Periodic Reporting for period 4 - BioAqua (Water as cosubstrate for biocatalytic redox reactions)
Periodo di rendicontazione: 2020-01-01 al 2020-06-30
Selective oxyfunctionalisation of non-activated C-H-bonds still represents a challenge for the organic chemist. Peroxygenases are a class of enzymes, which could significantly enrich the toolbox of organic chemistry and thereby contribute to more sustainable chemical synthesis.
However, to attain this goal, a range of challenges still have to be met. Peroxygenases necessitate balanced provision with the reactive oxidant (H2O2) used. With the work performed in BioAqua, we have provided the field with a range of practical in situ H2O2 generation systems that enable efficient use of peroxygenases; the turnover numbers achieved for peroxygenases in this project are the highest reported so far. Preparative-scale application of peroxygenases has now come into reach. In fact, a kg-scale demonstration had been scheduled for early 2020 and unfortunately had to be postponed to post-COVID times
However, to attain this goal, a range of challenges still have to be met. Peroxygenases necessitate balanced provision with the reactive oxidant (H2O2) used. With the work performed in BioAqua, we have provided the field with a range of practical in situ H2O2 generation systems that enable efficient use of peroxygenases; the turnover numbers achieved for peroxygenases in this project are the highest reported so far. Preparative-scale application of peroxygenases has now come into reach. In fact, a kg-scale demonstration had been scheduled for early 2020 and unfortunately had to be postponed to post-COVID times
Work performed in the project BioAqua covers various aspects of Biocatalysis: 1) Establishing peroxygenases as future benchmark catalysts for oxyfunctionalisation chemistry and 2) Debottlenecking peroxygenase catalysis en route to preparative application.
1) Peroxygenases as new P450 monooxygenses
P450 monooxygenases are widely recognised catalysts for the selective oxyfunctionalisation of non-activated C-H- or C=C-bonds. The Oxygen Dilemma (ChemBioChem 2016, DOI: 10.1002/cbic.201600176) however severely challenges their application for low-value added bulk chemicals. This is due to the loss of reducing equivalents in the complex electron transport chains of P450 monooxygenases. We therefore believe that fungal peroxygenases (UPOs) are a more promising alternative for synthetic organic chemistry. UPOs principally exhibit the same product scope of P450 monooxygenases but only rely on H2O2 as oxidant for catalysis (as compared to the complex, multi enzyme electron transport chains of P450 monooxygenses) (some overview articles from our group: Curr. Opin. Biotechnol. 2016, DOI: 10.1016/j.cbpa.2016.10.007; Angew. Chem. Int. Ed. 2018, DOI: 10.1002/anie.201800343; Green Chem. 2019, DOI: 10.1039/C9GC00633H). Our contributions on UPO catalysis are described below. In this context, however, also our works on oxidative lactonisation (ACS Sus. Chem. Eng., DOI: 10.1021/acssuschemeng.9b07494; ChemSusChem 2020, DOI: 10.1002/cssc.201902240) and the conversion of amino acids into nitrile building blocks (ChemCatChem 2020, DOI: 10.1002/cctc.201902194) are worth mentioning.
Given the enormous potential of UPOs and peroxidases in general, we have adjusted the focus of my research group on these promising catalysts.
2) Debottlenecking peroxygenase catalysis
UPOs have not been considered as oxyfunctionalisation catalysts for a long time yet. Therefore, it is not very astonishing, that prior widespread application, a range of fundamental issues had to be addressed; amongst them the oxidative inactivation by the stoichiometric oxidant; the current limitation of UPO catalysis on aqueous reaction media and the at-scale availability of UPOs:
On demand provision with H2O2: UPOs utilise H2O2 as stoichiometric oxidant (thereby circumventing the Oxygen Dilemma). H2O2, however, also irreversibly inactivates the prosthetic heme group. Therefore, careful provision with H2O2 is necessary to enable efficient catalysis while circumventing oxidative inactivation. One focus of BioAqua was to establish and characterise promising in situ H2O2 generation systems based on reductive activation of molecular oxygen. The various systems developed can be classified via the sacrificial electron donors used. Ideally, water would serve as sacrificial electron donor (abundant, O2 as sole by-product). The thermodynamic and kinetic stability of H2O, however, poses a significant challenge. Nevertheless, we were able to make H2O available for UPO-catalysis using modified TiO2 photocatalyst (Nature Catal. 2018, DOI: 10.1038/s41929-017-0001-5) lignin derivates (manuscript under consideration), using BiOCl catalysts and ultrasound (ACS Catal. 2020, DOI: 10.1021/acscatal.0c00188) or even utilising nuclear waste (manuscript under consideration). While we believe that these approached excel by their elegance their efficiency in terms of product formation rates and robustness still needs significant improvements to be of practical relevance.
