Chymotrypsin, Trypsin, and Related Enzymes

3 SERINE AND CYSTEINE PROTEASES FOR PEPTIDE SYNTHESIS 15.3.1 Chymotrypsin, Trypsin, and Related Enzymes 

Classical enzymes employed for peptide coupling of the serine hydrolase family are chymotrypsin, trypsin, and subtilisin. Chymotrypsin and trypsin are secreted in the mammalian gut as inactive precursors, which are activated by autoproteolysis and structural reorganization. The use of chymotrypsin for peptide synthesis has been reported since the 1930s [50]. Most early examples concern peptide s)mthesis using amides or (m)ethyl esters as acyl donors and free amino acids and their amides, short peptides, or short peptide amides as nucleophilic acyl acceptors [3]. These studies revealed that a high pH, high nucleophile concentration, and low product solubility stimulate the formation of synthetic product. Ethyl esters appeared suitable acyl donors in kinetically controlled conversions, and amino acid amides act better as nucleophilic acyl acceptors than the free amino acids [4]. Furthermore, tripeptides often performed better than dipeptides or amino acids as acyl acceptors. 

Example of bioactive peptide synthesis under kinetic control using a-chymotrypsin in water-cosolvent medium [5]. The gonadotropin-releasing hormone (PCA-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NHj) analogue [D-Phe ]GnRH was obtained in 97.5% yield. Abbreviations PCA, pyroglutamic acid. 

Example of bioactive peptide synthesis under thermodynamic control using trypsin in water-cosolvent medium. The a-melanocyte-stimulating hormone precursor was synthesized in 95% yield using trypsin in an almost dry mixture of DMF and hexafluoroisopropanol [6]. Protective groups CHO, formyl group CIZ, 2-chlorobenzyloxycarbonyl. 

TABLE 15.2 Strongly Activating Groups for Kinetic Peptide Coupling  

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