A general method for the synthesis of 2′-O-cyanoethylated oligoribonucleotides having promising hybridization affinity for DNA and RNA and enhanced nuclease resistance

Article abstract of DOI:10.1021/jo051741r

An effective method for the synthesis of 2′-O-cyanoethylated oligoribonucleotides as a new class of 2′-O-modified RNAs was developed. The reaction of appropriately protected ribonucleoside derivatives with acrylonitrile in t-BuOH in the presence of Cs

Full text of DOI:10.1021/jo051741r

A General Method for the Synthesis of 2′-O-Cyanoethylated
Oligoribonucleotides Having Promising Hybridization Affinity for
DNA and RNA and Enhanced Nuclease Resistance
Hisao Saneyoshi,† Kohji Seio,‡,§ and Mitsuo Sekine*,†,§
Department of Life Science, Tokyo Institute of Technology, Division of Collaborative Research for
Bioscience and Biotechnology, Frontier Collaborative Research Center, and CREST,
JST (Japan Science and Technology Agency), Nagatsuta, Midoriku, Yokohama 226-8501, Japan
msekine@bio.titech.ac.jp
Received August 18, 2005
An effective method for the synthesis of 2′-O-cyanoethylated oligoribonucleotides as a new class of
2′-O-modified RNAs was developed. The reaction of appropriately protected ribonucleoside
derivatives with acrylonitrile in t-BuOH in the presence of Cs2CO3 gave 2′-O-cyanoethylated
ribonucleoside derivatives in excellent yields, which were converted by a successive selective
deprotection/protection strategy to 2′-O-cyanoethylated 5′-O-dimethoxytritylribonucleoside 3′-
phosphoramidite derivatives in high yields. Fully 2′-O-cyanoethylated oligoribonucleotides, (Uce)12
and (GceAceCceUce)3, were successfully synthesized in the phosphoramidite approach by use of
the phosphoramidite building blocks. It was also found that oligoribonucleotides having a 2′-O-
cyanoethylated ribonucleoside (Uce, Cce, Ace, or Gce) could be obtained by the selective removal of
the TBDMS group from fully protected oligoribonucleotide intermediates without loss of the
cyanoethyl group by use of NEt3‚3HF as a desilylating reagent. The detailed Tm experiments revealed
that oligoribonucleotides containing 2′-O-cyanoethylated ribonucleosides have higher hybridization
affinity for both DNA and RNA than the corresponding unmodified and 2′-O-methylated oligo-
ribonucleotides. In addition, introduction of a cyanoethyl group into the 2′-position of RNA resulted
in significant increase of nuclease resistance toward snake venom and bovine spleen phosphodi-
esterases compared with that of the methyl group.
Introduction
powerful tool to control gene functions and has been used
for gene therapy targeting mRNAs.3,4 However, RNAs are
well-known to be rapidly degraded by cellular enzymes.
To mitigate this inherent disadvantage of RNAs as drugs
for clinical application, a large number of oligoribonucle-
otide derivatives having an ether-type skeleton at the 2′
position have been reported to date.5-21 In 1987, Inoue
and Ohtsuka reported that, among them, 2′-O-methyl
The chemical modification of natural RNAs has proved
to be of great importance to improve their original
physicochemical and biochemical properties such as
hybridization affinity and nuclease resistance. These
modified RNAs have been applied to the gene regulation
in antisense, antigene, and RNA interference (RNAi)
strategies.1-4 Particularly, RNAi mediated by small
interfering RNAs (siRNAs) has been recognized as a new
(3) (a) Manoharan M. Curr. Opin. Chem. Biol. 2004, 8, 570-579.
(b) Tang G. Trends Biochem. Sci. 2005, 30, 106-114.
(4) Dorsett, Y.; Tuschl, T. Nat. Rev. Drug Discovery 2004, 3, 318-
329.
(5) Manoharan, M. Biochim. Biophys. Acta 1999, 1489, 117-130.
(6) Earnshaw, D. J.; Gait, M. J. Biopolymers 1998, 48, 39-55.
(7) Freier, S. M.; Altmann, K.-H. Nucleic Acids Res. 1997, 25, 4429-
4443.
† Tokyo Institute of Technology.
‡ Frontier Collaborative Research Center.
§ CREST.
(1) Kurreck, J. Eur. J. Biochem. 2003, 270, 1628-1644.
(2) Buchini, S.; Leumann, C. J. Curr. Opin. Chem. Biol. 2003, 7,
717-726.
10.1021/jo051741r CCC: $30.25 © 2005 American Chemical Society
Published on Web 11/10/2005
J. Org. Chem. 2005, 70, 10453-10460
10453

Saneyoshi et al.
FIGURE 1. Modified ribonucleosides at the 2′-O-hydroxyl group.
RNAs exhibited enhanced chemical stability and affinity
for RNAs.9 Thereafter, it turned out that 2′-O-alkylated
RNAs having modified groups such as 2′-O-methoxy-
ethyl,11,12 2′-O-propargyl,15 2′-O-aminopropyl,16 2′-O-
dimethylaminooxyethyl,17a 2′-O-[2-(methylamino)-2-oxo-
ethyl],19c and 2′-O-[2-(guanidinium)ethyl]21 have excellent
nuclease resistance and affinity for the complementary
oligonucleotides (Figure 1).
nucleosides.13,17,18,20-23 Different approaches were often
required for the synthesis of pyrimidine and purine
ribonucleoside building blocks.19a,b As a typical procedure
for the synthesis of ribonucleoside building blocks, direct
2′-O-alkylation of unprotected or partially protected
ribonucleosides has been widely applied by use of alky-
lating reagents in the presence of NaH to give 2′-O-
modified ribonucleosides.11a,13,15-19,21,23 This type of modi-
fication is practically useful but often resulted in low yield
and required tedious separation of the desired 2′-O-
alkylated products and was not always the best choice
for all four canonical nucleosides.19a,b Particularly, 2′-O-
methoxyethyl ribonucleoside derivatives, which have
proved to be one of the most useful 2′-O-modified ribo-
nucleosides, have been synthesized from ribose via a
multistep reaction involving glycosylation and alkylation
of the once-generated 2′-free appropriately protected
ribonucleoside derivatives with methoxyethyl bromide.11a
Although a convenient route to 2′-O-methoxyethylribo-
nucleoside derivatives by the ring opening-mediated
etherification of 2,2′-cyclouridine derivatives nucleosides
was reported, this process is limited to a pyrimidine
series.24 More seriously, few articles are available for full
details of the synthetic procedure of four kinds of 2′-O-
modified ribonucleoside 3′-phosphoramidite building
blocks.11a,15,19b Among them, only 2′-O-methylated oligori-
bonucleotides are commercially available, and thus they
have been widely used as chemically stable RNA deriva-
tives for various studies.1-8
However, most of the articles related to 2′-O-modified
ribonucleosides were limited to pyrimidine ribo-
(8) Hall, R. H. The Modified Nucleosides in Nucleic Acids; Columbia
University Press: New York, 1971.
