Stereocontrolled solid-phase synthesis of phosphorothioate oligoribonucleotides using 2′-O-(2-cyanoethoxymethyl)-nucleoside 3′-O-oxazaphospholidine monomers

Article abstract of DOI:10.1021/jo301052v

A method for the synthesis of P-stereodefined phosphorothioate oligoribonucleotides (PS-ORNs) was developed. PS-ORNs of mixed sequence (up to 12mers) were successfully synthesized by this method with sufficient coupling efficiency (94-99%) and diastereose

Full text of DOI:10.1021/jo301052v

Article
pubs.acs.org/joc
Stereocontrolled Solid-Phase Synthesis of Phosphorothioate
Oligoribonucleotides Using 2′‑O‑(2-Cyanoethoxymethyl)-nucleoside
3′‑O‑Oxazaphospholidine Monomers
Yohei Nukaga,† Kohei Yamada,‡ Toshihiko Ogata,† Natsuhisa Oka,§ and Takeshi Wada*,†
†Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Bioscience Building 702,
5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan
‡Yamasa Corporation, 10-1 Araoi-cho 2-chome, Choshi, Chiba 288-0056, Japan
§Department of Chemistry, Faculty of Engineering, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan
S
* Supporting Information
ABSTRACT: A method for the synthesis of P-stereodefined
phosphorothioate oligoribonucleotides (PS-ORNs) was devel-
oped. PS-ORNs of mixed sequence (up to 12mers) were
successfully synthesized by this method with sufficient
coupling efficiency (94−99%) and diastereoselectivity
(≥98:2). The coupling efficiency was greatly improved by
the use of 2-cyanoethoxymethyl (CEM) groups in place of the
conventional TBS groups for the 2′-O-protection of nucleoside
3′-O-oxazaphospholidine monomers. The resultant diastereo-
pure PS-ORNs allowed us to clearly demonstrate that an ORN
containing an all-(Rp)-PS-backbone stabilizes its duplex with the complementary ORN, whereas its all-(Sp)-counterpart has a
destabilizing effect.
the functions of the pro-Rp and pro-Sp oxygen atoms of the
corresponding phosphodiesters in various RNA-related bio-
logical processes.4 However, this method is not applicable to
the preparation of ORNs with multiple stereodefined PS-
linkages, which are required for therapeutic studies. Diaster-
eopure dimer building blocks can be used to incorporate
multiple stereodefined PS-linkages into ORNs; however, this
method requires up to 32 types of building blocks (four types
of nucleosides for each of the 3′- and 5′-nucleosides and two P-
diastereomers) and yet cannot produce ORNs having
consecutive stereodefined PS-linkages.7a,b P-Stereodefined PS-
ORNs can also be synthesized using RNA polymerases, but
only those with (Rp)-PS-linkages are available, and this method
is not suitable for large-scale syntheses.7c,8 Although chemical
syntheses of P-stereodefined PS-ORNs using stereoselective or
stereospecific reactions have also been studied, the methods
reported to date suffer from low coupling efficiency or
stereoselectivity.9−11 Recently, an siRNA duplex having four
consecutive (Rp)-PS-linkages at both ends has been synthe-
sized using chromatographically separated diastereopure
nucleoside 3′-phosphorothioate triester derivatives as mono-
mers.12 To the best of our knowledge, this is the only report in
the literature describing the chemical synthesis of P-stereo-
defined PS-ORNs longer than 3mers with sufficient stereo-
INTRODUCTION
■
Post-transcriptional gene silencing, mediated by RNA mole-
cules such as short interfering RNAs (siRNAs) and microRNAs
(miRNAs), has been extensively studied for its therapeutic
potential in treating various diseases.1 Conversely, miRNA itself
has also emerged as a potential therapeutic target owing to its
susceptibility to being silenced by antisense oligonucleotides.2
In addition, gene silencing by siRNA has become a powerful
tool for functional genomics.3 Synthetic oligoribonucleotides
(ORNs) with appropriate chemical modifications are useful for
these applications, especially for therapeutic purposes that
require oligonucleotides with sufficient nuclease stability, cell
membrane permeability, and favorable pharmacokinetic proper-
ties.1,2 Among various chemically modified ORN analogues
developed so far, phosphorothioate oligoribonucleotide (PS-
ORN) is one of the most well studied analogues owing to its
sequence-specific hybridizing affinity for target RNAs and water
solubility, features that are comparable to those of natural
ORNs, as well as its high nuclease stability and lipophilicity.1
A PS-ORN has chiral phosphorus atoms, and its properties
are theoretically dependent on the configuration of these
phosphorus atoms because it functions by interacting with
chiral biomolecules such as nucleic acids and proteins.4 For this
reason, efforts have been made to develop methods for
synthesizing P-stereodefined PS-ORNs.4b,5 For example, ORNs
containing a single stereodefined PS-linkage at a specific
position have often been prepared by chromatographic
separation of diastereomixtures6 and used as probes to study
Received: June 7, 2012
Published: August 29, 2012
© 2012 American Chemical Society
7913
dx.doi.org/10.1021/jo301052v | J. Org. Chem. 2012, 77, 7913−7922

The Journal of Organic Chemistry
Article
specificity and selectivity. However, the details of the synthesis,
such as the coupling efficiency, are not described in this paper.
To overcome these limitations, we have recently developed a
method for synthesizing P-stereodefined PS-ORNs using
diastereopure 2′-O-TBS-protected nucleoside 3′-O-(1,3,2-ox-
azaphospholidine) monomer units.13 Using this method, we
have synthesized all-(Rp)- and all-(Sp)-PS-U10 with good
coupling yields (97−99%) and stereoselectivity (≥96:4) and
have shown that the duplex of the former with the
complementary ORN (rA10) was slightly more stable than
the unmodified duplex (ΔTm = ca. +0.4 °C per modification).
On the other hand, hybridization was not observed above 4 °C
for the all-(Sp)-PS-U10 and rA10. However, attempts to
synthesize P-stereodefined PS-ORNs of mixed sequence for a
thermal denaturing study have revealed that the coupling
efficiency was lower than that observed for the synthesis of PS-
U10, and only short oligomers up to 4mers were accessible in
satisfactory yields. To solve this problem, we sought to develop
new monomers with improved reactivity. Recently, significant
efforts have been devoted to the development of new methods
for synthesizing ORNs owing to their growing demand in
biological and medical studies, and a variety of new protecting
groups for the 2′-OH of ribonucleosides have been
developed.14−16 Among these new protecting groups, we
selected the 2-cyanoethoxymethyl (CEM)16 group because
the replacement of the conventional 2′-O-TBS group by this
new, less bulky protecting group has been demonstrated to
greatly improve the reactivity of the phosphoramidite
monomers, and very long ORNs (110−170mers) were
efficiently synthesized.16b,c It is also notable that CEM groups
can be removed under mild conditions using TBAF. In this
paper, we describe the development of novel 2′-O-CEM-
protected oxazaphospholidine monomers, their application to
the stereocontrolled synthesis of PS-ORNs, and a thermal
denaturing study on the duplexes of the resultant PS-ORNs.
Table 1. Synthesis of 2′-O-CEM-Nucleoside 3′-O-
Oxazaphospholidine Monomers 3a−d
a
entry
product
BPRO
yield (%)
trans:cis
1
2
3
4
5
6
7
8
(Sp)-3a
(Sp)-3b
(Sp)-3c
(Sp)-3d
(Rp)-3a
(Rp)-3b
(Rp)-3c
(Rp)-3d
U
71
75
51
54
62
53
49
42
>99:1
>99:1
>99:1
>99:1
>99:1
>99:1
>99:1
>99:1
Cac
Aac
Gce,pac
U
Cac
Aac
Gce,pac
a
Determined by 31P NMR.
Table 2. Stereocontrolled Solid-Phase Synthesis of 2′-O-
CEM-Protected Dinucleoside Phosphorothioates NSU 5a−
a b
,
d
RESULTS AND DISCUSSION
■
Stereoselective Synthesis of 2′-O-CEM-Protected 3′-
O-Oxazaphospholidine Monomers 3a−d. Table 1 summa-
rizes the synthesis of the 2′-O-CEM-protected nucleoside 3′-O-
oxazaphospholidine monomers 3a−d. The 2′-O-CEM-pro-
tected nucleosides having a free 3′-OH (1a−d)16 were allowed
to react with 2-chlorooxazaphospholidine derivatives D- and L-2,
which were synthesized from D- and L-proline, respectively.17
The reactions proceeded with complete stereoselectivity, and
only the trans-isomers were generated as was the case for their
2′-deoxy and 2′-O-TBS counterparts.13,17 Because the 2′-O-
CEM-monomers were less stable on silica gel than the 2′-O-
TBS-protected oxazaphospholidine monomers, 3-aminopropyl-
functionalized silica gel was used for purification.18 As a result,
the complete set of diastereopure monomer units required for
the synthesis of the P-stereodefined PS-ORNs of mixed
sequence were successfully isolated in modest to good yields
(Table 1), though partial decomposition of the monomers was
observed even with the 3-aminopropyl-functionalized silica gel.
The stability of monomers varied with the P-configuration and
nucleobase; the (Sp)- and pyrimidine monomers were more
stable than the (Rp)- and purine counterparts, respectively.
