Construction of giant glycosidase inhibitors from iminosugar-substituted fullerene macromonomers

Thi Minh Nguyet Trinh a, Michel Holler a, Jérémy P. Schneider b, M. Isabel García-Moreno c, José M. García Fernández d, Anne Bodlenner b, Philippe Compain *b, Carmen Ortiz Mellet *c and Jean-François Nierengarten *a
aLaboratoire 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:
bLaboratoire de Synthèse Organique et Molécules Bioactives, Université de Strasbourg et CNRS (UMR 7509), Ecole Européenne de Chimie, Polymères et Matériaux, 25 rue Becquerel, 67087 Strasbourg Cedex 2, France. E-mail:
cDepartment of Organic Chemistry, Faculty of Chemistry, University of Sevilla, c/Profesor García González 1, 41011 Sevilla, Spain. E-mail:
dInstituto de Investigaciones Químicas (IIQ), CSIC – Universidad de Sevilla, Avda. Américo Vespucio 49, E-41092 Sevilla, Spain

Received 17th April 2017 , Accepted 10th May 2017

First published on 10th May 2017

An ultra-fast synthetic procedure based on grafting of twelve fullerene macromonomers onto a fullerene hexa-adduct core was used for the preparation of a giant molecule with 120 peripheral iminosugar residues. The inhibition profile of this giant iminosugar ball was evaluated against various glycosidases. In the particular case of the Jack bean α-mannosidase, a dramatic enhancement of the glycosidase inhibitory effect was observed for the giant molecule with 120 peripheral subunits as compared to that of the corresponding mono- and dodecavalent model compounds.


Multivalent effects are of fundamental importance in many aspects of modern glycochemistry.1–3 The multivalent presentation of carbohydrates to protein receptors (lectins) is often essential to obtain ligands with high affinities.1,2 The glycoside cluster effect is actually used in nature to reach the affinities necessary for highly selective carbohydrate–protein interactions.1,2 Synthetic chemists have also taken advantage of these effects to prepare artificial ligands displaying high affinities for specific lectins.1,2 On the other hand, multivalent effects have also been observed for glycosidase3 and glycosyltransferase4 inhibition. The understanding of the enhanced inhibitory efficiency resulting from a multivalent presentation has focused substantial research efforts over the past few years;5–10 however, the role of these effects in biological systems is still not well understood.

This field has tremendously benefited from the development of dendrimer chemistry, allowing the preparation of sophisticated compounds displaying multiple carbohydrate entities.2 Despite several remarkable achievements, the synthesis of glycodendrimers with a large number of peripheral glycoside residues remains difficult due to a large number of synthetic steps, thus limiting both their availability and applicability. In recent years, the development of click reactions,11 in particular the copper-catalyzed alkyne–azide cycloaddition (CuAAC),12 has allowed for the simultaneous grafting of multiple carbohydrate moieties onto compact molecular scaffolds to efficiently generate glycoclusters in a limited number of synthetic steps. Examples include glycoclusters constructed from hexa-adducts of C60,13,14 polyhedral oligomeric silesquioxanes15 and macrocyclic nanoscaffolds,16–18 such as calixarenes,16 cyclodextrins17 or pillarenes.18 When compared to their analogous glycodendrimers, glycoclusters constructed in a single synthetic step by clicking peripheral sugars onto a nanoscaffold are by far easier to prepare and their affinities for specific lectins are in general similar if not higher.13–18 The limited number of synthetic steps is also a clear advantage when preparing multivalent systems from sophisticated glycoside building blocks.13 This has particularly been the case for the preparation of multivalent inhibitors for which the preparation of the glycomimetic moieties already requires a substantial number of synthetic steps.3,4 The valency of the resulting glycoclusters is, however, limited by the number of peripheral reactive subunits of the scaffold and the preparation of compounds with higher valencies requires the use of glycodendrons.19 Very recently, a synthetic approach using the grafting of fullerene-based macromonomers onto a fullerene scaffold has been reported.20 In this case, first generation compounds decorated with 120 peripheral mannose residues were prepared in a few synthetic steps. These giant glycoclusters have also shown remarkable anti-viral activity resulting from the very high number of peripheral carbohydrate subunits, thus rewarding our efforts towards developing efficient synthetic tools for their construction.21 As part of this research, we now report the preparation and the characterization of a giant molecule with 120 peripheral iminosugar residues. This ultra-fast synthetic procedure based on fullerene macromonomers is actually perfectly suited for the construction of multivalent systems from sophisticated building blocks such as iminosugars.22 The inhibition profile of this giant iminosugarball was evaluated against various glycosidases and compared to the corresponding mono- and dodecavalent model compounds. Remarkably, a dramatic enhancement of the glycosidase multivalent inhibitory effect was observed when going from the dodecavalent iminosugar balls to the giant molecule with 120 peripheral subunits.

Results and discussion

The design of the giant iminosugar ball was based on our previous system constructed on a hexa-substituted fullerene core.23 Dramatic multivalent inhibitory effects have been observed with compound 1 when compared to the monovalent model compound 2. For example, the inhibition constant values (Ki) obtained with 1 towards isomaltase of Baker's yeast and α-mannosidase of Jack bean were improved by two and three orders of magnitude, respectively, over that of monomer 2.
image file: c7tb01052d-u1.tif

In the present study, the same 1-deoxynojirimycin (DNJ) motif was used as peripheral group for the construction of the giant iminosugarball. It was however decided to introduce a –(CH2)9– linker rather than a –(CH2)6– one as in the case of compound 1. Indeed, studies on analogous derivatives with a cyclodextrin core have shown the importance of this structural parameter on the inhibitory activities.6 In order to also have an appropriate dodecavalent model compound, the –(CH2)9– analogue of 1 was also prepared.


