Synthesis of imidazole C1- and C3-ribonucleoside phosphoramidites for probing catalytic mechanism in ribozyme

Article abstract of DOI:10.3987/COM-11-12263

Synthesis of C4-linked imidazole N-pivaloyloxymethyl (POM)-2'-O- cyanoethylated (CE) C1- and C3-ribonucleoside phosphoramidites 1a and 1b is described. These phosphoramidite products were incorporated into RNA sequence through solid phase phosphoramidite

Full text of DOI:10.3987/COM-11-12263

HETEROCYCLES, Vol. 83, No. 9, 2011
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HETEROCYCLES, Vol. 83, No. 9, 2011, pp. 2041 - 2055. © The Japan Institute of Heterocyclic Chemistry
Received, 21st May, 2011, Accepted, 1st July, 2011, Published online, 6th July, 2011
DOI: 10.3987/COM-11-12263
SYNTHESIS OF IMIDAZOLE C1- AND C3-RIBONUCLEOSIDE
PHOSPHORAMIDITES FOR PROBING CATALYTIC MECHANISM IN
RIBOZYME
Shinya Harusawa,1* Kensuke Fujii,1 Masayoshi Nishiura,1 Lisa Araki,1
Yoshihide Usami,1 Zheng-yun Zhao,2 and David M. J. Lilley3
1Osaka University of Pharmaceutical Sciences, 4-20-1, Nasahara, Takatsuki,
Osaka 569-1094, Japan
2School of Chemistry, University of Birmingham, Edgbaston, Birmingham B15
2TT, UK
3CR-UK Nucleic Acid Structure Research Group, MSI/WTB Complex, University
of Dundee, Dundee DDI 5EH, UK
Abstract
–
Synthesis of C4-linked imidazole N-pivaloyloxymethyl
(POM)-2’-O-cyanoethylated (CE) C1- and C3-ribonucleoside phosphoramidites 1a
and 1b is described. These phosphoramidite products were incorporated into RNA
sequence through solid phase phosphoramidite approach, providing RNA with
imidazole linked through different length to sugar residue, to study the mechanism
of a ribozyme.
INTRODUCTION
RNA catalysis is important in the processing and translation of RNA molecules, yet the mechanisms of
catalysis are still unclear in most cases.1 The VS ribozyme is the largest of a group of nucleolytic
ribozymes that include hammerhead, hairpin, HDV, and GlmS, and catalyzes the site-specific cleavage of
a phosphodiester linkage to generate products containing 2’, 3’-cyclic phosphate and 5’-OH termini.2
Imidazole with a pKa of 7.1 is both a good donor and acceptor of proton. It can be placed in the normal
base position to study the general acid and base catalysis.3 We have recently developed a new
chemo-genetic strategy for examining the role of general acid-base catalysis in ribozyme function,

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HETEROCYCLES, Vol. 83, No. 9, 2011
O
O
N
N
N
NH
(POM)
DMTO
iPr2N
O
(CH2)n
(CH2)n
O
O
RNA synthesizer
O
OR
O
OH
P
-O P O
O
O
NC
1
1a 1b
: R = -CH2CH2CN, n = 1 ( ), n = 3 ( )
imidazole-containing ribozyme
(n = 0 ~3)
2 : R = -CH2CH2CN, n = 0 (2a), n = 2 (2b)
3
, n = 0
: R =
Si
Figure 1. Imidazole Cn-ribonucleoside phosphoramidites for solid-phase RNA synthesis
whereby C4-linked imidazole was covalently placed as a pseudonucleobase at position 756 (replacing
adenine) of the VS ribozyme2e and position 8 (replacing guanine) of the hairpin ribozyme5 respectively.
In the study, N-pivaloyloxymethyl (POM) 2’-O-tert-butyldimethylsilyl (t-BDMS)-imidazole
C0-phosphoramidite (PA) 34a-c was first employed to provide an imidazole-substituted ribozymes, which
catalyzed the almost-complete cleavage of substrate stem-loops at the correct position.2e,5 However, the
synthetic route of PA 3 lacks the selective introduction of t-BDMS group at 2’-hydroxy group and in
addition 3 itself has acid-labile character, eventually reducing the overall yield.4a-b
In our systematic studies on the catalytic mechanism of VS ribozyme,2 we have recently introduced a new
combination of protecting groups for the PAs 2a (n = 0)6b and a two-carbon-elongated homologue 2b (n =
2)6a of ribose-(CH2)n-imidazole species: POM for imidazole-N and cyanoethyl (CE) groups for the
2’-hydroxy group. They facilitated efficient synthesis of 2a and 2b and additionally provided
considerable stability compared to the first labile 2’-O-t-BDMS-imidazole C0-PA 3. Using 2b, a single
imidazole base-moiety with two carbon linker was inserted at position 638 of VS ribozyme in
trans-acting form to give a variant (G638C2Imz). G638 is one of the nucleobases proposed to act in
general acid-base catalysis in concert with A756 in the catalytic mechanism of the VS ribozyme.2a
It might be anticipated that the manner of linking the imidazole group to the ribose would affect its ability
to function in general acid base catalysis. If the linker is too short then its ring N cannot be superimposed

