Synthesis of 4′-C-aminoalkyl-2′-O-methyl modified RNA and their biological properties

Article abstract of DOI:10.1016/j.bmc.2018.05.025

In this paper, we describe the synthesis of 4′-C-aminoalkyl-2′-O-methylnucleosides and the properties of RNAs containing these analogs. Phosphoramidites of 4′-C-aminoethyl and 4′-C-aminopropyl-2′-O-methyluridines were prepared using glucose as starting ma

Full text of DOI:10.1016/j.bmc.2018.05.025

Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of 4′-C-aminoalkyl-2′-O-methyl modified RNA and their biological
properties
a,f
a,f
a
e
d
Kana Koizumi , Yusuke Maeda , Toshifumi Kano , Hisae Yoshida , Taiichi Sakamoto ,
e
a,b,c,⁎
Kenji Yamagishi , Yoshihito Ueno
Course of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, Japan
United Graduate School of Agricultural Science, Gifu University, Japan
Center for Highly Advanced Integration of Nano and Life Sciences (G-CHAIN), Gifu University 1-1 Yanagido, Gifu 501-1193, Japan
Faculty of Advanced Engineering, Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino, Chiba 275-0016, Japan
Department of Chemical Biology and Applied Chemistry, College of Engineering, Nihon University, 1 Aza-nakagawara, Tokusada, Tamuramachi, Koriyama, Fukushima
63-8642, Japan
a
b
c
d
e
9
A R T I C L E I N F O
A B S T R A C T
Keywords:
Oligonucleotide
RNA
RNA interference
Small interfering RNA
Aminoalkyl groups
Thermal stability
Nuclease resistance
In this paper, we describe the synthesis of 4′-C-aminoalkyl-2′-O-methylnucleosides and the properties of RNAs
containing these analogs. Phosphoramidites of 4′-C-aminoethyl and 4′-C-aminopropyl-2′-O-methyluridines were
prepared using glucose as starting material, and RNAs containing the analogs were synthesized using the
phosphoramidites. Thermal denaturation studies revealed that these nucleoside analogs decreased the thermal
stabilities of double-stranded RNAs (dsRNAs). Results of NMR, molecular modeling, and CD spectra measure-
ments suggested that 4′-C-aminoalkyl-2′-O-methyluridine adopts an C2′-endo sugar puckering in dsRNA. The 4′-
C-aminoalkyl modifications in the passenger strand and the guide strand outside the seed region were well
tolerated for RNAi activity of siRNAs. Single-stranded RNAs (ssRNAs) and siRNAs containing the 4′-C-ami-
noethyl and 4′-C-aminopropyl analogs showed high stability in buffer containing bovine serum. Thus, siRNAs
containing the 4′-C-aminoethyl and 4′-C-aminopropyl analogs are good candidates for the development of
therapeutic siRNA molecules.
1
. Introduction
present in the blood, tissues, and cells.12 Moreover, the hydrophilic
nature and negative charges of siRNAs prevent them from easily
The discovery of RNA interference (RNAi) by Fire et al. has focused
research on the potential use of small interfering RNA (siRNA) as
therapeutic agents.
stranded RNA (dsRNA) with two 3′ nucleotide overhangs. The guide
strand of the siRNA binds with Argonaute 2 protein (Ago2) to form the
RNA-induced silencing complex (RISC), which in turn causes en-
crossing the cell membrane.13 As a result, various chemical modifica-
1
4
tions for siRNAs have been attempted to overcome these problems.
1
–3
15,16
siRNAs comprise 19–21 base pairs of double
For example, siRNAs are commonly subjected to 2′-O-methyl
and
2′-fluoro1
7,18
modifications, which increase the stability of siRNAs in
serum. These modifications are also known to inhibit immune stimu-
lation of siRNAs and increase the thermal stability of double-stranded
4–7
15
donucleolytic cleavage of the complementary target mRNA.
siRNAs
RNAs (dsRNAs). Furthermore, so far, synthesis of many types of 2′-O-
aminoalkyl modified nucleic acids were reported.1
9–21
The positively
can be rationally designed and synthesized if the sequences of the target
disease-causing genes are known, so that siRNAs have been regarded as
novel potential therapeutic agents. Several siRNA drugs are currently
being evaluated in clinical trials for the treatment of diseases, such as
charged amino groups of the aminoalkyl linkers can neutralize the
negative charges of oligonucleotides (ONs) and interact with the ne-
gatively charged cell membrane, causing enhancement of the cell
8
,9
19
age-related macular degeneration (AMD),
diabetic macular edema
membrane permeability of ONs. However, Ago2 strictly recognizes
1
0
(
(
DME), glaucoma, hypercholesterolemia, and human solid tumor
melanoma).
However, clinical applications of siRNAs have been limited by
the backbone of siRNA, especially the phosphates and hydroxy groups
1
1
22,23
of the sugar moieties.
Therefore, incorporation of large functional
groups at the 2′-hydroxy group of siRNA will interfere with RISC for-
several challenges. In general, siRNAs are easily degraded by nucleases
mation.
⁎
Corresponding author.
E-mail address: uenoy@gifu-u.ac.jp (Y. Ueno).
These authors contributed equally to this work.
f
https://doi.org/10.1016/j.bmc.2018.05.025
Received 14 April 2018; Received in revised form 17 May 2018; Accepted 17 May 2018
0968-0896/ © 2018 Elsevier Ltd. All rights reserved.
Please cite this article as: Koizumi, K., Bioorganic & Medicinal Chemistry (2018), https://doi.org/10.1016/j.bmc.2018.05.025

K. Koizumi et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
following a standard procedure to produce the corresponding phos-
phoramidites 18a at 69% yield.32 In a similar manner, ribofuranose
derivative 7b was converted to 18b. The total yields of 18a and 18b
were 2.9% (after 22 steps) and 4.1% (after 22 steps), respectively. The
phosphoramidite of the aminomethyl analog 1 was synthesized fol-
lowing a previously reported method.27 To incorporate 17a and 17b
into the 3′-end of the RNA oligomer, 17a and 17b were further mod-
ified to produce the corresponding 3′-succinates 20a and 20b, which
were then reacted with controlled pore glass (CPG) to produce the solid
supports 21a and 21b containing 20a (44 µmol/g) and 20b (36 µmol/
g), respectively (Scheme 3).
Fig. 1. Structures of the nucleoside analogs.
Matsuda and his co-workers reported the synthesis of 4′-C-ami-
2
4,25
noalkyl-modified DNAs.
Incorporation of aminoalkyl groups,
especially the aminoethyl and aminopropyl groups, at the 4′-positions
of sugar moieties was found to markedly increase the resistance of
DNAs to nucleolytic degradation by both endonucleases and exonu-
2
.2. Synthesis of RNA oligomers
2
4,26
RNA oligomers containing nucleoside analogs 1, 2, and 3 were
cleases.
Recently, Pradeepkumar et al. reported the synthesis of 4′-
successfully synthesized via standard solid phase RNA synthesis using a
DNA/RNA synthesizer. Cleavage and deprotection of the oligomers
were carried out via treatment of CPG beads with 10% dimethylamine
C-aminomethyl-2′-O-methyluridine (1) and 4′-C-aminomethyl-2′-O-
2
7
methylcytidine (Fig. 1). The siRNAs containing these analogs were
less thermally stable but more nuclease-resistant than unmodified
siRNAs. Furthermore, siRNAs harboring modifications at the passenger
strands or the 3′-end of the guide strands still retained RNAi activity.
It was reported that the DNAs with longer aminoalkyl groups, such
as the aminoethyl and aminopropyl groups, at the 4′-positions of sugar
moieties were more nuclease-resistant than DNAs with shorter ami-
3
in MeCN, followed by conc. NH solution/40% methylamine (1:1, v/v)
to prevent the additional reaction of acrylonitrile with 4′-C-aminoalkyl
groups. After desilylation, oligomers were purified via 20% denaturing
polyacrylamide gel electrophoresis (PAGE). The oligonucleotide (ON)
sequences used in this study are depicted in Tables 1–3 and S1–S7.
2
6
noalkyl groups, such as the aminomethyl group. Thus, siRNAs in-
corporated with longer aminoalkyl groups at the 4′-position were ex-
pected to exhibit greater resistance to nucleolytic degradation by
nucleases.
2.3. Thermal denaturation study
First, we evaluated the effect of 4′-C-aminoalkyl-2′-O-methyl mod-
ifications on the thermal stability of dsRNAs (dsRNA 1–4). UV melting
experiments were performed in buffer containing 10 mM sodium
phosphate (pH 7.0) and 100 mM NaCl (Fig. S1). The melting tempera-
In the present study, we designed and synthesized RNAs and siRNAs
containing 4′-C-aminoethyl-2′-O-methyluridine (2) and 4′-C-amino-
propyl-2′-O-methyluridine (3) (Fig. 1). We then investigated the effects
of 4′-C-aminoalkyl modifications on thermal stability, stability in
serum, and RNAi activity of RNAs or siRNAs.
ture (T
was 43.5 °C, whereas that of dsRNA 2, dsRNA 3, and dsRNA 4 con-
taining 4′-C-aminoalkyl-2′-O-methyl modifications were 37.6, 37.3, and
6.6 °C, respectively. Incorporation of these modifications decreased
the T values of dsRNAs by 2.0–2.3 °C/modification. Then, incorpora-
m m
) values are shown in Table 1. The T of natural 11-mer dsRNA
1
3
2
. Results and discussion
.1. Synthesis of nucleoside analogs
′-C-aminoethyl-2′-O-methyl-modified uridine (2) and 4′-C-amino-
m
tion of 4′-C-aminoethyl-2′-O-methyl and 4′-C-aminopropyl-2′-O-me-
thyluridine was found to decrease the thermal stability of dsRNAs, si-
milar to the effect observed for 4′-C-aminomethyl-2′-O-methyluridine/
cytosine. These modifications might influence the conformation of the
sugar and hydration in the minor groove and disturb duplex forma-
2
4
propyl-2′-O-methyl-modified uridine (3) were synthesized from ribo-
furanose derivatives 4 and 7b as starting material following a pre-
tion.25 Longer side chain lengths were found to produce lower T
va-
m
2
8
viously reported method
(Schemes 1 and 2). The ribofuranose
lues.
derivative 4 prepared from D-glucose was converted to the hydro-
xyethyl derivative 6 via Wittig reaction and hydroboration, followed by
oxidation with 63% yield (after 2 steps). Tosylation of the hydroxy
group of 6 produced 7a at 81% yield. We attempted to convert com-
pound 7a to the azide derivative 9a via azidation, followed by acet-
olysis. However, this process generated many byproducts, which was
attributed to the reaction between the tosyl group and the 3′-benzyloxy
group of 7a. To solve this problem, acetolysis was carried out before
azidation. Acetolysis and azidation of 7a produced 9a at 74% yield
Modified siRNAs were also observed to exhibit reduced thermal
stability (Fig. S2 and Table 2). The T value of natural siRNA (siRNA 1)
was 79.5 °C, whereas those of siRNA 2, siRNA 3 and siRNA 4 were 65.7,
65.0 and 64.6 °C, respectively. The ΔT values of these siRNAs were
approximately −1.1 to −1.2 °C/modification, which were different
from those observed for dsRNAs and were found to be dependent on the
dsRNA sequence. Modification of dsRNAs with homopurine/homo-
pyrimidine sequence was found to decrease their thermal stabilities.
Destabilization of the DNA/RNA duplexes containing the 4′-C-ami-
noalkyl modification, which are composed of a mixed sequence, was
m
m
(
after two steps). Compound 9a was then subjected to modified Vor-
2
9
brüggen reaction using in situ silylation of uridine and subsequent
trimethylsilyl triflate-mediated coupling to produce 10a at 85% yield.
The acetate group of 10a was hydrolyzed using concentrated (conc.)
lower than those containing a homopurine/homopyrimidine se-
quence.23 These results suggested that the siRNAs with 4′-C-aminoalkyl
modifications can form stable duplexes under biological conditions.
Furthermore, we carefully examined the stability of the duplexes by
calculating the thermodynamic parameters of duplex formation based
3
NH solution in methanol to produce 11a at 98% yield. Then, the 2′-
hydroxy group of 11a was methylated via treatment with MeI and NaH
in THF at 0 °C to produce 12a at 67% yield. Debenzylation of 12a was
m T T
on the slope of the plot of 1/T vs. ln (C /4), where C is the total
carried out using BCl
3
in CH
2
Cl
2
at −78 °C to produce 13a at 78%
concentration of single-stranded RNAs (Fig. S3 and Table 3). The
ΔG°37°C value of dsRNA 5 was −10.6 kcal/mol, while those of dsRNA 9,
dsRNA 13 and dsRNA 17 were −10.1, −10.0, and −9.8 kcal/mol,
respectively. The ΔS° value of dsRNA 5 was −245.7 kcal/mol·K,
whereas those of dsRNA 9, dsRNA 13 and dsRNA 17 were −261.8,
−266.1, and −272.2 kcal/mol∙K, respectively. These results indicated
that the thermodynamic destabilization of the duplexes was caused by
disadvantages in terms of entropy (ΔS°). In addition, longer side chain
yield. The silyl group of 13a was removed via treatment with tetra-n-
butylammonium fluoride (TBAF) in THF to produce 14a at 97% yield.
The 5′-OH group of 14a was protected by a 4,4′-dimethoxytrityl (DMTr)
group to produce 15a at 96% yield. The azide group of 15a was re-
duced via treatment with PPh in THF at 45 °C, and the resulting amino
group was protected with a trifluoroacetyl (TFA) group to produce 17a
3
0
3
3
1
at 96% yield (after two steps). Compound 17a was phosphitylated
2