The synthesis of the fullerene-based macromonomer necessary for the construction of the giant iminosugar ball is depicted in Scheme 1. Malonate 5 was prepared in 65% yield via the monoesterification of pentaethyleneglycol (3) with ethyl malonyl chloride (4) in the presence of pyridine (py). Reaction of 5 with methanesulfonyl chloride (MsCl) followed by treatment of the resulting mesylate 6 with LiBr in THF gave bromide 7. Methanofullerene 8 was obtained from C60 and 7 under the modified Bingel conditions described by Diederich.24 In this case, the nucleophilic cyclopropanation of C60 was possible starting directly from the malonate reagent and the α-halomalonate is generated in situ via the direct treatment of the malonate with iodine in the presence of C60 and a base, namely 1,8-diaza-bicyclo[5.4.0]undec-7-ene (DBU). Under these conditions, the fullerene mono-adduct 8 was obtained in 48% yield. Treatment of 8 with an excess of malonate 9, CBr4 and DBU in o-dichlorobenzene (o-DCB) gave the [5[thin space (1/6-em)]:[thin space (1/6-em)]1]-hexaadduct of C60 bearing ten trimethylsilyl (TMS)-protected alkyne groups (10) in 42% yield. The ten peripheral N-nonyl DNJ units6 were then grafted onto scaffold 10 under the typical CuAAC conditions developed by Sharpless.12 It was notable that compound 10 was desilylated in situ with tetrabutylammonium fluoride (TBAF) to generate the corresponding intermediate bearing ten terminal alkyne functions. Specifically, treatment of 10 with an excess of azide 11 was carried out in the presence of CuSO4·5H2O, sodium ascorbate (NaAsc) and TBAF. A ternary solvent mixture (CH2Cl2/H2O/DMSO) was used for this reaction to ensure the solubility of the starting materials, the partially clicked intermediates formed over the course of the reaction and the final product.14a Compound 12 was thus obtained in 67% yield. Clickable macromonomer 13 was then prepared by treating bromide 12 with an excess of NaN3 in DMF. The presence of the azide function in 13 was confirmed by the diagnostic N[triple bond, length as m-dash]N asymmetric stretching absorption observed at 2105 cm−1 in the IR spectrum of 13. Giant iminosugar ball 15 was then constructed by grafting twelve copies of macromonomer 13 onto the fullerene hexa-adduct 14 (Scheme 2). This reaction was first achieved under the conditions used for the preparation of macromonomer 12 (CuSO4·5H2O, NaAsc, TBAF, CH2Cl2/H2O/DMSO), however, by-products were still present. Prolonged reaction times and higher temperatures were not efficient strategies to complete the reaction, most probably due to the concomitant degradation of the azide reagent. In contrast, the use of microwave heating allowed us to successfully complete the reaction. The purification of 15 was achieved by successive precipitation/centrifugation. The excess of azide starting material was conveniently eliminated by gel permeation chromatography on Sephadex G-25. Moreover, the remaining traces of the copper catalyst25 were eliminated by filtration through a QuadraSilTM Mercaptopropyl column. Compound 15 was finally isolated in 65% yield. The corresponding dodecavalent iminosugar ball (16) was also prepared. Reaction of an excess of azide 11 with 14 in the presence of CuSO4·5H2O, NaAsc and TBAF afforded 16 in 53% yield.
image file: c7tb01052d-s1.tif
Scheme 1 Reagents and conditions. (i) py, CH2Cl2, 0 °C to rt (65%); (ii) MsCl, Et3N, CH2Cl2, rt (91%); (iii) LiBr, THF, 60 °C (87%); (iv) C60, DBU, I2, PhMe, rt (48%); (v) DBU, CBr4, o-DCB, rt (42%); (vi) CuSO4·5H2O, NaAsc, TBAF, CH2Cl2/H2O/DMSO, 38 °C (67%); (vii) NaN3, DMF, 50 °C (62%).

image file: c7tb01052d-s2.tif
Scheme 2 Reagents and conditions. (i) 13, CuSO4·5H2O, NaAsc, TBAF, THF/CH2Cl2/H2O/DMSO, 80 °C (MW) (65%); (ii) 11, CuSO4·5H2O, NaAsc, TBAF, THF/CH2Cl2/H2O/DMSO, rt (53%).

