Multivalency in heterogeneous glycoenvironments: Hetero-glycoclusters, -glycopolymers and -glycoassemblies

4518 Chem.Soc.Rev.,2013, 42, 4518--4531 This journal is cThe Royal Society of Chemistry 2013 Cite this: Chem. Soc. Rev., 2013, 42, 4518 Multivalency in heterogeneous glycoenvironments: hetero-glycoclusters, -glycopolymers and -glycoassemblies† Jose ´L. Jime ´nez Blanco,* a Carmen Ortiz Mellet a and Jose ´M. Garcı ´a Ferna ´ndez* b Despite efficiently imitating functional ligand presentations in terms of valency and density, most of the reported multivalent carbohydrate prototypes barely reflect the inherent heterogeneity of biological systems, therefore underestimating the potential contribution of synergistic or antagonistic effects to molecular recognition events. To address this question, the design of novel molecular and supramolecular entities displaying different saccharide motifs in a controlled manner is of critical importance. In this review we highlight the current efforts made to synthesize heteromultivalent glycosystems on different platforms (peptides, dendrimers, polymers, oligonucleotides, calixarenes, cyclodextrins, microarrays, vesicles) and to evaluate the influence of heterogeneity in carbohydrate– protein (lectin, antibody) recognition phenomena. Although the number of publications on this topic is limited as compared to the huge volume of reports on homomultivalent sugar displays, the current body of results has already unravelled the existence of new binding mechanisms that operate in heterogeneous environments whose exact biological significance remains to be unveiled. 1. Introduction Molecular recognition phenomena occurring between carbohydrates and proteins are responsible for the initiation of critical events in many biological processes such as fertilization, cell– cell communication, host–pathogen interactions, immune response or cancer metastasis. 1 However, individual carbohydrates tend to bind weakly to their complementary proteins and stronger, biologically useful binding or enhanced inhibition is often ascribed to the interplay of multiple interactions by multivalent carbohydrates. Multivalency generally leads to greater affinity enhancements than predicted from the sum of the constitutive interactions. This phenomenon, first noted by Lee and co-workers 2 and referred as the ‘‘cluster’’ or ‘‘multivalent’’ glycoside effect, 3 has found a wide range of application in biology and medicine. 4–8 Synthetic polyconjugates with welldefined structures have contributed to unravel the mechanisms at work, 9–12 leading eventually to useful tools for biotechnological 13,14 or therapeutic purposes. 15 Typically, these systems incorporate several copies of identical sugar motifs attached to an appropriate scaffold (molecular, dendritic, polymeric) 10,11,16–18 or self-assembled in supramolecular constructs (nanoparticles, vesicles, microarrays). 11,19 It has been amply demonstrated that ligand multivalency increases protein-binding avidities dramatically. However, these models barely reflect the inherent heterogeneity of biological systems, therefore underestimating the potential contribution of synergistic or antagonistic effects to molecular recognition events. To investigate the significance of glycoheterogeneity in carbohydrate–protein binding, the development of efficient methodologies to build novel heteroglycoclusters (hGC) and heteroglycoassemblies (hGA) displaying different saccharide ligands in a controlled manner is mandatory (Fig. 1). Developing tools to tune heterogeneity and multivalency in artificial conjugates is of further interest to optimize carbohydrate binding to biomedically relevant receptor partners that, likewise, may be presented in combination in a given environment or be intrinsically heterotopic. In this review we highlight the current efforts made to synthesize heteromultivalent glycoentities and to evaluate their protein-recognition properties. Most of the synthetic approaches put forward to access hGC and hGA are based on those previously reported for homoglycoclusters and homoglycoassemblies. a Departamento de Quı ´mica Orga ´nica, Facultad de Quı ´mica, Universidad de Sevilla, Apartado 553, E-41071 Sevilla, Spain. E-mail: [email protected]; Fax: +34 954624960; Tel: +34 954559806 b Instituto de Investigaciones Quı ´micas (IIQ), CSIC – Universidad de Sevilla, Ame ´rico Vespucio 49, Isla de la Cartuja, E-41092 Sevilla, Spain. E-mail: [email protected]; Fax: +34-954460565; Tel: +34-954489559 † Part of the multivalent scaffolds in glycosciences themed issue. Received 19th June 2012 DOI: 10.1039/c2cs35219b www.rsc.org/csr Chem Soc Rev TUTORIAL REVIEW Published on 21 August 2012. Downloaded by Centro de Investigaciones