Novel trihydroxamate‐containing peptides: Design, synthesis, and metal coordination

Novel trihydroxamate‐containing peptides were designed to mimic desferrioxamine (Desferal®, DFO , a naturally occurring siderophore) but possess distinct conformational restrictions and varied lipophilicity to probe structure vs. metal coordination. The synthesis was performed via fragment condensation of hydroxamate‐containing oligopeptides such as Fmoc–Leu– Ψ[CON(OBz)]–Phe–Ala–Pro–OH and H–Leu–Ψ[CON(OBz)]–Phe–Ala–Pro–OBu t (Fmoc: 9‐fluor enylmethoxycarbonyl; OBz: benzyl; OBu t : tert ‐butyl) either in solution or on a solid support. The metal‐binding properties were studied by electrospray ionization–mass spectroscopy (ESI‐MS), ultraviolet (UV)‐visible spectroscopy, and 1 H nuclear magnetic resonance (NMR). Similar to the dihydroxamate analogs previously explored [ Biopolymers ( Peptide Science ), 2003, Vol. 71, pp. 489–515], the compounds with three hydroxamates arrayed at 10‐atom intervals, i.e., H–[Leu–Ψ[CON(OH)]–Phe–Ala–Pro] 3 –OH ( P1 ), cyclo[Leu–Ψ[CON(OH)]–Phe–Ala–Pro] 3 ( P2 ), and H–[Leu–Ψ(CONOH)–Phe–Ala–Pro] 2 –Leu–NHOH ( P7 ), exhibited high affinities for intramolecular coordination with Fe(III) and Ga(III). As expected, both P1 and P2 showed higher relative Fe(III)‐binding affinities than the corresponding dihydroxamate‐containing peptide analogs ( P11 and P12 ). Even though both P1 and P2 did not compete with DFO in the relative metal‐binding affinity in both solution and gas phases, P1 , P2 , and DFO exhibited similar relative binding selectivities to 11 different metal ions including Fe(III), Fe(II), Al(III), Ga(III), In(III), Zn(II), Cu(II), Co(II), Ni(II), Gd(III), and Mn(II). Compared to the other metal ions, they had higher relative binding affinities with Fe(III), Fe(II), Al(III), Ga(III), and In(III). The decreased metal‐binding affinities of P1 and P2 in comparison with DFO suggested the conformational restrictions of their backbones perturb their three hydroxamate groups from optimal hexadentate orientations for metal coordination. As detected by ESI‐MS, P2 was distinguished from both P1 and DFO by solvation of its Ga(III) and Fe(III) complexes (such as acetonitrile or water), thereby stabilizing the resulting complexes in the gas phase. Noteworthy, P2 led to 69% death rate in Hela cells at a concentration of 50 μ M , exhibiting higher cytotoxicity than DFO in vitro despite its much lower affinity for iron. This enhanced toxicity may simply reflect the increased lipophilicity of the cyclic trihydroxamate ( P2) together with the improvements in its cell penetration, and/or subsequent intracellular molecular recognition of both side chains and hydroxamate groups. The cytotoxicity was significantly suppressed by precoordination with Ga(III) or Fe(III), suggesting a mechanism of toxicity via sequestration of essential metal ions as well as the importance of curbing the metal coordination before targeting. The potential of such siderophore‐mimicking peptides in oncology needs further exploration. © 2006 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 84: 472–489, 2006 This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com