Efficient Conjugation of Oligosaccharides to Polymer Particles through Furan/Maleimide Diels–Alder Reaction: Application to the Capture of Carbohydrate‐Binding Proteins

Glycan–protein interactions play a crucial role in physiological and pathological events. Hence, improving the isolation of carbohydrate‐binding proteins (i.e., lectins and anti‐glycan antibodies) from complex media might not only lead to a better understanding of their function, but also provide solutions for public health issues, such as water contamination or the need for universal blood plasma. Here we report a rapid and efficient method for producing carbohydrate‐based affinity adsorbents combining enzymatic synthesis and metal‐free click chemistry. Both simple and complex glycans (maltose, blood group antigens A, B, and H) were readily modified by the addition of a furyl group at the reducing end without the need for protecting groups and were then efficiently conjugated to maleimide‐activated Sepharose particles through Diels–Alder cycloaddition. These neoglycoconjugates showed high efficiency for the purification of lectins (concanavalin A and Ulex europaeus agglutinin), as well as for the capture of anti‐A and anti‐B blood group antibodies, opening new prospects for glycoproteomics and for the development of universal blood plasma.     

Glycans in Medicinal Chemistry: An Underexploited Resource

The biological relevance of glycans as mediators of key physiological processes, including disease‐related mechanisms, makes them attractive targets for a wide range of medical applications. Despite their important biological roles, especially as molecular recognition elements, carbohydrates have not been fully exploited as therapeutics mainly due to the scarcity of structure–activity correlations and their non‐drug‐like properties. A more detailed understanding of the complex carbohydrate structures and their associated functions should contribute to the development of new glycan‐based pharmaceuticals. Recent significant progress in oligosaccharide synthesis and chemical glycobiology has renewed the interest of the medicinal chemistry community in carbohydrates. This promises to increase our possibilities to harness them in drug discovery efforts for the development of new and more effective, synthetic glycan‐based therapeutics and vaccines.     

Biological Evaluation of Multivalent Lewis X–MGL‐1 Interactions

Myeloid C‐type lectin receptors (CLRs) expressed by antigen‐presenting cells are pattern‐recognition receptors involved in the recognition of pathogens as well as of self‐antigens. The interaction of carbohydrate ligands with a CLR can trigger immune responses. Although several CLR ligands are known, there is limited insight into CLR targeting by carbohydrate ligands. The weak affinity of lectin–carbohydrate interactions often renders multivalent carbohydrate presentation necessary. Here, we have analyzed the impact of multivalent presentation of the trisaccharide Lewis X (Le^X) epitope on its interaction with the CLR macrophage galactose‐type lectin‐1 (MGL‐1). Glycan arrays, including N‐glycan structures with terminal Le^X, were prepared by enzymatic extension of immobilized synthetic core structures with two recombinant glycosyltransferases. Incubation of arrays with an MGL‐1‐hFc fusion protein showed up to tenfold increased binding to multiantennary N‐glycans displaying Le^X structures, compared to monovalent Le^X trisaccharide. Multivalent presentation of Le^X on the model antigen ovalbumin (OVA) led to increased cytokine production in a dendritic cell /T cell coculture system. Furthermore, immunization of mice with Le^X‐OVA conjugates modulated cytokine production and the humoral response, compared to OVA alone. This study provides insights into how multivalent carbohydrate–lectin interactions can be exploited to modulate immune responses.     

Antibody Induction Directed against the Tumor‐Associated MUC4 Glycoprotein

Mucin glycoproteins are important diagnostic and therapeutic targets for cancer treatment. Although several strategies have been developed to explore anti‐tumor vaccines based on MUC1 glycopeptides, only few studies have focused on vaccines directed against the tumor‐associated MUC4 glycoprotein. MUC4 is an important tumor marker overexpressed in lung cancer and uniquely expressed in pancreatic ductual adenocarcinoma. The aberrant glycosylation of MUC4 in tumor cells results in an exposure of its peptide backbone and the formation of tumor‐associated glycopeptide antigens. Due to the low immunogenicity of these endogenous structures, their conjugation with immune stimulating peptide or protein carriers are required. In this study, MUC4 tandem‐repeat glycopeptides were conjugated to the tetanus toxoid and used for vaccination of mice. Immunological evaluations showed that our MUC4‐based vaccines induced very strong antigen‐specific immune responses. In addition, antibody binding epitope analysis on glycopeptide microarrays, were demonstrating a clear glycosylation site dependence of the induced antibodies.     