Characterization of the iminosugar balls

Compounds 12, 15 and 16 were only moderately soluble in water and DMSO. It was however found that their solubility can be significantly improved by treatment with a small amount of aqueous HCl to partially protonate the peripheral N-nonyl DNJ subunits. Compounds 12 and 16 are also reasonably soluble in methanol, but this was not the case of 15. The characterization of 12, 15 and 16 was greatly facilitated by their symmetry and 13C NMR spectroscopy was proved to be a particularly useful analytical tool. As shown in Fig. 2, the 13C NMR spectrum of compound 12 was in full agreement with its T-symmetrical structure. The 15 expected resonances for the carbon atoms of the 12 equivalent peripheral N-nonyl DNJ moieties are clearly observed (C1–15, see Fig. 1) as well as the 3 expected signals arising from the 3C atoms of the 12 equivalent propylene linkers (C18–20, see Fig. 1). The diagnostic signals of the 12 equivalent 1,2,3-triazole rings are observed at δ = 145.9 (C-17) and 122.1 (C-16) ppm while the twelve equivalent malonic carbonyl C atoms give rise to a single resonance at δ = 163.2 ppm. Finally, 3 signals out of the 5 expected ones are observed for the fullerene C atoms (δ = 69.2 ppm for the sp3 C atoms; 145.4 and 141.2 ppm for the sp2 C atoms). As already observed for analogous fullerene sugar balls,14 the two pairs of diastereotopic sp2 C atoms are pseudo-equivalent and only 3 fullerene signals were observed. Actually, the long flexible spacer separating the fullerene core from the peripheral DNJ subunits prevent the transfer of their chiral information and the local Th symmetry influences the pattern observed for the fullerene signals in the 13C spectrum of 16. The 13C NMR spectrum of compound 12 was also strongly influenced by local symmetry effects. Due to the presence of the peripheral DNJ subunits, compound 12 is C1-symmetrical. However, on close inspection of the 13C NMR spectrum of 12 reveals that the five malonate groups bearing N-alkyl DNJ subunits are pseudo-equivalent and give rise to a single set of signals similar to the one observed in the spectrum of compound 16.
image file: c7tb01052d-f1.tif
Fig. 1 The 13C NMR spectra of compounds 12, 15 and 16 recorded in DMSO-d6 (25 °C, 100 MHz).

Moreover, three groups of signals appear for the fullerene C atoms reminiscent of those of the 3 non-equivalent C atoms in a Th-symmetrical hexa-adduct (δ = 145.4, 141.2 and 69.7 ppm). Finally, the expected resonances of the sixth malonate adduct of 12 are also observed. The 13C NMR spectrum of macromolecule 15 shows the characteristic features observed for its macromonomeric precursor (12). The twelve peripheral fullerene groups are equivalent and their peripheral N-alkyl DNJ subunits appear as pseudo-equivalent, as in the case of 12. It is indeed remarkable to observe such sharp signals for the 120 peripheral subunits in a macromolecule of a molecular weight higher than 6.8 kDaltons. It was also notable that no signals could be detected in the typical sp carbon region of the 13C NMR spectrum of 15, thus suggesting that no unreacted alkyne groups remain present in the isolated compound. The 1H NMR spectra of compounds 12, 15 and 16 were also fully consistent with the proposed structures. A substantial broadening of the signals was however observed in the spectra recorded in D2O or DMSO-d6. Broadening of some of the 1H NMR signals of the fullerene hexa-adducts was commonly observed and results from the limited molecular motion/rotation in the subunits located close to the central core due to confinement effects.14,21 In the particular case of 12 and 16, the DLS experiments also revealed some aggregation (vide infra) that may be also responsible for the broadening of the spectra. For compound 16, aggregation was limited in CD3OD and the 1H NMR spectrum recorded in this solvent presented a significantly improved resolution (see ESI). For this compound, the 1H NMR spectra were also recorded in DMSO-d6 at different temperatures. At the highest temperature (80 °C), the signals were substantially sharper but remain broadened.

The MALDI-TOF mass spectrum of compound 12 shows the expected molecular ion peak despite a high level of fragmentation resulting from retro-Bingel reactions and/or cleavage of the malonic ester subunits followed by decarboxylation. ESI-TOF analysis of 12 gave much better results, with very limited fragmentation and the expected molecular ion peak was clearly observed under these conditions. The mass spectrometric analysis of compound 15 and 16 exhibited a higher complexity. As a result of aggregation, the transfer of these molecules in the gas phase is difficult and requires harsher conditions leading to a very high level of fragmentation. Moreover, the formation of matrix-adducts was an additional problem26 and the molecular ion peaks of 15 or 16 could not be clearly detected. Their unambiguous structural characterization by NMR is however fully convincing.

Aggregation of iminosugar balls 15 and 16

As already mentioned, compounds 15 and 16 form aggregates in water and DMSO. Further characterization was therefore achieved using DLS. Regardless of the concentration, DLS measurements performed for compound 16 in H2O/DMSO solution (3[thin space (1/6-em)]:[thin space (1/6-em)]7 v/v) indicated the presence of three peaks centered at 100, 1000, and 5000 nm. The relative intensity of these peaks was concentration dependent, upon increasing the concentration, the largest aggregates were favored (ESI, Fig. S12). For this compound the DLS investigations in pure water were prevented due to the limited solubility. In contrast, the solubility of compound 15 was sufficient to perform DLS measurements in pure water. In this case, three peaks centered at 10, 100 and <1000 nm were observed. The first peak corresponds to single molecules of 15 while the others arise from the aggregation of several molecules (ESI, Fig. S13a). The relative intensity of the three peaks was also pH dependent and the addition of HCl 0.1 M (pH = 1–2) led to a significant increase in the 10 nm peak (ESI, Fig. S13b). This was in perfect agreement with the higher solubility of compound 15 in an acidic solution. These experiments revealed a much higher tendency to aggregate for compound 16 when compared to 15. This was also clearly shown by a comparison of the absorption spectra of 15 and 16 recorded under similar conditions (see Fig. S8d and S9h, ESI). Effectively, the UV-Vis spectrum of 16 was much broader as a result of aggregation.27 In the case of 15, the UV-Vis spectrum shows the typical features of fullerene hexa-adducts.28 Indeed, the spectrum of 15 was very similar to the one recorded for the hexa-adduct 10 for which no aggregation occurs. This shows also that under these conditions, the aggregation of 15 was rather limited.

