Synthesis and properties of novel l-isonucleoside modified oligonucleotides and siRNAs

Article abstract of DOI:10.1039/c2ob26219c

Antisense oligonucleotides and siRNAs are potential therapeutic agents and their chemical modifications play an important role to improve the properties and activities of oligonucleotides. Isonucleoside is a type of nucleoside analogue, in which the nucleobase is moved from C-1 to other positions of ribose. In this report, a novel isonucleoside 5 containing a 5′-CH ₂-extended chain at the sugar moiety was synthesized, thus isoadenosine 5a and isothymidine 5b were incorporated into a DNA single strand and siRNA. It was found that isonucleoside 5 modified oligonucleotides can form stable double helical structures with their complementary DNA and RNA and the stability towards nuclease and ability to activate RNase H are more promising compared with the unmodified, natural analogues. In siRNA, passenger strand modified with isonucleoside (5a/b) at 3′ or 5′ terminal can retain the silencing activity and minimize the passenger strand specific off-target effect.

Full text of DOI:10.1039/c2ob26219c

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PAPER
Synthesis and properties of novel L-isonucleoside modified oligonucleotides
and siRNAs†‡
Jun Zhang,§ Yue Chen,§ Ye Huang, Hong-Wei Jin, Ren-Ping Qiao, Lei Xing, Liang-Ren Zhang,
Zhen-Jun Yang* and Li-He Zhang*
Received 28th April 2012, Accepted 31st July 2012
DOI: 10.1039/c2ob26219c
Antisense oligonucleotides and siRNAs are potential therapeutic agents and their chemical modifications
play an important role to improve the properties and activities of oligonucleotides. Isonucleoside is a type
of nucleoside analogue, in which the nucleobase is moved from C-1 to other positions of ribose. In this
report, a novel isonucleoside 5 containing a 5′-CH2-extended chain at the sugar moiety was synthesized,
thus isoadenosine 5a and isothymidine 5b were incorporated into a DNA single strand and siRNA. It was
found that isonucleoside 5 modified oligonucleotides can form stable double helical structures with their
complementary DNA and RNA and the stability towards nuclease and ability to activate RNase H are
more promising compared with the unmodified, natural analogues. In siRNA, passenger strand modified
with isonucleoside (5a/b) at 3′ or 5′ terminal can retain the silencing activity and minimize the passenger
strand specific off-target effect.
delivery methods, and dosage changes.4 Phosphorothiate(PS)-
Introduction
modified ASONs are first generation ASONs, which exhibit high
stability against nuclease and promote RNase H activity.5
Antisense oligonucleotides (ASONs) and small interfering
RNAs (siRNAs) have attracted considerable attention of both
biologists and chemists due to their potent therapeutic activities.
An antisense oligonucleotide is a single strand of DNA which is
able to form a hybridized duplex with its complementary mRNA
resulting in the specific inhibition of gene expression by various
mechanisms.1 On the other hand, the later discovered RNAi
technique uses double stranded RNAs to inhibit the translation
of target gene through an activated RNA-induced silencing
complex (RISC*), which includes one single strand of siRNA
(the guide strand) and the endonuclease Argonaute 2 (Ago 2)
that promotes the cleavage of target mRNAs.2 These nucleic
acids target endogenous mRNA, and modulate gene expression
based on the Watson–Crick base pairing.
However, phosphorothiate modification produces non-specific tox-
icity. Another modification was reported with elongated phospho-
diester backbone, which increases ASONs’ stability toward
3′-exonuclease and causes a slightly lower Tm value of duplexes.6
Other modifications focused on the ribose moiety, that included
2′-O-modified ASONs, locked nucleic acid (LNA),7 peptide
nucleic acid (PNA),8 phosphoramidate morpolino oligomer
(PMO).9 These chemical modifications have also been applied to
siRNAs, and proved to be an efficient approach to improve the
biophysical and biochemical properties of siRNAs.10 A significant
modification of siRNA was at 2′-O-position of the sugar moiety
such as 2′-O-methyl (2′-O-Me)11 and 2′-deoxy-2′-fluoro (2′-F)12
substitutions, and locked nucleic acid (LNA).13,14
The main challenge for translating the antisense strategy into
clinical applications is how to solve the problems relating to the
stability of antisense oligonucleotides in blood and their delivery
to a target.3 Chemically synthesized antisense oligonucleotide
has great advantages in tolerating chemical modifications,
Besides the short half-life time (t1/2) in serum and poor pene-
tration through cell membrane, off-target silencing is a funda-
mental feature of siRNAs and cannot always be predicted and
eliminated.15,16 “Off-target” effect could be the immune
responses17 or knockdown of extra genes usually initiated by pas-
senger strand or ‘miRNA’ like activities.15 Appropriately modified
sense strand of siRNAs could significantly reduce the “off-target”
effect while maintaining silencing activity.18 It was reported that
thermodynamic stability and Watson–Crick base pairing as main
determinants of the siRNA-based off-target effects and an siRNA
with low thermal stability could show lower off-target effects.19
Our previous work indicated that the incorporation of isonu-
cleoside into the oligodeoxynucleotide might lead to some local
conformational change of the formed duplexes and lower Tm
State Key Laboratory of Natural and Biomimetic Drugs, School of
Pharmaceutical Sciences, Peking University, Beijing 100191, China.
E-mail: zdszlh@bjmu.edu.cn, yangzj@bjmu.edu.cn;
Tel: +86-10-82802503
†In memory of Professor Har Gobind Khorana (1922–2011), acknowl-
edging his legacy to the scientific community.
‡Electronic supplementary information (ESI) available. See DOI:
10.1039/c2ob26219c
§These authors contributed equally to this paper.
7566 | Org. Biomol. Chem., 2012, 10, 7566–7577
This journal is © The Royal Society of Chemistry 2012

Fig. 1 Isonucleoside 1,2,3,4 and the target compound 5.
value. Isonucleoside is a novel type of nucleoside analogue, in
the major product with minor 8b (Z-isomer, J = 11.5 Hz). Cata-
lytic hydrogenation of 8 gave 9, which was then converted to
epoxide 10 in high yield. Epoxide 10 was converted to the
desired isoadenosine 11 in 35% yield along with 12 in 10%
yield. It was presumed that compound 12 could be obtained by
the reaction of compound 11 and dimethylamine, which may be
formed by DMF in the presence of DBU at 110 °C (Scheme 1).
To solve this side reaction, reduction of the ester in 9 was
carried out using LiAlH4 to give 13 in 96% yield. After protec-
tion of the primary hydroxyl in 13 with TBDMSCl, the key
intermediate 14 was treated with nucleobases (adenine or
thymine) and DBU in dry DMF to obtain the desired isonucleo-
sides, isonucleoside 15a was separated with its C3-regioisomer
16a at 8 : 1 ratio. After deprotection of TBDMS group, 5a was
afforded in reasonable yield. 15b and its regioisomer 16b (6 : 1)
could not be separated by silica column chromatography directly.
After deprotection, pure 5b was separated (Scheme 2). The struc-
tures of the two desired isonucleosides 5a and 5b were also
confirmed by 1H NMR, COSY and NOESY spectra.
which the nucleobase is moved from C-1 to other positions of
ribose. Oligonucleotides consisting of isonucleosides (1–4,
Fig. 1) showed greatly increased stability towards nuclease and
sufficient affinity toward their complementary DNA or RNA.20
The DNA/RNA hybrid formed by an isonucleoside modified
oligodeoxynucleotide and its target RNA could activate RNase
H. The 3′-end modified antisense oligodeoxynucleotides inhib-
ited S-glycoprotein expression of SARS-CoV at the mRNA
levels in insect Sf 9 cells.21 When aminoisonucleoside was
incorporated into a luciferase gene-targeting siRNA, it was
found that sense strand modifications with aminoisonucleoside at
the 3′ or 5′ terminal have less effect on RNA duplex thermal and
serum stabilities, and their functional activities are also compar-
able to their native siRNAs. In contrast, antisense strand modifi-
cations with aminoisonucleoside at the corresponding positions
brought a strikingly negative effect on RNA duplex stability but
still maintained around 40–50% of gene knockdown.22
The novel isonucleoside 5 contains a 5′-CH2-extended chain
at the sugar moiety and the oligonucleotides incorporated with
isonucleoside 5 would provide an extended phosphate linkage
which may modulate the thermodynamic stability of the formed
duplex and initiate to reduce the off-target silencing in an un-
intended target. Here we report the synthesis of a novel type of
isonucleoside 5a/b (Fig. 1) from D-xylose. Isoadenosine 5a/
isothymidine 5b modified ODNs and siRNA were synthesized,
their stabilities against nuclease, ability to activate RNase H and
silencing activity of siRNA modified with isonucleosides (5a/b)
in either strand and at different positions were investigated. A
fragment of cdc2 gene was used as the target.23 The sequence of
siCdc2 was chosen from many variants because each strand of
this siRNA had considerable activity for its respective target
mRNA in an artificial RNAi assay. To evaluate the influence on
silencing activity and off-target effect, we constructed two
systems: each of this system contained one strand of siRNA as
target via siQuant vector, matched with the siRNA A strand
(sense strand) or B strand (antisense strand), and chemical
modification on each one strand of siRNA was carried out and
tested by these two systems respectively.
