[(M)C] 556.2997, found 556.2988.
4.1.16. [4-(2-O-Allyl-5-O-DMT-D-b-ribofuranosyl)imi-
dazolyl]methyl 2,2-dimethylpropiolate (20). By the
same procedure as used for the preparation of 9, a mixture
of allyl compound 19 (43 mg, 0.12 mmol), DMTCl (66 mg,
0.18 mmol), Et3N (0.03 ml, 0.18 mmol), and DMAP
(0.4 mg, 0.003 mmol) was stirred at rt for 19 h to give 20
(81 mg, quant) as an amorphous product; 1H NMR (CDCl3)
d 1.12 (s, 9H), 3.28 (dd, 1H, JZ10.0, 4.8 Hz), 3.38 (dd, 1H,
JZ10.0, 3.9 Hz), 3.78 (s, 6H), 4.00–4.28 (m, 5H), 4.97 (d,
1H, JZ3.8 Hz), 5.17 (dd, 1H, JZ10.3, 1.4 Hz), 5.26 (dd,
1H, JZ17.1, 3.0, 1.4 Hz), 5.69 (q, 2H, JZ10.8 Hz), 5.90
(ddt, 1H, JZ17.1, 15.7, 5.4 Hz), 6.81 (d, 4H, JZ9.0 Hz),
7.07 (br s, 1H), 7.14–7.50 (m, 9H), 7.62 (br d, 2H, JZ
1.3 Hz); 13C NMR (CDCl3) d 27.1, 55.3, 64.2, 67.7, 71.2,
71.4, 78.2, 82.1, 82.9, 86.0, 112.7, 117.2, 126.2, 127.3,
127.9, 129.8, 133.5, 135.6, 137.7, 141.6, 144.5, 157.8, 176.8
(CO); HRMS(EIMS) calcd for C38H44N2O8 [(M)C]
656.3095, found 656.3091.
4.1.14. {4-[2-O-Allyl-3,5-O-(1,1,3,3-tetraisopropyldi-
siloxane-1,3-diyl)-b-D-ribofuranosyl]imidazolyl}methyl
2,2-dimethylpropiolate (18). Compound 17 (481 mg,
0.87 mmol) was rendered anhydrous by coevaporation
with dry pyridine three times. The residue was repeated
coevaporated with dry toluene and finally dissolved in dry
THF (9 ml). Triphenylphosphine (45 mg, 0.17 mmol) and
tris(dibenzylideneacetone)-dipalladium (0) (16 mg,
0.017 mmol) were added. Finally, allyl ethyl carbonate
(0.2 ml, 1.74 mmol) was added dropwise with stirring, and
the reaction mixture was refluxed for 15 h and then
evaporated. The residue was purified by flash column
chromatography (10–30% EtOAc in hexanes) to obtain 18
(197 mg, 38%) as a colorless oil; 1H NMR (CDCl3) d 0.92–
1.12 (m, 28H), 1.16 (s, 9H), 3.97 (dd, 1H, JZ12.7, 2.4 Hz),
4.00–4.06 (m, 3H), 4.11 (dd, 1H, JZ12.7, 2.4 Hz), 4.16–
4.43 (m, 2H), 4.97 (s, 1H), 5.15 (dq, 1H, JZ10.1, 1.9 Hz),
5.32 (dq, 1H, JZ17.5, 1.9 Hz), 5.76 (dd, 1H, JZ15.9,
10.6 Hz), 5.87–6.01 (ddt, 1H, JZ17.5, 10.1, 5.0 Hz), 7.10
(s, 1H), 7.62 (s, 1H); HRMS(EIMS) calcd for
C29H52N2O7Si2 [(M)C] 596.3310, found 596.3308.