Transmission electron microscopy (TEM) images were also recorded for compound 15 (Fig. 2). Formvar-carbon coated grids (Cu, 200 mesh), previously made hydrophilic via glow discharge, were placed on top of small drops of the samples (2.15 mg mL−1 of the fullerene derivative in H2O). After 10 minutes of contact, the grids were removed from the drops and dried at rt for 24 h. Then, the grids were negatively stained with a 1% aqueous solution of uranyl acetate and further dried for 24 h. They were then observed using a Zeiss Libra 120 electron microscope operating at 80 kV. As shown in Fig. 2, the resulting TEM images revealed spherical nanoparticles with diameter ranging from ca. 4 to 10 nm corresponding to one or a few molecules. Higher aggregates were also observed. These observations are perfectly consistent with the DLS data.

image file: c7tb01052d-f2.tif
Fig. 2 TEM images of 15 upon deposition of a 2.15 mg mL−1 solution in H2O.

Glycosidase inhibition

The inhibition constant (Ki) values for 15 and 16 against a panel of commercial enzymes including α-glucosidase (Baker's yeast), amyloglucosidase (A. niger), β-glucosidase (almonds and bovine liver), β-galactosidase (E. coli), α-galactosidase (green coffee beans), α-mannosidase (Jack beans) and β-mannosidase (H. pomatia) are collected in Table 1. The corresponding data obtained for the monovalent derivative 17, previously reported,6 are also included for comparative purposes.
image file: c7tb01052d-u2.tif
Table 1 The glycosidase inhibitory activities Ki (μM) of multivalent compounds 15 and 16 over monovalent inhibitor 17 (in brackets)a
Valence 17b 16 15
1 12 120
a Inhibition was competitive in all cases (inhibition constant for the E-I binding) with the exception of that found against α-mannosidase from Jack bean for which a non-competitive-type inhibition mode was observed in one case. b Data from ref. 52. c (a) α-glycosidase (Baker's yeast), (b) amyloglucosidase (Aspergillus niger), (c) β-glucosidase (almonds, pH 7.3), (d) β-glucosidase (bovine liver), (e) β-galactosidase (Escherichia coli), (f) α-galactosidase (green coffee), (g) α-mannosidase (Jack bean) and (h) β-mannosidase (Helix pomatia). d NI, no inhibition observed at a 1 mM concentration of inhibitor (Ki > 1000 μM). e The inhibition constant for the competitive/uncompetitive binding modes.
(a)c 116 144 32
(b)c 1.7 3.00 0.14
(c)c 11 136 87
(d)c 23 NId 55
(e)c NId NId 60
(f)c 544 30 44
(g)c 188 0.099 0.0018e/0.0042
(h)c NId NId 30

Jack bean α-mannosidase has become the reference enzyme for multivalent enzyme inhibition studies.3a Although its crystal structure has not been solved yet, the data available for other α-mannosidases belonging to the same glycosyl hydrolase family29 (GH38) support that the glycone (−1) binding spot in the catalytic site was quite accessible and, to some extent, indifferent to steric constraints when faced to voluminous ligands,30,31 much like the carbohydrate binding domain in lectins.32,33 This scenario facilitates the sliding and rebinding processes of clustered inhitopes provided that the binding and dissociation events benefit from fast kinetics, which is generally the case for weakly binding motifs.34 Inhibition by multivalent species can be reinforced by additional interactions with secondary binding sites in the enzyme, including the aglycone (+1) and other regions in the vicinity of the active site.35 Recent mechanistic studies on multivalent interactions with Jack bean α-mannosidase using electron microscopy imaging and mass spectroscopy led to the proposal of an additional binding mode model where the multimeric inhibitor will cross-link two mannosidase molecules to form a strong chelate complex.8a This binding scenario is plausible considering the dimeric nature of Jack bean α-mannosidase. In full agreement with these considerations, a dramatic enhancement in the inhibition potency when compared with the monovalent control 17 (1899-fold) and a competitive inhibition mode (Ki 99 nM) was observed for the dodecavalent C60 conjugate 16, meaning a normalized relative inhibition potency of 158 (in DNJ molar basis). The inhibitory strength was further increased for the 120-valent DNJ-coated superball 15 (over 100[thin space (1/6-em)]000-fold compared to the monovalent control 17). Interestingly, the corresponding Lineweaver–Burk plot (ESI, Fig. S10b) was indicative of a mixed-type inhibition, that is, the macromolecular conjugate can bind to both the enzyme and the enzyme–substrate complex, preventing substrate hydrolysis in both cases. The competitive and uncompetitive components were characterized by Ki values of 1.9 and 4.2 nM, respectively. The first one stands for one of the lowest Ki reported for an inhibitor of this enzyme up to date.8a A direct comparison with the corresponding data for 16 reveals an additional over-50-fold enhancement in the inhibition potency, well over a statistic effect. On the other hand, the existence of an uncompetitive component represents the first evidence that strong inhibition of α-mannosidase can be achieved using nanosized multivalent inhibitors even when the catalytic site is fully occupied, a phenomenon that has only been previously observed for fullerene derivatives homogeneously coated with carbohydrates or heterogeneously conjugated with carbohydrates and glycomimetics of the sp2-iminosugar family36–40 against β-galactosidase.7

