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Development of Tunable Mechanism-Based Carbasugar Ligands that Stabilize Glycoside Hydrolases through the Formation of Transient Covalent Intermediates
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Cite this: ACS Catal. 2024, 14, 19, 14769–14779
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https://doi.org/10.1021/acscatal.4c04549
Published September 20, 2024

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Abstract

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Mutations in many members of the set of human lysosomal glycoside hydrolases cause a wide range of lysosomal storage diseases. As a result, much effort has been directed toward identifying pharmacological chaperones of these lysosomal enzymes. The majority of the candidate chaperones are active site-directed competitive iminosugar inhibitors but these have met with limited success. As a first step toward an alternative class of pharmacological chaperones we explored the potential of small molecule mechanism-based reversible covalent inhibitors to form transient enzyme–inhibitor adducts. By serial synthesis and kinetic analysis of candidate molecules, we show that rational tuning of the chemical reactivity of glucose-configured carbasugars delivers cyclohexenyl-based allylic carbasugar that react with the lysosomal enzyme β-glucocerebrosidase (GCase) to form covalent enzyme-adducts with different half-lives. X-ray structural analysis of these compounds bound noncovalently to GCase, along with the structures of the covalent adducts of compounds that reacted with the catalytic nucleophile of GCase, reveal unexpected reactivities of these compounds. Using differential scanning fluorimetry, we show that formation of a transient covalent intermediate stabilizes the folded enzyme against thermal denaturation. In addition, these covalent adducts break down to liberate the active enzyme and a product that is no longer inhibitory. We further show that the one compound, which reacts through an unprecedented SN1′-like mechanism, exhibits exceptional reactivity–illustrated by this compound also covalently labeling an α-glucosidase. We anticipate that such carbasugar-based single turnover covalent ligands may serve as pharmacological chaperones for lysosomal glycoside hydrolases and other disease-associated retaining glycosidases. The unusual reactivity of these molecules should also open the door to creation of new chemical biology probes to explore the biology of this important superfamily of glycoside hydrolases.

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Introduction

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As the number of glycoside hydrolases implicated in diseases continues to rise, the pursuit of ligands for these enzymes has gained increasing attention. (1,2) Ligands for this broad and diverse class of enzymes have entered the clinic (3,4) and several have gained clinical approval. (5,6) Many genetic diseases associated with glycoside hydrolases are driven by point mutations causing misfolding and consequent failure of the mutant enzyme to traffic properly to its target cellular compartment. Particular attention has been given to lysosomal glycoside hydrolases because once they exit the endoplasmic reticulum (ER) and reach the acidic environment of the lysosome they exhibit improved stability. Accordingly, one approach to therapeutic exploitation of active site-targeted ligands has been to explore their potential as pharmacological chaperones, which are generally high affinity iminosugars that have a weakly basic amine functionality. (6−8) Such pharmacological chaperones are able to bind to the enzyme within the ER, which leads to stabilization of the enzyme and a reduction in its premature degradation by the quality control mechanisms of the cell. This pharmacological stabilization accordingly enables increased trafficking of the chaperoned enzyme to lysosomes. Once in the lysosome, the pharmacological chaperone gradually diffuses out of this compartment allowing the enzyme to execute its essential function of degrading its glycoconjugates substrates.
While iminosugars continue to attract much attention as pharmacological chaperones, with one even gaining clinical approval, (5,6) an emerging class of alternative ligands for these enzymes include various elegant mechanism-based covalent inactivators including epoxides, (9) fluorosugars, (10) and aziridines. (11,12) All of these take advantage of the natural catalytic machinery of these enzymes to bind covalently to the catalytic nucleophile within the active site. Indeed, in a manner similar to iminosugars, irreversible binding of covalent inhibitors within the active site has been shown to stabilize the target protein against unfolding. (10,13) Such stabilization against unfolding has been found to correlate with improved trafficking of these lysosomal glycosidases, presumably by decreasing their misfolding. For example, a cyclophellitol-derived inactivator has been shown to increase the lysosomal quantity of the L444P variant β-glucocerebrosidase (GCase), a mutant that is prone to unfolding in the ER, but this did not translate into increased lysosomal activity of the enzyme. (14) Indeed, these three classes of covalent inactivators tend to either be irreversible or to result in inactive enzyme-adducts that have very long half-lives. For this reason, none of the currently known covalent ligands can serve as pharmacological chaperones because they cannot modulate enzyme activity in a useful controlled manner.
Nevertheless, we were intrigued by the idea of creating glycoside hydrolase covalent inhibitors that lead to formation of transient covalent glycosyl enzyme intermediates, but which can still stabilize the enzyme through favorable interactions within the active site (Figure 1c). (15) We noted that recently carbasugars, which resemble various bioactive natural products, have been modified to function as covalent inhibitors of various glycoside hydrolases. (16−18) This class of inhibitor makes use of strategic positioning of an alkene or cyclopropyl moiety to stabilize the carbocation that results from departure of a leaving group from the pseudoanomeric center, leading to formation of a covalent inhibitor-enzyme adduct (Figure 1d). However, unlike the aziridine and epoxide inactivators that form a dead-end enzyme–inhibitor complex, the carbasugar-enzyme adduct can be turned over by the enzyme leading to time dependent recovery of its activity. Notably, carbasugar derivatives exhibit differing behaviors in terms of their turnover by different enzymes. (16−18) These observations suggested to us that it may be possible to tune the reactivity of carbasugar analogues to create compounds that rapidly and covalently modify the enzyme within the active site but then turnover during a desirable time scale to liberate the active enzyme (Figure 1).

Figure 1

Figure 1. Small molecule covalent inhibitors and the mechanism of action of retaining glycosidases. (a) Mechanism used by glucocerebrosidase for hydrolysis of its natural β-glucoside substrate. (b) Kinetic scheme for GCase-catalyzed turnover of β-d-glucopyranoside substrates. (c) Mechanism of action for carbasugar covalent inhibitors. (d) Kinetic scheme for covalent carbasugar inhibitors.

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We decided to pursue this problem by initially focusing on the family 30 glycoside hydrolase (GH30) human β-glucocerebrosidase (GCase), which is of high interest. Homozygous loss-of-function mutations in the gene (GBA1) encoding GCase cause Gaucher Disease (GD), (19) a lysosomal storage disease that is characterized by the accumulation of glucosylceramide (GlcCer) within various organs of the body. Moreover, heterozygous destabilizing mutations in GBA1 that lead to premature degradation of GCase have been linked to Parkinson Disease (PD), (20) greatly raising interest in this enzyme. Here we describe the design and synthesis of a series of covalent inhibitors of GCase, demonstrate that these compounds can be tuned to have desirable kinetic properties, determine the structure of these ligands bound to GCase, and show that formation of the covalent enzyme intermediate leads to an increase in the thermal stability of this enzyme. We further show the ability of such carbasugar analogues to engage with enzymes having varied substrate requirements by examining a structurally distinct covalent inhibitor lacking a pseudoanomeric center leaving group against both GH30 GCase and the yeast GH13 α-glucosidase.

Results

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Most retaining glycosidases, including GCase and yeast α-glucosidase, act on their respective substrates using a two-step catalytic mechanism (15) involving formation of a transient covalent glycosyl enzyme intermediate, which typically has a half-life for hydrolysis in the millisecond time regime (21,22) (Figure 1a). The first step, known as glycosylation (kb, Figure 1b), involves departure of the aglycon leaving group and formation of the glycosyl enzyme intermediate. The second step, known as deglycosylation (kc, Figure 1b), involves breakdown of this intermediate by the attack of water at the anomeric center. The processing of substrates by these enzymes can be monitored by incorporating chromogenic or fluorogenic leaving groups.
Analogously, allylic carbasugars also form transient covalent adducts that break down to regenerate active enzyme (Figure 1c) at a rate that is governed by k3 (Figure 1d). (17,18)

Synthesis and Testing of First-Generation Allylic Carbasugar Covalent Inhibitors

As a first step toward our goal of creating allylic carbasugar covalent inhibitors that have a tunable reactivation rate constant (k3), we synthesized the known carbaglucose 1 (Figure 2), (18) which bears a chromogenic 2,4-dinitrophenolate (2,4DNP) leaving group, and analogue 2, which contains a fluorogenic 6,8-difluoro-4-methylumbelliferone (6,8DiFMU) (Scheme S1, Supporting Information). Both aglycons have similar leaving group abilities as judged by the pKa values of the corresponding phenols (2,4DNP pKa = 3.96, 6,8DiFMU pKa = 4.81). We found these carbaglucose compounds were good inhibitors with IC50 values of 0.17 ± 0.03 and 14.3 ± 1.7 μM for 1 and 2, respectively (Figure S1, Tables S1 and S2, Supporting Information), IC50 values that are comparable to many competitive inhibitors of GCase. (23) Nonlinear least-squares fitting of our kinetic data showed these compounds displayed classic signs of mixed inhibition with Ki,mixed values for 1 and 2 of 0.16 ± 0.03 and 17 ± 7 μM, respectively (Figure S2, Supporting Information). Such mixed inhibition is consistent with transient covalent labeling of an enzyme, (24) and we therefore monitored GCase-catalyzed turnover of these two carbasugars and determined that the rate constant governing their turnover (kcat) as being quite similar (∼0.013–0.018 s–1) (Figure S3, Supporting Information). We next checked for accumulation of a covalent intermediate on the enzyme using a series of classical “jump-dilution” experiments but could not observe any time dependent inhibition with either compound. Given the similarity of the observed kcat values for 1 and 2 and the relatively low measured IC50 values, we reasoned that both steps (k2 and k3) are likely kinetically significant and that processing of these carbasugars proceeds through a common pseudoglycosyl enzyme intermediate. To try and increase the rate of the pseudoglycosylation step we changed the leaving group to fluoride and synthesized carbasugar 3 (Scheme S1, Supporting Information). We reasoned that as fluoride ion is intrinsically a better leaving group than a phenoxide having a similar basicity (25) and that, within the confines of the active site of GCase, a fluoride leaving group receives H-bonding assistance whereas leaving groups such as 2,4-dinitrophenoxide do not. (26) Unfortunately, carbasugar 3 displayed similar inhibition of GCase (IC50 = 41 ± 15 μM) as carbasugars 1 and 2 (Figure S1 and Table S2, Supporting Information), with no observable time-dependent loss of GCase activity. Thus, pseudodeglycosylation is also not rate-limiting for carbasugar 3. We therefore considered approaches to modulate the relative rates of each enzymatic step to obtain a situation where the pseudodeglycosylation step (k3) becomes markedly slower than the pseudoglycosylation step (k2), which would lead to an increased lifetime of the GCase covalent intermediate.

Figure 2

Figure 2. Structures of carbasugars used in this study and their in vitro characterization. Carbasugar covalent inhibitors used in the current study. The pseudoanomeric carbons (shown by the asterisk on 1) are drawn in identical positions for all compounds.

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Synthesis and Testing of Second-Generation Allylic Carbasugar Covalent Inhibitors

Based on an examination of the literature, we reasoned that removing the hydroxymethyl (C6-CH2OH) group from the cyclohexene system should decrease both rate constants. In particular, we note that GCase cleaves both xylosylceramide and 4-methylumbelliferyl β-d-xylopyranoside, with kcat/Km values that are lower than the corresponding glucopyranoside substrates. (27) Given these observations we reasoned that an analogous change to the carbasugar structure, in which the hydroxymethyl group is removed, could lead to molecules having reactivity that would enable accumulation of a covalent intermediate. Accordingly, we set out to make the d-xylo configured carbasugar 4 containing a pseudoanomeric fluoride leaving group. We selected fluorine as a leaving group because it could benefit from H-bonding catalysis, which would accelerate the pseudoglycosylation step and thereby allow rapid formation of the pseudoglycosyl enzyme intermediate. In this way we expected that we might observe a situation where the rate of formation of the pseudoglycosyl enzyme intermediate was much faster than its breakdown.
To prepare carbasugar 4 we developed a synthetic route involving the protected cyclohex-2-en-1-ol 14 (Scheme 1) as a convenient intermediate. Periodate cleavage of the known (28) 3-O-benzyl-1,2-O-isopropylidene-α-d-glucofuranose 8 and subsequent Wittig homologation gave olefin 10. Standard functional group interconversion and ring closing metathesis of compound 12 provided us with 14, which was converted to allylic alcohol 16 by protecting the trans-diol as a butane 2,3-bisacetal (Scheme 1). (29) To install fluorine we used (diethylamino)sulfur trifluoride (DAST) given that it reacts with allylic cyclohexenols through a stereochemically desirable SNi-SNi′ reaction mechanism. (30) Specifically, allylic alcohol 16 reacted with DAST to afford two allylic fluorides (18:19, ratio 3:7). These protected carbasugar fluorides could not be separated, however, we found that removal of the bis-acetal protecting group enabled separation of the partly deprotected allylic fluorides 26 and 27.

Scheme 1

Scheme 1. Synthesis of d- and l-Carbaxylopyranosyl Halides; (a) Synthesis of Intermediates 14 and 15, (b) Synthesis of Intermediates 16 and 23, (c) Synthesis of Target Carbasugarsa
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aReagents: (i) NaIO4, H2O, rt; (ii) Ph3P+CH3, BuLi, 0 °C; (iii) dioxane/water, cat H+, 90 °C; (iv) CH2CHMgBr, THF, 0 °C; (v) Grubbs’ 2nd generation, CH2Cl2, heat; (vi) CH3COCOCH3, HC(OCH3)3, cat H+; (vii) DAST, diglyme, 0 °C; (viii) TFA, CH2Cl2 (for 4 and 5 from 20 and 21, respectively); (ix) Dess-Martin periodinane, CH2Cl2; (x) NaBD4 (MeOD); (xi) TFA, CH2Cl2, H2O; (xii) BCl3, CH2Cl2, −78 °C (for 6, 7, 6-D, and 7-D from 26 to 29, respectively). Abbreviations: DAST, (diethylamino)sulfur trifluoride; TFA, trifluoroacetic acid.

