# Borinic Acid-Based Fluorogenic Probes as an Alternative to the Amplex Red Assay for Real-Time H 2 O 2 Monitoring in Live Cells

Mathilde Pucher, Kirrthana Makenthirathasan, Hadrien Jalaber, Thomas

Lesaux, Oliver Nüsse, Gilles Doisneau, Yann Bourdreux, Blaise

Gatin-Fraudet, Ludovic Jullien, Boris Vauzeilles, et al.

► To cite this version:

Mathilde Pucher, Kirrthana Makenthirathasan, Hadrien Jalaber, Thomas Lesaux, Oliver Nüsse, et al. Borinic Acid-Based Fluorogenic Probes as an Alternative to the Amplex Red Assay for Real-Time H 2 O 2 Monitoring in Live Cells. ACS Chemical Biology, 2025, 20 (7), pp.1574-1583. 10.1021/ac- schembio.5c00156. hal-05250768

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Distributed under a Creative Commons Attribution 4.0 International License
Borinic acid-based fluorogenic probes as an alternative to Amplex® Red
assay for real-time H₂O₂ monitoring in live cells

Mathilde Pucher,[a] Kirrthana Makenthirathasan,[a,b] Hadrien Jalaber,[b] Thomas Le Saux,[c]
Oliver Nüsse,[b] Gilles Doisneau,[a] Yann Bourdreux,[a] Blaise Gatin-Fraudet,[a,d] Ludovic
Jullien,[c] Boris Vauzeilles,[d] Dominique Guianvarc'h,[a] Marie Erard,[b] and Dominique
Urban.*[a]

[a] Dr. M. Pucher, K. Makenthirathasan, Dr. G. Doisneau, Dr. Y. Bourdreux, Dr. B. Gatin-Fraudet, Prof. D. Guianvarc'h, Dr. D. Urban
Université Paris-Saclay, CNRS, Institut de Chimie Moléculaire et des Matériaux d'Orsay, UMR CNRS 8182, 91405 Orsay, France.
E-mail: dominique.urban@universite-paris-saclay.fr

[b] K. Makenthirathasan, H. Jalaber, Prof. M. Erard, Prof. O. Nüsse
Université Paris-Saclay, CNRS, Institut de Chimie Physique,
UMR CNRS 8000, 91405, Orsay, France.

[c] Dr. T. Le Saux, Prof. L. Jullien
CPCV, Département de chimie, École Normale Supérieure
PSL University, Sorbonne Université, CNRS, 75005 Paris, France

[d] Dr. B. Gatin-Fraudet, Dr. B. Vauzeilles
Université Paris-Saclay, CNRS, Institut de Chimie des Substances Naturelles, UPR 2301,
91198 Gif-sur-Yvette, France.

ABSTRACT

Hydrogen peroxide (H₂O₂) is a crucial reactive oxygen species (ROS) involved in regulating both physiological and pathological processes. Excessive H₂O₂ production can lead to oxidative stress, contributing to aging, cancer, and neurodegenerative diseases. In contrast to other ROS exhibiting short lifespans, H₂O₂ is relatively stable, and its spatial and temporal dynamics are central to understanding its pathophysiological role. Therefore, the development of fluorescent probes that are highly selective, sensitive, and capable of rapid response is still required. To date, numerous fluorescent probes have been developed. Among them, boronic acid triggers have attracted considerable attention but often suffer from limited reactivity preventing real-time H₂O₂ monitoring. To overcome this lack of reactivity, we report the design and the synthesis of new borinic acid-based fluorogenic probes for H₂O₂ detection in cellular environments. These probes are based on a hemicyanine scaffold functionalized with the borinic acid trigger, which demonstrated superior kinetics compared to its boronic counterpart. These probes enable efficient real-time monitoring of H₂O₂ in cellular models, both extracellularly and intracellularly. The kinetics of these enzyme-free chemical probes matched that of the gold standard Amplex™ UltraRed/HRP assay, representing a significant advancement in the field and offering a versatile and sensitive tool for studying H₂O₂-mediated cell signaling.
INTRODUCTION

