Protection-Free One-Pot Synthesis of 2′-Deoxynucleoside 5′-Triphosphates and DNA Polymerization

Abstract

By differentiating the functional groups on nucleosides, we have designed and developed a one-pot synthesis of deoxyribonucleoside 5′-triphosphates without any protections on the nucleosides. A facile synthesis is achieved by generating an in situ phosphitylating reagent that reacts selectively with the 5′-hydroxyl groups of the unprotected nucleosides. The synthesized triphosphates are of high quality and can be effectively incorporated into DNAs by DNA polymerase. This novel approach is straightforward and cost-effective for triphosphate synthesis.

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 was performed according to the general procedure described [41]. In general, the unprotected nucleoside: 2′-deoxyadenosine, 2′-deoxycytidine, 2′-deoxyguanosine, or thymidine (0.2 mmol, each), and tributylammonium pyrophosphate (182.4 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.…”

Phosphodeoxyribosyltransferases, Designed Enzymes for Deoxyribonucleotides Synthesis

“…Whereas chemical phosphorylation is widely used, it requires several steps of protection, deprotection, and purification (36). It is only recently that a one-pot synthesis of deoxyribonucleoside 5Ј-triphosphates without any protection on the nucleosides was reported (37). Enzymatic methods using dNMP have been attempted, but they require dNMP kinases and pyruvate kinases (38,39).…”

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.…”