Building blocks 19 and 21, suitable for solid-phase oligonu-
cleotide synthesis, were prepared from 5a/b respectively
(Scheme 3). The exocyclic amino and primary hydroxyl groups
in isoadenosine 5a were protected by benzoyl group using a one-
pot procedure26 to give 17, and then by dimethoxytrityl group to
give 18, which was converted to the phosphoramidite 19. The
corresponding phosphoramidite 21 was prepared from isothymi-
dine 5b by a similar procedure (Scheme 3).
Oligodeoxynucleotides (I–IV) containing 5a were synthesized
by solid phase phosphoramidite chemistry (DMT off) using an
Applied Biosystems model 381 DNA Synthesizer. For conven-
ience the synthesis was started with commercially available con-
trolled pore glass with cytidine-loaded or universal CPG (used
for III) and followed the standard protocol except for a pro-
longed isonucleosides’ coupling time of 900s to ensure adequate
coupling yields. The coupling efficiency was determined by the
release of DMT. Total yield of each oligonucleotide was around
40%. The isonucleoside coupling yield was about 93%, which
was slightly lower than native nucleoside coupling yield (∼96%
in our experiment). The structures of the modified oligonucleo-
tides were determined by MALDI-TOF mass spectrometry: I.
calcd 5945.9, found 5940.7; II. calcd 5945.9, found 5940.9; III,
calcd 6259.1, found 6253.9; IV, calcd 5974.0, found 5976.9.
Results
1
Synthesis of isonucleoside 5a/5b and 5a/5b modified
oligodeoxynucleotides24a
2
Stabilities of the formed DNA/DNA, DNA/RNA duplexes
Compound 6a obtained from D-xylose24b,25 was benzoylated and
then hydrolyzed to yield aldehyde 7. Compound 8 was obtained
from 7 under Wadsworth–Horner–Emmons reaction condition in
an overall 75% yield, 8a (E-isomer, J = 15.5 Hz) was yielded as
and activation of RNase H
The thermal stabilities of the formed DNA/DNA and DNA/RNA
duplexes are listed in Table 1. The results show that all the
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 7566–7577 | 7567

Scheme 1 Reagents and Conditions: i. BzCl, DMAP, Py, 98.1%; ii. TFA–H2O (v : v = 7 : 1); iii. (Et2O)2P(O)-CH2COOEt, NaH, THF, −30 °C; iv.
Pd-C, H2(0.4 MPa), THF, 67.9% from 7; v. K2CO3, MeOH, 96.0%; vi. Adenine, DBU, DMF, 34.7% for 11; 10% for 12; vii. LiAlH4, THF, 80.4%.
Scheme 2 Reagents and conditions: i. LiAlH4, THF, room temp, 96.2%; ii. TBDMSCl, imidazole, DMF, 94.6%; iii. Adenine or thymine, DBU,
DMF, 50.8% for 15a, 6.4% for 16a; 41.3% for 15b and 16b; iv. TBAF, THF, 96.9% for 5a, 93.2% for 5b.
Scheme 3 Reagents and Conditions: i. a. TMSCl, Py; b. BzCl, Py; c. NH3.H2O (pH 8–9); 94% for 17; ii. DMTrCl, Py; 79% for 18; 68% for 20; iii.
Tetrazole, NCCH2CH2OP[N(iPr)2]2, CH2Cl2; 91% for 19; 50% for 21.
oligomers can form the stable duplexes with their complemen-
tary DNA or RNA and give the very similar CD spectra (data
not shown). When 5a was incorporated in the center of
oligomers (I, IV), the Tm value of DNA/DNA duplexes
decreased significantly compared to their native counterpart, and
the Tm value of DNA/RNA duplexes decreased by 6 °C.
7568 | Org. Biomol. Chem., 2012, 10, 7566–7577
This journal is © The Royal Society of Chemistry 2012

Table 1 ODNs (I–IV) with isonucleoside 5a modified at 3′ or 5′ terminal or middle position, ODNs (M1, M2, M3) with single mismatched
nucleoside, ODN (Ds) with phosphorothiate modification. Thermal denaturation studies of these duplexes with complementary sequences (C1: 20-mer
complementary DNA of D1, C2: 21-mer complementary DNA of D2, Cr: 20-mer complementary RNA for natural and modified D1)
ODNs
ODN sequence synthesized
DNA/DNA Tm (°C)
ΔTm (°C)
DNA/RNA Tm (°C)
ΔTm (°C)
D1
D2
I
5′- d(ACATCT CCC GCATCC CAC TC) -3′
5′- d(ACATCT CCC GCATCC CAC TCA) -3′
5′- d(ACATCT CCC GC TCC CAC TC) -3′
5′- d( CATCT CCC GCATCC CAC TC) -3′
5′- d(ACATCT CCC GCATCC CAC TC ) -3′
5′- d(AC TCT CCC GCATCC C C TC) -3′
68.0a
69.0b
59.1a
67.2a
68.3a, 69.1b
57.1a
59.1a
67.1a
62.1a
58.2a
—
—
—
75.0c
—
—
—
−8.9
−0.8
69.1
75.1
76.1
69.2
67.0
76.1
71.1
70.2
—
−5.9c
0.1c
1.1c
−5.8c
−8.0c
1.1c
−3.9c
−4.8c
—
II
III
IV
Ds
M1
M2
M3
C1
C2
Cr
0.3a, 0.1b
−10.9
−8.9
−0.9
−5.9
−9.8
—
5′-d(ACATCT CCC GCATCC CACTC)-3′ (
5′-d( CATCT CCC GCATCC CAC TC)-3′
5′-d(ACATCT CCC GC TCC CAC TC)-3′
5′-d(AC TCT CCC GCATCC C C TC)-3′
5′- d(GAG TGG GAT GCG GGA GAT GT)-3′
5′- d(T GAG TGG GAT GCG GGA GAT GT)-3′
5′- GAG UGG GAU GCG GGA GAU GU -3′
)
—
—
—
—
—
—
—
—
a Tm value of ODN/D1 duplexes. b Tm value of ODN/D2 duplexes. c ΔTm : Compared to D1/Cr duplex’s Tm value (75.0 °C)
Fig. 2 a. PAGE analysis of the degradation of ODN I, III and D1 in 80% fetal bovine serum. Lanes 1 to 7 indicate the time points after 0, 15 and
30 min, 1, 2, 4 and 9 h of reaction, respectively. The percentage % of ODNs left after 9 h of incubation: 0% of D1, 0% of I and 18% of III; b. Time
course of ODN D1, I, III stability in 80% fetal bovine serum.
However, when 5a was incorporated in 5′-end (II) or 3′-end of
oligomers (III), the Tm values of DNA/DNA duplexes are
similar as the native oligomer, and the Tm value of DNA/RNA
duplexes increased by 1 °C. For comparison, nucleoside-
mismatched oligomers (M1, M2, M3) were used for the investi-
gation of Tm value of DNA/DNA or DNA/RNA duplexes. It was
found that mismatched oligomer at 3′ or 5′ or middle position
(M1, M2 and M3) makes the decrease of Tm value of the
formed double helix. It would indicate that the extended phos-
phodiester linkage in the modified strand might be more flexible
and could not influence the formation of DNA/RNA duplexes.
