Synthesis and hybridization of 2′-O-methyl-RNAs incorporating 2′-O-carbamoyluridine and unique participation of the carbamoyl group in U-G base pair

Article abstract of DOI:10.1016/j.bmc.2009.08.053

2′-O-Carbamoyluridine (U_cm) was synthesized and incorporated into DNAs and 2′-O-Me-RNAs. The oligonucleotides incorporating U_cm formed less stable duplexes with their complementary and U_cm-U, U_cm-C single-base m

Full text of DOI:10.1016/j.bmc.2009.08.053

Bioorganic & Medicinal Chemistry 17 (2009) 7275–7280
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and hybridization of 20-O-methyl-RNAs incorporating
20-O-carbamoyluridine and unique participation of the carbamoyl
group in U–G base pair
*
Kohji Seio , Ryuya Tawarada, Takeshi Sasami, Masashi Serizawa, Misako Ise, Akihiro Ohkubo,
*
Mitsuo Sekine
Department of Life Science, Tokyo Institute of Technology, 4259 Nagatsuta, Yokohama 226-8501, Japan
a r t i c l e i n f o
a b s t r a c t
20-O-Carbamoyluridine (Ucm) was synthesized and incorporated into DNAs and 20-O-Me-RNAs. The oligo-
nucleotides incorporating Ucm formed less stable duplexes with their complementary and Ucm–U, Ucm–C
single-base mismatched DNAs and RNAs in comparison with those without the carbamoyl group. On the
contrary, the Tm analyses revealed that the duplexes with a mismatched Ucm–G base pair showed almost
the same thermostability as the corresponding unmodified duplexes. Molecular dynamics (MD) simula-
tions of the Ucm-modified 20-O-Me-RNA/RNA duplexes with Ucm–G mismatched base pair suggested that
the carbamoyl group could participate in the Ucm–G base pair by an additional intermolecular hydrogen
bond between the carbamoyl oxygen and the H2 of the guanine base.
Article history:
Received 30 June 2009
Revised 20 August 2009
Accepted 21 August 2009
Available online 2 September 2009
Keywords:
20-O-Modified-RNA
Mismatched base pair
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
of the carbamoyl group into the 20-hydroxyl group could be
performed by simple reaction of an appropriately protected nucle-
20-O-Modified RNA molecules have been extensively used for
gene regulation such as antisense, antigene, and RNA interfer-
ence (RNAi) strategies1–4 and microRNA analysis.5 Modification
of RNAs could improve their stability toward hydrolysis by nuc-
leases and enhance their hybridization affinity for the target
RNAs.6 Most of the 20-O-substituents reported to date were
comprised of ether-type skeletons in which the 20-oxygen at-
taches to sp3-type carbon atoms of various functional groups
such as 20-O-methyl,7 20-O-methoxyethyl,8–15 20-O-propargyl,16
20-O-aminopropyl,17 20-O-dimethylaminopropyl,18 20-O-[2-(meth-
ylamino)-2-oxoethyl],19,20 and 20-O-(2-cyanoethyl).21
In this paper, we report the synthesis and hybridization proper-
ties of 20-O-methyl-RNA incorporating 20-O-carbamoyluridine. We
have been interested in this type of modification in consideration
of its potential utility because of the following reasons: (a) since
the polarized carbonyl part of the carbamoyl group is more hydro-
philic than the sp3-type alkyl carbon of 20-O-ethereal substituents,
such a modification might enhance a hydration network around
the minor groove.22–24 (b) Since the carbamoyl group contains a
carbonyl oxygen and two amino protons as hydrogen bond donor
and acceptor sites, respectively, it can form unique hydrogen bonds
both intramolecularly and intermolecularly.25–27 (c) Introduction
oside derivative with phenyl chloroformate followed by substitu-
tion with ammonia. In contrast, introduction of ether-type
substituents usually required proton abstraction from the 20-hy-
droxyl group by strong bases, which cause damage on the protect-
ing groups in the base and sugar moieties. (d) While most of the
acyl-type substituents at the 20-O-position are unstable under basic
conditions, and they easily migrate to the neighboring 30-hydroxyl
group even under the neutral conditions upon removal of the
neighboring 30-protecting group, the carbamoyl group is excep-
tionally stable under the same conditions.
In this paper, we report the details of the hybridization proper-
ties of DNA and 20-O-methyl-RNA (20-O-Me-RNA) oligomers incor-
porating Ucm. Molecular dynamics (MD) simulations of modified
20-O-Me-RNA/RNA duplexes indicated a unique hydrogen bond be-
tween the carbamoyl oxygen of Ucm and the H2 of G in the Ucm–G
mismatched base pair.
