Chemical synthesis of the galectin inhibitor

Synthesis of 1,1′‐sulphanediyl‐bis‐{3‐deoxy‐3‐[4‐(5‐fluoropyrid‐2‐yl)‐¹H‐1,2,3‐triazol‐1‐yl]‐β‐d‐galactopyranoside} was performed following procedures described in the literature.¹⁶ All reagents and solvents were dried before use according to standard methods. Commercial reagents were used without further purification. Purification of the compound was carried out by column chromatography on silica gel (40–60 μm, 60 Å) and preparative HPLC (Agilent 1260 infinity system, column SymmetryPrep‐C18, 17 ml/min H₂O‐MeCN gradient 10–100% 15 min with 0·1% formic acid). Specific rotations were measured on a PerkinElmer Model 341 Polarimeter. NMR spectra ¹H, ¹³C, 2D COSY and HMQC were recorded with a Bruker Avance II 400 MHz spectrometer (400 Hz for ¹H, 100 Hz for ¹³C) at ambient temperature (see Supplementary material, Fig. S1). HRMS was determined by direct infusion on a Waters XEVO‐G2 QTOF mass spectrometer using electrospray ionization. The purity of the inhibitor was > 95% as determined by HPLC analysis (Agilent series 1100 system, column Eclipse XDB‐C18, 0·8 ml/min H₂O‐MeCN gradient 5–95% 13 min with 0·1% trifluoroacetic acid). Galectin affinities were determined in a competitive fluorescence anisotropy assay as reported previously.¹⁶