Simple, highly fluorinated receptors are shown to function as highly effective transmembrane anion antiporters with the most active transporters rivalling the transport efficacy of natural anion transporter prodigiosin for bicarbonate

Gale, Philip A.

Haynes, Cally J. E.

Holder, Katie J.

Karagiannidis, Louise E.

Kirby, Isabelle L.

Moore, Stephen J.

Wells, Neil J.

English

Simple, highly fluorinated receptors are shown to function as effective transmembrane anion transporters with the most active transporters rivalling the transport efficacy of natural anion transporter prodigiosin for chloride/bicarbonate antiport.</p

Louise E. Karagiannidis

Cally J. E. Haynes

Katie J. Holder

Isabelle L. Kirby

Stephen J. Moore

Neil J. Wells

Philip A. Gale

Crossref

Chemical Communications

Highly effective yet simple transmembrane anion transporters based upon <i>ortho</i>-phenylenediamine bis-ureas

Southampton (e-Prints Soton)

This journal is©The Royal Society of Chemistry 2014 Chem. Commun.Cite this:DOI: 10.1039/c4cc05519eHighly eﬀective yet simple transmembrane aniontransporters based upon ortho-phenylenediaminebis-ureas†Louise E. Karagiannidis,a Cally J. E. Haynes,a Katie J. Holder,a Isabelle L. Kirby,aStephen J. Moore,a Neil J. Wellsa and Philip A. Gale*abSimple, highly fluorinated receptors are shown to function as highlyeﬀective transmembrane anion antiporters with the most activetransporters rivalling the transport eﬃcacy of natural anion transporterprodigiosin for bicarbonate.The transport of ions, and in particular chloride and bicarbonateanions, across cell membranes is important to numerous biologicalprocesses, and is often mediated by proteins embedded withincellular lipid bilayers.1 Malfunction of these proteins, throughgenetic mutation or otherwise, can lead to diseases such as cysticfibrosis.1 With this in mind, much focus is being placed on thedevelopment of small synthetic molecules that can mimic therole of such proteins. With the potential to bind or encapsulateions, such synthetic ‘‘carriers’’ could be developed for therapeuticapplications.2The prodigiosin family of compounds are highly potent naturalanionophores, produced by bacteria from the Streptomyces andSerratia genera.3 Prodigiosins have been shown to symport H+/Cl 4,5and function as Cl/NO3 6 and Cl/HCO3 7 antiporters, demon-strating an ability to act as antibiotic and anticancer agents, as well asexhibiting immunosuppressive behaviour.8 Prodigiosin itself, 8, isamongst the most potent anion transporters known.7Synthetic approaches to the design of anion transportersinclude the attachment of hydrogen bond donor groups to alipophilic scaﬀold, an approach to anion transporter designpioneered by Davis and co-workers, with the development ofcholic acid derived transporters and more recently systemsbased upon decalin and cyclohexane.9 Our approach has focusedon developing new hydrogen bonding arrays for transport, withsystems containing thiourea, squaramide and thiosquaramidegroups showing excellent anion transport properties.10 Recentlywe began to study the anion transport properties of bis-ureareceptors based on ortho-phenylenediamine, a system that wedeveloped as an anion receptor in 2005.11 We found that thesecompounds had excellent anion transport properties but in ourinitial study the compounds (including compounds 1 and 5)12 hadnot been optimised for transport. Here we report a more completeseries of ortho-phenylenediamine-based bis ureas functionalisedwith NO2, F, CF3 and SF513 groups that are structurally simple, canbe synthesised in one step and yet possess exceptional aniontransport properties (i.e. low EC50 values – see below) some ofwhich are more effective chloride/bicarbonate antiport agents thanthe natural product prodigiosin.7,14Wehave previously reported the synthesis and transport propertiesof receptors 1 and 5.11,12 Reaction of 1,10-carbonyl diimidazole (CDI)with 4-aminophenyl sulfur