2. Results and discussion
2.1. Synthesis of 20-O-carbamoyluridine and its derivatives
Synthesis of 20-O-carbamoyluridine 4 and its phosphoramidite
derivative 5 was performed as shown in Scheme 1. 30,50-O-(1,1,-
3,3-Tetraisopropyldisiloxane-1,3-diyl)uridine28 was converted to
the 20-O-phenoxycarbonyl derivative 1 with a 79% yield by treat-
ment with 1.2 equiv of phenyl chloroformate. The carbonate 1
* Corresponding authors. Tel.: +81 45 924 5706; fax: +81 45 924 5772.
E-mail addresses: kseio@bio.titech.ac.jp (K. Seio), msekine@bio.titech.ac.jp
(M. Sekine).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.08.053

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K. Seio et al. / Bioorg. Med. Chem. 17 (2009) 7275–7280
3.5 equiv.
NEt3·3HF
1.8 equiv.
NEt3
1.2 equiv.
PhOC(O)Cl
1.2 equiv.
Pyr
U
U
U
U
O
O
HO
6 equiv.
O
O
O
O
O
Si
O
NH3/EtOH
TIPDS
TIPDS
toluene
r.t. 3 h
O
O
OPh
O
O
NH2
OH
O
NH2
Si
Pyr
THF
O
OH
2
89%
r.t. 16 h
r.t. 2 h
1
O
O
O
79%
cm (4): R1 = R2 = H
(99% by procedure A)
1.5 equiv. DCA
1.5 equiv. NEt3
1.5 equiv. DMTrCl
U
U
U
R1O
R2
A) 1%TFA/CH2Cl2, r.t. 1 min
or
DMTrO
O
O
N(i-Pr)2
OCE
5: R1 = DMTr, R2
=
B) 1.2 equiv. CEOP-[N(i-Pr)2]2
0.5 equiv. HN(i-Pr)2
0.5 equiv. 1H-tetrazole
CH2Cl2, r.t. 2 h
P
OH
3
84%
O
NH2
O
O
NH2
Pyr
(51% by procedure B)
r.t. 24 h
O
O
Scheme 1. Synthesis of 20-O-carbamoyluridine derivatives.
was ammonolyzed to give the carbamate 2 with an 89% yield.
Then, the silyl-protecting group was removed using 3.5 equiv of
triethylamine–trihydrofluoride,29 and the resulting 50-hydroxyl
group was protected with a DMTr group without separation of
the triethylammonium salt to give 3. In this reaction, addition of
Cl2CHCOOH (DCA) and triethylamine30 to the deprotection mixture
proved to be effective. Finally, compound 3 was converted to the
nucleoside 4 and the phosphoramidite 5 by standard procedures.
oyl group. However, detailed hybridization properties such as the
base discrimination of the 20-O-NMC-T were not reported. In this
study, we carried out more detailed experiments for the hybridiza-
tion of DNA duplexes incorporating Ucm as well as the base recog-
nition properties of Ucm in these duplexes. We also studied the
stability of 20-O-Me-RNA/RNA duplexes incorporating Ucm. The
nucleotide sequences used in these experiments are shown in
Figure 1 and the Tm results are shown in Tables 2 and 3.
As shown in Table 2, the DNA–DNA duplexes consisted of DNA2
incorporating Ucm showed lower Tm than those consisting of the
unmodified DNA (DNA1) irrespective of the base pairing partner
at position X. This observation agreed with the hybridization prop-
erties of oligodeoxynucleotides containing 20-O-NMC-T, which
showed marked decrease in the affinity of the modified DNA due
to the dipole–dipole repulsion between the carbonyl groups. In
2.2. Sugar conformation of 20-O-carbamoyluridine
The sugar conformation property of 20-O-carbamoyluridine (4:
Ucm) was analyzed using the 1H–1H coupling constants between
the sugar protons. In general, the ribose conformation of nucleo-
sides could be estimated by the equilibrium between the N-type
and S-type conformers, and the population of the N-type confor-
the case of DNA2, the Tm decrease (
when Ucm and A formed a Watson–Crick base pair, as shown in
the row of DNAa. In contrast, the Tm values of the duplexes of
DNA2 with the target DNAs (DNAb, DNAc, and DNAd) containing
a mismatched base were much smaller (by ꢀ2 to ꢀ6 °C) than that
of the duplex with DNAa. Consequently, the base recognition accu-
racy of DNA2 containing Ucm was poorer than that of the unmod-
ified DNA1. Interestingly, among the mismatched base pairs, the
Ucm–G pair showed the smallest destabilization.
D
Tm) was the largest (ꢀ11 °C)
31
0
0
0
0
0
0
mation (%N) could be calculated by dividing J3 4 by J1 2 + J2 3
.
Ucm values are shown in Table 1 along with the coupling constants
of uridine (U) and 20-O-methyluridine (UOMe). It was revealed that
the %N values of the ribose moieties of U and Ucm were 56% and
57%, respectively, and almost identical. Moreover, the %N value
of Ucm differed from that of UOMe by only 3%.
