Synthesis and properties of morpholino chimeric oligonucleotides

Article abstract of DOI:10.1016/j.tetlet.2008.04.035

Chimeric oligonucleotides with the novel morpholino modification and the phosphoramidate linkers have been synthesized and characterized. These oligonucleotides showed moderate thermal stability with complementary RNA and DNA, and enhanced resistance towa

Full text of DOI:10.1016/j.tetlet.2008.04.035

Available online at www.sciencedirect.com
Tetrahedron Letters 49 (2008) 3570–3573
Synthesis and properties of morpholino chimeric oligonucleotides
a,b
b
b
c,b,
d
a
*
Nan Zhang , Chunyan Tan , Puqin Cai , Yuyang Jiang
, Peizhuo Zhang , Yufen Zhao
a
Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Ministry of Education, Department of Chemistry,
Tsinghua University, Beijing 100084, PR China
Key Laboratory of Chemical Biology, Guangdong Province, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, PR China
b
c
School of Medicine, Tsinghua University, Beijing 100084, PR China
Shanghai GenePharma Co., Ltd, 1011 Halley Road, Z.-J. High Tech Park, Shanghai 201203, PR China
d
Received 15 December 2007; revised 2 April 2008; accepted 7 April 2008
Available online 9 April 2008
Abstract
Chimeric oligonucleotides with the novel morpholino modification and the phosphoramidate linkers have been synthesized and
characterized. These oligonucleotides showed moderate thermal stability with complementary RNA and DNA, and enhanced resistance
toward the nuclease (t1/2 > 10 h). The phosphoramidate linker made the synthesis of such oligonucleotides applicable on a DNA
synthesizer. Under the acidic condition (pH 3.0), the phosphoramidate linkers were readily cleaved, and such property might be useful
for the DNA-sequence determination.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Morpholino oligonucleotides; Thermal stability; Nuclease resistance; Duplex conformation; Acid-catalyzed hydrolysis
Chemical-modified oligonucleotides are widely used in
1
biotechnology and gene therapy areas, acting as antisense
2
oligonucleotides, ribozymes, and siRNAs. An applicable
modified oligonucleotide needs high binding affinity to tar-
get RNA or DNA, high nuclease stability, high cellular
2
,3
uptake efficiency, and low toxicity. Morpholino oligonu-
loetides (Fig. 1a), with morpholino rings in subunits
instead of the ribose and a non-ionic intersubunit linkage
instead of the phosphodiester bond, have become one of
the promising candidates for in vivo and in vitro gene
4
function study. Morpholino oligonuloetides have many
advantages: (1) they inhibit the translation of target mRNA
by steric block, (2) they have superior binding affinity to
RNA; and(3) they have high resistance to enzymatic degra-
Fig. 1. Morpholino oligonucleotide with a neutral backbone (1a) and with
an anionic backbone (1b).
6
5
to deliver them into cells, such as microinjection, hybridi-
dations. However, the morpholino oligonucleotides have
zation of DNA with the morpholino oligonucleotide and
several limitations. Firstly, they cannot be transfected by
liposome and thus special methods have to be carried out
7
delivery with ethoxylated polyethylenimine, and conjuga-
8
tion with peptides. Secondly, the duplex formed by the
morpholino oligonucleotide and target RNA is not a sub-
strate for RNase H due to the heteroduplex conformation
change compared to normal DNA/RNA. Lastly, the mor-
pholino nucleoside monomer can not be incorporated into
*
Corresponding author. Tel.: +86 755 26036017; fax: +86 755
2
6036087.
E-mail address: jiangyy@sz.tsinghua.edu.cn (Y. Jiang).
0
040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.04.035