Stereocontrolled Solid-Phase Synthesis of PS-ORNs.
Next, we investigated the manual solid-phase synthesis of PS-
ORN 2mers by using the monomers described above (Table
2). Uridine anchored to a controlled-pore glass (CPG) support
via a succinate linker (4) was condensed with each of the
c
c
entry
monomer
product
(Rp)-USU
yield (%)
Rp:Sp
1
2
3
4
5
6
7
8
(Sp)-3a
(Sp)-3b
(Sp)-3c
(Sp)-3d
(Rp)-3a
(Rp)-3b
(Rp)-3c
(Rp)-3d
(Rp)-5a
(Rp)-5b
(Rp)-5c
(Rp)-5d
(Sp)-5a
(Sp)-5b
(Sp)-5c
(Sp)-5d
96
95
97
96
98
96
95
97
>99:1
>99:1
>99:1
>99:1
(Rp)-CSU
(Rp)-ASU
(Rp)-GSU
(Sp)-USU
(Sp)-CSU
(Sp)-ASU
(Sp)-GSU
>1:99
>1:99
>1:99
>1:99
a
Reagents and conditions: (i) (Sp)- or (Rp)-3a−d (0.13 M, 40 equiv),
CMPT (1 M, 300 equiv), CH3CN, rt, 5 min; (ii) DTD (0.3 M, 120
equiv), CH3CN, rt, 10 min; (iii) 3% DCA, CH2Cl2, rt, 4 × 15 s; (iv)
conc NH3 aq/ EtOH (3:1, v/v), rt, 3 h. Subscript S denotes PS-
b
c
linkage. Determined by RP-HPLC.
monomers in the presence of N-(cyanomethyl)pyrrolidinium
triflate (CMPT) that we developed for the stereospecific
condensation of oxazaphospholidine monomers.19 The resul-
tant dinucleoside phosphite intermediates were then sulfurized
with N,N′-dimethylthiuram disulfide (DTD)20 to give dinucleo-
side phosphorothioate triester intermediates. Finally, the 5′-O-
(4,4′-dimethoxytrityl) (DMTr) group was removed by treat-
7914
dx.doi.org/10.1021/jo301052v | J. Org. Chem. 2012, 77, 7913−7922

The Journal of Organic Chemistry
Article
Scheme 1. Synthetic Cycle for P-Stereodefined PS-ORNs
ment with 3% dichloroacetic acid (DCA) in CH2Cl2, and the
oligomer on the solid support was then treated with
concentrated aqueous ammonia to deprotect the base and the
PS-linkage and cleave the linker. HPLC analysis of the resultant
crude 2′-O-CEM-protected dinucleoside phosphorothioates
(Rp)- and (Sp)-5a−d21 showed that the efficiency of the
cycle (95−98%) and diastereopurity of the products (>99:1)
were good enough for the synthesis of oligomers (Table 2).
Encouraged by these results, we extended this method to the
synthesis of oligomers. The synthetic cycle is shown in Scheme
1. The cycle consists of the following steps: (1) chain
elongation by a condensation reaction between the 5′-OH of
a nucleoside or a PS-ORN on a solid support and one of the
oxazaphospholidine monomers (3a−d) in the presence of an
activator, (2) capping of any unreacted 5′-OH as well as the
liberated secondary amino group of intermediate 7 using
trifluoroacetylimidazole (CF3COIm) and 1,8-bis-
(dimethylamino)naphthalene (DMAN)13,17 and the subse-
quent P-sulfurization with DTD, and (3) 5′-O-detritylation
with 3% DCA. The capping step was added to the method
shown in Table 2 in order to facilitate the purification of the
products as well as to protect the secondary amino group
derived from the chiral auxiliary. Trifluoroacetylation is used for
capping in place of the conventional acetylation because the N-
acetylated chiral auxiliaries would be too stable to be removed
from the product.13,17 After repeating the cycle to assemble the
desired oligomer, the solid support is treated with a mixture of
concentrated aqueous ammonia and ethanol in order to remove
the protecting groups and chiral auxiliaries from the
nucleobases and PS-linkages, respectively, and to cleave the
succinate linker. The resultant PS-ORN bearing the 5′-O-
DMTr and 2′-O-CEM protecting groups is then treated with a
solution of TBAF in DMSO containing 0.5 vol % CH3NO2, a
scavenger of acrylonitrile to deprotect the 2′-O-CEM groups.16
Subsequent purification by RP-HPLC (DMTr-on and -off)
affords the desired P-stereodefined PS-ORN.
First, four kinds of PS-ORN 4mers, all-(Rp)-PS-U4 (9a), all-
(Sp)-PS-U4 (9b), all-(Rp)-PS-CAGU (9c), and all-(Sp)-CAGU
(9d) (Table 3, entries 1−5), were manually synthesized, and
the efficiency of the synthesis was evaluated from the average
coupling yields and the RP-HPLC profiles of the resulting PS-
ORNs21 (2′-O-CEM-on−5′-O-DMTr-off and fully depro-
tected). The results showed that the efficiency of the synthesis
was greatly improved by employing the 2′-O-CEM protection.
Thus, the desired PS-ORNs 9a−d were obtained in the average
coupling yields of 94−98% when the coupling time was
extended to 10−15 min (entries 2−5) from that used for the
synthesis of 2mers (5 min). In contrast, we have previously
reported that the average coupling yields were 67−90% when
the same oligomers were synthesized by using the 2′-O-TBS-
protected oxazaphospholidine monomers under similar con-
ditions with a longer coupling time (20 min).13 This
improvement can be attributed to the enhanced reactivity of
the new monomers bearing a less bulky CEM group as was the
case with the phosphoramidite monomers.16 Furthermore, the
HPLC analyses showed that the 2′-O-CEM-protecting groups
were removed from the resultant 4mers by treatment with
TBAF virtually quantitatively without observable side reac-
tions.21
Next, we applied the optimized reaction conditions for the
synthesis of 4mers to the synthesis of 12mers (entries 6−12).
As shown in entries 6 and 7, all-(Rp)-PS-U12 (10a) and all-
(Sp)-PS-U12 (10b) were manually synthesized in excellent
coupling yields under these conditions, although the isolated
yields were rather low owing to partial loss of the product
during a double purification procedure by RP-HPLC (DMTr-
on and -off) (Figures 1 and 2, A and B). However, attempts to
synthesize all-(Rp)-PS-(CAGU)3 (10c) and all-(Sp)-PS-
7915
dx.doi.org/10.1021/jo301052v | J. Org. Chem. 2012, 77, 7913−7922

The Journal of Organic Chemistry
Article
(CAGU)3 (10d) were completely digested with nP1, whereas
only ca. 34% of 10b and ca. 31% of 10d were digested with
svPDE. On the other hand, complete digestion was observed
when all-(Rp)-PS-U10 (10a) and all-(Rp)-PS-(CAGU)3 (10c)
were treated with svPDE, whereas ca. 32% of 10a and ca. 78%
of 10c were digested with nP1. The partial digestion is probably
owing to the lower specificity of the enzymes.23b,25 These
experiments confirmed that the stereochemistry of the PS-
ORNs synthesized using the new 2′-O-CEM oxazaphospholi-
dine monomers was as expected, that is, (Rp)- and (Sp)-PS-
linkages were formed from the (Sp)- and (Rp)-monomers,
respectively.
Hybridization Properties of the P-Stereodefined PS-
ORNs. Finally, the hybridization properties of the P-stereo-
defined all-(Rp)-PS-(CAGU)3 (10c) and all-(Sp)-PS-(CAGU)3
(10d) were studied. A UV denaturing study was carried out
with four kinds of duplexes consisting of 10c and 10d, stereo-
random-PS-(CAGU)3, or an unmodified (CAGU)3 and the
complementary ORNs. As shown in Figure 3, the Tm value of
the duplex containing of 10c was slightly higher than that with
unmodified (CAGU)3 (ΔTm = +3.7 °C). The stabilization
effect of an (Rp)-PS-linkage (ΔTm = ca. +0.4 °C per
modification) was virtually the same as that obtained previously
with all-(Rp)-PS-U10.13 In sharp contrast, the Tm values
obtained with all-(Sp)-PS-(CAGU)3 (10d) and stereo-
random-PS-(CAGU)3 were much lower than that of the
unmodified duplex (ΔTm = −10.8 and −4.2 °C, respectively).
We have also shown the destabilizing effect of (Sp)-PS-linkages
previously by using all-(Sp)-PS-U10; however, no distinct Tm
value was obtained.13 Thus, the destabilizing effect of (Sp)- and
stereo-random PS-linkages was evaluated more precisely in the
current study using P-stereodefined PS-ORNs of mixed
sequence.
