Ion Transport Across Membranes Mediated by a Dynamic Combinatorial Library

Ion Transport across Membranes Mediated by a Dynamic Combinatorial Library

Dissertation

Zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Christian-Albrechts-Universität zu Kiel

vorgelegt von Vittorio Saggiomo

Kiel 2010 Referent: Prof. Dr. Ulrich Lüning Korreferent: Prof. Dr. Rainer Herges Tag der mündlichen Prüfung: 29 / 04 / 2010 Zum Druck genehmigt: 25 / 05 /2010

gez. Prof. Dr. L. Kipp, Dekan Die vorliegende Arbeit wurde unter Anleitung von

Prof. Dr. Ulrich Lüning am Otto-Diels-Institut für Organische Chemie der Christian-Albrechts-Universität zu Kiel in der Zeit von Mai 2007 bis März 2010 angefertigt.

“The true delight is in the ﬁnding out rather than in the knowing.” Isaac Asimov (Chemist)

Acknowledgment

First of all to my ex-girlfriend (a.k.a. wife) Ilaria who is always encouraging and supporting me in almost everything I am doing. She is following me in the foolish idea of doing research somewhere else in the world. A huge thanks to my Ph.D. supervisor Prof. Dr. Ulrich Lüning, in these three years I learned more than I expected. I have learned great lessons not only in chemistry but also in “real” life. It will be hard to ﬁnd another kind supervisor like him. Thanks to Angelika Merten who took care of burEaUcracy on my behalf. That was not an easy job. A sincere thanks to all the working group. The passage from Italy to Germany was not so traumatic as I thought, mainly thanks to the kindness of all of them. In these three years I met very good friends. There are too many reasons to thanks Frank Schleef that I should write another three theses only to have three new pages for acknowledging him. Thanks to Tim (Mario) Reimers for his friendship, for the nice music selection during the lab time, funny going out in the night and for ﬁnding almost all the typos. Thanks to all the chemists that worked and are working in the lab 317 (Christiane Dethlefs and Mirja Oldefest over all with many other students) for the nice working environment. Dr. Ole Winkelmann is kindly acknowledged for the interesting chemistry conversations and the long video games nights. Dr. Dennis Stoltenberg is acknowledged for the nice meetings that we attended together. Thanks to Dr. Catrin Goeschen (Rainer Hergesʼ group), Jörg Klein (Jeremy Sandersʼ group in Cambridge), Michael Zengerle (Stefan Kubikʼs group in Kaiserslautern), Dr. Roberto Quesada (University of Burgos), and Jens Eckelmann for their collaboration in different projects. Working with them was interesting and funny. Thanks to Frank Sönnichsen and all the spectroscopic division, always fast and ready to help me for any problems with measurements. Thanks to Lisa Reck, the only brave student that did the OCF-3 in English with me. Thanks to Prof. Sijbren Otto for his great work as coordinator of the Marie Curie Network. Naturally thanks to the EU for its support through the Marie Curie Research Training Network MRTN-CT-2006-035614 Dynamic Combinatorial Chemistry (DCC). Without this grant, this thesis would never have seen the light. And always thanks to my parents and my brother. At the end, thanks to you reader. If you are reading this line after the others, you at least read one page of my thesis. Thank You.

Abstract

In this thesis, the proof of concept of using a Dynamic Combinatorial Library (DCL) directly for a function is presented. The transport of ions mediated by a carrier ampliﬁed from a DCL was successfully achieved. A carrier macrocycle is formed directly by a DCL and then it transports calcium ions from a water source phase to a water receiver phase passing through synthetic organic membranes.

OMe OMe

N H2N O NH2 CaCl2 N + N N 3 2+ O O Ca O O O

Proved for the ﬁrst time, this goal was achieved in a series of experiments. First, the possibility of using a library composed of imines in water was studied. Then, the same dynamic combinatorial experiment was screened in a biphasic system. Finally, the possibility of amplifying a macrocyclic carrier directly in a bulk membrane experiment was proven. A carrier was ampliﬁed from a DCL and the template was subsequently transported from the source phase to the receiver phase. The one step generation of a carrier from a library was successfully achieved.

The development of this idea was improved realizing a double screening methodology by a larger DCL. The template selects and ampliﬁes two related macrocycles from the DCL, and in a second step, the membrane itself selects the macrocycle with the best carrier activity. The proof of concept of DCL/transport was also tested using liposomes as mimic of a biological membrane. In order to maintain the electroneutrality during the transport of ions, the counter-ions should be transported as well. Transporting an ion duplet or triplet should be easier and faster than transporting only the related ion. Therefore, a macrocycle was synthesized and its ability to bind calcium chloride as an ion triplet was proven.

Zusammenfassung

In dieser Arbeit wird der Aufbau und die direkte Verwendung einer dynamischen kombinatorischen Bibliothek dargestellt. Dabei konnte gezeigt werden, dass ein makrocyclischer Carrier, der direkt aus einer DCL gebildet wurde, Calciumionen aus der wässrigen Phase durch eine organische Membran in eine wässrige Phase transportiert. Diese Funktion konnte in einer Reihe von Experimenten zum ersten Mal erzielt werden.

OMe OMe

N H2N O NH2 CaCl2 N + N N 3 2+ O O Ca O O O

Zudem konnte eine Imin-Bibliothek in einer wässrigen Phase realisiert und untersucht werden. Anschließend wurde das gleiche dynamisch-kombinatorische Experiment in einem zweiphasigen System untersucht. Schließlich konnte nachgewiesen werden, dass der makroyclische Carrier den Transport durch ein Membran unterstützt. Der Carrier wurde durch eine DCL verstärkt und das Templat konnte danach über eine Phasengrenze hinweg transportiert werden. Dabei gelang es, einen Carrier erfolgreich in einem Schritt aus einer Bibliothek zu erhalten. Diese Entwicklung konnte verbessert werden, indem eine größere DCL verwendet wurde. Dabei unterstützt das Templat die Bildung zweier verschiedener Makrocylen, zudem selektiert die Membran bevorzugt den Carrier mit der besten Aktivität.

Zum Nachweis der erstellten DCL bzw. des Transports wurden zusätzlich Liposome als Modell einer biologischen Membran verwendet. Um eine Elektroneutralität während des Transportes zu gewährleisten, müssen die Gegenionen ebenso transportiert werden. Der Transport eines Ionenpaars beziehungsweise eines Ionentripletts sollte einfacher und schneller erfolgen als der eines einfachen Ions. Hierzu wurde ein Makrocyclus synthetisiert, der in der Lage ist, Calciumdichlorid als ein Ionentriplet zu binden.

Table of Contents

1 Introduction 1

1.1 Supramolecular Chemistry 1

1.2 Dynamic Combinatorial Chemistry 3 1.2.1 Imines 5 1.2.2 The next step: Applications 8

2 Objective 10

3 Results and Discussion 12

3.1 Remarkable Stability of Imino Macrocycles in Water 13 3.2 On the Formation of Imines in Water, a Comparison 19 3.3 Transport of Calcium Ions through a Bulk Membrane by Use of a Dynamic Combinatorial Library 23

3.4 Ion Transport across Membranes Facilitated by a Dynamic Combinatorial Library 38 3.5 The First Supramolecular Ion Triplet Complex 58

4 Summary and Perspective 82

4.1 Summary 82 4.2 Perspective 84

5 References 86

1 Introduction 1 ______

1 Introduction

1.1 Supramolecular Chemistry

“Atoms are letters, molecules are the words, supramolecular entities are the sentences and the chapters”. 1 This brief sentence contains in itself the importance and the great fascination of supramolecular chemistry. The year was 1987. Jean- Marie Lehn, together with Donald J. Cram and Charles J. Pedersen, won the Nobel price “for their development and use of molecules with structure-speciﬁc interaction of high selectivity” (Fig. 1.1). Born by genial intuition and lucky accidental synthesis, 2 supramolecular chemistry has nowadays great importance in various and different

ﬁelds in chemistry and industry. C. J. Pedersen 503

Figure 1.1: Original drawing from Charles J. Pedersenʼs Nobel Prize Lecture. 3

Supramolecular chemistry was born as a branch of organic chemistry and slowly grew up asMy a highlyexcitement, interdisciplinary which had been subject. rising It during not only this attracts investigation, chemists now but also reached its peak and ideas swarmed in my brain. One of my first actions was 4 biochemists,motivated biologists, by esthetics engineers, more than physicists science. I derivedand theoreticians. great esthetic pleasure Moreover from it plays a crucial rolethe in three-dimensional the development structure of new as portrayed nanotechnologies. in the computer-simulated 5 In a more modelpoetic way we can say, withoutin Figure any 7. Whatdoubt, a simple,that supramolecular elegant and effective chemistry means “is for life”. the trapping of hitherto recalcitrant alkali cations! I applied the epithet “crown” to the first In biologicalmember life, of molecular this class recognition of macrocyclic plays polyethers a key because role in its almost molecular all processes model with looked like one and, with it, cations could be crowned and uncrowned without an amazingphysical selectivity. damage to eitherThis as high shown selectivity for the potassium is really complex difficult in Figure to 8. reproduce As in laboratoriesmy by studies synthetic progressed, hosts. I Host-guest created the system complexes of crown possess nomenclature great importance chiefly also in signalingbecause and thetransport, official names moving of the in crownand out ethers guests were soin complexdifferent and part hard of forcells. me to remember. It is a source of special satisfaction to me that this system of In everydayabbreviatedʼs life, supramolecular names, devised solely chemistry for the readyis even identification more present. of the macrocy-Plastic materials completelyclic changed polyethers, our has life, been and retained now bythis the branch scientific is establishment.evolving in polymer In Figure and 9 I material have illustrated how the nomenclature system is made up of the side-ring substituents, the total number of oxygen atoms in the main ring and the size of the ring. Another aspect of this discovery filled me with wonder. In ordinary organic reactions only rings of 5, 6, or 7 members form easily. Here a ring of 18 atoms had been formed in a single operation by the reaction of two molecules of catechol, which was present as a minor impurity, with two molecules of bis(2- chloroethyl)ether. Further experiments revealed that dibenzo-18-crown-6 can be synthesized from these intermediates in a 45 percent yield without resorting to high dilution techniques. This was most unexpected and some good reason must exist for such an unusual result. I concluded that the ring-closing step, either by a second molecule of catechol or a second molecule of bis(2-chloroethyl)ether, was facilitated by the sodium ion which, by ion-dipole interaction, “wrapped” the molecular pieces around itself to form a three-quarter circle and 2 1 Introduction ______science. High-tech materials such as thermoplastics, solar panels, adhesives or new and resistant materials for artificial organs are based on supramolecular and polymer chemistry. These new materials can drastically improve our daily life. Also in pharmaceutics, supramolecular chemistry is widely exploited. Cyclodextrines for example, thanks to their shapes and to the apolar binding site, are used since more than ten years in many pharmaceutical formulations in order to solubilize synthetic drugs. 6 Moreover “smart materials”, molecular shuttles, motors and rotors are, in this first part of the 21st century the new era of supramolecules thanks to the work of J. Fraser Stoddart, 7 David Leigh 8 and Ben L. Feringa. 9 Those are only a few applications of supramolecular chemistry. In an oversimplified way, supramolecular core concepts are: - Molecular Recognition: The formation of a complex takes place through recognition between guest species and host. The receptor should have, beyond a binding site, also a geometry and a three-dimensional structure complementary to the guest. - Non Covalent Bonds: The forces between host and guest are non-covalent interactions. Examples of those forces are: ion-ion, ion-dipole or dipole-dipole interaction, hydrogen bonding, π-π stacking, Van der Waals forces and hydrophobic effects. - Preorganisation: Often the host is rigidified in order to adopt the perfect conformation for the recognition of a specific guest. This preorganization leads to a high stability of the final host-guest complex. - Self Assembly: A new macromolecule can be generated by spontaneous association of single molecules. Since the birth of supramolecular chemistry, many host molecules have been synthesized. From crown and lariat ethers, podands, cryptands to calixarenes, cyclodextrines and then, to even more complex structures such catenanes, rotaxanes, foldamers or helicates.

