# Understanding multivalent effects in glycosidase inhibition using C-glycoside click clusters as molecular probes

Fabien Stauffert, Anne Bodlenner, Thi Minh Nguyen Trinh, M. Isabel García-Moreno, Carmen Ortiz Mellet, Jean-François Nierengarten, Philippe Compain

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## To cite this version:

Fabien Stauffert, Anne Bodlenner, Thi Minh Nguyen Trinh, M. Isabel García-Moreno, Carmen Ortiz Mellet, et al.. Understanding multivalent effects in glycosidase inhibition using C-glycoside click clusters as molecular probes. New Journal of Chemistry, 2016, 40 (9), pp.7421-7430. 10.1039/c6nj01311b. hal-03525592

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Cite this: *New J. Chem.*, 2016, 40, 7421

# Understanding multivalent effects in glycosidase inhibition using C-glycoside click clusters as molecular probes†

Fabien Stauffert,ª Anne Bodlenner,ª Thi Minh Nguyen Trinh,ᵇ

M. Isabel García-Moreno,ᶜ Carmen Ortiz Mellet,ᶜ Jean-François Nierengarten*ᵇ and Philippe Compain*ª

The synthesis of the first examples of multivalent C-glycosides based on C₆₀-fullerene or β-cyclodextrin cores by way of Cu(I)-catalyzed azide–alkyne cycloadditions is reported. These compounds were designed as molecular probes to understand the mechanisms underlying the outstanding multivalent effects observed in glycosidase inhibition. The inhibition results obtained support a multivalent-binding model based on two scenarios both involving nonspecific interactions and varying by the presence or the absence of active site specific interactions. The magnitude of the multivalent effect obtained depends on the identity of the glycosidase involved and more specifically on the accessibility of its catalytic active site. Large inhibitory multivalent effects can be obtained when both glycosidase active sites and non-catalytic sites at the protein surface are involved in binding events. On the other hand, nonspecific interactions alone are not sufficient to achieve relative affinity enhancements exceeding a simple statistical effect (i.e., a relative inhibition potency not better than one on a valence-corrected basis).

Received (in Montpellier, France) 25th April 2016,

Accepted 9th June 2016

DOI: 10.1039/c6nj01311b

www.rsc.org/njc

## Introduction

The recent discovery of multivalent glycosidase inhibitors showing outstanding binding enhancements up to five orders of magnitude over the corresponding monovalent ligands challenges and stimulates the imagination of (bio)chemists.¹⁻⁴ First, because these enzymes, which powerfully catalyse glycosidic cleavage in carbohydrates and glycoconjugates, do not exhibit the typical structural features required for multivalent design. Glycosidases indeed display generally a single, buried catalytic site⁵ and bind to their monovalent substrates with high affinity and selectivity. The main approach to reversible glycosidase inhibitors has been consequently based on natural substrate mimetics, such as iminosugars in which the endocyclic oxygen atom of the parent glycosides is replaced by a nitrogen atom.⁶,⁷ The high affinity of iminosugars for

glycosidases is commonly explained by their ability to become protonated in biological medium and to form ammonium cations that interact strongly with a nucleophilic catalytic residue, commonly a carboxylate, in the enzyme active site.⁸ In sharp contrast, lectins, an important class of proteins playing numerous roles in biological recognition phenomena, appear to be ideal candidates for multivalent design as they usually display accessible, multiple carbohydrate-binding pockets.⁹ In such systems, multivalent interactions are used by Nature as a method to increase affinity and modulate selectivity since monovalent carbohydrate ligands typically bind only weakly to lectin receptors. The bridging of several adjacent binding sites by multidentate ligands, i.e. the so-called chelate effect, is indeed at the basis of the largest multivalent effects known to date.⁹,¹⁰ For proteins with one binding site, as anticipated for most enzymes, the statistical rebinding effect, which is due to high local ligand concentration, is believed to be one of the major binding modes at play.¹⁰f In combination with chelation or non-chelation mechanisms, additional aggregation phenomena have also to be considered, further complicating the rationale behind the observed multivalent effects. It is worth noting that systems that do not allow chelation mechanisms typically show moderate multivalent effects and require ligands with very high valency (>$ca.$ 100–150) to reach significant affinity enhancements.¹⁰f Considering these points, how to explain that the multivalent effects observed in glycosidase

<footnote>ª Laboratoire de Synthèse Organique et Molécules Bioactives (SYBIO), Université de Strasbourg/CNRS (UMR 7509), Ecole Européenne de Chimie, Polymères et Matériaux (ECPM), 25 rue Becquerel, 67087 Strasbourg Cedex 2, France. E-mail: philippe.compain@unistra.fr</footnote>

<footnote>ᵇ Laboratoire de Chimie des Matériaux Moléculaires, Université de Strasbourg et CNRS (UMR 7509), Ecole Européenne de Chimie, Polymères et Matériaux, 25 rue Becquerel, 67087 Strasbourg Cedex 2, France. E-mail: nierengarten@unistra.fr</footnote>

<footnote>ᶜ Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla, Profesor García González 1, E-41012, Sevilla, Spain</footnote>

<footnote>† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nj01311b</footnote>
<figure><img src="image_4.png" /></figure>

inhibition with neoglycoclusters displaying up to 36 imino-
sugar ligands²,³ rival those encountered for carbohydrate-lectin
interactions? To answer this fundamental question, the mole-
cular basis of the rather counter-intuitive inhibitory multivalent
effect has been explored independently by several groups using
different strategies³,¹¹,¹²α,¹³,¹⁴ and complementary techniques
including isothermal titration calorimetry,¹²α mass spectrometry,³
electron microscopy imaging³ and atomic force microscopy.¹¹
Beyond fundamental interest in understanding these new
phenomena, these studies are also stimulated by the thera-
peutic relevance of glycosidase inhibitors⁶,⁷,¹⁵ and by the first
promising applications of the multivalent effect to cellular
glycosidases for correcting protein folding defects.¹⁶ Recently,
the dual lectin- and glycosidase binding abilities of designed
C₆₀-based sp²-iminosugar clusters¹⁷ were exploited to probe the
involvement of non-catalytic sites and active site interactions in
multivalent binding events. Competitive lectin-glycosidase
assays, which were performed in the presence or the absence
of a potent monovalent competitive inhibitor, suggested two
different multivalent binding modes depending on the nature
of the glycosidase involved. For glycosidases known to possess
relatively deep catalytic sites, the latter would be only marginally
involved in multivalent binding events, leading to poor response
to multivalent ligand presentation. Most of the stabilizing
interactions in the enzyme-inhibitor complexes would arise
from non-catalytic sites located at the protein surface and the
inhibition would be only due to the blockage of the catalytic site
entrance. This mechanistic model has been demonstrated
by competitive assays¹⁴ for yeast maltase which belongs
to the glycosyl hydrolase family GH13. It could be applied to
other glycosidases of the GH13 family or known to have buried
active sites such as GH1 or GH27 glycosyl hydrolases. Jack bean
α-mannosidase (JBα-man), which belongs to the glycosyl hydro-
lase GH38 family, is the glycosidase, being the most sensitive to
multivalent presentation known to date. Despite the fact that

the 3-D crystallographic structure of JBα-man is currently
unknown, it is believed, by analogy with other members of the
GH38 family, that this enzyme possesses an open active site cleft
with several sugar-binding subsites. Due to this structural feature,
interactions with non-catalytic sites and specific interactions
with a more accessible catalytic site would be simultaneously
involved in binding events leading to strong multivalent effects
via chelation mechanisms. The multivalent-binding model pre-
sented above is thus based on two scenarios involving either just
interactions at non-catalytic sites or the concerted occurrence of
both active site and non-catalytic site-interactions. Whereas
iminosugar glycomimetics are ideally suited to bind at the
catalytic site of glycosidases, it remains uncertain whether or
not the interactions with the non-catalytic sites involved in multi-
valent enzyme inhibition are governed by similar principles. To
explore this hypothesis, we have now designed neoglycoclusters
as molecular probes in which the iminosugar-based specific
active-site-directed inhibitory epitopes (inhitopes) were replaced
by C-glycoside motifs, with an expected weaker affinity for the
catalytic site. Here, we describe the full details of our study from
C-glycoside cluster synthesis to the contrasted inhibition results
obtained whether the glycosidase active-site is supposed to be
involved or not in the multivalent binding events.

