molecules

Review Multivalent Carbohydrate-Lectin Interactions: How Synthetic Chemistry Enables Insights into Nanometric Recognition

René Roy 1,*, Paul V. Murphy 2 and Hans-Joachim Gabius 3 1 Pharmaqam and Nanoqam, Department of Chemistry, University du Québec à Montréal, P. O. Box 8888, Succ. Centre-Ville, Montréal, QC H3C 3P8, Canada 2 School of Chemistry, National University of Ireland Galway, University Road, Galway, Ireland; [email protected] 3 Institute of Physiological Chemistry, Faculty of Veterinary Medicine, Ludwig-Maximilians-University Munich, Veterinärstr. 13, München 80539, Germany; [email protected] * Correspondence: [email protected]; Tel.: +1-514-987-3000 (ext. 2546)

Academic Editor: Trinidad Velasco-Torrijos Received: 7 April 2016; Accepted: 10 May 2016; Published: 13 May 2016 Abstract: Glycan recognition by sugar receptors (lectins) is intimately involved in many aspects of cell physiology. However, the factors explaining the exquisite selectivity of their functional pairing are not yet fully understood. Studies toward this aim will also help appraise the potential for lectin-directed drug design. With the network of adhesion/growth-regulatory galectins as therapeutic targets, the strategy to recruit synthetic chemistry to systematically elucidate structure-activity relationships is outlined, from monovalent compounds to glyco-clusters and glycodendrimers to biomimetic surfaces. The versatility of the synthetic procedures enables to take examining structural and spatial parameters, alone and in combination, to its limits, for example with the aim to produce inhibitors for distinct galectin(s) that exhibit minimal reactivity to other members of this group. Shaping spatial architectures similar to glycoconjugate aggregates, microdomains or vesicles provides attractive tools to disclose the often still hidden significance of nanometric aspects of the different modes of lectin design (sequence divergence at the lectin site, differences of spatial type of lectin-site presentation). Of note, testing the effectors alone or in combination simulating (patho)physiological conditions, is sure to bring about new insights into the cooperation between lectins and the regulation of their activity.

Keywords: agglutinin; galectin; glycocluster; glycodendrimer; glycophane; glycoprotein; liposomes; oligosaccharides; sugar code

1. The Third Alphabet of Life Synergy, by teaming up chemistry with biosciences, can be expected to arise when the topic on which to apply complementarity of the disciplines is clearly defined. In principle, the chemical platform of life includes molecular alphabets, whose ‘letters’ are a toolbox to form ‘messages’ (or ‘words’) in the form of oligomers and polymers. “To an observer trying to obtain a bird’s eye view of the present state of biochemistry, or what is sometimes referred to as molecular biology, life may until very recently have seemed to depend on only two classes of compounds: nucleic acids and proteins” [1]. Their broad natural occurrence is shared by a third group, i.e., glycans. Present in Nature as polysaccharides such as chitin, or as part of cellular glycoconjugates, i.e., glycoproteins and glycolipids [2–13], their ubiquity and abundance have attracted interest to characterize rules of structural design, starting with the composition. This systematic work on material from pro- and eukaryotes step by step led to compiling the list of all individual building blocks, which form cellular glycocompounds. With the analogy

Molecules 2016, 21, 629; doi:10.3390/molecules21050629 www.mdpi.com/journal/molecules Molecules 2016, 21, 629 2 of 35 Molecules 2016, 21, 629 2 of 36 eukaryotes step by step led to compiling the list of all individual building blocks, which form cellular glycocompounds. With the analogy to a molecular language in mind, this list constitutes the thirdto a molecular alphabet of language life, which in mind, complements this list constitutes the alphabets the of third nucleotides alphabet and of life, amino which acids complements (Figure 1). Asthe is alphabets common of for nucleotides amino acids and in amino proteins, acids substitutions (Figure1). As such is commonas phosphorylation for amino acids or sulfation in proteins, also broadensubstitutions the range such asof phosphorylationletters for glycans. or This sulfation use of also the broaden same principle the range of of post-synthetic letters for glycans. modifications This use underscoresof the same principlethe equivalence of post-synthetic of these modificationsalphabets with underscores respect to thetheir equivalence involvement of thesein the alphabets flow of withinformation respect from to their genes involvement to cellular in activities the flow [14,15]. of information from genes to cellular activities [14,15].

OH HO HO OH OH OH O O HO O Me O HO OH OH HO OH HO OH HO OH OH HO HO

D-Glucose (Glc) D-Galactose (Gal) D-Mannose (Man) -L-Fucose (Fuc)

OH HO OH O O HO O HO HO OH HO OH HO OH AcHN AcHN OH

D-N-Acetylglucosamine (GlcNAc) D-N-Acetylgalactosamine (GalNAc) D-Xylose (Xyl)

OH OH O CO2H HO C O HO2C O 2 HO OH HO OH OH OH HO HO HO O OH OH OH HO a b OH D-Glucuronic acid (GlcA) L-Iduronic acid (IdoA) L-Arabinose (Ara) HO H OH HO HO OH OH CO2H Me O HO O HO O OH HO CO2H OH AcHN OH HO OH

-L-Rhamnose (Rha) 5-N-Acetyl- 2-Keto-3-deoxy-D- neuraminic acid (Neu5Ac) manno-octulosonic acid (Kdo)

Figure 1. IllustrationIllustration of of the alphabet of thethe sugarsugar language.language. Structural representation, name and symbol as well as the set of known acceptor positionspositions (arrows) in glycoconjugates are given for each letter. Four sugars have L-configuration: fucose (6-deoxy-L-galactose), rhamnose (6-deoxy-L-mannose) letter. Four sugars have L-configuration: fucose (6-deoxy-L-galactose), rhamnose (6-deoxy-L-mannose) L and arabinose are introduced duringduring chain elongation, whereas L-iduronic acid (IdoA) results from post-synthetic epimerization of glucuronic acid at C-5. The 1C4 conformation1 of IdoA (a) is in equilibrium post-synthetic epimerization of glucuronic acid at C-5. The C4 conformation of IdoA (a) is in with the 2S0 form (b) 2in glycosaminoglycan chains, where this uronic acid can be 2-sulfated. All other equilibrium with the S0 form (b) in glycosaminoglycan chains, where this uronic acid can be 2-sulfated. Allletters other areletters D-sugars. are DNeu5Ac,-sugars. one Neu5Ac, of the onemore of than the more 50 sia thanlic acids, 50 sialic often acids, terminates often terminates sugar chains sugar in animalchains inglycoconjugates. animal glycoconjugates. Kdo is a constituent Kdo is a of constituent lipopolysaccharides of lipopolysaccharides in the cell walls in of the Gram-negative cell walls of bacteriaGram-negative and is bacteriaalso found and in is cell also wall found polysacchar in cell wallides polysaccharides of green algae of and green higher algae plants. and higher Foreign plants. to mammalianForeign to mammalian glycobiochemistry, glycobiochemistry, microbial polysacc microbialharides polysaccharides contain the containfuranose the ring furanose form of ring D-galactose form of andD-galactose also D/ andL-arabinose, also D/L -arabinose,then indicated then by indicated an italic by “ anf” italicderived “f” derivedfrom the from heterocyclus the heterocyclus furan. furan. The Theα-anomerα-anomer is prevalent is prevalent for the for pentose the pentose arabinose, arabinose, e.g., e.g.,in mycobacterial in mycobacterial cell wall cell wallarabinogalactan arabinogalactan and lipoarabinomannan.and lipoarabinomannan. β1-5/6-Linkedβ1-5/6-Linked galactofuranoside galactofuranoside is pr isesent present in inthe the arabinogalactan arabinogalactan and and the α1-3/6 linkage linkage in in lipopolysaccharides lipopolysaccharides (adapted (adapted from from [15]). [15]).

Drawing on textbook knowledge, the consideration of availability of several hydroxyl groups for building the glycosidic bond lets the special talent of carbohydrates to encode bioinformation become obvious. In contrast to the fixed connecting points of nucleotides and amino acids, two

Molecules 2016, 21, 629 3 of 36 monosaccharides can be covalently linked to yield a groups of isomers: the anomeric position and linkage points are the sources of variability. What initially appeared as a deterrent to work with glycans, that is the enormous structural variety unsurpassed among biopolymers that so fittingly is expressed in the term ‘complex carbohydrates’, was then realized to be the basis for enabling high-density coding. Ironically, what at first impeded smooth progress turned out to be a means to put signals for different aspects of cell sociology into a minimum of space, an amazing functional dimension of glycans [14,15]. Due to the growing realization of this potential developing tools for chemical analysis of glycans on all structural levels [16] is crucial to delineate structure-activity relationships. That “in many cases the real role of biological glycoconjugate glycans is still unknown and, in many cases, we are unable to answer the question: why are proteins and lipids glycosylated?” [17] and that “on the whole, the role of many glycans remains obscure and the subject of speculation or controversy” [17] is no longer valid. In fact, the sequence of glycans, as in proteins or nucleic acids, gives shape to these biomolecules, seemingly minor alterations acting like switches between conformations [18–22]. Of note, the often encountered limitations to intramolecular flexibility of biorelevant oligosaccharides, combined with their enormous potential to engage in intermolecular recognition via hydrogen bonding, C-H/π-interaction and apolar complementarity, make them well-suited as binding partners. They are like molecular keys fitting into appropriate locks, and such receptors are present in Nature, as previously outlined in detail in this journal for lectins [23,24].

2. Lectins: Translators of Sugar-Encoded Information Given the realized potential of carbohydrates to serve as a chemical platform for generating a large array of molecular signals, which defines the glycome, a functional implication could be expected if emergence of sugar receptor(s) is not a singular event in evolution. Strongly supporting the concept of significance of carbohydrate-protein binding, several families of proteins with this ability have developed, among them figuring prominently the lectins. They are separated from enzymes treating the bound glycan as substrate, immunoglobulins and transport proteins for free mono- or oligosaccharides [23–25]. In the case of animals and man, more than a dozen types of protein folds are capable to form sites to accommodate such ligands on the basis of mutual recognition (for a gallery of these lectins presenting folds and a snapshot of the molecular rendez-vous with a ligand, please see [26]), a sweet complementarity. Structurally, the architecture of the contact site spans the range from shallow groves to deep pockets, in certain cases recruiting the assistance of Ca2+ for structural organization or coordination bonds with hydroxyl groups of the sugar [26–30]. Physiologically, the architecture then determines the reactivity to glycans, generally with no or very limited promiscuity. From the perspective of the glycan, the same sugar epitope can become the target of several lectin types. As a means of implementing flexibility and responsiveness, these cellular signals can undergo a recoding without requiring neosynthesis. Enzymatic remodeling in situ can drastically alter a glycan’s reactivity its binding profile, opening the way to dynamic up- or down regulation of binding capacity. A classical example is the conversion of the hexasaccharide of the disialo-ganglioside GD1a to that of monosialo GM1 by a cell surface ganglioside sialidase (Neu3). Removal of one sialic acid moiety (for its structure, please see Figure1) from ganglioside GD1a generates a functional marker. It signals differentiation of neuroblastoma cells in vitro and acquired responsiveness of activated effector T cells to a control by regulatory T cells, in both cases sensed exerted by an endogenous lectin [31,32]. The formation of a complex between the produced ligand, i.e., the pentasaccharide of ganglioside GM1, and the lectin, i.e., the homodimeric galectin-1, is the molecular trigger for a signaling cascade, which results in the final response (growth/activity inhibition, setting limits to tumor cell proliferation and T cell-mediated activity causing auto-immune diseases). This connection between the glycan and the cell sociology justifies to call the human lectin a translator of the sugar (ganglioside GM1)-encoded message. What is in general so intriguing about the recognition process with endogenous receptors is the exquisite selectivity of a tissue lectin (receptor) for distinct cellular glycoconjugates (in terms of the Molecules 2016, 21, 629 4 of 36 scaffold and its glycosylation). As it turned out, often only few glycoproteins or glycolipids from the complexity on cells are binders, and lectin reactivity is temporally and spatially tightly regulated. Modulations of glycoconjugate synthesis, routing and/or degradation and of the structure of the presented glycan(s) open ways to make cells specifically prepared for carbohydrate-protein recognition. To give a further biomedically relevant example, the tumor suppressor p16INK4a is the master regulator for a glycan-based effector route to drive pancreatic carcinoma cells into programmed cell death: the underlying caspase-9 activation is achieved by clustering of α5β1-integrin and galectin-1 on the cell surface, a process favored by their concerted upregulation and the increase of galectin-1-suited integrin glycosylation through reducing α2,6-sialylation of its N-glycans [33,34]. That availability of an antagonist of galectin-1 in this context, i.e., galectin-3, is downregulated [35] lays open intricacies of fine-tuning this effector pathway. In addition to the direct contact possible and the complementarity between the cognate glycan determinant and the lectin, factors of spatial presentation, too, will most likely matter. Multivalency of glycans, achieved by branching or tight arrangement in a microdomain, also artificially established by a lectin and synthetic ligands [36], facilitates fractional high-affinity binding in the nM (and even below) range for galectins [37]. To give research to unravel the molecular origin of the selectivity of matchmaking direction, affinity regulation has been formally assigned to work on six levels [30,38]. After the target identification, the endogenous lectin in this case acts as a cross-linker of a glycoprotein, thus turning attention to the modes of structural design of tissue lectins are relevant for the outcome. As glycan structures vary in terms of branching and repeats, per glycan and per glycoconjugate, various modes are realized to present a carbohydrate recognition domain (CRD) (Figure2). Looking at the mentioned galectin-1 (bottom, left), it is a homodimer of two identical CRDs, ideal for cross-linking its counter receptor(s) to elicit the intracellular signaling cascade. Looking through the different sections of Figure2, and through its legend presenting further details on the respective lectins, it becomes clear that the topology of CRD presentation has gained a large degree of diversity, and there is more. Detailed analyses within lectin families based on database mining have underscored that the course of evolution has led to complexity on several levels: gene diversification in terms of variations of the number of genes in each family by duplications/losses and of the individual sequences, even among related species [39–44]. In overview, covalent and non-covalent modes of linking CRDs as well as the connection of CRDs with other types of modules are frequent among lectins, signaling a fundamental functionality to be disclosed by thorough structure-activity investigations. Here, using bioinspired synthetic ligands as sensors or glycoclusters as molecular rulers offers a promising perspective. To depict principles of this line of research we here describe instructive examples selected from work on a class of adhesion/growth-regulatory lectins. How carbohydrate chemistry can advance our level of understanding of aspects of how selectivity is achieved is thus examined for biomedically relevant effectors, two already mentioned above. These proteins are referred to as galectins (please see Figure2, left part and Figure3 for their modes of structural design in vertebrates). Molecules 2016, 21, 629 5 of 36 Molecules 2016, 21, 629 5 of 35

