Proteases are essential enzymes for cleaving peptide bonds in proteins and overtake critical roles in many and highly diverse cellular processes. While some proteases act mainly by non-specific degradation of proteins, others are highly selective and regulate numerous biological processes by selective substrate proteolysis. Accordingly, dysregulation of their expression or enzymatic activity may lead to various pathologies. Despite undisputable advances in our understanding of the molecular biology of many proteases, some proteases remain poorly characterized, e.g., regarding the cellular function, structure, or localization. The dipeptidyl peptidases (DPP) 8 and 9 (DPP8/9), which belong to the pharmacologically important dipeptidyl peptidase 4 (DPP4) gene family, are two examples of such understudied proteases, among other reasons due to a lack of suitable selective inhibitors for these proteases that can be used as chemical tools in functional studies. This is surprising as selective inhibitors for the homologous proteases DPP4 and fibroblast-activation protein α (FAP) exist and are even in clinical use since many years.

In this thesis, different approaches for the development of structurally new and selective DPP8/9 inhibitors were therefore investigated. First, studies on the derivatization of an established DPP8/9 inhibitor structure, aiming at an improvement of the DPP8 vs. DPP9 selectivity, were pursued. Besides the identification of novel DPP8/9 inhibitors, this study also led to the establishment of a straightforward and technically simple oxazolidinone synthesis route.

Subsequently, the natural product Sulphostin, a known potent DPP4 inhibitor, was investigated as a starting structure for the development of selective DPP8/9 inhibitors. To this end, Sulphostin as well as diverse synthetic analogs were thoroughly characterized via biochemical inhibition assays as well as activity-based protein profiling for determining their cellular activity and structural studies to deduce their molecular binding modes. These efforts led to the discovery of a suitable Sulphostin modification, consisting of the introduction of an alternative phospho residue that maintains potent inhibition but furthermore serves as an efficient selectivity filter for DPP8/9 vs. DPP4. As the developed inhibitor class also showed promising proteome-wide selectivity as well as cellular activity, these studies led to the establishment of a new class of selective DPP8/9 inhibitors that may find wider application in DPP8/9 research.

Finally, an additional biochemical tool for DPP8/9 research was also developed. To this end, the established DPP8/9 inhibitor scaffold was decorated with a VIS photocleavable moiety. Photoactivation then results in photocleavage of this moiety and thus liberation of the actual inhibitor in vitro and in situ, thereby generating a spatiotemporally controlled chemical tool. Moreover, these studies have helped to establish an alternative strategy for the systematic design of photocaged inhibitors.