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Activity-based probes for retaining β-glucosidases: Novel tools for research and diagnostics

Kallemeijn, W.W.

Publication date 2014

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Citation for published version (APA): Kallemeijn, W. W. (2014). Activity-based probes for retaining β-glucosidases: Novel tools for research and diagnostics.

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Download date:07 Oct 2021 Introduction, part I

Activity-based inhibitors of glycosidases: Design and applications

Wouter W. Kallemeijna, Martin D. Witteb, Tom Wennekesc, Johannes M. F. G. Aertsa and Hermen S. Overkleeftd

Prepared as invited review in Advances in Carbohydrate Chemistry and Biochemistry.

Graphical abstract – An activity-based probe can bind a target enzyme through mimicry of its substrate, form a covalent bond with the target via its catalytic mechanism, and allows detection of the bound enzyme by the implementation of a reporter tag.

9 Introduction, part I

Abstract This introductory chapter, to be published in revised form as invited review, covers recent developments in the design and application of activity-based probes (ABPs) for glycosidases, with emphasis on enzymes involved in glucosylceramide metabolism in humans. Described are the various catalytic reaction mechanisms employed by inverting and retaining glycosidases. Understanding of catalysis at the molecular level has stimulated the design of different types of ABPs for glycosidases. Such compounds range from oxocarbenium ion-like transition state-mimics tagged with reactive moieties, which associate with the target active-site – forming covalent bonds in a rather non-specific manner in or near the catalytic pocket – to probes that (partially) exploit the catalytic mechanism of target retaining glycosidases via the action of fluoroglycosides, epoxides and aziridines, which bind to the catalytic nucleophile. The design of highly specific ABPs for human retaining β-glucosidases, including the lysosomal glucocerebrosidase GBA, non- lysosomal β-glucosidase GBA2, cytosolic β-glucosidase GBA3 and lactase/phlorizin hydrolase LPH, is discussed. A broad spectrum of applications for the ABPs in research on various glycosidases is demonstrated, including specific quantitative visualization of active enzyme molecules in vitro and in vivo, and as tools to unambiguously identify catalytic residues in glycosidases in vitro, and rapid, specific introduction of glycosidase deficiencies in intact cells and animals.

Author affiliations aDepartment of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. bDepartment of Bio-organic Chemistry, Stratingh Institute for Chemistry, University of Groningen, Groningen, The Netherlands. cDepartment of Synthetic Organic Chemistry, Wageningen University, Wageningen, The Netherlands. dDepartment of Bio-organic Synthesis, Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands.

10 Activity-based probes for glycosidases

he human body contains a great diversity of glycoconjugates, located inside cells, their extracellular matrix and in bodily fluids. The essential building blocks of T glycoconjugates are carbohydrates, highly variable in composition, stereoisomerism and with the ability to connect to multiple moieties within a single molecule. These factors together result in an astounding level of combinatorial complexity: a simple hexasaccharide has at least 1012 possible isomers, far greater than achievable with amino acids or nucleotides1. Linkage of carbohydrates yields oligosaccharides, often named glycans. The number of distinct glycan structures in mammalian glycoconjugates is estimated to exceed 7,000+ structures, a diversity assembled from merely ten different carbohydrates: L-fucose, D-galactose, D-glucose, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, D-glucuronic acid, L-iduronic acid, D-mannose, N-acetyl-D-neuraminic acid and D-xylose (Fig 1)2−6. The generation of the full mammalian glycan structure diversity involves ~200 glycosyl- transferases2, using nucleotide− or lipid-linked sugars as activated donor substrates7. Vice versa, glycans are degraded via cleavage of their glycosidic bonds, a reaction catalyzed by specialized glycosylhydrolases (i.e. glycosidases)2. In total, 1−3% of the genes in the genome is believed to encode carbohydrate-modifying enzymes8, 9.

Glycosidases: proficient catalysts of glycoconjugate turnover − The glycosidic bond is very stable, far more than e.g. phosphodiester bonds in DNA and RNA, and peptide bonds in proteins10−13, glycosidases wield specialized active-sites to accelerate hydrolysis of the glycosidic bond. Glycosidase-catalyzed hydrolysis occurs up to 1017-fold faster than spontaneous auto-hydrolysis, calculated to occur once per ~5 million years, to mere microseconds14. To date, a plethora of glycosidases (> 6,300 individual entities)9, 15 from various organisms have been identified and categorized in 130+ glycosylhydrolase (GH) families, based on their homology in amino-acid sequence similarities, in the Carbohydrate Active enZYme database (CAZy)9. This classification system correlates with protein-fold and catalytic mechanism more than the enzymatic specificity towards substrates8, 16−18. Due to the conserved nature of glycosidases, the catalytic residues and overall mechanism of newly identified glycosidases can often be predicted8, 9, 16−18.

Figure 1 | Carbohydrates incorporated in mammalian glycoconjugates. The ten carbohydrate sugars required for synthesis of the full glycan diversity (estimated > 7,000 structures) in mammals, with α− and β-stereoisomer of C1-hydroxyl coordination depicted by a wavy bond (down or up, respectively).

11 Introduction, part I

Catalytic reaction mechanisms of glycosidases − Glycosidases hydrolyze the glycosidic bond between two moieties, via a reaction that results in inversion or retention of the anomeric stereochemistry, a mechanism first outlined by Daniel E. Koshland, Jr. in 195319. As shown in Fig 2a, inverting glycosidases employ two opposing carboxyl-exposing residues, typically spaced 6−11 Å apart on either side of the active site, to enable entry of both the substrate and a water molecule20−25. During the reaction, the carboxylate ion (i.e. the base) residue abstracts a proton from the incoming water molecule, thereby generating a nucleophilic hydroxyl, which in turn attacks the substrate’s anomeric C1 carbon20−25. Concomitantly, the opposing carboxylic acid protonates the inter-glycosidic oxygen, thereby assisting its departure from the anomeric center20−25. The reaction proceeds through a single oxocarbenium ion-like transition state, stabilized in part through hydrogen- bonding between the C2-hydroxyl and the carboxylate base (see Fig 2a, finely dotted line)20, 26−31. As the nucleophile can attack the C1 carbon from one side only, the oxocarbenium ion-like transition state causes the thereafter-formed hemi-acetal truncated sugar to exhibit inversed stereochemistry at the anomeric center19−31. In sharp contrast, retaining glycosidases employ a double-displacement mechanism proceeding by way of a glycosyl-enzyme intermediate, flanked by two oxocarbenium ion- like transition states, in order to preserve the initial anomeric oxygen configuration (see Fig 2b)19−31. This mechanism employs two adjacent carboxyl-exposing residues, spaced ~5.5 Å apart, with one functioning as the direct catalytic nucleophile and one as acid/base residue.

Figure 2 | Classic Koshland-type catalytic mechanisms of glycosidases. (a) Inverting β-glycosidase mechanism, with oxocarbenium ion-like transition state depicted between brackets (with asterisk; ❉). Transition state stabilized by hydrogen- bonding between substrate C2-hydroxyl and base residue (finely dotted line). (b) Catalytic mechanism of a retaining β- glycosidase, characterized by a covalent glycosyl-nucleophile adduct, flanked by two oxocarbenium ion-like transition states. Transition states stabilized by hydrogen-bonding between substrate C2-hydroxyl and nucleophile (finely dotted line).

12 Activity-based probes for glycosidases

Briefly, the deprotonated carboxylate ion of the nucleophile attacks the substrate’s anomeric C1 carbon, while the protonated carboxylate base of the acid/base donates a proton to the inter-glycosidic oxygen. Proceeding through an oxocarbenium ion-like transition state, the aglycone is expelled while concurrently a glycosyl-enzyme intermediate is formed with the nucleophile, exhibiting an inversed configuration at the anomeric center (Fig 2b)19−31. To liberate the nucleophile, the deprotonated carboxylate ion of the acid/base performs an inverting mechanism, characterized by the acid/base abstracting a proton from an incoming water molecule, forming a nucleophilic hydroxyl that attacks the anomeric center (C1) of the glycosyl-enzyme adduct, resulting in a second oxocarbenium ion-like transition state (Fig 2b). In turn, this reverses the changed anomeric configuration of the cleaved hemi-acetal to that present in the initial substrate19−31. Several variations on the classical Koshland-type mechanisms have been identified. Multiple glycosidases categorized in CAZy GH families 18, 20, 25, 56, 84 and 85 are able to hydrolyze the glycosidic bond at C1 of substrates containing an N-acetyl or N-glycolyl moiety at C2, through a mechanism involving neighboring group participation (Fig 3a, next page)25, 32, 33. For hydrolysis, a retaining N-acetyl-hexasominidase does not require a catalytic nucleophile: instead the C2-acetyl moiety executes the role as intra-molecular nucleophile. This leads to the formation of an oxazolinium intermediate, which is not covalently bound to the enzyme, as in retaining enzymes of the classic Koshland-type19, 34−38. Moreover, a carboxylate is generally present to stabilize the charge development during the transition state, thereby partially mimicking the role of the nucleophile in the stabilization of the oxocarbenium ion-like transition state through hydrogen-bonding (Fig 3a). The retaining mechanism identified in some sialidases (i.e. neuraminidases, in CAZy GH 33, 34) employs a tyrosine instead of a carboxylate as nucleophilic residue (Fig 3b)25, 39−41. As the anomeric center of the substrate is already negatively charged, the use of a similarly charged carboxylic acid would cause charge repulsion. A nearby base residue deprotonates the tyrosine to increase its nucleophilic character, thereby enabling the nucleophilic attack to the C1 carbon of sialic acid, forming a covalent glycosyl-enzyme intermediate with inversed anomeric C1 configuration. As the acid/base protonates the inter-glycosidic oxygen, it promotes deglycosylation of the nucleophile-adduct via generating a hydroxyl in a mechanism similar to inverting Koshland-type glycosidases39−41. Another catalytic mechanism has been described for α-glucan lyases (in GH 31), whom break the glycosidic bond through a two-step mechanism employing elimination (Fig 3c)25, 42. After a Koshland-type nucleophilic attack and concomitant protonation of the inter- glycosidic oxygen by the acid/base residue, a base residue in close proximity induces the hydrolysis of the formed glycosyl-enzyme intermediate. Withdrawal of the proton at C2 causes rearrangement inside the glycosyl-adduct, causing the formation of a 1,2-anhydro-D- fructose25, 42. Other unusual catalytic mechanisms include redox mechanisms, employing tightly- bound nicotinamide adenine dinucleotide (NAD+), which is found in several glycosidases

13 Introduction, part I

Figure 3 | Alternative glycosidase mechanisms. (a) Retaining mechanism of N-acetyl hexasominidase, involving neighboring group (C2-N-acetyl) participation of substrate during the double-displacement mechanism, with oxocarbenium ion-like transition states flanking the glycosyl-nucleophile adduct depicted between brackets (with asterisk; ❉). (b) Retaining mechanism of a sialidase, employing tyrosine residue as catalytic nucleophile, enhanced by a nearby nucleophile ʻboosterʼ. (c) Predicted catalytic mechanism of α-glucan lysases, driven by additional base residue (B). In all schemes, nucleophile (N), acid/base (A/B) and base (B) are abbreviated accordingly. categorized in CAZy families GH4 and 109, to facilitate glycoside cleavage via an oxidation, elimination, addition, reduction sequence42, and myrosinases (members of CAZy GH1), which utilize a exogenous base for hydrolysis25, 42, 43. These mechanisms will not be discussed in further detail in this review.

The concept of activity-based probes − Reducing the action of glycosidases through the use of inhibitory sugar-mimics44−47 has been used in research on (treatments for) cancer48−50, viral infections51−53, neurological disorders54−56, the metabolic syndrome57−59 and glycosphingolipid storage diseases60−63. Very potent (reversible) inhibitors for glycosidases can be obtained by the design of compounds that optimally mimic the oxocarbenium ion-like transition state of the target enzyme’s natural substrate during hydrolysis45, 46. They offer valuable research tools to study

14 Activity-based probes for glycosidases the physiological function of glycosidases. In addition, such type of reversible inhibitors may act as chemical chaperones promoting and/or stabilizing the correct folding of glycosidases64. The value of chemical chaperone therapy is presently investigated for patients suffering from lysosomal glycosidase deficiencies64−67. This review focuses on another class of inhibitors, which can assist in direct identification, e.g. through visualization, of enzyme molecules by specific covalent linkage in their catalytic pocket. These so-called activity-based probes (ABPs) enable the detection of the targeted glycosidase (or class of glycosidases) in complex mixtures, living cells and animals. In general, activity-based probes feature three structural elements, namely a warhead and a reporter moiety, linked together via a spacer (Fig 4a, left). Typically, the warhead comprises of a reactive group allowing the covalent attachment to the catalytic pocket of the enzyme. Employed for this are photo-crosslinkers and (latent) electrophiles, which bind to a(ny) nucleophile in the catalytic pocket. Grafting the warhead onto a natural substrate-mimicking molecular core exploits the exhibited specificity of, and affinity towards, the target enzyme’s active site. The composition of the reporter tag and spacer moiety bridging both subunits can further enhance ABP properties, including

Figure 4 | Concept of activity-based probes. (a) General activity-based probe (ABP) structure, depicted with reporter (lightbulb), spacer (curved line) and warhead (grey triangle) grafted onto a substrate mimick (white triangle), to gain affinity towards active-site of target enzymes. (b) ABP labeling of targeted enzyme in the complete proteome present in vitro, in intact cells or organisms in vivo, with various read-out possibilities. (c) Types of regularly employed reporter tags. From left to right: fluorescent moieties (BODIPY, fluorescein and rhodamine), affinity tag (biotin) and bio-orthogonal labeling handles (azide−, alkyne− and norbornene-moieties).

15 Introduction, part I augmentation of substrate mimicry and introduction of necessary spacing between the warhead-substrate core, tag and the target enzyme to minimize unfavorable interactions including steric hindrance. Optimally, exposure of the total proteome, either present in a lysate (in vitro), in living cells (in situ) or in intact laboratory animals (in vivo), results in the labeling of only the targeted enzyme or enzyme class (Fig 4b), where after the ABP- labeled proteins can be visualized through various techniques, including fluorescence scanning of SDS-PAGE gels, fluorescence microscopy, fluorescence-assisted cell sorting (FACS), positron emission tomography (PET), but labeling could also enable purification and subsequent MS/MS or NMR techniques. For detection of ABP-labeled enzyme, numerous reporter tags have been employed (Fig 4c)68, 69. Generally they include fluorescent moieties70 (BODIPY71−75, fluorescein70, 76−79, rhodamine80−82), radio-isotopes (3H, 19F, 125I)68, 69 and affinity tags such as biotin83−85. Since the inherent steric bulk of ABP reporter-tags may potentially sever recognition by the targeted enzyme(s), the covalent labeling mechanism or mitigate the cell-permeability of the ABP. An alternative strategy is to replace these reporters with markedly smaller bio- orthogonal ligation handles to enable two-step labeling strategies (Fig 4c)86−90. Bio-orthogonal ligation ideally occurs chemoselectively and without interference of the biological system. Several bio-orthogonal ligation strategies have been developed86−90, these include the Staudinger-Bertozzi ligation, copper(I)-catalyzed click reaction, strain- promoted click reaction and norbornene cycloaddition, which can be performed in the same sample (Fig 5)86−90.

Figure 5 | Bio-orthogonal labeling. Variants of azide with Staudinger-Bertozzi ligation (top), copper(I)-catalyzed click reaction (second) and strain-promoted click reaction (third), and tetrazine-norbornene cycloaddition (fourth). Presence of reporter tag, e.g. a fluorescent moiety (see also Fig 4c), is illustrated with a lightbulb.

16 Activity-based probes for glycosidases

Inhibitors of glycosidases, the road towards true activity-based probes − In recent years, different types of probes have been designed to label the catalytic pockets, or their close environment, in glycosidases. Some merely have affinity for the catalytic pocket and are subsequently covalently linked in rather non-specific manner, whilst true activity-based probes employ the catalytic mechanism of glycosidases for their linkage to the enzyme. A reversible inhibitor with sufficient affinity for the catalytic pocket of a target glycosidase may be transformed into a labeling probe by addition of a photo-activatable tag, e.g. a benzophenone, diazirine or aryl azide (Fig 6, next page)91−94. Optimally, photo-affinity tags are stable in the dark under various pH conditions, bear structural resemblance to the natural substrate of the target enzyme, are sterically non-congested, require a wavelength for photo-activation which does not cause undesired damage to other components in the biological sample, generate highly reactive, short-lived photo-intermediates upon irradiation and form a stable adduct with the target protein91−94.

