Development of Photoactivated Fluorescent N-Hydroxyoxindoles and Their Application for Cell-Selective Imaging

Abstract

Photoactivatable fluorophores are essential tools for studying the dynamic molecular interactions within important biological systems with high spatiotemporal resolution. However, currently developed photoactivatable fluorophores based on conventional dyes have several limitations including reduced photoactivation efficiency, cytotoxicity, large molecular size, and complicated organic synthesis. To overcome these challenges, we herein report a class of photoactivatable fluorescent N-hydroxyoxindoles formed through the intramolecular photocyclization of substituted o-nitrophenyl ethanol (ONPE). These oxindole fluorophores afford excellent photoactivation efficiency with ultra-high fluorescence enhancement (up to 800-fold) and are small in size. Furthermore, the oxindole derivatives show exceptional biocompatibility by generating water as the only photolytic side product. Moreover, structure-activity relationship analysis clearly revealed the strong correlation between the fluorescent properties and the substituent groups, which can serve as a guideline for the further development of ONPE-based fluorescent probes with desired photophysical and biological properties. As a proof-of-concept, we demonstrated the capability of a new substituted ONPE that has an uncaging wavelength of 365-405 nm and an excitation/emission at 515 and 620 nm, for the selective imaging of a cancer cell line (Hela cells) and a human neural stem cell line (hNSCs).

Keywords: cage compounds; dyes/pigments; fluorescence imaging; fluorescent probes; oxindole.

Figure 1

A) Photocyclization of ONPE to N-hydroxyoxindole. Inset shows the photographs of a solution of 1 under UV lamp before and after UV irradiation; B) Absorption and fluorescence spectra depicting the photolysis of 1 in MeOH/PBS (3:1, v/v) under UV light irradiation (254 nm); C) HPLC analysis of the photolytic reaction reveals progressive photolysis of 1 upon irradiation and formation of a major photolytic product (1 a). The signal was recorded with a UV-absorption detector at the wavelength of 299 nm, which is the isosbestic point of the absorption spectrum shown in (B); D) Content analysis of the 1 and 1 a during the photolysis shows 50 % symmetry indicating a clean photolysis reaction without any byproducts. Analysis was done using integrated area of 1 and 1 a on the HPLC chromatogram.

Figure 2

A) Absorption and fluorescence emission spectra of 1 a in MeOH/PBS (3:1, v/v, pH7.4, 10 mM PBS) solution; B) pH-dependent UV/Vis absorption spectrum of 1 a; C) pH-dependent fluorescence emission intensity of 1 a with different excitation wavelengths (detailed emission spectra were shown in Figure S15, the Supporting Information); D) Schematic illustration of the photo-induced deprotonation of the N-hydroxyl group of 1 a at the excited state; E) Electron density of HOMO and LUMO for 1 a computed by density functional theory (DFT).

Figure 3

Schematic illustration of the proposed excited-state keto–enol tautomerization of 1 a, whereas only the enol form is the fluorescent moiety. HOMO and LUMO calculations of the keto and enol forms computed by DFT calculations show that enol form of 1 a is more stable at the excited state than the keto form.

Figure 4

A) Molecular structures of the photoactivatable dye 11 and its corresponding photolysis product 11a; B) UV/Vis absorption and fluorescence emission spectra analysis of the photolysis of 11 in MeOH/PBS (3:1, v/v) under UV light irradiation (photolysis, 365 nm, 3 mW cm⁻²; excitation, 480 nm); C) HPLC monitoring revealed that the photolysis is clean and produces 11a as the major product; D) Analysis of viability and proliferation of HeLa cells and hNSCs in the presence of increasing concentrations of 11 and 11a.

Figure 5

A–H) Time-dependent photoactivation of 11 in HeLa cells. The photoactivation was performed on a Nikon Ti microscope using the DAPI excitation (Nikonen slight C-HGFI lamp, 130 W) with a 20 × objective lens. The fluorescent images were collected using Texas Red channel (excitation 550–580 nm, emission 600–650 nm). Dye concentration is 5 μM, scale bar is 100 μm; I–K) Spatially-controlled photoactivation of 11 in hNSCs, I) DIC, J) fluorescence, and K) merged images of a single hNSC with photoactivated fluorescence. The selected cell was first irradiated by a 405 nm excitation from the confocal microscope (5.95 mW, 15 s) and the fluorescence imaging was recorded with an excitation at 515 nm. Scale bar is 20 μm.