A new silyl ether-type linker useful for the automated synthesis of oligonucleotides having base-labile protective groups

Article abstract of DOI:10.1246/cl.2002.16

A new silyl-type linker for the automated solid-phase synthesis of oligodeoxynucleotides was developed. A hydrosilane-type reagent for introduction of the silyl linker was synthesized in an overall yield of 50% from 1,4-dibromobenzene. By using this linke

Full text of DOI:10.1246/cl.2002.16

16
Chemistry Letters 2002
A New Silyl Ether-Type Linker Useful for the Automated Synthesis of Oligonucleotides Having
Base-Labile Protective Groups
Akio Kobori, Kenichi Miyata, Masatoshi Ushioda, Kohji Seio, and Mitsuo SekineÃ
Department of Life Science, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 226-8501
(Received September 20, 2001; CL-010935)
A new silyl-type linker for the automated solid-phase synthesis
of oligodeoxynucleotides was developed. A hydrosilane-type
reagent for introduction of the silyl linker was synthesized in an
overall yield of 50% from 1,4-dibromobenzene. By using this
linker, an oligonucleotide having base-labile protecting groups
could be isolated from the controlled pore glass under neutral
conditions.
peptide
HN
O
6
O
H
N
CPG
poly-
styrene
O Si O
O
Si
T
O
1b
1a
Thy
DMTrO
O
Over the past several years, a number of approaches for gene
therapy have been explored by use of oligonucleotide analogues as
drugs.1 In particular, phosphorothioate (PS) oligonucleotides have
been well studied as the first-generation antisense molecules both in
vitro and in vivo, and their usefulness has been demonstrated by
many examples of successful inhibition of expression of the target
genes.2 However, side effects were observed in toxicity studies in
mice, rats and monkeys.3 Therefore, various oligonucleotides as the
second-generation antisense oligonucleotides were required to
overcome these problems. Furthermore, synthetic oligonucleotides
were widely used in the field of the analysis of single nucleotide
polymorphism (SNP).4 To develop the antisense therapy and DNA
diagnosis, large quantities of oligonucleotides with various
functional groups involving base-labile ones should be supplied
rapidly and inexpensively by the method of automated oligonucleo-
tides synthesis.5
O
O
Si
NH AP-CPG
1c
Figure 1.
b
a
Br
Br
HSi
Br
2
3
5
O
O
d
c
HSi
HSi
O-HN+Et3
OTBP
In the current automated solid-phase synthesis of DNA,
oligonucleotides bound to solid supports via a succinyl linker are
liberated by treatment with aqueous ammonia.6 However, DNA
oligomers having base-labile functional derivatives could not be
synthesized by the standard method. An approach to improve the
oligonucleotide synthesis involves the use of an oxalyl linker, which
was reported by Letsinger in 1991.7 By using this linker,
oligonucleotides could be released from the solid support by
treatment with propylamine under anhydrous conditions, and
oligodeoxynucleotides having N-benzoylated deoxycytidines were
synthesized. Fraser et al. reported a diisopropylsilanediyl linker8
(1a) which allowed the release of oligodeoxynucleotides from solid
supports under neutral conditions. They also synthesized oligo-
deoxynucleotides having N-benzoylated deoxycytidines. However,
partial cleavage of the silanediyl group of the linker was observed
during the detritylation step under acidic conditions.9 Recently,
Lipshutz et al. reported the silyl linker (1b) for the solid-phase
peptide synthesis using para-bromopolystyrene support (1%
crosslinked, 200-400 mesh, 2.6 ꢀmol/g).10 Therefore, we designed
a silyl-type linker (1c) bound to aminopropyl controlled pore glass
(AP-CPG) which was perfectly compatible with oligonucleotide
synthesizers, as shown in Figure 1.
4
T
DMTrO
O
O
O
Si
O
e
f
ClSi
1c
OTBP
OTBP
6
7
Scheme 1. a) BuLi (1 equiv), THF, À78 ꢀC, 5 min. then diisopropyl-
chlorosilane (1 equiv), 15 min. 86%. b) BuLi(1 equiv), THF, À78 ꢀC,
5 min. then excess dryice. 69% c) 2,4,6-tribromophenol(TBPOH)(2 equiv),
BOPCl (4 equiv), pyridine, rt, 30 min. quant. d) 1,3-dichloro-5,5-
dimethylhydantoine (2 equiv), CH2Cl2, rt, 1 h. e) DMTrT (2 equiv),
imidazole(10 equiv), CH2Cl2, rt, 1 h. 84%. f) aminopropyl-CPG, CH2Cl2, rt,
36 h.
reactions11 and purified by silica-gel column chromatography
containing 1% triethylamine. The tribromophenyl (TBP) ester 512
was obtained by condensation of 2 with tribromophenol in the
presence of BOPCl. Compound 5 was converted to a silyl chloride
derivative
6
by
treatment
with
1,3-dichloro-5,5-
dimethylhydantoine.13 The in situ generated silyl chloride was
allowed to react with 5’-O-DMTr-thymidine in the presence of
imidazole to give the anchor nucleoside 7 in an overall yield of 50%
from commercially available 1,4-dibromobenzene (2). When the
pentafluorophenyl ester was employed, it partially decomposed
during the 3’-O-silylation. Derivatization of aminopropyl-con-
Scheme 1 shows a simple and efficient synthetic route to the T-
loaded AP-CPG support bridged by the linker 1c. Triethylammo-
nium 4-(diisopropylsilyl)benzoate (4) was synthesized from 1,4-
dibromobenzene (2) by using stepwise halogen-metal exchange
Copyright Ó 2002 The Chemical Society of Japan

