
==== Front
Nat Commun
Nat Commun
Nature Communications
2041-1723
Nature Publishing Group UK London

51081
10.1038/s41467-024-51081-7
Article
Synthesis and screening of a library of Lewisx deoxyfluoro-analogues reveals differential recognition by glycan-binding partners
Hollingsworth Kristian 1
http://orcid.org/0000-0002-2740-9098
Di Maio Antonio 2
Richards Sarah-Jane 34
Vendeville Jean-Baptiste 5
http://orcid.org/0000-0002-5075-5554
Wheatley David E. 5
http://orcid.org/0000-0003-0600-4689
Council Claire E. 5
http://orcid.org/0000-0003-2466-6918
Keenan Tessa 6
Ledru Hélène 7
Chidwick Harriet 6
Huang Kun 4
http://orcid.org/0000-0001-5861-9269
Parmeggiani Fabio 4
Marchesi Andrea 4
http://orcid.org/0000-0003-2977-5347
Chai Wengang 2
McBerney Ryan 1
http://orcid.org/0000-0003-4823-3797
Kamiński Tomasz P. 1
Balmforth Matthew R. 1
Tamasanu Alexandra 1
http://orcid.org/0000-0002-8514-784X
Finnigan James D. 8
http://orcid.org/0009-0006-5195-2388
Young Carl 8
http://orcid.org/0000-0002-7589-6011
Warriner Stuart L. 1
http://orcid.org/0000-0003-3574-4686
Webb Michael E. 1
http://orcid.org/0000-0002-0066-4419
Fascione Martin A. 6
http://orcid.org/0000-0003-3974-646X
Flitsch Sabine 4
http://orcid.org/0000-0001-7307-2871
Galan M. Carmen 7
http://orcid.org/0000-0001-6495-0329
Feizi Ten t.feizi@imperial.ac.uk

2
http://orcid.org/0000-0002-8297-1278
Gibson Matthew I. matt.gibson@manchester.ac.uk

349
http://orcid.org/0000-0002-2566-6867
Liu Yan yan.liu2@imperial.ac.uk

2
http://orcid.org/0000-0002-7352-0360
Turnbull W. Bruce W.B.Turnbull@leeds.ac.uk

1
http://orcid.org/0000-0001-8762-0170
Linclau Bruno bruno.linclau@ugent.be

510
1 https://ror.org/024mrxd33 grid.9909.9 0000 0004 1936 8403 School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK
2 https://ror.org/041kmwe10 grid.7445.2 0000 0001 2113 8111 Glycosciences Laboratory, Department of Metabolism, Digestion and Reproduction, Imperial College London, London, UK
3 https://ror.org/01a77tt86 grid.7372.1 0000 0000 8809 1613 Department of Chemistry, University of Warwick, Coventry, UK
4 https://ror.org/027m9bs27 grid.5379.8 0000 0001 2166 2407 Manchester Institute of Biotechnology (MIB), Department of Chemistry, University of Manchester, Manchester, UK
5 https://ror.org/01ryk1543 grid.5491.9 0000 0004 1936 9297 School of Chemistry, University of Southampton, Highfield, Southampton, UK
6 https://ror.org/04m01e293 grid.5685.e 0000 0004 1936 9668 Department of Chemistry, University of York, Heslington, York UK
7 https://ror.org/0524sp257 grid.5337.2 0000 0004 1936 7603 School of Chemistry, Cantock’s Close, University of Bristol, Bristol, UK
8 grid.470604.1 Prozomix Limited, Haltwhistle Industrial Estate, Haltwhistle, Northumberland UK
9 https://ror.org/01a77tt86 grid.7372.1 0000 0000 8809 1613 Division of Biomedical Sciences, Warwick Medical School, University of Warwick, Coventry, UK
10 https://ror.org/00cv9y106 grid.5342.0 0000 0001 2069 7798 Department of Organic and Macromolecular Chemistry, Ghent University, Ghent, Belgium
13 9 2024
13 9 2024
2024
15 792522 5 2024
30 7 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by/4.0/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
Glycan-mediated interactions play a crucial role in biology and medicine, influencing signalling, immune responses, and disease pathogenesis. However, the use of glycans in biosensing and diagnostics is limited by cross-reactivity, as certain glycan motifs can be recognised by multiple biologically distinct protein receptors. To address this specificity challenge, we report the enzymatic synthesis of a 150-member library of site-specifically fluorinated Lewisx analogues (‘glycofluoroforms’) using naturally occurring enzymes and fluorinated monosaccharides. Subsequent incorporation of a subset of these glycans into nanoparticles or a microarray revealed a striking spectrum of distinct binding intensities across different proteins that recognise Lewisx. Notably, we show that for two proteins with unique binding sites for Lewisx, glycofluoroforms exhibited enhanced binding to one protein, whilst reduced binding to the other, with selectivity governed by fluorination patterns. We finally showcase the potential diagnostic utility of this approach in glycofluoroform-mediated bacterial toxin detection by lateral flow.

Distinguishing between different proteins that each bind to the same type of glycan is challenging. Here, the authors demonstrate that an enzymatically synthesised library of Lewisx ‘glycofluoroforms’ that feature site-specific fluorination can discriminate closely related proteins.

Subject terms

Carbohydrate chemistry
Biocatalysis
Carbohydrates
Glycobiology
https://doi.org/10.13039/501100000268 RCUK | Biotechnology and Biological Sciences Research Council (BBSRC) BB/M02878X/1 BB/M028852/1 BB/M028941/1 BB/M02847X/1 BB/M028976/1 BB/M028747/1 BB/M028836 BB/M011151/1 BB/V50953X/1 Linclau Bruno https://doi.org/10.13039/100004440 Wellcome Trust (Wellcome) WT099197/Z/12/Z 108430/Z/15/Z 218304/Z/19/Z 102576/Z/13/Z Linclau Bruno https://doi.org/10.13039/100010663 EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council) ERC-COG: 648239 ProgrES-ERC-2017-ADG Linclau Bruno https://doi.org/10.13039/501100000266 RCUK | Engineering and Physical Sciences Research Council (EPSRC) DTA1799721 Linclau Bruno issue-copyright-statement© Springer Nature Limited 2024
==== Body
pmcIntroduction

Cell surface carbohydrates (glycans) play many important roles in health and disease1, and significant advances have been made in understanding these processes through studies of the recognition of glycan sequences by glycan-binding proteins2. As the same glycan motifs are sometimes recognised by multiple biologically distinct protein partners (Fig. 1a), Nature deploys local glycan concentrations to generate selectivity, not only by varying 3D presentation and density3–6, but also spatial-segregation: e.g., the human bronchial and alveolar epithelia interact with and are colonised by different influenza viruses7. Biosensing or diagnostics using glycan sequences as targets is limited by glycan cross-reactivities, for example using sialic acids or heparan sulfates as attachment sites for COVID, which have lower selectivity than antibody-based systems8,9. This raises the challenge of how one can develop small molecule probes for diagnostics or therapeutics that can distinguish between the interactions of different proteins with the same glycan.Fig. 1 Aim of this work.

a A glycan motif such as Lewisx on surface of different cell types is bound by many different proteins, such as antibodies, lectins, and bacterial toxins. b Illustration of potentially favourable and unfavourable interactions upon deoxyfluorination/deoxygenation. c Structure of Lewisx. d) Depiction of chemo-enzymatic synthesis of Lewisx trisaccharide and a panel of deoxyfluoro and deoxy monosaccharide building blocks 2b–f, 3b–e, 4b–e used to construct a 150-member library of fluorinated Lewisx analogues. Monosaccharide symbols are in accord with the updated Symbol Nomenclature for Glycans (SNFG) convention81. The position of each fluorine substitution is indicated by the bond angle, and anomeric configuration is shown as solid (beta) or dashed (alpha) lines, both following the Oxford Nomenclature System82. GlcNAc N-acetyl glucosamine, Gal galactose, Fuc fucose, Ara arabinose, TFA trifluoroacetyl.

The contributions of individual non-covalent interactions of proteins are routinely studied using site-directed mutagenesis. The nearest equivalent for carbohydrates is the controlled replacement of individual OH-groups with hydrogen or fluorine to reveal which OH-groups are important for binding10,11. Whereas deoxygenation is almost always detrimental to binding, deoxy-fluorination (-OH to -F) can potentially provide a more diverse range of effects (Fig. 1b)12 as fluorine is a hydrogen-bond acceptor surrogate13,14. Furthermore, fluorination enhances CH–π interactions involving adjacent C-H bonds15–17, modifies lipophilicity18–20, and can facilitate attractive multipolar interactions21,22. OH to F replacement has a minimal steric effect and does not significantly alter monosaccharide conformation, as evidenced by emerging glycan 3D structures23,24. Fluorination can also lead to unfavourable interaction effects, such as loss of hydrogen bonding, or mismatched dipoles or partial charges. Hence, glycan fluorination has the potential to be a powerful strategy for introducing selectivity and differentiation between proteins that recognise the same glycan20,25.

Lewisx (Fig. 1c) is a glycan motif that is a ligand for recognition in numerous contexts with many different proteins. It is the trisaccharide capping sequence of developmentally regulated stage-specific embryonic antigen, SSEA-126, and the L5 antigen involved in early neural development27 The Lewisx motif is also expressed on tumour cells28,29 and occurs among human milk oligosaccharides30 where it has been shown to protect against toxins and pathogens involved in childhood diseases31. Lewisx is also expressed on the surface of pathogens such as gastric cancer-causing Helicobacter pylori32, and Schistosoma mansoni33, the causative agent of the life threatening parasitic disease schistosomiasis, affecting over 200 million people worldwide.

Given the biological importance of Lewisx, it would be desirable, for diagnostic and therapeutic purposes, to have chemical probes that could distinguish between each of the diverse proteins with which this glycan can interact. We envisioned that fingerprint profiles of protein binding could be generated using a library of site-specific deoxyfluorinated analogues or ‘glycofluoroforms’. However, the generation of such a library constitutes a formidable synthetic challenge.

Here we report the synthesis of a 150-member library of Lewisx ‘glycofluoroforms’ with site-specific fluorination on each monosaccharide component, by employing a diversity-orientated enzymatic assembly process (Fig. 1d), featuring a wide substrate scope. We investigate a 24-member subset of this library, for unique fluorination patterns that may be discerned by Lewisx binding proteins. These include antibodies and closely related glycan-binding proteins and bacterial toxins. Our design strategy involves adding azido-propyl handles to the glycans so that they can be converted into positively charged imidazolium-based tags (ITags) for reaction screening. These handles also enable facile conversion of a 24-member subset library into lipid-linked glycan probes (neoglycolipids, NGLs), or into glyconanoparticles (Fig. 2) for probing protein binding both in solid-phase high-throughput glycan microarray screening analyses34 and in solution-phase nanoparticle-based interaction studies4.Fig. 2 General enzymatic synthesis of Lewisx glycofluoroforms.

Mass spectrometry-based screening was performed using ITag derivatives before scaling up the synthesis of the azidopropyl glycans for preparation of neoglycolipid (NGL) and polyhydroxyethylacrylamide (PHEA) derivatives for use in binding assays. UDP Uridine diphosphate, ATP Adenosine 5’-triphosphate, GTP Guanosine 5’-triphosphate, DBCO-DH dibenzocycloctyne-functionalised DHPE, DHPE 1,2-Dihexadecyl-sn-glycero-3-phosphoethanolamine.

Results

Enzymatic synthesis

The glycan library was constructed using a panel of chemically synthesised monosaccharide derivatives based on glucosamine 2a, galactose 3a and fucose 4a. N-Acetyl/N-trifluoroacetyl glucosamine derivatives included mono-and di-deoxyfluorination at C-6 (Fig. 1), and galactose and fucose derivatives were monodeoxyfluorinated at positions 3, 4 or 6. d-Fucose 3e was also included as 6-deoxy-d-galactose, and d-arabinose 4e as a de-methylated l-fucose analogue.

