The complex interplay of chemo- and bio-catalysis for one-pot oxidation cascades – indole oxidation in focus

Abstract

The combination of chloroperoxidase (CPO), derived from the marine fungus Caldariomyces fumago, in combination with supported AuPd nanoalloys, has been demonstrated as a highly efficient one-pot system for the selective oxidation of indole to 2-oxindole. Peroxy-enzymatic oxygen transfer is driven via chemo-catalytic in situ H₂O₂ generation from H₂ and O₂ over AuPd surfaces, with the continuous supply of the oxidant at low-concentration essential in maintaining enzymatic activity. Indeed, coupling CPO and an optimised 1% Au₁Pd₁/TiO₂ formulation achieved 33 000 TTN for indole oxidation, which is among the highest reported when compared to alternative systems utilizing in situ generated H₂O₂, including electrochemical and co-enzymatic approaches. Exceptional tandem performance has been achieved upon adopting a holistic approach toward design and optimisation of the system, thus deconvoluting the complex array of parameters governing chemo- and bio-catalytic performance under operational conditions; including metal nanoparticle speciation, morphology, electronic structure, and leaching, in addition to enzyme deactivation and in situ physisorption. We consider such an approach to offer a case study and guide to the community, facilitating efficient optimisation of complex chemo-enzymatic systems for potential industrial applications.

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Green foundation

1. The selective oxidation of indole to 2-oxindole currently relies on the application of stoichiometric oxidants, resulting in the generation of large quantities of hazardous by-products and poor atom efficiency. Further complexity arises from the need to apply protecting/de-protecting protocols to control regio-selectivity. Our chemo-enzymatic approach for such chemical transformations offers significantly improved process efficiency and product selectivity under comparatively mild operating conditions.

2. Chloroperoxidase (CPO) offers exceptional performance towards selective indole oxidation, however its synthetic potential is limited by its susceptibility to peroxidative inactivation. Our work demonstrates that exceptional enzymatic performance can be achieved upon generating H₂O₂ in situ from H₂ and O₂ over supported AuPd nanoparticles, which facilitates the continuous supply of low concentrations of H₂O₂ required to maintain CPO activity, while avoiding the limitations of other approaches.

3. Further catalyst design is required to enhance AuPd nanoalloy stability under operating conditions, facilitating enhanced chemo-enzymatic cascade efficacy over extended lifetimes.

Introduction

The selective oxidation of indole to 2-oxindole is a highly valuable chemical transformation for the production of pharmaceutical products, with numerous cancer, Parkinson's disease, and pulmonary fibrosis therapeutics bearing this oxygenated heterocyclic moiety.^1–5 Conventional chemical indole oxidation approaches require stoichiometric consumption of toxic reagents (e.g. trichloroisocyanuric acid, N-bromosuccinimide, dimethyldioxirane, meta-chloroperoxybenzoic acid), resulting in the formation of hazardous by-products and poor atom economy.^6–9 Protecting groups and substituted indole rings are often necessary to control the regioselectivity for oxidation at the C-2 position under such protocols, owing to the competing activity at the electron-rich C-3 and N-centres, which hampers the overall process efficiency.¹⁰

The application of chloroperoxidase (CPO), derived from marine fungus Caldariomyces fumago, offers a highly attractive enzymatic alternative, avoiding many of the drawbacks associated with conventional chemical approaches. The heme-thiolate enzyme requires only H₂O₂ to maintain biocatalytic activity, which mitigates stoichiometric consumption of ancillary flavoproteins and cofactors that require regeneration, unlike the closely-related P450 monooxygenases.^11,12 In vivo, CPO utilizes H₂O₂ to perform oxidative halogenation reactions via hypohalous acid formation. In vitro, CPO exhibits a broad spectrum of synthetically valuable oxygen-transfer activity, including allylic, propargylic, and benzyl hydroxylation, asymmetric olefin epoxidation, and sulfoxidation with high regio- and enantio-selectivity.^13–21 As such, biocatalytic CPO applications in pharmaceutical synthesis, pollution remediation, and analytical diagnosis have been explored.^22–24 In the context of this study, CPO offers exceptional regioselectivity for the oxidation of indole derivatives to the corresponding 2-oxindole.^25–27

However, the synthetic potential of CPO has been limited by its susceptibility to oxidative inactivation in the presence of H₂O₂, even at modest concentrations (half-life of approximately 1 hour in 30 µM H₂O₂ in the absence of organic substrates).²⁸ Conventionally, this challenge has been circumvented via the continuous addition of pre-formed commercial H₂O₂ to the reaction media.^28–30 Sheldon and coworkers demonstrated this strategy can offer exceptional peroxy-enzymatic performance, with 860 000 TTNs offered for CPO indole oxidation.²⁸ Despite offering improved enzymatic stability, this approach precludes CPO-based chemical transformations from operating at scale, owing to the rapid dilution of valuable product streams and concurrent production of excessive quantities of contaminated aqueous waste.^28,31 There are additional concerns regarding the energy inefficiency and cost associated with the manufacture, storage, and transport of commercial H₂O₂ via the Anthraquinone Oxidation Process.³² As such, in situ H₂O₂ generation has the potential to offer a practical, atom-efficient, and environmentally benign solution to achieve the continuous low-concentration supply of oxidant required to maintain peroxy-enzymatic activity.³³

Numerous in situ approaches to H₂O₂ supply for peroxy-enzymes have been developed, including photocatalytic, electrochemical, and multi-enzymatic cascades. Co-enzymatic approaches represent a well-established approach, with H₂O₂ generation via glucose oxidase (GOx), formate oxidase (FOx), and choline oxidase (ChOx) previously reported.^24,34,35 Despite offering high H₂O₂ production rates under enzymatically compatible reaction conditions, co-enzymatic strategies offer poor atom efficiency and undesirable by-product formation associated with the stoichiometric consumption of sacrificial co-reagents. The application of FOx minimises these drawbacks, however continual monitoring of the reaction media pH is required via acid titration, which could limit the process at scale. Photocatalytic H₂O₂ generation suffers from similar drawbacks, which rely on stoichiometric quantities of redox cofactors.^31,36 Exposure of the aqueous reaction media to UV radiation also results in the formation of reactive oxygen species (e.g. ˙O₂⁻ and ˙OH), which are reported to contribute towards enzyme deactivation.³¹ Holtmann et al. reported a highly efficient electrochemical H₂O₂ generation approach which employed a gas-diffusion electrode for O₂ reduction, offering 1 150 000 TTN for CPO 2-chloro-5,5-dimethyl-1,3-cyclohexanedione chlorination.³⁷ This clearly demonstrates the potential to achieve exceptional peroxy-enzymatic performance via the rapid generation and utilization of H₂O₂ in situ, however, such electrochemical approaches offer limited compatibility with current industrial reactor designs.