The synthesis of host molecules for specific guests is usually long and difficult. Often the final product is only obtained by a multi-step synthesis, requiring various purification steps and many times also protection/deprotection steps. Once the host product is synthesized, it must be tested for the host-guest complexation. And this, unfortunately, is not always successful. In fact it is not rare, that the hard-won host does not bind the desired guest, or binds it with low affinity. An alternative strategy to obtain a host for a specific guest, with less synthetic effort, was developed during the last twenty years in an emerging field: Dynamic Combinatorial Chemistry. 1 Introduction 3 ______

1.2 Dynamic Combinatorial Chemistry Dynamic Combinatorial Chemistry (DCC) 10 is, by definition, combinatorial chemistry under thermodynamic control. In non-reversible combinatorial chemistry, a large Thermodynamically-controlled cyclisationnumber and of differentinterconversion related products of are synthesized from a mixture of different oligocholates: metal ion templated ‘living’starting macrolactonisation building blocks. The obtained products will not react with one another and the final composition of the library will not change. On the contrary, all members of a Dynamic Combinatorial Library (DCL) are in equilibrium and can be interconverted Paul A. Brady and Jeremy K. M. Sanders* into one another changing the library composition. In order to achieve the interconversion, the link between the components of the dynamic library must be Cambridge Centre for Molecular Recognition, Universityreversible. Chemical Pioneers Laboratory, with two different approaches were Jean-Marie Lehn and Lensfield Road, Cambridge, UK CB2 1EW Jeremy K. M. Sanders. Lehnʼs group in Strasbourg used DCC to cast a substrate (guest) for an enzyme (host). They describe “casting consist in the receptor-induced assembly of a substrate that fits the receptor”. 11 The use of DCC allows the guests in the library to A series of ester-linked macrocyclic oligomers (dimer–pentamer) of cholates equipped with a variety of interconvert into one another. The spontaneous guest diversity generation is then recognition and reporter elements has been prepared (a) by conventional irreversible chemistry, and (b) using methoxide-catalysed transesterification under reversibleshifted mainly equilibrium to one guest conditions. that will Templating fit inside the byenzyme pocket in what they call metal ions under these equilibrium conditions is also “demonstrated.sort of (supra)molecular These results Darwinism demonstrate” (Fig. 1.2, the a). In a few words, they discover an feasibility of creating a dynamic combinatorial libraryeasy of receptors.and efficient methodology to synthesize a perfectly fitting guest for a specific host. Chemistry: Huc and Lehn Proc. Natl. Acad. Sci. USA 94 (1997) 2107

Macrocyclic molecules have been central to the emergence of C1 C2 C3 ....Cn supramolecular chemistry.1 They have a defined cavity in which still imperfect and requiring improvement, the present results binding processes can take place as a result of the rigidity nevertheless illustrate the feasibility of the basic concept and and spatial arrangement of functional groups imposed by the T point out paths for future developments. cyclic structure. Furthermore the synthetic challenge of such a) large ring systems has stimulated numerous new preparative C2 T MATERIALS AND METHODS approaches. In particular, templates have proved useful in the Fig. 1 Changing a cyclisation equilibrium using a thermodynamic Materials. CAII from bovine erythrocytes was purchased promotion of cyclisation reactions and they can dramatically template T from Sigma and used without further purification. The con- 2 improve yields in many systems. Most reactions utilised in centration of CA stock solutions was estimated spectrophoto- 4 1 1 macrocycle syntheses have been performed under kinetic con- metrically with 280 5.7 10 M cm (24). para-and trol, so the template has acted by bringing the two ends of a meta-sulfamoylbenzaldehydes were prepared from the corre- particular intermediate into close proximity, thus favouring sponding nitriles according to a literature procedure (25). The their reaction. This effect results in an increase in the quantity starting 4-sulfamoylbenzonitrile was purchased from May- of the product, the precursor to which was bound effectively by bridge Chemical (Tintagel, UK) and 3-sulfamoylbenzonitrile b) the template. was synthesized from 3-carboxybenzenesulfonic acid (26). We, and others,3 have been interested in carrying out such Hexyl 4-sulfamoylbenzoate was prepared by heating the commercial acid in a HCl-saturated solution of 1-hexanol. Crystals templating in a thermodynamic regime. This approach is poten- FIG.1. Virtualcombinatoriallibraries.(Upper)Schematicrepre- were obtained upon addition of hexane to the cooled reaction tially more versatile since the template will promote formation sentation of the casting process: receptor-induced self-assembly of the mixture. of the product that it binds best, rather than just accelerating Fig.complementary 2 Schematic substrate picture from of a thermodynamic collection of components templatingfragments. for host generationThis process in a amounts complex to mixture the selection of building of the blocks optimal substrate from a Compounds 3a, 3b, 3c,and3d (Fig. 2) were synthesized the formation of a particular product. In a single componentFigure 1.2:virtual Original substrate drawings library. from ( L firstowe r experiments)Schematicrepresentationofthe of Dynamical Combinatorial according to the following general procedure. To a mixture of mixture such as that illustrated in Fig. 1, templating can biasChemistry.molding a) Casting process: of a substrate substrate-induced (guest) 11 and self-assembly b) selection of of a thehost. complemen- 12 4-sulfamoylbenzaldehyde (1.0 mmol), the hydrochloride salt of the equilibrium between different sized cyclic oligomers Cn tary receptor from a collection of structural componentsfragments. a, b, c,ord (2 equivalents), and Et3N(1equivalent)inMeOH This process amounts to the selection of the optimal receptor from a towards a particular product. If the idea is extended to the use (5 ml) at 0CNaBH3CN (3 equivalents) was added. After 24 h of a mixture of several different building blocks, thermo- virtual receptor library. The diverse potential constituents of the at 25C, the mixture was evaporated to dryness. For 3a, 3c,and libraries (Center)areeithercovalentlylinkedornoncovalentlybound 3d,theresiduewaschromatographedonsilicagelelutingwith dynamic templating could lead to the generation of a reversibly generated species that may or may not exist in significant mixed host that binds the template well (Fig. 2). In order to amount(s) in the free state, in absence of the partner. MeOHCH2Cl2 (1 to 102 of MeOH in CH2Cl2 depending on investigate this approach, we required an efficient thermo- compound). The final product was converted to its hydrochlo- dynamically-controlled method for the synthesis of macrocyclic set of components, (ii)theirreversiblecombinationforspon- ride salt. For compound 3b,theresiduewaschromatographed molecules. Fig.taneous 3 A general diversity building generation, block for (i hostii)theirrecognition-directed generation on C18 reversed-phase silica gel, eluting with a MeOHH2O (from 0 to 10% MeOH) gradient. The purity of these com- Many chemical reactions proceed under thermodynamic selection of one partner by the other one (in fact, both partners pounds was controlled by reversed-phase HPLC analysis. control and some of these have been utilised in macrocycle syn- could in principle be self-assembled supramolecular species). In addition one may wishStrategy to lock-in the self-assembled struc- Choice of the Library Generating Reaction. For the purpose theses. Cyclic polyesters of small ring lactones have been exten- of the present experiments, the reaction on which the library sively investigated by Seebach 4 and Roelens,5 and a theoretical Consideringtures by performing the process a chemical in Fig. reaction2, a broad that range irreversibly of products links together the components of the entity generated in the optimal is based has to be reversible and to take place in or near treatment of the product distribution expected in these systems will be formed if the mixture contains a variety of different combination. physiological conditions. This is the case for the fast and has been developed by Mandolini.6 Reversible imine formation building blocks. The utility of this approach will depend on the reversible condensation of amines and aldehydes to imines that 7 Both processes also amount to the generation of the fittest has been used in the synthesis of many cyclam ionophores, identificationand present adaptationof the products, and evolution so it will by be spontaneous necessary for recom- each allows the system to equilibrate in presence of the receptor. In though it is not clear that the reversibility has been fully buildingbination block under tochanges contain in the a unique partner(s) spectroscopic or in the environ- reporter addition, the imine(s) formed may subsequently be irreversibly exploited in most cases. The reversible formation of imines has group.mental In conditions. addition They to this, thus the embody building a sort blocks of (supra)mo- should be reduced by NaBH3CN (27), thus fixing the compound(s) been used by Lindsey and Mauzerall in the synthesis of macro- relativelylecular Darwinism! rigid and slightly curved, so as to define a cavity and generated in the form of amines, which also facilitates the isolation and analysis of the final equilibrated mixture ob- cyclic porphyrin–quinone adducts to give a high yield of the theyThe should VCL contain concept a describedrecognition herein element bears for relationinteraction to pro- with 8 tained (9). In addition, in view of the small steric difference structurally most stable adduct. Calix[4] arenes and calixresor- thecesses template and approaches or guest. Several such as possibilities the formation can ofbe clathrates imagined byfor cinarenes can be produced from larger ring analogues, demon- this:assembly an amide of a to solid-state hydrogen host bond structure to a guest around molecule; included a charged sub- strating reversible bond formation in their synthesis.9 Similarly, groupstrates to (6–8), attract the an template-directed oppositely-charged oligonucleotide site; or an synthesis aromatic isolated α-, β- or γ-cyclodextrins are found to re-equilibrate to group(9), the for generation π–π interactions. of RNA aptamers A cartoon (10), picture the computational of a suitable a mixture under the conditions of their formation, but enzymes buildingprogressive block build-up is shown of in aFig. host 3. in a binding site (11), the are required for this process.10 In most of these examples, the self-assemblyTo realise these of anprinciples, inorganic we catalyst turned (12)to derivatives or of ionophores of cholic use of thermodynamic control in macrocyclisations has led to acid.(13), These the template-induced bile acids have the formation general ofstructure a metallomacrocycle 1 and have the reactions generating a variety of products with a range of ring attractive(14) or offeature macrocyclic of combining lactams a rigid (15), concave the construction component of with a sizes. abinding flexible site chain. via metal Cholic ion acid coordination derivatives of the have components previously (16), been the equilibrated condensation of multiple components to yield macrobicyclic cryptands (17) or macrocyclic oligocholates (18), or theJ. formationChem. Soc., of Perkin a binding Trans. site 1, by 1997 noncovalent3237 self-assembly in a molecular monolayer (19) (see also section 9.4.1 in ref. 5). It is also implicit in the previously described self-recognition operating in helicate self-assembly, which involves the spontaneous selection of the correct partners in a mixture (ref. 20, see also section 9.5 in ref. 5). It has in particular been already exemplified in the self-assembly of a circular double helicate of specific size directed by the strong binding of a chloride ion in the central cavity, a process that amounts to a molding event (21). We present herein an implementation of the VCL concept involving a casting process based on the recognition directed assembly of inhibitors of carbonic anhydrase II (CAII, EC FIG.2.Precursoraminesa–d and aldehydes 1–3 and resulting 4.2.1.1). CAII is a well-characterized Zn(II) metalloenzyme components of the combinatorial library. The products are given as the (Mr 30,000) whose inhibition especially by para-substituted amines resulting from the hydride reduction of the initially formed sulfonamides (22, 23) has been extensively studied. Although imines. The alcohols 1–3 are obtained as side products. 4 1 Introduction ______

