Phosphate stereochemical course

Because of the earlier-mentioned error in configurational assignment of 120 (Fig. 74), the initial conclusion was that both steps involve retention and that the phosphate and phosphothioate reactions differ in stereochemical course 170). This conclusion was subsequently revised 168) as explained above (see also footnote 25 in Ref. 169). 

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

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. 

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. 

The first determination of the stereochemical course of enzymatic substitution in a phosphate was the study of pancreatic ribonuclease completed by Usher, Richardson and Eckstein and their associates in 1970 [31]. The enzyme had been shown to catalyze the endonucleolytic cleavage of RNA by the two-step catalytic pathway of Equation 11 ... 

A claim that the stereochemical course of cyclic AMP phosphodiesterase action on 3, 5 -cyclic AMPS and 3, 5 -cyclic AMP, l80 differed, inversion for the chiral cyclic phosphorothioate and retention for the chiral cyclic phosphate, resulted from... 

Finally, the decarboxylation of amino acids catalyzed by several pyridoxal phosphate-dependent enzymes has been shown to proceed by a retention of configuration at the Ca atom144. The stereochemical course of the decarboxylation of 5-hydroxy tryptophan to 5-hydroxytryptamine (serotonin) catalyzed by the pyridoxal phosphate-dependent aromatic L-amino acid decarboxylase (equation 15) exemplifies such studies145. 

Scheme 3. 3-Deoxygenation of NDP-L-ascarylose (7) with a pyridoxamine phosphate (PMP)-dependent enzyme, and its stereochemical course...

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-... 

Stereochemical data exist for a large number of displacement reactions on neutral phosphoric acid and thiophosphoric acid esters and amides (64). Many of these occur with retention of configuration, especially when the displacement reactions occur on five-membered ring compounds, and this stereochemical course is explained by pseudorotation of a pentacoordinate intermediate. In contrast, only one stereochemical study has been performed on an anionic phosphate ester with the intention of detecting the participation of a phosphorane intermediate. 

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. 

Prior to the X-ray analysis, the stereochemical course of the hydrolysis of the Sp diastereomer of thymidine 3 -(4-nitrophenyl [ 0, 0]phosphate) 5 -(4-nitrophenyl phosphate) in H2 0 was determined (26). This synthetic oligonucleotide analog is not a good substrate, and the Sp diastereomer of thymidine 3 -[ 0, 0, 0]phosphate 5 -(4-nitrophenyl phosphate) was obtained as product (recall that on double-stranded DNA the oligonucleotide products contain S -phosphate groups). The simplest explanation for this inversion of configuration is that the enzyme catalyzes the direct attack of water on the phosphodiester backbone of double-stranded DNA, presumably by general basic catalysis. 

We have directly identified the sugar product of the strand cleavage reaction catalyzed by endonuclease III and UV endonuclease V as the trans-a,/3-unsaturated aldehyde obtained by elimination of the 3 -phosphate 108, 109)-, with the determination that these endonucleases stereospecifically abstract the 2-pro-S hydrogen, the stereochemical course of the /3-elimination reaction is syn... 

The catalytic reactions of PPPs proceed via a single direct phosphoryl transfer without participation of a phosphoenzyme intermediate. Supporting evidence includes the finding that calcineurin does not exhibit phosphotransferase activity toward nucleophiles,that activity is inhibited by the products of the reaction with /)NPP, and that the enzyme cannot exchange 0-labeled water with phosphate. Further, the stereochemical course of the reaction catalyzed by the structurally similar PAPs proceeds with inversion of configuration at phosphorus. ... 

The stereochemical courses of many enzymic reactions involving phosphate derivatives have been investigated, and they have been reviewed by Westheimer (145) and Knowles (99). The individual steps in all enzymic reactions so far investigated have been found to proceed with inversion about the phosphorus center. 

Although it is convincing that metaphosphate ion is not a free intermediate in substitution reactions in aqueous solution, this does not exclude the possibility of reactions of related species proceeding via analogous intermediates. Reactions of thiophosphate monoesters are more rapid than those of phosphate monoesters. If three sulphur atoms replace three of the oxygens of metaphosphate, a stable salt can be isolated (Roesky et al, 1986). Harnett and Lowe (1988) examined the stereochemical course of the non-enzymic hydrolysis of adenosine (P- O, thio) diphosphate in In... 

In order to determine the stereochemical course of the nonenzymic hydrolysis of cyclic nucleotides, and in particular to examine the role of pseudorotation, Gerlt and coworkers carried out a chiral phosphate analysis of the base-catalysed hydrolysis of cyclic deoxyadenosine monophosphate (Mehdi et al., 1983). In 0.2 m Ba(OH)2 at lOO C, cdAMP hydrolyses to a mixture of 3 dAMP and 5 dAMP. The position of label incorporation on hydrolysis in labelled water indicates P—O as opposed to C—O bond cleavage. The overall stereochemistry of hydrolysis was followed by recycliz-ing and analysing both the ring-opened products of ( O, 0) cdAMP hydrolysis (Scheme 31). 

Fig. 7.3. Stereochemical course of the 5-enolpyruvylshikimate 3-phosphate synthase reaction (adapted with permission from Asano et al., 1985, copyright 1985, American Chemical Society).

Asano, Y., J. J. Lee, T. L. SHffiH, F. Spreahco, C. Kowal, and H. G. Floss, Stereochemical course of the enol pyruvylshimi-mate-3-phosphate synthase, chorismate mutase, and anthrinilate synthase, J. Am. Chem. Soc., 107, 4314-4320 (1985). 

The stereochemical course for the racemisation could be explained as follows. First, the oxidative addition to the propargylic phosphate gave an allenylpalla-dium(ii) through the back side attack of palladium(0). Thus, this enantio-enriched allenylpalladium(ii) was stereospecifically formed starting from the... 

Fig. 1. Reaction mechanism and stereochemical course of the conversion of SAM to ACC catalyzed by the pyridox-al phosphate (PLP)-dependent ACC synthase. C-3 ( ) of the L-methionine portion of SAM becomes the pro-(R) methylene group of ACC. whereas the two hydrogens attached to C-3.4 of the methionine moiety in the cis-configuration yield cis-ACC...

Seela, F. and Kretschmer, U. (1991) Diastereomerically pure Rp and 5p dinucleoside H-phosphonates. The stereochemical course of their conversion into P-methylphosphonates, phosphorothioates, and [ 0] chiral phosphates J. Org. Chem. 56,3861-3869... 

Potter, B. V. L., Connolly, B. A, and Eckstein, F. (1983) Synthesis and configurational analysis of a dinucleoside phosphate isotopically chiral at phosphorus Stereochemical course of penicillium citrum nuclease PI reaction Biochemistry 1369-1377. 

Use of Chiral [ < 0, 0,i 0]Phosphate Esters to Determine the Stereochemical Course of Enzymatic Phosphoryl Transfer Reactions... 

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