The role of hydrogen peroxide ($H_2O_2$) is crucial in the fine balance between physiological and pathological processes.¹ It is now well-admitted that $H_2O_2$ acts as a signaling molecule at low concentrations in numerous biological phenomena, including cell proliferation, differentiation, and migration.²⁻⁴ Conversely, abnormal regulation of this species can lead to oxidative stress conditions, which can cause lesions associated with aging, cancer⁵, and several neurodegenerative diseases, including Alzheimer's or Parkinson's.⁶⁻¹⁰ In contrast to other radical reactive oxygen species (ROS), such as the superoxide radical anion ($O_2^{\cdot-}$), the peroxyl radical (ROO·), and the hydroxyl radical (HO·), which have a short lifespan, $H_2O_2$ is the most stable and diffusible of these species.¹¹ Therefore, tracking $H_2O_2$ levels and analyzing its spatial and temporal dynamics is particularly meaningful as well as challenging due to its latter properties. Thus, the development of selective and sensitive reactive probes is still expected to improve the understanding of $H_2O_2$-mediated signaling in both physiological and pathological processes.

Fluorescence microscopy is a powerful tool for imaging hydrogen peroxide due to its non-invasiveness, ease of use, good biocompatibility, and high sensitivity. To date, numerous small molecular fluorogenic probes have been successively used for $H_2O_2$ visualization. Among them, commercially available Amplex™ Red and dichlorodihydrofluorescein diacetate ($H_2DCFDA$) are commonly used for real-time monitoring of extra- or intracellular $H_2O_2$, respectively. However, both probes have their limitations. For instance, Amplex™ Red requires the external addition of an enzyme, namely horseradish peroxidase (HRP), while the oxidation of $H_2DCF$ to fluorescent dichlorofluorescein (DCF) is promoted either by a peroxidase or a transition metal complex.¹² Thus, since the 2000s, new probes possessing specific triggers reacting directly with $H_2O_2$ have been developed.¹³⁻¹⁵ The most widely settled are boronic acid/ester-based probes, reported first by Chang¹⁶,¹⁷ and displayed in recent reviews.¹⁸,¹⁹ Most of these probes rely on introducing boronic acid/ester masking group in place of an alcohol on dyes, which disrupts the intramolecular charge transfer (ICT) mechanism, therefore turning off the fluorescence. Upon reaction with $H_2O_2$, an oxidative conversion into aryl borate esters that further hydrolyze into the corresponding phenols turns on the fluorescence. Significant efforts have been carried out to develop near-infrared (NIR) fluorescent probes for biological applications.²⁰⁻²⁶ Furthermore, the incorporation of organelle-targeting units, such as triphenylphosphonium, pyridinium, and morpholine, provided spatial insights into ROS distribution.²⁷⁻³⁰ Despite these improvements, $H_2O_2$-triggered conversion of boronic acids to phenols is relatively slow, which is still not completely satisfactory for real-time $H_2O_2$ monitoring in a biological context.³¹ In this context, we³² and others³³ have recently evaluated a borinic acid as a new $H_2O_2$-responsive trigger. Borinic acids feature two C–B bonds and one B–O bond, resulting in enhanced Lewis acidity compared to boronic acids and an expected higher reactivity toward $H_2O_2$. As a proof of concept, we thus designed and synthesized a prototype turn-on fluorescent probe possessing a coumarin scaffold conjugated to a borinic acid reporter.³² We have demonstrated the superior reactivity of the borinic acid-based probe compared to its boronic acid counterpart. This improvement also proved effective for in-cell detection of *exo*- as well as *endogenously* produced $H_2O_2$. Moreover, due to the probe's
unsymmetrical structure affording two oxidative pathways, we identified that the introduction of an *ortho*-electron-withdrawing and sterically hindered substituent (*o*-CF₃) onto the phenyl core directed the regioselectivity of the oxidative rearrangement, ensuring the exclusive release of the fluorophore upon H₂O₂ treatment (Scheme 1A).³⁴ In continuation of our work, we are now focusing on expanding the set of these new sensors by improving the photophysical properties of the released fluorophore for real-time imaging in a cellular context (Scheme 1B).

