Carbohydrate CuAAC click chemistry for therapy and diagnosis

Accepted Manuscript Title: Carbohydrate CuAAC click chemistry for therapy and diagnosis Author: Xiao-Peng He, Ya-Li Zeng, Yi Zang, Jia Li, Robert A Field, GuoRong Chen PII: DOI: Reference:

S0008-6215(16)30069-6 http://dx.doi.org/doi: 10.1016/j.carres.2016.03.022 CAR 7157

To appear in:

Carbohydrate Research

Received date: Revised date: Accepted date:

2-2-2016 22-3-2016 23-3-2016

Please cite this article as: Xiao-Peng He, Ya-Li Zeng, Yi Zang, Jia Li, Robert A Field, Guo-Rong Chen, Carbohydrate CuAAC click chemistry for therapy and diagnosis, Carbohydrate Research (2016), http://dx.doi.org/doi: 10.1016/j.carres.2016.03.022. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Carbohydrate CuAAC click chemistry for therapy and diagnosis Xiao-Peng He,a Ya-Li Zeng,a Yi Zang,b Jia Li,b,* Robert A Fieldc,* and Guo-Rong Chena,*

a

Key Laboratory for Advanced Materials & Institute of Fine Chemicals, East China University

of Science and Technology (ECUST), 130 Meilong Rd., Shanghai 200237, PR China b

National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai

Institute of Materia Medica (SIMM), Chinese Academy of Sciences (CAS), 189 Guo Shoujing Rd., Shanghai 201203, PR China c

Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich

NR4 7UH, UK

*Corresponding authors: [email protected] (J. Li) [email protected] (R. A. Field) [email protected] (G.-R. Chen)

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Graphical Abstract CuI Click

Pharmacophores Fluorophores Polymers Materials Surfaces

Click

CuI

…

We summarize the impressive progress since 2010 made in CuAAC-based carbohydrate click chemistry for therapy and diagnosis. Abstract Carbohydrates are important as signalling molecules and for cellular recognition events, therefore offering scope for the development of carbohydrate-mimetic diagnostics and drug candidates. As a consequence, the construction of carbohydrate-based bioactive compounds and sensors has become an active research area. While the advent of click chemistry has greatly accelerated the progress of medicinal chemistry and chemical biology, recent literature has seen an extensive use of such approaches to construct functionally diverse carbohydrate derivatives. Here we summarize some of the progress, covering the period 2010 to mid-2015, in CuI-catalyzed azide-alkyne 1,3-dipolar cycloaddition CuAAC “click chemistry” of carbohydrate derivatives, in the context of potential therapeutic and diagnostic tool development.

Keywords: Click chemistry, Carbohydrate, CuAAC, Therapy, Diagnosis

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1. Introduction Carbohydrates play diverse roles in the life cycle of nearly all living species. Of particular importance are the cell-surface glycan “antennae” that produce multivalent interactions with carbohydrate-recognition proteins (CRPs). These selective recognition events are responsible for a wide range of biological events, including the adhesion of white blood cells to sites of infection or tissue damage, sperm-egg interactions during mammalian fertilization, and the control of cell fate during differentiation.1-4 However, while malfunctional glycosylation correlates with fatal human diseases such as cancer,5 specific CRPs expressed on the surface of microbial pathogens can capture normal carbohydrates on human cells, leading to inflammation, bacterial adhesion or virus invasion.6,7 As a result, carbohydrates present many opportunities for intervention in disease diagnosis and therapy.8 For example, the anti-influenza agent Tamiflu (oseltamivir) – a sialic acid mimetic – that inhibits the activity of neuraminidase, acts by preventing completion of the virus life cycle (see also Fig 2 and associated text).9 Carbohydrates have also been employed as probes and sensors to detect carbohydrate-protein interactions10-12 while efficient solid-phase and solution-based methods have been developed, promoting the advancement of the “glycomics” as well as disease diagnosis opportunities.10-12 Despite the elegant carbohydrate chemistries devised by glycoscientists13-15, an effective and modular synthetic approach that meets the ever increasing interest in the preparation of functional carbohydrate derivatives is needed. Click chemistry, a synthetic concept coined by Sharpless et al. in 2001, encourages the discovery of simple and modular chemical tools for the effective construction of diverse compound libraries.16 The advent of the

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CuI-catalyzed

azide-alkyne

1,3-dipolar

cycloaddition

reaction

(CuAAC),

discovered

independently by Sharpless17 and Meldal18, has revolutionized the way in which chemists consider compound synthesis. Importantly, CuAAC has efficiently filled the gap between chemists and practitioners beyond the chemical community – including those who are focused on materials science, biomedicine, nanotechnology, chemical biology and cell biology19,20 – accelerating discovery and development in contemporary life sciences. A growing effort has been devoted to the employment of click chemistry (CuAAC and other click chemistries, such as the thiol-ene reaction21) for the production of functional carbohydrate derivatives over the last decade. While several comprehensive reviews of this topic are available covering the period up to 2010,22-25 here we summarize some of the impressive progress since that date in CuAAC-based carbohydrate click chemistry for therapy and diagnosis.

2. Carbohydrate click chemistry for therapy

2.1. Anticancer activity Triazolyl carbohydrate derivatives have been synthesized by CuAAC for evaluation of their anticancer activity, mainly against cancer cell lines. Carbonic anhydrase (CA) is a transmembrane enzyme that catalyzes the hydration of carbon dioxide to bicarbonate, a process that has proven to facilitate cancer cell survival in an acidic environment. A number of CA isozymes exist in human cells, and inhibition of the tumor cell-specific CAs (such as CA IX) has been suggested as a strategy for selective cancer therapy.26 In general, a

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carbohydrate is used as a hydrophilic tail to bind a hydrophilic pocket in CA, with an aryl sulfamides moiety used to target the catalytic domain (Fig. 1). By CuAAC, both 1,4-disubstituted (1)27 and 1,4,5-trisubstituted (2)28 triazoles with selective, low nanomolar CA inhibition activities have been synthesized. Co-crystal structures of the inhibitor-CA complexes revealed the motif by which the carbohydrate moiety associates with the enzyme.27 Interestingly, dual tail groups (a carbohydrate and an aromatic) were co-clicked to a CA inhibitor (3), interacting with the distinct hydrophilic and hydrophobic halves of a CA site.29 Heat shock protein 90 (Hsp90) is a chaperone for many proteins; the inhibition thereof might be expected to result in the degradation of multiple proteins related to cancer progression.30 A series of triazolyl glycocoumarin derivatives were synthesized (4), showing Hsp90-dependent anti-proliferative activities for breast cancer cells.31 CuAAC was also carried out on the side chain of carbohydrates to construct enzyme inhibitors with potential anticancer activities. Protein tyrosine phosphatases (PTPs) dephosphorylate the phosphotyrosine site of proteins and peptides. The overexpression and dysfunction of some PTPs is related with cancer.32,33 Considering the poor pharmacological properties of the highly charged phosphotyrosine (pTyr), alternative amino acids that might target the catalytic domain34,35 were used as a surrogate to decorate the side chain of several glycosides by CuAAC, producing bis-triazolyl (5)36,37 and mono-triazolyl (6)38,39 PTP inhibitors with micromolar inhibitory activities. Interestingly, decoration of the amino acids onto different side-chain positions of carbohydrates yielded distinct PTP inhibition activities,37-41 suggesting that carbohydrates might serve as suitable scaffolds to optimize lead compounds. Some such inhibitors also showed anti-proliferative activities for selected cancer cells.38

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Salicylate (7),42-44 α-ketoacid45,46 and other pharmacophores47,48 were alternatively used as the pTyr mimetic to modify the C1-, C6- or both positions of carbohydrates by CuAAC, and micromolar-range PTP inhibition activities were obtained. However, further evidence is needed to confirm that the anticancer properties observed are dependent upon PTP-inhibition.

