Synthesis of novel tetrathiafulvalene system containing redox-active ribonucleoside and oligoribonucleotide

Article abstract of DOI:10.1021/ol9910868

(matrix presented) RNA oligosynthesis block 8 was synthesized from selone 1 by successive silylation, acetylribosylation, coupling with selone 4, desacetylation, and treatment of obtained ribonucleoside 6 with monomethoxytrityl chloride, followed by o-chl

Full text of DOI:10.1021/ol9910868

ORGANIC
LETTERS
1
999
Vol. 1, No. 13
065-2067
Synthesis of Novel Tetrathiafulvalene
System Containing Redox-Active
2
Ribonucleoside and Oligoribonucleotide
,†
†
‡
Ojars Neilands,* Vilnis Liepinsh, and Baiba Turovska
Department of Organic Chemistry, Riga Technical UniVersity, 14 Azenes Street,
LV-1048, Riga, LatVia, and LatVian Institute of Organic Synthesis,
21 Aizkraukles Street, LV-1006, Riga, LatVia
neilands@ktf.rtu.lV
Received September 24, 1999
ABSTRACT
RNA oligosynthesis block 8 was synthesized from selone 1 by successive silylation, acetylribosylation, coupling with selone 4, desacetylation,
and treatment of obtained ribonucleoside 6 with monomethoxytrityl chloride, followed by o-chlorobenzoylation and phosphonylation. Solid-
phase oligosynthesis is being performed, and redox-active heptaribonucleotide phosphothioate 9 is obtained which has a lower oxidation
potential than ribonucleoside 6.
Redox-active nucleoside and nucleic base derivatives capable
of forming intermolecular complementary hydrogen bonds
ferrocene moiety. An electrochemical gene sensor system
has been developed using a redox-active ferrocene-modified
oligonucleotide anchored on a gold electrode by phospho-
thioate units on its 5′-terminus. We have aimed to investigate
the use of dioxopyrimidotetrathiafulvalenes containing di-
rectly fused uracil and tetrathiafulvalene systems for design
of oligonucleotides useful for nucleic acids and their
component recognition. This hybrid molecule is not only a
strong electron donor capable of forming stable cation radical
salts but also a target for CHB formation; therefore the close
location of the CHB center to the redox-active moiety can
promote a change in redox-properties and absorption spectra.
We were able to observe a change in oxidation potentials
and UV absorption spectra for dimethyl[1-butyl-2,4-dioxo-
(1H,3H-pyrimido]tetrathiafulvalene in the presence of 2,6-
(CHB) during recent years have received much attention due
to the recognition and detection possibility of nucleic acids
1
and their components. B a¨ uerle has investigated conducting
polythiophenes functionalized with uracil or adenine deriva-
tives. As was demonstrated in voltammetric and spectro-
electrochemical experiments, successive addition of comple-
mentary bases to polymers leads to specific modulation of
the electrochemical and optical properties due to specific
2
formation of CHB. Garnier used functionalized polypyrroles
and prepared a polypyrrole electrode functionalized with an
oligodesoxyribonucleoside and showed that the cyclic vol-
tammogram is significantly modified upon addition of a
3
a
complementary oligodesoxyribonucleoside target. Letsinger
3
b
4
and Ihara have synthesized oligonucleotides linked with a
di(acetylamino)pyridine. The goal of our investigation is
the design of oligonucleotides containing a tetrathiafulvalene
moiety.
†
Riga Technical University.
Latvian Institute of Organic Synthesis.
‡
(
1) (a) Emge, A.; B a¨ uerle, P. Synth. Met. 1997, 84, 213. (b) B a¨ uerle, P.;
Emge, A. AdV. Mater. 1998, 3, 324.
2) Garnier, F.; Korri-Youssoufi, H.; Srivastava, P.; Mandrand, B.; Delair,
Th. Synth. Met. 1999, 100, 89.
(3) (a) Mucic, C. R.; Herrlein, M. K.; Mirkin, C. A.; Letsinger, R. L.