K. Koizumi et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
Scheme 1. Synthesis 4′-C-aminoethyl-2′-O-methyluridine phosphoramidite 18a.
Scheme 2. Synthesis of 4′-C-aminopropyl-2′-O-methyluridine phosphoramidite 18b.
3

K. Koizumi et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
siRNA 4) were measured. As shown in Fig. 4, siRNAs exhibited positive
CD bands at around 270 nm and negative bands at 210 nm, which were
characteristic of an A-type duplex. However, the CD bands around
270 nm were slightly more shifted to red as the side chains of the
modified siRNAs became longer. These results suggested that although
the modified siRNAs formed A-type duplexes, incorporation of 4′-C-
aminoalkyl modifications induced slight conformational changes in the
siRNA duplexes.
Scheme 3. Synthesis of the controlled-pore glasses (CPGs) 21a and 21b car-
rying 17a and 17b.
2
.6. Molecular modeling study
lengths resulted in more unfavorable entropy, which suggested that 4′-
C-aminoalkyl modification induces conformational changes that inter-
fere with the duplex formation.
Thermal denaturation study, NMR measurements, and CD spectra
suggested that 4′-C-aminoalkyl-2′-O-methyl modifications caused con-
formational changes in the dsRNA. Therefore, to determine the effects
of 4′-C-aminoalkyl-2′-O-methyl modifications on sugar puckering, we
calculated the percentage of 4′-C-aminoalkyl-2′-O-methyluridines 1, 2
and 3 in the C3′-endo sugar conformation based on 100-ns MD trajec-
tories (Fig. S5 and Table 4). During MD simulation, sugar conforma-
tions at each nucleotide were determined based on the pseudo rota-
Next, we assessed the base-discriminating abilities of dsRNAs (Table
S1 and Fig. 2). The ΔT
of complementary duplex) values for the natural duplexes (dsRNA
–8) were −5.4 to −11.8 °C, whereas those for the duplexes harboring
m m
(T of duplex containing mismatched base pair
−T
m
6
the modifications (dsRNA 10–12, dsRNA 14–16 and dsRNA 18–20)
were −7.6 to −12.1 °C. Thus, the 4′-C-aminoalkyl-2′-O-methyluridines
had base-discriminating abilities in dsRNA.
36
tional angles. A previous study showed that the 4′-C-aminomethyl-2′-
O-methyl modification nucleotide drove the sugar conformation to the
south-type C2′-endo pucker. Similar results were observed for the 4′-C-
aminoethyl-2′-O-methyl and 4′-C-aminopropyl-2′-O-methyl modifica-
tions. Longer side chain lengths resulted in a higher percentage of the
population in the C2′-endo sugar conformation. Therefore, longer side
chains induced greater conformational changes in the dsRNA. These
results indicated that 4′-C-aminoalkyl modification of dsRNA caused
the conformational changes, such as sugar puckering, which in turn
resulted in the unfavorable entropy changes in the dsRNAs.
2
.4. NMR measurements
We performed NMR measurements to evaluate the sugar puckering
of 4′-C-aminoethyl-2′-O-methyluridine (2). First, the monomer popu-
lation in the C2′-endo conformation was calculated from J1′-2′. The
calculated percentages of 4′-C-azidoethyl-2′-O-methyluridine (14a) and
4
′-C-azidopropyl-2′-O-methyluridine (14b) in the C2′-endo conforma-
tion were 69.0% and 68.4%, respectively.
Next, the sugar puckering of 4′-C-amiNOEthyl-2′-O-methyluridine
(
2) in dsRNA was directly measured via NMR. The NMR signals of non-
2
.7. RNAi activity
exchangeable base protons (H2/H5/H6/H8) and ribose H1′ of 5′-CGC
GAAU2CGCG-3′ were assigned via the well-established sequential NOE
connectivity method (Fig. S4). NMR assignments were compared to
The RNAi activities of the modified and unmodified siRNAs were
3
3
assessed via dual luciferase reporter assays using a vector encoding
Renilla and firefly luciferases in HeLa cells. The siRNAs targeting the
Renilla luciferase gene were transfected using RNAiMAX. After 24 h, the
expression levels of both luciferase genes were analyzed. The relative
percentage of Renilla: firefly luciferase activity with respect to the no
siRNA control is shown in Tables S2–S7 and Figs. 5–8.
Most siRNAs (siRNA 6–29), which contained one analog at the
passenger strand, effectively downregulated the expression of the
Renilla luciferase gene both at 1 and 10 nM concentrations, which is
similar to the effects observed with the unmodified siRNA (siRNA 5)
(Fig. 5). Slight reduction in RNAi activity was observed only when
siRNA containing the analog with an aminopropyl group at position 11
from the 5′-end (siRNA 21) was used at a concentration of 1 nM, sug-
gesting that the 4′-C-aminopropyl modification located near the clea-
vage site slightly influences recognition by Ago2.37 Furthermore, we
examined the RNAi activity of siRNAs (siRNA 30–38), which contained
three analogs at the 5′-, middle, or 3′-regions of the passenger strand. As
3
4
previously reported NMR data. The H1′-H2′ cross peaks were ob-
served from the TOCSY spectra of C(1), 2(8), and G(12) (Fig. 3). The
H1′-H2′ cross peaks for C(1) and G(12) were attributed to the con-
formational equilibrium of the sugar pucker between C2′-endo and C3′-
endo because the cross peaks are often observed for residues at the ends
of the oligonucleotides. On the other hand, the observed H1′-H2′ cross
peaks for 2(8) in the middle of the oligonucleotide suggested that 2
prefers the C2′-endo conformation.
2
.5. Circular dichroism (CD) spectra
The global conformations of siRNAs are important for their RNAi
activity. The human immunodeficiency virus (HIV)-1 transactivating
response (TAR) RNA-binding protein (TRBP) has been reported to bind
A-form duplexes but not B-form duplexes. Thus, the CD spectra of
siRNAs with and without modifications (siRNA 1, siRNA 2, siRNA 3 and
3
5
Table 1
m
Sequences of ssRNAs, dsRNAs, and T values of dsRNAs.
Abbreviation of dsRNA
Abbreviation of ssRNA
Sequencea
T
m
(°C)
ΔT
m
(°C)b
ΔT
m
(°C)/mod.
dsRNA 1
RNA 1
RNA 9
F-5′-UUCUUCUUCUU-3′-S
3′-AAGAAGAAGAA-5′
43.5 ( ± 0.1)
37.6 ( ± 0.2)
37.3 ( ± 0.1)
36.6 ( ± 0.1)
–
–
dsRNA 2
dsRNA 3
dsRNA 4
RNA 2
RNA 9
F-5′-U1CUUC1UC1U-3′-S
3′-AAGAAGAAGAA-5′
−5.9 ( ± 0.3)
−6.3 ( ± 0.1)
−7.0 ( ± 0.1)
−2.0 ( ± 0.1)
−2.1 ( ± 0.0)
−2.3 ( ± 0.0)
RNA 3
RNA 9
F-5′-U2CUUC2UC2U-3′-S
3′-AAGAAGAAGAA-5′
RNA 4
RNA 9
F-5′-U3CUUC3UC3U-3′-S
3′-AAGAAGAAGAA-5′
m
The T values were determined using 3 µM dsRNA in a buffer containing 10 mM sodium phosphate (pH 7.0) and 100 mM NaCl.
a
b
F denotes a fluorescein. S shows an ethynyl linker. ΔT
m
represents [T
m
m
(dsRNAmod) – T (dsRNAunmod)].
4