Subsequent BCl3-promoted debenzylation of 26 and 27 led to the expected debenzylation but also an adventitious SNi reaction that yielded the corresponding d- and l-carbaxylosyl chlorides 6 and 7 in approximately 90% yields. We therefore changed our protecting group strategy to install a 4-methoxybenzyl (PMB) ether at C3 (9, Scheme 1). Following an identical reaction sequence to that described above, we obtained isomeric carbasugar fluorides (20:21, 1:2 ratio), which we readily separated. Acid-catalyzed deprotection proceeded without incident to give both 4 and 5 in ∼80% yields. With these desired fluorides in hand, we turned our attention to assessing their reactivity with GCase.
In contrast to the reactions of GCase with carbaglucoses 1–3, coincubation of these compounds with the fluorogenic substrate 4-methylumbelliferyl β-d-glucopyranoside (MU-Glc), revealed that d-carbaxylosyl fluoride 4 led to time-dependent inhibition of GCase, with steady state levels of activity being reached after ∼30–60 min. This time-dependent decrease in activity to a steady state level is consistent with transient covalent modification of GCase. To understand the kinetics governing formation of the pseudoxylosyl enzyme intermediate we fit the reaction progress curve (31) of fluorescence arising from turnover of MU-Glc versus time, at each inhibitor concentration (Figure S4, Supporting Information), where Ft is the initial fluorescence, νi is the initial velocity at t = 0, νf is the velocity at t = ∞, and kapp is the apparent rate constant (eq S1, Supporting Information). Next, we fit the resulting calculated kapp values as a function of the concentration of covalent inhibitor 4 and, taking into account the concentration of the substrate and its Km value (eqs S2 and S3, Supporting Information), obtained the inhibition parameters kinact = (2.0 ± 0.3) × 10–3 s–1, Ki = 3.4 ± 1.5 mM, and kinact/Ki = 0.59 ± 0.27 M–1 s–1 (Figure 3a and Table S3, Supporting Information).

Figure 3

Figure 3. In vitro characterization of carbasugars used in this study. (a) Nonlinear least-squares fit of kapp values to a modified Michaelis–Menten expression (eq S3) for the approach to steady-state GCase activity on incubation with d-carbaxylosyl fluoride 4. (b) Linear fit of kapp values for the approach to steady state for covalent inhibition of GCase by d-carbaxylosyl chloride 6 (blue) and l-carbaxylosyl chloride 7 (red). (c) Stabilization of folded GCase measured using differential scanning fluorimetry (DSF) in the presence of d-carbaxylosyl chloride 6 (blue), l-carbaxylosyl chloride 7 (red), or vehicle along (black). All lines are the best nonlinear least-squares fit to the appropriate equation. All experiments were carried out at T = 25 °C in McIlvaine buffer pH 5.2 containing 0.1% Triton X-100, 0.24% sodium taurodeoxycholate, and 0.1% BSA. (d) Mechanisms for the GCase pseudoglycosylation step by single turnover chaperones 6 and 7, which after diffusion of chloride out of the active site give identical allylic cation intermediates that are trapped by the enzymatic nucleophile (Glu340) to give the same covalent intermediate (E-αCarb).

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These inhibition parameters suggests that carbasugar 4 slowly labels GCase but also that the resulting covalent carbaxylosyl GCase intermediate must have a significantly longer half-life (t1/2) than the corresponding intermediate formed during the turnover of gluco-configured carbasugars 1, 2, and 3. Yet, the slow approach to steady state in combination with the comparatively similar rate for breakdown of the pseudoxylosyl enzyme intermediate make fluoride 4 ill suited for examining the potential stabilizing effects associated with formation of a transient covalent intermediate on folded β-glucocerebrosidase.

Synthesis and Testing of Third-Generation Allylic Carbasugar Covalent Inhibitors

To obtain a situation where greater steady state levels of the covalent carbaxylosyl intermediate could be obtained, we decided to speed the first step of the reaction by using a carbaxylose inhibitor having a better leaving group. We therefore turned to testing the adventitiously obtained carbaxylosyl chloride 6 (Figure S5, Supporting Information). By using the same kinetic analysis as described above, we observed that the covalent labeling of GCase (kinact/Ki = 6.9 ± 0.1 M–1 s–1) occurred ∼10–fold faster for chloride 6 as compared to fluoride 4 (Table 1 and Figures S4 and S5, Supporting Information). We also considered another possible strategy we could exploit using these carbaxylose compounds. In particular, we noted that glycoside hydrolases have been found to use catalytic mechanisms that generally involve significant charge delocalization. (15) This observation has led to development of substrates and inactivators that influence the stability of the transition state through installation of electron withdrawing groups at C5. (32) Moreover, we recently showed for an α-galactosidase acting on a carbasugar substrate that the enzyme efficiently stabilized positive charge development in the transition state at both the pseudoanomeric center (C1) as well as at C5. (33,34) Given this observation we decided to test the l-carbaxylosyl chloride 7 (Figure S6, Supporting Information), which owing to its leaving group being situated at C5, would necessarily react through a mechanism involving significant TS positive charge development at this center as the leaving group departs (Figure 3d). Remarkably, using the same series of kinetic analyses, we found that compound 7 covalently labels GCase and, moreover, does so more rapidly than d-carbasugar 6 (kinact/Ki = 41 ± 2 M–1 s–1; Figure 3b and Table 1). Notably, the rate constants we measured for breakdown of the carbaxylosyl enzyme intermediates formed by reaction of 6 and 7 with GCase were indistinguishable (t1/2 ≈ 50–55 min, Figure S7 and Table S3, Supporting Information), which suggests that the intermediates formed from labeling GCase with either compound are identical. However, the surprisingly greater rate constant governing formation of this common intermediate on GCase for l-carbaxylosyl chloride 7, as compared to d-carbaxylosyl chloride 6, drew into question our assignments for the structures of 6 and 7. We therefore synthesized the deuterated carbaxylosyl fluorides 28 and 29 (Scheme 1), and performed the BCl3 reaction. The resulting chloride products that we isolated (6-D and 7-D) exhibited NMR spectra in accord with the chlorination occurring solely via a SNi reaction with no evidence for a SNi′ process (Figure S8, Supporting Information). These data indicate that our original assignments were correct and confirm that GCase–and likely glycoside hydrolases in general–have the surprising ability to react through a novel SN1′ reaction with allylic carbasugar derivatives having a leaving group at C5. Collectively, these data show that the reactivity of carbasugar substrates of GCase can be tuned to obtain situations where the putative covalent intermediate can be rapidly formed and persist for varying lengths of time.
Table 1. Kinetic Parameters for the Covalent Inhibition and Reactivation of Human GCase by the Carbasugars 4–7c
inhibitorkinact/Ki (M–1 s–1)kreact (s–1)t1/2react (min)
40.59 ± 0.27a  
5N.I.b  
66.9 ± 0.1(2.2 ± 0.2) × 10–452.5 ± 4.8
741 ± 2(2.1 ± 0.1) × 10–455.0 ± 2.6
a

kinact = (2.0 ± 0.3) × 10–3 s–1, Ki = 3.4 ± 1.5 mM.

b

N.I. no time-dependent inhibition observed at 6 mM.

c

Conditions: T = 25 °C in McIlvaine buffer pH 5.2 containing 0.1% Triton X-100, 0.24% sodium taurodeoxycholate, and 0.1% BSA.

X-ray Structural Analysis of Michaelis Complexes and Covalent Intermediates of GCase

To better understand how these carbasugars interact with GCase and to clarify whether carbasugars 6 and 7 indeed reacted to give a common covalent enzyme adduct as our kinetic analyses suggested, we set out to obtain cocrystal structure of human GCase (35) in complex with these compounds. We therefore soaked crystals of GCase, produced recombinantly from insect cells, with carbasugar 6 with a view to capturing and visualizing the transient carbaxylosyl enzyme adduct. Following a 30 min soak, we obtained crystals that diffracted to 1.58 Å. Analysis of the diffraction data revealed unambiguous electron density showing 6 had reacted with the catalytic nucleophile (Glu340) to form a covalent bond to the pseudoanomeric carbon (Figure 4a). Analysis of the structure of the resulting enzyme–inhibitor complex showed this ester C–O bond has a length of 1.38 Å, which we modeled at 75% occupancy. This lower occupancy likely reflects hydrolytic turnover of the covalent complex (t1/2 ≈ 55 min in solution─Table S3, Supporting Information) during crystallization. The carbasugar ring of 6 in this complex adopts an envelope conformation (2E), presumably with the planarity enforced by the endocyclic double bond and the formation of hydrogen bonding interactions to active site residues Asp127, Trp179, Asn234, Glu340, and Trp381. Similar experiments using the l-carbasugar 7, led to a structure in which GCase has reacted to form the same adduct having the same ring conformation, hydroxyl stereochemistry, and hydrogen bonding network (Figure 4b). The observed enzyme–inhibitor complex, with a C–O covalent bond length of 1.40 Å, was modeled at 70% occupancy. These data supported our proposal that 7, being the enantiomer of 6, reacted through an alternative and unprecedented SN1′ mechanism (Figure 3d) to generate the same observed covalent adduct as seen when incubating 6 with GCase, which is consistent with the reactivation rate constant governing the breakdown of this intermediate being the same for both 6 and 7. We also tested whether the C2-symmetric tetraol 30, which lacks a good leaving group, affected GCase activity at 0.25 mM (Figure S9, Supporting Information) and found this compound showed neither competitive inhibition nor time dependent inhibition.

Figure 4

Figure 4. Structural characterization of covalent intermediates and Michaelis complex between GCase and carbasugars. (a) Crystal structure of the GCase-6 complex, showing two orientations of the covalent complex in which 6 adopts an envelope (2E) conformation. The maximum-likelihood σA-weighted 2Fo – Fc electron density map is contoured to 1 σ (0.37 e/Å3). (b) Crystal structure of the GCase-7 complex, showing two orientations of the covalent complex in which, the carbasugar adopts an envelope (2E) conformation, numbering of the ring starts at the covalent attachment to the enzyme. The maximum-likelihood σA-weighted 2Fo – Fc electron density map is contoured to 1 σ (0.36 e/Å3). Carbasugar average b-factor = 20 Å2. (c) Crystal structure of the GCase-5 complex, showing three orientations of l-carbasugar fluoride bound noncovalently in the active site in a half-chair (4H3) conformation. Green atom indicates fluorine. The maximum-likelihood σA-weighted 2Fo – Fc electron density map is contoured to 1 σ (0.37 e/Å3).

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To obtain a “Michaelis” complex with these carbasugars noncovalently bound by GCase, we performed structural studies using the less reactive l-carbaxylosyl fluoride 5. These experiments led to a cocrystal structure in which electron density for a single molecule of 5, bound noncovalently within the enzyme active site, was observed (Figure 4c). Although F and OH were indistinguishable within the electron density map, the absolute l-stereochemistry of the carbasugar bound within the active site shows unambiguously that 5 had not reacted. This “Michaelis” complex provides insight into how inhibitor 5 and, by extension, 7 bind prior to reacting within the active site of GCase. The electron density unambiguously shows that 5 was bound in a half-chair (4H3) conformation, which is enforced by the endocyclic double bond, that mimics the expected partial double bond character seen in the oxocarbenium ion-like transition states stabilized by this enzyme (Figures 1a and 4c). This carbasugar engages in hydrogen bonding interactions with active site residues Asp127, Trp179, Asn234, Glu340, and Trp396, showing an overall similar pattern of hydrogen bonds as seen for the covalent adduct of 7 linked to Glu340.
An additional coincidental, yet noteworthy, observation we made during structural studies using carbasugar 7 was that an additional molecule was also bound noncovalently to GCase at a site at the surface of the immunoglobulin-like domain of GCase. In contrast to the envelope conformation observed for the covalent adduct of 7 within the active site, this unreacted molecule of 7 molecule adopted a half-chair (3H4) conformation (Figure S10, Supporting Information), forming hydrogen bonding interactions with Arg47, Glu41, and a nearby water molecule. A potential aliphatic−π stacking interaction with the side chain of Arg39 was also seen, with a separation distance of 3.3 Å (Figure S10, Supporting Information). Supporting these observations, we also found l-carbaxylosyl fluoride 5 also bound noncovalently to this same remote binding site (Figure S11, Supporting Information). Carbasugar 5 at this site was also unreacted, based on its clear l-xylo configuration, and we readily modeled it in the same orientation as we had observed for 7. The electron density unambiguously showed that 5 at this site adopted the same half-chair conformation as 7 and formed the same hydrogen bonding network. Interestingly, we were never able to observe enantiomer 6 binding to this site, indicating the stereochemistry of the hydroxyl groups likely influences noncovalent binding to this site. The stereochemical requirements for binding to this site, coupled with reproducibly observing ligands in this location, suggests that this remote binding site may have a physiological function in binding some as yet unidentified ligand.
To probe whether transient formation of these covalent intermediates within the active site of GCase affects enzyme stability we performed differential scanning fluorimetry (DSF) in the presence and absence of both d,l-carbaxylosyl chlorides (6 and 7; Figure 2d). Incubation of GCase in the presence of either covalent inhibitor resulted in an indistinguishable 10 °C increase in melting temperature (6; 68.9 ± 0.1; 7 68.5 ± 0.1; control 59.0 ± 0.1 °C). These data again show that the enantiomeric pair of carbaxylosyl chlorides react with GCase to give an identical covalent intermediate, which confers a stabilizing effect on the enzyme.

