Thesis
Reference
Anion transport with anion-π interactions and halogen bonds
VARGAS JENTZSCH, Rodrigo Andreas
Abstract
Anion-π interactions and halogen bonds are applied to anion transport through lipids bilayer membranes. Calix[4]arenes and calix[4]pyrroles scaffolds, and minimalist π-acidic naphthalenediimides were explored for anion transport. Calix[4]arenes and octiphenyl rigid-rods scaffolds, and minimalistic perfluorinated halogen-bond donors were applied to anion transport with halogen bonds. The different systems were tested in large unilamelar vesicles to explore their ability to promote anion translocation. Anion-π interaction had been reported before for anion transport, here we extend its scope in anion-π moieties. In addition, by considerably varying the systems used, their general applicability to anion transport was assessed. Halogen bonds are introduced here to the field of anion transport; anion transporters based on this non-covalent interaction can be very simple due to their intrinsic lipophilicity as exemplified by the smallest organic anion-transport system:
trifluoroiodomethane. These results are confirmed by planar membrane conductance experiments.
VARGAS JENTZSCH, Rodrigo Andreas. Anion transport with anion-π interactions and halogen bonds . Thèse de doctorat : Univ. Genève, 2013, no. Sc. 4557
URN : urn:nbn:ch:unige-285134
DOI : 10.13097/archive-ouverte/unige:28513
Available at:
http://archive-ouverte.unige.ch/unige:28513
Disclaimer: layout of this document may differ from the published version.
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UNIVERSIT´E DE GEN`EVE Section de chimie et biochimie D´epartement de chimie organique
FACULT´E DES SCIENCES Professeur Stefan Matile
Anion Transport with Anion-π Interactions and Halogen Bonds
TH`ESE
pr´esent´ee `a la Facult´e des sciences de l’Universit´e de Gen`eve pour obtenir le grade de Docteur `es sciences, mention chimie
par
Rodrigo Andreas VARGAS JENTZSCH de
Cochabamba (Bolivie)
Th`ese N◦4557 GEN`EVE Atelier ReproMail
2013
To my parents and my brother and sister
You see, one thing is, I can live with doubt and uncertainty and not knowing. I think it’s much more interesting to live not knowing than to have answers which might be wrong. I have approximate answers and possible beliefs and different de- grees of certainty about different things but I’m not absolutely sure of anything and then many things I don’t know anything about, such as whether it means anything to ask, “Why we are here?” and what that question might mean. I might think about it a bit and then if I can’t figure it out then I go on to something else. But I don’t have to know an answer, I don’t have to. . . I don’t feel frightened by not knowing things, by being lost in the mysterious universe without having any purpose which is the way it really is as far as I can tell possibly. It doesn’t frighten me.
Richard Feynman
Ce travail de th`ese n’aurait pas ´et´e possible sans le soutient de nombreuses personnes :
Mes remerciements vont tr`es particuli`erement au Prof. Stefan Matile et au Dr. Naomi Sakai pour m’avoir accueilli dans leur groupe et surtout pour tout ce qu’ils m’ont appris au cours de ces ann´ees. Leur enthousiasme et leur d´evouement pour la chimie sont exemplaires.
Je suis tr`es reconnaissant au Prof. Christoph A. Schalley et au Dr. Jiri Mareda d’avoir accept´e de juger cette th`ese ainsi qu’au Dr. Adam Wilson,
`
a Ingeborg Jentzsch et `a Pierre Charbonnaz pour leurs contributions `a la correction de celle-ci.
Ce travail a ´et´e effectu´e au D´epartement de Chimie Organique de l’Univer- sit´e de Gen`eve, dont tous les membres ont fait de mon s´ejour un exp´erience tr`es plaisant. Je remercie aussi les ´equipes de RMN et spectroscopie de masse pour leur contribution `a cette th`ese.
Les membres du groupe Matile sont chaleureusement remerci´es pour les innombrables heures de discussions ainsi que les excellents moments pass´es en- sembles ; la richesse culturelle et scientifique partag´ee avec eux est ´eblouissante.
Il y a tellement d’autres personnes que j’aimerais remercier, je pense tr`es particuli`erement `a ma famille et `a mes amis. H´elas, je ne peux vraiment expri- mer l’ampleur de vos contributions, qui ont ´et´e essentielles ni combien vous ˆ
etre importants. Je veux croire que les personnes concern´ees sauront lire dans mes pens´ees mieux que dans mes mots la dette de gratitude que j’ai envers elles. Ceci est d’autant plus vrai qu’une bonne partie d’entre eux ne compren- dront jamais ce lignes. Je tiens juste `a leur dire, `a vous dire `a tous : MERCI !
List of Publications
• Montenegro, J.; Hennig, A.; Geotti-Bianchini, P.; Eggimann, G. A.; Jeannerat, D.;
Matile, S.; Misek, J.; Schuster, T.; Uhlich, N. A.; Vargas Jentzsch, A. Functional Biosupramolecular Systems.Chimia2009,63, 881-884.
• Misek, J.; Vargas Jentzsch, A.; Sakurai, S.-I.; Emery, D.; Mareda, J.; Matile, S. A Chiral and Colorful Redox Switch: Enhancedπ-Acidity in Action. Angew. Chem.
Int. Ed.2010,49, 7680-7683.
• Vargas Jentzsch, A.; Emery, D.; Mareda, J.; Metrangolo, P.; Resnati, G.; Matile, S.
Ditopic Ion Transport Systems: Anion-πInteractions and Halogen Bonds at Work.
Angew. Chem. Int. Ed.2011,50, 11675-11678.
• Matile, S.; Vargas Jentzsch, A.; Montenegro, J.; Fin, A. Recent Synthetic Transport Systems. Chem. Soc. Rev. 2011,40, 2453-2474.
• Alonso Doval, D.; Areephong, J.; Bang, E.-K.; Bertone, L.; Charbonnaz, P.; Fin, A.;
Lin, N.-T.; Lista, M.; Matile, S.; Montenegro, J.; Orentas, E.; Sakai, N.; Tran, D.- H.; Vargas Jentzsch, A. Recent Progress with Functional Biosupramolecular Systems.
Langmuir2011,27, 9696-9705.
• Lin, N.-T.; Vargas Jentzsch, A.; Guenee, L.; Neudorfl, J.-M.; Aziz, S.; Berkessel, A.;
Orentas, E.; Sakai, N.; Matile, S. Enantioselective Self-Sorting on Planar,π-Acidic Surfaces of Chiral Anion-πTransporters.Chem. Sci. 2012,3, 1121-1127.
• Vargas Jentzsch, A.; Emery, D.; Mareda, J.; Nayak, S. K.; Metrangolo, P.; Resnati, G.; Sakai, N.; Matile, S. Transmembrane Anion Transport Mediated by Halogen- Bond Donors.Nat. Commun. 2012,3, 905.
• Fin, A.; Vargas Jentzsch, A.; Sakai, N.; Matile, S. Oligothiophene Amphiphiles as Planarizable and Polarizable Fluorescent Membrane Probes.Angew. Chem. Int. Ed.
2012,51, 12736-12739.