K. Koizumi et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
Table 2
m
Sequences of ssRNAs, siRNAs, and T values of siRNAs.
Abbreviation of siRNA
Abbreviation of ssRNA
Sequencea
T
m
(°C)
ΔT
m
(°C)b
m
ΔT (°C)/mod.
siRNA 1
RNA 13
RNA 14
5′-GGCCUUUCACUACUCCUACUU-3′
3′-UUCCGGAAAGUGAUGAGGAUG-5′-F
79.5 ( ± 0.4)
65.7 ( ± 0.6)
65.0 ( ± 0.6)
64.6 ( ± 0.2)
–
–
siRNA 2
siRNA 3
siRNA 4
RNA 15
RNA 16
5′-GGCC111CAC1AC1CC1AC11-3′
3′-11CCGGAAAG1GA1GAGGA1G-5′-F
−13.7 ( ± 0.4)
−14.6 ( ± 0.2)
−14.8 ( ± 0.3)
−1.06 ( ± 0.03)
−1.11 ( ± 0.01)
−1.16 ( ± 0.04)
RNA 17
RNA 18
5′-GGCC222CAC2AC2CC2AC22-3′
3′-22CCGGAAAG2GA2GAGGA2G-5′-F
RNA 19
RNA 20
5′-GGCC333CAC3AC3CC3AC33-3′
3′-33CCGGAAAG3GA3GAGGA3G-5′-F
m
The T values were determined using 3 µM dsRNA in a buffer containing 10 mM sodium phosphate (pH 7.0) and 100 mM NaCl.
a
b
m m m
F denotes a fluorescein. ΔT represents [T (siRNAmod) – T (siRNAunmod)].
shown in Fig. 6, although slight decrease in RNAi activities was ob-
served at 1 nM, siRNAs harboring three modifications at the passenger
strand still preserved their RNAi activities.
As passenger strand modification at one or three positions did not
affect RNAi activity, we next examined the RNAi activities of siRNAs
harboring eight analogs in the passenger strand (siRNA 39–41). As
shown in Fig. 7, the RNAi activities of 1 nM siRNAs were lower than
those of the unmodified siRNAs. Longer aminoalkyl groups tended to
show reduced RNAi activities. The minor groove of the siRNA was
crowded with the aminoalkyl groups when the eight analogs were in-
2.8. Stability in serum
Natural RNA oligomers are easily degraded by nucleases that are
present both inside and outside of cells and thus limit the application of
RNA drugs. Therefore, chemical modifications are required to increase
the half-life of siRNAs and prevent nucleolytic degradation by these
nucleases. The 4′-C-aminoalkyl modifications are expected to enhance
the stability of the RNA oligomers. Thus, we assessed the effects of 4′-C-
aminoalkyl modifications on the stabilities of ssRNAs (RNA 1–4) and
siRNAs (siRNA 1–4) in serum. First, 11-mer RNA labeled at the 5′-end
with fluorescein was incubated in buffer containing 3% bovine serum
and subsequently analyzed via PAGE. As shown in Fig. 9, the un-
modified RNA was degraded by nucleases in serum within 1 h of in-
cubation. On the other hand, the modified RNAs exhibited considerably
longer half-lives than those of native RNA. The half-lives of RNA 1, RNA
2, RNA 3, and RNA 4 were determined to be 0.05, 2.1, 15 and 32 h,
respectively. Although the RNA harboring the 4′-C-aminomethyl-2′-O-
methyluridine modification exhibited longer half-life than the native
RNA, most of the full-length RNA was degraded after 6 h of incubation.
On the other hand, full length RNAs containing 4′-C-aminoethyl-2′-O-
methyl and 4′-O-aminopropyl-2′-O-methyl modifications were still in-
tact even after 6 h of incubation. We hypothesized that the ammonium
cations, which were produced via protonation of the terminal amino
functions of the aminoalkyl groups, interacted with the active centers of
the nucleases in the serum and inhibited the hydrolysis of the RNAs.
These results suggested that the ammonium cations of flexible and long
chains can effectively interact with nucleases and inhibit their activ-
ities.
troduced into the siRNA. Thus, steric hindrance by the aminoalkyl
groups might interfere with RISC formation.3
8,39
Furthermore, as the 4′-
C-aminoalkyl modification shifts the sugar conformation of the nu-
cleoside from C3′-endo to C2′-endo puckering, this conformational
change might influence Ago2 recognition. However, notably, siRNAs
harboring the eight analogs showed sufficient RNAi activities at 10 nM.
Finally, the modifications were introduced into the guide strand of
siRNAs (siRNA 42–50) (Fig. 8). The region spanning nucleoside posi-
tions 2–8 from the 5′-end of the guide strand is referred to as the seed
region. It is known that the formation of base pairs between the siRNA
and target mRNA in this region is crucial for eliciting the RNAi activity
3
8
of the siRNA. It was reported that incorporation of 4′-C-aminomethyl-
′-O-methyluridine (1) at the seed region inhibited the RNAi activity of
2
2
5
the siRNA. Similarly, incorporation of 4′-C-aminoethyl-2′-O-methy-
luridine (2) or 4′-C-aminopropyl-2′-O-methyluridine (3) at the nucleo-
side position 2 from the 5′-end (siRNA 49 and 50) was detrimental to
RNAi activity. However, surprisingly, the aminoalkyl modifications at
nucleoside position 8 from the 5′-end (siRNA 45–47) did not affect
RNAi activity although this position is within the seed region. Fur-
thermore, aminoalkyl modifications in the guide strand outside the seed
region (siRNA 42–44) did not abolish RNAi activity, although 1 nM
aminopropyl modification slightly reduced RNAi activity. These results
suggested that the 4′-C-aminoalkyl modifications at the passenger
strand and the guide strand outside the seed region did not significantly
affect Ago2 recognition, although Ago2 recognizes the minor groove of
siRNA.
Next, we examined the stabilities of the modified siRNAs in serum.
The modified and unmodified siRNAs, which were fluorescein-labeled
at the 5′-ends of the guide strands, were incubated in buffer containing
10% bovine serum. As shown in Fig. 10, the modified siRNAs showed
high stability in serum than the single-stranded RNAs. The unmodified
siRNAs were degraded by nucleases in serum within 3 h of incubation,
whereas the modified siRNAs remained intact after 6 h.
Table 3
Thermodynamic parameters of the dsRNAs.
Abbreviation of dsRNA
Abbreviation of ssRNA
Sequence
ΔH° (kcal/mol)
−86.8
ΔS° (cal/mol·K)
−245.7
ΔG°37°C (kcal/mol)
−10.6
dsRNA 5
RNA 5
RNA 9
5′-UUCUUCUUCUU-3′
3′-AAGAAGAAGAA-5′
dsRNA 9
dsRNA 13
dsRNA 17
RNA 6
RNA 9
5′-UUCUUC1UCUU-3′
3′-AAGAAGAAGAA-5′
−91.2
−92.5
−94.2
−261.8
−266.1
−272.2
−10.1
−10.0
−9.8
RNA 7
RNA 9
5′-UUCUUC2UCUU-3′
3′-AAGAAGAAGAA-5′
RNA 8
RNA 9
5′-UUCUUC3UCUU-3′
3′-AAGAAGAAGAA-5′
5

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Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
Table 4
Ratio of C3′-endo puckering of the unmodified
and modified uridine.
C3′-endo (%)
Uridine
2
3
38.5
16.4
16.1
Fig. 2. Base-discriminating abilities of RNAs containing 4′-C-aminoalk-
ylnucleoside analogs. Gray: unmodified dsRNAs. Green: dsRNAs containing the
aminomethyl analog 1. Red: dsRNAs containing the aminoethyl analog 2. Blue:
dsRNAs containing the aminopropyl analog 3.
Fig. 3. TOCSY spectra of 5′-CGCGAAU2CGCG-3′ at the 3.8–4.8 ppm and
2
5.6–6.1 ppm regions in D O at 45 °C.
Fig. 5. RNAi activities of siRNAs modified by one analog at the passenger
strands. Experimental conditions are described in the experimental section.
Fig. 4. CD spectra of the modified and unmodified siRNAs in a buffer con-
taining 10 mM sodium phosphate (pH 7.0) and 0.1 M NaCl at 15 °C. The con-
centration of duplexes was 4 μM.
the T
m
values of the siRNAs were around 60 °C, it was found that the
siRNAs with the 4′-C-aminoalkyl modifications could form stable du-
plexes under biological conditions. On the other hand, the 4′-C-ami-
noalkyl modifications significantly increased the nuclease resistance of
the single-stranded RNAs (ssRNAs) and siRNAs. Furthermore, the 4′-C-
aminoalkyl modifications at the passenger strand and the guide strand
outside the seed region were well tolerated for RNAi activities. Thus,
siRNAs containing the 4′-C-aminoethyl and 4′-C-aminopropyl analogs
are good candidates for the development of therapeutic siRNA mole-
cules.
3. Conclusions
In the present study, we successfully demonstrated the synthesis of
4
′-C-aminoethyl-2′-O-methyluridine (2) and 4′-C-aminopropyl-2′-O-
methyluridine (3) and the oligonucleotides (ONs) containing the ana-
logs. Incorporation of the analogs into dsRNAs and siRNAs decreased
the T values of dsRNAs and siRNAs by 1–2 °C/modification. However,
m
6

K. Koizumi et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
Fig. 6. RNAi activities of siRNAs modified by three analogs at the passenger strands. Experimental conditions are described in the experimental section.
(
CH
3
)
4
Si (δ = 0.00 for 1H NMR in CDCl
3
), or a solvent (for 13C NMR
) as an internal standard for coupling constants
J) in Hz. The abbreviations s, d, q and m signify singlet, doublet,
quadruplet and multiplet, respectively.
1
and H NMR in DMSO‑d
(
6
4.2. 5-O-[(1,1-Dimethylethyl)diphenylsilyl]-4-C-vinyl-3-O-benzyl-1,2-O-
isopropylidene-α-D-ribofuranose (5)
Sodium hydride (NaH, 60% in mineral, 2.24 g, 56.0 mmol) was
added to DMSO (40 mL) under argon atmosphere and mixture was
stirred for 30 min at 80 °C. after the mixture was cooled down to room
temperature,
a solution of methyltriphenylphosphonium bromide
(
3
Ph PMeBr, 20.0 g, 56.0 mmol) in anhydrous DMSO (60 mL) was added
dropwise to the mixture at 0 °C and the mixture was stirred for 30 min
at room temperature. A solution of 4 (23.5 g, 42.5 mmol) in anhydrous
DMSO (100 mL) was added dropwise and the mixture was stirred for
Fig. 7. RNAi activities of siRNAs modified by eight analogs at the passenger
strands. Experimental conditions are described in the experimental section.
3
.5 h at room temperature. The mixture was poured into H
bath and extracted with EtOAc. The organic layer was washed with
brine, dried over Na SO and concentrated in vacuo. The residue was
2
O in an ice-
2
4
4. Experimental section
purified by chromatography on silica gel (hexane/EtOAc = 10:1) to
give 5 as a colorless oil (16.6 g, 30.4 mmol, 72%). 1H NMR (600 MHz,
4.1. General remarks
CDCl
3
) δ 1.00 (s, 9H), 1.32 (s, 6H), 3.52 (dd, J = 11.7 Hz and 7.6 Hz,
2H), 4.46 (d, J = 4.8 Hz, 1H), 4.64–4.68 (m, 2H), 4.83 (d, J = 12.4 Hz,
1H), 5.19 (dd, J = 9.6 Hz and 2.0 Hz, 1H), 5.45 (d, J = 15.1 Hz, 1H),
5.81 (d, J = 4.1 Hz, 1H), 6.15–6.19 (m, 1H), 7.28–7.50 (m, 11H),
CDCl
3 6
or DMSO‑d was used as a solvent for obtaining NMR spectra.
Chemical shifts (δ) are given in parts per million (ppm) downfield from
Fig. 8. RNAi activities of siRNAs modified by one analog at the guaide strands. Experimental conditions are described in the experimental section.
7