Table 3. Stereocontrolled Solid-Phase Synthesis of PS-ORN
4−12mers
coupling
coupling
activator time (min)
yield
isolated
yield (%)
ab
,
entry
1
product
All-(Rp)-PS-
(%)
91
9a
CMPT
CMPT
CMPT
CMPT
CMPT
CMPT
CMPT
CMPT
CMPT
CMPT
PhIMT
PhIMT
5
10
10
15
15
15
15
15
15
15
15
15
U4
2
All-(Rp)-PS-
U4
9a
98
96
98
94
99
99
3
All-(Sp)-PS-
U4
9b
4
All-(Rp)-PS-
CAGU
9c
5
All-(Sp)-PS-
CAGU
9d
6
All-(Rp)-PS-
U12
10a
10b
10c
10c
10d
10c
10d
12
14
7
All-(Sp)-PS-
U12
8
All-(Rp)-PS-
(CAGU)3
90 (68)
c
9
All-(Rp)-PS-
(CAGU)3
92 (75 )
10
All-(Sp)-PS-
(CAGU)3
93 (80)
94 (86)
97 (92)
d
11
All-(Rp)-PS-
(CAGU)3
6
12
All-(Sp)-PS-
10
(CAGU)3
a
b
Average coupling yield. Average coupling yields of Cac-monomers
c
d
are given in parentheses. Double coupling. Synthesized on
automated DNA synthesizer.
(CAGU)3 (10d) under the same conditions resulted in lower
average coupling yields (entries 8−10). The DMTr+ assay
showed that the coupling yields of the Cac-monomers were
particularly lower than those of the other monomers. Double
coupling of the Cac-monomer improved the yield only slightly
(entry 9). The low coupling yield might be due to the relatively
high basicity of cytosine, which could be initially protonated
and thus hamper the protonation of its oxazaphospholidine
moiety. To solve this problem, we employed N-phenyl-
imidazolium triflate (PhIMT)22 as the activator for the
synthesis of 10c and 10d. As expected, the coupling yield of
the Cac-monomer improved, and the desired oligomers of
mixed sequence (10c and 10d) were isolated (Table 3, entries
11 and 12) (Figures 1 and 2, C and D). The diastereoselectivity
of the coupling reaction using PhIMT (98:2 for Rp; >99:1 for
Sp) was determined to be comparable or slightly lower than
that obtained using CMPT by independently synthesizing
(Rp)-USU and (Sp)-USU.21 We also applied the method to an
automated synthesis. The synthesis of all-(Rp)-PS-(CAGU)3
(10c) was performed on an automated DNA synthesizer to
demonstrate the applicability of the method to automation. The
desired all-(Rp)-PS-(CAGU)3 (10c) was obtained with average
coupling yield of 94% as shown in entry 11. All-(Sp)-PS-
(CAGU)3 (10d) was also synthesized on an automated
synthesizer with similar efficiency (av. 95%) to that obtained
by a manual solid-phase synthesis.21
CONCLUSION
■
In conclusion, we successfully developed a method for
synthesizing P-stereodefined PS-ORNs of mixed sequence by
employing 2′-O-CEM protection in the oxazaphospholidine
method. The coupling efficiency of the 2′-O-CEM-protected
oxazaphospholidine monomers was greater than that of the 2′-
O-TBS-protected monomers. The resultant P-stereodefined PS-
ORNs of mixed sequence enabled us to demonstrate that a PS-
ORN−ORN duplex was stabilized by incorporating (Rp)-PS-
linkages, whereas it was largely destabilized with (Sp)- or
stereo-random-PS-linkages. The expanded availability of P-
stereodefined PS-ORNs should promote their use in
therapeutic studies. Furthermore, the stabilizing effect of
(Rp)-PS-linkages can support the design of miRNA-based
drug candidates, which require a much higher hybridization
potential to target RNAs than that needed for siRNA.2
EXPERIMENTAL SECTION
■
O6-Cyanoethyl-N2-phenoxyacetyl-3′,5′-O-(1,1,3,3-tetraiso-
propyldisiloxane-1,3-diyl)-2′-O-(2-cyanoethoxymethyl)-gua-
nosine 11. N2-Phenoxyacetyl-3′,5′-O-(tetraisopropyldisiloxane-1,3-
diyl)-2′-O-(2-cyanoethoxymethyl)-guanosine (11.18 g, 14.9 mmol)
was dried by repeated coevaporations with dry toluene and dissolved
in dry CH2Cl2 (100 mL) under argon. DMAP (0.092 g, 0.75 mmol),
triethylamine (8.3 mL, 60 mmol), and 2-mesitylenesulfonyl chloride
(3.92 g, 17.9 mmol) were successively added, and the mixture was
stirred for 30 min at rt. A saturated NaHCO3 aqueous solution (50
mL) was then added. The organic layer was separated and washed with
saturated NaHCO3 aqueous solutions (2 × 50 mL). The washings
were combined and extracted with CHCl3 (2 × 30 mL). The organic
P-Configurational Assignment of P-Stereodefined PS-
ORNs by Enzymatic Digestion. Isolated PS-ORN 12mers
10a−d were treated with snake venom phosphodiesterase23
(svPDE) (Rp-specific) and nuclease P124 (nP1) (Sp-specific)
for configurational assignments. After being incubated with
svPDE or nP1 for 16 h at 37 °C, the PS-ORNs were analyzed
by RP-HPLC.21 All-(Sp)-PS-U10 (10b) and all-(Sp)-PS-
7916
dx.doi.org/10.1021/jo301052v | J. Org. Chem. 2012, 77, 7913−7922

The Journal of Organic Chemistry
Article
Figure 1. RP-HPLC profiles of PS-ORNs bearing 5′-O-DMTr and 2′-O-CEM groups: (A) crude all-(Rp)-PS-U12 (10a); (B) crude all-(Sp)-PS-U12
(10b); (C) crude all-(Rp)-PS-(CAGU)3 (10c); (D) crude all-(Sp)-PS-(CAGU)3 (10d). RP-HPLC was performed with a linear gradient of 0−60%
CH3CN in 0.1 M TEAA buffer (pH 7.0) over 60 min at 30 °C and a rate of 0.5 mL/min.
layers were combined, dried over Na2SO4, filtered, and concentrated
under reduced pressure. The residue was dried by repeated
coevaporations with dry toluene and dissolved in dry CH2Cl2 (100
mL) under argon. N-Methylpyrrolidine (15.9 mL, 149 mmol) was
added at 0 °C, and the mixture was stirred for 40 min at the same
temperature. 2-Cyanoethanol (10.1 mL, 149 mmol) and DBU (3.3
mL, 22 mmol) were then added, and the mixture was further stirred
for 25 min at 0 °C. A 1.0 M KH2PO4 aqueous solution (50 mL) was
then added, and the organic layer was separated and washed with 1.0
M KH2PO4 aqueous solutions (2 × 50 mL). The washings were
combined and extracted with CHCl3 (2 × 30 mL). The organic layers
were combined, dried over Na2SO4, filtered, and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography [ethyl acetate−hexane (50:50, v/v to 100:0, v/v)].
The fractions containing 11 were collected, washed with a saturated
NaHCO3 aqueous solution (100 mL), dried over Na2SO4, filtered, and
concentrated to dryness under reduced pressure to afford 11 (6.20 g,
7.8 mmol, 52%) as a pale yellow foam. 1H NMR (300 MHz, CDCl3) δ
8.92 (1H, brs, 2-NH), 8.31 (1H, s, 8-H), 7.40−7.38 (2H, dd, J = 8.0,
8.0 Hz, 3-H of Pac), 7.11−6.98 (3H, m, 2,4-H of Pac), 6.16 (1H, s, 1′-
H), 5.20 (1H, d, J = 7.2 Hz, OCH2O of CEM), 5.10 (1H, d, J = 7.2
Hz, OCH2O of CEM), 4.85−4.78 (2H, m, O6-OCH2CH2CN), 4.69
(2H, s, CH2 of Pac), 4.53 (1H, dd, J = 4.2, 9.3 Hz, 2′-H), 4.35−4.17
(3H, m, 3′-H, 5′-H), 4.08−4.01 (2H, m, 4′-H, OCH2CH2CN, of
CEM), 3.92−3.78 (1H, m, OCH2CH2CN, of CEM), 3.02 (2H, t, J =
6.8 Hz, O6-OCH2CH2CN), 2.66−2.61 (2H, t, J = 6.6 Hz,
OCH2CH2CN of CEM), 1.12−0.95 (28H, m, i-Pr). 13C NMR (75
MHz, CDCl3) δ 165.7, 159.6, 156.9, 152.0, 150.8, 139.8, 129.9, 122.6,
119.0, 117.8, 116.8, 114.9, 94.7, 88.4, 81.5, 78.6, 68.4, 67.9, 63.1, 61.7,
59.4, 18.8, 18.1, 17.5, 17.3, 17.3, 17.3, 17.1, 17.0, 16.9, 16.8, 13.3, 13.0,
+
12.9, 12.6. ESI-HRMS: m/z calcd for C37H54N7O9Si2 [(M + H)+]
796.3516, found 796.3527.
O6-Cyanoethyl-N2-phenoxyacetyl-2′-O-(2-cyanoethoxy-
methyl)-guanosine 12. Compound 11 (1.92 g, 2.4 mmol) was dried
by repeated coevaporations with dry toluene and dissolved in dry THF
(5.0 mL) under argon. Et3N·3HF (0.40 mL, 2.4 mmol) was added at
35 °C, and the mixture was stirred for 2 h at the same temperature.