pentafluoride and the appropriatelyfunctionalised ortho-phenylenediamine in dichloromethane yieldedreceptors 2 (86% yield) and 4 (23% yield). Reaction of the appropriateisocyanate with the appropriately substituted phenylenediamine in amixture of dichloromethane and pyridine gave compounds 3, 6 and7 in 68%, 59% and 32% yields respectively.a Chemistry, University of Southampton, Southampton, SO17 1BJ, UK.E-mail: philip.gale@soton.ac.ukb Department of Chemistry, Faculty of Science, King Abdulaziz University,Jeddah 21589, Saudi Arabia† Electronic supplementary information (ESI) available: Details of receptor synthesis,characterisation, anion transport and anion binding studies as well as additional X-raydiﬀraction information. CCDC 1004419–1004421. For ESI and crystallographic data inCIF or other electronic format see DOI: 10.1039/c4cc05519eReceived 17th July 2014,Accepted 23rd August 2014DOI: 10.1039/c4cc05519ewww.rsc.org/chemcommChemCommCOMMUNICATIONPublished on 02 September 2014. Downloaded by University of Southampton on 02/09/2014 09:50:06. View Article OnlineView JournalChem. Commun. This journal is©The Royal Society of Chemistry 2014X-ray crystal structures of the chloride complexes of compounds 2and 6 were elucidated using single crystal X-ray diﬀraction techni-ques. Crystals were obtained by slow evaporation from DMSOsolutions (a mix with MeOH in the case of receptor 6) in thepresence of an excess quantity of tetrabutylammonium chloride ortetramethylammonium chloride.Receptor 2 was found to form a 2 : 2 receptor : anion complexin the solid state with tetrabutylammonium chloride (N–Cl distances3.205(4)–3.270(4) Å, N–H  Cl angles 133.2(2)1–163.9(2)1). Eachchloride anion is bound by four hydrogen bonds, two from eachof the participating receptors (Fig. 1). Receptor 6 forms a 1 : 1receptor : anion complex with chloride (N–Cl distances 3.205(2)–3.315(2) Å, N–H  Cl angles 151.5(1)1–167.1(1)1). The chlorideanion is bound by each of the four possible hydrogen bondsfrom the receptor (Fig. 2).Proton NMR anion-binding studies (Table 1) were conductedfor receptors 1–7 with chloride, bicarbonate and nitrate anions,to determine their affinity and stoichiometry of binding. Stabilityconstants, K1, were determined by following the shift of the mostdeshielded N–H proton and fitting the data to a 1 : 1 receptor :anion binding isotherm using WinEQNMR2.15Stability constants measured for the interaction between thereceptors and chloride are quite moderate (30–100 M1). Manyof the measured stability constants between the receptors andbicarbonate were very large, particularly that of receptor 3(4104), and in some cases the data could not be fitted to abinding isotherm due to peak splitting and broadening within the1H NMR spectra. This behaviour can be attributed to strongbinding interactions and possible secondary equilibrium processesin solution. In general, the receptors did not significantly interactwith nitrate anions, and the majority of the stability constants weremeasured ato10 M1.Anion transport studies were carried out for receptors 2, 3, 4, 6,7 and prodigiosin 8 using vesicle based methods,16 using the sameprocedures employed to study compounds 1 and 5 previously.2To determine chloride/nitrate exchange transport capabilities asample of unilamellar POPC vesicles were prepared containing489 mM NaCl buﬀered to pH 7.2 with 5 mM sodium phosphatesalts. The vesicles were suspended at a lipid concentration of 1 mMin 489 mM NaNO3 buﬀered to pH 7.2 with 5 mM sodiumphosphate salts. The experiment was initiated by the addition ofa small amount of a DMSO solution of the receptor (mol% withFig. 