Determination of Carbohydrate‐Binding Preferences of Human Galectins with Carbohydrate Microarrays

Galectins are a class of carbohydrate‐binding proteins named for their galactose‐binding preference and are involved in a host of processes ranging from homeostasis of organisms to immune responses. As a first step towards correlating the carbohydrate‐binding preferences of the different galectins with their biological functions, we determined carbohydrate recognition fine‐specificities of galectins with the aid of carbohydrate microarrays. A focused set of oligosaccharides considered relevant to galectins was prepared by chemical synthesis. Structure–activity relationships for galectin–sugar interactions were determined, and these helped in the establishment of redundant and specific galectin actions by comparison of binding preferences. Distinct glycosylations on the basic lactosyl motifs proved to be key to galectin binding regulation—and therefore galectin action—as either high‐affinity ligands are produced or binding is blocked. High‐affinity ligands such as the blood group antigens that presumably mediate particular functions were identified.     

Non‐natural Peptide Triazole Antagonists of HIV‐1 Envelope gp120

We investigated the derivation of non‐natural peptide triazole dual receptor site antagonists of HIV‐1 Env gp120 to establish a pathway for developing peptidomimetic antiviral agents. Previously we found that the peptide triazole HNG‐156 [R‐I‐N‐N‐I‐X‐W‐S‐E‐A‐M‐M‐CONH₂, in which X=ferrocenyltriazole‐Pro (FtP)] has nanomolar binding affinity to gp120, inhibits gp120 binding to CD4 and the co‐receptor surrogate mAb 17b, and has potent antiviral activity in cell infection assays. Furthermore, truncated variants of HNG‐156, typified by UM‐24 (Cit‐N‐N‐I‐X‐W‐S‐CONH₂) and containing the critical central stereospecific ^LX‐^LW cluster, retain the functional characteristics of the parent peptide triazole. In the current work, we examined the possibility of replacing natural with unnatural residue components in UM‐24 to the greatest extent possible. The analogue with the critical “hot spot” residue Trp 6 replaced with L ‐3‐benzothienylalanine (Bta) (KR‐41), as well as a completely non‐natural analogue containing D ‐amino acid substitutions outside the central cluster (KR‐42, ^DCit‐^DN‐^DN‐^DI‐X‐Bta‐^DS‐CONH₂), retained the dual receptor site antagonism/antiviral activity signature. The results define differential functional roles of subdomains within the peptide triazole and provide a structural basis for the design of metabolically stable peptidomimetic inhibitors of HIV‐1 Env gp120.     

A Novel Chemoenzymatic Synthesis of Sulfated Type 2 Tumor‐Associated Carbohydrate Antigens by Transglycosylation of Sulfated Lewis X Oxazoline Catalyzed by Keratanase II

Sulfated type 2 carbohydrate chains are known tumor‐associated carbohydrate antigens (TACAs). Many reports on cancer vaccines employing TACAs as specific antigens have been published, but structurally specified sulfated TACAs have not been used because of the low natural abundance and difficulties in chemical synthesis. We demonstrate for the first time the synthesis of the sulfated type 2 TACAs with an l ‐fucose branch by keratanase‐II‐catalyzed transglycosylation of the sulfated Lewis X (Galβ(1→4)[Fucα(1→3)]GlcNAc(6‐OSO₃^?); su‐Le^x) oxazoline derivative. Two keratanase IIs (from Bacillus sp. Ks36 and Bacillus circulans KsT202) efficiently catalyzed the transglycosylation reaction of the su‐Le^x oxazoline derivative, thereby giving the su‐Le^x dimer as the main product in good yields. Structural analysis of the oligomers confirmed exclusive formation of the β(1→3) glycosidic bond.     