We have previously demonstrated the feasibility of a chemo-enzymatic approach, with supported AuPd nanoparticles employed to directly synthesise H₂O₂ in situ from molecular H₂ and O₂, coupled with an unspecific peroxygenase enzyme (UPO) from Agrocybe aegerita (PaDa-I variant), for selective C–H bond activation.^38–40 This approach offers highly atom efficient in situ H₂O₂ synthesis for biocatalytic utilization, while avoiding the use of sacrificial co-reagents and resulting side-product formation. However, high H₂O₂ direct synthesis reaction rates typically require sub-ambient temperatures, high pressure of gaseous reagents (>10 bar), and highly acidic reaction media (typically pH 4 or lower), which contrasts the near ambient conditions typically required to maintain enzyme activity.^41,42 Despite this, supported AuPd nanoparticles bridge this broad conditions gap, offering a continuous supply of H₂O₂ while maintaining peroxy-enzymatic activity, with both pressurized (2 bar) and atmospheric pressure reactor systems developed.^38–40

Herein, we demonstrate the applicability of the chemo-enzymatic approach for the selective oxidation of indole via the coupling of supported AuPd nanoparticles and CPO from Caldariomyces fumago (Fig. 1). Highly efficient cascade efficiency was achieved by tuning the rate of chemo-catalytic H₂O₂ production with respect to peroxy-enzymatic utilization. Detailed investigation into dynamic nature of the chemo-catalytic component and the parameters driving enzymatic deactivation under cascade reaction conditions have facilitated the design of a highly compatible tandem system. Indeed, we elucidate the highly intertwined nature of chemo- and bio-catalysts under one-pot reaction conditions, wherein optimisation of the tandem system requires detailed understanding of the complex interplay of the cooperating components.

Fig. 1 (A) Proposed reaction scheme for the chemo-enzymatic oxidation of indole oxidation with chloroperoxidase and supported AuPd nanoparticles. (B) Active pharmaceutical ingredients containing the 2-oxindole scaffold. Key: (A) chemo-catalytic pathways (blue), enzymatic pathways (green).

Experimental

Catalyst preparation

A series of 1 wt% Au_xPd_y/TiO₂ (where Au : Pd = x : y by wt.), catalysts have been prepared via a sol-immobilization procedure, based on methodology reported previously in the literature, which has been shown to result in highly dispersed metal species by limiting particle growth and agglomeration.⁴³ The experimental procedure to produce the 1% Au₁Pd₁/TiO₂ (1 g) catalysts is outlined below (where the Au :Pd wt. ratio is fixed at 1 : 1), with an analogous methodology employed to prepare the mono- and bi-metallic catalysts investigated in this study.

Aqueous metal salt precursor solutions of HAuCl₄ (0.454 mL, [Au] = 11 mg mL⁻¹, Strem Chemicals) and PdCl₂ (0.714 mL, [Pd] = 7 mg mL⁻¹, Merck) were added to 400 mL deionised water and stirred at 1000 rpm. The requisite amount of PVA solution (1.3 mL, 1 wt% aqueous solution, MW = 10 000, 80% hydrolysed, Merck), was added to achieve a weight ratio of PVA: [Au + Pd] = 1.3. A freshly prepared solution of NaBH₄ (3.618 mL, 0.1 M, >98%, Merck), was added to the solution such that the molar ratio of NaBH₄: [Au + Pd] = 5. The solution was stirred for 30 min to allow for colloidal formation, before steady addition of TiO₂ (P25, 0.99 g, Degussa), followed by acidification to pH 1 using H₂SO₄ (95%, Fisher Scientific). After a further 1 hour of stirring, the slurry was filtered under vacuum and the resulting catalyst was washed with 2 L of deionised water, ensuring washings of neutal pH, and then dried (110 °C, 16 h). The dried catalyst was then collected and ground before calcining in static air (400° C, 3 h, ramp rate = 10 °C min⁻¹).

Catalyst testing

Note 1: in all cases, reactions were run multiple times, over multiple batches of catalyst, with the data being presented as an average of these experiments. Catalytic activity was found to be consistent to within ±5% on the basis of multiple reactions.

Note 2: reaction conditions used within this study operate outside the flammability limits of gaseous mixtures of H₂ and O₂.⁴⁴

Chemo-catalytic H₂O₂ synthesis from H₂ and O₂

Reactions were conducted in 50 mL gas-tight round-bottom flasks rated to 4.1 bar and stirred using a Radleys 6 Plus Carousel equipped with a gas distribution system. The catalyst (0.001 g) was weighed directly into the round bottom flask before charging with reaction solution (10 mL). The reaction solvent was prepared by combining potassium phosphate buffer (KH₂PO₄/K₂HPO₄, Merck) with t-BuOH (>99%, Merck) in a 1 : 1 ratio by volume (10 mM, pH 5.0 K₂HPO₄/KH₂PO₄ buffer with 50 vol% t-BuOH). Subsequently, the flasks were sealed, purged, and pressurized to 2 bar with H₂ (1.6 bar) and air (0.4 bar) to give a reaction environment containing 80% H₂ in air. The reactions were stirred at 250 rpm at ambient temperature (20 °C) for a given reaction time (5–180 min). Upon completion of a reaction, the glassware was depressurized and the spent catalyst was removed by filtration, before H₂O₂ concentrations were determined via UV/vis spectroscopy. To determine H₂O₂ concentration, an aliquot (1.5 mL) of post-reaction was combined with potassium titanium oxalate dihydrate solution acidified with 30% H₂SO₄ (0.02 M, 1.5 mL), resulting in the formation of an orange pertitanic acid complex. The resulting solution was analysed using a Shimadzu UV/vis 1900i spectrophotometer at 400 nm, with H₂O₂ concentrations determined by comparison to a calibration curve.

Chemo-catalytic degradation of H₂O₂

Catalytic activity toward H₂O₂ degradation (via hydrogenation and decomposition pathways), was determined in a similar manner to the above outlined H₂O₂ synthesis testing protocol, based on a previously established procedure.³⁹ The 50 mL gas-tight round bottom flasks were charged with the reaction solvent (10 mL, 10 mM, pH 5.0 K₂HPO₄/KH₂PO₄ buffer with 50 vol% t-BuOH) and H₂O₂ (2000 ppm, Merck). From the solution, prior to the addition of the catalyst, two 0.05 g aliquots were removed to allow for the quantification of the initial H₂O₂ concentration via UV–vis spectroscopy. Subsequently, the catalyst (0.001 g) was added to the flask, which was then sealed, purged, and pressurized to 2 bar with H₂ (1.6 bar) and N₂ (0.4 bar) to give a reaction atmosphere containing 80% H₂ and 20% N₂. The reaction mixtures were stirred (250 rpm) at ambient temperature (20 °C) for 2 h. After the desired reaction time, the vessel was depressurized, the catalyst was removed via filtration, and the remaining H₂O₂ was quantified by UV–vis spectroscopy.