Other clicked carbohydrate derivatives with cytotoxicity for cancer cells, but without an identified molecular target, have been prepared (Fig. 1). Bis-triazolyl amino acids were noted to suppress the growth of multiple myeloma cells.49 Meanwhile, series of natural product-carbohydrate triazole conjugates50-52 showed toxicity for cancer cells, with a glycosyl glycyrrhetinic acid50 selectively inducing apoptosis of a human cervical cancer cell line over a normal cell line. Glycolipids are an important cell-surface component for signal transduction. They have been widely used as non-ionic surfactants due to their amphiphilic nature. Triazole-linked monosaccharide- (8)53,54 and nucleoside- (9)55 based glycolipid derivatives have been synthesized and identified to have micromolar range toxicity for a wide spectrum of cancer cell types. While increase of lipid chain length was found to increase the toxicity, change of the click conjugation site (anomeric or side chain) and monosaccharide (glucoside or galactoside) altered sharply the activity.53,54 Whereas these findings might provide an insight into the development of triazolyl glycolipids as potential anticancer agents, their mechanism of action needs to be better delineated – is the observed cytotoxicity simply a surfactant effect, for instance?56-58

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2.2. Antiviral and antimicrobial activity Pathogen infections often rely on the interaction of its surface proteins with the glycocalyx of human host cells. As a result, clicked carbohydrates that inhibit these processes have been developed as potential antiviral and antimicrobial compounds. Design and synthesis of pathogen surface glycosidase inhibitors by CuAAC represents the main strategy.

The neuraminidase (NA) family is an important class of glycosidases that hydrolyze the sialic acid terminus of glycans, modulating the interaction of host cell with extracellular stimuli. The most well-known is the surface-expressed NA family of influenza viruses, which have caused a series of flu pandemics over many decades. On the basis of the interaction between sialic acid and NA, zanamavir and oseltamivir (Fig. 2), two sialic acid glycomimetics, have been developed to inhibit NA, effectively combating influenza.8 CuAAC has been used to dimerize zanamavir, affording 1,4-triazole-linked zanamavir dimers (10).59 The dimers showed an up to 3000-fold increased inhibitory activity for influenza A and B viruses with respect to the monomer. Since multivalent interactions are critical to increase the generally low inherent binding affinity between a single carbohydrate and a protein, sialic acids have been polymerized. While a sialylthio-D-galactose fullerene conjugate synthesized by CuAAC click reaction showed moderate inhibitory activity against NA,60 a polyglycerol-based nanoparticle functionalized by triazolyl sialic acid inhibited binding and fusion between an influenza A virus and a human erythrocyte quite markably.61 The latter study suggested that

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both particle size and ligand density are important determinants of antiviral activity, adding insight into the design of multivalent nanoparticles for interactions at a biological interface. Clicked sialic acid derivatives have also been developed for other NAs. Children infected by human parainfluenza viruses (hPIVs) may experience respiratory tract diseases. However, no drugs have been approved for hPIVs to date. By combining computational and synthetic chemistries with NMR-based structural and biological techniques, a C4-triazole modified sialic acid derivative was developed (11).62 This compound efficiently blocked hPIV entry and progeny virion release by inhibiting the hPIV type 3 haemagglutinin-neuraminidase (HN), offering a potential preclinical candidate for combating the virus. Dysfunction of human neuraminidases (hNEUs) is implicated in sialic acid storage disorders and many other diseases. Triazolyl sialic acid derivatives with a CuAAC modification at the anomeric or at the side chain position have been prepared for inhibition of hNEUs. A click strategy was used to screen a focused library of aglycone-modified sialosides, leading to the identification of a hNEU2-selective, micromolar-range inhibitor (12).63 The CuAAC modification of the anomeric position was found to improve the Ki with minimal change in the t1/2 of the enzymatic activity. As for the side-chain click decoration, it was concluded that the C9-, but not the N5Ac-position, tolerated a triazole-linked hydrophobic group, producing selective hNEU3 inhibitors (13).64 Similarly, the introduction of a triazole to the N5Ac position of a sialyl lactoside resulted in the deactivation of the compound for hNEU3. In contrast, the presence of a lipid aglycone of the sialyl lactoside was critical for NEU3 binding (14).65 Iminosugar inhibitors for α-glucosidase, developed by assembling adamantane and

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iminosugars via CuAAC, were shown to disrupt viral glycoprotein processing; the increase of linker chain length of these inhibitors was found to be crucial for their antiviral activity (15).66 Interestingly, lipid chain length was also found to be crucial for the anticancer activity of triazolyl glycolipids.53,54 Nonetheless, the relationship between the micelle-forming ability of these amphiphiles and their bioactivity needs to be addressed.56-58 Pathogenic bacteria adhesion to host cells often relies on carbohydrate-protein recognition, as is typified by Escherichia coli which can cause urinary tract infections (UTIs). This bacterium uses the lectin domain of its type 1 fimbriae to bind the terminal α-mannosyl glycoproteins that are prevalent on epithelial cells of the urinary tract, leading to tissue colonization. Carbohydrate scaffolds that antagonize these recognition events have been developed (Fig. 3). Employing CuAAC,

β-cyclodextrin (CD) was used as a platform to

display mannosides in a multivalent manner.67 A heptyl mannoside grafted β-CD (16) synthesized by a twofold click reaction showed strong type 1 fimbriae (FimH) binding affinity and could rapidly reach mouse bladder following intravenous injection, with retention over 24 h preventing E. coli colonization. This study offers an example for the design of multivalent mannosides with good in vivo pharmacokinetics. Mannosides have also been introduced to nanodiamond particle (NDs) surfaces for multivalent display. Monomeric (17)68 and trimeric (18)69 mannosides were linked to NDs through triazoles formed by CuAAC, producing “first generation” and “second generation” mannosyl NDs, respectively. These nanoparticles were found to antagonize type 1 fimbriae-mediated bacterial adhesion to bladder cells, with the “second-generation” being more potent probably as a result of its increased mannoside valency as well as an increase of glycosidase stability due to the