N. Zhang et al. / Tetrahedron Letters 49 (2008) 3570–3573
Table 1
3571
chimeric oligonucleotide on a DNA synthesizer, which lim-
9
Oligonucleotides sequence and corresponding Tm values of complexes
with complementary DNA and RNA
its its applications in the ‘gapmer’ approach and thechem-
a
ical-modified siRNA.
0
0
No.
Sequence (5 -3 )
m
T /°C
Here we report our novel design of chimeric oligonucleo-
tides with anionic phosphate backbones containing
morpholino nucleoside analogues (Fig. 1b) and phosphor-
amidate linkers, which was aimed to maintain the good
enzymatic stability as well as to incorporate the modifica-
tion into oligonucleotides on a DNA synthesizer. Also
reported is their affinity toward DNA and RNA, resistance
to nuclease, and hydrolysis under acidic conditions.
The synthesis of the building block, phosphoramidite
monomer 7, is shown in Scheme 1. 5-Methyluridine (2)
was treated with DMTr–Cl in anhydrous pyridine over-
DNA
RNA
8
9
d(GCGTTTTTTGCT)
d(GCGTTTTTTGCT)
d(GCGTTTTTTGCT)
d(GCGTTTTTTGCT)
d(TTTTTTTTTT)
d(TTTTTTTTTT)
d(TTTTTTTTTT)
d(TTTTTTTTTT)
61.5
59.6
57.9
57.2
nd
nd
nd
nd
58.9
58.6
57.9
57.0
nd
nd
nd
nd
10
11
12
13
14
15
a
Morpholino modifications are in bold.
0
night under argon atmosphere to afford 5 -O-DMTr-5-
methyluridine (3) in 86% yield. Following published pro-
1
0
Thermal stability of modified oligonucleotides 9, 10, 11
1
1
toward DNA and RNA was studied by UV-Tm experi-
ments. As a result, the morpholino nucleosides modifica-
tion caused distabilization with RNA and DNA only to a
very limited extent. Specifically, the modification in oligo-
nucleotide unstabilized the duplex with DNA by ꢀ1.7 °C
per modification, whereas it only distabilized the duplex
with RNA by ꢀ0.5 °C per modification. The thermal sta-
bility of our morpholino-modified oligonucleotides toward
RNA is similar to that of the literature reported phos-
cedures with improvements, compound 3 was then sub-
jected to the construction of the morpholino ring in one
step by treating it with sodium periodate and then ammo-
0
0
nium biborate to afford 2 ,3 -dihydroxyl-5-methyl-morpho-
0
0
lino-uridine (compound 5). Reduction of 2 and 3 hydroxyl
groups on compound 5 gave compound 6. The total yield
of the 3 steps above was 84%. The phosphoramidite build-
ing block 7 was then obtained by the phosphitilation of
compound 6 under the conditions with 2-cyanoethyl-
1
4
0
0
phorothioate oligonucleotides. It is believed that such
slight distabilization effect will not affect the further appli-
cation of morpholino-modified oligonucleotides.
N,N,N ,N -tetraisopropylphosphorodiamidite and 4,5-di-
cyanoimidazole (DCI) in hydrous CH Cl for 4 h under
2
2
1
2
argon atmosphere in 90% yield. All the compounds were
1
13
31
The conformation of DNA/RNA duplex was reported
characterized by H NMR, C NMR, P NMR, and
HRMS.
1
5
to activate RNase H which degrades target RNA. The
CD spectroscopy was used to study the conformation of
duplexes formed by the modified oligonucleotide 9, 10,
and 11 with complementary RNA. As shown in Figure 2,
the spectra of the duplexes containing different numbers
of modified subunits are quite similar to that of the natural
DNA/RNA duplex, indicating that such modification did
not affect the helical conformation of the duplex.
The building block 7 was incorporated into oligonucleo-
tides (Table 1) on a DNA synthesizer, with 5-ethylthio-1H-
tetrazole (ETT) as the activator in 5 min coupling
time. After being removed from solid support, crude oligo-
nucleotides were purified by HPLC. The isolated yield of
modified oligonucleotides was approximately 30%. Oligo-
nucleotides were analyzed by HPLC and MALDI-TOF
1
3
We synthesized unmodified oligonucleotide 12 and three
modified oligonucleotides 13, 14 and 15 with the modifica-
mass spectroscopy.
0
tions on the 3 -terminal ends to investigate the stability of
0
O
O
N
O
N
our morpholino-modified oligonucleotides against 3 -exo-
NH
O
NH
O
NH
O
N
HO
(i)
DMTrO
(ii)
DMTrO
O
O
O
10
oligo 8/RNA
oligo 9/RNA
oligo 10/RNA
oligo 11/RNA
OH OH
OH OH
O
O
2
3
4
(
iii)
0
O
N
O
O
NH
O
NH
O
NH
O
DMTrO
iPr2
N
N
DMTrO
DMTrO
HO
O
(v)
(iv)
O
O
-
10
N
P
N
H
6
N
H
5
OH
CN
N
O
7
2
00
250
300
350
Scheme 1. Reagents and conditions: (a) DMTr–Cl, NEt
3
, Py, rt, Ar; (b)
, CH OH, rt; (e)
Cl
Wavelength / nm
and (c) NaIO
4
, (NH
4
0
)
2
0
B
4
O
7
, CH
3
OH, rt; (d) NaCNBH
3
3
2-cyanoethyl-N,N,N ,N-tetraisopropylphosphorodiamidite, DCI, CH
2
2
,
Fig. 2. CD spectra of the duplexs between modified oligonucleotides with
complementary RNA.
rt, Ar.

