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15.4: Another kind of electrophilic addition-elimination - shikimate to chorismate

A fascinating example of the richness of electrophilic mechanisms in biochemical pathways is found in the conversion of skikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP) to chorismate, a key phase of aromatic amino acid biosynthesis in plants and bacteria. This process has intrigued and perplexed enzymologists for decades, and is thought to proceed via a short-lived tetrahedral intermediate that is the result of an electrophilic addition of S3P to PEP.

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The enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase,  which catalyzes steps 1 and 2, is the target of the weed-killer glyphosate (better known by its trade name 'Roundup').  Glyphosate is a potent inhibitor of EPSP synthase, and thus plants exposed to it die because they are not able to synthesize aromatic amino acids. It does not effect humans and animals because we do not have this enzymatic pathway - we get our aromatic amino acids from our diet.

We have already discussed the mechanisms of steps 2 and 3, which are E1 eliminations (section 14.3). Now, we turn our attention to the first step, which is an electrophilic addition.  First of all, given that the addition is taking place on a double bond that is conjugated to a carbonyl group, why is this not characterized as a Michael (carbanion intermediate) addition?  Look very closely at what is happening: if this were indeed a Michael addition, the alcohol nucleophile on S3P would have to attack C3 of PEP, not C2, in order to allow the formation of a carbonyl-stabilized intermediate. 

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However, this would not lead to the correct connectivity observed in chorismate - the oxygen nucleophile needs to attack at C2 - but that does not represent a Michael addition!

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Rather, addition of the S3P nucleophile at C2 implies an electrophilic (carbocation intermediate) mechanism, with protonation occurring prior to nucleophilic attack by the S3P hydroxyl:

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Notice that the intermediate with a positive charge on C2 is the more stable of the two carbocation possibilities.  Also notice that this is an anti addition to the double bond.

Several lines of evidence point to the existence of the short-lived 'tetrahedral intermediate' in the EPSP synthesis reaction.  When the reaction was stopped midway by denaturing the enzyme with the mild base triethylamine, the intermediate was actually stable enough to be isolated and directly characterized by NMR (J. Am. Chem. Soc. 1988, 110, 6577).  More recently, both diastereomers of the proposed intermediate were chemically synthesized; the isomer with S stereoconfiguration at C2 was found to be converted by the enzyme to a mixture of starting compounds (S3P and PEP) and product (EPSP).   The R diastereomer was not reactive in the presence of the enzyme (J. Am. Chem, Soc. 2003, 125, 12759)

Additional evidence that the first step is a carbocation-intermediate electrophilic addition was provided by an experiment in which the reaction was run in D2O with PEP and a deoxy analog of S3P (Acc. Chem. Res. 1997, 30, 2).

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Although of course no condensation occurred (the nucleophile was removed!), deuterium was incorporated into the PEP substrate, suggesting that (reversible) protonation of the PEP double bond had occurred without participation by the S3P hydroxyl  - in other words, protonation appears to occur first, as expected for an electrophilic addition.

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