Proteins are important biomolecules for the regulation of quintessential mechanisms in living organisms. Through chemical modifications, their biophysical properties can be altered, leading to enhanced stability, higher bioactivity, and other beneficial alterations. In this doctoral thesis, the influence of site-specific fluorination on the activity of the bovine pancreatic trypsin inhibitor (BPTI), a well-studied serine protease inhibitor, was investigated. For this, new strategies for synthesizing a fluorinated amino acid and the chemical protein synthesis of BPTI were developed, followed by biological activity assays and structural investigations X-ray diffraction analysis. In the first part of this project, a novel synthesis in a gram scale for monofluoroethylglycine (MfeGly) was developed. This eight-step strategy started with aspartic acid and obtained the Fmoc-protected product in yields of up to 50 %. A particular focus of this study laid in the investigation of various nucleophilic fluoride sources. The second part of this project focused on incorporating the previously synthesized amino acid and several other derivatives into 58-amino acid long BPTI by solid-phase peptide synthesis (SPPS). Since fluorinated amino acids are of high value, special coupling cycles for their incorporation into amino acid sequence were developed, cutting usage of the material to 30 %. SPPS was followed by oxidative refolding the cysteine-rich protein to obtain the native conformation with correctly matched disulfide bridges. The inhibitory activity of fluorinated BPTI variants and their hydrocarbon derivatives was investigated towards the serine protease α-chymotrypsin. Difluoroethylglycine (DfeGly) showed enhanced inhibition, even better than wild-type BPTI. In contrast, inhibition activity drops by two orders of magnitude in non-fluorinated amino butanoic acid (Abu) containing BPTI. Lastly, BPTI variants containing Abu and its fluorinated derivatives MfeGly, DfeGly, and trifluoroethylglycine (TfeGly) were crystallized in complex with chymotrypsin and analyzed by X-ray diffraction. The results of this thesis will contribute to a better understanding of how fluorinated amino acids impact protein-protein interactions.
