Short-RNA selective binding of oligonucleotides modified using adenosine and guanosine derivatives that possess cyclohexyl phosphates as substituents

Article abstract of DOI:10.1039/c1ob06580g

We have developed new artificial oligonucleotides which distinguish short RNA targets from long ones. The modification of the 5′ termini of oligonucleotides by using adenosine derivatives that possess a bulky cyclohexyl phosphate moiety at their base moiety and a phosphate group at the position of their 5′-hydroxyl group maximized their short RNA selectivity. The 2′-O-methyl-RNA (5′-XC_mA_mA_mC _mC_mU_mA_mC_mU_m) having these modifications exhibits ca. 10 °C higher T_m in the duplexes with the complementary short RNA (3′-GUUGGAUGA-5′) than with the long RNA (3′-AUUAUAUGUUGGAUGAUGGUUA-5′). The oligodeoxynucleotides having the same modification exhibited similar selectivity. Such short-RNA selective binding of terminally modified oligonucleotides can be employed to distinguish between mature microRNAs and pre-microRNAs.

Full text of DOI:10.1039/c1ob06580g

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PAPER
Short-RNA selective binding of oligonucleotides modified using adenosine and
guanosine derivatives that possess cyclohexyl phosphates as substituents†
Kohji Seio,* Sayako Kurohagi, Erika Kodama, Yoshiaki Masaki, Hirosuke Tsunoda, Akihiro Ohkubo and
Mitsuo Sekine*
Received 15th September 2011, Accepted 27th October 2011
DOI: 10.1039/c1ob06580g
We have developed new artificial oligonucleotides which distinguish short RNA targets from long ones.
The modification of the 5¢ termini of oligonucleotides by using adenosine derivatives that possess a
bulky cyclohexyl phosphate moiety at their base moiety and a phosphate group at the position of their
5¢-hydroxyl group maximized their short RNA selectivity. The 2¢-O-methyl-RNA
(5¢-XCmAmAmCmCmUmAmCmUm) having these modifications exhibits ca. 10 ◦C higher Tm in the
duplexes with the complementary short RNA (3¢-GUUGGAUGA-5¢) than with the long RNA
(3¢-AUUAUAUGUUGGAUGAUGGUUA-5¢). The oligodeoxynucleotides having the same
modification exhibited similar selectivity. Such short-RNA selective binding of terminally modified
oligonucleotides can be employed to distinguish between mature microRNAs and pre-microRNAs.
precursors are sometimes more abundant than functionally mature
Introduction
miRNAs. Moreover, the biochemical mechanisms by which the
MicroRNAs (miRNAs) are noncoding RNAs approximately 22
processing of pre-miRNAs to mature miRNAs is regulated have
nt in length.1 Since the discovery of the miRNA let-7 in 1993,2
the presence of miRNAs in animals, plants, and other living
organisms has been revealed.3 Moreover, their involvement in var-
ious biological processes such as development, differentiation, cell
proliferation, apoptosis, and stress responses has been revealed.1
Recent bioinformatic data indicate that approximately one third
of all protein-coding genes are regulated by miRNAs,4 and that
they have been used as biomarkers by means of miRNA expression
profiling for various diseases such as rheumatic disease,5 cancer,6–9
and cardiovascular disease.10
been revealed in some cases.14–17
Because both pre-miRNAs and mature miRNAs co-exist in the
cytoplasm, distinguishing one from the other requires the use of
both sequence-specific and size-specific techniques.11,17
In order to selectively detect biologically active mature miRNAs,
several technologies have been developed.
The detection by using padlock probes with rolling circle
amplification18 and RNA-primed array-based Klenow enzyme
(RAKE) assay19 are the methods employed to selectively detect
mature miRNAs by using enzymatic primer extension.
It is well known that miRNAs are first transcribed from genomic
DNAs as pri-miRNAs, which are cleaved by the endogeneous
nuclease Drosha to give pre-miRNAs that are 60–70 nt in length.1
After being transported to the cytoplasm, pre-miRNAs are cleaved
by the nuclease Dicer to give mature miRNAs. Mature miRNAs
thus generated are the biologically active forms of miRNAs that
bind to target mRNAs to suppress translation processes.
Studies have recently shown that pri-miRNA and pre-miRNA
processing is regulated in various ways. For example, the expres-
sion levels of some mature miRNAs do not correlate with those
of pri-miRNA.11–13 In such cases, functionally inactive miRNA
A more convenient strategy for the selective detection of mature
miRNAs is to use oligonucleotides that demonstrate low affinity
toward long pre-miRNAs, but high affinity toward short mature
miRNAs. Such short-RNA selective binding can be achieved
by the use of hairpin oligonucleotides that interfere with the
binding of the long pre-miRNAs due to the steric repulsion of the
stem-loop region. Such hairpin probes are used in the RT-PCR
technique20 as well as in microarray devices.21
Yet another differentiation strategy, which we reported previ-
ously, is to develop oligonucleotide probes capable of short-RNA
selective binding by the simple modification of the 3¢ and/or 5¢
termini of the oligonucleotides.22 We developed deoxyadenosine
derivatives dAChcm and dAChcmP, possessing a cyclohexane ring and
a phosphorylated cyclohexane ring at the base moiety and as the
modification at the 3¢ and 5¢ termini (Fig. 1). The oligonucleotide
11mer, having two dAChcmP residues at the 5¢ and 3¢ termini,
demonstrated higher affinity toward the complementary shorter
RNA 9mer than toward the complementary longer RNA 15mer
(DTm = +9 ◦C). In addition, it was also proved that even the
Department of Life Science, Tokyo Institute of Technology, 4259
Nagatsuta, Midori-ku, Yokohama, Japan. E-mail: kseio@bio.
titech.ac.jp, msekine@bio.titech.ac.jp; Fax: +81-45-924-5144; Tel: +81-45-
924-5136
† Electronic supplementary information (ESI) available: Partial atomic
charges of dGCmcmP, synthetic schemes of ON-7, ON-9 and ODN-
1, and NMR charts of newly synthesized compounds. See DOI:
10.1039/c1ob06580g
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compared their hybridization properties with those of previously
reported dAChcmP-modified oligonucleotides. We also examined the
influence of the phosphorylation at the 5¢-hydroxyl group and of
the 2¢ methoxylation on the short-RNA selectivity by synthesizing
P
new adenosine-type modifications such as AmChcmP, dAChcmP, and
PAmChcmP (Fig. 1). We found the following: 1) Adenosine derivatives
improve the short-RNA selectivity more than guanosine deriva-
P
tives; and 2) dAChcmP possessing an additional phosphate group
at the 5¢-position of dAChcmP improves the short-RNA selectivity
even further (DTm > 10 ◦C).
Results and discussion
Preparation of oligonucleotides incorporating phosphoramidites of
dGChcm and dGCmcm
We first designed two deoxyguanosine derivatives, dGChcm and
dGCmcm, having carbamoyl and N-methylcarbamoyl linkages,
respectively, between the 2-amino group and the cyclohexyl group
(Fig. 1). It is well known that the 2-N-carbamoylguanine base
can assume two stable conformations: an open form and a closed
form (Fig. 3).23 In the case of N-alkylcarbamoylguanines such as
the base moiety of dGChcm, the carbonyl oxygen of the carbamoyl
group can form a hydrogen bond with the imino proton at
position 1, causing the carbamoyl group to block Watson–Crick-
type hydrogen bonding (Fig. 3A, closed form). In addition, the
carbamoyl group can form another type of hydrogen bond between
the terminal NH group and the nitrogen atom at position 3,
causing the carbamoyl group to extrude in the minor groove
(Fig. 3A, open form).
Fig. 1 Structures of adenosine-type and guanosine-type nucleoside
residues modified using N-cyclohexylcarbamoyl groups. Chcm, Cmcm
and P mean cyclohexylcarbamoyl, cyclohexyl(methyl)carbamoyl and
phosphate groups, respectively.
introduction of a single dAChcmP residue at the 5¢ terminus scarcely
affected this behavior (DTm = +7 ◦C). These results suggest that the
modification of the oligonucleotide termini by bulky, negatively
charged residues such as dAChcmP is an effective way to develop
oligonucleotides capable of short-RNA selective binding because
of the steric and electrostatic repulsion between long RNAs
(Fig. 2).
Fig. 3 Conformers: (A) Of 2-N-(N-alkylcarbamoyl)guanine; (B) Of
2-N-(N,N-dialkylcarbamoyl)guanine.
Fig. 2 Schematic illustration of the short-RNA selective binding of an
oligonucleotide modified using dAChcmP
.
On the basis of previous results, we approached the task
of improving the short-RNA selectivity of terminally modified
oligonucleotides by changing the structures of the terminal
residues. We synthesized and examined the short-RNA selective
binding of oligonucleotides possessing guanosine-type terminal
modifications such as dGChcm, dGCmcm, and dGCmcmP (Fig. 1), and
For the purpose of developing short-RNA selective hybridiza-
tion probes, we assumed that the closed form is stabilized for the
effective repulsive interaction with the counter strand because such
interaction is not expected from the open form conformation.
In addition, the formation of the open form conformation is
prohibited in N,N-dialkylcarbamoyl guanines such as the base
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moiety of dGCmcm because of the steric repulsion between the alkyl
group and the nitrogen atom at position 3 (Fig. 3B).
(Fig. 4A), where X and Y represent 5¢- and 3¢-terminal nucleoside
residues incorporated by 5 or 9. ON-2 incorporates dGChcm residues
at both the X and Y positions, ON-3 incorporates a dGCmcm residue
at these same positions, and ON-4 incorporates a dGCmcm residue
at only the 5¢ X position. We incorporated dGChcm and dGCmcm
residues at the 3¢ termini by using an automated DNA/RNA
synthesizer by means of the usual procedure with universal support
II.27 For comparison, we also synthesized ON-1 having unmodified
deoxyguanosine at the X and Y positions.
Thus, we speculated that N,N-dialkylcarbamoyl-type dGCmcm
would be more suitable to achieve the short-RNA selective
binding. To confirm this speculation, we synthesized two guano-
sine derivatives, dGChcm 3 and dGCmcm 7, and their respective
phosphoramidites 5 and 9 (Scheme 1). dGChcm 3 was synthesized
from 3¢,5¢-O-bis(tert-butyldimethylsilyl)deoxyguanosine 1a.24 The
6-O position was protected by treatment with trimethylsilyl
chloride, and then the amino group of 1a was acylated with
phenyl chloroformate. The phenoxycarbonyl intermediate was
converted by treatment with cyclohexylamine to the fully protected
dGChcm derivative 2 (74%). The TBDMS groups were removed
using triethylamine trihydrofluoride25 to give dGChcm 3 as the
intermediate. The 5¢-hydroxyl group of 3 was protected with a
DMTr group to give 4 (49%). Finally, phosphitylation of the 3¢-
hydroxyl group26 gave the phosphoramidite of dGChcm 5 (45%).