The fluorinated/deoxy UDP-Gal analogues were synthesised using a one-pot multienzyme system (Fig. 2)35, with each galactose analogue (3a-e) first converted to the corresponding sugar-1-phosphate using Streptococcus pneumoniae galactokinase (SpGalK)36,37, before transfer of UMP from UDP-Glc by E. coli galactose-1-phosphate uridylyltransferase (EcGalPU) and in-situ regeneration of UDP by Bifidobacterium longum UDP-sugar pyrophosphorylase (BlUSP) (Fig. 2)38. Crude preparations of each UDP derivative were used in subsequent glycosylation reactions. To aid assembly of the LacNAc disaccharide core, an electrospray-mass spectrometry (ESMS) screening protocol was established to investigate the substrate scope for enzymatic galactosylation of GlcNAc library 2a-f with UDP-Gal library UDP-3a-e using Homo sapiens β(1,4)-galactosyltransferase (Hsb4GalT1), which showed high tolerance of fluorination at the GlcNAc 6-position and trifluorination of the N-acetyl group, and could be easily expressed in E. coli as a fusion protein with maltose-binding protein. Conversion of the azidopropyl aglycon to a positively charged imidazolium-based tag (ITag, Fig. 2)39 enabled semi-quantitative MS-analysis by ensuring that both the starting material and product were fully ionised and thus detectable by ESMS with comparable efficiency, allowing an estimation of their relative conversion40,41. Formation of all thirty LacNAc analogues (Fig. 3a) was thus confirmed. All six GlcNAc derivatives were accepted as substrates by the Hsb4GalT1 enzyme, with high conversion when using the natural UDP-Gal donor; all other UDP-Gal analogues also accepted but with significant differences in conversion based on the position of fluorination/deoxygenation. Specifically, UDP-Gal, UDP-4FGal and UDP-6dGal were all substrates for HsB4GalT1 with non-optimised conversions generally over 60%, whilst UDP-3FGal and UDP-6FGal were poorer substrates with most conversions below 40%, which can be partially rationalised by crystal structures of β(1,4)-galactosyltransferase bound to UDP-Gal (Supplementary Fig. 1)42.Fig. 3 Enzymatic synthesis of Lewisx analogues.

The matrix depicts ESMS-derived conversion efficiencies of GlcNAc derivatives bearing ITags to (a) LacNAc analogues, and (b–f) Lewisx analogues containing fucose, 3FFuc, 4FFuc, 6FFuc, and Ara, respectively (See Supplementary Table 1 for numerical data). g A sub-library of 24 Lewisx analogues (R = azidopropyl), synthesised on a multi-milligram scale for binding assays, is also indicated in (b–f) with black boxes. The source data for Fig. 3 are provided in the supporting information file (Supplementary Figs. 18–197).

The library of thirty LacNAc acceptors was then glycosylated with fucoses 4a-e, to give a total of 150 possible Lewisx glycofluoroforms (Fig. 3b–f). A one-pot, two-step glycosylation reaction in which the crude product mixture from galactosylation of 2a–f was directly fucosylated using 1 molar equivalent of an α-(1,3)-fucosyltransferase (HpFucT) from H. pylori43 and in-situ generated GDP-fucose donors44. Fucosylation proved to be much more efficient than galactosylation, with the >80% relative conversion compared to LeX1, for fucosylation in 147 out of 149 reactions. Increased fluorination generally led to lower conversion although LacNAc analogues containing 6,6-diFGlcNTFA showed full comparative conversion in several cases. It was satisfying to note that the promiscuity of HpFucT44 facilitated turnover of our broad range of modified LacNAc acceptors. The crystal structure of HpFucT bound to GDP-Fuc shows that C-6 is surface exposed and therefore modifications at this position would be less likely to affect interactions between enzyme and substrate (Supplementary Fig. 2)45. In contrast, both the O-3/O-4 positions of GDP-Fuc form potential H-bonds to the enzyme so interruption of their ability to act as hydrogen bond donors would be expected to disrupt binding to the enzyme however both 3FFuc-GDP and 4FFuc-GDP were successfully turned over. The two notable exceptions to this observation were addition of 3FFuc and 4FFuc to 6dGal-β(1,4)-GlcNAcOR. Of the 150 Lewisx glycans identified by ESMS, 140 glycofluoroforms are novel compounds including, to the best of our knowledge, the first examples of fluorination to more than one monosaccharide in a Lewisx analogue.

Preparative scale synthesis and glycofluoroform characterisation

In order to obtain high-quality binding data, it was decided to use purified and fully characterised analogues instead of crude enzyme-synthesis reaction mixtures. Hence, a set of 24 compounds (Fig. 3g) was selected for preparative-scale synthesis, which included all single fluoro/deoxy monosaccharides employed in the screen and examples with fluorination in two and three of the monosaccharide residues. Following optimisation, synthetically useful conversion efficiencies were achieved, including examples for which screening had shown relatively poor conversion (LeX15, LeX17, and LeX23 which showed 30–50% conversion in the screen), demonstrating the scalability of our approach. Nuclear magnetic resonance (NMR) spectroscopic analysis of the scaled-up library of 24 trisaccharides confirmed that all of these had the correct Lewisx structure, with all analogues adopting the well-defined ‘closed conformation’ when in aqueous medium. This was deduced from the increased chemical shift of fucose H-5, which is known to result from a non-conventional CH-O hydrogen bond between fucose H-5 and the ring oxygen of the galactosyl residue (Supplementary Fig. 5)46. Another consequence of this ‘closed conformation’ is the proximity of the fucose H-6 and galactose H-2 positions, which is believed to provide hydrophobic stabilisation to the closed conformation of Lewisx 46,47. NMR analysis of the 24 glycans revealed a distinct increase, averaging 0.12 ppm, of the galactose H-2 chemical shift for those Lewisx analogues that contain a 6-fluorofucosyl residue, when compared to analogues with a normal fucosyl residue (Supplementary Fig. 6). It is postulated that this change results from an interaction of Fuc F-6 with Gal H-2, which is taken as further evidence of the preservation of the Lewisx 3D-conformation in aqueous medium upon fluorination.

Glycan microarray construction and screening

The 24 Lewisx trisaccharide analogues (LeX1 to LeX24) (Figs. 3g/4a) were converted into NGL probes to allow their non-covalent immobilisation on nitrocellulose-coated microarray slides. The clustered and flexible presentation of glycans in the NGL-based microarray system is advantageous in revealing binding by a diverse range of glycan binding systems, including endogenous carbohydrate binding receptors, adhesins of microbes, virus particles or their adhesins34,48. We used a new procedure for NGL preparation that takes advantage of the efficiency of the strain-promoted azide-alkyne cycloaddition (SPAAC, Fig. 2)49. The Lewisx analogues were conjugated to a new lipid reagent dibenzocycloctyne-functionalised DHPE (DBCO-DH) to generate the desired DBCO-DH-NGL probes in quantitative yields. A microarray was then constructed with the 24 Lewisx-based DBCO-DH-NGL probes which were prepared, quantified and arrayed in parallel (Fig. 4). The unmodified Lewisx trisaccharide (LeX1) served as the the control for the modified glycan probes including those with modification of the GlcNAc residue only (LeX2–LeX6); modification of Gal only (LeX7–LeX12), mono-fluorination of Fuc (LeX13 and LeX14); di-fluorination on two monosaccharide residues (LeX15–LeX17); multi-fluorinated structures with modification on two or more sugar residues (LeX18–LeX24). Four conventional NGLs derived from natural oligosaccharides were included as reference compounds to show that the the glycan-binding proteins analysed had the predcited binding activities. These were lacto-N-neotetraose (LNnT, position 25), its fucosylated analogue Lewisx pentasaccharide lacto-N-fucopentaose III (LNFP III, 26), pentasaccharide of GM1 ganglioside (27), and high-mannose N-glycan Man9GN2 (28) (Supplementary Table 2). The microarray was probed with nine glycan-binding proteins (Supplementary Table 3): three recombinant Fc-tagged C-type immune lectin receptors, three anti-Lewisx monoclonal antibodies, and three closely related bacterial toxins. The results of microarray analyses are shown in Fig. 4, Supplementary Figs. 7, 8, and in the Source Data file.Fig. 4 Microarray analyses of 24 Lewisx NGL probes.

a Symbolic forms of the 24 Lewisx analogues; b Binding signals with the six glycan-binding proteins presented as histogram charts. Positions 1 to 24 are NGLs corresponding to LeX1 to LeX24 shown in (a). The four control probes are at positions 25 to 28 (Supplementary Table 2). Numerical scores are average fluorescence intensities of the duplicate spots, with the two individual values displayed. The results shown are representative of at least three experiments. The differing effects of fluorination and other modifications at different sites of the Lewisx trisaccharide are summarised using colour shading: red, strong enhancement; blue, abolished binding. c Spider charts showing distinctive binding modes of Lewisx glycofluoroforms observed in microarray analyses. The signal intensities of the unnatural Lewisx probes (LeX2 to LeX24) are normalised against LeX1 and the difference is presented as percentage value (%) in the spider charts. Positive and negative values mean enhanced and diminished binding, respectively, compared to that of LeX1. Zero (red dotted circle line) means the same binding intensity as LeX1. DC-SIGN Dendritic cell-specific intracellular adhesion molecule-3-grabbing nonintegrin, CTB Vibrio cholerae toxin B-Subunit, LTBh Escherichia coli heat-labile toxin. The raw fluorescence intensities of the quantified microarray data are provided as Source data file (excel file).

Monoclonal anti-Lewisx antibodies: With high specificity for Lewisx among the histo-blood group carbohydrate determinants, anti-Lewisx antibodies are widely used as research tools for identifying Lewisx expressed on different organisms, tissues, and cells. The monoclonal antibodies anti-SSEA-126 and anti-L527 together with a commercial antibody BG-7 were analysed in the microarray, and all gave binding to native Lewisx probes LeX1 and the LNFPIII standard. Distinct binding patterns were observed with the three antibodies: anti-SSEA-1 showed the most restricted and anti-L5 the broadest binding profiles (Fig. 4c, Supplementary Fig. 10). Earlier studies have shown that anti-SSEA-1 recognises Lewisx epitope on a longer carbohydrate backbone compared to anti-L550, and this is corroborated by the weaker binding detected to the LeX1 than LNFPIII in the present study. A clear enhancement (almost 3-fold) of the binding of anti-SSEA-1 was observed with the 6F-Gal LeX9 compared to the native LeX1. The LeX9 was also the most potent ligand for BG-7 among all the Lex analogues. Interestingly, anti-L5 showed preferential binding to 6H-Gal (LeX10) compared to 6F-Gal (LeX9). The 3F-Gal, as in LeX7, abolished binding by all antibodies, indicating that the 3-OH group is likely a key hydrogen bond donor in the interactions of these antibodies. Most of the fluorine modifications on GlcNAc resulted in a reduction or abolition of antibody binding, except for C-6 di-fluorination as in LeX3, which was well tolerated by anti-L5 but not at all by anti-SSEA-1 and BG-7 (Fig. 4c, Supplementary Figs. 8–10). The GlcNAc N-TFA modification alone was tolerated by all of the antibodies, whereas little binding was detected when this modification occurred in combination with other fluorination in the Lewisx trisaccharide. Modifications on Fuc had differential effects on antibody binding: LeX11 with 3F-Fuc was strongly bound by anti-L5 but only moderately bound by BG-7 and not at all by anti-SSEA-1. The LeX14 which has arabinose instead of Fuc in the trisaccharide structure gave no binding with anti-SSEA-1 and BG-7 and very weak binding with anti-L5, highlighting the importance of methyl group at the Fuc C-5 position for binding by the two antibodies.

C-type immune lectins (CLRs): Dendritic cell-specific intracellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), its closely related protein DC-SIGNR and the Langerhans cell CLR Langerin were chosen for the microarray study. Whilst DC-SIGN is known to bind both to mannose- and fucose-terminating glycan sequences including Lewisx 51–54, the lack of Lewisx binding by the other two CLRs has been widely reported55–57; and thus DC-SIGNR and Langerin mainly served for comparison with DC-SIGN. As predicted, all the three CLRs gave strong binding signals with the N-glycan standard Man9GN2 (position 28) (Fig. 4b, Supplementary Fig. 10), and only human DC-SIGN (hDC-SIGN) showed significant binding to the unmodified Lewisx probes LeX1 and the LNFPIII standard (position 26). For DC-SIGN, 17 out of 23 unnatural Lewisx probes showed binding, among these there was superior binding to four probes compared to the unmodified LeX1. The positive influence on hDC-SIGN binding was mainly the fluorination on the GlcNAc residue: di-fluorination at C-6 position (LeX3) followed by the N-trifluoroacetyl (N-TFA) modification (LeX4), and to a lesser extent a combination of these two modifications as in LeX6. Fluorination at C-3 of Gal (as in LeX7) also resulted in an enhanced binding by hDC-SIGN, in contrast to the 4F-, 6F- and 6dH-Gal modifications which elicited a negative effect to various degrees. It is striking that 3F- and 4F-modification of the Fuc residue completely abolished hDC-SIGN binding; this is in full agreement with the essential roles of the OH groups at these two positions of the α1-3-linked Fuc in forming coordination bonds with Ca2+ 55,58. With hDC-SIGNR and rhesus CLR Langerin, only very weak binding was detected to selected fluorinated Lewisx probes (Supplementary Fig. 7) suggesting only a subtle gain in affinity effect through fluorination.