• Vargas Jentzsch, A.; Matile, S. Transmembrane Halogen-Bonding Cascades.J. Am.
Chem. Soc.2013,135, 5302-5303.
• Vargas Jentzsch, A.; Hennig, A.; Mareda, J.; Matile, S. Synthetic Ion Transporters that Work with Anion-πInteractions, Halogen Bonds and Anion-Macrodipole Inter- actions.Acc. Chem. Res.2013, in press.
• Adriaenssens, L.; Estarellas, C.; Vargas Jentzsch, A.; Martinez Belmonte, M.; Matile, S.; Ballester, P. Quantification of Nitrate-πInteractions and Selective Transport of Nitrate Using Calix[4]pyrroles with Two Aromatic Walls. J. Am. Chem. Soc.2013, in press.
Among the many processes essential for life, ion transport represents a supra- molecular function that is omnipresent from the simplest to the most complex forms of life populating Earth. This is the case because, on one hand, it is necessary to separate the cellular life from the rest of the world, a problem which was solved by nature early on with the introduction of the lipid bilayer membrane.
This 40 ˚A thick barrier is permeable to most of the small apolar molecules while the diffusion of polar solutes and macromolecules is prevented; this is essential for life as most molecules are required only for specific functions at specific places. On the other hand, complete isolation is not an option, because communication with the environment is required for life; the presence of several transporting units with specific functions is only logical. Ion transport systems represent a major part of this family, and they can be very simple, as occurs with small molecules fulfilling this task, or extremely complex as for the case of proteins.
Not surprisingly, scientists have paid particular attention to this field for several years and the design of synthetic transport systems, for curiosity and medical purposes, is a common practice. Any innovation in such a field is difficult and challenging. In this work, it is not the design of ion transport systems that will be addressed but the non-covalent interactions employed to achieve it.
Anion-π interactions and halogen bonding are two non-covalent interac- tions that have only scarcely beenused in supramolecular chemistry; this work demonstrates that their application to an old yet fundamental problem like anion transport is not only possible, but can be favoured in some cases.
The first part of the thesis is dedicated to the study of anion-πinteractions as applied to anion transport. To do this, several systems are employed:
naphthalenediimides (NDIs), calix[4]pyrroles and calix[4]arenes. To explore
Summary iv
the ability of the different systems to transport anions through the bilayer membrane, our main characterization method is the HPTS assay which is an experiment that employs fluorogenic vesicles to assess directly the ability of molecules or supramolecular assemblies to transport ions in this cellular membrane model system.
The first example is anion transport with naphthalenediimides. NDIs have already been reported to be able to promote anion transport in model mem- branes; in this work the use of differently decorated NDIs is explored. After demonstrating the high degree of flexibility that the naphthalenediimide scaf- fold can have when applied to anion transport, a more mechanism-directed study is undertaken.
This is accomplished by investigating the transport properties of structurally similar NDIs with specific self-assembly properties. It is only possible because anion transport is mainly dependent on the π-acidic NDI core. We demon- strate that NDIs promote anion transport by the spontaneous formation of a supramolecular assembly and that its stability can be correlated with the ability of the monomers to self-assemble into dimers. This is supported by the finding that the structure responsible for anion transport is no less than an octameric assembly.
The calix[4]pyrrole scaffold is explored next. Calixpyrroles are functional and clean anion transport systems; for this study, they were modified to have π-acidic or π-basic walls The presence of these walls, made up of substituted phenyl units, has been shown to have dramatic effects on the anion binding properties of the receptors. An excellent agreement between theπ-acidity and the observed transport activity in vesicles demonstrates that anion-π interac- tions can be used with the calixpyrrole scaffold to promote anion transport.
An important nitrate selectivity is observed and suggests that π-surfaces can interact better with flat π anionic systems like nitrate and lead to better transport.
The final system used to investigate the use of anion-πinteractions for anion transport is the calix[4]arene scaffold. In this case, classic π-acidic moieties were attached to the lower rim of a simple calix[4]arene. The resultingπ-acidic calix has been proved to be able to promote anion transport in the HPTS
assay but only in the presence of tetramethylammonium salts. Nevertheless, this ditopic system represents yet another example of anion transport with anion-π interactions. These findings are supported with numerous control experiments as well as by computational models.
The second part of the thesis is dedicated to the use of halogen bonding as applied to anion transport. In this case, a more systematic approach is used: first we explore anion transport with the calix[4]arene scaffold (a cyclic array), then we explore the properties of small halogen-bond donors as anion transporters; and lastly we optimize anion transport with halogen bonds by using the octiphenyl rigid-rod scaffold (a linear array) to access good levels of anion transport.
The calix[4]arenes applied to anion transport with halogen bonds are inves- tigated under the same conditions as the anion-πinteractions. This situation can be pictured as a cyclic array of binding units. For a first approach, per- fluorinated halogen-bond donor units are employed and the binding properties of these molecules are assessed by NMR techniques. Although binding takes place, anion transport is poor. The key to improving anion transport is the destabilization of the binding which is achieved with either weaker binders or less favorable conformational constraints.
The second approach consisted of the use of small halogen-bond donors in a conformationally free system. Indeed, small perfluorinated halogen-bond donors can self-assemble to form a protective shell in the presence of an anion to, thereafter, assist the anion in crossing the lipid bilayer membrane. The uncommon nature of this supramolecular transport system required a more careful investigation that was accomplished, mainly, with planar bilayer con- ductance experiments. The results from the latter give evidence of a clean, voltage-dependent and highly anion-selective transport system.
In fact, the use of small halogen-bond donors is not general and some of the constraints like the perfluorinated chain length are explored showing that an ideal length exists. A highlight of this system is the application of trifluoroiodomethane that, despite being a gas at room temperature, can pro- mote anion transport in this membrane model system; this is, by definition,
Summary vi
the smallest organic anion transport system possible, as it has only one car- bon. Anion transport at this level of reductionism is only possible due to the lipophilic nature of these halogen-dond donors. These findings are supported by computational models and solid state crystal structures.
The final example treated in this work concerns the optimization of anion transport with halogen bonds. To achieve a system with a good performance, we use the octiphenyl rigid-rod scaffold. This scaffold has been used very suc- cessfully before with more traditional non-covalent interactions (e.g. hydrogen bonding) and represents an excellent alternative as it represents a linear array of halogen-bond donors, a model that had not yet been explored. One inter- esting property of octiphenyl rigid rods is the parallel orientation they assume in the membrane, as a matter of fact, with eight phenyl units, these rigid rods can span the membrane.
We decorated oligophenyl rigid rods with increasing lengths of up to eight units with strong halogen-bond donors, in this case, perfluorinated iodophenyl units. The observed anion transport properties of this halogen-bonding cas- cade were clearly better than in all the previous cases, showing a stunning three orders of magnitude improvement as compared with the monomeric case. A strong cooperativity is observed as the transport activity increases exponen- tially with the number of units suggesting a hopping mechanism for the anion transport.
With these three different cases, this work represents the first use of halogen- bonding in synthetic anion transport systems.
