An investigation of the interaction of metal ions with cyclic octapeptides and cyclosporin A

Cusack, Rodney Michael (2001). An investigation of the interaction of metal ions with cyclic octapeptides and cyclosporin A PhD Thesis, School of Molecular and Microbial Sciences, The University of Queensland.

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Author Cusack, Rodney Michael
Thesis Title An investigation of the interaction of metal ions with cyclic octapeptides and cyclosporin A
School, Centre or Institute School of Molecular and Microbial Sciences
Institution The University of Queensland
Publication date 2001
Thesis type PhD Thesis
Supervisor Lawrence Gahan
Total pages 235
Collection year 2001
Language eng
Subjects L
259901 Organometallic Chemistry
780103 Chemical sciences
250204 Bioinorganic Chemistry
Formatted abstract

The aim of this Thesis was to investigate the binding of calcium(II), zinc(II) and copper(II) by the naturally occurring cyclic octapeptide patellamide D and some analogous cyclic octapeptides. The investigation was also extended to include cyclosporin A due to the biological relevance of this peptide and the dearth of research into its capacity to form metal complexes.

The solution structures of the cyclic peptides were investigated through the use of 1H NMR spectroscopy. 1D experiments, in conjunction with 2D TOCSY, DQF-COSY and NOESY experiments, allowed the conformations of the cyclic octapeptides to be determined. It was found that patellamide D maintains a "twisted-figure-eight", or Type III conformation that is independent of the solvent used (deuterated chloroform, d3-methanol and d3-acetonitrUe). This conformation was similar to the structure reported for the peptide in the solid state. The analogous cyclic octapeptides 4 (C(D-T-D-V(Thz)-I)2) and patellamide N (c(T-G(Thz)-I-S-G(Thz)-I)) were also found to adopt this "Type III" conformation in solution (d3-methanol and d3-acetonitrile). In contrast, 3 (c(T-D-V(Thz)-I)2) was found to adopt a chair conformation in solution (d6-acetone, d3-methanol and d3-acetonitrile). A further departure from the Type III conformation was discovered for 6 (c(T-D-V-aAbu-I)2), a cyclic octapeptide analogue of patellamide D where the four heterocyclic rings had been substituted with a-aminobutyric acid and threonine residues. 6 was found to be more conformationally flexible when compared to patellamide D, this fact being evident from the variation in the amide torsion angles on changing solvents (d3-methanol to d3-acetonitrile). Finally, cyclosporm A was notable for adopting multiple conformations in a variety of solvents, and consequently, no fiirther work was undertaken to elucidate the structures of these conformers.

Having elucidated the conformations of the cyclic octapeptides under investigation, the influence that the various structures and shapes of these peptides had upon their capacity to bind metal ions was investigated. In particular, studies focussed on calcium(II), zinc(II) and copper(II). Calcium(II), zinc(II) and copper(II) are biologically relevant metal ions involved in maintaining homeostasis and in enzymatic reactions in vivo.

Studies of calcium(II) binding revealed that the capacity to bind the metal increased upon the substitution of, firstly, the oxazolme groups (producing 3 with a logK1:1= 4.0, compared to a logK1:1 = 2.9 for ascidiacyclamide and patellamide D) and then the thiazole groups (producing 6, logK1:1 = 5.5). This trend suggests that the removal of constraints from the parent molecule, as well as the increase in available carbonyl oxygen donor atoms, results in an improved ability to bind metal ions. The influence of additional conformational flexibility was also evident from the binding constant for patellamide N (logK1:1 = 5.7), which, although still retaining the thiazole groups of the parent molecule, also replaces the o-valine residues of 3 with glycine residues that allow for extra conformational freedom. Furthermore, the steric hindrance applied to the cyclic octapeptide framework by the inclusion of D-threonine in place of L-threonine (generating 4 from 3) leads to a significant decrease in the magnitude of the binding constant (logK1:1 = 2.0 for 4). Finally, the binding of cyclosporin A with potassium, calcium(II) and magnesium was studied. It was found that there was little or no binding with potassium(I), whilst the binding constants for calcium(II) and magnesium(II) were logK1:1 = 5.0 and 4.6, respectively, indicating that the molecule binds preferentially to alkaline earth metals.

Zinc(II) binding was investigated using 1H NMR spectroscopy. When zinc(II) chloride was used as the source of zinc(II) ions, it was found that all peptides except 4 and 6 formed complexes. The trend established (in order of decreasing binding constant) was patellamide D = 3 = patellamide N > 4 = 6. The binding of zinc(II) ions by patellamide D was also investigated in the presence of four equivalents of triethylamine using CD and 1H NMR spectroscopy. The resulting binding constant obtained using CD spectroscopy was logK1:1 = 3.3, a value similar to that reported for ascidiacyclamide (logK1:1 = 3.0).

Finally, copper(II) complexation was investigated using electronic and EPR spectroscopy. Patellamide D, 3 and patellamide N formed mononuclear copper(II) complexes in the absence of triethylamine. No evidence for formation of similar complexes with 4, 6 and cyclosporin A could be obtained, Dinuclear copper(II) complexes were foimd for all of the cyclic octapeptides except 6 and cyclosporin A. The observation that neither 6 nor cyclosporin A formed dinuclear copper(II) complexes indicates that the presence of heterocyclic groups (or some other donor atom other than carbonyl oxygens and eimide nitrogens) are required within the peptide backbone for this type of complex to be formed.

Keyword Cyclic peptides
Additional Notes Variant title: Interaction of metal ions with cyclic octapeptides and cyclosporin A

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