Synthesis And Anionophoric Activities Of Dimeric Choloyl-spermine Conjugates Having A Rigid Core

Natural ion channels are large and complex proteins that span cell membranes and play an essential role in the transmembrane transport of ions across biological membranes.Abnormal or impaired channels have been implicated in causing a series of diseases that are known as "channelophaties”.Therefore,study on transmembrane ion transporting systems may not only enhance our understanding of the causes of these diseases,but also find wide applications,for example in new drug discovery.It is known that there are many types of natural ion channels and their structures and functions are complicated.Because natural ion channels function under physiological conditions,their practical applications have been largely restricted.Therefore,enormous attentions from medicinal chemists have been paid to the identification of small synthetic compounds that are capable of mimicking the structural features and functions of natural ion channels.Because of the potentials as therapeutic agents for the treatment of channelopathies and cancers,synthetic anionophores have become one of emerging topics in modern medicinal chemistry.To date,a wealth of synthetic anionophores based on discrete structures has been reported.Among them,cholic acid appears to be an attractive class of compounds.Firstly,the amphphilic framework is able to insert into phospholipid bilayers to form a relatively hydrophilic pathway in the membrane.Secondly,cholic acid can be functionalized by diverse groups that can assist ion flux across the lipid membranes.Finally,cholic acid matches lipid monolayers 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.However,most of them are cation selective and few dimeric sterol derivatives functioning as anionophores have been reported.Study on the structure-activity relationship of a variety of effective synthetic anionophores suggests that an anion-selective transporter should,in principle,have several structural features,including(1)it should have sufficient amphiphilicity;(2)the length of the transport-active species should match the thickness of phospholipid bilayers,or the carrier molecules can move freely within the membrane interior and(3)any additional functional groups,such as amide,thiourea and amino groups,are able to form hydrogen bonding,anion-π,electrostatic and/or anion-dipole interactions with the anions that are to be transported.Based on this principle,we have reported three tetrameric sterol-spermine conjugates that are capable of selectively transporting Cl-across lipoosomal membranes via an anion exchange process.Unfortunately,their anion-transporting activities are relatively low.This may be because(1)the highly flexible structures inhibited their effective insertion into the plasma membrane to form transport-active species;and(2)the linking position of polyamino groups on the choloyl subunits prevented polyamino groups from stablily distributing in phospholipid bilayers,which weakened the hydrogen-bonding and electrostatic interactions with the anions.In this thesis,we designed two types of dimeric choloyl-spermine conjugates bearing a rigid linker,with the aim at creating anionophores with potent activities and selectivity.The first type is compound 1 in which the two choloyl subunits are linked with p-bis(aminomethyl)benzene,and spermine is linked at the 3-position of the choloyl subunits.The rigid phenyl group is expected to enable compound 1 to span the entire lipid bilayers in its fully extended conformation,and the polyamino groups may promote the anion flow by forming hydrogen-bonding and/or electrostatic interactions.Compound 2 was synthesized as a control,where the choloyl-spermine subunits are linked to a flexible putrescine backbone.The other type is compound 3 that was prepared by placing the polyamino groups in between the two choloyl subunits.This spatial orientation may enable the polyamino groups to be located within the membrane interior,and thus is expected to enhance the interactions with the anions that are to be transported.Compounds 1-3 were synthesized as follows.First,activation of cholic acid methyl ester with di-(N-succinimidyl)carbonate(DSC)and subsequent reaction with spermine and protection of the remaining amino groups with Boc2O afforded Boc-protected choloyl-spermine conjugate.Mild hydrolysis of this conjugate with LiOH and acid iflcation with HCl afforded its acid form.The acid was then activated with N-hydroxylsuccinimide(NHS)and acylated with p-bis(aminomethyl)benzene and putrescine,respectively,to give Boc-protected compounds 1 and 2,respectively.Subsequent Boc-deprotection by trifluoroacetic acid(TFA)gave the desired compounds 1 and 2,respectively.For the synthesis of compound 3,reaction ofγ-benzyl-L-glutamate with cholic acid that was activated with NHS afforded cholic acid-glutamic acid conjugate.Activation of this conjugate with NHS,acylation with p-bis(aminomethyl)benzene and subsequent deprotection of the benzyl group via Pd/C catalytic hydrogenation gave bis(choloyl)conjugate having two carboxylic grotros.Acylation of tri-Boc spermine with the bis(choloyl)conjugate that was activated with NHS,followed by the deprotection of the Boc groups with TFA,gave compound 3.The structures of compounds 1-3 were confirmed on the basis of 1H-NMR,13C-NMR,ESI-MS and HRMS data.The anionophoric activities and ion selectivity of compounds 1-3 across liposomal membranes derived from egg-yolk L-α-phosphatidylcholine(EYPC),were studied by means of pyranine assay and chloride ion selective electrode technique.To determine whether compounds 1-3 are capable of mediating the transport of ions across lipid bilayers,we firstly carried out pH discharge experiments.For this purpose,a pH-sensitive dye,pyranine(pKa 7.2),was used as a fluorescence-responsive reporter of pH changes within the liposomal interior.The results indicate that compounds 1-3 are capable of inducing pH discharge across the membrane,and their ionophoric activities have strong dependence on the mol%concentrations of each compound in the membrane.Extensive Hill analyses indicate that the transport activities of co mpounds 1-3 follow the order of 1>3>2.Notably,they exhibit better ion-transporting activities than some effective synthetic anionophores reported to date.In addition,the Hill analyses also indicate that three to four molecules of compound 1 or 2 are needed to assemble into the transport-active species,whereas two to four molecules of compound 3 are needed.Chloride efflux experiments indicate that compounds 1-3 are capable of releasing chloride under the measuring conditions and that the rate of chloride efflux is found to be concentration dependent.When the chloride efflux experiments were conducted in an external Na2SO4 solution,the chloride efflux activity was significantly inhibited.This result suggests that anion transport is a dominant or rate-determining step and that compounds 1-3 mediate anions,most probably via a mechanism of anion-anion exchange.More interestingly,compounds 1-3 exhibit moderate selectivity among monoanionic ions,wherein compound 1 has the selectivity of NO3->Cl->Br-≈I-;compound 2 follows the order of I->Br-≈NO3->Cl-and compound 3 follws the sequence of I->>NO3->Br-≈ In addition,compound 3 shows no selectivity among alkali metal ions.These results further suggest that the transport process was dependent on the anions and compounds 1-3 function as anion-anion antiporters.The present results suggest that a bis(choloyl)conjugate having a rigid linker is able to span the entire lipid bilayers,and that a dimeric choloyl-spermine conjugate having appropriate lipophilicity may form the transport-active species more readily to improve the anionophoric activity.In addition,the anion selectivity of choloyl-spermine conjugates may be regulated by fine-tuning the linker and the linking position of polyamino groups on the cholooyl subunits.In future,the structure-activity relationships of bis(choloyl)conjugates of the type described in this study will be further clarified by altering the number of amino groups.The finding is expected to provide uesful guidance for the rational design of novel anionophores with high activities and ion selectivity.