Chiral thiophosphates

Available evidence (14,15) favors the pathway for pyruvate kinase by way of phosphorylation of pyruvate enol. Furthermore, J. Knowles and his coworkers (16,17), using chiral thiophosphates and chiral (160,170,180) phosphate have shown that pyruvate kinase transfers phosphate from phosphoenolpyruvate to ADP with stereochemical inversion at phosphorus. Since monomeric metaphosphate is presumably planar, a chemical reaction by way of that ion should proceed with racemization. In the active site of an enzyme, however, all components might be held so rigidly that racemization need not occur. Furthermore, no information is yet available on the detailed mechanism of reactions catalyzed by cytidine synthetase our own experiments, designed to distinguish among the mechanisms here discussed, are as yet incomplete. 

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). 

These comparative studies constituted the first example of an enzyme-catalyzed hydrolysis reaction whose stereochemical course was unaffected by sulfur substitution. At the time these experiments were performed, the stereochemical courses of the reactions catalyzed by glycerol kinase (83, 84) and by the bacterial adenylate cyclase (85, 86) had already been compared in the laboratories of Knowles and Gerlt, respectively, and these were also found to be unaffected by the sulfur substitution. A number of other comparisons of this type have been made, and in no case were the stereochemical consequences of the reactions studied with chiral phosphate esters and the chiral thiophosphate analogs found to differ. This agreement suggests that the necessary use of oxygen chiral thiophosphate monoesters to study the stereochemical course of phospho-monoesterases will provide pertinent results for ascertaining whether phosphory-lated intermediates are involved in the reaction mechanism. 

Inorganic chiral thiophosphate analysis of fructose bisphosphatase indicates overall inversion of stereochemistry (Domanico et ai, 1986). To distinguish between direct in-line displacement and the intermediacy of an acyl phosphate, a single-turnover experiment was performed. Isotopic equilibration of the putative enzyme-CO 2 with labelled water through multiple turnovers leads to label incorporation. A subsequent single turnover experiment with one equivalent of substrate in unlabelled water must lead to label... 

The hydrolysis of phosphonate 286 (R=Me) by wild-type PTE yielded mainly thioacids (5)-287, whereas the hydrolysis by mutant-type PTE led to the formation of (R)-thioacids 287. In contrast, the hydrolysis of thioacid triesters 286 by either wild- or mutant-type PTE in many cases yielded thioacids (S)-287 with 99% ee. The chiral thiophosphates synthesized by these enzymatic methods, were proposed as precursors for the synthesis of organic and organophosphorus compounds (Scheme 94) [190]. 

Inch and his co-workers continue to use carbohydrate derivatives in the synthesis of chiral esters of phosphorus acids and for studies of the displacement reactions of these esters. For example, the tetrahydro-l,3,2-oxazaphosphorine-2-ones (74) and (76) were synthesized and cleaved to give the chiral phosphonate (75) and the chiral phosphate (77), as illustrated in Schemes 35 and 36. 2-Thiones related to (74) and (76) were also prepared and used to obtain such chiral thiophosphates as (78) from (79). Attack on the acyclic S -methyl phos-... 

Use of Chiral Thiophosphates and the Stereochemistry of Enzymatic Phosphoryl Transfer... 

Stereochemical Problems Studied with Chiral Thiophosphates... 

The use of thiophosphate analogs of biophosphates in studying stereochemical problems was first introduced by Eckstein (1975) and subsequently widely applied to various problems. To illustrate the use of chiral thiophosphates in stereochemistry, consider the phosphoryl transfer reaction catalyzed by hexokinase (Scheme 1). Three types of problems can be studied by... 

Phosphorus-31 NMR is not the only method available for some problems, and this chapter does not intend to provide a comprehensive review of all chiral thiophosphate work. 

Because most biophosphate molecules contain at least one chiral carbon center, the two isomers la and lb are diastereomers and give distinguishable 3>P chemical shifts. Table II summarizes the chiral thiophosphates that belong to this category and the chemical shifts of the chind phosphorus of these isomers. 

The chiral thiophosphates la and lb can be used for two types of studies stereochemical course of enzymatic substitutions (type 1 in Section I) and stereospecificity of the two isomers as enzyme substrates (type 3 in Section I). A great number of enzyme reactions have been investigated by this approach and have been reviewed as described in Section I. An example in this category is the stereospecific hydrolysis of DPPsC by phospholipases A2 and C (Bruzik et ai, 1982). As shown in Scheme 6, when DPPsC(A + B)... 

P-NMR Analysis of the (R,)-ATPpS Derived from Chiral Thiophosphates... 

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