1 X-ray crystal structure of 2TBACl showing the packing arrangement.Counter ions, DMSO solvent and non-acidic hydrogen atoms have beenomitted for clarity.Fig. 2 X-ray crystal structure of 6TMACl.Table 1 EC50 (mol%) values for receptor 1–8 for the release of chloride from POPC vesicles in Cl/NO3 and Cl/HCO3 antiport systems at 270 s and390 s respectively. EC50 values are the average of three runs, unless otherwise stated, and Hill coefficient (n) values are also averaged. Stability constantK1 (M1) for receptors 1–7, measured in DMSO-d6/H2O 0.5% at 298 K. Guest anions Cl and NO3 were added as tetrabutylammonium salts, whileHCO3 was added as the tetraethylammonium salt. Stability constants were determined by 1H NMR titrations following the most deshielded N–Hresonance. All data sets were fitted to a 1 : 1 receptor : anion binding isotherm using WinEQNMR215EC50a (mol%) Cl/NO3 nb Cl/NO3 EC50c (mol%) Cl/HCO3 nd Cl/HCO3 K1, Cl i (M1) K1, HCO3 i (M1) K1, NO3 i (M1)1e 0.018 1.2 0.15 0.9 78 2090 o102 0.0099 1.1 0.077 1.0 65 g o103 0.0015 f 1.1 0.012 1.1 50 4104 o104 0.0030 1.1 0.012 1.0 71 g 135e 0.0048 0.9 0.038 1.1 78 7330 o106 0.0019 1.1 0.016 1.2 94 g o107 0.0016 f 1.1 0.011 1.3 h g o108 0.00064 1.3 0.014 1.0 Not determined Not determined Not determineda Concentration of receptor (mol% receptor to lipid) required to achieve 50% chloride eﬄux at 270 s during the Cl/NO3 exchange experiment.b Hill coeﬃcient for Cl/NO3 exchange experiment. c Concentration of receptor (mol% receptor to lipid) required to achieve 50% chloride eﬄux at390 s during the Cl/HCO3 exchange experiment. d Hill coeﬃcient for Cl/HCO3 exchange experiment. e From previously published results.12f Average of four runs. g Data could not be fitted due to resonance broadening upon addition of anions. h Data could not be adequately fitted to abinding model due to multiple equilibria in solution (see ESI). i See ESI for errors in stability constants.Communication ChemCommPublished on 02 September 2014. Downloaded by University of Southampton on 02/09/2014 09:50:06. View Article OnlineThis journal is©The Royal Society of Chemistry 2014 Chem. Commun.respect to lipid), and the resulting chloride eﬄux was monitoredusing a chloride ion selective electrode (ISE). At the end of theexperiment (300 s), the vesicles were lysed by adding a detergent,and the final electrode reading was used to calibrate 100% chloriderelease. Fig. 3 shows a comparison of the transport properties ofreceptors 1–8 (excluding 1 and 5) for chloride/bicarbonate antiportat 0.02 mol% loading of transporter.In order to determine the mechanism of chloride release bythe receptors, we performed a second experiment using POPCvesicles containing 451 mMNaCl buﬀered to pH 7.2 with 20 mMsodium phosphate salts. The vesicles were suspended in 150 mMNa2SO4 buﬀered to pH 7.2 with 20 mM sodium phosphate salts.On addition of a sample of receptor, there was little significantchloride release. This indicates that these receptors function byan anion exchange mechanism. In the first assay, the receptorswere able to facilitate Cl/NO3 exchange, but in this secondexperiment their activity was diminished due to the high hydro-philicity of the SO42 anion,17 which in most cases prevents itstransport by synthetic transporters.18 After 120 s, we added apulse of NaHCO3 solution, and found that the receptors weremediating chloride eﬄux, evidence in support of the hypothesisthat these receptors are also able to facilitate a Cl/HCO3antiport mechanism.In order to quantify these anion transport processes, weperformed a Hill analysis for chloride/nitrate and chloride/bicarbonate antiport by the receptors.19 These experimentsallowed us to calculate the EC50 for these processes, a measureof activity defined as the eﬀective receptor concentrationrequired to mediate 50% chloride eﬄux after a specified timeperiod. As is standard in our studies, this period is 270 s forchloride/nitrate experiments and 390 s for chloride/bicarbonateexperiments (corresponding to 270 s after the HCO3 spike).We were also able to determine the Hill coeﬃcient (n), whichcan be used to give an indication of the number of monomersthat form the active transport system.20 These values, and thepreviously reported data are shown in Table 