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ScienceDirect Carbohydrate nanotechnology: hierarchical assembly using nature’s other information carrying biopolymers Xu Han, Yeting Zheng, Catherine J Munro, Yiwen Ji and Adam B Braunschweig Despite their central role in directing some of the most complex biological processes, carbohydrates — nature’s other information carrying biopolymer — have been largely ignored as building blocks for synthetic hierarchical assemblies. The non-stoichiometric binding and astronomical diversity characteristic of carbohydrates could lead to tantalizingly complex assembly algorithms, but these attributes simultaneously increase the difficulty of preparing carbohydrate assemblies and anticipating their behavior. Convergences in biotechnology, nanotechnology, polymer chemistry, surface science, and supramolecular chemistry have led to many recent important breakthroughs in glycan microarrays and synthetic carbohydrate receptors, where the idiosyncrasies of carbohydrate structure and binding are increasingly considered. We hope to inspire more researchers to consider carbohydrate structure, diversity, and binding as attractive tools for constructing synthetic hierarchical assemblies. Addresses Department of Chemistry, University of Miami, 1301 Memorial Drive, Coral Gables, FL 33146, USA Corresponding author: Braunschweig, Adam B ([email protected])
Current Opinion in Biotechnology 2015, 34:41–47 This review comes from a themed issue on Nanobiotechnology Edited by Igor L Medintz and Matthew Tirrell
http://dx.doi.org/10.1016/j.copbio.2014.11.016 0958-1669/# 2014 Published by Elsevier Ltd.
Introduction Mimicking the bottom-up assembly of hierarchical biological structures — such as cell membranes, tissues, organs, and ultimately, organisms — is a major goal of biotechnology and nanotechnology. Within cells, the information that directs hierarchical assembly is encoded within oligonucleotides and oligopeptides as dictated by the central dogma of biology. Both oligonucleotides and oligopeptides can be prepared by automated solid-phase syntheses, and the combination of facile preparation and well-understood recognition contributes to their common www.sciencedirect.com
adoption by researchers seeking to achieve synthetic bottom-up assemblies. In DNA-based and RNA-based nanotechnolgies for example, information is encoded within well-known Watson–Crick or Hoogsteen base pairings along the (deoxy)ribophosphate backbones, and oligonucleotides are commonly employed in gene chips, but they are also more recently used for constructing other hierarchical assemblies, including exotic molecular topologies [1–3], molecular machines [4,5], sensors [6], drug delivery systems [7], and logical operators [8,9]. While the use of oligopeptides in protein chips [10] or in phage displays [11] is well known, the supramolecular recognition of oligopeptides is also used in templating nanoparticle assembly [12,13], or in medical applications, such as repairing spinal cord injuries [14]. These systems are remarkable in that they mimic the multi-length scale order, complex binding stoichiometries, stimuli-responsiveness, and emergent properties that are the hallmarks of biomolecular information, and their successful development demonstrates the importance of continuing to study and mimic the ways biology encodes information in molecular and polymeric scaffolds. Many of the most complex biological processes, including protein folding, cell–cell binding, cell motility and signal transduction, occur as a result of binding events on the glycocalyx, which is a 100 nm–1 mm thick layer of glycans (Figure 1) on the cell surface that includes glycolipids, glycopeptides, and glycopolymers [15]. The information containing elements in these glycans are oligosaccharides, but despite this fact carbohydrates have hardly been explored in the context of synthetic hierarchical assemblies or on biochips as a consequence of their challenging preparation, structural diversity, and complex binding, which is characterized by multivalency and cooperativity. Because information in carbohydrates is encoded so differently than the binary algorithms of DNA and the sequence diversity is infinitely greater than that achievable with oligopeptides, working with carbohydrates inevitably leads us to question our assumptions about biological recognition and information transfer at the most fundamental levels. Herein we discuss how the uniqueness of carbohydrates is increasingly considered as a central focus of two research areas at the interface of nanotechnology and biology — specifically glycan microarrays and synthetic carbohydrate receptors — where major recent advances could lead to the realization of complex carbohydrate directed hierarchical assemblies. In discussing their commonalities, we hope to provide readers with an appreciation of the concepts, challenges, and potential of Carbohydrate Nanotechnology. Current Opinion in Biotechnology 2015, 34:41–47
42 Nanobiotechnology
Figure 1
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Representation of the glycocalyx, which is a 100 nm–1 mm thick coating of glycoproteins, glycolipids, and glycopolymers on the surfaces of eukaryotic cells. Some of the most prevalent monosaccharides found on cell surface glycans, as well as their common symbolic representation, are shown.
Oligosaccharides are chains of monosaccharides that contain at least one carbonyl and multiple hydroxyl functions per residue, and often further functional group (Figure 2a–c). Each monosaccharide contains several stereocenters and typically occurs as an equilibrium mixture of 5-membered and 6-membered rings [16]. Because of the branching in oligosaccharide chains, the information density of oligosaccharides — which is the number of different oligomers that can be prepared from a chain of a given length — exceeds significantly what can be achieved with linear oligopeptides and oligonucleotides (Figure 2d) [17]. The carbohydrates in cell surface glycoconjugates can easily contain as many as 20 monosaccharides in an oligomer, enabling incredible structural variety arising from the number of monosaccharides in a chain and also from the different branching points and linkages (a or b). The preparation of oligosaccharides involves multistep routes that require selective functionalization of hydroxyl groups with nearly identical reactivity on scaffolds with tens of reactive hydroxyls. This complicates their preparation, such that carbohydrates are typically available only in small quantities, and just a small fraction of all the possible hexasaccharide combinations can be prepared with current synthetic methods. Additionally the specificity of glycan binding proteins (GBPs) is difficult to determine because the 1:1 association between carbohydrates and GBPs is weak (typically 102–103 M1) and GBPs are promiscuous, often binding many carbohydrates. To overcome low affinity, GBPs often possess multiple binding sites, and the glycans on cell surfaces present as multimeric assemblies, and these higher order binding modes increase affinity (up to 109 M1 increase in Ka, Current Opinion in Biotechnology 2015, 34:41–47
but typically 10–103 M1) and specificity, a phenomenon termed the cluster glycoside effect [18]. It is important to also note that binding between carbohydrates and GBPs frequently invokes cooperativity that arises from the interplay of multiple noncovalent interactions, making the binding strength and substrate specificity difficult to anticipate. In summary, carbohydrates are distinct from other information carrying biopolymers in terms of structure, synthesis, and recognition, and many fundamental questions and challenges remain that have delayed their incorporation into synthetic hierarchical assemblies. Elucidating how carbohydrates encode binding information within molecular structure could also lead to breakthroughs in disease detection, drug delivery, and therapeutics.
