Synthesis of 2′-O-[2-(N-methylcarbamoyl)ethyl]ribonucleosides using oxa-michael reaction and chemical and biological properties of oligonucleotide derivatives incorporating these modified ribonucleosides

Article abstract of DOI:10.1021/jo101963z

To develop oligonucleotides containing new 2′-O-modified ribonucleosides as nucleic acid drugs, we synthesized three types of ribonucleoside derivatives modified at the 2′-hydroxyl group with 2-(methoxycarbonyl)ethyl (MOCE), 2-(N-methylcarbamoyl)ethyl (MC

Full text of DOI:10.1021/jo101963z

ARTICLE
pubs.acs.org/joc
Synthesis of 20-O-[2-(N-Methylcarbamoyl)ethyl]ribonucleosides Using
Oxa-Michael Reaction and Chemical and Biological Properties of
Oligonucleotide Derivatives Incorporating These Modified
Ribonucleosides
Takeshi Yamada,† Natsuki Okaniwa,† Hisao Saneyoshi,† Akihiro Ohkubo,† Kohji Seio,† Tetsuya Nagata,‡
Yoshitsugu Aoki,‡ Shin’ichi Takeda,‡ and Mitsuo Sekine*,†
†Department of Life Science, Tokyo Institute of Technology, J2-12, 4259 Nagatsuta, Midoriku, Yokohama 226-8501, Japan
‡Department of Molecular Therapy, Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawa-Higashi,
Kodaira, Tokyo 187-8502, Japan
S Supporting Information
b
ABSTRACT: To develop oligonucleotides containing new 20-
O-modified ribonucleosides as nucleic acid drugs, we synthe-
sized three types of ribonucleoside derivatives modified at the
20-hydroxyl group with 2-(methoxycarbonyl)ethyl (MOCE),
2-(N-methylcarbamoyl)ethyl (MCE), and 2-(N,N-dimethylcar-
bamoyl)ethyl (DMCE) groups, as key intermediates, via the
oxa-Michael reaction of the appropriately protected ribonucleo-
side (U, C, A, and G) derivatives. Among them, the 20-O-MCE
ribonucleosides were found to be the most stable under basic
conditions. To study the effects of the 20-O-modification on the nuclease resistance of oligonucleotides incorporating the 20-O-
modified ribonucleosides and their hybridization affinities for the complementary RNA and DNA strands, 20-O-MCE-ribonucleo-
side phosphoramidite derivatives were successfully synthesized and subjected to the synthesis of 20-O-MCE-oligonucleotides and 20-
O-methyl-oligonucleotides incorporating 20-O-MCE-ribonucleosides. The 20-O-MCE-oligonucleotides and chimeric oligomers
with 20-O-MCE and 20-O-methyl groups thus obtained demonstrated complementary RNA strands and much higher nuclease
resistances than the corresponding 20-O-methylated species. Finally, we incorporated the 20-O-MCE-ribonucleosides into antisense
20-O-methyl-oligoribonucleotides to examine their exon-skipping activities in splicing reactions related to pre-mRNA of mouse
dystrophin. The exon-skipping assay of these 20-O-methyl-oligonucleotide incorporating 20-O-MCE-uridines showed better
efficacies than the corresponding 20-O-methylated oligoribonucleotide phosphorothioate derivatives.
’ INTRODUCTION
basic conditions.16 Meanwhile, we independently reported the
synthesis and properties of 20-O-cyanoethyl-modified (20-O-CE)
oligonucleotides17 (Figure 1). 20-O-CE oligoribonucleotides
demonstrated higher hybridization affinities for the cDNAs and
RNAs than the corresponding unmodified species; they also
demonstrated favorable nuclease resistances. These results in-
dicated that 20-O-CE oligonucleotides would be available for
oligonucleotide therapy; however, they were somewhat unstable
under basic conditions. The 20-O-CE group was gradually
removed via β-elimination under basic conditions such as aqu-
eous ammonia. We also found that the CE group could be used as
a protecting group of 20-OH in RNA synthesis, since this group
could be removed by treatment with 1 M Bu4NF in THF.18
On the basis of the previous results, we designed three types
of ribonucleosides modified at the 20-hydroxyl group with
2-(methoxycarbonyl)ethyl (MOCE) (1), 2-(N-methylcarba-
moyl)ethyl (MCE) (2), and 2-[(N,N-dimethylcarbamoyl)ethyl
A number of 20-O-modified ribonucleosides have been de-
signed and synthesized for oligonucleotide-based therapeutics,
represented by the silencing of mRNAs using siRNAs,1 antisense
RNAs,2 and shRNAs3 and the suppression of specific proteins
using RNA aptamers.4 Various modifications with substituent
groups, such as 20-O-methyl,5 20-O-methoxyethyl,6 20-O-
aminopropyl,7 and 20-O-[2-(methylamino)-2-oxoethyl],8 have
been reported to date. The incorporation of these 20-O-modified
nucleosides into oligoribonucleotides has proven to be useful for
improving their stability against hydrolysis by nucleases in
serum,9 controlling their biodistribution in cells and organs,10and
enhancing their hybridization affinities for the complementary
RNAs.11 20-O-Modification of ribonucleosides was also used for
the introduction of tethers with other small molecules such as
lipids,12 dyes,13 and peptides.14 Several 20-O-modified ribonu-
cleoside derivatives have been synthesized via the ring-opening
reaction of 2,20-anhydrouridine15 and the alkylation of appro-
priately protected ribonucleosides with alkyl halides under strong
Received: October 5, 2010
Published: March 23, 2011
r
2011 American Chemical Society
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Figure 1. The structures of 20-O-MOCE, 20-O-MCE, 20-O-DMCE, and 20-O-CE ribonucleosides.
Scheme 1. Oxa-Michael Reaction to Compound 5a
(DMCE) (3) groups as novel monomer components of 20-O-
modified oligoribonucleotides, as shown in Figure 1. We ex-
pected that these modifiers could be introduced via Michael
additions similar to the CsCO3-mediated 20-O-cyanoethylation
used for the synthesis of 20-O-CE ribonucleosides (4) followed
by amidation. It seemed that the transformation of the cyano
group in the CE group to weaker electron-withdrawing groups,
such as methoxycarbonyl, N-methylcarbamoyl, and N,N-di-
methylcarbamoyl, could suppress the loss of these 20-O-modifiers
due to β-elimination during the entire synthetic process invol-
ving ammonia treatment required for removal of base-protecting
groups. The MCE and DMCE groups have amide functions in their
structures. Prakash et al. previously reported that oligoribonucleo-
tides incorporating 20-O-[2-(methylamino)-2-oxoethyl]ribonucleo-
sides with an amide group similar to that of 20-O-MCE
ribonucleosides showed favorable nuclease resistance.9
a Conditions: acrylate ester derivatives, Cs2CO3, t-BuOH, rt, 2-18 h;
yield: 6 (71%), 7 (60%), 8 (73%).
Functional Group Transformation of 20-O-[2-(Alkoxy-
carbonyl)ethyl]uridine. Scheme 2 shows the synthesis of 50-
O-(4,40-dimethoxytrityl)uridine derivatives modified at the 20-
position with MOCE, MCE, and DMCE groups. The benzoyl
group of the methyl ester compound 6 was selectively removed
by treatment with 5 equiv of n-PrNH2 in THF to give the
deprotetected compound 9. The methyl ester group was
inert under these conditions. When compound 6 was treated
with 40% MeNH2 in MeOH, the monoalkyl amide 10 was
obtained in a good yield. However, treatment of compound 6
with 2 M Me2NH in THF did not give the desired dialkyl
amide 11 but instead gave the debenzoylated compound 9
quantitatively. The use of 50% Me2NH in H2O resulted in the
formation of a hydrolyzed compound as the main product
(data not shown). Therefore, we chose the more reactive ester
8 for the synthesis of 11. As a result, treatment of compound 8
with 2 M Me2NH in THF easily gave compound 11. Treatment
In this paper, we report the details of the synthesis of new 20-
O-modified ribonucleosides, which involved highly selective oxa-
Michael additions for purine nucleotides without alkylation of
the base residues, such as cytosine, adenine, and 2-aminoadenine,
and several unique properties of oligonucleotide derivatives
incorporating these 20-O-modified ribonucleosides. In addition
to these synthetic studies, promising biological properties of 20-
O-MCE/20-O-Me chimeric RNA oligomers, involving an effec-
tive exon skipping of the pre-mRNA of mouse dystrophin, are
also described.
’ RESULTS AND DISCUSSION
Oxa-Michael Addition of the Secondary 20-O-Hydroxyl
Group of Uridine to Acrylates. We tested various R,β-unsaturated
carbonyl compounds, such as alkyl acrylates (CH2dCHC(O)OR:
R = CH3, C2H5, n-C4H9, t-C4H9, and CF3CH2), acrylamide, N,N-
dimethylacrylamide, acrolein, and methyl vinyl ketone, as the accep-
tors of the oxa-Michael addition of N3-benzoyl-30,50-O-(1,1,3,3-
tetraisopropyldisiloxane-1,3-diyl)uridine (5)19in t-BuOH in the
presence of cesium carbonate at room temperature for 2-18
h, as shown in Scheme 1. Before compound 5 was selected for the
Michael addition donor, 30,50-O-(1,1,3,3-tetraisopropyldisil-
oxane-1,3-diyl)uridine20 was tested. However, the alkylation reac-
tion on the uridine moiety was faster than Michael addition to the
20-hydroxyl group. When methyl, ethyl, and 2,2,2-trifluoroethyl
esters of acrylic acid were used, the oxa-Michael reactions
proceeded smoothly to give 20-O-alkylated compounds 6, 7,
and 8, respectively. Other acceptors, such as n-butyl acrylates,
tert-butyl alkylate, acrylamide, N,N-dimethylacrylamide, acrolein,
and methyl vinyl ketone, were ineffective under these conditions.
The reaction rate increased by increasing the substrate concen-
tration from 0.1 to 0.2 M. The polymerization of the Michael
acceptors did not occur in most cases, except for the reaction of 5
with acrolein.
of compounds 9-11 with Et3N 3HF gave the resulting 30,50-
3
unprotected intermediates 12-14, which were allowed to react
with DMTrCl to give compounds 15-17, respectively.
Chemical Properties of the MOCE, MCE, and DMCE
Groups of 15-17 under Basic Conditions. We investigated
the stability of the MOCE, MCE, and DMCE groups of 15-17
under basic conditions; Table 1 summarizes these results.