Chemical Synthesis of GM2 Glycans,Bioconjugation with Bacteriophage Qβ, and the Induction of Anticancer Antibodies

The development of carbohydrate‐based antitumor vaccines is an attractive approach towards tumor prevention and treatment. Herein, we focused on the ganglioside GM2 tumor‐associated carbohydrate antigen (TACA), which is overexpressed in a wide range of tumor cells. GM2 was synthesized chemically and conjugated with a virus‐like particle derived from bacteriophage Qβ. Although the copper‐catalyzed azide–alkyne cycloaddition reaction efficiently introduced 237 copies of GM2 per Qβ, this construct failed to induce significant amounts of anti‐GM2 antibodies compared to the Qβ control. In contrast, GM2 immobilized on Qβ through a thiourea linker elicited high titers of IgG antibodies that recognized GM2‐positive tumor cells and effectively induced cell lysis through complement‐mediated cytotoxicity. Thus, bacteriophage Qβ is a suitable platform to boost antibody responses towards GM2, a representative member of an important class of TACA: the ganglioside.     

A Biocatalytic Nanomaterial for the Label‐Free Detection of Virus‐Like Particles

The design of nanomaterials that are capable of specific and sensitive biomolecular recognition is an on‐going challenge in the chemical and biochemical sciences. A number of sophisticated artificial systems have been designed to specifically recognize a variety of targets. However, methods based on natural biomolecular detection systems using antibodies are often superior. Besides greater affinity and selectivity, antibodies can be easily coupled to enzymatic systems that act as signal amplifiers, thus permitting impressively low detection limits. The possibility to translate this concept to artificial recognition systems remains limited due to design incompatibilities. Here we describe the synthesis of a synthetic nanomaterial capable of specific biomolecular detection by using an internal biocatalytic colorimetric detection and amplification system. The design of this nanomaterial relies on the ability to accurately grow hybrid protein‐organosilica layers at the surface of silica nanoparticles. The method allows for label‐free detection and quantification of targets at picomolar concentrations.     

Unravelling the Carbohydrate‐Binding Preferences of the Carbohydrate‐Binding Modules of AMP‐Activated Protein Kinase

The β subunit of adenosine monophosphate (AMP)‐activated protein kinase (AMPK), which exists as two isoforms (β1 and β2) in humans, has a carbohydrate‐binding module (CBM) that interacts with glycogen. Although the β1‐ and β2‐CBMs are structurally similar, with strictly conserved ligand‐contact residues, they show different carbohydrate affinities. β2‐CBM shows the strongest affinity for both branched and unbranched oligosaccharides and it has recently been shown that a Thr insertion into β2‐CBM (Thr101) forms a pocket to accommodate branches. This insertion does not explain why β2‐CBM binds all carbohydrates with stronger affinity. Herein, it is shown that residue 134 (Val for β2 and Thr for β1), which does not come into contact with a carbohydrate, appears to account for the affinity difference. Characterisation by NMR spectroscopy, however, suggests that mutant β2‐Thr101Δ/Val134Thr differs from that of β1‐CBM, and mutant β1‐Thr101ins/Thr134Val differs from that of β2‐CBM. Furthermore, these mutants are less stable to chemical denaturation, relative to that of wild‐type β‐CBMs, which confounds the affinity analyses. To support the importance of Thr101 and Val134, the ancestral CBM has been constructed. This CBM retains Thr101 and Val134, which suggests that the extant β1‐CBM has a modest loss of function in carbohydrate binding. Because the ancestor bound carbohydrate with equal affinity to that of β2‐CBM, it is concluded that residue 134 plays an indirect role in carbohydrate binding.     

PNA‐Encoded Synthesis (PES) of a 10 000‐Member Hetero‐Glycoconjugate Library and Microarray Analysis of Diverse Lectins

Identification of selective and synthetically tractable ligands to glycan‐binding proteins is important in glycoscience. Carbohydrate arrays have had a tremendous impact on profiling glycan‐binding proteins and as analytical tools. We report a highly miniaturized synthetic format to access nucleic‐acid‐encoded hetero‐glycoconjugate libraries with an unprecedented diversity in the combinations of glycans, linkers, and capping groups. Novel information about plant and bacterial lectin specificity was obtained by microarray profiling, and we show that a ligand identified on the array can be converted to a high‐affinity soluble ligand by straightforward chemistry.     

