Peptide hippuric acid

The azlactones of a-benzoylaminocinnamic acids have traditionally been prepared by the action of hippuric acid (1, Ri = Ph) and acetic anhydride upon aromatic aldehydes, usually in the presence of sodium acetate. The formation of the oxazolone (2) in Erlenmeyer-Plochl synthesis is supported by good evidence. The method is a way to important intermediate products used in the synthesis of a-amino acids, peptides and related compounds. The aldol condensation reaction of azlactones (2) with carbonyl compounds is often followed by hydrolysis to provide unsaturated a-acylamino acid (4). Reduction yields the corresponding amino acid (6), while drastic hydrolysis gives the a-0X0 acid (5). ... 

Note Primary amines yield fluorescent chromatogram zones even before the application of reagent 3. Secondary amines do not yield fluorescent derivatives until they have been treated with reagent 3. Hence, the reagent sequence allows the stepwise detection of primary and secondary amines. Taurine is preferred as the essential component of reagent 3 over the multiplicity of other possibilities because it produces intense fluorescence it is also not very volatile and is readily available. Amides and substances with peptide linkages, eg. hippuric acid, are not detected, neither are secondary amines that are volatile at high temperatures. 

Angiotensin-I converting enzyme (ACE) controls blood pressure by catalyzing the hydrolysis of two amino acids (His-Leu) at the C-terminus of angiotensin-I to produce a vasoconstrictor, angiotensin-II. The enzyme can also hydrolyze a synthetic substrate, hippuryl-L-histidyl-L-leucine (HHL), to hippuric acid (HA). At four different concentrations of HHL solutions (pH 8.3), the initial rates of HA formation (pmolmin" ) are obtained as shown in Table P3.8. Several small peptides (e.g., Ile-Lys-Tyr) can reversibly inhibit the ACE activity. The reaction rates of HA formation in the presence of 1.5 and 2.5 pmoll of an inhibitory peptide (Ile-Lys-Tyr) are also given in the table. 

This definition should be corrected by eliminating the word one, for it is impossible to consider as protides a large number of compounds which liberate upon hydrolysis one molecule of amino acid, such as glycocholic acid which yields glycine and cholic acid pantothenic acid which liberates -alanine and phospholipides containing serine. At the most, hippuric acid can be considered as a natural, somewhat special peptide. 

The unaccounted N includes some phospholipids, amino sugars, nucleotides, hippuric acid and orotic acid. The a-amino N includes free amino acids and small peptides almost a complete range of amino acids, including ornithine, has been identified in milk, but glutamic acid predominates. 

The ability to synthesize peptide bonds is an inherent propensity of hepatic cells and is a biological phenomenon associated with detoxification processes, in which the end products are generally water-soluble conjugates with amino acids. A classical example is the biosynthesis of hippuric acid by the conjugation of benzoic acid with glycine. 

After this introduction it would seem that no defined peptide bond had been produced prior to Fischers preparative work. However, he did not mention the experiments of Theodor Curtius (1857-1928) published from 1881 and 1882, who—unintentionally at first—had obtained and cleanly characterized benzoyl-glycylglycine (and higher homologs) by the interaction of benzoylchloride and the silver salt of hippuric acid (benzoylglycine). Fischer reported rather exactly on Curtius contributions to the development of peptide chemistry in his great... 

Most certainly, Curtius did not intend to enter protein chemistry when he started his work on hippuric acid and glycine. In the following years he was mainly fascinated by his discovery of diazo-fatty acid esters and the multitude of their reactions and so scarcely acquired interest in peptones and albuminoids. During the last decade of the past century, however, stimulated by E. Fischer s vigorous activity, Curtius resumed his studies, by which he contributed so much to modern peptide chemistry. It is somewhat ironical that Fischer s engagement, that had an immense echo at that time left less practical application for peptide chemistry than Curtius invention of the azide coupling method. Emil Fischer s great merit is to have drawn the attention of the whole scientific world to the field of proteins, whose mystery could be revealed by application of chemical methods and to lend trust to chemists that they are able to synthesize complicated natural substances like peptides. 

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). 

H. Chantrenne, Hippuric acid formation from glycine and dibenzoyl phosphate. Nature, 160, 603-604 (1947) Un modele de synthese peptidique. Proprietes due benzoylphosphate de phenyl. Biochim. Biophys. Acta 2 286-293 (1948) A new method of peptide synthesis. Nature 164 576-577 (1949) Peptide synthesis via glycyl phosphate. Biochim. Biophys. Acta 4 482-492 (1950)... 