Fig. 4 Sequences: (A) Of oligonucleotides ON-1 to ON-9 and ODN-1;
(B) Of the RNAs to be hybridized with them. Cm, Am, and Um denote
the corresponding 2¢-O-methylnucleotide residues; dC, dA, and dT denote
deoxynucleotide residues.
Hybridization properties of oligonucleotides incorporating dGchcm
and dGcmcm
We investigated the hybridization properties of ON-2 (X, Y =
dGChcm, dGChcm) and ON-3 (X, Y = dGCmcm, dGCmcm) by measuring
the UV melting temperature of their duplexes with the shorter
RNA-9mer and the longer RNA-22mer (Fig. 4B). The results are
shown in Table 1 together with associated Tm data of terminally
unmodified ON-1 (X, Y = dG, dG).
Scheme 1 Preparation of phosphoramidites of dGChcm (R2 = H) and
dGCmcm (R2 = CH3). a) Trimethylsilyl chloride, pyridine, then PhOC(O)Cl,
pyridine; b) cyclohexylamine for 2 or N-methylcyclohexylamine for 6;
c) NEt3·3HF, NEt3, pyridine; d) DMTrCl, pyridine; e) 1H-tetrazole,
diisopropylamine, CEO-P(N-iPr2)2.
Unmodified ON-1 (X, Y = dG, dG) showed higher affinity
toward RNA-22mer than toward RNA-9mer (Tm = 49 ◦C and
46 ◦C, respectively). In contrast, modified ON-2 (X, Y = dGChcm
,
dGChcm) showed yet higher affinity toward both RNA-22mer and
RNA-9mer (Tm = 53 ◦C and 50 ◦C, respectively). These results show
that the modification by dGChcm increased the thermal stability
of the duplexes with both RNA-9mer and with RNA-22mer in
comparison with the modification by dG. Neither ON-1 (X, Y =
dG, dG) nor ON-2 (X, Y = dGChcm, dGChcm) showed selectivity
toward the short RNA-9mer (DTm = -3 ◦C in both cases).
Similarly, the N-methylcyclohexyl derivative 6 (84%), interme-
diate 7, DMTr derivative 8 (77%), and its phosphoramidite 9
(70%) were prepared using N-methylcyclohexylamine in place of
the cyclohexylamine.
In order to study the affinities of oligonucleotides toward
the shorter RNA-9mer and the longer RNA-22mer, we used
phosphoramidite units of dGChcm (5) and dGCmcm (9) to synthe-
size terminally modified 2¢-O-methyl-RNAs (ON-2, -3, and -4)
having the common sequence 5¢-XCmAmAmCmCmUmAmCmUmY-3¢
In the case of the dGCmcm modified ON-3 (X, Y = dGCmcm
,
dGCmcm), the Tm with RNA-9mer was almost identical to that of
ON-2 (X, Y = dGChcm, dGChcm) with RNA-9mer giving Tm = 49 ◦C.
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Table 1
T
m [◦C] for the duplexes of modified oligonucleotides with RNA-
Hybridization properties of modified oligonucleotides
incorporating 5¢-terminal dGCmcmP
9mer and RNA-22mer
a
RNA-9mer
RNA-22mer
DTm
-3
To increase the selectivity of the dGCmcm-modified oligonucleotide
toward the shorter RNA-9mer, we investigated the effect of
introducing a phosphate group on the cyclohexane ring, expecting
ON-1
(X, Y = dG, dG)
46
50
49
46
47
47
50
51
50
38
49
53
47
45
42
40
41
40
41
29
ON-2
-3
to observe a similar improvement in the short-RNA selectivity,
(X, Y = dGChcm, dGChcm
ON-3
)
22
as was observed for phosphorylation of dAChcmP
.
In order to
+2
+1
+5
+7
+9
+11
+9
+9
incorporate the phosphorylated dGCmcm derivative dGCmcmP (Fig. 1),
we synthesized the corresponding phosphoramidite 13a (Scheme
2) as follows.
(X, Y = dGCmcm, dGCmcm
ON-4
)
(X = dGCmcm, Y = none)
ON-5
(X = dGCmcmP, Y = none)
ON-622
Compound 1a was treated with phenyl chloroformate, and
subsequently with trans-4-hydroxy-N-methylcyclohexylamine to
give the carbamoyl derivative 10a (67%). The protection of the
hydroxyl group by the levulinoyl (Lev) group gave 11a (65%).
The removal of the TBDMS group by NEt3·3HF and subsequent
protection of the 5¢-hydroxyl group by the DMTr group gave
12a (52%). Finally, the usual phosphitylation of the 3¢-hydroxyl
group26 gave the phosphoramidite 13a (97%).
(X = dAChcmP, Y = none)
ON-7
(X = AmChcmP, Y = none)
ON-8
P
(X = AmChcmP, Y = none)
ON-9
P
(X = dAChcmP, Y = none)
ODN-1
We synthesized modified oligonucleotide ON-5 (X = dGCmcmP
,
P
(X = dAChcmP, Y = none)
Y = none) having a dGChcmP residue at the 5¢ terminus (Scheme 3).
The protected 2¢-O-methyl-RNA precursor with its nucleobases
and the internucleotidic phosphates protected by acyl-type and
cyanoethyl protecting groups, respectively, was synthesized on
controlled pore glass (CPG). The phosphoramidite 13a was
coupled to the 5¢-hydroxyl group of the protected 2¢-O-methyl-
RNA precursor, and then oxidized in a 0.1 M I2 solution to
give the terminally modified oligonucleotide 14a. The levulinoyl
group of 14a was removed by treatment with hydrazine–acetic
acid solution to give 15a. The liberating hydroxyl group was
phosphorylated using a phosphoramidite-type phosphorylating
agent30 to give the fully protected precursor 16a of the 2¢-O-methyl-
RNA having dGCmcmP at the 5¢ termini. Finally, all protecting
groups were removed by successive treatment with aqueous NH3
and 2% aqueous CF3COOH to give ON-5 (X = dGCmcmP, Y = none).
a DTm = Tm of RNA-9mer - Tm of RNA-22mer .
In contrast, the Tm of the duplex of ON-3 (X, Y = dGCmcm, dGCmcm
)
with RNA-22mer was significantly decreased from 53 ◦C to 47 ◦C.
It should be noted that the structural difference between ON-2
(X, Y = dGChcm, dGChcm) and ON-3 (X, Y = dGCmcm, dGCmcm) is
just a methyl group at the guanine moiety at each terminus. This
result clearly suggests that this small difference, the presence of a
methyl group on the carbamoyl nitrogen, is essential for selective
destabilization of the duplex with RNA-22mer. Accordingly, low
selectivity (DTm = +2 ◦C) toward RNA-9mer was observed for
ON-3 (X, Y = dGCmcm, dGCmcm).
In the case of ON-1 (X, Y = dG, dG), its duplex with RNA-
22mer was more stable than its duplex with RNA-9mer, probably
because of the formation of G–U base pairs28,29 at the 5¢ and 3¢
termini. Similar base pairing is possible in the duplex of ON-2
(X, Y = dGChcm, dGChcm) with RNA-22mer. In contrast, dGCmcm of
ON-3 (X, Y = dGCmcm, dGCmcm) cannot pair with U in RNA-22mer
due to the closed conformation (Fig. 3B), which decreased the Tm.
In the cases of the previously reported dAChcm and dAChcmP
modification, the modification at the 5¢ terminus contributed more
to the selective hybridization toward RNA-9mer.22 Therefore, we
next measured the Tm of ON-4 (X = dGCmcm, Y = none) having a
single dGCmcm residue incorporated at the 5¢ terminus. Its value for
the duplex with RNA-9mer, Tm = 46 ◦C, is identical to the value for
the duplex of ON-1 (X, Y = dG, dG) with RNA-9mer. In contrast,
the value for ON-4 (X = dGCmcm, Y = none)/RNA-22mer duplex,
Tm = 45 ◦C, is 4 ◦C lower than the value for the ON-1 (X, Y = dG,
dG)/RNA-22mer duplex. This result indicates that ON-4 with a
single modification at the 5¢ termini by dGCmcm can still destabilize
the duplex with RNA-22mer.
We studied the hybridization behavior of ON-5 (X = dGCmcmP
,
Y = none) with the shorter RNA-9mer and the longer RNA-22mer.
Comparison with ON-4 (X = dGCmcm, Y = none) (Table 1) shows
that incorporation of a phosphate group on the cyclohexane ring
reduced the Tm for the duplex with RNA-22mer (from Tm = 45 ◦C
to 42 ◦C) but had essentially no effect on the Tm for the duplex with
RNA-9mer (Tm = 47 ◦C in both cases). As a result, the short-RNA
selectivity was improved (DTm = +5 ◦C).
Qualitatively, this trend is in accordance with that reported
previously for ON-6 (X = dAChcmP, Y = none) having a dAChcmP at
the 5¢ termini.22 Thus, we have clarified that the incorporation of
a phosphate group on the cyclohexane ring improves short-RNA
selective binding not only for adenosine-type dAChcmP modification
but also for guanosine-type dGCmcmP modification. However, as
shown in Table 1, selectivity is higher for ON-6 having dAChcmP than
that for ON-5 (X = dGCmcmP, Y = none) whose DTm were +7 ◦C and
+5 ◦C, respectively. Thus, the deoxyadenosine derivative dAChcmP is
superior to the deoxyguanosine derivative dGCmcmP for improving
the short-RNA selectivity.
Interestingly, the comparison of ON-3 (X, Y = dGCmcm, dGCmcm
)
The low affinities of ON-5 (X = dGCmcmP, Y = none) and ON-6
(X = dAChcmP, Y = none) toward the longer RNA-22mer can be
explained by considering the structure of the 5¢ termini of the
modified oligonucleotides in the duplex state. Shown in Fig. 5 are
the structures of the terminal residues of the previously reported
with ON-4 (X = dGCmcm, Y = none) suggests that dGCmcm at the
3¢ termini contributes to the stabilization of both duplexes with
RNA-9mer and RNA-22mer to a similar extent, i.e., 2–3 ◦C. A
contributing factor might be the hydrophobic interaction of the
Cmcm substituent and nucleobases in the counter strands.
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ChcmP
Scheme
2
Preparation of phosphoramidites of dGChcmP and Am
.
a) Trimethylsilyl chloride, pyridine, then PhOC(O)Cl, pyridine; b)
trans-4-hydroxy-N-methylcyclohexylamine; c) DCC, levulinic acid, DMAP, CH2Cl2; d) NEt3·3HF, THF; e) DMTrCl, pyridine; f) 1H-tetrazole,
diisopropylamine, CEO-P(N-iPr2)2, CH2Cl2 for 13a or diisopropylethylamine, CEO-P(Cl)(N-iPr2), CH2Cl2, for 13b.
Scheme
3
Preparation of ON-5 incorporating dGCmcmP
. a) 13a,
Fig. 5 Structure of the terminal residues of the modified oligonu-
cleotide/RNA-9mer duplexes obtained by molecular dynamics simula-
tions: A) dAChcmP-modified ON-6/RNA-9mer duplex; B) dGCmcmP-modified
ON-5/RNA-9mer duplex.