1.A study of retention times of the compounds by a reversed phaseHPLC21 showed that there is not a simple relationship betweenlipophilicity and the EC50 values of this series of compounds.An increase in transport activity is observed when going frombis-urea receptor 1 to receptor 2. These results may be rationalisedby considering the eﬀect of increased fluorination,10,22 in thatreceptor 2 is expected to have higher lipophilicity than receptor 1,and hence is expected to partition into the POPC membrane moreeﬀectively.Receptors 3 and 4 are analogous to receptors 1 and 2respectively, except that the central phenyl rings are fluorinated.Unlike their simpler counterparts, receptors 3 and 4 do notfollow the same trend in activity. The SF5 containing compound4 was found to be less active than the CF3 containing compound3 for chloride/nitrate antiport. An eﬃcacious transporter muststrike a balance between aqueous solubility and lipophilicity toallow for successful delivery to the POPC vesicles and to allowpartitioning into and out of the membrane to transport anionsfrom one side of the membrane to the other. In the case ofreceptor 4, one possibility is that the balance of aqueoussolubility and lipophilicity has been pushed too far in favourof lipophilicity, and so the receptor resides more within themembrane, hence chloride/nitrate transport is slightly hinderedwhen compared to that of receptor 3. The transport eﬃciency of3 and 4 are roughly equal for chloride/bicarbonate antiport, butperhaps the most surprising thing here is that this seeminglyminor structural change results in receptors that can outperformnatural product prodigiosin, 8, at chloride/bicarbonate antiport(Table 1).To explore this idea further we synthesised receptor 6.Attaching additional electron-withdrawing fluorine substituentsto the central ring boosts the transport properties of receptor 6relative to receptor 5. In fact receptor 6 is almost 2.5 times betterat promoting chloride/nitrate and chloride/bicarbonate antiportprocesses than the parent compound. However, unlike withreceptors 3 and 4, receptor 6 does not outperform prodigiosin8 at chloride/bicarbonate antiport.The strategy of introducing higher degrees of fluorinationonto a receptor scaﬀold to enhance its transport properties isfurther illustrated by compound 7 which contains a fluorinatedcentral ring and 3,5-bis(trifluoromethyl) substituents on itsterminal phenyl rings. This compound has a lower EC50 valuefor chloride/bicarbonate exchange than prodigiosin 8 (Table 1)and is thus a more eﬀective antiporter for this anion pair thanthe natural product (Fig. 3).We have demonstrated that fluorinated receptors based on thebisurea motif are highly eﬀective anion transporters and functionby an antiport mechanism of anion transport. Modification ofexisting transporters, by adding fluorination at the central phenylring or by increasing fluorination at the peripheral phenyl groups,yielded highly potent anion transporters capable of surpassing theactivity of the natural anion transporter prodigiosin, 8, for chloride/bicarbonate exchange. This may be due to the aﬃnity of thebisureas for carboxylates11 including bicarbonate.Fig. 3 Chloride eﬄux as a function of time, promoted by the addition of0.02 mol% (with respect to lipid) of receptors 2, 3, 4, 6, 7 and prodigiosin 8,from unilamellar POPC vesicles containing 451 mM NaCl buﬀered to pH7.2 with 20 mM sodium phosphate salts. The vesicles were dispersed in150 mM Na2SO4 buﬀered to pH 7.2 with 20 mM sodium phosphate salts.The receptor was loaded as a DMSO solution at 0 s, and a spike of NaHCO3(33 mM) added as 120 s. At the end of the experiment the vesicles werelysed to calibrate the ISE to 100% chloride eﬄux.ChemComm CommunicationPublished on 02 September 2014. Downloaded by University of Southampton on 02/09/2014 09:50:06. View Article OnlineChem. Commun. This journal is©The Royal Society of Chemistry 2014We thank the EPSRC for a studentship (LEK) and a post-doctoral fellowship (CJEH) (EP/J009687/1). PAG thanks theRoyal Society and the Wolfson Foundation for a Royal SocietyUniversity Research Merit Award. We thank Julie Herniman forconducting reverse phase HPLC on these compounds.Notes and references1 N. Busschaert and P. A. Gale, Angew. Chem., Int. Ed., 2013, 52,1374–1382.2 J. T. Davis, O. Okunola and R. Quesada, Chem. Soc. Rev., 2010, 39,3843–3862; S.