Chemo-enzymatic indole oxidation

Reactions were conducted in 50 mL gas-tight round-bottom flasks rated to 4.1 bar and stirred using a Radleys 6 Plus Carousel equipped with a gas distribution system. Indole (≥99%, Merck) was dissolved the reaction solvent (10 mL, 10 mM, pH 5.0 potassium phosphate buffer with 50 vol% t-BuOH) to give the desired substrate concentration (10 mM). The catalyst (0.001–0.004 g) was weighed directly into the 50 mL gas-tight round bottomed flasks, before charging with the prepared reaction mixture and CPO enzyme (15 U mL⁻¹, 600 nM, Merck). The flasks were immediately sealed and pressurized to 2 bar (80% H₂ in air), as described above. The reaction mixtures were stirred (250 rpm) at ambient temperature (20 °C) for the desired reaction time (15–180 minutes) before post-reaction analysis was conducted.

Filtered post-reaction aliquots were subjected to High-performance Liquid Chromatography (HPLC) analysis to monitor substrate conversion and product formation using an Agilent 1200 series equipped with an Agilent Poroshell 120 EC-C18 (2.7 μm 4.6 × 150 mm) column and Agilent 1260 series DAD detector at 278 nm. A water/acetonitrile mobile phase was employed with a gradient elution (0.75 mL min⁻¹, mobile phase ramped from 70 : 30 v/v to 50 : 50 v/v at 4–14 min). To quantify post-reaction concentrations of indole and 2-oxindole, calibrations were conducted with indole (≥99%, Merck) and 2-oxindole (97%, Merck) commercial standards, allowing concentration determination of indole and 2-oxindole by comparison to a known response factor. Residual H₂O₂ concentration was determined following extraction of organic components with ethyl acetate (2 × 10 mL), before subjecting aliquots of the aqueous later to UV/vis spectroscopy. Unfiltered post-reaction aliquots were subjected to monochlorodimedone (MCD) assays to determine post-reaction enzyme activity, followed by appropriate application of an assay inhibition model (see experimental methods).

CPO activity measurements – monochlorodimedone (MCD) assay

CPO activity was determined via the MCD (2-chloro-5,5-dimethyl-1,3-cyclohexanedione, Fisher Scientific, 98%) assay, based on a previously established procedure reported in the literature, using the Shimadzu UV/vis 1900i spectrophotometer.⁴⁵ CPO solution (100 μL) was added to the MCD assay reaction mixture (900 μL, 100 mM potassium phosphate buffer, pH 2.75, 0.1 mM MCD, 20 mM KCl, 2 mM H₂O₂) and substrate conversion was followed by measuring the absorbance of MCD at 278 nm (ε₂₇₈ = 12 200 M⁻¹ cm⁻¹) at 25 °C. The CPO solution was appropriately diluted to give linear enzyme kinetics. One activity unit (1 U) is defined as 1 μmol MCD converted in 1 min.

For more information regarding experimental details, including catalyst testing, analytical methods, and catalyst characterization, please see SI.

Results and discussion

Chemo-catalyst design for peroxy-enzymatic utilization

Our initial investigations aimed to establish chemo-enzymatic indole oxidation reaction conditions which optimized H₂O₂ synthesis rates, metal stability, and indole solubility (Fig. S1–10 and Tables S1, 2). Following this, we evaluated the efficacy of a 1% Au_xPd_y/TiO₂ (where Au : Pd = x : y by wt.) catalyst series, prepared by a previously established sol-immobilization method,⁴³ towards the direct synthesis and subsequent degradation of H₂O₂ under the optimized reaction conditions (Fig. 2A and B).⁴¹ Chemo-catalytic H₂O₂ production was assessed over extended reaction times (up to 3 h), with the initial rate of H₂O₂ synthesis determined following 5 minutes reactions, where it is possible to exclude contributions from competitive degradation pathways or reagent limitation concerns (Fig. 2A). We have previously identified the initial rate of H₂O₂ synthesis as a key performance metric to understand chemo-catalyst efficacy when utilized in peroxy-enzymatic tandem systems.^39,40 However, it is clear that one must consider the limitations of such an approach, given the potential for dynamic chemo-catalyst performance over extended reaction times.

Fig. 2 Catalytic activity of the 1% Au_xPd_y/TiO₂ series towards H₂O₂ synthesis and degradation. (A) H₂O₂ productivity and degradation determined over 5 minutes and 2 h reactions, respectively. (B) H₂O₂ production determined as a function of reaction time. H₂O₂ synthesis reaction conditions: catalyst (0.001 g), 2 bar (80% H₂ in air), potassium phosphate buffer (10 mM, 10 mL, pH 5.0) with 50 vol% t-BuOH, 250 rpm, 20 °C, 0–3 h. H₂O₂ degradation reaction conditions: catalyst (0.001 g), H₂O₂ (2000 ppm), 2 bar (80% H₂ in N₂), potassium phosphate buffer (10 mM, 10 mL, pH 5.0) with 50 vol% t-BuOH, 250 rpm, 20 °C, 2 h. Note: error bars within the boundary a given data point are not observable.

In keeping with previous reports, the incorporation of Au into Pd domains offered enhanced H₂O₂ synthesis rates (57–67 mmol_H2O2 g_cat⁻¹ h⁻¹ for bimetallic formulations), relative to the monometallic counterparts (3 and 24 mmol_H2O2 g_cat⁻¹ h⁻¹ for the Au and Pd monometallic analogues, respectively).^46–48 Such improvements have previously been attributed to the ability of Au to electronically modify Pd species and disrupt contiguous Pd ensembles.^49,50 Indeed, the performance of immobilized Pd-based catalysts towards H₂O₂ direct synthesis is known to be highly dependent on Pd speciation, with Pd²⁺ species typically reported to offer high selectivity while Pd⁰ offer higher activity.^51–53 XPS analysis of the as-prepared 1% Au_xPd_y/TiO₂ catalyst series (Fig. S11 and Table S3), revealed that the 1% Pd/TiO₂ formulation consisted of predominately Pd²⁺ surfaces, which can be attributed to the employed oxidative heat treatment (static air, 400 °C, 3 h, 10 °C min⁻¹).⁵⁴ Upon incorporation of Au a shift towards Pd⁰ was observed, indicating electronic modification of Pd species upon alloy formation, which correlates with the observed enhanced catalytic performance of the AuPd formulations. However, it is important to note that XPS analysis of the as-prepared formulations is not representative of the metal species in situ, particularly given the highly reducing nature of the reaction environment (80% H₂ in air).

