Carbohydrates in Supramolecular Chemistry - Chemical Reviews

Review pubs.acs.org/CR

Carbohydrates in Supramolecular Chemistry Martina Delbianco,† Priya Bharate,†,‡ Silvia Varela-Aramburu,†,‡ and Peter H. Seeberger*,†,‡ †

Department of Biomolecular Systems, Max-Planck-Institute of Colloids and Interfaces, Am Mühlenberg 1, 14476 Potsdam, Germany Institute of Chemistry and Biochemistry, Freie Universität Berlin, Arnimallee 22, 14195 Berlin, Germany

‡

ABSTRACT: Carbohydrates are involved in a variety of biological processes. The ability of sugars to form a large number of hydrogen bonds has made them important components for supramolecular chemistry. We discuss recent advances in the use of carbohydrates in supramolecular chemistry and reveal that carbohydrates are useful building blocks for the stabilization of complex architectures. Systems are presented according to the scaffold that supports the glyco-conjugate: organic macrocycles, dendrimers, nanomaterials, and polymers are considered. Glyco-conjugates can form host−guest complexes, and can self-assemble by using carbohydrate−carbohydrate interactions and other weak interactions such as π−π interactions. Finally, complex supramolecular architectures based on carbohydrate−protein interactions are discussed.

CONTENTS 1. Introduction 2. Organic Macrocycles 2.1. Cyclodextrins 2.1.1. CD-Based Glycoclusters 2.1.2. Interfaces 2.1.3. Self-Assembling Systems 2.2. Calixarenes 2.2.1. Host−Guest Interactions 2.2.2. Self-Assembling Systems 2.3. Pillararenes 2.3.1. Host−Guest Complexes 2.3.2. Microtube Formation 2.4. Cucurbiturils 2.4.1. Glyco-CBs Supramolecular Structures 2.4.2. Self-Assembling Polymers 3. Glycoclusters and Glycodendrimers 3.1. Self-Assembling Structures 3.1.1. Micelles, Vesicles, and Spherical Nanoparticles 3.1.2. Fibers 3.1.3. Gels 3.1.4. Metal-Mediated Self-Assembly 3.2. Aggregation upon Lectin Binding 3.2.1. Organic-Based Structures 3.2.2. Metal-Based Structures 3.3. Glycodendrimers as Host 3.4. Glycodendrimers as Stabilizing Agent 4. Glycosylated Nanomaterials 4.1. Glyconanoparticles for Biosensing 4.1.1. Biosensors in Solution 4.1.2. Surface-Based Sensors 4.2. Glyconanoparticles for Bacteria Sensing 4.3. Glyconanoparticles in Biomedicine © 2015 American Chemical Society

4.4. Carbon-Based Nanostructures 4.4.1. Graphene 4.4.2. Carbon Nanotubes 5. Polymers 5.1. Glycolipids 5.1.1. Cell Membrane-Like Structure 5.1.2. Self-Assembling Structures 5.2. Glycopeptides 5.2.1. Short Peptides 5.2.2. Longer Peptides 5.2.3. Sugar-Appended Peptides 5.3. Glycopolymers 5.3.1. Flexible Backbone 5.3.2. Aromatic Rigid Backbone 6. Conclusions and Outlook Author Information Corresponding Author Notes Biographies Acknowledgments References

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1. INTRODUCTION The importance of weak, noncovalent interactions in biological systems was first appreciated at the beginning of the 20th century with an improved understanding of hydrogen bonding and substrate−receptor interactions. These weak interactions Special Issue: Frontiers in Macromolecular and Supramolecular Science Received: September 1, 2015 Published: December 24, 2015 1693

DOI: 10.1021/acs.chemrev.5b00516 Chem. Rev. 2016, 116, 1693−1752

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Figure 1. Overview of the different scaffolds described in this Review.

were key to “host−guest” complexes that form well-defined architectures and were recognized with the 1987 Nobel Prize for supramolecular chemistry. Since then, a variety of supramolecular systems that respond to signals,1 molecular machines and switches,2,3 well-defined networks,4,5 and selfassembling macrostructures6,7 have been created and studied. All of these new noncovalent systems are built upon weak interactions such as hydrogen bonding,8,9 metal coordination,10,11 hydrophobic or hydrophilic forces,12,13 π−π interactions,14−16 and van der Waals forces.17,18 Stable systems, which are able to mimic nature and bind analytes with great specificity, have become accessible via multiple and directional interactions.19,20 Carbohydrates are the predominant class of biopolymers and are mainly used for energy production and as structural materials. These complex biomolecules can form many hydrogen bonds, a feature that renders them interesting for supramolecular systems. In nature, carbohydrate−carbohydrate (CCIs) and carbohydrate−protein interactions (CPIs) regulate a variety of biochemical processes, such as cell differentiation, proliferation and adhesion, inflammation, and the immune response.21 To increase binding strengths of CCIs and CPIs, many natural biomolecules exhibit multiple carbohydrates that engage in multivalent interactions. Synthetic chemists have

adopted nature’s strategies and developed multivalent systems that mimic natural supramolecular interactions.22,23 Systems displaying multiple identical carbohydrates present generally stronger affinities than the sum of the single contributions for each unit.24 Multivalency has been exploited for the design of high affinity ligands to be used as drugs and probes. Intense efforts have been devoted to better understanding this phenomenon as a first step toward predicting the optimal density of ligand display.25 We summarize the recent advances in carbohydrate supramolecular chemistry with a particular focus on illustrating the role of carbohydrates as stabilizers of complex architectures. We will focus on three main topics: (1) the use of multivalent glyco-conjugates for the formation of host−guest complexes that are used for sensing and drug delivery; (2) the combination of CCIs with other weak interactions, such as π−π interactions, for the formation of self-assembling glycoclusters; and (3) the formation of extended supramolecular networks upon binding to proteins. The scaffold at the center of the glyco-conjugate serves to classify the systems. Organic macrocycles22,26,27 dendrimers,28 nanoparticles,29 and polymeric backbones30 are used to delineate the classes of carbohydrate-based supramolecular assemblies (Figure 1). Each individual carbohydrate glyco1694


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Figure 2. General structures of cyclodextrin, calixarene, pillarene, and cucurbituril.

chemistry with particular attention on CDs as glycocluster scaffolds. 2.1.1. CD-Based Glycoclusters. Multivalent interactions between host and bacteria are important during infections and at various points of the immune response.46 To mimic and study these interactions, many molecular and supramolecular glycoclusters have been synthesized. The density of the sugar residues, the chemical structure of the linker, and the scaffold all play an important role in affinity and specificity regulation.51,52 CDs are a common scaffold for multivalent glycosystems as tools to study carbohydrate−protein (CPIs)44 and carbohydrate−carbohydrate (CCIs)53 interactions. CD-glycoclusters based on host−guest inclusion complexes, built around metallodendrimer cores and formations of rotaxanes, are discussed below. 2.1.1.1. Glycosylated CDs for the Study of CPIs and CCIs. C-type lectin receptors (CLRs) bind glycan structures displayed on pathogens. Multivalent carbohydrate systems with terminal sugar ligands tightly bind these lectins by glycoside clustering.24 The hydrophobic CD cavity and the carbohydrate ligand− lectin interaction result in “lectin−carbohydrate−ligand” guest complexes.50 A supramolecular pyrenyl-β-CD fluorescent probe was used to study lectin recognition.54 Truncated cone-shaped pyrenyl-βCDs were prepared using a Cu(I)-catalyzed Huisgen azide− alkyne cycloaddition reaction. N-Acetyl-galactosaminyl (GalNAc) and N-acetyl-glucosaminyl (GlcNA)-functionalized rhodamine B complexes were used as guests (Figure 3). The supramolecular complex formed between pyrenyl-β-CD and rhodamine B due to π−π interactions quenches the fluorescence of the system. Lectin agglutination by binding of the fluorescent probe via GalNac and GlcNac to soybean agglutinin and wheat germ agglutinin lectins recovers system fluorescence.

cluster has been reviewed with respect to synthesis, biological applications, and structural evaluation. We focus on the technologies that have been used to create supramolecular systems. We stress the importance of carbohydrates as building blocks for the synthesis and stabilization of supramolecular systems based on multiple weak interactions. The other big challenge for supramolecular chemists is sugar recognition. Discriminating between carbohydrates differing only in a single chiral center (e.g., Glc vs Man vs Gal) has proven difficult and is possible only in organic solvents. The few systems able to sense sugars in water that exist are based on two approaches: The first approach relies on the formation of a covalent bond between two hydroxyl groups of the selected sugar and a boronic acid present on the probe.31,32 The second approach combines the use of hydrogen bonding and apolar interactions to mimic sugar receptors existing in nature (i.e., lectins).33,34 Sugar recognition will not be examined in this Review, because the sugar is the analyte rather than part of the supramolecular architecture.

2. ORGANIC MACROCYCLES Purely organic macrocycles represent well-known scaffolds for the construction of supramolecular assemblies. Cyclodextrin, calixarenes, pillarenes, and cucurbiturils molecules (Figure 2) can be readily functionalized, while their cavities can be exploited as hosts for sensing and drug delivery applications. The shape and the properties of organic macrocycles render them good building blocks for the synthesis of self-assembling and supramolecular systems. 2.1. Cyclodextrins

Cyclodextrins (CDs) are a class of cyclic oligosaccharides, with a torus-like shape, built from 1 → 4 linked α-D-glucopyranose units. First described in 1891, CDs were named “cellulosine”. Three types of CDs with six, seven, or eight glucopyranose units are referred to as α-, β-, and γ-CD, with β-CD as the most common class.35 CDs have a hydrophilic exterior and a hydrophobic central cavity for the noncovalent inclusion of guest molecules.36 Different CDs can encapsulate a range of guests37 and have been employed for molecular recognition,38 catalysis,39 polymerization,40 material sciences, drug delivery,41 nanotechnology,42 and hydrolysis.43 The synthesis and biological applications of CDs as multivalent scaffold in carbohydrate chemistry have been reviewed before.35,44,45 Cyclodextrin glycoconjugates display the glycans in a multivalent form similar to the situation on the cell-surface.46 CDs have been used for drug delivery47 and sensing48,49 as well as developing saccharide-selective systems.50 We focus on the application of CDs in supramolecular

Figure 3. Fluorescent detection of a selective lectin using the CD− glycosyl rhodamine B complex. Reprinted with permission from ref 54. Copyright 2014 Royal Society of Chemistry. 1695


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Figure 4. Overview of protein separation by cross-linking and magnetic precipitation of CD appended magnetic nanoparticles and molecular structures of adamantine-functionalized glycans (1 and 2). Reprinted with permission from ref 55. Copyright 2014 John Wiley and Sons.

Figure 5. Preparation and self-assembly of glyco-QDs: (a) β-CD derivatives in ethanol−water (1:10). Adapted with permission from ref 56. Copyright 2012 Royal Society of Chemistry.

Another supramolecular system for carbohydrate−lectin recognition was based on magnetic iron oxide nanoparticles appended with CDs that host adamantyl-glycosides (mannose

and galactose). Fluorescently labeled lectins such as concanavalin A (ConA) and peanut agglutinin (PNA) interacted with the carbohydrates to create a ternary nanoparticle− 1696


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Figure 6. Synthesis of supramolecular complexes (T-1 and C-1). Reprinted with permission from ref 53. Copyright 2013 Royal Society of Chemistry.

carbohydrate−lectin complex. In a magnetic field, proteins can be selectively separated from a mixture and quantified by fluorescence emission intensity measurements (Figure 4).55 β-CD-Functionalized glycoclusters were appended with different sugars to explore CPIs and CCIs.56 Using a host− guest strategy, β-CD-quantum dots (β-CD-QDs) were prepared, and the cellular cytotoxicity was assessed. Synthetic β-CD-QDs were used to study CPIs in vitro showing and showed results similar to those of PEGylated QDs (Figure 5). ConA, Galanthus nivalis lectin (GNA), and PNA lectins are agglutinated by glyco-QDs. A photoswitchable glycocluster with distinct sugar arrangements helped to study CCIs with Ca2+ ions.53 Straightforward access to different sugar densities via self-assembly helped to tune intra- to intermolecular CCIs. Self-assembled photoswitchable glycoclusters of β-CDs appended with lactose and maltose formed complexes with 1,2-di(pyridin-4-yl) diazenes (Figure 6). Photoinduced isomerization yielded the cis-1 (C-1) and trans-1 (T-1) complexes and a change of glycocluster topology. Isothermal titration calorimetry (ITC) was used to determine the CCIs of the photoswitchable glycoclusters with Na+, K+, and Ca2+ cations. Significant changes in enthalpy were observed, indicating that the glycans do not bind each other in the presence of sodium or potassium ions. Calcium ions bind

glycocluster 6, in both cis and trans forms, following the trend C-1 > T-1 > 6. Intra- and inter-CCIs vary with sugar density. The CD-based glycoclusters were investigated concerning the self-inclusion process and the sugar−lectin recognition events in an extended conformation. A “reversible structuredependent binding” mimicked biological processes involving effector/antagonist molecules.57 Trimannose β-CD conjugates with and without aromatic amino acid were prepared (Figure 7A). In aqueous media, β-CD accommodates L-tyrosine via inclusion complexation and restricts mannose−lectin interactions. Addition of a competitive guest disrupts the inclusion complex and triggers lectin binding (Figure 7B). An enzymelinked lectin assay (ELLA) revealed that the affinity of the conjugate 11 (Man-tri−β-CD) for ConA was 6 times higher than that for conjugates 9 and 10. 2.1.1.2. Gene and Drug Delivery. Multivalent polyamidoamine (PAMAM) starburst CD dendrimers (called α, β, and γ CD conjugates-G2 generation) were used as gene delivery agents to mouse embryonic fibroblast and macrophage cell lines. PAMAM dendrimers functionalized with α-CD exhibit 100-fold higher transfection activity than PAMAM dendrimer alone.58 Casting α-CD conjugate as a nonviral vector of pDNA, further mannose- and galactose-functionalized α-CD dendrimers that incorporate a phenylisothiocyanate spacer were 1697


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Figure 7. (A) Structure of the Man-tri−β-CD conjugates 9, 10, and 11. (B) Possible carbohydrate−lectin binding through supramolecular interactions using “off/on” switching. Reprinted with permission from ref 57. Copyright 2006 John Wiley and Sons.

