Synthesis and hybridization properties of 2′-O-methylated oligoribonucleotides incorporating 2′-O-naphthyluridines

Article abstract of DOI:10.1039/c0ob00248h

2′-O-(1-Naphthyl)uridine and 2′-O-(2-naphthyl)uridine were synthesized by a microwave-mediated reaction of 2,2′-anhydrouridine with naphthols. Using the 3′-phosphoramidite building blocks, these 2′-O-aryluridine derivatives were incorporated into 2′-O-methylated oligoribonucleotides. Incorporation of five 2′-O-(2-naphthyl)uridines into a 2′-O-methylated RNA sense strand significantly increased the thermostability of the duplex with a 2′-O-methylated RNA antisense strand. Circular dichroism spectroscopy and molecular dynamic simulation of the duplexes formed between the modified RNAs and 2′-O-methyl RNAs suggested that there are π-π interactions between two neighboring naphthyl groups in a sequence of the five consecutively modified nucleosides.

Full text of DOI:10.1039/c0ob00248h

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PAPER
Synthesis and hybridization properties of 2¢-O-methylated
oligoribonucleotides incorporating 2¢-O-naphthyluridines†
Mitsuo Sekine,* Yusuke Oeda, Yoshihiro Iijima, Haruhiko Taguchi, Akihiro Ohkubo and Kohji Seio
Received 12th June 2010, Accepted 8th September 2010
DOI: 10.1039/c0ob00248h
2¢-O-(1-Naphthyl)uridine and 2¢-O-(2-naphthyl)uridine were synthesized by a microwave-mediated
reaction of 2,2¢-anhydrouridine with naphthols. Using the 3¢-phosphoramidite building blocks, these
2¢-O-aryluridine derivatives were incorporated into 2¢-O-methylated oligoribonucleotides.
Incorporation of five 2¢-O-(2-naphthyl)uridines into a 2¢-O-methylated RNA sense strand significantly
increased the thermostability of the duplex with a 2¢-O-methylated RNA antisense strand. Circular
dichroism spectroscopy and molecular dynamic simulation of the duplexes formed between the
modified RNAs and 2¢-O-methyl RNAs suggested that there are p–p interactions between two
neighboring naphthyl groups in a sequence of the five consecutively modified nucleosides.
RNA was based on the controlled reaction of RNA with F-DNP
to attain 70% modification, the real modified structure effective
Introduction
2¢-O-Modified ribonucleosides have been previously used as
monomer components of antisense molecules for thermal sta-
bilization of duplexes formed with complementary RNAs in
antisense/antigene and RNAi strategies.1–3 Among them, the 2¢-
O-alkylated species, exemplified by 2¢-O-methoxyethyl ribonucle-
osides, is one of the most frequently used.4 In contrast, only a few
2¢-O-aryl substituted ribonucleoside derivatives have been used
as monomer components for these approaches.5,6 It was reported
that oligodeoxynucleotide derivatives incorporating the simplest
2¢-O-phenyluridine lost a significant amount of the hybridization
affinity for the complementary DNA strands.5 On the other hand,
Wang and coworkers have extensively reported hybridization
and the biological properties of oligoribonucleotides incorpo-
rating 2¢-O-(2,4-dinitrophenyl)ribonucleosides.6a These 2¢-O-(2,4-
dinitrophenyl) (DNP)-modified RNA derivatives were obtained
by reacting RNA oligomers with 1-fluoro-2,4-dinitrobenzene (F-
DNP).6b They reported that DNP-RNA/RNA duplexes showed
higher thermostability than unmodified RNA/RNA duplexes,6c
and DNP-RNA acquired remarkable resistance to RNases and
phosphodiesterases6e as well as an excellent membrane perme-
ability even in the absence of transfection agents.6e–f They also
showed strong antisense effects on the expression of mRNA
of RIa/PKA in cancer cells6f–h and life-prolongation effects of
SCID mice bearing human breast cancer6f or mice infected with
Moloney murine leukemia virus.6i Since the synthesis of DNP-
for this observation was unclear. Therefore, it is desirable to
develop a general method for the synthesis of 2¢-O-arylated RNA
oligomers to clarify systematically the effect of aryl groups on
the hybridization ability and enzyme resistance of such materials
(Fig. 1). With this background, we studied the synthesis of
oligoribonucleotides modified with three kinds of 2¢-O-aryluridine
derivatives.
Fig. 1 Illustration of 2¢-O-arylated RNA oligomers capable of in-
tramolecular stacking interaction.
In this paper, we report the incorporation of these 2¢-O-modified
ribonucleosides into 2¢-O-methylated oligoribonucleotides as well
as their unique hybridization and enzymatic properties. In ad-
dition, the detailed structural analysis of these modified oligori-
bonucleotides is also reported based on circular dichroism (CD)
spectroscopy and molecular dynamic (MD) simulations of model
compounds.
Department of Life Science, Tokyo Institute of Technology, 4259, Nagatsuta,
Midoriku, Yokohama 226-8501, Japan. E-mail: msekine@bio.titech.ac.jp;
Fax: +81 45 924 5772; Tel: +81 45 924 5706
† Electronic supplementary information (ESI) available: The detailed
experimental section and the 1H and 13C data of all new products. See
DOI: 10.1039/c0ob00248h
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Table 1 Sequences of synthesized DNA and RNA oligomers incorporat-
ing 2¢-O-aryluridinesa
Results and discussion
Synthesis of 2¢-O-naphthyluridine 3¢-phosphoramidite building
blocks
RNA
sequence
Sense Strand
RNA 8
To expand the use of aromatic substituents as 2¢-O-modifiers,
we focused on the use of 1- and 2-naphthyl groups as the 2¢-O-
substituents. It seems that these groups interact with themselves
more effectively when 2¢-O-naphthylribonucleosides are arranged
in a consecutive base sequence. It is of great importance to
examine whether the resulting stacking interaction enhances
the hybridization affinity for complementary RNA strands as
well as whether increased bulkiness of the aromatic ring in-
creases the resistance to enzymatic digestion. Therefore, we
synthesized the 2¢-O-phenyluridine, 2¢-O-(1-naphthyl)uridine and
2¢-O-(2-naphthyl)uridine 3¢-phosphoramidite building blocks 4a–
c, as shown in Scheme 1.
