Evaluation of borinic acids as new,fast hydrogen peroxide–responsive triggers

Hydrogen peroxide (H₂O₂) is responsible for numerous damages when overproduced, and its detection is crucial for a better understanding of H₂O₂-mediated signaling in physiological and pathological processes. For this purpose, various “off–on” small fluorescent probes relying on a boronate trigger have been prepared, and this design has also been involved in the development of H₂O₂-activated prodrugs or theranostic tools. However, this design suffers from slow kinetics, preventing activation by H₂O₂ with a short response time. Therefore, faster H₂O₂-reactive groups are awaited. To address this issue, we have successfully developed and characterized a prototypic borinic-based fluorescent probe containing a coumarin scaffold. We determined its in vitro kinetic constants toward H₂O₂-promoted oxidation. We measured 1.9 × 10⁴ m⁻¹⋅s⁻¹ as a second-order rate constant, which is 10,000-fold faster than its well-established boronic counterpart (1.8 m⁻¹⋅s⁻¹). This improved reactivity was also effective in a cellular context, rendering borinic acids an advantageous trigger for H₂O₂-mediated release of effectors such as fluorescent moieties.

Reactive oxygen species (ROS) are involved in various physiological processes. In particular, hydrogen peroxide (H₂O₂) plays a critical role in the regulation of numerous biological activities as a signaling molecule (1, 2). However, aberrant production or accumulation of H₂O₂ leads to oxidative stress conditions, which can cause lesions associated with aging, cancer (3), and several neurodegenerative diseases such as Alzheimer’s or Parkinson’s (4, 5). Differentiation of physiological or abnormal conditions is closely connected with slight changes in H₂O₂ levels. However, the generation and degradation of H₂O₂ are variable within different cellular compartments, and this small molecule is highly diffusive, rendering the capture of small H₂O₂ fluctuations and the study of its spatial and temporal dynamics difficult. Therefore, the development of selective and sensitive H₂O₂-reactive tools for applications in a biological context represents a challenge for a better understanding of H₂O₂-mediated signaling in physiological and pathological processes or the use of H₂O₂ activation for the release of biological effectors (6).Numerous strategies have been developed to implement H₂O₂-reactive molecular triggers, as exemplified by “off–on” small fluorescent probes. Such probes have attracted particular attention due to their easy implementation, high expected signal-to-noise ratio, and compatibility with standard equipment present in cellular biology research environments (7–9). Activation in such a context is triggered or modified by H₂O₂-mediated transformation of a suitable chemical species. Several approaches have been explored including probes based on arylsulfonyl ester perhydrolysis (10), oxidation of arylboronates (11), Baeyer–Villiger oxidation of diketones (12), Tamao oxidation of silanes (13), a tandem Payne–Dakin reaction (14) or a seleno-Mislow–Evans rearrangement (15). Among them, designs based on the boronate esters oxidation pioneered by Chang are the most explored, due to their remarkable stability, low toxicity profile, ease of preparation, and specificity toward H₂O₂, as illustrated in recent reviews (16–18). Upon reaction with H₂O₂, these compounds undergo an oxidative conversion into aryl borate esters that further hydrolyze into the corresponding phenols along with borate esters or boric acid (Scheme 1A). This conversion turns on probe fluorescence or activates drug release either directly or via the degradation of a self-immolative spacer. This chemospecific and biologically compatible reaction allowed, for instance, developing highly selective fluorescent probes for H₂O₂ imaging in cells (19–23). However, H₂O₂-triggered conversion of boronic acids to phenols is still not completely satisfactory in a biological context (24) since most of these probes have second-order reaction rate constants of 0.1 to 1.0 m⁻¹⋅s⁻¹ (14). In cells, H₂O₂ is present in the 1 to 100 nm concentration range in physiological conditions and could reach up to 100 μm under oxidative stress conditions (25). Therefore, most of the boronate-based systems need an incubation time longer than 30 min for activation at an H₂O₂ concentration of 100 μm. At such a time scale, H₂O₂ typically diffuses over a distance of 2 mm (evaluated as (D_H2O2τ)^0.5 with D_H2O2 = 1.7 × 10⁻⁹ m²⋅s⁻¹ from ref. 26 and τ = 30 min). Hence, to improve spatial resolution for H₂O₂ imaging, alternative H₂O₂ triggers with rapid reaction rates allowing real-time activation by H₂O₂ are still required.Open in a separate windowScheme 1.(A) Current boronic acid (R = H) or boronate (R,R = tetramethylethylene) as H₂O₂-responsive group releasing a hydroxyaryl as effector and a boric acid or a borate ester respectively. (B) This study: a borinic acid as H₂O₂-responsive group releasing a hydroxyaryl as effector and a boronic acid.To address this issue, we envisioned the use of borinic acids, structures in which one of the boron–oxygen bonds of the boronic acid is replaced by a boron–carbon bond. Due to these electronic modifications, borinic acids exhibit more electrophilic properties (27–30) compared to their boronic acid counterparts and could be more prone to rapid oxidation. These structures have been mainly exploited as catalysts in various reactions such as epoxide ring opening (31), hydrosilylation (32), transamidation (33), aldol reaction (34, 35), C–H activation (36, 37), selective monoalkylation, acylation and sulfonation of diols (38, 39), or regioselective glycosylation reactions (40–42). Surprisingly, the reactivity of these borinic species remains underexplored (43–45), probably due to their limited synthetic access (46–49). They were usually obtained through the addition of strong organometallic reagents (RLi/RMgBr) onto boron-based electrophiles such as trialkylborates, boron halides, diborane, or boronate esters. To date, a detailed study of the reactivity of borinic acids toward oxidation including reaction with H₂O₂ has not been reported and their use as triggers for the direct release of a probe or an effector has not been considered.Herein, we report the design, synthesis, and evaluation of a borinic-triggered prototypic probe prone to direct and rapid activation by the H₂O₂ molecule (Scheme 1B). We establish a detailed kinetic analysis of the H₂O₂-promoted oxidation of this borinic acid as well as a comparative study with its corresponding boronic analog. Furthermore, we demonstrate the shorter response time of the borinic trigger compared to the boronic trigger against H₂O₂-mediated oxidation in a cellular environment.