Secondary amino acids, proline

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). 

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 search for improved pre-column derivatives for amino acid analysis is a continuing process. Dansyl derivatives are stable and have been used for amino acid analysis, but hydrolysis products (dansyl OH) of the dansyl reagent are difficult to eliminate and can interfere with the dansyl amino acid peaks (15). FMOC-Cl (9-fluor-enylmethyl chloroformate) is another derivative that has been used and that shows promise for the analysis of the secondary amino acids proline and hydroxyprollne as well as the primary amino acids (16). 

Another compound obtained from 3-deoxyosone via a relatively complex reaction is maltoxazine (VII in Formula 4.59), which has been identified in malt and beer. This compound could be formed from 3,4-dideoxyosone, which first undergoes a Streaker reaction with the secondary amino acid proline with decarboxylation to give the 1-pyrroline derivative (Formula 4.65). Enolization, formation of a five-membered carbocyclic compound and nucleophilic addition of the hydroxymethyl group to the pyrroline cation yields the tricyclic maltoxazine. In general, the formation of such carbocyclic compounds is favored in the presence of secondary amino acids like proline. 

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 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. 

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)... 

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. 

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. 

The proteins of the body are made up of some combination of twenty different subunits called a-amino acids. The general structure of an a-amino acid is shown in Figure 19.1. We find that nineteen of the twenty amino acids that are commonly isolated from proteins have this same general structure they are primary amines on the a-carbon. The remaining amino acid, proline, is a secondary amine. 

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). 

In the above examples, L-a-amino acids induce the natural 13P-chirality. Amines or amino acid derivatives (esters, amides) are much less effective, and a tertiary amino acid, hygrinic acid, was ineffective. For trione 13a, secondary amino acids (e.g., proline) are best for 13b and 13c [R > H, see also the Danishevsky (Scheme 8) and Tsuji (Scheme 10) syntheses below], a primary amino acid (e.g., L-phenylalanine) is preferred. The mechanism of the reaction has not yet been clarified. ... 

There is a relationship between the primary structures, or the amino acid content of many proteins, and the secondary structures [27]. The helical contents are inversely proportional to the amount of serine, threonine, valine, cysteine, and proline in the molecule. Conversely [28], valine, isoleucine, serine, cysteine, and threonine are non-helix-forming amino acids. Proline, due to its specific configuration, actually disrupts the helical structure when it is present in the polypeptide [29]. In addition, proteins that are composed of low ratios of polar to nonpolar amino acids have a tendency to aggregate [30]. Also, the globular protein, will, in an aqueous environment, tend to form shapes with nonpolar groups located inside the stmcture. This is due to the thermodynamic nature of the hydrophobic side chains. The polar ones, on the other hand, tend to be located outside, toward the water [31]. 

Globular proteins are mainly helical, but they have folds that permit the overall shape to be globular. One of the 20 amino acids, proline has a secondary amino group. Wherever a proline unit occurs in the primary peptide structure, there will be no N—H group available for intra-chain hydrogen bonding. 

The results of the labeling experiments with a primary and a secondary amino acids indicate that in both systems, same reactive fragments, interact similarly with the amino acids but produce structurally unrelated products (for example pyrazinone vs l,2-(r,T-dipyrrolidinyl)-l-propene) due to the nature of the secondary amino group of proline that prevents further cyclizations. 

A series of bradykinin analogs containing the simple secondary amino acid, sarcosine, in place of the cyclic secondary structure, proline, was prepared to determine whether modification of the biologic activity of bradykinin might be related to steric factors. 2-Sarcosine bradykinin was the most active analog in increasing hind limb blood flow in the dog, although its activity was at most only about 30% that of synthetic bradykinin. The 3-sarcosine derivative of bradykinin... 

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