Heparan Sulfate Microarray Reveals That Heparan Sulfate-Protein Binding Exhibits Different Ligand Requirements

Abstract

Heparan sulfates (HS) are linear sulfated polysaccharides that modulate a wide range of physiological and disease-processes. Variations in HS epimerization and sulfation provide enormous structural diversity, which is believed to underpin protein binding and regulatory properties. The ligand requirements of HS-binding proteins have, however, been defined in only a few cases. We describe here a synthetic methodology that can rapidly provide a library of well-defined HS oligosaccharides. It is based on the use of modular disaccharides to assemble several selectively protected tetrasaccharides that were subjected to selective chemical modifications such as regioselective O- and N-sulfation and selective de-sulfation. A number of the resulting compounds were subjected to enzymatic modifications by 3-O-sulfotransferases-1 (3-OST1) to provide 3-O-sulfated derivatives. The various approaches for diversification allowed one tetrasaccharide to be converted into 12 differently sulfated derivatives. By employing tetrasaccharides with different backbone compositions, a library of 47 HS-oligosaccharides was prepared and the resulting compounds were used to construct a HS microarray. The ligand requirements of a number of HS-binding proteins including fibroblast growth factor 2 (FGF-2), and the chemokines CCL2, CCL5, CCL7, CCL13, CXCL8, and CXCL10 were examined using the array. Although all proteins recognized multiple compounds, they exhibited clear differences in structure-binding characteristics. The HS microarray data guided the selection of compounds that could interfere in biological processes such as cell proliferation. Although the library does not cover the entire chemical space of HS-tetrasaccharides, the binding data support a notion that changes in cell surface HS composition can modulate protein function.

Figure 1

Modular synthesis of HS oligosaccharides. (a) Modular disaccharide building blocks. (b) Assembled tetrasaccharides.

Figure 2

Binding of synthetic heparan sulfate oligosaccharides to FGF-2. (a) Binding of FGF-2 (0.5 μg/mL) to the HS oligosaccharide microarray at 1 mM (n = 6). (b) SPR binding of immobilized FGF-2 to various tetrasaccharides (100 μM) used as analytes. (c) Inhibition of FGF-2 binding to mouse endothelium cell surface by various tetrasacharides and heparin. (d) Inhibition of FGF-2 induced cell proliferation by various tetrasacharides and heparin measured in real time by an xCELLigence RTCA DP system. Asterisks indicate statistically significant difference (** P < 0.01, *** P < 0.001) and ns indicates no significant difference. Bar graphs in panels a-d represent the mean ± SD. (e) Compound numbering and structures of the tetrasaccharide library.

Figure 3

Binding of different HS-binding proteins to the HS oligosaccharide microarray and subsequent biological examination. Microarray results of synthetic heparin sulfate tetrasaccharide library at 1 mM with (a) CCL2 (0.5 μg/mL); (b) CCL2 mutant R18AK19A (0.5 μg/mL; shown at same scale as CCL2); (c) CCL7 (1 μg/mL); and (d) CXCL10 (0.5 μg/mL). (e) Effect of various tetrasacharides on the inhibition of CXCL10 on cell proliferation. Asterisks indicate statistically significant difference (* P < 0.05, *** P < 0.001) and ns indicates no significant difference. Bar graphs in panels a–e represent the mean ± SD.

Scheme 1. Diversification of Tetrasaccharide 18^a

^aReagents and conditions: (I) (i) Et₃N, MeOH/DCM; (ii) Ac₂O, Pyr., DMAP; (iii) NH₂NH₂AcOH, DCM/MeOH, RT, 2 h, 77%; (II) Py.SO₃ excess, DMF, 2 h, 81%; (III) (i) LiOH, H₂O₂, THF, 4 h, then 4 M NaOH, MeOH, 12 h; (ii) PMe₃, THF, MeOH, 0.1 M NaOH, 1 h (24, 70%; 29, 78%; 30, 54%; 37, 67%; 38, 54%); (IV) (i) Ac₂O, MeOH, Et₃N, 30 min; (ii) Pd/C, H₂, MeOH, H₂O, 4 h, (iii) Pd(OH)₂, H₂, H₂O, 14 h (25, 85%; 31, 65%; 33, 72%; 39, 77%; 41, 72%, 45, 82%); (V) (i) Py.SO₃, MeOH, Et₃N, 0.1 M NaOH, 12 h; (ii) Pd/C, H₂, MeOH, H₂O, 4 h; (iii) Pd(OH)₂, H₂, H₂O, 14 h (26, 67%; 32, 68%; 34, 77%; 40, 69%; 42, 77%, 46, 71%); (VI) BTSA, Pyr., 60 °C, 2 h (35, 50%; 36, 27%); (VII) Py.SO₃, controlled addition, DMF (27, 48%; 28, 35%); (VIII) LiOH, H₂O₂, THF, 4 h, then 4 M NaOH, MeOH, 12 h, 84%; (IX) PMe₃, THF, MeOH, 0.1 M NaOH, 1 h, 87%. R^a=(CH₂)₅NBnCbz; R^b=(CH₂)₅NH₂.

Scheme 2. Tetrasaccharide Substrates for 3-OST1 and Their Modified Products^a

^aReagents and conditions: (I) 3-O-sulfotransferase 1, PAPS, MES buffer, MnCl₂, MgCl₂, (48, 19%; 50, 75%; 52, 25%; 54, 20%). R = (CH₂)₅NH₂.