-K. Ko, S. K. Kim, A. Share, V. M. Lynch, J. Park,W. Namkung, W. Van Rossom, N. Busschaert, P. A. Gale, J. L. Sesslerand I. Shin, Nat. Chem., 2014, DOI: 10.1038/nchem.2021.3 S. Ohkuma, T. Sato, M. Okamoto, H. Matsuya, K. Arai, T. Kataoka,K. Nagai and H. H. Wasserman, Biochem. J., 1998, 334, 731–741.4 T. Sato, H. Konno, Y. Tanaka, T. Kataoka, K. Nagai, H. H. Wassermanand S. Ohkuma, J. Biol. Chem., 1998, 273, 21455–21462.5 J. L. Sessler, L. R. Eller, W.-S. Cho, S. Nicolaou, A. Aguilar, J. T. Lee,V. M. Lynch and D. J. Magda, Angew. Chem., Int. Ed., 2005, 44, 5989–5992.6 J. L. Seganish and J. T. Davis, Chem. Commun., 2005, 5781–5783.7 J. T. Davis, P. A. Gale, O. A. Okunola, P. Prados, J. C. Iglesias-Sa´nchez, T. Torroba and R. Quesada, Nat. Chem., 2009, 1, 138–144.8 J. T. Davis, in Topics in Heterocyclic Chemistry, ed. P. A. Gale andW. Dahaen, Springer, New York, 2010, vol. 24, pp. 145–176.9 J. A. Cooper, S. T. G. Street and A. P. Davis, Angew. Chem., Int. Ed.,2014, 53, 5609–5613; S. Hussain, P. R. Brotherhood, L. W. Judd andA. P. Davis, J. Am. Chem. Soc., 2001, 133, 1614–1617; P. R. Brotherhoodand A. P. Davis, Chem. Soc. Rev., 2010, 39, 3633–3647.10 N. Busschaert, M. Wenzel, M. E. Light, P. Igesias-Hernandez, R. Perez-Tomas and P. A. Gale, J. Am. Chem. Soc., 2011, 133, 14136–14148;N. Busschaert, I. L. Kirby, S. Young, S. J. Coles, P. N. Horton,M. E. Light and P. A. Gale, Angew. Chem., Int. Ed., 2012, 51, 4426–4430;N. Busschaert, R. B. P. Elmes, D. D. Czech, X. Wu, I. L. Kirby, E. M. Peck,K. D. Hendzel, S. K. Shaw, B. Chan, B. D. Smith, K. A. Jolliffe andP. A. Gale, Chem. Sci., 2014, 5, 3617–3626.11 S. J. Brooks, P. R. Edwards, P. A. Gale and M. E. Light, New J. Chem.,2006, 30, 65–70; S. J. Brooks, P. A. Gale and M. E. Light, Chem.Commun., 2005, 4696–4698.12 S. J. Moore, C. J. E. Haynes, J. Gonzalez, J. L. Sutton, S. J. Brooks,M. E. Light, J. Herniman, G. J. Langley, V. Soto-Cerrato, R. Perez-Tomas,I. Marques, P. J. Costa, V. Felix and P. A. Gale,Chem. Sci., 2013, 4, 103–117.13 S. Altomonte and M. Zanda, J. Fluorine Chem., 2012, 143, 57–93;P. Kirsch, Modern Fluoroorganic Chemistry Synthesis, Reactivity,Applications, Wiley-VCH, Weinheim, 2004.14 P. I. Herna´ndez, D. Moreno, A. A. Javier, T. Torroba, R. Pe´rez-Toma´sand R. Quesada, Chem. Commun., 2012, 48, 1556–1558.15 M. J. Hynes, J. Chem. Soc., Dalton Trans., 1993, 311–312.16 B. D. Smith and T. N. Lambert, Chem. Commun., 2003, 2261–2268.17 Y. Marcus, J. Chem. Soc., Faraday Trans., 1991, 87, 2995–2999.18 N. Busschaert, L. E. Karagiannidis, M. Wenzel, C. J. E. Haynes,N. J. Wells, P. G. Young, D. Makuc, J. Plavec, K. A. Jolliﬀe andP. A. Gale, Chem. Sci., 2014, 5, 1118–1127.19 A. V. Hill, Biochemistry, 1913, 7, 471–480.20 S. Bhosale and S. Matile, Chirality, 2006, 18, 849–856.21 T. Braumann, J. Chromatogr. A, 1986, 373, 191–225.22 N. Busschaert, S. J. Bradberry, M. Wenzel, C. J. E. Haynes, J. R. Hiscock,I. L. Kirby, L. E. Karagiannidis, S. J. Moore, N. J. Wells, J. Herniman,G. J. Langley, P. N. Horton, M. E. Light, I. Marques, P. J. Costa, V. Felix,J. G. Frey and P. A. Gale, Chem. Sci., 2013, 4, 3036–3045.Communication ChemCommPublished on 02 September 2014. Downloaded by University of Southampton on 02/09/2014 09:50:06. View Article Online

Highly effective yet simple transmembrane anion transporters based upon ortho-phenylenediamine bis-ureas

Highly effective yet simple transmembrane anion transporters based upon ortho-phenylenediamine bis-ureas

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