Bacterial Toxins: The glycan-binding subunits of three structurally related bacterial toxins were also investigated: Vibrio cholerae toxin B-Subunit (CTB) of the O1 classical biotype (Classical CTB) and El Tor biotype (El Tor CTB), along with the enterotoxigenic Escherichia coli heat-labile toxin (LTBh). Cholera toxin, secreted by V. cholerae, is the major virulence factor in cholera infection, a life-threatening diarrhoeal disease. The cholera toxins and E. coli heat-labile toxin, which share 80% sequence identity, all bind strongly to GM1 ganglioside59,60. However recent studies have identified a secondary carbohydrate binding site that also recognises histo-blood group antigens, such as Lewisx61–65, and is important for cellular uptake and toxicity in epithelial cells in the small intestine66,67. As anticipated, all three toxins gave strong binding to the GM1 ganglioside standard (position 27, Fig. 4b) and the Gal-terminating tetrasaccharide LNnT NGL (position 25)68. Binding to the unmodified Lewisx trisaccharide LeX1 and LNFPIII standard was observed with classical CTB and LTBh, but not with El Tor CTB which was confirmed by isothermal titration calorimetry (Supplementary Fig. 11). With the library of modified Lewisx trisaccharide NGLs, unique binding profiles were observed with each toxin sample. Classical CTB showed dramatically enhanced binding to several fluorinated Lewisx probes that have modifications on Gal and Fuc. The most significant ‘enhancer modifications’ are 3-F and 4-F on Gal and 3-F on Fuc. LeX16 having both 4F-Gal and 6F-Fuc modifications was found to be the strongest ligand with almost four-fold increase of binding intensity compared to that of LeX1. In contrast, the N-TFA modification, 4F-Fuc and replacing Fuc with arabinose resulted in a substantial decrease in CTB binding. The observed changes in binding strength are consistent with structural information for the Classical CTB-Lewisx interaction (see supplementary discussion)65. With LTBh, most of the modifications had little effect or resulted in diminished binding; an exception was 3F-Fuc-modified LeX11 which showed marked signal enhancement. Although the unmodified Lewisx was not bound by the El Tor CTB, weak but detectable binding was observed to a few fluorinated probes including 4F-Gal (LeX8) and 3F-Fuc (LeX11).

Gold nanoparticle-linked assay

A colorimetric gold nanoparticle-linked assay was developed to provide a comparative validation for a subset of the microarray results, and to demonstrate that the discriminatory power of the fluorinated glycans is independent of biosensor architecture. Gold nanoparticles can be deployed in solution-based assays, where a target protein aggregates particles, leading to coupling of their surface plasmon resonance bands, and a macroscopic red-blue colour shift69,70. A subset of 10 azido-propyl Lewisx analogues were conjugated onto poly(hydroxyethyl acrylamide) (PHEA)71 by SPAAC (Fig. 2), which was then loaded onto 55 nm gold nanoparticles25. Glyconanoparticle aggregation on addition of the classical biotype V. cholerae toxin CTB was monitored by UV-Visible spectroscopy (Fig. 5b/c). Figure 5d shows complete dose-dependent binding responses of the Lewisx glycofluoroforms versus CTB. LeX8 and LeX16 show a 23-fold decrease in EC50 compared to the native Lewisx glycan LeX1. Lex11 and LeX24 show a 5-fold improvement in binding strength, while with LeX13 the binding was similar to that with LeX1. The results correlated very well with the microarray assay results (Fig. 5e).Fig. 5 Glyconanoparticle-based sensing of CTB.

a Polymer-tethered glyconanoparticles; b Principle of detection due to gold nanoparticle red-blue shift upon aggregation with CTB; c UV-Visible spectra for gold nanoparticles with native Lewisx (LeX1) and fluorinated LeX22; d Dose-dependent response of library of Lewisx glyconanoparticles to CTB. Data is presented as mean normalised Abs700 from UV-Visible spectroscopy ± standard deviation of 3 replicates. Control experiments are shown in Supplementary Fig. 17a–c. e Correlation of glycan array and glyconanoparticle binding data LeX22, which had a glycan array binding score of 55 ± 53, is omitted from the correlation graph as binding was too weak to be quantified in the nanoparticle assay. Pearson correlation analysis: r(7) = −0.96, p = 0.000041 (two-sided). Error bars on the x-axis correspond to values of duplicate measurements, and error bars on the y-axis correspond to the fitting uncertainty reported for EC50 values from the data shown in Fig. 5d. The gold nanoparticle binding data generated in this study have been deposited in the University of Manchester Figshare database (https://figshare.manchester.ac.uk).

Discussion

Overall, our microarray and nanoparticle binding studies reveal that different proteins with a Lewisx binding site have distinctive recognition modes for this important trisaccharide, which is illustrated by the contrasting binding preferences and tolerances of monoclonal Lewisx antibodies, DC-SIGN, and bacterial toxins (Fig. 4c, Supplementary Fig. 10). These Lewisx-binding proteins have different responses to the site-specific deoxy/fluorine modifications, as summarised for the 11 individual site modifications in Supplementary Table 4. Importantly, deoxygenations/deoxyfluorinations are able to either enhance or reduce affinities, as exemplified in classical CTB and hDC-SIGN binding studies. The minimal binding of fluorinated Lewisx analogues to DC-SIGNR, which is closely related to DC-SIGN but does not bind to Lewisx, indicates that enhancements in binding strength did not arise from non-specific interactions. The high levels of selectivity that can be achieved by introducing fluorine at multiple sites on the oligosaccharide is illustrated by LeX11 (fluorination at the fucose 3-position), which showed enhanced binding to the classical CTB bacterial toxin but was not tolerated by hDC-SIGN, and vice versa by LeX6 (fluorination at two GlcNAc positions) which showed enhanced binding to hDC-SIGN, but a much reduced binding to classical CTB. The difluorinated LeX16 (4F-Gal and 6F-Fuc modification) which was identified as the most potent and unique ligand for the classical CTB, but was only very weakly bound by the E. coli LTB. Notably LeX16 showed significantly reduced binding by hDC-SIGN and the anti-Lewisx antibodies compared to native LeX1. These fluorination patterns represent a promising framework for design of specific diagnostic or therapeutic agents, for example against pathogenic bacteria that do not interfere with endogenous immune lectins.

As a proof of concept towards the application of glycofluoroforms for diagnostic purposes, a lateral flow experiment using a low-cost glyco-assay was developed (Supplementary Figs. 271, 272). We demonstrated the discrimination between AuNP functionalised with a low-binding glycan (LeX4) vs those coated with a high-binding glycan (LeX16) for the detection of CTB. This unequivocally shows that the microarray and aggregation results can be translated into diagnostic tools.

Here we have demonstrated a practical and expedient diversity-oriented enzymatic strategy devised to generate a structurally diverse library of mono- and polyfluorinated Lewisx derivatives, termed ‘glycofluoroforms’. We have prepared a 24 member library of isolated and fully characterised glycans out of a total of 150 glycans that were generated and identified by mass spectrometry using a clickable ITag for reaction monitoring. Only wild-type forms of the enzymes were employed, and they showed high tolerance for nucleotide-activated monofluorinated sugar donors and their glycosylation of fluorinated acceptors, with all possible 150 Lewisx glycans generated from six GlcNAc acceptors, five Gal, and five Fuc donors. A third of the Lewisx derivatives were formed with conversions of at least 89%, half had conversions over 70% and almost two thirds had conversions over 40%. A total of 24 glycans, chosen to achieve structural diversity, were successfully upscaled and characterised, even for those cases that showed low (30–50%) conversion in the screening experiments.

Distinctive glycofluoroform fingerprint binding profiles for nine glycan-binding proteins were obtained by screening of a NGL-based microarray using the 24 analogues prepared in multi-milligram scale. A gold nanoparticle-based assay was successfully used to validate the relative binding affinities of a subset (10 glycans) of the microarray results. A number of examples of increased binding strength of Lewisx glycofluoroforms compared to the native glycan were also identified, notably between monoclonal Lewisx antibodies (3 examples) and bacterial toxins (3 examples) investigated, with different preferred fluorination patterns for different proteins. Thus, using wild-type enzymes, this study has established that the facile construction of extensive libraries of a given glycan structure with defined fluorination patterns across multiple monosaccharides, using wild-type enzymes, is a powerful approach to investigating protein-glycan binding specificities. This validated ability of glycan fluorination patterning to deliver significant changes in protein binding whilst maintaining glycan conformation, heralds great promise for the development of diagnostic kits or new anti-adhesion therapies to combat microbial infections. We anticipate that our preliminary data demonstrating application of the glycofluoroform approach for lateral flow detection of a bacterial toxin will accelerate the development of glycan-based diagnostics, and therapeutics.

Methods

Synthesis of the Lewisx trisaccharides

Materials and methods: 3-Deoxy-3-fluorogalactose, 4-deoxy-4-fluorogalactose, 6-deoxy-6-fluorogalactose, 6-deoxygalactose, UDP-galactose, fucose, arabinose, ATP, GTP and UTP were received as in-kind support from Biosynth Carbosynth. GlcNAc acceptors 2a-2f25, 3FFuc 4b72, 4FFuc 4c58, 6FFuc 4d73 were prepared by literature methods. 1H NMR spectra were recorded at 500 MHz on a Bruker AV4 NEO 11.75 T (500 MHz 1H) NMR spectrometer. 13C NMR spectra were recorded at 125 MHz on a Bruker AV4 NEO 11.75 T (500 MHz 1H) NMR spectrometer. 19F NMR spectra were recorded at 376.5 MHz on a Bruker AV3HD 9.4 T (400 MHz 1H) NMR spectrometer. Chemical shifts are given in parts per million downfield from tetramethylsilane. The following abbreviations are used in 1H NMR analysis: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = double doublet, dt = double triplet, td = triple doublet, ddd = double double doublet. Accurate Mass spectra were acquired on a Bruker Impact II QqTOF spectrometer equipped with a VIPHESI source using electrospray ionisation. Samples were introduced using an HTC PAL autosampler and Bruker Elute Pump. A 50:50 MeOH:water mix (0.1% formic acid) was used as the eluant. Samples passed through a Bruker Diode array uv-detector before entering the mass spectrometer. Calibration was performed by infusion of 5 mM sodium formate solution at the end of each acquisition. Silica chromatography was performed using a Biotage Isolera 4 with Ecopack basic D17 silica columns. Fractions were analysed by TLC using an DCE:MeOH:AcOH:H2O (50:30:25:10) resolving solvent and visualised with 0.2% orcinol and 5% sulfuric acid in MeOH with charring. Size exclusion was performed using an AKTA Prime with a 10 × 300 mm Tricorn column packed with Bio-rad Biogel P2. This method was run at 0.1 mL/min in water collecting 0.5 mL fractions with detection TLC. A 26 × 1000 mm Tricorn column packed with Bio-rad biogel P2 was used for more difficult separations and was run at 0.5 mL / min collecting 2 mL fractions. HPLC was performed on an Agilent 1260 Mass Directed Preparative HPLC using a Kinetex 5 um EVO C18 100 Å LC column 250 ×21.2 mm.