Figure 2. Illustration of the strategic ways that carbohydrate recognition domains (CRDs) in animal Figure 2. Illustration of the strategic ways that carbohydrate recognition domains (CRDs) in lectins are positioned to reach optimal ligand selection (for example, to separate self from non-self animal lectins are positioned to reach optimal ligand selection (for example, to separate self from glycan profiles in innate immunity) and topological complementarity. From left to right, the CRD non-selfdisplay glycan in the profiles three insubtypes innate within immunity) the galectin and topological family (chimera-type complementarity. (a), proto-type From left(b) and to right, the CRDtandem-repeat-type display in the (c three) arrangements subtypes binding within to the the galectinsaccharide family portion (chimera-type of a ganglioside ( aor); a proto-type branched (b) and tandem-repeat-typecomplex-type N-glycan ( c)without arrangements or with terminal binding α to2,3-sialylation; the saccharide please portion see also of Figure a ganglioside 3), the or a branchedpresentation complex-type of CRDs (C-typeN-glycan or fibrinogen-like without or with doma terminalin) in serumα2,3-sialylation; and surfactant please collectins see or also ficolins Figure 3), the presentation(d) connected of to CRDs their (C-typecollagenous or fibrinogen-like stalks and the domain)non-covalent in serumassociatio andn surfactantof binding collectinssites in or ficolinstransmembrane (d) connected C-type to their lectins collagenous by α-helical stalks coiled-coil and the stalks non-covalent (e) (for example association asialoglycoprotein of binding and sites in transmembraneKupffer cell receptors, C-type lectins the scavenger by α-helical receptor coiled-coil C-type lectin, stalks CD23, (e) (forDC-SIGN example or DC-SIGNR) asialoglycoprotein are given. and KupfferSimilar cell to receptors, tandem-repeat-type the scavenger galectins, receptor the C-type C-type family lectin, of lectins CD23, also DC-SIGN has a branch or DC-SIGNR) of members arewith given. this design, i.e., immulectins-1, -2 and -3. Next, the tandem-repeat display in the mannose-specific Similar to tandem-repeat-type galectins, the C-type family of lectins also has a branch of members with macrophage receptor (f) (also found on dendritic cells, hepatic endothelial cells, kidney mesangial this design, i.e., immulectins-1, -2 and -3. Next, the tandem-repeat display in the mannose-specific cells, retinal pigment epithelial cells and tracheal smooth muscle cells) and the related C-type lectin macrophage receptor (f) (also found on dendritic cells, hepatic endothelial cells, kidney mesangial cells, Endo180 with eight domains as well as in the cation-independent P-type lectin with 15 domains is retinalpresented. pigment Capacity epithelial for cells sugar and binding tracheal is confined smooth to muscle only few cells) domains and the as depicted. related C-type The occurrence lectin Endo180 of

with eightlectin domainsactivity for as wellGalNAc-4-SO as in the4-bearing cation-independent pituitary glycoprotein P-type lectin hormones with 15in domains the cysteine-rich is presented.

Capacitydomain, for sugar a member binding of the is confinedβ-trefoil fold to only family few with domains one (QxW) as depicted.3 domain The in the occurrence N-terminal of section lectin activityof for GalNAc-4-SOthe macrophage4-bearing mannose pituitary receptor glycoprotein (amino acids hormones8–128), which in theis linked cysteine-rich via a fibronectin-type-II- domain, a member of therepeat-containingβ-trefoil fold family module with to the one tandem-repeat (QxW)3 domain section, in is the alsoN included-terminal into section the schematic of the macrophagedrawing mannosefor these receptor lectins (amino with more acids than 8–128), one type which of CRD is pe linkedr protein via chain. a fibronectin-type-II-repeat-containing Moving further to the right side; module(g) tothe the association tandem-repeat of a distal section, CRD isin also selectins included (attached into theto an schematic epidermal-growth-factor drawing for these (EGF)-like lectins with moredomain than one and type two ofto nine CRD complement-binding per protein chain. consensu Movings furtherrepeats) toor thein the right siglec side; subfamily (g) the of association I-type lectins using 1–16 C2-set immunoglobulin-like units as spacer equivalents to let the CRD reach out to of a distal CRD in selectins (attached to an epidermal-growth-factor (EGF)-like domain and two to contact ligands and to modulate capacity to serve in cis- or trans-interactions on the cell surface is nine complement-binding consensus repeats) or in the siglec subfamily of I-type lectins using 1–16 shown. The force-dependent alterations of the topological arrangement of the two distal domains in C2-setselectins immunoglobulin-like accounts for catchunits bonds as of spacerselectins, equivalents a canonical toimmunoreceptor let the CRDreach tyrosine-based out to contact inhibitory ligands and tomotif modulate (ITIM) together capacity with to a serve putative in cistyrosine-based- or trans-interactions motif is frequently on the present cell surface in the intracellular is shown. The force-dependentportion of siglecs. alterations C2-set ofdomains the topological linked to fibron arrangementectin-type-III of therepeats two establish distal domains the extracellular in selectins accountssection for of catch the I-type bonds lectins of selectins, L1 and neural a canonical cell adhesion immunoreceptor molecule (NCAM) tyrosine-based (h). In the inhibitory matrix, the motif (ITIM)modular together proteoglycans with a putative (i) (hyalectans/lecticans: tyrosine-based motif aggrecan, is frequently brevican, present neurocan in and the versican) intracellular interact portion of siglecs.(i) with C2-set hyaluronan domains (and linkedalso link to protein) fibronectin-type-III via the link-protein-type repeats establish modules theof the extracellular N-terminal G1 section of thedomain I-type (and lectins an L1Ig-like and module); neural cell(ii) with adhesion receptors molecule binding (NCAM) to the glycosaminoglycan (h). In the matrix, chains the in modular the proteoglycanscentral region (i) (hyalectans/lecticans: and (iii) with carbohydrates aggrecan, or pr brevican,oteins (fibulins-1 neurocan and and-2 and versican) tenascin-R) interact via the (i) with C-type lectin-like domain flanked by EGF-like and complement-binding consensus repeat modules hyaluronan (and also link protein) via the link-protein-type modules of the N-terminal G1 domain (and (kindly provided by H. Kaltner) (from [38], with permission). an Ig-like module); (ii) with receptors binding to the glycosaminoglycan chains in the central region and (iii) with carbohydrates or proteins (fibulins-1 and -2 and tenascin-R) via the C-type lectin-like domain flanked by EGF-like and complement-binding consensus repeat modules (kindly provided by H. Kaltner) (from [38], with permission). Molecules 2016, 21, 629 6 of 36 Molecules 2016, 21, 629 6 of 35

Figure 3. TheThe three three basic basic types types of of modular modular design of galect galectins,ins, as given in the context of other lectins in Figure 22.. Proto-typeProto-type proteins proteins (e.g., (e.g., galectins-1, galectins-1, -2 -2 and and -7) -7) are are non-covalently non-covalently associated associated homodimers, homodimers, the thevertebrate vertebrate tandem-repeat-type tandem-repeat-type galectins galectins are formed are form byed two by different two different CRDs connectedCRDs connected by a linker by a (length linker (lengthcan vary can by vary alternative by alternative splicing andsplicing between and species)between and species) the chimera-type and the chimera-type galectin-3 hasgalectin-3 an N-terminal has an Ntail-terminal composed tail ofcomposed a peptide of witha peptide site(s) with for site(s) Ser phosphorylation for Ser phosphorylation and Gly/Pro-rich and Gly/Pro-rich sequence sequence repeats repeatsenabling enabling self-association self-association in the presence in the presence of multivalent of multivalent ligands. ligands.

3. Galectins: Galectins: Definition Definition and and Structures Structures To qualifyqualify to to become become a canonicala canonical member member of this of this family family a protein a protein must satisfymust satisfy the following the following criteria: criteria: (i) to have a CRD with a β-sandwich fold, first described in this field for the leguminous (i) to have a CRD with a β-sandwich fold, first described in this field for the leguminous lectin lectin concanavalin A and illustrated in the Gallery of Lectins [27], is a compact structural concanavalin A and illustrated in the Gallery of Lectins [27], is a compact structural platform platform consisting—in the case of human galectin-1—of two antiparallel β-sheets of five to six consisting—in the case of human galectin-1—of two antiparallel β-sheets of five to six strands, strands, respectively, respectively, (ii) to bind compounds from the group of β-galactosides and (ii) to bind compounds from the group of β-galactosides and (iii) to share a sequence signature (mostly the amino acids in contact with the ligand, especially (iii) to share a sequence signature (mostly the amino acids in contact with the ligand, especially a a central Trp for C-H/π-interaction with the B-face of galactose) [26,43,45–47]. central Trp for C-H/π-interaction with the B-face of galactose) [26,43,45–47]. In vertebrates the the CRD CRD is commonly presented in in three topological modes modes given given in in Figure Figure 33,, associated with a tail consisting mostly of non-triplenon-triple helical collagen-like repe repeatsats suited for aggregation (chimera type), as a non-covalently associated homodimer (proto (proto type) type) and as a covalently linked heterodimer (tandem-repeat type). type). Before Before synthetic synthetic wo workrk to to prepare prepare binders binders for for galectins galectins is is outlined, outlined, an overview of of diversity diversity of of galectin galectin representation representation among among species species is isgiven. given. Computational Computational mining mining of databases,of databases, together together with with experimental experimental validation validation of active of active genes, genes, accounts accounts for the for phylogenetic the phylogenetic tree thattree thatsummarizes summarizes the number the number of galectins of galectins in selected in selected species species (Figure (Figure 4). 4Reflecting). Reflecting the the dynamics dynamics of theof the evolutionary evolutionary process, process, the the galectin galectin display display is isnot not constant constant between between species, species, an an important fact precluding simplesimple extrapolations extrapolations and and calling calling for thoroughfor thorough analysis analysis of each of individual each individual protein. protein. Equally Equallyimportant, important, galectins galectins likely form likely a networkform a network of members of members of each groupof each (to group give a(to clinically give a clinically relevant relevantexample, example, colon cancer colon cells cancer express cells most express tested most galectins, tested galectins, responsive responsive to differentiation to differentiation and providing and providingprognostic prognostic information inform [48,49ation], posing [48,49], the challengeposing the to challenge define properties to define of properties the individual of the proteins individual and proteinscooperation/antagonism and cooperation/antagonismin situ. in situ. To start characterizingcharacterizing thethe ligandligand specificity,specificity, synthetic synthetic chemistry chemistry is is invaluable invaluable due due to to its its ability ability to tomake make lead lead compounds compounds available available for for affinity affinity testing testing (for (for a compilation a compilation of experimentalof experimental approaches approaches to tocharacterize characterize lectin lectin activity, activity, please please see see Table Table1 in1 in [ 26 [26]).]). Due Due to to the the advantage advantage ofof requiringrequiring onlyonly small amounts of lectin and ligand on a multi-assay scale, glycan microarrays microarrays have become instrumental for comparative analysis,analysis, settingsetting the the stage stage for for the the structural structural follow-up follow-up studies studies (for a(for flow a scheme,flow scheme, please pleasesee Figure see5 )[Figure50–54 5)]. This[50–54]. and This other and glycan-based other glycan-based techniques delineatedtechniques thedelineated grading ofthe bioactivity grading of bioactivitydiverse glycans, of diverse teaching glycans, the lessonteaching that the various lesson structural that various changes, structural e.g., changes, introduction e.g., ofintroduction sialylation of or sialylationsulfation (as or mentioned sulfation (as above) mentioned and extension above) toand oligosaccharides extension to oligosaccharides with branching andwith sequence branching repeats, and sequencecan matter repeats, markedly can for matter distinct markedly members for of distinct the family members (for examples, of the family please (for see [examples,55–65]). With please lactose see [55–65]).as reference, With significant lactose as affinityreference, enhancements significant affinity were found enhancements for the saccharides were found given for the in Figuresaccharides5, for givenexample in Figure with the 5, for synthetic examplep-nitrophenyl with the synthetic lactoside, p-nitrophenyl a classical lactoside, test case studied a classical up test to now, case recentlystudied upby NMRto now, spectroscopy recently by usingNMR15 spectroscopyN-labeled galectin-1 using 15N-labeled [66]. These galectin-1 structures, [66]. at theThese same structures, time, establish at the samea platform time, toestablish proceed a platform to address to theproceed issue to of addr selection.ess the One issue preferred of selection. galectin One ligand, preferred as depictedgalectin ligand,in Figure as6 depicted (bottom), in is Figure a repeat 6 (bottom), of the disaccharide is a repeat of [ 67the]. disaccharide Questions to [67]. be answered Questions are to whetherbe answered and are whether and to what extent homologous proteins have different binding properties and whether a target-specific blocking might be accomplished.

Molecules 2016, 21, 629 7 of 36 to what extent homologous proteins have different binding properties and whether a target-specific blocking might be accomplished. Molecules 2016, 21, 629 7 of 35 Molecules 2016, 21, 629 7 of 35

Figure 4. Canonical galectins (according to the classification given in Figure 2) in model organisms Figure 4. Canonical galectins (according to the classification given in Figure 2) in model organisms Figureon 4. theCanonical level of gene galectins (Roman (according number), transcript to the classification (Arabic number) given and in protein Figure (numerical2) in model information). organisms on on the level of gene (Roman number), transcript (Arabic number) and protein (numerical information). the levelFor details, of gene please (Roman see [42,43]. number), transcript (Arabic number) and protein (numerical information). For details,For details, please please see see [42 [42,43].,43].