(9) Inoue, H.; Hayase, Y.; Imura, A.; Iwai, S.; Miura, K.; Ohtsuka,
E. Nucleic Acids Res. 1987, 15, 6131-6148.
(10) (a) Lesnik, E. A.; Guinosso, C. J.; Kawasaki, A. M.; Sasmor,
H.; Zounes, M.; Cummins, L. L.; Ecker, D. J.; Cook, P. D.; Freier, S.
M. Biochemistry 1993, 32, 7832-7838. (b) Cummins, L. L.; Owens, S.
R.; Risen, L. M.; Lesnik, E. A.; Freier, S. M.; McGee, D.; Guinosso, C.
J.; Cook, P. D. Nucleic Acids Res. 1995, 23, 2019-2024.
(11) (a) Martin, P. Helv. Chim. Acta 1995, 78, 486-504. (b) Martin,
P. Helv. Chim. Acta 1996, 79, 1930-1938. (c) Martin, P. Helv. Chim.
Acta 2003, 86, 204-209.
(12) (a) Lesnik, E. A.; Sasmor, H. M.; Bennett, C. F. J. Biol. Chem.
1997, 272, 11994-12000. (b) Teplova, M.; Minasov, G.; Tereshko, V.;
Inamati, G. B.; Cook, P. D.; Manoharan, M.; Egli, M. Nat. Struct. Biol.
1999, 6, 535-539. (c) Maier, M. A.; Guzaev, A. P.; Manoharan, M. Org.
Lett. 2000, 2, 1819-1822. (d) Rajeev, K. G.; Prakash, T. P.; Manoharan,
M. Org. Lett. 2003, 5, 3005-3008. (e) Yu, R. Z.; Geary, R. S.; Monteith,
D. K.; Matson, J.; Truong, L.; Fitchett, J.; Levin, A. A. J. Pharm. Sci.
2004, 93, 48-59.
(13) Prakash, T. P.; Manoharan, M.; Kawasaki, A. M.; Fraser, A.
S.; Lesnik, E. A.; Sioufi, N.; Leeds, J. M.; Teplova, M.; Egli, M.
Biochemistry 2002, 41, 11642-11648.
(14) Karpeisky, A.; Gonzalez, C.; Burgin, A. B.; Beigelman, L.
Tetrahedron Lett. 1998, 39, 1131-1134.
In this article, we report a general and promising
method for the synthesis of 2′-O-cyanoethylated oligo-
ribonucleotide derivatives as a new class of 2′-O-modified
RNAs as well as a convenient and widely applicable
method for the 2′-O-cyanoethylation of ribonucleoside
derivatives by use of acrylonitrile in t-BuOH in the
presence of Cs2CO3.
(15) Grotli, M.; Douglas, M.; Eritja, R.; Sproat, B. S. Tetrahedron
1998, 54, 5899-5914.
(16) Griffey, R. H.; Monia, B. P.; Cummins, L. L.; Freier, S.; Greig,
M. J.; Guinosso, C. J.; Lesnik, E.; Manalili, S. M.; Mohan, V.; Owens,
S.; Ross, B. R.; Sasmor, H.; Wancewicz, E.; Weiler, K.; Wheeler, P. D.;
Cook, P. D. J. Med. Chem. 1996, 39, 5100-5109. Although this article
described the result of oligoribonucleotides containing four kinds of
ribonucleosides, no details were given for U, C, and G.
(17) (a) Prakash, T. P.; Manoharan, M.; Fraser, A. S.; Kawasaki, A.
M.; Lesnik, E. A.; Owens, S. R. Tetrahderon Lett. 2000, 41, 4855-
4859. (b) Prhavc, M.; Prakash, T. P.; Minasov, G.; Cook, P. D.; Egli,
M.; Manoharan, M. Org. Lett. 2003, 5, 2017-2020.
Results and Discussion
(18) Kawasaki, A. M.; Casper, M. D.; Prakash, T. P.; Manalili, S.;
Sasmor, H.; Manoharan, M.; Cook, P. D. Tetrahedron Lett. 1999, 40,
661-664.
Strategies for the Synthesis of 2′-O-Cyanoethy-
lated RNAs. Cyanoethylation of tRNAs with acryloni-
trile was reported in the early 1960s by Yoshida, Ukita,
and Ofengand.25 However, these reactions always gave
(19) (a) Prakash, T. P.; Manoharan, M.; Kawasaki, A. M.; Lesnik,
E. A.; Owens, S. R.; Vasquez, G. Org. Lett. 2000, 2, 3995-3998. (b)
Prakash, T. P.; Kawasaki, A. M.; Fraser, A. S.; Vasquez, G.; Manoha-
ran, M. J. Org Chem. 2002, 67, 357-369. (c) Prakash, T. P.; Kawasaki,
A. M.; Lesnik, E. A.; Owens, S. R.; Manoharan, M. Org. Lett. 2003, 5,
403-406. (d) Prakash, T. P.; Johnston, J. F.; Graham, M. J.; Condon,
T. P.; Manoharan, M. Nucleic Acids Res. 2004, 32, 828-833.
(20) Prhavc, M.; Lesnik, E. A.; Mohan, V.; Manoharan, M. Tetra-
hedron Lett. 2001, 42, 8777-8780.
(22) Prhavc, M.; Prakash, T. P.; Minasov, G.; Cook, P. D.; Egli, M.;
Manoharan, M. Org. Lett. 2003, 5, 2017-2020.
(23) Prakash, T. P.; Pu¨schl, A.; Lesnik, E.; Mohan, V.; Tereshko,
V.; Egli, M.; Manoharan, M. Org. Lett. 2004, 6, 1971-1974.
(24) Legorburu, U.; Reese, C. B.; Song, Q. Tetrahedron 1999, 55,
5635-5640.
(21) (a) Prakash, T. P.; Kawasaki, A. M.; Lesnik, E. A.; Owens, S.
R.; Manoharan, M. Org. Lett. 2003, 5, 403-406. (b) Pattanayek, R;
Sethaphong, L.; Pan, C.; Prhavc, M.; Prakash, T. P.; Manoharan, M.;
Egli, M. J. Am. Chem. Soc. 2004, 126, 15006-15007.
(25) (a) Yoshida, M.; Ukita, C. J. Biochem. (Tokyo) 1965, 58, 191-
193. (b) Ofengand, J. J. Biol. Chem. 1967, 242, 5034-5045.
