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Syl-Configured Cyclophellitol-Aziridines for Si- Multaneous Differential Fluorescent Labeling of Β-Glucosidases and Β- Galactosidases

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Syl-Configured Cyclophellitol-Aziridines for Si- Multaneous Differential Fluorescent Labeling of Β-Glucosidases and Β- Galactosidases UvA-DARE (Digital Academic Repository) Glycosphingolipids and the central regulation of metabolism Sugar analogues as research tools Herrera Moro Chao, D. Publication date 2017 Document Version Other version License Other Link to publication Citation for published version (APA): Herrera Moro Chao, D. (2017). Glycosphingolipids and the central regulation of metabolism: Sugar analogues as research tools. General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). 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UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl) Download date:28 Sep 2021 Chapter 3 Chapter Application of fluorescent β-glucopyranosyl-configured cyclophellitol-aziridines for simulta- neous differential fluorescent label- ing of β-glucosidases and β-galactosidases Marques A.R.A., Herrera Moro D., Artola M., Oussoren S.V., Flo- rea B.I., Willems L.I., Beenakker T., Li K.Y., Jiang J., Scheij S., van Roomen C.P.A.A., Ottenhoff R., Boot R.G., Overkleeft H.S., Aerts J.M.F.G. In preparation Application of fluorescent β-glucopyrano- syl-configured cyclophellitol-aziridines for si- multaneous differential fluorescent labeling of β-glucosidases and β- galactosidases. André R. A. Marques1, Daniela Herrera Moro1, Marta Artola2, Saskia V. Oussoren3, Bogdan I. Florea2, Lianne I. Willems2, Thomas Beenakker2, Kah-Yee Li2, Jianbing Jiang2, Saskia Scheij1, Cindy P. A. A. van Roomen1, Roelof Ottenhoff1, Rolf G. Boot3, Hermen S. Overkleeft2, Johannes M. F. G. Aerts1,3 1Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, 1105 AZ, Amsterdam, The Netherlands 2Department of Bioorganic Synthesis, Leiden Institute of Chemistry, Leiden University, 2333 CC, Leiden, The Netherlands 3Department of Medical Biochemistry, Leiden Institute of Chemistry, Leiden University, 2333 CC, Leiden, The Netherlands *Corresponding authors: H.S. Overkleeft ([email protected]) and J.M.F.G. Aerts ([email protected]) Einsteinweg 55, 2333 CC Leiden, The Netherlands ABSTRACT The natural product, cyclophellitol, modified at C6 with a BODIPY fluorophore specifical- ly binds to the catalytic nucleophile of lysosomal β-glucosylceramidase (GBA) in an ac- tivity-based manner. Likewise, fluorescent β-galactopyranosyl-configured cyclophellitol labels specifically lysosomal β- galactosylceramidase (GALC). In contrast, cyclophellitol aziridines equipped at the aziridine-nitrogen with a fluorophore, are in-class broad-spec- trum retaining β-glucosidase activity-based probes (ABPs). Here we document that the latter ABPs also labels with high affinity the retaining β-galactosidase GALC. With- cy clophellitol aziridine ABPs active GALC is detectedin vitro in mouse tissue homogenates and in vivo in cells and brain of mice upon intracerebroventricular administration of the probes. At elevated concentrations cyclophellitol aziridines also label lysosomal acid β-galactosidase (GLB1) as confirmed via chemical proteomics analysis with a biotinylat- ed analogue of the probe. We exploited the broad specificity of cyclophellitol aziridine to differentially visualize GBA1, GBA2, GALC and GLB1 by sequential labeling. Pre-incu- bations with cyclophellitol and high affinity GBA2 inhibitors can assist in the selective labeling of the enzyme of choice. In conclusion, cyclophellitol-aziridine derived ABPs can be employed for the simultaneous visualization of a broad spectrum of human retaining β-glycosidases. 100 INTRODUCTION The design and application of activity-based probes (ABPs) targeting human glycosidases that are implicated in inherited lysosomal storage diseases holds great promise (1–5). They offer valuable research tools to study the life cycle of targeted glycosidases in in- tact cells and organisms. Such ABPs can also be used in diagnosis of lysosomal storage disorders (LSDs) by visualizing the amount of residual active enzyme molecules in intact cells. Finally, they can be employed to study the efficacy of therapeutic interventions for LSDs. We have earlier designed different classes of ABPs for human retaining β- glu- cosidases. Taking into account that the natural product cyclophellitol is a specific and potent suicide inhibitor of lysosomal β-glucosylceramidase (glucocerebrosidase, GBA) (6), we designed cyclophellitol derivatives modified at C6 (glucopyranose numbering) with a fluorescent BODIPY (compound 2 and 3, Figure 1) (7). ABPs 2 and 3 were found 3 to covalently bind, with high specificity and in an activity-based manner, to the catalytic nucleophile residue E340 of GBA (7, 8). In situ labeling of active GBA in intact cells with ABPs 2 or 