HETEROCYCLES, Vol. 83, No. 9, 2011
2043
with that of N1 of adenine or guanine. On the other hand, if it is too long then it may spend too little time
in the position required for its potential catalytic function. It is interesting to note that the cleavage rate of
G638C2Imz was found to be 15-fold greater than that achieved with the ribozyme (G638C0Imz)
containing a C4-linked imidazole at the same position.6 The result suggests that imidzole nucleoside
analogues with flexible spacer lengths provide a valuable general methodology for further exploring the
catalytic mechanisms of ribozymes as well as other RNA functions as better structural mimics of purine
nucleobase. Further, the whole series of ribose-(CH2)n-imidazole PAs (n = 0 ~ 3) could make an
interesting series of compounds for systematically tracing out the geometry of the active site of a
ribozyme. As the VS ribozyme is the only member of the class of nucleolytic ribozymes for which there
is no crystal structure at the present time, mechanistic investigation cannot be guided by structural data.2d
We therefore required C1- and C3-PAs 1a and 1b in order to complete and extend our series, allowing us
to compare imidazole C1- and C3-ribozymes with those of the C0- and C2 forms. We herein describe the
chemical synthesis of novel C4-linked C1- and C3-imidazole ribonucleoside PAs 1a and 1b.
RESULTS AND DISCUSSION
Starting from commercially available 2,3,5-tri-O-benzyl-D-ribose 4,7 a two-step synthesis of methyl
3,6-anhydro-4,5,7-tri-O-benzyl-2-deoxy--D-allo-heptonate 6 was at first carried out according to the
synthetic procedure of Ohrui and co-workers.8 Wittig olefination of
4
with methyl
(triphenylphosphoranylidene)acetate afforded (Z)-alkene product 5 (60 %), together with (E)-isomer
(23%). Although the reaction required 6 hours (h) using the original procedure,8 use of benzoic acid (0.1
eq) as an additive shortened the reaction time to 50 min in our hands. Treatment of (Z)-5 with sodium
methoxide provided ester 6 (quant) and then DIBAL reduction of 6 afforded a new aldehyde 7 (94%)
which is a mutual intermediate for synthesis of C1- and C3-PAs 1a and 1b (Scheme 1 and 2).
We next tried to prepare the imidazole ring from aldehyde 7 by Büchi procedure.9 Reaction of 7 with
(p-tolylsulfonyl)methylisocyanide (TosMIC) in the presence of catalytic amount of NaCN in a [3+2]
cycloaddition led to the formation of the 4-tosyloxazoline 8, which was subsequently heated with a
o
solution of saturated ammonia in ethanol at 125 C in a stainless steel tube for 2 h to give a
one-carbon-elongated homo-C-nucleoside 9 in 35% yield from 7. Introduction of a POM group at the
imidazole-N position of 9 produced 2’,3’,5’-tri-O-benzyl-N-POM-nucleoside 10 (90%).4 The relative
-stereochemistry of 10 was confirmed from a NOESY experiment, as summarized in Scheme 1.
Debenzylation of 10 with Pd(OH)2-C/cyclohexene afforded imN-POM-imidazole C1-D-ribonucleoside 11a
(quant).

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HETEROCYCLES, Vol. 83, No. 9, 2011
BnO
BnO
BnO
CO2Me
OH
b
c
OH
a
O
O
CO2Me
[(Z)-5]
BnO OBn
BnO OBn
BnO OBn
-5 (60 %)
(Z)
6
(quant)
4
(E)-5 (23%)
N
NH
N
O
BnO
BnO
BnO
CHO
Tos
e
O
O
d
O
BnO OBn
BnO OBn
BnO OBn
9
7
(35% from )
8
7
(94 %)
O
+ NOE
O
N POM
N
N
N
H
BnO
HO
H
H
g
f
O
O
H
BnO OBn
HO OH
11a
(quant)
10 (90%)
11a
Scheme 1. Synthesis of imidazole C1 triol
.
Reagents and conditions: (a) Ph3P=CHCO2Me (1 eq), benzoic acid (0.1 eq), reflux 50 min, MeCN; (b)
NaOMe, rt, 10 min; (c) DIBAL, -78 oC; (d) TosMIC, NaCN (cat.), EtOH, 0.5h; (e) NH3 in EtOH, 125 oC, 2 h;
(f) NaH, POMCl, rt, 3 h, THF; (g) 20% Pd(OH)2-C, cyclohexene, EtOH, reflux, 4 h.
Alternatively, Wittig olefination (83%) of aldehyde 7 using N-trityl-4-imidazolylmethylphosphonium
chloride 126a followed by acid treatment to remove the trityl group gave three-carbon-elongated
imidazole 13 (88%) (Scheme 2). POM protection (63%) of 13 and subsequent debenzylation and
reduction of double bond with Pd(OH)2-C/cyclohexene (86%) afforded imN-POM-C3-imidazole 11b.
With C1-and C3-triol intermediates 11a and 11b in hand, we addressed the synthesis of imidazole-C1- and
C3-PAs 1a and 1b, as shown in Scheme 3. 3’,5’-O-TIPDS-protection (TIPDS = 1,1,3,3-tetra-
isopropyldisiloxanediyl) of 11a (n = 1) gave 14a (58%), which allowed selective introduction of the
2’-hydroxy protecting group in a moderate yield. Cyanoethylation of 14a with acrylonitrile did not
o
proceed at room temperature (rt),10 while the reaction gave fully protected intermediate 15a at 40 C in
50% yield, causing partial removal of POM group. The TIPDS group of 15a was selectively removed by
treatment with Et3N·3HF to give 3’,5’-O-unprotected ribonucleoside derivative 16a (80%). After

HETEROCYCLES, Vol. 83, No. 9, 2011
2045
N
N POM
N
NH
a (83%)
b (88%)
c (63%)
d (86%)
(CH2)3
BnO
BnO
HO
CHO
O
O
O
BnO OBn
HO OH
BnO OBn
11b
13
7
N-Tr
N
Cl -
+
Ph3P
12
11b
Scheme 2. Synthesis of imidazole C3 triol
.
Reagents and conditions: (a) 12, n-BuLi, -78 oC then rt; (b) 2N HCl, reflux, EtOH; (c) NaH, POMCl, rt, THF;
(d) 20% Pd(OH)2-C, cyclohexene, EtOH, reflux.
dimethoxytritylation (77 %) of 16a in the usual manner, 3’-free derivative 17a was subjected to
phosphitylation
condition.
Treatment
of
17a
with
2-O-cyanoethyl-N,N,N’,N’-
tetraisopropylphosphodiamidite in the presence of 4,5-dicyanoimidazole (DCI) in dichloroethane
o
proceeded smoothly at 40 C for 1h to give the final product imidazole-C1-PA 1a. Purification of the
crude product was readily carried out on basic silica gel column chromatography to yield
phosphoramidite 1a (60%). The overall yield of 1a is 1.9% in 12 steps from the starting tribenzyl D-ribose
4. 31P-NMR of 1a reaveled two P-diastereomers at  149.3 and 149.9 ppm (CDCl3). Although MS
measurement of PAs has been problematic owing to their labile properties, we recently reported MS
measurement of nucleoside and non-nucleoside PAs, with the new matrix system [triethanolamine
(TEOA)-NaCl] on LSIMS or FABMS using a double-focusing mass spectrometer.11 The present method
was consistent with the composition formula {C48H62N5O9Na [(M+Na)+]; calculated: 906.4182, observed:
906.4191}. In a similar manner, conversion of imN-POM-imidazole C3-ribonucleoside 11b into
imidazole-C3-PA 1b was performed according to that of C1-homologue 11a into 1a, as shown in Scheme
3. The structure of 1b was characterized by 31P-NMR [ 149.8 and 150.7 ppm (CDCl3)] and MS
measurement {C50H66N5O9Na [(M+Na)+]; calculated: 934.4495, observed: 934.4490}.11 The overall yield
of 1b is 2.5% in 12 steps from the starting material 4.
Studies of modified VS ribozymes and other imidazole oligonucleotides using 1a and 1b are under way,
and will be published in due course.

