Phenylthiohydantoin, Edman

Edman degradation (Section 27 13) Method for determining the N terminal amino acid of a peptide or protein It in volves treating the material with phenyl isothiocyanate (CgH5N=C=S) cleaving with acid and then identifying the phenylthiohydantoin (PTH derivative) produced Elastomer (Section 10 11) A synthetic polymer that possesses elasticity... 

Mass spectral fragmentation patterns of alkyl and phenyl hydantoins have been investigated by means of labeling techniques (28—30), and similar studies have also been carried out for thiohydantoins (31,32). In all cases, breakdown of the hydantoin ring occurs by a-ftssion at C-4 with concomitant loss of carbon monoxide and an isocyanate molecule. In the case of aryl derivatives, the ease of formation of Ar—NCO is related to the electronic properties of the aryl ring substituents (33). Mass spectrometry has been used for identification of the phenylthiohydantoin derivatives formed from amino acids during peptide sequence determination by the Edman method (34). 

FIGURE 5.19 N-Tertninal analysis using Edman s reagent, phenylisothiocyanate. Phenylisothiocyanate combines with the N-terminus of a peptide under mildly alkaline conditions to form a phenylthiocarbamoyl substitution. Upon treatment with TFA (trifluo-roacetic acid), this cyclizes to release the N-terminal amino acid residue as a thiazolinone derivative, but the other peptide bonds are not hydrolyzed. Organic extraction and treatment with aqueous acid yield the N-terminal amino acid as a phenylthiohydantoin (PTH) derivative. 

Figure 7.5 The Edman degradation method, by which the sequence of a peptide/polypeptide may be elucidated. The peptide is incubated with phenylisothiocyanate, which reacts specifically with the N-terminal amino acid of the peptide. Addition of 6 mol l-1 HCl results in liberation of a phenylthiohydantoin-amino acid derivative and a shorter peptide, as shown. The phenylthiohydantoin derivative can then be isolated and its constituent amino acid identified by comparison to phenylthiohydantoin derivatives of standard amino acid solutions. The shorter peptide is then subjected to a second round of treatment, such that its new amino terminus may be identified. This procedure is repeated until the entire amino acid sequence of the peptide has been established...

To sequence an entire polypeptide, a chemical method devised by Pehr Edman is usually employed. The Edman degradation procedure labels and removes only the amino-terminal residue from a peptide, leaving all other peptide bonds intact (Fig. 3-25b). The peptide is reacted with phenylisothiocyanate under mildly alkaline conditions, which converts the amino-terminal amino acid to a phenylthiocarbamoyl (PTC) adduct. The peptide bond next to the PTC adduct is then cleaved in a step carried out in anhydrous trifluo-roacetic acid, with removal of the amino-terminal amino acid as an anilinothiazolinone derivative. The deriva-tized amino acid is extracted with organic solvents, converted to the more stable phenylthiohydantoin derivative by treatment with aqueous acid, and then identified. The use of sequential reactions carried out under first basic and then acidic conditions provides control over... 

Ingenious protein sequenators have been devised to carry out the Edman degradation automatically.242 244-246 Each released phenylthiohydantoin is then identified by HPLC or other techniques. Commercial sequenators have often required 5-20 nmol of peptide but new microsequenators can be used with amounts as low as 5-10 picomoles or less.247 248... 

Step 3 Once formed, the thiazolone derivative isomerizes to a more stable phenylthiohydantoin (PTH) derivative, which is isolated and characterized, thereby providing identification of the N-terminal amino acid. The remainder of the peptide (formed in step 2) can be isolated and subjected to a second Edman degradation. 

Gas chromatography is used to analyze volatile derivatives of amino acids. Phenylthiohydantoins (products of Edman degradation) may be analyzed directly by GC but are better resolved if converted to their trimethylsilyl derivatives with N, O-bis(trimethylsilyl) acetamide. Free amino acids are generally converted to their 7V-trifluoroacetyl- -butyl esters or trimethylsilyl derivatives before GC analysis. For best results, all gas chromatography of amino acid derivatives should be done with a glass column and injection port, as contact with metals causes extensive decomposition of the derivatives. 