The mixture was then diluted with CHCl3 (50 mL) and washed with
saturated NaHCO3 aqueous solutions (3 × 30 mL). The washings
were combined and extracted with CHCl3 (2 × 20 mL). The organic
layers were combined, dried over Na2SO4, filtered, and concentrated
under reduced pressure. The residue was purified by silica gel column
chromatography [CHCl3−MeOH (100:3, v/v)] to afford 12 (0.86 g,
1
1.6 mmol, 64%) as a pale yellow foam. H NMR (300 MHz, DMSO-
d6) δ 10.73 (1H, brs, 2-NH), 8.57 (1H, s, 8-H), 7.33−7.28 (2H, dd,
7.8, 7.8 Hz, 3-H of Pac), 6.96−6.93 (3H, m, 2,4-H of Pac), 6.09 (1H,
d, J = 6.0 Hz, 1′-H), 5.38 (1H, d, J = 5.1 Hz, 3′-OH), 5.11−4.98 (3H,
m, 5′-OH, OCH2O of CEM), 4.78−4.65 (5H, m, 2′-H, O6-
OCH2CH2CN, CH2 of Pac), 4.36 (1H, dd, J = 3.6, 4.7 Hz, 3′-H),
3.98 (1H, d, J = 3.6 Hz, 4′-H), 3.72−3.51 (3H, m, 5′-H,
7917
dx.doi.org/10.1021/jo301052v | J. Org. Chem. 2012, 77, 7913−7922

The Journal of Organic Chemistry
Article
Figure 2. RP-HPLC profiles of fully deprotected and purified PS-ORNs: (A) all-(Rp)-PS-U12 (10a); (B) all-(Sp)-PS-U12 (10b); (C) all-(Rp)-PS-
(CAGU)3 (10c); (D) purified all-(Sp)-PS-(CAGU)3 (10d). RP-HPLC was performed with a linear gradient of 0−30% CH3CN in 0.1 M TEAA
buffer (pH 7.0) over 60 min at 30 °C and a rate of 0.5 mL/min.
O6-Cyanoethyl-N2-phenoxyacetyl-5′-O-(4,4′-dimethoxytri-
tyl)-2′-O-(2-cyanoethoxymethyl)-guanosine 1d. Compound 12
(0.86 g, 1.6 mmol) was dried by repeated coevaporations with dry
pyridine and dissolved in dry pyridine (5.0 mL) under argon. DMTrCl
(0.80 g, 2.4 mmol) was added at 0 °C, and the mixture was stirred for
9 h at rt. MeOH (5 mL) was then added, and the mixture was
concentrated under reduced pressure. The residue was dissolved in
CHCl3 (50 mL), and the solution was washed with saturated NaHCO3
aqueous solutions (3 × 30 mL). The washings were combined and
extracted with CHCl3 (3 × 20 mL). The organic layers were
combined, dried over Na2SO4, filtered, and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography [ethyl acetate−hexane−pyridine (50:50:0.5, v/v/v to
100:0:0.5, v/v/v)] to afford 1d (1.21 g, 1.4 mmol, 91%) as a pale
1
yellow foam. H NMR (300 MHz, CDCl3) δ 8.84 (1H, brs, 2-NH),
8.10 (1H, s, 8-H), 7.42−6.99 (14H, m, 2-H of p-An, Ph of Pac and
DMTr), 6.79 (4H, d, J = 8.1 Hz, 3-H of p-An), 6.24 (1H, d, J = 3.9 Hz,
Figure 3. UV melting curves of PS-(CAGU)3-PO-(ACUG)3.
1′-H), 5.03, 4.96 (2H, 2d, J = 7.1 Hz, OCH2O of CEM), 4.90−4.79
(3H, m, 2′-H, O6-OCH2CH2CN), 4.68−4.61 (3H, m, 3′-H, CH2 of
Pac), 4.26 (1H, d, J = 3.3 Hz, 4′-H), 3.78−3.65 (8H, m, OMe,
OCH2CH2CN of CEM), 3.49 (2H, d, J = 3.9 Hz, 5′-H), 3.02 (2H, t, J
= 6.6 Hz, O6-OCH2CH2CN), 2.62 (1H, d, J = 6.3 Hz, 3′-OH), 2.50
(2H, t, J = 6.2 Hz, OCH2CH2CN of CEM). 13C NMR (75 MHz,
CDCl3) δ 165.7, 159.7, 158.5, 156.9, 152.8, 150.8, 149.7, 144.4, 140.9,
OCH2CH2CN, of CEM), 3.47−3.25 (1H, m, OCH2CH2CN of CEM),
3.22−3.16 (2H, t, J = 5.9 Hz, O6-OCH2CH2CN), 2.66−2.50 (2H, m,
OCH2CH2CN of CEM). 13C NMR (75 MHz, CDCl3) δ 166.1, 160.0,
156.8, 152.1, 150.6, 142.6, 129.8, 122.5, 119.6, 117.6, 116.9, 114.8,
95.7, 88.4, 86.9, 79.9, 70.8, 67.5, 63.3, 62.4, 61.9, 18.7, 18.0. ESI-
+
HRMS: m/z calcd for C25H28N7O8 [(M + H)+] 554.1994, found
554.1996.
7918
dx.doi.org/10.1021/jo301052v | J. Org. Chem. 2012, 77, 7913−7922

The Journal of Organic Chemistry
Article
135.5, 135.4, 130.0, 129.8, 128.1, 127.9, 127.0, 122.5, 118.7, 117.6,
116.8, 114.9, 113.1, 95.6, 87.0, 86.6, 83.9, 80.2, 70.2, 67.7, 63.4, 63.1,
61.7, 55.2, 18.8, 18.0. ESI-HRMS: m/z calcd for C46H46N7O10+ [(M +
H)+] 856.3301, found 836.3300.
(Sp)-3c were collected, washed with a saturated NaHCO3 aqueous
solution (100 mL), dried over Na2SO4, filtered, and concentrated to
dryness under reduced pressure to afford (Sp)-3c (0.46 g, 0.51 mmol,
51%) as a colorless foam. 1H NMR (300 MHz, CDCl3) δ 8.60 (1H, s,
2-H), 8.51 (1H, brs, 6-NH), 8.21 (1H, s, 8-H), 7.42−7.22 (14H, m,
5″-Ph, 2-H of p-An, Ph of DMTr), 6.81 (4H, d, J = 6.9 Hz, 3-H of p-
An), 6.22 (1H, d, J = 5.1 Hz, 1′-H), 5.79 (1H, d, J = 6.6 Hz, 5″-H),
5.07−4.98 (2H, m, 2′-H, 3′-H), 4.84 (1H, d, J = 7.2 Hz, OCH2O of
CEM), 4.76 (1H, d, J = 7.2 Hz, OCH2O of CEM), 4.36 (1H, dd, J =
3.9, 8.1 Hz, 4′-H), 3.87 (1H, ddd, J = 6.0, 10.5, 10.5 Hz, 4″-H), 3.78
(6H, s, OMe), 3.64−3.44 (4H, m, 5′-H, 6″-H, OCH2CH2CN), 3.40
(1H, dd, J = 3.9, 10.8 Hz, 5′-H), 3.17−3.06 (1H, m, 6″-H), 2.61 (3H,
s, Ac), 2.37 (2H, t, J = 6.3 Hz, OCH2CH2CN), 1.72−1.56 (2H, m, 7″-
H), 1.28−1.19 (1H, m, 8″-H), 1.02−0.92 (1H, m, 8″-H). 13C NMR
(75 MHz, CDCl3) δ 170.3, 158.6, 152.4, 151.0, 149.1, 144.3, 141.7,
(Sp)-U-Monomer [(Sp)-3a]. A Typical Procedure for the
Synthesis of 3a−d. 5′-O-DMTr-2′-O-CEM-uridine (0.94 g, 1.5
mmol) was dried by repeated coevaporations with dry pyridine and
dry toluene and dissolved in freshly distilled THF (5.0 mL) under
argon. Triethylamine (1.5 mL, 10.5 mmol) and a 0.5 M solution of the
2-chloro-1,3,2-oxazaphospholidine derivative D-2 in freshly distilled
THF (9.3 mL, 4.7 mmol) were successively added at −78 °C, and the
mixture was stirred for 1.5 h at rt. The mixture was then diluted with
CHCl3 (400 mL) and washed with a saturated NaHCO3 aqueous
solutions (3 × 100 mL). The washings were combined and extracted
with CHCl3 (2 × 30 mL). The organic layers were combined, dried
over Na2SO4, filtered, and concentrated under reduced pressure. The
residue was then purified by silica gel column chromatography [NH
silica gel, hexane−ethyl acetate−triethylamine (20:10:0.03 to
10:20:0.03, v/v/v)]. The fractions containing (Sp)-3a were collected,
washed with a saturated NaHCO3 aqueous solution (100 mL), dried
over Na2SO4, filtered, and concentrated to dryness under reduced
pressure to afford (Sp)-3a (0.89 g, 1.1 mmol, 71%) as a colorless foam.