Benzophenone-type photo-affinity labels generate upon irradiation (λEX 350−360 nm) triplet carbonyl states, which can react with inactive C−H bonds (Fig 6a, next page)91−94. While stable, the photo-activatable group is rather bulky which potentially hinders probe- recognition and ultimately also binding to the target protein91−94. However, a bulky tag can also fortuitously improve specificity as is noted for extension of 1-deoxynojirimycin with a benzophenone-type tag, yielding the potent probe 1 (Fig 6b) for labeling of human acid β- glucosidase (GBA) and non-lysosomal β-glucosidase GBA295. Compared to benzophenones, aryl azides are relatively small molecules that are readily synthesized, relatively stable and highly reactive upon photolysis (Fig 6c). Irradiation with wavelengths of λEX < 300 nm is required for photolysis, which can induce severe damage to biological systems such as cells. Photolysis generates a singlet nitrene, able to generate a triplet nitrene via intersystem crossing. Importantly, the singlet nitrene can rearrange into bicyclic benzazirine, which generates 1,2-azacycloheptatetraene, which in turn can bind with nucleophiles further away, thereby reducing labeling efficiency and specificity91, 96−98. Overall, aryl azides allow binding to nearest molecules through multiple reaction mechanisms. For instance, development of a photo-affinity probe based on 1- deoxynojirimycin equipped with an aryl azide (probe 2, see Fig 6d) resulted in the successful labeling of human α-glucosidase99. Diazirines are currently the smallest photo-activatable moieties employed for photo- affinity labeling (Fig 6e), and are relatively inert towards nucleophilic attacks, acidic− and alkaline conditions. Photolysis of diazirines requires irradiation with λEX 350−380 nm, which concomitantly generate a highly reactive species, e.g. a carbene, able to form a covalent bond with the closest target molecule via C−C, C−H, O−H or C/N−H insertion (Fig 6e)100. As carbenes are quickly quenched by water, the efficiency of labeling with diaziridines is rather poor, but their half-life of nanoseconds101 enables more specific labeling of target proteins92, 96, 102, 103. Nevertheless, diazirine-wielding probe 3, developed by Kuhn et al. to label β-galactosidase from E.coli was unsuccessful in labeling its catalytic

17 Introduction, part I

Figure 6 | Photo-affinity probes. (a) Reaction mechanism of benzophenone during (nonspecific) labeling of nucleophilic residues, with R depicting residual probe architecture (e.g. substrate mimic, reporters). (b) Structure of probe 1, a 1- deoxynojirimycin decorated with benzophenone. (c) Reaction mechanism of aryl azide during (nonspecific) labeling of nearby residues, with R depicting residual probe architecture. (d) Structure of probe 2, a 1-deoxynojirimycin decorated with an aryl

azide. (e) Reaction mechanism of diazirine during (nonspecific) labeling of residues, with R1 and R2 depicting separate ends of the diazirine-wielding probe. (f) Structure of probe 3, a β-D-galactose decorated with a diazirine. pocket (Fig 6f)104, 105. In general, photo-affinity probes have been only successful to label enzymes such as α-glucosidases106, N-acetyl-hexosaminidases107, 108 and sialidases109, 110 in a non-specific manner. Photo-affinity probes also have great applicability in the field of lipid research: equipping lipids with these tags allows cross-linking to identify lipid-lipid as well as lipid-protein interactions111. While reversible inhibitors equipped with photo-affinity tags facilitate labeling of both inverting and retaining glycosidases, these probes cannot discriminate between inactive and active target enzyme molecules, and their covalent attachment after photolysis is rather unspecific. For this purpose, focus has shifted from the concept of employing reversible

18 Activity-based probes for glycosidases inhibitors, that mimic the oxocarbenium ion-like transition state (see Fig 2), to probes that exploit the catalytic mechanism intrinsic to the targeted glycosidases in order to become covalently attached, whilst mimicking the structure of the natural substrate or the oxocarbenium ion-like transition state. One such approach encompasses p-nitrophenyltriazene probes, such as probe 4 introduced by Sinnott112, 113 (Fig 7). After recognition and correct positioning of the probe in the active site, protonation of the aglycone subsequent to cleavage of the glycosidic bond, yields a reactive species that decomposes into nitrogen, arylamine and a highly reactive glycosyl methylcarbenium-like ion, which rapidly reacts with nearby nucleophiles68, 112, 113. Concomitantly, the probe is immobilized in the active site, thereby inhibiting the enzyme’s activity68, 112, 113. This approach has been applied successfully to several enzymes to identify the catalytic amino acid residues, such as in E. coli LacZ112, 113, and β-galactosidases from other origins114−116. Another approach is where the glycosidase cleaves the glycosidic bond between a sugar and an inherently non-reactive aglycone117. After hydrolysis, the expelled aglycone rearranges into a highly reactive moiety, which then non-specifically binds to other residues. Examples include glycosides containing a difluoroalkyl or difluorotolyl moiety, for instance probe 5118 (see Fig 7b, and c for reaction mechanism). After liberation, these moieties eliminate hydrogen fluoride and a highly reactive acyl fluoride or quinone methide, respectively118, 119. This mechanism-based activation allows the labeling of both inverting and retaining glycosidases, but as the reactive group is in the aglycone, i.e. the part of the product lowest affinity for the active site, covalent inactivation of the catalytic pocket is not necessarily very efficient. Accordingly, no studies have been reported on using these probes for active-site identification.

Figure 7 | Probes with reactive aglycones. (a) Reaction mechanism of triazene-type probes labeling the catalytic nucleophile (N). (b) Structure of probe 4, a β-D-glucose decorated with a p-nitrophenyltriazene and probe 5, a β-D-glucose decorated with difluorotolyl. (c) Reaction mechanism of difluorotolyl-wielding probe 5. During the retaining β-glucosidase double-displacement mechanism, involving nucleophile (N) and acid/base (A/B), rearrangements inside the released difluorotolyl aglycone result in semi-irreversible, nonspecific labeling of nearby nucleophilic residues (X).

19 Introduction, part I

2-Deoxy-2-fluoroglycosides as ABPs for retaining glycosidases − In the 1980s, the idea was conceived that one could temporarily trap retaining glycosidases in the glycosylated stage of their double-displacement mechanism. Initial experiments by Legler revealed that removal of the C2-hydroxyl in p-nitrophenyl β-D-glucoside, resulting in p-nitrophenyl β-D- 2-deoxyglucoside 6 (Fig 8a) resulted in an accumulation of the glycosyl-enzyme intermediate of a β-glucosidase, enabling the direct identification of its 2-deoxyglucoside- bound nucleophile120 (see Fig 8b for a similar reaction mechanism). Accumulation of the glycosylated nucleophile occurred due to the lack of key interactions between the C2-hydroxyl in the oxocarbenium ion-like transition state and the catalytic pocket of the enzyme120, 121 (see Fig 2 for proposed regular stabilization). Moreover, the replacement of the C2-hydroxyl group with a hydrogen has only moderate effects on binding, as expressed by the Michaelis constant Km, but is profoundly detrimental to the rate constant of product formation kcat. Absence of the C2-hydroxyl destabilized the transition state as such that the glycosyl-enzyme intermediate accumulated, due to a 106- fold decreased deglycosylation rate122, 123. It may well be that interactions with the C2- hydroxyl are not only required for stabilization, but also for the optimal orientation of the substrate to the catalytic residues, in respect to the to-be-cleaved glycosidic bond122, 123. Withers and co-workers subsequently developed activated 2-deoxy-2-fluoro- glycosides121−124, where the C2-hydroxyl was substituted for C2-fluorine, for instance in probes 7 and 8 (Fig 8a). Introduction of a fluorine onto the carbohydrate-ring destabilizes the charge development at O5 or C1 in the oxocarbenium ion-like transition state during both the glycosylation and deglycosylation steps, due to the strictly limited hydrogen bonding potential of fluorine (Fig 8b). In addition, fluorine stabilizes the glycosyl-enzyme intermediate through inductive destabilization of the electron-deficient transition states. As fluorine is more electronegative than a hydroxyl-group, it destabilizes the positive charge

Figure 8 | Fluoroglycoside-type probes. (a) Structures of 4-nitrophenyl 2-deoxyglucose probe 6, 2-deoxy-2-fluoroglucoside probes 7 and 8, 2-deoxy-2,2-difluoroglucoside probe 9 and 5-fluoroglucoside probe 10. (b) Trapping of the catalytic nucleophile (N) in retaining β-glucosidases after the initial step in the double-displacement mechanism, requiring the acid/base (A/B) for protonation of the interglycosidic oxygen linking to the aglycone (R). The oxocarbenium ion-like transition states flanking the glycosyl-nucleophile adduct are depicted between brackets (with asterisk; ❉).

20 Activity-based probes for glycosidases developed during the oxocarbenium ion-like transition state. Influence on both steps of the mechanism culminates in 106 to 107-fold reduction of the catalytic rates121−126. However, by introducing aglycones that are good leaving groups, i.e. aglycones with low pKa values, such as 2,4-dinitrophenyl (pKa ~4.1, probe 7) or fluorine (pKa ~3.2, probe 8), the formation of the glycosyl-enzyme intermediate proceeds faster, while the deglycosylation rate remains very poor due to the C2-fluorine destabilizing the subsequent oxocarbenium ion-like transition state123−127. Use of the natural aglycone in fluoroglycosides has also been 128, 129 described . A relatively slowly proceeding inhibition of enzyme activity by fluoroglycosides has been observed, as the result of temporary trapping of the glycosyl- enzyme intermediate129. The stability of the intermediate can vary considerably121−126, ranging from 1 to 600 hours, depending on the enzyme and the exact structure of the 124−129 fluoroglycoside employed . Retaining α-glycosidases are less effectively inhibited by 2-deoxy-2-fluoroglycosyl fluorides, as these commonly act as slow substrates in which the glycosylation step is rate limiting129. This has been attributed to the greater stability of the α-glycosyl enzyme intermediate formed on a β-glycosidase than the inverse situation. The rapid turn-over of the 2-deoxy-2-fluoroglycosyl intermediate in α-glycosidases could be overcome by development of 2,2-difluoroglycosides129, where a second fluorine at C2 further slowed both the glycosylation and deglycosylation steps, while further enhancing the reactivity of the leaving group. In the study of Braun et al.129, 2,4,6-trinitrophenyl 2-deoxy-2,2-difluoro-α-D- glucopyranoside 9 inhibited yeast α-glucosidase, and 2,4,6-trinitrophenyl 2-deoxy-2,2- difluoro-α-D-maltosyl fluoride potently inhibited human α-amylase. Enhanced accumulation of the glycosyl-enzyme intermediate can also be achieved by the introduction of 5-fluoroglycosyl probes such as 10130. These 5-fluoroglycosyl fluorides act as slow substrates due to the presence of the C2-hydroxyl. Unfortunately, 5-fluoroglycosides show a very poor KM, caused by the presence of the electron-withdrawing fluorine directly adjacent to the O5, which develops the greatest charge during the oxocarbenium ion-like transition state19−31, 131. Crystallographic analysis of the glycosyl-enzyme intermediates formed through the reaction of fluoroglycosides has yielded insight in the structure − and roles of − specific residues in the catalytic pocket of the enzymes132−135. Most potent tools to identify the nucleophile of glycosidases are 2-deoxy-2-fluoroglycosyl− and 5-fluoroglycosyl fluorides, and 2,4-dinitrophenyl 2-deoxy-2-fluoroglycosides136. The relatively stable, trapped glycosyl- enzyme intermediate also allowed analysis through 19F-NMR137 and/or MS/MS peptide sequencing138−143, assisting the identification of the nucleophile residue of various enzymes, including those of multiple α− and β-glycosidases144, 145, sialidases146 and a N-acetyl-β-D- glucosaminidase147. 2-Deoxy-2-fluoroglycosides enable the assignment of both the acid/base and nucleophile amino-acid residues, through detailed kinetic studies with mutant enzymes148−150. In this classic approach, the acid/base can be identified by the reduced

21 Introduction, part I catalysis of 2-deoxy-2-fluoroglycosides by enzyme lacking this amino acid residue. Complete absence of activity is seen for enzyme lacking the nucleophile residue148−151. Identification of acid/base and nucleophile residues can be confirmed by co-incubation with an alternative nucleophile such as azide148−151. In case of absence of the acid/base, the configuration of the anomeric carbon is retained upon rescue with azide (Fig 9a), whilst absence of the nucleophile causes subsequent stereochemical inversion (Fig 9b). In a similar approach, fluoroglycosides with inverted stereochemistry may be incubated with enzymes lacking the nucleophile to employ them as glycosynthases forming a glycosidic bond between two (complex) carbohydrate molecules (Fig 9c)25, 152. Besides identification of the catalytic residues, fluoroglycosides like on 2-deoxy-2- fluoro-β-D-glucopyranosyl and −β-D-mannopyranosyl compounds injected in rats labeled their target β-glucosidases and β-mannosidases in several organs, including the brain153. These data indicate that 18F-labeled fluoroglycosides might be used as imaging probes of

Figure 9 | Rescue of catalysis in mutant retaining glycosidases by azide as external nucleophile. (a) Catalytic mechanism of a retaining β-glucosidase lacking the acid/base keeps net retention of stereochemistry when rescued with azide. (b) Catalytic mechanism of a retaining β-glucosidase lacking the nucleophile inverts the anomeric configuration when rescued with azide. (c) Mechanism of trans-glycosylation with a nucleophile-lacking retaining β-glucosidase. In all panels, the aglycone is depicted as R and the oxocarbenium ion-like transition states are stated between brackets (with asterisk; ❉).

22 Activity-based probes for glycosidases glycosidases using positron emission tomography (PET)154. In animals injected with 18F-2- deoxy-2-fluoroglucoside labeled human acid β-glycosidase, its bio-distribution has been monitored via PET155.

Epoxides as ABPs for retaining glycosidases − Probes equipped with epoxides have been proven to be true mechanism-based inhibitors for numerous retaining glycosidases, as these ABPs require the target enzyme’s catalytic mechanism to bind covalently to the nucleophile68, 122. Typically, upon activation of the epoxide, i.e. after specific protonation of the oxirane by the acid/base residue, a reactive species is generated by the concomitant attack by the nucleophile to the anomeric C1 carbon, effectively opening the epoxide and the formation of a stable ester-bond with the nucleophile (Fig 10a, next page)122. The reactivity of the epoxide group reduces markedly when conjugated to a glycoside due to the electron-withdrawing effect of hydroxyl groups in the vicinity, consequently increasing both the inherent stability in solutio and the enzymatic requirements to open the oxirane122. Covalent enzyme inhibition only occurs with epoxides with an appropriate orientation allowing protonation of the epoxide oxygen atom by the acid/base, i.e. by a carboxylic acid group in close proximity122. Conduritol epoxides incorporate an endocyclic epoxide grafted to a cyclitol scaffold that mimics the carbohydrate ring of xylose. The most common conduritol epoxide stereoisomer is conduritol β-epoxide (CBE 12, see Fig 10b), or DL-1,2-anhydro-myo- inositol, which was discovered by Legler122 and meticulously characterized and reviewed68, 156. CBE 12 is a potent mechanism-based inhibitor for several retaining glucosidases present in various organisms122, 156−159. It has been employed to identify the catalytic nucleophiles in several glucosidases122, 159−163. The symmetry in CBE 12 at the C2 axis allows it to irreversibly inhibit both retaining α− and β-glucosidases164−170. Inhibition of retaining α-glucosidases by CBE 12 occurs with reduced reaction kinetics as compared for β-glucosidases171. This is due to the alternative orientation the molecule must be in to open the β-epoxide: in β-glucosidases it preferentially opens trans-diaxially (Fig 10a), but in α-glucosidase it opens trans- equatorially (Fig 10c)172−176. The trans-diaxial opening of the β-epoxide of CBE 12 in β- glucosidases was confirmed by cleaving the 14C radio-labeled CBE 12-nucleophile adduct with hydroxylamine, which revealed that only D-1,2-anhydro-myo-inositol reacted with the glucosidase176, closely mimicking the structure of D-xylose and D-glucose. No β-glucosidase inhibition was observed for L-1,2-anhydro-myo-inositol177. Alternatively, analogues of CBE 12, including L-1,2-anhydro-myo-inositol, conduritol F epoxide 13, conduritol C cis- epoxide 14 and trans-epoxide 15 were found to irreversibly inhibit a β-fructosidase, α- mannosidases, β-galactosidases, α-galactosidases and a α-fucosidase, respectively177−181. Symmetry in CBE 12 furthermore permits binding into some catalytic pockets with different orientations, thereby covalently binding catalytic residues other than the nucleophile. This is illustrated by the labeling of alternative residues with conduritol C cis-