Results and discussion

Neoglycycluster design

The mechanistic probes we wanted to construct needed first to
be close analogues of iminosugar click clusters such as **1** which
are known to display significant inhibitory multivalent effects
(Fig. 1).¹,² Secondly, these neoglycoclusters have to be based on
inhitopes that should be hydrolytically stable¹⁸ and structurally
as close as possible as iminosugars without displaying their
selectivity towards glycosidase active-sites. To achieve this objective,

<figure><img src="image_5.png" /><figcaption>Fig. 1 Mono- and multivalent glycomimetics.</figcaption></figure>
we selected a C-glycosidic motif. The absence of a basic nitrogen atom was indeed expected to suppress key electrostatic interactions with nucleophilic catalytic residues and thus inhitope affinity with minimal structural modifications.

[60]Fullerene (C₆₀) and β-cyclodextrin (β-CD) were selected since these platforms have led, in previous studies, to clusters displaying large multivalent effects with relative inhibition potency on molar basis (rp/n) up to 620-fold.¹,²

## Neoglycocluster synthesis

Based on these design criteria, we targeted multivalent C-glycosides 2 differing by the nature of their central core (Fig. 1). As shown in Scheme 1, the synthesis began with the preparation of the azide-armed C-glycosides 9 and 10 from methyl tetra-O-benzyl-D-glucopyranoside (5). Following the protocol of Hosomi and Sakurai,¹⁹ compound 5 was reacted with allyltrimethylsilane in the presence of TMSOTf. This efficient process afforded α-C-allyl glycoside 6a²⁰ in 73% yield after purification on silica gel, the minor β-epimer 6b being obtained in 7% yield.

Our objective was then to perform a cross-metathesis reaction to complete the construction of the linker and functionalize the terminal position with a tosyl group. Unfortunately, our first attempts towards this goal were unsuccessful. The reaction of 4-penten-1-yl tosylate²¹ with α-C-allyl glycoside 6a in the presence of the Hoveyda-Grubbs II catalyst led to an inseparable mixture of the expected product along with the two corresponding analogues bearing a C₅ or a C₇ alkyl spacer as judged by NMR and MS analysis. This product distribution resulted from olefin isomerization/migration in the reactants during the cross-metathesis step.²² After optimization, compound 7 was obtained in 68% yield using an additive to the cross-metathesis ruthenium catalyst (Table S1 in the ESI⁺). Specifically, treatment of 6a and 4-penten-1-yl tosylate with

the Grubbs II catalyst (15 mol%) in the presence of 1,4-benzoquinone (1,4-BQ, 20 mol%) at room temperature was found to prevent the formation of the C₅ and C₇ byproducts. Under these conditions, 1,4-BQ is believed to oxidize ruthenium hydrides resulting from the decomposition of the metathesis catalyst and which are responsible for the olefin isomerization side-reactions.²²,²³ The selective one-step removal of the benzyl protecting groups and reduction of the double bond in 7 were performed by catalytic hydrogenolysis with H₂/Pd-C. The resulting crude tetrol product obtained was directly converted to the corresponding tetraacetate. Compound 8 was thus obtained in 68% yield from 7 (two steps). The displacement of the tosyl group with sodium azide was achieved in 91% yield in DMF to provide the desired protected substrate 9 for azide-alkyne cycloaddition coupling with the β-CD-based scaffold 12²⁴ (Scheme 2). Compound 10, the fully deprotected analogue of 9, prepared for the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) with fullerene hexa-adduct 13, was obtained using anion exchange Amberlite IRA-400 (OH⁻) resin. To assess any possible multivalent effect, C-glycoside 9 was reacted with ethynylcyclopropane in the presence of CuSO₄·5H₂O and sodium ascorbate to yield, after deprotection, the corresponding adduct 4b used as monovalent control. It can be noted that a cyclopropyl residue was used for 4b and not an n-propyl one as in the case of 4a. Actually, the reaction between 9 and 1-pentyne was attempted but it led to side-products¹²b arising from prior aerobic copper-catalyzed oxygenation at the propargylic methylene group. We found that this problem could be avoided by using the cyclopropylated alkyne as starting material for the preparation of the C-glycoside model compound. Having in hands the key azide-armed C-glycosides 9 and 10, we then performed the final steps of the cluster synthesis (Schemes 2 and 3).

<figure><img src="image_6.png" /><figcaption>Scheme 1 Synthesis of azide-armed C-glycosides 9–10 and of the monovalent model 4b.</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>Scheme 2 Synthesis of C-glycoside cluster 2b.</figcaption></figure>

<figure><img src="image_8.png" /></figure>
<figure><img src="image_9.png" /><figcaption>Scheme 3 Synthesis of C-glycoside cluster 2a.</figcaption></figure>

Microwave-assisted click conjugation of 9 and heptakis(2,6-di-O-propargyl) β-cyclodextrin 12²⁴ led to the desired tetradecavalent C-glycoside 3 in 83% yield (Scheme 2). Copper residues were removed at this stage following a simple protocol based on the filtration of the crude product on a pad of silica gel using ammonia as a complexing agent in the eluent before purification by flash chromatography. This purification process have previously demonstrated its efficiency for the synthesis of iminosugar click clusters leading to low content of residual copper ions (60 ppm) as measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES).³ Subsequent O-deacetylation of adduct 3 using anion exchange Amberlite IRA-400 (OH⁻) resin afforded the expected cluster 2b in high yield. Compound 2b showed good solubility in water. For the preparation of 2a, the fullerene scaffold 13²⁵ had to be directly functionalized with the unprotected C-glycoside as the presence of the malonic esters on the C₆₀ core is not compatible with the reaction conditions used for the deacetylation of a protected analogue. The click reaction between 10 and 13 was performed in a ternary solvent system (CH₂Cl₂/DMSO/H₂O) allowing the solubilisation of all the reagents but also of the final product. Importantly, no precipitation of partially clicked intermediates occurred during the course of the reaction thus allowing the complete functionalization of the scaffold.²⁶

At the end of the reaction, the product was precipitated with methanol and dried. The work-up and purification process proved difficult because of solubility problems and copper contamination leading to modest yields of the desired product. Purification of 2a was first achieved by gel permeation chromatography (Sephadex G-25, H₂O). Although this purification protocol was typically sufficient to remove the remaining amount of copper catalyst for related glycosylated fullerene derivatives, it was not the case here and ICP-AES analysis revealed 19 000 ppm

of copper at this stage. This prompted us to further filter the compound over a QuadraSil™ Mercaptopropyl column. Compound 2a was finally isolated in 34% yield and ICP-AES analysis revealed a low level of residual copper (994 ppm, i.e. a maximal concentration of 35 µM during inhibition assays).²⁷ The structure of 2a was confirmed by its T-symmetry deduced from the ¹³C NMR spectrum recorded in DMSO-d₆ as well as by mass spectrometry.

