A new RNA synthetic method with a 2′-O-(2-cyanoethoxymethyl) protecting group

Article abstract of DOI:10.1021/ol051151f

(Chemical Equation Presented) A novel method for the synthesis of RNA oligomers with 2-cyanoethoxymethyl (CEM) as the 2′-hydroxyl protecting group has been developed. The new method allows the synthesis of oligoribonucleotides with an efficiency and final

Full text of DOI:10.1021/ol051151f

ORGANIC
LETTERS
2005
Vol. 7, No. 16
3477-3480
A New RNA Synthetic Method with a
-O-(2-Cyanoethoxymethyl) Protecting
2′
Group
Tadaaki Ohgi,* Yutaka Masutomi, Kouichi Ishiyama, Hidetoshi Kitagawa,
Yoshinobu Shiba, and Junichi Yano
DiscoVery Research Laboratories, Nippon Shinyaku Co. Ltd., 3-14-1 Sakura,
Tsukuba City, Ibaraki 305-0003, Japan
t.ohgi@po.nippon-shinyaku.co.jp
Received May 17, 2005
ABSTRACT
A novel method for the synthesis of RNA oligomers with 2-cyanoethoxymethyl (CEM) as the 2
′
-hydroxyl protecting group has been developed.
The new method allows the synthesis of oligoribonucleotides with an efficiency and final purity comparable to that obtained in DNA synthesis.
The recent discovery of RNA interference (RNAi) has
opened new windows on the biology and potential therapeutic
applications of short RNA molecules such as small interfer-
ing RNA (siRNA).1,2 This in turn has led to increased interest
in the chemical synthesis of RNA. From the outset of
research on RNA synthesis, it has been appreciated that the
single most demanding problem is the selection of an
appropriate protecting group for the 2′-hydroxyl function.
This protecting group must be stable throughout the solid-
phase synthetic cycle, yet it must be readily removable under
conditions under which the final RNA product is completely
stable.3 The conventional synthetic strategy is to use a 4,4′-
dimethoxytrityl (DMTr) group or a silyl derivative4 to protect
the 5′-hydroxyl position and then to select a 2′-hydroxyl
protecting group compatible with this, such as a fluoride-
cleavable silyl ether,5 a photolabile moiety,6 or an acid-
cleavable acetal.7 In particular, tert-butyldimethylsilyl
(TBDMS), a silyl ether group, is a popular protecting group
whose amidite, which is used in solid-phase synthesis, has
been commercially available for several years.
Although the TBDMS method gives RNA of modest purity
in modest yield, it is not a robust method in the sense that
both the purity and the yield are sensitive to small variations
in the experimental conditions. Furthermore, the TBDMS
group is associated with relatively long coupling times8a and
insufficiently high coupling yields,8b though improvements
(5) (a) Ogilvie, K. K.; Beaucage, S. L.; Schifman, A. L.; Theriault, N.
Y.; Sadana, K. L. Can. J. Chem. 1978, 56, 2768-2780. (b) Usman, N.;
Ogilvie, K. K.; Jiang, M.-Y.; Cedergren, R. J. J. Am. Chem. Soc. 1987,
109, 7845-7854.
(6) (a) Tanaka, T.; Tamatsukuri, S.; Ikehara, M. Nucleic Acids Res. 1986,
14, 6265-6279. (b) Hayes, J. A.; Brunden, M. J.; Gilham, P. T.; Gough,
G. R. Tetrahedron Lett. 1985, 26, 2407-2410. (c) Pitsch, S.; Weiss, P. A.;
Wu, X.; Ackermann, D.; Honegger, T. HelV. Chim. Acta 1999, 82, 1753-
1761.
(7) (a) Griffin, B. E.; Reese, C. B. Tetrahedron Lett. 1964, 5, 2925-
2931. (b) Rao, M. V.; Reese, C. B.; Schehlmann, V.; Yu, P. S. J. Chem.
Soc., Perkin Trans. 1 1993, 43-55.
(8) (a) Gough, G. R.; Miller, T. J.; Mantick, N. A. Tetrahedron Lett.
1996, 37, 981-982. (b) Welz, R.; Mu¨ller, S. Tetrahedron Lett. 2002, 43,
795-797.
(1) Fire, A.; Xu, S.; Montgomery, M. K.; Kostas, S. A.; Driver, S. E.;
Mello C. C. Nature 1998, 391, 806-811.
(2) Elbashir, S. M.; Harborth, J.; Lendeckel, W.; Yalcin, A.; Weber, K.;
Tuschl, T. Nature 2001, 411, 494-498.
(3) Reese, C. B. Tetrahedron 2002, 58, 8893-8920.
(4) Scaringe, S. A.; Wincott, F. E.; Caruthers, M. H. J. Am. Chem. Soc.
1998, 120, 11820-11821.
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© 2005 American Chemical Society
Published on Web 07/07/2005

