Sialic acids in infection and their potential use in detection and protection against pathogens.

In structural terms, the sialic acids are a large family of nine carbon sugars based around an alpha-keto acid core. They are widely spread in nature, where they are often found to be involved in molecular recognition processes, including in development, immunology, health and disease. The prominence of sialic acids in infection is a result of their exposure at the non-reducing terminus of glycans in diverse glycolipids and glycoproteins. Herein, we survey representative aspects of sialic acid structure, recognition and exploitation in relation to infectious diseases, their diagnosis and prevention or treatment. Examples covered span influenza virus and Covid-19, Leishmania and Trypanosoma, algal viruses, Campylobacter, Streptococci and Helicobacter, and commensal Ruminococci.

Targeted photodynamic therapy for breast cancer: the potential of glyconanoparticles.

Photodynamic therapy (PDT) uses a non-toxic light sensitive molecule, a photosensitiser, that releases cytotoxic reactive oxygen species upon activation with light of a specific wavelength. Here, glycan-modified 16 nm gold nanoparticles (glycoAuNPs) were explored for their use in targeted PDT, where the photosensitiser was localised to the target cell through selective glycan-lectin interactions. Polyacrylamide (PAA)-glycans were chosen to assess glycan binding to the cell lines. These PAA-glycans indicated the selective uptake of a galactose-derivative PAA by two breast cancer cell lines, SK-BR-3 and MDA-MD-231. Subsequently, AuNPs were modified with a galactose-derivative ligand and an amine derivate of the photosensitiser chlorin e6 was incorporated to the nanoparticle surface via amide bond formation using EDC/NHS coupling chemistry. The dual modified nanoparticles were investigated for the targeted cell killing of breast cancer cells, demonstrating the versatility of using glycoAuNPs for selective binding to different cancer cells and their potential use for targeted PDT.

ß-1,2-Oligomannan phosphorylase-mediated synthesis of potential oligosaccharide vaccine candidates.

ß-(1,2)-Mannan antigens incorporated into vaccines candidates for immunization studies, showed that antibodies raised against ß-(1,2)-mannotriose antigens can protect against disseminated candidiasis. Until recently, ß-(1,2)- mannans could only be obtained by isolation from microbial cultures, or by lengthy synthetic strategies involving protecting group manipulation. The discovery of two ß-(1,2)-mannoside phosphorylases, Teth514_1788 and Teth514_1789, allowed efficient access to these compounds. In this work, Teth514_1788 was utilised to generate ß-(1,2)-mannan antigens, tri- and tetra-saccharides, decorated with a conjugation tether at the reducing end, suitable to be incorporated on a carrier en-route to novel vaccine candidates, illustrated here by conjugation of the trisaccharide to BSA.

Synthesis of cholera toxin B subunit glycoconjugates using site-specific orthogonal oxime and sortase ligation reactions.

The chemoenzymatic synthesis of a series of dual N- and C-terminal-functionalized cholera toxin B subunit (CTB) glycoconjugates is described. Mucin 1 peptides bearing different levels of Tn antigen glycosylation [MUC1(Tn)] were prepared via solid-phase peptide synthesis. Using sortase-mediated ligation, the MUC1(Tn) epitopes were conjugated to the C-terminus of CTB in a well-defined manner allowing for high-density display of the MUC1(Tn) epitopes. This work explores the challenges of using sortase-mediated ligation in combination with glycopeptides and the practical considerations to obtain high levels of conjugation. Furthermore, we describe methods to combine two orthogonal labeling methodologies, oxime- and sortase-mediated ligation, to expand the biochemical toolkit and produce dual N- and C-terminal-labeled conjugates.

Glycosylated gold nanoparticles in point of care diagnostics: from aggregation to lateral flow.

