Two-photon ESIPT-based fluorescent probe using 4-hydroxyisoindoline-1,3-dione for the detection of peroxynitrite

Abstract

Excited-state intramolecular proton transfer (ESIPT)-based fluorophores with two-photon excitation fluorescence (TPEF) are rare. Our aim with this research was to develop ESIPT-based fluorophores exhibiting TPEF. Herein, we used 4-hydroxyisoindoline-1,3-dione as a scaffold to develop a two-photon fluorescent probe BHID-Bpin, for the detection of peroxynitrite (ONOO⁻). BHID-Bpin exhibits excellent selectivity, sensitivity, and fast response towards ONOO⁻ in PBS buffer solution (10 mM, pH = 7.40). Additionally, BHID-Bpin displays high photo-stability under two-photon irradiation at 750 nm. Furthermore, the probe can image endogenous ONOO⁻ in HeLa cells and exogenous ONOO⁻ in rat hippocampal slices at a depth of 110 μm.

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Peroxynitrite (ONOO⁻) is formed by the combination of nitric oxide (NO) and superoxide anion radical (O₂˙⁻) under diffusion control without enzymatic catalysis.^1–3 It acts as a strong reactive nitrogen species (RNS) which can react with wide array of critical bioactive species, such as proteins, nucleic acids, lipids, and transition-metal enzyme centers.⁴ The concentrations of ONOO⁻in vivo vary and have been estimated to be around 50–100 μM,⁵ with concentrations up to 500 μM having been reported in macrophages.⁶ In addition, hydrogen peroxide (H₂O₂) is an important reactive oxygen species (ROS), which has a key role in physiological processes, including host defense, cell proliferation, cell growth, and signaling pathways.^7–9 Previous studies have revealed that H₂O₂ can be endogenously produced in various cells and tissues via the activation of NADPH oxidase.^10,11 The exact range of H₂O₂ varies depending on the cell line, and the intracellular homeostatic concentration ranges from 1 to approximately 100 nM.¹² Excessive production of ONOO⁻ and H₂O₂ are implicated in many pathological and physiological process associated with multiple oxidative stress-related diseases, including neurodegenerative, inflammatory, and cardiovascular diseases as well as cancer.^13–16 Therefore, methods for the detection of ONOO⁻/H₂O₂ in biological systems are of considerable importance for both early diagnosis of human disease and exploration of various pathophysiologies.

Fluorescence-based probes have been developed as powerful tools for the detection of ONOO⁻/H₂O₂, due to their good selectivity, high sensitivity, minimal invasiveness and convenience^17–24 However, traditional one-photon probes require short excitation wavelengths ranging from ultraviolet to the visible range, limiting their use in tissues or animals owing to cellular autofluorescence, and shallow tissue penetration depth.^19,25 Over the last three decades, two-photon microscopy (TPM) has attracted particular interest since it employs two photons of low energy, near-infrared light to excite a fluorescent dye. As such TPM can overcome the above-mentioned disadvantages to a certain extent.^26–29 Excited-state intramolecular proton transfer (ESIPT) fluorophores are attractive for developing reaction-based fluorescent probes. Since ESIPT fluorophores usually have large Stokes shift, high quantum yield, good photostability, and are biocompatible^30–32 Two-photon ESIPT-based probes often incorporate: 2-(2′-hydroxyphenyl)benzimidazole (HBI) or 2-(2-hydroxyphenyl)-4(3H)-quinazolinone (HPQ) as a framework. For example, two ESIPT-based fluorescent probes (1 and 2) based on HBI and HPQ respectively as the core fluorophore are illustrated in Fig. 1. James et al. designed probe 1 by incorporating HBI and boronate ester. The probe enabled the imaging of endogenous ONOO⁻ in RAW 264.7 macrophages and rat hippocampus tissue under two-photon excitation.³³ Zhou and colleagues developed probe 2 which used HPQ as a scaffold for the detection of H₂O₂ in HeLa cells and muscle tissues under two-photon excitation at 720 nm.³⁴ In order to extend the range of suitable ESIPT fluorophores for the construction of two-photon probes. Herein, the two-photon fluorescence properties of 2-butyl-4-hydroxyisoindoline-1,3-dione (BHID, a 4-hydroxyisoindoline-1,3-dione (HID) derivative) are explored in the development of probe BHID-Bpin.