The tolerance of the modified ODNs I and III toward an exo-
nuclease (snake venom phosphodiesterase, SVPDE) was also
investigated. It is expected that 5a at 3′-end of ODN makes the
oligomer more stable: the native D1 was completely degraded in
20 min of incubation, while 100% of ODN-III was left after
20 min incubation with the enzyme (date not shown). Stabilities
of ODN I and III in serum were also tested. It was found that
ODN III showed a better stability than that of ODN D1 (native)
and I in 80% fetal bovine serum (Fig. 2).
RNase H is an endogenous enzyme, which hydrolyses the
RNA strand in an RNA/DNA hybrid in a catalytic manner.27
Activation of RNase H activity by ODN I and ODN III was
investigated in comparison with native ODN D1 and PS-
modified ODN Ds. In Fig. 3-a, the RNA in duplex III/RNA had
almost the same susceptibility to RNase H compared to native
duplex. It seemed that RNA in I/RNA showed a little more sus-
ceptibility than in Ds/RNA (Fig. 3-b). These results show that
duplex of RNA and ODN I with 5a in the middle is a good sub-
strate for RNase H. It may be that the enhancement of RNase H
activity arises due to distortion of the conformation of ODN I,
which makes the enzyme to hydrolyze the RNA strand.
3
Synthesis of 5a/5b modified siRNAs
Using standard phosphoramidite method (DMT-off), building
blocks 19 and 21 were incorporated into the sense or antisense
strand of the designed siRNA. Controlled pore glass with
thymine-loaded CPG was used for the synthesis of each strand,
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 7566–7577 | 7569

Fig. 3 (a) Autoradiograms of 20% denaturing PAGE, showing the cleavage kinetics of 5′-32P-labeled target RNA by E. coli RNase H1 in the native
20 mer D1 /RNA, the native 20 mer Ds/RNA, III/RNA and I/RNA hybrid duplexes. Lanes 1 to 5 indicate the aliquots of the digest taken after 0, 5,
10, 20, and 30 min, respectively. Conditions of cleavage reaction: RNA (0.8 μM) and the oligodeoxynucleotides (4 μM) in buffer, containing 20 mM
Tris-HCl (pH 8.0), 20 mM KCl, 10 mM MgCl2 and 0.1 mM DTT at 21 °C; 0.8 U of RNase H. Total reaction volume was 11 μL. (b) Time course of
RNase H cleavage of hybrid duplexes. The digestion (%) shows the extent of cleavage at time points.
Table 2 Cdc2-siRNA and modified single RNA strand
activity of these modified siRNAs. A siQuantTM vector, the
MW
MW
fusion luciferase reporter plasmids carrying the target site of the
functional cdc2-siRNA (target sequence complementary to B
strand in Fig. 4, upper), was constructed, and the dual-luciferase
assay was carried out to determine the percentage of intact target
mRNA. According to the normalized FL/RL signals in our
experiment, it was found that sense strand (passenger strand) of
siRNAs modified with isonucleoside at the 3′ or 5′ terminal pos-
ition showed comparative silencing activity (AI/B; AII/B,
93–95%) as the native one (95%) (Fig. 4, upper). It was
confirmed that the passenger strand generally had better tolera-
tion toward isonucleoside modification.22 Isonucleoside incor-
porating in antisense strand (guide strand) adversely affected the
siRNAs silencing activity to various degrees. With single iso-
nucleoside at the 5′-terminal position of guide strand, siRNAs
(A/BI, A/BIII) silencing activity dropped, but the 3′-terminal
modified guide strand (antisense) siRNA(A/BII) remained
similar silencing activity (91%) toward cdc2 mRNA as native
siRNA. This indicated that isonucleoside 5a/5b modification on
guide strand has a lesser influence on the silencing activity.
The main off-target silencing is mediated by a guide strand
and the passenger strand of a siRNA can assemble into RISC
and serve as the guide strand, thus initiating passenger strand-
specific off-targeting. It would be interesting to test these
modified siRNAs on the off-target silencing activities mediated
by a guide strand. A siQuantTM vector containing luciferase
reporter and the target sequence complementary to strand A in
cdc2-siRNAwas also constructed (Fig. 4, bottom). Here the nor-
malized FL/RL signals indicated the inhibitory efficiency of
siRNA toward to passenger strand specific off-target effect.
Interestingly, AI/B and AII/B showed only less than 15% and
35% activity, respectively, in comparison with native siRNA
which showed 80% silencing activity. Obviously, AI/B and AII/
B show a significant selectivity between the siRNA silencing
and off-target effect mediated by guide strand. On the contrary,
A/BII and A/BIII still retain about 90% silencing activity. The
Synthesized RNAs
(calcd)
(found)
Native
A: 5′- UCG GGA AAU UUC UCU
AUU Att -3′
B: 3′- tt AGC CCU UUA AAG
AGA UAA U -5′
A strand modification:
AI
5′- CG GGA AAU UUC UCU
AUU Att -3′
5′- UCG GGA AAU UUC UCU
AUU tt -3′
6604.99
6604.99
6601.12
6604.42
AII
B strand modification:
BI
3′- tt AGC CCU UUA AAG
AGA AA U -5′
3′- tt GC CCU UUA AAG AGA
UAA U -5′
3′- tt AGC CCU UUA AAG AGA
UA U -5′
6674.11
6674.11
6674.11
6673.03
6672.21
6673.03
BII
BIII
t = dT.
and coupling time for isonucleoside building block was
increased to 15 min to ensure satisfactory reaction yields. The
yield of each single RNA strand was determined according to
the release of DMT. For comparison, oligonucleotides incorpor-
ated by 5a and 5b at 3′ or 5′ terminal of both strands were syn-
thesized respectively (Table 2, t = dT). After the synthesis and
purification, 5 single stranded RNAs were obtained and ident-
ified by MALDI-TOF MS.
4
Silencing activities of L-isonucleoside modified siRNAs on
guide strand specific and passenger strand specific targets
Isonucleoside 5a/b modified ODNs could increase the stability
of oligomers and alter the local conformation of hybridized
duplexes. It would also be interesting to investigate the silencing
7570 | Org. Biomol. Chem., 2012, 10, 7566–7577
This journal is © The Royal Society of Chemistry 2012

Fig. 4 Inhibitory effects of HEK-293 cells were expressed in normalized ratios between the Renilla luciferase and the firefly luciferase activities.
(Upper: target sequence complementary to strand B, Bottom: target sequence complementary to strand A.).
data indicate that L-isonucleoside 5a/5b modified at 3′ or 5′
terminal of passenger strand of siRNA could retain silencing
activities and reduce the off-target effect mediated by guide
strand.
exhibits irregularities in base pairing, which suggest that the
helical conformation of the hybrid is very flexible. Furthermore,
the minor groove width for this region of the hybrid is greatly
diminished and the depth is increased compared to canonical
A-conformation, which facilitates contacts between RNase H
residues and the hybrid duplex. Our data show that duplex of 5a
modified ODN I with RNA is a good substrate for RNase
H. According to computer simulation, the averaged structures
over the last 1500 ps of MD for hybrid D1/RNA, Ds/RNA,
I/RNA, and III/RNA duplexes show conformational character-
istics ranging between the canonical A-form and B-form, and
more close to the A-form. CURVES program was used to
Discussion
A number of studies have indicated that flexibility and minor
groove width are important structural factors governing DNA/
RNA hybrid duplex recognition by RNase H.28 The hybrid
DNA/RNA duplex in the vicinity of the RNase H active site
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 7566–7577 | 7571

calculate the minor groove width of the four DNA/RNA
duplexes studied. The results indicate that the minor groove
width of the duplexes is greatly reduced compared to A-confor-
mation and lie intermediate between A-form and B-form geome-
tries (ESI‡). In I/RNA duplex, the minor groove width in the
modified region becomes narrow, the incorporating of isonucleo-
side 5a in ODN I caused large deviations in some helical para-
meters and makes the duplex structure bent. This modified area
of the duplex may correspond to the region interacting with the
RNase H. The high variability of helical parameters is also
found in the structure of Ds/RNA duplex.
with isonucleoside (5a/b) can retain the silencing activity and
minimize the passenger strand specific off-target effect.
