Sulfonium derivatives of thioxanthenone, a new class of photodetritylating agents for microarray oligonucleotide synthesis

Article abstract of DOI:10.1134/S1068162010010152

The usability of a new class of photo acids, namely, sulfonium hexaphosphates based on thioxanthenone, for the removal of the dimethoxytrityl protective group in the process of oligonucleotide synthesis has been studied in order to search for new detritylating agents for microarray oligodeoxyribonucleotide synthesis. 2,4-Diethyl-9-oxo-10-(4-heptyloxyphenyl)-9H- thioxanthenium hexafluorophosphate has been successfully used for the solid-phase synthesis of (dT)₁₀.

Full text of DOI:10.1134/S1068162010010152

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2010, Vol. 36, No. 1, pp. 130–132. © Pleiades Publishing, Ltd., 2010.
Original Russian Text © A.N. Sinyakov, A.A. Ryabinin, G.A. Maksakova, V.V. Shelkovnikov, V.A. Loskutov, E.V. Vasil’ev, N.V. Shekleina, 2010, published in Bioorganicheskaya
Khimiya, 2010, Vol. 36, No. 1, pp. 139–141.
LETTERS TO THE EDITOR
Sulfonium Derivatives of Thioxanthenone, a New Class
of Photodetritylating Agents for Microarray
Oligonucleotide Synthesis
a,1
a
a
b
b
A. N. Sinyakov , A. A. Ryabinin , G. A. Maksakova , V. V. Shelkovnikov , V. A. Loskutov ,
b
b
E. V. Vasil’ev , and N. V. Shekleina
a
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences,
pr. Akademika Lavrent’eva 8, Novosibirsk, 630090 Russia
Institute of Organic Chemistry, Siberian Branch, Russian Academy of Sciences, Novosibirsk, 630090 Russia
b
Received July 15, 2009; in final form, July 17, 2009
Abstract—The usability of a new class of photo acids, namely, sulfonium hexaphosphates based on thioxanꢀ
thenone, for the removal of the dimethoxytrityl protective group in the process of oligonucleotide synthesis
has been studied in order to search for new detritylating agents for microarray oligodeoxyribonucleotide synꢀ
thesis. 2,4ꢀDiethylꢀ9ꢀoxoꢀ10ꢀ(4ꢀheptyloxyphenyl)ꢀ9Hꢀthioxanthenium hexafluorophosphate has been sucꢀ
cessfully used for the solidꢀphase synthesis of (dT) .
10
Key words: photogenerators of acids, oligodeoxyribonucleotides, thioxanthenone, trityl cation
DOI: 10.1134/S1068162010010152
1
The development of the method of DNA microarꢀ fonium hexafluorophosphates on the basis of thioxanꢀ
ray oligonucleotide synthesis has opened up vast thenone derivatives, for the removal of the
opportunities for the biochemical identification of trimethoxytrityl protective group during the process of
biological molecules and mass using synthetic oligoꢀ oligonucleotide synthesis.
nucleotides in gene engineering. Microarray oligonuꢀ
We have synthesized and studied eight representaꢀ
cleotide synthesis developed by Affimetrix has been
tives of this class of compounds in model experiments
known for a rather long time [1]; however, it was
on the deprotection of 5'ꢀ
midine. One of these derivatives, 2,4ꢀdiethylꢀ9ꢀoxoꢀ
0ꢀ(4ꢀheptyloxyphenyl)ꢀ9 ꢀthioxanthenium hexaꢀ
Oꢀ(4,4'ꢀdimethoxytrityl)thyꢀ
impossible to use these oligonucleotides for gene engiꢀ
neering purposes because of their heterogeneity and
low availability due to the insufficient yield at the stage
of synthesis.
1
Н
fluorophosphate (Fig. 1), was further used for the
model synthesis of decathymidylate (dT)10 in an autoꢀ
The developers of modern methods of microarray mated DNA synthesizer.
synthesis succeeded in the improvement of the quality
The synthesis of (dT)10 was carried out using a
of oligodeoxynucleotides synthesized [2] by the use of
the approaches earlier applied in photolithography
and new protective groups.
modified ASMꢀ800 (Biosett, Russia) synthesizer.
The synthesizer was modified for the use of a semiꢀ
automated protocol for the 0.5ꢀmmol scale photoꢀ
In our opinion, the approach using photoactivated synthesis of oligonucleotides on porous glass in the
detritylation [3] is the most promising scheme for oliꢀ flow of reagents. A solution of 2,4ꢀdiethylꢀ9ꢀoxoꢀ
gonucleotide microarray synthesis, as all other steps of 10ꢀ(4ꢀheptyloxyphenyl)ꢀ
9Нꢀthioxanthenium hexaꢀ
oligonucleotide chain elongation are traditional and fluorophosphate (1%) [5] was loaded into the synꢀ
have been sufficiently exhausted. At the same time, thesizer for the removal of the protection of the
using triarylsulfonium hexafluoroantimonates as acid 5'ꢀtrityl group instead of trifluoroacetic acid. The
photogenerators [4] cannot be considered as optimum oligonucleotide synthesis was performed in quartz
because of the potential possibility of the apurinizaꢀ columns containing porous glass covalently bound
tion of the oligonucleotide chain synthesized.
to the first nucleotide unit. The synthesizer was supꢀ
plied with a mercuryꢀvapor lamp (BKL 120) and an
optical block with a shutter, a holder for filters, and
lenses for separating and focusing the emission of
the mercuryꢀvapor lamp at 365 nm on the reaction
column. Detritylation was performed three times,
the exposure time for each photo acid portion being
In order to optimize the scheme for oligonucleꢀ
otide microarray synthesis, we have studied the possiꢀ
bility of using a new class of photo acids, namely, sulꢀ
1
Corresponding author; phone: +7 (383) 330ꢀ4653; fax: +7 (383)
333ꢀ3677; eꢀmail: sinyakov@niboch.nsc.ru.
130

