# Electron Relay Boosts Ruthenium-Laccase System Photo-Epoxidation

Claudio Righetti, Antonio Recupido, Mehdi Yemloul, Pierre Rousselot-pailley, Ally Aukauloo, Winfried Leibl, Elise Courvoisier-dezord, A. Jalila Simaan, Thierry Tron, Yasmina Mekmouche

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Claudio Righetti, Antonio Recupido, Mehdi Yemloul, Pierre Rousselot-pailley, Ally Aukauloo, et al. Electron Relay Boosts Ruthenium-Laccase System Photo-Epoxidation. ChemCatChem, 2025, 10.1002/cctc.202501086. hal-05245155

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# Electron Relay Boosts Ruthenium-Laccase System Photo-Epoxidation

Claudio Righetti,[a, b] Antonio Recupido,[a] Mehdi Yemloul,[a] Pierre Rousselot-Pailley,[a] Ally Aukauloo,[c, d] Winfried Leibl,[d] Elise Courvoisier-Dezord,[a] A. Jalila Simaan,[a] Thierry Tron,[*a] and Yasmina Mekmouche*[a]

We report here the light-driven oxidation of olefin to epoxide using a [Ru(bipyridine)₃]²⁺/laccase (Ru¹¹/LAC3)-based system, enhanced by the presence of methyl viologen (MV²⁺) as an electron relay. This approach leads to a fivefold increase in the conversion yield of sodium *p*-styrene sulfonate (pSS) oxidation (from 3% to 14% in the presence of MV²⁺), and favors chemoselectivity toward epoxidation. We demonstrate that laccase plays a crucial role in steering product selectivity, favoring

epoxidation due to its ability to scavenge superoxide (O₂⁻⁻). In contrast, in the absence of laccase, the reaction predominantly yields sodium *p*-benzaldehyde sulfonate (A) and the sodium α-hydroxyketone *p*-phenyl sulfonate (α-HK). Mechanistic insights, supported by reactive intermediate trapping, isotope labeling, and product distribution analysis, suggest a reaction pathway involving both molecular oxygen and water as the oxygen sources.

## 1. Introduction

Light-driven multi-electron catalysis for fuel or chemical synthesis from renewable feedstocks is a key area of research, explored from fundamental to technological perspectives.[1] Inspired by natural photosynthesis, researchers are developing artificial systems to store solar energy in fuels or convert it into high-value chemical feedstocks.[12] In this context, photocatalysis has emerged as a powerful strategy that enables unique reactivity under visible light, allowing broader substrate scope under milder conditions than traditional light-independent methods.[3-5] Enzymatic catalysts are increasingly used in industrial processes due to their advantages, including high chemical and enantioselectivity, and operation under mild, environmentally friendly conditions.[6,7] Consequently, the combination of

photocatalysis and enzymatic catalysis has recently emerged as a promising strategy for sustainable organic synthesis.[8,9]

Various classes of photo-biocatalytic reactions have been developed including: (i) photocatalytic regeneration cascades in which light triggers redox transformations;[10,11] (ii) photoenzymatic reactions involving either natural[12] or artificial cofactor-dependent photoenzyme;[13,14] and (iii) photoenzymatic systems in which photochemical conversion of small molecules is followed by enzyme catalyzed transformation.[15] However, photo-biocatalytic conversions still face challenging issues in order to reach viable synthetic procedures in organic synthesis both on a laboratory or technological level. For instance, the unavoidable necessity of high concentrations of sacrificial electron donors or acceptors, as well as the requirement for controlled atmospheres. Additionally, although highly selective, enzymatic photoinduced processes often rely on costly cofactors such as NADH or FADH₂.[16-18] With the aim of addressing these challenges, we previously demonstrated that a [Ru(bpy)₃]²⁺/laccase/O₂ (Ru¹¹/laccase/O₂) system can effectively couple alkene photooxidation with the light-driven reductive activation of dioxygen.[19,20] In these initial studies, we showed that this approach enables the oxidation of organic substrates using molecular oxygen as the sole oxygen source and the use of reversible electron carrier thereby eliminating the need for a sacrificial electron donor for O₂ activation.[19,20]

Laccase (EC1.10.3.2) is a robust and extensively studied copper-containing oxidoreductase.[21] Substrate oxidation occurs at a surface-exposed blue T1 copper site (I₆₁₀nm = 5600 M⁻¹ cm⁻¹) from which electrons are transferred to a buried trinuclear copper cluster (TNC). This cluster consists of a T2 copper site and an antiferromagnetically coupled T3 copper pair, which together catalyze the reduction of O₂ to water (Equation 1).[22]

$$O_2 + 4 Cu^+ + 4H^+ \rightarrow 4 Cu^{II+} + 2H_2O \quad (1)$$

[a] C. Righetti, A. Recupido, M. Yemloul, P. Rousselot-Pailley, E. Courvoisier-Dezord, A. J. Simaan, T. Tron, Y. Mekmouche
Aix Marseille Univ, CNRS, Centrale Marseille, iSm2, Marseille, France
E-mail: thierry.tron@univ-amu.fr
y.mekmouche@univ-amu.fr

[b] C. Righetti
Université Grenoble Alpes, CNRS, CEA, IRIG, Laboratoire de Chimie et Biologie des Métaux, Grenoble 38000, France

[c] A. Aukauloo
Université Paris-Saclay, UMR 8182, CNRS, Institut de Chimie Moléculaires et des Matériaux d'Orsay, Orsay 91405, France

