## 2 Chemical Synthesis of Nucleoside Di- and Triphosphates
### Synthesis of Nucleoside Di- and Triphosphates via Cyclic Phosphite Trister Intermediates  
The previously discussed methods for di- and triphosphate synthesis were based on the use of reagents containing the phosphorus atom in the V oxidation state (P(V) compounds). At the end of the 1980s, [PERSON] and [PERSON] developed a different approach for the preparation of nucleoside triphosphates and theirs \(\alpha\)-thio-analogs, which was based on the reactivity of P(III)-P(V) mixed anhydrides (Scheme 11). In this method, nucleosides must be properly protected due to their non-regiosellective phosphorylation with salicylchlorophosphate **15**. In the first stage of the synthesis, nucleoside cyclic phosphite **16** is formed, which undergoes a reaction with tributammonium salt of pyrophosphate and is transformed into mixed P(III)-P(V) \"trimeta\" anhydride **17**, a key intermediate for the whole process. Its oxidation, followed by hydrolysis and deprotection, gives the final product, the corresponding nucleoside 5\({}^{\prime}\)-triphosphate (Scheme 11) [78].  
**Scheme 11. Synthesis of NTP and NTP\(\alpha\)S using the Ludwig and Eckstein method [78].**It is postulated that the first intermediate **16** undergoes a nucleophilic attack at the phosphorus center by pyrophosphate with the departure of a carboxylate, followed by the ring's closure as a result of an intramolecular substitution of the aryloxy group [78]. The formed mixed anhydride **17** is then oxidized with iodine/water or sulfur, leading to nucleoside cyclic triphosphates **18** or their thio derivatives. Hydrolysis of these trimetaphosphate intermediates, followed by their deprotection (under basic or acidic conditions, depending on the protecting groups used), leads to the formation of nucleoside triphosphates in the ribo- and deoxy- series (oxidation with I\({}_{2}\)/H\({}_{2}\)O system) or their \(\alpha\)-thio-analogs (oxidation with elemental sulfur). The ring opening proceeds with full regioselectivity towards NTPs/NTP\(\alpha\)Ss without the formation of branched isomers \(\gamma\)NTPs/\(\gamma\)NTPs(Scheme 11). After DEAE-Sephadex chromatography, the products were obtained in 60-75% yields. Due to the high reactivity of salicylchiorophosphate **15** it is important to maintain strictly anhydrous conditions to minimize the formation of undesired side-products [65].  
Exploring the further synthetic possibilities of this method, [PERSON] and [PERSON] proposed the use of thiopyrophosphate salts (Figure 2) as a nucleophilic for the reaction with cyclic intermediate **16**. This, combined with the oxidation of the P(III) center by sulfur, provided access to the P,P\({}_{\beta}\)-dithio analogs of nucleoside 5\({}^{\prime}\)-triphosphates. The ring opening was not fully chemoselective, and the formation of ca. 15% of P\({}_{\alpha}\),P\({}_{\gamma}\)-dithio isomer was also observed. This by-product could be removed by selective acidic hydrolysis toward nucleoside thiodiphosphate, followed by DEAE-Sephadex chromatography [79].  
The above-described one-pot, three-steps protocol is currently one of the most popular methods for the preparation of nucleoside 5\({}^{\prime}\)-triphosphates NTPs and theirs various analogs [32].