Synthesis and screening of a library of Lewisx deoxyfluoro-analogues reveals differential recognition by glycan-binding partners

Abstract

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 Lewis^x 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 Lewis^x. Notably, we show that for two proteins with unique binding sites for Lewis^x, 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.

Fig. 1. Aim of this work.

a A glycan motif such as Lewis^x 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 Lewis^x. d) Depiction of chemo-enzymatic synthesis of Lewis^x 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 Lewis^x analogues. Monosaccharide symbols are in accord with the updated Symbol Nomenclature for Glycans (SNFG) convention. 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 System. GlcNAc N-acetyl glucosamine, Gal galactose, Fuc fucose, Ara arabinose, TFA trifluoroacetyl.

Fig. 2. General enzymatic synthesis of Lewis^x 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.

Fig. 3. Enzymatic synthesis of Lewis^x analogues.

The matrix depicts ESMS-derived conversion efficiencies of GlcNAc derivatives bearing ITags to (a) LacNAc analogues, and (b–f) Lewis^x analogues containing fucose, 3FFuc, 4FFuc, 6FFuc, and Ara, respectively (See Supplementary Table 1 for numerical data). g A sub-library of 24 Lewis^x 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).

Fig. 4. Microarray analyses of 24 Lewis^x NGL probes.

a Symbolic forms of the 24 Lewis^x 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 Lewis^x trisaccharide are summarised using colour shading: red, strong enhancement; blue, abolished binding. c Spider charts showing distinctive binding modes of Lewis^x glycofluoroforms observed in microarray analyses. The signal intensities of the unnatural Lewis^x 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).

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 Lewis^x (LeX1) and fluorinated LeX22; d Dose-dependent response of library of Lewis^x 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).