Synthesis and properties of cationic 2′-O-[N-(4-aminobutyl)carbamoyl] modified oligonucleotides

Article abstract of DOI:10.1016/j.bmcl.2012.02.012

2′-O-[N-(4-Aminobutylcarbamoyl)]uridine (U_abcm) was synthesized and incorporated into oligonucleotides. The oligonucleotides incorporating U_abcm formed more stable duplexes with their complementary and mismatched RNAs than those cont

Full text of DOI:10.1016/j.bmcl.2012.02.012

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2470–2473
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and properties of cationic 20-O-[N-(4-aminobutyl)carbamoyl]
modified oligonucleotides
⇑
Kohji Seio , Munefumi Tokugawa, Takashi Kanamori, Hirosuke Tsunoda, Akihiro Ohkubo,
⇑
Mitsuo Sekine
Department of Life Science, Tokyo Institute of Technology, J2-16, 4259 Nagatsuta, Midoriku, 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-[N-(4-Aminobutylcarbamoyl)]uridine (Uabcm) was synthesized and incorporated into oligonucleo-
tides. The oligonucleotides incorporating Uabcm formed more stable duplexes with their complementary
and mismatched RNAs than those containing 20-O-carbamoyluridine (Ucm). The stability of duplex with a
Article history:
Available online 13 February 2012
Keywords:
U
abcm-rG base pair showed higher thermostability than the duplex having unmodified U-rG base pair. The
20-O-Modified RNA
Cationic residue
Uabcm residue showed enhanced resistance to snake venome phosphodiesterase.
Ó 2012 Elsevier Ltd. All rights reserved.
Hybridization properties
Nuclease resistance
20-O-Modified RNA molecules have been extensively used for
gene regulation such as antisense,1 antigene,2 and RNA interfer-
ence (RNAi).3 20-O-Modification of RNAs can improve their stability
toward hydrolysis4 and enhance the hybridization affinity for the
target RNAs.5
modified nucleoside, 20-O-[N-(4-aminobutyl)carbamoyl]uridine
(Uabcm). It is well known that the amino groups incorporating into
oligonucleotides neutralize the negative charges of the phosphate
backbone. Therefore, it is expected that the oligonucleotides hav-
ing Uabcm form more stable duplexes with their complementary
strands than those containing Ucm. We report the synthesis of
the oligonucleotides having Uabcm and the properties of the 20-O-
As one of the 20-modified RNAs, several research groups have
reported the synthesis and properties of oligonucleotides contain-
ing 20-O-carbamoyl and 20-O-N-alkylcarbamoyl groups.6–8 In these
studies, it was reported that various functional groups, such as the
propargyl group, the dansyl-6-sulphonamidohexyl group and 4-
(pyren-1-ylethynyl)phenylmethyl group, could be easily intro-
duced into the 20-position of RNAs through the carbamoyl group.6
methyl RNA oligomers incorporating Ucm and Uabcm
.
To introduce Uabcm into the oligonucleotides, the phosphorami-
dite unit 5 was synthesized, as shown Scheme 1.
30,50-O-(1,1,3,3-Tetraisopropyldisiloxane-1,3-diyl)uridine 1 was
treated with 1.1 equiv of phenyl chloroformate. Subsequently,
compound 2 was treated in situ with 1,4-diaminobutane at 0 °C.
The NH2 group was protected with a trifluoroacetyl group to give
3 in 70% yield in three steps. The silyl-protecting group was
removed by using 3.5 equiv of triethylamine-tri(hydrofluororide).
The triethylammonium salts were removed by treatment with
ethoxytrimethylsilane,9 and the resulting 50-hydroxyl group was
In addition, we have reported the uridine derivative having the
7
simplest carbamoyl group (Ucm
)
to study the intrinsic properties
of the carbamoyl modifications and found that the carbonyl could
participate in the wobble-type uracil-guanine base pair forming a
hydrogen bond with the amino group of guanine at position 2.
These results indicated the unique character of carbamoyl modifi-
cations for the development of the artificial nucleic acids having
useful functional groups and unique base pairing properties. How-
ever, we and other groups also found a drawback of the carbamoyl
group that the incorporation of the carbamoyl modification de-
creased the stabilities of the duplexes probably due to the close
contact between the carbonyl oxygen of the 20-O-carbamoyl sub-
stituent and the O2 of nucleobase.8
protected with
a DMTr group to give 4. Phosphitylation
with chloro-(2-cyanoethoxy)-(N,N-diisopropylamino)phosphine
furnished the phosphoramitite unit 5.
Before synthesis of the oligonucleotides, we synthesized the di-
mer 6 incorporating Uabcm in the usual manner by using 1H-tetra-
zole as an activator (Scheme 2).10 However, the coupling yield was
surprisingly too low (<3%) to obtain the target dimer as judged by
the trityl cation assay.
In order to improve the coupling yield, we optimized the condi-
tions of the coupling reactions by measuring the coupling yield at
the Tr cation assay step. First, we tested 5-[3,5-bis(trifluoro-
methyl)phenyl]-1H-tetrazole (Activator 42),11 N-phenylimidazo-
lium triflate (N-PhIMT)12 and 5-benzylthiotetrazole (BTT)13 which
In this paper, in order to improve the hybridization affinity
and the nuclease resistance, we designed new carbamoyl-type
⇑
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).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.02.012

