of β-Glucosidase Inhibitor.

: Gluco-azoles competitively inhibit glucosidases by transition-state mimicry and their ability to interact with catalytic acid residues in glucosidase active sites. We noted that no azole-type inhibitors described, to date, possess a protic nitrogen characteristic for 1 H -imidazoles. Here, we present gluco-1 H imidazole, a gluco-azole bearing a 1 H -imidazole fused to a gluco-pyranose-configured cyclitol core, and three close analogues as new glucosidase inhibitors. All compounds inhibit human retaining (cid:533) -glucosidase, GBA1, with the most potent ones inhibiting this enzyme (deficient in Gaucher disease) on a par with glucoimidazole. None inhibit glucosylceramide synthase, cytosolic (cid:533) - glucosidase GBA2 or (cid:302) -glucosidase GAA. Structural, physical

by transition-state mimicry and their ability to interact with catalytic acid residues in glucosidase active sites. We noted that no azole-type inhibitors described, to date, possess a protic nitrogen characteristic for 1H-imidazoles. Here, we present gluco-1Himidazole, a gluco-azole bearing a 1H-imidazole fused to a glucopyranose-configured cyclitol core, and three close analogues as new glucosidase inhibitors. All compounds inhibit human retaining -glucosidase, GBA1, with the most potent ones inhibiting this enzyme (deficient in Gaucher disease) on a par with glucoimidazole. None inhibit glucosylceramide synthase, cytosolicglucosidase GBA2 or -glucosidase GAA. Structural, physical and computational studies provide first insights into the binding mode of this conceptually new class of retaining -glucosidase inhibitors.

Scheme 1. Synthesis of 1H-imidazoles 6 and 7. a a
Reagents and conditions: a) RuCl 3 á3H 2 O (7 mol%), NaIO 4 , We determined competitive kinetic constants and IC 50 values for 1H-imidazoles 6-9 as compared with glucoimidazole 5 for inhibition of the retaining glucosidases TmGH1 from Thermotoga maritima, 20 TxGH116 from Thermoanaerobacterium xylanolyticum, 21 sweet almond -glucosidase (GH1), human lysosomal acid -glucosylceramidase (GBA1, GH30), human cytosolicglucosylceramidase (GBA2, GH116), murine glucosylceramide synthase (GCS) and human acid -glucosidase (GAA, GH31). Gluco-1H-imidazole 6 displayed micromolar inhibitory activity against most -glucosidases tested, with K i values ranging from ~3.9 µM against GBA1 to ~69 µM against TxGH116 (Table 1). Remarkably, given their apparent structural similarity, 1Himidazole 6 proved to be a weaker -glucosidase inhibitor than glucoimidazole 5, which inhibited -glucosidases at nanomolar concentrations. Attachment of a butyl moiety, as in 7, increased the inhibitory potency of the gluco-1H-imidazole scaffold. Whilst this increase in potency was modest for TmGH1 and TxGH116, 7 inhibited sweet almond -glucosidase and GBA1 with nanomolar potency. Notably, the biological role of GBA1 is breakdown of amphiphilic glucosylceramide. 22 Conduritol-1H-imidazole 8 did not effectively inhibit any glucosidases tested except for GBA1, consistent with a loss of interactions at the O6 position, but also with the fact that GBA1 is effectively inhibited by conduritol B epoxide (CBE). 23 As with the gluco-1H-imidazoles, addition of a butyl moiety to the conduritol-1H-imidazole scaffold increased activity against sweet almond -glucosidase and GBA1, rendering 9 a nanomolar inhibitor of these enzymes. No compounds displayed any inhibitory activity against GBA2, GCS orglucosidase GAA under our assay conditions. Competitive, selective GBA1 inhibitors are considered attractive starting points for the development of pharmacological chaperones: compounds that stabilize the fold of mutant, partially malfunctioning GBA1 in Gaucher patients. 24,25 Compounds 7 and 9 (as well as known glucoimidazole 5) are in our opinion attractive starting points for this purpose.
One striking result displayed in Table 1 is that 1H-imidazole 6 is a comparatively much weaker retaining -glucosidase inhibitor than glucoimidazole 5. Since the structures are closely related, we elected to investigate this result in more depth. Literature studies have shown that the inherent basicity of the azole ring in compounds 2-5 correlates to the inhibitory potency. 26 Basicity plays a negligible role here, since the experimental pK AH values of 5 and 6 are almost identical (5: pK AH 6.2, lit. 6.1 26 ; 6: pK AH 6.0; Figure  S1, S2). We also explored the interaction of gluco-1H-imidazoles with TmGH1 and TxGH116 by isothermal titration calorimetry (ITC) at pH 5.8 (activity optimum 11,21 ) and pH 6.8 (where 5 shows strongest inhibition) 11 . In line with the K i data (Table 1), ITC titrations at both pH values showed nanomolar binding affinities for 5, and micromolar affinities for 6 and 7 (Table S3). The enthalpic contributions in active site binding for 1H-azoles are smaller than those observed for 5, and only partially compensated for by increased entropic contributions to the binding energy. In line with what has been reported for 5, 11 the presence of a hydrophobic moiety in 7 reduced its enthalpy of binding compared to 6, which was counterbalanced by an increase in favorable entropic contributions to binding. Values are mean ± SD from triplicate experiments. Assays measured at a pH 6.8 using -p-NPG substrate; b pH 5.2 using -2,4-DNPG substrate; c Apparent IC 50 . d Lit. K i = 0.100 µM. 6 Next, the X-ray structure of 6 in complex with TmGH1 was determined to 1.7 Å (PDB: 5OSS), and compared to the structure of the same enzyme complexed with 5 (PDB: 2CES). 11 These structures revealed the binding mode of both compounds to be similar. We observed a single molecule of 6 in the active site after ligand soaking, adopting a 4 E conformation, similar to the complex of 5 and TmGH1 (Figure 2a). 11 H-bonding interactions made by 6 to active site residues were identical to those observed with 5, albeit with a slight 'upwards' tilt for 6 compared to 5 (~0.4 or 0.5 Å 'upward' shifts at the apical imidazole carbon, compared with ligands in chains A or B of 2CES respectively; Figure 2b, Figure  S3). Crystal structures in TxGH116 at 2.1 Å resolution ( Figure  2c,d) also revealed similar binding modes 21 and a ~0.5 Å 'upwards' tilt for 6 compared to 5 (PDB: 5OST and 5BX4, respectively). For both TmGH1 and TxGH116 complexes, B-factors in the 'imidazole' portion of 6 were markedly higher when compared to the 'glucose' portion of the molecule, indicating the imidazole of 6 was more disordered in the crystal structure and may be bound less strongly. No strong B-factor trend was observed for complexes with 5 ( Figure S3).