3¢-(CUGAAAAACUGA)-5¢
3¢-(CUGAAAAACUGA)-5¢
RNA 9
Antisense Strand
RNA 10
RNA 5a
RNA 5b
RNA 5c
RNA 6a
RNA 6b
RNA 6c
5¢-(GACUUUUUGACU)-3¢
5¢-(GACUU Ph UUGACU)-3¢
5¢-(GACU PhPhPh UGACU)-3¢
5¢-(GAC PhPhPhPhPh GACU)-3¢
5¢-(GACUU 1Np UUGACU)-3¢
5¢-(GACU 1Np1Np1Np UGACU)-3¢
5¢-(GAC 1Np1Np1Np1Np1Np GACU)-3¢
5¢-(GACUU 2Np UUGACU)-3¢
5¢-(GACU 2Np2Np2Np UGACU)-3¢
5¢-(GAC 2Np2Np2Np2Np2Np GACU)-3¢
RNA 7a
RNA 7b
RNA 7c
a Ph: 2¢-O-phenyluridine, 1Np: 2¢-O-(1-naphthyl)uridine, 2Np: 2¢-O-(2-
naphthyl)uridine, underlined letter: 2¢-O-methylribonucleoside
Fig. 2 2¢-O-Methylated RNA 12-mers (RNAs 5a–c, RNAs 6a–c, and
RNAs 7a–c) incorporating 2¢-O-aryluridines; a: n = 1, b: n = 3, c: n = 5
(for the full structures see Table 1).
Hybridization ability of 2¢-O-methyl-RNA 12mers incorporating
2¢-O-aryluridines
Scheme 1 Synthesis of 2¢-O-aryluridine 3¢-phosphoramidite derivatives
4a–c.
As mRNAs are targets in the antisense or RNAi strategies,1
we examined the hybridization ability of 2¢-O-methylated olig-
oribonucleotides (RNAs Xa–c: X = 5–7) incorporating 2¢-O-
aryluridines (2a–c) toward the complementary RNA 12mer [3¢-
(CUGAAAAACUGA)-5¢] (RNA 8). When RNAs 5a, 5b and
5c were hybridized with RNA 8, the resulting duplexes showed
markedly lower Tm values with DTm per one modification of -2.3,
-3.4 and -3.7 ◦C, respectively, than the unmodified RNA 12-mer
(RNA 10), as shown in Fig. 3A. A similar tendency was obtained in
the case of the 2¢-O-(1-naphthyl) modified RNAs 6a–c that showed
DTm values per one modification of -1.9, -4.4 and -4.4 ◦C for
RNAs 6a, 6b and 6c, respectively (Fig. 3B).
Quite recently, we reported a convenient method for the
synthesis of 2¢-O-aryluridines from 2,2¢-anhydrouridine by
microwave-mediated reactions.7 2¢-O-Phenyluridine (2a: Ph), 2¢-
O-(1-naphthyl)uridine (2b: 1Np) and 2¢-O-(2-naphthyl)uridine (2c:
2Np) were synthesized by this method. The usual tritylation of
2a–c gave 5¢-O-dimethoxytritylated products (3a–c). The phos-
phoramidite derivatives 4a–c could be synthesized easily by the
usual 3¢-phosphitylation. Purification using silica gel column
chromatography was very easy as the lipophilicity of these
products is very high.
Interestingly, it was found that the hybridization affinity of 2¢-
O-(2-naphthyl) modified RNAs 7a–c for the complementary RNA
8 did not decrease as significantly as it did for RNAs 6a–c. Since
partial incorporation of 2¢-O-methylribonucleosides into the sense
strands of siRNAs did not affect the RNAi activity, as reported
by Tuschl,9 the sense strand was changed from RNA 8 to 2¢-O-
methylated RNA strand (RNA 9). It is also known that the 5¢-
terminal site of antisense strands of siRNAs should have weaker
binding ability to the sense sequence.1,2 Therefore, it is of interest to
see if oligoribonucleotides incorporating 2¢-O-arylribonucleosides
Synthesis of oligoribonucleotides incorporating
2¢-O-naphthyluridine derivatives
By the general procedure of the phosphoramidite approach,8
we synthesized 2¢-O-methylated oligoribonucleotides (RNAs 5a–
c, RNAs 6a–c and RNAs 7a–c) incorporating these modified
nucleoside derivatives (Fig. 2 and Table 1) and then purified them
by HPLC. In the nucleotide sequence, 2¢-O-methylribonucleosides
were used, except for the modified ribonucleosides.
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Table 2 Coupling constants of J1¢,2¢ and J3¢,4¢ and N (%) of 2a–c
Compound
J1¢,2¢
J3¢,4¢
N (%)
2a (Ph)
2b (1Np)
2c (2Np)
5.0
5.1
4.9
5.0
5.0
4.9
50
49.5
50
incorporation of 1Nps (when RNA 6c was used) resulted in a
significant recovery of the Tm value, exceeding the◦original Tm of
the unmodified RNA 10, with an increase of 2.2 C (DTm value
per one modification = 0.4 ◦C).