10454 J. Org. Chem., Vol. 70, No. 25, 2005

Synthesis of 2′-O-Cyanoethylated Oligoribonucleotides
TABLE 1. Results of 2′-O-Cyanoethylation of 1a under
SCHEME 1. Cyanoethylation of the 2′-Hydroxyl
Group of Uridine Derivative
Various Conditionsa
base
solvent
time
yield of 2a
entry (1.0 equiv) (reagent or cosolvent) (h)
(%)
1
Et3N
CH2dCHCN
24
2
no reaction
24
2
Triton B CH2dCHCN
3
DBU
MgO
CaO
CH2dCHCN
CH2dCHCN
CH2dCHCN
24
24
24
24
24
24
24
24
24
50
4
no reaction
no reaction
no reaction
0
5
6
N-MeIm CH2dCHCN
DMAP CH2dCHCN
DABCO CH2dCHCN
Na2CO3 CH2dCHCN
K2CO3
Cs2CO3
Cs2CO3
7
8
no reaction
no reaction
no reaction
54
9
10
11
12
CH2dCHCN
CH2dCHCN
base-modified products. Particularly, they used the
Michael reaction of acrylonitrile to identify modified
bases such as pseudouridine, 4-thiouridine, and inosine
in tRNAs. No studies of cyanoethylation of hydroxyl
groups of nucleosides, nucleotides, and nucleic acid
derivatives have been reported to date. Our interest was
focused on the 2-cyanoethyl group as a new type of 2′-
substituent since it has a simple structure with less steric
hindrance and a polarized cyano group that seems to
stabilize water bridge structures around the 2′-position.
In general, however, the Michael reaction of alcohols
with acrylonitrile giving rise to 1:1 adducts was sluggish,
requiring use of strong bases26 such as sodium hydroxide,
alcoholates, and benzyltrimethylammonium hydroxide
(Triton B). Apparently, these basic conditions could not
be applied to the synthesis of 2′-O-cyanoethylated ribo-
nucleoside derivatives as the key synthetic intermediates
required for the synthesis of 2′-O-cyanoethylated oligo-
ribonucleotides.
MeOH-CH2dCHCN 24
complex mixture
(20 equiv)
13
14
15
16
Cs2CO3
Cs2CO3
Cs2CO3
Cs2CO3
THF-CH2dCHCN
24
34
72
(20 equiv)
CH2dCHCN-t-BuOH 12
(10 equiv)
CH2dCHCN-t-BuOH 2.5 93
(40 equiv)
t-BuOH-CH2dCHCN
1
95
(20 equiv)
a
A 0.2 M solution of 1a was used under these conditions.
carbanion. On the other hand, the formation of 2a
requires the protonation of the carbanion. Therefore,
addition of suitable proton sources to the carbanion
should be effective to prevent the undesirable polymer-
ization.
In consideration of this mechanism, we selected metha-
nol as not only a proton source but also the cosolvent. In
this case, unfortunately, debenzoylation of 2a was ob-
served to give methyl benzoate. This result indicated that
a kind of methoxide anion generated by Cs2CO3 worked
as a nucleophile to attack the benzoyl group. Therefore,
we next selected tert-butyl alcohol as a proton source. As
shown in entry 16 of Table 1, the combined use of Cs2-
CO3 and tert-butyl alcohol gave the desired product 2a
in an excellent yield of 95%. Moreover, orange precipi-
tates were not observed under these conditions. The
effectiveness of the tert-butyl alcohol could be attributed
to its efficient proton-donor property capable of proto-
nating the once-generated carbanion. Since the acidity
of tert-butyl alcohol (pKa ) 18) is rather lower than that
of methanol (pKa ) 15.5), it is more difficult to generate
a basic species such as the t-butoxide ion directly by the
action of Cs2CO3. Even if such an alchoxide-like species
is generated, the benzoyl group would remain stable
because of the poor nucleophilicity of the sterically
hindered tert-butoxide ion. Furthermore, Cs2CO3 is
scarcely soluble in tert-butyl alcohol so that the super-
natant of the resulting heterogeneous reaction mixture
can be kept as a nonbasic medium, which is favorable
for the 2′-O-cyanoethylation of 1a without damaging the
base labile-protecting groups.
To overcome this problem, we have extensively searched
for mild conditions for the 2′-O-cyanoethylation of an
appropriately protected uridine derivative 1a (Scheme
1).27 These results are summarized in Table 1.
The 2′-O-cyanoethylation of N3-benzoyl-(1,1,3,3-tetra-
isopropyldisiloxane-1,3-diyl)uridine (1a) was studied by
use of various base catalysts and acrylonitrile as a
solvent. As shown in Table 1, triethylamine did not give
the Michael reaction product 2a. As expected, the use of
strong organic bases such as DBU and Triton B gave 2a,
but the reaction was not completed. In these reactions,
considerable polymerization of acrylonitrile occurred with
formation of significant amounts of orange materials.
DABCO, a strong organic base, as well as N-methylimi-
dazole and 4-(dimethylamino)pyridine, well-known nu-
cleophilic catalysts, did not give the Michael adduct. It
was found that, among the metal carbonates and oxides
tested, only Cs2CO3 gave the Michael reaction adduct 2a
in 54% yield. In this case, the undesirable polymerization
of acrylonitrile was also observed. To avoid this serious
side reaction, the mechanism of the polymerization was
considered. It is likely that the first step of the polym-
erization is the addition of the 2′-hydroxyl group to
acrylonitrile that gives a carbanion on the R-carbon atom
next to the cyano group, and the second step is the
reaction of another acrylonitrile molecule on this
Our new method for the O-cyanoethylation was applied
to the synthesis of 2′-O-cyanoethylated cytidine, adeno-
sine, and guanosine derivatives (2b-d). In the case of
2b and 2c, the amino groups on the base moieties were
protected with the dimethylaminomethylene group.28 The
(26) (a) Krishna, T. R.; Jayaraman, N. J. Org. Chem. 2003, 68,
9694-9704. (b) Chung, H.-H.; Harms, G.; Seong, C. M.; Choi, B. H.;
Min, C.; Taulane, J. P.; Goodman, M. Biopolymers 2004, 76, 83-96.
(c) Li, X.; Goh, S. H.; Lai, Y. H.; Deng, S.-M. J. Appl. Polym. Sci. 1999,
73, 2771-2777. (d) Pearson, W. H.; Guo, L.; Jewell, T. M. Tetrahedron
Lett. 2002, 43, 2175-2178. (e) Simonot, B.; Rousseau, G. Synth.
Commun. 1993, 23, 549-560.
(28) McBride, L. J.; Kierzek, R.; Beaucage, S. L.; Caruthers, M. H.
J. Am. Chem. Soc. 1986, 108, 2040-2048.
(27) Sekine, M. J. Org Chem. 1989, 54, 2321-2326.
J. Org. Chem, Vol. 70, No. 25, 2005 10455

Saneyoshi et al.