1H NMR (300 MHz, CDCl3) δ 8.42 (1H, brs, 3-NH), 8.11 (1H, d, J =
3
138.0 (d, JPC = 4.0 Hz), 135.5, 135.4, 130.16, 128.3, 128.2, 127.9,
127.6, 127.0, 126.6, 126.0, 125.4, 122.2, 117.5, 113.1, 94.9, 87.1, 86.7,
3
2
2
83.6 (d, JPC = 3.7 Hz), 82.4 (d, JPC = 9.5 Hz), 78.0, 70.7 (d, JPC
=
12.0 Hz), 67.3 (d, 2JPC = 3.2 Hz), 62.9, 62.2, 55.2, 53.1, 50.7, 47.1 (d,
2JPC = 34.7 Hz), 39.6, 27.9, 26.6, 26.0, 25.8 (d, JPC = 18.6 Hz), 20.6,
3
18.6. 31P NMR (121 MHz, CDCl3) δ 156.1. ESI-HRMS: m/z calcd for
C48H51N7O9P+ [(M + H)+] 900.3480, found 900.3479.
(Sp)-Gce,pac-Monomer [(Sp)-3d]. Crude (Sp)-3d was synthesized
from 5′-O-DMTr-2′-O-CEM-O6-cyanoethyl-N2-phenoxyacetylguano-
sine 1d (1.28 g, 1.5 mmol) following the typical procedure described
above and purified by silica gel column chromatography [NH silica gel,
hexane−ethyl acetate−triethylamine (30:10:0.04 to 10:20:0.03, v/v/
v)]. The fractions containing (Sp)-3d were collected, washed with a
saturated NaHCO3 aqueous solution (100 mL), dried over Na2SO4,
filtered, and concentrated to dryness under reduced pressure to afford
(Sp)-3d (0.87 g, 0.82 mmol, 54%) as a colorless foam. 1H NMR (300
MHz, CDCl3) δ 8.77 (1H, brs, 2-NH), 8.13 (1H, s, 8-H), 7.45−6.98
(19H, m, 5″-Ph, 2-H of p-An, Ph of DMTr, Ph of Pac), 6.79 (4H, d, J
= 6.6 Hz, 3-H of p-An), 6.23 (1H, d, J = 4.8 Hz, 1′-H), 5.76 (1H, d, J =
6.6 Hz, 5″-H), 4.99−4.75 (6H, m, 2′-H, 3′-H, OCH2O of CEM, O6-
OCH2CH2CN), 4.62 (2H, s, CH2 of Pac), 4.39 (1H, d, J = 3.3 Hz, 4′-
H), 3.85 (1H, ddd, J = 6.0, 10.4, 10.4 Hz, 4″-H), 3.77 (6H, s, OMe),
3.62−3.42 (5H, m, 5′-H, 6″-H, OCH2CH2CN of CEM), 3.19−3.03
(3H, m, 6″-H, O6-OCH2CH2CN), 2.34 (2H, t, J = 6.3 Hz,
OCH2CH2CN of CEM), 1.75−1.59 (2H, m, 7″-H), 1.28−1.17 (1H,
8.4 Hz, 6-H), 7.41−7.16 (14H, m, 5″-Ph, 2-H of p-An, Ph of DMTr),
6.85 (4H, dd, J = 2.1, 9.0 Hz, 3-H of p-An), 5.98 (1H, d, J = 1.5 Hz, 1′-
H), 5.79 (1H, d, J = 6.3 Hz, 5″-H), 5.20 (1H, d, J = 8.4 Hz, 5-H),
4.89−4.81 (3H, m, 3′-H, OCH2O of CEM), 4.32−4.25 (2H, m, 2′-H,
4′-H), 3.87−3.68 (10H, m, 5′-H, 4″-H, OCH2CH2CN, OMe), 3.54−
3.41 (2H, m, 5′-H, 6″-H), 3.14−3.03 (1H, m, 6″-H), 2.47 (2H, ddd, J
= 1.8, 6.0, 6.0 Hz, OCH2CH2CN), 1.68−1.59 (2H, m, 7″-H), 1.28−
1.17 (1H, m, 8″-H), 1.01−0.88 (1H, m, 8″-H). 13C NMR (75 MHz,
CDCl3) δ 163.0, 158.7, 158.7, 150.1, 144.0, 139.8, 137.7 (d, 3JPC = 4.0
Hz), 135.0, 134.8, 130.3, 129.0, 128.4, 128.3, 128.2, 128.0, 127.7,
127.2, 125.3, 117.9, 113.2, 113.2, 102.2, 94.6, 88.4, 87.1, 82.3 (d, 2JPC
=
9.5 Hz), 81.8 (d, 3JPC = 4.3 Hz), 78.5, 69.3 (d, 2JPC = 15.5 Hz), 67.3 (d,
2JPC = 3.2 Hz), 62.9, 60.2, 55.2, 47.0 (d, 2JPC = 34.4 Hz), 27.9, 25.9 (d,
3JPC = 3.4 Hz), 18.6. 31P NMR (121 MHz, CDCl3) δ 159.4. ESI-
HRMS: m/z calcd for C47H51N5O10P+ [(M + H)+] 835.3103, found
835.3104.
(Sp)-Cac-Monomer [(Sp)-3b]. Crude (Sp)-3b was synthesized
from 5′-O-DMTr-2′-O-CEM-N4-acetylcytidine 1b (0.67 g, 1.0 mmol)
following the typical procedure described above and purified by silica
gel column chromatography [NH silica gel, toluene−ethyl acetate−
triethylamine (10:20:0.03, v/v/v)]. The fractions containing (Sp)-3b
were collected, washed with a saturated NaHCO3 aqueous solution
(100 mL), dried over Na2SO4, filtered, and concentrated to dryness
under reduced pressure to afford (Sp)-3b (0.66 g, 0.75 mmol, 75%) as
m, 8″-H), 1.01−0.89 (1H, m, 8″-H). 13C NMR (75 MHz, CDCl3) δ
3
165.7, 159.7, 158.6, 157.0, 153.0, 151.0, 144.3, 140.8, 137.9 (d, JPC
=
3.8 Hz), 135.4, 135.4, 130.1, 129.8, 128.5, 128.3, 128.2, 127.9, 127.6,
127.0, 125.4, 122.4, 118.6, 117.6, 116.8, 114.9, 113.2, 94.8, 86.8, 86.5,
3
2
2
83.8 (d, JPC = 3.7 Hz), 82.3 (d, JPC = 9.5 Hz), 78.0, 70.6 (d, JPC
=
2
12.9 Hz), 67.7, 67.3 (d, JPC = 3.2 Hz), 63.0, 62.6, 61.8, 55.2, 47.1 (d,
2JPC = 34.9 Hz), 27.9, 25.9 (d, JPC = 3.5 Hz), 18.6, 18.0. 31P NMR
3
1
a pale yellow foam. H NMR (300 MHz, CDCl3) δ 9.07 (1H, brs, 4-
(121 MHz, CDCl3) δ 157.5. ESI-HRMS: m/z calcd for
NH), 8.61 (1H, d, J = 7.5 Hz, 6-H), 7.44−7.16 (14H, m, 5″-Ph, 2-H of
p-An, Ph of DMTr), 6.99 (1H, d, J = 7.5 Hz, 5-H), 6.86 (4H, dd, J =
1.8, 8.7 Hz, 3-H of p-An), 5.98 (1H, s, 1′-H), 5.78 (1H, d, J = 6.3 Hz,
5″-H), 5.04 (1H, d, J = 6.9 Hz, OCH2O of CEM), 4.95 (1H, d, J = 6.9
Hz, OCH2O of CEM), 4.78 (1H, ddd, J = 4.8, 9.3, 9.3 Hz, 3′-H),
4.34−4.27 (2H, m, 2′-H, 4′-H), 3.88−3.73 (10H, m, 5′-H, 4″-H,
OCH2CH2CN, OMe), 3.56−3.39 (2H, m, 5′-H, 6″-H), 3.12−3.00
(1H, m, 6″-H), 2.49 (2H, t, J = 6.9 Hz, OCH2CH2CN), 2.24 (3H, s,
Ac), 1.78−1.58 (2H, m, 7″-H), 1.28−1.11 (1H, m, 8″-H), 0.99−0.86
(1H, m, 8″-H). 13C NMR (75 MHz, CDCl3) δ 170.2, 162.6, 158.7,
C57H58N8O11P+ [(M + H)+] 1061.3957, found 1061.3977.
