The smallest known naturally occurring trypsin inhibitor SFTI-1 (14 amino acid residues head-to-tail cyclic peptide containing one disulfide bridge) and its two analogues with one cycle each were synthesized by the solid phase method. Their trypsin inhibitory activity was determined as association equilibrium constants (K.). Additionally, hydrolysis rates with bovine beta-trypsin were measured. Among all three peptides, the wild SFTI-1 and the analogue with the disulfide bridge only had, within the experimental error, the same activity (the K-a values 1.1 X 10(10) and 9.9 x 10(9) M-1, respectively). Both peptides displayed unchanged inhibitory activity up to 6 h. The trypsin inhibitory activity of the analogue with the head-to-tail cycle only was 2.4-fold lower. It was also remarkably faster hydrolyzed (k = 1.1 X 10(-4) mol(peptide) X mol(enzyme)(-1) X s(-1)) upon the incubation with the enzyme than the other two peptides. This indicates that the head-to-tail cyclization is significantly less important than the disulfide bridge for maintaining trypsin inhibitory activity. (C) 2002 Elsevier Science (USA).