Enzymes

BlUSP: Bifidobacterium longum UDP-sugar pyrophosphorylase (Uniprot code—C2GXC3)

SpGalK: Streptoccocus pneumoniae galactokinase (Uniprot code—B1I864)

EcGalPU: Escherichia coli galactose-1-phosphate uridylyltransferase (Uniprot code—P07902)

BfFKP: Bacteroides fragilis l-fucokinase/l-fucose-1-P guanylyltransferase (Uniprot code—Q58T34)

HpFucT: Helicobacter pylori α(1,3/4)-fucosyltransferase (Uniprot code—O30511)

Hsb4GalT1. Homo sapiens β(1,4)-galactosyltransferase (Uniprot code—P15291).

HpFucT43, BfFKP74, spGalK36, EcGalPU35 and BlUSP38 were obtained as in-kind support from Prozomix. They were expressed in E. coli BL21 as His-tagged proteins, and stored as ammonium sulfate precipitates following purification by nickel affinity chromatography.

Hsb4GalT1 did not express in a soluble form when expression was attempted using the same approach employed for the other enzymes. Instead it was expressed as follows: a synthetic gene coding for residues 61-398 of human B4GalT1, codon-optimised for expression in E. coli, and with an N-terminal TEV protease recognition sequence (ENLYFQ/G), was cloned between the BamHI and HindIII sites of a modified version of pDB.His.MBP75, in which the nucleotides encoding the original TEV sequence and the multiple cloning site prior to the BamHI site had been deleted. Shuffle T7 Express E. coli pRARE2 (New England Biolabs) were transformed with the resulting plasmid. An overnight culture of the strain in LB media was diluted 400-fold into autoinduction media (Formedium) supplemented with kanamycin (50 mg/L), the culture was grown at 37 °C for 2 h, before transfer to 18 °C for 44 h. Cells were harvested at 10000 g for 10 min, resuspended in lysis buffer (100 mM Tris-HCl, pH 7.5, 100 mM NaCl, 40 mM imidazole, 2 units/mL DNase, 1 Complete EDTA-free protease inhibitor mixture tablet) and lysed using a Constant Systems cell disrupter (20 kpsi). Cell lysates were cleared by centrifugation at 30,000 g for 45 min before purification by nickel affinity chromatography, washing with 50 mM imidazole before elution with imidazole (500 mM). Protein was dialysed into 100 mM Tris-HCl, 100 mM NaCl and stored at 4 °C either as a solution (ca. 150 μM) or an ammonium sulfate precipitate (3.2 M NH4SO4).

Enzymatic synthesis: general procedures

General procedure for the synthesis of the I-Tagged GlcNAc acceptors

ITagged GlcNAc acceptors were synthesised in the following way: Azido GlcNAc (10 mM), Alkyne ITag (10 mM), sodium ascorbate (15 mM) and copper (II) sulfate (5 mM) in H2O were incubated at 37 °C for 10 min before LCMS showed the reaction to be complete. The ITagged acceptors were used crude in the HRMS screening assay.

HRMS screening assay general procedure

GlcNAc acceptor 2a-2f (R = ITag) (0.2 mM), UDP-donor derived from 3a-3e (1 mM), MnCl2 (10 mM), Tris (pH 8.0, 50 mM), BSA (1 mg/mL), HsB4GalT1 (30 μM) in a total volume of 60 μl was incubated at 37 °C for 16 h. The samples for LacNAc conversion were then frozen until analysis by Electrospray Mass Spectrometry (ESMS). Crude LacNAc derivatives (Fig. 2b; R = ITag) (0.1 mM), Tris (pH 8.0, 100 mM), MgCl2 (10 mM), ATP (4 mM), GTP (2 mM), monosaccharide 4a-4e (2 mM), HpFucT (0.59 mg/mL), BfFKP (0.78 mg/mL) in a total volume of 10 μl was incubated for 16 h at 37 °C. The samples were then frozen. The samples were analysed by HRMS through direct injection of the crude reaction mixture. The percentage conversion for each reaction featuring ITag-label substrates was calculated automatically by comparison of the integration of the ITag-labelled starting material and product ESMS peaks using Bruker DataAnalysis 4.3. A replicate of n = 1 was used for this study.

In situ one-pot synthesis of sugar nucleotides

Fluorinated/deoxy UDP-Gal analogues were synthesised using a one-pot multienzyme system. Briefly, the Gal analogue (8 mM), ATP (10 mM), UTP (10 mM), MgCl2 (10 mM), Tris buffer (50 mM, pH 8.0), SpGalK (300 μg/mL), BlUSP (75 μg/mL), EcGalPU (32.5 μg/mL) and pyrophosphatase (50 U/mL) was incubated at 37 °C overnight as described previously (Fig. 2)35. GDP-fucose donors were generated in situ from monosaccharides 4a-4e by a bifunctional l-fucokinase/l-fucose-1-P guanylyltransferase from Bacteroides fragilis (BfFKP)44.

One pot, preparative scale fluorinated Lewisx glycan synthesis

Reactions were performed following the one-pot, two-step enzymatic synthesis protocol, on azidopropyl GlcNAc analogues 2a-2f. In general, the concentration of the acceptors was increased from the 0.1–0.2 mM that was used in the screening experiments to 10–20 mM in the scale up (variations in enzyme and substrate concentrations depending on the efficiency of reaction are detailed in the SI). The molar equivalents of enzyme were also reduced by a factor of 10 compared to the galactosylation screening. GlcNAc derivative 2a-2f (R = azidopropyl) (10 mM), UDP-Gal derived from 3a-3e (11 mM), MnCl2 (10 mM), BSA (1 mg/mL), HsB4GalT1 (30 μM) in Tris (100 mM, pH 8.0) in a total volume of 3.25 mL were incubated overnight at 37 °C. The fucosylation reaction components were added to achieve the following final concentrations: Tris, pH 7.0 (200 mM), MgCl2 (10 mM), ATP (16 mM), GTP (8 mM), Fuc derivative 4a-4e (8 mM), HpFucT (8 μM) and BfFKP (5 μM) in a total volume of 8.12 mL, and the mixture was incubated overnight at 37 °C. The reaction mixture was passed through a 10 kDa molecular weight cut off spin concentrator to remove proteins and the filtrate was concentrated to dryness. The product was purified by silica gel chromatography (20:80 → 50:50 MeOH:EtOAc), followed by size exclusion chromatography using a Biogel P2 column equilibrated with ammonium formate (20 mM) in water.

Glycan microarrays

Materials

1,2-Dihexadecyl-sn-glycero-3-phosphoethanolamine (DHPE) was purchased from Fluka (Dorset, UK). Dibenzocyclooctyne-N-hydroxysuccinimidyl ester (DBCO-NHS) was purchased from Conju-Probe, LLC (California, USA). N,N-Diisopropylethylamine (DIPEA), high performance thin layer chromatography (HPTLC) plates (60 F254) were from MERCK. Solvents, with HPLC grade purity, were purchased from Fisher scientific (Massachusetts, USA). H2O was purified with a Milli‐Q purification system from Millipore (18.3 MΩ‐cm). Sep-Pak silica cartridges (500 mg) were from Waters (Milford, US).

Synthesis of the new lipid reagent

Dibenzocyclooctyne-N-1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine (DBCO-DH): The dibenzocyclooctyne (DBCO) functionality was incorporated into the amino-phospholipid DHPE via amide coupling of DBCO-NHS and DHPE (Supplementary Fig. 1). DBCO-NHS (8.0 mg, 20 μmol) with DIPEA (7 μL, 40 μmol) were dissolved in CHCl3 (1 mL) and added dropwise to a solution of DHPE (6.6 mg, 10 μmol) in CHCl3:MeOH 2:1 (3 mL). The reaction mixture was stirred at room temperature for 21 h. HPTLC analysis of the mixture in solvent CHCl3:MeOH:NH3 100:30:1 visualised under longwave UV light after primulin staining76, showed the complete conversion of DHPE to DBCO-DH (Rf 0.63). The product was purified by preparative-HPTLC (CHCl3:MeOH:H2O 100:30:1) with a yield of 91% (8.6 mg). MALDI-MS analysis of the purified DBCO-DH gave a [M-H]- ion at m/z 949.8 as predicted (C56H91N2O8P, calculated 950.6513 [M]). HR-ESI-MS, m/z: 949.6464 ([M-H]-, calc. 949.6435).

General procedure for the preparation of DBCO-DH NGLs

A total of 24 azidopropyl Lewisx trisaccharide analogues were converted into DBCO-DH NGLs via micro-scale SPAAC reaction (Supplementary Fig. 2). with DBCO-DH in quantitative yields using the general procedure described below. To a solution of DBCO-DH (400 nmol) in CHCl3:MeOH 1:1 (200 μL), a solution of 3-azido propyl glycan (100 nmol) in water (30 μL) was added. The reaction mixture was shaken at room temperature for 4 h. HPTLC analysis of the mixture (1 nmol of glycan starting material) in solvent CHCl3:MeOH:H2O 130:50:9 visualised under longwave UV light after primulin staining76 showed the formation Glycan-DBCO-DH NGL, as a mixture of regioisomers (Supplementary Fig. 12) with an efficiency greater than 90%. The products were purified by semi-preparative HPTLC (CHCl3:MeOH:H2O 60:25:4) or Silica cartridge using established conditions for conventional NGLs76. The purified NGLs were analysed by HPTLC (Supplementary Fig. 12) and MALDI-MS. The molecular masses detected were in accord with theoretical values (Supplementary Table 5). The 24 DBCO-DH NGLs were quantified using established protocol as described for conventional DH-NGLs77. The quantified NGL stock solutions were stored at −20 °C in CHCl3:MeOH:H2O 25:25:8.

For analysis of the NGL probes, matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) was carried out on Shimadzu AXIMA Assurance Resonance instrument with a QIT-TOF configuration (Shimadzu, Milton Keynes, England). The NGLs were dissolved in CHCl3:MeOH:H2O 25:25:8 at a concentration of ~10 pmol/μL, and 0.5 μL was deposited on the sample target together with a matrix solution of 2′,4′,6′-trihydroxyacetophenone in MeOH (1 μg/μL) for analysis. A nitrogen laser was used to irradiate the samples at 337 nm with a laser energy at 100% (coarse) and 50% (fine); resolution was at 1000. For high resolution MS, electrospray (ESI-MS) was used at a resolution of 10,000 on a Waters Synapt G2-S instrument (Waters, Wilmslow, England). Samples (1 μL) were introduced by flow injection and cone voltage was set at 80 V.

Glycan microarray screening analyses

Microarray analyses were carried out using the NGL-based microarray system78. Details of the glycan probe library, the generation of the microarrays, the glycan binding protein samples and detection systems and assay protocol used in the microarray binding experiments, imaging, and data analysis are in Supplementary Table 3 in accordance with the Minimum Information Required for A Glycomics Experiment (MIRAGE) guidelines for reporting glycan microarray-based data77. These analyses serve to measure the relative strengths of interaction with the proteins (relative avidities) rather than binding affinities. Microarray data at the protein concentration with the optimal signals for the Lewisx glycan library are presented to demonstrate that differing binding patterns are obtained for different Lewisx binding proteins.

Gold nanoparticle platform

Materials

N-Hydroxyethyl acrylamide (97%), 4,4′-azobis(4-cyanovaleric acid) (98%), mesitylene (reagent grade), triethylamine ( > 99%), sodium citrate tribasic dihydrate ( > 99%), gold(III) chloride trihydrate (99.9%), dibenzocyclooctyne-amine and cholera toxin B subunit were all purchased from Sigma-Aldrich and used without further purification. 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid pentafluorophenyl ester was synthesised as previously outlined by Richards et al.25. HEPES buffer with 0.15 M NaCl, 0.1 mM CaCl2 and 0.01 mM MnCl2 (pH 7.4) was used for the aggregation studies.