Fig. 1 Structures of previously reported two-photon ESIPT-based fluorescent probes. Where HBI and HPQ are used as the fluorophores.

BHID-Bpin can be readily prepared and is non-fluorescent. However, upon addition of ONOO⁻/H₂O₂, the phenylboronic acid pinacol ester is oxidized by ONOO⁻/H₂O₂ which leads to the release of phenol moiety (Scheme 1). The BHID unit is fluorescent, since the ESIPT process is restored. The boronate pinacol ester can respond to both ONOO⁻ and H₂O₂. However, it is much more sensitive to reaction with ONOO⁻ than H₂O₂, because the enhanced nucleophilicity of ONOO⁻ leads to a reaction rate ∼10⁶ times faster than that for H₂O₂.³⁵ Furthermore, different concentrations of ONOO⁻/H₂O₂ also affects the selectivity.²² Given the possible dual reactivity, we evaluated the concentration-dependent and time-dependent reactivity of BHID-Bpin towards both H₂O₂ and ONOO⁻ in aqueous solutions. In addition, the probe was used to image ONOO⁻ in cells and tissues with satisfactory results, demonstrating its practical applicability.

Scheme 1 (a) Reaction between probe BHID-Bpin and ONOO⁻/H₂O₂. (b) Diagram of ESIPT process of BHID.

BHID-Bpin was synthesized using a simple two-step sequence (Scheme S1, ESI†). Initially, BHID was synthesized utilizing commercially available 3-hydroxyphthalic anhydride and butylamine. Then a mixture of BHID, 4-bromomethylphenylboronic acid pinacol ester, and K₂CO₃ in dry DMF afforded BHID-Bpin in 61% yield. It is worth noting that 3-hydroxyphthalic anhydride has two sites that can be functionalized. The phenol which serves as a unit that can be used to block the ESIPT process upon attachment of a receptor. Additionally its anhydride unit can be used to construct dual-responsive probes or organelle-targeting probes by varying the amine used. With BHID-Bpin in hand we then evaluated the optical properties of the probe in PBS buffer solution (10 mM, pH = 7.40). The probe BHID-Bpin (10 μM) exhibits no absorption peak, however, upon addition of ONOO⁻ (20 μM)/H₂O₂ (95 μM), the probe displays a weak absorption at about 400 nm (Fig. S1 and S6, ESI†). Therefore, excitation at 400 nm was used to investigate the fluorescence response of the probe towards ONOO⁻/H₂O₂ (Fig. 2 and 3).

Fig. 2 (a) One-photon fluorescence spectra of BHID-Bpin (10 μM) after the addition of various concentrations of ONOO⁻ (0–20 μM) in PBS buffer solution (10 mM, pH = 7.40) after 1 min. λ_ex = 400 nm (b) Linear relationship between fluorescence intensity of BHID-Bpin (10 μM) and concentration of ONOO⁻ (0–6 μM) after 1 min. λ_ex/em = 400/515 nm.

Fig. 3 (a) One-photon fluorescence spectra of BHID-Bpin (10 μM) with addition of various concentrations of H₂O₂ (0–95 μM) in PBS buffer solution (10 mM, pH = 7.40) after 80 min. λ_ex = 400 nm. (b) Linear relationship between fluorescence intensity of BHID-Bpin (10 μM) and concentration of H₂O₂ (0–15 μM) after 80 min. λ_ex/em = 400/515 nm.