Current point-of-care lateral flow immunoassays, such as the home pregnancy test, rely on proteins as detection units (e.g. antibodies) to sense for analytes. Glycans play a fundamental role in biological signalling and recognition events such as pathogen adhesion and hence they are promising future alternatives to antibody-based biosensing and diagnostics. Here we introduce the potential of glycans coupled to gold nanoparticles as recognition agents for lateral flow diagnostics. We first introduce the concept of lateral flow, including a case study of lateral flow use in the field compared to other diagnostic tools. We then introduce glycosylated materials, the affinity gains achieved by the cluster glycoside effect and the current use of these in aggregation based assays. Finally, the potential role of glycans in lateral flow are explained, and examples of their successful use given.

End-Functionalized Poly(vinylpyrrolidone) for Ligand Display in Lateral Flow Device Test Lines.

Lateral flow devices are rapid (and often low cost) point-of-care diagnostics-the classic example being the home pregnancy test. A test line (the stationary phase) is typically prepared by the physisorption of an antibody, which binds to analytes/antigens such as viruses, toxins, or hormones. However, there is no intrinsic requirement for the detection unit to be an antibody, and incorporating other ligand classes may bring new functionalities or detection capabilities. To enable other (nonprotein) ligands to be deployed in lateral flow devices, they must be physiosorbed to the stationary phase as a conjugate, which currently would be a high-molecular-weight carrier protein, which requires (challenging) chemoselective modifications and purification. Here, we demonstrate that poly(vinylpyrrolidone), PVP, is a candidate for a polymeric, protein-free test line, owing to its unique balance of water solubility (for printing) and adhesion to the nitrocellulose stationary phase. End-functionalized PVPs were prepared by RAFT polymerization, and the model capture ligands of biotin and galactosamine were installed on PVP and subsequently immobilized on nitrocellulose. This polymeric test line was validated in both flow-through and full lateral flow formats using streptavidin and soybean agglutinin and is the first demonstration of an "all-polymer" approach for installation of capture units. This work illustrates the potential of polymeric scaffolds as anchoring agents for small-molecule capture agents in the next generation of robust and modular lateral flow devices and that macromolecular engineering may provide real benefit.

Lateral Flow Glyco-Assays for the Rapid and Low-Cost Detection of Lectins-Polymeric Linkers and Particle Engineering Are Essential for Selectivity and Performance.

Lateral flow immuno-assays, such as the home pregnancy test, are rapid point-of-care diagnostics that use antibody-coated nanoparticles to bind antigens/analytes (e.g., viruses, toxins or hormones). Ease of use, no need for centralized infrastructure and low-cost, makes these devices appealing for rapid disease identification, especially in low-resource environments. Here glycosylated polymer-coated nanoparticles are demonstrated for the sensitive, label-free detection of lectins in lateral flow and flow-through. The systems introduced here use glycans, not antibodies, to provide recognition: a "lateral flow glyco-assay," providing unique biosensing opportunities. Glycans are installed onto polymer termini and immobilized onto gold nanoparticles, providing colloidal stability but crucially also introducing assay tunability and selectivity. Using soybean agglutinin and Ricinus communis agglutinin I (RCA120 ) as model analytes, the impact of polymer chain length and nanoparticle core size are evaluated, with chain length found to have a significant effect on signal generation-highlighting the need to control the macromolecular architecture to tune response. With optimized systems, lectins are detectable at subnanomolar concentrations, comparable to antibody-based systems. Complete lateral flow devices are also assembled to show how these devices can be deployed in the "real world." This work shows that glycan-binding can be a valuable tool in rapid diagnostics.

Glycan-Based Flow-Through Device for the Detection of SARS-COV-2.