On the other hand, RNA 7a with one 2Np gave a slightly
increased Tm value (Fig. 4A) and RNA 7b with three 2Nps gave
almost the same Tm value as that of RNA 10 (Fig. 4B). In contrast
to these results, RNA 7c incorporating five 2NPs showed a more
significant increase (DTm = 5.9 ◦C, DTm per one modification =
1.2 ◦C) in the binding affinity for fully 2¢-O-methylated RNA 9
(Fig. 4C).
Fig. 3 Tm values of duplexes of RNA 8 with RNA 10, RNAs 5a–c, RNAs
6a–c and RNAs 7a–c.
Sugar puckering of modified nucleosides
can have increased binding affinity for complementary 2¢-O-
methylated RNA strands. The results obtained by hybridization
experiments using modified RNAs 5a–c, RNAs 6a–c and RNAs
7a–c, are shown in Fig. 4.
Since sugar puckering is an essential factor in regulation of RNA
duplex stability,10 we also measured the ratio of the C3¢-endo (N)
and C2¢-endo (S) conformers in 2¢-O-modified nucleosides 2a–c
by NMR analysis. N (%) values were calculated by the following
equation: N (%) = 100 ¥ J3¢,4¢/(J1¢,2¢ + J3¢,4¢).11 These results are
summarized in Table 2.
To our surprise, these 2¢-O-arylated uridine derivatives ex-
ist in an almost 1 : 1 ratio of the two conformers, showing
conformational flexibility. Apparently, modified RNA oligomers
containing these 2¢-O-aryluridines require conformational change
to A-type sugar puckering (C3¢-endo) when hybridized with
the target complementary RNA oligomer. This conformational
change induces energy loss. Nonetheless, it is of great interest
that the duplex formed between 2¢-O-methylated RNA 9 and 2¢-
O-methylated RNA 7c containing five 2¢-O-(2-naphthyl)uridines
showed remarkably higher thermostability than that derived from
RNA 9 and RNA 10.
CD spectra of single stranded modified RNAs and their double
stranded duplexes with RNA 8 and 2¢-O-methylated RNA 9
To understand why the 2¢-O-methylated RNA 9/RNA 7c duplex
where five 2¢-O-(2-naphthyl)uridines were incorporated into the
antisense strand showed unexpectedly high thermostability com-
pared with the unmodified RNA/RNA duplexes, we conducted
detailed experiments on modified RNA single strands by CD
spectroscopy.12
As shown in Fig. 5, when one or more 2¢-O-phenyluridines
were incorporated into the RNA antisense strand, the intensity
of the positive and negative Cotton effects changed although the
total shape remained almost unchanged (Fig. 5A). Therefore, there
is no significant interaction of the phenyl group with itself or
with other nucleobases in the single strand. In contrast to this
result, RNAs 6a–c containing 2¢-O-(1-naphthyl)uridines exhibited
characteristic strong negative Cotton effects at around 240 nm
(Fig. 5B). These effects might be due to some interactions between
the neighboring 1-naphthyl groups or between this group and the
nearby base residues. In addition to this characteristic peak, RNA
Fig. 4 Tm values of duplexes of 2¢-O-methylated RNA 9 with RNA 10,
RNAs 5a–c, RNAs 6a–c and RNAs 7a–c.
In the case of duplexes of RNAs 5a–c, 6a–c and 7a–c with 2¢-
O-unmodified RNA 8, Tm values decreased unexceptionally with
an increase in the number of modified nucleosides, as shown in
Fig. 3A–C. However, RNAs 5–7 showed strange behavior when
hybridized with RNA 9. Tm values of RNA 9/RNAs 5a–c duplexes
decreased with an increase in the number of Ph, as shown in
Fig. 4A, but the degree of the drop of Tm was moderate compared
with that of RNA 8/RNAs 5a–c (see Fig. 3A). RNAs 5a, 5b
and 5c showed DTm values per one modification of -1.8, -2.4
and -2.0 ◦C, respectively. In a series of RNAs 6a–c, unexpected
behavior was observed as shown in Fig. 4B: RNA 6a incorporating
one 1Np showed a slightly increased Tm compared with that of the
unmodified RNA 10, while incorporation of three 1Nps (when
RNA 6b was used) resulted in a sharp drop in Tm, but five-point
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shown in Fig. 7, but the peak intensities at 260 nm increased more
markedly than those seen in Fig. 6. It seems that this result suggests
the conformational change in the base residues becomes somewhat
more restricted.
Fig. 5 CD spectra of single stranded 2¢-O-methylated RNAs 5a–c,
6a–c and 7a–c incorporating Ph, 1Np and 2Np, respectively, where
2¢-O-methyluridines were replaced by 2¢-O-aryluridines (X).
6c showed a clear positive Cotton effect at 215 nm. Since 2¢-O-
(1-naphthyl)uridine (2b) has a strong UV lmax at 215 nm, this
eminent peak might be based on the geometrically regulated 1-
naphthyl group in the single strand. In the case of RNAs 7a–c
containing 2¢-O-(2-naphthyl)uridines (2Np), broad monotonous
negative Cotton effects were observed at around 230–240 nm
(Fig. 5C).
Fig.
7
CD spectra of RNA–RNA duplexes formed between
2¢-O-methylated RNA 9 and 2¢-O-methylated RNAs 5a–c, 6a–c and 7a–c
incorporating Ph, 1Np and 2Np, respectively, where 2¢-O-methyluridines
were replaced by 2¢-O-aryluridines (X).
The duplexes of the complementary unmodified RNA 8 with 2¢-
O-phenyl- and 2¢-O-(1-naphthyl) modified RNAs 5a–c and RNAs
6a–c showed roughly similar whole CD patterns, although the
peak intensities significantly changed at 270 nm and 220–240 nm,
as shown in Fig. 6A and B, respectively.