23 Introduction, part I

Figure 10 | Catalytic nucleophile-binding by epoxide-wielding probes. (a) Reaction mechanism of (conduritol) β-epoxides labeling the catalytic nucleophile (N) of retaining β-glucosidases (through trans-diaxial ring opening) requires the acid/base (A/B) for activation. (b) Structures of 2,3-epoxypropyl β-D-xyloside probe 11, conduritol β-epoxide 12, conduritol F epoxide 13, conduritol C cis-epoxide 14, conduritol C trans-epoxide 15, cyclophellitol β-epoxide 16, and diastereomers 17−19. (c) Reaction mechanism of β-epoxides labeling the nucleophile of a retaining α-glucosidase (through trans-equatorial ring opening). In all panels, the oxocarbenium ion-like transition states are depicted between brackets (with asterisk; ❉). epoxide 14 in E. coli LacZ β-galactosidase182, and conduritol β-epoxide 12 for both human GBA183 and almond β-glucosidase184. The correct residues were later identified with 2- deoxy-2-fluoroglycosides185−187. The symmetry of CBE 12 permits multiple binding modes, thereby enabling irreversible inhibition of both α− and β-glucosidases through different interactions. Loss of symmetry dramatically restricts the number of orientations of the inhibitor in a catalytic pocket. Introduction of a structural group in the cyclitol ring of CBE 12, which breaks its symmetry, results in superior target specificity and inhibitory potential towards specific retaining β-glucosidases. The asymmetric cyclophellitol β-epoxide 16, isolated from the Phellinus sp. mushroom, indeed inhibits β-glucosidases several orders of magnitude more potently188−190. Its covalent binding to the catalytic nucleophile was demonstrated firstly through crystallography of a β-glucosidase from Thermotoga maritima191. The substitution of the C5-hydroxyl group of CBE 12 with a 5-hydroxymethyl group makes the inhibitor 16 resemble β-D-glucose more closely than β-D-xylose189. Importantly, this β-epoxide 16 has a reduced affinity for α- glucosidases188−191. Specific targeting of α-glucosidases can be achieved by synthesis of the

24 Activity-based probes for glycosidases diastereomer 1,6-epi-cyclophellitol 17192, which potently inactivated α-glucosidases in cancer cells. Diastereomers with α-glucose 17, β-mannose 18 and α-mannose 19 configurations were expectedly potent inhibitors of the corresponding α-glucosidases, β- mannosidases and α-mannosidases190−193. Exo-alkyl epoxide-glycosides, i.e. compounds with the epoxide-group attached via an alkyl-spacer to a sugar (see e.g. probe 11, in Fig 10a) deserve also discussion. These compounds lack the structural rigidity of conduritol− and cyclophellitol structures and, due to their flexibility, can also label catalytic carboxylates of several glycosidases194−206, including exoglucosidases and endoglycosidases such as cellulases194, 199−202. Interestingly, 2,3-epoxypropyl β-D-xyloside probe 11 labeled the catalytic nucleophile of a retaining 1,4-xylanase, while the 3,4-epoxybutyl form covalently bound to the enzyme’s acid/base204. Apparently, the length of alkyl chains causes in this case alternative orientations of the epoxide in the catalytic pocket206, a flexibility that can be exploited. By variation of the alkyl chain length and configurations of the epoxide, specific ABPs can be tailored for a particular enzyme201, 207. Another class of irreversible, mechanism-based inhibitors encompasses compounds equipped with an aziridine as warhead (Fig 11). Glycosyl aziridines have received far less attention than epoxide-containing inhibitors. Typically, the nitrogen in the aziridine head- group is protonated by the acid/base residue. The protonated nitrogen thereby becomes an optimal leaving group, allowing aziridine-ring opening following a nucleophilic attack, resulting in the formation of a stable ester-bond with the nucleophile, similar as observed/proposed for epoxide-containing probes (Fig 11a). Theoretically, aziridines have a high affinity for the rather negatively charged catalytic pockets of glycosidases due to the positive charge of the protonated nitrogen atom in the aziridine moiety. Aziridines with particular configurations are highly specific inhibitors of corresponding retaining glycosidases, similarly as observed for epoxide-containing inhibitors. Of note, conduritol β- aziridine 20 (Fig 11b) inhibits both β-glucosidases and α-glucosidases due to its symmetry at the C2 axis, which is similar as in CBE 12208, 209.

Figure 11 | Catalytic nucleophile-binding by aziridine-wielding probes. (a) Reaction mechanism of (conduritol) β-azidirine labeling the nucleophile (N) of a retaining β-glucosidase (through trans-diaxial ring opening), after activation of the warhead by the acid/base (A/B). The oxocarbenium ion-like transition state is depicted between brackets (with asterisk; ❉). (b) Structure of conduritol β-aziridine 20.

ABPs for the human glucosylceramide-metabolizing glycosidases − Conduritol β- epoxide 12 and cyclophellitol β-epoxide 16 are irreversible inhibitors of the human

25 Introduction, part I lysosomal acid β-glucosidase, named glucocerebrosidase (GBA)210−213. GBA is responsible for the breakdown of glucosylceramide, by catalyzing the hydrolytic cleavage of the glycosidic bond between β-D-glucose and ceramide, which is the final step in the degradation of glycosphingolipids. This important class of glycoconjugates was discovered in 1884 by Johan L. W. Thudichum, who coined their name ‘glycosphingolipids’, after the Sphinx and its riddle in his book214 entitled ‘A Treatise on the Chemical Constitution of Brain’. Glycosphingolipids are currently known to be involved in a myriad of biological processes, including the formation of membrane domains215, function as bioactive molecules involved in regulation of the cell-cycle216, differentiation217, senescence218, necrosis219, proliferation220, apoptosis221, and act as structural components required for cell adhesion222. As a consequence, glycosphingolipids are implicated in multiple pathological processes223, including cancer224−226, the metabolic syndrome227−229 and glycosphingolipid storage disorders230−233. Glycolipids make up a predicted 80% of all glycoconjugates, with the class of glycosphingolipids estimated to be the largest (> 9,000 species)234. In human plasma alone, more than 200 distinct glycosphingolipids have been identified recently235.

Glycosphingolipid metabolism – The structure of glycosphingolipids is comprised of several elements (Fig 12, next page), first of which is a long-chain sphingoid base, represented by sphingosine, (2S,3R,4E)-2-aminooctadec-4-ene-1,3-diol, also referred to as (E)-sphing-4-enine, the major sphingoid base found in mammals234, 236, 237. The majority of sphingoid bases are amide-linked with a long− or very-long-chain fatty acid (second structural feature) to form ceramides, which (third structural feature) can be further derivatized by addition of a head-group to form (glyco)sphingolipids, including sphingomyelin, glucosylceramide, galactosylceramide and more complex glycosphingolipids containing numerous sugar residues234, 237−239. See for excellent overviews of (glyco)sphingolipid chemistry references234, 237, 239. The biosynthesis and degradation of glycosphingolipids is highly complex, as it requires the expression and correct localization of an array of enzymes234, 237. Namely, after synthesis of dihydroceramide from L-serine and Coenzyme A-activated fatty acids at the cytosolic leaflet of the ER membrane240−242, dihydroceramide desaturase produces ceramide. Additionally, ceramide can be produced by acylation of sphingosine, freed during lysosomal degradation of (glyco)sphingolipids234. Subsequent transport of ceramide to the membrane of the cis-Golgi apparatus243−245 allows glucosylceramide synthase (GCS) to glycosylate ceramide with UDP-activated α-D-glucose to produce β-D-glucosylceramide, one of the simplest known glycosphingolipids (Fig 12a)246−249. Additionally, ceramide can be equipped with a α-D-galactose, resulting in β-D-galactosylceramide, which serves as the basis for different glycosphingolipid species, or ceramide is employed to synthesize sphingomyelin234. Creation of more complex glucosphingolipids occurs upon flipping of glucosylceramide to the luminal side of the Golgi apparatus250−253, where other UDP-

26 Activity-based probes for glycosidases

Figure 12 | Glycosphingolipid metabolism. (a) Synthesis of glucosylceramide by glucosylceramide synthase (GCS) and subsequent formation of lactosylceramide by galactosyltransferase (GalT1), enabling the further synthesis of other classes of complex glycosphingolipids (Globo−, ganglio−, muco−, lacto−, neolacto− and isoglobo−series). (b) Structure of the more complex glycosphingolipids Gb4 (left) and GM1 (right); each glycosphingolipid is annotated with the various individual glycosidases required for the step-wise hydrolysis of each specific glycosidic bond. Figure partially modified from ref283. activated sugars can be added to through a step-wise process. As a first step, glucosylceramide is extended with UDP-activated galactose by GalT1 to form lactosylceramide (Fig 12a)234. Next, highly complex glycosphingolipids containing multiple branches and comprising of different sugar moieties are formed234, 237, with each biosynthetic step tightly regulated through localization of the glycosyltransferase, availability of appropriate glycosphingolipid substrate and the presence of activated glycosyl donor substrate. Vice versa, during the catabolism of complex glycosphingolipids, each glycoside is removed in a stepwise fashion, requiring an array of specialized glycosidases (Fig 12b)234, 237. Breakdown occurs predominantly in the endosomal− and lysosomal compartment, of which the membrane is protected against glycosidase-executed degradation through the presence of a glycocalyx consisting of heavily glycosylated membrane proteins254. Glycosphingolipids destined for degradation may enter the endosomal/lysosomal apparatus through receptor-mediated endocytosis of lipoproteins, endocytosis of the plasma membrane or phagocytosis of membrane structures and/or senescent− and apoptotic cells. The latter process occurs predominantly in macrophages, which, amongst others, phagocytose senescent erythrocytes containing high levels of (blood-group)

27 Introduction, part I glycosphingolipids235, 255−257. In the lumen of lysosomes, glycosidases require specific (glyco)sphingolipid activator proteins (SAPs) to assist in the interaction with the membrane-bound glycosphingolipid substrate, in order to remove sequentially the carbohydrates from the glycosphingolipids, starting at the non-reducing end of the molecule237, 254, 258, 259. Inability of cells to successfully catalyze the cleavage of all glycosidic bonds encountered in glycosphingolipids inevitably results in the accumulation of the retained, uncleavable glycosphingolipid, thereby eventually causing lysosomal dysfunction and a glycosphingo- lipid storage disorder. The most common lysosomal glycosphingolipid storage disorder is Gaucher disease, named after the French clinician Philippe C. E. Gaucher, who firstly authored a detailed case report in his doctoral thesis260 − ‘De l’Epithelioma Primitif de la Rate, Hypertrophie Idiopathique de la Rate sans Leucemie’ − in 1882. This thesis included a description of the clinical manifestations of a 32-year old woman presenting an unexplained hepatosplenomegaly. After it became clear that macrophages in Gaucher disease accumulated a lipoid structure261, the French chemist Aghion identified this chemical structure as glucosylceramide262, one of the simplest glycosphingolipids234, 262−265. Roscoe O. Brady and co-workers discovered in the 1960s that the activity of the lysosomal glycosylhydrolas glucocerebrosidase (GBA) was deficient in Gaucher disease, causing the hallmark accumulation of glucosylceramide266−268. The gene GBA encoding the GBA enzyme was independently cloned in the early 1980s by the research groups of Barranger269, Beutler270 and Horowitz271. Almost three decades later the existence of another, non- lysosomal, β-glucosidase (GBA2) was firstly described272. The gene encoding this membrane-bound enzyme was independently cloned by Yildiz273 and Boot274 in 2007. Other human enzymes that (may) also be able to hydrolyze glucosylceramide are the intestinal lactase/phlorizin hydrolase (LPH)275, 276 and the cytosolic non-specific β-glucosidase GBA3, albeit the latter with very low affinity277, 278.

Design of ABPs for glucocerebrosidase – Recently, a 2-deoxy-2-fluoro-D-glucoside was designed with an imidate as optimized leaving group (Fig 13)279. This probe 21 was found to avidly label GBA in vitro and in living cells. As discussed previously, the binding of 2- deoxy-2-fluoro-D-glucoside to glutamate E340 is not irreversible. Reactivation of GBA, and loss of labeling, occurs spontaneously also at a low rate (Chapter 3). In parallel, CBE 12 and cyclophellitol β-epoxide 16 were utilized as leads for the In In In

Figure 13 | Fluorescent 2-deoxy-2-fluoro-D-glucoside. Structure of fluorescent β-2-deoxy-2-fluoro-D-glucoside probe 21, containing imidate as leaving group for optimal in situ labeling of GBA. See also Chapter 3, or ref279.

28 Activity-based probes for glycosidases

In parallel, CBE 12 and cyclophellitol β-epoxide 16 were utilized as leads for the development of specific fluorescent activity-based probes for GBA. An azide-group was grafted to the C6 position of cyclophellitol β-epoxide 16, yielding azido-cyclophellitol probe 22 (Fig 14a). Azido-cyclophellitol 22 was 100-fold more potent than CBE 12. It was also observed that when 22 was covalently extended at C6 with BODIPY fluorophore moieties, these ABPs became even more potent inhibitors for GBA280, 281. These fluorescent ABPs, green fluorescent β-epoxide 23 and red fluorescent β-epoxide 24, proved superior probes to azido-cyclophellitol 22, with an approximately 5,000-fold improved inhibitory potency when compared to CBE 12 (1.24 and 1.94 nM versus 9,497 nM). The inhibition of GBA with β-epoxide 23 occurred most efficiently at its pH optimum, indicating that only active enzyme molecules are labeled with the probe (Fig 14b). This was expected since the β-epoxide warhead should only open after activation by the acid/base and concomitant nucleophilic attack (see Fig 10 for reaction mechanism). Analysis of labeled GBA with MS/MS indeed validated that azido-cyclophellitol 22 bound to the nucleophile E340281. Moreover, direct attachment of the BODIPY tag allows for immediate fluorescence scanning of β-epoxide 23-labeled proteins on for instance SDS-PAGE gels, and in turn revealed that direct labeling can be ablated by pre-incubation with known active-site inhibitors or denaturing conditions (Fig 14c). Moreover, the detection of the BODIPY-emitted fluorescence proved ultra-sensitive, with visualization achievable of 20 attomoles of GBA molecules (20 × 10−18 mol, or ~12

Figure 14 | GBA specific cyclophellitol epoxide-type ABPs. (a) Structures of cyclophellitol β-epoxide 16, azido-cyclophellitol 22 (KY170), and fluorescent β-epoxide 23 (MDW933, green fluorescence) and β-epoxide 24 (MDW941, red). (b) Effect of pH

on relative glucocerebrosidase (GBA) activity towards artificial 4MU-β-D-Glc substrate (open circles; ¢–¢) compared to the IC50 of β-epoxide 23 (closed squares; n–n) across the pH-range. (c) Labeling of GBA was blocked by incubating with irreversible inhibitor CBE 12, competitive reversible inhibitor AMP-DNM or by denaturing conditions (1% (w/v) SDS for 4 min at 100 °C) prior to labeling with β-epoxide 23. (d) Sensitivity of detection and labeling. GBA was incubated with an excess of β-epoxide 23 and dilutions applied on gel. (e) Fluorescent labeling of murine liver and duodenum lysates exposed to β-epoxide 23. Arrows indicate molecular weight of recombinant GBA (~59 kDa). Figure is a combination of excerpts from Chapter 1, see also ref281.