Chem. Commun. 1996, 555. (b) Ihara, T.; Nakayama, M.; Murata, M.;
Nakano, K.; Maeda, M. Chem. Commun. 1997, 1609.
(
1
0.1021/ol9910868 CCC: $18.00 © 1999 American Chemical Society
Published on Web 12/02/1999

8
The key compound for oligoribonucleotide synthesis is
ribonucleoside 6. In this Letter we describe the synthesis
2
confirmed by NMR spectra. In UV spectra (MeCN/H O 1:1)
5
a broad absorption maximum was observed at 400-410 nm
(ꢀ ) 2000) which is characteristic of dioxopyrimidotetra-
thiafulvalenes. Cyclic voltammetric investigation confirmed
two stages of reversible redox behavior in MeCN solution
independently of the electrode used, Epa ) 0.54, Epa ) 0.88
V (vs SCE), ∆E ) 0.08 V. In MeCN/H O (1:1) solution,
only the first redox couple can be registered, Eox ) 0.44 V,
and the separation of cathodic/anodic peaks is increased until
0.15 V.
and properties of compound 6, transformation of 6 to RNA
oligosynthesis building block 8, and preparation of the first
heptaribonucleotide phosphothioate 9 bearing the tetrathia-
fulvalene moiety. The starting compound 1 has been
4
1
2
6
synthesized from barbituric acid. The acetylribosylation of
p
2
1
1
has been performed by trimethylsilylation and interaction
with tetraacetylribose in the presence of trimethylsilyl triflate
Scheme 1). The acetylribosyl product 3 was purified by
(
9
RNA synthesis building block 8 has been synthesized
from nucleoside 6 (Scheme 2) by monomethoxytritylation
Scheme 1
Scheme 2
in position 5′ and successive o-chlorobenzoylation and
1
0
11
phosphonylation in the same reacton mixture using the
1
2
3
PCl /imidazole reagent of 5′ O-protected nucleoside 7.
silica gel chromatography using gradient of toluene (0-35%) in ethyl acetate
as eluent. Product 5 was obtained as a red foam. Yield: 0.33 g, 25%. 5
(0.31 g, 0.54 mmol) was dissolved in saturated ethanolic ammonia (2.5
mL), and saturated aqueous ammonia was added (3 mL). The mixture was
left at rt for 6 h, evaporated to dryness, washed with EtOAc, and filtered,
and the precipitate was dissolved in hot ethanol (100 mL), filtered,
evaporated to a small volume (ca 4 mL), and left at +4 °C overnight.
Compound 6 was obtained as a red crystalline solid. Yield: 0.13 g, 54%.
column chromatography and reacted with selone 4 in the
presence of triphenylphosphine. Deprotection of purified
compound 5 by an ethanolic ammonia solution gave ribo-
7
nucleoside 6 as light red crystals in 9% isolated yield
8
(
calculated on 1). The structure of compound 6 has been
1
H NMR (DMSO-d6, 270 MHz) δ: 11.84 (bs, 1H, NH), 5.70 (d, J ) 5.77
(
(
(
4) Goldenberg, L.; Neilands, O. J. Electroanal. Chem. 1999, 463, 212.
5) Neilands, O.; Liepinsh, V. Chimia 1997, 392.
6) Neiland, O. Ya.; Khodorkovsky, V. Yu.; Tilika, V. Zh. Khim.
Hz, 1H, H1′), 5.30 (d, J ) 5.77 Hz, 1H, OH), 5.05 (d, J ) 5.49 Hz, 1H,
OH), 4.81 (t, J ) 5.77 Hz, 1H, OH), 4.31 (m, 1H, H2′), 4.01 (m, 1H, H3′),
1
3
3.81 (m, 1H, H4′), 3.65 (m, 2H, H5′ and H5′′), 1.98 (s, 6H, 2 × CH3).
