Proline, secondary amino

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. 

Ninhydrin Proline Yellow chromophore Simplified reaction between ninhydrin and secondary amino adds... 

Tris(2,2 -bipyridine)ruthenium(UI) complex ions (44) produce a chemiluminescence in the presence of amino acids in a FTA system. Amino acids containing secondary amino groups have the strongest response LOD 20 pmol for proline to 50 nmol for... 

The earliest report on such lactim ether formation was from Sammes [72JCS(P1)2494], who converted piperazine-2,5-dione to 2,5-diethoxy-3,6-dihydropyrazine (173) with an excess of triethyloxonium fluoroborate. Subsequently, Rajappa and Advani (73T1299) converted proline-based piperazine-2,5-diones into the corresponding monolactim ethers. The starting material was a piperazinedione in which one of the amino acid units was the secondary amino acid proline, and the other a primary amino acid. This naturally led to the regiospecific formation of a monolactim ether (169) (on O-alkylation) from the secondary amide, whereas the tertiary amide remained intact. This was later extended to piperazine-2,5-diones in which the secondary amino acid was sarcosine [74JCS(P 1)2122], leading to the monolactim ethers (170). 

Proline and hydroxyproline differ from the other amino acids in that they contain a secondary amino group in a pyrrolidine ring therefore, they do not produce aminoketones and Strecker aldehydes in the reaction with dicarbonyls, ffowever, nitrogen heterocyclics are produced, including 1-pyrroline, pyrrolidine, 2-acetyl-1-pyrroline and 2-acetyltetrahydropyridine (Scheme 12.5) [19]. 

Detection of amino acids is typically by UV absorption after postcolumn reaction with nin-hydrin. Precolumn derivatization with ninhydrin is not possible, because the amino acids do not actually form an adduct with the ninhydrin. Rather, the reaction of all primary amino acids results in the formation of a chromophoric compound named Ruhemann s purple. This chro-mophore has an absorption maximum at 570 nm. The secondary amino acid, proline, is not able to react in the same fashion and results in an intermediate reaction product with an absorption maximum at 440 nm. See Fig. 5. Detection limits afforded by postcolumn reaction with ninhydrin are typically in the range of over 100 picomoles injected. Lower detection limits can be realized with postcolumn reaction with fluorescamine (115) or o-phthalaldehyde (OPA) (116). Detection limits down to 5 picomoles are possible. However, the detection limits afforded by ninhydrin are sufficient for the overwhelming majority of applications in food analysis. 

The N-chlorosuccinimide is introduced by means of an auxiliary pumping system after the elution of glutamic acid and is removed after the elution of proline. Without this treatment, proline would not appear in the chromatographic results. The decrease in baseline is due to a change in the reagents and the total flow-rate of the cell. The limit of detection for secondary amino acids is ca. 0.25 nmole. 

All proteins are made up from the same set of 20 standard amino acids. A typical amino acid has a primary amino group, a carboxyl group, a hydrogen atom and a side-chain (R group) attached to a central a-carbon atom (Ca). Proline is the exception to the rule in that it has a secondary amino group. 

For the proline- and proline congener-catalyzed aldol reaction [23, 24], a mechanism based on enamine formation is proposed [25], Scheme 7. The catalytic process starts with condensation of the secondary amino group of proline with a carbonyl substrate leading to a nucleophilic enamine intermediate, which mimics the condensation of the active-site lysine residue with a carbonyl substrate in type I aldolases. The adjacent carboxylic acid group of the enamine intermediate... 

The R represents the side chain that is different for each of the amino acids that are commonly found in proteins. However, all 20 amino acids have a free carboxylic acid group and a free amino (primary amine) group, except proline which has a cyclic side chain and a secondary amino group. 

Ornithine (a) and hydroxyproline (d) are both a-amino acids, because an amino group is attached to the same carbon atom as that which carries a carboxyl. Although ornithine is not used in protein synthesis, it is an intermediate in the urea cycle (Chap. 15). /3-Alanine (b) and y-aminobutyrate (c) have their amino group attached to a carbon atom different from that bearing the carboxyl, and are a /3- and a y-amino acid, respectively. Strictly speaking proline is a secondary amino acid, because the amino nitrogen is covalently connected to the side chain. It is sometimes referred to as an imino acid. 

