Chemical synthesis of RNA via 2′-O-cyanoethylated intermediates

Article abstract of DOI:10.1016/j.tet.2007.07.102

It was found that 2′-O-cyanoethyl group could be removed from 2′-O-cyanoethylated ribonucleoside derivatives by treatment with Bu₄NF. This finding was successfully applied to the synthesis of oligoribonucleotides via their 2′-O-cyanoethylated derivatives as key intermediates where a cyanoethyl group was used as the 2′-hydroxyl protecting group. The rate of condensation using this protecting group in the presence of various activators was generally faster than that observed when a TBDMS group was used as the protecting group.

Full text of DOI:10.1016/j.tet.2007.07.102

Tetrahedron 63 (2007) 11195–11203
Chemical synthesis of RNA via 20-O-cyanoethylated intermediates
Hisao Saneyoshi,a,c Kaori Ando,d Kohji Seiob,c and Mitsuo Sekinea,c,
*
aDepartment of Life Science, Tokyo Institute of Technology, Yokohama 226-8501, Japan
bDivision of Collaborative Research for Bioscience and Biotechnology, Frontier Collaborative Research Center,
Yokohama 226-8503, Japan
cCREST, JST (Japan Science and Technology Agency), Nagatsuta, Midoriku, Yokohama 226-8501, Japan
dDepartment of Applied Chemistry, Gifu University, Yanagito, Gifu 501-1193, Japan
Received 14 June 2007; accepted 25 July 2007
Available online 6 August 2007
Abstract—It was found that 20-O-cyanoethyl group could be removed from 20-O-cyanoethylated ribonucleoside derivatives by treatment with
Bu4NF. This finding was successfully applied to the synthesis of oligoribonucleotides via their 20-O-cyanoethylated derivatives as key inter-
mediates where a cyanoethyl group was used as the 20-hydroxyl protecting group. The rate of condensation using this protecting group in the
presence of various activators was generally faster than that observed when a TBDMS group was used as the protecting group.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
Independently, we have recently established a facile method
for the synthesis of 20-O-cyanoethylated RNA derivatives as
a new type of 20-O-modified RNA. 20-O-Cyanoethylated
RNA derivatives could be synthesized using appropriately
protected 20-O-cyanoethylribonucleoside 30-phosphorami-
dite derivatives.23 These monomer building blocks were
conveniently obtained by a series of reactions involving
the Michael reaction of the 20-hydroxyl group of ribo-
nucleoside derivatives with acrylonitrile in t-BuOH in the
presence of Cs2CO3. Interestingly, in the study of the synthe-
sis of 20-O-cyanoethylated RNAs, we observed that the
2-cyanoethyl group remained intact upon treatment with
Et3N$3HF but was immediately cleaved upon treatment
with Bu4NF, the reagent, which has been widely used
in the deprotection of the protecting groups for the 20-O-
hydroxyl groups.23 These unexpected but intriguing results
led us to the idea that the 20-O-cyanoethylated RNAs, which
are useful as 20-O-modified functional RNAs, can be, at the
same time, useful as intermediates for natural-type RNA
synthesis.
Naturally occurring and chemically modified oligoribonu-
cleotides have been synthesized for a wide variety of studies
in molecular biology, gene diagnosis, and gene therapy.1
Recently, new strategies for gene regulation using small
interfering RNA (siRNA)2 have attracted growing interest
among molecular biologists and medical researchers.3 To
respond to the current strong demand for synthetic RNAs
in the biological and medicinal fields, much attention has
been paid to the development of new strategies for the chem-
ical synthesis of RNA.4–10
Because the chemical synthesis of RNA requires an addi-
tional protecting group for the 20-hydroxyl group compared
with that of DNA, several problems have been pointed out.
For example, the necessity of prolonged coupling times
due to the steric hindrance of the 20-protecting group,5 diffi-
culty in deprotection of the 20-protecting group,4c,11 30/20
migration of internucleotidic phosphate groups,12 difficulty
in purification of crude 20-O-protected intermediates,13 and
instability of RNA.14 Various kinds of 20-protecting groups
using silyl,15 acetal,16–19 orthoester,20,21 and o-nitrobenzyl
ether,22 triisopropylsilyloxymethyl,5 tert-butyldithiomethyl,
phenylsulfonylethoxymethyl,9 and p-toluenesulfonyloxy-
ethoxymethyl10 functions have been reported over the past
50 years.