Figure 5. Sequence of steps to identify high-affinity carbohydrate ligands for galectin binding. Galectin Figure 5. Sequence of steps to identify high-affinity carbohydrate ligands for galectin binding. Galectin Figureinteraction 5. Sequence with of the steps most to potent identify binders high-affinity based on carbohydrate this screening ligandscan then for be galectinstudied further binding. on Galectinthe interaction with the most potent binders based on this screening can then be studied further on the interactionstructural with level. the most potent binders based on this screening can then be studied further on the structuralstructural level. level.

Molecules 2016, 21, 629 8 of 36 Molecules 2016, 21, 629 8 of 35

Figure 6. Representative structures of galectin ligands. Figure 6. Representative structures of galectin ligands.

Table 1. Amino acids of the CRDs of six galectins directly interacting with lactose residues are shown Table 1. Amino acids of the CRDs of six galectins directly interacting with lactose residues are shown in in red, and amino acids that interact with lactose via water-mediated hydrogen bonds are shown in red, and amino acids that interact with lactose via water-mediated hydrogen bonds are shown in blue. blue. Comparison with galectin-1 (PDB-1GZW), galectin-2 (PDB-1HLC), galectin-3 (PDB-1KJL), Comparison with galectin-1 (PDB-1GZW), galectin-2 (PDB-1HLC), galectin-3 (PDB-1KJL), galectin-4 galectin-4 (PDB-1X50), galectin-7 (PDB-4GAL), and galectin-8 (PDB 3AP9). Amino acids letter codes (PDB-1X50), galectin-7 (PDB-4GAL), and galectin-8 (PDB 3AP9). Amino acids letter codes are indicated are indicated in parentheses for Gal-3 [26]. in parentheses for Gal-3 [26]. CRD-Subsite Galectin-1 Galectin-2 Galectin-3 Galectin-4C Galectin-7 Galectin-8N CRD-SubsiteA Galectin-1Ser29 Galectin-2Gly30 Arg144 Galectin-3 (R) Galectin-4CSer220 Galectin-7Arg31 Galectin-8NArg45 AB Ser29Val31 Gly30Val32 Ala146 Arg144 (A) (R) Ala222 Ser220 His33 Arg31 Gln47 Arg45 BC Val31Asn33 Val32Asn34 Asp148 Ala146 (A)(D) Asn224 Ala222 Asn35 His33 Asp49 Gln47 CD Asn33Gly35 Asn34Gly36 Asp148Gln150 (Q) (D) Lys226 Asn224 Leu37 Asn35 Gln51 Asp49 DE Gly35- Gly36- Gln150- (Q) Lys226- Leu37- Arg59 Gln51 EF His44 - His45 - His158 - (H) His236 - His49 - His65 Arg59 GF His44Asn46 His45Asn47 Asn160 His158 (H)(N) Asn238 His236 Asn51 His49 Asn67 His65 G Asn46 Asn47 Asn160 (N) Asn238 Asn51 Asn67 H Arg48 Arg49 Arg162 (R) Arg240 Arg53 Arg69 H Arg48 Arg49 Arg162 (R) Arg240 Arg53 Arg69 I His52 - - - - - I His52 - - - - - JJ Asp54Asp54 Gln52Gln52 Glu165 (E) ------K Asn61Asn61 Asn58Asn58 Asn174 (N) Asn249 Asn249 Asn62 Asn62 Asn79 Asn79 L Trp68Trp68 Trp65Trp65 Trp181 (W) Trp256 Trp256 Trp69 Trp69 Trp86 Trp86 M Glu71Glu71 Glu68Glu68 Glu184 (E (E)) Glu259 Glu259 Glu72 Glu72 Glu89 Glu89 N Arg73Arg73 Arg70Arg70 Arg186 (R (R)) Lys261 Lys261 Arg74 Arg74 Ile91 Ile91 O -- Arg120Arg120 Ser237 (S) Gln313 Gln313 Gly124 Gly124 Tyr141 Tyr141

In that case, a a high-affinity high-affinity and specific specific carbohyd carbohydraterate derivative would need to be identified identified for each galectin. Because Because the the CRDs CRDs of of the galectins ar aree rather conserved, this represents a formidable synthetic challenge. However, However, if if one takes the N-terminal CRD of galectin-8 as an example, distinct preferences have indeed developed [[68–74].68–74]. Load Loadeded with either LNF-III (Figure 77 left)left) oror 331′-sulfated lactose (Figure (Figure 77 right),right), thethe structuresstructures ofof thethe complexescomplexes andand thethe intimateintimate interactionsinteractions (shown in Figure 88)) reveal thatthat itit becomesbecomes conceivable conceivable that that achieving achieving selectivity selectivity gains gains is not is not an insurmountable an insurmountable task. task. This Thisexample example clearly clearly demonstrates demonstrates that th suitablyat suitably functionalized functionalized carbohydrate carbohydrate derivatives derivatives cancan eveneven take the place of of rather complex complex glycans. glycans. Indeed, Indeed, alread alreadyy a arationally rationally derivatized derivatized disaccharide disaccharide can can offer offer a substantiala substantial selectivity selectivity gain, gain, despite despite the the overall overall rather rather conserved conserved contact contact pattern pattern of of galectins for lactose (Table 1 1).).

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Molecules 2016, 21, 629 9 of 35

FigureFigure 7.7. Comparison of structuresstructures ofof LNF-IIILNF-III ((leftleft)) andand 331′-sulfated-sulfated lactoselactose derivativesderivatives ((rightright)) thatthat Figure 7. Comparison of structures of LNF-III (left) and 3′-sulfated lactose derivatives (right) that bindbind almostalmost equallyequally well well to to human human galectin-8N. galectin-8N. Suggested Suggested additional additional modifications modifications of the of sugar the sugar head bind almost equally well to human galectin-8N. Suggested additional modifications of the sugar groupshead groups together together with their with substituents, their substituents, which mightwhich increase might increase the affinity the of affi glycansnity of toward glycans galectins, toward head groups together with their substituents, which might increase the affinity of glycans toward whilegalectins, limiting while synthetic limiting effortssynthetic towards efforts this towards achievement, this achievement, are highlighted. are highlighted. galectins, while limiting synthetic efforts towards this achievement, are highlighted.

Figure 8. Human galectin-8N (Connolly surfaces and binding sites) loaded with lacto-N-fucopentaose-III Figure 8. Human galectin-8N (Connolly surfaces and binding sites) loaded with lacto-N-fucopentaose-III (top) and 3′-sulfated lactose (bottom) (PDB 3AP9). (top) and 31-sulfated lactose (bottom) (PDB 3AP9). In view of Figure 3, the structural design of the galectins will then also be an incentive for Figure 8. Human galectin-8N (Connolly surfaces and binding sites) loaded with lacto-N-fucopentaose-III tailoring spatial aspects of binding partners, to optimize selectivity and to unravel structure-activity In(top view) and of3′-sulfated Figure3 lactose, the structural (bottom) (PDB design 3AP9). of the galectins will then also be an incentive for tailoringrelationships, spatial aspects as the protein, of binding too, can partners, be tailored, tooptimize for example selectivity engineering and new to homodimers unravel structure-activity (Figure 9). relationships, In view asof theFigure protein, 3, the too, structural can be tailored, design forof examplethe galectins engineering will then new also homodimers be an incentive (Figure for9). tailoring spatial aspects of binding partners, to optimize selectivity and to unravel structure-activity relationships, as the protein, too, can be tailored, for example engineering new homodimers (Figure 9).

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Strategically,Strategically, the ligandthe ligand structure structure can can be be modified modified forfor optimal fit fit at at different different sites, sites, with with the aglycone, the aglycone, as theas case the ofcasep-nitrophenyl, of p-nitrophenyl, as startingas starting point, point, a a strategy strategy defineddefined as as subsite-assisted subsite-assisted binding binding [75]. [ 75].

Figure 9. Relative spatial orientation of CRDs of galectins, which can be differently responsible for Figure 9. Relative spatial orientation of CRDs of galectins, which can be differently responsible for intra/inter-cellular cross-linking (left: face view; right: lateral view). Blue arrows point the positioning intra/inter-cellular cross-linking (left: face view; right: lateral view). Blue arrows point the positioning of two CRDs. of two CRDs. 4. Tailoring Monovalent Ligands for Selectivity 4. Tailoring Monovalent Ligands for Selectivity In chemical terms, each structural part of the ligand can be examined for its contribution to Inselectivity. chemical Thus, terms, the aglycone each structural (in glycoclusters part of the linker ligand to canthe scaffold), be examined the atom for at its the contribution glycosidic to selectivity.linkage Thus, and the the sugar aglycone (sequence (in glycoclusters and substitutions) the linker are subject to the to scaffold), changes theon the atom drawing at the board, glycosidic linkageproviding and the interesting sugar (sequence compounds and to be substitutions) synthesized and are tested. subject to changes on the drawing board, providing interesting compounds to be synthesized and tested. 4.1. Modifications of the Aglycone 4.1. ModificationsThe classical of the example Aglycone is the aromatic extension by a p-nitrophenyl group, reported for galectin-1 (formerly called galaptin) [57,58]. In detail, a 2.5-fold enhancement was seen by adding this group Theto lactose classical [57], example and even is athe 19-fold aromatic enhancement extension wa bys observed a p-nitrophenyl when it was group, used reported as an extension for galectin-1 to (formerlygalactose called [58]. galaptin) That alterations [57,58]. in In the detail, aglycone a 2.5-fold part have enhancement the capacity was to generate seen by contributions adding this to group to lactoseselectivity [57], has and been even delineated a 19-fold by enhancement testing a series was of β observed-lactosides when with aromatic it was used substituents as an extension [76]. to galactoseβ-Naphthyl [58]. and That 2-benzothiazolyl alterations in proved the aglycone especially part active, have in theterms capacity of affinity to to generate galectin-3 contributions relative to selectivityto galectin-1 has beenin isothermal delineated titration by testing calorimetry a series and of inβ comparative-lactosides withinhibition aromatic assays substituents [76–78]. As [76]. β-Naphthylillustrated and in 2-benzothiazolylScheme 1, the synthesis proved of especiallythe lactosides active, started in from terms readily of affinity available to galectin-3 peracetylated relative to galectin-1lactosyl bromide in isothermal 1, which titrationwas obtained calorimetry in quantitative and yiel ind comparative by treatment inhibitionof peracetylated assays lactose [76 –78]. As illustratedwith HBr inin SchemeAcOH. As1, theexpected, synthesis the phase of the transf lactosideser-catalyzed started (PTC) from nucleophilic readily available displacements peracetylated of lactosyl bromides by the aryl alcohols or aryl thiols occurred with complete anomeric inversion to lactosyl bromide 1, which was obtained in quantitative yield by treatment of peracetylated lactose with afford exclusively the corresponding β-glycosides in good to excellent yields (78% for 2a and 75% HBr infor AcOH. 3) [76]. As They expected, were ready the phase to be transfer-catalyzed evaluated for ligand (PTC) activity nucleophilic with galectins displacements after Zemplén of lactosyl bromidesde-O-acetylation by the aryl (NaOMe, alcohols MeOH). or aryl With thiols β-napthyl occurred thioglycoside with complete such as anomericthe peracetylated inversion derivative to afford exclusively2a in hand, the correspondingsynthesis of anomericβ-glycosides sulfones by in goodoxidation to excellent of the sulfide yields groups (78% using for 2amCPBAand 75% (2.1 equiv, for 3)[ 76]. TheyCH were2Cl2 ready) generated to be sulfones evaluated 2b (92%) for ligand after de- activityO-acetylation. with galectinsMost O-aryl after lactosides Zemplén were de- estimatedO-acetylation as (NaOMe,being MeOH).3-fold better With thanβ lactose-napthyl in activity thioglycoside assays, thus such underscoring as the peracetylated a preference for derivative aromatic aglycons.2a in hand, What’s more, the data reveal this region to be of conspicuous relevance for selectivity [76–78]. synthesis of anomeric sulfones by oxidation of the sulfide groups using mCPBA (2.1 equiv, CH2Cl2) generated sulfones 2b (92%) after de-O-acetylation. Most O-aryl lactosides were estimated as being 3-fold better than lactose in activity assays, thus underscoring a preference for aromatic aglycons. What’s more, the data reveal this region to be of conspicuous relevance for selectivity [76–78]. Molecules 2016, 21, 629 11 of 36 Molecules 2016, 21, 629 11 of 35

