Periodic Reporting for period 1 - SusECAS - DemoPlant (Metaraminol Demonstration Plant as Commercialisation Blueprint for Sustainable Production of Pharmaceuticals using Enzyme Cascades)
Okres sprawozdawczy: 2022-10-01 do 2024-03-31
The development of more environmentally friendly synthesis methods is urgently needed if humanity wants to maintain an environment worth living in. The establishment of more biocatalytic steps in chemical synthesis is a possible solution, as enzymes and whole cells offer sustainable advantages such as biodegradability, toxicity, high selectivity and much more.
Amino alcohols and 2-hydroxy ketones are valuable, optically active, compounds with various applications in the chemical and pharmaceutical industry with market prices >1000 €/kg, but challenging synthesis strategies. As a result of classical synthesis options with disastrous footprints, these molecules are often produced in countries with poor working conditions and significantly lower safety and environmental standards than those accepted in the EU.
The idea of 'SusECAS - DemoPlant' was to enable an economic and ecological competitive process for the production sustainable production of 2-hydroxy ketones and thus amino alcohols in Europe. In the ERC Starting Grant project 'LightCas', Prof. Rother laid the base with an atomic and step-efficient enzymatic 2-step reaction for amino alcohols from inexpensive starting materials (<10-100 €/kg). In 'SusECAS' these innovative synthesis idea should be brought to a new level of industrial application. A multi-gram demonstration process to the drug metaraminol should enable a clear indication of the ecological and economic potential.
Amino alcohols and 2-hydroxy ketones are valuable, optically active, compounds with various applications in the chemical and pharmaceutical industry with market prices >1000 €/kg, but challenging synthesis strategies. As a result of classical synthesis options with disastrous footprints, these molecules are often produced in countries with poor working conditions and significantly lower safety and environmental standards than those accepted in the EU.
The idea of 'SusECAS - DemoPlant' was to enable an economic and ecological competitive process for the production sustainable production of 2-hydroxy ketones and thus amino alcohols in Europe. In the ERC Starting Grant project 'LightCas', Prof. Rother laid the base with an atomic and step-efficient enzymatic 2-step reaction for amino alcohols from inexpensive starting materials (<10-100 €/kg). In 'SusECAS' these innovative synthesis idea should be brought to a new level of industrial application. A multi-gram demonstration process to the drug metaraminol should enable a clear indication of the ecological and economic potential.
The amino alcohol Metaraminol is a valuable drug required to treat hypotension, which is a risk factor for serious diseases. An intermediate of the Metaraminol production is (R)-3-hydroxy-phenylacetylcarbinol ((R)-3-OH-PAC). In 'SusECAS', the synthesis of (R)-3-OH-PAC was developed based on the inexpensive starting materials 3-OH-benzaldehyde (3-OH-BA) and pyruvate. In principle, this type of reaction can be carried out by enzymes called carboligases. In the beginning of the project, we screened and identified a carboligase that could do this job. Once the feasibility had been fundamentally demonstrated, an important focus was on optimizing the reaction from an economic and ecological point of view. In order to survive in a possible industrial competition, the highest possible product concentrations and yields had to be achieved in the process. Thus, we performed a performed a muti-parameter reaction optimization and compared the several aspects. On the one hand, experiments with different catalyst production and formulation strategies were performed. Moreover, reactions with substrates from different manufacturers and reactions with different substrate feeding strategies have been carried out. After optimization of all these parameters, we could increase the substrate (and thus the product) concentration to >0.3-0.4M with a very high conversion value of up to >95%. Moreover, by establishing a downstream processing protocol, we could isolate the product with a very high chemical purity of >99 % and an extremely high enantiomeric purity of >99.9%. In order to show the scalability of this reaction, a synthesis with a reaction volume of 2.5 L has been successfully conducted. In discussion with our industrial partners, our developed process represents a very good and competitive technology to produce this valuable intermediate (R)-3-OH-PAC of metaraminol synthesis.
