Solid‐Phase Synthesis of Oligonucleotide 5′‐(α‐P‐Thio)triphosphates and 5′‐(α‐P‐Thio)(β,γ‐methylene)triphosphates

Abstract

A robust solid‐phase synthesis was developed to obtain original oligonucleotides (ONs) functionalized at their 5′ end with modified triphosphate (TP) moieties, in which a nonbridging oxygen atom of the α phosphorus atom was replaced by a sulfur atom and the labile P–O–P linkage was changed into a methylene bridge between the β and γ phosphorus atoms. The efficient method is based on solid‐supported ON assembly followed by 5′‐H‐phosphonylation, oxidation to the thiophosphate subsequently activated as a phosphoanhydride with diphenyl phosphoryl chloride, then nucleophilic substitution with the alkylammonium salt of pyrophosphate or its β,γ‐methylene analogue. After deprotection and release from the solid support under basic conditions, 5′‐(α‐P‐thio)TP and 5′‐(α‐P‐thio)(β,γ‐methylene)TP oligonucleotides were obtained in satisfactory yields, and they were isolated with high purity. These hydrolysis‐resistant 5′‐TP ONs will be useful in biological research to elucidate the mechanism of enzymes involved in mRNA processing and maturation.

Chemical Synthesis of the Antiviral Nucleotide Analogue ddhCTP

“…Silyl ether 10 , methyltriphenoxy­phosphonium iodide, difluorenyl N , N -diisopropyl­phosphoramidite, tributylammonium phosphate, and bis­(tributylammonium) pyrophosphate were prepared according to literature procedures. Reactions requiring anhydrous conditions were carried out in flame-dried glassware under a positive pressure of argon in anhydrous solvents using standard Schlenk techniques.…”

Biocatalytic Synthesis of Antiviral Nucleosides, Cyclic Dinucleotides, and Oligonucleotide Therapies

“…Chemical phosphorylation of nucleosides is challenging, so to access scalable quantities of stereopure S p -2’F-thio-ATP and S p -3′F-thio-GTP substrates, engineered kinases were employed to generate the required NTPs from the corresponding nucleoside monothiophosphates. − Saccharomyces cerevisiae adenylate kinase ( Sc -AdK) and Branchistoma floridae guanylate kinase ( Bf -GK) were used to produce the nucleotide α-thio-diphosphates S p -2’F-thio-ADP and S p -3′F-thio-GDP, respectively, which were subsequently converted to the analogous α-thiotriphosphates using Thermotoga maritima acetate kinase ( Tm -AK). ATP is the canonical phosphate source used by Sc -AdK and Bf -GK; however, ATP can also react in the cGAS mediated coupling/cyclization step to form undesired side products.…”

Phosphonate analogues of nucleoside polyphosphates

“…Diphenyl chlorophosphate [40][41][42][43] , N,N´-carbonyldiimidazole (CDI) 31,44,45 , imidazole/2,2´-dithiopyridine/Ph3P system [46][47][48][49][50] , dicyclohexylcarbodiimide (DCC) 51,52 , and trifluoracetic anhydride 53,54 are the most widely used activating reagents for the synthesis of methylene-modified nucleoside tri-and polyphosphates. All coupling methods have the same strategy in common: one nucleotide subunit (usually nucleoside monophosphate or bisphosphonate) is converted via an activation process into an electrophilic substrate and then reacted with a second phosphate or phosphonate subunit acting as a nucleophile.…”