MD simulation of duplexes incorporating modified nucleosides
To confirm whether the 2¢-O-methyl group in the sense strand
affects the thermostability of modified RNA–RNA duplexes, MD
simulation of RNA duplexes containing modified nucleosides was
carried out using the standard AMBER system13,14 (for the details
of the procedure see Experimental Section) on a supercomputer
(Tsubame, Tokyo Tech.).
In
the
case
of
5¢-(GACUU1NpUUGACU)-3¢/3¢-
(CUGAAAAACUGA)-5¢ where the underline denotes 2¢-
O-methylribonucleosides, we selected the typical structures
during a 5000 ps MD simulation after 50 ps equilibrium. These
structures showed that the stabilized forms were of two main
structures; 1-naphthyl group extruded to the outer space (Fig. 8B)
in the first, and in the second one it covered the minor groove
(Fig. 8C).
When MD simulation of the duplex of 5¢-(GACU1Np1Np
1NpUGACU)-3¢/3¢-(CUGAAAAACUGA)-5¢ containing three
1Nps was performed, each 1-naphthyl group existed indepen-
dently in conformations similar to those observed in the above
simulation. No p–p stacking between two 1-naphthyl groups could
be seen (Fig. 8F) but a CH–p-type interaction-like structure15 was
often observed in the snapshots (Fig. 8E).
Fig. 6 CD spectra of RNA–RNA duplexes formed between unmodified
RNA 8 and 2¢-O-methylated RNAs 5a–c, 6a–c and 7a–c incorporating Ph,
1Np and 2Np, respectively, where 2¢-O-methyluridines were replaced by
2¢-O-aryluridines (X).
In the duplex of 5¢-(GACUU2NpUUGACU)-3¢/3¢-
(CUGAAAAACUGA)-5¢ containing a 2Np, two types of
In sharp contrast, the CD spectra of the duplexes containing
2¢-O-(2-naphthyl) modified RNAs 7a–c showed dramatically dif-
ferent CD patterns at around 230 nm (Fig. 6C). In particular, the
strong negative Cotton effect at 234 nm observed in RNAs 7b and
7c was characteristic. This result suggests there is an interaction
between two neighboring 2-naphthyl groups since 2Np has a strong
lmax at 225 nm in its UV spectrum (Fig. S1†).
When the modified RNAs 5a–c and RNAs 6a–c were hybridized
with the 2¢-O-methylated RNA 9, the overall CD patterns were
basically similar to those observed in the unmodified RNA 8, as
structures (Fig. 9B and C) were observed as described in the case
of the 1-naphthyl group. When the three modified nucleosides
were arranged in the RNA duplex, the apparent p–p interaction
between two 2-naphthyl groups in the double strand was
observed, as shown by the red circle in Fig. 9E and F, but the
three 2-naphthyl groups never stack in a consecutive manner.
Only neighboring two of the three 2-naphthyl groups allow p–p
stacking interaction. This interaction might increase the duplex
stability.
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Fig. 10 Snapshots of the typical orientation of 2-naphthyl groups in
a fully 2¢-O-methyl modified RNA duplex containing three consecutive
2Nps after MD simulation.
the duplex. In conclusion, p–p interaction and the hydrophobic
effect would be essential for stabilization of RNA–RNA duplexes
incorporating 2¢-O-(2-naphthyl)uridines.
Nuclease resistance of modified oligomers
Fig. 8 MD simulation of duplexes of an unmodified RNA 12-mer with
a 2¢-O-methyl-RNA 12-mer incorporating one and three 1Nps. Panel A:
Initial structure before MD simulation. Panels B and C: Representative
snapshots during MD simulation of the duplex incorporating one 1Np
where the 1Np group was seen in two forms: protruded (B) and covered
(C). Panel D: Initial structure before MD simulation. Panels E and F:
Representative snapshots of MD simulation of the duplex incorporating
three 1Nps where 1-naphthyl groups were orientated in different directions
without stacking interactions.
We examined the enzymatic property of 2¢-O-naphthyl modified
oligoribonucleotide derivatives using snake venom phosphodi-
esterase (SVP) and spleen phosphodiesterase (SPD). As substrates,
four dinucleoside monophosphate derivatives 11–14 were synthe-
sized using a liquid-phase synthesis (Fig. 11).
Fig. 11 Structures of 2¢-O-modified UpU derivatives as substrates for
phosphodiesterases.
Results of enzymatic reactions of these substrates are shown
in Fig. 12. In the digestion of 11–14 with SVP, surprisingly, 2¢-
O-phenyl- and 2¢-O-(2-naphthyl) modified dimers 12 and 14 were
Fig. 9 MD simulation of duplexes of an unmodified RNA 12-mer
with
a 2¢-O-methyl-RNA 12-mer incorporating one and three
2¢-O-(2-naphthyl)uridines. Panel A: Initial structure before MD simula-
tion. Panels B and C: Representative snapshots during MD simulation
of the duplex incorporating one 2Np where 2-naphthyl groups were seen
in two forms: protruded (B) and covered (C). Panel D: Initial structure
before MD simulation. Panels E and F: Representative snapshots during
MD simulation of the duplex incorporating three 2Nps where two of three
2-naphthyl groups were stacked, as shown by the red circle.