In order to go one step further in value creation, not only the synthesis of the intermediate 3-OH-PAC was considered, but also the synthesis of metaraminol itself. In the literature, purely chemical routes starting from (R)-3-OH-PAC are known and established in pharmaceutical manufacturing companies. We looked at the enzymatic synthesis of (R)-3-OH-PAC to metaraminol and wanted to demonstrate this first on a laboratory scale and then optimize and scale it up analogously to the synthesis of (R)-3-OH-PAC. Theoretically, this type of reaction can be carried out by so-called amine transaminases. We tested a large number of enzymes and initially found some positive screening hits. The results of the screening rounds gradually showed that there are enzymes that can produce metaraminol, but only product concentrations of less than 20mM could be achieved. Even after a multi-parameter optimization of the best screening hits, the product concentration could not be further increased. We initially thought of a classical enzyme inhibition caused by the substrate and/or the product. After various investigations, it turned out that the co-factor required for these enzymes (PLP) is irreversibly destroyed by the substrate (R)-3-OH-PAC and the product metaraminol after a short time and the reaction therefore comes to a standstill. The problem could not be solved by adding new enzymes or addition of new co-factor. We also tried to precipitate the metaraminol product from the reaction while it was still forming (in-situ product isolation) and/or to find other enzymes. So far, no option has led to success. The low product concentrations of approx. 10mM are not competitive (in coordination with our industrial partners) compared to the existing chemical synthesis routes.
In order to go one step further in value creation, not only the synthesis of the intermediate 3-OH-PAC was considered, but also the synthesis of metaraminol itself. In the literature, purely chemical routes starting from (R)-3-OH-PAC are known and established in pharmaceutical manufacturing companies. We looked at the enzymatic synthesis of (R)-3-OH-PAC to metaraminol and wanted to demonstrate this first on a laboratory scale and then optimize and scale it up analogously to the synthesis of (R)-3-OH-PAC. Theoretically, this type of reaction can be carried out by so-called amine transaminases. We tested a large number of enzymes and initially found some positive screening hits. The results of the screening rounds gradually showed that there are enzymes that can produce metaraminol, but only product concentrations of less than 20mM could be achieved. Even after a multi-parameter optimization of the best screening hits, the product concentration could not be further increased. We initially thought of a classical enzyme inhibition caused by the substrate and/or the product. After various investigations, it turned out that the co-factor required for these enzymes (PLP) is irreversibly destroyed by the substrate (R)-3-OH-PAC and the product metaraminol after a short time and the reaction therefore comes to a standstill. The problem could not be solved by adding new enzymes or addition of new co-factor. We also tried to precipitate the metaraminol product from the reaction while it was still forming (in-situ product isolation) and/or to find other enzymes. So far, no option has led to success. The low product concentrations of approx. 10mM are not competitive (in coordination with our industrial partners) compared to the existing chemical synthesis routes.
The enzymatic synthesis of the hydroxyl-ketone (R)-3-OH-PAC was established and optimised under industrial aspects. In addition, it was demonstrated that a scale-up to 2.5L was possible. By establishing downstream processing, the product could be isolate with a very high chemical purity of >99 % and an extremely high enantiomeric purity of >99.9%. These successes represent a valuable basis for possible commercialization in future. The manufacture of intermediates, which are used as pharmaceutical products in a synthesis, is subject to strict regulations (e.g. GMP and ISO certifications). For this reason, it is not possible to produce and sell these products at the Forschungszentrum Jülich. However, the process for manufacturing the product could be commercialized together with industrial partners. Various licensing concepts are established at Forschungszentrum Jülich. Commercialisation partners might be companies that produce Metaraminol from (R)-3-OH-PAC themselves or companies that sell (R)-3-OH-PAC. In addition, a spin-off is currently planned at the Forschungszentrum Jülich based on different ideas based on biocatalytic synthesis concepts. This could potentially result in synergies in the exploitation strategy in the future.
In contrast to the (R)-3-OH-PAC synthesis, the enzymatic production of Metaraminol from (R)-3-OH-PAC was demonstrated within 'SusECAS' only in principle. Although we have found enzymes that perform the reaction in principle, we have not managed to establish the process at a level that would be industrially competitive. Unfortunately, it has been shown that the co-factor is irreversibly damaged by the substrate and the product. This is a new phenomenon for this type of, which had not been described before. In order to be able to produce Metaraminol in an industrially competitive manner, the problem needs to be better understood and solutions developed for it. Contrary to expectations, more research is needed here. In order to draw attention to this new phenomenon, we will publish our data to sensitize other researchers to this problem and perhaps find a solution in future.
In contrast to the (R)-3-OH-PAC synthesis, the enzymatic production of Metaraminol from (R)-3-OH-PAC was demonstrated within 'SusECAS' only in principle. Although we have found enzymes that perform the reaction in principle, we have not managed to establish the process at a level that would be industrially competitive. Unfortunately, it has been shown that the co-factor is irreversibly damaged by the substrate and the product. This is a new phenomenon for this type of, which had not been described before. In order to be able to produce Metaraminol in an industrially competitive manner, the problem needs to be better understood and solutions developed for it. Contrary to expectations, more research is needed here. In order to draw attention to this new phenomenon, we will publish our data to sensitize other researchers to this problem and perhaps find a solution in future.