Biologically Active Heteroglycoclusters Constructed on a Pillar[5]arene-Containing [2]Rotaxane Scaffold.

A synthetic approach combining recent concepts for the preparation of multifunctional nanomolecules (click chemistry on multifunctional scaffolds) with supramolecular chemistry (self-assembly to prepare rotaxanes) gave easy access to a large variety of sophisticated [2]rotaxane heteroglycoclusters. Specifically, compounds combining galactose and fucose have been prepared to target the two bacterial lectins (LecA and LecB) from the opportunistic pathogen Pseudomonas aeruginosa.

The synthesis of complex multifunctional molecules exhibiting specific properties for applications in materials science or biology is at the forefront of modern synthetic chemistry. The availability of these compounds is, however, a critical aspect for their applicability. For the synthetic chemist, the challenge is often to increase the complexity of the molecular structures without being limited by the synthetic route. In this respect, the use of pre‐constructed nanoscaffolds allowing the successive grafting of different molecular entities through the use of efficient orthogonal synthetic strategies is very attractive.1 Functionalizable scaffolds have been particularly useful for the efficient preparation of new materials for various applications, including liquid–crystalline materials2 or photo‐ and electro‐active molecular devices.3 As part of this research, we became interested in the use of pillar[5]arene cores as scaffolds for the preparation of nanomaterials.4, 5 The existing pillar[5]arenes prepared so far have, however, some limitations. Actually, whereas building blocks with 10 identical peripheral groups are conveniently prepared, pillar[5]arenes combining different monomeric moieties are only accessible under statistical conditions.6 A controlled synthesis appears difficult as cleavage of the Ar—CH2 bonds occurs under the Friedel–Crafts conditions used for their preparation and scrambling cannot be avoided.7 In order to overcome this problem, we now propose to take profit of the capability of pillar[5]arenes to form host–guest complexes with alkyl chains to build [2]rotaxane scaffolds.8 The macrocyclic pilar[5]arene component can carry ten copies of a first functional group and the molecular axis can be substituted with additional functional subunits. The structure of the [2]rotaxane scaffold provides a perfect control of the spatial organization of the different functional subunits as the ten peripheral groups attached on the macrocyclic core generate an equatorial belt, whereas the two stoppers are located at the opposite poles of the ring. As a proof of concept, we have applied this strategy for the construction of heteroglycoclusters.

In order to mimic the multivalent presentation of carbohydrates on glycoprotein or cell surfaces, multivalent glycoconjugates have been built using a large variety of scaffolds.9 Heteromultivalent glycosystems presenting two or more carbohydrates have also been prepared.10 Multivalent glycoconjugates can be used as high avidity competitors for blocking the binding of bacterial receptors to glycoconjugates on the cell surface. Among others, major targets of synthetic glycoclusters are the two soluble lectins of the opportunistic pathogen Pseudomonas aeruginosa, LecA and LecB,9a,c,h, 10a,b that are virulent factors responsible for cell adhesion, internalization, and biofilm formation.11 As part of this research, we report herein [2]rotaxanes12 combining two different sugar residues, namely galactose and fucose, in order to target simultaneously the two bacterial lectins from P. aeruginosa with a single supermolecule.

In order to show that sugar moieties are suitable stoppers for the construction of pillar[5]arene‐containing [2]rotaxanes, the preparation of model compound 7 was attempted first (Scheme 1).

Scheme 1

a) CuBr⋅SMe2, CHCl3, RT (75 %); b) 1, 4 (4 equiv), CuBr⋅SMe2, CHCl3, −20 °C to RT (75 %); c) 1, CuBr⋅SMe2, CHCl3, RT (62 %); d) MeOH, MeONa (from 5: 7 (85 %); from 6: 8 (84 %)).

