Total synthesis of bovine pancreatic trypsin inhibitor and the protein diastereomer [Gly37D-Ala]BPTI using Boc chemistry solid phase peptide synthesis

Bovine pancreatic trypsin inhibitor (BPTI) is a well-studied model for investigation of protein folding and stability. Here, we report the synthesis and characterization of wild-type BPTI and a diastereomeric protein analogue [Gly37D-Ala]BPTI. Each 58-residue polypeptide chain was made by native chemical ligation of two peptide segments, BPTI[1-29]-(alpha)thioester and the appropriate version of the Cys(30)-58 BPTI peptide segment. Boc chemistry in situ neutralization solid phase synthesis was used to prepare the peptide segment reactants. The resulting full-length polypeptide chains were folded in a cysteine/cystine redox buffer to give synthetic protein molecules containing three disulfide bonds. The diastereomeric analogue [Gly37D-Ala] BPTI folded as efficiently as the native protein. Synthetic proteins were characterized by analytical LCMS and by natural-abundance H-1-N-15 HSQC NMR fingerprinting. These results illustrate the power of Boc chemistry peptide synthesis and its utility for the total chemical synthesis of protein molecules.