SULFONIUM DERIVATIVES OF THIOXANTHENONE
O
131
+
S
PF6–
O
Fig. 1. .The structure of 2,4ꢀdiethylꢀ9ꢀoxoꢀ10ꢀ(4ꢀheptyloxyphenyl)ꢀ9
Hꢀthioxanthenium hexafluorophosphate used in oligonuꢀ
cleotide synthesis.
1
min. The remaining steps of the oligonucleotide invariable. After the synthesis was over, the carrier
chain elongation when using the phosphoramidite was treated with concentrated aqueous ammonia for
approach, including blocking the unreacted 5'ꢀ 2 h and the reaction mixture was analyzed with
hydroxyl groups with acetic anhydride, remained HPLC and PAGE (Fig. 2). The yields for the elonꢀ
(а)
(
b)
0.75 AU
2
60 nm
80 nm
2
1
2
3
4
5
6
7
8
9
min
1
2
Fig. 2. The control of (dT)10 synthesis. (a) Electrophoregram in 15% PAG (denaturing conditions) of (
upon synthesis, (
AQ column with the particle size of 5
for 10 min, flow rate is 150 l/min.
1
), the reaction mixture
2
) (dT)10 reference; (b) reverseꢀphase HPLC of the reaction mixture: Millikhrom Aꢀ02, ProntoSilꢀ120ꢀ5ꢀC18
µ
m, 2–20% linear gradient of acetonitrile in 0.05 M triethylammonium acetate (pH 7.5)
µ
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 36
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2010

132
SINYAKOV et al.
gation step by both methods using either photogeꢀ Basics of Fundamental Studies in Nanotechnologies
nerated or added acid were identical and came to and Nanomaterials.
9
8%.
Thus, 2,4ꢀdiethylꢀ9ꢀoxoꢀ10ꢀ(4ꢀheptyloxyphenyl)ꢀ
Нꢀthioxanthenium hexafluorophosphate can be sucꢀ
cessfully used for the generation of hexafluorophosꢀ
phoric acid during oligonucleotide synthesis, opening
up perspectives for its application in microarray nucleꢀ
otide synthesis.
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