Phenylthiohydantoine

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). 

Step 3 Once formed, the thiazolone derivative isornerizes 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 Edrnan degradation. 

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. 

The ATZ rearranges in the presence of aqueous acid to an isomeric AZ-phenylthiohydantoin (PTH) as the final product. 

Apply sample solution followed by heptafluoro- [100] butync acid, heat to 140 °C for 10 min, rapidly cool to room temperature and develop the phenylthiohydantoins that are formed No reaction occurs with, for example, threomne, senne, tryptophan or glutamic aad... 

Hayakawa, K., Hirano, M., Yoshikawa, K., Katsumata, N, and Tanaka, T., Separation of phenylthiohydantoin-amino acids by temperature-controlled reversed-phase high-performance liquid chromatography, /. Chromatogr. A, 846, 73, 1999. 

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...

Reaction with phenylisothiocyanate (PITC) in alkaline conditions produces stable phenylthiocarbamyl (PTC) adducts which can be detected either in the ultraviolet below 250 nm or electrochemically. However, this method involves a complex derivatization procedure and offers poorer sensitivity than the alternatives available for individual amino acids. It is useful, however, in conjunction with the automated analysis of peptides when single derivatized residues can be cleaved and analysed after conversion in acidic conditions to phenylthiohydantoins. 

Neben der quantitativen Bestimmung der freien Aminosauren spielt auch die quantitative Analyse der N-2,4-Dinitrophenylaminosiiuren und der AminosiLure-Phenylthiohydantoine eine Rolle (vgl. II/4 und Gas-... 

Die Aminosaure-Phenylthiohydantoine (kurz PTH-Aminosauren) wer-den abgetrennt und qualitativ odcr quantitativ identifiziert. Der Rest des Peptides wird vor neuerlicher Reaktion mit I oftmals quantitativ analysiert. 

Sequence analysis is one of the most useful tools for determining disulfide connectivities by detecting the phenylthiohydantoins (PTH) of either cystine or cysteine derivatives. Sequencing can be carried out on the intact peptide, although most frequently disulfide-bridged fragments are subjected to this type of analysis. 

Finally, in a third stage — conversion — the unstable thiazolinone is converted into a phenylthiohydantoine (PTH) derivative which can be identified. 

So far, only the coupling and the cleavage reactions have been automated. Future developments might provide automated conversion of the liberated thiazolinone derivatives into phenylthiohydantoins and their automated identification. Alternatively, mass spectrometry which has as yet rarely been employed for identification may be applied directly to the thiazolinones dispensing altogether with the conversion step... 

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