FULL PAPER DOI: 10.1002/chem.201303158

Fullerene-sp2-Iminosugar Balls as Multimodal Ligands for Lectins and Glycosidases: A Mechanistic Hypothesis for the Inhibitory Multivalent Effect Roco Rsquez-Cuadro,[a] Jos M. Garca Fernndez,*[b] Jean-FranÅois Nierengarten,*[c] and Carmen Ortiz Mellet*[a] Abstract: Concerted functioning of lectins and carbohydrate-processing enzymes, mainly glycosidases, is essential in maintaining life. It was commonly assumed that the mechanisms by which each class of protein recognizes their cognate sugar partners are intrinsically different: multivalency is a characteristic feature of carbohydrate–lectin interactions, whereas glycosidases bind to their substrates or substrate-analogue inhibitors in monovalent form. Recent observations on the glycosidase inhibitory potential of multivalent glycomimetics have questioned this paradigm and led to postulate an inhibitory multivalent effect. Here the mechanisms at the origin of this phenomenon have been investigated. A d-gluco-configured sp2-iminosugar glycomimetic motif, namely 1-amino-5N,6O-oxomethylydenenojirimycin (1N-ONJ), behaving, simultaneously, as a ligand of

peanut agglutinin (PNA) lectin and as an inhibitor of several glycosidases, has been identified. Both the 1N-ONJ– lectin- and 1N-ONJ–glycosidase-recognition processes have been found to be sensitive to multivalency, which has been exploited in the design of a lectin–glycosidase competitive assay to explore the implication of catalytic and non-glycone sites in enzyme binding. A set of isotropic dodecavalent C60-fullerene–sp2-iminosugar balls incorporating matching or mismatching motifs towards several glycosidases (inhitopes) was synthesized for that purpose, thereby preventing differences in binding modes arising from orientational preferences. The data supports Keywords: fullerenes · glycosidases · iminosugars · inhibitors · multivalency

Introduction Glycoside hydrolases, the enzymes that catalyze the cleavage of glycosidic bonds in oligosaccharides and glycoconju[a] R. Rsquez-Cuadro, Prof. C. Ortiz Mellet Departamento de Qumica Orgnica, Facultad de Qumica Universidad de Sevilla, C/Prof. Garca Gonzlez 1 41012 Sevilla (Spain) E-mail: [email protected] [b] Prof. J. M. Garca Fernndez Instituto de Investigaciones Qumicas (IIQ) CSIC - Universidad de Sevilla, Av. Amrico Vespucio 49 Isla de la Cartuja, 41092 Sevilla (Spain) E-mail: [email protected] [c] Prof. J.-F. Nierengarten Laboratoire de Chimie des Matriaux Molculaires Universit de Strasbourg et CNRS (UMR 7509) Ecole Europenne de Chimie, Polymres et Matriaux 25 rue Becquerel, 67087 Strasbourg (France) E-mail: [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201303158.

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that: 1) multivalency allows modulating the affinity and selectivity of a given inhitope towards glycosidases; 2) multivalent presentation can switch on the inhibitory capacity for some inhitope– glycosidase pairs, and 3) interactions of the multivalent inhibitors with non-glycone sites is critical for glycosidase recognition. The ensemble of results point to a shift in the binding mode on going from monovalent to multivalent systems: in the first case a typical ’’key– lock’’ model involving, essentially, the high-affinity active site can be assumed, whereas in the second, a lectinlike behavior implying low-affinity non-glycone sites probably operates. The differences in responsiveness to multivalency for different glycosidases can then be rationalized in terms of the structure and accessibility of the corresponding carbohydrate-binding regions.

gates, play important roles in biological processes that result in the maintenance of life, including the degradation of polysaccharides, the lysosomal catabolism of glycoconjugates, and the biosynthesis of the oligosaccharide units in glycoproteins and glycolipids, which in turn are involved in a plethora of cell communication events.[1–3] In addition to the catalytic domain, some glycosidases also contain noncatalytic carbohydrate-binding modules (CBMs) that have important substrate-targeting, site-specific directing or membrane-anchoring functions.[4] CBMs have also been found not directly appended to enzymatically active modules;[5] this blurs their distinction from lectins, namely nonenzymatic proteins, which are specific for the mono- or oligosaccharide motifs present in glycans that are displayed on glycoproteins.[6] Studies in the field of carbohydrate-binding proteins and glycosidases are quickly developing and there are many examples of inter- and intramolecular direct co-functioning, with extended prospects in medicine and biotechnology.[7–9] In spite of the close relationships between carbohydratebinding proteins, particularly lectins and glycosidases, as interprets of the information stored in oligosaccharide sequen-

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ces and their frequently cooperative operation, the mechanism by which these two families of carbohydrate-interacting proteins bind to their putative sugar partners is commonly considered to be starkly different. Glycosidases usually bind to single oligosaccharides sequences with high affinity and selectivity, which is followed by conformational distortion and enzymatic hydrolysis of the critical glycosidic linkage.[10] On the contrary, many lectins depend upon multivalent interactions, with typical monovalent carbohydrate glycotopes binding only weakly.[11, 12] This dogma has largely dominated research on artificial glycosidase and lectin ligands for fundamental studies or biomedical applications through the last twenty years. Thus, many synthetic polyconjugates with various copies of an identical individual sugar recognition motif onto molecular, dendritic, polymeric, selfassembled or nanometric scaffolds have been developed aimed at mimicking and matching the arrangement of complementary lectin receptors in their natural mode of affinity enhancement.[13–21] The design of glycosidase inhibitors has instead been focused on monovalent glycomimetics with a resemblance to the natural substrate or to the corresponding transition state.[22–24] In contrast to most lectins, glycosidases may appear to be unpromising targets for multivalent binding because the active site, which is normally unique, is generally buried at the interior of the protein and not very accessible, preventing quelation and cross-linking phenomena and making sliding and rebinding of multitopic ligands improbable.[25] In principle, the increase in molecular size associated with multibranched architectures coated with a given inhibitory epitope (inihitope) is more likely to negatively affect the formation of the enzyme–inhibitor complex by sterically affecting the intricate hydrogen bonding and hydrophobic contacts governing binding at the active site. In the particular case of Zanamivir, a potent influenza neuraminidase inhibitor,[26] multivalency has been shown to impart enhanced pharmacological effects in vivo, but this is despite a comparatively weak inhibition of the enzyme.[27] Notwithstanding, an early attempt to modulate the activity of the iminosugartype glycomimetic 1-deoxynojirimycin (DNJ), a potent inhibitor of a- and b-glucosidases, by grafting up to three copies onto a dendritic core (compound 1) afforded a strongly enzyme-dependent response, leading in some cases to enhanced inhibitory potencies (Figure 1).[28] This raised the question of whether or not multivalency could represent a new tool to tailor the glycosidase inhibitory profile of a given glycomimetic in a manner that would be reminiscent of the multivalent or cluster effect operating in carbohydrate–lectin recognition processes.[11, 12] We recently provided evidence that the inhibitory strength of DNJ towards a-mannosidase was dramatically enhanced (2147-fold; 179 per DNJ unit) when twelve iminosugar motifs were symmetrically displayed onto a C60-fullerene scaffold (e.g., compound 3 compared with 2; Figure 1).[29] Further work using b-cyclodextrin as the central platform (e.g., compound 4; Figure 1) demonstrated the importance of the valency, the length of the segment connect-

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Figure 1. General structure of multivalent iminosugar systems displaying 1-deoxynojirimycin (DNJ) inhitopes onto a dendritic (1), fullerene (3), and a b-cyclodextrin core (4).

ing the iminosugar motif to the core, and the overall architecture of the conjugate in the glycosidase inhibition properties.[30] Preliminary data on the inhibitory activity of a multivalent b-cyclodextrin–DNJ conjugate towards human b-glu-

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cocerebrosidase and its ability to promote the correct folding in a mutant associated to type 1 Gaucher disease, acting as a pharmacological chaperone, further challenged the current notion that site-specific glycosidase ligands must necessarily be small molecules mimicking mono or oligosaccharide epitopes.[31] In all, those results point to the need of a deep revision of the field that should unveil the relevance of multivalency in glycomimetic-enzyme inhibition and the mechanisms at play. Recent isothermal titration calorimetry studies on the association of multivalent DNJ or its C-2 epimer 1-deoxymannojirimycin (DMJ) derivatives and Jack bean a-mannosidase[32] were consistent with a 1:1 stoichiometry and revealed a thermodynamic signature dominated by a favorable enthalpic term, with an either unfavorable or close to 0 entropic contribution, as well as a characteristic enthalpy–entropy compensation trend.[33] This is not very different from that reported for many carbohydrate–lectin recognition processes.[34] The fact that multivalent sugars have been also shown to be able to bind and inhibit a glycosyltransferase contributes further to blur the line between carbohydrateprocessing enzyme inhibitors and lectin ligands,[35] suggesting that multivalency may exert a multimodal switcher/modulator function in a much broader context than previously considered. Here, we have probed this concept by developing a new family of glycomimetic conjugates, namely fullerene– sp2-iminosugar balls, with the ability to bind to peanut (Arachis hypogaea) agglutinin (PNA), a lectin broadly used in fundamental carbohydrate–protein interaction studies,[36] and/or to several glycosidases. By comparing the respective affinity data with data for monovalent controls, the influence of multivalency in either type of molecular recognition event has been assessed. Most interestingly, the availability of dual lectin/glycosidase ligands provided a unique tool to explore the molecular basis of the inhibitory multivalent effect that led to the formulation of a general hypothesis on the operating mechanisms.

second strongly promoted inhibition of a-mannosidase (e.g., 6; Figure 2).[37] By presenting them in a multivalent manner (11–14) and evaluating their glycosidase inhibition abilities in comparison with monovalent references (7–10) we intended to scrutinize the scope of the inhibitory multivalent effect and how it is affected by the individual affinity of the ligands towards the target enzyme. The preference for 1N-ONJ in place of DNJ was additionally motivated by previous observations indicating that dgluco-configured derivatives with (pseudo)amide functionalities behaved as ligands for galactose-specific lectins,[44] which can be exploited in the design of competitive lectin– glycosidase assays after multivalent presentation (see hereinafter). The isotropic C60-fullerene platform used to generate multivalency prevents orientational effects that might influence the binding mode for different enzyme–ligand pairs. Linear six-carbon (C6) and nine-carbon (C9) spacers were inserted to explore how inter-inhitope distances affect the binding processes. The copper(I)-catalyzed azide–alkyne cy-

Results and Discussion Design criteria: All previous evidences for the glycosidase multivalent inhibitory effect come from iminosugar motifs, DNJ or DMJ.[26–30] Surprisingly, the highest increase in the inhibitory potency after multivalent displaying was always observed for the DNJ/a-mannosidase pair, in spite of their mismatching configurational relationship, independently of the valency or the nature of the core. In this study we have instead selected the 1-amino-5N,6O-oxomethylydenenojirimycin (1N-ONJ) and its C2 epimer 1-amino-5N,6O-oxomethylydenemannnojirimycin (1N-OMJ),]37–39] two representatives of the so-called sp2-iminosugar glycosidase inhibitors family[40–43] with ’’mismatching’’ and ’’matching’’ relationships with a-mannosidase, respectively, as the inhitope motifs. In agreement with their hydroxylation profiles, the first one has proven to impart selective inhibitory properties towards a-glucosidase in monovalent form (e.g., 5),[37–39] whereas the

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Figure 2. Structures of the 1-amino-5N,6O-oxomethylydenenojirimycin (1N-ONJ) and 1-amino-5N,6O-oxomethylydenemannojirimycin (1NOMJ) derivatives 5–14.