Interestingly, evaluating H₂O₂ production over extended reaction times (3 h) revealed that the 1% Pd/TiO₂ formulation offered enhanced steady state activity towards H₂O₂ direct synthesis (6.3 mmol_H2O2 g_cat⁻¹ h⁻¹), relative to the bimetallic AuPd analogues (1.6–2.8 mmol_H2O2 g_cat⁻¹ h⁻¹) (Fig. 2B and Table S4). While this was not expected based on our initial rate measurements (Fig. 2A), this does align with the higher H₂O₂ degradation rates observed for the bi-metallic formulations, relative to the mono-metallic counterparts (0–13 and 284–315 mmol_H2O2 g_cat⁻¹ h⁻¹ for the mono- and bi-metallic catalysts, respectively) (Fig. 2A). However, given the rapid peroxy-enzymatic utilization, relative to H₂O₂ generation, chemo-catalytic H₂O₂ selectivity may be considered to be of limited concern in the tandem system. Indeed, we have previously demonstrated that under conditions where chemo-catalytic activity is rate-limiting, that is the rate of in situ H₂O₂ generation will govern the overall process efficiency of the tandem system.^39,40

We subsequently evaluated the catalytic performance of the 1% Au_xPd_y/TiO₂ series when used in conjunction with CPO (600 nM, 15 U mL⁻¹) for the selective oxidation of indole to 2-oxindole via in situ H₂O₂ synthesis (Fig. 3). The highest chemo-enzymatic cascade efficacy was offered by the 1% Au₁Pd₁/TiO₂ catalyst, reflecting the enhanced initial rate of H₂O₂ synthesis offered by this formulation (Fig. 2A and Table S1). Notably, complete substrate conversion (>99% at 3 h) and exceptional selectivity towards 2-oxindole (93%) was achieved upon employing the 1% Au₁Pd₁/TiO₂ catalyst (Fig. S12). Importantly, each chemo-catalyst formulation offered 2-oxindole selectivity comparable to reported values for CPO with pre-formed H₂O₂, indicating the presence of the metal catalyst does not influence enzymatic selectivity under one-pot reaction conditions (Fig. S12).²⁶

Fig. 3 Catalytic activity of the 1% Au_xPd_y/TiO₂ series towards indole oxidation in conjunction with CPO, as a function of catalyst formulation. Indole oxidation reaction conditions: catalyst (0.001 g), CPO (600 nM, 15 U mL⁻¹), indole (10 mM), potassium phosphate buffer (10 mM, 10 mL, pH 5.0) with 50 vol% t-BuOH, 2 bar (80% H₂ in air), 250 rpm, 20 °C, 0–3 h.

To investigate the selectivity of the tandem system further, we deconvoluted chemo-catalytic and enzymatic contributions towards unselective reaction pathways (Fig. S13, 14 and Table S5). Chemo-catalytic experiments (in the absence of CPO) revealed the 1% Au_xPd_y/TiO₂ catalyst series offered negligible activity towards indole oxidation and hydrogenation pathways, as well as 2-oxindole over-oxidation. While chemo-enzymatic experiments conducted with 2-oxindole revealed CPO contributions towards over-oxidation pathways (5% 2-oxindole conversion), indicating that the selectivity of the tandem system was enzymatically controlled (Fig. S14). Further control experiments established the significant improvement in indole oxidation activity achieved in the presence of H₂ and O₂, in comparison to that when either gaseous reagent (H₂ and O₂) was used separately (Table S6).

Notably, significantly higher indole oxidation rates were also observed via the chemo-enzymatic approach than could be achieved when utilizing pre-formed H₂O₂ at concentrations comparable to that offered by the chemo-catalyst (Table S7), where H₂O₂ was charged in a single injection to initiate the reaction. Further experiments were conducted via the continuous addition of ex situ H₂O₂ from an external reserviour (1.36 mM) using a perastaltic pump (50 mL h⁻¹), where oxidant supply rates were aligned with chemo-catalytic H₂O₂ synthesis rates over the 1% Au₁Pd₁/TiO₂ (1.1 µmol_H2O2 min⁻¹) (Fig. S15A and B). The pump rate was selected in alignment with previous studies which utilize continuous oxidant addition, allowing appropriate benchmarking of chemo-enzymatic system.²⁸ Despite offering improved enzymatic performance and substrate turnover rates, relative to the single injection of H₂O₂, the rapid dilution of valuable reaction products and generation of excessive solvent waste is of significant concern when considering the green credentials and economic viability of this stategy (Fig. S15B). Additional experiments were conducted which employed GOx for in situ H₂O₂ generation (Fig. S15C), however enzymatic turnover rapidly reached extinction. Such deactivation can be associated with gluconic acid formation at 1 : 1 molar ratio with H₂O₂, resulting in a rapid acidification of the reaction media under the weakly buffered reaction conditions (pH 5.0, 10 mM potassium phosphate buffer). This further highlights the improved co-catalyst stability offered by the chemo-catalytic approach, hence facillitating a broader operating window for in situ H₂O₂ synthesis.

Evolving chemo- and bio-catalytic activity under reaction conditions

Upon closer inspection of the chemo-enzymatic indole oxidation rates offered by the 1% Au_xPd_y/TiO₂ catalysts (Fig. 3), the Pd-rich formulations were found to offer significantly higher cascade activity, relative to the Au-rich analogues, than could have been predicted based on measurements of initial H₂O₂ production rates (Fig. 2A and Table S1). Given the disparate reaction times at which H₂O₂ synthesis (5 minutes) and indole oxidation (3 h) experiments were conducted, we were motivated to investigate the potential evolution of chemo-catalytic activity under reaction conditions. We subsequently measured the initial rate of H₂O₂ synthesis of the 1% Au_xPd_y/TiO₂ catalyst series over multiple uses (Fig. S16), revealing increased activity of the Pd-rich formulations following exposure to reaction conditions, which correlates with the exceptional tandem performance offered by these catalysts. Interestingly, the 1% Au₃Pd₁/TiO₂ formulation exhibited a significant reduction towards H₂O₂ synthesis rates over multiple uses (57% activity reduction upon third use), which aligns with the comparatively poor chemo-enzymatic efficacy offered by this formulation. ICP-MS analysis of post-reaction solutions (Table S.2) demonstrated minimal leaching of metal species (≤0.1% Pd leaching for the 1% Au_xPd_y/TiO₂ series, Au leaching was not observed), suggesting chemo-catalytic deactivation cannot be attributed to loss of active metal species. Interestingly, XPS analysis of the 1% Au_xPd_y/TiO₂ catalysts revealed a complete shift towards Pd⁰ species following model H₂O₂ synthesis reactions, which were conducted with ten times the typical catalyst mass (0.01 g), and in the absence of CPO and indole (Fig. S17). Such findings highlight the dependency of tandem performance on the dynamics of chemo-catalytic activity under reaction conditions.

