# Recent Advances in Biocatalysis for Drug Synthesis
## 2 Biocatalysis for Drug Synthesis
### Expanding Biocatalytic Capabilities in Diversification and Late-Stage Functionalization
Purified model enzymes, _AaeUPO_ from _Agrocybe aegita_, and _Mro_UPO from _Marasmius rotula_, hydroxylated _trans_-stilbene to the resveratrol analogue 4,4\({}^{\prime}\)-dihydroxy-trans-stilbene with product yields of 94-96% [111], while high yields were also obtained in the conversion of antithrombotic prodrugs clopidogrel or 2-oxo-prasugrel to bioactive metabolites by both enzymes [112]. Since protein engineering is also a powerful toolbox in UPO research [132], the Alcalde group further evolved JaWa, an evolved variant originated from _Aae_UPO, to SoLo (containing four mutations in the signal peptide and eight in the mature protein) by directed evolution in order to synthesize the human drug metabolite 5\({}^{\prime}\)-hydroxypropanolol from the betablocker propranolol on a semi-preparative scale [113]. Furthermore, they could also show that the evolved _Aae_UPO variants PaDa-I, JaWa, SoLo, and SoLo-D241G are suitable biocatalysts with different substrate scopes to synthesize known and novel metabolites of the pharmaceutical agents dextromethorphan, naproxen, and tolbutamide [114]. One major bottleneck of UPO application is its irreversible inactivation at stoichiometric concentrations of H\({}_{2}\)O\({}_{2}\)[133]. Besides combination with photo- or electrocatalysis, enzyme cascades are a promising strategy to control the in situ supply of H\({}_{2}\)O\({}_{2}\). Recently, a self-sufficient fusion enzyme, consisting of an evolved aryl-alcohol oxidase from _Pleurotus erysgii_ and the SoLo variant, was constructed and successfully used for the synthesis of dextrorphan, a metabolite of dextromethorphan, with a product yield of \(\sim\)25% [131]. In addition to P450s and UPOs, the strategies of genome mining, enzyme screening and protein engineering have also been successfully applied to other enzyme families. Very recently, a new promiscuous FAD-dependent halogenase, DkLH, from _Frankia ahti_ was identified for the modification of flavonoid compounds by heterologous expression of a biosynthetic secondary metabolite gene cluster in _Streptomyces albus_ (Figure 12B) [115; 134]. DkLH was able to chlorinate, dichlorinate or brominate 14 flavonoids, such as bioactive chrysin, apigenin, and luteolin, with high yields of up to 86% [115]. In a different approach, [PERSON] and colleagues evolved the non-heme iron halogenase WelOS* from _Hapalsoibon welwitschia_ using structure-guided evolution. Catalytic activity of the engineered WelOS* variant towards chlorination of a promising martinelline-derived substrate, which showed anticancer activity, was efficiently improved with a 290-fold higher total turnover number [116]. Moreover, they demonstrated the potential of algorithm-assisted enzyme evolution by designing a WelOS* variant that is capable of halogenating the bulky  
Figure 12: Functionalization of mentioned substrates by wild-type or engineered UPOs (**A**) and halogenases (**B**). Functionalization sites are highlighted with a colored arrow representing hydroxylation (blue), demethylation (red), dealkylation (green), C-S bond cleavage (pink), chlorination (grey) or bromination (brown). acetyl-coenzyme A carboxylase inhibitor soraphen A, a new target of pharmaceutical interest, due to reshaping of the active site [117]. As displayed here, many functionalization reactions catalyzed by enzymes possess great application potential. Limitations, such as substrate solubility [135], co-substrate supply [136], and cofactor regeneration [137], need to be addressed in future work, in which combined approaches such as enzymatic and chemoenzymatic cascades or photobiocatalytic strategies are promising [88; 138].