12.5: Peptide Synthesis- Solution-Phase

Objectives

After completing this section, you should be able to

1. describe why it is necessary to protect certain amino and carboxyl groups during the synthesis of a peptide.
2. describe, using appropriate equations, how carboxyl groups are protected by ester formation and amino groups are protected by the formation of their tert‑butoxycarbonyl amide derivatives.
3. describe, using appropriate equations, the formation of a peptide link between an amino acid with a protected amino group and an amino acid with a protected carboxyl group using dicyclohexylcarbodiimide.
4. outline the five steps required to form a dipeptide from two given amino acids.

Solution-phase synthesis was the first developed and the only method for peptide synthesis until the introduction of solid-phase peptide synthesis (SPPS) by Merrifield revolutionized the way peptides and their analogues are prepared today. However, some peptides, due to their chemical structure, cannot be synthesized by SPPS, and the “old school” technique remains favorable for making them.

Once the structure of a peptide is known, its synthesis can be undertaken—perhaps to obtain a larger amount for biological evaluation. A simple amide might be formed by treating an amine and a carboxylic acid with a carbodiimide (either DCC or EDC; Section 7.4), but in solution, peptide synthesis is a more difficult problem because many different amide bonds must be formed in a specific order, rather than at random.

The solution to the specificity problem is the use of protecting groups. The protective groups should be specific for amines (PgN) or carboxylic acids (PgC) and should be easy to remove under mild conditions. 

If we want to couple alanine with leucine to synthesize Ala-Leu, for instance, we could protect the –NH₂ group of alanine and the –CO₂H group of leucine to shield them from reacting, then form the desired Ala-Leu amide bond by reaction with EDC or DCC, and then remove the protecting groups.

A number of different amino- and carboxyl-protecting groups have been devised, but only a few are used in peptide synthesis. Carboxyl groups are often protected simply by converting them into methyl or benzyl esters. Both groups are easily introduced by standard methods of ester formation (Section 7.7) and are easily removed by mild hydrolysis with aqueous NaOH. Benzyl esters can also be cleaved by catalytic hydrogenolysis of the weak benzylic C–O bond  \(\ce{( RCO2–CH2Ph+H2→RCO2H+PhCH3
 )}\).

Amino groups are sometimes protected as their tert-butyloxycarbonyl amide (Boc) or, more commonly, as their fluorenylmethyloxycarbonyl amide (Fmoc). The Boc protecting group is introduced by reaction of the amino acid with di-tert-butyl dicarbonate in a nucleophilic acyl substitution reaction and is removed by brief treatment with a strong acid such as trifluoroacetic acid, CF₃CO₂H. The Fmoc protecting group is introduced by reaction with fluorenylmethyloxycarbonyl chloride and is removed by treatment with a 20% solution of the amine piperidine in dimethylformamide as solvent.

Thus, five steps are needed to synthesize a dipeptide such as Ala-Leu:

These steps can be repeated to add one amino acid at a time to the growing chain or to link two peptide chains together. Many remarkable achievements in peptide synthesis have been reported, including a complete synthesis of human insulin. Insulin is composed of two chains totaling 51 amino acids linked by two disulfide bridges. The three-dimensional structure of insulin, shown previously, was determined by Dorothy Crowfoot Hodgkin, a British chemist who received the 1964 Nobel Prize in Chemistry for her work on this and other complex biological molecules.

Exercise \(\PageIndex{1}\)

Show the mechanism of the formation of a Boc derivative by reaction of an amino acid with di-tert-butyl dicarbonate.

Answer

This is a typical nucleophilic acyl substitution reaction, with the amine of the amino acid as the nucleophile and tert-butyl carbonate as the leaving group. The tert-butyl carbonate then loses CO₂, giving tert-butoxide, which is subsequently protonated.

Exercise \(\PageIndex{2}\)

Write all five steps required for the synthesis of Leu-Ala from alanine and leucine.

Answer

1. Protect the amino group of leucine.
2. Protect the carboxylic acid group of alanine.
3. Couple the protected amino acids with DCC.
4. Remove the leucine protecting group.
5. Remove the alanine protecting group.