Next, MD simulation was performed after the unmodified
RNA strand was replaced with fully 2¢-O-methylated RNA. A
similar MD simulation of 5¢-(GACU2Np2Np2Np UGACU)-
3¢/3¢-(CUGAAAAACUGA)-5¢ suggested that the 2-naphthyl
group interacts with the nearby 2¢-O-methyl groups of the
complementary strand using hydrophobic interaction, as shown
in Fig. 10. This effect also contributes to further stabilization of
Fig. 12 Enzyme resistance of modified UpU toward snake venom
phosphodiesterase (Panel A) and calf-spleen phosphodiesterase (Panel B).
Red: compound 11, green: compound 12, blue: compound 13 and orange:
compound 14.
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digested most rapidly, as shown in Fig. 12A. The digestion was ca.
three time faster than that of the simplest 2¢-O-methylated species
11. 2¢-O-(1-naphthyl) modified species 13 exhibited a degradation
rate similar to that of 11.
On the other hand, enzymatic reactions of 11–14 with SPD
gave entirely different results, as shown in Fig. 12B. At 140 min,
ca. 80% of the 2¢-O-methylated UpU 11 was digested while 2¢-O-
phenyl- and 2¢-O-(1-naphthyl) modified UpU derivatives 11 and
13 proved to be the most resistant among all the derivatives to this
enzyme showing ca. 20% digestion at 140 min, and ca. 40% of the
2¢-O-(2-naphthyl) modified dimer 14 was digested at this moment.
From the above mentioned results, 2¢-O-(1-naphthyl) modified
UpU proved to be most resistant to the two phosphodiesterases.
Conclusions
Aromatic groups have an inherent property capable of a potential
p–p stacking interaction with themselves. For this interaction, 1-
naphthyl and 2-naphthyl groups are more accessible than a phenyl
group because the former have planar structures two times as
wide as the latter. The results of MD simulation of modified
RNA/RNA duplexes suggested that there might be a p–p
interaction in the double-stranded duplex.
The significant increase or recovery in Tm value in the modified
RNA/2¢-O-methylated RNA duplexes should lead us to recon-
sider the synthetic value of 2¢-O-arylated RNAs as alternatives to
widespread 2¢-O-alkyl substituted siRNA or antisense molecules.
Our present research also implies that, since incorporation of 1Np
or 2Np into 2¢-O-methylated RNA oligomers did not affect its base
recognition ability, such a 2¢-O-aryluridine block would be used as
a new site for modification of oligonucleotides with a wide variety
of functional groups that can be attached to the aromatic ring
using halogenated or aminated aromatic substituents. We are now
studying further applications using these new 2¢-O-aryluridine
derivatives.
Base discrimination ability of 2¢-O-naphthylribonucleoside
derivatives in RNA oligomers
To examine whether one-point modification of an RNA oligomer,
5¢-(GACUUXUUGACU)-3¢, affects the base recognition ability
of A against G, C and U, we measured Tm values of the duplexes of
modified RNA 12-mers (RNAs 5a, 6a and 7a) with complementary
RNA 8 and mismatched RNA 12-mers (RNAs 15–17) having G,
C, and U, respectively, at the central position. These results are
summarized in Fig. 13.
Experimental
General remarks
1H, 13C and 31P NMR spectra were obtained at 500, 126 and
203 MHz, respectively. Chemical shifts were measured from
tetramethylsilane (0.0 ppm) or DMSO-d6 (2.49 ppm) for 1H NMR,
CDCl3 (77.0 ppm) or DMSO-d6 (39.7 ppm) for 13C NMR and
85% phosphoric acid (0.0 ppm) for 31P NMR. UV spectra were
recorded on a U-2000 spectrometer. Column chromatography was
performed with silica gel C-200 purchased from Wako Co. Ltd.,
and a minipump of a goldfish bowl was conveniently used to attain
sufficient pressure for rapid chromatographic separation. HPLC
was performed using the following systems. Reversed-exchange
HPLC was done on a Waters Alliance system with a Waters 3D
UV detector and a Waters XTerra MS C18 column (4.6 ¥ 150 mm).
A linear gradient (0–10%) of solvent I (0.03 M ammonium acetate
buffer (pH 7.0)) in solvent II (CH3CN) was used at 50 ◦C at a
rate of 1.0 mL min-1 for 30 min. Anion-exchange HPLC was done
on a Shimadzu SLC-10A connected with LC-10 AD VP, CTO-
10A, SPD-M10A, and a Gen-PakTM FAX column (Waters, 4.6 ¥
100 mm). A linear gradient (0–30%) of solvent III (1 M NaCl
in 25 mM phosphate buffer (pH 6.0) containing 10% CH3CN)
in solvent IV (25 mM phosphate buffer (pH 6.0) containing 10%
CH3CN) was used at 50 ◦C at a flow rate of 1.0 mL min-1 for 30 min.
For a large-scale synthesis, preparative HPLC was performed with
a JAIGEL GS-310 column using LC-9021R with a UV detector
S-3110 at the flow rate of 5 ml min-1. ESI mass spectrometry
was performed using MarinerTM (PerSeptive Biosystems Inc.).
MALDI-TOF mass spectrometry was performed using a Bruker
Daltonics [Matrix: 3-hydoroxypicolinic acid (100 mg ml-1) in
H2O–diammonium hydrogen citrate (100 mg ml-1) in H2O (10 : 1,
v/v)]. MD simulation was carried out with Sun Dire X4600 using
AMBER ver 7.0.16
Fig. 13 Base discrimination ability of 2¢-O-aryl one-point modified RNA
12-mers.
The base-discrimination ability of modified RNAs was eval-
uated using the difference between the Tm value of the duplex
of fully 2¢-O-methylated RNA 10 with complementary RNA 8
and the next largest Tm observed among the three mismatched
duplexes. As shown in Fig. 13, the recognition patterns of all
four RNAs 10 and 5a–7a were basically similar to each other, but
2¢-O-aryl modified RNA 5a–7a exhibited better base recognition
abilities than 2¢-O-methyl modified RNA 10, which showed the
poorest discrimination ability with DTm = 6.1 ◦C. In particular,
RNA 6a showed the biggest difference (DTm = 10.3 ◦C) in
discrimination between RNA 8 (Y = A) and RNA 15 (Y = G).
