Terminal Amino Acids in Peptides and

The identification of N-terminal amino acids in peptides and proteins is of considerable practical importance because it constitutes an essential step in the process of sequential analysis of peptide structures. 

Carboxypeptidase A catalyzes the hydrolysis of the C-terminal amino acids in peptides and proteins it shows a preference for aromatic and branched aliphatic side chains at the C-terminal end. The enzyme also acts as an esterase. The X-ray structure of the native form of carboxypeptidase A, was solved by Rees and Lipscomb to 1.5 A... 

The identification of N-terminal amino acids in peptides and proteins is of considerable practical importance because it constitutes an essential step in the process of sequential analysis of peptide structures. Many N-amino acid derivatives have been proposed for this purpose and the ones most commonly studied by TLC are 2,4-dinitrophenyl (DNP)-and 5-dimethylaminonaphthalene-l-sulfonyl (dansyl, Dns)-amino acids, and 3-phenyl-2-thiohydantoins (PTH-amino acids). Recently, 4-(dimethylamino)azobenzaie-4/-isothio-cyanate (DABITC) and phenyl-isothiocyanate (PITC) have also been investigated as derivatizing agents of amino acids. 

The high reactivity of di- and trinitrophenyl fluorides towards nucleophiles has been used for the arylation of various N-nucleophiles. A method was developed for the determination of N-terminal amino acids in peptides. Thus, nucleophilic attack of the amino acid nitrogen at Sanger s reagent (2,4-dinitrophenyl fluoride, 4), hydrolysis and subsequent analysis of the N-(2,4-dinitrophenyl)amino acid allows determination of the amino acid.162,163 Although this method has been replaced by more efficient procedures, it marked a milestone in the elucidation of peptide structures (Nobel Prize 1958). A variety of N-nuclcophilcs (no amino acids) which have been used in the nucleophilic substitution of 2,4-dinitrophcnyl fluoride is listed. 

Hitherto we have concentrated on electrophilic aromatic substitution. However, certain n-deficient aromatic rings are deactivated towards electrophilic attack but are susceptible to nucleophilic addition and a subsequent elimination. A particular example is 2,4-dinitrochloroben-zene. The electron-withdrawing nitro groups facilitate a Michael-type addition of a nucleophile to give a so-called Meisenheimer intermediate (Scheme 4.8). Collapse of the Meisenheimer intermediate and reversion to the aromatic system may lead to expulsion of the halide ion, as exemplified by the preparation of 2,4-dinitrophenylhydrazine. 2,4-Dinitrofluorobenzene is known as Sanger s reagent and is used for the detection of the N-terminal amino acids in peptides. 

Co(in) complexes promote similar reactions. When four of the six octahedral positions are occupied by amine ligands and two cis positions are available for further reactions, it is possible to study not only the hydrolysis itself, but the steric preferences of the complexes. In general, these compounds catalyze the hydrolysis of N-terminal amino acids from peptides, and the amino acid that is removed remains as part of the complex. The reactions apparently proceed by coordination of the free amine to cobalt, followed either by coordination of the carbonyl to cobalt and subsequent reaction with OH or H2O from the solution (path A in Figure 12-15) or reaction of the carbonyl carbon with coordinated hydroxide (path B). As a result, the N-terminal amino acid is removed from the peptide and left as part of the cobalt complex in which the a-amino nitrogen and the carbonyl oxygen are bonded to the cobalt. Esters and amides are also hydrolyzed by the same mechanism, with the relative importance of the two pathways dependent on the specific compoimds used. 

Halogen bound to an aromatic ring is rendered considerably more reactive by nitro groups in the para- or, especially, the ortho-position to it carboxyl, sulfonyl, and cyano groups have considerably less effect. Among such activated halo aromatic compounds the relative reactivities of the halogens are reversed, and fluorine is much the most reactive. This is used for, e.g.9 determination of A-terminal amino acids in peptides by means of l-fluoro-2,4-dinitrobenzene.547 The ease of reaction is illustrated in the following example ... 

These compounds are obtained in acid medium via dimethylaminoazobenzenethiocarbamyl (DABTC) derivatives formed by the reaction in basic medium of DABITC with the primary amino group of N-terminal amino acids in peptides. The color differences among DABITC, DABTC derivatives, and dimethylaminoazobenzenethiohydantoin (DABTH)-amino acids facilitate identification on TLC. These derivatives are colored compounds and, because of their stability and sensitivity, are usually used for qualitative and quantitative analyses of amino compounds such as amino acids and amines. 

The reader is also referred to the article by S. W. Fox in vol. 2, pp. 155-177, Advances in Protein Chemistry, in which he discusses certain reactions including aryl sulfonylation of terminal amino acids of peptides and proteins. 