Molecules 2016, 21, 629 11 of 35

Scheme 1. Synthetic strategy leading to O- or S-lactosides under phase transfer-catalyzed (PTC) conditions. Scheme 1. Synthetic strategy leading to O- or S-lactosides under phase transfer-catalyzed (PTC) conditions. On the structural level, the O-3 hydroxyl group of the glucose moiety of lactose is the main OncontactScheme the of structural 1. this Synthetic part of strategy level, lactose the leading withO -3galect to hydroxyl O-ins, or S as-lactosides first group detected under of the byphase chemical glucose transfer-catalyzed mapping moiety ofβ (PTC)-methyl lactose conditions. lactoside is the main contactderivatives of this part (deoxy of lactose and fluoro) with [79]. galectins, Semi-empirical as first detected calculations by chemical correlated mapping well withβ -methylthe chemical lactoside derivativesmodificationsOn (deoxythe structural done and at fluoro) thelevel, anomeric the [79 ].O-3 Semi-empiricalposition hydroxyl with group the effect calculations of the on glucose charge correlated densitymoiety atof wellOlactose-3 of with the is glucosidethe the main chemical modificationscontactresidue ofof this donelactose. part at of theThe lactose anomericO-3 withelectron galect position densityins, as with calculationsfirst the detected effect were by on chemicalcharge made on densitymapping the lactoside at β-methylO-3 ofderivatives thelactoside glucoside residuederivativesevaluated of lactose. on (deoxy galectin-1 The andO in -3fluoro) the electron study [79]. [80–82]. densitySemi-empirical Electr calculationson-withdrawing calculations were correlatedaglycones, made on wellfor the example with lactoside the the chemical sulfone derivatives evaluatedmodificationsmoiety on in 2b galectin-1, decreaseddone at the in the theanomeric O-3 study electronic position [80– 82densities with]. Electron-withdrawing the and effect favored on charge interactions density aglycones, withat O -3Glu71, of the for which glucoside example was the residueaccounted of lactose.for substantially The O-3 electronby hydrogen density bonding calculations with werethe human made onlectin the [83].lactoside Hence, derivatives a direct sulfone moiety in 2b, decreased the O-3 electronic densities and favored interactions with Glu71, evaluatedcorrelation on exists galectin-1 between in the the study inhibitory [80–82]. potenciesElectron-withdrawing and the charge aglycones, density for at example O-3 calculated the sulfone on which was accounted for substantially by hydrogen bonding with the human lectin [83]. Hence, moietyelectrostatic in 2b potential., decreased Lac- theO O-p-3NP electronic has a theoretical densities electronand favored density interactions of −0.346 withcompared Glu71, to which that of was its a directaccountedo-nitrophenyl correlation for analogsubstantially exists (− between0.322). by This hy the differencedrogen inhibitory bonding of electron potencies with densit the and yhuman could the charge explainlectin density[83].the relative Hence, at O inhibitory -3a calculateddirect on electrostaticcorrelationpotency variation exists potential. onbetween galectin-1. Lac- theO inhibitoryCompound-pNP has potencies a2b theoretical, having and the the electronlowest charge O-3 densitydensity electron at ofdensity, O´-30.346 calculated also compared has theon to thatelectrostatic ofhighest its o-nitrophenyl inhibitory potential. potency Lac- analogO [76].-pNP ( ´ Amonghas0.322). a theoretical the This compou difference electronnds testeddensity of electronby of isothermal−0.346 density compared calorimetry could to that explain (ITC), of its the relativeo-nitrophenylbenzothiazole inhibitory analog3 potencyshowed (−0.322). the variation best This thermodynamic difference on galectin-1. of electron performance Compound densit y(Δ couldG2b of, −explain having6.7 kcal/mol; the the relative lowest Ka 65 inhibitory× O10-3−3 M electron−1 ) density,potencyfor galectin-3 also variation has [76]. the higheston galectin-1. inhibitory Compound potency 2b, having [76]. Among the lowest the O compounds-3 electron density, tested also by has isothermal the calorimetryhighestWhen (ITC),inhibitory assaying benzothiazole potency derivatives [76].3 showed harboringAmong the the a best4-substitu compou thermodynamicndsted 1,2,3-triazoletested by performance isothermal unit, derived calorimetry (∆G from of ´6.7 lactosyl (ITC), kcal/mol; azide 4 using copper-catalyzed azide–alkyne cycloaddition (Scheme 2) to afford heteroaryl lactoside−3 −1 Ka 65benzothiazoleˆ 10´3 M´1 3) showed for galectin-3 the best [76 thermodynamic]. performance (ΔG of −6.7 kcal/mol; Ka 65 × 10 M ) for6 in galectin-3 essentially [76]. quantitative yield after Zemplén deprotection, the potential of substitution at this When assaying derivatives harboring a 4-substituted 1,2,3-triazole unit, derived from lactosyl site wasWhen reinforced assaying [66]. derivatives harboring a 4-substituted 1,2,3-triazole unit, derived from lactosyl 4 6 azideazideusing 4 using copper-catalyzed copper-catalyzed azide–alkyne azide–alkyne cycloaddition cycloaddition (Scheme 2)2) to to afford afford heteroaryl heteroaryl lactoside lactoside in essentially quantitative yield after Zemplén deprotection, the potential of substitution at this site 6 in essentially quantitative yield afterO ZemplénCN deprotection, the potential of substitution at this AcO HO OH was reinforcedOAc [66]. 5 OH site was reinforcedO [66].OAc O O 1. CuSO vit C, DMF O N N AcO O 4, HO O O N microwave, 70 oC HO N OAc AcO 3 OH CN 30 min OH OAc O CN AcO HO OH OAc 5 4 OAc 2. MeONa/MeOH O 6 OH O quant. N N O 1. CuSO4, vit C, DMF O AcO O HO O O N microwave, 70 oC HO N OAc AcO 3 OH CN Scheme 2. RepresentativeOAc synthesis30 of min regioselectively 4-substituted 1,2,3-triazoleOH lactoside 6 using

copper-catalyzed4 azide–alkyne cycloaddition2. MeONa/MeOH (CuAAC, click chemistry).6 quant. 4.2. ModificationsScheme 2. Representative at the Glycosidic synthesis Linkage of regioselectively 4-substituted 1,2,3-triazole lactoside 6 using Scheme 2. Representative synthesis of regioselectively 4-substituted 1,2,3-triazole lactoside 6 using copper-catalyzed azide–alkyne cycloaddition (CuAAC, click chemistry). copper-catalyzedThe introduction azide–alkyne of S, Se or cycloaddition C atoms into (CuAAC,the glycosidic click linkage chemistry). makes the glycoside resistant to glycosidases. Also, stereoelectronic aspects, bond angles and flexibility are altered [84–86]. Testing the 4.2. Modifications at the Glycosidic Linkage S/Se-bridged digalactosides (TDG, SeDG), rather small discriminatory effects were noted whereas 4.2. Modifications at the Glycosidic Linkage dithio/selenoThe introduction bridging of was S, Se unfavorable or C atoms for into galectins the glycosidic [87]. However, linkage makes S/Se-digalactoside the glycoside activity resistant to to a Theglycosidases.plant introduction toxin opens Also, the stereoelectronic of possibility S, Se or C for atoms aspects, avoiding into bond thecross-re an glycosidicglesactivity, and flexibility linkageif desiring are makes toaltered block the [84–86]. the glycoside toxin Testing [87]. resistant The the to glycosidases.S/Se-bridgedsynthesis Also,of thedigalactosides stereoelectronicSeDG is summarised (TDG aspects,, SeDG), in Scheme bondrather angles 3.small The peracetylated anddiscrimina flexibilitytory galactosyl effects are altered were bromide [noted84–86 is ].whereas treated Testing the S/Se-bridgeddithio/selenowith selenourea digalactosides bridging in acetone was (TDG,unfavorable to generate SeDG), for a rathersalt.galectins Pr smalloduction [87]. discriminatoryHowever, of the selenolate S/Se-digalactoside effects anion were from activity noted this to whereassalt a dithio/selenoplantfollowed toxin by bridging opens reaction the waswithpossibility unfavorable7 gave for SeDG. avoiding A for related galectins cross-re approach,activity, [87]. However,using if desiring thiourea, S/Se-digalactosideto block gives the TDG toxin via [87].glycosyl activity The to synthesisthiol intermediates, of the SeDG which is summarised are generally in usefulScheme in 3.S-glycoside The peracetylated synthesis. galactosyl bromide is treated a plant toxin opens the possibility for avoiding cross-reactivity, if desiring to block the toxin [87]. The with selenourea in acetone to generate a salt. Production of the selenolate anion from this salt synthesis of the SeDG is summarised in Scheme3. The peracetylated galactosyl bromide is treated followed by reaction with 7 gave SeDG. A related approach, using thiourea, gives TDG via glycosyl withthiol selenourea intermediates, in acetone which to generateare generally a salt. useful Production in S-glycoside of the synthesis. selenolate anion from this salt followed by reaction with 7 gave SeDG. A related approach, using thiourea, gives TDG via glycosyl thiol intermediates, which are generally useful in S-glycoside synthesis.

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Scheme 3. Structures of O-digalactoside (ODG) and its S- and Se- mimics (TDG and SeDG), with Scheme 3. Structures of O-digalactoside (ODG) and its S- and Se- mimics (TDG and SeDG), with synthesissynthesis ofof SeDG.SeDG.

Proceeding from the reducing end, the next part that can be altered is the sugar headgroup. Proceeding from the reducing end, the next part that can be altered is the sugar headgroup. Here, Here, a brief look at Figure 6 indicates that certain positions are preferred sites for increasing a brief look at Figure6 indicates that certain positions are preferred sites for increasing reactivity to reactivity to galectins, inspiring exploitation. galectins, inspiring exploitation. 4.3. Modification at Bioinspired Positions 4.3. Modification at Bioinspired Positions The synthetic extensions of disaccharides to natural oligosaccharides has enabled to map contact The synthetic extensions of disaccharides to natural oligosaccharides has enabled to map contact sites in the CRDs beyond lactose. An example for the synthesis of relevant structures for galectins is sites in the CRDs beyond lactose. An example for the synthesis of relevant structures for galectins the addition of a fucose moiety in an α1,2-linkage to lactose, constituting the histo-blood group is the addition of a fucose moiety in an α1,2-linkage to lactose, constituting the histo-blood group H-trisaccharide, with general principles of oligosaccharide synthesis outlined in [88]. The synthesis H-trisaccharide, with general principles of oligosaccharide synthesis outlined in [88]. The synthesis of oligosaccharides enables generation of stereochemically pure and homogeneous substances for of oligosaccharides enables generation of stereochemically pure and homogeneous substances for evaluation of their binding to lectins. It also enables the incorporation of these substances onto evaluation of their binding to lectins. It also enables the incorporation of these substances onto microarrays for identification of ligands for lectins (Figure 5). Chemical synthesis of glycoside bonds microarrays for identification of ligands for lectins (Figure5). Chemical synthesis of glycoside bonds relies on having both suitably protected donors and acceptors available. The synthesis of 2′-fucosyl relies on having both suitably protected donors and acceptors available. The synthesis of 21-fucosyl lactose exemplifies an approach to generating a lactos derivative [89]. Matta and coworkers have lactose exemplifies an approach to generating a lactos derivative [89]. Matta and coworkers have shown that lactose can be converted to 9 via acetonide formation and benzoylation [90] and used this shown that lactose can be converted to 9 via acetonide formation and benzoylation [90] and used this in a synthesis of 2′-fucosyl lactose. This leaves the 2′-OH free to be a nucleophile in a glycosidation in a synthesis of 21-fucosyl lactose. This leaves the 21-OH free to be a nucleophile in a glycosidation reaction. In a variation of Matta’s synthesis, a benzylated thiofucopyranoside 10 was shown to act as reaction. In a variation of Matta’s synthesis, a benzylated thiofucopyranoside 10 was shown to act a donor using Crich and Smith’s activation protocol [91]. The use of benzyl protecting groups, which as a donor using Crich and Smith’s activation protocol [91]. The use of benzyl protecting groups, do not participate in the glycosidation reaction, ensure that the required α-stereoisomer 11 is formed which do not participate in the glycosidation reaction, ensure that the required α-stereoisomer 11 after activation of the thioglycoside. Most likely, the undesired β-anomer would have resulted if is formed after activation of the thioglycoside. Most likely, the undesired β-anomer would have acylated protecting groups had been used, unless anomerisation could occur or be induced in situ [92]. resulted if acylated protecting groups had been used, unless anomerisation could occur or be induced Subsequent removal of the benzoyl and acetonide protecting groups generates 13 and, finally, in situ [92]. Subsequent removal of the benzoyl and acetonide protecting groups generates 13 and, 2′-fucosyl lactose is obtained by hydrogenolytic removal of the benzyl groups. finally, 21-fucosyl lactose is obtained by hydrogenolytic removal of the benzyl groups. As noted above with respect to Scheme 4, potent binding partners for galectins present distinct As noted above with respect to Scheme4, potent binding partners for galectins present distinct substitutions at the N-acetyllactosamine core, for example the α1,3-linked N-acetylgalactosamine substitutions at the N-acetyllactosamine core, for example the α1,3-linked N-acetylgalactosamine residue, which turns the histo-blood group H-type trisaccharide to the A-type tetrasaccharide. Such a residue, which turns the histo-blood group H-type trisaccharide to the A-type tetrasaccharide. site for a substitution is referred to as a rational design based on structural information (bioinspiration) Such a site for a substitution is referred to as a rational design based on structural information Scheme 5 documenting typical reaction sequences toward the production of compounds following (bioinspiration) Scheme5 documenting typical reaction sequences toward the production of this bioinspiration. compounds following this bioinspiration. In general, irrespective of the aglycone residues on unprotected β-D-lactopyranosides, it is possible In general, irrespective of the aglycone residues on unprotected β-D-lactopyranosides, it is to use the well-known behavior of tin-ether or tin-acetal activation that greatly and regioselectively possible to use the well-known behavior of tin-ether or tin-acetal activation that greatly and potentiate the nucleophilicity of the O-3′ oxygen to modify this particular position. For example, regioselectively potentiate the nucleophilicity of the O-31 oxygen to modify this particular position. For lactoside 14 can be selectively propargylated to give compound 15 in very good yield after acetylation example, lactoside 14 can be selectively propargylated to give compound 15 in very good yield after [76]. The propargyl ether of 8 was then treated by a [1–3]-dipolar cycloaddition [93] between the acetylation [76]. The propargyl ether of 8 was then treated by a [1,3]-dipolar cycloaddition [93] between alkyne and nitrile oxide, generated in situ from benzaldehyde oxime and N-chlorosuccinimide (through intermediate benzhydroximoyl chloride) to afford 16 in 91% yield, which after Zemplén

Molecules 2016, 21, 629 13 of 36 the alkyne and nitrile oxide, generated in situ from benzaldehyde oxime and N-chlorosuccinimide (throughMolecules intermediate 2016, 21, 629 benzhydroximoyl chloride) to afford 16 in 91% yield, which after13 Zemplén of 35 deprotectionMolecules 2016 provided, 21, 629 the desired ligand 17 almost quantitatively. An analogous strategy was13 of used 35 to deprotection provided the desired ligand 17 almost quantitatively. An analogous strategy was used to give the corresponding 31-sulfated O- and S-naphthyl lactosides 21 and 22 from 19 and 20, respectively. give the corresponding 3′-sulfated O- and S-naphthyl lactosides 21 and 22 from 19 and 20, respectively. It is alsodeprotection worth noting provided that the the desired above chemicalligand 17 almost sequences quantitatively. provided An compounds analogous reactivestrategy was with used galectins-1 to Itgive is thealso corresponding worth noting 3 ′-sulfatedthat the Oabove- and Schemic-naphthylal sequences lactosides 21provided and 22 from compounds 19 and 20 reactive, respectively. with and -3 in a much faster route than the one previously described for the synthesis of 18 showing a K galectins-1It is also worth and -3 noting in a much that fasterthe above route chemicthan theal onesequences previously provided described compounds for the synthesis reactive ofwith 18 D of 320 nM against galectin-3 [94]. showinggalectins-1 a KandD of -3 320 in nM a much against faster galectin-3 route than [94]. the one previously described for the synthesis of 18 showing a KD of 320 nM against galectin-3 [94].