Glycan microarrays Glycan arrays consist of solid surfaces that display a library of glycans spatially encoded into micrometer scale spots, and the resulting surfaces are used to rapidly and combinatorially assess the affinity and selectivity of GBPs to the immobilized glycans [19]. The microarray format is particularly useful in the context of glycosciences because carbohydrate recognition, much more so than oligonucleotide or oligopeptide, is strongly dependent on cooperativity and multivalency — parameters that can be controlled and studied by varying the density and orientation of the immobilized glycans. Glycan arrays are an important step in the path towards more sophisticated carbohydrate nanotechnologies because they clarify how all the contributors to glycan binding determine affinity and selectivity. The two challenges that remain to increase the adoption of glycan arrays involve improving surface chemistry to access multivalent and cooperative binding interaction and increasing the diversity of the glycan libraries within the arrays, which is limited by the difficulty of preparing carbohydrates or obtaining natural samples of glycans. The immobilization chemistry — the chemical bonding and interactions that hold the glycans onto the substrates — has a major impact on glycan array performance, such that it has been shown that glycans printed using different immobilization strategies display different GBP selectivities [20]. These paradoxical data likely reflect both the promiscuity of GBPs and the poorly understand role of nanoscale phenomena, such as orientation and density, on glycan surface binding. In an effort to investigate the role of immobilization chemistry on binding and to improve array performance, researchers have used printing strategies based on noncovalent, covalent, and polymer chemistry, and in doing so, have simultaneously developed ingenious approaches for increasing array diversity. Noncovalent printing strategies bind glycans onto surfaces using supramolecular interactions such as Van der Waals, ionic, hydrogen bonding, and solvophobic interactions. The advantages of noncovalent approaches are that they can reduce the number of synthetic steps for www.sciencedirect.com
Carbohydrate nanotechnology Han et al. 43
Figure 2
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(a) The 4C1 conformation of the monosaccharide a-mannose (a-Man) with the anomeric carbon labeled as C1, following conventional notation. (b) The trisaccharide contains both a and b linkages. (c) The pentasaccharide contains multiple hydroxyl groups and further functional groups. (d) Table presents the number of different oligomers, N, that can be created by all possible combinations of a given oligomer size, n. Calculations are based on a4 nucleotides, N = 4n, b20 common amino acids, N = 20n, and c10 common mammalian monosaccharides that can adopt only a-pyranoside, or Q b-pyranoside conformations, N ¼ 20n nk¼1 ð3n 2Þ.
preparing glycans for printing, the surfaces are easy to fabricate, and the success of attachment is generally high. The first glycan array was prepared using such a noncovalent approach, whereby glycans bearing long alkyl chains were immobilized onto hydrophobic surfaces [21]. Several recent creative advances in this field have vastly improved noncovalent glycan arrays. In 2005, the Pohl group successfully immobilized fluorous-tagged carbohydrates onto fluorinated solid supports with a robotic spotter. This approach simplifies fabrication steps by directly attaching a single C8F17 tail to carbohydrates, www.sciencedirect.com
and importantly strong binding affinity between immobilized glycan and GBPs noncovalent bind pairs are observed [22]. Later glycan arrays were made by the attachment of sulfonated polysaccharides to poly-Llysine coated surfaces by Hsieh-Wilson et al. [23] to elucidate the effects of the glycoaminoglycans on GBP binding. In another recent example, Chevolot and co-workers built a new glycan array platform by immobilizing oligonucleotide-modified glycans onto surfaces printed with complementary DNA sequences [24] to vary precisely the distance between carbohydrate residues and the surfaces. These advances have resulted in control of orientation and significantly improved binding, but purely adsorptive approaches still require the preparation and isolation of carbohydrates to make the array. An alternate approach towards immobilizing glycans involves covalent attachment to a substrate, whereby a new covalent bond is formed between the glycans and the surfaces, and this strategy offers the potential to control glycan orientation and density by accessing the selectivity of organic chemistry. Covalent immobilization was demonstrated by Blixt, Wong, Paulson and coworkers, who attached amine containing glycans to N-hydroxysuccinimide activated slides, and they used this array to discover the specificity of a variety of GBPs including plant lectins, human GBPs, glycan-specific antibodies, and other proteins [25]. Different binding pairs, like erythrina cristagalli agglutinin to N-acetyllactosamine and differentiation-22 to Siaa-6-Galb1-4-GlcNAc were observed. This method however does not necessarily control orientation because complex glycans can contain multiple amine residues, and subsequent covalent strategies focused on bioorthogonal reactions that do not interfere with the functional groups common to glycans, and also proceed quickly and in high yield. The first example of such an approach involved the use of Diels– Alder cycloaddition [26] and hetero-Michael addition [27] reactions to form glycan chips. Other reactions that have been employed subsequently include the Staudinger Ligation [28,29], CuI-catalyzed azide-alkyne click reaction [29,30,31,32], and the light-induced thiol-ene reaction [33,34,35]. In these examples, the immobilized glycans bind successfully to the target GBPs, and the reactions can be carried out in the presence of spacers to explore how glycan density affects binding [36]. The limitations to purely covalent strategies are that the surface-reactive functional groups must be attached to already difficult to obtain glycans, and that cluster glycoside effects are not operating fully in monolayers, so binding specificity may not reflect accurately the conditions on cell surfaces. By leveraging ideas from polymer chemistry, researchers have recently made breakthrough advances that simplify the preparation of glycan chips, increase library diversity, and clarify the subtle interplay between carbohydrate Current Opinion in Biotechnology 2015, 34:41–47
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density and binding affinity. In polymer-based strategies, the backbone of either a naturally derived biopolymer or a synthetic polymer is used as a scaffold to prepare a multivalent saccharide display that extends away from the substrate, and in doing so create a binding environment that closely resembles the cell surface, and diversity can be increased by varying chain length, spacing, and carbohydrate presentation. Gildersleeve and coworkers have demonstrated the potential of this approach using the protein bovine serum albumin (BSA) as the polymer scaffold, where saccharides first attached to different linkers, like 6-aminohexanoic acid and 3-[(2-hydroxyethyl)thio]propanamide, are attached to the BSA lysine residues using EDC coupling reactions [37]. The BSA presents multiple attachment sites, and by varying saccharide concentration when printing onto a BSA-coated surface, they are able to change the degree of functionalization of the BSA to capture affects of multivalency. By