As expected, BHID-Bpin displayed weak fluorescence due to the ESIPT process being blocked by the boronate ester moiety. However, with incremental addition of ONOO⁻ (0–20 μM)/H₂O₂ (0–95 μM), the fluorescence spectra of BHID-Bpin displayed a continuous increase in emission intensity at 515 nm (Fig. 2a and 3a), that reached a plateaue when 2 equiv. of ONOO⁻/9.5 equiv. of H₂O₂ were added, causing a 31/29-fold enhancement in the fluorescence emission intensity (Fig. S2 and S7, ESI†). Plots of the emission intensity at 515 nm versus concentration of ONOO⁻/H₂O₂ were used to determine the limit of detection to be 73 nM for ONOO⁻ and 221 nM for H₂O₂ (linear equation: y₁ = 448.67 + 1000.74 × [ONOO⁻] (μM), R² = 0.999, y₁ is the intensity at 515 nm, Fig. 2b; y₂ = 632.99 + 331.02 × [H₂O₂] (μM), R² = 0.986, y₂ is the intensity at 515 nm, Fig. 3b). To determine the selectivity towards ONOO⁻ and H₂O₂, various interfering species were evaluated. As shown in Fig. S3 (ESI†), BHID-Bpin was particularly responsive towards ONOO⁻ over H₂O₂, ROO˙, ˙OH, ¹O₂, and ClO⁻ when the solutions were incubated for 1 min. However, the existence of ONOO⁻ or H₂O₂ could induce significant fluorescence enhancements of the probe BHID-Bpin when the solutions were incubated for 80 min and compared with other interfering species. The dynamic behaviour of probe BHID-Bpin was recorded in the presence of ONOO⁻/H₂O₂. As depicted in Fig. S5 and S9 (ESI†). Upon addition of 2 equiv. of ONOO⁻/9.5 equiv. of H₂O₂, a fluorescence enhancement was observed with maximum fluorescence observed at 1/80 min. These results indicated that probe BHID-Bpin has a much faster response and was more sensitive for ONOO⁻ than H₂O₂.

To evaluate the practicability of probe BHID-Bpin under physiological conditions, the effect of pH on the fluorescence response of the probe in the absence and presence of ONOO⁻/H₂O₂ at various pH values was determined. The fluorescence intensities of probe BHID-Bpin changed over a pH range from 3.00 to 12.00. In the presence of ONOO⁻/H₂O₂, there were notable changes in fluorescence of probe BHID-Bpin over a pH range from 7.40 to 9.00, (Fig. S4 and S8, ESI†) which indicated that the probe could be used to monitor ONOO⁻/H₂O₂ under physiological conditions. The fluorescence intensity at 515 nm decreased significantly when the pH was higher than 10, probably due to inhibition of proton transfer by deprotonation of phenol groups. Decomposition of ONOO⁻ in an acidic environment is likely responsible for the decreased emission intensities at low pH.³⁶ In addition, LC-MS experiments were performed to confirm the proposed fluorescence mechanism (Fig. S10–S13, ESI†).

Furthermore, the two-photon action cross section (Φδ) of BHID-Bpin and BHID was measured using rhodamine-6G as a reference (Fig. S14, ESI†).³⁷ The maxima Φδ value of BHID-Bpin and BHID were calculated to be 0.7 GM and 1.9 GM, respectively, under two-photon excitation at 750 nm. Moreover, a cell counting kit-8 (CCK-8) method using HeLa cells was performed with different concentrations of BHID and BHID-Bpin to determine the cytotoxicity. As shown in Fig. S15 (ESI†), both BHID and BHID-Bpin had negligible impact on cell survival. These data indicate that BHID-Bpin is suitable for measuring ONOO⁻ concentrations in a cellular environment. In addition, no photobleaching occurred after BHID-Bpin was irradiated for 10 min under two-photon excitation at 750 nm, which implied high photo-stability of the probe for real-time imaging (Fig. S16, ESI†). Next, BHID-Bpin was used to image exogenous ONOO⁻ and H₂O₂ respectively using the same concentration (300 μM) and incubation time (20 min) in HeLa cells. The two-photon imaging indicates that BHID-Bpin has higher sensitivity towards ONOO⁻ over H₂O₂ (Fig. S17, ESI†). We then evaluated the use of probe BHID-Bpin for the detection ONOO⁻ using other stimulus in live cells using TPM. TPM images of probe BHID-Bpin (5 μM) labelled HeLa cells exhibit weak fluorescence intensity (Fig. S18a, ESI†). The two-photon excitation fluorescence (TPEF) intensity was markedly increased when the cells are treated with 300 μM ONOO⁻ (Fig. S18b, ESI†). In addition, an increase in the intensity was observed when 3-morpholinosydnonimine (SIN-1), a well-known ONOO⁻ donor, was added (Fig. S18c, ESI†).^38,39 However, the fluorescence decreased upon pre-treatment with aminoguanidine (an inhibitor of nitric oxide synthase)⁴⁰ or ebselen (an organoselenium compound, which is a known ONOO⁻ scavenger that rapidly catalyzes the reduction of ONOO⁻) (Fig. S18e and f respectively, ESI†).⁴¹BHID-Bpin was then used to measure endogenous ONOO⁻. Using 50 ng mL⁻¹ interferon-γ (IFN-γ) and 500 ng mL⁻¹ lipopolysaccharide (LPS) resulted in the production of endogenous ONOO⁻,^42,43 leading to a significant enhancement in fluorescence, compared to the control group (Fig. S18, ESI†). Next, we determined whether probe BHID-Bpin can be used to detect ONOO⁻ in mice tissue (Fig. 4). TPM images of a portion of BHID-Bpin-labeled fresh rat hippocampal slices were obtained. The accumulated image at depths of 90–160 μm exhibited weak fluorescence intensity (Fig. 4a); however, the TPEF intensity of the image was markedly increased when the tissues were pre-treated with 50 μM SIN-1 for 20 min (Fig. 4b). The images collected at a higher magnification clearly indicate the ONOO⁻ levels in the individual cells (Fig. 4c–e). In addition, the TPEF intensity decreased upon treatment with 150 μM ebselen for 40 min (Fig. 4e). These results confirm that BHID-Bpin can be used as a tool for the imaging of exogenous ONOO⁻ in living systems.