Scheme 4. Example of oligosaccharide synthesis to produce naturally occurring galectin ligands. Scheme 4. Example of oligosaccharide synthesis to produce naturally occurring galectin ligands. Scheme 4. Example of oligosaccharide synthesis to produce naturally occurring galectin ligands.

Scheme 5. Chemical modifications of lactosides at both the aglycones and at the 3′-position.

Scheme 5. Chemical modifications of lactosides at both the aglycones and at the 3′-position.1 SchemeTo initiate 5. Chemicalto explain modifications its marked reactivity of lactosides to galectins-3, at both the aglycones-4 and -8, andthe isoxazole at the 3 -position. lactoside 17 was dockedTo initiate into to the explain active its site marked of human reactivity galectin-3. to galectins-3, Possible -4interactions and -8, the with isoxazole all 13 lactosideamino acid 17 Toresidueswas initiate docked listed to into explain in theTable active its 1 markedcan site thus of reactivity humanbe monitored. galectin-3. to galectins-3, It can Possible be clearly -4 interactions and seen -8, the that with isoxazole 17 allmay 13 lactosideformamino strong acid17 was dockedhydrogenresidues intothe listed bonding active in Table with site ofAsp148,1 humancan thus H158, galectin-3. be and monitored. possibly Possible Itthe can key interactions be remote clearly Arg144 seen with thatwith all 17the 13 may aminoisoxazole form acid moietystrong residues listed(Figurehydrogen in Table 10). bonding1 Evidently,can thus with bethorough Asp148, monitored. H158,study and of It structur canpossibly bee-activity clearly the key seenremote relationships that Arg14417 (SmaywithARs) the form offers isoxazole strong remarkable moiety hydrogen potential to understand selectivity and push an enhancement to its inherent limits. bonding(Figure with 10). Asp148, Evidently, H158, thorough and possibly study of the structur key remotee-activity Arg144 relationships with the (S isoxazoleARs) offers moiety remarkable (Figure 10). potential to understand selectivity and push an enhancement to its inherent limits. Evidently, thorough study of structure-activity relationships (SARs) offers remarkable potential to understand selectivity and push an enhancement to its inherent limits.

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Molecules 2016, 21, 629 14 of 35

Figure 10. Schematic representation of the CRD site of human galectin-3 (PDB 3AYE) showing all Figure 10.aminoSchematic acids involved representation as per Table 1 of and the interacting CRD site with of compound human galectin-3 17 (docked (PDB in by superposition 3AYE) showing all amino acidswith natural involved lactose as ligand); per Table left1 Connolly and interacting surface re withpresentation compound with red17 surface(docked (anionic) in by and superposition blue with naturalsurface lactose (cationic ligand); amino acids). left Connolly surface representation with red surface (anionic) and blue surface (cationic amino acids). In summary, a binding partner of a (ga)lectin has been formally separated into distinct sections. The advantage of this approach is that any structural change introduced by synthesis can be In summary,evaluated and a bindingoptimized. partner Combinatorial of a (ga)lectin integration has can been then formally fully realize separated the potential into of distinct tailoring sections. The advantagethe monovalent of this structure. approach Of is note, that activity any structural testing should change include introduced the natural by panel synthesis of lectins can (of be the evaluated and optimized.same family Combinatorialand of other families integration sharing the same can thennominal fully glycan realize specificity) the to potential preclude premature of tailoring the monovalentconclusions structure. on selectivity. Of note, Hence, activity keeping testing an eye should on the size include of the the lectin natural network, panel as illustrated of lectins in (of the Figure 4, and on cross-reactive activities is essential. With this part done, the next source of same family and of other families sharing the same nominal glycan specificity) to preclude premature inspirations for tailoring synthetic binders of lectins comes from the natural presentation of glycans conclusionsas multivalent on selectivity. clusters Hence,up to microdomains. keeping an eyeAffinity on theand sizeselectivity of the are lectin increased network, when asproperly illustrated in Figure4,taking and on spatial cross-reactive factors of the activities presentation is essential. into acco Withunt, the this fine part work done, on the the glycoside next source cluster of inspirationseffect for tailoringwith pioneering synthetic bindersexperiments of lectins on the comes hepatic from asialoglycoprotein the natural presentation receptor, a C-type of glycans lectin asshown multivalent clustersschematically up to microdomains. in Figure 2, and Affinity synthetic and glycoc selectivitylusters serving are increasedas excellent whenprecedent properly [95,96]. taking spatial factors of the presentation into account, the fine work on the glycoside cluster effect with pioneering 5. Tailoring Spatial Aspects for Selectivity experiments on the hepatic asialoglycoprotein receptor, a C-type lectin shown schematically in Figure2, A wide variety of scaffolds can be used to present glycans as bioactive ligands, generating and synthetic glycoclusters serving as excellent precedent [95,96]. glycoclusters or glycodendrimers [97–101]. Of course, any favorable structural feature found on the level of testing monovalent compounds can now be implemented. The following schemes can thus 5. Tailoring Spatial Aspects for Selectivity be considered as examples tested with galectins, starting with a selection of bivalent compounds. A wideMacrocyclic variety of scaffolds scaffolds derived can from be used glucuronic to present acid and glycans phenylene-1,4-diamine as bioactive ligands,(glycophanes; generating glycoclusters[102,103]) or had glycodendrimers been used as fragment [97–101s in]. building Of course, bivalent any mannosides favorable structural23 and 24 (Scheme feature 6) found [104]. on the This inspired the use of such chiral scaffolds based on p-xylylenediamine to prepare structurally level of testing monovalent compounds can now be implemented. The following schemes can thus be defined bivalent lactoside 26 [105]. Compound 26 is considered rigid when compared with 25. Molecular consideredmodeling as examples can be used tested to investigate with galectins, distance startingprofiles between with a galactose selection headgroups of bivalent in compounds.such bivalent Macrocycliccompounds. The scaffolds use of macromodel derived for fromexample, glucuronic indicated that acidthe intergalactoside and phenylene-1,4-diamine distance in 26 (glycophanes;was 24.5 [Å.102 While,103] )both had 25 beenand 26 used showed as similar fragments affinity in for building galectin 3 bivalent in a solid mannosidesphase binding 23 and 24 (Schemeassay,6 )[the104 glycophane]. This 26 inspired was >10-fold the usemore of potent such than chiral 25 towards scaffolds the basedtruncated on galectinp-xylylenediamine 3. to prepare structurallyVarious different defined frameworks bivalent (scaffolds) lactoside can26 be[105 incorporated]. Compound in bivalent26 is glycocluster considered design rigid when and synthesis and a selection are shown in Scheme 7 [105–107]. While topology within this series can comparedbe considered with 25. Molecular the same (i.e. modeling, they are can all bivalent), be used tothe investigate spatial relationships distance are profiles different, between allowing galactose headgroupsthis property in such tobivalent be investigated compounds. through bioassays. The use ofInteractions macromodel of the forscaffold example, with the indicated lectin that that the intergalactosidemay contribute distance to, or inhinder,26 was binding 24.5 cannot Å. While be di bothscounted25 and given,26 forshowed example, similar activity affinity observed for for galectin 3 in a solidcompounds phase such binding as 6 (Scheme assay, 2). the Inglycophane the series shown26 thewas dist >10-foldances between more the potent galactose than residues25 towards can the truncatedvary galectin from 15.5 3. Å to 35 Å, as estimated by molecular modeling. A variety of scaffold types have been investigated including both tertiary (27–29) and secondary (30, 31) diamides, diglycosyldiamides Various different frameworks (scaffolds) can be incorporated in bivalent glycocluster design and synthesis and a selection are shown in Scheme7[ 105–107]. While topology within this series can be considered the same (i.e., they are all bivalent), the spatial relationships are different, allowing this property to be investigated through bioassays. Interactions of the scaffold with the lectin that may contribute to, or hinder, binding cannot be discounted given, for example, activity observed for compounds such as 6 (Scheme2). In the series shown the distances between the galactose residues can vary from 15.5 Å to 35 Å, as estimated by molecular modeling. A variety of scaffold types have Molecules 2016, 21, 629 15 of 36 been investigated including both tertiary (27–29) and secondary (30, 31) diamides, diglycosyldiamides (33, 34), ditriazoles (32) and the previously mentioned glycophane 26 together with a C-glycosyl version 35 and a more flexible variant of the latter 36. Highlights from biological study of such a series include observations that the tertiary diamide 27 was 5-fold more selective for proteolytically truncated Molecules 2016, 21, 629 15 of 35 galectin-3 (complete removal of the non-CRD sections, shown in Figure3) than 26 [104] and that the more flexible(33, 34 glycocluster), ditriazoles (3236) andshowed the previously 100-fold mentioned selectivity glycophane for the chicken 26 together galectin-3 with a comparedC-glycosyl with chicken proto-typeversion 35 and galectin-1A a more flexible [106 ].variant of the latter 36. Highlights from biological study of such a Theseries synthesis include of observations25 and 26 thatis the summarised tertiary diamide in Scheme 27 was 5-fold8[ 104 more]. Theselective glycosidation for proteolytically reaction of truncated galectin-3 (complete removal of the non-CRD sections, shown in Figure 3) than 26 [104] 1,6-anhydro donor 37 with 1C conformation, which is more reactive than a related donor with 4C and that the more flexible4 glycocluster 36 showed 100-fold selectivity for the chicken galectin-3 1 conformation,compared occurs with inchicken the presence proto-type of galectin-1A triethylsilyloxyprop-1-ene [106]. to initially give a β-glycoside but this species undergoesThe synthesis rapid of 25in and situ 26anomerisation is summarised inin theScheme presence 8 [104]. of The SnCl glycosidation4 to give the reactionα-glucuronide of 38 [108,1091,6-anhydro]. This wasdonor converted 37 with 1C4 toconformation,39 by removal which ofis more the acetatereactive than protecting a related groups donor with followed 4C1 by an acetylationconformation, reaction occurs that in gavethe presence a 6,3-lactone of triethylsilyloxyprop-1-ene intermediate and to subsequent initially give a reaction β-glycoside of but this with this species undergoes rapid in situ anomerisation in the presence of SnCl4 to give the α-glucuronide allyl alcohol gave 39. The glycosidation reaction between benzoylated trichloroacetimidate 40 and 38 [108,109]. This was converted to 39 by removal of the acetate protecting groups followed by an 39 and subsequentacetylation reaction removal that of gave the a allyl 6,3-lactone esterusing intermediate Pd(0) gaveand subsequent41. Next reaction the coupling of this ofwith acid allyl41 with p-xylylenediaminealcohol gave gave39. The42 glycosidation. Ring closure reaction metathesis between usingbenzoylated the Grubbs-I trichloroacetimidate catalyst followed 40 and 39 and by alkene reductionsubsequent and acyl removal group removalof the allyl gave ester glycophane using Pd(0) 26gave, whereas 41. Next alkene the coupling reduction of acid and 41 deacylation with under similarp-xylylenediamine conditions gave 4225. .Ring The closure use of metathesis the benzoyl using protecting the Grubbs-I groups catalyst in 40followedwas important by alkene as the analogousreduction per-O -acetylatedand acyl group donor removal gave gave none glycophane of the desired 26, whereas glycoside alkene butreduction only anand orthoester, deacylation which under similar conditions gave 25. The use of the benzoyl protecting groups in 40 was important as can be observed in the course of glycosidation. the analogous per-O-acetylated donor gave none of the desired glycoside but only an orthoester, Synthesiswhich can of be35 observedwith thein thep course-xylylenediamine of glycosidation. based scaffold is shown in Scheme9[ 106]. The propargylSynthesis derivative of 3544 withwas the first p-xylylenediamine prepared from based benzylidene scaffold is43 shownviaa in partial Scheme reductive 9 [106]. The cleavage of the benzylidenepropargyl derivative followed 44 bywas 4- firstO-propargylation. prepared from benzylidene Next formation 43 via a partial of the reductiveC-glycosyl cleavage derivative of 45 was carriedthe benzylidene by anomeric followed allylation by 4-O [110-propargylation.], followed Next by selective formation removal of the C-glycosyl of the 6- derivativeO-benzyl 45group was and subsequentcarried oxidation. by anomeric The allylation copper (I)-promoted [110], followed triazole by selective formation removal with of the azide 6-O-benzyl4 gave group46. Subsequent and subsequent oxidation. The copper (I)-promoted triazole formation with azide 4 gave 46. Subsequent couplingcoupling to p-xyxylenediamine to p-xyxylenediamine followed followed by by deacetylation deacetylation gave gave 3636, whereas, whereas ring ring closure closure metathesis metathesis using Grubbs-IIusing Grubbs-II catalyst catalyst followed followed by de-by de-O-acetylationO-acetylation gavegave 3535. .