Synthetic biology approach for the development of conditionally replicating HIV‐1 vaccine

While the combined antiretroviral therapy has resulted in a significant decrease in HIV‐1 related morbidity and mortality, the HIV‐1 pandemic has not been substantially averted. To curtail the 2.4 million new infections each year, a prophylactic HIV‐1 vaccine is urgently needed. This review first summarizes four major completed clinical efficacy trials of prophylactic HIV‐1 vaccine and their outcomes. Next, it discusses several other approaches that have not yet advanced to clinical efficacy trials, but provided valuable insights into vaccine design. Among them, live‐attenuated vaccines (LAVs) provided excellent protection in a non‐human primate model. However, safety concerns have precluded the current version of LAVs from clinical application. As the major component of this review, two synthetic biology approaches for improving the safety of HIV‐1 LAVs through controlling HIV‐1 replication are discussed. Particular focus is on a novel approach that uses unnatural amino acid‐mediated suppression of amber nonsense codon to generate conditionally replicating HIV‐1 variants. The objective is to attract more attention towards this promising research field and to provoke creative designs and innovative utilization of the two control strategies. © 2016 Society of Chemical Industry     

Lectin‐Based Drug Design: Combined Strategy to Identify Lead Compounds using STD NMR Spectroscopy,Solid‐Phase Assays and Cell Binding for a Plant Toxin Model

The growing awareness of the sugar code—i.e. the biological functionality of glycans—is leading to increased interest in lectins as drug targets. The aim of this study was to establish a strategic combination of screening procedures with increased biorelevance. As a model, we used a potent plant toxin (viscumin) and lactosides synthetically modified at the C6/C6′ positions and the reducing end aglycan. Changes in the saturation transfer difference (STD) in NMR spectroscopy, applied in inhibition assays, yielded evidence for ligand activity and affinity differences. Inhibitory potency was confirmed by the blocking of lectin binding to a glycoprotein‐bearing matrix. In cell‐based assays, iodo/azido‐substituted lactose derivatives were comparatively active. Interestingly, cell‐type dependence was observed, indicating the potential of synthetic carbohydrate derivative to interact with lectins in a cell‐type (glycan profile)‐specific manner. These results are relevent to research into human lectins, glycosciences, and beyond.     

Development of Benzophenone‐Alkyne Bifunctional Sigma Receptor Ligands

Sigma (σ) receptors are unique non‐opioid binding sites that are associated with a broad range of disease states. Sigma‐2 receptors provide a promising target for diagnostic imaging and pharmacological interventions to curb tumor progression. Most recently, the progesterone receptor (PGRMC1, 25 kDa) has been shown to have σ2 receptor‐like binding properties, thus highlighting the need to understand the biological function of an 18 kDa protein that exhibits σ2‐like photoaffinity labeling (denoted here as σ2‐18k) but the amino acid sequence of which is not known. In order to provide new tools for the study of the σ2‐18k protein, we have developed bifunctional σ receptor ligands each bearing a benzophenone photo‐crosslinking moiety and an alkyne group to which an azide‐containing biotin affinity tag can be covalently attached through click chemistry after photo‐crosslinking. Although several compounds showed favorable σ2 binding properties, the highest affinity (2 nM ) and the greatest potency in blocking photolabeling of σ2‐18k by a radioactive photoaffinity ligand was shown by compound 22 . These benzophenone‐alkyne σ receptor ligands might therefore be amenable for studying the σ2‐18k protein through chemical biology approaches. To the best of our knowledge, these compounds represent the first reported benzophenone‐containing clickable σ receptor ligands, which might potentially have broad applications based on the “plugging in” of various tags.     

