Synthesis of Insulin

Polypeptide synthesis is one of the exciting and challenging areas in modern chemistry. Here we describe the two most well-known polypeptide syntheses the synthesis of insulin and the synthesis of ribonuclease. 

The synthesis of insulin was reported by three groups Zahn (1963), Katsoyannis and Dixon (1964), and Niu et al. (1965). Insulin consists of an A chain and a B chain  

FIGURE 2.2 Coupling of polypeptides. [Source Wingrove and Caret (1981).] 

Abbreviations. BZL, benzyl OBU, butyl ester OME, methyl ester A, amino acid Z, benzyloxy-carbonyl. 

FIGURE 2.4 (a) First steps of ribonuclear synthesis, (b) General diagram. [Source Merrifield (1963).] 

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

It has been known for years that the activated residues of acyl- and peptidylamino acids enantiomerize during coupling (1.9). However, the racemization tests available (see section 4.9) did not allow for a valid comparison of the tendency of residues to isomerize because they incorporated a variety of aminolyzing residues and N-substituents. Valid demonstration of the different sensitivities of residues was provided by classical work on the synthesis of insulin. It was found that a 16-residue segment with O-tert-butyltyrosine at the carboxy terminus produced 25% of epimer in HOBt-assisted DCC-mediated coupling in dimethylformamide, and the same segment with leucine at the carboxy terminus produced no epimer. Only when series such as Z-Gly-Xaa-OH coupled with valine benzyl ester became available was it possible to compare many residues with confidence. Unfortunately, it transpires that the issue is extremely complex. 

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. 

FIGURE 6.23 The synthesis of insulin, starting with a cystine-containing peptide. [Kamber et al., 1977]. Moc = methoxycarbonyl, Bpoc = biphenylisopropoxycarbonyl, Trt = trityl, Acm = acetamidomethyl. (a) HOBt-assisted carbodiimide-mediated coupling (b) removal of Trt by HC1 in CF3CH2OH-CH2Cl2 (9 1) at pH 3.5 (c) removal of Bpoc by CF3CH2OH-CH2 Cl2 (9 1) at 60°C (d) removal of Acm and oxidation by iodine. 

In some cases, such as the synthesis of insulin, the recombination mixture is added to a host organism, here E. coli. This infected mixture is then plated out and the individual colonies tested for insulin production. Those colonies that produce insulin are further plated out and grown for mass insulin production. Cells that accept the recombinant DNA are called transformed. More specialized sequences have been developed to increase the probability of gene incorporation and its successful reproduction. 

Figure 4.2. cloning of insulin from cDNA isolated from pancreas, the main tissue responsible for synthesis of insulin, and inserted into plasmid vectors that permit expression in host cells. 

The anterior pituitary releases GH in 6 to 8 pulsatile bursts over a 24 h period the major portion is released just prior to deep sleep. Its secretion is controlled by hypothalamic peptides GH-releasing hormone stimulates GH secretion, while somatostatin inhibits it. GH stimulates the synthesis of insulin-like growth factor-1 (IGF-1 somatomedin C) mainly, but not solely, in the liver. GH and IGF-1 receptors are widely scattered throughout the body, and both hormones exert important metabolic actions in various tissues, especially muscle and bone. 

In states of chronically elevated blood levels of insulin, there is a substantive decrease in the density of insulin receptors in insulin-dependent cells due to a decrease in the synthesis of insulin receptors. This phenomenon, which is referred to as downregulation, represents a means by which a cell autoregulates its receptivity to the hormone, and is also detected by other hormones such as the catecholamines, endorphins, and GnRH. The precise mechanisms involved in downregulation are not known. 

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. 

Nearly all peptide hormones are synthesized as inactive precursors and then converted to active hormones by proteolytic processing. Studies of the synthesis of insulin provided the first evidence of this phenomenon (see Figure 5.21). Insulin contains two polypeptide chains, of 21 and 30 residues, with two interchain disulfide bridges and one intrachain bridge (Figure 5.15). 

Scheme 17 Synthesis of insulin lispro via an ester-insulin intermediate...

A plasmid, which is a circular DNA molecule separate from the chromosomal DNA, is obtained from bacterial cells such as Escherischia coli and treated with a restriction enzyme to snip the DNA at a specific site (Figure 26.15). The human DNA sequence that codes for the synthesis of insulin is then inserted into the plasmid to give a recombinant DNA molecule. The new DNA is the result of the recombination of DNA from the plasmid plus the sequence that codes for human insulin. The new plasmid is termed a chimeric plasmid because it contains DNA from two sources, bacterial and human. The plasmid is taken up by growing bacterial cells through a process called transformation. The chimeric plasma serves as a cloning vector because it serves as a vehicle to carry the recombinant DNA into E. coli. Transcription and translation of the insulin DNA then occur to produce human insulin. When the cells divide, the plasmids are divided between the daughter cells and they continue to produce clones. Insulin produced by recombinant DNA technology is commercially sold as Humulin. 

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