Nucleoside phosphoramidates

Methodologies to create functional and topological diversity in libraries of nucleoside phosphoramidates have been improved by the use of parallel solid-phase synthesis. A representative 600-member library of dinucleoside phosphoramidates (16) and tri-nucleoside phosphoramidates (17) was synthesised from CGP-support-bound di- and tri-nucleoside H-phosphonates (5 -DMT protected) and selected amines. The library was screened for antiviral activity against HBV and some of its members showed potent activity. ... 

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 deamination of phosphoramidates by isoamyl nitrite has been mentioned in previous Reports and is the basis of a method for the protection of 5 -phosphoryl groups in the synthesis of oligonucleotides. The oxidation of nucleoside phosphoramidates with bromine in the presence of alcohols leads to phosphodiesters. The AT-bromophosphoramidate which is presumably an intermediate should break down by a pathway which is similar to that of the acid-catalysed decomposition of phosphoramidates. 

Tobias SC, Borch RF (2001) Synthesis and biological studies of novel nucleoside phosphoramidate prodrugs. J Med Chem 44 4475-4480... 

Herdewijn and co-workers have been for some time investigating alternative nucleotide building blocks suitable for synthesis of oligonucleotides based on nucleoside phosphoramidates. Here they have shown that the iminodipropionic acid derivative (5) is a good substrate for HIV-1 RT resulting in -fl primer extension of 91% at 50 pM concentration. Similar results were found for the pyrophosphate mimic (6) with HIV-1 RT. They have also shown that the L-Ala phosphoramidate derivatives are also substrates for HIV-1 RT and for various DNA polymerases. ... 

Nucleoside phosphoramidates are the oxidised form of nucleoside phosphoramidites, but they are designed for different purposes. Nucleoside phosphoramidites are very reactive and used as the monomers for chemical synthesis of oligomers while nucleoside phosphoramidates are relatively stable and used primarily as prodrugs since the amidate moiety is often constructed by linking with a natural amino acid to enhance the cellular uptake of a phosphoramidate which is then hydrolysed to generate the desired nucleoside monophosphate. Cho et al. developed an efficient method for the synthesis of exo-N-carbamoyl nucleosides and their phosphoramidate prodrugs (51) in excellent yields. 

Recent developments in DNA/RNA chemical synthesis have allowed us to attach some functional groups covalently to nucleic acids, thus permitting the introduction of a functionality or properties not normally present in the native biomolecule The use of non-nucleosidic linkers is probably the most popular approach for the 5 -terminal modification of chemically synthesized nucleic acid oligonucleotides and a number of such linkers are commercially available. The linker shown in Fig. 2 is designed as a phosphoramidate derivative so that it can be incorporated into the 5 -terminus of the sequence as the last... 

A more complex pathway of activation is seen in N-amino acid derivative of phosphoramidic acid diesters of antiviral nucleosides, as exemplified by prodrugs of stavudine (9.79, Fig. 9.14) [153 -155], The activation begins with a carboxylesterase-mediated hydrolysis of the terminal carboxylate. This is followed by a spontaneous nucleophilic cyclization-elimination, which forms a mixed-anhydride pentacycle (9.80, Fig. 9.14). The latter hydrolyzes spontaneously and rapidly to the corresponding phosphoramidic acid monoester (9.81, Fig. 9.14), which can then be processed by phosphodiesterase to the nucleoside 5 -monophosphate, and by possible further hydrolysis to the nucleoside. 

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). 

Extensive work has been reported on the chemistry of polyphosphates, in particular that of dinucleoside and sugar nucleoside pyrophosphates. This reflects the reliability and flexibility of the phosphoramidate methods which have been developed over the past few years. Similarly, a wide range of oligonucleotide building blocks, incorporating extensive structural modifications when compared to the natural nucleoside structures, have been described. 

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