Reactivity of Carbaxylosyl Chlorides with Other Glycoside Hydrolases

Given the remarkable observation that β-glucocerebrosidase is covalently labeled by l-carbaxylosyl chloride 7, which possesses a sp2 center at the pseudoanomeric carbon atom, we reasoned that in the absence of an anomeric substituent this compound may also be able to covalently modify mechanistically distinct α-glucosidases. To investigate this idea, we used yeast GH13 α-glucosidase as a test case. Gratifyingly, we note that 7 labeled yeast α-glucosidase in a time-dependent manner (Table 2 and Figures S12 and S13, Supporting Information). Given that GH13 yeast α-glucosidase is a strikingly stable enzyme we reasoned that we could reliably examine the pH-dependence of the inactivation process leading to the covalent intermediate and, in this manner, examine the active protonation states of this retaining α-glycoside hydrolase (Figure 1c). (15,36) We therefore measured the second-order rate constants for the covalent inhibition of yeast α-glucosidase by 7 over a range of pH values (Figures 5 and S14, S15) and found a bell-shaped pH-rate profile, albeit with the best fit being for three ionization events, which is similar to profiles observed for other glycoside hydrolase-catalyzed reactions. (37) These data show that these cyclohexene-derived carbasugars lacking an anomeric substituent can covalent label anomerically distinct glycoside hydrolases in a mechanism-based manner that depends on the enzyme having a protonation state that is also required for turnover of natural substrates.
Table 2. Kinetic Parameters for the Covalent Inhibition and Reactivation of Yeast α-Glucosidase by the Carbasugar 7a
inhibitorkinact/Ki (M–1 s–1)kreact (s–1)t1/2react (h)
77.70 ± 0.07(2.35 ± 0.17) × 10–681.8 ± 5.9
a

Conditions: experiments were carried out at T = 37 °C in 50 mM sodium phosphate buffer, pH 7.0 containing 0.1% BSA.

Figure 5

Figure 5. pH-rate profile for the logarithm of the inactivation second-order rate constants (kinact/Ki) for yeast α-glucosidase by 7. All experiments were performed at T = 37 °C in 50 mM buffer (acetic acid-sodium acetate buffer for pH 5.0–5.5, sodium phosphate buffer for pH 6.0–7.5, TAPS buffer for pH 7.9) containing 0.1% BSA. The line through the points is the best fit to a model where two protonation events (low pH) lead to an inactive enzyme, while a single deprotonation at higher pH values gives inactive enzyme.

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Discussion

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By rationally tuning the reactivity of a glucose-configured carbasugar we have created a new class of reversible covalent glycoside hydrolase inhibitors. These inhibitors derive their ability to transiently inactivate glycoside hydrolases by retaining features of the natural substrate essential for binding, which are suitably oriented hydroxyl groups positioned on a six-membered ring. However, the mechanisms by which these cyclohexenyl inhibitors act are distinct, involving stabilization of the cationic transition states, not by an endocyclic ring oxygen as seen for natural substrates, but rather by a suitably positioned alkene moiety that enables cation stabilization through charge delocalization. Indeed, we suggest that GCase-catalyzed covalent labeling occurs via SN1 reactions for fluoride 4, chloride 6, and, most unusually, an atypical mechanism that we describe here as SN1′ for chloride 7 (Figure 3d). Specifically, we propose that GCase-catalyzed pseudoglycosylation with d-carbaxylosyl fluoride 4, chloride 6, and the more reactive l-carbaxylosyl chloride 7, occurs via such dissociative processes, which give rise to a common enzyme bound allylic cation intermediate (Figure 3d), based on the following reasoning:
(i)

Both experimental (38−40) and theoretical (41) studies indicate that an SN2 displacement has a significantly lower TS free energy than the corresponding SN2′ counterpart. As a result, if covalent labeling involved SN2 and SN2′ mechanisms inhibitor 6 should covalently label GCase more rapidly than does 7, a situation that is contrary to our observations

(ii)

Contrary to the commonly held view, fluoride is a viable leaving group in bona fide SN1 reactions performed in polar H-bonding environments. For instance, the difference in SN1 reactivity of 1-adamantyl chloride (1-AdCl) (42) and 1-adamantyl fluoride (1-AdF) (43) in trifluoroethanol is approximately 760-fold at 25 °C. Moreover, this ratio is almost independent of temperature as the major difference in the corresponding activation parameters is a larger negative entropy (ΔS⧧) for the solvolysis of 1-AdF, likely caused by the obligatory strong H-bonding that is needed to assist fluoride ion departure for formation of the 1-adamantyl carbocation (44,45)

(iii)

The ratio of the second order rate constants (kinact/Ki) governing the GCase-catalyzed reactions of 6 and 4 is 12 ± 5, a ratio that is consistent with SN1 reactions in which the presence of an optimally positioned H-bonding donor only assists departure of fluoride to give enzyme-bound allylic cations, (34) and

(iv)

l-Carbaxylosyl chloride 7 undergoes pseudoglycosylation faster than does the d-enantiomer 6 because 7 binds in the conformation of the transition state for GCase-catalyzed cleavage of natural substrate glucosidic bonds (4H3), a conformation that enables the most efficient stabilization of positive charge development at the allylic cation-like transition state (Figure 3d).

These collective points, along with detailed QM/MM computational studies on a retaining galactosidase, (34) support such allylic covalent inhibitors reacting with GCase by SN1 (4 and 6) and SN1′ (7) mechanisms for these reactions. We expect that this mechanistic knowledge can be exploited in the generation of such carbasugar labeling agents for GCase as well as other enzymes of interest. Further, the ability of GCase to catalyze an SN1′ mechanism for 7 is intriguing and suggests the possibility of designing other substrates or inhibitors that exploit this mechanistic promiscuity for other members of the large class of glycoside hydrolases. Indeed, 7 bears a chloride leaving group that requires little to no catalytic assistance for departure from C5 but, once bound into an active site that has evolved to stabilize the development of positive charge, undergoes rapid pseudoglycosylation and ultimately hydrolytic turnover of this intermediate. As a result, such allylic carbasugar inhibitors lacking a pseudoanomeric leaving group possess the exceptional reactivity needed to inhibit both α- and β-glucosidases.
Further support for these carbasugars functioning as mechanism-based labeling agents is seen in the standard bell-shaped pH-rate profile for the inactivation processes (kinact/Ki) that is similar to that seen for the hydrolysis of natural substrates (kcat/Km). (37) This bell-shaped profile arises from ionization of two key carboxylic acid residues that are needed in the appropriate protonation state (46) to effect the first chemical step involving pseudoglycosylation. (37) This observation may initially appear inconsistent with our conclusion that neither general-acid nor nucleophilic catalysis is required for departure of the chloride leaving group. However, though yeast α-glucosidase did not evolve to provide catalytic assistance for leaving group departure at C5 of 7, the correct protonation state of the enzyme is still needed to provide the correct negatively charged environment that stabilizes positive charge development within the allyl cation-like transition states at C5, C5a, and C1 for pseudoglycosylation. (33)
In summary, we have demonstrated the ability to tailor the reactivity of carbasugar inhibitors, and we anticipate that such principles can be applied, in principle, to refine the properties of such molecules for any glycoside hydrolase that operates via a double displacement mechanism. Such variations can be tailored for the enzyme of interest to generate useful chemical biology tools. As well as being exploited in the development of next generation small molecule pharmacological chaperones that stabilize both wild-type and misfolding variant lysosomal glycoside hydrolases (Figure 3c, covalent adduct), yet yield active enzyme following dealkylation (Figures 1c,d and 3d, liberated enzyme). More broadly, the design of such tunable reversible covalent inhibitors should be generally applicable to the larger set of active-site directed reversible covalent ligands for other enzymes.

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acscatal.4c04549.

  • General methods and reagents, eqs S1–S3, Tables S1–S3, Scheme S1, Figures S1–S78 (PDF)

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Author Information

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  • Corresponding Authors
  • Authors
    • Sandeep Bhosale - Department of Chemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada;  Orcidhttps://orcid.org/0000-0002-1850-1271
    • Sachin Kandalkar - Department of Chemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada
    • Pierre-André Gilormini - Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada;  Orcidhttps://orcid.org/0000-0003-1491-1174
    • Oluwafemi Akintola - Department of Chemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada;  Orcidhttps://orcid.org/0000-0001-9790-7132
    • Rhianna Rowland - Department of Chemistry, University of York, York YO10 5DD, U.K.;  Orcidhttps://orcid.org/0000-0002-8717-346X
    • Pal John Pal Adabala - Department of Chemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada
    • Dustin King - Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada;  Orcidhttps://orcid.org/0000-0003-1775-2885
    • Matthew C. Deen - Department of Chemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada
    • Xi Chen - Department of Chemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada
    • Gideon J. Davies - Department of Chemistry, University of York, York YO10 5DD, U.K.;  Orcidhttps://orcid.org/0000-0002-7343-776X
  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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The authors are grateful for support from the Canadian Institutes of Health Research (A.J.B.: grant-173228) and the Natural Sciences and Engineering Council of Canada (A.J.B.: RGPIN-04910 and CRDPJ-480335, and D.J.V.: RGPIN-05426). We thank Diamond Light Source for access to beamline I03 and I04 (proposal numbers mx18598 and mx24948). G.J.D. thanks the Royal Society for the Ken Murray Research Professorship and RJR thanks the BBSRC for White Rose Doctoral Training Partnership funding (BB/M011151/1). D.J.V. thanks the Canada Research Chairs program for support as a Tier I Canada Research Chair in Chemical Biology.

