Investigation of integrated crystallization methods for the biocatalytic preparation of pharmaceutically relevant compounds and fine chemicals

Abstract

Throughout the last three decades, the transition towards a sustainable and “green” approach to chemistry has been set as a future goal for many states and industrial players. In pursuit of this goal, the research on enzymes as biocatalysts and on enzymatically driven synthesis of industrially relevant compounds has intensified. During that time, first industrial-scale applications containing enzymatic reaction steps have been adopted by pharmaceutical and agrochemical manufacturers. Enzymatic catalysis possesses many advantages compared to conventional organic chemistry synthesis pathways, yet it also has its drawbacks, which often prevents enzymes from replacing their conventional counterparts in the manufacturing process. Nevertheless, some of those drawbacks, like product inhibitions and unfavorable reaction equilibria, can be circumvented by reaction engineering towards combining the reaction with a simultaneous separation process, removing the product to another phase. This process is called in situ product removal (ISPR). This work investigates ISPR approaches based on product removal to the solid phase, namely reactive crystallization, focusing on their application for the (chemo)enzymatic synthesis of the compound classes of chiral amines and chiral carboxylic acids. In the first part, a previously developed concept for the reactive crystallization of chiral amines from transaminase-catalyzed reactions i1s reverted to a simple downstream processing concept to test its selectivity towards the high molecular weight amino products compared to low molecular weight amino donors. A model compound 1s crystallized selectively as an ammonium-carboxylate salt with very high purity and high yield recovered from the reaction mixtures. In the process, this crystallization technique 1s further adopted for another class of enzymatic reaction systems (amine dehydrogenases), broadening the application field for this separation method to other enzymatic methods of amine synthesis. The selectivity of this product crystallization system 1s tested under duress with varying crystallization parameters and surpluses of low molecular weight amino donors, showing robustness and reproducibility of high purities and yields of the crystallized amino product salts. In the second part of this work, the amine product salt crystallization concept, previously developed for reactive crystallization of primary a-chiral amines, is adopted for the transaminase-catalyzed synthesis of [-chiral amines. As a representative B-chiral amine model compound, (R)-B-methylphenethylamine 1s chosen. A dynamic kinetic resolution of its precursor, 2-phenylpropanal, 1s performed, using a highly stereoselective transaminase as the enzymatic catalyst. Reactions, which are augmented by the described reactive amine product salt crystallization concept, show much higher yields, than the controls without reactive crystallization, reaching productivities of 16 g/(1-d) on preparative scale. Therefore, in the second part of this work, the concept for continuous reactive crystallization of amino product salts from transaminase-catalyzed reactions 1s proven to be applicable in the synthesis of -chiral amines, a compound class represented among important pharmaceuticals. In the third part of this work, a novel reactive crystallization concept is established for the (chemo)enzymatic dynamic kinetic resolution of chiral racemic compounds. On the basis of mandelic acid as a model substrate, enzymatically driven racemization (via a mandelate racemase) was successfully combined with diastereomeric salt crystallization. The resolution of the racemic mandelic acid towards (R)-mandelic acid was achieved via diastereomeric salt crystallization with an enantiopure ammonium counterion. On preparative scale, the developed concept led to yields of up to 60 % (based on the total amount of the racemate) and a very high enantiomeric excess of 95 % for the isolated mandelic acid enantiomer, underlining the enantioselectivity of the crystallization. Remarkably, this concept was realized in an aqueous medium, although the initial solubility of mandelic acid in water is fairly high (1 M).