Scheme 6. Structures of bivalent glycoclusters based on glycophane and an acyclic variant. Space-filling Scheme 6.modelStructures of 26 is shown of bivalent on right glycoclusters with distance based between on galactose glycophane anomeric and ancarbon acyclic atoms variant. indicated. Space-filling model of 26 is shown on right with distance between galactose anomeric carbon atoms indicated.

Molecules 2016, 21, 629 16 of 36 Molecules 2016, 21, 629 16 of 35

Scheme 7. A panel of synthetic lactose-presenting bivalent compounds with inter-galactoside distances Scheme 7. A panel of synthetic lactose-presenting bivalent compounds with inter-galactoside distances are shown. Various types of scaffolds have been used including secondary and tertiary diamides, are shown. Various types of scaffolds have been used including secondary and tertiary diamides, diglycosyldiamides, ditriazoles and glycophanes. diglycosyldiamides, ditriazoles and glycophanes.

Molecules 2016, 21, 629 17 of 36 Molecules 2016, 21, 629 17 of 35 Molecules 2016, 21, 629 17 of 35

Scheme 8. Synthesis of glycophane 26 and the acyclic analogue 25. Scheme 8. Synthesis of glycophane 26 and the acyclic analogue 25. Scheme 8. Synthesis of glycophane 26 and the acyclic analogue 25.

Scheme 9. Synthesis of glycophane 36 and the acyclic analogue 35. Scheme 9. Synthesis of glycophane 36 and the acyclic analogue 35. Scheme 9. Synthesis of glycophane 36 and the acyclic analogue 35.

Molecules 2016, 21, 629 18 of 36

Molecules 2016, 21, 629 18 of 35 The chemistry shown in Scheme 10 exemplifies an approach to the synthesis of diamides where 1 2 -fucosylThe chemistry lactose headgroups shown in Scheme are incorporated 10 exemplifies [107]. an Diamides approach can to bethe prepared synthesis from of diamides corresponding where 2glycosylamines′-fucosyl lactose such headgrou as 49.ps The are Ugiincorporated reaction is[107]. a four Diamides component can be reaction prepared involving from corresponding amine, acid, glycosylaminesaldehyde and isocyanidesuch as 49. that The is Ugi suitable reaction for is tertiary a four amidecomponent generation. reactionWhen involving a diacid amine, such acid, as aldehydeterephthalic and acid isocyanide is used with that49 ,is it suitable efficiently for gives tertiary the divalent amide glycoclustergeneration. 50Whenafter a protecting diacid such group as terephthalicremoval. Alternatively, acid is used reduction with 49 of, it the efficiently azide 51 ingives the presencethe divalent of Pd-C glycocluster and hydrogen 50 after gave protecting a glycosyl groupamine removal. and subsequent Alternatively, coupling reduction of this of with the azide terephthaloyl 51 in the chloridepresence andof Pd-C then and deacetylation hydrogen gave gave a glycosyldiamide 52amine. The and secondary subsequent amides coupling in 52 prefer of this a transwith-arrangement terephthaloyl whereas chloride those and then in 51 deacetylationprefer cis and gavethis difference diamide 52 gives. The rise secondary to different amides spatial in arrangements52 prefer a trans- betweenarrangement the carbohydrate whereas those headgroups in 51 prefer and ciscan and account this fordifference differences gives in preferences rise to different for lectins. spatial The arrangements direct copper-promoted between triazolethe carbohydrate formation 1 headgroupsfrom 51, prepared and can from account 2 -fucosyl for differences azide (Scheme in pref 11),erences and subsequent for lectins. protecting The direct group copper-promoted removal gave triazoleditriazole formation53. This latterfrom compound51, prepared showed from 2 high′-fucosyl affinity azide (IC 50(Scheme= 8 µM) 11), for and galectin-4 subsequent and was protecting ~6-fold groupmore selective removal to gave this lectinditriazole than 5352. [107This]. latter compound showed high affinity (IC50 = 8 μM) for galectin-4Valency and increaseswas ~6-fold give more rise selective to new to this glycocluster lectin than 52 topologies [107]. and can be taken to the levelValency of tri- increases and tetravalent give rise glycoclusters, to new glycocluster as shown topologies next and with can notable be taken enhancements to the level of tri- for and the tetravalentchimera-type glycoclusters, galectin-3 [111 as ].shown Synthetically, next with 2-propynyl notable enhancements lactosides were for conjugated the chimera-type under Sonogashira galectin-3 [111].palladium-catalyzed Synthetically, 2-propynyl cross-coupling lactosides conditions were conjugated with triiodobenzene under Sonogashira and suitablypalladium-catalyzed derivatized cross-couplingpentaerythritol conditions cores (Schemes with 12 triiodobenzene and 13). Starting and withsuitably 2-propynyl derivatized lactoside pentaerythritol55, a series ofcores tri- (Schemesand tetra-valent 12 and 13). derivatives Starting 56with, 59 2-propynyl, and 60 were lactoside thus prepared55, a series using of tri- a and common tetra-valent general derivatives protocol 56((Ph, 593P), and2PdCl 602 ,were DMF:Et thus3N prepared (1:1, v/v ),using CuI, a rt, common 3–5 h). The general simultaneous protocol Sonogashira((Ph3P)2PdCl2 cross-coupling, DMF:Et3N (1:1, of v55/vto), CuI, 1,3,5-triiodobenzene rt, 3–5 h). The simultaneous54 (Scheme 12 Sonogashira) and to pentaerythritol cross-coupling tetrakis( of 55m -to and 1,3,5-triiodobenzenep-iodobenzyl)ether 5457 (Schemeor 58 (Scheme 12) and 13) to afforded pentaerythritol an efficacious tetrakis( entrym- into and this p-iodobenzyl)ether family of carbohydrate 57 or 58 clusters (Scheme56 (trimer13) afforded 80%), anand efficacious tetramers entry59 (meta into) and this60 family(para )of in carbohydrate 78 and 75% yields, clusters respectively. 56 (trimer 80%), Complete and de-tetramersO-acetylation 59 (meta of) andthese 60 clusters (para) in was 78 uneventful, and 75% yields, resulting respectively. in freely water-solubleComplete de-O clusters-acetylation56, 59 ,of and these60 inclusters more thanwas uneventful,90% yields. resulting in freely water-soluble clusters 56, 59, and 60 in more than 90% yields.

Scheme 10. Synthesis of diamides with the histo-blood H trisaccharide headgroup. Scheme 10. Synthesis of diamides with the histo-blood H trisaccharide headgroup.

Molecules 2016, 21, 629 19 of 36 Molecules 2016, 21, 629 19 of 35 Molecules 2016, 21, 629 19 of 35

Scheme 11. Synthesis of the bivalent compound 53. SchemeScheme 11. 11.Synthesis Synthesis of of the the bivalent bivalent compound compound53 53. .

Scheme 12. Trivalent lactose-presenting cluster 56 with equivalent inter-galactoside distances Scheme 12. Trivalent lactose-presenting cluster 56 with equivalent inter-galactoside distances Schemewas obtained 12. Trivalent using the lactose-presenting core 54 and 2-propynyl cluster 56lactosidewith equivalent 55. It showed inter-galactoside remarkable reactivity distances with was was obtained using the core 54 and 2-propynyl lactoside 55. It showed remarkable reactivity with obtainedgalectin-3 using[111]. the core 54 and 2-propynyl lactoside 55. It showed remarkable reactivity with galectin-3galectin-3 [111 [111].].

Molecules 2016, 21, 629 20 of 36 Molecules 2016, 21, 629 20 of 35

Scheme 13. Tetrameric lactoside clusters 59 and 60 with different inter-galactoside distances Scheme 13. Tetrameric lactoside clusters 59 and 60 with different inter-galactoside distances prepared prepared using palladium-catalyzed cross coupling Sonogashira reactions. using palladium-catalyzed cross coupling Sonogashira reactions. The click-chemistry reaction can also be used to give trivalent glycoclusters [89]. Hence, the reaction of 2′-fucosylThe click-chemistry azide 51 (Scheme reaction 14) with can alsophloroglucinol be used to derivative give trivalent 61, and glycoclusters subsequent [protecting89]. Hence, group the 1 reactionremoval ofgave 2 -fucosyltritriazole azide 62. The51 (Schemedistances 14 between) with phloroglucinolgalactose residues derivative in 62 was61 ,23.5 and Å. subsequent This latter protectingcompound groupshowed removal >100-fold gave selectivity tritriazole for62. the The chicken distances galectin-3 between relative galactose to residues the homodimeric in 62 was 23.5galectin-1A, Å. This and latter was compound 10-fold more showed potent >100-fold for th selectivitye chimera-type for the protein chicken than galectin-3 the corresponding relative to thetrilactoside, homodimeric which galectin-1A, was in turn and more was potent 10-fold than more a potentrelated fordilactoside. the chimera-type As scaffold protein with than similar the correspondingdegree of rigidity, trilactoside, other choices which have was been in turn realiz moreed, e.g., potent metal-ion than a coordinated related dilactoside. complexes As [112,113] scaffold withor cyclopeptides similar degree [114,1 of15], rigidity, the latter other especially choices suitable have beento block realized, binding e.g., of galectin-4. metal-ion coordinated complexesTurning [112 to ,113applications,] or cyclopeptides relative affinity [114,115 values], the of latter glycoclusters especially for suitablegalectins to can block discriminate binding ofbetween galectin-4. family members, as is the case between galectins-1 and -3. When combining the oligovalent displayTurning with toa bioinspire applications,d substitution relative affinity at the values3′-position, of glycoclusters affinity to galectins-3 for galectins and can -4 discriminate was further betweenenhanced, family increasing members, selectivity as is the against case between galectin-1 galectins-1 [116,117]. and As -3. a Whendifferent combining type of the application, oligovalent a 1 displaysynthetic with glycocluster a bioinspired has proven substitution as potent at the tool 3 to-position, determine affinity the stoichiometry to galectins-3 of and ligand-dependent -4 was further enhanced,galectin-3 oligomerization, increasing selectivity which againstdepends galectin-1 on a critical [116 length,117]. of Asthe N a-terminal different tail type on of the application, side of the aprotein synthetic [118,119]. glycocluster The feasibility has proven to asincrease potent the tool level to determine of branching the stoichiometry made it tempting of ligand-dependent to further raise galectin-3the valency, oligomerization, entering the realm which of dependsglycodendrimers. on a critical Poly(amidoamine) length of the N-terminal starburst tail (PAMAM) on the side scaffold of the protein(Scheme [ 11815) ,serves119]. The as an feasibility impressive to increaseexample. theSuch level a lactosylated of branching glycodendrimer made it tempting (65) up to to further generation raise thefive valency,(G5) (128 entering lactosides), the realm anchored of glycodendrimers. through an aromatic Poly(amidoamine) p-isothiocyanatophenyl starburst (PAMAM)β-D-lactoside scaffold 63 as (Schemeligand (Scheme 15) serves 15), as too, an impressive reacted differentially example. Such with a lactosylateda galactoside-specific glycodendrimer plant toxin (65) up and to galectins-1 generation β fiveand (G5)-3 [120]. (128 Of lactosides), note, the numbers anchored of through generation an aromaticin designp progress-isothiocyanatophenyl had a strong impact-D-lactoside on reactivity.63 as ligandIn (Schemespite of 15the), too,numerous reacted strategies differentially leading with to a galactoside-specificthe syntheses of glycodendrimers, plant toxin and galectins-1it became andobvious -3 [120 that]. Ofthe note,chemical the numbers procedures of generationleading to inthem design would progress greatly had benefit a strong by using impact core on molecules reactivity. alreadyIn spitepossessing of the numerousthe highest strategies possible leadingnumber toof theexposed syntheses surface of glycodendrimers,functionalities. In itaddition, became obviousin orderthat to better the chemical control procedures the physicochemical leading to them properties would of greatly dendrimers, benefit by while using accelerating core molecules the alreadyincrease possessingof surface groups, the highest it was possible suggested number that di offferent exposed building surface blocks functionalities. be used in between In addition, each ingeneration. order to This better approach control led the to physicochemical the general concep propertiest of “onion of peel” dendrimers, strategy while [121–125] accelerating For instance, the a glycodendrimer having 18-exposed lactoside residue was readily build-up in a few steps by using

Molecules 2016, 21, 629 21 of 36 increase of surface groups, it was suggested that different building blocks be used in between each generation.Molecules 2016, This21, 629 approach led to the general concept of “onion peel” strategy [121–125] For instance,21 of 35 a glycodendrimer having 18-exposed lactoside residue was readily build-up in a few steps by using a hexaphenylbenzenehexaphenylbenzene core such as 6969 andand aa tris(hydroxymethyl)aminomethanetris(hydroxymethyl)aminomethane (TRIS) precursor suitably substituted (Scheme 77).). TheThe syntheticsynthetic sequencesequence leadingleading toto thisthis typetype ofof glycodendrimerglycodendrimer was initiated by by a adouble double Stille Stille coupling coupling reac reactiontion involving involving bis(tributylstannyl)acetylene bis(tributylstannyl)acetylene (66) and (66) andpara-iodoanisolepara-iodoanisole (67) (67in) the in the presence presence of of tetrakis(triphen tetrakis(triphenylphosphine)palladium(0)ylphosphine)palladium(0) to to give give the corresponding symmetrical diarylalkynediarylalkyne (68) inin 71%71% yield.yield. AA [2+2+2][2+2+2] cyclotrimerizationcyclotrimerization of 68 catalyzedcatalyzed by dicobalt octacarbonyl octacarbonyl [Co [Co2(CO)2(CO)8] and8] and subsequent subsequent methyl methyl ether ether deprotection deprotection with withBBr3 BBrprovided3 provided rapid rapidaccess accessto perhydoxylated to perhydoxylated 69 in 87%69 inyield. 87% Propargylation yield. Propargylation of 69 with of propargyl69 with propargyl bromide and bromide K2CO and3 in ˝ KDMF2CO 653 in °C DMF afforded 65 C hexapropargylated afforded hexapropargylated ether 70 in ether 82%70 yield.in 82% Using yield. the Using orthogonal the orthogonal AB3 synthon AB3 synthon2-bromoacetamido-tris[(propargyloxy)methyl]-aminomethane 2-bromoacetamido-tris[(propargyloxy)methyl]-aminomethane (71) gave the (71 )targeted gave the 18-mer targeted precursor 18-mer precursor72 (79%). 72The(79%). introduction The introduction of the modified of the modified lactopyranoside lactopyranoside termini termini was efficiently was efficiently achieved achieved using usingCu(I)-catalyzed Cu(I)-catalyzed azide-alkyne azide-alkyne [1,3]- [1,3]-dipolardipolar cycloaddition cycloaddition reaction reaction (Sch (Schemeeme 8).8 ).To To this this end, lactosyllactosyl azide 4 was “clicked” on either 70 or 72 toto affordafford hexa-lactosylatedhexa-lactosylated dendrons (not shown) and 18-mer dendrimer 73,, respectively,respectively, inin goodgood overalloverall yieldsyields afterafter usualusual transesterificationtransesterification (MeONa(MeONa in in MeOH). MeOH).

