Convenient synthesis of nucleoside 5′-triphosphates for RNA transcription

Abstract

By generating a selective phosphitylating reagent in situ, nucleoside 5'-triphosphates can be conveniently synthesized in one pot. This novel strategy without nucleoside protection has been developed to largely simplify synthesis of the nucleoside triphosphates. This demonstrated principle can be applied to the 5'-triphosphate synthesis of both native and modified nucleosides.

Convenient synthesis of nucleoside 5′-(α-P-thio)triphosphates and phosphorothioate nucleic acids (DNA and RNA)

“…To address the need for a clean 5′-phosphitylation reaction, we have developed a strategy involving the use of an in situ phosphitylating reagent 2 formed by reacting 2-chloro-4H-1,3,2-benzo-dioxaphosphorin-4-one (salicyl phosphorochloridite, 1) and tributylammonium pyrophosphate in the presence of DMF and tributylamine to synthesize 5′-triphosphates without the need for protection of the starting nucleosides. We have extended this synthesis strategy, further exploring its potential in the preparation of 5′-dNTPαS and 5′-NTPαS where the oxidation step utilizes a sulfurizing reagent as the oxidant instead of iodine, which affords the native counterpart [41,42], or 3H-1, 2-benzothaselenol-3-one (BTSe) [50], which offers the 5′-NTPαSe [43]. Sulfurization of the nucleoside cyclic phosphate 3 was performed using 3-((dimethylamino-methylidene)amino)-3H-1,2,4,dithiazole-3-thione, (sulfurizing Reagent II), following hydrolysis to afford the crude S p and R p diastereomers of the 5′-dNTPαS (4) and 5′-NTPαS (5).…”

“…The synthesis follows the general procedure described in section 2.2 [42,43]. In brief, the unprotected nucleoside: adenosine, cytidine, guanosine, or uridine (0.2 mmol, each), and tributylammonium pyrophosphate (184.4 mg, 0.4 mmol, 2 eq.)…”

“…Our research group has recently developed a convenient one-pot synthetic methodology to prepare natural and Se-modified 5′-triphosphates (dNTPs and NTPs) [41][42][43] without protecting either the base or sugar moiety of the nucleosides. We found that a mild phosphitylating agent (2) could react selectively with the 5′-hydroxyl over the 2′-and 3′-hydroxyl groups of the nucleosides, offering satisfactory yields of the 5′-triphosphates.…”

Antiviral Nucleoside and Nucleotide Analogs: A Review

“…Because of the drawbacks of enzymatic synthesis, such as the substrate specificity, yield and cost, chemical synthesis is still a better choice for preparing a large quantity of nucleoside triphosphates, especially those with modifications. 30 In spite of many synthetic strategies developed including one-pot synthesis such as Yoshikawa protocol, [31][32][33] Ludwig and Eckstein protocol 34,35 and Borch protocol, 36 a convenient synthesis of the 5`-triphosphates directly from unprotected nucleosides with high regioselectivity still remains a challenge. 30 …”

PPN Pyrophosphate: A New Reagent for the Preparation of Nucleoside Triphosphates

“…1 Over the last ~25-50 years, a small number of key methods have been adopted for the preparation of NTPs and other phosphoanhydrides, 2 although new methods are now becoming available. [3][4][5][6][7][8][9][10][11][12][13][14] Often, these approaches rely on triand tetralkylammonium salts as organic-solvent-soluble sources of nucleophilic phosphate. Unfortunately, these salts are extremely hygroscopic, [15][16][17][18][19][20] with deleterious consequences on the water-sensitive reactions that use them.…”