Experimental section
Chemistry
All solvents were dried and distilled prior to use. Thin layer
chromatography (TLC) was performed using silica gel GF-254
(Qing-Dao Chemical Co., China) plates with detection by UV or
dyed by phosphomolybdic acid. Column chromatography was
performed on silica gel (200–300 mesh, Qing-Dao Chemical
Co.). NMR spectra were recorded on Varian Inova VXR-500 or
Avance 500 Bruker spectrometer with TMS as an internal stan-
dard. Mass spectra (ESI-TOF MS) were obtained on a MDS
SCLEX QSTAR instrument. High-resolution mass spectra
(ESI-TOF HRMS) were obtained on a Bruker DALTONICS
APEX IV 70e, and the data were reported in m/e. MALDI-TOF
mass spectra was recorded on Bruker BIFLEX III instrument.
Elemental analyses were obtained by Vario EL III instrument.
UV spectra were recorded with a Pharmacia LKB Biochrom
4060 spectrophotometer. Optical rotations were recorded on a
Perkin-Elmer 243B polarimeter. All the intermediates were dried
in vacuum over P2O5 at room temperature, for expoxide opening
reaction, adenine or thymine was dried in vacuum over P2O5 at
78 °C.
In general, the loading of the siRNA into the RISC is accom-
plished by the formation of RISC-loading complex (RLC), and
during the activation of the RISC, the designated sense strand is
cleaved.29 On the other hand, the antisense strand played vital
role for the RISC formation and activity, and its modification
may seriously influence the silencing activity, especially the
5′-terminal position which is important for the 5′-phosphorylation.
Off-target effects are strictly sequence-specific and are caused
either by near-perfect complementary between the central region
of the siRNA and its targets or by seed-sequence complementary
between the siRNA and the target 3′-UTR.30–32 It would be a
competition between passenger strand and guide strand to the
number of B-RISC*.33 It was proposed that the thermodynamic
characteristics of the siRNA, that is, the relative stability of the
two ends, determine the asymmetric incorporation of the two
strands into the RISC.34 Many approaches were reported to
reduce the off-target effect mediated by guide strand including
2′-OMe, 5′-OMe-modified siRNA, DNA substitution in the seed
region of siRNA.16,35,36 Recently, unlocked nucleobase analogs
modification on siRNA was reported and showed a significant
reduction on global off-target events.37,38 Our experiments show
that as expected the sense strand is much better tolerated toward
L-isonucleoside 5a/5b modification and 3′-terminal L-isonucleo-
side 5a/5b modified antisense strand seems to produce no
adverse effect on the silencing activity. In our experiment, isonu-
cleoside modified at 3′ or 5′ terminal of oligonucleotide can
maintain or slightly increase the thermodynamic stability
(Table 1); L-isonucleoside modified at 3′ or 5′ terminal of passen-
ger strand of siRNA could retain silencing activities and reduce
the off-target effect mediated by guide strand. From the exper-
imental data reported here, it is proposed that a 5′–CH2–
extended chain isonucleoside makes the modified passenger
strand more flexible and thereby could not influence the potency
of guide strand to form the RISC, however this modification
could inhibit the entrance of passenger strand and minimize the
passenger specific off-target effect.
[2S-(2-Ethoxycarbonyl-vinyl)-3R-p-toluenesulfonyl-4R-benzoxy]-
tetrahydrofuran (8). Compound 6b (3.00 g, 6.87 mmol) was dis-
solved in trifluoroacetic acid (7 mL) and water (1 mL), and the
solution was stirred for 5 h at room temperature. After evapor-
ation of most of solvent, the residue was dissolved in CH2Cl2,
and the solution was washed with saturated NaHCO3 and brine,
dried over anhydrous Na2SO4. Solvent was removed to afford
white foam (crude aldehyde). After dried in vacuum for 2 days
at room temperature, NaH (60%, 275 mg, 6.88 mmol) was sus-
pended in anhydrous THF (19 mL) under an inert atmosphere at
−30 °C and triethyl phosphonoacetate (1.43 mL, 6.99 mmol)
was added. After the mixture was stirred for 30 min at −30 °C,
crude aldehyde was added. The solution was stirred for 4 h at
room temperature. Saturated NH4Cl solution was added to
quench the reaction, and the aqueous system was extracted with
EtOAc. After drying over anhydrous Na2SO4, the organic
extracts were evaporated and the residue was purified by silica
gel column chromatography (P.E.–EtOAc) to give pure 8a and a
mixture of 8a/b (2.36 g, 74.6%) as a white solid.
1
Compound 8a. [α]2D0 −82.00° (c = 0.050, MeOH). H NMR
(500 MHz, CDCl3) δ 1.32 (t, J = 7.0 Hz, 3H, –CH3), 2.41 (s,
3H, Ts-CH3), 3.94 (dd, J5a,5b = 11.0 Hz, J5a,4 = 2.0 Hz, 1H,
5a-H), 4.22 (q, 2H, –COOCH2–), 4.41 (dd, J5b,4 = 5.0 Hz, 1H,
5b-H), 4.76 (m, 1H, 2-H), 5.11 (dd, J2,3 = 4.0 Hz, J3,4 = 1.5 Hz,
Conclusion
1H, 3-H), 5.48 (m, 1H, 4-H), 6.09 (dd, J1′,2′ = 15.5 Hz, J2′,2
=
A novel isonucleoside 5 containing a 5′–CH2 extended chain at
the sugar moiety was synthesized and several oligonucleotides
incorporated with 5 have been shown to form stable double
helical structures with complementary DNA and RNA. The stabi-
lity of these modified oligonucleotides towards nuclease and
their ability to activate RNase H are more promising compared
with the natural analogues. In siRNA, passenger strand modified
2.0 Hz, 1H, 2′-H), 6.73 (dd, J1′,2 = 5.0 Hz, 1H, 1′-H), 7.31 (d,
2H, Bz-H), 7.44–7.47 (m, 2H, Bz-H), 7.59–7.62 (m, 1H, Bz-H),
7.80 (d, 2H, Ts), 7.97–7.99 (m, 2H, Ts); 13C NMR (125 MHz,
CDCl3) δ 14.2, 21.7, 60.6, 71.5, 77.6, 78.8, 83.3, 124.4, 128.1,
128.5, 128.8, 129.8, 130.0, 133.7, 139.7, 145.5, 165.0, 165.4;
Anal. Calcd for C23H24O8S: C, 59.99; H, 5.25. Found: C, 59.93;
H, 5.27.
7572 | Org. Biomol. Chem., 2012, 10, 7566–7577
This journal is © The Royal Society of Chemistry 2012

1
[2S-(2-Ethoxycarboxyethyl)-3R-p-toluenesulfonyl-4R-benzoxy]-
tetrahydrofuran (9). The solution of 8 (661 mg, 1.44 mmol) in
THF (15 mL) was stirred under hydrogen (0.4 MPa) in the pres-
ence of 10% Pd-C at room temperature overnight, and the cata-
lyst was filtered and the solution was evaporated to residue.
Recrystallization from EtOAc and petroleum ether (60–90 °C)
provided pure 9 (313 mg, 96.0%) as a white crystalline solid.
[α]2D0 −35.71° (c = 0.154, MeOH). 1H NMR (500 MHz,
CDCl3) δ 1.26 (t, J = 7.0 Hz, 3H, –CH3), 1.87–1.94 (m, 1H, 1′–
CH2–), 1.97–2.02 (m, 1H, 1′–CH2–), 2.38 (s, 3H, Ts-CH3),
Compound 12, H NMR (500 MHz, DMSO-d6) δ 1.71–1.78
(m, 1H, –CH2–), 1.86–1.93 (m, 1H, –CH2–), 2.36–2.45 (m, 2H,
–CH2–), 2.80 (s, 3H, –N(CH3)2), 2.94 (s, 3H, –N(CH3)2), 3.66
(m, 1H, 5-H), 4.09–4.16 (m, 2H, 2-H), 4.28 (m, 1H, 4-H), 4.84
(m, 1H, 3-H), 5.73 (d, J = 5.5 Hz, 1H, 4-OH), 7.23 (s, 2H,
–NH2), 8.14 (s, 1H, 2-H in adenine), 8.16 (s, 1H, 8-H in
adenine); ESI-TOF MS m/z: 321.2 (M + H)+, 343.25 (M + Na)+.
