Oxidative decarboxylation of hydroxycarboxylic acids

As mentioned in the introductory part, stereochemical course of the conversion of isocitric acid to a-ketoglutaric acid in TCA cycle is completely enantiose-lective although the reaction does not form an asymmetric carbon in the usual metabolic path. If such type of oxidative decarboxylation can be applied to synthetic compounds, it is expected that an entirely new type of asymmetric biotransformation will be developed.

Aiming at the feasibility study of such type of asymmetric decarboxylation, we screened microorganisms which are able to grow on the medium containing tropic acid as the sole source of carbon. It is expected that at least one of the major metabolic pathway of tropic acid is the oxidation of the hydroxyl group followed by decarboxylation and further oxidation of the resulting aldehyde (Fig. 20). If

this enzyme system is also active to a-methyl derivative, optically active aldehyde or carboxylic acid will be obtained.

Fortunately, some soil microorganisms were found to grow utilizing tropic acid as the sole source of carbon. Among them, a bacterium identified as Rhodococ-cus sp. was also active to the methyl derivative. The isolated product was (R)-a-phenylproionate as shown in Fig. 20.

Under the optimum condition, present microbial oxidation was extended to other tropic acid derivatives. As shown in Table 9, when R group was changed to ethyl (entry 2), the yield of the product decreased compared to the case of methyl group (entry 1). This must be due to the difference of the steric bulkiness of ethyl and methyl groups. The effect of variation of aromatic part was also examined. When Ar group was 4-methoxyphenyl, 4-chlorophenyl and 2-naphthyl, the oxidation reaction proceeded smoothly and the corresponding esters were obtained in good yields after esterification of the primary product with TMS-diazomethane. The configuration of the products was R except for the case of the 4-chlorophenyl derivative, in which the product was racemic. Although it seems difficult at present to interpret consistently the effect of the structure of Ar and R on the reactivity, it is certain that electron-donating substituents are favorable for this enzyme system.

Table 9

Microbial oxidation of tropate derivatives




Reaction time (d)


61 R

25 R

61 R

To obtain a better understanding of the reaction mechanism, some compounds that are considered to be intermediates were subjected to the reaction. Various reaction courses can be considered as illustrated in Fig. 21. Path A: a-Methyltropic acid is oxidized to a-phenyl-a-methylmalonic acid. Then, the malonate is converted to optically active a-phenylpropionate by arylmalonate decarboxylase.8 9 In order to confirm this assumption, incubation of the malonic acid with Rhodococcus sp. was carried out. The result obtained was the total recovery of the substrate, indicating that no decarboxylase is present in this bacterium. Path B: a-Methyltropic acid is converted to racemic a-phenylpropionic acid, which is deracemized to optically active propionic acid.8182 To examine the possibility of this route, racemic a-phenylpropionic acid was subjected to the reaction to observe

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Figure 21: Possible reaction paths of a-methyltropate to optically active a-phenylpropionate.

Figure 21: Possible reaction paths of a-methyltropate to optically active a-phenylpropionate.

no deracemization at all. Path C: a-methyltropic acid is converted to racemic 2-phenylpropanal. If this aldehyde can racemize under the reaction conditions and (R)-enantiomer is preferentially oxidized to (R)-phenylpropionic acid, the final product can be optically active. Actually, racemic a-phenylpropanal gave almost racemic a-phenylpropionic acid. Thus, it is clear that the intermediate aldehyde should be optically active. In this way, the most possible reaction path is the following. Path D: Both enantiomers of a-methyltropic acid are oxidized to racemic malonic semialdehyde, which is converted by decarboxylation to enol-type intermediate in the active site of the enzyme. The enolate is immediately protonated in an enantioface-selective manner to result in optically active aldehyde, which in turn is oxidized to a-phenylpropionic acid without losing its enantiomeric excess. In this way, the (R)-enantiomer of the final product would be obtained utilizing both enantiomers of a-methyltropic acid.

The enzymatic reaction was performed using optically active a-methyltropic acid at 30° C for 24 h. As expected, both enantiomers were converted to the final product, (R)-a-phenylpropionic acid, with similar enantioselectivity. Also, there was no significant difference in the rate of reaction between two enantiomers. Accordingly, it can be concluded that both enantiomers of a-methyltropic acid are non-selectively oxidized to the corresponding malonic semialdehyde. The semialdehyde is converted by decarboxylation to enol-type intermediate. Because the final product is optically active, the protonation to this intermediate should be enantioface-selective to give (R)-aldehyde as shown in Path D of Fig. 21. Thus similar tandem reactions with that of isocitric acid into 2-oxoglutaric acid by the NAD+-linked dehydrogenase1 and L-malic acid into pyruvic acid by the malate dehydrogenase83 could be achieved using a synthetic substrate.84 Stereochemistry of isocitrate dehydrogenase-catalyzed decarboxylation of isocitric acid has been reported. Thus, it is thought that the mechanism which we presumed is also sufficiently possible.85-87

In summary, a new type of activation of a carboxyl group has been realized by incubation with a microorganism resulting in the formation of an optically active compound. This type of reactions is also expected to develop more in the future.

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