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HETEROCYCLES, Vol. 83, No. 9, 2011
N
N POM
N
N POM
b
N POM
a
N
(CH2)n
(CH2)n
(CH2)n
O
O
HO
O
O
O
Si
O
Si
O
Si
O
O
Si
O
OH
HO OH
CN
11a (n =1)
b (n =3)
15a
b
14a
b
(n =1, 50%)
(n =3, 67%)
(n =1, 58%)
(n =3, 46%)
N POM
N POM
N
N
(CH2)n
d
(CH2)n
HO
DMTO
c
O
O
HO
O
HO
O
CN
CN
16a (n =1, 80%)
b (n =3, 85%)
17a (n =1, 77%)
b (n =3, 84%)
N
N POM
(CH2)n
O
DMTO
e
1a (n =1, 60%)
b (n =3, 51%)
N
O
O
P
CN
O
NC
Scheme 3. Synthesis of 2'-O-cyanoethylimidazole C1 and C3-nucleoside phosphoramidites 1a and 1b.
.
Reagents and conditions: (a) TIPDSCl2, py; (b) CH2=CHCN, CsCO3, tBuOH; (c) Et3N 3HF, Et3N, THF;
(d) DMTCl, DMAP, Et3N, py; (e) (iPr2N)2POCH2CH2CN, 4,5-DCI, ClCH2CH2Cl, 40 oC.
EXPERIMENTAL
General
IR spectra were recorded using a Shimadzu IR-435 spectrometer. 1H- and 13C-spectra were measured with
tetramethylsilane as an internal standard on a Varian Mercury-300 or Varian UNITY INOVA-500
spectrometers. 31P-NMR spectra were recorded at 121 MHz (Varian UNITY INOVA-500) and the
chemical shifts were measured relative to 85% H3PO4 as an external standard. Fast atom bombardment
mass spectrometric studies (FAB-MS) were performed using a JMS-700 (JEOL Ltd.) instrument in the
positive ion mode with a TEOA-NaCl or 3-nitrobenzylalcohol (NBA) matrix. Reactions with air- and

HETEROCYCLES, Vol. 83, No. 9, 2011
2047
moisture-sensitive compounds were carried out under an argon atmosphere. Unless otherwise noted, all
extracts were dried over Na2SO4, and the solvents were removed in a rotary evaporator under reduced
pressure. Anhydrous solvents were purchased from Wako Chemical Co. and Nacalai Chemical Co. Fuji
Silysia FL-60D silica gel, Fuji Silysia BW-127ZH silica gel, and Merck 60F254 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 Silysia Chemical Ltd.) was used.
2-(2,3,5-Tri-O-benzyl--D-ribofuranosyl)acetaldehyde (7)
To a solution of 6 (88 mg, 0.19 mmol) in dry toluene (2 mL) at -70 oC was added dropwise a 1M solution
of DIBAL in toluene (0.56 mL, 0.56 mmol) over 5 min. After stirring for 0.5 h at the same temperature,
the reaction mixture was quenched with MeOH (0.6 mL) and further stirred for 0.5 h at rt. Saturated aq.
NaHCO3 (0.6 mL) was added and the reaction mixture was stirred for another 0.5 h. After anhydrous
magnesium sulfate was added to the resulting suspension, the reaction mixture was filtered through Celite
pad, and washed with AcOEt. The filtrate was evaporated to give a crude oil, which was purified by
column chromatography (25% AcOEt in hexane) to give 7 (80 mg, 94%) as a colorless oil. IR (film) cm-1
1
1720 (CO); H-NMR (CDCl3)  2.54 (ddd, 1H, J = 16.3, 7.8, 2.2 Hz), 2.64 (ddd, 1H, J = 16.3, 5.2, 1.9
Hz), 3.47 (d, 2H, J = 4.4 Hz), 3.65 (dd, 1H, J = 7.7, 5.5 Hz), 3.92 (dd, 1H, J = 5.5, 3.3 Hz), 4.17-4.25 (m,
13
1H), 4.40-4.65 (m, 7H), 7.20-7.40 (m, 15H), 9.74 (t, 1H, J = 2.3 Hz); C-NMR (CDCl3)  47.1, 70.1,
71.7, 72.1, 73.4, 75.5, 76.6, 80.6, 82.1, 127.5, 127.6, 127.7, 127.8, 127.9, 128.0, 128.1, 128.3,128.4, 137.4,
137.6, 137.9, 200.6; MS of 7 could not be measured owing to its thermal instability.
4-(2,3,5-Tri-O-benzyl--D-ribofuranosyl)methyl-1H-imidazole (9)
To a suspension of TosMIC (372 mg, 1.87 mmol) and 7 (837 mg, 1.87 mmol) in EtOH (4 mL) was added
NaCN (14 mg, 0.28 mmol) at rt, and the mixture was stirred for 30 min. The resulting solution was
transferred to a saturated NH3 in EtOH (30 mL) in a stainless steel tube at 0 oC, and the sealed system was
then heated at 125 oC for 2 h. After the mixture was cooled, the solvent was evaporated to give a residue
which was purified by column chromatography (15% MeOH in AcOEt) to give the imidazole compound
9 (295 mg, 35%) as a pale brown oil. Rf (15% MeOH in AcOEt): 0.67; 1H NMR(CDCl3)  2.67 (dd, 1H,
J = 16.0, 4.8 Hz), 2.92 (dd, 1H, J = 16.0, 4.8 Hz), 3.50-3.58 (m, 2H), 3.62 (t, 1H, J = 6.8 Hz), 3.80 (dd,
1H, J = 10.6, 2.9 Hz), 4.02-4.17 (m, 1H), 4.26-4.64 (m, 7H), 6.57 (s, 1H), 6.66 (s, 1H), 7.15-7.47 (m,
15H). 13C-NMR (CDCl3)  28.4, 68.7, 70.2, 72.3, 73.2, 73.7, 76.4, 77.2, 77.8, 78.1, 79.8, 80.1, 82.0,
124.9, 127.0, 127.6, 127.8, 127.9 (127.89), 127.9 (127.94), 128.1 (128.06), 128.1 (128.12), 128.3, 128.4
(128.38), 128.4 (128.41), 128.6, 134.9, 137.1, 137.5, 137.6. HRMS (FABMS) calcd for C30H33N2O4

