1,6-Cyclophellitol Cyclosulfates: A New Class of Irreversible Glycosidase Inhibitor

Abstract

The essential biological roles played by glycosidases, coupled to the diverse therapeutic benefits of pharmacologically targeting these enzymes, provide considerable motivation for the development of new inhibitor classes. Cyclophellitol epoxides and aziridines are recently established covalent glycosidase inactivators. Inspired by the application of cyclic sulfates as electrophilic equivalents of epoxides in organic synthesis, we sought to test whether cyclophellitol cyclosulfates would similarly act as irreversible glycosidase inhibitors. Here we present the synthesis, conformational analysis, and application of novel 1,6-cyclophellitol cyclosulfates. We show that 1,6-epi-cyclophellitol cyclosulfate (α-cyclosulfate) is a rapidly reacting α-glucosidase inhibitor whose ⁴C₁ chair conformation matches that adopted by α-glucosidase Michaelis complexes. The 1,6-cyclophellitol cyclosulfate (β-cyclosulfate) reacts more slowly, likely reflecting its conformational restrictions. Selective glycosidase inhibitors are invaluable as mechanistic probes and therapeutic agents, and we propose cyclophellitol cyclosulfates as a valuable new class of carbohydrate mimetics for application in these directions.

Figure 1

Conformational itinerary of retaining glucosidase reaction pathways and conformation of covalent inhibitors. (a) Reaction itineraries of retaining β-glucosidases and retaining α-glucosidases, showing conformations of the Michaelis complex, transition state, and covalent intermediates. (b) Structures of cyclophellitol (1), cyclophellitol aziridine (2), 1,6-epi-cyclophellitol (3), 1,6-epi-cyclophellitol aziridine (4), α-cyclosulfate (5), and β-cyclosulfate (6).

Figure 2

Conformational free energy landscapes of cyclosulfates 5 and 6. Cyclosulfates 5 (a) and 6 (b) adopt ⁴C₁ ground state conformations, with a broad energy minimum extending toward ⁴H₃. The x and y axes of each graph correspond to the φ and θ Cremer–Pople puckering coordinates (in degrees), respectively. Isolines are 1 kcal/mol.

Figure 3

Synthesis of cyclophellitol cyclosulfates 5 and 6, and inactivation of GAA by compound 5. (a) Compounds 5 and 6 were prepared from cyclohexene 11. Reagents and conditions: (i) BnBr, TBAI, NaH, DMF, rt, 18 h, 70%; (ii) RuCl₃, NaIO₄, EtOAc, ACN, 0 °C, 2 h (13, 39%; 14, 26%); (iii) (a) SOCl₂, Et₃N, DCM, 0 °C, (b) RuCl₃, NaIO₄, CCl₄, ACN, 0 °C, 3 h (15, 59%; 16, 62%); (iv) H₂, Pd/C, MeOH, rt, 18 h (5, 71%; 6, 72%). (b) Semilogarithmic plots of residual activity of GAA versus time at 9, 8, 7, 6, 5, 4, 3, and 2 μM α-cyclosulfate 5. (c) Plot of pseudo first order rate constants from panel c vs concentration of 5.

Figure 4

Structures of reacted 4 and 5 bound to wild type CjAgd31B and reacted 6 bound to wild type TxGH116. (a) Unreacted (left) and reacted (right) 5 in complex with CjAgd31B D412N nucleophile mutant and wt CjAgd31B respectively. Unreacted 5 adopts a ⁴C₁ ring conformation in the active site of CjAgd31B, mimicking the Michaelis complex conformation of GH31 α-glucosidase substrates. Reacted 5 adopts a ¹S₃ covalent intermediate conformation. (b) Unreacted (left) and reacted (right) 4 in complex with CjAgd31B D412N nucleophile mutant and wt CjAgd31B respectively. Unreacted 4 adopts a ⁴H₃ TS conformation. Reacted 4 adopts the same ¹S₃ intermediate conformation as observed for 5. (c) Unreacted (left) and reacted (right) β-cyclosulfate 6 with TxGH116. Unreacted 6 in complex with GH116 β-glucosidase TxGH116 adopts a ⁴C₁ conformation in the enzyme active site, which is poorly poised for attack by the enzyme nucleophile Glu441, and thus reacts extremely slowly. (Two conformations for the enzyme catalytic acid/base Asp593 can be seen in this complex.) Reacted 6 can be observed after extended soaking, and also adopts a ⁴C₁ conformation, covalently bound to the enzyme nucleophile. Electron density for protein side chains is REFMAC maximum-likelihood/σ_A-weighted 2F_o – F_c contoured to 0.44–0.51 and 0.40–0.42 electron/ų for CjAgd31B and TxGH116 complexes, respectively. Electron density for ligand is F_o – F_c maps calculated just prior to building in ligand, contoured to 0.17–0.26 and 0.27–0.35 electron/ų for CjAgd31B and TxGH116 complexes, respectively. nuc. = nucleophile; a./b. = acid/base.

Figure 5

In vitro and in situ inhibition of GAA and GANAB. (a) 5 inhibits labeling of GAA and GANAB by fluorescent ABP 13 in fibroblast lysates in a concentration and time dependent manner. (b) 5 inhibits labeling of several α-glucosidases in mouse intestine lysate by 13. Sucrase-isomaltase (Sis), maltase-glucoamylase (MGAM), GAA, and GANAB are labeled by 13, as well as some off-target β-glucosidase labeling (LPH and GBA). α-Glucosidase labeling can be abrogated by preincubation with 5, while β-glucosidase labeling persists, demonstrating the superior selectivity of 5 compared to 13. (c) In situ inhibition of GAA and GANAB in fibroblasts at pH 4.0 and 7.0 respectively by 5 at incubation times of 2 and 24 h, followed by labeling of GAA and GANAB by cyclophellitol aziridine Cy5 probe 13. (d) Apparent IC₅₀s for in situ inhibition of GAA and GANAB enzyme activity by 5. Reported IC₅₀s are mean ± standard deviation from two biological replicates, each with three technical replicates.