Periodic Reporting for period 1 - cassaFLOW (Cascades for Stereoselective Synthesis of Amino Acids)
Berichtszeitraum: 2024-01-01 bis 2024-12-31
Non-natural amino acids are essential building blocks for pharmaceuticals. While the preparation of these compounds with a single stereocenter is routinely performed in industry, the synthesis of non-natural amino acids containing two stereocenters is particularly challenging. However, these compounds show great promise, as they are building blocks of several peptide-based drugs such as Paxlovid™, used for the treatment of COVID-19, and Retosiban, used for preterm labor prevention. Developing an effective synthetic route for these amino acids is thus key to further the development of new peptide-based drugs.
In cassaFLOW, we will develop a concise, 3-step stereoselective synthesis for amino acids with 2 stereocenters, focusing on the amino acid Isoleucine and its isomers as a user case. This target compound is a key component of several antibiotics and is in high demand in the market. It is currently produced by our partner ChiralVision, but the lengthy and inefficient 8-step synthetic procedure does not allow for scaling-up to meet current demand levels. We will shorten the current synthesis while improving efficiency by utilizing the technology developed in our previous FET-OPEN ONE-FLOW project. In ONE-FLOW, we demonstrated the integration of several steps without intermediate downstream processing, as well as the combination of cofactor recycling with an immobilized enzyme system, at TRL-4 level. With cassaFLOW, we build on this technology to bring our production to TRL-6. We will install 2 stereocenters in 1 biocatalytic step and by applying the flow technology previously developed we telescope long reaction paths with multiple work-up steps into short and clean processes. Our innovative technology will be combined with the reaction engineering and commercial expertise of the two SME’s ChiralVision and SpinChem. The outcome of cassaFLOW will be a 3-step TRL 6 synthesis of an isoleucine that will replace the current 8- step production and can be directly placed on the market. Furthermore, the developed process will be applicable as a modular technology platform: by changing the initial starting product of the synthesis, we can obtain a wide range of different amino acids with 2 stereocenters.
Importantly, the cassaFLOW outcomes will contribute to the EU Strategic Autonomy 2013-2023 Plan that aims to achieve a greater independence from delivery chains, and is in line with the “Pharmaceutical Strategy for Europe” that aims to produce pharmaceuticals with short production chains in Europe. The 3-step synthesis can be performed in-house in a single production center, avoiding lengthy and vulnerable international logistical chains. Moreover, by shortening the synthesis and reducing the intermediate processing, the environmental impact is also greatly reduced. The technology developed in the project, such as the enzyme carriers for enzyme immobilization, will consist of renewable, biodegradable materials to reduce waste pollution.
In cassaFLOW, we will develop a concise, 3-step stereoselective synthesis for amino acids with 2 stereocenters, focusing on the amino acid Isoleucine and its isomers as a user case. This target compound is a key component of several antibiotics and is in high demand in the market. It is currently produced by our partner ChiralVision, but the lengthy and inefficient 8-step synthetic procedure does not allow for scaling-up to meet current demand levels. We will shorten the current synthesis while improving efficiency by utilizing the technology developed in our previous FET-OPEN ONE-FLOW project. In ONE-FLOW, we demonstrated the integration of several steps without intermediate downstream processing, as well as the combination of cofactor recycling with an immobilized enzyme system, at TRL-4 level. With cassaFLOW, we build on this technology to bring our production to TRL-6. We will install 2 stereocenters in 1 biocatalytic step and by applying the flow technology previously developed we telescope long reaction paths with multiple work-up steps into short and clean processes. Our innovative technology will be combined with the reaction engineering and commercial expertise of the two SME’s ChiralVision and SpinChem. The outcome of cassaFLOW will be a 3-step TRL 6 synthesis of an isoleucine that will replace the current 8- step production and can be directly placed on the market. Furthermore, the developed process will be applicable as a modular technology platform: by changing the initial starting product of the synthesis, we can obtain a wide range of different amino acids with 2 stereocenters.
Importantly, the cassaFLOW outcomes will contribute to the EU Strategic Autonomy 2013-2023 Plan that aims to achieve a greater independence from delivery chains, and is in line with the “Pharmaceutical Strategy for Europe” that aims to produce pharmaceuticals with short production chains in Europe. The 3-step synthesis can be performed in-house in a single production center, avoiding lengthy and vulnerable international logistical chains. Moreover, by shortening the synthesis and reducing the intermediate processing, the environmental impact is also greatly reduced. The technology developed in the project, such as the enzyme carriers for enzyme immobilization, will consist of renewable, biodegradable materials to reduce waste pollution.
The synthethic routes developed in this proposal are based on the introduction of two stereocenters in a single catalytic step. Two routes are developed in parallel, each using a different enzyme family. The protocol for the production of these enzymes has been optimized and standardized, with the enzymes now produced, characterized and tested for proof of activity. New enzyme carriers and immobilization systems have also been developed and tested for the enzymes developed in the project. Optimization to determine the most suitable system is currently underway. The first milestones of the project has been achieved, representing the obtention of all enzyme formulations at a 10g wet biomass scale. The second milestone, obtention of the target isoleucine stereomer, is currently underway.
Two different enzyme families have been selected for the evaluation as biocatalysts for the formation of 2 stereocenters in a single step. A total of 34 enzymes were selected from a broad range of clades, produced, characterized and tested for proof of activity. Production has been achieved at a liter scale, and can be easily upscaled as needed. These results will be jointly patented by the partners developing the technology and will then be followed by adaptation to a scientific manuscript.
Furthermore, sustainable and mutlifunctional enzyme carriers have been developed with over 15 different functional combinations. Two different technologies for enzyme encapsulation have been developed. Both technologies have been thus far been successfully applied to the enzymes developed in this project, and will be further optimized to select the best system for the target enzymes.
Furthermore, sustainable and mutlifunctional enzyme carriers have been developed with over 15 different functional combinations. Two different technologies for enzyme encapsulation have been developed. Both technologies have been thus far been successfully applied to the enzymes developed in this project, and will be further optimized to select the best system for the target enzymes.