Lactonization spontaneous, mechanism

An experiment was designed to discriminate between the possible mechanisms. Biosynthesis of ra-butyryl lactone was carried out in the presence of (2/iV)- [2-2H]methy 1 ma-lonyl-CoA and NADPH. In the absence of spontaneous loss of the deuterium, the resulting labeling pattern in the ra-butyryl lactone was diagnostic for a particular mechanism. Any epimerization step in a mechanism acted to remove the label from the corresponding center. Structural analysis of the lactone product demonstrated labeling consistent with mechanism in. 

Fig. 6.22. Spontaneous lactonizations according to the Aac2 mechanism of Figure 6.19.

The 2-pyrone synthase (2-PS) found in several plant species is the simplest Type III PKS.ii i 2-PS condenses three malonyl-CoAs and yields a spontaneously cyclized triketide lactone (TKL). The first malonyl-CoA is decarboxylated and serves as the acetate primer, analogous to the decarboxylative mechanism observed in Type II PKSs. The 2-PS is unable to accept larger starter units due to a considerable smaller active site than that of CHS. Mutating the residues that surround the CHS-binding pocket to those found in 2-PS yielded a mutant CHS that is biochemically indistinguishable from 2-PS." ... 

Such cases are extremely rare, as the normal mode of action involves CGA mechanisms. However, Trost et al. have shown (equation 87) that in cases where the normal CGA reaction is prohibited due to ring strain, HGA ring closure under Mitsunobu conditions may be accomplished, e.g. from (248) to (249). In contrast, hydroxy acid (250) has been found to undergo CGA ring closure to (251) spontaneously, as well as under Mitsunobu treatment. However, HGA reactions can be accomplished (equation 88) by preparing the mesylate (252) with sulfene, and by subsequent 5n2 cyclization with base to form (252). An acid-catalyzed HGA lactonization has been reported for the hydroxypyrrolidines (254) and (256), both of which, under acid treatment, form the lactone (255), presumably via (257 equation 89). ... 

The last step In the mechanism, the formation of the carboxylate anion, a weaker base than hydroxide ion, drives the equilibrium to GHB. When acid is added, GHB is protonated. The hydroxy group and the carboxylic acid group undergo spontaneous ester (lactone) formation because an ester and a carboxylic acid are of similar reactivities and intramolecular reactions are more favorable. 

The CHMO from Acinetobacter sp. NCIMB 9871 has been studied quite extensively. The enzyme is a monomer (M, 66,000) contains a noncovalently bound FAD molecule and uses NADPH as the electron donor. This enzyme was cloned and expressed in E. coli [18]. The mechanism of action of CHMO was proposed by Walsh et al. [19] (Fig. 6). It proposes that the enzyme-FAD-4a-OOH is the nucleophilic peroxide equivalent that produces the tetrahedral adduct with bound substrate. The adduct then decomposes by the migration of carbon-carbon bond to the proximal peroxide oxygen as the 0-0 bond breaks. This produces the ring expansion to lactone and the enzyme-FAD-4a-OH spontaneously eliminates H2O to regenerate the oxidized FAD. 

---