SCHEME 2. Cyanoethylation of Ribonucleosides and Their Nucleoside Derivativesa
a Reagents and conditions: (a) acrylonitrile, tert-butyl alcohol, rt; (b) Et3N‚3HF, Et3N, THF, rt; (c) deprotection of the base moieties.
cytidine and adenosine derivatives 1b29 and 1c30
underwent smooth 2′-O-cyanoethylation with acryloni-
trile under conditions similar to those described for the
synthesis of 2a to give 2b and 2c in 89 and 90% yields,
respectively. For the synthesis of the guanosine deriva-
tive 2d, the 6-O-position was protected with a 2,4,6-
triisopropylbenzenesulfonyl group31 and the 2-amino
group was masked with the dimethylaminomethylene
group. Thus, a similar Michael reaction of 1d with
acrylonitrile gave 2d in 83% yield, as shown in Scheme
2. It should be noted that during the reaction the 1,1,3,3-
tetraisopropyldisiloxane-1,3-diyl (TIPS) group and the
other base-protecting groups remained intact. Particu-
larly, it was found that the TPS group could be conve-
niently used as the 6-O-protecting group of the guanine
residue. The TIPS groups of 2a-d were selectively
deprotected by treatment with Et3N‚3HF to give the 3′,5′-
O-unprotected ribonucleoside derivatives (3a-3d) in high
yields. In the case of 3d, the TPS group was selectively
removed by an oximate reagent of o-nitrobenzaldoxime
and tetramethylguanidine before the Et3N‚3HF treat-
ment. Finally, treatment of 3a and 3b with NH4OH-
EtOH (1:1 and 3:1, respectively, v/v) at room temperature
for 1 h gave 4a and 4b in 95 and 92% yields, respectively,
as shown in Scheme 2. Treatment of 3c with hydrazine
monohydrate in CH3CN at room temperature for 3 h gave
4c in 82% yield. The reaction of 3d with cyclohexylamine
in THF gave the desired product 4d in 85% yield.
Next, the sugar puckering modes of the new modified
ribonucleosides 4a-4d were studied by use of 1H NMR.
The J1′,2′ and J3′,4′ values and the percentage (%N) of the
C3′-endo form are summarized, as shown in Table 2, and
compared with those of the corresponding 2′-O-methy-
lated derivatives. The %N values were calculated accord-
ing to the equation of %N(C3′-endo) ) {J3′,4′ (Hz)/J1′,2′ (Hz)
+ J3′,4′ (Hz)} × 100.32
TABLE 2. Sugar Puckering Modes of
2′-O-Cyanoethylated Ribonucleosides and
2′-O-Methylated Ribonucleosides (25 °C, 500 MHz NMR in
D2O)
UOCE
COCE
AOCE
GOCE
UOMe 4a COMe 4b AOMe 4c GOMe 4d
J1′,2′
3.9
5.8
9.7
3.7
6.1
3.4
6.6
3.2
7.1
6.4
3.2
9.6
6.4
3.2
9.6 10.0
33 39
6.1
3.9
6.1
3.7
9.8
J3′,4′
J1′,2′ + J3′,4′
%N
(C3′-endo)
9.8 10.0 10.3
63 66 69
60
33
38
a These values were calculated according to the equation
%N(C3′-endo) ) {J3′,4′ (Hz)/J1′,2′ (Hz) + J3′,4′ (Hz)} × 100.32
As a result, it was found that the %N values of the
2′-O-cyanomethylated purine ribonucleosides 4c,d were
essentially similar to those of 2′-O-methylated ribo-
nucleosides, but in the 2′-O-cyanoethylated pyrimidine
ribonucleosides 4a,b the C3′-endo form was increased
slightly to a degree of 3%. It is interesting that, although
the 2-cyanoethyl group has two methylene groups and a
cyano group, the 2′-O-cyanoethylated ribonucleosides
maintain the ratio of the N- and S-type conformers sugar
puckering mode in the same manner as 2′-O-methylated
ribonucleosides.
Oligoribonucleotides Synthesis. For the synthesis
of 2′-O-cyanoethylated oligoribonucleotides, the acetyl
group was chosen as the protecting group for the cytosine
base. 4-N-Acetyl-2′-O-(2-cyanoethyl)cytidine (4b′) was
obtained by reaction of 4b with acetic anhydride in the
presence of EtOH. For the adenine and guanine bases,
the dimethylaminomethylene group was used.28 The
uracil base was not protected, as reported usually.33
Compounds 4a, 4b′, 3c, and 3d thus obtained were
converted to the phosphoramidite derivatives 6a-d via
the 5′-O-dimethoxytritylated products 5a-d according to
(29) Matthews, D. P.; Persichetti, R. A.; Sabol, J. S.; Stewart, K. T.;
McCarthy, J. R. Nucleosides Nucleotides 1993, 12, 115-123.
(30) Vu, H.; McCollum, C.; Jacobson, K.; Theisen, P.; Vinayak, R.;
Spiess, E.; Andrus, A. Tetrahedron Lett. 1990, 31, 7269-7272.
(31) (a) Daskalov, H. P.; Sekine, M.; Hata, T. Bull. Chem. Soc. Jpn.
1981, 54, 3076-3083. (b) Daskalov, H. P.; Sekine, M.; Hata, T.
Tetrahedron Lett. 1980, 21, 3899-3903.
(32) (a) Davies, D. B.; Danyluk, S. S. Biochemistry 1974, 13, 4417-
4434. (b) Davies, D. B. Prog. Nucl. Magn. Reson. Spectrosc. 1978, 12,
135-225. (c) Altona, C.; Sundaralingham, M. J. Am. Chem. Soc. 1973,
95, 2333-2344.
(33) Wincott, F. E. In Current Protocols in Nucleic Acid Chemistry;
Beaucage, S. L., Bergstrom, D. E., Glick, G. D., Jones, R. A., Eds.; Wiley
& Sons: New York, 2000; pp 3.5.1-3.5.12.
10456 J. Org. Chem., Vol. 70, No. 25, 2005

Synthesis of 2′-O-Cyanoethylated Oligoribonucleotides
SCHEME 3. Preparation of Phosphoramidite Building Blocks and Polymer Supporta
a Reagents and conditions: (a) DMTrCl, pyridine, rt; (b) Chloro(2-cyanoethyl)(N, N-diisopropylamino)phosphine, ethyldiisopropylamine,
CH2Cl2, rt; (c) succinic anhydride, DMAP, CH2Cl2, rt; LCAA-CPG, DCC, CH2Cl2, rt.
FIGURE 2. Anion exchange HPLC profile of crude cyanoethylated RNAs. (Left) (Uce)12. (Right) (GceAceCceUce)3.
the standard procedure (Scheme 3).33 The 5′-tritylated
compound 5a was attached to long-chain aminoalkyl CPG
via a succinyl linker to give the solid support.
of the dmf group. Thus, the fully 2′-O-cyanoethylated
dodecaoligoribonucleotide (GceAceCceUce)3 was success-
fully synthesized. It was also found that the use of
n-propylamine-THF (1:1, v/v) as the deblocking reagent
of the Pac (A), isopropyl-pac (G), and acetyl (C) groups
resulted in the selective deprotecton of the acyl-type
protecting groups [Pac (A), isopropyl-Pac (G), and acetyl
(C)] without damage to the cyanoethyl and TBDMS
groups at the 2′-position. Moreover, the successive treat-
ment of the resulting N-unprotected RNA species with
Et3N‚3HF gave the desired RNA oligomers containing a
2′-O-cyanoethylated ribonucleoside. Under these condi-
tions, the 2′-O-cyanoethyl group remained intact. Con-
trary to this result, tetrabutylammonium fluoride could
not be used as the desilylating reagent. Surprisingly, in
this case, the 2-cyanoethyl ether was considerably depro-
tected.34 These products were purified by use of reverse-
phase C18 cartridge and anion exchange HPLC. The
purified products were characterized by MALDI-TOF
mass (Table 3).