4.1.17. {4-[2-O-Allyl-5-O-DMT-3-O-(2-CE-N,N-diiso-
propylphosphoramidite)-b-D-ribofuranosyl]imidazolyl}-
methyl 2,2-dimethylpropiolate (1c). By the same
procedure for 1a, compound 20 (43 mg, 0.07 mmol) was
dissolved in dichloromethane (0.3 ml). To the solution was
added a solution of DCI (6 mg, 0.05 mmol) in CH3CN
(0.05 ml) followed by CETPA (21 ml, 0.07 mmol). After the
mixture was stirred at rt for 12 h, DCI (6 mg, 0.050mmol) in
CH3CN (0.05 ml) and 2-cyanoethyl N,N,N0,N -tetraiso-
propylphosphodiamidite (21 ml, 0.07 mmol) were added.
The resulting mixture was further stirred for 12 h at rt and
then evaporated. The residual oil was chromatographed
using benzene by NH-silica gel chromatography to give
partially purified 1c (33 mg, 60%). The semi-purified 1c was
further purified by NH-silica gel (35% EtOAc in hexane) to
give 1c (15 mg, 27%) as a colorless oil; 1H NMR (500 MHz,
CDCl3) d 1.00 (s, 2H), 1.08–1.40 (m, 19H), 2.32 (t, 1H, JZ
6.5 Hz), 2.61 (q, 1H, JZ5.9 Hz), 3.20 (td, 2H, JZ8.8,
3.8 Hz), 3.33–3.65 (m, 5H), 3.78 (s, 6H), 4.00–4.45 (m, 5H),
4.98 (t, 1H, JZ5.3 Hz), 5.11 (br d, 1H, JZ9.5 Hz), 5.23 (d,
1H, JZ17.3 Hz), 5.60–5.77 (m, 2H), 5.80–5.95 (m, 1H),
6.80 (dd, 4H, JZ8.3, 6.4 Hz), 7.10 (br d, 1H, JZ3.7 Hz),
7.16–7.30 (m, 3H), 7.31–7.39 (m, 4H), 7.44–7.50 (m, 2H),
7.61 (s, 1H); 31P NMR (202 MHz, CDCl3) d 149.7, 150.4;
HRMS(LSIMS)19 calcd for C47H62N4O9P [(MCH)C]
857.4251, found 857.4252; calcd for C47H61N4O9PNa
[(MCNa)C] 879.4070, found 879.4069; calcd for
C47H61N4O9PK [(MCK)C] 895.3810, found 895.3811.
4.1.15. [4-(2-O-Allyl-b-D-ribofuranosyl)imidazolyl]
methyl 2,2-dimethylpropiolate (19). A solution of
TMEDA (0.3 ml, 1.98 mmol) in CH3CN (0.3 ml) and HF
(46% aqueous solution, 57 ml) was added to a small round-
bottom flask at 0 8C. The HF/TMEDA mixture was stirred at
0 8C for 10 min, and a solution of 18 (197 mg, 0.33 mmol)
in CH3CN (2.3 ml) was added dropwise over 5 min. The

L. Araki et al. / Tetrahedron 61 (2005) 11976–11985
11985
7845–7854. (c) Wincott, F.; DiRenzo, A.; Shaffer, C.;
Grimm, S.; Tracz, D.; Workman, C.; Sweedler, D.;
Gonzalez, C.; Scaringe, S.; Usman, N. Nucleic Acids Res.
1995, 23, 2677–2684.
Acknowledgements
We are grateful to Dr. Y. Hari at Graduate School of
Pharmaceutical Sciences, Nagoya City University, for
helpful discussions. The authors thank Mr. K. Minoura for
help with measurements of NMR. We also thank Mr. A.
Miwa at Fuji Silysia Chemical Ltd for his kind advice on the
purification of the products. We acknowledge financial
support [Grant No. 16590024 (S. H.), No. 17790024 (L. A),
and No 16590089 (T. K.)] and a Grant-in-Aid for High
Technology Research from the Ministry of Education,
Science, Sports, and Culture, Japan.
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