Phosphorothioates, stereospecific

R0P032 is pra-prochiral, since two oxygen atoms must be substituted to produce a chiral compound. Chiral phosphates have been synthesized de novo by using stereospecific chemical and enzymatic reactions with isotopic and/or atomic substitutions. For example, a chiral phosphorothioate may be synthesized from a prochiral phosphate by replacing an oxygen atom with a sulfur atom. Similarly, what would otherwise be a pro-prochiral phosphate has been synthesized as a chiral product by replacing one oxygen atom with sulfur and another... 

With regard to the phosphorus stereochemistry, a new stereospecific synthesis of chiral 0,0-dialkyl thiophosphoric acids (6,7) has been developed which is based on the Horner-Wittig reaction of the optically active phosphonothionate carbanions containing the sulfoxide or dithioacetal moieties. The transformation of (R)-(+) 0-methyl 0-isopropyl methanephosphonothionate (10)(8) into (R)-(-) 0-isopropyl phosphorothioic acid (H) best Illustrates this method. 

Stereospecific Synthesis and Assignment of Absolute Configuration at Phosphorus in Nucleoside 3 - and 5 -0-Arylphosphorothioates and Nucleoside Cyclic 3, 5 -Phosphorothioates... 

ATPases. A few pyrophosphotransferases catalyze substitution at Pg. P0 and Pg are prochiral centers and can be made chiral by stereospecific replacement of one or the other diastereotopic oxygen with sulfur or enrichment with lsO or nO. Py is an achiral center which can be made chiral either by replacement of one of the three equivalent oxygens with sulfur and stereospecific enrichment of another with 180 to give a chiral [180]phosphorothioate, or by stereospecific enrichment of one with nO and another with lsO to give a chiral [160, nO, lsO]phosphate. 

The first chiral phosphates to be used for stereochemical analyses were chiral phosphorothioates, which were used to determine the stereochemical courses of ribonuclease, UDP-glucose pyrophosphorylase, adenylate kinase and several other kinases and synthetases. The chiral phosphorothioates either had sulfur in place of an oxygen at an otherwise prochiral center of a phosphodiester or phosphoanhydride or stereospecifically placed sulfur and 180 (or nO) in a terminal phosphoryl group. The syntheses and configurational analyses of the most important of these compounds are outlined in the following. 

The chiral y-[l80]phosphorothioate of ATP, (Pp)-ATPyS, yl80(/3, y)lsO, was synthesized by the procedure outlined in Fig. 8 [25]. (Sp)-ADPaS, alsO(a,/3)l80 was prepared by stereospecific phosphorylation of AMPaS, al802 using the adenylate and pyruvate kinase system followed by dephosphorylation with glucose and hexokinase. This was the starting material for the synthesis, with the chiral a-... 

Fig. 15. Configurational analysis of chiral [ lsO]phosphorothioates by stereospecific phosphorylation.

The early attempts to use relatively easily available diastereomerically pure nucleoside 3 -0-(2-cyanoethyl-AT,AT-diisopropylphosphoramidite) monomers for the stereospecific synthesis of PS-Oligos failed because of inevitable racemiza-tion of Pm intermediate caused by an excess of lff-tetrazole necessary for efficient elongation of oligonucleotide chain [13]. An idea to use for that purpose appropriately protected nucleosides functionalized at 3 -0 position with 2-thio-1,3,2-oxathiaphospholane moiety arose from the studies on the reactions of di-substituted phosphorothioates with oxiranes [14,15], and in particular from the observation that PS-PO exchange in 0,0-diphenyl phosphorothioate (8) upon treatment with ethylene oxide in methanol solution resulted in formation of... 

Much more advanced and effective appeared to be the method reported recently by Beaucage et al. [127]. Starting from ( )-2-amino-l-phenylethanol (88) the authors performed chemoselective N-acylation with ethyl fluoroacetate (89) providing 90, followed by its reaction with hexaethylphosphorous triamide (Scheme 23). Cyclic N-acylphosphoramidite 91 has been obtained as a mixture of diastereomeric rotamers. Condensation of AT4-benzoyl-5 -0-DMT-2 -deoxy-cytidine 92 with 91 in the presence of lH-tetrazole gave, after silica gel chromatography, pure Rc,RP-93 and Sc,SP-93.31P NMR studies indicated that when Rp-93 or Sp-93 was reacted with 3 -0-acetyl-AT-benzoylcytidine (94) and A ATjN jN -tetramethylguanidine (TMG), the dinucleoside phosphotriester was formed in nearly quantitative yield with full P-stereospecificity. After subsequent sulfurization, the P-stereodefined dinucleoside phosphorothioate tri-... 