Using the opposite approach, Sandersʼ group in Cambridge used DCC in order to obtain a host for a specific guest. 12 In this case, the reaction of different building blocks gave various linear and macrocyclic products. To this resulting library was then added a guest. Perturbing the equilibrium by addition of a guest resulted in a different library composition. Thus the macrocycles that better bound the given guest were “amplified” to the detriment of other library members (Fig. 1.2, b). Since then, dynamic combinatorial chemistry has evolved discovering and using different reversible covalent reactions [mainly used are imines, 13 hydrazones 14 and disulfides 15 (Fig. 1.3), in our group there are also examples of boronic ester exchange 16 ,17], as well as non-covalent bonds (examples: metal-ligand exchange 18 or hydrogen bond). 19

basic pH 2 S R2 3 - S R 1 - R1 S + R S R3 S + R S

H H H H acidic pH H H H H C N + C N 4 C N + C N R1 N R2 R3 N R R1 N R4 R3 N R2

Figure 1.3: Two different reversible reactions: disulﬁde exchange (top) and hydrazone exchange (bottom). The disulﬁde reaction is reversible only at basic pH. Decreasing the pH will freeze the equilibration of the library due to the protonation of the thiolate to thiol. The hydrazone exchange, on the contrary, is reversible only at acidic pH. Increasing the pH will result in the frozen library. The residues Rn are coloured only to enable the readersʼ eyes to see the exchange. However it is important to remember that the whole thiolic and hydrazide groups are involved in the exchange and not only the residues.

Moreover it was proven that two different reversible reactions can be carried out at the same time giving an “orthogonal” screening. 20 Without any doubt, the idea to synthesize perfect fitting guests for a specific host in one step (or other way round: to synthesize a perfect host for a specific guest) is intriguing and fascinating. Naturally in a novel field like this many problems arise. The DCL is in thermodynamic equilibrium, and small physical or chemical changes can affect the library distribution. In addition to this, it should be noted that the more complex the library is, the more difficult is its screening, and small changes in the library composition can sometimes be overlooked. However thanks to DCC many novel hosts have been discovered in the last years. 1 Introduction 5 ______

It is interesting to note that also only one building block can be used for setting up a dynamic combinatorial library. Various products can be ampliﬁed from this dynamic library using different templates. Starting from only one building block, it is possible to amplify various products (Fig. 1.4).

Figure 1.4: In Dynamic Combinatorial Chemistry, only one building block can react to give a Dynamic Combinatorial Library. All members of the dynamic library are in equilibrium one with another. The addition of a guest (solid black) shifts this equilibrium to the products that best bind it. Note that if only one building block is used, it must be a bifunctional molecule (for example dithiols).

Due to this great interest the European Commission fully granted a Marie Curie Research Training Network for Dynamic Combinatorial Chemistry. This Ph.D. research work and the author were funded through this European grant. Together with Kiel, nine other laboratories, in different European countries, were associated in the network with a common goal: bringing Dynamic Combinatorial Chemistry to the next step and train young European scientists in this novel ﬁeld.

1.2.1 Imines

Imine exchange, with disulfide and hydrazone exchange, is one of the most used reversible reaction in Dynamic Combinatorial Chemistry. The condensation between aldehydes and amines is a reversible reaction. In order to obtain imines in good yield, 6 1 Introduction ______it is usually important to remove water. In fact the imine formation (from amine and aldehydes or ketones) is accompanied with the release of one molecule of water for each imine formed. In the DCC specific case hydrolysis plays an important role for the reversibility of the reaction. The freshly formed imines can be hydrolyzed and the starting material will be free to react again forming a different imine. Imines can also be interconverted by transimination. 21 Beyond DCC, imines were and are still widely used for several reasons. Since their discovery by Hugo Schiff in 1864 (imines are also known as Schiffʼs bases) 22 they found broad applications in organic and inorganic chemistry. The reason for this interest by organic chemists lies in the fact that condensation between aldehydes and imines is considered an easy reaction and that the generated imines can then be used as versatile key intermediates in various synthetic routes: from reductive amination 23, 24 to multicomponent reactions like the Mannich 25 or the Ugi reaction. 26 The latter was also one of the first reaction used in (non reversible) combinatorial chemistry. 27 Inorganic imino-complexes with different transition metal ions are used since a long time as catalysts. Salen, salen-like complexes 28 and bis(imino)pyridines 29 are doing lionʼs share within the large plethora of related catalysts. Moreover imine formation is extensively used in macrocycle syntheses. 30, 31 Even when this reaction was exploited in a large number of syntheses, it has only recently become an essential part of Dynamic Combinatorial Chemistry. In 2002, Ole Storm elegantly demonstrated that imino-macrocycles can efficiently be synthesized from a Dynamic Combinatorial Library (Fig. 1.5). 13 The reaction of a dialdehyde with diamino chains of different lengths produces various imino-macrocycles, oligo- and polymers. Then using different alkaline earth metal ions, it is possible to shift the equilibria to mainly only one of the various products. Cations act as templates for the formation of the macrocycle amplifying the best binder and shifting all the other products of the library to it. Several products can selectively be obtained starting from a single Dynamic Combinatorial Library. At the end of equilibration, imines are reduced to amines in order to “freeze” the dynamic library and to screen it. This step is necessary because imines usually are not stable enough to purification steps, some of them could easily be hydrolyzed giving back a different library composition during the screening. Unfortunately, once the imino-macrocycle is reduced to an amino-macrocycle it will not exactly be the amplified binder anymore as the imine is reduced to an amine. FULL PAPER U. L¸ning and O. Storm

oligomers and polymers into the desired macrocycle. It would therefore be advantageous to force the desired macrocycle to be the most stable product in order to exploit the repair mechanisms in an equilibrium of a thermodynamically controlled reaction. Thus the desired macrocycle has to be stabilized relative to the oligomers and polymers. Template molecules or template ions fulfill this job, and such a template effect has already been exploited for the synthesis of many macrocycles and other target molecules from a complex reaction mixture (Fig- ure 1).[17±23]

Scheme 1. The reaction system examined, which consisted of dialdehydes 2 1 or 2, diamines 3a± c and alkaline earth metal ions M .

FULL PAPER use of different templates. FinallyU. L¸ning the and parallel O. Storm synthesis of more than one macrocycle by the simultaneous use of more 1 Introduction U. L¸ning and O. Storm 7 FULL PAPERFULL PAPER ______oligomers and polymers into the desiredU. L¸ning macrocycle. and O. Storm It would than one template has been investigated. therefore be advantageous to force the desired macrocycle to oligomers and polymers into the desired macrocycle. It would oligomers and polymers into the desired macrocycle. It wouldbe the most stable product in order to exploit the repair therefore be advantageoustherefore to force be advantageous the desired to macrocycle force the desired to macrocycle to Alkaline earth be the most stable productbe the most in order stable to product exploit in the order repair to exploit the repairmechanisms in an equilibrium of a thermodynamically con- mechanisms in an equilibriummechanisms of in a an thermodynamically equilibrium of a thermodynamically con- con-trolled reaction. metal ions Results and Discussion

