## 2 Chemical Synthesis of Nucleoside Di- and Triphosphates
### Synthesis of Nucleoside Di- and Triphosphates via Phosphoramidate Intermediates  
During studies on the activation of phosphate monoesters, nucleoside phosphoramidates emerged as potential synthetically useful intermediates. Preliminary studies by [PERSON] and [PERSON] showed that phosphoramidic acid rapidly hydrolyzed to phosphoric acid in an acidic environment [50]. Under these conditions, the protonation of the amino function apparently transformed it into a good leaving group (ammonia), significantly increasing the electrophilicity of the phosphorus center. This could open up new synthetic possibilities. The expected advantage of the phosphoramidate strategy in the synthesis of di- and triphosphates compared to the chlorophosphate method described above was the possibility of using unprotected phosphate groups, which would reduce the number of possible side reactions, mainly di- and triphosphate hydrolysis, during the deprotection process. An important point was also the relatively low reactivity of phosphoramidates towards alcohols, which should alleviate the need to protect the 2\({}^{\prime}\)- and 3\({}^{\prime}\)-OH groups in the ribose ring [43; 47]. In the initial attempts to synthesize di- and triphosphates by the phosphoramidate method, an unsubstituted amide group P-NH\({}_{2}\) was used. However, the low reactivity of such compounds has limited their use to only a few cases, e.g., ADP, ATP and UDP [51; 52]. In order to increase the reactivity of phosphoramidates as well as their solubility in organic  
Figure 4: A mechanism for the formation of nucleoside 5\({}^{\prime}\)-diphosphates using dicyclohexylcarbodiimide (DCC) as a condensing agent. solvents, derivatives of 2\({}^{\circ}\) amines were investigated, mainly phosphoromorpholidates and phosphorimidazoles, generated from the corresponding NMPs. The first method made use of phosphoromorpholidates of type 7 (Scheme 5) as reactive intermediates generated in situ, which were further reacted with tetra-_n_-butylammonium salts of mono- or diphosphates in pyridine to provide nucleoside di- or triphosphates, respectively [51], although the latter were obtained only in moderate yields (<60%). Further studies have showed that pyridine used as a solvent stimulates equilibrium between nucleoside triphosphate (NTP) and diphosphate (NDP), and its replacement by DMSO raised the yields of NMPs to >80% (Scheme 5) [53]. This method of synthesizing di- and triphosphates, developed by [PERSON] and [PERSON] in the early 1960s, was rather universal and is still in use today; it works well with a variety of nucleoside substrates, and is also used to make phosphonate mimics of the triphosphate group [54; 55; 56]. It is worth noting that in 2013, [PERSON] et al. described a novel variant of this approach, in which the reactive phosphoramidate intermediate **8** was obtained from a suitably protected nucleoside and benzyl \(N\),_N_-diisopropylchlorophosphoramidite **9** (Scheme 6). The phosphorylation was promoted by 1_H_-tetrazole and the obtained phosphoramidite intermediate (structure not shown) was hydrolyzed to benzyl nucleoside H-phosphonate diester, which was subjected to oxidative coupling using the CCl4/Et3N/piperidine system to afford phosphoropiperidate **8**. Then, all benzyl groups (Bn and Cbz) were removed by hydrogenation, and the unprotected phosphoramidate was treated with tris(tetrabutylammonium) salts of phosphoric or pyrophosphate acids in the presence of 4,5-dicyanoimidazole (DCI), which acted as a mild proton donor that catalyzed the substitution reaction at the phosphorus center. This chemistry was used for the preparation of ribo- and deoxynucleoside di- and triphosphates in good yields (68-81%) [57]. Another route to nucleoside di- and triphosphates was proposed by [PERSON] et al., who showed that phosphorimidazoles **10** (Scheme 7) could also be easily generated by the reaction of nucleoside phosphates with carbonyldiimidazole (CDI) (Scheme 7), and may be useful intermediates in the synthesis of nucleoside pyrophosphates [58; 59; 60].