1H-tetrazole, CH3CN, then 0.1 M DMAP, Ac2O–pyridine (1 : 9, v/v);
b) 0.1 M I2, pyridine–H2O; c) 0.5 M NH2NH2/pyridine–AcOH (3 : 2,
v/v); d) (i-Pr)2N-P(OCE)(OCH2CH2SO2CH2CH2ODMTr), 1H-tetrazole,
CH3CN, then 0.1 M I2/pyridine–H2O (9 : 1, v/v); e) 28% aq. NH3, then
2% aq. TFA on a C-18-cartridge column.
having additional phosphodiester backbone extended from this
3¢-hydroxyl group, strong steric and electrostatic repulsions are
expected between the phosphate of dAChcmP and the phosphodiester
backbone of RNA-22mer. In contrast, in the case of the ON-5
ON-6 (X = dAChcmP, Y = none)/RNA-9mer (Fig. 5A) and ON-5
(X = dGCmcmP, Y = none)/RNA-9mer duplex (Fig. 5B) obtained by
molecular dynamics simulation by using the AMBER31 program
and the parameters for the 2¢-O-methyl backbone.32 The phosphate
group of the dAChcmP interacts with the 3¢-terminal hydroxyl group
of RNA-9mer (Fig. 5A). Thus, in the duplex with RNA-22mer
(X = dGCmcmP, Y = none)/RNA-9mer duplex modified by dGCmcmP
,
the phosphate group interacts more strongly with the 2¢-OH of
the terminal residue (Fig. 5B). Comparison of Fig. 5A and 5B
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suggests that we should expect dAChcmP to destabilize the duplex
with RNA-22mer more effectively than dGCmcmP because of the
effective repulsion between the phosphate on the cyclohexane ring
and the phosphodiester backbone of RNA-22mer extended from
the terminal 3¢-hydroxyl group.
Improvement of short-RNA selective binding by 2¢-methoxylation
and 5¢-phosphorylation of dAChcmP
As mentioned above, we clarified that adenosine-type dAChcmP
effectively improves the short-RNA selectivity more than guanine-
type dGCmcmP. Therefore, we investigated further the effect on the
short-RNA selectivity of oligonucleotides modified with dAChcmP
derivatives.
We designed Am
ChcmP
(Fig. 1) having the 2¢-O-methylribose
instead of the 2¢-deoxyribose of dAChcmP. In addition, we designed
the 5¢-phosphate derivatives PdAChcmP and PAm
(Fig. 1).
ChcmP
Scheme 4 Preparation of base and 5¢-bisphosphorylated ON-8. a)
3% dichloroacetic acid/CH2Cl2; b) (i-Pr)2N-P(OCE)(OCH2CH2SO2-
CH2CH2ODMTr), 1H-tetrazole, CH3CN, then 0.1 M I2/pyridine–H2O
(9 : 1, v/v); c) 28% aq. NH3, then 2% aq. TFA on a C-18-cartridge column.
It is well known that 2¢-O-methyladenosine assumes predom-
inantly the N-type sugar conformation.33,34 It has also been
suggested that the phosphorylation of the 5¢-hydroxyl group of
adenosine reduces the flexibility of the glycosidic conformation.35
Thus, we expected that the 5¢-terminal nucleotide residues such as
moiety increased the stability of the duplex with RNA-9mer by 3 ◦C
(from Tm = 47 ◦C to Tm = 50 ◦C) but left the stability of the duplex
with RNA-22mer essentially unchanged (Tm = 40–41 ◦C). As a
P
P
AmChcmP, dAChcmP, and AmChcmP, having relatively inflexible sugar
and/or glycosidic conformations, should interact effectively with
the counter strand.
In order to introduce Am
result, the short-RNA selectivity of ON-7 (X = AmChcmP) (DTm
=
and PAmChcmP, we prepared the
+9 ◦C) increased.
ChcmP
2¢-O-methyladenosine phosphoramidite 13b (Scheme 2) in a
manner that is almost identical to that for the guanosine deriva-
tive 13a. Starting from 3¢,5¢-O-bis-TBDMS-2¢-O-methyladenosine
1b,36 a (trans-4-hydroxylcyclohexylamino)carbonyl group was
introduced to the amino group of the adenine moiety to
give 10b. The protection of the hydroxyl group by the Lev
group gave 11b. Further conversion to the DMTr-protected
derivative 12b and phosphitylation with 2-cyanoethyl-N,N-
diisopropylphosphorochloridite gave the phosphoramidite deriva-
tive 13b.
We then evaluated the effect of 5¢ phosphorylation by comparing
P
ON-7 (X = AmChcmP, Y = none) and ON-8 (X = AmChcmP, Y = none).
For ON-7 (X = AmChcmP, Y = none) and ON-8 (X = PAm
,
ChcmP
Y = none), the 5¢ phosphorylation of the 2¢-O-methyladenosine
residue changed the Tm of their duplexes with both RNA-9mer
◦
and RNA-22mer slightly within 1 C; the short-RNA selectivity
for the 5¢-phosphorylated ON-8 (X = PAmChcmP, Y = none) was
comparable to or somewhat larger ◦than that for ON-7 (X =
◦
AmChcmP, Y = none) with DTm = +11 C and +9 C, respectively.
In contrast, in the comparison of ON-6 (X = dAChcmP, Y = none)
P
We prepared the oligonucleotide ON-7 (X = AmChcmP, Y = none)
as described for ON-5 (X = dGChcmP, Y = none) but by using
phosphoramidite 13b in place of 13a (Scheme S1†).
and ON-9 (X = dAChcmP, Y = none), the 5¢ phosphorylation of the
deoxyadenosine-type terminal residue increased the Tm of their
duplexes with RNA-9mer by 3 ◦C (from Tm = 47 ◦C to Tm = 50 ◦C)
but had little effect on the Tm of their duplexes with RNA-22mer.
P
We also synthesized 5¢-phosphorylated ON-8 (X = AmChcmP, Y =
P
none) as follows (Scheme 4). Starting from the 5¢-DMTr oligonu-
cleotide 15b, which is the synthetic intermediate of ON-7, the
DMTr group was removed by treatment with 3% DCA/CH2Cl2
to give 17b. The hydroxy groups at the 5¢ position and the
cyclohexane ring were phosphitylated simultaneously by using
the aforementioned phosphoramidite-type phosphitylating agent
to give 18b. Finally, deprotection and cleavage from the solid
supports were carried out by treatment with 3% DCA/CH2Cl2
Thus, the short-RNA selectivity of ON-9 (X = dAChcmP, Y = none)
(DTm = +9 ◦C) became comparable to that of ON-7 (X = Am
,
ChcmP
Y = none) and ON-8 (X = PAmChcmP, Y = none) modified with
2¢-O-methyladenosine derivatives.
Among AmChcmP-, PAmChcmP-, and PdAChcmP-modified oligonu-
cleotides, all of which demonstrated comparable short-RNA
selectivity, we selected PdAChcmP for further studies because its
starting material is 2¢-deoxyadenosine which is more conveniently
available than 2¢-O-methyladenosine, the starting material of
and aqueous ammonia to give ON-8 (X = AmChcmP, Y = none).
P
We prepared ON-9 (X = PdAChcmP, Y = none) having 5¢-
phosphorylated deoxyadenosine-type modification at the 5¢ ter-
minus (Scheme S2†), as described for ON-8, but using previously
Am
and PAm
.
ChcmP
ChcmP
First, we evaluated the short-RNA selectivity of PdAChcmP
incorporated into the DNA backbone. We synthesized
the oligodeoxynucleotide ODN-1 having the PdAChcmPd-
(CAACCTACUT) sequence (Scheme S2†) in a manner similar
to that described for ON-8 and ON-9 but by using the canonical
deoxynucleoside phosphoramidites in place of the 2¢-O-methyl-
RNA phosphoramidites.
reported phosphoramidite of dAChcmP (13c in Scheme 2).22
ChcmP
We evaluated the hybridization properties of ON-7 (X = Am
,
,
Y = none), ON-8 (X = AmChcmP, Y = none), and ON-9 (X = dAChcmP
P
P
Y = none) by examining their duplexes with the shorter RNA-9mer
and the longer RNA-22mer (Table 1). Comparison of the data for
ON-6 (X = dAChcmP, Y = none) and ON-7 (X = AmChcmP, Y = none)
shows that the introduction of the 2¢-methoxy group to the ribose
As shown in Table 1, the Tm of the duplexes of oligodeoxynu-
cleotide ODN-1 (X = PdAChcmP, Y = none) with RNA-9mer
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Table 2 Tm [◦C] of the duplexes of modified oligodeoxynucleotides, 5¢-
PdAChcmPN1N2ACCTACT-3¢, and the corresponding RNA-9mer-X1X2 or
RNA-22mer-X1X2 having the common sequence 3¢-X1X2UGGAUGA-5¢
(Tm = 38 ◦C) and RNA-22mer (Tm = 29 ◦C) are lower than those
of the 2¢-O-methyl-RNA derivative ON-9 (X = PdAChcmP, Y =
none) by more than 10 ◦C. However, the short-RNA selectivity
P
of ODN-1 (X = dAChcmP, Y = none) is identical to that of ON-9
RNA-9mer
RNA-22mer
(X = dAChcmP, Y = none) (DTm = +9 ◦C in both cases). Thus, the
(X1X2)
(X1X2)
DTm
+9
+5
+7
+8
+9
+7
+7
P
a
PdAChcmP modification is clearly useful for the development of both
2¢-O-methyl-RNA-type and DNA-type oligonucleotides that are
capable of short-RNA selective binding.
ODN-1
38
(GU)
30
(AU)
40
(GA)
44
(GC)
47
(GG)
33
(AA)
40
29
(GU)
25
(AU)
33
(GA)
36
(GC)
38
(GG)
26
(AA)
33
N1N2 = d(CA)
ODN-2
N1N2 = d(TA)
ODN-3
Sequence dependence of short-RNA selective binding for
oligodeoxynucleotides modified by PdAChcmP
N1N2 = d(CT)
ODN-4
N1N2 = d(CG)
ODN-5
N1N2 = d(CC)
ODN-6
In order to expand the applicability of the PdAChcmP-modified
oligonucleotide, we evaluated the sequence dependence of the
short-RNA selective binding by using oligodeoxynucleotides
modified by PdAChcmP. We focused on the two base pairs adjacent to
the terminal PdAChcmP, expecting that the structures and stabilities
of these base pairs should affect interactions between PdAChcmP
and the counter strands. We synthesized ODN-2 through to
ODN-7 (Fig. 6A) by changing the one or two bases adjacent to
PdAChcmP and investigated the hybridization properties with the
corresponding target 9mer-X1X2 and 22mer-X1X2 (Fig. 6B), where
X1X2 were designed to be complementary to each ODN.
N1N2 = d(TT)
ODN-7
N1N2 = d(TG)
(AC)
(AC)
a DTm = Tm of RNA-9mer-X1X2 - Tm of RNA-22mer-X1X2.
the collective data for ODN-1 to ODN-7 show that their short-
RNA selectivities, as measured by DTm, clearly depend on the
sequence at least at the N1N2 position, and are all significantly
large, between +5 ◦C and +9 ◦C.
