Peptides stepwise degradation

The stepwise chemical degradation of peptide chains with Edman s method represents one of the truly significant additions to the inventory of methods in modem biochemistry. Without this procedure the rapid development within recent years of our knowledge about stmcture and function of proteins would have been impossible (see Attempts based on different chemical reactions have not yet yielded practical methods 56-62)... 

The reaction is stoichiometric rather than catalytic and goes to completion within a few minutes at pH 7-8 at 60°-65°C. When four equivalents of chelate react with tetraglycine, four equivalents of the glycine-metal complex are formed. In view of the specificity as well as the rapidity of the reaction under rather mild conditions, it is evident that metal chelates of this type may prove useful for stepwise degradation of peptides. 

Acid treatment of the substituted thioureas resulting from the treatment of esters, amides, or peptides of a-amino acids (10) with isothiocyanates (usually phenyl isothiocyanate) also affords 2-thiohydantoins (11). These reactions furnish the basis of the Edman s method for the stepwise degradation of peptides.3... 

A modification of the stepwise degradation of peptides involves their N-terminal cyclization with N,7V -carbonyl- or N,N -thiocarbonyldiimid-azole.120 Acetylation of N-hydroxyphenylurethane (67) yields the diester 68, which by simply heating in refluxing ethanol furnishes the JV-acetoxy-hydantoin 69.121... 

Several partially reduced pyrrolo[l,2-a]quinoxalines have been obtained as derivatives of proline in studies on the stepwise degradation of peptides. These compounds (25) are obtained when a peptide (22) with an N-terminal proline residue is condensed with an appropriately activated aryl halide, and the resultant N-aryl derivative 23 or 24 is cyclized with expulsion of the peptide residue 26. 

P. Edman, Determination of the amino acid sequence in peptides. Arch. Biochem. 22 475-476 (1949) also Method for the determination of the amino acid sequence in peptides. Acta Chem. Scand. 4 283-293 (1950) Stepwise degradation of peptides via phenylthiohydantoins. Acta Chem. Scand. 7 700-701 (1953)... 

Edman, Pehr Victor, 1916-1977 (pp. 118,240, Plate 15) born in Stockholm in 1916, matriculation examination in 1935, studied medicine at the Karolinska Institute Medical School in Stockholm from 1935, Bachelor of Medicine in 1938, graduation as a physician in 1946. Concurrently with his studies in medicine he started his training in biochemistry with Erik Jorpes, for a short time also with Hugo Theorell, and soon started a project on angiotensin that led to a MD thesis. Then he widened his experience in protein chemistry during one year at the Rockefeller Institute in Princeton with Northrop and Kunitz (crystallization of proteolytic enzymes). On his return to Sweden, Edman was awarded an associate professorship in Lund in 1947 where he conducted his stepwise peptide degradation work (p. 118) between 1950 and 1956. In 1957 Pehr Edman accepted an offer to be Director of Research at St. Vincent s School of Medical Research in Melbourne, Australia, where he remained for 15 years, during which the work on an automated sequence analyzer was finished in 1967. From 1972 until his death from a brain tumor in 1977 he was Director of the Department of Protein Chemistry of the Max-Planck-Institute for Biochemistry in Martinsried near Munich. 

The classical method is the Edman degradation reaction. It involves stepwise degradation of peptides with phenylisothiocyanate (cf. 1.2.4.2.3) or suitable derivatives, e. g. dimethylaminoa-zobenzene isothiocyanate (DABITC). The resultant phenylthiohydantoin is either identified directly or the amino acid is recovered. The stepwise reactions are performed in solution or on peptide bound to a carrier, i.e. to a sohd phase. Both approaches have been automated ( sequencer ). Carriers used include resins containing amino groups (e. g. amino polystyrene) or glass beads treated with amino alkylsiloxane ... 

Bergmann has employed the azides of acylated amino acids for the stepwise degradation of peptides, as illustrated by the conversion of hippurylalanine to hippuramide and acetaldehyde. Hippurylalanine is converted through its ester and hydrazide to its azide, which is heated with benzyl alcohol to give 1-hippuramido-l-carbobenzoxyaminoethmie. This upon hydrogenation is cleaved to hippuramide mid acetaldehyde. ... 

Hans Fischer and his students, has been adapted to the stepwise degradation of peptides. The advantage of this method is that the conditions can be made almost completely non-hydrolytic. Benzyl urethans also appear to be more readily hydrolyzed by conventional methods than methyl and ethyl urethans. ... 

Glycyl-dl-phenylalanine allowed to react 25 min. at 65° and pH 7.5 with cis-hydroxoaquotriethylenetetraminecobalt(III) ions in aq. soln. -> dl-phenyl-alanine. Y ca. 100%.—The N-terminal aminoacid moiety is selectively hydrolyzed and converted into an inert metal complex. This reaction may prove useful for the stepwise degradation of peptides. F. e. s. J. P. Collman and D. A. Buckingham, Am. Soc. 85, 3039 (1963). 

