Photogenerator of trichloroacetic acid as a promising detritylation agent for oligonucleotide microarray synthesis

Article abstract of DOI:10.1134/S1068162014050124

[[4-(4-Methoxyphenyl)-2,6-dinitro-phenyl](phenyl)methyl]-2,2,2-trichloroacetate, which generates trichloroacetic acid when irradiated with light of 405 nm wavelength, has been studied as a detritylation agent in the oligonucleotide microarray synthesis. This agent was shown to be suitable for the deprotection of the 4,4'-dimethoxytrityl group during the solid phase oligonucleotide synthesis.

Full text of DOI:10.1134/S1068162014050124

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2014, Vol. 40, No. 5, pp. 586–588. © Pleiades Publishing, Ltd., 2014.
Original Russian Text © A.N. Sinyakov, E.B. Nikolaenkova, I.A. Os’kina, V.A. Savel’ev, V.A. Samsonov, A.Y. Tikhonov, M.Yu. Palatkina, D.E. Zaytsev, 2014, published in
Bioorganicheskaya Khimiya, 2014, Vol. 40, No. 5, pp. 636–638.
Photogenerator of Trichloroacetic Acid as a Promising Detritylation
Agent for Oligonucleotide Microarray Synthesis
, 1
A. N. Sinyakova , E. B. Nikolaenkovab, I. A. Os’kinab, V. A. Savel’evb, V. A. Samsonovb,
A. Y. Tikhonovb, M. Yu. Palatkinac, and D. E. Zaytsevc
a Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy
of Sciences, pr. Lavrent’eva 8, Novosibirsk, 630090 Russia
b Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch
of Russian Academy of Sciences, Novosibirsk Russia
c Novosibirsk State University, Novosibirsk Russia
Received March 5, 2014; in final form, April 22, 2014
Abstract—[[4ꢀ(4ꢀMethoxyphenyl)ꢀ2,6ꢀdinitroꢀphenyl](phenyl)methyl]ꢀ2,2,2ꢀtrichloroacetate, which genꢀ
erates trichloroacetic acid when irradiated with light of 405 nm wavelength, has been studied as a detritylation
agent in the oligonucleotide microarray synthesis. This agent was shown to be suitable for the deprotection of
the 4,4'ꢀdimethoxytrityl group during the solid phase oligonucleotide synthesis.
Keywords: photogenerated acids, trityl cation, oligodeoxyribonucleotides
DOI: 10.1134/S1068162014050124
1
Development of the oligonucleotide synthesis on mediate of MDPM·TCA, i.e., 4ꢀ(4ꢀmethoxyphenyl)ꢀ
2,6ꢀdinitrobezaldehyde [6]. This allowed us to study
MDPM·TCA as the detritylation agent in the oligodeꢀ
oxyribonucleotides synthesis. This compound may be
a convenient detritylation agent in the oligonucleotide
microarray synthesis because trichloroacetic acid
formed during its photolysis leads to minimal depuriꢀ
nation. Moreover, the ability of MDPM·TCA to be
degraded during irradiation at 405 nm allows the use of
sufficiently powerful industrial laser diodes. In addiꢀ
tion, transition from the near ultraviolet (365 nm)
(usually used for photolysis [3]) to longer wavelengths
(405 nm) for the acid generation reduces the opportuꢀ
nity of the involvement of the growing oligonucleotide
chain in side photochemical reactions.
microarrays enabled us to obtain desired oligonucleꢀ
otides for the construction of genetic elements for varꢀ
ious tasks in synthetic biology and to create a broad
range of diagnostic tests based on the methods of
nucleic hybridization and minisequencing of nucleic
acids [1, 2]. Photoactivated detritylation is a promisꢀ
ing approach to oligonucleotide microarray synthesis
[3], with all other stages of the elongation of the oligoꢀ
nucleotide chain being standard and wellꢀelaborated.
Photosensitive compounds used, as a rule, for photoꢀ
activated detritylation generate strong mineral acids,
which can cause depurination of oligonucleotides [4].
At the same time, substituted 2ꢀnitrobenzyl trichloroꢀ
acetates have been recently proposed to remove the
4,4'ꢀdimethoxytrityl group during the oligonucleotide
synthesis on thin films [5]. Photolysis of these comꢀ
pounds leads to the formation of trichloroacetic acid,
which is used in the solid phase oligonucleotide synꢀ
thesis. It was found that 2ꢀnitrobenzyl trichloroaceꢀ
tates can be applicable for detritylation on thin polyꢀ
mer films when irradiating with UV light at 365 nm.
Only one of 2ꢀnitrobenzyl trichloroacetates, i.e., [[4ꢀ
(4ꢀmethoxyphenyl)ꢀ2,6ꢀdinitrophenyl](phenyl)methyl]ꢀ
2,2,2ꢀtrichloroacetate (MDPM·TCA) degraded durꢀ
ing the photolysis by visible light at 405 nm. The appliꢀ
cability of MDPM·TCA for the detritylation was not
examined presumably because of problems with its
synthesis. We improved the synthesis of the key interꢀ
NO2
O
O
O
NO2
Cl
Cl
Cl
Structure of [[4ꢀ(4ꢀMethoxyphenyl)ꢀ
2,6ꢀdinitroꢀphenyl](phenyl)methyl]ꢀ2,2,2ꢀtrichloroacetate
(MDPM·TCA) used in oligonucleotide
synthesis as a photoacid
.
It was found in the model experiments that detrityꢀ
lation of 5'ꢀ ꢀ(4,4'ꢀdimethoxytritylꢀ2'ꢀdeoxythymiꢀ
dine at a concentration of
10–4 M with 2 mM
O
2
×
1
MDPM TCA in methylene chloride occurs for less
than 20 s (figure) under LED irradiation with a power
Corresponding author: phone: +7(383)363ꢀ51ꢀ73, fax:
+7(383)363ꢀ51ꢀ53; eꢀmail: sinyakov@niboch.nsc.ru.
586