To explore the temporal dependence of Pd speciation under reaction conditions, further model H₂O₂ synthesis experiments were conducted over a range of reaction times (5–60 minutes), with a particular focus on the 1% Au₁Pd₁/TiO₂ and 1% Pd/TiO₂ formulations (Fig. 4B and Fig. S18). Interestingly, a rapid shift towards Pd⁰ was observed within 5 minutes of reaction for both catalysts, revealing that Pd⁰ is the prevailing chemo-catalytically active species under cascade conditions. Such findings indicate the enhanced CPO turnover rates achieved in conjunction with the Pd-rich formulations (Fig. 3), relative to the Au-rich catalyst, correspond with an enhanced proportion of surface Pd⁰ species and consequently promoted H₂O₂ direct synthesis activity. However, under circumstances where H₂O₂ productions rates are solely governed by surface Pd⁰ species, one would predict the 1% Pd/TiO₂ formulation to offer the highest efficacy in the tandem system. Therefore, the role of AuPd species in maintaining high H₂O₂ synthesis rates is considered critical, as highlighted by the enhanced indole oxidation rates offered by the 1% Au₁Pd₁/TiO₂ formulation, relative to the 0.5% Pd/TiO₂ monometallic counterpart (83% and 33% indole conversion for 1% Au₁Pd₁/TiO₂ and 0.5% Pd/TiO₂ catalyst at 2 h, respectively) (Table S6). Such findings underpin the critical requirement to design chemo-catalysts which offer high H₂O₂ synthesis activity, rather than high H₂O₂ selectivity, under tandem conditions which impose chemo-catalytic activity as rate-limiting, relative to peroxy-enzymatic utilization.

Fig. 4 Structure and morphology of the 1% Au₁Pd₁/TiO₂ catalyst. (A) HAADF AC-STEM X-EDS imaging of the as-prepared (i) and used (ii) catalyst, following a model H₂O₂ synthesis reaction over 2 h. (B) Surface atomic compositions in the Pd (3d) region of the as-prepared (i) and used catalyst (ii–iii), following a model H₂O₂ synthesis reaction over 5 minutes (ii) and 2 h (iii). Key: Au⁰ (orange), Pd⁰ (blue), Pd²⁺ (green), Ca²⁺ (pink). Model H₂O₂ direct synthesis reaction conditions: catalyst (0.01 g), 2 bar (80% H₂ in air), potassium phosphate buffer with 50 vol% t-BuOH (10 mM, pH 5.0, 10 mL), 250 rpm, 20 °C, 0.083–2 h.

Building further on these studies, and with a focus on the 1% Au₁Pd₁/TiO₂ formulation, we next investigated the influence of evolving chemo-catalytic activity towards tandem performance (Fig. S19). Such studies first exposed the chemo-catalyst to model H₂O₂ synthesis reaction conditions over a standard 2 h reaction, prior to subsequent use in the chemo-enzymatic cascade. Notably, these studies revealed the instability of the chemo-catalyst with a considerable loss in indole conversion, compared to that observed when utilising the as-prepared catalyst (4660 h⁻¹ and 2860 h⁻¹ for the fresh and used catalyst at 2 h, respectively). This contrasts our earlier studies, which established that chemo-catalytic activity towards H₂O₂ was maintained upon exposure to reaction conditions (Fig. S16). However, it is important to note H₂O₂ direct synthesis experiments conducted over extended reaction times also revealed a reduction towards H₂O₂ production upon chemo-catalyst reuse (Fig. S20). ICP-MS analysis of post-reaction solutions revealed the increased Pd leaching of the 1% Au₁Pd₁/TiO₂ catalyst upon second use (Table S8), relative to that observed upon initial exposure to the reaction conditions (0.1% and 0.7% Pd leaching upon first and second use for the catalyst at 2 h, respectively, Au leaching was not observed). While such findings clearly indicate increased mobility of the supported nanoparticles, such extensive chemo-catalytic deactivation cannot be entirely attributed to the observed Pd leaching.

To probe the surface morphology and structure of the 1% Au₁Pd₁/TiO₂ formulation, HAADF AC-STEM X-EDS analysis was conducted on the fresh and used catalyst, following a model H₂O₂ synthesis reaction over 2 h (Fig. 4A, with additional data reported in Fig. S21–28). X-EDS imaging revealed the presence of well-defined AuPd nanoalloy structures in the fresh catalyst, with particle sizes ranging from 2–10 nm, in addition to a population of Pd-rich nanoparticles ranging from 4–11 nm and highly dispersed sub-nano monometallic Pd species (Fig. S21–23). In contrast, the used 1% Au₁Pd₁/TiO₂ catalyst exhibited populations of large aggregates (≤50 nm), with the highly dispersed (<1 nm) monometallic Pd species absent, demonstrating significant nanoparticle agglomeration under reaction conditions (Fig. S24–28). Unsurprisingly, X-EDS imaging also revealed deposited K and P on the chemo-catalyst surface associated with surface adsorption from the potassium phosphate buffer (Fig. S27). As such, we attribute the observed loss of chemo-catalytic activity, and therefore chemo-enzymatic performance, to changes in nanoparticle structure and morphology under reaction conditions. These studies clearly highlight the need for improved approaches to chemo-catalyst design, with a particular focus on stability.

We subsequently investigated the evolution of enzymatic activity under reaction conditions, with monochlorodimedone (MCD) assays employed to determine CPO activity following chemo-enzymatic indole oxidation experiments with the 1% Au_xPd_y/TiO₂ catalyst series (Fig. S29). Given peroxidative inactivation proceeds concurrently with the conversion of substrate, CPO deactivation has been evaluated with respect to enzyme turnovers (Fig. 5A).^55,56 Interestingly, the extent of CPO deactivation was observed to be highly dependent on the chemo-catalyst formulation, with the Pd-containing and monometallic Au catalysts resulting in disparate CPO deactivation profiles. Unsurprisingly, CPO deactivation proceeded steadily with substrate turnover when used in conjunction with Pd-containing formulations, associated with peroxy-enzymatic turnover. However, the extensive CPO deactivation observed upon utilizing the 1% Au/TiO₂ catalyst cannot be attributed to peroxidative inactivation, owing to the minimal H₂O₂ production rates and consequently poor cascade efficacy offered by this formulation. Such findings highlight the potential for CPO deactivation to proceed via non-peroxy-mediated pathways under chemo-enzymatic cascade reaction conditions. Importantly, contributions of the reaction substrates and products towards the measured CPO activity have been accounted for via an assay inhibition model (see experimental methods in the SI).

