# Fluorogenic heptazines functionalized with tetrazine rings for conjugation through inverse Electron Demand Diels-Alder reaction

Mewenn Yvin, Elise Michel, Rémi Métivier, Laurent Galmiche, Lucas

Frédéric, Clémence Allain, Pierre Audebert

► To cite this version:

Mewenn Yvin, Elise Michel, Rémi Métivier, Laurent Galmiche, Lucas Frédéric, et al. Fluorogenic heptazines functionalized with tetrazine rings for conjugation through inverse Electron Demand Diels-Alder reaction. Dyes and Pigments, 2025, 236, pp.112666. 10.1016/j.dyepig.2025.112666. hal-05397487

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# Fluorogenic heptazines functionalized with tetrazine rings for conjugation through inverse Electron Demand Diels-Alder reaction

Mewenn Yvin<sup>a</sup>, Elise Michel<sup>a</sup>, Rémi Métivier<sup>a</sup>, Laurent Galmiche<sup>a</sup>, Lucas Frédéric<sup>a,** ID</sup>, Clémence Allain<sup>a,* ID</sup>, Pierre Audebert<sup>a,b</sup>

<figure><img src="image_3.png" /></figure>

## ARTICLE INFO

Keywords:

Heptazine

Tetrazine

Click chemistry

Diels-Alder reaction

Fluorogenic probes

## ABSTRACT

Fluorogenic probes that can selectively target molecules or materials thanks to click chemistry are promising tools for labelling or functionalization. Heptazines are heterocycles that gather appealing electrochemical and photophysical properties, combining high photooxidation potentials with long fluorescence lifetimes. However, selective grafting of molecular heptazines on a substrate is still underdeveloped. In this work, we report the synthesis, photophysical and electrochemical properties of two heptazine derivatives conjugated with one and three tetrazine rings respectively. These heptazines conjugated to tetrazines are very weakly fluorescent. We demonstrate that they can react in a fast manner with a strained alkyne, through an Inverse Electron Demand Diels-Alder reaction. This reaction has been followed thanks to a home-built photokinetics setup: in a timecale of a few seconds blue emitting heptazine pyridazine conjugates are generated with an 8.6 to 13.6 fluorescence turn-on.

## 1. Introduction

Heptazines are triangular tricyclic aromatic derivatives with high nitrogen content. As indicated by their name, they merge seven nitrogen atoms distributed in an alternate pattern in the periphery of the structure and in the central position, along with six carbon atoms [1]. Due to the aromatic character of the core, heptazines are fluorescent, emitting in the UV or visible part of the electromagnetic spectrum depending on their substituents [1]. Furthermore, due to the high electron-deficient character of these polycyclic derivatives, the production of quite stable radical anions can occur with low reduction potentials reaching –1, 23 V (vs SCE) [2]. Several applications have been developed based on these properties, such as catalysis [3], water splitting [4], liquid crystal assemblies [5] or optoelectronics [6]. Nonetheless, the synthesis of molecules bearing highly functionalized heptazine cores has been impeded by the few building blocks available. Until 2019, trichloroheptazine was the only starting material for electrophilic and nucleophilic aromatic substitutions ($S_E$Ar and $S_N$Ar) [7]. However, it requires harsh and dangerous synthesis conditions ($PCl_5$ at 140 °C) and has low stability, especially in the presence of water [8]. Recently, our

group has reported a new platform named TDPH (for 2,5,8-Tris(3, 5-Diethyl-Pyrazolyl)-Heptazine), which can be prepared under mild conditions (ball milling, in the presence of a catalytic amount of PTSA) [2]. This intermediate is bench-stable for several weeks when protected from light and enables to perform $S_N$Ar reactions. A number of new molecules have been derived from TDPH, with a variety of linking heteroatoms like sulfur, nitrogen or oxygen [2,9].