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HETEROCYCLES, Vol. 83, No. 9, 2011
(M+H)+ 485.2440, found 485.2448.
[4-(2,3,5-Tri-O-benzyl--D-ribofuranosyl)methylimidazolyl]methyl 2,2-dimethylpropionate (10)
Under stirring, 60% NaH (37 mg, 0.91 mmol) in mineral oil was added to THF (3 ml) to give a
suspension. A solution of 9 (295 mg, 0.61 mmol) in THF (5 mL) was added to the suspension, and the
resulting mixture was stirred at rt for 0.5 h. Then, a solution of chloromethyl pivaloate (138 mg, 0.91
mmol) in THF (4 mL) was added. After 3 h, H2O (1 mL) was added and the mixture was evaporated to
give a residue, which was subsequently dissolved in AcOEt. The organic layer was washed with water,
brine, dried, and evaporated. The residue was purified by column chromatography on silica gel using
AcOEt in hexane (50% to 100%) to give compound 10 (328 mg, 90%) as an oil; 1H-NMR (CDCl3)  1.14
(s, 9H), 2.78 (dd, 1H, J = 14.3, 6.5 Hz), 2.86 (dd, 1H, J = 14.3, 6.5 Hz), 3.49 (dd, 1H, J = 10.4, 3.9 Hz),
3.55 (dd, 1H, J = 10.4, 3.9 Hz), 3.76-3.83 (m, 2H), 4.20 (dd, 1H, J = 9.1, 3.9 Hz), 4.32 (dd, 1H, J = 11.7,
5.2 Hz), 4.38-4.62 (m, 6H), 5.65 (q, 2H, J = 9.5 Hz), 6.83 (s, 1H), 7.16-7.40 (m, 15H), 7.53 (s, 1H);
13C-NMR (CDCl3)  : 26.8, 32.8, 38.7, 67.6, 70.2, 71.7, 73.3, 77.3, 79.7, 80.7, 81.0, 117.0, 127.6, 127.9,
128.0, 128.3, 137.3, 137.9, 138.0, 138.2, 139.5; HRMS(FABMS) calcd for C36H43N2O6 (M+H)+ 599.3121,
found 599.3123.
[4-(-D-Ribofuranosyl)methylimidazolyl]methyl 2,2-dimethylpropionate (11a)
A mixture of 10 (328 mg, 0.55 mmol), 20% Pd(OH)2-C (197 mg), and cyclohexene (1.7 mL, 16.43
mmol) in EtOH (12 mL) was refluxed for 4 h. After filtration through Celite, the filtrate was evaporated
to give a residue, which was purified by column chromatography (20% MeOH-EtOAc) to give 11a (180
mg, quant) as a colorless oil; 1H-NMR (CD3OD)  1.17 (s, 9H), 2.72 (dd, 1H, J = 15.5, 7.1 Hz), 2.86 (dd,
1H, J = 15.5, 5.2 Hz), 3.51 (dd, 1H, J = 17.8, 6.6 Hz), 3.65 (dd, 1H, J = 17.8, 6.6 Hz), 3.71-4.03 (m, 4H),
5.89 (s, 2H), 7.09 (s, 1H), 7.76 (s, 1H); 13C-NMR (CD3OD)  27.2, 32.8, 39.7, 63.4, 69.3, 72.6, 75.7, 83.7,
85.5, 118.8, 139.1, 179.0; HRMS (FABMS) calcd for C15H25N2O6: 329.1712 (M+H)+, found 329.1711.
4-[(EZ)-3-(2,3,5-Tri-O-benzyl--D-ribofuranos-1-yl)prop-1-enyl]-1H-imidazole [(EZ)-13]
A 1.6 M BuLi solution in hexane (2.9 ml, 4.6 mmol) was added dropwise over a period of 15 min to a
white suspension of phosphonium salt 12 (2.90 g, 4.6 mmol) in dry THF (20 mL) at -70 °C. The resulting
yellow suspension was stirred for 30 min at the same temperature, and a solution of aldehyde 7 (1.02 g,
2.30 mmol) in THF (10 mL) was added slowly to keep the temperature of the suspension at
approximately -70 °C. The reaction mixture was elevated to rt and continued to stir for 3 h. The reaction
was quenched by the addition of H2O and was evaporated. CHCl3 was added to the residue, and the