De tous les m´ecanismes qui sont essentiels `a la vie, le transport d’ions repr´e- sente la fonction supramol´eculaire qui est toujours pr´esente, ceci des formes de vie les plus primitives jusqu’aux plus ´evolu´ees qui peuvent ˆetre retrouv´ees sur terre. Deux raisons `a cela : Premi`erement, la vie cellulaire doit ˆetre s´epar´e du monde ext´erieur, ce qui a ´et´e accompli tr`es tˆot dans l’´evolution par la nature en introduisant de la membrane cellulaire lipidique. Cette barri`ere de 40 ˚A d’´epaisseur est perm´eable aux petites mol´ecules apolaires tout en res- tant imperm´eable aux macromol´ecules et, surtout, aux mol´ecules charg´ees et polaires. Ce point est critique puisque chaque mol´ecule n’est n´ecessaire qu’`a certains endroits sp´ecifiques dans la cellule.
Deuxi`ement, une compl`ete isolation n’est pas envisageable. En cons´equence, l’existence de plusieurs unit´es de transport sp´ecifiques est logique et parmi elles, bon nombre sont des transporteurs d’ions. Ils peuvent ˆetre tr`es simples et des exemple de petites mol´ecules remplissant ce rˆole existent, ou bien tr`es complexes, comme des prot´eines.
Il n’est pas ´etonnant que les scientifiques aient prˆet´e beaucoup d’int´erˆet aux transporteurs membranaires depuis plusieurs ann´ees. Des nos jours, la conception de syst`emes de transport membranaires est une pratique courante.
L’innovation est d`es lors tr`es difficile. Pour ce travail ce n’est pas la concep- tion de syst`emes de transport membranaires qui sera abord´ee mais plutˆot les moyens pour y arriver.
Les interactions anion-π et les liaisons halog`ene sont deux liaisons non- covalentes qui n’ont ´et´e que tr`es peu utilis´ees en chimie supramol´eculaire. Le pr´esent travail d´emontre qu’elles peuvent non seulement ˆetre appliqu´ees `a ce domaine de recherche mais qu’elles offrent des avantages dans certains cas.
La premi`ere partie de cette th`ese concerne l’application des interactions anion-πau transport d’anions. Pour ce faire, trois syst`emes de transport mem- branaire sont ´etudi´es : les naphtal`enediimides (NDIs), les calix[4]pyrroles et les calix[4]ar`enes. A fin d’´etudier les propri´et´es de nos syst`emes en tant que trans-
R´esum´e viii
porteurs membranaires d’ions, la principale m´ethode de caract´erisation utilis´ee se base sur des v´esicules charg´ees d’un chromophore sensitive au pH, le HPTS, qui permet de quantifier le transport d’ions par la mol´ecule ou supramol´ecule concern´ee.
Le premier exemple concerne les naphtal`enediimides, d´ej`a d´ecrits dans la litt´erature. Ici on ´etudie des NDIs diff´erents et on d´emontre qu’une grande diversit´e est possible parce que seule la surface π-acide est responsable du transport des anions.
Un ´etude ciblant le m´ecanisme du transport d’anions est faite par la suite.
Ceci a ´et´e possible grˆace `a l’utilisation des NDIs structurellement similaires mais avec des propri´et´es d’auto-assemblage diff´erentes. Il en sort que le trans- port d’anions avec des naphtal`enediimides est inversement corr´el´e avec la ten- dance des NDIs de s’assembler en dim`eres. Ceci est confirm´e par le fait que le transport d’anions se fait avec des assemblage d’au moins huit mol´ecules.
Les calix[4]pyrroles, qui sont des transporteurs membranaires connus et se comportent tr`es bien dans des mod`eles de membranes ont ´et´e ´etudi´es ensuite.
A cette fin, ils ont ´et´e substitu´es avec des “murs”π-acides ouπ-basiques, en fait, des ph´enyle substitu´es. Une tr`es bonne corr´elation a ´et´e trouv´ee entre l’ac- tivit´e de transport et l’acidit´e du syst`eme aromatique. En plus, une s´electivit´e important pour les anions nitrate a ´et´e observ´ee ce qui sugg`ere que des inter- actions du typeπ-πpeuvent avoir une influence favorable.
Le dernier syst`eme `a ˆetre investigu´e sous l’angle des interactions anion-πest celui des calix[4]ar`enes. Pour ce faire, ces mol´ecules ont ´et´e substitu´ees avec des unit´esπ-acides classiques : des ph´enyles perfluorin´es. Ce “calices”π-acides sont capables de transporter des anions `a travers la membrane lipidique, et, bien qu’ils ne le fassent qu’en pr´esence de sels de tetrabutyleammonium, c’est encore un exemple o`u des interactions du type anion-π sont appliqu´ees au transport des anions dans des membranes lipidiques. Tous ces ces r´esultants exp´erimentaux sont ´etay´es par des ´etudes computationelles.
La deuxi`eme partie de la th`ese concerne les liaison halog`enes. Dans ce cas, l’´etude a ´et´e plus syst´ematique. Dans un premier temps le transport par un ar- rangement cyclique de donneurs de liaison halog`ene est ´etudi´e en se basant sur
des calixar`enes. Ensuite, des donneurs de liaison halog`ene sans pr´eorganisation sont utilis´es. Enfin, un transporteur membranaire plus performant est obtenu avec un arrangement linaire de donneurs de liaison halog`ene.
Dans le premier cas, on a utilis´e le mˆeme syst`eme qu’avec les interactions du type anion-π bas´e sur les calix[4]ar`enes. Ici, il peut ˆetre assimil´e a un ar- rangement cyclique de donneurs de liaison halog`enes. D’abord, des donneurs plutˆot forts on ´et´e utilis´es, des substituants iodoperfluoroph´enyles. Malheu- reusement ce syst`eme n’a donn´e qu’un transport d’anions faible. La cl´e pour avoir un transporteur membranaire plus performant est d’affaiblir la liaison halog`ene soit en employant des donneurs de liaison halog`ene plus faibles, soit en imposant une g´eom´etrie moins ad´equate.
Le deuxi`eme approche impliqu´e un syst`eme moins restreint avec des petits, mais forts, donneurs de liaison halog`enes (des iodoperfluoroalcanes et ar`enes).
Ces donneurs de liaisons halog`ene peuvent s’auto-assembler en pr´esence d’un anion pour former une couche protectrice. Par la suite, l’anion peut ˆetre trans- port´e `a travers la membrane lipidique. Vu la nouveaut´e de ce syst`eme, des
´
etudes plus pouss´ees ont ´et´e requises. Il a `a d`es lors ´et´e caract´eris´e par des exp´eriences de conductance sur des membrane planes. Il en ressort que ce syst`eme est exceptionnellement propre, d´epend du voltage appliqu´e et est tr`es s´electif pour les anions.
On ne peut cependant g´en´eraliser ce cas, tout donneur de liaison halog`ene ne peut pas transporter des anions `a travers la membrane lipidique comme le montrent les pertes de performance associ´ees `a l’allongement de la chaine al- cane perfluorin´ee. Il est int´eressant de mentionner que le trifluoroiodomethane lui aussi est capable de transporter des anions, ceci malgr´e son ´etat gazeux `a temp´erature ambiante. Il s’agit en fait du plus petit transporteur membranaire organique qui soit, avec un seul carbone. L`a encore, ces r´esultas sont ´etay´es par des structures cristallines et des ´etudes computationelles.
