Synthesis and properties of siRNAs containing 5′-amino-2′,5′-dideoxy-2′α-fluororibonucleosides

Article abstract of DOI:10.1016/j.tet.2008.08.117

Short interfering RNAs (siRNAs) containing P3′→N5′ phosphoramidate linkages were successfully synthesized by introducing 2′-deoxy-2′-fluororibonucleoside and 5′-amino-2′,5′-dideoxy-2′α-fluororibonucleoside in succession. It was found that the introduction

Full text of DOI:10.1016/j.tet.2008.08.117

Tetrahedron 64 (2008) 11328–11334
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis and properties of siRNAs containing 50-amino-20,50-dideoxy-
20
a-fluororibonucleosides
,
b
,
d
,
e
,
a
a
a
a,d
Yoshihito Ueno a
, Miki Hirai , Kayo Yoshikawa , Yoshiaki Kitamura , Yoko Hirata
,
*
Kazutoshi Kiuchi a d, Yukio Kitade a
,
c
,
,b,c,d,
*
a Department of Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
b Center for Emerging Infectious Diseases, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
c Center for Advanced Drug Research, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
d United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
e PRESTO, JST (Japan Science and Technology Agency), 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan
a r t i c l e i n f o
a b s t r a c t
Short interfering RNAs (siRNAs) containing P30/N50 phosphoramidate linkages were successfully
synthesized by introducing 20-deoxy-20-fluororibonucleoside and 50-amino-20,50-dideoxy-20
-fluoror-
ibonucleoside in succession. It was found that the introduction of 50-amino-20,50-dideoxy-20
-fluoro-
ribonucleosides into siRNAs improved the nuclease-resistant properties of the siRNAs without loss of
their silencing efficacy.
Article history:
Received 16 June 2008
Received in revised form 27 August 2008
Accepted 27 August 2008
Available online 4 October 2008
a
a
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
RNAi
siRNA
Phosphoramidate
Fluoride
HeLa cells
1. Introduction
RNA interference (RNAi) induced by small interfering RNA
(siRNA) has emerged as a powerful technique to silence gene ex-
pression post-transcriptionally.1,2 Several research groups have
demonstrated the efficacy of siRNA-mediated inhibition of clini-
cally relevant genes not only in vitro but also in vivo.2–4 An im-
provement in the nuclease stability of siRNA is an important issue
in the therapeutic application of synthetic siRNA. Thus far, several
types of siRNAs modified at the base, sugar, or phosphate moieties
have been synthesized, and their nuclease-resistant properties and
RNAi-inducing activities have been studied.5–26 Among the
B
O
P
HN
O-
O
O
B
O
HN
P
O
O
O-
P3' N5'
phosphoramidate
N3' P5'
phosphoramidate
Figure 1. Structures of phosphoramidates.
abovementioned methods, the introduction of 20-deoxy-20
a-fluo-
roribonucleosides instead of ribonucleosides into siRNAs is an at-
tractive modification because it enhances both the chemical and
biological stabilities of siRNAs without largely reducing their RNAi-
inducing activities.11,17,22
On the other hand, it has been reported that DNAs possessing
phosphoramidate linkages are more nuclease-resistant than the
unmodified DNAs. Till date, two types of DNAs containing phos-
phoramidate linkages have been reported (Fig. 1):27–36 DNA pos-
sessing a P30/N50 phosphoramidate27–29 and DNA containing an
N30/P50 phosphoramidate.30–37 The DNA possessing the N30/P50
phosphoramidate increases the thermal stability of duplexes with
DNA and RNA complements. On the other hand, the DNA containing
the P30/N50 phosphoramidate largely decreases the thermal sta-
bility of duplexes with DNA and RNA complements. Thus, the DNA
containing the N30/P50 phosphoramidate has been extensively
used for antisense studies.38,39
* Corresponding authors. Tel.: þ81 58 293 2639; fax: þ81 58 293 2749.
E-mail addresses: uenoy@gifu-u.ac.jp (Y. Ueno), ykkitade@gifu-u.ac.jp
(Y. Kitade).
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.08.117

Y. Ueno et al. / Tetrahedron 64 (2008) 11328–11334
11329
O
N
NH2
N
2. Results
NH
2.1. Synthesis of phosphoramidite units
O
N
O
HO
HO
O
O
In order to synthesize oligoribonucleotides (ONs) containing 3
and 4 using a DNA/RNA synthesizer, phosphoramidites of 3 and 4
were prepared according to the routes shown in Schemes 1 and 2.
HO
F
HO
F
20-Deoxy-20- -fluorouridine (1)40 was treated with NaN3, Ph3P, and
a
1
2
CBr4 in DMF to afford a 50-azide derivative 5 in 40% yield. Catalytic
hydrogenation of 5 in MeOH and subsequent protection of the
O
N
NH2
N
NH
amino group with a 4-monomethoxytrityl (MMTr) group afforded
0
an N5 -MMTr derivative 6 in 61% yield. Compound 6 was phosphi-
O
N
O
H2N
H2N
tylated by the standard procedure41 to afford the corresponding
phosphoramidite 7 in 54% yield, which was used as the nucleotide
unit for a DNA/RNA synthesizer. To incorporate 3 into the 30-ends of
the ONs, 6 was further modified to afford the corresponding
30-succinate, which was then reacted with controlled pore glass
O
O
HO
F
HO
F
3
4
Figure 2. Structures of modified nucleosides.
(CPG) to afford a solid support containing 3 (20 mmol/g).
The cytidine derivative 10 was derived from the uridine de-
rivative 6. After protection of the 30-hydroxyl function of 6 with
a tert-butyldimethylsilyl (TBDMS) group, the O-TBDMS derivative 9
was treated with 2,4,6-triisopropylbenzenesulfonylchloride (TPSCl)
in the presence of DMAP and Et3N, followed by treatment with
NH4OH to afford the cytidine derivative 10 in 96% yield. After
protection of the exo-amino function of 10 with a benzoyl (Bz)
group, the silyl group was removed by treating with TBAF to afford
N5-MMTr derivative 11 in 65% yield. Compound 11 was phosphi-
tylated by the standard procedure41 to produce the corresponding
phosphoramidite 12 in 46% yield.
However, from the synthetic point of view, the introduction of
the P30/N50 phosphoramidate linkage in the DNA moiety is an
attractive modification because the DNA containing the same can
be easily synthesized by using 30-O-phosphoramidites of 50-amino-
20,50-dideoxyribonucleosides and the normal commercially avail-
able 30-O-phosphoramidite units.