HETEROCYCLES, Vol. 83, No. 9, 2011
2049
organic layer was washed with H2O and brine, dried, and then evaporated to give a residue. It was again
diluted with AcOEt, mixed with a small amount of silica gel, and evaporated to obtain coated silica gel
for use in column chromatography. Chromatography with 60% AcOEt-hexane as eluent gave a pale
yellow foam (1.42 g, 83%) of 4-[(EZ)-2-(2,3,5-tri-O-benzyl--D-ribofuranos-1-yl)prop-1-enyl]-1-trityl-
1H-imidazole; 1H-NMR (CDCl3)  2.30-2.48 (m, 2H), 3.46 (d, 0.33H, J = 4.3 Hz), 3.51 (d, 1.67H, J = 4.3
Hz), 3.63-3.70 (m, 1H), 3.85 (t, 1H, J = 5.1 Hz), 4.07-4.22 (m, 2H), 4.42-4.58 (m, 6H), 6.19-6.35 (m, 2H),
6.68 (s, 0.83H), 6.78 (s, 0.17H), 7.08-7.40 (m, 31H); 13C-NMR (CDCl3)  14.1, 21.0, 32.9, 36.9, 60.3,
70.3, 70.4, 71.7 (71.66), 71.7 (71.71), 73.3, 75.1, 77.3, 77.6, 79.5, 79.7, 80.8, 80.9, 81.0; HRMS
(FABMS) calcd for C51H49N2O4 (M+H)+: 753.3692, found 753.3687. Aqueous 2N HCl (12 mL) was
added to the solution of the propenylimidazole (1.49 g, 1.89 mmol) in EtOH (15 mL) and the solution
was refluxed for 2.5 h. After cooling to rt, the precipitated material was removed by filtration. The filtrate
was evaporated to give a residue that was subsequently diluted with water and neutralized by the addition
of saturated aq. NaHCO3 solution. The aqueous was extracted with AcOEt, and the combined organic
layers were washed with H2O, dried, and evaporated to yield a residue. Chromatography purification on
silica with 10% MeOH in AcOEt as eluent gave a mixture (0.85 g, 88%) of 13 as a yellow foam.
1H-NMR (CDCl3)  2.26-2.64 (m, 2H), 3.40-3.74 (m, 3H), 3.82-3.97 (m, 1H), 3.98-4.26 (m, 2H),
4.40-4.67 (m, 6H), 5.53 (dt, 0.3H, J = 10.8, 8.4 Hz), 6.01 (dt, 0.7H, J = 18.1, 8.4 Hz), 6.23 (d, 0.7H, J =
18.1 Hz), 6.40 (d, 0.3H, J = 10.8 Hz), 6.81 (s, 0.7H), 6.92 (s, 0.3H), 7.19-7.48 (m, 16H); 13C-NMR
(CDCl3)  32.5, 36.8, 69.8, 70.4, 71.8, 72.0 (71.95), 72.0 (72.03), 72.3, 73.4 (73.38), 73.4 (73.44), 77.1,
120.9, 121.7, 123.8, 124.7, 127.6, 127.7, 127.8, 127.9, 128.0, 128.1 (128.09), 128.1 (128.12), 128.3,
128.4, 128.5, 134.9, 135.3, 137.5, 137.7 (137.68), 137.7 (137.73), 138.1; HRMS (FABMS) calcd for
C32H34N2O4 (M+H)+: 511.2597, found 511.2592.
{4-[3-(-D-Ribofuranosyl)propyl]imidazolyl}methyl 2,2-dimethylpropionate (11b)
Imidazole 13 (220 mg, 0.43 mmol) was first converted into (EZ)-imN-POM-propenylimidazoles (168 mg,
63%, E/Z = 20:1) as a colorless oil, according to the synthetic procedure of 10. Although the separation
(E) and (Z)-POM products was not required for the following reduction, they could be resolved by silica
gel chromatography (50% AcOEt in hexane); {4-[(E)-3-(2,3,5-Tri-O-benzyl--D-ribofuranosyl)prop-
1-enyl]imidazolyl}methyl 2,2-dimethylpropionate; colorless oil; Rf (60% AcOEt in hexane); 0.28;
1H-NMR (CDCl3)  1.15 (s, 9H), 2.70-2.90 (m, 2H), 3.48-3.60 (m, 2H), 3.73 (t, 1H, J = 5.7 Hz), 3.92 (t,
1H, J = 5.0 Hz), 4.17-4.27 (m, 2H), 4.46-4.60 (m, 6H), 5.63-5.74 (m, 3H), 6.35 (d, 1H, J = 11.7 Hz), 7.04
13
(s, 1H), 7.22-7.37 (m, 15H), 7.59 (s, 1H); C-NMR (CDCl3)  26.8, 33.0, 38.7, 67.6, 70.5, 71.7, 71.8,
73.3, 77.5, 80.1, 80.8, 81.2, 118.0, 122.9, 126.1, 127.5 (127.49), 127.5 (127.52), 127.6, 127.7, 127.8,