based on the use of more powerful activation reagents or
sterically less demanding 2′-hydroxyl protecting groups have
been proposed.8b Recently, to resolve these problems, two
new protecting groups, bis(2-acetoxyethyloxy)methyl (ACE)4
and triisopropylsilyloxymethyl (TOM),9 were developed.
However, though these protecting groups represent major
improvements in the synthesis of RNA oligonucleotides,10
they still leave a certain amount to be desired. Thus, the
synthesis of ACE-amidites is relatively complex3 (though
they are now commercially available), and automated
synthesizers require special modification for use with them
because of the incompatibility of glass materials with
triethylamine trihydrofluoride, the 5′-desilylation reagent.
TOM-protected oligonucleotides, meanwhile, are not readily
amenable to routine analysis and purification by HPLC
because of the hydrophobic nature of the silyl group.
Pfleiderer and co-workers11 investigated a wide variety of
acid-cleavable acetal derivatives as protecting groups for the
2′-hydroxyl function. In particular, they identified benzyl
acetals as promising protecting groups. They also observed
that acetal derivatives with electron-withdrawing substituents,
for example, 1-(2-cyanoethoxy)ethyl, were cleaved by fluo-
ride anion under aprotic conditions in a side reaction during
the synthesis of the 2′-O-protected nucleoside, though both
these workers and Wada’s group12 have described a way to
suppress this side reaction by the addition of AcOH.
Furthermore, Gough et al.8 have introduced the fluoride-
cleavable 4-nitrobenzyloxymethyl protecting group, which
allows analysis and purification of the protected oligonucle-
otides by HPLC.
In our search for a synthetic method that would exclude
the side reaction, minimize steric hindrance, avoid the
generation of asymmetric centers, and allow ready cleavage
of the protecting group from the final product by fluoride
anion, we decided to focus on the introduction of electron-
withdrawing substituents into formaldehyde acetal type
protecting groups. This approach led to the development of
a novel protecting group, 2-cyanoethoxymethyl (CEM). In
the present communication, we report on the use of CEM-
amidite chemistry to synthesize homo- and mixed-base RNA
oligomers up to 55 bases in length, and we show that the
RNA product is obtained in high yield and high purity. Our
syntheses proceeded about as readily and efficiently as DNA
synthesis, demonstrating the potential usefulness of the CEM
protecting group in solid-phase RNA synthesis.
Scheme 1
derivatization was carried out via the 2′,3′-O-dibutylstan-
nylidene intermediate,9 which was treated with the novel
alkylating agent 2-cyanoethyl chloromethyl ether (6; CEM-
Cl) to give a mixture of the 3′-O- and 2′-O-CEM derivatives
(3a-d and 4a-d). By using 1.0-1.3 equiv of 6, the desired
compound 4a-d was obtained in 29-51% yield, and for
all compounds except 4b the 2′-isomer 4 was obtained in
higher yield than the undesired 3′-isomer 3 (see Supporting
Information). With G as the base, the ratio of the 2′-isomer
(4d) to the 3′-isomer (3d) was the highest, at 3.0. After
isolating the 2′-O-CEM derivative 4a-d by silica gel column
chromatography, we carried out phosphitylation of the 3′-
hydroxyl group to obtain the corresponding amidite 5a-d.
The yields of the amidites have not yet been optimized, and
this route is still under investigation to try to improve the
regioselectivity of the alkylation reaction. CEM-Cl itself was
prepared via the Pummerer reaction. Briefly, 3-hydroxypro-
pionitrile and dimethyl sulfoxide were reacted to give the
methylthiomethyl ether, which was then treated with sulfuryl
chloride to give CEM-Cl. Both steps proceeded in reasonable
yield (70-85%; see Supporting Information).
Turning to the synthesis of oligomers, we initially syn-
thesized a uridine homo-oligomer, U40 (7), by our CEM
method. Commercially available 2′- or 3′-O-benzoyl-rU
controlled-pore glass (CPG) was used as the solid support
and 5-ethylthiotetrazole as the activator. Solid-phase syn-
thesis was carried out on an Applied Biosystems Expedite
model 8909 DNA synthesizer on a 1-µmol scale with a
coupling time of 150 s. Cleavage from the resin and
deprotection of the phosphate moiety were carried out by
treatment with concentrated ammonia in EtOH at 40 °C for
4 h. At this stage, the 2′-O-CEM-protected U40 can be
monitored by HPLC (Figure 1A), because the 2′-O-CEM
protecting group is relatively hydrophilic compared with the
TBDMS group. (We initially tried MeNH2 in EtOH/water
for the cleavage/deprotection step, because this is the
deprotecting reagent usually used. However, under these
conditions we observed substantial loss of the CEM group
First, we synthesized the phosphoramidite 5a-d according
to Scheme 1. Starting with a suitable base-protected nucleo-
side, 1a-d,9,13 we derivatized the 5′-hydroxyl group with
DMTr and then the 2′-hydroxyl group with CEM. CEM
(9) Pitsch, S.; Weiss, P. A.; Jenny, L.; Stutz, A.; Wu, X. HelV. Chim.
Acta 2001, 84, 3773-3795.
(10) Micura, R. Angew. Chem. Int. Ed. 2002, 41, 2265-2269.
(11) Matysiak, S.; Fitznar, H.-P.; Schnell, R.; Pfleiderer, W. HelV. Chim.
Acta 1998, 81, 1545-1566.
(12) Umemoto, T.; Wada, T. Tetrahedron Lett. 2004, 45, 9529-9531.
(13) (a) Ti, G. S.; Gaffney, B. L.; Jones, R. A. J. Am. Chem. Soc. 1982,
104, 1316-1319. (b) Chaix, C.; Molko, D.; Teoule, R. Tetrahedron Lett.
1989, 30, 71-74.
3478
Org. Lett., Vol. 7, No. 16, 2005