Polymerisation of hydroxyethyl acrylamide using PFP-DMP

Polymerisation of hydroxyethyl acrylamide using PFP-DMP as a chain transfer agent was carried out as previously outlined25,71. Monomer N-hydroxyethyl acrylamide (HEA) (0.5 g, 4.34 mmol), chain transfer agent pentafluorophenyl 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid (PFP-DMP) (0.092 g, 0.17 mmol), and intiator 4,4′-azobis(4-cyanovaleric acid) (ACVA) (0.0097 g, 0.034 mmol were dissolved in 1:1 mixture of toluene and methanol (4 mL). Mesitylene (150 μL) was added as an internal reference for NMR. A 25 μL aliquot was taken for 1H NMR analysis of the conversion in CDCl3. The solution was degassed under a stream N2 for 30 mins. The reaction was stirred at 70 °C for 90 mins. Another 25 μL aliquot was taken for the NMR analysis of conversion in MeOD. The reaction was quenched in liquid nitrogen and precipitated into diethyl ether (50 mL). The polymer was reprecipitated into diethyl ether from methanol twice more to yield a yellow polymer product that was dried under vacuum. 96% conversion by NMR, Mn (Theoretical) = 3400 g.mol−1 Mn (SEC) = 5800 g.mol−1 Mn/Mw (SEC) = 1.16. (SEC = Size Exclusion Chromatography).

Gold nanoparticle synthesis

55 nm gold nanoparticles were synthesised by a modified step growth method developed by Bastús et al.79 as outlined previously25. A seed solution was made as follows: 150 mL of 2.2 mM sodium citrate in MilliQ water was heated under reflux for 15 min with vigorous stirring. Once the solution as boiling, 1 mL of HAuCl4 (25 mM) was added. The colour of the solution changed from yellow to blue-ish grey initially and then to pale pink within 10 min, 1 mL was taken for DLS (Dynamic Light Scattering) and UV-Vis analysis. This Au seed solution was cooled to 90 °C. Once cooled, 1 mL HAuCl4 (25 mM) was added. After stirring for 20 min, two further additions of 1 mL HAuCl4 (25 mM) were added with 20 min between each addition. 1 mL was taken for DLS and UV/Vis analysis. Following this, the sample was diluted by adding 85 mL of MilliQ water and 3.1 mL of 60 mM sodium citrate was added. This solution was then used as a seed solution, and three further portions of 1.6 mL of 25 mM HAuCl4 were added with 20 min between each addition. Following completion of this step 1 mL was taken for DLS and UV/Vis analysis. The sample was diluted by adding 135 mL of MilliQ water and 4.9 mL of 60 mM sodium citrate was added. This solution was then used as a seed solution, and the process was repeated with three further additions of 2.5 mL of 25 mM HAuCl4 with 20 min waiting period between addition. Following completion of this step 1 mL was taken for DLS and UV/Vis analysis. The sample was diluted by adding 215 mL of MilliQ water and 7.8 mL of 60 mM sodium citrate was added. This solution was then used as a seed solution, and the process was repeated with three further additions of 3.9 mL of 25 mM HAuCl4 with 20 min between additions, this solution was analysed by DLS and UV/Vis and target size of 55 nm was reached, and the solution was cooled. AuNPs had an OD (optical density) of 4.16 @ SPRmax = 533 nm. This solution was stored in the dark and used without further purification.

Functionalisation of PHEA with DBCO

PFP-PHEA (500 mg, 0.15 mmol), dibenzocylclooctyne-amine (DBCO) (81 mg, 0.29 mmol) were dissolved in 2 mL dimethyl formamide (DMF). The reaction was stirred at room temperature for 16 h. The polymer was precipitated into diethyl ether from methanol three times and dried under vacuum. The resulting polymer was an off white solid. IR indicated loss of C = O stretch corresponding to the PFP ester. 19F NMR also indicated the removal of the PFP ester.

Capture of Lex derivatives onto DBCO-PHEA

In a typical reaction, DBCO-PHEA (1 mg, 0.32 μmol) and azidopropyl-linked glycan (2 equiv.) was dissolved in 1 mL MilliQ water and left to react overnight on a tube roller. The solution was used immediately for immobilisation onto AuNPs.

Gold nanoparticle functionalisation using Lex-functionalised PHEAs

100 μL of 1 mg.mL−1 of polymer solution was added to 1 mL of ~OD533 4 particles and left for 30 min at room temperature on a tube roller. After 30 min, particles were centrifuged at 6000 rpm (3381 g), the resulting supernatant was removed, and the particles were resuspended in 1 mL MilliQ H2O. This was repeated a further two times to ensure complete removal of any unattached polymer. Stability was confirmed by incubating in 10 mM HEPES buffer for 30 min and measuring UV-Vis and comparing to AuNPs in MilliQ H2O. Particles were diluted to ODmax 1 for testing against CTB.

Nanoparticle characterisation

Particles were characterised by UV-Vis (Supplementary Fig. 13) and DLS (Supplementary Fig. 14), the analyses of these are summarised in Supplementary Table 6.

Absence of aggregation was confirmed with TEM analysis (Supplementary Figs. 15)

AuNPs (OD540 = 1) were drop cast on Holey Carbon Film 300 mesh copper grids (Agar Scientific). TEM images were obtained with a Thermo Fisher Scientific—Talos F200X microscope equipped with an X-FEG electron source. The experiment was performed using an acceleration voltage of 200 kV and a beam current of approximately 5 nA. Images were recorded with a Thermo Scientific—CETA 4k x 4k CMOS camera. Analysis of particle diameter was conducted in Image J2 Version 2.14.0/1.54 f.

Cholera toxin-induced aggregation studies by Absorbance

A stock solution of CTB was made up 0.2 mg.mL−1 in 10 mM HEPES buffer with 0.15 M NaCl, 0.1 mM CaCl2 and 0.01 mM MnCl2. 25 μL 2-fold serial dilution was made up in the same buffer in a clear, flat bottom, half-area 96-well microtitre plate (7 dilutions and a buffer control). 25 μL of the glycoAuNP were added to each well and incubated at room temperature for 30 min. After 30 min, an absorbance spectrum was recorded from 450 nm -700 nm with 10 nm intervals (Supplementary Fig. 16). LeX8-, LeX11-, LeX16- and LeX24-funtionalised AuNPs required further serial dilutions to realise the full isotherm. Three repeats for each Lex-derivative-functionalised AuNPs were carried out and average binding isotherms were plotted in origin and EC50 were derived from the Hill1 function (Eq. 1).1 y=START+(END−START)xnkn+xn

Lateral flow strip production and running

Protocol for manufacturing lateral flow dipsticks

Dipsticks were made using the same procedure as outlined by Baker et al.80. Briefly, 20 mm of the backing cards (part for the sample pad) were removed using a guillotine. Nitrocellulose was added to the backing card using the self-adhesive on the card. The wick material was then added to the backing card with an overlap with the nitrocellulose of ~5 mm. The lateral flow strips were cut to size of width 2–3 mm using a guillotine. A small “v” ( ~ 3 mm) was cut into the test strips at the non-wick end to aid in fluid flow.

Protocol for test line addition to the lateral flow dipsticks

1 µL of 10 mg.mL−1 GM1 was applied to the dipstick ~1 cm from the non-wick end of the strip. The strips were dried at 50 °C in an oven for 5 min. The tests strips were allowed to cool to room temperature before testing.

Lateral flow assay buffer

10× HEPES buffer (20% PVP400) in 100 mL H2O. 2.38 g (100 mmol.dm−3) of HEPES, 8.77 g (1.50 mol.dm−3) of NaCl, 0.011 g (1.0 mmol.dm−3) of CaCl2, 0.8 g (0.8% w/v., 123 mmol.dm−3) of NaN3, 0.5 g (0.5% w/v., 4.07 mmol.dm−3) of Tween-20 and 20 g (20% w/v.) of poly(vinyl pyrrolidone)40 (PVP40, Average Mw ~40,000) were dissolved in 100 mL of water. The buffer was not pH adjusted.

Protocol for running lateral flow test

5 µL Lex-functionalised AuNPs (OD10), 5 µL lateral flow assay buffer – 10 × HEPES buffer, 5 µL 10 × desired concentration of CTB (or an extra 5 µL water for negative control) and 35 µL of H2O in a well of a half area 96 well plate. The strips were added to the wells, one test per well. All tests were run in triplicate. The tests were run for 20 min before removal from the wells. The test strips were allowed to dry at room temperature for ~5 min. The test strips were mounted test-face down onto a clear and colourless piece of acetate sheeting and scanned.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Supplementary information

Supplementary Information

Peer Review File

Reporting Summary

Source data

Source Data

Supplementary information

The online version contains supplementary material available at 10.1038/s41467-024-51081-7.

Acknowledgements

This project has been funded by the Industrial Biotechnology Catalyst (Innovate UK, BBSRC, EPSRC) to support the translation, development and commercialisation of innovative Industrial Biotechnology processes (BB/M02878X/1 (MIG), BB/M028852/1 (T.F., Y.L.), BB/M028941/1 (B.L.), BB/M02847X/1 (M.A.F.), BB/M028976/1 (M.C.G.), BB/M028747/1 (W.B.T.), BB/M028836/1 (S.F.)) with additional support from Carbosynth, Dextra, GSK, Ludger, and Prozomix. T.F. and Y.L. acknowledge the Wellcome Trust Biomedical Resource Grants WT099197/Z/12/Z, 108430/Z/15/Z and 218304/Z/19/Z, as well as the March of Dimes Prematurity Research Centre grant 22-FY18-82 which provided financial support to the Imperial College Glycosciences Laboratory. M.R.B. thanks the Wellcome Trust for a studentship (102576/Z/13/Z). M.C.G. and S.F. thank the European Research Council for funding (ERC-COG: 648239 and 788231- ProgrES-ERC-2017-ADG). R.Mc.B. thanks EPSRC for a DTA studentship (1799721) and T.P.K. and A.T. thank BBSRC for PhD studentships (BB/M011151/1 and BB/V50953X/1). We thank Dr Adrian Higson, Prof Rob Field, Prof Anne Imberty and Prof Wim Soetaert for discussions.

Author contributions

B.L., C.E.C., M.A.F., M.C.G., M.E.W., M.I.G., S.F., S.L.W., T.F., W.B.T., Y.L. designed the research and analysed the data; A.D.M., A.M., A.T., C.Y., D.E.W., F.P., H.C., J.D.F., H.L., J.B.V., K.Ho, K.Hu, M.R.B., R.Mc.B., S.J.R., T.K., T.P.K., W.C. performed the research; A.D.M., B.L., K.Ho., M.A.F., M.C.G., S.F., T.F., W.B.T., Y.L. wrote the manuscript.

Peer review

Peer review information

Nature Communications thanks the anonymous reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

Data availability

Supplementary information (Supplementary Figs., tables, supplementary discussion about bacterial toxin-Lewis glycan complexes, detailed experimental procedures for ITag, Lewisx and glycofluoroform synthesis, copies of HRMS screening assays of both LacNAc and Lewisx synthesis, NMR spectra of the Lewisx and the glycofluoroforms, and MALDI-MS spectra of the 24 Lewisx NGLs). Source data files of NMR spectroscopy and mass spectrometry data associated with glycan systhesis are available through the Leeds Research Data Repository (10.5518/1412). The glycan array datasets will be deposited and shared via the GlyGen Glycan Array Repository currently under its final testing phase as part of the NIH-funded GlyGen initiative (https://www.glygen.org/). As of now there is no publicly accessible glycan array data repository. The GlyGen Glycan Array Data Repository is undergoing its final testing phase as part of the NIH-funded GlyGen initiative https://glygen.ccrc.uga.edu/array/. Once the repository is officially launched, we will share our data through our Facility web portal (https://glycosciences.med.ic.ac.uk/data.html). In the interim, the Source data of microarray analyses (quantified raw fluorescence intensities) are provided as a Source Data file. The gold nanoparticle binding data generated in this study have been deposited in the University of Manchester Figshare database https://figshare.manchester.ac.uk. Correspondence and requests for materials should be addressed to Bruno Linclau. Source data are provided with this paper.