Conclusion
We investigated the effect on the oligonucleotide binding of
guanosine-type terminal modifications by dGChcm and dGCmcm. The
incorporation of a single methyl group on the 2-N position of
the guanine ring significantly reduced the oligonucleotide affinity
toward the longer RNA-22mer but had essentially no effect on
its affinity toward the shorter RNA-9mer. This result suggests the
importance of conformational restriction around the carbamoyl
group of dGCmcm
.
We also investigated the effect on the oligonucleotide binding
of guanosine-type terminal modifications by dGCmcmP with a
phosphate moiety on the cyclohexane ring. dGCmcmP improved
the short-RNA selectivity of the modified oligonucleotide in
comparison with dGCmcm, but lowered the short-RNA selectivity
to a value lower than that previously reported for adenosine-type
Fig. 6 Sequences for evaluation of the sequence dependence of short-
RNA selectivity: A) Of ODNs modified with PdAChcmP; B) Of target RNAs.
Nucleotide residues different from those of ODN-1 are underlined.
dAChcmP
.
We further modified the termini by introducing a 2¢-methoxy
or 5¢-phosphate group onto the adenosine-type modification. The
5¢-phosphate derivative PdAChcmP significantly improved the short-
RNA selectivity of the modified oligonucleotide in comparison
with dAChcmP, raising the short-RNA selectivity to as high as DTm =
10 ◦C.
The modification by PdAChcmP at the 5¢ terminus of both 2¢-
O-methyl-RNA and oligodeoxynucleotide increases the relative
binding affinity toward short RNA. Currently, the chain length
of the 5¢-modified oligonucleotides is 9nt. Oligonucleotide probes
of this length should be capable of recognizing the 9 nucleotide
residues of the 3¢ terminal of miRNAs.
It is well known that the 5¢-terminal sequence of a miRNA
family is the seed sequence that binds to a target mRNA and is
common to all family members.37 Thus, oligonucleotide probes
that recognize the 3¢-terminal region are expected to be useful for
detection of mature miRNAs in a variant-specific manner. Because
The Tm data are shown in Table 2; for comparison, they include
data for ODN-1, already shown in Table 1. First, we examined the
influence of the nucleotide residue N1 adjacent to PdAChcmP. When
N1 changed from dC to T, as in the case ofODN-2 {N1N2 = d(TA)},
Tm of the duplexes with 9mer-AU and 22mer-AU decreased (from
Tm = 30 ◦C to 25 ◦C), reducing the short-RNA selectivity (DTm
=
+5 ◦C). This result clearly suggests that the base pair at the N1-X1
position affects both the Tm and the DTm values.
Next, we examined the influence of N2, the second neighbor of
PdAChcmP, by fixing N1 to the dC of ODN-1. The data of ODN-
3 {N1N2 = d(CT)}, -4 {N1N2 = d(CG)}, and -5 {N1N2 = d(CC)}
(DTm = +7, +8, and +9 ◦C, respectively) show that duplexes having
different base pairs at the second neighbor base–base pair N2–X2
exhibited significant short-RNA selectivities.
The data for ODN-6 {N1N2 = TT} and ODN-7 {N1N2 = d(TG)}
◦
show comparable short-RNA selectivities (DTm = +7 C). Thus,
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both 2¢-O-methyl-RNA and oligodeoxynucleotides modified by
PdAChcmP maintain the value of short-RNA selectivity, these probes
should be useful in the development of microarray-related and RT-
PCR related techniques for detecting miRNA. Such applications
of our probes will be reported elsewhere.
(3H, s), 3.73–3.74 (1H, m), 4.16 (1H, m), 4.64 (1H, m), 6.27 (1H,
m), 6.70–6.72 (4H, m), 7.10–7.24 (7H, m), 7.34 (2H, m), 7.79 (1H,
s); 13C NMR (CDCl3, 126 MHz): d = 24.9, 25.6, 29.7, 32.9, 41.0,
49.0, 55.1, 64.1, 71.6, 77.4, 84.2, 86.3, 113.0, 119.4, 126.8, 127.8,
128.2, 130.0, 135.7, 144.6, 149.2, 155.1, 158.4; HRMS (ESI): m/z
calcd for C38H42N6NaO7 [M+Na]+: 717.3007, found 717.3027.
+
Experimental section
5¢-O-(4, 4¢-Dimethoxytrityl)-2-N-cyclohexylcarbamoyl-
deoxyguanosine 3¢-(2-cyanoethyl
3¢,5¢-O-Bis(tert-butyldimethylsilyl)-2-N-
N,N-diisopropylphosphoramidite) (5)
cyclohexylcarbamoyldeoxyguanosine (2)
3¢,5¢-O-Bis(tert-butyldimethylsilyl)deoxyguanosine (1a)24 (2.5 g,
5.0 mmol) was rendered anhydrous by repeated co-evaporation
three times with dry pyridine, and finally dissolved in dry pyridine
(50 mL). Trimethylsilyl chloride (1.9 mL, 15 mmol) was added
and the solution was stirred at ambient temperature for 10 min.
Phenyl chloroformate (953 mL, 7.6 mmol) was added and the
solution was stirred at ambient temperature for 3 h. Subsequently,
cyclohexylamine (3.4 mL, 30 mmol) was added and the mixture
was stirred for 2 h. The solvents were removed under reduced
pressure, and the residue was diluted with ethyl acetate (150 mL),
washed three times with saturated aq. NaHCO3 (100 mL each),
dried over Na2SO4, filtered and concentrated under reduced
pressure. The residue was chromatographed on a column of silica
gel using chloroform–methanol (30 : 1, v/v) as eluent to give 2 (2.3
Compound 4 (400 mg, 0.58 mmol) was rendered anhydrous
by repeated co-evaporation three times each with dry pyri-
dine, dry toluene and dry CH2Cl2 and finally dissolved in dry
CH2Cl2 (5.8 mL). 1H-Tetrazole (25 mg, 0.35 mmol), diiso-
propylamine (50 mL, 0.35 mmol) and 2-cyanoethyl N,N,N¢,N¢-
tetraisopropylphosphorodiamidite (203 mL, 0.64 mmol) were
added, and the solution was stirred at ambient temperature for 22
h. The reaction was quenched by addition of methanol (2 mL), and
the solvents were removed under reduced pressure. The residue was
dissolved in diethylether (30 mL), and washed three times with 0.1
M NaOH (20 mL), dried over Na2SO4, filtered and concentrated
under reduced pressure. The residue was chromatographed on a
column of silica gel using chloroform–methanol (200 : 1, v/v) to
give 5 (230 mg, 45%). 1H NMR (CDCl3, 500 MHz): d = 0.97–1.08
(12H, m), 1.23–1.34 (5H, m), 1.47 (1H, m), 1.61 (2H, m), 1.87
(2H, m), 2.25 (1H, m), 2.32 (1H, m), 2.51 (1H, m), 2.60 (1H, m),
3.11–3.12 (2H, m), 3.17–3.25 (1H, m), 3.33–3.56 (1H, m), 3.48–
3.49 (2H, m), 3.67 (6H, m), 4.12–4.16 (1H, m), 4.63 (1H, m), 6.20
(1H, m), 6.69–6.71 (4H, m), 7.09–7.21 (7H, m), 7.29–7.31 (2H, m),
7.72–7.75 (1H, m); 13C NMR (CDCl3, 126 MHz): d = 20.3, 20.3,
20.5, 20.5, 24.6, 24.7, 24.7, 25.8, 29.8, 33.3, 40.1, 43.3, 43.3, 43.4,
43.4, 45.9, 48.6, 55.3, 55.4, 58.1, 58.3, 63.5, 73.2, 73.7, 73.8, 84.0,
85.6, 85.8, 86.5, 113.2, 113.2, 117.9, 120.0, 127.0, 127.9, 128.2,
128.3, 129.1, 130.1, 130.2, 130.2, 135.7, 135.7, 135.8, 144.6, 144.6,
148.7, 148.9, 154.5, 158.6; 31P NMR (CDCl3, 203 MHz): d = 150.0;
HRMS (ESI): m/z calcd for C47H58N8O8- [M-H]-: 893.4121, found
893.4094.
1
g, 74%). H NMR (DMSO-d6, 500 MHz): d = 0.03–0.09 (12H,
m), 0.85–0.87 (18H, m), 1.21–1.23 (3H, m), 1.31–1.34 (2H, m),
1.51 (1H, m), 1.63 (2H, m), 1.80–1.82 (2H, m), 2.28–2.30 (1H, m),
2.65–2.68 (1H, m), 3.54 (1H, br), 3.63–3.71 (2H, m), 3.84 (1H,
m), 4.48 (1H, m), 6.12–6.13 (1H, m), 7.14 (1H, s br), 8.09 (1H,
s), 9.76 (1H, s br), 11.79 (1H, s br); 13C NMR (DMSO-d6, 126
MHz): d = -5.5, -5.5, -5.0, -4.8, 17.7, 18.0, 24.0, 25.1, 25.6, 25.8,
32.3, 47.9, 62.6, 72.0, 82.7, 87.2, 115.2, 119.2, 129.4, 136.4, 148.8,
153.8; HRMS (ESI): m/z calcd for C29H52N6NaO5Si2 [M+Na]+:
+
643.3430, found 643.3428.
5¢-O-(4,4¢-Dimethoxytrityl)-2-N-
cyclohexylcarbamoyldeoxyguanosine (4)
Compound 2 (1.8 g, 2.9 mmol) was rendered anhydrous by
repeated co-evaporation three times with dry pyridine, and finally
dissolved in dry pyridine (14 mL). Triethylamine trihydrofluoride
(1.90 mL, 11.6 mmol) and triethylamine (1.60 mL, 11.6 mmol)
were added, and the solution was stirred at ambient temperature
for 23 h. After the solvents were removed under reduced pressure,
the residue was chromatographed on a column of silica gel
using chloroform–methanol (9 : 1, v/v) as eluent to give the
crude intermediate 3 (1.95 g). A portion (480 mg) of crude
3 was collected and dissolved in dry pyridine (12 mL). 4,4¢-
Dimethoxytrityl chloride (624 mg, 1.84 mmol) was added and the
mixture was stirred at ambient temperature for 1.5 h. The reaction
was quenched by addition of methanol (3 mL), and the mixture
was diluted with ethyl acetate (60 mL). The solution was washed
three times with saturated aq. NaHCO3 (40 mL each), dried over
Na2SO4, filtered and concentrated under reduced pressure. The
residue was chromatographed on a column of silica gel using
chloroform–methanol (49 : 1, v/v) as eluent to give 4 (742 mg,
49%). 1H NMR (CDCl3, 500 MHz): d = 1.34 (5H, m), 1.52 (1H,
m), 1.68 (2H, m), 1.93 (2H, m), 2.61 (2H, m), 3.28 (2H, m), 3.69
3¢,5¢-O-Bis(tert-butyldimethylsilyl)-2-N-(N-cyclohexyl-N-
methylcarbamoyl)deoxyguanosine (6)
Compound 1a (2.5 g, 5.0 mmol) was rendered anhydrous by
repeated co-evaporation three times with dry pyridine, and finally
dissolved in dry pyridine (50 mL). Trimethylsilyl chloride (1.6 mL,
13 mmol) was added and the solution was stirred at ambient
temperature for 1 min. Phenyl chloroformate (953 mL, 7.6 mmol)
was added and the solution was stirred at ambient temperature for
4 h. Subsequently, N-methylcyclohexylamine (6.7 mL, 50 mmol)
was added and the mixture was stirred for 24 h. The solvents
were removed under reduced pressure, and the residue was diluted
with ethyl acetate (100 ml), washed three times with saturated aq.
