Preparation of alkylamine and 125I-radiolabeled derivatives of hyaluronic acid uniquely modified at the reducing end

Abstract

Present procedures to obtain radiolabeled hyaluronic acid derivatives are limited to low-specific-activity isotopes and small amounts of material, and often involve multiple points of chemical modification within the polymer. A synthesis has been developed which affords large quantities of a unique, chemically modified derivative of hyaluronic acid containing a single hydroxyphenyl group at the reducing end, which can be radioiodinated to high specific activity. Very little alteration in oligosaccharide structure is expected since only the terminal reducing sugar is modified. Oligosaccharides of hyaluronic acid, which have no free amino groups, were first converted to alkylamine derivatives to allow subsequent reaction with the Bolton-Hunter reagent, N-succinimidyl-3(4-hydroxyphenyl)propionate. Synthesis of the hyaluronate-amine was achieved by (i) reduction of the terminal reducing sugar with sodium borohydride, (ii) controlled sodium periodate oxidation to generate an aldehyde group only at the reduced end, and (iii) coupling this aldehyde to an α,ω-alkyldiamine (e.g., 1,6-hexanediamine) in the presence of sodium cyanoborohydride. Purified hyaluronate-amine oligosaccharides were then reacted with the Bolton-Hunter reagent, and the hydroxyphenyl derivative thus obtained was radioiodinated with Na 125I. Specific activities up to 8 × 10 9 cpm/nmol oligosaccharide can be obtained. This approach yields a uniquely modified, highly radioactive probe which will be useful in studies of cellular and extracellular matrix interactions with hyaluronic acid. In addition, the uniquely modified alkylamine derivative of hyaluronic acid has been used to prepare affinity chromatography media and synthetic cell culture surfaces.