Competing interests

S.J.R. and M.I.G. are inventors on glycan-diagnostic patent application US 17330986, GB 2007895. The remaining authors declare no competing interests.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Kristian Hollingsworth, Antonio Di Maio.
==== Refs
References

1. Varki A Biological roles of glycans Glycobiology 2017 27 3 49 10.1093/glycob/cww086 27558841
Varki, A. Biological roles of glycans. Glycobiology 27, 3–49 (2017).27558841 10.1093/glycob/cww086
2. Solis D A guide into glycosciences: how chemistry, biochemistry and biology cooperate to crack the sugar code Biochim. Biophys. Acta 2015 1850 186 235 10.1016/j.bbagen.2014.03.016 24685397
Solis, D. et al. A guide into glycosciences: how chemistry, biochemistry and biology cooperate to crack the sugar code. Biochim. Biophys. Acta 1850, 186–235 (2015).24685397 10.1016/j.bbagen.2014.03.016
3. Larkin M Spectrum of sialylated and nonsialylated fuco-oligosaccharides bound by the endothelial-leukocyte adhesion molecule E-selectin. Dependence of the carbohydrate binding activity on E-selectin density J. Biol. Chem. 1992 267 13661 13668 10.1016/S0021-9258(18)42264-8 1377689
Larkin, M. et al. Spectrum of sialylated and nonsialylated fuco-oligosaccharides bound by the endothelial-leukocyte adhesion molecule E-selectin. Dependence of the carbohydrate binding activity on E-selectin density. J. Biol. Chem. 267, 13661–13668 (1992).1377689 10.1016/S0021-9258(18)42264-8
4. Richards S-J Gibson MI Toward glycomaterials with selectivity as well as affinity JACS Au 2021 1 2089 2099 10.1021/jacsau.1c00352 34984416
Richards, S.-J. & Gibson, M. I. Toward glycomaterials with selectivity as well as affinity. JACS Au 1, 2089–2099 (2021).34984416 10.1021/jacsau.1c00352
5. Wang Z A general strategy for the chemoenzymatic synthesis of asymmetrically branched N-glycans Science 2013 341 379 383 10.1126/science.1236231 23888036
Wang, Z. et al. A general strategy for the chemoenzymatic synthesis of asymmetrically branched N-glycans. Science 341, 379–383 (2013).23888036 10.1126/science.1236231
6. Dubacheva GV Curk T Richter RP Determinants of superselectivity─practical concepts for application in biology and medicine Acc. Chem. Res. 2023 56 729 739 10.1021/acs.accounts.2c00672 36916901
Dubacheva, G. V., Curk, T. & Richter, R. P. Determinants of superselectivity─practical concepts for application in biology and medicine. Acc. Chem. Res. 56, 729–739 (2023).36916901 10.1021/acs.accounts.2c00672
7. Shinya K Influenza virus receptors in the human airway Nature 2006 440 435 436 10.1038/440435a 16554799
Shinya, K. et al. Influenza virus receptors in the human airway. Nature 440, 435–436 (2006).16554799 10.1038/440435a
8. Baker AN Glycan-based flow-through device for the detection of SARS-COV-2 ACS Sens. 2021 6 3696 3705 10.1021/acssensors.1c01470 34634204
Baker, A. N. et al. Glycan-based flow-through device for the detection of SARS-COV-2. ACS Sens. 6, 3696–3705 (2021).34634204 10.1021/acssensors.1c01470
9. Kim SH GlycoGrip: cell surface-inspired universal sensor for betacoronaviruses ACS Cent. Sci. 2022 8 22 42 10.1021/acscentsci.1c01080 35106370
Kim, S. H. et al. GlycoGrip: cell surface-inspired universal sensor for betacoronaviruses. ACS Cent. Sci. 8, 22–42 (2022).35106370 10.1021/acscentsci.1c01080
10. Glaudemans CPJ Mapping of subsites of monoclonal, anti-carbohydrate antibodies using deoxy and deoxyfluoro sugars Chem. Rev. 1991 91 25 33 10.1021/cr00001a002
Glaudemans, C. P. J. Mapping of subsites of monoclonal, anti-carbohydrate antibodies using deoxy and deoxyfluoro sugars. Chem. Rev. 91, 25–33 (1991).10.1021/cr00001a002
11. Maximilian R Anja H-R Antibody recognition of fluorinated haptens and antigens Curr. Top. Med. Chem. 2014 14 840 854 10.2174/1568026614666140202203811 24484428
Maximilian, R. & Anja, H.-R. Antibody recognition of fluorinated haptens and antigens. Curr. Top. Med. Chem. 14, 840–854 (2014).24484428 10.2174/1568026614666140202203811
12. Linclau B Fluorinated carbohydrates as chemical probes for molecular recognition studies. current status and perspectives Chem. Soc. Rev. 2020 49 3863 3888 10.1039/C9CS00099B 32520059
Linclau, B. et al. Fluorinated carbohydrates as chemical probes for molecular recognition studies. current status and perspectives. Chem. Soc. Rev. 49, 3863–3888 (2020).32520059 10.1039/C9CS00099B
13. Dalvit C Vulpetti A Weak intermolecular hydrogen bonds with fluorine: detection and implications for enzymatic/chemical reactions, chemical properties, and ligand/protein fluorine NMR screening Chem. Eur. J. 2016 22 7592 7601 10.1002/chem.201600446 27112430
Dalvit, C. & Vulpetti, A. Weak intermolecular hydrogen bonds with fluorine: detection and implications for enzymatic/chemical reactions, chemical properties, and ligand/protein fluorine NMR screening. Chem. Eur. J. 22, 7592–7601 (2016).27112430 10.1002/chem.201600446
14. Paquin J-F Champagne P Desroches J Organic fluorine as a hydrogen-bond acceptor: recent examples and applications Synthesis 2015 47 306 322 10.1055/s-0034-1379537
Paquin, J.-F., Champagne, P. & Desroches, J. Organic fluorine as a hydrogen-bond acceptor: recent examples and applications. Synthesis 47, 306–322 (2015).10.1055/s-0034-1379537
15. Santana AG A dynamic combinatorial approach for the analysis of weak carbohydrate/aromatic complexes: dissecting facial selectivity in CH/pi stacking interactions J. Am. Chem. Soc. 2013 135 3347 3350 10.1021/ja3120218 23418701
Santana, A. G. et al. A dynamic combinatorial approach for the analysis of weak carbohydrate/aromatic complexes: dissecting facial selectivity in CH/pi stacking interactions. J. Am. Chem. Soc. 135, 3347–3350 (2013).23418701 10.1021/ja3120218
16. Calle LP Monitoring glycan–protein interactions by NMR spectroscopic analysis: a simple chemical tag that mimics natural CH–π interactions Chem. Eur. J. 2015 21 11408 11416 10.1002/chem.201501248 26177718
Calle, L. P. et al. Monitoring glycan–protein interactions by NMR spectroscopic analysis: a simple chemical tag that mimics natural CH–π interactions. Chem. Eur. J. 21, 11408–11416 (2015).26177718 10.1002/chem.201501248
17. Unione L Fluoroacetamide Moieties as NMR Probes for molecular recognition of GlcNAc-containing sugars: Modulation of the CH-pi stacking interactions by different fluorination patterns Chem. Eur. J. 2017 23 3957 3965 10.1002/chem.201605573 28124793
Unione, L. et al. Fluoroacetamide Moieties as NMR Probes for molecular recognition of GlcNAc-containing sugars: Modulation of the CH-pi stacking interactions by different fluorination patterns. Chem. Eur. J. 23, 3957–3965 (2017).28124793 10.1002/chem.201605573
18. Linclau B Investigating the Influence of (Deoxy)fluorination on the Lipophilicity of Non-UV-Active Fluorinated Alkanols and Carbohydrates by a new log P determination method Angew. Chem. Int. Ed. Engl. 2016 55 674 678 10.1002/anie.201509460 26592706
Linclau, B. et al. Investigating the Influence of (Deoxy)fluorination on the Lipophilicity of Non-UV-Active Fluorinated Alkanols and Carbohydrates by a new log P determination method. Angew. Chem. Int. Ed. Engl. 55, 674–678 (2016).26592706 10.1002/anie.201509460
19. St-Gelais J Côté É Lainé D Johnson PA Giguère D Addressing the structural complexity of fluorinated glucose analogues: insight into lipophilicities and solvation effects Chem. Eur. J. 2020 26 13499 13506 10.1002/chem.202002825 32652740
St-Gelais, J., Côté, É., Lainé, D., Johnson, P. A. & Giguère, D. Addressing the structural complexity of fluorinated glucose analogues: insight into lipophilicities and solvation effects. Chem. Eur. J. 26, 13499–13506 (2020).32652740 10.1002/chem.202002825
20. Kurfirt M Selectively deoxyfluorinated N-acetyllactosamine analogues as 19 F NMR probes to study carbohydrate-galectin interactions Chem. Eur. J. 2021 27 13040 13051 10.1002/chem.202101752 34216419
Kurfirt, M. et al. Selectively deoxyfluorinated N-acetyllactosamine analogues as 19 F NMR probes to study carbohydrate-galectin interactions. Chem. Eur. J. 27, 13040–13051 (2021).34216419 10.1002/chem.202101752
21. Paulini R Müller K Diederich F Orthogonal multipolar interactions in structural chemistry and biology Angew. Chem. Int. Ed. 2005 44 1788 1805 10.1002/anie.200462213
Paulini, R., Müller, K. & Diederich, F. Orthogonal multipolar interactions in structural chemistry and biology. Angew. Chem. Int. Ed. 44, 1788–1805 (2005).10.1002/anie.200462213
22. Dohi H Stereoselective glycal fluorophosphorylation: synthesis of ADP-2-fluoroheptose, an inhibitor of the LPS biosynthesis Chem. Eur. J. 2008 14 9530 9539 10.1002/chem.200801279 18833547
Dohi, H. et al. Stereoselective glycal fluorophosphorylation: synthesis of ADP-2-fluoroheptose, an inhibitor of the LPS biosynthesis. Chem. Eur. J. 14, 9530–9539 (2008).18833547 10.1002/chem.200801279
23. Yu Y Delbianco M Conformational studies of oligosaccharides Chem. Eur. J. 2020 26 9814 9825 10.1002/chem.202001370 32329095
Yu, Y. & Delbianco, M. Conformational studies of oligosaccharides. Chem. Eur. J. 26, 9814–9825 (2020).32329095 10.1002/chem.202001370
24. Anggara K Identifying the origin of local flexibility in a carbohydrate polymer Proc. Natl. Acad. Sci. USA 2021 118 e2102168118 10.1073/pnas.2102168118 34074784
Anggara, K. et al. Identifying the origin of local flexibility in a carbohydrate polymer. Proc. Natl. Acad. Sci. USA 118, e2102168118 (2021).34074784 10.1073/pnas.2102168118
25. Richards S-J Introducing affinity and selectivity into galectin-targeting nanoparticles with fluorinated glycan ligands Chem. Sci. 2021 12 905 910 10.1039/D0SC05360K
Richards, S.-J. et al. Introducing affinity and selectivity into galectin-targeting nanoparticles with fluorinated glycan ligands. Chem. Sci. 12, 905–910 (2021).10.1039/D0SC05360K
26. Gooi HC Stage-specific embryonic antigen involves alpha 1 goes to 3 fucosylated type 2 blood group chains Nature 1981 292 156 158 10.1038/292156a0 6165896
Gooi, H. C. et al. Stage-specific embryonic antigen involves alpha 1 goes to 3 fucosylated type 2 blood group chains. Nature 292, 156–158 (1981).6165896 10.1038/292156a0
27. Streit A The lex carbohydrate sequence is recognized by antibody to L5, a functional antigen in early neural development J. Neurochem. 1996 66 834 844 10.1046/j.1471-4159.1996.66020834.x 8592159
Streit, A. et al. The lex carbohydrate sequence is recognized by antibody to L5, a functional antigen in early neural development. J. Neurochem. 66, 834–844 (1996).8592159 10.1046/j.1471-4159.1996.66020834.x
28. Feizi T Demonstration by monoclonal antibodies that carbohydrate structures of glycoproteins and glycolipids are onco-developmental antigens Nature 1985 314 53 57 10.1038/314053a0 2579340