These results suggested that replacement of ribonucleosides with
2¢-O-aryluridines could enhance the base recognition ability of
original RNA sequences. It is interesting if the formation of the
mismatched base pair G–U could be suppressed by 2¢-O-aryl
modification. Further studies are needed to generalize this effect.
Tm analysis of duplexes
Tm analysis was performed using a UV spectrometer. An appro-
priate oligomer (1 mM) and its complementary strand (1 mM) were
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dissolved in 10 mM phosphate buffer (pH 7.0) containing 0.1 M
NaCl and 0.1 mM EDTA. The solution was kept at 85◦◦C or 70 ◦C
for 5 min and the temperature was decreased to 10 C at a rate
102.8, 110.1, 113.6, 118.7, 124.9, 127.1, 127.2, 127.5, 127.9, 128.3,
128.4, 130.0, 130.3, 130.5, 134.3, 135.1, 135.3, 140.3, 144.3, 150.0,
154.7, 159.0, 159.0, 162.5. ESI-MS m/z calcd for C40H36N2NaO8
[M+Na] 695.2364, found 695.2579.
◦
of 0.5 C min-1. After that, UV absorbance was measured at an
interval of 1 ◦C by increasing the temperature to 85 ◦C or 70 ◦C. An
UV melting curve was obtained by smoothing the original data
7 times using Dtavitzky–Golay method (25 points). Differentiation
of this UV melting curve gave the Tm value. concentration of
modified oligonucleotides was calculated using the e values of the
unmodified oligonucleotides. The average data of the Tm values
thus obtained was calculated and used for discussion.
2¢-O-Phenyl-5¢-O-(4,4¢-dimethoxytrityl)uridine-3¢-(2-cyanoethyl
N,N-diisopropyl)phosphoramidite (4a)
Compound 3a (100 mg, 0.16 mmol) was coevaporated thrice
with each of anhydrous pyridine and anhydrous toluene, and
then dissolved in anhydrous CH2Cl2 (400 ml, 0.4 M) under
argon atmosphere. Diisopropylamine (14 ml, 0.10 mmol), 1H-
tetrazole (6.7 mg, 0.10 mmol) and 2-cyanoethyl N,N,N¢,N¢-
tetraisopropylphosphorodiamidite (60.3 mg, 0.20 mmol) were
added to this solution. After the reaction, the mixture was diluted
with CH2Cl2 and quenched by NaHCO3 (aq). The solution was
extracted with CH2Cl2 and washed with brine. The organic layer
was dried over anhydrous Na2SO4. After filtration, the filtrate was
evaporated in vacuo. The residue was purified by RP-HPLC using
acetonitrile as an eluent to yield compound 4a (75%).
2¢-O-Phenyl-5¢-O-(4,4¢-dimethoxytrityl)uridine (3a)
Compound 2a (100 mg, 0.32 mmol) was coevaporated 3 times with
anhydrous pyridine and dissolved in anhydrous pyridine (800 ml,
0.4 M) under argon atmosphere. 4,4¢-Dimethoxytrityl chloride
(126.9 mg, 0.37 mmol) was added to the above solution, and the
mixture was stirred for 4 h. After the reaction, NaHCO3 (aq)
was added to the mixture and the solution was extracted with
ethyl acetate. The organic layer was washed thrice with brine
and dried over anhydrous Na2SO4. After filtration, the filtrate
was evaporated in vacuo. The residue was purified by N60 silica
gel column chromatography (hexane/CHCl3 50–75%) to give
compound 3a (91%).
1H NMR (CDCl3, 500 MHz) d 0.99 (6H, d, J = 6.6 Hz), 1.09
(6H, t, J = 7.6 Hz), 1.27 (2H, J = 6.8 Hz), 2.45 (2H, m), 3.43–3.75
(6H, m), 3.80 (6H, d, J = 4.4 Hz), 4.35 (1H, t, J = 2.7 Hz), 4.74
(1H, m), 5.00 (1H, d, J = 4.2 Hz), 5.28 (1H, t, J = 9.3 Hz), 6.21 (1H,
d, J = 3.7 Hz), 6.85 (4H, t, J = 8.1 Hz), 7.03 (3H, m), 7.24–7.33
(9H, m), 7.41 (2H, m), 7.95(1H, t, J = 8.3 Hz);13C NMR (CDCl3,
125 MHz) d 20.3, 20.4, 24.6, 24.7, 43.2, 43.3, 55.4, 55.4, 55.4, 58.3,
58.5, 61.9, 80.2, 83.0, 87.4, 88.0, 102.6, 113.5, 116.1, 116.3, 117.8,
122.1, 122.2, 127.4, 128.2, 128.4, 128.4, 129.7, 130.4, 130.4, 135.1,
135.2, 140.2, 144.3, 150.2, 157.9, 158.9, 162.9; 31P NMR (CDCl3,
203 MHz) d 151.9, 151.7. ESI-MS m/z calcd for C45H52N4O9P
[M+H] 823.3466, found 823.3455.