K. Koizumi et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
Fig. 9. PAGE of ssRNAs treated in buffer containing 3% bovine serum. Experimental conditions are described in the experimental section.
7
6
1
1
5
.59–7.65 (m, 4H). 13C NMR (101 MHz, CDCl
6.5, 72.6, 77.0, 77.1, 77.4, 78.9, 87.5, 104.1, 113.6, 116.4, 127.8,
27.8, 127.9, 128.0, 128.6, 129.8, 129.8, 133.1, 133.6, 135.7, 135.8,
38.1. HRMS (ESI-TOF) m/z Calcd for
83.2282, Found 583.2292.
3
) δ 19.4, 26.0, 26.4, 26.9,
15.3 g, 80.0 mmol) was added to the solution of 6 (13.1 g, 23.3 mmol)
in anhydrous CH Cl (120 mL) under argon atmosphere at 0 °C and the
mixture was stirred for 20 h at room temperature. The mixture was
extracted with saturated NaHCO solution and CH Cl . The organic
layer was washed with brine, dried over Na SO and concentrated in
2
2
C
33
H
40KO
5
Si [M+Na]+
3
2
2
2
4
vacuo. The residue was purified by chromatography on silica gel
(hexane/EtOAc = 4:1) to give 7a as a colorless oil (13.5 g, 18.9 mmol,
4
1
.3. 5-O-[(1,1-Dimethylethyl)diphenylsilyl]-4-C-hydroxyethyl-3-O-benzyl-
,2-O-isopropylidene-α-D-ribofuranose (6)
81%). 1H NMR (500 MHz, CDCl
) δ 0.98 (s, 9H), 1.31 (s, 3H), 1.47 (s,
H), 1.85–1.93 (m, 1H), 2.38 (s, 3H), 2.51–2.54 (m, 1H), 3.35 (d,
3
3
A 0.5 M in THF solution of 9-borabicyclo[3.3.1]nonane (9-BBN,
82 mL, 90.8 mmol) was added dropwise to a solution of 5 (16.6 g,
0.4 mmol) in anhydrous THF (100 mL) under argon atmosphere and
the mixture was stirring for 13.5 h at room temperature·H O was added
2
to the reaction until evolution of gas ceased. 3 N NaOH solution (60 mL)
was added and then slowly 30% aqueous hydrogen peroxide solution
J = 10.9 Hz, 1H), 3.58 (d, J = 10.9 Hz, 1H), 4.11–4.18 (m, 1H), 4.20
(d, J = 5.8 Hz, 1H), 4.26–4.31 (m, 1H), 4.49 (d, J = 12.0 Hz, 1H),
4.59–4.62 (m, 1H), 4.74 (d, J = 12.0 Hz, 1H), 5.71 (d, J = 4.00 Hz,
1H), 7.19 (d, J = 8.0 Hz, 2H), 7.28–7.45 (m, 11H), 7.55–7.64 (m, 6H).
C NMR (101 MHz, CDCl ) δ 19.3, 21.7, 26.2, 26.7, 26.9, 31.3, 67.1,
3
67.6, 72.5, 78.0, 79.1, 86.4, 104.5, 113.4, 127.8, 127.9, 127.9, 127.9,
1
3
13
(
5
30 mL) was added while keeping the temperature between 30 and
0 °C. The mixture was stirred for 3 h and extracted with H O and
EtOAc. The organic layer was washed with neutral phosphate buffer
solution and brine, dried over Na SO and concentrated in vacuo. The
128.0, 128.6, 129.8, 132.0, 133.2, 133.4, 135.7, 135.7, 137.8, 144.4.
+
HRMS (ESI-TOF) m/z Calcd for C40
H48NaO
8
SSi [M+Na] 739.2737,
2
Found 739.2746.
2
4
residue was purified by chromatography on silica gel (hexane/
EtOAc = 10:1) to give 5 as a colorless oil (14.9 g, 26.4 mmol, 87%). H
4.5. 5-O-[(1,1-Dimethylethyl)diphenylsilyl]-4-C-(p-
toluenesulfonyloxyethyl)-3-O-benzyl-1,2-di-O-acetyl-α-D-ribofuranose (8)
1
NMR (400 MHz, CDCl
.71–1.72 (m, 1H), 2.43–2.48 (m, 1H), 2.80–2.82 (m, 1H), 3.42 (d,
J = 11.0 Hz, 1H), 3.63–3.72 (m, 2H), 3.73 (d, J = 11.0 Hz, 1H), 4.29
3
) δ 0.99 (s, 9H), 1.36 (s, 3H), 1.68 (s, 3H),
1
Acetic anhydride (Ac O, 17.5 mL, 189 mmol) and 1% H SO in
2
2
4
AcOH (5.1 mL) was added to a solution of 7a (13.5 g, 18.9 mmol) in
(
(
d, J = 5.5 Hz, 1H), 4.56 (d, J = 12.4 Hz, 1H), 4.68–4.70 (m, 1H), 4.81
d, J = 11.9 Hz, 1H), 5.80 (d, J = 4.12 Hz, 1H), 7.30–7.45 (m, 11H),
AcOH (40 mL) at 0 °C and the mixture was stirred for 30 min at room
temperature. The mixture was extracted with H O and EtOAc. The or-
2
1
3
7
2
1
1
6
.57–7.64 (m, 4H). C NMR (101 MHz, CDCl
7.0, 27.3, 33.7, 35.0, 59.0, 66.9, 72.6, 78.0, 79.4, 88.4, 104.5, 113.9,
27.9, 127.9, 128.1, 128.6, 129.9, 130.0, 133.0, 133.2, 135.7, 135.8,
3
) δ 19.4, 22.9, 26.3, 26.6,
ganic layer was washed with saturated NaHCO3 solution and brine,
dried over Na SO and concentrated in vacuo. The residue was purified
2
4
by chromatography on silica gel (hexane/EtOAc = 4:1) to give 8 as a
33 6
C H42KO
Si [M+Na]+
colorless oil (13.0 g, 17.1 mmol, 90%). 1H NMR (500 MHz, CDCl ) δ
37.8. HRMS (ESI-TOF) m/z Calcd for
3
01.2388, Found 601.2395.
1.02 (s, 9H), 1.75 (s, 3H), 2.03 (s, 3H), 2.06–2.15 (m, 1H), 2.24–2.36
(
m, 1H), 2.39 (s, 3H), 3.47–3.52 (m, 2H), 4.15–4.20 (m, 1H), 4.25 (d,
4
.4. 5-O-[(1,1-Dimethylethyl)diphenylsilyl]-4-C-(p-
J = 5.2 Hz, 1H), 4.28–4.33 (m, 1H), 4.48 (d, J = 10.9 Hz, 1H), 4.54 (d,
J = 10.9 Hz, 1H), 5.29 (d, J = 5.2 Hz, 1H), 6.04 (s, 1H), 7.22–7.24 (m,
4H), 7.30–7.48 (m, 10H), 7.58–7.72 (m, 5H). 13C NMR (126 MHz,
toluenesulfonyloxyethyl)-3-O-benzyl-1,2-O-isopropylidene-α-D-ribofuranose
(
7a)
CDCl
3
) δ 19.4, 20.8, 21.0, 21.7, 27.0, 31.1, 67.6, 67.5, 73.6, 74.3, 79.1,
Pyridine (12.9 mL, 160 mmol) and p-toluenesulfonyl chloride (TsCl,
86.8, 97.7, 127.6, 127.9, 128.0, 128.6, 129.9, 130.0, 130.1, 132.7,
Fig. 10. PAGE of siRNAs treated in buffer containing 10% bovine serum. Experimental conditions are described in the experimental section.
8

K. Koizumi et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
133.1, 133.4, 135.6, 135.7, 137.3, 144.5. HRMS (ESI-TOF) m/z Calcd
[M+Na]+ 664.2567, Found 664.2547.
+
for C41H48KO10SSi [M+Na] 799.2375, Found 799.2395.
4
1
.6. 5-O-[(1,1-Dimethylethyl)diphenylsilyl]-4-C-azidoethyl-3-O-benzyl-
,2-di-O-acetyl-α-D-ribofuranose (9a)
4.9. 5′-O-[(1,1-Dimethylethyl)diphenylsilyl]-4′-C-azidoethyl-3′-O-benzyl-
2′-O-methyluridine (12a)
NaN
3
(1.04 g, 16.0 mmol) was added to a solution of 8 (3.48 g,
NaH (1.14 g, 28.4 mmol) was added to a solution of 11a (6.06 g,
9.46 mmol) in THF (60 mL) at 0 °C under argon atmosphere and the
mixture was stirred 10 min at 0 °C. Then methyl iodide (MeI, 2.94 mL,
47.3 mmol) was added dropwise to the mixture and stirred 8 h at 0 °C.
4
.57 mmol) in DMF (35 mL) under argon atmosphere and the mixture
was stirred overnight at 50 °C. The mixture was extracted with brine
and EtOAc. The organic layer was washed with brine, dried over
Na
2
SO
4
and concentrated in vacuo. The residue was purified by chro-
Saturated NaHCO
mixture was extracted with saturated NaHCO
organic layer was washed with brine, dried over Na
3
solution was added to the mixture at 0 °C. The
solution and EtOAc. The
SO and con-
matography on silica gel (hexane/EtOAc = 7:1) to give 9a as a colorless
3
1
oil (2.37 g, 3.75 mmol, 82%). H NMR (400 MHz, CDCl
3
) δ 1.07 (s, 9H),
.81 (s, 3H), 1.97–2.05 (m, 1H), 2.10 (s, 3H), 2.14–2.21 (m, 1H),
.25–3.31 (m, 1H), 3.39–3.46 (m, 1H), 3.60 (s, 2H), 4.32 (d,
2
4
1
3
centrated in vacuo. The residue was purified by chromatography on
silica gel (hexane/EtOAc = 2:1) to give 12a as a white solid (4.18 g,
1
J = 5.0 Hz, 1H), 4.52 (d, J = 11.5 Hz, 1H), 4.59 (d, J = 11.4 Hz, 1H),
.35 (d, J = 5.0 Hz, 1H), 6.14 (s, 1H), 7.27–7.46 (m, 10H), 7.62–7.65
6.37 mmol, 67%). H NMR (400 MHz, CDCl
3
) δ 1.09 (s, 9H), 1.71–1.77
5
(m, 1H), 2.29–2.35 (m, 1H), 3.25–3.31 (m, 1H), 3.34–3.39 (m, 1H),
3.52 (s, 3H), 3.68 (d, J = 11.5 Hz, 1H), 3.75 (dd, J = 5.7 Hz and 2.3 Hz,
1H), 3.98 (d, J = 11.5 Hz, 1H), 4.35 (d, J = 6.3 Hz, 1H), 4.51 (d,
J = 11.5 Hz, 1H), 4.71 (d, J = 11.5 Hz, 1H), 5.09 (dd, J = 8.0 Hz and
1.7 Hz, 1H), 6.08 (d, J = 2.3 Hz, 1H), 7.34–7.40 (m, 9H), 7.44–7.47 (m,
2H), 7.51 (d, J = 7.5 Hz, 2H), 7.61 (d, J = 6.9 Hz, 2H), 7.79 (d,
1
3
(
4
1
m, 5H). C NMR (151 MHz, CDCl
3
) δ 19.5, 21.0, 21.1, 27.0, 31.6,
6.8, 68.0, 73.7, 74.6, 79.3, 87.1, 97.8, 127.6, 127.9, 128.0, 128.1,
28.6, 130.0, 130.1, 132.7, 133.1, 135.7, 135.8, 137.5, 169.3, 169.8.
+
41 3 7
HRMS (ESI-TOF) m/z Calcd for C34H N NaO Si [M+Na] 654.2612,
Found 654.2632.
J = 8.1 Hz, 1H), 8.96 (s, 1H). 13C NMR (126 MHz, CDCl
) δ 19.6, 27.3,
31.0, 46.6, 59.45, 65.5, 73.2, 75.8, 84.0, 87.3, 88.3, 102.6, 128.0,
128.2, 128.3, 128.3, 128.7, 130.3, 130.4, 132.0, 132.9, 135.4, 135.6,
3
4.7. 5′-O-[(1,1-Dimethylethyl)diphenylsilyl]-4′-C-azidoethyl-3′-O-benzyl-
2
′-O-acetyluridine (10a)
1
C
37.3, 140.0, 150.0, 163.1. HRMS (ESI-TOF) m/z Calcd for
Uracil (3.95 g, 35.2 mmol) and N,O-bis(trimethylsilyl)acetamide
35
H
41
N
5
NaO
6
Si [M+Na]+ 678.2724, Found 678.2704.
(
1
BSA, 34.4 mL, 141 mmol) was added to a solution of 9a (11.1 g,
7.6 mmol) in MeCN (100 mL) under argon atmosphere and the mix-
ture was stirred 1 h at 95 °C. The mixture was cooled to room tem-
perature, trimethylsilyl trifluoromethanesulfonate (TMSOTf, 6.36 mL,
4.10. 5′-O-[(1,1-Dimethylethyl)diphenylsilyl]-4′-C-azidoethyl-2′-O-
methyluridine (13a)
3
5.2 mmol) was added dropwise to the mixture at 0 °C and the mixture
was stirred for 3 h at 50 °C. Saturated NaHCO solution was added to
the mixture at 0 °C. The mixture was extracted with saturated NaHCO
solution and CHCl . The organic layer was washed with saturated
NaHCO solution, dried over Na SO and concentrated in vacuo. The
3
M of BCl
solution of 12a (1.06 g, 1.62 mmol) in CH
argon atmosphere and the mixture was stirred for 3 h at −78 °C, then
warmed to −30 °C and stirred 5 h. A solution of MeOH/CH Cl (1:1, v/
3
solution in CH
2
Cl
2
(11.0 mL, 11.0 mmol) was added to a
3
2
Cl (42 mL) at −78 °C under
2
3
3
2
4
2
2
residue was purified by chromatography on silica gel (hexane/
EtOAc = 2:1) to give 10a as a white solid (10.2 g, 14.9 mmol, 85%). H
v) was added to the mixture. The mixture was concentrated in vacuo.
The residue was purified by chromatography on silica gel (hexane/
EtOAc = 1:1) to give 13a as a white solid (0.713 g, 1.26 mmol, 78%).
1
NMR (400 MHz, CDCl
3
) δ 1.10 (s, 9H), 1.65–1.73 (m, 1H), 2.05–2.14
1
(
m, 4H), 3.23–3.30 (m, 1H), 3.35–3.41 (m, 1H), 3.56 (d, J = 11.5 Hz,
H NMR (600 MHz, CDCl3) δ 1.11 (s, 9H), 1.73–1.78 (m, 1H),
1H), 3.86 (d, J = 11.5 Hz, 1H), 4.39–4.42 (m, 2H), 4.61 (d,
2.05–2.10 (m, 1H), 2.89 (d, J = 5.5 Hz, 1H), 3.28–3.33 (m, 1H),
3.36–3.40 (m, 1H), 3.53 (s, 3H), 3.71 (d, J = 11.7 Hz, 1H), 3.89–3.93
(m, 2H), 4.49 (t, J = 6.2 Hz, 1H), 5.33 (d, J = 6.2 Hz, 1H), 6.08 (s,
J = 4.1 Hz, 1H), 7.40–7.43 (m, 4H), 7.46–7.48 (m, 2H), 7.61 (d,
J = 7.6 Hz, 2H), 7.65 (d, J = 7.6 Hz, 2H), 7.78 (d, J = 8.2 Hz, 1H), 9.11
(s, 1H). 13C NMR (151 MHz, CDCl3) δ 19. 5, 27.2, 30.9, 46.6, 59.4,
66.8, 70.1, 84.4, 86.7, 87.8, 103.0, 128.2, 128.3, 130.4, 130.5, 131.8,
132.7, 135.4, 135.7, 139.9, 150.3, 163.4. HRMS (ESI-TOF) m/z Calcd
J = 11.0 Hz, 1H), 5.32–5.39 (m, 2H), 6.13 (d, J = 5.04 Hz, 1H),
1
3
7
.33–7.49 (m, 10H), 7.57–7.64 (m, 6H), 8.02 (s, 1H). C NMR
(
8
1
151 MHz, CDCl
3
) δ 19.5, 20.9, 27.18, 3.09, 46.5, 66.5, 74.6, 74.9,
6.9, 87.4, 103.1, 128.0, 128.2, 128.3, 128.4, 128.7, 130.4, 130.5,
31.9, 132.6, 135.5, 135.8, 137.1, 139.9, 150.2, 162.8, 170.1. HRMS
ESI-TOF) m/z Calcd for C36
+
(
H
41
N
5
NaO
7
Si [M+Na] 706.2673, Found
706.2676.
35 5 6
for 28H N NaO
Si [M+Na]+ 588.2254, Found 588.2266.
4.8. 5′-O-[(1,1-Dimethylethyl)diphenylsilyl]-4′-C-azidoethyl-3′-O-
benzyluridine (11a)
4.11. 4′-C-Azidoethyl-2′-O-methyluridine (14a)
NH
3
solution (83 mL) was added to a solution of 10a (10.2 g,
14.9 mmol) in MeOH (83 mL) and the mixture was stirred overnight at
1 M of tetrabutylammonium fluoride (TBAF) solution in THF
room temperature. The mixture was concentrated in vacuo. The residue
(1.85 mL, 1.85 mmol) was added to a solution of 13a (0.693 g,
1.23 mmol) in THF (7.0 mL) under argon atmosphere and the mixture
was stirred for 21 h at room temperature. The mixture was concentrated
in vacuo. The residue was purified by chromatography on silica gel
was purified by chromatography on silica gel (hexane/EtOAc = 1:1) to
1
give 11a as a white solid (9.39 g, 14.6 mmol, 98%). H NMR (500 MHz,
CDCl
3
) δ 1.09 (s, 9H), 1.67–1.73 (m, 1H), 2.15–2.21 (m, 1H), 3.20–3.26
(
m, 1H), 3.33–3.38 (m, 1H), 3.47–3.55 (m, 2H), 3.80 (d, J = 10.9 Hz,
3
(CHCl /MeOH = 8:1) to give 14a as a white solid (0.390 g, 1.19 mmol,
1
1H), 4.19 (d, J = 6.3 Hz, 1H), 4.30 (q, J = 5.8 Hz, 1H), 4.59 (d,
6
97%). H NMR (500 MHz, DMSO‑d ) δ 1.78–1.81 (m, 1H), 1.95–1.98
J = 11.5 Hz, 1H), 4.72 (d, J = 11.5 Hz, 1H), 5.39 (d, J = 8.6 Hz, 1H),
(m, 1H), 3.30 (s, 3H), 3.35–3.42 (m, 4H), 3.99 (t, J = 6.3 Hz, 1H), 4.18
(t, J = 5.8 Hz, 1H), 5.29–5.33 (m, 2H), 5.68 (d, J = 8.1 Hz, 1H), 5.92
5
7
2
1
1
.90 (d, J = 5.7 Hz, 1H), 7.32–7.42 (m. 9H), 7.45–7.48 (m, 2H),
1
3
13
.57–7.62 (m, 5H), 9.20 (s, 1H). C NMR (151 MHz, CDCl
7.2, 31.2, 46.6, 66.9, 74.7, 75.0, 78.8, 87.2, 89.4, 102.8, 128.2, 128.3,
28.4, 128.6, 128.9, 130.4, 130.5, 132.0, 132.6, 135.5, 135.7, 136.9,
3
) δ 19.4,
(d, J = 6.9 Hz, 1H), 7.90 (d, 8.1 Hz, 1H), 11.4 (s, 1H). C NMR
(101 MHz, DMSO‑d
102.3, 140. 6, 150.7, 163.0. HRMS (ESI-TOF) m/z Calcd for
[M+K]+ 366.0816, Found 366.0813.
6
) δ 31.0, 46.4, 57.5, 64.3, 69.7, 82.3, 85.0, 87.2,
40.0, 150.8, 163.0. HRMS (ESI-TOF) m/z Calcd for C34
H
39
N
5
NaO
6
Si
12 17 5 6
C H N O
9