Table 1. Inhibition constants (K i ) and IC 50 values in µM for compounds 5-9.
The underlying cause for the reduced potency of gluco-1Himidazole 6 compared to 5 is most likely the combination of a number of factors. We propose that repositioning of the N1 atom (from the bridgehead position in 5 to the position in 6) brings two major consequences that together reduce the binding affinity of 6 compared to 5. Firstly, considering the situation where the imidazole is in a neutral state 28 : the free lone pair of the N2 atom in 5 can laterally coordinate to the acid/base residue of the bound glucosidase in typical anti-protonating glucosidases 14 (although TxGH116 lacks this interaction due to the unusual placement of its acid/base residue 21 ). This lateral positioning of N2 is main-tained in 6, as observed in its complex with TmGH1 ( Figure 2a). However, and in contrast to 5, 1H-imidazole 6 may undergo prototropic tautomerism (Figure 3a). Thus, whilst the overall pK AH values of 5 and 6 are similar, the N2 lone pair of 6 may be less available for interaction with the glucosidase acid/base, reducing the binding affinity of 6 compared to 5. Protonation of the imidazole in turn (either in solution or by proton abstraction from the acid/base residue) 28 results in positive charge delocalization. Resulting charge-charge interactions with enzyme active site carboxylates are thought to contribute substantially to enzyme binding energy of azole-type inhibitors. 29 We calculated the Mulliken charge on all atoms for protonated 5 and 6 by DFT. Protonation of the azole ring in 5 produces a + charge on the 'anomeric' carbon, which is ideally located for a charge-charge interaction with a retaining glucosidase active site nucleophile. Conversely, protonation of 6 leads to a + charge largely delocalized onto the 'apical' carbon atom of the imidazole, with the overall + charge also being less pronounced (Figure 3b). This apical + charge is located distal from the catalytic nucleophile and thus poorly positioned for charge-charge interactions, which may explain the reduced binding enthalpy observed in ITC for gluco-1Himidazoles 6 compared to 5. The small 'upwards' shift and increased imidazole B-factors, observed in crystal structure complexes of 6 compared to 5 is also consistent with a weaker chargecharge interaction of the imidazole portion of 6 with the enzyme catalytic nucleophile. Interestingly, in contrast to neutral 6, glucoimidazole 5 also contains a significant + character (+0.306 Mulliken charge) on the 'anomeric' carbon in its neutral state (see SI).
In conclusion, we have described a new class of competitiveglucosidase inhibitors: the 1H-gluco-azoles. The synthetic route is flexible regarding substituents on the imidazole ring, and can likely be transferred to configurational isomers by applying this route to configurational isomers of cyclohexene 10. 30 Our compounds resemble to some extent the 1H-imidazoles reported by Li and Byers 31 ,and Field et al.,32 whose simple, achiral molecules inhibit several glucosidases as well, though likely by a different mode of action. 1H-Imidazole 6 appeared a poorer inhibitor than compound 5, and we hypothesize this is caused by delocalization  of the lone pair on the nitrogen atom due to tautomerism and/or impaired + charge development at the anomeric center. Introduction of an alkyl substituent on the 1H-imidazole ring (as in 7) as well as 'deletion' of the methylene carbon (as in 9) yielded potent competitive inhibitors of the human lysosomal glucosylceramidase GBA1, selective over human cytosolic GBA2 and GCS. These and related compounds we believe have potential to be developed into pharmacological chaperone candidates for Gaucher disease and perhaps also neuropathological disorders (in particular, Parkinsonism) that are characterized by partial deficiency in GBA. 33,34 ASSOCIATED CONTENT

Supporting Information
The Supporting Information is available free of charge on the ACS Publications website.
Experimental data and procedures, enzyme kinetics, DFT calculations, crystallographic data (PDF).

Notes
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