On the other hand, s-tetrazines are six-membered heterocyclic rings that are also redox active and sometimes fluorescent depending on their substitution pattern [10]. This family of molecules have attracted considerable attention for their ability to react with various alkenes and alkynes through Inverse Electron Demand Diels-Alder (IEDDA) reaction. This reaction was originally developed to access various pyridazine motifs and has been more recently recognized as a « click » reaction that allows conjugation to different molecules or materials [11,12]. This has led to the development of several fluorophore-tetrazine conjugates such as aza-Bodipy [13] or two-photon excitable AcriPy conjugates [14]: when conjugated to the tetrazine ring, the fluorophore is usually quenched either by intramolecular energy transfer or by photoinduced electron transfer. A strong fluorescence enhancement upon consumption

<footnote><sup>a</sup> Université Paris-Saclay, ENS Paris-Saclay, CNRS, PPSM, Gif-sur-Yvette, 91190, France</footnote>

<footnote><sup>b</sup> XLIM, UMR CNRS, 7252 123, avenue Albert Thomas, 87060, Limoges CEDEX, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>** Corresponding author.</footnote>

<footnote>E-mail addresses: lucas.frederic@ens-paris-saclay.fr (L. Frédéric), clemence.allain@ens-paris-saclay.fr (C. Allain).</footnote>
of the tetrazine by IEDDA reaction allows specific imaging of the labeled
targets. Besides biological chemistry, IEDDA reactions with tetrazine
derivatives have been used in the field of polymers and materials science
[15]. In particular, some of us have synthesized a molecule bearing two
tetrazine anchoring groups and a tetrazine redox unit and successfully
grafted it onto reduced graphene oxide by IEDDA reaction. The resulting
composite material exhibits an enhanced capacitance compared to
pristine materials [16].

In order to study the influence of the redox unit used and of the
number of anchoring tetrazines used for grafting, our group recently
prepared an heptazine functionalized with three chloro-alkoxy-tetrazine
derivatives, but this resulted in non-covalent interactions between
heptazine-tetrazine dyads and graphene oxide [17]. In this context, we
decided to develop new conjugates bearing tetrazines that are known to
be highly reactive for IEDDA reactions [18]. Thus, we herein report the
synthesis and study of heptazines functionalized with phenyltetrazine
(s). These molecules present a significant decrease in luminescence in-
tensity, however, the pending tetrazine arms are able to react with cyclic
strained alkyne through IEDDA reaction in a rapid manner to give the
corresponding pyridazine. Thanks to this click reaction, an upsurge of
fluorescence arising from the heptazine core can be noted confirming
the fluorogenic character of these new conjugates.

## 2. Results and discussion

### a. Synthesis

Our previous studies have highlighted TDPH as a reagent in S<sub>N</sub>Ar reactions [2,9]. Nevertheless, in order to improve the rate of the reaction, tests have been performed with imidazole. In this case, imidazole would first substitute the pyrazole of TDPH, leading to an intermediate that can react through the S<sub>N</sub>Ar reaction. This strategy was considered for reactions with weak nucleophiles that could difficultly react directly with TDPH. As a proof of concept, a first attempt was realized in the presence of imidazole and benzylic alcohol in dichloromethane under reflux (see Scheme 1 and SI for details), giving the product Hept [OCH<sub>2</sub>Ph]<sub>3</sub> in moderate yield (33 %, Scheme 1 Conditions B). From this promising result, we envisaged to work with the already reported HOCH<sub>2</sub>PhTz [19]. Although the expected product Hept [OCH<sub>2</sub>PhTz]<sub>3</sub> was observed by NMR analysis of the crude reaction, insolubility issues as well as imidazole co-elution prevented isolation. Then, solvent modification was considered to increase the reaction temperature and chloroform was used instead of dichloromethane. Nonetheless, the

formation of a side-product was noticed and isolated (see Scheme 1, Conditions A). After careful investigation by ¹H and ¹³C NMR as well as mass spectrometry, we identified two products corresponding to the Hept [OCH₂PhTz](OEt)₂ (31 % yield) and Hept(OEt)₃ (39 % yield). These peculiar structures with pendant ethoxy arms arise from the reaction with ethanol, which is used as a stabilizing agent in the solvent of the synthesis. In order to circumvent insolubility and purification issues, and to obtain Hept [OCH₂PhTz]₃, the reaction conditions were modified by using N,N-dimethylaminopyridine as base at the dichloromethane boiling point (see Scheme 1, Conditions C). This last strategy enabled to obtain the expected product in 30 % yield after purification using only a moderate excess of alcohol reagent.

