Structure-activity studies on C-terminal hirudin peptides containing sulfated tyrosine residues

To clarify the role of the negative charge of the C-terminal region of hirudin, we chemically synthesized the C-terminal peptide of hirudin variant-1 (HV-1), HV-1-(54-65), and its analogs, [E61Y,E62Y]HV-1-(54-65) and [E62Y]HV-1-(54-65), and then sulfated the Tyr residue(s) in these peptides by both enzymic and chemical methods. Enzymic O-sulfation of Tyr residues in the peptides by use of sulfotransferase isolated from Eubacterium A-44 allowed us to produce four kinds of the sulfated peptide, whose C-terminal sequences were -PEY(SO₃H)YLQ, -PYY(SO₃H)YLQ, -PYYY(SO₃H)LQ and -PYY(SO₃H)Y(SO₃H)LQ. On the other hand, all Tyr residues in the peptides were successfully sulfated by chemical reaction with N, N′-dicyclohexylcarbodiimide in the presence of sulfuric acid. Based on the analysis of structure-activity relationships of these sulfated peptides for thrombin inhibition, the Tyr62 and Tyr63 bisulfated peptide GDFEEIPEY(SO₃H)Y(SO₃H)LQ was found to be the most potent inhibitor of thrombin among the products tested. No increase in potency was observed by further substitution of Glu61 with Tyr(SO₃H). The inhibitory activity by substitution with Tyr(SO₃H) at position 63 was greater than that obtained by the substitution at position 62. © Munksgaard 1996.