From these background information, we planned to synthesize
siRNAs containing 50-amino-20,50-dideoxy-20
a-fluororibonucleo-
sides (Fig. 2). We envisioned that the siRNAs would be more nu-
clease-resistant after the introduction of the phosphoramidate
linkage, and the introduction of a fluorine atom into the 20
a-posi-
tion of the analogs would enhance the thermal stability of the
siRNAs. In this paper, we report the synthesis and properties of
2.2. Sugar conformation
siRNAs containing 50-amino-20,50-dideoxy-20
a
-fluorouridine (3)
Before 3 and 4 were introduced into RNAs, the conformation of
the sugar moiety of 3 was studied by 1H NMR. The fractional
populations of the N-conformers of 3, thymidine, and 50-amino-50-
and 50-amino-20,50-dideoxy-20
a
-fluorocytidine (4).
0
0
deoxythymidine were calculated by the formulae, %S¼(J1 ,2 ꢀ1)/
O
O
N
O
N
6.9ꢁ100 and %N¼100ꢀ%S.42 The J1 ,2 values of 3, thymidine, and
50-amino-50-deoxythymidine were 2.6 Hz, 6.9 Hz, and 6.9 Hz,
respectively. Thus, the %N values of 3, thymidine, and 50-amino-
50-deoxythymidine were estimated to be 77%, 14%, and 14%, re-
spectively. From these results, it was found that the introduction
a
of a fluorine atom to the 20 -position of 50-amino-50-deoxypyr-
imidine shifts the conformational equilibrium of the sugar moiety
0
0
NH
NH
NH
N
O
O
O
HO
N3
H2N
O
O
O
a
b
HO
F
HO
F
HO
F
1
5
3
to a C30-endo conformation.
O
N
c
NH
O
H
N
2.3. Synthesis of ONs
O
MMTr
O
N
NH
d
All ONs were synthesized using a DNA/RNA synthesizer. It is
reported that the P30/N50 phosphoramidate linkage in RNA is
very unstable and is easily hydrolyzed.43 The lability of the
linkage has been interpreted by the neighboring participation
effect of the 20-hydroxyl group adjacent to the N-protonated
phosphoramidate. To avoid this effect, 20-fluoro-20-deoxynucleo-
H
N
N
O
MMTr
O
P
F
O
NC
O
HO
F
7
O
N
6
NH
e
side and 50-amino-20,50-dideoxy-20
troduced into the ONs in succession (Table 1). The fully protected
ONs (1.0 mol each) linked to solid supports were treated with
a-fluoronucleoside were in-
H
O
MMTr
O
N
O
m
8
concentrated NH4OH/EtOH (3:1, v/v) at room temperature for
12 h and then with 1.0 M TBAF/THF at room temperature for 12 h.
Released ONs were purified by denaturing 20% polyacrylamide
gel electrophoresis (20% PAGE) to afford deprotected ONs 23–28.
These ONs were analyzed by matrix-assisted laser desorption/
ionization time-of-flight mass spectrometry (MALDI-TOF/MS),
and their observed molecular weights were in agreement with
their structures.
O
F
N
H
O
controlled pore glass
(CPG)
Scheme 1. Reagents and conditions: (a) NaN3, Ph3P, CBr4, DMF, rt, 28 h, 40%; (b) H2, Pd/C,
MeOH, rt, 12 h, 95%; (c) MMTrCl, DMAP, pyridine, rt, 18 h, 61%; (d) chloro-
(2-cyanoethoxy)(N,N-diisopropylamino)phosphine, i-Pr2NEt, THF, rt, 1 h, 54%; and (e)
(1) succinic anhydride, DMAP, pyridine, rt, 20 h; (2) CPG, WSCI, DMF, rt, 72 h, 20 mmol/g.

11330
Y. Ueno et al. / Tetrahedron 64 (2008) 11328–11334
O
N
O
NH2
N
NH
NH
H
N
H
H
N
O
N
O
N
O
MMTr
MMTr
N
MMTr
O
O
O
a
b
HO
F
TBDMSO
F
TBDMSO
F
6
9
10
c
Bz
N
HN
N
Bz
N
HN
N
H
O
H
N
MMTr
NC
N
O
MMTr
O
O
d
O
P
F
HO
F
O
N
11
12
Scheme 2. Reagents and conditions: (a) TBDMSCl, imidazole, DMF, rt, 12 h, 92%; (b) (1) 2,4,6,-triisopropylbenzenesulfonyl chloride, Et3N, DMAP, CH3CN, rt, 1 h, then concd NH4OH,
ꢂC to rt, 2 h, 96%; (c) (1) BzCl, pyridine, rt, 30 min, (2) TBAF, THF, rt, 19 h, 65%; and (d) chloro(2-cyanoethoxy)(N,N-diisopropylamino)phosphine, i-Pr2NEt, THF, rt, 1 h, 46%.
0
2.4. Thermal stabilities of siRNAs
imply that the global conformation of siRNA 14 containing analog 4
is not significantly different from that of the normal siRNA 13.
The thermal stability of the siRNAs was studied by thermal
denaturation in a buffer of 0.1 M sodium phosphate (pH 7.0) con-
taining 0.01 M NaCl. The melting temperatures (Tms) of siRNAs 13,
14, and 15 were 76.0 ꢂC, 78.5 ꢂC, and 77.0 ꢂC, respectively. From
these results, it was found that although the thermal stability of
siRNA 15 containing 1 and 4 is lower than that of siRNA 14 con-
taining 1 and 2, the introduction of the analogs 1 and 4 slightly
enhances the thermal stability of the duplex as compared to that of
normal siRNA 13.
2.6. Dual-luciferase assay
The ability of the modified siRNAs to suppress gene expression
was studied by a dual-luciferase assay using a psiCHECK-2 vector,
which contained Renilla and firefly luciferase genes. The siRNA
sequences were designed to target Renilla luciferase. HeLa cells
were co-transfected with the vector and indicated the amounts of
siRNAs. The signals of Renilla luciferase were normalized to those of
the firefly luciferase.
As shown in Figure 4, the silencing activity of siRNA 15 con-
taining the phosphoramidate linkage was slightly weaker than that
of siRNA 13 at each concentration. However, the RNAi-inducing
ability of siRNA 15 containing the phosphoramidate linkage was
almost equal to that of siRNA 14.
