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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, 2010Analogues of Trypsin Inhibitor SFTI-1 Modified inAbsolutely Conserved P1’ Position by Synthetic orNon-Proteinogenic Amino AcidsRafał Łukajtis, Anna Łęgowska, Dawid Dębowski,Magdalena Wysocka, Adam Lesner, and Krzysztof RolkaFaculty of Chemistry, University of Gdansk, Sobieskiego 18, Gdansk, 80-952, PolandIntroductionThe inhibitors of Bowman-Birk family are canonical inhibitors found in various plantsources. One of the most studied inhibitor of this family is trypsin inhibitor SFTI-1 isolatedfrom sunflower seeds [1]. The amino acid sequences of BBIs indicate that Ser at P1’ andcis-Pro at P3’ are absolutely conserved in this familyof inhibitors. In the case of SFTI-1, these aminoacid residues are located in the positions 6 and 8,respectively (see Figure 1). There are several reportsthat substitution of Ser located in the P1’ position byAla preserved trypsin inhibitory activity. On theother hand, according to Mc Bride, et al. [2], theinteraction between Ser6 and Thr4 appears to beFig. 1. Primary structure of SFTI-1.instrumental in projecting the P 1 side chainoutwards for the interaction with the enzyme S 1pocket. Bearing in mind the results of our previousstudies on peptomeric SFTI-1 analogues and the limited experimental evidence supportingthe statement about the role played by the inhibitor’s P1’ position, we decided to focus ourattention on the role of hydroxyl group of Ser6 in the inhibitor – enzyme interaction.Herein we report chemical synthesis and determination of -chymotrypsin and trypsininhibitory activity of a series of linear and monocyclic analogues of SFTI-1 modified in theP1’ position by Ala, Sar, Pro, Hyp, Hpr (L-homoproline), Hse (L-homoserine) and Nhse(N-(2-hydroxyethyl)glycine), Aze (L-azetidine-2-carboxylic acit) and Oic (L-octahydroindole-2-carboxylicacid). Consequently, either Phe, Nphe or Lys were introduced in thesubstrate specificity P 1 position.Results and DiscussionAll 21 SFTI-1 analogues were synthesized by the solid phase method. Associationequilibrium constants (K a ) of analogues synthesized with bovine β-trypsin and bovine -chymotrypsin as well as their proteolytic stability were also determined. Details ofsynthetic methods and kinetic investigations were described in our previous work [3].The Ka values determined for most active analogues modified in discussed position(Table 1) proved that absolutely conserved in BBI Ser residue located in the P1’ position isnot essential for inhibitory activity. Analogues with Ala, Hse, Pro, Hyp and Azeadditionally modified in substrate specificity P1’ position are able to inhibit both ofexperimental enzymes. On the other hand, the introduction of Hpr residue in discussedposition decreased the Ka values of such modified analogues by at least three orders ofmagnitude whereas Oic yielded inactive analogues. Interestingly enough, introduction ofthe peptoid monomer Nhse in position P1’ produced analogues 3 and 5 that were almostequipotent with that containing in this position naturally occurring Ser. This is the firstevidence that the absolutely conserved Ser present in the BBI inhibitor’s P1’ position canbe successfully replaced by a synthetic derivative. Studies on proteolytic resistance on theseactive monocyclic analogues of SFTI-1 displayed full proteolytic resistance of analogues 4and 5 whereas other like 2, 3 and 13 with Phe5 and the non-proteinogenic Hse, Nhse andAze in position 6 were slowly hydrolyzed by the cognate enzyme. It should be emphasizedthat linear analogues containing aforementioned modifications were inactive due to theirhigh proteolytic susceptibility.470