## 2 Enzymatic Cascade Reactions to Produce Natural and Modified 5\({}^{\prime}\)-NTPs
### Biocatalytic Synthesis of 5\({}^{\prime}\)-NtPs Using Polyphosphate Kinases  
Due to the cost-effective phosphate donor, polyphosphate, polyphosphate kinases (PPKs) have gained significant importance in recent years. This is evident not only in ATP regeneration but also in 5\({}^{\prime}\)-NTP synthesis. PPK-based cascades, like NMPK-based cascades, begin with the nucleobase or nucleoside. To date, either two different PPKs have been used to convert 5\({}^{\prime}\)-NMPs to 5\({}^{\prime}\)-NDPs or 5\({}^{\prime}\)-NDPs to 5\({}^{\prime}\)-NNPs or a single PPK has been utilized to catalyze both reaction steps.  
Figure 5: Biocatalytic synthesis of 5\({}^{\prime}\)-riphosphorylated C-nucleotides. (**A**) Reaction scheme. (**B**) Reported product yields [94]. RbsK: ribokinase, YeiN: C-glycosidase, CMPK: CMP kinase, NDPK: NDP kinase, PK: pyruvate kinase, PEP: phosphoenolpyruvate, PYR: pyruvate.  
When synthesis starts from nucleobases, PRPP is used as an activated sugar, and a PRT serves as the biocatalyst (Figure 6A). Afterwards, the 5\({}^{\prime}\)-riboNTP was synthesized using _Meiothermus ruber_ PPK or an optimized variant, and subsequently, the 5\({}^{\prime}\)-dNTP was formed through an RNR [80; 95]. Using this approach, compounds such as cladribine-5\({}^{\prime}\)-TP and 6-mercapto-5\({}^{\prime}\)-dGTP have been formed, with conversions of 80% and 12%, respectively (Figure 6B). A 5\({}^{\prime}\)-NMP screening was also conducted for the PPKs used, confirming that both the _Meiothermus ruber_ wildtype enzyme and the engineered version showed a broad substrate spectrum, converting compounds such as (d)AMP, (d)GMP, (d)CMP, 8-bromoAMP, and etheno-AMP into the respective 5\({}^{\prime}\)-NTPs [80]. A PRT-PPK-based cascade, without RNR, was also employed for the synthesis of 5-aminoimidazole-4-carboxamide 5\({}^{\prime}\)-NTP (5\({}^{\prime}\)-NTP of AICA) (Figure 6). Using the _Meiothermus ruber_ PPK, a 65% yield was achieved in a two-enzyme approach [96].  
Starting with nucleosides, phosphorylation by an NK is first performed, followed by the PPK-mediated conversion of 5\({}^{\prime}\)-NMP to 5\({}^{\prime}\)-NTP (Figure 7A). [PERSON] et al. successfully synthesized ATP using this approach (Figure 7B) [97], while [PERSON] et al. investigated a range of compounds, including all natural 5\({}^{\prime}\)-NTPs and base-modified variants, achieving yields from 8% to 72% (Figure 7B) [79].  
NK-PPK cascades have also been used in larger cascades. For example, ATP and GTP building blocks were efficiently produced for the subsequent synthesis of 2\({}^{\prime}\),3\({}^{\prime}\)-cGAMP [98; 99]. Furthermore, the route was employed to establish a SAM regeneration system, where ATP was efficiently generated from adenosine in a cascade involving _Saccharomyces cerevisiae_ adenosine kinase, _Acinetobacter jolnsonii_ PPK, and _Sinorhizobium melidi_ PPK [100; 101].  
In summary, these studies demonstrate the great potential of PPK-containing enzyme cascades, as PPKs possess a broad substrate spectrum. However, it remains to be seen how well this system can be applied to sugar-modified compounds or whether the PPK-typical hyperphosphorylation of nucleotides will present a challenge in downstream processing.  
Figure 6: Biocatalytic synthesis of 5\({}^{\prime}\)-(d)NTPs in a PRT-PPK cascade. (**A**) Reaction scheme. (**B**) Reported conversions [80; 95; 96]. EeATP: _Escherichia coli_ adenine PRT, _Ec_APT: _Escherichia coli_ hypoxanthine PRT, _Mr_PPK: _Meiothermus ruber_ wildtype PPK, _Mr_PPK D1275: engineered _Meiothermus ruber_ PPK, TVNrdjm: RNR of _Thermus virus_ TV74-23.