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127.9, 128.0, 128.1 (128.09), 128.1 (128.14), 128.2 (128.18), 128.2 (128.24), 128.3 (128.26), 128.3
(128.28), 137.6, 137.9, 138.0, 138.2, 140.4, 177.6; HRMS(FABMS) calcd for C38H46N2O6 (M+H)+
625.3277, found 625.3279. {4-[(Z)-3-(2,3,5-Tri-O-benzyl--D-ribofuranosyl)prop-1-enyl]imidazolyl}-
methyl 2,2-dimethylpropionate; colorless oil; Rf (60% AcOEt in hexane); 0.14. 1H-NMR (CDCl3)  1.17
(s, 9H), 2.34-2.53 (m, 2H), 3.52 (d, 2H, J = 4.2 Hz), 3.67 (t, 1H, J = 6.0 Hz), 3.87 (t, 1H, J = 4.8 Hz),
4.06-4.25 (m, 2H), 4.44-4.65 (m, 6H), 5.78 (s, 2H), 6.27 (d, 1H, J = 15.6 Hz), 6.28-6.40 (m, 1H), 6.89 (s,
1H), 7.19-7.40 (m, 15H), 7.60 (s, 1H); 13C-NMR (CDCl3)  26.6, 29.5, 30.1, 36.7, 38.5, 67.4, 70.2, 71.5,
71.6, 73.2, 77.2, 79.5, 80.6, 80.9, 115.7, 123.8, 124.9, 127.3, 127.4, 127.5, 127.6, 127.8, 127.9 (127.88),
127.9 (127.94), 128.1, 128.2, 137.7, 138.0, 138.1, 141.0, 177.5; HRMS(FABMS: NBA) calcd for
C38H46N2O6 (M+H)+ 625.3277, found 625.3276. Next, a mixture of (EZ)-imN-POM-propenylimidazole
(173 mg, 0.28 mmol), 20% Pd(OH)2-C (104 mg), and cyclohexene (1.0 mL, 8.40 mmol) in EtOH (9 mL)
was refluxed for 2.5 h. After filtration through Celite, the filtrate was evaporated to give a residue, which
was purified by silica column chromatography (20% MeOH in AcOEt) to give 11b (86 mg, 86%) as a
colorless oil. 1H-NMR (CD3OD)  1.16 (s, 9H), 1.44-1.89 (m, 4H), 2.57 (t, 2H, J = 9.6 Hz), 3.50-3.83 (m,
13
5H), 3.87 (t, 1H, J = 6.0 Hz), 5.90 (s, 2H), 6.98 (s, 1H), 7.73 (s, 1H); C-NMR (CD3OD)  26.7, 27.4,
28.9, 34.3, 39.9, 48.6, 48.9, 49.5, 49.8, 63.8, 69.4, 73.0, 76.5, 84.1, 85.7, 117.4, 139.4, 143.9, 179.1;
HRMS(FABMS) calcd for C17H29N2O6 (M+H)+ 357.2025, found 357.2029.
{4-[(3,5-O-TIPDS--D-ribofuranosyl)methyl]imidazolyl}methyl 2,2-dimethypropionate (14a)
1,3-Dichloro-1,1,3,3-tetraisopropyldisiloxane (0.14 mL, 0.43 mmol) was added dropwise to a solution of
o
11a (141 mg, 0.43 mmol) in pyridine (10 mL) at 0 C. The resulting mixture was stirred for 1 h at the
same temperature and evaporated to a crude product (white wax), which was subsequently purified by
column chromatography (AcOEt then 10% MeOH in AcOEt) to yield 14a (141 mg, 58%) as an oil.
1H-NMR (CD3OD)  0.80-1.17 (m, 28H), 1.98 (s, 9H), 2.75-3.20 (m, 2H), 3.50-4.20 (m, 6H), 6.09 (s,
2H), 7.57 (brs, 1H), 9.04 (s, 1H); 13C-NMR (CD3OD)  13.8, 13.9, 14.0, 14.1, 14.4, 14.7, 17.4, 17.5, 17.6
(17.57), 17.6 (17.64), 17.8, 27.1, 30.1, 39.8, 63.1, 64.3, 70.7, 73.0, 75.1, 79.5, 81.1, 83.5, 83.8, 86.9,
120.6, 137.9, 178.7; HRMS(FABMS) calcd for C27H51N2O7Si2 (M+H)+ 571.3234, found 571.3240.
{4-[(2-O-CE-3,5-O-TIPDS--D-ribofuranosyl)methyl]imidazolyl}methyl
2,2-dimethylpropionate
(15a)
Acrylonitrile (0.12 mL, 1.8 mmol) and cesium carbonate (29 mg, 0.09 mmol) were added to the solution
o
of 14a (50 mg, 0.09 mmol) in t-BuOH (0.4 mL). After being vigorously stirred at 40 C for 15 h, the
mixture was diluted with AcOEt (25 mL). Filtration through Celite and evaporation gave a residue, which

HETEROCYCLES, Vol. 83, No. 9, 2011
2051
was purified by column chromatography (50% AcOEt in hexane) to obtain 15a (27 mg, 50%) as a
colorless oil. 1H-NMR (CDCl3)  0.89-1.13 (m, 28H), 1.19 (s, 9H), 2.40-3.00 (m, 4H), 3.50-4.25 (m, 8H),
5.79 (s, 2H), 6.93 (s, 1H), 7.60 (s, 1H); 13C-NMR (CDCl3)  12.6, 12.7, 13.0, 13.4, 17.0, 17.1, 17.3
(17.27), 17.3 (17.32), 18.8, 19.1, 26.8, 27.1, 33.1, 38.7, 60.2, 65.4, 65.9, 67.7, 71.6, 80.1, 82.2, 83.0,
117.0, 117.9, 137.7, 138.8; HRMS(FABMS) calcd for C30H54N3O7Si2(M+H)+ 624.3500, found 624.3501.
{4-[(2-O-CE--D-ribofuranosyl)methyl]imidazolyl}methyl 2,2-dimethylpropionate (16a)
Compound 15a (53 mg, 0.085 mmol) was dissolved in anhydrous THF (0.5 mL). To the solution were
added Et3N･3HF (50 L, 0.30 mmol) and triethylamine (21 L, 0.15 mmol). After stirring at rt for 1.5 h,
the reaction mixture was evaporated. The residue was subjected to chromatography on a silica gel column
using MeOH-CHCl3 (3:97 to 5:95 v/v) as eluent to give compound 16a (26 mg, 80%) as a colorless oil.
1H-NMR (CDCl3)  1.19 (s, 9H), 2.63 (t, 2H, J = 7.3 Hz), 2.83 (dd, 1H, J = 16.9, 6.0 Hz), 2.95 (dd, 1H, J
= 16.9, 6.0 Hz), 3.56-4.07 (m, 6H), 4.15-4.27 (m, 2H), 5.79 (s, 2H), 6.96 (s, 1H), 7.62 (s, 1H); 13C-NMR
(CDCl3)  19.1, 26.8, 30.5, 38.7, 62.0, 64.9, 67.7, 70.8, 79.7, 81.9, 84.6, 117.5, 138.0, 138.5;
HRMS(FABMS) calcd for C18H28N3O6 (M+H)+ 382.1978, found 382.1981.
[4-(2-O-CE-5-O-DMT--D-ribofuranosyl)methylimidazolyl]methyl 2,2-dimethylpropionate (17a)
Compound 16a (26 mg, 0.068 mmol) was co-evaporated three times with pyridine (1 mL) and
redissolved in dry pyridine (0.3 mL). 95% DMTCl (36 mg, 0.10 mmol), Et3N (14 L, 0.10 mmol), and
DMAP (0.2 mg, 0.0017 mmol) were added to the solution, and the mixture stirred at rt for 20 h. MeOH (2
mL) was added and the mixture stirred for further 5 min. The solvents were removed to leave a residue
that was purified by the column chromatography (NH-silica gel) with CHCl3-benzene (0:100, 1:1, 2:1, to
1
100:0 v/v) as eluent to give the foam of compound 17a (36 mg, 77%). H NMR (CDCl3)  1.16 (s, 9H),
2.64 (td, 2H, J = 5.8, 2.0 Hz), 2.86 (dd, 1H, J = 14.6, 6.6 Hz), 2.96 (dd, 1H, J = 14.6, 5.9 Hz), 3.14 (dd,
1H, J = 10.1, 4.4 Hz), 3.30 (dd, 1H, J = 10.1, 4.0 Hz), 3.68-3.76 (m, 2H), 3.78 (s, 6H), 3.89 (t, 1H, J = 4.9
Hz), 3.92-3.97 (m, 1H), 4.01 (t, 1H, J = 5.3 Hz), 4.21 (q, 1H, J = 4.4 Hz), 5.65 (s, 2H), 6.82 (d, 4H, J =
8.8 Hz), 6.92 (s, 1H), 7.19-7.36 (m, 7H), 7.43 (d, 2H, J = 7.3 Hz), 7.56 (s, 1H) ; 13C NMR (CDCl3)  18.9,
26.8, 32.4, 38.7, 55.2, 64.0, 64.8, 67.9, 71.4, 77.4, 80.5, 82.0, 82.7, 86.0, 113.0, 117.3, 117.5, 138.9, 144.9,
158.4, 177.6; HRMS(FABMS) calcd for C39H46N3O8 (M+H)+ 684.3284, found 684.3284.
[4-{5-O-DMT-2-O-CE-3-O-(2-cyanoethoxy-N,N-diisopropylaminophosphino)--D-ribofuranos-1-yl}-
methylimidazol-1-yl]methyl 2,2-dimethylpropionate (1a)
Compound 17a (36 mg, 0.053 mmol) was dissolved in dry dichloroethane (0.5 mL) and 4,5-DCI (8.0 mg,