We treated 10 mg of each compound with 1 mL of 1 M NaOH,
28% NH4OH, and1MTBAFinTHF, andcheckedthetimecourse
of the reaction by silica gel thin layer chromatography analysis.
Treatment with 1 M NaOH easily hydrolyzed the methyl ester
15 to give the carboxylic acid 18 in 65% yield. The ammonia
treatment of compound 15 also resulted in rapid ammonolysis,
giving rise to the amide compound 19. These results showed that
hydrolysis and ammonolysis were much faster than β-elimina-
tion, and once the ester was transformed to the amide or the
carboxylic acid, further β-elimination was well suppressed.
We isolated and identified the hydrolyzed product 18 and the
amidate derivative 19 by 1H NMR and HRMS. Rapid removal of
the MOCE group took place when compound 15 was treated
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Table 1. Chemical Properties of Compound 15-17
conditionsa
compd 15
compd 16
compd 17
1 M NaOH
hydrolysis within 5 min
amidation within 5 min
elimination within 5 min
stable for 24 h
stable for 24 h
stable for 24 h
hydrolysis in 18 h
stable for 24 h
eliminationb
28% NH4OH
1 M TBAF/THF
a All reactions were done at room temperature. b t1/2 = 2 h. The reaction was completed at 40 °C for 2 h.
Scheme 2. Preparation of Compound 15-19a
Figure 2. The structures of 20-O-MCE-RNA-phosphoramidites 20-
23.
Scheme 3. Selective Oxa-Michael Reactions to Compounds
26, 27, and 29a
a Conditions: (i) Cs2CO3, methyl acrylate, t-BuOH, rt, 6-18 h, yield: 26
(72%), 27 (75%), 29 (79%).
and guanosine derivatives such as 6-N-benzoyl-30,50-(1,1,3,3-tetra-
isopropyldisilane-1,3-diyl)adenosine,20 4-N-benzoyl-30,50-(1,1,3,3-
tetraisopropyldisilane-1,3-diyl)cytidine,20 and 2-N-phenoxyacetyl-
30,50-(1,1,3,3-tetraisopropyldisilane-1,3-diyl)guanosine.21 However,
these reactions gave complex mixtures in all cases. These results
indicated that there were side reactions on the N-protected base
moieties that had amido protons.
a Conditions: (i) n-PrNH2, THF, rt, 4 h, yield: 9 (71%); (ii) 40%
MeNH2 in MeOH, rt, 4 h, yield: 10 (66%); (iii) 2 M Me2NH in THF, rt,
2 h, yield: 11 (81%); (iv) Et3N 3HF, THF, rt; (v) DMTrCl, pyridine, rt,
3
yield: 15 (90%, 2 steps), 16 (51%, 2 steps), 17 (70%, 2 steps); (vi) 0.1 M
NaOHaq, rt, 30 min, yield: 18 (65%); (vii) 28% NH4OH, rt, 30 min,
yield: 19 (99%).
Several research groups have reported the selective alkylation
of the 20-hydroxyl group of ribonucleosides, such as adenosine
and 2-aminoadenosine, with alkylating reagents under basic
conditions.22 On the basis of these results, we studied whether
a Michael reaction would be available in the presence of N-free
nucleobases.
with 1 M TBAF in THF. In our previous study, we reported that
the cyanoethyl group was removed from 20-O-cyanoethyluridine
by using this reagent.18
The monoalkyl amide 16 was very stable under all tested
conditions. In addition, it was also stable under the conditions
of 28% NH4OH at 55 °C for 5 h, which are the conditions
for the oligonucleotide synthesis in this study. The amide
proton of the MCE group was supposed to be dissociated
faster than the R-proton under basic conditions, so that the
dissociated species suppressed further deprotonation at the
R-position.
The results are shown in Scheme 3. We found that the N-
unmasked adenosine derivative 24 underwent smooth regiose-
lective Michael additions with methyl acrylate to give the desired
20-O-MOCE-adenosine derivative 26 in 72% yield. Similar con-
ditions were also tested on N-unmasked cytidine derivative 25.
Alkylation on the nucleobase moiety was observed as time passed
at room temperature, but Michael addition to the 20-hydroxyl
group was much faster than the side reaction so that the desired
20-O-MOCE-cytidine derivative 27 was selectively obtained in
75% yield.
The N,N-dialkyl amide 17 demonstrated the most unique
properties. The DMCE group was stable under ammonia treat-
ment but could be removed by treatment with 1 M TBAF.
Table 1 shows that the MCE group is the most stable among
the three modified groups. Therefore, it is tolerable in the
standard RNA synthesis. This result led us to synthesize the 20-
O-MCE-ribonucleoside 30-phosphoramidite derivatives 20-23,
as shown in Figure 2.
The reaction of 30,50-O-TIPDS-guanosine20 was also tested
under Cs2CO3 conditions, but the reaction resulted in formation
of a complex mixture. The fully base-protected species, i.e., 6-O-
diphenylcarbamoyl-2-N-dimethylaminomethylene-30,50-O-TIPDS-
guanosine,23 was also examined, but the rate of Michael reaction was
very slow. The dimethylaminomethylene (dmf) and diphenylcar-
bamoyl (dpc) group were gradually degraded so that the reaction
Synthesis of 20-O-MCE-Ribonucleoside 30-O-Phosphora-
midite Derivatives 20-23. For the synthesis of 20-O-MCE-
ribonucleoside 30-phosphoramidite derivatives, we applied the
Michael addition to the N-protected cytidine, adenosine,
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Scheme 4. Synthesis of Phosphoramidites 20-22a
33 and cytidine derivative 34 were treated with DMTrCl to give
the 50-O-protected adenosine derivative 35 and cytidine deriva-
tive 36 in 73% and 52% yields, respectively. The 30-phosphityla-
tion of 50-O-DMTr derivatives 16, 35, and 36 with 2-cyanoethyl-
N,N,N0,N0-tetraisopropylphosphordiamidite28 in the presence of
diisopropylammonium 1H-tetrazolide gave the phosphoramidite
uridine derivatives 20, adenosine derivative 21, and cytidine
derivative 22 in 66%, 76%, and 48% yields, respectively.
For the guanosine 30-phosphoramidite building block 23, we
succeeded in synthesizing this compound via a six-step reaction
from the 2-aminoadenosine derivative 29, as shown in Scheme 5.
The treatment of 20-MOCE derivative 29 with methylamine
yielded the 20-O-MCE derivative compound 37 in good yield
(77%). In this reaction, the selective conversion of 29 to 37
occurred without the β-elimination of the MCE group. We
attempted the selective acylation of the 2-amino group of
compound 37 by using isobutyryl chloride and phenoxyacetyl
chloride. The latter gave a better result, and the 2-N-phenox-
yacetyl derivative 38 could be easily obtained by reprecipitation.
The deamination of compound 38 with NaNO2 in the presence
of AcOH gave the desired guanosine derivative 39. Since we
observed the partial elimination of the TIPDS group during this
a Conditions: (i) 40% MeNH2 in MeOH, EtOH, rt; (ii) AcCl, pyridine,
rt (for 31 and 32); (c) Et3N 3HF, Et3N, THF, rt, yield: 13 (73%, 2
3
steps), 33 (60%, 3 steps), 34 (53%, 3 steps); (iii) DMTrCl, pyridine, rt,
yield: 35 (73%) and 36 (52%); (iv) 2-cyanoethyl-N,N,N0,N0-tetraiso-
propylphosphorodiamidite, diisopropylammonium 1H-tetrazolide,
MeCN, rt, yield: 20 (60%), 21 (76%), 22 (48%).
reaction, we treated the resulting product 39 with Et3N 3HF in
3
THF in situ to give the 30,50-unprotected product 30 in an overall
yield of 71%. The usual dimethoxytritylation of compound 30
followed by the 30-phosphitylation of the resulting 50-protected
compound 40 gave the guanosine 30-O-phosphoramidite deriva-
tive 23 in a good yield (80%).
gave a complex mixture. Finally, based on the successful results
using the N-free adenosine and cytidine derivatives 24 and 25, we
chose 30,50-O-TIPDS-2-aminoadenosine 2824 as a precursor for
the synthesis of 20-O-MCE-guanosine derivative 30 (see
Scheme 5) since the 2-aminoadenine moiety can be converted
to the guanosine one.24 Consequently, it was found the reaction
of N-unmasked 2-aminoadenosine derivative 28 with methyl
acrylate in t-BuOH gave the desired 20-O-MOCE-2-aminoade-
nosine product 29 in 78% yield without base modification.
The imino proton of uridine (pKa = 9.25)25 is apparently more
acidic than the amino protons of adenosine or cytidine (probably
more than that of aniline (pKa = 30.6)26) and the 20-OH proton
(pKa = 12.14-12.86).27 Therefore, it was considered that the
imino proton of uridine would be easily dissociated by cesium
carbonate as a base so that, as compared to the nondissociated
species, the dissociated one became more reactive toward the
Michael acceptors. In contrast to the dissociation property of the
uracil base, the amino protons of adenosine and cytidine could
not be dissociated by CsCO3. Therefore, it was expected that this
property was dependent on the inherent nucleophilicity of the
nucleobases when a Michael reaction competitively occurs on the
unprotected nucleobase sites toward a CsCO3-mediated disso-
ciated 20-hydroxyl group.
Synthesis of 20-O-MCE/20-O-Me-Chimeric Oligonucleo-
tides. 20-O-MCE-oligonucleotides and chimeric oligonucleo-
tides partially modified with 20-O-MCE-ribonucleosides were
synthesized by using the ribonucleoside 30-phosphoramidite
building blocks 20-23, according to the standard
phosphoramidite40,41 procedure. We carried out the synthesis
of these modified oligonucleotide using an ABI-392 DNA/RNA
synthesizer and CPG resins. Either 5-benzylthio-1H-tetrazole
(BTT)29 or 5-[bis(3,5-trifluoromethyl)phenyl]-1H-tetrazole30
was used as an activator. We used BTT for the synthesis of
ORNs 1-5 (Table 2). BTT was also used for the synthesis of
ORN 6; however, the coupling reaction with compound 23 did
not proceed well. Compound 23 was less reactive than the other
phosphoramidites 20-22. Therefore, we tested several activa-
tors to obtain an optimized coupling reaction using compound
23 at room temperature for 10 min. When 1H-tetrazole or
BTT was used as the activator, the coupling efficiency was
lower than 10%. On the other hand, the use of 5-(bis-3,5-
trifluoromethylphenyl)-1H-tetrazole gave a much better result
with more than 90% coupling efficiency, and thereby, 5-(bis-3,5-
trifluoromethylphenyl)-1H-tetrazole was the best choice for our
reagent. The release of oligonucleotides from the resin and
simultaneous deprotection of the protecting groups were carried
out by using 28% NH4OH at 55 °C for 5 h. In all cases, we
obtained the desired products as the main peaks. The side
products derived from β-elimination and transamidation were
not observed. 3-[(Dimethylaminomethylidene)amino]-3H-
1,2,4-dithiazole-5-thione (DDTT)31 was used as a sulfurization
agent for the synthesis of ORNs 10 and 12. We characterized the
synthesized oligonucleotides by MALDI-TOF.