The Edman degradation method for polypeptide sequence determination. The sequence is determined one amino acid at a time, starting from the amino-terminal end of the polypeptide. First the polypeptide is reacted with phenylisothiocyanate to form a polypeptidyl phenylthiocarbamyl derivative. Gentle hydrolysis releases the amino-terminal amino acid as a phenylthiohydantoin (PTH), which can be separated and detected spectrophoto-metrically. The remaining intact polypeptide, shortened by one amino acid, is then ready for further cycles of this procedure. A more sensitive reagent, dimethylaminoazobenzene isothiocyanate, can be used in place of phenylisothiocyanate. The chemistry is the same. 

The formation of methyl- (MTH) or phenylthiohydantoin (PTH) amino acids is a valuable technique for sequencing of amino acids in peptides and proteins by the Edman degradation procedure [1], HPLC is very useful for the separation of MTH- or PTH-amino acids as adsorption [2,3], reversed-phase [4] and ion-exchange [S] chromatography. 

In 1991, we first introduced the one-bead one-compound (OBOC ) combinatorial library method.1 Since then, it has been successfully applied to the identification of ligands for a large number of biological targets.2,3 Using well-established on-bead binding or functional assays, the OBOC method is highly efficient and practical. A random library of millions of beads can be rapidly screened in parallel for a specific acceptor molecule (receptor, antibody, enzyme, virus, etc.). The amount of acceptor needed is minute compared to solution phase assay in microtiter plates. The positive beads with active compounds are easily isolated and subjected to structural determination. For peptides that contain natural amino acids and have a free N-terminus, we routinely use an automatic protein sequencer with Edman chemistry, which converts each a-amino acid sequentially to its phenylthiohydantoin (PTH) derivatives, to determine the structure of peptide on the positive beads. 

The structure of peptides containing 20 eukaryotic natural amino acids is now routinely determined by the use of automatic protein microsequencer, which uses Edman chemistry to convert each a-amino acid sequentially to its phenylthiohydantoin (PTH) derivative. The formed PTH-amino acids can be identified by their retention times on HPLC systems by comparison with reference standards derived from the 20 natural amino acids. For an OBOC peptide library composed of natural amino acids, the sequencing protocols of the automatic sequencer are well developed and standardized. However, structure determination of peptides composed of unnatural a-amino acids requires modification of the standard sequencing program.32 For peptides composed of non-a-amino acids, one can use an encoding strategy or mass spectrometry if a cleavable linker is employed. In this chapter, we shall focus on the new sequencing method we have developed for unnatural a-amino acids. 

The Edman degradation removes the N-terminal amino acid, which is identified as a phenylthiohy-dantoin derivative. The first Edman degradation of Val-Phe-Gly-Ala gives the phenylthiohydantoin derived from valine the second gives the phenylthiohydantoin derived from phenylalanine. 

The first cycle of the Edman degradation of Phe-Ala-Ser will give the phenylthiohydantoin of Phe because it is the N-terminal amino acid. 

When insulin is subjected to the Edman degradation, the N-terminal amino acids of the A and B chains (glycine and phenylalanine, respectively) will both be converted to their phenylthiohydantoins. Their structures are ... 

The N-terminal amino acid of a protein can be determined by reacting the protein with dansyl chloride or fluorodinitrobenzene prior to acid hydrolysis. The amino acid sequence of a protein can be determined by Edman degradation which sequentially removes one residue at a time from the N terminus. This uses phenyl isothiocyanate to label the N-terminal amino acid prior to its release from the protein as a cyclic phenylthiohydantoin amino acid. 

The most useful method of N-terminal analysis is called the Edman degradation. This method allows the N-terminal amino acid to be removed and its identity to be determined without hydrolyzing the other peptide bonds. The reaction initially produces a thiazolinone, which is rearranged by aqueous acid to a phenylthiohydantoin for identification by high-performance liquid chromatography ... 

Lequin and Niall [282] described GC analysis of more volatile analogues, pentafluoro-phenylthiohydantoins, which were prepared by modifying the Edman degradation using pentafluorophenylisothiocyanate as a reagent. Except for Arg and His, the derivatives of all amino acids could be chromatographed and separated satisfactorily in a simple column (1.22 m X 2 mm I.D.) packed with either 10% of DC-560 or 2% of OV-25 on Chromo-... 

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