Oxygen chiral thiophosphate

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

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. 

Methods for Syntheses and Configurational Analyses of Oxygen Chiral Phosphate and Thiophosphate Esters... 

Given the inability to implicate free metaphosphate anion in these studies, Cullis and Rous examined the stereochemical consequence of an alcoholysis reaction (55). When [ 0, 0, 0]P -0-ethyl-P -thiopyrophosphate is treated with methyl iodide in ethanol, the S-methyl derivative rapidly decomposes to form 0-ethyl-S-methyl thiophosphate and ethyl [ 0, 0, 0]phosphate this reaction is thought to proceed via a mechanism involving metaphosphate anion, but, as would be expected on the basis of the observations by Knowles, the stereochemical consequence is nearly quantitative inversion of configuration. In dichloromethane solution, the decomposition of the oxygen chiral substrate yields ethyl [ 0, 0, 0]phosphate that has suffered extensive racemization (approximately 70%). This stereochemical result can be rationalized by the intermediacy of free metaphosphate anion in the absence of nucleophile acceptors. 

An additional method to determine the nature of the metal-nucleotide complex that is preferred by the enzyme is in the study of the effects of cation substitution on the stereoselectivity of chiral-specific thiophosphate nucleotides. Thiophosphate derivatives of cAMP, AMP, ADP, and ATP at the a-, /3-, and/or y-positions were initially introduced by Eckstein and co-workers (66, 67) and expanded to several other nucleotides. Substitution of a phosphate oxygen by sulfur at the a-position of cAMP, ADP, or ATP or at the /3-position of ATP gives rise to a new chiral center, Substitution in the a-position of AMP, j8-position of ADP, and the y-position of ATP changes an achiral center to a prochiral center. This substitution gives rise to a small decrease in p a for the phosphate, and with many enzymes these thiophosphate analogs have a decreased substrate ac-... 

B. Synthesis of Thiophosphate Monoesters Chiral by Virtue of Oxygen Isotopes. 102... 

C. Synthesis of Phosphate Diesters Chiral by Virtue OF Oxygen Isotopes and of Thiophosphate Diesters... 

Two new techniques have been introduced that are especially useful when no stable intermediate can be isolated. Thiophosphates or phosphates with isotopically labeled oxygen atoms are chiral and can thus be us to probe the stereochemistry of the phosphoryl transfer event (Eckstein, 1979 Knowles, 1980). One in-line displacement leads to an inversion of configuration on the phosphorus atom. If one covalent intermediate is involved, retention of configuration should result from the double-displacement reaction. Using this technique, the existence of the acetate kinase acyl phosphate intermediate has been challenged (Knowles, 1980), whereas others (Spector, 1980) have interpreted the same results as being indicative of a triple-displacement mechanism. 

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