### b. Photophysical characterizations

With these products in hand, their photophysical properties were
studied in dichloromethane solution at room temperature. As depicted

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Absorption spectra of HOCH₂PhTz (black), Hept[OCH₂Ph]₃ (red), Hept [OCH₂PhTz](OEt)₂ (purple) and Hept [OCH₂PhTz]₃ (blue) in dichloromethane solution. Inset: zoom between 300 and 600 nm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>**Scheme 1.** Synthesis of tri-substituted heptazines. Conditions A: 1) 1H-imidazole (3.3 equiv.), CHCl₃ stab. EtOH, 60 °C, 20 min, 2) Benzylic alcohol 4-tetrazine (4.5 equiv.), 60 °C, 24 h. Conditions B: 1) 1H-imidazole (3.3 equiv.), CH₂Cl₂, 40 °C, 20 min, 2) Benzylic alcohol (4.6 equiv.), 40 °C, 4 h. Conditions C: DMAP (6.3 equiv.), Benzylic alcohol 4-tetrazine (4.5 equiv.), CH₂Cl₂ 40 °C, 6 h.</figcaption></figure>
in Fig. 1, for the starting materials HOCH₂PhTz, Hept[OCH₂PhTz](OEt)₂ and Hept[OCH₂PhTz]₃, a weak and structured band centered at 545 nm can be observed. It corresponds to the characteristic n-π* transition of tetrazine and is not observed for Hept[OCH₂Ph]₃. As expected, the molar absorption coefficient is higher at this wavelength for Hept [OCH₂PhTz]₃, reaching 1450 L·mol⁻¹·cm⁻¹ compared to Hept [OCH₂PhTz](OEt)₂ (630 L·mol⁻¹·cm⁻¹) and HOCH₂PhTz (750 L·mol⁻¹·cm⁻¹), resulting from the three pendant tetrazines attached to the heptazine core. For Hept[OCH₂Ph]₃, Hept[OCH₂PhTz](OEt)₂ and Hept[OCH₂PhTz]₃, the heptazine part displays a first absorption band located around 330 nm corresponding to the forbidden n-π* transition of the heptazine core, overlapping with the π-π* transition arising from tetrazine part(s) for Hept[OCH₂PhTz](OEt)₂ and Hept[OCH₂PhTz]₃ (see Table 1). A second intense band originating from the heptazine can be observed around 275 nm, overlapping with the band resulting from the 4-phenyl-tetrazine unit and explaining the important increase in the molar absorption coefficient for Hept[OCH₂PhTz]₃. For Hept [OCH₂Ph]₃, TD-DFT calculations predict (see Fig. S24 and Table S3) a first forbidden transition at 320 nm corresponding to a HOMO-LUMO transition, which is in good agreement with the experimental absorption spectrum. Concerning Hept[OCH₂PhTz]₃ the first three electronic transitions are computed by TD-DFT at 556 nm (see Table S4). Analysis of the orbitals implied in these transitions (Fig. S25) reveals that they correspond to the n-π* transitions on the three tetrazine rings. In this compound, the n-π* transition purely located on the heptazine core is also computed but at a higher energy (321 nm), which is fully consistent with the experimental absorption spectrum. This is also fully consistent with the fact that Hept[OCH₂PhTz]₃ has the same dark excited state as HOCH₂PhTz (see below).

The fluorescence was then investigated in the same solvent for each heptazine bearing benzylic alcohol moieties. As expected, and in regard to the previous results obtained with alkoxy derivatives, Hept [OCH₂Ph]₃ exhibits blue fluorescence with a maximum at 416 nm, quantum yield (PLQY) of 11 % and a long fluorescence decay time (average fluorescence lifetime of 90.7 ns, see Table 1). However, for Hept[OCH₂PhTz](OEt)₂ and Hept[OCH₂PhTz]₃, the fluorescence intensity is very weak (PLQY < 1 %) but two signals can be measured (see Fig. 2). A first emission band attributed to the heptazine core is centered around 405 nm and 421 nm for Hept[OCH₂PhTz](OEt)₂ and Hept [OCH₂PhTz]₃ respectively. This slight shift probably results from the number and the nature of arms linked to the heptazine core. A second weak structured emission band, attributed to the tetrazine, is measured between 550 nm and 650 nm for both compounds, and is also observed for HOCH₂PhTz. The position and weak intensity of this emission band are characteristic of 3-aryl-1,2,4,5-tetrazine derivatives [19,20]. The difference in intensity observed for heptazine emission between Hept [OCH₂Ph]₃ on one side and Hept[OCH₂PhTz](OEt)₂ and Hept [OCH₂PhTz]₃ on the other side could be due to the presence of intramolecular energy transfer from heptazine to tetrazine. This assumption is supported by the spectral overlap between heptazine emission and

<caption>**Table 1**</caption>

Photophysical properties of investigated compounds in dichloromethane solution<sup>a</sup> average fluorescence lifetimes (see ESI).

