Mechanisms Edman degradation

MECHANISM Edman Degradation—Cleavage of an A/-Teiminal Amino Acid... 

Figure 26.4 MECHANISM Mechanism of the Edman degradation for N-terminal analysis of peptides.

The critical feature of the Edman degradation is that it allows the N-terminal amino acid to be removed without cleaving any of the other peptide bonds. Let s see how this occurs. The mechanism of the reaction is shown in Figure 26.3. First the nucleophilic nitrogen of the N-terminal amino acid attacks the electrophilic carbon of phenyl isothiocyanate. When anhydrous HF is added in the next step, the sulfur of the thiourea acts as an intramolecular nucleophile and attacks the carbonyl carbon of the closest peptide bond. II is the intramolecular nature of this step and the formation of a five-membered ring that result in the selective cleavage of only the N-terminal amino acid. The mechanism for this part of the reaction is very similar to that for acid-catalyzed hydrolysis of an amide (see Section 19.5). However, because no water is present, only the sulfur is available to act as a nucleophile. The sulfur is ideally positioned for intramolecular attack at the carbonyl carbon of the N-terminal amino acid, so only this amide bond is broken. 

Show all of the steps for the mechanism of the first reaction in the Edman degradation ... 

Step 1 of the Edman degradation is the addition of the NH2 group of the N-ter-minal amino acid to the C=N double bond of phenyl isothiocyanate. Step 2 (B —> C) is an intramolecular SN reaction of an S nucleophile on the carboxyl carbon of a pro-tonated amide. It follows the substitution mechanism shown in Figure 6.5. The substitution product C is a heterocyclic derivative of the N-terminal amino acid. The simultaneously formed second reaction product, the oligopeptide D, which has been shortened by one amino acid, is ejected as the leaving group. 

Refer to Figure 26.2 and propose a mechanism for the final step in the Edman degradation—the acid-catalyzed rearrangement of the ATZ derivativ e to the PTH derivative. 

Edman degradation. Alanine amide was treated with phenyl isothiocyanate to form PTH-alanine. Write a mechanism for this reaction. 

The Sattger end-group determination is sometimes used as an alternative to the Edman degradation. In the Sanger method, a peptide is allowed to react with 2,4-dinitrofluorobensene, the peptide is hydrolyzed, and the N-terminal amino acid is identified by separation as its iV-2,4-dinitrophenyl derivative. Propose a mechanism to account for the initial reaction between peptide and dinitrofluorobenzene. 

A major advance was devised by Pehr Edman (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 Mechanism 25.3. 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 (Edman used nitromethane saturated with hydrogen chloride) to cleave the amide bond between the N-terminal amino 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 an internal nucleophile, and the only amide bond cleaved under these conditions is the one to the N-terminal amino acid. The product of this cleavage, called a thiazolone, is unstable under the conditions of its formation and rearranges to a phenylthiohydantoin (PTH), which is isolated and identified by comparing it with standard samples of PTH derivatives of known amino acids. This is normally done by chromatographic methods, but mass spectrometry has also been used. 

Edman Degradation (Section 27.4B) Treatment with phenyl isothiocyanate followed by acid removes the N-terminal amino acid as a substituted phenylthiohydan-toin, which is then separated and identified. The mechanism involves reaction of the electrophilic C atom of phenyl isothiocyanate with the nucleophilic terminal amino group to give an N-phenylthiourea intermediate that decomposes upon heating by cy-clization to give a thiazoKnone intermediate as the C-terminal peptide bond is cleaved. The thiazolinone intermediate isomerizes to the phenylthiohydantoin product. 

Mechanism 28.2 illustrates some of the key steps of the Edman degradation. The nucleophilic N-terminal NH2 group adds to the electrophilic carbon of phenyl isothiocyanate to form an A-phenylthiourea, the product of nucleophilic addition (Part [1]). Intramolecular cyclization followed by elimination results in cleavage of the terminal amide bond in Part [2] to form a new peptide with one fewer amino acid. A sulfur heterocycle, called a thiazolinone, is also formed, which rearranges by a multistep pathway (Part [3]) to form an A-phenylthiohydantoin. The R group in this product identifies the amino acid located at the N-terminal end. 

As shown in Mechanism 28.2, the final steps in the Edman degradation result in rearrangement of a thiazolinone to an A/-phenylthiohydantoin. Draw a stepwise mechanism for this acid-catalyzed reaction. 

Mechanism ofthe Edman degradation for N-terminai anaiysis of peptides. 

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