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cloaddition (CuAAC) reaction, the paradigmatic ’’click’’ ligation chemistry, which is already amply exploited in the glycosciences, was used for multiple conjugation purposes.[45] Synthesis: The synthesis of the azide-armed N-hexyl- and Nnonyl-1N-ONJ derivatives 25 and 26, respectively, was accomplished in three steps from the reducing bicyclic nojirimycin precursor 15.[46] Incubation of 15 with mono-tert-butoxycarbonyl (Boc)-protected 1,6-diaminohexane or 1,9-diACHTUNGREaminononane afforded the gem-diamine pseudo-N-glycosides 17 and 18 in 72–79 % yield. Exclusively, the diastereomer having the pseudo-anomeric substituent in an axial orientation, matching the configuration of a-d-glucopyranosides, was detected in both cases, which is in agreement with the strong orbital contribution to the generalized anomeric effect[47] previously observed in related sp2-iminosugar gemdiamines.[37–39] The clickable azide-armed derivatives 25 and 26 were accessed by Boc hydrolysis (!21 and 22) followed by diazo-transfer reaction.[48] To prepare the monovalent 1N-ONJ clicked adducts 7 and 8 as control compounds, heterogeneous CuAAC with 1-pentine was next effected by using CuI as the catalyst (Scheme 1). The same reaction sequence starting from the bicyclic mannojirimycin derivative 16[46] afforded the monovalent 1N-MNJ sp2-iminosugar controls 9 and 10 (Scheme 1). The synthesis of the dodecavalent fullerene–sp2-iminosugar balls 11–14 was accomplished by a click reaction of the C60 hexakis-adduct 29[49–51] and the corresponding unprotected azide-armed sp2-iminosugars 25– 28 under the CuAAC conditions previously optimized for the preparation of fullerene sugar balls (Scheme 1).[49–51] They involve the use of tetra-n-butylammonium fluoride to generate the reactive terminal alkyne functions in situ. The homogeneity and T-symmetry of the dodecavalent adducts were confirmed by spectroscopic and microanalytical techniques. Glycosidase inhibitory activity: The inhibitory activities of the new sp2-iminosugar conjugates against selected glycosidases are summarized in Table 1. Data for the previously reported DNJ conjugates 2 and 3 are also included for comparative purposes.[29] The monovalent 1N-ONJ derivatives 7 and 8 behaved as potent inhibitors of the yeast a-glucosidases (a-glcases) maltase and isomaltase, as expected from the configurational complementarity with the natural a-d-glucopyranoside substrates (Ki = (5.1  0.5) and (2.2  0.2) mm). They also inhibited b-glucosidase (b-Glc, bovine liver; Ki = (60  7) and (29  3) mm), but with about tenfold weaker potencies. No inhibition was detected towards a- or b-galactosidase (a-galase) and b-manase, whereas a weak affinity towards a-manase (Jack beans) was observed (Ki = (596  60) and (451  46) mm). The epimeric 1N-OMJ congeners 9 and 10 exhibited even higher selectivities towards the corresponding matching enzyme a-manase, with Ki values of (4.5  0.5) and (1.8  0.2) mm, respectively, and no affinity or over 100-fold weaker affinities for other mismatching glycosidases (Table 1).

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Scheme 1. Synthesis of the dodecavalent fullerene–sp2-iminosugar balls 11–14 and the monovalent control conjugates 7–10.

As already demonstrated for the DNJ conjugates 2 and 3,[29] multivalent presentation of the sp2-iminosugar inhitopes onto the fullerene platform in 11–14 affected the inhibition properties in an enzyme-dependent manner. For maltase, isomaltase, b-glcase, and a-galase, inhibition constants in the range 4.5–193 mm (Table 1) were determined. Rather than a real inhibitory multivalent effect, that is, an increase in the inhibition potency over that expected on statistic basis when compared with the corresponding monovalent reference, the data seem to reflect a change in the binding mode on going from mono to multivalent conjugates. Thus, the Ki values for the dodecavalent DNJ (3) and 1N-ONJ derivatives (11 and 12) against maltase or isomaltase are comparable even though the Ki values for the corresponding monovalent compounds differ by two orders of magnitude (Table 1). For

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Table 1. Glycosidase inhibitory activities (Ki, mm) for 1N-ONJ and 1N-OMJ monovalent sp2-iminosugar derivatives (7–10) and dodecavalent fullerene conjugates (11–14). Data for the previously reported homologous DNJ conjugates 2 (monovalent) and 3 (dodecavalent) are included for comparative purposes.[a] Enzyme maltase isomaltase b-glcase a-galase a-manase

Monovalent derivatives 8 9

2

7

152  12 943  75 482  30 N.I.[c] 322  20

2.6  0.3 5.1  0.5 60  7 N.I.[c] 596  60

5.3  0.6 2.2  0.2 29  3 N.I.[c] 451  46

N.I.[c] N.I.[c] 685  67 N.I.[c] 4.5  0.5

10

3

227  23 518  52 352  36 N.I.[c] 1.8  0.2

18  1 10.5  0.5 247  11 84  6 0.15  0.02

Dodecavalent fullerene conjugates 11 12 13 18  2 4.5  0.5[b] 52  5 N.I.[c] 2.0  0.2

49  5 20  2 42  5 41  4 0.81  0.09

67  7 25  3 65  6 78  8 0.085  0.005

14 104  10 193  20 24  2 104  11 0.66  0.07

[a] Inhibition, when detected, was competitive in all cases excepting for 11 against isomaltase, for which a mixed-type inhibition mode was observed. None of the compounds showed inhibitory activity at a concentration of 2 mm against b-galactosidase (E. coli), b-mannosidase (Helix pomatia) and trehalase (pig kidney). [b] The Ki value corresponding to the competitive term is given; for the non-competitive contribution, the corresponding apparent inhibition constant (Ki’) was (20.4  2) mm. [c] No inhibition detected at 2 mm.

a given enzyme (e.g., maltase) the observed differences can be ascribed to structural considerations regarding the spacer (i.e., for a given inhitope, increasing the length from C6 to C9 has a detrimental effect) or the inhitope (i.e., for a given spacer length, d-gluco-configured inhitopes lead to lower Ki values as compared with d-manno inhitopes). The fact that in some cases (e.g., for compounds 12–14 against a-galase) multivalency acts as a switcher, activating the inhibition of an enzyme that was not responsive to the monovalent inhitope motif, further supports the hypothesis of a shift in the binding mechanisms at play. a-Manase showed a singular behavior among the assayed glycosidases inasmuch as the impact of multivalency in the inhibition potency was very much acute. Affinity enhancements on inhitope molar basis, taking the corresponding monovalent conjugate as the reference, are much higher for the weakly binding mismatching DNJ or 1N-ONJ motifs than for the matching 1N-OMJ motif. In the first case, a real inhibitory multivalent effect can thus be invoked, which should be the consequence of distinct cooperative effects. PNA Lectin binding and cross-linking capabilities: The enzyme-linked lectin assay (ELLA) test measures the ability of a soluble saccharide to inhibit the association between a lectin (here PNA) labeled with horseradish peroxidase (HRP–PNA), and a ligand immobilized on the microtiter well (here a lactosylated glycopolymer). The presence of the relatively large HRP protein label (40 kDa) prevents two lectin molecules from approaching each other, avoiding aggregation. The corresponding IC50 values are then assumed to be proportional to the corresponding binding affinities, providing information on the intrinsic multivalent effect.[12] Neither of the mono (7–10) or dodecavalent conjugates (11– 14) was recognized by the mannose/glucose specific lectin concanavalin A (ConA) in ELLA. However, the monovalent 1N-ONJ derivatives were found to bind to the galactose-specific lectin PNA, although with a two- to threefold weaker affinity compared with lactose. Upon multivalent presentation onto the fullerene core, the binding affinity was greatly enhanced, with IC50 values in the low micromolar range (Figure 3). To assess the cross-linking capabilities of the dodecavalent 1N-ONJ glycomimetic clusters, a two-site ’’sandwich’’ ELLA

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Figure 3. ELLA plots (logarithm scale) and IC50 values [mm] for the inhibition of HRP–PNA binding to poly(acrylamide-co-(N-[2-(b-lactosylACHTUNGREthio)ethyl)acrylamide) by increasing concentrations of the 1N-ONJ fullerene balls 11 and 12 in comparison with the monovalent conjugates 7 and 8. The corresponding data for lactose is included for reference. Data represent the mean of three independent determinations (S.D. 10–15 %).

was additionally conducted. Unlabeled (therefore cross-linkable) PNA was first adsorbed onto a microtiter well coated with the lactosylated glycopolymer. Pre-formed complexes of 11 and 12 at different concentrations with HRP-labeled PNA were then added. The plots of the relative amount of HRP–PNA bound to the plate against the concentration of ligand evidenced the capacity of the 1N-ONJ fullerene balls to bridge two PNA lectin molecules, the conjugate having the longest C9 spacer being slightly more efficient than the C6 homologue in this respect (Figure 4). Overall, the data demonstrate the behavior of the 1N-ONJ motif as glycotope-like partner for PNA binding and underlines its character as multivalency-sensitive ligand for glycosidase and lectin recognition. Mapping the involvement of the catalytic site of glycosidases in binding to fullerene-sp2-iminosugar balls: The unique dual lectin–glycosidase ligand features of the 1N-ONJ-fullerene conjugates was exploited to develop a modified two-site ELLA test in which HRP–PNA and a-manase compete to bind to the dodecavalent sp2-iminosugar balls 11 or 12 in solution. Although yeast a-manase is an N-glycoprotein,[52] the carbohydrate chains are not accessible for lectin binding and do not interfere in the competition assay, as confirmed in a control experiment. By performing the experiment in the

