A Chemo-enzymatic Route Towards Nucleoside Triphosphate Synthesis

Nucleoside 5’-triphosphates (NTPs) are ubiquitous throughout the natural world. As such investigations into their interactions within organisms is essential. Cur- rently, commercially available NTPs are expensive due to the synthetic challenges associated with the formation of phosphoanhydride bonds. Chemo-enzymatic approaches to NTP synthesis are readily applicable to natu- rally occuring NTPs, as such these methods have gained traction over recent years. We have investigated a nucleoside diphosphate kinase (NDPK H122G) mediated chemo-enzymatic method for the phosphorylation of nucleoside- 5’-diphosphates (NDPs), using phosphoimidazole as a phosphoryl donor. We developed a synthetic route and a purification procedure, which allowed for the production and partial purification of phosphoimidazole using inexpensive chemical precursors. The opti- misation investigations undertaken during the development of the phosphoimidazole synthetic protocol resulted in an increase in phosphoimidazole production from 45% to 55% in the crude reaction solution. Optimisation of the purification procedure led to the production of phosphoimidazole of 98% purity. NDPK H122G catalysed phosphorylation reactions of adenosine 5’-diphosphate (ADP), were investigated over a range of conditions. The effect of both acetoni- trile and imidazole impurities on the total conversion of ADP to adenosine 5’- triphosphate (ATP) was investigated. It was found that both acetonitrile and imi- dazole impurities prompted a decrease in the % conversion to ATP of 17% and 22%, respectively. The effect of phosphoimidazole concentration on the initial rate of phosphorylation and total % conversion after 24 h was also investigated. The initial rate of phosphorylation and the % conversion to ATP after 24 h, were both found to increase with the initial concentration of phosphoimidazole. Initial phosphoimi- dazole concentrations of 5, 10, 20 and 50 mM were investigated; total % conversion to ATP after 24 h was found to be 12%, 14%, 30% and 43%, the initial rate of phosphorylation was 0.752 × 108, 1.263 × 108, 1.627 × 108 and 2.588 × 108 M s−1, respectively. The kinetics of the NDP phosphorylation reactions were monitored using high performance liquid chromatography (HPLC). Phosphorylation of ADP was moni- tored on a SUPELCOSIL-LC-18-T reversed-phase analytical column, while GDP phosphorylation was monitored on a SIELC-Primesep SB mixed mode analytical column. Protocols were developed for both of the columns to ensure that the nu- cleotides had significant retentions times and the peaks were well separated with little tailing.