Question

Dinitrofluorobenzene, very often known as Sanger's reagent after the English chemist Frederick Sanger who popularized its use, reacts selectively with the \(N\)-terminal amino group of a polypeptide chain. Sanger was awarded the 1958 Nobel Prize in Chemistry for his work in determining the primary structure of bovine insulin. One of the few people to be awarded two Nobel Prizes, he also shared the 1980 award in chemistry with American chemists Paul Berg and Walter Gilbert for the development of chemical and biological analyses of DNA. Following reaction with 2,4 -dinitrofluorobenzene, all amide bonds of the polypeptide chain are hydrolyzed and the amino acid labeled with a 2,4-dinitrophenyl group is separated by either paper or column chromatography and identified. (a) Write a structural formula for the product formed by treatment of the \(N\)-terminal amino group with Sanger's reagent and propose a mechanism for its formation. (b) When bovine insulin is treated with Sanger's reagent followed by hydrolysis of all peptide bonds, two labeled amino acids are detected: glycine and phenylalanine. What conclusions can be drawn from this information about the primary structure of bovine insulin? (c) Compare and contrast the structural information that can be obtained from use of Sanger's reagent with that from use of the Edman degradation.

Short answer

Answer: The reaction of bovine insulin with Sanger's reagent and subsequent detection of labeled glycine and phenylalanine amino acids indicate that bovine insulin has two polypeptide chains, with the N-terminal amino acids of the chains being glycine and phenylalanine, respectively.

Step-by-step solution

Reaction of N-terminal amino group with Sanger's reagent

2,4-Dinitrofluorobenzene (Sanger's reagent) reacts with the N-terminal amino group of a polypeptide chain and forms a 2,4-dinitrophenyl group. To write the structural formula for the product, first, we need the structure of 2,4-dinitrofluorobenzene: 2,4-Dinitrofluorobenzene: \(\displaystyle \underset{2,4-\textnormal{NO}_{2}\textnormal{ groups}}{(\textnormal{C}_{6}\textnormal{H}_{3}\textnormal{F}\textnormal{(NO}_{2})_{2})}\) Now let's consider the reaction of the N-terminal amino group of an amino acid with Sanger's reagent (using a generic amino acid R-NH2): Product formed: \(\displaystyle \overset{R^{1}-\textnormal{NH}-(2,4-\textnormal{NO}_{2}\textnormal{ groups})}{\textnormal{C}_{6}\textnormal{H}_{3}\textnormal{F}\textnormal{(NO}_{2})_{2}\textnormal{NH}R^{1}}\) The proposed mechanism for its formation is the nucleophilic attack of the N-terminal amino group at the fluorine atom of 2,4-dinitrofluorobenzene, followed by the release of a fluoride ion.

Analyzing bovine insulin structure

Bovine insulin was treated with Sanger's reagent and after hydrolysis of all peptide bonds, two labeled amino acids, glycine and phenylalanine, were detected. This observation indicates that bovine insulin has two polypeptide chains, with the N-terminal amino acids of the chains being glycine and phenylalanine respectively.

Comparing Sanger's reagent and Edman degradation

Both Sanger's reagent and Edman degradation methods are used for the determination of N-terminal amino acids in polypeptide chains. Sanger's reagent: - Involves the reaction of the N-terminal amino group with 2,4-dinitrofluorobenzene - Labels the N-terminus with a 2,4-dinitrophenyl group - Requires hydrolysis of the entire polypeptide chain - Useful for detecting the presence of multiple polypeptide chains with different N-terminal amino acids in proteins Edman degradation: - Involves the reaction of the N-terminal amino group with phenyl isothiocyanate - Repeatedly removes and identifies the N-terminal amino acid in a stepwise manner - Does not require hydrolysis of the entire polypeptide chain - Useful for determining the N-terminal amino acid sequence of a polypeptide chain up to about 50-60 residues In summary, Sanger's reagent is useful for detecting multiple polypeptide chains with different N-terminal amino acids in proteins, while the Edman degradation method allows for the determination of the N-terminal amino acid sequence in a polypeptide chain.