Fig. 5 Extent of CPO deactivation, as determined via the MCD assay. (A) Chemo-enzymatic indole oxidation reactions with 1% Au_xPd_y/TiO₂ catalysts. (B) CPO deactivation as a function of reaction conditions, as determined via the MCD assay, in the absence of the chemo-catalyst and indole. (A) Indole oxidation reaction conditions: catalyst (0.001 g), CPO (600 nM, 15 U mL⁻¹), indole (10 mM), potassium phosphate buffer (10 mM, 10 mL, pH 5.0) with 50 vol% t-BuOH, 2 bar (80% H₂ in air), 250 rpm, 20 °C, 0–3 h. (B) Reaction conditions: (i) CPO (600 nM, 15 U mL⁻¹), potassium phosphate buffer (10 mL, 10 mM, pH 5.0), ambient pressure, 20 °C, 250 rpm, 0–3 h. (ii) CPO (600 nM, 15 U mL⁻¹), potassium phosphate buffer (10 mL, 10 mM, pH 5.0) with 50 vol% t-BuOH, ambient pressure, 20 °C, 250 rpm, 0–3 h. (iii) CPO (600 nM, 15 U mL⁻¹), potassium phosphate buffer (10 mL, 10 mM, pH 5.0), 2 bar (80% H₂ in air), 20 °C, 250 rpm, 0–3 h. (iv) CPO (600 nM, 15 U mL⁻¹), potassium phosphate buffer (10 mL, 10 mM, pH 5.0) with 50 vol% t-BuOH, 2 bar (80% H₂ in air), 20 °C, 250 rpm, 0–3 h. MCD assay reaction conditions: post-reaction aliquot (100 μL), assay solution (900 μL, 0.1 mM MCD, 20 mM KCl in 100 mM, pH 2.75 potassium phosphate buffer), absorbance monitored at 278 nm (ε₂₇₈ = 12 200 M⁻¹ cm⁻¹), 25 °C.

To begin deconvoluting contributions towards enzyme deactivation, CPO was exposed to the employed reaction parameters in a stepwise manner, in the absence of chemo-catalyst and indole substrate (Fig. 5B). While the key reaction parameters contributed minimal CPO deactivation when assessed individually (Fig. 5B, i–iii), the combination of the pressurized reaction environment (2 bar, 80% H₂ in air) and organic cosolvent (50 vol% t-BuOH) resulted in extensive CPO deactivation (77%, 3 h). Pressure-induced enzyme deactivation mechanisms have been reported to proceed via promoted exchange between protein bound water molecules and the bulk solvent environment.^57–59 We postulate such effects perpetuate CPO deactivation in this study, with enhanced t-BuOH penetration into the protein bonding network at elevated pressure driving the observed hydrophobic/pressure co-action effect.

To investigate the potential for metal species to contribute towards enzyme deactivation, model studies were conducted to expose CPO to leached metal species from the 1% Au₁Pd₁/TiO₂ catalyst and metal chloride salts at various concentrations (PdCl₂, HAuCl₄) (Table S9). Such experiments were conducted in an aqueous buffer system (10 mL, 10 mM, pH 5.0 potassium phosphate buffer) at ambient pressure, thus removing pressure and organic solvent contributions towards CPO deactivation. We previously reported the capacity of homogenous metal species to deactivate a closely-related UPO enzyme,⁴⁰ however, CPO exhibited excellent stability in the presence of aqueous metal species.

Optimization of the tandem system and substrate scope

Following our initial studies, the 1% Au₁Pd₁/TiO₂ formulation emerged as the most promising chemo-catalyst candidate for application in the tandem system. To enable further improvements of the overall process efficiency, we optimised the kinetic balance between in situ H₂O₂ production rates and subsequent peroxy-enzymatic utilization. With a particular focus on the 1% Au₁Pd₁/TiO₂ formulation, the chemo-catalyst: enzyme ratio was modified by increasing the mass of chemo-catalyst employed, facilitating a simple approach to promote in situ H₂O₂ synthesis rates (Fig. 6A and Fig. S30). Interestingly, enzymatic TOF scaled linearly with increased chemo-catalyst mass from 1 to 3 mg (Fig. S31), reaching a maxima of 18 600⁻¹ when utilising the highest catalyst mass, with near full indole conversion achieved (98% conversion, 92% selectivity to 2-oxindole) after 1 h of reaction. These observations further highlight the high oxidant utilization efficiency of the enzyme, as well as the limited contribution of unselective H₂O₂ consumption via chemo-catalytic degradation pathways.

Fig. 6 (A) Catalytic activity of 1% Au₁Pd₁/TiO₂ towards indole oxidation, when used in conjunction with CPO, as function of chemo-catalyst mass. (B) Extent of CPO deactivation, as a function of CPO turnovers, following indole oxidation reactions with varying chemo-catalyst mass. Indole oxidation reaction conditions: catalyst (0.001–0.004 g), CPO (600 nM, 15 U mL⁻¹), indole (10 mM), 2 bar (80% H₂ in air), potassium phosphate buffer (10 mM, 10 mL, pH 5.0) with 50 vol% t-BuOH, 250 rpm, 20 °C, 0–3 h. MCD assay reaction conditions: post-reaction aliquot (100 μL), assay solution (900 μL, 0.1 mM MCD, 20 mM KCl in 100 mM, pH 2.75 potassium phosphate buffer), absorbance monitored at 278 nm (ε₂₇₈ = 12 200 M⁻¹ cm⁻¹), 25 °C. Note: Red circle indicates complete enzyme deactivation upon utilizing 4 mg chemo-catalyst.

Further increasing chemo-catalyst mass to 4 mg offered the highest initial enzymatic TOF (23 300 h⁻¹ at 0.5 h), however substrate turnover could not be maintained and rapidly reached extinction (76% conversion, 0.75 h). Evaluation of post-reaction enzyme activity (Fig. 6B) revealed this was accompanied with complete enzyme deactivation (100% deactivation, 0.75 h), indicating in situ H₂O₂ production rates exceeded that of peroxy-enzymatic utilization, resulting in rapid oxidative inactivation. This aligns with findings from chemo-enzymatic experiments conducted in the absence of indole, which revealed significantly increased CPO deactivation rates upon switching-off enzymatic oxygen transfer pathways (Fig. S32). Such effects are associated with the characteristic catalase (H₂O₂ degradation) activity of CPO, which is prone to malfunction in the presence of excess oxidant, resulting in enzyme active site denaturing via ‘heme-bleaching’.^55,60,61 As such, this work demonstrates that the promotion of cascade efficacy via enhanced H₂O₂ synthesis rates must operate under chemo-catalytic rate limitations to maintain enzymatic activity.