Chemistry Letters 2002
17
Figure 2. a) Anion exchange HPLCprofiles of TTTTTTTT synthesized from the anchor nucleoside 1. b) Reversed-phase HPLCprofiles of
i) d(CÃAastGÃT)3 synthesized from the anchor nucleoside 1 ii) d(CÃAÃGÃT)3 after NH3 treatments iii) d(CAGT)3 synthesized from the
standard succinate linker. c) Reversed-phase HPLCprofiles of the mixture of T, dC Ã, dGÃ and dAÃ which was obtained after treatment of
d(CÃAÃGÃT)3 with SVPD and AP.
trolled pore glass (AP-CPG, 89.2 ꢀmol/g) was performed with 7 at
room temperature for 36 h. After capping of the unreacted amino
functions with Ac2O in the presence of DMAP, the DMTrT-loaded
resin (1: 20.8 ꢀmol/g) was obtained. When this resin was treated
with 1 M TBAF-AcOH in THF for 1 h, 90% of the nucleoside was
released from the solid support, as evidenced by the DMTr assay.
To demonstrate the practical value of the silyl linker in the
automated DNA synthesis, TTTTTTTT was prepared from 1c in the
phosphoramidite approach14 on an Applied Biosystems 392 DNA/
RNA synthesizer. As the result, the average coupling yield was
>98% which was measured bythe DMTrcation assay. Treatment of
10% DBU/CH3CN for 30 s for deprotection of the cyanoethyl
group was required before the cleavage of the oligonucleotide using
1 M TBAF-AcOH in THF for 1 h. After SepPak chromatography
purification,15 TTTTTTTT was obtained in an overall yield of 73%
from the T-loaded resin having the linker 1c. The anion exchange
HPLCprofile of the final product is shown in Figure 2(a).
Furthermore, an oligodeoxynucleotide 12mer including N-acet-
yldeoxycytidine dCÃ,16 N-benzoyldeoxyadenosine dAÃ and N-
isobutyryldeoxyguanosine dGÃ whose acyl groups are known to be
easily eliminated under basic conditions were similarly synthesized
from the T-loaded resin with the average coupling yield of >98%.
The reversed-phase HPLCprofile of d(C ÃAÃGÃT)3 thus obtained is
shown in Figure 2b-(i). Figure 2b-(ii) shows the HPLCprofiles of
the reaction mixtures obtained by treatment of d(CÃAÃGÃT)3 with
This work was supported by a Grant from ‘‘Research for the
Future’’ Program of the Japan Society for the Promotion of Science
(JSPS-RFTF97I00301) and a Grant-in-Aid for Scientific Research
from the Ministry of Education, Culture, Sports, Science and
Technology, Japan.
This paper is dedicated to Professor Teruaki Mukaiyama on the
occasion of his 75th birthday
References and Notes
1
2
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Studies on the side reaction are now under way. The results will be
reported elsewhere.
3
4
5
6
7
8
9
ꢀ
NH3aq for 12 h at 60 C. It was shown that the N-acylated DNA
oligomer could be converted to the natural-type DNA oligomer
under basic conditions. The structures of these purified products
were further confirmed by MALDI-TOF mass. The N-acylated
oligonucleotide d(CÃAÃGÃT)3 was treated successively with snake
venom phosphodiesterase (SVPD) and calf intestinal alkaline
phosphatase (AP) to give T, dCÃ, dGÃ and dAÃ in the expected
ratio, as shown in Figure 2c. From these results, it was found that
d(CÃAÃGÃT)3 was synthesized without elimination of the acyl
group through the DNA synthesis.
10 B. H. Lipshutz and Y-J. Shin, Tetrahedron Lett., 42, 5629 (2001).
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¨
In conclusion, we have demonstrated the utility of the silyl
linker for DNA solid-phase synthesis. The linker was fully matched
to the present automated DNA synthesizer, and could be used for the
synthesis of oligonucleotides having various base-labile functional
groups. Further studies are now under way.
15 C18 SepPak cartridge was obtained from Millipore corporation.
16 M. P. Reddy, N. B. Hanna, and F. Forooqui, Tetrahedron Lett., 35, 4311
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17 MALDI-TOF MS Calcd for C156H183N45O79P11 [M-H]À : 4290.9.
Found: 4291.2.