Plaquet et al. (PI) found in the urine of rachitic children peptides consisting of proline, hydroxyproline, and glycine, which they believed to be the products of collagen degradation. Two similar peptides containing considerable amounts of proline and hydroxyproline were isolated from the urine of a patient with rheumatoid arthritis by Mechanic et al. (Ml). One of these peptides consisted of three proline, two hydroxyproline, and nine glutamic acid residues, the second one consisted of four proline, four hydroxyproline, and one glutamic acid residues. The N-terminal amino acid in the first peptide was demonstrated to be hydroxyproline. 

S ATP -I- peptide <3> (<3> synthetic [9,13,14] <3> e.g. Leu-Glu-Glu-Ser-Ser-Ser-Ser-Asp-His-Ala-Glu-Arg-Pro-Pro-Gly or Arg-Arg-Arg-Glu-Glu-Glu-Glu-Glu-Ser-Ala-Ala-Ala, role of acidic amino acids in peptide substrates, preference for negatively charged amino acids localized to the N-terminal side of a Ser- or Thr-residue, Ser-containing peptides are 4fold better than Thr-containing [9] <3> /1-ARK 1 and 2 prefer peptide substrates with acidic amino acids N-terminal to a Ser-residue [13] <3> /)-ARK 1 prefers peptides containing acidic residues on the N-terminal side of a serine or threonine, presence of activated receptor enhances peptide phosphorylation [14]) (Reversibility <3> [9,13,14]) [9, 13, 14]... 

Identify the N-terminal and the C-terminal amino acid in the original peptide and in each fragment. 

Sequential analysis of amino acids in purified peptides and proteins is best initiated by analysis of the terminal amino acids. A peptide has one amino acid with a free a-amino group (NH2-terminus) and one amino acid with a free a-carboxyl group (COOH-terminus). Many chemical methods have been developed to selectively tag and identify these terminal amino acids. 

A related reaction is that of 2,4-dinitrofluorobenzene with the amino groups of peptides and proteins, and this reaction provides a means for analysis of the N-terminal amino acids in polypeptide chains. (See Section 25-7B.)... 

The retention times of peptides with fewer than 20 residues in reversed-phase chromatography can be predicted with a high degree of accuracy based on their amino acid composition and the characteristics of their N-terminal and C-terminal amino acids. A number of researchers (66 -75) have studied the role of amino acids in peptide retention and have established retention coefficients for the different amino acids. The retention coefficient value of each amino acid is normally calculated by regression analysis of the retention times for peptides of known composition. 

Carboxy terminal amino acid or peptide thiols are prepared from various p-amino alcohols by conversion into a thioacetate (R2NHCHR1CH2SAc) via a tosylate followed by saponification.Several methods have been used to prepare N-terminal peptide thiols, the most common procedure is the coupling of (acetylsulfanyl)- or (benzoylsulfanyl)alkanoic acids or add chlorides with a-amino esters or peptide esters, followed by deprotection of the sulfanyl and carboxy groups. 8 16 Other synthetic methods include deprotection of (trit-ylsulfanyl)alkanoyl peptides, 1718 alkaline treatment of the thiolactones from protected a-sulfanyl acids, 19 and preparation of P-sulfanylamides (HSCH2CHR1NHCOR2, retro-thior-phan derivatives) from N-protected amino acids by reaction of P-amine disulfides with carboxylic acid derivatives, followed by reduction. 20,21 In many cases, the amino acid or peptide thiols are synthesized as the disulfides and reduced to the corresponding thiols by the addition of dithiothreitol prior to use. 

The three orthogonally removable lysine protecting groups we use here are Fmoc (9-fluorenyl methoxy carbonyl), cleavable with 20% base, preferably with 20% piperidine or 3% DBU (l,8-diazabicyclo[5.4.0]undec-7-ene) (18), Dde (1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-ethyl), cleavable with 2% hydrazine, and Aloe (allyloxy carbonyl), cleavable with palladium. Hydrazine also removes Fmoc, and thus it can be applied only if no Fmoc groups are present on the growing peptide chain. Acid-sensitive amino protecting groups available are Boc (tert-butyloxy carbonyl), cleavable with 90% TFA, and Mtt (4-methyl-trityl), cleavable with 1% TFA. We use Boc for the protection of the N-terminal moieties of N-terminal amino acids in each peptide chain as well as at the N-terminus of the scaffold. 

Although all proteinogenic amino acids form predominantly anti peptide bonds, a search in the Brookhaven Protein Database revealed that approximately 6-7% of all X-prolyl peptide bonds are found in the syn conformation in the native state of proteins [8]. The reason for this relatively frequent occurrence of syn-prolyl peptide bonds lies in steric repulsion of the proline 3 protons and the adjacent N-terminal amino acid in the anti conformation, resulting in a low barrier of rotation and energetically similar syn and anti isomers (Figure 1.2.3). 

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