# Enantioselective synthesis of ( R )-citronellal from geraniol with an immobilised copper alcohol oxidase and ene reductase

Beatrice Tagliabue, Christian Heckmann, Rocio Villa, Sacha Grisel, Jean-Guy

Berrin, Mickael Lafond, David Ribeaucourt, Caroline Paul

► To cite this version:

Beatrice Tagliabue, Christian Heckmann, Rocio Villa, Sacha Grisel, Jean-Guy Berrin, et al.. Enantioselective synthesis of ( R )-citronellal from geraniol with an immobilised copper alcohol oxidase and ene reductase. Reaction Chemistry & Engineering, 2025, 10.1039/d5re00034c. hal-05052061

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ARTICLE

# Enantioselective synthesis of (R)-citronellal from geraniol by an immobilised copper alcohol oxidase and ene reductase

Beatrice Tagliabue,ª Christian M. Heckmann,ª Rocio Villa,ª,ᵇ Sacha Grisel,ᶜ,ᵈ Jean-Guy Berrin,ᶜ Mickael Lafond,ᶜ,ᵉ David Ribeaucourt,ᶜ Caroline E. Paulª,\*

Received 00th January 20xx,
Accepted 00th January 20xx

DOI: 10.1039/x0xx00000x

(R)-Citronellal is one of the key chiral intermediates in the synthesis of the isomer (−)-menthol, one of the most commercialised terpenoid flavours worldwide. Enzymatic approaches could represent a less energy-demanding alternative for its synthesis, such as a previously reported bienzymatic cascade starting from inexpensive, commercially available geraniol. A copper radical oxidase (CgrAlcOx) followed by a flavin-dependent ene reductase (OYE2) were used to obtain (R)-citronellal. Here, we use a metal-affinity immobilisation strategy on the His-tagged enzymes for the cascade and we study enzyme recovery and reusability as well as increased solvent tolerance. After screening a panel of resins for enzyme immobilisation and water-immiscible co-solvents, we successfully obtained 95% of (R)-citronellal with 96.9% enantiomeric excess (ee) in a concurrent cascade after 7 h of reaction time, starting from 10 mM of geraniol.

## Introduction

The cyclic terpene (−)-menthol is one of the primary constituent of essential oils from peppermint (Mentha x piperita L.) and corn mint (Mentha arvensis).¹, ² Thanks to its minty aroma, refreshing flavour, cooling and analgesic proprieties,³ this compound has a wide range of applications, spanning from the food and fragrance industries⁴ to the cosmetic⁵ and pharmaceutical sectors.⁶ Synthetic menthol accounts for 60% of the 34,000 metric tons produced per year, with three main chemical routes used for its industrial production (SI Scheme S1).⁶ (R)-Citronellal is a key chiral intermediate in two of these processes to obtain the enantiomer associated with the pleasant minty aroma, (1R,2S,5R)-(-)-menthol.⁶⁻⁸ Among these, the process developed by BASF starts from citral, a mixture of E- and Z-isomers, referred to as geranial and neral, respectively (Scheme S1C). In order to obtain the desired enantiomer of citronellal, an energy intensive distillation of citral to isolated neral is required. This step is followed by asymmetric reduction with a rhodium complex with chiral ligands, achieving only ~87% ee of (R)-citronellal.⁶ An enzymatic process offers a promising alternative, providing higher enantioselectivity at mild conditions.⁹⁻¹¹ Recently, an in vitro enzymatic cascade for the production of (R)-citronellal has been reported by our group,¹² starting from the relatively inexpensive substrate geraniol.

In the first step, geraniol 1 is oxidised to geranial (E)-2 by a copper radical alcohol oxidase (CRO) from Colletotrichum graminicola (CgrAlcOx). This is followed by a reduction step to (R)-citronellal 3 catalysed by an ene reductase of the old yellow enzyme family (OYE2 from Saccharomyces cerevisiae). This one-pot, two-step cascade resulted in a 95.1% conversion of geraniol to (R)-citronellal with 95.9% ee.¹² Formation of the undesired enantiomer likely being due to the known isomerisation of geranial to neral.¹³ Furthermore, the CRO is inhibited by the final product citronellal, requiring a two-step process.

