Protease-Catalyzed Peptide Synthesis for the Site-Specific Incorporation of α-Fluoroalkyl Amino Acids into Peptides

Abstract

Substitution of native amino acids by fluoroalkyl analogues represents a new approach for the design of biologically active peptides with increased metabolic stability as well as defined secondary structure and provides a powerful label for spectroscopic investigations. Here, we introduce a methodology for the incorporation of sterically demanding C(alpha)-fluoroalkyl amino acids into the P(1) position of peptides catalyzed by the commercially available proteases trypsin and alpha-chymotrypsin. The combination of 4-guanidinophenyl ester of C(alpha)-fluoroalkyl amino acids as substrate mimetics with frozen-state reaction conditions provided the most efficient strategy for protease-catalyzed site-specific introduction of this kind of nonnatural amino acids into peptide sequences. Consequently, a library of di-, tri-, and tetrapeptides containing alpha-methyl, alpha-difluoromethyl, and alpha-trifluoromethyl alanine, leucine, and phenylalanine in the P(1) position was synthesized catalyzed by trypsin as well as alpha-chymotrypsin. Trypsin was shown to be the more versatile protease.