K. Koizumi et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
4.12. 5′-O-[Bis(4-methoxyphenyl)phenylmethyl]-4′-C-azidoethyl-2′-O-
4.15. 5-O-[(1,1-Dimethylethyl)diphenylsilyl]-4-C-azidopropyl-3-O-benzyl-
methyluridine (15a)
1,2-O-(1-methylethylidene)-α-D-ribofuranose (19)
4,4′-Dimethoxytrityl chloride (DMTrCl, 0.569 g, 1.68 mmol) was
3
NaN (6.59 g, 12.1 mmol) was added to a solution of 7b (8.82 g,
added to a solution of 14a (0.365 g, 1.12 mmol) in pyridine (4.0 mL)
under argon atmosphere and the mixture was stirred for 20 h at room
12.1 mmol) in DMF (90 mL) under argon atmosphere and the mixture
was stirred overnight at 60 °C. The mixture was extracted with brine
and EtOAc. The organic layer was washed with brine, dried over
temperature. The mixture was extracted with H
ganic layer was washed with saturated NaHCO
dried over Na SO and concentrated in vacuo. The residue was purified
by chromatography on silica gel (hexane/EtOAc = 1:1) to give 15a as a
2
O and EtOAc. The or-
3
solution and brine,
Na
matography on silica gel (hexane/EtOAc = 15:1) to give 19 as a col-
orless oil (5.98 g, 9.94 mmol, 82%). 1H NMR (400 MHz, CDCl
) δ 0.98
2 4
SO and concentrated in vacuo. The residue was purified by chro-
2
4
3
1
white solid (0.679 g, 1.08 mmol, 96%). H NMR (400 MHz, CDCl
1
3
3
) δ
.74–1.81 (m, 1H), 2.08–2.11 (m, 1H), 2.87 (d, J = 6.4 Hz, 1H),
.18–3.23 (m, 1H), 3.26–3.30 (m, 1H), 3.35 (s, 2H), 3.58 (s, 3H), 3.80
(s, 9H), 1.36 (s, 3H), 1.35–1.41 (m, 1H), 1.55–1.57 (m, 1H), 1.62 (s,
3H), 1.73–1.78 (m, 1H), 2.09–2.13 (m, 1H), 3.18–3.23 (m, 2H), 3.41 (d,
J = 11.0 Hz), 3.65 (d, J = 11.0 Hz, 1H), 4.30 (d, J = 5.5 Hz, 1H), 4.59
(d, J = 12.4 Hz, 1H), 4.67 (dd, J = 5.5 Hz and 3.6 Hz, 1H), 4.82 (d,
J = 12.4 Hz, 1H), 5.79 (d, J = 3.7 Hz, 1H), 7.30–7.46 (m, 10H),
(
(
s, 6H), 3.93 (dd, J = 6.0 Hz and 3.6 Hz, 1H), 4.10–4.15 (m, 1H), 4.61
t, J = 6.4 Hz, 1H), 5.22 (d, J = 8.2 Hz, 1H), 6.03 (d, J = 3.6 Hz, 1H),
6
(
4
1
1
6
.85 (d, J = 9.2 Hz, 4H), 7.24–7.25 (m, 2H), 7.26–7.35 (m, 7H), 7.81
7.59–7.64 (m, 5H). 13C NMR (101 MHz, CDCl
29.0, 52.2, 66.5, 72.6, 78.1, 79.5, 87.5, 104.3, 113.3, 127.8, 127.9,
3
) δ 19.3, 23.3, 26.3, 26.9,
d, J = 8.2 Hz, 1H), 8.50 (s, 1H). 13C NMR (151 MHz, CDCl
) δ 31.2,
3
6.6, 55.4, 59.5, 65.4, 70.5, 84.5, 87.0, 87.2, 87.7, 102.7, 113.5, 123.4,
128.6, 129.8, 129.9, 133.0, 133.3, 135.7, 135.8, 138.1. HRMS (ESI) m/
43 3 5
H N O
SiNa [M+Na]+ 624.2870, found 624.2899.
28.2, 128.3, 130.3, 130.4, 134.8, 135.0, 140.3, 144.2, 150.3, 158.9,
z Calcd for C34
35 5 8
58.9, 163.2. HRMS (ESI-TOF) m/z Calcd for C33H N NaO
[M+Na]+
52.2383, Found 652.2379.
4.16. 5-O-[(1,1-Dimethylethyl)diphenylsilyl]-4-C-azidopropyl-3-O-benzyl-
1,2-di-O-acetyl-α-D-ribofuranose (9b)
4.13. 5′-O-[Bis(4-methoxyphenyl)phenylmethyl]-4′-C-
A solution of 19 (0.40 g, 0.665 mmol) in 50% AcOH (5.70 mL) was
trifluoroacetylaminoethyl-2′-O-methyluridine(17a)
warmed to 120 °C and stirred for 1 h. The mixture was concentrated in
vacuo and dried by co-evaporation with EtOH. The residue was dis-
Triphenylphosphine (PPh , 2.46 g, 9.38 mmol) and H
3
2
O (2.70 mL)
solved in pyridine (1.43 mL) and Ac
under argon atmosphere. The mixture was stirred overnight at room
temperature. The mixture was poured into H O in an ice-bath and ex-
tracted with EtOAc. The organic layer was washed with saturated
NaHCO solution and brine, dried over Na SO and concentrated in
vacuo. The residue was purified by chromatography on silica gel
(hexane/EtOAc = 5:1) to give 9b as colorless oil (0.314 g,
0.486 mmol, 74%). 1H NMR (400 MHz, CDCl
1.06 (s, 9H),
2
O (0.95 mL, 10.2 mmol) was added
was added to a solution of 15a (2.36 g, 3.75 mmol) in THF (100 mL)
under argon atmosphere and the mixture was stirred for 18 h at 45 °C.
The mixture was concentrated in vacuo. The residue was purified by
2
chromatography on silica gel (CHCl
6a. CF CO Et (1.25 mL, 10.5 mmol) and Et
were added to a solution of 16a in CH Cl (20 mL) and the mixture was
stirred overnight at room temperature. The mixture was extracted with
O and EtOAc. The organic layer was washed with brine, dried over
Na SO and concentrated in vacuo. The residue was purified by chro-
matography on silica gel (hexane/EtOAc = 2:3) to give 17a as a white
3
/MeOH = 30:1) to give compound
3
2
4
1
3
2
3
N (0.728 mL, 5.25 mmol)
2
2
a
3
) δ
H
2
1.10–1.14 (m, 1H), 1.54–1.58 (m, 1H), 1.74–1.77 (m, 1H), 1.82 (s, 3H),
1.84–1.90 (m, 1H), 2.10 (s, 3H), 3.19–3.23 (m, 2H), 3.59 (dd,
J = 10.6 Hz and 13.3 Hz, 2H), 4.38 (d, J = 5.5 Hz, 1H), 4.54 (d,
J = 11.4 Hz, 1H), 4.60 (d, J = 11.4 Hz, 1H), 5.36 (d, J = 5.5 Hz, 1H),
2
4
1
solid (2.44 g, 3.49 mmol, 93%). H NMR (600 MHz, CDCl
3
) δ 1.92–1.94
(
m, 1H), 2.00–2.05 (m, 1H), 3.09 (s, 1H), 3.27 (s, 1H), 3.31–3.40 (m,
6.13 (s, 1H), 7.27–7.64 (m, 15H). 13C NMR (101 MHz, CDCl
3
) δ 19.5,
3
5
4
H), 3.53 (s, 3H), 3.80 (s, 6H), 4.03 (t, J = 4.8 Hz, 1H), 4.48 (s, 1H),
.30 (d, J = 8.2 Hz, 1H), 6.06 (d, J = 4.8 Hz, 1H), 6.85 (d, J = 8.9 Hz,
H), 7.13 (s, 1H), 7.23–7.25 (m, 4H), 7.29–7.35 (m, 5H), 7.69 (d,
20.9, 22.9, 27.1, 29.5, 52.1, 67.4, 73.6, 74.9, 79.3, 88.1, 97.9, 127.6,
127.7, 127.9, 128.0, 130.1, 132.9, 133.2, 135.7, 135.8, 169.5, 169.9.
43 3 7
HRMS (ESI) m/z Calcd for C35H N O
SiNa [M+Na]+ 668.2768, found
1
3
J = 8.3 Hz, 1H), 9.02 (s, 1H). C NMR (151 MHz, CDCl
3
) δ 31.0, 35.5,
5.4, 59.4, 65.9, 70.9, 83.9, 86.6, 87.7, 87.9, 103.0, 113.5, 127.6,
28.2, 128.3, 130.2, 130.3, 134.6, 134.8, 140.1, 144.0, 150.4, 159.0,
668.2747.
5
1
1
7
4.17. 5′-O-[(1,1-Dimethylethyl)diphenylsilyl]-4′-C-azidoepropyl-3′-O-
benzyl-2′-O-acetyluridine (10b)
36 3 3 9
63.0. HRMS (ESI-TOF) m/z Calcd for C35H F N O
[M+Na]+
22.2301, Found 722.2315.
Uracil (0.957 g, 8.54 mmol) and BSA (11.1 mL, 34.2 mmol) was
added to a solution of 9b (2.76 g, 4.27 mmol) in MeCN (28 mL) under
argon atmosphere and the mixture was stirred 1 h at 95 °C. The mixture
was cooled to room temperature, TMSOTf (1.55 mL, 8.54 mmol) was
added dropwise to the mixture at 0 °C and the mixture was stirred for
4
.14. 5′-O-[Bis(4-methoxyphenyl)phenylmethyl]-4′-C-
trifluoroacetylaminoethyl-2′-O-methyl-3′-[2-cyanoethyl-N,N-bis(1-
methylethyl)phosphoramidite]uridine (18a)
1
5 min at 95 °C. Saturated NaHCO
at 0 °C. The mixture was extracted with saturated NaHCO
CHCl . The organic layer was washed with saturated NaHCO
dried over Na SO and concentrated in vacuo. The residue was purified
by chromatography on silica gel (hexane/EtOAc = 2:1) to give 10b as a
3
solution was added to the mixture
solution and
solution,
Chloro(2-cyanoethoxy)(N,N-diisopropylamino)phosphine (CEP-Cl,
3
0
1
1
.638 mL, 2.86 mmol) and N,N-diisopropylethylamine (DIPEA.
.25 mL, 7.15 mmol) were added to solution of 17a (1.00 g,
.43 mmol) in THF (10 mL) under argon atmosphere and the mixture
3
3
a
2
4
1
was stirred for 1.5 h at room temperature. The mixture was extracted
with saturated NaHCO solution and CHCl . The organic layer was
washed with brine, dried over Na SO and concentrated in vacuo. The
3
white solid (2.27 g, 3.26 mmol, 76%). H NMR (400 MHz, CDCl ) δ 1.09
3
3
(s, 9H), 1.45–1.51 (m, 2H), 1.64–1.69 (m, 1H), 1.82–1.89 (m, 1H), 2.11
(s, 3H), 3.18–3.24 (m, 2H), 3.56 (d, J = 11.4 Hz, 1H), 3.84 (d,
J = 10.9 Hz, 1H), 4.39–4.44 (m, 2H), 4.61 (d, J = 11.0 Hz, 1H),
2
4
residue was purified by chromatography on silica gel (hexane/
EtOAc = 1:1) to give 18a as a white solid (0.884 g, 0.983 mmol, 69%).
5.32–5.48 (m, 2H), 6.18 (d, J = 5.0 Hz, 1H), 7.28–7.57 (m, 15H), 7.67
(d, J = 8.2 Hz, 1H), 8.66 (s, 1H). C NMR (101 MHz, CDCl ) δ 19.5,
3
20.9, 23.0, 23.1, 27.0, 27.2, 29.2, 51.9, 66.5, 74.6, 75.1, 77.8, 86.3,
3
1
13
P NMR (162 MHz, CDCl
3
) δ 150.94, 151.53. HRMS (ESI-TOF) m/z
+
Calcd for C44 10P [M+Na] 922.3380, Found 922.3371.
H
53
F N
3 5
O
10