adding multiple different saccharides to the ink, they can attach different saccharides onto the same BSA chain and in effect create a new glycan and increase array diversity. In changing both carbohydrate density and composition on the biopolymer backbone, they have successfully developed a strategy to increase library diversity and have produced microarrays with as many as 537 glycans [37]. Wang et al. was the first to deposit a synthetic glycopolymer by printing onto a nitrocellulose coated surface using a grafted-to noncovalent strategy [38]. The Bertozzi group immobilized a synthetic glycopolymer with a poly (methylvinylketone) backbone and pendent N-acetylgalactosamine (GalNAc) residues to serve as mucin mimics that were subsequently attached to azide terminated surfaces using the CuI-catalyzed azide-alkyne click reaction. By adding spacer groups during the surface immobilization step, they were able to alter interchain spacing, and thereby distinguish how glycan valency and
Figure 3
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(a) A nanophotolithography platform was used to create grafted-from glycopolymers. (i) Beam pen tips coated with an ink mixture composed of 2,2-dimethoxy-2-phenylacetophenone and methacrylate-terminated carbohydrates in a polyethylene glycol matrix, deposits inks onto thiol terminated surfaces (ii) UV light passes through beam pen tips to initiate the polymerization reactions. (iii) Washing the surface removes unreacted chemicals, so only the covalently immobilized glycopolymers remain. (b) Fluorescence microscopy image of Cy3 labeled concanavalin A bound to glucose-bearing glycopolymers prepared by the process described in (a). (c) Fluorescence intensity of printed features decreases with decreasing chain length. The x-axis corresponds to the white line from left to right in the inset figure of (b). (d) Plot indicates the relationship between expose time and fluorescence intensity or polymer height. (e) Plot shows how different surface chemistries including the CuAAC (from Ref [32]), thiol acrylate (from Ref [35]), or thiol-ene polymerization (from Ref [35]), alter binding sensitivity towards concanavalin A.Reproduced by permission of The Royal Society of Chemistry. Current Opinion in Biotechnology 2015, 34:41–47
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Carbohydrate nanotechnology Han et al. 45
organization determine interactions with helix promatia agglutinin [39]. Applying a grafted-from approach, the Braunschweig group used tip-based nanophotolithography to create arrays of glycopolymer brushes by radical polymerization of glucose methacrylate directly from a thiol-terminated surface, where the light exposure time during printing was used to control polymer chain length [35]. They demonstrated a 100-fold increase in sensitivity towards GBPs compared to glycan monolayers, and they also show that GBP binding is dependent on polymer height (Figure 3). Thus glycan microarrays are a rapidly advancing field of biotechnology, where noncovalent interactions, surface science, and polymer chemistry are combining to accelerate our understanding of glycan recognition.
Synthetic lectins An equally important step towards carbohydrate nanotechnology is the development of carbohydrate-binding molecules, which, in addition to hierarchical assembly, could be used for delivering drugs to specific cell types, detecting diseases, or even as therapeutic agents by targeting and binding cell-surface glycans. The term synthetic-lectin has been coined to refer to man-made molecular receptors that bind carbohydrates, but glycans are amongst the most challenging natural targets for synthetic receptors that must distinguish between monosaccharides that may differ by only the orientation of a single stereocenter in a very competitive solvent. Despite these obstacles, researchers have made major advances in the preparation of synthetic lectins using traditional supramolecular approaches, and more recently, by creating new receptor designs inspired by the promiscuity and unconventional binding motifs of traditional GBPs.
Following Cram’s rules of preorganization and complementarity, a number of synthetic lectins have been developed with a preference for binding all equatorial glucosides. Cram’s rules state that to achieve high affinity, the receptors must be preorganized to accommodate the target size and shape and must be electronically complementary to the target molecule [40]. Thus, researchers first sought to bind carbohydrates using strong interactions such as boronate ester formation with the vicinal diols present in glucose, and boronic acid based synthetic lectins have achieved 1:1 Kas with glucose as high as 104 M1 in aqueous solvents and selectivity over other monosaccharide epimers [41]. Rigid receptors with preorganization also successfully bind all equatorial glucosides using only noncovalent interactions, with noteworthy examples being the tripodal benzene receptors developed by Mazik [42] and Roellens [43] and the temple series developed by Davis [44]. Temple receptors contain hydrophobic groups to bind axial regions and polar groups along the sides groups, and are thus ideal for binding all-equatorial carbohydrates, such as b-cellobiosyl with a Ka of 103 M1 in aqueous solvent [44]. Alternatively, the tripodal receptors achieve preorganization with 1,3,5-triethylbenzenes modified with H-bonding groups, like aminopyrroles, and bind to octyl-b-D-glucopyranoside with a Ka of 4.8 104 M1 in CDCl3 [43]. By introducing additional acetalic substituents to the tripodal receptors, selectivity for b-mannoside in CDCl3 over b-glucose was observed [45]. The terminal residues of cell surface glycans, however, are rarely composed of glucosides, and galactose, glucosamine, fucose, N-acetylglucosamine, N-acetylneuraminic acid, and mannose are prevalent [15,46], and these monosaccharides are appropriate targets for synthetic lectin drug delivery vehicles. Inspired by the complex
Figure 4
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(a) Synthetic lectin 1 that has multiple H-bonding groups and unrestricted rotation along several chemical bonds. (b) Representation of the equilibria between carbohydrate synthetic receptor 1 and b-Man at 258C in CDCl3. The receptor initially dimerizes and displays 1:1 binding, K1, with most monosaccharides. However, selectivity for b-Man is achieved as a result of 2:1 receptor:b-Man, K3, and 1:2 receptor: b-Man, K2, binding stoichiometrics that arise only with b-Man (from Ref [50]).Reproduced by permission of The Royal Society of Chemistry. www.sciencedirect.com
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binding modes utilized by natural GBPs, researchers have followed alternate strategies for developing synthetic lectins. The promiscuity of GBPs motivated work by Anslyn and coworkers [47], who showed how a nonspecific mixture of carbohydrate binding molecules subjected to principal component analysis could be used to determine the presence of sucralose by a boronic acid based pentapeptide. The success of these receptors is such that they are now employed commercially for removing excess glucose from blood [48]. Rather than attempt to design a carbohydrate receptor, the Waters group used dynamic covalent chemistry to discover a synthetic receptor specific for acetyl protected glucose (Ac4Glc) [49]. Recently, the Braunschweig group reported a synthetic lectin that achieves high specificity for a-octyl mannoside (a-OctMan) in CDCl3 using multivalent and cooperative binding modes [50]. Specifically, they created a small molecule replete with H-bonding groups, but with flexibility atypical of molecular receptors. Like many GBPs, this receptor bound to most monosaccharides weakly but achieves high selectivity for mannose as a result of 2:1 and 1:2 receptor:carbohydrate stoichiometries that arose only with a-OctMan, and a 16.8:1 selectivity over b-OctGal was observed (Figure 4). These new synthetic lectin design approaches highlight the importance of understanding the idiosyncrasies of glycan binding in approaching the challenge of synthetic lectins, which could lead to new synthetic lectins with alternate selectivities or novel recognition motifs.