Cientificas Isla de la Cartuja (CICIC) on 25/06/2014 09:01:59. View Article Online View Journal | View Issue This journal is cThe Royal Society of Chemistry 2013 Chem. Soc. Rev., 2013, 42, 4518--4531 4519 Nevertheless, heterogeneity implies increased structural complexity and requires the development of more sophisticated strategies for the elaboration of suitable models. For the sake of clarity, the different heteroglycosystems described have been categorized by increasing ligand valency and density, which are the main parameters influencing their recognition behaviour. The aim is to provide a general view of the current state-of-art on these novel architectures and the information they provide on supplementary effects upon evaluation of their recognition abilities as compared to homoglycosylated constructs. 2. Low-density heteroglycoconjugates The general prototype of multivalent sugar constructs comprises a core molecule serving as an oligovalent scaffold, a variable number of peripheral carbohydrate epitopes and suitable spacers to link the sugar moieties to the central core. When installing more than a glycotope in a given platform, two general arrangements can be considered, namely a mixed-up (‘‘shuffled’’) distribution of the motifs or their clustering in a multidomain architecture. The choice of one or the other is Fig. 1 Schematic representation of heteroglycoclusters (hGC) and heteroglycoassemblies (hGA). Carmen Ortiz Mellet Carmen Ortiz Mellet received her PhD degree in Chemistry from the University of Seville (Spain) in 1984, where she was appointed Tenure Professor of Organic Chemistry in 1987. In 1990 and 1995 she joined the group of Professor Jacques Defaye (Centre d’Etudes de Grenoble, France) to work in the synthesis of complex thiooligosaccharides and pursue synthetic and supramolecular studies on cyclodextrins. Since 1998 she is responsible for the Carbohydrate Bioorganic Chemistry Group at the University of Seville, being promoted to Full Professor in 2008. Ongoing projects include the implementation of the concept of multivalency to glycosidase inhibition, the development of pharmacological chaperones for the treatment of lysosomal storage disorders and the design of self-assembled glycomaterials with dual nucleic acid and lectin recognition abilities for site-specific gene delivery. The laboratory also develops a research line on prebiotic oligosaccharides. Jose ´M. Garcı ´a Ferna ´ndez Jose Manuel Garcı ´a Ferna ´ndez received his Doctor of Chemistry degree from the university of Seville (Spain) in 1988. Between 1990 and 1995 he pursued postdoctoral research at the Centre d’Etudes de Grenoble, entering the field of cyclodextrins under the guidance of Dr Jacques Defaye. In 1996 he joined the Spanish National Research Council (CSIC) at the Institute for Chemical Research (CSIC – University of Seville), where he currently serves as Research Professor and Director of this Institute. He authored above 150 scientific articles in peerreviewed journals, review articles and book chapters and is coinventor of 16 patents. Current targets of the laboratory include the implementation of carbohydrate–protein and carbohydrate–nucleic acid interactions in the design of glycocarriers and glycodrugs for applications in nanomedicine, gene delivery, anticancer therapy, the treatment of lysosomal storage disorders and the management of inflammatory bowel diseases. Jose ´L. Jime ´nez Blanco Jose ´Luis Jime ´nez Blanco studied chemistry at the University of Seville (Spain) where he received the MSc degree in 1992. After finishing his PhD studies in the field of carbohydrate chemistry (1996), he joined the research group of Prof. J. F. G. Vliegenthart and J. P. Kamerling at the University of Utrecht (Netherlands), being awarded a Marie Curie Fellowship. In 2000, he returned to the University of Seville to join Prof. Ortiz Mellet’s group, working on pseudooligosaccharides of biological interest. He was appointed as university lecturer at the Department of Organic Chemistry of this university in 2002 and promoted to Tenure Professor in 2008. His current research focuses on the design, synthesis, and characterization of novel glycosystems to probe carbohydrate–protein and carbohydrate–nucleic acid interactions, aiming at developing applications in site-specific drug and gene delivery. Tutorial Review Chem Soc Rev Published on 21 August 2012. Downloaded by Centro de Investigaciones Cientificas Isla de la Cartuja (CICIC) on 25/06/2014 09:01:59. View Article Online 4520 Chem.Soc.Rev.,2013, 42, 4518--4531 This journal is cThe Royal Society of Chemistry 2013 intuitively expected to have strong consequences in the recognition properties of the ensemble. (a) ‘‘Shuffled’’ heteroglycoconjugates The preparation of molecular multiglycoligand architectures was first realized in the context of the synthesis of multiantigenic glycoconjugates for vaccine development. Thus, Danishefsky’s group developed a general strategy consisting of the preparation of oligosaccharide tumor antigens from nonnatural glycoamino acids that could be assembled into unimolecular multivalent oligopeptides. The final heteroconjugates were armed for conjugation to a carrier protein. 