## Biological results

The inhibitory properties of C-glycosides clusters 2 as well as the monovalent control 4b were assayed towards a panel of commercial glycosidases, including JBα-man and yeast maltase (Table 1). As their corresponding analogues in the iminosugar series,¹,² the mono- and multivalent C-glycosides were found to display no inhibition towards β-mannosidase and β-galactosidase. In contrast, iminosugar clusters 1 and C-glycoside clusters 2 drastically differed on their behavior against β-glucosidase (from almonds) and α-galactosidase (from green coffee beans): whereas 1a and 1b displayed moderate to good inhibitory properties, compounds 2a and 2b showed no inhibition at concentrations up to 2 mM. In the case of yeast maltase the inhibition profile were very similar irrespective of the C-glycoside or iminosugar nature of the inhibitope. The 12-valent fullerene clusters 1a and 2a were at the limit of exhibiting formal multivalent effects, with relative inhibition potencies relative to the monovalent references 4a and 4b, on a valence-corrected basis (rp/n) close to one. The inhibitory efficiency was significantly decreased for the β-CD-based 14-valent clusters 1b and 2b. Considering that β-glucosidase (almonds), α-galactosidase (green coffee beans) and yeast maltase possess deep catalytic sites, a priori not accessible for multivalent conjugates, the above results can be interpreted in terms of the relative affinity of the different constructs for the non-catalytic sites involved in multivalent enzyme inhibition and their ability to block access of the substrate to the catalytic site. In the case of β-glucosidase (almonds) and α-galactosidase, the presence of the iminosugar motif in the clusters is essential for binding, both fullerene and β-CD architectures being equally efficient. In the case of maltase, however, the iminosugar and the C-glycoside inhibitope are equally efficient, but the enzyme was very sensitive to the topology of the conjugates, the fullerene conjugates affording much stronger inhibition as compared to the β-CD counterparts. The inhibitory performance of glycomimetic clusters towards JBα-man, an enzyme for which the active site and non-catalytic sites are simultaneously involved in multivalent enzyme inhibition,¹⁴ was found to be strongly dependent on the nature of the inhibitope. As observed with yeast maltase, both monovalent models 4a and 4b produced weak inhibition. Yet, replacing the iminosugar inhibitope by the C-glycoside motif had a drastic impact on the inhibition properties. Strong multivalent effects were indeed produced for the multivalent iminosugars 1 with rp/n up to 178, whereas no modest relative affinity enhancements (rp/n ~ 0.5) were observed with 2, the corresponding C-glycoside analogues. The higher differences in JBα-man inhibition potencies for multivalent than for

<figure><img src="image_10.png" /></figure>
<table><caption>**Table 1** Glycosidase inhibitory activities ($K_i$, µM) for the monovalent derivative (4) as well as multivalent glycomimetics (1–2)ᵃᵇ</caption><thead><tr><th rowspan="2">Enzyme</th><th colspan="3">Iminosugar derivatives</th><th colspan="3">C-Glycoside derivatives</th></tr><tr><th>Mono<br>DNJ 4a<sup>1,2</sup></th><th>12-Valent<br>DNJ 1a<sup>1</sup></th><th>14-Valent<br>DNJ 1b<sup>2</sup></th><th>Mono<br>C-glycoside 4b</th><th>12-Valent<br>C-glycoside 2a</th><th>14-Valent<br>C-glycoside 2b</th></tr></thead><tbody><tr><td><strong>α-Glucosidase</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Yeast maltase</td><td>152</td><td><strong>18</strong> (0.7)</td><td>270</td><td><strong>217</strong> ± <strong>40</strong></td><td><strong>19</strong> ± <strong>3</strong> (0.9)<sup>b</sup></td><td><strong>NI</strong><sup>a</sup></td></tr><tr><td>Amyloglucosidase<sup>c</sup></td><td>0.71</td><td>0.69</td><td>0.2</td><td>160 ± 32</td><td><strong>NI</strong><sup>a</sup></td><td>378 ± 29</td></tr><tr><td><strong>β-Glucosidase</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Almonds</td><td>11</td><td>95</td><td>111</td><td><strong>NI</strong><sup>a</sup></td><td><strong>NI</strong><sup>a</sup></td><td><strong>NI</strong><sup>a</sup></td></tr><tr><td>Bovine liver</td><td>482</td><td>247</td><td>25 (1.4)<sup>b</sup></td><td>282 ± 45</td><td><strong>NI</strong><sup>a</sup></td><td>201 ± 10</td></tr><tr><td><strong>α-Galactosidase</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Green coffee bean</td><td>NI</td><td>84</td><td>28</td><td><strong>NI</strong><sup>a</sup></td><td><strong>NI</strong><sup>a</sup></td><td><strong>NI</strong><sup>a</sup></td></tr><tr><td><strong>β-Galactosidase</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td><em>E. coli</em></td><td><strong>NI</strong><sup>a</sup></td><td><strong>NI</strong><sup>a</sup></td><td><strong>NI</strong><sup>a</sup></td><td><strong>NI</strong><sup>a</sup></td><td><strong>NI</strong><sup>a</sup></td><td><strong>NI</strong><sup>a</sup></td></tr><tr><td><strong>α-Mannosidase</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Jack Bean</td><td>322</td><td><strong>0.15</strong> (178)<sup>b</sup></td><td><strong>0.5</strong> (46)<sup>b</sup></td><td>524 ± 35</td><td><strong>79</strong> ± <strong>10</strong> (0.5)<sup>b</sup></td><td><strong>NI</strong><sup>a</sup></td></tr><tr><td><strong>β-Mannosidase</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Helix pomatia</td><td><strong>NI</strong><sup>a</sup></td><td><strong>NI</strong><sup>a</sup></td><td><strong>NI</strong><sup>a</sup></td><td><strong>NI</strong><sup>a</sup></td><td><strong>NI</strong><sup>a</sup></td><td><strong>NI</strong><sup>a</sup></td></tr></tbody></table>

monovalent derivatives in the iminosugar and C-glycoside series suggest that interactions at non-catalytic sites contribute significantly to the binding affinity of the multivalent conjugates and that the iminosugar motif is better suited for such binding mode. As for maltase, a notable influence of the scaffold architecture in the inhibition performance was additionally observed, fullerene conjugates being much more efficient than β-CD derivatives. β-Glucosidase from bovine liver and amyloglucosidase from *Aspergillus niger* offer further examples of inhitope-dependent and scaffold-dependent multivalent enzyme inhibition. As the almond enzyme, bovine liver β-glucosidase belongs to the GH1 family and the catalytic site is not expected to participate in binding to multivalent conjugates. Whereas in monovalent form the C-glycoside derivative **4b** was a two-fold better inhibitor that the iminosugar derivative **4a**, after multivalent presentation the iminosugar turned to be a much better inhitope, the β-CD conjugate **1b** becoming about 10-fold more efficient than the corresponding C-glycoside analogue **2b**. Amyloglucosidase, a GH15 enzyme, also possesses a relatively deep glycone (−1) binding site, but the aglycone (+1) binding site has been shown to be relatively accessible²⁸ and, interestingly, to bind 1-deoxynojirimycin.²⁹ This probably explains the strong inhibition properties of the multivalent derivatives **1a** and **1b**, similar to that of the monovalent reference **4a**, in spite of the expected shift in the binding mode. A preference for the β-CD architecture was observed in this case.

