Bioorganic & Medicinal Chemistry Letters
Thiol-responsive pro-fluorophore labeling: Synthesis of a pro-fluorescent
labeled oligonucleotide for monitoring cellular uptake
a,b,⁎
a
a
c
a,⁎
Hisao Saneyoshi
b
c
, Yuta Yamamoto , Takayuki Ohta , Shoji Akai , Akira Ono
a
Department of Material and Life Chemistry, Faculty of Engineering, Kanagawa University, 3-27-1 Rokkakubashi, Kanagawa-ku, Yokohama 221-8686, Japan
Department of Chemistry, Shiga University of Medical Science, Seta Tsukinowa-cho, Otsu, Shiga 520-2192, Japan
Laboratory of Synthetic Organic Chemistry, Kagawa Nutrition University, 3-9-21 Chiyoda, Sakado, Saitama 350-0288, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
Pro-fluorescent labeled oligonucleotides are potential alternative tools to classical fluorescently labeled oligo-
nucleotides for monitoring cellular uptake. Here, we report the design and synthesis of a thiol-responsive pro-
fluorophore labeled oligonucleotide, and its fluorescence responsivity to glutathione in the test tube and live
cells.
Oligonucleotides
Fluorogenic reaction
Thiol
Cellular uptake
Conjugation
Intracellular delivery of oligonucleotides is very important for the
tag that are activated by an esterase have been reported for visualizing
1
–4
8
development of oligonucleotide therapeutics and diagnostics.
Vi-
Fluorescent-labeled oligonucleotides have
intracellular esterase activity. We consider glutathione (GSH) as an
sualization and evaluation of cellular uptake of desired oligonucleotides
ideal internal trigger for fluorescence activation. GSH exists universally
in high concentrations in the cytosol, which is the site of action of
5
–7
are also critical steps.
9
,10
been used widely for fluorescence imaging of cellular uptake. However,
fluorescent-labeled oligonucleotides bearing a standard fluorophore
such as fluorescein are constantly fluorescing. To visualize cellular
uptake of oligonucleotides, excess fluorescent labeled oligonucleotides
in the medium must be removed by a washing step prior to fluorescence
imaging. For this reason, this method is not applicable to real-time
monitoring of cellular uptake in live cells. Based on this issue, we have
focused on the use of pro-fluorescent labeled oligonucleotides bearing a
pro-fluorophore for simple evaluation of cellular uptake (Fig. 1). This
kind of materials does not fluoresce in the medium and after cellular
uptake the pro-fluorescent moiety switches to the fluorescent form by
internal triggers. These advantages enable the use of a non-washing
protocol for live cell imaging. Oligonucleotides bearing a fluorogenic
oligonucleotide drugs.
After pro-fluorescent labeled oligonucleo-
tides enter the cytosol, the thiol-labile protecting group on the pro-
fluorescent moiety is deprotected by intracellular GSH to convert to the
fluorescent form (Fig. 1B). This strategy enables real-time monitoring of
cellular uptake of oligonucleotide drug candidates in a variety of cell
lines.
In this paper, synthesis of a GSH-activated pro-fluorescent rhoda-
mine derivative as a pro-fluorophore and its conjugation to an oligo-
nucleotide to generate a pro-fluorescent labeled oligonucleotide are
reported. The fluorescence properties of the synthesized oligonucleo-
tide in the test tube and living cells are also reported.
The use of rhodamine-type fluorophores to design pro-fluorescent
labeled oligonucleotides is promising because these fluorophores have
⁎
Corresponding authors at: Department of Chemistry, Shiga University of Medical Science, Seta Tsukinowa-cho, Otsu, Shiga 520-2192, Japan (H. Saneyoshi).
Department of Material and Life Chemistry, Faculty of Engineering, Kanagawa University, 3-27-1 Rokkakubashi, Kanagawa-ku, Yokohama 221-8686, Japan (A.
Ono).
Received 23 March 2020; Received in revised form 21 April 2020; Accepted 25 April 2020
Available online 27 April 2020
0960-894X/ © 2020 Elsevier Ltd. All rights reserved.