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From Mechanism-Based Retaining Glycosidase Inhibitors to Activity-Based Glycosidase Profiling
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Cite this: J. Am. Chem. Soc. 2024, 146, 36, 24729–24741
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https://doi.org/10.1021/jacs.4c08840
Published August 30, 2024

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Abstract

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Activity-based protein profiling (ABPP) is an effective technology for the identification and functional annotation of enzymes in complex biological samples. ABP designs are normally directed to an enzyme active site nucleophile, and within the field of Carbohydrate-Active Enzymes (CAZymes), ABPP has been most successful for those enzymes that feature such a residue: retaining glycosidases (GHs). Several mechanism-based covalent and irreversible retaining GH inhibitors have emerged over the past sixty years. ABP designs based on these inhibitor chemistries appeared since the turn of the millennium, and we contributed to the field by designing a suite of retaining GH ABPs modeled on the structure and mode of action of the natural product, cyclophellitol. These ABPs enable the study of both exo- and endo-acting retaining GHs in human health and disease, for instance in genetic metabolic disorders in which retaining GHs are deficient. They are also finding increasing use in the study of GHs in gut microbiota and environmental microorganisms, both in the context of drug (de)toxification in the gut and that of biomass polysaccharide processing for future sustainable energy and chemistries. This account comprises the authors’ view on the history of mechanism-based retaining GH inhibitor design and discovery, on how these inhibitors served as blueprints for retaining GH ABP design, and on some current and future developments on how cyclophellitol-based ABPs may drive the discovery of retaining GHs and their inhibitors.

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Introduction

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Sugars comprise the most abundant and among the structurally most diverse class of biomolecules on earth. Their structural diversity and the countless biological functions in which they partake is reflected by the enzymes that have evolved to create and break down glycans and that are collectively referred to as Carbohydrate-Active Enzymes (CAZymes). Glycosyltransferases (GTs), transglycosidases and phosphorylases catalyze the formation of glycosidic linkages, the breakdown of which is done by several enzyme families that are characterized by distinct chemistries. The largest family of glycoside-degrading enzymes are the glycoside hydrolases (glycosidases, GHs), classified in the CAZy database (1,2) into almost two hundred families based on primary sequence (and thus predictive of structure/fold and mechanism), with two main types when looking at the stereochemical fate of the anomeric carbon at which hydrolysis takes place. (3) These follow the mechanisms first proposed by Daniel Koshland in 1953: (4) single displacement for inverting GHs, and double displacement for retaining GHs. Inverting GHs (illustrated in Figure 1A for an inverting β-glucosidase) catalyze substrate hydrolysis with inversion of anomeric configuration. In this process, the general acid–base residue (normally an aspartic acid or glutamic acid) residing in the inverting GH active site protonates the exocyclic acetal oxygen, thereby turning it into a good leaving group. This process coincides with deprotonation of an active site-residing water molecule, effected by an active site aspartate/glutamate, leading to overall substitution of a β-glucoside into α-glucose – thus substrate hydrolysis with net inversion of anomeric configuration. In this reaction, the substrate glucoside proceeds through an oxocarbenium ion-like transition state, with the endocyclic C-O bond developing double bond character with a partial positive charge (δ+) distributed over this C-O bond.

Figure 1

Figure 1. Mechanism of action of inverting β-glucosidases (A) and retaining β-glucosidases (B).

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Retaining glycosidases (illustrated in Figure 1B for a retaining β-glucosidase) catalyze substrate hydrolysis with overall retention of anomeric configuration: a β-glucoside substrate is hydrolyzed to give β-glucose. As with inverting glucosidases, two active site carboxyl residues make up the catalytic machinery, but in contrast to the inverting glucosidase situation there is no room for a water residue within the enzyme active site upon substrate binding. Rather, the Asp/Glu residue residing at the bottom face of the substrate β-glucoside is situated closer to the substrate such that, upon protonation this residue substitutes the aglycon to form a covalent enzyme–substrate intermediate, via an oxocarbenium ion like transition state. Water then enters the enzyme active site and in a reversal of steps the glycosyl-enzyme linkage is hydrolyzed to form β-glucose, again through an oxocarbenium like transition state. Both steps proceed with inversion of configuration at the substrate anomeric carbon, and the net result is therefore retention of anomeric configuration. The existence of a covalent intermediate such as emerges during retaining β-glucosidase catalysis provides an opportunity for the design of mechanism-based inhibitors, (5) and from there, activity-based probes. In contrast, ABP designs for inverting glycosidases are not so obvious, and for this reason, CAZyme ABPP has focused almost exclusively on studies of retaining glycosidases. The retaining mechanism is conserved over many GH families, both exo-acting (recognizing a chain-end and removing a sugar of defined length, usually a single monosaccharide but disaccharide and trisaccharide liberating exoenzymes are also known) and endo-acting (cleaving in the middle of a glycan). Most retaining glycosidases feature two Asp/Glu active site residues as general acid/base catalyst (for protonation of the aglycon) and catalytic nucleophile, respectively. Variations in general acid/base residue (His in, among others, fucoidanases (6)) and nucleophilic residue (Tyr in retaining neuraminidases, (7) Cys in some retaining arabinofuranosidases (8)) have been identified but also these enzymes process their substrate through a covalent glycosyl-enzyme intermediate and are therefore amenable to ABPP. Notable exceptions of retaining glycosidases are hexosaminidases and chitinases that utilize substrate assisted catalysis, in which a substrate residue engages as a nucleophile (9) and for which no suitable ABP designs have yet emerged. GH99 α-endomannanases as well utilize neighboring group participation and catalyze substrate hydrolysis through a 1,2-anhydromannose intermediate, again posing challenges for a classical ABPP approach. (10)

Mechanism-Based Retaining Glycosidase Inhibitor Designs

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Several mechanism-based, covalent and irreversible retaining GH inhibitor designs have emerged over the past decades, some of which have in later years also been adapted for ABP designs. The two most widely applied design principles are based on the two classical retaining glycosidase inactivators depicted in Figure 2A. 2-Deoxy-2-fluoroglucoside 1 was developed by Withers and co-workers in 1987 as a mechanism-based retaining β-glucosidase inhibitor. (11) The presence of an electron-withdrawing fluorine substituent (the 2-F group in 1) was envisaged to slow down the reaction rate through raising the energy of the oxocarbenium ion-like transition states in both steps of the retaining glycosidase mechanism (see Figure 1B). However, the inclusion of a highly reactive 2,4-dinitrophenyl selectively accelerates the first step relative to the second step. The resulting 2-deoxy-2-fluoroglycosyl-enzyme adduct 2 accumulates and depending on the enzyme can have a half-life of minutes, hours or even days, resulting in inhibition of the enzyme. The deoxyfluoro glycoside design, also termed ‘activated fluorinated glycosides’, has been used in the following years in the establishment of such nucleophiles in a range of retaining glycosidases. (12) One seminal study (13) demonstrates, by X-ray crystallography and mass spectrometry, that hen egg white lysozyme employs the Koshland two-step double displacement mechanism (4) and that the active site residue, Asp52, acts as a nucleophile rather than an oxocarbenium ion-stabilizing carboxylate, forming adduct 3 (Figure 1A, insert).

Figure 2

Figure 2. Two archetypal retaining glycosidase inhibitor designs: deoxyfluoroglycosides (A) and cyclitol epoxides (B).

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Preceding the activated deoxyfluoro glycoside design is Legler’s work on conduritol B epoxide (CBE, 4, Figure 2B). (14−17) CBE is pseudosymmetric, and mimicks β-glucosides (Figure 2B) and α-glucosides (Figure 2A) through rotation by 180° around the axis dissecting the C1-C2 and C3-C4 bonds. This feature of CBE explains the ability of this molecule to inhibit retaining β-glucosidases and retaining α-glucosidases. Treatment of an Aspergillus wentii retaining β-glucosidase with 4 followed by reaction of the formed enzyme–inhibitor adduct 5 with hydroxylamine released (+)-chiro-inositol 6. (16) In contrast, treatment of rabbit intestine sucrase-isomaltase, a retaining α-glucosidase, with CBE 4 followed by reaction of the adduct with hydroxylamine, returned scyllo-inositol 8. (17) These results strongly suggested enzyme-mediated, regioselective opening of the epoxides at the carbon atom assuming, within the active site of the respective enzymes, the position of the anomeric carbon of the β- and α-glucoside substrates, respectively, providing supporting evidence for the two-step double displacement mechanism proposed by Koshland. (4)
CBE (4) is a member of a series of carbohydrate-mimetic epoxides and aziridines that have been used for many decades as GH inactivators to study the mechanism of (retaining) GHs. Besides cyclitol-fused epoxides, also linear, glycosylated epoxy alcohols have contributed to unearthing retaining GH active site nucleophiles. (18,19) The example shown in Figure 3A pertains an X-ray study revealing that both diastereomeric epoxides from a mixture of 3,4-epoxybutyl-β-cellobioside 9 reacted within the Fusarium oxysporum cellulase, EG I, to form a covalent ester bond with the active site nucleophile (E197). (20) Such epoxy-alkylglycosides have been applied to inactivate retaining glycosidases since 1969, when Thomas (21) used GlcNAc-epoxides 11 for the irreversible inactivation of hen egg white lysozyme (HEWL) in a study aimed to gather evidence for the Koshland two-step mechanism invoking an active site nucleophile employed by retaining glycosidases. (4) Although the mode of action (reaction of either of the epoxide carbons with either of the active site carboxylates E35 or D52) was not revealed in these studies, HEWL was shown to be inhibited irreversibly. Perhaps because linear epoxides such as 9 are rather weak GH inhibitors, and because later studies revealed they may alkylate both nucleophile and general acid base residues at the same time, (22) these compounds have not been used as a starting point for glycosidase ABP design, even though grafting a reporter moiety on either terminus of the molecule appears a relatively simple manner. It may also be that, when in 1999 the concept of ABPP emerged, (23) better design blueprints had already been identified. Besides epoxides, haloacetyl aminoglycosides have been used as mechanism-based retaining GH inactivators since the early seventies of the last century as well. Escherichia coli retaining β-galactosidase inactivator 12 comprises the first example of this compound class, (24) which has been adapted in later years in ABP designs.

Figure 3

Figure 3. Glycomimetic epoxides, aziridines and episulfides as mechanism-based, covalent and irreversible retaining glycosidase inhibitors.

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Returning to the theme of cyclic epoxides and aziridines, Caron and Withers in 1989 reported conduritol B aziridine 13 as a mechanism-based inactivator of both almond β-glucosidase and yeast α-glucosidase, thus emulating the activity profile also exhibited by CBE 4. (25) This study was preceded by one year by the design, by Tong and Ganem, of the coffee bean α-galactosidase inactivator 15. (26) Although the proposed mechanism of inactivation (the formation of 16) as shown in Figure 3C was not proven, the compound blocked the enzyme in an irreversible manner and proved to be selective over yeast α-glucosidase and jack bean α-mannosidase. In their work on conduritol B aziridine 13, Caron and Withers predicted (25) that breaking the pseudosymmetry in CBE (or conduritol B aziridine) by inserting an extra methylene in the C3-OH or C6-OH bonds would yield compounds both more potent and more selective for retaining α and β-glucosidases, respectively. The discovery, (27) one year later, by Umezawa and co-workers, of the natural product, cyclophellitol (17) proved this idea to be valid. Cyclophellitol, the CBE analogue having the added methylene (the blue bulb) inserted in the C6-OH bond, is a highly potent and selective (also with respect to retaining α-glucosidases) retaining β-glucosidase inhibitor, and X-ray studies by Gloster, Madsen and Davies revealed that the mode of action is indeed by active site nucleophile modification, in particular the Thermotoga maritima retaining β-glucosidase (TmGH1) active site nucleophile, Glu351. (28) Shortly following the discovery of cyclophellitol, 1,6-epi-cyclophellitol (18, with a methylene now inserted in the indicated C3-OH bond in CBE) was synthesized and shown to be an effective and selective retaining α-glucosidase inactivator. (29) As part of these studies also the corresponding aziridines 19 and 20 were synthesized and shown to be potent retaining β-glucosidase (19) and retaining α-glucosidase (20) inactivators. Episulfides 21 and 22 in contrast proved much less active, while configurational isosters such as β- and α-mannose-configured cyclophellitols 23 and 24 were put forward as potential inactivators of the corresponding retaining GHs. (30)

The Advent of Activity-Based Retaining Glycosidase Profiling

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Cravatt and co-workers introduced the concept of ABPP 25 years ago (23) when they used a biotin-fluorophosphonate construct (termed FP-biotin) to capture and identify serine hydrolases from complex biological samples. In 2000, Bogyo and co-workers demonstrated the generality of ABPP by using tagged peptide epoxysuccinates to capture cysteine proteases of the cathepsin family. (31) Mechanism-based, covalent and irreversible retaining GH inhibitors had been known for several decades at that time, and it is therefore no surprise that the first reports on retaining GH ABPs followed shortly after these two foundational ABPP studies on serine hydrolases and cysteine proteases. The first activity-based GH probe design (2002) was not rooted in the above-described retaining GH inhibitors but in altogether different chemistries: the GH-catalyzed generation of tagged, reactive electrophiles. Inspired by the strategy (32) of Wong and Lerner for selecting, from large pools, catalytic antibodies with glycosidase activity, Lo and co-workers designed β-glucosidase substrate 25 (Figure 4A) containing a latent quinone methide. (33) Exposure of 25 to recombinant Flavobacterium meningospectum β-glucosidase generated phenolate 26 which after fluoride expulsion led to in situ formation of quinone methide 27. The test enzyme used here is a retaining GH, but the strategy should work as well for an inverting one: compound 27 is a reactive electrophile designed to react with any nucleophile within or nearby the GH active site to form a covalent and irreversible adduct, thereby attaching a biotin moiety to the protein. Upon SDS-PAGE of the denatured protein mixture from the experiment in which the test GH was treated with 25, Coomassie staining then revealed the emergence of higher molecular weight protein bands (shown are graphic representations of the original gels; for this and the ensuing examples, the reader is directed to the original papers), suggesting that reaction with one or multiple quinone methides have occurred (lane 2). The same image is returned in a streptavidin–biotin Western blot (lane 4), demonstrating that the 25-treated GH, but not the nontreated one (lane 3) has acquired biotin residues. The authors expanded their strategy to labeling, with the latent quinone methide 28 (yielding a more reactive electrophile compared to 25), of recombinant Athrobacter ureafaciens neuraminidase, (34) but had not progressed to ABPP in cell extracts or more complex biological samples at that time. Withers and co-workers observed that such experiments may lead to diffusion from the active site of the generated quinone methide to indiscriminately label nearby proteins and capitalized on this idea by staining cells in a GH-activity-dependent manner. (35)

Figure 4

Figure 4. Latent quinone methides (A) and activated fluorinated glycosides (B) as activity-based (retaining) GH probes. For the original images of the SDS-PAGE gels that are shown here and in the remainder of this account, please see the papers referred to in the text.

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Vocadlo and Bertozzi were the first to capitalize on the activated fluorinated glycoside design in their development of an activity-based retaining GH probe targeting the Escherichia coli retaining β-galactosidase, LacZ (Figure 4B). (36) In their strategy they combined bioorthogonal chemistry with mechanism-based retaining GH inhibition, and designed 1,2,6-trideoxy-1,2-difluoro-6-azido-β-galactoside 29 based on previous findings (37) that the 6-OH analogue covalently and irreversibly modifies the LacZ nucleophile, Glu537. They then cultured Escherichia coli in the absence or presence of the LacZ inducing agent, isopropyl-β-D-thiogalactoside 33, and treated extracts of these cells with 29, and then with the Staudinger-Bertozzi phosphine 31 carrying a FLAG tag. Besides some background labeling a major protein band in the ensuing anti-FLAG Western blot of SDS-PAGE separated proteins was returned (lane 4) only when cells were treated with 33, then 29 and finally the bioorthogonal reagent, 31. Two-step ABP 29 proved in-class active toward a few retaining β-galactosidases and modified almond β-glucosidase as well. The strategy was expanded (38) a few years later using the bioorthogonal activated fluorinated GlcNAc analogue 34 to label, in Pseudomonas aeruginosa extracts, the retaining β-glucosaminidase, NagZ (formation and bioorthogonal detection of adduct 35) and influenza neuraminidases. (39) As well, Hekmat and Withers expanded the strategy to capture and identify endoglycosidases in their work on the identification of a new xylanase expressed by Cellulomonas fimi. (40) Following these studies, suites of GH ABPs composed of latent quinone methides, activated deoxyfluoro glycosides and haloacetyl aminoglycosides were used by Wright and co-workers (41) to study cellulose-degrading enzymes produced by Clostridium thermocellum. Many carbohydrate-active proteins from various cellulosomes were captured in this manner, but the number of off-targets and promiscuous amino acid side chain labeling made data interpretation somewhat arduous.

Cyclophellitol Aziridine Isosters in Activity-Based Retaining Exoglycosidase Profiling

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In the year following the conception of ABPP when we started work on the design of GH ABPs, none of the ABP designs in Figure 4 had been reported. To us, the activated deoxyfluoro glycosides and the cyclophellitols/cyclophellitol aziridines appeared most suited for retaining GH ABPP. They present an ‘anomeric’ electrophilic carbon (instead on one a few atoms removed) to the enzyme active site nucleophile, which we thought may impact activity and active side nucleophile-selectivity. Moreover, glycoside/glycomimetic configuration in both designs appeared to match well with retaining GH selectivity. Of the two design blueprints, we considered the cyclophellitol one the most attractive. In contrast to activated deoxyfluoro glycosides, (42) enzyme reactivation by hydrolysis of reacted epoxides had not been shown, and substitution of the epoxide for an aziridine would create room for installing a reporter moiety without the need to sacrifice another of the hydroxyl functionalities. We felt this may be important for initial enzyme active site binding and noted that in most activated deoxyfluoro glycoside designs OH-2 is already sacrificed for fluorine. The downside of this choice we thought would be the lengthier and more complicated synthesis schemes required for the preparation of cyclophellitol-based ABPs as opposed to activated, fluorinated glycoside ones. This turned out to be true and it was only after Robert Madsen published (43) his total synthesis of cyclophellitol (17) that we were able to generate our first retaining GH ABPs.
The Madsen route proved reliable, scalable, and adaptable. A key step in Madsen’s synthesis (Figure 5A) is the lanthanum triflate-catalyzed, indium-mediated Barbier addition of ethyl-4-bromocrotonate 38 to xylose-derived 4-pentenal 37. Reduction of the methyl ester in the resultant 1,7-diene 39 and ring-closing metathesis gives cyclohexene 40 with the four chiral carbon centers emulating the glucopyranose configuration. The homoallylic primary alcohol in 40 allows for stereoselective epoxidation of the double bond to yield 41, and palladium-catalyzed hydrogenolysis of the two benzyl ethers then gives cyclophellitol (17).

Figure 5

Figure 5. Retaining GH ABPs based on cyclophellitol and cyclophellitol aziridine. A) Adaptation of the Madsen cyclophellitol synthesis to give cyclophellitol and cyclophellitol aziridine ABPs 44 and 48. B) Comparative ABPP of the human retaining β-glucosidases, GBA1, GBA2 and GBA3. C) Head-to-head comparison of activated fluorinated glucosides 49-52 and cyclophellitol 53 as GBA1 ABPs.

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For our first retaining GH ABP design, (44) we took advantage of partially protected cyclohexene 40, the primary alcohol of which we transformed into the corresponding azide by first selective tosylation and then azide displacement of the resultant tosylate. (45) Protecting group manipulation and epoxidation of the double bond then gave two-step ABP 42, and copper(I)-catalyzed azide-alkyne click ligation Bodipy-FL-tagged direct ABP 43. This probe (which we referred to as MDW933) and its red-fluorescent analogue 53 (Figure 5C, MDW941) we then applied in our first forays into activity-based retaining GH profiling studies. Cyclophellitol 44 proved to be highly active and very selective in labeling the human lysosomal retaining β-glucosidase, glucosylceramide (glucocerebroside, GBA1). (44) Genetic deficiency in GBA1 is at the basis of the lysosomal storage disorder, Gaucher disease, and a variety of mutations in the gene encoding for GBA1 can lead to various disease states ranging from mild to severe. Mutations in the GBA1 gene impact ER quality control, rather than catalytic activity, and disease states therefore correlate with the number of active enzymes within lysosomes. This can be quantified by ABPP, and this is what we have done with ABP 44: we took extracts of macrophages from individuals carrying different GBA1 mutations, treated these at pH 5 with our probe, resolved the protein content by SDS-PAGE and scanned the wet gel slabs for in-gel fluorescence (Figure 5B). Compared to the sample derived from a healthy individual, the one from the N370S mutant returned a slightly weaker signal (GBA1 comes in several glycoforms leading to separated signals). The L444P mutant yielded a much weaker signal while the RECNCI collodion sample gave no signal at all. This pattern coincides with the severeness of disease, ranging from mild (N370S) to severe (L444P) to lethal (collodion). The right lane comprises a sample of recombinant GBA1 (Cerezyme) used in the clinical treatment of Gaucher disease as enzyme replacement therapy.
Our publication, (44) in 2010, on cyclophellitol ABPs 44 and 53 was accompanied by an editorial entitled ‘getting lucky in the lysosome’. (46) It is fortuitous that GBA1, in contrast to most other exoglycosidases, allows for substantial modification of the carbohydrate (mimetic) core. But there was no luck involved in our design. We had already established that 6-O-alkyl-deoxynojirimycins inhibit GBA1, the natural substrate of which is glucosylceramide (so, a glucosylated lipid) almost equally potently as its N- or C1-alkyl counterparts. (47) We also knew that substitution of the epoxide for an aziridine yields equally potent retaining GH inhibitors and anticipated that grafting a fluorophore onto the aziridine nitrogen would yield ABPs with the bulky reporter moiety in the direction of a substrate aglycon, a direction that should be generally tolerant of large groups. Many exoglycosidases are particular to the nature (configuration, substitution pattern) of their substrate monosaccharide but considerably less so to the aglycon and we felt that such a design would yield a general template for retaining exo-GH ABPs not offered by the other design blueprints. To test this hypothesis, we made N-acyl-cyclophellitol aziridine 48, for which we again turned to the Madsen intermediate 40. (43,45) Key step in this synthesis comprises iodocyclization of imidate 45 to deliver the nitrogen to the top face of the alkene. Hydrolysis of the resultant iminal was then followed by intramolecular iodine displacement to give, after Birch reduction of the two benzyl ethers, cyclophellitol aziridine 19. N-Acylation (in most following studies we opted for N-alkylation) and click ligation then gave ABP 48. This probe indeed proved reactive to all three retaining β-glucosidases expressed constitutively in mice and man: GBA1, GBA2 and GBA3. This we demonstrated (Figure 5B) in a comparative study, (48) in which we treated mice intravenously with varying concentrations of either the GBA1-selective ABP 44 or with ABP 48, after which the animals were sacrificed, their tissues harvested, homogenized, and the resultant protein mixtures resolved by SDS-PAGE. Scanning of the resultant gel obtained from liver homogenates shows one major, concentration-dependent band for animals treated with 44, and three concentration-dependent bands for 48-treated mice. These bands correspond with the molecular weight of GBA3 (lowest), GBA1 (middle) and GBA2 (highest) and substitution of the fluorophore in 48 for a biotin indeed allowed for identification of GBA1, GBA2 and GBA3 by chemical proteomics (pull down of biotinylated proteins with streptavidin-coated magnetic beads, then on-bead trypsin digestion and LC-MSMS analysis of the resultant trypsin digest peptides). Finally on our first ventures into retaining GH ABPP, we directly compared cyclophellitol ABPs with their activated deoxyfluoro glycoside counterparts. (49) The latter design does not allow grafting a reporter in the aglycon direction and therefore relies on bioorthogonal chemistries, thus on two-step ABPP approaches, excepting situations where carbohydrate (mimetic) core modification with a fluorophore or biotin is tolerated. Such as with GBA1, which allowed us to compare the labeling efficiency (concentration at which labeling of recombinant GBA1 when resolved by SDS-PAGE is still detected) of the series of activated fluorinated glucosides 49-52 with red fluorescent cyclophellitol 53. ABP 53 clearly emerges as the most potent ABP from these studies. As well, the nature of the activation of fluorinated glycosides can have a major impact on potency, with the Biao Yu trifluoroimidate 51 (50) being the clear winner in this respect, and the 2,4-dinitrophenylglucoside in these studies surprisingly inactive. We did this comparison for retaining β-glucosidases only, both for direct ABPP as shown and (using 42 and the corresponding 6-azido-2-fluoroglucosides) in two-step bioorthogonal ABPP. (51) We therefore do not wish to claim the cyclophellitol (aziridine) design blueprint is the superior one in all instances. But neither did we turn to other designs in our subsequent studies.
The Madsen cyclophellitol synthesis scheme proved not only effective for the synthesis of modified cyclophellitols and cyclophellitol aziridines, but also for the synthesis of configurational isosters. Llebaria and co-workers reported that, besides partaking in a Barbier allylation, pentenal 37 can undergo an asymmetric aldol condensation with an Evans-templated acrylate. (52,53) This led to a precursor 1,7-diene resembling 39 but that yields, upon further processing rather like the Madsen synthesis, the β-galacto-configured cyclophellitol aziridine. Adaptation of either of these routes, combined with a separate route of synthesis we developed (54) of a fully orthogonally protected cyclohexene analogue of 40, allowed us to synthesize an array of cyclophellitol isosteres (α-glucose, (55) α/β-mannose, (56,57) α-fucose, (58) α/β-galactose, (59,60) α-L-iduronic acid, (61) β-glucuronic acid (62)) that all proved in-class selective for their corresponding retaining GHs, some more active than others, some also more selective than others, but with the exception of the α-L-iduronic acid (61) probe (which detects recombinant enzyme), all able to identify their target GHs in cell extracts.