In this paper, we examined the usefulness of 20-O-cyano-
ethylated RNA as a synthetic intermediate for unmodified
RNAs. There are several papers on the 20-O-protecting
groups containing a 2-cyanoethyl group such as the 1-(2-
cyanoethoxy)ethyl group6 and more recently, the (2-cyano-
ethoxy)methyl group,7,8 which can be removed by Bu4NF.19
In comparison to these protecting groups, our 20-O-
cyanoethyl group has two advantages; first, the group can
be introduced directly using very cheap acrylonitrile, and sec-
ond, cyanoethylated RNAs can be used as either functional
RNAs or synthetic intermediates.
Keywords: RNA synthesis; Hydroxyl protecting group; b-Elimination;
siRNA.
* Corresponding author. Tel.: +81 45 924 5706; fax: +81 45 924 5772;
e-mail: msekine@bio.titech.ac.jp
0040–4020/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2007.07.102

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H. Saneyoshi et al. / Tetrahedron 63 (2007) 11195–11203
2. Results and discussion
in 50% yield after 6 days with K2CO3 and 70% yield in 24 h
with DBU. In contrast, the cyanoethyl group of 1a was com-
pletely cleaved within 10 min by treatment with 1 M Bu4NF
in THF. The milder fluoride reagents such as 1 M Bu4NF–
AcOH and 1 M Et3N$3HF were not effective in cleaving
the cyanoethyl group.
2.1. Stability of 20-O-cyanoethyl group toward various
basic reagents
As reported in our previous paper,23 we have synthesized
various 20-O-cyanoethylated ribonucleosides 1a–1d and
a partially 20-O-cyanoethylated UpU 2a (Scheme 1) via
the Michael reaction of 20-O-free ribonucleoside derivatives
with acrylonitrile in the presence of Cs2CO3 in t-BuOH.
Next, we examined the influences of nucleobase structures
and the presence of internucleotidic phosphate on this de-
protection using 1b–d and 2a. As shown in Table 1, the
cytidine (1b), adenosine (1c), and guanosine (1d) deriva-
tives23 were slightly more labile than the uridine derivatives
and cyanoethyl group was removed completely within
5 min.
NH2
N
O
N
N
N
NH
N
1c
O
B
We also checked the stability of dimer 2a against 1 M
Bu4NF and revealed that the removal of cyanoethyl group
took 30 min to give UpU (3a), as shown in Panels A and B
of Figure 1, and in Table 1. Even when the reaction time
was prolonged to 24 h, no decomposition or migration of
the phosphodiester linkage was observed (data not shown).
This result indicated that the lability of 20-O-cyanoethyl de-
pends on the base moieties and the presence of 30-phosphate,
but it can be removed within 30 min. These results clearly
showed the possibility of 20-O-cyanoethyl group as the
20-O-protecting group.
1a
HO
O
O
NH2
N
B =
N
NH
NH2
HO
O
CN
N
N
N
O
1b
1d
Ura
O
HO
O
O
O
O
-O
O
CN
Ura
P
2.2. Effects of 20-hydroxyl protecting group
on condensation
HO
OH
2a
Next, we examined the effect of 20-O-protecting group on
the kinetics of internucleotidic bond formation as shown in
Scheme 2. Generally, 20-O-protecting groups significantly
affect the coupling efficiency because of their own steric
effect. The effect of 20-O-protecting group on the coupling
reaction was studied using the 20-O-cyanoethyluridine
30-phosphoramidite derivatives 4a and the commercially
available 20-O-(tert-butyldimethylsilyl)uridine 30-phosphor-
amidite derivatives 5.
