Nucleosides phosphotriester

Nucleoside Acyclic Phosphates - It is well established that most nucleoside analogues become biologically active as a result of cellular conversion to phosphomonoesters. For this reason there has been increasing interest in the synthesis of nucleoside phosphotriesters and in some cases, i osphodiesters which might act as membrane soluble pro-drugs of the bioactive nucleotides. The diethyl-, dipropyl- and dibutyl-phosphates (1) of 2, 3 -dideoxy-3-deaza-adenosine have been synthesis and shown to possess anti-HIV-1 activity at non-cytotoxic doses. Phosphate triester derivatives of the anti-leukaemic nucleoside nmbi/io-cytidine have... 

Diastereoselective synthesis of cyc/o-saligenyl nucleoside phosphotriesters has been achieved using two diastereopure 3-methyl-cyc/o-Sal-phospho-triesters, e.g. (1, antiviral activity was between five and 20-fold different between the individual diastereoisomers. A series of C5-substituted alkenyl- alkynyl- and aryl-dUMP derivatives was synthesised and assayed against the flavin-dependent thymidylate synthase ThyX from Mycobacterium tuberculosis. Of these some were found to be active at submicromolar concentration with specificity for ThyX compared with a bacterial thymidylate synthase. Eleven triazole nucleoside monophosphates (2) were synthesised, with inhibitory effects against bacterial NAD-dependent malic enzyme. ... 

Using phosphotriester methods, dinucleoside (3 - 50-monophosphates containing 6-methyl-2,-deoxyuridine at the 3 - or 5 -end have been prepared.44 N.m.r. spectroscopy indicates that this nucleoside possesses the syn conformation in these compounds, and, on treatment with snake venom phosphodiesterase, d(m6UpT) is degraded, while d(Apm6U) is not, indicating that this enzyme, a 3 -exonuclease, requires the anti conformation to be present in the substrate. Two modified nucleo-side-5 -monophosphates, (20) and (21), which are resistant to 5 -nucleotidase, have been isolated from tRNA snake venom hydrolysates.45 A synthesis of (20) has been reported.46... 

Example 63 Meier in his studies on potential prodrugs derived from biologically active nucleoside monophosphates used the cyclic chlorophosphites 2-chloro-4-ff-l,3,2,-benzodioxa-phosphinines for the synthesis of the cyclic phosphotriesters having two ligands of different hydrolytic stability benzy-loxy and aryloxy [102]. 

In the 1,3,2-dioxaphosphole method a bis(2-butene-2,3-diyl) pyrophosphate is used as the condensing agent. It allows two successive esterifications of one phosphate group to be performed without additional activation. First a 5 -O-protected nucleoside is added in methylene chloride in the second reaction an unprotected nucleoside can be used, since only the 3 OH group is able to attack the cyclic enediol 3 -nucleosidyl phosphotriester. Protected dinucleoside triesters are obtained in 80% yield. Removals of protective groups, methoxytrityl by means of trifluoroacetic acid in methylene chloride and 1-methylacetonyl by aqueous triethyl-amine, also give about 80% yield (F. Ramirez, 1975, 1977). 

PhosphorylationThis reagent can be used for 3 -and/or 5 -phosphorylation of nucleosides in the phosphotriester approach to oligonucleotides. Deblocking by / -elimination proceeds cleanly with DBU in pyridine at 25" in 24 hours. 

There are three widely used chemistries for oligodeoxynucleotide synthesis, and all have been successfully transferred onto SP. The first is the so-called phosphotriester approach (71), which is shown in Fig. 2.9. In this method a 5 -protected nucleoside... 

Phosphorus oxychloride is a suitable reagent for preparation of the symmetrically substituted phospho-triesters of type (RO)3PO. The preparation is easily achieved by treatment of phosphorus oxychloride with 3 equiv. of alcohols or their metal salts. The reaction is generally promoted by a base or acid. Titanium trichloride is a particularly effective catalyst for the reaction. Conversion of POCI3 to unsymmetri-cally substituted phosphotriesters is achievable with difficulty. Phosphorochloridates and phosphorodichloridates have been used for the preparation of mixed tertiary phosphoric esters of type (ROlmPOfOROn (ffi = 1, n = 2, or m = 2, n = 1) in a very wide variety. Reaction of phosphorus oxychloride and 1 or 2 equiv. of alcohols followed by hydrolysis forms phosphomonoesters or phosphodi-esters, respectively. The hydrolysis may be generally effected by dilute aqueous alkali. Some phosphoFodichlori te intermediates are easily hydrolyzed by water. For example, the phosphorylation of a ribonucleoside (1 equation 4) with phosphorus oxychloride in an aqueous pyridine-acetonitrile mixture furnishes the nucleoside S -monophosphate (2) in excellent yield. ... 

A phosphotriester method has been developed for rapid synthesis of oligodeoxynucleoside phosphorodithioates in solution. Couplings are performed by the chemoselective oxygen activation of protected nucleoside dithiophosphate anions (56) with 4-nitro-6-trifluoromethylbenzotriazol-l-yl-oxy-tris(pyrrolidine)-phosphonium hexafluorophosphate (PyFNOP) (57) or 6-nitrotriazol-l-yl-oxy-tris(pyrrolidine)-phosphonium hexafluorophosphate (PyNOP) (58). Under optimised conditions, coupling yields above 95% were achieved in between 10 and 20... 

However, the best results were obtained when the first nucleoside was attached via the succinyl linker onto the ethylenediamine-treated polydimethylacrylamide resin (Figure 19.4) [178,179] or Enzacryl Gel K-2 [177]. The polydimethylacrylamide support has been utilized by Gait et al. [180] in the automated solid phase oligonucleotide synthesis by the phosphotriester method on a peptide synthesizer. 

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