To overcome these issues, we explored immobilisation to enable enzyme recovery and reusability, reduce sensitivity to temperature and pH, and enable use of water-immiscible organic solvents (Scheme 1).¹⁴, ¹⁵ Metal affinity immobilisation is very effective, due to the high affinity of His-tagged enzymes for the metal-chelated resin. This method is commonly employed for protein purification, often affording up to 95% purity in just one purification step.¹⁶

<figure><img src="image_2.png" /><figcaption>**Scheme 1.** CRO-OYE2 enzymatic cascade for the synthesis of (R)-citronellal 3 starting from geraniol 1. The first step consists in the oxidation of geraniol 1 to geranial (E)-2, further reduced in the second step to (R)-citronellal 3. CRO = copper radical alcohol oxidase (CgrAlcOx); HRP = horseradish peroxidase, CAT = catalase, GDH = glucose dehydrogenase, NAD(P) = nicotinamide adenine dinucleotide (phosphate).</figcaption></figure>

<footnote>ª Biocatalysis section, Department of Biotechnology, Delft University of Technology, van der Maasweg 9, 2629HZ Delft, The Netherlands; c.e.paul@tudelft.nl</footnote>

<footnote>ᵇ Current affiliation: Department of Biochemistry and Molecular Biology B and Immunology, Faculty of Chemistry, University of Murcia, 30100 Murcia, Spain</footnote>

<footnote>ᶜ INRAE, Aix Marseille Univ, BBF, Biodiversité et Biotechnologie Fongiques, Marseille, France</footnote>

<footnote>ᵈ INRAE, Aix Marseille Univ, 3PE, Marseille, France</footnote>

<footnote>ᵉ Marseille Univ, CNRS, Centrale Marseille, iSm2, 13013 Marseille, France.</footnote>

<footnote>Supplementary Information available: [details of any supplementary information available should be included here]. See DOI: 10.1039/x0xx00000x</footnote>
Compared to other immobilisation approaches, immobilising via metal affinity has less impact on the enzyme structure, resulting in a higher retained activity.¹⁴ The cost-effectiveness and straightforward implementation make it an attractive option for immobilising enzymes for industrial applications, and has been previously reported as an effective method for both batch and flow biocatalytic processes,¹⁷⁻¹⁹ including in the recently reported enzymatic synthesis of islatravir by Merck.²⁰ Considering these advantages, we developed a metal-affinity immobilisation approach to facilitate a one-pot concurrent cascade for geraniol conversion to (R)-citronellal (Scheme 1), using a biphasic system with heptane as both substrate reservoir and product sink.

## Results and discussion

The overall enzymatic cascade can be seen in **Scheme 1**. CgrAlcOx is mostly inactive in its resting state, empirically, CROs are generally activated using peroxidases, commonly from horseradish (HRP). Of note, in their natural context, the CRO is most likely activated by a natural fungal peroxidase partner.²¹ Catalase (CAT) is also required for preventing the accumulation of H₂O₂, which is deleterious to enzymes, by catalysing its dismutation into oxygen and water.²² Additionally, glucose dehydrogenase (GDH) is required to recycle the nicotinamide adenine dinucleotide (NADPH) cofactor in the OYE-catalysed step. To avoid potential limitations in the HRP-mediated CgrAlcOx activation, HRP was not immobilised for the first step. For the second step, OYE2 was co-immobilised with GDH for cofactor recycling, as already reported for other ene reductases.²³

We first produced and purified (Figure S1) or purchased all enzymes involved, and measured their individual activities (Table S1). We then proceeded to screen different polyacrylic carriers from Purolite, Sunresin, and ChiralVision (Table S2) for the immobilisation of CgrAlcOx as well as of OYE2 with GDH. The obtained immobilisation efficiency was above 70% in all cases except OYE2-GDH on IB-His-4 after a 2-hour immobilisation (Figure S2). We then turned to reaction performance to select a suitable resin for the CRO and OYE2-GDH. To this end, we evaluated the relative amount of desired product formed under the reaction conditions, considering separately the CRO-catalysed geraniol oxidation step and the OYE-catalysed geranial reduction step (Figure S3). Commercial citral (62% geranial (E)-2 and 38% neral (Z)-2, see GC chromatogram Figure S7) was used as the substrate for the second step. We favoured this method over a direct activity assay because of difficulties in finding an effective and reproducible way to remove the beads, as reported in other studies,¹⁸ and because of the lack of an accurate quantification method, especially for co-immobilised enzymes.

