Table of Contents:
The first part of this thesis (chapters 1-7) presents a synthetic and biophysical study of a congeneric series of D-Phe/D-DiPhe-Pro based inhibitors for thrombin and trypsin. Both proteins are structurally highly related serine proteases which show differences in their preferred substrate recognition. Thrombin is Arg-specific, whereas trypsin cleaves peptide chains after Lys and Arg. They recognize the basic substrate head group via Asp189, located at the bottom of the S1 pocket. The synthesized inhibitors are crystallographically analyzed for both serine proteases and thermodynamically investigated by isothermal titration calorimetry (ITC) to describe and compare their binding properties in the S1 pocket. An enzyme-kinetic fluorescence assay also supports the biophysical characterization of these compounds toward both proteins. In order to elucidate selectivity characteristics of thrombin and trypsin more precisely, the protonation effects were investigated in the S1 pocket. Although both proteases have a highly similar S1 pocket, our results suggest a difference in the electrostatic properties of Asp189 which may correlate with their selectivity in substrate recognition. It is surprising that one ligand of the series binds to trypsin in protonated state at its P1 head group while it remains unprotonated in case of thrombin. For this ligand, a slight difference in the residual solvation structures is observed. Our data show that these differences may be caused by the specific sodium binding site which is only present in thrombin, but absent in trypsin. This may be further pronounced by the charged Glu192 at the rim of the S1 pocket in thrombin and is not affected by the uncharged Gln192 in trypsin. Here, the consideration of different pKa values of the involved functional groups also play an essential role.
Furthermore, various fragments as well as D-Phe-Pro derivatives are presented in both proteins whose binding properties in the S1 pocket are characterized and compared.
The second part of this thesis (chapters 8-12) is a synthetic study of aldose reductase inhibitors with a (2-arylcarbamoyl-phenoxy)-acetic acid scaffold by varying benzyl moieties with different m-substituted withdrawing groups (here: sulfoxide and boronic acid). They are prepared for the investigation of the transient specificity pocket in aldose reductase. Electron-deficient aromatic systems can trigger an opening of the specificity pocket thus lead to favored interactions with Trp111. In order to understand the opening mechanism of this transient binding pocket, these synthesized compounds should be also used in mutagenesis studies. Our first crystallographic results and ITC experiments did not reveal any clear interpretation regarding this mechanism so far. Different desolvation properties of the inhibitors may also have an impact on this mechanism.
The third part of this thesis (chapters 13-17) is another synthetic study of a series of p-alkylated benzenesulfonamides and their ether derivatives as human carbonic anhydrase II (hCAII) inhibitors which are varied in their alkyl chain length. While the sulfonamide group binds anionically to Zn2+, the aliphatic alkyl chain addresses the hydrophobic pocket in the active site. The binding events of these compounds in hCAII were investigated crystallographically, kinetically and thermodynamically by ITC. The results demonstrate the conformational changes and reveal effects of the importance of shape complementarity for the structure-binding relationship.
Additionally in this part, the synthesis of perfluorinated derivatives are presented which exhibit a different binding pattern in hCAII. A kinetic and thermodynamic study was also performed analogously according to the investigation of the non-fluorinated series. The acidity of the sulfonamide group is influenced by the strongly electron-withdrawing effects of the fluorine substituents which lead to putative different binding characteristics of the aromatic system in the active site of this protein.