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presence of S-mannosides. More interestingly, these carbohydrate-grafted nanomaterials reduced E. coli biofilm formation, an unprecedented property amongst all multivalent glycans reported to date. Inhibition of trans-glycosidase activities using clicked carbohydrate derivatives has been developed as a therapeutic strategy for protozoal infection. Trypanosoma cruzi trans-sialidase (TcTS) is implicated in the pathogenesis of Chagas’ disease – a parasitic blood-borne parasite infection that occurs widely in Central and South America.70-74 A triazole-linked sialyl galactose (19) was produced by CuAAC, and then was identified to have sub-millimolar inhibitory activity for TcTS and an anti-parasite activity.70 The use of CuAAC oligomerization of azido-alkyne functionalized galactose building blocks was shown to give rise to macrocycles and linear oligomers, the former capable of serving as TcTS substrates and of blocking macrophage invasion by T. cruzi (20).71,72 Tuberculosis, caused by Mycobacterium tuberculosis, is a leading cause of mortality in many countries. Its’ carbohydrate-based cell wall plays a major role in drug resistance – blocking drug uptake – and persistence of the organism in infected macrophages. A number of potential glycobiology drug targets for tuberculosis therapy are therefore evident.

Click chemistry has been used to produce 1,6-oligmannosides, of which the hexa-mannoside and octa-mannoside (21) showed the highest activity against mycobacterial α-1,6-mannosyltransferases.75 This suggests that the length of the linear oligomerization impacts the activity of the resulting oligo-mannosides. Alternatively, a focused small molecule library was prepared by CuAAC for M. tuberculosis, among which a per-acetylated

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glycosyl quinoline hybrids showed better activity than a carbohydrate-free control compound.76 In addition to the discussed triazolyl carbohydrates with antimicrobial activities, click derivatization was carried out on conventional antibiotics scaffolds, such as amino sugars,77,78 macrolides and aminoglycosides.79 While a clicked tobramycin-based cationic amphiphile (22) suppressed the growth of both gram-positive and gram-negative bacterial strains,80 the presence of a triazolyl glycolipid derivative (23) enhanced the drug susceptibility of a superbug, MRSA (Staphylococcus aureus).81 Interestingly, the combined use of 23 lowered the MIC (minimal inhibitory concentration) of a range of commercial β-lactam antibiotics, upward of 256-fold, for both ATCC and clinical MRSA isolates. Preliminary mechanistic investigations showed that the combination lowered the expression of a protein (penicillin binding protein 2a) that resists β-lactam drugs. In addition, the fact that a triazole-free glycolipid analogue did not show a similar synergistic effect highlighted the importance of the triazolyl moiety for the activity. This research provides a new glycolipid weapon for disarming superbugs. Collectively, while multivalent display of carbohydrates that are recognized by bacterial glycosidases is an effective tactic to antagonize bacterial adhesion, click glycosylation of conventional antibiotics might be a promising strategy to enhance the pharmacological properties of the resulting glycosyl derivatives.

2.3. Other glycosidase and glycosyltransferase inhibitors In addition to their involvement in infectious processes, many glycosidases and

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glycosyltransferases are implicated in other pathological events (Fig. 4). Gaucher’s disease (GD) is a lysosomal storage disorder caused by mutations in, and therefore deficient activity of, glucocerebrosidase (GCase), which hydrolyzes glucosylceramide. Iminosugars are carbohydrate mimetics where the endocyclic oxygen is replaced by a nitrogen atom. CuAAC was carried out to connect iminosugars with adamantane; the resulting conjugates (24) showed sub-micromolar inhibitory activity against GCase, being at least 10-fold more active than unmodified iminosugars.82,83 As glycomimetics of iminosugars, aminocyclitols were conjugated with lipids of different chain length by CuAAC, producing GCase inhibitors with nanomolar inhibitory activity (25).84,85 A computational docking study suggested the crucial role of triazole for enzyme inhibition and thermal stabilization. Meanwhile, click chemistry proved applicable for preparing triazolyl carbohydrate libraries as broad range glycosidase and glycosyltransferase inhibitors. Building on earlier work,86 both aglycone- and sidechain-modified carbohydrate triazoles were synthesized by CuAAC, showing activities for glucosidases87-91 and mannosidases (26, 27, Fig. 4).92,93

In contrast to glycosidases, glycosyltransferases catalyze the formation of glycosidic bonds. During these processes, a nucleotide diphosphate sugar (NDP) is typically used as the substrate, with transfer of a carbohydrate to an acceptor substrate. As a result, phosphate or pyrophosphate mimetics are frequently involved in glycosyltransferase inhibitor design, in

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order to decrease polarity. Therefore, neutral carbohydrate inhibitors have been developed by CuAAC. Pyridine was used as a pyrophosphate surrogate to click with carbohydrates, producing galactosyltransferase inhibitor (28)94 and N-acetylglucosaminyltransferase (OGT) inhibitor (29).95 However, both crystallographic and docking analyses suggested that the carbohydrate head groups do not occupy the catalytic site, with little interaction with the enzymes.

These

triazoles

probably

have

the

potential

to

be

developed

as

carbohydrate-based metal chelators for sensors design (see later section). Triazole-linked glycosyl naphthalimide was prepared for OGT inhibition, and the aglycone was proposed to generate π-stacking with a tryptophan residue of the enzyme.96 Series of sialyltransferase inhibitors, exemplified by compound (30), with different molecular shapes and properties were also synthesized by CuAAC.97 It was determined that the charge and hydrophobicity of these compounds contributed predominantly to their inhibitory activity. In an anti-bacterial discovery program, lactose was clicked to a microplate, followed by poly-sialylation with poly-sialyltransferase from Neisseria meningitidis. This platform has proven suitable for screening inhibitors of this glycosyltransferase.98 To prevent infection by human pathogen Helicobacter pylori (H. pylori), triazole linked GDP-fucose conjugates were synthesized, some of which showed inhibitory activity for H. pylori α1,3-fucosyltransferase.99 Interestingly, triazole-linked mannosyl oligomers prepared by CuAAC were used as an acceptor for a mycobacterial mannosyltransferase, establishing a useful small molecule assay for the enzymatic activity.100 Glycogen phosphorylase (GP), which catalyzes the phosphorolysis of glycogen, is a validated therapeutic target for type 2 diabetes mellitus. To the C1-position of the glucose

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scaffold that docks into the catalytic site, a variety of aromatic aglycones were introduced, by CuAAC, to target a hydrophobic site of GP (31 as a representative example).101-104 However, these triazolyl inhibitors showed moderate activities, suggesting that a more elaborate selection of hydrophobic aglycone as well as the use of glycomimetics, beyond glucose, might be needed to associate with the catalytic site of GP with a higher affinity.

