Prephenate, intermediates

The Shikimate pathway is responsible for biosynthesis of aromatic amino acids in bacteria, fungi and plants [28], and the absence of this pathway in mammals makes it an interesting target for designing novel antibiotics, fungicides and herbicides. After the production of chorismate the pathway branches and, via specific internal pathways, the chorismate intermediate is converted to the three aromatic amino acids, in addition to a number of other aromatic compounds [29], The enzyme chorismate mutase (CM) is a key enzyme responsible for the Claisen rearrangement of chorismate to prephenate (Scheme 1-1), the first step in the branch that ultimately leads to production of tyrosine and phenylalanine. 

Aryl side chain containing L-a-amino acids, such as phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp), are derived through the shikimate pathway. The enzymatic transformation of phosphoenolpyr-uvate (PEP) and erythro-4-phosphate, through a series of reactions, yields shikimate (Scheme 2). Although shikimate is an important biosynthetic intermediate for a number of secondary metabolites, this chapter only describes the conversion of shikimate to amino acids containing aryl side chains. In the second part of the biosynthesis, shikimate is converted into chorismate by the addition of PEP to the hydroxyl group at the C5 position. Chorismate is then transformed into prephenate by the enzyme chorismate mutase (Scheme 3). 

The preparation of a key intermediate in an imaginative synthesis of prephenic acid is depicted below. Write a series of equations showing the important steps and intermediates in this process. Indicate the reagents required to bring about the desired... 

Figure 1. Hypothetical mechanism for shuttling of intermediates of the common aromatic pathway between plastidic and cytosolic compartments. Enzymes denoted with an asterisk (DAHP synthase-Co, chorismate mutase-2, and cytosolic anthranilate synthase) have been demonstrated to be isozymes located in the cytosol. DAHP molecules from the cytosol are shown to be shuttled into the plastid compartment in exchange for EPSP molecules synthesized within the plastid. Abbreviations C3, phosphoenolpyruvate C4, erythrose 4-P DAHP, 3-deoxy-D-arabino-heptulosonate 7-phosphate EPSP, 5-enolpyruvylshikimate 3-phosphate CHA, chorismate ANT, anthranilate TRP, L-tryptophan PPA, prephenate AGN, L-arogenate TYR, L-tyrosine and PHE, L-phenylalanine.

Claisen rearrangement plays an important part in the biosynthesis of several natural products. For example, the chorismate ion is rearranged to the prephenate ion by the Claisen rearrangement, which is catalysed by the enzyme chorismate mutase. This prephenate ion is a key intermediate in the shikimic acid pathway for the biosynthesis of phenylalanine, tyrosine and many other biologically important natural products. 

In plants and bacteria, phenylalanine and tyrosine are synthesized from chorismate in pathways much less complex than the tryptophan pathway. The common intermediate is prephenate (Fig. 22-19). The final step in both cases is transamination with glutamate. 

Finally, the possible biogenesis of echitamine remains for discussion (88). This can readily be rationalized, using as an intermediate a base such as geissoschizine (LX), one of the hydrolytic fission products of the alkaloid geissospermine. Geissoschizine can clearly be obtained from tryptamine and dihydroxyphenylalanine or prephenic acid by a route which has many analogies in indole alkaloid chemistry. Dehydrogenation... 

Structural considerations and known enzymic reactions suggest that the reaction of shikimate 5-phosphate with enolpyruvate phosphate is related to prephenate formation, and that the reactions of shikimate 5-phosphate with glutamine are involved in the synthesis of anthranilate and p-amino-benzoate. A common intermediate for these two branches of the main pathway, after shikimate 5-phosphate, appears to be unlikely, since it would require the further transformation of the latter compound before reaction with enolpyruvate phosphate. If shikimate 5-phosphate is the branch-point intermediate, quintuple auxotrophs should be completely blocked in any one reaction before shikimate 5-phosphate. In the absence of secondary metabolic effects, mutants blocked immediately after it should not be quintuple auxotrophs. They should show a requirement for either phenylalanine plus tyrosine, or tryptophan plus p-aminobenzoate (since anthranilate and p-aminobenzoate may have a common intermediate ). p-Hydroxybenzoate might be derived from this intermediate, or, independently, from shikimate 5-phosphate. This possibility is illustrated on page 264, X being the possible common intermediate. 

The Claiscn rearrangement of chorismate 1, the last common intermediate in the biosynthesis of aromatic amino acids via the shikimate pathway, to prephenate 2 proceeds readily without catalysis263 and is greatly accelerated by a factor of 1.9 xlO6 by the enzyme chorismate mutase655-656. 

To fully establish whether arogenate (41), phenylpyruvate (39), or both were pathway intermediates to Phe (1) in vascular plants, it was essential to unambiguously identify all enzymatic processes (and encoding genes) needed for conversion of prephenate (38) into Phe (1) both in vivo and in vitro. Moreover, it was also essential to obtain rigorous enzymatic kinetic data using highly purified recombinant enzymes in vitro for all potential substrates, in order to compare and contrast relative efficacies/feedback inhibition properties and so forth. 