Figure 1. HPLC analysis of homo RNA and DNA. (A) Unpurified 2′-O-CEM-protected U40, DNA Pac PA-100 anion-exchange column
(4.6 mm × 250 mm; Dionex); buffer A, 10% CH3CN, 25 mM Tris-HCl, pH 8.0; buffer B, 10% CH3CN, 25 mM Tris-HCl, pH 8.0,
containing 700 mM NaClO4; gradient from 5% to 50% buffer B in 20 min; flow rate, 1.5 mL/min; 50 °C. (B) Unpurified fully deprotected
U40 (7), gradient 5% to 50% buffer B in 20 min. (C) Unpurified fully deprotected dT40 (DNA). HPLC conditions: buffer A, 25 mM
Tris-HCl, pH 8.0; buffer B, 25 mM Tris-HCl, pH 8.0, containing 700 mM NaClO4; gradient from 10% to 30% buffer B in 20 min; flow
rate, 1 mL/min; 40 °C. UV detection was at 260 nm.
accompanied by chain cleavage. When we used ammonia
instead, the loss of CEM was less than 5% and no chain
cleavage was observed.) In the next step, the 2′-O-CEM
protecting group was completely removed by treatment with
1 M TBAF/THF for several hours. (With other deprotecting
reagents that we tried, for example, Et3N‚3HF, the CEM
group was not removed.) HPLC profiles of U40 after
deprotection are shown for the fully deprotected crude RNA
prepared by the CEM method (Figure 1B) and for DNA of
the same chain length (dT40; Figure 1C). Comparison of the
HPLC profiles of the entire crude reaction mixtures shows
that the CEM method gave RNA of more than 80% purity,
as obtained in DNA synthesis.
We next synthesized RNA oligonucleotides incorporating
all four bases (Table 1). The phenoxyacetyl group was used
as the base-protecting group for G and the acetyl group as
the base-protecting group for A and C. For capping,
phenoxyacetic anhydride was used instead of acetic anhy-
dride to prevent replacement of the phenoxyacetyl group of
protected G by the acetyl group, a side reaction reported by
Chaix et al.13b
n-propylamine and 1% bis(2-mercaptoethyl) ether in 1 M
TBAF/THF gave the desired fully deprotected adduct-free
product. A HPLC profile of 8 (55mer) in the entire crude
reaction mixture from the CEM method is shown in Figure
2A. We emphasize that this HPLC profile shows absolutely
unpurified RNA oligomer. As observed for the synthesis of
the uridine homo-oligomer (7), the CEM method also yielded
a high-purity, high-yield product in the synthesis of mixed-
base RNA oligomers. Anion-exchange chromatography of
the reaction mixture resulting from the CEM method with
broad pooling of the main peak fractions followed by
desalting by dialysis was sufficient to give highly purified
RNA oligomer (Figure 2B). No modified bases were detected
on HPLC analysis of enzymatically digested RNA oligomer
8 (Figure 2C). The identity of 8 was confirmed when its
mass was measured by MALDI-TOF MS (positive-ion mode)
at 17474.6 ([M + H]+; calcd, 17476.6). As far as we know,
a 55mer is the longest synthetic RNA oligomer whose
structure has been confirmed by physicochemical data.
In conclusion, we have found that 2-cyanoethoxymethyl
(CEM), a new 2′-hydroxyl protecting group that is readily
removable under TBAF deprotection conditions, allowed
RNA synthesis to be carried out much more simply and
easily than by the conventional TBDMS methodology. RNA
synthesis by the CEM method proceeded with coupling
yields greater than 99% at each step while giving a high
overall yield and a high purity, and the method is comparable
in efficiency with DNA synthesis. We would like to
The RNA attached to CPG was initially treated similarly
to the way we treated U40 (7) at this stage. However, during
subsequent removal of the CEM group, a side reaction
occurred that MALDI-TOF MS evidence suggested was the
formation of cyanoethyl adducts (data not shown). To try to
suppress the formation of these adducts, we tested various
scavengers in various combinations and found that 10%
Table 1. Isolated Yields in the Synthesis of Oligonucleotides
entry
sequence
yield
1
2
3
4
5
5′-UUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUU-3′ (7; 40mer)
254 ODU (65%)
83 ODU (15%)
75 ODU (19%)
92 ODU (31%)
112 ODU (58%)
5′-UGAAUACAAAUCACAGAAUCGUCGUAUGCAGUGAAAACUCUCUUCAAUUCUUUAdT-3′ (8; 55mer)
5′-AAUCACAGAAUCGUCGUAUGCAGUGAAAACUCUCUUCAAdT-3′ (9; 40mer)
5′-ACAUCACUUACGCUGAGUACUUCGAAAUGU-3′ (10; 30mer)
5′-CUUACGCUGAGUACUUCGAU-3′ (11; 20mer)
Org. Lett., Vol. 7, No. 16, 2005
3479