References

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    Ben Bdira, F.; Kallemeijn, W. W.; Oussoren, S. V.; Scheij, S.; Bleijlevens, B.; Florea, B. I.; van Roomen, C.; Ottenhoff, R.; van Kooten, M.; Walvoort, M. T. C.; Witte, M. D.; Boot, R. G.; Ubbink, M.; Overkleeft, H. S.; Aerts, J. Stabilization of Glucocerebrosidase by Active Site Occupancy. ACS Chem. Biol. 2017, 12, 1830– 1841,  DOI: 10.1021/acschembio.7b00276
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    Vocadlo, D. J.; Davies, G. J. Mechanistic insights into glycosidase chemistry. Curr. Opin. Chem. Biol. 2008, 12, 539– 555,  DOI: 10.1016/j.cbpa.2008.05.010
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    Mechanistic insights into glycosidase chemistry
    Vocadlo, David J.; Davies, Gideon J.
    Current Opinion in Chemical Biology (2008), 12 (5), 539-555CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)
    A review. The enzymic hydrolysis of the glycosidic bond continues to gain importance, reflecting the critically important roles complex glycans play in health and disease as well as the rekindled interest in enzymic biomass conversion. Recent advances include the broadening of our understanding of enzyme reaction coordinates, through both computational and structural studies, improved understanding of enzyme inhibition through transition state mimicry and fascinating insights into mechanism yielded by phys. org. chem. approaches.
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    Adamson, C.; Pengelly, R.; Shamsi Kazem Abadi, S.; Chakladar, S.; Draper, J.; Britton, R.; Gloster, T.; Bennet, A. J. Structural snapshots for mechanism-based inactivation of a glycoside hydrolase by cyclopropyl-carbasugars. Angew. Chem., Int. Ed. 2016, 55, 14978– 14982,  DOI: 10.1002/anie.201607431
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    Shamsi Kazem Abadi, S.; Tran, M.; Yadav, A. K.; Adabala, P. J. P.; Chakladar, S.; Bennet, A. J. New class of glycoside hydrolase mechanism-based covalent inhibitors: Glycosylation transition state conformations. J. Am. Chem. Soc. 2017, 139, 10625– 10628,  DOI: 10.1021/jacs.7b05065
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    New Class of Glycoside Hydrolase Mechanism-Based Covalent Inhibitors: Glycosylation Transition State Conformations
    Shamsi Kazem Abadi, Saeideh; Tran, Michael; Yadav, Anuj K.; Adabala, Pal John Pal; Chakladar, Saswati; Bennet, Andrew J.
    Journal of the American Chemical Society (2017), 139 (31), 10625-10628CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The design of covalent inhibitors in glycoscience research is important for the development of chem. biol. probes. Here we report the synthesis of a new carbocyclic mechanism-based covalent inhibitor of an α-glucosidase. The enzyme efficiently catalyzes its alkylation via either an allylic cation or a cationic transition state. We show this allylic covalent inhibitor has different catalytic proficiencies for pseudoglycosylation and deglycosylation. Such inhibitors have the potential to be useful chem. biol. tools.
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    Danby, P. M.; Withers, S. G. Glycosyl cations versus allylic cations in spontaneous and enzymatic hydrolysis. J. Am. Chem. Soc. 2017, 139, 10629– 10632,  DOI: 10.1021/jacs.7b05628
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    Glycosyl cations versus allylic cations in spontaneous and enzymatic hydrolysis
    Danby, Phillip M.; Withers, Stephen G.
    Journal of the American Chemical Society (2017), 139 (31), 10629-10632CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Enzymic prenyl and glycosyl transfer are seemingly unrelated reactions that yield mols. and protein modifications with disparate biol. functions. However, both reactions employ diphosphate-activated donors and each proceed via cationic species: allylic cations and oxocarbenium ions, resp. Here, we explored the relation between these processes by prepg. valienyl ethers to serve as glycoside mimics that were capable of allylic rather than oxocarbenium cation stabilization. Rate consts. for the spontaneous hydrolysis of aryl glycosides and their analogous valienyl ethers were found to be almost identical, as were the corresponding activation enthalpies and entropies. This close similarity extended to the assocd. secondary kinetic isotope effects (KIEs), indicating very similar transition state stabilities and structures. Screening a library of >100 β-glucosidases identified a no. of enzymes that catalyzed the hydrolysis of these valienyl ethers with kcat values of ≤20 s-1. Detailed anal. of one such enzyme showed that ether hydrolysis occurred via the analogous mechanisms found for glycosides, and through a very similar transition state. This suggested that the generally lower rates of enzymic cleavage of the cyclitol ethers reflects evolutionary specialization of these enzymes toward glycosides rather than inherent reactivity differences.
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    Parenti, G. Treating lysosomal storage diseases with pharmacological chaperones: from concept to clinics. EMBO Mol. Med. 2009, 1, 268– 279,  DOI: 10.1002/emmm.200900036
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    Treating lysosomal storage diseases with pharmacological chaperones: from concept to clinics
    Parenti, Giancarlo
    EMBO Molecular Medicine (2009), 1 (5), 268-279CODEN: EMMMAM; ISSN:1757-4684. (John Wiley & Sons Ltd.)
    A review. Lysosomal storage diseases (LSDs) are a group of genetic disorders due to defects in any aspect of lysosomal biol. During the past two decades, different approaches have been introduced for the treatment of these conditions. Among them, enzyme replacement therapy (ERT) represented a major advance and is used successfully in the treatment of some of these disorders. However, ERT has limitations such as insufficient biodistribution of recombinant enzymes and high costs. An emerging strategy for the treatment of LSDs is pharmacol. chaperone therapy (PCT), based on the use of chaperone mols. that assist the folding of mutated enzymes and improve their stability and lysosomal trafficking. After proof-of-concept studies, PCT is now being translated into clin. applications for Fabry, Gaucher and Pompe disease. This approach, however, can only be applied to patients carrying chaperone-responsive mutations. The recent demonstration of a synergistic effect of chaperones and ERT expands the applications of PCT and prompts a re-evaluation of their therapeutic use and potential. This review discusses the strengths and drawbacks of the potential therapies available for LSDs and proposes that future research should be directed towards the development of treatment protocols based on the combination of different therapies to improve the clin. outcome of LSD patients.
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  20. 20
    Sidransky, E.; Lopez, G. The link between the GBA gene and parkinsonism. Lancet Neurol. 2012, 11, 986– 998,  DOI: 10.1016/S1474-4422(12)70190-4
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    The link between the GBA gene and parkinsonism
    Sidransky, Ellen; Lopez, Grisel
    Lancet Neurology (2012), 11 (11), 986-998CODEN: LNAEAM; ISSN:1474-4422. (Elsevier Ltd.)
    A review. Summary: Mutations in the glucocerebrosidase (GBA) gene, which encodes the lysosomal enzyme that is deficient in Gaucher's disease, are important and common risk factors for Parkinson's disease and related disorders. This assocn. was first recognized in the clinic, where parkinsonism was noted, albeit rarely, in patients with Gaucher's disease and more frequently in relatives who were obligate carriers. Subsequently, findings from large studies showed that patients with Parkinson's disease and assocd. Lewy body disorders had an increased frequency of GBA mutations when compared with control individuals. Patients with GBA-assocd. parkinsonism exhibit varying parkinsonian phenotypes but tend to have an earlier age of onset and more assocd. cognitive changes than patients with parkinsonism without GBA mutations. Hypotheses proposed to explain this assocn. include a gain-of-function due to mutations in glucocerebrosidase that promotes α-synuclein aggregation; substrate accumulation due to enzymic loss-of-function, which affects α-synuclein processing and clearance; and a bidirectional feedback loop. Identification of the pathol. mechanisms underlying GBA-assocd. parkinsonism will improve our understanding of the genetics, pathophysiol., and treatment for both rare and common neurol. diseases.
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    Sinnott, M. L.; Souchard, I. J. The mechanism of action of β-galactosidase. Effect of aglycone nature and α-deuterium substitution on the hydrolysis of aryl galactosides. Biochem. J. 1973, 133, 89– 98,  DOI: 10.1042/bj1330089
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    Mechanism of action of β-galactosidase. Effect of aglycone nature and α-deuterium substitution on the hydrolysis of aryl galactosides
    Sinnott, Michael L.; Souchard, Jan J. L.
    Biochemical Journal (1973), 133 (1), 89-98CODEN: BIJOAK; ISSN:0264-6021.
    Steady-state kinetic parameters for the hydrolysis of 13 aryl β-D-galactopyranosides catalyzed by β-galactosidase (I) showed no simple dependence on aglycone acidity. The α-deuterium kinetic isotope effects (kH/kD) for 7 substrates and the effects of methanolysis of galactosyl-I in 1.5M MeOH on kH/kD were incompatible with a 2-step mechanism. A conformational change, liberation of a galactopyranosyl cation in an intimate ion-pair, nonproductive but preferential collapse of the ion pair to a covalent species, and reaction of the galactosyl-I through the ion-paired form was proposed as a reaction mechanism for β-galactosidase.
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    Kempton, J. B.; Withers, S. G. Mechanism of Agrobacterium .beta.-glucosidase: kinetic studies. Biochemistry 1992, 31, 9961– 9969,  DOI: 10.1021/bi00156a015
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    Mechanism of Agrobacterium β-glucosidase: kinetic studies
    Kempton, Julie B.; Withers, Stephen G.
    Biochemistry (1992), 31 (41), 9961-9CODEN: BICHAW; ISSN:0006-2960.
    β-Glucosidase from A. faecalis (previously Alcaligenes faecalis) was subjected to a detailed kinetic investigation with a range of substrates to probe its specificity and mechanism. It had a relatively broad specificity for the substrate sugar moiety and exhibited a classical pH dependence for its kinetic parameters with 3 different substrates and an identical pH dependence for its inactivation by a mechanism-based inactivator, cyclophellitol. Measurement of kcat and Km values for a series of aryl glucoside substrates allowed construction of a Broensted plot, the concave-downward shape of which was consistent with the anticipated 2-step mechanism involving a glucosyl-enzyme intermediate which is formed and hydrolyzed via oxocarbonium ion-like transition states. The slope of the leaving group-dependent portion of the Broensted plot (β1g = -0.7) indicated a large degree of bond cleavage at the transition state. Secondary deuterium kinetic isotope effects measured for 5 different aryl glucosides were also consistent with this mechanism and further suggested that the transition state for formation of the glucosyl-enzyme intermediate, probed with the slower substrates for which kH/kD = 1.06, is more SN2-like than that for its hydrolysis (for which kH/kD - 1.11). Reasons for this difference are proposed, and values of Ki for several ground-state and transition-state analog inhibitors are presented which support the concept of sp2-hybridized transition states.
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    Breen, I. Z.; Artola, M.; Wu, L.; Beenakker, T. J. M.; Offen, W. A.; Aerts, J. M. F. G.; Davies, G. J.; Overkleeft, H. S. Competitive and covalent inhibitors of human lysosomal retaining exoglucosidases. eLS; Wiley & Sons Ltd.: Chichester, 2018.
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    Segel, I. H. Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady State Enzyme Systems; Wiley: New York, 1975.
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    Jones, C. C.; Sinnott, M. L. Leaving ability and basicity of leaving groups attached by first-row elements. J. Chem. Soc., Chem. Commun. 1977, 767– 768,  DOI: 10.1039/c39770000767
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    Farren-Dai, M.; Sannikova, N.; Świderek, K.; Moliner, V.; Bennet, A. J. Fundamental insight into glycoside hydrolase-catalyzed hydrolysis of the universal Koshland substrates–glycopyranosyl fluorides. ACS Catal. 2021, 11, 10383– 10393,  DOI: 10.1021/acscatal.1c01918
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    Fundamental Insight into Glycoside Hydrolase-Catalyzed Hydrolysis of the Universal Koshland Substrates-Glycopyranosyl Fluorides
    Farren-Dai, Marco; Sannikova, Natalia; Swiderek, Katarzyna; Moliner, Vicent; Bennet, Andrew J.
    ACS Catalysis (2021), 11 (16), 10383-10393CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)
    The authors measured a panel of five kinetic isotope effects [KIEs; three secondary deuterium (1-, 2-, and 5-2H) and two primary (1-13C and 5-18O) effects] for the catalyzed hydrolysis of α-D-glucopyranosyl fluoride by two inverting glycoside hydrolases (GHs). The exptl. KIEs were compared to av. values computed with multiscale QM/MM methods for the hydrolysis of α-D-glucopyranosyl fluoride promoted by an inverting α-glucosidase (Aspergillus niger, GH family 15) to give β-D-glucopyranose, which the authors explored by the generation of free energy surfaces. The authors also measured the same KIEs for catalysis of α-D-glucopyranosyl fluoride by the Trichoderma virens GH55 inverting β-glucosidase using the authors' panel of isotopologues; this reaction occurs via the "Hehre resynthesis-hydrolysis mechanism" to give, by two inversions of configuration, the hydrolysis product α-D-glucopyranose. The transition states for both enzymic reactions are essentially identical with fluoride ion departure occurring within a glycoside hydrolase active site that stabilizes pyranosylium ion-like TSs, and with catalysis driven solely by H-bonding assistance from an enzymic carboxylic acid residue. That is, no assistance is required from the bound nucleophile, which in these two cases is either a water mol. (GH15) or a sugar hydroxyl group (GH55).
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    Boer, D. E.; Mirzaian, M.; Ferraz, M. J.; Zwiers, K. C.; Baks, M. V.; Hazeu, M. D.; Ottenhoff, R.; Marques, A. R. A.; Meijer, R.; Roos, J. C. P.; Cox, T. M.; Boot, R. G.; Pannu, N.; Overkleeft, H. S.; Artola, M.; Aerts, J. M. Human glucocerebrosidase mediates formation of xylosyl-cholesterol by β-xylosidase and transxylosidase reactions. J. Lipid Res. 2021, 62, 100018,  DOI: 10.1194/jlr.RA120001043
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    Beauhaire, J.; Ducrot, P. H. An epoxide derived from D-glucose as the key intermediate for penaresidine and sphingolipids synthesis. Synth. Commun. 1998, 28, 2443– 2456,  DOI: 10.1080/00397919808004296
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    An epoxide derived from D-glucose as the key intermediate for penaresidine and sphingolipids synthesis
    Beauhaire, Josiane; Ducrot, Paul-Henri
    Synthetic Communications (1998), 28 (13), 2443-2456CODEN: SYNCAV; ISSN:0039-7911. (Marcel Dekker, Inc.)
    A multigram-scale synthesis of 3R,4R,5R-3,5-dibenzyloxy-4-p-methoxybenzyl-1,2-epoxypentane and its use as intermediate for sphingolipids, penazeridine and penazetidine synthesis are described.
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    Ley, S. V.; Baeschlin, D. K.; Dixon, D. J.; Foster, A. C.; Ince, S. J.; Priepke, W. M.; Reynolds, D. J. 1,2-Diacetals: A new opportunity for organic synthesis. Chem. Rev. 2001, 101, 53– 80,  DOI: 10.1021/cr990101j
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    1,2-Diacetals: A New Opportunity for Organic Synthesis
    Ley, Steven V.; Baeschlin, Daniel K.; Dixon, Darren J.; Foster, Alison C.; Ince, Stuart J.; Priepke, Henning W. M.; Reynolds, Dominic J.
    Chemical Reviews (Washington, D. C.) (2001), 101 (1), 53-80CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
    A review with 127 refs. 1,2-Diacetals offer diol protection.
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    Shih, T. L.; Liao, W. Y.; Yen, W. C. Regioselective fluorination in synthesis of deoxyfluoro quercitols from D-(−)-quinic acid. Tetrahedron 2014, 70, 9621– 9627,  DOI: 10.1016/j.tet.2014.11.001
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    Baici, A.; Schenker, P.; Wachter, M.; Ruedi, P. 3-Fluoro-2,4-dioxa-3-phosphadecalins as inhibitors of acetylcholinesterase. A reappraisal of kinetic mechanisms and diagnostic methods. Chem. Biodivers. 2009, 6, 261– 282,  DOI: 10.1002/cbdv.200800334
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    McCarter, J. D.; Withers, S. G. 5-Fluoro Glycosides:  A New Class of Mechanism-Based Inhibitors of Both α- and β-Glucosidases. J. Am. Chem. Soc. 1996, 118, 241– 242,  DOI: 10.1021/ja952732a
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    5-Fluoro Glycosides: A New Class of Mechanism-Based Inhibitors of Both α- and β-Glucosidases
    McCarter, John D.; Withers, Stephen G.
    Journal of the American Chemical Society (1996), 118 (1), 241-2CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Mechanism-based inhibitors of retaining glycosidases are of considerable interest, both academically and as potential therapeutics. 5-Fluoroglycosyl fluorides I represent a new class of such inhibitors, functioning via the formation of a stabilized 5-fluoroglycosyl-enzyme intermediate that turns over only very slowly. Synthesis is achieved via radical bromination of the corresponding protected glycosyl fluoride, displacement of the bromide by fluoride and deprotonation. 5-Fluoro-β-glucosyl fluoride functions as a time-dependent inactivator of Agrobacterium faecalis β-glucosidase with a second order rate consts. for inactivation of ki/Ki = 660 min-1 mM-1. Turnover of the 5-fluoro α-glucosyl-enzyme intermediate occurs with a half life of 8.5 min. 5-Fluoro α-glucosyl fluoride functions similarly as an inactivator of yeast α-glucosidase, but inactivation is too rapid to allow detn. of kinetic parameters for the inactivation process. Turnover is also more rapid, occurring with a rate const. of 6.6 min-1. Steady state apparent Ki values of 0.3 μM and 1.4 μM are detd. for β-glucosidase and α-glucosidase resp.
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    Ren, W.; Farren-Dai, M.; Sannikova, N.; Świderek, K.; Wang, Y.; Akintola, O.; Britton, R.; Moliner, V.; Bennet, A. J. Glycoside hydrolase stabilization of transition state charge: New directions for inhibitor design. Chem. Sci. 2020, 11, 10488– 10495,  DOI: 10.1039/D0SC04401F
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    Glycoside hydrolase stabilization of transition state charge: new directions for inhibitor design
    Ren, Weiwu; Farren-Dai, Marco; Sannikova, Natalia; Swiderek, Katarzyna; Wang, Yang; Akintola, Oluwafemi; Britton, Robert; Moliner, Vicent; Bennet, Andrew J.
    Chemical Science (2020), 11 (38), 10488-10495CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
    Carbasugars are structural mimics of naturally occurring carbohydrates that can interact with and inhibit enzymes involved in carbohydrate processing. In particular, carbasugars have attracted attention as inhibitors of glycoside hydrolases (GHs) and as therapeutic leads in several disease areas. However, it is unclear how the carbasugars are recognized and processed by GHs. Here, we report the synthesis of three carbasugar isotopologues and provide a detailed transition state (TS) anal. for the formation of the initial GH-carbasugar covalent intermediate, as well as for hydrolysis of this intermediate, using a combination of exptl. measured kinetic isotope effects and hybrid QM/MM calcns. We find that the α-galactosidase from Thermotoga maritima effectively stabilizes TS charge development on a remote C5-allylic center acting in concert with the reacting carbasugar, and catalysis proceeds via an exploded, or loose, SN2 transition state with no discrete enzyme-bound cationic intermediate. We conclude that, in complement to what we know about the TS structures of enzyme-natural substrate complexes, knowledge of the TS structures of enzymes reacting with non-natural carbasugar substrates shows that GHs can stabilize a wider range of pos. charged TS structures than previously thought. Furthermore, this enhanced understanding will enable the design of new carbasugar GH transition state analogs to be used as, for example, chem. biol. tools and pharmaceutical lead compds.
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    Akintola, O.; Farren-Dai, M.; Ren, W.; Bhosale, S.; Britton, R.; Świderek, K.; Moliner, V.; Bennet, A. J. Glycoside hydrolase catalysis: Do substrates and mechanism-based covalent inhibitors react via matching transition states?. ACS Catal. 2022, 12, 14667– 14678,  DOI: 10.1021/acscatal.2c04027
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    Rowland, R. J.; Wu, L.; Liu, F.; Davies, G. J. A baculoviral system for the production of human β-glucocerebrosidase enables atomic resolution analysis. Acta Crystallogr. Sect. D-Struct. Biol. 2020, 76, 565– 580,  DOI: 10.1107/S205979832000501X
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    Speciale, G.; Thompson, A. J.; Davies, G. J.; Williams, S. J. Dissecting conformational contributions to glycosidase catalysis and inhibition. Curr. Opin. Struct. Biol. 2014, 28, 1– 13,  DOI: 10.1016/j.sbi.2014.06.003
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    Dissecting conformational contributions to glycosidase catalysis and inhibition
    Speciale, Gaetano; Thompson, Andrew J.; Davies, Gideon J.; Williams, Spencer J.
    Current Opinion in Structural Biology (2014), 28 (), 1-13CODEN: COSBEF; ISSN:0959-440X. (Elsevier Ltd.)
    A review. Glycoside hydrolases (GHs) are classified into >100 sequence-based families. These enzymes process a wide variety of complex carbohydrates with varying stereochem. at the anomeric and other ring positions. The shapes that these sugars adopt upon binding to their cognate GHs, and the conformational changes that occur along the catalysis reaction coordinate is termed the conformational itinerary. Efforts to define the conformational itineraries of GHs have focussed upon the crit. points of the reaction: substrate-bound (Michaelis), transition state, intermediate (if relevant) and product-bound. Recent approaches to defining conformational itineraries that marry X-ray crystallog. of enzymes bound to ligands that mimic the crit. points, along with advanced computational methods and kinetic isotope effects are discussed.
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    Matsusaka, K.; Chiba, S.; Shimomura, T. Purification and substrate specificity of brewer’s yeast .ALPHA.-glucosidase. Agric. Biol. Chem. 1977, 41, 1917– 1923,  DOI: 10.1271/bbb1961.41.1917
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    Purification and substrate specificity of Brewers' yeast α-glucosidase
    Matsusaka, Kouetsu; Chiba, Seiya; Shimomura, Tokuji
    Agricultural and Biological Chemistry (1977), 41 (10), 1917-23CODEN: ABCHA6; ISSN:0002-1369.
    Three kinds of α-glucosidase (I, II and III) were isolated from brewers' yeast. α-Glucosidases I and II were homogeneous in disc electrophoresis, but α-glucosidase III was not. Their pH optima were 6.3-7.1. Each of them was a typical α-glucosidase showing a preferential activity on Ph α-glucoside (I). α-Glucosidase I was isomaltase, which can hydrolyze isomaltose but not maltose. α-Glucosidases II and III showed no activity on isomaltose. The ratio of velocity of hydrolysis for I and isomaltose of α-glucosidase I was 100:9, and those for I and maltose of α-glucosidases II and III were 100:17 and 100:18, resp.
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    Bordwell, F. G. Are nucleophilic bimolecular concerted reactions involving four or more bonds a myth?. Acc. Chem. Res. 1970, 3, 281– 290,  DOI: 10.1021/ar50033a001
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  1. Oluwafemi Akintola, Sandeep Bhosale, Andrew J. Bennet. Mechanism-Based Allylic Carbasugar Chlorides That Form Covalent Intermediates with α- and β-Galactosidases. Molecules 2024, 29 (20) , 4870. https://doi.org/10.3390/molecules29204870
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  • Abstract