NaHCO3 (100 mL each), dried over Na2SO4, filtered and concen-
trated under reduced pressure. The residue was chromatographed
on a column of silica gel using hexane–ethyl acetate (3 : 2, v/v) as
1
eluent to give 6 (2.7 g, 84%). H NMR (CDCl3, 500 MHz): d =
-0.07 to -0.05 (12H, m), 0.75–0.77 (18H, m), 0.95–0.97 (1H, m),
1.27 (4H, m), 1.53–1.55 (3H, m), 1.69 (2H, m), 2.18–2.21 (1H,
m), 2.28–2.32 (1H, m), 2.82 (3H, s), 3.62–3.67 (2H, m), 3.78–3.79
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(1H, m), 3.93 (1H, br), 4.42–4.43 (1H, m), 6.01–6.03 (1H, t, J =
6.0 Hz), 7.79 (1H, s), 8.13 (1H, s br), 12.38 (1H, s br); 13C NMR
(CDCl3, 126 MHz): d = -5.7, -5.6, -5.1, -4.9, 17.7, 18.2, 25.1,
25.3, 25.5, 25.7, 28.5, 29.9, 41.1, 54.1, 62.3, 71.0, 77.4, 83.0, 83.0,
87.3, 120.2, 135.9, 148.4, 148.9, 154.0, 155.7; HRMS (ESI): m/z
calcd for C30H54N6NaO5Si2+ [M+Na]+: 657.3586, found 657.3604.
2¢-O-methyluridine (0.1 mmol) for ON-4. The standard DMTr-
ON RNA synthesis protocol consisting of chain elongation,
removal of the DMTr group, capping and oxidation was used.
The oligonucleotides were first cleaved from the solid support by
using 2.0 M ammonia/methanol (2 mL) for 1 h. After the solution
was collected, the solvents were removed under reduced pressure
and the residue was treated with 28% aqueous ammonia (2 mL) for
8 h. The ammonia was removed under reduced pressure, and the
solution was diluted by the addition of 0.1 M ammonium acetate
(10 mL). The solution was charged on a C-18 cartridge column,
washed with 10% acetonitrile–0.1 M ammonium acetate, treated
with aqueous 2% TFA and washed with 0.1 M ammonium acetate.
After an additional wash with water, the oligonucleotides were
eluted by using 30% acetonitrile–water. The pure oligonucleotides
were obtained by purification on an anion-exchange HPLC
(gradient of 0–62% of 1 M NaCl in 25 mM NaH2PO4, pH 6.0
in 35 min). Finally, the phosphate salts were removed on a C-
18 cartridge column by using water as eluent. Finally, the pure
oligonucleotides were eluted by using 30% acetonitrile–water.
The yields of oligonucleotides were calculated assuming that the
extinction coefficients of the oligonucleotides were identical to
those of oligoribonucleotides having ribonucleotide residues in
place of 2¢-O-methyl-ribonucleotide residues and guanosine in
5¢-O-(4,4¢-Dimethoxytrityl)-2-N-(N-cyclohexyl-N-
methylcarbamoyl)deoxyguanosine (8)
According to a similar procedure for 4, compound 6 (2.0 g,
3.15 mmol) was first converted to the crude intermediate 7 (1.42
g) by treatment with triethylamine trihydrofluoride (2.1 mL,
12.6 mmol) for 6.5 h, and column chromatography by using
chloroform–methanol (50 : 1, v/v). Subsequently, 7 (1.0 g) was
converted to 8 (1.2 g, 77%) by treatment with 4,4¢-dimethoxytrityl
chloride (1.12 g, 3.31 mmol) for 1 h and purification by col-
umn chromatography using chloroform–methanol (99 : 1, v/v).1H
NMR (CDCl3, 500 MHz): d = 1.04 (1H, m), 1.31 (4H, m), 1.62
(3H, m), 1.76 (2H, m), 2.49–2.55 (2H, br), 2.64 (3H, s), 3.26–3.30
(2H, br), 3.69 (7H, m), 4.08 (1H, m), 4.23 (1H, m), 4.67 (1H, m),
5.43 (1H, m), 6.20 (1H, m), 6.72–6.74 (4H, br), 7.10–7.38 (9H, br),
7.76 (1H, s), 12.76 (1H, s); 13C NMR (CDCl3, 126 MHz): d = 25.2,
25.4, 28.3, 29.8, 40.6, 54.2, 55.0, 63.9, 71.6, 77.4, 83.9, 86.1, 86.5,
113.0, 120.0, 123.8, 126.7,127.7, 129.8, 135.6, 136.2, 136.7,144.6,
149.0, 149.2, 149.3, 154.6, 156.1, 158.3; HRMS (ESI): m/z calcd
place of the terminal modified deoxyguanosines. ON-2: e260
=
104 200, MALDI-TOF mass [M + H+] calcd 3792.8, found 3794.7;
ON-3: e260 = 104 200, MALDI-TOF mass [M + H+] calcd 3820.9,
found 3821.0; ON-4: e260 = 94 100; MALDI-TOF mass [M + H+]
calcd 3352.7, found 3354.0.
for C39H44N6NaO7 [M+Na]+: 731.3164, found 731.3177.
+
5¢-O-(4,4¢-Dimethoxytrityl)-2-N-(N-cyclohexyl-N-
methylcarbamoyl)deoxyguanosine 3¢-(2-cyanoethyl-N,N-
diisopropylphosphoramidite) (9)
3¢,5¢-O-Bis(tert-butyldimethylsilyl)-2-N-[N-(trans-4-
According to a similar procedure for 5, compound 8 (710 mg,
1.0 mmol) was converted to the phosphoramidite 9 (642 mg,
70%) by treatment with 1H-tetrazole (42 mg, 0.60 mmol), di-
isopropylamine (85 mL, 0.60 mmol) and 2-cyanoethyl N,N,N¢,N¢-
tetraisopropylphosphorodiamidite (350 mL, 1.1 mmol) for 5 h,
and column chromatography using chloroform–methanol (200 : 1,
v/v). 1H NMR (CDCl3, 500 MHz): d = 1.10–1.17 (12H, m), 1.20–
1.42 (5H, m), 1.60–1.65 (3H, m), 1.68 (3H, s), 1.79–1.81 (2H, m),
2.17 (1H, m), 2.39 (1H, m), 2.44 (1H, m), 2.62–2.64 (1H, m), 3.17–
3.22 (1H, m), 3.32–3.41 (1H, m),3.55–3.62 (2H, m), 3.68–3.73
(1H, m), 3.77 (6H, s), 3.83–3.92 (1H, m), 4.00 (1H, br), 4.22–4.27
(1H, m), 4.68–4.72 (1H, m), 6.20 (1H, m), 6.80 (4H, m), 7.19–
hydroxycyclohexyl)-N-methylcarbamoyl]deoxyguanosine (10a)
Compound 1a (2.5 g, 5.0 mmol) was rendered anhydrous by
repeated co-evaporation three times with dry pyridine, and finally
dissolved in dry pyridine (100 mL). Trimethylsilyl chloride (5.1
ml, 40 mmol) was added and the solution was stirred at ambient
temperature for 20 min. Phenyl chloroformate (953 mL, 7.5 mmol)
was added and the solution was stirred at ambient temperature for
3 h. The solution was diluted with ethyl acetate (200 mL) and then
washed once with saturated aq. NaHCO3 (150 mL). The organic
layer was collected and concentrated under reduced pressure. The
residue was dissolved in pyridine (100 mL), and trans-4-hydroxy-
N-methylcyclohexylamine (4.9 g, 38 mmol) was added. After being
stirred at ambient temperature for 16 h, the solvent was removed
under reduced pressure and the residue was diluted with ethyl
acetate (200 ml), washed three times with saturated aq. NaHCO3
(150 ml), dried over Na2SO4, filtered and concentrated under
reduced pressure. The residue was chromatographed on a silica gel
column with chloroform–methanol (50 : 1, v/v) to give 10a (6.57 g,
67%). 1H NMR (CDCl3, 500 MHz): d = 0.03 (12H, m), 0.84–0.86
(18H, m), 1.48 (4H, m), 1.65 (2H, m), 2.04 (2H, m), 2.28–2.30 (1H,
m), 2.35–2.41 (1H, m), 2.85 (3H, s), 3.55 (1H, br), 3.71 (2H, m),
3.89–3.90 (1H, m), 4.02 (1H, br), 4.50 (1H, m), 6.13–6.15 (1H, t,
J = 6.3 Hz), 7.87 (1H, s); 13C NMR (CDCl3, 126 MHz): d = -5.5,
-5.4, -4.8, -4.7, 18.0, 18.4, 25.7, 25.8, 26.0, 28.2, 34.2, 41.4, 53.5,
62.7, 69.4, 71.7, 77.4, 83.4, 87.8, 120.4, 136.2, 148.7, 149.0, 154.2,
7.38 (7H, m), 7.44–7.48 (2H, m), 7.73 (1H, s), 12.34 (1H, br); 13
C
NMR (CDCl3, 126 MHz): d = 20.1, 20.3, 21.3, 22.4, 22.8, 23.5,
23.8, 24.4, 25.2, 27.6, 27.8, 29.5, 29.8, 39.5, 39.7, 43.0, 54.0, 54.5,
55.0, 55.3, 55.7, 58.0, 58.2, 63.0, 63.3, 73.3, 73.9, 84.0, 84.3, 84.5,
85.6, 86.2, 112.9, 113.2, 117.5, 117.7, 120.8, 121.0, 126.7, 126.9,
127.9, 129.9, 135.6, 136.3, 136.5, 136.7, 144.6, 148.5, 148.8, 153.8,
154.0, 155.8, 158.4; 31P NMR (CDCl3, 203 MHz): d = 149.4, 149.7;
HRMS (ESI): m/z calcd for C48H62N8O8P+ [M+H]+: 909.4423,
found 909.4424.
Synthesis of modified 2¢-O-methyl-RNA (ON-2, ON-3 and ON-4)
The phosphoramidites of dGChcm (5) or dGCmcm (9) were dissolved
in dry acetonitrile (0.1 M) and loaded on the automated RNA
synthesizer. The synthesis was initiated from the universal support
II (1.0 mmol) for ON-2 and ON-3 or from the solid support of
156.0; HRMS (ESI): m/z calcd for C30H54N6NaO6Si2 [M+Na]+:
+
673.3536, found 673.3526.