Scheme 4 shows the synthesis of phosphoramidite compounds
20-22. We converted the 20-O-MOCE derivatives 6, 26, and 27
to the N-free 20-O-MCE-uridine derivative 10, adenosine deri-
vative 31, and cytidine derivative 32 via treatment with methy-
lamine. Under these conditions, the N3-benzoyl group of
the uridine derivative 6 was simultaneously removed to give
the 20-O-MCE disilylated derivative 10, which in turn was
allowed to react with Et3N 3HF in THF to give 20-O-MCE-
3
urdine (13) in 73% yield. In the case of cytidine derivative 27, a
trace amount of the β-elimination product 25 was formed by
methylamine treatment. The N-free intermediates 31 and 32
thus obtained from 26 and 27 were treated in situ with acetyl
Hybridization Properties of 20-O-MCE-Oligonucleotides.
We measured the Tm values of the duplexes of 20-O-MCE-
oligonucleotides with the cDNA and RNA strands in 10 mM
sodium phosphate buffer (pH 7.0) in the presence of 100 mM
chloride in pyridine followed by Et3N 3HF in THF to give the
3
20-O-MCE N-acetyl adenosine (33) and cytidine (34) in 51%
and 53% yields, respectively. 20-O-MCE N-acetyl derivatives
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Scheme 5. Synthesis of Phosphoramidite 23a
a Conditions: (i) MeNH2 in MeOH, EtOH, rt, yield: 37 (77%); (ii) phenoxyacetyl chloride, pyridine, -10 °C, yield: 38 (73%); (iii) NaNO2, AcOH,
H2O, rt; (iv) Et3N 3HF, Et3N, THF, rt; yield: 30 (71%); (v) DMTrCl, pyridine, rt, yield: 40 (75%); (vi) 2-cyanoethyl-N,N,N0,N0-tetraisopropylpho-
3
sphorodiamidite, diisopropylammonium 1H-tetrazolide, MeCN, rt, yield: 23 (80%).
Table 2. Synthesized Oligonucleotides Used in This Study and Mass Data
entry
sequencea
backboneb
calcd mass
obsd mass
ORN 1
ORN 2
ORN 3
ORN 4
ORN 5
ORN 6
ORN 7
ORN 8
ORN 9
ORN 10
ORN 11
ORN 12
50-UUU UUU UUU UUU-30
50-(20-O-CE)-UUU UUU UUU UUU-30
50-(20-O-Me)-UUU UUU UUU UUU-30
50-AAA AAA AAA AAA-30
50-(20-O-Me)-CGU AGA CUA UCU C-30
50-(20-O-Me)-CGU AGA CUA UCU C-30
50-(20-O-Me)-CGU AGA CUA UCU C-30
50-(20-O-Me)-CGU AGA CUA UCU C-30
50-(20-O-Me)-CUC CAA CAG CAA AGA AGA UGG CAU UUC UAG-30
50-(20-O-Me)-CUC CAA CAG CAA AGA AGA UGG CAU UUC UAG-30
50-(20-O-Me)-CUC CAA CAG CAA AGA AGA UGG CAU UUC UAG-30
50-(20-O-Me)-CUC CAA CAG CAA AGA AGA UGG CAU UUC UAG-30
PO
PO
PO
PO
PO
PO
PO
PO
PO
PS
4630.0
4236.4
3779.5
4908.8
4241.7
4315.8
4315.8
4315.8
10019.8
10483.2
10446.0
10909.4
4628.8
4238.4
3779.5
4908.3
4240.3
4314.8
4315.2
4315.3
10025.9
10492.2
10453.6
10915.1
PO
PS
a B: 20-O-MCE-RNA. b PO, PS means phosphate backbone, and phosphorothioate backbone, respectively.
NaCl and 0.1 mM EDTA. These results are summarized in
Table 3. The data were compared with those of natural RNAs and
20-O-methyl RNAs. When 20-O-MCE-U was incorporated into
all U’s of U12 (ORN 1, entry 2), the Tm value of the duplex of 20-
O-MCE-U12 with the complementary RNA strand was 12 °C
higher than that of the duplex derived from natural RNA (entry 1).
The ΔTm value was 1 °C per modification. The degree of this
Tm increase was the same as that of 20-O-Me-U12.17 As opposed
to this result, when we incorporated 20-O-MCE-A into all A’s of
A12 (entry 4), we did not observe such a significant Tm increase.
Next, the effect of one point 20-O-MCE-ribonucleoside modifi-
cation on the thermostabilities of the duplexes was examined. We
incorporated 20-O-MCE-G, A, and C into the fifth G (ORN 6,
entry 6), sixth A (ORN 7, entry 7), and seventh C (ORN 8,
entry 8), respectively, of 50-(20-O-Me)-CGUAGACUAUCUC-30
(ORN 5, entry 5). The differences in Tm between the modified
and unmodified duplexes were -2, þ1, and 0 °C for the
complementary RNA strand, respectively. Meanwhile, the ΔTm
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Table 3. Hybridization Affinity of 20-O-MCE-Oligonucleotides
entry
sequence
Tm (°C) vs RNAa
Tm (°C) vs DNAa
1
2
50-UUU UUU UUU UUU-30
50-UUU UUU UUU UUU-30 (ORN 1)
50-AAA AAA AAA AAA-30
50-AAA AAA AAA AAA-30 (ORN 4)
50-(20-O-Me)-CGU AGA CUA UCU C-30 (ORN 5)
50-(20-O-Me)-CGU AGA CUA UCU C-30 (ORN 6)
50-(20-O-Me)-CGU AGA CUA UCU C-30 (ORN 7)
50-(20-O-Me)-CGU AGA CUA UCU C-30 (ORN 8)
50-(20-O-Me)-CUC CAA CAG CAA AGA AGA UGG CAU UUC UAG-30 (PO backbone, ORN 9)
50-(20-O-Me)-CUC CAA CAG CAA AGA AGA UGG CAU UUC UAG-30 (PS backbone, ORN 10)
50-(20-O-Me)-CUC CAA CAG CAA AGA AGA UGG CAU UUC UAG-30 (PO backbone, ORN 11)
50-(20-O-Me)-CUC CAA CAG CAA AGA AGA UGG CAU UUC UAG-30 (PS backbone, ORN 12)
14
26
14
15
67
65
68
67
85
81
85
81
9
n.d.
n.d.
n.d.
49
45
48
48
74
69
71
73
3
4
5
6
7
8
9
10
11
12
a Tm values were measured in 2 μM oligonucleotide, 10 mM sodium phosphate buffer (pH 7.0), 100 mM NaCl, and 0.1 mM EDTA.
analysis and nuclease resistance assay of 20-O-MCE oligonucleo-
tides described above demonstrated their potential availability for
oligonucleotide therapy targeting specific RNAs, we examined the
biological activities of the exon skipping of pre-mRNA of mouse
dystrophin.33 Exon skipping is currently the most promising
approach to the oligonucleotide therapy of Duchenne muscular
dystrophy.34 Because antisense oligonucleotides for exon skipping
must be delivered to the cellular nucleus in order to modulate the
splicing of pre-mRNA, they must possess sufficient stabilities
against nucleases in blood and cytoplasm. Once antisense oligo-
nucleotides are delivered into the nucleus successfully, they must
hybridize with the targeting pre-mRNA selectively. It was reported
that phosphorothioate (PS) oligonucleotides with nuclease
resistance35 were easily delivered into the nucleus once they
permeated thecells.36 Weconsidered that20-O-MCE-oligonucleo-
tides with phosphorothioate backbones would improve the effi-
cacy of exon skipping by adding further nuclease resistance to the
phosphorothioate 20-O-methyl-oligonucleotides and improve the
Figure 3. Time courses of degradation of 20-O-MCE, 20-O-CE, and 20-
binding toward the complementary RNA strands. Recently, 20-O-
O-methyl oligonucleotides by snake venom phosphodiesterase.
methyl-PS-oligonucleotides and morpholino oligonucleotides
values of the same 20-O-MCE oligonucleotides for the cDNA
strand were -4, -1, and -1 °C, respectively. The 20-O-MCE
oligonucleotides exhibited stronger binding affinities for RNAs
than DNAs, as shown in Table 3 similar to the 20-O-methyl
RNAs. Six-point modification of ORNs 9 and 10 with 20-O-
MCE-U showed these modified ORNs 11 and 12 could maintain
the original binding affinity for the complementary RNA and
DNA strands, as shown in entries 9-12. This property should be
suitable for oligonucleotide therapeutics targeting RNAs.
have been proven to be effective for exon skipping, as exemplified
by successful mdx mice in vivo experiments. Therefore, we
synthesized a 20-O-MCE-PS-oligonucleotide of ORN 12 that
corresponded to a 30 base sequence (mB30) reported by Are-
chevala-Gomeza et al.37 The mB30 sequence was reported to be
effective for exon skipping when incorporated into not only 20-O-
methyloligoribonucleotides with phosphorothioate linkages but
also morpholino oligonucleotides. We incorporated 20-O-MCE-
Us into the U residues of 20-O-methyl-phosphorothioate ORN 10.
There are a total of 6 uridine residues in mB30, and they are mainly
localized at the 30-end of the strand. To evaluate the influence of
the phosphorothioate backbone, we also synthesized phosphate
derivatives ORNs 9 and 11. We purchased the antisense morpholino
ORN 13 from Gene Tools.