Scheme 1. Examples of the 20-O-cyanoethylnucleoside derivatives.23
Initially, we checked the stability of the uridine derivative 1a
toward various basic conditions using TLC analysis. First of
all, the stability toward K2CO3–MeOH solution used for
oligonucleotide synthesis and 0.5 M DBU as an example
of highly basic amines was examined. As a result, the cya-
noethyl group of 1a was cleaved very slowly to give uridine
In this study, we have chosen 1H-tetrazole,24,25 5-ben-
zylthio-1H-tetrazole (BnS-tet),24 benzimidazolium triflate
(BIT),26,27N-phenylimidazolium triflate (PhIMT),26 2-
sulfobenzimide-N-methylimidazolium salt (SBIM),28 which
have been widely used as activators of the P–N bond of
phosphoramidite building blocks. The reaction of 20,30-
O,2-N-triacetylguanosine (6) with 4a or 5 in the presence
of these activators was monitored by 31P NMR. The coupling
efficiency was calculated by the amount of the phosphite
Table 1. Stability of the cyanoethyl group of 1a–1d and 2a
Compound Conditions
Results
1a
1a
1a
1a
0.05 M K2CO3–MeOH
0.5 M DBU–CH3CN
1 M Bu4NF–THF
50% deprotected (6 days)
70% deprotected (24 h)
100% deprotected (10 min)
1 M Bu4NF–AcOH–THF 24 h stable
1a
1b–1d
2a
1 M Et3N$3HF–THF
1 M Bu4NF–THF
1 M Bu4NF–THF
24 h stable
100% deprotected (5 min)
100% deprotected (30 min)
Figure 1. Reversed-phase HPLC profiles of UcepU (2a) and the mixture obtained after removal of 2-cyanoethyl group from 2a by treatment with Bu4NF. Panel
A: purified 2a; Panel B: crude UpU (3a) after decyanoethylation.

H. Saneyoshi et al. / Tetrahedron 63 (2007) 11195–11203
11197
O
N
NH
Ura
DMTrO
ECO
N
N
NHAc
O
O
HO
O
O
N
O
P
O R1
N
N
activator
Ura
AcO
OAc
NH
NHAc
DMTrO
ECO
O
6
O
O
O R1
P
N(iPr)2
AcO
OAc
4a: R1 = 2-cyanoethyl
7: R1 = 2-cyanoethyl
5: R1 = t-butyldimethylsilyl
8: R1 = t-butyldimethylsilyl
activator
O
H
N
Ph
N
CH3
N
H
N
H
N
BnS
-OTf
-OTf
N
N
N-
S
N
N
N
H
BIT
N
N
N
H
N
O
O
H
PhIMT
BnSTet
Tet
SBIM
Scheme 2. Effects of the protecting groups on the coupling reaction at the 30-reaction site.
intermediate 7 or 8. The results obtained when 1H-tetrazole
and 5-benzylthio-1H-tetrazole were used are shown in
Figure 2. Panel A shows the results of the comparison of
the condensation of the TBDMS building unit 5 in aceto-
nitrile in the presence of 1H-tetrazole with that of the cya-
noethyl building unit 4a. Apparently, the cyanoethyl
derivative was activated more quickly than the TBDMS
unit. The same is true for the use of 5-benzylthio-1H-
tetrazole, as shown in Panel B. Similar experiments were
performed using other activators described above and the
Figure 3. Coupling efficiency in the synthesis of 7 and 8 using various
activators. The coupling yields after 3 min were estimated by 31P NMR.
results are summarized in Figure 3 as the percentage forma-
tion of dimers 7 and 8 within 3 min. In all cases, the cya-
noethyl building block proved superior to the one having
a TBDMS group in terms of the kinetics of phosphite triester
bond formation.
2.3. Deprotection of cyanoethyl group from XcepU
(2b–2d: X=C, A, G) using Bu4NF
To examine if cyanoethyl group could be removed at the
level of oligoribonucleotides, the dimers XcepU (2b–2d:
X¼C, A, and G) were synthesized using the previously
reported phosphoramidites 4b–4d by solid-phase synthesis
using CPG, as shown in Scheme 3, and treated with 1 M
Bu4NF solutions to remove the 20-O-cyanoethyl group.
The deprotection of AcepU (2c) and GcepU (2d) was tested
(Fig. 4). In this case, the fully protected dimers, which were
Figure 2. Effects of TBDMS and cyanoethyl groups as 20-protecting groups
on the coupling efficiency in the synthesis of 7 and 8. Panel A: 3 equiv of
1H-tetrazole was used as the activator; Panel B: 3 equiv of 5-benzylthio-
1H-tetrazole was used as the activator.