= trolled reaction. trolled reaction. Thus the= desired macrocycle has to be stabilized relative to Thus the desired macrocycleThus the has desired to be macrocycle stabilized has relative to be stabilized to relative tothe oligomers and polymers. Template molecules or template The general procedure for the reaction of dialdehyde 1 with the oligomers and polymers.the oligomers Template and polymers. molecules Template or template molecules or template diamines 3a± c was chosen to be comparable with established ions fulfill this job, and such a template effect has already beenions fulfill this job, and such a template effect has already been ions fulfill this job, and such a template effect has already been [31±33] exploited for the synthesis of many macrocycles and otherexploited for the synthesis of many macrocycles and other synthetic procedures. These former experiments describe exploited for the synthesis of many macrocycles and other A B M2+ target molecules from a complex reaction mixture (Fig-target molecules from a complex reaction mixture (Fig- the use of a single template ion to obtain a single macrocycle target molecules from a[17±23] complex reaction mixture (Fig- ure 1). [17±23] 2 2 2 ure 1).[17±23] ure 1). (Mg for 6a,Ca for 6b, and Sr for 6c). The reaction times were now standardized for all experiments and therefore, in Figure 1. Representation of some possible members of the virtual combi- general, were longer than in the original procedure. The natorial library examined. amount of solvent was also standardized at a high level; this resulted in lower overall concentrations. In all cases, the The equilibria discussed above form virtual combinatorial reduction of the diimines 4 was achieved by the same large libraries (VCL).[24±27] All members of such a library are excess of sodium borohydride (13.5 mmol). In contrast to Scheme 1. The reaction system examined, which consisted of dialdehydes constantly undergoing interconversion2 into each other. The former experiments, several alkaline earth salts were used at 1 or 2, diamines 3a± c and alkaline earth metal ions M . Scheme 1. Theuse reaction of a system feasible examined, template which consisted can of select dialdehydes the proper macrocycle the same time. The yields were determined from the crude 2 1 or 2, diamines 3a± c and alkaline earth metal ions M . fromuse such of different a library. templates. The application Finally the parallel of the synthesis metal-ion-template of Schemereaction 1. The reaction mixtures system by examined,1H NMR which spectroscopy consisted of dialdehydes after addition of a 1 or 2, diamines 3a± c and alkaline earth metal ions M2 . effectmore to than the one formation macrocycle of by macrocyclic the simultaneous pyridine use of more diimines dates standard. All results are listed in Tables 1 ± 4, below. use of differentthan templates. one template Finally has the been parallel investigated. synthesis of back to the work of Nelson.[28±30] Here, we would like to The reaction of dialdehyde 1 with all three diamines 3a± c more than one macrocycle by the simultaneous use of more use of different templates. Finally the parallel synthesis of than one templatedemonstrate has been the investigated. selection of a macrocycle from a VCL for the in the absence of any template salt and final reduction yielded more than one macrocycle by the simultaneous use of more formation of macrocyclicResults and Discussion diimines 4a± c and macrocyclic only 9% of 6b and literally no 6a or 6c (Table 1, entry 1). diamines 6a± c, which can easily be obtained from 4a± c bythan onePresumably template hasall reactants been investigated. form some imine structures includ- reductionThe general (Scheme procedure 1). for The the resulting reaction of diaminesdialdehyde 16withare less potent ing oligo- and polymeric ones. However, none of these is diaminesResults3a and± c was Discussion chosen to be comparable with established ligandssynthetic for procedures. alkaline[31±33] earthThese ions former than experiments diimines describe4, and thus metal especially favored, and this results in a large variety of The generalfree procedurethe macrocycles use of for a single the reaction6 templatecan be of ion isolated. dialdehyde to obtain Thisa1 singlewith template macrocycle reaction has different productsResults andafter Discussion reduction. On addition of alkaline 2 2 2 diamines 3a± c (Mgwas chosen for 6a,Ca to be for comparable6b, and Sr with for established6c). The reaction times synthetic procedures.alreadywere now been[31±33] standardizedThese utilized former for experiments all the experiments synthesis describe and of therefore, a single in macrocycle earth ions of increasing size to a mixture of dialdehyde 1 and Figure 1. Representation of some possible members of the virtual combi- The general procedure for the reaction of dialdehyde 1 with Figurefrom 1.5:general, Onthe top, dialdehyde were starting longer materials than1 and in used the a diamine originaland their procedure. schematic3, for instance, The representation. in the diamine 3a, the resulting yield of 6a decreases as the ionic natorial library examined. the use of a single template ion to obtain a single macrocycle 2 Dialdehyde A2 (R = OMe and H),2 diamine B (n = 2,3,4) and various alkaline earth metal[31±33] diamines 3a± c was chosen to be comparable with established2 (Mg for 6areaction,Caamount for sequence6b of, solvent and Sr was forfor also6c bimacrocyclic). standardized The reaction at times a concave high level; this pyridines, radii increase (entries 2 ± 4). The small Mg ion fits well into 2+ 2+ 2+ 2+ [31±33] were nowions standardized (Mwhich=resulted Mg possess, Ca in for, lower all Ba appealing experiments).overall On bottom, concentrations. andselectivities schematic therefore, representation In all in in cases, the of the acylation the dynamic ofsyntheticthe procedures.macrocycle 4a,These the larger former ions experiments are less suitable describe for forming Figure 1. Representation ofThe some equilibria possible members discussed of abovethe virtual form combi- virtual combinatorialcombinatorialreduction library. The of the equilibration diimines forms4 was different achieved products. by the The same addition large of a guest general, were longer than in the original procedure. The [34±36] the use of a single template ion to obtain2 a single macrocycle natorial library examined.libraries (VCL).[24±27] All members of such a library arealcoholsexcess ofand sodium monosaccharides borohydride (13.5 mmol). with ketenes. In contrast to We have a [1 1] diimine complex 4a¥M . Therefore the [2 2] dimer amount(solid of solvent black) shifts was alsothe library standardized towards the at a macrocycle high level; that this better binds it. Next, by 2 2 2 constantly undergoing interconversion into each other. The former experiments, several alkaline earth salts were used at (Mg for6a,Ca for 6b, and Sr for 6c). The reaction times resultedreducing innow lower the investigatedimines overall to amines concentrations. and the removing possibility the In guest, all of cases, free controlling macrocycles the theare obtained. selection of 8a could be found in notable quantities when larger tem- use of a feasible template can select the proper macrocycle the same time. The yields were determined from the crude were now standardized for all experiments and therefore, in Figuredifferent 1. Representation macrocycles of some from possible the same membersreaction of the virtual mixture combi- by the plate ions were used (entries 3 and 4). The yield of The equilibria discussedfrom such above a library. form The virtual application combinatorial of the metal-ion-templatereduction of thereaction diimines mixtures4 was by achieved1H NMR byspectroscopy the same after large addition of a general, were longer than in the original procedure. The libraries (VCL).[24±27] All members of such a library are excess ofnatorial sodium library borohydride examined. (13.5 mmol). In contrast to effect to the formation of macrocyclic pyridine diimines dates standard. All results are listed in Tables 1 ± 4, below. amount of solvent was also standardized at a high level; this [28±30] Since Ole Stormʼs paper appeared in the literature, imine formation grows in constantly undergoingback interconversion to the work of into Nelson. each other.Here, The we wouldformer like experiments, to794 The several reaction alkaline of dialdehyde¹ earthWILEY-VCH1 saltswith were all Verlag three used diamines GmbH, at 694513a± c Weinheim, Germany, 2002 0947-6539/02/0804-0794 $ 17.50+.50/0 Chem. Eur. J. 2002, 8, No. 4 use of a feasible templatedemonstrate can select the selection the proper of a macrocycle macrocycle from a VCLtheimportance same for the time. asin The a the reversible yields absence were ofreaction any determined template in DCC. salt from and In the final the last reductioncrude decade, yielded many publicationsresulted in lower overall concentrations. In all cases, the from such a library. Theformation application of macrocyclic of the metal-ion-template diimines 4a± c andreaction macrocyclicappeared mixtures regardingTheonly equilibriaby 9%1 HDCLs NMR of 6bmade discussed spectroscopyand of literally imines. above no after 326a,33, additionform34or Related6c (Table virtual of work a 1, combinatorialentry was 1). done by Vicentereduction of the diimines 4 was achieved by the same large effect to the formationdiamines of macrocyclic6a± c, which pyridine can easily diimines be obtained dates fromstandard.4a± c bylibraries All resultsPresumably (VCL). are listed all[24±27] reactants in TablesAll form 1 members ± 4, some below. imine of structures such a includ- library are excess of sodium borohydride (13.5 mmol). In contrast to back to the work ofreduction Nelson. (Scheme[28±30] Here, 1). The we resulting would diamines like to 6 are lessThe potent reactionconstantlying of dialdehyde oligo- undergoing and polymeric1 with interconversion all threeones. However, diamines into3a none± c each of these other. is The former experiments, several alkaline earth salts were used at demonstrate the selectionligands of for a macrocycle alkaline earth from ions a than VCL diimines for the 4, andin thus the metal absence ofespecially any template favored, salt and final this results reduction in ayielded large variety of free macrocycles 6 can be isolated. This template reaction hasuse ofdifferent a feasible products template after reduction. can select On addition the proper of alkaline macrocycle the same time. The yields were determined from the crude formation of macrocyclic diimines 4a± c and macrocyclic only 9% of 6b and literally no 6a or 6c (Table 1, entry 1). 1 already been utilized for the synthesis of a single macrocyclefromearth such ions a library. of increasing The application size to a mixture of ofthe dialdehyde metal-ion-template1 and reaction mixtures by H NMR spectroscopy after addition of a diamines 6a± c, which can easily be obtained from 4a± c by Presumably all reactants form some imine structures includ- from the dialdehyde 1 and a diamine 3, for instance, in theeffectdiamine to the3a formation, the resulting of macrocyclic yield of 6a decreases pyridine as diiminesthe ionic dates standard. All results are listed in Tables 1 ± 4, below. reduction (Scheme 1). The resulting diamines 6 are less potent ing oligo-[31±33] and polymeric ones. However, none of these2 is reaction sequence for bimacrocyclic concave pyridines, backradii to theincrease work (entries of Nelson.2 ± 4). The[28±30] smallHere, Mg ion we fits would well into like to The reaction of dialdehyde 1 with all three diamines 3a± c ligands for alkaline earthwhich ions possess than diimines appealing4, selectivities and thus metal in the acylationespecially of favored,the macrocycle and this4a results, the larger ina ions large are less variety suitable of for forming [34±36] demonstrate the selection of a macrocycle2 from a VCL for the in the absence of any template salt and final reduction yielded free macrocycles 6 canalcohols be isolated. and monosaccharides This template reaction with ketenes. has differentWe have productsa [1 after1] diimine reduction. complex On4a¥ additionM . Therefore of alkaline the [2 2] dimer already been utilizednow for theinvestigated synthesis the of possibility a single macrocycle of controlling theearth selection ions offormation of increasing8a could of size be macrocyclic found to a mixture in notable diiminesof dialdehyde quantities4a when1± andc and larger macrocyclic tem- only 9% of 6b and literally no 6a or 6c (Table 1, entry 1). from the dialdehyde different1 and a macrocycles diamine 3, from for instance, the same inreaction the mixturediamine by the3adiamines, theplate resulting6a ions± c yield were, which of used6a candecreases (entries easily 3 be andas obtainedthe 4). ionic The from yield4a of ± c by Presumably all reactants form some imine structures includ- [31±33] 2 reaction sequence for bimacrocyclic concave pyridines, radii increasereduction (entries (Scheme 2 ± 4). The 1). small The Mg resulting ion fits diamines well into6 are less potent ing oligo- and polymeric ones. However, none of these is 794 ¹ WILEY-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002 0947-6539/02/0804-0794 $ 17.50+.50/0 Chem. Eur. J. 2002, 8, No. 4 which possess appealing selectivities in the acylation of the macrocycleligands4a, for the alkaline larger ions earth are less ions suitable than diiminesfor forming4, and thus metal especially favored, and this results in a large variety of [34±36] 2 alcohols and monosaccharides with ketenes. We have a [1 1] diimine complex 4a¥M . Therefore the [2 2] dimer free macrocycles 6 can be isolated. This template reaction has different products after reduction. On addition of alkaline now investigated the possibility of controlling the selection of 8a could be found in notable quantities when larger tem- different macrocycles from the same reaction mixture by the plate ionsalready were been used utilized (entries 3 for and the 4). synthesis The yield of a ofsingle macrocycle earth ions of increasing size to a mixture of dialdehyde 1 and from the dialdehyde 1 and a diamine 3, for instance, in the diamine 3a, the resulting yield of 6a decreases as the ionic [31±33] 2 794 ¹ WILEY-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002 0947-6539/02/0804-0794reaction sequence $ 17.50+.50/0 for bimacrocyclicChem. Eur. J. 2002 concave, 8, No. 4 pyridines, radii increase (entries 2 ± 4). The small Mg ion fits well into which possess appealing selectivities in the acylation of the macrocycle 4a, the larger ions are less suitable for forming [34±36] 2 alcohols and monosaccharides with ketenes. We have a [1 1] diimine complex 4a¥M . Therefore the [2 2] dimer now investigated the possibility of controlling the selection of 8a could be found in notable quantities when larger tem- different macrocycles from the same reaction mixture by the plate ions were used (entries 3 and 4). The yield of