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    Figure 1

    Figure 1. Small molecule covalent inhibitors and the mechanism of action of retaining glycosidases. (a) Mechanism used by glucocerebrosidase for hydrolysis of its natural β-glucoside substrate. (b) Kinetic scheme for GCase-catalyzed turnover of β-d-glucopyranoside substrates. (c) Mechanism of action for carbasugar covalent inhibitors. (d) Kinetic scheme for covalent carbasugar inhibitors.

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    Figure 2

    Figure 2. Structures of carbasugars used in this study and their in vitro characterization. Carbasugar covalent inhibitors used in the current study. The pseudoanomeric carbons (shown by the asterisk on 1) are drawn in identical positions for all compounds.

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    Scheme 1

    Scheme 1. Synthesis of d- and l-Carbaxylopyranosyl Halides; (a) Synthesis of Intermediates 14 and 15, (b) Synthesis of Intermediates 16 and 23, (c) Synthesis of Target Carbasugarsa
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    aReagents: (i) NaIO4, H2O, rt; (ii) Ph3P+CH3, BuLi, 0 °C; (iii) dioxane/water, cat H+, 90 °C; (iv) CH2CHMgBr, THF, 0 °C; (v) Grubbs’ 2nd generation, CH2Cl2, heat; (vi) CH3COCOCH3, HC(OCH3)3, cat H+; (vii) DAST, diglyme, 0 °C; (viii) TFA, CH2Cl2 (for 4 and 5 from 20 and 21, respectively); (ix) Dess-Martin periodinane, CH2Cl2; (x) NaBD4 (MeOD); (xi) TFA, CH2Cl2, H2O; (xii) BCl3, CH2Cl2, −78 °C (for 6, 7, 6-D, and 7-D from 26 to 29, respectively). Abbreviations: DAST, (diethylamino)sulfur trifluoride; TFA, trifluoroacetic acid.

    Figure 3

    Figure 3. In vitro characterization of carbasugars used in this study. (a) Nonlinear least-squares fit of kapp values to a modified Michaelis–Menten expression (eq S3) for the approach to steady-state GCase activity on incubation with d-carbaxylosyl fluoride 4. (b) Linear fit of kapp values for the approach to steady state for covalent inhibition of GCase by d-carbaxylosyl chloride 6 (blue) and l-carbaxylosyl chloride 7 (red). (c) Stabilization of folded GCase measured using differential scanning fluorimetry (DSF) in the presence of d-carbaxylosyl chloride 6 (blue), l-carbaxylosyl chloride 7 (red), or vehicle along (black). All lines are the best nonlinear least-squares fit to the appropriate equation. All experiments were carried out at T = 25 °C in McIlvaine buffer pH 5.2 containing 0.1% Triton X-100, 0.24% sodium taurodeoxycholate, and 0.1% BSA. (d) Mechanisms for the GCase pseudoglycosylation step by single turnover chaperones 6 and 7, which after diffusion of chloride out of the active site give identical allylic cation intermediates that are trapped by the enzymatic nucleophile (Glu340) to give the same covalent intermediate (E-αCarb).

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    Figure 4

    Figure 4. Structural characterization of covalent intermediates and Michaelis complex between GCase and carbasugars. (a) Crystal structure of the GCase-6 complex, showing two orientations of the covalent complex in which 6 adopts an envelope (2E) conformation. The maximum-likelihood σA-weighted 2Fo – Fc electron density map is contoured to 1 σ (0.37 e/Å3). (b) Crystal structure of the GCase-7 complex, showing two orientations of the covalent complex in which, the carbasugar adopts an envelope (2E) conformation, numbering of the ring starts at the covalent attachment to the enzyme. The maximum-likelihood σA-weighted 2Fo – Fc electron density map is contoured to 1 σ (0.36 e/Å3). Carbasugar average b-factor = 20 Å2. (c) Crystal structure of the GCase-5 complex, showing three orientations of l-carbasugar fluoride bound noncovalently in the active site in a half-chair (4H3) conformation. Green atom indicates fluorine. The maximum-likelihood σA-weighted 2Fo – Fc electron density map is contoured to 1 σ (0.37 e/Å3).

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    Figure 5

    Figure 5. pH-rate profile for the logarithm of the inactivation second-order rate constants (kinact/Ki) for yeast α-glucosidase by 7. All experiments were performed at T = 37 °C in 50 mM buffer (acetic acid-sodium acetate buffer for pH 5.0–5.5, sodium phosphate buffer for pH 6.0–7.5, TAPS buffer for pH 7.9) containing 0.1% BSA. The line through the points is the best fit to a model where two protonation events (low pH) lead to an inactive enzyme, while a single deprotonation at higher pH values gives inactive enzyme.