1 Scheme 14. Trivalent 2′-fucosyl lactose-presenting cluster 62 prepared using click-chemistry reaction from 51 and phloroglucinolphloroglucinol derivative.derivative. In thisthis case,case, inter-galactosideinter-galactoside distancesdistances areare shortershorter thanthan inin 5656..

Molecules 2016, 21, 629 22 of 36 Molecules 2016, 21, 629 22 of 35

Scheme 15. Poly(amidoamine) dendrimer of generation G2 (65) incorporating 16 lactoside residues Scheme 15. Poly(amidoamine) dendrimer of generation G2 (65) incorporating 16 lactoside residues linked through thiourea linkages. linked through thiourea linkages. Another type of glycodendrimer with ligand radiating from propargylated hexaphenylbenzene coresAnother and containing type of glycodendrimer up to 54 peripheral with ligand sugar radiatingligands has from been propargylated synthesized hexaphenylbenzene by the powerful coresCu(I)-catalyzed and containing [1,3]-dipolar up to 54cycloadditions peripheral sugar using ligands both convergent has been synthesized and divergent by theapproaches. powerful Cu(I)-catalyzedAn expeditive [1,3]-dipolarand systematic cycloadditions route toward using the bothsynthesis convergent of innovative and divergent glycoclusters approaches. and AnG(1)-glycodendrimers expeditive and systematic built from circular route toward and aromatic the synthesis polypropargylated of innovative scaffolds glycoclusters have thus been and G(1)-glycodendrimersdeveloped (Scheme 16). built The multivalent from circular glycoconjugate and aromatics were polypropargylated obtained with si scaffoldsmple and havegenerally thus high been developedyielding reactions (Scheme including 16). The nucleophilic multivalent substitutions glycoconjugates to generate were synthons obtained and with copper-catalyzed simple and generally azide highalkyne yielding cycloaddition reactions reaction including to covalently nucleophilic attach substitutions the minimally to generate modified synthons galectin and ligand copper-catalyzed as lactoside azidemoieties. alkyne Interestingly, cycloaddition the hydrophobic reaction to covalentlycharacter of attach the inner the minimallyaromatic core modified was counterbalanced galectin ligand asby lactoside the hydrophilic moieties. sugar Interestingly, derivatives, affording the hydrophobic fully water-soluble character derivatives. of the inner aromatic core was counterbalancedBased on hexachlorocyclotriphosphazene by the hydrophilic sugar derivatives, (N3P3Cl affording6) as a central fully molecule water-soluble (Scheme derivatives. 17), which is knownBased to be on nearly hexachlorocyclotriphosphazene planar having three up and (Nthree3P3 downCl6) as substituents a central molecule lactosylated (Scheme dendrons 17), and which a isdumbbell-shape known to be nearly architecture planar were having obtained three up(Sch andeme three18) [126]. down In substituentsdetail, a functionalized lactosylated AB dendrons3 trimer, andderived a dumbbell-shape from TRIS (71 architecture, Scheme 16), were together obtained with (Scheme an AB 518 building)[126]. Inblock detail, (76 a), functionalizedderived from ABthe3 trimer,commercially derived available from TRIS N (371P3,Cl Scheme6, were 16used.), together Lactosylation with an was AB5 achievedbuilding blockusing (click76), derivedchemistry from with the commerciallylactosyl azide available75 possessing N3P3 aCl tetraethylene6, were used. glycol Lactosylation spacer. As was also achieved used in usingglycodendrimer click chemistry synthesis with lactosylfor glycodendrimersome azide 75 possessing surface a tetraethylene programming glycol [127], spacer. to give As alsotrimer used 77 and in glycodendrimer pentamer 78 in synthesisgood to forexcellent glycodendrimersome yields. Transformation surface of programming the bromides at [127 both], to dendron’s give trimer focal77 poandint pentamerwas readily78 performedin good to excellentby an SN yields.2 substitution Transformation using sodium of the azide bromides to afford at both compounds dendron’s 79focal and 80 point, respectively. was readily Pentavalent performed hypermonomer 80 was then used toward the decavalent dumbbell-shape lactosylated compound 82 by an SN2 substitution using sodium azide to afford compounds 79 and 80, respectively. Pentavalent hypermonomerby the above double80 was click then reaction used toward with dipropargylated the decavalent dumbbell-shape tetraethylene glycol lactosylated 81. Standard compound Zemplén82 by thede- aboveO-acetylation double (MeONa, click reaction MeOH) with generated dipropargylated 79 (3-mer), tetraethylene 80 (5-mer) glycol and 8183 .(10-mer) Standard in Zemplén almost quantitative yields in each case.

Molecules 2016, 21, 629 23 of 36 de-O-acetylation (MeONa, MeOH) generated 79 (3-mer), 80 (5-mer) and 83 (10-mer) in almost quantitativeMolecules 2016, 21 yields, 629 in each case. 23 of 35 Molecules 2016, 21, 629 23 of 35

Scheme 16. Synthetic procedures leading to hexaphenylbenzene core (69) which was either directly SchemeScheme 16.16. SyntheticSynthetic proceduresprocedures leadingleading toto hexaphenylbenzenehexaphenylbenzene core (69)) whichwhich waswas eithereither directlydirectly propargylated to give 70 or treated with a propargylated TRIS intermediate (71) to afford a scaffold propargylatedpropargylated toto givegive 7070 oror treatedtreated withwith aa propargylatedpropargylated TRISTRIS intermediateintermediate (71)) toto affordafford aa scaffoldscaffold harboring 18-propargyl residues (72). harboringharboring 18-propargyl18-propargyl residuesresidues ((7272).).

Scheme 17. Accelerated synthesis of multivalent lactosides using click chemistry with scaffold 72 Scheme 17. Accelerated synthesis of multivalent lactosides using click chemistry with scaffold 72 andScheme a lactosyl 17. Accelerated azide (4) to synthesis give a glycodendrimer of multivalent lactosideswith 18-exposed using click lactoside chemistry residues. with scaffold 72 and and a lactosyl azide (4) to give a glycodendrimer with 18-exposed lactoside residues. a lactosyl azide (4) to give a glycodendrimer with 18-exposed lactoside residues.

Molecules 2016, 21, 629 24 of 36 Molecules 2016, 21, 629 24 of 35

SchemeScheme 18. 18.Lactose-presenting Lactose-presenting glycoclusters glycoclusters withwith an azide functionality functionality at at the the focal focal point point were were used used for the convergent synthesis of highly substituted glycodendrimers 79 (3-mer), 80 (5-mer), and 83 for the convergent synthesis of highly substituted glycodendrimers 79 (3-mer), 80 (5-mer), and 83 (10-mer) possessing an extended tetraethylene glycol spacer for accessibility to galectins. (10-mer) possessing an extended tetraethylene glycol spacer for accessibility to galectins. Alternatively, globular-shaped glycodendrimers having six (6-mer, 87), eighteen (18-mer, 88), andAlternatively, thirty six (36-mer, globular-shaped 90) were constructed glycodendrimers using the having extended six (6-mer,lactoside87 residues), eighteen (Scheme (18-mer, 19) 88[126].), and thirtyThus, six (36-mer,using a 90hexapropargylated) were constructed cyclotriphosphazene using the extended derivative lactoside residues(84) and (Schemethe copper-catalyzed 19)[126]. Thus, using[1–3]-dipolar a hexapropargylated cycloaddition cyclotriphosphazene together with azide derivative 75, the three (84) andcompounds the copper-catalyzed were prepared [1,3]-dipolar in 82%, cycloaddition65%, and 77% together yields, withrespectively. azide 75 After, the Zemplén three compounds trans esterification, were prepared the unprotected in 82%, 65%,lactosylated and 77% yields,dendrimers respectively. were obtained. After Zemplén Interestingly, trans high-field esterification, 1H-NMR the spectra unprotected clearly lactosylated probed completion dendrimers of werethe obtained. multiple click Interestingly, processes, high-fieldas judged by1H-NMR the complete spectra disappearance clearly probed of the completion critical alkyne of the protons multiple clickat δ processes, 2.64 ppm. asIn addition, judged by all thethe relative complete inte disappearancegrations of each of triazole the critical protons alkyne around protons δ 8 ppm at wereδ 2.64 ppm.in perfect In addition, agreement all thewith relative those of integrations the newly formed of each internal triazole region protons (18H aroundvs. 6H, respectivelyδ 8 ppm were for in perfect18-mer agreement 88). Moreover, with thoseMALDI-TOF of the experiments newly formed affo internalrded typical region isotopic (18H patternsvs. 6H, for respectively 90 with the for expected molecular weight signal ([M + Na]+ adduct) at m/z 23,750 Da. Using an analogous strategy, 18-mer 88). Moreover, MALDI-TOF experiments afforded typical isotopic patterns for 90 with lactodendrimers harboring 20-mer, 30-mer, 60-mer, and finally 90-mer were also constructed (Scheme 20) the expected molecular weight signal ([M + Na]+ adduct) at m/z 23,750 Da. Using an analogous starting from thiolated pentaerythritol and hexasubstituted benzene derivatives [126]. The above strategy, lactodendrimers harboring 20-mer, 30-mer, 60-mer, and finally 90-mer were also constructed

Molecules 2016, 21, 629 25 of 36

Molecules 2016, 21, 629 25 of 35 (Scheme 20) starting from thiolated pentaerythritol and hexasubstituted benzene derivatives [126]. Thestructures above structures were shown were to shown have low to have polydispersity low polydispersity PDI (Mw/Mn) PDI (Mw/Mn) (<1.08) with (<1.08) molecular with molecularweight weightranging ranging from from5 kDa 5to kDa more to than more 75 than kDa 75[126]. kDa [126].

O O O O O O O O O O O O O O O O O O O N N N N N N N N O O N N N O O O N O O O O N N O N N N O O N O O O HN O O O O HN O N N N O N N O N N N N N N N O O O O O O O N O O O O N N O H N N O O N O N N N O O N N P P N O O O O O O N N O N N O N N N N O O O P P O O O N P O O N N O N N N N N N O O O N P O O O H N N O O O O N N N N O O O O N O O O O N O O O O O O O O O N N N N N N N O N N N N N O O NH O O O NH O N O N O O N N O O N N O O O O O O O O N N N N N N N O N N N N N O O O O O MeONa , MeOH 86 R=Ac O O O MeONa , MeOH 85 R=Ac O r.t. , 18h 90% 88 R=H O O r.t.,18h 87 R=H O O 82% O O O O O O 84% O O

THF/H2O 50oC (3h.) , r.t. (18h.) 75 79 THF/H2O o CuSO4×5H2O,NaAsc 50 C (3h.) , r.t. (18h.) CuSO4×5H2O,NaAsc 65%

O O

RO OR OR O O O N O P P O O O O O RO N N RO RO P RO O O

89 R=Ac NaOMe, MeOH O O r.t., 18 h 84 90 R=H 93%

80 CuSO4×5H2O,NaAsc THF/H2O 50oC, 3h, r.t. 18h 77%

O O O O O O O O O O O O O O O O O O O O O O O O O

N N N N N N N O O N N N O N N N O O N N N O O N N O O N O O O O N N O N N O O N O O O O N O N O N P P O O O O P N N N N N O O P O O O N O O P P N O O N O O N O O O

NH O O O O O O NH O N N O N N N O O N N N N N O O O N N N O N O O O O N O O N O O H N N O N N P N O O N N O O N O N N O O O O N N P P O O N O O P P O O O N O P P O N O N N N O O O N P O N N N O N O O N P O O N H O O O N N N O O N O O O O O N O O N N N N N O N N N O N N N N O N O HN O O O O O O HN O N O O O O O N O O N O N O O P P O P O N N O O O N N O P O P P N O N O O O O N O N O N O N N O O O O N O N O N N O O O O N N N N N O N N N O O N N N N N N O N N N O O O O O O O O O O O O O O O O O O O O O O O

O O O

Scheme 19. Convergent synthesis of a globular glycodendrimer with six (6-mer, 87), eighteen Scheme 19. Convergent synthesis of a globular glycodendrimer with six (6-mer, 87), eighteen (18-mer, 88), and thirty six (30-mer, 90) sugar headgroups. (18-mer, 88), and thirty six (30-mer, 90) sugar headgroups.