1002 | Org. Biomol. Chem., 2012, 10, 994–1006
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3¢,5¢-O-Bis(tert-butyldimethylsilyl)-2-N-[N-(trans-4-
levulinyloxycyclohexyl)-N-methylcarbamoyl]deoxyguanosine (11a)
30.4, 38.0, 38.8, 40.6, 52.8, 55.3, 64.0, 68.2, 72.0, 72.1, 77.4, 84.2,
86.3, 86.5, 113.3, 120.7, 127.0, 128.0, 128.1, 128.9, 130.1, 131.0,
135.8, 135.9, 137.1, 144.9, 149.0, 154.5, 156.1, 158.6, 172.3, 206.9;
HRMS (ESI): m/z calcd for C44H50N6NaO10+ [M+Na]+: 845.3481,
found 845.3476.
Compound 10a (4.2 g, 6.45 mmol) was rendered anhydrous
by repeated co-evaporation with dry pyridine, and the residue
was dissolved in dry CH2Cl2 (65 mL). Levulinic acid (1.31 mL,
12.9 mmol), N, N¢-dicyclohexylcarbodiimide (2.66 g, 12.9 mmol)
and 4-dimethylaminopyridine (79 mg, 0.645 mmol) were added
and the resulting solution was stirred at ambient temperature
for 1 h. The precipitates were removed by filtration and washed
with CH2Cl2 (50 mL). The filtrate was washed three times with
saturated aq. NaHCO3 (100 mL each), dried over Na2SO4,
filtered and concentrated under reduced pressure. The residue
was chromatographed on a silica gel column with chloroform–
methanol (99 : 1, v/v) to give 11a (3.15 g, 65%). 1H NMR (CDCl3,
500 MHz): d = 0.01–0.04 (12H, m), 0.84–0.85 (18H, m), 1.51 (4H,
m), 1.71 (2H, m), 2.02 (2H, m), 2.15 (3H, s), 2.26–2.31 (1H, m),
2.35–2.40 (1H, m), 2.49–2.52 (2H, m), 2.69–2.71 (2H, m), 2.86 (3H,
s), 3.70 (2H, m), 3.88 (1H, d, J = 3.0 Hz), 4.12 (1H, br), 4.50 (1H,
m), 4.58 (1H, br), 6.11 (1H, t, J = 6.3 Hz), 7.85 (1H, s); 13C NMR
(CDCl3, 126 MHz): d = -5.5, -5.4, -4.8, -4.7, 18.0, 18.4, 25.7,
25.9, 27.5, 28.3, 28.7, 29.9, 30.3, 37.9, 41.3, 52.8, 62.6, 71.6, 72.0,
77.4, 83.3, 87.8, 120.5, 136.2, 148.4, 148.8, 154.1, 155.8, 172.3,
5¢-O-(4,4¢-Dimethoxytrityl)-2-N-[N-(trans-4-
levulinyloxycyclohexyl)-N-methylcarbamoyl]deoxyguanosine
3¢-(2-cyanoethyl-N,N-diisopropylphosphoramidite) (13a)
Compound 12a (740 mg, 0.90 mmol) was rendered anhydrous
by repeated co-evaporation three times each with dry pyridine,
dry toluene, dry CH2Cl2 and finally dissolved in dry CH2Cl2
(9 mL). To this solution, 1H-tetrazole (38 mg, 0.54 mmol), di-
isopropylamine (76 mL, 0.54 mmol) and 2-cyanoethyl-N,N,N¢,N¢-
tetraisopropylphosphorodiamidite (314 mL, 0.99 mmol) were
added, and the solution was stirred at ambient temperature for
3.5 h. After the reaction was quenched with methanol (2 mL),
the solution was washed three times with saturated aq. NaHCO3
(50 mL each), dried over Na2SO4, filtered and concentrated under
reduced pressure. The residue was dissolved in chloroform (2 mL)
and then poured into diisopropyl ether–hexane (20 mL; 1 : 3, v/v).
The resulting precipitates were collected by filtration to give 13a
(895 mg, 97%). 1H NMR (CDCl3, 500 MHz): d = 1.10–1.24 (12H,
m), 1.37–1.71 (7H, m), 1.95–2.08 (3H, m), 2.20 (3H, s), 2.22–2.32
(1H, br), 2.41–2.44 (1H, m), 2.48–2.56 (2H, m), 2.60–2.65 (1H, m),
2.74–2.90 (2H, m), 3.16–3.22 (1H, m), 3.31–3.43 (1H, m), 3.54–
3.60 (2H, m), 3.68–3.73 (1H, m), 3.76 (6H, s), 3.81–3.90 (1H, m),
4.08 (1H, br), 4.21–4.25 (1H, m), 4.58–4.60 (1H, m), 4.65–4.70
(1H, m), 6.18 (1H, m), 6.80 (4H, m), 7.20–7.39 (7H, m), 7.42–7.50
(2H, m), 7.75 (1H, s), 12.25 (1H, br); 13C NMR (CDCl3 126 MHz):
d = 20.4, 20.6, 24.7, 27.4, 27.5, 27.9, 28.4, 30.0, 30.4, 38.1, 39.6,
40.0, 43.3, 43.4, 52.7, 55.4, 58.1, 58.3, 58.4, 63.6, 72.1, 73.6, 73.7,
74.3, 74.5, 84.7, 84.9, 85.8, 86.0, 86.4, 86.5, 113.4, 117.5, 117.7,
121.6, 121.9, 127.1, 128.1, 130.1, 135.8, 135.9, 136.1, 136.9, 137.3,
145.0, 145.2, 148.5, 148.6, 148.8, 153.9, 154.2, 155.9, 158.8, 172.4,
206.8; 31P NMR (CDCl3, 203 MHz): d = 149.2, 149.6; HRMS
(ESI): m/z calcd for C53H68N8O11P+: 1023.4740, found 1023.4745.
206.8; HRMS (ESI): m/z calcd for C35H60N6NaO8Si2 [M+Na]+:
771.3903, found 771.3906.
+
5¢-O-(4,4¢-Dimethoxytrityl)-2-N-[N-(trans-4-
levulinyloxycyclohexyl)-N-methylcarbamoyl]deoxyguanosine (12a)
Compound 11a (3.0 g, 4.0 mmol) was dissolved in tetrahydrofuran
(20 mL). Triethylamine trihydrofluoride (2.6 mL, 16 mmol) was
added and the resulting solution was stirred at ambient tempera-
ture for 20 h. The solvents were removed under reduced pressure,
and the residue was dissolved in chloroform–methanol (60 mL;
5 : 1, v/v). The solution was washed once with 5% aq. ammonium
chloride (40 mL), dried over Na2SO4, filtered and concentrated
under reduced pressure. The residue was chromatographed on a
silica gel column with chloroform–methanol (19 : 1, v/v) to give
the crude material of the 3¢,5¢-unprotected intermediate (1.23
g). This material was used for the synthesis of 12a without
further purification. 540 mg of the crude material was rendered
anhydrous by repeated co-evaporation with dry pyridine, and
finally dissolved in dry pyridine (10 mL). 4,4¢-Dimethoxytrityl
chloride (491 mg, 1.45 mmol) was added and the solution was
stirred at ambient temperature for 2 h. The reaction was quenched
by addition of water (2 ml), and the solution was diluted with
ethyl acetate (50 mL). The solution was washed three times
with saturated aq. NaHCO3 (50 mL each), dried over Na2SO4,
filtered and concentrated under reduced pressure. The residue
was chromatographed on a silica gel column with chloroform–
methanol (99 : 1, v/v) to give the crude material of 12a (751 mg,
3¢,5¢-O-Bis(tert-butyldimethylsilyl)-6-N-[N-(trans-4-
hydroxycyclohexyl)carbamoyl]-2¢-O-methyladenosine (10b)
Compound 1b36 (2.6 g, 5.2 mmol) was rendered anhydrous by
repeated co-evaporation three times with dry pyridine, and finally
dissolved in dry pyridine (50 mL). Phenyl chloroformate (1.4 mL,
11.4 mmol) was added and the solution was stirred at ambient
temperature for 45 min, and trans-4-hydroxycyclohexylamine (3.0
g, 25.8 mmol) was added. After being stirred at 85 ◦C for 2 h, the
solvent was removed under reduced pressure and the residue was
diluted with chloroform (50 ml). The filtrate was washed three
times with saturated aq. NaHCO3 (30 ml), dried over Na2SO4,
filtered and concentrated under reduced pressure. The residue
was chromatographed on a silica gel column with chloroform–
methanol (100 : 1, v/v) to give 10b (2.6 g, 76%). 1H NMR (CDCl3,
500 MHz): d = 0.03–0.07 (12H, m), 0.85–0.87 (18H, m), 1.34–1.46
(4H, m), 1.98–2.00 (2H, br), 2.09–2.11 (2H, br), 3.22 (1H, s), 3.43
(3H, s), 3.67–3.72 (1H, m), 3.74–3,77 (2H, m), 3.92–3.95 (1H, m),
4.06–4.07 (1H, m), 4.16 (1H, t, J = 4.5 Hz), 4.50 (1H, t, J = 5.0 Hz),
6.14 (1H, d, J = 4.0 Hz), 8.43 (1H, s), 8.46 (1H, s), 8.98 (1H, s),
1
52% from 11a). H NMR (CDCl3, 500 MHz): d = 1.45 (4H, m),
1.65 (2H, m), 1.99 (2H, m), 2.17 (3H, s), 2.34 (4H, m), 2.51–2.54
(2H, t, J = 6.0 Hz), 2.61 (1H, m), 2.71–2.73 (2H, t, J = 6.3 Hz),
3.20–3.21 (1H, d, J = 6.0 Hz), 3.29–3.30 (1H, d, J = 8.0 Hz), 3.71
(6H, s), 4.14 (1H, m), 4.30 (1H, m), 4.57 (1H, m), 4.63 (1H, m),
6.14 (1H, m), 6.73–6.74 (4H, m), 7.10–7.13 (1H, m), 7.17–7.19
(2H, m), 7.26 (4H, m), 7.38–7.40 (2H, m), 7.71 (1H, s), 12.49 (1H,
s br); 13C NMR (CDCl3, 126 MHz): d = 27.4, 28.1, 28.4, 29.0, 30.0,
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9.45 (1H, d, J = 7.5 Hz); 13C NMR (CDCl3, 126 MHz): d = -5.3,
-5.2, -4.6, -4.5, 18.3, 18.6, 25.9, 26.2, 31.0, 33.9, 43.6, 58.6, 62.1,
69.6, 70.0, 83.9, 85.3, 86.9, 120.9, 141.8, 150.1, 150.8, 151.2, 153.7;
2.77 (2H, m), 3.42–3.44 (1H, m), 3.51–3.52 (1H, m), 3.57 (3H, s),
3.79 (6H, s), 4.20 (1H, s), 4.39–4.40 (1H, m), 4.50–4.51 (1H, m),
4.79 (1H, m), 6.17 (1H, d, J = 3.42), 6.81 (4H, d, J = 8.30), 7.22–
7.43 (9H, m), 7.87 (1H, s), 8.14 (1H, s), 8.45 (1H, s), 9.37 (1H,
d, J = 7.57); 13C NMR (CDCl3, 126 MHz): d = 28.6, 29.9, 30.1,
30.5, 38.2, 48.2, 55.4, 59.1, 63.2, 70.0, 72.3, 83.5, 84.4, 86.8, 86.9,
113.4, 121.1, 127.2, 128.1, 128.4, 130.3, 135.8, 135.9, 144.8, 150.1,
150.6, 151.3, 153.4, 158.8, 172.5, 206.9; HRMS (ESI): m/z calcd
HRMS (ESI): m/z calcd for C30H53N6O6Si2 [M–H]-: 649.3571,
-
found 649.3562.