Initial reactions carried out in buffer revealed that both product and substrate were absorbed by the beads, raising concerns about product recovery from the carrier and making quantitative analysis challenging (see GC chromatograms Figure S8-9). To address this issue, a biphasic system was selected based on the hydrophobic properties of the substrate and

products. In this system, the organic solvent serves as a substrate reservoir and product sink, and preventing absorption by the resin. In addition, the biphasic system might also help avoiding the inhibition of CgrAlcOx by the final product (R)-citronellal, as previously reported,¹² allowing for both steps of the cascade to be carried out concurrently.

Taking this into account, a screening for geranial and citronellal formation with immobilised enzymes was carried out in a biphasic system using 20% v/v heptane (Figure 1). The highest amount of geranial (E)-2 obtained from geraniol 1 oxidation was 81% with CgrAlcOx (5 mgCgrAlcOx / gdry resin) immobilised on Seplife/Ni after 30 min of reaction time. Starting from citral, the highest amount of (R,S)-citronellal 3 obtained was 43% with OYE2 and GDH (10 mgOYE2 and 3 mgGDH / gdry resin) co-immobilised on Chromalite/Co after 5 h of reaction time. OYE2 can also catalyse the reduction of neral (Z)-2 to (S)-citronellal, resulting in a decrease of ee over time (Figure S4).

<figure><img src="image_3.png" /><figcaption>**Figure 1.** Resin screening: monitoring of products formation for the reaction catalysed by the immobilised enzymes; each enzymatic system evaluated separately (system a: CgrAlcOx, system b: OYE2 + GDH). The relative amount of geranial obtained from geraniol oxidation and the amount of (R,S)-citronellal obtained from citral reduction were used to select the carrier. Conditions a (CRO-catalysed geraniol oxidation): 50 mg<sub>dry</sub> resin immobilised CgrAlcOx, 10 mM geraniol (as 1% v/v in acetone), 360 U/mL catalase, 12 µM HRP in 50 mM NaPi buffer pH 8.0 and 20% v/v heptane, 30 min, 23 °C, 200 rpm. Conditions b (OYE2-catalysed citral reduction): 50 mg<sub>dry resin</sub> co-immobilised OYE2 and GDH, 40 mM glucose, 1 mM NADP<sup>+</sup>, 20 mM citral in acetone (as 1% v/v in acetone) in 100 mM KPi buffer at pH 8.0 and 30% v/v MTBE, 5 h, 25 °C, 150 rpm.</figcaption></figure>

To improve the conversion of the second step, different water-immiscible co-solvents (Table S3) were screened for the OYE2-catalysed step, (Figure 2A). Among the co-solvents tested, heptane showed the best performance, reaching a relative
amount of 57% of citronellal 3, after 5 h, starting from citral and the highest *ee* of 81%. This solvent seems thus well-tolerated by the enzymes for the second step of the cascade, and is suitable for minimising the conversion of neral (Z)-2 to the undesired (S)-citronellal compared to the other solvents tested. In addition, the influence of the amount of heptane was evaluated (Figure 2B). Using a lower concentration of 20% v/v heptane (Figure 2B), the relative amount of citronellal formed was consistently higher for three runs carried out with the same batch of immobilised enzyme.