1H NMR (CDCl3, 500 MHz) d 3.56–3.63 (2H, m), 3.80 (6H, s),
4.24–4.25 (1H, m), 4.68 (1H, q, J = 5.9 Hz), 4.88 (1H, t, J = 4.0
Hz), 5.30 (1H,d, J = 8.1 Hz), 6.19 (1H, d, J = 3.9 Hz), 6.85 (4H,
d, J = 8.6 Hz), 7.08 (3H, d, J = 7.6 Hz), 7.26–7.35 (9H, m), 7.40
(2H, d, J = 7.6 Hz), 7.95 (1H, d, J = 8.3 Hz), 8.30 (1H, s); 13C
NMR (CDCl3) d 55.4, 62.3, 69.9, 81.3, 83.7, 87.3, 102.7, 113.5,
116.6, 123.3, 127.4, 128.2, 128.3, 130.1, 130.3, 130.4, 135.1, 135.3,
140.3, 144.4, 150.0, 157.0, 158.9, 159.0, 162.7. ESI-MS m/z calcd
for C36H34N2NaO8 [M+Na] 645.2207, found 645.2041.
2¢-O-(1-Naphthyl)-5¢-O-(4,4¢-dimethoxytrityl)-3¢-(2-cyanoethyl
N,N-diisopropyl)phosphoramidite (4b)
2¢-O-(1-Naphthyl)-5¢-O-(4,4¢-dimethoxytrityl)uridine (3b)
Compound 4b was synthesized from 3b according to the same
procedure as that described for compound 4a.
Compound 3b was synthesized from 2b according to the same
procedure as that described for compound 3a.
1H NMR (CDCl3, 500 MHz) d 0.72 (3H, d, J = 6.6 Hz), 0.93
(3H, d, J = 6.6 Hz), 0.99 (3H, d, J = 6.6 Hz), 1.28 (3H, t, J = 6.8
Hz), 2.15 (1H, m), 2.35 (1H, m), 3.38 (2H, m), 3.49–3.62 (3H, m),
3.69 (1H, t, J = 9.2 Hz), 3.80 (6H, d, J = 4.6 Hz), 4.53 (1H, m),
4.90 (1H, m), 5.15 (1H, m), 5.32 (1H, dd, J = 8.2, 16.5 Hz), 6.41
(1H, dd, J = 4.0, 8.2 Hz), 6.86 (4H, t, J = 8.2 Hz), 7.10 (1H, dd, J =
7.6, 12.5 Hz), 7.26–7.49 (14H, m), 7.78 (1H, dd, J = 7.6, 15.6 Hz),
1H NMR (CDCl3, 500 MHz) d 3.60–3.68 (2H, m), 3.80 (6H, s),
4.39–4.40 (1H, m), 4.80 (1H, t, J = 4.9 Hz), 5.08 (1H, t, J = 4.4 Hz),
5.30 (1H, d, J = 8.1 Hz), 6.37 (1H, d, J = 3.9 Hz), 6.86 (4H, dd, J =
2.7 Hz, 9.2 Hz), 7.12 (1H, d, 7.8 Hz), 7.26–7.33 (7H, m), 7.38–7.42
(3H, m), 7.52–7.58 (3H, m), 7.84–7.86 (1H, m), 7.93–7.95 (1H, d,
J = 8.3 Hz), 8.19–8.20 (1H, d, 9.3 Hz); 13C NMR (CDCl3) d 55.4,
55.5, 62.6, 70.1, 81.5, 84.0, 87.1, 87.5, 102.8, 108.1, 113.6, 121.5,
122.9, 125.7, 125.8, 126.3, 127.0, 127.5, 128.0, 128.3, 130.3, 134.9,
135.1, 135.3, 140.2, 144.3, 150.0, 152.6, 159.0, 162.5. ESI-MS m/z
calcd for C40H36N2NaO8 [M+Na] 695.2364, found 695.2382.
7.99 (1H, dd, J = 8.2, 13.3 Hz), 8.26 (1H, dd, J = 3.8, 7.7 Hz); 13
C
NMR (CDCl3, 125 MHz) d 20.0, 20.4, 24.1, 24.6, 43.2, 43.3, 43.4,
55.4, 57.7, 57.8, 58.1, 58.2, 62.2, 70.5, 70.6, 70.8, 70.9, 79.4, 80.0,
83.4, 83.7, 87.4, 87.5, 87.7, 102.8, 106.8, 113.3, 113.5, 117.6, 117.7,
121.5, 121.6, 122.3, 122.5, 125.5, 125.6, 125.8, 126.0, 126.6, 126.7,
127.4, 127.6, 128.2, 128.4, 129.3, 130.3, 134.7, 135.1, 135.3, 140.2,
144.3, 144.4, 150.5, 153.1, 153.2, 158.9, 163.2; 31P NMR (CDCl3,
203 MHz) d 151.8. ESI-MS m/z calcd for C49H54N4O9P [M+H]
873.3623, found 873.3533.
2¢-O-(2-Naphthyl)-5¢-O-(4,4¢-dimethoxytrityl)uridine (3c)
Compound 3c was synthesized from 2c according to the same
procedure as that described for compound 3a.
1H NMR (CDCl3, 500 MHz) d 3.57–3.66 (2H, m), 3.79 (6H, s),
4.29–4.30 (1H, m), 4.76 (1H, t, J = 5.0 Hz), 5.06 (1H, t, J = 4.2 Hz),
5.33 (1H, d, J = 8.1 Hz), 6.28 (1H, d, J = 3.7 Hz), 6.86 (4H, d, J =
7.8 Hz), 7.24–7.28 (2H, m), 7.31–7.34 (6H, m), 7.38–7.47 (5H, m),
7.66 (1H, d, J = 8.3 Hz), 7.79–7.81 (2H, m), 8.00 (1H, d, J = 8.3
Hz); 13C NMR (CDCl3) d 55.4, 62.5, 69.9, 81.1, 83.7, 87.2, 87.6,
2¢-O-(2-Naphthyl)-5¢-O-(4,4¢-dimethoxytrityl)uridine-3¢-(2-
cyanoethyl N,N-diisopropyl)phosphoramidite (4c)
Compound 4c was synthesized from 3c using the same procedure
as that described for compound 4a.