Le dernier exemple concerne l’optimisation du transport d’anions grˆace `a des liaison halog`enes. Pour ceci on utilise desp-octiph´enyles, ou “baguettes rigides”, ces baguettes rigides ont la propri´et´e d’adopter une position parall`ele au chaines lipidiques dans la membrane et, avec huit unit´es ph´enyls, elles sont assez longues pour la traverser.
R´esum´e x
Desp-oligo-ph´enyles de longueurs allant de deux `a huit unit´es ont ´et´e sub- stitu´es par des donneurs de liaison halog`ene, ici des iodoperfluoroph´enyles.
Ceci donne un arrangement linaire de donneurs de liaison halog`ene qui tra- verse la membrane de part en part. Un transport d’anions clairement sup´erieur aux cas pr´ec´edents `a ´et´e observ´e, jusqu’au mille fois plus performant que le monom`ere correspondant. Une tr`es forte coop´erativit´e a ´et´e observ´ee ce car l’activit´e augmente de fa¸con exponentielle avec le nombre d’unit´es. De plus, ceci sugg`ere qu’un m´echanisme de petits sauts est probable.
Illustr´e par les trois cas, qui viennent d’ˆetre ´evoqu´es, ce travail de th`ese repr´esente le premier example de syst`emes synth´etiques de transport membra- naires utilisant des liaisons halog`ene.
Remerciements i
List of Publications ii
Summary iii
R´esum´e vii
Contents xi
1 Introduction 1
1.1 General . . . 1
1.2 Ion Transport Systems . . . 1
1.2.1 Classification . . . 2
1.2.2 Examples from Nature . . . 3
1.2.3 Synthetic Ion Transport Systems . . . 8
1.3 How to Evaluate Ion Transport . . . 9
1.3.1 Fluorogenic Vesicles: The HPTS Assay . . . 9
Ion Selectivity Topologies . . . 11
Further Possibilities . . . 13
1.3.2 Fluorogenic Vesicles: The Lucigenin Assay . . . 13
1.3.3 Experiments with Vesicles: Ion-Selective Electrodes . . 14
1.3.4 Experiments with Vesicles: NMR . . . 15
1.3.5 Hill Coefficient . . . 15
1.3.6 Planar Membrane Conductance Experiments . . . 17
1.3.7 Examples of Synthetic Transport Systems . . . 18
1.4 Anion-πInteractions . . . 27
1.4.1 Generalities . . . 27
1.4.2 Characteristics of Anion-πInteractions . . . 29
1.4.3 Typical Molecules withπ-Acidic Surfaces . . . 30
1.4.4 In Solution Quantification of Anion-πInteractions . . . 35
Contents xii
1.5 Halogen Bonds . . . 36
1.5.1 Definition . . . 37
1.5.2 Characteristics . . . 38
1.5.3 Theσ-Hole . . . 38
1.5.4 About Halogen Bonding Strength . . . 40
1.5.5 Geometry of Halogen Bonds . . . 40
1.5.6 Halogen Bonding in Crystal Engineering . . . 41
1.5.7 Halogen Bonding in Anion Binding . . . 41
1.5.8 Halogen Bonding in Catalysis . . . 45
1.5.9 Halogen Bonding in Medicinal Chemistry . . . 47
1.5.10 Anion-πInteraction Enhanced by Halogen Bonding . . 48
2 Objectives 50 3 Results and Discussion 52 3.1 Anion Transport with Anion-πInteractions . . . 52
3.1.1 General Considerations and Design . . . 52
3.1.2 Sulfur-Containing NDIs: The Role ofπ-Acidity . . . . 52
Diisopropylphenyl Sulfur-Containing NDIs . . . 54
Trimethylphenyl Sulfur-Containing NDIs . . . 55
Tetrasubstituted Trimethylphenyl Sulfur-Containing NDIs 57 Ion Selectivity . . . 57
The Lithium Anomaly . . . 60
3.1.3 Sulfur-Containing NDIs: The Role of Self-Sorting . . . 64
tert-Butylphenyl Sulfur Containing NDIs . . . 65
Self-Sorting Properties . . . 65
Anion Transport in Fluorogenic Vesicles . . . 67
3.1.4 Computational Studies . . . 69
3.1.5 Conclusions About Anion Transport withπ-Acidic NDIs 70 3.1.6 Calix[4]pyrroles: The Role ofπ-Acidity . . . 71
General Considerations and Design . . . 71
Anion Transport in Fluorogenic Vesicles . . . 74
Ion Selectivity: Nitrate Selectivity with Anion-π Inter- actions . . . 77
Anion Transport Mediated by Calixpyrroles withπ-Acidic
Walls . . . 79
3.1.7 Calix[4]arenes . . . 79
Synthesis . . . 80
Anion Transport in Fluorogenic Vesicles . . . 80
Anion and Cation Selectivities . . . 82
3.1.8 Anion Transport with Anion-πInteractions: Conclusions 83 3.2 Anion Transport with Halogen Bonds . . . 84
3.2.1 Calixarenes: General Considerations and Design . . . . 85
Synthesis . . . 85
Ion Selectivity . . . 86
Anion Transport in Fluorogenic Vesicles . . . 88
NMR Titrations: Dissociation Constants . . . 90
Anion Binding vs Anion Transport . . . 94
Computational Models . . . 95
Conclusions About Anion Transport with Anion-π In- teractions and Halogen Bonds . . . 95
3.2.2 Anion Transport with Small Halogen-Bond Donors . . 97
The Arene Series . . . 98
The Alkane Series . . . 100
Planar Bilayer Conductance Experiments . . . 101
The Effect of Membrane Charge . . . 105
The Effect of Membrane Polarization . . . 106
The Effect of the Membrane Fluidity . . . 108
The Role of the Counterion . . . 112
Ion Selectivity . . . 114
Smallest Possible Organic Anion Transport System . . 115
Discussion and Proposed Anion Transport Mechanism . 117 Crystal Structures and Computational Models . . . 120
Small Halogen-Bond Donors as Anion Transport Sys- tems: Conclusions . . . 121
3.2.3 Anion Transport with Halogen-Bonding Cascades . . . 122
General Considerations and Design . . . 122
Contents xiv
Synthesis . . . 124
Anion Transport and Cooperative Effects . . . 124
Ion Selectivity . . . 128
Halogen-Bonding Cascades: Conclusions . . . 129
3.2.4 Non-Specific Leakage: CF assay . . . 129
3.2.5 Anion Transport with Halogen Bonds: Conclusions . . 130
4 Perspectives 133 4.1 General - Anion Transport . . . 133
4.1.1 Optimization . . . 133
4.1.2 Innovative Design . . . 135
4.1.3 About Hill Coefficients . . . 136
4.2 Halogen Bond in Medicinal Chemistry . . . 136
4.3 Halogen Bond Fundamental Understanding . . . 137
4.3.1 The Effect of Perfluorination . . . 137
4.3.2 Trivalent Binding in Solution: The Negative Ring . . . 138
4.4 Bottom-Line . . . 138
5 Experimental Section 139 5.1 General . . . 139
5.1.1 Reagents, Solvents and Equipment . . . 139
5.1.2 Equipment for Characterization and Purification . . . . 139
5.1.3 Equipment for Experiments . . . 140
5.2 Synthesis . . . 141
5.2.1 Substituted Benzyl Units . . . 142
5.2.2 Calix[4]arenes . . . 144
5.2.3 Synthesis of Bi, p-Quarter, p-Sexi and p-Octiphenyl Cascades . . . 156
5.3 Methods . . . 167
5.3.1 NMR Titrations . . . 167
5.4 Studies in Lipid Bilayer Membranes . . . 171
5.4.1 Vesicles Preparation . . . 171
5.4.2 Transport Experiments in Fluorogenic Vesicles . . . 174
Determination of Transport Activity with the HPTS
Assay . . . 174
Determination of the Temperature Dependent Trans- port Activity with the HPTS Assay . . . 177
Determination of Transport Activity with the HPTS Assay Doped with valinomycin . . . 179
Membrane Potential: Calibration . . . 179
Determination of Transport Activity in Polarized Vesi- cles with the HPTS Assay . . . 180
Determination of Non-Specific Leakage with the CF Assay . . . 181
Ion Selectivity . . . 181
5.4.3 Planar Bilayer Membranes (BLM’s) . . . 182
5.5 Abbreviations . . . 184
6 Bibliography 191
If I have seen further it is by standing on the shoulder of giants.