ORNs 9-13 were administrated to the exon 52-deleted mdx
mouse (mdx52) by intramuscular injection.38 We evaluated the
efficacy of exon skipping by the RT-PCR of the total RNAs as
described previously.39 Figure 4 displays the results. The experi-
ments were performed two times. As shown in part a, six-point
substitution of 20-O-MCE-U (ORN 12, exon-skipping efficacy =
62%) dramatically enhanced the exon-skipping efficacy com-
pared with that of the 20-O-methyl-PS oligonucleotides (ORN
10, exon-skipping efficacy = 34%). Its efficacy was almost the
Nuclease Resistance of 20-O-MCE RNA. We examined the 30-
exonuclease resistance of 20-O-MCE oligonucleotides using
snake venom phosphodiesterase (SVPD).32 The 20-O-MCE-
oligonucleotide ORN 1 was subjected to the digestion reaction
using SVPD. The time course of the remaining full-length ORN 1
was measured by RP-HPLC, as shown in Figure 3. The 30-
exonuclease resistance properties of 20-O-CE oligonucleotide
(ORN 2) and 20-O-Me oligonucleotide (ORN 3) were also
studied to compare with that of ORN 1. As a result, the 30-
exonuclease resistance of ORN 1 was much higher than those of
ORN 2 and ORN 3. Although t1/2 of ORN 1 was about 120 min,
it was six times larger than those of ORN 2 and ORN 3.
Exon-Skipping Activity of 20-O-MCE RNAs in the Splicing
of the Pre-mRNA of Mouse Dystrophin. Because the Tm
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appropriate residual undeuterated solvent. 31P NMR was externally
referenced with 85% H3PO4. Column chromatography was performed
with silica gel C-200. Reversed phase HPLC on a C18 column was
performed using a linear gradient of acetonitrile in 0.1 M ammonium
acetate buffer. Anion-exchange HPLC was performed using a linear
gradient of 0-100% solution A (20% CH3CN in 0.5 M KH2PO4) in
solution B (20% CH3CN in 0.005 M KH2PO4). Thin-layer chromatog-
raphy was performed using 60F-254 (0.25 mm). MALDI-TOF was
measured in reflectron positive mode. The matrix condition was
3-hydroxypicolinic acid (HPA) 50 mg/mL in MeCN:H2O (1:1, v/v),
diammonium hydrogen citrate (AHC) 100 mg/mL in H2O.
N3-Benzoyl-20-O-(2-methoxycarbonylethyl)-30,50-O-(1,1,3,
3-tetraisopropyldisiloxane-1,3-diyl)uridine (6). To a solution
of N3-benzoyl-30,50-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)uridine
(5) (1.80 g, 3.03 mmol) in t-BuOH (15 mL) were added methyl acrylate
(5.40 mL, 60.2 mmol) and cesium carbonate (489 mg, 1.39 mmol). The
solution was stirred at ambient temperature for 4 h. The solution was
extracted with EtOAc and saturated NH4Cl. The organic layer was
collected, dried over sodium sulfate, and concentrated under reduced
pressure. The residue was chromatographed on silica gel. Elution with
hexane/EtOAc (9:1, v/v) gave compound 6 (1.47 g, 2.17 mmol, 71%) as
a white foam: 1H NMR (500 MHz, DMSO-d6) δ 8.00 (d, J = 8.0 Hz,
1H), 7.94-7.48 (m, 5H), 5.77 (d, J = 8.0 Hz, 1H), 5.75 (s, 1H), 4.27-
4.04 (m, 6H), 4.00 (dd, J = 2.5, 13.5 Hz, 1H), 3.88 (d, J = 4.0 Hz, 1H),
3.62 (s, 3H), 2.60-2.58 (m, 2H), 1.11-0.97 (m, 28H); 13C NMR (125
MHz, CDCl3) δ 171.7, 168.9, 162.3, 149.1, 139.3, 135.4, 131.4, 130.7,
129.3, 101.6, 89.2, 82.6, 81.9, 68.2, 66.7, 59.5, 51.7, 35.2, 17.6, 17.4, 17.3,
17.1,17.0, 13.6, 13.2, 13.0, 12.6. HRMS m/z calcd for C32H49N2O10Si2
[M þ H]þ 677.2926, found 677.2931.
Figure 4. (a) One canonical example of the detection of antisence-
mediated exon-skipping in mdx52 mice by reverse transcriptase poly-
merase chain reaction (RT-PCR). (b) Immunohistochemical staining of
dystrophin in TA muscle of untreated and treated mdx52 mice (ORN 12
and ORN 13). There was no expression of dystrophin around the rim of
any muscle fibers from untreated mdx52 mice. In contrast, restoration of
dystrophin around the rim of muscle fibers was observed in both muscles
from treated mdx52 by ORN12 and ORN 13. Dystrophin was detected
with with dystrophin C-terminal antibody (Dys-2). Bar = 100 μm.
N3-Benzoyl-20-O-(2-ethoxycarbonylethyl)-30,50-O-(1,1,3,3-
tetraisopropyldisiloxane-1,3-diyl)uridine (7). To a solution of
compound 5 (120 mg, 0.20 mmol) in t-BuOH (1 mL) were added
cesium carbonate (65 mg, 0.20 mmol) and ethyl acrylate (0.43 mL, 4.0
mmol). The solution was vigorously stirred at ambient temperature for
18 h. The solution was extracted with EtOAc and saturated NH4Cl aq.
The organic layer was collected, dried over sodium sulfate, and
concentrated under reduced pressure. The residue was chromato-
graphed on silica gel. Elution with hexane/EtOAc (5:1, v/v) gave
compound 7 (83 mg, 0.12 mmol, 60%): 1H NMR (500 MHz, CDCl3)
δ 8.00 (d, J = 8.0 Hz, 1H), 7.93 (d, J = 7.5 Hz, 2H), 7.65 (dd, J = 7.5, 7.5
Hz, 1H), 7.50 (dd, J = 7.5, 7.5 Hz, 1H), 5.77 (d, J = 8.0 Hz, 1H), 5.75 (s,
1H), 4.09-3.88 (m, 9H), 2.65-2.55 (m, 2H), 1.09-0.85 (m, 31H).
HRMS m/z calcd for C33H51N2O10Si2 [M þ H]þ 691.3078, found
691.2997.
same as that of the corresponding morpholino nucleic acid
(ORN 13, exon-skipping efficacy = 65%). We also evaluated
the efficacy of ORN 12 by immunohistochemistry with dystro-
phin C-terminal antibody (Dys-2) as shown in part b. The
number of dystrophin positive fibers was almost the same as
that which resulted from ORN 13. These results indicated that
the incorporation of 20-O-MCE-Us into antisense RNA was
effective for exon skipping.
N3-Benzoyl-20-O-[2-[(2,2,2-trifluoroethoxy)carbonyl]ethyl]-
30,50-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)uridine (8).
To a solution of compound 5 (9.00 g, 15.23 mmol) in t-BuOH (75 mL)
were added 2,2,2-trifluoroethyl acrylate (30.2 g, 304.67 mmol) and cesium
carbonate (4.96 g, 15.23 mmol). The solution was stirred at ambient
temperature for 2 h. Then the solution was extracted with EtOAc and
saturated NH4Cl aq. The organic layer was collected, dried over sodium
sulfate, and concentrated under reduced pressure. The residue was
chromatographed on silica. Elution with hexane/EtOAc (6:1, v/v) gave
compound 8 (8.29 g, 11.12 mmol, 73%): 1H NMR (500 MHz, DMSO-d6)
δ 8.02 (d, J = 7.5 Hz, 2H), 7.85 (d, J = 8.0 Hz, 1H), 7.78 (dd, J = 7.0 Hz,
2H), 7.60 (dd, J = 7.5, 2 Hz), 5.82 (d, J = 8.0, 1 Hz), 5.64 (s, 1H), 4.66 (dd,
J = 9.0, 13.0 Hz, 2H), 4.28-4.25 (m, 1H), 4.19-4.17 (m, 2H), 3.99-3.93
(m, 4H), 2.72 (dd, J = 6.0, 2 Hz), 1.05-0.94 (m, 27H); 13C NMR (125
MHz, DMSO-d6) δ 169.5, 169.4, 161.9, 148.6, 140.1, 135.5, 131.1, 130.3,
129.4, 100.6, 89.0, 81.2, 68.4, 65.9, 59.7, 59.5, 59.4, 34.3, 17.3, 17.2, 17.1,
17.0, 16.9, 16.84, 16.82, 16.7, 12.8, 12.3, 12.2, 11.9. HRMS m/z calcd for
C33H48F3N2O10Si2 [M þ H]þ 745.2798, found 745.2781.
’ CONCLUSIONS
In conclusion, we have developed an effective method for the
synthesis of 20-O-alkoxycarbonyl-ribonucleosides via Michael
addition by using acrylate esters. We easily converted the MOCE
group to the chemically stable MCE group by aminolysis. The
amide group of the MCE group was not detrimental to the
hybridization and enhanced metabolic stability. The phosphor-
othioate derivative of a 20-O-Me-oligonucleotide incorporating
six 20-O-MCE-Us with a base sequence of mB30 proved to
exhibit effective exon skipping in the splicing of the pre-mRNA of
mouse dystrophin.
’ EXPERIMENTAL SECTION
Materials and Methods. All reagents and solvents obtained from
commercial suppliers were used without further purification. All reac-
tions were carried out under argon atmosphere in oven-dried glassware.
NMR chemical shifts are given in parts per million (ppm); J values are
given in hertz (Hz). 1H and 13C spectra were internally referenced to the
20-O-(2-Methoxycarbonylethyl)-30,50-O-(1,1,3,3-tetraisopro-
pyldisiloxane-1,3-diyl)uridine (9). To a solution of compound 6
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(1.71 g, 2.53 mmol) in anhydrous THF (20 mL) was added n-PrNH2
(1.5 mL, 18.0 mmol). The solution was vigorously stirred for 4 h. The
solution was evaporated under reduced pressure. The residue was chromato-
graphed on silica gel. Elution with hexane/EtOAc (1:4, v/v) gave compound
9 (1.03 g, 1.79 mmol, 71%) as a white foam: 1H NMR (500 MHz, CDCl3)
δ 9.49 (s, 1H), 7.89 (d, J = 5.0 Hz, 1H), 5.73 (s, 1H), 5.67 (d, J = 5.0 Hz,
1H), 4.24-4.09 (m, 6H), 3.96-3.84 (m, 1H), 3.84 (s, 1H), 2.66-2.65
(m, 1H), 1.09-0.92 (m, 28H). HRMS m/z calcd for C25H45N2O9Si2
[M þ H]þ 573.2663, found 573.2619.
3H), 2.70-2.56 (m, 2H). HRMS m/z calcd for C34H36N2NaO10 [M þ
Na]þ 655.2268, found 655.2269.
(4,40-Dimethoxytrityl)-20-O-[2-(N-methylcarbamoyl)ethyl]-
uridine (16). To a solution of compound 13 (553 mg, 1.67 mmol) in
dry pyridine (15 mL) was added DMTrCl (622 mg, 1.84 mmol). The
solution was stirred at ambient temperature for 4 h. The solution was
diluted with EtOAc and washed with saturated NaHCO3 aq. The
organic layer was collected, dried over sodium sulfate, and concentrated
under reduced pressure. The residue was chromatographed on silica gel.