Next, in order to examine the difference in the effects of modi-
fications between passenger strands (sense strands) and guide
strands (antisense strands), the silencing activities of siRNAs 16 and
17 were tested. siRNA 16 contains the modified nucleoside 4 in the
guide strand, whereas siRNA 17 involves the modified nucleoside 4
in the passenger strand. As shown in Figure 5, the silencing activity
of siRNA 16 containing the analog in the guide strand was slightly
2.5. Circular dichroism (CD)
To study the global conformation of the siRNAs containing the
analogs, the CD spectra of the siRNAs were measured in a buffer of
0.1 M sodium phosphate (pH 7.0) containing 0.01 M NaCl. As shown
in Figure 3, negative and positive CD bands at w209 nm and
w263 nm, respectively, which were attributable to A-type duplexes,
were observed. Although the intensity of the positive band of siRNA
14 containing analog 4 was slightly smaller than that of normal
siRNA 13, the shapes of their spectra were similar. These results
Table 1
Sequences of oligonucleotides (ONs) used in this study
1.5X106
No. of siRNA
No. of ON
sequence
siRNA13
siRNA13
ON 18
ON 19
ON 20
ON 21
ON 22
ON 23
ON 22
ON 19
ON 18
ON 23
ON 24
ON 25
ON 26
ON 27
ON 28
ON 29
50-CUUCUUCGUCGAGACCAUGtt-30
30-ttGAAGAAGCAGCUCUGGUAC-50
50-CUUCU1 2GUCGAGACCAUGtt-30
30-ttGAAGAAGCAG2 1CUGGUAC-50
50-CUUCU1 4GUCGAGACCAUGtt-30
30-ttGAAGAAGCAG4 1CUGGUAC-50
50-CUUCUUCGUCGAGACCAUGtt-30
30-ttGAAGAAGCAG4 1CUGGUAC-50
50-CUUCU1 4GUCGAGACCAUGtt-30
30-ttGAAGAAGCAGCUCUGGUAC-50
F-50-AAAAAAAAAAAAAAAUU-30
F-50-AAAAAAAAAAAAAAA1 1-30
F-50-AAAAAAAAAAAAAAA1 3-30
F-50-AAAAAAAAUUAAAAAAA-30
F-50-AAAAAAAA1 1AAAAAAA-30
F-50-AAAAAAAA1 3AAAAAAA-30
siRNA14
siRNA15
siRNA14
siRNA15
siRNA16
siRNA17
0
d
d
d
d
d
d
-1.5X106
200
250
300
350
Wavelength (nm)
The uppercased letters indicate ribonucleosides and lowercased italicized letters
represent 20-deoxyribonucleosides. F indicates fluorescein.
Figure 3. Circular dichroism (CD) spectra.

Y. Ueno et al. / Tetrahedron 64 (2008) 11328–11334
11331
1.5
1.0
0.5
0
(a)
(b)
(c)
0.1 nM
1.0 nM
10 nM
1
2 3 4 5
6
7 8
9
1
2 3 4
5
6
7 8 9 1 2 3 4
5
6
7 8
9
Figure 6. 20% PAGE of ONs hydrolyzed by SVPD. (a) ON 24; (b) ON 25; and (c) ON 26.
ONs were incubated with SVPD for 0 min (lane 1), 1 min (lane 2), 5 min (lane 3), 10 min
(lane 4), 30 min (lane 5), 1 h (lane 6), 3 h (lane 7), 6 h (lane 8), and 15 h (lane 9). Ex-
perimental conditions are described in Section 4.
the band corresponding to the ON 29 remained stable under the
same conditions (lane 16). Thus, it was found that ON 29 containing
the phosphoramidate linkage was more resistant to RNase A than
ON 28.
siRNA13
siRNA14
siRNA15
Figure 4. Dual-luciferase assay (1). Experimental conditions are described in Section 4.
Finally, the stability of the ONs in bovine serum was examined.
ONs 24, 25, and 26 were incubated in PBS containing 5% bovine
serum and analyzed by 20% PAGE under denaturing conditions
(Fig. 8). The unmodified ON 27 was completely digested after 1 min
incubation, and a band attributable to the cleavage around the
uridine residues was also observed. On the other hand, ONs 28 and
29 were gradually digested from the 30-ends of the strands. The
results imply that the ONs containing analogs 1 and 3 are highly
resistant to the activity of endonuclease in bovine serum.
weaker than that of siRNA 17 containing the analog in the pas-
senger strand at each concentration. From these results, it was
suggested that the modification at the guide strand influences the
silencing activities of siRNAs more significantly than the modifi-
cation at the passenger strand dose.
2.7. Nuclease resistance
We examined the susceptibility of the ONs containing the ana-
logs to nucleolytic digestion. In this study, we used two types of
nucleases, snake venom phosphodiesterase (SVPD) and RNase A, as
models for a 30-exonuclease and an endonuclease, respectively. The
stability of the ONs in bovine serum was also examined.
ONs 24, 25, and 26 labeled at their 50-ends with fluorescein were
incubated with SVPD. ONs 25 and 26 contain the modified nucle-
osides at their 30-ends. The reactions were analyzed by PAGE under
denaturing conditions. As shown in Figure 6, after 1 h of incubation,
all the ONs were completely hydrolyzed. Thus, it was found that the
ON containing the phosphoramidate linkage was not resistant to
30-exonuclease.
Next, the susceptibility of ONs 27, 28, and 29 to nucleolytic di-
gestion by RNase A was studied. RNase A preferentially cleaves ONs
on the 30-side of the pyrimidine nucleosides. ONs 28 and 29 contain
the modified nucleosides at the middle of polyadenylate sequences.
As shown in Figure 7a, ONs 28 and 29 were highly resistant to the
nuclease under the conditions where the unmodified ON was
completely digested. In order to compare the nuclease-resistant
property of ON 29 containing the phosphoramidate linkage with
that of ON 28, the ONs were incubated with the enzyme at a high
concentration. As shown in Figure 7b, though the band corre-
sponding to the ON 28 disappeared after 3 h incubation (lane 10),
3. Discussion
We have designed and synthesized siRNA possessing P30/N50
phosphoramidate linkages. It has been reported that P30/N50
phosphoramidate linkages in RNA are very unstable and easily
hydrolyzed.43 The lability of the linkages has been explained on the
basis of the neighboring group participation effect of the 20-hy-
droxyl group adjacent to the N-protonated phosphoramidate. We
have succeeded in synthesizing RNA possessing phosphoramidate
linkages by the successive introduction of 20-deoxy-20-fluoror-
ibonucleoside and 50-amino-20,50-dideoxy-20
side into the RNA.