Isaac Newton
1 Introduction
1.1 General
Nature: this was, is, and will be, forevermore, the main source of inspiration in chemical research. Working at incredible levels of precision, exploiting structures that remain, most of the time, unclear, nature does a great job.
Ironically, more often than not, it is when nature fails to deliver that research flourishes.
For example, several diseases are known to be caused by the dysfunction of ion channels or “channelopathies” like muscle disorder myotonia, bone disease osteopetrosis, Dent’s disease, Bartter’s syndrome and many more; the best known of this family is cystic fibrosis, which is caused by the dysfunction of the transmembrane regulator anion channel.1
Either motivated by this need or by curiosity, research on ion transport systems started more than 50 years ago2and is now an active field of research as indicated by awarding of the Nobel prize for this topic in 2003.3
1.2 Ion Transport Systems
The lipid bilayer membrane is essential for life, not only it is responsible for separating regions and therefore allowing multiple processes to take place, but it is also where the chemical energy is used/produced.4 In fact, there is little chance that life, even in its simplest form, could exist without a lipid membrane. On the other hand, lipid bilayer membranes are extremely good at their function and decrease ion permeability by several orders of magnitude, as proven more than 40 year ago.5 Even so, the extent to which an ion can diffuse in a lipid bilayer membrane would not be measured for decades, since it needed for the field of ion transport to be developed. It is known now that sodium can travel up to 11 ˚A into the hydrophobic media of membranes.4
For any organism, complete isolation from its environment would be just as lethal as the absence of a membrane; consequently, nature solved this problem
by the use of transport systems either for ions or whole molecules. Here we will focus on ion transport systems and already in this subdivision there is a wide variety of sizes and complexity depending, mainly, on function.
1.2.1 Classification
Here we will introduce a main classification of transport systems based only on their working mechanism without any further consideration. This is not an exhaustive account.
Channels and pores. Their official description would want them to be molecules or supramolecular systems that are able to translocate ions in a discrete manner without significant movement. As a matter of facts, charac- terization of transport systems is challenging and as a consequence experts would rather define them in a functional way: a compound or supramolecular structure that shows single-molecule currents in planar bilayer conductance experiments.6
Carriers. In contrast to the previous category, these systems travel across the bilayer membrane and are limited by diffusion.
Detergents, fusogens, endovesiculators. These systems disturb the struc- ture of the membrane and transport is a consequence of this; they do not necessarily break the membrane. It is worth mentionning that under the right conditions, some detergents can show “channel” behavior.7
Polymeric structures. This family is particular, indeed, reports usually are mainly focused on their function, and few mechanistic studies exist. For rea- sons of consistency they are assigned to a special category.
In general, independently of their nature, they are better characterized as
“transport systems” without further designation, unless there is ground to support a claim on the transport mechanism. In the following sections, some
1 Introduction 3
examples of natural and synthetic transport systems will be discussed to illus- trate the field and, in the case of synthetic transport systems, an idea of the actual state-of-the-art is also intended; several excellent reviews on this field exist.7–14
1.2.2 Examples from Nature
Naturally occurring transport systems are the most interesting ones because the majority of them are actually proteins. There are also minimalistic channels and carriers.
The term “natural ion channel” is somehow bound with the work of the MacKinnon group.3,15–17 This group has extensively studied K+ channels by the means of crystallography. Several discoveries in this field come from this group. Arguably the most important contribution concerns the identification of the “channel” binding site in the middle of the protein bundle (Figure 1).
These perfectly aligned peptides allow the mimicking of the water coordina- tion. In fact, the protein mimics the potassium coordination with water and, as a consequence, sodium could enter but would collapse the structure. This selectivity creates a perfect K+ channel that filters other cations. Figure 1 depicts two crystal structures that belong to two different K+ channels, but there is no need to specify these K+channels as the “K+filter” is a common structure found ubiquitously.
In addition, the K+at the bottom of the filter is not held by a coordination to any carbonyl group, because at this stage K+is held in place and directed by the cooperative action ofα-helix bundles that “coordinate” the K+cation with cation-macrodipole interactions.
This short description should give an idea of the complexity of protein trans- port systems. Voltage gating was not addressed in the discussion but has also been explored.17Selectivity as well as stimulus-responsiveness are achieved by conformational changes of thewhole protein with the corresponding changes in the “filter” region.
Another example for natural “channels” can be found with the anthrax toxin. Actually, in order to fulfill its function, in this case to kill the cell, the toxin needs to deliver either the lethal factor (LF, 90 kDa) or/and the edema
Figure 1:Crystal structure of the KvAP K+channel (axial view)17(left) and zoom- in into the crystal structure of the KcsA K+channel with K+coordination (side view)16(right).
factor (EF, 89 kDa) into the cytoplasm. This is achieved by the protective antigen (PA, 83 kDa) which forms a heptameric structure as shown in Figure 2.
This heptameric structure is aβ-barrel pore with a pore size of 20 - 35 ˚A. At this point, the LF can traverse the lipid membrane. The action of the anthrax toxin does not end here and other steps are needed. In addition, an alternative path that involves endocytosis of the PA (modified) bind with both LF and EF followed by heptamerization in the late endosome and delivery of both factors into the lumen.18,19
This example of a pore should illustrate the degrees of complexity that gov- erns natural systems as well as illustrate, in the extreme case, the differences between channels and pores.
Of course, not all transport systems that nature uses are based in proteins:
simple peptides can also promote transport. Given the context, the description of another toxin is fitted: melittin.