<figure><img src="image_4.png" /><figcaption>**Figure 2.** Influence of solvents. (A) Solvent screening for the OYE-catalysed citral reduction. Reaction conditions: 50 mg<sub>dry resin</sub> co-immobilised OYE2 and GDH, 40 mM glucose, 1 mM NADP+, 20 mM citral (as 1% v/v in acetone) in 100 mM KPi buffer at pH 8.0 and 30% of organic solvent, 5 h, 25 °C, 150 rpm. (B) Reusability of OYE2 and GDH immobilised on Chromalite/Co with different heptane concentrations. Reaction conditions: 50 mg<sub>dry resin</sub> co-immobilised OYE2 and GDH, 100 mM glucose, 1 mM NADP+, 20 mM citral (as 1% v/v in acetone) in 100 mM KPi buffer at pH 8.0 and 20, 30 or 50% v/v heptane, 5 h, 25 °C, 150 rpm</figcaption></figure>

Enzyme recovery and reuse is one of the main advantages of using immobilisation techniques, making the process more cost-effective and appealing for industrial applications.²⁴ With the same amount of solvent, the reusability of the immobilised CgrAlcOx on Seplife/Ni was also tested, but proved to be lower, with the amount of geranial formed decreasing from 80% to

37% after the second run (II) and to 32% after three runs (III) (Figure 3).

<figure><img src="image_5.png" /><figcaption>**Figure 3.** Reusability of CgrAlcOx immobilised on Seplife/Ni. Reaction conditions: 50 mg<sub>dry resin</sub> of immobilised CgrAlcOx, 10 mM of geraniol (as 1% v/v in acetone), 360 U/mL of catalase, 3 µM of HRP in 100 mM KPi buffer at pH 8.0 and 20% v/v of heptane, 1 mL volume, 25 °C, 30 min, 200 rpm</figcaption></figure>

Immobilising enzymes using their His-tag allows for both purification and immobilisation in a single step,²⁵ avoiding chromatography steps for enzyme purification, that can increase the overall process costs of around one order of magnitude.²⁶ After measuring enzyme specific activity of the cell-free extracts (CFEs) of OYE2 and GDH and comparing them to the purified enzymes (Table S1), we approximated that around 10% of the proteins in the CFE were the protein of interest. To reproduce the ratio used for co-immobilisation of purified enzymes (i.e., 10 mg OYE2, 3 mg GDH), we performed immobilisation with 100 mg of CFE containing OYE2 and 30 mg of CFE containing GDH. Using this resin, we achieved the same product quantity in a 5-h reaction starting from citral (Figure S5): 57% of citronellal with 85% *ee* with immobilised CFEs compared to 58% of citronellal with 80% *ee*, with the purified enzymes. The successful one-step purification and co-immobilisation of OYE2 and GDH demonstrated the possibility of using the immobilised CFE for the cascade.

When combining the two steps with CgrAlcOx immobilised on Seplife/Ni and OYE2-GDH immobilised on Chromalite/Co, we obtained 95% of citronellal with 96.9% *ee* in a 7-hour one-pot concurrent cascade (Figure 4). This result suggests that the previously reported CgrAlcOx inhibition⁸ can be avoided using immobilised enzymes in a biphasic system with 20% v/v heptane. This improvement might be explained by a protecting effect of heptane in which citronellal preferentially resides. In this hydrophobic solvent, the hydration rate of citronellal is likely to be lower, limiting the formation of the *geminal*-diol that we thought caused CgrAlcOx inhibition.¹², ²⁷ Over time, minor (R)-citronellol formation was observed, which appears to be catalysed by the GDH (see GC chromatogram Figure S15).
<figure><img src="image_6.png" /><figcaption>**Figure 4.** One-pot enzymatic CRO-OYE2 cascade time course. Reaction conditions: 50 mg<sub>dry resin</sub> immobilised CgrAlcOx and 50 mg<sub>dry resin</sub> co-immobilised OYE2<sub>CFE</sub> and GDH<sub>CFE</sub>, 360 U/mL catalase, 3 µM HRP, 10 mM geraniol (as 1% v/v in acetone), 100 mM glucose, 1 mM NADP<sup>+</sup>, in 100 mM KPi buffer at pH 8.0, and 20% v/v heptane, 25 °C, 180 rpm</figcaption></figure>

Lastly, we evaluated the reusability of the immobilised enzymes for the full cascade (Figure 5A). After the second run (II), the relative amount of (R+S)-citronellal dropped to 35%, and a further decrease to 12% and 9% after three (III) and four (IV) runs, respectively. It should also be noted that the reusability of CgrAlcOx was lower than for OYE2-GDH (Figures 2B and 3), indicating that CgrAlcOx is likely contributing more to the lack of enzyme re-usability for the full cascade. The poor reusability of the system could be due to the reversible nature of metal affinity immobilisation, rendering this type of immobilisation more prone to leaching compared to other methods, such as covalent immobilisation.¹² It is likely that leaching of the enzymes from the solid support is responsible for this decrease in the relative amount product formed, although a lack of enzyme stability (due to enzyme inactivation or copper leaching) during the reaction cannot be ruled out.