2.4. Immunostimulating activity The fact that carbohydrates play a pivotal role in the mammalian immune system has spurred extensive efforts in constructing carbohydrate-based immunostimulating agents.105 Natural killer T (NKT) cells express both a T cell receptor (TCR) and natural killer cell receptors. They recognize glycolipid antigen/CD1d complex by the TCR, rapidly releasing cytokines, such as interleukins (ILs) and interferon-γ (IFN-γ). The released cytokines eventually lead to activation of innate and adaptive immune responses.106,107 The first and most well-studied TCR agonist is an α-galactosyl ceramide (α-GalCer or KRN7000) (Fig. 5).108 On the basis of this scaffold, triazolyl derivatives synthesized by CuAAC have been developed. The click strategy can be divided into the following three aspects: 1) Cer (ceramide)-modification, 2) Gal (galactose)-modification and 3) oligomerization (Fig. 5).

The amide linkage of Cer has been replaced by a bioisosteric triazole, coupling a set of alkyl chains of different length.109 The replacement was determined to upregulate the release of both IL-4 and IFN-γ from an NKT cell line, with long-chain triazole analogues (32)

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being more potent than their short-chain counterparts. A docking study suggested that the triazole moiety might form hydrogen bonds with amino acid residues of CD1d, thereby influencing the cytokine release profile. A preliminary in vivo test suggested that the triazolyl α-GalCer derivatives induced greater IL-4 secretion into the serum of a mouse model than a control.109 This pioneering study established a basis for the development of α-GalCer derivatives by the CuAAC click chemistry. In addition to the lipid chain and anomeric carbon of α-GalCer,109-111 the Gal residue itself has also been modified by click chemistry. Coupling of aliphatics, but not aromatics, to the C6-position of Gal, as in

(33), by CuAAC produced triazolyl α-GalCer derivatives with

increased ability to release cytokines both in vitro and in vivo compared to α-GalCer.112,113 Considering that CD1d molecules show localized clustering properties, a click dimerization was carried out to crosslink the C6-positions of two independent α-GalCers, producing a homo-dimeric α-GalCer (34),114 Although no obvious improvement in immunostimulating effect was observed for the dimers (which precluded a plausible bivalent effect), this study encourages the multivalent display of α-GalCer for high avidity binding with clustered CD1d. A similar strategy was developed to form a water-soluble β-GalCer-containing dendritic polymer by CuAAC for enhancing the interaction with HIV-1 gp120 (35).115 Finally, analysis of the crystal structure of a TCR-α-GalCer-CD1d ternary complex identified that the α-methylene unit of the fatty acid moiety could be amenable to introducing a label for biochemical studies. Biotinylated and fluorophore-labelled α-GalCer (36) were thus synthesized, both showing a comparable activity to unlabeled α-GalCer. This study offers a useful labelling approach for tracking the in vivo behavior of CD1d agonists.116 Since the

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stability of the TCR-α-GalCer-CD1d ternary complex is crucial for the immunostimulating effect, development of α-GalCer (the cross-linker between TCR and CD1d) derivatives has become the main strategy for more tightly associating the two proteins. In this sense, click chemistry has successfully made possible the flexible functionalization of α-GalCer at both the Gal (TCR binder) and the Cer (CD1d binder) ends. The development of carbohydrate-based vaccines has become a popular topic in recent years because of the existence of specific carbohydrate antigens on tumor cells. However, the major issue related to this subject is the low immunogenicity of the endogenous carbohydrate antigens. Click chemistry has been used to conjugate the poorly active carbohydrate antigens with a vaccine adjuvant, or to display the antigens in a multivalent manner.120-124 The most researched examples have been the Tn and sialyl-Tn (STn) tumor-associated antigens (Fig. 6). On the basis of the sophisticated syntheses of triazolyl glycopeptide, glycoprotein and glycolipid derivatives by CuAAC,117-120 Tn or STn peptide was coupled to a lipopeptide through click chemistry. It was determined that the vaccine with four copies of antigen (37) showed a remarkable cluster effect, without the need for use of external adjuvant, inducing antibodies that suppress the viability of MCF-7 breast tumor cells.121 Interestingly, the Tn antigen has also been attached to bacteriophage Qβ virus-like particles (38),122 eliciting much higher levels of antibodies than other virus-like particles. The antibodies produced strongly reacted with native Tn antigens on human leukemia cells, proving the effectiveness of dense multivalent display of carbohydrate antigens on a particle surface for vaccine design. In an alternative approach, a safe and potent immunostimulant, monophosphoryl

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lipid A, was used as a built-in adjuvant for click conjugation with a tumor-associated carbohydrate antigen, GM3 (39).123,124 The vaccine elicited robust antibody responses, and the antibodies produced bound selectively to target tumor cells. As a combined strategy, the multivalent display of carbohydrate antigens with a built-in immunostimulant by click chemistry can be anticipated.

2.5. Multivalent and small molecule carbohydrates for high-affinity lectin association Carbohydrates often require multivalent display to associate strongly with their cognate, oligomeric binding proteins due to the typically poor affinity of monovalent carbohydrate-lectin associations. On the one hand, click chemistry has proven a modular tool to couple carbohydrates to a variety of multivalent backbones due to its regio- and stereo-specificity and functional group tolerance. On the other hand, small-molecule carbohydrates with enhanced binding affinity with a lectin pair have been discovered from click-based high-throughput libraries. A comprehensive review with respect to this specific subject has been published recently.125 The frequently selected backbones on which carbohydrates are presented in multivalent format include dendritic scaffolds, peptides, polymers as well as carbohydrates. Dendrimers have emerged as a promising material for imaging and drug delivery applications because of their structural homogeneity, controllable degradation and the ability to entrap bioactive compounds. Dendritic presentation of carbohydrates has proven effective to increase remarkably the binding avidity of the resulting glycodendrimers for lectins. The relative

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potency (r.p.) was used to measure the enhancement of lectin binding affinity of a multivalent glycol-scaffold comparing to a monovalent carbohydrate (Fig. 7). Both low valency and high valency glycodendrimers have been prepared by CuAAC.126-131 For example, trivalent presentation of a LeX trisaccharide (Lewis X determinant) by CuAAC on a carbosilane core yielded a r.p. of 92 for lotus lectin compared to the monomer (40).126 An “onion peel strategy” was developed by combining the thiol-ene and the CuAAC click chemistry to display N-acetylactosamine (LacNAc) at each layer of a dendritic growth. A glycodendrimer with 29 triazolyl LacNAc copies showed a remarkable r.p. of 1168 for a LacNAc-specific leguminous lectin.128 By combining glycomimetic and multivalent strategies, a C-galactoside was clicked to a dendritic backbone, showing a 409 r.p. for PA-IL lectin (41).129 Moreover, a strain-promoted azide-alkyne cycloaddition (SPAAC) was used to assembly two independent dendrons, leading to a heterobifunctional dendrimer (42).130