29 Introduction, part I million molecules), or less (Fig 14d). Incubation of complex lysates such as homogenates from murine liver (Fig 14e) revealed that β-epoxide 23 labeled GBA highly specifically. This was observed in numerous lysates, except in those of small intestine, where β-epoxide 23 additionally bound to LPH, and fragments thereof281. Importantly, cell-types as including hepatocytes, present in the liver, contain besides GBA also the aforementioned retaining β-glucosidases GBA2 and GBA3. However, these remained evidently unlabeled by β-epoxide 23. This was confirmed by separate enzyme assays, showing β-epoxide 23 very 281, 282 poorly inhibits GBA2 and GBA3 (IC50 > 100 μM, for both enzymes) . The amphiphilic nature of β-epoxide 23 also allows passage of the ABP across membranes and results in efficient in situ labeling of active GBA molecules in living cells and mice. Intriguingly, the attachment of the bulky BODIPY moiety at the C6 position in cyclophellitol β-epoxide 16 further enhanced and skewed the specificity of the inhibitor for GBA and not to GBA2, GBA3 or LPH. Albeit this finding was unexpected, this effect can be explained by the degree of acceptance toward substrates exhibited by the different retaining β-glucosidases expressed in man and mice. While GBA, GBA2, GBA3 and LPH are able to catalyze the hydrolysis of the glycosidic bond between β-D-glucose and an aglycone (e.g. ceramide or 4-methylumbelliferone), they are categorized into different CAZy glycosyl hydrolase families: GBA in GH30, GBA2 in GH116 and GBA3/LPH in GH1. The specificity of β-epoxide 23 for GBA is likely due to its partial resemblance of the structure of β-1,6- glucan, as can be seen in Fig 15a (next page). Along with GBA, several retaining β-1,6- glucanases are categorized in GH30, indicating still some resemblance between GBA and this class of glucosidases is remaining in its structure and therefore bulky groups at C6 are readily accepted. To create potent ABPs for all retaining β-glucosidases, an ABP structure was envisioned that resembled significantly more that of glucosylceramide (Fig 15a). For this purpose, conduritol β-aziridine 20 was identified as putative scaffold208. The nitrogen of the aziridine can be acylated to yield an ABP structure containing a large bulky BODIPY group close to the C1 position, resembling the positioning of the ceramide aglycone in glucosylceramide. Moreover, the C6-position of conduritol β-aziridine 20 was changed from an exocyclic hydroxyl to an exocyclic hydroxylmethyl, yielding cyclophellitol β-aziridine 25, which improved specificity similar as observed for the cyclophellitol β-epoxides 16 and 22−24282. Acylation of the nitrogen in the aziridine with an alkyl-group allowed extension of the β- aziridine (probe 26), to yield both the green fluorescent β-aziridine 27 and a biotin- containing β-aziridine 28282.Incubation of GBA in vitro with β-aziridine 27 revealed that it could still bind GBA at high pH values where the enzymatic activity was lost (Fig 15b). This indicated β-aziridine 27 can bind regardless of the action/state of the acid/base, in contrast to β-epoxides such as 23. The acid/base-independent binding mechanism of β-aziridine 27 was verified by investigations with GBA with mutated acid/base (E235) or nucleophile (E340). In absence of the acid/base, β-aziridine 27 still binds GBA, whilst β-epoxide 23 cannot (Fig 15c). The acid/base-independent binding mechanism of β-aziridines 25−28 is

30 Activity-based probes for glycosidases

Figure 15 | Broad-spectrum aziridine-type ABPs for retaining β-glucosidases. (a) Structure of fluorescent cyclophellitol β- epoxide 23 aligned with those of β-1,6-glucan, glucosylceramide, cyclophellitol β-aziridine 25 and novel β-aziridine probes 26−28. (b) Quantified labeling β-epoxide 23 (open circles; ¢–¢) and β-aziridine 27 (closed squares; n–n), compared to GBA β- glucosidase activity profile towards artificial 4MU-β-D-Glc substrate across the pH-range (open triangles; r –r). Data are average of triplicates ± SD, normalized to conditions at pH 5.0. (c) Labeling of GBA active-site mutants. Over-expressed myc/His-tagged GBA, either as wild-type or lacking the acid/base (E235G or E235Q), or nucleophile (E340G or E340Q) labeled with β-aziridine 27 in total lysate (top), or isolated from labeling with β-epoxide 23 or β-aziridine 27 in total lysate after pull-down (middle), with α-myc as loading control (bottom). (d) Labeling of endogenously expressed retaining β-glucosidases in homogenates of murine brain and using β-epoxide 23 and β-aziridine 27. Molecular weights are marked of recombinant H. sapiens GBA (59 kDa, imiglucerase, asterisk; ❉), endogenous murine GBA (circle; ●), GBA2 (open diamond; ♢), GBA3 (square; 282 n), LPH and its degradation fragments (triangle; p). Figure is a combination of excerpts from Chapter 2, see also ref . extraordinarily fast, hampering determination of kinetic rate constants. As discussed below,

31 Introduction, part I extraordinarily fast, hampering the determination of kinetic rate constants. As discussed below, β-aziridines 27 and 28 prove to be broad-spectrum ABPs, labeling also the retaining β-glucosidases GBA2, GBA3 and LPH (see Fig 15d)282 and from various other organisms, such as bacteria and fungi282. Other retaining glycosidases, including fungal cellulases or xylosidases, were not labeled by either ABP, indicating cross-species selectivity for retaining β-glucosidases282.

Use of ABPs to investigate retaining β-glucosidases − For some decades, GBA properties have been studied with irreversible inhibitors. For example, through labeling of GBA with β-epoxide 23 and subsequent MS/MS analysis, the catalytic nucleophile could be correctly identified as E340. Using the novel fluorescent β-epoxide− and β-aziridine ABPs in parallel, the acid/base and nucleophile residues of GBA can be easily mapped. GBA lacking the catalytic nucleophile E340 cannot bind fluorescent β-epoxide 23 or β-aziridine 27. Where labeling of GBA by β-epoxide ABPs requires activation through protonation by the acid/base that by β-aziridine 27 does not282. As a result, GBA with an inactivated acid/base residue E235 labels avidly with β-aziridine 27 but not with β-epoxide 23, as can be conveniently detected with SDS-PAGE and fluorescence scanning. In the case other retaining β-glucosidases than GBA are used, the catalytic nucleophile can be mapped by identification of the amino acid to which an epoxide or aziridine ABP is covalently bound, either directly by mass spectrometry or indirectly using site-directed mutagenesis of putative nucleophile residues. The acid/base residues of these enzymes can be identified using external nucleophiles such as azide, able to substitute a lacking acid/base and thus rescue activity and labeling by a β-epoxide ABP148−151. The latter can be very sensitively detected using SDS-PAGE and fluorescence scanning. In Chapter 4, this approach is applied to identify the key catalytic residues of all known retaining β-glucosidases in man. Another use of CBE 12 and other epoxide ABPs is to differentiate retaining β-glucosidases in complex (tissue) homogenates283, 284. Selective inhibitors have for example aided in the discovery of non-lysosomal β-glucosidase GBA2272. An important application for β-epoxide 23 and β-aziridine 27 is laid in the visualization and quantification of (active) GBA and other retaining β-glucosidases. Incubation of wild- type fibroblasts with fluorescent β-epoxide 25 results in in vivo labeling of active GBA and allows in situ detection with (time-lapse) confocal fluorescence microscopy (Fig 16a, next page). After harvesting of ABP-labeled cells and fixation, the pattern of in vivo labeled enzyme overlaps with GBA detected with monoclonal α-GBA antibody 8E4, albeit the latter is markedly less sensitive. In fibroblasts of Gaucher patients completely lacking (active) GBA, no labeling with β-epoxide 24 occurs281. β-Epoxide 23 selectively labeling active GBA can be used for diagnosis of Gaucher disease. Following incubation of cells or lysates thereof with 23, GBA molecules can be visualized by fluorescence scanning after SDS- PAGE. A clear inverse relationship between the cellular amount of labeled GBA and the severity of Gaucher disease has been observed for fibroblasts (Fig 16b).

32 Activity-based probes for glycosidases

Figure 16 | Applications of ABPs for retaining β-glucosidases. (a) Representative fluorescence micrographs of GBA wild- type fibroblasts treated with red β-epoxide 25 and subsequent immunohistochemistric detection of GBA peptide. After spectral imaging, channels were unmixed into AlexaFluor488 fluorescence of GBA visualized with α-GBA antibody (green), red β- epoxide 24 BODIPY fluorescence of GBA (red), overlay of the latter two where equal green and red fluorescence yields yellow overlay. In all pictures, nuclei are stained with DAPI (blue). Scale bar represents 20 μm. (b) Detection of GBA in lysates of wild- type, homozygous N370S, L444P and RecNCI fibroblasts with β-epoxide 23 (top row) and GBA peptide detected by α-GBA 8E4 (bottom row). Imiglucerase labeled with β-epoxide 23 (asterisk; ❉) serves as loading control. (c) Pulse-chase of wild-type fibroblasts. After overnight incubation with red β-epoxide 24, cells were chased with green β-aziridine 27 for 0–152 h. (d) In vivo labeling of retaining β-glucosidases in mice. Fluorescently labeled proteins were analyzed in tissue homogenates of duodenum (left) and liver (right). From left to right: Animals treated in vivo with vehicle or 10–1000 pmol β-epoxide 23 compared to maximal in vitro labeling with excess 23, animals treated with 10–1000 pmol β-aziridine 27 compared to maximal in vitro labeling with excess 27. Marked is recombinant H. sapiens GBA (59 kDa, imiglucerase, asterisk; ❉), endogenous GBA (circle; ●), GBA2 (open diamond; ♢), GBA3 (square; n), LPH and its degradation fragments (triangle; p). Figure is a combination of excerpts from 281, 282 Chapter 1 & 2, see also refs .

The fact that GBA is labeled specifically by β-epoxides 23 and 24 can be exploited for determination of the half-life of GBA in living cells (Fig 16c). An overnight pulse with red β-epoxide 24 followed by a continuous chase with green β-aziridine 27, allows analysis of the rate of formation of de novo red 24-labeled GBA. Similar results were obtained with the combination of green and red β-epoxides 23 and 24. In addition, one can follow the modifications in newly formed enzyme: newly synthesized GBA in the ER contains four high mannose-type N-linked glycans (apparent molecular weight ~62 kDa)285−289; in the Golgi apparatus these are modified to sialylated complex-type glycans resulting in an increased molecular weight (62 to 66 kDa); finally, in the lysosome the glycans of GBA are trimmed by the sequential action of exoglycosidases resulting in a decrease in molecular weight (66 kDa reduced back down to the bare 58 kDa peptide)285−289.

33 Introduction, part I

The selectivity of CBE 12 for GBA has been employed to specifically ablate its activity in intact cells and animals, causing the accumulation of glucosylceramide and glucosylsphingosine290, 291. Incubation of macrophages with CBE 12 results in altered morphology292 and formation of glucosylceramide-accumulating Gaucher-like cells293, 294. Loading of CBE 12-treated macrophages with lysed red blood cells has been argued to closely mimic the formation of Gaucher cells294. Inhibition of GBA by CBE 12 has also been used to obtain insight in the role of glycosphingolipids in the epidermis295−298, most particularly the importance of hydrolysis glucosylceramide to ceramide in the stratum corneum295−298. Based on such studies, amongst others, GBA is thought to play a crucial role in maintaining the barrier function of the skin, e.g. prevent excessive water loss and absorption of exogenous molecules295, 296, 299−304. The most severe Gaucher patients, so-called collodion babies, suffer from a compromised barrier function of the skin due to a (near) total absence of GBA activity259. CBE 12 has also been used to study the role of GBA in neuronal cells. Inhibition of GBA was found to result in significantly increased neuronal axon length and branching305, 306. Finally, CBE 12 has been employed to induce GBA deficiency in wild-type mice to attempt to generate a genuine Gaucher disease model. Indeed, repeated administration of CBE 12 results in accumulation of glucosylceramide, albeit much more moderate than observed in Gaucher patients307, 308. CBE 12-treated mice are short-lived as the result of lethal neurological complications. Nevertheless, CBE 12-treated mice have been used as a short- lived animal model to test gene therapy as treatment for Gaucher disease322. Titration studies with CBE 12 revealed that GBA could be inhibited about 85% in mice, without causing Gaucher symptoms309. This suggests that therapies who elevate GBA activity to at least ~15% of levels observed in control individuals, might render beneficial clinical responses64−67, 310−312. Such a partial increase in GBA activity is aimed at in current studies with small molecular chaperones, which may stabilize the ‘native’ fold of mutant GBA in the endoplasmic reticulum (ER), thereby reduce degradation by the ER-associated degradation system and thereby enhance GBA delivery to lysosomes64−67, 313−316. A major drawback of the use of CBE 12 to induce Gaucher disease in animal models, is its ability to permeate into the brain and irreversibly inhibit GBA there, and as a consequence, induce fatal neurological complications. Cyclophellitol β-epoxide 16 has also been used to inactivate GBA in living cells and mice190, 290, 291, 295. Again, 16 also inhibits GBA activity in the brain of animals treated in vivo, and thus does not allow generation of a long- lived Gaucher mouse model. Neither a complete GBA knockout renders a completely genuine Gaucher model in mice, since it is embryonically lethal317, 318. More sophisticated inducible and/or cell-type specific deficiency of GBA and knock-ins of specific mutations occurring in Gaucher patients, have been employed to generate a variety of mouse models319−326. The newly developed β-epoxide 23 offers an exciting new possibility to generate a long- lived Gaucher mouse model with prominent visceral symptomatology. This is based on the

34 Activity-based probes for glycosidases ability of β-epoxide 23 to inhibit GBA irreversibly in all tissues except for the brain, skin and eye. Intravenous administration of the β-epoxide 23 is well tolerated to dosages up to at least 1 nmol/mouse (20 μg kg−1)282. Intravenous administration of wild-type mice with β- epoxide 23 results in specific, dose-dependent labeling of GBA in various organs (Fig 16d). Besides GBA, no other retaining β-glucosidases are labeled except LPH in the small intestine. Repeated intravenous administration to mice of β-epoxide 23 generates a Gaucher-like phenotype, exhibiting classic hallmarks such as splenomegaly, infiltration of tissue with macrophages expressing markers of Gaucher cells such as gpNMB, and increased tissue and plasma concentrations of glucosylceramide and glucosylsphingosine (see also Chapter 7). In the case of β-aziridine 27, intravenous administration results not only in prominent in vivo ABP-labeling, and thus inactivation, of GBA in various organs but also in that of GBA2 and GBA3, as well as LPH in the small intestine (Fig 16d). Again, β-aziridine 27 does not label β-glucosidases in brain and eye, most likely through its active removal by P-glycoproteins present in the blood brain barrier282. Finally, ABPs such as β-epoxide 23 and β-aziridine 27 can be used to elegantly, fluorescently label therapeutic enzymes in vitro. The fate of pre-labeled enzymes following administration to cultured cells or after intravenous infusion into mice can be sensitively monitored. Enzyme therapy is presently the first choice treatment for type I Gaucher disease. This non-neuropathic variant of the disorder is thought to be a macrophage disorder in which glucosylceramide-laden phagocytes (Gaucher cells) underlie the visceral symptoms327−329. Prevention and/or removal of these lipid-laden macrophages is the rational treatment goal for type I Gaucher disease330, 331. Brady and colleagues developed an enzyme therapy approach to specifically supplement visceral macrophages with exogenous GBA332, 333, exploiting the presence of mannose-lectin(s) in this cell-type334−339. To date, several GBA preparations have been produced with exposed terminal mannose-moieties mediating recognition and uptake by mannose-lectin(s). Chronic two-weekly intravenous administration of therapeutic GBAs, such as alglucerase340, imiglucerase341, velaglucerase342 and taliglucerase343, results in impressive clinical responses of type I Gaucher patients344, 345. Direct comparisons of the different therapeutic GBAs, including bio-distribution studies, are scarce342, 346−351. Differential labeling of different therapeutic GBAs with ABPs with distinguishable tags, for instance with green β-epoxide 23 and red β-epoxide 24, and their equimolar administration to cell− and animal models enables a head-to-head analysis of the binding and uptake by cells and bodily distribution of these therapeutir GBAs, visualized by methods as SDS-PAGE, FACS, or fluorescence microscopy (see also Chapter 6). An exciting future development is the development of ABPs carrying specialized tags that allow non-invasive imaging and bodily distribution in animal models and individual Gaucher patients, when trace-labeled onto therapeutic enzymes. Another potential future application of ABPs may be laid in the development of more potent chemical chaperones to stabilize the ‘native’ fold(ing) and lysosomal delivery of mutant GBA enzymes in Gaucher patients. Semi-irreversible ABPs such as 2-deoxy-2-

35 Introduction, part I

fluoro-D-glucosides, equipped with optimized leaving groups as discussed previously appear attractive. These types of ABPs should only transiently bind to the nucleophile of GBA, thereby promoting the critical process of folding in the ER and sorting to lysosomes. Inside lysosomes the rescued enzyme should be reactivated for glucosylceramide hydrolysis, following the slow irreversible release of 2-deoxy-2-fluoro-D-glucose (see Chapter 5). Obviously, the use of appropriate concentrations of such probes is of eminent importance: too little probe will be without effect and too much will backfire by inhibiting all residual enzyme activity. Animal studies will have to reveal whether an optimal concentration of such chemical chaperons can be reached to in order to result in a sustained clinical benefit.

Concluding remarks Recently designed activity-based probes for retaining β-glucosidases offer extraordinarily versatile tools that can be broadly applied. Applications are found in fundamental characterization of the catalytic mechanism of these enzymes, diagnosis of patients suffering from enzyme deficiencies, determination of the in situ half-life of individual enzymes, their processing and subcellular trafficking, induction of long-lived Gaucher- disease in animal models and the in vivo monitoring of enzyme therapy efficacy in individual patients by imaging of therapeutic enzyme trace-labeled with ABPs. The potential application of semi-irreversible activity-based probes as transient chemical chaperones promoting successful folding in the ER of mutant GBA and consequently improving cellular enzymatic activity warrants further investigation.

