# Recent Advances in Biocatalysis for Drug Synthesis
## 2 Biocatalysis for Drug Synthesis
### Synthetic Enzyme Cascades
The fluorine derivative was shown to confer improved STING binding, attesting its pharmacological relevance [162]. To further optimize the synthesis of CDNs and to lower the substrate costs, enzymes were implemented to deliver a suitable substrate _in situ_. In this regard, [PERSON] _et al._ developed an enzyme cascade to produce the substrate ATP for a single-step transformation by cGAS to form the CDN 2'3'-cGAMP. The multi-enzymatic reaction was developed to form ATP by phosphorylating adenosine, which is subsequently cyclized with GTP to synthesize the cyclic dinucleotide (Figure 15) [153]. In the first step of the cascade, adenosine kinase (ADK) phosphorylates the nucleoside to AMP by consuming ATP. AMP is then converted stepwise to ATP by two homologues of polyphosphate kinase 2 (PPK2). This enables the recycling of the formed ADP by the ADK and the chosen PPK2s allows the usage of cheap polyphosphate (polyP) as the substrate. The formed nucleotide triphosphate serves with GTP as the substrate cGAS, which produces 2'3'-cGAMP. To increase synthesis rates, yield, and enzyme usage, substrate concentrations, as well as the ratios of the enzyme concentrations, were adjusted according to their specific activities. Several attempts were conducted to find the best composition. The 2'3'-cGAMP production could be increased \(\sim\)2,5-fold and ultimately, the product was gained by the four-enzyme cascade with 0.08 mol per mol adenosine, which is in the same range as the chemical synthesis [153]. Recently, Merck & Co and Codexis published the multi-enzymatic synthesis of the derivative ML-1454 (Ulevostinag), a more potent fluorinated phosphorothioate CDN analogue [154]. The cyclization of thioGTP and thioATP was also performed by cGAS, though, the enzyme was evolved towards improved reactivity in seven rounds. The challenge to form the correct CDN diastereomer with achiral thiophosphategroups was solved by an enzymatic desymmetrization. Thiomonophosphates were phosphorylated by adenylate kinase (AK) or guanylate kinase (GK) and the resulting thiodiphosphates were subsequently stereoselectively phosphorylated by an Ack. AK, GK, and Ack were evolved in 2-5 rounds, leading to good diastereomeric ratios and high yields. The merging of the enzymatic reactions to a one-pot cascade resulted in a 62% isolated yield from nucleotide monothiophenstates [154]. This is a remarkable production, especially in comparison to chemical synthesis, where yields around 5% for \(2^{\prime}3^{\prime}\)-GAMP within 3 days are achieved [160; 163]. In addition, these examples highlight the difficulties of using enzyme cascades. The composition of the reaction, such as enzyme ratios and substrate concentrations, plays a vital role for the performance of the systems. Engineering the enzymes towards tailored properties can improve the interplay of the catalysts but it is time- and cost-intensive. Chemoenzymatic cascades combine the advantages of biocatalysis with classical chemistry to broaden the reaction scope. However, mutual inactivation is a problem in these synthesis approaches since enzymes have to tolerate harsher conditions such as solvents [164] and higher temperatures, and chemical catalysts can be inhibited in aqueous solutions as well. Nonetheless, lipases and serine proteases were among the first enzymes  
Figure 14: (**A**) Enzyme cascades for the synthesis of GSK-2879552 [34], (**B**) Molinupiravir [149], (**C**) Islatravir [141], and (**D**) cannabinoid acid and cannabinoid acid (CBGA) [152].