20 Immunological evaluation of these constructs (e.g. 1, Fig. 2) for multiantigenic carbohydrate-based cancer vaccines demonstrated their potential to stimulate a multifaceted immune response. 21,22 In 2002, Lindhorst and co-workers proposed an approach based on the orthogonal derivatization of D-galactopyranose to attach different sugars (a-D-mannose = aMan, a-L-fucose = aFuc and b-lactose = bLact), thereby accessing novel ‘‘mixed’’- or ‘‘hetero’’-glycoclusters (e.g.,2). 23 The different coating saccharides were sequentially incorporated, after activation of amine or carboxylic acid functional groups, through amide or thiourea ligation chemistries. Amide bond formation was also privileged for the construction of the heterodivalent glycoconjugate 3, bearing a mannose trisaccharide and a monomeric mannosyl unit in separate branches (Fig. 3). 24 Compound 3was designed to explore whether or not the mannose-specific bacterial lectin found on type 1 fimbriae (FimH), for which the presence of a monovalent carbohydrate recognition domain has been characterized, possesses additional carbohydrate binding sites that might contribute to bacterial adhesion in a multi-epitope scenario. However, anti-adhesion assays showed no significant increase in the inhibition of type 1 fimbriae-mediated bacterial adhesion in comparison to the standard inhibitor methyl a-D-mannopyranoside. The same research group has reported the preparation of polyether heteroglycodendrons by exploiting the nucleophilic addition of hydroxyl groups to methallyldichloride (4). The reaction proceeds sequentially, thereby allowing the incorporation of two successive sugar motifs, e.g. b-D-galactose (bGal) and aMan. The alkene functionality at the focal point can be then elaborated to produce hydroxyl-armed divalent dendrons (e.g. 5) that can enter the cycle to produce second generation tetravalent compounds (e.g. 6; Scheme 1). Unfortunately, the approach is limited to the use of ketal protecting groups and failed to afford higher generations of heteroglycodendrimers in acceptable yields and purity. 25 Katajisto, Lo ¨nnberg and co-workers combined the benefits of solid-phase synthesis and the efficiency of parallel synthesis for the generation of a short library of triantennary peptide heteroglycoclusters (Scheme 2). 26 The key building block is the a,a-bis(aminomethyl)-b-alanine derivative 7, bearing conventional N0-Fmoc, N0-Boc and N0-Alloc protecting groups on the three amino functions and a free carboxylic acid group for the attachment to the solid support (-8). The different glycotopes (bGlc, bGal, aMan, and b-D-ribopyranose) were incorporated sequentially by removal of the amino protections of the solidsupported amino acid core and subsequent coupling with the corresponding O-glycosylated, N-Fmoc-protected, pentafluorophenyl ester-activated serine derivatives (e.g.,-9). Lehn and co-workers proposed dynamic combinatorial chemistry (DCC) as a suitable strategy to investigate the affinity Fig. 2 Multiepitope vaccine 1designed by Danishefsky’s group. TF: ThomsenFriedenreich antigen; Tn: tumor-associated a-N-acetylgalactosaminyl epitope; STn: sialyl-a(2-6)Tn. 21,22 Fig. 3 Examples of ‘‘mixed type’’ oligosaccharide mimetics based on carbohydrate (2) and peptide (3) scaffolds. 23,24 Scheme 1 Preparation of heteroglycodendron 6. 25 Chem Soc Rev Tutorial Review Published on 21 August 2012. Downloaded by Centro de Investigaciones Cientificas Isla de la Cartuja (CICIC) on 25/06/2014 09:01:59. View Article Online This journal is cThe Royal Society of Chemistry 2013 Chem. Soc. Rev., 2013, 42, 4518--4531 4521 of lectins when faced to a multi-epitope pool. 27 The concept is based on the creation of reversible connections between suitable building blocks, leading to spontaneous assembly of all their possible combinations and allowing for the simple one-step generation of extended libraries. A dynamic library of bis-carbohydrate ligands 10–15 based on covalent disulfide bond formation between thiol-derivatized carbohydrates, including aMan, bGal, bGlc, b-L-arabinopyranosyl (bAra) and b-D-xylopyranosyl (bXyl) was thus generated (Scheme 3). 