These results indicated that conformation property of Ucm was
essentially identical to that of U and UOMe, and oligonucleotides
incorporating Ucm were expected to preferably form A-type du-
plexes upon hybridization with the complementary DNA and
RNA oligomers.
D
Next, we examined the hybridization properties of 20-O-Me-
RNA (Me-RNA2) incorporating Ucm. As shown in Table 3, in the
case of DNAa–d duplexes, the Tm values of the duplexes of Me-
RNA2/DNAa (X = A), DNAb (X = T), and DNAd (X = C) were de-
creased by 8, 10, and 5 °C, respectively, compared with that of
the unmodified RNA (Me-RNA1). Interestingly, the Tm drop was
2.3. Hybridization properties of DNAs and 20-O-Me-RNAs
incorporating Ucm
much smaller (DTm = –2 °C) for the Ucm–G mismatched duplex
(Me-RNA2/DNAc). We also studied the thermostability of
Next, we examined the hybridization properties of DNA and 20-
O-Me-RNA incorporating Ucm. Previously, studies on the synthesis,
hybridization properties, and structure of DNAs incorporating a
20-O-(N-methylcarbamoyl)ribothymidine (20-O-NMC-T) were re-
ported,20,32 and it was concluded that incorporation of 20-O-
NMC-T into DNA reduced the affinity of the modified DNA for
the complementary RNA due to the dipole–dipole interaction be-
tween the carbonyl oxygen at position 2 of uracil and the carbam-
DNA1: 5'-d(GTACCTTTCCGG)-3'
DNA2: 5'-d(GTACC)[Ucm]d(TTCCGG)-3'
DNAa-d: 5'-d(CCGGAAXGGTAC)-3'
a: X = A; b: X = T; c: X = G; d: X = C
Me-RNA1: 5'-GUACCUUUCCGG-3'
Table 1
Ribose puckering (%N) of uridine derivatives
Me-RNA2: 5'-GUACC[Ucm]UUCCGG-3'
0
0
0
0
0
0
0 0
J1 2
J3 4
J1 2 + J2 3
%N
RNAa-d: 5'-r(CCGGAAXGGUAC)-3'
a: X = A; b: X = U; c: X = G; d: X = C
U
UOMe
Ucm
4.4
3.9
4.4
5.5
5.8
5.9
9.9
9.7
10.3
56
60
57
a
Figure 1. Nucleotide sequences incorporating Ucm. The 20-O-Me-ribonucleotide
residues are underlined.
a
Data from Ref. 21.

K. Seio et al. / Bioorg. Med. Chem. 17 (2009) 7275–7280
7277
Table 2
Tm (°C) and
DTm (°C) of the duplexes of unmodified DNA1 or Ucm-modified DNA2 and
counter strands DNAa–DNAd
DNA1
DNA2
DTm
DNAa
X = A
DNAb
X = T
DNAc
Y = G
DNAd
X = C
49
38
34
24
38
ꢀ11
ꢀ6
32
32
18
ꢀ2
ꢀ6
D
Tm = Tm of DNA2 ꢀ Tm of DNA1.
Table 3
Tm (°C) and
DTm (°C) of the duplexes of unmodified Me-RNA1 or Ucm-modified
Me-RNA2 and the counter strands DNAa–DNAd and RNAa–RNAd
Me-RNA1
Me-RNA2
DTm
DNAa
X = A
DNAb
X = T
DNAc
X = G
DNAd
X = C
RNAa
X = A
RNAb
X = U
RNA-c
X = G
RNA-d
X = C
39
31
ꢀ8
ꢀ10
ꢀ2
ꢀ5
ꢀ5
ꢀ4
0
31
21
21
70
58
63
57
21
19
16
65
54
63
52
Figure 2. CD spectra of the fully matched (panel A) and U–G mismatched (panel B)
duplexes. RD, RR, and DD indicate the category of the duplex types, 20-O-Me-RNA/
DNA, 20-O-Me-RNA/RNA, and DNA/DNA duplexes, respectively. The red lines in each
category refer to carbamoyl-modified duplexes and the blue lines refer to
unmodified duplexes. (panel A) DD-blue: DNA1/DNAa, DD-red: DNA2/DNAa;
RD-blue: Me-RNA1/DNAa, RD-red: Me-RNA2/DNAa; RR-blue: Me-RNA1/RNAa,
RR-red: Me-RNA2/RNAa. (panel B) DD-blue: DNA1/DNAc, DD-red: DNA2/DNAc;
RD-blue: Me-RNA1/DNAc, RD-red: Me-RNA2/DNAc; RR-blue: Me-RNA1/RNAc,
RR-red: Me-RNA2/RNAc.
ꢀ5
Me-RNA2/RNA duplexes. In this case, incorporation of Ucm
decreased the Tm by 5, 5, and 4 °C when X was A, C, and U, respec-
tively. However, the Tm of Me-RNA2/RNA-c with a Ucm–G mis-
match was identical to that of Me-RNA1/RNA-c with an
unmodified U–G mismatch.
