Leucine anilide

An alternative to the synthesis of proteins by classical fragment synthesis in solution or by solid-phase synthesis on a support is the use of enzyme-catalyzed condensation of amino acids or peptides. This possibility was first demonstrated in 1938 91 with the synthesis of poorly soluble benzoyl-leucyl-leucine anilide by papain catalysis. After many years, this approach was extended to the preparation of peptide hormones such as Leu-enkephalin 92 and dynorphin(l -8).[93 This was made possible by the use of highly purified enzymes and by careful control of reaction conditions. The basic principles of protease-catalyzed peptide bond formation have been discussed.194 ... 

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). 

In plastein synthesis the free energy of formation of the peptide bonds is small, as attested to by the reversal of hydrolysis merely by concentrating certain enzymatic hydrolytic products the synthetic product is insoluble, which tends to drive the reaction toivards synthesis. In these two respects plastein formation resembles reactions of the type benzoyl-L-leucine - - L-leucine anilide to benzoyl-L-leucyUeucine anilide. 

Benzoyl-L-leucine + n-Leucine anilide-------- Benzoyl-L-leucine anilide... 

The free energy of formation of the peptide bonds in plastein synthesis must be small this is attested to by the possibility of inducing peptide bond synthesis merely by concentrating an enzymatic hydrolyzate. The synthetic product is practically insoluble and this drives the reaction toward synthesis. It is in these two respiects that plastein formation resembles reactions of the type benzoyl-L-leucine + L-leucine anilide —> benzoyl-L-leucylleucine anilide. 

Application and Principle This procedure is used to determine leucine aminopeptidase activity in enzyme preparations derived from Lactococcus lactis. The assay is based on the rate of absorbance change over 5 min at 30° the change in absorbance is due to liberated p-nitroaniline from the hydrolysis of leucine p-nitro anilide. 

Substrate Solution Dissolve 0.0200 g of leucine p-nitro-anilide hydrochloride (Sigma Chemical Co., Catalog No. L2158) in 100 mL of pH 7.0 Phosphate Buffer. 

Finally, libraries aimed to chiral resolution of racemates will be covered here in particular, the use of chiral stationary phases (CSPs) has recently been reported for the identification of materials to be used for chiral separation of racemates by HPLC. The group of Frechet reported the selection of two macroporous poly methacrylate-supported 4-aryl-1,4-dihydropyrimidines (DHPs) as CSPs for the separation of amino acid, anti-inflammatory drugs, and DHP racemates from an 140-member discrete DHP library (214,215) as well as a deconvolutive approach for the identification of the best selector phase from a 36-member pool library of macroporous polymethacrylate-grafted amino acid anilides (216,217). Welch and co-workers (218,219) reported the selection of the best CSP for the separation of a racemic amino acid amide from a 50-member discrete dipeptide iV-3,5-dinitrobenzoyl amide hbrary and the follow-up, focused 71-member library (220). Wang and Li (221) reported the synthesis and the Circular Dichroism- (CD) based screening of a 16-member library of CSPs for the HPLC resolution of a leucine ester. Welch et al. recentiy reviewed the field of combinatorial libraries for the discovery of novel CSPs (222). Dyer et al. (223) reported an automated synthetic and screening procedure based on Differential Scanning Calorimetry (DSC) for the selection of chiral diastereomeric salts to resolve racemic mixtures by crystallization. Clark Still rejxrrted another example which is discussed in detail in Section 9.5.4. 

Despite this, there are examples of molecularly imprinted chiral stationary phases that are capable of resolving more than the raceme corresponding to the template. In these cases, minor structural differences are possible without compromising the separation. For example, a polymer imprinted with L-phenylalanine anilide efficiently separated the protected amino acids with different side chains or amide substituents [36]. Anilides of all aromatic amino acids were resolved as well as p-naphthylamides and -nitroanilides of leucine and alanine [40]. 

Fig. 3. Chromatography of subtilisin DY on bacitracin-cellulose (prepared with 2,4,6-trichlorotriazine). 200 mg of subtilisin DY dissolved in 3 ml of 50 mM Tris-HCl buffer, pH 8.3, containing 1 mM CaCl2> was applied on a column (30 x 2 cm) of bacitracin-cellulose (inhibitor content 14.9 jjmol/g of dry carrier) equilibrated with 50 mM Tris-HCl buffer, pH 8.3, containing 1 mM CaCl2- After washing the column with the equilibrium buffer, 20% isopropyl alcohol in 1 M NaCl, buffered at pH 8.3 was applied at the position marked with arrow. Fractions (6 ml) were taken at 5-min intervals. Full line, protein dashed line, activity determined with benzyloxycarbonyl-L-alanyl-l-alanyl-L-leucine p-nitro-anilide (Lyublinskaya et al., 1974 1977). 

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 ... 

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. 

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