Cyclophellitol ABPs Act as Transition State Analogues in Respect of Conformation

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GH-catalyzed hydrolysis of glycosidic bonds proceeds through transition states having a considerable oxocarbenium ion character. Michael Sinnott predicted the emergence of chair- or boat-like transition states to accommodate the developing sp2-character around the C-O oxocarbenium bond during GH catalysis. (3) Building on this, and supported by experimental and computational (63) evidence, Davies, Rovira and Planas in their 2011 Account entitled ‘conformational analysis of the reaction coordinate of glycosidases’ posed that GHs (inverting and retaining alike) would distort their substrates away from their lowest energy conformation in a predictable manner to allow the formation of such half-chair or boat-like transition states. (64) According to this evaluation, retaining β-glucosidases in general follow the 1S3-4H3-4C1 reaction itinerary as depicted in Figure 6A.

Figure 6

Figure 6. A, B) Reaction coordinates by which retaining β-glucosidases (A) and retaining α-glucosidases (B) process their substrates. C) Computed free energy landscapes (FELs) of selected compounds.

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In this pathway, the substrate β-glucoside adopts a 1S3 skew boat conformation in the initial Michaelis complex such that the leaving group (aglycon) is positioned axially, allowing the formation of an endocyclic C═O double bond and thus the transition state oxocarbenium ion. This is a short-lived species, whose 4H3 half-chair conformation is then trapped by the active site nucleophile to complete the first half of the catalytic cycle, with the substrate glucoside now α-linked within the enzyme active site and having adopted a 4C1 chair conformation. It is this latter conformation (4C1) that is revealed in the structure of the TmGH1 retaining β-glucosidase bound to cyclophellitol (17), and mechanism-based inhibitors can thus assist in determining substrate processing pathways, just as (as is put forward in the aforementioned Account, and before that also by Andrea Vasella (65)) reaction itineraries may assist in the design of GH-selective inhibitors. As evidenced by their computed (63) free energy landscapes, cyclophellitol (17), just like cyclophellitol aziridines 19 and 48, adopts a lowest energy 4H3 conformation. In this conformation, the epoxide/aziridine heteroatom is ideally placed for general acid–base protonation, and the ‘anomeric’ carbon for subsequent attack by the active site nucleophile. A structure of an unreacted cyclophellitol derivative within the TmGH1 active site reveals this transition state conformation (66) and together with the reacted one reveals part of the reaction itinerary shown in Figure 6A, and this conformational positioning of the electrophile for nucleophilic displacement may explain why cyclophellitol-based ABPs inhibit retaining β-glucosidases so well. It also points the way for the development of ABPs targeting retaining GHs that process their substrate through a similar set of reaction coordinates but that take on glycans other than β-glucosides. Among others, several retaining β-galactosidase and retaining β-glucuronidase families follow the 1S3-4H3-4H1 itinerary and are readily trapped by the corresponding β-galacto (60) and β-glucurono-cyclophellitol aziridines. (62) The latter is demonstrated in Figure 6A, showing that ABP 54 captures from platelet extract the human β-exoglucuronidase, GUSB (present in several isoforms) and surprisingly also the endoglucuronidase, heparanase (HPSE). To date this is the only retaining endoglycosidase we found to react with a monosaccharidic cyclophellitol aziridine and this finding demonstrates the power of ABPP: one may design a probe to react with a certain enzyme (family), but the unbiased nature of the technology allows for detection (through biotin-ABPs and by chemical proteomics) also of its off-targets. These may be of interest by themselves, and we realized that HPSE has been seen as a potential antitumor target for many years. It is upregulated in, and excreted by, many metastatic cancers and is thought to facilitate metastasis by degradation of the extracellular matrix (of which heparan sulfate proteoglycans make up a major component), facilitating metastatic dissemination and releasing mitogens that drive cellular proliferation. Four competitive HPSE inhibitors have undergone clinical trials, but none have reached the clinic yet. These inhibitors are all large, mostly heterogeneous and highly negatively charged to match the substrate and the extensive active site pocket, a match that cannot be met with small molecule competitive HPSE inhibitors. Mechanism-based inhibitors can overcome weak initial affinities by forming a covalent bond – a strategy that for instance has met with success in the clinical development of proteasome inhibitors. With this in mind, we prepared α-1,4-GlcNAc-glucuronic cyclophellitol 55 and showed this to be at least equally potent as the current best-in-class (large, heterogeneous, strongly anionic) competitive HPSE inhibitor in blocking cancer metastasis in three in vivo tumor models. (67)
Retaining α-glucosidases often process their substrates through an 1C4-4H3-1S3 itinerary (Figure 6B), thus the opposite pathway to that employed by retaining β-glucosidases and sharing the half-chair oxocarbenium ion transition state. (64) The lowest energy conformation adopted by 1,6-epicyclophellitol aziridine 56 is 4H3 as well and fits therefore well within the enzyme active site but now (compared to the situation depicted in Figure 6A for cyclophellitol aziridine 48) with the aziridine pointing downward for nucleophilic displacement from the top face. ABP 56 is an effective label of lysosomal α-glucosidase (GAA) and ER α-glucosidase II (ER-II), the two retaining α-glucosidases encoded in the human genome. (55) Deficiency in GAA is at the basis of the lysosomal glycogen storage disorder, Pompe disease and the lack of GAA, compared to ER-II, in Pompe patients (P) is revealed by comparative ABPP using 56 (Figure 6B). Retaining α-glucosidases bind their substrate in 4C1 conformation in the initial Michaelis complex (compare the 1S3-conformer in retaining β-glucosidases), which allows the design of a new and selective class of mechanism-based inhibitors. We noted some cross-reactivity of ABP 56 toward retaining β-glucosidases. This is not unexpected given that both transition states in Figures 6A and 6B are alike, and the close analogue conduritol B aziridine 13 inhibits (Figure 3B) both retaining α-and β-glucosidases. 1,6-Epi-cyclophellitol cyclosulfate 57 in turn emulates the 4C1 Michaelis complex conformation. This compound turned out to be a highly potent, and highly selective, inhibitor of GAA and ER-II in a study (68) demonstrating that evaluation of reaction itineraries can indeed assist in the rational design of GH inhibitors. The 1C4-4H3-1S3 itinerary is also used by several retaining α-galactosidases, which are trapped by ABP 58, and sulfoquinovosidases (SQases, trapped by 59). (69)

Activity-Based Profiling of Retaining GHs Involved in Biomass Polysaccharide Turnover

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Sulfoquinovosyl diacylglycerol (SQDG) is produced by plants, algae and cyanobacteria and constitutes one of the major natural organosulfur species. The annual production of SQDG is speculated to be on a scale commensurate of the sulfur-containing amino acids cysteine and methionine. SQDG degradation is therefore an important process in the natural organosulfur cycle and control over this process may impact sulfur nutrition and the carbon cycle. GH31 sulfoquinovosidases, enzymes that hydrolyze SQDG and sulfoquinovosylglycerol, are retaining GHs that can be labeled by appropriately configured cyclophellitol aziridines such as 59 (Figure 6B). Using ABP 59 we demonstrated that SQases are expressed, by both Escherichia coli and Pseudomonas putida, when exposed to sulfoquinovose but not in the absence thereof. (69) We also showed that, once expressed, these SQases have a relatively long lifetime and remain active for hours after their encoding mRNA has disappeared.
The Wright (40) laboratory has used suites of GH ABPs in their studies on microbial biomass polysaccharide degradation. In line with the SQase trapping work, we felt that our cyclophellitol design blueprint would be of use in this area as well. Some of our exploits in this direction are presented in Figure 7 and pertain the study of retaining exo- and endoglycosidases in bacterial and fungal secretions of proteins (termed secretomes) when grown on specific glycan sources. In one experiment, we treated various basidiomycete strains grown on α-L-arabinofuranoside-containing polysaccharides with α-L-arabinofuranose-configured aziridine 61. (70) Fluorescence scanning of the resultant SDS-PAGE gels rapidly identifies proteins that have reacted with 61 and that by this virtue may also be retaining GHs responsible for cleavage of the respective glycosidic bond, and one may for instance select strain 1557 (one major activity) for further studies.

Figure 7

Figure 7. A), Activity-based secretomes profiling of glucuronoarabinoxylan-degrading retaining arabinofuranosidases and xylanases. B) Xyloglucan-degrading retaining exoglucosidases, cellulases and xyloglucanases. Shown in the gel are samples of intact cells (C), cell extracts (L) and supernatant/secretomes (S) with ABPs 63-65.

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Xylobiose-configured aziridine 62 comprises the first retaining endoglycosidase ABP we designed and emulates part of the backbone structure of the major biomass polysaccharide, glucuronoarabinoxylan. (71) Treatment of secretomes of Aspergillus niger grown on this polysaccharide with 62 yielded two fluorescent bands (Figure 7A) that were revealed by chemical proteomics (using the biotin counterpart of 62) as a GH10 xylanase (the lower band) and a GH3 exoxylosidase (the upper band). The latter likely first removes the nonreducing β-xyloside to be then confronted by a β-xylose-configured cyclophellitol aziridine, with which it then reacts to form a covalent and irreversible adduct. Beyond usage of 62 in the discovery of xylan-processing enzymes it can also be applied to study their resilience toward harsh conditions that may be required in their (industrial) biotechnological application: pH, salt concentrations and as illustrated in Figure 7A, temperature. Both GH3 and GH10 proved to retain activity after exposure to 50 °C for up to two hours (shown in the graphic representation of the original gel is up to one hour). GH3 (the exoxylosidase) retained full activity also at 60 °C and partial activity at 70 °C, at which temperature GH10 (the xylanase) was completely inactivated already after 5 min. One lesson we learned from these studies is that, in contrast to exo-acting GHs, ABPs directed to endo-acting ones are perhaps better designed to have the (fluorescent) reporter at the nonreducing end, at the position where the backbone of the natural polysaccharide extents. We took this into account when we designed the set of ABPs 63-65 bearing orthogonal fluorophores to characterize the xyloglucan-degrading GH system excreted by the soil saprophyte, Cellvibrio japonicus, when grown on various glycan sources (Figure 7B). (72) Xyloglucanase activity (the red band), which we by chemical proteomics identified to be the Cel5D gene product to which cellulase activity was ascribed, was found to be expressed and excreted only in the samples of Cellvibrio japonicus grown on xyloglucan. Growing these bacteria on arabinoxylan yielded an unexpected yellow signal with an apparently unique molecular weight, and if a single protein causes this signal, it must be reactive to both probes 63 and 64, even though its expression is elicited by a polysaccharide source that does not contain these structural elements.

Conclusion and Outlook

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This year marks the 25th birthday of activity-based protein profiling, with the seminal paper on serine hydrolases (22) dating back to 1999. Glycosidase ABPP emerged not much later but initially evolved at a much slower pace. This may be because glycosidases are more particular to their substrates, defying the design of a one-size-fits-all ABP. This in contrast to for instance the Cravatt serine hydrolase ABPs (22) that at once capture hundreds of serine hydrolases. The increasing number of cyclophellitol/cyclophellitol aziridine ABPs becoming available allows for multiplexing ABPP studies to capture multiple retaining GH families at once, and the field is therefore catching up in this aspect. At the same time, they have proven to be high precision instruments that allow for detailed in situ and sometimes in vivo (73) ABPP studies less easily accomplished by broad-spectrum probes. While at times they show some cross-reactivity toward retaining GHs they were not designed for, nonspecific reactions with other proteins occur only rarely, much less frequent than what is observed when working with ABPs targeting other enzyme families. Retaining GH ABPP is therefore low-tech with targets easily visualized by SDS-PAGE. It is also compatible with high-tech proteomics platforms.
The remarkable selectivity of cyclophellitol-cyclophellitol aziridine ABPs may in part be because these are relatively polar compounds not prone to binding to protein surfaces. As well, they appear not to be very reactive electrophiles. One of the surprising observations we made in synthesizing endoglycosidase probes like 64 and 65 (72) is that one can in fact chemically glycosylate partially protected cyclophellitols/aziridines, thus exposing these functionalities to (Lewis) acidic chemical glycosylation conditions. (74) Possibly the electron-withdrawing nature of the remaining cyclitol substituents dampen the reactivity of the epoxide/aziridine, at least for SN1 substitutions (destabilization of carbocation intermediates, like oxocarbenium ion destabilization in deoxyfluoro glycosides). Once within a retaining GH active site, though, they may benefit from protonation by the general acid/base residue to develop a positive charge which is stabilized by the active site. This may hold true especially for the aziridines, with an estimated (based on the value for the nonsubstituted cyclohexylaziridine) pKa of around 8 which is like that of the widely used competitive retaining and inverting GH inhibitors: deoxynojirimycins.
Variation of configuration and substitution pattern has allowed us to capture a range of retaining exo- and endoglycosidases, of either biomedical or biotechnological relevance. We are assembling focused libraries of retaining GH ABPs and have shared our reagents with numerous colleagues who are now using these in their own studies. Redinbo and collaborators apply glucuronic cyclophellitol aziridines 54 to map individual gut microbiota retaining β-glucuronidases, both their nature and abundance, and correlates this to intestinal therapeutics (de)activation with the aim to stratify patient populations for drug toxicity/efficacy. (75) Fleishman and co-workers have used xylobiose aziridine 62 for the identification of potential new xylanase activities, out of a pool of close to one million in silico-generated proteins. (76) Retaining GH ABPs, like ABPs in general, start to prove their worth in (high) throughput settings, which may be for the discovery or engineering of new GH activities but also for the discovery or design of competitive GH inhibitors. (77) Future research will see more efforts in this direction, as well as in further expansion of the current pool of retaining exo- and endo-GH probes. Retaining neuraminidases for instance employ a tyrosine as nucleophile (7) which should be reactive toward an appropriately configured cyclophellitol/aziridine as well. Fucoidan comprises a major marine biomass polysaccharide composed of branched fucose backbone highly dotted with sulfate groups, and fucoidan-degrading secretomes contain retaining GHs that can be annotated by approaches as shown in Figure 7B. (6) Rhamnogalacturonan II, one of our dietary fibers, is a highly complex polysaccharide composed of 21 unique interglycosidic linkages. One single species of gut microbiota, Bacteroides thetaiotaomicron, was shown to be able to hydrolyze 20 of these, 13 of which by retaining GHs. (78) The development of ABPs for such enzymes may require new chemistries, possibly also in the design of the nature of the electrophilic trap. This is an ongoing activity, besides our work most recently for instance in the work (79) by Andrew Bennet on allylic, carbacyclic glycomimetics.
Inverting GHs have almost completely defied all ABP designs, this while this family is about equal in size as that of the retaining GHs. Brumer and co-workers in their studies on mixed-linkage endoglucanases showed that haloacetyl aminoglycoside-based ABPs may react with the general acid–base residue in retaining GHs (but surprisingly not with the corresponding nucleophile). (80) Building on this finding, glycomimetic designs that fit in inverting GH active sites to present a suitable electrophile to either of the two active site carboxylates appear feasible. The same holds true for retaining GHs utilizing neighboring group participation, such as the epoxide-forming α-mannanases, (10) for which we designed spiro-epoxides as a new class of mechanism-based inhibitors and ABPs with promising affinity. (81) Finally, cyclophellitol-inspired designs may find use in a biomedical context beyond ABPP. Mechanism-based HPSE inhibitor 55 may have potential for the development of new antimetastatic cancer agents. (67) Cyclophellitol cyclosulfate 57 (68) in turn halts SARS-CoV-2 proliferation in infected lung cells equally effective as the best-in-class N-alkyl-deoxynojirimycins. (82) This, while 57 selectively inhibits ER α-glucosidase II and does not inactivate the inverting GH, ER α-glucosidase I. These compounds comprise early stage drug candidates, at most. Yet they do demonstrate the potential of mechanism-based inhibitors, beyond serving as blueprints for ABP design, as promising starting points for therapeutics development.

Author Information

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Acknowledgments

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The work described in this review was supported by The Netherlands Organization for Scientific Research (NWO TOP grant 2018-714.018.002, to H.S.O.), the European Research Council (ERC-2011-AdG 290836 Chembiosphing, to H.S.O and ERC-2020-SyG 951231 Carbocentre, to H.S.O, C.R and G.J.D.), the Spanish Ministry of Science, Innovation and Universities (grants PID2020-118893GB-100MICIU/AEI /10.13039/501100011033 to C.R.), the Spanish Structures of Excellence María de Maeztu (CEX2021-001202-M, to C.R.), the Agency for Management of University and Research Grants of Catalonia (AGAUR, 2021-SGR-00680, to C.R.), and the Royal Society (Ken Murray Research Professorship to G.J.D.). We thank all undergraduate students, PhD students and postdoctoral researchers who did the actual work over the past decades and whose names are found on the cited papers. We thank Spencer J. Williams for his insightful and critical review of the manuscript. H.S.O. is thankful to the University of Melbourne (Australia) and the Norman Thomas Mortimer Wilsmore Research Fund for a Wilsmore Fellowship which enabled him to contemplate on mechanistic aspects on glycoside hydrolases and their inhibition. The Graphical abstract was created with Biorender. com.