3572
N. Zhang et al. / Tetrahedron Letters 49 (2008) 3570–3573
nuclease (snake venom phosphodiesterase, SVPDE), which
is the major cause of oligonucleotides degradation in
In conclusion, we have synthesizedmorpholino-modified
chimeric oligonucleotides containing anionic phosphate
backbones, along with moderate binding affinity to com-
plementary RNA and high resistance of nuclease. Incorpo-
ration of modifications into oligonucleotides does not affect
the conformation of duplex with complementary RNA.
The phosphoramidate linkage can be readily cleaved under
acidic condition. These favorable properties make morpho-
lino modification a good candidate for antisense therapeu-
tics and the gene function studies.
1
6
serum. Hydrolysis was carried out at near physiological
conditions, and the degradation of oligonucleotides was
analyzed by HPLC at several time points. As shown in
Figure 3, the unmodified oligonucleotide 12 degraded rap-
idly with a half-life t1/2 of 20 min, whereas in contrast, half-
lives of modified oligonucleotides 13, 14 and 15 are 10 h,
1
2 h, and 15 h, respectively. As a result, all the modified
oligonucleotides demonstrated significantly improved sta-
bility, which was gradually enhanced with the increasing
number of modifications.
Additionally, the acid-catalyzed hydrolysis properties of
these modified oligonucleotides were evaluated. Morpho-
lino-modified oligonucleotide 9 was rapidly cleaved at pH
Acknowledgments
The authors would like to thank the financial supports
from the Ministry of Science and Technology of China
3
.0, while it was stable at pH 7.0 (Fig. 4). For example,
(2005CCA03400, 2007AA02Z160), the Chinese National
after 30 min, about 50% modified oligonucleotide 9 was
cleaved at pH 3, while no obvious cleavage was observed
at pH 7. In addition, no cleavage of unmodified oligonu-
cleotide 8 was detected at pH 3.0. This acid labile phos-
phoramidate linkage can be used to produce short
polynucleotide fragments, which can be easily analyzed
by MALDI-TOF mass spectrometry and thereby be appli-
Natural Science Foundation (20572060, 20472043), and
the Department of Science and Technology of Guangdong
Province (2005A11601008).
Supplementary data
1
7
Experimental procedures and spectral data for com-
pounds in Scheme 1, synthesis of oligonucleotides and pro-
cable for DNA-sequence determination.
cedures used for T , CD, nuclease study and acid-catayzed
m
1
00
hydrolysis are available. Supplementary data associated
with this article can be found, in the online version, at
doi:10.1016/j.tetlet.2008.04.035.
oligo 13
oligo 14
oligo 15
oligo 12
7
5
2
5
0
5
0
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ꢀ
1
3. MALDI-TOF data of oligonucleotides: Compound 8 [MꢀH] 3632.30
ꢀ
ꢀ
(
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