**A. Previous studies**

<figure><img src="image_2.png" /></figure>

**B. This Study**

<figure><img src="image_3.png" /><figcaption>**Scheme 1.** A. Previous work: prototype borinic acid-based probes containing a coumarin scaffold. B. This work: structure of new sensors with improved photophysical properties.</figcaption></figure>

Hemicyanine dyes (HD) have attracted considerable attention since they exhibit good water solubility, cell permeability, and high photostability. Typically, HD are constituted of a positively charged nitrogen heterocyclic moiety (electron acceptor) connected to a terminal hydroxyl or amino group (electron donor) through a conjugated alkene. Due to their interesting properties, these scaffolds were already conjugated to a boronic acid/ester trigger for sensing hydrogen peroxide.³⁵ In addition, modulation of the donor-π-acceptor system allows fine-tuning of emission wavelengths up to the near-infrared region, providing valuable devices for biomedical applications.³⁶⁻³⁸ These probes are also capable of detecting mitochondrial H₂O₂ due to the electronic interaction of the positively charged indolium moiety with the negative inner membrane potential of mitochondria.³⁹⁻⁴¹ Considering the advantages of HD, we thought it would be interesting to combine our borinic acid trigger with this class of fluorescent agents.

Herein, we report the design and the synthesis of two new turn-on fluorescent probes based on the hemicyanine scaffold connected for the first time to a borinic acid trigger (HEtBori and HSBori). These compounds were further engaged in detecting endogenously produced hydrogen peroxide in cells. We demonstrated that these probes can selectively detect either
extra- or intracellular hydrogen peroxide depending on the nature of the alkyl chain on the indolenine core, thus providing versatile tools for cellular H₂O₂ monitoring. We also compared the efficacy of these probes with Amplex™ UltraRed/Horseradish Peroxidase (HRP) and demonstrated that HSBori provides an effective, enzyme-free alternative for Amplex™ UltraRed/HRP, which typically requires an external peroxidase. This breakthrough highlights the potential of these borinic acid-based probes as powerful tools for real-time detection of extracellular H₂O₂.

## RESULTS AND DISCUSSION

### Synthesis of fluorogenic probes HEtBori and HSBori

We have synthesized two hemicyanine-based probes, which differ by the nature of the alkyl chain (ethyl or sulfonated propyl) on the indolium moiety. The introduction of a sulfonated propyl group should increase water solubility and avoid aggregation, rendering it suitable for sensing and imaging applications. Moreover, as mentioned previously, the presence of an *ortho*- (trifluoromethyl)phenyl group next to the boron atom is essential to allow the complete release of the fluorophore.³⁴ Hence, probes **10** and **11** were synthesized following a common strategy from aldehyde **5** as a valuable building block (Scheme 2A). In brief, after the protection of 4-bromobenzyl alcohol, compound **1** was subjected to a Miyaura⁴² coupling reaction to afford the boronic acid pinacol ester **2** in 86% yield. This compound was then treated with *ortho*- (trifluoromethyl)phenyllithium, resulting from the reaction of 2-bromobenzotrifluoride with *sec*-butyllithium at -78°C, followed by acidic treatment and subsequent treatment with picolinic acid⁴³ providing a mixture of borinic esters **3** and **4**, separable by flash column chromatography, in 58 and 32% yield, respectively. THP-protected alcohol **4** was easily converted to unprotected compound **3** under mild acidic conditions. Finally, Dess-Martin oxidation⁴⁴ of alcohol **3** yielded the desired aldehyde **5** in a good 90% yield. Indolium species **6**⁴⁵ and **7**⁴⁶ were then subjected to a Knoevenagel-type condensation with **5** to afford the expected borinic esters **8** and **9** in good yields. Displacement of the picolinic acid ligand was performed using zinc(II) acetate dihydrate⁴⁷ in CH₂Cl₂/MeOH, affording the two borinic acid-based fluorogenic probes **10** (*HEtBori*) and **11** (*HSBori*) in a moderate 42% and a good 84% yield, respectively.
<figure><img src="image_4.png" /></figure>