(Rp)-U-Monomer [(Rp)-3a]. Crude (Rp)-3a was synthesized from
5′-O-DMTr-2′-O-CEM-uridine 1a (0.63 g, 1.0 mmol) following the
typical procedure described above and purified by silica gel column
chromatography [NH silica gel, toluene−ethyl acetate−triethylamine
(60:40:0.1, v/v/v)]. The fractions containing (Rp)-3a were collected,
washed with a saturated NaHCO3 aqueous solution (100 mL), dried
over Na2SO4, filtered, and concentrated to dryness under reduced
pressure to afford (Rp)-3a (0.52 g, 0.62 mmol, 62%) as a colorless
foam. 1H NMR (300 MHz, CDCl3) δ 8.98 (1H, brs, 3-NH), 8.07 (1H,
d, J = 8.1 Hz, 6-H), 7.41−7.19 (14H, m, 5″-Ph, 2-H of p-An, Ph of
DMTr), 6.79 (4H, dd, J = 8.1, 8.1 Hz, 3-H of p-An), 5.95 (1H, d, J =
1.2 Hz, 1′-H), 5.75 (1H, d, J = 6.6 Hz, 5″-H), 5.16 (1H, d, J = 8.1 Hz,
5-H), 5.01, 4.94 (2H, 2d, J = 7.2 Hz, OCH2O of CEM), 4.89 (1H,
ddd, J = 6.9, 8.4, 8.4 Hz, 3′-H), 4.35 (1H, dd, J = 1.2, 4.8 Hz, 2′-H),
4.21 (1H, d, J = 8.1 Hz, 4′-H), 3.94−3.87 (3H, m, 4″-H,
OCH2CH2CN), 3.77, 3.74 (6H, 2s, OMe), 3.63−3.52 (3H, m, 5′-H,
6″-H), 3.09−3.03 (1H, m, 6″-H), 2.67 (2H, ddd, J = 2.7, 6.5, 6.5 Hz,
OCH2CH2CN), 1.65−1.56 (2H, m, 7″-H), 1.25−1.19 (1H, m, 8″-H),
1.03−0.91 (1H, m, 8″-H). 13C NMR (75 MHz, CDCl3) δ 163.0,
3
154.8, 144.7, 144.0, 137.8 (d, JPC = 4.3 Hz), 135.1, 135.0, 135.0,
130.3, 128.3, 128.3, 128.0, 127.6, 127.2, 125.3, 118.0, 113.2, 113.2,
113.2, 96.5, 94.6, 90.0, 87.1, 82.4 (d, 2JPC = 9.5 Hz), 81.3 (d, 3JPC = 4.0
Hz), 78.6, 68.6 (d, 2JPC = 14.9 Hz), 67.2 (d, 2JPC = 3.2 Hz), 63.0, 59.7,
55.2, 46.9 (d, 2JPC = 35.4 Hz), 29.7, 27.9, 25.9 (d, 3JPC = 3.8 Hz), 24.9,
18.6. 31P NMR (121 MHz, CDCl3) δ 159.0. ESI-HRMS: m/z calcd for
C47H51N5O10P+ [(M + H)+] 876.3368, found 876.3365.
(Sp)-Aac-Monomer [(Sp)-3c]. Crude (Sp)-3c was synthesized
from 5′-O-DMTr-2′-O-CEM-N6-acetyladenosine 1c (0.70 g, 1.0
mmol) following the typical procedure described above and purified
by silica gel column chromatography [NH silica gel, toluene−ethyl
acetate−triethylamine (50:50:0.1, v/v/v)]. The fractions containing
3
158.7, 158.6, 150.1, 144.3, 140.0, 138.0 (d, JPC = 4.0 Hz), 135.0,
130.3, 130.1, 128.3, 128.2, 128.0, 127.6, 127.2, 125.5, 117.9, 113.2,
7919
dx.doi.org/10.1021/jo301052v | J. Org. Chem. 2012, 77, 7913−7922

The Journal of Organic Chemistry
Article
2
113.2, 102.1, 94.6, 88.5, 87.1, 82.6 (d, JPC = 9.6 Hz), 81.8, 78.4 (d,
OCH2CH2CN), 4.63 (2H, s, CH2 of Pac), 4.35 (1H, d, J = 2.7 Hz, 4′-
H), 3.90−3.42 (12H, m, 5′-H, 4″-H, 6″-H, OMe, OCH2CH2CN),
3.19−3.00 (3H, m, 6″-H, O6-OCH2CH2CN), 2.48 (2H, t, J = 6.3 Hz,
OCH2CH2CN of CEM), 1.73−1.59 (2H, m, 7″-H), 1.29−1.12 (1H,
m, 8″-H), 1.01−0.87 (1H, m, 8″-H). 13C NMR (75 MHz, CDCl3) δ
165.7, 159.7, 158.5, 158.5, 157.0, 153.0, 151.0, 144.4, 140.8, 138.0 (d,
3JPC = 4.1 Hz), 135.5, 135.4, 130.1, 130.0, 129.8, 128.2, 128.1, 127.9,
127.6, 127.0, 125.5, 122.4, 118.7, 117.6, 116.8, 114.9, 113.1, 95.1, 86.7,
86.6, 83.7, 82.7 (d, 2JPC = 9.5 Hz), 78.2 (d, 3JPC = 3.5 Hz), 71.1 (d, 2JPC
= 12.0 Hz), 67.7, 67.3 (d, 2JPC = 3.2 Hz), 63.2, 62.6, 61.8, 55.2, 47.0 (d,
3JPC = 3.2 Hz), 69.4 (d, 2JPC = 14.4 Hz), 67.3 (d, 2JPC = 3.2 Hz), 63.1,
60.3, 55.2, 55.2, 47.1 (d, 2JPC = 34.7 Hz), 28.1, 26.0 (d, 3JPC = 3.7 Hz),
18.7. 31P NMR (121 MHz, CDCl3) δ 158.2. ESI-HRMS: m/z calcd for
C45H48N4O10P+ [(M + H)+] 835.3103, found 835.3104.
(Rp)-Cac-Monomer [(Rp)-3b]. Crude (Rp)-3b was synthesized
from 5′-O-DMTr-2′-O-CEM-N4-acetylcytidine 1b (1.01 g, 1.5 mmol)
following the typical procedure described above and purified by silica
gel column chromatography [NH silica gel, hexane−ethyl acetate−
triethylamine (20:10:0.03 to 10:30:0.03, v/v/v)]. The fractions
containing (Rp)-3b were collected, washed with a saturated
NaHCO3 aqueous solution (100 mL), dried over Na2SO4, filtered,
and concentrated to dryness under reduced pressure to afford (Rp)-3b
(0.69 g, 0.79 mmol, 53%) as a pale yellow foam. 1H NMR (300 MHz,
CDCl3) δ 9.39 (1H, brs, 4-NH), 8.54 (1H, d, J = 7.5 Hz, 6-H), 7.42−
7.16 (14H, m, 5″-Ph, 2-H of p-An, Ph of DMTr), 6.88 (1H, d, J = 7.5
Hz, 5-H), 6.84−6.77 (4H, dd, J = 6.0, 9.0 Hz, 3-H of p-An), 5.94 (1H,
s, 1′-H), 5.72 (1H, d, J = 6.3 Hz, 5″-H), 5.15 (1H, d, J = 6.9 Hz,
OCH2O of CEM), 4.98, (1H, d, J = 6.9 Hz, OCH2O of CEM), 4.84
(1H, ddd, J = 4.8, 9.3, 9.3 Hz, 3′-H), 4.33−4.26 (2H, m, 2′-H, 4′-H),
3.96−3.86 (3H, m, 4″-H, OCH2CH2CN), 3.77, 3.75 (6H, 2s, OMe),
3.69−3.50 (3H, m, 5′-H, 6″-H), 3.15−3.04 (1H, m, 6″-H), 2.78−2.59
(2H, m, OCH2CH2CN), 2.24 (3H, s, Ac), 1.67−1.51 (2H, m, 7″-H),
1.28−1.17 (1H, m, 8″-H), 1.01−0.93 (1H, m, 8″-H). 13C NMR (75
MHz, CDCl3) δ 170.1, 162.6, 158.7, 158.6, 154.9, 144.8, 144.2, 138.0
3
2JPC = 34.7 Hz), 28.0, 25.9 (d, JPC = 3.5 Hz), 18.6, 18.0. 31P NMR
(121 MHz, CDCl3) δ 156.8. ESI-HRMS: m/z calcd for
C57H58N8O11P+ [(M + H)+] 1061.3957, found 1061.3964.
A General Procedure for Solid-Phase Synthesis of PS-ORNs.
Manual solid-phase synthesis of P-stereodefined PS-ORNs was
performed according to the procedure given in Table 4 by using 5′-
Table 4. Procedure for Manual Solid-Phase Synthesis of PS-
ORNs
step
operation
reagents and solvents
3% DCA in CH2Cl2
time
1
2
detritylation
washing
4 × 15 s
(i) CH2Cl2, (ii) dry CH3CN, (iii)
drying in vacuo
3
condensation 0.13 M monomer 3a−d, 1 M
5, 10, or
15 min
3
(d, JPC = 4.0 Hz), 135.1, 130.3, 130.1, 128.3, 128.2, 128.0, 127.6,
127.2, 125.5, 118.1, 113.2, 98.4, 96.4, 94.4, 90.1, 87.1, 82.6 (d, JPC
activator, dry CH3CN
2
=
4
5
washing
capping
(i) dry CH3CN, (ii) drying in vacuo
2
2
9.5 Hz), 81.4, 78.2, 68.8 (d, JPC = 14.6 Hz), 67.3 (d, JPC = 3.5 Hz),
63.0, 59.9, 55.2, 47.1 (d, 2JPC = 34.7 Hz), 28.0, 25.9 (d, 3JPC = 3.5 Hz),
24.9, 18.7. 31P NMR (121 MHz, CDCl3) δ 158.0. ESI-HRMS: m/z
calcd for C47H51N4O10P+ [(M + H)+] 876.3368, found 876.3367.