Structure-Based Reverse Vaccinology Failed in the Case of HIV Because it Disregarded Accepted Immunological Theory

Two types of reverse vaccinology (RV) should be distinguished: genome-based RV for bacterial vaccines and structure-based RV for viral vaccines. Structure-based RV consists in trying to generate a vaccine by first determining the crystallographic structure of a complex between a viral epitope and a neutralizing monoclonal antibody (nMab) and then reconstructing the epitope by reverse molecular engineering outside the context of the native viral protein. It is based on the unwarranted assumption that the epitope designed to fit the nMab will have acquired the immunogenic capacity to elicit a polyclonal antibody response with the same protective capacity as the nMab. After more than a decade of intensive research using this type of RV, this approach has failed to deliver an effective, preventive HIV-1 vaccine. The structure and dynamics of different types of HIV-1 epitopes and of paratopes are described. The rational design of an anti-HIV-1 vaccine is shown to be a misnomer since investigators who claim that they design a vaccine are actually only improving the antigenic binding capacity of one epitope with respect to only one paratope and not the immunogenic capacity of an epitope to elicit neutralizing antibodies. Because of the degeneracy of the immune system and the polyspecificity of antibodies, each epitope studied by the structure-based RV procedure is only one of the many epitopes that the particular nMab is able to recognize and there is no reason to assume that this nMab must have been elicited by this one epitope of known structure. Recent evidence is presented that the trimeric Env spikes of the virus possess such an enormous plasticity and intrinsic structural flexibility that it is it extremely difficult to determine which Env regions are the best candidate vaccine immunogens most likely to elicit protective antibodies.     

Effects of Hapten Density on the Induced Antibody Repertoire

Small peptides and oligosaccharides are important antigens for the development of vaccines and the production of monoclonal antibodies. Because of their small size, peptides and oligosaccharides are non‐immunogenic on their own and typically must be conjugated to a larger carrier protein to elicit an immune response. Selection of a suitable carrier protein, conjugation method, and hapten density are critical for generating an optimal immune response. We used a glycan array to compare the repertoire of antibodies induced after immunizing with either low or high‐density conjugates of the tumor‐associated Tn antigen. At high hapten density, a broader range of antibodies was induced, and reactivity to the clustered Tn antigen was observed. In contrast, antibodies induced by the low‐density conjugate had narrower reactivity and did not bind the clustered Tn antigen.     

Recent Advances in the Development of Indazole‐based Anticancer Agents

Cancer is one of the leading causes of human mortality globally; therefore, intensive efforts have been made to seek new active drugs with improved anticancer efficacy. Indazole‐containing derivatives are endowed with a broad range of biological properties, including anti‐inflammatory, antimicrobial, anti‐HIV, antihypertensive, and anticancer activities. In recent years, the development of anticancer drugs has given rise to a wide range of indazole derivatives, some of which exhibit outstanding activity against various tumor types. The aim of this review is to outline recent developments concerning the anticancer activity of indazole derivatives, as well as to summarize the design strategies and structure–activity relationships of these compounds.     

Synthesis and Structural Characterisation of Selective Non‐Carbohydrate‐Based Inhibitors of Bacterial Sialidases

The major human pathogen Streptococcus pneumoniae plays a key role in several disease states including septicaemia, meningitis and community‐acquired pneumonia. Although vaccines against S. pneumoniae are available as prophylactics, there remains a need to identify and characterise novel chemical entities that can treat the diseases caused by this pathogen. S. pneumoniae expresses three sialidases, enzymes that cleave sialic acid from carbohydrate‐based surface molecules. Two of these enzymes, NanA and NanB, have been implicated in the pathogenesis of S. pneumoniae and are considered to be validated drug targets. Here we report our studies on the synthesis and structural characterisation of novel NanB‐selective inhibitors that are inspired by the β‐amino‐sulfonic acid family of buffers.     

Intramolecular Base‐Free Sonogashira Reaction for the Synthesis of Benzannulated Chiral Macrocycles Embedded in Carbohydrate Templates

A base‐free, intramolecular Sonogashira reaction‐based general approach has been established for the diversity‐oriented synthesis (DOS) of fused bi‐ and tricyclic systems containing benzannulated, 10‐ to 13‐membered chiral macrocycles embedded in carbohydrate templates. Macrocycles with different ring sizes have been prepared by pre‐designing the chiral building blocks. Base‐sensitive groups like acetyl and TBDPS survived the reaction conditions.