27 Screening of the library by adding concanavalin A (Con A), an aMan specific lectin, to the equilibrating mixture of library components did not evidenced significant differences in the binding affinity within the heterodimer series (Scheme 3). A second family of dynamic carbohydrate libraries was generated from a pool of carbohydrate aldehydes (16–21) and dior tritopic hydrazide components (A–H) through reversible acylhydrazone exchange (Scheme 4). 28 The library members can thus incorporate up to three different glycotopes (e.g. 22) Deconvolution analysis of the diand trivalent glycolibraries, using enzyme-linked lectin assay (ELLA), allowed the efficient identification of the best binder to Con A lectin, namely the trimannoside cluster having core G. Removal of the aMan building block fully abolished binding to the lectin, clearly indicating that this glycotope is required for molecular recognition. Much smaller effects were observed when other structural components were removed, preventing any conclusion about possible cooperative phenomena. Jime ´nez Blanco, Benito and co-workers reported the synthesis of trivalent glycodendrons incorporating aMan/bGlc or aMan/bLact substituents (28). 29,30 The synthetic scheme exploited the radical addition of the corresponding per-Oacetylated 1-thiosugars (23–25) to a tri-O-allylated pentaerythritol derivative 26. Reaction conditions were optimized to favour either single-, doubleor triple addition, which allows the incorporation of the different glycotopes in a sequential manner. In a second series of compounds, the resulting glycodendrons were armed with an isothiocyanate group and conjugated with the amine functionalized methyl a-D-mannopyranoside derivative 27 through the thiourea-forming reaction (-29; Scheme 5). Evaluation of the binding abilities towards the mannosespecific lectin Con A and the b-galactose/lactose-specific lectin peanut agglutinin (PNA) by ELLA indicated that the presence of the second sugar was irrelevant regarding binding of the primary ligand to its complementary lectin receptor. The potential of the copper(I)-catalysed alkyne-azide cycloaddition (CuAAC), the archetypal ‘‘click’’-type reaction, 31,32 to create heteromultivalent glycodisplays has been exploited by Santoyo-Gonza ´lez and co-workers 33 to develop a modular synthesis of neoglycoconjugates incorporating two different monosaccharides among D-mannose, D-glucose and D-glucosamine Scheme 2 Katajisto’s strategy to generate a short library of triantennary peptide heteroglycoclusters (e.g.,9). 26 Scheme 3 Dynamic library generation using disulfide interchange. 27 Scheme 4 Dynamic library generation using reversible acylhydrazone formation. Hydrazide compounds A–I are combined with six carbohydrate benzaldehydes, simultaneously. 28 Tutorial Review Chem Soc Rev Published on 21 August 2012. Downloaded by Centro de Investigaciones Cientificas Isla de la Cartuja (CICIC) on 25/06/2014 09:01:59. View Article Online 4522 Chem.Soc.Rev.,2013, 42, 4518--4531 This journal is cThe Royal Society of Chemistry 2013 onto a variety of scaffolds (methylene, ethylidene, erythritol, methyl-a-D-glucopyranoside, methyl-a-D-galactopyranoside and trehalose). Structural parameters such as the total and relative valencies, the anomeric configuration (aor b) of the coating sugars, the grafting pattern or the length of the spacers linking the peripheral glycotopes to the central core were systematically varied. The binding properties of all the library members towards Con A were evaluated by ELLA. By comparing the data for derivatives sharing the same scaffold (pentaerythritol; total valency 4) the authors observed a cooperative effect only in the case of heteroconjugates bearing aMan and aGal. Thus, the relative potency per aMan unit was 1.5-fold higher for the (aMan) 2 (aGlc) 2 derivative as compared to the (aMan) 4 homoconjugate, even though aMan is a much better ligand for Con A than aGlc (Fig. 4). Morvan and co-workers 34 have implemented the CuAAC ligation strategy to access glycooligonucleotide conjugates exhibiting two aMan and two bGal residues (31, Scheme 6) intended to be incorporated in novel heteroglycoarrays for lectin affinity investigation upon DNA-directed immobilization. The methodology involves the use of two functionalized phosphoramidite derivatives, one bearing a bromoalkyl group as precursor of azide and another one that bears a clickable propargyl group. Both were incorporated into an oligonucleotide by phosphoramidite chemistry on a DNA synthesizer (30). After a first CuAAC cycle with a monosaccharide–azide derivative, the bromo groups are substituted by azide anion and a second CuAAC reaction with a different propargylated sugar was performed. Alternatively, 50-bis-conjugation of oligonucleotides (-36) was performed by combining amidative oxidation and CuAAC chemistries. In that case, a propargylated phosphoramidite is incorporated in the oligonucleotide on the solid support (32) and then subjected to reaction with 3-bromopropylalanine (-33). Sequential CuAAC (-34), nucleophilic displacement of bromo by azide (-35) and a second CuAAC allows installing aMan and bGal motifs in the same phosphorous functionality (Scheme 7). 