Synthesis And Transmembrane Cation/Anion Symport Activities Of Functionalized Bis(Choloyl) Conjugates

Natural ion channels are proteins embedded within the cell membranes and play a crucial role in the transmembrane transport of ions across biological membranes. Defects in anion transport proteins may lead to a number of diseases known as "channelopathies". Because of this, great efforts have been made to identify small organic compounds that have promising transmembrane anion transporting activities. To date, some elegant non-peptidic synthetic compounds, including prodigiosin analogs, squaramides, calix[4]pyrroles, (thio)ureas and others, have been reported to exhibit potent anionophoric activities. Some of them also show moderate antibacterial and/or antitumor activities.In these aspects, cholic acid is an attractive building motif given its structural features. In previous work we have shown that bis(choloyl) conjugate bearing appended amino groups is capable of promoting ion transport activity across egg-yolk L,-α-phosphatidylcholine (EYPC)-based liposomal membranes, presumably via a cation/proton antiport mechanism. However, its analogue without additional groups exhibits much lower activity.With the aim to optimize the transmember anionphoric activities of bis(choloyl) conjugates as well as to clarify the effects of functional groups and/or linkers on anion transport ability, in this study we designed three types of functionalized bis(choloyl) conjugates. The first type is compounds 1 and 2 that are tethered with guanidino and additional hydroxyl groups, respectively. It is known that guanidino groups have powerful ability to form H-bonding interactions with anions. Thus compound 1 is expected to show anionphoric activity. Compound 2 is polyols and expected to be capable of having multiple hydrogen bonding interactions with both proton and anions.The second type is squaramide-linked bis(choloyl) conjugate 3. The rigid phenyl groups enable compound 3 to be capable of spanning the entire lipid bilayers in its fully extended configuration. It is reported that squaramide-based molecules are able to bind both cations and anions and that squaramide derivatives bearing trifluoromethylphenyl substituents can transport anions across lipid bilayers. Thus, compound 3 is expected to act as a potent anionophore having promising activity.Compounds 4 and 5 are rigid bis(choloyl) conjugates bearing two squaramido groups with methyl and ethyl substituents, respectively. The rigid phenyl group enables compounds 4 and 5 to span the entire lipid bilayers in their fully extended configuration. This spatial orientation would drive the squaramido groups into the membrane interior to regulate ion flow. Compound 5 is expected to be more active than compound 4, because it has higher lipophilicity that enables more portions of it to reside within the membrane to form the transport-active species.Compounds 1-5 were synthesized and fully characterized on the basis of ESI MS, HR-ESI MS,]H NMR and 13C NMR data.The anionophoric activities and ion selectivity of compounds 1-5 across liposomal membranes derived from EYPC, were studied by means of chloride ion selective electrode technique and pyranine assay. The data indicate that compounds 1-5 are capable of releasing chloride under the measuring conditions and that the rate of chloride efflux is concentration dependent.Compound 1 is capable of promoting the transport of anions, presumably via a cation/anion symport process, whereas compound 2 acts as a proton/anion symporter. Two molecules of compound 1 are assembled into the membrane to form the transport-active species, whereas only one molecule is needed for compound 2. Compounds 1-2 show moderate selectivity among monoanionic ions, wherein compound 1 has the selectivity of I-> Br-≈NO3-> Cl- and compound 2 follows the order of I-> NO3-> Br-≈Cl-. In addition, the transport activity of compound 2 toward alkali metal ions follows the sequence of Li+> Na+> K+≈Rb+> Cs+.Compound 3 acts as an anion-modulating anion-cation symporter. Extensive Hill analyses have indicated that two to four molecules of compound 3 are assembled to form the transport-active species. The transport activity of compound 3 toward monoanionic ions follows the order of F-> CV-> Br-> NO-.Compounds 4 and 5 are capable of mediating the transport of anions, presumably via a mechanism of cation/anion symport. One molecule is needed to assemble into the membrane to form the transport-active species for compounds 4 and 5. Compound 5 is more active than compound 4 under the same assay conditions. This might be attributed to the higher lipophilicity of compound 5 that enables more portions of it to reside within the membrane to form the transport-active species.The present results highlight the findings that the nature of the additional functional groups and the linkers play a crucial role in regulating the ionophoric activity and ion selectivity of a bis(choloyl) conjugate.In future, the structure-activity relationships of compounds 4 and 5 will be further clarified by systematically varying the alkyl substituents. The study is expected to provide useful guidance for the rational design of novel anionophores with high activities and ion selectivity.