a-fluororibonucleo-
The thermal stability of siRNA possessing phosphoramidate
linkages was studied by thermal denaturation. It has been reported
that DNA containing P30/N50 phosphoramidate significantly
(a)
1
2
3
4
5
6
7
8 9 10 11 12 13 14 15 16 17 18 19 20 21
1.5
0.1 nM
1.0 nM
10 nM
(b)
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18
1.0
0.5
0
Figure 7. 20% PAGE of ONs hydrolyzed by RNase A. (a) ON 27 (lanes 1–7); ON 28 (lanes
8–14); ON 29 (lanes 15–21). ONs were incubated with RNase A (10 ng) for 0 min (lanes
1, 8, and 15), 5 min (lanes 2, 9, and 16), 10 min (lanes 3, 10, and 17), 20 min (lanes 4, 11,
and 18), 30 min (lanes 5, 12, and 19), 60 min (lanes 6, 13, and 20), and 90 min (lanes 7,
14, and 21). (b) ON 27 (lanes 1–6); ON 28 (lanes 7–12); ON 29 (lanes 13–18). ONs were
incubated with RNase A (100 ng) for 0 min (lanes 1, 7, and 13), 1 h (lanes 2, 8, and 14),
2 h (lanes 3, 9, and 15), 3 h (lanes 4, 10, and 16), 5 h (lanes 5, 11, and 17), and 7 h (lanes
6, 12, and 18). Experimental conditions are described in Section 4.
siRNA13
siRNA15
siRNA16
siRNA17
Figure 5. Dual-luciferase assay (2). Experimental conditions are described in Section 4.

11332
Y. Ueno et al. / Tetrahedron 64 (2008) 11328–11334
(a)
(b)
(c)
4.2. 50-Azide-20,50-dideoxy-20-fluorouridine (5)
mixture of 20,50-dideoxy-20-fluorouridine (1) (1.96 g,
1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10
A
7.96 mmol), NaN3 (2.59 g, 39.8 mmol), Ph3P (2.71 g, 10.3 mmol),
and CBr4 (3.96 g, 11.9 mmol) in DMF (40 mL) was stirred at room
temperature. After 4 h, Ph3P (1.04 g, 4.00 mmol) and CBr4 (2.64 g,
7.96 mmol) were added to the mixture, and then the entire mixture
was stirred at room temperature. After 24 h, the solvent was
evaporated in vacuo, and the residue was partitioned between
EtOAc and H2O. The organic layer was washed with brine, dried
(Na2SO4), and concentrated. The residue was purified by column
chromatography (SiO2, 5–6% MeOH in CHCl3) to afford 5 (0.86 g,
Figure 8. 20% PAGE of ONs incubated in PBS containing 5% bovine serum. (a) ON 24;
(b) ON 25; (c) ON 26. ONs were incubated for 0 min (lane 1), 1 min (lane 2), 5 min (lane
3), 10 min (lane 4), 30 min (lane 5), 1 h (lane 6), 3 h (lane 7), 6 h (lane 8), 13 h (lane 9),
and 24 h (lane 10). Experimental conditions are described in Section 4.
3.17 mmol) in 40% yield: 1H NMR (DMSO-d6)
d
3.52 (dd, 1H, J¼5.6
and 13.7, 50-H), 3.73 (dd, 1H, J¼2.8 and 13.7, 50-H), 3.93 (m, 1H,
40-H), 4.23 (m, 1H, 30-H), 5.19 (ddd, 1H, J¼1.2, 4.7 and 52.4, 20-H),
5.65 (d, 1H, J¼8.0, 5-H), 5.74 (d, 1H, J¼6.5, 30-OH), 5.85 (dd,1H, J¼1.2
and 21.7, 10-H), 7.66 (d, 1H, J¼8.0, 6-H), 11.44 (br s, 1H, 3-NH); 13C
decreases the thermal stability of duplexes with DNA and RNA
complements.27–29 The Tm values of normal siRNA 13, siRNA 14
containing 20-deoxy-20-fluororibonucleosides 1 and 2, and siRNA
15 containing
1 a-fluoror-
and the 50-amino-20,50-dideoxy-20
NMR (DMSO-d6)
d
50.61, 68.81 (d, J¼16.4), 80.56, 89.42 (d, J¼36.0),
ibonucleoside 4 were 76.0 ꢂC, 78.5 ꢂC, and 77.0 ꢂC, respectively.
Although the thermal stability of siRNA 15, containing 1 and 4, was
lower than that of siRNA 14, containing 1 and 2, the introduction of
analogs 1 and 4 to siRNA 15 did result in the enhancement of the
thermal stability of the duplex to a level slightly above that of siRNA
92.79 (d, J¼184.2), 102.02, 141.82, 150.15, 163.15; LRMS (FAB) m/z
272 (MHþ); HRMS (FAB) calcd for C9H11N5FO4 (MHþ) 272.0795,
found 272.0804. Anal. Calcd for C9H10N5FO5: C, 39.86; H, 3.72; N,
25.82. Found: C, 39.81; H, 3.70; N, 25.89.
4.3. 50-Amino-20,50-dideoxy-20-fluorouridine (3)
13. The %N values of 50-amino-20,50-dideoxy-20
a-fluorouridine (3)
was estimated to be 77%, which was similar to that of natural ri-
bonucleosides. Therefore, it was suggested that the introduction of
A mixture 5 (0.21 g, 0.77 mmol) and Pd–C (10%, 51 mg) in MeOH
(3.8 mL) was stirred under atmospheric pressure of H2 at room
temperature. After 12 h, the catalyst was filtered off with Celite. The
filtrate was evaporated under reduced pressure and dried in vacuo
a fluorine atom to the 20 -position of 50-amino-50-deoxypyrimidine
a
shifted the conformational equilibrium of the sugar moiety to a C30-
endo conformation, thus enhancing the thermal stability of the
A-type duplex even though it contained the phosphoramidate
linkage.
to give 3 (0.18 g, 0.73 mmol) in 95% yield: 1H NMR (DMSO-d6)
d 2.75
(dd, 1H, J¼4.6 and 13.9, 50-H), 2.88 (dd, 1H, J¼3.0 and 13.9, 50-H),
3.76 (m, 1H, 40-H), 4.11 (m, 1H, 30-H), 5.07 (dd, 1H, J¼2.6 and 53.3,
20-H), 5.60 (d, 1H, J¼8.0, 5-H), 5.86 (dd, 1H, J¼2.6 and 18.4, 10-H),
7.94 (d, 1H, J¼8.0, 6-H); LRMS (FAB) m/z 246 (MHþ); HRMS (FAB)
calcd for C9H13N3FO4 (MHþ) 246.0890, found 246.0884.
