A novel synthesis of S6-cyanoethyl-2'-deoxy-6-thioguanosine and its incorporation into triple helix forming oligonucleotides

Article abstract of DOI:10.1016/0040-4039(93)88007-6

A simple and expeditious synthesis of S⁶-cyanoethyl-2'-deoxy-6-thioguanosine from 2'-deoxyguanosine has been accomplished in good yield and incorporated into several triple helix forming oligonucleotides using the solid-phase phosphoramidite ch

Full text of DOI:10.1016/0040-4039(93)88007-6

Vol. 33. No. 50. pp. 7651-7654.1992
$5.00 +
in Great Br i t ai n
A Novel Synthesis of
Incorporation into Triple Helix Forming
and its
T. Sudhakar Rao, Krishna Jayaraman, Ross H. Durland,
and Ganapathi R.
Triplex Pharmaceutical Corporation,
9391 Grogans Mill Road, The Woodlands, TX 77380, U.S.A.
ABSTRACT: A simple and expeditious synthesis of
from has been accomplished in good yield and incorporated into several
triple helix forming oligonucleotides using the solid-phase phosphoramidite chemistry.
Recently it has been shown that in the presence of
metal ions, short guanine-
at
Evidence has also been provided to show that the formation of such
rich oligonucleotides can bind to specific sites in duplex DNA to form triple
physiological
sequence-specific triple
can inhibit DNA
and block transcription
initiation,* thus resulting in the specific inhibition of the synthesis of target protein.
Therefore, the potential therapeutic significance of these triplex forming oligonucleotides
is obvious.
The preponderance of guanine residues in these
can promote self-association and
lead to stable by the formation of eight H-bonds and by coordination of the four
atoms of guanine with alkali metal ions bound to the center of the quadruplex. Chemical
modification of the guanine moiety that disrupts the multiple hydrogen bonding of the
tetrad can reduce or eliminate its formation. Replacement of all or some of the guanine
residues in these
with
is expected to reduce self-association by interfering
with the coordination of alkali metal and by reducing the strength of H-bonds to that position.
In addition, the increased atomic size of sulfur relative to oxygen may lead to significant steric
repulsion in the tetrad. However, this modification is not expected to affect the groups
believed to be involved in the H-bonding of GGC triplets. We now report a novel and simple
synthesis of protected
and its incorporation into several
using the automated solid-phase phosphoramidite chemistry.
Although a few reports have appeared describing the synthesis of
containing
none of these syntheses considered protecting the thione group of
which is of paramount importance to prevent oxidative hydrolysis that is expected in
sulfur-containing nucleotides. Our own attempts to use
in the solid-phase oligonucleotide synthesis resulted not
only in lower coupling yields but also in the formation of multiple products. Therefore, we
7651

7652
decided to protect the thione functionality of
cyanoethyl group can be readily removed under mild alkaline conditions. During the course
of the present work, Christopherson and
containing using the cyanoethyl group for the protection of the thione moiety. Their
with a cyanoethyl group, since the
reported the synthesis of oligonucleotides
approach involved the synthesis of S-protected building block from the preformed
R.-P<
In our first approach,
was transformed to the
corresponding intermediate using trifluoroacetic anhydride in
Since the
pyridyl group attached to the
the
of purine being very susceptible to nucleophilic
intermediate was reacted with
This
one-flask, two-step procedure gave a homogeneous product (85% yield), which was identified
as
The
group of
was selectively protected as the
ether by the treatment with
dimethoxytrityl chloride in pyridine. The pure product was isolated in a 76% yield after
silica gel column chromatography. Compound was conveniently converted into the
by reaction w ith
correspond ing
tetraisopropylphosphoro-diamidite in the presence of tetrazole and diisopropylamine in
dichloromethane. The yield of pure after silica gel column chromatography was 83%.
In this approach, during the preparation of the
group was displaced by a
trifluoroacetyl moiety. Although the exact mechanism of this displacement is unknown, it
may be due to the high reactivity of trifluoroacetic anhydride, which is reacting with the basic
NI-I functionality displacing the isobutyryl group.
R

