USING "SMALL" MOLECULES AS TRANSMEMBRANE ANION TRANSPORTERS
Abstract
Functional, "small" molecule anion transporters have been identified and developed from natural products and synthetic organic compounds. The major discoveries include the design of a transmembrane Cl<super>-</super> transporter whose activity is pH-tunable, a NO<sub>3</sub><super>-</super> transporter that displays unique selectivity for NO<sub>3</sub><super>-</super> over Cl<super>-</super> anions, and a series of small molecules that efficiently transport HCO<sub>3</sub><super>-</super> across liposomal membranes via a HCO<sub>3</sub><super>-</super>/Cl<super>-</super> exchange mechanism. An assay for detecting transmembrane HCO<sub>3</sub><super>-</super> transport using paramagnetic Mn<super>2+</super> and <super>13</super>C NMR is also described.
Modulated Cl<super>-</super> transport was achieved by lipophilic calix[4]arene amides <bold>2.2-2.4</bold>, all in the cone conformation. Modulation was achieved through functional group modification to one of the four side-chains. The cone conformation was confirmed by both <super>1</super>H NMR and X-ray crystallography. Significantly, Cl<super>-</super> transport was gated by pH in the presence of triamido calixarene TAC-OH <bold>2.3</bold>, which possesses a phenolic hydroxyl group. Using fluorescence assays, the rate of Cl<super>-</super> transport by TAC-OH <bold>2.3</bold> across liposomal membranes decreased with increasing pH, while transport rate by cone-H <bold>2.2a</bold>, lacking an OH group, was not affected by pH.
Nitrate was selectively transported over Cl<super>-</super> in the presence of nitro tripod <bold>3.1</bold>, a small molecule receptor for both anions. The selective transport of NO<sub>3</sub><super>-</super> by <bold>3.1</bold> is a significant discovery as most known synthetic Cl<super>-</super> transporters also transport NO<sub>3</sub><super>-</super> ions and vice versa. Nitrate transport across liposomal membranes was confirmed by enzyme-coupled and fluorescence assays. Tripod <bold>3.1</bold> induced an increase in the intravesicular pH of liposomes that were not experiencing a pH gradient, while no pH changes occurred in the presence of calixarene <bold>2.1</bold> a known Cl<super>-</super> and NO<sub>3</sub><super>-</super> transporter. This result suggests that <bold>3.1</bold> is an H<super>+</super>/NO<sub>3</sub><super>-</super> symporter.
Transmembrane HCO<sub>3</sub><super>-</super> transport was achieved using the natural product, prodigiosin, <bold>4.1</bold>, and synthetic isophthalamides <bold>4.2-4.4</bold>. The Cl<super>-</super>/HCO<sub>3</sub><super>-</super> exchange mechanism by which compounds <bold>4.1-4.4</bold> transport HCO<sub>3</sub><super>-</super> was elucidated by ISE and NMR assays. The <super>13</super>C NMR assay provided direct evidence for HCO<sub>3</sub><super>-</super> transport in the presence of paramagnetic Mn<super>2+</super> ions, and was adaptable to various assay conditions.