The RNAi-inducing ability of siRNA containing phosphor-
amidates was studied using a dual-luciferase assay. Although the
introduction of phosphoramidate linkages into siRNA marginally
reduced its silencing activity, the RNAi-inducing ability of siRNA
with phosphoramidates was almost equal to that of siRNA con-
taining the corresponding 20-deoxy-20-fluororibonucleosides. CD
spectroscopy revealed that the global conformation of siRNA 14
4.4. 50-Amino-20,50-dideoxy-20-fluoro-50-N-(40-
monomethoxytrityl)uridine (6)
containing 50-amino-20,50-dideoxy-20
a-fluorocytidine (4) was not
A mixture of 3 (0.18 g, 0.73 mmol), MMTrCl (0.30 g, 0.97 mmol),
significantly different from that of siRNA 13. Thus, it was shown
that the introduction of 4 into siRNA influences neither the global
conformation nor the RNAi-inducing ability of siRNA to a significant
extent.
and DMAP (9 mg, 74
temperature. After 6 h, MMTrCl (0.23 g, 0.74 mmol) and DMAP
(9 mg, 74 mol) were added to the mixture, and the whole mixture
mmol) in pyridine (3.7 mL) was stirred at room
m
was stirred at room temperature. After 12 h, the mixture was par-
titioned between EtOAc and aqueous NaHCO3 (saturated). The or-
ganic layer was washed with brine, dried (Na2SO4), and
concentrated. The residue was purified by column chromatography
(SiO2, 50–100% EtOAc in hexane) to give 6 (0.23 g, 0.45 mmol) in 61%
The susceptibility to nucleolytic digestion of the RNA containing
the analogs was also studied. It was found that although RNA that
contains phosphoramidate linkages was not resistant to snake
venom phosphodiesterase (30-exonuclease), it was more resistant
to nucleolytic hydrolysis by RNase A (an endonuclease) than nor-
mal RNA or RNA containing the corresponding 20-deoxy-20-fluo-
roribonucleosides. These results suggest that the introduction of
these phosphoramidate linkages into siRNA would be a useful
modification for improving the nuclease-resistant property of
siRNA.
yield: 1H NMR (CDCl3)
d
2.46 (dd,1H, J¼6.0 and 13.0, 50-H), 2.71 (dd,
1H, J¼3.4 and 13.0, 50-H), 3.79 (s, 3H, OCH3), 4.06 (m, 1H, 40-H), 4.34
(m,1H, 30-H), 5.10 (dd,1H, J¼4.6 and 53.3, 20-H), 5.69 (d,1H, J¼8.0, 5-
H), 5.79 (d, 1H, J¼19.1, 10-H), 6.81–7.46 (m, 18H, 6-H and MMTr); 13C
NMR (CDCl3)
d
44.66, 55.21, 70.24, 70.79 (d, J¼17.2), 82.42, 90.29 (d,
J¼36.0), 93.50 (d, J¼185.0), 94.43, 102.83, 113.26, 126.49, 127.97,
128.45, 129.75, 137.54, 140.64, 145.75, 149.52, 158.03, 162.67; LRMS
(FAB) m/z 518 (MHþ); HRMS (FAB) calcd for C29H29N3FO5 (MHþ)
518.2091, found 518.2082. Anal. Calcd for C29H28N3FO5$1/4H2O: C,
66.72; H, 5.50; N, 8.05. Found: C, 66.77; H, 5.50; N, 7.95.
4. Experimental section
4.1. General remarks
The NMR spectra were recorded at 400 MHz (1H) and 100 MHz
4.5. 50-Amino-30-O-[(2-cyanoethoxy)(N,N-diisopropylamino)-
phosphinyl]-20,50-dideoxy-20-fluoro-50-N-(40-
monomethoxytrityl)uridine (7)
(
13C), and were reported in parts per million downfield from tet-
ramethylsilane. The coupling constants (J) are expressed in hertz.
The mass spectra were obtained by fast atom bombardment (FAB).
Thin-layer chromatography was performed on Merck coated plates
60F254. Silica gel column chromatography was carried out on
Wakogel C-300.
A mixture of 6 (0.49 g, 0.94 mmol), N,N-diisopropylethylamine
(0.96 mL, 5.63 mmol), and chloro(2-cyanoethoxy)(N,N-diisopropyl-
amino)phosphine (0.42 mL, 1.88 mmol) in THF (4.7 mL) was stirred

Y. Ueno et al. / Tetrahedron 64 (2008) 11328–11334
11333
at room temperature for 1 h. The mixture was partitioned between
CHCl3 and aqueous NaHCO3 (saturated). The organic layer was
washed with brine, dried (Na2SO4), and concentrated. The residue
was purified by column chromatography (a neutralized SiO2, 50–
100% EtOAc in hexane) to give 7 (0.36 g, 0.51 mmol) in 54% yield:
MMTr); 13C NMR (CDCl3)
d
ꢀ5.09, ꢀ4.70, 17.98, 25.56, 45.36, 55.20,
70.32, 71.31 (d, J¼15.8), 82.40, 91.92 (d, J¼36.0), 93.17 (d, J¼188.0),
99.17, 113.20, 126.34, 127.89, 128.54, 129.83, 137.85, 141.94, 145.89,
155.22, 165.87. Anal. Calcd for C35H43N4FO4Si$6/5H2O: C, 64.43; H,
7.01; N, 8.59. Found: C, 64.08; H, 6.61; N, 8.28.
31P NMR (CDCl3)
d 151.2, 151.6.