K. Koizumi et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
8
1
8.2, 103.0, 128.0, 128.1, 128.2, 128.3, 128.7, 130.3, 130.5, 132.0,
132.8, 135.4, 135.7, 140.0, 150.4, 163.1. HRMS (ESI) m/z Calcd for
29 37 5 6
C H N O
SiNa [M+Na]+ 602.2411, found 602.2396.
32.7, 135.5, 135.7, 135.78, 137.3, 139.8, 150.3, 162.8, 170.2. HRMS
+
(
ESI) m/z Calcd for C37
H
43
N
5
O
7
SiNa [M+Na] 720.2829, found
720.2848.
4.21. 4′-C-Azidopropyl-2′-O-methyluridine (14b)
4
.18. 5′-O-[(1,1-Dimethylethyl)diphenylsilyl]-4′-C-azidopropyl-3′-O-
1
M of TBAF solution in THF (7.74 mL, 7.74 mmol) was added to a
benzyluridine (11b)
solution of 13b (2.99 g, 5.16 mmol) in THF (30 mL) under argon at-
mosphere and the mixture was stirred overnight at room temperature.
The mixture was concentrated in vacuo. The residue was purified by
chromatography on silica gel (CHCl
white solid (1.68 g, 4.94 mmol, 96%). H NMR (600 MHz, DMSO‑d
1.55–1.67 (m, 4H), 3.27–3.29 (m, 2H), 3.32 (s, 3H), 3.42–3.45 (m, 2H),
.98 (dd, J = 7.6 Hz and 4.8 Hz, 1H), 4.16 (t, J = 5.5 Hz, 1H), 5.13 (d,
J = 6.2 Hz, 1H), 5.20 (d, J = 5.5 Hz, 1H), 5.67 (d, J = 8.3 Hz, 1H), 5.90
NH
3
solution (16 mL) was added to a solution of 10b (1.57 g,
2
.24 mmol) in MeOH (16 mL) and the mixture was stirred overnight at
3
/MeOH = 10:1) to give 14b as a
room temperature. The mixture was concentrated in vacuo. The residue
was purified by chromatography on silica gel (hexane/EtOAc = 2:1) to
give 11b as a white solid (1.44 g, 2.19 mmol, 98%). H NMR (400 MHz,
CDCl
1
6
) δ
1
3
3
) δ 1.09 (s, 9H), 1.37–1.41 (m, 1H), 1.51–1.55 (m, 1H), 1.61–1.68
(
m, 1H), 1.85–1.89 (m, 1H), 3.13–3.19 (m, 2H), 3.55 (d, J = 11.0 Hz,
1
3
(
(
8
d, J = 6.8 Hz, 1H), 7.89 (d, J = 8.2 Hz, 1H), 11.34 (s, 1H). C NMR
101 MHz, DMSO‑d ) δ 22.8, 29.1, 51.3, 57.3, 64.2, 69.6, 82.5, 84.7,
8.0, 102.3, 140.7,+150.7, 163.0. HRMS (ESI) m/z Calcd for
K [M+K] 380.0972, found 380.0976.
1
H), 3.60 (d, J = 7.80 Hz, 1H), 3.78 (d, J = 11.0 Hz, 1H), 4.19 (d,
6
J = 6.0 Hz, 1H), 4.29 (dd, J = 6.0 Hz and 12.4 Hz, 1H), 4.59 (d,
J = 11.4 Hz, 1H), 4.74 (d, J = 11.5 Hz, 1H), 5.39 (d, J = 8.2 Hz, 1H),
13 19 5 6
C H N O
5
9
6
1
1
6
.94 (d, J = 5.5 Hz, 1H), 7.34–7.60 (m, 15H), 7.69 (d, J = 7.8 Hz, 1H),
1
3
.37 (s, 1H). C NMR (101 MHz, CDCl ) δ 19.4, 23.0, 27.2, 29.4, 51.9,
3
6.8, 74.7, 75.2, 78.8, 87.9, 88.9, 102.8, 128.1, 128.2, 128.3, 128.5,
28.8, 130.3, 130.4, 132.1, 132.6, 135.5, 135.7, 137.1, 139.9, 151.0,
63.2. HRMS (ESI) m/z Calcd for
4
.22. 5′-O-[Bis(4-methoxyphenyl)phenylmethyl]-4′-C-azidopropyl-2′-O-
methyluridine (15b)
35 41 5 6
C H N O
SiNa [M+Na]+
78.2724, found 678.2703.
DMTrCl (2.51 g, 7.41 mmol) was added to a solution of 14b (1.68 g,
4
.94 mmol) in pyridine (17 mL) under argon atmosphere and the mix-
ture was stirred for 5 h at room temperature. The mixture was extracted
with H O and EtOAc. The organic layer was washed with saturated
NaHCO solution and brine, dried over Na SO and concentrated in
vacuo. The residue was purified by chromatography on silica gel
hexane/EtOAc = 2:1) to give 15b as a white solid (3.12 g, 4.85 mmol,
4.19. 5′-O-[(1,1-Dimethylethyl)diphenylsilyl]-4′-C-azidopropyl-3′-O-
benzyl-2′-O-methyluridine (12b)
2
3
2
4
NaH (60% in mineral, 0.972 g, 24.3 mmol) was added to a solution
of 11b (5.33 g, 8.10 mmol) in THF (53 mL) at 0 °C under argon atmo-
sphere and the mixture was stirred 10 min at 0 °C. Then MeI (3.02 mL,
(
8%). 1H NMR (400 MHz, CDCl
) δ 1.38–1.44 (m, 1H), 1.54–1.64 (m,
9
2
3
5
3
4
8.6 mmol) was added dropwise to the mixture and stirred 8 h at 0 °C.
Saturated NaHCO solution was added to the mixture at 0 °C. The
mixture was extracted with saturated NaHCO solution and EtOAc. The
organic layer was washed with saturated NaHCO solution and brine,
dried over Na SO and concentrated in vacuo. The residue was purified
by chromatography on silica gel (hexane/EtOAc = 3:1) to give 12b as a
H), 1.78–1.84 (m, 1H), 2.77 (d, J = 6.4 Hz, 1H), 3.17–3.22 (m, 2H),
.34 (s, 2H), 3.57 (s, 3H), 3.80 (s, 6H), 3.92 (dd, J = 4.1 Hz and
.96 Hz, 1H), 4.60 (t, J = 6.0 Hz, 1H), 5.22 (d, J = 8.2 Hz, 1H), 6.02 (d,
3
3
3
J = 4.1 Hz, 1H), 6.84–7.36 (m, 13H), 7.82 (d, J = 8.2 Hz, 1H), 8.09 (s,
2
4
13
1
6
1
1
3
H). C NMR (101 MHz, CDCl ) δ 14.3, 23.0, 29.3, 51.8, 55.4, 59.5,
0.5, 65.4, 70.6, 84.7, 86.8, 87.6, 88.0, 102.7, 113.5, 127.4, 128.2,
28.3, 130.3, 130.4, 134.9, 135.2, 140.3, 144.3, 150.3, 158.9, 159.0,
63.10. HRMS (ESI) m/z Calcd for C34
1
white solid (4.00 g, 5.98 mmol, 74%). H NMR (400 MHz, CDCl
3
) δ 1.08
(
s, 9H), 1.39–1.46 (m, 1H), 1.52–1.60 (m, 1H), 1.65–1.70 (m, 1H),
+
H
37
N
5
O
8
Na [M+Na] 666.2540,
1.97–2.05 (m, 1H), 3.19–3.26 (m, 2H), 3.51 (s, 3H), 3.68 (d,
found 666.2527.
J = 11.5 Hz, 1H), 3.74 (m, 1H), 3.95 (d, J = 11.5 Hz, 1H), 4.35 (d,
J = 4.6 Hz, 1H), 4.52 (d, J = 11.5 Hz, 1H), 4.73 (d, J = 11.9 Hz, 1H),
5
.12 (d, J = 8.2 Hz, 1H), 6.09 (s, 1H), 7.35–7.63 (m, 15H), 7.78 (d,
4.23. 5′-O-[Bis(4-methoxyphenyl)phenylmethyl]-4′-C-
trifluoroacetylaminopropyl-2′-O-methyluridine (17b)
1
3
J = 8.2 Hz, 1H), 9.04 (s, 1H). C NMR (101 MHz, CDCl
3
) δ 19.6, 22.8,
7.3, 29.0, 51.8, 59.4, 65.4, 73.5, 75.9, 84.2, 87.9, 88.1, 102.6, 128.0,
28.1, 128.2, 128.3, 128.7, 130.3, 130.4, 132.1, 133.0, 135.4, 135.6,
2
1
1
3 2
PPh (3.18 g, 12.1 mmol) and H O (3.50 mL) was added to a solu-
tion of 15b (3.12 g, 4.85 mmol) in THF (62.4 mL) under argon atmo-
sphere and the mixture was stirred for 20 h at 45 °C. The mixture was
37.5, 140.0, 150.1, 163.2. HRMS (ESI) m/z Calcd for C36
H
43
N
5
O
6
SiNa
+
[
M+Na] 692.2880, found 692.2897.
concentrated in vacuo to give crude 16b. CF
14.5 mmol) and Et N (1.00 mL, 7.28 mmol) were added to a solution of
16b in CH Cl (30 mL) and the mixture was stirred 24 h at room tem-
perature. The mixture was extracted with H O and EtOAc. The organic
layer was washed with brine, dried over Na SO and concentrated in
3 2
CO Et (1.74 mL,
4
.20. 5′-O-[(1,1-Dimethylethyl)diphenylsilyl]-4′-C-azidopropyl-2′-O-
3
methyluridine (13b)
2
2
2
1
M of BCl solution in CH
3
2
Cl
2
(35.9 mL, 35.9 mmol) was added to a
2
4
solution of 12b (4.00 g, 5.98 mmol) in CH
argon atmosphere and the mixture was stirred for 3 h at −78 °C, then
warmed to −30 °C and stirred 5 h. A solution of MeOH/CH Cl (1:1, v/
2
Cl
2
(60 mL) at −78 °C under
vacuo. The residue was purified by chromatography on silica gel
(hexane/EtOAc = 2:3) to give 17b as a white solid (quant.). H NMR
(400 MHz, CDCl ) δ 1.40–1.45 (m, 1H), 1.51–1.54 (m, 1H), 1.63–1.67
3
(m, 1H), 1.72–1.78 (m, 1H), 2.88 (d, J = 4.6 Hz, 1H), 3.25–3.28 (m,
2H), 3.30 (d, J = 10.5 Hz, 1H), 3.35 (d, J = 10.1 Hz, 1H), 3.54 (s, 3H),
3.80 (s, 6H), 4.03 (t, J = 5.0 Hz, 1H), 4.54 (t, J = 5.0 Hz, 1H), 5.26 (d,
J = 8.2 Hz, 1H), 6.03 (d, J = 5.0 Hz, 1H), 6.66 (m, 1H), 6.84–7.55 (m,
13H), 7.74 (d, J = 8.2 Hz, 1H), 8.17 (s, 1H). 13C NMR (101 MHz,
1
2
2
v) was added to the mixture. The mixture was concentrated in vacuo.
The residue was purified by chromatography on silica gel (hexane/
1
EtOAc = 1:1) to give 13b as a white solid (2.99 g, 5.16 mmol, 86%). H
NMR (600 MHz, CDCl
3
) δ 1.11 (s, 9H), 1.49–1.56 (m, 2H), 1.58–1.64
(
m, 1H), 1.76–1.80 (m, 1H), 2.74 (d, J = 5.5 Hz, 1H), 3.21–3.25 (m,
2
3
H), 3.52 (s, 3H), 3.70 (d, J = 11.0 Hz, 1H), 3.88 (d, J = 11.0 Hz, 1H),
.93 (m, 1H), 4.49 (t, J = 5.5 Hz, 1H), 5.31 (d, J = 8.3 Hz, 1H), 6.06 (d,
3
CDCl ) δ 14.3, 22.8, 29.4, 40.2, 55.4, 59.3, 60.5, 65.9, 70.7, 84.3, 86.4,
87.7, 87.9, 102.8, 113.5, 127.5, 128.2, 128.3, 130.2, 130.3, 132.1,
133.1, 134.8, 135.0, 140.2, 144.1, 150.4, 158.9, 159.0, 163.0. HRMS
J = 4.8 Hz, 1H), 7.41–7.66 (m, 10H), 7.08 (d, J = 7.6 Hz, 1H), 8.04 (s,
1
3
36 38 3 3 9
(ESI) m/z Calcd for C H F N O K
[M+K]+ 752.2197, found
752.2207.
1H). C NMR (101 MHz, CDCl
3
) δ 19.5, 23.1, 27.2, 29.0, 51.9, 59.4,
6
6.8, 70.1, 84.6, 86.5, 88.5, 102.9, 128.2, 128.3, 130.4, 130.5, 132.0,
11

