Peptide reaction with phenylisothiocyanate

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. 

Even more versatile than the dansyl method is the Edman method (Figure E2.4). The NH2-terminal amino acid is removed as its phenylthiohydan-toin (PTH) derivative under anhydrous acid conditions, while all other amide bonds in the peptide remain intact. The derivatized amino acid is then extracted from the reaction mixture and identified by paper, thin-layer, gas, or high-performance liquid chromatography. The intact peptide (minus the original NH2-terminal amino acid) may be isolated and recycled by reaction with phenylisothiocyanate. Since this method is nondestructive to the remaining peptide (aqueous acid hydrolysis is not required) and results in good yield, it can be used for stepwise sequential analysis of peptides. The method is now automated. 

The N-terminal residue, i.e., the first amino acid in the sequence of a peptide, can be determined by reaction with phenylisothiocyanate. At neutral pH, this compound reacts with the a-amino group. After mild acid hydrolysis, the reaction product cydizes, releasing the terminal residue as a phenylthiohydantoin (PTH) derivative (the Edman degradation. Fig. 4-1). The derivative can be analyzed to determine its parent amino acid and its quantity. 

The sequencing of a peptide (26) uses the well-known Edman degradation (27) of peptides, as shown in Fig. 2.2. The reaction cycle consists of reaction with phenylisothiocyanate followed by treatment with anhydrous TFA to promote cycliza-tion of the intermediate thiourea. Rearrangement induced by treatment with aqueous TFA produces the phenylthiohydantoin (PTH) of the N-terminal amino acid 2.2 and the tmncated peptide 2.3. The sequence is repeated through n cycles until the whole peptide is degraded. The PTHs produced are detected by HPLC-UV and their retention times are compared to those of 20 standard PTHs, one for each natural amino acid. 

Figure 5-2. Chemistry of the Edman degradation. In the Edman degradation, peptides undergo reaction with phenylisothiocyanate which generates a phenylthiocarbamylpeptide adduct. This adduct is cleaved to release the...

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... 

Dansyl chloride and phenylisothiocyanate (PITC) are the derivatizating agents most used in UV detection. Dansyl chloride reacts with the primary and secondary amino groups of peptides in a basic medium (pH 9.5), forming dansylated derivatives that are very stable to hydrolysis but are photosensitive. The derivatives are detectable in UV at 254 nm and by fluorescence. Dansyl sulfonic acid is formed as a by-product of the reaction, and excess reagent reacts with the dansyl derivatives to form dansyl amide the conditions of derivatization must therefore be optimized in order to avoid the formation of such by-products to the extent possible. The conditions of the reaction with dansyl chloride and of the separation of the derivatives thus formed have been thoroughly studied (83,84). Martin et al. (85) carried out derivatization using an excess concentration of dansyl chloride of 5 -10-fold in a basic medium (lithium carbonate, pH 9.5) in darkness for 1 h. 

This reaction takes place in three stages. First, the free amino group of the N-terminal amino acid reacts with phenylisothiocyanate to form a phenylthiourea. Second, the phenylthiourea cyclizes to a thiazolinone and expels the shortened peptide chain. Third, the thiazolinone isomerizes to the more stable phenylthiohydantoin. 

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 ... 

When the peptide is treated with phenylisothiocyanate, reaction at the amino terminus occurs to produce the corresponding thiourea derivative of the peptide. 

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 ... 

It might be noticed that under acidic conditions (i.e., anhydrous trifluoro-acetic acid) the hydroxyl (protonated) becomes a leaving group, and so it is expected an amine (amide linkage) could also leave. Thus, the reaction of a protein with phenylisothiocyanate provides an important method for the determination of the N-terminal amino acid and subsequent determination of the amino acid sequence of the protein. Remembering that the monomeric units of the protein are connected by amide (so-called peptide) linkages, this may be illustrated for the simple dipeptide glycylalanine. 

Figure 4-6. The Edman reaction. Phenylisothiocyanate derivatizes the amino-terminal residue of a peptide as a phenylthiohydantoic acid. Treatment with acid in a nonhydroxylic solvent releases a phenyithiohydantoin, which is subsequently identified by its chromatographic mobility, and a peptide one residue shorter. The process is then repeated.

If the phenylisothiocyanate method is used, the cyclization and release of the N-terminal derivative occur under mild conditions that leave the rest of the chain intact. It is therefore possible to repeat the procedure on this shortened peptide to determine the second residue in the sequence, and so on. Like most chemical reactions, Edman degradation is not 100% efficient, meaning that at each step there is a chance of additional peptide hydrolysis or incomplete reaction thus the method is not reliable for peptides with more than 60 residues. However, this can be overcome by generating a series of peptides by specific protease pretreatment (see next section) and then separating the peptide fragments prior to using Edman analysis. 

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