**Scheme 2.** A. Synthesis of borinic acid-based fluorogenic probes. *Reagents and conditions:* (a) DHP (1.2 equiv), CSA, CH₂Cl₂, rt, 18 h, quant.; (b) (PinB)₂, Pd(dppf)Cl₂·CH₂Cl₂ (0.1 equiv), AcOK (3 equiv), 1,4-dioxane, reflux, 18 h, 86%; (c) sec-BuLi (1.8 equiv), o-CF₃PhBr (1.2 equiv), THF, -78°C, 2 h, then HCl 2 M in Et₂O (9 equiv), then picolinic acid (1.2 equiv), THF, rt, overnight; (d) p-TsOH·H₂O (10 mol%), MeOH, 7 h, 71%; (e) Dess-Martin periodinane (1.2 equiv), CH₂Cl₂, rt, 1.5 h, 90%; (f) EtI (2 equiv), CH₃CN, 60°C, 3 days, 91% or 1,3-propanesultone (1 equiv), toluene, reflux, 24 h, 72%; (g) **5** (1.1 equiv), piperidine (0.5 equiv), EtOH, reflux, overnight; (h) Zn(OAc)₂·2H₂O (1.5 equiv), CH₂Cl₂/MeOH 95:5, rt, 6 h. **B.** Synthesis of boronic acid-based probes and fluorophores. *Reagents and conditions:* (a) 4-formylphenylboronic acid (1.2 equiv), AcONa (1.5 equiv), Ac₂O, 45°C, 18 h; (b) 4-hydroxybenzaldehyde (1.2 equiv), piperidine (0.5 equiv), EtOH, reflux, overnight.

Fluorophores **14**<sup>48</sup> and **15**<sup>49</sup>, which should result from oxidation of borinic acids **10** and **11**, respectively, were also synthesized as controls according to the same procedure (i.e., piperidine in absolute ethanol) involving 4-hydroxybenzaldehyde in 94 and 52% yield, respectively (Scheme 2B). The corresponding boronic acid derivatives **12** (**HEtBoro**) and **13** (**HSBoro**) were also synthesized. Applying these previous conditions from 4-formylphenylboronic acid proved less effective to get **12** and **13**. However, reactions of indolium **6** and **7** with 4-formylphenylboronic acid in the presence of sodium acetate and acetic acid anhydride afforded the expected products in good yields (Scheme 2B).

## Kinetic studies, stability and selectivity of probes toward ROS

In order to investigate the response of probes **HEtBori** and **HSBori** toward hydrogen peroxide, UV-absorption and fluorescence emission spectra were recorded in the presence of increasing concentrations of H₂O₂ (Figure 1). In the absence of H₂O₂, both probes displayed characteristic absorption bands at 420-425 nm. However, upon the addition of H₂O₂, these absorption bands gradually decreased, while a new red-shifted absorption band at 520-530 nm emerged (Figure 1, A and B). Concomitantly, upon excitation at 500 nm, a fluorescence emission band at 560 nm gradually increased, revealing the formation of the corresponding fluorophores **14** and **15** (Figure 1, C and D). We observed no significant changes in fluorescence emission when more
than 1 equivalent of H₂O₂ was used, indicating the complete consumption of probes and the full release of fluorophores.