36 Activity-based probes for glycosidases

References 1. Laine RA (1994) A calculation of all possible oligosaccharide isomers both branched and linear yields 1.05 x 10(12) structures for a reducing hexasaccharide: the Isomer Barrier to development of single-method saccharide sequencing or synthesis systems. Glycobiology. 4, 759−767. 2. Moremen KW, Tiemeyer M & Nairn AV (2012) Vertebrate protein glycosylation: diversity, synthesis and function. Nat Rev Mol Cell Biol. 13, 448−462. 3. Cummings RD (2009) The repertoire of glycan determinants in the human glycome. Mol. Biosyst. 5, 1087−1104. 4. Nairn AV & Moremen KW (2009) in Handbook of Glycomics (Cummings R & Pierce JM, Academic Press), 95−136. 5. Nairn AV et al. (2008) Regulation of glycan structures in animal tissues: transcript profiling of glycan-related genes. J Biol Chem. 283, 17298−17313. 6. Spiro RG (2002) Protein glycosylation: nature, distribution, enzymatic formation, and disease implications of glycopeptide bonds. Glycobiology. 12, 43R−56R. 7. Lairson LL, Henrissat B, Davies GJ & Withers SG (2008) Glycosyltransferases: structures, functions, and mechanisms. Annu Rev Biochem. 77, 521−555. 8. Davies GJ, Gloster TM & Henrissat B (2005) Recent structural insights into the expanding world of carbohydrate-active enzymes. Curr Opin Struct Biol. 15, 637−645. 9. Cantarel BL, et al. (2009) The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics. Nucleic Acids Res. 37, D233−238. 10. Wolfenden R, Ridgway C & Young G (1998) Spontaneous hydrolysis of glycosides. J Am Chem Soc. 120, 833−834. 11. Carmi N & Breaker RR (2001) Characterization of a DNA-cleaving deoxyribozyme. Bioorg Med Chem. 9, 2589−2600. 12. Breaker RR (1999) Catalytic DNA: in training and seeking employment. Nat Biotechnol. 17, 422−423. 13. Li & Breaker RR (1999) Kinetics of RNA degradation by specific base catalysis of transesterification involving the 2’-hydroxyl group. J Am Chem Soc. 121, 5364−5372. 14. Wolfenden R, Lu X & Young G (1998) Spontaneous hydrolysis of glycosides. J Am Chem Soc. 120, 6814−6815. 15. Naumoff DG (2011) Hierarchical classification of glycoside hydrolases. Biochemistry (Mosc). 76, 622−635. 16. Henrissat B (1991) A classification of glycosyl hydrolases based on amino-acid-sequence similarities. Biochem J. 280, 309−316. 17. Henrissat B & Bairoch A (1993) New families in the classification of glycosyl hydrolases based on amino- acid-sequence similarities. Biochem J. 293, 781−788. 18. Davies GJ & Sinnott ML (2008) Sorting the diverse: The sequence-based classifications of carbohydrate- active enzymes. Biochem J. 1–5, 10.1042/B J20080382 (online paper). 19. Koshland DE (1953) Stereochemistry and mechanism of enzymatic reactions. Biol Rev Camb Philos Soc. 28, 416−436. 20. McCarter JD & Withers SG (1994) Mechanisms of enzymatic glycoside hydrolysis. Curr Opin Struct Biol. 4, 885−892. 21. Zechel DL & Withers SG (2000) Glycosidase mechanisms: anatomy of a finely tuned catalyst. Acc Chem Res. 33, 11−18. 22. Sinnott ML (1990) Catalytic mechanisms of enzymatic glycosyl transfer. Chem Rev. 90, 1171−1202. 23. Vasella A, Davies GJ & Bohm M (2002) Glycosidase mechanisms. Curr Opin Chem Biol. 6, 619−629. 24. Yip VL & Withers SG (2004) Nature's many mechanisms for the degradation of oligosaccharides. Org Biomol Chem. 2, 2707−2713. 25. Rye CS & Withers SG (2000) Glycosidase mechanisms. Curr Opin Chem Biol. 4, 573−580. 26. Lai EC, Morris SA, Street IP & Withers SG (1996) Substituted glycals as probes of glycosidase mechanisms. Bioorg Med Chem. 4, 1929−1937.

37 Introduction, part I

27. Sidhu G, et al. (1999) Sugar ring distortion in the glycosyl-enzyme intermediate of a family GH11 xylanase. Biochemistry. 38, 5346−5354. 28. Sabini E, et al. (1999) Catalysis and specificity in enzymatic glycoside hydrolysis: a 2,5B conformation for the glycosyl-enzyme intermediate revealed by the structure of the Bacillus agaradhaerens family 11 xylanase. Chem Biol. 6, 483−492. 29. Davies GJ, Ducros VM, Varrot A & Zechel DL (2003) Mapping the conformational itinerary of β- glycosidases by X-ray crystallography. Biochem Soc Trans. 31, 523−527. 30. Gloster TM, et al. (2004) Structural studies of the β-glycosidase from Sulfolobus solfataricus in complex with covalently and noncovalently bound inhibitors. Biochemistry. 43, 6101−6109. 31. Lawson SL, Wakarchuk WW & Withers SG (1997) Positioning the acid/base catalyst in a glycosidase: studies with Bacillus circulans xylanase. Biochemistry. 36, 2257−2265. 32. Mark BL, et al. (2001) Crystallographic evidence for substrate-assisted catalysis in a bacterial β- hexosaminidase. J Biol Chem. 276, 10330–10337. 33. Piszkiewicz D & Bruice TC (1968) Glycoside hydrolysis. II. Intramolecular carboxyl and acetamido group catalysis in β-glycoside hydrolysis. J Am Chem Soc. 90, 2156−2163. 34. Macauley MS, Whitworth GE, Debowski AW, Chin D & Vocadlo DJ (2005) O-GlcNAcase uses substrate- assisted catalysis − Kinetic analysis and development of highly selective mechanism-inspired inhibitors. J Biol Chem. 280, 25313–25322. 35. Terwisscha van Scheltinga AC, et al. (1995) Stereochemistry of chitin hydrolysis by a plant chitinase/lysozyme and X-ray structure of a complex with allosamidin: evidence for substrate assisted catalysis. Biochemistry. 34, 15619−15623. 36. Knapp S, et al. (1996) NAGthiazoline, an N-acetyl-β-hexosaminidase inhibitor that implicates acetamido participation. J Am Chem Soc. 118, 6804–6805. 37. Vocadlo DJ & Withers SG (2005) Detailed comparative analysis of the catalytic mechanisms of β-acetyl- glucosaminidases from families 3 and 20 of glycoside hydrolases. Biochemistry. 44, 12809−12818. 38. Drouillard S, Armand S, Davies GJ, Vorgias CE & Henrissat B (1997) Serratia marcescens chitobiase is a retaining glycosidase utilizing substrate acetamido group participation. Biochem J. 328, 945−949. 39. Amaya MF, et al. (2004) Structural insights into the catalytic mechanism of Trypanosoma cruzi trans- sialidase. Structure. 12, 775−784. 40. Watts AG, et al. (2003) Trypanosoma cruzi trans-sialidase operates through a covalent sialyl-enzyme intermediate: tyrosine is the catalytic nucleophile. J Am Chem Soc. 125, 7532−7533. 41. Watson JN, Dookhun V, Borgford TJ & Bennet AJ (2003) Mutagenesis of the conserved active-site tyrosine changes a retaining sialidase into an inverting sialidase. Biochemistry. 42, 12682−12690. 42. Yip VL & Withers SG (2006) Breakdown of oligosaccharides by the process of elimination. Curr Opin Chem Biol. 10, 147–155. 43. Burmeister WP, Cottaz Rollin P, Vasella A & Henrissat B (2000) High resolution X-ray crystallography shows that ascorbate is a cofactor for myrosinase and substitutes for the function of the catalytic base. J Biol Chem. 275, 39385−39393. 44. Roseman S (2001) Reflections on glycobiology. J Biol Chem. 276, 41527−41542. 45. Asano N, Nash RJ, Molyneux RJ & Fleet GWJ (2000) Sugar-mimic glycosidase inhibitors: Natural occurrence, biological activity and prospects for therapeutic application. Tetrahedron Asymmetry. 11, 1645−1680. 46. Asano N (2003) Glycosidase inhibitors: Update and perspectives on practical use. Glycobiology. 13, 93R−104R. 47. de Melo EB, Gomes AD & Carvalho I (2006) α− and β-glucosidase inhibitors: Chemical structure and biological activity. Tetrahedron. 62, 10277−10302. 48. Alper J (2003) Glycobiology. Turning sweet on cancer. Science. 301, 159−160. 49. Tiwari VK, Mishra RC, Sharma A & Tripathi RP (2012) Carbohydrate based potential chemotherapeutic agents: recent developments and their scope in future drug discovery. Mini Rev Med Chem. 12, 1497−1519. 50. Humphries MJ, Matsumoto K, White SL & Olden K (1986) Oligosaccharide modification by swainsonine

38 Activity-based probes for glycosidases

treatment inhibits pulmonary colonization by B16-F10 murine melanoma cells. Proc Natl Acad Sci U S A. 83, 1752−1756. 51. Robina I, Moreno-Vargas AJ, Carmona AT & Vogel P (2004) Glycosidase inhibitors as potential HIV entry inhibitors? Curr Drug Metab. 5, 329−361. 52. Chairat K, Tarning J, White NJ, Lindegardh N (2013) Pharmacokinetic properties of anti-influenza neuraminidase inhibitors. J Clin Pharmacol. 53, 119−139. 53. Block TM, et al. (1994) Secretion of human hepatitis B virus is inhibited by the imino sugar N- butyldeoxynojirimycin. Proc Natl Acad Sci U S A. 91, 2235−2239. 54. Nietupski JB, et al. (2012) Iminosugar-based inhibitors of glucosylceramide synthase prolong survival but paradoxically increase brain glucosylceramide levels in Niemann-Pick C mice. Mol Genet Metab. 105, 621−628. 55. Ashe KM, et al. (2011) Iminosugar-based inhibitors of glucosylceramide synthase increase brain glycosphingolipids and survival in a mouse model of Sandhoff disease. PLoS One. 6, e21758. 56. Yuzwa SA, et al. (2012) Increasing O-GlcNAc slows neurodegeneration and stabilizes tau against aggregation. Nat Chem Biol. 8, 393−399. 57. Lombardo E, et al. (2012) Correction of liver steatosis by a hydrophobic iminosugar modulating glycosphingolipids metabolism. PLoS One. 7, e38520. 58. Bietrix F, et al. (2010) Inhibition of glycosphingolipid synthesis induces a profound reduction of plasma cholesterol and inhibits atherosclerosis development in APOE*3 Leiden and low-density lipoprotein receptor−/− mice. Arterioscler Thromb Vasc Biol. 30, 931−937. 59. Bijl N, et al. (2009) Modulation of glycosphingolipid metabolism significantly improves hepatic insulin sensitivity and reverses hepatic steatosis in mice. Hepatology. 50, 1431−1441. 60. Venier RE & Igdoura SA (2012) Miglustat as a therapeutic agent: prospects and caveats. J Med Genet. 49, 591−597. 61. Benito JM, García Fernández JM & Ortiz Mellet C (2011) Pharmacological chaperone therapy for Gaucher disease: a patent review. Expert Opin Ther Pat. 21, 885−903. 62. Radin NS (1996) Treatment of Gaucher disease with an enzyme inhibitor. Glycoconj J. 13, 153−157. 63. Aerts JM, Hollak CE, Boot RG, Groener JE & Maas M (2006) Substrate reduction therapy of glycosphingolipid storage disorders. J Inherit Metab Dis. 29, 449−456. 64. Fan JQ (2003) A contradictory treatment for lysosomal storage disorders: inhibitors enhance mutant enzyme activity. Trends Pharmacol Sci. 24, 355–360. 65. Brooks DA (2007) Getting into the fold. Nat Chem Biol. 3, 84–85. 66. Boyd RE, et al. (2013) Pharmacological chaperones as therapeutics for lysosomal storage diseases. J Med Chem. 56, 2705–2725. 67. Benito JM, García Fernández JM & Ortiz Mellet C (2011) Pharmacological chaperone therapy for Gaucher disease: a patent review. Expert Opin Ther Pat. 21, 885–903. 68. Witte MD, van der Marel GA, Aerts JM & Overkleeft HS (2011) Irreversible inhibitors and activity-based probes as research tools in chemical glycobiology. Org Biomol Chem. 9, 5908−5926. 69. Sadaghiani AM, Verhelst SH & Bogyo M (2007) Tagging and detection strategies for activity-based proteomics. Curr Opin Chem Biol. 11, 20−28. 70. Shi W & Ma H (2012) Spectroscopic probes with changeable π-conjugated systems. Chem Commun (Camb). 48, 8732−8744. 71. Treibs A & Kreuzer FH (1968) Difluorboryl-Komplexe von Di− und Tripyrrylmethenen. Justus Liebigs Annalen der Chemie. 718, 208−223 72. Schmitt A, Hinkeldey B, Wild M & Jung G (2009) Synthesis of the core compound of the BODIPY dye class: 4,4′-difluoro-4-bora-(3a,4a)-diaza-s-indacene. J Fluoresc. 19, 755−759. 73. Tram K, Yan H, Jenkins HA, Vassiliev S & Bruce D (2009) The synthesis and crystal structure of unsubstituted 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)". Dyes and Pigments 82, 392−395. 74. Arroyo IJ, Hu R, Merino G, Tang BZ & Peña-Cabrera E (2009) The smallest and one of the brightest. Efficient preparation and optical description of the parent borondipyrromethene system. J Org Chem. 74,

39 Introduction, part I

5719−5722. 75. Loudet A & Burgess K (2007) BODIPY dyes and their derivatives: syntheses and spectroscopic properties. Chem. Rev. 107, 4891−4932. 76. Lopatin DE & Voss EW Jr (1971) Fluorescein. Hapten and antibody active-site probe. Biochemistry. 10, 208−213. 77. Ohkuma S & Poole B (1978) Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents. Proc Natl Acad Sci U S A. 75, 3327−3331. 78. Best QA, Sattenapally N, Dyer DJ, Scott CN & McCarroll ME (2013) pH-Dependent Si-Fluorescein Hypochlorous Acid Fluorescent Probe: Spirocycle Ring-Opening and Excess Hypochlorous Acid-Induced Chlorination. J Am Chem Soc. 135, 13365−13370. 79. Huang R, et al. (2013) Multifunctional fluorescent probe for sequential detections of glutathione and caspase-3 in vitro and in cells. Anal Chem. 85, 6203−6207. 80. Arbeloa IL & Ojeda PR (1981) Molecular forms of rhodamine B. Chem Phys Lett. 79, 347−350. 81. Walsh RJ, et al. (2002) Nonphotochemical hole burning spectroscopy of a mitochondrial selective rhodamine dye molecule in normal and cancerous ovarian surface epithelial cells. J Lumin. 98, 115−121. 82. Abugo OO, Nair R & Lakowicz JR (2000) Fluorescence properties of rhodamine 800 in whole blood and plasma. Anal Biochem. 279, 142−150. 83. Miller AK & Tausig F (1964) Biotin-binding by parenterally-administered streptavidin or avidin. Biochem Biophys Res Commun. 14, 210−214. 84. Hofmann K, Wood SW, Brinton CC, Montibeller JA & Finn FM (1980) Iminobiotin affinity columns and their application to retrieval of streptavidin. Proc Natl Acad Sci U S A. 77, 4666−4668. 85. Tsai CS, et al. (2013) Cell-permeable probe for identification and imaging of sialidases. Proc Natl Acad Sci U S A. 110, 2466−2471. 86. Vocadlo DJ, Hang HC, Kim EJ, Hanover JA & Bertozzi CR (2003) A chemical approach for identifying O- GlcNAc-modified proteins in cells. Proc Natl Acad Sci U S A. 100, 9116−9121. 87. Martin DD, et al. (2008) Rapid detection, discovery, and identification of post-translationally myristoylated proteins during apoptosis using a bio-orthogonal azidomyristate analog. FASEB J. 22, 797−806. 88. Kele P, et al. (2009) Clickable fluorophores for biological labeling − with or without copper. Org Biomol Chem. 7, 3486−3490. 89. Willems LI, et al. (2012) Triple bio-orthogonal ligation strategy for simultaneous labeling of multiple enzymatic activities. Angew Chem Int Ed Engl. 51, 4431−4434. 90. Willems LI, et al. (2011) Bioorthogonal chemistry: applications in activity-based protein profiling. Acc Chem Res. 44, 718−729. 91. Fleming SA (1995) Chemical reagents in photoaffinity labeling. Tetrahedron. 51, 12479−12520. 92. Das J (2011) Aliphatic diazirines as photoaffinity probes for proteins: Recent developments. Chem Rev. 111, 4405–4444. 93. Vodovozova EL (2007) Photoaffinity labeling and its application in structural biology. Biochemistry-Moscow 72, 1−20. 94. Sumranjit J & Chung SJ (2013) Recent advances in target characterization and identification by photoaffinity probes. Molecules. 18, 10425−10451. 95. van Scherpenzeel M, et al. (2010) Nanomolar affinity, iminosugar-based chemical probes for specific labeling of lysosomal glucocerebrosidase. Bioorg Med Chem. 18, 267−273. 96. Dubinsky L, Krom BP & Meijler MM (2012) Diazirine based photoaffinity labeling. Bioorg Med Chem. 20, 554−570. 97. Leyva E, de Loera D & Leyva S (2008) Photochemistry of 7-azide-1-ethyl-3-carboxylate-6,8- difluoroquinolone: A novel reagent for photoaffinity labeling. Tetrahedron Lett. 49, 6759−6761. 98. Peng Q, et al. (2005) Synthesis of a photoactivatable phospholipidic probe containing tetrafluorophenylazide. Tetrahedron Lett. 46, 5893−5897. 99. Romaniouk AV, Silva A, Feng J & Vijay IK (2004) Synthesis of a novel photoaffinity derivative of 1- deoxynojirimycin for active site-directed labeling of glucosidase I. Glycobiology. 14, 301−310.