794 ¹ WILEY-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002 0947-6539/02/0804-0794 $ 17.50+.50/0 Chem. Eur. J. 2002, 8, No. 4 8 1 Introduction ______

Gotorʼs group in Oviedo. Starting from a library composed of pyridine-2,6- dicarbaldehyde and trans-cyclohexane-1,2-diamine they ampliﬁed different macrocycles (dimer and trimer) using various cations. 35 A few years later they discovered a diastereoselective ampliﬁcation from a DCL of macrocyclic oligoimines. Using cadmium ions they were able to shift the equilibria to one major product as a single diastereomer from a mixture of stereochemically different species. 36

Although many different DCLs were exploited and many receptors were discovered and synthesized using this methodology, the “road to fulﬁlling the promise” of using DCC for new areas of application is still long. 37

1.2.2 The next step: Applications

At the beginning of this thesis, DCLs were mainly applied in order to synthesize the “best binder” for a specific guest. It was time to move on to the next step, namely applications. Few publications appeared in recent years describing the use of a Dynamic Combinatorial Library directly for a function instead of single purified products. 38,39,40,41 This perspective is growing in importance day after day. 42 Without spoiling the rest of this thesis during the introduction, it is gratifying to state here that we were the first, 43 in parallel to Jeremy Sandersʼ group in Cambridge, 44 to use directly a dynamic combinatorial library for the screening of a transport carrier. Transport of molecules from a source phase to a receiver phase passing through a membrane is a remarkable task but usually difficult to achieve. In nature this task is done in various ways, from channels to carriers (Fig. 1.6) and by various mechanisms like passive or active transport. 45 Those channels and carriers are relevant in cells, where the concentrations of metabolites, salts and pH make the difference between life and death. Chemists have, since a long time, tried to mimic and understand transport in nature by studying synthetic membranes and carriers or channels. Various biological membrane mimics were used in order to understand how the transport works. Nowadays a number of membranes have been used and studied: bulk membrane, supported liquid membrane and liposomes (see pg. 34 for bulk membrane experiment set-up, and pg. 46 for supported liquid membrane experiment set-up). Each of those membranes has its weak and strong points. For example, if it is possible to use a bulk membrane for months, it will be difficult doing the same with the more fragile liposomes. 1 Introduction 9 ______Bilayer mechanisms

Carriers: Channels: Disrupting valinomycin, gramicidin, Agents: crown ethers. amphotericin. mellitin, detergents.

Figure 1.6: Schematic representation of carriers, two different self assembled channels,

!""#$%!!!$&"'$()*+,-).$46 78 and disrupting/012*340$5*34,.)*6 agents.

Parallel to studying different membranes, the focus laid also on synthesizing artiﬁcial channels and carriers.

Channels and carriers have great importance in many different fields. Ion sensors are mainly based on carriers immobilised in a polymeric film. When in contact with a fitting ion, it will be transported across the polymeric film to the detector, signalling the presence of the ion. Using a specific carrier for a given ion is also helpful in the cleaning of wastewater (for example to remove toxic transition metal ions or radioactive particles) or for blood dialysis. Moreover as stated a couple of pages above (Ch. 1.1), drug delivery is one of the major research lines in pharmaceutical industries with a special focus on targeting. Even if we do have effective drugs against cancer, this is still one of the worst diseases nowadays. The main problem of those anti-cancer medicines is that they are not target specific. Those drugs will simply “kill” indistinctly different cells, both diseased and normal cells. For in vivo imaging, small molecules are easily accessible but it is important to deliver them to the right cell that needs to be screened, otherwise they become useless. For those reasons, it is essential to focus on the synthesis of water soluble carriers for a specific guest. It is also notable to modify it in order to recognize or be recognized by a targeted cell. These two tasks are usually translated into hard bench work in order to synthesize the carrier step by step and to optimize it. To overcome and simplify this task, this thesis shall explore the possibility of using directly a Dynamic Combinatorial Library in order to screen for a specific carrier. 10 2 Objective ______

2 Objective

By the use of Dynamic Combinatorial Chemistry (DCC), it is possible to synthesize in one step the host that best binds a speciﬁc guest used as a template. Moreover from a single Dynamic Combinatorial Library (DCL), a number of products can be ampliﬁed using various templates. The advantages of using the DCC instead of a step by step synthesis is clear. In a schematic summary, the differences between the two approaches are even more evident (Fig. 2.1).

Step by Step DCC Synthesis Building Blocks / DCL

Template

n Puriﬁcation Ampliﬁcation

Optimization “Stop DCL”

Test Puriﬁcation

Function Function

Figure 2.1: Schematic representation of a step by step synthesis (left) for n synthetic steps, and Dynamic Combinatorial Chemistry (right). This thesis will study whether it will be possible to screen for a function directly by the use of a dynamic library (right, dashed arrow).

In a step by step synthesis (Fig 2.1, left), every single intermediate of the synthetic route must be synthesized and purified. Usually the first reaction tried does not work with 90% of yield immediately. It is then necessary to carry out some optimization work to find out the best reaction conditions. All of these steps must be done n times in a multi-step synthesis. At the end of hard bench-work, it is finally possible to test the product for an application. 2 Objective 11 ______

On the contrary in the DCC approach (Fig 2.1, right), the building blocks are mixed, the template is added and the mixture is left to equilibrate. When, and if, an amplification occurs, it is necessary to stop the equilibration and to purify the final amplified product. At this point, the product is ready to be tested for an application. The main goal of this thesis is bringing DCC one step further: to use a DCL directly to screen for a function. In this way it should be possible to skip several steps in the DCC flowchart (Fig. 2.1, right, dashed arrow).

Using the dynamic library directly instead of a purified product should result not only in an amplification of a product, but in an amplification of a product able to perform a task. The benefit of synthesizing a product for an application in only one step is, without any doubt, attractive. Many functions are not yet screened by a DCL and this thesis focuses on transport as the function. DCC is still young and there are no proofs of using a library in order to amplify a carrier for transport. To achieve this goal it is wise starting from a well known DCL. It is based on imines and it was used in our laboratory. 13 As a first guest to be transported, calcium ion was chosen. This ion was used in a previous investigation and its ability to shift a specific imine DCL was already proved. 13 In general, this proof of concept can be possible for many ions and guest molecules. For a good and full study of using a DCL to amplify a carrier, various test must be done. First of all, it is necessary to study the stability of imines in water and how the ionic guest influences this stability. After this, the dynamic library must be tested directly in presence of different synthetic membranes such as bulk membranes, supported liquid membranes and liposomes. Another interesting task will be the set up of a double screening experiment: When more than only one product is amplified from a DCL which one will perform a better transport? Moreover, in order to prove once more the simplicity of the proposed methodology, only a few building blocks (commercially available or easily synthesizable) shall be used to set up the libraries. 12 3 Results and Discussion ______

3 Results and Discussion

Dynamic Combinatorial Chemistry (DCC) has gained large interest since its discovery. Using Dynamic Combinatorial Libraries (DCLs), it is possible to synthesize a host for a template guest in only one step. However the direct application of a DCL instead of a single isolated product is hardly investigated at all. This Ph.D. thesis shall investigate the proof of concept of using a DCL for the screening and the ampliﬁcation of a carrier. The main question is if this latter, directly obtained from the DCL, will be able to transport ions from a water source phase to a water receiver phase through synthetic organic membranes? The methodology of this screening is hitherto unknown in literature and it must be proved for the ﬁrst time in a series of different experiments.

This chapter summarizes five publications obtained as part of the Ph.D. thesis work. Each publication has its own introduction page. The first two publications treat the role of water in imine synthesis. In the first one, the stability of various imine products is tested and the influence of a template ion on their stability is discussed (Ch. 3.1). During this work, a big discrepancy about synthesis of imines in pure water was found in the literature. The explanation of this discrepancy and its solution is described in chapter 3.2. The third publication presents the first example of using a Dynamic Combinatorial Library to screen for a carrier. Using a bulk membrane and a dynamic library, it is possible to amplify a macrocyclic carrier. This latter transports the template ion from an aqueous source phase to an aqueous receiver phase passing through an organic solvent membrane (Ch. 3.3). A further development is in the fourth publication: the double screening methodology. In a first stage, the template amplifies two similar macrocycles from the dynamic library. Subsequently, the membrane itself discriminates between these related macrocyclic carriers to find the best one (Ch. 3.4). When cations are transported, one way to keep the electroneutrality of the system, is transporting anions simultaneously. If a macrocycle is designed for complexing cations, the transport of the counter-ions is not easy. Mainly because this latter, during the transport, is not shielded at all from organic solvent. For this reason a study was started by using a macrocycle that binds both ion and counter-ion. The fifth publication is the first description of an ion triplet complex (Ch. 3.5). 3 Results and Discussion 13 ______

3.1 Remarkable Stability of Imino Macrocycles in Water V. Saggiomo, U. Lüning, Eur. J. Org. Chem., 2008, 4329-4333.

Water plays an important role in the DCC of imines. Hydrolysis of imines is one of the possible pathways for an imine exchange: the reaction of the imine with water to give back the aldehyde which then can react with a second amine. The study of the following publication was set up to test the stability of imines at different percentages of water. Three different compounds were synthesized and left in methanol to equilibrate using calcium ions as template. After equilibration, various amounts of water were added. The NMR spectra were recorder after each addition in order to check the stability to hydrolysis. Three different amines were chosen. One has the proper length to form a good host for complexing calcium ions (Fig. 3.1, a). One is a longer diamine but with the same number of donor atoms (Fig. 3.1, b). The last one forms a product that has almost no afﬁnity to the template (Fig 3.1, c). All NMR experiments show that one (bis)imino pyridine (Fig. 3.1, a) is extremely stable to water at room temperature also for one week. In pure water, this macrocycle can be obtained in good yield when 2 equivalents of template are used.

