Carboxylated glucuronic poly-amido-saccharides as protein stabilizing agents

Abstract

The synthesis of novel carbohydrate-based polymers allows the structure to be tailored at the monomer level for a specific property and expands the range of available structures beyond those found in nature. Using a controlled anionic polymerization, a new type of carbohydrate polymer is synthesized in which glucose-derived monomers are joined by an α-1,2 amide linkage to give enantiopure poly-amido-saccharides (PASs). To investigate the effect of adding ionizable carboxylic acid groups, such as those found in natural polysaccharides containing glucuronic acid, the oxidation of the primary alcohol at the C6-position of the repeat unit to a carboxylic acid is reported. TEMPO-mediated oxidation provides control over the degree of oxidation in excellent yield. Based on circular dichroism, the oxidized polymers possess an ordered helical secondary structure in aqueous solution. Finally, oxidized PASs stabilize lysozyme toward dehydration and freezing stresses better than a current, widely used protein stabilizing agent, trehalose.

Scheme 1. Polymer Synthesis

Scheme 2. Sodium Periodate Oxidation and Tautomerization

Figure 1

CD spectrum in 7 mM phosphate buffer of unoxidized PAS, 4, 5, and 8 (DP = 20).

Figure 2

CD spectrum of oxPAS 4 (DP = 20) in 7 mM phosphate buffer at pH values of 2, 7, and 12.

Figure 3

Lysozyme activity following 10 lyophilization cycles versus untreated controls (N = 5 for all samples).