Fig. 4 TPM rat hippocampal slice images acquired after incubation of 20 μM BHID-Bpin for 1 h and obtained at ×40 magnification. (a and b) The TPM images are implemented by combining images in the z-direction, which correspond to depths of 90–160 μm and detected in the (a) absence and (b) presence of 50 μM SIN-1. (c–e) Enlarged images show a white box part of panels a and b at a depth of 110 μm and were acquired (c) before and (d and e) after the addition of (d) SIN-1 (50 μM) for 20 min, (e) 150 μM ebselen with 50 μM SIN-1 for 40 min. (f) Average TPEF intensity in panels c–e. Images were acquired at the range of 400–600 nm upon excitation at 750 nm. Scale bar: (a) 300 and (c) 50 μm, respectively.

In summary, we have developed a fluorescent probe, BHID-Bpin, where HID was used for the first time as part of an ESIPT framework to detect ONOO⁻ using TPM. Our approach used a simple two-step synthetic route to prepare the two-photon activatable probe. More importantly, the probe was used to measure ONOO⁻ in HeLa cells with low cytotoxicity. Furthermore, the probe exhibited good two-photon properties and could be used to image exogenous ONOO⁻ in rat hippocampal slices at a depth up to 110 μm. Thus, our probe can be regarded as a powerful imaging tool to investigate ONOO⁻ in living biological systems. The success of BHID-Bpin in imaging ONOO⁻ using two-photon excitation indicates that HID is suitable for the construction of novel ESIPT-based two-photon fluorescent probes. Finally, we anticipate that the short emission wavelengths of BHID can be overcome by employing BHID as a Förster resonance energy transfer donor.⁴⁴

LW wishes to thank China Scholarship Council and the University of Bath for supporting his PhD work in the UK. XT wishes to thank the University of Bath for supporting her PhD work in the UK. We would like to thank the EPSRC and the University of Bath for funding. DJL, CSL and HMK thank the National Leading Research Lab Program of the National Research Foundation of Korea (NRF), funded by the Korean government (MSIP) (NRF-2019R1A2B5B03100278, NRF-2021R1C1C1013187), Center for Convergence Research of Neurological Disorders (NRF-2019R1A5A2026045). TDJ wishes to thank the Royal Society for a Wolfson Research Merit Award and the Open Research Fund of the School of Chemistry and Chemical Engineering, Henan Normal University for support (2020ZD01). NMR and Mass Spectroscopic (MS) Characterisation facilities were provided by the Material and Chemical Characterisation Facility (MC²) at the University of Bath (http://go.bath.ac.uk/mc2). All data supporting this study are provided as ESI† accompanying this paper.

Conflicts of interest

There are no conflicts to declare.

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/d1cc03160k

‡ These authors contributed equally to this work.

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