Figure 2. HPLC analysis of mixed-base RNA. (A) Unpurified fully deprotected RNA (8; 55mer). (B) Purified fully deprotected RNA (8;
55mer). HPLC conditions were the same as in Figure 1A except that the linear gradient was from 5% to 40% buffer B in 20 min. (C) HPLC
analysis of RNA (8; 55mer) after enzymatic digestion: Develosil ODS-UG-5 reverse-phase column (4.6 mm × 250 mm); buffer, 5%
MeOH, 5 mM (nBu)4NHSO4, 50 mM phosphate, pH 7.5; flow rate, 1 mL/min; 35 °C. UV detection was at 260 nm. As expected, a small
dT peak derived from the 3′ end of 8 was observed.
emphasize that, unlike ACE chemistry, CEM chemistry is
compatible with standard unmodified DNA synthesizer
equipment. Furthermore, our results suggest that the CEM
method, after it is suitably optimized, will have the potential
for application to the synthesis of very long RNA oligo-
nucleotides. An additional attractive feature of the method
is that the CEM alkylating agent used can be synthesized
by a simple two-step procedure from a moderately priced
starting material, 3-hydroxypropionitrile, and so the method
may be scaled up at reasonable cost.
Acknowledgment. We thank Dr. G. E. Smyth, Discovery
Research Laboratories, Nippon Shinyaku Co., for helpful
discussions and suggestions concerning this manuscript.
Supporting Information Available: Data on the other
RNA oligomers in Table 1, as well as a detailed description
of the experimental procedures. This material is available
free of charge via the Internet at http://pubs.acs.org.
OL051151F
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