Conclusions Herein our aim was to highlight two areas of glycosciences, namely glycan microarrays and synthetic lectins, where rapid developments are occurring from the convergence of biotechnology, supramolecular chemistry, surface science, polymer chemistry, and nanotechnology. Only a small fraction of work in the fields was chosen to identify current challenges in studying glycan recognition and to illustrate some particularly creative work. Multivalency, cooperativity, and surface effects imbue cell surface glycans with the ability to control some of the most sophisticated processes in biology, and understanding how these operate could not only suggest new approaches to addressing pressing health challenges but also inspire researchers to access the complexity of carbohydrate recognition to create stimuli–responsive hierarchical assemblies.
Acknowledgements The authors are grateful for generous financial support from the Air Force Office of Scientific Research (FA9550-12-1-0280) and the National Science Foundation (DBI1353823).
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest Current Opinion in Biotechnology 2015, 34:41–47
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chemotactic proteins using microarrays. Chem Biol 2007, 14:195-208. 24. Chevolot Y, Bouillon C, Vidal S, Morvan F, Meyer A, Cloarec JP, Jochum A, Praly JP, Vasseur JJ, Souteyrand E: DNA-based carbohydrate biochips: a platform for surface glycoengineering. Angew Chem Int Ed Engl 2007, 46:2398-2402. 25. Blixt O, Head S, Mondala T, Scanlan C, Huflejt ME, Alvarez R, Bryan MC, Fazio F, Calarese D, Stevens J et al.: Printed covalent glycan array for ligand profiling of diverse glycan binding proteins. Proc Natl Acad Sci U S A 2004, 101:17033-17038. 26. Houseman BT, Mrksich M: Carbohydrate arrays for the evaluation of protein binding and enzymatic modification. Chem Biol 2002, 9:443-454. 27. Park SJ, Shin IJ: Fabrication of carbohydrate chips for studying protein–carbohydrate interactions. Angew Chem Int Ed Engl 2002, 41:3180-3182. 28. Kohn M, Wacker R, Peters C, Schroder H, Soulere L, Breinbauer R, Niemeyer CM, Waldmann H: Staudinger ligation: a new immobilization strategy for the preparation of small-molecule arrays. Angew Chem Int Ed Engl 2003, 42:5830-5834. 29. Kalia J, Raines RT: Advances in bioconjugation. Curr Org Chem 2010, 14:138-147. 30. Fazio F, Bryan MC, Blixt O, Paulson JC, Wong CH: Synthesis of sugar arrays in microtiter plate. J Am Chem Soc 2002, 124:14397-14402. 31. Godula K, Rabuka D, Nam KT, Bertozzi CR: Synthesis and microcontact printing of dual end-functionalized mucin-like glycopolymers for microarray applications. Angew Chem Int Ed Engl 2012, 51:7881. Glycopolymers were covalently bound to surfaces using CuAAc and microcontact printing. The densities and orientations of the glycan ligands can be determined by altering backbone sturcture. 32. Bian SD, He JJ, Schesing KB, Braunschweig AB: Polymer pen lithography (PPL)-induced site-specific click chemistry for the formation of functional glycan arrays. Small 2012, 8:2000-2005. The authors developed a tool for creating glycan arrays by polymer pen lithography. The CuAAc reaction was used to covalently bind alkyne terminated mannose to azide coated glass surfaces. The glycan array demonstrated in the paper shows binding affinity to Cy3 modified con A. 33. Mehlich J, Ravoo BJ: Click chemistry by microcontact printing on self-assembled monolayers: a structure–reactivity study by fluorescence microscopy. Org Biomol Chem 2011, 9:4108-4115. 34. Wendeln C, Rinnen S, Schulz C, Arlinghaus HF, Ravoo BJ: Photochemical microcontact printing by thiol-ene and thiolyne click chemistry. Langmuir 2010, 26:15966-15971. 35. Bian SD, Zieba SB, Morris W, Han X, Richter DC, Brown KA, Mirkin CA, Braunschweig AB: Beam pen lithography as a new tool for spatially controlled photochemistry, and its utilization in the synthesis of multivalent glycan arrays. Chem Sci 2014, 5:2023-2030. The authors fabricated a grafted-form glycopolymers array using beam pen lithography. The 3D architecture can be controlled by the printing exposure time. The glycopolymers described in this paper show high binding affinity to Cy3 modified GBPs. 36. Godula K, Bertozzi CR: Density variant glycan microarray for evaluating cross-linking of mucin-like glycoconjugates by lectins. J Am Chem Soc 2012, 134:15732-15742. The authors created a glycopolymer array to study the role of glycan valency and spatial orientation on the binding to four different GBPs.
