Enolpyruvylshikimic acid

Phosphoenblpyruvic acid (D 2) and erythrose-4-phosphate serve as precursors. 3-Deoxy-D-arabinoheptulosonic acid-7-phosphate is built as a key intermediate. This compound cyclizes to 5-dehydroquinic acid, which is transformed to 5-de-hydroshikimic acid and shikimic acid. After phosphorylation shikimic acid reacts with phosphoenolpyruvate. The formed 3-enolpyruvylshikimic acid-5-phosphate yields chorismic acid by an anti-elimination of a proton and the phosphate group. 

When the entire chain of seven common intermediates had been elucidated, the surprising fact emerged that this sequence oscillates back and forth between phosphorylated and phosphorus-free stages. It begins with 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP), continues with the three phosphorus-free metabolites 3-dehydroquinic acid, 3-dehydroshikimic acid, and shikimic acid, proceeds next through the two phosphorylated intermediates shikimic acid 3-phosphate and 5-enolpyruvylshikimic acid 3-phosphate, and ends with the phosphorus-free chorismic acid. 

The early studies concerning this compound centred on its dephosphorylated form 5-enolpyruvylshikimic acid (48) which was shown to accumulate, in addition to (—)-shikimic acid and shikimic acid-3-phosphate, in the media of several multiple aromatic bacterial auxotrophs blocked beyond shikimate . The compound... 

Enolpyruvylshikimic acid-3-phosphate (49) is best prepared enzymically from shikimic aci(i-3-phosphate and phosphoenolpyruvate using an ammonium sulpWe fraction from Escherichia coli K-12 mutant 58-278 and is normally isolated as its barium salt. Its proton magnetic resonance spectrum (potassium salt in deuterium oxide) has been reported by Sprinson and his collaborators and analysis of the various coupling constants indicated that... 

The effects of glyphosate on phenolic compound production are two-fold 1) accumulation of phenolic compounds that are derivatives of aromatic amino acids is reduced and 2) pools of phenolic compounds derived from constituents of the shikimate pathway prior to 5-enolpyruvylshikimate-3-phosphate become larger. Assays that do not distinguish between effects on these two groups, such as that for hydroxyphenolics of Singleton and Rossi (18), can lead to equivocal and difficult to interpret results (e.g. 3-5). 

Jaworski (4) reported that growth inhibition of both plant and microbes by glyphosate could be reversed by aromatic amino acids. Further work of Amrhein and his coworkers revealed that glyphosate inhibits the shikimate pathway enzyme, 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase (5). This enzyme catalyzes the reaction shown in Figure 1. Glyphosate-treated plant and bacterial cultures accumulate shikimate and/or shikimate 3-phosphate (S3P), confirming that inhibition of EPSPS is at least a part of the in vivo mechanism of action of this herbicide (6, 7). 

Trimethylbromosilane has been used to deesterify phosphate esters. Two such examples are shown here Chouinard and Bartlett s conversion of shikimic acid to 5-enolpyruvylshikimate 3-phosphate9 (equation 6) and the Degenhardt and Burdsall preparation of ethylenylidenebis(phosphonic acid)10 (equation 7). 

For example, glyphosate inhibits the enzyme, EPSP (5-enolpyruvylshikimate 3-phosphate) synthase, that catalyzes a step in the synthesis of the aromatic amino acids. Similarly, both the imidazolinones and sulfonylureas inhibit acetolactate synthase (ALS), the enzyme that catalyzes the first step in the formation of branched-chain amino acids (11). Triazine herbicides act by binding to a specific protein in the thylakoid membranes of the chloroplasts, preventing the flow of electrons and inhibiting photosynthesis (12). 

Plants synthesize all 20 common amino acids de novo. Glyphosate, a weed killer sold under the trade name Roundup, is an analog of phosphoenolpyruvate that specifically inhibits 3-enolpyruvylshikimate 5-phosphate synthase, a key enzyme of the pathway for chorismate biosynthesis. This compound is a very effective plant herbicide, but has virtually no effect on mammals. Why ... 

A 1,4-conjugate elimination of phosphoric acid then transforms 5-enolpyruvylshikimate-3-phosphate (8) into chorismate (9) (Fig. 7.1). The 6-pro-R hydrogen atom is labile in the chorismate synthetase reaction hence, the reaction occurs with overall trans-g ometry. 

Deoxy-D-arabinoheptulosonic acid-7-phosphate (DAHP) synthase 2 5-dehydroquinate synthase 3 quinate dehydrogenase 4 5-dehydroquinate dehydratase 5 shikimate dehydrogenase 6 shikimate kinase 7 3-enolpyruvylshikimate-5-phosphate synthase 8 chorismate synthase... 

Research in David Sprinson s laboratory at Columbia University, in part contemporary with the research outlined in the preceding pages, has unraveled the earliest stages of the sequence and the derivation of the first, non-cyclic intermediate, DAMP. In the same laboratory, Judith Levin discovered 5-enolpyruvylshikimate 3-phosphate and predicted the existence and correct structure of the next, and last one, of the common intermediates, the compound from which the pathways to the individual primary aromatic products branch off. Isolation and structure proof of this branchpoint intermediate, chorismic acid, by the Australian workers F. Gibson, L.M. Jackman, and J.M. Edwards completed the elucidation of the general pathway. 

In this chapter, the discussion will concentrate on two inhibitors with a reasonable claim to selective action on enz3ones related to the shikimate pathway glyphosate, which inhibits 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase and L-a-aminooxy-3 phenylpropionic acid (L-AOPP), an inhibitor of phenylalanine ammonia-lyase (PAL) (Fig. 2). In addition to introducing a novel inhibitor of PAL, (R)-(l-amino-2-phenylethyl)phosphonic acid (APEP), previous and current efforts to design inhibitors of other shikimate pathway enzymes will be described. The treatment presented here will show that the deductions and predictions made on the basis of the abstract scheme in Figure 1 can be, and have been, tested on the basis of the real pathway presented in Figure 2. 

The information obtained from the application of glyphosate to complex systems strongly pointed to one of the following three enzymes as the target of the inhibitor in the shikimate pathway shikimate kinase (EC 2.7.1.71), 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase (EC 2.5.1.19), and chorismate synthase (EC 4.6.1.4). Jointly, these three enzymes convert shikimic acid to chorismic acid in a series of interesting reactions >(Fig. 2). A defined system" had therefore to be found in which the conversion of shikimic acid to chorismic acid could be conveniently studied. 

When we began our synthetic program, shikimic acid had been prepared by a number of routes, and the groups of Danishefsky and Plieninger had reported syntheses of prephenic acid. The intervening intermediates, shikimate-3-phosphate (S-3-P), 5-enolpyruvylshikimate-3-phosphate (EPSP), and chorismate, were only available from natural sources or by enz)onatic transformations. 

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