RNA1/DNAc, and Me-RNA1/RNAc without carbamoyl modification
(Fig. 2B).
Considering the observations shown in Tables 2 and 3, the base
pairing property of Ucm could be summarized as follows. The incor-
poration of Ucm mostly destabilized DNA/DNA, 20-O-methyl-RNA/
DNA, and 20-O-methyl-RNA/RNA duplexes probably due to di-
pole–dipole repulsion20 between the carbonyl oxygen at position
2 of the uracil and the carbamoyl group, as suggested in the case
of 20-O-NMC-T. Second, although the Ucm residue destabilized the
duplexes in most cases, the Tm decrease was the smallest in the
case of the Ucm–G pair in the DNA/DNA and 20-O-Me-RNA/DNA du-
plexes. In particular, in the case of the 20-O-Me-RNA/RNA duplexes,
the duplexes with a Ucm–G pair were as stable as those with a U–G
pair. These results could be explained by assuming some specific
stabilization mechanism of the Ucm–G pair by the carbamoyl
group. Therefore, we next studied the structure of the duplexes
with the Ucm–G pair in more detail using their CD spectra and
MD simulations.
As shown in Fig. 2A, the fully matched DNA/DNA-type (DD) du-
plexes, such as DNA1/DNA-a (blue) and DNA2/DNA-a (red),
showed almost identical CD spectra that corresponded to the B-
form irrespective of the presence of the carbamoyl group. Such
conformational similarities were also observed between Me-
RNA1/DNAa and Me-RNA2/DNAa in the category of the 20-O-Me-
RNA/DNA-type (RD) duplexes, and between Me-RNA1/RNAa and
Me-RNA2/RNAa in the 20-O-Me-RNA/RNA-type duplexes. As
indicated by the shape of the CD spectra,33 the structures of the
DD-, RR-, and RD-type duplexes are considered to exist in B-form,
A-form, and one between the A-form and B-form duplexes, respec-
tively, independent of the presence of the carbamoyl group.
Similar conformation properties independent of the carbamoyl
modification were also observed in the case of the duplexes con-
taining a G–U mismatch base pair (Fig. 2B). These results suggested
that the incorporation of a single carbamoyl group did not affect
the structure of these duplexes.
2.4. CD spectra of duplexes with Ucm
2.5. MD simulation of 20-O-Me-RNA with Ucm–G mismatch
The structures of the fully matched modified duplexes, DNA2/
DNAa, Me-RNA2/DNAa, and Me-RNA2/RNAa, were studied by
measuring their CD spectra, which were then compared with those
of the duplexes, DNA1/DNAa, Me-RNA1/DNAa, and Me-RNA1/
RNAa, without the carbamoyl modification (Fig. 2A). We also mea-
sured the CD spectra of the duplexes, DNA2/DNAc, Me-RNA2/
DNAc, and Me-RNA2/RNAc, with a Ucm–G mismatch. These CD
spectra were also compared with those of DNA1/DNAc, Me-
MD simulations of the 20-O-Me-RNA-/RNA duplexes incorporat-
ing Ucm were performed using the AMBER94 force field.34 The
atomic charges for Ucm was derived from the ab initio calculations
*
at the HF/6-31G level. This level of theory was the same as that
used in derivatization of the atomic charges of the original AM-
BER94 parameters. The calculation was performed for Me-RNA2/
RNA-b duplex incorporating Ucm–G base pair.

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K. Seio et al. / Bioorg. Med. Chem. 17 (2009) 7275–7280
The average structure of the Ucm–G pair from the last 200 ps
Because the introduction of the carbamoyl group into the du-
plexes reduced the stability of sequence-matched duplexes in all
of the DNA/DNA, the 20-O-Me-RNA/DNA, and the 20-O-Me-RNA/
RNA cases, the oligonucleotides fully modified with 20-O-carbam-
oyl groups did not exhibit strong hybridization affinity and hence
do not seem applicable. However, we found an interesting ability
of the 20-O-carbamoyl group to directly interact with the opposite
guanine base to stabilize the Ucm–G base pair. These results indi-
cate a new possibility that appropriately designed 20-O-substitu-
ents in artificial RNAs can participate in the base pairing with the
opposite strands and modulate the base pair stability and selectiv-
ity. To the best of our knowledge, such a possibility has not been
proposed yet. Therefore, the carbamoyl modification might be use-
ful for nucleotide units that are to be incorporated into appropriate
positions of functional nucleic acids, and to modulate their struc-
tures and functions with its unique interactions.