K. Seio et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2470–2473
2471
ammonia (Scheme 3). However, we found the formation of consid-
erable amount of N-cyanoethylated dimer 8 as the by-product
(Fig. 2b). Similar cyanoethylation of the amino groups was also re-
ported by Buchini and Griffey in the synthesis of 20-O-aminoethyl
and 20-O-aminopropyl ribonucleotides.2,16 To prevent the
cyanoethylation of the terminal amino group, we treated the
oligonucleotides first with 1,8-diazabicyclo[5,4,0]undec-7-ene
(DBU)–acetonitrile (1:9, v/v)2,17 for 1 min to selectively remove
the cyanoethyl group on the phosphate, and the subsequent
cleavage of the oligonucleotides from the solid supports and the
deprotection of the nucleobase and the terminal amino group were
performed by treatment with 28% aqueous ammonia. Figure 2a
shows the effect of DBU treatment in the deprotection and
cleavage of the oligonucleotide having Uabcm. The oligonucleotides
were characterized by ESI mass.
U
U
U
O
TIPDS
O
TIPDS
O
TIPDS
O
O
O
a
b, c
O
OH
O
O
O
O
O O
NHR
OPh
1
2
3
d, e, f
U
U
DMTrO
DMTrO
O
O
g
(iPr)2N
O
O
O
NHR
OH O
O
P
O
NHR
NC
5
4
O
R =
By using above mentioned coupling and deprotection
N
H
CF3
conditions, we synthesized the 12mer 20-O-methyl RNAs (ON2,
ON4 and ON6 in Table 2) having
a
50-GmU2AmCmCmU6Um
Scheme 1. Synthesis of phosphoramidite unit 5 of Uabcm: (a) PhOC(O)Cl (1.1 equiv),
pyridine, rt, 3 h; (b) 1,4-diaminobutane (1.2 equiv), pyridine, 0 °C, 3 h; (c) CF3COOEt
(2.0 equiv). TEA (1.0 equiv), EtOH, rt, overnight, 70% (3 steps); (d) TEA-3HF
(3.5 equiv), TEA (1.8 equiv), THF, rt, 3 h; (e) TMSOEt (10 equiv), THF, rt, overnight;
(f) DMTrCl (1.2 equiv), pyridine, rt, 8 h, 75% (3 steps); (g) NCCH2CH2OPClN(iPr)2
(1.2 equiv), (iPr)2NEt (1.5 equiv), dichloromethane, 1 h, rt, 72%.
U8CmCmGmGm-30 sequence. ON2 has Uabcm at the U6 position.
ON4 and ON6 have two Uabcm residues at U6/U8 andU2/U8 posi-
tions, respectively. We studied the hybridization properties of
them with sequence matched RNA strand 30-CAUGAAAGGCC-50
(Table 3). For comparison, we also prepared the 20-O-methyl RNAs
such as ON1, ON3 and ON5 incorporating Ucm (Table 2).
As shown in Table 3, ON1 to ON6 having Ucm or Uabcm showed
the lower Tm than ON7 composed of 20-O-methyl RNAs due to
the destabilization effect of 20-O-carbamoyl group. While ON2
incorporating a Uabcm at U6 position showed the Tm of 69 °C which
is 4 °C higher than ON1 having a Ucm residue at the same position
probably due to the presence of the cationic aminobutyl group.
Interestingly, the Tm difference became more significant when
two Ucm and Uabcm groups were incorporated at position U6 and
U8 as in the case of ON3 and ON4, respectively. In this case, the
ON4 incorporated two Uabcm at U6 and U8 positions showed Tm of
61 °C that was higher than that of ON3 incorporating two Ucm res-
idues by 18 °C. Similarly, ON6 having two Uabcm group at U2 and U8
U
HO
O
Scheme 2. Synthesis of dimer 6.
aq. NH3
or
O
O
O
10% DBU/CH3CN
then aq. NH3
O P O
O
NHR
C
are frequently used stronger activator for the oligonucleotide syn-
thesis, but the coupling yields were not improved even after the
reaction time prolonged to 40 min (data not shown).
6
O
We also tested 4,5-dicyanoimidazole (DCI)14 known as a strong
nucleophilic activator. In the activation with DCI for 40 min, the
coupling yield increased to 57% (Table1). For further improvement
of the reaction, doubling the equivalent of DCI to 160 equiv mar-
ginally improved the coupling yield to 62%. Finally, we found that
the coupling yield could be increased to 87% by repeating the
40 min coupling twice before the capping. These results indicate
that the phosphoramidite having lower reactivity due to the mod-
ification at the 20-position can be efficiently introduced into oligo-
nucleotides by use of DCI.15
OH OMe
+
7: R = (CH2)4NH3
8: R = (CH2)4NH2+(CH2)2CN
Scheme 3. Synthesis of dimer UabcmCm (7).
Table 2
Oligonucleotide sequences incorporating Uabcm or Ucm
Sequences
Next, we tested the deprotection of the protecting groups on the
fully protected dimer 6 attached on the solid supports. We first
tried the simultaneous removal of the trifluoroacetyl group, the
cyanoethyl group and the acetyl group by treatment with aqueous
ON1
ON2
ON3
ON4
ON5
ON6
ON7
50-GUACCUcmUUCCGG-30
50-GUACCUabcmUUCCGG-30
50-GUACCUcmUUcmCCGG-30
50-GUACCUabcmUUabcmCCGG-30
50-GUcmACCUUUcmCCGG-30
50-GUabcmACCUUUabcmCCGG-30
50-GUACCUUUCCGG-30
Table 1
Optimization of conditions for coupling reaction of the dimer 6
ON8
ON9
ON10
50-UUdT-30
50-UcmUcmdT-30
50-UabcmUabcmdT-30
Activator (equiv)
Coupling tine (min)
Coupling yield (%)
DCI (80)
DCI (160)
DCI (80)
40
40
40 ꢀ 2
57
62
87
The 20-O-methyl ribonucleotide residues are
italicized.