K. Koizumi et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
−
4.24. 5′-O-[Bis(4-methoxyphenyl)phenylmethyl]-4′-C-
for C103
(calcd for
z = 3582.40 (calcd for C109
1 m/z = 3583.38 (calcd for C109
RNA 12 m/z = 3622.50 (calcd for
H
134
N
26
O
H
83
P
10 [M−H] , 3374.03); RNA 9 m/z = 3606.81
−
trifluoroaminopropyl-2′-O-methyl-3′-[2-cyanoethyl-N,N-bis(1-methylethyl)
phosphoramidite]uridine (18b)
C
110
133
N
55
O
67
P
H
10 [M−H]
,
3607.30); RNA 10 m/
−
132
N
53
O
68
P
10 [M−H] , 3581.60); RNA
O
−
1
H
131
N
52
69
P
10 [M−H] , 3582.58);
−
CEP-Cl (0.97 mL, 4.34 mmol) and DIPEA (1.90 mL, 10.9 mmol) were
added to a solution of 17b (1.55 g, 2.17 mmol) in THF (15 mL) under
argon atmosphere and the mixture was stirred for 1.5 h at room tem-
C
110
H
132
N
55
O
68
P
10 [M−H]
,
3621.60); RNA 13 m/z = 6506.12 (calcd for C194
H
245
N
65
O
150
P
20
−
[M−H]
C
,
6505.84); RNA 14 m/z = 7353.56 (calcd for
−
perature. The mixture was extracted with saturated NaHCO
and CHCl . The organic layer was washed with brine, dried over
Na SO and concentrated in vacuo. The residue was purified by chro-
matography on silica gel (hexane/EtOAc = 1:1) to give 18b as a white
3
solution
230
H
272
N
87
O
153
P
H
21 [M−H]
,
7354.57)−; RNA 15 m/z = 6851.94
3
(calcd for
z = 7569.94 (calcd for C240
17 m/z = 6963.35 (calcd for C218
C
210
285
N
73
O
150
P
20 [M−H] 6851.44); RNA 16 m/
,
−
2
4
H
297
N
H
92
O
153
P
21 [M−H] , 7569.94); RNA
−
301
N
73
O
150
P
20 [M−H] , 6962.62);
3
1
−
solid (1.57 g, 1.71 mmol, 79%). P NMR (243 MHz, CDCl
3
) δ 150.57,
RNA 18 m/z = 7638.62 (calcd for C245
H
307
N
92
O
153
P
21 [M−H]
,
151.44. HRMS (ESI) m/z Calcd for
C
45
H
55
3
F N
5
O
10PK [M+K]+
7639.06); RNA 19 m/z = 7074.20 (calcd for C226
H
317
N
73
O
150
P
20
−
952.3276, found 952.3286.
[M−H]
C
,
7075.83); RNA 20 m/z = 7709.50 (calcd for
−
250
H
317
N
92
O
153
P
H
21 [M−H]
,
7710.19)−; RNA 21 m/z = 6814.82
4.25. Synthesis of the controlled pore glass support (21a) or (21b)
(calcd for
z = 6520.22 (calcd for C195
23 m/z = 6549.02 (calcd for C196
C
203
248
N
86
O
144
P20 [M−H] 6816.11); RNA 22 m/
,
−
H
247
N
H
65
O
150
P
20 [M−H] , 6520.87); RNA
−
N,N-Dimethyl-4-aminopyridine (DMAP, 26.4 mg, 0.216 mmol) and
250
N
66
O
150
P
20 [M−H] , 6549.92);
−
succinic anhydride (43.2 mg, 0.432 mmol) were added to a solution of
7a (74.5 mg, 0.108 mmol) in pyridine (1.0 mL) under argon atmo-
sphere and the mixture was stirred for 27 h at room temperature. The
mixture was extracted with H O and EtOAc. The organic layer was
washed with sat. NaHCO solution, brine, dried over Na SO and con-
centrated in vacuo. The residue was purified by chromatography on
silica gel (CHCl /MeOH = 15:1) to give 20a as white solid.
Aminopropyl controlled pore glass (171 mg, CPG) and 1-ethyl-3-[3-
dimethylamino)propyl]carbodiimide hydrochloride (EDC, 21.9 mg,
.114 mmol) were added to a solution of 20a, and the mixture was kept
at room temperature for 5 days. After the resin was washed with pyr-
idine, 15 mL of capping solution (0.1 M DMAP in pyridine: Ac O 9:1)
RNA 24 m/z = 6560.70 (calcd for C197
H
252
N
66
O
150
P
20 [M−H]
,
1
6563.94); RNA 25 m/z = 6577.41 (calcd for C198
H
254
N
66
O
150
P
20
−
[M−H]
C
,
6577.97); RNA 26 m/z = 6521.16 (calcd for
−
2
195
H
247
N
65
O
150
P
H
20 [M−H]
,
6520.87)−; RNA 27 m/z = 6548.98
3
2
4
(calcd for
z = 6562.83 (calcd for C197
29 m/z = 6577.88 (calcd for C198
C
196
250
N
66
O
150
P20 [M−H] 6549.92); RNA 28 m/
,
−
H
252
N
H
66
O
150
P
20 [M−H] , 6562.93); RNA
−
3
a
254
N
66
O
150
P
20 [M−H] , 6577.97);
−
RNA 30 m/z = 6520.52 (calcd for C195
H
247
N
65
O
150
P
20 [M−H]
,
(
0
6520.87); RNA 31 m/z = 6547.96 (calcd for C196
H
250
N
66
O
150
P
20
−
[M−H]
C
,
6549.92); RNA 32 m/z = 6560.41 (calcd for
−
197
H
252
N
66
O
150
P
H
20 [M−H]
,
6563.94)−; RNA 33 m/z = 6578.14
2
(calcd for
z = 6519.83 (calcd for C195
35 m/z = 6549.74 (calcd for C196
C
198
254
N
66
O
150
P20 [M−H] 6577.97); RNA 34 m/
,
−
were added to the resin and the mixture was kept at room temperature
for 2 days. The resin was washed with EtOH and acetone and was dried
under vacuum to give solid support 21a. The amount loaded nucleoside
H
247
N
H
65
O
150
P
20 [M−H] , 6520.87); RNA
−
250
N
66
O
150
P
20 [M−H] , 6549.92);
−
RNA 36 m/z = 6562.89 (calcd for C197
H
252
N
66
O
150
P
20 [M−H]
,
2
0a to the solid support is 44.1 µmol/g from calculation of released
dimethoxytrityl cation by a solution of 70% HClO :EtOH (3:2, v/v). In a
similar manner, the solid supports 21b with 2.b were obtained in
6562.93); RNA 37 m/z = 6577.59 (calcd for C198
H
254
N
66
O
150
P
20
−
4
[M−H]
,
N
6577.97); RNA 38 m/z = 6520.51 (calcd for
−
C
195
H
247
65
O
150
P
H
20 [M−H]
,
6520.87)−; RNA 39 m/z = 6549.27
35.8 µmol/g of loading amounts.
(calcd for
z = 6560.44 (calcd for C197
41 m/z = 6577.96 (calcd for C198
C
196
250
N
66
O
150
P
20 [M−H] 6549.92); RNA 40 m/
,
−
H
252
N
66
O
150
P
20 [M−H] , 6563.94); RNA
−
4.26. Oligomer synthesis
H
254
N
66
O
150
P20 [M−H] , 6577.97);
−
RNA 42 m/z = 6520.23 (calcd for C195
H
247
N
65
O
150
P
20 [M−H]
,
The synthesis was carried out with DNA/RNA synthesizer by the
6520.87); RNA 43 m/z = 6548.33 (calcd for C196
H
250
N
66
O
150
P
20
−
phosphoramidite method. After the synthesis, the CPG beads were
treated with 10% dimethylamine in MeCN for 5 min followed by rinse
with MeCN to selectively remove cyanoethyl groups. Then, the oligo-
mers were cleaved from CPG beads and deprotected by treated with
[M−H]
,
N
6549.92); RNA 44 m/z = 6562.89 (calcd for
−
C
197
H
252
66
O
150
P
H
20 [M−H]
,
6562.93)−; RNA 45 m/z = 6577.47
(calcd for
z = 6635.92 (calcd for C200
47 m/z = 6678.36 (calcd for C203
C
198
254
N
66
O
150
P
20 [M−H] 6577.97); RNA 46 m/
,
−
H
260
N
68
O
150
P
20 [M−H] , 6636.07); RNA
−
concentrated NH
5 °C. 2′-O-TBDMS groups were removed by Et
3
solution/40% methylamine (1:1, v/v) for 15 min at
N·3HF in DMSO at 65 °C
H
266
N
68
O
150
P20 [M−H] , 6678.14);
−
6
3
RNA 48 m/z = 6720.48 (calcd for C206
H
272
N
68
O
150
P
20 [M−H]
,
for 1.5 h. The reaction quenched with 0.1 M TEAA buffer (pH 7.0) and
the mixture desalted using Sep-Pak C18 cartridge. The oligonucleotides
were purified by 20% PAGE containing 7 M urea to give highly purified
ONs.
6720.22); RNA 49 m/z = 6636.11 (calcd for C200
H
260
N
68
O
150
P
20
−
[M−H]
,
N
6636.07); RNA 50 m/z = 6678.36 (calcd for
−
C
203
H
266
68
O
150
P
H
20 [M−H]
,
6678.14)−; RNA 51 m/z = 6721.18
(calcd for
z = 6635.31 (calcd for C200
53 m/z = 6678.83 (calcd for C203
C
206
272
N
68
O
150
P
20 [M−H] 6720.22); RNA 52 m/
,
−
H
260
N
68
O
150
P
20 [M−H] , 6636.07); RNA
−
4.27. MALDI-TOF/MS analysis of ONs
H
266
N
68
O
150
P20 [M−H] , 6678.14);
−
RNA 54 m/z = 6719.67 (calcd for C206
H
272
N
68
O
150
P
20 [M−H]
,
The spectra were obtained with a time-of-flight mass spectrometer
6720.22); RNA 55 m/z = 6859.23 (calcd for C205
H
253
N
87
O
144
P
20
−
equipped with nitrogen laser (337 nm, 3 ns pulse). A solution of 3-hy-
droxypicolinic acid (3-HPA) and diammonium hydrogen citrate in H
was used as the matrix. Data of synthetic ONs: RNA 1 m/z = 4174.4
[M−H]
,
N
6859.18); RNA 56 m/z = 6873.75 (calcd for
−
2
O
C
206
H
255
87
O
144
P
H
20 [M−H]
,
6873.21)−; RNA 57 m/z = 6887.80
(calcd for
z = 6859.09 (calcd for C205
59 m/z = 6872.19 (calcd for C206
C
207
257
N
87
O
144
P
20 [M−H] 6887.23); RNA 58 m/
,
−
−
(
calcd for
z = 4304.30 (calcd for C144
m/z = 4345.86 (calcd for C147
C
138
H
168
N
28
O
99
P
12 [M−H]
,
4175.63); RNA
2
m/
H
253
N
87
O
144
P
20 [M−H] , 6859.18); RNA
−
−
H
183
N
31
O
N
H
99
31
195
P
O
N
12 [M−H] , 4304.86); RNA 3
H
255
N
87
O
144
P20 [M−H] , 6873.21);
−
−
H
189
99
31
99
P
O
12 [M−H] , 4346.93); RNA
RNA 60 m/z = 6887.74 (calcd for C207
H
257
N
87
O
144
P
20 [M−H]
N
,
−
4
m/z = 4388.00 (calcd for C150
99
P
12 [M−H] , 4389.01);
6887.23); RNA 61 m/z = 6859.28 (calcd for C205
H
253
87
O
144
P
20
−
−
RNA
5
m/z = 3301.99 (calcd for
m/z = 3345.31 (calcd for
3345.98); RNA m/z = 3359.41
10 [M−H] , 3360.01); RNA 8 m/z = 3373.47 (calcd
C
H
125
N
25
O
83
P10 [M−H]
,
[M−H]
,
6859.18); RNA 62 m/z = 6872.97 (calcd for
−
3
[
C
302.91); RNA
6
C
101
H
130
N
26
O
83
P
10
C
206
H
255
N
87
O
144
P
20 [M−H]
, 6873.21); RNA 63 m/z = 6887.55
−
−
M−H]
,
7
(calcd
for
(calcd for C207
257 87 144
H N O P
20 [M−H] , 6887.23).
−
102
H
132
N
26
O
83
P
12