trajectories are shown in Figure 3A. The torsion angle of C30–C20–
O20–C(O)NH2 was in the anti-periplanar conformation and the
C20–O20–C–NH2 torsion was in the trans-conformation. This confor-
mation around the carbamoyl group was identical to those found
in the X-ray structure of the (20-O-NMC-T)-A pair in a DNA/RNA
duplex.20
Interestingly, in this Ucm–G pair, in addition to the usual wob-
ble-type base pair between the uracil and the guanine moiety sta-
bilized by the hydrogen bonds between NH3 of uracil and O6 of
guanine, and O2 of uracil and NH1 of guanine, the carbonyl oxygen
of the carbamoyl group formed an additional third hydrogen bond
with NH2 of guanine. This additional hydrogen bond might partly
compensate the duplex destabilization effect of the carbamoyl
group and contribute to the stability of this Ucm–G base pair, as
shown in Tables 2 and 3. In several X-ray crystallographic struc-
tures of RNA duplexes incorporating G–U mismatches,35–37 a water
molecule bridges the N2 atom of guanine and the O2 atom of ura-
cil. An example of the G–U mismatch pair mediated by a water
molecule was reported in Ref. 36 and is shown in Figure 3B. There-
fore, the carbamoyl group of Ucm–G could be considered to mimic
the hydrogen bonding of such a water molecule in the usual U–G
mismatch base pair. The geometries of the hydrogen bonds be-
tween the carbamoyl group and the nucleobases were not exactly
the same as but similar to those of the water molecule as shown in
Figure 3. For example, the interatomic distance between the uracil
O2 atom and the carbamoyl oxygen was 2.9 Å and that between
the oxygen of water was 3.1 Å, as shown in Figure 3A and B, respec-
tively. The same analyses between the N2 atom of guanine and the
carbamoyl oxygen (2.8 Å) and the oxygen of water (3.1 Å) also sug-
gested a similar but not identical geometry.
Studies on the other 20-O-carbamoyl nucleosides and the duplex
stabilization mechanism of the carbamoyl group are currently
underway, in addition to the design of new functional groups for
the 20-O-position on the basis of the unique results of the present
study.
4. Experimental procedures
4.1. General procedures
The dry solvents were purchased and stored over molecular
sieves 4 A. 1H, 13C and 31P NMR spectra were obtained at 500,
126 and 203 MHz, respectively. The chemical shifts were measured
from tetramethylsilane (0.0 ppm) or DMSO-d6 (2.49 ppm) for 1H
NMR, CDCl3 (77.0 ppm), DMSO-d6 (39.7 ppm) for 13C NMR and
85% phosphoric acid (0.0 ppm) for 31P NMR. MALDI-TOF and ESI-
TOF mass spectra were obtained in the positive ion mode.
3. Conclusions
We studied the synthesis and properties of Ucm and oligonucle-
otides incorporating Ucm. The carbamoyl group did not signifi-
cantly affect the sugar conformation compared with uridine and
20-O-methyluridine. The introduction of Ucm into the duplexes de-
creased the duplex stability, as suggested previously. The MD sim-
ulation revealed some interesting participations of the carbamoyl
group in the Ucm–G base pair, which could partly explain the sta-
bility of the Ucm–G base pair suggested in the Tm analysis.
4.2. Synthesis of phosphoramidite unit
4.2.1. 20-O-Phenoxycarbonyl-30,50-O-(1,1,3,3-tetraisopropyldisil-
oxane-1,3-diyl)uridine 1
30,50-O-(1,1,3,3-Tetraisopropyldisiloxane-1,3-diyl)uridine (6.1 g,
13 mmol) was dissolved in anhydrous toluene (126 mL) followed
by addition of pyridine (1.2 mL, 13 mmol). Next, phenyl chlorofor-
mate (1.9 mL. 15 mmol) was added dropwise, and the resulting
mixture was stirred for 3 h. The reaction was quenched by addition
of water (10 mL), and the solution was diluted with ethyl acetate
(200 mL). The organic layer was washed thrice with brine
(200 mL ꢁ 3), dried over Na2SO4, filtered, and evaporated to dry-
ness. The residue was chromatographed on a neutralized silica
gel column (180 g) with chloroform–hexane (6:4–4:6, v/v) to give
1 (6.1 g, 79%). 1H NMR (CDCl3) d 0.97–1.12 (28H, m), 4.03 (1H, dd,
J = 2.6 Hz, 11.0 Hz), 4.11 (1H, dd, J = 1.3 Hz, 8.1 Hz), 4.26 (1H, d,
J = 3.4 Hz), 4.47 (1H, dd, J = 4.4 Hz, 4.9 Hz), 5.31 (1H, d, J = 4.6 Hz),
5.71 (1H, d, J = 8.0 Hz), 5.93 (1H, s), 7.17 (2H, m), 7.26 (1H, m),
7.38 (2H, m), 7.73 (1H, d, J = 8.0 Hz), 8.28 (1H, s); 13C NMR (CDCl3)
d 13.1, 17.4, 59.6, 68.1, 79.7, 82.2, 88.6, 102.5, 121.1, 126.5, 129.8,
139.5, 149.7, 151.3, 152.4, 162.8. MS m/z calculated for
C28H43N2O9Si2 [M+H]+: 607.2502, found: 607.2596.
