Humans insulin synthesis

It is often important to control the CSD of pharmaceutical compounds, eg, in the synthesis of human insulin, which is made by recombinant DNA techniques (1). The most favored size distribution is one that is monodisperse, ie, all crystals are of the same size, so that the rate at which the crystals dissolve and are taken up by the body is known and reproducible. Such uniformity can be achieved by screening or otherwise separating the desired size from a broader distribution or by devising a crystallization process that will produce insulin in the desired form. The latter of these options is preferable, and considerable effort has been expended in that regard. 

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. 

Blood sugar (blood glucose) in human beings is controlled by the secretion of (—>) insulin by the beta (B- or (3-) cells of the islands of Langerhans in the pancreas. Loss of insulin synthesis leads to (—>) diabetes. Type 1 diabetes (insulin dependent diabetes mellitus, EDDM) begins in juveniles as an organ-specific autoimmune reaction, the destructive insulitis. 

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... 

P Sieber, B Kamber, J Hartmann, A Johl, B Riniker, W Rittel. 4. Total synthesis of human insulin. IV Description of the final product. Helv Chim Acta 60, 27, 1977. 

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]. 

K. Morihara, T. Oka, H. Tsuzuki, Semi-Synthesis of Human Insulin by Trypsin-Catalyzed Replacement of AlaB30 by Thr in Porcine Insulin , Nature 1979, 280, 412-413. 

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). 

Once the desired gene (such as the one that directs the synthesis of human insulin) has been located, some mechanism must be found for inserting it into the host animal (the bacterium, cow, pig. 

An octapeptide, corresponding in sequence to the C-terminal octapeptide found in the human insulin B-chain, is prepared by chemical synthesis. This octapeptide is then coupled to the shortened insulin using a well-established chemical method, thus yielding a semi-synthetic human insulin product ( human insulin emp Box 8.2). 

Typically, 7.0 g of human insulin can be prepared from 10 g of purified porcine insulin. The process allows large-scale production of human insulin simply and inexpensively. The complete de novo synthesis of human insulin, while technically possible, is economically unattractive. 

Scheme 5 Total Synthesis of Human Insulin by Selective Formation of Disulfides at Different Stages of a Fragment Condensation Scheme 1... 

There is a tendency to reserve semisynthetic and totally synthetic methods for the introduction of bonds and residues that cannot be specified by the genetic code. The present chapter will concentrate on these aspects. However, semisynthesis can have a role to play even when building structures that are completely accessible to the genetic code. The first industrial challenge for the emerging technologies of total chemical synthesis, recombinant protein expression, and semisynthesis was the economic production of human insulin in pharmaceutically usable quantity and quality. The semisynthetic human insulin that was made from porcine insulin proved exceptionally convenient to produce, and was the first introduction to human insulin for very many patients. 

Scheme 21 Synthesis of Human Insulin by the Fragment Condensation Strategy in Solution1184183 ...

Scheme 24 Synthesis of Human Insulin by Regioselective Disulfide Bridging of the A- and B-Chains1 1 31...

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, RMM about 6000, is made up of two chains of amino adds joined by disulfide linkages. The sequence of amino acids in the two chains (termed A for acidic and B for basic) and the arrangement of the three disulfide bridges were worked out by Sanger and associates in the period 1945-1955.1229 The complete synthesis of both ovine and human insulin was achieved in 1963.1230,1231... 

Replacement Therapy. If the endogenous production of a hormone is deficient or absent, therapeutic administration of the hormone can be used to restore normal endocrine function.30 35 The exogenous hormone can be obtained from natural sources, such as extracts from animal tissues, or from chemical synthesis. In addition, new recombinant DNA techniques are being used to produce hormones from cell cultures, and these techniques have shown great promise in being able to generate hormones like human 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. 

Many preparations are available for the treatment of diabetics with insulin. Rittel et al.128) described total synthesis of human insulin in 1974. Since that time new ways of obtaining insulin have been developed, namely the E. Lilly Co. biotechnological synthesis by genetically modified microorganisms l29) (DNA recombination)... 

Total synthesis of peptides is rarely an economical method for their commercial production. Important peptides are usually derived from biological sources. For example, insulin for diabetics was originally taken from pork pancreas. Now, recombinant DNA techniques have improved the quality and availability of peptide pharmaceuticals. It is possible to extract the piece of DNA that contains the code for a particular protein, insert it into a bacterium, and induce the bacterium to produce the protein. Strains of Escherichia coli have been developed to produce human insulin that avoids dangerous reactions in people who are allergic to pork products. 

Contributions to the biotechnology industry Commercialization of human insulin Interferon isolation Gene synthesis Protein analysis Peptide isolation Peptide mapping Amino-acid sequencing Total amino-acid composition Summary of LC benefits to biotechnology Contributions to environment research. / Air quality v Water quality... 

The drug development process starts with the synthesis of novel chemical compounds. Substances with complex structures may be obtained from various sources, e.g., plants (cardiac glycosides), animal tissues (heparin), microbial cultures (penicillin G) or cultures of human cells (urokinase), or by means of gene technology (human insulin). As more insight is gained into structure-activity relationships, the search for new agents becomes more clearly focused. 

Human insulin is produced by two methods biosynthetically from porcine insulin, by exchanging the wrong amino acid or by gene technology involving synthesis in E. coli bacteria. 

Fig. 3.8. Recombinant production of human insulin in E. coli. A. Independent synthesis of chains followed hy assembly. B. Synthesis as proinsulin followed by protease digestion.

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