Syntheses of defined sulfated oligohyaluronans reveal structural effects, diversity and thermodynamics of GAG-protein binding

Abstract

Binding of sulfated glycosaminoglycans (GAG) to a wide spectrum of extracellular regulatory proteins is crucial for physiological processes such as cell growth, migration, tissue homeostasis and repair. Thus, GAG derivatives exhibit great relevance in the development of innovative biomaterials for tissue regeneration therapies. We present a synthetic strategy for the preparation of libraries of defined sulfated oligohyaluronans as model GAG systematically varied in length, sulfation pattern and anomeric substitution in order to elucidate the effects of these parameters on GAG recognition by regulatory proteins. Through an experimental and computational approach using fluorescence polarization, ITC, docking and molecular dynamics simulations we investigate the binding of these functionalized GAG derivatives to ten representative regulatory proteins including IL-8, IL-10, BMP-2, sclerostin, TIMP-3, CXCL-12, TGF-β, FGF-1, FGF-2, and AT-III, and we establish structure-activity relationships for GAG recognition. Binding is mainly driven by enthalpy with only minor entropic contributions. In several cases binding is determined by GAG length, and in all cases by the position and number of sulfates. Affinities strongly depend on the anomeric modification of the GAG. Highest binding affinities are effected by anomeric functionalization with large fluorophores and by GAG dimerization. Our experimental and theoretical results suggest that the diversity of GAG binding sites and modes is responsible for the observed high affinities and other binding features. The presented new insights into GAG-protein recognition will be of relevance to guide the design of GAG derivatives with customized functions for the engineering of new biomaterials.

Scheme 1. Synthesis of polysulfated hyaluronan oligosaccharides with a variety of anomeric substituents. Reaction conditions: (a) DMC, N-methylmorpholine, NaN₃, H₂O, 0 °C to RT, 30 h, 59% (4), 72% (5) and 48% (6); (b) (i) DMC, DIPEA, D₂O/MeCN 2 : 1, 0 °C to RT, 90 min; (ii) thiobenzoic acid in MeCN, 5 min; 74%; (c) (i) ethynyltrimethylsilane, TBTA, CuSO₄·5H₂O, Na-ascorbate in MeOH/H₂O 3 : 1, 2 h; (ii) TBAF in THF/AcOH/H₂O 12 : 1:4, 16 h, 76%; (d) TBTA, CuSO₄·5H₂O, Na-ascorbate in MeOH/H₂O 3 : 1, 2 h, 93% (8) and 79% (9); (e) MeI, NaOMe in MeOH, 16 h, 63%; (f) 2-[2-[2-[(triphenylmethyl)-thio]ethoxy]ethyl]-bromide, NaOMe, TCEP in MeOH, 36 h, 50 °C, 93%; (g) (i) TFA/DCM/TES/H₂O 10 : 10 : 1 : 1, 5 min; (ii) 0.1 M NaOH, O₂, 35 °C, 4 h, 91%; (h) NaOMe in MeOH, DOWEX-H⁺, then TCEP, 2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone in MeOH/H₂O 4 : 1, hν, 1 h, 71%; (i) SO₃·Py, DMF, 6 h, 61–82% (15), 61% (16), 47% (17), 72% (18), 61–76% (19), 74% (20); DMC = 2-chloro-1,3-dimethyl-imidazolium chloride, DIPEA = N,N-diisopropylethylamine, TBTA = tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, TCEP = tris(2-carboxyethyl)phosphine, TFA = trifluoroacetic acid, TES = triethylsilane.

Scheme 2. Synthesis of selectively sulfated oligohyaluronan azides. Reaction conditions (a) SO₃·Py, DMF, 0 °C, 1 h, 35%; (b) (i) PhB(OH)₂, DMF, 80 °C, 4 h; (ii) SO₃·Py, 60 °C, 1 h, 45%; (c) (i) DMTrt-Cl, Py, 85 °C, 48 h; (ii) SO₃·Py, 40 °C, 4 h, (iii) HFIP, water/CHCl₃, 38%; (d) SO₃·Py, DMF, 6 h, 72% (24) 6 h, 74% (25) 6 h, 83% (26); DMTrt-Cl = 4,4′-dimethoxytriphenylmethyl chloride, HFIP = 1,1,1,3,3,3-hexafluoro-2-propanol.

Scheme 3. Summary of the recognition of four defined sulfated hyaluronanes by ten regulatory proteins obtained experimentally by fluorescence polarization. Affinity ratios of fluorophor-labeled tetra- and hexahyaluronans 15 and 19 (top) versus the azide-functionalized tetra- and hexahyaluoronans 25 and 26 (bottom) are represented schematically by arrows. Each of the ten regulatory proteins analyzed is labeled with a different color, which is also used for the arrows representing its affinity ratios. The length and thickness of each arrow correspond to the ratio of the experimentally determined K_D values for this case. A ratio of < = 10 is illustrated by the length of the arrow and a ratio >10 is represented by the thickness of the arrow. Each arrow is labeled with a number representing the determined affinity ratio for this case.

Fig. 1. Isothermal titration calorimetry of a 50 μM solution of (A) 9s-HA-4-N₃ (25), (B) (9s-HA-4-S-PEG-S-)₂ (20), and a 100 μM solution of (C) 9s-HA-4-TAMRA (15) titrated to IL-8 (10 μM) yielded dissociation constants (K_D) of 34 ± 10 nM, 16 ± 10 and 9 ± 4 nM, respectively, determined as the average of three independent experiments.

Fig. 2. Predicted diversity in binding sites and modes of highly sulfated hyaluronan derivatives on IL-8 (PDB ID: 1IL8). IL-8 is depicted in gray cartoon (light and dark representing each monomer unit). In outlined boxes, schematic representation of secondary structure of IL-8 (grey; left), and summary of all binding sites and modes predicted for TAMRA (green; right), for azide (light brown) and for the half-bivalent molecule (dark brown). In full color boxes, representative snapshots from 100 ns MD simulations of most favorable binding sites and modes of 15 (in green) on IL-8 monomer (m, left), and of 15 and 19 on IL-8 dimer (d, middle and right), 25 (in light brown) on IL-8 monomer (m, left), and of 25 and 26 on IL-8 dimer (d, middle and right) and the bivalent 20 (in dark brown) on IL-8 dimer (d). sHA molecules are shown in sticks and colored by atom type. IL-8 interacting residues are highlighted in yellow and numbered (indicating those belonging to the second monomer unit with a comma). Length and thickness of the blue arrows correspond to the increase on binding obtained computationally.