þ
4.2.2. 20-O-Carbamoyl-30,50-O-(1,1,3,3-tetraisopropyldisiloxane-
1,3-diyl)uridine 2
To the solution of 1 (1.8 g, 3.0 mmol) in anhydrous pyridine
(30 mL) was added 2 M NH3/EtOH (8.9 mL, 17.8 mmol), and the
resulting solution was stirred for 16 h. The solution was diluted
with ethyl acetate (100 mL) and washed thrice with brine
(100 mL ꢁ 3), dried over Na2SO4, filtered, and evaporated under re-
duced pressure. The residue was chromatographed on a NH-silica
Figure 3. Average structures of the (A) Ucm–G base pair. Panel (B) shows the G(13)–
U(4) pair mediated by a water molecule found in a crystal.36 Numbers indicate the
distances between the atoms in Å.

K. Seio et al. / Bioorg. Med. Chem. 17 (2009) 7275–7280
7279
gel column (50 g) with chloroform–methanol (100:0–100:2, v/v) to
give 2 (1.4 g, 89%). 1H NMR (CDCl3) d 0.93–1.11 (28H, m), 4.00 (2H,
m), 4.20 (1H, d, J = 3.5 Hz), 4.39 (1H, dd, J = 3.9 Hz, 5.2 Hz), 4.90
(2H, s), 5.27 (1H, d, J = 5.1 Hz), 5.69 (1H, dd, J = 8.3 Hz), 5.83 (1H,
s), 7.64 (1H, d, J = 8.3 Hz), 8.76 (1H, s); 13C NMR (CDCl3) d 13.2,
17.3, 60.0, 68.1, 76.3, 82.4, 89.0, 102.5, 139.7, 149.9, 155.2, 163.1.
give 5 (0.89 g, 51%). 1H NMR (CDCl3) d 1.06–1.27 (14H, m), 2.42
(1H, m, CH2 of cyanoethyl), 2.69 (1H, m), 3.42–3.53 (2H, m),
3.57–3.70 (2H, m), 3.79 (6H, d, J = 2.9 Hz), 4.12 (1H, m), 4.21–
4.31 (1H, m), 4.67 (1H, m), 4.69 (2H, d, J = 3.2 Hz), 5.30–5.43
(2H, m), 6.22 (1H, m), 6.84 (4H, m), 7.24–7.40 (9H, m), 7.70 (1H
m), 8.11 (1H, m); 13C NMR (CDCl3) d 14.5, 20.4, 24.8, 43.5, 55.5,
58.2, 60.6, 63.1, 71.3, 75.5, 84.5, 86.2, 87.5, 103.1, 113.6, 127.5,
128.5, 130.5, 135.2, 140.3, 144.3, 150.7, 155.5, 159.0, 162.8; 31P
NMR (CDCl3) d 151.2, 151.5. MS m/z calculated for C40H49N5O10P+
[M+H]+: 790.2312, found: 790.3297.
MS m/z calculated for C22H40N3O8Si2 [M+H]+: 530.2348, found:
þ
530.2370.
4.2.3. 20-O-Carbamoyl-50-O-(4,40-dimethoxytrityl)uridine 3
Compound 2 (6.0 g, 11 mmol) was dissolved in anhydrous THF
(57 mL). To this solution were added triethylamine (2.7 mL,
20 mmol) and triethylamine trihydrofluoride (6.3 mL, 38 mmol).
The resulting solution was stirred for 2 h. The solvents were re-
moved under reduced pressure, and the residual triethylamine
and THF were removed by co-evaporation once with toluene and
thrice with pyridine, and the residue was finally dissolved in anhy-
drous pyridine (17 mL). To this solution were added 4,40-dime-
thoxytrityl chloride (5.7 g, 17 mmol), triethylamine (2.4 mL,
17 mmol), and dichloroacetic acid (1.4 mL, 17 mmol). After the
resulting mixture was stirred for 23 h, the reaction was quenched
by addition of methanol (5 mL). The solution was diluted with
ethyl acetate (200 mL), washed thrice with brine (200 mL ꢁ 3),
dried over Na2SO4, filtered, and concentrated under reduced pres-
sure. The residual pyridine was removed by co-evaporation with
toluene, and the residue was chromatographed on a silica gel col-
umn with chloroform–methanol (100:0–100:2, v/v) containing
0.5% triethylamine to give 3 (5.5 g, 84%). 1H NMR (CDCl3) d 3.44
(2H, m), 3.73 (6H, s), 3.96 (1H, m), 4.16 (1H, d, J = 3.9 Hz), 4.57
(1H, d, J = 4.2 Hz), 5.26 (1H, m), 5.34 (1H, d, J = 8.1 Hz), 5.65 (2H,
s), 6.14 (1H, d, J = 5.1 Hz), 6.80 (4H, m) 7.15–7.37 (9H, m), 7.76
(1H, d, J = 8.1 Hz), 9.71 (1H, s); 13C NMR (CDCl3) d 55.5, 62.8,
70.4, 84.1, 86.8, 87.4, 103.1, 113.6, 127.4, 128.3, 128.4, 130.4,
135.3, 135.5, 140.3, 144.4, 151.2, 156.5, 158.9, 163.6. MS m/z calcu-
4.3. Synthesis of oligonucleotides incorporating 20-O-
carbamoyluridine
Oligodeoxynucleotides and 20-O-Me-oligoribonucleotides incor-
porating 20-O-carbamoyluridine, 50-d(GTACC)[Ucm]d(TTCCGG)-30,
