Reaction-Based Fluorescent Probes for the Detection and Imaging of Reactive Oxygen, Nitrogen, and Sulfur Species

Abstract

This Account describes a range of strategies for the development of fluorescent probes for detecting reactive oxygen species (ROS), reactive nitrogen species (RNS), and reactive (redox-active) sulfur species (RSS). Many ROS/RNS have been implicated in pathological processes such as Alzheimer's disease, cancer, diabetes mellitus, cardiovascular disease, and aging, while many RSS play important roles in maintaining redox homeostasis, serving as antioxidants and acting as free radical scavengers. Fluorescence-based systems have emerged as one of the best ways to monitor the concentrations and locations of these often very short lived species. Because of the high levels of sensitivity and in particular their ability to be used for temporal and spatial sampling for in vivo imaging applications. As a direct result, there has been a huge surge in the development of fluorescent probes for sensitive and selective detection of ROS, RNS, and RSS within cellular environments. However, cellular environments are extremely complex, often with more than one species involved in a given biochemical process. As a result, there has been a rise in the development of dual-responsive fluorescent probes (AND-logic probes) that can monitor the presence of more than one species in a biological environment. Our aim with this Account is to introduce the fluorescent probes that we have developed for in vitro and in vivo measurement of ROS, RNS, and RSS. Fluorescence-based sensing mechanisms used in the construction of the probes include photoinduced electron transfer, intramolecular charge transfer, excited-state intramolecular proton transfer (ESIPT), and fluorescence resonance energy transfer. In particular, probes for hydrogen peroxide, hypochlorous acid, superoxide, peroxynitrite, glutathione, cysteine, homocysteine, and hydrogen sulfide are discussed. In addition, we describe the development of AND-logic-based systems capable of detecting two species, such as peroxynitrite and glutathione. One of the most interesting advances contained in this Account is our extension of indicator displacement assays (IDAs) to reaction-based indicator displacement assays (RIAs). In an IDA system, an indicator is allowed to bind reversibly to a receptor. Then a competitive analyte is introduced into the system, resulting in displacement of the indicator from the host, which in turn modulates the optical signal. With an RIA-based system, the indicator is cleaved from a preformed receptor-indicator complex rather than being displaced by the analyte. Nevertheless, without a doubt the most significant result contained in this Account is the use of an ESIPT-based probe for the simultaneous sensing of fibrous proteins/peptides AND environmental ROS/RNS.

Scheme 1. (a) 1 and 1–d-Fructose Complex for Detecting H₂O₂; (b) 2 and 2–d-Fructose Complex for Detecting H₂O₂

Scheme 2. Boronate Probes for Detecting H₂O₂

Scheme 3. Mechanism and Fluorescence Spectra of the ARS–PBA Complex for Detecting H₂O₂

Adapted from ref (35). Published by The Royal Society of Chemistry.

Scheme 4. ESIPT-Based Probe TCBT-OMe for Detecting HOCl/ClO^–

Scheme 5. ESIPT-Based Probe C7 for Detecting HOCl

Scheme 6. (a) ESIPT-Based Probe HMBT-LW for Detecting O₂^•–; (b) Changes in Fluorescence Emission Intensity of HMBT-LW (5 μM) with Increasing O₂^•– in Phosphate-Buffered Saline (PBS) (10 mM v/v, 1:1 DMSO/PBS, pH 7.4) after 3 min at λ_ex = 310 nm

Adapted with permission from ref (40). Published by The Royal Society of Chemistry (RSC) on behalf of the Centre National de la Recherche Scientifique (CNRS) and the RSC.

Scheme 7. Probe 3 for Detecting ONOO^– in the Presence of d-Fructose

Scheme 8. ARS–NBA Complex for Detecting ONOO^–

Figure 1

Probe 4 for detecting ONOO^–.

Scheme 9. (a) Probe ABAH-LW for Detecting ONOO^–; (b) Changes in Fluorescence Emission of ABAH-LW (3 μM) with Increasing Addition of ONOO^– in PBS (pH 8.2, Containing 8% DMSO, 1 mM CTAB) after 1 min at λ_ex = 370 nm

Adapted with permission from ref (46). Copyright 2018 Royal Society of Chemistry.

Figure 2

TCFB1 and TCFB2 for detecting ONOO^–.

Scheme 10. PR1 for Detecting ONOO^–

Scheme 11. 1–copper(II) Complex for Detecting NO and HNO

Scheme 12. Probe 5 for Detecting GSH

Figure 3

TCF-GSH and TCFCl-GSH for detecting GSH.

Figure 4

Theranostic prodrug DCM-S-CPT.

Figure 5

(a) Probe 6 for detecting thiols. (b) Fluorescence emission spectra of probe 6 (10 μM) before and after addition of l-cysteine (300 μM) in 4:1 v/v MeOH/H₂O at λ_ex = 505 nm at 37 °C. Adapted with permission from ref (55). Copyright 2012 Royal Society of Chemistry.

Scheme 13. DT-Gal for Detecting H₂S

Scheme 14. OPD for Detecting Na₂S

Scheme 15. (a) GSH-PF3 for Detecting ONOO^– AND GSH; (b, c) Fluorescence Spectra of GSH-PF3 (0.5 μM) upon Addition of (a) ONOO^– (10 μM) Followed by GSH (0–80 μM) with a 5 min Wait between Additions and (c) GSH (200 μM) Followed by Addition of ONOO^– (0–10 μM) with a 10 min Wait between Additions (52 wt % Methanol, pH 8.21, λ_ex = 488 nm, 25 °C)

Adapted with permission from ref (59). Copyright 2018 Royal Society of Chemistry.

Scheme 16. (a) GSH-ABAH for Detecting ONOO^– AND GSH; (b, c) Fluorescence Spectra of GSH-ABAH (2 μM) upon Addition of (a) ONOO^– (4 μM) Followed by GSH (0–2 μM) after a 1 min Wait and (c) GSH (5 μM) Followed by ONOO^– (0–14 μM) after a 1 min Wait (8% DMSO, 1 mM CTAB, pH 8.20, λ_ex = 390 nm, 25 °C)

Adapted with permission from ref (60). Copyright 2018 Royal Society of Chemistry.

Scheme 17. Pinkment-OH for Detecting ONOO^–, Pinkment-OTBS for Detecting ONOO^– AND Fluoride, and Pinkment-OAc for Detecting ONOO^– AND Esterase Activity

Scheme 18. Dual-Enzyme-Activated PF3-Glc

Figure 6

(a) ESIPT probes 3-HF-X (X = OMe, Me, H) for detecting ONOO^–. The normal (N) and phototautomeric (T*) forms are shown. (b, c) Fluorescence imaging of a brain section of a transgenic mouse treated with 3-HF-OMe (20 μM) (b) without and (c) with ONOO^– (30 μM). The excitation/emission wavelengths for the blue (N-state), green (T*-state), and red (anti-Aβ antibody) channels are 404/425–475, 404/500–550, and 561/640–730 nm, respectively. The white arrows indicate stained Aβ aggregates. Reprinted from ref (64). Copyright 2018 American Chemical Society.