More activated electron donors such as formic acid (Angew. Chem. Int. Ed. 2017, DOI: 10.1002/ange.201708668; Angew. Chem. Int. Ed. 2019, DOI: 10.1002/anie.201902380) or methanol (ACS Catal. 2020, DOI: 10.1021/acscatal.0c01958) result in more productive and robust production systems. Also using natural deep eutectic solvents as performance solvents also serving as sacrificial electron donors is an interesting alternative (ChemCatChem 2020, DOI: 10.1002/cctc.201901842).
Therefore we are convinced that the issue of suitable H2O2 provision for UPO catalysis has been solved in the course of BioAqua. We and other research groups worldwide are busy in further characterising and optimising these H2O2 generation systems.
Peroxygenase reactions in non-aqueous reaction media.
A large part of the reagents of interest are rather hydrophobic and therefore only poorly soluble in aqueous media. Unfortunately, many (if not most) biocatalytic oxyfunctionalisation reactions therefore rely on rather dilute reagent concentrations (below 20 mM) and therefor are very unattractive from a preparative point-of-view (up to 1000 times more water waste produced than product of interest). We therefore have recently started investigating UPO reactions in non-aqueous (ideally neat) reaction mixtures. Either using so-called two-liquid phase systems (Mol. Catal. 2016, DOI: 10.1016/j.molcatb.2016.09.013) or neat systems (ChemCatChem 2019, DOI: 10.1002/cctc.201901142) we could demonstrate that product concentrations of significantly higher than 100 mM can be achieved. Higher concentrations are feasible but currently, we are limited by the rather poor immobilisation yield of UPOs on heterogeneous carriers. Further investigations addressing this are currently underway.
1) Peroxygenases as new P450 monooxygenses
P450 monooxygenases are widely recognised catalysts for the selective oxyfunctionalisation of non-activated C-H- or C=C-bonds. The Oxygen Dilemma (ChemBioChem 2016, DOI: 10.1002/cbic.201600176) however severely challenges their application for low-value added bulk chemicals. This is due to the loss of reducing equivalents in the complex electron transport chains of P450 monooxygenases. We therefore believe that fungal peroxygenases (UPOs) are a more promising alternative for synthetic organic chemistry. UPOs principally exhibit the same product scope of P450 monooxygenases but only rely on H2O2 as oxidant for catalysis (as compared to the complex, multi enzyme electron transport chains of P450 monooxygenses) (some overview articles from our group: Curr. Opin. Biotechnol. 2016, DOI: 10.1016/j.cbpa.2016.10.007; Angew. Chem. Int. Ed. 2018, DOI: 10.1002/anie.201800343; Green Chem. 2019, DOI: 10.1039/C9GC00633H). Our contributions on UPO catalysis are described below. In this context, however, also our works on oxidative lactonisation (ACS Sus. Chem. Eng., DOI: 10.1021/acssuschemeng.9b07494; ChemSusChem 2020, DOI: 10.1002/cssc.201902240) and the conversion of amino acids into nitrile building blocks (ChemCatChem 2020, DOI: 10.1002/cctc.201902194) are worth mentioning.
Given the enormous potential of UPOs and peroxidases in general, we have adjusted the focus of my research group on these promising catalysts.