Yeast α-glucosidase (yeast maltase), a GH13 glycosyl hydrolase, has been previously found to be also responsive to multivalent inhibition. Differently from α-mannosidase, the glycone (−1) site, which is located in a deep pocket and provides most of the binding energy in the case of monovalent ligands,41 is only marginally involved when the enzyme is faced to multiconjugates exposing several copies of the inhitope, the aglycone (+1) and other nonglycone sites then take a leading role.5 This shift in the binding mode prevents a straightforward quantification of the multivalent effect.35 In any case, moving from 16 (Ki 144 μM) to 15 (Ki 32 μM) translates into a significant (4.5-fold) enhancement in the inhibition potency. Even though the percentage increase is below the statistic effect, this implies that an increase in valency/molecular size has the potential to elicit a relevant biological effect by decreasing the inhibition constant below a critical value. An analogous analysis can be argued when considering the data collected in Table 1 for other enzymes having catalytic pockets sharing a deep location for the −1 site and relatively accessible aglycone subsites, as it is the case for amyloglucosidase (GH15),42,43 β-glucosidase (GH1)44 or α-galactosidase (GH27).45,46

The sharp difference encountered in the susceptibility of the β-galactosidase (GH2) and β-mannosidase (not yet annotated) enzymes included in this study towards inhibition by the dodecavalent fullerene-DNJ conjugate 16 or the superball homolog 15 is noteworthy. Both glycosidases possess active sites that are deeply buried in the protein and bear nonglycone binding sites potentially reachable by multivalent inhibitors.47–50 Yet, multivalent DNJ conjugates built on molecular scaffolds, such as fullerene C60 or cyclodextrins,51 have systematically been found inactive against these two glycosidases, strongly suggesting that the affinity of the DNJ inhitope for such subsites is negligible.8,23,52 It is interesting to speculate that a larger surface area of the enzyme must interact with the nanosized species in order to achieve efficient inhibition in these cases. This binding mode, relying on complementary surfaces moieties, is indeed similar to that operating in nature for many protein enzyme inhibitors and can be thus considered as biomimetic.53 Although the affinity of such nanoparticle-interacting enzyme region towards different recognition motifs is unknown, its implication in the inhibition of β-galactosidase by inorganic nanoparticles has been documented.54 The fact that most β-mannosidases belong to the same GH2 family49 further suggest that a similar binding mode may operate in this case.


A fullerene macromonomer with ten peripheral DNJ groups has been prepared and successfully grafted onto a hexa-substituted fullerene scaffold to generate a giant iminosugar ball. The resulting tridecafullerene with 120 peripheral DNJ moieties (15) was assayed against a panel of commercial enzymes. A corresponding mono-fullerene analogue decorated with 12 DNJ moieties (16) and a monovalent reference compound (17) were also tested for comparison purposes. Altogether, the differences in the glycosidase inhibitory profile between the fullerene- (16) and superball-scaffolded (15) multivalent DNJ-displaying inhibitors can be interpreted by invoking the interplay of valency and size effects. The valency feature is particularly relevant for α-mannosidase inhibition, in agreement with the body of evidence previously accumulated in the field.3 This glycosidase is inhibited by compound 15 from 100 to 10[thin space (1/6-em)]000 times more strongly than any of the other enzymes assayed. The size effect seems to be the origin of the activation of inhibition against β-galactosidase and β-mannosidase (Ki values in the μM range), since both enzymes are unresponsive to monovalent controls or to high valency, yet smaller than 16, DNJ conjugates. The overall consequence is an increase in the promiscuity on moving from 16 to 15 as a result of the capacity of the bigger conjugate to interact with different enzymes through a variety of binding modes. Whereas this behavior may limit applications requiring a strict specificity of action, it also opens new possibilities beyond those previously foreseen. For instance, nanoparticles inhibiting a variety of glycosidases have shown promise as antimicrobial55,56 and antidiabetic agents,57–60 and evident advantages are expected for molecularly well-defined, tailorable nanosized species.

Experimental section

Solvents were of reagent grade and further dried when necessary. Dichloromethane (CH2Cl2) was distilled over CaH2 under argon. Dry acetonitrile and dry DMF (both over molecular sieves) were purchased from commercial vendors and used as received. Compounds 9,21b1152 and 1421b were prepared according to literature 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 F254 purchased from Merck KGaA. Visualization was accomplished with UV light (at 254 nm) and exposure to TLC stains, phosphomolybdic acid or potassium permanganate, followed by heating. Flash column chromatography was 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 the residual solvent signals used as an internal reference. The assignment of the 1H and 13C signals were made using DEPT, 1H–1H COSY, HSQC and HMBC experiments. Infrared (IR) spectra were recorded neat on a Perkin-Elmer Spectrum Two FT-IR spectrometer.

Compound 5

A solution of 4 (0.25 mL, 1.95 mmol) in CH2Cl2 (40 mL) was added dropwise over 1 h to a solution of 3 (0.82 mL, 3.90 mmol) and pyridine (0.17 mL, 2.15 mmol) in CH2Cl2 (30 mL) at 0 °C. The resulting mixture was allowed to slowly warm to room temperature (over 1 h). The mixture was then stirred for 2 h at room temperature, filtered through SiO2 (CH2Cl2/MeOH 100[thin space (1/6-em)]:[thin space (1/6-em)]5) and the solvents evaporated. Column chromatography (SiO2, CH2Cl2/ether/MeOH 100[thin space (1/6-em)]:[thin space (1/6-em)]10[thin space (1/6-em)]:[thin space (1/6-em)]0 → 100[thin space (1/6-em)]:[thin space (1/6-em)]10[thin space (1/6-em)]:[thin space (1/6-em)]3) gave 5 (444 mg, 65%) as a colorless oil. IR (neat): 3456 (broad, –OH), 1730 (C[double bond, length as m-dash]O). 1H NMR (400 MHz, CDCl3): 4.33 (t, J = 4 Hz, 2H), 4.22 (q, J = 7 Hz, 2H), 3.74 (t, J = 4 Hz, 4H), 3.71–3.66 (m, 12H), 3.43 (s, 2H), 2.66 (broad t, 1H), 1.30 (t, J = 7 Hz, 3H). 13C NMR (100 MHz, CDCl3): 166.7, 166.5, 72.5, 70.6, 70.5, 70.4, 68.8, 64.6, 61.8, 61.6, 41.5, 14.1.