Elution with 2% MeOH in CHCl3 containing 0.5% triethylamine gave
20-O-[2-(N-Methylcarbomoyl)ethyl]-30,50-O-(1,1,3,3-tetrai-
sopropyldisiloxane-1,3-diyl)uridine (10). To a solution of com-
pound 5 (1.80 g, 3.03 mmol) in t-BuOH (15 mL) were added cesium
carbonate (489 mg, 1.39 mmol) and methyl acrylate (5.4 mL, 60.2
mmol). The solution was vigorously stirred at ambient temperature for
4 h. The suspension was diluted with EtOAc, and extracted with EtOAc
and saturated NaHCO3 aq. The organic layer was collected, dried over
sodium sulfate, and concentrated under reduced pressure. The residue
was treated with 40% MeNH2 in MeOH (30 mL) at ambient tempera-
ture for 2 h. The solution was concentrated under reduced pressure. The
residue was chromatographed on silica gel. Elution with hexane/EtOAc
1
compound 16 (740 mg, 1.17 mmol, 70%) as a white foam: H NMR
(500 MHz, CDCl3) δ 7.95 (d, J = 8.0 Hz, 1H), 7.39-7.22 (9H, m),
6.86-6.83 (m, 4H), 5.93 (d, J = 3.0 Hz, 1H), 5.86 (br, 1H), 5.23 (d, J =
8.0 Hz, 1H), 3.97-3.90 (m, 2H), 3.79 (s, 3H), 3.54-3.48 (m, 2H), 2.81
(m, 3H), 2.61-2.43 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 172.2,
163.6, 158.8, 150.8, 144.5, 140.3, 135.5, 135.3, 130.3, 130.2, 128.1, 127.3,
113.4, 102.4, 87.9, 87.2, 83.7, 82.9, 69.2, 66.6, 62.1, 55.4, 35.9, 26.5. HRMS
m/z calcd for C34H37N3NaO9 [M þ Na]þ 654.2427, found 654.2446.
50-O-(4,40-Dimethoxytrityl)-20-O-[2-(N,N-dimethylcarbamoyl)-
ethyl]uridine (17). To a solution of compound 11 (3.61 g, 6.18
1
mmol) in THF (60 mL) was added Et3N 3HF (3.4 mL, 21.6 mmol).
3
(1:1, v/v) gave compound 10 (1.14 g, 66%): H NMR (500 MHz,
The solution was vigorously stirred for 4 h. The solution was evaporated
under reduced pressure. The residue was chromatographed on silica gel.
Elution with 10% MeOH in CHCl3 gave compound 14 as an inter-
mediate, which was coevaporated three times with anhydrous pyridine
three times. The residue was dissolved in anhydrous pyridine (60 mL).
To the solution was added DMTrCl (2.30 g, 6.80 mmol). The solution
was vigorously stirred overnight. The solution was evaporated under
reduced pressure. The residue was chromatographed on silica gel.
Elution with 5% MeOH in CHCl3 containing 0.5% Et3N gave com-
pound 17 (2.78 g, 4.32 mmol, 70%): 1H NMR (500 MHz, CDCl3) δ
8.72 (br, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.29-7.21 (m, 9H), 6.84-6.82
(m, 4H), 5.96 (d, J = 3.5 Hz, 1H), 5.36 (d, J = 3.5 Hz, 1H), 5.28 (d, J = 8.0
Hz, 1H), 4.59-4.56 (m, 1H), 4.11-4.07 (m, 2H), 4.00-3.98 (m, 1H),
3.90-3.87 (m, 1H), 3.79 (s, 6H), 3.54-3.44 (m, 2H), 3.00 (s, 3H), 2.96
(s, 3H), 2.78-2.75 (m, 1H), 2.45-2.41 (m, 1H). HRMS m/z calcd for
C35H39N3NaO9 [M þ Na]þ 668.2584, found 668.2582.
CDCl3) δ 9.90 (br, 1H), 7.91 (d, J = 8.0 Hz, 1H), 6.90 (br, 1H), 5.73-5.71
(m, 2H), 4.29-3.85 (7H, m), 2.81-2.79 (m, 3H), 2.63-2.47 (m, 2H).
HRMS m/zcalcd for C25H46N3O8Si2 [Mþ H]þ 572.2823, found 572.2826.
20-O-[2-(N,N-Dimethylcarbamoyl)ethyl]30,50-O-(tetraisopro-
pyldisiloxane-1,3-diyl)uridine (11). To a solution of compound 8
(240 mg, 0.32 mmol) in THF (3 mL) was added 2 M Me2NH (2 mL).
The solution was stirred for 2 h. The solution was concentrated under
reduced pressure. The residue was chromatographed on silica gel. Elution
with 5% MeOHinCHCl3 gave compound11(152 mg, 0.26 mmol, 81%):
1H NMR (500 MHz, CDCl3) δ 9.56 (br, 1H), 7.85 (d, J = 5.0 Hz, 1H),
5.74 (s, 1H), 5.65 (d, J = 5.0 Hz, 1H), 4.19-4.06 (m, 5H), 3.92 (m, 1H),
3.85 (s, 1H), 3.03 (s, 3H), 2.91 (s, 3H), 1.01-0.90 (m, 28H). HRMS m/z
C26H48N3O8Si2 [M þ H]þ 586.2980, found 586.2921.
20-O-[2-(N-Methylcarbomoyl)ethyl]uridine (13). A solution
of compound 10 (563 mg, 0.98 mmol) in dry THF (10 mL) was treated
with triethylamine trihydrofluoride (570 μL, 3.50 mmol) and triethyla-
mine (253 μL, 1.80 mmol) at ambient temperature for 1 h. The solution
was concentrated under reduced pressure. The residue was chromato-
graphed on silica gel. Elution with 10% MeOH in CHCl3 gave
compound 13 (250 mg, 0.75 mmol, 76%): 1H NMR (500 MHz,
D2O) δ 5.95 (d, J = 4.5 Hz, 1H), 5.92 (d, J = 8.5 Hz, 1H), 4.33 (t, J =
5.5 Hz, 1H), 4.17-4.12 (m, 2H), 3.98-3.88 (m, 3H), 3.79 (dd, J = 4.5,
13.0, 1H), 2.72-2.68 (m, 3H), 2.57-2.53 (m, 2H); 13C NMR (125
MHz, D2O) δ 175.0, 166.7, 152.0, 142.3, 102.9, 88.2, 85.1, 81.8, 68.9,
67.2, 61.1, 36.5, 26.4. HRMS m/z calcd for C13H20N3O7 [M þ H]þ
330.1301, found 330.1307.
Triethylamine Salt of 50-O-(4,40-Dimethoxytrityl)-20-O-(2-
carboxyethyl)uridine (18). To a solution of compound 15 (10 mg,
0.015 mmol) in pyridine (1 mL) was added 0.1 M NaOH (1 mL). The
solution was stirred at ambient temperature for 30 min. The solution was
extracted with CHCl3 and 10% citric acid in H2O. To the organic
solution was added Et3N (1 mL). The organic layer was dried over
sodium sulfate and concentrated under reduced pressure to give
1
compound 18 (7 mg, 0.0097 mmol, 65%): H NMR (CDCl3, 500
MHz) 8.08-7.16 (m, 14H), 6.83 (d, J = 10.0 Hz, 1H), 6.00 (d, J = 5.0
Hz, 1H), 5.31 (d, J = 10.0 Hz, 1H), 4.52-4.53 (m, 1H) 4.10-3.92 (m,
5H), 3.78 (s, 6H), 3.64-3.63 (m, 2H), 3.12-3.08 (m, 6H), 2.67-2.51
(m, 2H), 1.29-1.27 (m, 9H). HRMS m/z calcd for C33H34N2NaO10
[M þ Na]þ 641.2111, found 641.2174.
50-O-(4,40-Dimethoxytrityl)-20-O-[2-(methoxycarbonyl)-
ethyl]uridine (15). To a solution of compound 9 (780 mg, 1.37
mmol) in anhydrous THF (13 mL) was added Et3N 3HF (780 μL, 4.77
3
50-O-(4,40-Dimethoxytrityl)-20-O-(2-carbamoylethyl)uridine-
(19). To a solution of compound 15 (10 mg, 0.015 mmol) in pyridine
(1 mL) was added 28% NH4OH (1 mL). The solution was stirred at
ambient temperature for 30 min. The solution was concentrated under
reduced pressure to give compound 19 (10 mg, 0.015 mmol, 99%): 1H
NMR (CD3CN, 500 MHz) δ 7.79 (d, J = 8.0 Hz, 1H), 7.53-7.34 (m,
11H), 6.98-6.96 (m, 11H), 6.50 (br, 1H), 5.99 (br, 1H), 5.89 (d, J = 4.0
Hz, 1H), 5.53 (s, 1H), 5.38 (d, J = 8.0 Hz, 1H), 4.48-4.46 (m, 1H),
4.08-4.05 (m, 2H), 3.92-3.86 (m, 2H), 3.50 (s, 6H), 3.41-3.39 (m,
2H), 2.60-2.51 (m, 2H); 13C NMR (CD3CN, 125 MHz) δ 174.9,
163.9, 159.7, 159.7, 151.4, 145.8, 141.1, 136.6, 136.4, 131.0, 131.0, 129.0,
128.9, 127.9, 118.3, 114.1, 114.1, 102.5, 88.3, 87.5, 84.2, 82.8, 70.0, 66.9,
63.4, 55.9, 35.7. HRMS m/z C33H35N3NaO9 [M þ Na]þ 640.2271,
found 640.2257.
mmol). The solution was stirred at ambient temperature for 2 h. The
solution was concentrated under reduced pressure. The residue was
chromatographed on silica gel. Elution with 10% MeOH in CHCl3 gave
compound 12 as an intermediate, which was coevaporated three times
with dry pyridine and finally dissolved in dry pyridine (13 mL). To the
solution was added DMTrCl (510 mg, 1.50 mmol). The solution was
stirred vigorously for 4 h. Then the solution was extracted with EtOAc
and brine. The organic layer was collected, dried over sodium sulfate,
and concentrated under reduced pressure. The residue was chromato-
graphed on silica gel. Elution with EtOAc containing 0.5% Et3N gave
compound 15 (780 mg, 1.23 mmol, 90%): 1H NMR (500 MHz, CDCl3)
δ 8.78 (br, 1H), 7.95 (d, J = 8.0 Hz, 1H), 7.28-7.21 (m, 9H), 6.82-6.80
(m, 4H), 5.91 (d, J = 1.5 Hz, 1H), 5.26 (d, J = 8.0 Hz, 1H), 4.49-4.45
(m, 1H), 4.04-3.92 (m, 4H), 3.76 (s, 6H), 3.68 (s, 3H), 3.50-3.45 (m,
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20-O-[2-(Methoxycarbonyl)ethyl]-30,50-O-(1,1,3,3-tetraiso-
propyldisiloxane-1,3-diyl)adenosine (26). A solution of 30,50-
O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)adenosine (24) (10.0 g,
19.6 mmol) in t-BuOH (100 mL) was treated with cesium carbonate
(6.4 g, 19.6 mmol) at ambient temperature for 30 min. To the
suspension was added methyl acrylate (35.5 mL, 392 mmol). The
suspension was vigorously stirred for 9 h. The suspension was extracted
with EtOAc and saturated NH4Cl aq. The organic layer was collected,
dried over sodium sulfate, and concentrated under reduced pressure.
