Transaminases immobilized on a membrane reactor for the production of chiral amines

Details

Supervisors

Debecker, Damien P.

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en chimie et bioindustries, à finalité spécialisée

Abstract

The production of Active Pharmaceutical Ingredient (APIs) such as chiral amines still requires energy- and reagent- intensive homogeneous chemocatalytic methods implying low-yield, high waste (high E-factor) and involving poorly enantioselective organometallic catalysts based on toxic and depleted heavy metals. It is therefore crucial to develop more efficient and sustainable alternative for their synthesis. In that context, transaminases (TAs) could represent interesting candidates. They are the enzymes that naturally catalyze the production of chiral amines from keto-precursor using cheap and readily available amino donor with excellent enantioselectivity, and under mild conditions. Their immobilization on functionalized polypropylene (PP) membranes support not only would increase their stability, but also would allow their recoverability and reusability. Such heterogeneous biocatalyst is therefore targeted and investigated for an industrially relevant reaction : the asymmetric synthesis of R-fluoromethylbenzylamine (R-FMBA) from 2’-fluoroacetophenone (FAP), using isopropylamine (ISO) as amino donor. However, one of the main challenges to tackle when studying such asymmetric syntheses, is to overcome their unfavorable thermodynamics. By employing an excess of ISO and utilizing a physical product separation strategy, namely in situ product crystallization (ISPC) with 3-diphenylpropionic acid (DPPA), substrate conversion was successfully enhanced towards the desired product, R-FMBA. This strategy allowed to significantly improve the amine yields beyond the thermodynamic limit while directly generating pure API crystals. 1H NMR analysis revealed a 99% purity for our produced R-FMBA-DPPA crystals. These were easily recovered by straightforward filtration at the end of the transamination reactions with high yield (82 %). The productivity of the present crystallization-assisted transamination batch process was optimized by enhancing the reaction kinetics, precisely by overcoming the enzyme inhibition by the FAP substrate, which would allow to work at higher substrate concentration. In this aim, the main strategy consisted in the use of multiphasic medium (e.g., using supersaturated FAP substrate, creating FAP emulsions as external substrate reservoirs). This approach allowed to maintain a constant concentration of FAP at its solubility limit in the aqueous phase, ensuring acceptable activity while counteracting enzyme inhibition issue. Transfer of this technology from batch to continuous flow mode was also studied, in the aim to boost the productivity of the presented process. Not only the targeted transamination reaction, but also the TA immobilization process, were implemented in a flow reactor. Interestingly, these conditions allowed to boost the immobilization yield (i.e. a three-fold increase of TA loading was obtained, with respect to the batch immobilization). Additionally, higher productivity was obtained in flow mode (in absence of product crystallization), as the TA specific activity was doubled with respect to the batch process. However, due to pore clogging issues, no equilibrium shifting strategy could be implemented in continuous flow mode. Eventually, different keto substrates were also screened in an attempt to expand the scope of enantiopure amine production via crystallization-assisted transamination in batch mode. Although the multiphasic strategy was not directly applicable to all substrates, the screening highlighted the versatility and limitations of the immobilized TAs. This exploration revealed promising keto substrate candidates for further development, such as phenoxyacetone, phenylbutanone, and phenylpropanone (an amphetamine precursor), paving the way for new API production via this biocatalytic process.