Glycoclusters based on structurally defined, rigid cores such have also been constructed by CuAAC.131-139 Lactose-appended calix[4]arene glycoclusters showed a 625 r.p. for galectin-1 compared to a monovalent counterpart.135 Ganglioside GM1 derivative (43), produced by CuAAC with a calix[5]arene, exhibited a remarkable 100,000 r.p. for cholera toxin compared to

a control monomer.136 Chromogenic core scaffolds, such as

porphyrin137,138 and perylene bisimide139 dyes, have also been employed to display carbohydrates, imparting the resulting glycodendrimers with an inherent optical signal for biological assays. Interestingly, in two systematic studies, glycoclusters with different linker arms (with varied rigidity and hydrophilicity) coupling carbohydrate epitopes to the above

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scaffolds were prepared by CuAAC.137,138 It was concluded that the geometry and topology of the core scaffold and linker rigidity had a large impact on the binding of the glycoclusters to lectins. Overall, these studies highlight the power of click chemistry in the concise construction of diverse and complex carbohydrate structures for elaboration of structure-lectin binding activity relationships. Click chemistry-based glycol-polymerization has been carried out as another strategy to obtain multivalent carbohydrate presentation.140-142 (Readers are directed to a recent review on the construction of glycopolymers143) Besides the conventional chain transfer polymerization followed by CuAAC approach to clustered carbohydrates,143 peptides have been used extensively as the backbone to produce polymeric carbohydrate-containing architectures, since glycopeptides/proteins and proteoglycans are prevalent glyco-structures in nature. CuAAC has been shown to be effective for linking carbohydrates to various functional peptide and protein backbones with a defined secondary structure.144-151 These glycopeptides showed enhanced cellular uptake145 and potential for use in tissue engineering (44).146 Given the importance of peptide conformation towards its biological functions, glycopeptidomimetics with unnatural peptide bonds have been developed by click chemistry (45).148,149 It was found that subtle conformational change of the glycopeptide mimetics impacted largely their association with lectins.148 Towards a similar end, carbohydrates themselves have been used as the core scaffold for multivalent carbohydrate display (Fig. 8). Through CuAAC conjugation of multiple mannoside copies to mannoside cores, triazole-connected high-mannose glycan mimetics have been developed (46).152 Beyond a simple multivalent presentation, X-ray crystallography analysis suggested that

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these compounds are real “structural mimetics” of the high-mannose oligosaccharides. In addition to CuAAC, ruthenium catalysis was found effective towards the synthesis of a thiolactoside based glycocluster.153 Carbohydrates have also been clicked to cyclic and linear polyglycosyl scaffolds. Both cyclic glucosamines (47)154 and cyclodextrins (CDs) (48)155 were determined to be suitable centers to improve lectin binding, with the latter addressing high loading capacity of hydrophobic anti-cancer and anti-HIV drugs. A Lactose-clicked inulin (β-2,1-fructan) glycopolymer was synthesized by CuAAC, and the inulin sidechain-modified click adduct lowered the critical gelation concentration with respect to that of natural inulin.156 In contrast, triazole-linked carbohydrate repeating units prepared by CuAAC were used to space galactoside units; compared to a conventional flexible linkage, the rigidity of the triazolyl carbohydrate spacer was determined to greatly facilitate a lectin binding, with an r.p. of up to 7555 for (49).157,158 This observation allows for the development of multivalent glycoscaffolds with a pre-organized structure using the biocompatible and rigid carbohydrates as the spacer.

Contrary to the multivalent strategy, identification of small molecule glycomimetics from click libraries has also proven suitable for drastically improving lectin binding affinity (Fig. 9). The siglec family comprises a number of sialic acid-binding proteins expressed on cells of the mammalian immune system.159 Sialic acid derivatives that target these lectins might thereby facilitate cell type-specific therapies. Thanks to the modularity and effectiveness of CuAAC click chemistry, series of sialoside libraries have been prepared in a high-throughput manner.

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The resulting crude mixtures were further immobilized onto a solid phase and screened for their ability to bind siglecs. On the basis of this straightforward approach, triazolyl sialosides with excellent selectivity for homologous siglecs have been identified.160-162 Interestingly, click decoration of different substituents on the same position of sialic acid led to distinct specificity of the resulting triazoles for different siglec members, including siglec-7 (50),161 siglec-9 (51),160 siglec-10 (52)160 and human CD22 and CD33 associated with myeloid leukemia and B cell lymphoma.162 Other sialoside mimetics have similarly been prepared by click chemistry. In particular, a sialyl LewisX mimetic (53) showed nanomolar E-selectin antagonistic affinity with a significantly prolonged t1/2 with respect to its’ natural counterpart.163 Galectins are implicated in the progression of cancer, inflammation and heart disease.6 Both lactoside and galacto-coumarin derivatives have been prepared by CuAAC for galectin binding at both the molecular and cellular levels.163-166 However, aglycone modification was noted to also convey cytotoxicity.164 Clicked galactosides have been further developed for cholera toxin inhibition167,168 and for targeting a galactose-selective C-type lectin, the asialoglycoprotein receptor (ASGPr).169 The ASGPr is highly expressed on hepatocytes, modulating the clearance of serum asialoglycoproteins. High affinity agents that target this receptor may facilitate liver-specific drug delivery. A C6-triazole-modified galactomimetic (54)169 showed a 55-fold better binding affinity for the ASGPr than the parent carbohydrate, offering a useful warhead molecule for drug delivering systems.

While the multivalent glycoscaffolds constructed by click chemistry might provide insight

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into the construction of functional macromolecules for biomedical applications, the success in high-throughput screening of bioactive small-molecule carbohydrate derivatives by CuAAC encourages the use of carbohydrates as starting materials to optimize bioactive compounds.160-170 The latter strategy has not been limited to screening lectin antagonists, as highlighted by a recent study on the development of a heparan sulfate click library for binding fibroblast growth factors (55), for instance.171 3. Carbohydrate click chemistry and diagnostics Given their ability to selectively interact with cell surface receptors, carbohydrates have been used as a warhead to construct glycoprobes for the detection of pathogenic targets. In general, these probes are composed of a carbohydrate as recognition/targeting group, a signaling tag as a reporter and, in some cases, a third component as a receptor for specific intracellular species such as ions, small molecules and biomacromolecules. The most extensively researched constructs have been the fluorescence glycoprobes because of the sensitivity and ease of manipulation of fluorescence sensors (here readers are directed to a themed issue guest edited by Chang, Gunnlaugsson and James).172 Click-conjugated glyco-nanoparticles and electrochemically-active glycoprobes have also been developed. Thanks to the modularity of CuAAC click chemistry, these diagnostic systems could be constructed in a facile manner.