<figure><img src="image_5.png" /><figcaption>Figure 1. Evolution of UV-Vis absorption spectra of HEtBori (A) and HSBori (B) probes (30 µM) in the presence of different concentrations of H₂O₂ (0-40 µM) in HBSS buffer (pH 7.4, 0.3% DMSO). Absorbance intensities were recorded from 350 to 600 nm in HBSS buffer (pH 7.4, 0.2% DMSO) after 2 min. Fluorescence emission spectra of HEtBori (C) and HSBori (D) probes (15 µM) in the presence of different concentrations of H₂O₂ (0-40 µM). Fluorescence emission intensities were recorded from 530 to 700 nm at λ<sub>ex</sub> = 500 nm in HBSS buffer (pH 7.4, 0.2% DMSO) after 2 min.</figcaption></figure>

To confirm these observations, the selectivity of the probe oxidation reaction upon exposure to H₂O₂ was then examined using ¹H NMR spectroscopy. For an illustrative purpose, the results of **HEtBori** oxidation are displayed in Figure 2. Upon the addition of one equivalent of H₂O₂, the complete consumption of the probe was observed within the first 2 minutes. As expected, only a single oxidative rearrangement occurred, leading to the exclusive formation of the corresponding fluorophore **14** and 2-(trifluoromethyl)phenylboronic acid. Neither 2-(trifluoromethyl)phenol nor compound **12**, resulting from the other oxidative rearrangement, were detected within the ¹H NMR detection limit.
<figure><img src="image_6.png" /><figcaption>**Figure 2.** Oxidation of **HEtBori** upon addition of one equivalent of **H₂O₂** in deuterated PBS buffer 1X (pH 7.4) monitored by ¹H NMR spectroscopy (600 MHz). ¹H NMR spectra of **HEtBori** (Bottom), **HEtBori** with **H₂O₂** (1 equiv) after 2 minutes, **14**, 2-(trifluoromethyl)phenylboronic acid, 2-(trifluoromethyl)phenol and **12** as expected compounds.</figcaption></figure>

We further determined the second-order rate constants associated with the borinic acid oxidation as 8.4×10³ ± 0.1 M⁻¹.s⁻¹ for **HEtBori** and 5.6×10³ ± 0.2 M⁻¹.s⁻¹ for **HSBori** at 293K (see Table S1, *SI Appendix*). These values illustrate the high reactivity of the borinic acid trigger toward H₂O₂ and are of a similar order of magnitude to those we have previously reported.³²,³⁴

The stability was also evaluated under cell culture conditions and was monitored in Hank's balanced salt buffer (HBSS) at different times by HPLC chromatography (see Figure S5, *SI Appendix*). Both probes remained stable for up to 2.5 hours, which is significantly longer than the period typically required for cellular experiments.

Subsequently, we evaluated the selectivity of **HEtBori** and **HSBori** probes toward different reactive oxygen species. Fluorescence emission intensity was recorded at 560 nm upon treatment with numerous relevant ROS, including H₂O₂, *tert*-butyl hydroperoxide (TBHP), superoxide radical anion or hydroxyl and *tert*-butoxy radical (Figure 3). The fluorescence
intensity did not significantly increase in the presence of all these species, except for H₂O₂, revealing the good selectivity of borinic acid trigger with respect to H₂O₂. In addition, we observed a good linear relationship between the fluorescence intensity and the concentration of H₂O₂ from 0 to 10 µM. The limit of detection (LOD) values for **HEtBori** and **HSBori** were estimated to be 1.15 and 0.63 µM, respectively (see Figure S7, *SI Appendix*).

