Nucleoside 5 -phosphoramidates, preparation

Nucleoside S -phosphoramidates have proved to be useful intermediates in the synthesis of nucleoside 5 -pyrophosphates and nucleotide coenzymes. Nucleoside phosphoramidates may also be prepared by treatment of a nucleoside 5 -phosphate with ammonia in the presence of N, IV -dioyclohexylcarbodiimide [R. W. Chambers and J. G. Moffatt, J. Am. Chem. Soc., 80, 3752 (1958)]. 

The choice of these derivatives, namely, those formed from amines of moderate strength, appears to be the best compromise for the synthesis to be effective. Although the susceptibility of phosphor-amidates to attack by nucleophiles increases with enhancement of the basic strength of the parent amine,279 280 the reverse trend is observed for the yields of nucleoside 5 -phosphoramidates when they are prepared from nucleoside 5 -phosphates by condensation with the appropriate amines through the action of N,N -dicyclohexylcarbodi-imide.279... 

The synthetic protocols used for the preparation of oligonucleotides on supports can also be used to prepare oligomers from diols other than nucleosides. Symmetric or unsymmetric diols, such as N-acylated 4-hydroxyprolinol [268] or cyclopentane-derived diols (carbocyclic deoxyribose analogs [269]), can be selectively mono-trity-lated and then converted into a phosphoramidite that is suitable for the solid-phase synthesis of oligophosphates. An illustrative synthesis of protected //-phosphonates from diols, as well as their conversion on CPG into oligomeric phosphoramidates, are outlined in Figure 16.28. 

Deoxynucleoside phosphoramidates of 4-benzoylpyrazolo[3,4-d]pyrimidine-5-amines were prepared and utilized in automatic DNA synthesis yielding DNA fragments having an isosterically modified nucleoside base <91MI 712-02). 

PolynucleotidesThese compounds are now generally prepared by use of a bifunctional phosphitylating agent such as o-chlorophenylphosphorodichloridite (6, 114-115) or methoxydichlorophosphine, CH30PC12. The intermediate nucleoside phosphites from these reagents tend to be unstable. This difficulty can be alleviated by use of 1, which reacts with suitably protected nucleosides to form stable phosphoramidates 2 in good yield. These products can be activated for formation of a dinucleotide phosphite (4) by treatment with a weak acid such as N,N-dimethylaniline hydrochloride or 1 //-tetrazole (5). 

Phosphorylation. Chambers et al. found the reagent useful for the preparation of nucleoside diphosphoric acids (3, R —adenine, uracil, etc.). A nucleotide (I) is converted into the 5 -phosphoramidate (2) by reaction with ammonia and dicyclo-hexylcarbodiimide (DCC), and the amidate (2) is treated with dioxane diphosphate in o-chlorophenol (3 hrs..0 ). The diphosphoric acid (3) is precipitated with petroleum ether and purified as the ammonium salt. Yields are around 90%, whereas when 8.5% phosphoric add was used yields were in the order of 50-60%. However, anhydrous... 

The conformationally-locked 3 -amino thymidine derivative (47) was prepared and incorporated into DNA as a dimer unit, attaching the amino group via a carbamate or phosphoramidate linkage.Both modifications were found to be destabilising towards both RNA and DNA targets. The hybridisation properties of an amino-modified bicyclic thymine nucleotide (48) have been exam-ined. Using alternate T and (48) residues there is an increase in duplex stability with d(A)io, with no pH dependence. When the amino group is acetylated, the duplex stability is decreased. The locked C-nucleoside oxazole derivative (49) has been incorporated into TFOs where it was found to selectively enhance the stability with a C.G base pair. ... 

A new method for the preparation of 5 -amino-5 -deox)mucleotides has been described, based on the reaction between phosphorus triesters and phenyl azide. Treatment of protected 5 -azido-5 -deoxynucleosides with trimethyl or triphenyl phosphite leads, after removal of the nucleoside protecting groups and one of the esterifying groups from the phosphoryl residue, to the phosphoramidates (9). Snake venom phosphodiesterase hydrolyses (9) to 5 -aminonucleosides. 

Analogues of cyclic AMP derived from the carbocyclic nucleosides aristeromycin and neplanocin have been prepared by intramolecular phosphodiester bond formation catalysed by divalent lead ions, and studies have been reported on the synthesis and biological activity of nucleoside 3, 5 -cyclic phosphotriesters and phosphoramidates. ... 

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