PHOTOGENERATOR OF TRICHLOROACETIC ACID
587
density of 40 mW/cm2 (Collimated LED Light
(a)
(b)
Source for Leica DMI Microscopes M405L2ꢀC2,
ThORLABS).
1
2
3
MDPM TCA was examined as the deprotection
agent in the column oligonucleotide synthesis on a
modified oligonucleotide synthesizer ASMꢀ800 (Bioꢀ
sset, Russia). The synthesizer was modified for the
semiautomatic photosynthesis of oligonucleotides on
the 0.5 millimolar scale. The 5'ꢀhydroxyl group was
deprotected with 3% MDPM TCA placed in the synꢀ
thesizer instead of trichloroacetic acid. The synthesis
was carried out in quartz columns containing the
porous CPG glass bearing the first nucleotide unit.
The synthesizer was fitted with LED and the optical
unit for focusing light on the reaction column. Detrityꢀ
lation was performed in triplicate, the exposure time for
each portion of the photoacid was 1 min. All other
stages of the elongation of the oligonucleotide chain
during the phosphoroamidite synthesis were unchanged
including the protection of the unreacted 5'ꢀhydroxyl
groups with acetic anhydride. After the completion of
the synthesis, the solid support was treated with conꢀ
centrated aqueous ammonia for 4 h at 60°C. The reacꢀ
tion mixtures were analyzed by HPLC and gel electroꢀ
phoresis in PAAG. The target oligonucleotides, i.e.
TGTCCTA,
TCTGTCCTA,
ATCCTTGGTC,
AAAAAAAA, TTTTTTTTTT (5'ꢀ3') were isolated by
RP HPLC.
The structures of the synthesized oligonucleotides
were confirmed by ESI mass spectrometry on an Agiꢀ
lent 6410 TrippleQuadLC/MS mass spectrometer
(Agilent Technologies, United States). MS (ESI) [
M –
H]: TGTCCTA: calc. 2069.374, found 2069.380;
TCTGTCCTA: calc. 2662.466, found 2662.986;
ATCCTTGGTC: calc. 2991.518, found 2991.966;
AAAAAAAA: calc. 2441.494, found 2441.374;
TTTTTTTTTT: calc. 2977.766, found 2977.493.
The yields for one elongation stage were 97%,
which is satisfactory for the solid phase synthesis.
It should be noted that the time of the deprotection
of the 5'ꢀhydroxyl group of the growing oligonucleꢀ
otide chain, which comprised the formation of
trichloroacetic acid from the photoacid precursor, its
diffusion into the CPG pores, and the detritylation
itself, was 3 min. One can assume that this time will be
much less in the case of the microarray synthesis due
to the absence of diffusion processes.
Analysis of the reaction mixture after the synthesis of
AAAAAAAA. (a) Electrophoregram in 20% PAAG (denaꢀ
turing conditions):
tion mixture after the synthesis of AAAAAAAA,
1
, TTTTTTTTTT (standard),
2
3
, reacꢀ
, broꢀ
mophenol blue; (b) RP HPLC of the reaction mixture in a
linear gradient of acetonitrile (2–20%) in 0.05 M triethyꢀ
lammonium acetate (pH 7.5) for 10 min at a flow rate of
150
µ
L/min. Chromatograph Millichrom Aꢀ02, column
m).
ProntoSilꢀ120ꢀ5ꢀC18 AQ (5
µ
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 40
No. 5
2014

588
SINYAKOV et al.
Kraft, A.J., Weisburg, W.G., and Mather, E.L., J. Clin.
Thus, it was found that [[4ꢀ(4ꢀmethoxyphenyl)ꢀ
Microbiol., 2009, pp. 390–396.
2,6ꢀdinitroꢀphenyl](phenyl)methyl]ꢀ2,2,2ꢀtrichloroꢀ
acetate can be successfully used as the detritylation
agent in the solid phase oligonucleotide synthesis and
be promising for the oligonucleotide microarray synꢀ
thesis.
2. Borovkov, A.Y., Loskutov, A.V., Robida, M.D., Day, K.M.,
Cano, J.A., Le Olson, T., Patel, H., Brown, K.,
Hunter, P.D., and Sykes, K.F., Nucleic Acids Res.
2010, vol. 38, p. e180.
,
3. Gao, X., Yu, P., LeProust, E., Sonigo, L., Pellois, J.P.,
and Zhang, H., J. Am. Chem. Soc., 1998, vol. 120,
pp. 12698–12699.
This work was supported by the Integration Project
of the Siberian Branch of the Russian Academy of Sciꢀ
ences No. 138 “Technological Platform for Synthetic
Biology” and Project No. 24.60 “Development of the
Method of Nonfluorescent Microarray Diagnostics”
of the Program of RAS No 24 “Fundamentals of
Technology of Nanostructures and Nanomaterials”
4. LeProust, E., Pellois, J.P., Yu, P., Zhang, H., and Gao, X.,
J. Comb. Chem., 2000, vol. 2, pp. 349–354.
5. Serafinowski, P.J. and Garland, P.B., Org. Biomol.
Chem., 2008, vol. 6, pp. 3284–3291.
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Translated by A. Levina
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 40
No. 5
2014