[d] A. Aukauloo, W. Leibl
Université Paris Saclay, CEA, CNRS, Institut de Biologie Intégrative de la Cellule (I2BC), Gif-sur-Yvette 91198, France

Supporting information for this article is available on the WWW under https://doi.org/10.1002/cctc.202501086

© 2025 The Author(s). ChemCatChem published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
<figure><img src="image_3.png" /><figcaption>**Figure 1.** Hybrid photocatalytic system developed for epoxidation. ribbon model of laccase: copper ions are depicted as spheres. Tested olefins: styrene, sodium *p*-styrene sulfonate (pSS), cyclohexene, cyclooctene; Photocatalyst Ru¹=[Ru(bpy)₃]²⁺, methyl viologen MV²⁺ is the electronic relay used in this study.</figcaption></figure>

Laccases can oxidize a wide range of substrates, including aromatic and inorganic compounds, though their reactivity is constrained by the redox potential of the T1 copper site, which typically ranges from 420 to 790 mV versus NHE. We previously reported on a photo-biocatalytic system combining a recombinant laccase (LAC3 from *Trametes* sp. C30) [23] with Ru-based photosensitive polypyridyl complexes, either in a bimolecular interaction or covalently linked, which resulted in one of the first sustainable photocatalytic platforms for the oxidation of high-redox potential olefins (styrene, *p*-styrene sulfonate (pSS), cyclohexene, and cyclooctene), coupled to the light-driven reduction of O₂ to water (Figure 1). [19,20] Using pSS as a model this system achieved only of 3–5% yield, with selectivity toward epoxide formation. Two key features were noted: (i) LAC3 played a crucial role in steering product selectivity toward epoxidation; (ii) dioxygen acts as both the terminal sustainable electron acceptor and as an oxygen atom donor (Figure 1). Overall, the Ru¹/LAC3/O₂ system behaves as self-regenerating hybrid photocatalyst that operates through a light-driven electron cascade. In this configuration, the ruthenium photosensitizer serves both as an oxidant, generating an alkenyl radical cation, and as a reductant that drives LAC3 reduction.

Photophysical studies revealed that despite a high driving force (E(RuIII/RuII*) - E(CuII/CuI)) = (-0.82 V - 0.68) V versus NHE, ΔG = -1.5 eV) [24] the quantum yield of formation of the charge-separated state (CSS) between the RuIII and the reduced LAC3 (LAC3red) remained very low (below 1%), leading to poor olefin conversion (typically less than 3%–5% for pSS). [19] Under anaerobic conditions, two diffusion-controlled mechanisms have been proposed to account for this limitation: (i) a strong overlap

<figure><img src="image_4.png" /><figcaption>**Scheme 1.** Identified oxidation products. E: epoxide; D: diol; A: aldehyde; and α-HK: α-hydroxyketone.</figcaption></figure>

between [Ru(bpy)₃]²⁺ (RuII*) emission and the oxidized T1 copper site absorption accounting for a large unproductive energy transfer (FRET) and; (ii) a fast charge-recombination process between the two activated species (i.e., LAC3red and RuIII). [24]

To circumvent these limitations, methyl viologen (MV²⁺) was used as an electron relay to enhance the light-driven electron transfer from the excited photosensitizer to the enzyme. MV²⁺ is a well-known oxidative quencher of photoexcited sensitizers; resulting in a long-lived reduced species (MV⁺), which can deliver electrons to the enzyme. [24–27] Almost quantitative photo reduction of laccase was achieved upon addition of MV²⁺ (300 µM), with a > 10-fold increase in reaction rate, highlighting the critical role of this electron relay in managing electron transfer from a sacrificial e-donor (i.e., EDTA) to the enzyme (Equation 2). [24] Introducing air into this system presented challenges, because dioxygen can readily react with MV⁺ to generate superoxide radicals (O₂⁻⁻), [28] which can then disproportionate to form hydrogen peroxide (H₂O₂) (Equations 3 and 4). Under these conditions (EDTA/RuII/MV²⁺/LAC3/O₂), laccase was found to efficiently scavenge O₂⁻⁻ radicals, thereby redirecting the reaction toward harmless H₂O (Equations 5 and 6):

$$4 \text{MV}^{*+} + 4 \text{Cu}^{\text{II}} \rightarrow 4 \text{MV}^{2+} + 4 \text{Cu}^{\text{I}} \quad (2)$$

$$\text{MV}^{*+} + \text{O}_2 \rightarrow \text{MV}^{2+} + \text{O}_2^{*-} \quad (3)$$

$$2 \text{O}_2^{*-} + 2 \text{H}^{+} \rightarrow \text{H}_2\text{O}_2 \quad (4)$$

$$4 \text{O}_2^{*-} + 4 \text{Cu}^{\text{II}} \rightarrow 4 \text{O}_2 + 4 \text{Cu}^{\text{I}} \quad (5)$$

$$4 \text{MV}^{*+} + \text{O}_2 + 4 \text{H}^{+} \rightarrow 4 \text{MV}^{2+} + 2 \text{H}_2\text{O} \quad (6)$$

Building on these previous results, we now investigate the RuII/MV²⁺/LAC3/O₂ system operating under visible light irradiation for the selective oxidation of sodium *p*-styrene sulfonate (pSS) under mild, aqueous conditions. We demonstrate that incorporating MV²⁺ as an electron relay significantly enhances the overall olefin oxidation efficiency (Figure 1). While sodium *p*-benzaldehyde sulfonate (A) and sodium *a*-hydroxyketone *p*-phenyl sulfonate (α-HK), are predominantly formed in the absence of the enzyme (Scheme 1), laccase's presence, owing to its O₂⁻⁻-scavenging properties, leads to a change in product
<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>**Figure 2.** Photoreduction of the laccase under white light irradiation in the absence of O₂. [LAC3] = [RuII] = 30 µM; [p-styrene sulfonate] = 10 mM in Britton-Robinson (B and R) buffer pH 4.0 in presence of increasing amounts of MV²⁺: 0 mM (black line); 30 µM (red line); 150 µM (blue line) and 4 mM (green line). Irradiation was performed with a Dolan-Jenner Mi-LED A7, 235 mW cm⁻², 450 < λ < 700 nm.</figcaption></figure>

selectivity toward formation of the epoxide and related derivatives. Mechanistic insights are further supported by isotope labeling experiments and detailed product analysis.