The residue was chromagraphed on silica gel. Elution with 0.5% MeOH
in CHCl3 gave compound 26 (8.40 g, 14.1 mmol, 72%): 1H NMR (500
MHz, DMSO-d6) δ 8.19 (s, 1H), 8.07 (s, 1H), 7.35-7.25 (br, 2H), 5.92
(s, 1H), 4.33 (dd, J = 4.0 Hz, 6.5 Hz, 1H), 4.47 (d, J = 4.0 Hz, 1H), 4.04-
4.02 (m, 3H), 3.92-3.90 (m, 3H), 2.65-2.55 (m, 2H), 1.06 -0.95 (m,
28 H); 13C NMR (125 MHz, CDCl3) δ 171.3, 156.0, 152.5, 148.5,
139.2, 119.2, 87.5, 81.3, 80.6, 70.0, 66.5, 60.3, 51.2, 34.7, 17.2, 17.14,
17.10, 17.0, 16.9, 16.8, 16.7, 12.7, 12.3, 12.1, 12.1. HRMS m/z calcd for
C26H45N5NaO7Si2 [M þ Na]þ 618.2758, found 618.2772.
NaHCO3 aq. The organic layer was collected, dried over sodium sulfate,
and concentrated under reduced pressure to give compound 31 as an
intermediate. The residue was coevaporated with toluene and dissolved
in dry THF. To the solution were added triethylamine and triethylamine
trihydrofluoride. The solution was vigorously stirred for 18 h. The
solution was concentrated under reduced pressure. The residue was
chromatographed on silica gel without aqueous workup. Elution with
3.5% MeOH in CHCl3 gave compound 33 (1.87 g, 4.74 mmol, 60%): 1H
NMR (500 MHz, DMSO-d6) δ 10.75-10.65 (br, 1H), 8.69 (s, 1H),
8.65 (s, 1H), 7.83 (d, J = 4.5 Hz, 1H), 6.06 (d, J = 6.0 Hz, 1H), 5.34 (d, J =
3.5 Hz, 1H), 5.18 (dd, J = 5.0 Hz), 4.57 (dd, J = 6.0 Hz, 1H), 4.41 (d, J =
3.0 Hz, 1H), 3.99 (d, J = 3.0 Hz, 1H), 3.08-3.76 (m, 1H), 3.69-3.33
(m, 3H), 2.51-2.52 (d, J = 4.5 Hz, 3H), 2.33-2.30 (m, 2H), 2.25 (s,
3H); 13C NMR (125 MHz, DMSO-d6) δ 170.8, 168.8, 151.7, 151.6,
149.6, 142.7, 123.7, 86.1, 85.6, 80.9, 68.9, 65.6, 61.2, 35.2, 25.4, 24.3.
HRMS m/z calcd for C16H22N6NaO6 [M þ Na]þ 417.1499, found
417.1498.
4-N-Acetyl-20-O-[2-(N-methylcarbamoyl)ethyl]cytidine
(34). Compound 27 (2.90 g, 5.08 mmol) was treated with 40% MeNH2
in MeOH (50 mL) at ambient temperature for 6 h. The solution was
concentrated under reduced pressure and coevaporated three times with
dry pyridine. The residue was dissolved with dry pyridine (50 mL), and
to the solution was added acetyl chloride (398 μL, 5.58 mmol). The
solution was vigorously stirred for 4 h. The solution was concentrated
under reduced pressure, and coevaporated with toluene to give com-
pound 32 as an intermediate. The residue was dissolved in THF, and to
20-O-[2-(Methoxycarbonyl)ethyl]-30,50-O-(1,1,3,3-tetraiso-
propyldisiloxane-1,3-diyl)cytidine (27). To the solution of 30,50-
O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)cytidine (25) (5.0 g, 10.3
mmol) in t-BuOH (50 mL) was added cesium carbonate (3.33 g, 10.3
mmol). The suspension was stirred at ambient temperature for 30 min.
To the suspension was added methyl acrylate (22.5 mL, 392 mmol). The
suspension was vigorously stirred for 6 h. The suspension was extracted
with EtOAc and saturated NH4Cl aq. The organic layer was collected,
dried over sodium sulfate, and concentrated under reduced pressure.
The residue was chromatographed on silica gel. Elution with 2% MeOH
in CHCl3 gave compound 27 (4.41 g, 7.73 mmol, 75%): 1H NMR (500
MHz, DMSO-d6) δ 7.68 (d, J = 7.5 Hz, 1H), 7.19 (br, 1H), 5.68 (d, J =
7.5 Hz, 1H), 5.59 (s, 1H), 4.16-4.12 (m, 2H), 4.01-3.83 (m, 5H), 3.58
(s, 3H), 2.63-2.60 (t, J = 6.5 Hz, 2H), 1.06-0.92 (m, 28H); 13C NMR
(125 MHz, CDCl3) δ 172.1, 166.3, 155.8, 140.8, 93.9, 89.4, 82.4, 81.4,
68.1, 66.5, 59.7, 51.7, 35.2, 17.6, 17.6, 17.4, 17.4, 17.2, 17.14, 17.11, 17.0,
13.5, 13.1, 13.0, 12.7. HRMS m/z calcd for C25H46N3O8Si2 [M þ H]þ
572.2832, found 572.2738.
the solution were added TEA (1.25 mL, 8.63 mmol) and TEA 3HF
3
(2.92 mL, 17.77 mmol). The solution was vigorously stirred for 18 h.
The solution was concentrated under reduced pressure. The residue was
chromatographed on silica gel. Elution with 7% MeOH in CHCl3 gave
compound 34 (994 mg, 2.68 mmol, 53%) as white foam: 1H NMR (500
MHz, DMSO-d6) δ 10.88 (s, 1H), 8.43 (d, J = 7.5 Hz, 1H), 7.84 (d, J =
4.0 Hz, 1H), 7.18 (d, J = 7.5 Hz, 1H), 5.80 (d, J = 2.5 Hz, 1H), 5.21 (dd,
J = 5.0 Hz, 1H), 5.17 (d, J = 6.0 Hz, 1H), 4.08 (dd, J = 4.0, 6.0 Hz, 1H),
3.86-3.39 (m, 6H), 2.56 (d, J = 4.0 Hz, 1H), 2.39-2.35 (m, 2H), 2.09
(s, 3H). HRMS m/z calcd for C15H22N4O7 [M þ H]þ 371.1556, found
371.1516.
20-O-[2-(Methoxycarbonyl)ethyl]-30,50-O-(1,1,3,3-tetraiso-
propyldisiloxane-1,3-diyl)-2-aminoadenosine (29). To a solu-
tion of 30,50-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-2-aminoadenosine
(28) (11.0 g, 21.0 mmol) in t-BuOH (110 mL) were added cesium
carbonate (7.03 g, 21.6 mmol) and methyl acrylate (39.1 mL, 431
mmol). The solution was stirred at ambient temperature for 18 h. The
suspension was extracted with CHCl3 and saturated NH4Cl aq. The
organic layer was collected, dried over sodium sulfate, and concentrated
under reduced pressure. The residue was dissolved in CHCl3 and
absorbed with silica gel. The silica gel was subjected to a column.
Elution with 3% MeOH in CHCl3 gave compound 29 (10.1 g, 16.5
mmol, 79%): 1H NMR (500 MHz, DMSO-d6) δ 7.74 (s, 1H), 6.82-
6.78 (br, 2H), 5.75 (s, 1H), 5.72 (s, 2H), 4.56 (dd, J = 5.0, 10 Hz, 1H),
4.30 (d, J = 5.0 Hz, 1H), 4.06-4.02 (m, 2H), 3.93-3.88 (m, 3H), 3.55
(s, 3H), 2.63-2,60 (m, 2H), 1.08-0,94 (m, 28H); 13C NMR (125
MHz, DMSO-d6) δ 171.3, 160.3, 156.1, 151.0, 134.5, 113.3, 86.3, 81.2,
80.5, 69.9, 66.3, 60.2, 51.3, 34.7, 17.3, 17.2, 17.1, 17.0, 16.9, 16.87, 16.80,
12,8, 12.4, 12.2, 12.1. HRMS m/z calcd for C26H47N6O7Si2 [M þ H]þ
611.3039, found 611.3038.
6-N-Acetyl-50-O-(4,40-dimethoxytrityl)-20-O-[2-(N-methyl-
carbamoyl)ethyl]adenosine (35). To a solution of compound 33
(1.50 g, 3.80 mmol) in dry pyridine was added DMTrCl (1.05 g, 3.24
mmol). The solution was stirred at ambient temperature for 5 h. The
solution was extracted with CHCl3 and brine. The organic layer was
collected, dried over sodium sulfate, and concentrated under reduced
pressure. The residue was chromatographed on silica gel. Elution with
3% MeOH in CHCl3 containing 0.5% Et3N gave compound 35 (1.95 g,
2.80 mmol, 73%): 1H NMR (500 MHz, DMSO-d6) δ 10.70 (br, 1H),
8.57 (s, 1H), 8.56 (s, 1H), 7.83-7.82 (m, 1H), 7.33-7.17 (m, 9H),
6.84-6.80 (m, 4H), 6.09 (d, J = 5.0 Hz, 1H), 5.38 (d, J = 5.0 Hz, 1H),
4.71-4.69 (m, 1H), 4.49-4.47 (m, 1H), 4.09-4.08 (m, 1H), 3.82-
3.67 (m, 8H), 3.21-3.16 (m, 2H), 2.50 (s, 3H), 2.33-2.25 (m, 2H),
2.11 (s, 3H). HRMS m/z calcd for C37H41N6O8 [M þ H]þ 697.2986,
found 697.2946.