OMe

O H2N 3 NH2 N a) N N

O O O

OMe

OMe H2N O NH2 3 N b) N N N O O O O 1 O

OMe

O H N NH 2 nBuNH 3 2 N a) 2 c) N NN

O O O Me Me

Figure 3.1: Schematic representation of the experiments. The synthesesOMe were done in

MeOD and then, different aliquots of D2O were added to test the stability of the three OMe H N O NH different products (on the right). 2For the two macrocycles,2 different amounts of CaCl2 as 3 N template wereb) used. N N N O O O O 1 O

OMe

N nBuNH2 c) N N

Me Me 14 3 Results and Discussion ______FULL PAPER

DOI: 10.1002/ejoc.200800462

Remarkable Stability of Imino Macrocycles in Water[‡]

Vittorio Saggiomo[a] and Ulrich Lüning*[a]

Keywords: Combinatorial chemistry / Imines / Macrocycles / Pyridine / Templates

Imines 3, 5 and 7 generated from 4-methoxypyridine-2,6-di- anol (50:50) without any template effect. In a mixture of pyr- carbaldehyde (1) and several amines 2, 4 and 6 show a re- idinedicarbaldehyde 1 and two glycol-derived diamines 4 markable stability in water. The 18-membered macrocyclic and 6, a calcium template ion selects the 18-membered diimine 5 is stable and could be synthesized in good yield by macrocycle 5 over the 20-membered one (7). using 2 equiv. of calcium ions as template in pure water, and (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, even the non-macrocyclic diimine 3 survives in water/meth- Germany, 2008)

Introduction leads to an increased stability of the imines even in water.[15] But in general, imines are rather unstable and are hy- Macrocyclic structures have gained enormous attention drolyzed quickly if water is present.[16–18] However, converse [1] over the past decades. However, macrocycles are not as results have also been reported.[19] In the DCC synthesis of [2] easily prepared as five- or six-membered rings, and thus macrocyclic imines, too, the sensitivity against water has to special approaches have been developed for better synthe- be investigated for several reasons: (i) every formation of an ses. For kinetically controlled macrocyclizations, the use of imine from an aldehyde and an amine forms a water mole- [3] the high-dilution technique often increases yields con- cule, (ii) the template salts often contain water,[14,20] (iii) the siderably, whereas in thermodynamically controlled reac- reactions are usually not carried out under strict exclusion tions, the template effect has proven to be the proper tool of moisture and (iv) the hydrolysis plays an important rule [4–6] to shift the equilibrium to the desired product. Many in the reversibility of the imine bond formation.[21] applications of macrocycles including the metal-ion-complexing abilities of crown ethers make use of endo functionalities. Therefore, it is self-evident that these functionalities Results and Discussion can be exploited in the macrocyclic assembly process.[7] Un- like in a kinetically controlled reaction, the products of a Due to the instability of imines under the conditions of thermodynamically controlled reaction are interconverted many standard purification techniques, the analysis of an [22] into one another constantly. The composition of the prod- imine library is rarely performed on the imine mixture uct mixture is depending on the relative thermodynamic itself but usually done by investigating the amine mixture [14] stability of the products. The mixture is dynamic. If poly- obtained by reduction of the imines. Besides HPLC- [22] functional molecules or several starting materials are used, MS, one other direct method to monitor the equilibrium numerous different combinations are conceivable, and usu- is the observation of the imines by NMR spectroscopy. In ally mixtures are formed. For these situations, the term this work, dynamic combinatorial libraries (DCL) derived dynamic combinatorial chemistry (DCC) has been from pyridine-2,6-dicarbaldehyde 1 and various diamines 1 coined.[8–11] Since decades,[4] the imine formation has been such as 2, 4 or 6 have been investigated by H NMR spec- used to synthesize macrocycles, and templates have been troscopy. One prerequisite for the analysis is the use of a 4- found to shift the equilibria of the dynamic combinatorial substituted pyridine-2,6-dicarbaldehyde such as 4-methoxy- libraries towards one product in good yield. Dynamic com- pyridine-2,6-dicarbaldehyde (1) because the pyridine hydro- binatorial libraries have been investigated by starting from gen atoms of 2,4,6-trisubstituted pyridines show a sharp a dialdehyde and one[12,13] or several[14] diamines. The coor- singlet in the NMR spectra. If the dynamic combinatorial dinative bond between a transition-metal ion and an imine library gives a manageable number of products, their relative ratio can be determined by integration of these pyridine [‡] Dynamic Combinatorial Chemistry, 2. Part 1: Ref.[14] 3,5-H peaks. Quantitative measurements are possible if an [a] Otto-Diels-Institut für Organische Chemie, internal standard (for instance dimethyl terephthalate, Christian-Albrechts-Universität zu Kiel, DMT) is added. Many components of a library can also be Olshausenstr. 40, 24098 Kiel, Germany Fax: +49-431-880-1558 detected by ESI mass spectrometry, although no quantita- E-mail: [email protected] tive analysis is possible by this analytic technique. In the

Eur. J. Org. Chem. 2008, 4329–4333 © 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 4329 3 Results and Discussion 15 ______FULL PAPER V. Saggiomo, U. Lüning present study, three different amines [n-butylamine (2), a be detected. With 50% of water, still 90% of macrocycle 5 triethylene glycol derived diamine 4 and a bis(homo) ana- exists. This surprising stability led us to an experiment in logue 6] have been used. From the resulting imines 3, 5 and pure water by using various amounts of templating calcium 7, only the largest macrocycle 7 was well detected by ESI ions. In pure water, the quantitative analysis of the NMR [M + H+], whereas the smaller macrocycle 5 appeared as signals against the standard DMT cannot be carried out an [M + Ca2+]/2 signal, and the butyl compounds could anymore due to DMT’s insolubility, but, to calculate the not be detected at all (Figure 1). yield, the intensities of the imine peak and the aldehyde peak can be compared according to literature.[16] With 1 or less equiv. of calcium ions, the reaction mixture was not a solution, but a suspension, due to the fact that aldehyde 1 does not have sufficient solubility in water.

Figure 2. 1H NMR spectrum (500 MHz, 298 K) of imine 3 in: (a) CD3OD, (b) CD3OD/D2O, 98:2, (c) CD3OD/D2O, 90:10, (d) CD3OD/D2O, 50:50. All ratios are expressed in v/v. Capital letters indicate prominent resonances: I = imine, S = DMT, P = pyridine, A = free amine chain.

Figure 1. (a) CD3OD/D2O in various ratios, 12 h, room temp.; (b) CD3OD/D2O in various ratios with 1 equiv. of CaCl2, 12 h, room However, with 2 equiv. of calcium ions, the solution be- temp., and pure D2O with various equiv. of CaCl2, 12 h, room came clear, and only one product 5, as the calcium complex, temp.; (c) CD3OD/D2O in various ratios with 1 equiv. of CaCl2 12 h, room temp. was detected (Figure 3). Thus, even in water, a calcium ion stabilizes the macrocyclic diimine 5 to such an extent that it becomes the only product! In contrast to the stabilization However, all three compounds gave nice and clear signals of a macrocycle by transition-metal ions, which bind donor in the NMR spectra, well distinguishable from each other atoms by coordinative bonds, here, the interactions between or from oligomer signals. The individual imine synthesis, the calcium ion and the donor atoms of the macrocycle are as well the imine-based libraries, usually is carried out in predominantly dipol–ion interactions. But these interac- methanol,[6,20,14] and consequently the reaction of 4-meth- tions are strong enough to withstand the competing sol- oxypyridine-2,6-dicarbaldehyde (1) with n-butylamine (2) vation by 55  water, and macrocycle 5 is found exclusively. gave the respective diimine 3 in good yield. Increasing per- The reaction with the longest diamine 6, which contains centages of water reduced the yield of 3 as could be deduced two aminopropylene groups instead of aminoethylene ones, from the integrations of the respective signals in the NMR gave less clear results. Also in methanol, the macrocyclic spectrum (Figure 2, for instance NH2CH2 of the free butyl diimine 7 was formed in good yield (Figure 4a) and could chains at δ = 2.7 ppm, and CH of the aldehyde in its hydrate be analyzed by ESI mass spectrometry or NMR spec- or hemiacetal form at δ ≈ 5.5 ppm). If more than 50% of troscopy. However, in contrast to the macrocycle formation water was added, the solvent became too polar to dissolve of 5, the addition of water disturbs the formation of the the starting materials completely. However, the diimine 3 macrocyclic diimine 7 (Figure 4). The NMR signals proved to be rather stable, and 68% of it was still detected broaden, and increasing amounts of free diamine 6 appear. when the solvent contained 50% of water. Next, the forma- But also in this case, the addition of templating calcium tion of macrocyclic diimine 5 was investigated in the pres- ions favors the formation of the macrocycle, and for in- ence of 1 equiv. of calcium ions. In methanol with various stance with 10 equiv. of CaCl2 in CD3OD/D2O (50:50), only percentages of water, no change in the composition could the signals of the macrocycle 7 were observed again.

Remarkable Stability of Imino Macrocycles in Water

1 Figure 3. H NMR spectrum (500 MHz, D2O, 298 K). Synthesis of Figure 5. Exchange experiment. (a) 1 equiv. of dialdehyde 1, imine 5 in pure D2O with: (a) no CaCl2, (b) 1 equiv. of CaCl2, (c) 1 equiv. of diamine 6, and 1 equiv. of CaCl2 in CD3OD give the 2 equiv. of CaCl2. Capital letters indicate prominent resonances: I = imine, P = pyridine. macrocyclic diimine 7; (b) addition of 5% of D2O interfers with the exclusive formation of 7; (c) after addition of diamine 4 and equilibration for 12 h, the most stable diimine complex 5 is exclusively formed on the cost of the larger macrocycle 7 with liberation of diamine 6. Capital letters indicate prominent resonances: I = imine, S = DMT, P = pyridine, A = free amine chain.

