Bioorganic & Medicinal Chemistry Letters
Bioreductive deprotection of 4-nitrobenzyl group on thymine base
in oligonucleotides for the activation of duplex formation
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Hisao Saneyoshi , Yuki Hiyoshi, Koichi Iketani, Kazuhiko Kondo, Akira Ono
Department of Material and Life Chemistry, Faculty of Engineering, Kanagawa University, 3-27-1 Rokkakubashi, Kanagawa-ku, Yokohama 221-8686, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Oligonucleotides containing 4-O-(4-NO2-benzyl)thymine residues were synthesized to assess potential
prodrug-type action against hypoxic cells. These modified oligonucleotides were incapable of stable
duplex formation under non-hypoxic conditions. However, following deprotection of the thymine resi-
dues under bioreductive conditions, the deprotected oligonucleotides were able to form stable duplexes
with target oligonucleotides.
Received 29 August 2015
Revised 7 October 2015
Accepted 10 October 2015
Available online 22 October 2015
Ó 2015 Elsevier Ltd. All rights reserved.
Keywords:
Nucleic acid-based therapeutics
Prodrug
Protecting group
Hypoxia
Hybridization
Various nucleobase-protecting groups have been used in the
chemical synthesis of oligonucleotides.1 In recent years, the appli-
cation of protecting groups has expanded to include the control of
nucleic acid functions.2 For example, in the pioneering work by
Kröck and Heckel, 2-nitrobenzyl and its congeners were installed
onto thymine residues to generate light-activatable oligonu-
cleotides.3 Their study inspired our use of protected nucleobases
to produce hypoxia-activatable oligonucleotides. Hypoxia is a
characteristic property of locally advanced solid tumors resulting
from the insufficient supply of oxygen from the poorly developed
vasculature. Hypoxia induces increased resistance to both
chemotherapy and radiation therapy.4 Approaches to treating
hypoxic tumors with drugs activated under such conditions
have been reported.5 These prodrugs are typically protected
with hypoxia-labile protecting groups that mask binding sites to
the target molecules. Under hypoxic conditions, these protecting
groups are removed and the drugs are converted into their active
forms.
nucleobases. Specifically, oligonucleotides containing 4-O-nitrobenzy-
lated thymine residues as the prototype nucleobase were
synthesized. The 4-NO2-benzyl group is frequently used for
hypoxia-activated prodrugs or to switch fluorescent probes on
and off.6–20 In normal cells (non-hypoxic), oligonucleotides con-
taining 4-O-nitrobenzylated thymine residues (pro-oligos) do not
hybridize to target RNA sequences (Fig. 1a). In hypoxic tumors,
the 4-nitrobenzyl groups are deprotected via reduction of the nitro
group to amino (or hydroxyamino) followed by a 1,6-elimination
process to expel the active oligos (Fig. 1a and b). Thus, in the
hypoxic tumor cell, a pro-oligo will be converted to an active
oligonucleotide which hybridizes to the target sequence, thereby
inhibiting mRNA translation.
For this study, we synthesized oligonucleotides containing 4-O-
(4-NO2-Bn)thymine residues, which were treated with nitroreduc-
tase for conversion into free oligonucleotides. The hybridization
properties of the protected and the free oligonucleotides were
investigated by thermal denaturation experiments.
In this report, we describe the synthesis of an oligonucleotide
Briefly, a protected thymidine 1 was reacted with triisopropyl-
benzenesulfonyl chloride to produce a 4-O-sulfonylated intermedi-
ate, which was subsequently substituted with 4-NO2 benzyl
alcohol to yield 4-benzylated derivative 2. Acetyl groups in 2 were
deprotected with NH4OH to produce 3 which was treated with
4,40-dimethoxytrityl chloride to give 4; then, phosphitylation of
the free 30-hydroxyl group with 2-(cyanoethoxy)-N,N-diisopropy-
laminochlorophosphine yielded the desired phosphoramidite
derivative 5 (Scheme 1). Using the monomer unit 5, oligonucleotides
with
a hypoxia-labile protecting group on one of the four
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0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.