3.1. Clicked fluorescence glycoprobes and fluorogenic composite materials for lectin detection and live cell imaging Recently, series of carbohydrate-fluorescent dye conjugates (glycodyes) have been

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synthesized by CuAAC click chemistry (Fig. 10) for the detection of lectins and carbohydrate receptors on the surface of live cells. A rhodamine-GalNAc adduct (56) was prepared for targeting the ASGPr that exists on both hepatocytes and hepatoma cells.173,174 Although the physiological function of ASGPr is to clear excess or aging galactose-terminated proteins in circulating blood, the receptor has also been identified as an invasion site for hepatic viruses; the ASGPr is overexpressed during liver inflammation.175 As a consequence, high-affinity probes that antagonize ASGPr-ligand interactions might be useful tools for disease theranostics (i.e. combined diagnostic and therapeutic).173-175 Using 56 as a fluorescence probe,149 the antagonist effect of a glycopeptidomimetic (45) for GalNAc-ASGPr interactions was determined, providing a unique tool for the analysis of receptor-glycoligand recognition directly at the cellular level.173 On the basis of the affinity of 56 for transmembrane ASGPr, a fluorogenic composite material (FCM)174 was constructed using graphene oxide (GO) as a base substrate for ligand presentation. GO, owing to its broad adsorption band, water solubility and large surface area, has been widely used as a quenching material (by the Förster Resonance Energy Transfer [FRET] mechanism) for optical biosensors.175-180 56 was used to self-assemble with GO, presumably by π-stacking and electrostatic contacts, forming a GalNAc-coated FCM with quenched fluorescence.174 The resulting material was shown to be suitable for the imaging of liver cancer cells overexpressing ASGPr (Fig. 11), whereas change of the carbohydrate warhead to GlcNAc or knock-down of the receptor led to a clearly decreased signal. As such, this study establishes a new glycomaterial platform for probing cancer cells. More interestingly, because of the large surface area of GO, the glycodyes are presented in a

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multivalent manner, suggesting an alternative, non-covalent self-assembly strategy for acquiring carbohydrate multivalency, in contrast to covalent approaches.125,143 Considering that multiple carbohydrate receptors may co-exist on a single cell, a duplexed FCM was constructed for the simultaneous detection of two lectin receptors.181-184 A Gal-DCM (dicyanomethylene, 57) and a Man-coumarin (58) were co-assembled on a single GO surface, quenching the fluorescence of both. It was observed that the presence of a single lectin receptor recovered the fluorescence of the cognate glycodye, whereas the dual emission could be observed simultaneously when both lectin receptors were present. This research offers a versatile tool for the simultaneous detection of multiple disease biomarkers. It was also determined that the size of GO had a large impact on both the lectin sensing183 and cellular imaging184 effect of fluorophore-labeled carbohydrates. Using GO with an optimal size, a supramolecular system where a glycoprobe and an anticancer drug were co-assembled to a single GO surface has been developed for receptor-targeting theranostics.184

A tetrameric Man-FBT (benzothiadiazole, 59) was constructed to assemble with GO for detection of a Man-selective lectin as well as a Gram-negative bacterium (E. coli) that expresses a cell surface mannose receptor (see also earlier section).185 Porphyrin was also shown to be a suitable scaffold to click with Gal, producing a glycodye, (60), to be installed in a field-effect transistor device.186 It was found that, comparing to GO, the use of carbon nanotubes enhanced the sensitivity of the device, probably because of a morphological

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effect. These studies highlighted the versatility of glycodyes for the construction of carbon material-based diagnostic tools, with the dye molecules as the essential material binder to non-covalently present the carbohydrate ligands. In addition to the non-covalent assembling strategies, we note that covalent coupling of carbohydrates with GO have also been prepared by CuAAC.187 Other supramolecular strategies have also been developed, including the self-assembly between a clicked triazolyl pyrenyl CD (61) and 56 was carried out for the fluorogenic detection of lectins.188 Spectroscopic analyses suggested that the structure of the CD is tubular, thus facilitating the stacking of the rhodamine moiety of 56 to the pyrene cluster. The fluorescence of 56 was quenched upon supramolecular assembly, probably due to a static quenching mechanism (that 56 and 61 form a ground-state complex before excitation). Subsequently, the presence of a lectin selective for the sugar ligand recovered the fluorescence, suggesting the competitive dissociation of the complex formed between 56 and 61 (Fig. 12). This research provides an all-carbohydrate-based, supramolecular sensing system for the homogenous detection of lectins.

Aggregation-induced-emission

(AIE),

which

is

contrary

to

the

conventional

aggregation-induced quenching, is a unique photophysical phenomenon where the emission of certain weakly fluorescent dyes intensifies upon aggregation.189-193 A red-emitting diketopyrrolopyrrole (DPP), which is a typical AIE dye, was used to couple with carbohydrates through CuAAC, producing a glycodye with a weak emission (62).194 It was

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observed that supramolecular self-assembly of 62 with a selective lectin produced strong red emission arising from AIE, setting a basis for the development of AIE-based lectin probes (Fig. 13).

Other

small

molecule

glycodyes

based

on

pyrene,195

porphyrin196

and

boron-dipyrromethene (BODIPY)197-199 with the ability to sense biomolecules or to image live cells, and glyco-photochromic compounds,200 have similarly been developed by CuAAC chemistry.

3.2. Clicked fluorescence glycoprobes for ions and small molecules In addition to probing lectin receptors, carbohydrate based small-molecule glycodyes grafted with an additional receptor site for an ionic analyte, have also been developed (Fig. 14). In these glycodye ion probes, the triazole ring serves as a linker and, sometimes a chelator for a metal ion because of its’ ability to coordinate with cations.201-206 Despite the physiological role of the copper (II) ions (Cu2+), their accumulation in the human body may cause disease.207 A bis-triazolyl per-acetyl glucosyl anthraquinone was synthesized by CuAAC (63). The strong coordination of the bis-triazolyl motif with Cu2+ induced the fluorescence quenching of the glycodye, possibly due to an intramolecular charge transfer (ICT) process.208 Meanwhile, because of the electrochemical activity of quinone, a voltammetric method was also used for Cu2+ detection. Some fluorescence “Off-On”-type glycodyes have also been developed, since quenching-based probes are not