Galactose-funtionalized β-CDs were designed for hepatocytespecific drug delivery and prepared via “click chemistry” (Figure 9). Encapsulation of rhodamine in the cyclodextrin cavity allowed for targeting of hepatocellular carcinoma cell line HepG2 as determined by flow cytometry.61 A trivalent mannosylated β-CD conjugate was used to encapsulate and deliver pharmacological chaperones to macrophages via the macrophage mannose receptor (MMR) (Figure 10).62 Carbohydrate-based targeting approaches take advantage of several CLRs including the ASGPR on liver cells and DC-SIGN expressed by dendritic cells.63 A CD-based glycoconjugate was created for HIV drug delivery (Figure 11). Glycoclusters and star glycopolymers were obtained by combining click chemistry and copper-mediated living radical polymerization. The resulting glycoconjugate bound to DC-SIGN and inhibited its interaction with HIV gp120 at nanomolar concentrations. In addition, the drug was delivered and released in the cells.64 2.1.1.3. Metal Complexes. Glycoclusters that contain a metal complex in the framework are called metalloglycoclusters.65 Metallo-glycodendrimers bearing transition metals or lanthanide ions have been prepared by self-assembly.66−69 The inherent physical properties of metallodendrimers, such as fluorescent emission, render them ideal for biological applications.70 CD-based glycoclusters have been exploited for the synthesis of multivalent probes built via host−guest

prepared. Man-α-CDs and Gal-α-CDs have remarkable gene transfer activities for various cells except to macrophage receptor (MR) or asialoglycoprotein receptor (ASGPR)mediated gene cells.59 Lactose appended α-CD(G2) dendrimers have ASGPR-mediated hepatocyte-selective gene transfer activity in vitro and in vivo (Figure 8).60

Figure 8. α-CD conjugates appended with mannose, galactose, and lactose. Reprinted with permission from ref 59. Copyright 2005 Elsevier. 1698


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Figure 9. Schematic representation of β-CD glycodendrimer consisting of β-CD, fluorescent dye rhodamine B, and terminal mannose or galactose (12 and 13) residues.

bacteria bind the Ru-dendrimer in a star-shaped complex as the orientation of the ligands also determines the shape of the dendrimer−bacteria complexes (Figure 12B). Ferrocene [Fe(C5H5)2]-based adamantane lactose-modified β-CD glycoodendrimers logic operations (fuzzy logic FL and Boolean logic BL) were applied to optimize calcium-mediated CPIs (Figure 13). The association constants between glycocluster and PNA lectin were determined using ITC to conclude that fuzzy logic is the appropriate model for sensing.71 2.1.1.4. Rotaxanes. α-CDs were first used as rotors for rotaxane in 1978.72 The rigidity, water solubility, lack of toxicity, low cost, and ability to bind low molecular weight compounds render CDs attractive rotaxane building blocks that gave rise to materials and devices.73,74 Poly pseudorotaxanes contain multiple rotors that were created first via a “beads on a string” strategy in 1990. CDs (beads) form pseudopoly rotaxanes with polymers (string) such as poly(ethylene)glycol (PEG),75−77 poly(propylene) glycol (PPG),78 and poly(tetrahydropyran) (PTHP).79 The materials show great potential as scaffold for tissue engineering due to their gelation properties.80 Carbohydrate-displaying (pseudo)polyrotaxanes were used to study CPIs. A variety of mechanically interlocked structures, where the CDs form inclusion complexes with the polymeric chain, were explored for macromolecular recognition.75,76,78,81,82 Polyrotaxanes bearing maltose-appended α-CDs along the PEG chain were synthesized exploiting the strong interactions between the hydrophobic cavity of the α-CD and the −CH2OCH2− moieties of PEG.77 The mobility of maltoseCD units along the polyrotaxane facilitates ConA recognition (Figure 14A). In fact, the lectin binding affinity of the system is increased by the maltose ligands along the polyrotaxanes.83

Figure 10. Schematic representation of macrophage-specific delivery of GD pharmacological chaperones by MMR-mediated internalization of inclusion complexes. Reprinted with permission from ref 62. Copyright 2014 Royal Society of Chemistry.

assembly around a metal core. Metallo-glycodendrimers have been reviewed recently.68 Metallo-glycodendrimers based on a Ru(bipy)3 fluorescent scaffold and appended with two, four, or six adamantyl groups were adorned with mannose-functionalized CD-glycoclusters by host−guest interactions.48 The inherent emission of the Ru(II) core was the basis for biosensing applications of these complexes (Figure 12A). The glycodendrimers bind the lectin ConA depending on the mannose density. SPR sensor chips containing different densities of ConA protein revealed that complexes 14 and 15 bind better to surfaces that contain more lectin. Different E. coli strains were employed to visualize specific and shape-dependent interactions with the supramolecular glycodendrimers. Confocal microscopy showed that 1699


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Figure 11. Evolution from glycoclusters to star diblock glycopolymers. Reprinted with permission from ref 64. Copyright 2014 American Chemical Society.

Figure 12. (A) Structures of RuCDMan glycodendrimers. (B) Confocal laser scanning microscopy images for the incubation of (a,b) bacteria E. coli strain ORN178 with RuCDMan6 16. Reprinted with permission from ref 48. Copyright 2011 American Chemical Society.

Lactose-bearing α-CD and β-CD polyrotaxanes threaded

To target galectin-1 (Gal-1), stable pseudopolyrotaxanes bearing lactose-functionalized α-CDs on polyviologen were synthesized (Figure 15). The CDs spin around the polymer axis and move back and forth along the backbone. Adjustable ligand presentation provides the stereochemistry required for lectin binding. Pseudopolyrotaxanes rapidly and efficiently bind Gal-1

onto hydrophobic polymers such as PTHP and PPG (Figure 14B) orient the saccharide ligands for maximal overall receptor binding efficiency, as confirmed by one- and two-dimensional NMR spectroscopy.84 1700


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Figure 13. (A) Lactose glycodendrimers 18 and 19; and (B) schematic diagram of the mechanism of interactions. Adapted with permission from ref 71. Copyright 2013 Royal Society of Chemistry.

Figure 14. (A) Formation of maltose appended α-CD−polyrotaxanes. Reprinted with permission from ref 83. Copyright 2003 American Chemical Society. (B) Lactose bearing α-CD threaded on polymeric chain forming polyrotaxanes. Reprinted with permission from ref 84. Copyright 2003 American Chemical Society.

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Figure 15. Formation of pseudopolyrotaxane. Reprinted with permission from ref 108. Copyright 2004 American Chemical Society.

Figure 16. Structures and schematic representation of the immobilization of glycan-modified β-CD scaffolds on glass slides. Reprinted with permission from ref 88. Copyright 2015 Royal Society of Chemistry.

Mannosylated and fucosylated β-CD host glycodendrimers have been attached to ferrocene-functionalized silylepoxidecoated glass surfaces via host−guest interactions (Figure 16).88 E. coli and P. aeruginosa bacteria bound to these surfaces. A photosensitive supramolecular system based on highly ordered monolayers of β-CD immobilized on gold or glass surfaces was produced using azobenzene-functionalized mannose or galactose glycoconjugates that bind to the CDs surface and dissociate upon light irradiation.89 Binding was measured using the azobenzene derivative fluorescence to quantify the

in a T-cell agglutination assay, to provide 20-fold enhancement over free lactose displaying CD.85,86 2.1.2. Interfaces. Surface techniques have been used to study complex host−guest systems. Docking of (bio) molecules on functionalized solid surfaces by self-assembly processes helps to tailor the surface properties. Glycosylated surfaces have been developed to study multivalent glyco-interfaces. β-CD receptors immobilized on a gold surface act as host for azobenzene−oligoglycerol conjugates.87 Host−guest responsive systems were immobilized on glass substrates. 1702


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CD vesicle surface.97 Host−guest assembly occurs via orthogonal noncovalent interactions due to metal-coordination complexes.95 A ternary system using vesicles, carbohydrates, and lectin was used to study the kinetics of the orthogonal multivalent interfacial interactions. Maltose and lactose decorated vesicles aggregate with the lectins ConA and PNA in a sugar-dependent manner.98 Amphiphilic CD vesicles can form supramolecular systems that are able to capture and release proteins in response to light. Noncovalent cross-linkers containing azobenzene and a carbohydrate decorate the surface of the vesicle for lectin binding. Upon irradiation, the azobenzene unit transits from a high-affinity to a low-affinity state, resulting in the disruption of the supramolecular vesicle and protein release (Figure 19).99 Layer-by-layer deposition creates a mimic of cellular assembly. High density mannose or biotin surface-based sensors were used in dynamic systems for ConA detection. CD vesicles decorated with guest molecules form supramolecular aggregates via noncovalent intervesicular links on the surface in the presence of Con A or streptavidin (Figure 20).100 The influence of multivalency and glycan surface density was explored further by amphiphilic CD vesicles involving an adamantane guest that carries mannose and CD vesicle hosts. Mannose−adamantane conjugates carrying one, two, or three adamantanes and mannose ligands were prepared (Figure 21). Agglutination experiments and ITC revealed that divalent guests with two mannose units bind better to CD vesicles and ConA than others.101

binding affinity of azo-Man to ConA using a quartz crystal microbalance (QCM) and confocal microscopy (Figure 17).

Figure 17. Schematic representation of photoswitchable ligands on a β-CD layer. Reprinted with permission from ref 89. Copyright 2014 Royal Society of Chemistry.

A supramolecular carbohydrate-functionalized two-dimensional (2D) surface was prepared by decorating graphene sheets with multivalent sugar ligands.90 Adamantyl-functionalized graphene (AD-TRGO) was equipped with mannose-βcyclodextrin (ManCD) for bacterial binding assays, exploiting CPIs (Figure 18A). The self-assembled sensor system (ManCD-TRGO) binds bacteria reversibly, due to the interaction of mannose and E. coli strain ORN178 (Figure 18B). IR-laser irradiation91 of the ManCD-TRGO−E. coli complex kills the captured bacteria. 2.1.3. Self-Assembling Systems. Spontaneous processes forming ordered structures are key to some signaling pathways.92,93 Self-assembled surfaces have been used to study multivalency and stimuli responsive systems.94 Liposome and vesicles mimic noncovalent interactions involved at the interface between cell membrane and aqueous solution phase.95,96 Amphiphilic CD vesicles show enhanced host−guest interactions when different guest polymers are placed at the

2.2. Calixarenes

Calixarenes are synthetic macrocycles that were identified in the early 1970s and are generally synthesized by condensing phenol or resorcinol (calixresorcarene) with formaldehyde. The lower and upper rims of calixarenes can be functionalized, while the typical basket-like shape provides a hydrophobic cavity that can be exploited for host−guest interaction. Differently substituted calixarenes host guests ranging from small ions to more complex biomolecules.102

Figure 18. (A) Schematic representation of supramolecular carbohydrate-functionalized graphene complexes. (B) Confocal laser scanning microscopy images of ManCD@AG4 incubated with E. coli bacteria ORN178, mannose binding strain (top), and ORN208, nonbinding strain (bottom). Reprinted with permission from ref 90. Copyright 2015 American Chemical Society. 1703


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Figure 19. An amphiphilic α-CD bilayer vesicle enables photoresponsive capture and release of lectins based on azobenzene−carbohydrate crosslinkers. Reprinted with permission from ref 99. Copyright 2012 American Chemical Society.

Figure 20. Overview of layer-by-layer deposition of vesicle multilayers using supramolecular interactions. Reprinted with permission from ref 100. Copyright 2013 American Chemical Society.

concerning the synthesis and biological applications of “calixsugars” were provided by Stoddard26 and Casnati.27 We focus on the use of glycocalixarenes in supramolecular systems. 2.2.1. Host−Guest Interactions. Calixsugars based on calix[4]resorcarene ([4] indicates the number of repeating units in the macrocycle) containing monosaccharides fixed to the upper rim and the lower rim presenting aliphatic chains were described in the late 1990s.106 This system forms in water stable 1:1 complexes with guests, such as 8-anilinonaphthalene1-sulfonate (ANS) and fluorescent dyes. The complex absorbs onto a silica surface by virtue of the hydrogen-bonding forming sugars. Glucose-based calixarenes bind to a ConA-Sepharose gel (Figure 22). Cells recognize the system as shown for the uptake of galactose-modified calix[4]resorcarene into hepatocytes, mediated by the asialoglycoprotein receptor that recognizes terminal galactoses.107 The Gal-based system, with a fluorescent dye as guest, can be delivered to hepatocytes that express asialoglycoprotein receptors on the cell surface that recognize the terminal galactose. Fluorescence microscopy confirmed the binding specificity. 2.2.2. Self-Assembling Systems. The calix[4]resorcarene scaffold contains, in addition to the carbohydrates, four alkyl chains oriented in the same direction that create an amphiphilic structure. These molecules self-assemble to form micelles,

Figure 21. Schematic presentation of the binding of (A) a monovalent, (B) a divalent, or (C) a trivalent guest onto the surface of CD vesicles and their interaction with ConA (d CH , average spacing of carbohydrates; dBS, effective binding-site separation of ConA). Reprinted with permission from ref 101. Copyright 2012 BeilsteinInstitut.

