Peptide Sequencing: Edman Degradation: Videos & Practice Problems

Edman degradation is a vital chemical method used for sequencing peptides from the N-terminal end. The primary reagent in this process is Phenylisothiocyanate (PITC), which plays a crucial role in the sequencing mechanism. Each cycle of Edman degradation consists of three key reactions that cleave one amino acid residue from the peptide chain.

The first step is nucleophilic addition, where PITC reacts with the N-terminal amino group of the peptide, resulting in the formation of a thiourea or urea derivative. This reaction is essential as it sets the stage for the subsequent steps.

In the second step, known as cyclization and cleavage, the thiourea derivative undergoes cyclization to form a thioazolinone derivative. During this process, the N-terminal amino acid is cleaved from the peptide chain, effectively shortening the peptide.

The final step involves rearrangement through acidic hydrolysis. The thioazolinone derivative rearranges to produce a more stable phenylthiohydantoin (PTH) derivative. This PTH derivative is crucial for identifying the internal amino acid, which can be achieved through chromatographic techniques.

Overall, Edman degradation provides a systematic approach to peptide sequencing, allowing for the identification of amino acid sequences through the careful manipulation of chemical reactions involving PITC and the resulting derivatives.

In the process of determining the structure of the PTH (phenylthiohydantoin) derivative formed from the Edmond degradation of a tetrapeptide, it is essential to identify the amino acids involved. The tetrapeptide begins with Tyrosine, which has a specific side chain that plays a crucial role in the formation of the derivative.

Tyrosine's side chain, or R group, consists of a methylene carbon (–CH₂) linked to a phenolic group (–C₆H₄OH). When constructing the PTH derivative, this R group must be attached to the thiohydantoin structure. The attachment of Tyrosine's R group to the thiohydantoin is what ultimately yields the final PTH derivative.

Thus, the final structure of the PTH derivative incorporates the methylene carbon and the phenolic group from Tyrosine, resulting in a unique compound that reflects the original tetrapeptide's composition.

In the context of Edman degradation, the initial phase involves nucleophilic addition, which can be likened to the reaction of an amino group with phenylisothiocyanate (PITC). This process begins with a nucleophilic attack, where the amino group, specifically the nitrogen atom, utilizes its lone pairs to target the carbon atom of the PITC. This interaction resembles the behavior of a carbonyl group, although it is important to note that the nitrogen cannot form five bonds. Consequently, the bond connecting the nitrogen to the carbon breaks, allowing the nitrogen to form four bonds while acquiring an additional lone pair. This results in a structure where the nitrogen carries a positive charge due to its four bonds, while the carbon becomes negatively charged due to the shift of the pi bond.

Following this, the second step involves a proton transfer. In this step, a proton (H⁺) is transferred from the protonated amino group to the negatively charged nitrogen of the PITC. This transfer results in both nitrogen atoms having one hydrogen atom and retaining a lone pair. The overall outcome of these two steps in the nucleophilic addition process is the formation of a new structure that balances the charges, leading to a more stable configuration. Thus, the nucleophilic addition in Edman degradation can be effectively summarized as comprising a nucleophilic attack followed by a proton transfer, both of which are crucial for the successful progression of the reaction.

In the context of nucleophilic addition reactions involving peptides and phenyl isothiocyanate (PITC), the process begins with the nucleophilic attack of the amino group from the N-terminal amino acid on the carbon atom of PITC. This initial step is crucial as it sets the stage for subsequent reactions.

Following the nucleophilic attack, a resonance-stabilized ion is formed. In this scenario, the nitrogen atom, which carries a negative charge, is bonded to a carbon that is double-bonded to sulfur. This configuration allows for resonance, where the negative charge can delocalize, enhancing the stability of the intermediate formed.

Next, a proton transfer occurs, leading to the formation of a thiourea derivative. This step is significant as it results in the conversion of the initial nucleophilic addition product into a more stable thiourea structure, which is a common outcome in reactions involving isothiocyanates.

However, it is important to note that an unlikely step in this process would be the nucleophilic attack of the sulfur atom of PITC on the carbonyl group of the N-terminal amino acid. This is not a typical pathway for nucleophilic addition, as the initial attack is specifically carried out by the amino group on the carbon, not the sulfur. Therefore, the correct identification of the unlikely step in this reaction is essential for understanding the mechanism of nucleophilic addition involving PITC.

In the Edmond degradation, reaction 2 involves the formation of a thioazolelionone derivative through a nucleophilic acyl substitution mechanism, which can be broken down into five key steps: protonation, nucleophilic attack, proton transfer, loss of the leaving group, and deprotonation.

In the first step, the carbonyl oxygen of the N-terminal amino acid undergoes protonation. This process involves the addition of a proton (H⁺) to the carbonyl oxygen, resulting in a positively charged intermediate where the oxygen forms three bonds.

Step two features an intramolecular nucleophilic attack that leads to the formation of a cyclic tetrahedral intermediate. The positively charged oxygen, seeking to stabilize itself, facilitates a nucleophilic attack by sulfur on the carbonyl carbon. This action breaks the pi bond, allowing the nitrogen to form a new pi bond, ultimately creating a five-membered cyclic structure.

