Boronate-based Fluorescent Probe Research Articles

Selective imaging of hydrogen peroxide over peroxynitrite by a boronate-based fluorescent probe engineered via a doubly activated electrophilicity-increasing strategy

Boronate-based fluorescent probes are widely used for the imaging of hydrogen peroxide (H2O2). However, their selectivity might be subjected to the interference of other reactive oxygen species, especially peroxynitrite (ONOO-), due to the reaction of boronates with ONOO- being several orders of magnitude faster than with H2O2. This work highlights a doubly activated electrophilicity-increasing strategy to develop a boronate-based fluorescent probe THMP for selective imaging of H2O2 over ONOO-, where a boronate-modified pyridiniumylacrylonitrile is grafted on the 2-(2′-hydroxy-3′-methoxyphenyl) benzothiazole scaffold. Specifically, the boronate oxidation of THMP by H2O2 leads to the release of the free fluorophore THMP-N, triggering a ratiometric fluorescence response from red to green. The presence of a doubly activated electrophilic site on the carbon-carbon double bond of THMP, by both the strong electron-withdrawing cyano group and the pyridinium moiety, allows selective oxidative cleavage of the double bond by ONOO- to an aldehyde HMBT-CHO, thereby excluding the interference of ONOO- in monitoring H2O2. With the aid of the probe, we successfully visualized increased levels of H2O2 during ferroptosis of HepG2 cells, and burst of H2O2 in brains of live mice during cerebral ischemia reperfusion injury.

Boronate-Based Fluorescent Probes as a Prominent Tool for H2O2 Sensing and Recognition.

Given the crucial association of hydrogen peroxide with a wide range of human diseases, this compound has currently earned the reputation of being a popular biomolecular target. Although various analytical methods have attracted our attention, fluorescent probes have been used as prominent tools to determine H2O2 to reflect the physiological and pathological conditions of biological systems. The sensitive responsive part of these probes is the boronate ester and boronic acid groups, which are important reporters for H2O2 recognition. In this review, we summarize boronate ester/boronic acid group-based fluorescent probes for H2O2 reported from 2012 to 2020, and we have generally classified the fluorophores into six categories to exhaustively elaborate the design strategy and comprehensive systematic performance. We hope that this review will inspire the exploration of new fluorescent probes based on boronate ester/boronic acid groups for the detection of H2O2 and other relevant analytes.

Boronate probe-based hydrogen peroxide detection with AlGaN/GaN HEMT sensor.

The results from this study demonstrate the potential of an AlGaN/GaN high electron mobility transistor sensor for the detection of reactive and transient biological molecules such as hydrogen peroxide. A boronate-based fluorescent probe was used with this device to detect the presence of micromolar levels of hydrogen peroxide typically associated with intracellular processes. The real-time electrical response of the high electron mobility transistor sensor showed a gradual decrease in the two-dimensional electron gas current as the reaction proceeded over time. A corresponding increase in the emission intensity was measured from the fluorescent probe with the progression of the reaction. The fluorescence from the boronate probe was used as an indicator to confirm the detection of hydrogen peroxide. These results demonstrate the dynamic measurement capability of AlGaN/GaN high electron mobility transistor sensors in monitoring real-time reactions of reactive oxygen species such as hydrogen peroxide.

Boronate based sensitive fluorescent probe for the detection of endogenous peroxynitrite in living cells.

In this work, a novel boronate-based fluorescent probe (FAM) for the endogenous detection of peroxynitrite (ONOO-) has been developed by using anthracycline as the fluorophore, arlyboronate as the recognition moiety, lipophilic cation as the mitochondrial targeting moiety. Upon reaction of the probe with ONOO-, the oxidation and subsequent hydrolysis of ONOO- to arlyboronate triggers quite rapid fluorescence off-on response, providing a sensitive and highly selective method for the detection of ONOO-. In addition, probe holds high sensitivity with the detection limit of 3.2nM and excellent specificity including a series of biologically relevant reactive oxygen species. Importantly, FAM with good water solubility displays excellent performances for imaging endogenous peroxynitrite produced by RAW264.7 cells.

105 - Development and characterization of a mitochondria-targeted boronate-based fluorescent probe for peroxynitrite detection

The unambiguous detection, formation rates and steady-state concentrations of peroxynitrite (ONOO‒/ONOOH) in biological systems is a great challenge due to its high reactivity towards several biomolecules. The accurate cellular detection of peroxynitrite in different cellular compartments and the estimation of its formation rates, represent fundamental steps to understand how nitric oxide-derived oxidants affect biological processes, including mitochondrial dysfunction and cell death. Aryl-boronates react rapidly and stoichiometrically with ONOO− to yield the corresponding hydroxylated compound as the major reaction product. It has been demonstrated by others and us, that in cellular systems where several reactive oxygen and nitrogen species coexist, boronates are kinetically selective for peroxynitrite. We have recently reported a boronate-based fluorescent probe for the direct and sensitive detection and quantitation of endogenously-produced peroxynitrite [1]. Furthermore, those data allowed us to tailor boronate-derivatives targeted to identify subcellular sites and rates of peroxynitrite generation. Herein, we communicate the design, synthesis and characterization of new organelle-targeted boronate-based fluorescent probe MitoCT. MitoCT chemical structure consist of a boronic ester attached to a modified coumarin in order to enhance spectroscopic properties, and supporting a triphenylphosphonium cation to target and concentrate in mitochondria matrix. The synthetic route involves a convergent route and results in reasonable product yields. Kinetic and mechanistic characterization of the reaction of MitoCT with peroxynitrite and other oxidants, including a side-by-side comparison with the commercially-available and putative H2O2 mitochondrial probe MitoPy1, are underway.