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37. Oyelaran O, Li Q, Farnsworth D, Gildersleeve JC: Microarrays with varying carbohydrate density reveal distinct subpopulations of serum antibodies. J Proteome Res 2009, 8:3529-3538. 38. Wang DN, Liu SY, Trummer BJ, Deng C, Wang AL: Carbohydrate microarrays for the recognition of cross-reactive molecular markers of microbes and host cells. Nat Biotechnol 2002, 20:275-281. 39. Godula K, Rabuka D, Nam KT, Bertozzi CR: Synthesis and microcontact printing of dual end-functionalized mucin-like glycopolymers for microarray applications. Angew Chem Int Ed Engl 2009, 48:4973-4976. 40. Cram DJ, Cram JM: Container Molecules and their Guests. Royal Society of Chemistry; 1997. 41. James TD, Sandanayake KRAS, Shinkai S: Saccharide sensing with molecular receptors based on boronic acid. Angew Chem Int Ed Engl 1996, 35:1910-1922. 42. Mazik M: Recent developments in the molecular recognition of carbohydrates by artificial receptors. RSC Adv 2012, 2:2630-2642. This review provides an overview of carbohydrate receptors developed recently through noncovalent interactions, including binding studies both in organic media and aqueous media. 43. Francesconi O, Ienco A, Moneti G, Nativi C, Roelens S: A selfassembled pyrrolic cage receptor specifically recognizes beta-glucopyranosides. Angew Chem Int Ed Engl 2006, 45:6693-6696. 44. Ke CF, Destecroix H, Crump MP, Davis AP: A simple and accessible synthetic lectin for glucose recognition and sensing. Nat Chem 2012, 4:718-723. The synthetic lectin developed in this paper shows specific binding to glucose in water. This approach suggests the potential for practical glucose monitoring in biological and environmental systems. 45. Nativi C, Cacciarini M, Francesconi O, Moneti G, Roelens S: A beta-mannoside-selective pyrrolic tripodal receptor. Org Lett 2007, 9:4685-4688. 46. Werz DB, Ranzinger R, Herget S, Adibekian A, von der Lieth CW, Seeberger PH: Exploring the structural diversity of mammalian carbohydrates (‘‘Glycospace’’) by statistical databank analysis. ACS Chem Biol 2007, 2:685-691. 47. Umali AP, Anslyn EV: A general approach to differential sensing using synthetic molecular receptors. Curr Opin Chem Biol 2010, 14:685-692. 48. Edwards NY, Sager TW, McDevitt JT, Anslyn EV: Boronic acid based peptidic receptors for pattern-based saccharide sensing in neutral aqueous media, an application in real-life samples. J Am Chem Soc 2007, 129:13575-13583. 49. Laughrey ZR, Kiehna SE, Riemen AJ, Waters ML: Carbohydrate– pi interactions: what are they worth? J Am Chem Soc 2008, 130:14625-14633. 50. Rieth S, Miner MR, Chang CM, Hurlocker B, Braunschweig AB: Saccharide receptor achieves concentration dependent mannoside selectivity through two distinct cooperative binding pathways. Chem Sci 2013, 4:357-367. A synthetic lectin which shows specific binding to b-Man was developed. The association between the carbohydrate receptor and saccharides was studied by variable temperature 1H NMR titrations. Cooperativity, multivalency, and concentration dependent selectivity were observed.
Current Opinion in Biotechnology 2015, 34:41–47
ScienceDirect Carbohydrate nanotechnology: hierarchical assembly using nature’s other information carrying biopolymers Xu Han, Yeting Zheng, Catherine J Munro, Yiwen Ji and Adam B Braunschweig Despite their central role in directing some of the most complex biological processes, carbohydrates — nature’s other information carrying biopolymer — have been largely ignored as building blocks for synthetic hierarchical assemblies. The non-stoichiometric binding and astronomical diversity characteristic of carbohydrates could lead to tantalizingly complex assembly algorithms, but these attributes simultaneously increase the difficulty of preparing carbohydrate assemblies and anticipating their behavior. Convergences in biotechnology, nanotechnology, polymer chemistry, surface science, and supramolecular chemistry have led to many recent important breakthroughs in glycan microarrays and synthetic carbohydrate receptors, where the idiosyncrasies of carbohydrate structure and binding are increasingly considered. We hope to inspire more researchers to consider carbohydrate structure, diversity, and binding as attractive tools for constructing synthetic hierarchical assemblies. Addresses Department of Chemistry, University of Miami, 1301 Memorial Drive, Coral Gables, FL 33146, USA Corresponding author: Braunschweig, Adam B ([email protected])
Current Opinion in Biotechnology 2015, 34:41–47 This review comes from a themed issue on Nanobiotechnology Edited by Igor L Medintz and Matthew Tirrell
http://dx.doi.org/10.1016/j.copbio.2014.11.016 0958-1669/# 2014 Published by Elsevier Ltd.
