Insulin, solid phase synthesis

Three main categories of processes are used for the synthesis of peptides chemical synthesis, extraction from natural substances, and biosynthesis. On further scrutiny, almost 10 distinct synthetic methods can be distinguished (see Table 4.2). For the synthesis of some peptides, more than one method is used, including chemical (solution or solid phase) synthesis and recombinant biotechnology for salmon calcitonin, extraction from pancreas, semisynthesis, and recombinant biotechnology for insulin. 

Jansson, A., Meldal, M., and Bock, K. (1992) Solid-phase synthesis and characterization of O-dimannosylated heptadecapeptide analogues of human insulin-like growth factor 1 (IGF-1). J. Am. Chem. Soc. Perkin Trans I., 1699-1707. 

Another solid phase fragment condensation with CDI and 1-hydroxybenzotriazole in the synthesis of the human insulin B-chain afforded the oligopeptide in 75% yield. The reaction time with the coupling pair CDI/HOBt was shorter than in the case of the DCC/ HOBt system.136 The CDI/HOBt activation method was also applied to the synthesis of a... 

Recombinant insulin precursor MI3 Study of the target protein per se and selection of appropriate binding sites Solid-phase combinatorial chemistry (64 ligands) solution-phase synthesis of a sub-library Affinity chromatography SPR 15,18,19... 

The first synthesis reported by Merrifield produced the desired tetrapeptide (Leu-Ala-Gly-Val).f l Amino acids, dipeptides, and tripeptides were all detected in the crude product released from the resin. Through continued improvements of the method, the high speed of the amino acid incorporation and automation, the solid-phase peptide synthetic methodology has become the method of choice for most laboratories synthesizing peptides. Shortly after the introduction of the solid-phase procedure it was used to synthesize insulin. This impressive achievement awakened the biochemical community to the promise of synthetic chemistry and initiated a period in which large numbers of peptide analogues were prepared and analyzed. 

Synthesis of inositol phosphoglycans (IPGs) (71), analogues to second messengers of insulin, has been described. These derivatives contain the glucosamine (a 1-6) myo-inositol 1,2 cyclic phosphate motif and the thiol-terminated spacers for efficient coupling to maleimide functionalized solid phases or proteins. ... 

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. 

A. Marglin, R.B. Merrifield, The synthesis of bovine insulin by the solid phase method. J. Amer. Chem. Soc. 88 5051-5052 (1966)... 

Early production of insulin in this country stimulated the development of peptide research at two institutions, Nordisk Insulinlaboratorium and NOVO Pharmaceutical Company. At Nordisk, particularly after the formation of the Protein Chemistry Laboratory under the auspices of The Danish Academy of Technical Science, K. Brunfeldt initiated considerable effort toward improvements in solid phase peptide synthesis. 

Hammerstrom et al. [120] studied the synthesis of the B]. 3 sequence of insulin using the solid-phase technique. In these experiments ten different S-protective groups for the cysteine residue at B7 were evaluated including benzyl, p-methoxy-benzyl, benzhydryl, trityl, tetrahydropyranyl, benzylthio-methyl, ethylcarbamoyl, carbobenzyloxy, acetyl, and benzoyl. The benzyl, p-methoxybenzyl, benzhydryl, benzylthiomethyl, and ethylcarbamoyl withstood all synthetic operations but each, with the exception of the benzyl, was cleaved to a small extent when the peptide was removed from the resin with hydrogen bromide in trifluoroacetic acid. The S-tetrahydro-pyranyl was, on the other hand, cleanly removed by treatment with hydrogen bromide in trifluoroacetic acid and better overall... 

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). 

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