# Recent Advances in Biocatalysis for Drug Synthesis
## 2 Biocatalysis for Drug Synthesis
### Synthetic Enzyme Cascades  
Along with the intriguing properties of enzymes, with their high specificity and selectivity, efficiency under mild reaction conditions, and sustainable catalysis, several reaction steps using various isolated enzymes can also be combined in one pot. Since enzymes mostly prefer similar reaction conditions such as an aqueous buffer and physiological pH and temperatures, it is suitable to catalyze their reactions simultaneously in the same reactor (Figure 13). The additional advantages of such multi-enzymatic reactions, also called enzyme cascades, include the reduced demand for intermediate isolation, which decreases the need for large amounts of solvents, the equilibrium-shift towards the product side in these systems, and complex molecules can be built up with simple starting materials [139]. This is especially interesting for drug synthesis, since the compound structures are often complex and the usage ofbulk materials as educts reduces the costs of their production. Furthermore, using enzyme cascades gives the advantage of being able to design the reaction steps to a customized and efficient synthesis route. However, several parameters, such as reaction conditions or inhibitory effects, can influence the interplay of the cascade components. Therefore, the complex systems often suffer from low performances at the beginning and optimization effort has to be put in [140]. By engineering the biocatalysts, the adjustment towards tailored properties enables both precising and optimizing of the specificities of the enzyme thus enlarging the reaction scope even further. To tackle the challenging task of creating a functional multi-enzymatic pathway for API synthesis and improving the performance, several approaches have been published, ranging from bioretrosynthetic to chemoenzymatic strategies [141; 142]. Numerous examples exist for the impressive synthetic possibilities for drugs enabled by enzyme cascades, some of the most recent of which are presented here (Table 4). As mentioned, IREDs are exiting enzymes for the insertion of chiral amines. Reactions resulting in these groups are, for example, reductive animations, which in turn can be catalyzed by some IREDs with a carbonyl group and an amine as substrates. Such an enzyme was utilized in the two-enzyme cascade for the synthesis of the investigational drug GSK-2879552 (Figure 14A) [34]. GSK-2879552 is a lysine-specific histone demethylase-1A (LSD1 or lysine demethylase 1A, KDM1A) inhibitor and was investigated for its activity against small cell lung cancer (SCLC), acute myeloid leukemia (AML), and myelodysplastic syndrome (MDS), however clinical trials were terminated [143; 144]. In 2019, GlaxoSmithK-line (GSK) published an enzyme cascade for the production of the protected precursor of  
Figure 13: Schematic representation of an enzyme cascade for drug synthesis. Two plus n enzymes can participate in product production (\(n\geq 0\)). E, educt; I, intermediate; P, product. GSK-2879552. For the synthesis of this intermediate, a suitable IRED was found that was even able to convert its substrate, a racemic _trans_ amine compound with the stereogenic centers already included, with excellent enantioselectivity. To further improve its usability for a commercially viable process, the enzyme was evolved for better pH tolerance, increased substrate concentrations and product yield, and reduced biocatalyst loading. After three rounds of evolution, a 38,000-fold improvement in the turnover number (TON) was reached and the other criteria were either met or exceeded. A KRED was then implemented to receive the aldehyde substrate from its corresponding alcohol in situ. Furthermore, this enzyme allowed the recycling of the cofactor nicotinamide adenine dinucleotide phosphate(H) (NADP(H)) in a hydrogen borrowing manner resulting in a redox-neutral cascade. GSK-2879552 precursor production was run at a 5 g scale with a 48.3% yield, 99.5% _ee_ and 97.9% purity [34].