C
Geterots. Soedin. 1992, 1667 (Engl. Ed. Chem. Heterocycl. Comp. 1992,
NMR (DMSO-d6, 67.9 MHz) δ: 155.31 (C4), 149.59 (C2), 145.84 (C6),
122.91 and 122.72 (C-Me), 108.26 and 99.58 (CdC), 116.97 (C5), 91.83
(C1′), 85.70 (C4′), 70.89(C2′), 69.27 (C3′), 61.28 (C5′), 13.34 (2 × Me).
(9) Synthesis of 8: 6 was alkylated in dry pyridine at 0 °C by
monomethoxytrityl chloride and purified by silica gel column chromatog-
raphy using chloroform-methanol (0-1.5%) containing 0.2% Et3N as
eluent. Product 7 was obtained as a red amorphous foam in a yield of 40%.
Nucleoside 7 underwent acylation by o-chlorobenzoyl chloride in a CH2Cl2/
pyridine solution at -78 °C and without isolation of the acyl product was
phosphonylated by PCl3 in the presence of imidazole and triethylamine at
-78 °C. The reaction mixture was poured onto and extracted with 1.0 M
aqueous triethylammonium bicarbonate, pH 7.5. The organic layer was dried
and evaporated, and the product was purified by silica gel chromatography
using chloroform-methanol (0-7%) containing 0.1% Et3N as eluent and
the reprecipitated from a chloroform solution by light petroleum ether. The
1
432).
(7) Neiland, O. Ya.; Tilika, V..Zh.; Edzhina, A. S. Khim. Geterots. Soedin.
1
994, 1285 (Engl. Ed. Chem. Heterocycl. Comp. 1994, 1116).
(8) Synthesis of 6: 1 (3 g, 11.32 mmol) was refluxed with 25 mL of
HMDS and 0.2 mL of TMS-Cl for 22 h. The mixture was evaporated to
dryness, coevaporated with dry toluene (3 × 50 mL), and dried in a vacuum
for 1 h. The crystalline product was dissolved in MeCN (70 mL), tetra-O-
acetylribose 2 was added (3.06 g, 9.62 mmol), and the solution was cooled
to -50 °C in an acetone-dry ice bath. Trimethylsilyl triflate (2.36 mL, 13
mmol) was added during 10 min via septum with vigorous stirring. Mixture
was warmed to rt in 2 h, stirred at rt for 37 h, poured into saturated NaHCO3
(
aqueous) (200 mL), and stirred for 1 h. The resulting mixture was filtered,
the precipitate was washed with chloroform, and the aqueous layer was
extracted with chloroform (400 mL). After silica gel column chromatography
using a gradient of methanol (0-1.5%) in dichloromethane as eluent, product
1
yield of orange solid 8 was 58%. H NMR (CDCl3, 400.13 MHz) δ: 10.34
3
was obtained as a red amorphous foam and characterized by NMR.
(bs, 1H, NH), 8.21, 7.90, 7.61-7.0 (m, 16H, Ar), 6.82 (d, J ) 645.63 Hz,
1H, PH), 6, 77 (m, 2H, Ar), 6.01 (s, 1H, H1′), 5.57 (m, 1H, H2′), 5.34 (m,
1H, H3′), 4.23 (m, 1H, H4′), 3.71 (s, 3H, OMe), 3.60 and 3.44 (2m, 2H,
H5′ and H5′′), 2.98 (q, J ) 7.24 Hz, 6H, CH2N), 1.92 (s, 6H, 2 × Me).
1.25 (t, 9H, NCH2CH3). 31P NMR (CDCl3, 162.0 MHz) δ: 2.83.
Yield: 3.3 g, 65%. 3 (1.2 g, 2.29 mmol) and selone 4 (1.2 g, 5.73 mmol)
were dissolved in dry toluene (20 mL). A solution of triphenylphosphine
(
3.0 g, 11.47 mmol) in toluene (15 mL) was added and stirred at rt for 3 h.