Melittin is one of the components of bee toxin. It is a relative simple pep- tide with for sequence Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu- Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln-NH2and, aside from the physiological effects that make it a very toxic molecule, it is able to form
1 Introduction 5
Figure 2:Crystal structure of the (PA63)7 prepore structure monomer (left) and heptamer of the (PA63)7prepore structure from the bottom (right).18
channels when interacting with the lipid bilayer membrane.20
Melittin form channels (or pores) in the lipid bilayer membrane as shown by the characteristic signature in planar membrane conductance experiments, but these are not unimolecular but rather the association of around eight α-helix units as shown by several spectroscopic methods like circular dichroism.21 In addition, the aggregation of melittin units does disrupt the membrane, which, at high concentrations, leads to micellar aggregates and therefore its channel formation mechanism is said to be “micellar” or “toroidal”.7,20
The arrangement of the melittin units is parallel between each other, and parallel to the phospholipids. The presence of positive charges near to the C-terminus make it more selective towards anions than cations.
Similar to melittin is the also naturalalamethicin.
Isolated from fungus and with the peptide sequence Ac-Aib-Pro-Aib-Ala- Aib-Ala-Gln-Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Glu-Gln-Phl, alamethi- cin contains amino acids that are not part of the twenty main amino acids and their presence and the presence of two proline units, strongly induces an α-helix formation.
Theseα-helices which different from melittin, interact nicely with the lipid bilayer membrane; the channel formation, following a barrel-staves model, does
not disturb the membrane. It also requires the aggregation of around six units and leads to well-behaved channels in planar bilayer conductance experiments.
It also displays voltage dependance, which is attributed to conformational changes of the α-helix to expose a more important hydrophilic part, and a minor cation selectivity.21,22
In both cases, even if a helical structure exist, it is not directly promoting transport by itself and the active barrel-staves model is strongly reminiscent of protein folding in the case of the anthrax toxin, for example. This is not necessarily the case and helical peptides can also promote ion transport by themselves. A good example for this is gramicidin A.
Gramicidin A, which is the major component of gramicidin D, an antibiotic produced by Bacillus brevis, has for its peptide sequence formyl-L-X-Gly-L- Ala-D-Leu-L-Ala-D-Val-L-Val-D-Val-L-Trp-D-Leu-L-Trp-D-Leu-L-Trp-D-Leu- L-Trp-ethanolamine with X being either Val or Ile.
Where the structure described for Alameticin contains uncommon amino acids, gramicidin alternates D and L amino-acids which allows the formation of a so-calledβ-helix. The active structure of gramicidin A has been a subject of discussion for a long time and the two proposed models are illustrated in Figure 3.23–25
Figure 3:Crystal structures of gramicidin A (A: right-handed double helix from Cs+/methanol; B: left-handed double helix from ethanol) (left); Structure of gramicidin A in the membrane(right).26
The main reason for this controversy is that, in solution, gramicidin A forms a double-stranded helical structure and its helicity depends on cation
1 Introduction 7
complexation. It is now known that the active structure of gramicidin A in the lipid bilayer membrane is a double single-strandedβ-helix with both formyl moieties in the middle of the membrane. Such a channel requires, therefore, the right arrangement of both parts of the active structure to be assembled.27 The channel, which can be perceived as a longβ-helix, is sodium-selective, highly cation-selective, and can be blocked by the presence of divalent cations.
Several modifications of this channel have been reported.26
Already at this point, the dimeric structure of gramicidin A can barely span the lipid membrane and smaller molecules would, most likely, fall into the category of carrier systems (or a higher degree of aggregation).
Natural carriers are somehow less common but several famous examples exist like valinomycin which is produced by many Streptomyces strains and is a cyclic dodecadepsipeptide with the formula [-L-Lac-L-Val-D-Hiv-D-Val-]3
(Figure 4). Along with those amide groups on the main chain, there are ester groups. The common point is that the twelve carboxylic oxygens are available for binding at the center of the cycle.28
Given the geometrical constraints of the macrocycle, this carrier is potassium- selective. Binding to valinomycin is highly selective, displaying a difference as high as four orders of magnitude between its association constants for potas- sium and sodium.29
HN O
O O O
O NH O O O
NH O O O O
O NH
O
HN O
O O
O HN
1
N H
N H
N OCH3
H3C C5H11 2
Figure 4:Chemical structures of valinomycin (left) and prodigiosin (right).
As previously mentioned, in order for the cation to travel through the mem- brane, the whole complex must shuttle through the membrane and this implies
that in most of the cases the process will be limited by the rate of diffusion.30 Valinomycin is smaller than the aforementioned peptides and proteins, but it still has a considerable size. A better example of the reductionism that is possible in nature would beprodigiosin
Prodigiosin is part of the family of prodigiosines and is produced by Ser- ratia marcescens. It is a small non-peptidic anion carrier (Figure 4) that has been known for over one thousand years. Its transport mechanism is fairly well understood: coordination of chloride with the protonated form of prodi- giosin allows the symport transport of HCl. Recently, it has been shown that bicarbonate can be transported too, and thus prodigiosin can also act as a chloride/bicarbonate anion exchanger following an antiport mechanism. Given the importance of these anions, this is biologically relevant.31
Moreover, if prodigiosin research has developed in the last years, it is because it shows high activity as a drug, especially against cancer cell lines. This has somehow obscured the research of prodigiosin analogues as anion transport systems.32–34Nevertheless, prodigiosin analogues have been studied and many structural variations are possible to improve its transport properties. This will be discussed in the following section.
1.2.3 Synthetic Ion Transport Systems
The field of synthetic channels began more than 30 years ago with the seminal paper by Tabushi.35 The development of ion carriers started well before, but mainly on ion carriers in “bulk” membranes; the theoretical background for ion carriers is mostly the same. A a consequence, a real starting point for synthetic ion transport systems is difficult to define.
Nevertheless, with the rapid development of the field, several methods to observe and quantify ion transport were created. Although no real ranking could possibly be given for them, some ion transport systems, and especially the ones that will be used in this work, will be presented here. Some important characteristics of ion transport systems will be discussed thereafter to conclude with several representative examples of synthetic transport systems.
1 Introduction 9
1.3 How to Evaluate Ion Transport
Evaluation of ion transport is a major issue and over the years several methods have been proposed. To this date, no method has proven to be better than others and most of them are currently being exploited.
1.3.1 Fluorogenic Vesicles: The HPTS Assay
Ion transport activity can be easily followed in model systems with the HPTS assay.36 In this assay, vesicles containing 8-Hydroxy-1,3,6-pyrenetrisulfonate (HPTS, Figure 10) are prepared and then suspended in an isomolar buffer so- lution. Since HPTS is a pH-sensitive fluorophore, the pH in the interior of the vesicles can be followed by fluorescence in a standard spectrofluorometer. If a pH change occurs outside the vesicles, only minor changes are to be expected in the fluorescence signal. In the presence of a molecule, macromolecule or supramolecule able to promote the translocation of ions through the mem- brane, the situation is different; indeed, the transport of anions or cations entails a change in pH that ultimately leads to the equilibration of the pHs inside and outside of the membrane.