To probe the potential for in situ enzyme physisorption onto the chemo-catalyst surface, post-reaction CPO activity was assessed in filtered and unfiltered post-reaction aliquots, allowing free and immobilized enzyme contributions to be deconvoluted (Fig. S33). With a particular focus on the 1% Au₁Pd₁/TiO₂ catalyst at loadings of 1 mg and 3 mg, CPO was exposed to key reaction parameters (2 bar N₂, 10 mL, 10 mM, pH 5.0 potassium phosphate buffer with 50 vol% t-BuOH), in the absence of indole and H₂O₂ synthesis reagent gases, which switched-off peroxy-enzymatic turnover. Interestingly, immobilized enzyme accounted for 23% of post-reaction CPO activity when utilizing 1 mg chemo-catalyst, which raised to 58% at 3 mg. Notably, increased enzyme immobilization was accompanied with suppressed CPO deactivation, suggesting in situ physisorption effects mediate enzyme stabilization under the employed reaction conditions. Muñoz-Guerrero et al. reported similar findings, demonstrating enhanced operational stability of CPO following physical immobilization on TiO₂ nanotubes, with the immobilized enzyme offering a 69% improvement in TTN, relative to the free enzyme.⁶² Such findings highlight the complex interplay between peroxidative inactivation and physical stabilization upon varying chemo-catalyst mass, wherein an optimized tandem system carefully balances such effects.

Hitherto, enzymatic turnovers have been limited to 16 670 under chemo-enzymatic cascade conditions, owing to the selected substrate concentration (indole, 10 mM). To demonstrate the capacity of the tandem system to achieve higher TTN, a series of substrate recharging experiments were performed, with a continued focus on the 1% Au₁Pd₁/TiO₂ formulation (Fig. 7 and Fig. S34). Such experiments were carried out with 1, 2, and 3 mg chemo-catalyst, allowing the TTN to be probed as a function of in situ H₂O₂ production rate. For each of the systems investigated, substrate recharging (2 bar, 80% H₂ in air, 10 mM indole) was conducted at intervals equal to the reaction time required to reach near total conversion of indole (as reported in Fig. 6A, e.g. the 1 mg chemo-catalyst experiment was re-charged at 3 h intervals). Interestingly, the resulting enzymatic TTN was highly dependent on the rate of in situ H₂O₂ production, with 2 mg of chemo-catalyst offering the highest enzymatic efficacy (25 000, 33 000, and 25 900 TTN for 1, 2, and 3 mg chemo-catalyst, respectively), corresponding to 19.6 mM of 2-oxindole formation in 3 h. As such, the reported chemo-enzymatic cascade offers highly competitive CPO TTN for indole oxidation, particularly when compared to previously reported systems which exploit in situ generated H₂O₂ via co-enzymatic and electrochemical approaches (Table S10).^63,64 The relative performance of the chemo-catalyst masses investigated is rationalized by considering the trade-off between enzyme TOF and peroxidative inactivation, as a function of H₂O₂ production rate, while the extent of physical stabilization at varying chemo-catalyst masses should also be considered. Indeed, this study further highlights the enhanced enzymatic performance that can be offered via the chemo-enzymatic approach, owing to the highly tuneable nature of in situ H₂O₂ production rates.

Fig. 7 Catalytic activity of 1% Au₁Pd₁/TiO₂ towards indole oxidation, when used in conjunction with CPO, as a function of chemo-catalyst mass, probed via substrate recharging experiments. Indole oxidation reaction conditions: 1% Au₁Pd₁/TiO₂ (0.001–0.003 g), CPO (600 nM, 15 U mL⁻¹), indole (10 mM), potassium phosphate buffer (10 mL, 10 mM, pH 5.0) with 50 vol% t-BuOH, 2 bar (80% H₂ in air), 20 °C, 250 rpm, 0–3 h. Note: Reaction time at which substrate recharging (indole and reagent gases) was conducted indicated by hollow data points.

The developed chemo-enzymatic system was subsequently utilized for the selective oxidation of a range of substituted indoles, chosen for their unique electronic and structural characteristics, allowing the specificity of the tandem system to be assessed. Such experiments were conducted with the 1% Au₁Pd₁/TiO₂ (1 mg) formulation over a standard 2 h reaction (Table 1 and Table S11). Interestingly, CPO offered reduced oxidation activity towards substituted substrates. Van Deurzen et al. reported analogous findings upon utilizing pre-formed H₂O₂ for CPO-mediated synthesis of substituted oxindoles.²⁶ Indeed, the presence of substituent at the indole C-3–7 positions has been reported to sterically hinder access to the heme active site via the narrow hydrophobic channel, relative to non-substituted indole. It is also important to consider the electronic effects of substituents on the resonance stabilized radical cation indole intermediate species in the oxidative mechanism.²⁵ In keeping with our findings thus far, both the chemo-catalytic activity towards the indole-derivatives was not observed and the supply of ex situ H₂O₂ offered significantly lower enzymatic efficacy than in situ generated oxidant (Table S11). Such findings highlight that the versatile nature of CPO-based biocatalysis is maintained when coupled with supported AuPd catalysts under one-pot reaction conditions. However, the reduced chemo-enzymatic efficacy upon utilizing substituted indole is considered critical when designing synthetic routes towards target substrates.

Table 1 Substrate scope of the chemo-enzymatic cascade for the synthesis of substituted 2-oxindoles

Substrate	Substrate conversion/%	2-Oxindole derivative selectivity/%	TON
Substituted Indole oxidation reaction conditions: 1% Au1Pd1/TiO2 (0.001 g), CPO (600 nM, 15 U mL^−1), substrate (10 mM), potassium phosphate buffer (10 mM, 10 mL, pH 5.0) with 50 vol% t-BuOH, 2 bar (80% H2 in air), 250 rpm, 20 °C, 2 h. Note: enzymatic performance reported as TON as experiments did not reach catalytic extinction.
	83	95	13 970
	55	98	9 260
	61	86	8 830
	24	99	4 040
	32	83	4 470

