Synthetic Ion Channels from Lipophilic Guanosine Derivatives

Title of Dissertation: SYNTHETIC ION CHANNELS FROM LIPOPHILIC GUANOSINE DERIVATIVES Ling Ma, Doctor of Philosophy, 2009 Dissertation directed by: Professor Jeffery T. Davis Department of Chemistry and Biochemistry Synthetic ion channels and pores not only represent models of natural transmembrane ion channels, but also demonstrate their potential applications in the areas of drug delivery, biosensors, antimicrobial agents and other molecular devices. In this thesis, lipophilic guanosine derivatives that combine both “molecular recognition” and “membrane soluble” features are utilized for the development of the self-assembled synthetic ion channels. The potential of lipophilic G-quadruplexes to function as synthetic ion channels has been investigated by tracing the cation exchange process between free cations and Gquadruplex bound cations. Cation exchange between bulk cations (K, NH4 ) in solution and the bound cations in G-quadruplexes (G 1)16•4Na •4DNP was investigated by electrospray ionization mass spectrometry and by H , N NMR spectroscopy. The ESIMS and H NMR data showed that G-quadruplexes containing “mixed cations” formed through a sequential ion exchange process. The use of NMR-“visible” NH4 + cations in the NMR titration experiments allowed the determination of two “mixed-cation” intermediates by N-filtered H NMR and selective NOE spectroscopy. A “central insertion” pathway was proposed for the cation exchange process from (G 1)16• 4Na • 4DNP to (G 1)16• 4NH4 • 4DNP. In the lipophilic G-quadruplex, the “central” Na, bound between the 2 symmetry related G8-Na + octamers, is bound less strongly than are the 2 “outer” Na ions sandwiched within the G8-octamers. These results demonstrated the dynamic nature of lipophilic G-quadruplex in solution and directed the design of a ditopic guanosine-sterol conjugate as an approach toward making synthetic ion channels. Guanosine-sterol conjugate 3-1 was prepared by coupling 2’, 3’-bis-TBDMS, 5’amino guanosine with a bis-lithocholic acid derivative. Voltage clamp experiments demonstrated a series of stable, single ion channel conductances when compound 3-1 was incorporated into a planar phospholipid membrane. These channels are large; with nanoSiemens conductance values and they last for seconds of “open” time. This feature distinguishes them from most synthetic channels, which typically conduct in the picosiemens range with millisecond lifetimes. The structural studies using the bislithocholamide linker demonstrated that the guanosine moiety plays an essential role in the self-assembly of the transmembrane ion channels. The sizes of the most prevalent single channels calculated by Hille’s equation are much larger than the diameter of a Gquartet, which suggested that the ion transport proceeded through larger pore(s) that form upon self-assembly of lipophilic guanosine-lithocholate 3-1 within the phospholipid membrane. The large transmembrane pore(s) could be envisioned as a supramolecular structure with hydrophobic walls of bis-lithocholate linker and a central pillar of a cationfilled G-quadruplex. The use of a bis-urea functionality in the bis-lithocholic acid linker generated guanosine-sterol conjugate 4-1. The ion channel activity of 4-1 was demonstrated by voltage clamp experiment. Large ion channels formed from 4-1 had longer life-times than those formed from compound 3-1. The extra stabilization of self-assembled ion channels attributed to the bisurea hydrogen bonding is consistent with the structural hypothesis of ion channels. The stable large transmembrane ion channels self-assembled by lipophilic guanosine derivatives have potential for delivery of drugs or biomolecules. Synthetic Ion Channels From Lipophilic Guanosine Derivatives