The synthesis of fully and partially 2′-O-cyanoethylated
oligoribonucleotides was performed by the phosphor-
amidite approach using the phosphoramidite building
blocks 6a-d and the widely used 2′-O-TBDMS-ribo-
nucleoside phosphoramidite units by use of an ABI 392
DNA/RNA automated synthesizer. After the chain elon-
gation for the synthesis of the fully 2′-O-cyanoethylated
dodecauridylate, simple treatment of the protected oli-
gomer on the resin with concentrated NH3 gave the
desired product (Uce)12 as the main peak, as shown in
Figure 2. However, in the case of the synthesis of
(GceAceCceUce)3 having all four ribonucleosides, the
ammonia treatment was somewhat detrimental since the
cyanoethyl group was not stable during base deprotec-
tion. Therefore, we extensively studied more suitable
conditions for the full deprotection of the base- and
phosphate-protecting groups. Consequently, it was found
that treatment of fully protected oligoribonucleotides on
CPG with concentrated NH4OH-NH4OAc (10:1, w/w)28
at room temperature for 90 min was very effective for
the selective removal of the dmf and acetyl groups on the
base moieties. When only concentrated NH3 was used,
part of the cyanoethyl group was eliminated and the dmf
was much more slowly deprotected. Addition of NH4OAc
to concentrated NH4OH was essential to avoid elimina-
tion of the cyanoethyl group and to accelerate removal
(34) This result suggests that the cyanoethyl group can be used as
a new promising 2′-hydroxyl protecting group for the RNA synthesis.
The details of this study will be shortly reported by us.
(35) Xu, Y.; Kool, E. T. Nucleic Acids Res. 1998, 26, 3159-3164.
(36) Matthews, D. P.; Persichetti, R. A.; Sabol, J. S.; Stewart, K. T.;
McCarthy, J. R. Nucleosides Nucleotides 1993, 12, 115-123.
(37) Markiewicz, W. T. J. Chem. Res., Synop. 1979, 24-25.
(38) (a) Hagen, M. D.; Chladek, S. J. Org. Chem. 1989, 54, 3189-
3195. (b) Grotli, M.; Douglas, M.; Beijer B.; Garcia, G. R.; Eritja, R.;
Sproat, B. J. Chem. Soc., Perkin Trans. 1 1997, 2779-2788.
J. Org. Chem, Vol. 70, No. 25, 2005 10457

Saneyoshi et al.
TABLE 3. Oligoribonucleotides Synthesized by Use of 2′-O-Cyanoethylated Ribonucleosides
MALDI-TOF mass
oligoribonucleotides sequencea
conditions for deprotectionb
calcd
found
UU
NH4OH
602.11
4236.51
1434.31
4428.86
4135.59
4135.59
4135.59
4135.59
5474.80
602.85
4238.41
1434.12
4428.55
4134.27
4135.38
4133.90
4135.37
5473.90
UUUUUUUUUUUU
GACU
NH4OH
NH4OH-NH4OAc
NH4OH-NH4OAc
n-PrNH2 and then Et3N‚3HF
n-PrNH2 and then Et3N‚3HF
n-PrNH2 and then Et3N‚3HF
n-PrNH2 and then Et3N‚3HF
n-PrNH2 and then Et3N‚3HF
GACUGACUGACU
GCUAGACUAUCUA
GCUAGACUAUCUA
GCUAGACUAUCUA
GCUAGACUAUCUA
GAGCCAAGCIUCGGCUC
a Conditions for release of oligonucleotides from CPG and removal of the protecting groups of the base moieties and the 2′-hydroxyl
group. b None: Natural ribonucleoside, bold: 2′-O-Cyanoethylribonucleoside.
TABLE 4. Hybridization Properties of 2′-O-Cyanoethylated RNA Derivatives
sequence and complement
vs DNA
∆Tm
∆Tm/mod.
vs RNA
∆Tm
∆Tm/mod.
1
2
UUUUUUUUUUUU
UUUUUUUUUUUU
UUUUUUUUUUUU
GACUGACUGACU
GACUGACUGACU
GACUGACUGACU
CGUAGACUAUCUA
CGUAGACUAUCUA
CGUAGACUAUCUA
CGUAGACUAUCUA
CGUAGACUAUCUA
CGUAGACUAUCUA
CGUAGACUAUCUA
CGUAGACUAUCUA
CGUAGACUAUCUA
8.5
10.3
17.1
47.9
46.9
53.8
41.5
41.0
42.6
40.3
42.9
37.6
42.3
38.2
42.4
14.1
26.1
33.5
59.1
63.6
68.4
54.8
54.9
55.9
54.3
56.9
50.5
55.5
51.3
56.1
+1.8
+8.6
+12.0
+19.4
+1.0
+1.6
3
+0.6
+0.5
4
5
-1.0
+5.9
+4.5
+9.3
+0.4
+0.8
6
7
8
-0.5
+1.1
-1.2
+1.4
-3.9
+0.8
-3.3
+0.9
-0.5
+1.1
-1.2
+1.4
-3.9
+0.8
-3.3
+0.9
+0.1
+1.1
-0.5
+2.1
-4.3
+0.7
-3.5
+1.3
+0.1
+1.1
-0.5
+2.1
-4.3
+0.7
-3.5
+1.3
9
10
11
12
13
14
15
a Conditions: 10 mM sodium phosphate buffer (pH 7.0), 100 mM NaCl, 0.1 mM EDTA, and 2.0 µM duplex. b None: Natural
ribonucleoside, italic: 2′-O-methylribonucleoside, bold: 2′-O-cyanoethylribonucleoside.
Hybridization Properties of 2′-O-Cyanoethylated
Oligoribonucleotides. Hybridization properties of oli-
goribonucleotides containing 2′-O-cyanoethylated nucleo-
sides with the complementary DNA or RNA strands were
studied. 2′-O-Methylated oligoribonucleotides were used
as the control. The hybridization properties of 2′-O-
cyanoethylated RNA derivatives are summarized in
Table 4. The hybridization affinity of the 2′-O-cyano-
ethylated dodecauridylate (Uce)12 for dA12 or A12 was
compared with that of the unmodified U12 and 2′-O-
methylated dodecauridylate (Um)12. As a result, it turned
out that the thermodynamic stability of (Uce)12-dA12 (Tm
) 17.1 °C) was considerably higher by 8.6 °C and 6.8 °C
than that of U12-dA12 and (Um)12-dA12, respectively, as
shown in Table 4. Similarly, the RNA-RNA duplex
(Uce)12-A12 exhibited a Tm value (Tm ) 33.5 °C) higher
than that of U12-A12 (Tm ) 14.1 °C) and (Um)12-A12 (Tm
) 26.1 °C). These results clearly suggested that the
duplex stabilizing effect of the 2′-O-cyanoethyl modifica-
tion is stronger than that of the 2′-O-methyl modification.