and 50-GOMeUOMeAOMeCOMeCOMe[Ucm]UOMeUOMeCOMeCOMeGOMeGOMe-30,
respectively, were synthesized as follows. First, 30-half sequences,
50-d(TTCCGG)-30, and UOMeUOMeCOMeCOMeGOMeGOMe, were synthe-
sized using an Applied Biosystems 394 automated DNA/RNA
synthesizer by the standard 1.0-lmol-scale phosphoramidite ap-
proach for DNA and RNA, respectively, which consists of detrityla-
tion, coupling, capping, and iodine oxidation steps. The CPG
support was removed from a cartridge column and placed on a
glass syringe having a glass filter. Then, a Ucm unit was introduced
into the 50-terminal position by use of 20 equiv of the phospho-
ramidite derivative 5 and 80 equiv of 1H-tetrazole by use of
250 lL of dry CH3CN. The coupling time was 5 min. After this man-
ual procedure, the CPG support was placed back in the cartridge
column, and the remaining 50-half sequences, 50-d(GTACC)-30 and
50-GOMeUOMeAOMeCOMeCOMe-30, were synthesized in the automated
synthesizer. Release of the resulting protected 50-O-DMTr-oligonu-
cleotide from the solid support and deprotection of the phosphate
and base-protecting groups were performed by treatment with
28% aqueous ammonia at room temperature for 24 h. The solution
was evaporated under reduced pressure at room temperature to
remove ammonia, and the residue was diluted with 0.1 M ammo-
nium acetate (50 mL). The solution was placed on a C18 cartridge
column, and the failure sequences were eluted using 10% CH3CN/
0.1 M ammonium acetate as an eluent. After washing with 0.1 M
ammonium acetate and water, the column was treated with aque-
ous 2% TFA to remove the DMTr group and further washed with
0.1 M ammonium acetate and water. The target oligonucleotide
was eluted using 20% CH3CN/water, and the fractions containing
the target were lyophilized to give the crude oligonucleotide. Pure
material was obtained by anion-exchange HPLC using a 0–50% gra-
dient of 1 M NaCl in 25 mM sodium phosphate–10% CH3CN. The
salts were removed using a C18 cartridge column to give the pure
oligonucleotide after being lyophilized to dryness. The yields of the
pure materials were 26% for the DNA and 33% for the 20-O-Me-RNA.
These yields were calculated assuming that the molar extinction
lated for C31H31N3NaO9 [M+Na]+: 612.1925, found: 612.2367.
þ
4.2.4. 20-O-Carbamoyluridine 4
Compound 3 (200 mg, 0.34 mmol) was dissolved in 1% trifluoro-
acetic acid/CH2Cl2 (13 mL). After 1 min, the solution was poured
into a suspension of dimethylamino-polystyrene (1 g) in methanol
(20 mL). The solids were removed by filtration, and the filtrate was
diluted with chloroform (50 mL). The product was extracted with
water (50 mL), and the aqueous solution was lyophilized to give
4 (96 mg, 99%). 1H NMR (D2O) d 3.84 (1H, dd, J = 4.6 Hz, 10.0 Hz),
3.95 (1H, dd, J = 2.7 Hz, 10.0 Hz), 4.10–4.14 (1H, m), 4.35–4.45
(1H, dd, J = 5.9 Hz, 5.6 Hz), 5.21 (1H, dd, J = 4.4 Hz, 5.6 Hz), 5.92
(1H, d, J = 8.1 Hz), 6.03 (1H, d, J = 4.4 Hz), 7.85 (1H, d, J = 8.1 Hz);
13C NMR (D2O) d 60.8, 68.8, 75.6, 84.4, 88.6, 102.7, 142.5, 151.7,
þ
157.8, 166.4. MS m/z calculated for C10H14N3O7
[M+H]+:
288.0826, found: 288.0881.
4.2.5. 20-O-Carbamoyl-50-O-(4,40-dimethoxytrityl)uridine 30-(2-
cyanoethyl N,N-diisopropylphosphoramidite) 5
Compound 3 (1.3 g, 2.2 mmol) was rendered anhydrous by re-
peated co-evaporation thrice each with anhydrous pyridine,
anhydrous toluene, and anhydrous CH2Cl2, and the residue was fi-
nally dissolved in anhydrous CH2Cl2 (22 mL). To this solution
coefficients of 50-GTACC[Ucm]TTCCGG-30 and 50-GOMeUOMeAOMeCOMe
-
COMe[Ucm]UOMeUOMeCOMeCOMeGOMeGOMe-30 were identical to those
of 50-GTACCTTTCCGG-30 and 50-GUACCUUUCCGG-30, respectively.