2472
K. Seio et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2470–2473
Table 3
Tm (°C) and
D
Tm (°C) of 20-O-methyl RNA/ RNA duplexes, 50-GmU2AmCmCmU6
UmU8CmCmGmGm-30/ 30-CAUGGAAAGGCC-50, incorporating Uabcm or Ucm
Me-RNA
U2, U6, U8
m, Ucm, Um
Um, Uabcm, Um
m, Ucm, Ucm
Um, Uabcm, Uabcm
=
Tm (°C)
DTm (°C)
ON1
ON2
ON3
ON4
ON5
ON6
ON7
U
65*
69
43
61
47
61
70*
+4
U
+18
+14
Ucm, Um, Ucm
Uabcm, Um, Uabcm
Um, Um, Um
Conditions: 10 mM phosphate buffer (pH 7.0), 150 mM NaCl, 0.1 mM EDTA, 2
duplex.
Data from Ref. 7.
lM
*
positions showed Tm of 61 °C that was higher by 14 °C than ON5
having Ucm whose Tm was 47 °C.
These Tm results exhibited that although an increasing number
of carbamoyl groups in the oligonucleotide considerably decreased
the hybridization stabilities of the oligonucleotide, the cationic res-
idues moderated the destabilization effects of Ucm residues.
Next, we evaluated the base discrimination abilities of Uabcm
(Table 4) by using the singly modified ON2 and the counter strand
RNA 30-CAUGGXAAGGCC-50 changing the base of position X to A, U,
G and C. As shown in Table 4, ON2 containing a Uabcm showed Tm of
69 °C with fully matched RNA (X = A) which is 3 °C lower than the
fully matched duplex of ON7 without Uabcm and same counter-
strand RNA whose Tm was 72 °C. On the other hand, in the cases
of the mismatched duplexes where nucleotides at position X are
U, G and C, the duplexes incorporating Uabcm were equally or more
stable than the corresponding duplexes incorporating Um. Espe-
cially, the duplex containing G-Uabcm was significantly more stable
than those incorporating Um by 5 °C, probably due to the participa-
tion of the carbamoyl group to the wobble-type guanine-uracil
base pair as suggested in Figure 1.
Figure 2. RP-HPLC profiles of the crude mixtures obtained by the treatment with aq
ammonia (a) with and (b) without DBU treatment. Cm = 20-O-methylcytidine.
100
80
ON8
60
ON9
40
20
ON10
Finally, we evaluated the stability of the Uabcm modified oligo-
nucleotides toward the enzymatic digestion. We synthesized Uabcm
modified trimer ON10 (50-UabcmUabcmdT) and digested ON10 by
snake venome phosphodiesterase (SVPDE). The time course of
the digestion was compared with the 20-O-methyl modified trimer
ON8 (50-UmUmdT-30) and the Ucm modified ON9 (50-UcmUcmdT). The
results are shown in Figure 3.
0
0
30
60
90
120
Time (min)
Table 4
Figure 3. Nuclease resistance of ON8 (closed circle), ON9 (open box) and ON10
(closed trigona) against SVPDE. Hydrolysis of the ONs (50 M) was carried out at
Tm (°C) and
D
Tm (°C) of 50-GmUmAmCm
U6 = Um (ON7)
C
mU6UmUmCmCmGmGm-30/30-CAUGG-
l
XAAGGCC-50
37 °C in 50 mM Tris–HCl buffer (pH 8.5) containing 72 mM NaCl, 14 mM MgCl2 and
2.5 ꢀ 10ꢁ4 U/ml SVPDE.
U6 = Uabcm (ON2)
DTm (°C)
X = A
X = U
X = G
X = C
70*
58
63
57
69
59
68
57
ꢁ3
+1
+5
0
As shown in Figure 3, the ON10 modified with Uabcm was the
most stable to SVPDE among the three trimers. This stability was
partially due to the cationic modification that prevents binding
of a metal ion that is known to be required for the enzyme to effi-
ciently catalyze the phosphoryl transfer reaction.18
Conditions: 10 mM phosphate buffer (pH 7.0), 150 mM NaCl, 0.1 mM EDTA, 2
duplex. Tm values were determined from Tm difference of ON2 and ON7.
Data from Ref. 7.
lM
D
*
In summary, we established the practical protocol for the syn-
thesis of oligonucleotides incorporating Uabcm. By using this car-
bamoyl-type modification, the cationic aminoalkyl group can be
readily incorporated to the 20-position of oligonucleotides. In addi-
tion, the Uabcm modified oligonucleotide showed improved hybrid-
ization affinity in comparison with previously reported Ucm
keeping its unique base pairing properties. Moreover, the cationic
Uabcm modification was proved to be effective to increase the enzy-
matic stability of oligonucleotide. These properties of Uabcm sug-
gested its usefulness for the development of functional
Figure 1. Base pair between guanosine and 20-O-carbamoyl modified uridine. Ucm
:
+
R = H. Uabcm: R = CH2CH2CH2CH2NH3
.

