Decarboxylation of acetoacetic acid

A third early mechanism for enzymic processes involves the formation of imines between the amino group of a lysine residue on an enzyme and the carbonyl group of a substrate, followed by standard imine chemistry. The first example concerned the decarboxylation of acetoacetic acid (Hamilton and Westheimer, 1959). The mechanism was based on the non-enzymic physical organic chemistry of Kai Pedersen (Pedersen, 1934). He postulated that the catalysis by aniline of the decarboxylation of dimethylacetoacetic acid proceeds by a mechanism parallel to that shown in Scheme 7 for acetoacetic acid itself (Pedersen, 1938). 

Another example where mechanism and model have been developed is that for the decarboxylation of acetoacetic acid here no coenzyme is required, and the chemistry involves the enzyme itself. The mechanism for the enzymic decarboxylation with crystalline decarboxylase from Clostridium acetobutylicum has been worked out in some detail it is presented below (20, 21). The initial work, carried out in the author s laboratory by G. Hamilton (22) and I. Fridovich (23, 24) proved that the essential intermediate is a ketimine much of the subsequent development of the enzymic system resulted from the researches of W. Tagaki (25). 

The decarboxylation of acetoacetic acid [Eq. (16)] has been studied extensively by Westheimer and associates (93, 94). 

The rate of the enzymic decarboxylation of acetoacetic acid exceeds the rate of the spontaneous reaction by perhaps a billionfold. However, simple amines of appropriate pK also catalyze the reaction, and the difference in rate between catalysis by the enzyme and catalysis by cyanomethylamine (pK 5.5) is only about 100-fold (]00). 

Decarboxylation of acetoacetic acid involves transfer of the acidic hydrogen to the keto group. Note that compound B must be acetoacetic acid because that is the only possible manner in which we can attach a carboxyl group to acetone. 

The decarboxylation of acetoacetic acid, a P-keto acid, occurs by way of a cyclic transition state in which a proton is transferred from the carboxylate atom to the carbonyl oxygen to give an enol that rapidly tautomerizes to give acetone. 

The roots of iminium activation can be traced back to the pioneering works of Knoevenagel [4]. The Knoevenagel condensation became the first reaction that might proceed via iminium catalysis. Next, Pollack reported that several proteins and amino acids catalyzed the decarboxylation of acetoacetic acid. The mechanism suggested by Petersen involves an imine intermediate [5]. 

It is also possible to use the dilithium derivative of acetoacetic acid as the synthetic equivalent of acetone enolate.49 In this case, the hydrolysis step is unnecessary and decarboxylation can be done directly on the alkylation product. 

Examples of this approach to the synthesis of ketones and carboxylic acids are presented in Scheme 1.6. In these procedures, an ester group is removed by hydrolysis and decarboxylation after the alkylation step. The malonate and acetoacetate carbanions are the synthetic equivalents of the simpler carbanions lacking the ester substituents. In the preparation of 2-heptanone (entries 1, Schemes 1.5 and 1.6), for example, ethyl acetoacetate functions as the synthetic equivalent of acetone. It is also possible to use the dilithium derivative of acetoacetic acid as the synthetic equivalent of acetone enolate.29 In this case, the hydrolysis step is unnecessary, and decarboxylation can be done directly on the alkylation product. 

Hamilton marked the carbonyl group of acetoacetic acid with ieO, and then carried out the enzymic decarboxylation (Hamilton and Westheimer, 1959). The product of the decarboxylation, acetone, contained none of the label. This result is demanded by the ketimine mechanism, whereas the mechanism of uncatalyzed decarboxylation would have required that the label appear intact in the product. Of course, in order to make these statements we had to carry out an elaborate set of control experiments, since 180 is washed out of both acetone and acetoacetic acid by buffers and even more... 