synthesized on CPG supports were first treated with NH3–
AcONH4
23
to cleave the protecting groups on the

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H. Saneyoshi et al. / Tetrahedron 63 (2007) 11195–11203
B2
O
HO
O
B2
O
DMTrO
aq.NH3-AcONH4
(10:1, w/w)
0.1 M 0.25 M
O
capping
Ura
HO
4b-4d, BTT
oxidation
O
O
O
O
2b: B2 = Cyt
2c: B2 = Ade
2d: B2 = Gua
CN
Ura
CH3CN
10 min
O
O
O
P
O
CN
Ura
-
O
O
O
O
P
ECO
O
, Ac
HN
OH OH
O
O
O
O
CPG
, Ac
HN
O
CPG
B2
HO
O
O
1 M Bu4NF/THF or 1M Bu4N-n-PrNH2 (20:1,v/v)
O
OH
3b: B2 = Cyt
P
H
Ura
3b': B2 = 4-N-cyanoethylcytosine
3c: B2 = Ade
-
O
O
O
3d: B2 = Gua
OH OH
NMe2
B1
Ac
N
N
N
4d: B1
O
=
DMTrO
O
N
N
4c: B1
=
N
NH
N
4b: B1
=
N
O
O
CN
O
N
N
N
NMe2
(i-Pr)2N
P
N
OCE
Scheme 3. Solid-phase synthesis of UcepU, CcepU, AcepU, and GcepU.
Figure 4. Reversed-phase HPLC profiles of ApU 3c (Panel A), GpU 3d (Panel B), and CpU 3b (Panel C) obtained by treatment of AcepU, GcepU, and CcepU,
respectively, with Bu4NF. Panel D: 3b obtained in the presence of PrNH2.
nucleobase, phosphates, and the linker between 30 terminal
nucleotide residues and CPG supports. As described in the
previous paper, 20-O-cyanoethyl group remained intact in
this buffered aqueous ammonia solution. After this ammonia
treatment, the dimers were subjected to 1 M Bu4NF solution.
Similarly, it was confirmed that AcepU (2c) and GcepU (2d)
were converted successfully to ApU (3c) and GpU (3d),
respectively.
cyanoethylated species 3b0 of the cytosine moiety, in 32%
yield. This result is explained in terms of the alkylation of
once-formed acrylonitrile on the cytosine residue under
basic conditions (Fig. 4C).
The mechanism was supported by the fact that the addition
of n-PrNH2, which trapped the acrylonitrile effectively re-
duced the side reaction. Thus, when 1 M Bu4NF–n-PrNH2
(20:1, v/v) was used instead of 1 M Bu4NF, the N-cyano-
ethylated product 3b0 could not be observed as shown in
Figure 4D. Side reactions on the cytosine moiety concerning
However, it was observed that treatment of CcepU
with Bu4NF was accompanied by a byproduct, a 4-N-

H. Saneyoshi et al. / Tetrahedron 63 (2007) 11195–11203
11199
the N-cyanoethylation have already been reported by some
research groups.29
methylene (dmf) group of the adenine base did not have
sufficient stability during the oligonucleotide synthesis.
Therefore, the protecting group for the exo-amino group of
adenosine was changed to a phenoxyacetyl group. A new
adenosine building block 4c0 was synthesized, as shown in
Scheme 4. The N-dmf protected derivative 924 was converted
to the N-unprotected adenosine derivative 10 by the action of
hydrazine monohydratewithout loss of the cyanoethyl group.
Compound 10 was allowed to react successively with
phenoxyacetic anhydride, Et3N$3HF, and DMTrCl to give
the 30-free derivative 11. The building block 5c0 was synthe-
sized by phosphitylation of 11 in the usual manner.
2.4. Synthesis of longer RNA fragments using
20-O-cyanoethyl intermediates
We first studied the synthesis of a RNA fragment of U30.
A 20-O-cyanoethylated intermediate of oligouridylate was
synthesized using the phosphoramidite 4a and BnS-Tet as
an activator. Unexpectedly, a significant amount of a 29mer
was observed with HPLC (Fig. 5A) probably due to incom-
plete detritylation process repeated at each coupling cycle.
As expected, this problem was resolved by changing detrity-
lation time from 15 s (5 sꢀ3) to 30 s (5 sꢀ6), as shown in
Figure 5B. Subsequently, the cyanoethyl group was removed
by treatment with 1 M Bu4NF–THF to givethe desired 30mer
whose structure was confirmed by MALDI TOF mass analy-
ses and enzymatic digestion as shown in Figure 6C.
For further demonstration of the chemical synthesis of RNAs
using our strategy, an RNA 21mer (GCUUUCAAAAUGAU
CUCACUG) known as an antisense strand of miR-
bantam,30 one of the microRNAs, was synthesized using
our improved adenosine building block and BnS-Tet. It
should be noted that a 20-O-cyanoethylated oligoribonucleo-
tide intermediate with a 50-terminal DMTr group could be
separated from the failure sequences without the DMTr
When we tried to synthesize longer oligoribonucleotides
having mixed sequences, we noticed that the dimethylamino-
Figure 5. Anion-exchange HPLC profiles of crude (Uce)30 obtained using a detritylation time of 15 s (Panel A) and prolonged time of 30 s (Panel B).
