Philipp Reiß

Effect of Conformational Preorganization of a Three‐Armed Host on Anion Binding and Selectivity

A set of three-armed urea-containing anion receptors was prepared. The receptors all have the same binding topology but differ in the level of conformational preorganization with respect to the arrangement of the side-arms relative to the platform and within the side arms themselves. This is mirrored in a specific increase (x 2.5) in the binding constant for chloride and in a 12-fold increase in the chloride/nitrate-selectivity.

Ion-channels: goals for function-oriented synthesis.

Ion channels provide a conductance pathway for the passive transport of ions across membranes. These functional molecules perform key tasks in biological systems such as neuronal signaling, muscular control, and sensing. Recently, function-oriented synthesis researchers began to focus on ion channels with the goal of modifying the function of existing ion channels (ion selectivity, gating) or creating new channels with novel functions. Both approaches, ion channel engineering and de novo design, have involved synthetic chemists, biochemists, structural biologists, and neurochemists. Researchers characterize the function of ion channels by measuring their conductance in samples of biological membranes (patch clamp) or artificial membranes (planar lipid bilayers). At the single molecule level, these measurements require special attention to the purity of the sample, a challenge that synthetic chemists should be aware of. Ideally, researchers study the function of channels while also acquiring structural data (X-ray, NMR) to understand and predict how synthetic modifications alter channel function. Long-term oriented researchers would like to apply synthetic ion channels to single molecule sensing and to implantat these synthetic systems in living organisms as tools or for the treatment of channelopathies. In this Account, we discuss our own work on synthetic ion channels and explain the shift of our research focus from a de novo design of oligo-THFs and oligo-THF-amino acids to ion channel engineering. We introduce details about two biological lead structures for ion channel engineering: the gramicidin β(6,3) helix as an example of a channel with a narrow ion conductance pathway and the outer membrane porins (OmpF, OmpG) with their open β-barrel structure. The increase and the reversal of ion selectivity of these systems and the hydrophobic match/mismatch of the channel with the phospholipid bilayer are of particular interest. For engineering ion channels, we need to supplement the single-point attachment of a synthetic modulator with the synthesis of a more challenging two-point attachment. The successful function-oriented synthesis of ion channels will require interdisciplinary efforts that include new electrophysiology techniques, efficient synthesis (peptide/protein/organic), and good structural analysis.

Expanding the scope of protein trans-splicing to fragment ligation of an integral membrane protein: towards modulation of porin-based ion channels by chemical modification.

Its raining, its porin: Fragment ligation of OmpF ion channels was achieved by using the split Psp‐GBD Pol intein; this allowed reconstitution of active trimeric porin. In combination with cysteine modification at an internal position, the porins conductance properties were altered.

A voltage-responding ion channel derived by C-terminal modification of gramicidin A.

The proper function of biological ion channels requires defined modes of control (gating). Voltage gating is the response of the ionic flux through the channel pore upon a change of the membrane potential. Neuronal signal propagation relies on voltage-gated ion channels. Progress in the structural biology of voltage gated ion channels and the design and functional studies of voltagemodulated model channels helps in understanding voltage gating, and is a first step towards the implantation of synthetic voltage-modulated channels into neurons. In a continuation of our studies on the implantation of synthetic gramicidin-hybrid channels into cells (trabecular meshwork cells, CHO cells), we turned to synthetic voltage-gated channels. Terminal-charged amphiphilic compounds, can self-assemble into voltage-dependent pores and rigid push–pull rods with permanent axial macrodipoles to give voltage-sensitive pores. Compared with these self-assembled pores, the channel-active b-helix of gramicidin A (gA) is structurally well defined. (Figure 1) The attachment of a positive charge at the C-terminal end of gA as a voltage-modulating element resulted in gA derivatives of type 1; this work was pioneered by L<uger and Woolley. A schematic description for the voltage modulation is the ball-and-chain model that is shown in Figure 2. A heterodimer channel of 1 and gA should lead to an open state if the positive end group of 1 is located at a negative-potential membrane site (Figure 2A). Upon switching to a positive membrane potential, electrostatic repelling forces should direct the positive charge into the channel entrance like a stopper in a bottle neck. The steric demand and the rigidity of the linker (in blue) between the C terminus and the positive charge is crucial for successful voltage gating. A sterically demanding linker allows the positive charge to come only near to the entrance, which results in

Structural and functional characterization of a synthetically modified OmpG

Chemical modification of ion channels has recently attracted attention due to their potential use in stochastic sensing and neurobiology. Among the available channel templates stable β-barrel proteins have shown their potential for large scale chemical modifications due to their wide pore lumen. Ion-channel hybrids using the outer membrane protein OmpG were generated by S-alkylation with a synthetic modulator and functionally as well as structurally characterized. The dansyl moiety of the used modulator resulted in partial blockage of current though the OmpG channel with its gating characteristics mainly unaffected. The crystal structure of an OmpG-dansyl hybrid at 2.4Å resolution correlates this finding by showing that the modulator lines the inner walling of the OmpG pore. These results underline the suitability of OmpG as a structural base for the construction of stochastic sensors.

Synthesis and biological evaluation of gramicidin S dimers

The design and synthesis of analogues of the cyclic beta-sheet gramicidin S (GS), having additional functionalities in their turn regions, is reported. The monomeric GS analogues were transformed into dimers and their activities towards biological membranes, through antimicriobial and hemolytic assays, were evaluated. Finally, conductivity measurements have been performed to elucidate ion channel forming properties.

Functional Studies of Synthetic Gramicidin Hybrid Ion Channels in CHO Cells

The function of a gramicidin hybrid ion channel in living Chinese hamster ovary (CHO) cells was investigated by the patch clamp method. The synthetic ion channel 1 consists of two cyclohexyl ether amino acids that link two minigramicidin strands. With 1 at a concentration of 1.0 μM, an increase in the whole‐cell membrane conductance was observed after 1.37 min. The conductance showed larger currents when Cs+ was used as charge carrier than when Na+ and K+ were used. In single‐channel recordings with Cs+ as charge carrier, the substance showed comparable single‐channel amplitudes in the membrane of living cells and artificial black lipid bilayers. In addition to functioning as a cation channel, compound 1 appeared to be a water channel. Exposure of the CHO cells to an extracellular hypoosmotic solution did not substantially change the cell volume. Extracellular hypoosmotic conditions in the presence of 1 increased the cell size to 146.5 % that of the control. Thus, the synthetic hybrid channel 1 can function as a cation channel with some Cs+ specificity, and as a water channel in CHO cells.

Conformation of Isobutyl, 2,2-Dibromoethyl, and 2-Methoxypropyl Side Chains on Cyclohexane and Tetrahydropyran Ring Systems

An isobutyl group placed equatorially in the 2-position of a 1-equatorially substituted cyclohexane adopts a preferred conformation (cf. 5). This also holds when it is placed in the 2-position on a 3-equatorially substituted tetrahydropyran (cf. 6). The same conformational preference is found for 2-methoxypropyl residues in the 2-position of 3-substituted tetrahydropyrans (cf. 8 and 10). The latter compounds chelate lithium cations as analogues of 1,2-dimethoxyethane. Through this complexation, it is possible to effect a change in the side chain conformation.

Synthetic efficiency and functional selectivity: two goals for synthetic ion channels

Abstract Recent progress in the synthesis of membrane-bound ion channels is reported. Important results from several groups are highlighted and contributions from the authors group are summarized. Topics covered include the synthetic approach, the importance of self-assembly systems, the functional analysis (ion selectivity, gating, asymmetric insertion and asymmetric function) and the structural characterization of the channel.