Chiral thiophosphates configurational analysis

The enzymes in this category inlcude phosphatases, nucleotidases, ATPases, and so on. Except for the phosphatases, which also catalyse the transfer reaction in addition to the hydrolysis reaction (112, 166), the stereochemistry has to be studied by use of a chiral inorganic phosphate (Pj, a proproprochiral center). To make a Pj chiral, it is necessary to use 160,170,180 and sulfur. The synthesis and configurational analysis of chiral [160,170,180]thiophosphate (Psj) have been described in previous sections the first application of chiral [160,170,180]Psj in the reaction type c is the elucidation of the stereochemistry... 

A sensitive probe applied to understand the nature of the reaction mechanism of group transfer is the stereochemistry of the overall reaction. The reaction at a phosphoryl center normally is a degenerate question, since a monosubstituted phosphate ester or anhydride is proprochiral at the phosphate center. Phosphate centers at a diester or disubstituted anhydride are prochiral. Two related methods to analyze the stereochemistry at a phosphate center have been developed by the generation of chirality at the phosphorus center. The first approach was developed by Usher et al. (24) and gave rise to the formation of isotopi-cally chiral [ 0, 0]thiophosphate esters and anhydrides (I). Isotopically chiral [ 0, 0, 0]phosphates (II) have also been synthesized and the absolute configurations determined. Two primary problems must first be addressed with respect to both of the methods that have been developed the synthesis of the isotopically pure chiral thiophosphates and phosphates and the analysis of the isotopic chirality of the products. An example of the chiral starting substrates, as developed for ATP, is schematically demonstrated. Ad = adenosine. 

F. Configurational Analysis of Oxygen Chiral Thiophosphate Monoesters. 112... 

As noted previously, studies of the mechanisms of phosphate monoester solvolysis have been extended to the mechanisms of the analogous phosphorothioate ester solvolysis because the thiometaphosphate anion is believed to be more stable than the metaphosphate anion. Thus, a general method based upon P NMR spectroscopy for the configurational analysis of chiral thiophosphate monoesters (see Fig. 10) was described recently by Cullis and co-workers (38). 

The mechanism of an actual hydrolysis reaction catalyzed by this prototype phosphomonoesterase has never been studied stereochemically. This apparent omission is presumably explained by the very low catalytic efficiency of the enzyme toward phosphorothioate monoesters as compared to phosphate monoesters (75) certainly, chiral [ O, 0]phosphorothioate 0-ester substrates already exist, and methodology is available for the configurational analysis of the chiral [ 0, 0, 0]thiophosphate that would be produced if the chiral substrate were hydrolyzed in H2. In fact, the low catalytic reactivity of phosphorothioate O-esters and the high reactivity of phosphorothioate S-esters has been explained by the enzyme utilizing nucleophilic catalysis (an associate mechanism) to achieve hydrolysis of the phosphate ester bond 40). 

The definitive work on the mechanism of PEPC is the chiral thiophos-phate analysis of Hansen and Knowles (1985), employing [ 0, 0]thio-phosphoenol pyruvate as substrate. The inorganic thiophosphate product generated by the enzyme reaction was analysed and indicated overall inversion of configuration at phosphorus, a result consistent with the stepwise mechanism via carboxyphosphate. 

There are, however, no alternatives in the configurational analysis of the chiral [ 0, 0, 0]thiophosphate (P, ) discussed in the next section. The only method available is a P-NMR method based on the combined effects of O and 0. 

To illustrate the application and configurational analysis of chiral [> 0, 0, 0]thiophosphate, we describe the stereochemical study of 5 -nu-... 

---