<figure><img src="image_7.png" /><figcaption>**Figure 3.** Selectivity of probes **HEtBori** (A) and **HSBori** (B) toward various reactive oxygen species (ROS). Fluorescence emission intensity of probes (5 µM) to 100 µM of various ROS in HBSS buffer (pH 7.4 containing 0.05% of DMSO) was recorded at λ<sub>em</sub> = 560 nm (λ<sub>ex</sub> = 500 nm) at 2, 5, 10, 20, 30 and 60 min at 25°C. Experiments were performed in triplicate and the results were averaged. H₂O₂ and *tert*-butyl hydroperoxide (TBHP) were delivered from solid H₂O₂.urea and from 70% aqueous solution, respectively. Hydroxyl radical (*HO*') and *tert*-butoxy radical (*t*-BuO') were generated by the Fenton reaction of 2.5 mM *Fe*²⁺ with 100 µM H₂O₂ or 100 µM TBHP, respectively. Superoxide (*O*₂²⁻) was produced from xanthine/xanthine oxidase system.</figcaption></figure>

It is worth mentioning that with bisulfite (HSO₃⁻) or peroxynitrite (ONOO⁻), we observed the disappearance of the absorption band at 420-425 nm at high concentration, but no characteristic absorption band at 520 nm assigned to the corresponding fluorophores was detected (see Figure S6, *SI Appendix*). We believe that bisulfite ion proceeds to a Michael addition on the hemicyanine moiety as recently stated in the literature,⁵⁰⁻⁵² while ONOO⁻ oxidizes the activated double bond to furnish predominantly non-fluorescent aldehydes.⁵³⁻⁵⁵

*Detection of extracellular hydrogen peroxide - Comparison with AmplexTM UltraRed*

We then turned our attention to the evaluation of **HEtBori** and **HSBori** probes for detecting extracellular endogenously produced hydrogen peroxide. For this purpose, we used the PLB985 myeloid cell line differentiated in neutrophil-like cells.⁵⁶ In those cells, activating the NADPH oxidase (NOX2) at the plasma membrane can be induced by adding a soluble stimulus such as phorbol 12-myristate 13-acetate (PMA). PMA induces phosphorylation pathways, which trigger the assembly of NADPH oxidase and the endogenous production of superoxide radical anion (O₂⁻) in a few minutes. Once produced, O₂⁻ dismutates to form H₂O₂ in the extracellular medium either spontaneously or through a reaction catalyzed by superoxide dismutase (SOD). We first compared the relative oxidation kinetic rates of **HSBori** and the corresponding boronic acid derivative **HSBoro 13** in the presence of PMA-activated PLB985 cells in HBSS buffer. As anticipated, the oxidation of the borinic acid trigger is faster than for the boronic acid one (Figure 4A).
Furthermore, we compared the real-time responses of **HEtBori** and **HSBori** probes with the standard Amplex™ UltraRed assay (AR), commonly used to detect extracellular hydrogen peroxide produced endogenously in biological environments. This assay relies on the H₂O₂-mediated oxidation of 10-acetyl-3,47-dihydropenoxazine, which is catalyzed by horseradish peroxidase (HRP), resulting in the formation of resorufin as a red fluorescent product. However, the main drawback is that the efficiency of this sensor relies on the stability and the activity of HRP enzyme. We believed that developing tools capable of achieving rapid response kinetics without requiring the use of any enzyme would present an interesting alternative. Hence, fluorescence signals of **HEtBori**, **HSBori**, and Amplex™ UltraRed/HRP were collected (see Figure S7, *SI Appendix*) and normalized upon PMA addition (Figure 4B). In order to record fluorescence intensities in the same range of values for all probes, the gain of the detector has been set at a lower value for the Amplex™ UltraRed assay than for **HSBori** and **HEtBori** probes, because fluorophores **14** ($\Phi_F$ = 0.19) and **15** ($\Phi_F$ = 0.21) display lower brightness than resorufin ($\Phi_F$ = 0.74).⁵⁷ Nevertheless, after normalization, all the kinetics are superimposable and the nature of the alkyl chain on the indolenine moiety does not affect the oxidation rate (Figure 4B). These observations suggest that probes **HSBori** and **HEtBori** are suitable surrogates for the Amplex™ UltraRed/HRP assay, and they provide the significant advantage of detecting extracellular H₂O₂ without the need for any enzyme.
<figure><img src="image_8.png" /><figcaption>**Figure 4.** Representative experiment of extracellular H₂O₂ detection upon activation of 10,000 PLB985 cells by PMA in 200 µL of HBSS buffer (pH 7.4) at 37°C. Time-dependent variation of fluorescence intensities of 15 µM of **HSBori** and **HSBoro** (A), **HSBori** and **HEtBori** compared to a mixture Amplex™ UltraRed (15 µM)/HRP (0.1 U.mL⁻¹) after normalization (B) (non-normalized data are available in Figure S8, *SI Appendix*). [PMA] = 1 µM. Lines are not true fits and are here for eye guidance only. Time-dependent variation of fluorescence intensities of **HSBori** (15 µM) or a mixture Amplex™ UltraRed (15 µM)/HRP (0.1 U.mL⁻¹) upon addition of DPI (50 µM) in (C) and (D), catalase (2 mg.mL⁻¹) or SOD (50 µM)/catalase (2 mg.mL⁻¹) in (E) and (F) (Figure S9, *SI Appendix*) displayed the average of 3 experiments). [PMA] = 100 nM. Arrows indicate the time of addition.</figcaption></figure>

