Hydroxyproline oxidation

Hydroxyproline assay. Hydroxyproline was determined according to Jamall et al. (1981). The assay employs p-dimethylaminobenzaldehyde (Ehrlich s reagent), which forms colored products with pyrroles originating from hydroxyproline oxidation. The values thus determined for hydroxyproline mass were multiplied by 8.0 to obtain the corresponding collagen mass. 

B) Isatin chromophore of hydroxyproline oxidation products [Lang, 1933 (L3) McFarlane and Guest, 1939 (M2)]... 

Knowledge of hydroxyproline oxidation is even more advanced than that for proline. Formation of a carbonyl compound on enzymic oxidation of L-hydroxyproline had been observed by Taggart and Krakaur 173), and it was postulated to be y-hydroxyglutamic-y-semialdehyde in equilibrium with A -pyrroline-3-hydroxy-5-carboxylic acid. 

The advantages of this method are a short reaction time and the nonfluorescence of the OPA reagent. Therefore, excess reagent must not be removed before the chromatography stage. Using this method, it is possible to measure tryptophan, but not secondary amino acids such as proline or hydroxyproline. Cysteine and cystine can be measured, but because of the low fluorescence of their derivatives, they must be detected using an UV system, or alternatively oxidized to cysteic acid before reaction. 

Homochiral hydroxyproline 139 served as the starting material for the synthesis of various bicyclic[5.5]hydantoins 140 (Scheme 19). The corresponding amino derivatives 142 were also available by oxidation of the alcohol 141 and reductive amination of the ketone followed by separation of diastereomers by silica gel column chromatography (Equation 19) <2005BML1161>. 

Primary amino acids will react with o-phthalaldehyde in the presence of the strongly reducing 2-mercaptoethanol (pH 9-11) to yield a fluorescent product (emission maximum, 455 nm excitation maximum, 340 nm). Peptides are less reactive than a-amino acids and secondary amines do not react at all. As a result, proline and hydroxyproline must first be treated with a suitable oxidizing agent such as chloramine T (sodium A-chloro-p-toluene-sulphonamide) or sodium hypochlorite, to convert them into compounds which will react. Similarly cystine and cysteine should also be first oxidized to cysteic acid. 

Collagen determination. In order to calculate the amount of collagen in a sample, hydroxyproline was measured in acid hydrolyzates (Jamall et al., 1981). After oxidation, hydroxyproline reacts with p-dimethyl-aminobenzaldehyde to give a red colour, which is measured by spectrophotometry. For bovine dentin, collagen mass was estimated as 8.0 x the hydroxyproline value as calculated from previous data (Volpin and Veis, 1973). To calculate the molar quantities of collagen, a molecular mass of 300 kDa was used. 

Ascorbic acid or vitamin C is found in fruits, especially citrus fruits, and in fresh vegetables. Man is one of the few mammals unable to manufacture vitamin C in the liver. It is essential for the formation of collagen as it is a cofactor for the conversion of proline and lysine residues to hydroxyproline and hydroxylysine. It is also a cofactor for carnitine synthesis, for the conversion of folic acid to folinic acid and for the hydroxylation of dopamine to form norepinephrine. Being a lactone with two hydroxyl groups which can be oxidized to two keto groups forming dehydroascorbic acid, ascorbic acid is also an anti-oxidant. By reducing ferric iron to the ferrous state in the stomach, ascorbic acid promotes iron absorption. 

Most of the fluoro derivatives of proline described in the literature are fluorinated in the 4 position. 4-Fluoroprolines are able to mimic 4-hydroxyproline, present in some proteins and polypeptides. On the other hand, 4-fluorination could suppress oxidative metabolism or modified ring conformation of ligands.The labeled analogues may be used as probes in PET (positron emission tomography) for localization of tumors... 

An early example of this type of transformation was described in 1964 by Drehfahl and Horhold (310). These authors prepared racemic 4-hydroxyproline, albeit with low diastereoselectivity for the isoxazoline reduction step [Scheme 6.75, (1)] (310). Much higher selectivity was achieved using 5-halomethylisoxazolines bearing a 3-(l-oxyalkyl) side chain, which was introduced from the nitrile oxide portion. The examples presented in Scheme 6.75 outline the synthesis of 4-hydroxy-pyrrolidines, which contain a dioxyethyl or trioxypropyl side chain. Both of these substrates were converted into the corresponding imino acids of 4-hydroxyproline (23,225,234) and 4, 1 -dihydroxyhomoproline, respectively (23,207,225) (Eq. 2, 3). The latter compound is part of an N-Val dipeptide structure, that was mistakenly proposed for the antibiotic Tii 1718B (311,312). 

Several deadly species of the genus Amanita produce colorless toxic octapeptides, the amani-tins.a b Two residues of glycine, one of L-isoleucine, one of the unusual L-dihydroxyisoleucine, one of L-asparagine, and one of L-hydroxyproline are present in a-amanitin. In the center a modified tryptophan residue has been combined oxidatively with an SH group of a cysteine residue. If the dihy-droxyisoleucine residue of a-amanitin is replaced with unhydroxylated leucine, the resulting compound, known as amanullin, is nontoxic. The LD50 for mice is 0.3 mg kg 1 and 50 g of fresh Amanita phalloides may be sufficient to kill a person. Arnan-itins act slowly, and it is impossible to kill mice in less than 15 h, no matter how high the dose. 

Ninhydrin Reaction. A neutral solution of an amino acid will react with ninhydrin (triketohydrindene hydrate) by heating to cause oxidative decarboxylation. The central carbonyl of the tnketone is reduced to an alcohol. This alcohol further reacts with ammonia formed from the amino add and causes a red-purplish color. Since the reaction is quantitative, measurement of the optical density of the color produced is an indication of amino add concentration. Imino acids, such as hydroxyproline and proline, develop a yellow color in the same type of reaction. 

In an attempt to learn whether collagen hydroxyproline synthesized in granuloma minces incubated with tritiated proline contained tritium at the 4 position, we converted such tritiated hydroxyproline to the corresponding N-carbobenzoxy derivative and then oxidized this derivative with chromic acid to N-carbobenzoxy-4-ketoproline according to the procedure of Patchett and Witkop(29). In most of these experiments very little tritiated water was formed although some tritiated water was detected in several experiments, the small amount formed could logically be attributed to adventitious oxidative reactions or perhaps to enolization of ketoproline. The evidence was therefore consistent with the absence of tritium at carbon atom 4 of the isolated hydroxyproline. 

According to this proposal, proline is converted to collagen pro-line and collagen hydroxyproline by two separate and distinct pathways. Proline destined to become hydroxyproline is oxidized to a bound hydroxyproline intermediate, which does not equilibrate significantly with free hydroxyproline and is not yet in peptide linkage with proline. The marked drop in the specific activity of hydroxyproline in scorbutic collagen may then be explained by the accumulation of an intermediate or intermediates in the hydroxyproline pathway. Such accumulation would... 

Hydroxyproline is a very unusual amino acid. There is no genetic codon for the insertion of Hyp into a growing protein because collagen is not made that way. The collagen molecule is first assembled with Pro where Hyp ends up. Then some proline residues are oxidized to... 

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