3¢,5¢-O-Bis(tert-butyldimethylsilyl)-6-N-[N-(trans-4-
levulinyloxycyclohexyl)carbamoyl]-2¢-O-methyladenosine (11b)
for C44H50N6NaO10 [M+Na]+: 845.3481, found 845.3507.
+
Compound 10b (2.1 g, 3.2 mmol) was rendered anhydrous by
repeated co-evaporation with dry pyridine, and the residue was
dissolved in dry CH2Cl2 (30 mL). Levulinic acid (0.72 mL,
6.3 mmol), N,N¢-dicyclohexylcarbodiimide (1.3 g, 6.3 mmol) and
4-dimethylaminopyridine (40 mg, 0.32 mmol) were added and
the resulting solution was stirred at ambient temperature for
21 h. The precipitates were removed by filtration and washed
with chloroform (50 mL). The filtrate was washed three times
with saturated aq. NaHCO3 (30 mL each), dried over Na2SO4,
filtered and concentrated under reduced pressure. The residue
was chromatographed on a silica gel column with chloroform–
methanol (4 : 1, v/v) to give 11b (1.56 g, 65%). 1H NMR (CDCl3,
500 MHz): d = 0.10–0.11 (12H, m), 0.93–0.94 (18H, m), 1.45–
1.53 (4H, m), 2.01–2.03 (2H, m), 2.15–2.17 (2H, m), 2.19 (3H,
m), 2.57–2.58 (2H, m), 2.74–2.76 (2H, m), 3.48 (3H, s), 3.77–3.80
(1H, m), 3.84 (1H, m), 3.98–4.01 (1H, m), 4.12–4.15 (2H, m),
4.52 (1H, t, J = 4.9 Hz), 4.77–4.81 (1H, m), 6.17 (1H, d, J = 3.9
Hz), 7.84 (1H, s), 8.31 (1H, s), 8.51 (1H, s), 9.39 (1H, d, J = 7.6
Hz); 13C NMR (CDCl3, 126 MHz): d = -5.2, -4.6, -4.4, 18.4,
26.0, 26.2, 28.6, 29.9, 30.1, 30.6, 38.2, 58.7, 62.1, 69.9, 72.3, 85.5,
86.9, 101.4, 121.1, 141.3, 150.1, 151.4; HRMS (ESI): m/z calcd
5¢-O-(4,4¢-Dimethoxytrityl)-6-N-[N-(trans-4-
levulinyloxycyclohexyl)carbamoyl]- 2¢-O-methyladenosine
3¢-(2-cyanoethyl N,N-diisopropylphosphoramidite) (13b)
Compound 12b (250 mg, 0.30 mmol) was rendered anhydrous by
repeated co-evaporation three times each with dry pyridine, dry
toluene, dry CH2Cl2 and finally dissolved in dry CH2Cl2 (4.4 mL).
To this solution, diisopropylethylamine (261 mL, 1.5 mmol)
and chloro-2-cyanoethoxydiisopropylaminophosphine (177 mg,
0.75 mmol) were added, and the solution was stirred at ambient
temperature for 6 h. The reaction was quenched by addition
of water (1 mL), and the solution was diluted with chloroform
(50 mL). The solution was washed three times with saturated
aq. Na2CO3 (30 mL each), dried over Na2SO4, filtered and
concentrated under reduced pressure. The residue was trituated
with diisopropyl ether–diethyl ether (50 mL; 1 : 1, v/v), and the
resulting precipitates were collected by filtration to give 13b (223
mg, 73%). 1H NMR (CDCl3, 500 MHz): d = 1.06–1.25 (12H, m),
1.45–1.56 (4H, m), 2.01–2.03 (2H, m), 2.15–2.17 (2H, m), 2.20
(3H, m), 2.38 (1H, t, J = 6.10), 2.56–2.57 (2H, m), 2.64 (1H, t, J =
6.35), 2.74–2.76 (2H, m), 2.33–2.36 (1H, m), 2.37–2.51 (3H, m),
3.54–3.67 (4H, m), 3.78–3.79 (6H, m), 3.85–3.92 (1H, m), 4.34–
4.40 (1H, m), 4.56–4.57 (1H, m), 4.60–4.64 (1H, m), 4.79 (1H, m),
6.11–6.13 (1H, m), 6.78–6.81 (4H, m), 7.22–7.44 (9H, m), 7.84 (1H,
s), 8.08–8.13 (1H, m), 8.42–8.43 (1H, m), 9.37 (1H, d, J = 7.08);
13C NMR (CDCl3 126 MHz): d = 24.9, 28.6, 29.9, 30.1, 30.5, 38.2,
43.5, 48.3, 55.5, 63.3, 72.3, 84.1, 86.9, 113.4, 121.2, 127.2, 128.1,
128.4, 128.5, 130.3, 130.4, 130.4, 135.8, 144.7, 150.5, 151.3, 153.4,
158.8, 172.5, 206.9; 31P NMR (CDCl3, 203 MHz): d = 151.5, 152.2;
HRMS calcd. for C53H67N8NaO11P+ [M+Na]+: 1045.4559; found,
1045.4590.
for C35H59N6O8Si2 [M–H]-: 747.3938, found 747.3943.
-
5¢-O-(4, 4¢-Dimethoxytrityl)-6-N-[N-(trans-4-
levulinyloxycyclohexyl)carbamoyl]-2¢-O-methyladenosine (12b)
Compound 11b (1.6 g, 2.1 mmol) was rendered anhydrous by
repeated co-evaporation with dry pyridine, and the residue was
dissolved in pyridine (20 mL). Triethylamine trihydrofluoride
(1.8 mL, 11 mmol) and triethylamine (1.5 mL, 11 mmol) were
added and the resulting solution was stirred at ambient tempera-
ture for 3 days. The solvents were removed under reduced pressure,
and the residue was dissolved in chloroform (50 mL). The solution
was washed three times with aq. NaHCO3 (30 mL each), dried
over Na2SO4, filtered and concentrated under reduced pressure.
The residue was chromatographed on a silica gel column with
chloroform–methanol (98 : 2, v/v) to give the crude material of the
3¢,5¢-free derivative (0.98 g, 98%). This material (0.98 g, 1.9 mmol)
was rendered anhydrous by repeated co-evaporation with dry
pyridine, and finally dissolved in dry pyridine (10 mL). 4,4¢-
Dimethoxytrityl chloride (0.77 g, 2.3 mmol) was added and the
solution was stirred at ambient temperature for 3 h. The reaction
was quenched by addition of water (1 mL), and the solution
was diluted with chloroform (50 mL). The solution was washed
three times with saturated aq. NaHCO3 (30 mL each), dried over
Na2SO4, filtered and concentrated under reduced pressure. The
residue was chromatographed on a silica gel column with hexane–
chloroform (1 : 4, v/v) to give the crude material of 12b (1.1 mg,
70% from 11b).1H NMR (CDCl3, 500 MHz): d = 1.47–1.53 (4H,
m), 2.01–2.03 (2H, m), 2.15–2.17 (2H, m), 2.56–2.61 (3H, m), 2.74–
Synthesis of ON-2, ON-3 and ON-4
The terminally modified 2¢-O-methyl-oligoribonucleotides were
synthesized on a DNA/RNA synthesizer in 1 mmol scale by using
the commercially available 2¢-O-methyl-RNA phosphoramidites,
phosphoramidite 5 and 9, and the solid supports. For ON-2 and
ON-3, the universal supports II (Glen Research Inc.) was used.
The standard RNA synthesis protocol implemented in the DNA
synthesizer was used. The cleavage of the synthesized 5¢-DMTr-
oligonucleotide from the solid support and the deprotection
of the nucleobases were carried out by treatment with 28%
aqueous ammonia at room temperature for 8 h. The solution was
evaporated under reduced pressure at room temperature to remove
ammonia, and the residue was diluted with 0.1 M ammonium
acetate (50 mL). The solution was placed on a C-18 cartridge
column and the failure sequences were eluted by use of 10%
CH3CN/0.1 M ammonium acetate as an eluent. After being
1004 | Org. Biomol. Chem., 2012, 10, 994–1006
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washed with 0.1 M ammonium acetate and water, the column
was treated with aqueous 2% TFA to remove the DMTr group,
washed with 0.1 M ammonium acetate and water. The target
oligonucleotide was eluted by use of 30% CH3CN/water and the
fractions containing the target were lyophilized to give the crude
oligonucleotide. Pure material was obtained by purification on
an anion-exchange HPLC column by use of 0–50% gradient of
1 M NaCl in 25 mM sodium phosphate–10% CH3CN. The salts
were removed by use of the C-18 cartridge column to give the
pure oligonucleotide after being lyophilized to dryness. The yields
were calculated by assuming the molar extinction coefficients of
dGChcm and dGCmcm are identical to those of deoxyguanosine. ON-
2: MALDI-TOF mass [M+H]+ calcd 3792.8, found 3794.7; ON-3:
MALDI-TOF mass [M+H]+ calcd 3820.9, found 3821.0; ON-4:
MALDI-TOF mass [M+H]+ calcd 3352.7, found 3354.0.
Tm measurement
Each oligonucleotide was dissolved in 10 mM sodium phosphate
(pH 7.0) containing 0.1 M NaCl and 0.1 mM EDTA so that
the final concentration of each oligonucleotide was 1.0 mM. The
solution was separated into quartz cells (10 mm) and incubated
at 85 ◦C. After 800 s, the solution was cooled to 5 ◦C at the rate
of 0.5 ◦C min-1 and heated to 85 ◦C at the same rate. During this
annealing and melting, the absorption at 260 nm was recorded at
every 1 ◦C and used to draw UV-melting curves. The Tm value was
calculated as the temperature that gave the maximum of the first
derivative of the UV-melting curve. The Tm values are accurate
within 1 ◦C.
