Phosphate monoester, chiral

A. Synthesis of Phosphate Monoesters Chiral by Virtue of Oxygen Isotopes. 99... 

Although phosphate monoesters chiral by virtue of oxygen isotope substitutions cannot be used in stereochemical studies of phosphate monoester hydrolysis (since there are only three stable isotopes of oxygen), they have been used profitably in studies of phosphoryl transfer reactions relevant to the question of the intermediacy of monomeric metaphosphate anion in phosphoryl transfer reactions (see Section III,A). The laboratories of Knowles and Lowe have reported general methods for the synthesis of phosphate monoesters chiral by virtue of oxygen isotope substitution, and these syntheses are summarized in this section. 

Chiral [160,170,180] Phosphate Monoesters for Determining the Stereochemical Course of Phosphokinases... 

The general route developed for the synthesis of chiral [160,170, 80]phosphate monoesters of known absolute configuration is shown in Scheme 1 (1). ... 

The stereochemical analysis depends on converting a chiral[160,170,180]phosphate monoester into two diastereoisomeric conformationally locked six membered cyclic phosphate triesters (2 ). In the cyclization step any one of the peripheral oxygen isotopes will be lost with equal probability, and the residual oxygen isotopes will take up axial and equatorial positions. Methylation of the isotopically labelled cyclic phosphate... 

In the following sections we discuss the methods that have been developed to study the steric course of reactions at proprochiral and proproprochiral centers on carbon and phosphorus, and some of the biochemical applications of these methods. Specifically, we consider chiral methyl groups, chiral malonic acid, chiral phosphate monoesters, and chiral inorganic phosphate. 

Simultaneously and independently, Cullis and Lowe developed a second general methodology for the synthesis of, 0, 0-labeled chiral phosphate monoesters (76, 77). This synthesis relies upon the synthesis of a cyclic hydrobenzoin triester of the alcohol or phosphoric acid followed by hydrogenolysis to liberate the isotopically labeled monoester product (Fig. 2). Hydrobenzoin, chiral by virtue of stereospecific labeling with 0 and 0, is the source of the two specified oxygen isotopes, and O is derived from H2 0 via P OCL. The reader is referred to the articles by Cullis and Lowe for details of the synthesis. 

Lowe, Cullis, and co-workers have described general methods for the synthesis of oxygen chiral phosphorothioate monoesters (see Figs. 3 and 4). The synthesis by Cullis is strictly analogous to the synthesis of chiral phosphate monoesters by Knowles except that (- )-ephedrine is thiophosphorylated with PSCI3... 

Whereas the 0 effect on P NMR chemical shifts is sufficient for determining the configurations of chirally labeled samples of prochiral phosphorus atoms in a diastereomeric environment, additional use of the quadrupolar effect of O on P NMR resonances is required for configurational analyses of oxygen chiral phosphate monoesters. The basic strategy for the configurational analyses of phosphate monoesters is the same, i.e., the enantiomeric center in the monoester must be converted to a diastereomeric center in a cyclic phosphodiester so that... 

A conceptually similar approach can be used to determine the configurations of chiral phosphate monoesters of chiral alcohols without the necessity for transferring the chiral phosphoryl group to 1,3-butanediol. For example, in a number of stereochemical studies of hydrolysis reactions performed in the author s laboratory, 3 - or 5 -nucleotides were obtained as reaction products. In the case of samples of 5 -AMP, these can be enzymically converted to isotopically labeled cyclic 3, 5 -nucleotides by initial pyrophosphorylation with adenylate kinase and pyruvate kinase, followed by enzymic cyclization with inversion of configuration by a bacterial adenylate cyclase (20) (see Figs. 6 and 7). In the case of samples of 3 -nucleotides or 5 -nucleotides other than 5 -AMP, the cyclization reactions can be accomplished chemically (23). In either case, following chemical con-... 

As noted previously, the stereochemical course of the hydrolysis of a phosphate monoester can be studied only if an oxygen chiral phosphorothioate ester is used a substrate so that an enantiomer of 0-labeled chiral thiophos-... 

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). 

All of these studies are consistent with the solvolyses of phosphate monoesters involving some form of metaphosphate anion however, on the basis of these experiments, the question of whether the anion exists symmetrically solvated and, therefore, can be considered to be a free intermediate in solution remains uncertain. Knowles, Cullis, and co-workers have used stereochemical techniques to examine the intermediacy of free metaphosphate anion in hydrolyses and alcoholyses of chiral phosphate monoesters. These experiments are important since they place significant constraints on the lifetime of the reactive intermediate and, therefore, clarify the large number of observations made in other laboratories on the question of whether metaphosphate anion is sufficiently stable that it can be considered a mechanistically significant intermediate. 

The stereochemical consequences of the methanolyses of the monoanion of phenyl [ 0, 0, 0]phosphate, the dianion of 4-nitrophenyl [" 0, 0, 0] phosphate, and [ 0, 0, 0]phosphocreatine have been determined by Knowles and co-workers (35). The monoanion of phenyl phosphate behaves as a typical phosphate monoester in that its rate of hydrolysis is maximal at pH 4, where an intramolecular proton transfer is possible. The dianion of 4-nitrophenyl phosphate is highly reactive since protonation of the leaving group is not necessary. Finally, A-phosphoguanidines have been reported to be the most reactive phosphoryl compound (the chiral phosphocreatine can be enzymically synthesized from [ y- 0, 0, 0]ATP). Thus, the solvolyses of all three of these compounds are believed to involve the participation of metaphosphate anion. The meth-anolysis of each of these compounds proceeds with quantitative inversion of configuration. 

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). 

This elegant kinetic and stereochemical analysis provides two important firsts (1) the demonstration of retention via pseudorotation, employing chiral phosphate analysis (2) direct evidence for a pseudorotation mechanism in the reaction of a phosphate monoester. 

In 1979 Knowles, McLafferty and their co-workers made dramatic use of metastable ion mass spectrometry in solving the stereochemistry of phosphate monoesters R0P 0 0 02 that are chiral by virtue of substitution with oxygen isotopes. Reaction with optically active 1,2-propanediol affords the mixture of three isotopomeric cyclic triesters shown to the left in Scheme 2. One R0P 0 0 0 enantiomer gives the mixture depicted the other ROP O O O enantiomer gives products in which and are transposed. 

The rationale of configurational analysis for chiral is illustrated by Scheme 11. The same principle applies to chiral phosphate monoesters... 

Thus, determination of whether 0 is at the pro-R or pro-5 position would tell the configuration of chiral P,i or chiral phosphate monoesters. A general way to achieve this is to deiivatize stereospecifically the pro-R or pro-5 oxygen. The P( 0)-NMR method can then be used to distinguish the bridging and nonbridging on the basis of the different m nitude of... 

Given the low natural abundance of O and 0, chemical methods for the synthesis of phosphate esters that are chiral by virtue of oxygen-isotope substitution must allow for the introduction of any oxygen isotope from (ideally) the commercially available forms of the heavy isotopes H2O, CO2, or O2. In addition, the substrates of the phosphoryl and nucleotidyl transfer reactions include three types of structurally and chemically different phosphates, almost all of which are polyhydroxylic phosphate monoesters, such as sugar phosphates and mononucleotides, phosphate diesters, such as 3, 5 -cyclic nucleotides and oligonucleotides, and phosphate anhydrides... 

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