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Table 1. Biological evaluation of natural and synthetic phakellistatin 19Phakellistatin 19GI 50 (M)MDA-MB-231GI 50 (M)A549GI 50 (M)HT29Natural 5.15 10 -7 4.62 10 -7 4.41 10 -7Synthetic n.d. n.d. n.d.n.d. Not detectedA low energy structure for phakellistatin 19 was obtained performing a standard protocol ofrestrained simulated annealing (SA) using ROESY data (Figure 1). As it can be seen in theresulting structure, the presence of a -turn involving Pro 1 -Leu 8 is confirmed. On the otherhand, as suggested by monodimensional NMR, a second intramolecular hydrogen bondstabilizes a -turn involving the residues Phe 2 -Trp 3 -Pro 4 and leaving the indole moiety ofthe tryptophan completely solvent-exposed.Some hypotheses that may explain the surprising biological behavior shown byphakellistatin 19 (Table 1) were investigated.- An epimer that co-elutes with synthetic phakellistatin 19 or that corresponds with oneof the extra peaks detected in the chromatogram of the natural cyclopeptide could beresponsible of the biological activity.The ten epimers, including those replacing L-Ile and L-Thr with L-allo-Ile and L-allo-Thr respectively, were synthesized. None of the ten epimers displayed a GI 50 below 10 -6 Min biological assays, allowing us to rule out the first hypothesis.- The presence of several proline residues capable of cis-trans isomerism in aconstrained macrocycle provides structures with a complex conformational profile.Individual conformers at proline linkages bearing different biological properties could bestabilized in different conditions (i.e. solvents).A detailed NMR analysis of cis-trans isomerism was therefore carried out. Syntheticphakellistatin 19 was studied by NMR in three different deuterated solvents: DMSO-d 6 ,CD 3 OD and CDCl 3 . A spectrally comparable and H 2 O-soluble analogue obtained byreplacement of Leu 8 with Orn 8 was also studied in DMSO-d 6 , H 2 O:D 2 O (9:1), pH = 5.95and H 2 O:D 2 O (9:1), pH = 8.12. Trans geometry was detected for all the Xaa-Pro x amidebonds in all the solvents. Only when working with CD 3 OD and CDCl 3 other conformers(10% approx.) were detected. No assignment of Pro linkages geometry was achieved forthe minor conformers.- Preparations of natural phakellistatins could contain a spectrally undetectable amountof highly potent cytotoxic agents.Due to the impossibility of working on the last hypothesis, the synthesis and biologicalevaluation of the seven possible cis-trans conformers of phakellistatin 19 using prolinederivatives capable of fixing cis-configuration at proline linkages, could eventually shedsome light on the unexpected biological behavior of phakellistatins family.AcknowledgmentsThis work was funded by CICYT (Grant CTQ2009-07758), Generalitat de Catalunya (2009SGR1024), Pharma Mar S.A., the Institute for Research in Biomedicine and Barcelona Science Park.M.P.G thanks Ministerio de Educación y Ciencia for a FPU PhD fellowship.References1. Pettit, G.R., Cichacz, Z., Barkoczy, J., Dorsaz, A.C., Herald, D.L., Schmidt, J.M., Tackett, L.P.,Brune, D.C. J. Nat. Prod. 56, 260-267 (1993).2. Pettit, G.R., Rhodes, M.R., Tan, R. J. Nat. Prod. 62, 409-414 (1999).3. Napolitano, A., Bruno, I., Riccio, R., Gomez-Paloma, L. Tetrahedron 61, 6808-6815 (2005).4. Tabudravu, J.N., Jaspars, M., Morris, L., Al, Kettenes-van den Bosch, J.J., Smith, N. J. Org. Chem.67, 8593-8601 (2002).5. Pettit, G.R., Lippert III, J.W., Taylor, S.R., Tan, R., Williams, M.D. J. Nat. Prod. 64, 883-891(2001).491