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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, 2010Phakellistatins: Are They True Active Natural Products?Marta Pelay-Gimeno 1,2* , Judit Tulla-Puche 1,2 , Andrés M. Francesch 3 ,Carmen Cuevas 3 , and Fernando Albericio 1,2,41 Institute for Research in Biomedicine (IRB Barcelona), Barcelona Science Park, BaldiriReixac 10, 08028, Barcelona, Spain; 2 CIBER-BBN, Networking Centre on Bioengineering,Biomaterials and Nanomedicine, Barcelona Science Park, Baldiri Reixac 10, 08028,Barcelona, Spain; 3 Pharma Mar S.A., Avda de los Reyes 1, E-28770, Colmenar Viejo,Madrid, Spain; 4 Department of Organic Chemistry, University of Barcelona, Martí iFranqués 1-11, 08028, Barcelona, SpainIntroductionPhakellistatins [1] are members of a family of proline-rich cyclic peptides isolated frommarine sponges of the genus Phakellia displaying interesting biological activities, such ascytotoxic and antiproliferative effects, when isolated from the natural extracts. Surprisingly,although they proved to be chemically identical to their natural counterparts, the majorityof the synthesized cyclopeptides showed significantly different biological properties [2,3].Conformational differences [4] or a highly bioactive contaminant [5] retained in very smallamounts in phakellistatins samples would account for this unexpected behavior.Results and DiscussionThe synthetic approach for phakellistatin 19 follows a Fmoc/tBu strategy with chlorotritylchloride resin (CTC), which minimizes the formation of DKPs and allows retention of theprotecting groups after cleavage, which is crucial for the head-to-tail solution-phasecyclization step (Figure 2). Once the synthesis waswell established, chemical equivalence between thesynthetic and the natural cyclopeptide was validated byco-elution in a reverse-phase C18 column with a lineargradient over 30 minutes of 0.036% TFA in ACN and0.045% TFA in H 2 O from 35:65 to 55:45.A detailed NMR analysis was performed.Complete 1 H and 13 C assignment in DMSO-d 6 wasachieved by using 1D and homo- and heteronuclear 2DFig. 1. Low energy structure ofphakellistatin 19 obtained usingrestrained SA.experiments. The ROESY spectrum cross-peaks Phe 2 -H /Pro 1 -H , Ile 5 -H /Pro 4 -H and Thr 7 -H /Pro 6 -Hprovided evidence of the trans geometry of all theXaa-Pro x amide bonds, also supported by the smalldifference between and13 C NMR chemical shifts ofprolines. Finally, the hydrogen bonding pattern in DMSO-d 6 was studied by calculating thetemperature coefficients of the amide protons. The low values found for HN-Phe 2 (2.18ppb/K) and HN-Thr 7 (0.20 ppb/K) strongly suggest its participation in two intramolecularhydrogen bonds. This would be consistent with the existence of two -turns, one involvingPro 1 -Leu 8 and the other Pro 4 -Trp 3 .Fig. 2. Synthetic strategy for phakellistatin 19.490