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    O-GlcNAc is an abundant post-translational modification of serine and threonine residues of nucleocytoplasmic proteins. This modification, found only within higher eukaryotes, is a dynamic modification that is often reciprocal to phosphorylation. In a manner analogous to phosphatases, a glycoside hydrolase termed O-GlcNAcase cleaves O-GlcNAc from modified proteins. Enzymes with high sequence similarity to human O-GlcNAcase are also found in human pathogens and symbionts. We report the three-dimensional structure of O-GlcNAcase from the human gut symbiont Bacteroides thetaiotaomicron both in its native form and in complex with a mimic of the reaction intermediate. Mutagenesis and kinetics studies show that the bacterial enzyme, very similarly to its human counterpart, operates via an unusual 'substrate-assisted' catalytic mechanism, which will inform the rational design of enzyme inhibitors.
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  10. 10
    Sobala, L. F.; Speciale, G.; Zhu, S.; Raich, L.; Sannikova, N.; Thompson, A. J.; Hakki, Z.; Lu, D.; Shansi Kazem Abadi, S.; Lewis, A. R.; Rojas-Cervellera, V.; Bernardo-Seisdedos, G.; Zhang, Y.; Millet, O.; Jiménez-Barbero, J.; Bennet, A. J.; Sollogoub, M.; Rovira, C.; Davies, G. J.; Williams, S. J. An epoxide intermediate in glycosidase catalysis. ACS Cent. Sci. 2020, 6, 760– 770,  DOI: 10.1021/acscentsci.0c00111
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    10
    An epoxide intermediate in glycosidase catalysis
    Sobala, Lukasz F.; Speciale, Gaetano; Zhu, Sha; Raich, Lluis; Sannikova, Natalia; Thompson, Andrew J.; Hakki, Zalihe; Lu, Dan; Shamsi Kazem Abadi, Saeideh; Lewis, Andrew R.; Rojas-Cervellera, Victor; Bernardo-Seisdedos, Ganeko; Zhang, Yongmin; Millet, Oscar; Jimenez-Barbero, Jesus; Bennet, Andrew J.; Sollogoub, Matthieu; Rovira, Carme; Davies, Gideon J.; Williams, Spencer J.
    ACS Central Science (2020), 6 (5), 760-770CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)
    Retaining glycoside hydrolases cleave their substrates through stereochem. retention at the anomeric position. Typically, this involves two-step mechanisms using either an enzymic nucleophile via a covalent glycosyl enzyme intermediate or neighboring-group participation by a substrate-borne 2-acetamido neighboring group via an oxazoline intermediate; no enzymic mechanism with participation of the sugar 2-hydroxyl has been reported. Here, we detail structural, computational, and kinetic evidence for neighboring-group participation by a mannose 2-hydroxyl in glycoside hydrolase family 99 endo-α-1,2-mannanases. We present a series of crystallog. snapshots of key species along the reaction coordinate: a Michaelis complex with a tetrasaccharide substrate; complexes with intermediate mimics, a sugar-shaped cyclitol β-1,2-aziridine and β-1,2-epoxide; and a product complex. The 1,2-epoxide intermediate mimic displayed hydrolytic and transfer reactivity analogous to that expected for the 1,2-anhydro sugar intermediate supporting its catalytic equivalence. Quantum mechanics/mol. mechanics modeling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar via a transition state in an unusual flattened, envelope (E3) conformation. Kinetic isotope effects (kcat/Km) for anomeric-2H and anomeric-13C support an oxocarbenium ion-like transition state, and that for C2-18O (1.052 ± 0.006) directly implicates nucleophilic participation by the C2-hydroxyl. Collectively, these data substantiate this unprecedented and long-imagined enzymic mechanism. Mannosidases of glycoside hydrolase family 99 use a neighboring-group participation mechanism involving the substrate 2-hydroxyl.
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  11. 11
    Withers, S. G.; Street, I. P.; Bird, P.; Dolphin, D. H. 2-Deoxy-2-fluoroglucosides: a novel class of mechanism-based glucosidase inhibitors. J. Am. Chem. Soc. 1987, 109, 7530– 7531,  DOI: 10.1021/ja00258a047
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    2-Deoxy-2-fluoroglucosides: a novel class of mechanism-based glucosidase inhibitors
    Withers, Stephen G.; Street, Ian P.; Bird, Paul; Dolphin, David H.
    Journal of the American Chemical Society (1987), 109 (24), 7530-1CODEN: JACSAT; ISSN:0002-7863.
    2,4-Dinitrophenyl 2-deoxy-2-fluoro β-D-glucopyranoside was synthesized and demonstrated to be a specific mechanism-based inhibitor for β-glucosidase from Alcaligenes faecalis. The inactivation of the enzyme followed pseudo-1st-order kinetics with a rate const. of 25 min-1 and a dissocn. const. of 0.05 mM. Protection against inactivation was shown by the competitive inhibitor isopropylthio β-D-glucopyranoside. Reactivation of the enzyme was obsd. as expected after removal of free inhibitor and incubation in the presence of substrate. The use of such inhibitors to investigate the intermediacy of a glucosyl enzyme intermediate and to identify the active site residue involved is projected.
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  12. 12
    Rempel, B. P.; Withers, S. G. Covalent inhibitors of glycosidases and their applications in biochemistry and biology. Glycobiol. 2008, 18, 570– 586,  DOI: 10.1093/glycob/cwn041
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    Vocadlo, D. J.; Davies, G. J.; Laine, R.; Withers, S. G. Catalysis by hen egg-white lysozyme proceeds via a covalent intermediate. Nature 2001, 412, 835– 838,  DOI: 10.1038/35090602
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    Catalysis by hen egg-white lysozyme proceeds via a covalent intermediate
    Vocadlo, David J.; Davies, Gideon J.; Laine, Roger; Withers, Stephen G.
    Nature (London, United Kingdom) (2001), 412 (6849), 835-839CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Hen egg-white lysozyme (HEWL) was the first enzyme to have its three-dimensional structure detd. by x-ray diffraction techniques. A catalytic mechanism, featuring a long-lived oxo-carbenium-ion intermediate, was proposed on the basis of model-building studies. The 'Phillips' mechanism is widely held as the paradigm for the catalytic mechanism of β-glycosidases that cleave glycosidic linkages with net retention of configuration of the anomeric center. Studies with other retaining β-glycosidases, however, provide strong evidence pointing to a common mechanism for these enzymes that involves a covalent glycosyl-enzyme intermediate, as previously postulated. Here the authors show, in three different cases using electrospray ionization mass spectrometry, a catalytically competent covalent glycosyl-enzyme intermediate during the catalytic cycle of HEWL. The authors also show the three-dimensional structure of this intermediate as detd. by x-ray diffraction. The authors formulate a general catalytic mechanism for all retaining β-glycosidases that includes substrate distortion, formation of a covalent intermediate, and the electrophilic migration of C1 along the reaction coordinate.
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    Legler, G. Untersuchungen zum Wirkunsmechanismus glycosidspaltender Enzyme, I. Darstellung und Eigenschaften spezifischer Inaktivatoren. Hoppe-Seyler’s Z. Physiol. Chem. 1966, 345, 197– 214,  DOI: 10.1515/bchm2.1966.345.1.197
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    Legler, G. Untersuchungen zum Wirkunsmechanismus glycosidspaltender Enzyme, II. Isolierung und enzymatische Eigenschaften from zwei β-Glucosidasen aus Aspergillus wentii. Hoppe-Seyler’s Z. Physiol. Chem. 1967, 348, 1359– 1366,  DOI: 10.1515/bchm2.1967.348.1.1359
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    Legler, G. Untersuchungen zum Wirkunsmechanismus glycosidspaltender Enzyme, III. Markierung des aktiven Zentrums einer β-Glucosidase aus Aspergillus wentii mit [14C]Condurit-B-epoxid. Hoppe-Seyler’s Z. Physiol. Chem. 1968, 349, 767– 774,  DOI: 10.1515/bchm2.1968.349.1.767
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    Braun, H.; Legler, G.; Deshusses, J.; Semenza, G. Stereoselective ring opening of conduritol-B-epoxide by an active site aspartate residue of sucrose-isomaltase. Biochim. Biophys. Acta 1977, 483, 135– 140,  DOI: 10.1016/0005-2744(77)90015-8
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    Legler, G.; Bause, E. Epoxy-alkyl oligo-(1–4)-β-D-glucosides as active-site-directed inhibitors of cellulases. Carbohydr. Res. 1973, 28, 45– 52,  DOI: 10.1016/S0008-6215(00)82855-4
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    Rodriguez, E. B.; Scally, G. D.; Stick, R. V. The synthesis of optically pure epoxy-alkyl β-D-glucosides and β-cellobiosides as active-site directed inhibitors of some β-glucan hydrolases. Aust. J. Chem. 1990, 43, 1391– 1405,  DOI: 10.1071/CH9901391
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    Sulzenbacher, G.; Schülein, M.; Davies, G. J. Structure of the endoglucanase I from Fusarium oxysporum: native, cellobiose, and 3,4-epoxybutyl β-D-cellobioside-inhibited forms, at 2.3 Å resolution. Biochemistry 1997, 36, 5902– 5911,  DOI: 10.1021/bi962963+
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    20
    Structure of the endoglucanase I from Fusarium oxysporum: Native, cellobiose, and 3,4-epoxybutyl β-D-cellobioside-inhibited forms, at 2.3 Å resolution
    Sulzenbacher, Gerlind; Schuelein, Martin; Davies, Gideon J.
    Biochemistry (1997), 36 (19), 5902-5911CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    The crystal structure of F. oxysporum cellulase endoglucanase I (I) of glycosyl hydrolase family 7 was solved at 2.3 Å resoln. In addn. to the native enzyme, structures were also detd. of I complexed with both the affinity label, 3,4-epoxybutyl β-D-cellobioside, and the reaction product, cellobiose. The affinity label was covalently bound, as expected, to the catalytic nucleophile, Glu-197, with clear evidence for binding of both the R and S stereoisomers. Cellobiose was found bound to the -2 and -1 subsites of I. In marked contrast to the previously reported crystal structure of I complexed with a nonhydrolyzable thiosaccharide analog, which spanned the -2, -1, and +1 subsites and which had a skew-boat conformation for the -1 subsite sugar, the I-cellobiose complex showed no pyranoside ring distortion in the -1 subsite, implying that strain is induced primarily by the addnl. +1 subsite interactions and that the product is found, as expected, in its unstrained conformation.
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    Thomas, E. W.; McKelvy, J. F.; Sharon, N. Specific and irreversible inhibition of lysozyme by 2′,3′-epoxypropyl β-glycosides of N-acetyl-D-glucosamine oligomers. Nature 1969, 222, 485– 486,  DOI: 10.1038/222485a0
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    Havukainen, R.; Törrönen, A.; Laitinen, T.; Rouvinen, J. Covalent binding of three epoxyalkyl xylosides to the active site of endo-1,4-xylanase II from Trichoderma reesei. Biochemistry 1996, 35, 9617– 9624,  DOI: 10.1021/bi953052n
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    22
    Covalent binding of three epoxyalkyl xylosides to the active site of endo-1,4-xylanase II from Trichoderma reesei
    Havukainen, Riikka; Torronen, Anneli; Laitinen, Tuomo; Rouvinen, Juha
    Biochemistry (1996), 35 (29), 9617-9624CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    The three-dimensional structures of endo-1,4-xylanase II from (XYNII) Trichoderma reesei complexed with 4,5-epoxypentyl β-D-xyloside (X-O-C5), 3,4-epoxybutyl β-D-xyloside (X-O-C4), and 2,3-epoxypropyl β-D-xyloside (X-O-C3) were detd. by x-ray crystallog. High-resoln. measurement revealed clear electron densities for each ligand. Both X-O-C5 and X-O-C3 were found to form a covalent bond with the putative nucleophile Glu86. Unexpectedly, X-O-C4 was found to bind to the putative acid/base catalyst Glu177. In all three complexes, clear conformational changes were found in XYNII compared to the native structure. These changes were largest in the X-O-C3 complex structure.
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    Liu, L.; Patricelli, M. P.; Cravatt, B. F. Activity-based protein profiling: the serine hydrolases. Proc. Natl. Acad. Sci. USA 1999, 96, 14694– 14699,  DOI: 10.1073/pnas.96.26.14694
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    Activity-based protein profiling: the serine hydrolases
    Liu, Yongsheng; Patricelli, Matthew P.; Cravatt, Benjamin F.
    Proceedings of the National Academy of Sciences of the United States of America (1999), 96 (26), 14694-14699CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    With the postgenome era rapidly approaching, new strategies for the functional anal. of proteins are needed. To date, proteomics efforts have primarily been confined to recording variations in protein level rather than activity. The ability to profile classes of proteins on the basis of changes in their activity would greatly accelerate both the assignment of protein function and the identification of potential pharmaceutical targets. Here, we describe the chem. synthesis and utility of an active-site directed probe for visualizing dynamics in the expression and function of an entire enzyme family, the serine hydrolases. By reacting this probe, a biotinylated fluorophosphonate referred to as FP-biotin, with crude tissue exts., we quickly and with high sensitivity detect numerous serine hydrolases, many of which display tissue-restricted patterns of expression. Addnl., we show that FP-biotin labels these proteins in an activity-dependent manner that can be followed kinetically, offering a powerful means to monitor dynamics simultaneously in both protein function and expression.
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    Yariv, J.; Wilson, K. J.; Hildesheim, J.; Blumberg, S. Labelling of the active site of β-galactosidase by N-bromoacetyl β-D-galactopyranosylamine. FEBS Lett. 1971, 15, 24– 26,  DOI: 10.1016/0014-5793(71)80070-4
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    24
    Labeling of the active site of β-galactosidase by N-bromoacetyl β-D-galactopyranosylamine
    Yariv, Joseph; Wilson, Kenneth J.; Hildesheim, Jean; Blumberg, Shmaryahu
    FEBS Letters (1971), 15 (1), 24-6CODEN: FEBLAL; ISSN:0014-5793.
    The hydrolysis of o-nitrophenyl β-galactopyranoside by β-galactosidase was inhibited by incubating with labeled N-bromoacetyl β-D-galactopyranosylamine. The reaction followed 1st order kinetics leading to complete inactivation and 1 mole of reagent was bound to the enzyme per mole of site inactivated. The corresponding L-fucose deriv. did not inactivate the enzyme.
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    Caron, G.; Withers, S. G. Conduritol aziridine: a new mechanism-based glucosidase inactivator. Biochem. Biophys. Res. Commun. 1989, 163, 495– 499,  DOI: 10.1016/0006-291X(89)92164-5
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    Tong, M. K.; Ganem, B. A potent new class of active-site-directed glycosidase inactivators. J. Am. Chem. Soc. 1988, 110, 312– 313,  DOI: 10.1021/ja00209a062
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    A potent new class of active-site-directed glycosidase inactivators
    Tong, Michael K.; Ganem, Bruce
    Journal of the American Chemical Society (1988), 110 (1), 312-13CODEN: JACSAT; ISSN:0002-7863.
    Aziridine I was prepd. from piperidine II in 6 steps. I showed potent inhibition of green coffee bean α-galactosidase, but had little or no effect on yeast α-glucosidase, jackbean α-mannosidase, or bovine β-galactosidase.
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    Atsumi, S.; Umezawa, K.; Iinuma, H.; Naganawa, H.; Nakamura, H.; Iitaka, Y.; Takeuchi, T. Production, isolation and structure determination of a novel β-glucosidase inhibitor, cyclophellitol, from Phellinus sp. J. Antibiot. 1990, 43, 49– 53,  DOI: 10.7164/antibiotics.43.49
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    Production, isolation and structure determination of a novel β-glucosidase inhibitor, cyclophellitol, from Phellinus sp
    Atsumi, Sonoko; Umezawa, Kazuo; Iinuma, Hironobu; Naganawa, Hiroshi; Nakamura, Hikaru; Iitaka, Yoichi; Takeuchi, Tomio
    Journal of Antibiotics (1990), 43 (1), 49-53CODEN: JANTAJ; ISSN:0021-8820.
    A culture filtrate of a mushroom, Phellinus species, strongly inhibited β-glucosidase. The active substance was isolated through charcoal sepn., column chromatog. and crystn. Spectroscopic and crystallog. anal. revealed that it had a novel cyclitol structure, (1S,2R,3S,4R,5R,6R)-5-hydroxymethyl-7-oxabicyclo[4.1.0]heptane-2,3,4-triol; the compd. was named cyclophellitol (I). It inhibited almond-derived β-glucosidase with an IC50 of 0.8 μg/mL.
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    Gloster, T. M.; Madsen, R.; Davies, G. J. Structural basis for cyclophellitol inhibition of a β-glucosidase. Org. Biomol. Chem. 2007, 5, 444– 446,  DOI: 10.1039/B616590G
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    Structural basis for cyclophellitol inhibition of a β-glucosidase
    Gloster, Tracey M.; Madsen, Robert; Davies, Gideon J.
    Organic & Biomolecular Chemistry (2007), 5 (3), 444-446CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
    The structural basis for β-glucosidase inhibition by cyclophellitol was demonstrated using x-ray crystallog., enzyme kinetics and mass spectrometry.
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    Nakata, M.; Chong, C.; Niwata, Y.; Toshima, K.; Tatsuta, K. A family of cyclophellitol analogues: synthesis and evaluation. J. Antibiot. 1993, 46, 1919– 1922,  DOI: 10.7164/antibiotics.46.1919
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    Shing, T. K. M.; Tai, V. W.-F. (-)-Quinic acid in organic synthesis. Part 4. Synthesis of cyclophellitol and its (1R,6S)-, (2S)-, (1R,2S,6S)-diastereomers. J. Chem. Soc. Perkin Trans 1. 1994, 2017– 2025,  DOI: 10.1039/P19940002017
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    Greenbaum, F.; Medzihradszky, K. F.; Burlingame, A.; Bogyo, M. Epoxide electrophiles as activity-dependent cysteine protease profiling and discovery tools. Chem. Biol. 2000, 7, 569– 581,  DOI: 10.1016/S1074-5521(00)00014-4
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    Epoxide electrophiles as activity-dependent cysteine protease profiling and discovery tools
    Greenbaum, Doron; Medzihradszky, Katalin F.; Burlingame, Alma; Bogyo, Matthew
    Chemistry & Biology (2000), 7 (8), 569-581CODEN: CBOLE2; ISSN:1074-5521. (Elsevier Science Ltd.)
    Background: Anal. of global changes in gene transcription and translation by systems-based genomics and proteomics approaches provides only indirect information about protein function. In many cases, enzymic activity fails to correlate with transcription or translation levels. Therefore, a direct method for broadly detg. activities of an entire class of enzymes on a genome-wide scale would be of great utility. Results: We have engineered chem. probes that can be used to broadly track activity of cysteine proteases. The structure of the general cysteine protease inhibitor E-64 was used as a scaffold. Analogs were synthesized by varying the core peptide recognition portion while adding affinity tags (biotin and radio-iodine) at distal sites. The resulting probes contg. a P2 leucine residue (DCG-03 and DCG-04) targeted the same broad set of cysteine proteases as E-64 and were used to profile these proteases during the progression of a normal skin cell to a carcinoma. A library of DCG-04 derivs. was constructed in which the leucine residue was replaced with all natural amino acids. This library was used to obtain inhibitor activity profiles for multiple protease targets in crude cellular exts. Finally, the affinity tag of DCG-04 allowed purifn. of modified proteases and identification by mass spectrometry. Conclusions: We have created a simple and flexible method for functionally identifying cysteine proteases while simultaneously tracking their relative activity levels in crude protein mixts. These probes were used to det. relative activities of multiple proteases throughout a defined model system for cancer progression. Furthermore, information obtained from libraries of affinity probes provides a rapid method for obtaining detailed functional information without the need for prior purifn./identification of targets.
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    Janda, K. D.; Lo, L.-C.; Lo, C.-H. L.; Sim, M.-M.; Wang, R.; Wong, C.-H.; Lerner, R. A. Chemical selection for catalysis in combinatorial libraries. Science 1997, 275, 945– 948,  DOI: 10.1126/science.275.5302.945
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    Chemical selection for catalysis in combinatorial antibody libraries
    Janda, Kim D.; Lo, Lee-Chiang; Lo, Chih-Hung L.; Sim, Mui-Mui; Wang, Ruo; Wong, Chi-Huey; Lerner, Richard A.
    Science (Washington, D. C.) (1997), 275 (5302), 945-948CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    For the past decade the immune system has been exploited as a rich source of de novo catalysts. Catalytic antibodies have been shown to have chemoselectivity, enantioselectivity, large rate accelerations, and even an ability to reroute chem. reactions. In many instances catalysts have been made for reactions for which there are no known natural or man-made enzymes. Yet, the full power of this combinatorial system can only be exploited if there was a system that allows for the direct selection of a particular function. A method that allows for the direct chem. selection for catalysis from antibody libraries was so devised, whereby the pos. aspects of hybridoma technol. were preserved and re-formatted in the filamentous phage system to allow direct selection of catalysis. This methodol. is based on a purely chem. selection process, making it more general than biol. based selection systems because it is not limited to reaction products that perturb cellular machinery.
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    Tsai, C.-S.; Li, Y.-K.; Lo, L.-C. Design and synthesis of activity probes for glycosidases. Org. Lett. 2002, 4, 3607– 3610,  DOI: 10.1021/ol0265315
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    Design and Synthesis of Activity Probes for Glycosidases
    Tsai, Charng-Sheng; Li, Yaw-Kuen; Lo, Lee-Chiang
    Organic Letters (2002), 4 (21), 3607-3610CODEN: ORLEF7; ISSN:1523-7060. (American Chemical Society)
    A new synthetic route was developed for the prepn. of glycoside I activity probe for β-glucosidase in this study. The key glycosidation step begins with benzyl p-hydroxyphenylacetate. Benzylic functionalization for the construction of the trapping device was achieved at later stages. Probe I was shown to be able to label the target enzyme. This cassette-like design offers great flexibility for future alterations. It would allow the synthetic scheme to expand to other glycosidase probes with different linker/reporter combinations.
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    Lu, C.-P.; Ren, C.-T.; Lai, Y.-N.; Wu, S.-H.; Wang, W.-M.; Chen, J.-Y.; Lo, L.-C. Design of a mechanism-based probe for neuraminidase to capture influenza viruses. Angew. Chem., Int. Ed. 2005, 44, 6888– 6892,  DOI: 10.1002/anie.200501738
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    Design of a mechanism-based probe for neuraminidase to capture influenza viruses
    Lu, Chun-Ping; Ren, Chien-Tai; Lai, Yi-Ning; Wu, Shih-Hsiung; Wang, Wei-Man; Chen, Jean-Yin; Lo, Lee-Chiang
    Angewandte Chemie, International Edition (2005), 44 (42), 6888-6892CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A mechanism-based probe for neuraminidase was developed. The probe consisted of a recognition head (N-acetylneuraminic acid), a trapping device (o-difluoromethylphenyl group), a linker, and a reporter group (biotin). It is capable of forming a biotinylated adduct with neuraminidase (NA) from Arthrobacter ureafaciens. It also displays an inhibitory effect on a no. of NA activities. ELISA expts. successfully demonstrated that influenza viruses can be selectively captured with this probe.
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  35. 35
    Kwan, D. H.; Chen, H.-M.; Ratananikom, K.; Hancock, S. M.; Watanabe, Y.; Kongsaeree, P. T.; Samuels, A. L.; Withers, S. G. Self-immobilizing fluorogenic imaging agents of enzyme activity. Angew. Chem., Int. Ed. 2011, 50, 300– 303,  DOI: 10.1002/anie.201005705
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    Self-Immobilizing Fluorogenic Imaging Agents of Enzyme Activity
    Kwan, David H.; Chen, Hong-Ming; Ratananikom, Khakhanang; Hancock, Susan M.; Watanabe, Yoichiro; Kongsaeree, Prachumporn T.; Samuels, A. Lacey; Withers, Stephen G.
    Angewandte Chemie, International Edition (2011), 50 (1), 300-303, S300/1-S300/35CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    We have demonstrated a novel application for enzyme substrates that generate quinone methides. Such substrates have previously been used as enzyme inactivators and abused as proteomics probes; however, these compds. may find greater value as histol. or cell-labeling agents for the investigation of a wide variety of organisms, as illustrated by our results with plants, yeast, and bacteria.
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    Vocadlo, D. J.; Bertozzi, C. R. A strategy for functional proteomic analysis of glycosidase activity from cell lysates. Angew. Chem., Int. Ed. 2004, 43, 5338– 5342,  DOI: 10.1002/anie.200454235
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    A strategy for functional proteomic analysis of glycosidase activity from cell lysates
    Vocadlo, David J.; Bertozzi, Carolyn R.
    Angewandte Chemie, International Edition (2004), 43 (40), 5338-5342CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    We have developed a strategy for activity-based labeling of retaining glycosidases by using the azide group as a sterically unobtrusive chem. tag. We prepd. 6-azido-2,6-dideoxy-2-fluoro-β-D-galactosyl fluoride (6Az2FgalF, 10, Scheme 2) as an activity-based probe for β-galactosidase. The high selectivity of both the inactivation with fluorosugars and the Staudinger ligation with phosphine probes allows detection of glycosidases in complex mixts. and the strategy can be used for profiling these enzyme activities in cell lysates. We have demonstrated that the approach can be used to tag several glycosidases from different glycoside hydrolase families. We anticipate that the strategy will find broad utility in proteomic anal. of these enzymes in prokaryotic and eukaryotic proteomes. The azide group might also be useful as a chem. tag within inactivators of other enzymes from entirely different families with sterically confining active sites.
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  37. 37
    Gebler, J. C.; Aebersold, R.; Withers, S. G. Glu-537, not Glu-461, is the nucleophile in the active site of (LacZ) b-galactosidase from Escherichia coli. J. Biol. Chem. 1992, 267, 11126– 11130,  DOI: 10.1016/S0021-9258(19)49884-0
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    Glu-537, not Glu-461, is the nucleophile in the active site of (lac Z) β-galactosidase from Escherichia coli
    Gebler, John C.; Aebersold, Ruedi; Withers, Stephen G.
    Journal of Biological Chemistry (1992), 267 (16), 11126-30CODEN: JBCHA3; ISSN:0021-9258.
    The covalent intermediate formed during catalysis by the lac Z β-galactosidase from E. coli can be trapped by reaction of the enzyme with 2',4'-dinitrophenyl-2-deoxy-2-fluoro-β-D-galactopyranoside, thereby inactivating the enzyme. Kinetic parameters for this inactivation process with the holo- and apoenzymes have been detd. The nucleophilic amino acid involved has been identified as Glu-537 by using a tritium-labeled inactivator to label the enzyme, then cleaving the labeled protein into peptides and purifying and sequencing the labeled peptide. This residue is conserved in five homologous β-galactosidases and is different from that (Glu-461) proposed to be the nucleophile (Herrchen, M.; Legler, G., 1984) on the basis of affinity labeling studies with conduritol C cis-epoxide. A role for glutamic acid residue 461 as the acid/base catalyst is proposed and justified.
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  38. 38
    Stubbs, K. A.; Scaffidi, A.; Debowski, A. W.; Mark, B. L.; Stick, R. V.; Vocadlo, D. J. Synthesis and use of mechanism-based protein profiling probes for retaining b-D-glucosaminidases facilitate identification of Pseudomonas aeruginosa NagZ. J. Am. Chem. Soc. 2008, 130, 327– 335,  DOI: 10.1021/ja0763605
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    38
    Synthesis and Use of Mechanism-Based Protein-Profiling Probes for Retaining β-D-Glucosaminidases Facilitate Identification of Pseudomonas aeruginosa NagZ
    Stubbs, Keith A.; Scaffidi, Adrian; Debowski, Aleksandra W.; Mark, Brian L.; Stick, Robert V.; Vocadlo, David J.
    Journal of the American Chemical Society (2008), 130 (1), 327-335CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The NagZ class of retaining exo-glucosaminidases play a crit. role in peptidoglycan recycling in Gram-neg. bacteria and the induction of resistance to β-lactams. Here we describe the concise synthesis of 2-azidoacetyl-2-deoxy-5-fluoro-β-D-glucopyranosyl fluoride as an activity-based proteomics probe for profiling these exo-glycosidases. This active-site directed reagent covalently inactivates this class of retaining N-acetylglucosaminidases with exquisite selectivity by stabilizing the glycosyl-enzyme intermediate. Inactivated Vibrio cholerae NagZ can be elaborated with biotin or a FLAG-peptide epitope using the Staudinger ligation or the Sharpless-Meldal click reaction and detected at nanogram levels. This ABPP enabled the profiling of the Pseudomonas aeruginosa proteome and identification at endogenous levels of a tagged protein with properties consistent with those of PA3005. Cloning of the gene encoding this hypothetical protein and biochem. characterization enabled unambiguous assignment of this hypothetical protein as a NagZ. The identification and cloning of this NagZ may facilitate the development of strategies to circumvent resistance to β-lactams in this human pathogen. As well, this general strategy, involving such 5-fluoro inactivators, may prove to be of general use for profiling proteomes and identifying glycoside hydrolases of medical importance or having desirable properties for biotechnol.
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  39. 39
    Tsai, C.-S.; Yen, H.-Y.; Lin, M.-I.; Tsai, T.-I.; Wang, S.-Y.; Huang, W.-I.; Hsu, T.-L.; Cheng, Y. S. E.; Fang, J.-M.; Wong, C.-H. Cell-permeable probe for identification and imaging of sialidases. Proc. Natl. Acad. Sci. USA 2013, 110, 2466– 2471,  DOI: 10.1073/pnas.1222183110
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    Hekmat, O.; Kim, Y.-W.; Williams, S. J.; He, S.; Withers, S. G. Active-site “fingerprinting” of glycosidases in complex mixture by mass spectrometry. Discovery of a novel retaining b-1–4-glycanase in Cellulomonas fimi. J. Biol. Chem. 2005, 280, 35126– 35135,  DOI: 10.1074/jbc.M508434200
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    Chauvigné-Hines, L. M.; Anderson, L. N.; Weaver, H. M.; Brown, J. N.; Koech, P. K.; Nicora, C. D.; Hofstad, B. A.; Smith, R. D.; Wilkins, M. J.; Callister, S. J.; Wright, A. T. Suite of activity-based probes for cellulose-degrading enzymes. J. Am. Chem. Soc. 2012, 134, 20521– 20532,  DOI: 10.1021/ja309790w
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    Suite of Activity-Based Probes for Cellulose-Degrading Enzymes
    Chauvigne-Hines, Lacie M.; Anderson, Lindsey N.; Weaver, Holly M.; Brown, Joseph N.; Koech, Phillip K.; Nicora, Carrie D.; Hofstad, Beth A.; Smith, Richard D.; Wilkins, Michael J.; Callister, Stephen J.; Wright, Aaron T.
    Journal of the American Chemical Society (2012), 134 (50), 20521-20532CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Microbial glycoside hydrolases play a dominant role in the biochem. conversion of cellulosic biomass to high-value biofuels. Anaerobic cellulolytic bacteria are capable of producing multicomplex catalytic subunits contg. cell-adherent cellulases, hemicellulases, xylanases, and other glycoside hydrolases to facilitate the degrdn. of highly recalcitrant cellulose and other related plant cell wall polysaccharides. Clostridium thermocellum is a cellulosome-producing bacterium that couples rapid reprodn. rates to highly efficient degrdn. of cryst. cellulose. Herein, we have developed and applied a suite of difluoromethylphenyl aglycon, N-halogenated glycosylamine, and 2-deoxy-2-fluoroglycoside activity-based protein profiling (ABPP) probes to the direct labeling of the C. thermocellum cellulosomal secretome. These activity-based probes (ABPs) were synthesized with alkynes to harness the utility and multimodal possibilities of click chem. and to increase enzyme active site inclusion for liq. chromatog.-mass spectrometry (LC-MS) anal. We directly analyzed ABP-labeled and unlabeled global MS data, revealing ABP selectivity for glycoside hydrolase (GH) enzymes, in addn. to a large collection of integral cellulosome-contg. proteins. By identifying reactivity and selectivity profiles for each ABP, we demonstrate our ability to widely profile the functional cellulose-degrading machinery of the bacterium. Derivatization of the ABPs, including reactive groups, acetylation of the glycoside binding groups, and mono- and disaccharide binding groups, resulted in considerable variability in protein labeling. Our probe suite is applicable to aerobic and anaerobic microbial cellulose-degrading systems and facilitates a greater understanding of the organismal role assocd. with biofuel development.
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    Street, I. P.; Kempton, J. B.; Withers, S. G. Inactivation of a b-glucosidase through the accumulation of a stable 2-deoxy-2-fluoro-a-D-glucopyranosyl-enzyme intermediate: a detailed investigation. Biochemistry 1992, 31, 9970– 9978,  DOI: 10.1021/bi00156a016
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    Hansen, F. G.; Bundgaard, E.; Madsen, R. A short synthesis of (+)-cyclophellitol. J. Org. Chem. 2005, 70, 10139– 10142,  DOI: 10.1021/jo051645q
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    A Short Synthesis of (+)-Cyclophellitol
    Hansen, Flemming Gundorph; Bundgaard, Eva; Madsen, Robert
    Journal of Organic Chemistry (2005), 70 (24), 10139-10142CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)
    A new synthesis of (+)-cyclophellitol, a potent β-glucosidase inhibitor, has been completed in nine steps from D-xylose. The key transformations involve a zinc-mediated fragmentation of benzyl-protected Me 5-deoxy-5-iodo-xylofuranoside followed by a highly diastereoselective indium-mediated coupling with Et 4-bromocrotonate. Subsequent ring-closing olefin metathesis, ester redn., olefin epoxidn., and deprotection then afford the natural product. This constitutes the shortest synthesis of (+)-cyclophellitol reported to date.