35 Karskela, Lo ¨nnberg and co-workers have extended the battery of oligonucleotide heteroglycoconjugates by preparing compounds having two dissimilar trivalent glycodendrons exposing either aMan or aGlc motifs (37). 36 In this case, the peracetylated methyl glycosides were first linked through their primary C-6 positions to a tripropargylated pentaerythritol core, armed with a benzaldehyde moiety, by CuACC. The aldehyde group of one of those glycodendrons was then engaged in oxime ligation with an oligonucleotide into which an aminooxy-modified Scheme 5 Preparation of trivalent homoand heteroglycoconjugates using thiol-ene and thiourea-forming reactions. 29,30 Fig. 4 Tetravalent ‘‘click’’ heteroglycoconjugates and their relative binding affinities towards Con A lectin. 33 Scheme 6 General procedure of bi-click strategy to obtain Man/Galglycooligonucleotides. 35 Scheme 7 General procedure of combined amidative oxidation-click strategy to obtain Man/Gal-glycooligonucleotides. 35 Chem Soc Rev Tutorial Review Published on 21 August 2012. Downloaded by Centro de Investigaciones Cientificas Isla de la Cartuja (CICIC) on 25/06/2014 09:01:59. View Article Online This journal is cThe Royal Society of Chemistry 2013 Chem. Soc. Rev., 2013, 42, 4518--4531 4523 building block had been incorporated on the DNA synthesizer (38). This cycle was repeated a second time with the next glycodendron to afford a (Man) 3 (Glc) 3 conjugate (39, Scheme 8). 36 Unfortunately, no data on lectin binding properties of the oligonucleotide heteroglycoconjugates appear to be available up to date. The group of Dumy and Renaudet proposed a combinatorial approach that allowed the rapid generation and screening of a structurally diverse library of tetravalent hGC combining various sugar motifs among aMan, a-galactosamine (aGalNAc), bLact and a-L-fucopyranoside (aFuc). They implemented the so-called template-assembled synthetic protein (TASP) concept by using the topological cyclodecapeptide scaffold 40, bearing four aldehyde groups, as a regioselectively addressable functionalized template (RAFT), to couple multiple carbohydrate units in a parallel disposition through oxime-based ligation chemistry. 37 This strategy secures a quantitative coupling of biomolecules with a randomized and statistical distribution of each expected library species (Fig. 5). Various hGC libraries combining up to four carbohydrates or carbohydrate and amino acid units were thus generated. 38 The composition and binding potency of each library was screened by HPLC with a Con A binding affinity column. Then, the libraries exhibiting the higher lectin affinities were subjected to separation by semipreparative HPLC to individually study the affinity of their components by surface plasmon resonance (SPR). The results indicated that the presence of hydrophobic residues, such as tyrosine, instead of a sugar in mannoside clusters improve the interaction with Con A. Competition studies suggested that the hydrophobic residue does no interact with the specific mannose-binding pocket, but with an independent binding site. The data evidenced the expected decrease in binding affinity when a mannose residue is replaced by any of the other sugars. Nevertheless, they revealed differences in binding strength depending on the secondary glycotope nature; e.g. (aMan) 3 (aFuc) binds Con A stronger than (aMan) 3 (bLact), and the latter is a better ligand that a trimannoside in which the forth position is occupied by an aspartic acid residue. A quantitative determination of the binding affinity on a per a-Man basis was not carried out, however. The above combinatorial procedure leads to the formation of inseparable mixtures of regioisomers, which precludes their utilisation for further assays with relevant biological targets. The same group designed a novel synthetic protocol to prepare hGC keeping the same cyclopeptide template but in a regioselectively controlled manner. 39 The methodology consisted of the application of two successive chemoselective reactions, namely oxime ligation and CuAAC, to incorporate different carbohydrates onto cyclodecapeptides containing either two aldehyde and two azide or one aldehyde and three azide functionalities, respectively (Fig. 6). 