<figure><img src="image_7.png" /><figcaption>**Figure 5.** Evaluation of (A) reusability of the immobilised enzymatic system and (B) substrate scale up, in one-pot cascade. Reaction conditions: 50 mg<sub>dry resin</sub> immobilised CgrAlcOx and 50 mg<sub>dry resin</sub> co-immobilised OYE2<sub>CFE</sub> and GDH<sub>CFE</sub>, 360 U/mL catalase, 3 µM HRP, 10 or 20 mM geraniol in acetone (final concentration, 1% v/v), 100 mM glucose, 1 mM NADP<sup>+</sup>, in 100 mM KPi buffer at pH 8.0, and heptane (20% v/v), 7 or 18 h, 25°C, 180 rpm</figcaption></figure>

Finally, we successfully scaled up the substrate concentration to 20 mM of geraniol, achieving 76% conversion to citronellal with 96.7% *ee* after 7 h. Extending the reaction time to 18 h (overnight) further increased the relative amount of citronellal to 86% with 96.6% *ee* (Figure 5B and Figure S6). These findings confirm that CgrAlcOx is not fully inhibited by the presence of (R)-citronellal. A small amount of neral was consistently observed throughout the reaction, likely due to its presence as an impurity in the geraniol substrate, contributing to the formation of (S)-citronellal. Additionally, geraniol may undergo isomerisation to neral during the course of the reaction,¹³ which could further explain the slight decrease in *ee* values over time.

## Conclusions

With the aim of using an immobilisation approach to enhance enzyme stability and reusability, the selected system consisted of CgrAlcOx immobilised on Seplife® Chelex 7350/Ni from Sunresin, and OYE2 and GDH co-immobilised on Chromalite MIDA/M/Co from Purolite®. Using 20% (v/v) heptane as a co-solvent, we obtained 95% (R)-citronellal with 96.9% *ee* after 7 h of reaction time in a one-pot concurrent cascade starting from 10 mM geraniol. Increasing geraniol concentration from 10 to 20 mM resulted in 76% and 86% (R)-citronellal after 7 and 18 h, respectively. This indicates the potential of the system in avoiding the previously reported CgrAlcOx inhibition by the final product,¹² also suggesting the feasibility of further substrate scale-up. Unfortunately, the reusability of this system was low, with a steep decrease in product formation observed after two or three runs, potentially due to enzyme leaching from the resin, suggesting the need to explore different immobilisation approaches, such as covalent immobilisation.²⁸

## Author contributions

C.E.P. conceptualised the study. B.T. and C.M.H. produced enzymes, performed the immobilisation and experiments. R.V. performed CRO
activity measurements. S.G. produced and purified the CRO enzyme. B.T. processed the experimental data, performed the analysis, drafted the manuscript and designed the figures. C.M.H. and C.E.P. supervised the study and worked on the manuscript. J.-G.B., M.L. and D.R. contributed to supervision, aided in analysing and interpreting the results and worked on the manuscript. All authors discussed the results and gave input on the manuscript.

## Conflicts of interest

There are no conflicts to declare.

## Data availability

The data supporting this article have been included as part of the Supplementary Information.

## Acknowledgements

B.T. acknowledges an Erasmus+ grant. This project has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement n°949910). C.M.H. was funded by the European Union (MSCA, grant agreement n°101062327). Views and opinions expressed are however those of the authors only and do not necessarily reflect those of the European Union or European Research Council. Neither the European Union nor the granting authority can be held responsible for them. R.V. acknowledges the grant "Recualificación del Sistema Universitario Español durante el trienio 2021-2023 (Modalidad Margarita Salas)", University of Murcia (Spain).

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