Figure 6. Anion-exchange HPLC profiles of crude U30 (Panel A), purified U30 (Panel B) and reversed-phase HPLC profile of enzymatic digestion of U30 with
snake venom phosphodiesterase and phosphatase (Panel C).

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H. Saneyoshi et al. / Tetrahedron 63 (2007) 11195–11203
NHPac
2.5 equiv
CH3
X
N
1.5 equiv
(i-Pr)2NEt
1.1 equiv
(i-Pr)2NP(OCE)Cl
NHPac
N
N
N
N
N
N
N
2.0 equiv
N
N
N
N
N
1)
Pac2O
DMTrO
CEO
N
O
O
O
DMTrO
CH3CN, 2 h
2) 3.5 equiv
Si
O
O
CH2Cl2
2 h
1.8 equiv
Et3N·3HF NEt3
THF, 1 h
1 equiv
O
O
CN
P
O
O
Si
OH
11
O
CN
N(CH3)2
CN
N(i-Pr)2
4c'
3) DMTrCl, Pyr, 2 h
X =
X =
9:
N
67%
85%
6 equiv
NH2NH2
/CH3CN
10:
NH2
93%
Scheme 4. Synthesis of N-phenoxyacetyladenosine phosphoramidite derivative.
Figure 7. Anion-exchange HPLC profiles of the 20-O-cyanoetylated RNA 21mer (Panel A) and the decyanoethylated RNA 21mer (Panel B) obtained using BnS-
Tet and reversed-phase HPLC profile of enzymatic digestion of the RNA 21mer with snake venom phosphodiesterase and phosphatase.
group using a simple reversed-phase cartridge column be-
cause the very polar cyanoethyl group did not interact with
C18 column surfaces.
a protecting group for the 20-hydroxyl group. When
Bu4NF was used as a reagent for removal of the cyanoethyl
group, it was immediately deprotected. The present finding
that the cyanoethyl group can be removed by treatment
with Bu4NF would be useful for the synthesis of naturally
occurring products since this protecting group can be easily
introduced by reaction of alcoholic functions with acryloni-
trile in t-BuOH in the presence of Cs2CO3 as reported
previously.23 These results clearly demonstrated that the
20-O-cyanoethylated RNA previously reported as a new
20-O-modified functional RNA could also be used as a
synthetic intermediate for natural-type RNA. Further devel-
opment of the cyanoethyl protection strategy for RNA syn-
thesis will be reported in the near future to achieve the
synthesis of much longer and purer natural-type RNAs.
We are now studying the mechanism of elimination of the
cyanoethyl group by Bu4NF using theoretical calculations.
These results will be also reported in the near future.
At this stage, the desired 20-O-cyanoethylated oligoribonu-
cleotide could be obtained as the main peak in HPLC
(Fig. 7A). Treatment of this intermediate with 1 M Bu4NF
in the presence of n-propylamine resulted in fully
deprotected RNA, isolated in 34% yield (Fig. 7B). The
structure of this product was characterized by MALDI
TOF mass analyses and enzymatic digestion analysis as
shown in Figure 7C.
3. Conclusion
In this study, we developed a new method for the synthesis
of oligoribonucleotides using the cyanoethyl group as

H. Saneyoshi et al. / Tetrahedron 63 (2007) 11195–11203
11201
4. Experimental section
4.3. 6-N-Phenoxyacetyl-50-O-dimethoxytrityl-20-O-
cyanoethyladenosine (11)
4.1. General
Compound 10 (563 mg, 1.0 mmol) was dissolved in anhy-
drous CH3CN (5 mL). To the solution were added phenoxy-
acetic anhydride (573 mg, 2 mmol) and N-methylimidazole
(0.2 mL, 2.5 mmol). The mixture was stirred at room tem-
perature for 2 h and evaporated. The residue was dissolved
in THF–MeOH–Et3N–H2O (5.5 mL, 2:2:1:0.5, v/v). The so-
lution was stirred at room temperature for 30 min. The solu-
tion was evaporated in vacuo and diluted with CHCl3. The
solution was washed with brine, dried over Na2SO4 and fil-
tered. The solution was evaporated in vacuo. The residue was
dissolved in anhydrous THF (5 mL). To the solution were
added Et3N$3HF (570 mL, 3.5 mmol) and Et3N (246 mL,
1.8 mmol). The mixture was stirred at room temperature
for 1 h. The solution was evaporated. The residue was chro-
matographed on a silica gel column (CHCl3–MeOH, 100:0
to 95:5, v/v). The residue was coevaporated five times with
anhydrous pyridine and dissolved in anhydrous pyridine
(100 mL). To the solution was added DMTrCl (338 mg,
1 mmol). The mixture was stirred at room temperature for
2 h. The reaction was quenched with H2O and evaporated.
