Hippuric anilide

A simple case of the general transpeptidation reaction was the trans-amidation resulting in the formation of hippuric anilide from aniline and hippuric amide in the presence of papain. Since this reaction proceeded much faster than the enzymatic synthesis of hippuric anilide from hippuric acid and aniline, it seems reasonable to infer that exchange, in the former reaction, took place between the aniline and ammonia. Waley and Watson subjected L-lysyl-L-tyrosyl-L-lysine and L-lysyl-L-tyrosyl-L-leucine to treatment with chymotrypsin and trypsin at pH 7.8. In the hydrolysis mixture of either of these substrates they were able to identify lysyllysine which could have arisen only by rearrangement of the amino acids in peptide bond. The peptide may have reacted with the lysine liberated by hydrolysis ... 

The problem of the enzymatic synthesis of peptide bonds assumed a new aspect in around 1937, when Heinz Fraenkel-Conrat [30], then in the laboratory of M. Bergmann, demonstrated the papain-catalyzed formation at pH 5 of sparingly soluble benzoylglycine anilide from benzoylglycine amide or from benzoylglycine (hippuric acid) and aniline as well as the condensation of benzoylleucine with leucine anilide yielding the nearly insoluble dipeptide benzoyldileucine anilide (Fig. 6). 

Waldschmidt-Leitz and Kuhn studied in detail the synthesis of hippurylanilide—the type of synthesis first foimd by Bergmann and associates. Using nearly equivalent amounts of hippuric acid and aniline, with papain as enzyme, there was 94% synthesis of the anilide. The equilibrium was approached from both sides. They could not obtain synthesis of hippurylamide from hippuric acid and ammonia the amide was completely and rapidly hydrolyzed. The condensation with hippuric acid occurred with aniline, o- m-, and p-toluidine, o- and p-aminophenol, o-anisidine, p-aminobenzoic acid, sulfanilamide, and o- and p-phenylene-diamine. The following compounds were inactive JV-methylaniline, o-aminobenzoic acid, sulfanilic acid, a-aminopyridine, adenine, benzyl-amine, cyclohexamine, and ammonia. 

From the equilibrium data it is possible to calculate the free energy of formation of hippurylanilide under their conditions. The value is approximately 5000 cal. at 37°C., whereas the free energy of formation of small peptides and of amides is of the order of magnitude of —3600 cal. In other words, the formation of the anilide proceeds spontaneously, as they found, but the formation of analogous peptides and amides does not. This is the explanation of the failure to observe condensation of hippuric... 

An example in this category is as follows (8,9) 4.2% carbobenzoxy-glycine and 3.7% aniline incubated with papain at 40° and at pH 4.6 gave an 80% yield of carbobenzoxyglycine anilide. The optimum pH and the necessity for activation by cysteine, glutathione, or HCl were the same as for the hydrolytic action of the enzyme. Acetyl, benzoyl, and carbobenzoxy derivatives of alanine, leucine, and phenylalanine yielded with aniline or phenylhydrazine the corresponding anilides or phenyl-hydrazides. Similar reactions were catalyzed by bromelin and cathepsin, proteolytic enzymes obtained respectively from pineapple and pig liver. Under the conditions which promoted the above syntheses, hippurylamide was completely hydrolyzed there was no synthrais of the amide from hippuric acid and ammonia. 

Waldschmidt-Leitz and Kfihn (134) studied the synthesis of hippuryl-anilide in detail. With nearly equivalent concentrations of hippuric acid and aniline and papain as enzyme they found 94% synthesis of the anilide. The equilibrium was approached from both sides. They could not observe any synthesis of hippurylamide from hippuric acid and... 

Hippuric acid (Benzoylglycine) 187 p-Nitrobenzyl ester, 136. p-Bromophenacyl ester, 151. Anilide, 208. 

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