[2S-(3-Hydroxypropyl)-3S,4R-epoxy]-tetrahydrofuran (13). The
solution of 9 (180 mg, 0.39 mmol) in dry THF (5 mL) was
cooled to 0 °C, and LiAlH4 (25 mg, 0.62 mmol) was added in
batches. The mixture was stirred for 10 h at room temperature,
quenched with water, stirred overnight, and then filtered. After
removing the solvents the mixture was purified by silica gel
column chromatography (CH2Cl2–MeOH) to give 13 (54 mg,
96.2%) as a colorless syrup.
2.40–2.46 (m, 2H, 2′–CH2–), 3.72 (dd, J5a,5b = 11.0 Hz, J5a,4
3.0 Hz, 1H, 5a-H), 4.10–4.17 (m, 3H, 2-H, –COOCH2–), 4.33
(dd, J5b,4 = 5.5 Hz, 1H, 5b-H), 5.05 (dd, J3,4 = 1.0 Hz, J2,3
=
=
3.5 Hz, 1H, 3-H), 5.30 (m, 1H, 4-H), 7.31 (d, 2H, Bz-H),
7.44–7.47 (m, 2H, Bz-H), 7.58–7.61 (m, 1H, Bz-H), 7.84 (d,
2H, Ts), 7.94–7.96 (m, 2H, Ts); 13C NMR (125 MHz, CDCl3):
δ 14.2, 21.6, 23.9, 30.4, 60.4, 71.0, 77.9, 78.9, 79.3, 82.4, 83.0,
128.0, 128.5, 128.9, 129.7, 130.0, 133.1, 133.6, 145.4, 165.0,
172.9; Anal. Calcd for C23H26O8S: C, 59.73; H, 5.67. Found: C,
59.73; H, 5.63.
[α]2D0 −24.66 (c = 0.073, MeOH). 1H NMR (500 MHz,
CDCl3) δ 1.41–1.48 (m, 1H, 1′–CH2), 1.55–1.62 (m, 1H,
1′–CH2), 1.64–1.75 (m, 2H, 2′–CH2), 2.53 (brs, 1H, –OH), 3.61
(d, J3,4 = 3.0 Hz, 1H, 3-H), 3.65 (m, 2H, 3′–CH2–), 3.73 (d,
J5a,5b = 10.5 Hz, 1H, 5a-H), 3.78 (d, 1H, 4-H), 3.98 (d, 1H, 5b-
H), 4.10 (dd, J2,1′a = 4.5 Hz, J2,1′b = 8.0 Hz, 1H, 2-H); 13C NMR
(125 MHz, CDCl3) δ 27.4, 28.7, 55.7, 58.7, 62.2, 65.8, 77.4;
Anal. Calcd for C7H12O3: C, 58.32; H, 8.39. Found: C, 58.24;
H, 8.12.
[2S-(2-Methoxycarboxyethyl)-3S,4R-epoxy]-tetrahydrofuran
(10). To the solution of 9 (870 mg, 1.88 mol) in dry methanol
(25 mL) was added anhydrous K2CO3 (1.10 g, 7.96 mmol), and
the resulting suspension was stirred for 1 h at room temperature.
The mixture was neutralized with acetic acid and evaporated.
The residue was purified by silica gel column chromatography
(P.E.–EtOAc) to give 10 (313 mg, 96.0%) as a colorless syrup.
[α]2D0 −32.72° (c = 0.162, MeOH). 1H NMR (500 MHz,
CDCl3) δ1.64–1.72 (m, 1H, 1′–CH2–), 1.76–1.83 (m, 1H, 1′–
CH2–), 2.39–2.53 (m, 2H, 2′–CH2–), 3.61 (d, J3,4 = 3.0 Hz, 1H,
3-H), 3.68 (s, 3H, –COOCH3), 3.69 (dd, 1H, 5a-H), 3.77 (d, 1H,
4-H), 3.97 (d, J5a,5b = 11.0 Hz, 1H, 5b-H), 4.08 (dd, J2,1′a = 10.0
Hz, J2,1′b = 3.5 Hz, 1H, 2-H); 13C NMR (125 MHz, CDCl3): δ
25.9, 29.7, 30.1, 51.7, 55.6, 58.7, 65.9, 173.6; Anal. Calcd for
C8H12O4: C, 55.81; H, 7.02. Found: C, 55.94; H, 6.97.
[2S-(3-O-t-Butyldimethylsilyl-propyl)-3S,4R-epoxy]-tetrahydro-
furan (14). To a solution of compound 13 (458 mg, 3.18 mmol)
in dry DMF (15 mL) was added TBDMSCl (764 mg,
5.09 mmol) and imidazole (75 mg, 11.07 mol), and the mixture
was stirred for 3 h at room temperature. Water (∼30 mL) was
added to quench the reaction, and the solvent was evaporated.
The residue was purified by silica gel column chromatography
(P.E.–EtOAc) to yield 14 (777 mg, 94.6%) as a colorless syrup.
[α]2D0 −13.21° (c = 0.106, MeOH). 1H NMR (500 MHz,
CDCl3)
δ
0.05 (s, 6H, TBDMS-CH3), 0.89 (s, 9H,
TBDMS-tBu), 1.40–1.71 (m, 4H, 1′,2′–CH2–), 3.60 (d, J3,4
3.0 Hz, 1H, 3-H), 3.62–3.68 (m, 2H, 3′–CH2–), 3.71 (d, J5a,5b
=
=
[5S-(2-Methoxycarboxyethyl)-4R-hydroxy-3S-(adenin-9-yl)]-
tetrahydrofuran (11) {5S-[2-(N,N-dimethyl amino-carboxy-
ethyl)]-4R-hydroxy-3S-(adenin-9-yl)}-tetrahydrofuran (12). To
a solution of 10 (971 mg, 5.64 mmol) and adenine (1.54 g,
11.40 mmol) in DMF (32 mL) was added DBU (2.53 mL,
16.92 mmol), and the mixture was heated to 105 °C for 46 h.
After the mixture cooled to room temperature, the solvent was
removed and residue purified by silica gel column chromato-
graphy (CH2Cl2–MeOH) to yield 11 (600 mg, 34.7%) and 12
(55 mg, 10.0%) as a white solid, respectively.
10.5 Hz, 1H, 5a-H), 3.76 (d, 1H, 4-H), 3.97 (d, 1H, 5b-H), 4.09
(dd, J2,1′a = 5.5 Hz, J2,1′b = 7.5 Hz, 1H, 2-H); 13C NMR
(125 MHz, CDCl3) δ −5.4, 18.3, 25.9, 27.3, 28.7, 55.8, 58.9,
62.6, 65.9, 77.4; Anal. Calcd for C13H26O3Si: C, 60.42; H,
10.14. Found: C, 60.13; H, 10.32.
[5S-(3-O-t-Butyldimethylsilyl-propyl)-4R-hydroxyl-3S-(adenin-
9-yl)]-tetrahydrofuran (15a). To a solution of compound 14
(491 mg, 1.90 mmol) and dry adenine (515 mg, 3.79 mmol) in
dry DMF (18 mL) was added DBU (0.87 mL, 5.70 mmol), and
the solution was heated to 110 °C for 5d. After the mixture
cooled to room temperature, the solvent was evaporated and the
residue was purified by silica gel column chromatography
(CH2Cl2–MeOH) to give 15a (376 mg, 50.8%) and 16a (37 mg,
6.4%) as a white solid, respectively.