40 Activity-based probes for glycosidases

100. Ford F, Yuzawa T, Platz MS, Matzinger S & Fülscher M (1998) Rearrangement of dimethylcarbene to propene: Study by laser flash photolysis and ab initio molecular orbital theory. J Am Chem Soc. 120, 4430−4438. 101. Toscano JP, Platz MS & Nikolaev V (1995) Lifetimes of simple ketocarbenes. J Am Chem Soc. 117, 4712−4713. 102. Kumar NS & Young RN (2009) Design and synthesis of an all-in-one 3-(1,1-difluoroprop-2-ynyl)-3H- diazirin-3-yl functional group for photo-affinity labeling. Bioorg. Med. Chem. 17, 5388−5395. 103. Platz MS (1995) Comparison of phenylcarbene and phenylnitrene. Acc Chem Res. 28, 487−492. 104. Kuhn CS, Lehmann J, Jung G & Stevanovic S (1992) Investigation of the active-site of Escherichia coli β-D- galactosidase by photoaffinity-labeling. Carbohydr Res. 232, 227−233. 105. Jacobson RH, Zhang XJ, Dubose RF & Matthews BW (1994) Three-dimensional structure of β-galactosidase from Escherichia coli. Nature. 369, 761−766. 106. Rawlings AJ, et al. (2009) Synthesis and biological characterisation of novel N-alkyl-deoxynojirimycin α- glucosidase inhibitors. Chembiochem. 10, 1101−1105. 107. Kuhn CS, Lehmann JC & Sandhoff K (1992) Efficient photoaffinity-labeling of human β-hexosaminidase-α − Synthesis and application of 3-Azi-1-[(2-acetamido-2-deoxy-1-β-D-glucopyranosyl)Thio]butane and 3-Azi- 1-[(2-acetamido-2-deoxy-1-β-D-galactopyranosyl)Thio]butane. Bioconjug Chem. 3, 230−233. 108. Liessem B, et al. (1995) Photoaffinity-labeling of human lysosomal β-hexosaminidase-B—Identification of Glu-355 at the substrate binding site. J Biol Chem. 270, 23693−23699. 109. Vanderhorst GTJ, Rose U, Brossmer R & Verheijen FW (1990) Photoaffinity-labeling of the lysosomal neuraminidase from bovine testis. Febs Lett. 277, 42−44. 110. Kopitz J, Sinz K, Brossmer R, Cantz M (1997) Partial characterization and enrichment of a membrane-bound sialidase specific for gangliosides from human brain tissue. Eur J Biochem. 248, 527−534. 111. Xia Y & Peng L (2013) Photoactivatable lipid probes for studying biomembranes by photoaffinity labeling. Chem Rev. 113, 7880−7929. 112. Sinnott ML & Smith PJ (1976) Active-site-directed irreversible inhibition of E. coli β-galactosidase by the “hot” carbonium ion precursor, β-D-galactopyranosylmethyl-p-nitrophenyltriazene. J Chem Soc Chem Commun. 6, 223−224. 113. Marshall PJ, Sinnott ML, Smith PJ & Widdows D (1981) Active-site-directed irreversible inhibition of glycosidases by the corresponding glycosylmethyl-(p-nitropheny1)triazenes. J Chem Soc Perkin Trans. 2, 366−376. 114. Mega T, Nishijima T & lkenaka T (1990) Active-site-directed inactivation of Aspergillus oryzae β- galactosidase with β-D-galactopyranosyl-methyl-p-nitrophenyltriazene. J Biochem. 107, 641−644. 115. Fowler AV, Zabin I, Sinnott ML & Smith PJ (1978) Methionine 500, the site of covalent attachment of an active site-directed reagent of β-galactosidase. J Biol Chem. 253, 5283−5285. 116. Fowler AV & Smith PJ (1983) The active site regions of lacZ and ebg β-galactosidases are homologous. J Biol Chem 258, 10204−10207. 117. Rempel BP & Withers SG (2008) Covalent inhibitors of glycosidases and their applications in biochemistry and biology. Glycobiology. 18, 570−86. 118. Halazy S, Danzin C, Ehrhard A & Gerhart F (1989) 1,L-Difluoroalkyl glucosides: A new class of enzyme- activated irreversible inhibitors of α-glucosidases. J Am Chem Soc. 111, 3484−3485. 119. Halazy S, Berges V, Ehrhard E & Danzin C (1990) Ortho− and para-(difluoro-methyl) aryl-B-o-glucosides: A new class of enzyme-activated irreversible inhibitors of β-glucosidases. Bioorg Chem. 18, 330−344. 120. Roeser KR & Legler G (1981) Role of sugar hydroxyl groups in glycoside hydrolysis. Cleavage mechanism of deoxyglucosides and related substrates by β-glucosidase A3 from Aspergillus wentii. Biochim Biophys Acta. 657, 321−333. 121. McCarter J, Adam M & Withers SG (1992) Binding energy and catalysis: Fluorinated and deoxygenated glycosides as mechanistic probes of Escherichia coli (lacZ) β-galactosidase. Biochem J. 286, 721−727. 122. Legler G (1990) Glycoside hydrolases: mechanistic information from studies with reversible and irreversible inhibitors. Adv Carbohydr Chem Biochem. 48, 319−384.

41 Introduction, part I

123. Withers SG, Street IP, Bird P & Dolphin DH (1987) 2-Deoxy-2-fluoro-glucosides: A novel class of mechanism-based glucosidase inhibitors. J Am Chem Soc. 109, 7530−7531. 124. Withers SG, Rupitz K & Street IP (1988) 2-Deoxy-2-fluoro-D-glycosyl fluorides. A new class of specific mechanism-based glycosidase inhibitors. J Biol Chem. 263, 7929−7932. 125. Street IP, Rupitz K & Withers SG (1989) Fluorinated and deoxygenated substrates as probes of transition state structure in glycogen phosphorylase. Biochemistry. 28, 1581−1587. 126. Street IP, Kempton JB, Withers SG (1992) Inactivation of a β-glucosidase through the accumulation of a stable 2-deoxy-2-fluoro-α-D-glucopyranosyl-enzyme intermediate: A detailed investigation. Biochemistry. 31, 9970−9978. 127. Cottaz S, Henrissat B & Driguez H (1996) Mechanism-based inhibition and stereochemistry of glucosinolate hydrolysis by myrosinase. Biochemistry. 35, 15256−15259. 128. McCarter JD, Yeung W, Chow J, Dolphin D & Withers SG (1997) Design and synthesis of 2′-deoxy-2′- fluorodisaccharides as mechanism-based glycosidase inhibitors that exploit aglycone specificity. J Am Chem Soc. 119, 5792−5797. 129. Braun C, Brayer GD & Withers SG (1995) Mechanism-based inhibition of yeast α-glucosidase and human pancreatic α-amylase by a new class of inhibitors − 2-Deoxy-2,2-difluoro-α-glycosides. J Biol Chem. 270, 26778−26781. 130. McCarter JD & Withers SG (1996) Unequivocal identification of Asp-214 as the catalytic nucleophile of Saccharomyces cerevisiae α-glucosidase using 5-fluoro glycosyl fluorides. J Biol Chem. 271, 6889−94. 131. Kajimoto T, et al. (1991) Enzyme-catalyzed aldol condensation for asymmetric synthesis of azasugars: synthesis, evaluation, and modeling of glycosidase inhibitors. J Am Chem Soc. 113, 6187−6196. 132. White A, Tull D, Johns K, Withers SG & Rose DR (1996) Crystallographic observation of a covalent catalytic intermediate in a β-glycosidase. Nat Struct Biol. 3, 149−154. 133. Notenboom V, et al. (1998) Insights into transition state stabilization of the β-1,4-glycosidase Cex by covalent intermediate accumulation in active site mutants. Nat Struct Biol. 5, 812−818. 134. Shaikh FA, Müllegger J, He S & Withers SG (2007) Identification of the catalytic nucleophile in Family 42 β- galactosidases by intermediate trapping and peptide mapping: YesZ from Bacillus subtilis. FEBS Lett. 581, 2441−2446. 135. Tarling CA, et al. (2003) Identification of the catalytic nucleophile of the family 29 α-L-fucosidase from Thermotoga maritima through trapping of a covalent glycosyl-enzyme intermediate and mutagenesis. J Biol Chem. 278, 47394−47399. 136. Withers SG & Aebersold R (1995) Approaches to labeling and identification of active-site residues in glycosidases. Protein Sci. 4, 361−372. 137. Withers SG & Street IP (1988) Identification of a covalent α-D-glucopyranosyl enzyme intermediate formed on a β-glucosidase. J Am Chem Soc. 110, 8551−8553. 138. Tull D, Burgoyne DL, Chow DT, Withers SG & Aebersold R (1996) A mass spectrometry-based approach for probing enzyme active sites: Identification of Glu 127 in Cellulomonas fimi exoglycanase as the residue modified by N-bromoacetyl cellobiosylamine. Anal Biochem. 234, 119−125. 139. Michikawa M, et al. (2012) Structural and biochemical characterization of glycoside hydrolase family 79 β- glucuronidase from Acidobacterium capsulatum. J Biol Chem. 287, 14069−14077. 140. Gebler JC. Aebersold R & Withers SG (1992) Glu-537, not Glu-461, is the nucleophile in the active site of (lacZ) β-galactosidase from Escherichia coli. J Biol Chem. 267, 11126−11130. 141. Wang Q, et al. (1993) Glu-280 is the nucleophile in the active site of Clostridium thermocellum Cel C, a Family A endoglucanase. J Biol Chem. 268, 14096−14103. 142. Miao S, et al. (1994) Identification of Glu 340 as the active site nucleophile in human glucocerebrosidase by use of electrospray tandem mass spectrometry. J Biol Chem 269, 10975−10978. 143. Miao S, Ziser L, Aebersold R & Withers SG (1994) Identification of glutamic acid 78 as the active site nucleophile in Bacillus subtilis xylanase using electrospray tandem mass spectrometry. Biochemistry. 33, 7027−7032. 144. Withers SG, et al. (1990) Unequivocal demonstration of the involvement of a glutamate residue as a

42 Activity-based probes for glycosidases

nucleophile in the mechanism of a retaining glycosidase. J Am Chem Soc. 112, 5887−5889. 145. Ly HD, et al. (2000) The synthesis, testing and use of 5-fluoro-α-D-galactosyl fluoride to trap an intermediate on green coffee bean α-galactosidase and identify the catalytic nucleophile. Carbohydr Res. 329, 539−547. 146. Amaya MF, et al. (2004) Structural insights into the catalytic mechanism of Trypanosoma cruzi trans- sialidase. Structure. 12, 775−784. 147. Vocadlo DJ, Mayer C, He S & Withers SG (2000) Mechanism of action and identification of Asp242 as the catalytic nucleophile of Vibrio furnisii N-acetyl-β-D-glucosaminidase using 2-acetamido-2-deoxy-5-fluoro-α- L-idopyranosyl fluoride. Biochemistry. 39, 117−126. 148. Zhang Y, Bommuswamy J & Sinnott ML (1994) Kinetic isotope effect study of transitions states for the hydrolysis of α− and β-glucopyranosyl fluorides. J Am Chem Soc. 116, 7557−7563. 149. Banair NS & Jencks WP (1991) Reactions of anionic nucleophiles with α-D-glucopyranosyl fluoride in

aqueous solution through concerted, ANDN (SN2) mechanism. J Am Chem Soc. 113, 7951−7958. 150. Ly HD & Withers SG (1999) Mutagenesis of glycosidases. Annu Rev Biochem. 68, 487−522 151. Knegtel RMK, et al. (1995) Crystallographic studies of the interaction of cyclodextrin glycosyltransferase from Bacillus circulans strain-251 with natural substrates and products. J Biol Chem. 270, 29256−29264. 152. Mosi R, He S, Uitdehaag J, Dijkstra BW & Withers SG (1997) Trapping and characterization of the reaction intermediate in cyclodextrin glycosyltransferase by use of activated substrates and a mutant enzyme. Biochemistry. 36, 9927−9934. 153. McCarter JD, Adam MJ, Hartman NG & Withers SG (1994) In vivo inhibition of β-glucosidase and β- mannosidase activity in rats by 2-deoxy-2-fluoro-β-glycosyl fluorides and recovery of activity in vivo and in vitro. Biochem J. 301, 343−348. 154. McCarter JD, Adam MJ & Withers SG (1992) Binding energy and catalysis. Fluorinated and deoxygenated glycosides as mechanistic probes of Escherichia coli (lacZ) β-galactosidase. Biochem J. 286, 721−727. 155. Phenix CP, et al. (2010) Imaging of enzyme replacement therapy using PET. Proc Natl Acad Sci U S A. 107, 10842−10847. 156. Legler G (1966) Studies on the action mechanism of glycoside splitting enzymes, I. Presentation and properties of specific inhibitors. Hoppe Seylers Z Physiol Chem. 345, 197−214. 157. Legler G (1968) Untersuchungen zum Wirkungsmechanismus glykosidspaltender Enzyme. III. Markierung des aktiven Zentrums einer β-Glucosidase aus Aspergillus wentii mit [14C]Condurit-B-epoxid. Hoppe-Seyler’s Z. Physiol. Chem. 349, 767−774. 158. Legler G & Hasnain SN (1975) Untersuchungen zum Wirkungsmechanismus glykosidspaltender Enzyme. V. Markierung des aktiven Zentrums der β-Glucosidasen A und B aus dem Süßmandel-Emulsin mit [3H]6- Brom-6-desoxy-condurit-B-epoxid. Hoppe-Seyler’s Z. Physiol. Chem. 351, 25−31. 159. Kanfer JN, Legler G, Sullivan J, Raghavan SS & Mumford RA (1975) Gaucher mouse. Biochem Biophys Res Commun. 67, 85−90. 160. Quaroni A, Gershon-Quaroni E & Semenza G (1975) Tryptic digestion of native small-intestinal sucrase- isomaltase complex: isolation of the sucrase subunit. Eur J Biochem. 52, 481−486. 161. Quaroni A & Semenza G (1976) Partial amino-acid sequences around essential carboxylate in active-sites of intestinal sucrase-isomaltase complex. J Biol Chem. 251, 3250−3253. 162. Quaroni A, Gershon E & Semenza G (1974) Affinity labeling of active-sites in sucrase-isomaltase complex from small-intestine. J Biol Chem. 249, 6424−6433. 163. Hermans MMP, Kroos MA, van Beeumen J, Oostra BA & Reuser AJJ (1991) Human lysosomal α-glucosidase − Characterization of the catalytic site. J Biol Chem. 266, 13507−13512. 164. Quaroni A, Gershon-Quaroni E & Semenza G (1975) Tryptic digestion of native small-intestinal sucrase- isomaltase complex: isolation of the sucrase subunit. Eur J Biochem. 52, 481−486. 165. Quaroni A & Semenza G (1976) Partial amino-acid sequences around essential carboxylate in active-sites of intestinal sucrase-isomaltase complex. J Biol Chem. 251, 3250−3253. 166. Quaroni A, Gershon E & Semenza G (1974) Affinity labeling of active-sites in sucrase-isomaltase complex from small-intestine. J Biol Chem. 249, 6424−6433. 167. Hermans MMP, Kroos MA, van Beeumen J, Oostra BA & Reuser AJJ (1991) Human lysosomal α-glucosidase