Many investigators have studied the incorporation of labeled amino acids into proteins. The system involved in mammalian cells seems to be associated with microsomes, but requires factors from the soluble portion of the cell. Both ATP and GTP are required. The function of the ATP may be to form activated amino acids. Several systems have been found with relative specificity for various amino acids that catalyze the exchange of pyrophosphate with ATP in the presence of the amino acid, and presumably form adenyl-amino acid compounds. The activated amino acids react with hydroxylamine to form hydroxamic acids. Several reactions have been considered as models for peptide bond formation. These include the formation of hippuric acid, in which benzoyl CoA condenses with glycine in a reaction similar to the acetylation of... 

ATP is involved in the synthesis of hippuric acid and its analogs. The role of ATP is to form the aromatic acyl-Co A which then condenses with glycine to form the quasi-peptide. In the synthesis of glutamine from glutamic acid and ammonia Co A is not required, but ATP is. And in the synthesis of glutathione, the one case so far elucidated of an enzymatic... 

In animals hippuric acid is formed from benzoic acid and glycine which are joined together by a secondary amide linkage similar to a peptide link. The reaction has been well studied, ATP is required as an energy-donor and the benzoic acid must be activated by being first combined with coenzyme A. 

Although it has not yet been possible to study the synthesis of proteins in purified enzyme systems, a considerable number of model reactions which involve the formation of peptide or amide linkages have been investigated. These include the synthesis of glutamine, hippuric acid, acetylated amines, glutathione, and urea. It is of significance that every one of these model reactions, studied in isolated enzyme systems, has been shown to require ATP. In addition, two of these reactions, the formation of hippuric acid and the acetylation of amines, have been shown to require coenzyme A. 

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 ... 

Substituted-2-oxazoline-5-ones are extremely useful for the synthesis of a-aminoacids and peptides. A report on their formation using a water-soluble carbodi-imide for the dehydration-cyclization step has now appeared (Scheme 49). Previous methods for cyclization of hippuric acid (356) and its derivatives include use of AC2O, PX3, and DCC. The water-soluble carbodi-imide method gave a good yield and made work-up simpler. 

At the time ATP was implicated in the synthesis of hippuric acid, a more searching study had been undertaken independently by Cohen and McGilvery (38-40) of an analogous reaction, the synthesis of p-aminohippuric (PAH) acid from p-aminobenzoic acid (PAB) and glycine. PAB is a constituent of naturally occurring peptides (5,95). The discovery of the role of Co A in the synthesis of hippuric acid was made in Cohen s laboratory and was, indeed, led up to by the findings there on the synthesis of p-aminohippuric acid. 

Another important reaction is acylation, which yields again acid amides. The acetyl and benzoyl derivatives of glycine (the latter is called hippuric acid) occur naturally, in addition to a few other compounds of similar composition. If the acylating agent is another amino acid, the result is a peptide. 

The NBT-PABA test of pancreatic function is based on the hydrolysis, by chymotrypsin, of a synthetic tripeptide— N-benzoyl-l-tyrosyl-p-aminobenzoic acid. The tripeptide, variously called NBT-PABA, BTP, or bentiromide, is administered orally together with a test meal to stimulate pancreatic secretion. BTP is specifically hydrolyzed by chymotrypsin in the duodenum to release PABA, which is subsequently absorbed in the intestinal tract and metabolized in the liver to hippurate and to PABA glucuronide and PABA acetylate. These arylamines are then excreted by the Iddney. In the presence of low chymotrypsin, as found in pancreatic insufficiency, less peptide is hydrolyzed, and therefore less chromogen is excreted in the urine or found in serum. Thus the amount of PABA detected in serum or urine is an indirect measure of chymotrypsin activity in duodenal content. 

Protein Hydrolysates. Instead of ethyl hippurate, a peptic hydrolysate of ovalbumin was used as substrate for the resynthesis reaction (64). This substrate (300 mg) was dissolved in water, adjusted to pH 6.0 with NaOH and to 0.9 ml with additional water. An amino acid ester was added to produce a 22.2mM solution and the mixture preincubated at 37°C for 15 min. Papain (3 mg), dissolved in 0.1M L-cysteine (0.1 ml), was combined with the above-mentioned preincubation mixture and incubation carried out at 37°C. After 2 hr, 0.1N NaOH (10 ml) was added to stop the enzymatic reaction and the resulting solution allowed to stand for 3 hr to hydrolyze completely the remaining amino acid ester as well as the ester group from the peptide product. The free amino acid produced from the base-catalyzed hydrolysis of the amino acid ester was determined with an amino acid analyzer. The amount of the amino acid incorporated was obtained by subtracting the determined value from the initial concentration of amino acid ester. The data obtained with the same L-amino acid esters as used in the model experiment (above) are plotted along the ordinate of Figure 3. An excellent correlation is found between the data from the model experiment and those from this experiment using a protein hydrolysate. In Table III data are shown for the extent of covalent incorporation after 2 hr of various amino acid ethyl esters into the protein hydrolysate. There is a close relationship between... 

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