K. Seio et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2470–2473
2473
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This study was supported by a grant-in-aid for Scientific Re-
search from the Ministry of Education, Culture, Sports, Science
and Technology, Japan, and Adaptable and Seamless Technology
Transfer Program of JST. The authors thank center for Advanced
Materials Analysis (Suzukakedai), Technical Department, Tokyo
Institute of Technology, for MALDI-TOF mass analysis.
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An acCm-loaded CPG solid phase support (1
lmol) having a succinyl linker was
treated with 3% TCA in CH2Cl2 (1 mL) for 1 min at room temperature. After
washing [CH3CN 1 mL ꢀ 3] and drying under reduced pressure, the
condensation of the acCm-loaded solid phase support with phosphoramidite
unit 5 was performed as follows: (1) coupling [phosphoramidite unit 5 (19 mg,
20
(400
[phosphoramidite unit 5 (19 mg, 20
(10 mg, 80 mol) in dry CH3CN (400
l
mol) and 4,5-dicyanoimidazole (DCI) (10 mg, 80
L)] for 40 min, (2) washing [CH3CN 1 mL ꢀ 3], (3) drying, (4) coupling
mol) and 4,5-dicyanoimidazole (DCI)
L)] for 40 min, (5) washing [CH3CN
lmol) in dry CH3CN
l
l
l
l
1 mL ꢀ 3], (6) drying, (7) capping [2.5% phenoxyacetic anhydride and 5% 1-
methylimidazole in THF-pyridine 1 mL] for 5 min, (8) washing [CH3CN
1 mL ꢀ 3], (9) oxidation [0.02 M I2 in THF–pyridine–H2O 1 mL] for 1 min,
(10) washing [CH3CN 1 mL ꢀ 3], (11) drying, (12) 3% TCA in CH2Cl2, (13)
washing [CH3CN 1 mL ꢀ 3], (12) drying. Subsequently, the CPG solid phase
support was treated with 10% DBU in CH3CN (1 mL) for 1 min. After washing
[CH3CN 1 mL ꢀ 3] and drying under reduced pressure, the solid phase support
was treated with 28% aqueous ammonia at room temperature for 24 h. The
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CPG support was removed by filtration and washed with H2O (500
filtrate was purified by reversed phase HPLC to give UabcmCm 7 in 35% yield.
abcmCm 7; ESI-mass m/z Mass calcd for [C24H36N7O14P+H]+ 678.2, found 678.5.
lL ꢀ 3). The
U
18. Teplova, M.; Wallace, S. T.; Tereshko, V.; Minasov, G.; Symons, A. M.; Cook, P.
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