The modification of different positions on the calixarene platform furnishes multivalent glycoconjugates. The upper rim as well as both rims have been decorated with glycans.103 Initially, sugars were added simply to increase the solubility of calixarenes in water104 and to provide hydrogen-bonding contacts for guest stabilization.105 Rigid multivalent glycocalixarenes were employed to study carbohydrate−protein and carbohydrate−carbohydrate interactions. Excellent summaries 1704


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Lac > α-Glc > Gal > β-Glc). Because of their size, monomeric viruses can undergo pinocytosis and trigger cell transfection.112 The size-dependence of this process was investigated with quantum dots coated with amphiphilic calixarenes to form small nanoparticles (15 nm).113 Fluorescence microscopy studies confirmed that endocytosis followed a size-specific mechanism (50 ≫ 15 ≫ 5 nm) and proved that bigger aggregates (100 nm) were not internalized. Because of their low toxicity and the size-dependent internalization, these macromolecular structures are potentially carriers for gene delivery. In addition to spherical structures, filaments with a defined structure can be formed by taking advantage of carbohydrate− protein interactions.114 Symmetrically substituted galactocalixarenes provide the right geometry for the inhibition of the Pseudomonas aeruginosa lectin LecA/PA-IL that is implicated in lung infections. LecA/PA-IL is a rectangular shaped tetramer with four Ca2+-dependent binding sites on the short sides and a high affinity for galactosides (Figure 24). Tetravalent 1,3-

Figure 22. Host−guest interaction and guest delivery of a glucosesubstituted calix[4]resorcarene.

called glycocluster nanoparticles (GNPs). Aggregates of six units and diameters of around 3 nm in water were analyzed by dynamic light scattering (DLS), gel permeation chromatography (GPC), and transmission electron microscopy (TEM).108 Addition of phosphate buffer resulted in aggregation of small micelles to bigger structures of diameters between 60 and 100 nm, using phosphate ions as glue (Figure 23, top). Immobilization of the amphiphilic calixarene on the hydrophobic surface of a sensor chip created a closely packed monolayer where the saccharide moieties are exposed to water. Subsequent, alternating addition of phosphate and micelles helped to form phosphate-mediated multilayered structures. Alternatively, carbohydrate-binding proteins (lectins) can help to agglutinate small micelles. GNPs can be formed from calix[4]arene-based glycoconjugates, presenting terminal Nacetyl-D-glucosamine and alkyl chains on the opposite rim.109 The lectin wheat germ agglutinin (WGA) specifically binds Nacetyl-D-glucosamine on the calix[4]arene scaffold. Following the same principle, binding of the lectins ConA and PNA to GNPs carrying Glc and Gal was studied.110 This supramolecular assembly bound and inhibited lectin receptors that are otherwise involved in pathological events. Artificial glyco-viruses were formed by self-assembly of amphiphilic calixarenes.111 The addition of an anionic polymer, such as DNA, resulted in monodisperse spherical nanoparticles, composed of several GNPs (Figure 23, bottom). These new nanoparticles had diameters of around 50 nm, neutral surface potential, and encapsulated a single DNA plasmid. The glycoviruses self-aggregate in a saccharide-dependent manner (Mal >

Figure 24. Molecular model and schematic representation of the lectin-calixarene filaments. Reprinted with permission from ref 114. Copyright 2011 Royal Society of Chemistry.

alternated galactocalixarenes bound most efficiently, because their geometry permits the chelation of two adjacent binding sites on the same lectin. In addition, the other two sugar moieties on the opposite calixarene rim bind another lectin. A monodimensional filament arose from the repeating selfassembly of these lectin-ligand units confirmed by the rectangular pattern observed by atomic force microscopy (AFM). These filaments may give rise to interesting biomaterials. 2.3. Pillararenes

Pillararenes are a class of organic macrocycles similar to calixarenes that are condensation products of 1,4-dimethoxybenzene (DMB) with paraformaldehyde.115 The cylindrical

Figure 23. Schematic representation of micelles formation (GNPs) and phosphate-induced agglutination using sugar-substituted calixarenes building blocks (top). GNPs self-assembly after the addition of DNA, to form glyco-viruses (bottom). 1705


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Figure 25. Formation of nanotubes and vesicles by self-assembly of amphiphilic pillararenes. Adapted with permission from ref 119. Copyright 2013 American Chemical Society.

Figure 26. Synthesis of the supramolecular (FITC)-sperimine conjugate β-Gal-CB[6] complex through host−guest interactions.

amphiphilic calixarenes (section 2.2.2).119 Galactose functionalization on one and placement of aliphatic chains on the other rim result in van der Waals interactions and hydrogen-bonding stabilized nanotubes of 100−200 nm diameter and several micrometers length. Upon bilayer formation, vesicles formed that rearranged into nanotubes (Figure 25). These biocompatible clusters agglutinate E. coli due to the presence of sugar.

geometry of pillararenes is a result of the methylene bridges between the DMB units. The facile synthesis and functionalization, combined with a rigid, symmetric, and π-rich cavity, rendered pillararenes an attractive basis for applications in host−guest chemistry. The superb selectivity toward special guests and the formation of supramolecular networks and molecular machines such as artificial ion channels are promising.116,117 Sugar-functionalized pillararenes increase the water solubility and the formation of supramolecular structures.118,119 2.3.1. Host−Guest Complexes. The rigid cavity of pillararenes binds various guests highly selectively. Glycofunctionalized pillar[5]arenes can host fullerene.118 Placement of 10 glucoses (five on each rim) on the backbone provided a neutral, water-soluble ligand. The bulky sugar substituents rigidify the system and improve host−guest interactions. By monitoring the intrinsic fluorescence of the pillararene ligand as a function of fullerene concentration (fluorescence quenching due to electron transfer to the guest), it was possible to calculate the association constant for the two species in an organic solvent. The host−guest complex forms even in water and solubilizes fullerene. Molecular dynamics suggested the formation of a 1:1 complex arising from two possible binding conformations; in one case, the fullerene is located in front of the cavity, and in the second it stands on the side. 2.3.2. Microtube Formation. Amphiphilic pillar[5]arenes can self-assemble into supramolecular structures similarly to

2.4. Cucurbiturils

The structure of cucurbiturils (CBs) that were first synthesized in the early 1900s was elucidated only in 1981. CB[n]s are produced by condensation of glycoluril and formaldehyde to give macrocyclic structures of glycoluril repeating units (most common 5 ≤ n ≤ 8) linked via methylene groups. The rigid hydrophobic cavity gives rise to various supramolecular systems.120 The cavity size depends on the number of repeating units and permits the encapsulation of different substances. Because of the different cavity sizes, CBs are interesting substrates for drug delivery, molecular recognition, and selfassembly,121,122 and CBs have been explored as multivalent scaffolds.123 In this section, we summarize the use of CB building blocks for the formation of glyco-supramolecular structures. 2.4.1. Glyco-CBs Supramolecular Structures. A CB[6]based scaffold was adorned with multiple carbohydrates to bind proteins with high selectivity (Figure 26).124 This glycosylatedCB formed stable complexes with different guests. Fluorescein 1706


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Figure 27. Synthesis of glyco-poly rotaxanes with different sugar densities. Reprinted with permission from ref 125. Copyright 2010 John Wiley and Sons.

Figure 28. (a) Reversible formation of supramolecular glycopolymers stabilized by CB[8]. Reprinted with permission from ref 126. Copyright 2011 American Chemical Society. (b) Supramolecular hydrogel formation based on polymer 21 and 22. Adapted with permission from ref 127. Copyright 2012 American Chemical Society.

Cucurbit[8]uril “supramolecular handcuffs” have resulted in a responsive ultrahigh-water-content hydrogel.127 Cellulose 21 and poly(vinyl alcohol) 22 derivatives were functionalized with cucurbit[8]uril guests and, upon addition of CB[8] to the mixture, a hydrogel that is responsive to external stimuli, such as temperature changes and competing guests, formed (Figure 28b). Cucurbit[8]uril can form supramolecular glycolipids.128 Mixing of naphthyl glucosamine (GlcNap) and alkyl viologel (RV8) in the presence of CB[8] forms redox responsive glycolipids (Figure 29). TEM and DLS analysis showed that these lipids self-assembled further to vesicle-like structures with a diameter of 200 nm. These assemblies interact with ConA to confirm the carbohydrate presence on the vesicle surface, suggesting potential application for biological applications.

isothiocyanate (FITC)−spermine conjugate was used as a guest for the β-Gal-CB[6] scaffold. Delivery of the complex to hepatocellular carcinoma cells that overexpress galactose receptors resulted in internalization. A series of pseudorotaxanes based on ManCB[6] and polyviolongel (PV) strings bearing 3, 5, or 10 carbohydrate wheels were synthesized (Figure 27).125 These water-stable poly rotaxanes bound E. coli more specifically and efficiently than an individual wheel. In a model of urinary infection, E. coli adhesion was inhibited. 2.4.2. Self-Assembling Polymers. In addition to covalently glycosylated CBs (section 2.4.1), host−guest interactions between glyco-functionalized guests and CB-derivatives are also possible. A water-soluble supramolecular glycopolymer based on a polymeric scaffold substituted with pendant moieties was found to interact with α-mannoside viologen (Figure 28a).126 The system was stabilized by cucurbit[8]uril that acts as “supramolecular handcuffs”. This reversible supramolecular glycopolymer associates in the presence of cucurbit[8]uril and dissociates upon addition of Na2S2O4. This flexible system adapts its topology when cell surface lectins are bound. This supramolecular approach can give rise to a library of glycopolymers for the recognition of different proteins.

Figure 29. Schematic representation of self-assembling glycolipid. Reprinted with permission from ref 128. Copyright 2013 American Chemical Society. 1707


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3.1.1. Micelles, Vesicles, and Spherical Nanoparticles. The first class of self-assembling systems that we consider comprehends micelles, vesicles, and spherical nanoparticles. To form supramolecular aggregates, CCIs and hydrophobic interactions (e.g., π−π interactions) are required to form simultaneously. The use of asymmetric building blocks (Janus dendrimers), which link together a lipophilic and a hydrophilic part, has been described as a strategy for the formation of selfassembling systems.135 Self-assembling amphiphilic rods have been explored by Lee and co-workers that reported the formation of stable carbohydrate-coated nanocapsules by self-assembly of tetra(pphenylene)and oligo(ethylene oxide) substituted with mannose.136 These water-stable spherical nanocapsules incorporate a guest molecule such as the fluorescent dye calcein. The mannoses stabilize the nanocapsules via CCIs and permit the specific targeting of E. coli mannose receptors. By slightly varying the building blocks, precise control over the aggregation pattern was possible.137 Vesicles (40 nm diameters), spherical micelles (20 nm), or cylindrical micelles were obtained by varying the length of the oligo(ethylene oxide) fragment or the number of rods attached to the sugar (Figure 31). All aggregates maintained a high affinity for the mannose receptor on E. coli, with higher values for the spherical micelle.

3. GLYCOCLUSTERS AND GLYCODENDRIMERS Dendrimer refers usually to a class of branched compounds with a regular and symmetric spherical shape. Different terminologies are adopted: clusters are small molecules with only few repeating arms, whereas the term dendrimer describes more complex, branched structures (Figure 30). Here, we treat the two classes together as platforms for the synthesis of multivalent systems.

Figure 30. General structures of glycoclusters, glycodendrimers, and glycodendrons.

Following the first dendritic structure reported in 1978 by Vögtle,129 this field developed quickly in the 1980s.130 Many dendrimeric structures based on different cores have been reported: polymers, peptides, sugars, aromatic or aliphatic molecules, and metal complexes have been used as a starting point to build branched structures with different geometries.131,132 Synthetic strategies such as Michael reaction, click chemistry, and Diels−Alder reaction are important strategies for dendrimer synthesis that were reviewed in 2012.133 Dendrimers provide a useful platform for the creation of multivalent systems. The facile functionalization of the outer sphere permits the conjugation of different molecules on the surface, while the inner sphere can be used as a container for gene or drug delivery.131 Multivalent display enhances carbohydrate−protein interactions and has prompted the synthesis of a variety of synthetic glycocluster and glycodendrimers. These systems found applications as vaccines, antibacterial agents, anticancer drugs, and imaging reporters.28,134 In addition, the hydrophilic glycodendrimers’ surface makes them water-soluble, while the hydrophobic core can encapsulate small molecules, to enhance their solubility. 3.1. Self-Assembling Structures

Multivalent glycoclusters can be synthesized through the covalent conjugation of monomers to a branched core, through complexation around a metal, or through a self-assembly process. In the latter case, the hydrophilic sugar is generally attached to a lipophilic unit creating an amphiphilic compound. These amphiphilic units are able to self-organize in defined structures such as micelle, vesicle, and wires or into gels. Multiple CCIs can be established between single monomers to further stabilize the systems. Self-assembly offers two key advantages: multivalent scaffolds can be prepared with relatively small synthetic effort as compared to covalent-based analogues and well-defined structures with tunable sugar distribution. We describe recent advances in the field of self-organizing structures bearing carbohydrate moieties; the systems are presented depending on the type of supramolecular architecture resulting from self-assembly.

Figure 31. Self-assembly of amphiphilic rods. Reprinted with permission from ref 136. Copyright 2005 Royal Society of Chemistry.

Modification of the rod resulted in a new amphiphile that acted as a responsive system and self-aggregated into either cylindrical or lamellar structures.138 The cylindrical architectures were responsive to external stimuli as the addition of the fluorescent dye Nile Red triggered the rearrangement of the cylindrical objects into spherical micelles upon dye incorporation (Figure 32). These spherical micelles bound and labeled the mannose receptors on E. coli. Percec contributed fundamentally to the field of selfassembly and dendrimers.139 Many sugar-substituted Janus dendrimers were prepared and shown to form different 1708


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Figure 32. Reversible transformation of cylindrical into spherical micelles in response to Nile Red addition. Reprinted with permission from ref 138. Copyright 2007 American Chemical Society.

Figure 33. Janus dendrimers self-assemble into glycodedndrimersomes and agglutinate in the presence of sugar-binding proteins. Reprinted with permission from ref 140. Copyright 2013 American Chemical Society.