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Figure 4. Relative cross-linking efficiencies of the 1N-ONJ fullerene balls 11 and 12 at different concentrations. Data, in arbitrary units, are obtained from the readout of the optical density (410 nm relative to 570 nm) after addition of a suitable substrate of HRP (see the Supporting Information) and represent the mean of three independent determinations (S.D. 12–15 %).

absence and presence of a potent active-site-directed monovalent inhibitor (i.e., 10, Ki = 1.8 mm, Table 1), the involvement of the glycone catalytic site in the multivalent inhibitory effect can be mapped. Alternatively, a multivalent inhibitor of the enzyme can be used to assess the contribution of non-glycone sites to the binding process (Figure 5). Figure 6 depicts the two-site ELLA cross-linking inhibition plot for conjugate 11 upon adding increasing amounts of a-manase. Blocking the active site of the enzyme with an excess of the monovalent a-manase inhibitor 10 reverted this process only partially, suggesting that the a-manase–10 complex is still able to bind to 11 to form a ternary species, although with lower affinity than the free enzyme. In contrast, the dodecavalent fullerene–1N-OMJ ball 13 (or its homologue 14) virtually prevented inhibition of 11-mediated PNA–HRP–PNA cross-linking by a-manase. An analogous set of data was obtained for compound 12. Altogether, these results strongly suggest that the dodecavalent fullerene balls 11–14 bind to a-manase through mechanisms that involve the catalytic site and non-glycone sites in the enzyme simultaneously. A second set of competitive lectin/enzyme experiments replacing a-manase by the a-glcase maltase evidenced a significantly different scenario. Whereas the enzyme was also able to inhibit the PNA–HRP–PNA cross-linking mediated by the dodecavalent 1N-ONJ conjugates 11 or 12, addition of the monovalent active site-directed maltase inhibitor 5 (Ki = 0.54 mm)[38] now had a very weak influence in the equilibrium at play. On the contrary, the multivalent conjugate 13, even though a relatively modest inhibitor of maltase (Ki = 67 mm, Table 1), was very efficient at preventing the lectin cross-linking inhibition in this assay format (Figure 7). The data are consistent with a marginal implication of the maltase active site in the formation of the corresponding complexes with the fullerene-sp2-iminosugar balls, independently of the length of the spacer. Most of the stabilizing interactions should then arise from non-glycone sites in the enzyme.

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Figure 5. Schematic representation of the HRP–PNA/a-manase competitive ELLA test developed to assess the contribution of the active (glycone) site and of non-glycone sites in the enzyme to the a-manase–sp2iminosugar-fullerene balls 11 or 12. By keeping the concentrations of HRP–PNA and sp2-iminosugar conjugate constant, the proportion of cross-linked lectin decreases with increasing concentrations of the enzyme. This process is affected by the presence of an active site-directed monovalent inhibitor and of a multivalent inhibitor to an extent that depends on the implication of the catalytic and non-glycone sites in the binding event.

Figure 6. Plots of the inhibition of the PNA–HRP–PNA cross-linking mediated the 1N-ONJ fullerene conjugates 11 (A) and 12 (B) in two-site ELLA tests by increasing amounts of a-manase. Data were collected in the absence and presence of excess of the monovalent inhibitor 10 or the 1N-OMJ multivalent inhibitor 13 and represent the mean of three independent determinations (S.D. 15–20 %).

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FULL PAPER explanation for the mutivalency-dependent ’’on–off’’ switching effect observed for some inhitope–glycosidase pairs, for example, for a-galase with 12, 13, or 14. This can be rationalized if assuming that the d-gluco or d-manno-configured inhitopes do not fit in the active site of the mismatching enzyme but are able to bind to some extent to non-glycone sites, thus then benefiting from a multivalent effect and, in principle, blocking the access to the catalytic site (Figure 8 A).

Figure 7. Plots of the inhibition of the PNA–HRP–PNA cross-linking mediated the 1N-ONJ fullerene conjugates 11 (A) and 12 (B) in two-site ELLA tests by increasing amounts of maltase. Data were collected in the absence and presence of excess of the monovalent inhibitor 10 or the 1NOMJ multivalent inhibitor 13 and represent the mean of three independent determinations (S.D. 15–20 %).

Molecular basis for the inhibitory multivalent effect: The ensemble of results discussed above strongly suggests that the binding modes for monovalent and multivalent iminosugar-type glycomimetics towards glycosidases are starkly different. Monovalent competitive inhibitors sit at the glycone binding site in the catalytic pocket, whereas glycosidase inhibition by multivalent inhibitors seems to depend critically on interactions involving non-glycone binding sites. The fact that the multivalent 1N-ONJ conjugates 11 and 12 can bind to yeast maltase even when the catalytic site is occupied by an active-site-directed inhibitor is much revealing in this respect. Yeast maltase belongs to the family GH13 in the CAZy classification,[53] also called the a-amylase family. Although the mechanisms of substrate binding in these enzymes is not fully elucidated, crystallographic and computational data indicate that, upon binding, the nonreducing end of glucose residue that will be cleaved off is almost completely buried in the protein and most of the binding energy comes from interactions between this residue and amino acids at the 1 site in the enzyme, a topology a priori rather unfavorable for interactions with sterically demanding multivalent derivatives.[54] However, the aglycon binding site is known to host the distal oligosaccharide portion in a relatively shallow pocket, which is much better suited to act as a lectin-like domain towards multivalent ligands.[54] This mechanistic model likely applies also to the isomaltase, bglcase, and a-galase enzymes, all of them belonging to glycolsyl hydrolase families known to possess relatively deep catalytic sites (GH13, GH1, and GH27, respectively).[54–58] The monovalent/multivalent shift in the binding mode inferred from the data presented here provides an immediate

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Figure 8. Schematic representations of the shift in binding modes after multivalent presentation of inhitopes for enzymes having a deep catalytic site but accessible lectin-like aglycone sites (e.g., maltase; A) or accessible catalytic as well as aglyconic sites (e.g., a-manase; B). Note that in both cases the presence of a potent active site-directed monovalent inhibitor at the active site does not necessarily prevent further binding to the multivalent congener, although in the second case the affinity would be more largely affected.

Compared with the other assayed glycosidases, Jack bean a-manase is much more susceptible to experience significant affinity enhancements towards competitive inhibitor motifs when those are presented in a multivalent manner. The enzyme has a heterodimeric structure, yet it is considered to possess a single catalytically competent unit.[59] Nevertheless, some controversy at this point still persists and the formation of aggregates with multivalent inhibitors cannot be fully discarded.[60] In our case, however, no precipitation occurred under the conditions used for Ki or competitive ELLA determinations as established by turbidimetric analysis. The formation of soluble a-manase aggregates in the presence of 11–14 was also discarded after dynamic light-scattering (DLS) measurements. Like all class II a-mannosidases, Jack bean a-manase belongs to the glycoside hydrolase family GH38.[53] Although the X-ray structure of this particular enzyme has not been solved yet, it is known that GH38 amannosidase active sites can be quite long and open,[61–63]

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which must favor sliding and rebinding processes when faced with multivalent presentations of relatively weakly binding inhitopes such as DNJ or 1N-ONJ. Moreover, it has been shown that GH38 a-manases possess binding sites for additional monosaccharide subunits near the catalytic site.[64] A multivalent ligand could then bind simultaneously at the catalytic site and at secondary binding sites, benefiting from a chelate-like effect (Figure 8 B). The later mechanisms may explain the fact that an enhancement in the inhibitory potency is observed even in the case of the matching 1N-OMJ multivalent inhibitors 13 and 14. The outstandingly low Ki value for 13 (85 nm), indicative of an eightfold higher affinity towards a-manase as compared with 14, point to the sixcarbon-length spacer as the optimal spacer to benefit from cooperative binding at the catalytic and non-glycone sites in this enzyme.

Conclusion Lessons from the knowledge accumulated over the years on glycosidase inhibitors teach that subtle structural alterations in substrate or transition-state analogues may have profound consequences in enzyme-binding affinities. Generally, only limited modifications are tolerated. The recent observation that multivalency does not cancel but rather can even increase the inhibitory potency was, thus, highly intriguing. Although the possibility of a lectin-like behavior was intuitively advanced, the idea challenged the current notions on glycosidase active-site-fitting requirements. By using isotropic C60-fullerene-centred sp2-iminosugar balls with dual lectin (PNA) and glycosidase (maltase, isomaltase, b-glcase, agalase, a-manase) -binding abilities, we have investigated the scope of the so-called inhibitory multivalent effect and the possible mechanisms at play. Three general scenarios can be identified for those inhitope/glycosidase pairs that are responsive to multivalency: 1) different matching inhitopes that strongly differ in their inhibitory potencies in monovalent form (e.g., 2 and 7) exhibit similar modest-tostrong affinities when displayed in a multivalent manner (e.g., 3 and 11 against maltase); 2) inhitopes are not recognized by a mismatching enzyme but inhibition is switched ’’on’’ by multivalency (e.g., 12 against a-galase); and 3) multivalency promotes a very significant glycosidase inhibition potency enhancement for both mismatching and matching inhitopes (11–14 against a-manase). The first two cases can be rationalized assuming that a shift in the binding mode takes place upon multivalent presentation of the inhitopes, which has been corroborated by competitive lectin–glycosidase assays. The active site for those enzymes is only marginally involved in binding of the multivalent fullerene balls. Non-glycone binding sites with lectin-like abilities are probably implicated in this process, which ultimately lead to blockage of the catalytic site entrance, impairing the access and processing of the substrate. The third situation results from the interplay of interactions involving simultaneously the catalytic site and non-glycone binding sites, enabling

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positive cooperativity between inhitopes, and is responsible for the repeated observation of the remarkable susceptibility of a-manase to multivalency. It is important to stress that although the evidence here presented supports the concept of multivalency as a tool to modulate glycosidase inhibition, it questions the idea of a multivalent inhibitory effect quantifiable from the direct comparison of the Ki values for homologous mono and multivalent conjugates, as previously formulated. Such an analysis would require determining the affinity of the monovalent ligand towards the aglycone binding sites operating in the case of multivalent inhibitors. There is a double relevance of this work in the field of glycobiology. First, it provides glycomimetics with dual lectin–glycosidase-binding capabilities as valuable tools to investigate the molecular basis of mutivalency in glycosidase inhibition. Second, it strongly suggests that glycosidases may behave as lectins against multivalent glycodisplays through their aglycon binding sites. It is interesting to speculate that appropriate glycotopes could also bind to those lectin-like domains after multivalent presentation, which would imply unforeseen biological consequences. Confirmation of this hypothesis will concentrate, with no doubt, many efforts in the near future.