39 (b) Multidomain heteroglycoconjugates Dondoni, Marra and co-workers 40 described a calix[4]arene glycoconjugate (41) in which two different sugars (bGlc and bGal) are installed at the upper and lower rims of the calix[4]arene macrocycle, respectively, via sequential CuAAC and photoinduced thiol-ene coupling (TEC). 41 In this heteroglycoconjugate prototype the two monosaccharides do not Scheme 8 Oligonucleotide heteroglycoclusters. 36 Fig. 5 General strategy and structure of carbohydrates and aminoacids used for the generation of hGC libraries from cyclopeptide template 40 displaying four glyoxoaldehyde anchoring sites. 38 Fig. 6 Schematic representation of cyclopeptide-scaffolded 3 : 1 and 2 : 2 bLac/ aMan and aMan/aFuc heteroglycoclusters, respectively. 39 Tutorial Review Chem Soc Rev Published on 21 August 2012. Downloaded by Centro de Investigaciones Cientificas Isla de la Cartuja (CICIC) on 25/06/2014 09:01:59. View Article Online 4524 Chem.Soc.Rev.,2013, 42, 4518--4531 This journal is cThe Royal Society of Chemistry 2013 share the same space regions, but are instead homogeneously occupying opposite domains (Fig. 7). In principle, heteromultivalent glycoconjugate prototypes consisting of spatially separated homoglycoclusters are likely to be incompatible with heterocooperativity upon binding to a given lectin. However, they might be well suited to cross-link two different lectins, each specific for one of the clusterized sugars. This hypothesis was explored by Roy and co-workers 42 in an attempt to develop efficient antiadhesion therapeutics against pathogenic Pseudomonas aeruginosa. These bacteria express intracellular and outer membrane lectins, PA-IL (LecA) and PA-IIL (LecB), which are specific for D-Gal and L-Fuc residues, respectively. The authors demonstrated, using a turbidimetric assay, that the heterobifunctional ‘‘click’’ glycodendrimer 42, possessing four aFuc and four bGal residues in opposite hemispheres (Fig. 8), displayed fast cross-linking abilities with both PA-IL and PA-IIL simultaneously as planned. No interference in the binding of the bGal/PA-IL and aFuc/PA-IIL pairs due to the presence of the second sugar was observed. The ensemble of the above commented results indicate that low-density heteroglycoclusters display a moderate glycoside cluster effect against lectin partners exclusively dependent on the valency of the ‘‘active’’ saccharide epitopes. If two sugar motifs exhibiting different affinity for the same lectin are presented together on the scaffold, the resulting heteroglycocluster may bind more efficiently than the respective homogeneous multivalent conjugates. However, no significant influence of ‘‘non-recognizable’’ residues in heteroglycoligand– protein recognition has been clearly established so far in that type of architecture. On the other hand, these molecularly welldefined multiglycoligand derivatives have a strong potential in the development of multiantigenic vaccines and multilectintargeted inhibitors of bacterial infection. 3. High-density heteroglycoconjugates Carbohydrate–protein recognition in biological environments generally involves high density regions of the glycocalix. Varying the relative expression of the primary glycotope can then switch on or off a given process, e.g. inflammation. 43 Although increasing the valency of the primary recognition motif results in enhanced affinities for a complementary lectin even in low valency glycoconjugate models, it is conceivable that the observation of any supplementary effect due to the presence of other sugar motifs which themselves are not ligands for that lectin might require the involvement of heavily dense heteromultivalent glycodisplays. Heteroglycopolymers, hyperbranched heteroglycoclusters and heteroglycoassemblies have been designed for that purpose. (a) Heteroglycopolymers Haddlenton and co-workers 44 designed a synthetic protocol for the preparation of hetero-neoglycopolymers 46 based on ‘‘coclicking’’ two different sugar azides by CuAAC with a propargyl-functionalized polymer. The clickable polymeric scaffold was obtained by transition metal-mediated living radical polymerization (TMM LRP) of trimethylsilyl (TMS)-protected propargyl metacrylate precursors (e.g.,43) and a benzyl abromoester (e.g.,44) as polymerization initiator (-45). Small libraries of heteroglycopolymers bearing variable relative proportions of aMan and bGal motifs were prepared in this manner. The library components were assayed for their binding capacity to Con A by turbidimetry and quantitative precipitation. Interestingly, a 75 : 25 mannose : galactose ratio was found as efficient as the homomannosylated polymer in clusterizing the lectin, implying a 1.5-fold higher efficiency in a mannose molar basis. Although a saturation effect cannot be discarded, the results are consistent with the existence of synergic interactions involving the bGal residues (Scheme 9). 