4.9. 50-Amino-4-N-benzoyl-20,50-dideoxy-20-fluoro-50-N-(40-
4.6. Solid support synthesis
monomethoxytrityl)cytidine (11)
A mixture of 7 (0.20 g, 0.39 mmol), succinic anhydride (0.12 g,
1.20 mmol), and DMAP (12 mg, 0.10 mmol) in pyridine (4 mL) was
stirred at room temperature. After 20 h, the solution was parti-
tioned between CHCl3 and H2O, and the organic layer was washed
with H2O and brine. The separated organic phase was dried
(Na2SO4) and concentrated to give a succinate. Aminopropyl con-
A mixture of 10 (77 mg, 0.12 mmol) and BzCl (18 ml, 0.16 mmol)
in pyridine (1 mL) was stirred at room temperature. After 30 min,
the mixture was partitioned between EtOAc and H2O. The organic
layer was washed with aqueous NaHCO3 (saturated), brine, dried
(Na2SO4), and concentrated in vacuo. The residue was stirred with
TBAF (1 M in THF, 0.17 mL, 0.17 mmol) in THF (1.1 mL) at room
temperature. After 19 h, the solvent was evaporated, and the
resulting residue was purified with column chromatography (SiO2,
trolled pore glass (0.49 g, 96 mmol) was added to a solution of the
succinate and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (90 mg, 0.47 mmol) in DMF (10 mL), and the mixture
was kept for 72 h at room temperature. After the resin was washed
with pyridine, a capping solution (10 mL, 0.1 M DMAP in pyridine/
Ac2O¼9:1, v/v) was added and the whole mixture was kept for 24 h
at room temperature. The resin was washed with MeOH and ace-
tone, and dried in vacuo. Amount of loaded compound 7 to the solid
2% MeOH in CHCl3) to give 11 (45 mg, 73 m
mol) in 65% yield: 1H
NMR (CDCl3)
d
2.47 (dd,1H, J¼6.2 and 13.0, 50-H), 2.84 (m, 1H, 50-H),
3.81 (s, 3H, OCH3), 4.12 (d, 1H, J¼21.7, 30-H), 4.21 (m, 1H, 40-H), 5.12
(dd, 1H, J¼4.2 and 52.6, 20-H), 5.96 (d, 1H, J¼17.8, 10-H), 6.84–7.90
(m, 18H, 6-H and MMTr), 8.72 (br s, 1H, NH); 13C NMR (CDCl3)
d
45.12, 55.23, 70.37, 70.87 (d, J¼17.2), 82.38, 90.55 (d, J¼34.4),
support was 20
mmol/g from calculation of released dimethoxy-
93.28 (d, J¼186.5), 99.44, 113.31, 126.55, 127.59, 128.02, 128.46,
128.49, 129.08, 129.75, 133.34, 137.54, 145.70, 145.72, 158.06. Anal.
Calcd for C36H33N4FO5Si$H2O: C, 67.70; H, 5.52; N, 8.77. Found: C,
67.60; H, 5.42; N, 8.56.
trityl cation by a solution of 70% HClO4/EtOH (3:2, v/v).
4.7. 50-Amino-30-O-(tert-butyldimethylsilyl)-20,50-dideoxy-20-
fluoro-50-N-(40-monomethoxytrityl)uridine (9)
4.10. 50-Amino-N-4-benzoyl-30-O-[(2-cyanoethoxy)(N,N-
diisopropylamino)phosphinyl]-20-fluoro-20,50-dideoxy-
50-N-(40-monomethoxytrityl)cytidine (12)
A mixture of 6 (3.00 g, 5.80 mmol), TBDMSCl (1.31 g, 8.69 mmol),
and imidazole (1.18 g, 17.4 mmol) in DMF (19 mL) was stirred at
room temperature. After 12 h, EtOH (8 mL) was added to the mix-
ture, and then the whole mixture was stirred at room temperature.
After 30 min, the mixture was partitioned between EtOAc and
aqueous NaHCO3 (saturated). The organic layer was washed with
brine, dried (Na2SO4), and concentrated. The residue was purified
by column chromatography (SiO2, 30–40% EtOAc in hexane) to give
A mixture of 11 (0.19 g, 0.31 mmol), N,N-diisopropylethylamine
(0.32 mL, 1.84 mmol), and chloro(2-cyanoethoxy)(N,N-diisopropy-
lamino)phosphine (0.14 mL, 0.61 mmol) in THF (1.5 mL) was stirred
at room temperature for 1 h. The mixture was partitioned between
CHCl3 and aqueous NaHCO3 (saturated). The organic layer was
washed with brine, dried (Na2SO4), and concentrated. The residue
was purified by column chromatography (a neutralized SiO2, 50–
100% EtOAc in hexane) to give 12 (0.12 g, 0.51 mmol) in 46% yield:
9 (3.37 g, 5.32 mmol) in 92% yield: 1H NMR (CDCl3)
d 0.00–0.06 (m,
6H, Si–CH3), 0.85 (s, 9H, Si–t-Bu), 2.31 (dd, 1H, J¼6.8 and 12.8, 50-H),
2.65 (dd, 1H, J¼2.8 and 12.8, 50-H), 3.80 (s, 3H, OCH3), 4.09 (m, 1H,
40-H), 4.16 (m, 1H, 30-H), 4.96 (ddd, 1H, J¼1.8, 4.4, and 53.3, 20-H),
5.71 (d, 1H, J¼7.7, 5-H), 5.77 (dd, 1H, J¼1.8 and 18.6, 10-H), 6.82–7.48
(m, 18H, 6-H and MMTr), 8.21 (s, 1H, 3-NH); 13C NMR (CDCl3)
31P NMR (CDCl3)
d 151.4, 151.7.
4.11. RNA synthesis
d
ꢀ5.07, ꢀ4.71, 18.02, 25.56, 45.27, 55.22, 70.29, 71.40 (d, J¼16.4),
83.12, 90.97 (d, J¼35.2), 92.19 (d, J¼192.3), 102.71, 113.25, 126.46,
127.95,128.48,129.76, 137.64,140.89, 145.77, 149.48,158.03, 162.54;
LRMS (FAB) m/z 633 (MHþ).
Synthesis was carried out with a DNA/RNA synthesizer by
phosphoramidite method. Deprotection of bases and phosphates
was performed in concentrated NH4OH/EtOH (3:1, v/v) at room
temperature for 12 h. 20-TBDMS groups were removed by 1.0 M
tetrabutylammonium fluoride (TBAF, Aldrich) in THF at room
temperature for 12 h. The reaction was quenched with 0.1 M TEAA
buffer (pH 7.0) and desalted on a Sep-Pak C18 cartridge. Depro-
tected ONs were purified by 20% PAGE containing 7 M urea to give
the highly purified ON 20 (3), ON 21 (6), ON 22 (7), ON 23 (6), ON 25
(3), ON 26 (9), ON 28 (11), ON 29 (19). The yields are indicated in
4.8. 50-Amino-30-O-(tert-butyldimethylsilyl)-20,50-dideoxy-20-
fluoro-50-N-(40-monomethoxytrityl)cytidine (10)
A mixture of 9 (3.36 g, 5.32 mmol), Et3N (1.48 mL, 10.6 mmol),
DMAP (1.30 g, 10.6 mmol), and 2,4,6-triisopropylbenzenesulfonyl-
chloride (3.22 g, 10.6 mmol) in CH3CN (27 mL) was stirred at room
temperature for 1 h. The mixture was cooled in an ice-bath. Con-
centrated NH4OH (12.5 mL) was added, and the whole mixture was
stirred at room temperature for 2 h. The mixture was partitioned
between EtOAc and H2O. The organic layer was washed with
aqueous NaHCO3 (saturated), brine, dried (Na2SO4), and concen-
trated. The residue was purified by column chromatography (SiO2,
3% MeOH in CHCl3) to give 10 (3.22 g, 5.09 mmol) in 96% yield: 1H
parentheses as OD units at 260 nm starting from 1.0 mmol scale.