## Conclusions

In conclusion, we have synthesized unprecedented multivalent C-glycosides based on $C_{60}$-fullerene or β-cyclodextrin cores. These compounds were designed as mechanistic probes to understand the molecular basis of strong inhibition of glycosidases by multivalent constructs. The inhibition results further confirmed

the hypothesis that large inhibitory multivalent effects can be obtained when both glycosidase active sites and non-catalytic sites are involved in binding events, as it is the case for JBz-man. For glycosyl hydrolases with less accessible active sites, multivalent presentation of inhibitory motifs may lead to cancellation, conservation or enhancement of the inhibition capabilities. In the last two cases, binding of the multivalent conjugate occurs at non-catalytic sites of the enzyme, which makes a direct comparison with the corresponding monovalent model questionable. Instead, comparison of analogous multivalent structures led us to evaluate the efficiency of different inhitope motifs and topologies in promoting the inhibition of glycosidases. Indeed, the present body of work revealed that enzymes sensitive to multivalent enzyme inhibition can exhibit marked differences in their response to different inhitopes as well as to different architectural presentations. It is expected that the implications of these findings will be exploited in the design of potent multivalent glycosidase inhibitors.

## Experimental section

Solvents were of reagent grade and further dried when necessary. Dichloromethane ($CH_2Cl_2$) was distilled over $CaH_2$ under argon. Pyridine was distilled over KOH under argon and stored over KOH. Dry acetonitrile and dry DMF (both over molecular sieves) were purchased from commercial vendors and used as such. 4-Penten-1-yl tosylates,²¹ **12**²⁴ and **13**²⁵ were prepared according to previously reported procedures. All reactions were performed in standard glassware and microwave reactions were carried out using Biotage microwave reactor vials and an initiator microwave synthesizer. The reactions were monitored by thin layer chromatography (TLC) on aluminium sheets coated with silica gel 60 $F_{254}$ purchased from Merck KGaA. Visualization was accomplished using UV light (at 254 nm) and exposure to

<figure><img src="image_11.png" /></figure>

<footnote>ᵃ NI: no inhibition detected at 2 mM. ᵇ Whenever relevant, rp/n values are given in brackets: rp = $K_i$ (monovalent reference)/$K_i$(glycocluster), n = number of inhitope units. ᶜ From *A. Niger*.</footnote>
<figure><img src="image_12.png" /></figure>

TLC stains, phosphomolybdic acid or potassium permanganate, followed by heating. Flash column chromatographies were carried out on silica gel 60 (230–400 mesh, 40–63 µm) purchased from Merck KGaA. ¹H and ¹³C NMR experiments were carried out at 298 K on either a Bruker Avance 300 MHz or a Bruker Avance III HD 400 MHz spectrometer. The chemical shifts are reported as δ values in parts per million (ppm) relative to residual solvent signals used as an internal reference. The exponents “A” or “B” will be used for diastereotopic protons, “A” is assigned to the proton with the lowest chemical shift and “B” is assigned to the proton with the highest chemical shift. Assignments of ¹H and ¹³C signals were made by DEPT, ¹H–¹H COSY, HSQC and HMBC experiments. For convenience the assignment of ¹H and ¹³C for all the molecules was based on the same numbering (see ESI†). Optical rotations were measured at 589 nm (sodium lamp) and 20 °C on either a Perkin-Elmer 341 polarimeter or an Anton Paar MCP 200 polarimeter with a path length of 1 dm. The concentration (c) is indicated in gram per deciliter. Infrared (IR) spectra were recorded neat on a Perkin-Elmer Spectrum Two FT-IR spectrometer. High-resolution (HRMS) electrospray ionization-time-of-flight (ESI-TOF) mass spectra were recorded on a Bruker micrOTOF® mass spectrometer. Residual copper ions traces were measured on an Inductively Coupled Plasma Optical Emission Spectrometer (Varian 720-ES) at 324.754 nm.

### α-1-C-Allyl-2,3,4,6-tetra-O-benzyl-1-deoxy-D-glucopyranose 6a

At rt, allyltrimethylsilane (1.75 mL, 11.01 mmol, 3 eq.) was added to a solution of 5 (2.01 g, 3.62 mmol) in dry MeCN (20 mL) and the solution was stirred for 30 min. The solution was then cooled to 0 °C and TMSOTf (0.66 mL, 3.65 mmol, 1 eq.) was added dropwise. The reaction mixture was allowed to warm to rt and stirred for 19 h. Et₂O was added and the organic layer was successively washed with sat. aqueous NaHCO₃, water and brine, dried over Na₂SO₄, filtered and concentrated under reduced pressure. The crude residue was purified by flash column chromatography (Et₂O/petroleum ether, 20:80) to afford 6a (1.49 g, 73%) and 6b (150 mg, 7%) as white solids. The analytical data of 6a were in complete agreement with those reported in the literature.²⁰

$R_f$ 0.42 (Et₂O/petroleum ether, 20:80); ¹H-NMR (400 MHz, CDCl₃) $\delta$ 2.46–2.58 (m, 2H; CH₂-7), 3.61–3.86 (m, 6H; H-2, H-3, H-4, H-5 and CH₂-6), 4.16 (ddd, $J$ = 10.2, 5.4, 4.8 Hz, 1H; H-1), 4.50 (d, $J$ = 12.2 Hz, 1H; CH₂Ph), 4.50 (d, $J$ = 10.6 Hz, 1H; CH₂Ph), 4.65 (d, $J$ = 12.2 Hz, 1H; CH₂Ph), 4.66 (d, $J$ = 11.6 Hz, 1H; CH₂Ph), 4.72 (d, $J$ = 11.6 Hz, 1H; CH₂Ph), 4.83 (d, $J$ = 11.0 Hz, 1H; CH₂Ph), 4.84 (d, $J$ = 10.6 Hz, 1H; CH₂Ph), 4.96 (d, $J$ = 11.0 Hz, 1H; CH₂Ph), 5.10 (dd, $J$ = 10.0, 1.5 Hz, 1H; H<sup>B-9</sup>), 5.14 (dd, $J$ = 17.1, 1.5 Hz, 1H; H<sup>A-9</sup>), 5.85 (ddt, $J$ = 17.1, 10.0 and 7.0 Hz, 1H; H-8), 7.12–7.18 (m, 2H; H<sub>Ar</sub>), 7.26–7.39 (m, 18H; H<sub>Ar</sub>); ¹³C-NMR (100 MHz, CDCl₃) $\delta$ 30.0 (C-7), 69.1 (C-6), 71.3 (C-5), 73.2 (CH₂Ph), 73.6 (CH₂Ph), 73.9 (C-1), 75.2 (CH₂Ph), 75.6 (CH₂Ph), 78.3 (C-4), 80.2 (C-2), 82.6 (C-3), 117.0 (C-9), 127.7, 127.8, 127.9, 127.96, 128.01, 128.07, 128.12, 128.47, 128.53, 128.6 (20 × CH<sub>Ar</sub>), 134.9 (C-8), 138.2, 138.3, 138.4, 138.9 (4 × C<sub>Ar</sub>).