The residue was diluted with ethyl acetate and washed
with brine and aq NaHCO3. The solution was dried over
Na2SO4 and evaporated. The residue was chromatographed
on a silica gel column (CHCl3–hexane, 1:1 to 1:0, v/v) to
give 11 (505 mg, 67%). 1H NMR (CDCl3, 500 MHz)
d 2.64–2.75 (3H, m), 3.43 (1H, dd, J¼4.15, 10.74), 3.55
(1H, dd, J¼3.17, 10.74), 3.78 (6H, s), 3.91–3.95 (1H, m),
4.02–4.07 (1H, m), 4.23–4.25 (1H, m), 4.52–4.55 (1H, m),
4.63 (1H, dd, J¼3.17, 4.59), 6.19 (1H, d, J¼3.17), 6.80–
7.44 (18H, m), 8.28 (1H, s), 8.72 (1H, s), 9.49 (1H, br);
13C NMR (CDCl3, 500 MHz) d 19.2, 55.4, 62.7, 65.9,
68.2, 69.9, 82.2, 83.8, 86.8, 87.5 113.4, 115.1, 117.4,
122.6, 123.4, 127.1, 128.1, 128.2, 128.3, 129.2, 130.0,
135.6, 135.7, 142.1, 144.6, 148.6, 151.3, 152.7, 157.1,
158.7, 166.8; HRMS calcd for C42H40N6O8 (M+H+)
757.2986, found 757.2947.
1H NMR spectra were recorded at 500 MHz and the chemi-
cal shifts were measured from the solvent peak as an internal
standard (in CDCl3) or HDO (in D2O) as an external stan-
dard. 13C NMR spectra were recorded at 500 MHz and the
chemical shifts were measured from the solvent peak and di-
oxane (D2O) as an internal standard. 31P NMR spectra were
recorded at 109 MHz and the chemical shifts were measured
from 85% H3PO4 as an external standard. Pyridine was dis-
tilled twice from p-toluenesulfonylchloride and CaH2 after
being reluxed for several hours and stored over molecular
˚
sieves 4 A. Triethylamine was distilled from CaH2 and
˚
stored over molecular sieves 4 A. TLC was performed on
Merck Kieseigel 60 F254 precoated glass plates. Column
chromatography was performed with silica gel C-200,
C-300 (Wako Co. Ltd), 60N (Kanto Chemical, Co., Inc.), and
NH (Fuji Silysia Chemical Ltd), and a minipump for a gold-
fish bowl was conveniently used to attain sufficient pressure
for rapid chromatographic separation. The solid-phase syn-
thesis of oligonucleotides was carried out on a DNA/RNA
synthesizer (ABI 392) using normal phosphoramidite proto-
col. HPLC was performed using the following systems. Sys-
tem A: reversed-phase HPLC was performed on a Waters
Aliance system with a Waters 3D UV detector and a mBonda-
˚
sphere 5m C18 100 A column (Waters, 3.9ꢀ150 mm). A lin-
ear gradient (0–30% or 0–10%) starting from 0.1 M
NH4OAc and applying CH3CN was used at a flow rate of
1 mL/min for 30 min at 50 ꢁC. System B: anion-exchange
HPLC was performed on a Waters Aliance system with a
Waters 3D UV detector and a Gen-Pak FAX column (Waters,
4.6ꢀ100 mm). A linear gradient (0–60%, A; 10–80%, C, C0)
starting from 25 mM sodium phosphate buffer (pH 6.0) and
applying 25 mM sodium phosphate buffer (pH 6.0) contain-
ing 1 M NaCl (pH 6.0) was used at a flow rate of 1 mL/min
for 50 min (A) or 45 min at 50 ꢁC (B, C) or 80 ꢁC (C0). High
resolution ESI mass spectrometry was performed by use of
a Mariner (PerSeptive Biosystems, Inc.). The MALDI
TOF mass spectrometry was carried out by use of a Voyger
RP (PerSeptive Biosystems, Inc.).