Extinction coefficients of the ONs were calculated from those of
mononucleotides and dinucleotides according to the nearest-
neighbor approximation method.44
4.12. MALDI-TOF/MS analysis of RNAs
NMR (CDCl3)
d
0.00 (s, 3H, Si–CH3), 0.08 (s, 3H, Si–CH3), 0.86 (s, 9H,
Spectra were obtained with a time-of-flight mass spectrometer.
ON20: calculated mass, 6583.9; observed mass, 6582.7. ON 21:
calculated mass, 6733.0; observed mass, 6732.2. ON 22: calculated
mass, 6581.9; observed mass, 6585.1. ON 23: calculated mass,
6731.0; observed mass, 6736.9. ON 25: calculated mass, 6056.3;
Si–t-Bu), 2.37 (dd, 1H, J¼7.0 and 12.7, 50-H), 2.71 (dd, 1H, J¼2.3 and
12.7, 50-H), 3.84 (s, 3H, OCH3), 4.05 (ddd, 1H, J¼4.5, 8.6, and 20.9, 30-
H), 4.24 (m,1H, 40-H), 5.03 (dd,1H, J¼4.5 and 53.3, 20-H), 5.67 (d,1H,
J¼7.2, 5-H), 5.81 (d, 1H, J¼18.8, 1-H), 6.86–7.54 (m, 18H, 6-H and

11334
Y. Ueno et al. / Tetrahedron 64 (2008) 11328–11334
observed mass, 6053.8. ON 26: calculated mass, 6055.3; observed
mass, 6057.3. ON 28: calculated mass, 6056.3; observed mass,
6052.3. ON 29: calculated mass, 6055.3; observed mass, 6057.3.
appropriate periods, aliquots (5
separated and added to a solution of 9 M urea (15
were analyzed by gel electrophoresis described above.
m
L) of the reaction mixture were
mL). The mixtures
4.13. Thermal denaturation and CD spectroscopy
Acknowledgements
Each solution containing each siRNA (3 mM) in a buffer com-
This study was supported by a grant from PRESTO of the Japan
ScienceandTechnologyAgency(JST)anda Grant-in-AidforScientific
Research from the Japan Society for the Promotion of Science (JSPS).
posed of 10 mM Na2HPO4/NaH2PO4 (pH 7.0) and 100 mM NaCl was
heated at 95 ꢂC for 5 min, then cooled gradually to an appropriate
temperature, and used for the thermal denaturation studies.
Thermal-induced transitions of each mixture were monitored at
260 nm with a spectrophotometer. The sample temperature was
increased by 0.5 ꢂC/min. CD spectra were measured by a spec-
tropolarimeter. Samples for CD spectroscopy were prepared by the
same procedure used in the thermal denaturation study, and
spectra were measured at 15 ꢂC. The molar ellipticity was calcu-
References and notes
1. Fire, A.; Xu, S.; Montgomery, M. K.; Kostas, S. A.; Driver, S. E.; Mello, C. C. Nature
1998, 391, 806.
2. Bumcrot, D.; Manoharan, M.; Koteliansky, V.; Sah, D. W. Y. Nat. Chem. Biol. 2006,
2, 711.
3. Behlke, M. A. Mol. Ther. 2006, 13, 644.
4. Kim, D. A.; Rossi, J. J. Nat. Rev. Genet. 2007, 8, 173.
5. Parrish, S.; Fleenor, J.; Xu, S.; Mello, C.; Fire, A. Mol. Cells 2000, 6, 1077.
6. Schwarz, D. S.; Hutva´gner, G.; Haley, B.; Zamore, P. D. Mol. Cells 2002, 10, 537.
7. Hamada, M.; Ohtsuka, T.; Kawaida, R.; Koizumi, M.; Morita, K.; Furukawa, H.;
Imanishi, T.; Miyagishi, M.; Taira, K. Antisense Nucleic Acid Drug Dev. 2002,12, 301.
8. Harborth, J.; Elbashir, S. M.; Vandenburgh, K.; Manninga, H.; Scaringe, S. A.;
Weber, K.; Tuschl, T. Antisense Nucleic Acid Drug Dev. 2003, 13, 83.
9. Chiu, Y.-L.; Rana, T. M. Mol. Cells 2002, 10, 549.
10. Chiu, Y.-L.; Rana, T. M. RNA 2003, 9, 1034.
11. Braasch, D. A.; Jensen, S.; Liu, Y.; Kaur, K.; Arar, K.; White, M. A.; Corey, D. R.
Biochemistry 2003, 42, 7967.
12. Amarzguioui, M.; Holen, T.; Babaie, E.; Prydz, H. Nucleic Acids Res. 2003, 31, 589.
13. Czauderna, F.; Fechtner, M.; Dames, S.; Aygu¨n, H.; Klippel, A.; Pronk, G. J.; Giese,
K.; Kaufmann, J. Nucleic Acids Res. 2003, 31, 2705.
14. Gru¨nweller, A.; Wyszko, E.; Bieber, B.; Jahnel, R.; Erdmann, V. A.; Kurreck, J.
Nucleic Acids Res. 2003, 31, 3185.
15. Hall, A. H. S.; Wan, J.; Shaughnessy, E. E.; Shaw, B. R.; Alexander, K. A. Nucleic
Acids Res. 2004, 32, 5991.
16. Hall, A. H. S.; Wan, J.; Spesock, A.; Sergueeva, Z.; Shaw, B. R.; Alexander, K. A.
Nucleic Acids Res. 2006, 34, 2773.
17. Prakash, T. P.; Allerson, C. R.; Dande, P.; Vickers, T. A.; Sioufi, N.; Jarres, R.; Baker,
B. F.; Swayze, E. E.; Griffey, R. H.; Bhat, B. J. Med. Chem. 2005, 48, 4247.
18. Hoshika, S.; Minakawa, N.; Kamiya, H.; Harashima, H.; Matsuda, A. FEBS Lett.
2005, 579, 3115.
lated from the equation [
c the sample concentration, and l the cell path length in centimeters.
q
]¼
q/cl, where q is the relative intensity,
4.14. Dual-luciferase assay
HeLa cells were grown at 37 ꢂC in a humidified atmosphere of
5% CO2 in air in minimum essential medium (MEM) (Invitrogen)
supplemented with 10% fetal bovine serum (FBS). Twenty-four
hours before transfection, HeLa cells (4ꢁ104/mL) were transferred
to 96-well plates (100 mL/well). They were transfected, using
TransFast (Promega), according to instructions for transfection of
adherent cell lines. Cells in each well were transfected with a so-
lution (35
dicated amounts of siRNAs, and 0.3
Reduced-Serum Medium (Invitrogen), and incubated at 37 ꢂC. After
1 h, MEM (100 L) containing 10% FBS and antibiotics was added to
mL) of 20 ng of psiCHECK-2 vector (Promega), the in-
mg of TransFast in Opti-MEM I
m
each well, and the whole mixture was further incubated at 37 ꢂC.