The Edman degradation permits one to remove the N-terminal amino acid in the form of a hydantoin derivative the procedure may then be repeated on the remaining peptide, which is thus degraded stepwise. 

In contrast to the stepwise addition of amino acids to a protein, much of the semisynthesis of proteins has been concerned with the covalent combination of synthetic peptides with natural proteins or their partially degraded fragments. 

Peptides containing serine or threonine may undergo an N—>0 acyl shift upon exposure to strong acids (Scheme 40). 592,594 This reaction has been exploited in the structure elucidation of cyclosporin A, since the acid-catalyzed acyl shift with formation of an ester allowed its selective hydrolysis to the linear peptide for further stepwise degradation. 593 ... 

The present article will be devoted in its first chapters to the chemical basis of the stepwise degradation procedure and to a comparative description of design and function of the two instruments, and in its later chapters to the results so far achieved. Finally, present limitations of the automated method as well as novel approaches to the sequence analysis of peptides will be discussed. 

Several enzymes catalyze stepwise removal of amino acids from one or the other end of a peptide chain. Carboxypeptidases232 remove amino acids from the carboxyl-terminal end, while aminopeptidases attack the opposite end. Using chromatographic methods, the amino acids released by these enzymes may be examined at various times and some idea of the sequence of amino acids at the chain ends may be obtained. A dipeptidyl aminopeptidase from bovine spleen cuts dipeptides one at a time from the amino terminus of a chain. These can be converted to volatile trimethylsilyl derivatives and identified by mass spectrometry.233 If the chain is shortened by one residue using the Edman degradation (Section 3) and the dipeptidyl aminopeptidase is again used, a different set of dipeptides that overlaps the first will be obtained and a sequence can be deduced. Carboxypeptidase Y can be used with MALDI mass spectrometry to deduce the C-terminal amino acid sequence for a peptide. However, He and Leu cannot be distinquished. 

One reason for an otherwise apparently excessive interest in Co(trien)X2+ systems is the use of ds-Co(OH)(trien)(OH2)2+ in the hydrolysis of amino acid esters, amino acid amides and peptides785 to form cis-px- and cis-/J2-Co(trien)(aa)2+ (aa = amino acid) complexes.16 In principle, a peptide could be degraded in a stepwise manner and each amino acid residue successively characterized. By the introduction of a chiral center into the backbone of the trien moiety, it was hoped to make such reactions stereoselective. Consequently, while fully A-alkylated trien systems have been widely investigated for M11 central ions, the C-alkylated trien systems have been almost exclusively the reserve of the Co111 chemist (Table 11). 

The primary structure (i.e., the amino acid sequence) of a protein can be determined by stepwise chemical degradation of the purified protein. By far the most powerful and commonly used technique for doing this is the automated Edman degradation. The amino terminal amino acid residue of the polypeptide is reacted with Edman s reagent (phenylisothiocyanate) to form the phenylthiocar-bamyl derivative, which is removed without hydrolysis of the other peptide bonds by cyclization in anhydrous acid. The amino acid derivative is converted to the more stable phenylthiohydantoin and identified by HPLC. The process can be repeated many times, removing the amino acids from the amino terminus of the polypeptide one residue at a time and identifying them until the entire sequence... 

Transport peptides can be synthesized using either t-Boc or Fmoc solid phase peptide synthesis strategies with a synthesizer or manually. We routinely synthesize CPPs in a stepwise manner on solid support using an Applied Biosystems Model 431A peptide synthesizer. tert-Butyloxycarbonyl amino acids are coupled as 1-hydroxybenzotriazole (HOBt) esters to a p-methylbenzylhydryl-amine (MBHA) resin (65). C-terminally amidated CPPs are less prone to degradation and show higher internalization efficiency than carboxylic acid derivatives. 

Edman s method of stepwise degradation (Edman, 1949, 1950) (Scheme 5.3) involves reaction of the a-amino group at the TV-terminus of a peptide with phenyl isothiocyanate under slightly basic conditions. Excess reagent is extracted and the... 

Unfortunately, the 3-phenyl-2-thiohydantoins formed in the Edman stepwise degradation suffer racemisation (Davies and Mohammed, 1984) so that the method cannot be used to determine the configuration of amino acids in a peptide. This is not usually a serious limitation, but enantiomerisation is a perpetual hazard in peptide synthesis (Chapter 7). It is therefore desirable to determine if enantiomerisation has occurred at any residue. Such information could be important, for example, in dating bone proteins obtained in archaeological excavations. Examination of the chiral purity of the amino acids in a total acid hydrolysate is not satisfactory. 

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