4-N-Acetyl-50-O-(4,40-dimethoxytrityl)-20-O-[2-(N-methyl-
carbamoyl)ethyl]cytidine (36). To a solution of compound 34
(374 mg, 1.00 mmol) in dry pyridine (6 mL) was added DMTrCl (244
mg, 0.72 mmol). The solution was stirred at ambient temperature for
5 h. The solution was extracted with CHCl3 and brine. The organic layer
was collected, dried over sodium sulfate, and concentrated under
reduced pressure. The residue was chromatographed on silica gel.
Elution with 6% MeOH in CHCl3 containing 0.5% Et3N gave com-
pound 36 (350 mg, 0.52 mmol, 52%): 1H NMR (500 MHz, CDCl3) δ
9.49 (br, 1H), 8.48 (d, J = 7.5 Hz, 1H), 7.41-7.23 (m, 9H), 6.85-6.83
(m, 4H), 6.52 (br, 1H), 5.89 (s, 1H), 4.49-4.45 (m, 2H), 4.15-3.92
(m, 4H), 3.79 (s, 3H), 3.79 (s, 3H), 3.54-3.54 (m, 2H), 2.76 (d, 3H),
6-N-Acetyl-20-O-[2-(N-methylcarbamoyl)ethyl]adenosine
(33). To a solution of compound 26 (4.70 g, 7.90 mmol) in EtOH
(90 mL) was added 40% MeNH2 in MeOH (90 mL). The solution was
stirred at ambient temperature for 18 h. Then the solution was
concentrated under reduced pressure. The residue was coevaporated
three times with dry pyridine and finally dissolved in dry pyridine
(90 mL). To the solution was added acetyl chloride (790 μL, 11.1
mmol). The solution was vigorously stirred for 6 h. The solution was
diluted with EtOAc, and then extracted with EtOAc and saturated
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ARTICLE
2.62-2.39 (m, 2H), 2.18 (s, 3H). HRMS m/z calcd for C36H40N4NaO9
[M þ Na]þ 695.2693, found 695.2694.
20-O-[2-(N-Methylcarbamoyl)ethyl]-2-N-phenoxyacetyl-30,50-
O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-2-aminoadenosine
(38). To a solution of compound 37 (500 mg, 0.82 mmol) in dry pyridine
(20 mL) at -10 °C was dropwise added phenoxyacetyl chloride (124 μL,
0.90 mmol) over 5 min. The solution was vigorously stirred at -10 °C for2 h
and then at ambient temperature for 1 h. The solution was diluted with
CHCl3 and extracted with brine. The organic layer was collected, dried over
sodium sulfate, and concentrated under reduced pressure. Elution with 5%
MeOH in CHCl3 gave compound 38 (442 mg, 0.60 mmol, 73%): 1H NMR
(500 MHz, DMSO-d6) δ 10.00 (s, 1H), 8.04 (s, 1H), 7.68 (d, J = 4.0 Hz,
1H), 7.50-7.38 (br, 2H), 7.29-7.26 (m, 2H), 6.37-6.91 (m, 3H), 5.88 (s,
1H), 5.02 (s, 2H), 4.53 (dd, J = 5.0, 8 Hz, 1H), 4.38 (d, J = 8.0 Hz, 1H),
4.11-4.08 (m, 1H), 4.03-3.86 (m, 4H), 2.49 (m, 3H), 2.33 (dd, J = 7.0, 7.0
Hz, 2H), 1.04-0.93 (m, 28H); 13C NMR (125 MHz, DMSO-d6) δ 170.1,
158.0, 156.1, 152.5, 149.4, 137.2, 129.4, 120.8, 116.1, 114.5, 86.8, 81.0, 80.9,
69.7, 67.4, 67.1, 60.4, 40.0, 36.1, 25.3, 17.3, 17.16, 17.12, 17.0, 16.9 16.8, 12.7,
12.3, 12,1, 12.0. HRMS m/z calcd for C34H53N7NaO8Si2 [M þ Na]þ
766.3392, found 766.3400.
50-O-(4,40-Dimethoxytrityl)-20-O-[2-(N-methylcarbamoyl)-
ethyl]uridine-3-(2-cyanoethyl-N,N-diisopropylphosphora-
midite) (20). To a solution of compound 16 (490 mg, 0.77 mmol) in
dry MeCN (2 mL) were added diisopropylammonium 1H-tetrazolide
(101 mg, 1.19 mmol) and 2-cyanoethyl-N,N,N0,N0-tetraisopropylpho-
sphordiamidite (358 mg, 1.19 mmol). The solution was stirred under
argon atmosphere at ambient temperature for 8 h. The solution was
diluted with EtOAc and washed with saturated NaHCO3 aq. The
organic layer was collected, dried over sodium sulfate, and concentrated
under reduced pressure. The residue was chromatographed on silica gel.
Elution with 1% MeOH in CHCl3 containing 0.5% triethylamine gave
1
compound 20 (384 mg, 0.46 mmol, 60%) as a white foam: H NMR
(500 MHz, CDCl3) δ 8.09-8.01 (m, 1H), 7.47-7.27 (m, 9H), 6.86-
6.82 (m, 4H), 6.49 (br, 1H), 5.90 (m, 1H), 5.28-5.21 (m, 1H), 4.61-
4.48 (m, 1H), 4.25-3.86 (m, 4H), 3.80 -3.79 (m, 1H), 3.75-3.43 (m,
6H), 2.80-2.77 (m, 3H), 2.64-2.42 (m, 4H), 1.29-1.03 (m, 12H); 31P
NMR (125 MHz, CDCl3) δ 151.32, 150.48. HRMS m/z calcd for
C43H55N5O10P [M þ H]þ 832.3687, found 832.3650.
20-O-[2-(N-Methylcarbamoyl)ethyl]-2-N-phenoxyacetylgua-
nosine (30). To a stirred solution of compound 38 (1.00 g, 1.34 mmol)
in AcOH (100 mL) were added H2O (40 mL) and NaNO2 (752 mg,
10.75 mmol). After 30 min another portion of NaNO2 (752 mg, 10.75
mmol) was added. The solution was stirred at ambient temperature for
48 h. The solution was concentrated under reduced pressure to give a
yellow syrup. The syrup was extracted once with brine and three times
with saturated NaHCO3 aq. The organic layer was dried, collected over
sodium sulfate, and concentrated under reduced pressure to give
compound 39 as an intermediate. The residue was coevaporated with
dry pyridine. The residue was dissolved in anhydrous THF (130 mL).
To the solution were added triethylamine (340 μL) and triethylamine
trihydrofluoride (650 μL). The solution was stirred vigorously at
ambient temperature for 6 h. The solution was concentrated under
reduced pressure. The residue was extracted with H2O and CHCl3. After
several minutes, white precipitates appeared in the aqueous layer. The
aqueous layer was concentrated to reduce its volume. The precipitate was
collected by filtrtion and dried. Finally, it was dried by vacuum and gentle
heating to give compound 30 (490 mg, 0.97 mmol, 71%): 1H NMR (500
MHz, DMSO-d6) δ 11.84 (s, 1H), 11.78 (s, 1H), 8.29 (s, 1H), 7.89 (d,
J = 4.0 Hz, 1H), 7.32-7.29 (m, 2H), 6.99-6.97 (m, 3H), 5.87 (d, J = 6.5
Hz, 1H), 5.30 (d, J = 3.5 Hz, 1H, diminished with D2O), 5.09 (dd, J = 5.5,
5.5 Hz, 1H, diminished with D2O), 4.86 (s, 2H), 4.44-4.42 (m, 1H),
4.38-4.37 (m, 1H), 3.95 (d, J = 2.0 Hz, 1H), 3.79-3.76 (m, 1H), 3.66-
3.55 (m, 3H), 2.54 (d, J = 4.0 Hz, 3H), 2.33 (s, 2H); 13C NMR (125
MHz, DMSO-d6) δ 171.05, 171.01, 157.6, 154.8, 148.7, 147.4, 137.8,
129.5, 121.3, 120.3, 114.5, 86.1, 84.5, 81.2, 68.9, 66.2, 65.4, 61.3, 35.1,
25.0. HRMS m/z calcd for C22H26N6NaO8 [M þ Na]þ 525.1710,
found 525.1715.
6-N-Acetyl-50-O-(4,40-dimethoxytrityl)-20-O-[2-(N-methyl-
carbamoyl)ethyl]adenosine-30-(2-cyanoethyl-N,N-diisopro-
pylphosphoramidite) (21). To a solution of compound 35 (1.00 g,
1.44 mmol) indry MeCN (150 mL) were added 2-cyanoethyl-N,N,N0,N0-
tetraisopropylphosphordiamidite (920 μL, 2.87 mmol) and diisopropy-
lammonium 1H-tetrazolide (147 mg, 0.86 mmol) at ambient temperature
for 18 h. The solution was concentrated under reduced pressure. The
residue was chromatographed on silica gel. Elution with 3% MeOH in
CHCl3 gave compound 21 (980 mg, 1.09 mmol, 76%): 1H NMR (500
MHz, CDCl3) δ 8.63-8.61 (m, 2H), 8.26-8.22 (m, 1H), 7.42-7.21 (m,
9H), 6.82-6.79 (m, 4H), 6.41-6.38 (m, 1H), 6.16-6.15 (m, 1H),
4.69-4.59 (m, 2H), 4.40-4.33 (m, 1H), 4.04-3.97 (m, 1H), 3.90-3.85
(m, 2H), 3.79-3.62 (m, 6H), 3.60-3.52 (s, 4H), 3.39-3.35 (m, 1H);
31P NMR (125 MHz, CDCl3) δ 151.34, 151.30. HRMS m/z calcd for
C46H58N8O9P [M þ H]þ 897.4064, found 897.4022.