Compound 6

Methanesulfonyl chloride (0.18 mL, 2.27 mmol) was added to a solution of 5 (400 mg, 1.14 mmol) and triethylamine (0.32 mL, 2.27 mmol) in CH2Cl2 (20 mL). The resulting mixture was stirred for 1 h at room temperature. The reaction mixture was filtered through SiO2 (CH2Cl2/MeOH 100[thin space (1/6-em)]:[thin space (1/6-em)]5) and the solvents evaporated. Column chromatography (SiO2, CH2Cl2/ether/MeOH 100[thin space (1/6-em)]:[thin space (1/6-em)]7[thin space (1/6-em)]:[thin space (1/6-em)]0 → 100[thin space (1/6-em)]:[thin space (1/6-em)]10[thin space (1/6-em)]:[thin space (1/6-em)]2) gave 6 (462 mg, 91%) as a colorless oil. IR (neat): 1730 (C[double bond, length as m-dash]O). 1H NMR (400 MHz, CDCl3): 4.38 (t, J = 4 Hz, 2H), 4.30 (t, J = 5 Hz, 2H), 4.20 (q, J = 7 Hz, 2H), 3.76 (t, J = 4 Hz, 2H), 3.71 (t, J = 4 Hz, 2H), 3.68–3.62 (m, 12H), 3.40 (s, 2H), 3.08 (s, 3H), 1.27 (t, J = 7 Hz, 3H). 13C NMR (100 MHz, CDCl3): 166.7, 166.5, 70.65, 70.64, 70.61, 70.59, 70.57, 70.53, 69.3, 69.0, 68.9, 64.5, 61.6, 41.5, 37.7, 14.1.

Compound 7

A mixture of 6 (458 mg, 1.06 mmol) and LiBr (185 mg, 2.28 mmol) in THF was stirred at 60 °C for 4 h. The reaction mixture was filtered through SiO2 (CH2Cl2/MeOH 100[thin space (1/6-em)]:[thin space (1/6-em)]5) and the solvents evaporated. Column chromatography (SiO2, CH2Cl2/ether/MeOH 100[thin space (1/6-em)]:[thin space (1/6-em)]10[thin space (1/6-em)]:[thin space (1/6-em)]0 → 100[thin space (1/6-em)]:[thin space (1/6-em)]10[thin space (1/6-em)]:[thin space (1/6-em)]1) gave 7 (386 mg, 87%) as a colorless oil. IR (neat): 1731 (C[double bond, length as m-dash]O). 1H NMR (400 MHz, CDCl3): 4.30 (t, J = 5 Hz, 2H), 4.20 (q, J = 7 Hz, 2H), 3.81 (t, J = 6 Hz, 2H), 3.71 (t, J = 5 Hz, 2H), 3.68–3.63 (m, 12H), 3.47 (t, J = 6 Hz, 2H), 3.40 (s, 2H), 1.28 (t, J = 7 Hz, 3H). 13C NMR (100 MHz, CDCl3): 166.7, 166.5, 71.2, 70.67, 70.65, 70.63, 70.60, 70.59, 70.55, 68.9, 64.6, 61.6, 41.5, 30.0, 14.1.

Compound 8

DBU (0.35 mL, 2.29 mmol) was added to a solution of C60 (659 mg, 0.92 mmol), 7 (380 mg, 0.92 mmol) and I2 (279 mg, 1.10 mmol) in toluene (1320 mL). The resulting mixture was stirred for 1 h at room temperature, filtered through a plug of SiO2 (CH2Cl2) and concentrated. Column chromatography (SiO2, CH2Cl2/THF 100[thin space (1/6-em)]:[thin space (1/6-em)]0 → 100[thin space (1/6-em)]:[thin space (1/6-em)]3) gave 8 (502 mg, 48%) as a brown solid. IR (neat): 1743 (C[double bond, length as m-dash]O). UV/Vis (CH2Cl2): λmax(ε) = 260 (128[thin space (1/6-em)]000), 330 (37[thin space (1/6-em)]900), 394 (sh, 48[thin space (1/6-em)]000), 402 (sh, 3600), 414 (sh, 2600), 426 (2800), 498 (1600), 693 (180) nm. 1H NMR (400 MHz, CDCl3): 4.68 (t, J = 5 Hz, 2H), 4.56 (q, J = 7 Hz, 2H), 3.88 (t, J = 5 Hz, 2H), 3.81 (t, J = 6 Hz, 2H), 3.74–3.67 (m, 12H), 3.47 (t, J = 6 Hz, 2H), 1.52 (t, J = 7 Hz, 3H). 13C NMR (100 MHz, CDCl3): 166.7, 166.5, 145.4, 145.3, 145.23, 145.19, 145.18, 144.9, 144.70, 144.68, 144.63, 144.62, 143.90, 143.88, 143.1, 143.03, 143.01, 142.23, 142.20, 141.91, 141.89, 141.0, 140.9, 139.2, 139.0, 71.5, 71.2, 70.75, 70.70, 70.69, 70.62, 70.57, 68.8, 66.2, 63.5, 52.2, 30.4, 14.3. MALDI-TOF-MS: 1134.1 ([M]+, calcd. for C75H25BrO8: 1134.1).