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1H NMR (CDCl3, 500 MHz) d 0.90–1.19 (12H, m), 1.98 (4H,
dd, J = 5.4, 10.5 Hz), 2.18 (1H, d, J = 2.9 Hz), 2.35-2.45 (1H,
m), 3.36–3.41 (1H, m), 3.46–3.70 (5H, m), 3.77–3.82 (7H, m), 4.43
(1H, m), 4.86 (1H, m), 5.15 (1H, m), 5.33 (1H, dd, J = 8.1, 12.5
Hz), 6.30 (1H, m), 6.85 (4H, m), 7.21–7.47 (13H, m), 7.61 (1H, dd,
J = 3.7, 8.1 Hz), 7.75 (1H, m), 8.02 (1H, d, J = 8.3 Hz);
methyl-RNAs.17 The MD simulations were performed using the
duplexes S1/S2, S1/S3. The MD simulations were performed
using AMBER 7 program with the ff94 force field18 and the
TIP3P water model.19 The partial atomic charges for 2¢-O-Aryl-U
were determined at the HF/6-31G level of calculations,20 and
are described in the ESI.† The starting structures of A-type
RNAs were generated using the NUCGEN module of AMBER
software. The starting structure was contained in a solvent box
consisting of water, sodium ions, and chloride ions. The solvent
13C NMR (CDCl3, 125 MHz) d 20.4, 24.5, 24.7, 25.4, 43.2, 43.3,
43.3, 43.4, 55.4, 55.4, 57.9, 58.4, 62.0, 62.2, 70.5, 70.7, 76.9, 77.2,
77.4, 79.2, 80.0, 83.4, 87.5, 87.9, 102.7, 102.7, 109.3, 113.5, 117.6,
117.8, 119.0, 119.3, 124.3, 126.6, 126.7, 127.1, 127.1, 127.4, 127.7,
128.2, 128.3, 128.4, 129.6, 129.7, 130.4, 134.4, 135.1, 135.3, 140.2,
140.4, 144.3, 144.4, 150.3, 155.4, 155.6, 158.9; 31P NMR (CDCl3,
203 MHz) d 152.0, 151.8. ESI-MS m/z calcd for C49H53N4NaO9P
[M+Na] 895.3442, found 895.3304.
˚
box was extended by 12 A from the non-hydrogen atoms of the
nucleic acids. To neutralize the negative charges of the duplexes,
sodium cations were placed near each phosphate group using the
additions command of the Leap module. In addition, another
8 sodium and 8 chloride ions were added at random positions
to yield approximately 0.1 M NaCl concentrations. Molecular
dynamics simulations were performed using the SANDER
module of AMBER software. Minimization of the starting
models was performed with the positional restraint of 500 kcal
mol-1 on the non-hydrogen atoms of the oligonucleotides. After
200 steps of minimization, equilibrations were performed by eight
50 ps MD calculations during which the positional restraints
were reduced to 20, 10, 5, 2, 1, 0.5, 0.1, and 0.01 kcal mol-1.
The integration time step was 1 fs, and SHAKE21 was used to
constrain all covalent bonds involving hydrogen atoms. In all
calculations, electrostatic interactions were treated by the particle
mesh Ewald method,22,23 and the cut-off of distance was set as
Synthesis of 2¢-O-methylated oligoribonucleotides
2¢-O-Methylated oligoribonucleotides were synthesized using an
Applied Biosystems 392 oligonucleotide synthesizer, starting from
CPG-supported 2¢-OMe-U derivative (1 mmol). Chain extension
was carried out with 2¢-O-aryluridine phosphoramidite units (4a–
c) or 2¢-O-methylribonucleoside phosphoramidite units (PacA,
isopropyl-Pac-G, and acetyl-C) purchased from Glen Research.
Coupling time was set to 10 min. A 0.45 M solution of 1H-tetrazole
in CH3CN was used as the reaction activator. After the extension,
deprotection and excision from CPG were carried out with NH3
(aq) for 12 h, and the mixture was filtered. The excess ammonia was
removed from the filtrate in vacuo, and the residue was purified
by a C18 cartridge column and anion-exchange HPLC to yield
2¢-O-methylated oligoribonucleotides 5a–c, 6a–c, and 7a–c.
˚
9.0 A for Lennard-Jones interactions. After equilibrations, the
final 5 ns calculation was performed for each of the duplexes
without the positional restraints on the non-hydrogen atoms.
Acknowledgements
Nuclease resistance assay
This work was supported by a Grant-in-Aid for Scientific Research
from the Ministry of Education, Culture, Sports, Science and
Technology, Japan, and in part by Health Sciences Research
Grants for Research on Psychiatric and Neurological Diseases and
Mental Health from the Ministry of Health, Labor and Welfare
of Japan and the global COE project.
The nuclease stability of 2¢-O-arylated dinucleotide was evaluated
by treatment with SVP or bovine SPD (purchased from Sigma).
The SVP assay (5 ¥ 6 U/ml) was performed in a 50 mM Tris-
HCl buffer (pH 8.5, 72 mM NaCl, 14 mM MgCl2) at 37 ◦C. The
bovine SPD assay (0.2 U/ml) was performed in a 30 mM NaOAc
buffer (pH 6.0) at 37 ◦C. A 50 mmol sample of oligonucleotide was
used in each assay. After digestion, the enzyme was deactivated by
heating at 100 ◦C for 5 min. The solution was filtered and analyzed
by reverse-phase HPLC.
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