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

Abstract

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 × 104m−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.

Guidelines for measuring reactive oxygen species and oxidative damage in cells and in vivo

“…Within cells, H 2 O 2 detection by phenylboronate-based probes is more reliable 38 although these may lack sufficient sensitivity because they react only slowly with H 2 O 2 , which can make it difficult to detect small or localized changes in H 2 O 2 levels 39 . However, recent studies suggest that borinic acids, which react more rapidly with H 2 O 2 , may be more sensitive detectors 40 . The mechanism of oxidation of phenylboronates to phenols requires a two-electron oxidant, such as H 2 O 2 .…”

“…The mechanism of oxidation of phenylboronates to phenols requires a two-electron oxidant, such as H 2 O 2 . Because H 2 O 2 is typically generated at higher concentrations than other ROS, boronate probes can be selective for H 2 O 2 detection subject to proper controls 39,40 . However, boronate probes react with ONOO − /ONOOH or HOCl much more rapidly than they do with H 2 O 2 which can sometimes complicate measurements, and orthogonal approaches or the use of inhibitors can aid validation 41 .…”

Hydrogen Peroxide‐Responsive Triggers Based on Borinic Acids: Molecular Insights into the Control of Oxidative Rearrangement

“…It is important to note that the reaction proceeded smoothly to afford the corresponding substituted phenols with a much slower oxidation rate than borinic acids, as we have previously observed. [13] In addition, only 10% of the ortho- (trifluoromethyl)phenylboronic acid was converted to phenol within one hour, while the reaction required 35 minutes to reach completion with its para-isomer. These results support a slow 1,2-migration of the o-CF3-phenyl group from boron atom to oxygen atom, which could explain the total selectivity observed during oxidation of 3i.…”

“….4 ± 0.1 [13] 1.8 [13] 2 R = Ph 20 80 5.3 ± 0.1 [13] 1.9´10 4 [13] 3 R = o-CF3Ph 100 0 6.0 ± 0.1 3.2´10 3 We then briefly studied the oxidation rates of the two SIS-containing fluorogenic probes 10 and 15. From a mechanistic perspective, H2O2-urea-promoted oxidation generates a phenol, which spontaneously undergoes a fragmentation process through a 1,6-benzyl elimination resulting in the release of a quinone methide together with (in the case of probe 15) or without (in the case of probe 10) a decarboxylation process, to finally elicit turn-on fluorescence (Figure 2A). )…”

Hydrogen Peroxide‐triggered Chemical Strategy for Controlling CRISPR systems

“…Hydrogen peroxide is a signaling molecule that plays a critical role in the regulation of numerous biological activities [6i,j,8] . Based on studies of boronic acid as a small molecule probe in organisms, we believe that boronic acid will become an effective chemical tool especially due to its high reactivity with hydrogen peroxide [8a,c,f,9] . Here, we synthesized a compound named 4‐(4,4,5,5‐tetramethyl‐1,3,2‐dioxaborolan‐2‐yl)benzyl 1H‐imidazole‐1‐carboxylate (TDYBIC, Figure S1, S2) and report the synthesis of a boronic‐triggered RNA structure prototypic probe (Masked RNA, Scheme 1a, Scheme S1) prone to direct and rapid activation by the hydrogen peroxide molecule to control the CRISPR system (Scheme 1b).…”