4-N-Acetyl-50-O-(4,40-dimethoxytrityl)-20-O-[2-(N-methyl-
carbamoyl)ethyl]cytidine-30-(2-cyanoethyl-N,N-diisopropy-
lphosphoramidite) (22). To a solution of compound 36 (480 mg,
0.71 mmol) in dry MeCN (2 mL) were added diisopropylammonium
1H-tetrazolide (92 mg, 0.54 mmol) and 2-cyanoethyl-N,N,N0,N0-tetra-
isopropylphosphorodiamidite (343 μL, 1.07 mmol). The solution was
stirred at ambient temperature for 10 h. The solution was extracted with
CHCl3 and brine. The organic layer was dried, collected over sodium
sulfate, and concentrated under reduced pressure. The residue was
chromatographed on silica gel. Elution with 1.5% MeOH in CHCl3
containing 0.5% Et3N gave compound 22 (300 mg, 0.34 mmol, 48%):
1H NMR (500 MHz, CDCl3) δ 9.70-9.67 (m, 1H), 8.63-8.56 (m,
1H), 7.34-6.86 (m. 11H), 5.93 (s, 1H), 4.57-4.48 (m, 1H), 4.30-4.02
(m 4H), 3.84-3.83 (m, 6H), 3.73-3.46 (m, 6H), 2.81-2.62 (m, 4H),
2.43-2.37 (m, 2H), 2.26 (s, 3H), 1.27-1.03 (m, 12H); 31P NMR (125
MHz, CDCl3) δ 151.43, 150.23. HRMS m/z calcd for C45H58N6O10P
[M þ H]þ 873.3952, found 872.3911.
50-O-(4,40-Dimethoxytrityl)-20-O-[2-(N-methylcarbamoyl)-
ethyl]-2-N-phenoxyacetylguanosine (40). To a solution of
compound 30 (330 mg, 0.66 mmol) in pyridine (6 mL) was added
DMTrCl (244 mg, 0.72 mmol). The solution was stirred at ambient
temperature for 3 h. The solution was diluted with CHCl3 and extracted
with saturated NaHCO3 aq. The organic layer was dried, collected over
sodium sulfate, and concentrated under reduced pressure. The residue
was chromatographed on silica gel. Elution with TEA/MeOH/CHCl3
(0.5:2:98, v/v/v) gave compound 40 (401 mg, 0.50 mmol, 75%):
1H NMR (500 MHz, DMSO-d6) δ 11.95-11.85 (m, 2H), 8.13 (s,
1H), 7.89 (d, J = 4.5 Hz, 1H), 7.36-7.21 (m, 11H), 7.00-6.97 (m, 3H),
6.85-6.82 (m, 4H), 5.92 (d, J = 5.5 Hz, 1H), 5.36 (d, J = 4.5 Hz, 1H,
diminished with D2O), 4.86 (dd, J = 15.5, 22.5 Hz, 2H), 4.50 (dd, J = 5.5,
10.0 Hz, 1H), 4.39-4.36 (m, 1H), 4.05-4.04 (m, 1H), 3.84-3.82 (m,
1H), 3.76-3.68 (m, 7H), 3.30-3.27 (m, 1H), 3.17-3.15 (m, 1H), 2.55
(d, J = 4.5 Hz, 3H), 2.38-2.34 (m, 2H); 13C NMR (125 MHz, DMSO-
d6) δ 172.42, 170.78, 158.49, 156.97, 148.39, 144.44, 137.31, 135.53,
20-O-[2-(N-Methylcarbamoyl)ethyl]-30,50-O-(1,1,3,3-tetrai-
sopropyldisiloxane-1,3-diyl)-2-aminoadenosine (37). To a
solution of compound 29 (1.0 g, 1.64 mmol) in EtOH (8 mL) was
added 40% methylamine in MeOH (8 mL) at ambient temperature for
18 h. The solution was concentrated under reduced pressure. The
residue was chromatographed on silica gel. Elution with 4% MeOH in
CHCl3 gave compound 37 (770 mg, 1.26 mmol, 77%): 1H NMR (500
MHz, DMSO-d6) δ 7.76-7.74 (m, 2H), 6.82-6.76 (br, 2H), 5.75 (s,
1H), 5.73 (s, 2H), 4.54 (dd, J = 5.0, 8.0 Hz, 1H), 4.27 (d, J = 5.0 Hz, 1H),
4.06-3.98 (m, 2H), 3.94-3.90 (m, 2H), 3.87-3.82 (m, 1H), 2.53 (d,
J = 4.0 Hz, 3H), 2.37 (dd, J = 6 Hz, 2H), 1.04-1.02 (m, 28H). HRMS
m/z calcd for C26H48N7O6Si2 [M þ H]þ 610.3198, found 610.3183.
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The Journal of Organic Chemistry
ARTICLE
135.48, 130.00, 129.61, 128.08, 127.80, 126.88, 122.16, 121.26, 114.64,
114.53, 113.12, 86.50, 84.19, 82.24, 69.67, 66.94, 66.18, 63.34, 60.33,
55.12, 45.98, 35.61, 26.22, 14.12, 9.03. HRMS m/z calcd for
C43H45N6O10 [M þ H]þ 805.3119, found 805.3112.
primer sequences were Ex50F 50-TTTACTTCGGGAGCTGAGGA-30
and Ex53R 50-ACCTGTTCGGCTTCTTCCTT-30 for amplification of
cDNA from exons 50-53. The PCR conditions were 95 °C for 4 min,
then 35 cycles of 94 °C for 1 min, 60 °C for 1 min, 72 °C for 1 min, and
finally 72 °C for 7 min. The intensity of PCR bands was analyzed by
using ImageJ software (http://rsbweb.nih.gov/ij/), and skipping effi-
ciency was calculated by using the following formula [(the intensity
of skipped band)/(the intensity of skipped band þ the intensity of
unskippedband)], after the resulting PCR bands were extracted using a
gel extraction kit.
Immunohistochemistry. Eight micrometer cryosections were cut
from flash-frozen muscle, then placed on poly-L-lysine-coated slides and
air-dried. The sections were stained with monoclonal mouse antibody
Dys-2 against C-terminus of cardiac muscular dystrophin from normal
mouse used as a secondary antibody. 40,6-Diamidino-2-phenylindole
containing a mounting agent was used for nuclear counterstaining.
50-O-(4,40-Dimethoxytrityl)-20-O-[2-(N-methylcarbamoyl)-
ethyl]-2-N-phenoxyacetylguanosine-30-(2-cyanoethyl N,N-
diisopropylphosphoramidite) (23). To a solution of compound
40 (500 mg, 0.62 mmol) in dry MeCN (50 mL) were added
2-cyanoethyl-N,N,N0,N0-tetraisopropylphosphordiamidite (477 μL,
1.49 mmol) and diisopropylammonium 1H-tetrazolide (64 mg, 0.37
mmol). The solution was stirred at ambient temperature for 18 h. The
solution was concentrated under reduced pressure. The residue was
chromatographed on silica gel. Elution with 2% MeOH in CHCl3 gave
compound 23 (498 mg, 0.49 mmol, 80%): 1H NMR (500 MHz, DMSO-
d6) δ 7.91-7.87 (m, 1H), 7.47-7.26, (m, 13H), 7.23-7.20, (m, 1H),
7.07-7.04 (m, 1H), 6.99-6.95 (m, 2H), 6.83-6.80 (m, 2H), 6.16-
6.00 (m, 1H), 5.99-5.93 (m, 1H), 4.74-4.70 (m, 2H), 4.46-3.84 (m,
3H), 3.78-3.77 (m, 6H), 3.67-3.32 (m, 4H), 2.74-2.73 (m, 3H),
2.66-2.35 (m, 5H), 1.29-1.00 (m, 12H). HRMS m/z calcd for
C52H61N8O11PNa [M þ Na]þ 1027.4095, found 1027.4098.
’ ASSOCIATED CONTENT
Supporting Information. 1H NMR, 13C NMR, and 31
P
S
b
Oligonucleotide Synthesis. RNA oligonucleotides (Table 2)
were synthesized on an Applied Biosystems 392 oligonucleotide synthe-
sizer on a 1 μmol scale, using a 0.1 M solution each of 20, 21, 22,
and 23 with commercially available 50-O-(4,40-dimethoxytrityl)-20-O-
methyl-30-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite mono-
mers of uridine, 4-N-benzoylcytidine, 6-N-benzoyladenosine (ABz),
and 2-N-isobutyrylguanosine (GiBu). A 0.25 M solution of
5-ethiothio-1H-tetrazole in dry acetonitrile or a 0.25 M solution of
5-(bis-3,5-trifluoromethylphenyl)-1H-tetrazole in dry acetonitrile was
used as the activator for the coupling reaction. Coupling time was set to
be 10 min. [(Dimethylaminomethylidene)amino]-3H-1,2,4-dithiazole-
3-thione (DDTT)42 was used as the sulfurization agent for the phos-
phorothioate synthesis. After completion of the synthesis, the release of
20-O-modified oligonucleotides from the support and deprotection of all
protecting groups were simultaneously carried out in NH4OH (1 mL) at
ambient temperature for 2 h, and then CPG was filtrated. The filtrate was
heated at 55 °C for 5 h. The filtrate was concentrated under reduced
pressure and then subjected to C-18 cartridge purification. Each product
was purified by using anion-exchange chromatography.
NMR charts. This material is available free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Email: msekine@bio.titech.ac.jp.
’ ACKNOWLEDGMENT
This work was supported by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Culture, Sports,
Science and Technology, Japan, and in part by Health Sciences
Research Grants for Research on Psychiatric and Neurological
Diseases and Mental Health from the Ministry of Health, Labor
and Welfare of Japan and the global COE project.
’ REFERENCES
Tm Experiments. Each oligonucleotide (1.0 nmol) was dissolved in
10 mM sodium phosphate buffer (pH 7.0, containing 150 mM NaCl and
0.1 mM EDTA) (500 μL) to arrange the final concentration of each
oligonucleotide to be 2 μM. The solution was separated into quartz cells
(10 mm) and incubated at 85 °C. After 10 min, the solution was cooled
to 5 °C at a rate of annealing and melting, the absorption at 260 nm was
recorded and used to draw UV-melting curves. Tm values were calculated
as the temperature that gave the maximum of the first derivative of the
UV-melting curve.
Nuclease Resistance Assay. Each oligonucleotide (10.0 nmol)
was dissolved in Tris-HCl buffer (at pH 8.5, 72 mM NaCl, 14 mM
MgCl2 at 37 °C) (990 μL). To the solution was added snake venom
phosphodiesterase (5 ꢀ 10-4 unit/mL) (10 μL) to arrange the final
concentration of each oligonucleotide to be 10 μM. Aliquots of the
reaction solution were removed at indicated times, quenched by
heating at 100 °C for 2 min. The mixture was analyzed by anion
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in saline. Ten micrograms of antisense oligonucleotide was injected into
each tibialis anterior muscle with lipofectamine 2000. Two weeks after
injection, mice were sacrificed and muscles were dissected. Total RNA
was extracted from frozen tissue and 50 ng of total RNA was used for
one-step RT-PCR according to the manufacturer’s instructions. The
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