However, recent studies with bacteria other than E. coli and the availability and analysis of genomic sequences from many prokaryotic and eukaryotic microorganisms have established that there are three different routes to the soluble intermediate 4-HB (Figure 12). The three routes to 4-HB are (1) the CPL reaction, (2) the tyrosine-4-hydroxyphenylpynivate (THP) pathway, and (3) the chorismate-prephenate- 4-hydroxyphenylpyruvate (CPHP) pathway. 

Figure 3. Schematic outline of various intermediates and products including enzymes of the phenolic pathway in plants. Enzymes 1, 3-deoxy-2-oxo-D-arabino-heptulosate-7-phosphate synthase 2, 5-dehydroquinate synthase 3, shikimate dehydrogenase 4, shikimate kinase 5, 5-enolpyruvylshikimate-3-phosphate synthase 6, chorismate synthase 7, chorismate mutase 8, prephenate dehydrogenase 9, tyrosine aminotransferase 10, prephenate dehydratase 11, phenylalanine aminotransferase 12, anthranilate synthase 13, tryptophan synthase 14, phenylalanine ammonia-lyase 15, tyrosine ammonia-lyase and 16, polyphenol oxidase.

This new route to cyclohexadienones has been used in a total synthesis of the disodium salt of prephenic acid (2j, an unstable intermediate in biosynthesis of various phenols. The first attempt used the dienophile 3 in which the potential... 

Fig. 2 (1)]. A key intermediate in the pathway is chorismate from which branched pathways lead to tryptophan, phenylalanine, tyrosine, 4-amino benzoate, isoprenoid quinones, and metacarboxyphenylalanine. A secondary branch also occurs at prephenate leading to phenylalanine and tyrosine. A representative number of non amino acid compounds in relation to their shikimic acid pathway precursors are shown in Fig. 1. One side branch leads to quinate which participates in the formation of depsides. 

Danishefsky and Hirama have published a neat total synthesis of the disodium salt of prephenic acid (24), a central intermediate in the shikimic acid biosynthetic pathway.The key step involves a Diels-Alder reaction between the diene (22) and the unsaturated lactone (23). A crucial feature of this synthesis is the simultaneous protection of both the C-lO-carboxy and C-8-keto functions as a methoxy-lactone, allowing umasking in a single step by alkaline hydrolysis. 

The conversion of chorismate into prephenate is an example of a biologically relevant Claisen rearrangement. It is the key intermediate in the biosynthesis of aromatic amino acids (tyrosine, phenylalanine, and tryptophan) in bacteria, fungi, and higher plants (Figure 1.23) [23]. 

In both bacteria and plants, two additional amino acids, phenylalanine and tyrosine, are formed from chorismic acid. From chorismate, two separate routes diverge and lead to the amino acids L-phenylalanine and L-tyrosine. However, the pathways in bacteria and plants are distinct and involve different intermediates. Both of these pathways pass through the same intermediate, prephenic acid (26) (Fig. 7.9) (Floss,... 

Phenylalanine (31) may be formed by conversion of chorismate (9) to prephenate (as an enzyme-bound intermediate) and subsequent formation of phenylpyruvate (28). Transamination of this intermediate produces phenylalanine (31). 

In higher plants, phenylalanine seems to be formed in an alternative manner by formation of prephenate (26) and conversion of this intermediate into arogenic acid (32). Chorismate mutase occurs as two isozymes which have been purified to homogeneity from mung bean and sorghum. All chorismate mutase isozymes show allosteric activation by chorismate (Poulsen and Verpoorte, 1991). One chorismate mutase isozyme is inhibited by phenylalanine or tyrosine and activated by tryprophan, whereas the second is not affected by any of the aromatic amino acids. 

Chorismic acid is the key branch point intermediate in the biosynthesis of aromatic amino acids in microorganisms and plants (Scheme 1.1a) [1]. In the branch that leads to the production of tyrosine and phenylalanine, chorismate mutase (CM, chorismate-pyruvate mutase, EC 5.4.99.5) is a key enzyme that catalyzes the isomerization of chorismate to prephenate (Scheme 1.1b) with a rate enhancement of about lO -lO -fold. This reaction is one of few pericyclic processes in biology and provides a rare opportunity for understanding how Nature promotes such unusual transformations. The biological importance of the conversion from chorismate to prephenate and the synthetic value of the Claisen rearrangement have led to extensive experimental investigations [2-43]. 

Chorismic acid serves as the branch point intermediate in the biosynthesis of many important aromatic products. These metabolites include anthranilate (required for the end product tryptophan), prephenate (tyrosine and phenylalanine), PABA (folic acid and aromatic polyenes), isochorismate (encerochelin, menaquinone), and p hydioxyben> zoatc (ubiquinone) (33). 

---