### 2,3,4,6-Tetra-O-benzyl-1-deoxy-1-C-[6-(tosyloxy)hex]-α-D-glucopyranose 7

An oven-dried Schlenk tube was charged with 6a (52 mg, 0.0921 mmol), 4-penten-1-yl tosylate (120 mg, 0.4993 mmol, 5.4 eq.), 1,4-benzoquinone (2 mg, 0.0185 mmol, 0.2 eq.) and the Grubbs II catalyst (7.7 mg, 0.0091 mmol, 0.1 eq.). The Schlenk tube was evacuated under vacuum and backfilled with argon (three cycles). Dry and degassed CH₂Cl₂ (1.2 mL) was added and the reaction mixture was stirred at rt under argon for 6 h. Additional Grubbs 2nd generation catalyst (3.9 mg, 0.0046 mmol, 0.05 eq.) was added to the reaction mixture, followed by additional stirring for 64 h. The reaction mixture was concentrated under reduced pressure. The crude residue was purified by flash column chromatography (EtOAc/petroleum ether, 15:85 to 20:80; solid deposition) to afford 7 (49 mg, 68%) as a mixture of E/Z isomers (colorless oil).

$R_f$ 0.34 (EtOAc/petroleum ether, 20:80); IR (neat) 1175, 1360 cm⁻¹; ¹H-NMR (400 MHz, CDCl₃) $\delta$ 1.62–1.72 (m, 2H; CH₂-11), 1.96–2.08 (m, 2H; CH₂-10), 2.31–2.46 (m, 5H; CH₂-7 and CH₃), 3.53–3.81 (m, 6H; H-2, H-3, H-4, H-5 and CH₂-6), 3.96–4.06 (m, 3H; H-1 and CH₂-12), 4.43–4.50 (m, 2H; CH₂Ph), 4.57–4.64 (m, 2H; CH₂Ph), 4.65–4.71 (m, 1H; CH₂Ph), 4.77–4.83 (m, 2H; CH₂Ph), 4.90–4.96 (m, 1H; CH₂Ph), 5.30–5.45 (m, 2H; H-8 and H-9), 7.10–7.15 (m, 2H; H<sub>Ar</sub>), 7.22–7.36 (m, 20H; H<sub>Ar</sub>), 7.76–7.80 (m, 2H; H<sub>Ar</sub>); ¹³C-NMR (100 MHz, CDCl₃) $\delta$ 21.8 (CH₃), 28.5, 28.6, 28.7 (C-7, C-10, C-11), 69.2 (C-6), 70.1 (C-12), 71.2 (C-5), 73.2 (CH₂Ph), 73.6 (CH₂Ph), 74.0 (C-1), 75.2 (CH₂Ph), 75.6 (CH₂Ph), 78.3 (C-4), 80.3 (C-2), 82.5 (C-3), 127.6, 127.7, 127.8, 127.88, 127.93, 128.01, 128.03, 128.05, 128.12, 128.47, 128.54, 128.6 (C-8, C-9, 24 × CH<sub>Ar</sub>), 134.9 (C-8), 133.4, 138.2, 138.3, 138.4, 138.9, 144.8 (6 × C<sub>Ar</sub>); HRMS (ESI) *m/z* 799.3279 ([M + Na]⁺, calcd for C₄₇H₅₂O₈Nₐ: 799.3275).

### 2,3,4,6-Tetra-O-acetyl-1-deoxy-1-C-[6-(tosyloxy)hex]-α-D-glucopyranose 8

To a solution of 7 (144 mg, 0.1853 mmol) in THF (4 mL) and MeOH (2 mL) were added 1 M aqueous HCl (0.25 mL) and Pd/C (20 mg, 0.0188 mmol, 10% Pd on C, 0.1 eq.). The flask was evacuated under vacuum and backfilled with argon (four cycles), and then evacuated under vacuum and backfilled with H₂ (four cycles). The reaction mixture was stirred under an atmosphere of H₂ (balloon) at rt for 4 h. The reaction mixture was then filtered through a pad of Celite, previously washed first with 1M aqueous HCl (at least 250 mL) and then with water. The catalyst was washed with MeOH, and the filtrate was concentrated under reduced pressure. The crude residue was dissolved in a mixture of dry pyridine (4 mL) and Ac₂O (4 mL), and then DMAP (4.5 mg, 0.0368 mmol, 0.2 eq.) was added to the solution. The reaction mixture was stirred at rt for 5 h. Solvents were evaporated under reduced pressure and the crude residue was purified by flash column chromatography (EtOAc/petroleum ether, 30:70) to afford 8 (74 mg, 68%) as a colorless oil.

$R_f$ 0.43 (EtOAc/petroleum ether, 40:60); [x]ₚD²⁰ + 40 (c 1.0, CHCl₃); IR (neat) 1175, 1365, 1744 cm⁻¹; ¹H-NMR (400 MHz, CDCl₃) $\delta$ 1.22–1.48 (m, 7H; H<sup>A-7</sup>, CH₂-8, CH₂-9 and CH₂-10),
<figure><img src="image_13.png" /></figure>

1.59–1.68 (m, 2H; CH₂-11), 1.69–1.80 (m, 1H; H<sup>B</sup>-7), 2.02 (s, 3H; C(O)CH₃), 2.03 (s, 3H; C(O)CH₃), 2.04 (s, 3H; C(O)CH₃), 2.07 (s, 3H; C(O)CH₃), 2.45 (s, 3H; CH₃), 3.79 (ddd, *J* = 9.4, 5.3 and 2.3 Hz, 1H; H-5), 4.01 (*t*, *J* = 6.4 Hz, 2H; CH₂-12), 4.07 (*dd*, *J* = 12.1, 2.3 Hz, 1H; H<sup>A</sup>-6), 4.09–4.16 (m, 1H; H-1), 4.22 (*dd*, *J* = 12.1, 5.3 Hz, 1H; H<sup>B</sup>-6), 4.96 (*dd*, *J* = 9.4, 8.8 Hz, 1H; H-4), 5.05 (*dd*, *J* = 9.5, 5.7 Hz, 1H; H-2), 5.30 (*dd*, *J* = 9.5, 8.8 Hz, 1H; H-3), 7.35 (*d*, *J* = 8.1 Hz, 2H; H<sub>Ar</sub>), 7.78 (*d*, *J* = 8.1 Hz, 2H; H<sub>Ar</sub>); <sup>13</sup>C-NMR (100 MHz, CDCl₃) δ 20.7 (C(O)CH₃), 20.75 (2 × C(O)CH₃), 20.77 (C(O)CH₃), 21.7 (CH₃), 24.8, 25.2, 25.4, 28.6, 28.8 (C-7, C-8, C-9, C-10, C-11), 62.5 (C-6), 68.6 (C-5), 69.0 (C-4), 70.48, 70.52 (C-2, C-3, C-12), 72.5 (C-1), 127.9 (2 × CH<sub>Ar</sub>), 129.9 (2 × CH<sub>Ar</sub>), 133.2 (C<sub>Ar</sub>), 144.8 (C<sub>Ar</sub>), 169.6, 169.7, 170.2, 170.7 (4 × C(O)CH₃); HRMS (ESI) *m/z* 609.1897 ([M + Na]<sup>+</sup>, calcd for C₂₇H₃₈O₁₂SNa: 609.1976).

