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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, 2010Side-Chain to Side-Chain Cyclization of Opioid PeptidesEnhances Proteolytic Stability of Their Exocyclic Peptide Bonds% unreacted peptide10080604020Marek Cebrat 1 , Piotr Stefanowicz 1 , Alicja Kluczyk 1 ,Zbigniew Szewczuk 1 , Katarzyna Filip 2 , Małgorzata Ciszewska 2 ,and Jan Izdebski 21 Faculty of Chemistry, University of Wrocław, Wrocław, 50-383, Poland2 Department of Chemistry, Warsaw University, Warsaw, 02-093, PolandIntroductionSide-chain to side-chain cyclization can significantly affect proteolytic stability, which isan important factor in designing the peptide-based drugs [1,2].Previously we described the synthesis and biological activity of deltorphin andenkephalin analogs restricted by cyclization via the urea bridge. The analogs contained acarbonyl bridge which linked two side-chain amino groups to form an ureido moiety.Several of these compounds showed a very high -receptor agonist potency [3].The goal of our present work was to analyze proteolytic stability of the cyclic peptides1-9 incubated in the presence of chymotrypsin and pepsin and to identify degradationproducts of the peptides by electrospray mass spectrometry (ESI-MS).Results and DiscussionWe studied the proteolytic stability of peptides in the presence of chymotrypsin and pepsinby analysis of ESI-MS spectra of the reaction mixture obtained after various incubationtimes, as shown on the example of peptide 1 (Figure 1).Identification of the degradation products00 50 100 150time [min]Fig. 1. The percentage of peptide 1hydrolyzed by chymotrypsin estimatedfrom the peak areas in the ESI-MSspectra.was based on the analysis of the high resolutionmass spectra (HR-MS) and tandem massspectrometry (MS/MS) of the main digestionproducts.Cyclization via the urea bridge increased theresistance of the peptides incubated with pepsinand chymotrypsin to proteolytic digestion.The observed stability depends not only onthe ring size but also on localization of the ureabridge within the ring. One of the intriguingobservations is the proteolytic stability of apotent opioid peptide 2, whereas its isomer 1hydrolyzed rapidly in the presence ofchymotrypsin (Figure 2).Our studies indicate that the cyclization viathe urea bridge formation influences not only thepotency and selectivity of the peptide but also itsenzymatic stability. A high stability againstproteolytic enzymes was detected for peptide 3 which also showed a high activity as the -receptor agonist [3]. The combination of the high stability, potency, and selectivity of thiscompound makes it an attractive lead compound for the design of new analgetic drugs.Presented results indicate the usefulness of mass spectrometric procedure for the fastscreening of the proteolytic stability of bioactive peptides. The advantage of the presentedapproach is its speed as well as the possibility of the simultaneous identification ofproteolytic fragments by MS/MS method.440