Phenylthiohydantoin derivative

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 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... 

In the paper chromatographic systems which were employed it was not possible to discriminate clearly between the phenylthiohydantoin derivatives of leucine and isoleucine. 

Thin-layer chromatography of amino acid-phenylthiohydantoin derivatives on silica gel plates, (a) Separation is done in a 98 2 mixture of chloroform and ethanol. (b) This is followed by further separation using an 88 2 10 mixture of chloroform, ethanol, and methanol. More sophisticated procedures, using column chromatography, give superior resolution and improved sensitivity. Automated sequencers always use such procedures. A general description of the use of columns is given in chapter 6. 

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 phenylthiohydantoin derivative is identified by chromatography, by comparing it with phenylthiohydantoin derivatives of the standard amino acids. This gives the identity of the original N-terminal amino acid. The rest of the peptide is cleaved intact, and further Edman degradations are used to identify additional amino acids in the... 

The first two steps in sequencing oxytocin. Each Edman degradation cleaves the N-terminal amino acid and forms its phenylthiohydantoin derivative. The shortened peptide is available... 

The first 34 residues of human parathyroid hormone (PTH) [190] were identified from the Cl mass spectra of their phenylthiohydantoin derivatives by repeated Edman degradations on a Beckmann Sequencer [163]. In a similar way as part of the characterisation of ovine hypothalmic luteinizing hormone-releasing factor (LRF), the trimethylsilyi derivatives of the phenylthiohydantoins from Edman degradation were confirmed by MS [191]. 

Automated Edman degradation of protein and peptide samples were performed using an Applied Biosystems sequencer (Model 470A or 477A) or a Hewlett Packard G1005A sequencer. Each sequencer was fitted with an on-line HPLC analyzer for the identification of phenylthiohydantoin derivatives. 

Contained ala + gly + cys + glu + arg + ile + N HT Carboxypeptidase A liberated isoleucine Treatment with phenylisothiocyanate (PITC, the Edman reagent) yielded the phenylthiohydantoin derivative of glycine (PTH-glycine)... 

In the Edman procedure, PITC reacts under basic conditions with the free a-amino group to form a phenylthiocarbamoyl peptide (Figure 3-9). Treatment with anhydrous acid yields the labeled terminal amino residue plus the remainder of the peptide. In this process, the terminal amino acid is cyclized to the corresponding phenylthiohydantoin derivative (PTH-amino... 

Phenylthiohydantoin derivative of N-terminal amino acid (PTH-amino acid)... 

Amino acid Methylthiohydantoin derivatives Phenylthiohydantoin derivatives ... 

Draw the structure of the phenylthiohydantoin derivatives of (a) alanine (b) valine (c) lysine (d) proline... 

Edman degradation A method for removing and identifying the N-terminal amino acid from a peptide without destroying the rest of the peptide chain. The peptide is treated with phenyliso-thiocyanate, followed by a mild acid hydrolysis to convert the N-terminal amino acid to its phenylthiohydantoin derivative. The Edman degradation can be used repeatedly to determine the sequence of many residues beginning at the N terminus, (p. 1176)... 

The TLC method was developed for amino acids therefore, in principle, it is equally applicable to peptides. Peptides, such as amino acids, are generally hydrophilic. There are, however, limits to this analogy. Dinitrophenylamino acid derivatives (DNP amino acids) and phenylthiohydantoin derivatives (PTH amino acids) are obtained when reaction of peptides or proteins with dinitrofluorobenzene or phenyl mustards are properly degraded. Their separation from reaction mixtures and their identification are considerable practical importance because they constitute essential steps... 

Cyclic degradation of peptides based on the reaction of phenylisothiocyanate with the free amino group of the TV-terminal residue such that amino acids are removed one at a time and identified as their phenylthiohydantoin derivatives ... 

PROBLEM 17.16 Write the structure of the phenylthiohydantoin derived from the first cycle of Edman degradation of Phe—Ala—Ser. 

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