Overcoming yield limitations when resolving racemates by combination of crystallization and/or chromatography with racemization

The major drawback of using enantioseparation techniques when resolving racemates is a yield of maximum 50%. To prevail over this disadvantage new approaches are explored for manufacturing single enantiomers. The work presented in this thesis is dedicated in particular to this challenge. In this work two integrated processes for production of optically pure enantiomers are studied. Each one couples a separation technique with a racemization reaction. The principle of work consists of resolution of the racemate and racemization of the unwanted enantiomer, whereby the yield of the target enantiomer can be significantly increased - at best to 100%. The engagement of the racemization which should offer minimum of no loss of optically active material should considerably contribute to the process economics. Based on the information obtained from studying each single unit, more detailed scenarios for both integrated processes are suggested and general outlook is given regarding their potentials and critical aspects. The applicability of one of the examined integrated processes is experimentally proven

Enzyme-assisted physicochemical enantioseparation processes - part III: Overcoming yield limitations by dynamic kinetic resolution of asparagine via preferential crystallization and enzymatic racemization

The application of enantioseparation methods alone can only yield up to 50% of the desired chiral product. Thus enantioseparation becomes more attractive when accompanied by the racemization of the counter-enantiomer. Here we present first results of dynamic kinetic resolution of L-asparagine (L-Asn) via preferential crystallization and enzymatic racemization from a racemic, supersaturated solution on a 20 mL scale. An enzyme lyophilisate (WT amino acid racemase from P. putida KT2440 (E.C. 5.1.1.10), overexpressed in E. coli BL21(DE3)) was used for in situ racemization (enzyme concentrations varying from 0 to 1 mg/mL). When preferential crystallization was applied without any enzyme, a total of 31 mg of L-Asn monohydrate could be crystallized, before crystal formation of d-Asn started. Crystallization experiments accompanied by enzymatic racemization led to a significant increase of crystallized L-Asn (198 mg L-Asn monohydrate; >92%ee) giving the first experimental proof for this new process concept of dynamic kinetic resolution via preferential crystallization and enzymatic racemization. Measurements of the racemase activity before and after the crystallization process showed no significant differences, which would allow for enzyme recovery and recycling

Preferential Crystallization of L-Asparagine in Water

Because of its simplicity and cost-effectiveness, preferential crystallization (PC) can be considered as one of the most attractive techniques available for enantioseparation. In this paper,the enantioseparation of the nonessential amino acid DL-asparagine (DL-Asn), which belongs to the group of conglomerate forming systems, is studied experimentally and theoretically. Goals of this work are to investigate the applicability of PC of L-Asn·H2O from an aqueous solution of racemic DL-Asn using simple isothermal batch preferential crystallization (SIB-PC) and to provide a reliable database for model validation based on essential model parameters identified. To further improve the performance of PC, two crystallizers can be connected in order to exchange continuously the mother liquors (CIB-PC, coupled isothermal batch preferential crystallization). This new configuration is tested and assessed. Copyright © 2011 American Chemical Society [accessed 27th May 2011

Enzyme-assisted physicochemical enantioseparation processes - part II: Solid-liquid equilibria, preferential crystallization, chromatography and reacemization reaction

This contribution addresses the design and investigation of two hybrid enantioseparation processes including an enzymatic racemization step in order to enhance the overall performance. Complementary to part I where the manufacturing and the characterization of an amino acid racemase (EC 5.1.1.10) was emphasized [Wurges, K., Petrusevska, K., Serci, S., Wilhelm, S., Wandrey, C., Seidel-Morgenstern, A., Elsner, M.P.. Lutz, S., 2009. Enzyme-assisted physicochemical enantioseparation processes-part I: production and characterization of a recombinant amino acid racemase. J. Mol. Cat. B (in print). online available: doi:10.1016/j.molcatb.2008.10.006], the work presented in this paper tends more towards developing a data base for potential process schemes for the manufacture of selected amino acids.The first proposed process concept (P-I) couples preferential crystallization (PC) and racemization for the production of I-asparagine (I-Asn) using racemic mixture of dl-asparagine (conglomerate-forming system) as a starting material, while the second concept (P-II) integrates chromatography and racemization for the preparation of I-methionine (I-Met) starting with racemic mixture of dl-methionine (compound-forming system). As mentioned in part I, a racemization unit, where the unwanted enantiomer will be converted into racemate, is incorporated into the hybrid processes for the sake of 100% yield, theoretically. Besides the basic investigation according to the solid-liquid equilibria, PC and chromatography, the focus of this paper is mainly on the kinetic studies of the racemization reaction. Initially, the solubility ternary phase diagrams of both examined systems were determined, leading into the idea of combination of the proposed process schemes. For P-I the concept of PC of I-Asn was experimentally proven and the kinetics of the racemization was examined for d- and I-Asn in water using purified lyophilizate (PL). Concerning P-II, for the chromatographic unit the impact on the separation of dl-Met on eremomycin based stationary phase using KPi buffer and MeOH as mobile phase was evaluated in terms of resolution and selectivity at three different temperatures by varying the content of methanol (MeOH) in the mobile phase and the pH. The experiments for determination of the racemization kinetics were done for a compromised parameter set using crude lyophilizate (CL). In both cases a Michaelis-Menten three-step model was used to describe the enzymatic reaction. (C) 2009 Elsevier Ltd. All rights reserved