Feizi, T. Demonstration by monoclonal antibodies that carbohydrate structures of glycoproteins and glycolipids are onco-developmental antigens. Nature 314, 53–57 (1985).2579340 10.1038/314053a0
29. Hakomori, S.-i. in Advances in Cancer Research, 52. (eds. G. F. Vande Woude & G. Klein) 257–331 (Academic Press, 1989).
30. Watkins WM Biochemistry and Genetics of the ABO, Lewis, and P blood group systems Adv. Hum. Genet 1980 10 1 136 6156588
Watkins, W. M. Biochemistry and Genetics of the ABO, Lewis, and P blood group systems. Adv. Hum. Genet 10, 1–136, 379-185 (1980).6156588
31. Ayechu-Muruzabal V Diversity of human milk oligosaccharides and effects on early life immune development Front Pediatr. 2018 6 239 10.3389/fped.2018.00239 30250836
Ayechu-Muruzabal, V. et al. Diversity of human milk oligosaccharides and effects on early life immune development. Front Pediatr. 6, 239 (2018).30250836 10.3389/fped.2018.00239
32. Aspinall GO Monteiro MA Pang H Walsh EJ Moran AP Lipopolysaccharide of the helicobacter pylori type strain NCTC 11637 (ATCC 43504): structure of the O antigen chain and core oligosaccharide regions Biochemistry 1996 35 2489 2497 10.1021/bi951852s 8652593
Aspinall, G. O., Monteiro, M. A., Pang, H., Walsh, E. J. & Moran, A. P. Lipopolysaccharide of the helicobacter pylori type strain NCTC 11637 (ATCC 43504): structure of the O antigen chain and core oligosaccharide regions. Biochemistry 35, 2489–2497 (1996).8652593 10.1021/bi951852s
33. Hokke CH Deelder AM Schistosome glycoconjugates in host-parasite interplay Glycoconj. J. 2001 18 573 587 10.1023/A:1020634602161 12376724
Hokke, C. H. & Deelder, A. M. Schistosome glycoconjugates in host-parasite interplay. Glycoconj. J. 18, 573–587 (2001).12376724 10.1023/A:1020634602161
34. Palma AS Feizi T Childs RA Chai W Liu Y The neoglycolipid (NGL)-based oligosaccharide microarray system poised to decipher the meta-glycome Curr. Opin. Chem. Biol. 2014 18 87 94 10.1016/j.cbpa.2014.01.007 24508828
Palma, A. S., Feizi, T., Childs, R. A., Chai, W. & Liu, Y. The neoglycolipid (NGL)-based oligosaccharide microarray system poised to decipher the meta-glycome. Curr. Opin. Chem. Biol. 18, 87–94 (2014).24508828 10.1016/j.cbpa.2014.01.007
35. Huang K Biochemical characterisation of an α1,4 galactosyltransferase from Neisseria weaveri for the synthesis of α1,4-linked galactosides Org. Biomol. Chem. 2020 18 3142 3148 10.1039/D0OB00407C 32255449
Huang, K. et al. Biochemical characterisation of an α1,4 galactosyltransferase from Neisseria weaveri for the synthesis of α1,4-linked galactosides. Org. Biomol. Chem. 18, 3142–3148 (2020).32255449 10.1039/D0OB00407C
36. Chen M Wide sugar substrate specificity of galactokinase from Streptococcus pneumoniae TIGR4 Carbohydr. Res. 2011 346 2421 2425 10.1016/j.carres.2011.08.014 21903203
Chen, M. et al. Wide sugar substrate specificity of galactokinase from Streptococcus pneumoniae TIGR4. Carbohydr. Res. 346, 2421–2425 (2011).21903203 10.1016/j.carres.2011.08.014
37. Saito S Ozutsumi M Kurahashi K Galactose l-phosphate uridylyltransferase of escherichia coli: II. Further purification and characterization J. Biol. Chem. 1967 242 2362 2368 10.1016/S0021-9258(18)95970-3 5338129
Saito, S., Ozutsumi, M. & Kurahashi, K. Galactose l-phosphate uridylyltransferase of escherichia coli: II. Further purification and characterization. J. Biol. Chem. 242, 2362–2368 (1967).5338129 10.1016/S0021-9258(18)95970-3
38. Muthana MM Efficient one-pot multienzyme synthesis of UDP-sugars using a promiscuous UDP-sugar pyrophosphorylase from Bifidobacterium longum (BLUSP) Chem. Commun. 2012 48 2728 2730 10.1039/c2cc17577k
Muthana, M. M. et al. Efficient one-pot multienzyme synthesis of UDP-sugars using a promiscuous UDP-sugar pyrophosphorylase from Bifidobacterium longum (BLUSP). Chem. Commun. 48, 2728–2730 (2012).10.1039/c2cc17577k
39. Ghirardello M Zhang Y-Y Voglmeir J Galan MC Recent applications of ionic liquid-based tags in glycoscience Carbohydr. Res. 2022 520 108643 10.1016/j.carres.2022.108643 35977445
Ghirardello, M., Zhang, Y.-Y., Voglmeir, J. & Galan, M. C. Recent applications of ionic liquid-based tags in glycoscience. Carbohydr. Res. 520, 108643 (2022).35977445 10.1016/j.carres.2022.108643
40. Galan MC Anh Tuan T Bromfield K Rabbani S Ernst B Ionic-liquid-based MS probes for the chemo-enzymatic synthesis of oligosaccharides Org. Biomol. Chem. 2012 10 7091 7097 10.1039/c2ob25855b 22875034
Galan, M. C., Anh Tuan, T., Bromfield, K., Rabbani, S. & Ernst, B. Ionic-liquid-based MS probes for the chemo-enzymatic synthesis of oligosaccharides. Org. Biomol. Chem. 10, 7091–7097 (2012).22875034 10.1039/c2ob25855b
41. Sittel I Galan MC Chemo-enzymatic synthesis of imidazolium-tagged sialyllactosamine probes Bioorg. Med. Chem. Lett. 2015 25 4329 4332 10.1016/j.bmcl.2015.07.049 26318990
Sittel, I. & Galan, M. C. Chemo-enzymatic synthesis of imidazolium-tagged sialyllactosamine probes. Bioorg. Med. Chem. Lett. 25, 4329–4332 (2015).26318990 10.1016/j.bmcl.2015.07.049
42. Ramakrishnan B Ramasamy V Qasba PK Structural snapshots of β-1,4-galactosyltransferase-I along the kinetic pathway J. Mol. Biol. 2006 357 1619 1633 10.1016/j.jmb.2006.01.088 16497331
Ramakrishnan, B., Ramasamy, V. & Qasba, P. K. Structural snapshots of β-1,4-galactosyltransferase-I along the kinetic pathway. J. Mol. Biol. 357, 1619–1633 (2006).16497331 10.1016/j.jmb.2006.01.088
43. Ge Z Chan NWC Palcic MM Taylor DE Cloning and heterologous expression of an α1,3-fucosyltransferase gene from the gastric PathogenHelicobacter pylori J. Biol. Chem. 1997 272 21357 21363 10.1074/jbc.272.34.21357 9261149
Ge, Z., Chan, N. W. C., Palcic, M. M. & Taylor, D. E. Cloning and heterologous expression of an α1,3-fucosyltransferase gene from the gastric PathogenHelicobacter pylori. J. Biol. Chem. 272, 21357–21363 (1997).9261149 10.1074/jbc.272.34.21357
44. Wang W Chemoenzymatic synthesis of GDP-L-fucose and the Lewis X glycan derivatives Proc. Natl. Acad. Sci. USA 2009 106 16096 16101 10.1073/pnas.0908248106 19805264
Wang, W. et al. Chemoenzymatic synthesis of GDP-L-fucose and the Lewis X glycan derivatives. Proc. Natl. Acad. Sci. USA 106, 16096–16101 (2009).19805264 10.1073/pnas.0908248106
45. Sun H-Y Structure and Mechanism of Helicobacter pylori Fucosyltransferase: A Basis for Lipopolysaccharide Variation and Inhibitor Design J. Biol. Chem. 2007 282 9973 9982 10.1074/jbc.M610285200 17251184
Sun, H.-Y. et al. Structure and Mechanism of Helicobacter pylori Fucosyltransferase: A Basis for Lipopolysaccharide Variation and Inhibitor Design. J. Biol. Chem. 282, 9973–9982 (2007).17251184 10.1074/jbc.M610285200
46. Zierke M Stabilization of branched oligosaccharides: Lewisx benefits from a nonconventional C–H···O hydrogen bond J. Am. Chem. Soc. 2013 135 13464 13472 10.1021/ja4054702 24001318
Zierke, M. et al. Stabilization of branched oligosaccharides: Lewisx benefits from a nonconventional C–H···O hydrogen bond. J. Am. Chem. Soc. 135, 13464–13472 (2013).24001318 10.1021/ja4054702
47. Imberty A Pérez S Structure, conformation, and dynamics of bioactive oligosaccharides: theoretical approaches and experimental validations Chem. Rev. 2000 100 4567 4588 10.1021/cr990343j 11749358
Imberty, A. & Pérez, S. Structure, conformation, and dynamics of bioactive oligosaccharides: theoretical approaches and experimental validations. Chem. Rev. 100, 4567–4588 (2000).11749358 10.1021/cr990343j
48. Feizi T Chai W Oligosaccharide microarrays to decipher the glyco code Nat. Rev. Mol. Cell Biol. 2004 5 582 588 10.1038/nrm1428 15232576
Feizi, T. & Chai, W. Oligosaccharide microarrays to decipher the glyco code. Nat. Rev. Mol. Cell Biol. 5, 582–588 (2004).15232576 10.1038/nrm1428
49. Hu Y Schomaker JM Recent developments and strategies for mutually orthogonal bioorthogonal reactions ChemBioChem 2021 22 3254 3262 10.1002/cbic.202100164 34261195
Hu, Y. & Schomaker, J. M. Recent developments and strategies for mutually orthogonal bioorthogonal reactions. ChemBioChem 22, 3254–3262 (2021).34261195 10.1002/cbic.202100164
50. Fukui S Feizi T Galustian C Lawson AM Chai W Oligosaccharide microarrays for high-throughput detection and specificity assignments of carbohydrate-protein interactions Nat. Biotechnol. 2002 20 1011 1017 10.1038/nbt735 12219077
Fukui, S., Feizi, T., Galustian, C., Lawson, A. M. & Chai, W. Oligosaccharide microarrays for high-throughput detection and specificity assignments of carbohydrate-protein interactions. Nat. Biotechnol. 20, 1011–1017 (2002).12219077 10.1038/nbt735
51. Geijtenbeek TBH Van Kooyk Y Pathogens target DC-SIGN to influence their fate DC-SIGN functions as a pathogen receptor with broad specificity APMIS 2003 111 698 714 10.1034/j.1600-0463.2003.11107803.x 12974773
Geijtenbeek, T. B. H. & Van Kooyk, Y. Pathogens target DC-SIGN to influence their fate DC-SIGN functions as a pathogen receptor with broad specificity. APMIS 111, 698–714 (2003).12974773 10.1034/j.1600-0463.2003.11107803.x
52. Appelmelk BJ Cutting edge: carbohydrate profiling identifies new pathogens that interact with dendritic cell-specific ICAM-3-grabbing nonintegrin on dendritic cells J. Immunol. 2003 170 1635 1639 10.4049/jimmunol.170.4.1635 12574325
Appelmelk, B. J. et al. Cutting edge: carbohydrate profiling identifies new pathogens that interact with dendritic cell-specific ICAM-3-grabbing nonintegrin on dendritic cells. J. Immunol. 170, 1635–1639 (2003).12574325 10.4049/jimmunol.170.4.1635
53. Gringhuis SI Kaptein TM Wevers BA Mesman AW Geijtenbeek TB Fucose-specific DC-SIGN signalling directs T helper cell type-2 responses via IKKε- and CYLD-dependent Bcl3 activation Nat. Commun. 2014 5 3898 10.1038/ncomms4898 24867235
Gringhuis, S. I., Kaptein, T. M., Wevers, B. A., Mesman, A. W. & Geijtenbeek, T. B. Fucose-specific DC-SIGN signalling directs T helper cell type-2 responses via IKKε- and CYLD-dependent Bcl3 activation. Nat. Commun. 5, 3898 (2014).24867235 10.1038/ncomms4898
54. Valverde P Martinez JD Canada FJ Arda A Jimenez-Barbero J Molecular recognition in C-Type Lectins: the cases of DC-SIGN, Langerin, MGL, and L-Sectin ChemBioChem 2020 21 2999 3025 10.1002/cbic.202000238 32426893
Valverde, P., Martinez, J. D., Canada, F. J., Arda, A. & Jimenez-Barbero, J. Molecular recognition in C-Type Lectins: the cases of DC-SIGN, Langerin, MGL, and L-Sectin. ChemBioChem 21, 2999–3025 (2020).32426893 10.1002/cbic.202000238
55. Guo Y Structural basis for distinct ligand-binding and targeting properties of the receptors DC-SIGN and DC-SIGNR Nat. Struct. Mol. Biol. 2004 11 591 598 10.1038/nsmb784 15195147
Guo, Y. et al. Structural basis for distinct ligand-binding and targeting properties of the receptors DC-SIGN and DC-SIGNR. Nat. Struct. Mol. Biol. 11, 591–598 (2004).15195147 10.1038/nsmb784