Treatment of bis‐azide 2 with alkyne 1 under copper‐catalyzed alkyne azide cycloaddition (CuAAC) conditions provided 3 in 75 % yield. [2]Rotaxane 5 was then prepared from 1, 3, and 4. The reaction was carried out in CHCl3, a solvent unable to form an inclusion complex with pillar[5]arene 4 thus preventing any competition for the binding of reagent 3. To further favor the threading of reagent 3 and, therefore, the formation of [2]rotaxane 5, the reaction was carried out at low temperature (−20 °C) and at the highest possible concentration. Moreover, the macrocyclic reagent was used in large excess (4 equiv). Under these conditions, the threading/stoppering process using the CuAAC reaction was efficient. Effectively, the targeted [2]rotaxane (5) was thus obtained in 75 % yield. Owing to the D 5‐symmetry of the pillar[5]arene ring, it is worth noting that compound 5 was obtained as an inseparable 1:1 mixture of two diastereomers differing by the absolute configuration of the macrocyclic component. For comparison purposes, axle 6 was also prepared by reaction of 1 with 3 in the presence of CuBr⋅SMe2. Inspection of the 1H NMR spectra of compounds 5 and 6 revealed a dramatic shielding for all the signals arising from the —(CH2)10— chain of [2]rotaxane 5 when compared to axle 6 (Figure S1, Supporting Information). This effect results from the ring current effect of the pillar[5]arene aromatic moieties on the methylene groups of the axle in 5 and is a diagnostic signature for a [2]rotaxane structure.8 Finally, deacetylation of 5 and 6 under Zemplén conditions (MeOH/MeONa) afforded 7 and 8, respectively. Importantly, the chemical shifts of the signals corresponding to the methylene groups of the axle moiety in 7 are typical of a [2]rotaxane structure (Figure S2, Supporting Information). Moreover, no evolution of the 1H NMR spectrum of 7 is observed even after several days in solution thus showing that both components of 7, namely the pillar[5]arene macrocycle and the fucosylated molecular axle, remain associated through mechanical bonding. In other words, the deprotected carbohydrate residues of 7 are large enough to prevent the dissociation of the [2]rotaxane.

In order to prepare [2]rotaxane derivatives incorporating additional sugar residues on their pillar[5]arene subunit, pillar[5]arene 9 bearing 10 brominated alkyl chains was used as the starting material (Scheme 2). [2]Rotaxane 13 a incorporating acetylated fucose residues was obtained from 1, 3, and 9 under the conditions developed for the preparation of 5. The yield in [2]rotaxane was, however, lower, most probably due to negative steric effects resulting from the presence of the bromides on the macrocycle.13

Scheme 2

a) 1 and 3 or 10 and 11, CuBr⋅SMe2, CHCl3, −20 °C to RT (from 1 and 3: 13 a (32 %); from 10 and 11: 13 b (26 %)); b) NaN3, DMF, RT (from 13 a: 14 a (85 %); from 13 b: 14 b (84 %)); c) 1, 10, or 15, CuSO4 ⋅5 H2O, sodium ascorbate, CH2Cl2/H2O, RT (from 13 a and 10: 16 (85 %); from 13 b and 1: 17 (82 %); from 13 a and 15: 18 (65 %); from 13 b and 15: 19 (79 %)); d) MeOH, MeONa, RT (from 16: 20 (86 %); from 17: 21 (91 %), from 18: 22 (89 %); from 19: 23 (88 %)); e) CuBr⋅SMe2, CHCl3, RT (62 %); f) 10, CuBr⋅SMe2, CHCl3, RT then MeOH, MeONa, RT (62 %).

The corresponding [2]rotaxane derivative with acetylated galactosyl stoppers (13 b) was prepared by following a similar synthetic approach. Treatment of 2 with alkyne 10 and subsequent reaction of the resulting azide (11) with 10 in the presence of 9 afforded [2]rotaxane 13 b. For comparison purposes, compound 12 was also prepared from 11. Treatment of 13 a and 13 b with NaN3 in DMF afforded polyazides 14 a and 14 b, respectively. Post‐functionalization of the macrocyclic component of [2]rotaxanes 14 a–b was then efficiently achieved under CuAAC conditions. In both cases, the complementary sugar subunits were introduced onto the [2]rotaxane scaffolds to generate the corresponding heteroglycoclusters (16 and 17). In the aromatic region of the 1H NMR spectra of 16 and 17, a diagnostic signal is observed at δ=7.76–7.95 ppm for the 10 1,2,3‐triazole rings generated in the last step. The signals arising from the 1,2,3‐triazole rings of the stoppers are also observed in the spectra (δ=7.35–7.68 ppm) as well as the typical resonances of the pillar[5]arene aromatic protons (δ=6.56–6.70 ppm). The structure of 16 and 17 was further confirmed by their mass spectra showing the expected molecular ion peak at m/z: 6045.4 (16; calcd for C265H340N36O126: 6045.71) and 5581.3 (17; calcd for C249H324N36O110: 5581.42).

Model [2]rotaxanes 18 and 19 were prepared by introducing acetylated glycerol moieties onto the macrocyclic moiety. Finally, treatment of compounds 16–19 with MeOH/MeONa gave the corresponding unprotected [2]rotaxanes 20–23 in good yields.

