Amino acid hydroxyproline

The many (possibly more than 30) types of collagens found in human connective tissues have substantially the same chemical structure consisting mainly of glycine with smaller amounts of proline and some lysine and alanine. In addition, there are two unusual amino acids, hydroxyproline and hydroxylysine, neither of which has a corresponding base-triplet or codon within the genetic code. There is therefore, extensive post-translational modification of the protein by hydroxylation and also by glycosylation reactions. 

Darifenacin 63 is Pfizer s treatment for urinary urge incontinence. Disconnection at the C-N bond with some amine synthesis in mind (chapter 8) gives a much smaller heterocycle 64 that can again be disconnected in the middle with the idea of alkylating some enolate such as 65 with the derivative of an alcohol 66. This is attractive because 66 is available as a single enantiomer cheaply from the amino acid hydroxyproline.19... 

Question The amino acids hydroxyproline and hydroxylysine are absent from Table 17.1, which describes the genetic code. How do these amino acids arise in some proteins ... 

Recent studies on separation optimization showed that accurate control of mobile phase pH was essential to successfully resolve a number of important non-hydrolysate amino acids. With good control of a complex gradient profile the system could resolve a mixture of amino acids including, Asn, Gin, cysteine derivatives carboxymethyl cysteine and pyridylethyl cysteine, and the hydroxylated amino acids hydroxyproline (Hyp) and hydroxylysine (Hyl) as well as the hydrolysate amino acids (4). However, the required precision in the control of eluent pH unnecessarily complicated transfer of the method between laboratories. The method also lacked the ability to separate Orn from the hydrolysate amino acids. The current study demonstrates the utility of quaternary HPLC gradient systems for facilitating methods development and simplifying routine eluent preparation with excellent pH control. 

Reflect and Apply The amino acid hydroxyproline is found in collagen. There is no codon for hydroxyproline. Explain the occurrence of this amino acid in a common protein. 

Amino acids other than the usual 20 are produced by modification of one of the common amino acids. See Figure 3.5 for the structures of some modified amino acids. Hydroxyproline and hydroxylysine are found in collagen thyroxine is found in thyroglobulin. 

Arginine is > 5% of the cell amino acids. Histidine is > 5% of the cell amino acids. Phenylalanine is > 5% of the cell amino acids. Tyrosine is > 5% of the cell amino acids. Proline is > 5% of the cell amino acids. Hydroxyproline is > 5% of the cell amino acids. 

Collagens are the main proteins of connective tissues and constitute about 30 per cent of the total proteins in the mammalian body. As mentioned earlier (p. 54), the amino acid hydroxyproline is an important component of collagen. Hydroxylation of proline to hydroxyproline involves vitamin C if this vitamin is deficient, collagen fibres are weakened and may give rise to gum and skin lesions (see p. 100). The indispensable amino acid tryptophan is not found in these proteins. 

Several adequate methods for hydroxyproline are available. Generally, a method for detection of protein-bound hydroxyproline is required, inasmuch as collagenases break down collagen into peptides and not free amino acids. Hydroxyproline assays are useful on many occasions, such as providing quantitative determinations of the degree of lysis of collagen gel plates. 

Hydroxylated amino acids (eg, 4-hydroxyproline, 5-hydroxylysine) and A/-methylated amino acids (eg, /V-methylhistidine) are obtained by the acid hydrolysis of proteins. y-Carboxyglutamic acid occurs as a component of some sections of protein molecules it decarboxylates spontaneously to L-glutamate at low pH. These examples are formed upon the nontranslational modification of protein and are often called secondary protein amino acids... 

Thin-Layer Chromatography (tic). Tic (126) is used widely for quahtative analysis and micro-quantity separation of amino acid mixtures. The amino acids detected are developed by ninhydrin coloring, except for proline and hydroxyproline. Isatia has been recommended for specific coloring of pToline (127). 

Figure 14.1 Each polypeptide chain in the collagen molecule folds into an extended polyproline type II helix with a rise per turn along the helix of 9.6 A comprising 3.3 residues. In the collagen molecule three such chains are supercoiled about a common axis to form a 3000-A-long rod-like molecule. The amino acid sequence contains repeats of -Gly-X-Y- where X is often proline and Y is often hydroxyproline. (a) Ball and stick model of two turns of one polypeptide chain.