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  44. 44
    Witte, M. D.; Kallemeijn, W. W.; Aten, J.; Li, K.-Y.; Strijland, A.; Donker-Koopman, W. E.; Blijlevens, B.; Kramer, G.; van den Nieuwendijk, A. M. C. H.; Florea, B. I.; Hooibrink, B.; Hollak, C. E. M.; Ottenhoff, R.; Boot, R. G.; van der Marel, G. A.; Overkleeft, H. S.; Aerts, J. M. F. G. Ultrasensitive in situ visualization of active glucocerebrosidase molecules. Nat. Chem. Biol. 2010, 6, 907– 913,  DOI: 10.1038/nchembio.466
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    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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    Li, K.-Y.; Jiang, J.; Witte, M. D.; Kallemeijn, W. W.; van den Elst, H.; Wong, C.-S.; Chander, S. D.; Hoogendoorn, S.; Beenakker, T. J. M.; Codée, J. D. C.; Aerts, J. M. F. G.; van der Marel, G. A.; Overkleeft, H. S. Synthesis of cyclophellitol, cyclophellitol aziridine, and their tagged derivatives. Eur. J. Org. Chem. 2014, 2014, 6030– 6043,  DOI: 10.1002/ejoc.201402588
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    Goddard-Borger, E. D.; Wennekes, T.; Withers, S. G. Getting lucky in the lysosome. Nat. Chem. Biol. 2010, 6, 881– 883,  DOI: 10.1038/nchembio.470
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    Wennekes, T.; van den Berg, R. J. B. H. N.; Donker, W.; van der Marel, G. A.; Strijland, A.; Aerts, J. M. F. G.; Overkleeft, H. S. Development of adamantan-1-yl-methoxy-functionalized 1-deoxynojirimycin derivatives as selective inhibitors of glucosylceramide metabolism in man. J. Org. Chem. 2007, 72, 1088– 1097,  DOI: 10.1021/jo061280p
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    Development of Adamantan-1-yl-methoxy-Functionalized 1-Deoxynojirimycin Derivatives as Selective Inhibitors of Glucosylceramide Metabolism in Man
    Wennekes, Tom; Van den Berg, Richard J. B. H. N.; Donker, Wilma; van der Marel, Gijsbert A.; Strijland, Anneke; Aerts, Johannes M. F. G.; Overkleeft, Herman S.
    Journal of Organic Chemistry (2007), 72 (4), 1088-1097CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)
    The synthesis of adamantan-1-yl-methoxy-functionalized 1-deoxynojirimycin derivs., e.g. I, via reductive ring opening, Swern oxidn. and reductive amination, is reported. The compds. reported are lipophilic iminosugar based on lead compd. II, a potent inhibitor of the three enzymes involved in the metab. of the glycosphingolipid glucosylceramide. The results demonstrate that relocating the lipophilic moiety from the nitrogen atom to other positions on the 1-deoxynojirimycin ring system does not lead to a more potent or selective inhibitor of glucosylceramide synthase. The β-aza-C-glycoside analog I retained the best inhibitory potency for glucosylceramide synthase and is a more potent inhibitor than the therapeutic agent N-butyl-1-deoxynojirimycin, marketed as treatment for Gaucher disease under the com. name Zavesca.
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    Kallemeijn, W. W.; Li, K.-Y.; Witte, M. D.; Marques, A. R. A.; Aten, J.; Scheij, S.; Jiang, J.-B; 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. C.; Florea, B. I.; Codée, J. D. C.; van der Marel, G. A.; Aerts, J. M. F. G.; Overkleeft, H. S. Novel activity-based probes for broad-spectrum profiling of retaining beta-exoglucosidases in situ and in vivo. Angew. Chem., Int. Ed. 2012, 51, 12529– 12533,  DOI: 10.1002/anie.201207771
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    Novel Activity-Based Probes for Broad-Spectrum Profiling of Retaining β-Exoglucosidases In Situ and In Vivo
    Kallemeijn, Wouter W.; Li, Kah-Yee; Witte, Martin D.; Marques, Andre R. A.; Aten, Jan; Scheij, Saskia; Jiang, Jianbing; Willems, Lianne I.; Voorn-Brouwer, Tineke M.; van Roomen, Cindy P. A. A.; Ottenhoff, Roelof; Boot, Rolf G.; van den Elst, Hans; Walvoort, Marthe T. C.; Florea, Bogdan I.; Codee, Jeroen D. C.; van der Marel, Gijsbert A.; Aerts, Johannes M. F. G.; Overkleeft, Herman S.
    Angewandte Chemie, International Edition (2012), 51 (50), 12529-12533CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Cyclophellitol aziridine-type activity-based probes allow for ultra-sensitive visualization of mammalian β-glucosidases (GBA1, GBA2, GBA3, and LPH) as well as several non-mammalian β-glucosidases. These probes offer new ways to study β-exoglucosidases, and configurational isomers of the cyclophellitol aziridine core may give activity-based probes targeting other retaining glycosidase families.
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    Walvoort, M. T. C.; Kallemeijn, W. W.; Willems, L. I.; Witte, M. D.; Aerts, J. M. F. G.; van der Marel, G. A.; Codée, J. D. C.; Overkleeft, H. S. Tuning the leaving group in 2-deoxy-2-fluoroglucoside results in improved activity-based retaining beta-glucosidase probes. Chem. Commun. 2012, 48, 10386– 10388,  DOI: 10.1039/c2cc35653h
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    Yu, B.; Tao, H. Glycosyl trifluoroacetimidates. Part 1: preparation and application as new glycosyl donors. Tetrahedron Lett. 2001, 42, 2405– 2407,  DOI: 10.1016/S0040-4039(01)00157-5
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    Glycosyl trifluoroacetimidates. Part 1: Preparation and application as new glycosyl donors
    Yu, B.; Tao, H.
    Tetrahedron Letters (2001), 42 (12), 2405-2407CODEN: TELEAY; ISSN:0040-4039. (Elsevier Science Ltd.)
    Glycosyl (N-phenyl)trifluoroacetimidates, readily prepd. from 1-hydroxyl sugars by treatment with (N-phenyl)trifluoroacetimidoyl chloride in the presence of K2CO3, were demonstrated to be effective glycosyl donors. Glycosidation of these glycosyl fluoroacetimidates with alcs. is also reported.
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    Witte, M. D.; Walvoort, M. T. C.; Li, K.-Y.; Kallemeijn, W. W.; Donker-Koopman, W. E.; Boot, R. G.; Aerts, J. M. F. G.; Codée, J. D. C.; van der Marel, G. A.; Overkleeft, H. S. Activity-based profiling of retaining beta-glucosidases: a comparative study. ChemBioChem 2011, 12, 1263– 1269,  DOI: 10.1002/cbic.201000773
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    Harrak, Y.; Barra, C. M.; Delgado, A.; Castaño, A. R.; Llebaria, A. Galacto-configured aminocyclitol phytoceramides are potent in vivo invariant natural killer T cell stimulators. J. Am. Chem. Soc. 2011, 133, 12079– 12084,  DOI: 10.1021/ja202610x
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    Galacto-Configured Aminocyclitol Phytoceramides Are Potent in Vivo Invariant Natural Killer T Cell Stimulators
    Harrak, Youssef; Barra, Carolina M.; Delgado, Antonio; Castano, A. Raul; Llebaria, Amadeu
    Journal of the American Chemical Society (2011), 133 (31), 12079-12084CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    A new class of α-galactosylceramide (αGC) non-glycosidic analogs bearing galacto-configured aminocyclitols as sugar surrogates, e.g. I (R = H, OH), have been obtained. The aminocyclohexane having a hydroxyl substitution pattern similar to an α-galactoside is efficiently obtained by a sequence involving Evans aldol reaction and ring-closing metathesis with a Grubbs catalyst to give a key intermediate cyclohexene, which has been converted in galacto-aminocyclohexanes that are linked through a secondary amine to a phyto-ceramide lipid having a cerotyl N-acyl group. Natural Killer T (NKT) cellular assays have resulted in the identification of an active compd., HS161, which has been found to promote NKT cell expansion in vitro in a similar fashion but more weakly than αGC. This compd. stimulates the release of Interferon-γ (IFNγ) and Interleukin-4 (IL-4) in iNKT cell culture but with lower potency than αGC. The activation of Invariant Natural Killer T (iNKT) cells by this compd. has been confirmed in flow cytometry expts. Remarkably, when tested in mice, HS161 selectively induces a very strong prodn. of IFN-γ indicative of a potent Th1 cytokine profile. Overall, these data confirm the agonist activity of αGC lipid analogs having charged amino-substituted polar heads and their capacity to modulate the response arising from iNKT cell activation in vivo.
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    Alcaide, A.; Trapero, A.; Pérez, Y.; Llebaria, A. Galacto configured N-aminoaziridines: a new type of irreversible inhibitor of b-galactosidases. Org. Biomol. Chem. 2015, 13, 5690– 5697,  DOI: 10.1039/C5OB00532A
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    Ofman, T. P.; Küllmer, F.; van der Marel, G. A.; Codée, J. D. C.; Overkleeft, H. S. An orthogonally protected cyclitol for the construction of nigerose- and dextran-mimetic cyclophellitols. Org. Lett. 2021, 23, 9516,  DOI: 10.1021/acs.orglett.1c03723
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    Jiang, J.; Kuo, C.-L.; Wu, L.; Franke, C.; Kallemeijn, W. W.; Florea, B. I.; van Meel, E.; van der Marel, G. A.; Codée, J. D. C.; Boot, R. G.; Davies, G. J.; Overkleeft, H. S.; Aerts, J. M. F. G. Detection of active mammalian GH31 alpha-glucosidases in health and disease using in-class, broad-spectrum activity-based probes. ACS Cent. Sci. 2016, 2, 351– 358,  DOI: 10.1021/acscentsci.6b00057
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    Armstrong, Z.; Kuo, C.-L.; Lahav, D.; Liu, B.; Johnson, R.; Beenakker, T. J. M.; de Boer, C.; Wong, C.-S.; van Rijssel, E. R.; Debets, M. F.; Florea, B. I.; Hissink, C.; Boot, R. G.; Geurink, P. P.; Ovaa, H.; van der Stelt, M.; van der Marel, G. A.; Codée, J. D. C.; Aerts, J. M. F. G.; Wu, L.; Overkleeft, H. S.; Davies, G. J. Manno-epi-cyclophellitols enable activity-based protein profiling of human α-mannosidases and discovery of new Golgi mannosidase II inhibitors. J. Am. Chem. Soc. 2020, 142, 13021– 13029,  DOI: 10.1021/jacs.0c03880
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    56
    Manno-epi-cyclophellitols EnableActivity-Based Protein Profiling of Human α-Mannosidasesand Discovery of New Golgi Mannosidase II Inhibitors
    Armstrong, Zachary; Kuo, Chi-Lin; Lahav, Daniel; Liu, Bing; Johnson, Rachel; Beenakker, Thomas J. M.; de Boer, Casper; Wong, Chung-Sing; van Rijssel, Erwin R.; Debets, Marjoke F.; Florea, Bogdan I.; Hissink, Colin; Boot, Rolf G.; Geurink, Paul P.; Ovaa, Huib; van der Stelt, Mario; van der Marel, Gijsbert M.; Codee, Jeroen D. C.; Aerts, Johannes M. F. G.; Wu, Liang; Overkleeft, Herman S.; Davies, Gideon J.
    Journal of the American Chemical Society (2020), 142 (30), 13021-13029CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Golgi mannosidase II (GMII) catalyzes the sequential hydrolysisof two mannosyl residues from GlcNAcMan5GlcNAc2 to produce GlcNAcMan3GlcNAc2, the precursor for all complex N-glycans, including the branched N-glycans assocd. with cancer. Inhibitors of GMII are potential cancer therapeutics, but their usefulness is limited by off-target effects, which produce α-mannosidosis-like symptoms.Despite many structural and mechanistic studies of GMII, we still lack a potent and selective inhibitor of this enzyme. Here, we synthesized manno-epi-cyclophellitol epoxide and aziridines and demonstrate their covalent modification and time-dependent inhibition of GMII. Application of fluorescent manno-epi-cyclophellitolaziridine derivs. enabled activity-based protein profiling ofα-mannosidases from both human cell lysate and mouse tissue exts. Synthesized probes also facilitated a fluorescence polarization-based screen for dGMII inhibitors. We identified seven previously unknown inhibitors of GMII from a library of over 350 iminosugars and investigated their binding modalities through X-ray crystallog. Our results reveal previously unobserved inhibitor binding modes and promising scaffolds for the generation of selective GMII inhibitors.
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  57. 57
    McGregor, N. G. S.; Kuo, C.-L.; Beenakker, T. J. M.; Wong, C.-S.; Armstrong, Z.; Florea, B. I.; Codée, J. D. C.; Overkleeft, H. S.; Aerts, J. M. F. G.; Davies, G. J. Synthesis of broad-specificity activity-based probes for exo-β-mannosidases. Org. Biomol. Chem. 2022, 20, 877– 886,  DOI: 10.1039/D1OB02287C
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    57
    Synthesis of broad-specificity activity-based probes for exo-β-mannosidases
    McGregor, Nicholas G. S.; Kuo, Chi-Lin; Beenakker, Thomas J. M.; Wong, Chun-Sing; Offen, Wendy A.; Armstrong, Zachary; Florea, Bogdan I.; Codee, Jeroen D. C.; Overkleeft, Herman S.; Aerts, Johannes M. F. G.; Davies, Gideon J.
    Organic & Biomolecular Chemistry (2022), 20 (4), 877-886CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
    Exo-β-mannosidases are a broad class of stereochem. retaining hydrolases that are essential for the breakdown of complex carbohydrate substrates found in all kingdoms of life. Yet the detection of exo-β-mannosidases in complex biol. samples remains challenging, necessitating the development of new methodologies. Cyclophellitol and its analogs selectively label the catalytic nucleophiles of retaining glycoside hydrolases, making them valuable tool compds. Furthermore, cyclophellitol can be readily redesigned to enable the incorporation of a detection tag, generating activity-based probes (ABPs) that can be used to detect and identify specific glycosidases in complex biol. samples. Towards the development of ABPs for exo-β-mannosidases, we present a concise synthesis of β-manno-configured cyclophellitol, cyclophellitol aziridine, and N-alkyl cyclophellitol aziridines. We show that these probes covalently label exo-β-mannosidases from GH families 2, 5, and 164. Structural studies of the resulting complexes support a canonical mechanism-based mode of action in which the active site nucleophile attacks the pseudoanomeric center to form a stable ester linkage, mimicking the glycosyl enzyme intermediate. Furthermore, we demonstrate activity-based protein profiling using an N-alkyl aziridine deriv. by specifically labeling MANBA in mouse kidney tissue. Together, these results show that synthetic manno-configured cyclophellitol analogs hold promise for detecting exo-β-mannosidases in biol. and biomedical research.
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  58. 58
    Jiang, J.; Kallemeijn, W. W.; Wright, D. W.; van den Nieuwendijk, A. M. C. H.; Coco Rohde, V.; Folch, E. C.; van den Elst, H.; Florea, B. I.; Scheij, S.; Donker-Koopman, W. E.; Verhoek, M.; Li, N.; Schürmann, M.; Mink, D.; Boot, R. G.; Codée, J. D. C.; van der Marel, G. A.; Davies, G. J.; Aerts, J. M. F. G.; Overkleeft, H. S. In vitro and in vivo comparative and competitive activity-based protein profiling of GH29 alpha-L-fucosidases. Chem. Sci. 2015, 6, 2782– 2789,  DOI: 10.1039/C4SC03739A
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    58
    In vitro and in vivo comparative and competitive activity-based protein profiling of GH29 α-L-fucosidases
    Jiang, Jianbing; Kallemeijn, Wouter W.; Wright, Daniel W.; van den Nieuwendijk, Adrianus M. C. H.; Rohde, Veronica Coco; Folch, Elisa Colomina; van den Elst, Hans; Florea, Bogdan I.; Scheij, Saskia; Donker-Koopman, Wilma E.; Verhoek, Marri; Li, Nan; Schuermann, Martin; Mink, Daniel; Boot, Rolf G.; Codee, Jeroen D. C.; van der Marel, Gijsbert A.; Davies, Gideon J.; Aerts, Johannes M. F. G.; Overkleeft, Herman S.
    Chemical Science (2015), 6 (5), 2782-2789CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
    GH29 α-L-fucosidases catalyze the hydrolysis of α-L-fucosidic linkages. Deficiency in human lysosomal α-L-fucosidase (FUCA1) leads to the recessively inherited disorder, fucosidosis. Herein, we describe the development of fucopyranose-configured cyclophellitol aziridines as activity-based probes (ABPs) for selective in vitro and in vivo labeling of GH29 α-L-fucosidases from bacteria, mice and man. Crystallog. anal. on bacterial α-L-fucosidase confirms that the ABPs act by covalent modification of the active site nucleophile. Competitive activity-based protein profiling identified L-fuconojirimycin as the single GH29 α-L-fucosidase inhibitor from eight configurational isomers.
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  59. 59
    Willems, L. I.; Beenakker, T. J. M.; Murray, B.; Scheij, S.; Kallemeijn, W. W.; Boot, R. G.; Verhoek, M.; Donker-Koopman, W. E.; Ferraz, M. J.; van Rijssel, E. R.; Florea, B. I.; Codée, J. D. C.; van der Marel, G. A.; Aerts, J. M. F. G.; Overkleeft, H. S. Potent and selective activity-based probes for GH27 human retaining alpha-galactosidases. J. Am. Chem. Soc. 2014, 136, 11622– 11625,  DOI: 10.1021/ja507040n
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    59
    Potent and Selective Activity-Based Probes for GH27 Human Retaining α-Galactosidases
    Willems, Lianne I.; Beenakker, Thomas J. M.; Murray, Benjamin; Scheij, Saskia; Kallemeijn, Wouter W.; Boot, Rolf G.; Verhoek, Marri; Donker-Koopman, Wilma E.; Ferraz, Maria J.; van Rijssel, Erwin R.; Florea, Bogdan I.; Codee, Jeroen D. C.; van der Marel, Gijsbert A.; Aerts, Johannes M. F. G.; Overkleeft, Herman S.
    Journal of the American Chemical Society (2014), 136 (33), 11622-11625CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Lysosomal degrdn. of glycosphingolipids is mediated by the consecutive action of several glycosidases. Malfunctioning of one of these hydrolases can lead to a lysosomal storage disorder such as Fabry disease, which is caused by a deficiency in α-galactosidase A. Herein we describe the development of potent and selective activity-based probes that target retaining α-galactosidases. The fluorescently labeled aziridine-based probes inhibit the two human retaining α-galactosidases αGal A and αGal B covalently and with high affinity. Moreover, they enable the visualization of the endogenous activity of both α-galactosidases in cell exts., thereby providing a means to study the presence and location of active enzyme levels in different cell types, such as healthy cells vs. those derived from Fabry patients.
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  60. 60
    Kuo, C.-L.; Su, Q.; van den Nieuwendijk, A. M. C. H.; Beenakker, T. J. M.; Offen, W. A.; Willems, L. I.; Boot, R. G.; Sarris, A. J.; Marques, A. R. A.; Codée, J. D. C.; van der Marel, G. A.; Florea, B. I.; Davies, G. J.; Overkleeft, H. S.; Aerts, J. M. F. G. The development of a broad-spectrum retaining beta-exogalactosidase activity-based probe. Org. Biomol. Chem. 2023, 21, 7813– 7820,  DOI: 10.1039/D3OB01261A
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    Artola, M.; Kuo, C.-L.; McMahon, S. A.; Hansen, T.; van der Lienden, M.; He, X.; van den Elst, H.; Florea, B. I.; Kermode, A. R.; van der Marel, G. A.; Gloster, T.; Codée, J. D. C.; Overkleeft, H. S.; Aerts, J. M. F. G. New irreversible α-L-iduronidase inhibitors and activity-based probes. Chem. Eur. J. 2018, 24, 19081– 19088,  DOI: 10.1002/chem.201804662
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    Wu, L.; Jiang, J.; Jin, Y.; Kallemeijn, W. W.; Kuo, C.-L.; Artola, M.; Dai, W.; van Elk, C.; van Eijk, M.; van der Marel, G. A.; Codée, J. D. C.; Florea, B. I.; Aerts, J. M. F. G.; Overkleeft, H. S.; Davies, G. J. Activity-based probes for functional interrogation of retaining beta-glucuronidases. Nat. Chem. Biol. 2017, 13, 867– 873,  DOI: 10.1038/nchembio.2395
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    Biarnés, X.; Ardèvol, A.; Planas, A.; Rovira, C.; Laio, A.; Parrinello, M. The conformational free energy landscape of b-D-glucopyranose. Implications for substrate preactivation in b-glucoside hydrolysis. J. Am. Chem. Soc. 2007, 129, 10686– 10693,  DOI: 10.1021/ja068411o
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    63
    The Conformational Free Energy Landscape of β-D-Glucopyranose. Implications for Substrate Preactivation in β-Glucoside Hydrolases
    Biarnes, Xevi; Ardevol, Albert; Planas, Antoni; Rovira, Carme; Laio, Alessandro; Parrinello, Michele
    Journal of the American Chemical Society (2007), 129 (35), 10686-10693CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Using ab initio metadynamics the conformational free energy landscape of β-D-glucopyranose as a function of the puckering coordinates has been computed. It is shown that the correspondence between the free energy and the Stoddard's pseudorotational itinerary for the system is rather poor. The no. of free energy min. (9) is smaller than the no. of ideal structures (13). Moreover, only six min. correspond to a canonical conformation. The structural features, the electronic properties, and the relative stability of the predicted conformers permit the rationalization of the occurrence of distorted sugar conformations in all the available X-ray structures of β-glucoside hydrolase Michaelis complexes. It is shown that these enzymes recognize the most stable distorted conformers of the isolated substrate and at the same time the ones better prepd. for catalysis in terms of bond elongation/shrinking and charge distribution. This suggests that the factors governing the distortions present in these complexes are largely dictated by the intrinsic properties of a single glucose unit.
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    Davies, G. J.; Planas, R.; Rovira, C. Conformational analysis of the reaction coordinate of glycosidases. Acc. Chem. Res. 2012, 45, 308– 316,  DOI: 10.1021/ar2001765
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    Conformational Analyses of the Reaction Coordinate of Glycosidases
    Davies, Gideon J.; Planas, Antoni; Rovira, Carme
    Accounts of Chemical Research (2012), 45 (2), 308-316CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
    A review. The enzymic hydrolysis of the glycosidic bond is catalyzed by diverse enzymes generically termed glycoside hydrolases (hereafter GHs) or glycosidases. The many sequence-based families of glycosidases have served as a rich hunting ground for enzymologists for years. Not only are these enzymes of fundamental interest, providing paradigms for enzymic catalysis that extend beyond the bounds of carbohydrate chem., but the enzymes themselves play myriad essential roles in diverse biol. processes. The wide utility of glycosidases, from their industrial harnessing in the hydrolysis of plant biomass to their roles in human physiol. and disease, has engendered a large scientific constituency with an interest in glycosidase chem. A fascinating thread of this research, and one with major impact on the design of enzyme inhibitors, is the conformational anal. of reaction pathways within the diverse families. These GH families provide a large pallet of enzymes with which chemists have attempted to depict the conformational landscape of glycosidase action. In this Account, we review three-dimensional insight into the conformational changes directed by glycosidases, primarily from structural observations of the stable enzyme-ligand species adjacent to the transition state (or states) and of enzyme-inhibitor complexes. We further show how recent computational advances dovetail with structural insight to provide a quantum mech. basis for glycosidase action. The glycosidase-mediated hydrolysis of the acetal or ketal bond in a glycoside may occur with either inversion or retention of the configuration of the anomeric carbon. Inversion involves a single step and transition state, whereas retention, often referred to as the double displacement, is a two-step process with two transition states. The single transition state for the inverting enzymes and the two transition states (those flanking the covalent intermediate) in the double displacement have been shown to have substantial oxocarbenium ion character. The dissociative nature of these transition states results in significant relative pos. charge accumulation on the pyranose ring. The delocalization of lone-pair electrons from the ring oxygen that stabilizes the cationic transition state implies that at, or close to, the transition states the pyranose will be distorted away from its lowest energy conformation to one that favors orbital overlap. Over the preceding decade, research has highlighted the harnessing of noncovalent interactions to aid this distortion of the sugar substrates from their lowest energy chair conformation to a variety of different boat, skew boat, and half-chair forms, each of which favors catalysis with a given enzyme and substrate. Crystallog. observation of stable species that flank the transition state (or states), of both retaining and inverting glycosidases, has allowed a description of their conformational itineraries, illustrating how enzymes facilitate the "electrophilic migration" of the anomeric center along the reaction coordinate. The blossom of computational approaches, such as ab initio metadynamics, has underscored the quantum mech. basis for glycoside hydrolysis. Conformational analyses highlight not only the itineraries used by enzymes, enabling their inhibition, but are also reflected in the nonenzymic synthesis of glycosides, wherein chemists mimic strategies found in nature.
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  65. 65
    Vasella, A.; Davies, G. J.; Böhm, M. Glycosidase mechanisms. Curr. Opin. Chem. Biol. 2002, 6, 619– 629,  DOI: 10.1016/S1367-5931(02)00380-0
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    Glycosidase mechanisms
    Vasella, Andrea; Davies, Gideon J.; Boehm, Matthias
    Current Opinion in Chemical Biology (2002), 6 (5), 619-629CODEN: COCBF4; ISSN:1367-5931. (Elsevier Science Ltd.)
    A review with 99 refs. The 3-dimensional structure of glycosidases and of their complexes and the study of transition-state mimics reveal structural details that correlate with their catalytic mechanism. Of particular interest are the transition-state conformations harnessed by individual enzymes and the substrate distortion obsd. in enzyme-ligand complexes. The 3-dimensional structure in synergy with transition-state mimicry opens the way for mechanistic interpretation of enzyme inhibition and for the development of therapeutic agents.
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  66. 66
    Beenakker, T. J. M.; Wander, D. P. A.; Offen, W. A.; Artola, M.; Raich, L.; Ferraz, M. J.; Li, K.-Y.; Houben, J. H. P. M.; van Rijssel, E. R.; Hansen, T.; van der Marel, G. A.; Codée, J. D. C.; Aerts, J. M. F. G.; Rovira, C.; Davies, G. J.; Overkleeft, H. S. Carba-cyclophellitols are neutral retaining-glucosidase inhibitors. J. Am. Chem. Soc. 2017, 139, 6534– 6537,  DOI: 10.1021/jacs.7b01773
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    Carba-cyclophellitols are neutral retaining-glucosidase inhibitors
    Beenakker, Thomas J. M.; Wander, Dennis P. A.; Offen, Wendy A.; Artola, Marta; Raich, Lluis; Ferraz, Maria J.; Li, Kah-Yee; Houben, Judith H. P. M.; van Rijssel, Erwin R.; Hansen, Thomas; van der Marel, Gijsbert A.; Codee, Jeroen D. C.; Aerts, Johannes M. F. G.; Rovira, Carme; Davies, Gideon J.; Overkleeft, Herman S.
    Journal of the American Chemical Society (2017), 139 (19), 6534-6537CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The conformational anal. of glycosidases affords a route to their specific inhibition through transition-state mimicry. Inspired by the rapid reaction rates of cyclophellitol and cyclophellitol aziridine, both covalent retaining-β-glucosidase inhibitors, we postulated that the corresponding carba "cyclopropyl" analog would be a potent retaining β-glucosidase inhibitor for those enzymes reacting through the 4H3 transition-state conformation. Ab initio metadynamics simulations of the conformational free energy landscape for the cyclopropyl inhibitors showed a strong bias for the 4H3 conformation, and carba-cyclophellitol, with an N-(4-azidobutyl)carboxamide moiety, proved to be a potent inhibitor (Ki = 8.2 nM) of Thermotoga maritima (TmGH1) β-glucosidase. Three-dimensional structural anal. and comparison with unreacted epoxides showed that this compd. indeed bound in the 4H3 conformation, suggesting that conformational strain induced through a cyclopropyl unit may add to the armory of tight-binding inhibitor designs.
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    de Boer, C.; Armstrong, Z.; Lit, V. A. J.; Barash, U.; Ruijgrok, G.; Boyango, I.; Weitzenberg, M. M.; Schröder, S. P.; Sarris, A. J. C.; Meeuwenoord, N. J.; Bule, P.; Kayal, Y.; Ilan, N.; Codée, J. D. C.; Vlodavsky, I.; Overkleeft, H. S.; Davies, G. J.; Wu, L. Mechanism based heparanase inhibitors reduce cancer metastasis in vivo. Proc. Natl. Acad. Sci. USA 2022, 119, e2203167119  DOI: 10.1073/pnas.2203167119
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    Artola, M.; Wu, L.; Ferraz, M. J.; Kuo, C.-L.; Raich, L.; Breen, I. Z.; Offen, W. A.; Codée, J. D. C.; van der Marel, G. A.; Rovira, C.; Aerts, J. M. F. G.; Davies, G. J.; Overkleeft, H. S. 1,6-Cyclophellitol cyclosulfates: a new class of irreversible glycosidase inhibitor. ACS Cent. Sci. 2017, 3, 784– 793,  DOI: 10.1021/acscentsci.7b00214
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    68
    1,6-Cyclophellitol cyclosulfates: A new class of irreversible glycosidase inhibitor
    Artola, Marta; Wu, Liang; Ferraz, Maria J.; Kuo, Chi-Lin; Raich, Lluis; Breen, Imogen Z.; Offen, Wendy A.; Codee, Jeroen D. C.; van der Marel, Gijsbert A.; Rovira, Carme; Aerts, Johannes M. F. G.; Davies, Gideon J.; Overkleeft, Herman S.
    ACS Central Science (2017), 3 (7), 784-793CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)
    The essential biol. roles played by glycosidases, coupled with the diverse therapeutic benefits of pharmacol. targeting of these enzymes, provide considerable motivation for the development of new inhibitor classes. Cyclophellitol epoxides and aziridines are recently established covalent glycosidase inactivators. Inspired by the application of cyclic sulfates as electrophilic equivs. of epoxides in org. synthesis, we sought to test whether cyclophellitol cyclosulfates would similarly act as irreversible glycosidase inhibitors. Here, we present the synthesis, conformational anal., and application of novel 1,6-cyclophellitol cyclosulfates. We showed that 1,6-epi-cyclophellitol cyclosulfate (α-cyclosulfate) is a rapidly reacting α-glucosidase inhibitor whose 4C1 chair conformation matched that adopted by α-glucosidase Michaelis complexes. The 1,6-cyclophellitol cyclosulfate (β-cyclosulfate) reacted more slowly, likely reflecting its conformational restrictions. Selective glycosidase inhibitors are invaluable as mechanistic probes and therapeutic agents, and we propose cyclophellitol cyclosulfates as a valuable new class of carbohydrate mimetics for application in these directions.
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    Li, Z.; Pickles, I. B.; Sharma, M.; Melling, B.; Pallasdies, L.; Codée, J. D. C.; Williams, S. J.; Overkleeft, H. S.; Davies, G. J. Detection of sulfoquinovosidase activity in cell lysates using activity-based probes. Angew. Chem., Int. Ed. 2024, 63, e202401358  DOI: 10.1002/anie.202401358
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    McGregor, N.; Artola, M.; Nin-Hill, A.; Linzel, D.; Haon, M.; Reijngoud, J.; Ram, A. F. J.; Rosso, M.-N.; van der Marel, G. A.; Codée, J. D. C.; van Wezel, G. P.; Berrin, J.-G.; Rovira, C.; Overkleeft, H. S.; Davies, G. J. Rational design of mechanism-based inhibitors and activity-based probes for the identification of retaining α-L-arabinofuranosidases. J. Am. Chem. Soc. 2020, 142, 4648– 4662,  DOI: 10.1021/jacs.9b11351
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    Rational Design of Mechanism-Based Inhibitors and Activity-Based Probes for the Identification of Retaining α-L-Arabinofuranosidases
    McGregor, Nicholas G. S.; Artola, Marta; Nin-Hill, Alba; Linzel, Daniel; Haon, Mireille; Reijngoud, Jos; Ram, Arthur; Rosso, Marie-Noelle; van der Marel, Gijsbert A.; Codee, Jeroen D. C.; van Wezel, Gilles P.; Berrin, Jean-Guy; Rovira, Carme; Overkleeft, Herman S.; Davies, Gideon J.
    Journal of the American Chemical Society (2020), 142 (10), 4648-4662CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Identifying and characterizing the enzymes responsible for an obsd. activity within a complex eukaryotic catabolic system remains one of the most significant challenges in the study of biomass-degrading systems. The debranching of both complex hemicellulosic and pectinaceous polysaccharides requires the prodn. of α-L-arabinofuranosidases among a wide variety of coexpressed carbohydrate-active enzymes. To selectively detect and identify α-L-arabinofuranosidases produced by fungi grown on complex biomass, potential covalent inhibitors and probes which mimic α-L-arabinofuranosides were sought. The conformational free energy landscapes of free α-L-arabinofuranose and several rationally designed covalent α-L-arabinofuranosidase inhibitors were analyzed. A synthetic route to these inhibitors was subsequently developed based on a key Wittig-Still rearrangement. Through a combination of kinetic measurements, intact mass spectrometry, and structural expts., the designed inhibitors were shown to efficiently label the catalytic nucleophiles of retaining GH51 and GH54 α-L-arabinofuranosidases. Activity-based probes elaborated from an inhibitor with an aziridine warhead were applied to the identification and characterization of α-L-arabinofuranosidases within the secretome of A. niger grown on arabinan. This method was extended to the detection and identification of α-L-arabinofuranosidases produced by eight biomass-degrading basidiomycete fungi grown on complex biomass. The broad applicability of the cyclophellitol-derived activity-based probes and inhibitors presented here make them a valuable new tool in the characterization of complex eukaryotic carbohydrate-degrading systems and in the high-throughput discovery of α-L-arabinofuranosidases.
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  71. 71
    Schröder, S. P.; de Boer, C.; McGregor, N. G. S.; Rowland, R. J.; Moroz, O.; Blagova, E.; Reijngoud, J.; Arentshorst, M.; Osborn, D.; Morant, M. D.; Abbate, E.; Stringer, M. A.; Krogh, K. B. R. M.; Raich, L.; Rovira, C.; Berrin, J.-G.; van Wezel, G. P.; Ram, A. F. J.; Florea, B. I.; van der Marel, G. A.; Codée, J. D. C.; Wilson, K. S.; Wu, L.; Davies, G. J.; Overkleeft, H. S. Dynamic and functional profiling of xylan-degrading enzymes in Aspergillus secretomes using activity-based probes. ACS Cent. Sci. 2019, 5, 1067– 1078,  DOI: 10.1021/acscentsci.9b00221
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    Dynamic and Functional Profiling of Xylan-Degrading Enzymes in Aspergillus Secretomes Using Activity-Based Probes
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    ACS central science (2019), 5 (6), 1067-1078 ISSN:2374-7943.
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  • Abstract