## 2. Results and Discussion

### 2.1. Photoinduced Electron Transfer to the Laccase

Prior to assessing the photocatalytic performance of the RuII/MV²⁺/LAC3/O₂ system in olefin oxidation, we first confirmed the existence of photoinduced electron transfer between the different partners using the starting substrate, pSS, as the electron donor. The blue color of the enzyme, which reflects its oxidized state (λ<sub>610 nm</sub> = 5600 L.mol⁻¹.cm⁻¹), serves as a convenient probe to monitor redox changes. The decrease in absorbance at 610 nm, corresponding to the T1 copper center, was used as a direct indicator of laccase photoreduction. Visible light irradiation (450 < λ < 700 nm) of a dioxygen-free laccase (30 µM) buffered solution (B&R, pH 4.0) containing RuII (30 µM) as photocatalyst, pSS (10 mM) as electron donor, and increasing amounts of MV²⁺ (0 to 4 mM) led to the time-dependent bleaching of the 610 nm peak in the absorption spectrum (Figure 2). As previously observed for EDTA,[24] the kinetics of enzyme's photoreduction were strongly dependent on MV²⁺ concentration. The yield of MV²⁺ reduction follows a saturation curve (Figure S1). The half-life (t₁/₂) decreased sharply from 60 min in the absence to 2 min at 4 mM. This observation highlights the enhanced light-driven electron transfer to laccase in the presence of the electron relay. When the system was exposed to air, a marked increase in O₂ consumption was observed, which correlated with the concentrations of both laccase (Equation 1) and MV²⁺ (Equation 3) (Figure S2). Overall, in the presence of pSS, the RuII/MV²⁺/LAC3 (30 µM/ 4 mM/ 90 µM) system exhibited a fourfold increase in O₂ reduction efficiency compared to the RuII/LAC3 system.

### 2.2. Catalysis, Products Identification, and Optimization

In previous experiments, we studied the oxidation of *p*-styrene sulfonate (pSS) under irradiation with a Ru/LAC3 hybrid system in the presence of air. Although the overall conversion yield remained modest (3–5%), three main products were clearly identified: the sodium *p*-styrene sulfonate oxirane (epoxide) (E), the sodium *p*-styrene sulfonate gem-diol (D), and the sodium *p*-benzaldehyde sulfonate (A), all derived from pSS (Scheme 1).[19,20,29,30,31] Notably, the product distribution showed a strong preference toward epoxide formation, with a molar ratio of (E + D) to A close to 4:1.[19]

#### 2.2.1. Catalysis and Products Identification

The oxidation of pSS coupled with the reduction of dioxygen by the RuII/MV²⁺/LAC3/O₂ system under visible light irradiation at acidic pH (B&R, pH 4.0) was monitored by ¹H NMR, HPLC and mass spectrometry analysis (supplementary materials). As previously reported,[19] no product formation is detected in the absence of light or air. Under the above-mentioned experimental conditions, and light irradiation (filtered white LED), four products were identified: the epoxide (E), the diol (D), the aldehyde (A) and an *a*-hydroxyketone (*α*-HK) (Scheme 1). The detection of *α*-HK in this system, not previously observed, likely reflects the increased oxidation efficiency in the presence of MV²⁺ (Figure S3). The evolution of these products can be either spontaneous (ring opening of E to form D at acidic pH) or consecutive to an oxidation (D can be further oxidized into A or *α*-HK; see below). Control experiments in which D or E were used as starting materials (10 mM each, under irradiation) showed no formation of oxidized species beyond the conversion of E into D through hydration under acidic conditions. With the previous system (RuII/LAC3/O₂), laccase had been shown to influence the product selectivity by favoring the formation of the epoxide E and subsequently D, suggesting that alternative mechanistic pathways are responsible for the formation of A and *α*-HK. To estimate an apparent selectivity, we use below a factor (Sf) defined as the ratio between (E + D) and (A + *α*-HK).

