Phenylthiocarbamoyl derivatives

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. 

The examples mentioned here for RP-HPLC analy sis of monosaccharides in the form of perbenzoates and amino adds as V-phenylthiocarbamoyl derivatives (Table 1) satisfy both prindpal criteria introducing the chromophores into molecules of the analytes... 

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) ... 

Figure 9. The formation of phenylthiocarbamoyl derivatives of amino acids.

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. 

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 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... 

Dihydrazide 231 was also used in the synthesis of thienothiophenes containing triazole substituents. For example, the reaction with phenyl isothiocyanate in ethanol produces di(A -phenylthiocarbamoyl)carbohydrazide 238, whose treatment with KOH followed by acidification affords compound 239. Diazotization of dihydrazide 231 with 10% sodium nitrite in acetic acid gives diazide 240. The reactions of the latter with compounds containing the active methylene group, viz., ethyl cyanoacetate, malononitrile or diethyl malonate, in the presence of sodium ethoxide, yield the corresponding ditriazole derivatives 241-243. 

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... 

Derivatization with PITC may alternatively be used to detect primary and secondary amino acids as the phenylthiocarbamoyl (PTC) derivatives (Figure 9) [58-62]. The PITC derivatization reagent, made up fresh each day, consists of ethanol/triethylamine/water/PITC (7 1 1 1 by vol) and is stored at —20 °C under nitrogen to prevent degradation. The derivatization procedure is identical for all dried samples (standard mixtures, hydrolysates, serum and urine extracts). PITC reagent (100 pi) is mixed throughly with the dried sample and allowed to react for 5 min at room temperature. The mixture is evaporated to dryness under vacuum and the... 

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