After 24 h, cell extracts were prepared in Passive Lysis Buffer
(Promega). Activities of firefly and Renilla luciferases in cell lysates
were determined with a dual-luciferase assay system (Promega)
according to a manufacturer’s protocol. The results were confirmed
by at least three independent transfection experiments with two
cultures each and are expressed as the average from four experi-
ments as meanꢃSD.
´
19. Elmen, J.; Thoberg, H.; Ljungberg, K.; Frieden, M.; Westergaard, M.; Xu, Y.;
Wahren, B.; Liang, Z.; Ørum, H.; Koch, T.; Wahlestedt, C. Nucleic Acids Res. 2005,
33, 439.
20. Dowler, T.; Bergeron, D.; Tedeschi, A.-L.; Paquet, L.; Ferrari, N.; Damha, M. J.
Nucleic Acids Res. 2006, 34, 1669.
21. Watts, J. K.; Choubdar, N.; Sadalapure, K.; Robert, F.; Wahba, A. S.; Pelletier, J.;
Pinto, B. M.; Damha, M. J. Nucleic Acids Res. 2007, 35, 1441.
22. Choung, S.; Kim, Y. J.; Kim, S.; Park, H.-O.; Choi, Y.-C. Biochem. Biophys. Res.
Commun. 2006, 342, 919.
23. Abe, N.; Abe, H.; Ito, Y. J. Am. Chem. Soc. 2007, 129, 15108.
24. Fisher, M.; Abramov, M.; Aerschot, A. V.; Xu, D.; Juliano, R. L.; Herdewijn, P.
Nucleic Acids Res. 2007, 35, 1064.
4.15. Partial hydrolysis of ONs with snake venom
phosphodiesterase
25. Bramsen, J. B.; Laursen, M. B.; Damgaard, C. K.; Lena, S. W.; Babu, B. R.; Wengel,
J.; Kjems, J. Nucleic Acids Res. 2007, 35, 5886.
26. Odadzic, D.; Bramsen, J. B.; Smicius, R.; Bus, C.; Kjems, J.; Engels, J. W. Bioorg.
Med. Chem. 2008, 16, 518.
27. Gryaznov, S. M.; Letsinger, R. L. Nucleic Acids Res. 1992, 20, 3403.
28. Gryaznov, S. M.; Lloyd, D. A.; Chen, J.-K.; Schultz, R. G.; DeDionisio, L. A.; Rat-
meyer, L.; Wilson, W. D. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 5798.
29. Obika, S.; Nakagawa, O.; Hiroto, A.; Hari, Y.; Imanishi, T. Chem. Commun. 2003,
2202.
30. Gryaznov, S. M.; Chen, J.-K. J. Am. Chem. Soc. 1994, 116, 3143.
31. Chen, J.-K.; Schultz, R. G.; Lloyd, D. H.; Gryaznov, S. M. Nucleic Acids Res. 1995,
23, 2611.
Each ON (600 pmol) labeled with fluorescein at 50-end was in-
cubated with snake venom phosphodiesterase (5ꢁ10ꢀ3 units) in
a buffer containing 33 mM Tris–HCl (pH 8.0) and 7 mM MgCl2 (total
300
action mixture were separated and added to a solution of 9 M urea
(15 L). The mixtures were analyzed by electrophoresis on 20%
m mL) of the re-
L) at 37 ꢂC. At appropriate periods, aliquots (5
m
polyacrylamide gel containing 7 M urea. The labeled ON in the gel
was visualized by a Typhoon system (Amersham Biosciences).
32. Schultz, R. G.; Gryaznov, S. M. Nucleic Acids Res. 1996, 24, 2966.
33. Mignet, N.; Gryaznov, S. M. Nucleic Acids Res. 1998, 26, 431.
34. Pongracz, K.; Gryaznov, S. M. Nucleic Acids Res. 1998, 26, 1099.
35. Gryaznov, S. M.; Winter, H. Nucleic Acids Res. 1998, 26, 4160.
36. Matray, T. J.; Gryaznov, S. M. Nucleic Acids Res. 1999, 27, 3976.
37. Schultz, R. G.; Gryaznov, S. M. Tetrahedron Lett. 2000, 41, 1895.
38. Gryaznov, S. M.; Skorski, T.; Cucco, C.; Nieborowska-Skorska, M.; Chiu, C. Y.;
Lloyd, D.; Chen, J.-K.; Koziolkiewicz, M.; Calabretta, B. Nucleic Acids Res. 1996,
24, 1508.
39. Heidenreich, O.; Gryaznov, S.; Nerenberg, M. Nucleic Acids Res. 1997, 25, 776.
40. Williams, D. M.; Benseler, F.; Eckstein, F. Biochemistry 1991, 30, 4001.
41. Beaucage, S. L.; Caruthers, M. H. Tetrahedron Lett. 1981, 22, 1859.
42. Altona, C.; Sundaralingam, M. J. Am. Chem. Soc. 1973, 95, 2333.
43. Wolfe, J. L.; Wang, B. H.; Kawate, T.; Stanton, V. P., Jr. J. Am. Chem. Soc. 2003, 125,
10500.
4.16. Partial hydrolysis of ONs with RNase A
Each ON (450 pmol) labeled with fluorescein at 50-end was in-
cubated with RNase A (10 ng or 100 ng) in a buffer of 10 mM Tris–
HCl (pH 8.0) containing 10 mM NaCl (total 300
appropriate periods, aliquots (5 L) of the reaction mixture were
separated and added to a solution of 9 M urea (15 L). The mixtures
m
L) at 37 ꢂC. At
m
m
were analyzed by gel electrophoresis described above.
4.17. Stability of ON in the PBS containing bovine serum
Each ON (600 pmol) labeled with fluorescein at 50-end was in-
44. Puglisi, J. D.; Tinoco, I., Jr. In Methods in Enzymology; Dahlberg, J. E., Abelson, J.
N., Eds.; Academic: San Diego, CA, 1989; Vol. 180, pp 304–325.
cubated in PBS (300 m
L) containing 5% bovine serum at 37 ꢂC. At