To assess the nature of the fluorescence emission turn-on, we conducted several assays including the inhibition of NADPH oxidase activity by introducing diphenyleneiodonium
chloride (DPI), the dismutation of superoxide radical anion to hydrogen peroxide using SOD, and the conversion of hydrogen peroxide into water using catalase. These assays were first carried out with Amplex™ UltraRed/HRP system as control experiments. As expected, in all cases, the fluorescence emission intensity was stopped upon the addition of these scavengers, confirming the turn-on fluorescence accurately reflects the kinetics of hydrogen peroxide production by the NADPH oxidase (Figure 4, D and F). Then, analogous experiments were carried out using **HSBori** probe (Figure 5, C and E). To our delight, similar trends were observed *i.e.*, the addition of DPI, extracellular catalase, or SOD/catalase also disrupted the fluorescence signal increase. All these results clearly support the fact that the borinic acid trigger is a valuable tool for real-time and specific monitoring of H₂O₂ produced upon NADPH oxidase activation under physiological conditions, and can effectively compete with the Amplex™ UltraRed/HRP assay gold standard. In addition, during the optimization of these experiments, we also observed that the fluorescence emission was not completely suppressed upon addition of increasing amounts of catalase, showing that the reaction kinetics of **HSBori** with H₂O₂ can compete with the kinetics of catalase (see Figure S10, *SI Appendix*). This highlights the extremely high reaction rates of the borinic acid-based probes synthesized in this study.

## Detection of intracellular hydrogen peroxide

With these probe designs, we expected specific detection of intracellular H₂O₂ since hemicyanine scaffolds have been proven to target mitochondria.³⁶⁻³⁸ To evaluate intracellular H₂O₂ detection, we performed flow cytometry experiments. For this purpose, PLB985 cells were incubated with probes and PMA for 60 min. We detected a very weak signal for **HSBori** probe, barely superior to the autofluorescence of the cells. However, a 22-fold change in green fluorescence emission intensity was observed in the presence of **HEtBori** probe (Figure 5). These results suggest that **HSBori** predominantly detects extracellular H₂O₂ like the Amplex™ UltraRed/HRP system, while **HEtBori** is more effective for sensing intracellular H₂O₂. This difference could be explained by the chemical structure of probes. Specifically, the introduction of a sulfonate group for increasing water solubility of **HSBori** could reduce its lipophilicity and, consequently, the cell permeability of this sensor compared to **HEtBori**. Interestingly, a similar 20-fold amplification in fluorescence emission was obtained for **HEtBori** after 5 minutes even without PMA-mediated activation of NADPH oxidase.
<figure><img src="image_9.png" /><figcaption>**Figure 5.** Flow cytometry analysis of the fluorescence of PLB985 cells exposed at 15 µM of **HEtBori** or **HSBori** upon activation by PMA (average of 2 experiments ± SD, [PMA] = 100 nM). The last bar represents the median of the cell fluorescence intensity 5 min after the addition of **HEtBori**.</figcaption></figure>