### 2,3,4,6-Tetra-O-acetyl-1-deoxy-1-C-(6-azidohexyl)-α-D-glucopyranose 9

NaN₃ (41 mg, 0.6307 mmol, 4.6 eq.) was added to a solution of 8 (80 mg, 0.1364 mmol) in dry DMF (2 mL), and the reaction mixture was stirred at rt under argon for 17 h. The solvent was evaporated under reduced pressure and the residue was diluted with EtOAc and washed with water (2×) and brine. The organic layer was dried over Na₂SO₄, filtered and concentrated under reduced pressure. The crude residue was purified by flash column chromatography (EtOAc/petroleum ether, 30:70) to afford 9 (57 mg, 91%) as a colorless solid.

$R_f$ 0.30 (EtOAc/petroleum ether, 30:70); [$\alpha$]<sup>20</sup><sub>D</sub> + 63 (c 2.0, CHCl<sub>3</sub>); IR (neat) 1743, 2096 cm<sup>-1</sup>; <sup>1</sup>H-NMR (400 MHz, CDCl<sub>3</sub>) δ 1.22–1.50 (m, 7H; H<sup>A</sup>-7, CH<sub>2</sub>-8, CH<sub>2</sub>-9 and CH<sub>2</sub>-10), 1.53–1.63 (m, 2H; CH<sub>2</sub>-11), 1.70–1.81 (m, 1H; H<sup>B</sup>-7), 2.00 (s, 3H; C(O)CH<sub>3</sub>), 2.01 (s, 3H; C(O)CH<sub>3</sub>), 2.02 (s, 3H; C(O)CH<sub>3</sub>), 2.06 (s, 3H; C(O)CH<sub>3</sub>), 3.25 (*t*, *J* = 6.8 Hz, 2H; CH<sub>2</sub>-12), 3.78 (ddd, *J* = 9.4, 5.3 and 2.4 Hz, 1H; H-5), 4.05 (*dd*, *J* = 12.1, 2.4 Hz, 1H; H<sup>A</sup>-6), 4.10–4.17 (m, 1H; H-1), 4.21 (*dd*, *J* = 12.1, 5.3 Hz, 1H; H<sup>B</sup>-6), 4.95 (*dd*, *J* = 9.4, 8.9 Hz, 1H; H-4), 5.04 (*dd*, *J* = 9.5, 5.8 Hz, 1H; H-2), 5.29 (*dd*, *J* = 9.5, 8.9 Hz, 1H; H-3); <sup>13</sup>C-NMR (100 MHz, CDCl<sub>3</sub>) δ 20.7, 20.8 (4 × C(O)CH<sub>3</sub>), 24.9, 25.2, 26.8, 28.8, 28.9 (C-7, C-8, C-9, C-10, C-11), 51.4 (C-12), 62.5 (C-6), 68.7 (C-5), 69.0 (C-4), 70.5 (C-3), 70.6 (C-2), 72.6 (C-1), 169.6, 169.7, 170.2, 170.7 (4 × C(O)CH<sub>3</sub>); HRMS (ESI) *m/z* 480.1976 ([M + Na]<sup>+</sup>, calcd for C₂₀H₃₁N₃O₉Na: 480.1953).

### α-1-C-(6-Azidohexyl)-1-deoxy-D-glucopyranose 10

To a solution of 9 (130 mg, 0.2842 mmol) in a mixture of MeOH (7 mL) and H₂O (7 mL) was added Amberlite IRA400 (OH⁻) resin (3.5 g). The mixture was stirred using a rotavapor at rt for 18 h. The resin was then filtered, rinsed with MeOH and the filtrate was concentrated under reduced pressure. The crude residue was purified by flash column chromatography (CH₂Cl₂/MeOH, 90:10) to afford 10 (70 mg, 85%) as a colorless oil.

$R_f$ 0.28 (CH₂Cl₂/MeOH, 90:10); [$\alpha$]<sup>20</sup><sub>D</sub> + 69 (c 2.0, MeOH); IR (neat) 2096, 3353 cm<sup>-1</sup>; <sup>1</sup>H-NMR (400 MHz, CD₃OD) δ 1.26–1.48 (m, 5H; H<sup>A</sup>-7, CH₂-9 and CH₂-10), 1.49–1.75 (m, 5H; H<sup>B</sup>-7, CH₂-8 and CH₂-11), 3.21–3.34 (m, 3H; H-4 and CH₂-12), 3.35–3.43 (m, 1H; H-5), 3.50–3.68 (m, 3H; H-2, H-3 and H<sup>A</sup>-6), 3.75–3.82 (m, 1H; H<sup>B</sup>-6), 3.84–3.92 (m, 1H; H-1); <sup>13</sup>C-NMR (100 MHz, CD₃OD)

δ 25.2, 26.5, 27.8, 29.8, 30.0 (C-7, C-8, C-9, C-10, C-11), 52.4 (C-12), 63.1 (C-6), 72.4 (C-4), 73.1 (C-2), 74.3 (C-5), 75.3 (C-3), 77.2 (C-1); HRMS (ESI) *m/z* 312.1556 ([M + Na]<sup>+</sup>, calcd for C₁₂H₂₃N₃O₅Na: 312.1530).

### 2,3,4,6-Tetra-O-acetyl-1-C-[6-(4-cyclopropyl-1H-1,2,3-triazol-1-yl)hexyl]-1-deoxy-α-D-glucopyranose 11

To a solution of 9 (29 mg, 0.0634 mmol) and ethynylcyclopropane (0.02 mL, 0.2363 mmol, 3.7 eq.) in DMF (1.7 mL) in a microwave vial was added a yellow suspension of CuSO₄·5H₂O (2 mg, 0.0080 mmol, 0.13 eq.) and sodium ascorbate (3 mg, 0.0151 mmol, 0.24 eq.) in water (0.45 mL). The resulting suspension was stirred and heated under microwave irradiation at 80 °C for 30 min. Additional ethynylcyclopropane (0.02 mL, 0.2363 mmol, 3.7 eq.) was added and the reaction mixture was stirred and heated under microwave irradiation at 80 °C for another 30 min. The reaction mixture was diluted with water and extracted with EtOAc (3×). The combined organic extracts were washed with water (3×), dried over Na₂SO₄, filtered and concentrated under reduced pressure. The residue was diluted with a mixture of MeCN/H₂O/NH₄OH (10:1:1) and filtered through a pad of silica gel, using the same mixture as the eluent. Copper salts precipitated as a blue powder and remained at the top of the silica gel pad. The filtrate was concentrated under reduced pressure and the crude residue was purified by flash column chromatography (EtOAc/petroleum ether, 70:30) to afford 11 (30 mg, 90%) as a colorless solid.

