Linkage Isomerism in the Binding of Pentapeptide Ac-His(Ala)3His-NH2to (Ethylenediamine)Palladium(II): Effect of the Binding Mode on Peptide Conformation

Abstract

The reaction of the pentapeptide Ac-His1-Ala2-Ala3-Ala4-His5-NH2 (AcHAAAHNH2) (1) with [Pd(en)(ONO2)2] (en = NH2CH2CH2NH2) in either DMF-d(7) or H2O:D2O (90%:10%) gave three linkage isomers of [Pd(en)(AcHAAAHNH2)](2+) (2), 2a, 2b, and 2c, which differ only in which pair of imidazole nitrogen atoms bind to Pd. In the most abundant isomer, 2a, Pd is bound by N1 from each of the two imidazole rings. In the minor isomers 2b and 2c, Pd is bound by N1(His1) and N3(His5) and by N3(His1) and N1(His5), respectively. The reactions of [Pd(en)(ONO2)2] with the N-methylated peptides Ac-(N3-MeHis)-Ala-Ala-Ala-(N3-MeHis)-NH2 (AcH*AAAH*NH2) (3), Ac-(N3-MeHis)-Ala-Ala-Ala-(N1-MeHis)-NH2 (AcH(*)AAAH(#)NH2) (4), and Ac-(N1-MeHis)-Ala-Ala-Ala-(N3-Me-His)-NH2 (AcH(#)AAAH(*)NH2) (5) each gave a single species [Pd(en)(peptide)](2+) in N,N-dimethylformamide (DMF) or aqueous solution, 7, 8, and 9, respectively, with Pd bound by the two nonmethylated imidazole nitrogen atoms in each case. These complexes were analogous to 2a, 2b, and 2c, respectively. Ac-(N1-MeHis)-Ala-Ala-Ala-(N1-MeHis)-NH2 (AcH(#)AAAH(#)NH2) (6) with [Pd(en)(ONO2)2] in DMF slowly gave a single product, [Pd(en)(AcH(#)AAAH(#)NH2)](2+) (10), in which Pd was bound by the N3 of each imidazole ring. The corresponding linkage isomer of 2 was not observed. Complex 10 was also the major product in aqueous solution, but other species were also present. All compounds were exhaustively characterized in solution by multinuclear 1D ((1)H , (13)C, and, with (15)N-labeled ethylenediamine, (15)N) and 2D (correlation spectroscopy, total correlation spectroscopy, transverse rotating-frame Overhauser effect spectroscopy (T-ROESY), heteronuclear multiple-bond correlation, and heteronuclear single quantum coherence) NMR spectra, circular dichroism (CD) spectra, electrospray mass spectroscopy, and reversed-phase high-performance liquid chromatography. ROESY spectra were used to calculate the structure of 2a, which contained a single turn of a peptide alpha helix in both DMF and water, the helix being better defined in DMF. The Pd(en)(2+) moiety was not used in structure calculations, but its location and coordination by one imidazole N1 from each histidine to form a 22-membered metallocycle were unambiguously established. Convergence of the structures was greatest when calculated with two hydrogen-bond constraints (Ala4 peptide NH...OC acetyl and His5 peptide NH...OC-His1) that were indicated by the low temperature dependence of these NH chemical shifts. Vicinal HN-CHalpha coupling constants and chemical shifts of alpha-H atoms were also consistent with a helical conformation. Similar long-range ROE correlations were observed for [Pd(en)(AcH(*)AAAH(*)NH2)](2+) (7), which displayed a CD spectrum in aqueous solution that suggested the presence of some helicity. Long-range ROE correlations were not observed for 8, 9, or 10, but a combination of NMR data and CD spectroscopy was interpreted in terms of the conformational behavior of the coordinated pentapeptide. Only for the linkage isomer [Pd(en)(AcH(*)AAAH(#)NH2)](2+) (8) was there evidence of a contribution from a helical conformation. The data for 8 were interpreted as interconversion between the helix and random coil conformations. Zn(2+) with peptides gave broad NMR peaks attributed to lability of this metal ion, while reactions of cis-[Pt(NH3)2(ONO2)2] were slow, giving a complex mixture of products rather than the macrochelate ring observed with Pd(en)(2+). In summary, these studies indicate that Pd(en)(2+) coordinates to histidine with similar preference for each of the two imidazole nitrogens, enabling the formation of up to four linkage isomers in its complexes with pentapeptides His-xxx-His. Only the N1-N1 linkage isomer that forms a 22-membered macrochelate ring is able to induce an alpha-helical peptide conformation, whereas the 20- and 21-membered rings of linkage isomers do not. This suggests that linkage isomeric mixtures may compromise histidine coordination to metal ions and reduce alpha-helicity.