44 The above heteroglycopolymers were further conjugated to bovine serum albumin (BSA). The coclicking strategy was extended in that case to incorporate a fluorescent probe (rhodamine B) in addition to the aMan and bGal motifs (47 -48; Scheme 10). 45 The binding abilities of these BSA-neoglycopolymer hybrid materials towards the human dendritic cell associated lectin Fig. 7 Tetravalent dual glucosylated and galactosylated calix[4]arene cluster 41. 40 Fig. 8 Octavalent aFuc/bGal heteroglycodendrimer 42. 42 Chem Soc Rev Tutorial Review Published on 21 August 2012. Downloaded by Centro de Investigaciones Cientificas Isla de la Cartuja (CICIC) on 25/06/2014 09:01:59. View Article Online This journal is cThe Royal Society of Chemistry 2013 Chem. Soc. Rev., 2013, 42, 4518--4531 4525 (DC-SIGN), known to bind mannoside residues, were evaluated by SPR. 46 The binding affinity and the relative affinity per mannose unit increased with the density of the aMan ligand, as expected. The experiments were designed to maintain constant the total sugar density by varying the aMan : bGal ratio, which prevents the evaluation of the effect of the bGal moieties for compounds having identical aMan density, however. In a work aiming at the generation of libraries of polymeric cholera toxin (CT) antagonists, the synthesis and activities of a series of heterobifunctional ligands conjugated to two polymer carriers (polyacrylamide and dextran) were described. 47 Since multivalent presentations of bGal residues have demonstrated to exhibit exceptional high activity towards the B subunit of CT (CTB), all the conjugates contained an invariable bGal fragment and variable non-galactose fragments incorporated by CuACC reaction. Considering that the principal ligand for CTB is the ganglioside GM 1 (Gal-b(1–3)-GalNAc-b(1–4)-[NeuNAc-a(2–3)]- Gal-b(1–4)-Glc-b(1–3)-ceramide) and that, in addition to the keystone bGal residue, the neuraminic acid moiety (NeuNAc) is an important fragment for affinity and selectivity, the corresponding heterobifunctional neoglycopolymers were first assayed (Fig. 9). They actually showed partial inhibition in a competitive ELLA experiment, while galactose-only progenitors showed no detectable activity. Nevertheless, the IC 50 values were deceivingly high (0.5–0.8 mM), much higher than those obtained for non-carbohydrate fragments. The length of the linker is sufficient for both bGal and NeuAc to reach their respective positions in the GM 1 binding site on the surface of cholera toxin; however, entropy loss due to linker flexibility offsets the contribution from this additional interaction. Kobayashi and Nishida designed a facile synthetic way to obtain acrylamide biand terpolymers as Pand L-selectin blockers by applying copolymerization synthetic strategies, involving vinyl monomers of a-L-fucoside and 3or 6-sulfoor 3,6-disulfo-b-D-galactoside as key carbohydrate modules to mimic sulfated sLe x tetrasaccharide 48,49 (Fig. 10). Binding assays showed that acrylamide bipolymers carrying only 3-sulfogalactoside did not display activity for any selectin while the fucosylated terpolymers showed potent activity to block both Pand L-selectin/sLe x binding in an ELISA experiment at a concentration of a few micrograms per millilitre. 48 The enhanced activity is ascribed to the cooperative binding effects of the fucoside and the sulfogalactoside residues. Deepening in this concept, this research group generalized the so-called ‘‘carbohydrate module method’’, which involves three steps: segmentation of a targeted oligosaccharide into smaller sugars, synthesis of the corresponding glycosylated monomers and the reassembly of oligosaccharide mimics by copolymerization of the modules. 49 The utility of the carbohydrate module method Scheme 9 Synthesis of the heteroglycopolymer 46. 44 Scheme 10 Synthetic procedure to obtain a library of BSA-neoheteroglycopolymers containing a fluorescent tag (rhodamine B). 45 Fig. 9 General design of a focused library of heterobifunctional polymers as CTB ligands. 47 Fig. 10 Artificial selectin blockers: heterocopolymers carrying 6-sulfo sialyl Lewis x tetrasaccharide as key carbohydrate modules. 48 Tutorial Review Chem Soc Rev Published on 21 August 2012. Downloaded by Centro de Investigaciones Cientificas Isla de la Cartuja (CICIC) on 25/06/2014 09:01:59. View Article Online 4526 Chem.Soc.Rev.,2013, 42, 4518--4531 This journal is cThe Royal Society of Chemistry 2013 as a tool to assemble oligosaccharide mimics of high biological significance was further supported by the development of new heteroglycopolymers that combined aFuc, 3-sulfo-b-Gal and 6-sulfo-b-GlcNAc exhibiting a significant increase in binding affinity and selectivity towards L-selectin. 