<table><thead><tr><th></th><th>λ<sub>abs</sub>/<br>nm</th><th>ε/L·<br>mol<sup>-1</sup>·cm<sup>-1</sup></th><th>λ<sub>em</sub>/<br>nm</th><th>Stokes<br>shift/<br>cm<sup>-1</sup></th><th>Φ</th><th>τ/ns<sup>a</sup></th></tr></thead><tbody><tr><td>HOCH<sub>2</sub>PhTz</td><td>540</td><td>795</td><td>577</td><td>1190</td><td>&lt;0.01</td><td>0.64</td></tr><tr><td>Hept<br>[OCH<sub>2</sub>Ph]<sub>3</sub></td><td>342<br>(sh)</td><td>610</td><td>408</td><td>4730</td><td>0.11</td><td>90.7</td></tr><tr><td>Hept<br>[OCH<sub>2</sub>PhTz]<br>(OEt)<sub>2</sub></td><td>540<br>330<br>(sh)</td><td>660<br>1780</td><td>579<br>405</td><td>1250<br>5610</td><td>&lt;0.01</td><td>12.9<br>1.04</td></tr><tr><td>Hept<br>[OCH<sub>2</sub>PhTz]<sub>3</sub></td><td>540<br>330<br>(sh)</td><td>1580<br>4615</td><td>578<br>421</td><td>1220<br>6550</td><td>&lt;0.01</td><td>5.26<br>0.81</td></tr></tbody></table>

<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Normalized fluorescence spectra of Hept[OCH₂Ph]₃ (red), Hept [OCH₂PhTz](OEt)₂ (purple) and Hept[OCH₂PhTz]₃ (blue) in dichloromethane solution. Excitation wavelength: 290 nm. Dichloromethane Raman peak can be observed at 319 nm for Hept[OCH₂PhTz](OEt)₂ and Hept[OCH₂PhTz]₃ due to low intensity of the fluorescent signals. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

tetrazine absorption (see Supporting Information). Furthermore, intermolecular energy transfer is unlikely to happen in the measurement conditions (dilute solutions). Finally, it is important to note that the presence of only one pendant tetrazine unit appears to be sufficient to induce a significant energy transfer and thus drastically decrease the fluorescence intensity arising from the heptazine core.

### c. Electrochemical characterizations

Electrochemical features were also measured for all compounds (see Fig. 3). The electrochemical characteristics of Hept[OCH₂PhTz](OEt)₂

<figure><img src="image_7.png" /><figcaption>**Fig. 3.** Cyclic voltammetry curves of Hept[OCH₂Ph]₃ (red), Hept[OCH₂PhTz] (OEt)₂ (purple) and Hept[OCH₂PhTz]₃ (blue) recorded at glassy carbon electrode at a concentration of 1 mM in dichloromethane solution under inert atmosphere using [nBu₄N][PF₆] 10⁻¹ M as supporting electrolyte. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>
display both reductions of the tetrazine and heptazine rings with equal currents, as expected. While the tetrazine reduction is reversible, the heptazine reduction is not, probably due to dimerization as already proposed for trialkoxyheptazines [9]. On the other hand, for Hept [OCH₂PhTz]₃ the reduction of the three tetrazine rings is observed as a broad peak, because of the spanning of the reduction potentials due to charge repulsion, as long as the three tetrazine rings are reduced one after the other. On the other hand, the heptazine ring reduction is not observable, certainly because of the very slow electron transfer induced by the triple negative charge already present on the three tetrazine rings. The triple negative charge renders the heptazine reduction very slow, with as a probable consequence a very flat signal close to the electrolyte reduction wall, and thus unobservable.

