Phosphorothioates as substrates

To date phosphorothioates have with very few exceptions, been found to be acceptable substrates for stereochemical experiments. They generally react more slowly than naturally occurring substrates, from 1 to 10% of the rates for phosphates in many cases of phosphotransferases and 10 to 100% of the normal rates in the cases of nucleotidyltransferases acting on a-thionucleotides. In a very few cases, notably alkaline phosphatase and phosphoglycerate mutase, phosphorothioates are unacceptable as substrates, necessitating the development of methods for synthesiz- 

Despite the broad utility of chiral phosphorothioates, certain enzymes, such as alkaline phosphatase and phosphoglycerate mutase, do not accept phosphorothioates as substrates. Stereochemical studies of these enzymes awaited the development of methods to synthesize and assign configurations to chiral phosphates. Chiral [l60, l70,180]phosphomonoesters and [l8OJphosphodiesters have been elegantly 

Chiral [160, l70, l80]phosphomonoesters and ATPy[l60, l70, lsO] have been synthesized by Knowles and associates, who devised the procedure outlined in Fig. 19 [51-55], The procedure has been used to synthesize phenyl[160, l70, l80]phos-phate and 2-[160,170,180]phospho-D-glycerate as well as the propylene glycol ester shown. The starting cyclic adduct was prepared by reaction of (— )-ephedrine with P17OCl3, giving a separable mixture of 2-chloro-l,3,2-oxazaphospholidin-2-ones whose chemistry had been described [56], The major isomer was converted to (/ p)-l-[160, nO,180]phospho-1,2-propanediol and (Sp)-ATPy[l60, nO, lsO] by the reactions shown. The stereochemistry at each step of the synthesis was well prece-dented in the literature nevertheless, the configurations were verified by independent methods described in the next section. 

Knowles and associates have devised two methods for analyzing the configurations of the chirally enriched phosphate group in (S)- 1,2-propanediol-1 -[l6Ot l70, lsO]phosphate [58,59], They have described their methods in detail elsewhere [5], so the present discussion has been abridged, with the intention of conveying the principles upon which the analyses are based, while avoiding an extended discussion of factors which complicate them. 

The syn- isomers from the (Rp) and (Sp) epimers of S- 1,2-propanediol-1-[160, l70, lsO]phosphate are compared in Fig. 22, and can be seen to differ. For example, the m + 2 species differ in that the one derived from the (Rp) epimer has an [l60]methoxyl group while that from (Sp) epimer has an [l80]methoxyl group. This is not alone sufficient to distinguish the (Rp) and (Sp) epimers, however, since the m + 3 species from the (Sp) epimer also has an [180]methoxyl group. It is necessary in identifying the Sp epimer to show that it is the m + 2 species that contains an [180]methoxyl group. Similarly, the m + 1 species contains an [160]methoxyl and the m + 3 contains an [l70]methoxyl. 

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