Karsten Von Dem Bruch

Valinomycin-mediated transport of alkali cations through solid supported membranes

Abstract Highly flexible lipid bilayers were immobilized on gold surfaces via self-assembly of thiolipids on gold in order to obtain an appropriate matrix for the carrier valinomycin. The bilayers were created by chemisorption of thiolipids to form the first hydrophobic monolayer on the gold substrate and subsequent fusion of unilamellar 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine vesicles resulted in the second physisorbed monolayer. The synthesis of the thiolipid is based on the functionalization of phosphatidylethanolamine with a linker consisting of succinic acid hooked on a thiolated tetraethyleneglycol serving as the hydrophilic linker responsible for the flexibility of the monolayer and the anchor group. The solid supported membranes were characterized by X-ray photoelectron spectroscopy and impedance spectroscopy. The latter technique revealed that the bilayers form a considerable barrier against ions in solution. The capacitance and resistivity of the prepared bilayers amount to C m =1.0±0.2 μ F/cm 2 and R m =11 000±1000 Ω cm 2 in 10 mM Tris, 50 mM N(CH 3 ) 4 Cl, pH 7.0. Ion transport of sodium and potassium ions through the bilayers in the absence and presence of valinomycin was investigated by impedance spectroscopy in the frequency range of 10 −1 –10 6 s −1 . Valinomycin was dissolved in dimethyl sulfoxide and added to the bilayer. Data evaluation was performed using a modified model established by de Levie for carrier-mediated ion transport through free-standing lipid bilayers making use of the continuum equation. The membrane resistance showed the expected linear relation to the reciprocal of the valinomycin concentration in solution. The conductivity of the membrane in the presence of valinomycin corrected for the conductivity in the absence of the carrier vs. the concentration of alkali ions in solution showed a tenfold larger slope for potassium than for sodium ions.

Synthesis of 20‐O‐linked 20(S)‐Camptothecin Glycoconjugates: Impact of the Side Chain of the Ester‐linked Amino Acid on Epimerization During the Acylation Reaction and on Hydrolytic Stability of the Final Glycoconjugates

To improve solubility and tumor selectivity of 20(S)-camptothecin the synthesis of 20-O-linked glycoconjugates 11A—G is described. Particular focus of the paper is the utilization of N-tert-butoxycarbonyl protected amino acid N-carboxy anhydrides (UNCAs) 2a—f for an efficient acylation of the sterically hindered and deactivated tertiary 20-hydroxy group of 20(S)-camptothecin 1. Depending on the solvent and on the side chain of the amino acid different extents of epimerization of the amino acids during the coupling reaction are observed; however, the epimers can easily be separated after removal of the tert-butoxycarbonyl protecting group and camptothecin amino acid conjugates 4B—E with L- and D- configured amino acids, respectively, are obtained. Particularly, bulky and β-branched amino acids can be attached to camptothecin in high yields and with low epimerization. Starting from the camptothecin amino acid conjugates 4B—E the synthesis of the glycoconjugates 11A—G is straightforward following standard procedures. The glycoconjugate hydrochlorides 11A—G show good water solubility (> 5mg- ml) and hydrolytic stability of the ester bond which again depends on the side chain of the amino acid residue linked to camptothecin. Particularly, glycoconjugates 11B—E with a bulky and β-branched amino acid at the ester linkage show high hydrolytic stability in aqueous solutions with less than 2.5% of 20(S)-camptothecin cleaved within 24 h. These results provide a basis for the selection of appropriate spacer groups for delivery systems of 20(S)-camptothecin for therapeutic use.