Phosphorothioate, synthesis

Phosphorothioates. All three synthetic approaches appHcable to unmodified oligonucleotides can be adapted for synthesis of phosphorothioates (11) (33,46). If all of the phosphodiester linkages in an oligonucleotide are to be replaced with phosphorothioates, the ff-phosphonate method for coupling, followed by oxidation with Sg in carbon disulfide and triethylamine in the final step, is the most straightforward method. 

Chirahty at the phosphoms is an unavoidable problem in all phosphorothioate syntheses. The phosphoramidite method produces a mixture of both the and the diastereomers having a small excess of the isomer (53). Although some progress has been made in the chiral synthesis of dinucleoside phosphorothioates, low yields have limited the utility of these approaches. The chiral center may be eliminated by replacing the other, nonbridging oxygen with sulfur. Avoidance of the chirahty problem is one reason for the interest in phosphorodithioates. 

These groups, along with a number of other trialkylsilylethyl derivatives, were examined for protection of phosphorothioates. Only the phenyl-substituted silyl derivative was useful, because simple trialkylsilyl derivatives were prone to acid-catalyzed thiono-thiolo rearrangement. Other trialkylsilylethyl derivatives also suffer from inherent instability upon storage,but the trimethylsilylethyl group has been used successfully in the synthesis of the very sensitive agrocin 84 and for intemucleotide phosphate protection with the phosphoramidite approach. 

The 4-chloro-2-nitrobenzyl group was useful in the synthesis of dithymidine phosphorothioates. It could be cleaved with a minimun of side reactions with PhSH, TEA. Pyr. ... 

Furthermore, the GPO procedure can also be used for a preparative synthesis of the corresponding phosphorothioate (37), phosphoramidate (38), and methylene phosphonate (39) analogs of (25) (Figure 10.20) from suitable diol precursors [106] to be used as aldolase substrates [102]. In fact, such isosteric replacements of the phosphate ester oxygen were found to be tolerable by a number of class I and class II aldolases, and only some specific enzymes failed to accept the less polar phosphonate (39) [107]. Thus, sugar phosphonates (e.g. (71)/(72)) that mimic metabolic intermediates but are hydrolytically stable to phosphatase degradation can be rapidly synthesized (Figure 10.28). 

Figure 10.25 Inverted approach for aldose synthesis using FruA catalysis, and application ofthe strategy for deoxysugar synthesis based on a phosphorothioate analog synthesis of 1-deoxysugars by FSA catalyzed addition of hydroxyacetone.

The use of triphenylphosphine and 2,2 -bipyridyl disulphide in oxidation-reduction condensations has been extended to the phosphorylation of alcohols and phosphates, and to the preparation of 5 -(2-pyridyl) phosphorothioates (60) which have been used for the synthesis of pyrophosphates (see Chapter 6, Section 1). 

An alternative approach for the synthesis of 2, 3 -0,0-cyclic //-phosphonate 20a was based on the condensation of a mixture of uridine 3 - and 2 -//-phosphonates (23 and 24 respectively) induced by pivaloyl chloride (Scheme 9) [24], Its reaction with elemental sulfur in carbon disulfide gave 5 -0-DMT-uridine 2, 3 -cyclic phosphorothioate (21a) which after final deprotection afforded the desired 2, 3 -cyclic phosphorothioate 22a (Scheme 9). Its Sp and Rp diastereomers were separated by HPLC [24],... 

In this context, it is interesting to note that the first synthesis of 2, 3 -0,0-cyclic phosphorothioate 22a was reported by Eckstein in 1968 [25], He also isolated pure Rp diastereomer by fractional crystallization of the triethylammonium salts [26] and used it as reference to determine the absolute configurations of the other phosphorothioate analogues [27], 2, 3 -0,0-Cyclic H-phosphonate 20a was used as a key substrate for the synthesis of uridine 2, 3 -0,0-cyclic boranophosphate 27. Silylation of H-phosphate 20a gave the phosphite triester 25 (two diastereomers). Its boronation, with simultaneous removal of the trimethylsilyl group, was achieved by its reaction with borane-A.A-diisopropylethylamine complex (DIPEA-BH3). 

Scheme 9 Synthesis of 2 ,3 -0,0-cyclic phosphorothioate 22a via 2 ,3 -0,0-cyclic Ff-phosphonate 20a...

Scheme 69 Synthesis of deoxycytidine cyclic 3 5 -0,0-phosphorothioates 298a and 298b...

Rigterink, R.H. and E.E. Kenaga. 1966. Synthesis and insecticidal activity of some 0,0-dialkyl O-3,5,6-trichloro-2-pyridyl phosphates and phosphorothioates. Jour. Agric. Food Chem. 14 304-306. 

This improvement allowed achieving the synthesis of 2-deoxy disaccharide 103 with high stereoselectivity and good yield in short reaction times. The composition of the reaction mixture was found to be practically independent of the configuration of the S-(2-deoxy-D-glucopyranosyl)phosphorothioate. These results seem to provide an evidence that this glycosylation procedure proceeds via the SN1 displacement reaction mechanism. 

Phosphorite deposits, 17 688, 691 Phosphorite uranium deposits, 17 520 Phosphorochloridate synthesis, 19 28 Phosphorodithioate DNA, 17 630 Phosphorodithioates, 17 630 Phosphorothioates, 17 629-630 synthesis of, 17 630 Phosphorous acid, 19 52 Phosphorous donor ligands, thorium and, 24 768 Phosphors... 

Example 4 Wada et al. have been successful in developing a new class of activators dialkyl (cyanomethyl) ammonium tetrafluoroborates [21]. These activators have high proton-donating ability and a counteranion of low nu-cleophilicity. With this class of salts a remarkably efficient diastereocon-trolled synthesis of dinucleoside phosphorothioates has been devised. The power of this method lies in the fact that stereopure monomers can be ob-... 

Nucleoside 5 -phosphorothioates have also been employed as activated nucleotide derivatives for synthesis of pyrophosphates.321 The interaction of tributylammonium 2, 3 -di-0-benzoyluridine 5 -phosphorothioate (73) with silver a-D-glucopyranosyl and a-D-galac-topyranosyl phosphates in pyridine solution, with subsequent de-benzoylation, gave the corresponding glycosyl esters in 60-70% yield. This procedure can probably be classified as a variant of the mixed-anhydride method, the driving force of the reaction being the formation of insoluble silver sulfide. 

A supported sulfide source has been demonstrated by Zhang et al.16 to synthesize oligonucleotide phosphorothioates (entry 12). Aminodithiazole-thione attached to a methacrylate-ethyleneglycol copolymer is used to efficiently convert a nucleotide phosphite to a phosphorothioate. The product nucleotide possesses protecting groups suitable for solid-phase oligonucleotide synthesis and thus is a valuable building block for nucleic acid therapeutics. 

To study the stability of 5-methyl-1,2,4-dithiazole-3-one 27 in solution at room temperature, the sulfurization efficiency of the freshly prepared compound was compared with a 3-week solution in the synthesis of 25-mer oligodeoxyribonucleotide phosphorothioate <1999TL2095>. The result obtained showed that in both cases the same sulfur transfer efficiency (>99.5%) was achieved. 

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. 

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