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0.064 mmol) and 2-cyanoethyl N,N,N’,N’-tetraisopropylphosphordiamidite (36 L, 0.11 mmol) were
added. The resulting mixture was stirred at 40 °C for 1 h and then evaporated. The residual oil was
subjected to chromatography on NH-silica gel with the solvent system; AcOEt in hexane (0:100, 30:70,
40:60 to 50:50 v/v) to give 1a (28 mg, 60%) as a white foam. 1H NMR (CDCl3)  0.92 (d, 4H, J = 6.6 Hz),
1.04-1.12 (m, 19H), 2.21 (t, 1H, J = 6.6 Hz), 2.49-2.59 (m, 3H), 2.88 (m, 2H), 2.96 (dd, 1H, J = 10.2, 4.2
Hz), 3.17 (dd, 0.5H, J = 10.2, 4.2 Hz), 3.26 (dd, 0.5H, J = 9.9, 3.3Hz), 3.39-3.66 (m, 4H), 3.73 (s, 6H),
3.75-3.83 (m, 1H), 4.01-4.20 (m, 3H), 5.58 (s, 2H), 6.71-6.80 (m, 4H), 6.86 (s, 1H), 7.13-7.27 (m, 7H),
13
7.33-7.38 (m, 2H), 7.49 (s, 1H); C NMR (CDCl3)  18.77, 18.83, 19.94, 20.03, 20.35, 20.42, 24.47,
24.56, 24.68, 26.78, 29.65, 32.42, 38.65, 42.87, 43.03, 43.22, 43.28, 55.12, 57.81, 58.22, 58.45, 63.51,
64.75, 65.03, 67.66, 71.95, 72.17, 72.38, 80.00, 80.03, 81.87, 82.22, 82.50, 82.79, 85.91, 85.99, 112.99,
117.26, 117.55, 117.89, 126.67, 127.71, 127.84, 128.21, 128.27, 130.11, 135.89, 136.04, 136.15, 137.52,
139.14, 144.81, 144.87, 158.38, 158.41, 177.64; 31P NMR (CDCl3)  149.3 and 149.9. HRMS (FABMS:
TEOA+NaCl) calcd for C48H62N5O9P+Na [(M+Na)+] 906.4182, found 906.4191.
{4-[3-(3,5-O-TIPDS--D-ribofuranosyl)propyl]imidazolyl}methyl 2,2-dimethylpropionate (14b)
By the same procedure as used for the preparation of 14a, 11b (118 mg, 0.33 mmol) was converted to
14b (60 mg, 46%) as a colorless oil. 1H-NMR (CDCl3)  0.92-1.13 (m, 28H), 1.98 (s, 9H), 1.50-1.87 (brs,
4H), 2.59 (t, 2H, J = 8.4 Hz), 3.70-3.90 (m, 4H), 4.01 (dd, 1H, J = 11.6, 2.9 Hz), 4.18 (t, 1H, J = 6.7 Hz),
13
5.77 (s, 2H), 6.79 (s, 1H), 7.57 (s, 1H); C-NMR (CDCl3)  12.6, 12.7, 13.2, 13.4, 16.9, 17.0 (17.00),
17.0 (17.03), 17.2, 17.3, 17.4, 25.1, 26.8, 28.1, 33.2, 38.7, 63.0, 67.7, 72.5, 74.5, 77.2, 82.2, 84.0, 115.4,
137.5, 143.5, 177.7; HRMS (FABMS:NBA) calcd for C29H55N2O7Si2 (M+H)+ 599.3547, found 599.3549.
{4-[3-(2-O-CE-3,5-O-TIPDS--D-ribofuranosyl)propyl]imidazolyl}methyl 2,2-dimethylpropionate
(15b)
By the same procedure as used for the preparation of 15a, 14b (121 mg, 0.2 mmol) was converted to 15b
1
(87 mg, 67%) as an oil. IR (film) cm-1: 2200 (CN), 1735 (COO); H-NMR (CDCl3)  0.90-1.13 (brs,
28H), 1.18 (s, 9H), 1.50-1.83 (m, 4H), 2.50-2.69 (m, 4H), 3.55-4.21 (m, 8H), 5.78 (s, 2H), 6.79 (s, 1H),
7.60 (s, 1H); 13C-NMR (CDCl3)  12.5, 12.7, 13.0, 13.4, 16.9, 17.0, 17.2 (17.19), 17.2 (17.23), 17.3, 18.8,
19.3, 24.9, 26.8, 27.3, 27.9, 28.5, 34.0, 38.7, 60.3, 62.3, 65.5, 65.8, 67.6, 72.0, 80.0, 83.2, 90.0, 115.5,
117.7, 137.5, 143.0, 177.6; HRMS (FABMS) calcd for C32H58N3O7Si2 (M+H)+ 652.3813, found
652.3817.
{4-[3-(2-O-CE--D-ribofuranosyl)propyl]imidazolyl}methyl 2,2-dimethylpropionate (16b)