(Rp)-Aac-Monomer [(Rp)-3c]. Crude (Rp)-3c was synthesized
from 5′-O-DMTr-2′-O-CEM-N6-acetyladenosine 1c (0.70 g, 1.0
mmol) following the typical procedure described above and purified
by silica gel column chromatography [NH silica gel, toluene−ethyl
acetate−triethylamine (20:10:0.03, v/v/v)]. The fractions containing
(Rp)-3c were collected, washed with a saturated NaHCO3 aqueous
solution (100 mL), dried over Na2SO4, filtered, and concentrated to
dryness under reduced pressure to afford (Rp)-3c (0.44 g, 0.49 mmol,
0.5 M CF3COIm and 1 M DMAN in 30 s
dry THF
6
washing
(i) dry THF, (ii) dry CH3CN, (iii)
drying in vacuo
7
8
sulfurization
washing
0.3 M DTD in dry CH3CN
10 min
(i) dry CH3CN, (ii) drying in vacuo
O-DMTr-uridine-loaded HCP or CPG (0.5 μmol). Automated solid-
phase synthesis (10c,d) was performed according to the procedure
given in Table 5 by using 5′-O-DMTr-uridine-loaded HCP (0.25
1
Table 5. Procedure for Automated Solid-Phase Synthesis of
PS-ORNs
49%) as a colorless foam. H NMR (300 MHz, CDCl3) δ 8.64 (1H,
brs, 6-NH), 8.61 (1H, s, 2-H), 8.26 (1H, s, 8-H), 7.45−7.16 (14H, m,
5″-Ph, 2-H of p-An, Ph of DMTr), 6.76 (4H, d, J = 9.0 Hz, 3-H of p-
An), 6.24 (1H, d, J = 4.8 Hz, 1′-H), 5.77 (1H, d, J = 6.3 Hz, 5″-H),
5.06−4.95 (2H, m, 2′-H, 3′-H), 4.90, 4.84 (2H, 2d, J = 7.2 Hz,
OCH2O of CEM), 4.36 (1H, dd, J = 3.0, 6.0 Hz, 4′-H), 3.88 (1H, ddd,
J = 6.3, 10.5, 10.5 Hz, 4″-H), 3.81−3.51 (10H, m, 5′-H, 6″-H, OMe,
OCH2CH2CN), 3.41 (1H, dd, J = 3.6, 10.5 Hz, 5′-H), 3.18−3.06 (1H,
m, 6″-H), 2.60 (3H, s, Ac), 2.49 (2H, t, J = 6.3 Hz, OCH2CH2CN),
1.69−1.59 (2H, m, 7″-H), 1.28−1.18 (1H, m, 8″-H), 0.98−0.91 (1H,
m, 8″-H). 13C NMR (75 MHz, CDCl33) δ 170.3, 158.5, 158.5, 152.3,
150.9, 149.2, 144.4, 141.9, 138.0 (d, JPC = 3.7 Hz), 135.5, 135.4,
130.1, 130.1, 128.3, 128.2, 127.8, 127.6, 126.9, 125.5, 122.2, 117.5,
step
operation
reagents and solvents
3% DCA in CH2Cl2
dry CH3CN
time
49 s
1
2
3
detritylation
washing
condensation 0.15 M monomer 3a−d, 1 M activator,
15 min
dry CH3CN
4
5
washing
capping
dry CH3CN
0.5 M CF3COIm and 1 M DMAN in dry 30 s
THF
6
7
8
washing
dry CH3CN
sulfurization
washing
0.3 M DTD in dry CH3CN
dry CH3CN
6 min
2
113.1, 95.2, 87.3, 86.7, 83.6, 82.8 (d, JPC = 10.0 Hz), 78.0, 71.0 (d,
2JPC = 10.6 Hz), 67.3 (d, 2JPC = 3.2 Hz), 63.1, 62.4, 55.2, 47.0 (d, 2JPC
=
34.9 Hz), 28.1, 25.8 (d, JPC = 18.1 Hz), 18.6. 31P NMR (121 MHz,
CDCl3) δ 157.0. ESI-HRMS: m/z calcd for C48H51N7O9P+ [(M +
H)+] 900.3480, found 900.3480.
3
μmol). After the chain elongation by repeating steps 1−8 in Table 4 or
5, the 5′-O-DMTr group was removed by treatment with 3% DCA in
CH2Cl2 except for those isolated ones, for which the DMTr group was
left as a purification handle. The deprotection of the bases and the PS-
linkages and the cleavage of the linker were performed by treatment
with a 25% NH3 aqueous solution−EtOH (3:1, v/v) (6 mL) for 3 h
(for 2mers), 12 h (for 4mers), or 48 h (for 12mers) at rt. The resultant
crude products were analyzed (2−4mers) or purified (12mers) by RP-
HPLC. Fractions containing the desired PS-ORN 12mers (2′-O-CEM-
on−5′-O-DMTr-on) were collected and lyophilized. The residue was
then treated with a 0.5 M TBAF solution in dry DMSO containing
0.5% CH3NO2 (400 μL) for 5 h at rt and diluted with a 0.1 M TEAA
buffer solution (pH 7.0) (40 mL). The mixture was purified with a
Sep-pak C18 cartridge. The Sep-pak was washed twice with a 0.1 M
TEAA buffer solution (pH 7.0) (5.0 mL) to remove DMSO, TBAF,
(Rp)-Gce,pac-Monomer [(Rp)-3d]. Crude (Rp)-3d was synthesized
from 5′-O-DMTr-2′-O-CEM-O6-cyanoethyl-N2-phenoxyacetylguano-
sine 1d (0.86 g, 1.0 mmol) following the typical procedure described
above and purified by silica gel column chromatography [NH silica gel,
toluene−ethyl acetate−triethylamine (80:20:0.1, v/v/v)]. The frac-
tions containing (Rp)-3d were collected, washed with a saturated
NaHCO3 aqueous solution (100 mL), dried over Na2SO4, filtered, and
concentrated to dryness under reduced pressure to afford (Rp)-3d
1
(0.45 g, 0.42 mmol, 42%) as a colorless foam. H NMR (300 MHz,
CDCl3) δ 8.79 (1H, brs, 2-NH), 8.10 (1H, s, 8-H), 7.42−7.00 (19H,
m, 5″-Ph, 2-H of p-An, Ph of DMTr, Ph of Pac), 6.76 (4H, d, J = 6.6
Hz, 3-H of p-An), 6.23 (1H, d, J = 5.1 Hz, 1′-H), 5.74 (1H, d, J = 6.3
Hz, 5″-H), 5.01−4.82 (6H, m, 2′-H, 3′-H, OCH2O of CEM, O6-
7920
dx.doi.org/10.1021/jo301052v | J. Org. Chem. 2012, 77, 7913−7922

The Journal of Organic Chemistry
Article
and CH3NO2, and then the desired PS-ORNs were eluted with 80%
CH3CN and lyophilized. The residue was then treated with an 80%
AcOH aqueous solution (500 μL) for 1 h at rt and diluted with a 2 M
TEAA buffer solution (pH 7.0) (20 mL). The mixture was desalted
with a Sep-pak C18 cartridge. The Sep-pak was washed twice with a
0.1 M TEAA buffer solution (pH 7.0) (5.0 mL), and then the product
was eluted with 40% CH3CN. The eluate was concentrated under
reduced pressure and purified by RP-HPLC to afford the desired PS-
ORNs. Isolated yields were determined by UV quantitation at 260 nm.
All-(Rp)-PS-U12 10a, 12% isolated yield, MALDI-TOF MS: m/z calcd
REFERENCES
■
(1) (a) Bumcrot, D.; Manoharan, M.; Koteliansky, V.; Sah, D. W. Y.
Nat. Chem. Biol. 2006, 2, 711−719. (b) Shukla, S.; Sumaria, C. S.;
Pradeepkumar, P. I. ChemMedChem 2010, 5, 328−349.
(2) Seto, A. G. Int. J. Biochem. Cell Biol. 2010, 42, 1298−1305.
(3) Echeverri, C. J.; Perrimon, N. Nat. Rev. Genet. 2006, 7, 373−384.
(4) (a) Verma, S.; Eckstein, F. Annu. Rev. Biochem. 1998, 67, 99−134.
(b) Guga, P.; Koziołkiewicz, M. Chem. Biodiversity 2011, 8, 1642−
1681.
(5) Oka, N.; Wada, T. Chem. Soc. Rev. 2011, 40, 5829−5843.
(6) Frederiksen, J. K.; Piccirilli, J. A. Methods Enzymol. 2009, 468,
289−309.
(7) (a) Yano, J.; Ohgi, T.; Ueda, T. Patent WO2006/043521-A1,
CAN: 144:450874. (b) Nawrot, B.; Rębowska, B.; Michalak, O.;
Bulkowski, M.; Błaziak, D.; Guga, P.; Stec, W. J. Pure Appl. Chem.
2008, 80, 1859−1871. (c) Wrzesinski, J.; Wichłacz, A.; Nijakowska, D.;
Rebowska, B.; Nawrot, B.; Ciesiołka, J. New J. Chem. 2010, 34, 1018−
1026.
