Phenylthiocarbamoyl peptides

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... 

Step 1 A peptide is treated with phenyl isothiocyanate to give a phenylthiocarbamoyl (PTC) derivative... 

A major advance was devised by Pehr Edrnan (University of Lund, Sweden) that has become the standard method for N-terminal residue analysis. The Edman degradation is based on the chemistry shown in Figure 27.12. A peptide reacts with phenyl isothiocyanate to give a phenylthiocarbamoyl (PTC) derivative, as shown in the first step. This PTC derivative is then treated with an acid in an anhydrous medium (Edrnan used nitrornethane saturated with hydrogen chloride) to cleave the amide bond between the N-terminal anino acid and the remainder of the peptide. No other peptide bonds are cleaved in this step as amide bond hydrolysis requires water. When the PTC derivative is treated with acid in an anhydrous medium, the sulfur atom of the C=S unit acts as... 

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. 

Phenylthiocarbamoyl derivatives of 18 chiral amino acids were separated on a C8 column connected in series to a phenylcarbamoylated (3-cyclodextrin column (Iida et al., 1997). The Cg column separated the derivatized amino acids from one another entering the chiral column. Under this configuration several enantiomers of adjacent amino acids coeluted resulting in poor resolution. However, this configuration was successful in determining the amino acid sequence and chirality of the amino acids in a D-amino acid containing peptide. 

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... 

Numerous chemical derivatization strategies have been developed also to direct the fragmentation reactions of protonated peptides toward the selective formation of single characteristic sequence-type product ions [85-87]. For example, Sununerfield and coworkers have demonstrated that N-terminal derivatization, with phenyliso-thiocyanate to form the corresponding phenylthiocarbamoyl (PTC) derivative, results in exclusive fragmentation of the amide bond between the first two amino... 

Summerfield, S.G. Bolgar, M.S. Gaskell, S.J. Promotion and stabilization of ions in peptide phenylthiocarbamoyl derivatives analogies with condensed-phase chemistry. J. Mass Spectrom. 1997, 32, 225-231. 

The new N-terminal residue can then be removed as a phenylhydantoin in a second cycle of phenylcarbamoylation and cyclization. This method of stepwise degradation, while discovered several decades earlier (Bergmann et al. 1927), reached practicality only in 1950 when Edman modified the reagent and applied chromatographic procedures for identification of the cyclic products. The improved reagent, phenylisothiocyanate, smoothly converts the peptide to the phenylthiocarbamoyl derivative which is cyclized and cleaved by the action of hydrochloric acid (dissolved in an organic solvent such as dioxane) ... 

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