Individuals with either type of diabetes are unable to take up glucose efficiently from the blood recall that insulin triggers the movement of GLUT4 glucose transporters to the plasma membrane of muscle and adipose tissue (see Fig. 12-8). Another characteristic metabolic change in diabetes is excessive but incomplete oxidation of fatty acids in the liver. The acetyl-CoA produced by JS oxidation cannot be completely oxidized by the citric acid cycle, because the high [NADH]/[NAD+] ratio produced by JS oxidation inhibits the cycle (recall that three steps convert NAD+ to NADH). Accumulation of acetyl-CoA leads to overproduction of the ketone bodies acetoacetate and /3-hydroxybutyrate, which cannot be used by extrahepatic tissues as fast as they are made in the liver. In addition to /3-hydroxybutyrate and acetoacetate, the blood of diabetics also contains acetone, which results from the spontaneous decarboxylation of acetoacetate ... 

It was shown that an enol intermediate was initially formed in the decarboxylation of l -ketoacids and presumably in the decarboxylation of malonic acids. It was found that the rate of decarboxylation of a,a-dimethylacetoacetic acid equalled the rate of disappearance of added bromine or iodine. Yet the reaction was zero order in the halogen . Qualitative rate studies in bicyclic systems support the need for orbital overlap in the transition state between the developing p-orbital on the carbon atom bearing the carboxyl group and the p-orbital on the i -carbonyl carbon atom . It was also demonstrated that the keto, not the enol form, of p ketoacids is responsible for decarboxylation of the free acids from the observa-tion that the rate of decarboxylation of a,a-dimethylacetoacetic acid k cid = 12.1 xlO sec ) is greater than that of acetoacetic acid (fcacw = 2.68x10 sec ) in water at 18 °C. Enolization is not possible for the former acid, but is permissible for the latter. Presumably this conclusion can be extended to malonic acids. 

D. Decreased insulin levels cause fatty acid synthesis to decrease and glucagon levels to increase. Adipose triacylglycerols are degraded. Fatty acids are converted to ketone bodies in liver a ketoacidosis can occur. There is increased decarboxylation of acetoacetate to form acetone, which causes the odor associated with diabetic ketoacidosis. 

Ketone body formation, which occurs within the matrix of liver mitochondria, begins with the condensation of two acetyl-CoAs to form acetoacetyl-CoA. Then acetoacetyl-CoA condenses with another acetyl-CoA to form /3-hydroxy-/3-methylglutaryl-CoA (HMG-CoA). In the next reaction, HMG-CoA is cleaved to form acetoacetate and acetyl-CoA. Acetoacetate is then reduced to form /3-hydroxybutyrate. Acetone is formed by the spontaneous decarboxylation of acetoacetate when the latter molecule s concentration is high. (This condition, referred to as ketosis, occurs in uncontrolled diabetes, a metabolic disease discussed in Special Interest Box 16.3, and during starvation. In both of these conditions there is a heavy reliance on fat stores and /3-oxidation of fatty acids to supply energy.)... 

The decarboxylation of acetoacetate is acid catalyzed (20). Metals do not catalyze the spontaneous decarboxylation of acetoacetate, presumably because the substrate and the product acetone enol are poor ligands for the metal (27). Primary amines catalyze the decarboxylation of acetoacetate by a Schiff base mechanism (Scheme VII), and this provides the best model for acetoacetate decarboxylase (94). 

A variety of metal ions have been found to increase markedly the rate of decarboxylation of several jS-keto acids, but to have no effect on the decarboxylation of ketomonocarboxylic acids such as acetoacetic acid. Moreover, only those fl-keto acids having a second carboxylic acid group adjacent to the /3-keto group are affected by the presence of metal ions, e.g., oxaloacetic or oxalosuccinic acids (90, 116, 166, 170, 190, 191, 208). 

The decarboxylation of y8-keto acids by both primary amines and by acetoacetate decarboxylase has been studied by the method of carbon isotope effects [90,91]. A typical isotope effect for the amine-catalyzed reaction is 1.03 for the cyanomethylamine-catalyzed decarboxylation of acetoacetate at pH 5.0. This demonstrates that the carbon-carbon bond is being cleaved in the rate-determining decarboxylation step. The comparable carbon isotope effect for the enzymatic decarboxylation is = 1.018 and is pH-independent over the range pH... 

The thermal decarboxylation of -keto acids is the last step in a ketone synthesis known as the acetoacetic ester synthesis. The acetoacetic ester synthesis is discussed in Section 21.6. 

