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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, 2010Dendrimeric Peptides with Affinity to Opioid Receptors –Complexation StudiesMarta Sowińska 1 , Anna Leśniak 2 , Andrzej W. Lipkowski 2 , andZofia Urbanczyk-Lipkowska 21 Institute of Organic Chemistry, PAS, Warsaw 01-224, Poland;2 Medical Research Centre, PAS, Warsaw, 02-106, PolandIntroductionA pharmaceutical formulation improving bioavailability or cellular uptake gained recentlya lot of attention [1]. Biphalin, dimeric enkephalin analogue (Tyr-D-Ala-Gly-Phe-NH) 2 hasunique properties, i.e. high antinociceptive effects. It has limited permeability of bloodbrainbarrier [2]. Several reports were published on design of analogs or conjugates ofbiphalin with improved bioavailability [3,4]. Here we present preparation of (Lys) 2 Lysdendrons functionalized with terminal Tyr-Gly- and investigate complex formation withbiphalin. ESI MS studies have been applied to study complex stoichiometry. Opioidreceptor binding was performed for these derivatives alone and in the complex.Results and DiscussionIt has been discovered recently, that short amphipatic peptides act as an efficient carriers ofoligonucleotides due to formation of noncovalent complex between the two species [4].Analogously, it is expected that properties of any biologically active peptide (i.e. 3Dstructure, hydrophobicity, solubility, etc.) canbe modified by formation of molecularRHNOHNA-1: R = BocTyr-Gly-A-2: R = H 2NTyr-Gly-OOHNOHNHN OOHNONHRFig. 1. Structure of peptide dendrimers.complex with another peptide that has affinityto the same receptor. To check this hypothesis,two peptides with branched structure,functionalized with enkephalin fragment weresynthesized using convergent strategy. Initiallysynthesized (Lys) 2 Lys dendron A (Figure 1)containing phenylethylamino group at C-endwas coupled with two equivalents of dipeptideBoc-Tyr-Gly-OH yielding neutral compoundA-1, that after deprotection of amino groupsyielded compound A-2, bearing double charge.Dendrimers were purified with size exclusionchromatography using Sephadex LH-20 (in MeOH). Their structure was confirmed by ESIMS and NMR spectroscopy. Possibility of formation of the dendrimer/biphalin (BIPH)complex was checked by taking ESI MS spectra of the mixtures containing 0.5, 1, 1.5 and2.0 molar dendrimer/biphalin ratios in water. ESI MS spectra revealed ions belonging to [A+ BIPH + 2H] 2+ ions, thus both dendrimers form in gas phase associates with 1:1stoichiometry. Fragmentation study of the complex between neutral A-1 and biphalin isshown in Figure 2.Fig. 2. MS/MS spectra of the 1:1 complex between A-1 and biphalin, measured at variouscollision energies (CE).592