Experimental Section General methods: Reagents and solvents were purchased from commercial sources and used without further purification. Optical rotations were measured with a JASCO P-2000 polarimeter, using a sodium lamp (l = 589 nm) at 22 8C in 1 cm or 1 dm tubes. Thin-layer chromatography was performed on E. Merck precoated TLC plates, silica gel 30F-245, with visualization by UV light and by staining with 10 % H2SO4 or 0.2 % w/v cerium(IV) sulfate 5 % ammonium molybdate in 2 m H2SO4, or 0.1 % ninhydrin in EtOH. Column chromatography was performed on Chromagel (SDS silica 60 AC.C 70–200 mm). NMR experiments were performed at 300 (75.5) and 500 (125.7) MHz using Bruker DMX300 and DRX500, respectively. The notation of atoms for NMR in the case of the fullerene adducts is indicated in Figure 2 and Scheme 1. Copies of the spectra are provided in Figure S1 to S24 (the Supporting Information). 1-D TOCSY as well as 2-D COSY and HMQC experiments were carried out to assist in signal assignment. In the FABMS spectra, the primary beam consisted of Xe atoms with maximum energy of 8 keV. The samples were dissolved in m-nitrobenzyl alcohol or thioglycerol as the matrices and the positive ions were separated and accelerated over a potential of 7 keV. NaI was added as cationizing agent. Electrospray mass spectra (ESI-MS) were obtained with a Bruker Esquire6000 instrument. MALDI-TOF-mass spectra were carried out on a Bruker BIFLEX matrix-assisted laser desorption time-of-flight mass spectrometer. Elemental analyses were performed at the Servicio de Microanlisis del Instituto de Investigaciones Qumicas de Sevilla, Spain. 5,6-Oxomethylidenenojirimycin (15) and 5,6-oxomethylidenemannojirimycin (16) were prepared according to literature procedures.[46] The preparation of (C60-Th)-[5,6] hexa-adduct 27 was carried out by using the cyclopropanation method.[48]

Synthesis General procedure: The general procedure for the preparation of spacerarmed 5,6-oxomethylidenenojirimycin (1N-ONJ) and 5,6-oxomethylidenemannojirimycin (1N-OMJ) derivatives (17–20) is as follows. A solution

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Fullerene-sp2-Iminosugar Balls

FULL PAPER

of 5,6-oxomethylidenenojirimycin (15) or 5,6-oxomethylidenemannojirimycin (16; 1.72 mmol) and N-Boc-1,6-diaminohexane (578 mL, 2.58 mmol) or N-Boc-1,9-diaminononane (667 mg, 2.58 mmol) in MeOH (7.2 mL) was stirred, under Ar atmosphere, at 65 8C for 24–48 h. The solvent was removed under reduced pressure, and the resulting residue was purified by column chromatography using the eluent indicated in each case. 1-Amino-1-deoxy-N-(6-N-tert-butoxycarbonylhexyl)-5,6-oxomethylidenenojirimycin (17): Column chromatography, eluent 100:10:1 CH2Cl2/ MeOH/H2O. Yield: 500 mg (72 %). [a]D = + 56 (c = 1.0, MeOH); Rf = 0.57 (50:10:1 CH2Cl2/MeOH/H2O); 1H NMR (500 MHz, CD3OD): d = 4.63 (d, 1 H, J1,2 = 5.1 Hz, H-1), 4.48 (t, 1 H, J6a,6b = J5,6a = 8.9 Hz, H-6a), 4.27 (dd, 1 H, J5,6b = 4.9 Hz, H-6b), 3.82 (ddd, 1 H, J4,5 = 9.6 Hz, H-5), 3.61 (t, 1 H, J2,3 = J3,4 = 9.6 Hz, H-3), 3.47 (dd, 1 H, H-2), 3.26 (t, 1 H, H-4), 3.02 (t, 2 H, JH,H = 7.1 Hz, CH2NHBoc), 2.58 (m, 2 H, CH2NH), 1.49 (m, 4 H, 2CH2), 1.43 (bs, 9 H, CMe3), 1.37 ppm (m, 4 H, 2CH2); 13C NMR (125.7 MHz, CD3OD): d = 159.2 (CON), 158.5 (CO carbamate), 79.8 (CMe3), 75.6 (C-4), 74.5 (C-3), 72.4 (C-2), 69.5 (C-1), 67.9 (C-6), 54.6 (C5), 47.4 (CH2NH), 41.3 (CH2NHBoc), 30.9, 30.5 (CH2), 28.8 (CMe3), 28.0, 27.7 ppm (CH2); ESI-MS: m/z: 829.8 [2M+Na] + , 426.4 [M+Na] + , 404.4 [M+H] + ; elemental analysis calcd (%) for C18H33N3O7: C 53.58, H 8.24, N 10.41; found: C 53.64, H 8.329, N 10.53. 1-Amino-1-deoxy-N-(9-N-tert-butoxycarbonylnonyl)-5,6-oxomethylidenenojirimycin (18): Column chromatography, eluent 100:10:1 CH2Cl2/ MeOH/H2O. Yield: 605 mg (79 %). [a]D = + 48 (c = 1.0, MeOH); Rf = 0.53 (50:10:1 CH2Cl2/MeOH/H2O); 1H NMR (500 MHz, CD3OD): d = 4.62 (d, 1 H, J1,2 = 5.1 Hz, H-1), 4.48 (t, 1 H, J6a,6b = J5,6a = 8.9 Hz, H-6a), 4.27 (dd, 1 H, J5,6b = 4.9 Hz, H-6b), 3.82 (ddd, 1 H, J4,5 = 9.6 Hz, H-5), 3.61 (t, 1 H, J2,3 = J3,4 = 9.6 Hz, H-3), 3.47 (dd, 1 H, H-2), 3.26 (t, 1 H, H-4), 3.01 (t, 2 H, JH,H = 7.1 Hz, CH2NHBoc), 2.57 (m, 2 H, CH2NH), 1.49 (m, 2 H, CH2), 1.43 (bs, 9 H, CMe3), 1.29 ppm (bs, 12 H, 6CH2); 13C NMR (125.7 MHz, CD3OD): d = 159.2 (CON), 158.5 (CO carbamate), 79.8 (CMe3), 75.7 (C-4), 74.5 (C-3), 72.4 (C-2), 69.6 (C-1), 68.0 (C-6), 54.6 (C5), 47.5 (CH2NH), 41.4 (CH2NHBoc), 31.0, 30.6, 30.6, 30.5, 30.4, 30.1 (CH2), 28.8 (CMe3), 28.3, 27.9 ppm (CH2); ESI-MS: m/z: (468.4 [M+Na] + ); elemental analysis calcd (%) for C21H39N3O7: C 56.61, H 8.82, N 9.43; found: C 56.75, H 8.763, N 9.49. 1-Amino-1-deoxy-N-(6-N-tert-butoxycarbonylhexyl)-5,6-oxomethylidenemannojirimycin (19): Column chromatography, eluent 100:10:1 CH2Cl2/ MeOH/H2O: Yield: 296 mg (94 %). [a]D = + 16.4 (c = 1.0, MeOH); Rf = 0.47 (30:10:1 CH2Cl2/MeOH/H2O); 1H NMR (500 MHz, CD3OD): d = 4.57 (d, 1 H, J1,2 = 2.0 Hz, H-1), 4.49 (t, 1 H, J6a,6b = J5,6a = 8.7 Hz, H-6a), 4.28 (dd, 1 H, J5,6b = 4.4 Hz, H-6b), 3.95 (t, 1 H, J2,3 = 2.0 Hz, H-2), 3.72 (m, 1 H, H-5), 3.66 (m, 2 H, H-3, H-4), 3.04 (m, 2 H, CH2NHBoc), 2.59 (m, 2 H, CH2NH), 1.45 (m, 13 H, Boc, 2 CH2), 1.33 ppm (m, 4 H, 2CH2); 13 C NMR (125.7 MHz, CD3OD): d = 160.3 (CO), 158.5 (CO carbamate), 79.8 (CMe3), 73.2 (C-2), 72.4 (C-3), 72.1 (C-1), 72.0 (C-4), 67.9 (C-6), 55.7 (C-5), 47.0 (CH2NH), 41.3 (CH2NHBoc), 30.9, 30.4 (CH2), 28.8 (CMe3), 28.1, 27.7 ppm (CH2); ESI-MS: m/z: 829 [2M+Na] + , 426 [M+Na] + ; elemental analysis calcd (%) for C18H33N3O7: C 53.58, H 8.24, N 10.41; found: C 53.27, H 7.87, N 10.54. 1-Amino-1-deoxy-N-(9-N-tert-butoxycarbonylnonyl)-5,6-oxomethylidenemannojirimycin (20): Column chromatography, eluent 100:10:1 CH2Cl2/ MeOH/H2O; Yield: 278 mg (80 %). 18 % of starting material was recovered. [a]D = + 14.8 (c = 0.8, MeOH); Rf = 0.77 (50:10:1 CH2Cl2/MeOH/ H2O); 1H NMR (400 MHz, CD3OD): d = 4.57 (d, 1 H, J1,2 = 2.0 Hz, H-1), 4.49 (dd, 1 H, J6a,6b = 8.8 Hz, J5,6a = 8.1 Hz, H-6a), 4.29 (dd, 1 H, J5,6b = 4.3 Hz, H-6b), 3.95 (t, 1 H, J2,3 = 2.0 Hz, H-2), 3.76–3.67 (m, 3 H, H-5, H-3, H-4), 3.01 (t, 2 H, JH,H = 7.0 Hz, CH2NHBoc), 2.58 (m, 2 H, CH2NH), 1.42 (bs, 14 H, Boc, 2CH2), 1.32 ppm (bs, 10 H, 5CH2); 13C NMR (75.5 MHz, CD3OD): d = 160.3 (CO), 158.5 (CO carbamate), 79.8 (CMe3), 73.1 (C-2), 72.4 (C-3), 72.0 (C-1), 71.9 (C-4), 67.9 (C-6), 55.6 (C-5), 47.1 (CH2NH), 41.3 (CH2NHBoc), 30.9, 30.6, 30.5, 30.4, 30.3 (CH2), 28.8 (CMe3), 28.4, 27.8 ppm (CH2); ESI-MS: m/z: 913.9 [2M+Na] + , 468.5 [M+Na] + , 446.4 [M+H] + ; elemental analysis calcd (%) for C21H39N3O7: C 56.61, H 8.82, N 9.43; found: C 56.44, H 8.704, N 9.64. General procedure for the preparation of amide-armed 1N-ONJ and 1NOMJ conjugates (21–24): Treatment of 17–20 (1.66 mmol) with TFA/

