Synthesis and Biological Evaluation of Deoxycyclophellitols as Human Retaining β-Glucosidase Inhibitors

Abstract

Cyclophellitol is a potent and selective mechanism-based retaining β-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 β-glucosidases, GBA1, GBA2 and GBA3. We show that 3,6-dideoxy-β-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 β-glucosidases: GBA3.

Keywords: Carbasugar; Cyclophellitol; Deoxygenation; Glucosidase; Inhibitors and activity-based probes (ABPs).

Figure 1

Structures of cyclophellitol‐based ABPs 1–5 reported previously.

Scheme 1

Reagents and conditions: (a) i. MeOH, AcCl; ii. PhCH(OCH₃)₂, CSA, MeCN; iii. NapBr, NaH, TBAI, DMF, −5 °C→r.t.; iv. pTSA, TFA, MeOH; v. TrCl, TEA, DMAP, DMF; vi. BnBr, NaH, TBAI, DMF, −5 °C→r.t.; vii. pTSA, TFA, DCM/MeOH, 41 % over 7 steps. (b) I₂, PPh₃, imidazole, THF, reflux, 95 %. (c) Zn dust, THF/H₂O, sonication, 92 %. (d) Ethyl 4‐bromocrotonate, La(OTf)₃, In powder, H₂O, 28 %. (e) 2nd generation Grubbs catalyst, DCM, reflux, 96 % for 11, 85 % for 16. (f) DIBAL−H, 0 °C→r.t., NaBH₄, THF, 84 %. (g) 13, Bu₂BOTf, TEA, −78 °C→−5 °C, H₂O₂, THF, 89 %. (h) LiBH₄, THF, 0 °C, 88 %.

Scheme 2

(A) Synthesis of glucose‐configured 3,6‐dideoxycyclophellitols. Reagents and conditions: (a) i. TsCl, TEA, DCM, 0 °C→r.t., 89 %. (b) LiAlH₄, THF, 0 °C→r.t., 76 %. (c) BnBr, NaH, TBAI, DMF, 0 °C→r.t., 95 %. (d) DDQ, β‐pinene, DCM/H₂O, 0 °C→r.t., 91 %. (e) i‐Pr₂NH, n‐BuLi, 20, CS₂, MeI, THF, −78 °C →4 °C. (f) Bu₃SnH, AIBN, toluene, reflux, 56 % over 2 steps. (g) m‐CPBA, DCM, 23: 69 %, 24: 19 %, or Oxone^®, NaHCO₃, Na₂EDTA, AcCF₃, MeCN, 0 °C, 23: 71 %, 24: 17 %. (h) Pd(OH)₂/C, H₂ 1 atm., 1,4‐dioxane, Milli‐Q, MeOH, 50 % for 25, 52 % for 26. (B) Synthesis of glucose‐configured 3‐deoxycyclophellitols. Reagents and conditions: (a) BnBr, NaH, TBAI, DMF, 0 °C→r.t., 98 %. (b) DDQ, DCM/H₂O, 0 °C, 89 %. (c) CS₂, LDA, MeI, THF, −78 °C →3 °C, 63 %. (d) Bu₃SnH, AIBN, toluene, reflux, 89 %. (e) Oxone^®, NaHCO₃, Na₂EDTA, AcCF₃, MeCN, 0 °C, 31: 39 %, 32: 36 %. (f) Pd(OH)₂/C, H₂ 1 atm., 1,4‐dioxane, Milli‐Q, MeOH, 69 % for 33, 76 % for 34.

Scheme 3

Synthesis of glucose‐configured 3‐deoxy‐β‐aziridines. Reagents and conditions: (a) NaN₃, LiClO₄, DMF, 95 °C, 88 % in total. (b) PPh₃ (beads), MeCN, 60 °C, 91 %. (c) 8‐Azido‐1‐octanol, pyridine, Tf₂O, DCM, 37, DiPEA, 83 %. (d) PPh₃ (beads), H₂O, MeCN, 70 °C, quant. (e) Li, NH₃(liq.), −70 °C→−55 °C, 58 %. (f) Cy5COOH, PFPOC(O)CF₃, DiPEA, DMF, 40, 32 % for 41, or biotin‐OSu, DiPEA, 37 % for 42.

Figure 2

Overview all compounds subjected to the GBA1, GBA2, and GBA3 enzyme inhibition assays.

Figure 3

In vitro profiling of deoxygenated cyclophellitol aziridines. (A) IC₅₀ values for inhibition of rhGBA1, GBA2 and/or GBA3 as measured in fluorogenic substrate assays (incubation time 30 min). (B) ABP labeling in lysates of GBA2/GBA3 overexpressing HEK293T cells at pH 6.0. KO: lysates of GBA1/GBA2 KO HEK293T cells were incubated with the specified ABP concentration. Lower panels: Coomassie brilliant blue (CBB) loading controls.

Figure 4

ABP labeling with 50 followed by cABPP with 2 (300 nM) in lysates of HEK293T GBA2/GBA3 overexpressing cells (incubation time (A) 30 min; (B) 3 h); (C) HEK293T WT cells (incubation time 30 min). (D) ABP labeling with 50 in rhGBA1 at pH 6.0 supplemented with 0.1 % (w/v) BSA. (E) cABPP. The sample for lane 2 was pre‐incubated with 2 (300 nM) followed by 50 (10 μM). Samples for lanes 3–9 were incubated with 50 followed by 2 (300 nM). (F) Effect of pH value (3.0–8.0) on labeling with 50 (3.5 μM) in lysates of HEK293T GBA2/GBA3 overexpressing cells. (G) Detection limit. rhGBA3 (2 pmol) was incubated with a decreasing amount of 50. (H) Comparison of labeling selectivity with 50 (top) and 1 (bottom) in lysates of HEK293T GBA2/GBA3 overexpressing cells at varying ABP concentrations (1–5 μM). KO ‐ lysates of GBA1/GBA2 KO HEK293T cells were incubated the specified ABP concentration. Lower panels: Coomassie brilliant blue (CBB) loading controls.