### d. Click chemistry and kinetics

To complete this study, and as a proof of concept of the reactivity in IEDDA reactions, Hept[OCH₂PhTz](OEt)₂ and Hept[OCH₂PhTz]₃ were reacted with bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN-OH) (see Scheme 2). In the presence of a slight excess of BCN-OH (1.5 equiv. and 4.5 equiv. for Hept[OCH₂PhTz](OEt)₂ and Hept[OCH₂PhTz]₃ respectively) in dichloromethane at room temperature, the resulting pyridazines were observed and characterized by mass spectrometry (see SI), confirming that the reaction can occur under mild conditions.

Thus, care was taken to monitor the reaction progress by UV-vis spectroscopy and spectrofluorometric analysis. First, experiments were carried out with Hept[OCH₂PhTz](OEt)₂, and the absorption spectra show important differences before and after addition of BCN-OH. Indeed, the band attributed to the tetrazine ring (500–560 nm) completely disappears at the end of the reaction. In addition, a moderate decrease in absorbance is observed for the band located between 300 and 350 nm, confirming the overlap of the heptazine and tetrazine

electronic transitions in this spectral region. Fluorescence spectra display an upsurge of intensity signal, multiplied by a factor reaching 13.6. In the meantime, fluorescence average lifetime also increases to reach 79.5 ns. This recovery can be explained by the consumption of tetrazine unit. The formation of pyridazine ring prevents energy transfer due to absence of overlap between absorption of pyridazine and emission of heptazine. It then results in an emission arising from heptazine ring comparable to the one observed with Hept[OCH₂Ph]₃.

Kinetic measurements were also performed thanks to a customized optical set-up allowing the acquisition of absorption and emission spectra at high rates (see Supporting Information for details) to follow the IEDDA reaction in real time. Absorption and emission time profiles were then plotted at the tetrazine maximum absorption wavelengths (533 nm for Hept[OCH₂PhTz](OEt)₂) in order to follow the consumption of this part of the molecule, and in emission at the maximum fluorescence wavelength of the heptazine core (401 nm). These plots show a simultaneous decrease in the tetrazine absorption band and increase in the heptazine fluorescence, as depicted in Fig. 4. The absorption time evolution shows that upon addition of the strained alkyne, half of the starting material is consumed after 15 s and 90 % after a bit more than 1 min (65 s). Similarly, the fluorescence intensity reaches half of the maximum emission after 18 s and 90 % after 79 s of reaction. The kinetic values obtained through absorption and fluorescence measurements are consistent and confirm that the disappearance of the tetrazine absorption correlates with the enhancement of the heptazine fluorescence. It is worth noting that these experiments were performed at room temperature with only a slight excess of BCN-OH (1.5 equiv. per tetrazine ring) showing that the reaction can proceed efficiently in smooth conditions. In these conditions, the absorbance of Hept[OCH₂PhTz](OEt)₂ is best fitted by a second order kinetic model, with the approximation that the concentration in tetrazine is equal to the concentration in BCN-OH at all times, as expected for a bimolecular reaction with a partial first order in

<figure><img src="image_8.png" /><figcaption>**Scheme 2.** Synthesis of click-heptazine derivatives.</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>**Fig. 4.** Top: Absorption and fluorescence spectra of Hept[OCH₂PhTz](OEt)₂ (full line and dashed purple respectively) and Hept[OCH₂PhPyridazine](OEt)₂ (full line and dashed light purple respectively) in dichloromethane solution, before and after addition of BCN-OH. Bottom: Time dependent evolution of absorbance at 533 nm of Hept[OCH₂PhTz](OEt)₂ (purple) and fluorescence at 401 nm of Hept[OCH₂PhPyridazine](OEt)₂ (faded purple) upon addition of BCN-OH in dichloromethane solution. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

tetrazine and strained alkyne (see Supporting Information). The experiments were then repeated with a large excess of BCN-OH (20, 30 and 50 equivalents) and the corresponding data could be fitted using a pseudo-first order model (assuming that [BCN-OH] remains constant throughout the whole experiment, see Fig. S23 and Table S2), despite solubility problems that limit the precision of the determination of the second-order kinetic rate constant. Kinetics measurements were also performed in these conditions (excess of BCN-OH) with the parent HOCH₂PhTz yielding very similar kinetics traces and kinetic rate constant, thus showing that the conjugation to the heptazine core does not significantly affect the tetrazine reactivity in the IEDDA reaction.