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CH2Cl2 (1:1, 9 mL) at 0 8C!RT for 1 h, followed by co-evaporation several times with water until neutral pH, and purification by column chromatography using the eluent indicated in each case, gave the aminearmed conjugates, which were characterized as the corresponding dihydrochloride salts 21–24 after freeze-drying from aqueous HCl solutions. 1-Amino-1-deoxy-N-(6-aminohexyl)-5,6-oxomethylidenenojirimycin dihydrochloride (21): Column chromatography: 50:10:1! 30:10:1 CH2Cl2/ MeOH/H2O. Yield: 575 mg (92 %). [a]D = + 12.9 (c = 1.0, H2O); Rf = 0.30 (30:10:1 CH2Cl2/MeOH/H2O); 1H NMR (500 MHz, CD3OD) d = 5.14 (d, 1 H, J1,2 = 6.1 Hz, H-1), 4.63 (t, 1 H, J6a,6b = J5,6a = 8.7 Hz, H-6a), 4.36 (dd, 1 H, J5,6b = 4.9 Hz, H-6b), 3.98 (m, 1 H, H-5), 3.85 (dd, 1 H, J2,3 = 9.4 Hz, H-2), 3.73 (t, 1 H, J3,4 = 9.4 Hz, H-3), 3.39 (t, 1 H, H-4), 3.11 (td, 1 H, CHNH), 2.99 (td, 1 H, CHNH), 2.93 (t, 2 H, CH2NH2), 1.44 (bs, 4 H, 2CH2), 1.29 ppm (bs, 4 H, 2CH2); 13C NMR (75.5 MHz, CD3OD) d = 158.7 (CO), 74.3 (C-4), 74.0 (C-3), 69.7 (C-2), 68.4 (C-6), 67.3 (C-1), 55.1 (C-5), 46.0 (CH2NH), 40.5 (CH2NH2), 28.2, 27.1, 26.8, 25.9 ppm (CH2); ESI-MS: m/z: 304.1 [M+H] + ; elemental analysis calcd (%) for C13H27Cl2N3O5 : C 41.50, H 7.23, N 11.17; found: C 41.33, H 7.15, N 10.89. 1-Amino-1-deoxy-N-(9-aminononyl)-5,6-oxomethylidenenojirimycin dihydrochloride (22): Column chromatography: 50:10:1!30:10:1 CH2Cl2/ MeOH/H2O. Yield: 573 mg (quant.); [a]D = + 52.7 (c = 0.62, MeOH); Rf = 0.46 (30:10:1 CH2Cl2/MeOH/H2O); 1H NMR (500 MHz, CD3OD): d = 5.15 (d, 1 H, J1,2 = 6.1 Hz, H-1), 4.63 (t, 1 H, J6a,6b = J5,6a = 8.8 Hz, H-6a), 4.37 (dd, 1 H, J5,6b = 4.8 Hz, H-6b), 3.93 (ddd, 1 H, J4,5 = 9.4 Hz, H-5), 3.87 (dd, 1 H, J2,3 = 9.4 Hz, H-2), 3.70 (t, 1 H, J3,4 = 9.4 Hz, H-3), 3.40 (t, 1 H, H4), 3.10 (td, 1 H, CHNH), 2.99 (td, 1 H, CHNH), 2.93 (t, 2 H, CH2NH2), 1.85 (m, 1 H, CHCH2NH), 1.73 (m, 1 H, CHCH2NH), 1.66 (m, 1 H, CHCH2NH2), 1.38 ppm (bs, 10 H, 5CH2); 13C NMR (125.7 MHz, CD3OD) d = 158.7 (CO), 74.3 (C-4), 74.0 (C-3), 69.6 (C-2), 68.5 (C-6), 67.3 (C-1), 55.1 (C-5), 46.3 (CH2NH), 40.7 (CH2NH2), 30.1, 29.9, 29.9, 28.5, 27.6, 27.3, 26.1 ppm (CH2); ESI-MS: m/z 346.3 [M+H] + ; elemental analysis calcd (%) for C16H33Cl2N3O5 : C 45.93, H 7.95, N 10.04; found: C 45.58, H 7.602, N 9.81. 1-Amino-1-deoxy-N-(6-aminohexyl)-5,6-oxomethylidenemannojirimycin dihydrochloride (23): Column chromatography: 50:10:1 CH2Cl2/MeOH/ H2O!MeOH. Yield: 472 mg (94 %); [a]D = + 5.0 (c 1.0, H2O); Rf = 0.27 (30:10:1 CH2Cl2/MeOH/H2O); 1H NMR (500 MHz, D2O): d = 5.09 (d, 1 H, J1,2 = 2.0 Hz, H-1), 4.72 (t, 1 H, J6a,6b = J5,6a = 9.1 Hz, H-6a), 4.52 (dd, 1 H, J5,6b = 4.5 Hz, H-6b), 4.36 (t, 1 H, J2,3 = 2.0 Hz, H-2), 3.99 (td, 1 H, J4,5 = 4.5 Hz, H-5), 3.99 (dd, 1 H, J3.4 = 9.2 Hz, H-3), 3.91 (m, 1 H, H-4), 3.23 (m, 1 H, CH2NH), 3.10 (m, 1 H, CH2NH), 3.09 (t, 2 H, J = 7.6 Hz, CH2NH2), 1.77 (m, 2 H, CH2), 1.69 (m, 2 H, CH2), 1.44 ppm (m, 4 H, 2CH2); 13C NMR (125.7 MHz, D2O): d = 158.9 (CO), 69.6 (C-4), 69.1 (C3), 68.5 (C-1), 68.2 (C-2), 67.6 (C-6), 54.4 (C-5), 45.6 (CH2NH), 39.3 (CH2NH2), 26.4, 25.3, 25.0, 24.8 ppm (4CH2); FABMS: m/z 326 [M2HCl + Na] + , 304 [M+H] + ; elemental analysis calcd (%) for C13H27Cl2N3O5 : C 41.50, H 7.23, N 11.17; found: C 41.21, H 7.17, N 10.87. 1-Amino-1-deoxy-N-(9-aminononyl)-5,6-oxomethylidenemannojirimycin dihydrochloride (24): Column chromatography: 50:10:1!30:10:1 CH2Cl2/MeOH/H2O; Yield: 567 mg (99 %). [a]D = + 7.4 (c = 1.0, H2O); Rf = 0.53 (30:10:1 CH2Cl2/MeOH/H2O); 1H NMR (500 MHz, D2O): d = 5.08 (d, 1 H, J1,2 = 2.2 Hz, H-1), 4.71 (t, 1 H, J6a,6b = J5,6a = 9.0 Hz, H-6a), 4.51 (dd, 1 H, J5,6b = 4.5 Hz, H-6b), 4.35 (t, 1 H, J2,3 = 2.1 Hz, H-2), 3.97 (td, 1 H, J4,5 = 4.5 Hz, H-5), 3.90 (dd, 1 H, J3.4 = 9.2 Hz, H-3), 3.87 (m, 1 H, H-4), 3.21 (m, 1 H, CHNH), 3.08 (m, 1 H, CHNH), 2.99 (t, 2 H, J = 7.6 Hz, CH2NH2), 1.74 (m, 2 H, CH2), 1.66 (m, 2 H, CH2), 1.34 ppm (m, 10 H, CH2); 13C NMR (125.7 MHz, D2O): d = 158.9 (CO), 69.6 (C-4), 69.1 (C-3), 68.5 (C-1), 68.2 (C-2), 67.5 (C-6), 54.4 (C-5), 45.8 (CH2NH), 39.5 (CH2NH2), 28.1, 28.0, 27.9, 26.7, 25.7, 24.9 ppm (CH2); ESI-MS: m/z: 346.3 [M+H] + ; elemental analysis calcd (%) for C16H33Cl2N3O5 : C 45.93, H 7.95, N 10.04; found: C 45.80, H 8.046, N 9.91. General procedure for the preparation of azide-armed conjugates by the diazo-transfer reaction (25–28): The corresponding 1N-ONJ (21, 22) or 1N-OMJ (23, 24) w-aminoalkyl pseudo-N-glycoside (0.62 mmol), NaHCO3 (2.48 mmol) and copper(II) sulfate pentahydrate (0.03 mmol) were dissolved in water (820 mL). Freshly prepared triflic azide (1– 2 equiv in toluene) and MeOH (6.1 mL) were added and the blue reaction mixture was stirred vigorously at room temperature for 14 h moni-