#### 2.2.2. Effect of MV²⁺ and LAC3 on Catalytic Performance

Both LAC3 and MV²⁺ were found to play a decisive role in determining both the yield and the product selectivity. Irradiation of an aerated enzyme-free-buffered solution containing 10 mM of pSS, 30 mM of [Ru(bpy)₃]²⁺ and increasing amount of MV²⁺ (up to 5 mM), yielded a maximum of 2.8% conversion of pSS into D, A, and *α*-HK (Sf = 0.4) after 20 h (Figure 3 and Table S1), with a predominance of the aldehyde production. Upon the addition of laccase to this system, the conversion yield reached 13.5 % (Figure 3 and Table S1) and the product distribution shifted toward epoxide formation (Sf = 1.5). This shift in selectivity was clearly dependent on laccase concentration (Figure S4). Below 10 µM enzyme, the aldehyde A remained the major product. As the concentration of laccase increased (e.g., 20 µM of LAC3), selectivity progressively favored epoxide E, reaching a plateau at 60 µM LAC3 (Sf = 1.6, Figure S4). Based on these observations, a
<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /><figcaption>**Figure 3.** Yield (%) of different products in photocatalytic experiments in the presence of pSS and various combinations of RuII/MV2+/LAC3 or in the presence of CuSO4. Blue numbers indicate selectivity expressed as Sf = (E + D)/(A + α-HK). Conditions: aerated B and R pH 4.0 at 25 °C, 20 h irradiation. [RuII] = 30 µM; [MV2+] = 4 mM; [LAC3] = 90 µM; [pSS] = 10 mM; [CuSO4] = 360 µM corresponding to the maximum concentration of copper that could be expected consecutive to a release from 90 µM LAC3. n.d. = not determined.</figcaption></figure>

laccase concentration of 90 µM was selected to maximize both conversion and selectivity toward epoxide E formation under optimized conditions with MV2+ (4 mM).

### 2.2.3. Stability of the Enzyme Under Continuous White Light Irradiation

Monitoring the photocatalytic reaction over time revealed that pSS conversion reached a maximum after 20 h (Figure 4). Reaction selectivity initially favored the formation of epoxide E, with the selectivity factor (Sf) peaking at 2.1 after 16 h (Figure 4). Beyond that point, selectivity gradually shifted toward the formation of aldehyde A and α-HK, indicating a change in the catalytic regime, as reflected by a decrease in Sf to 1.5 at 20 h) (Figure 3).

Concomitant monitoring of laccase activity during the photocatalytic reaction revealed a significant decline, with the enzyme retaining less than 15% of its initial activity by the end of the process (Figure 4). In comparison, when laccase was irradiated with the system components individually, laccase exhibited only minor deactivation (less than 20% loss after 18 h, Figure S5). Enzyme integrity was further evaluated using UV-vis, EPR, and SDS-PAGE, which collectively indicated time-dependent structural modifications such as oligomerization and partial degradation during catalysis (Figure S6). These alterations are likely linked to the generation of superoxide during continuous irradiation. With a continuous production of superoxide radicals during irradiation, an oxidative modification of the protein scaffold is possible, despite a reported ability of the enzyme to

<figure><img src="image_9.png" /><figcaption>**Figure 4.** Comparison of the catalytic performance of the RuII/MV2+/LAC3 system (yield and selectivity), and the catalytic activity of LAC3 as a function of time in B&R buffer pH 4.0 at 25 °C. Sf is the selectivity factor defined as the molar ratio of (E + D)/(A+α-HK). [Rull] = 30 µM; [MV2+] = 4 mM; [LAC3] = 90 µM; [pSS] = 10 mM.</figcaption></figure>

use O₂⁻ as electron donor for dioxygen reduction.[24] Enzyme degradation raises the possibility of free copper ion release, confirmed by post-irradiation EPR spectra, which could, in turn, lead to nonenzymatic catalysis. To evaluate this, a control experiment in the absence of laccase but including CuSO₄, [Ru(bpy)₃]²⁺ and MV²⁺ was conducted under irradiation (Figure 3 and Table S1). This system yielded 2% conversion of pSS into A and α-HK exclusively, suggesting that free copper ions may contribute marginally to product formation. These findings emphasize the role of the enzyme itself, whose loss of activity correlates with both reduced conversion of pSS and shift in product selectivity toward (A+α-HK) formation.

### 2.2.4. Effect of O₂ Supply

Insufficient dioxygen availability is expected to limit product formation, as O₂ serves both as terminal electron acceptor and as an oxygen atom donor in the oxygen atom transfer reaction (OATR), as previously demonstrated.[19] Consistently, reducing O₂ concentration in the system led to a significant decrease in pSS conversion: from nearly 14% under continuous aeration to less than 7% when O₂ was limited by the initial concentration of 250 µM (Figures 3 and S7a, Table S1). Notably, the total amount of oxidized products under O₂-limiting conditions (640 µM) exceeded the theoretical maximum for an O atom insertion based on the available O₂, raising questions about the actual source of oxygen in the products. This observation strongly suggests the involvement of an alternative oxygen donor, likely water, under the tested conditions (buffered aqueous solution at 25 °C with 250 µM O₂). Increasing dioxygen concentration fivefold (i. e., 1.25 mM, corresponding to a O₂ saturated solution at atmospheric pressure) resulted in a twofold increase in conver-
<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /><figcaption>**Scheme 2.** Proposed mechanism for (a) Ru<sup>III</sup> mediated O<sub>2</sub> dependent reaction and subsequent water dependent ring opening hydration. (b) Ru<sup>III</sup> mediated H<sub>2</sub>O dependent reaction. (c) The intermediate pSS-dioxetane undergoes rearrangement to produce A and formaldehyde. (d) Proposed pSS-dioxetane mediated D-oxidation into α-HK.</figcaption></figure>

sion yield (up to 14%), albeit with a slight decrease in selectivity toward E + D formation (S<sup>f</sup> = 1.3) (Figure S7a and Table S1).

Taken together, these results describe a picture in which MV<sup>2+</sup> facilitates the OATR reaction by mediating electron transfer from the Ru-based photosensitizer to the enzyme, likely via superoxide generation, leading to a fourfold increase in pSS oxidation and a predominance of epoxide as product.