−
for C108H132N24O83P11S11 [(M − H)−] 3785.09, found 3788.62. All-
(Sp)-PS-U12 10b, 14% isolated yield, MALDI-TOF MS: m/z calcd for
−
C108H132N24O83P11S11 [(M − H)−] 3785.09, found 3789.25. All-
(Rp)-PS-(CAGU)3 10c, 6% isolated yield, MALDI-TOF MS: m/z
calcd for C114H141N45O71P11S11− [(M − H)−] 3968.29, found 3971.19.
All-(Sp)-PS-(CAGU)3 10d, 10% isolated yield, MALDI-TOF MS: m/z
calcd for C114H141N45O71P11S11− [(M − H)−] 3968.29, found 3972.53.
Enzymatic Digestion of P-Stereodefined PS-ORNs. Digestion
with svPDE. An aqueous solution (20 μL, pH 8.5) containing a
purified PS-ORN (10a−d) (1.0 nmol), svPDE (0.1 × 10−3 unit), 100
mM Tris-HCl, and 15 mM MgCl2 was incubated for 16 h at 37 °C.
The mixture was then diluted with 0.1 M TEAA buffer (pH 7.0) (80
μL), heated for 1 min at 100 °C to inactivate the enzyme, filtrated, and
analyzed by RP-HPLC.
(8) Matzura, H.; Eckstein, F. Eur. J. Biochem. 1968, 3, 448−452.
(9) Sierzchała, A.; Okruszek, A.; Stec, W. J. J. Org. Chem. 1996, 61,
6713−6716.
(10) Lesnikowski, Z. J. Nucleosides Nucleotides 1992, 11, 1621−1638.
(11) (a) Almer, H.; Stawinski, J.; Stromberg, R. J. Chem. Soc., Chem.
̈
Commun. 1994, 1459−1460. (b) Almer, H.; Stawinski, J.; Stromberg,
R. Nucleic Acids Res. 1996, 24, 3811−3820.
̈
Digestion with nP1. An aqueous solution (20 μL, pH 7.2)
containing a purified PS-ORN (10a−d) (1.0 nmol), nuclease P1 (1
unit), 50 mM CH3COONa, and 1 mM ZnCl2 was incubated for 16 h
at 37 °C. The mixture was then diluted with 0.1 M TEAA buffer (pH
7.0) (80 μL), heated at 100 °C for 1 min to inactivate the enzyme,
filtrated, and analyzed by RP-HPLC.
Thermal Denaturating Experiment. An aqueous solution (200 μL,
pH 7.0) containing a 1:1 ratio of complementary ORNs (0.45 nmol
each), 10 mM phosphate, 100 mM NaCl, and 0.1 mM EDTA was
deaerated for 10 min under reduced pressure. An aliquot (165 μL) was
placed in a 1 cm path length quartz cell. The solution was then heated
at a rate of 5 °C/min from rt to 90 °C, kept for 10 min at 90 °C, and
cooled at a rate of −2 °C/min from 90 to 0 °C to hybridize the ORNs.
After being kept for 90 min at 0 °C, the solution was gradually heated
for denaturating experiments. UV absorbance values (260 nm) were
recorded at intervals of 0.5 °C while the temperature was ramped at a
rate of 0.5 °C/min from 0 to 90 °C under Ar.
(12) Efimov, V. A.; Molchanova, N. S.; Chakhmakhcheva, O. G.
Nucleosides Nucleotides Nucleic Acids 2007, 26, 1087−1093.
(13) Oka, N.; Kondo, T.; Fujiwara, S.; Maizuru, Y.; Wada, T. Org.
Lett. 2009, 11, 967−970.
́
(14) Somoza, A. Chem. Soc. Rev. 2008, 37, 2668−2675.
(15) (a) Scaringe, S. A.; Wincott, F. E.; Caruthers, M. H. J. Am.
Chem. Soc. 1998, 120, 11820−11821. (b) Dellinger, D. J.; Timar
Myerson, J.; Sierzchala, A. B.; Turner, J.; Ferreira, F.; Kupihar, Z.;
́
, Z.;
́
Dellinger, G.; Hill, K. W.; Powell, J. A.; Sampson, J. R.; Caruthers, M.
H. J. Am. Chem. Soc. 2011, 133, 11540−11556. (c) Umemoto, T.;
Wada, T. Tetrahedron Lett. 2004, 45, 9529−9531. (d) Semenyuk, A.;
Foldesi, A.; Johansson, T.; Estmer-Nilsson, C.; Blomgren, P.;
̈
Brannvall, M.; Kirsebom, L. A.; Kwiatkowski, M. J. Am. Chem. Soc.
̈
2006, 128, 12356−12357. (e) Martin, A. R.; Lavergne, T.; Vasseur, J.-
J.; Debart, F. Bioorg. Med. Chem. Lett. 2009, 19, 4046−4049.
(f) Lavergne, T.; Bertrand, J.-R.; Vasseur, J.-J.; Debart, F. Chem.
Eur. J. 2008, 14, 9135−9138. (g) Saneyoshi, H.; Ando, K.; Seio, K.;
Sekine, M. Tetrahedron 2007, 63, 11195−11203. (h) Zhou, C.;
Pathmasiri, W.; Honcharenko, D.; Chatterjee, S.; Barman, J.;
Chattopadhyaya, J. Can. J. Chem. 2007, 85, 293−301. (i) Lackey, J.
G.; Mitra, D.; Somoza, M. M.; Cerrina, F.; Damha, M. J. J. Am. Chem.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H, 13C, and 31P NMR spectra, HPLC profiles and UV melting
curves. This material is available free of charge via the Internet
at http://pubs.acs.org.
́
Soc. 2009, 131, 8496−8502. (j) Cieslak, J.; Grajkowski, A.; Kauffman,
J. S.; Duff, R. J.; Beaucage, S. L. J. Org. Chem. 2008, 73, 2774−2783.
(16) (a) Ohgi, T.; Masutomi, Y.; Ishiyama, K.; Kitagawa, H.; Shiba,
Y.; Yano, J. Org. Lett. 2005, 7, 3477−3480. (b) Shiba, Y.; Masuda, H.;
Watanabe, N.; Ego, T.; Takagaki, K.; Ishiyama, K.; Ohgi, T.; Yano, J.
Nucleic Acids Res. 2007, 35, 3287−3296. (c) Nagata, S.; Hamasaki, T.;
Uetake, K.; Masuda, H.; Takagaki, K.; Oka, N.; Wada, T.; Ohgi, T.;
Yano, J. Nucleic Acids Res. 2010, 38, 7845−7857.
AUTHOR INFORMATION
Corresponding Author
*E-mail: wada@k.u-tokyo.ac.jp.
Notes
■
The authors declare no competing financial interest.
(17) Oka, N.; Yamamoto, M.; Sato, T.; Wada, T. J. Am. Chem. Soc.
2008, 130, 16031−16037.
ACKNOWLEDGMENTS
■
(18) Ueda, T.; Nagata, S. Patent JP2011-121881A, CAN: 155:68060.
(19) (a) Oka, N.; Wada, T.; Saigo, K. J. Am. Chem. Soc. 2002, 124,
4962−4963. (b) Oka, N.; Wada, T.; Saigo, K. J. Am. Chem. Soc. 2003,
125, 8307−8317.
(20) Song, Q.; Wang, Z.; Sanghvi, Y. S. Nucleosides Nucleotides Nucleic
Acids 2003, 22, 629−633.
(21) See the Supporting Information.
(22) Hayakawa, Y.; Kawai, R.; Hirata, A.; Sugimoto, J.; Kataoka, M.;
Sakakura, A.; Hirose, M.; Noyori, R. J. Am. Chem. Soc. 2001, 123,
8165−8176.
(23) (a) Burgers, P. M. J.; Eckstein, F. Proc. Natl. Acad. Sci. U.S.A.
1978, 75, 4798−4801. (b) Bryant, F. R.; Benkovic, S. J. Biochemistry
1979, 18, 2825−2829.
We thank Professor Kazuhiko Saigo (Kochi University of
Technology) and Mr. Seigo Nagata (Nippon Shinyaku Co.,
Ltd.) for helpful suggestions. We also thank Nippon Shinyaku
Co., Ltd. for providing 2′-O-CEM nucleosides and Dr. Daisuke
Nakajima (Kazusa DNA Research Institute) for measurement
of mass spectra. This research was supported by grants from the
Ministry of Education, Culture, Sports, Science, and Technol-
ogy (MEXT) Japan, the New Energy and Industrial
Technology Development Organization (NEDO) Japan for
its Functionalized RNA Project and Grant-in-Aid for Scientific
Research on Innovative Areas (21115008).
7921
dx.doi.org/10.1021/jo301052v | J. Org. Chem. 2012, 77, 7913−7922

The Journal of Organic Chemistry
Article
(24) Potter, B. V. L; Connolly, B. A.; Eckstein, F. Biochemistry 1983,
22, 1369−1377.
(25) Griffiths, A. D.; Potter, B. V. L; Eperon, I. C. Nucleic Acids Res.
1987, 15, 4145−4162.
NOTE ADDED AFTER ASAP PUBLICATION
■
There were errors in two of the chemical formulas in the
version published ASAP August 29, 2012. The correct version
reposted September 5, 2012.
7922
dx.doi.org/10.1021/jo301052v | J. Org. Chem. 2012, 77, 7913−7922