Conclusions Even in water, the macrocyclic diimine 5 can be formed in excellent yields, provided that enough calcium ions are present. These have two functions: they stabilize this product by their template effect and they solubilize the complex by the introduction of charge. But not only the templating of the macrocycle contributes to the remarkable stability of diimine 5 in water. Even the non-macrocyclic diimine 3 is still formed in good yield in a solvent mixture containing 50% of water and 50% of methanol. This stability is proba- 1 Figure 4. H NMR (500 MHz, 298 K) of imine 7 in: (a) CD3OD, bly caused by the pyridine unit and its conjugation with the [23] (b) CD3OD/D2O, 98:2, (c) CD3OD/D2O, 90:10, (d) CD3OD/D2O, imine groups. 50:50, (e) CD3OD/D2O, 50:50, and 10 equiv. of CaCl2. All ratios are expressed in v/v. Capital letters indicate prominent resonances: I = imine, S = DMT, P = pyridine. Experimental Section General Remarks: n-Butylamine (2), 4,7,10-trioxa-1,13-tridecanediamine (6), and sodium cyanoborohydride were obtained commer- Finally, we investigated a dynamic combinatorial library cially from Fluka and used without further purification. 4-Meth- derived from the dialdehyde 1 and both diamines 4 and 6. oxypyridine-2,6-dicarbaldehyde (1)[20] and 3,6,9-trioxa-1,11-unde- [20] In the first experiment, a mixture of CaCl2, dialdehyde 1 canediamine (4) were synthesized according to literature pro- and the propylenediamine 6 was prepared, and the respec- cedures. NMR spectra were recorded with Bruker DRX 500 or AV tive imines were allowed to form. Then, 5% of water and 600 instruments. Assignments are supported by COSY, HSQC and 1 equiv. of ethylenediamine 4 were added one after the HMBC. All chemical shifts were referenced to the residual proton (1H) or carbon (13C) signal of the solvent [CD OD, δ = 3.35 (1H), other, and each mixture was analyzed by NMR spec- 3 49.0 (13C) ppm]. Mass spectra were recorded with a Finnigan MAT troscopy (Figure 5). First, the typical broadening of the sig- 8200 or MAT 8230. ESI mass spectra were recorded with an Ap- nals of the propylenemacrocycle 7 appeared (see Figures 4 plied Biosystems Mariner Spectrometry Workstation. and 5b), and after addition of 4, the most stable macro- General Procedure for the Preparation of Stock Solutions: To solu- cyclic diimine 5 was formed exclusively (shift of imine peak tions of 4-methoxypyridine-2,6-dicarbaldehyde (1, 9.6 mg, from δ = 8.60 to 8.64 ppm), and free propylenediamine 6 0.058 mmol), dimethyl terephthalate (DMT, 5.6 mg, 0.029 mmol) was detected (see for instance CH2CH2CH2 of the free and CaCl2 (6.4 mg, 0.058 mmol) in CD3OD (5 mL), various diamine chain: δ = 1.75 ppm), proving the formation of the amines 2, 4 or 6 (1.05 to 1.5 equiv.) were added. The solutions were more stable 18-membered macrocycle in this dynamic com- stirred at room temp. for 12 h and were then transferred into NMR binatorial library. tubes with various percentages of D2O. These solutions were al-

Eur. J. Org. Chem. 2008, 4329–4333 © 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.eurjoc.org 4331 3 Results and Discussion 17 ______FULL PAPER V. Saggiomo, U. Lüning lowed to stand for at least 4 h in order to equilibrate. In another DMT [δ = 8.15 (s, 4 H), 3.97 (s, 6 H) ppm] and imine protons: pure 1 set of experiments, water was added before the amines to start the CD3OD: 88%. H NMR (600 MHz, 298 K, CD3OD) of 7 in the reaction in aqueous media. The final volume for all NMR experi- mixture: δ = 8.60 (t, J = 1.2 Hz, 2 H, CH=N), 7.58 (s, 2 H, Py), ments was 600 µL, the tube’s atmosphere was replaced with nitro- 4.11 (s, 3 H, OMe), 4.01–3.92 [overlapping signals, 16 H, gen, and the tubes were capped with Teflon caps. CH2(CH2OCH2)3CH2, CH=NCH2], 2.19 (quint, J = 6.6 Hz, 4 H, 13 CH2CH2CH2) ppm. C NMR (150 MHz, 298 K, CD3OD): δ = 2,6-Bis(n-butyliminomethyl)4-methoxypyridine (3): A stock solution 171.58 [C-2,6 (Py)], 165.41 (CH=N), 154.59 [C-4 (Py)], 115.49 was prepared as follows: 4-methoxypyridine-2,6-dicarbaldehyde (1, [C-3,6 (Py)], 71.54, 71.21, 70.92, 70.45, 69.64, 68.94, 58.14 5 mg, 0.03 mmol) and DMT (2.15 mg, 0.011 mmol) were dissolved [CH (CH OCH ) CH , N=CH ], 57.39 (OMe), 29.04 in CD OD (2.5 mL). n-Butylamine (2, 6.2 µL, 0.063 mmol) was 2 2 2 3 2 2 3 (CH CH CH ) ppm. ESI-MS (positive ions) of the solution with added, and the solution was stirred at room temp. for 12 h. Then, 2 2 2 10% of water: calcd. for C18H27N3O4 349.42, found 350.29 [M + various percentages of water (2%, 10%, 50% v/v) were added, and + 1 H ]. HRMS: calcd. for C18H27N3O4 349.20016, found 349.20015; H NMR spectra were recorded after 4 h. Yields calcd. from the calcd. for C 13CH N O 350.20352, found 350.20405. ratio between signals of aromatic protons of DMT [δ = 8.15 (s, 4 17 27 3 4 Exchange Reaction between the Macrocyclic Diimines 7 and 5: A H), 3.97 (s, 6 H) ppm] and imine protons: pure CD3OD: 91%, 2% 1 stock solution was prepared as follows: 4-methoxypyridine-2,6-di- D2O: 88%, 10% D2O: 71%, 50% D2O: 68%. H NMR (600 MHz, carbaldehyde (1, 8.7 mg, 0.052 mmol), DMT (6.6 mg, 0.034 mmol), 298 K, CD3OD) of 3 in the mixture: δ = 8.40 (t, J = 1.2 Hz, 2 H, CH=N), 7.62 (s, 2 H, Py), 4.00 (s, 3 H, OMe), 3.77 (m, 4 H, and CaCl2 (5.7 mg, 0.052 mmol) were dissolved in CD3OD (5 mL). 4,7,10-Trioxa-1,13-tridecanediamine (6, 17.1 µL, 0.078 mmol) was CH=NCH2), 1.76 (quint, J = 6 Hz, 4 H, N=CH2CH2CH2CH3), added, and the solution was stirred for 12 h. Then, the solution 1.45 (m, 4 H, N=CH2CH2CH2CH3), 0.98 (t, J = 7.8 Hz, 6 H, CH3) 13 was transferred into an NMR tube with 5% of water and left at ppm. C NMR (150 MHz, 298 K, CD3OD): δ = 168.79 [C-2,6 (Py)], 163.09 (CH=N), 157.04 [C-4 (Py)], 109.43 [C-3,5 (Py)], 61.88 room temperature for 4 h. Next, 3,6,9-trioxa-1,11-undecanediamine (4, 1 equiv.) was added into the tube, and the reaction was (N=CH2), 56.38 (OMe), 33.77 (CH2CH2CH2), 21.38 (CH2CH3), left to equilibrate for 12 h. 1H NMR spectra were recorded in each 14.12 (CH2CH3) ppm. HRMS: calcd. for C16H25N3O 275.19977, 13 step. found 275.19992; calcd. for C15 CH25N3O 276.20313, found 176.20323. 14-Methoxy-6,9,12-trioxa-3,15-diaza-1(2,6)-pyridinahexadecacyclo- Acknowledgments phan-2,15-diene (5). (a): A stock solution was prepared as follows: 4-methoxypyridine-2,6-dicarbaldehyde (1, 8.7 mg, 0.052 mmol), We are grateful for the support by the Marie Curie Research Train- ing Network (MRTN-CT-2006-035614), Dynamic Combinatorial DMT (6.2 mg, 0.032 mmol), and CaCl2 (5.7 mg, 0.052 mmol) were Chemistry (DCC). dissolved in CD3OD (5 mL). 3,6,9-Trioxa-1,11-undecanediamine (4, 10.9 mg, 0.057 mmol) was added, and the solution was stirred for 12 h. Various percentages of water (2%, 10%, 50%) were added [1] a) Encyclopedia of Supramolecular Chemistry (Eds.: J. L. At- to different tubes, and 1H NMR spectra were recorded after 5 h. wood, J. W. Steed), Marcel Dekker, New York, 2004, p. 311– Yields calcd. from the ratio between signals of aromatic protons of 318; b) J. W. Steed, J. L. Atwood, Supramolecular Chemistry, DMT [δ = 8.15 (s, 4 H), 3.97 (s, 6 H) ppm] and imine protons: Wiley, Chichester, New York, Weinheim, Brisbane, Singapore, CD OD: 92%, 2% D O: 90%, 10% D O: 90%, 50% D O: 90%. Toronto, 2005, reprinted; c) Core Concepts in Supramolecular 3 2 2 2 Chemistry and Nanochemistry (Eds.: J. W. Steed, D. R. Turner, 1H NMR (600 MHz, 298 K, CD OD) of 5 in the mixture: δ = 8.64 3 K. J. Wallace), Wiley, Chichester, New York, Weinheim, Bris- (t, J = 1.2 Hz, 2 H, CH=N), 7.58 (s, 2 H, Py), 4.11 (s, 3 H, OMe), bane, Singapore, Toronto, 2007. 4.05 (t, J = 4 Hz, 4 H, CH=NCH2), 3.96–3.91 [overlapping signals, [2] Third Schiemenz “law” of organic chemistry: “whenever five- 13 12 H, (CH2OCH2)3] ppm. C NMR (150 MHz, 298 K, CD3OD): or six-membered rings may be formed, they will”. For an illus- δ = 169.87 [C-2,6 (Py)], 163.45 (CH=N), 153.05 [C-4 (Py)], 113.82 tration of the three “laws” see: U. Lüning, Organische Reak-

