Stereospecific phosphorylation

This process is stereospecific and has the advantage of giving a quantitative conversion. Although AMP, the usual acceptor substrate for adenylate kinase, is achiral at phosphorus and so not subject to stereospecific phosphorylation, the phosphorus in AMPS is a prochiral center. Adenylate kinase catalyzes the phosphorylation of the pro-R oxygen exclusively to produce the (Sp) configuration. 

The chiral y-[l80]phosphorothioate of ATP, (Pp)-ATPyS, yl80(/3, y)lsO, was synthesized by the procedure outlined in Fig. 8 [25]. (Sp)-ADPaS, alsO(a,/3)l80 was prepared by stereospecific phosphorylation of AMPaS, al802 using the adenylate and pyruvate kinase system followed by dephosphorylation with glucose and hexokinase. This was the starting material for the synthesis, with the chiral a-... 

Fig. 15. Configurational analysis of chiral [ lsO]phosphorothioates by stereospecific phosphorylation.

The stereochemical course of 3, 5 -cyclic AMP phosphodiesterase was shown to involve inversion of configuration. This was first concluded by Eckstein and Stec and their associates, who synthesized (Sp)-3, 5 -cyclic AMPS and carried out its cyclic AMP phosphodiesterase-catalyzed hydrolysis in H O. The resulting AMPS, lsO was subjected to stereospecific phosphorylation to (Sp)-ATPaS, al80 by the... 

The configuration of AMPS, lsO was shown to be (Rp), corresponding to inversion of configuration, by stereospecific phosphorylation, using the adenylate kinase-pyruvate kinase system, and analysis for bridging and nonbridging l80. Similar approaches have been applied to amino acyl-tRNA synthetases, which also catalyze inversion of P of ATP [79]. 

On the basis of these results, possible mechanisms would be those suggested for alpha-amylase (see p. 328), that is, a front-side displacement, a double displacement, or the stereospecific phosphorylation of a carbonium ion (during polymer degradation). 

Chiral [7-180]ATPyS was first synthesized by Richard and Frey (124) as shown in Scheme 35. The (S)-[a-18O,a0-18O]ADPaS (46), prepared from enzyme-catalyzed stereospecific phosphorylation of 31, was condensed with 2, 3 -methoxymethylidene-AMP (41) to give the dinucleotide 47. Chemical re-... 

It was found that the enzyme is specific for (/ )-ATPaS but does not react with (S)-ATPaS. As shown in Scheme 43, when (/ )-ATPatS and l70-acetate are used as substrates, the 170 from acetate will be incorporated into the pro-5 position of AMPS if the reaction proceeds with retention of configuration or into the pro-/ position if inversion occurs. To determine the configuration of the 170-labeled AMPS (compound type 4), it is converted to (S)-ATPoS by stereospecific phosphorylation at the pro-/ oxygen catalyzed by adenylate kinase, followed by a second phosphorylation catalyzed by pyruvate kinase (144,145). By such a conversion, 170 should be incorporated into the nonbridging position of (5)-ATPaS if the step of acetate activation proceeds with retention of configuration. On the other hand, 170 should be located at the P—O—P bridging... 

Despite the aromatic character, the arylphospholes could also participate in Diels-Alder reactions to give new type of 7-phosphanorbomenes. These products together with phosphanorbomenes obtained by the regio- and stereospecific dimerization of arylphosphole oxides were useful model compounds in the UV light mediated fragmentation-related phosphorylation of alcohols. A novel mechanism was substantiated. 

Recent years have also seen the development of a variety of com-plexation reagents incorporating C-P bonds and their associated functionalities, such as phosphoryl linkages. For example, substituted calixarenes (Figure 1.4) have been developed for extraction of radionuclides, and phosphorus-derivatized cyclodextrins for stereospecific inclusion interactions. 