Figure 34. Fluorescent Janus dendrimer.

systems provide a homogeneous and controlled distribution of sugar on the glycosendrimersome surface; this is not possible in the case of glycodendrimers forming upon covalent attachment of carbohydrate to a dendritic core, where only a statistical distribution can be obtained. Thus, these systems represent an ideal substrate for the study of molecular aspects of cell interactions, providing a better understanding of lectins.142,143 Other fluorescent organic nanoparticles functionalized with mannose were based on the self-assembly of Janus dendrimerlike monomers (Figure 34).144 A fluorescent dye was introduced into the core of the dendrimer for intrinsic fluorescence of the aggregate. Mannose-bearing nanoparticles bound to E. coli and were used for optical sensing. Self-assembling Janus dendrimers were used for drug delivery. 145 A hydrophobic PAMAM dendron bearing diazanaphtoquinone (DNQ) was attached, via click chemistry, to a hydrophilic PAMAM dendron functionalized with lactose

supramolecular structures depending on their chemical structures (Figure 33). The supramolecular structure formed upon addition of dendrimer dissolved in THF or ethanol solution to water or buffer. Solid lamellae as well as solid or hard vesicles and hard micelles were obtained depending on the monomer structures and the way of injection. Size and shape of the aggregates were investigated by DLS and revealed that the membrane thickness was approximately equal to the length of two Janus glycodendrimers. The formation of differently shaped and sized aggregates enabled many applications in drug delivery and molecular recognition. The interaction with lectins was affected by the morphology of the aggregate: the smaller was the glycodendrimersome, the faster was the agglutination, probably due to the highest surface/volume ratio.140 Agglutination of these structures, in the presence of lectins, depends on the amount of sugar displayed on the surface as well as the topological conformation.141 These 1709


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with saccharides and hydrophobic adamantyl or phenyl groups (Figure 37).148 The amphiphilic shell caused self-assembly in water. Only the phenyl-substituted dendrimer aggregated into 160 nm particles. Sonication of these small aggregates produced after 5 min smaller aggregates, but after equilibration (24 h at room temperature) necklace- and doughnut-like structures (180 nm diameter and 40 nm cavity) formed by aggregation of the small particles. 3.1.2. Fibers. Like spherical aggregates, linear nanofibers have found many applications in biology149 and materials chemistry.150 The self-assembly into linear one-dimensional structures can be exploited for the synthesis of multivalent ligands and for structural investigations. A scaffold based on mono- and di(2,2′:6′,2′′-terpyridinyl)arenes functionalized with β-D-glucopyranosyl isocyanate formed linear and branched sugar-functionalized twisted nanofibers with diameters of around 4 nm.151 Molecular modeling studies point to aggregates with the terpyridine stack in the interior of the fiber and the sugars pointing out (Figure 38). Aromatic π−π interactions, together with CCIs and Hbonding between urea moieties, stabilize the system. Control over fiber length is required to use nanofibers as biomaterials. Co-assembly of two building blocks, one with a highly crystalline aromatic core (23) and one with a less crystalline aromatic core (24), allows for control of the π−π interactions (Figure 39).152 Fiber length was controlled by varying the amounts of the two components: shorter fibers (70 nm to a few micrometers) resulted when more of the less crystalline aromatic core compound was used. Thereby, novel size-controlled mannose-functionalized fibers were synthesized. These new materials bound mannose receptors on E. coli, and the agglutination force was dependent on the fiber length: only longer fibers inhibited bacterial proliferation. In the previous examples, every monomer was carbohydrate functionalized. Because not only valency, but also aggregate topology affects binding affinity, self-assembling systems that integrate unfunctionalized and sugar-bearing monomers have

(Figure 35). These amphiphilic structures self-assembled into micelles exposing the galactoses. Encapsulation of doxorubicin

Figure 35. Janus dendrimers for drug delivery. Doxorubicin is encapsulated and internalized by the cells. Upon IR irradiation DNQ reacts releasing the drug. Reprinted with permission from ref 145. Copyright 2012 American Chemical Society.

(DOX) into the lipophilic core yielded drug-loaded micelles for specific RCA120 lectin-mediated internalization. The intracellular DOX release was triggered by IR irradiation. Wolff rearrangement of DNQ induced by IR light destabilizes the micelles and releases the drug into the cytosol. A different sugar-capped dendridic skeleton was formed by self-assembly of the asymmetric monomers in water (Figure 36).146 The size of the aggregates was strongly dependent on the dendrimer core, but not on the sugar substitution. The particle size decreased with increasing monomer molecular weight. Bigger monomers presented more sugars and adopted a more globular shape, thus shielding the hydrophobic core and allowing for fewer building blocks to interact. The only self-assembling spherical glycodendrimer was based on a poly(propyleneimine) (PPI) core, randomly substituted

Figure 36. Structure of glycodendrimer that self-assembles into micelles. Reprinted with permission from ref 147. Copyright 2006 John Wiley and Sons. 1710


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Figure 37. First spherical glycodendrimer that forms micelles and, upon sonication, larger aggregates. Adapted with permission from ref 148. Copyright 2012 Royal Society of Chemistry.

in the polymer affected binding. Surprisingly, fewer mannoses enhanced bacterial aggregation, probably due to reduced steric hindrance, confirming the importance of topology for multivalent interactions. Another supramolecular mannose-functionalized fiber was based on a poly(benzyl ether) dendron functionalized with DNA.154 These scaffolds self-assembled into a fiber with DNA strands pointing out, due to electrostatic repulsion. The introduction of mannose-functionalized oligonucleotides was achieved via DNA hybridization. These fibers bound and clustered E. coli bacteria. Fewer mannoses in the fiber still resulted in bacterial agglutination to confirm that multivalency combined with topology achieves higher affinities. 3.1.2.1. Chiral Helices. Because aggregate morphology influences the binding properties of multivalent structures, better insight into how the self-assembly conditions (solvent, temperature, and concentration) affect the aggregation of different substrates is key. In addition, the ability to form chiral structures can provide information on a range of biological processes. Amphiphilic glycoclusters, based on perylene diimides, form supramolecular nanofibers due to the strong π−π stacking in different solvents.155,156 Depending on the solvent mixture, leftor right-handed helices were observed (Figure 41a). Righthanded helices were observed in organic solvent (chloroform/ n-octane), but in the presence of excess water, left-handed helices formed. In the former case, the helix created a structure built around the saccharide functionalities, to shield the hydrophilic core from the organic solvent. In the latter case, the sugars were exposed to the bulk water, stabilizing the structure via hydrogen bonding. Further aggregation, due to CCIs, was possible, allowing for the formation of a superhelical

Figure 38. Molecular modeling of glyco nanofibers stabilized by the combination of π−π interactions and H-bonding. Reprinted with permission from ref 151. Copyright 2009 Royal Society of Chemistry.

been developed. A new supramolecular fluorescent polymer that binds bacteria was based on a columnar polymer composed of unfunctionalized discotic monomers intercalating mannosefunctionalized units (Figure 40).153 Upon aggregation, these polymers were highly fluorescent and were used to sense E. coli. Fluorescence microscopy showed that the amount of mannose 1711


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Figure 39. Glycoclusters used to control fiber length. Shorter fibers were synthesized by increasing the amount of 24.

structure. The helicity can be modulated by modifying the length of the alkyne chains.157 Placement of two β-D-lactosides on the perylene diimide core increases the water solubility of the system (Figure 41b).158 These glyco-functionalized building blocks self-assembled in water into right-handed helices that selectively bound to PNA due to the presence of the lactosides on the helix exterior. A perylene diimide scaffold bearing six mannosides gave rise to right- or left-handed helices that bind ConA.159 Many chiral aggregates containing different sugars were synthesized. Different stacking was observed in the presence of monosaccharide (left-handed helix) or disaccharide (right-handed helix), probably by combining steric effects and H-bonding.160 3.1.3. Gels. Gels are another type of supramolecular aggregate that require aromatic structures, which engage in strong π−π interactions, combined with groups that provide directional and rigid hydrogen bonding (e.g., sugars).161 Many small glycoconjugates have been reported as gelators and have been applied in biomedicine.

Figure 40. Multivalent supramolecular polymer for bacteria sensing. Bacterial agglutination is affected by the mannose density along the polymer. Reprinted with permission from ref 153. Copyright 2009 John Wiley and Sons.

Figure 41. (a) Helicity modulation depending on the solvent used: left-handed helices are obtained in organic solvent, whereas right-handed helices form in the presence of water. Reprinted with permission from ref 155. Copyright 2011 Royal Society of Chemistry. (b) Formation of water-soluble right-handed helix that binds PNA. Adapted with permission from ref 158. Copyright 2012 Royal Society of Chemistry. 1712


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Figure 42. Chemical structures of common sugar-based gelators.

The azobenzene undergoes cis−trans isomerization upon light irradiation and has been used widely for responsive gels.166 Different azobenzene-based gelators that are symmetrically functionalized with sugars exhibited gelation properties that are strongly affected by the sugar confirmation (29).167 Three asymmetric systems based on azobenzene functionalized with lactose 30 or maltose disaccharides (31 and 32) formed hydrogels.168 Nanofibers with opposite chiroptical properties were observed for the lactose and maltose analogues. Lectin binding studies indicated that the carbohydrates are solvent exposed. The gels possess a reversible sol−gel transition upon temperature change or UV irradiation, due to the presence of the azobenzene. Glyco-functionalized triazoles have been used as LMMGs because triazole provides aromatic as well as hydrogen-bonding interactions. The dimeric peracetylated glucose 34169 and glucosamine 33169 structures were effective gelators in many different solvents. The latter showed gelation properties in water, even at low concentrations (0.2−1.0 wt %). Methyl 4,6-O-benzylidene monosaccharide derivatives are commonly used to create glyco-based gels (Figure 43). These structures are readily accessible and can be further functionalized.170 Many compounds have been synthesized with modifications on the benzylidine ring (35, 37, and 38)171 and on the sugar backbone (36).172 Some derivatives act as “supergelators” that gelate solvent even at very low concentrations (0.03−0.05 wt %).173 Porphyrin rings are other gel-forming building blocks. Substitution of the porphyrin ring with four galactopyranosides gave rise to one-dimensional aggregates.174 These monodimensional fibers were stabilized by porphyrin ring stacking and sugar hydrogen bonding on the exterior of the fiber (Figure 44). The compounds form right- or left-handed helices

Many gelators, bearing sugars substituted in different positions with aromatic groups, have been reported. Usually, these small molecules are capable of trapping various solvents into a 3D-network (low molecular mass gelators, LMMGs). Because the supramolecular structure is based on weak π−π and hydrogen-bonding interactions, network aggregation and disruption of responsive systems is fairly easy. An overview of small aromatic-based glycoclusters is presented. 4-(4′-Butoxyphenyl)phenyl glucoside 25 is a gelator able to immobilize large quantities of solvents ranging from water to organic solvents (Figure 42).162 Depending on the solvent, different morphologies are obtained: planar ribbons form in water, helical fibers in toluene, and sheets in chloroform. Substituted glucosamines act as gelator and form a biocompatible hydrogel that is used for wound healing.163 Two diastereoisomers that differ in the aromatic unit (26 and 27) yielded similar gel morphologies. Aromatic groups can be exploited to ordain the gel with fluorescent properties that can in turn be used to study the aggregation process. Different fluorescent LMMGs based on glucose and naphthalene linked with a spacer formed organogel or hydrogels depending on the spacer length.164 A notable increase in fluorescence was observed upon gel formation. Chiroptical properties were also observed after aggregation. Fluorescence changes, upon sol−gel transition, were exploited for responsive systems such as a sugar-based hydrogel able to detect insulin.165 The gelator incorporated a pyrene with high fluorescence at 393 nm, and a saccharide part for insulin binding (28). Upon binding to insulin, the gel fiber pattern changed. By monitoring pyrene fluorescence quenching, insulin was detected at the microgram level. 1713


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glycodendrimers upon addition of FeCl3. The sugar coating was essential for the selective target of a specific strain of E. coli bacteria. After recognition of the selected receptor, these dendrimers delivered iron via a mannose−lectin-mediated process. Poly metallic systems enable the construction of highly multivalent structures, although their synthesis is often tedious. Self-organization by self-assembly of smaller monomers into polymetallic supramolecular architectures helps to overcome these problems. A collection of spherical poly-Pd(II) clusters coated with 24 saccharides was synthesized from small precursors (Figure 47).180 Each ligand, bearing one sugar, self-assembled upon addition of Pd(II), to form 4 to 8 nm sized M12L24 molecules. These saccharide-appended structures formed colloidal aggregates in the presence of lectins and may act as novel biosensors. Another multivalent poly metallic supramolecular architecture is based on a well-defined grid-shaped tetrazinc core bearing eight saccharides (Figure 48).181 A one-pot process brought together monovalent building blocks. The carbohydrates that decorate the grid bind ConA to form a precipitating polymeric network. Even at very low lectin concentrations phase separation by precipitation was achieved.