The COVID-19 pandemic, and future pandemics, require diagnostic tools to track disease spread and guide the isolation of (a)symptomatic individuals. Lateral-flow diagnostics (LFDs) are rapid and of lower cost than molecular (genetic) tests, with current LFDs using antibodies as their recognition units. Herein, we develop a prototype flow-through device (related, but distinct to LFDs), utilizing N-acetyl neuraminic acid-functionalized, polymer-coated, gold nanoparticles as the detection/capture unit for SARS-COV-2, by targeting the sialic acid-binding site of the spike protein. The prototype device can give rapid results, with higher viral loads being faster than lower viral loads. The prototype's effectiveness is demonstrated using spike protein, lentiviral models, and a panel of heat-inactivated primary patient nasal swabs. The device was also shown to retain detection capability toward recombinant spike proteins from several variants (mutants) of concern. This study provides the proof of principle that glyco-lateral-flow devices could be developed to be used in the tracking monitoring of infectious agents, to complement, or as alternatives to antibody-based systems.

Recent Developments in the Use of Glyconanoparticles and Related Quantum Dots for the Detection of Lectins, Viruses, Bacteria and Cancer Cells.

Carbohydrate-coated nanoparticles-glyconanoparticles-are finding increased interest as tools in biomedicine. This compilation, mainly covering the past five years, comprises the use of gold, silver and ferrite (magnetic) nanoparticles, silicon-based and cadmium-based quantum dots. Applications in the detection of lectins/protein toxins, viruses and bacteria are covered, as well as advances in detection of cancer cells. The role of the carbohydrate moieties in stabilising nanoparticles and providing selectivity in bioassays is discussed, the issue of cytotoxicity encountered in some systems, especially semiconductor quantum dots, is also considered. Efforts to overcome the latter problem by using other types of nanoparticles, based on gold or silicon, are also presented.

Correction to "The SARS-COV-2 Spike Protein Binds Sialic Acids, and Enables Rapid Detection in a Lateral Flow Point of Care Diagnostic Device".

[This corrects the article DOI: 10.1021/acscentsci.0c00855.].

The SARS-COV-2 Spike Protein Binds Sialic Acids and Enables Rapid Detection in a Lateral Flow Point of Care Diagnostic Device.

There is an urgent need to understand the behavior of the novel coronavirus (SARS-COV-2), which is the causative agent of COVID-19, and to develop point-of-care diagnostics. Here, a glyconanoparticle platform is used to discover that N-acetyl neuraminic acid has affinity toward the SARS-COV-2 spike glycoprotein, demonstrating its glycan-binding function. Optimization of the particle size and coating enabled detection of the spike glycoprotein in lateral flow and showed selectivity over the SARS-COV-1 spike protein. Using a virus-like particle and a pseudotyped lentivirus model, paper-based lateral flow detection was demonstrated in under 30 min, showing the potential of this system as a low-cost detection platform.

Revisiting the Language of Glycoscience: Readers, Writers and Erasers in Carbohydrate Biochemistry.

The roles of carbohydrates in nature are many and varied. However, the lack of template encoding in glycoscience distances carbohydrate structure, and hence function, from gene sequence. This challenging situation is compounded by descriptors of carbohydrate structure and function that have tended to emphasise their complexity. Herein, we suggest that revising the language of glycoscience could make interdisciplinary discourse more accessible to all interested parties.

Membrane-enclosed multienzyme (MEME) synthesis of 2,7-anhydro-sialic acid derivatives.

Naturally occurring 2,7-anhydro-alpha-N-acetylneuraminic acid (2,7-anhydro-Neu5Ac) is a transglycosylation product of bacterial intramolecular trans-sialidases (IT-sialidases). A facile one-pot two-enzyme approach has been established for the synthesis of 2,7-anhydro-sialic acid derivatives including those containing different sialic acid forms such as Neu5Ac and N-glycolylneuraminic acid (Neu5Gc). The approach is based on the use of Ruminoccocus gnavus IT-sialidase for the release of 2,7-anhydro-sialic acid from glycoproteins, and the conversion of free sialic acid by a sialic acid aldolase. This synthetic method, which is based on a membrane-enclosed enzymatic synthesis, can be performed on a preparative scale. Using fetuin as a substrate, high-yield and cost-effective production of 2,7-anhydro-Neu5Ac was obtained to high-purity. This method was also applied to the synthesis of 2,7-anhydro-Neu5Gc. The membrane-enclosed multienzyme (MEME) strategy reported here provides an efficient approach to produce a variety of sialic acid derivatives.

