# Recent Advances in Biocatalysis for Drug Synthesis
## 2 Biocatalysis for Drug Synthesis
### Expanding Biocatalytic Capabilities in Diversification and Late-Stage Functionalization
Using a different strategy, the elucidation of biosynthetic pathways for natural products exhibited the involvement of previously unknown P450s, which may be of interest for future investigations [100; 101; 102; 103; 104; 105]. For instance, unraveling the biosynthesis pathway of the antibiotic chuangxinmycin in _Actinoplanes tsianensis_ led to the identification and characterization of the C-S bond-forming P450 CxnD [105]. Figure 11: Functionalization of mentioned substrates by wild-type or engineered P450s. Oxidation sites are highlighted with a colored arrow representing hydroxylation (blue), demethylation (red), dealkylation (green), decarboxylation (black), C-S bond formation (yellow) or epoxidation (purple). Instead of searching for a native enzyme with the desired catalytic activity, rational protein design and directed evolution are effective tools to get the enzyme of interest (Figure 11) [92]. For more than two decades, numerous approaches of protein engineering have targeted the CYP102A1 (P450 BM3) from Bacillus megaterium due to beneficial properties such as its self-sufficiency based on the heme and flavin mononucleotide (FMN)/ flavin adenine dinucleotide (FAD)-containing reductase domain on a single basis [108]. are capable of producing desired drug metabolites and new analogues [92; 106; 107; 121]. For the synthesis of metabolites of the opium poppy alkaloid noscapine, a drug with anticancer activity, site-directed mutagenesis was used to generate a library of 18 variants with mutations located within or close to the active site and the access channel. Five mutations (R47L/A74G/F87A/L188Q/E267V) led to the highly regioselective N-demethylation of noscapine (88% regioselectivity and 50% conversion), whereas no methylation activity could be detected with the wild-type enzyme [106]. In a different approach, site-directed mutagenesis, and especially glycine mutagenesis, have provided access to diverse mono- and dihydroxylated steroids (androstenedione, dehydroepiandrorestorne, testosterone) due to altered steroid binding [107]. Almost a decade ago, the [PERSON] group bioengineered an efficient variant of P450 BM3, P411, by substitution of the conserved axial-ligand cysteine to serine [122]. Further directed evolution resulted in the selective primary amination activity of primary, secondary, and tertiary C-H bonds with yields of up to 93%, which may provide a platform for C-N bond formation of drug molecules [123]. In addition to P450 BM3 and its variants, other P450s have also been selected for protein engineering in recent years, leading to successful selective functionalization of substrates such as the antidepressant (\(R\))-ketamine [108], immunosuppressive drug molecule cyclospor A [109] and natural product triandamycin [110]. In the last two decades, the exclusively fungal family of robust C-H oxidizing UPOs have emerged as a promising alternative for the oxyfunctionalization of organic substrates [124]. Similar to the \"peroxygenase\" mode of some P450s, the heme-thiolate enzymes catalyze a broad range of H\({}_{2}\)O\({}_{2}\)-dependent oxidations but do not require NAD(P)H or electron transfer proteins [125]. Because UPO secretion often occurs after 1-3 weeks of fungal cultivation during secondary metabolism, published screening approaches for new wild-type enzymes were mainly performed using heterologous expression rather than the original fungus [126; 127]. To date, comprehensive screening data for the functionalization of drug molecules by UPOs are not available, as earlier described, for P450 screenings. However, to bring UPOs out of their infancy and into pharmaceutical application, several groups aimed to elucidate the catalytic properties of both described wild-type UPOs [111; 112; 128; 129] as well as engineered biocatalysts [113; 114; 130; 131] towards relevant bioactive compounds (Figure 12A).