## 2 Enzymatic Cascade Reactions to Produce Natural and Modified 5\({}^{\prime}\)-NTPs
### Biocatalytic Synthesis of 5\({}^{\prime}\)-NtPs Starting from Nucleobases and Using NMP Kinases  
In addition to nucleosides, nucleobases are an affordable starting material. PRTs are commonly used as catalysts to couple the nucleobase with the activated sugar moiety PRPP. However, this method cannot be used to synthesize cytosine derivatives, as no PRT is available for this purpose. Additionally, the synthesis of _C_-nucleotides requires the use of different enzymes, specifically PUS. The following sections describe cascades involving PRT/NMPK and PUS/NMPK.  
By combining a PRT with adenylate kinase and CK, 2F-ATP was synthesized in 96 h with a 90% yield, starting with a nucleobase and ribose as substrates [66] (Figure 4). Ribose was first phosphorylated by ribokinase to form ribose-5-phosphate, which was then converted to PRPP by PRPP synthetase (Figure 4A). Similar cascades have also been used for the production of 5F-UTP [92] and 8-aza-GTP [64], yielding 80% and 60% product, respectively (Figure 4). In both studies, PK was employed to interconvert the 5\({}^{\prime}\)-NDP to the corresponding 5\({}^{\prime}\)-NTP. To ensure efficient conversion from PRPP to 5\({}^{\prime}\)-NMP, substrate-specific PRTs were utilized in each case. Adenine PRT was used for 2F-ATP synthesis [66], while uracil PRT and guanine PRT were applied to produce 5F-UTP [92] and 8-aza-GTP [64], respectively (Figure 4A).  
The advantages of this approach include the potential for very high (>80%) conversions, particularly when phosphate donor regeneration systems are employed. Additionally, ribose and nucleobases are inexpensive starting materials. With the identification of 4-(D-ribofuranosyl)aminobenzene synthases (8-RFAS), _C_-nucleosides could also be produced using this approach. 8-RFAS catalyze the formation of a \(\beta\)-C-ribosidic bond between PRPP and differing acceptor substrates. Depending on the enzyme, substrates may contain  
Figure 4: Biocatalytic cascades for 5\({}^{\prime}\)-NTP synthesis using PRTs and NMPKs as biocatalysts. Reactions were started from ribose and a nucleobase. (**A**) Reaction scheme. (**B**) Reported isolated yields [64; 66; 92]. RbsK: ribokinase, PrsA: PRPP synthase, AprT: adenine PRT, UraP: uracil PRT, glyD: guanine PRT, AMPK: AMP kinase, NMPK: NMP kinase, GMPK: GMP kinase, CK: creatine kinase, PK, pyruvate kinase.  
a carboxylic group. Examples are aromatic acceptors (e.g., para-aminobenzoic acid) or pyrazole-derived carboxylic acids. Detailed information on this enzyme class is available from [PERSON] and [PERSON] (2023) [93]. However, the drawbacks include a comparably long route requiring five enzymes and the fact that these protocols are currently limited to ribose as the sugar moiety. Moreover, PRPP synthases are often inhibited by purine 5\({}^{\prime}\)-NDPs.  
Another approach to access C-nucleotides is based on the preplacement of PRT with PUS (Figure 5). Several sugar- and base-modified pseudouridine-5\({}^{\prime}\)-monophosphate variants were produced and then phosphorylated to NTPs applying _Escherichia coli_ pseudouridylate synthase YeiN, CMPK, and NDPK in a one-pot, multi-step process with yields of up to 85% [94] (Figure 5B). A PK-based ATP regeneration system was incorporated into the reaction. The process started from ribose, which was initially converted to ribose-5-phosphate, the substrate for PUS, by ribokinase (Figure 5A). Unlike other approaches, this method is specific to C-nucleotides and has so far only been applied to uracil derivatives [94].