Molecules 2016, 21, 629 26 of 36 Molecules 2016, 21, 629 26 of 35

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O N O O O N N N N O O O N N N N N N O O O N N N O O O N N N N N O NN N N N N N N N N N N N N N N N O N N N N N O N N O O N N O N N N N N N N O N O O N N O O N N N O N O O N O O O O O N O O O O O N O N O N O O O N O O N O N N O HN O N O O N O O O O O O N N O O O N N O HN NH O N O O N O O HN O O O O O P P N N O N O P N N O O O N O N O N O P N N O N N N N HN O O P O O O N O N N N N N NN N N N N N O P N O H O O N O N N N N O N O O N O O N N O O N O O N O N N O HN N O O O HN O O O O N O N O O N N HN S O N N N O O S O N O N N N N H O O N O O O S H O O N O N S O N N P P O O N O O N O O N O O N P N O N N N N N O O O N P P N NH O N O O NP O O N P O O O O N O O O O O O P N O O N N N O O O O N O O O N P O O N NH HN O N N O P O N O N N N N N N O O N O N O O N O HN O N O O O P P O O O N N O O O O N O O S O O N O O O N O N S HN N N N O N O O O N O O N N N O N O O O N O O O O O NN N N O N N O N O N O O N 93 (60-mer) N N O O N O N N O O O N N S O N O NH S N O O O N N N O O N N O O O O NH O O O O O O O O N O O N N HN N O O O N N O NH O O O O O O N N O O O N O N O O O O O O O O O O O O P N O P N N O O N P N N N O O O O O N N O O N PNO O P P O N O N O O O 91 (20-mer) N O O O H N O O O N O O N N N N N N O O O NH N N N O O O O O O N O N O O N O N O O N O N N O N O O N N O N N N H O O O N N N N N N O N N N N N N N N N O HO OH O NH N N O O OH O N O O N O N O O O NH HN O N N O O N O O NH O O O O HO O N O NH O HO HO N O N HO O N N O O O O O O N N O O O O N N O N N O O O N N N N N O N N N N N N O O N N N N N N N N N N N O O N N N N N N N O O O N N N N N N N N O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O NN O O N N N N O O N N O O N O O O O O N O O O O N NN O NN N N N O N N N O O O O O O O N O N N O O N N O O O N O N P O N O N N O O P N N O O N P NN O O N O O O O O O O O N O O O HN N O O N N N S O O N N O N OP N O N O O N O O N N O O N P O O O N P N O HN P N O O HN N O O O O O N O O O O O O O O N P O N OP N O N N P O O N S O N O N N H S HN O P N O O N O O O N P N O S N O O N N O O NN O S O N O N O N O O O O N O O N O NH H 94 (90-mer) O S S O NN O N HN N O N N O N O N O O N P O N N P N O O O O N O N N O O O O O O N P NH O O O O O N P O N O O O P N O O N N N O N P O N N N O N O O N N N N HN N O O N N O O N N O O O O O N O O O O O O N N O P N O HN O O O N N P O N HN N P N O O NN O N O O N N O N N O N O O O O O O O O O O O N N N O N O N N N O O N N N N N O NN O N O O N N O O O N O O N N N N O O N N N N O O O O N O N N O N O N N N N O NN N N N O O O O O O N N O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 92 (30-mer) O 6 Scheme 20. Illustration of the structures of variations of the theme to build glycodendrimers with the Scheme 20. Illustration of the structures of variations of the theme to build glycodendrimers with the same core as in Scheme 18 harboring 20 (91), 30 (92), 60 (93), and 90 (94) lactoside residues. same core as in Scheme 18 harboring 20 (91), 30 (92), 60 (93), and 90 (94) lactoside residues. The size of the glycodendrimers containing both the acetylated and the deprotected functionalities, andThe more size particularly of the glycodendrimers their solvodynamic containing radii, both was the estimated acetylated by andpulsed-field-gradient the deprotected functionalities, stimulated andecho more (PFG-STE) particularly NMR their experiments solvodynamic using radii, bipolar was pulse estimated pairs by longitudinal-eddy-current pulsed-field-gradient stimulated delay echo(BPP-LED) (PFG-STE) in CDCl NMR3 and experiments D2O, respectively, using at bipolar 25 °C, pulseas described pairs longitudinal-eddy-currentin detail [126]. Diffusion values delay ˝ (BPP-LED)were determined in CDCl by3 and the Daverage2O, respectively, of individual at 25valuesC, ascorresponding described in to detail the decay [126]. of Diffusion the signal values intensity were determinedof different by protons the average located of individualat different valueslevel in corresponding the molecule. toThe the resulting decay of measurements the signal intensity were of differentconsistent protons with locatedspherical at differentand unimolecular level in the character molecule. of the The glyc resultingodendrimers. measurements The Stokes–Einstein were consistent withequation spherical directly and yielded unimolecular the corresponding character of solvodynamic the glycodendrimers. diameters using The Stokes–Einstein viscosities determined equation directlyfor pure yielded deuterated the corresponding solvents. As expected, solvodynamic diffusion diameters data revealed using viscositiesthe increase determined of solvodynamic for pure deuterateddiameters solvents.as the number As expected, of peripheral diffusion epitopes data was revealed enhanced, the increase under similar of solvodynamic solvent conditions, diameters showing dendrimers with solvodynamic diameters ranging from 2.6 to 10.1 nm. Systematic monitoring as the number of peripheral epitopes was enhanced, under similar solvent conditions, showing of bioactivity with the CRD of galectin-3, physiologically a product of proteolytic processing that dendrimers with solvodynamic diameters ranging from 2.6 to 10.1 nm. Systematic monitoring of removes the N-terminal tail (please see Figure 3), revealed ligand property for all compounds. As bioactivity with the CRD of galectin-3, physiologically a product of proteolytic processing that removes noted above for starburst glycodendrimers, the relative potency per sugar unit was highest at low the N-terminal tail (please see Figure3), revealed ligand property for all compounds. As noted above to medium level of valency, here for the dumbbell-shapes decavalent 83 (r.p./sugar = 53) of a diameter forof starburst 5.3 nm [126]. glycodendrimers, These examples the document relative potency the inge pernuity sugar of unitchemical was highestdesign atto lowreach to the medium level of level ofgiant valency, clusters here with for the a core. dumbbell-shapes To take design decavalent even further83 (r.p./sugarinto the direction = 53) ofof aengineering diameter of vesicle-like 5.3 nm [126 ]. Thesestructures, examples a different document strategy the ingenuityhas been successfully of chemical implemented. design to reach the level of giant clusters with a core. To take design even further into the direction of engineering vesicle-like structures, a different strategy has been successfully implemented.

Molecules 2016, 21, 629 27 of 36

The way (glyco)lipids self-organize into monolayers or liposomes has been equally efficiently achieved with amphiphilic Janus glycodendrimers (for a comparison of a glycolipid and the synthetic analogue,Molecules please 2016, see 21, 629 Scheme 21)[127]. The products are glycodendrimersomes or aggregates27 of 35 of other types of design such as cubosomes with fully programmable properties in size and surface properties [127The, 128way]. (glyco)lipids For example, self-organize ligand into density monolaye onrs micro- or liposomes and macroscaleshas been equally can efficiently be changed in achieved with amphiphilic Janus glycodendrimers (for a comparison of a glycolipid and the synthetic a definedanalogue, manner, please as branching see Scheme in 21) glycodendrimers [127]. The products is. Theseare glycodendrimersomes products are ideal or for aggregates interaction of studies with lectins.other Here, types theof design influence such ofas anycubosomes parameter with onfully both programmable sides on aggregation properties in cansizebe and examined. surface These assays withproperties human [127,128]. galectins For haveexample, already ligand answered density on fundamentalmicro- and macroscales questions can on be galectin changed functionalityin a in trans-interactionsdefined manner, and as branching as a function in glycodendrimers of carbohydrate is. Thesedensity products andare ideal headgroup for interaction display studies [129 –131]. Galectin-1’swith designlectins. asHere, non-covalently the influence of associated any paramete homodimerr on both sides (please on aggregation see Figure can3) appearsbe examined. more suited These assays with human galectins have already answered fundamental questions on galectin for cross-linkingfunctionality counter in trans receptors-interactions on and the as surface a function than of tocarbohydrate act as bridge density between and headgroup glycodendrimersomes, display as indicated[129–131]. by comparative Galectin-1’s analysisdesign as with non-covalently engineered associated variants thathomodimer have covalent (please see connections Figure 3) between the twoappears CRDs more [129 ].suited Fittingly, for cross-linking atomic counter force recept measurementsors on the surface have than revealed to act as bridge a comparatively between low ruptureglycodendrimersomes, of 37 ˘ 3 pN at a loadingas indicated rate by ofcomparative 3 nN/s, whenanalysis galectin-1 with engineered interacted variants with that Nhave-glycans of the glycoproteincovalent connections asialofetuin between [132 the]. two An CRDs emerging [129]. Fittingly, aspect atomic for endogenous force measurements lectins have is therevealed occurrence a of comparatively low rupture of 37 ± 3 pN at a loading rate of 3 nN/s, when galectin-1 interacted with genetic polymorphisms.N-glycans of the glycoprotein It is a pertinent asialofetuin question [132]. whetherAn emerging they aspect have for pathophysiological endogenous lectins is significance.the As biomedicaloccurrence effector, of genetic it is polymorphisms. noteworthy thatIt is a this pertinent lectin question can have whether anti- they and have pro-inflammatory/tumoral pathophysiological activitiessignificance. depending As on thebiomedical context, effector, for example it is noteworthy stimulating that tissue this invasion lectin can (glioblastoma) have anti- and or cartilage degenerationpro-inflammatory/tumoral (osteoarthritis) or actingactivities as depending negative regulatoron the context, in chronic for example inflammation stimulating or carcinomatissue cell invasion (glioblastoma) or cartilage degeneration (osteoarthritis) or acting as negative regulator in cycle progression [133–139]. chronic inflammation or carcinoma cell cycle progression [133–139].

Scheme 21. Typical structure of a natural glycolipid (LacCer) compared to a member of the recently Schemesynthesized 21. Typical family structure of self-assembling of a natural amphiphilic glycolipid Janus (LacCer) glycodendrimers compared capable to a memberof forming of stable the recently synthesizedliposomes family (glycodendrimersomes). of self-assembling amphiphilic Janus glycodendrimers capable of forming stable liposomes (glycodendrimersomes). For the first time, a functional correlation of a single nucleotide polymorphism in a CRD of a galectin could be discerned with this assay system, here for galectin-8’s F19Y variant whose Forexpression the first time,is strongly a functional associated with correlation occurrence of of a rheumatoid single nucleotide arthritis and polymorphism early age at disease in a CRD of a galectinonset could [130,131,140]. be discerned This approach with thisthus takes assay activity system, testing here to the for level galectin-8’s of vesicle-like F19Y structures, variant whose expressionand isthe strongly recently accomplished associated withhybrid occurrence formation with of bacterial rheumatoid membranes arthritis [141] andlet the early perspective age at disease onset [130become,131,140 envisioned]. This approachto combine thus natural takes glycoconju activitygates testing with to these the levelsynthetic of vesicle-likeglycodendrimers structures, to and tailor surface properties in a so far unsurpassed manner according to the biomedical question to be the recentlyaddressed. accomplished Design of hybrid monolayers formation with the with same bacterial toolbox membranes will flank the [141 analysis] let the of perspectiveaggregation become envisioned(trans to-interaction). combine natural In such glycoconjugatesa setting, cis-bridging with can these be monitored, synthetic as glycodendrimers exemplified by monitoring to tailor surface propertiesgalectin-1/ganglioside in a so far unsurpassed GM1 interplay manner [142]. according to the biomedical question to be addressed. Design of monolayers with the same toolbox will flank the analysis of aggregation (trans-interaction). In such a setting, cis-bridging can be monitored, as exemplified by monitoring galectin-1/ganglioside GM1 interplay [142]. Molecules 2016, 21, 629 28 of 36

6. Conclusions The increasingly unraveled physiological significance of carbohydrate-lectin recognition poses challenging questions on the nanometric characteristics underlying the specificity and selectivity of the functional pairing. As the example of the adhesion/growth-regulatory galectins attests, delineating structure-activity relationships with respect to the intrafamily divergence in CRD sequence and its spatial presentation requires sophisticated tools and transsectional cooperation. Here, the individual parts of the toolbox provided by synthetic chemistry are invaluable sensors, e.g., to determine the capacity for multivalent binding and induction of cell signaling that may cause harmful effects. Actually, glycoclusters had been helpful to ascertain a reduction in cross-linking, leading to undesired T cell activation, within the process of engineering an antiviral lectin for further testing of its potential for therapeutic application [143]. Along the route to obtain insights into the biofunctionality of the nanometric features, bringing test systems for activity assessment of the synthetic products as close as possible to the in vivo conditions, e.g., by testing their effect on lectin binding to tissue sections [144], will team up with the design of vesicle-like structures with increasing degree of resemblance to cell surfaces. By rational tailoring on both sides, i.e., the ligand and the lectin, as hypothesis-driven process, these advances bring understanding how endogenous lectins ‘read’ sugar-encoded information into reach. The monovalent [145,146] as well as the multivalent glycomimetic lactoside derivatives presented herein offer several avenues to identify high affinity galectin-selective antagonists for the treatment of various diseases. Nanometric glycodendrimers in particular, with their size ranging from 2 to 10 nm and their high water-solubility, constitute a valuable alternative to other types of insoluble nanoparticles [147]. Of great interest in this regard, is the new family of glycodendrimersomes described in the last section which are also nanometric (100–150 nm, for liposomes) or 100–200 µm when forming giant vesicles. These biomimetics of cell membranes have the fortuitous possibility to adapt their flexible inter-carbohydrate distances in response to the galectin receptors they encounter, thus adding an additional advantage over other nanoparticles.

Acknowledgments: R.R. thanks the Natural Sciences and Engineering Research Council of Canada (NSERC) for a Canadian Research Chair and the Canadian Glycomics Network for financial support. P.V.M. thanks Science Foundation Ireland for financial support (08/SRC/B1393 & 12/IA/1398) which was co-funded under the European Regional Development Fund under Grant No. 14/SP/2710. P.V.M. also thanks the European Commission for Marie Curie IntraEuropean Fellowships to Sebastien G. Gouin, Trinidad Velasco-Torrijos and Dilip V. Jarikote, which supported some of the research reviewed herein. H.-J.G. thanks the excellence program of the Ludwig-Maximilians-University Munich and the EC (for ITN network funding) for generous support. Conflicts of Interest: The authors declare no conflict of interest.

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