Direct assay for endo-α-mannosidase substrate preference on correctly folded and misfolded model glycoproteins.

We previously reported a unique assay system for UDP-glucose glycoprotein glucosyltransferase (UGGT) toward glycoprotein folding intermediates during the folding process. The assay involved the in vitro folding of both high-mannose type oligosaccharyl crambin, which yielded only the correctly folded glycoprotein form (M9-glycosyl-native-crambin), and its mutant, which yielded misfolded glycoproteins (M9-glycosyl-misfolded-crambin), in the presence of UGGT. The process successfully yielded both mono-glucosylated M9-glycosyl-native-crambin (G1M9-glycosyl-native-crambin) and M9-glycosyl-misfolded-crambin (G1M9-glycosyl-misfolded-crambin). Here, we report the use of our in vitro folding system to evaluate the substrate preference of Golgi endo-α-mannosidase against G1M9-native and -misfolded glycoprotein forms. In our assay Golgi endo-α-mannosidase removed Glc-α-1-3-Man unit from G1M9-native and -misfolded-crambins clearly proving that Golgi endo-α-mannosidase does not have specific preference for correctly folded or misfolded protein structure.

Evaluation of the effect of post-translational modification toward protein structure: Chemical synthesis of glycosyl crambins having either a high mannose-type or a complex-type oligosaccharide.

Glycoproteins are assembled and folded in the endoplasmic reticulum (ER) and transported to the Golgi for further processing of their oligosaccharides. During these processes, two types of oligosaccharides are used: that is, high mannose-type oligosaccharide in the ER and complex-type oligosaccharide in the Golgi. We were interested to know how two different types of oligosaccharides could influence the folding pathway or the final three-dimensional structure of the glycoproteins. For this purpose, we synthesized a new glycosyl crambin having complex-type oligosaccharide and evaluated the folding process, the final protein structure analyzed by NMR, and compared the CD spectra with previously synthesized glycosyl crambin bearing high mannose-type oligosaccharides. From our analysis, we found that the two different oligosaccharides do not influence the folding pathway in vitro and the final structure of the small glycoproteins. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 446-452, 2016.

Folding of synthetic homogeneous glycoproteins in the presence of a glycoprotein folding sensor enzyme.

UDP-glucose:glycoprotein glucosyltransferase (UGGT) plays a key role in recognizing folded and misfolded glycoproteins in the glycoprotein quality control system of the endoplasmic reticulum. UGGT detects misfolded glycoproteins and re-glucosylates them as a tag for misfolded glycoproteins. A flexible model to reproduce in vitro folding of a glycoprotein in the presence of UGGT in a mixture containing correctly folded, folding intermediates, and misfolded glycoproteins is described. The data demonstrates that UGGT can re-glucosylate all intermediates in the in vitro folding experiments, thus indicating that UGGT inspects not only final folded products, but also the glycoprotein folding intermediates.

Efficient synthesis of glycopeptide-α-thioesters with a high-mannose type oligosaccharide by means of tert-Boc-solid phase peptide synthesis.

