Abstract

Immobilized Enzymes in Novel Continuous-Flow Reactor Systems

Laszlo Poppe, Professor, Budapest University of Technology & Economics

Phenylalanine ammonia-lyase (PAL) can catalyze ammonia elimination from a wide selection of aromatic amino acids. PAL was used as biocatalyst for preparation of a-D-arylalanines by kinetic resolutions from racemic arylalanines or for synthesis of a-L-arylalanines by addition of ammonia onto the corresponding arylacrylates. By our epoxide-based immobilization strategy PAL could be attached to nanostructured supports such as carbon nanotubes (SwCNT-PAL) or magnetic nanoparticles (MNP-PAL).

SwCNT-PAL was successfully applied in continuous-flow packed-bed microreactor to produce enantiopure (S)-2-amino-3-(thiophen-2-yl)propanoic acid by the ammonia addition reaction. 

MNP-PAL was applied for biotransformation of L-phenylalanine and five further arylalanines in a MagneChip device comprising microliter volume reaction cells filled with MNP-PAL and equipped with in-line UV detection. Furthermore, by using MNP-PAL in the MagneChip device, we first demonstrated that PAL can catalyze the ammonia elimination from the acyclic propargylglycine indicating new opportunities to extend the application of PAL in biotransformation of acyclic substrates.

A continuous-flow cascade reactor system was applied to perform a protease (Alcalase)-catalyzed dynamic kinetic resolution (DKR) of N-Boc-phenylalanine ethyl thioester with benzylamine. The thioester was transformed in a serial cascade system of six biocatalyst-filled columns at 50 °C (KR) and five silica gel-filled racemization columns at 150 °C yielding the product in 79% conversion and 98% ee. This was the first example of a DKR for an amino acid derivative in continuous-flow mode using an alternating cascade of packed-bed enzyme reactors and racemization reactors kept at different temperatures.