This result was further confirmed using microscopy (Figure 6A and Figure S11A, *SI Appendix*). The addition of **HEtBori** probe to non-activated cells led to an increase in fluorescence emission intensity within 3 minutes. Additionally, we observed higher fluorescence intensity in the cellular compartment rather than in the culture buffer, suggesting that the lipophilic cationic **HEtBori** probe effectively penetrates cell membranes. Since the cells are not PMA-stimulated, we hypothesize that, once inside, the probe may accumulate in mitochondria due to its ammonium moiety and be subsequently oxidized by mitochondrial respiratory chain-produced H₂O₂. Co-localization experiments were therefore performed with MitoView™ 633, a far-red fluorescent dye suitable for staining mitochondria. Results showed that fluorescence of both probes were mainly distributed in the same region within cells (Figure 6B and Figure S11B, *SI Appendix*) and suggest **HEtBori** can detect basal levels of hydrogen peroxide in cells.
<figure><img src="image_10.png" /><figcaption>**Figure 6.** (A) Wide-field time-lapse images of PLB985 cells in HBSS buffer (pH 7.4) with 30 µM **HEtBori**, fluorescence images were acquired using a YFP filter set ($\lambda_{em} = 500/25$ nm, $\lambda_{ex} = 542/27$ nm, top row), bright field (bottom row). Zoom on the two white regions of interest Figure S11A. (B) Confocal images of PLB985 cells in HBSS buffer (pH 7.4) incubated with **HEtBori** (30 µM) and **MitoView™ 633** (0.2 µM). For controls, see Figure S11B.</figcaption></figure>

## CONCLUSION

In this study, we designed new selective H₂O₂-sensitive probes relying on a borinic acid trigger featuring photophysical properties suitable for cellular studies. We chose a hemicyanine scaffold, which was successfully functionalized with the borinic trigger, providing two new fluorogenic probes in a few synthetic steps. Reactivity, photophysical characterization, and selectivity studies were carried out and confirmed that these probes retained: i) superior kinetic performance over their boronic acid counterparts, ii) regioselective cleavage leading to exclusive release of the fluorophore with photophysical properties advantageous for cellular imaging, and iii) high selectivity for H₂O₂. Application of these probes in cellular models demonstrated their efficacy in real-time H₂O₂ monitoring. Notably, the kinetics of these probes was comparable to that of Amplex™ UltraRed/HRP, the current gold standard for such studies, rendering these new probes the first enzyme-free chemical alternatives to match the performance of the Amplex™ UltraRed/HRP system. Furthermore, the results showed an ability to detect hydrogen peroxide both extracellularly and intracellularly, depending on the structure of the alkyl chain on the indolenine core. Finally, one of the probes (**HEtBori**) is sensitive enough to detect basal levels of intracellular H₂O₂. We believe these borinic acid-based probes represent a new and efficient tool allowing the development of new devices for a better understanding of H₂O₂-mediated signaling processes.

## SUPPORTING INFORMATION

The Supporting Information is available free of charge via the Internet.
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ACKNOWLEDGMENTS

We thank the Ministère de la Recherche et de l'Enseignement Supérieur for a PhD grant to M.P.
This work was partially funded by the French Agency for Research (Grant ANR21-CE07-0047-
03 Boriprobe) and Bioprobe, an interdisciplinary program of the University of Paris Saclay for
fundings. We also acknowledge the Graduate School of Chemistry University Paris Saclay for
financial support to K. M and A. Chevalier for kindly providing us MitoView™ 633 for
confocal experiments. The present work has benefited from Imagerie-Gif core facility
supported by the French Agency for Research (ANR-10-INBS-04/FranceBioImaging; ANR-
11-IDEX-0003-02/ Saclay Plant Sciences)