Mechanism of action studies on new ligands for soluble guanylate cyclase.

This thesis will comprise two topics. The first part relates to the application of photoaffinity labelling to soluble guanylate cyclase (sGC), which belongs to a family of enzymes that catalyse the conversion of guanosine-5'-triphosphate (GTP) to cyclic guanosine-3', 5'- monophosphate (cGMP). By formation of the intracellular signalling molecule cGMP, sGC plays a key role in smooth muscle relaxation and inhibition of platelet aggregation and in many signal transduction pathways. Although there are two types of activators of sGC, NO-dependent (nitrovasodilators such as sodium nitroprusside, DEANO, etc.) and NO-independent (YC-1, l-benzyl-3- 5'-hydroxymethyl-2'-furyl indazole), the mechanism of action of only the former is understood. This thesis describes an attempt to locate residues that make up the binding sites for these activators. This comprises the design, synthesis and biological evaluation of small molecule activators, and their photolabeled analogues, followed by photoafiinity labelling experiments using tryptic digestion and mass spectroscopy (MALDI) for the identification of peptide fragments covalently bonded to activator analogue. The second part involves the investigation of a traceless linker designed for use in the solid phase synthesis of sGC modulators. The resulting method could also be applied in the synthesis of a variety of biologically active molecules. We wanted to design a linker which would combine the advantages of a traceless and a "safety-catch" linker. Therefore we investigated the application of a benzoisothiazol-based linker as this heterocycle is stable until activated by oxidation of the sulphur atom, allowing facile cleavage in the last step of the synthesis