High-mannose type oligosaccharides consist of nine mannose and two N-acetylglucosamine residues (Man(9)GlcNAc(2):M9) and play an important role in protein folding processes in the endoplasmic reticulum. A highly efficient preparation method of this asparaginyl-M9-oligosaccharide from hen egg yolk was established by a two-step proteolysis with commercially available proteases and subsequent purification using high performance liquid chromatography (HPLC). To avoid the hydrolysis of the desired M9-oligosaccharide during the proteolysis steps, several commercially available proteases were screened for their contamination with mannosidases. The α-amino group of the resultant H(2)N-Asn-(M9-oligosaccharide)-OH was protected with 9-fluorenylmethyloxycarbonyl (Fmoc) group for convenient separation by HPLC. The structure of Fmoc-Asn-(M9-oligosaccharide)-OH thus obtained was confirmed by ESI-MS spectrometry and several NMR experiments. Using this Fmoc-Asn-(M9-oligosaccharide)-OH, the synthesis of the M9-glycopeptide-α-thioester was demonstrated by means of tert-Boc-solid phase peptide synthesis. These tert-Boc conditions afforded the M9-glycopeptide-α-thioester in moderate yield.

Chemical synthesis of intentionally misfolded homogeneous glycoprotein: a unique approach for the study of glycoprotein quality control.

Biosynthesis of glycoproteins in the endoplasmic reticulum employs a quality control system, which discriminates and excludes misfolded malfunctional glycoproteins from a correctly folded one. As chemical tools to study the glycoprotein quality control system, we systematically synthesized misfolded homogeneous glycoproteins bearing a high-mannose type oligosaccharide via oxidative misfolding of a chemically synthesized homogeneous glycopeptide. The endoplasmic reticulum folding sensor enzyme, UDP-glucose:glycoprotein glucosyltransferase (UGGT), recognizes a specific folding intermediate, which exhibits a molten globule-like hydrophobic nature.

Synthesis of alpha- and beta-D-glucopyranosyl triazoles by CuAAC 'click chemistry': reactant tolerance, reaction rate, product structure and glucosidase inhibitory properties.

Cu(I)-catalysed azide alkyne 1,3-dipolar cycloaddition (CuAAC) 'click chemistry' was used to assemble a library of 21 alpha-D- and beta-D-glucopyranosyl triazoles, which were assessed as potential glycosidase inhibitors. In the course of this work, different reactivities of isomeric alpha- and beta-glucopyranosyl azides under CuAAC conditions were noted. This difference was further investigated using competition reactions and rationalised on the basis of X-ray crystallographic data, which revealed significant differences in bond lengths within the azido groups of the alpha- and beta-anomers. Structural studies also revealed a preference for perpendicular orientation of the sugar and triazole rings in both the alpha- and beta-glucosyl triazoles in the solid state. The triazole library was assayed for inhibition of sweet almond beta-glucosidase (GH1) and yeast alpha-glucosidase (GH13), which led to the identification of a set of glucosidase inhibitors effective in the 100 microM range. The preference for inhibition of one enzyme over the other proved to be dependent on the anomeric configuration of the inhibitor, as expected.

'Click chemistry' synthesis of a library of 1,2,3-triazole-substituted galactose derivatives and their evaluation against Trypanosoma cruzi and its cell surface trans-sialidase.

Trypanosoma cruzi trans-sialidase (TcTS) plays a key role in the recognition and invasion of host cells and in enabling the parasite to escape the human immune response. To explore this potential drug target, we have synthesized a small library of substrate analogues based on 1,4-disubstituted 1,2,3-triazole derivatives of galactose modified at either the C-1 or C-6 positions. This was achieved by coupling the appropriate azido-sugars with a panel of 23 structurally diverse terminal alkynes by using the copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction, giving a library of 46 derivatives in good to excellent yield and with complete regioselectivity. The sugar triazoles showed weak inhibition towards TcTS-catalyzed hydrolysis of 2'-(4-methylumbelliferyl)-alpha-d-N-acetylneuraminic acid in vitro (<40% inhibition at 1mM concentration); many of the compounds assessed proved to be acceptor substrates for the enzyme. Despite this modest inhibitory activity, in vitro trypanocidal activity assays against the trypomastigote form of T. cruzi Y strain revealed several compounds active in the low 100s of muM range. Further assessment of these compounds against cultured mouse spleen cells suggests a specific mode of anti-parasite action rather than a generic cytotoxic effect.