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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXmtF2muw%253D%253D&md5=dbc82fd0fae5c7e33f99add6f956bb98
    8. 8
      Parenti, G.; Andria, G.; Valenzano, K. J. Pharmacological chaperone therapy: Preclinical development, clinical translation, and prospects for the treatment of lysosomal storage disorders. Mol. Ther. 2015, 23, 1138– 1148,  DOI: 10.1038/mt.2015.62
      8
      Pharmacological Chaperone Therapy: Preclinical Development, Clinical Translation, and Prospects for the Treatment of Lysosomal Storage Disorders
      Parenti, Giancarlo; Andria, Generoso; Valenzano, Kenneth J.
      Molecular Therapy (2015), 23 (7), 1138-1148CODEN: MTOHCK; ISSN:1525-0016. (Nature Publishing Group)
      A review. Lysosomal storage disorders (LSDs) are a group of inborn metabolic diseases caused by mutations in genes that encode proteins involved in different lysosomal functions, in most instances acidic hydrolases. Different therapeutic approaches have been developed to treat these disorders. Pharmacol. chaperone therapy (PCT) is an emerging approach based on small-mol. ligands that selectively bind and stabilize mutant enzymes, increase their cellular levels, and improve lysosomal trafficking and activity. Compared to other approaches, PCT shows advantages, particularly in terms of oral administration, broad biodistribution, and pos. impact on patients' quality of life. After preclin. in vitro and in vivo studies, PCT is now being translated in the first clin. trials, either as monotherapy or in combination with enzyme replacement therapy, for some of the most prevalent LSDs. For some LSDs, the results of the first clin. trials are encouraging and warrant further development. Future research in the field of PCT will be directed toward the identification of novel chaperones, including new allosteric drugs, and the exploitation of synergies between chaperone treatment and other therapeutic approaches.
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    9. 9
      Witte, M. D.; Kallemeijn, W. W.; Aten, J.; Li, K. Y.; Strijland, A.; Donker-Koopman, W. E.; van den Nieuwendijk, A.; Bleijlevens, B.; Kramer, G.; Florea, B. I.; Hooibrink, B.; Hollak, C. E. M.; Ottenhoff, R.; Boot, R. G.; van der Marel, G. A.; Overkleeft, H. S.; Aerts, J. Ultrasensitive in situ visualization of active glucocerebrosidase molecules. Nat. Chem. Biol. 2010, 6, 907– 913,  DOI: 10.1038/nchembio.466
      9
      Ultrasensitive in situ visualization of active glucocerebrosidase molecules
      Witte, Martin D.; Kallemeijn, Wouter W.; Aten, Jan; Li, Kah-Yee; Strijland, Anneke; Donker-Koopman, Wilma E.; van den Nieuwendijk, Adrianus M. C. H.; Bleijlevens, Boris; Kramer, Gertjan; Florea, Bogdan I.; Hooibrink, Berend; Hollak, Carla E. M.; Ottenhoff, Roelof; Boot, Rolf G.; van der Marel, Gijsbert A.; Overkleeft, Herman S.; Aerts, Johannes M. F. G.
      Nature Chemical Biology (2010), 6 (12), 907-913CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
      Deficiency of glucocerebrosidase (GBA) underlies Gaucher disease, a common lysosomal storage disorder. Carriership for Gaucher disease has recently been identified as major risk for parkinsonism. Presently, no method exists to visualize active GBA mols. in situ. The authors here report the design, synthesis and application of two fluorescent activity-based probes allowing highly specific labeling of active GBA mols. in vitro and in cultured cells and mice in vivo. Detection of in vitro labeled recombinant GBA on slab gels after electrophoresis is in the low attomolar range. Using cell or tissue lysates, the authors obtained exclusive labeling of GBA mols. The authors present evidence from fluorescence-activated cell sorting anal., fluorescence microscopy and pulse-chase expts. of highly efficient labeling of GBA mols. in intact cells as well as tissues of mice. In addn., the authors illustrate the use of the fluorescent probes to study inhibitors and tentative chaperones in living cells.
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    10. 10
      Scherer, M.; Santana, A. G.; Robinson, K.; Zhou, S.; Overkleeft, H. S.; Clarke, L.; Withers, S. G. Lipid-mimicking phosphorus-based glycosidase inactivators as pharmacological chaperones for the treatment of Gaucher’s disease. Chem. Sci. 2021, 12, 13909– 13913,  DOI: 10.1039/D1SC03831A
      10
      Lipid-mimicking phosphorus-based glycosidase inactivators as pharmacological chaperones for the treatment of Gaucher's disease
      Scherer, Manuel; Santana, Andres G.; Robinson, Kyle; Zhou, Steven; Overkleeft, Hermen S.; Clarke, Lorne; Withers, Stephen G.
      Chemical Science (2021), 12 (41), 13909-13913CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
      Gaucher's disease, the most prevalent lysosomal storage disorder, is caused by missense mutation of the GBA gene, ultimately resulting in deficient GCase activity, hence the excessive build-up of cellular glucosylceramide. Among different therapeutic strategies, pharmacol. chaperoning of mutant GCase represents an attractive approach that relies on small org. mols. acting as protein stabilizers. Herein, we expand upon a new class of transient GCase inactivators based on a reactive 2-deoxy-2-fluoro-β-D-glucoside tethered to an array of lipid-mimicking phosphorus-based aglycons, which not only improve the selectivity and inactivation efficiency, but also the stability of these compds. in aq. media. This hypothesis was further validated with kinetic and cellular studies confirming restoration of catalytic activity in Gaucher cells after treatment with these pharmacol. chaperones.
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    11. 11
      Adams, B. T.; Niccoli, S.; Chowdhury, M. A.; Esarik, A. N. K.; Lees, S. J.; Rempel, B. P.; Phenix, C. P. N-Alkylated aziridines are easily-prepared, potent, specific and cell-permeable covalent inhibitors of human β-glucocerebrosidase. Chem. Commun. 2015, 51, 11390– 11393,  DOI: 10.1039/C5CC03828F
      11
      N-Alkylated aziridines are easily-prepared, potent, specific and cell-permeable covalent inhibitors of human β-glucocerebrosidase
      Adams, B. T.; Niccoli, S.; Chowdhury, M. A.; Esarik, A. N. K.; Lees, S. J.; Rempel, B. P.; Phenix, C. P.
      Chemical Communications (Cambridge, United Kingdom) (2015), 51 (57), 11390-11393CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      β-Glucocerebrosidase deficiency leads to Gaucher disease and is a potential marker of Parkinson's disease. The authors have identified N-octyl conduritol aziridine as a potent and specific covalent inactivator of GBA1 in living cells. This compd. is a promising lead towards a positron emission tomog. probe intended to image GBA1 activity.
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    12. 12
      Kallemeijn, W. W.; Li, K. Y.; Witte, M. D.; Marques, A. R.; Aten, J.; Scheij, S.; Jiang, J.; Willems, L. I.; Voorn-Brouwer, T. M.; van Roomen, C. P. A. A.; Ottenhoff, R.; Boot, R. G.; van den Elst, H.; Walvoort, M. T.; Florea, B. I.; Codee, J. D.; van der Marel, G. A.; Aerts, J. M.; Overkleeft, H. S. Novel Activity-Based Probes for Broad-Spectrum Profiling of Retaining β-Exoglucosidases In Situ and In Vivo. Angew. Chem. Int. Ed., Engl. 2012, 51, 12529– 12533,  DOI: 10.1002/anie.201207771
      There is no corresponding record for this reference.
    13. 13
      Do, J.; McKinney, C.; Sharma, P.; Sidransky, E. Glucocerebrosidase and its relevance to Parkinson disease. Mol. Neurodegener. 2019, 14, 36,  DOI: 10.1186/s13024-019-0336-2
      13
      Glucocerebrosidase and its relevance to Parkinson disease
      Do Jenny; McKinney Cindy; Sharma Pankaj; Sidransky Ellen
      Molecular neurodegeneration (2019), 14 (1), 36 ISSN:.
      Mutations in GBA1, the gene encoding the lysosomal enzyme glucocerebrosidase, are among the most common known genetic risk factors for the development of Parkinson disease and related synucleinopathies. A great deal is known about GBA1, as mutations in GBA1 are causal for the rare autosomal storage disorder Gaucher disease. Over the past decades, significant progress has been made in understanding the genetics and cell biology of glucocerebrosidase. A least 495 different mutations, found throughout the 11 exons of the gene are reported, including both common and rare variants. Mutations in GBA1 may lead to degradation of the protein, disruptions in lysosomal targeting and diminished performance of the enzyme in the lysosome.Gaucher disease is phenotypically diverse and has both neuronopathic and non-neuronopathic forms. Both patients with Gaucher disease and heterozygous carriers are at increased risk of developing Parkinson disease and Dementia with Lewy Bodies, although our understanding of the mechanism for this association remains incomplete. There appears to be an inverse relationship between glucocerebrosidase and α-synuclein levels, and even patients with sporadic Parkinson disease have decreased glucocerebrosidase. Glucocerebrosidase may interact with α-synuclein to maintain basic cellular functions, or impaired glucocerebrosidase could contribute to Parkinson pathogenesis by disrupting lysosomal homeostasis, enhancing endoplasmic reticulum stress or contributing to mitochondrial impairment. However, the majority of patients with GBA1 mutations never develop parkinsonism, so clearly other risk factors play a role. Treatments for Gaucher disease have been developed that increase visceral glucocerebrosidase levels and decrease lipid storage, although they have yet to properly address the neurological defects associated with impaired glucocerebrosidase. Mouse and induced pluripotent stem cell derived models have improved our understanding of glucocerebrosidase function and the consequences of its deficiency. These models have been used to test novel therapies including chaperone proteins, histone deacetylase inhibitors, and gene therapy approaches that enhance glucocerebrosidase levels and could prove efficacious in the treatment of forms of parkinsonism. Consequently, this rare monogenic disorder, Gaucher disease, provides unique insights directly applicable to our understanding and treatment of Parkinson disease, a common and complex neurodegenerative disorder.
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    14. 14
      Ben Bdira, F.; Kallemeijn, W. W.; Oussoren, S. V.; Scheij, S.; Bleijlevens, B.; Florea, B. I.; van Roomen, C.; Ottenhoff, R.; van Kooten, M.; Walvoort, M. T. C.; Witte, M. D.; Boot, R. G.; Ubbink, M.; Overkleeft, H. S.; Aerts, J. Stabilization of Glucocerebrosidase by Active Site Occupancy. ACS Chem. Biol. 2017, 12, 1830– 1841,  DOI: 10.1021/acschembio.7b00276
      There is no corresponding record for this reference.
    15. 15
      Vocadlo, D. J.; Davies, G. J. Mechanistic insights into glycosidase chemistry. Curr. Opin. Chem. Biol. 2008, 12, 539– 555,  DOI: 10.1016/j.cbpa.2008.05.010
      15
      Mechanistic insights into glycosidase chemistry
      Vocadlo, David J.; Davies, Gideon J.
      Current Opinion in Chemical Biology (2008), 12 (5), 539-555CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)
      A review. The enzymic hydrolysis of the glycosidic bond continues to gain importance, reflecting the critically important roles complex glycans play in health and disease as well as the rekindled interest in enzymic biomass conversion. Recent advances include the broadening of our understanding of enzyme reaction coordinates, through both computational and structural studies, improved understanding of enzyme inhibition through transition state mimicry and fascinating insights into mechanism yielded by phys. org. chem. approaches.
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    16. 16
      Adamson, C.; Pengelly, R.; Shamsi Kazem Abadi, S.; Chakladar, S.; Draper, J.; Britton, R.; Gloster, T.; Bennet, A. J. Structural snapshots for mechanism-based inactivation of a glycoside hydrolase by cyclopropyl-carbasugars. Angew. Chem., Int. Ed. 2016, 55, 14978– 14982,  DOI: 10.1002/anie.201607431
      There is no corresponding record for this reference.
    17. 17
      Shamsi Kazem Abadi, S.; Tran, M.; Yadav, A. K.; Adabala, P. J. P.; Chakladar, S.; Bennet, A. J. New class of glycoside hydrolase mechanism-based covalent inhibitors: Glycosylation transition state conformations. J. Am. Chem. Soc. 2017, 139, 10625– 10628,  DOI: 10.1021/jacs.7b05065
      17
      New Class of Glycoside Hydrolase Mechanism-Based Covalent Inhibitors: Glycosylation Transition State Conformations
      Shamsi Kazem Abadi, Saeideh; Tran, Michael; Yadav, Anuj K.; Adabala, Pal John Pal; Chakladar, Saswati; Bennet, Andrew J.
      Journal of the American Chemical Society (2017), 139 (31), 10625-10628CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The design of covalent inhibitors in glycoscience research is important for the development of chem. biol. probes. Here we report the synthesis of a new carbocyclic mechanism-based covalent inhibitor of an α-glucosidase. The enzyme efficiently catalyzes its alkylation via either an allylic cation or a cationic transition state. We show this allylic covalent inhibitor has different catalytic proficiencies for pseudoglycosylation and deglycosylation. Such inhibitors have the potential to be useful chem. biol. tools.
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    18. 18
      Danby, P. M.; Withers, S. G. Glycosyl cations versus allylic cations in spontaneous and enzymatic hydrolysis. J. Am. Chem. Soc. 2017, 139, 10629– 10632,  DOI: 10.1021/jacs.7b05628
      18
      Glycosyl cations versus allylic cations in spontaneous and enzymatic hydrolysis
      Danby, Phillip M.; Withers, Stephen G.
      Journal of the American Chemical Society (2017), 139 (31), 10629-10632CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Enzymic prenyl and glycosyl transfer are seemingly unrelated reactions that yield mols. and protein modifications with disparate biol. functions. However, both reactions employ diphosphate-activated donors and each proceed via cationic species: allylic cations and oxocarbenium ions, resp. Here, we explored the relation between these processes by prepg. valienyl ethers to serve as glycoside mimics that were capable of allylic rather than oxocarbenium cation stabilization. Rate consts. for the spontaneous hydrolysis of aryl glycosides and their analogous valienyl ethers were found to be almost identical, as were the corresponding activation enthalpies and entropies. This close similarity extended to the assocd. secondary kinetic isotope effects (KIEs), indicating very similar transition state stabilities and structures. Screening a library of >100 β-glucosidases identified a no. of enzymes that catalyzed the hydrolysis of these valienyl ethers with kcat values of ≤20 s-1. Detailed anal. of one such enzyme showed that ether hydrolysis occurred via the analogous mechanisms found for glycosides, and through a very similar transition state. This suggested that the generally lower rates of enzymic cleavage of the cyclitol ethers reflects evolutionary specialization of these enzymes toward glycosides rather than inherent reactivity differences.
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    19. 19
