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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010The Influence of Disulfide Bridge of Trypsin Inhibitor SFTI-1for Enzyme – Inhibitor InteractionAnna Łęgowska, Dawid Dębowski, Magdalena Wysocka,Adam Lesner, and Krzysztof RolkaFaculty of Chemistry, University of Gdansk, Sobieskiego 18, Gdansk, 80-952, PolandIntroductionThe trypsin inhibitor SFTI-1 isolated in 1999 from the sunflower seeds by Luckett, et al.[1] is currently the smallest naturally occurring peptidic proteinase inhibitor. This peptidedisplays the strongest trypsin inhibitory activity among the Bowman-Birk family ofinhibitors. Owing to its small size (14 amino acid residues) and the well-defined structure(stabilized by head-to-tail cyclization and disulfide bridge), SFTI-1 has been chosen byseveral research teams to be a lead structure in the design of new inhibitors of serineproteinases. In order to investigate in detail the role of disulfide bridge in inhibitoryactivity, two series of SFTI-1 analogues were synthesized. In the first series of monocyclicSFTI-1 analogues, the disulfide bridge was formed by combination of Cys, Hcy, Pen andNhcy (N-sulfanylethylglycine) introduced at positions 3 and/or 11, originally occupied bythe Cys residues. In addition, in the substrate specificity P 1 position, peptoid monomersNlys and Nphe, resembling proteinogenic Lys and Phe, respectively were introducedinstead of Lys5. We have already proved [2] that both peptoid monomers areaccommodated well in the substrate pockets of trypsin and chymotrypsin, respectively. Inthe second series of monocyclic SFTI-1 analogues, a ring formation was achieved via aureido group incorporating the side-chain amino groups of L-2,3-diaminopropionic acid(Dap), L-2,4-diaminobutyric acid (Dab), Orn and Lys placed in positions originallyoccupied by Cys residues (Figure 1). The objective of the introduction of the set of dibasicamino acid residues starting with Dap (one CH 2 group) up to Lys (four CH 2 groups) in thepositions 3 and 11 was the determination of the optimal size of the SFTI-1 cycle/ring forthe inhibitor – trypsin interaction. In addition, unlike disulfide bridge, N-(ureidoethyl)-amide moieties are redox stable. This is a very important factor in case of introducing suchpeptides into the biological system.Fig. 1 Chemical structure of SFTI-1 analogues with carbonyl bridge, (x, y = 1 for Dap, 2for Dab, 3 for Orn and 4 for Lys).Results and DiscussionAll SFTI-1 analogues were synthesized by the solid phase method. Peptoid monomers(Nphe, Nlys(Boc) and Nhcy(Trt)) were introduced into the peptide chain by thesubmonomeric approach [3]. The carbonyl bridge was introduced in two step procedure.Dde and ivDde protecting groups were removed from side-chain amino functions of Orn,Lys, Dab and Dap, followed by reaction of bis(4-nitrophenyl) carbonate with free sidechainamino groups of peptidyl-resin [4]. Association equilibrium constants (K a ) ofsynthesized analogues with bovine -trypsin and bovine -chymotrypsin as well as theirproteolytic stability were determined. Details on synthetic methods and kineticinvestigations were described in our previous work [5].The results presented in Table 1 clearly indicated that Pen and Nhcy were notacceptable at the position 3, yielding inactive analogues, whereas another residue (Cys11)could be substituted without any significant impact on the affinity towards both ofexperimental enzymes. On the other hand, elongation of the Cys3 side chain by472