Fig. 1 Reflectance scan (A) and fluorescence scan (B) of a chromatogram track with 50 ng of each amino acid per chromatogram zone hydroxyproline (1), proline (2).

The chromatograms stained with ninhydrin are immersed in the reagent solution for 1 s or sprayed evenly with it and then placed in the free half of a twin-trough chamber containing 25% ammonia solution. Apart from proline and hydroxyproline, which yield yellow copper complexes, all the amino acids yield reddish-colored chromatogram zones [3],... 

Ornithine, proline, hydroxyproline, pipecolic acid and sarcosine yield red zones, glycine greenish-brown and the other amino acids weakly brown ones [3]. The colors of the zones are different if an alcoholic solution of potassium carbonate is used for basification instead of dipping solution II. 

The tendencies of the amino acids to stabilize or destabilize a-helices are different in typical proteins than in polyamino acids. The occurrence of the common amino acids in helices is summarized in Table 6.1. Notably, proline (and hydroxyproline) act as helix breakers due to their unique structure, which fixes the value of the —N—C bond angle. Helices can be formed from either... 

Early examples of enantioselective extractions are the resolution of a-aminoalco-hol salts, such as norephedrine, with lipophilic anions (hexafluorophosphate ion) [184-186] by partition between aqueous and lipophilic phases containing esters of tartaric acid [184-188]. Alkyl derivatives of proline and hydroxyproline with cupric ions showed chiral discrimination abilities for the resolution of neutral amino acid enantiomers in n-butanol/water systems [121, 178, 189-192]. On the other hand, chiral crown ethers are classical selectors utilized for enantioseparations, due to their interesting recognition abilities [171, 178]. However, the large number of steps often required for their synthesis [182] and, consequently, their cost as well as their limited loadability makes them not very suitable for preparative purposes. Examples of ligand-exchange [193] or anion-exchange selectors [183] able to discriminate amino acid derivatives have also been described. 

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. 

The amino acids proline and hydroxyproline exert a stabilizing influence on the triple helix as described in detail in Sect. 4.5. By examining the CB peptides of collagen, a structural stability which is directly proportional to the itnino acid content may thus be found. It has, however, not been possible to synthesize model peptides displaying structural stability comparable to that of the native peptides having corresponding amino acid contents. 

Note It is possible to differentiate amino acids by color on the basis of the markedly different shades produced [2, 3]. Proline and hydroxyproline, that only react weakly with ninhydrin, also yield pink-red colored derivatives [2]. Ergot alkaloids and LSD are detected by spraying with 10% hydrochloric add and then heating to 110°C for 20 min after they have been treated with the reagent [9]. Ergot alkaloids and LSD yield red to purple zones when treated in this manner other alkaloids, e.g. reserpine, emetine, quinine, strychnine, pilocarpine, atropine, scopolamine, cocaine and opium alkaloids, do not give a reaction [9]. 

Schoeninger, M.J. and DeNiro, M.J. 1984 Nitrogen and carbon isotopic composition of bone collagen from marine and terrestrial animals. Geochimica et Cosmochimica Acta 48 625-639. Schuette, S. A., Hegsted, M., Zemel, B. and Linkswiler, H.M. 1981 Renal acid, urinary cyclic AMP, and hydroxyproline excretion as affected by level of protein, sulfur amino acids and phosphorus intake. Journal of Nutrition 111 2106-2116. 

Table 1 presents the chemical composition and some properties of both gums reported by Osman et al., 1993 and Williams Phillips, et al., 2000. Despite having different protein content, amino acid composition is similar in both gums. Recently, Mahendran et al., 2008, reported the GA amino acid composition in Acacia Senegal, being rich in hydroxyproline, serine, threonine, leucine, glycine, histidine. Table 2. 

Fig. 4. Helical structure of collagen, typical amino acid sequence within a collagen strand, and exchangeable versus non-exchangeable hydrogen atoms in an individual leucine molecule (Gly - glycine. Pro - Proline, Leu - leucine. Hyp - hydroxyproline.

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