50 The same concept was applied to the synthesis of galactotrehalose (GT) acrylamide polymers. The binding abilities of such polymers were evaluated against BSI-B 4 lectin (Bandeiraea simplicifolia), which is specific to a-galactoside-carrying oligosaccharides including Gb3 ceramide and human blood B determinants. 51 The results supported that both a,a-and a,b-GT-polymers have binding activity towards this lectin, whereas homoglycopolymers bearing bGal or bGlcNAc, used as negative references, did not show any binding. The binding activity increased with the aGal density in the polymer and could be integrated as the result of multivalent binding and/or carbohydrate cluster effects. The effect of bGlc and bGlcNAc residues added as second sugars in the heteroglycopolymers was next examined. Unexpectedly, a terpolymer carrying both a,a-GT and bGlcNAc was found to exhibit the strongest affinity to this lectin. In contrast, bGlc residues brought about no positive effect in any of the polymers and it was even detrimental in the case of the a,b-GT polymer. A similar trend was observed for Shiga toxin-1, another aGal specific binding protein. Thus, heteroglycopolymer 49 carrying both a,a-GT and bGlcNAc (Fig. 11) along the polymer chain displayed significantly higher detoxifying activity than the a,a-GT homopolymer. The authors invoked a ‘‘module effect’’ to explain the notable role of bGlcNAc in enhancing interactions with these carbohydrate binding proteins in a supplementary way, but they admitted that it is hard to imagine such a molecular packing geometry in which the a,a-GT and GlcNAc residues are fused along a polymer chain to make a Gb3 ceramide mimetic. The exact mechanism by which GlcNAc assists the carbohydrate–protein interaction in these particular systems remains, thus, mysterious. Wolfenden and Cloninger 52,53 functionalized poly(amidoamine) (PAMAM) dendrimers of generations G3, G4, G5 and G6 with aMan, aGlc and aGal glycotopes (Scheme 11) to investigate Whitesides’ relationship between association constants for monovalent and multivalent associations given in eqn (1), where Nis the number of receptor–ligand interactions and a is the cooperativity factor. 54 K poly N =(K mono ) aN (1) Based on size and architectural considerations, they assumed a divalent interaction (N= 2) between tetrameric Con A and aMan-containing PAMAM glycodendrimers, with a positive cooperativity factor a= 1. Considering that monomeric aMan is recognized by the lectin with a 4-fold higher affinity than aGlc, a 16-fold decrease would be expected when going from 50% aMan loaded PAMAM homoglycodendrimers to heteroanalogues keeping the same total carbohydrate loading but with an 1 : 1 aMan : aGlc composition. The experimental values obtained from hemagglutination experiments were very close to the theoretical ones for the G4 and G5 glycodendrimers, which led the author to conclude that the activity of heteroglycoligands can be modulated for those systems in a predictable manner. However, significantly lower decreases in binding affinity were observed for the sixth-generation aMan:aGlc dendrimer and, especially, in the case of aGlc:aGal conjugates. It was advanced that in systems having either higher flexibility or lower affinity, proximity/statistical effects are more important to binding. Thus, dendrimers with more glucose residues appear to compensate for the steric downfall of full functionalization by relying more on proximity enhancements than mannose functionalized dendrimers do. 53 Nevertheless, the existence of synergistic interactions involving the second sugar that compensate, in part, the diminution of the primary ligand concentration cannot be discarded. (b) Heteroglycoclusters The use of polymeric scaffolds to build highly dense glycoarchitectures implies an intrinsic polydispersity and lack of conformational control that hampers a rigorous evaluation of the influence of architectural parameters on the binding affinity to protein receptors, a fact that is exacerbated for multiligand-coated derivatives. In order to get a deeper insight into the issues related to heteromultivalency, Ortiz Mellet, Defaye and Garcı ´a Ferna ´ndez designed a series of hyperbranched heteroglycoclusters that comply with the requirements for polyvalency, high density and monodispersity. An efficient procedure based on multiple coupling of isothiocyanate-armed glycoligands or heteroglycodendrons to a per(C-6)cysteaminyl Fig. 11 Structure of polyvalent a,a-GT and b-GlcNAc acrylamide polymer 49 mimicking globosyl 3 (Gb3). 51 Scheme 11 Mannose/glucose/galactose-coated PAMAM dendrimers. 52,53 Chem Soc Rev Tutorial Review Published on 21 August 2012. 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