0 0.2 0.4 0.6 0.8 1
0 50 100 150 200 250 300 350 t / s
IF
NaOH Transporter
Detergent
Figure 5:Typical time-dependent fluorescence trace for the HPTS assay.
Practically, the change in pH is induced by the addition of a base, followed by
the transporter and, at the end of the experiment, the addition of a detergent that destroys the vesicles thus equilibrating the pH. For a given concentration of transporter, the ion transport kinetics would make it possible to observe the changes in fluorescence as a function of a macroscopic time-scale. A typical fluorescence kinetic trace is presented in Figure 5.
0 0.2 0.4 0.6 0.8 1
0 50 100 150 200 250 300 350 100 µM
0 µM
t / s IF
0 0.2 0.4 0.6 0.8 1
1 10 100
Y
c / µM
Figure 6:Typical analysis for transport activity with the HPTS assay. Time- dependent fluorescence traces at different concentrations (left) and dose response curve with reported fractional activities (Y) (right).
For different concentrations, the macroscopic response does follow the ex- pected trend, as a higher concentration gives better transport, but it does not follow a linear behavior. The dependence of the transport activity with respect to the transporter concentration can very often be characterized with the Hill equation (Equation (12)); this will be discussed in detail in Chap- ter 5. A typical example is shown in Figure 6. Briefly, the transport activity is characterized at different concentrations and the transmembrane activity is represented as a function of the transporter concentration. The Hill analysis yields the EC50, that is the concentration needed to reach 50% of transport activity under the given conditions and, the Hill coefficient nwhich can give information about the cooperativity of the active transport structure. This will be discussed in a later section.
The use of HPTS as a pH-sensitive fluorophore is motivated by the fact that with two absorption maxima depending on the protonation state, a ra- tiometric analysis of the fluorescence traces is less sensitive to artifacts and
1 Introduction 11
systematically eliminates pH errors by giving an absolute value for the pH change.
Ion Selectivity Topologies
One consequence of following the change of pH is that the transport of anions or cations is measured indirectly, that is a priori anion transport and cation transport as well as symport/antiport mechanism are not distinguishable in a single experiment. This is an important advantage as it provides a systematic method that reports on most of the possible transport events.
To evaluate a specific process, further analysis is required and this is achieved by evaluating the effects of anion and cation exchange. Assuming that the cation transport is the rate limiting step in the transport process, which is the case with cation transporters, exchange of the cation would result in a proportional enhancement or inhibition of the transport rate. If the transport mechanism is an antiport, the simultaneous transport of a cation and proton is expected to be responsible for the change in pH; if the mechanism is rather a symport, a co-transport of the cation and the hydroxy group takes place (Figure 7a,b).37
For anions the situation is a bit more complicated as a result of the external base pulse that is applied; in general, it is assumed that the required supple- mentary steps that ultimately lead to the situation in Figure 7c,d are constant and only the transport of the anion, either by an antiport or a symport mech- anism, limits the rate of the transport; the situation thereafter is similar to that of the cations.37
The different ion selectivity topologies that can be observed are described in the literature, mainly known as the Eisenman cation selectivity sequences38 and the Hofmeister anion selectivity.39
The Eisenman cation selectivity is based mainly on two factors: the hy- dration energies and the size of the cation. Indeed, the smaller a dehydrated cation is, the better it can bind; on the other hand, the bigger the cation, the smaller the hydration energy. Clearly, for a cation to be transported, partial or total dehydration is needed and, therefore, the basic sequence Cs+>Rb+>
K+ > Na+ >Li+ (Eisenman I) depends only on the hydration energies. If
H+ Na+ Cl-
OH- H+
X- Na+
OH- d
H+
Na+ Cl- OH-
H+
OH- X+
Na+ c
H+ Na+ Cl-
OH- H+
Cl- OH- M+ b
H+ Na+ Cl-
OH- H+
Cl- OH-
M+ a
X- M+
X- = OH- or Cl- M+ = H+ or Na+
Figure 7:Some different situations in ion selectivity determination; a) Cation-proton antiport, b) Cation-anion symport, c) Anion-hydroxy antiport, d) Anion- cation symport.
the binding is the most important factor, the opposite is observed (Eisenman XI). For the five alkaline cations, 120 possibilities exist. Out of these, Eisen- man selected eleven on the basis of ”natural” occurrence and these series are known as the Eisenman cation selectivity sequences.38
The Hofmeister ion selectivity sequence derives historically from the exper- iments of Prof. Hofmeister in 1888 about protein stability;40 as a matter of fact, proteins are more or less stable depending on the ions present in the aque- ous media. The original Hofmeister series for anions and cations are shown in Figure 8.
SO42- > CO32- > OAc- > F- > Cl- > NO2- > Br- > NO3- > SCN- > I- >ClO4-
Al3+ > Mg2+ > Ca2+ > H+ > Li+ > Na+ > K+ > Rb+ > Cs+ > NH4+ > N(CH3)4+
Figure 8:Original Hofmeister anion and cation series in decreasing order of protein stabilization (from left to right).
In its original meaning, the Hofmeister ion selectivity sequence correlates well with the hydration energies; the highest hydration energy corresponds to the ions that best stabilize a protein. In the past years, researchers have disputed this sequence and some modifications have been proposed.39
For cations and anions, both the Eisenman or the Hofmeister sequences are closely related to the hydration energies and, as a consequence, the ion selectivity topologies are represented as a function of the hydration energies of
1 Introduction 13
the ions. In Figure 9 some typical fluorescence traces and a Hofmeister anion selectivity topology are presented.
0 0.2 0.4 0.6 0.8 1
0 50 100 150 200 250 300 350 t / s
IF
F- OAc- Cl- Br- ClO4
-
I- NO3
-
0 0.2 0.4 0.6 0.8 1
-450 -400 -350 -300
F- OAc-
Cl- Br-
ClO4 -
I- NO3
-
Ynorm
∆Ghyd / kJ·mol-1
Figure 9:Typical anion selectivity as observed in the HPTS assay (left) and typical anion selectivity topology displaying a Hofmeister selectivity (right).
Further Possibilities
Other than allowing the possibility of easily detecting ion selectivities, the HPTS assay utilization is possible with vesicles of different lipid compositions.
This gives access to information about the effect of charges on the membrane surface. It also can report on lipid viscosity or even provide information on the effect of different lipid membrane phases.
Finally, under certain conditions that will be discussed later in the exper- imental part, the influence of very specific external factors like membrane polarization, which normally can only be accessed with more complex tech- niques, can also be measured.41
1.3.2 Fluorogenic Vesicles: The Lucigenin Assay
In the previous section, the advantages of using a non-selective fluorophore as detection method were discussed. That method is based mainly on the generation of a pH gradient by the addition of a base which acts as the driving force for anion transport. It is also possible to detect the target ion
O O
COOH HOOC
HO
N
N NO3-
NO3- -O3S SO3- SO3- HO
Na+
Na+ Na+
3 4 5
Figure 10: Structure of carboxyfluorescein (3), lucigenin (4) and HPTS (5)
directly with a selective fluorophore that reacts in its presence or absence. One example of this case is the Lucigenin assay.