4.4. 6-N-Phenoxyacetyl-50-O-dimethoxytrityl-20-O-
cyanoethyl adenosine 30-(2-cyanoethyl N,N-diisopropyl-
phosphoramidite) (4c0)
4.2. 20-O-(2-Cyanoethyl)-30,50-O-(1,1,3,3-tetraisopropyl-
disiloxane-1,3-diyl)adenosine (10)
Compound 11 (412 mg, 0.54 mmol) was coevaporated five
times with anhydrous toluene and replaced into argon atmo-
sphere and dissolved in anhydrous CH2Cl2 (4.4 mL). To the
solution were added ethyldiisopropylamine (142 mg, 0.82 mmol)
and chloro(2-cyanoethyl)(N,N-diisopropylamino)phosphine
(142 mg, 0.60 mmol) in CH2Cl2. The mixture was stirred at
room temperature for 2 h and diluted with Et2O (30 mL).
The solution was washed with brine three times, satd aq
NaHCO3, and finally brine. The solution was dried over
Na2SO4 and filtered. The solution was evaporated in vacuo.
The residue was chromatographed on a silica gel column
(hexane–EtOAc, 2:1 to 1:1 to 1:2, v/v) to give 4c0
Compound 923 (618 mg, 1.00 mmol) was dissolved in anhy-
drous CH3CN (5 mL). To the solution was added NH2NH2
monohydrate (291 mL, 6.00 mmol). After being vigorously
stirred at room temperature for 2 h, the mixture was diluted
with ethyl acetate. The mixture was washed six times with
brine and dried over Na2SO4. The mixture was evaporated
in vacuo. The residue was precipitated from diisopropyl
ether and CHCl3 to give compound 10 as a white solid
1
(521 mg, 93%). H NMR (CDCl3, 500 MHz) d 0.98–1.13
(28H, m), 2.72–2.75 (2H, m), 3.95–4.04 (2H, m), 4.15–
4.18 (1H, m), 4.21–4.26 (2H, m), 4.30 (1H, d, J¼4.40),
4.75 (1H, dd, J¼4.40, 9.28), 5.67 (2H, br), 5.99 (1H, s),
8.10 (1H, s), 8.30 (1H, s); 13C NMR (CDCl3, 500 MHz)
d 12.7, 12.9, 13.0, 13.5, 17.0, 17.1, 17.2, 17.3, 17.4, 17.6,
19.3, 59.7, 66.2, 69.7, 81.3, 82.6, 88.6, 117.7, 120.3,
139.0, 149.0, 153.2, 155.6; HRMS calcd for C25H42N6O5Si2
(M+H+) 563.2833, found 563.2787.
1
(440 mg, 85%). H NMR (CDCl3, 500 MHz) d 1.06–1.19
(12H, m), 2.59–2.65 (4H, m), 3.34–3.37 (1H, m), 3.55–
3.76 (4H, m), 3.77–4.07 (10H, m), 4.37–4.38 (1H, m),
4.59–4.63 (1H, m), 4.82–4.87 (3H, m), 6.16–6.17 (1H, m),
6.78–6.82 (4H, m), 7.03–7.06 (3H, m), 7.19–7.43 (11H,
m), 8.26–8.29 (1H, m), 8.70–8.71 (1H, m), 9.51 (1H, br);
13C NMR (CDCl3, 500 MHz) d 19.0, 20.2, 20.3, 20.5,

11202
H. Saneyoshi et al. / Tetrahedron 63 (2007) 11195–11203
24.6, 24.7, 24.8, 24.9, 43.3, 43.4, 43.5, 55.3, 57.9, 58.1, 58.3,
58.4, 62.6, 65.5, 65.7, 68.2, 71.0, 71.1, 80.7, 81.1, 83.3, 83.5,
83.6, 86.7, 87.7, 87.8, 113.2, 114.9, 115.0, 117.2, 117.5,
117.6, 117.9, 122.1, 122.4, 123.4, 127.0, 127.1, 128.0,
128.3, 129.8, 129.9, 130.2, 135.6, 135.7, 142.5, 142.6,
144.5, 144.6, 148.5, 151.5, 152.6, 157.1, 158.7, 166.8; 31P
lyophilized. The residue was dissolved in 0.1 M NH4OAc
buffer (10 mL). The solution was subjected to a C18-car-
tridge. After the cartridge was washed with 0.1 M NH4OAc
buffer (10 mL), H2O (10 mL), elution was performed with
20% CH3CN–H2O (10 mL). The fractions containing the
product were combined and lyophilized: U30, 15%, MALDI
TOF mass calcd for C270H332N60O238P29 9119.81, found
NMR (CDCl3)
d 150.8, 150.7; HRMS calcd for
C51H57N8O9P (M+H+) 957.4064, found 957.4066.