The hybridization affinity of a fully cyanoethylated RNA
derivative of (GceAceCceUce)3 with a mixed sequence for
the complementary DNA oligomer was also much higher
(Tm ) 53.8 °C) than the unmodified one (Tm ) 47.9 °C)
and the 2′-O-methyl oligomer (Tm ) 46.9 °C). The same
is true for the hybridization affinity of (GceAceCceUce)3
for the complementary RNA strand, as shown in entries
4-6 of Table 4, but this modified RNA-RNA duplex
resulted in a more significant stabilizing effect than the
modified RNA-DNA duplex.
To examine the relationship between the stabilizing
effect and the nucleobase moiety, comparative hybridiza-
tion experiments were carried out by use of various RNA
strands incorporating one of the four 2′-O-cyanoethyl or
2′-O-methylnucleosides (Table 4, entries 8-15).
Interestingly, the stabilizing effect of the one-point
modification was dependent on the structure of the base
moieties. Incorporation of a pyrimidine nucleoside Uce
in place of U slightly increased the Tm value versus DNA
and RNA by +1.1 and +1.1 °C, respectively. One-point
replacement of C with Cce also affected the Tm value to
a degree of +1.4 and +2.1 °C versus DNA and RNA,
respectively. On the other hand, incorporation of Um and
Cm into RNA resulted in a significant decrease of the
Tm values of the duplexes with the complementary DNA
strands by -0.5 and -1.2 °C, respectively, as shown in
entries 7, 8, and 10 of Table 4. The RNA oligomers
incorporating Um and Cm showed the same level and
weaker hybridization affinity (∆Tm ) +0.1 and -0.5 °C,
respectively) for the complementary RNA strand com-
pared with those of the unmodified RNA oligomer, as
shown in entries 7, 8, and 10 of Table 4. On the other
hand, it was found that the incorporation of the purine
nucleosides Ace and Gce resulted in a more equivocal
increase of the Tm value. The former affected the stability
by +0.8 versus DNA and +0.7 °C versus RNA, and the
latter showed a similar tendency with the ∆Tm values of
+0.9 versus DNA and 1.3 °C versus RNA. Surprisingly,
it was also found that incorporation of Am and Gm into
RNA caused marked destabilization of the duplexes with
10458 J. Org. Chem., Vol. 70, No. 25, 2005

Synthesis of 2′-O-Cyanoethylated Oligoribonucleotides
the conditions suitable for selective deprotection of the
base- and phosphate-protecting groups. It was empha-
sized that the 2′-O-cyanoethyl group could remain abso-
lutely intact when a mixture of concentrated NH4OH-
NH4OAc was employed as the deblocking reagent.
Furthermore, it turned out that oligoribonucleotides
incorporating 2′-O-cyanoethylated nucleosides enhanced
not only the hybridization affinity for DNA and RNA but
also nuclease resistance compared with the 2′-O-methyl
oligoribonucleotides. These promising properties of 2′-O-
cyanoethylated RNA oligomers would provide new insight
into RNA nanotechnotogy as well as RNAi and antisense
strategies since all four common ribonucleoside 3′-phos-
phoramidite building blocks can be easily obtained by a
series of simple reactions as described here. Further
applications of this new material are now under study.
Experimental Section
Synthesis 2′-O-Cyanoethylated Nucleoside and Its
Phosphoramidite. Example of Uridine Derivatives. N3-
Benzoyl-2′-O-(2-cyanoethyl)-3′,5′-O-(1,1,3,3,-tetraiso-
propyldisiloxane-1,3-diyl)uridine (2a). Compound 1a27 (60
mg, 0.102 mmol) was dissolved in t-butanol (500 µL). To the
solution were added acrylonitrile (131 µL, 2 mmol) and cesium
carbonate (35 mg, 0.1 mmol). After being vigorously stirred
at room temperature for 1 h, the mixture was filtered by use
of Celite. The solvent and excess volatile reagents were
evaporated in vacuo. The residue was chromatographed on a
silica gel column with hexane-ethyl acetate (3:1, v/v) to give
compound 2a as a white solid (61 mg, 95%): mp 159 °C
FIGURE 3. Time course of degradation of 2′-O-cyanoethylated
diuridylic acids UCEpU and UOMepU with two kinds of phos-
phodiesterases. (Top) Snake venom. (Bottom) Bovine spleen.
-3.9 versus DNA and -4.3 °C versus RNA, and -3.3
versus DNA and -3.5 °C versus RNA, respectively.
From these results, it was suggested that the 2′-O-
cyanoethylation has a unique effect on the hybridization,
which is different from that of 2′-O-methylation. More-
over, the cyanoethylation also proved to contribute to
stabilization of duplexes when both pyrimidine and
purine nucleosides are incorporated into RNA. The
equivocal stabilization effect of the 2′-O-cyanoethylation
would provide new insight into the creation of 2′-O-
modified RNA derivatives, and its mechanism should be
clarified in due course.
Nuclease Stabilities of 2′-O-Cyanoethylated Oli-
gouridylates. Nuclease resistance of 2′-O-cyanoethy-
lated oligoribonucleotides was tested by use of snake
venom phosphodiesterase10b and bovine spleen phos-
phodiesterase (Figure 3).33
The higher nuclease resistance of UCEpU was clearly
observed in the snake venom phosphodiesterase assay
compared with that of UOMepU. The half-lives of UCEpU
and UOMepU were determined to be 4 and 2 h, respec-
tively.
It was also found that UCEpU was markedly resistant
to bovine spleen phosphodiesterase, a major mammalian
phosphodiesterase. More than 90% of UCEpU remained
intact after 10 days, while only 2% of UOMepU remained
under the same conditions.
1
(CHCl3-i-Pr2O); H NMR (CDCl3, 500 MHz) δ 0.94-1.12 (28
H, m), 2.61-2.63 (2 H, m), 3.91-4.05 (4 H, m), 4.18-4.29 (3
H, m), 5.70 (1 H, s), 5.79 (1 H, d, J ) 8.30), 7.49-7.94 (5 H,
m), 8.00 (1H, d, J ) 8.3); 13C NMR (CDCl3, 500 MHz) δ 12.5,
12.8, 13.0, 13.4, 16.8, 16.9, 17.0, 17.2, 17.4, 19.1, 59.2, 65.8,
68.1, 81.8, 82.7, 89.0, 101.6, 117.4, 129.2, 130.5, 131.2, 135.3,
138.9, 149.0, 162.1, 168.7. Anal. Calcd for C31H45N3O8Si2: C,
57.83; H, 7.04; N, 6.53. Found: C, 58.02; H, 6.78; N, 6.51.