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ideal for cellular imaging purposes due to the high background signal. Rhodamine-based small molecule fluorescence probes have been extensively reported in the literature due to their sensitive lactam-ring opening property upon recognition of an analyte, producing both a strong fluorescence and a colorimetric change.209,210 On the basis of this simple rationale, series of rhodamine-based glycodyes were prepared by CuAAC. Mercury ion (Hg2+) is highly neurotoxic to mammals; the glycorhodamine probes (64-66) developed were principally selective for Hg2+.211-213 A per-acetyl galactoside was used as an FRET donor to conjugate with rhodamine as an FRET acceptor because of the overlap of the emission band of the former with the absorbance band of the latter (64). Upon chelation with Hg2+, FRET occurred between the two fluorophores, leading to quenching of the acetyl

galactoside but fluorescence emission of rhodamine.211 On the basis of the above study, a bis-triazolyl galactosyl rhodamine (66) was developed,213 in which the galactose served as a targeting agent for ASGPr of a liver cancer cell. It was determined that, among a series of cancer cells pre-incubated with Hg2+, only a hepatoma cell with ASGPr expression showed selective probe endocytosis, and thus fluorescence emission due to chelation of the probe with intracellular Hg2+. While knockdown of the receptor led to suppression of the fluorescence signal, the change of the galactoside to a non-carbohydrate poly(ethylene glycerol) (PEG) chain led to unselective fluorescence generation in all cells tested.214 This suggests that glycosylation of small molecule probes could substantially enhance their targeting ability for cells with a cognate carbohydrate receptor. Similar observations have been reported using the “click glycosylation” strategy to modify known zinc ion (67)215 and

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H2S (68)216 probes, leading to their enhanced hepatocellular selectivity. These studies demonstrate the generality of the click glycosylation strategy, in combination with the effectiveness of CuAAC, setting a simple basis for the target-specific imaging of intracellular species. Alternatively, the addition of a carbohydrate warhead to a thiophenol (Tp) probe (69) improved its water solubility for the transient determination of Tp in environmental water samples.217 The use of carbohydrate targeting strategy has also facilitated the selective detection of intracellular pH218 and enzymatic activities.219

3.3. Clicked fluorescence glycoligands Considering their precisely oriented hydroxyl groups, carbohydrates have also been employed as a scaffold on which to install diverse fluorophores by CuAAC click chemistry, producing so-called glycoligands (Fig. 15).220 Furanosyl carbohydrate scaffolds were first used to conjugate with pyridine (70)221 and benzothiadiazole (71)222 by a twofold click reaction, producing conformationally-constrained glycoligands for Cu2+. A ligand was proven to passively cross membranes of living cells by fluorescence imaging.223 Informed by these studies, pyranosyl carbohydrate scaffolds modified by CuAAC were also employed to display a diverse range of fluorophores. Bis-triazolyl coumarin glycoligands developed showed a selective fluorescence response to the toxic silver ions (Ag+).224-226 Notably, it was determined that the introduction of the bis-triazolyl to different positions of an identical glucopyranosyl scaffold could lead to the production of completely reverse fluorescence signals upon chelation with Ag+. Whereas the C3,4-disubstituted glycoligand (72) showed a quenched fluorescence upon chelation with Ag+,224 the C2,3-disubstituted counterpart (73)

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exhibited a remarkably enhanced fluorescence in the presence of the ion.226 The latter observation was tentatively ascribed to a chelation-enhanced fluorescence mechanism, suggesting the versatility of using the carbohydrate scaffold for sensor design.

The introduction of two different fluorophore molecules to the same carbohydrate pyranosyl scaffold was found to render diversity in metal ion selectivity.227-229 By modifying the adjacent hydroxyl groups of the side chain of glycosyl scaffolds, fluorophore excimers (excited dimer) could be formed.229 The eximer fluorescence was used to detect heavy metals in a selective manner. Macrocyclic carbohydrate based glycoligands were also developed through intramolecular CuAAC reactions,230,231 producing interesting cyclic glycoligands for guesting metal ions (75).232 Instead of using organic fluorescence dyes as the signal reporter, nanoparticle materials have also been functionalized by clicked carbohydrates for the colorimetric detection of lectins.233-238 Polydiacetylene (PDA) was clicked with mannosides and lactosides, and the resulting glyco-PDAs were converted to liposomes by photopolymerization. The nanoparticles formed showed a subtle colorimetric transition upon binding with selective lectins with an ene-yne polymeric reporter responsible for the chromatic transitions.233 Interestingly, considering their sensitive colorimetric change upon aggregation, gold nanoparticles (AuNPs) were also coated with carbohydrates (Fig. 16).235-238 Seasonal influenza causes a number of human deaths each year, and the pandemic as a result of the influenza virus crossing from animal species is of particular concern. The hemagglutinin on the influenza virus particle surface can recognize a sialic acid-terminated trisaccharide on

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host cells, thereby leading to virus invasion. A trimeric glycomimetic (76) of the trisaccharide coupled with an alkanethiol chain by CuAAC was coated to AuNP, and the resulting glyco-AuNPs showed a rapid colorimetric change with non-purified influenza virus in allantoic fluid as a result of virus particle-induced aggregation. Importantly, the probe was also able to discriminate human and avian strains of influenza virus.237 A recent study also suggested the possibility of using facile strain-promoted click chemistry (77) for the self-assembly of core-shell glyco-AuNPs for specific detection of galactose-binding lectins.238 3.4. Click chemistry for electrochemically active glycoprobes Electrochemistry is a sensitive technique that has been widely applied for the construction of biosensors.238-245 To complement several other surface/interface-based techniques by click chemistry,246-252 electrochemically active carbohydrates have been synthesized and assembled to different electrode materials for detection of lectin and live cells (Fig. 17 and Fig. 18). While previous studies mainly made use of the classic alkanethiol-gold electrode self-assembly for the fabrication of electroactive carbohydrate monolayers,238,253 new approaches using other electrode materials have been developed. GO, because of its good molecular adsorption ability and unique electric properties, has been established as an effective alternative.254-256

An anthraquinone (AQ)-labelled galactoside (78) was synthesized by CuAAC. The AQ redox center served not only as an electrochemical reporter but also a binder to graphene surfaces through π-stacking.257 It was determined that 78 could self-assemble to the surface of the graphene working electrode area of a screen-printed electrode,258,259 producing an electrical

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signal. However, replacement of the graphene electrode by graphite resulted in a much weakened signal, suggesting the importance of graphene for signal amplification. The system constructed was successfully used for the voltammetric detection of lectins as well as liver cancer cells highly expressing ASGPr; RNAi knockdown of the receptor led to suppressed voltammetric response.257

Subsequent investigations using a longer PEG linker connecting the AQ and glycoside, (79), suggested the suitability of clicked glyco-AQs, in combination with graphene, for the impedance detection of lectins.260,261 To reinforce the association between the AQ-labelled carbohydrates with graphene, an additional pyrene group was introduced to the electroactive probe (80). Based on the strong π-stacking of pyrene with graphene, the resulting graphene composite electrode enabled the dynamic tracking of the expression level of pathogenic receptors expressed on different live cells.262 Notably, the above studies only required a portable and miniaturized electrochemical workstation in connection with a personal laptop, making possible a simple new technique for the economic point-of-care disease diagnosis. In addition to AQ as the electroactive species, ferrocene was used to couple with propargyl mannoside by CuAAC. The resulting electroactive mannosyl ferrocene with a thiol anchor was coated onto an AuNP for the multivalent display of mannose (81). The glyco-AuNPs showed much better electrochemical sensitivity than a single mannosyl ferrocene counterpart, suggesting the importance of AuNPs for signal amplification.263 This study suggests that the construction of hybrid material-based electrode architectures might

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further improve the sensitivity of the carbohydrate biosensors developed to date.