In this assay, vesicles are prepared containing nitrate salt inside and chloride salts outside of the vesicles always at isoosmotic pressures and in presence of a buffer. The vesicles also contain Lucigenin (Figure 10), which is a halide selective fluorophore and, in particular, a very sensitive fluorophore with re- gards to chloride.42–44 Indeed, the fluorescence of Lucigenin is quenched by the presence of chloride.
The experiment works in a similar manner to the HPTS assay; the time de- pendent fluorescence traces are acquired upon the addition of the transporter and the decrease in fluorescence is monitored. At the end of the experiment, the vesicles are lysed and the chloride concentrations are equilibrated at a concentration high enough to show a significant quenching of the Lucigenin fluorescence. The obtained data are treated in a way similar way to the HPTS experiment, obtaining the same parameters,i.e. theEC50 and the Hill coeffi- cientn.
The main advantage of the use of Lucigenin is the selectivity of the detec- tion. However, a cation transport for instance would not be detected.
1.3.3 Experiments with Vesicles: Ion-Selective Electrodes
An alternative to the use of fluorescent probes to detect transport is the use of ion-selective electrodes. In such an experiment, the target ion, usually chloride, is encapsulated inside vesicles with similar characteristics to the ones
1 Introduction 15
used for the HPTS assay and as long as the ion remains inside the vesicles, the electrode is unable to detect their presence in the solution.
The addition of a transporter would, as before, allow the release of the target ion and thus the measurement of the ion’s concentration. Thereafter, with a similar data treatment, the characterization of the transport is possible.
Unfortunately, this method requires specific ion-selective electrodes for every ion of interest and the time of response of the electrodes usually restricts the sampling to long intervals.45,46
1.3.4 Experiments with Vesicles: NMR
NMR spectroscopy has also been used as a method to quantify ion transport, mainly in the case of sodium. The idea is to prepare vesicles, very stable vesicles and at high concentrations, containing an NMR shift reagent. The escape of sodium can then be measured by standard NMR methods and the data treated as before. This has been reported as successful when applied to sodium,47 lithium,48 and chloride.49
Unfortunately, the experiments under these conditions are measured in a completely different timescale (hours rather than minutes) and this allows many other factors to influence the observed transport. In addition, mea- surements at different concentrations require much time and thus are only reported very infrequently nowadays.
1.3.5 Hill Coefficient
Hill coefficients require a separate section because they are largely overlooked when analyzing transport data. The reason for this is the difficulty of their interpretation. The Hill coefficient is usually said to report on the cooperativity of the system: if a molecule is not able to transport by itself but requires the assembly of two or more molecules in order to promote transport, the Hill coefficient should consequently increase.
This is true only if certain conditions are fulfilled and was nicely described in the literature:50
Assume a model system with T the transporter with a [T] concentration;
ion transport of ion I in concentration [I] happens at a certain “rate”. Under these conditions, the rate can be expressed as follows:
rate = ko·[I] (1)
kois the observed rate constant. This constant depends on two factors: the active structure AS that is present in a concentration [AS] that is determined byKASandk the real rate constant for the ion transport:
ko= k·[AS] (2)
KAS= [T]n
[AS] (3)
Equation (3) is derived directly from the thermodynamic equilibrium andn is the stoichiometric value of the transporters necessary for the generation of to generate an active structure. From these equations we derive:
ko= k·[T]n
KAS (4)
If KAS is big, then [AS][T] and [AS] can be approximated by[AS]/nand Equation (2) becomes:
ko= k·[AS]
n (5)
Where ko is linearly proportional to [T]. On the other hand, if [T][AS]
then Equation (4) is to be applied and kois exponentially proportional to [T].
“n” is the cooperativity and is proportional to the Hill coefficient. Unfortu- nately, only in rare cases do extreme situations occur and most often middle values ofnare observed.
Then, if n reports on the cooperativity, this is only true as long as the value for k is small enough as for the “monomer” to be in excess but sufficiently high as to allow for the formation of the required active structure.
The dependence of the Hill coefficient on the association constant as defined for the active structure implies that if a Hill coefficient is lower than “expected”
1 Introduction 17
due to this higher stability, induced destabilization by thermal or chemical denaturation allows the measurement of a meaningful value of cooperativity.51 Unfortunately, this is not always possible, but its implications are to be kept in mind when analyzing supramolecular structures.
1.3.6 Planar Membrane Conductance Experiments
“Black lipid membrane” (BLM) experiments by their historical name, or even
“voltage-clamp technique” as a more method-based name, are the main ex- periments for the characterization of ion channels. As a matter of fact and as mentioned before, the very definition of ion channels is based on this tech- nique, as only a functional definition of ion channels is possible at this point.
The origin of this method can be traced back to 1962,52 but its main devel- opment has to be attributed to the “patch-clamp” techniques in biology as used for the studyin situof natural channels.53
~ A
!"#
$%&'#
Figure 11: Representation of a typical set-up for planar membrane conductance ex- periments. Insert: changes in current for a gramicidin A channel.6
In these experiments, two separated chambers are connected by a small hole (typically a diameter of around 200µm). Both chambers are filled with an electrolyte and connected to electrodes by salt bridges. Over the hole a bilayer membrane can be “painted”; given the high capacitance of lipid bilayer membranes, the two chambers are formally isolated.
In presence of a transporter, a flux of ions can be detected by the appearance of a current. Typically, the incorporation of a single channel will allow the observation of an increase in current, due to a single channel transporting
ions (insert Figure 11). Many parameters can be obtained with this method, especially in the case of channels where, for instance, the size of the pore can be assessed,54and its main advantage comes from the fact that the obtained data are generally comparable to that obtained for other putative transporters regardless of their structures.6
One excellent example is the information concerning ion selectivity: in the HPTS assay for example, a single measurement gives no information at all on the ion selectivity and although this information can be obtained with further experiments, doing so adds more parameters, and certainty can not be achieved; planar membrane conductance experiments can, because the current flow is known and controlled, determine, beyond doubt, anion and cation selectivities and even quantify them.
This and historical reasons make it the method of choice when confronting challenging systems that require clean characterization beyond the typical structure to function relationships.6
Its main limitation is the requirement of specialised equipment and long measurement times. Practical details about this method are given in Chap- ter 5.
1.3.7 Examples of Synthetic Transport Systems
In this section an overview of synthetic transport systems will be given. The aim is not to describe what can be found in the literature, mainly because it would be too extensive, but to give key examples to illustrate both historical and structural properties of synthetic transport systems. Examples especially relevant to this study will be described.
The first example concerns a period where “nature-inspired” channels were developed; the β-peptides by the DeGrado group are nice examples.55,56
Several of these β-peptides have been reported (some are depicted in Fig- ure 12). Ion transport in this case was explored with regard to the antimicrobial activity againstE. coli and hemolysis. Independently, the formation of helical structures was monitored by CD spectroscopy and the presence of the helical structures confirmed, thus correlating it to the observed activity.
Compounds 6 and 7 for example would show activity at around 25 mM.