MD simulation
MD calculation was carried out by using the AMBER program31
with parameters for 2¢-O-methyl-RNA and backbone parameters
of parm99.parmbsc0 force field.32 First, the A-type duplex struc-
ture of 2¢-O-methyl-(ACAACCUACUA)/r(UAGUAGGUUGU)
was constructed by using the NUCGEN module. The 5¢- and 3¢-
terminal U residues were removed and the 5¢- and 3¢-terminal
2¢-O-methyl-A residues were changed to the phosphorylated
cyclohexylcarbamoyldeoxyguanosine (dGCmcmP) residues to give
the model structure of ON-5/RNA-9mer duplex. 29 Na+ ions
and 7 Cl- ions were added by using the addions command, and
the systems were surrounded by 3856 TIP3P model waters. The
cut-off distance for the non-bonding interactions was set to 9.0
Synthesis of ON-5, ON-7, ON-8, ON-9, and ODN-1 to ODN-10
By use of phosphoramidite 13a, 13b or 13c, 2¢-O-methyl-RNA
phosphoramidites, the appropriate fully protected oligonucleotide
XCmAmAmCmCmUmAmCmUm or Xd(N1N2ACCTACT), was syn-
thesized on the CPG supports. Here X represents the Lev protected
nucleoside residue introduced by using 13a, 13b or 13c. For the
synthesis of ON-5 and ON-7, the CPG supports were dried under
reduced pressure and then transferred to a glass syringe equipped
with a glass filter. 0.5 M NH2NH2/pyridine–acetic acid (60 mL : 40
mL) was added. After 15 min, the solution was eluted and the CPG
supports were washed three times with anhydrous acetonitrile and
then dried under reduced pressure. After 10 min argon gas was
introduced to the syringe, and 1H-tetrazole, (3.5 mg, 50 mmol) 2-
cyanoethyl [2-(4,4¢-dimethoxytrityloxy)ethyl]sulfonylethyl-N,N-
diisopropylphosphoramidite (32 mg, 50 mmol) and anhydrous
acetonitrile (100 mL) were added. After 1 min, the solution
was eluted and the CPG supports were washed three times
with acetonitrile. Subsequently, the phosphite intermediate was
oxidized by treatment with 0.1 M I2/pyridine–H2O (500 mL, 9 : 1,
v/v) for 2 min. For the syntheses of ON-8, ON-9 and ODNs, the
terminal 5¢-O-DMTr group was removed by treatment with 1%
TFA/CH2Cl2 for 30 s prior to the NH2NH2 treatment.
˚
A and the electrostatic interactions were treated with the PME
method. After the 5000 steps of minimization, the systems were
headed to 300 K within 0.1 ns with the positional restraints of
-2
˚
A
10 kcal mol-1
on the heavy atoms during which the volume
was kept constant. Afterwards, the system was equilibrated under
the constant pressure of 1 atm for 0.2 ns at 300 K without
any positional constraints. The simulation was continued for
additional 10 ns at 300 K. The data of the last 5 ns were used
for the analyses. The atomic charges of the aglycon part of dGChcmP
were calculated at the HF/6-31G* level.
The solution was eluted and the CPG supports were washed
three times with acetonitrile, dried under reduced pressure and
then treated with 2.0 M ammonia/methanol (2.0 ml) for 2 h. The
solution was eluted and the eluent was concentrated under reduced
pressure. The residue was dissolved in 28% aq. NH3 and the
reaction mixture was left to stand for 18 h. Ammonia was removed
under reduced pressure and the material was further purified by
anion-exchange HPLC. ON-5: MALDI-TOF mass [M+H]+ calcd
3448.7, found 3448.8; ON-7: MALDI-TOF mass [M+H]+ calcd
3448.68, found 3449.57; ON-8: MALDI-TOF mass [M+H]+ calcd
3528.65, found 3530.32; ON-9: MALDI-TOF mass [M+H]+ calcd
3498.63, found 3500.03; ODN-1: MALDI-TOF mass [M+H]+
calcd 3256.57, found 3258.81; ODN-2: MALDI-TOF mass
[M+H]+ calcd 3271.57, found 3277.29; ODN-3: MALDI-TOF
mass [M+H]+ calcd 3247.56, found 3247.56; ODN-4: MALDI-
TOF mass [M+H]+ calcd 3272.57, found 3274.48; ODN-5:
MALDI-TOF mass [M+H]+ calcd 3232.56, found 3234.88; ODN-
6: MALDI-TOF mass [M+H]+ calcd 3262.56, found 3262.45;
ODN-7: MALDI-TOF mass [M+H]+ calcd 3287.57, found
3289.11.
Acknowledgements
This study was supported by a grant-in-aid for Scientific Research
from the Ministry of Education, Culture, Sports, Science and
Technology, Japan, and Adaptable and Seamless Technology
Transfer Program of JST.
Notes and references
1 D. P. Bartel, Cell, 2004, 116, 281–297.
2 R. C. Lee, R. L. Feinbaum and V. Ambros, Cell, 1993, 75, 843–854.
3 S. Griffiths-Jones, H. K. Saini, S. V. Dongen and A. J. Enright, Nucleic
Acids Res., 2008, 36, D154–D158.
4 B. P. Lewis, C. B. Burge and D. P. Bartel, Cell, 2005, 120, 15–20.
5 I. Alevizos and G. G. Illei, Nat. Rev. Rheumatol., 2010, 6, 391–398.
6 M. Ferracin, A. Veronese and M. Negrini, Expert Rev. Mol. Diagn.,
2010, 10, 297–308.
7 Q. Z. Wang, W. Xu, N. Habib and R. Xu, Curr. Cancer Drug Targets,
2009, 9, 572–594.
8 C. L. Bartels and G. J. Tsongalis, Clin. Chem., 2009, 55, 623–31.
9 K. M. Nelson and G. Weiss, Mol. Cancer Ther., 2008, 7, 3655–60.
10 P. K. Mishra, N. Tyagi, M. Kumar and S. C. Tyagi, J. Cell. Mol. Med.,
2009, 13, 778–789.
This journal is
The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 994–1006 | 1005
©

View Article Online
11 G. Obernosterer, P. J. F. Leuschner, M. Alenius and J. Martinez, RNA,
2006, 12, 1161–1167.
12 J. M. Thomson, M. Newman, J. S. Parker, E. M. Morin-Kensicki, T.
Wright and S. M. Hammond, Genes Dev., 2006, 20, 2202–2207.
13 F. G. Wulczyn, L. Smirnova, A. Rybak, C. Brandt, E. Kwidzinski,
O. Ninnemann, M. Strehle, A. Seiler, S. Schumacher and R. Nitsch,
FASEB J., 2007, 21, 415–426.
14 H. Siomi and M. Siomi, Mol. Cell, 2010, 38, 323–332.
15 B. R. Cullen, Mol. Cell, 2004, 16, 861–865.
16 M. A. Newman and S. M. Hammond, Genes Dev., 2010, 24, 1086–
1092.
28 S. M. Freier, R. Kierzek, M. H. Caruthers, T. Neilson and D. H. Turner,
Biochemistry, 1986, 25, 3209–3213.
29 N. Sugimoto, R. Kierzek and D. H. Turner, Biochemistry, 1987, 26,
4559–1562.
30 T. Horn and M. S. Urdea, Tetrahedron Lett., 1986, 27, 4705–4708.
31 D. A. Case, T. A. Darden, T. E. Cheatham, III, C. L. Simmerling, J.
Wang, R. E. Duke, R. Luo, R. C. Walker, W. Zhang, K. M. Merz, B.
Roberts, B. Wang, S. Hayik, A. Roitberg, G. Seabra, I. Kolossva´ry,
K. F. Wong, F. Paesani, J. Vanicek, J. Liu, X. Wu, S. R. Brozell,
T. Steinbrecher, H. Gohlke, Q. Cai, X. Ye, J. Wang, M.-J. Hsieh, G.
Cui, D. R. Roe, D. H. Mathews, M. G. Seetin, C. Sagui, V. Babin, T.
Luchko, S. Gusarov, A. Kovalenko and P. A. Kollman, 2010, AMBER
11, University of California, San Francisco.
17 I. Heo, C. Joo, J. Cho, M. Ha, J. Han and V. N. Kim, Mol. Cell, 2008,
32, 276–284.
18 S. P. Jonstrup, J. Koch and J. Kjems, RNA, 2006, 12, 1747–1752.
19 P. T. Nelson, D. A. Baldwin, L. M. Scearce, J. C. Oberholtzer, J. W.
Tobias and Z. Mourelatos, Nat. Methods, 2004, 1, 155–161.
20 C. Chen, D. A. Ridzon, A. J. Broomer, Z. Zhou, D. H. Lee, J. T. Nguyen,
M. Barbisin, N. L. Xu, V. R. Mahuvakar, M. R. Andersen, K. Q. Lao,
K. J. Livak and K. J. Guegler, Nucleic Acids Res., 2005, 33, e179.
21 H. Wang, R. A. Ach and B. Curry, RNA, 2007, 13, 151–159.
22 K. Seio, Y. Takaku, K. Miyazaki, S. Kurohagi, Y. Masaki, A. Ohkubo
and M. Sekine, Org. Biomol. Chem., 2009, 7, 2440–2451.
23 T. Sasami, Y. Odawara, A. Ohkubo, M. Sekine and K. Seio, Tetrahedron
Lett., 2007, 48, 5325–5329.
32 (a) P. Auffinger and E. Westhof, Angew. Chem., Int. Ed., 2001, 40,
4648–4650; (b) C. I. Bayly, P. Cieplak, W. Cornell and P. A. Kollman,
J. Phys. Chem., 1993, 97, 10269–10280; (c) R. Aduri, B. T. Psciuk, P.
Saro, H. Taniga, H. B. Schlegel and J. SantaLucia, Jr., J. Chem. Theory
Comput., 2007, 3, 1464–1475; (d) Q. Zhang and T. Schlick, Biophys. J.,
2006, 90, 1865–1877; (e) A. Pe´rez, I. Marcha´n, D. Svozil, J. Sponer, T.
E. Cheatham, C. A. Laughton III and M. Orozco, Biophys. J., 2007,
92, 3817–3829.
33 S. Uesugi, H. Miki, M. Ikehara, H. Iwahashi and Y. Kyogoku,
Tetrahedron Lett., 1979, 20, 4073–4076.
34 Y. Masaki, R. Miyasaka, A. Ohkubo, K. Seio and M. Sekine, J. Phys.
Chem. B, 2010, 114, 2517–2524.
24 K. K. Ogilvie, Can. J. Chem., 1973, 51, 3799–3807.
25 E. Westman and R. Stro¨mberg, Nucleic Acids Res., 1994, 22, 2430–
2431.
35 R. Stolarski, L. Dudycz and D. Shugar, Eur. J. Biochem., 1980, 108,
111–121.
26 J. Nielse, M. Taagaard, J. E. Marugg, J. H. van Boom and O. Dahl,
Nucleic Acids Res., 1986, 14, 7391–7403.
27 A. V. Azhayev and M. L. Antopolsky, Tetrahedron, 2001, 57, 4977–
4986.
36 M. A. Peterson, B. L. Nilsson, S. Sarker, B. Doboszewski, W. Zhang
and M. J. Robins, J. Org. Chem., 1999, 64, 8183–8192.
37 (a) P. Maziere and A. J. Enright, Drug Discovery Today, 2007, 12, 452–
458; (b) U. A. Ørom and A. H. Lund, Gene, 2009, 451, 1–5.
1006 | Org. Biomol. Chem., 2012, 10, 994–1006
This journal is
The Royal Society of Chemistry 2012
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