Figure 43. Gelators based on the methyl 4,6-O-benzylidene skeleton.

depending on the chirality of the sugar. Helical-silica structures are based on these templates.175 3.1.4. Metal-Mediated Self-Assembly. Besides combining hydrophobic and carbohydrate−carbohydrate interactions (see above), supramolecular glycoclusters can self-assemble around a metal. In the past decade, metal-based multivalent glycodendrimers have been explored.68,176 The metal can induce a rigid and defined geometry for the cluster. The probe can be detected by exploiting the inherent emission or redox activity of certain metals. A one-pot supramolecular synthesis of a series of fluorescent glycodendrimers was established.177 Hydroxyquinoline-functionalized dendrons 39 capped with Man, Glc, or Gal selfassemble around a variety of metals (Zn(II), Al(III), Gd(III), Figure 45). Quick access to a series of highly functionalized dendrimers showed that these assemblies are fluorescent and have high affinities and specificities for lectins. Cu(II) bipyridyl-glycoclusters 41 were prepared from dendrons covalently linked to four or eight TN-antigens (aGalNAc-OR), a carbohydrate-based cancer marker (Figure 46).178 These building blocks self-assembled upon addition of copper(II) sulfate to form complexes with square planar geometry. Solid-phase competition assays with asialoglycophorin, a glycoprotein found on human erythrocyte membranes, showed the inhibitory potential of the tetra-substituted complex toward horseradish peroxidase-labeled lectin Vicia villosa. Glyco-functionalized Fe(III) dendrimers 40 are biomimetic analogues of Fe(III) delivery (Figure 45).179 Mannose and galactose glycodendrons functionalized with catechol provide a binding site for Fe(III). Self-assembly formed Fe(III)

3.2. Aggregation upon Lectin Binding

In the previous section, we described the synthesis of multivalent glycoclusters by self-assembly. Here, we summarize the formation of supramolecular networks arising from the interaction of multiple glycoclusters with multiple lectins. Depending on the geometry and the core of the glycocluster or glycodendrimer, the formation of supramolecular aggregates by cross-linking various lectins is possible. 3.2.1. Organic-Based Structures. Organic-based glycoclusters are the best studied multivalent systems. Small organic cores,160 polymeric structures,182 cyclopeptides,183 porphyrins,184 and sugars185 have served as scaffolds for the construction of multivalent ligands. 3.2.1.1. Responsive Systems. Systems that change their photophysical properties upon aggregation are good substrates for the production of responsive systems. In this section, we describe glycoconjugate systems that form aggregates in the presence of lectins, reporting changes in their photophysical properties. Fluorescence is a quick and facile method to monitor binding. A phosphole oxide 42-based glycocluster186 showed no emission in solution, and was intensely fluorescent in the aggregate state, due to aggregation-induced emission (AIE) (Figure 49).187 The presence of ConA triggered α-mannopyranoside aggregation to turn the probe “on”. The system is

Figure 44. Porphyrin-based gel composed of monodimensional fibers. 1714


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Figure 45. Glycodendrimers based on hydroxyquinoline 39 and catechol 40 ligand.

Figure 48. Self-assembly of multivalent metallic-based glycoclusters around zinc cations (gray spheres). Reprinted with permission from ref 181. Copyright 2014 John Wiley and Sons.

Figure 46. Cu(II) glycocluster linked to four TN-antigens.

reversible, and the addition of excess mannan dissociates the aggregate and “turns off” fluorescence. Another responsive AIE system is based on mannose derivatives attached to a tetraphenylethane (TPE) core (43 and 44).188 TPE emission is “turned off” in solution and “turned on” in the presence of ConA. This system is specific for ConA and responds linearly to ConA concentration. In solution, the molecules have a certain mobility that causes nonradiative quenching. Aggregation by CPIs restricts intramolecular rotation and “turns on” fluorescence. A “turn on” system based on a porphyrin glycodendrimer (Figure 50) formed supramolecular aggregates in aqueous solution that quenched the emission of the porphyrin ring.189 ConA addition destroyed the fluorescent protein−porphyrin complex. Addition of mannose showed the reversibility of the system. A triazatruxene scaffold 45 responds to solvent polarity with changing fluorescence where higher polarity results in lower emission.190 Binding of ConA to the mannose-modified probe results in increased fluorescence. Alternatively, circular dichroism (CD) was used to study this interaction (Figure 51). Because of its secondary structure, ConA presents a distinct CD spectrum. Upon addition of the probe, the CD

Figure 47. Self-assembly of poly-Pd(II) glycoclusters M12L24. Reprinted with permission from ref 180. Copyright 2007 American Chemical Society.

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Figure 49. Schematic representation of AIE-based system and chemical structures of reported AIE-based probes.

Figure 50. Cartoon explaining the glycoporphyrin-based sensor system.

All probes listed above result in fluorescence “turn on” or “off” but do not allow for ratiometric analysis such that the concentration of the probe has to be known to obtain quantitative information. This problem is common for fluorescent probes used for in vivo analysis. Monitoring lectin intrinsic fluorescence lifetime, which is concentration independent, as a function of dendrimer concentration is not affected by lectin precipitation.195 Thereby, the inhibition efficiency of a series of glycodendrimers is used to characterize the process of glycodendrimer-mediated aggregation.

intensity decreased and its maximum shifts, as formation of a ConA−probe complex modifies the protein’s secondary structure. The responsive systems based on structures 46 and 47 serve as representative examples for perylene-based probes (Figure 52).191 Those generally highly fluorescent sugar-functionalized perylene bisimide derivatives are “turned off”192 by lectin binding. Nonemissive derivatives that are “turned on” upon lectin binding193 or that affect the ConA CD signal194 have been reported as well. 1716


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Figure 51. CD investigation of the binding of compound 45 to ConA. Upon addition of 45, the ConA CD spectrum intensity decreased. Reprinted with permission from ref 190. Copyright 2013 John Wiley and Sons.

Figure 52. Perylene-based sensors that report fluorescence “turn off” upon lectin binding.

Figure 53. General principle of bacteria sensing and antiadhesion therapy.

3.2.1.2. Sensing Bacteria. Pathogenic bacteria express high levels of carbohydrate binding lectins that recognize and bind host cells. Multivalent glycoclusters are interesting substrates for the inhibition of bacterial adhesion (Figure 53). To promote bacterial agglutination and inhibit infections, the cross-linking ability of some dendrimers was determined.196 The formation of supramolecular complexes between dendrimer and multiple lectins, on one or more bacteria, inhibits bacterial infection.197 We summarize recent achievements with respect to the formation of supramolecular aggregates in a vibrant field. A tetramannoside glycocluster based on tetrapropargyl pentaerythritol can cross-link ConA (Figure 54).198 Because of its geometry, this cluster was 1000 times more potent than mannose and inhibited binding of E. coli to erythrocytes in vitro.

A dendronized polymer based on a polylysine scaffold prepared by flow chemistry is a flexible platform for bacterial detection (Figure 55).199 The mannose derivative clusters E. coli by binding lectins on the bacteria surface as determined by fluorescence microscopy and atomic force microscopy (AFM). New cyclopeptide-based glycodendrimers with different geometry and flexibility were synthesized on the basis of a hexadecavalent scaffold.200 These dendrimers bound with high affinity to the lectin LecB from Pseudomonas aeruginosa. Molecular modeling suggested aggregative chelate binding, and ITC confirmed that each compound binds up to six lectin monomers. 3.2.1.3. Microarrays. A quick way to evaluate the effect of multivalency is offered by the use of microarrays. Glycodendrimers with different valencies can be immobilized directly 1717


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Figure 54. Glycocluster with cross-link ability toward E. coli (left). Model showing the cross-linking ability of the glycocluster. Reprinted with permission from ref 198. Copyright 2007 John Wiley and Sons.

Figure 55. Dendronized polymer for bacteria sensing.

fluorescent lectin.203 Thus, problems associated with analyte diffusion were avoided, and real time measurements were possible as the fluorescent protein is readily removed from the plate. This technology was optimized for the study of different

onto an array surface, and different lectins can be screened simultaneously.201,202 A porous aluminum oxide chip was used to immobilize different dendrimeric structures and pump a solution of 1718


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Figure 56. Microarray for the direct and quick comparison of different glycodendrimeric structures. Reprinted with permission from ref 204. Copyright 2010 John Wiley and Sons.

Figure 57. Ru(II)-based glycocluster for ConA detection based on PET (A) and its application on microarrays (B). Adapted with permission from ref 208. Copyright 2010 American Chemical Society.

These dendrimers were printed on microarrays, and their interaction with lectins was monitored with fluorescence spectroscopy and electrochemical analysis (Figure 57B).208 ConA was immobilized on a microarray prior to incubation with the dendrimers. Fluorescence analysis showed the binding only for the mannosylated complexes because ConA does not bind galactose. ConA immobilization on a gold surface, before incubation with the dendrimers, permitted electrochemical monitoring of CPIs electrochemically, due to the redox activity of the Ru(II) complexes. A concentration-dependent decreased current was observed upon incubation with different sugars. Displacement of Ru(II) dendrimers from the gold chip by sugars with higher affinities was observed. This platform allows for easy and cheap analysis of many complex sugars in parallel. Gd(III)-based glycodendrimers were studied as MRI contrast agents. A series of DOTA (1,4,7,10-tetraazacyclododecane1,4,7,10-tetraacetic acid)-based glycodendrimers exhibit an enhanced relaxivity upon binding to lectins.209 Lectin binding prolonged tumbling time of the complex lectin-dendrimer, resulting in an enhanced relaxivity and consequently high contrast. These Gd(III)-based glycodendrimers were later used in vivo.210

dendrimers, bearing various sugars such as Man, Glc, GlcNAc, Gal, and Galα1,4Gal (Figure 56).204 The flow-through microarray proved a quick and simple screening method to gain information on CPIs. Other glycodendrimer microarrays were prepared by attaching dendrimers to a gold surface via click chemistry to study the interactions with various lectins with XPS, cyclic voltammetry, and contact angle goniometry.205 3.2.2. Metal-Based Structures. In this section, we summarize work on metal dendrimers that, upon interaction with lectins, form supramolecular networks. A series of saccharide-capped dendrimers based on the Ru(II) tris(bipyridine) core have intense emission from the MLCT (metal to ligand charge transfer) excited state.206,207 Upon addition of the boronic acid of the methyl viologel dication (BBV), a dye that shows high affinity for the glycosylated dendrimers, quenching of the Ru(II) emission was observed, due to PET (photoinduced electron transfer). The addition of a lectin with high affinity for the sugars reestablished the Ru(II) fluorescence by displacing the BBV from the complex (Figure 57A). By monitoring PET quenching disruption, the interaction between lectins and the glycodendrimers presenting different branched structures was quantified. 1719


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Primaquine phosphate (PP) was encapsulated into a glycodendrimer, coated with galactose.213 The covalent nature of the dendrimer rendered this system more stable than other self-assembled vesicles. The saccharides enhance the solubility and direct the dendrimer−drug complex to the liver. Liver tissue presents high levels of the asialo-glycoprotein (ASGP) receptor that binds galactose. Drug release is prolonged up to 6 days more efficient and specific due to receptor targeting.

An electroactive poly(amidoamine) (PAMAM)-based dendrimer was based on a central PAMAM-polymer that was decorated with mannose-functionalized ferrocenyl groups (Figure 58).211 The presence of multiple ferrocenes amplified

3.4. Glycodendrimers as Stabilizing Agent

3.3. Glycodendrimers as Host

Glycodendrimers can act as glue for nanoparticle stabilization as the carbohydrate shell controls the stability and size of the nanoparticle. This area holds much potential; the use of sugarbased compounds to stabilize heterogeneous catalysts requires much work beyond the beginnings summarized here. The hyperbranched polyethylenimine (PEI) core was functionalized with various carbohydrates and used to support metal nanoparticles in water.214 Pt(IV) was anchored to the support prior to reduction with NaHB4 in water. The platinum nanoparticles, stabilized with the glycodendrimers, are effective in the selective hydrogenation of isophorone in water. Water-soluble glycodendrimers containing nine xylose branches stabilize Pd, Pt, and Au nanoparticles (Figure 60A). All glycodendrimer-stabilized platinum nanoparticles showed good activity catalyzing olefin hydrogenation albeit without stereoselectivity.215 The palladium analogues catalyzed the reduction of nitro to amine groups (in the presence of NaBH4) and the Suzuki−Miyaura cross-coupling.216 The nitro group reduction was also possible in the presence of gold nanoparticles.217 A similar system showed faster rates of reduction (Figure 60B).218 Quantum dots can be stabilized with glycodendrimers.219 The addition of maltose-modified dendrimers rendered QDs water-soluble and biocompatible while enhancing stability.

Glycodendrimers present a hydrophilic outside shell that protects the core. Depending on dendrimer shape and size, the interior may encapsulate guest molecules (Figure 59). The guest becomes more water-soluble and stable toward quenching in case of organic dyes. The core microenvironment can favorably affect catalysis reactions. Glucose-substituted poly(amidoamino) (PAMAM) glycodendrimers can encapsulate guest molecules and behave like unimolecular micelles in water.212 Lipophilic compounds, such as pyrene or aromatic ketones, dissolve in aqueous solution thanks to encapsulation in dendrimer microcavities. The concentration of solubilized compound correlates with dendrimer concentration. The carbohydrate-based shell creates a chiral interface that can be exploited for asymmetric synthesis. The asymmetric reduction of prochiral ketones with sodium borohydride in water yielded the product in 50% ee.

4. GLYCOSYLATED NANOMATERIALS Nanomaterials, materials with a size between 1 and 100 nm in at least one dimension, have been frequently glycosylated. Glycosylated nanomaterials can assemble into supramolecular networks for biosensing CPI and CCI, interaction studies, and biomedical applications. Gold and silver nanoparticles (NPs) have been used commonly due to their size-dependent optical properties that arise from the collective oscillation of conduction electrons.220 A tunable plasmon band (absorption) results from this oscillation. A direct synthesis of gold NPs using a biphasic water−toluene reduction of a gold salt by sodium borohydride in the presence of an alkanethiol as a stabilizing agent221 was later adapted to the synthesis of silver NPs.222,223 This method was used to prepare glycosylated NPs using thiol-functionalized

Figure 58. Polyferrocenyl glycocluster and its aggregation with ConA. The formation of the cross-linked network caused a decrease in the current peak. Reprinted with permission from ref 211. Copyright 2013 American Chemical Society.

the electrochemical signal into a single voltammetric wave. ITC and differential pulse voltammetry (DPV) indicates the formation of a cross-linked network upon ConA addition. The peak current decreased with increasing ConA concentration such that this system may serve as a multielectron redox probe for ConA detection.