Fujii. M.. Ozaki, K., Kume, A., Sekine, M.. and Hata. T. Acylphosphonates. Part 5. A new method for stereospecific generation of phosphorothioate via aioylphosphonale intermediate. Tetrahedron Lett., 2J, 935. 1986. 

Stereospecific syntheses have been reported for ethyl isopropyl methyl phosphate, O-ethyl 0,S-dimethyl phosphorothioate, and ethyl methyl methyl-phosphonate,"" " and optically active alkyl S-methyl methyl phosphonothioates and dialkyl S-methyl methyl phosphorothioates have been prepared from ephedrine-RPSCU starting materials."" Finally, optically active phosphines can be obtained when a racemic phosphine oxide such as MePr"PhPO or (97) is reduced with a chiral, non-racemic aluminium hydride." "... 

Picoline 77-oxide was used as an intramolecular catalytic group to secure stereochemical integrity of the phosphorus center in a stereospecific synthesis of dinucleoside phosphorothioate diesters <04CC290>. 

Oligonucleotides containing phosphorothioate intemucleoside linkages (122) have been of interest for several years now. Full details describing the stereospecific synthesis of phosphorothioates using the oxathiaphospholane monomers... 

It is a well established property that many enzymes interact in a stereospecific manner with the different diastereoisomers of nucleoside phosphorothioates. For example, when AMP is substituted by adenosine S -t7-monopho phorothioate (AMPS), adenylate kinase-catalysed phosphorylation occurs specifically at the pro-R oxygen atom to give (5ip)-ADPaS. The stereospecificity results from the restricted orientation of the P-O and P-S bonds in the enzyme active site. While it is known that changing the metal ion can perturb the stereoselectivity, Tsai and... 

A crucial point in the synthesis of optically pure dinucleoside phosphorothioates is the separation of the precursors of the final products that differ only in their configuration around phosphorus. Column chromatography on silica gel is an efficient and convenient way to separate the diastereomers of H-phosphonate diesters. The results obtained reveal that the sulfurization of suitable protected diribonucleoside H-phosphonates with elemental sulfur is a stereospecific reaction, which is most likely proceeding with the retention of the configuration at the phosphorus center. 

Lesnikowski, Z J and Jaworska, M M (1989) Studies on stereospecific formation of p-chiral internucleotide linkage. Synthesis of (Rp,Rp)-annitro-benzyl group as a new S-protection Tetrahedron Lett. 30,3821-3824... 

The method developed in the authors laboratory is based on the stereochemical correlation between three classes of analogs of dinucleotides, namely phosphorothioates, 0-ethyl phosphorothioates, and 0-ethyl-phosphates (9). Stereospecific dealkylation of diastereoisomers of the dinucleoside 0-alkyl phosphorothioate allows the correlation of their configuration with phosphorothioates. On the other hand, stereospecific oxidation leads to 0-alkyl phosphates (Fig. 4). 

Much more promising seems to be another, so far hypothetical, assumption that an access to stereo-defined oligo (nucleoside phosphoro-thioates) will allow stereospecific conversion of phosphorothioate function to 0-alkyl functions, for example, by selective 5-alkylation and conversion of 5-alkyl phosphates into 0-alkyl phosphates (24). However, one has to take into account potential problems with selective 5-alkylation in the presence of other nucleophilic centers, mostly on the nucleic bases. 

Stereospecificities of the Formation of Dinucleoside (3 -5 ) Phosphorothioates (10) in Reaction Between Corresponding 8 and 5 -OH Nucleosides Bound to Solid Support, with DBU as the Catalyst... 

The discovery of antiviral activity of oligo(nucleoside methane-phosphonate)s (44) and oligo(nucleotide phosphorothioate)s (45-47), so far chemically prepared by the nonstereocontrolled methods, attracted the attention of several research establishments to the search for stereospecific synthesis of those classes of oligonucleotide analogs. Since their routine synthesis via phosphoramidite or any other approach leads to the mixture of m diastereoisomers, the question may be asked whether desired antiviral activity is owing to all m components of diastereo-isomeric mixture or to the fraction possessing the proper sense of chirality at each modified phosphate. Moreover, since the seminal works of... 

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