      Parenti, G. Treating lysosomal storage diseases with pharmacological chaperones: from concept to clinics. EMBO Mol. Med. 2009, 1, 268– 279,  DOI: 10.1002/emmm.200900036
      19
      Treating lysosomal storage diseases with pharmacological chaperones: from concept to clinics
      Parenti, Giancarlo
      EMBO Molecular Medicine (2009), 1 (5), 268-279CODEN: EMMMAM; ISSN:1757-4684. (John Wiley & Sons Ltd.)
      A review. Lysosomal storage diseases (LSDs) are a group of genetic disorders due to defects in any aspect of lysosomal biol. During the past two decades, different approaches have been introduced for the treatment of these conditions. Among them, enzyme replacement therapy (ERT) represented a major advance and is used successfully in the treatment of some of these disorders. However, ERT has limitations such as insufficient biodistribution of recombinant enzymes and high costs. An emerging strategy for the treatment of LSDs is pharmacol. chaperone therapy (PCT), based on the use of chaperone mols. that assist the folding of mutated enzymes and improve their stability and lysosomal trafficking. After proof-of-concept studies, PCT is now being translated into clin. applications for Fabry, Gaucher and Pompe disease. This approach, however, can only be applied to patients carrying chaperone-responsive mutations. The recent demonstration of a synergistic effect of chaperones and ERT expands the applications of PCT and prompts a re-evaluation of their therapeutic use and potential. This review discusses the strengths and drawbacks of the potential therapies available for LSDs and proposes that future research should be directed towards the development of treatment protocols based on the combination of different therapies to improve the clin. outcome of LSD patients.
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    20. 20
      Sidransky, E.; Lopez, G. The link between the GBA gene and parkinsonism. Lancet Neurol. 2012, 11, 986– 998,  DOI: 10.1016/S1474-4422(12)70190-4
      20
      The link between the GBA gene and parkinsonism
      Sidransky, Ellen; Lopez, Grisel
      Lancet Neurology (2012), 11 (11), 986-998CODEN: LNAEAM; ISSN:1474-4422. (Elsevier Ltd.)
      A review. Summary: Mutations in the glucocerebrosidase (GBA) gene, which encodes the lysosomal enzyme that is deficient in Gaucher's disease, are important and common risk factors for Parkinson's disease and related disorders. This assocn. was first recognized in the clinic, where parkinsonism was noted, albeit rarely, in patients with Gaucher's disease and more frequently in relatives who were obligate carriers. Subsequently, findings from large studies showed that patients with Parkinson's disease and assocd. Lewy body disorders had an increased frequency of GBA mutations when compared with control individuals. Patients with GBA-assocd. parkinsonism exhibit varying parkinsonian phenotypes but tend to have an earlier age of onset and more assocd. cognitive changes than patients with parkinsonism without GBA mutations. Hypotheses proposed to explain this assocn. include a gain-of-function due to mutations in glucocerebrosidase that promotes α-synuclein aggregation; substrate accumulation due to enzymic loss-of-function, which affects α-synuclein processing and clearance; and a bidirectional feedback loop. Identification of the pathol. mechanisms underlying GBA-assocd. parkinsonism will improve our understanding of the genetics, pathophysiol., and treatment for both rare and common neurol. diseases.
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    21. 21
      Sinnott, M. L.; Souchard, I. J. The mechanism of action of β-galactosidase. Effect of aglycone nature and α-deuterium substitution on the hydrolysis of aryl galactosides. Biochem. J. 1973, 133, 89– 98,  DOI: 10.1042/bj1330089
      21
      Mechanism of action of β-galactosidase. Effect of aglycone nature and α-deuterium substitution on the hydrolysis of aryl galactosides
      Sinnott, Michael L.; Souchard, Jan J. L.
      Biochemical Journal (1973), 133 (1), 89-98CODEN: BIJOAK; ISSN:0264-6021.
      Steady-state kinetic parameters for the hydrolysis of 13 aryl β-D-galactopyranosides catalyzed by β-galactosidase (I) showed no simple dependence on aglycone acidity. The α-deuterium kinetic isotope effects (kH/kD) for 7 substrates and the effects of methanolysis of galactosyl-I in 1.5M MeOH on kH/kD were incompatible with a 2-step mechanism. A conformational change, liberation of a galactopyranosyl cation in an intimate ion-pair, nonproductive but preferential collapse of the ion pair to a covalent species, and reaction of the galactosyl-I through the ion-paired form was proposed as a reaction mechanism for β-galactosidase.
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    22. 22
      Kempton, J. B.; Withers, S. G. Mechanism of Agrobacterium .beta.-glucosidase: kinetic studies. Biochemistry 1992, 31, 9961– 9969,  DOI: 10.1021/bi00156a015
      22
      Mechanism of Agrobacterium β-glucosidase: kinetic studies
      Kempton, Julie B.; Withers, Stephen G.
      Biochemistry (1992), 31 (41), 9961-9CODEN: BICHAW; ISSN:0006-2960.
      β-Glucosidase from A. faecalis (previously Alcaligenes faecalis) was subjected to a detailed kinetic investigation with a range of substrates to probe its specificity and mechanism. It had a relatively broad specificity for the substrate sugar moiety and exhibited a classical pH dependence for its kinetic parameters with 3 different substrates and an identical pH dependence for its inactivation by a mechanism-based inactivator, cyclophellitol. Measurement of kcat and Km values for a series of aryl glucoside substrates allowed construction of a Broensted plot, the concave-downward shape of which was consistent with the anticipated 2-step mechanism involving a glucosyl-enzyme intermediate which is formed and hydrolyzed via oxocarbonium ion-like transition states. The slope of the leaving group-dependent portion of the Broensted plot (β1g = -0.7) indicated a large degree of bond cleavage at the transition state. Secondary deuterium kinetic isotope effects measured for 5 different aryl glucosides were also consistent with this mechanism and further suggested that the transition state for formation of the glucosyl-enzyme intermediate, probed with the slower substrates for which kH/kD = 1.06, is more SN2-like than that for its hydrolysis (for which kH/kD - 1.11). Reasons for this difference are proposed, and values of Ki for several ground-state and transition-state analog inhibitors are presented which support the concept of sp2-hybridized transition states.
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    23. 23
      Breen, I. Z.; Artola, M.; Wu, L.; Beenakker, T. J. M.; Offen, W. A.; Aerts, J. M. F. G.; Davies, G. J.; Overkleeft, H. S. Competitive and covalent inhibitors of human lysosomal retaining exoglucosidases. eLS; Wiley & Sons Ltd.: Chichester, 2018.
      There is no corresponding record for this reference.
    24. 24
      Segel, I. H. Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady State Enzyme Systems; Wiley: New York, 1975.
      There is no corresponding record for this reference.
    25. 25
      Jones, C. C.; Sinnott, M. L. Leaving ability and basicity of leaving groups attached by first-row elements. J. Chem. Soc., Chem. Commun. 1977, 767– 768,  DOI: 10.1039/c39770000767
      There is no corresponding record for this reference.
    26. 26
      Farren-Dai, M.; Sannikova, N.; Świderek, K.; Moliner, V.; Bennet, A. J. Fundamental insight into glycoside hydrolase-catalyzed hydrolysis of the universal Koshland substrates–glycopyranosyl fluorides. ACS Catal. 2021, 11, 10383– 10393,  DOI: 10.1021/acscatal.1c01918
      26
      Fundamental Insight into Glycoside Hydrolase-Catalyzed Hydrolysis of the Universal Koshland Substrates-Glycopyranosyl Fluorides
      Farren-Dai, Marco; Sannikova, Natalia; Swiderek, Katarzyna; Moliner, Vicent; Bennet, Andrew J.
      ACS Catalysis (2021), 11 (16), 10383-10393CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)
      The authors measured a panel of five kinetic isotope effects [KIEs; three secondary deuterium (1-, 2-, and 5-2H) and two primary (1-13C and 5-18O) effects] for the catalyzed hydrolysis of α-D-glucopyranosyl fluoride by two inverting glycoside hydrolases (GHs). The exptl. KIEs were compared to av. values computed with multiscale QM/MM methods for the hydrolysis of α-D-glucopyranosyl fluoride promoted by an inverting α-glucosidase (Aspergillus niger, GH family 15) to give β-D-glucopyranose, which the authors explored by the generation of free energy surfaces. The authors also measured the same KIEs for catalysis of α-D-glucopyranosyl fluoride by the Trichoderma virens GH55 inverting β-glucosidase using the authors' panel of isotopologues; this reaction occurs via the "Hehre resynthesis-hydrolysis mechanism" to give, by two inversions of configuration, the hydrolysis product α-D-glucopyranose. The transition states for both enzymic reactions are essentially identical with fluoride ion departure occurring within a glycoside hydrolase active site that stabilizes pyranosylium ion-like TSs, and with catalysis driven solely by H-bonding assistance from an enzymic carboxylic acid residue. That is, no assistance is required from the bound nucleophile, which in these two cases is either a water mol. (GH15) or a sugar hydroxyl group (GH55).
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    27. 27
      Boer, D. E.; Mirzaian, M.; Ferraz, M. J.; Zwiers, K. C.; Baks, M. V.; Hazeu, M. D.; Ottenhoff, R.; Marques, A. R. A.; Meijer, R.; Roos, J. C. P.; Cox, T. M.; Boot, R. G.; Pannu, N.; Overkleeft, H. S.; Artola, M.; Aerts, J. M. Human glucocerebrosidase mediates formation of xylosyl-cholesterol by β-xylosidase and transxylosidase reactions. J. Lipid Res. 2021, 62, 100018,  DOI: 10.1194/jlr.RA120001043
      There is no corresponding record for this reference.
    28. 28
      Beauhaire, J.; Ducrot, P. H. An epoxide derived from D-glucose as the key intermediate for penaresidine and sphingolipids synthesis. Synth. Commun. 1998, 28, 2443– 2456,  DOI: 10.1080/00397919808004296
      28
      An epoxide derived from D-glucose as the key intermediate for penaresidine and sphingolipids synthesis
      Beauhaire, Josiane; Ducrot, Paul-Henri
      Synthetic Communications (1998), 28 (13), 2443-2456CODEN: SYNCAV; ISSN:0039-7911. (Marcel Dekker, Inc.)
      A multigram-scale synthesis of 3R,4R,5R-3,5-dibenzyloxy-4-p-methoxybenzyl-1,2-epoxypentane and its use as intermediate for sphingolipids, penazeridine and penazetidine synthesis are described.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXktFCitrk%253D&md5=80b7b7320718c1c3e7e70efab3b2295f
    29. 29
      Ley, S. V.; Baeschlin, D. K.; Dixon, D. J.; Foster, A. C.; Ince, S. J.; Priepke, W. M.; Reynolds, D. J. 1,2-Diacetals: A new opportunity for organic synthesis. Chem. Rev. 2001, 101, 53– 80,  DOI: 10.1021/cr990101j
      29
      1,2-Diacetals: A New Opportunity for Organic Synthesis
      Ley, Steven V.; Baeschlin, Daniel K.; Dixon, Darren J.; Foster, Alison C.; Ince, Stuart J.; Priepke, Henning W. M.; Reynolds, Dominic J.
      Chemical Reviews (Washington, D. C.) (2001), 101 (1), 53-80CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      A review with 127 refs. 1,2-Diacetals offer diol protection.
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    30. 30
      Shih, T. L.; Liao, W. Y.; Yen, W. C. Regioselective fluorination in synthesis of deoxyfluoro quercitols from D-(−)-quinic acid. Tetrahedron 2014, 70, 9621– 9627,  DOI: 10.1016/j.tet.2014.11.001
      There is no corresponding record for this reference.
    31. 31
      Baici, A.; Schenker, P.; Wachter, M.; Ruedi, P. 3-Fluoro-2,4-dioxa-3-phosphadecalins as inhibitors of acetylcholinesterase. A reappraisal of kinetic mechanisms and diagnostic methods. Chem. Biodivers. 2009, 6, 261– 282,  DOI: 10.1002/cbdv.200800334
      There is no corresponding record for this reference.
    32. 32
      McCarter, J. D.; Withers, S. G. 5-Fluoro Glycosides:  A New Class of Mechanism-Based Inhibitors of Both α- and β-Glucosidases. J. Am. Chem. Soc. 1996, 118, 241– 242,  DOI: 10.1021/ja952732a
      32
      5-Fluoro Glycosides: A New Class of Mechanism-Based Inhibitors of Both α- and β-Glucosidases
      McCarter, John D.; Withers, Stephen G.
      Journal of the American Chemical Society (1996), 118 (1), 241-2CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Mechanism-based inhibitors of retaining glycosidases are of considerable interest, both academically and as potential therapeutics. 5-Fluoroglycosyl fluorides I represent a new class of such inhibitors, functioning via the formation of a stabilized 5-fluoroglycosyl-enzyme intermediate that turns over only very slowly. Synthesis is achieved via radical bromination of the corresponding protected glycosyl fluoride, displacement of the bromide by fluoride and deprotonation. 5-Fluoro-β-glucosyl fluoride functions as a time-dependent inactivator of Agrobacterium faecalis β-glucosidase with a second order rate consts. for inactivation of ki/Ki = 660 min-1 mM-1. Turnover of the 5-fluoro α-glucosyl-enzyme intermediate occurs with a half life of 8.5 min. 5-Fluoro α-glucosyl fluoride functions similarly as an inactivator of yeast α-glucosidase, but inactivation is too rapid to allow detn. of kinetic parameters for the inactivation process. Turnover is also more rapid, occurring with a rate const. of 6.6 min-1. Steady state apparent Ki values of 0.3 μM and 1.4 μM are detd. for β-glucosidase and α-glucosidase resp.
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    33. 33
      Ren, W.; Farren-Dai, M.; Sannikova, N.; Świderek, K.; Wang, Y.; Akintola, O.; Britton, R.; Moliner, V.; Bennet, A. J. Glycoside hydrolase stabilization of transition state charge: New directions for inhibitor design. Chem. Sci. 2020, 11, 10488– 10495,  DOI: 10.1039/D0SC04401F
      33
      Glycoside hydrolase stabilization of transition state charge: new directions for inhibitor design
      Ren, Weiwu; Farren-Dai, Marco; Sannikova, Natalia; Swiderek, Katarzyna; Wang, Yang; Akintola, Oluwafemi; Britton, Robert; Moliner, Vicent; Bennet, Andrew J.
      Chemical Science (2020), 11 (38), 10488-10495CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
      Carbasugars are structural mimics of naturally occurring carbohydrates that can interact with and inhibit enzymes involved in carbohydrate processing. In particular, carbasugars have attracted attention as inhibitors of glycoside hydrolases (GHs) and as therapeutic leads in several disease areas. However, it is unclear how the carbasugars are recognized and processed by GHs. Here, we report the synthesis of three carbasugar isotopologues and provide a detailed transition state (TS) anal. for the formation of the initial GH-carbasugar covalent intermediate, as well as for hydrolysis of this intermediate, using a combination of exptl. measured kinetic isotope effects and hybrid QM/MM calcns. We find that the α-galactosidase from Thermotoga maritima effectively stabilizes TS charge development on a remote C5-allylic center acting in concert with the reacting carbasugar, and catalysis proceeds via an exploded, or loose, SN2 transition state with no discrete enzyme-bound cationic intermediate. We conclude that, in complement to what we know about the TS structures of enzyme-natural substrate complexes, knowledge of the TS structures of enzymes reacting with non-natural carbasugar substrates shows that GHs can stabilize a wider range of pos. charged TS structures than previously thought. Furthermore, this enhanced understanding will enable the design of new carbasugar GH transition state analogs to be used as, for example, chem. biol. tools and pharmaceutical lead compds.