Introduction Mimicking the bottom-up assembly of hierarchical biological structures — such as cell membranes, tissues, organs, and ultimately, organisms — is a major goal of biotechnology and nanotechnology. Within cells, the information that directs hierarchical assembly is encoded within oligonucleotides and oligopeptides as dictated by the central dogma of biology. Both oligonucleotides and oligopeptides can be prepared by automated solid-phase syntheses, and the combination of facile preparation and well-understood recognition contributes to their common www.sciencedirect.com
adoption by researchers seeking to achieve synthetic bottom-up assemblies. In DNA-based and RNA-based nanotechnolgies for example, information is encoded within well-known Watson–Crick or Hoogsteen base pairings along the (deoxy)ribophosphate backbones, and oligonucleotides are commonly employed in gene chips, but they are also more recently used for constructing other hierarchical assemblies, including exotic molecular topologies [1–3], molecular machines [4,5], sensors [6], drug delivery systems [7], and logical operators [8,9]. While the use of oligopeptides in protein chips [10] or in phage displays [11] is well known, the supramolecular recognition of oligopeptides is also used in templating nanoparticle assembly [12,13], or in medical applications, such as repairing spinal cord injuries [14]. These systems are remarkable in that they mimic the multi-length scale order, complex binding stoichiometries, stimuli-responsiveness, and emergent properties that are the hallmarks of biomolecular information, and their successful development demonstrates the importance of continuing to study and mimic the ways biology encodes information in molecular and polymeric scaffolds. Many of the most complex biological processes, including protein folding, cell–cell binding, cell motility and signal transduction, occur as a result of binding events on the glycocalyx, which is a 100 nm–1 mm thick layer of glycans (Figure 1) on the cell surface that includes glycolipids, glycopeptides, and glycopolymers [15]. The information containing elements in these glycans are oligosaccharides, but despite this fact carbohydrates have hardly been explored in the context of synthetic hierarchical assemblies or on biochips as a consequence of their challenging preparation, structural diversity, and complex binding, which is characterized by multivalency and cooperativity. Because information in carbohydrates is encoded so differently than the binary algorithms of DNA and the sequence diversity is infinitely greater than that achievable with oligopeptides, working with carbohydrates inevitably leads us to question our assumptions about biological recognition and information transfer at the most fundamental levels. Herein we discuss how the uniqueness of carbohydrates is increasingly considered as a central focus of two research areas at the interface of nanotechnology and biology — specifically glycan microarrays and synthetic carbohydrate receptors — where major recent advances could lead to the realization of complex carbohydrate directed hierarchical assemblies. In discussing their commonalities, we hope to provide readers with an appreciation of the concepts, challenges, and potential of Carbohydrate Nanotechnology. Current Opinion in Biotechnology 2015, 34:41–47
42 Nanobiotechnology
Figure 1
Glycoprotein
Glycopolymer Glycolipid
Asn
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Glucosamine (GlcN)
Galactose (Gal)
N-Acetylglucosamine (GlcNAc) N-Acetylneuraminic acid (Neu5Ac)
Fucose (Fuc)
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Representation of the glycocalyx, which is a 100 nm–1 mm thick coating of glycoproteins, glycolipids, and glycopolymers on the surfaces of eukaryotic cells. Some of the most prevalent monosaccharides found on cell surface glycans, as well as their common symbolic representation, are shown.
Oligosaccharides are chains of monosaccharides that contain at least one carbonyl and multiple hydroxyl functions per residue, and often further functional group (Figure 2a–c). Each monosaccharide contains several stereocenters and typically occurs as an equilibrium mixture of 5-membered and 6-membered rings [16]. Because of the branching in oligosaccharide chains, the information density of oligosaccharides — which is the number of different oligomers that can be prepared from a chain of a given length — exceeds significantly what can be achieved with linear oligopeptides and oligonucleotides (Figure 2d) [17]. The carbohydrates in cell surface glycoconjugates can easily contain as many as 20 monosaccharides in an oligomer, enabling incredible structural variety arising from the number of monosaccharides in a chain and also from the different branching points and linkages (a or b). The preparation of oligosaccharides involves multistep routes that require selective functionalization of hydroxyl groups with nearly identical reactivity on scaffolds with tens of reactive hydroxyls. This complicates their preparation, such that carbohydrates are typically available only in small quantities, and just a small fraction of all the possible hexasaccharide combinations can be prepared with current synthetic methods. Additionally the specificity of glycan binding proteins (GBPs) is difficult to determine because the 1:1 association between carbohydrates and GBPs is weak (typically 102–103 M1) and GBPs are promiscuous, often binding many carbohydrates. To overcome low affinity, GBPs often possess multiple binding sites, and the glycans on cell surfaces present as multimeric assemblies, and these higher order binding modes increase affinity (up to 109 M1 increase in Ka, Current Opinion in Biotechnology 2015, 34:41–47
but typically 10–103 M1) and specificity, a phenomenon termed the cluster glycoside effect [18]. It is important to also note that binding between carbohydrates and GBPs frequently invokes cooperativity that arises from the interplay of multiple noncovalent interactions, making the binding strength and substrate specificity difficult to anticipate. In summary, carbohydrates are distinct from other information carrying biopolymers in terms of structure, synthesis, and recognition, and many fundamental questions and challenges remain that have delayed their incorporation into synthetic hierarchical assemblies. Elucidating how carbohydrates encode binding information within molecular structure could also lead to breakthroughs in disease detection, drug delivery, and therapeutics.
Glycan microarrays Glycan arrays consist of solid surfaces that display a library of glycans spatially encoded into micrometer scale spots, and the resulting surfaces are used to rapidly and combinatorially assess the affinity and selectivity of GBPs to the immobilized glycans [19]. The microarray format is particularly useful in the context of glycosciences because carbohydrate recognition, much more so than oligonucleotide or oligopeptide, is strongly dependent on cooperativity and multivalency — parameters that can be controlled and studied by varying the density and orientation of the immobilized glycans. Glycan arrays are an important step in the path towards more sophisticated carbohydrate nanotechnologies because they clarify how all the contributors to glycan binding determine affinity and selectivity. The two challenges that remain to increase the adoption of glycan arrays involve improving surface chemistry to access multivalent and cooperative binding interaction and increasing the diversity of the glycan libraries within the arrays, which is limited by the difficulty of preparing carbohydrates or obtaining natural samples of glycans. The immobilization chemistry — the chemical bonding and interactions that hold the glycans onto the substrates — has a major impact on glycan array performance, such that it has been shown that glycans printed using different immobilization strategies display different GBP selectivities [20]. These paradoxical data likely reflect both the promiscuity of GBPs and the poorly understand role of nanoscale phenomena, such as orientation and density, on glycan surface binding. In an effort to investigate the role of immobilization chemistry on binding and to improve array performance, researchers have used printing strategies based on noncovalent, covalent, and polymer chemistry, and in doing so, have simultaneously developed ingenious approaches for increasing array diversity. Noncovalent printing strategies bind glycans onto surfaces using supramolecular interactions such as Van der Waals, ionic, hydrogen bonding, and solvophobic interactions. The advantages of noncovalent approaches are that they can reduce the number of synthetic steps for www.sciencedirect.com
Carbohydrate nanotechnology Han et al. 43
Figure 2
(a)
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(a) The 4C1 conformation of the monosaccharide a-mannose (a-Man) with the anomeric carbon labeled as C1, following conventional notation. (b) The trisaccharide contains both a and b linkages. (c) The pentasaccharide contains multiple hydroxyl groups and further functional groups. (d) Table presents the number of different oligomers, N, that can be created by all possible combinations of a given oligomer size, n. Calculations are based on a4 nucleotides, N = 4n, b20 common amino acids, N = 20n, and c10 common mammalian monosaccharides that can adopt only a-pyranoside, or Q b-pyranoside conformations, N ¼ 20n nk¼1 ð3n 2Þ.