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    Figure 1

    Figure 1. Mechanism of action of inverting β-glucosidases (A) and retaining β-glucosidases (B).

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    Figure 2

    Figure 2. Two archetypal retaining glycosidase inhibitor designs: deoxyfluoroglycosides (A) and cyclitol epoxides (B).

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    Figure 3

    Figure 3. Glycomimetic epoxides, aziridines and episulfides as mechanism-based, covalent and irreversible retaining glycosidase inhibitors.

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    Figure 4

    Figure 4. Latent quinone methides (A) and activated fluorinated glycosides (B) as activity-based (retaining) GH probes. For the original images of the SDS-PAGE gels that are shown here and in the remainder of this account, please see the papers referred to in the text.

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    Figure 5

    Figure 5. Retaining GH ABPs based on cyclophellitol and cyclophellitol aziridine. A) Adaptation of the Madsen cyclophellitol synthesis to give cyclophellitol and cyclophellitol aziridine ABPs 44 and 48. B) Comparative ABPP of the human retaining β-glucosidases, GBA1, GBA2 and GBA3. C) Head-to-head comparison of activated fluorinated glucosides 49-52 and cyclophellitol 53 as GBA1 ABPs.

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    Figure 6

    Figure 6. A, B) Reaction coordinates by which retaining β-glucosidases (A) and retaining α-glucosidases (B) process their substrates. C) Computed free energy landscapes (FELs) of selected compounds.

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    Figure 7

    Figure 7. A), Activity-based secretomes profiling of glucuronoarabinoxylan-degrading retaining arabinofuranosidases and xylanases. B) Xyloglucan-degrading retaining exoglucosidases, cellulases and xyloglucanases. Shown in the gel are samples of intact cells (C), cell extracts (L) and supernatant/secretomes (S) with ABPs 63-65.