The heptazine functionalized with three tetrazines, Hept[OCH₂PhTz]₃, was reacted with BCN-OH and followed through absorption and fluorescence acquisition. As expected, the same trends are observed. On the one hand, a decrease of the absorbance at 539 nm is observed, along with a slight decrease between 300 and 350 nm, resulting from the disappearance of the tetrazine moiety. On the other hand, a slight hypochromic shift of the emission (around 30 nm) is observed together with an increase in fluorescence (by a factor of 8.6) after the click reaction, together with an increase of the average

fluorescence lifetime that reaches 35.7 ns. From a kinetic point of view, the absorbance follows a similar decay observed for Hept[OCH₂PhTz](OEt)₂: 50 % of the starting material is consumed after 14 s and 90 % after 49 s. Similarly to Hept[OCH₂PhTz](OEt)₂, the absorbance can be fitted with a second order kinetic model (see Supporting Information). Surprisingly, the fluorescence intensity does not follow a comparable increase. As shown in Fig. 5 the fluorescence intensity increases more slowly, reaching 50 % of its maximum 37 s after addition of the strained alkyne and 90 % in 104 s. This difference may be due to the fact that all tetrazine units must have reacted with BCN-OH to observe the fluorescence enhancement of the heptazine.

### 3. Conclusion

To conclude, a new series of heptazines bearing tetrazine arms has been synthesized by nucleophilic aromatic substitutions. Different synthetic approaches have been demonstrated by the introduction of an intermediate leaving group or by direct substitution. Absorption and fluorescence studies show the contribution of both heptazine and tetrazine units. Of note, a significant decrease in fluorescence emission can be observed for Hept[OCH₂PhTz](OEt)₂ and Hept[OCH₂PhTz]₃, resulting from energy transfer processes from the heptazine core to the

<figure><img src="image_10.png" /><figcaption>**Fig. 5.** Top: Absorption and fluorescence spectra of Hept[OCH₂PhTz]₃ (full line and dashed blue respectively) and Hept[OCH₂PhPyridazine]₃ (full line and dashed light blue respectively) in dichloromethane solution, before and after addition of BCN-OH. Bottom: Time dependent evolution of absorbance at 539 nm of Hept[OCH₂PhTz]₃ (blue) and fluorescence at 401 nm of Hept[OCH₂PhPyridazine]₃ (faded blue) upon addition of BCN-OH in dichloromethane solution. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>
tetrazine arm(s), leading to non-radiative deactivation pathways. These molecules react through Inverse Electron Demand Diels-Alder reaction with a strained alkyne, proving the potential coupling with various molecules and materials, in particular graphene oxide. In this context, grafting of Hept[OCH₂PhTz](OEt)₂ with one reactive tetrazine and grafting of Hept[OCH₂PhTz]₃ with three reactive arms, can lead to functional materials with distinct properties. This reaction is fast and efficient with these substrates and can be followed by both absorbance and fluorescence. Finally, these molecules can also act as fluorogenic probes, since an increase in fluorescence can be observed upon addition with a strained alkyne paving the way for further applications in labelling.

### CRediT authorship contribution statement

**Mewenn Yvin:** Visualization, Investigation, Formal analysis. **Elise Michel:** Investigation. **Rémi Métivier:** Writing – review & editing, Software, Investigation. **Laurent Galmiche:** Writing – review & editing, Supervision. **Lucas Frédéric:** Writing – original draft, Visualization, Supervision, Formal analysis. **Clémence Allain:** Writing – review & editing, Supervision, Project administration, Conceptualization. **Pierre Audebert:** Project administration, Funding acquisition, Conceptualization.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgments

this work was funded by the ANR (Heptacat grant ANR-21-CE07-0049). We gratefully acknowledge the mass spectrometry platform of ICMMO (Université Paris-Saclay) for MS analysis. We gratefully acknowledge Jean-Frédéric Audibert (PPSM laboratory) for his help with the acquisition of fluorescence decays. This work was performed using computational resources from the “Mésocentre” computing center of Université Paris-Saclay, CentraleSupélec and École Normale Supérieure Paris-Saclay supported by CNRS and Région Île-de-France (https://mesocentre.universite-paris-saclay.fr/).

### Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.dyepeg.2025.112666.

### Data availability

Data will be made available on request.

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