2) Debottlenecking peroxygenase catalysis
UPOs have not been considered as oxyfunctionalisation catalysts for a long time yet. Therefore, it is not very astonishing, that prior widespread application, a range of fundamental issues had to be addressed; amongst them the oxidative inactivation by the stoichiometric oxidant; the current limitation of UPO catalysis on aqueous reaction media and the at-scale availability of UPOs:
On demand provision with H2O2: UPOs utilise H2O2 as stoichiometric oxidant (thereby circumventing the Oxygen Dilemma). H2O2, however, also irreversibly inactivates the prosthetic heme group. Therefore, careful provision with H2O2 is necessary to enable efficient catalysis while circumventing oxidative inactivation. One focus of BioAqua was to establish and characterise promising in situ H2O2 generation systems based on reductive activation of molecular oxygen. The various systems developed can be classified via the sacrificial electron donors used. Ideally, water would serve as sacrificial electron donor (abundant, O2 as sole by-product). The thermodynamic and kinetic stability of H2O, however, poses a significant challenge. Nevertheless, we were able to make H2O available for UPO-catalysis using modified TiO2 photocatalyst (Nature Catal. 2018, DOI: 10.1038/s41929-017-0001-5) lignin derivates (manuscript under consideration), using BiOCl catalysts and ultrasound (ACS Catal. 2020, DOI: 10.1021/acscatal.0c00188) or even utilising nuclear waste (manuscript under consideration). While we believe that these approached excel by their elegance their efficiency in terms of product formation rates and robustness still needs significant improvements to be of practical relevance.
More activated electron donors such as formic acid (Angew. Chem. Int. Ed. 2017, DOI: 10.1002/ange.201708668; Angew. Chem. Int. Ed. 2019, DOI: 10.1002/anie.201902380) or methanol (ACS Catal. 2020, DOI: 10.1021/acscatal.0c01958) result in more productive and robust production systems. Also using natural deep eutectic solvents as performance solvents also serving as sacrificial electron donors is an interesting alternative (ChemCatChem 2020, DOI: 10.1002/cctc.201901842).
Therefore we are convinced that the issue of suitable H2O2 provision for UPO catalysis has been solved in the course of BioAqua. We and other research groups worldwide are busy in further characterising and optimising these H2O2 generation systems.
Peroxygenase reactions in non-aqueous reaction media.
A large part of the reagents of interest are rather hydrophobic and therefore only poorly soluble in aqueous media. Unfortunately, many (if not most) biocatalytic oxyfunctionalisation reactions therefore rely on rather dilute reagent concentrations (below 20 mM) and therefor are very unattractive from a preparative point-of-view (up to 1000 times more water waste produced than product of interest). We therefore have recently started investigating UPO reactions in non-aqueous (ideally neat) reaction mixtures. Either using so-called two-liquid phase systems (Mol. Catal. 2016, DOI: 10.1016/j.molcatb.2016.09.013) or neat systems (ChemCatChem 2019, DOI: 10.1002/cctc.201901142) we could demonstrate that product concentrations of significantly higher than 100 mM can be achieved. Higher concentrations are feasible but currently, we are limited by the rather poor immobilisation yield of UPOs on heterogeneous carriers. Further investigations addressing this are currently underway.
Peroxygenases. In 2015, peroxygenases had been known to only a handful of (mostly biologically oriented) research groups. We therefore take pride in the fact that our contributions on peroxygenases have helped to popularise this very exciting class of enzymes. In addition to our publications, conference contributions and further active promotion we have shared samples with chemically oriented research groups and companies worldwide. Now, several research groups worldwide have started working on various aspects of peroxygenase catalysis (engineering of enzyme mutants with tailored properties, new H2O2 generation systems and exploration of the synthetic possibilities).
We are convinced that soon, the ‘critical mass’ of research groups will be reached leading to an explosion of new reactions and applications of peroxygenases.
Photobiocatalysis. Similar to peroxygenases, photobiocatalysis was a rather peripheral area in biocatalysis with few groups showing interest in it. Today, we can attest that this situation is changing rapidly and we believe to have our share in this development.
We are convinced that soon, the ‘critical mass’ of research groups will be reached leading to an explosion of new reactions and applications of peroxygenases.
Photobiocatalysis. Similar to peroxygenases, photobiocatalysis was a rather peripheral area in biocatalysis with few groups showing interest in it. Today, we can attest that this situation is changing rapidly and we believe to have our share in this development.