Lysosomal acid glucosylceramidase (GBA1) is a lysosomal enzyme that degrades glucolipids with its main substrate being glucosylceramide (GlcCer). Defects in the GBA1 gene lead to... Show moreLysosomal acid glucosylceramidase (GBA1) is a lysosomal enzyme that degrades glucolipids with its main substrate being glucosylceramide (GlcCer). Defects in the GBA1 gene lead to glycosphingolipidosis Gaucher disease (GD), in which the hydrolysis of GlcCer is impaired and therefore, it accumulates in the lysosome. GD has a wide range of phenotypes that reaches from asymptomatic to neuropathologic manifestations, which are lethal within the first years of life. Although various GBA1 mutations have been identified, they cannot be systematically matched with GD phenotypes. Therefore, this thesis aims to get a better understanding of GBA1 and its role in GD by identifying GBA1’s cellular interaction partners with the aid of an enrichment assay. For this assay, photocleavable activity-based probes (ABPs) were developed, which ought to be able to isolate GBA1 from a cell lysate together with its interaction partners with the aid of streptavidin-coated beads. By photocleaving the complex off the beads, the background of the GBA1-containing samples can be lowered. So far, GBA1 could successfully be isolated from a cell lysate with the developed ABPs. Next, the assay conditions need to be optimized for isolation of GBA1 together with its interaction partners. Show less
This thesis describes the synthesis and biochemical evaluation of a variety of cyclophellitol based activity-based probes and inhibitors targeting various endo- and exo-acting retaining... Show moreThis thesis describes the synthesis and biochemical evaluation of a variety of cyclophellitol based activity-based probes and inhibitors targeting various endo- and exo-acting retaining glycosidases. In the last two decades a variety of probes and inhibitors for (hemi)cellulose degrading enzymes have been developed. However, at the onset of the work described in this thesis the focus has been mainly on cellulases, xylanases, xyloglucanases and glycanases. Chapter 2 describes the design and synthesis of a first generation of inhibitors and probes for β-mannanases. The general scaffold is composed of a β-1,4-linked mannobioside featuring either an epoxide or an aziridine warhead. All synthesized probes feature a linker attached to the nonreducing end and are condensed with either biotin or Cy5. Biochemical evaluation in an Aspergillus niger secretome revealed the utility of these compounds but also the limitations. The probes react efficiently with some endo-β-mannanases yet lack reactivity with others. Chapter 3 describes the design and synthesis of a second generation of β-mannanase probes and inhibitors designed to overcome the limitations of the first generation by changing the general scaffold. In this chapter two conceptually distinct ABP scaffolds are presented: a β-1,4-linked mannotriose and a β-1,4-linked manno-gluco configured scaffold. The second generation Cy5 probes are evaluated in Aspergillus niger secretomes and compared to the first generation. Chapter 4 describes studies towards the design and synthesis of inhibitors and ABPS for both α-N-acetylglucosaminidase and α-Nacetylgalactosaminidase. The chapter introduces an orthogonally protected ribose building block as common intermediate from which both mannoseand talose configured cyclohexenes are synthesized. Furthermore it describes a late state stereoselective C-2 inversion with an azide to install a 20 nitrogen atom at C-2. Chapter 5 presents a summary and suggestions forfuture research. Show less
