Insulin peptide synthesis

Other than the biochemical methods typified by the synthesis of insulin there are two major approaches to peptide synthesis... 

Although equihbrium-controUed peptide synthesis has been successfully used on a number of occasions, including thermolysin-catalyzed synthesis of aspartame (126) and semisynthesis of insulin (127), the method has a significant drawback a water-miscible organic cosolvent added to the reaction medium to suppress the ionization of unactivated carboxy components significantly reduces the reaction rate. 

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. Its structure was determined by Frederick Sanger, who received the 1958 Nobel Prize in chemistry for his work. 

E Bayer, M Dengler. B Hemmasi. Peptide synthesis on the new polyoxyethylene-polystyrene graft copolymer, synthesis of insulin B21.30. btt J Pept Prot Res 25, 178, 1985. 

It is interesting to note that serine peptidases can, under special conditions in vitro, catalyze the reverse reaction, namely the formation of a peptide bond (Fig. 3.4). The overall mechanism of peptide-bond synthesis by peptidases is represented by the reverse sequence f-a in Fig. 3.3. The nucleophilic amino group of an amino acid residue competes with H20 and reacts with the acyl-enzyme intermediate to form a new peptide bond (Steps d-c in Fig. 3.3). This mechanism is not relevant to the in vivo biosynthesis of proteins but has proved useful for preparative peptide synthesis in vitro [17]. An interesting application of the peptidase-catalyzed peptide synthesis is the enzymatic conversion of porcine insulin to human insulin [18][19]. 

Use of Proteases in Peptide Synthesis. Typically peptides are synthesized the standard solid or liquid phase methodologies (56, 57). However, both of these techniques require harsh chemical reactions which are detrimental to certain amino acids. Furthermore, in practical terms most peptide syntheses are limited to the range of 30 to 50 amino acid residues. Hence, peptide synthesis is still somewhat problematic in many cases. In certain situations, the alternative method of peptide synthesis using proteases is an attractive choice. With this form of synthesis, one can avoid the use of the noxious and hazardous chemicals used in solid or liquid phase peptide synthesis. Since the reactions are enzyme catalyzed, racemization of the peptide bond does not occur. This technique has been used with success in the synthesis and semisynthesis of several important peptides including human insulin (55,59). 

There are several other examples of enzyme peptide synthesis. The conversion of porcine insulin into a human insulin precursor, by replacing the B chain C-terminal with threonine, is still an important alternative to microbially produced human insulin. Peptides that are produced from ethyl esters of L-amino acids by BioEurope are used as ingredients of cosmetics. 

Insulin peptides. X. Synthesis of the B-chain of insulin and its combination with natural or synthetic A-chain to generate insulin activity. J. Am. Chem. Soc. 1964 86 930-932. 113. 

Phosgene has been used as a condensing agent in peptide synthesis [2183], and its effect on the poiypeptide hormone, insulin, has been examined [1422,1423]. It is assumed that the COClj reacts preferentially with the amino groups of the insulin, and that inter- or intra-molecular linkages are created [1423] ... 

Three groups of workers independently reported the total chemical synthesis of insulin during the 1960s. The methods used were to synthesize both chains separately and then to couple them. Since coupling was predictably random, the yields were low. The preparations took years. It is ironic that, utilizing Merrifield s development of solid-phase peptide synthesis with present automated peptide synthesis machines, insulin could be synthesized today in less than 200 hours. 

Biochemistry continues to have a major influence on the development of peptide synthesis. Peptide bond formation via catalysis with proteolytic enzymes has the promise of products with absolute chiral purity and should also be free from many side reactions encountered in synthesis by the methods of organic chemistry. Therefore coupling with the help of enzymes (cf page 57) is receiving growing attention. Perhaps even more exciting is the exploitation of ribosomal protein synthesis for the production of selected target peptides, such as insulin. In recent years preparation of the necessary DNAs became almost routine and hence this avenue of peptide synthesis broadened to a major area that transcends the boundaries of this book. 

---