preparing glycans for printing, the surfaces are easy to fabricate, and the success of attachment is generally high. The first glycan array was prepared using such a noncovalent approach, whereby glycans bearing long alkyl chains were immobilized onto hydrophobic surfaces [21]. Several recent creative advances in this field have vastly improved noncovalent glycan arrays. In 2005, the Pohl group successfully immobilized fluorous-tagged carbohydrates onto fluorinated solid supports with a robotic spotter. This approach simplifies fabrication steps by directly attaching a single C8F17 tail to carbohydrates, www.sciencedirect.com
and importantly strong binding affinity between immobilized glycan and GBPs noncovalent bind pairs are observed [22]. Later glycan arrays were made by the attachment of sulfonated polysaccharides to poly-Llysine coated surfaces by Hsieh-Wilson et al. [23] to elucidate the effects of the glycoaminoglycans on GBP binding. In another recent example, Chevolot and co-workers built a new glycan array platform by immobilizing oligonucleotide-modified glycans onto surfaces printed with complementary DNA sequences [24] to vary precisely the distance between carbohydrate residues and the surfaces. These advances have resulted in control of orientation and significantly improved binding, but purely adsorptive approaches still require the preparation and isolation of carbohydrates to make the array. An alternate approach towards immobilizing glycans involves covalent attachment to a substrate, whereby a new covalent bond is formed between the glycans and the surfaces, and this strategy offers the potential to control glycan orientation and density by accessing the selectivity of organic chemistry. Covalent immobilization was demonstrated by Blixt, Wong, Paulson and coworkers, who attached amine containing glycans to N-hydroxysuccinimide activated slides, and they used this array to discover the specificity of a variety of GBPs including plant lectins, human GBPs, glycan-specific antibodies, and other proteins [25]. Different binding pairs, like erythrina cristagalli agglutinin to N-acetyllactosamine and differentiation-22 to Siaa-6-Galb1-4-GlcNAc were observed. This method however does not necessarily control orientation because complex glycans can contain multiple amine residues, and subsequent covalent strategies focused on bioorthogonal reactions that do not interfere with the functional groups common to glycans, and also proceed quickly and in high yield. The first example of such an approach involved the use of Diels– Alder cycloaddition [26] and hetero-Michael addition [27] reactions to form glycan chips. Other reactions that have been employed subsequently include the Staudinger Ligation [28,29], CuI-catalyzed azide-alkyne click reaction [29,30,31,32], and the light-induced thiol-ene reaction [33,34,35]. In these examples, the immobilized glycans bind successfully to the target GBPs, and the reactions can be carried out in the presence of spacers to explore how glycan density affects binding [36]. The limitations to purely covalent strategies are that the surface-reactive functional groups must be attached to already difficult to obtain glycans, and that cluster glycoside effects are not operating fully in monolayers, so binding specificity may not reflect accurately the conditions on cell surfaces. By leveraging ideas from polymer chemistry, researchers have recently made breakthrough advances that simplify the preparation of glycan chips, increase library diversity, and clarify the subtle interplay between carbohydrate Current Opinion in Biotechnology 2015, 34:41–47
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density and binding affinity. In polymer-based strategies, the backbone of either a naturally derived biopolymer or a synthetic polymer is used as a scaffold to prepare a multivalent saccharide display that extends away from the substrate, and in doing so create a binding environment that closely resembles the cell surface, and diversity can be increased by varying chain length, spacing, and carbohydrate presentation. Gildersleeve and coworkers have demonstrated the potential of this approach using the protein bovine serum albumin (BSA) as the polymer scaffold, where saccharides first attached to different linkers, like 6-aminohexanoic acid and 3-[(2-hydroxyethyl)thio]propanamide, are attached to the BSA lysine residues using EDC coupling reactions [37]. The BSA presents multiple attachment sites, and by varying saccharide concentration when printing onto a BSA-coated surface, they are able to change the degree of functionalization of the BSA to capture affects of multivalency. By
adding multiple different saccharides to the ink, they can attach different saccharides onto the same BSA chain and in effect create a new glycan and increase array diversity. In changing both carbohydrate density and composition on the biopolymer backbone, they have successfully developed a strategy to increase library diversity and have produced microarrays with as many as 537 glycans [37]. Wang et al. was the first to deposit a synthetic glycopolymer by printing onto a nitrocellulose coated surface using a grafted-to noncovalent strategy [38]. The Bertozzi group immobilized a synthetic glycopolymer with a poly (methylvinylketone) backbone and pendent N-acetylgalactosamine (GalNAc) residues to serve as mucin mimics that were subsequently attached to azide terminated surfaces using the CuI-catalyzed azide-alkyne click reaction. By adding spacer groups during the surface immobilization step, they were able to alter interchain spacing, and thereby distinguish how glycan valency and
Figure 3
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(a) A nanophotolithography platform was used to create grafted-from glycopolymers. (i) Beam pen tips coated with an ink mixture composed of 2,2-dimethoxy-2-phenylacetophenone and methacrylate-terminated carbohydrates in a polyethylene glycol matrix, deposits inks onto thiol terminated surfaces (ii) UV light passes through beam pen tips to initiate the polymerization reactions. (iii) Washing the surface removes unreacted chemicals, so only the covalently immobilized glycopolymers remain. (b) Fluorescence microscopy image of Cy3 labeled concanavalin A bound to glucose-bearing glycopolymers prepared by the process described in (a). (c) Fluorescence intensity of printed features decreases with decreasing chain length. The x-axis corresponds to the white line from left to right in the inset figure of (b). (d) Plot indicates the relationship between expose time and fluorescence intensity or polymer height. (e) Plot shows how different surface chemistries including the CuAAC (from Ref [32]), thiol acrylate (from Ref [35]), or thiol-ene polymerization (from Ref [35]), alter binding sensitivity towards concanavalin A.Reproduced by permission of The Royal Society of Chemistry. Current Opinion in Biotechnology 2015, 34:41–47
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Carbohydrate nanotechnology Han et al. 45
organization determine interactions with helix promatia agglutinin [39]. Applying a grafted-from approach, the Braunschweig group used tip-based nanophotolithography to create arrays of glycopolymer brushes by radical polymerization of glucose methacrylate directly from a thiol-terminated surface, where the light exposure time during printing was used to control polymer chain length [35]. They demonstrated a 100-fold increase in sensitivity towards GBPs compared to glycan monolayers, and they also show that GBP binding is dependent on polymer height (Figure 3). Thus glycan microarrays are a rapidly advancing field of biotechnology, where noncovalent interactions, surface science, and polymer chemistry are combining to accelerate our understanding of glycan recognition.