N3-Benzoyl-2′-O-(2-cyanoethyl)uridine (3a). Compound
2a (322 mg, 0.50 mmol) was dissolved in dry THF (5 mL). To
the solution were added Et3N‚3HF (285 µL, 1.75 mmol) and
triethylamine (125 µL, 0.897 mmol). After being stirred at
room temperature for 1 h, the reaction mixture was evaporated
in vacuo. The residue was chromatographed on a silica gel
column with CHCl3-MeOH (98:2-96:4, v/v) to give compound
3a (199 mg, 99%) as white foam: 1H NMR (CDCl3, 500 MHz)
δ 2.63-2.66 (2H, m), 3.83-3.90 (2H, m), 4.04-4.09 (4H, m),
4.31 (1H, dd, J ) 5.4, 7.3), 5.81 (1H, d, J ) 1.7), 5.83 (1H, d,
J ) 8.3), 7.50-7.94 (5H, m), 8.10 (1H, d, J ) 8.3); 13C NMR
(CDCl3) δ 19.0, 60.1, 65.3, 67.8, 82.5, 84.2, 88.7, 102.0, 117.7,
129.3, 130.5, 131.1, 135.5, 140.4, 149.4, 162.3, 168.7; HRMS
calcd for C19H19N3O7 (M + H+) 402.1301, found 402.1315.
2′-O-(2-Cyanoethyl)uridine (4a). Compound 3a (80 mg,
0.20 mmol) was dissolved in EtOH-28% NH3 aq (2 mL, 1:1,
v/v). After being stirred at room temperature for 1 h, the
mixture was evaporated in vacuo. The residue was dissolved
in MeOH-ether (11 mL, 1:10, v/v), and the solution was
extracted three times with H2O (3 mL). The aqueous extracts
were combined and evaporated in vacuo. The residue was
chromatographed on a column of silica gel with CHCl3-MeOH
(5:1, v/v) to give compound 4a as a white solid (57 mg, 95%):
Conclusion
In this study, we developed a general method for the
synthesis of 2′-O-cyanoethylated ribonucleosides and
oligoribonucleotides. As a result, the cyanoethyl group
could be introduced readily into the 2′-hydroxyl group of
the appropriately protected canonical ribonucleosides by
use of Michael addition using the acrylonitrile-t-BuOH-
Cs2CO3 system. We succeeded in establishing a depro-
tection procedure for the synthesis of the fully and
partially 2′-O-cyanoethylated RNA oligomers by choosing
1
mp 119 °C (EtOH); H NMR (D2O, 500 MHz) δ 2.70-2.72 (2
H, m), 3.69 (1 H, dd, J ) 4.2, 12.9), 3.82-3.85 (3 H, m), 4.02-
4.04 (1 H, m), 4.08 (1 H, dd, J ) 3.7, 5.2), 4.19 (1 H, t, J )
6.1), 5.77 (1 H, d, J ) 8.1), 5.87 (1 H, d, J ) 3.7), 7.80 (1 H, d,
8.06); 13C NMR (D2O) δ 19.0, 60.9, 66.0, 68.8, 82.2, 84.7, 88.6,
102.8, 120.3, 142.2, 152.1, 166.8; HRMS calcd for C12H15N3O6
(M + H+) 298.1039, found 298.1033. Anal. Calcd for C12H15-
N3O6: C, 47.91; H, 5.16; N, 13.97. Found: C, 47.66; H, 5.27;
N, 13.48.
J. Org. Chem, Vol. 70, No. 25, 2005 10459

Saneyoshi et al.
2′-O-(2-Cyanoethyl)-5′-O-(4,4′-dimethoxytrityl)-
uridine (5a). Compound 4a (1.97 g, 6.63 mmol) was coevapo-
rated five times with dry toluene and finally dissolved in dry
pyridine (70 mL). To the solution was added 4,4′-dimethoxy-
trityl chloride (2.47 g, 7.29 mmol). After being stirred at room
temperature for 4 h, the mixture was quenched by addition of
water and evaporated in vacuo. The residue was dissolved with
CHCl3. The solution was washed with brine and aqueous
NaHCO3. The organic layer was dried over Na2SO4 and
concentrated in vacuo. The residue was chromatographed on
a column of silica gel with CHCl3-MeOH (95:5, v/v) containing
0.5% triethylamine to give compound 5a as white foam (3.91
g, 98%): 1H NMR (CDCl3, 500 MHz) δ 2.68-2.71 (2 H, m),
3.53-3.58 (2 H, m), 3.90-3.98 (2 H, m), 4.03-4.06 (1 H, m),
4.17-4.22 (1 H, m), 4.49 (1 H, dd, J ) 5.1, 8.8), 5.31 (1 H, d,
J ) 8.1), 5.89 (1 H, s), 6.84-6.86 (4H, m), 7.21-7.40 (9H, m),
8.06 (1 H, d, J ) 8.1); 13C NMR (CDCl3) δ 19.0, 55.3, 60.8,
65.4, 68.2, 83.0, 87.1, 87.8, 102.3, 113.4, 117.7, 127.2, 128.1,
128.2, 130.1, 130.2, 135.1, 135.3, 139.8, 144.4, 150.7, 158.7,
158.8, 163.9; HRMS calcd for C33H33N3O8 (M + Na+) 622.2165,
found 622.2162.
Loading of 2′-O-(2-Cyanoethyl)uridine on CPG Resin.
Compound 5a (180 mg, 0.30 mmol) was dissolved in dry CH2-
Cl2 (30 mL). To the solution were added 4-(dimethylamino)-
pyridine (48 mg, 0.39 mmol) and succinic anhydride (60 mg,
0.60 mmol). After being stirred at room temperature for 20 h,
the mixture was washed with aqueous 10% citric acid and H2O.
The organic layer was dried over Na2SO4 and concentrated in
vacuo. The residue was chromatographed on a column of silica
gel with CHCl3-MeOH (90:10, v/v) to give the 3′-O-succinate
as white foam (151 mg, 73%): 1H NMR (CDCl3, 500 MHz) δ
2.38-2.42 (1 H, m), 2.51-2.55 (1 H, m), 2.54-2.93 (4 H, m),
3.38 (1 H, dd, J ) 2.0, 11.7), 3.75-3.80 (7 H, m), 3.90-3.94 (1
H, m), 4.02-4.06 (1 H, m), 4.38-4.41 (1 H, m), 5.13 (1H, dd,
J ) 4.9, 9.8), 5.32 (1 H, dd, J ) 1.7, 8.1), 5.79 (1 H, s), 6.81-
6.85 (4H, m), 7.22-7.38 (9H, m), 8.21 (1 H, d, J ) 8.1), 11.17
(1H, br); 13C NMR (CDCl3) δ 19.4, 28.0, 28.2, 55.4, 59.9, 67.2,
68.5, 80.1, 81.7, 87.4, 89.7, 101.0, 113.4, 113.5, 118.7, 127.3,
128.2, 130.3, 135.1, 140.8, 144.3, 149.4, 158.9, 166.7, 172.0,
177.4.
