Selectively Modified Lactose and N-Acetyllactosamine Analogs at Three Key Positions to Afford Effective Galectin-3 Ligands

Abstract

Galectins constitute a family of galactose-binding lectins overly expressed in the tumor microenvironment as well as in innate and adaptive immune cells, in inflammatory diseases. Lactose ((β-D-galactopyranosyl)-(1→4)-β-D-glucopyranose, Lac) and N-Acetyllactosamine (2-acetamido-2-deoxy-4-O-β-D-galactopyranosyl-D-glucopyranose, LacNAc) have been widely exploited as ligands for a wide range of galectins, sometimes with modest selectivity. Even though several chemical modifications at single positions of the sugar rings have been applied to these ligands, very few examples combined the simultaneous modifications at key positions known to increase both affinity and selectivity. We report herein combined modifications at the anomeric position, C-2, and O-3' of each of the two sugars, resulting in a 3'-O-sulfated LacNAc analog having a Kd of 14.7 µM against human Gal-3 as measured by isothermal titration calorimetry (ITC). This represents a six-fold increase in affinity when compared to methyl β-D-lactoside having a Kd of 91 µM. The three best compounds contained sulfate groups at the O-3' position of the galactoside moieties, which were perfectly in line with the observed highly cationic character of the human Gal-3 binding site shown by the co-crystal of one of the best candidates of the LacNAc series.

Keywords: X-ray; galectin; isothermal titration calorimetry (ITC); lactoside.

Scheme 4

Tin acetal catalyzed regioselective 3′-O-sulfation (30), 3′-O- etherification (32), and 3′-O sialylation following a recently described strategy (33) of intermediate 28 [32].

Scheme 1

Synthetic route to propargyl and para-nitrophenyl-based lactosides (4, 6) and their 3-O′-sulfated derivatives (11, 12).

Scheme 2

Synthetic route to para-nitrophenyl-based LacNAc (20) and its 3-O′-sulfated derivative (23).

Scheme 3

Synthesis of propargyl LacNAc (27) and key intermediate 28.

Figure 1

(A) Binding of hGal-3 to analog 12 as measured by isothermal titration calorimetry (ITC) at 27 °C and pH 7.4. The top panel shows data obtained for automatic injections (4 μL each) of analog 12. The integrated curve showing experimental points and the best fit are shown in the bottom panel. (B) Binding of hGal-3 to analog 30 as measured by isothermal titration calorimetry (ITC) at 27 °C and pH 7.4. The top panel shows data obtained for automatic injections (4 μL each) of analog 30. The integrated curve showing experimental points and the best fit are shown in the bottom panel.

Figure 2

Structure of the Gal-3 CRD in complex with 23. (a) Electron density map (2Fo-Fc) displayed at 1 σ around the sugar. (b) Representation of the B factor of the sugar atoms, from dark blue (low) to red (high).

Figure 3

Location of important waters involved in binding the sugar to the protein. The protein is shown as a cartoon; residues involved directly or through waters in binding the sugar, as well as the sugar, are shown as sticks. (a) CRD of Gal-3 in complex with 23. (b) Superposition of the Gal-3 CRD structures with lactose (3ZSJ), LacNAc (1KJL), LacNAcNAc (5NF7) and 23. (c) Panel b rotated 90 degrees.