Figure 59. Dendrimer as host. 1720


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Figure 60. (A) First generation of dendrimer-stabilized nanoparticles for catalysis. Reprinted with permission from ref 215. Copyright 2011 Elsevier. (B) Improved ligand showing faster rate of reduction. Reprinted with permission from ref 218. Copyright 2015 Royal Society of Chemistry.

ymethyldextran and polylysine were used to study carbohydrate−protein interactions.229 We describe carbon-based nanomaterials that exhibit unique electrical, chemical, thermal, and mechanical properties and focus on the use of glycosylated graphene and carbon nanotubes as scaffolds for supramolecular structures. Many supramolecular systems based on nanomaterials have been described. Glyconanomaterials are a suitable platform to detect lectins or study CCIs and CPIs. Here, we provide an overview of some systems as well as applications as biosensors or as tools for CCIs and CPIs studies and as biomedical nanocarriers. Only a few of these materials have been applied to biomedicine, and much work remains before glycosylated nanomaterials can become scaffolds for in vivo assays.

glycoconjugates and improved to prepare glycoNPs in one phase.224 Alternatively, a place-exchange reaction of the stabilizing group of synthetic metal NPs yielded the desired product.225 Other nanomaterials are based on quantum dots (QDs). QDs are luminescent semiconducting nanomaterials. Typically, QDs are made of binary compounds such as selenides or sulfides of metals such as cadmium or zinc. QDs can emit in the entire spectrum, and their optical properties are tunable depending on the size.226 As compared to organic dyes, QDs have a broader excitation spectrum and sharper emission bands, allowing for multicomponent analysis with a single excitation source (Figure 61).227 QDs are usually prepared at high temperature in the presence of thiol derivatives containing hydrophilic groups that improve the QDs stability and solubility.228 The first glyco-QDs functionalized with carbox-

4.1. Glyconanoparticles for Biosensing

GlycoNPs have been used extensively for biosensing due to their capability to form supramolecular networks that report a measurable signal.230 Different glycosylated nanomaterials that create supramolecular structures by interacting with lectins, through CPIs, or with other carbohydrates, through CCIs, have been reported. Gold NPs are useful biosensors for their aggregationdependent optical properties.231 Small gold NPs in solution are ruby red because of the strong absorption of light at around 520 nm. Upon aggregation, gold NPs come in close proximity, resulting in absorption of red wavelength light causing a change in color (Figure 62).232 This property has been exploited extensively in biosensing as well as CCI and CPI studies. 4.1.1. Biosensors in Solution. CCIs have been studied using gold NPs functionalized with two different glycoconjugates: thiol-derivatized disaccharide lactose (Galβ(1 → 4)Glcβ1-OR) and trisaccharide LeX (Galβ(1 → 4)[Fucα(1 → 3)]GlcNAcβ1-OR) (Figure 63).224 Upon addition of calcium cations, transmission electron microscope (TEM) images revealed that the Lex-functionalized glycoNPs aggregated, whereas the lactose-functionalized NPs remained well dispersed. Self-recognition of LeX molecules in the presence of

Figure 61. Absorbance and emission profiles of QDs with different sizes. Typically, QDs show a broad excitation band and a narrow and tunable emission band that shifts to longer wavelengths as a function of the QD size. 1721


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Figure 62. (A) General scheme of glyconanoparticles forming a supramolecular network upon lectin addition. (B) Absorbance spectra of welldispersed (red) and aggregates (blue) glyconanoparticles.

Figure 63. Lactose and LeX structures for the synthesis of glyconanoparticles for interaction studies between the two carbohydrates. Reprinted with permission from ref 224. Copyright 2001 John Wiley and Sons.

wavelengths. This aggregation was a result of each lectin binding four NPs and each nanoparticle binding more than one lectin. The absorbance of the shifted band increased upon an increase in ConA concentration. Gold and silver glycoNPs functionalized with mannose and their interaction with ConA lectin were compared.235 The silver glycoNPs responded faster than the gold analogues and presented a longer linear dynamic range. Nevertheless, the gold glycoNPs were more sensitive. To explore the selectivity of the bioassay, lactose-stabilized gold NPs and mannosestabilized silver NPs were mixed and tested with both ConA and RCA120, showing that each ligand is specific for the respective lectin. Gold NPs functionalized with lactose serve to detect cholera toxin that binds the ganglioside GM1 of mucosal cells.236 With increasing amounts of cholera toxin, the surface plasmon band shifts, and the color changes from red to deep purple.

calcium ions and specific interactions were responsible for this observation. Addition of EDTA to the solution containing aggregates resulted in the redispersion of the glycoNPs. PEGylated gold NPs functionalized with lactose and mannose in different ratios (5:5 and 2:8) were prepared.233 On incubation of the glycoNPs with R. communis agglutinin (RCA120), a lectin that binds specifically to galactose, the NPs containing a higher ratio of lactose aggregated (Figure 64). The lectin−nanoparticle network resulted in a concentrationdependent red-shift and in broadening of the plasmon band. Addition of galactose completely redispersed the NPs even after several repeated cycles. Mannose-functionalized gold NPs were used to study CPIs in the presence of ConA.234 The NPs were functionalized with a short hydrocarbon anchored mannose via a place-exchange reaction. Incubation of the glycoNPs with ConA resulted in a broadening and shift of the plasmon band to longer 1722


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Figure 64. Lactose-modified glycoNPs aggregate in the presence of RCA120 and redisperse upon addition of galactose. Reprinted with permission from ref 233. Copyright 2001 American Chemical Society.

Figure 65. Aggregation of glycoNPs in the presence of (A) ConA, and (B) RCA120 depending on the carbohydrate density on the nanoparticles. For the ConA the optimal density of mannose was 100%, whereas for RCA120 the optimal galactose coating was 70%. Reprinted with permission from ref 237. Copyright 2008 Royal Society of Chemistry.

Figure 66. Aggregation of mannose-modified glycoNPs in the presence of ConA (X) and redispersion by addition of other proteins (Y) that interact with ConA through protein−protein interactions. Reprinted with permission from ref 238. Copyright 2005 Royal Society of Chemistry.

The carbohydrate density on the gold NPs modifies the aggregation upon lectin binding.237 For these studies, galactose and mannose were used to detect RCA120 and ConA,

respectively. The optimal density of carbohydrates on gold NPs was 100% for mannose and 70% for galactose, possibly due to the structural difference of the lectins (Figure 65). NPs 1723


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with proper coating can be synthesized to optimize binding affinity. A competitive colorimetric assay based on mannosefunctionalized gold NPs was developed.238 The glycoNPs aggregated in the presence of ConA and were redispersed by addition of proteins that interacted with ConA through protein−protein interactions (Figure 66). Using thyroglobulin, BS-I, SBA, and MAL, the nanoparticle aggregates were redispersed. This process was visible via a color change from blue to purple and burgundy red. This method assesses protein−protein interactions through a qualitative assay. Gold NPs were functionalized by using perfluorophenyl azide (PFPA)-thiol followed by coupling with the carbohydrate to produce glycoNPs.239 Fluorescently labeled ConA and mannose as a free competing ligand were used to study the equilibria between ConA and glycoNPs as well as ConA with mannose. The mixture was centrifuged, and the fluorescence of nonbinding labeled-ConA was measured. Interactions of NPbound carbohydrates with lectins were significantly enhanced when compared to free ligands. The influence of nanoparticle size, spacer length, ligand size, and density on binding affinity was studied. Previously described NPs were used for interaction studies using ITC.240 By titrating the lectins with the glycoNPs, the dissociation constant, thermodynamic parameters, and number of binding sites can be obtained, depending on the heat release. A turn-off/turn-on biosensor for drug delivery was designed by functionalizing gold NPs with mannose and incubating with the boronic acid derivative of fluorescein.241 The fluorescence was quenched when the glycoNP complex was formed. Addition of ConA replaced the boronic acid derivative and re-established fluorescence. The glycocalyx was mimicked for CPIs studies in an artificial cell system by using multiple carbohydrates. To better understand the biological behavior, the mannose-functionalized NPs together with boronic acid were incubated with Jurkat human T lymphoma cells and analyzed by confocal microscopy (Figure 67). Boronic acid was released due to the presence of ConA on the cell surface that interacted with the mannosides. The hydrophobic dye was then internalized by the cell. A new method for the functionalization of gold NPs relied on cyclooctyne substituted with a lipid chain that was coupled via a copper-free click reaction to an azido galactoside.242 This amphiphilic glycolipid was embedded into the PEG on the nanoparticle surface through self-assembly. The glycoNPs aggregated with PNA lectin. CdSe-ZnS QDs were functionalized with three carbohydrates, melibiose (Gal-1-α-4-Glc), lactose (Gal-1-β-4-Glc), and maltotriose (Glc-1-α-4-Glc-1-α-4-Glc), for lectin biosensing.243 Binding of soybean agglutinin (SBA), a galactose binding lectin, was assessed using the glyco-QDs identifying melibiose QDs as the best binder. Fluorescent QDs were highly sensitive as nanomolar concentrations of lectins were detected. Dextran-functionalized glycosylated NPs based on Ag, Fe3O4, and ZnS-CdSe QDs coated with a silica layer were prepared by introducing a primary amine on the silica surface, followed by a coupling reaction with a succinimidyl active ester of dextran.244 Upon aggregation with ConA, a red-shift of the surface plasmon band was observed for silver NPs, whereas QDs and Fe3O4 NPs decreased in fluorescence and absorbance (Figure 68A,B). The Fe3O4 NPs were separated using a magnetic field (Figure 68C).

Figure 67. Turn off/turn on biosensor based on mannose-functionalized gold nanoparticles containing fluorescent boronic acid for ConA detection. Upon the addition of ConA, the boronic acid derivative is replaced, re-establishing its fluorescence. Reprinted with permission from ref 241. Copyright 2012 American Chemical Society.

NaGdF4:Er3+,Yb3+ QDs doped with lanthanide ions, through upconversion upon near-infrared (NIR) excitation, can emit UV, visible, and NIR light.245 The NPs were first coated with poly(amidoamine) (PAMAM) dendrimers and then functionalized with mannose. This system can detect tetramethylrhodamine (RITC) labeled ConA using luminescence resonance energy transfer (LRET). The donor (NPs) used the upconverted light to excite the RITC acceptor. This process resulted in a decrease of the donor luminescence and an increase in the acceptor fluorescence. In the presence of mannose, the LRET signal decreased, showing the reversibility of the system. QDs functionalized with mixtures of glucosamine or galactosamine can detect ConA and PNA simultaneously because different QDs can be excited at one wavelength and give a different fluorescent signal (Figure 68D,E).246 In the presence of lectins, the fluorescence signal decreased due to the formation of noncovalent cross-links between the glyco-QDs. At increasing concentrations of one lectin, the fluorescence signal of the corresponding QDs decreased, allowing the system to detect both lectins in the same solution. 4.1.2. Surface-Based Sensors. Surface immobilized glycans have been used extensively to study CPIs and CCIs. SPR was used to analyze CCIs247 by employing gold nanoparticles carrying different glycans224 (Figure 69). The sensors were functionalized with a self-assembled monolayer of LeX and lactose with a thiol linker. The same groups were present on the NPs. LeX NPs bind to LeX monolayers in the presence of calcium ions slowly, and the dissociation phase is moderate. Multivalency is important as the free ligand binds very weakly. CdS QDs functionalized with carbohydrates interact with immobilized lectins on a gold surface for CPI studies (Figure 70).248 The voltammetric response was measured for different carbohydrates that interact with PNA. The cadmium stripping peak decreased for GalNAc, Gal, and the disaccharide β-D-Gal1724


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Figure 68. Lectin detection using different materials: (A) silver, (B) magnetite, and (C) QDs functionalized with dextran. Reprinted with permission from ref 244. Copyright 2008 American Chemical Society. Detection of (D) ConA or (E) PNA in a mixture using different QDs functionalized with carbohydrates by detecting changes in absorbance. Reprinted with permission from ref 246. Copyright 2013 American Chemical Society.

Figure 69. SPR measurements of LeX-functionalized gold NPs on a LeX-functionalized surface for CCIs studies. Reprinted with permission from ref 247. Copyright 2003 John Wiley and Sons.

Figure 70. Voltammetric measurements using QDs functionalized with galactoses that interact with immobilized PNA for CPIs studies. Reprinted with permission from ref 248. Copyright 2006 American Chemical Society.

[1 → 3]-D-GalNAc when the carbohydrate−lectin affinity increased. A chip-based system to detect protein glycosylation was based on measuring the energy transfer between ConAfunctionalized QDs and dextran-modified gold NPs (Figure 71A).249 The QDs were immobilized on an amine-reactive glass, permitting irradiation from the back of the glass to induce photoluminescence of the lectin-QDs. When the gold glycoNPs interact with the lectin, the luminescence was quenched. When glycoproteins were added to the solution, the carbohydrates on the glycoproteins competed with glycoNPs and re-established a FRET signal. Gold-coated iron oxide NPs were used to measure interactions of proteins glycan arrays (Figure 71B).250 Different

glycans were immobilized onto an amine-reactive glass surface, and the NPs were functionalized with ConA and Antihuman/ Mouse SSEA-3. Silver was deposited onto the array to amplify the signal after a magnetic field was applied, showing gray spots that can be imaged with a flatbed scanner. The antibody specifically bound its antigen. ConA bound glycans better the more mannoses they contained. Gold NPs functionalized with glycopolymers were synthesized by the reversible addition−fragmentation chain transfer (RAFT) process and contained biotin and either GlcNAc or Man and were used to sense proteins in solution and on surfaces (Figure 72).251 Diffraction optics technology (DOT) was used to determine the binding affinities. The surfaces were modified with avidin that binds the biotin on the glycoNPs. 1725


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Figure 71. (A) Energy transfer measurement between immobilized QDs modified with ConA with dextran AuNPs. When glycoNPs were added, the fluorescence was quenched, and also when glycoproteins were added the energy transfer was re-established. Reprinted with permission from ref 249. Copyright 2009 Elsevier. (B) CPIs studies using gold-coated iron oxide NPs functionalized with ConA on a glycan array. Silver was deposited on the surface for signal enhancement. Reprinted with permission from ref 250. Copyright 2009 American Chemical Society.