43 Introduction, part I

− Characterization of the catalytic site. J Biol Chem. 266, 13507−13512. 168. Iwanami S, et al. (1995) Chemical modification and amino-acid-sequence of active-site in sugar-beet α- glucosidase. Biosci Biotechnol Biochem. 59, 459−463. 169. Kimura A, et al. (1997) A catalytic amino acid and primary structure of active site in Aspergillus niger α- glucosidase. Biosci Biotechnol Biochem. 61, 1091−1098. 170. Okuyama M et al. (2001) Carboxyl group of residue Asp647 as possible proton donor in catalytic reaction of α-glucosidase from Schizosaccharomyces pombe. Eur J Biochem. 268, 2270−2280. 171. Braun H, Legler G, Deshusses J & Semenza G (1977) Stereospecific ring opening of conduritol-B-epoxide by an active site aspartate residue of sucrase-isomaltase. Biochim Biophys Acta. 483, 135−140. 172. Hermans MM, Kroos MA, van Beeumen J, Oostra BA & Reuser AJ (1991) Human lysosomal α-glucosidase. Characterization of the catalytic site. J Biol Chem. 266, 13507−13512. 173. Yang SJ, Ge SG, Zeng YC & Zhang SZ (1985) Inactivation of α-glucosidase by the active-site-directed inhibitor, conduritol B epoxide. Biochim Biophys Acta. 828, 236−240. 174. Iwanami S, et al. (1995) Chemical modification and amino-acid-sequence of active-site in sugar-beet α- glucosidase. Biosci Biotechnol Biochem. 59, 459−463. 175. Lovering AL, Lee SS, Kim YW, Withers SG & Strynadka NCJ (2005) Mechanistic and structural analysis of a family 31 α-glycosidase and its glycosyl-enzyme intermediate. J Biol Chem. 280, 2105−2115. 176. Legler G (1968) Labelling of the active centre of a β-glucosidase. Biochim. Biophys. Acta. 151, 728−729. 177. Mercier D, Olesker A, Gero SD & Barnett JEG (1971) The synthesis of 1L-1,2-anhydro-myo-inositol and 1L− and DL-1,2-anhydro-chiro-inositol. Carbohydr Res. 18, 227−231. 178. Braun H (1976) External yeast β-fructosidase. Affinity labeling of the active site. Biochim Biophys Acta. 452, 452−457. 179. Cavanagh KT, et al. (1985) Goat liver β-mannosidases: molecular properties, inhibition and inactivation of the lysosomal and nonlysosomal forms. Enzyme. 34, 75−82. 180. Legler G & Herrchen M (1981) Active site-directed inhibition of galactosidases by conduritol C epoxides (1,2-anhydro-epi− and neo-inositol). FEBS Lett. 135, 139−144. 181. White WJ Jr, Schray KJ, Legler G, Alhadeff JA (1986) Active-site-directed inactivation of human liver α-L- fucosidase by conduritol C trans-epoxide. Biochim Biophys Acta. 873, 198−203. 182. Herrchen M & Legler G (1984) Identification of an essential carboxylate group at the active-site of lacZ β- galactosidase from Escherichia coli. Eur J Biochem. 138, 527−531. 183. Dinur T, et al. (1986) Human acid β-glucosidase − Isolation and amino-acid-sequence of a peptide containing the catalytic site. Proc Natl Acad Sci USA. 83, 1660−1664. 184. Legler G & Harder A (1978) Amino-acid sequence at the active-site of β-glucosidase A from bitter almonds. Biochim Biophys Acta. 524, 102−108. 185. Gebler JC. Aebersold R & Withers SG (1992) Glu-537, not Glu-461, is the nucleophile in the active site of (lacZ) β-galactosidase from Escherichia coli. J Biol Chem. 267, 11126−11130. 186. Miao SC, et al. (1994) Identification of Glu(340) as the active-site nucleophile in human glucocerebrosidase by use of electrospray tandem mass-spectrometry. J Biol Chem. 269, 10975−10978. 187. He SM & Withers SG (1997) Assignment of sweet almond β-glucosidase as a family 1 glycosidase and identification of its active site nucleophile. J Biol Chem. 272, 24864−24867. 188. Withers SG, Umezawa K (1991) Cyclophellitol − A naturally-occurring mechanism-based inactivator of β- glucosidases. Biochem Biophys Res Commun. 177, 532−537. 189. Atsumi S, Iinuma H, Nosaka C & Umezawa K (1990) Biological-activities of cyclophellitol. J Antibiot. 43, 1579–1585. 190. Atsumi S, et al. (1990) Production, isolation and structure determination of a novel β-glucosidase inhibitor, cyclophellitol, from Phellinus sp. J Antibiot. 43, 49−53. 191. Gloster TM, Madsen R & Davies GJ (2007) Structural basis for cyclophellitol inhibition of a β-glucosidase. Org Biomol Chem. 5, 444−446. 192. Atsumi S, Nosaka C, Ochi Y, Iinuma H & Umezawa K (1993) Inhibition of experimental metastasis by an α- glucosidase inhibitor, 1,6-epi-cyclophellitol. Cancer Res. 53, 4896−4899.

44 Activity-based probes for glycosidases

193. Tai VWF, Fung PH, Wong YS & Shing TKM (1995) Kinetic-studies on cyclophellitol analogs − Mechanism- based inactivators. Biochem Biophys Res Commun. 213, 175−180. 194. Legler G & Bause E (1973) Epoxyalkyl oligo-(1–4)-β-D-glucosides as active-site-directed inhibitors of cellulases. Carbohydr Res. 28, 45−52. 195. Shulman ML, Shiyan SD & Khorlin AY (1976) Specific irreversible inhibition of sweet-almond β-glucosidase by some β-glycopyranosylepoxyalkanes and β-D-glucopyranosyl isothiocyanate. Biochim Biophys Acta. 445, 169−181. 196. Eshdat Y, McKeIvy JF & Sharon N (1973) Identification of aspartic acid 52 as the point of an affinity label in hen egg lysozyme. J Biol Chem 248, 5892−5898. 197. Moult J, Eshdat Y & Sharon N (1973) The identification by X-ray crystallography of the site of attachment of an affinity label to hen egg-white lysozyme. J Mol Biol 75, 1−4. 198. Eshdat Y, Dunn A & Sharon N (1974) Chemical conversion of aspartic acid 52, a catalytic residue in hen egg- white lysozyme, to homoserine. Proc Nail Acad Sci USA 71, 1658−1663. 199. Clarke AJ, Strating H (1989) Affinity labeling of Schizophyllum commune cellulase with [3H]-4,5-epoxypentyl β-cellobioside: Synthesis of inhibitor and stoichiometry of interaction. Carbohydr Res 188, 245−250. 200. Hoj PB, Condron R, Traeger JC, McAuliffe JC & Stone BA (1992) Identification of glutamic-acid 105 at the active-site of Bacillus amyloliquefaciens 1,3-1,4-β-D-glucan 4-glucanohydrolase using epoxide-based inhibitors. J Biol Chem. 267, 25059−25066. 201. Hoj PB, Rodriguez EB, Iser JR, Stick RV & Stone BA (1991) Active site-directed inhibition by optically pure epoxyalkyl cellobiosides reveals differences in active-site geometry of two 1,3-1,4-β-D-glucan 4- glucanohydrolases − The importance of epoxide stereochemistry for enzyme inactivation. J Biol Chem. 266, 11628−11631. 202. Keitel T, Simon O, Borriss R & Heinemann U (1993) Molecular and active-site structure of a Bacillus 1,3-1,4- β-glucanase. Proc Natl Acad Sci USA. 90, 5287−5291. 203. Macarron R, Vanbeeumen J, Henrissat B, Delamata I & Claeyssens M (1993) Identification of an essential glutamate residue in the active-site of endoglucanase-III from Trichoderma reesei. Febs Lett. 316, 137−140. 204. Havukainen R, Torronen A, Laitinen T & Rouvinen J (1996) Covalent binding of three epoxyalkyl xylosides to the active site of endo-1,4-xylanase II from Trichoderma reesei. Biochemistry. 35, 9617−9624. 205. Sulzenbacher G, Schulein M & Davies GJ (1997) Structure of the endoglucanase I from Fusarium oxysporum: Native, cellobiose, and 3,4-epoxybutyl β-D-cellobioside-inhibited forms, at 2.3 Ångstrom resolution. Biochemistry. 36, 5902−5911. 206. Laitinen T, Rouvinen J & Perakyla M (2000) Inversion of the roles of the nucleophile and acid/base catalysts in the covalent binding of epoxyalkyl xyloside inhibitor to the catalytic glutamates of endo-1,4-β-xylanase (XYNII): A molecular dynamics study. Protein Eng. 13, 247−252. 207. Shulman ML, Shiyan SD & Khorlin AY (1976) Specific irreversible inhibition of sweet-almond β-glucosidase by some β-glycopyranosylepoxyalkanes and β-D-glucopyranosyl isothiocyanate. Biochim Biophys Acta. 445, 169−181. 208. Caron G & Withers SG (1989) Conduritol aziridine − A new mechanism-based glucosidase inactivator. Biochem Biophys Res Commun. 163, 495−499. 209. Tong MK, Ganem B (1988) A potent new class of active-site-directed glycosidase inactivators. J Am Chem Soc. 110, 312−313. 210. Ho MW, O'Brien JS, Radin NS & Erickson JS (1973) Glucocerebrosidase: reconstitution of activity from macromolecular components. Biochem J. 131, 173−176. 211. Pentchev PG, Brady RO, Hibbert SR, Gal AE & Shapiro D (1973) Isolation and characterization of glucocerebrosidase from human placental tissue. J Biol Chem. 248, 5256−5261. 212. Ho MW (1973) Identity of 'acid' β-glucosidase and glucocerebrosidase in human spleen. Biochem J. 136, 721−729. 213. Glew RH, Christopher AR, Schnure FW & Lee RE (1974) The occurrence of β-glucocerebrosidase activity in the glucocerebroside-rich deposits of Gaucher's disease. Arch Biochem Biophys. 160, 162−167.

45 Introduction, part I

214. Thudichum JLW (1884) A Treatise on the Chemical Constitution of Brain. Bailliere, Tindall, and Cox: London (United Kingdom). 215. Sonnino S & Prinetti A (2013) Membrane domains and the "lipid raft" concept. Curr Med Chem. 20, 4−21. 216. Chatterjee S, Sweeley CC & Velicer LF (1975) Glycosphingolipids of human KB cells grown in monolayer, suspension, and synchronized cultures. J Biol Chem 250, 61−66. 217. Okazaki T, Bell RM & Hannun YA (1989) Sphingomyelin turnover induced by vitamin D3 in HL-60 cells. Role in cell differentiation. J Biol Chem. 264, 19076−19080. 218. Venable ME, Lee JY, Smyth MJ, Bielawska A& Obeid LM (1995) Role of ceramide in cellular senescence. J Biol Chem. 270, 30701−30708. 219. Hetz CA, et al. (2002) Caspase-dependent initiation of apoptosis and necrosis by the Fas receptor in lymphoid cells: onset of necrosis is associated with delayed ceramide increase. J Cell Sci. 115, 4671−4683. 220. Adam D, Heinrich M, Kabelitz D & Schutze S (2002). Ceramide: does it matter for T cells? Trends Immunol. 23, 1−4. 221. Obeid LM, Linardic CM, Karolak LA & Hannun YA (1993). Programmed cell death induced by ceramide. Science. 259, 1769−1771. 222. Iwabuchi K, Yamamura S, Prinetti A, Handa K & Hakomori S (1998) GM3-enriched microdomain involved in cell adhesion and signal transduction through carbohydrate-carbohydrate interaction in mouse melanoma B16 cells. J Biol Chem 273, 9130−9138. 223. Zhang X & Kiechle FL (2004) Glycosphingolipids in health and disease. Ann Clin Lab Sci. 34, 3−13. 224. Liang YJ, et al. (2013) Differential expression profiles of glycosphingolipids in human breast cancer stem cells vs. cancer non-stem cells. Proc Natl Acad Sci U S A. 110, 4968−4973. 225. Beckham TH, Cheng JC, Marrison ST, Norris JS & Liu X (2013) Interdiction of sphingolipid metabolism to improve standard cancer therapies. Adv Cancer Res. 117, 1−36. 226. Morad SA & Cabot MC (2013) Ceramide-orchestrated signalling in cancer cells. Nat Rev Cancer. 13, 51−65. 227. Aerts JM, et al. (2011) Glycosphingolipids and insulin resistance. Adv Exp Med Biol.721, 99−119. 228. Samad F, Badeanlou L, Shah C & Yang G (2011) Adipose tissue and ceramide biosynthesis in the pathogenesis of obesity. Adv Exp Med Biol. 721, 67−86. 229. Brice SE & Cowart LA (2011) Sphingolipid metabolism and analysis in metabolic disease. Adv Exp Med Biol. 721, 1−17. 230. Neufeld EF (1991) Lysosomal storage diseases. Annu Rev Biochem. 60, 257−280. 231. Aerts JM, Hollak C, Boot RG & Groener A (2003) Biochemistry of glycosphingolipid storage disorders: implications for therapeutic intervention. Phil Trans R Soc Lond B. 358, doi 10.1098/rstb.2003. 232. Ballabio A & Gieselmann V (2009) Lysosomal disorders: From storage to cellular damage. Biochim Biophys Acta 1793, 684−696. 233. Schulze H & Sandhoff K (2011) Lysosomal lipid storage diseases. Cold Spring Harb Perspect Biol. 3, a004804. 234. Merrill AH Jr (2011) Sphingolipid and glycosphingolipid metabolic pathways in the era of sphingolipidomics. Chem Rev. 111, 6387−6422. 235. Quehenberger O, et al. (2010) Lipidomics reveals a remarkable diversity of lipids in human plasma. J Lipid Res. 51, 3299−3305. 236. Breathnach CS (2001) Johann Ludwig Wilhelm Thudichum 1829−1901, bane of the Protagonisers. Hist Psychiatry. 12, 283−296. 237. Wennekes T, et al. (2009) Glycosphingolipids − nature, function, and pharmacological modulation. Angew Chem Int Ed Engl. 48, 8848−8869. 238. Merrill AH Jr, Wang MD, Park M & Sullards MC (2007) (Glyco)sphingolipidology: an amazing challenge and opportunity for systems biology. Trends Biochem. Sci. 32, 457−468. 239. Sud M, et al. (2006) LMSD: LIPID MAPS structure database. Nucleic Acids Res. 35, D527−532. 240. Pruett ST, et al. (2008) Biodiversity of sphingoid bases ("sphingosines") and related amino alcohols. J Lipid Res. 49, 1621−1639. 241. Zheng W, et al. (2006) Ceramides and other bioactive sphingolipid backbones in health and disease: lipidomic analysis, metabolism and roles in membrane structure, dynamics, signaling and autophagy.

46 Activity-based probes for glycosidases

Biochim Biophys Acta. 1758, 1864−1884. 242. Pewzner-Jung Y, Ben-Dor S & Futerman AH (2006) When do Lasses (longevity assurance genes) become CerS (ceramide synthases)?: Insights into the regulation of ceramide synthesis. J Biol Chem. 281, 25001−25005. 243. De Matteis MA, Di Campli A & D'Angelo G (2007) Lipid-transfer proteins in membrane trafficking at the Golgi complex. Biochim Biophys Acta. 1771, 761−7688. 244. Hanada K, Kumagai K, Tomishige N & Yamaji T (2009) CERT-mediated trafficking of ceramide. Biochim Biophys Acta. 1791, 684−691 245. Giussani P, et al. (2008) Ceramide traffic in C6 glioma cells: evidence for CERT-dependent and independent transport from ER to the Golgi apparatus. Biochim Biophys Acta. 1781, 40−51. 246. Coutinho PM & Henrissat B (1999) Life with no sugars? J Mol Microbiol Biotechnol. 1, 307−308. 247. Ichikawa S, Sakiyama H, Suzuki G, Hidari KI & Hirabayashi Y (1996) Expression cloning of a cDNA for human ceramide glucosyltransferase that catalyzes the first glycosylation step of glycosphingolipid synthesis. Proc Natl Acad Sci U S A. 93, 4638−4643. Erratum in: Proc Natl Acad Sci U S A. 93, 12654. 248. Ichikawa S & Hirabayashi Y (1998) Glucosylceramide synthase and glycosphingolipid synthesis. Trends Cell Biol. 8, 198−202. 249. Marks DL, et al. (1999) Oligomerization and topology of the Golgi membrane protein glucosylceramide synthase. J Biol Chem. 274, 451−456. 250. Buton X, et al. (2002) Transbilayer movement of monohexosylsphingolipids in endoplasmic reticulum and Golgi membranes. Biochemistry. 41, 13106−13115. 251. Eckford PD & Sharom FJ (2005) The reconstituted P-glycoprotein multidrug transporter is a flippase for glucosylceramide and other simple glycosphingolipids. Biochem J. 389, 517−526. 252. D'Angelo G, et al. (2007) Glycosphingolipid synthesis requires FAPP2 transfer of glucosylceramide. Nature. 449, 62−67. 253. De Matteis MA & Luini A (2008) Exiting the Golgi complex. Nat Rev Mol Cell Biol. 9, 273−284. 254. Kolter T & Sandhoff K (2006) Sphingolipid metabolism diseases. Biochim Biophys Acta. 1758, 2057−2079. 255. Mayor S & Pagano RE (2007) Pathways of clathrin-independent endocytosis. Nat Rev Mol Cell Biol. 8, 603−612. 256. Singh RD, et al. (2003) Selective caveolin-1-dependent endocytosis of glycosphingolipids. Mol Biol Cell. 14, 3254−3565. 257. Schulze H, Kolter T & Sandhoff K (2009) Principles of lysosomal membrane degradation: Cellular topology and biochemistry of lysosomal lipid degradation. Biochim Biophys Acta. 1793, 674−683. 258. Kolter T & Sandhoff K (2005) Principles of lysosomal membrane digestion: stimulation of sphingolipid degradation by sphingolipid activator proteins and anionic lysosomal lipids. Annu Rev Cell Dev Biol. 21, 81−103. 259. Grabowski GA (2012) Gaucher disease and other storage disorders. Hematology Am Soc Hematol Educ Program. 2012, 13−18. 260. Gaucher PCE (1882) De l'epithelioma primitif de la rate, hypertrophie idiopathique de la rate sans leucemie. PhD thesis. Paris (France). 261. Lieb H (1924). Cerebrosidspeicherung bei Splenomegalie, Typus Gaucher. Hoppe-Seylers Z. physiol. Chem. 140, 305−315. 262. Aghion H (1934) La maladie de Gaucher dans l’enfance. PhD Thesis. Paris (France). 263. Rosenberg A & Chargaff E (1958) A re-investigation of the cerebroside deposited in Gaucher's disease. J Biol Chem. 233, 1323−1326. 264. Agranoff BW, Radin N & Suomi W (1962) Enzymic oxidation of cerebrosides: studies on Gaucher's disease. Biochim Biophys Acta. 57, 194−196. 265. Trams EG & Brady RO (1960) Cerebroside synthesis in Gaucher's disease. J Clin Invest. 39, 1546−1550. 266. Brady RO, Kanfer J & Shapiro D (1965) The metabolism of glucocerebrosides. Purification and properties of a glucocerebroside cleaving enzyme from spleen tissue. J Biol Chem. 240, 39−43.

