SYNTHESIS AND ION BINDING PROPERTIES OF CYCLIC HOMOPEPTIDES

Synthesis and ion binding properties of cyclic homopeptides Starting from the isolation of the antibiotic Gramicidin S, the first biologically active cyclopeptide identified in 1944, cyclic peptides have attracted increasing interest due to their unique chemical and biological properties. In cyclic peptides the amino acidic chains are closed in the ring to give structures with reduced conformational freedom compared to linear precursors. As a consequence, cyclic peptides generally have greater resistance to in vivo enzymatic degradation, greater bioavailability and ultimately greater binding affinity and selectivity toward substrates. Synthesis of cyclic peptides, in particular head-to-tail cyclisation, represents a significant synthetic challenge and cyclization reaction requires particular conditions. For instance, high dilution is still the main route applicable to minimize unwanted intermolecular processes. The success or failure of macrocyclization relies on the ability of a linear precursor to conformationally pre-organize its reactive ends bringing them close to each other before ring closure. Many features have been found to favor cyclization such as introduction of cis-amide bond in the middle of the peptide chain, the presence of both D- and L- residues in the termini, introduction of N-methyl amino acids etc. In this thesis the synthesis of a series of linear homopeptides containing lysine, serine and aminoalanine were performed in solution following a classical orthogonal protection scheme. Macrocyclization reactions were carried out on completely protected peptide precursors under high dilution conditions in the presence of different salts (NaCl, LiCl, NaTPB and TEACl) to promote the head-to-tail condensation. Microcalorimetric titrations using linear tetra-, penta- and hexalysine containing peptides as ligands and NaClO4 and TEACl as salts indicated a very weak binding of the peptides with Na+ and a strong binding with Cl-. The binding constants for the complexation of chloride ions were evaluated to be Log K=1.51, Log K=1.47 and Log K=1.68, for tetra-, penta- and hexapeptide, respectively. These findings suggest that the mechanism commonly accepted for the metal-ion assisted cyclization has to be revised indicating that coordination of chloride ions (and not alkali metals) with amide groups along the chain is the factor that predominantly brings N- and C-termini closer, forcing them to react. Accordingly, the highest cyclization yields were obtained when TEACl was used as the salt. Moreover, MD simulations show the formation of stable complexes of linear peptides with Cl- while in complexes with sodium dissociation of the cation occurs. Binding properties of cyclic pentalysine toward different anions were investigated by means of microcalorimetric titrations in CH3CN. In complexation reactions with halides (Cl-, Br-, I-) as well as with NO3-, inflection was observed at a ratio n(A-)/n(L) ≈ 1 which indicates that the stoichiometry of the resulting complex is 1:1. In contrast, with H2PO4- we observed inflection at the ratio n(L)/n(A-) ≈ 2 i.e. two ligands are needed to coordinate one H2PO4- anion. Calorimetric titrations showed also that the most efficient binding of cyclopentalysine is with Cl- with the binding constant of Log K=5.72 while that of cyclohexalysine is with OAc- (Log K > 4, obtained by NMR titrations). In general, cyclic pentapeptide binds stronger all investigated anions than the corresponding cyclic hexapeptide. Complexation reactions of cyclic penta- and hexalysine were also investigated by 1H NMR and ESI-MS titrations. The results are in agreement with those obtained by microcalorimetric titrations. Linear H2N-(Leu)5-COOH and H2N-Phe-Leu-Leu-Phe-Leu-Leu-COOH were also prepared in order to confirm the role of chloride anion in assisting macrocyclization of peptides. With the ultimate goal to prepare a novel class of anion receptors, namely calixarene homo-cyclopeptide derivatives, work is in progress to set-up a simple and efficient method to directly conjugate the prepared homocyclopeptides to calixarenes by exploiting side chain functional groups.