Angewandte
Communications
Chemie
DNA Modification
2-Substituted dATP Derivatives as Building Blocks for Polymerase-
Catalyzed Synthesis of DNA Modified in the Minor Groove
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Jꢀn Matyasovsky, Pavla Perlꢁkovꢀ, Vincent Malnuit, Radek Pohl, and Michal Hocek*
Abstract: 2’-Deoxyadenosine triphosphate (dATP) derivatives
bearing diverse substituents (Cl, NH2, CH3, vinyl, ethynyl, and
phenyl) at position 2 were prepared and tested as substrates for
DNA polymerases. The 2-phenyl-dATP was not a substrate for
DNA polymerases, but the dATPs bearing smaller substituents
were good substrates in primer-extension experiments, produc-
ing DNA substituted in the minor groove. The vinyl-modified
DNA was applied in thiol–ene addition and the ethynyl-
modified DNA was applied in a CuAAC click reaction to form
DNA labelled with fluorescent dyes in the minor groove
polymerases, whereas 2-arylamino-dATP derivatives were
found to act as polymerase inhibitors.[16] The minor groove
sites of the nucleobases are difficult to modify since they are
crucial both for Watson–Crick base pairing and for key minor-
groove interactions with DNA polymerase that are important
for extension of the chain.[17] On the other hand, 2-ethynyl-
pyridone-C-nucleotide incorporated into DNA[18] formed
a stable base pair with adenine, and 2-(imidazolylalkylami-
no)purines in ONs also stabilized duplexes.[19] Because the
possibility of minor-groove base labelling would be attractive
for many prospective applications, for example, the mapping
of DNA–protein interactions, we envisaged that a small
substituent at position 2 of a purine may not fully disturb the
key H-bonding interactions with the opposite base and the
polymerase, and we report herein the first enzymatic syn-
thesis of minor-groove base-modified DNA.
A series of six 2-substituted dATP derivatives bearing Cl,
NH2, CH3, vinyl, ethynyl and phenyl substituents (dRATPs)
was designed to study the effect of substituents of different
bulkiness at position 2 of adenine on polymerase-mediated
incorporation. While dClATP[14,20] and dNH2ATP[15] were
known, the others were prepared through triphosphoryla-
tion[21] of the corresponding 2’-deoxy-ribonucleosides (dRAs,
Scheme 1), which were synthesized through cross-coupling
reactions of the 2-iodo-2’-deoxyadenosine (for details of the
synthesis, see the Supporting Information).
B
ase-modified oligonucleotides (ONs) or DNA are widely
used as tools in chemical biology, diagnostics, or materials
science.[1] The modification is mostly attached to position 5 of
pyrimidines or position 7 of 7-deazapurines, not only because
it then points out into the major groove of DNA and thus does
not destabilize the duplex, but because in most cases, the
corresponding substituted 2’-deoxyribonucleoside triphos-
phates (dNTPs) are good substrates for DNA polymerases
and can be used in the polymerase-catalyzed synthesis of
modified DNA.[2,3] Diverse modifications, including fluoro-
phores,[4] redox[5] or spin labels,[6] reactive groups for con-
jugations,[7] and biomolecules (e.g., oligonucleotides[8] or
proteins[9]), have been introduced into the major groove
through the enzymatic incorporation of modified nucleotides
and applied in different fields. Modification or labelling of the
minor groove has mostly been reported with 2’- and 4’-sugar-
modified derivatives.[10–13] 2-Chloroadenine[14] and 2,6-diami-
nopurine[15] dNTPs are the only minor-groove base-modified
nucleotides that have been reported as substrates for DNA
The dRATPs were then tested as substrates for DNA
polymerases in primer extension (PEX) experiments. First,
we performed PEX in presence of KOD XL, Vent(exo-), or
Bst DNA polymerase, using a 15-nt primer (prim248short) and
19-nt template (tempoligo1A) designed for the incorporation of
one modified nucleotide, and the outcome was analyzed by
denaturing polyacrylamide gel electrophoresis (PAGE). All
three DNA polymerases (Figure 1a and Figure S1 in the
Supporting Information) incorporated the 2-substituted deox-
yadenosine nucleotides, giving clean full-length DNA prod-
ucts (DNA1RA). The only exception was the 2-phenyl
derivative dPhATP, which apparently was not a substrate for
DNA polymerases since almost no extension was observed.
Then PEX was conducted using a longer 31-nt template
(tempPrb4basII, which is modified with TINA at 3’-end to
prevent non-templated incorporation,[22] Figure 1b and Fig-
ure S2) designed for the incorporation of 4 RA modifications.
Most of the modified dRATPs were good substrates, giving
full-length products (DNA4RA). Only the PEX product from
ethynylated dEATP and KOD XL DNA polymerase was
partially halted at the nÀ1 position (but Vent(exo-) and Bst
DNA polymerases gave clean full-length products; see Fig-
ure S2), while dPhATP did not give PEX with any of the tested
DNA polymerases. All of the PEX experiments (with all of
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[*] J. Matyasovsky, Dr. P. Perlꢀkovꢁ, Dr. V. Malnuit, Dr. R. Pohl,
Prof. Dr. M. Hocek
Institute of Organic Chemistry and Biochemistry
Czech Academy of Sciences
Flemingovo nam. 2, 16610 Prague 6 (Czech Republic)
E-mail: hocek@uochb.cas.cz
Prof. Dr. M. Hocek
Department of Organic Chemistry, Faculty of Science
Charles University in Prague
Hlavova 8, 12843 Prague 2 (Czech Republic)
Supporting information and the ORCID identification number(s) for
ꢂ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co.
KGaA. This is an open access article under the terms of the Creative
Commons Attribution Non-Commercial NoDerivs License, which
permits use and distribution in any medium, provided the original
work is properly cited, the use is non-commercial, and no
modifications or adaptations are made.
Angew. Chem. Int. Ed. 2016, 55, 1 – 5
ꢀ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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