Human Biochemistry, Volume 1 - Murray R. 1993

Structure and Functions of Proteins and Enzymes
Peptides
Automated Peptide Synthesis

Classical chemical Methods successfully yielded the Synthesis of the octapeptides Vasopressin and Oxytocin, and later bradykinin. However, the final product yields were so low that there was little hope of synthesizing longer Polypeptides or Proteins. This challenge was finally overcome through the automated solid-phase Peptide Synthesis method developed by Merrifield. The entire process takes place in a single reaction vessel, where Reagents are automatically added and products removed at scheduled intervals according to a predetermined program. The Procedure consists of the following stages.

1. The amino acid destined for the C-terminus of the polypeptide is anchored to an insoluble resin particle.

2. A second amino acid, bearing a temporarily blocked amino group, is introduced, and a peptide bond is formed in the presence of the dehydrating agent dicyclohexylcarbodiimide.

3. The blocking group is cleaved off using an acid, releasing gaseous byproducts that are subsequently removed.

4. Steps 2 and 3 are repeated with the next incoming (second) amino acid, then with the third, and so forth, until the fully synthesized polypeptide is covalently attached to the resin particle.

5. The polypeptide is cleaved from the resin support.

The process proceeds rapidly and with excellent yields. Each peptide bond formation takes approximately 3 hours. Using this method, the Insulin A-chain (21 residues) was synthesized in 8 days, and the B-chain (30 residues) in 11 days. The most remarkable achievement was the total synthesis of pancreatic Ribonuclease (124 residues; Fig. 5.10) with an overall yield of 18%, representing the first synthesized enzyme. This milestone heralded a new era not only in Cell/13.html">Protein Structure research, but also in related fields such as immunology, vaccine production, and potentially in the medical Treatment of diseases linked to inborn errors of METABOLISM. A number of physiologically crucial peptides were synthesized from L-Amino Acids using racemization-free methods, and the resulting products exhibited full physiological activity. Examples include the octapeptides oxytocin and vasopressin, adrenocorticotropic hormone (ACTH), and melanocyte-stimulating hormone (Chap. 45).

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