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      Watts, A. F.; Damager, I.; Amaya, M. L.; Buschiazzo, A.; Alzari, P.; Frasch, A. C.; Withers, S. G. Trypanosoma cruzi trans-sialidase operates through a covalent sialyl-enzyme intermediate. J. Am. Chem. Soc. 2003, 125, 7532– 7533,  DOI: 10.1021/ja0344967
      7
      Trypanosoma cruzi trans-sialidase operates through a covalent sialyl-enzyme intermediate: Tyrosine is the catalytic nucleophile
      Watts, Andrew G.; Damager, Iben; Amaya, Maria L.; Buschiazzo, Alejandro; Alzari, Pedro; Frasch, Alberto C.; Withers, Stephen G.
      Journal of the American Chemical Society (2003), 125 (25), 7532-7533CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Modified sialic acid substrates have been used to label Trypanosoma cruzi trans-sialidase, demonstrating that the enzyme catalyzes the transfer of sialic acid through a covalent glycosyl-enzyme intermediate, a mechanism common to most retaining glycosidases. Peptic digestion of labeled protein, followed by LC-MS/MS anal. of the digest, identified Tyr 342 as the catalytic nucleophile. This is the first such example of a retaining glycosidase utilizing an aryl glycoside intermediate. It is suggested that this alternative choice of nucleophile is a consequence of the chem. nature of sialic acid. A Tyr/Glu couple is invoked to relay charge from a remote glutamic acid, thereby avoiding electrostatic repulsion with the sialic acid carboxylate group.
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    8. 8
      McGregor, N. G. S.; Coines, J.; Borlandelli, V.; Amaki, S.; Artola, M.; Nin-Hill, A.; Linzel, D.; Yamada, C.; Arakawa, T; Ishiwata, A.; Ito, Y.; van der Marel, G. A.; Codée, J. D. C.; Fushinobu, S.; Overkleeft, H. S.; Rovira, C.; Davies, G. J. Cysteine nucleophiles in glycosidase catalysis: application of a covalent β-L-arabinofuranosidase inhibitor. Angew. Chem., Int. Ed. 2021, 60, 5754– 5758,  DOI: 10.1002/anie.202013920
      8
      Cysteine Nucleophiles in Glycosidase Catalysis: Application of a Covalent β-L-Arabinofuranosidase Inhibitor
      McGregor, Nicholas G. S.; Coines, Joan; Borlandelli, Valentina; Amaki, Satoko; Artola, Marta; Nin-Hill, Alba; Linzel, Daniel; Yamada, Chihaya; Arakawa, Takatoshi; Ishiwata, Akihiro; Ito, Yukishige; van der Marel, Gijsbert A.; Codee, Jeroen D. C.; Fushinobu, Shinya; Overkleeft, Herman S.; Rovira, Carme; Davies, Gideon J.
      Angewandte Chemie, International Edition (2021), 60 (11), 5754-5758CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The recent discovery of zinc-dependent retaining glycoside hydrolases (GHs), with active sites built around a Zn(Cys)3(Glu) coordination complex, has presented unresolved mechanistic questions. In particular, the proposed mechanism, depending on a Zn-coordinated cysteine nucleophile and passing through a thioglycosyl enzyme intermediate, remains controversial. This is primarily due to the expected stability of the intermediate C-S bond. To facilitate the study of this atypical mechanism, we report the synthesis of a cyclophellitol-derived β-L-arabinofuranosidase inhibitor, hypothesised to react with the catalytic nucleophile to form a non-hydrolysable adduct analogous to the mechanistic covalent intermediate. This β-L-arabinofuranosidase inhibitor reacts exclusively with the proposed cysteine thiol catalytic nucleophiles of representatives of GH families 127 and 146. X-ray crystal structures detd. for the resulting adducts enable MD and QM/MM simulations, which provide insight into the mechanism of thioglycosyl enzyme intermediate breakdown. Leveraging the unique chem. of cyclophellitol derivs., the structures and simulations presented here support the assignment of a zinc-coordinated cysteine as the catalytic nucleophile and illuminate the finely tuned energetics of this remarkable metalloenzyme clan.
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    9. 9
      Dennis, R. J.; Taylor, E. J.; Macauley, M. S.; Stubbs, K. A.; Turkenburg, J. P.; Hart, S. J.; Black, G. N.; Vocadlo, D. J.; Davies, G. J. Structure and mechanism of a bacterial β-glucosaminidase having O-GlcNAcase activity. Nat. Struct. Mol. Biol. 2006, 13, 365– 371,  DOI: 10.1038/nsmb1079
      9
      Structure and mechanism of a bacterial β-glucosaminidase having O-GlcNAcase activity
      Dennis, Rebecca J.; Taylor, Edward J.; Macauley, Matthew S.; Stubbs, Keith A.; Turkenburg, Johan P.; Hart, Samuel J.; Black, Gary N.; Vocadlo, David J.; Davies, Gideon J.
      Nature Structural & Molecular Biology (2006), 13 (4), 365-371CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)
      O-GlcNAc is an abundant post-translational modification of serine and threonine residues of nucleocytoplasmic proteins. This modification, found only within higher eukaryotes, is a dynamic modification that is often reciprocal to phosphorylation. In a manner analogous to phosphatases, a glycoside hydrolase termed O-GlcNAcase cleaves O-GlcNAc from modified proteins. Enzymes with high sequence similarity to human O-GlcNAcase are also found in human pathogens and symbionts. We report the three-dimensional structure of O-GlcNAcase from the human gut symbiont Bacteroides thetaiotaomicron both in its native form and in complex with a mimic of the reaction intermediate. Mutagenesis and kinetics studies show that the bacterial enzyme, very similarly to its human counterpart, operates via an unusual 'substrate-assisted' catalytic mechanism, which will inform the rational design of enzyme inhibitors.
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    10. 10
      Sobala, L. F.; Speciale, G.; Zhu, S.; Raich, L.; Sannikova, N.; Thompson, A. J.; Hakki, Z.; Lu, D.; Shansi Kazem Abadi, S.; Lewis, A. R.; Rojas-Cervellera, V.; Bernardo-Seisdedos, G.; Zhang, Y.; Millet, O.; Jiménez-Barbero, J.; Bennet, A. J.; Sollogoub, M.; Rovira, C.; Davies, G. J.; Williams, S. J. An epoxide intermediate in glycosidase catalysis. ACS Cent. Sci. 2020, 6, 760– 770,  DOI: 10.1021/acscentsci.0c00111
      10
      An epoxide intermediate in glycosidase catalysis
      Sobala, Lukasz F.; Speciale, Gaetano; Zhu, Sha; Raich, Lluis; Sannikova, Natalia; Thompson, Andrew J.; Hakki, Zalihe; Lu, Dan; Shamsi Kazem Abadi, Saeideh; Lewis, Andrew R.; Rojas-Cervellera, Victor; Bernardo-Seisdedos, Ganeko; Zhang, Yongmin; Millet, Oscar; Jimenez-Barbero, Jesus; Bennet, Andrew J.; Sollogoub, Matthieu; Rovira, Carme; Davies, Gideon J.; Williams, Spencer J.
      ACS Central Science (2020), 6 (5), 760-770CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)
      Retaining glycoside hydrolases cleave their substrates through stereochem. retention at the anomeric position. Typically, this involves two-step mechanisms using either an enzymic nucleophile via a covalent glycosyl enzyme intermediate or neighboring-group participation by a substrate-borne 2-acetamido neighboring group via an oxazoline intermediate; no enzymic mechanism with participation of the sugar 2-hydroxyl has been reported. Here, we detail structural, computational, and kinetic evidence for neighboring-group participation by a mannose 2-hydroxyl in glycoside hydrolase family 99 endo-α-1,2-mannanases. We present a series of crystallog. snapshots of key species along the reaction coordinate: a Michaelis complex with a tetrasaccharide substrate; complexes with intermediate mimics, a sugar-shaped cyclitol β-1,2-aziridine and β-1,2-epoxide; and a product complex. The 1,2-epoxide intermediate mimic displayed hydrolytic and transfer reactivity analogous to that expected for the 1,2-anhydro sugar intermediate supporting its catalytic equivalence. Quantum mechanics/mol. mechanics modeling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar via a transition state in an unusual flattened, envelope (E3) conformation. Kinetic isotope effects (kcat/Km) for anomeric-2H and anomeric-13C support an oxocarbenium ion-like transition state, and that for C2-18O (1.052 ± 0.006) directly implicates nucleophilic participation by the C2-hydroxyl. Collectively, these data substantiate this unprecedented and long-imagined enzymic mechanism. Mannosidases of glycoside hydrolase family 99 use a neighboring-group participation mechanism involving the substrate 2-hydroxyl.
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    11. 11
      Withers, S. G.; Street, I. P.; Bird, P.; Dolphin, D. H. 2-Deoxy-2-fluoroglucosides: a novel class of mechanism-based glucosidase inhibitors. J. Am. Chem. Soc. 1987, 109, 7530– 7531,  DOI: 10.1021/ja00258a047
      11
      2-Deoxy-2-fluoroglucosides: a novel class of mechanism-based glucosidase inhibitors
      Withers, Stephen G.; Street, Ian P.; Bird, Paul; Dolphin, David H.
      Journal of the American Chemical Society (1987), 109 (24), 7530-1CODEN: JACSAT; ISSN:0002-7863.
      2,4-Dinitrophenyl 2-deoxy-2-fluoro β-D-glucopyranoside was synthesized and demonstrated to be a specific mechanism-based inhibitor for β-glucosidase from Alcaligenes faecalis. The inactivation of the enzyme followed pseudo-1st-order kinetics with a rate const. of 25 min-1 and a dissocn. const. of 0.05 mM. Protection against inactivation was shown by the competitive inhibitor isopropylthio β-D-glucopyranoside. Reactivation of the enzyme was obsd. as expected after removal of free inhibitor and incubation in the presence of substrate. The use of such inhibitors to investigate the intermediacy of a glucosyl enzyme intermediate and to identify the active site residue involved is projected.
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    12. 12
      Rempel, B. P.; Withers, S. G. Covalent inhibitors of glycosidases and their applications in biochemistry and biology. Glycobiol. 2008, 18, 570– 586,  DOI: 10.1093/glycob/cwn041
      There is no corresponding record for this reference.
    13. 13
      Vocadlo, D. J.; Davies, G. J.; Laine, R.; Withers, S. G. Catalysis by hen egg-white lysozyme proceeds via a covalent intermediate. Nature 2001, 412, 835– 838,  DOI: 10.1038/35090602
      13
      Catalysis by hen egg-white lysozyme proceeds via a covalent intermediate
      Vocadlo, David J.; Davies, Gideon J.; Laine, Roger; Withers, Stephen G.
      Nature (London, United Kingdom) (2001), 412 (6849), 835-839CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Hen egg-white lysozyme (HEWL) was the first enzyme to have its three-dimensional structure detd. by x-ray diffraction techniques. A catalytic mechanism, featuring a long-lived oxo-carbenium-ion intermediate, was proposed on the basis of model-building studies. The 'Phillips' mechanism is widely held as the paradigm for the catalytic mechanism of β-glycosidases that cleave glycosidic linkages with net retention of configuration of the anomeric center. Studies with other retaining β-glycosidases, however, provide strong evidence pointing to a common mechanism for these enzymes that involves a covalent glycosyl-enzyme intermediate, as previously postulated. Here the authors show, in three different cases using electrospray ionization mass spectrometry, a catalytically competent covalent glycosyl-enzyme intermediate during the catalytic cycle of HEWL. The authors also show the three-dimensional structure of this intermediate as detd. by x-ray diffraction. The authors formulate a general catalytic mechanism for all retaining β-glycosidases that includes substrate distortion, formation of a covalent intermediate, and the electrophilic migration of C1 along the reaction coordinate.
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    14. 14
      Legler, G. Untersuchungen zum Wirkunsmechanismus glycosidspaltender Enzyme, I. Darstellung und Eigenschaften spezifischer Inaktivatoren. Hoppe-Seyler’s Z. Physiol. Chem. 1966, 345, 197– 214,  DOI: 10.1515/bchm2.1966.345.1.197
      There is no corresponding record for this reference.
    15. 15
      Legler, G. Untersuchungen zum Wirkunsmechanismus glycosidspaltender Enzyme, II. Isolierung und enzymatische Eigenschaften from zwei β-Glucosidasen aus Aspergillus wentii. Hoppe-Seyler’s Z. Physiol. Chem. 1967, 348, 1359– 1366,  DOI: 10.1515/bchm2.1967.348.1.1359
      There is no corresponding record for this reference.
    16. 16
      Legler, G. Untersuchungen zum Wirkunsmechanismus glycosidspaltender Enzyme, III. Markierung des aktiven Zentrums einer β-Glucosidase aus Aspergillus wentii mit [14C]Condurit-B-epoxid. Hoppe-Seyler’s Z. Physiol. Chem. 1968, 349, 767– 774,  DOI: 10.1515/bchm2.1968.349.1.767
      There is no corresponding record for this reference.
    17. 17
      Braun, H.; Legler, G.; Deshusses, J.; Semenza, G. Stereoselective ring opening of conduritol-B-epoxide by an active site aspartate residue of sucrose-isomaltase. Biochim. Biophys. Acta 1977, 483, 135– 140,  DOI: 10.1016/0005-2744(77)90015-8
      There is no corresponding record for this reference.
    18. 18
      Legler, G.; Bause, E. Epoxy-alkyl oligo-(1–4)-β-D-glucosides as active-site-directed inhibitors of cellulases. Carbohydr. Res. 1973, 28, 45– 52,  DOI: 10.1016/S0008-6215(00)82855-4
      There is no corresponding record for this reference.
    19. 19
      Rodriguez, E. B.; Scally, G. D.; Stick, R. V. The synthesis of optically pure epoxy-alkyl β-D-glucosides and β-cellobiosides as active-site directed inhibitors of some β-glucan hydrolases. Aust. J. Chem. 1990, 43, 1391– 1405,  DOI: 10.1071/CH9901391
      There is no corresponding record for this reference.
    20. 20
      Sulzenbacher, G.; Schülein, M.; Davies, G. J. Structure of the endoglucanase I from Fusarium oxysporum: native, cellobiose, and 3,4-epoxybutyl β-D-cellobioside-inhibited forms, at 2.3 Å resolution. Biochemistry 1997, 36, 5902– 5911,  DOI: 10.1021/bi962963+
      20
      Structure of the endoglucanase I from Fusarium oxysporum: Native, cellobiose, and 3,4-epoxybutyl β-D-cellobioside-inhibited forms, at 2.3 Å resolution
      Sulzenbacher, Gerlind; Schuelein, Martin; Davies, Gideon J.
      Biochemistry (1997), 36 (19), 5902-5911CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      The crystal structure of F. oxysporum cellulase endoglucanase I (I) of glycosyl hydrolase family 7 was solved at 2.3 Å resoln. In addn. to the native enzyme, structures were also detd. of I complexed with both the affinity label, 3,4-epoxybutyl β-D-cellobioside, and the reaction product, cellobiose. The affinity label was covalently bound, as expected, to the catalytic nucleophile, Glu-197, with clear evidence for binding of both the R and S stereoisomers. Cellobiose was found bound to the -2 and -1 subsites of I. In marked contrast to the previously reported crystal structure of I complexed with a nonhydrolyzable thiosaccharide analog, which spanned the -2, -1, and +1 subsites and which had a skew-boat conformation for the -1 subsite sugar, the I-cellobiose complex showed no pyranoside ring distortion in the -1 subsite, implying that strain is induced primarily by the addnl. +1 subsite interactions and that the product is found, as expected, in its unstrained conformation.
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    21. 21
      Thomas, E. W.; McKelvy, J. F.; Sharon, N. Specific and irreversible inhibition of lysozyme by 2′,3′-epoxypropyl β-glycosides of N-acetyl-D-glucosamine oligomers. Nature 1969, 222, 485– 486,  DOI: 10.1038/222485a0
      There is no corresponding record for this reference.
    22. 22
      Havukainen, R.; Törrönen, A.; Laitinen, T.; Rouvinen, J. Covalent binding of three epoxyalkyl xylosides to the active site of endo-1,4-xylanase II from Trichoderma reesei. Biochemistry 1996, 35, 9617– 9624,  DOI: 10.1021/bi953052n
      22
      Covalent binding of three epoxyalkyl xylosides to the active site of endo-1,4-xylanase II from Trichoderma reesei
      Havukainen, Riikka; Torronen, Anneli; Laitinen, Tuomo; Rouvinen, Juha
      Biochemistry (1996), 35 (29), 9617-9624CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      The three-dimensional structures of endo-1,4-xylanase II from (XYNII) Trichoderma reesei complexed with 4,5-epoxypentyl β-D-xyloside (X-O-C5), 3,4-epoxybutyl β-D-xyloside (X-O-C4), and 2,3-epoxypropyl β-D-xyloside (X-O-C3) were detd. by x-ray crystallog. High-resoln. measurement revealed clear electron densities for each ligand. Both X-O-C5 and X-O-C3 were found to form a covalent bond with the putative nucleophile Glu86. Unexpectedly, X-O-C4 was found to bind to the putative acid/base catalyst Glu177. In all three complexes, clear conformational changes were found in XYNII compared to the native structure. These changes were largest in the X-O-C3 complex structure.
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    23. 23
      Liu, L.; Patricelli, M. P.; Cravatt, B. F. Activity-based protein profiling: the serine hydrolases. Proc. Natl. Acad. Sci. USA 1999, 96, 14694– 14699,  DOI: 10.1073/pnas.96.26.14694
      23
      Activity-based protein profiling: the serine hydrolases
      Liu, Yongsheng; Patricelli, Matthew P.; Cravatt, Benjamin F.
      Proceedings of the National Academy of Sciences of the United States of America (1999), 96 (26), 14694-14699CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      With the postgenome era rapidly approaching, new strategies for the functional anal. of proteins are needed. To date, proteomics efforts have primarily been confined to recording variations in protein level rather than activity. The ability to profile classes of proteins on the basis of changes in their activity would greatly accelerate both the assignment of protein function and the identification of potential pharmaceutical targets. Here, we describe the chem. synthesis and utility of an active-site directed probe for visualizing dynamics in the expression and function of an entire enzyme family, the serine hydrolases. By reacting this probe, a biotinylated fluorophosphonate referred to as FP-biotin, with crude tissue exts., we quickly and with high sensitivity detect numerous serine hydrolases, many of which display tissue-restricted patterns of expression. Addnl., we show that FP-biotin labels these proteins in an activity-dependent manner that can be followed kinetically, offering a powerful means to monitor dynamics simultaneously in both protein function and expression.
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    24. 24
      Yariv, J.; Wilson, K. J.; Hildesheim, J.; Blumberg, S. Labelling of the active site of β-galactosidase by N-bromoacetyl β-D-galactopyranosylamine. FEBS Lett. 1971, 15, 24– 26,  DOI: 10.1016/0014-5793(71)80070-4
      24
      Labeling of the active site of β-galactosidase by N-bromoacetyl β-D-galactopyranosylamine
      Yariv, Joseph; Wilson, Kenneth J.; Hildesheim, Jean; Blumberg, Shmaryahu
      FEBS Letters (1971), 15 (1), 24-6CODEN: FEBLAL; ISSN:0014-5793.
      The hydrolysis of o-nitrophenyl β-galactopyranoside by β-galactosidase was inhibited by incubating with labeled N-bromoacetyl β-D-galactopyranosylamine. The reaction followed 1st order kinetics leading to complete inactivation and 1 mole of reagent was bound to the enzyme per mole of site inactivated. The corresponding L-fucose deriv. did not inactivate the enzyme.
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    25. 25
      Caron, G.; Withers, S. G. Conduritol aziridine: a new mechanism-based glucosidase inactivator. Biochem. Biophys. Res. Commun. 1989, 163, 495– 499,  DOI: 10.1016/0006-291X(89)92164-5
      There is no corresponding record for this reference.
    26. 26
      Tong, M. K.; Ganem, B. A potent new class of active-site-directed glycosidase inactivators. J. Am. Chem. Soc. 1988, 110, 312– 313,  DOI: 10.1021/ja00209a062
      26
      A potent new class of active-site-directed glycosidase inactivators
      Tong, Michael K.; Ganem, Bruce
      Journal of the American Chemical Society (1988), 110 (1), 312-13CODEN: JACSAT; ISSN:0002-7863.
      Aziridine I was prepd. from piperidine II in 6 steps. I showed potent inhibition of green coffee bean α-galactosidase, but had little or no effect on yeast α-glucosidase, jackbean α-mannosidase, or bovine β-galactosidase.
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    27. 27
      Atsumi, S.; Umezawa, K.; Iinuma, H.; Naganawa, H.; Nakamura, H.; Iitaka, Y.; Takeuchi, T. Production, isolation and structure determination of a novel β-glucosidase inhibitor, cyclophellitol, from Phellinus sp. J. Antibiot. 1990, 43, 49– 53,  DOI: 10.7164/antibiotics.43.49
      27
      Production, isolation and structure determination of a novel β-glucosidase inhibitor, cyclophellitol, from Phellinus sp
      Atsumi, Sonoko; Umezawa, Kazuo; Iinuma, Hironobu; Naganawa, Hiroshi; Nakamura, Hikaru; Iitaka, Yoichi; Takeuchi, Tomio
      Journal of Antibiotics (1990), 43 (1), 49-53CODEN: JANTAJ; ISSN:0021-8820.
      A culture filtrate of a mushroom, Phellinus species, strongly inhibited β-glucosidase. The active substance was isolated through charcoal sepn., column chromatog. and crystn. Spectroscopic and crystallog. anal. revealed that it had a novel cyclitol structure, (1S,2R,3S,4R,5R,6R)-5-hydroxymethyl-7-oxabicyclo[4.1.0]heptane-2,3,4-triol; the compd. was named cyclophellitol (I). It inhibited almond-derived β-glucosidase with an IC50 of 0.8 μg/mL.
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    28. 28
      Gloster, T. M.; Madsen, R.; Davies, G. J. Structural basis for cyclophellitol inhibition of a β-glucosidase. Org. Biomol. Chem. 2007, 5, 444– 446,  DOI: 10.1039/B616590G
      28
      Structural basis for cyclophellitol inhibition of a β-glucosidase
      Gloster, Tracey M.; Madsen, Robert; Davies, Gideon J.
      Organic & Biomolecular Chemistry (2007), 5 (3), 444-446CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
      The structural basis for β-glucosidase inhibition by cyclophellitol was demonstrated using x-ray crystallog., enzyme kinetics and mass spectrometry.
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    29. 29
      Nakata, M.; Chong, C.; Niwata, Y.; Toshima, K.; Tatsuta, K. A family of cyclophellitol analogues: synthesis and evaluation. J. Antibiot. 1993, 46, 1919– 1922,  DOI: 10.7164/antibiotics.46.1919
      There is no corresponding record for this reference.
    30. 30
      Shing, T. K. M.; Tai, V. W.-F. (-)-Quinic acid in organic synthesis. Part 4. Synthesis of cyclophellitol and its (1R,6S)-, (2S)-, (1R,2S,6S)-diastereomers. J. Chem. Soc. Perkin Trans 1. 1994, 2017– 2025,  DOI: 10.1039/P19940002017
      There is no corresponding record for this reference.
    31. 31
      Greenbaum, F.; Medzihradszky, K. F.; Burlingame, A.; Bogyo, M. Epoxide electrophiles as activity-dependent cysteine protease profiling and discovery tools. Chem. Biol. 2000, 7, 569– 581,  DOI: 10.1016/S1074-5521(00)00014-4
      31
      Epoxide electrophiles as activity-dependent cysteine protease profiling and discovery tools
      Greenbaum, Doron; Medzihradszky, Katalin F.; Burlingame, Alma; Bogyo, Matthew
      Chemistry & Biology (2000), 7 (8), 569-581CODEN: CBOLE2; ISSN:1074-5521. (Elsevier Science Ltd.)
      Background: Anal. of global changes in gene transcription and translation by systems-based genomics and proteomics approaches provides only indirect information about protein function. In many cases, enzymic activity fails to correlate with transcription or translation levels. Therefore, a direct method for broadly detg. activities of an entire class of enzymes on a genome-wide scale would be of great utility. Results: We have engineered chem. probes that can be used to broadly track activity of cysteine proteases. The structure of the general cysteine protease inhibitor E-64 was used as a scaffold. Analogs were synthesized by varying the core peptide recognition portion while adding affinity tags (biotin and radio-iodine) at distal sites. The resulting probes contg. a P2 leucine residue (DCG-03 and DCG-04) targeted the same broad set of cysteine proteases as E-64 and were used to profile these proteases during the progression of a normal skin cell to a carcinoma. A library of DCG-04 derivs. was constructed in which the leucine residue was replaced with all natural amino acids. This library was used to obtain inhibitor activity profiles for multiple protease targets in crude cellular exts. Finally, the affinity tag of DCG-04 allowed purifn. of modified proteases and identification by mass spectrometry. Conclusions: We have created a simple and flexible method for functionally identifying cysteine proteases while simultaneously tracking their relative activity levels in crude protein mixts. These probes were used to det. relative activities of multiple proteases throughout a defined model system for cancer progression. Furthermore, information obtained from libraries of affinity probes provides a rapid method for obtaining detailed functional information without the need for prior purifn./identification of targets.
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    32. 32
      Janda, K. D.; Lo, L.-C.; Lo, C.-H. L.; Sim, M.-M.; Wang, R.; Wong, C.-H.; Lerner, R. A. Chemical selection for catalysis in combinatorial libraries. Science 1997, 275, 945– 948,  DOI: 10.1126/science.275.5302.945
      32
      Chemical selection for catalysis in combinatorial antibody libraries
      Janda, Kim D.; Lo, Lee-Chiang; Lo, Chih-Hung L.; Sim, Mui-Mui; Wang, Ruo; Wong, Chi-Huey; Lerner, Richard A.
      Science (Washington, D. C.) (1997), 275 (5302), 945-948CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      For the past decade the immune system has been exploited as a rich source of de novo catalysts. Catalytic antibodies have been shown to have chemoselectivity, enantioselectivity, large rate accelerations, and even an ability to reroute chem. reactions. In many instances catalysts have been made for reactions for which there are no known natural or man-made enzymes. Yet, the full power of this combinatorial system can only be exploited if there was a system that allows for the direct selection of a particular function. A method that allows for the direct chem. selection for catalysis from antibody libraries was so devised, whereby the pos. aspects of hybridoma technol. were preserved and re-formatted in the filamentous phage system to allow direct selection of catalysis. This methodol. is based on a purely chem. selection process, making it more general than biol. based selection systems because it is not limited to reaction products that perturb cellular machinery.
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    33. 33
      Tsai, C.-S.; Li, Y.-K.; Lo, L.-C. Design and synthesis of activity probes for glycosidases. Org. Lett. 2002, 4, 3607– 3610,  DOI: 10.1021/ol0265315
      33
      Design and Synthesis of Activity Probes for Glycosidases
      Tsai, Charng-Sheng; Li, Yaw-Kuen; Lo, Lee-Chiang
      Organic Letters (2002), 4 (21), 3607-3610CODEN: ORLEF7; ISSN:1523-7060. (American Chemical Society)
      A new synthetic route was developed for the prepn. of glycoside I activity probe for β-glucosidase in this study. The key glycosidation step begins with benzyl p-hydroxyphenylacetate. Benzylic functionalization for the construction of the trapping device was achieved at later stages. Probe I was shown to be able to label the target enzyme. This cassette-like design offers great flexibility for future alterations. It would allow the synthetic scheme to expand to other glycosidase probes with different linker/reporter combinations.
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    34. 34
      Lu, C.-P.; Ren, C.-T.; Lai, Y.-N.; Wu, S.-H.; Wang, W.-M.; Chen, J.-Y.; Lo, L.-C. Design of a mechanism-based probe for neuraminidase to capture influenza viruses. Angew. Chem., Int. Ed. 2005, 44, 6888– 6892,  DOI: 10.1002/anie.200501738
      34
      Design of a mechanism-based probe for neuraminidase to capture influenza viruses
      Lu, Chun-Ping; Ren, Chien-Tai; Lai, Yi-Ning; Wu, Shih-Hsiung; Wang, Wei-Man; Chen, Jean-Yin; Lo, Lee-Chiang
      Angewandte Chemie, International Edition (2005), 44 (42), 6888-6892CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A mechanism-based probe for neuraminidase was developed. The probe consisted of a recognition head (N-acetylneuraminic acid), a trapping device (o-difluoromethylphenyl group), a linker, and a reporter group (biotin). It is capable of forming a biotinylated adduct with neuraminidase (NA) from Arthrobacter ureafaciens. It also displays an inhibitory effect on a no. of NA activities. ELISA expts. successfully demonstrated that influenza viruses can be selectively captured with this probe.
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    35. 35
      Kwan, D. H.; Chen, H.-M.; Ratananikom, K.; Hancock, S. M.; Watanabe, Y.; Kongsaeree, P. T.; Samuels, A. L.; Withers, S. G. Self-immobilizing fluorogenic imaging agents of enzyme activity. Angew. Chem., Int. Ed. 2011, 50, 300– 303,  DOI: 10.1002/anie.201005705
      35
      Self-Immobilizing Fluorogenic Imaging Agents of Enzyme Activity
      Kwan, David H.; Chen, Hong-Ming; Ratananikom, Khakhanang; Hancock, Susan M.; Watanabe, Yoichiro; Kongsaeree, Prachumporn T.; Samuels, A. Lacey; Withers, Stephen G.