$R_f$ 0.36 (EtOAc/petroleum ether, 70:30); [$\alpha$]<sup>20</sup><sub>D</sub> + 54 (c 1.0, CHCl₃); IR (neat) 1746 cm<sup>-1</sup>; <sup>1</sup>H-NMR (400 MHz, CDCl₃) δ 0.78–0.96 (m, 4H; CH₂-16 and CH₂-17), 1.18–1.49 (m, 7H; H<sup>A</sup>-7, CH₂-8, CH₂-9 and CH₂-10), 1.67–1.80 (m, 1H; H<sup>B</sup>-7), 1.81–1.96 (m, 3H; CH₂-11 and H-15), 2.01 (s, 6H; 2 × C(O)CH₃), 2.03 (s, 3H; C(O)CH₃), 2.05 (s, 3H; C(O)CH₃), 3.73–3.81 (m, 1H; H-5), 4.02–4.09 (m, 1H; H<sup>A</sup>-6), 4.09–4.16 (m, 1H; H-1), 4.17–4.31 (m, 3H; H<sup>B</sup>-6 and CH₂-12), 4.91–4.99 (m, 1H; H-4), 5.00–5.08 (m, 1H; H-2), 5.24–5.32 (*dd*, *J* = 9.2 Hz, 1H; H-3), 7.20 (s, 1H; H-13); <sup>13</sup>C-NMR (100 MHz, CDCl₃) δ 6.8 (C-15), 7.8 (C-16, C-17), 20.76, 20.82, 20.9 (4 × C(O)CH₃), 24.9, 25.3, 26.6, 28.8 (C-7, C-8, C-9, C-10), 30.3 (C-11), 50.1 (C-12), 62.5 (C-6), 68.7 (C-5), 69.0 (C-4), 70.5, 70.6 (C-2, C-3), 72.6 (C-1), 119.5 (C-13), 150.3 (C-14), 169.6, 169.8, 170.3, 170.7 (4 × C(O)CH₃); HRMS (ESI) *m/z* 524.2628 ([M + H]<sup>+</sup>, calcd for C₂₅H₃₈N₃O₉: 524.2603).

### α-1-C-[6-(4-Cyclopropyl-1H-1,2,3-triazol-1-yl)hexyl]-1-deoxy-D-glucopyranose 4b

To a solution of 11 (28 mg, 0.0535 mmol) in MeOH (3 mL) was added Amberlite IRA400 (OH⁻) resin (1.0 g). The mixture was stirred using a rotavapor at rt for 16 h. The resin was then filtered, rinsed with MeOH and the filtrate was concentrated under reduced pressure. The crude residue was purified by flash column chromatography (CH₂Cl₂/MeOH, 90:10 to 85:15) to afford 4b (17 mg, 89%) as a colorless oil.

$R_f$ 0.20 (CH₂Cl₂/MeOH, 90:10); [$\alpha$]<sup>20</sup><sub>D</sub> + 53 (c 1.0, MeOH); IR (neat) 3340 cm<sup>-1</sup>; <sup>1</sup>H-NMR (400 MHz, CD₃OD) δ 0.72–0.79 (m, 2H; CH₂-16 or CH₂-17), 0.91–1.00 (m, 2H; CH₂-16 or CH₂-17), 1.24–1.45 (m, 5H; H<sup>A</sup>-8, CH₂-9 and CH₂-10), 1.47–1.71 (m, 3H; CH₂-7 and H<sup>B</sup>-8), 1.83–1.99 (m, 3H; CH₂-11 and H-15), 3.22
<figure><img src="image_14.png" /></figure>

(dd, *J* = 9.5, 8.5 Hz, 1H; H-4), 3.34–3.41 (m, 1H; H-5), 3.50 (dd, *J* = 9.4, 8.5 Hz, 1H; H-3), 3.57 (dd, *J* = 9.4, 5.6 Hz, 1H; H-2), 3.61 (dd, *J* = 11.5, 5.6 Hz, 1H; H-A-6), 3.77 (dd, *J* = 11.5, 2.3 Hz, 1H; H-B-6), 3.86 (ddd, *J* = 10.3, 5.6 and 3.9 Hz, 1H; H-1), 4.32 (t, *J* = 7.0 Hz, 2H; CH₂-12), 7.67 (s, 1H; H-13); ¹³C-NMR (100 MHz, CD₃OD) δ 7.3 (C-15), 8.2 (C-16, C-17), 25.3 (C-7), 26.4 (C-8), 27.4 (C-10), 29.8 (C-9), 31.2 (C-11), 51.2 (C-12), 63.2 (C-6), 72.5 (C-4), 73.1 (C-2), 74.4 (C-5), 75.3 (C-3), 77.2 (C-1), 121.8 (C-13), 151.4 (C-14); HRMS (ESI) *m/z* 356.2200 [(M + H)⁺], calcd for C₁₇H₃₀N₃O₅: 356.2180).

## Compound 3

To a solution of heptakis(2,6-di-O-propargyl)cyclo maltoheptaose ¹² (13 mg, 0.0078 mmol) and ⁹ (55 mg, 0.1202 mmol, 15.4 eq.) in DMF (1.2 mL) in a microwave vial was added a yellow suspension of CuSO₄·5H₂O (3 mg, 0.0120 mmol, 1.5 eq.) and sodium ascorbate (5 mg, 0.0252 mmol, 3.2 eq.) in water (0.3 mL). The resulting suspension was stirred and heated under microwave irradiation at 80 °C for 30 min. The reaction mixture was diluted with water and extracted with EtOAc (3 × 10 mL). The combined organic extracts were washed with water, dried over Na₂SO₄, filtered and concentrated under reduced pressure. The residue was diluted with a mixture of MeCN/H₂O/NH₄OH (15:0.5:0.5) and filtered through a pad of silica gel, using the same mixture as the eluent. Copper salts precipitated as a blue powder and remained at the top of the silica gel pad. The filtrate was concentrated under reduced pressure and the crude residue was purified by flash column chromatography (CH₂Cl₂/MeOH, 98:2 to 95:5) to afford 3 (52 mg, 83%) as a pale yellow oil.