The [2]rotaxane heteroglycoclusters (20 and 21) were tested by isothermal titration calorimetry (ITC) as ligands of the two bacterial lectins from Pseudomonas aeruginosa, namely galactolectin LecA and fucolectin LecB. These compounds were compared to [2]rotaxanes 22 and 23 displaying only carbohydrate stoppers and to monovalent references β‐GalOMe (methyl‐β‐d‐galactopyranoside)14 and α‐FucOMe (methyl‐α‐l‐fucopyranoside).15 The ITC data are summarized in Table 1.

Table 1

The publisher did not receive permission from the copyright owner to include this object in this version of this product. Please refer either to the publisher's own online version of this product or the printed product where one exists.

The stoichiometry derived for the binding of LecA and LecB to rotaxanes with Gal (21 and 23) and Fuc (20 and 22) stoppers, respectively, is close to 0.5 (0.43 to 0.66) and similar to the binding of the axle alone (8 and 12). This is in agreement with the binding of one divalent molecule to two lectin monomers thus indicating an effective binding of both stoppers onto the target in all the cases. Concerning the peripheral sugar residues grafted onto the pillar[5]arene moiety, the stoichiometry towards the targeted lectin varies from 0.15 (20) to 0.28 (21) indicating that 3 (21) to 6 (20) out of the 10 monosaccharides reach a binding site. Altogether, the stoichiometry values obtained for 20 and 21 towards LecA and LecB indicate that these compounds are able to bind simultaneously to the two lectins.

Comparison of 20 with the corresponding model compound bearing the same stoppers (22) reveals similar dissociation constants for LecB. Similar K D values are also observed for the binding of 21 and 23 to LecA. In both cases, the nature of the substituents of the pillar[5]arene unit has no significant influence on the binding of the stoppers to LecA and LecB.

Fucolectin LecB has a unique mode of binding involving two bridging calcium ions and thus a high affinity for monovalent fucose derivatives such as α‐FucOMe.14a The tetrameric structure presents the binding sites in opposite orientation in space14b and therefore this particular lectin is also not very sensitive to multivalent effects.16 In the particular case of 21, a significant decrease in affinity is even observed when compared to monovalent ligand α‐FucOMe thus suggesting that negative steric effects are certainly playing a role for the binding of fucose moieties of decavalent compound 21 to LecB. Conversely, comparison of the K D values of the Fuc‐stoppered rotaxanes (20 and 22) with the corresponding divalent model compound (8) reveals only limited steric effects resulting from the presence of the macrocyclic component in 20 and 22 (Figure S3, Supporting Information).

Owing to the close presentation of two neighboring galactose binding sites in LecA, this lectin is very sensitive to multivalent effects.17 The K D value is already increased by more than two orders of magnitude when going from the monovalent model compound (β‐GalOMe) to divalent model compound 12. As schematically represented in Figure 1 A, the two Gal residues of 12 are most probably bound to the same LecA. In contrast, the lower affinity of divalent Gal derivatives 21 and 23 suggests that their macrocyclic subunit prevents the folding of the axel and thus the simultaneous binding of their two Gal residues onto the same lectin (Figure 1 B and Figure S4, Supporting Information). For [2]rotaxane 20 bearing ten Gal residues, the affinity for LecA is further increased. A 268‐fold increase is effectively observed when going from β‐GalOMe to 20. Importantly, the combination of ten Gal residues with two Fuc stoppers in [2]rotaxane 20 is perfectly suited to achieve high affinities for both LecA and LecB. Compound 20 appears to be more efficient than the only known heteroglycocompound directed against P.aeruginosa lectins: an octoglycodendrimer presenting galactose and fucose that could precipitate LecB but with weak activity for LecA.10b As schematically shown in Figure 1 C, the high affinity of 20 results from the clustering of LecA and the aggregation of LecB.

Figure 1

Schematic representations showing possible binding modes of divalent ligands 12 (A) and 23 (B) to LecA (ligands and proteins are not represented at real scale). In the case of heteroglycorotaxane 20 (C), clustering of LecA to the decavalent macrocyclic component and aggregation of LecB to the divalent axle moiety provide high affinity for both lectins.

In conclusion, we have developed a synthetic approach combining recent concepts for the preparation of multifunctional nanomolecules (click chemistry on multifunctional scaffolds) with supramolecular chemistry (self‐assembly to prepare rotaxanes) allowing the synthesis of sophisticated supramolecular heteroglycoclusters for biological applications. As the CuAAC reaction conditions used for the functionalization of the rotaxane scaffold are tolerant to a large variety of functional units, this synthetic approach will allow for the easy preparation of unprecedented multifunctional materials for various applications.

As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

miscellaneous_information

Click here for additional data file.