Compound 10

DBU (0.96 mL, 6.35 mmol) was added to a solution of 8 (480 mg, 0.42 mmol), 9 (1.21 g, 3.18 mmol) and CBr4 (10.5 g, 31.75 mmol) in o-DCB (70 mL). The resulting mixture was stirred for 16 h at room temperature, filtered through a plug of SiO2 (CH2Cl2/THF 100[thin space (1/6-em)]:[thin space (1/6-em)]5) and concentrated. Column chromatography (SiO2, CH2Cl2/Et2O 100[thin space (1/6-em)]:[thin space (1/6-em)]0 → 100[thin space (1/6-em)]:[thin space (1/6-em)]4) gave 10 (540 mg, 42%) as an orange glassy solid. IR (neat): 2176 (CC), 1745 (C[double bond, length as m-dash]O). UV/Vis (CH2Cl2): λmax(ε) = 246 (148[thin space (1/6-em)]000), 267 (109[thin space (1/6-em)]000), 283 (108[thin space (1/6-em)]000), 338 (sh, 45[thin space (1/6-em)]600). 1H NMR (400 MHz, CDCl3): 4.36 (m, 24H), 3.80 (t, J = 6 Hz, 2H), 3.73 (m, 2H), 3.69–3.60 (m, 12H), 3.46 (t, J = 6 Hz, 2H), 2.32 (m, 20H), 1.91 (m, 20H), 1.31 (t, J = 7 Hz), 0.00 (s, 30H). 13C NMR (100 MHz, CDCl3): 163.46, 163.44, 145.71, 145.68, 145.63, 140.88, 105.00, 104.95, 85.6, 71.1, 70.52, 70.46, 70.41 68.9, 68.5, 65.5, 62.9, 45.1, 30.2, 27.4, 16.4, 14.0, 0.0. MALDI-TOF-MS: 3026.4 ([M]+, calcd for C170H175BrO28 Si10: 3026.9).

Compound 12

A 1 M solution of TBAF in THF (0.68 mL, 0.68 mmol) was added to a mixture of 10 (186 mg, 0.061 mmol), 11 (223 mg, 0.676 mmol), CuSO4·5H2O (5 mg, 0.018 mmol) and sodium ascorbate (12 mg, 0.061 mmol) in CH2Cl2/H2O/DMSO (2[thin space (1/6-em)]:[thin space (1/6-em)]1.5[thin space (1/6-em)]:[thin space (1/6-em)]1.2 mL). The resulting mixture was vigorously stirred at 38 °C under argon for 3 days. The crude product was precipitated in H2O and filtered. The precipitate was dissolved in water acidified using 2 N HCl. Gel permeation chromatography (Sephadex G-25, H2O) followed by filtration through a QuadraSilTM Mercaptopropyl column gave 12 (232 mg, 67%) as a brown glassy product. IR (neat): 3380 (br, OH), 1741 (C[double bond, length as m-dash]O). UV/Vis (H2O/DMSO 10[thin space (1/6-em)]:[thin space (1/6-em)]0.2): 238 (89[thin space (1/6-em)]700), 274 (65[thin space (1/6-em)]300), 284 (sh, 62[thin space (1/6-em)]100), 331 (sh, 30[thin space (1/6-em)]500). 1H-NMR (400 MHz, DMSO-d6): 7.79 (s, 10H), 4.25 (m, 44H), 3.71 (m, 22H), 3.56 (m, 16H), 3.23 (m, 10H), 3.06 (m, 10H), 2.94 (m, 10H), 2.80 (m, 10H), 2.70 (m, 10H), 2.55 (m, 20H), 2.39 (m, 10H), 1.96 (m, 40H), 1.75 (m, 20H), 1.35–1.09 (m, 143H). 13C-NMR (100 MHz, DMSO-d6): 163.3, 145.9, 145.4, 141.2, 122.1, 79.5, 71.0, 70.8, 70.2, 70.0, 69.7, 69.2, 66.9, 59.2, 57.2, 55.4, 52.5, 49.7, 46.0, 30.6, 30.2, 29.4, 28.8, 28.2, 27.4, 26.4, 24.7, 21.8, 14.2. ESI-MS: 5610.7 ([M + H]+, calcd for C290H396BrN40O68: 5609.8).

Compound 13

A mixture of 12 (140 mg, 0.025 mmol) and NaN3 (8 mg, 0.125 mmol) in DMF (0.9 mL) was stirred at 50 °C for 16 h. Gel permeation chromatography (Sephadex G-25, H2O) gave 13 (86 mg, 62%) as a brown-orange glassy solid. IR (neat): 3360 (br, OH), 2105 (–N3), 1740 (C[double bond, length as m-dash]O). 1H-NMR (400 MHz, DMSO-d6): 7.84 (s, 10H), 4.28 (m, 44H), 3.90 (m, 10H), 3.80 (m, 10H), 3.67 (m, 10H), 3.57 (m, 2H), 3.49 (m, 10H), 3.42 (t, J = 6 Hz, 10H), 3.21 (m, 16H), 3.09 (m, 10H), 2.95 (m, 10H), 2.87 (m, 10H), 2.64 (m, 20H), 1.94 (m, 40H), 1.76 (m, 20H), 1.64 (m, 20H), 1.24 (s, 123H).

