RESUMEN
Cyclophellitol is a potent and selective mechanism-based retaining ß-glucosidase inhibitor that has served as a versatile starting point for the development of activity-based glycosidase probes (ABPs). We developed routes of synthesis of eight mono- and dideoxycyclophellitols and cyclophellitol aziridines, the latter as ABPs carrying either a biotin or fluorophore linked to the aziridine nitrogen. We reveal the potency of these 24 compounds as inhibitors of the three human retaining ß-glucosidases, GBA1, GBA2 and GBA3. We show that 3,6-dideoxy-ß-galacto-cyclophellitol aziridine selectively captures GBA3 over GBA1 and GBA2 in extracts of cells overexpressing both GBA2 and GBA3. We also identify a probe that selectively labels GBA1 and GBA2 over GBA3 at lower concentrations. In sum, the here-presented studies reveal new chemistries to prepare chiral, substituted cyclitol epoxides and aziridines, add to the growing suite of cyclophellitols varying in configuration and substitution pattern, and yielded a reagent that may find use to investigate the physiological role and therapeutic relevance of the most elusive of the three retaining ß-glucosidases: GBA3.
RESUMEN
ADP-ribosylation (ADPr), as a post-translational modification, plays a crucial role in DNA-repair, immunity and many other cellular and physiological processes. Serine is the main acceptor for ADPr in DNA damage response, whereas the physiological impact of less common ADPr-modifications of cysteine and threonine side chains is less clear. Generally, gaining molecular insights into ADPr recognition and turn-over is hampered by the availability of homogeneous, ADP-ribosylated material, such as mono-ADP-ribosylated (MARylated) peptides. Here, a new and efficient solid-phase strategy for the synthesis of Ser-, Thr- and Cys-MARylated peptides is described. ADP-ribosylated cysteine, apart from being a native post-translational modification in its own right, proved to be suitable as a stabile bioisostere for ADP-ribosylated serine making it a useful tool to further biochemical research on serine ADP-ribosylation. In addition, it was discovered that the Streptococcus pyogenes encoded protein, SpyMacroD, acts as a Cys-(ADP-ribosyl) hydrolase.