Proteins play a critical role in all living organisms and are responsible for the regulation of various biological processes. Their properties and functions result directly from their primary sequence, which is dictated by the DNA that encodes them. Mutations of the primary sequence can alter properties such as stability or activity. This can be used in chemical protein synthesis with canonical as well as non-canonical building blocks to rationally design or modify peptides and proteins to specifically change their properties. In this thesis, the influence of fluorinated amino acids in particular on the activity of an enzyme and the interaction between it and its natural inhibitor barstar was investigated. For this purpose, different fluorinated variants of aminobutyric acid were incorporated at site Lys27 of the enzyme sequence. The first major hurdle of this project was the development of suitable synthesis methods to produce barnase, which consists of 110 amino acids. In order to minimize the consumption of the fluorinated amino acids, which are only accessible to a limited extent, during protein synthesis, special cycles were used. However, it was found that due to the position of the amino acid to be incorporated in the synthesis sequence, a reduction in equivalents used reduced achieved yields. The purified proteins were then subjected to various methods of structure elucidation to ensure purity and native folding. All barnase variants were analyzed for their enzymatic activity, as well as interaction with the natural inhibitor barstar. It was found that although all variants showed activity, non-natural variants showed significantly reduced activity compared to natural barnase. Nevertheless, calculated values for kcat/kM were in line with literature reported values for all variants. In future research, inhibitory efficiency and affinity of the different complexes can be investigated by means of fluorescence based inhibitory assays and ITC measurements. Furthermore, the herein presented results can be used as a basis for the characterization of precise complex structures.