An LCAA CPG resin (500 mg, 101.9 µmol/g) was suspended
in CH2Cl2 (5 mL). To the solution were added 3′-O-succinate
(18 mg, 26 µmol) and DCC (16 mg, 78 µmol). After being stirred
at room temperature for 15 h, the resin was filtered and
washed with dry CH2Cl2 and dry CH3CN. To the resin was
added a capping solution (pyridine-Ac2O, 9:1, v/v) in advance
and stirred at room temperature for 12 h. The resin was
washed with dry CH2Cl2 and dry CH3CN and dried in vacuo.
The amount of Uce loaded to the solid support was calculated
to be 16 µmol/g from calculation of the released dimethoxytrityl
cation by use of a solution of 60% HClO4-EtOH (3:2, v/v).
2-Cyanoethyl 2′-O-(2-Cyanoethyl)-5′-O-(4,4′-dimeth-
oxytrityl)uridine 3′-(N,N-Diisopropyl)phosphoramidite
(6a). Compound 5a (1.20 g, 2.00 mmol) was coevaporated 5
times with dry toluene and dissolved in dry CH2Cl2 (10 mL)
under argon atmosphere. To the solution were added ethyl-
diisopropylamine (0.5 mL, 2.87 mmol) and a solution of chloro-
(2-cyanoethoxy)(N,N-diisopropylamino)phosphine (521 mg, 2.20
mmol) in dry CH2Cl2 (2 mL). After being stirred at room
temperature for 2 h, the mixture was diluted with CHCl3. The
solution was washed with brine and aqueous NaHCO3. The
organic layer was dried over Na2SO4 and filtered. The solution
was evaporated in vacuo. The residue was chromatographed
on a column of silica gel with CHCl3-MeOH (98:2, v/v)
containing 0.5% triethylamine to give compound 6a as white
foam (1.33 g, 83%): 1H NMR CDCl3, 500 MHz) δ 1.11-1.29
(12 H, m), 2.42-2.73 (4 H, m), 3.42-4.66 (16 H, m), 5.19-
5.26 (1 H, m), 5.87-5.88 (1 H, m), 6.82-6.87 (4 H, m), 7.23-
7.43 (9 H, m), 8.05-8.11 (1 H, m); 13C NMR (CDCl3) δ 19.0,
19.1, 20.6, 20.7, 22.0, 22.8, 23.1, 24.6, 24.7, 24.8, 43.3, 43.4,
45.4, 55.4, 58.0, 59.4, 60.5, 65.6, 65.9, 69.0, 69.7, 69.8, 80.5,
80.6, 81.7, 82.3, 82.8, 87.2, 88.9, 89.2, 102.2, 102.3, 113.4, 113.5,
117.8, 118.1, 127.3, 127.5, 128.1, 128.2, 128.4, 130.4, 134.7,
135.1, 135.2, 139.4, 139.9, 143.81, 144.3, 150.3, 150.4, 158.8,
158.9, 159.0, 163.3, 163.4; 31P NMR (CDCl3) δ 151.1, 150.0;
HRMS calcd for C42H50N5O9P (M + H+) 800.3424, found
800.3419
Oligonucleotide Synthesis. Oligoribonucleotides were
synthesized on an Applied Biosystems 392 oligonucleotide
synthesizer on a 1 µmol, using 2′-O-cyanoethylated phosphor-
amidite building blocks 6a-6d and/or 2′-O-TBDMS PAC
phosphoramidites (PacA, isopropyl-PAC-G, and Acetyl-C) from
Glen Research. A 0.1 M solution of each 2′-O-Ce or 2′-O-
TBDMS nucleoside phosphoramidite was used, and the time
for coupling time was set to be 10 min. 1H-Tetrazole (0.45 M;
for fully 2′-O-cyanoethylated RNA) and 5-ethylthio-1H-tetra-
zole (0.25 M; for one point modified RNAs) were used as the
activator. Deprotection was carried out by use of ammonium
hydroxide at room temperature for 20 min (for fully 2′-O-
cyanoethylated oligouridylate), NH4OH-NH4OAc (10:1, w/w)
at room temperature for 90 min (for fully cyanoethylated RNAs
containing U, C, A, and G), propylamine-THF (1:1, v/v) at 40
°C for 24 h, and Et3N‚3HF at room temperature for 24 h (one-
point modified RNAs).
The products were analyzed after C18 cartridge purification
by use of anion-exchange HPLC as shown in Figure 2
(chromatographic conditions: a linear gradient of 25 mM
sodium phosphate buffer containing 1 M NaCl (pH 6.0), 0-50%
for 50 min, in 25 mM sodium phosphate buffer, pH 6.0, at a
flow rate of 1 mL/min at 50 °C). The pure materials of the
following oligoribonucleotides were obtained after additional
reversed-phase HPLC (chromatographic conditions: a linear
gradient of acetonitrile, 0-30% for 30 min, in 100 mM
ammonium acetate, pH 7.0, at a flow rate of 1 mL/min at 50
°C) for UceU or anion-exchange HPLC for the other oligo-
nucleotides in the following yields. UceU (80%), (Uce)12 (21%),
GceAceCceUce
(58%),
GceAceCceUceGceAceCceUce-
GceAceCceUce (6%), GCUAGceACUAUCUA (10%), GCUA-
GAceCUAUCUA (12%), GCUAGACceUAUCUA (13%), GC-
UAGACUceAUCUA (14%), GAGCCAceAGCIUCGGCUC (33%).
Nuclease Resistance Assay. The nuclease stability of the
2′-O-cyanoethylated oligonucleotides was evaluated by treat-
ment with snake venom phosphodiesterase (Purchased from
Sigma) or bovine spleen phosphodiesterase (Purchased from
Sigma). The enzyme assay using snake venom phosphodi-
esterase (5 × 10-4 u/mL) was performed in a buffer of 50 mM
Tris-HCl at pH 8.5, 72 mM NaCl and 14 mM MgCl2 at 37 °C
by use of 50 µM oligonucleotide. The enzyme assay using
bovine spleen phosphodiesterase (0.2 U/mL) was performed
in a buffer 30 mM NaOAc at pH 6.0 at 37 °C by use of 50 µM
oligonucleotide. After the enzyme was deactivated by heating
at 100 °C for 2 min, the solution was diluted and filtered by a
0.45-µm filter (Millex-HV, Millipore). The mixture was ana-
lyzed by reverse-phase HPLC or anion exchange HPLC.
Acknowledgment. This work was supported by a
Grant from CREST of JST (Japan Science and Technol-
ogy Agency) and a Grant-in-Aid for Scientific Research
from the Ministry of Education, Culture, Sports, Science
and Technology, Japan. This work was also supported
by the COE21 project. This work was supported in part
by a grant of the Genome Network Project from the
Ministry of Education, Culture, Sports, Science and
Technology, Japan.
Supporting Information Available: General procedure
and the procedures for the synthesis of 2′-O-cyanoethylated
adenosine, guanosine, cytidine, and their phosphoroamidites.
1H NMR, 13C NMR, and 31P NMR spectra of all new com-
pounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
JO051741R
10460 J. Org. Chem., Vol. 70, No. 25, 2005