HETEROCYCLES, Vol. 83, No. 9, 2011
2053
By the same procedure as used for the preparation of 16a, 15b (87 mg, 0.13 mmol) was converted to 16b
(47 mg, 85%) as an oil.1H-NMR (CDCl3)  1.18 (s, 9H), 1.58-1.90 (m, 4H), 2.47-2.73 (m, 4H), 3.59-3.70
(m, 2H), 3.71-3.88 (m, 4H), 3.94 (q, 1H, J = 6.0 Hz), 4.16 (t, 1H, J = 6.0 Hz), 5.78 (s, 2H), 6.80 (s, 1H),
13
7.60 (s, 1H). C-NMR (CDCl3)  19.0, 25.3, 26.8, 27.6, 32.6, 38.7, 62.0, 65.0, 67.7, 70.7, 77.2, 81.2,
83.3, 83.5, 84.1, 115.6, 117.6, 137.6, 143.0, 177.7; HRMS (FABMS) calcd for C20H32N3O6 (M+H)+
410.2293, found 410.2293.
{4-[2-(5-DMT-2-O-CE--D-ribofuranosyl)propyl]imidazolyl}methyl 2,2-dimethylpropionate (17b)
By the same procedure as used for the preparation of 17a, 16b (47 mg, 0.11 mmol) was converted to 17b
(68 mg, 84%) as a foam. 1H-NMR (CDCl3)  1.14 (s, 9H), 1.60-1.93 (m, 4H), 2.55-2.68 (m, 4H), 3.14 (dd,
1H, J = 10.3, 4.3 Hz), 3.28 (dd, 1H, J = 11.1, 3.4 Hz), 3.62 (t, 1H, J = 6.0 Hz), 3.68-3.84 (m, 2H), 3.78 (s,
6H), 3.86-3.99 (m, 2H), 4.06 (t, 1H, J = 6.0 Hz), 5.69 (s, 2H), 6.78 (s, 1H), 6.81 (d, 4H, J = 8.6 Hz),
7.15-7.38 (m, 7H), 7.40-7.48 (m, 2H), 7.55 (s, 1H); 13C-NMR (CDCl3)  19.0, 25.3, 26.7, 28.1, 33.6, 38.6,
55.1, 64.2, 65.0, 67.6, 71.6, 77.2, 80.4, 82.9, 83.6, 86.0, 113.0, 115.5, 117.4, 126.6, 127.7, 128.1, 128.3,
130.0, 135.9, 136.0, 137.5, 143.2, 144.9, 149.0, 158.3, 177.6; HRMS (FABMS) calcd for C41H50N3O8
(M+H)+ 712.3597, found 712.3599.
(4-{3-[5-O-DMT-2-O-CE-3-O-(2-cyanoethoxy-N,N-diisopropylaminophosphino)--D-ribofuranos-1-
yl]propyl}imidazol-1-yl)methyl 2,2-dimethylpropionate (1b)
By the same procedure as used for the preparation of 1a, 17b (20 mg, 0.028 mmol) was converted to 1b
(13 mg, 51%) as a colorless oil. Rf (AcOEt) 0.57; 1H-NMR (CDCl3)  1.01 (d, 4H, J = 6.9 Hz), 1.09-1.18
(m, 20H), 1.94-2.08 (m, 4H), 2.27 (t, 1H, J = 6.5 Hz), 2.55-2.70 (m, 5H), 3.07 (dt, 1H, J = 10.3, 3.4 Hz),
3.24 (dd, 0.7H, J = 10.4, 3.9 Hz), 3.32 (dd, 0.3H, J = 10.4, 2.6 Hz), 3.46-4.04 (m, 6H), 3.78 (s, 6H),
4.06-4.17 (m, 1H), 4.17-4.28 (m, 1H), 5.69 (s, 2H), 6.75-6.84 (m, 5H), 7.17-7.39 (m, 7H), 7.39-7.49 (m,
2H), 7.57 (s, 1H); 13C-NMR (CDCl3)  18.9, 19.9, 20.3, 20.4, 24.4, 24.5, 24.6, 24.7, 25.4, 26.8, 28.2, 33.6,
38.7, 42.9, 43.0, 43.1, 43.3, 55.2, 57.8, 58.1, 58.3, 63.9, 65.0, 67.7, 72.3, 72.6, 72.8, 77.2, 80.1, 80.2, 82.6,
83.0, 83.5, 85.9, 86.0, 113.0, 115.4, 117.5, 117.8, 117.9, 126.7, 127.7, 128.2, 128.3, 130.1, 135.9, 136.1,
137.5, 143.5, 144.8, 158.4, 177.7; 31P-NMR (CDCl3)  149.8, 150.7; HRMS (FABMS: TEOA+NaCl)
calcd for C50H66N5O9P+Na [(M+Na)+] 934.4495, found 934.4490.
ACKNOWLEDGEMENTS
We are grateful to Dr. K. Minoura and Ms. M. Fujitake at Osaka University of Pharmaceutical Sciences
for NMR and MS measurements, respectively. This work was supported in part by a Grant-in-Aid for

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Scientific Research [Grant No.21590130 (to S.H.)] from JSPS, 2009-2013. We acknowledge a
Grant-in-Aid for “High Technology Research Center” Project for Private Universities: matching fund
subsidy from MEXT (Ministry of Education, Sciences, Sports and Culture), 2007-2009, Japan. We also
acknowledge the financial support of Cancer Research UK.
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