Compound 11, [α]2D0 +21.57° (c = 0.102, MeOH); UV
(MeOH): λmax = 260.5 nm (ε 11 754). 1H NMR (500 MHz,
DMSO-d6) δ 1.78–1.86 (m, 1H, 1′–CH2–), 1.92–1.98 (m, 1H,
1′–CH2–), 2.42–2.47 (m, 2H, 2′–CH2–), 3.60 (s, 3H,
–COOCH3), 3.65 (m, 1H, 5-H), 4.11 (dd, J2a,2b = 9.5 Hz, J2a,3
=
6.0 Hz, 1H, 2a-H), 4.16 (dd, J2b,3 = 7.5 Hz, 1H, 2b-H), 4.30 (m,
1H, 4-H), 4.86 (m, 1H, 3-H), 5.77 (d, J = 5.5 Hz, 1H, 4-OH),
7.24 (s, 2H, –NH2), 8.15 (s, 1H, 2-H in adenine), 8.17 (s, 1H, 8-
H in adenine); 13C NMR (125 MHz, DMSO-d6) δ 27.9, 29.8,
51.3, 61.8, 68.7, 78.5, 82.6, 119.0, 139.3, 149.4, 152.3, 156.0,
173.0; Anal. Calcd for C13H17N5O4: C, 50.81; H, 5.58; N,
22.79. Found: C, 50.97; H, 5.54; N, 22.72.
Compound 15a, [α]2D0 +18.18° (c = 0.055, MeOH); UV
(MeOH): λmax = 260.5 nm (ε 11 684). 1H NMR (500 MHz,
DMSO-d6) δ 0.01 (s, 6H, TBDMS-CH3), 0.84 (s, 9H,
TBDMS-tBu), 1.52–1.69 (m, 4H, –CH2CH2–), 3.57–3.64 (m,
3H, 5-H, –CH2–O–), 4.12 (m, 2H, 2-H), 4.23 (m, 1H, 4-H), 4.83
(m, 1H, 3-H), 5.71 (d, J = 5.5 Hz, 1H, 4-OH), 7.22 (br s, 2H,
–NH2), 8.13 (s, 2H, 2-H, 8-H in adenine); 13C NMR (125 MHz,
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 7566–7577 | 7573

[5S-(2-Hydroxypropyl)-4R-hydroxyl-3S-(thymin-1-yl)]-tetra-
hydrofuran (5b). A solution of mixture 15b and 16b (224 mg,
0.58 mmol) and TBAF (1 M in THF, 1.2 mL, 1.2 mmol) in THF
(8.5 mL) was stirred for 3 h at room temperature. The resulting
mixture was evaporated, and the residue was purified by silica
gel column chromatography (CH2Cl2–MeOH) to give 5b
(147 mg, 93.2%) as a white solid.
DMSO-d6) δ −5.3, 17.9, 25.8, 28.8, 29.1, 62.0, 62.4, 68.6, 78.9,
83.6, 119.0, 139.2, 149.4, 152.3, 156.0; Anal. Calcd for
C18H31N5O3Si: C, 54.93; H, 7.94; N, 17.80. Found: C, 54.94;
H, 7.75; N, 17.73.
1
Compound 16a, H NMR (500 MHz, DMSO-d6) δ −0.18 (d,
3H, TBDMS-CH3), −0.16 (d, 3H, TBDMS-CH3), 0.70 (s, 9H,
TBDMS-tBu), 1.30–1.65 (m, 4H, –CH2CH2–), 3.45–3.65 (m,
2H, –CH2–O–), 4.07 (m, 1H, 2a-H), 4.46–4.50 (m, 2H, 2b-H,
3-H), 4.82 (m, 1H, 4-H), 5.77 (d, J = 3.5 Hz, 1H, 3-OH), 7.22 (s,
2H, –NH2), 8.13 (s, 2H, 2-H, 8-H); ESI-TOF MS m/z: 394.34
(M + H)+, 416.33 (M + Na)+.
[α]2D0 +4.17° (c = 0.048, MeOH); UV (MeOH): λmax
=
262.0 nm (ε 8040). 1H NMR (500 MHz, DMSO-d6)
δ 1.45–1.61 (m, 3H, 1′–CH2–, 2′–CH2–), 1.65–1.72 (m, 1H, 1′–
CH2–), 1.79 (s, 3H, thymidinyl-CH3), 3.41 (m, 2H, 3′–CH2),
3.48 (m, 1H, 5-H),3.78 (dd, J2a,2b = 10.0 Hz, J2a,3 = 5.0 Hz, 1H,
2a-H), 3.87 (m, 1H, 4-H), 3.97 (dd, J2b,3 = 8.0 Hz, 1H, 2b-H),
4.39 (t, 1H, 3′–OH), 4.78 (m, 1H, 3-H), 5.55 (d, J = 5.5 Hz, 1H,
4-OH), 7.45 (s, 1H, 6-H in thymine), 11.27 (s, 1H, –NH–); 13C
NMR (125 MHz, DMSO-d6) δ21.1, 29.0, 60.6, 62.6, 68.1, 78.7,
83.2, 109.4, 137.7, 151.0, 163.7; Anal. Calcd for C12H18N2O5:
C, 53.33; H, 6.71; N, 10.36. Found: C, 53.30; H, 6.49; N, 10.24.
[5S-(3-O-t-Butyldimethylsilyl-propyl)-4R-hydroxyl-3S-(thymin-
1-yl)]-tetrahydrofuran (15b). To the solution of 14 (514 mg,
1.99 mmol) and dry thymine (559 mg, 4.39 mmol) in anhydrous
DMF (27 mL) was added DBU (0.9 mL, 5.90 mmol), and the
solution was heated to 110 °C for 5d. After the mixture cooled
to room temperature, the solution was evaporated and the residue
was purified by silica gel column chromatography (CH2Cl2–
MeOH) to give mixture of 15b and 16b (6 : 1 by 1H NMR,
316 mg, 41.3%) as a white foam, respectively.
[5S-(3-Hydroxypropyl)-4R-hydroxyl-3S-(6′-N-benzoyl-adenine-
9′-yl)]-tetrahydrofuran (17). A solution of 5a (90 mg,
0.32 mmol) in pyridine (3 mL) was added TMSCl (0.43 mL,
3.35 mmol). After stirring at room temperature for 3 h, BzCl
(0.2 mL, 1.74 mmol) was added at 0 °C, and the mixture was
kept on stirring at room temperature for 4 h. Conc. aq. ammonia
was added to adjust the solution pH to 8–9 at 0 °C. After
another 2 h stirring at room temperature, the solvent was
removed, and the residue was purified by silica gel column
chromatography (CH2Cl2–MeOH) to give 17 (90 mg, 93.6%) as
a white foam.
[α]2D0 −18.75° (c = 0.016, MeOH); UV (MeOH): λmax
=
1
272.0 nm (ε 8918). H NMR (500 MHz, DMSO-d6) δ 0.02 (s,
6H, TBDMS-CH3), 0.85 (s, 9H, TBDMS-tBu), 1.48–1.69 (m,
4H, –CH2CH2–), 1.77 [2s (6 : 1), 3H, Thymine-CH3], 3.48 (m,
1H, 5-H), 3.59 (m, 2H, –CH2–O–), 3.78 (dd, J2a,2b = 10.0 Hz,
J2a,3 = 5.0 Hz, 1H, 2a-H), 3.86 (m, 1H, 4-H), 3.95 (dd, J2b,3
=
7.5 Hz, 1H, 2b-H), 4.75 (m, 1H, 3-H), 5.56 (d, J = 6.0 Hz, 1H,
4-OH), 7.43 (d, J = 1.0 Hz, 1H, 6-H in thymine), 11.26 (s, 1H,
–NH–); 13C NMR (125 MHz, DMSO-d6) δ −5.3, 12.1, 17.9,
25.7, 28.8, 62.4, 62.6, 68.1, 78.7, 83.1, 109.4, 137.7, 151.0,
163.7; HRMS (TOF) calcd for C18H33N2O5Si (M + H)+:
385.2080; found: 385.2153.
[α]2D0 +121.67° (c = 0.120, MeOH). 1H NMR (500 MHz,
DMSO-d6) δ 1.50–1.61 (m, 3H, 1′′–CH2–, 2′′–CH2–), 1.62–1.73
(m, 1H, 1′′–CH2–), 3.43 (m, 2H, 3′′–CH2–), 3.67 (m, 1H, 5-H),
4.17–4.23 (m, 2H, 2-H), 4.29 (m, 1H, 4-H), 4.40 (t, J = 5.0 Hz,
1H, 3′′–OH), 5.01 (m, 1H, 3-H), 5.78 (d, J = 5.0 Hz, 1H, 4-OH),
7.54–7.57 (m, 2H, Bz), 7.63–7.66 (m, 1H, Bz), 8.04–8.05 (m,
2H, Bz), 8.51 (s, 1H, 2′-H), 8.76 (s, 1H, 8′-H), 11.16 (s, 1H,
–NH–); 13C NMR (125 MHz, DMSO-d6) δ 29.0, 29.4, 60.6,
62.3, 68.5, 79.0, 83.7, 125.7, 128.4, 132.4, 133.4, 143.1, 150.2,
151.4, 152.4, 165.6; Anal. Calcd for C19H21N5O4(0.8 H2O): C,
57.36; H, 5.73; N, 17.60. Found: C, 57.51; H, 5.66; N, 17.83.