Compound 15

A mixture of 14 (5.2 mg, 25 μmol), 13 (220 mg, 0.040 mmol), CuSO4·5H2O (1 mg, 5 μmol) and sodium ascorbate (3 mg, 15 μmol) in THF/H2O/DMSO (0.45[thin space (1/6-em)]:[thin space (1/6-em)]0.7[thin space (1/6-em)]:[thin space (1/6-em)]1.2 mL) was heated at 80 °C under microwave irradiation in a sealed tube for 2 h. Gel permeation chromatography (Sephadex G-25, H2O) followed by filtration through a QuadraSilTM Mercaptopropyl column gave 15 (111 mg, 65%) as a brown-orange glassy solid. IR (neat): 3267 (br, OH), 1738 (C[double bond, length as m-dash]O). UV/Vis (H2O/DMSO 10[thin space (1/6-em)]:[thin space (1/6-em)]0.2): 245 (1[thin space (1/6-em)]262[thin space (1/6-em)]000), 331 (sh, 962[thin space (1/6-em)]000). 1H-NMR (400 MHz, DMSO-d6): 7.83 (m, 132H), 5.65 (m, 120H), 5.52 (m, 240H), 5.37 (m, 240H), 4.27 (m, 432H), 3.89 (m, 120H), 3.79 (m, 120H), 3.64 (m, 120H), 3.47 (m, 120H), 3.22 (m, 192H), 3.07 (m, 120H), 2.95 (m, 120H), 2.87 (m, 120H), 2.66 (m, 120H), 1.97 (m, 480H), 1.77 (m, 264H), 1.63 (m, 240H), 1.24 (m, 1476H). 13C-NMR (100 MHz, DMSO-d6): 163.3, 146.21, 146.04, 145.95, 145.59, 145.54, 145.49, 145.42, 141.30, 141.20, 141.14, 76.93, 72.95, 72.76, 70.21, 70.12, 70.05, 69.7, 69.2, 67.8, 66.97, 66.90, 66.8, 66.2, 63.7, 63.5, 55.4, 55.2, 53.8, 52.6, 50.4, 49.6, 46.0, 30.2, 29.1, 28.9, 28.8, 28.2, 26.6, 26.3, 22.6, 21.8, 14.2.

Compound 16

A 1 M solution of TBAF in THF (28 μL, 28 μmol) was added to a mixture of 14 (64 mg, 22 μmol), 11 (93 mg, 28 μmol), CuSO4·5H2O (1.3 mg, 5 μmol) and sodium ascorbate (1.5 mg, 8 μmol) in CH2Cl2/H2O/DMSO (0.7[thin space (1/6-em)]:[thin space (1/6-em)]0.6[thin space (1/6-em)]:[thin space (1/6-em)]0.4 mL). The resulting mixture was vigorously stirred at rt under argon. After 6 days, water (10 mL) was added to the mixture and the resulting brown precipitate filtered, extensively washed with acetone and dried under high vacuum to give 16 (69 mg, 53%) as a brown-orange glassy product. IR (neat): ν = 3369 (br, OH), 1740 (C[double bond, length as m-dash]O) cm−1. UV/Vis (H2O/DMSO 10[thin space (1/6-em)]:[thin space (1/6-em)]0.2): λmax(ε) = 270 (80[thin space (1/6-em)]000), 283 (78[thin space (1/6-em)]000), 321 (sh, 62[thin space (1/6-em)]100), 337 (sh, 56[thin space (1/6-em)]600), 386 (sh, 36[thin space (1/6-em)]400) nm. 1H NMR (400 MHz, CD3OD): δ = 7.68 (s, 12H), 4.34 (m, 48H), 3.84 (s, 24H), 3.47 (m, 12H), 3.35 (t, J = 9 Hz, 12H), 3.14 (t, J = 9 Hz, 12H), 2.98 (dd, J = 5 Hz, 12H), 2.75 (m, 36H), 2.55 (m, 12H), 2.20–2.03 (m, 48H), 1.86 (m, 24H), 1.46 (m, 24H), 1.28 (s, 120H) ppm. 13C NMR (100 MHz, DMSO-d6): δ = 163.2, 145.9, 145.4, 141.2, 122.1, 79.6, 71.2, 69.9, 69.2, 67.05, 66.95, 59.5, 57.3, 52.6, 49.7, 46.0, 30.2, 29.4, 28.9, 28.2, 27.5, 26.4, 24.9, 21.8 ppm. MALDI-TOF-MS: 6094 ([M + H]+, calcd for C318H445N48O72: 6092.2).

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 appropriate inhibitor. Each assay was performed in phosphate buffer or phosphate–citrate buffer (for α- or β-mannosidase and amyloglucosidase) at the optimal pH for each enzyme. The reactions were initiated upon 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 the addition of 1 M Na2CO3. The reaction times were appropriated 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 the different glycosidases) and various concentrations of inhibitor. Full Ki determinations and enzyme inhibition mode were determined from Dixon plots.


We acknowledge the financial support from the Spanish Ministerio de Economía y Competitividad (MINECO; contracts SAF2016-76083-R and CTQ2015-64425-C2-1-R), the Junta de Andalucía (contract FQM2012-1467), the CITIUS, the Biomolecular Interactions Platform (CicCartuja), the CNRS, the University of Strasbourg, the International Center for Frontier Research in Chemistry and the LabEx “Chimie des Systèmes Complexes”. We further thank M. Schmitt for high-field NMR measurements and J.-M. Strub for the mass spectra.

Notes and references

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Electronic supplementary information (ESI) available. See DOI: 10.1039/c7tb01052d

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