# Recent Advances in Biocatalysis for Drug Synthesis
## 2 Biocatalysis for Drug Synthesis
### Expansion of Product Spectrum
#### 2.1.1 Natural Metabolism as Source of Enzymes and Biochemical Pathways
It is unlikely that a chemist would give the predicate for a pro-fane esterification using a leaving group, although this is also carried out in biosynthesis (e.g., Ac-CoA); it is more likely that something is described as biomimetic when the key step involves the so-called scaffold formation [49; 50; 51; 52]. Scaffold formation, as an early phase of secondary metabolism, not only adds or transforms functional groups but also determines the basic carbon structure of a natural substance (Figure 1). In the past decade, the targeted use of complex scaffold-forming enzymes has made it possible to shorten natural and active agent syntheses and also to make them more precise stereochemically. One example is the synthesis of the tetrahydroiosquinolines scaffold, which is the basis for numerous alkaloids such as quinine, strychnine or vinblastine, and is formed by a Pictet-Spengler reaction between an amine like dopamine and a carbonyl [53; 54]. The newly formed \(N\)-heterocycle moiety is built up by both, a C-N- and a C-C-coupling, including a newly formed stereogenic center. The natural alkaloids are formed from aldehydes. However, [PERSON] and colleagues developed mutants of the norcocalaurine synthase that also accept ketones or even cyclic ketones, resulting finally in _spiro_-tetrahydroiosquinolines  
Figure 5: Biocatalytic reactions of the L-proline _cis_-4-hydroxylase SmP4H from _Sinorhizobium meliliot_. (**A**) Engineered enzymatic reaction for hydroxylation or desaturation of L-homophenylalanine [20]. (**B**) Engineered enzymatic reaction with chlorination activity [22]. bearing a quaternary stereogenic center (Figure 6) [23]. This complex ring structure is formed within one step with good to moderate yields and the enantiomeric excesses were still sufficiently controlled by these enzyme variants. Methylations occur rather often in secondary metabolism, usually as late-stage functionalization. In the case of the physostigmine biosynthesis, the methylation of tryptamine leads to the formation of an additional pyrrolidine ring as a subsequent reaction. The most striking feature here is the formation of a C-C-coupling yielding, simultaneously, a quaternary stereogenic center with high stereoselectivity (Figure 7) [24]. [PERSON] et al. were able to demonstrate the flexibility of this methylase for various substrates. The methyl group originates from \(S\)-adenosyl methionine, which is both, unstable and expensive. This drawback for an in vitro reaction could be circumvented by applying a cofactor recycling that employs iodomethane, rather than ATP or other costly co-substrates [55]. This reaction scheme allows for the synthesis of various physostigmine congeners, while physostigmine is known to be an acetylcholine esterase inhibitor [56; 57; 58]. We have already seen, in the case of the norcocalurine synthase, for the formation of tetrahydroroisoquinolines [23], that sometimes adaptions of the enzyme must be made in terms of mutagenesis in order to realize the substrate acceptance. [PERSON] and colleagues applied the strategy of mutagenesis to channel the substrate formation towards a monoclinic, rather than the bicyclic, product (Figure 8) [25]. Naturally  
Figure 6: Formation of tetrahydroroisoquinolines by Pictet-Spenglerase mutants and the wild-type of the norcocalurine synthase (NCS) [23]. Figure 7: The methylation of the indole causes the cyclisation towards compound. \(S\)-Adenosyl methionine (SAM) is recycled from \(S\)-adenosyl homocysteine (SAH) and methyl iodide by the auxiliary enzyme CfHMT [24]. are rather unselective and generate a bouquet of compounds [15], which can be evolutionary advantageous. _[PERSON]_ and colleagues engineered the active site of the terpene cyclase _Aac_SHC from _Alicyclobacillus acidocalarius_ in a sophisticated, semi-rational manner to exclusively yield the terpenoid compound with a very good yield and an excellent enantiomeric excess.