Acetylphosphate is a mixed anhydride of phosphoric acid and acetic acid, one of the high-energy phosphates. It is important as an intermediate metabolite for bacteria to synthesize or utilize acetic acid. It is generated from acetyl-CoA under the action of phosphotransacetylase or from acetic acid and ATP under the action of acetate kinase.
C-nucleosides are an important class of synthetic target molecules as building blocks of anti-infective agents and therapeutic nucleic acids. However, the reported synthesis methods of C-nucleosides face challenges such as limited raw material sources, low yields, and small reaction ranges. Studies have evaluated the various steps of the phosphorylation-condensation cascade reaction using pentose and uracil analogs as raw materials. For the phosphorylation reaction, the reactions catalyzed by phosphatases and kinases were investigated, and the effectiveness of acetyl phosphate (AcP) as a phosphate donor was discussed and confirmed. Furthermore, through the combination of biocatalysis and reaction engineering, the phosphorylation reaction and the condensation reaction were successfully integrated, and Ψ5P was efficiently synthesized in one pot. The reaction boundary of this process far exceeds (30 times) the limit of the soluble substrate concentration of the nucleobase and maximizes the use of the AcP donor. In addition, a variety of N-nucleosides have also been synthesized through the phosphorylation-condensation cascade reaction.
First, the researchers tried the phosphorylation reaction catalyzed by phosphatase, using phosphatases from various biological sources and acetyl phosphate (AcP) as the phosphate donor substrate for research. However, the nuclear magnetic resonance NMR spectrum results of the product showed that when AcP was fully converted, the yields of D-ribose-1-phosphate and Rib5P were similar, indicating that the phosphorylation reaction of Rib catalyzed by phosphatase was not selective, which may be due to the lack of site selectivity of phosphatase for Rib. Subsequently, the researchers selected the RbsK reaction that is selective for the Rib O5 site to synthesize Rib5P, and coupled the AcK reaction to achieve ATP regeneration. The reaction is catalyzed by kinase and carried out under the conditions of pH 7.5 and 30°C in aqueous phase. ATP phosphorylates Rib to obtain Rib5 with high yield (≥ 90%), which is the combined optimal condition for two kinases and C-glycosidase.
After confirming that the coupled kinase reaction is an effective reaction type for phosphorylation, the authors further significantly improved the efficiency of the phosphorylation reaction by adjusting the substrate concentration and Mg2+ concentration. When the Rib concentration was adjusted to ~ 1M, its conversion rate was as high as about 97%; and when Mg2+ was adjusted to close to its saturation concentration (~ 60 mM), the generation rate of Rib5P increased by about 20 times.
After optimizing the reaction of phosphorylating Rib to synthesize Rib5P, the authors then systematically explored the C-C condensation involved in the cascade reaction. The study used C-glycosidase YeiN from Escherichia coli, which can maintain its reaction activity in the Rib5P reaction system and realize the C-C condensation reaction of Rib5P and Ura without the addition of Mn2+. Considering that the solubility of Ura in water is about 1/30 of the concentration of Rib5P used in the reaction, the researchers adopted a step-by-step feeding strategy of adding Ura, with a single input amount between 50-670 mM, and applied stirring to jointly overcome the solubility and mass transfer limitations. By adjusting the concentration of the YeiN enzyme used, the yield of Ura and Rib5P condensation conversion to generate Ψ5P exceeded 97%, and the output was as high as 52 g/L/h.
Subsequently, the researchers expanded the one-pot reaction scale from 0.5 mL to 10 mL by 20 times. The phosphorylation step and the condensation step in the cascade reaction are compatible, but the yield dropped by about 30% because the activity of YeiN under the reaction conditions was affected by the alkaline environment. Under optimized conditions, about 2.2 g of Ψ5P can be obtained in a 10 mL reaction volume, with a yield of 38 g/L/h.
For various pentoses, such as D-arabinose (Ara), 2-deoxy-Rib (2dRib), and D-xylose (Xyl), as well as uracil analogs, the researchers demonstrated the synthetic flexibility of the developed cascade synthesis reaction in the construction of nucleosides from pentoses and Ura analogs by optimizing conditions.
The researchers compared two cascade reactions for the synthesis of Ψ5P using Rib/Ura as raw materials and U as raw materials reported in the literature, and found that although both had high yields, the Ψ5P product concentration obtained from the Rib/Ura raw material cascade reaction was lower, which was due to the limited available concentration of AcP and the dilution caused by pH control.
Given that kinases have sufficient site and stereo control over substrate phosphorylation, the challenge of reaction design and engineering lies in the efficient regeneration of nucleoside 5'-triphosphate (NTP) phosphate donors. Previously reported kinase reactions face problems such as complex reaction control factors, high cost, difficulty in scale-up, low product selectivity and difficulty in separation. Therefore, the use of AcK reactions using AcP has unique advantages and excellent yields for phosphorylation reactions. The synthesis requirements of AcP raw materials, the need for pH control and sensitivity to hydrolysis are its main disadvantages. For Ψ5P synthesis, based on the principle of economy in reaction engineering, it is preferred to perform AcK reactions directly from AcP. Related calculations show that the yield of the Rib phosphorylation process demonstrated in the current study is close to the upper limit of the physical OTR. Overall, this study achieved record product concentrations and yields in reaction types with AcP as the phosphate donor by optimizing the Rib phosphorylation process. The performance results show that NTP-dependent phosphorylation reactions can match the technical and economic goals of industrial biocatalysis.