Synthetic lectins An equally important step towards carbohydrate nanotechnology is the development of carbohydrate-binding molecules, which, in addition to hierarchical assembly, could be used for delivering drugs to specific cell types, detecting diseases, or even as therapeutic agents by targeting and binding cell-surface glycans. The term synthetic-lectin has been coined to refer to man-made molecular receptors that bind carbohydrates, but glycans are amongst the most challenging natural targets for synthetic receptors that must distinguish between monosaccharides that may differ by only the orientation of a single stereocenter in a very competitive solvent. Despite these obstacles, researchers have made major advances in the preparation of synthetic lectins using traditional supramolecular approaches, and more recently, by creating new receptor designs inspired by the promiscuity and unconventional binding motifs of traditional GBPs.
Following Cram’s rules of preorganization and complementarity, a number of synthetic lectins have been developed with a preference for binding all equatorial glucosides. Cram’s rules state that to achieve high affinity, the receptors must be preorganized to accommodate the target size and shape and must be electronically complementary to the target molecule [40]. Thus, researchers first sought to bind carbohydrates using strong interactions such as boronate ester formation with the vicinal diols present in glucose, and boronic acid based synthetic lectins have achieved 1:1 Kas with glucose as high as 104 M1 in aqueous solvents and selectivity over other monosaccharide epimers [41]. Rigid receptors with preorganization also successfully bind all equatorial glucosides using only noncovalent interactions, with noteworthy examples being the tripodal benzene receptors developed by Mazik [42] and Roellens [43] and the temple series developed by Davis [44]. Temple receptors contain hydrophobic groups to bind axial regions and polar groups along the sides groups, and are thus ideal for binding all-equatorial carbohydrates, such as b-cellobiosyl with a Ka of 103 M1 in aqueous solvent [44]. Alternatively, the tripodal receptors achieve preorganization with 1,3,5-triethylbenzenes modified with H-bonding groups, like aminopyrroles, and bind to octyl-b-D-glucopyranoside with a Ka of 4.8 104 M1 in CDCl3 [43]. By introducing additional acetalic substituents to the tripodal receptors, selectivity for b-mannoside in CDCl3 over b-glucose was observed [45]. The terminal residues of cell surface glycans, however, are rarely composed of glucosides, and galactose, glucosamine, fucose, N-acetylglucosamine, N-acetylneuraminic acid, and mannose are prevalent [15,46], and these monosaccharides are appropriate targets for synthetic lectin drug delivery vehicles. Inspired by the complex
Figure 4
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(a) Synthetic lectin 1 that has multiple H-bonding groups and unrestricted rotation along several chemical bonds. (b) Representation of the equilibria between carbohydrate synthetic receptor 1 and b-Man at 258C in CDCl3. The receptor initially dimerizes and displays 1:1 binding, K1, with most monosaccharides. However, selectivity for b-Man is achieved as a result of 2:1 receptor:b-Man, K3, and 1:2 receptor: b-Man, K2, binding stoichiometrics that arise only with b-Man (from Ref [50]).Reproduced by permission of The Royal Society of Chemistry. www.sciencedirect.com
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binding modes utilized by natural GBPs, researchers have followed alternate strategies for developing synthetic lectins. The promiscuity of GBPs motivated work by Anslyn and coworkers [47], who showed how a nonspecific mixture of carbohydrate binding molecules subjected to principal component analysis could be used to determine the presence of sucralose by a boronic acid based pentapeptide. The success of these receptors is such that they are now employed commercially for removing excess glucose from blood [48]. Rather than attempt to design a carbohydrate receptor, the Waters group used dynamic covalent chemistry to discover a synthetic receptor specific for acetyl protected glucose (Ac4Glc) [49]. Recently, the Braunschweig group reported a synthetic lectin that achieves high specificity for a-octyl mannoside (a-OctMan) in CDCl3 using multivalent and cooperative binding modes [50]. Specifically, they created a small molecule replete with H-bonding groups, but with flexibility atypical of molecular receptors. Like many GBPs, this receptor bound to most monosaccharides weakly but achieves high selectivity for mannose as a result of 2:1 and 1:2 receptor:carbohydrate stoichiometries that arose only with a-OctMan, and a 16.8:1 selectivity over b-OctGal was observed (Figure 4). These new synthetic lectin design approaches highlight the importance of understanding the idiosyncrasies of glycan binding in approaching the challenge of synthetic lectins, which could lead to new synthetic lectins with alternate selectivities or novel recognition motifs.
Conclusions Herein our aim was to highlight two areas of glycosciences, namely glycan microarrays and synthetic lectins, where rapid developments are occurring from the convergence of biotechnology, supramolecular chemistry, surface science, polymer chemistry, and nanotechnology. Only a small fraction of work in the fields was chosen to identify current challenges in studying glycan recognition and to illustrate some particularly creative work. Multivalency, cooperativity, and surface effects imbue cell surface glycans with the ability to control some of the most sophisticated processes in biology, and understanding how these operate could not only suggest new approaches to addressing pressing health challenges but also inspire researchers to access the complexity of carbohydrate recognition to create stimuli–responsive hierarchical assemblies.
Acknowledgements The authors are grateful for generous financial support from the Air Force Office of Scientific Research (FA9550-12-1-0280) and the National Science Foundation (DBI1353823).
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest Current Opinion in Biotechnology 2015, 34:41–47
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