3 is feasible, and allows the visualization of the intralysosomal location of the enzyme. Intravenous infusion of mice with these ABPs results in specific labeling of GBA in most tissue, but not in the brain and eye (9). Moreover, intracerebroventricular ad- ministration of ABPs2 and 3 to mice allows visualization of active GBA in brain with high spatial resolution (10). GBA is deficient in patients suffering from Gaucher disease (GD) and a reduction in the amount of active enzyme can be detected upon incubation of cultured GD fibroblasts and subsequent labeled protein analysis by gel electrophoresis and quantitative fluorescence scanning (7). The approach for GBA was then successful- ly adapted to target the lysosomal enzyme β-galactosylceramidase (galactocerebrosi- dase, GALC). A β- galactopyranosyl-configured cyclophellitol analogue modified at C6 with a BODIPY (compound 6) was synthesized and found to specifically label GALC (11) (Marques et al. submitted for publication). The latter enzyme is deficient in Krabbe dis- ease, a LSD with devastating neuropathology (12). Next, in order to generate a broad spectrum ABP, we designed N-substituted cyclophellitol aziridines (compounds 7 and 8) (9, 13) in which the fluorophore is situated roughly at the position also occupied by the aglycon in β-glucosidase substrates. Indeed ABP 8 was found to covalently and irre- versibly react with all known human retaining β-glucosidases, namely, GBA, GBA2, GBA3 and lactase-phloridzin hydrolase (9). The cytosolic enzyme GBA3 has broad substrate specificity, and is able to hydrolyze artificial β-glucosyl and β-galactosyl substrates with about equal efficiency (14). Using low nanomolar concentrations of compounds7 and 8, we were able to specifically visualize the retaining β-glucosidases GBA, GBA2, GBA3 and LPH in cell and tissue context. Next, Hoornet al. used biotinylated β-glucopyranosyl-con- figured cyclophellitol-aziridine (compound 9) in an affinity purification experiment with an Arabidopsis thaliana lysate. Besides retaining β-glucosidases, several β-xylosidases, β-glucuronidases and β-galactosidases were also identified. A concentration of 2 μM was used in the affinity purification experiment, which suggested to us that at higher concentrations also β-galactosidases may react with β-glucopyranosyl- configured -cy clophellitol-aziridine. There are two human lysosomal β-galactosidases, GALC and acid β- galactosidase (GLB1). GALC is deficient in Krabbe disease and GLB1 in GM1 ganglio- sidosis, two neuronopathic lysosomal storage disorders. With this in mind we re-exam- ined the target specificity of β-glucopyranosyl-configured cyclophellitol-aziridine ABPs (7, 8 and 9). We found that GALC is labeled at high nanomolar ABP concentration and GLB1 at micromolar concentration. The findings sparked the development of a single assay to simultaneously visualize β-glucosidases and β-galactosidases in biological sam- ples. The experimental evidence for this is here described and discussed. 101 O O O R R HO OH HO OH F N OH OH F B O N 1: R = N3 5: R = H N 2: R = BODIPY red 6: R = BODIPY red N N : = reen 2 R BODIPY g BODIPY red 4: R = Cy5 F N B O R F N N 7 OH N N N R HO NH BODIPY green HO OH HO OH OH 7: R = BODIPY red 10 O 8: R = BODIPY reen g N 9: R = Biotin N H N N N Cy5 HO HO HO NH N O N O HO 7 HO Cl S H OH OH HN N N N H 5 11 12 NH O N N O Biotin Figure 1. Structure of compounds 1-12 used in study. Compound 1: β-glucopyranose az- ido-cyclophellitol; compound 2: β-glucopyranose cyclophellitol BODIPY red; compound 3: β-glucopyranose cyclophellitol BODIPY green; compound 4: β-glucopyranose cyclophellitol Cy5 blue; compound 5: β-galactopyranose cyclophellitol; compound 6: β-galactopyranose cyclophellitol BODIPY red; compound 7: β-glucopyranose cyclophellitol-aziridine BODIPY red; compound 8: β-glucopyranose cyclophellitol-aziridine BODIPY green; compound 9: biotinylated β- glucopyranose cyclophellitol-aziridine; compound 10: ABB166; compound 11: α-1-C-nonyl-DIX (anDIX); compound 12: N-[5’-adamantane-1’-yl-methoxy]-pen- tyl-1-deoxynojirimycin. MATERIALS AND METHODS Synthesis of compounds The synthesis of cyclophellitol derivatives 1-3 (7), β-galactopyranose-configured cy- clophellitol derivatives5 -6 (11), β-glucopyranose-configured cyclophellitol aziridines7 -9 (9, 13), competitive GALC inhibitor ABB166 10 (15), GBA3 inhibitor anDIX 11 (16) and GBA2 inhibitor AMP-DNM 12 (17) was reported previously. β-Glucopyranose cyclophelli- tol Cy5 4 was synthesized by copper–catalyzed click chemistry of azidocyclophellitol 1 with Cy5 alkyne. β-Glucopyranose cyclophellitol Cy5 4: Azidocyclophellitol 1 (24.2 mg, 0.12 mmol) and the desired Cy5- alkyne (84 mg, 0.15 mmol) were dissolved in BuOH/ toluene/H2O (6 mL, 1:1:1, v/v/v). CuSO4 (0.024 mL, 1 M in H2O) and sodium ascorbate (0.024 mL, 1 M in H2O) were added and the reaction mixture was heated at 80 ºC for 18 h.
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