Iminium mechanism, covalent

As noted above, many decarboxylases are known to exploit Schiff base formation in the active-site as a source of catalysis. Shostack and Jones explored this possibility in the case of ODCase [8]. They found that when the enzymatic reaction is performed in 0 water, the product does not incorporate from bulk solvent. For this reason, a covalent iminium mechanism for ODCase was abandoned, in spite of its attractive similarities to other decarboxylase mechanisms. 

The enantioselective Michael addition of malononitrile CH2(CN)2 to trans-chalcones R CH=CHCOR catalysed by diarylprolinols (151), has been studied in detail. Both experimental and computational results are consistent with a bifunctional non-covalent activation of the reactants (152). The latter mechanism correctly predicts formation of the (R)-configured products (<75% eef- but is in conflict with the generally accepted iminium mechanism. Furthermore, (151) is likely to form the corresponding oxazolidine derivative as an intermediate, which has not been taken into account. 

Rosenthaler et al. [106] purified histidine decarboxylase from Lactobacillus 30A and demonstrated that there was no pyridoxal phosphate, as had been suggested by Rodwell [107]. Treatment with [ C]phenylhydrazine labeled the protein, but did not if the protein was first reduced with borohydride. Chymotrypsin digestion of the [ C]phenylhydrazone treated enzyme resulted in a labeled fragment identified as A -pyruvoylphenylalanine [100]. Borohydride reduction of the native enzyme resulted in lactate production after hydrolysis. Thus it was established that a pyruvoyl group is covalently bound as an amide to the NH 2-terminal phenylalanine. As is consistent with this proposed mechanism the enzyme is also inhibited by cyanide and by hydroxylamine. The iminium ion predicted by the mechanism above was trapped with borohydride in the presence of substrate and identified [108]. 

With respect to the covalent activation in conjugate additions, the catalyst, usually a primary or a secondary amine, can reversibly form a chiral enamine [ 11 ] to activate the nucleophile (D, Fig. 2.2) or a chiral iminium ion [12] to activate the acceptor (E, Fig. 2.2). The detection of enamine intermediates in asymmetric oiganocatalysis has been for a long time the missing piece of evidence for the commonly accepted mechanism of enamine catalysis. This gap has been recently solved with the first detection and structnral characterization of enamine intermediates in proUne-cata-lyzed aldol reactions by real-time NMR spectroscopy [13] and the direct observation of an enamine intermediate in the crystal strnctnre of an aldolase antibody [14]. 

The mechanism of thymidylate synthase in both its normal mode and when it is about to be blocked by the inhibitor involves attack of an enolate ion on an iminium cation. This process is closely analogous to the Mannich reaction discussed in Section 19.8. The enolate ion in this attack arises by conjugate addition of a thiol group from thymidylate synthase to the a,/3-unsaturated carbonyl group of the substrate. This process is analogous to the way an enolate intermediate occurs in a Michael addition. The iminium ion that is attacked in this process derives from the coenzyme N, l / °-methylenetetrahydrofolate (N, l / °-methylene-THF). Attack by the enolate in this step forms the bond that covalently links the substrate to the enzyme. It is this bond that cannot be broken when the fluorinated inhibitor is used. The mechanism of inhibition is shown at right. 

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