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toring by TLC. The solvent was removed under reduced pressure below 25 8C and the resulting residue was purified by column chromatography using the eluent indicated in each case to afford the target azide-armed derivative 25–28. 1-Amino-1-deoxy-N-(6-azidohexyl)-5,6-oxomethylidenenojirimycin (25): Column chromatography, eluent 100:10:1! 50:10:1 CH2Cl2/MeOH/H2O; Yield: 190 mg (93 %). [a]D = + 49 (c = 1.0, MeOH); Rf = 0.48 (50:10:1 CH2Cl2/MeOH/H2O); 1H NMR (500 MHz, CD3OD): d = 4.63 (d, 1 H, J1,2 = 8.5 Hz, H-1), 4.48 (t, 1 H, J5,6a = J6a,6b = 8.3 Hz, H-6a), 4.27 (dd, 1 H, J5,6b = 4.9 Hz, H-6b), 3.82 (m, 1 H, H-5), 3.61 (t, 1 H, H-3), 3.47 (dd, 1 H, J2,3 = 9.2 Hz, H-2), 3.26 (m, 3 H, H-4, CH2N3), 2.59 (m, 2 H, CH2NH), 1.60 (m, 2 H, CH2), 1.52 (m, 2 H, CH2), 1.40 ppm (m, 4 H, 2CH2); 13C NMR (125.7 MHz, CD3OD): d = 159.3 (CO), 75.7 (C-4), 74.5 (C-3), 72.3 (C-2), 69.6 (C-1), 68.0 (C-6), 54.6 (C-5), 52.4 (CH2N3), 47.4 (CH2NH), 30.4, 29.9, 27.8, 27.6 ppm (CH2); ESIMS: m/z: 681.4 [2M+Na] + , 352.2 [M+Na] + , 330.2 [M+H] + ; elemental analysis calcd (%) for C13H23N5O5 : C 47.41, H 7.04, N 21.26; found: C 47.23, H 6.875, N 21.01. 1-Amino-1-deoxy-N-(9-azidononyl)-5,6-oxomethylidenenojirimycin (26): Column chromatography, eluent 80:10:1 CH2Cl2/MeOH/H2O; Yield: 228 mg (99 %). [a]D = + 42 (c = 1.0, MeOH); Rf = 0.64 (50:10:1 CH2Cl2/ MeOH/H2O); 1H NMR (500 MHz, CD3OD): d = 4.63 (d, 1 H, J1,2 = 5.0 Hz, H-1), 4.48 (t, 1 H, J6a,6b = J5,6a = 8.7 Hz, H-6a), 4.27 (dd, 1 H, J5,6b = 4.9 Hz, H-6b), 3.82 (m, 1 H, H-5), 3.61 (t, 1 H, J2,3 = J3,4 = 9.3 Hz, H-3), 3.47 (dd, 1 H, H-2), 3.27 (m, 1 H, H-4), 2.58 (m, 2 H, CH2NH), 1.58 (m, 2 H, CH2), 1.51 (m, 2 H, CH2), 1.32 ppm (m, 10 H, 5CH2); 13C NMR (125.7 MHz, CD3OD): d = 159.2 (CO), 75.7 (C-4), 74.5 (C-3), 72.3 (C-2), 69.6 (C-1), 68.0 (C-6), 54.6 (C-5), 52.4 (CH2N3), 47.5 (CH2NH), 30.6, 30.5, 30.4, 30.2, 29.9, 28.3, 27.8 ppm (CH2). ESI-MS: m/z 765.7 [2M+Na] + , 394.3 [M+Na] + , 372.3 [M+H] + ; elemental analysis calcd (%) for C16H29N5O5 ; C 51.74, H 7.87, N 18.86; found: C 53.27, H 7.87, N 10.54. 1-Amino-1-deoxy-N-(6-azidohexyl)-5,6-oxomethylidenemannojirimycin (27): Column chromatography, eluent 100:10:1! 50:10:1CH2Cl2/MeOH/ H2O. Yield: 157 mg (77 %); [a]D = + 28 (c = 1.0, MeOH); Rf = 0.43 (50:10:1 CH2Cl2/MeOH/H2O); 1H NMR (500 MHz, CD3OD): d = 4.62 (d, 1 H, J1,2 = 2.0 Hz, H-1), 4.51 (dd, 1 H, J5,6a = 8.2 Hz, J6a,6b = 8.8 Hz, H-6a), 4.31 (dd, 1 H, J5,6b = 4.5 Hz, H-6b), 3.97 (t, 1 H, J2,3 = 2.0 Hz, H-2), 3.75 (m, 1 H, H-5), 3.71 (m, 2 H, H-3, H-4), 3.30 (t, 2 H, CH2N3), 2.64 (m, 2 H, CH2NH), 1.64 (m, 2 H, CH2), 1.56 (m, 2 H, CH2), 1.43 ppm (m, 4 H, 2CH2); 13C NMR (125.7 MHz, CD3OD): d = 160.3 (CO), 73.1 (C-2), 72.4 (C-3), 72.0 (C-1), 71.9 (C-4), 67.9 (C-6), 55.6 (C-5), 52.4 (CH2N3), 46.9 (CH2NH), 30.3, 29.8, 27.9, 27.7 ppm (CH2); ESI-MS: m/z: 681.4 [2M+Na] + , 352.2 [M+Na] + , 330.2 [M+H] + ; elemental analysis calcd (%) for C13H23N5O5 : C 47.41, H 7.04, N 21.26; found: C 47.38, H 7.027, N 21.10. 1-Amino-1-deoxy-N-(9-azidononyl)-5,6-oxomethylidenemannojirimycin (28): Column chromatography, eluent 100:10:1!50:10:1 CH2Cl2/MeOH/ H2O; Yield: 230 mg (99 %); [a]D = + 19.3 (c = 1.0, MeOH); Rf = 0.43 (50:10:1 CH2Cl2/MeOH/H2O); 1H NMR (400 MHz, CD3OD): d = 4.57 (d, 1 H, J1,2 = 2.0 Hz, H-1), 4.48 (dd, 1 H, J6a,6b = 8.8 Hz, J5,6a = 8.1 Hz, H-6a), 4.28 (dd, 1 H, J5,6b = 4.3 Hz, H-6b), 3.95 (t, 1 H, J2,3 = 2.0 Hz, H-2), 3.73 (m, 1 H, H-5), 3.67 (m, 2 H, H-3, H-4), 3.27 (t, 2 H, JH,H = 6.8 Hz, CH2N3), 2.57 (m, 2 H, CH2NH), 1.59 (m, 2 H, CH2), 1.49 (m, 2 H, CH2), 1.34 ppm (m, 10 H, 5CH2); 13C NMR (75.5 MHz, CD3OD): d = 160.3 (CO), 73.1 (C-2), 72.4 (C-3), 72.0 (C-1), 71.9 (C-4), 67.9 (C-6), 55.6 (C-5), 52.4 (CH2N3), 47.1 (CH2NH), 30.6, 30.5, 30.4, 30.2, 29.9, 28.4, 27.8 ppm (CH2); ESI-MS: m/z: 765.7 [2M+Na] + , 394.3 [M+Na] + , 372.3 [M+H] + ; elemental analysis calcd (%) for C16H29N5O5 : C. 51.74, H 7.87, N 18.86; found: C 53.27, H 7.87, N 10.54. General procedure for the synthesis of monovalent 1N-ONJ and 1NOMJ conjugates by the ’’click’’ reation of azide-armed precursors and 1pentine (7–10): N,N-Diisopropylethylamine (DIPEA; 0.19 mmol) and CuI (0.038 mmol) were added to a solution of the corresponding azido derivative (25–28; 0.19 mmol) and 1-pentine (1.9 mmol) in acetone (4.5 mL), and the reaction mixture was heated at reflux for 8–24 h. The solvent was removed under reduced pressure and the resulting residue was purified by column chromatography using the eluent indicated in each case to give the respective triazol adducts 7–10.

