P H A R M AC EU T I CAL S
Efficient Chiral Chemistry by Application of Stereoselective Biocatalysts in Micro-Aqueous Reaction Systems Dr Rainer Wardenga of Enzymicals and Dr Dörte Rother of Forschungszentrum Jülich discuss the applicability of diverse enzymes in micro-aqueous reaction systems, enabling the conversion of hydrophobic or water-unstable substrates while maintaining the stereoselectivity of the biocatalysts. Introduction In the search for novel techniques that lead us towards a sustainable and biobased economy, recent advances in biocatalysis have strongly boosted its recognition as a valuable addition to traditional chemical synthetic routes. Primarily, biocatalysis contributes to the highly selective synthesis of fine chemicals and pharma products.1 Apart from a few exceptions, enzymes show highest activities in buffer, mimicking their natural environments. However, when hydrophobic or water unstable substrates are involved, pure aqueous solutions are more difficult to apply – especially when high substrate concentrations are added and a second phase formation needs to be avoided. In 1987 Yamane et al introduced the concept of micro-aqueous reaction systems (MARS, Figure 1).2 Highlighting the fact that a certain amount of water is essential for a biocatalyst to display its full catalytic activity, the authors proposed the term ‘micro-aqueous’ in order to emphasize the importance of water for biocatalysis in organic solvents, and to cover all possible forms or states of biocatalysis in organic media. The term implies that the system is neither aqueous nor non-aqueous; nor is it anhydrous. Rather, the term ‘micro-aqueous’ refers to a state between the two extremes of aqueous and anhydrous, in which there is a small (‘micro’) amount of water. The optimal control of moisture content is the keystone of micro-aqueous biocatalysis systems, as water affects not only reaction rates and stereoselectivities, but also the stability of biocatalysts.2
MARS are also an alternative when the high substrate loads of neat substrate systems are prohibited, e.g. due to catalyst inactivation by substrate/product inhibitions or safety concerns. Still, the advantages of high substrate loads, minimum water addition, and working in a monophasic system remain when diluting the substrates in organic solvent. Successful biocatalytic applications of in MARS show substrate concentrations up to 0.5M and simplified downstream processing.3–7
Applicability of diverse enzymes Case studies are used here to present the applicability of diverse enzymes in micro-aqueous reaction systems. Subjects of individual studies were stereoselective lyases exhibiting carboligation activity and reductases for the synthesis of α-hydroxy ketones, dioles as well as chiral amines. By applying a flexible enzymatic cascade with thiamine-diphosphate (ThDP)-dependent enzymes and alcohol dehydrogenases for the synthesis of all stereo-isomers of 1-phenylpropane-1,2-diol (PPD), as well as by using imine reductases for the reduction of hydrophobic β-carboline and isoquinoline substrates, excellent conversions and stereoselectives may be achieved.
Case study I: Optically pure 1,2-diols are versatile building blocks for the manufacturing of pharmaceuticals, agrochemicals, or chiral catalysts. Among them, PPD serves by itself as an agent against neurodegenerative disease or as a building block for anti-inflammatory drugs and cardiovascular agents.8 By the combination of a carboligation and a reduction step, PPD could be gained from benzaldehyde and acetaldehyde via a 2-step enzyme cascade. For the (S)-selective carboligation towards the intermediate 2 hydroxy-1-phenyl-propanone (HPP) we used a rationally designed enzyme variant of Pseudomonas putida benzoylformate decarboxylase (PpBFD). For (R)-selective C-C coupling, the benzaldehyde lyase from Pseudomonas fluorescens (PfBAL) was applied. In combination with alcohol dehydrogenases (ADHs) exhibiting different stereoselecivities, namely Ralstonia sp. ADH (RADH) or Lactobacillus brevis ADH (LbADH), these cascades grant access towards all four diastereomers of PPD (Figure 2).
Figure 1: Graphical representation of the micro-aqueous reaction system (MARS) Applications in MARS are especially desirable for hydrophobic substrates, as the originally proclaimed inherent greenness of biocatalytic reactions under aqueous conditions has been replaced by a new perception: unconventional media can be ecologically advantageous, due to less generation of solvent waste during the reaction (less dilution) and during downstream processing (no extraction). Furthermore, such reactions become economically favourable due to higher possible product titres in the presence of high substrate concentrations.
16 Speciality Chemicals Magazine 37.01 February 2017
Figure 2: Synthetic routes to all four stereoisomers of 1-phenyl-1,2propanediol (PPD) by combina-tion of two ThDP-dependent enzymes with carboligase activity and two oxidoreductases. HPP = hydroxypropiophenone; PfBAL = Pseudomonas fluorescens benzaldehyde lyase; PpBFD = Pseudomonas putida benzoylformate decarboxylase variant; RADH = Ralstonia spec. alcohol dehydrogasne; LbADH = Lactibacillus brevis aldohol dehydrogenase