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    34. 34
      Akintola, O.; Farren-Dai, M.; Ren, W.; Bhosale, S.; Britton, R.; Świderek, K.; Moliner, V.; Bennet, A. J. Glycoside hydrolase catalysis: Do substrates and mechanism-based covalent inhibitors react via matching transition states?. ACS Catal. 2022, 12, 14667– 14678,  DOI: 10.1021/acscatal.2c04027
      There is no corresponding record for this reference.
    35. 35
      Rowland, R. J.; Wu, L.; Liu, F.; Davies, G. J. A baculoviral system for the production of human β-glucocerebrosidase enables atomic resolution analysis. Acta Crystallogr. Sect. D-Struct. Biol. 2020, 76, 565– 580,  DOI: 10.1107/S205979832000501X
      There is no corresponding record for this reference.
    36. 36
      Speciale, G.; Thompson, A. J.; Davies, G. J.; Williams, S. J. Dissecting conformational contributions to glycosidase catalysis and inhibition. Curr. Opin. Struct. Biol. 2014, 28, 1– 13,  DOI: 10.1016/j.sbi.2014.06.003
      36
      Dissecting conformational contributions to glycosidase catalysis and inhibition
      Speciale, Gaetano; Thompson, Andrew J.; Davies, Gideon J.; Williams, Spencer J.
      Current Opinion in Structural Biology (2014), 28 (), 1-13CODEN: COSBEF; ISSN:0959-440X. (Elsevier Ltd.)
      A review. Glycoside hydrolases (GHs) are classified into >100 sequence-based families. These enzymes process a wide variety of complex carbohydrates with varying stereochem. at the anomeric and other ring positions. The shapes that these sugars adopt upon binding to their cognate GHs, and the conformational changes that occur along the catalysis reaction coordinate is termed the conformational itinerary. Efforts to define the conformational itineraries of GHs have focussed upon the crit. points of the reaction: substrate-bound (Michaelis), transition state, intermediate (if relevant) and product-bound. Recent approaches to defining conformational itineraries that marry X-ray crystallog. of enzymes bound to ligands that mimic the crit. points, along with advanced computational methods and kinetic isotope effects are discussed.
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    37. 37
      Matsusaka, K.; Chiba, S.; Shimomura, T. Purification and substrate specificity of brewer’s yeast .ALPHA.-glucosidase. Agric. Biol. Chem. 1977, 41, 1917– 1923,  DOI: 10.1271/bbb1961.41.1917
      37
      Purification and substrate specificity of Brewers' yeast α-glucosidase
      Matsusaka, Kouetsu; Chiba, Seiya; Shimomura, Tokuji
      Agricultural and Biological Chemistry (1977), 41 (10), 1917-23CODEN: ABCHA6; ISSN:0002-1369.
      Three kinds of α-glucosidase (I, II and III) were isolated from brewers' yeast. α-Glucosidases I and II were homogeneous in disc electrophoresis, but α-glucosidase III was not. Their pH optima were 6.3-7.1. Each of them was a typical α-glucosidase showing a preferential activity on Ph α-glucoside (I). α-Glucosidase I was isomaltase, which can hydrolyze isomaltose but not maltose. α-Glucosidases II and III showed no activity on isomaltose. The ratio of velocity of hydrolysis for I and isomaltose of α-glucosidase I was 100:9, and those for I and maltose of α-glucosidases II and III were 100:17 and 100:18, resp.
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    38. 38
      Bordwell, F. G. Are nucleophilic bimolecular concerted reactions involving four or more bonds a myth?. Acc. Chem. Res. 1970, 3, 281– 290,  DOI: 10.1021/ar50033a001
      There is no corresponding record for this reference.
    39. 39
      Bordwell, F. G.; Mecca, T. G. Nucleophilic substitutions in allylic systems. Further evidence against existence of concerted SN2’ mechanism. J. Am. Chem. Soc. 1972, 94, 5829– 5837,  DOI: 10.1021/ja00771a048
      There is no corresponding record for this reference.
    40. 40
      Kantner, S. S.; Humski, K.; Goering, H. L. On the solvolysis of 2-cyclohexenyl 3,5-dinitrobenzoate and p-nitrobenzoate in aqueous acetone. Introduction of acyl-oxygen cleavage by basic buffer systems. J. Am. Chem. Soc. 1982, 104, 1693– 1697,  DOI: 10.1021/ja00370a040
      There is no corresponding record for this reference.
    41. 41
      Streitwieser, A.; Jayasree, E. G.; Hasanayn, F.; Leung, S. S. H. A theoretical study of SN2’ reactions of allylic halides: Role of ion pairs. J. Org. Chem. 2008, 73, 9426– 9434,  DOI: 10.1021/jo8020743
      41
      A Theoretical Study of SN2' Reactions of Allylic Halides: Role of Ion Pairs
      Streitwieser, A.; Jayasree, E. G.; Hasanayn, F.; Leung, S. S.-H.
      Journal of Organic Chemistry (2008), 73 (23), 9426-9434CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)
      Various disparate exptl. results are explained by the hypothesis that reactions of anionic nucleophiles with allylic halides are generally SN2. The SN2' reactions that do occur proceed generally with anti stereochem. Reactions with ion pair nucleophiles occur preferentially as SN2' reactions with syn stereochem. This hypothesis is consistent with a variety of computations at the HF, B3LYP, mPW1PW91 and MP2 levels with the 6-31+G(d) basis set of reactions of Li and Na fluoride and chloride with allyl halides and 4-halo-2-pentenes. Solvation is considered by a combination of coordination of di-Me ether to the lithium and sodium cations and "dielec. solvation" with a polarized continuum model.
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    42. 42
      Bentley, T. W.; Carter, G. E. The SN2-SN1 spectrum. 4. The SN2 (intermediate) mechanism for aolvolysis of tert-butyl chloride: A revised Y scale of solvent ionizing power based on solvolysis of 1-adamantyl chloride. J. Am. Chem. Soc. 1982, 104, 5741– 5747,  DOI: 10.1021/ja00385a031
      42
      The SN2-SN1 spectrum. 4. The SN2 (intermediate) mechanism for solvolyses of tert-butyl chloride: a revised Y scale of solvent ionizing power based on solvolyses of 1-adamantyl chloride
      Bentley, T. William; Carter, Gillian E.
      Journal of the American Chemical Society (1982), 104 (21), 5741-7CODEN: JACSAT; ISSN:0002-7863.
      New kinetic data for solvolysis of 1-adamantyl chloride and bromide were detd. in several solvents. The reliability of the results is supported by the consistent activation parameters and by bromide-chloride rate ratios, which closely parallel the corresponding ratios obtained for solvolysis of tert-Bu halides. The effects of added salts on solvolyzes are given. A new scale of solvent ionizing power for chlorides (YCl) was defined as log (k/k0) = mYCl (k = solvolysis rate const. for 1-adamantyl chloride in any solvent at 25°; k0 = same for 80 vol.% aq. EtOH; m = 1); an analogous scale is defined using data for 1-adamantyl bromide. These Y values were used to correlate rate data for tert-Bu and 1-adamantyl halides.
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    43. 43
      Ohga, Y.; Munakata, M.; Kitagawa, T.; Kinoshita, T.; Takeuchi, K.; Oishi, Y.; Fujimoto, H. Solvolyses of bicyclo[2.2.2]oct-1-yl and 1-adamantyl systems containing an ethylidene substituent on the 2-position: typical examples of rate enhancements ascribed to relief of F-strain. J. Org. Chem. 1994, 59, 4056– 4067,  DOI: 10.1021/jo00094a012
      There is no corresponding record for this reference.
    44. 44
      Chan, J.; Tang, A.; Bennet, A. J. A stepwise solvent-promoted SNi reaction of α-D-glucopyranosyl fluoride: Mechanistic implications for retaining glycosyltransferases. J. Am. Chem. Soc. 2012, 134, 1212– 1220,  DOI: 10.1021/ja209339j
      44
      A Stepwise Solvent-Promoted SNi Reaction of α-D-Glucopyranosyl Fluoride: Mechanistic Implications for Retaining Glycosyltransferases
      Chan, Jefferson; Tang, Ariel; Bennet, Andrew J.
      Journal of the American Chemical Society (2012), 134 (2), 1212-1220CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The solvolysis of α-d-glucopyranosyl fluoride in hexafluoro-2-propanol gives two products, 1,1,1,3,3,3-hexafluoropropan-2-yl α-d-glucopyranoside and 1,6-anhydro-β-d-glucopyranose. The ratio of these two products is essentially unchanged for reactions that are performed between 56 and 100 °C. The activation parameters for the solvolysis reaction are as follows: ΔH⧺ = 81.4 ± 1.7 kJ mol-1, and ΔS⧺ = -90.3 ± 4.6 J mol-1 K-1. To characterize, by use of multiple kinetic isotope effect (KIE) measurements, the TS for the solvolysis reaction in hexafluoro-2-propanol, we synthesized a series of isotopically labeled α-d-glucopyranosyl fluorides. The measured KIEs for the C1 deuterium, C2 deuterium, C5 deuterium, anomeric carbon, ring oxygen, O6, and solvent deuterium are 1.185 ± 0.006, 1.080 ± 0.010, 0.987 ± 0.007, 1.008 ± 0.007, 0.997 ± 0.006, 1.003 ± 0.007, and 1.68 ± 0.07, resp. The transition state for the solvolysis reaction was modeled computationally using the exptl. KIE values as constraints. Taken together, the reported data are consistent with the retained solvolysis product being formed in an SNi (DN⧺*ANss) reaction with a late transition state in which cleavage of the glycosidic bond is coupled to the transfer of a proton from a solvating hexafluoro-2-propanol mol. In comparison, the inverted product, 1,6-anhydro-β-d-glucopyranose, is formed by intramol. capture of a solvent-equilibrated glucopyranosylium ion, which results from dissocn. of the solvent-sepd. ion pair formed in the rate-limiting ionization reaction (DN⧺ + AN). The implications that this model reaction have for the mode of action of retaining glycosyltransferases are discussed.
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    45. 45
      Sinnott, M. L.; Jencks, W. P. Solvolysis of D-glucopyranosyl derivatives in mixtures of ethanol and 2,2,2-trifluoroethanol. J. Am. Chem. Soc. 1980, 102, 2026– 2032,  DOI: 10.1021/ja00526a043
      45
      Solvolysis of D-glucopyranosyl derivatives in mixtures of ethanol and 2,2,2-trifluoroethanol
      Sinnott, Michael L.; Jencks, William P.
      Journal of the American Chemical Society (1980), 102 (6), 2026-32CODEN: JACSAT; ISSN:0002-7863.
      The products of solvolysis of α- and β-D-glucopyranosyl fluorides, 2,4-dinitrophenyl β-D-glucopyranoside, and the trifluoromethanesulfonates of the β-D-glucopyranosyl 3-bromopyridinium and α-D-glucopyranosyl 4-methylpyridinium ions in an equimolar mixt. of ethanol and trifluoroethanol buffered with ∼2 equiv of 2,6-lutidine have been examd. by gas-liq. chromatog. of their trimethylsilyl ethers. The initial products of the solvolyses of Ph α- and β-D-glucopyranosides catalyzed by CF3SO3H in an equimolar mixt. of ethanol and trifluoroethanol, and the products of uncatalyzed solvolysis of β-D-glucopyranosyl-p-nitrophenyltriazene, have been likewise examd. The compn. of the medium for solvolysis of the glucosyl fluorides has also been systematically varied from pure ethanol to pure trifluoroethanol. The percentage of products with the same anomeric configuration as the starting material is in the range 8.1-88.5%; change of leaving group, at const. anomeric configuration, or of anomeric configuration, at const. leaving group, yields different product distributions. Therefore the transition state of the product-detg. step contains the leaving group. The preference for attack by ethanol as compared with trifluoroethanol varies from 0.9 to 20 in a way which shows no general systematic distinction between pathways for retention or inversion. The nucleophilic selectivity for retention is lowered by anionic leaving groups, esp. fluoride, which preferentially stabilize the transition state contg. trifluoroethanol by H bonding. Nucleophilic attack at the α face is preferred over nucleophilic attack at the β face, and exhibits a lower selectivity: this is ascribed to H bonding between the O atom of the 2-hydroxyl group and the hydroxyl group of the approaching alc. A model for solvolysis involving a reversibly formed ion pair or encounter complex is incompatible with the selectivities still obsd. with leaving groups less nucleophilic than the solvent components: a model involving selection between the components of a pool of solvent mols. by an irreversibly formed ion pair or encounter complex requires an implausibly large pool to explain obsd. specificities. The obsd. selectivities are a consequence of the facilitation of the departure of the leaving group by the solvent, from either side of the reaction center.
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    46. 46
      Wan, Q.; Parks, J. M.; Hanson, B. L.; Fisher, S. Z.; Ostermann, A.; Schrader, T. E.; Graham, D. E.; Coates, L.; Langan, P.; Kovalevsky, A. Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography. Proc. Natl. Acad. Sci. U.S.A. 2015, 112, 12384– 12389,  DOI: 10.1073/pnas.1504986112
      46
      Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography
      Wan, Qun; Parks, Jerry M.; Hanson, B. Leif; Fisher, Suzanne Zoe; Ostermann, Andreas; Schrader, Tobias E.; Graham, David E.; Coates, Leighton; Langan, Paul; Kovalevsky, Andrey
      Proceedings of the National Academy of Sciences of the United States of America (2015), 112 (40), 12384-12389CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Glycoside hydrolases (GHs) apply acid/base chem. to catalyze the decompn. of complex carbohydrates. These ubiquitous enzymes accept protons from solvent and donate them to substrates at close to neutral pH by modulating the pKa values of key side-chains during catalysis. However, it is not known how the catalytic acid residue acquires a proton and transfers it efficiently to the substrate. To better understand GH chem., we used macromol. neutron crystallog. to directly det. protonation and ionization states of the active site residues of a family 11 GH, Trichoderma reesei xylanase II (XynII), at multiple pD (pD = pH + 0.4) values. The general acid (Glu residue) cycled between 2 conformations, upward and downward, but was protonated only in the downward orientation. The authors performed continuum electrostatics calcns. to est. the pKa values of the catalytic Glu residues in both the apoenzyme and substrate-bound states of XynII. The calcd. pKa of the Glu residue increased substantially when the side-chain moved down. The energy barrier required to rotate the catalytic Glu residue back to the upward conformation, where it could protonate the glycosidic O atom of the substrate, was 4.3 kcal/mol according to free energy simulations. These findings shed light on the initial stage of the glycoside hydrolysis reaction in which mol. motion enables the general acid catalyst to obtain a proton from the bulk solvent and deliver it to the glycosidic O atom.
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  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acscatal.4c04549.

    • General methods and reagents, eqs S1–S3, Tables S1–S3, Scheme S1, Figures S1–S78 (PDF)


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