[5S-(2-Hydroxypropyl)-4R-hydroxyl-3S-(adenin-9-yl)]-tetra-
hydrofuran (5a). (1) 11a (56 mg, 0.18 mmol) was dissolved in
dry THF (15 mL), and LiAlH4 (20 mg, 0.50 mmol) was added
stepwise at 0 °C. After stirring over night at room temperature,
the reaction mixture was quenched with water, filtered, and evap-
orated. The residue was purified by silica gel column chromato-
graphy (CH2Cl2–MeOH) to yield 5a (41 mg, 80.4%) as a white
foam.
{5S-[3-O-(4′,4-Dimethoxytriphenylmethyl)-propyl]-4R-hydroxyl-
3S-(6′-N-benzoyl-adenine-9′-yl)}-tetrahydrofuran (18). Compound
17 (235 mg, 0.61 mmol) was dissolved in dry pyridine (9 mL),
and dimethoxytrityl chloride (234 mg, 0.68 mmol) was added.
The solution was stirred at room temperature for 26 h. After
evaporation the mixture was purified by silica gel column chrom-
atography (CH2Cl2–MeOH) to give 18 (333 mg, 79.3%) as a
white foam.
(2) A solution of 15a (452 mg, 1.15 mmol) and TBAF (1 M
in THF, 2.3 mL, 2.3 mmol) in THF (30 mL) was stirred for 3 h
at room temperature. The resulting mixture was evaporated, and
the residue was purified by silica gel column chromatography
(CH2Cl2–MeOH) to give 5a (white foam, 311 mg, 96.9%).
[α]2D0 +37.89° (c = 0.095, MeOH); UV (MeOH): λmax
=
260.5 nm (ε 12 035). 1H NMR (500 MHz, DMSO-d6)
δ 1.48–1.62 (m, 3H, 1′–CH2–, 2′–CH2–), 1.66–1.71 (m, 1H,
1′–CH2–), 3.42 (m, 2H, 3′–CH2–), 3.62 (m, 1H, 5-H), 4.11 (dd,
J2a,2b = 9.5 Hz, J2a,3 = 5.5 Hz, 1H, 2a-H), 4.15 (dd, J2b,3 = 7.0
Hz, 1H, 2b-H), 4.23 (m, 1H, 4-H), 4.40 (t, J = 5.0 Hz, 1H,
3′-OH), 4.85 (m, 1H, 3-H), 5.72 (d, J = 5.5 Hz, 1H, 4-OH), 7.23
(s, 2H, –NH2), 8.15 (s, 2H, 2-H, 8-H in adenine). 13C NMR
(125 MHz, DMSO-d6) δ 29.0, 29.4, 60.6, 62.0, 68.6, 79.0, 83.7,
119.0, 139.2, 149.5, 152.4, 156.0; Anal. Calcd for C12H17N5O3:
C, 51.60; H, 6.14; N, 25.08. Found: C, 51.66; H, 6.21; N, 24.96.
1H NMR (500 MHz, DMSO-d6) δ 1.64–1.78 (m, 4H, 1′′–
CH2–, 2′′–CH2–), 3.00 (m, 2H, 3′′–CH2–), 3.64 (m, 1H, 5-H),
3.73 (s, 6H, –OCH3), 4.16–4.22 (m, 2H, 2-H), 4.32 (m, 1H,
4-H), 5.01 (m, 1H, 3-H), 5.79 (d, J = 5.5 Hz, 1H, 4-OH),
6.88–6.91 (m, 4H, DMT), 7.20–7.26 (m, 5H, DMT), 7.30–7.33
(m, 2H, DMT), 7.37–7.38 (m, 2H, DMT), 7.54–7.57 (m, 2H,
Bz), 7.63–7.66 (m, 1H, Bz), 8.04–8.05 (m, 2H, Bz), 8.52 (s, 1H,
2′-H), 8.75 (s, 1H, 8′-H), 11.16 (s, 1H, –NH–); 13C NMR
(125 MHz, DMSO-d6) δ 26.0, 29.6, 55.0, 62.2, 62.7, 68.6, 78.8,
7574 | Org. Biomol. Chem., 2012, 10, 7566–7577
This journal is © The Royal Society of Chemistry 2012

83.4, 85.2, 113.1, 125.6, 126.5, 127.6, 127.8, 128.4, 129.6,
132.4, 133.4, 136.0, 143.1, 145.2, 150.2, 151.4, 152.4, 158.0,
165.5; Anal. Calcd for C40H39N5O6: C, 70.06; H, 5.73; N,
10.21. Found: C, 70.09; H, 5.78; N, 10.30.
(III, V) were performed in mixed solution of conc. aq. ammonia
and 0.5 M NaCl (v : v = 5 : 1) at 55 °C for 12 h. The crude oligo-
mers were purified by HPLC (ZORBAX Bio Series Oligo
Column, 6.2 mm ID × 80 mm), gradient eluting with eluants A
(MeCN/0.02 M NaH2PO4, 1 : 4) and B (1.0 M NaCl in A) and
1.0 ml min−1 flow rate. The fraction containing pure oligonu-
cleotides were desalted by a Sephadex G-25 column. The pure
oligonucleotides were lyophilized and stored at −20 °C. Total
yields were about 35% after purification. Other natural oligonu-
cleotides were purchased from TaKaRa Biotechnology (Dalian)
Co., Ltd.
{5S-[3-O-(4′,4-Dimethoxytriphenylmethyl)-propyl]-4R-O-[(2-
cyanoethyl-N,N′-diisopropyl)-phosphoramidite]-3S-(6′-N-benzoyl-
adenine-9′-yl)}-tetrahydrofuran (19). A solution of compound
18 (291 mg, 0.42 mmol) and 1H-tetrazole (28 mg, 0.40 mmol)
in dry CH2Cl2 (8 mL) was added NCCH2CH2OP[N(iPr)2]2
(0.19 mL, 0.60 mmol) under inert atmosphere. The mixture was
stirred at room temperature for 2 h, and was diluted with
CH2Cl2. The organic layer was washed with 5% aq. NaHCO3,
followed by saturated aq. NaCl, dried over Na2SO4, evaporated,
and purified by silica gel column chromatography [P.E.
(60–90 °C)–CH2Cl2–EtOAc, 0.5% Et3N] to give 19 (344 mg,
91.4%) as a white foam.
Synthesis of siRNAs
siRNAs (21 nt, Table 2) were chemically synthesized by the
automatic DNA synthesizer (Applied Biosystems model 381)
using commercial phosphoramidites as building blocks. Isonu-
cleoside-modified siRNAs were synthesized by the same method
but replacing the regular A and T unit with building blocks 19
and 21 at the defined positions (Table 2) (synthesis scale,
1 μmol; coupling time, 15 min). Cleavage and deprotection of
RNA oligos were carried out according to the standard protocol
(firstly treated with 29% NH3–H2O and ethanol (v : v = 3 : 1) in
sealed vessel at 55 °C for 12 h, and then treated with TBAF
(1.0 M in THF) to remove TBDMS). The synthetic oligoribonucleo-
tides were then purified by HPLC using ion-exchange column
(ZORBAX Bio Series Oligo Column, 6.2 mm ID* 80 mm), gra-
dient eluting with eluants A (MeCN/0.02 M NaH2PO4, 1 : 4)
and B (1.0 M NaCl in A) with 1.0 mL min−1 flow rate [Clarity
10u Oligo-WAX Column 300A (150 × 10.0 mm), Phenomenex,
gradient eluting with eluants A (10% CH3CN: 90% 0.02 M Tris-
HCl(H2O), pH = 8.0) and B (2.0 M NaCl in solvent A) with
3.0 mL min−1 flow rate], and the pure oligonucleotides were lyo-
philized and stored at −78 °C in about 20% yield. By mixing
complementary sense- and antisense-strand RNAs at equal
20 μM in MQ water, incubating them in boiling water for 1 min,
and then gradually decreasing temperature to room temperature
and then 4 °C, the siRNA duplexes were efficiently formed. The
quality of the RNA duplexes was assessed on a 15% PAGE gel.