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1-Amino-1-deoxy-N-(6-(4-propyl-1H-1,2,3-triazol-1-yl)hexyl)-5,6-oxomethylidenenojirimycin (7): Column chromatography, eluent 100:10:1! 50:10:1 CH2Cl2/MeOH/H2O; Yield: 57 mg (75 %). [a]D = + 59 (c = 1.0, MeOH); Rf = 0.53 (50:10:1 CH2Cl2/MeOH/H2O); 1H NMR (500 MHz, CD3OD): d = 7.73 (s, 1 H, CHtriazole), 4.62 (d, 1 H, J1,2 = 4.8 Hz, H-1), 4.47 (t, 1 H, J5,6a = J6a,6b = 8.6 Hz, H-6a), 4.36 (t, 2 H, CH2Ntriazole), 4.26 (dd, 1 H, J5,6b = 5.0 Hz, H-6b), 3.81 (m, 1 H, H-5), 3.61 (t, 1 H, J2,3 = J3,4 = 9.3 Hz, H2), 3.26 (t, 1 H, J3,4 = J4,5 = 9.3 Hz, H-4), 2.66 (t, 2 H, JH,H = 7.5 Hz, CH2C), 2.57 (m, 2 H, JH,H = 7.3 Hz, CH2NH), 1.69 (m, 2 H, CH2CH3), 1.49 (m, 2 H, CH2), 1.39 (m, 2 H, CH2), 1.31 (m, 2 H, CH2), 0.96 ppm (t, 3 H, JH,H = 7.3 Hz, CH3); 13C NMR (125.7 MHz, CD3OD): d = 159.1 (CO), 123.2 (C4triazole), 111, 3 (C-5triazole), 75.8 (C-4), 74.3 (C-3), 72.3 (C-2), 69.5 (C-1), 67.7 (C-6), 54.6 (C-5), 51.5 (CH2N), 47.3 (CH2NH), 31.1, 30.2 (CH2), 28.3 (CH2C), 27.4, 27.4 (CH2), 23.7 (CH2CH3), 14.0 ppm (CH3); ESI-MS: m/z: 817.5 [2M+Na] + , 420.2 [M+Na] + , 398.3 [M+H] + ; elemental analysis calcd (%) for C18H31N5O5 : C 54.39, H 7.86, N 17.62; found: C 54.22, H 7.663, N 17.26. 1-Amino-1-deoxy-N-(9-(4-propyl-1H-1,2,3-triazol-1-yl)nonyl)-5,6-oxomethylidenenojirimycin (8): Column chromatography, eluent 100:10:1 CH2Cl2/MeOH/H2O. Yield: 95 mg (88 %); [a]D = + 44 (c = 1.0, MeOH); Rf = 0.46 (50:10:1 CH2Cl2/MeOH/H2O); 1H NMR (500 MHz, CD3OD): d = 7.74 (s, 1 H, CHtriazole), 4.63 (bs, 1 H, H-1), 4.47 (t, 1 H, J5,6a = J6a,6b = 8.5 Hz, H-6a), 4.35 (t, 2 H, JH,H = 7.1 Hz, CH2Ntriazole), 4.26 (dd, 1 H, J5,6b = 4.5 Hz, H-6b), 3.81 (m, 1 H, H-5), 3.61 (m, 1 H, H-3), 3.48 (bs, 1 H, H-2), 3.26 (m, 1 H, H-4), 2.65 (tm 2 H, JH,H = 7.5 Hz, CH2C), 2.57 (bs, 2 H, CH2NH), 1.89 (m, 2 H, CH2), 1.69 (m, 2 H, CH2CH3), 1.47 (m, 2 H, CH2), 1.25 (m, 10 H, 5CH2), 0.96 ppm (t, 3 H, JH,H = 7.4 Hz, CH3); 13C NMR (125.7 MHz, CD3OD): d = 159.2 (CO), 123.2 (C-4triazole), 118.5 (C-5triazole), 75.7 (C-4), 74.5 (C-3), 72.3 (C-2), 69.5 (C-1), 68.0 (C-6), 54.6 (C-5), 51.2 (CH2N), 47.3 (CH2NH), 31.2, 30.4, 30.4, 29.9, 28.3 (CH2), 27.4 (CH2C), 23.8 (CH2CH3), 14.0 ppm (CH3); ESI-MS: m/z: 902 [2 M+Na] + , 462.5 [M+Na] + , 440.5 [M+H] + ; elemental analysis calcd (%) for C21H37N5O5 : C 57.38, H 8.48, N 15.93; found: C 57.24, H 8.34, N 16.09. 1-Amino-1-deoxy-N-(6-(4-propyl-1 H-1,2,3-triazol-1-yl)hexyla)-5,6-oxomethylidenemannojirimycin (9): Column chromatography, eluent 100:10:1!50:10:1 CH2Cl2/MeOH/H2O. Yield: 65 mg (86 %); [a]D = + 20 (c = 1.0, MeOH); Rf = 0.48 (50:10:1 CH2Cl2/MeOH/H2O); 1H NMR (500 MHz, CD3OD): d = 7.73 (s, 1 H, CHtriazole), 4.57 (d, 1 H, J1,2 = 1.9 Hz, H-1), 4.48 (t, 1 H, J5,6a = J6a,6b = 8.4 Hz, H-6a), 4.36 (t, 2 H, CH2Ntriazole), 4.28 (dd, 1 H, J5,6b = 4.4 Hz, H-6b), 3.95 (t, 1 H, J2,3 = 2.1 Hz, H-2), 3.72 (m, 1 H, H-5), 3.68 (m, 2 H, H-3, H-4), 2.66 (t, 2 H, JH,H = 7.5 Hz, CH2C), 2.56 (m, 2 H, CH2NH), 1.90 (q, 2 H, JH,H = 7.2 Hz, CH2CH2N), 1.69 (m, 2 H, CH2CH3), 1.49 (m, 2 H, CH2), 1.39 (m, 2 H, CH2), 1.32 (m, 2 H, CH2), 0.96 ppm (t, 3 H, JH,H = 7.4 Hz, CH3); 13C NMR (125.7 MHz, CD3OD): d = 160.3 (CO), 123.2 (C-4triazole), 110.2 (C-5triazole), 73.1 (C-2), 72.4 (C-3), 72.0 (C-4), 72.0 (C-1), 67.9 (C-6), 55.6 (C-5), 51.1 (CH2N), 46.8 (CH2NH), 31.2, 30.1 (CH2), 28.3 (CH2C), 27.6, 27.3 (CH2), 23.8 (CH2), 14.0 ppm (CH3); ESIMS: m/z: 817.3 [2M+Na] + , 420.1 [M+Na] + , 398.2 [M+H] + ; elemental analysis calcd (%) for C18H31N5O5 : C 54.39, H 7.86, N 17.62; found: C 54.22, H 7.663, N 17.26. 1-Amino-1-deoxy-N-(9-(4-propyl-1H-1,2,3-triazol-1-yl)nonyl)-5,6-oxomethylidenemannojirimycin (10): Column chromatography, eluent 100:10:1 CH2Cl2/MeOH/H2O; Yield: 59 mg (71 %); [a]D = + 23 (c 1.0, MeOH); Rf = 0.44 (50:10:1 CH2Cl2-MeOH-H2O); 1H NMR (500 MHz, CD3OD) d = 7.73 (s, 1 H, CHtriazole), 4.58 (d, 1 H, J1,2 = 1.8 Hz, H-1), 4.49 (t, 1 H, J5,6a = J6a,6b = 8.8 Hz, H-6a), 4.35 (t, 2 H, JH,H = 7.1 Hz, CH2Ntriazole), 4.29 (dd, 1 H, J5,6b = 4.5 Hz, H-6b), 3.96 (bs, 1 H, H-2), 3.73 (m, 1 H, H-5), 3.68 (m, 2 H, H-3, H-4), 2.66 (t, 2 H, JH,H = 7.5 Hz, CH2C), 2.58 (m, 2 H, CH2NH), 1.88 (m, 2 H, CH2), 1.68 (m, 2 H, CH2CH3), 1.49 (m, 2 H, CH2), 1.38 (m, 2 H, CH2), 1.25 (m, 10 H, 5CH2), 0.96 ppm (t, 3 H, JH,H = 7.4 Hz, CH3); 13C NMR (125.7 MHz, CD3OD): d = 160.3 (CO), 123.2 (C-4triazole), 114.9 (C-5triazole), 73.0 (C-2), 72.4 (C-3), 72.0 (C-1), 71.9 (C-1), 68.0 (C-6), 55.7 (C-5), 51.2 ACHTUNGRE(CH2N), 47.1 (CH2NH), 31.2, 30.4, 30.4, 30.2, 29.9, 28.3, 28.3, 27.4, 23.8 (CH2), 13.9 ppm (CH3); ESI-MS: m/z: 902 [2M+Na] + , 462.5 [M+Na] + , 440.5 [M+H] + ; elemental analysis calcd (%) for C21H37N5O5 : C 57.38 H 8.48 N 15.93; found: C 57.06, H 8.18, N 15.75. General procedure for the synthesis of C60-fullerene-sp2-iminosugar balls (11–14): The fullerene hexa-adduct 29 (83 mg, 0.028 mmol) and the corre-

 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Chem. Eur. J. 2013, 19, 16791 – 16803

Fullerene-sp2-Iminosugar Balls

FULL PAPER

sponding azide-armed sp2-iminosugar derivative 21–24 (0.36 mmol) were dissolved in 1:1 CH2Cl2/DMSO (2 mL). A solution of CuSO4·5 H2O (4.8 mg, 0.03 mmol) and sodium ascorbate (19.2 mg, 0.1 mmol) in distilled water (1 mL) was added. After a few minutes of stirring, 1 m TBAF in THF (415 mL, 0.42 mmol) was added and the reaction mixture was vigorously stirred at room temperature for 2 days. After evaporation of CH2Cl2, DMSO (1.5 mL) was added and the resulting solution was further for 2–4 days. The reaction mixture was concentrated and the product (11–14) was purified by precipitation using (50:10:1 CH2Cl2/CH3OH/ H2O)/H2O and washed several times with water. Fullerene–1N-ONJ (C6) ball 11: Yield: 148 mg (87 %); Rf = 0.69 (50:10:1 CH2Cl2/CH3OH/H2O); 1H NMR (300 MHz, [D6]DMSO): d = 8.03 (s, 12 H), 5.46 (m, 12 H), 5.39 (m, 12 H), 5.30 (m, 12 H), 5.19 (m, 12 H), 4.71 (m, 12 H), 4.24 (m, 24 H), 3.75 (m, 24 H), 2.65 (m, 24 H), 1.96 ppm (m, 24 H); 13C NMR (100.6 MHz, [D6]DMSO): d = 163.0 (c), 156.2 (CO; carbamate), 146.6 (g), 145.0 (sp2 C; C60), 140.7 (sp2 C; C60), 121.7 (h), 74.1 (C-4), 72.7 (C-3), 70.9 (C-2, d), 67.9 (C-1), 66.0 (C-6, a), 52.8 (C-5), 49.2 (CH2N, b), 45.7 (CH2NH), 29.7, 29.0 (CH2), 27.7 (f), 26.1, 25.8 (CH2), 21.4 ppm (e); IR (neat): n˜ = 3323 (O-H), 1733 cm1 (C=O); UV/Vis (H2O): lmax (e) = 269 (75 200), 283 (73 100), 319 (sh, 46 100), 337 nm (sh, 34 100 mol1 dm3 cm1); elemental analysis calcd (%) for C249H360N60O84 : C 58.09, H 5.97, N 13.83; found: C 57.86 H 5.68 N 13.45. Fullerene–1N-ONJ (C9) ball 12: Yield: 135 mg, 73 %; Rf = 0.66 (50:10:1 CH2Cl2 :CH3OH:H2O); 1H NMR (300 MHz, [D6]DMSO): d = 8.03 (s, 12 H), 5.46 (m, 12 H), 5.39 (m, 12 H), 5.30 (m, 12 H), 5.19 (m, 12 H), 4.71 (m, 12 H), 4.24 (m, 24 H), 3.75 (m, 24 H), 2.65 (m, 24 H), 1.96 ppm (m, 24 H); 13C NMR (100.6 MHz, [D6]DMSO): d = 163.1 (c), 156.1 (CO; carbamate), 145.1 (g), 144.9 (sp2 C; C60), 140.9 (sp2 C; C60), 121.7 (h), 74.1 (C-4), 72.8 (C-3), 71.8 (d), 70.9 (C-2), 67.9, (C-1), 66.0 (C-6), 65.6 (a), 52.8 (C-5), 49.2 (CH2N, b), 45.8 (CH2NH), 29.7, 29.2, 28.9, 28.4 (CH2), 27.7 (f), 26.7, 25.9, 23.1 (CH2), 21.3 ppm (e); IR (neat): n˜ = 3324 (O-H), 1737 cm1 (C=O); UV/Vis (H2O): lmax (e) = 269 (75 200), 283 (73 100), 319 (sh, 46 100), 337 nm (sh, 34 100 mol1 dm3 cm1); elemental analysis calcd (%) for C330H432N60O84 : C 60.21, H 6.61, N 12.77; found: C 59.89, H 6.43, N 12.40. Fullerene–1N-OMJ (C6) ball 13: Yield: 116 mg (68 %). Rf = 0.45 (50:10:1 CH2Cl2/CH3OH/H2O); 1H NMR (300 MHz, [D6]DMSO): d = 8.03 (s, 12 H), 5.46 (m, 12 H), 5.39 (m, 12 H), 5.30 (m, 12 H), 5.19 (m, 12 H), 4.71 (m, 12 H), 4.24 (m, 24 H), 3.75 (m, 24 H), 2.65 (m, 24 H), 1.96 ppm (m, 24 H); 13C NMR (125.7 MHz, [D6]DMSO): d = 162.9 (c), 157.0 (CO; carbamate), 145.5 (g), 145.0 (sp2 C; C60), 140.7 (sp2 C; C60), 121.7 (h), 71.8 (d), 71.5 (C-2), 70.6 (C-3), 70.4 (C-4, C-1), 66.4 (a), 65.8 (C-6), 54.9 (C-5), 49.2 (CH2N), 48.2 (b), 45.4 (CH2NH), 29.8, 28.9 (CH2), 27.7 (f), 26.2, 25.8 (CH2), 21.4 ppm (e); IR (neat): n˜ = 3323 (O-H), 1733 cm1 (C=O); UV/ Vis (H2O): lmax (e) = 269 (75 200), 283 (73 100), 319 (sh, 46 100), 337 nm (sh, 34 100 mol1 dm3 cm1); elemental analysis calcd (%) for C249H360N60O84 : C 58.09, H 5.97, N 13.83; found: C 57.71, H 5.76, N 13.49. Fullerene–1N-OMJ (C9) ball 14: Yield: 94 mg (51 %). Rf = 0.63 (50:10:1 CH2Cl2/CH3OH/H2O); 1H NMR (300 MHz, [D6]DMSO): d = 8.03 (s, 12 H), 5.46 (m, 12 H), 5.39 (m, 12 H), 5.30 (m, 12 H), 5.19 (m, 12 H), 4.71 (m, 12 H), 4.24 (m, 24 H), 3.75 (m, 24 H), 2.65 (m, 24 H), 1.96 ppm (m, 24 H); 13C NMR (100.6 MHz, [D6]DMSO): d = 163.0 (c), 157.0 (CO; carbamate), 145.7 (g), 145.5 (sp2 C; C60), 140.9 (sp2 C; C60), 121.8 (h), 71.5 (C-2), 70.6 (C-3, C-1), 70.4 (C-4, d), 65.8 (C-6, a), 53.8 (C-5), 49.2 (CH2N, b), 45.5 (CH2NH), 31.3, 29.7, 28.8 (CH2), 27.8 (f), 26.7, 25.9, 23.1, 22.1 (CH2), 19.2 ppm (e); IR: (neat): n˜ = 3298 (O-H), 1733 cm1 (C=O); UV/ Vis (H2O): lmax (e) = 269 (75 200), 283 (73 100), 319 (sh, 46 100), 337 nm (sh, 34 100 mol1 dm3 cm1); elemental analysis calcd (%) for C330H432N60O84 : C 60.21, H 6.61, N 12.77; found: C 59.90, H 6.34, N 12.37. General procedure for the inhibition assays: Inhibition constant (Ki) values were determined by spectrophotometrically measuring the residual hydrolytic activities of the glycosidases against the respective o- (for bglucosidase from bovine liver) or p-nitrophenyl a- or b-d-glycopyranoside (for other glycosidases) or a,a’-trehalose (for trehalase). Each assay was performed in phosphate buffer or phosphate-citrate buffer (for a- or b-mannosidase) at the optimal pH for the enzymes. The reactions were initiated by addition of enzyme to a solution of the substrate in the absence or presence of various concentrations of inhibitor. The mixture was