R<sub>f</sub> 0.34 (CH<sub>2</sub>Cl<sub>2</sub>/MeOH, 96:4); [α]<sup>20</sup><sub>D</sub> + 57 (c 2.0, CHCl<sub>3</sub>); IR (neat) 1744, 3424 cm<sup>-1</sup>; ¹H-NMR (400 MHz, CDCl<sub>3</sub>) δ 1.19–1.51 (m, 98H; H<sup>A</sup>-7, CH<sub>2</sub>-8, CH<sub>2</sub>-9 and CH<sub>2</sub>-10), 1.68–1.81 (m, 14H; H<sup>B</sup>-7), 1.81–1.95 (m, 28H; CH<sub>2</sub>-11), 1.95–2.07 (several s, 168H; 56 × C(O)CH<sub>3</sub>), 3.28–3.54 (m, 14H; H-2' and H-4'), 3.56–3.73 (m, 7H; H-5'), 3.73–3.83 (m, 14H; H-5), 3.91 (dd, *J* = 8.9 Hz, 7H; H-3'), 4.04 (dt, *J* = 12.1, 2.3 Hz, 14H; H-A-6), 4.08–4.15 (m, 14H; H-1), 4.21 (dd, *J* = 12.1, 4.9 Hz, 14H; H<sup>B</sup>-6), 4.30 (t, *J* = 7.3 Hz, 14H; CH<sub>2</sub>-12), 4.35 (t, *J* = 7.3 Hz, 14H; CH<sub>2</sub>-12), 4.42–4.68 (m, 14H; CH<sub>2</sub>-6'), 4.69–4.81 (m, 7H; H-1'), 4.84–5.02 (m, 14H; CH<sub>2</sub>-7' or CH<sub>2</sub>-10'), 4.95 (dd, *J* = 9.1 Hz, 14H; H-4), 5.02 (dd, *J* = 9.5, 5.7 Hz, 14H; H-2), 5.23–3.31 (m, 28H; H-3 and CH<sub>2</sub>-7' or CH<sub>2</sub>-10'), 7.54–7.80 (m, 14H; H-9' and H-12'); ¹³C-NMR (100 MHz, CDCl<sub>3</sub>) δ 20.7, 20.78, 20.82 (CH<sub>3</sub>), 25.0, 25.3, 26.7, 28.8 (C-7, C-8, C-9, C-10), 30.4 (C-11), 50.26, 50.35 (C-12), 53.5 (C-7'or C-10'), 62.4 (C-6), 64.9, 65.2 (C-6' and C-7' or C-10'), 68.67 (C-5 and C-5'), 68.99 (C-4), 70.52, 70.55 (C-2 and C-3), 72.6 (C-1), 73.4 (C-3'), 79.1 (C-2'), 83.0 (C-4'), 101.7 (C-1'), 122.9, 123.6 (C-9', C-12'), 144.1, 144.7 (C-8', C-11'), 169.6, 169.7, 170.2, 170.7 (C(O)CH<sub>3</sub>); HRMS (ESI) *m/z* 2713.4838 [(M + 3Na)³⁺], calcd for C₃₆₄H₅₃₂N₄₂O₁₆₁Na₃: 2713.4844).

## Compound 2b

To a solution of 3 (50 mg, 0.0062 mmol) in a mixture of MeOH (1 mL) and water (1 mL) was added Amberlite IRA400 (OH⁻) resin (2.20 g). The mixture was stirred using the rotavapor at rt for 20 h. The resin was then filtered, rinsed with MeOH and

water, and the filtrate was concentrated under reduced pressure. The solvents were evaporated under reduced pressure to afford 2b (33 mg, 93%) as a pale yellow oil.

[α]<sup>20</sup><sub>D</sub> + 80 (c 1.0, H₂O); IR (neat) 3369 cm<sup>-1</sup>; ¹H-NMR (300 MHz, D₂O) δ 1.10–1.94 (m, 140H; CH₂-7, CH₂-8, CH₂-9, CH₂-10 and CH₂-11), 3.30–3.99 (m, 140H; H-1, H-2, H-2', H-3, H-3', H-4, H-4', H-5, H-5', CH₂-6 and CH₂-6'), 4.15–4.65 (m, 42H; CH₂-7' or CH₂-10', CH₂-12), 4.80–5.04 (m, 21H; H-1', CH₂-7' or CH₂-10'), 7.92 (br s, 7H; H-9' or H-12'), 8.09 (s, 7H; H-9' or H-12'); ¹³C-NMR (75.5 MHz, D₂O) δ 23.4, 23.5, 24.4, 24.5, 25.55, 25.62, 27.8, 27.9, 29.5, 29.6 (C-7, C-8, C-9, C-10, C-11), 50.2, 50.4 (C-12), 61.1, 61.2 (C-6), 63.4, 64.5 (C-7', C-10'), 68.6 (C-6'), 70.32, 70.35 (C-4, C-5'), 71.4 (C-2), 72.4 (C-5), 72.8 (C-3'), 73.5 (C-3), 75.7 (C-1), 79.4 (C-2'), 81.9 (C-4'), 100.4 (C-1'), 124.6, 125.2 (C-9', C-12'), 143.4, 143.9 (C-8', C-11'); HRMS (ESI) *m/z* 1430.4807 [(M + 4H)⁴⁺], calcd for C₂₅₂H₄₂₄N₄₂O₁₀₅: 1430.4799).

## Compound 2a

A mixture of ¹³ (37.3 mg, 0.018 mmol), ⁴b (68 mg, 0.235 mmol), CuSO₄·5H₂O (1.3 mg, 52 µmol) and sodium ascorbate (3.5 mg, 0.018 mmol) in CH₂Cl₂/H₂O/DMSO (0.6:0.4:0.4 mL) was stirred at 30 °C under argon for 3 days. MeOH (10 mL) was added. The orange-brown precipitate was filtered and washed with MeOH and acetone. Two successive gel permeation chromatographies (Sephadex G-25, H₂O) followed by filtration over a QuadraSil™ Mercaptopropyl column gave 2a (33 mg, 34%) as an orange-brown glassy product. IR (neat) 3340 (br, OH), 1738 (C=O) cm⁻¹; UV/vis (H₂O/DMSO 10:0.2) λ<sub>max</sub>(ε) = 235 (179 900), 273 (sh, 66 400), 337 (sh, 31 700) nm; ¹H NMR (400 MHz, D₂O/DMSO-d₆: 4:1) δ 7.84 (s, 12H), 4.80 (m, 36H), 4.42–4.02 (m, 60H), 3.66 (m, 12H), 3.17 (m, 12H), 3.01 (m, 12H), 2.62 (m, 24H), 2.05–1.57 (m, 60H), 1.54–1.03 (m, 84H); ¹³C NMR (100 MHz, DMSO-d₆) δ 163.2, 146.2, 145.4, 141.2, 122.5, 75.5, 74.2, 73.8, 71.9, 71.3, 69.2, 67.0, 61.9, 49.9, 46.0, 30.1, 28.8, 28.2, 26.4, 25.4, 24.3, 21.9; ES-TOF-MS *m/z* 1889.04 [(M + 3Na)³⁺], calcd for C₂₈₂H₃₆₀N₃₆O₈₄Na₃: 1889.02).

## Inhibition studies with commercial enzymes

Inhibition constant (Kᵢ) values were determined by spectrophotometrically measuring the residual hydrolytic activities of the glycosidases against the respective *p*-nitrophenyl *α*- or *β*-d-glycopyranoside, *o*-nitrophenyl *β*-d-galactopyranoside (for *β*-galactosidases) in the presence of the *C*-glycosides. Each assay was performed in phosphate buffer or phosphate-citrate buffer (for *α*- or *β*-mannosidase and amyloglucosidase) at the optimal pH of each enzyme. The reactions were initiated by addition of the enzyme to a solution of the substrate in the absence or presence of various concentrations of inhibitor. The mixture was incubated for 10–30 min at 37 °C or 55 °C (for amyloglucosidase) and the reaction was quenched by addition of 1 M Na₂CO₃. Reaction times were approximated 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. Approximate values of Kᵢ were determined using a fixed concentration of substrate (around the Kₘ value for different glycosidases) and
<figure><img src="image_15.png" /></figure>

various concentrations of inhibitor. Full Kᵢ and enzyme inhibition modes were determined from Dixon plots.

## Acknowledgements

The authors are grateful to financial support from the Institut Universitaire de France (IUF), the CNRS (UMR 7509), the University of Strasbourg and the International Centre for Frontier Research in Chemistry (icFRC). FS thanks the French Department of Research for a doctoral fellowship. The Spanish Ministerio de Economía y Competitividad (MINECO, contract numbers SAF2013-44021-R) and cofinancing from the European Regional Development Funds (FEDER and FSE) are also thanked.

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