Carbohydrate Protease Conjugates: Stabilized Proteases for Peptide Synthesis

Article abstract of DOI:10.1021/jo00112a049

The synthesis of oligopeptides using stable carbohydrate protease conjugates (CPCs) was examined in acetonitrile solvent systems.CPC<α-chymotrypsin> was used for the preparation of peptides containing histidine, phenylalanine, tyrosine, and tryptophan in the P₁ position in 60-93percent yield.The CPC<α-chymotrypsin)-catalyzed synthesis of octamer Z-Gly-Gly-Phe-Gly-Gly-Phe-Gly-Gly-OEt from Z-Gly-Gly-Phe-Gly-Gly-Phe-OMe was achieved in 71percent yield demonstrating that synthesis was the dominant kinetic process relative to amide hydrolysis.CPC was used to synthesize peptides containing both hydrophilic and hydrophobic amino acids.The P₂ specificity of papain for aromatic residues was utilized for the 2 + 3 coupling of Z-Tyr-Gly-OMe to H2N-Gly-Phe-Leu-OH to generate the leucine enkephalin derivative in 79percent yield.Although papain is nonspecific for the hydrolysis of N-benzyloxycarbonyl amino acid methyl esters in aqueous solution, the rates of synthesis for these derivatives with nucleophile leucine tert-butyl ester differed by nearly 2 orders of magnitude.CPC was used to prepare the aspartame precursor Z-Asp-Phe-OMe in 90percent yield.The increased stability of CPCs prepared from periodate-modified poly(2-methacrylamido-2-deoxy-D-glucose), poly(2-methacrylamido-2-deoxy-D-galactose), and poly(5-methacrylamido-5-deoxy-D-ribose), carbohydrate materials designed to increase the aldehyde concentration in aqueous solution, suggests that the stability of CPCs is directly related to the aldehyde concentration of the carbohydrate material.Periodate oxidation of poly(2-methacrylamido-2-deoxy-D-glucose) followed by covalent attachment to α-chymotrypsin gave a CPC with catalytic activity in potassium phosphate buffer at 90 deg C for 2 h.