9119.32;
GCUUUCAAAAUGAUCUCACUG,
34%
MALDI TOF mass calcd for C198H247N74O146P20 6615.89,
found 6620.11.
4.5. Steric effects of the 20-O-protecting group
in the coupling reaction
20,30-O,2-N-Triacetylguanosine (6)31 (25 mg, 0.06 mmol) and
a protected phosphoramidite 4a or 5 (0.03 mmol) were co-
evaporated three times each with anhydrous pyridine, anhy-
drous toluene, and anhydrous CH3CN under reduced
pressure. The dried mixture was dissolved in CD3CN
(1 mL or 840 mL, 800 mL), and to the solution was added a
0.25 M solution of 1H-tetrazole, 5-benzylthio-1H-tetrazole,
0.2 M benzimidazolium triflate, N-phenylimidazolium tri-
flate, or o-sulphobenzimide N-methylimidazolium salt in
CH3CN. The reaction was monitored by 31P NMR. These
results are summarized in Figures 2 and 3.
Acknowledgements
The authors thank Dr. Akihiro Ohkubo and Dr. Itaru
Okamoto for their helpful discussions throughout this study.
This work was supported by a Grant from CREST of JST
(Japan Science and Technology Agency). This work was
supported in part by a grant of the Genome Network and
by the COE21 project.
Supplementary data
4.6. Oligonucleotide synthesis
The 1H, 13C, and 31P NMR data of all new products. Supple-
mentary data associated with this article can be found in the
online version, at doi:10.1016/j.tet.2007.07.102.
Oligoribonucleotides were synthesized on an Applied Bio-
systems 392 DNA synthesizer on a 1 mmol scale, using
20-O-cyanoethylated ribonucleoside 30-phosphoramidite
building blocks 4a, 4b, 4c0, and 4d. A 0.1 M solution of
each 20-O-cyanoethylated ribonucleoside 30-phosphorami-
dite was used and the time for condensation was set to be
10 min. A 0.25 M 5-benzylthio-1H-tetrazole in acetonitrile
was used. Release of the oligonucleotide from the resin
and deprotection of the protecting groups of the phosphate
and base moieties were carried out by use of NH4OH–
NH4OAc (10:1, w/w, 2 mL, System A) or n-PrNH2–THF
(1:1, v/v, 2 mL, System B) at room temperature for 60–
120 min (CpU, ApU, GpU, and U30, Method A) and for
15 h (RNA21mer: GCUUUCAAAAUGAUCUCACUG
Method B). The mixture was diluted in 0.1 M NH4OAc
buffer (10 mL) and the solution was subjected to a C18-
cartridge (DMTr-On). The cartridge was washed with 10–
20% CH3CN–0.1 M NH4OAc buffer (10 mL), detritylation
with 2% aq TFA (5 mLꢀ3), washed with 0.1 M NH4OAc
buffer (10 mL), H2O (10 mL) and elution with 20%
CH3CN–H2O (10 mL) was done. The eluate was lyophi-
lized. The powder of the 20-O-cyanoethylated oligomer
thus obtained was treated with a 1 M solution of TBAF in
THF–n-PrNH2 (20:1, v/v, 210 mL for 2mer, 630 mL for
12mer, 2 mL for 21mer) at room temperature for 0.5 h
(2mer), 12 h (12mer, U30mer), 15 h (21mer). The mixture
was diluted with 0.1 M ammonium acetate buffer (10–
20 mL), and THF component of the solution was evaporated
under reduced pressure. The solution was subjected to a cat-
ion exchange cartridge (OASISMCX cartridge) pre-washed
with CH3CN and 0.1 M NH4OAc buffer, passed through it.
Further purification was carried out with C18-cartridge,
which was washed with 0.1 M NH4OAc buffer and eluted
with 20% CH3CN–H2O (10 mL). The eluate was lyophi-
lized. The residue was dissolved in H2O and purified with
anion-exchange HPLC. The fractions containing the product
were combined. For desalt, the obtained fraction was
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