Phosphate esters, configurational analysis

In order to solve this mechanistic problem, a method for analysing the stereochemical course of phosphokinases has been developed using chiralf160,170,180]phosphate esters. Stereochemical analysis should allow a clear mechanistic distinction to be made, since inversion of configuration at phosphorus implies a direct in-line phosphoryl transfer mechanism whereas retention of configuration suggests a doubledisplacement mechanism with a phosphoryl-enzyme intermediate on the reaction pathway. 

Stereochemical analysis of the products using [ieO, 170, lsO] phosphate ester methods show that the PGM reaction proceeds with overall retention of configuration at phosphorus,200 which indicates that an even number of phosphoryl transfers are involved. The catalytic reactions of both a- and /i-PGM proceed via a phosphoenzyme intermediate, formed by the reaction of an active-site nucleophile with Gl,6-diP. The a-PGM utilizes an active-site Ser nucleophile, while /i-PGM uses an active-site Asp. The phosphorylated PGM binds either G1P or G6P and transfers the phosphoryl group to the C(6)OH or C(l)OH, respectively (Scheme 3). 

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. 

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 first account of the synthesis of an oxygen chiral phosphate monoester was reported in 1978 by Knowles and co-workers (Abbott et ai, 1978, 1979). This ester, a diastereomer of l-phospho-(5)-1,2-propanediol, was selected as the synthetic target because the general method of configurational analysis that Knowles devised for monoesters was based on the ability to assign the configuration of the diastereomers at phosphorus of this particular phosphate monoester. The chemical steps in this synthesis are shown in Fig. 1. Briefly, [ OJPOCls, prepared in hi yield from and... 

The success of the synthetic methods described in the previous section was tied closely to the availability of techniques to determine the absolute configurations of the synthetic materials. As previously indicated, the configurational differences of phosphate esters that are enantiomeric or diaster-eomeric at phosphorus are nearly always cyrptic, so that chemical modification reactions must be performed before any physical technique can be used to perform the configurational analysis. 

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