The structures were confirmed by MALDI-TOF mass spectroscopy.
MALDI-TOF mass of 50-GTACC[Ucm]TTCCGG-30 [M + H]+: calculated
3657.5, found: 3656.6; MALDI-TOF mass of 50-GOMeUOMeAOMeCOMe-
COMe[Ucm]UOMeUOMeCOMeCOMeGOMeGOMe-30 [M+H]+: calculated
3946.1, found: 3947.1.
were added diisopropylamine (155
(77 mg, 1.1 mmol), and bis(N,N-diisopropylamino)phosphine
(839 L, 2.6 mmol). After the resulting mixture was stirred for
lL, 1.1 mmol), 1H-tetrazole
l
2 h, the reaction was quenched by adding water (1 mL), and the
mixture was diluted with ethyl acetate (50 mL). The organic layer
was washed five times with 5% Na2CO3, dried over Na2SO4, fil-
tered, and concentrated to dryness. The residue was chromato-
4.4. CD spectra
CD spectra were measured using 2 lM solutions of the duplexes
in 10 mM sodium phosphate (pH 7.0) containing 150 mM NaCl and
graphed on
a silica gel column (26 g) with chloroform–
0.1 mM EDTA at 10 °C.
methanol (100:2–100:4, v/v) containing 0.5% triethylamine to

7280
K. Seio et al. / Bioorg. Med. Chem. 17 (2009) 7275–7280
3. Jackson, A.; Burchard, J.; Leake, D.; Reynold, A.; Schelter, J.; Guo, J.; Johnson, J.
4.5. Tm measurement
M.; Lim, L.; Karpilow, J.; Nichols, K.; Marshall, W.; Khvorova, A.; Linsley, P. S.
RNA 2006, 12, 1197.
Each oligonucleotide was dissolved in 10 mM sodium phos-
phate (pH 7.0) containing 150 mM NaCl and 0.1 mM EDTA so that
the final concentration of each oligonucleotide became 2 lM. The
solution was separated into quartz cells (10 mm) and incubated
at 85 °C. After 10 min, the solution was cooled to 5 °C at a rate of
0.5 °C/min and heated to 85 °C at the same rate. During this
annealing and melting, the absorption at 260 nm was recorded
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.
4. Bryan, A.; Kraynack, B. A.; Baker, A. F. RNA 2006, 12, 163.
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4.6. MD simulation
The atomic charges of 20-O-carbamoyluridine were estimated
*
from the ab initio calculation at the HF/6-31G level performed
using Gaussian03.38 The structures obtained by two 5.4-ns MD
simulations of the 20-O-Me RNA and RNA duplex containing the
20-O-CONH2 group at the central position, at constant temperature
(300 K) and pressure (1 atm), were generated using the AMBER 9.0
molecular simulation package.39
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tures were generated using the NUCGEN module of AMBER.
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nell et al. was employed.34 The box dimensions were chosen
such that they ensured a 10-Å solvation shell around the
duplexes.
(ii) MD simulation
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atoms were fixed at the initial positions. Then, the next
seven 50-ps MD runs were performed with positional con-
straints on the nucleic acid atoms of 10, 5, 2, 1, 0.5, 0.1,
and 0.01 kcal/mol. After the 400-ps equilibration phase, a
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Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.;
Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao,
O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken,
V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A.
J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G.
A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.;
Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.;
Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.;
Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.;
Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe,
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Gaussian 03, Revision C.02, 2004.
Acknowledgments
This study was supported by Industrial Technology Research
Grant Program in 005 from the New Energy and Industrial Technol-
ogy Development Organization (NEDO) of Japan. This study was
also supported by a Grant-in-Aid for Scientific Research (B)
20350074.
39. Case, A.; Darden, T. A.; Cheatham, T. E., III, Simmerling, C. L.; Wang, J.; Duke, R.
E.; Luo, R.; Merz, K. M.; Pearlman, D. A.; Crowley, M.; Walker, R. C.; Zhang, W.;
Wang, B.; Hayik, S.; Roitberg, A.; Seabra, G.; Wong, K. F.; Paesani, F.; Wu, X.;
Brozell, S.; Tsui, V.; Gohlke, H.; Yang, L.; Tan, C.; Mongan, J.; Hornak, V.; Cui, G.;
Beroza, P.; Mathews, D. H.; Schafmeister, C.; Ross, W. S. and Kollman, P. A.
AMBER 9.0.
References and notes
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K.; Kaufmann, J. Nucleic Acid Res. 2003, 31, 2705.
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