Universal Approach Towards r‐Hirudin Derivatives with High Anti‐Thrombin Activity Based on Chemical Differentiation of Primary Amino Groups

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‐hirudin. 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.