47 Introduction, part I

267. Brady RO, Kanfer JN & Shapiro D (1965) Metabolism of glucocerebrosides. Evidence of an enzymatic deficiency in Gaucher's disease. Biochem Biophys Res Commun. 18, 221−225. 268. Brady RO, Kanfer JN, Bradley RM & Shapiro D (1966) Demonstration of a deficiency of glucocerebroside- cleaving enzyme in Gaucher's disease. J Clin Invest. 45, 1112−1115. 269. Tsuji S, et al. (1986) Nucleotide sequence of cDNA containing the complete coding sequence for human lysosomal glucocerebrosidase. J Biol Chem. 261, 50−53. 270. Sorge J, West C, Westwood B & Beutler E (1985) Molecular cloning and nucleotide sequence of human glucocerebrosidase cDNA. Proc Natl Acad Sci U S A. 82, 7289−7293. 271. Reiner O, Wilder S, Givol D & Horowitz M (1987) Efficient in vitro and in vivo expression of human glucocerebrosidase cDNA. DNA. 6, 101−108. 272. van Weely S, Brandsma M, Strijland A, Tager JM & Aerts JM (1993) Demonstration of the existence of a second, non-lysosomal glucocerebrosidase that is not deficient in Gaucher disease. Biochim Biophys Acta. 1181, 55−62. 273. Yildiz Y, et al. (2006) Mutation of β-glucosidase 2 causes glycolipid storage disease and impaired male fertility. J Clin Invest. 116, 2985−2994 274. Boot RG, et al. (2007) Identification of the non-lysosomal glucosylceramidase as β-glucosidase 2. J Biol Chem. 282, 1305−1312. 275. Skovbjerg H, Sjöström H & Norén O (1981) Purification and characterisation of amphiphilic lactase/phlorizin hydrolase from human small intestine. Eur J Biochem. 114, 653–661. 276. Wacker H, Keller P, Falchetto R, Legler G & Semenza G (1992) Location of the two catalytic sites in intestinal lactase-phlorizin hydrolase. Comparison with sucrase-isomaltase and with other glycosidases, the membrane anchor of lactase-phlorizin hydrolase. J Biol Chem. 267, 18744–18752. 277. Gopalan V, Pastuszyn A, Galey WR Jr & Glew RH (1992) Exolytic hydrolysis of toxic plant glucosides by guinea pig liver cytosolic β-glucosidase. J Biol Chem. 267, 14027−14032. 278. Yahata K, et al. (2000) Molecular cloning and expression of a novel klotho-related protein. J Mol Med (Berl). 78, 389−394. 279. Walvoort MT, et al. (2012) Tuning the leaving group in 2-deoxy-2-fluoroglucoside results in improved activity-based retaining β-glucosidase probes. Chem Commun (Camb). 48, 10386−10388. 280. Witte MD, et al. (2011) Activity-based profiling of retaining β-glucosidases: a comparative study. Chembiochem. 12, 1263−1269. 281. Witte MD, et al. (2010) Ultrasensitive in situ visualization of active glucocerebrosidase molecules. Nat Chem Biol. 6, 907−913. 282. Kallemeijn WW, et al. (2012) Novel activity-based probes for broad-spectrum profiling of retaining β- exoglucosidases in situ and in vivo. Angew Chem Int Ed Engl. 51, 12529−12533. 283. Daniels LB, Glew RH, Radin NS & Vunnam RR (1980) A revised fluorometric assay for Gauchers-disease using conduritol-β-epoxide with liver as the source of β-glucosidase. Clin Chim Acta. 106, 155−163. 284. Ridley CM, et al. (2013) β-Glucosidase 2 (GBA2) activity and imino sugar pharmacology. J Biol Chem. 288, 26052−26066. 285. Jonsson LM, et al (1987) Biosynthesis and maturation of glucocerebrosidase in Gaucher fibroblasts. Eur J Biochem 164, 171–179. 286. Aerts JM, et al. (1986). Efficient routing of glucocerebrosidase to lysosomes requires complex oligosaccharide chain formation. Biochem Biophys Res Commun. 141, 452−458. 287. Aerts JM, et al. (1988) Glucocerebrosidase, a lysosomal enzyme that does not undergo oligosaccharide phosphorylation. Biochim Biophys Acta. 964, 303−308. 288. Aerts JM, et al. (1988) Characterization of glucocerebrosidase in peripheral blood cells and cultured blastoid cells. Exp Cell Res. 177, 391−398. 289. Van Weely S, et al. (1990) Function of oligosaccharide modification in glucocerebrosidase, a membrane- associated lysosomal hydrolase. Eur J Biochem. 191, 669−677. 290. Atsumi S, Nosaka C, Iinuma H & Umezawa K (1992) Inhibition of glucocerebrosidase and induction of neural abnormality by cyclophellitol in mice. Arch Biochem Biophys. 297, 362−367.

48 Activity-based probes for glycosidases

291. Atsumi S, Nosaka C, Iinuma H & Umezawa K (1993) Accumulation of tissue glucosylsphingosine in Gaucher-like mouse induced by the glucosylceramidase inhibitor cyclophellitol. Arch Biochem Biophys. 304, 302−304. 292. Yatziv S, Newburg DS, Livni N, Barfi G & Kolodny EH (1988) Gaucher-like changes in human-blood derived macrophages induced by β-glucocerebrosidase inhibition. J Lab Clin Med. 111, 416−420. 293. Das PK, Murray GJ, Gal AE & Barranger JA (1987) Glucocerebrosidase deficiency and lysosomal storage of glucocerebroside induced in cultured macrophages. Exp Cell Res. 168, 463−474. 294. Schueler UH, et al. (2004) Correlation between enzyme activity and substrate storage in a cell culture model system for Gaucher disease. J Inherit Metab Dis. 27, 649−658. 295. Takagi Y, Kriehuber E, Imokawa G, Elias PM & Holleran WM (1999) β-Glucocerebrosidase activity in mammalian stratum corneum. J Lipid Res. 40, 861−869. 296. Holleran WM, Takagi Y & Uchida Y (2006) Epidermal sphingolipids: Metabolism, function, and roles in skin disorders. Febs Lett. 580, 5456−5466. 297. Yoshida N, Sawada E & Imokawa G (2012) A reconstructed human epidermal keratinization culture model to characterize ceramide metabolism in the stratum corneum. Arch Dermatol Res. 304, 563−577. 298. Lecourt S, et al. (2012) Bone marrow microenvironment in an in vitro model of Gaucher disease: consequences of glucocerebrosidase deficiency. Stem Cells Dev. 21, 239−248. 299. Imokawa G, Akasaki S, Hattori M & Yoshizuka N (1986) Selective recovery of deranged water-holding properties by stratum corneum lipids. J Invest Dermatol. 87, 758–761. 300. Imokawa G, Akasaki S, Minematsu Y & Kawai M (1989) Importance of intercellular lipids in water-retention properties of the stratum corneum: induction and recovery study of surfactant dry skin. Arch Dermatol Res. 281, 45–51. 301. Imokawa G & Hattori M (1985) A possible function of structural lipids in the water-holding properties of the stratum corneum. J Invest Dermatol. 84, 282–284. 302. Grubauer G, Feingold KR, Harris RM & Elias PM (1989) Lipid content and lipid type as determinants of the epidermal permeability barrier. J Lipid Res. 30, 89–96. 303. Holleran WM, et al. (1991) Regulation of epidermal sphingolipid synthesis by permeability barrier function. J Lipid Res. 32, 1151–1158. 304. Lampe MA, et al. (1983) Human stratum corneum lipids: characterization and regional variations. J Lipid Res. 24, 120–130. 305. Schwarz A, Rapaport E, Hirschberg K & Futerman AH (1995) A regulatory role for sphingolipids in neuronal growth − inhibition of sphingolipid synthesis and degradation have opposite effects on axonal branching. J Biol Chem. 270, 10990−10998. 306. Prence EM, Chaturvedi P & Newburg DS (1996) In vitro accumulation of glucocerebroside in neuroblastoma cells: A model for study of Gaucher disease pathobiology. J Neurosci Res. 43, 365−371. 307. Kanfer JN, Legler G, Sullivan J, Raghavan SS & Mumford RA (1975) Gaucher mouse. Biochem Biophys Res Commun. 67, 85−90. 308. Kanfer JN, Stephens MC, Singh H & Legler G (1982) The Gaucher mouse. Prog Clin Biol Res. 95, 627−644. 309. Stephens MC, Bernatsky A, Burachinsky V, Legler G & Kanfer JN (1978) Gaucher mouse − Differential action of conduritol-B epoxide and reversibility of its effects. J Neurochem. 30, 1023−1027. 310. Trapero A & Llebaria A (2013) Glucocerebrosidase inhibitors for the treatment of Gaucher disease. Future Med Chem. 5, 573−590. 311. Sawkar AR, D’Haeze W & Kelly JW (2006) Therapeutic strategies to ameliorate lysosomal storage disorders − A focus on Gaucher disease. Cell Mol Life Sci. 63, 1179−1192. 312. Butters TD (2007) Gaucher disease. Curr Opin Chem Biol. 11, 412−418. 313. Araki K & Nagata K (2011) Protein folding and quality control in the ER. Cold Spring Harb Perspect Biol. 3, a007526. 314. Wang F, Song W, Brancati G & Segatori L (2011) Inhibition of endoplasmic reticulum-associated degradation rescues native folding in loss of function protein misfolding diseases. J Biol Chem. 286, 43454−43464.

49 Introduction, part I

315. Hruska KS, LaMarca ME, Scott CR & Sidransky E (2008) Gaucher disease: mutation and polymorphism spectrum in the glucocerebrosidase gene (GBA). Hum Mutat. 29, 567−583. 316. Ron I & Horowitz M (2005) ER retention and degradation as the molecular basis underlying Gaucher disease heterogeneity. Hum Mol Genet. 14, 2387−2398. 317. Tybulewicz VLJ, et al. (1992) Animal-model of Gaucher’s disease from targeted disruption of the mouse glucocerebrosidase gene. Nature. 357, 407−410. 318. Liu YJ, et al. (1998) Mice with type 2 and 3 Gaucher disease point mutations generated by a single insertion mutagenesis procedure (SIMP). Proc Natl Acad Sci USA. 95, 2503−2508. 319. Beutler E, West C, Torbett BE & Deguchi H (2002) A chimeric mouse model of Gaucher disease. Mol Med. 8, 247−250. 320. Mizukami H, et al. (2002) Systemic inflammation in glucocerebrosidase-deficient mice with minimal glucosylceramide storage. J Clin Invest. 109, 1215−1221. 321. Xu YH, Quinn B, Witte D & Grabowski GA (2003) Viable mouse models of acid β-glucosidase deficiency − The defect in Gaucher disease. Am J Pathol. 163, 2093−2101. 322. Sun Y, Quinn B, Witte DP & Grabowski GA (2005) Gaucher disease mouse models: Point mutations at the acid β-glucosidase locus combined with low-level prosaposin expression lead to disease variants. J Lipid Res. 46, 2102−2113. 323. Sinclair GB, et al. (2007) Generation of a conditional knockout of murine glucocerebrosidase: Utility for the study of Gaucher disease. Mol Genet Metab. 90, 148−156. 324. Enquist IB, et al. (2007) Murine models of acute neuronopathic Gaucher disease. Proc Natl Acad Sci USA. 104, 17483−17488. 325. Mistry PK, et al. (2010) Glucocerebrosidase gene-deficient mouse recapitulates Gaucher disease displaying cellular and molecular dysregulation beyond the macrophage. Proc Natl Acad Sci U S A. 107, 19473−19478. 326. Sanders A, et al. (2013) Transgenic mice expressing human glucocerebrosidase variants: utility for the study of Gaucher disease. Blood Cells Mol Dis. 51, 109−115. 327. Beutler E (1988) Gaucher Disease. Blood Rev. 2, 59−70. 328. Morales LE (1996) Gaucher’s Disease: A review. Ann Pharmacother. 30, 381–388. 329. Cox TM & Schofield JP (1997) Gaucher's disease: clinical features and natural history. Baillieres Clin Haematol. 10, 657−689. 330. Ihler GM, Glew RH & Schnure FW (1973) Enzyme loading of erythrocytes. Proc Natl Acad Sci U S A. 70, 2663−2666. 331. Brady RO, Pentchev PG & Gal AG (1975) Investigations in enzyme replacement therapy in lipid storage diseases. Fed Proc. 34, 1310−1315. 332. Brady RO, Pentchev PG, Gal AE, Hibbert SR & Dekaban AS (1974) Replacement therapy for inherited enzyme deficiency. Use of purified glucocerebrosidase in Gaucher's disease. N Engl J Med. 291, 989−993. 333. Pentchev PG, Brady RO, Hibbert SR, Gal AE & Shapiro D (1973) Isolation and characterization of glucocerebrosidase from human placental tissue. J Biol Chem. 248, 5256−5256 334. Ezekowitz, RAB & Stahl PD (1988) The structure and function of vertebrate mannose lectin-like proteins. J Cell Sci. 9, 121−133. 335. Wileman TE, Lennartz MR & Stahl PD (1986) Identification of the macrophage mannose receptor as a 175- kDa membrane protein. Proc Natl Acad Sci U S A. 83, 2501−2505. 336. Lennartz MR, Cole FS, Shepherd VL, Wileman TE & Stahl PD (1987) Isolation and characterization of a mannose-specific endocytosis receptor from human placenta. J Biol Chem. 262, 9942−9944. 337. Magnusson S & Berg T (1989) Extremely rapid endocytosis mediated by the mannose receptor of sinusoidal endothelial rat liver cells. Biochem J. 257, 651−656. 338. Stang E, Kindberg GM, Berg T & Roos N (1990) Endocytosis mediated by the mannose receptor in liver endothelial cells. An immunocytochemical study. Eur J Cell Biol. 52, 67−76. 339. Takahashi K, Donovan MJ, Rogers RA & Ezekowitz RA (1998) Distribution of murine mannose receptor expression from early embryogenesis through to adulthood. Cell Tissue Res. 292, 311−323.

50 Activity-based probes for glycosidases

340. Doebber TW, et al. (1982) Enhanced macrophage uptake of synthetically glycosylated human placental β- glucocerebrosidase. J Biol Chem. 257, 2193−2199. 341. Barton NW, et al. (1991) Replacement therapy for inherited enzyme deficiency–macrophage-targeted glucocerebrosidase for Gaucher’s disease. N Engl J Med. 324, 1464−1470. 342. Brumshtein B, et al. (2010) Characterization of gene-activated human acid-β-glucosidase: Crystal structure, glycan composition, and internalization into macrophages. Glycobiology. 20, 24−32. 343. Aviezer D, et al. (2009) A plant-derived recombinant human glucocerebrosidase enzyme--a preclinical and phase I investigation. PLoS One. 4, e4792. 344. Zimran A, et al. (1995) Replacement therapy with imiglucerase for type 1 Gaucher's disease. Lancet. 345, 1479−1480. 345. Grabowski GA, Leslie N & Wenstrup R (1998) Enzyme therapy for Gaucher disease: the first 5 years. Blood Rev. 12, 115−133. 346. Tekoah Y, et al. (2013) Glycosylation and functionality of recombinant β-glucocerebrosidase from various production systems. Biosci Rep. [Epub ahead of print] 347. Berger J, et al. (2012) The uptake of recombinant glucocerebrosidases by blood monocytes from type 1 Gaucher disease patients is variable. Br J Haematol. 157, 274−277. 348. Xu YH, Sun Y, Barnes S & Grabowski GA (2010) Comparative therapeutic effects of velaglucerase alfa and imiglucerase in a Gaucher disease mouse model. PLoS One. 5, e10750. 349. Phenix CP, et al. (2010) Imaging of enzyme replacement therapy using PET. Proc Natl Acad Sci U S A. 107, 10842−10847. 350. Van Patten SM, et al. (2007) Effect of mannose chain length on targeting of glucocerebrosidase for enzyme replacement therapy of Gaucher disease. Glycobiology. 17, 467−478. 351. Piepenhagen PA, et al. (2010) Use of direct fluorescence labeling and confocal microscopy to determine the biodistribution of two protein therapeutics, Cerezyme and Ceredase. Microsc Res Tech. 73, 694−703.

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