An ESIPT Probe for the Ratiometric Imaging of Peroxynitrite Facilitated by Binding to Aβ-Aggregates.

A series of 3-hydroxyflavone (3-HF) ESIPT (excited-state intramolecular proton transfer) boronate-based fluorescent probes have been developed for the detection of peroxynitrite (ONOO-). The dyes are environmentally sensitive, and each probe exhibited a ratiometric response toward ONOO- in a micellar environment. The probes were used to image different aggregation states of amyloid-β (Aβ) in the presence of ONOO-. The 3-HF-OMe probe was found to produce a ratiometric response toward ONOO- when bound to Aβ aggregates, resulting in a novel host-guest ensemble, which adds insight into the development of other ESIPT-based probes for the simultaneous sensing of fibrous proteins/peptides and environmental ROS/RNS.

In vitro and in vivo imaging of peroxynitrite by a ratiometric boronate-based fluorescent probe

Peroxynitrite (ONOO−) is an important species involved in many physiopathological processes. Progresses have been made in developing novel fluorescent probes to detect peroxynitrite with relatively high sensitivity and specificity. Herein, we report the synthesis, characterization and biological applications of a new boronate-based fluorescent probe, 4-MB. The studies showed that 4-MB exhibits a dual ratiometric and calorimetric response toward peroxynitrite due to ONOO−-triggered oxidative reaction. A possible mechanism of the oxidation reaction was proposed and the reaction product was isolated and characterized using different spectroscopic methods. We have thoroughly demonstrated the utility of 4-MB for intracellular peroxynitrite imaging. Further, we showed that 4-MB can be potentially employed to visualize exogenous and endogenous peroxynitrite in RAW264.7 macrophages, EAhy926 cells, zebrafish and in live tissues from a high-fat diet-induced obese mouse model.

A water-soluble boronate-based fluorescent probe for the selective detection of peroxynitrite and imaging in living cells

A water-soluble boronate-based fluorescent probe was evaluated for the detection of peroxynitrite (ONOO−) in the presence of a monosaccharide. The enhanced fluorescence of the probe when bound with D-fructose was switched off in the presence of peroxynitrite. In contrast, other reactive oxygen/nitrogen species (ROS/RNS) led to only slight fluorescence decreases due to protection by an internal N–B interaction. The interaction of the probe with D-fructose not only strengthens the fluorescence signal, but also protects the boronic acid from oxidation by other ROS/RNS. Therefore, under conditions generating various ROS/RNS, the boronate-based saccharide complex preferentially reacts with peroxynitrite. The probe was used in cell imaging experiments for the detection of endogenous and exogenous peroxynitrite. The sensor displays good “on–off” responses towards peroxynitrite, both in RAW 264.7 cells and HeLa cells.

A boronate-based fluorescent probe for the selective detection of cellular peroxynitrite.

A boronate-based fluorescent probe 1 for the selective monitoring of intracellular peroxynitrite has been developed. The probe takes advantage of the fast reaction of an arylboronate group with peroxynitrite, yielding a corresponding phenol that undergoes spontaneous subsequent reactions to produce a strongly fluorescent product associated with a large turn-on signal.

“Integrated” and “insulated” boronate-based fluorescent probes for the detection of hydrogen peroxide

Integrated and insulated boronate-based fluorescent probes have been evaluated for the detection of hydrogen peroxide in the presence of saccharides.

Peroxynitrite Is the Major Species Formed from Different Flux Ratios of Co-generated Nitric Oxide and Superoxide: DIRECT REACTION WITH BORONATE-BASED FLUORESCENT PROBE

There is much interest in the nitration and oxidation reaction mechanisms initiated by superoxide radical anion (O(2)()) and nitric oxide ((*)NO). It is well known that O(2) and (*)NO rapidly react to form a potent oxidant, peroxynitrite anion (ONOO(-)). However, indirect measurements with the existing probes (e.g. dihydrorhodamine) previously revealed a bell-shaped response to co-generated (*)NO and O(2) fluxes, with the maximal yield of the oxidation or nitration product occurring at a 1:1 ratio. These results raised doubts on the formation of ONOO(-) per se at various fluxes of (*)NO and O(2). Using a novel fluorogenic probe, coumarin-7-boronic acid, that reacts stoichiometrically and rapidly with ONOO(-) (k = 1.1 x 10(6) m(-1)s(-1)), we report that ONOO(-) formation increased linearly and began to plateau after reaching a 1:1 ratio of co-generated (*)NO and O(2) fluxes. We conclude that ONOO(-) is formed as the primary intermediate during the reaction between (*)NO and O(2) co-generated at different fluxes.

Boronate-Based Fluorescent Probes for Imaging Cellular Hydrogen Peroxide

The syntheses, properties, and biological applications of the Peroxysensor family, a new class of fluorescent probes for hydrogen peroxide, are presented. These reagents utilize a boronate deprotection mechanism to provide high selectivity and optical dynamic range for detecting H2O2 in aqueous solution over similar reactive oxygen species (ROS) including superoxide, nitric oxide, tert-butyl hydroperoxide, hypochlorite, singlet oxygen, ozone, and hydroxyl radical. Peroxyresorufin-1 (PR1), Peroxyfluor-1 (PF1), and Peroxyxanthone-1 (PX1) are first-generation probes that respond to H2O2 by an increase in red, green, and blue fluorescence, respectively. The boronate dyes are cell-permeable and can detect micromolar changes in H2O2 concentrations in living cells, including hippocampal neurons, using confocal microscopy and two-photon microscopy. The unique combination of ROS selectivity, membrane permeability, and a range of available excitation/emission colors establishes the potential value of PR1, PF1, PX1, and related probes for interrogating the physiology and pathology of cellular H2O2.