      Angewandte Chemie, International Edition (2011), 50 (1), 300-303, S300/1-S300/35CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      We have demonstrated a novel application for enzyme substrates that generate quinone methides. Such substrates have previously been used as enzyme inactivators and abused as proteomics probes; however, these compds. may find greater value as histol. or cell-labeling agents for the investigation of a wide variety of organisms, as illustrated by our results with plants, yeast, and bacteria.
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    36. 36
      Vocadlo, D. J.; Bertozzi, C. R. A strategy for functional proteomic analysis of glycosidase activity from cell lysates. Angew. Chem., Int. Ed. 2004, 43, 5338– 5342,  DOI: 10.1002/anie.200454235
      36
      A strategy for functional proteomic analysis of glycosidase activity from cell lysates
      Vocadlo, David J.; Bertozzi, Carolyn R.
      Angewandte Chemie, International Edition (2004), 43 (40), 5338-5342CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      We have developed a strategy for activity-based labeling of retaining glycosidases by using the azide group as a sterically unobtrusive chem. tag. We prepd. 6-azido-2,6-dideoxy-2-fluoro-β-D-galactosyl fluoride (6Az2FgalF, 10, Scheme 2) as an activity-based probe for β-galactosidase. The high selectivity of both the inactivation with fluorosugars and the Staudinger ligation with phosphine probes allows detection of glycosidases in complex mixts. and the strategy can be used for profiling these enzyme activities in cell lysates. We have demonstrated that the approach can be used to tag several glycosidases from different glycoside hydrolase families. We anticipate that the strategy will find broad utility in proteomic anal. of these enzymes in prokaryotic and eukaryotic proteomes. The azide group might also be useful as a chem. tag within inactivators of other enzymes from entirely different families with sterically confining active sites.
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    37. 37
      Gebler, J. C.; Aebersold, R.; Withers, S. G. Glu-537, not Glu-461, is the nucleophile in the active site of (LacZ) b-galactosidase from Escherichia coli. J. Biol. Chem. 1992, 267, 11126– 11130,  DOI: 10.1016/S0021-9258(19)49884-0
      37
      Glu-537, not Glu-461, is the nucleophile in the active site of (lac Z) β-galactosidase from Escherichia coli
      Gebler, John C.; Aebersold, Ruedi; Withers, Stephen G.
      Journal of Biological Chemistry (1992), 267 (16), 11126-30CODEN: JBCHA3; ISSN:0021-9258.
      The covalent intermediate formed during catalysis by the lac Z β-galactosidase from E. coli can be trapped by reaction of the enzyme with 2',4'-dinitrophenyl-2-deoxy-2-fluoro-β-D-galactopyranoside, thereby inactivating the enzyme. Kinetic parameters for this inactivation process with the holo- and apoenzymes have been detd. The nucleophilic amino acid involved has been identified as Glu-537 by using a tritium-labeled inactivator to label the enzyme, then cleaving the labeled protein into peptides and purifying and sequencing the labeled peptide. This residue is conserved in five homologous β-galactosidases and is different from that (Glu-461) proposed to be the nucleophile (Herrchen, M.; Legler, G., 1984) on the basis of affinity labeling studies with conduritol C cis-epoxide. A role for glutamic acid residue 461 as the acid/base catalyst is proposed and justified.
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    38. 38
      Stubbs, K. A.; Scaffidi, A.; Debowski, A. W.; Mark, B. L.; Stick, R. V.; Vocadlo, D. J. Synthesis and use of mechanism-based protein profiling probes for retaining b-D-glucosaminidases facilitate identification of Pseudomonas aeruginosa NagZ. J. Am. Chem. Soc. 2008, 130, 327– 335,  DOI: 10.1021/ja0763605
      38
      Synthesis and Use of Mechanism-Based Protein-Profiling Probes for Retaining β-D-Glucosaminidases Facilitate Identification of Pseudomonas aeruginosa NagZ
      Stubbs, Keith A.; Scaffidi, Adrian; Debowski, Aleksandra W.; Mark, Brian L.; Stick, Robert V.; Vocadlo, David J.
      Journal of the American Chemical Society (2008), 130 (1), 327-335CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The NagZ class of retaining exo-glucosaminidases play a crit. role in peptidoglycan recycling in Gram-neg. bacteria and the induction of resistance to β-lactams. Here we describe the concise synthesis of 2-azidoacetyl-2-deoxy-5-fluoro-β-D-glucopyranosyl fluoride as an activity-based proteomics probe for profiling these exo-glycosidases. This active-site directed reagent covalently inactivates this class of retaining N-acetylglucosaminidases with exquisite selectivity by stabilizing the glycosyl-enzyme intermediate. Inactivated Vibrio cholerae NagZ can be elaborated with biotin or a FLAG-peptide epitope using the Staudinger ligation or the Sharpless-Meldal click reaction and detected at nanogram levels. This ABPP enabled the profiling of the Pseudomonas aeruginosa proteome and identification at endogenous levels of a tagged protein with properties consistent with those of PA3005. Cloning of the gene encoding this hypothetical protein and biochem. characterization enabled unambiguous assignment of this hypothetical protein as a NagZ. The identification and cloning of this NagZ may facilitate the development of strategies to circumvent resistance to β-lactams in this human pathogen. As well, this general strategy, involving such 5-fluoro inactivators, may prove to be of general use for profiling proteomes and identifying glycoside hydrolases of medical importance or having desirable properties for biotechnol.
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    39. 39
      Tsai, C.-S.; Yen, H.-Y.; Lin, M.-I.; Tsai, T.-I.; Wang, S.-Y.; Huang, W.-I.; Hsu, T.-L.; Cheng, Y. S. E.; Fang, J.-M.; Wong, C.-H. Cell-permeable probe for identification and imaging of sialidases. Proc. Natl. Acad. Sci. USA 2013, 110, 2466– 2471,  DOI: 10.1073/pnas.1222183110
      There is no corresponding record for this reference.
    40. 40
      Hekmat, O.; Kim, Y.-W.; Williams, S. J.; He, S.; Withers, S. G. Active-site “fingerprinting” of glycosidases in complex mixture by mass spectrometry. Discovery of a novel retaining b-1–4-glycanase in Cellulomonas fimi. J. Biol. Chem. 2005, 280, 35126– 35135,  DOI: 10.1074/jbc.M508434200
      There is no corresponding record for this reference.
    41. 41
      Chauvigné-Hines, L. M.; Anderson, L. N.; Weaver, H. M.; Brown, J. N.; Koech, P. K.; Nicora, C. D.; Hofstad, B. A.; Smith, R. D.; Wilkins, M. J.; Callister, S. J.; Wright, A. T. Suite of activity-based probes for cellulose-degrading enzymes. J. Am. Chem. Soc. 2012, 134, 20521– 20532,  DOI: 10.1021/ja309790w
      41
      Suite of Activity-Based Probes for Cellulose-Degrading Enzymes
      Chauvigne-Hines, Lacie M.; Anderson, Lindsey N.; Weaver, Holly M.; Brown, Joseph N.; Koech, Phillip K.; Nicora, Carrie D.; Hofstad, Beth A.; Smith, Richard D.; Wilkins, Michael J.; Callister, Stephen J.; Wright, Aaron T.
      Journal of the American Chemical Society (2012), 134 (50), 20521-20532CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Microbial glycoside hydrolases play a dominant role in the biochem. conversion of cellulosic biomass to high-value biofuels. Anaerobic cellulolytic bacteria are capable of producing multicomplex catalytic subunits contg. cell-adherent cellulases, hemicellulases, xylanases, and other glycoside hydrolases to facilitate the degrdn. of highly recalcitrant cellulose and other related plant cell wall polysaccharides. Clostridium thermocellum is a cellulosome-producing bacterium that couples rapid reprodn. rates to highly efficient degrdn. of cryst. cellulose. Herein, we have developed and applied a suite of difluoromethylphenyl aglycon, N-halogenated glycosylamine, and 2-deoxy-2-fluoroglycoside activity-based protein profiling (ABPP) probes to the direct labeling of the C. thermocellum cellulosomal secretome. These activity-based probes (ABPs) were synthesized with alkynes to harness the utility and multimodal possibilities of click chem. and to increase enzyme active site inclusion for liq. chromatog.-mass spectrometry (LC-MS) anal. We directly analyzed ABP-labeled and unlabeled global MS data, revealing ABP selectivity for glycoside hydrolase (GH) enzymes, in addn. to a large collection of integral cellulosome-contg. proteins. By identifying reactivity and selectivity profiles for each ABP, we demonstrate our ability to widely profile the functional cellulose-degrading machinery of the bacterium. Derivatization of the ABPs, including reactive groups, acetylation of the glycoside binding groups, and mono- and disaccharide binding groups, resulted in considerable variability in protein labeling. Our probe suite is applicable to aerobic and anaerobic microbial cellulose-degrading systems and facilitates a greater understanding of the organismal role assocd. with biofuel development.
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    42. 42
      Street, I. P.; Kempton, J. B.; Withers, S. G. Inactivation of a b-glucosidase through the accumulation of a stable 2-deoxy-2-fluoro-a-D-glucopyranosyl-enzyme intermediate: a detailed investigation. Biochemistry 1992, 31, 9970– 9978,  DOI: 10.1021/bi00156a016
      There is no corresponding record for this reference.
    43. 43
      Hansen, F. G.; Bundgaard, E.; Madsen, R. A short synthesis of (+)-cyclophellitol. J. Org. Chem. 2005, 70, 10139– 10142,  DOI: 10.1021/jo051645q
      43
      A Short Synthesis of (+)-Cyclophellitol
      Hansen, Flemming Gundorph; Bundgaard, Eva; Madsen, Robert
      Journal of Organic Chemistry (2005), 70 (24), 10139-10142CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)
      A new synthesis of (+)-cyclophellitol, a potent β-glucosidase inhibitor, has been completed in nine steps from D-xylose. The key transformations involve a zinc-mediated fragmentation of benzyl-protected Me 5-deoxy-5-iodo-xylofuranoside followed by a highly diastereoselective indium-mediated coupling with Et 4-bromocrotonate. Subsequent ring-closing olefin metathesis, ester redn., olefin epoxidn., and deprotection then afford the natural product. This constitutes the shortest synthesis of (+)-cyclophellitol reported to date.
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    44. 44
      Witte, M. D.; Kallemeijn, W. W.; Aten, J.; Li, K.-Y.; Strijland, A.; Donker-Koopman, W. E.; Blijlevens, B.; Kramer, G.; van den Nieuwendijk, A. M. C. H.; Florea, B. I.; Hooibrink, B.; Hollak, C. E. M.; Ottenhoff, R.; Boot, R. G.; van der Marel, G. A.; Overkleeft, H. S.; Aerts, J. M. F. G. Ultrasensitive in situ visualization of active glucocerebrosidase molecules. Nat. Chem. Biol. 2010, 6, 907– 913,  DOI: 10.1038/nchembio.466
      44
      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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    45. 45
      Li, K.-Y.; Jiang, J.; Witte, M. D.; Kallemeijn, W. W.; van den Elst, H.; Wong, C.-S.; Chander, S. D.; Hoogendoorn, S.; Beenakker, T. J. M.; Codée, J. D. C.; Aerts, J. M. F. G.; van der Marel, G. A.; Overkleeft, H. S. Synthesis of cyclophellitol, cyclophellitol aziridine, and their tagged derivatives. Eur. J. Org. Chem. 2014, 2014, 6030– 6043,  DOI: 10.1002/ejoc.201402588
      There is no corresponding record for this reference.
    46. 46
      Goddard-Borger, E. D.; Wennekes, T.; Withers, S. G. Getting lucky in the lysosome. Nat. Chem. Biol. 2010, 6, 881– 883,  DOI: 10.1038/nchembio.470
      There is no corresponding record for this reference.
    47. 47
      Wennekes, T.; van den Berg, R. J. B. H. N.; Donker, W.; van der Marel, G. A.; Strijland, A.; Aerts, J. M. F. G.; Overkleeft, H. S. Development of adamantan-1-yl-methoxy-functionalized 1-deoxynojirimycin derivatives as selective inhibitors of glucosylceramide metabolism in man. J. Org. Chem. 2007, 72, 1088– 1097,  DOI: 10.1021/jo061280p
      47
      Development of Adamantan-1-yl-methoxy-Functionalized 1-Deoxynojirimycin Derivatives as Selective Inhibitors of Glucosylceramide Metabolism in Man
      Wennekes, Tom; Van den Berg, Richard J. B. H. N.; Donker, Wilma; van der Marel, Gijsbert A.; Strijland, Anneke; Aerts, Johannes M. F. G.; Overkleeft, Herman S.
      Journal of Organic Chemistry (2007), 72 (4), 1088-1097CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)
      The synthesis of adamantan-1-yl-methoxy-functionalized 1-deoxynojirimycin derivs., e.g. I, via reductive ring opening, Swern oxidn. and reductive amination, is reported. The compds. reported are lipophilic iminosugar based on lead compd. II, a potent inhibitor of the three enzymes involved in the metab. of the glycosphingolipid glucosylceramide. The results demonstrate that relocating the lipophilic moiety from the nitrogen atom to other positions on the 1-deoxynojirimycin ring system does not lead to a more potent or selective inhibitor of glucosylceramide synthase. The β-aza-C-glycoside analog I retained the best inhibitory potency for glucosylceramide synthase and is a more potent inhibitor than the therapeutic agent N-butyl-1-deoxynojirimycin, marketed as treatment for Gaucher disease under the com. name Zavesca.
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    48. 48
      Kallemeijn, W. W.; Li, K.-Y.; Witte, M. D.; Marques, A. R. A.; Aten, J.; Scheij, S.; Jiang, J.-B; 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. C.; Florea, B. I.; Codée, J. D. C.; van der Marel, G. A.; Aerts, J. M. F. G.; Overkleeft, H. S. Novel activity-based probes for broad-spectrum profiling of retaining beta-exoglucosidases in situ and in vivo. Angew. Chem., Int. Ed. 2012, 51, 12529– 12533,  DOI: 10.1002/anie.201207771
      48
      Novel Activity-Based Probes for Broad-Spectrum Profiling of Retaining β-Exoglucosidases In Situ and In Vivo
      Kallemeijn, Wouter W.; Li, Kah-Yee; Witte, Martin D.; Marques, Andre R. A.; Aten, Jan; Scheij, Saskia; Jiang, Jianbing; Willems, Lianne I.; Voorn-Brouwer, Tineke M.; van Roomen, Cindy P. A. A.; Ottenhoff, Roelof; Boot, Rolf G.; van den Elst, Hans; Walvoort, Marthe T. C.; Florea, Bogdan I.; Codee, Jeroen D. C.; van der Marel, Gijsbert A.; Aerts, Johannes M. F. G.; Overkleeft, Herman S.
      Angewandte Chemie, International Edition (2012), 51 (50), 12529-12533CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Cyclophellitol aziridine-type activity-based probes allow for ultra-sensitive visualization of mammalian β-glucosidases (GBA1, GBA2, GBA3, and LPH) as well as several non-mammalian β-glucosidases. These probes offer new ways to study β-exoglucosidases, and configurational isomers of the cyclophellitol aziridine core may give activity-based probes targeting other retaining glycosidase families.
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    49. 49
      Walvoort, M. T. C.; Kallemeijn, W. W.; Willems, L. I.; Witte, M. D.; Aerts, J. M. F. G.; van der Marel, G. A.; Codée, J. D. C.; Overkleeft, H. S. Tuning the leaving group in 2-deoxy-2-fluoroglucoside results in improved activity-based retaining beta-glucosidase probes. Chem. Commun. 2012, 48, 10386– 10388,  DOI: 10.1039/c2cc35653h
      There is no corresponding record for this reference.
    50. 50
      Yu, B.; Tao, H. Glycosyl trifluoroacetimidates. Part 1: preparation and application as new glycosyl donors. Tetrahedron Lett. 2001, 42, 2405– 2407,  DOI: 10.1016/S0040-4039(01)00157-5
      50
      Glycosyl trifluoroacetimidates. Part 1: Preparation and application as new glycosyl donors
      Yu, B.; Tao, H.
      Tetrahedron Letters (2001), 42 (12), 2405-2407CODEN: TELEAY; ISSN:0040-4039. (Elsevier Science Ltd.)
      Glycosyl (N-phenyl)trifluoroacetimidates, readily prepd. from 1-hydroxyl sugars by treatment with (N-phenyl)trifluoroacetimidoyl chloride in the presence of K2CO3, were demonstrated to be effective glycosyl donors. Glycosidation of these glycosyl fluoroacetimidates with alcs. is also reported.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXhslOgs7w%253D&md5=187fb1007ca9f8cf9dcbc2aa4ffe0fbe
    51. 51
      Witte, M. D.; Walvoort, M. T. C.; Li, K.-Y.; Kallemeijn, W. W.; Donker-Koopman, W. E.; Boot, R. G.; Aerts, J. M. F. G.; Codée, J. D. C.; van der Marel, G. A.; Overkleeft, H. S. Activity-based profiling of retaining beta-glucosidases: a comparative study. ChemBioChem 2011, 12, 1263– 1269,  DOI: 10.1002/cbic.201000773
      There is no corresponding record for this reference.
    52. 52
      Harrak, Y.; Barra, C. M.; Delgado, A.; Castaño, A. R.; Llebaria, A. Galacto-configured aminocyclitol phytoceramides are potent in vivo invariant natural killer T cell stimulators. J. Am. Chem. Soc. 2011, 133, 12079– 12084,  DOI: 10.1021/ja202610x
      52
      Galacto-Configured Aminocyclitol Phytoceramides Are Potent in Vivo Invariant Natural Killer T Cell Stimulators
      Harrak, Youssef; Barra, Carolina M.; Delgado, Antonio; Castano, A. Raul; Llebaria, Amadeu
      Journal of the American Chemical Society (2011), 133 (31), 12079-12084CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      A new class of α-galactosylceramide (αGC) non-glycosidic analogs bearing galacto-configured aminocyclitols as sugar surrogates, e.g. I (R = H, OH), have been obtained. The aminocyclohexane having a hydroxyl substitution pattern similar to an α-galactoside is efficiently obtained by a sequence involving Evans aldol reaction and ring-closing metathesis with a Grubbs catalyst to give a key intermediate cyclohexene, which has been converted in galacto-aminocyclohexanes that are linked through a secondary amine to a phyto-ceramide lipid having a cerotyl N-acyl group. Natural Killer T (NKT) cellular assays have resulted in the identification of an active compd., HS161, which has been found to promote NKT cell expansion in vitro in a similar fashion but more weakly than αGC. This compd. stimulates the release of Interferon-γ (IFNγ) and Interleukin-4 (IL-4) in iNKT cell culture but with lower potency than αGC. The activation of Invariant Natural Killer T (iNKT) cells by this compd. has been confirmed in flow cytometry expts. Remarkably, when tested in mice, HS161 selectively induces a very strong prodn. of IFN-γ indicative of a potent Th1 cytokine profile. Overall, these data confirm the agonist activity of αGC lipid analogs having charged amino-substituted polar heads and their capacity to modulate the response arising from iNKT cell activation in vivo.
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    53. 53
      Alcaide, A.; Trapero, A.; Pérez, Y.; Llebaria, A. Galacto configured N-aminoaziridines: a new type of irreversible inhibitor of b-galactosidases. Org. Biomol. Chem. 2015, 13, 5690– 5697,  DOI: 10.1039/C5OB00532A
      There is no corresponding record for this reference.
    54. 54
      Ofman, T. P.; Küllmer, F.; van der Marel, G. A.; Codée, J. D. C.; Overkleeft, H. S. An orthogonally protected cyclitol for the construction of nigerose- and dextran-mimetic cyclophellitols. Org. Lett. 2021, 23, 9516,  DOI: 10.1021/acs.orglett.1c03723
      There is no corresponding record for this reference.
    55. 55
      Jiang, J.; Kuo, C.-L.; Wu, L.; Franke, C.; Kallemeijn, W. W.; Florea, B. I.; van Meel, E.; van der Marel, G. A.; Codée, J. D. C.; Boot, R. G.; Davies, G. J.; Overkleeft, H. S.; Aerts, J. M. F. G. Detection of active mammalian GH31 alpha-glucosidases in health and disease using in-class, broad-spectrum activity-based probes. ACS Cent. Sci. 2016, 2, 351– 358,  DOI: 10.1021/acscentsci.6b00057
      There is no corresponding record for this reference.
    56. 56
      Armstrong, Z.; Kuo, C.-L.; Lahav, D.; Liu, B.; Johnson, R.; Beenakker, T. J. M.; de Boer, C.; Wong, C.-S.; van Rijssel, E. R.; Debets, M. F.; Florea, B. I.; Hissink, C.; Boot, R. G.; Geurink, P. P.; Ovaa, H.; van der Stelt, M.; van der Marel, G. A.; Codée, J. D. C.; Aerts, J. M. F. G.; Wu, L.; Overkleeft, H. S.; Davies, G. J. Manno-epi-cyclophellitols enable activity-based protein profiling of human α-mannosidases and discovery of new Golgi mannosidase II inhibitors. J. Am. Chem. Soc. 2020, 142, 13021– 13029,  DOI: 10.1021/jacs.0c03880
      56
      Manno-epi-cyclophellitols EnableActivity-Based Protein Profiling of Human α-Mannosidasesand Discovery of New Golgi Mannosidase II Inhibitors
      Armstrong, Zachary; Kuo, Chi-Lin; Lahav, Daniel; Liu, Bing; Johnson, Rachel; Beenakker, Thomas J. M.; de Boer, Casper; Wong, Chung-Sing; van Rijssel, Erwin R.; Debets, Marjoke F.; Florea, Bogdan I.; Hissink, Colin; Boot, Rolf G.; Geurink, Paul P.; Ovaa, Huib; van der Stelt, Mario; van der Marel, Gijsbert M.; Codee, Jeroen D. C.; Aerts, Johannes M. F. G.; Wu, Liang; Overkleeft, Herman S.; Davies, Gideon J.
      Journal of the American Chemical Society (2020), 142 (30), 13021-13029CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Golgi mannosidase II (GMII) catalyzes the sequential hydrolysisof two mannosyl residues from GlcNAcMan5GlcNAc2 to produce GlcNAcMan3GlcNAc2, the precursor for all complex N-glycans, including the branched N-glycans assocd. with cancer. Inhibitors of GMII are potential cancer therapeutics, but their usefulness is limited by off-target effects, which produce α-mannosidosis-like symptoms.Despite many structural and mechanistic studies of GMII, we still lack a potent and selective inhibitor of this enzyme. Here, we synthesized manno-epi-cyclophellitol epoxide and aziridines and demonstrate their covalent modification and time-dependent inhibition of GMII. Application of fluorescent manno-epi-cyclophellitolaziridine derivs. enabled activity-based protein profiling ofα-mannosidases from both human cell lysate and mouse tissue exts. Synthesized probes also facilitated a fluorescence polarization-based screen for dGMII inhibitors. We identified seven previously unknown inhibitors of GMII from a library of over 350 iminosugars and investigated their binding modalities through X-ray crystallog. Our results reveal previously unobserved inhibitor binding modes and promising scaffolds for the generation of selective GMII inhibitors.
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    57. 57
      McGregor, N. G. S.; Kuo, C.-L.; Beenakker, T. J. M.; Wong, C.-S.; Armstrong, Z.; Florea, B. I.; Codée, J. D. C.; Overkleeft, H. S.; Aerts, J. M. F. G.; Davies, G. J. Synthesis of broad-specificity activity-based probes for exo-β-mannosidases. Org. Biomol. Chem. 2022, 20, 877– 886,  DOI: 10.1039/D1OB02287C
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      Synthesis of broad-specificity activity-based probes for exo-β-mannosidases
      McGregor, Nicholas G. S.; Kuo, Chi-Lin; Beenakker, Thomas J. M.; Wong, Chun-Sing; Offen, Wendy A.; Armstrong, Zachary; Florea, Bogdan I.; Codee, Jeroen D. C.; Overkleeft, Herman S.; Aerts, Johannes M. F. G.; Davies, Gideon J.
      Organic & Biomolecular Chemistry (2022), 20 (4), 877-886CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
      Exo-β-mannosidases are a broad class of stereochem. retaining hydrolases that are essential for the breakdown of complex carbohydrate substrates found in all kingdoms of life. Yet the detection of exo-β-mannosidases in complex biol. samples remains challenging, necessitating the development of new methodologies. Cyclophellitol and its analogs selectively label the catalytic nucleophiles of retaining glycoside hydrolases, making them valuable tool compds. Furthermore, cyclophellitol can be readily redesigned to enable the incorporation of a detection tag, generating activity-based probes (ABPs) that can be used to detect and identify specific glycosidases in complex biol. samples. Towards the development of ABPs for exo-β-mannosidases, we present a concise synthesis of β-manno-configured cyclophellitol, cyclophellitol aziridine, and N-alkyl cyclophellitol aziridines. We show that these probes covalently label exo-β-mannosidases from GH families 2, 5, and 164. Structural studies of the resulting complexes support a canonical mechanism-based mode of action in which the active site nucleophile attacks the pseudoanomeric center to form a stable ester linkage, mimicking the glycosyl enzyme intermediate. Furthermore, we demonstrate activity-based protein profiling using an N-alkyl aziridine deriv. by specifically labeling MANBA in mouse kidney tissue. Together, these results show that synthetic manno-configured cyclophellitol analogs hold promise for detecting exo-β-mannosidases in biol. and biomedical research.
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    58. 58
      Jiang, J.; Kallemeijn, W. W.; Wright, D. W.; van den Nieuwendijk, A. M. C. H.; Coco Rohde, V.; Folch, E. C.; van den Elst, H.; Florea, B. I.; Scheij, S.; Donker-Koopman, W. E.; Verhoek, M.; Li, N.; Schürmann, M.; Mink, D.; Boot, R. G.; Codée, J. D. C.; van der Marel, G. A.; Davies, G. J.; Aerts, J. M. F. G.; Overkleeft, H. S. In vitro and in vivo comparative and competitive activity-based protein profiling of GH29 alpha-L-fucosidases. Chem. Sci. 2015, 6, 2782– 2789,  DOI: 10.1039/C4SC03739A
      58
      In vitro and in vivo comparative and competitive activity-based protein profiling of GH29 α-L-fucosidases
      Jiang, Jianbing; Kallemeijn, Wouter W.; Wright, Daniel W.; van den Nieuwendijk, Adrianus M. C. H.; Rohde, Veronica Coco; Folch, Elisa Colomina; van den Elst, Hans; Florea, Bogdan I.; Scheij, Saskia; Donker-Koopman, Wilma E.; Verhoek, Marri; Li, Nan; Schuermann, Martin; Mink, Daniel; Boot, Rolf G.; Codee, Jeroen D. C.; van der Marel, Gijsbert A.; Davies, Gideon J.; Aerts, Johannes M. F. G.; Overkleeft, Herman S.
      Chemical Science (2015), 6 (5), 2782-2789CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
      GH29 α-L-fucosidases catalyze the hydrolysis of α-L-fucosidic linkages. Deficiency in human lysosomal α-L-fucosidase (FUCA1) leads to the recessively inherited disorder, fucosidosis. Herein, we describe the development of fucopyranose-configured cyclophellitol aziridines as activity-based probes (ABPs) for selective in vitro and in vivo labeling of GH29 α-L-fucosidases from bacteria, mice and man. Crystallog. anal. on bacterial α-L-fucosidase confirms that the ABPs act by covalent modification of the active site nucleophile. Competitive activity-based protein profiling identified L-fuconojirimycin as the single GH29 α-L-fucosidase inhibitor from eight configurational isomers.
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    59. 59
      Willems, L. I.; Beenakker, T. J. M.; Murray, B.; Scheij, S.; Kallemeijn, W. W.; Boot, R. G.; Verhoek, M.; Donker-Koopman, W. E.; Ferraz, M. J.; van Rijssel, E. R.; Florea, B. I.; Codée, J. D. C.; van der Marel, G. A.; Aerts, J. M. F. G.; Overkleeft, H. S. Potent and selective activity-based probes for GH27 human retaining alpha-galactosidases. J. Am. Chem. Soc. 2014, 136, 11622– 11625,  DOI: 10.1021/ja507040n
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      Potent and Selective Activity-Based Probes for GH27 Human Retaining α-Galactosidases
      Willems, Lianne I.; Beenakker, Thomas J. M.; Murray, Benjamin; Scheij, Saskia; Kallemeijn, Wouter W.; Boot, Rolf G.; Verhoek, Marri; Donker-Koopman, Wilma E.; Ferraz, Maria J.; van Rijssel, Erwin R.; Florea, Bogdan I.; Codee, Jeroen D. C.; van der Marel, Gijsbert A.; Aerts, Johannes M. F. G.; Overkleeft, Herman S.
      Journal of the American Chemical Society (2014), 136 (33), 11622-11625CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Lysosomal degrdn. of glycosphingolipids is mediated by the consecutive action of several glycosidases. Malfunctioning of one of these hydrolases can lead to a lysosomal storage disorder such as Fabry disease, which is caused by a deficiency in α-galactosidase A. Herein we describe the development of potent and selective activity-based probes that target retaining α-galactosidases. The fluorescently labeled aziridine-based probes inhibit the two human retaining α-galactosidases αGal A and αGal B covalently and with high affinity. Moreover, they enable the visualization of the endogenous activity of both α-galactosidases in cell exts., thereby providing a means to study the presence and location of active enzyme levels in different cell types, such as healthy cells vs. those derived from Fabry patients.
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    60. 60
      Kuo, C.-L.; Su, Q.; van den Nieuwendijk, A. M. C. H.; Beenakker, T. J. M.; Offen, W. A.; Willems, L. I.; Boot, R. G.; Sarris, A. J.; Marques, A. R. A.; Codée, J. D. C.; van der Marel, G. A.; Florea, B. I.; Davies, G. J.; Overkleeft, H. S.; Aerts, J. M. F. G. The development of a broad-spectrum retaining beta-exogalactosidase activity-based probe. Org. Biomol. Chem. 2023, 21, 7813– 7820,  DOI: 10.1039/D3OB01261A
      There is no corresponding record for this reference.
    61. 61
      Artola, M.; Kuo, C.-L.; McMahon, S. A.; Hansen, T.; van der Lienden, M.; He, X.; van den Elst, H.; Florea, B. I.; Kermode, A. R.; van der Marel, G. A.; Gloster, T.; Codée, J. D. C.; Overkleeft, H. S.; Aerts, J. M. F. G. New irreversible α-L-iduronidase inhibitors and activity-based probes. Chem. Eur. J. 2018, 24, 19081– 19088,  DOI: 10.1002/chem.201804662