As the amino acids come off the column they are accessed by the ninhydrin reagent. The ninhydrin-amino acid complexes (Schemes 7.3 and 7.4) are detected spectrophotometrically at 570 nm (primary amino acids) and 440 nm (secondary amino acids, such as proline) and recorded as a series of peaks (Fig. 7.6). The retention time of the peak on the chart identifies the amino acid, the area under the peak indicating the quantity of the amino acid present. 

List ° and Barbas investigated the effect of different potential catalysts on the reaction of acetone with p-nitrobenzaldehyde (Table 6.11). The important conclusions drawn from this work are that primary and acyclic secondary amino acids do not catalyze the reaction. Cyclic secondary amino acids do catalyze the aldol reaction, the best being proline. Converting proline into either a tertiary amine (A -methylproline) or an amide destroys its catalytic behavior. It is clear that the catalyst must provide both basic and acidic sites. 

The participation of secondary amino acids, e.g., of proline, and the intermediate formation of quaternary iminolactones (CIII) (Craig, 1952)... 

When a Boc amino acid is being coupled to a N-tenninal proline, hydroxyproline, or other secondary amino group, the Kaiser test is negative or gives an inconclusive brownish color. Such N-terminal residues give a good blue color with isatin.P ... 

Amino acid analysis. There are some 20 amino acids found in proteins and these are released by overnight hydrolysis in 6M HCl. Plasma and urine contain an even larger number of amino acids or related compounds. At low pH, amino acids are cations and for 40 years have been separated by cation exchange column, chromatography. The problem with amino adds is that in general they possess no chromophores by which they can all be detected. In the traditional amino add analyser, their detection was accomplished by a post-column reaction with nin-hydrin which forms a purple colour on heating with an amino acid at pH 5.5. This colour, Ruhemann s purple, is formed with all primary amino acids and can be detected at 570 nm. Secondary amino acids such as proline form a yellow chromophore measurable at 440 nm. 

Only certain a-amino acids are present in any protein structure, and they have the general formula R-CH(NH2)-COOH, with the exception of proline and hydroxyproline that have secondary amino groups. With few exceptions, amino acids have chiral molecules with L (S) configuration at the chiral a carbon ... 

Amino acids with secondary amino groups, such as proline or iV-methylglycine, also generate active copper catalysts for the coupling of aryl iodides with aromatic amines, although the scope of this method for reactions of aromatic amines has not been explored in detail254. No examples of the coupling of arylamines with aryl bromides were reported. 

Secondary amino acids, imino acids , such as proline and hydroxyproline, do not possess an a-amino group, and react with ninhydrin to form a yellow product which is usually detected at 440 nm. Therefore, amino acid analyzers are equipped with a photometer capable of measuring at two different wavelengths (570 nm and 440 nm). The sensitivity of this detection method is about 200 pmol. 

Proteins are formed from a-amino acids which, with the exception of proline, have a primary amino group and a carboxylate group on the same carbon (proline is an a-imino acid, with a secondary amino group). With the exception of glycine, they all have the L (or R) conhguration, and are joined by peptide (amide) bonds (Figure 3.4) to form polypeptide chains. Since, at pH 7, they are present as dipolar ions, it follows that proteins... 

Note Proline is commonly called an imino acid, although it does not contain an imino group, C=NH, but a secondary amino group,... 

The stractures of the 20 most common naturally occurring amino acids and the frequency with which each occurs in proteins are shown in Table 23.1. Other amino acids occur in nature, but only infrequently. All amino acids except proline contain a primary amino group. Proline contains a secondary amino group incorporated into a five-mem-bered ring. The amino acids differ only in the substituent (R) attached to the a-carbon. The wide variation in these substituents (called side chains) is what gives proteins their great stractural diversity and, as a consequence, their great functional diversity. 

Proline, histidine, and tryptophan are heterocyclic amino acids. Proline has its nitrogen incorporated into a five-membered ring—it is the only amino acid that contains a secondary amino group. Histidine is an imidazole-substituted alanine. Imidazole is an aromatic compound because it is cyclic and planar and has three pairs of delocalized tt electrons (Section 21.11). The pA a of a protonated imidazole ring is 6.0, so the ring will be protonated in acidic solutions and nonprotonated in basic solutions (Section 23.3). 

The nitrosation of -amino acids is especially interesting because of the biological importance of N-nitrosoamines (see Sect. 4.2). The nitrosation mechanism was investigated first with amino acids containing a secondary amino group, namely proline (4.1, X = H), 4-hydroxyproline (4.1, X = OH), and sarcosine (4.2), but also with cysteine (4.3). 

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