## 2.3. Mechanistic Considerations

Initially, performing photocatalysis with a Ru<sup>II</sup>/LAC3/O<sub>2</sub> system we concluded that the oxidation of pSS proceeds via a radical-mediated mechanism (Figure 1).<sup>[19]</sup> In this scheme, after photoinduced electron transfer from the Ru<sup>II</sup>* to the laccase generates Ru<sup>III</sup>, which then oxidizes pSS by single-election transfer, forming *p*-styrene sulfonate radical cation (pSS<sup>+</sup>). This organic radical can subsequently engage with O<sub>2</sub> and reactive oxygen species (O<sub>2</sub><sup>•</sup> and O<sub>2</sub><sup>•−</sup>) to yield oxygenated products (Scheme 2). In the present study, where catalytic efficiency was markedly improved, we were able to trap reactive intermediates and investigate the origin of the oxygen atoms incorporated in the products providing deeper mechanistic Insights.

### 2.3.1. Formation and Reactivity of pSS•+

In a previous paper, we showed that Ru<sup>III</sup> reacts with pSS under anaerobic conditions to generate a carbon centered

radical.<sup>[30,31]</sup> The EPR signature is similar to that a styrene-derived radical species (carbon-centered radical) formed upon gamma-irradiation of polystyrene and was assigned to the pSS radical cation (pSS<sup>+</sup>).<sup>[32]</sup> We reproduced these experiments and investigated the effect of dioxygen on the nature of the species formed. We therefore synthesized the Ru<sup>III</sup>-polypyridyl complex, Ru(bpy)<sub>3</sub>(PF<sub>6</sub>)<sub>3</sub> by oxidation of Ru<sup>II</sup> parent complex as previously reported.<sup>[30,33]</sup>

Under aerobic conditions, the reaction of the Ru<sup>III</sup> complex with the substrate generates a different EPR signal (Figure 5). This signal closely resembles those of known oxygen-centered radicals formed upon reaction of protein-based radicals with O<sub>2</sub><sup>[34,35]</sup> or DNA with peroxyl radicals.<sup>[36]</sup> This species can therefore be assigned to a [pSS-OO]<sup>+</sup> (peroxyl) species. Hence, these experiments support the following reaction events for the oxidation of pSS by Ru<sup>III</sup>: (i) an intermolecular single-electron transfer yielding an alkenyl radical cation (pSS<sup>+</sup>) and (ii) subsequent reaction of pSS<sup>+</sup> with an oxygen donor, likely forming a peroxyl-type radical species ([pSS-OO]<sup>•+</sup>).

Interestingly, analysis of the reaction mixture by NMR reveals that pSS is exclusively converted into the diol D (yield ~80%), irrespective of the presence or absence of O<sub>2</sub>. Although caution should be taken when comparing this chemical system (i. e., 10 mM [Ru(bpy)<sub>3</sub>]<sup>3+</sup> ± 250 µM O<sub>2</sub>, extemporaneously) with the photocatalytic conditions (20 h under continuous irradiation). The results suggest that both O<sub>2</sub> and H<sub>2</sub>O may serve as oxygen donors (Scheme 2a,b). By extrapolation, it is possi-
<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /><figcaption>**Figure 5.** X-band EPR spectrum (120 K) of the radical trapped after reaction of [Ru<sup>II</sup>(bpy)<sub>3</sub>(PF<sub>6</sub>)<sub>3</sub>] (10 mM) with pSS (100 mM) in B&R buffer at pH = 4.0 under air. Dotted line: simulation with the following parameters: g<sub>1</sub> = 2.033 g<sub>2</sub> = 2.010, and g<sub>3</sub> = 2.003.</figcaption></figure>

<table><caption>**Table 1.** Labelling experiments with <sup>18</sup>O<sub>2</sub> or H<sub>2</sub><sup>18</sup>O for the optimized photocatalytic experiment.<sup>a)</sup></caption><thead><tr><th>Entry</th><th>Product</th><th>H<sub>2</sub>O Oxygen Source (%)</th><th>O<sub>2</sub> Oxygen Source (%)</th></tr></thead><tbody><tr><td>1</td><td><img src="image_0_0.png" alt="Chemical structure of 1,3-butadiene with a hydroxyl group on the phenyl ring and a methoxycarbonyl group on the phenyl ring."></td><td>9.0 ± 2.0</td><td>86.0 ± 7.0</td></tr></tbody></table>

<sup>a)</sup> Ru<sup>II</sup>/MV<sup>2+</sup>/LAC3 (30 µM/ 4 mM/ 90µM) in B&R buffer, pH 4.0.

ble to propose that peroxyl-type radical species ([pSS–OO]<sup>•+</sup>) are formed in our photocatalytic experiments by reaction with a Ru<sup>II</sup> species formed after electron transfer from photogenerated excited Ru<sup>II</sup>* complex. These peroxyl radicals could rearrange to yield epoxides and, subsequently, diols as previously proposed (Scheme 2a).<sup>[19,20,30,37]</sup>