56. Feinberg H Structural basis for langerin recognition of diverse pathogen and mammalian glycans through a single binding site J. Mol. Biol. 2011 405 1027 1039 10.1016/j.jmb.2010.11.039 21112338
Feinberg, H. et al. Structural basis for langerin recognition of diverse pathogen and mammalian glycans through a single binding site. J. Mol. Biol. 405, 1027–1039 (2011).21112338 10.1016/j.jmb.2010.11.039
57. Galustian C High and low affinity carbohydrate ligands revealed for murine SIGN‐R1 by carbohydrate array and cell binding approaches, and differing specificities for SIGN‐R3 and langerin Int. Immunol. 2004 16 853 866 10.1093/intimm/dxh089 15136555
Galustian, C. et al. High and low affinity carbohydrate ligands revealed for murine SIGN‐R1 by carbohydrate array and cell binding approaches, and differing specificities for SIGN‐R3 and langerin. Int. Immunol. 16, 853–866 (2004).15136555 10.1093/intimm/dxh089
58. Valverde P Molecular insights into DC-SIGN binding to self-antigens: the interaction with the blood group A/B antigens ACS Chem. Biol. 2019 14 1660 1671 10.1021/acschembio.9b00458 31283166
Valverde, P. et al. Molecular insights into DC-SIGN binding to self-antigens: the interaction with the blood group A/B antigens. ACS Chem. Biol. 14, 1660–1671 (2019).31283166 10.1021/acschembio.9b00458
59. Turnbull WB Precious BL Homans SW Dissecting the cholera toxin-ganglioside GM1 interaction by isothermal titration calorimetry J. Am. Chem. Soc. 2004 126 1047 1054 10.1021/ja0378207 14746472
Turnbull, W. B., Precious, B. L. & Homans, S. W. Dissecting the cholera toxin-ganglioside GM1 interaction by isothermal titration calorimetry. J. Am. Chem. Soc. 126, 1047–1054 (2004).14746472 10.1021/ja0378207
60. Holmgren J Comparison of the tissue receptors for vibrio cholerae and escherichia coli enterotoxins by means of gangliosides and natural cholera toxoid Infect. Immun. 1973 8 851 859 10.1128/iai.8.6.851-859.1973 4206342
Holmgren, J. Comparison of the tissue receptors for vibrio cholerae and escherichia coli enterotoxins by means of gangliosides and natural cholera toxoid. Infect. Immun. 8, 851–859 (1973).4206342 10.1128/iai.8.6.851-859.1973
61. Holmner Å Askarieh G Ökvist M Krengel U Blood group antigen recognition by escherichia coli heat-labile enterotoxin J. Mol. Biol. 2007 371 754 764 10.1016/j.jmb.2007.05.064 17586525
Holmner, Å., Askarieh, G., Ökvist, M. & Krengel, U. Blood group antigen recognition by escherichia coli heat-labile enterotoxin. J. Mol. Biol. 371, 754–764 (2007).17586525 10.1016/j.jmb.2007.05.064
62. Mandal PK Towards a structural basis for the relationship between blood group and the severity of El Tor cholera Angew. Chem. Int. Ed. Engl. 2012 51 5143 5146 10.1002/anie.201109068 22488789
Mandal, P. K. et al. Towards a structural basis for the relationship between blood group and the severity of El Tor cholera. Angew. Chem. Int. Ed. Engl. 51, 5143–5146 (2012).22488789 10.1002/anie.201109068
63. Holmner Å Novel binding site identified in a hybrid between cholera toxin and heat-labile enterotoxin: 1.9 Å Crystal Struct. Reveals Details Struct. 2004 12 1655 1667
Holmner, Å. et al. Novel binding site identified in a hybrid between cholera toxin and heat-labile enterotoxin: 1.9 Å Crystal. Struct. Reveals Details Struct. 12, 1655–1667 (2004).
64. Heggelund JE High-resolution crystal structures elucidate the molecular basis of cholera blood group dependence PLOS Pathog. 2016 12 e1005567 10.1371/journal.ppat.1005567 27082955
Heggelund, J. E. et al. High-resolution crystal structures elucidate the molecular basis of cholera blood group dependence. PLOS Pathog. 12, e1005567 (2016).27082955 10.1371/journal.ppat.1005567
65. Heim JB Hodnik V Heggelund JE Anderluh G Krengel U Crystal structures of cholera toxin in complex with fucosylated receptors point to importance of secondary binding site Sci. Rep. 2019 9 12243 10.1038/s41598-019-48579-2 31439922
Heim, J. B., Hodnik, V., Heggelund, J. E., Anderluh, G. & Krengel, U. Crystal structures of cholera toxin in complex with fucosylated receptors point to importance of secondary binding site. Sci. Rep. 9, 12243 (2019).31439922 10.1038/s41598-019-48579-2
66. Wands AM Fucosylated molecules competitively interfere with cholera toxin binding to host cells ACS Infect. Dis. 2018 4 758 770 10.1021/acsinfecdis.7b00085 29411974
Wands, A. M. et al. Fucosylated molecules competitively interfere with cholera toxin binding to host cells. ACS Infect. Dis. 4, 758–770 (2018).29411974 10.1021/acsinfecdis.7b00085
67. Cervin J GM1 ganglioside-independent intoxication by Cholera toxin PLOS Pathog. 2018 14 e1006862 10.1371/journal.ppat.1006862 29432456
Cervin, J. et al. GM1 ganglioside-independent intoxication by Cholera toxin. PLOS Pathog. 14, e1006862 (2018).29432456 10.1371/journal.ppat.1006862
68. Heggelund JE Specificity of escherichia coli heat-labile enterotoxin investigated by single-site mutagenesis and crystallography Int. J. Mol. Sci. 2019 20 703 10.3390/ijms20030703 30736336
Heggelund, J. E. et al. Specificity of escherichia coli heat-labile enterotoxin investigated by single-site mutagenesis and crystallography. Int. J. Mol. Sci. 20, 703 (2019).30736336 10.3390/ijms20030703
69. Elghanian R Storhoff JJ Mucic RC Letsinger RL Mirkin CA Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles Science 1997 277 1078 1081 10.1126/science.277.5329.1078 9262471
Elghanian, R., Storhoff, J. J., Mucic, R. C., Letsinger, R. L. & Mirkin, C. A. Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles. Science 277, 1078–1081 (1997).9262471 10.1126/science.277.5329.1078
70. Schofield CL Haines AH Field RA Russell DA Silver and gold glyconanoparticles for colorimetric bioassays Langmuir 2006 22 6707 6711 10.1021/la060288r 16831017
Schofield, C. L., Haines, A. H., Field, R. A. & Russell, D. A. Silver and gold glyconanoparticles for colorimetric bioassays. Langmuir 22, 6707–6711 (2006).16831017 10.1021/la060288r
71. Richards S-J Gibson MI Optimization of the polymer coating for glycosylated gold nanoparticle biosensors to ensure stability and rapid optical readouts ACS Macro Lett. 2014 3 1004 1008 10.1021/mz5004882 35610783
Richards, S.-J. & Gibson, M. I. Optimization of the polymer coating for glycosylated gold nanoparticle biosensors to ensure stability and rapid optical readouts. ACS Macro Lett. 3, 1004–1008 (2014).35610783 10.1021/mz5004882
72. Valverde P Chemoenzymatic synthesis of 3-deoxy-3-fluoro-L-fucose and its enzymatic incorporation into glycoconjugates Chem. Commun. 2020 56 6408 6411 10.1039/D0CC02209H
Valverde, P. et al. Chemoenzymatic synthesis of 3-deoxy-3-fluoro-L-fucose and its enzymatic incorporation into glycoconjugates. Chem. Commun. 56, 6408–6411 (2020).10.1039/D0CC02209H
73. Burkart MD Chemo-enzymatic synthesis of fluorinated sugar nucleotide: useful mechanistic probes for glycosyltransferases Biorg. Med. Chem. 2000 8 1937 1946 10.1016/S0968-0896(00)00139-5
Burkart, M. D. et al. Chemo-enzymatic synthesis of fluorinated sugar nucleotide: useful mechanistic probes for glycosyltransferases. Biorg. Med. Chem. 8, 1937–1946 (2000).10.1016/S0968-0896(00)00139-5
74. Yi W Remodeling bacterial polysaccharides by metabolic pathway engineering Proc. Natl. Acad. Sci. USA 2009 106 4207 4212 10.1073/pnas.0812432106 19251666
Yi, W. et al. Remodeling bacterial polysaccharides by metabolic pathway engineering. Proc. Natl. Acad. Sci. USA 106, 4207–4212 (2009).19251666 10.1073/pnas.0812432106
75. Cormier CY Protein Structure Initiative Material Repository: an open shared public resource of structural genomics plasmids for the biological community Nucleic Acids Res. 2009 38 D743 D749 10.1093/nar/gkp999 19906724
Cormier, C. Y. et al. Protein Structure Initiative Material Repository: an open shared public resource of structural genomics plasmids for the biological community. Nucleic Acids Res. 38, D743–D749 (2009).19906724 10.1093/nar/gkp999
76. Chai, W., Stoll, M. S., Galustian, C., Lawson, A. M. & Feizi, T. in Methods in Enzymology, 362 160–195 (Academic Press, 2003).
77. Liu Y The minimum information required for a glycomics experiment (MIRAGE) project: improving the standards for reporting glycan microarray-based data Glycobiology 2017 27 280 284 27993942
Liu, Y. et al. The minimum information required for a glycomics experiment (MIRAGE) project: improving the standards for reporting glycan microarray-based data. Glycobiology 27, 280–284 (2017).27993942
78. Liu Y Neoglycolipid-based oligosaccharide microarray system: preparation of NGLs and their noncovalent immobilization on nitrocellulose-coated glass slides for microarray analyses Methods Mol. Biol. 2012 808 117 136 10.1007/978-1-61779-373-8_8 22057521
Liu, Y. et al. Neoglycolipid-based oligosaccharide microarray system: preparation of NGLs and their noncovalent immobilization on nitrocellulose-coated glass slides for microarray analyses. Methods Mol. Biol. 808, 117–136 (2012).22057521 10.1007/978-1-61779-373-8_8
79. Bastús NG Comenge J Puntes V Kinetically controlled seeded growth synthesis of citrate-stabilized gold nanoparticles of up to 200 nm: size focusing versus ostwald ripening Langmuir 2011 27 11098 11105 10.1021/la201938u 21728302
Bastús, N. G., Comenge, J. & Puntes, V. Kinetically controlled seeded growth synthesis of citrate-stabilized gold nanoparticles of up to 200 nm: size focusing versus ostwald ripening. Langmuir 27, 11098–11105 (2011).21728302 10.1021/la201938u
80. Baker AN Lateral flow glyco-assays for the rapid and low-cost detection of lectins–polymeric linkers and particle engineering are essential for selectivity and performance Adv. Healthc. Mater. 2022 11 2101784 10.1002/adhm.202101784
Baker, A. N. et al. Lateral flow glyco-assays for the rapid and low-cost detection of lectins–polymeric linkers and particle engineering are essential for selectivity and performance. Adv. Healthc. Mater. 11, 2101784 (2022).10.1002/adhm.202101784
81. Neelamegham S Updates to the symbol nomenclature for glycans guidelines Glycobiology 2019 29 620 624 10.1093/glycob/cwz045 31184695
Neelamegham, S. et al. Updates to the symbol nomenclature for glycans guidelines. Glycobiology 29, 620–624 (2019).31184695 10.1093/glycob/cwz045
82. Harvey DJ Proposal for a standard system for drawing structural diagrams of N- and O-linked carbohydrates and related compounds Proteomics 2009 9 3796 3801 10.1002/pmic.200900096 19670245
Harvey, D. J. et al. Proposal for a standard system for drawing structural diagrams of N- and O-linked carbohydrates and related compounds. Proteomics 9, 3796–3801 (2009).19670245 10.1002/pmic.200900096
