Decoration of Chondroitin Polysaccharide with Threonine: Synthesis, Conformational Study, and Ice-Recrystallization Inhibition Activity

Abstract

Several threonine (Thr)- and alanine (Ala)-rich antifreeze glycoproteins (AFGPs) and polysaccharides act in nature as ice recrystallization inhibitors. Among them, the Thr-decorated capsular polysaccharide (CPS) from the cold-adapted Colwellia psychrerythraea 34H bacterium was recently investigated for its cryoprotectant activity. A semisynthetic mimic thereof was here prepared from microbial sourced chondroitin through a four-step strategy, involving a partial protection of the chondroitin polysaccharide as a key step for gaining an unprecedented quantitative amidation of its glucuronic acid units. In-depth NMR and computational analysis suggested a fairly linear conformation for the semisynthetic polysaccharide, for which the antifreeze activity by a quantitative ice recrystallization inhibition assay was measured. We compared the structure-activity relationships for the Thr-derivatized chondroitin and the natural Thr-decorated CPS from C. psychrerythraea.

Figure 1

Repeating units of (a) CS and unsulfated chondroitin, and (b) CPS from C. psychrerythraea 34H

Figure 2

¹H-NMR spectra (600 MHz, D₂O, 298K) of polysaccharides 2a-d (I = Integral area)

Figure 3

¹H and HSQC-DEPT NMR spectra of polysaccharide 2d (600 MHz, D₂O, 298K; CH and CH₃ signals in black colour, CH₂ signal in red colour)

Figure 4

Zimm plot for the molecular weight determination of 2c

Figure 5

Front (A) and zoomed (B) view of the representative structure of the inspected polysaccharide as calculated by restrained SA calculations. GlcA(Thr) and GalNAc are represented as green and purple sticks. H-bonds are represented as dashed blue lines. C) rmsd fluctuations of the studied of the polysaccharide heavy atoms, along the last 10 ns of the MD simulation with respect to the conformation reported in (A). D) Superimposition between the structure of inspected polysaccharide 2d, as calculated by restrained SA calculations (green sticks), and the published solution structure of unsulfated chondroitin (orange sticks, PDB 2KQO).

Figure 6

IRI activity of the polysaccharides 2a, 2b, 2c, and 2d (0.14, 0.19, 0.43 and 1.00 indicate their DSs of Thr amidation), chondroitin, PEG (negative control) and dextran (positive control). MLGS is expressed as a percentage of PBS buffer, and small MLGS values indicate increased IRI activity

Scheme 1

Decoration of chondroitin polysaccharide with Thr amides