### 2.3.2. Origin of Oxygen in the Oxidation Products

Isotopic labeling experiments were conducted using <sup>18</sup>O<sub>2</sub> and H<sub>2</sub><sup>18</sup>O to identify the origin of the oxygen atoms incorporated into the reaction products. The photocatalytic products were analyzed by mass spectrometry (Table 1, Figures S8 and S9). Since aldehydes can exist in equilibrium with their hydrated forms in aqueous media, and α-HK may arise from subsequent transformations (see below), the discussion of isotope incorporation primarily focuses on the epoxide (E) and diol (D) products. Although detected in low amounts, both <sup>18</sup>O- and <sup>16</sup>O-labeled epoxides were observed under both labeling conditions (H<sub>2</sub><sup>16</sup>O/<sup>18</sup>O<sub>2</sub> and H<sub>2</sub><sup>18</sup>O/<sup>16</sup>O<sub>2</sub>), confirming that oxygen insertion can originate from either O<sub>2</sub> or H<sub>2</sub>O (Figure S7). When performed in the presence of H<sub>2</sub><sup>18</sup>O and unlabeled dioxygen from air (<sup>16</sup>O<sub>2</sub>), photocatalysis generated the <sup>16</sup>O<sup>16</sup>O and the doubly <sup>18</sup>O<sup>18</sup>O labelled diol D as well as its <sup>18</sup>O<sup>16</sup>O singly labelled version. In the presence of H<sub>2</sub><sup>16</sup>O and under an <sup>18</sup>O<sub>2</sub> atmosphere, a similar trend was observed. If the presence of

mixed-labeled diols (<sup>16</sup>O/<sup>18</sup>O), is consistent with epoxide ring opening at acidic pH, the simultaneous presence of isotopically pure and mixed (<sup>16</sup>O/<sup>18</sup>O) molecules highlights that O<sub>2</sub> and H<sub>2</sub>O can both serve as source of oxygen atom in our photocatalytic experiments (Figures S9 and S10). These findings align with the presence of oxygenated products even in the absence of dioxygen (see section “Formation and reactivity of pSS•⁺”).

That said, O-incorporation was found to be predominantly from O<sub>2</sub> (accounting for 86% of insertion, Table 1) with a minor contribution coming from water.

### 2.3.3. ROS Contribution

Reactive oxygen species (ROS), including O<sub>2</sub><sup>•−</sup> (Equation 3) and <sup>1</sup>O<sub>2</sub> (Equation 7) can be produced in greater or lesser quantities and, more importantly, continuously during the photocatalytic reaction. Both O<sub>2</sub><sup>•−</sup> and <sup>1</sup>O<sub>2</sub> are known to react with pSS<sup>•+</sup> and pSS itself, respectively, leading to the formation of a transient dioxetane.<sup>[38–40]</sup> This one can subsequently decompose into A and formaldehyde, as illustrated in Scheme 2c. As previously discussed, laccase has also been shown to interact with O<sub>2</sub><sup>•−</sup>, further implicating ROS in the overall reaction pathway (Equation 5).<sup>[24]</sup>

To assess the role of O<sub>2</sub><sup>•−</sup> in the catalytic system, superoxide dismutase (SOD) and subsequently catalase (CAT) were introduced. The reaction pH was adjusted to 6.0 to ensure SOD activity, even though LAC3 operates most efficiently at acidic pH (pH = 3–5).<sup>[23]</sup> As anticipated, raising the pH resulted in a twofold decrease in the overall yield of pSS oxidation products, which is consistent with the laccase’s pH-dependent activity. The selectivity remained in favor of (E + D) formation (Sf = 1.4 at pH 6.0 versus 1.5 at pH 4.0, Figure S7b and Table S1), with E predominating due to reduced ring-opening at higher pH. The addition of SOD (and CAT) to the Ru<sup>II</sup>/Mv<sup>2+</sup>/LAC3 system at pH 6.0 had little effect on the total pSS conversion yield (Figure S7b and Table S1), but led to a marked shift in product distribution. The Sf increased from 1.4 to 1.7 in the presence of SOD, suggesting the involvement of superoxide radicals under our photocatalytic conditions. These findings further support the hypothesis that laccase loses its ability to scavenge O<sub>2</sub><sup>•−</sup> during the reaction.

Given the well-established reactivity of singlet oxygen (<sup>1</sup>O<sub>2</sub>) in alkene oxidation,<sup>[41,42]</sup> its involvement in the present photocatalytic system, via energy transfer from the excited state of [Ru(bpy)<sub>3</sub>]<sup>2+</sup> (Equation 7), could not be ruled out. To assess the ability of <sup>1</sup>O<sub>2</sub> to oxidize pSS, Rose Bengal, a well-known <sup>1</sup>O<sub>2</sub>-generating photosensitizer, was used under irradiation (Equation 8).<sup>[43–45]</sup> Under conditions mimicking our optimized system (10 mM pSS, 30 or 90 µM Rose Bengal) no pSS conversion was observed. These findings suggest that <sup>1</sup>O<sub>2</sub> does not significantly contribute to pSS oxidation under our experimental conditions.

$$Ru^{II*} + O_2 \rightarrow Ru^{II} + ^1O_2 \quad (7)$$

$$\text{Rose Bengal} * + \text{O}_2 \rightarrow \text{RoseBengal} + ^1\text{O}_2 \quad (8)$$
<figure><img src="image_14.png" /><figcaption>Scheme 3. Proposed mechanism for the oxidation of pSS by laccase LAC3 in the presence of Ru and excess MV²⁺ as electron relay.</figcaption></figure>

### 2.3.4. Origin of A and α-HK

Among the experimental facts reported in this study two experimental observations deserve to be highlighted: (i) A and α-HK are produced only when the Ru/MV²⁺ system is irradiated in the presence of O₂ (Figure 3 and Table S1); (ii) the formation of A and α-HK in the presence of laccase correlates with the progressive loss of the enzymatic activity (Figure 4). Few examples of oxidation of vicinal diols into α-hydroxyketones have been reported.[46,47] Mecozzi et al. mentioned a Mn-based catalyst able to perform vicinal diols oxidation in the presence of H₂O₂.[46] Bovicelli et al. described a catalytic system in which dimethyloxirane (DMD) acts as the oxidant.[47] As mentioned, compounds E and D did not undergo oxidation into α-HK under our photocatalytic conditions, ruling out a direct oxidation pathway. However, one can consider the transient dioxetane, formed during photocatalysis, as a stoichiometric oxidant capable of promoting the selective oxidation of D into α-HK as depicted in Scheme 2d. Dioxetanes have indeed been proposed as stoichiometric oxidants in the formation of disulfide bridges.[48,49] Altogether, these findings support the hypothesis that ROS, either through direct oxidation of pSS or via transient intermediates such as dioxetanes, contribute to the formation of compounds A and/or α-HK (Scheme 2c,d). The exact mechanisms of A and/or α-HK formation remain under active investigation.

