Universal approach towards r-hirudin derivatives with high anti-thrombin activity based on chemical differentiation of primary amino groups

Full Paper: Chemical modification of recombinant hirudin (r-hirudin) is necessary whenever surface-confinement to a biomaterial or biotinylation for subsequent conjugation with carriers is intended. Here, we report a modification strategy that permits chemical discrimination between r-hirudin's amino groups and preserves its thrombin inhibitor activity. By reaction with Msc-ONSu, protective groups were successively introduced in r-hirudin yielding four derivatives (Msc) x -hirudin (1 ≤ x ≤ 4) and pure fractions were isolated by ion exchange chromatography. Structure-function relationships were studied for all derivatives and revealed a decrease in activity of more than 90% as compared to unprotected r-hirduin. MALDI-TOF MS was used to determine the locations of the Msc groups. Furthermore, evidence was provided that r-hirudin's N-terminal amino group is highly important for its anti-thrombin activity. Selective modification of the lysine residues which maintained the free N-terminal amino group preserved the anti-thrombin activity of r-hirudin even after biotinylation and subsequent linkage to streptavidin or confinement to a polymer surface. Anti-thrombin activity of r-hirudin derivatives and r-hirudin as determined by amidolytic assay. Low extinction expresses high anti-thrombin activity of the r-hirudin derivatives.