CNDO/2 Theoretical calculations have been used to predict the favoured conformations of creatine and creatine derivatives, including phosphocreatine. These calculations predict that phosphocreatine may adopt conformation (35) to avoid unnecessary steric and electrostatic repulsions.110 The possibility also exists that creatine kinase phosphorylates creatine stereospecifically to form the favoured conformation (35) at the active site of the enzyme. When chicks are fed a diet containing cyclocreatine (l-carboxymethyl-2-iminoimidazole), the phosphorylated... 

R0)2P-S—S—P(0R)2. A number of these products and others from reactions of dithiophosphoric acids with oxidants are listed in Table 2 since they are some of the impurities to be anticipated. Thiophosphoryl (P=S) compounds are rapidly, quantitatively, and stereospecifically converted to phosphoryl (P=0) compounds by organic peroxy acids under mild conditions. The reactions of peroxy acids and dithiophosphoric acids and salts have apparently not been characterized. 

Additional information (for the enzymes described in references 3-11 information concerning the stereoisomer of the phosphorylated form is lacking. These are included in EC 2.7.3.11 but may as well be classified as EC 2.7.3.12. Detailed organism-specific information is summerized in EC 2.7.3.11-12. These enzymes cannot be classified precisely since information on the stereospecificity towards histidine is lacking)... 

In the present case, alkaline solvolysis forms almost completely racemic product in contrast with that from the neutral hydrolysis which is stereospecific. It is clear that the alkaline solvolysis involves a symmetrical intermediate, a metaphosphorimidothioate. which reasonably may be expected to be planar (isoelectronic with S03). The close similarity in reactions of thiophosphoryl and phosphoryl centers is well established for bimolecular displacements on phosphorus and the resemblance apparently extends to the metaphosphate eliminations. 

Much of the earlier work on hexokinase has been summarized in two reviews that appeared in 1973 (25,26). The emphasis here will be on information that has appeared since that time. Significant advances have been made with respect to kinetic mechanism, stereospecificity of phosphoryl transfer, and x-ray structure. 

Single alkene diastereomers are accessible through a Wittig-Homer reaction only if it is performed in two steps (Figure 11.10). A 1 1 mixture of the phosphorylated lithium alkoxides syn- and anti-D is still formed but if the mixture is protonated at this point, the resulting phosphorylated alcohol diastereomers C can usually be separated without difficulty. The suitable diastereomer will be deprotonated with potassium-ferf-butoxide in the second step and then be converted into the stereouniform trans- or cis-alkene E via stereospecific oxaphosphetane formation and fragmentation. 

During the last two decades, the mechanisms of many enzymic processes have been established, and model systems have been developed that effectively mimic their action. In particular, the roles of thiamin, NAD, pyridoxal, flavins, Bl2, ferridoxin, and metals in many enzymic processes now are understood. Model systems have been developed to imitate the action of decarboxylases and esterases, to imitate the action of enzymes in binding their substrates, and to approach the stereospecificity of enzymes. Our laboratory recently has found phosphorylating agents that release monomeric methyl metaphosphate, which in turn carries out phosphorylation reactions, including some at carbonyl oxygen atoms, that suggest the actions of ATP. The ideas of biomimetic chemistry are illustrated briefly in terms of the processes mentioned above. 

The first chiral phosphates to be used for stereochemical analyses were chiral phosphorothioates, which were used to determine the stereochemical courses of ribonuclease, UDP-glucose pyrophosphorylase, adenylate kinase and several other kinases and synthetases. The chiral phosphorothioates either had sulfur in place of an oxygen at an otherwise prochiral center of a phosphodiester or phosphoanhydride or stereospecifically placed sulfur and 180 (or nO) in a terminal phosphoryl group. The syntheses and configurational analyses of the most important of these compounds are outlined in the following. 

Baseline separations of ADPaS can also be achieved by reversed-phase high-performance liquid chromatography [22], which is the method of choice because enzymatic phosphorylation at the -phosphorus is stereoselective rather than stereospecific with respect to Pa configurations, so that the separations are not complete. 

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