Chem. Eur. J. 2013, 19, 16791 – 16803

incubated for 10–30 min at 37 8C and the reaction was quenched by addition of 1 m Na2CO3. For trehalase the reaction was stopped by placing the mixture over boiling water for 3 min, cooled in ice/water, and centrifuged at 12 000 rpm for 5 min for remove the denatured protein. The concentration of d-glucose in the supernatant was determined by the glucose oxidase–peroxidase method (Glucose Trinder 100, from Sigma). Reaction times were appropriate to obtain 10–20 % conversion of the substrate in order to achieve linear rates. The absorbance of the resulting mixture was determined at 405 nm or 505 nm (for trehalase). Approximate values of Ki were determined using a fixed concentration of substrate (around the KM value for the different glycosidases) and various concentrations of inhibitor. Full Ki determinations and enzyme inhibition mode were determined from the slope of Lineweaver–Burk plots and double reciprocal analysis. Representative examples of the Lineweaver–Burk plots, with typical profile for competitive inhibition mode, are shown in the Supporting Information. Enzyme-linked lectin assay (ELLA): Nunc-Inmuno plates (MaxiSorp) were coated overnight with click lactose-polystyrene glycopolymer[65] at 100 mL per well diluted from a stock solution of 10 mg mL1 in 0.01 m phosphate buffer saline (PBS, pH 7.3 containing 0.1 mm Ca2 + and 0.1 mm Mn2 + ) at room temperature. The wells were then washed three times with 300 mL of washing buffer (containing 0.05 % (v/v) Tween 20; PBST). The washing procedure was repeated after each of the incubations throughout the assay. The wells were then blocked by incubation with 150 mL per well of 0.5 % polyvinyl alcohol/PBS[66] for 1 h at 37 8C and washed again. After washing, the wells were filled with 100 mL of serial dilutions of horseradish peroxidase labeled peanut (Arachis hypogaea) lectin (HRP–PNA) from 101 to 105 mg mL1 in PBS, and incubated at 37 8C for 1 h. The plates were washed and 50 mL per well of 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS; 0.25 mg mL1) in citrate buffer (0.2 m, pH 4.0 with 0.015 % H2O2) was added. The reaction was stopped after 20 min by adding 50 mL per well of 1 m H2SO4 and the absorbance was measured at 405 nm. Blank wells contained citrate-phosphate buffer. The concentration of lectin–enzyme conjugate that displayed an absorbance between 0.8 and 1.0 was used for inhibition experiments. To carry out the inhibition experiments, each ligand was added in a serial of twofold dilutions (60 mL per well) in PBS with of the desired PNA-peroxidase conjugate concentration (60 mL) on Nunclon (Delta) microtiter plates and incubated for 1 h at 37 8C. The above solutions (100 mL) were then transferred to the lactose polymer-coated microplates, which were incubated for 1 h at 37 8C. The plates were washed and the ABTS substrate was added (50 mL per well). Color development was stopped after 20 min and the absorbance was measured. The percent of inhibition was calculated as follows: Inhibition ð%Þ ¼ ðAðno inhibitorÞ Aðwith inhibitorÞ Þ=Aðno inhibitorÞ  100

ð1Þ

Results in triplicate were used for the plotting the inhibition curves for each individual ELLA experiment. Typically, the IC50 values (concentration required for 50 % inhibition of the Con A-coating lactose polymer association) obtained from several independently performed tests were in the range of  15 %. Nevertheless, the relative inhibition values calculated from independent series of data were highly reproducible. Two-site ELLA (sandwich assay): Nunc-Inmuno plates (MaxiSorp) microtitration plates were coated with peanut lectin at 100 mL per well of a stock solution of 5 mg mL1 in 0.01 m phosphate buffer (PBS, pH 7.3) for 2 h at 37 8C. The synthesized multivalent lactosides and lactose as a negative control were used as a stock solution of of PBS (3.4 mmol mL1). The ligands were added in serial two- to tenfold dilutions (50 mL per well) in PBS and incubated at 37 8C. After 1 h, horseradish peroxidase-labeled peanut lectin (50 mL per well of 200-fold dilution of a 1 mg mL1 stock solution in PBS) was added to the microtiter plates which were incubated for another hour at 37 8C. The plates were washed with PBS, and 50 mL per well of ABTS (1 mg/4 mL) in citrate-phosphate buffer (0.2 m, pH 4.0 with 0.015 % H2O2) was added. The reactions were stopped after 30 min by adding 50 mL per well of 1 m H2SO4, and the optical density was measured at 410 nm relative to 570 nm.

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Two-site competitive lectin–glycosidase ELLA: The lactosyl glypolymercoated microplates prepared as above described were further coated with PNA lectin at 100 mL per well of a stock solution of 5 mg mL1 in 0.01 m phosphate buffer (PBS, pH 7.3) for 2 h at 37 8C. The synthesized dodecavalent fullerene–1N-ONJ balls 11 and 12, used as 62 mm solutions in PBS, were then added (50 mL per well) and incubated at 37 8C. At this concentration, a classical two-site ELLA provided optical density values of 0.61 and 0.45 (absorbance units; A.U.; see Figure 4), respectively, which were normalized at 100 % cross-linking for the lectin–glycosidase competition experiments. The corresponding glycosidase (a-manase or maltase) in serial twofold dilutions (50 mL per well) from a stock solution of 40 U per mL in PBS and HRP–PNA lectin (50 mL per well of 100-fold dilution of a 1 mg mL1 stock solution in PBS) were then added and the microplates were incubated at 37 8C. The plates were washed with PBS, and 50 mL per well of 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS, 1 mg per 4 mL) in citrate-phosphate buffer (0.2 m, pH 4.0 with 0.015 % H2O2) was added. The reactions were stopped after 30 min by adding 50 mL per well of 1 m H2SO4, and the optical density was measured at 410 nm relative to 570 nm. Control experiments were conducted to confirm that the enzyme itself did not interact with PNA and that it retained its catalytic activity under the conditions of the assay. Two-site competitive lectin–glycosidase ELLA in the presence of monoand multivalent inhibitors: To get information about the involvement of the catalytic and non-glycone sites in the binding of the enzyme to the fullerene–1N-ONJ balls, the about protocol was repeated using in the last incubation step a solution of HRP–PNA lectin (50 mL per well of 100fold dilution of a 1 mg mL1 stock solution in PBS) that contained a mono or multivalent inhibitor of the corresponding glycosidase (10 or 13 for a-manase; 5 or 13 for maltase; 500 mm). Control experiments demonstrated that neither of the inhibitors affected PNA–HRP–PNA crosslinking in the absence of the enzyme.

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Acknowledgements

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The Spanish Ministerio de Economia y Competitividad (contract numbers SAF2010-15670 and CTQ2010–15848), the Fondo Europeo de Desarrollo Regional (FEDER), the Fondo Social Europeo (FSE), the Fundacin Ramn Areces, the Junta de Andaluca, and the Agence Nationale de la Recherche (ANR, Programme Blanc 2011, Sweet60s) are gratefully acknowledged for funding. R.R.-C. is recipient of a FPU grant. We further thank Dr. M. Holler and Dr. R. Caballero for helpful discussions.

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