# Recent Advances in Biocatalysis for Drug Synthesis
## 2 Biocatalysis for Drug Synthesis
### Synthetic Enzyme Cascades
Metaraminol, an API used to treat hypotension during anesthesia [145], also comprises a chiral amine [146]. The synthesis from 3-hydroxybenzaldehyde (3-OH-BA) and L-alanine as an amine donor is catalyzed by a pyruvate decarboxylase (PDC) and a transaminase (TA). Often, TAs have unfavorable equilibria for transamination reactions and the challenge is to shift it towards the product side. [PERSON] et al. exploited the solubility of metaraminol in organic solvents to pull the equilibrium by the in situ product removal (ISPR) [146]. To include liquid-liquid extraction, a suitable organic solvent was sought, measured on transaminase activity and conversion. With additional optimization of the pH for the reaction, an increased yield of 29%, compared to a 14% conversion, could be achieved [146]. Other enzyme cascades were developed to synthesize relevant pharmaceutical products such as molnupiravir (MK-4482, formerly EIDD-2801) from simple starting materials. Molnupiravir is a compound which is currently investigated for the treatment of the coronavirus disease 2019 (COVID-19) with promising results during clinical trials to reduce the risk of hospitalization [147]. Due to the urgent need of the API during the pandemic, there was considerable interest in the development of synthetic routes. Chemical routes suffer from drawbacks such as low yields and harsh reaction conditions, which is why biocatalytic alternatives were sought. To introduce the \(N\)-hydroxy group at the base, the enzyme cytidine deaminase was engineered to transform cytidine to \(N\)-hydroxy-cytidine at high concentrations, with high conversion and low biocatalyst loadings [148]. The resulting intermediate could be further acylated to the final product by the enzyme Novozym(r) 435, an enzyme that is used in an enzyme cascade developed by Merck & Co in 2021 (Figure 14B) [149]. This route was designed rationally and enzymes were sought to catalyze the necessary reactions to use the simple starting materials ribose and uracil. The first enzyme, Novozym(r) 435, acrylates the primary alcohol of ribose at the 5\({}^{\prime}\) position. Afterwards, 5-5-methylthiophene (MTR) kinase first activates the anomeric center at the 1\({}^{\prime}\) position by phosphorylation and then uridine phosphorylase (UP) couples uracil to this position. The MTR kinase and UP were evolved for improved activity on the non-natural substrates leading to an 80- and a 100-fold improvement. In addition, an ATP regeneration system consisting of pyruvate oxidase, acetate kinase, and a catalase was included to shift the equilibrium towards the product. The resulting molnupiravir precursor was isolated and finalized by a chemical step to convert the amidic carbonyl of uracil to an oxime. Molnupiravir was gained with a 69% overall yield and notably, the whole cascade with enzyme discovery and evolution, as well as the reaction development and implementation on a large scale, was developed in only 6 months [149]. Furthermore, the patented chemical synthesis consisting of 10 steps yields molnupiravir in much lower overall yields, at around <10% [150]. Besides this forward approach for pathway design, an imposing synthesis route for islatravir was developed using a bioretrosynthetic method by Merck & Co. and Codexis in 2019 (Figure 14C) [141]. Islatravir was in clinical trials for the treatment of HIV but the study was paused in 2021 due to the reduction of important immune cells [155]. For the synthetic route, they used the bacterial nucleoside salvage pathway as a scheme. With this scheme, they chose the reactions and substrates needed to build the nucleoside analog, starting at the product [141]. The reactions, in reversed order, are the attachment of the nucleobase, the phosphorylation at this position, and the assembly of the deoxyribose out of a glyceraldehyde 3-phosphate derivative and acetaldehyde. The enzymes purine nucleoside phosphorylase (PNP), phosphopentumatase (PPM), and deoxyribose 5-phosphate aldolase (DERA) were suitable for these reactions, respectively.