K. Koizumi et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
4
.28. Thermal denaturation study
expressing both Renilla and firefly luciferases were grown in Dulbecco's
Modified Eagle Medium (d-MEM) supplemented with 10% bovine
serum (BS) and 0.25 mg/mL hygromycin at 37 °C. HeLa-psiCHECK-2
The solution containing 3.0 µM duplex in a buffer of 10 mM sodium
4
phosphate (pH 7.0) containing 100 mM NaCl was heated at 100 °C, then
cooled gradually to room temperature and used for the thermal dena-
turation study. Thermally induced transitions were monitored at
cells (8.0 × 10 /mL) were cultured on a 96-well plate (100 µL/well) for
24 h and transfected with siRNA targeting the Renilla luciferase gene
using lipofectamine RNAi max in Opti-MEM I reduced serum medium.
Transfection without siRNA was used as a control. After 1 h, d-MEM (50
µL) containing 10% BS was added to each well and cells was further
incubated for another 24 h. The activities of Renilla and firefly luci-
ferases in the cells were determined with Dual-Luciferase Reporter
Assay System (Promega) according to a manufacture’s protocol. The
activity of Renilla luciferase was normalized by the firefly luciferase
activity. The results were confirmed by at least three independent
transfection experiments with two cultures each and are expressed as
the average from four experiments as mean ± SD.
2
60 nm with a UV/Vis spectrometer fitted with temperature controller
in quartz cuvettes with a path length of 1.0 cm. The sample temperature
was increased by 0.5 °C/min. The thermodynamic parameters of the
duplexes on duplex formation were determined by calculations based
on the slope of a 1/T
1
m
vs. ln (C
T T
/4) plot, where C (1, 3, 6, 12, 15, 21,
6, 30, and 60 µM) is the total concentration of single strands.
4.29. CD spectroscopy
All CD spectra were recorded at 25 °C. The following instrument
settings were used: resolution, 0.1 nm; response, 1.0 s; speed, 50 nm/
min; accumulation, 10.
4.33. Stability in serum
Fluorescein labeled ONs and siRNA (600 pmol) were dissolved in
10 µL buffer of 10 mM sodium phosphate (pH 7.0) containing 100 mM
NaCl and the solutions were heated at 100 °C, then cooled gradually to
room temperature and used for the serum stability test. 90 µL bovine
serum was added and the mixture was incubated at 37 °C for the re-
quired time. Aliquots of 10 μL were diluted with a stop solution (65 mM
EDTA 12% glycerol 8.0 μL). Samples were subjected to electrophoresis
in 15% polyacrylamide-TBE under non-denaturing conditions and
quantified by Luminescent Image analyzer LAS-4000 (Fujifilm).
4
.30. NMR measurements
The ON7 was dissolved in 10 mM sodium phosphate (pH 7.0) con-
taining 100 mM NaCl. The final concentration of the RNA was 0.5 mM.
NMR spectra were measured using Bruker AVANCE 600 spectrometer.
Spectra were recorded at probe temperature of 45 °C. Non-exchange-
able proton resonances were assigned by TOCSY (mixing time of 50 ms)
and NOESY (mixing times of 100 and 400 ms) in D
.31. Molecular modeling study
The initial structure of uridine and three 4′-C-amino-alkyl-2′-O-
2
O.
4
Acknowledgement
This work was supported by the Japan Agency for Medical Research
and Development (AMED) through its Funding Program for Basic
Science and Platform Technology Program for Innovative Biological
Medicine, development of siRNA conjugates with tissue-specific de-
livery functions. The authors thank Professor Yoko Hirata (Gifu
University) for supplying cells and giving technical advice.
methyl-uridine nucleosides 1, 2 and 3 were modeled using the BIOP-
OYMER module implemented in SYBYL package. The geometry of each
4
0
∗
nucleoside was optimized using Gaussian09 at the HF/6-31G level of
theory and RESP charges4 were derived using the Antechamber RESP
1
4
2
fitting procedure. The general AMBER force field (GAFF) adopted to
obtain the force field parameters of each nucleosides. Each nucleotide
was solvated in TIP3P water model using the LEaP module of AMBER
4
3
A. Supplementary data
4
4
1
4
and placed with periodic boundary conditions in a cubic box with
side length 42.0 Å. Counter-ion (Cl−) was added into the solution to
neutralize the charge of the systems. The solvent was energy minimized
by 2500 steps of the steepest descent method and then followed by
Supplementary data (UV melting profiles and graphical data of 1/
T
m
vs. ln (C
T
/4) plots. Additional data from molecular modeling stu-
dies. 2D NOESY spectrum of 5′-CGCGAAU2CGCG-3′. Sequences of
1
13
2
500 steps of the conjugate gradient method, applying a restraint force
siRNAs and their abilities to suppress gene expression. H and C NMR
2
31
constant of 2 kcal/(mol Å ) to the nucleoside. After that, the entire
system was relaxed with 5000 steps of energy minimization without
any restrains. Initially, the temperature of system was increased gra-
dually from 0 to 300 K over a period of 100 ps of NVT dynamics. This
was followed by 50 ps of NPT equilibration at 300 K and 1 atm pressure.
After the equilibration phase, 100 ns productive MD simulations were
performed in an NPT ensemble at 1 atom and 300 K without any re-
strains. During all MD simulations, a time step of 2 fs was used. The cut-
off of the van der Waals interaction was set to be 10 Å, the SHAKE
algorithm was used to constrain all bonds involving hydrogen atoms
and particle-mesh Ewald (PME) was used long-range electrostatic
interactions. The temperature is regulated by the Langevin dynamics
with a collision frequency of 1.0 ps . All of the MD simulations were
performed using the SANDER module of AMBER 14 package. Trajectory
analysis was done using the cpptraj module in AMBER 14 and examined
visually using VMD 1.9.2.
spectra of compounds 5–19 and P NMR spectra of compounds 18a
and 18b) associated with this article can be found, in the online ver-
sion, at http://dx.doi.org/10.1016/j.bmc.2018.05.025.
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