The solution was filtered through silica gel, evaporated, and purified by
2066
Org. Lett., Vol. 1, No. 13, 1999

The obtained building block 8 was used in an automated
solid-phase H-phosphonate method for RNA synthesis and
′-O-o-ClBz protection10 using a modified Pharmacia Gene
oligonucleotide and cleavage from the polystyrene support
with an ethanolic ammonia solution, the crude oligonucleo-
tide phosphothioate was purified by reversed phase HPLC.
Pure heptaribonucleotide 9 was obtained in the form of a
yellow solid as a mixture of diastereomers, unsoluble in
13
2
Assembler Plus oligonucleotide synthesizer.
Our first attempts involved standard oxidation conditions
with 2% iodine solution in wet pyridine after the final
coupling step in order to get phosphate internucleotidic
bonds. These attempts, however, were not successful due to
instability and breakage of the tetrathiafulvalene system in
these oxidative reaction conditions. Therefore, we decided
to oxidize the internucleotidic bonds with elemental sulfur
2 2 2
MeCN or CH Cl . In UV spectra (MeCN/H O 1:1) absorp-
tion at 418 nm (ꢀ ) 2700) has been observed. The spectra
confirmed the molecular composition of oligoribonucleotide
9, containing dioxopyrimido-TTF and U moieties. In MeCN/
2
H O solution CVA measurements on a glassy carbon
electrode showed that 9 is redox-active: the first quasi-
1
4
1
in pyridine in order to obtain the desired oligonucleotide
phosphothioate as a mixture of diastereoisomers (chirality
of P atoms). The synthesized sequence was 5′-UsUsUs(U-
TTF)sUsUsU-3′ 9 (Scheme 3). After the deprotection of the
reversible oxidation takes place at Epa ) 0.29 V; the second
redox-stage cannot be registered in this media.
It should be noted that in the UV spectra of 9 the
absorption maximum at 418 nm is bathochromic shifted in
comparison with that of ribonucleotide 6 (410 nm), but the
E
pa of 9 is lower than that of 6 (0.29 and 0.44 V). This
Scheme 3
phenomenon could be explained by intramolecular interaction
in this unusual molecule 9 that reduces the electron acceptor
ability of the uracil fragment.
To the best of our knowledge this is the first case where
the tetrathiafulvalene system has been incorporated into an
oligoribonucleotide in a manner that preserves the system’s
potential to form complementary hydrogen bonds. The
investigation of oligonucleotide 9 properties in the presence
of complementary nucleobases and its binding on the gold
surface is in progress.
Acknowledgment. We thank Prof. Roger Str o¨ mberg of
Karolinska Institute (Stockholm) for help with the oligo-
nucleotide synthesizer and HPLC instruments.
Supporting Information Available: Experimental pro-
cedures for compounds 3, 5, 6, 7, 8, and 9 and physical
measurements. This information is available free of charge
via the Internet at http://pubs.acs.org.
OL9910868
(
10) Rozners, E.; Str o¨ mberg, R.; Bizdena, E. Nucleosides Nucleotides
995, 14, 855. (b) Rozners, E.; Renhofa, R.; Petrova, M.; Popelis, J.;
Kumpins, V.; Bizdena, E. Nucleosides Nucleotides 1992, 11, 1579.
1
(
11) Rozners, E.; Str o¨ mberg, R. J. Org. Chem. 1997, 62, 1846.
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986, 26, 59. (b) Stawinski, J.; Str o¨ mberg, R.; Thelin, M.; Westman, E.
Nucleic Acids Res. 1988, 16, 9285.
13) (a) Garegg, P. J.; Lindh, I.; Regberg, T.; Stawinski, J.; Str o¨ mberg,
(
R.; Henrichson, C. Tetrahedron Lett. 1986, 27, 4055. (b) Westman, E.;
Sigurdson, S.; Stawinski, J.; Str o¨ mberg, R. Nucleic Acids Res. Symp. Ser.
No. 31 1994, 25.
(
14) Stec, W. J.; Zon, G.; Egan, W.; Stec, B. J. Am. Chem. Soc. 1984,
1
06, 6077.
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