Aniline papain

R=Me or COOEt) failed.2-Amino-4-phosphonobutanoic acid, the phosphonic acid analogue of glutamic acid, has been obtained in resolved forms by the action of papain, followed by aniline, on 2-benzoylamino-4-(diethoxyphosphinyl)butanoic acid when anilide occurs preferentially for the L- compound. Enantiomers of 4-amino-4-phosphonobutanoic acid were obtained by resolution... 

In the other method, I-labeled aniline was prepared by the chlora-mine-T method, diazotized, and allowed to react with the protein under basic conditions (Fig. 1). Presumably it couples to the phenol moiety of tyrosine residues. If this is the case, the product is analogous to that obtained by direct iodination by the chloramine-T or lactoperoxidase procedures. Papain and certain plant lectins in which cysteine residues are part of the active site were labeled without loss of activity. When variations of the chloramine-T or lactoperoxidase methods were used, 64-83% of the activity was lost. 

Papain, Bandeiraea simplicifolia lectin, and lima bean lectin have been labeled with diazotized [ I]aniline according to the procedure described... 

Transpeptidation, transamidation, a reaction involving the transfer of one or more amino acids from one peptide chain to another. This term was first coined by Fruton, in 1950, by analogy with transglycosidation for the papain-catalyzed displacement reaction between Bz-Gly-NH2 and aniline forming Bz-Gly-NHPh. Of special importance in relation to protease-catalyzed transpeptidation reactions in a preparative scale is the one-step tryptic conversion of porcine insulin into human insulin, despite the controversial interpretation of the mechanism involved. A bacterial transpeptidase, serim-type u-Ala-u-Ala carhoxypeptidase (EC... 

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. 

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 ... 

N-Benzyloxycarbonyl-DL-alanide amide and aniline in acetic acid buffer soln. containing hexamethylphosphoramide, L-cysteine hydrochloride, and papain incubated 48 hrs. at ca. 40 and pH 4.25 -> N-benzyloxycarbonyl-L-alanine anilide. Y 61.8%. F. e. s. J. L. Abernethy et al., Tetrahedron 31, 2659 (1975). 

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... 

Formation of a thiol ester is consistent with the spatial and chemical specificity of papain which is toward the amino acid residue bearing the carbonyl group of the sensitive bond. The moiety which is displaced from, or transferred to, the carbon of this carbonyl group may be any of the compounds mentioned above, e.g., a peptide, an amino acid, ammonia, aniline, hydrazine, hydroxylamine, an alcohol,... 

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