Detection of WGA with GlcNAc and ConA with Man was demonstrated. To study CPIs using localized surface plasmon resonance (LSPR), gold island transducers on glass substrates were functionalized with ConA, and gold NPs were modified with mannose (Figure 73).252 The interaction between ConA and mannose created an enhanced LSPR signal due to a change in refractive index. The association and dissociation rate constants for the interactions were determined under flow conditions.

Figure 72. CPIs studies using biotinylated glycopolymers-functionalized AuNPs using DOT to obtain the binding affinities between WGA and GlcNAc and between ConA and Man. Reprinted with permission from ref 251. Copyright 2010 American Chemical Society. Figure 73. CPI studies between Man-modified AuNPs and ConAfunctionalized gold islands using LSPR. Reprinted with permission from ref 252. Copyright 2012 American Chemical Society.

Exposure of the surfaces to coherent light produced diffractive images upon increasing height of the diffraction pattern. 1726


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Figure 74. Sialic acid detection using an electrochemical biosensor based on an immobilized monolayer of gold NPs modified with SNA I. Reprinted with permission from ref 254. Copyright 2013 Elsevier.

Figure 75. CPIs studies using dextran-functionalized AuNPs and ConA deposited onto a SPR chip modified with graphene. Reprinted with permission from ref 255. Copyright 2013 Elsevier.

4.2. Glyconanoparticles for Bacteria Sensing

High selectivity for protein−sugar recognition was obtained, even in the presence of other proteins. Silica NPs encapsulating a fluorescent dye were used to determine Gal and 3-sulfogalactose (SGal) interactions.253 Glycolipids were immobilized on a multiwell plate, and NPs functionalized with sugars were added. Fluorescence was observed only for the NPs containing SGal when galactolipids were added. TEM confirmed the formation of Gal- and SGalfunctionalized NP aggregates. An electrochemical biosensor was based on an immobilized monolayer of gold NPs functionalized with Sambucus nigra agglutinin (SNA I), which recognized 2,6-linked sialic acids (Figure 74).254 The change in charge transfer resistance was studied using electrochemical impedance spectroscopy (EIS). This biosensor detected glycoproteins containing sialic acid such as fetuin and asialofetiun down to attomolar levels for early disease diagnoses. A graphene oxide-based system to study CPIs using gold glycoNPs was created by depositing graphene oxide onto a SPR gold film followed by addition of phenoxy-derivatized dextran via π−π interactions.255 The dextran captured ConA on the surface before dextran-modified gold NPs bound to ConA (Figure 75). This system is more sensitive than a direct assay when quantitating ConA due to the increased adsorption of the dextran on graphene.

The first gold glycoNPs for bacterial detection employed mannose-functionalized gold NPs that were incubated with two different E. coli strains, ORN178 and ORN208.256 The ORN178 strain expresses type 1 pili with mannose-specific f imH, whereas the ORN208 strain does not contain f imH gene and does not bind mannose. TEM revealed that mannosefunctionalized gold NPs bound selectively only to the ORN178 strain (Figure 76). Excess mannose did not interfere significantly with the binding of the glycoNPs to the bacterial pili. On the basis of this principle, an improved method to capture and detect E. coli was developed.257 Magnetite NPs coated with a silica layer were synthesized and functionalized with mannose. Bacteria were captured in a magnetic field with 88% efficiency. Dextran-coated gold NPs that bind ConA served to sense the metabolic state of bacteria.258 Bacteria proliferate without antibiotic or at very low (noninhibitory) antibiotic concentrations and reduce the carbohydrate levels in the medium. The carbohydrates present on the surface of growing bacteria bind to ConA and assemble glycoNPs (Figure 77). In the presence of an effective antibiotic, carbohydrate uptake was inhibited and ConA bound mostly to the glycoNPs, thus producing a red shift in the plasmon band. Thereby the success of antibiotic effectivity was tested. 1727


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layer of glycoconjugates that mimic the glycocalyx and are a useful tool for biomedicine. Gold glycoNPs have been employed for antiadhesive therapy.260,261 GlycoNPs were designed to interfere with several glycosphingolipids and glycoproteins involved in the initial step of metastasis. Because melanoma cells express the lactosylceramide antigen during the cell adhesion process, lactose-functionalized gold NPs were exploited to inhibit melanoma cell binding to the endothelium (Figure 78). For the ex vivo experiment, mice were treated with melanoma cells containing lactose-functionalized NPs. After the metastatic process, analysis of mice lungs revealed 70% tumor inhibition as compared to the controls. Silica mesoporous supports (SMPS) coated with lactose were selectively recognized by the enzyme galactosidase.262 This enzyme, present on the brush border membrane of the simple columnar epithelium of the small intestine, hydrolyzes the lactose disaccharide to glucose and galactose. SMPS was used as a drug container (pore diameter: 2.4 nm) for controlled drug delivery (Figure 79). Loading with [Ru(bipy)3]Cl2 showed that upon addition of the enzyme, the guest was released by uncapping.

Figure 76. Detection of bacteria using Man-modified AuNps using (A) ORN178 and (B) ORN208 strains with TEM. Reprinted with permission from ref 256. Copyright 2002 American Chemical Society.

Figure 77. Dextran-modified AuNPs detect the metabolic state of bacteria by measuring the carbohydrate levels in the medium using ConA. Reprinted with permission from ref 258. Copyright 2008 American Chemical Society.

Figure 79. Silica mesoporous supports coated with lactose for enzymebased drug delivery. The drug is released upon the hydrolysis of lactose. Reprinted with permission from ref 262. Copyright 2009 John Wiley and Sons.

Mannose-functionalized CdS QDs were prepared in one pot and used for the detection of the E. coli strains ORN178 and ORN208.259 Luminescent aggregates were observed for the ORN178 strain only.

Capping of the silica container was tested with hydrolyzed starch derivatives (Glucidex 47, Glucidex 39, and Glucidex 29), where the degree of starch hydrolysis degree is presented as dextrose equivalent.263 Pancreatin hydrolyzes the 1 → 4 glucosidic bond in starch. Again, cargo is released in the presence of the enzyme. Doxorubicin was used as a cargo in the

4.3. Glyconanoparticles in Biomedicine

The glycocalyx covers the surface of mammalian cells with carbohydrates. GlycoNPs are multivalent tools that contain a

Figure 78. Antiadhesion therapy using gold glycoNPs during the initial steps of metastasis of melanoma. Reprinted with permission from ref 256. Copyright 2004 Springer. 1728


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Figure 80. (A) ConA detection using maltose-modified graphene detected through a fluorescence switch. Reprinted with permission from ref 269. Copyright 2011 Elsevier. (B) Mannose-modified FBT structure and ConA detection using FBT modified graphene. The lectin is detected through the fluorescence activation of FBT when ConA was added. Reprinted with permission from ref 270. Copyright 2011 John Wiley and Sons.

Figure 81. Supramolecular network formed via self-assembly of glyco-graphene through hydrogen bonds. Reprinted with permission from ref 271. Copyright 2012 American Chemical Society.

carbon nanotubes.264 The monocrystalline graphitic films265

glucidex 47 system, showing cell internalization and pore opening upon enzyme hydrolysis, leading to cell death.

have been functionalized and used for a host of biomedical

4.4. Carbon-Based Nanostructures

applications.266−268 Graphene oxide is soluble in water and

4.4.1. Graphene. Graphene is a two-dimensional carbon allotrope and the basic structure of fullerenes, graphite, and

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Figure 82. Hydrogel formation based on chitosan-modified graphene. At high concentration of graphene the hydrogel was formed at room temperature, whereas at low concentrations high temperature was required. Reprinted with permission from ref 272. Copyright 2014 American Chemical Society.

due to hydrogen bonds, was created (Figure 82). Heating increases the free motion of the chains and allows for more interactions between the two components. A drug delivery nanocarrier based on graphene coated with carbohydrates to improve colloidal stability and functionalized with folate was developed.273 Folate was chosen to target cancer cells overexpressing folate receptors. Several hydrophobic and hydrophilic drugs were loaded by adsorption in the aromatic plain of the graphene. In HeLa cells, the system entered the cytoplasm, and cell destruction was more efficient when compared to the free drugs in similar dose. 4.4.2. Carbon Nanotubes. Carbon nanotubes (CNTs) are carbon allotropes with a cylindrical shape. Two different types of CNTs, single-walled CNTs (SWCNTs) and multiwalled CNTs (MWCNTs), are distinguished. CNTs are frequently functionalized via a 1,3-dipolar cycloaddition of azomethine ylides.274,275 Carbohydrate-functionalized CNTs enhance solubility, biocompatibility, and bioactivity.268,274,276 The solubility of SWCNTs upon supramolecular encapsulation of the SWCNT with helical amylose was tested.277 Two analogues of amylose, pullulan and carboxymethyl amylose, did not improve solubility. Cut SWCNTs (1−2 μm long) and as-grown SWCNTs (sometimes pieces of as-grown CNTs) were functionalized with schizophyllan and Curdlan.278 The former is a natural polysaccharide containing three β-(1 → 3) glucoses and one β(1 → 6) glucose side-chain linked at every third main-chain, whereas the latter is a single polysaccharide consisting of β-1 → 3 glucan without side-chains. Both polysaccharides wrap the SWCNTs by formation of a helix pattern, as shown by TEM and AFM. Galactose-functionalized SWCNTs were more water-soluble and used for E. coli capturing by cell agglutination.279 CNTs functionalized with glycopolymers composed of αGalNAc containing a poly(methyl vinylketone) [poly(MVK)] backbone mimic cell surface mucin glycoproteins (Figure 83).280 The α-GalNAc was recognized by the lectin Helix pomatia agglutinin (HPA). Cell surface interactions were promoted in two ways: either functionalized CNTs bind to

A biosensor based on maltose-grafted-aminopyrene (MalApy) was self-assembled on graphene via π-stacking interactions (Figure 80A).269 Because of FRET, when Mal-Apy was on the graphene, the pyrene fluorescence was quenched. In the presence of ConA, the π−π interactions were destroyed because of the competitive binding of ConA and glucose, and reactivation of the pyrene fluorescence was observed. Detection of ConA relied on a selective fluorescence switch. In addition, the π-stacking interactions improved the water solubility and graphene stability. Another graphene oxide-based bioassay utilizing a conjugated oligomer, 4,7-bis(9,9-bis(2-(2-(2-(2,3,4,5,6-pentahydroxyhexanal)-ethoxy)ethyl)fluorenyl)benzothiadiazole) (FBT).270 FBT was substituted with mannose residues to increase the water solubility of the system and detect ConA as well as E. coli. FBT fluorescence was quenched by graphene oxide due to the strong π−π interactions between FBT and graphene. Upon addition of ConA, the π−π interactions decreased leading to a reactivation of FBT fluorescence (Figure 80B). Moreover, the proximity of the chromophore to ConA resulted in a more hydrophobic environment, causing a fluorescence blue shift. This method permitted the quantification of ConA to a subnanomolar detection limit. A three-dimensional graphene oxide-based gel network resulted from the self-assembly of amphiphilic molecules containing lactopyranosyl and cellopyranosyl head-groups attached to a hydrophobic pyrene (Figure 81).271 With cellopyranosyl the mechanical properties of the gels decreased. Dye-loading properties were explored using basic fuchsine as model dye. This system holds promise for catalysis or biosensor applications. Supramolecular hydrogels based on chitosan and graphene oxide were prepared by self-assembly through electrostatic interactions.272 The graphene worked as a two-dimensional cross-linker thanks to multifunctional groups on both sides. High concentration of graphene oxide forms a hydrogel at room temperature, whereas at low graphene concentrations the hydrogel was formed only at 95 °C. A graphene−chitosan complex, where the chitosan chains are in a compressed state 1730


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physical studies that investigate structure-aggregation behaviors were reviewed,283−285 we focus on a few illustrative examples. 5.1.1. Cell Membrane-Like Structure. Liposomes and micelles spontaneously aggregate from amphiphilic structures, generally lipophilic chains with a polar head, and readily incorporate glycolipids (Figure 84). Depending on the size and the chemical composition of the alkyl chain, aggregates with different shapes including lamellar, columnar, and spherical structures are obtained (Figure 85).286 Biocompatible liposomes and micelles have been explored as conveniently prepared multivalent systems that incorporate multiple monomers.287 We summarize some key elements of glycoliposomes and micelles for the study of CPIs and CCIs as the area has been reviewed.282 Glycolipids connected through a phosphate linker (Figure 86) are probably the most common building blocks for incorporation into liposomes.288−290 Most glycolipids contain an alkyl or PEG chain linker that connects the sugar with the sterically stabilizing lipid.291 The phosphate group can link one or two alkyl chains. Mono-, di-, and polysaccharides have been incorporated. Because of its important role in cell recognition, sialyl Lewis × derivatives have been incorporated frequently to mimic the cell membrane.288,292 Amides293,294 and aromatic structures295 used for conjugation provide additional stabilizing interactions to the aggregates and influence the overall geometry. Aggregate geometry is also influenced by the degree of unsaturation of the alkyl chain296 and the bulkiness of the sugar.297 To achieve high affinity for the desired protein, the sugar density on the aggregate surface has to be taken into account.293 5.1.2. Self-Assembling Structures. With an aliphatic chain attached to a polar head, glycopolymers are good building blocks for the formation of supramolecular architectures. Lipid nanotubes, nanofibers, gels, and liquid crystals have been studied extensively the over the past 20 years.298 The first anisotropic chiral glycolipid aggregates (aldonamide) were described in 1985,299 and the quadruple helix structure composed of cylindrical micelles was elucidated (Figure 87).300 Modifications of the sugar component impacted stabilization and solubility of the supramolecular structure. A variety of glycolipids have been used to form nanotubes, and differences in aggregation patterns depend on the building block structure.286 Unsaturation in different positions of the alkyl chain, variation in the linking point of the two components, and different sugar moieties all affected the supramolecular network. A systematic analysis of glycolipid assembly, differing in the position and number of unsaturation, showed that many different structures such as twisted nanofibers, helical ribbon, and nanotubular structures depending on double bond unsaturation can be formed (Figure 88).301 Glycolipid selfassembly is controlled by a combination of hydrogen-bonding,

Figure 83. (a) Cell surface mimic based on glyco CNTs. (b) Possible pathways promoting interaction with HPA. Reprinted with permission from ref 280. Copyright 2006 American Chemical Society.

the lectin and afterward to the cell surface glycoconjugates, or HPA binds first to the cell surface glycoconjugates and then the free lectin binding sites on the CNTs. Functionalized CNTs were less toxic than uncoated CNTs, thus rendering these nanomaterials interesting for biological studies. Pyrene-functionalized glycodendrimers adsorbed onto SWCNTs through π−π interactions and improved the solubility while reducing the cytotoxicity.281 The system accommodates different carbohydrates for a broad range of bioassays.

