Angewandte
Chemie
DOI: 10.1002/anie.201305555
Base Pairing
A Nucleobase Analogue that Pairs Strongly with Adenine**
Marco Minuth and Clemens Richert*
Base pairing is a pivotal process in nature. Genetic informa-
tion is passed on by the formation of pairs between
complementary nucleobases.[1,2] The detection of DNA or
RNA sequences through hybridization also relies on base
pairing, with multiple base pairs being formed simultane-
ously.[3–5] The two canonical base pairs (A:T and G:C) are of
different strength, though, so that the affinity of strands for
their target sequence depends strongly on the G/C content.
Differences in duplex stability between one sequence and
another are desirable for genetic material, for example to
facilitate strand separation at a TATA box, but are undesir-
able for detecting nucleic acid sequences in parallel or in
standardized fashion.[6] For example, the failure of A/T-rich
sequences to hybridize under conditions appropriate for
mixed sequences causes false negative signals in diagnostic
tests, and the ability of G/C-rich sequences to form duplexes
with mismatches can lead to false positive calls.[7]
Figure 1. Structure of thymidine and thymidine analogues.
leads to a pyrimidinium species when maintaining the
aromatic character of the nucleobase (1, Figure 1). The
instability of such species can be avoided when replacing
a natural deoxynucleoside with a C-nucleoside, so that
a neutral and more stable glycosidic bond arrangement
results. We have previously reported a 2-ethynyl derivative
of difluorotoluene (2), an isostere of thymidine,[14] in the form
of ethynylfluorobenzene nucleoside 3, and we were able to
show that it pairs more strongly with adenine when incorpo-
rated in an oligonucleotide than its fluoro counterpart 2.[15]
Still, the overall base pairing strength of 3 was considerably
weaker than that of natural thymidine, as evidenced by
a depression in the UV-melting point of a dodecamer duplex
by 12.78C, compared with the natural T-containing duplex.
Herein, we present 6-ethynylpyridone C-nucleoside 4, which
combines the hydrogen bonding capabilities of Tat the 3- and
4-positions with the ability of an ethynyl substituent to engage
in stacking and van der Waals interactions. In duplexes, E
pairs more strongly and more selectively with adenine than
thymidine. In fact, 4 pairs with almost equal strength as a C
does when it binds to G, as detected in UV-melting experi-
ments.
Several routes to a building block containing 4 were
tested. Key intermediates of the preferred, but not yet
optimized route are shown in Scheme 1. Details of the
synthesis and spectroscopic data are given in the Supporting
Information. Glycal 5[16] was prepared from deoxyribose in
four steps and 50% overall yield, by a route that is shorter and
higher yielding than earlier routes.[17,18] A Heck coupling was
used to couple 5 to diazonium salt 6 in diastereoselective
fashion, in an extension of elegant work by Hocek et al. on
pyridyl C-nucleosides that employs less reactive aglycons,[19,20]
producing enol ether 7. Compound 6 was accessible from 2-
chloro-4-nitropyridine 8 in four steps and 59% overall yield.
Desilylation of 7 to ketone 9 was followed by reduction with
NaBH(OAc)3 to give C-nucleoside 10, whose stereochemical
Nature tunes base pairing strength, where needed, either
through varying G/C content or through chemical modifica-
tion, as best exemplified by modifications in tRNAs.[8]
Synthetic chemists have also developed nucleobase analogues
whose affinity for complementary bases exceeds that of their
natural counterparts,[9–11] including bases with alkynyl sub-
stituents.[12] Among the two natural bases that pair weakly (A
and T/U), adenine is the one more readily converted into
a high affinity analogue. A well-established high affinity
derivative of adenine is 2,6-diaminopurine, which forms three
hydrogen bonds with thymine, resulting in more stable
duplexes.[13] Binding to A with a high-affinity analogue of T
is more difficult, though, as adenine does not offer a third
functional group for hydrogen bonding on its Watson–Crick
face, making it necessary to reach into the major or minor
groove to engage more distant hydrogen bond acceptors or to
provide other interactions. To the best of our knowledge, no
nucleobase analogue that pairs with A with greater affinity
and greater selectivity than thymine, when incorporated into
DNA strands, is known.
Starting from thymidine as the lead, one possible way to
engage in additional molecular interactions with adenine is to
place an alkynyl group at the 2-position. Unfortunately, this
[*] Dipl.-Chem. M. Minuth, Prof. C. Richert
Institut fꢀr Organische Chemie, Universitꢁt Stuttgart
70569 Stuttgart (Germany)
E-mail: lehrstuhl-2@oc.uni-stuttgart.de
[**] This work was supported by the DFG (RI 1063/9-1 and RI 1063/13-
1 to C.R.) The authors thank Dr. N. Griesang and Dr. K. Mꢀller for
sharing results, D. Gçhringer for skilled technical assistance, H.
Griesser for a review of the manuscript, Dr. W. Frey for X-ray
crystallography, and C. Krçner, S. Vollmer, and A. Gçckel for help
with acquiring spectra.
Supporting information for this article is available on the WWW
Angew. Chem. Int. Ed. 2013, 52, 1 – 5
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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