Date of Degree
PhD (Doctor of Philosophy)
Lee E. Kirsch
The objectives of this research were to identify the individual ionization constants (pKa values) of lipopeptide (daptomycin), evaluate the factors of pH, concentration, temperature, and calcium ions on daptomycin aggregation in aqueous solutions, and elucidate the effects of conformation and aggregation on ionization and the interaction mechanism between polyamidoamine (PAMAM) dendrimers and daptomycin.
Daptomycin is a cyclic anionic lipopeptide antibiotic. It is composed of 13 amino acids with six ionizable groups, four side-chain carboxylic acids and two side-chain amine residues. The pKa values for individual daptomycin residues have not been elucidated. The sequence-specific pKa values for the four acidic residues and one aromatic amine (Kyn-13) in daptomycin were determined in the monomeric state by TOCSY 2D 1H NMR. From the NMR pH titration, the estimated pKa values for Asp-3, Asp-9, and mGlu-12 were determined to be 4.15, 3.85, and 4.55 in the absence of salt, and 4.07, 3.83, and 4.39 in the presence of 150 mM NaCl, respectively. The pKa value for Asp-7 is estimated to be ~1.01 in the absence of salt and 1.31 in the presence of salt. The estimated Hill coefficients for Asp-7 were 0.72 and 1.31 in the absence and presence of salt, respectively. The increase in Hill coefficients from 0.72 to 1.31 with increasing salt concentration is consistent with the estimated lower pKa in the absence of salt and suggests that a salt bridge is formed in solution possibly between Asp-7 acidic group and the neighboring Orn-6 basic group. The pKa value of the aromatic amine (Kyn-13) was confirmed using UV and fluorescence spectroscopic titrations.
Aggregation behavior and critical aggregation concentration (CAC) values of daptomycin were evaluated in the different pH aqueous solutions by using the complementary analytical techniques, fluorescence, dynamic and static light scattering, and NMR spectroscopy. Based on fluorescence resonance energy transfer (FRET) from donor Trp-1 to acceptor Kyn-13, the CAC values were determined by an upward inflection of the intrinsic fluorescence emission from Kyn-13 at 460 nm as a function of increasing daptomycin concentration. The pH-dependent CAC values were determined to be 0.14 mM at pH 3.0, 0.12 mM 4.0, and 0.20 mM at pH 2.5 and 5.0. The CAC values obtained by fluorescence spectroscopy were confirmed by dynamic light scattering and NMR spectroscopy. The effects of temperature and calcium ion on daptomycin aggregation were also discussed.
The interaction mechanism between daptomycin and PAMAM dendrimers generation 5 and 6 was studied using fluorescence spectroscopy. The shapes of binding isotherms daptomycin were quantitatively described by one- and two-site binding models to estimate binding capacity and dissociation constants. Both solvent pH values and PAMAM generation size were shown to affect the binding model and parameters. The interaction between daptomycin and PAMAM dendrimer was proposed wherein the ionized Asp-3 and Asp-9 residues of daptomycin interact with PAMAM cationic surface amine.
Daptomycin, Dendrimers, Fluorescence spectroscopy, NMR, Peptide aggregation
xii, 139 pages
Includes bibliographical references (pages 133-139).
Copyright 2013 Jiang Qiu