      There is no corresponding record for this reference.
    62. 62
      Wu, L.; Jiang, J.; Jin, Y.; Kallemeijn, W. W.; Kuo, C.-L.; Artola, M.; Dai, W.; van Elk, C.; van Eijk, M.; van der Marel, G. A.; Codée, J. D. C.; Florea, B. I.; Aerts, J. M. F. G.; Overkleeft, H. S.; Davies, G. J. Activity-based probes for functional interrogation of retaining beta-glucuronidases. Nat. Chem. Biol. 2017, 13, 867– 873,  DOI: 10.1038/nchembio.2395
      There is no corresponding record for this reference.
    63. 63
      Biarnés, X.; Ardèvol, A.; Planas, A.; Rovira, C.; Laio, A.; Parrinello, M. The conformational free energy landscape of b-D-glucopyranose. Implications for substrate preactivation in b-glucoside hydrolysis. J. Am. Chem. Soc. 2007, 129, 10686– 10693,  DOI: 10.1021/ja068411o
      63
      The Conformational Free Energy Landscape of β-D-Glucopyranose. Implications for Substrate Preactivation in β-Glucoside Hydrolases
      Biarnes, Xevi; Ardevol, Albert; Planas, Antoni; Rovira, Carme; Laio, Alessandro; Parrinello, Michele
      Journal of the American Chemical Society (2007), 129 (35), 10686-10693CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Using ab initio metadynamics the conformational free energy landscape of β-D-glucopyranose as a function of the puckering coordinates has been computed. It is shown that the correspondence between the free energy and the Stoddard's pseudorotational itinerary for the system is rather poor. The no. of free energy min. (9) is smaller than the no. of ideal structures (13). Moreover, only six min. correspond to a canonical conformation. The structural features, the electronic properties, and the relative stability of the predicted conformers permit the rationalization of the occurrence of distorted sugar conformations in all the available X-ray structures of β-glucoside hydrolase Michaelis complexes. It is shown that these enzymes recognize the most stable distorted conformers of the isolated substrate and at the same time the ones better prepd. for catalysis in terms of bond elongation/shrinking and charge distribution. This suggests that the factors governing the distortions present in these complexes are largely dictated by the intrinsic properties of a single glucose unit.
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    64. 64
      Davies, G. J.; Planas, R.; Rovira, C. Conformational analysis of the reaction coordinate of glycosidases. Acc. Chem. Res. 2012, 45, 308– 316,  DOI: 10.1021/ar2001765
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      Conformational Analyses of the Reaction Coordinate of Glycosidases
      Davies, Gideon J.; Planas, Antoni; Rovira, Carme
      Accounts of Chemical Research (2012), 45 (2), 308-316CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
      A review. The enzymic hydrolysis of the glycosidic bond is catalyzed by diverse enzymes generically termed glycoside hydrolases (hereafter GHs) or glycosidases. The many sequence-based families of glycosidases have served as a rich hunting ground for enzymologists for years. Not only are these enzymes of fundamental interest, providing paradigms for enzymic catalysis that extend beyond the bounds of carbohydrate chem., but the enzymes themselves play myriad essential roles in diverse biol. processes. The wide utility of glycosidases, from their industrial harnessing in the hydrolysis of plant biomass to their roles in human physiol. and disease, has engendered a large scientific constituency with an interest in glycosidase chem. A fascinating thread of this research, and one with major impact on the design of enzyme inhibitors, is the conformational anal. of reaction pathways within the diverse families. These GH families provide a large pallet of enzymes with which chemists have attempted to depict the conformational landscape of glycosidase action. In this Account, we review three-dimensional insight into the conformational changes directed by glycosidases, primarily from structural observations of the stable enzyme-ligand species adjacent to the transition state (or states) and of enzyme-inhibitor complexes. We further show how recent computational advances dovetail with structural insight to provide a quantum mech. basis for glycosidase action. The glycosidase-mediated hydrolysis of the acetal or ketal bond in a glycoside may occur with either inversion or retention of the configuration of the anomeric carbon. Inversion involves a single step and transition state, whereas retention, often referred to as the double displacement, is a two-step process with two transition states. The single transition state for the inverting enzymes and the two transition states (those flanking the covalent intermediate) in the double displacement have been shown to have substantial oxocarbenium ion character. The dissociative nature of these transition states results in significant relative pos. charge accumulation on the pyranose ring. The delocalization of lone-pair electrons from the ring oxygen that stabilizes the cationic transition state implies that at, or close to, the transition states the pyranose will be distorted away from its lowest energy conformation to one that favors orbital overlap. Over the preceding decade, research has highlighted the harnessing of noncovalent interactions to aid this distortion of the sugar substrates from their lowest energy chair conformation to a variety of different boat, skew boat, and half-chair forms, each of which favors catalysis with a given enzyme and substrate. Crystallog. observation of stable species that flank the transition state (or states), of both retaining and inverting glycosidases, has allowed a description of their conformational itineraries, illustrating how enzymes facilitate the "electrophilic migration" of the anomeric center along the reaction coordinate. The blossom of computational approaches, such as ab initio metadynamics, has underscored the quantum mech. basis for glycoside hydrolysis. Conformational analyses highlight not only the itineraries used by enzymes, enabling their inhibition, but are also reflected in the nonenzymic synthesis of glycosides, wherein chemists mimic strategies found in nature.
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    65. 65
      Vasella, A.; Davies, G. J.; Böhm, M. Glycosidase mechanisms. Curr. Opin. Chem. Biol. 2002, 6, 619– 629,  DOI: 10.1016/S1367-5931(02)00380-0
      65
      Glycosidase mechanisms
      Vasella, Andrea; Davies, Gideon J.; Boehm, Matthias
      Current Opinion in Chemical Biology (2002), 6 (5), 619-629CODEN: COCBF4; ISSN:1367-5931. (Elsevier Science Ltd.)
      A review with 99 refs. The 3-dimensional structure of glycosidases and of their complexes and the study of transition-state mimics reveal structural details that correlate with their catalytic mechanism. Of particular interest are the transition-state conformations harnessed by individual enzymes and the substrate distortion obsd. in enzyme-ligand complexes. The 3-dimensional structure in synergy with transition-state mimicry opens the way for mechanistic interpretation of enzyme inhibition and for the development of therapeutic agents.
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    66. 66
      Beenakker, T. J. M.; Wander, D. P. A.; Offen, W. A.; Artola, M.; Raich, L.; Ferraz, M. J.; Li, K.-Y.; Houben, J. H. P. M.; van Rijssel, E. R.; Hansen, T.; van der Marel, G. A.; Codée, J. D. C.; Aerts, J. M. F. G.; Rovira, C.; Davies, G. J.; Overkleeft, H. S. Carba-cyclophellitols are neutral retaining-glucosidase inhibitors. J. Am. Chem. Soc. 2017, 139, 6534– 6537,  DOI: 10.1021/jacs.7b01773
      66
      Carba-cyclophellitols are neutral retaining-glucosidase inhibitors
      Beenakker, Thomas J. M.; Wander, Dennis P. A.; Offen, Wendy A.; Artola, Marta; Raich, Lluis; Ferraz, Maria J.; Li, Kah-Yee; Houben, Judith H. P. M.; van Rijssel, Erwin R.; Hansen, Thomas; van der Marel, Gijsbert A.; Codee, Jeroen D. C.; Aerts, Johannes M. F. G.; Rovira, Carme; Davies, Gideon J.; Overkleeft, Herman S.
      Journal of the American Chemical Society (2017), 139 (19), 6534-6537CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The conformational anal. of glycosidases affords a route to their specific inhibition through transition-state mimicry. Inspired by the rapid reaction rates of cyclophellitol and cyclophellitol aziridine, both covalent retaining-β-glucosidase inhibitors, we postulated that the corresponding carba "cyclopropyl" analog would be a potent retaining β-glucosidase inhibitor for those enzymes reacting through the 4H3 transition-state conformation. Ab initio metadynamics simulations of the conformational free energy landscape for the cyclopropyl inhibitors showed a strong bias for the 4H3 conformation, and carba-cyclophellitol, with an N-(4-azidobutyl)carboxamide moiety, proved to be a potent inhibitor (Ki = 8.2 nM) of Thermotoga maritima (TmGH1) β-glucosidase. Three-dimensional structural anal. and comparison with unreacted epoxides showed that this compd. indeed bound in the 4H3 conformation, suggesting that conformational strain induced through a cyclopropyl unit may add to the armory of tight-binding inhibitor designs.
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    67. 67
      de Boer, C.; Armstrong, Z.; Lit, V. A. J.; Barash, U.; Ruijgrok, G.; Boyango, I.; Weitzenberg, M. M.; Schröder, S. P.; Sarris, A. J. C.; Meeuwenoord, N. J.; Bule, P.; Kayal, Y.; Ilan, N.; Codée, J. D. C.; Vlodavsky, I.; Overkleeft, H. S.; Davies, G. J.; Wu, L. Mechanism based heparanase inhibitors reduce cancer metastasis in vivo. Proc. Natl. Acad. Sci. USA 2022, 119, e2203167119  DOI: 10.1073/pnas.2203167119
      There is no corresponding record for this reference.
    68. 68
      Artola, M.; Wu, L.; Ferraz, M. J.; Kuo, C.-L.; Raich, L.; Breen, I. Z.; Offen, W. A.; Codée, J. D. C.; van der Marel, G. A.; Rovira, C.; Aerts, J. M. F. G.; Davies, G. J.; Overkleeft, H. S. 1,6-Cyclophellitol cyclosulfates: a new class of irreversible glycosidase inhibitor. ACS Cent. Sci. 2017, 3, 784– 793,  DOI: 10.1021/acscentsci.7b00214
      68
      1,6-Cyclophellitol cyclosulfates: A new class of irreversible glycosidase inhibitor
      Artola, Marta; Wu, Liang; Ferraz, Maria J.; Kuo, Chi-Lin; Raich, Lluis; Breen, Imogen Z.; Offen, Wendy A.; Codee, Jeroen D. C.; van der Marel, Gijsbert A.; Rovira, Carme; Aerts, Johannes M. F. G.; Davies, Gideon J.; Overkleeft, Herman S.
      ACS Central Science (2017), 3 (7), 784-793CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)
      The essential biol. roles played by glycosidases, coupled with the diverse therapeutic benefits of pharmacol. targeting of these enzymes, provide considerable motivation for the development of new inhibitor classes. Cyclophellitol epoxides and aziridines are recently established covalent glycosidase inactivators. Inspired by the application of cyclic sulfates as electrophilic equivs. of epoxides in org. synthesis, we sought to test whether cyclophellitol cyclosulfates would similarly act as irreversible glycosidase inhibitors. Here, we present the synthesis, conformational anal., and application of novel 1,6-cyclophellitol cyclosulfates. We showed that 1,6-epi-cyclophellitol cyclosulfate (α-cyclosulfate) is a rapidly reacting α-glucosidase inhibitor whose 4C1 chair conformation matched that adopted by α-glucosidase Michaelis complexes. The 1,6-cyclophellitol cyclosulfate (β-cyclosulfate) reacted more slowly, likely reflecting its conformational restrictions. Selective glycosidase inhibitors are invaluable as mechanistic probes and therapeutic agents, and we propose cyclophellitol cyclosulfates as a valuable new class of carbohydrate mimetics for application in these directions.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtFGltrfK&md5=b8aaf5f955c0d78618aded623375f92e
    69. 69
      Li, Z.; Pickles, I. B.; Sharma, M.; Melling, B.; Pallasdies, L.; Codée, J. D. C.; Williams, S. J.; Overkleeft, H. S.; Davies, G. J. Detection of sulfoquinovosidase activity in cell lysates using activity-based probes. Angew. Chem., Int. Ed. 2024, 63, e202401358  DOI: 10.1002/anie.202401358
      There is no corresponding record for this reference.
    70. 70
      McGregor, N.; Artola, M.; Nin-Hill, A.; Linzel, D.; Haon, M.; Reijngoud, J.; Ram, A. F. J.; Rosso, M.-N.; van der Marel, G. A.; Codée, J. D. C.; van Wezel, G. P.; Berrin, J.-G.; Rovira, C.; Overkleeft, H. S.; Davies, G. J. Rational design of mechanism-based inhibitors and activity-based probes for the identification of retaining α-L-arabinofuranosidases. J. Am. Chem. Soc. 2020, 142, 4648– 4662,  DOI: 10.1021/jacs.9b11351
      70
      Rational Design of Mechanism-Based Inhibitors and Activity-Based Probes for the Identification of Retaining α-L-Arabinofuranosidases
      McGregor, Nicholas G. S.; Artola, Marta; Nin-Hill, Alba; Linzel, Daniel; Haon, Mireille; Reijngoud, Jos; Ram, Arthur; Rosso, Marie-Noelle; van der Marel, Gijsbert A.; Codee, Jeroen D. C.; van Wezel, Gilles P.; Berrin, Jean-Guy; Rovira, Carme; Overkleeft, Herman S.; Davies, Gideon J.
      Journal of the American Chemical Society (2020), 142 (10), 4648-4662CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Identifying and characterizing the enzymes responsible for an obsd. activity within a complex eukaryotic catabolic system remains one of the most significant challenges in the study of biomass-degrading systems. The debranching of both complex hemicellulosic and pectinaceous polysaccharides requires the prodn. of α-L-arabinofuranosidases among a wide variety of coexpressed carbohydrate-active enzymes. To selectively detect and identify α-L-arabinofuranosidases produced by fungi grown on complex biomass, potential covalent inhibitors and probes which mimic α-L-arabinofuranosides were sought. The conformational free energy landscapes of free α-L-arabinofuranose and several rationally designed covalent α-L-arabinofuranosidase inhibitors were analyzed. A synthetic route to these inhibitors was subsequently developed based on a key Wittig-Still rearrangement. Through a combination of kinetic measurements, intact mass spectrometry, and structural expts., the designed inhibitors were shown to efficiently label the catalytic nucleophiles of retaining GH51 and GH54 α-L-arabinofuranosidases. Activity-based probes elaborated from an inhibitor with an aziridine warhead were applied to the identification and characterization of α-L-arabinofuranosidases within the secretome of A. niger grown on arabinan. This method was extended to the detection and identification of α-L-arabinofuranosidases produced by eight biomass-degrading basidiomycete fungi grown on complex biomass. The broad applicability of the cyclophellitol-derived activity-based probes and inhibitors presented here make them a valuable new tool in the characterization of complex eukaryotic carbohydrate-degrading systems and in the high-throughput discovery of α-L-arabinofuranosidases.
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    71. 71
      Schröder, S. P.; de Boer, C.; McGregor, N. G. S.; Rowland, R. J.; Moroz, O.; Blagova, E.; Reijngoud, J.; Arentshorst, M.; Osborn, D.; Morant, M. D.; Abbate, E.; Stringer, M. A.; Krogh, K. B. R. M.; Raich, L.; Rovira, C.; Berrin, J.-G.; van Wezel, G. P.; Ram, A. F. J.; Florea, B. I.; van der Marel, G. A.; Codée, J. D. C.; Wilson, K. S.; Wu, L.; Davies, G. J.; Overkleeft, H. S. Dynamic and functional profiling of xylan-degrading enzymes in Aspergillus secretomes using activity-based probes. ACS Cent. Sci. 2019, 5, 1067– 1078,  DOI: 10.1021/acscentsci.9b00221
      71
      Dynamic and Functional Profiling of Xylan-Degrading Enzymes in Aspergillus Secretomes Using Activity-Based Probes
      Schroder Sybrin P; de Boer Casper; Florea Bogdan I; van der Marel Gijsbert A; Codee Jeroen D C; Overkleeft Herman S; McGregor Nicholas G S; Rowland Rhianna J; Moroz Olga; Blagova Elena; Wilson Keith S; Wu Liang; Davies Gideon J; Reijngoud Jos; Arentshorst Mark; van Wezel Gilles P; Ram Arthur F J; Osborn David; Abbate Eric; Morant Marc D; Stringer Mary A; Krogh Kristian B R M; Raich Lluis; Rovira Carme; Rovira Carme; Berrin Jean-Guy
      ACS central science (2019), 5 (6), 1067-1078 ISSN:2374-7943.
      Plant polysaccharides represent a virtually unlimited feedstock for the generation of biofuels and other commodities. However, the extraordinary recalcitrance of plant polysaccharides toward breakdown necessitates a continued search for enzymes that degrade these materials efficiently under defined conditions. Activity-based protein profiling provides a route for the functional discovery of such enzymes in complex mixtures and under industrially relevant conditions. Here, we show the detection and identification of β-xylosidases and endo-β-1,4-xylanases in the secretomes of Aspergillus niger, by the use of chemical probes inspired by the β-glucosidase inhibitor cyclophellitol. Furthermore, we demonstrate the use of these activity-based probes (ABPs) to assess enzyme-substrate specificities, thermal stabilities, and other biotechnologically relevant parameters. Our experiments highlight the utility of ABPs as promising tools for the discovery of relevant enzymes useful for biomass breakdown.
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    72. 72
      McGregor, N. G. S.; de Boer, C.; Foucart, Q. P. O.; Beenakker, T.; Offen, W. A.; Codée, J. D. C.; Willems, L. I.; Overkleeft, H. S.; Davies, G. J. A multiplexing activity-based protein profiling platform for dissection of a native bacterial xyloglucan-degrading system. ACS Cent. Sci. 2023, 9, 2306– 2314,  DOI: 10.1021/acscentsci.3c00831
      There is no corresponding record for this reference.
    73. 73
      van Meel, E.; Bos, E.; van den Lienden, M. J. C.; Overkleeft, H. S.; van Kasteren, S. I.; Koster, A. J.; Aerts, J. M. F. G. Localization of active endogenous and exogenous beta-glucocerebrosidase by correlative light-electron microscopy in human fibroblasts. Traffic 2019, 20, 346– 356,  DOI: 10.1111/tra.12641
      There is no corresponding record for this reference.
    74. 74
      Schröder, S. P.; Petracca, R.; Minnee, H.; Artola, M.; Aerts, J. M. F. G.; Codée, J. D. C.; van der Marel, G. A.; Overkleeft, H. S. A divergent synthesis of L-arabino and D-xylo-configured cyclophellitol epoxides and aziridines. Eur. J. Org. Chem. 2016, 2016, 4787– 4794,  DOI: 10.1002/ejoc.201600983
      There is no corresponding record for this reference.
    75. 75
      Jariwala, P. B.; Pellock, S. J.; Goldfarb, D.; Cloer, E. C.; Artola, M.; Simpson, J. B.; Bhatt, A. P.; Walton, W. G.; Roberts, L. R.; Major, M. B.; Davies, G. J.; Overkleeft, H. S.; Redinbo, M. R. Discovering the microbial enzymes driving drug toxicity with activity-based protein profiling. ACS Chem. Biol. 2020, 15, 217– 225,  DOI: 10.1021/acschembio.9b00788
      75
      Discovering the Microbial Enzymes Driving Drug Toxicity with Activity-Based Protein Profiling
      Jariwala, Parth B.; Pellock, Samuel J.; Goldfarb, Dennis; Cloer, Erica W.; Artola, Marta; Simpson, Joshua B.; Bhatt, Aadra P.; Walton, William G.; Roberts, Lee R.; Major, Michael B.; Davies, Gideon J.; Overkleeft, Herman S.; Redinbo, Matthew R.
      ACS Chemical Biology (2020), 15 (1), 217-225CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
      It is increasingly clear that interindividual variability in human gut microbial compn. contributes to differential drug responses. For example, gastrointestinal (GI) toxicity is not obsd. in all patients treated with the anticancer drug irinotecan, and it has been suggested that this variability is a result of differences in the types and levels of gut bacterial β-glucuronidases (GUSs). GUS enzymes promote drug toxicity by hydrolyzing the inactive drug-glucuronide conjugate back to the active drug, which damages the GI epithelium. Proteomics-based identification of the exact GUS enzymes responsible for drug reactivation from the complexity of the human microbiota has not been accomplished, however. Here, we discover the specific bacterial GUS enzymes that generate SN-38, the active and toxic metabolite of irinotecan, from human fecal samples using a unique activity-based protein profiling (ABPP) platform. We identify and quantify gut bacterial GUS enzymes from human feces with an ABPP-enabled proteomics pipeline and then integrate this information with ex vivo kinetics to pinpoint the specific GUS enzymes responsible for SN-38 reactivation. Furthermore, the same approach also reveals the mol. basis for differential gut bacterial GUS inhibition obsd. between human fecal samples. Taken together, this work provides an unprecedented tech. and bioinformatics pipeline to discover the microbial enzymes responsible for specific reactions from the complexity of human feces. Identifying such microbial enzymes may lead to precision biomarkers and novel drug targets to advance the promise of personalized medicine.
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    76. 76
      Lipsh-Sokolik, R.; Khersonsky, O.; Schröder, S. P.; de Boer, C.; Hoch, S.-Y.; Davies, G. J.; Overkleeft, H. S.; Fleishman, S. J. Combinatorial assembly and design of enzymes. Science 2023, 379, 195– 201,  DOI: 10.1126/science.ade9434
      There is no corresponding record for this reference.
    77. 77
      Lahav, D.; Liu, B.; van den Berg, R. J. B. H. N.; van den Nieuwendijk, A. M. C. H.; Wennekes, T.; Ghisaidoobe, A. T.; Breen, I.; Ferraz, M. J.; Kuo, C.-L.; Wu, L.; Geurink, P. P.; Ovaa, H.; van der Marel, G. A.; van der Stelt, M.; Boot, R. G.; Davies, G. J.; Aerts, J. M. F. G.; Overkleeft, H. S. A fluorescence polarization activity-based protein profiling assay in the discovery of potent, selective inhibitors for human non-lysosomal glucosylceramidase. J. Am. Chem. Soc. 2017, 139, 14192– 14197,  DOI: 10.1021/jacs.7b07352
      77
      A fluorescence polarization activity-based protein profiling assay in the discovery of potent, selective inhibitors for human nonlysosomal glucosylceramidase
      Lahav, Daniel; Liu, Bing; van den Berg, Richard J. B. H. N.; van den Nieuwendijk, Adrianus M. C. H.; Wennekes, Tom; Ghisaidoobe, Amar T.; Breen, Imogen; Ferraz, Maria J.; Kuo, Chi-Lin; Wu, Liang; Geurink, Paul P.; Ovaa, Huib; van der Marel, Gijsbert A.; van der Stelt, Mario; Boot, Rolf G.; Davies, Gideon J.; Aerts, Johannes M. F. G.; Overkleeft, Herman S.
      Journal of the American Chemical Society (2017), 139 (40), 14192-14197CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Human nonlysosomal glucosylceramidase (GBA2) is one of several enzymes that controls levels of glycolipids and whose activity is linked to several human disease states. There is a major need to design or discover selective GBA2 inhibitors both as chem. tools and as potential therapeutic agents. Here, we describe the development of a fluorescence polarization activity-based protein profiling (FluoPol-ABPP) assay for the rapid identification, from a 350+ library of iminosugars, of GBA2 inhibitors. A focused library is generated based on leads from the FluoPol-ABPP screen and assessed on GBA2 selectivity offset against the other glucosylceramide metabolizing enzymes, glucosylceramide synthase (GCS), lysosomal glucosylceramidase (GBA), and the cytosolic retaining β-glucosidase, GBA3. Our work, yielding potent and selective GBA2 inhibitors, also provides a roadmap for the development of high-throughput assays for identifying retaining glycosidase inhibitors by FluoPol-ABPP on cell exts. contg. recombinant, overexpressed glycosidase as the easily accessible enzyme source.
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    78. 78
      Ndeh, D.; Rogowski, A.; Cartmell, A.; Luis, A. S.; Baslé, A.; Gray, J.; Venditto, I.; Briggs, J.; Zhang, X.; Labourel, A.; Terrapon, N.; Buffetto, F.; Nepogodiev, S.; Xiao, Y.; Field, R. A.; Zhu, Y.; O’Neil, M.; Urbanowicz, B. R.; York, W. S.; Davies, G. J.; Abbott, D. W.; Ralet, M.-C.; Martens, E. C.; Henrissat, B.; Gilbert, H. J. Complex pectin metabolism by gut bacteria reveals novel catalytic functions. Nature 2017, 544, 65– 70,  DOI: 10.1038/nature21725
      78
      Complex pectin metabolism by gut bacteria reveals novel catalytic functions
      Ndeh, Didier; Rogowski, Artur; Cartmell, Alan; Luis, Ana S.; Basle, Arnaud; Gray, Joseph; Venditto, Immacolata; Briggs, Jonathon; Zhang, Xiaoyang; Labourel, Aurore; Terrapon, Nicolas; Buffetto, Fanny; Nepogodiev, Sergey; Xiao, Yao; Field, Robert A.; Zhu, Yanping; O'Neill, Malcolm A.; Urbanowicz, Breeanna R.; York, William S.; Davies, Gideon J.; Abbott, D. Wade; Ralet, Marie-Christine; Martens, Eric C.; Henrissat, Bernard; Gilbert, Harry J.
      Nature (London, United Kingdom) (2017), 544 (7648), 65-70CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      The metab. of carbohydrate polymers drives microbial diversity in the human gut microbiota. It is unclear, however, whether bacterial consortia or single organisms are required to depolymerize highly complex glycans. Here we show that the gut bacterium Bacteroides thetaiotaomicron uses the most structurally complex glycan known: the plant pectic polysaccharide rhamnogalacturonan-II, cleaving all but 1 of its 21 distinct glycosidic linkages. The deconstruction of rhamnogalacturonan-II side chains and backbone are coordinated to overcome steric constraints, and the degrdn. involves previously undiscovered enzyme families and catalytic activities. The degrdn. system informs revision of the current structural model of rhamnogalacturonan-II and highlights how individual gut bacteria orchestrate manifold enzymes to metabolize the most challenging glycan in the human diet.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXltVWnurY%253D&md5=8a779b1beb1d7d394bcae22b635488cf
    79. 79
      Ren, W.; Pengelly, R.; Farren-Dai, M.; Shamsi Kazem Abadi, S.; Oehiler, V.; Akintola, O.; Draper, J.; Meanwell, M.; Chakladar, S.; Swiderek, K.; Moliner, V.; Britton, R.; Gloster, T. M.; Bennet, A. J. Revealing the mechanism for covalent inhibition of glycoside hydrolases by carbasugars at an atomic level. Nat. Commun. 2018, 9, 3243,  DOI: 10.1038/s41467-018-05702-7
      79
      Revealing the mechanism for covalent inhibition of glycoside hydrolases by carbasugars at an atomic level
      Ren Weiwu; Farren-Dai Marco; Akintola Oluwafemi; Draper Jason; Meanwell Michael; Chakladar Saswati; Britton Robert; Bennet Andrew J; Pengelly Robert; Oehler Verena; Gloster Tracey M; Shamsi Kazem Abadi Saeideh; Swiderek Katarzyna; Moliner Vicent
      Nature communications (2018), 9 (1), 3243 ISSN:.
      Mechanism-based glycoside hydrolase inhibitors are carbohydrate analogs that mimic the natural substrate's structure. Their covalent bond formation with the glycoside hydrolase makes these compounds excellent tools for chemical biology and potential drug candidates. Here we report the synthesis of cyclohexene-based α-galactopyranoside mimics and the kinetic and structural characterization of their inhibitory activity toward an α-galactosidase from Thermotoga maritima (TmGalA). By solving the structures of several enzyme-bound species during mechanism-based covalent inhibition of TmGalA, we show that the Michaelis complexes for intact inhibitor and product have half-chair ((2)H3) conformations for the cyclohexene fragment, while the covalently linked intermediate adopts a flattened half-chair ((2)H3) conformation. Hybrid QM/MM calculations confirm the structural and electronic properties of the enzyme-bound species and provide insight into key interactions in the enzyme-active site. These insights should stimulate the design of mechanism-based glycoside hydrolase inhibitors with tailored chemical properties.
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    80. 80
      Jain, N.; Tamura, K.; Déjean, G.; van Petegem, F.; Brumer, H. Orthogonal active-site labels for mixed-linkage endo-b-glucanases. ACS Chem. Biol. 2021, 16, 1968– 1984,  DOI: 10.1021/acschembio.1c00063
      There is no corresponding record for this reference.
    81. 81
      Schröder, S. P.; Kallemeijn, W. W.; Debets, M. F.; Hansen, T.; Sobala, L. F.; Hakki, Z.; Williams, S. J.; Beenakker, T. J. M.; Aerts, J. M. F. G.; van der Marel, G. A.; Codée, J. D. C.; Davies, G. J.; Overkleeft, H. S. Spiro-epoxyglycosides as activity-based probes for glycoside hydrolase family 99 endomannosidase/endomannanase. Chem. Eur. J. 2018, 24, 9983– 9992,  DOI: 10.1002/chem.201801902
      There is no corresponding record for this reference.
    82. 82
      Thaler, M.; Ofman, T. P.; Kok, K.; Heming, J. J. A.; Moran, E.; Pickles, I.; Leijs, A. A.; van den Nieuwendijk, A. M. C. H.; van den Berg, R. J. B. H. N.; Ruijgrok, G.; Armstrong, Z.; Salgado-Benvindo, C.; Ninaber, D. K.; Snijder, E. J.; van Boeckel, C. A. A.; Artola, A.; Davies, G. J.; Overkleeft, H. S.; van Hemert, M. J. Epi-cyclophellitol cyclosulfate, a mechanism-based ER α-glucosidase II inhibitor, blocks replication of SARS-CoV-2 and other coronaviruses. ACS Cent. Sci. 2024, 10, 1594,  DOI: 10.1021/acscentsci.4c00506
      There is no corresponding record for this reference.