Synthesis And Ionophoric Activities Of Functionalized Bis(Choloyl)Conjugates With A Rigid Core

Ion transport across a membrane plays an essential role in the natural functioning of biological systems. Some channelopathies, for example, are a result of impaired transepithelial transport or vesicular function. Therefore, the agents that are capable of mediating ion transport, that is, ionophores can not only enhance our understanding of the biological role of natural ionophores, but also find wide applications, in particular in new drug discovery.It is known that there are many types of natural ion channels and their structures and functions are complex. Because they fuction under physiological conditions, this restricts their wide practical application. Many attentions have been paid to the identification of small synthetic compounds that are capable of mimicing the structural features and functions of natural ion channels. Primarily, the approaches to create synthetic ion transporters are essentially aimed at clarifying the structural requirements for large ion flow across a membrane, and at acquiring the skills of regulating ionophoric activities with ion selectivity.To date, a wealth of non-peptidic synthetic ion transporters has been reported based on the use of amphiphilic molecules, including cyclodextrins, crown ethers, calixarenes and others. Among these building motifs, hydroxylated steroids, in particular cholic acid, appear to be an attractive class of compounds. The flat lipophilic choloyl nucleus, by interacting with the alkyl chains of lipids, provides a rigid framework for the formation of stable pores in a membrane, whereas the inward-directed hydroxyl groups define a relatively hydrophilic pathway for large ion flux. In addition, cholic acid matches lipid monolayer in length. Thus, a bis(choloyl) conjugate that is linked by an appropriate spacer is able to span the whole lipid bilayers in its fully extended conformation. As a consequence, several pore-forming bis(hydroxylated sterol) conjugates have been reported, and found to exhibit promising ionophoric activities. But except the hydroxyl groups of the hydroxylated sterol framework and the ester/carbamate linking groups, they have no additional functional groups to interact with the ions that are to be transported. Thus, we reason that a transmembrane bis(choloyl)-based conjugate having additional functional groups in between the two end choloyl subunits may exhibit a unique ionophoric activity.In this thesis, we describe the design, synthesis and ionorphoric activities of bis(choloyl) conjugates1-3bearing a rigid linker and amino/acetamido groups that are placed in between the two choloyl subunits. Here, a small, rigid and planar phenyl group is used to connect the two choloyl units so as to enable compounds1-3to span the entire lipid bilayers. Because the functional groups are located in the middle of the molecules, they are always located within the membrane interior and expected to be capable of regulating the ion flow. To assess the effect of the additional functional groups in compounds1-3on the ionophoric activity, compound4having no additional functional groups was also synthesized.Thus, acylation of p-phenylenediamine and p-bis(aminomethyl)benzene with N-a-choloyl-N-ε-(tert-butyloxycarbonyl)-L-lysine that was activated with1-hydroxybenzotriazole (HOBT), and subsequent Boc-deprotection by TFA, afforded compounds1and2, respectively. Acylation of compound2with acetic anhydride in aqueous THF gave compound3. Compound4was prepared from the reaction of p-bis(aminomethyl)benzene with cholic acid that was activated with N-hydroxysuccinimide (NHS). The structures of compounds1-4were confirmed by 1H NMR, ESI-MS and HRMS.The ionophoric activities of compounds1-4across egg-yolk L-α-phosphatidylcholine (EYPC)-based liposomal membranes, were studied by means of fluorescence pH discharge assay. For this purpose, a pH-sensitive dye,8-hydroxypyrene-1,3,6-trisulfonate (HPTS, or pyranine, pKa7.2) was used as a fluorescence-responsive reporter of pH changes within the vesicle interior. The results indicate that the ionophoric activities of compounds1-4had a strong dependence on the mol%concentrations in the membrane. Of these compounds, compounds1,2and3bearing additional functional groups were more active than compound4without functionalized groups. In addition, we found from the concentration-dependent experiments that four molecules are needed to assemble into the transport-active species for compounds1,2and4, whereas only two molecules are needed for compound3. Interestingly, the transport activities of compounds1and2toward alkali metal ions followed the order of Na+> Li+> K+≈Rb+≈Cs+. When the amino groups of compound2were acetylated to give compound3, the transport activity followed the order of Li+> Na+> K+≈Rb+≈Cs+. Compound4showed similar ion selectivity with compound3.These results suggest that the cation selectivity of functionalized bis(choloyl) conjugates of the type described in this study may be regulated by fine tuning the structures. In addition, these results suggest that alkali metal ion transport is a dominant or rate-determining step and compounds1-4may function as Na+/H+antiporters. This was further supported by the chloride transport experiments.