CONTROL OF PEPTIDE DISULFIDE REGIOISOMER FORMATION BY MIXED CYSTEINE PENICILLAMINE BRIDGES - APPLICATION TO ENDOTHELIN-1

While incorporation of penicillamine residues (Pen; beta,beta-dimethyl cysteine) into a peptide can cause dramatic changes in biological act ivity, the tendency of Pen to form mixed disulfides should also allow the exploitation of the steric bulk of the beta-methyls as a synthetic device to control the production of disulfide isomers. That is, oxida tion of a peptide containing an equal number of Cys and Pen residues s hould predominantly form products which contain mixed Cys-Pen disulfid es. Endothelin (ET) is a 21 amino acid peptide which contains Cys at p ositions 1, 3, 11 and 15. While oxidation of ET tetrathiol has been re ported to produce a 3:1 ratio of the natural 1-15, 3-11 to the unnatur al 1-11, 3-15 isomers, we show that oxidation of ET analogs containing two cysteines and two penicillamines predominantly formed products co ntaining Cys-Pen disulfides. Random oxidation (air, aqueous NH4OH) of the tetrathiols of [Pen1,11, Nle7]-ET-1 or [Pen3,15, Nle7]-ET-1 produc ed the correct 1-15, 3-11 isomer in > 12:1 and > 22:1 ratios, respecti vely. Oxidation of the tetrathiol of [Pen1,11, Nle7]-ET-1 favored the unnatural 1-11, 3-15 isomer by a 4:1 ratio, indicating that a normally contrathermodynamic disulfide isomer can become the favored product a s a result of the driving force for penicillamine mixed disulfide form ation. Disulfide isomers were identified using ion-spray mass spectrom etry in conjunction with enzymatic and acid hydrolysis. [Pen1,11, Nle7 ]-ET-1 was a partial agonist at the ET(A) receptor (EC50 = 7.5 nm in r abbit carotid artery rings; K(d) = 4.5 nm in rat A10 cell membranes) w hile [Pen3,15, Nle7]-ET-1 (EC50 = 0.9 nm; K(d) = 0.7 nm) was a full ag onist with similar potency to ET-1. (C) Munksgaard 1993.