The second classical reaction mentioned above is the acetoacetic ester synthesis. this reaction, an ester of acetoacetic acid (3-oxobutanoic acid) such as ethyl acetoacetate is treated with base under thermodynamic control conditions and alkylated, as with the malonic ester synthesis. Reaction with sodium ethoxide in ethanol (since an ethyl ester is being used) generated the enolate and quenching with benzyl bromide led to 84. Saponification and decarboxylation (as above) gave a substituted ketone (85). Although the malonic ester synthesis and the acetoacetic ester synthesis are fundamentally similar, the different substrates lead to formation of either a highly substituted acid or a ketone. The reaction is not restricted to acetoacetate derivatives, and any p-keto-ester can be used (ethyl 3-oxopentanoate for example). ... 

Ethyl acetoacetate is frequently used in the Knorr synthesis, which gives rise to a pyrrole with a 3-carbethoxy group. However, this can be removed easily if desired, first by hydrolysis of the ester to the carboxylic acid and then decarboxylation of the acid group. 2,4-Dimethypyrrole is a well-known compound easily made by the Knorr process and elimination of the carbethoxy group (Scheme 4.33). 

The concentration of ketone bodies in the blood of healthy, well-fed humans is approximately 0.01 mM/L. However, in persons suffering from starvation or diabetes mellitus, the concentration of ketone bodies may increase to as much as 500 times normal. Under these conditions, the concentration of acetoacetic acid increases to the point where it undergoes spontaneous decarboxylation to form acetone and carbon dioxide. Acetone is not... 

P-Hydroxybutyrate dehydrogenase (located in mitochondria) catalyses the conversion of acetoacetate to P-hydroxybutyrate. Acetone is formed by the spontaneous decarboxylation of acetoacetate (Fig. 1). Acetoacetate is also produced by degradation of the keto-plastic amino acids, leucine, isoleucine, phenylalanine and tyrosine. 

As another example, consider the decarboxylation of acetoacetate. This compound will decarboxylate under acidic conditions with heating. However, the addition of aniline catalyzes the reaction, so it occurs at less acidic pH and ambient temperature (Eq. 9.10). Formation of the imine between aniline and the p-keto acid leads to a species that is protonated and can act as a good electron sink during the decarboxylation. Hydrolysis of the enamine product gives the ketone and regenerates the catalyst, thus leading to turnover. 

FIGURE 19.56 The acid-catalyzed hydrolysis and decarboxylation of acetoacetic esters. 

The question arises as to whether acetone is a primary product of the oxidation of isovalerate or is formed secondarily by decarboxylation of acetoacetate. The experimental results of Zabin and Bloch are in accord with a reaction mechanism in which isovaleric acid is oxidized initially at the carbon 2-position to yield a 3-carbon and a 2-carbon fragment. This conclusion is based on the high absolute C concentrations found in the methyl carbons of acetone and also in comparison to that in the methyl or methylene carbons of acetoacetate. Secondly, the C C ratios were significantly greater in the acetone fractions than in the corresponding carbon atoms of acetoacetate. This finding can be explained only if acetone is formed directly from isovaleric acid, but is at variance with the assumption that acetone arose exclusively by decarboxylation of acetoacetate. The authors also point out that it is possible that the postulated 3-carbon intermediate is not acetone, but that acetone is formed in a side reaction from a more labile 3-carbon precursor. 

Foods frequently contain saturated and unsaturated aliphatic ketones with between 3 and 17 carbon atoms in the molecule. These ketones are formed by several different mechanisms. Frequently occurring aliphatic ketones are methylketones. The most common methylketone is acetone (propanone, 8-46, = 0). Acetone is present, usually in small quantities, in all biological substrates, where it arises by decarboxylation of acetoacetic (3-oxobutanoic) acid. Acetoacetic acid is formed as an intermediate during degradation of fatty acids by -oxidation. Acetone in the skins of apples, for example, is produced from pyruvic acid via citramalic acid (Figure 8.16). The relatively large amount of acetone is generated by acetone-butanol fermentation (see Section 8.2.2.1.1). Many other saturated and unsaturated methylketones occur as odour-active components of essential oils. For example, a component of cinnamon and star anise essential oils is heptane-2-one, also known as methyl pentyl ketone (8-46, = 4). 

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