5. POLYMERS Polymeric structures offer a useful platform for the synthesis of multivalent glyco-systems. By combining mono- and polysaccharides with other biomolecules such as lipids and peptides, systems that mimic nature evolve. In this section, we present supramolecular systems that form upon interaction of polymers. The systems described in this section adopt interesting architectures, but display sugars randomly. Current efforts are directed at understanding the ideal sugar density and distribution. Fundamental insights in this area are required to advance this field. 5.1. Glycolipids

Glycolipids contain a sugar moiety substituted with a long alkyl chain. Because of their amphiphilic nature, glycolipids often self-assemble into supramolecular structures. The stabilization of the supramolecular structure is due to CCIs combined with hydrophobic interactions of the lipid component. Natural incorporation of glycolipids has been applied in many ways.282 Typically, the saccharide portion is pointing out of the membrane to target-specific proteins. Because glycolipids and

Figure 84. Formation of bilayers or spheres. 1731


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Figure 85. Membrane-like self-assembling structures.

Gemini surfactant-based aggregates were used for DNA transfection (Figure 90).308 Upon gradual acidification a lamellar phase, a condensed lamellar phase, and an inverted hexagonal columnar phase were traversed. Acidities comparable with the endosomal pH range corresponded to an inverted hexagonal columnar phase. DNA can be incorporated into the lamellar phase and carried into the cell by endocytosis. Morphology changes at endosomal pH destabilize the endosome and release DNA into the cytoplasm.

Figure 86. General glycolipid structure based on phosphate linker.

π−π stacking, and hydrophobic forces.302 In addition, nanofiber association and network formation induced efficient gelation of organic solvents.303 In addition to gelation of organic solvents, the introduction of unsaturation in the aliphatic chain affects gelation properties in water.304 Glycolipids bearing lipophilic diacetylene groups (48) can gelate water efficiently even at low concentrations (0.05 wt %) (Figure 89). Modification of the linker connecting the aliphatic chain and the sugar residue can affect the gelating properties. Glycolipids bearing a urea (49 and 50)305 or a triazole (51)306 linker are efficient hydrogelators. The formation of micelles and vesicles was explored in aqueous solution at neutral pH using glucose-based gemini surfactants (Figure 90), molecules that contain two head groups and two aliphatic chains.307 A transition from vesicles to large cylindrical micelles was observed, decreasing the pH of the solution (pH 6). A further increase in the acidity of the solution resulted in the formation of small globular micelles.

5.2. Glycopeptides

Glycopeptides are an important class of biomolecules composed of a saccharide part covalently attached to a peptide chain. These natural products are involved in a variety of biological events, such as recognition, cell proliferation, and inflammatory reactions.309 The production of synthetic analogues has the potential to generate new biomaterials and tools for the study of CPIs and CCIs. In addition, peptide secondary structure, combined with the possibility of CCIs, can create high-order structures.310 Responsive systems, which change their assembling state in relation to external stimuli, have also been studied. The drawback of these compounds is the complexity of the synthesis. Up to now, only a few examples of self-assembling systems based on glycopeptides have been reported. 5.2.1. Short Peptides. The addition of glycans to amino acids or dipeptides has been exploited for the formation of

Figure 87. First chiral glycolipid aggregate observed. Reprinted with permission from ref 300. Copyright 1993 American Chemical Society. 1732


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Figure 88. Different assemblies forming depending on the unsaturation pattern of the glycolipid. Reprinted with permission from ref 302. Copyright 2002 John Wiley and Sons.

ties, but formed sonication stable vesicles. Upon sonication, encapsulation of DNA into the spherical aggregates was observed.312 The galactose analogue of 52 was used to target hepatocytes.313 The surface sugars were oxidized by galactose oxidase (GO) to form C6 aldehydes. These aldehydefunctionalized aggregates were cross-linked via a diamine to afford an interconnected, dynamic network. Glycosylated vesicles loaded with fluorescent gold nanoclusters were delivered into hepatocytes, which express galactose binding receptors, and imaged by confocal microscopy. 5.2.1.2. Systems Based on Diphenylalanine. Nature, where glycosylation influences peptide and protein self-assembly, inspired the study of diphenylalanine scaffold self-assembly.314 Small differences in glycosylation were responsible for different supramolecular patterns (Figure 92): the α-glycosylated dipeptides 54 and 55 assembled into discrete tubular structures, whereas the β-analogue 56 gave no ordered structures. The introduction of more complex saccharides (57 and 58) increased the solubility of the fragment and reduced selfassembly. Small glycosylated diphenylalanine-based peptides (59) aggregate into nanostructure.315 The amphiphilic structures formed hydrogels or nanostructures depending on the concentration. These aggregates incorporate hydrophobic molecules such as fluorescent dyes and stabilized gold nanoparticles. This multivalent system displayed antimicrobial activity against Micrococcus flavus, Bacillus subtilis, and Pseudomonas aeruginosa. 5.2.2. Longer Peptides. In this section, we describe systems that exploit the secondary structure of polypeptides to create highly ordered glycopeptide structures.

Figure 89. Examples of efficient glycolipid gelators.

multivalent glycoclusters. While these are not really polymeric scaffolds, we describe them in the glycopeptide section. 5.2.1.1. Lysine-Based Systems. Dimannosylated lysine conjugates 52 assemble into soft spherical hollow structures (diameter 700 nm) where the mannose groups are solvent exposed (Figure 91).311 The hollow structures are able to encapsulate alkaline phosphatase, plasmid DNA, and a GFP reporter gene. Mild sonication induces supramolecular disassembly and release of the encapsulated guest. These aggregates served as drug carriers to transfect COS-7 cells with plasmid DNA. A tetrapeptide was coupled to the lysine carboxylic acids (53) and showed similar aggregation proper-

Figure 90. DNA transfection with gemini surfactants-based micelles. 1733


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Figure 91. Lysine-based glycopeptides that aggregate into vesicles.

Figure 92. Diphenylalanine-based glycopetides that give rise to different assemblies.

formation of cone-like structures (Figure 94B).319 Direct solubilization in water resulted in the spontaneous formation of small (50 nm) and polydisperse nano-objects. 5.2.3. Sugar-Appended Peptides. In the examples described above, the glycopeptide secondary structure was governed by the peptide. Here, we describe how the side-chain glycosylation influences the polymer conformation. A diblock copolypeptide of poly(L-leucine) attached to a glycosylated segment, poly(α-D-galactopyranosyl-L-lysine) and poly(α-D-galactopyranosyl-L-cysteine sulfone), was tested.320 The purely peptide block forms an α-helix, whereas the glycosylated block depends on its backbone. Lysine formed an α-helical structure, whereas the presence of cysteine gave rise to a disordered conformation (Figure 95). This difference in secondary structure affected the supramolecular self-assembly of the polymers: in the lysine case plate-like objects were observed, as the more flexible cysteine system permitted the formation of spherical vesicles (100 nm diameter). Another α-helical glycopolymer consisted of amphiphilic pendant arms (carbohydrate substituted with a C6 alkyl chain) that were attached to a poly(L-lysine) backbone that forms multimicellar aggregates in water (Figure 96).321 These aggregates incorporate both hydrophobic and hydrophilic dyes. Random polypeptides containing 10−20% mannose recognized and bound ConA. The length and dendron structure of a glucose-appended poly(L-lysine) connected with a PEG chain to a hydrophobic dendron affected its supramolecular assembly (Figure 97).322 In DMSO, an organogel was obtained, while nanorods or micellar

Carbohydrate-coated-β-sheet nanoribbons 60 aggregate in solution to give ribbons and encapsulate hydrophobic compounds between the hydrophobic interface formed by the bilayer of β-tapes (Figure 93).316 These compounds specifically and sensitively inhibit bacterial agglutination. Two glycoconjugates bearing the same peptide units, one substituted with a short linear coil block 61 and the other with a bulky dendritic block 62, both terminating with mannose, were reported.317 The introduction of a bulky dendritic block affected the formation of the β-sheet, as observed by circular dichroism. The two compounds formed nanoribbons of different length where the bulky substituent formed shorter structures. Both compounds were capable of binding E. coli, but only the longer nanoribbons formed bacterial clusters. Amphiphilic copolypeptides composed of a poly(γ-benzyl-Lglutamate) component attached to a poly(galactosylated propargilglycine) block self-assemble (Figure 94A).318 In every case, the peptide adopted an α-helix secondary structure, whereas the galactose prevented the formation of a defined conformation. Following the nanoprecipitation method using slow addition of a nonsolvent, all copolymers formed supramolecular aggregates. The morphology of the aggregates changed with the composition of the copolymer and the precipitation conditions. Worm-like and spherical micelles or polymersomes were obtained. All structures resulted from kinetic trapping induced by the rigidity of the polyglutamate component and exposed galactose on the surface. Novel, “tree-like” copolymers were synthesized by substituting the galactose residues with polysaccharide such as dextran and hyaluronal to provide the bulkiness required for the 1734


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Figure 93. Carbohydrate-coated copolymers that form monoribbons in solution. Control over the ribbon length can be obtained depending on the bulkiness of the substituent.

Figure 94. Linear amphiphilic copolypeptides (A) and “tree-like” amphiphilic copolypeptides (B). Reprinted with permission from refs 318 (A) and 319 (B). Copyright 2012 American Chemical Society and 2012 Royal Society of Chemistry.

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Figure 95. Diblock copolypeptide based on poly(L-leucine) that aggregates into plate-like objects or spherical vesicles depending on the glycosylated block. Reprinted with permission from ref 320. Copyright 2013 Royal Society of Chemistry.

transfer radical polymerization (ATRP),327 reversible additionfragment chain transfer (RAFT),328 and ring-opening metathesis polymerization (ROMP)329 have been employed to access these structures. These techniques provide facile and fast access to analogues with controlled shape and structural features.324 Variation in valency, sugar spacing, and overall architecture are possible by modification of monomer or polymerization conditions. Because of the straightforward synthesis, these molecules have emerged as attractive glycan analogues for biological and medical applications.30 5.3.1. Flexible Backbone. Polymeric structures with a flexible backbone can adapt their structure to maximize the binding affinity to the receptor. Long polymers can reach multiple binding sites on the same protein or can cluster different receptors on the same or different cells.30 In addition, copolymers bearing a glycopolymer block are useful substrates for self-assembly. 5.3.1.1. Self-Assembling Systems. Many glycopolymers able to self-assemble into nanoparticles and micelles, vesicles, or tubular aggregates, with exposed sugars, have been prepared. Control over the morphology of these nanoparticles is possible depending on the polymer structure and on the aggregationinduced mechanism. Because aggregation is generally promoted by the amphiphilic nature of the polymers, diblock glycopolymers or polymers substituted with aromatic groups have been studied. Responsive materials, fluorescent aggregates, and bioactive nanoparticles have been reported. We can focus on some representative examples because glycopolymer-based nanoparticle synthesis was reviewed recently.330 Amphiphilic diblock glycopolymers appended with mannoses were compared for their binding efficiencies of linear glycopolymers and dendrimer appended polymers.331 Both structures self-assembled into micelles with surface sugars and form aggregates, but the affinity of the dendritic analogues was higher. Branching is important even for macromolecular aggregates (Figure 98).332 Linear or branched hydrophilic poly(galactose acrylate) blocks were attached to a hydrophobic linear polymethyl acrylate, and nanoprecipitation formed nanoparticles. Galactose binding lectins interact much more tightly with the branched system.

Figure 96. Structure of α-helical glycopolymer consisting of amphiphilic pendant arms. Reprinted with permission from ref 321. Copyright 2013 American Chemical Society.

aggregates that can encapsulate Nile Red were observed in water. 5.3. Glycopolymers

Glycopolymers are synthetic polymers bearing pendant carbohydrates. Since the first example in 1998,323 a variety of glycopolymers based on different backbones has been reported.324,325 Free radical polymerization (FRO),326 atom

Figure 97. Dendron-copolypeptide that forms micelles or nanorods in water depending on the dendron structure. Reprinted with permission from ref 322. Copyright 2012 American Chemical Society. 1736


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the saccharide. Self-assembly of these polymers resulted in aggregates with different size and morphology depending on the length of the amine chain. Biocompatible micelles (30 nm) were obtained from amines with shorter chain length (propylamine). Large vesicles (200−600 nm) and micelles with low bioactivity were formed with hexylamine and dodecylamine. Another approach permitted the spontaneous formation of glycosylated nano-objects in concentrated aqueous solution (Scheme 2).335 Polymerization-induced self-assembly (PISA) relied on the efficient and regioselective thia-Michael addition to acrylates and enabled the formation of well-defined block aggregates directly at 25% solid (usual dilution

Carbohydrates in Supramolecular Chemistry - Chemical Reviews

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