7653
Although the phosphoramidite
was found to be suitable for solid-phase DNA
synthesis, this methodology limits the possibility of using a very labile protecting group on
the exocyclic amino function. In view of this we have also synthesized the fully protected
building block
a
intermediate. Thus, treatment of
with 1-methylpyrrolidine (10 eq) and
dichloromethane for 2 h at room temperature gave
(10 eq) in
ether in a 89%
yield. Selective removal of the sugar protecting groups using HF/tetrabutylammonium
fluoride in dry pyridine afforded (Z), mp
Although this approach allows the usage of any type of protecting groups for the
amino function, in the present study it is demonstrated only by using a isobutyryl group.
Compound
was converted to the corresponding
derivative
by the
conventional procedure, which on treatment with
propylphosphorodiamidite in the presence of tetrazole and diisopropylamine gave the target
building block
containing
by coupling
additional 30 seconds on a ABI 3808 DNA synthesizer. Deprotection was performed with
concentrated and the TFO was purified by ion-exchange HPLC using a Q-Sepharose
column (Pharmacia). The purified product was desalted and analyzed on a 20% denaturing
polyacrylamide gel after labeling with using polynucleotide kinase. Unsubstituted
oligonucleotide was used as the standard to compare its mobility and purity.
residues were prepared with a
coupling efficiency of
three times to the solid support and increasing the reaction time for an
A series of
were prepared based on the following model sequence, which was
testing of triplex formation
designed for in
The test TFO is a 26-mer containing 20
and 6 T residues. The
Three, 11 and all 20
units. The ratio of absorptions at 345 and 260 nm is consistent with
residues, e.g. the TFO containing three residues had a
residues exhibit a characteristic UV absorption at 345
residues
were replaced by
the increasing number of
ratio of 0.13 and for 11 and 20 residues, the ratios were 0.6 and 1.8, respectively.
In conclusion, this communication describes a simple and novel synthetic strategy
which can be used for the synthesis of containing without the possibility of
expected oxidative hydrolysis of the thione function.

7654
References and Notes
1.
2.
3.
This research was supported in part by the National Institutes of Health, National Cancer
Institute, SBIR Grant R43
Cooney, M.; Czernuszewicz, G.; Postel, E. H.; Flint, S.
456459.
Hogan, M. E. Science, 1988,242,
Du rlan d ,
Biochemistry,
H.; Kessler, D. J.;
9246-9255.
S.; Duvic, M.; Pettitt, B. M.; Hogan, M.E.
4.
5.
D. S.; Levenson, C.; Shafer, R. H.
Birg, F.; Praseuth, D.; A.; Thuong, N.;
Acids Res., 2901-2908.
L. J.; Dervan, P.B.;
U.;
T.; Helene, C. Nucleic
8820-8826.
6.
7.
a.
9.
B. J. Biochemistry,
Sen, D.; Gilbert, W. Nature,
Sundquist, W. I.;
A. Nature, 1989,342, 825829.
Jin, R.; Breslauer, K. J.; Jones,
A.; Gaffney,
L. Science, 1998,250, 543-546.
10. Kim, Cheong, C.; Moore, P. B. Nature,
11. Bronstein, S. M.; Richardson, F. C.; Skopek, T. R.; Swenberg, J. A.
Res., 1401.
12. Rappaport, H. P. Nucleic Acids Res.,
Am. Assoc. Cancer
7253-7267.
Richardson, F. C.; Bronstein, S. M. Intl. Patent Publication, No.
1989).
778 (Oct. 19,
13.
14.
Christopherson, M. S.; Broom, A. D. Nucleic Acids Res., 1991,
L.; Jones, R. A. Am. 1982,104, 1316-1319.
B.; P-P.; Gaffney, B. L.; Jones, R. A. Tetrahedron
5719-5724.
15. Ti, G. S.; Gaffney,
16. R.;
319-322.
17. Kung, P-P.; Jones, A. Tetrahedron
Compound had mp of
39193922.
(dec.).
3.15 (t, J = 6.6
4.44 (m, 1
8.69
NMR
2 H,
s, 1
and 12.17
2.27-2.52 (m, 1 H,
3.58 (m, 4 H, and SC&-),
5.33 s, 1 H,
1 H, CONK).
2.69-2.84 (m, 1 H,
3.85 (m, 1 H,
J = 6.4 Hz, 1 H,
6.38 (t,
Calcd. for
1 H,
0.25
C, 41.24; H, 3.56; N, 19.26; F, 13.06. Found: C, 41.08; H, 3.43; N,
18.94; F, 13.05.
19.
:
1.00-1.35 [m,
3.03 (t, 2 H,
1.95 [m, 6 H,
3.10-3.70 (m, 4 H,
6.37 (t, J = 6.4 Hz, 1 H,
149.38 ppm.
2.40-2.80 (m, 2
3.73 (s, 6 H, 2
6.60-7.40 (m, 13
Calcd. for
H,
and
4.20 (m, 1 H,
H, DMT) and 8.18 (s, 1 H,
and
4.85 (m, 1 H,
NMR
:
C, 57.80; H, 5.39; N, 11.98; S, 3.43. Found: C, 57.51; H, 5.74; N, 12.01; S,
3.30.
Gaffney, B. L.; Marky, L. A.; Jones, R. A. Tetrahedron,
20.
21.
NMR
1.15 (m, 18 H, isopropyl and isobutyryl protons), 1.95 (m, 1 H,
(m, 2 H,
4.20 (m, 1 H,
and
3.00-3.90 (m, 6 H,
4.89 (m, 1 H, 6.34 (t, J = 6.40
and 8.75 s, 1 H, NMR
and
3.73
6.67-
149.29
(s, 6 H, 2
7.38 (m, 13 H,
1 H,
8.10 (s, 1 H,
(Received in USA 10 July 1992; accepted 15 September 1992)