### 2.4. Proposed Mechanism

Based on these studies, we propose a mechanism in which MV²⁺ facilitates the photo-accumulation of Ru¹¹ by mediating electron transfer from the Ru¹¹* to either laccase or molecular oxygen (Scheme 3). The resulting Ru¹¹ species then oxidizes pSS to pSS⁺, thereby regenerating Ru¹¹. The pSS⁺ can either react with O₂, O₂⁻ or H₂O leading to oxidized products. labeling data indicate that the inserted oxygen atom predominantly originates from O₂ and that, although possible, insertion of oxygen atom from H₂O into epoxide-derived products remains a minor pathway. We further suggest that laccase, thanks to its ability to scavenge superoxide (O₂⁻), directs the reactivity of pSS⁺ toward O₂ or H₂O, ultimately yielding the epoxide E and diol D. Isotopic labeling

data indicate that the inserted oxygen atom predominantly originates from O₂. In contrast, the formation of aldehyde A and α-hydroxyketone α-HK is attributed to O₂⁻-mediated pathways, potentially involving a dioxetane intermediate. Therefore, superoxide radicals appear to compromise the chemoselectivity of the reaction, by favoring alternative oxidation routes over epoxidation or dihydroxylation.

## 3. Conclusion

In conclusion, we report a novel light-driven epoxidation of pSS by a Ru¹¹/MV²⁺/LAC3 system, where laccase governs product selectivity and MV²⁺ enhances electron transfer, either directly or via O₂⁻. The conversion yield increased fivefold rising from 3% in absence of MV²⁺ to 14% when it is present, while the selectivity toward the epoxide is favored. Isotopic labeling and product distribution analyses suggest that this selectivity arises from the superoxide-scavenging ability of laccase. However, continuous light irradiation of the system compromises the integrity of the enzyme, highlighting the need for strategies to preserve enzymatic function integrity. Future work should focus on enhancing direct electron transfer to the enzyme while minimizing undesired ROS-mediated side reactions. These findings highlight the critical role of ROS scavenging in fully harnessing laccase-mediated alkene epoxidation. Additionally, designing electron relays that facilitate efficient charge transfer without unintended reactivity with O₂ will be essential. Furthermore, the use of olefins lacking electron-withdrawing substituents may improve both the yield and selectivity of the process. Collectively, these approaches will be keys to optimizing the selectivity, efficiency, and robustness of laccase-driven photocatalytic epoxidation.

## 4. Experimental Section

All other chemicals and reagents used in this study are of analytical grade and are used without any further purification (unless specified). Chemicals purchased from Sigma-Aldrich: tris(2,2'-bipyridyl)dichlororuthenium(II) hexahydrate (99.5%) ([Ru(bpy)₃]²⁺), methyl viologen dichloride hydrate (98%; 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS); sodium 4-vinylbenzenesulfonate (>90 1-Phenyl-1,2-ethanediol 97%, acetic acid glacial (≥99%), boric acid (≥99.5%), phosphoric acid 85 wt. % in H₂O (99.99%), hydrochloric acid 37%, sodium hydroxide (99%), deuterium oxide 99.9 atom % D, trimethylsilylpropanoic acid (99%), SOD enzyme (CAS: 9003–99–0), and catalase from bovine liver (CAS: 001–05–2). All buffer solutions were made with deionized water. Britton and Robinson buffer (B&R) was prepared as a mixture of equimolar 0.1 M acetic acid, boric acid and phosphoric acid adjusted to pH4.0 or 6.0 with 15 M NaOH.[50] LAC3 originally from the organism *Trametes* sp C30 was expressed in *Aspergillus niger* and purified according to previously published procedures.[23] Protein concentration was determined by UV-vis spectroscopy (ε₆₁₀nm = 5600 M⁻¹ cm⁻¹ attributed to the T1 oxidized copper). The preparation of [Ru(III)(bpy)₃(PF₆)₃] was performed according to a published procedure by oxidation of the [Ru(bpy)₃Cl₂] with MnO₂.[34] A Dolan-Jenner Mi-LED A7, 235 mW cm⁻² prior filtration with optic fibers (diameter of 0.8 cm), equipped with a Wratten 2B filter (λ > 420 nm) was used for polarography or UV-vis experiments. Light-Oxygen consumption was measured with polarography using
a Hansatech Oxygraph system with a micro-Clark electrode fitted to a temperature-controlled (25 °C) glass chamber (1 mL). Irradiation was performed through the glass chamber. The photochemical reaction was operated on a flat white LED panel 5 x 15 cm, ≈5 mW cm⁻², Metaphase Technologies) equipped with a Wratten 2B filter (λ > 420 nm) in a temperature-controlled room 25 °C. cw-X-band EPR spectra were obtained using a BRUKER Elexsys E500 spectrometer operating at X-band equipped with a B-VT 3000 digital temperature controller (100-400 K). Simulations were performed using the easyspin package.[51]