4. Summary and perspective In this review, we have highlighted recent progress in the use of the CuAAC click chemistry for the effective construction of a variety of functional carbohydrate structures towards the development of new therapeutic and diagnostic tools. The 1,4-disubstituted 1,2,3-triazole formed by CuAAC acts as both a robust linker between a carbohydrate and a functional molecule and, in some cases, a pharmacophore itself to enhance bioactivity. The “clicked” triazolyl carbohydrates show a diverse range of bioactivities, including anti-cancer, anti-microbial,

anti-viral

and

immunostimulating

effects,

and

the

inhibition

of

disease-related glycosyltransferases and glycosidases. In addition, click chemistry has proven to be efficient approach for the multivalent display of carbohydrates (glycoligands) on a variety of molecular scaffolds for high avidity lectin association. CuAAC has also served as a versatile tool for the modification of carbohydrates with fluorescently- or electrochemically-active labels, producing triazolyl glycoprobes for the detection of lectins, ions (where the triazole serves as a metal ion chelator) and small molecules, as well as for investigating the glycobiology of live cancer cells and pathogens. Interestingly, the clicked glycoprobes could be used to self-assemble with different material substrates, such as graphene and gold nanoparticles, to construct composite sensing materials for serum diagnosis264 and drug delivery.265 In addition to these diverse applications, CuAAC click chemistry is also amenable to live cell imaging, a dynamic area which has been extensively reviewed previously.266-271 Noteworthy within this particular field

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is recent progress in the use of bio-orthogonal CuAAC for the species-specific fluorogenic labelling of the lipopolysaccharide of bacteria.272,273 Despite the promise of carbohydrate click chemistry, several concerns need to be addressed in future investigations. First, the majority of the clicked bioactive carbohydrate derivatives developed to date lack a systematic evaluation of their pharmacology and pharmacokinetics. This is of particular importance for potential drug lead compounds. Comparative tests are needed with both glycosylated and carbohydrate-free molecules to elucidate the role the carbohydrate plays in improving the activity, whether directly as “warhead” or indirectly as an aid to solubility, distribution or cellular uptake. A recent study also shows that the presence of triazole may alter the physicochemical properties of bioactive glycolipids, thereby increasing their antimicrobial activities.81 The latter point also suggests a unique role for triazoles in the bioactivity of carbohydrate click adducts. On a second front, the use of glycomimetics with improved binding affinity and/or increased multi-valency for a lectin receptor could be a good tactic for the design of glycoprobes for use in cell biology or diagnostics. CuAAC click chemistry proves to be well-suited for this type of application due to its’ modularity, opening up opportunities for the construction of compound libraries by parallel synthesis. Since cell surface lectins frequently recognize more than one carbohydrate ligand, the use of glycomimetics with an enhanced specificity for a given target receptor might increase the accuracy of both cellular/tissue imaging and drug delivery. Last but not the least, going forward standardization should be a focus of glycoprobe-incorporated sensing materials, since the size and structure of graphene and gold nanoparticles must be uniform from batch to batch

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to ensure the reproducibility of the glyco-composite materials. In addition, more sophisticated tests with clinical samples (such as whole blood, serum and tissue) should be carried out to examine the potential of these diagnostic tools in real life diagnostics applications. In summary, “click glycosylation” has the potential to become a general strategy for drug development and sensors design, allowing the generation development and deployment of “sweet” clinical tools for in many areas of therapy and diagnosis.

Additional note For a recent broader overview of CuAAC click reactions in carbohydrate chemistry, see reference 274.

Acknowledgements This research is supported by the 973 project (2013CB733700), the National Natural Science Foundation of China (21572058 and 21576088), Key Project of the Shanghai Science and Technology Commission (13NM1400900), Fundamental Research Funds for Central Universities (222201414010), the UK BBSRC Institute Strategic Programme Grant on Understanding and Exploiting Metabolism (MET) [BB/J004561/1] and the John Innes Foundation.

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Figure 1. Triazolyl carbohydrate derivatives prepared by CuAAC with potential anti-cancer activities.

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Figure 2. Triazolyl carbohydrate derivatives prepared by CuAAC with potential antiviral activities.

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Figure 3. Triazolyl carbohydrate derivatives prepared by CuAAC with potential antimicrobial activities.

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Figure 4. Triazolyl carbohydrate derivatives prepared by CuAAC for glycosidases and glycosyltransferases.

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Figure 5. Triazolyl carbohydrate derivatives prepared by CuAAC as α-galactosyl ceramide (α-GalCer) mimetics.

Figure 6. Triazolyl carbohydrate derivatives prepared by CuAAC as vaccines.

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Figure 7. Multivalent triazolyl carbohydrate derivatives prepared by CuAAC based on various scaffolds.

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Figure 8. Multivalent triazolyl carbohydrate derivatives prepared by CuAAC based on carbohydrate scaffolds.

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Figure 9. Triazolyl glycomimetics prepared by CuAAC for lectin binding (the dashed lines of compounds 50-52 mean that they are bound to a surface).

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Figure 10. Triazolyl glycodyes for the probing of lectins and/or live cells.

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Glycoligand

Lectin receptor

Self-assembly (Multivalent display)

Fluorophore

FRET (Fluorescence quenched)

GO

Disassociation (Fluorescence recovered)

Figure 11. Supramolecular self-assembly between glycodyes and graphene oxide (GO) as substrate for the fluorogenic detection of lectin receptors.

Glycoligand 56

Supramolecular assembly

Fluorophore 61

=

Lectin receptor competition

Figure 12. Supramolecular self-assembly between a glycodye and pyrenyl cyclodextrin (CD) for the fluorogenic detection of lectin receptors.

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Supramolecular assembly with lectin receptor

AIE!

62 (Gal-DPP)

=

Weak emission

Figure 13. Supramolecular self-assembly between a glycodye and a lectin receptor for aggregation-induced-emission (AIE).

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Figure 14. Triazolyl glycodyes with an additional ionic receptor site.

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Figure 15. Triazolyl glycoligands prepared by the CuAAC click chemistry.

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Figure 16. Carbohydrate derivatives prepared by click chemistry for the functionalization with glyco-nanoparticles (AuNPs).

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Figure 17. Electrochemically-active carbohydrate derivatives prepared by the CuAAC click chemistry.

Live cell

Graphene

Receptor

Glyco-AQ

Receptor expressed

Live cell Without receptor

Impeded Screen Printed Electrode

Redox active

Screen Printed Electrode

Figure 18. Electrochemical detection of live cells that express a carbohydrate receptor with glyco-anthraquinone (AQ) assembled graphene electrodes.

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