---

Techno-economic analysis

We subsequently conducted a detailed techno-economic analysis of the materials employed for the chemo-enzymatic, co-enzymatic (GOx), and continuous H₂O₂ addition strategies (Fig. 8, additional data reported in Tables S12–15). Importantly, material cost has been determined for the key components in the generation of 1 ton of 2-oxindole, with the cost associated with post-reaction purification and solvent processing not accounted for in this analysis. Interestingly, the chemo-catalytic approach offered significantly improved economic potential, relative to the co-enzymatic and continuous addition approaches. Unsurprisingly, the co-enzymatic approach suffers from the combined high cost and poor stability of GOx under reaction conditions, rendering this approach economically unviable. Commercial H₂O₂ offers lower cost than that of the chemo-catalyst and H₂ usage combined, however the requirement for excessive solvent to maintain dilute H₂O₂ streams hampers the economic potential of this approach. These drawbacks are further exacerbated by the high cost associated with downstream processing of contaminated solvent waste. Interestingly, this analysis aligns with the resulting E-factors (mass waste/mass product) for each of systems investigated (670, 11 470, and 3250 for the chemo-catalytic, continuous H₂O₂ addition, and co-enzymatic approaches, respectively) (Table S16), with further improvements obtained for the chemo-catalytic approach via substrate recharging experiments (E-factor = 340) (Fig. 7). In summary, the moderate cost of chemo-catalyst preparation is significantly offset by the comparatively low cost of H₂ and significantly reduced solvent usage, which exemplifies the exceptional green credentials and economic viability of this technology, relative to previously established H₂O₂ supply strategies for biocatalytic applications.

Fig. 8 Continuous H₂O₂ addition reaction conditions: H₂O₂ reservoir (1.36 mM), pump (50 mL h⁻¹), CPO (15 U mL⁻¹, 600 nM), indole (10 mM), potassium phosphate buffer (10 mL, 10 mM, pH 5.0) with 50 vol% t-BuOH, ambient pressure, 250 rpm, 20 °C, 0–3 h. Co-enzymatic reaction conditions: glucose oxidase (0.2 U mL⁻¹), glucose (100 mM), CPO (15 U mL⁻¹, 600 nM), indole (10 mM), potassium phosphate buffer (10 mL, 10 mM, pH 5.0) with 50 vol% t-BuOH, ambient pressure, 250 rpm, 20 °C, 0–3 h. Chemo-enzymatic reaction conditions: catalyst (0.001 g), CPO (15 U mL⁻¹, 600 nM), indole (10 mM), potassium phosphate buffer (10 mL, 10 mM, pH 5.0) with 50 vol% t-BuOH, 2 bar (80% H₂ in air), ambient pressure, 250 rpm, 20 °C, 0–3 h. Note 1: * indicates H₂ consumption has been calculated based on estimated H₂ utilization efficiencies with respect to 2-oxindole formation. Note 2: H₂O₂ (407 USD) and H₂ (30–120 USD) cost are not visually observable on the scale provided in the figure.

Conclusion

We have demonstrated the applicability of coupling chemo-catalytic generation of H₂O₂ and CPO to selectively oxidize indoles as an attractive route for the synthesis of pharmaceutically valuable products. The incorporation of Au into Pd nanoparticles offered significantly enhanced H₂O₂ synthesis rates, which facilitated kinetic optimization of in situ oxidant generation and peroxy-enzymatic utilization. Indeed, the 1% Au₁Pd₁/TiO₂ offered 33 000 TTN for selective indole oxidation to 2-oxindole, which is among the most efficacious CPO-based indole oxidation systems reported which utilize in situ generated H₂O₂.^63,64 Indeed, when compared to alternative in situ H₂O₂ production approaches for peroxy-enzymatic utilization, the chemo-enzymatic approach has exceptional potential to be scaled for industrial applications, offering high atom efficiency, economic viability, and compatibility with current reactor technologies. Importantly, the characteristically exceptional regio- and chemo-selectivity of CPO was maintained under one-pot reaction conditions.

Despite offering highly efficient H₂O₂ generation and utilization, we have revealed the potential for chemo- and bio-catalytic deactivation under the tandem system reaction conditions. We have also demonstrated the dynamic nature of metal species, which is proposed to contribute towards the observed loss of chemo-catalytic activity over multiple uses. Therefore, for the chemo-enzymatic system to rival current industrial processes, further improvements in catalyst stability are required to maintain high chemo-catalytic activity over extended lifetimes.

We have also revealed the combination of a pressurized reaction environment and highly organic solvent environment results in extensive enzyme deactivation through an observed co-action effect. As such, efforts to improve the scalability and economic viability of the chemo-enzymatic cascade system should focus on enhancing enzyme stability under operating conditions. Numerous strategies to achieve such effects have been widely reported, with protein engineering, enzyme immobilization, and utilization of extremophilic enzymes representing viable approaches.^65–67 Indeed, this work has demonstrated improved enzyme stability following in situ physisorption at the chemo-catalyst surface, highlighting the potential for further enhancements via robust immobilization strategies, such as covalent anchoring. The development of a heterogeneous chemo-enzymatic tandem system would offer significant industrial and economic advantages, owing to improved ease of catalyst separation from post-reaction mixtures, as well as facilitating the design of continuous/semi-continuous reactor systems.^68,69

In summary, through a holistic and detailed approach we have elucidated a complex array of critical chemo- and bio-catalytic parameters which must be considered to optimise chemo-enzymatic performance (as summarised in Fig. 9). As Gröger and coworkers describes, chemo-enzymatic catalysis imposes the selection of reaction conditions as crucial and evidently nontrivial, owing to the complexities associated with overcoming the conditions gap between chemo- and bio-catalytic components, which must be achieved to realise the vast synthetic potential of such tandem systems.⁷⁰ Indeed, this work highlights the dynamic nature of such catalytic phenomena under reaction conditions, with such understanding considered critical towards developing highly active, stable, and industrially feasible chemo-enzymatic tandem systems.

Fig. 9 Schematic representation of the critical parameters to optimise when combining chemo- and bio-catalysts for tandem system chemical transformations.

Author contributions

A. S. and J. P. conducted catalyst synthesis, testing, and corresponding data processing. A. S., R. J. L., D. J. M., T. E. D. conducted catalyst characterization and corresponding data processing. A. S., R. J. L., and G. J. H. contributed to the design of the study and interpretation. A. S. wrote the manuscript and SI, with all authors commenting on and amending both documents. All authors discussed and contributed to this work.

Conflicts of interest

The authors declare no conflict of interest.

Data availability

All data is available in the manuscript and the supplementary information (SI), which is available at DOI: https://doi.org/10.1039/d5gc05367f.

Acknowledgements

The authors would like to thank the CCI-Electron Microscopy Facility which has been funded in part by the European Regional Development Fund through the Welsh Government, and The Wolfson Foundation. XPS data collection was performed at the EPSRC National Facility for XPS (“HarwellXPS”). A. S., R. J. L. and G. J. H. gratefully acknowledge Cardiff University and the Max Planck Centre for Fundamental Heterogeneous Catalysis (FUNCAT) for financial support.

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