**Light dependent bleaching experiments:** Enzyme photoreduction was performed as previously reported by us.[19]

**Typical procedure for photochemical reactions:** Photocatalytic tests were performed under air or under N₂ atmosphere in a glove box, in 0.5 mL total volume and placed in 2 mL flat bottom transparent glass HPLC vials closed using screw caps with a PTFE septum and equipped with a stir bar. The vials were placed on flat white LED panel. The reaction was stopped after 20 h of irradiation (unless specifically reported). For O₂ concentration experiments: (i) **250 µM O₂:** An air saturated buffer solution was placed in a gas-tight Hamilton syringe and injected in previously N₂-sealed empty vials (prepared in N₂-atmosphere glove box) and then placed upon irradiation for 20 h; (ii) **100% O₂ experiments:** after O₂-saturating a buffer solution with an O₂ tank, the photocatalytic experiment was performed directly connecting this oxygen tank (1 bar pressure). **SOD and CAT effects** were conducted at pH 6.0, where both enzymes exhibit reasonable activity (ca. 80 % of their optimal activities), adding 1 µM of SOD and/or 1 µM of CAT to the photocatalytic medium were conducted at pH 6.0, where both enzymes are active. **Rose Bengal photocatalytic control** was conducted in B&R pH 4.0 adding 90 µM of Rose Bengal to a 10 mM buffered solution of pSS in the same irradiation solutions. Prior to performing NMR analysis, **LAC3** (when present) was removed by ultrafiltration with a 30-kDa cutoff ultrafiltration membrane (centrifuging for 45 min at 13.5 rpm) until less than 20 µL of the retentate was remaining. 50 µL of TMSp in D₂O (10 mM) were added to 450 µL of the filtrate, homogenized and introduced into an NMR tube for analysis. TMSp was used as an internal standard for quantification.[29]

**Oxidation of pSS by Ru<sup>II</sup>:** 10 mM freshly prepared [Ru<sup>III</sup>(bpy)<sub>3</sub>(PF<sub>6</sub>)<sub>3</sub>] were placed in the presence of 100 mM of pSS in B&R buffer set at pH 4.0, either anaerobically (in a glove box) of under air. Aliquots of the reaction mixtures were immediately transferred in hermetically sealed EPR tubes and subsequently frozen. The remaining reaction mixtures were left to react during 24 h and further analyzed by NMR.

**Labeling experiments:** Samples were prepared and analyzed by low- and high-resolution mass spectrometry and ¹H NMR. The reaction mixtures analyzed were: (a) Ru<sup>II</sup>/MV<sup>2+</sup>/LAC3/O₂ in H₂<sup>16</sup>O in air (¹⁶O₂); (b) Ru<sup>II</sup>/MV<sup>2+</sup>/LAC3 in H₂<sup>16</sup>O with <sup>18</sup>O₂, and (c) Ru<sup>II</sup>/MV<sup>2+</sup>/LAC3/O₂ in H₂<sup>18</sup>O in air (¹⁶O₂). Except for (a) - prepared as before and used as a control reaction, the other reactions (b and c) slightly differed from the classical preparation. <sup>18</sup>O₂ experiments (reaction b). A <sup>18</sup>O₂-enriched dioxygen tank (>90% enriched) was used for all the labelled dioxygen experiments performed. The reaction mixture was prepared in an inert atmosphere (N₂, in a glove box), and three purge cycles of labeled dioxygen were performed to guarantee enough dioxygen for carrying out the reaction. H₂<sup>18</sup>O experiments (reaction c). Labeled reaction was performed in 80.6 % H₂<sup>18</sup>O and 19.4 % H₂<sup>16</sup>O. For both cases, reactions were stopped after 20 h of irradiation, followed by ¹H NMR analysis as previously. Two independent experiments were prepared. Diol D was considered to come from E ring opening for calculations.

**Laccase activity:** Aliquots of photocatalytic experiments within time were withdrawn to measure the activity. Laccase activity was assayed by following the oxidation of ABTS as substrate at 420 nm

(ε<sub>420nm</sub> = 3.6 × 10<sup>4</sup> M<sup>-1</sup> cm<sup>-1</sup>) The reaction was performed in 1 mL 0.1 M B&R buffer set at pH 4.0 over 5 min at 30 °C. The reaction was initiated by the addition of 10 µL of appropriately diluted enzyme sample to 990 µL of 2.5 mM ABTS solution. One unit (U) of laccase (M.W. = 84 kDa) is defined as one µmol of substrate oxidized per minute.

## Supporting Information

Dioxygen consumption with *p*-styrene sulfonate oxidation products as substrate. Characterization of the photo-oxidation products. Synthesis of Ru(Bpy)₃(PF₆)₃. *p*-Styrene sulfonate oxidation as function O₂ concentration and pH. *p*-Styrene sulfonate oxidation reaction carried out with H₂¹⁸O or in the presence of ¹⁸O₂. Effect of light on laccase (activity profile, UV-vis, EPR, and SDS PAGE).

## Acknowledgments

The authors thank ANR Multiplet (ANR-15-CE07-0021-01) for financial support. Labelling studies were performed with the help and support of Dr. Sabine Chevallier-Michaud of our institute.

## Conflict of Interests

The authors declare no conflict of interest.

## Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

**Keywords:** Epoxidation · Laccase · Methyl viologen · Photocatalysis · Reactive oxygen species

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Manuscript received: July 7, 2025

Revised manuscript received: August 18, 2025

Accepted manuscript online: August 18, 2025

Version of record online: ■■, ■■