Exchange of one L-Valin by an L-Lysine

1. Lysine-modified valinomycin as a Biosensor Julia Braunagel, Ingo Köper Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany http://www.mpip-mainz.mpg.de e-mail: braunagel@mpip-mainz.mpg.de Introduction The ionophore valinomycin is a cyclododecadepsipeptide, that was first synthesized by Shemyakin, et al.,(1) in 1963. They showed that this depsipeptide is identical with natural valinomycin by testing the antimicrobial and ion transport inducing properties.(2) Depsipeptides are oligomers that are similar to peptides but some of the amino acids are replaced by hydroxyl acids, so that amide and ester bonds are present along the chain.(3) Valinomycin contains two amino acids (L-Valine and D‑Valine) and two hydroxyl acids (D-α-Hydroxy-isovaleric acid and L-Lactic acid), which are arranged in a 36‑membered ring regularly alternating between amino and hydroxyl acids. It presents a cyclic structure with the polar groups oriented towards the central cavity, whereas the rest of the molecule is relatively nonpolar. This enables it to complex ions and act as selective ion transporter (K+) through cellular membranes.(4) Exchange of one L-Valin by an L-Lysine possibility to bind e.g. biotin to valinomycin Natural valinomycin Lysine-valinomycin Synthesis(5) 2. Synthesis of the tetradepsipeptides 1. Synthesis of the didepsipeptides A) 80 - 90% Deprotection of A) and B) Deprotection of A) and C) B) + + 80 - 90% DMAP, DCC (DCM) 60 - 70% DMAP, DCC (DCM) C) 60% 3. Synthesis of the octadepsipeptide 4. Synthesis of the dodecadepsipeptide 5. Cyclisation of the dodecadepsipeptide + DMAP, DCC 60% (DCM) Lysine-modified valinomycin Boc-L-Lys(Fmoc)-D-Hyiv-D-Val-L-Lac-L-Val-D-Hyiv-D-Val-L-Lac-L-Val-D-Hyiv-D-Val-L-Lac-Bn Application and Outlook Cornell´s Biosensor based on the gramicidin ion channel(7) Tethered bilayer lipid membrane (tBLM)(6) Lysine-valinomycin as a Biosensor Streptavidin tBLMs mimic properties of a natural biomembrane. They consist of a lipid bilayer, the proximal leaflet is covalently attached to a solid support. In a biosensing application, the membrane serves as a matrix that allows for the functional incorporation of membrane proteins, e.g. ion channels. Biotin Lysine-valinomycin linked to biotin tBLM Tethered membrane-spanning lipids • Advantages: • easy synthesis on a multi-milligram scale • very specific to potassium • no tethered membrane-spanning lipids necessary • no anchorage on the surface necessary The analyte (streptavidin) prevents the gramicidin ion channel forming dimers so that the ion current is switched off. tBLM on a gold surface Literature (1) Shemyakin, M., N. A. Aldanova, E. I. Vinogradova, M. Y. Feigina, 1963, The structure and total synthesis of valinomycin, Tetr. Letters 4: 1921-1925 (2) Shemyakin, M., E. I. Vinograd, M. Y. Feigina, N. A. Aldanova, N. F. Loginova, I. D. Ryabova, I. A. Pavlenko, 1965, Structure-Antimicrobial Relation for Valinomycin Depsipeptides, Experientia 21: 548 (3) Kuisle, O., E. Quinoa, R. Riguera, 1999, A General Methodology for Automated Solid-Phase Synthesis of Depsides and Depsipeptides. Preparation of a Valinomycin Analogue, J. Org. Chem. 64: 8063-8075 (4) Ovchinnikov, Y. A., 1974, Membrane Active Complexones. Chemistry and Biological Function, FEBS Letters 44: 1-21 (5) Dory, Y. L., J. M. Mellor, J. F. McAleer, 1996, Applications of Pentafluorophenylester Coupling in the Synthesis of Cyclodepsipeptides Related to Valinomycins. Tetrahedron 52: 1343-1360 (6) Atanasov, V., P. P. Atanasova, I. K. Vockenroth, N. Knorr, I. Köper, 2006, A Molecular Toolkit for Highly Insulating Tethered Bilayer Lipid Membranes on Various Substrates, Bioconjugate Chem.17: 631-637 (7) Cornell, B. A., V. L. B. Braach-Maksvytis, L. G. King, P. D. J. Osman, B. Raguse, L. Wieczorek, R. J. Pace, 1997, A Biosensor that uses Ion-Channel Switches, Nature 387: 580-583