Hydrolysis of peptide esters by different enzymes.
The combined use in peptide synthesis of the Fmoc-group with methyl, benzyl or p-nitro benzyl esters is not practical because of the elimination of the Fmoc-group under basic conditions and by catalytic hydrogenation. Nevertheless the solution synthesis of peptides requires those combinations in some cases. For this purpose we have investigated enzymatic hydrolysis of some tri and tetrapeptide esters. The hydrolysis were carried out under pH-control. We measured deprotection of the carboxyl group by thermitase, porcine liver esterase, carboxypeptidase A and alpha-chymotrypsin. The main problems are to suppress proteolytic degradation of the peptide bond and to bring the protected peptides into solution. To solve both problems we used dimethylformamide and dimethylsulfoxide as cosolvents. The ratios between esterolytic and proteolytic activity were estimated under various cosolvent concentrations. Advantages of this method are to avoid side reactions of alkaline instable side chains (e.g. asparagine, glutamine), cleavage of base labile protecting groups and racemization by alkaline saponification. The enzymatic deprotection was followed by HPLC, HPTLC and titration. On a preparative scale this method gives good yields and sufficiently pure products.
Reissmann
S
S
Institute of Biochemistry and Biophysics, Friedrich Schiller University of Jena, Germany.
Greiner
G
G
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D002268:Carboxypeptidases / Q000378:metabolism; D043422:Carboxypeptidases A; D002918:Chymotrypsin / Q000378:metabolism; D004121:Dimethyl Sulfoxide; D004126:Dimethylformamide; D004950:Esterases / Q000378:metabolism; D004952:Esters / Q000737:chemistry*; D005449:Fluorenes / Q000737:chemistry*; D006868:Hydrolysis; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D012697:Serine Endopeptidases / Q000378:metabolism*; D012995:Solubility; D013237:Stereoisomerism; D013552:Swine; D013696:Temperature
D000596:Amino Acids; D004952:Esters; D005449:Fluorenes; C016456:N(alpha)-fluorenylmethyloxycarbonylamino acids; D010455:Peptides; D004126:Dimethylformamide; D004950:Esterases; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D012697:Serine Endopeptidases; C022728:thermitase; D002918:Chymotrypsin; D004121:Dimethyl Sulfoxide