31P NMR (121.5 MHz, DMSO-d6): δ 148.5, 148.8
{2S-[3-O-(4′,4-Dimethoxytriyl)-propyl]-3R-hydroxyl-4S-(thymin-
1′-yl)}-tetrahydrofuran
(20). Compound
5b
(320
mg,
1.18 mmol) was dissolved in dry pyridine (8 mL), and
dimethoxytrityl chloride (400 mg, 1.16 mmol) was added. The
solution was stirred at room temperature for 26 h. After evapor-
ation the mixture was purified by silica gel column chromato-
graphy (CH2Cl2–MeOH) to give 20 (461 mg, 68.0%) as a white
foam.
1H NMR (500 MHz, DMSO-d6): δ 1.56–1.74 (m, 4H,
1′–CH2–, 2′–CH2–), 1.76 (s, 3H, T-CH3), 2.98 (m, 2H, 3′–
CH2–), 3.47 (m, 1H, 2-H), 3.74 (s, 6H, –OCH3), 3.77 (dd, 1H,
J5a,5b = 10.0 Hz, 5a-H), 3.89 (m, 1H, 3-H), 3.95 (dd, 1H, 5b-H),
4.77 (m, 1H, 4-H), 5.58 (br s, 1H, 3-OH), 6.88 (d, 4H, Ph),
7.20–7.38 (m, 9H, Ph), 7.43 (s, 1H, 6-H in thymine), 11.28 (br
s, 1H, –NH–); 13C NMR (125 MHz, DMSO-d6): δ 12.2, 26.0,
29.4, 55.0, 62.5, 62.7, 68.1, 78.5, 83.0, 85.1, 109.3, 113.1,
126.5, 127.6, 127.7, 129.5, 136.0, 137.5, 145.2, 151.4, 158.0,
164.2; HRMS (TOF) calcd for C33H36N2O7 (M + Na)+:
595.2415; found: 595.2443.
{2S-[3-O-(4′,4-Dimethoxytriyl)-propyl]-3R-O-[(2-cyanoethyl-
N,N′-diisopropyl)-phosphoramidite]-4S-(thymin-1′-yl)}-tetra-
hydrofuran (21). A solution of compound 20 (291 mg,
0.42 mmol) and 1H-tetrazole (28 mg, 0.40 mmol) in dry
CH2Cl2 (8 mL) was added NCCH2CH2OP[N(iPr)2]2 (0.19 mL,
0.60 mmol) under inert atmosphere. The mixture was stirred at
room temperature for 2 h, and was diluted with CH2Cl2. The
organic layer was washed with 5% aq. NaHCO3, followed by
saturated aq. NaCl, dried over Na2SO4, evaporated, and purified
by silica gel column chromatography [P.E. (60–90 °C)–CH2Cl2–
EtOAc, 0.5% Et3N] to give 21 (344 mg, 50.0%) as a white
foam.
UV Melting experiments
Determination of the Tm of the ODNs/DNA or ODNs/RNA
hybrids was carried out in the following buffer: 57 mM Tris-HCl
(pH 7.5), 57 mM KCl, 1 mM MgCl2. Absorbance was moni-
tored at 260 nm in the temperature range from 20 °C to 90 °C
using UV-visible spectrophotometer with the heating rate of
0.5 °C per minute. Prior to measurements, the samples (0.85 μM
of ODNs and 0.75 μM of DNA mixture) were preannealed by
heating to 90 °C for 5 min followed by slow cooling to 4 °C and
keeping this temperature overnight.
31P NMR (121.5 MHz, DMSO-d6): δ 148.5
Synthesis of oligonucleotides
CD Spectra
Oligonucleotides synthesis was carried out on a 1.0 μM scale by
an Applied Biosystems model 381 DNA Synthesizer with regular
phosphoramidite chemistry (DMT off). Cleavage and deprotec-
tion of the oligonucleotides (I, II, IV) were performed in conc.
aq. ammonia soln. at 55 °C for 12 h, and oligonucleotides
CD Spectra were recorded from 300 to 200 nm in 1 cm path
length cuvettes. Spectra were obtained with an ASON/DNA or
ASON/RNA duplex concentration of 0.75 μM in buffer contain-
ing 57 mM Tris-HCl (pH 7.5), 57 mM KCl and 1 mM MgCl2.
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 7566–7577 | 7575

All the spectra were measured at 20 °C with a J715 CD spectro-
photometer (JAC).
glycerol with 0.14 nM bromophenol blue, and 0.19 nM xylene
cyanol FF) was added, and samples were loaded on a 20% de-
naturing polyacrylamide gel and run at room temperature and
recorded by autoradiography.
Exonuclease degradation studies
Stability of the ASONs toward 3′-enonuclease was tested using
snake venom phosphodiesterase (SVPED). All reactions were
performed at 7.1 μM DNA concentration in 56 mM Tris-HCl
(pH 7.9) and 4.4 mM MgCl2 at 37 °C. Exonuclease concen-
tration of 28.6 ng μL−1 was used for digestion of oligonucleo-
tides. Total reaction volume was 14 μL. Aliquots were taken at 0,
10, 20, 40, 60 min and quenched by addition of the same
volume of 50 mM EDTA in 95% formamide. Reaction progress
was monitored by 20% denaturing (7 M urea) PAGE and was
visualized by staining with SYBR gold and quantified by Model
& Storm 860 hardware and Imagequant software (Amersham
Biosciences, PKU, China).
Transfection and dual-luciferase assay
Human embryonic kidney cells (HEK293) were grown in
DMEM (Life Technologies, Gibco) and seeded in 24-well plates.
After the cells reached about 50% confluence, the culture
medium was changed to OPTIMEM (Gibco) and then trans-
fected with plasmids and siRNA duplex in the presence of
0.17% Lipofectamine 2000 (Invitrogen). For each well, 0.17 g of
recombination plasmid and 0.017 g pRL-TK were used. The
final concentration of siRNA is 13 nM. The transfection medium
was changed to culture medium (1 mL) after 4 h. All exper-
iments were performed in triplicate and repeated at least twice.
Cells were harvested 24 h after transfection and lyzed by passive
cell lysis. Dual-luciferase assay was conducted. Luciferase
activities were determined with 10 μL cell lysate using the Dual-
Luciferase Assay System (Promega) by NOVOStar (BMG Lab-
technologies Gmbh, Germany). The ratio between firefly and
Renilla luciferase readings was generated for each well, and the
inhibition efficiency of each siRNA was calculated by normaliz-
ing to respective buffer control.
Endonuclease degradation studies
Stability of ASONs toward endonuclease was tested using
DNase I form bovine pancreas. Reactions were carried out at
10 μM DNA concentration in 100 mM Tris-HCl (pH 7.5) and
10 mM MgCl2 at 37 °C using 15 unit of DNase I. Total reaction
volume was 10 μL. Aliquots were taken at 0, 2, 4, 10, 20, 40 and
60 min and quenched with the same volume of 50 mM EDTA in
95% formamide. They were resolved in 20% polyacrylamide
denaturing (7 M urea) gel electrophoresis and visualized by
staining with SYBR gold and quantified by Model & Storm
860 hardware and Imagequant software (Amersham Biosciences,
PKU, China).
Acknowledgements
We thank Prof. Zi-Cai Liang, Quan Du and Fan Yi in Peking
University, for providing siRNA sequence and assistance with
biological assay experiments. This work was supported by the
National Natural Science Foundation of China (Grant No.
20932001), the Ministry of Science and Technology of China
(Grant No. 2006AA02Z144, 2009ZX09503).
Stability studies in bovine fetal serum
To investigate the stability in 80% bovine fetal serum, the AONs
(8 μL) at 10 μM concentration were incubated in 32 μL of fetal
bovine serum at 37 °C (total reaction volume was 40 μL). Ali-
quots (5 μL) were taken at 0, 15 and 30 min, 1, 2 and 9 h, and
quenched with 10 μL of solution containing 8 M urea and
50 mM EDTA, resolved in 20% polyacrylamide denaturing (7 M
urea) gel electrophoresis and visualized by staining with SYBR
gold and quantified by Model & Storm 860 hardware and Image-
quant software (Amersham Biosciences, PKU, China).
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