In step three, a proton transfer occurs, enhancing the leaving group ability of the peptide chain. The nitrogen, now making four bonds and carrying a positive charge, transfers a proton to become neutral, thus improving its capacity to leave in the subsequent step.

Step four involves the hydroxyl group (OH) pushing its electrons to reform the carbonyl, which results in the expulsion of the peptide chain. The oxygen, now making three bonds, becomes positively charged again as it forms a double bond with the carbonyl carbon.

Finally, in step five, the carbonyl oxygen is deprotonated, yielding the final thioazolelionone derivative. The nitrogen utilizes its lone pair to capture the proton, allowing the oxygen to stabilize as a neutral carbonyl group.

Through these five steps, the reaction successfully transforms the initial substrate into the desired derivative, illustrating the intricate mechanisms of nucleophilic acyl substitution in organic chemistry.

In the context of thiazolidin formation during the Edman degradation process, it is essential to understand the sequence of reactions involved. The formation of thiazolidin occurs through an intramolecular nucleophilic attack, which is a critical step in the reaction mechanism. This step is followed by the formation of a tetrahedral intermediate, which indeed requires a proton transfer to convert into thiazolidin. This proton transfer is a necessary part of the process, confirming its validity.

However, a common misconception arises regarding the timing of the cleavage of the N-terminal amino acid from the peptide chain. It is incorrect to state that this cleavage occurs before the ring formation. Instead, the intramolecular attack must take place first to facilitate the cyclization into thiazolidin, after which the peptide chain can be cleaved. This highlights the importance of the reaction order in the Edman degradation process.

Additionally, the nucleophilicity of sulfur compared to nitrogen plays a significant role in the reaction. The sulfur atom from phenyl isothiocyanate (PITC) is indeed more nucleophilic than the nitrogen atom, primarily due to its larger atomic size, which allows it to better stabilize the negative charge during the nucleophilic attack on the carbonyl group. This characteristic further supports the validity of the reaction mechanism.

In summary, the incorrect statement regarding the Edman degradation process is that the N-terminal amino acid is cleaved before the ring formation occurs. Understanding the correct sequence of these reactions is crucial for grasping the overall mechanism of thiazolidin formation.

The rearrangement of thiazolidin to thiohytonin occurs through a series of four key steps: ring opening, tautomerization, rotation, and ring closure. The process begins with the ring opening, which is facilitated by an acid-catalyzed nucleophilic acyl substitution (NAS) hydrolysis reaction. In this step, the carbonyl oxygen is protonated by the acid catalyst, represented as H₃O⁺. This protonation activates the carbonyl carbon for nucleophilic attack by water, leading to the breaking of the pi bond and the formation of a positively charged intermediate with two hydroxyl (–OH) groups.

To stabilize this intermediate, another water molecule deprotonates the positively charged carbonyl oxygen, resulting in the formation of a carboxylic acid and a thiol group (–SH) from the protonation of sulfur. The next step, tautomerization, involves the conversion of the intermediate into an enol-like form, where the sulfur is double-bonded to carbon, transitioning to a keto form with a double bond to sulfur.

Following tautomerization, rotation occurs around the carbon-nitrogen bond, allowing the structure to adjust and prepare for the final step. The last step is ring closure, which also involves an acid-catalyzed intramolecular NAS reaction. Here, the carbonyl oxygen is again protonated, leading to a positively charged oxygen that facilitates cyclization. The nitrogen atom attacks the carbonyl carbon, resulting in the formation of a cyclic structure.

During this cyclization, a proton transfer occurs, allowing the migration of a hydrogen atom to one of the hydroxyl groups, which generates a water molecule as a leaving group. The carbonyl group is reformed as the oxygen forms a pi bond, expelling the water molecule. Finally, the protonated carbonyl oxygen is deprotonated by water, yielding the final cyclic product of the rearrangement reaction.

In the context of peptide sequencing, acetylation at the N-terminal is a significant post-translational modification that can impact the sequencing process. When considering the Edman degradation method, which is used to sequentially remove amino acids from the N-terminal end of a peptide, it is essential to understand the structural implications of acetylation.

During Edman degradation, phenylisothiocyanate (PITC) reacts with the N-terminal amino acid to form a thiohydantoin derivative. This reaction is crucial for the identification of the amino acid sequence. However, if the N-terminal amino acid is acetylated, the acetyl group prevents the PITC from effectively reacting with the amino group of the N-terminal residue. Consequently, this modification hinders the ability to cleave and identify the amino acid through Edman degradation.

To illustrate this, consider the structure of the thioazolinone formed during the Edman degradation process. The reaction involves the PITC and the amino acid, represented generically as R, where R denotes the side chain of the amino acid. The presence of an acetyl group at the N-terminal would lead to cyclization, which further complicates the cleavage process, making it impossible to sequence the peptide accurately.

In summary, the acetylation at the N-terminal prevents the necessary reactions for Edman degradation, thereby obstructing the sequencing of the peptide. Understanding these structural modifications is crucial for interpreting peptide sequences and their functional implications in biological systems.