Porcine insulin enzymatic conversion

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]. 

Semisynthesis has become a suitable technique for chemical variations of large polypeptides or proteins from natural sources. Human insulin differs from porcine insulin only in position B30, where alanine is the C-terminal B-chain residue of porcine insulin but threonine is the C-terminal of the human insulin B-chain. Among the various ways to obtain human insulin from porcine insulin by protease-catalyzed semisynthesis, the one-step conversion by exchange of the C-terminal alanine B of porcine insulin with a threonine residue seems to be the method of choice. Several proteases, e.g. trypsin, achromobacter protease, and car-boxypeptidase Y, are able to catalyze the conversion and various groups have independently described an essential improvement of the enzymatic semisynthesis of insulin.The Hoechst procedure,which was developed as an industrial process, is described below as an example of a large-scale conversion of porcine insulin to human insulin in kilogram amounts for therapeutic application. Based on this type of transamidation reaction, it is easy to prepare various B30-insulin analogues. 

Although the kinetic approach should be preferable, the decision must depend on the overriding total synthesis concept. The largest industrial scale application of the equilibrium approach is probably the enzymatic synthesis of Z-Asp-Phe-OMe, the precursor of the peptide sweetener aspartame 115l The best known use of trans-peptidation technology is the large scale conversion of porcine insulin into human insulin by trypsin[U6 or Achromobacter lyticus protease 117. ... 

A more general use of proteolytic enzymes in peptide synthesis became feasible with the discovery (Sealock and Laskowsky 1969) of the effect of water miscible organic solvents on the equilibrium in enzyme catalyzed peptide bond hydrolysis and synthesis. In the presence of isopropanol (or dimethylfor-mamide, etc.) the dissociation of the carboxyl group is suppressed and, at least in a selected pH region, the equilibrium is shifted toward synthesis. A notable case is the conversion of porcine insulin to human insulin. Enzymatic cleavage of the C-terminal residue of the B-chain (alanine) with carboxypeptidase yields desalanino pork insulin. This cleavage is followed by the incorporation of... 

Scheme 3.27 Enzymatic conversion of porcine into human insulin...

A trypsin-related bacterial enzyme that was tested in coupling reactions is the lysine-specific serine protease I from Achromobacter lyticus. The enzyme is secreted and widely used in sequence analysis of proteins [55]. It was also used in a chemo-enzymatic route for the production of human insulin from porcine insulin. Since this endopeptidase cleaves only after Lys, it could be applied for replacing the C-terminus of the insulin B-chain from -Lys-Ala (porcine C-terminal sequence) to -Lys-Thr (human C-terminus) in a two-step or a single-step reaction. Using the B-chain as the acyl donor and Thr-OBu in DMF-ethanol mixtures as the nucleophile, a high conversion (85-90%) was obtained [9,56]. Trypsin could also be applied in this biotransformation but required higher enzyme loading. 

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