In vitro and in vivo comparative and competitive activity-based protein profiling of GH29 α-l-fucosidases

Abstract

GH29 α-l-fucosidases catalyze the hydrolysis of α-l-fucosidic linkages. Deficiency in human lysosomal α-l-fucosidase (FUCA1) leads to the recessively inherited disorder, fucosidosis. Herein we describe the development of fucopyranose-configured cyclophellitol aziridines as activity-based probes (ABPs) for selective in vitro and in vivo labeling of GH29 α-l-fucosidases from bacteria, mice and man. Crystallographic analysis on bacterial α-l-fucosidase confirms that the ABPs act by covalent modification of the active site nucleophile. Competitive activity-based protein profiling identified l-fuconojirimycin as the single GH29 α-l-fucosidase inhibitor from eight configurational isomers.

Fig. 1. (A) Double-displacement mechanism of retaining α-l-fucosidases. (B) Comparative and competitive activity-based profiling of GH29 α-l-fucosidases presented here.

Fig. 2. Structures of inhibitors and ABPs presented in this study.

Scheme 1. Synthesis of aziridine ABPs 1, 2, 3 and inhibitors 4, 5. Reagents and conditions: (a) DBBT, Et₃N, CH₂Cl₂, –78 °C, 71%; (b) (i) LiBH₄, THF, 83%; (ii) Grubbs 2^nd generation, CH₂Cl₂, 95%; (c) p-TsCl, Et₃N, CH₂Cl₂, 87%; (d) LiAlH₄, THF, 0 °C to rt, 87%; (e) (i) Li, NH₃ (l), THF, –60 °C, 73%, (ii) 2,2-dimethoxypropane, CSA, rt, 60%; (f) (i) CCl₃CN, DBU, CH₂Cl₂, rt; (ii) NaHCO₃, I₂, H₂O, rt, 46%; (g) (i) 37% HCl (aq.), MeOH, 60 °C; (ii) NaHCO₃, MeOH, rt, 65%; (h) EEDQ, benzoic acid or acetic acid, DMF, 0 °C, 7% 4, 28% 5; (i) EEDQ, 24, DMF, 0 °C 25%; (j) CuSO₄, sodium ascorbate, 25, 26 or 27, rt, 19% 1, 12% 2, 13% 3.

Fig. 3. Structures of 1-deoxy-l-fuconojirimycin 6 and configurational isomers 7–13.

Scheme 2. Synthesis of 1-deoxy-l-fuconojirimycin 6. Reagents and conditions: (a) (i) Et₂O, DIBAL-H, –80 °C, (ii) MeOH, –90 °C, (iii) amine 29, NaOMe, (iv) NaBH₄, –15 °C to rt, 88%; (b) Boc₂O, 50 °C, 100%; (c) Grubbs 1^st generation, CH₂Cl₂, 88%; (d) (i) K₂OSO₄·2H₂O, NMO, acetone–H₂O (1 : 1), –10 °C, (ii) Ac₂O, pyridine, DMAP, 0 °C, 88% (33 : 34 = 1 : 3); (e) (i) K₂CO₃, MeOH, (ii) TBAF, THF, (iii) 6M HCl, MeOH, 71%.

Fig. 4. In vitro activity-based protein profiling of GH29 α-l-fucosidases. (A) Labeling with ABP 1 of recombinant α-l-fucosidase from Bacteroides thetaiotaomicron 2970. (B) In vitro labeling of lysate of spleen from a Gaucher disease patient, α-(1-2,3,4)-fucosidase from Xanthomonas sp. and α-(1-6)-fucosidase from Elizabethkingia miricola with ABP 2. (C) In vitro labeling of human healthy and Gaucher disease spleen. (D) Direct labeling of GH29 α-l-fucosidases with green-fluorescent ABP 1 and retaining β-glucosidases GBA, GBA2 and GBA3 with red-fluorescent JJB75. The location of albumin autofluorescence is designated on each gel.

Fig. 5. In vivo labeling of α-l-fucosidases in mice with various concentrations of ABP 1 during 2 hours. In vivo labeling compared to maximal labeling with excess ABP 1 of matched homogenates (Ctrl). The location of albumin autofluorescence is designated on each gel.

Fig. 6. Competitive ABPP on recombinant FUCA1 with deoxyfuconojirimycin 6 and configurational analogues 7–13 towards α-l-fucosidases, with ABP 1 labeling as readout.

Fig. 7. Crystal structure of α-l-fucosidase from Bacteroides thetaiotaomicron in complex with 5. Catalytic residues are annotated: Asp 229 (nucleophile) and Glu 288 (acid/base). Electron density displayed is F_o–F_c density from phases calculated prior to the inclusion of 5 in refinement, contoured at 3σ. Figure was prepared using CCP4MG.