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 all the three CLRs gave strong binding signals with the N-glycan standard Man9GN2 (#28) (Figures and), only human DC-SIGN (hDC-SIGN) showed significant binding to the unmodified Lewis X probes LeX1 (#1) and the LNFPIII standard (#26). This is consistent with the known specificity of DC-SIGN to bind both to mannose-and fucose-terminating glycan sequences. For DC-SIGN, 17 out of 23 unnatural Lewis X probes showed binding, among these there was superior binding to four probes compared to the unmodified LeX1. In contrast, only some very weak binding was detected to certain fluorinated Lewis X probes by hDC-SIGNR and the Rhesus CLR Langerin (Figure ). 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 Ca 2+ . 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 Vibrio cholerae, is the major virulence factor in cholera infection, a life-threatening diarrhoeal disease. E. coli heat-labile toxin, sharing 80% sequence identity with cholera toxin, and all three proteins bound with high affinity to the GM1 ganglioside. Recent studies have identified a secondary carbohydrate binding site that recognises that histo-blood group antigens, such as Lewis x , that is also important for cellular uptake and toxicity in epithelial cells in the small intestine. All three toxins gave strong binding to the GM1 ganglioside standard (#27, Figure ) and the Gal-terminating tetrasaccharide LNnT NGL (#26), consistent with a previous report. Binding to the unmodified Lewis X trisaccharide LeX1 (#1) and LNFPIII standard (#26) was observed with Classical CTB and LTBh, but not with El Tor CTB which was confirmed by isothermal titration calorimetry (Figure ). With the library of modified Lewis X trisaccharide NGLs, unique binding profiles were observed with each toxin sample. Classical CTB showed dramatically enhanced binding to several fluorinated Lewis X probes that have modifications on Gal and Fuc. The most significant 'enhancer modifications' are 3F and 4F on Gal and 3F on Fuc. LeX16 having both 4F-Gal and 6F-Fuc modifications was found to be the best 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 caused a substantial decrease in CTB binding. With LTBh, most of the modifications had little effect or gave diminished binding; an exception was 3F-Fuc-modified LeX11 which showed marked signal enhancement. Although the unmodified Lewis X 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).