[C-3,5 (Py)], 70.92, 70.04, 69.74, 69.36, 69.05, 68.72 [(CH2OCH2)3], tionen – Eine Einführung in Reaktionswege und Mechanismen, Elsevier – Spektrum, Spektrum Akademischer Verlag, Heidel- 56.90 (N=CH2), 55.92 (OMe) ppm. HRMS: calcd. for C16H23N3O4 321.16885, found 321.16899; calcd. for C 13CH N O 322.17221, berg, 2007. 15 23 3 4 [3] See, e.g.: F. Vögtle, 1972, , 396–403. found 322.17218. (b): A stock solution was prepared as follows: Chem.-Ztg. 96 [4] Probably the first example of a metal-ion template for the for- 3,6,9-trioxa-1,11-undecanediamine (4, 10.9 mg, 0.057 mmol) was mation of imino macrocycles: N. F. Curtis, D. A. House, Chem. added to a solution of 4-methoxypyridine-2,6-dicarbaldehyde (1, Ind. 1961, 42, 1708. 8.7 mg, 0.052 mmol) in D2O (5 mL). Then various equivalents of [5] D. H. Cook, D. E. Fenton, M. G. B. Drew, S. G. McFall, S. M. CaCl2 were added to the solution in different NMR tubes, and the Nelson, J. Chem. Soc., Dalton Trans. 1977, 5, 446–449. solutions were left to equilibrate for 12 h. Then the 1H NMR spec- [6] U. Lüning, Liebigs Ann. Chem. 1987, 11, 949–955. tra were recorded. ESI-MS (positive ions) of the water solution [7] A recent example for the use of organic molecules as templates for the formation of a family of endo-functionalized molecules: with 2 equiv. of CaCl2: calcd. for C16H23CaN3O4 361.13, found 180.554 [M + Ca2+]/2. S. Lüthje, C. Bornholt, U. Lüning, Eur. J. Org. Chem. 2006, 909–915. 14-Methoxy-7,10,13-trioxa-3,17-diaza-1(2,6)-pyridinaoctadecacyclo- [8] Imine formation in the presence of a carbonic anhydrase as phan-2,17-diene (7): A stock solution was prepared as follows: 4- template: I. Huc, J.-M. Lehn, Proc. Natl. Acad. Sci. USA 1997, methoxypyridine-2,6-dicarbaldehyde (1, 8.7 mg, 0.052 mmol), 94, 2106–2110. [9] Steroid oligomers by transesterification: P. A. Brady, J. K. M. DMT (6.6 mg, 0.034 mmol), and CaCl (5.7 mg, 0.052 mmol) were 2 Sanders, J. Chem. Soc. Perkin Trans. 1 1997, 21, 3237–3253. dissolved in CD3OD (5 mL). 4,7,10-Trioxa-1,13-tridecanediamine [10] S. J. Rowan, S. J. Cantrill, G. R. L. Cousins, J. K. M. Sanders, (6, 17.1 µL, 0.078 mmol) was added, and the solution was stirred J. F. Stoddart, Angew. Chem. 2002, 114, 938–993; Angew. for 12 h. In different tubes, various percentages of water (2%, 10%, Chem. Int. Ed. 2002, 41, 898–952. 50%) were added, and 1H NMR spectra were recorded after 5 h. [11] P. T. Corbett, J. Leclaire, L. Vial, K. R. West, J.-L. Wietor, Yield calcd. from the ratio between signals of aromatic protons of J. K. M. Sanders, S. Otto, Chem. Rev. 2006, 106, 3652–3711.

Remarkable Stability of Imino Macrocycles in Water

[12] A. González-Álvarez, I. Alfonso, F. López-Ortiz, A. Aguirre, [19] A. Simion, C. Simion, T. Kanda, S. Nagashima, Y. Mitoma, T. S. Garcia-Granda, V. Gotor, Eur. J. Org. Chem. 2004, 1117– Yamada, K. Mimura, M. Tashiro, J. Chem. Soc. Perkin Trans. 1127. 1 2001, 2071–2078. [13] For a diastereoselective amplification of imine macrocyles, see: [20] U. Lüning, R. Baumstark, K. Peters, H. G. v. Schnering, Lie- A. González-Álvarez, I. Alfonso, V. Gotor, Chem. Commun. bigs Ann. Chem. 1990, 2, 129–143. 2006, 21, 2224–2226. [21] C. D. Meyer, S. Joiner, F. Stoddart, Chem. Soc. Rev. 2007, 36, 1705–1723. [14] O. Storm, U. Lüning, 2002, , 793–798. Chem. Eur. J. 8 [22] S. Zameo, B. Vauzeilles, J. M. Beau, Eur. J. Org. Chem. 2006, [15] A recent example for the stability of an imine in water when 24, 5441–5444. forming a coordinative bond to a transition-metal ion: M. Hu- [23] In preliminary experiments, the imine formation of isophthalic tin, C. A. Schalley, G. Bernardinelli, J. R. Nitschke, Chem. Eur. dialdehyde with amines 2 and 4 has been investigated in meth- J. 2006, 12, 4069–4076, and cited references. anol/water mixtures, too. Due to the endo-hydrogen atom in 2- [16] C. Godoy-Alcantar, A. K. Yatsimirsky, J.-M. Lehn, J. Phys. position of the isophthalic dialdehyde, macrocycles do not Org. Chem. 2005, 18, 979–985. form as good as with pyridine-2,6-dicarbaldehyde 1. But in the [17] W. Lu, T. H. Chang, J. Org. Chem. 2001, 66, 3467–3473: the reaction of isophthalic dialdehyde with n-butylamine (2), imine (sulfonimino)pyridine compound 27 (more stable than an signals can also be detected, and the amount of imine formed imine) was hydrolyzed in THF/H2O (1:1). is comparable with that of diimine 3 obtained from pyridinedi- [18] For an example, see: T. Hirashita, Y. Hayashi, K. Mitsui, S. aldehyde 1. This result shows the importance of conjugation in Araki, J. Org. Chem. 2003, 68, 1309–1313. Product 2a was hy- the imines 3, 5 and 7. drolyzed with a small amount of water in the solvent; in Received: May 9, 2008 MeOH/H2O (1:1), the product was fully hydrolyzed. Published Online: July 15, 2008

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On the formation of imines in water—a comparison Tetrahedron Letters 50 (2009) 4663–4665 * Vittorio Saggiomo, Ulrich Lüning Tetrahedron Letters 50 (2009) 4663–4665 Tetrahedron Letters 50 (2009)Contents 4663–4665 lists available at ScienceDirect Otto-Diels-Institut für Organische Chemie, Christian-Albrechts-Universität zu Kiel, Olshausenstr. 40, D-24098 Kiel, Germany Contents lists available at ScienceDirect Contents lists available at ScienceDirect TetrahedronTetrahedron Letters 50 (2009) Letters 4663–4665 Tetrahedron Letters article info abstractTetrahedronContents Letters lists available at ScienceDirect journal homepage: www.elsevier.com/locate/tetlet journal homepage: www.elsevier.com/locate/tetlet Article history: journalThe reaction homepage: of aniline www.elsevier.com/locate/tetlet withTetrahedron aryl aldehydes in Letters water has been investigated in the past, but contradictory Received 13 March 2009 results have been published. While only small amounts of imines 3 were detected by NMR analysis, iso- Accepted 29 May 2009 lation afforded highjournal imine homepage: yields. A www.elsevier.com/locate/tetlet reinvestigation of the reaction of benzaldehyde (1a) and salicylalde- Available online 6 June 2009 On the formation of imines in water—a comparison On thehyde formation (1b) with aniline of imines (2) revealed in water—a two important comparison factors which explain the putative contradiction: (i) On the formationVittorio ofSaggiomo, iminesNMR Ulrich only in revealswater—a Lüning the* fraction comparison of products which is soluble in water, and (ii) imines 3 form during or after Keywords: Vittorioworkup. Saggiomo, Ulrich Lüning * Imines Otto-Diels-Institut für Organische* Chemie, Christian-Albrechts-Universität zu Kiel, Olshausenstr. 40, D-24098 Kiel, Germany Vittorio Saggiomo, UlrichOn the Lüning formation of imines in water—a comparison Ó 2009 Elsevier Ltd. All rights reserved. Solvent-free reactions Otto-Diels-Institut für Organische Chemie, Christian-Albrechts-Universität zu Kiel, Olshausenstr. 40, D-24098 Kiel, Germany Otto-Diels-Institut für Organische Chemie, Christian-Albrechts-Universität zu Kiel, Olshausenstr. 40, D-24098 Kiel, Germany Schiff bases Vittorio Saggiomo, Ulrich Lüning * article info abstract Water Otto-Diels-Institutarticle für Organische info Chemie, Christian-Albrechts-Universitätabstract zu Kiel, Olshausenstr. 40, D-24098 Kiel, Germany article info abstract Article history: The reaction of aniline with aryl aldehydes in water has been investigated in the past, but contradictory Article history: Received 13 March 2009 The reaction of aniline with aryl aldehydes in water has been investigated in the past, but contradictory Article history: Received 13 March 2009 results have been published. While only small amounts of imines 3 were detected by NMR analysis, iso- Acceptedarticle 29 May 2009 infoThe reaction of aniline withabstract aryl aldehydesresults have in water been has published. been investigated While only in small the past, amounts but contradictory of imines 3 were detected by NMR analysis, iso- Received 13 March 2009 Accepted 29 May 2009 lation afforded high3 Results imine yields.and Discussion A reinvestigation of the reaction of benzaldehyde (1a) and salicylalde-19 Available online 6 June 2009results have been published. Whilelation only small afforded______amounts high imine of imines yields.3 were A reinvestigation detected by NMR of the analysis, reaction iso- of benzaldehyde (1a) and salicylalde- Accepted 29 May 2009 Available online 6 June 2009 hyde (1b) with aniline (2) revealed two important factors which explain the putative contradiction: (i) Article history: lation afforded high imine yields.The reaction Ahyde reinvestigation of (1b aniline) with with aniline of the aryl reaction ( aldehydes2) revealed of benzaldehydein two water important has been (1a factors)investigated and salicylalde- which in explain the past, the but putative contradictory contradiction: (i) Available online 6 June 2009 NMR only reveals the fraction of products which is soluble in water, and (ii) imines 3 form during or after 1. Introduction Received 13 March 2009hyde (1b) with aniline (2) revealedresults haveNMR two beenamounts only important3.2 published. reveals On factorsthe the of WhileFormation fraction imineswhich only of explain small products of can Imines amounts the which be putative in of detectedWater, iminesis soluble contradiction: 3a wereComparison in water, in detected water. (i) and by (ii) NMR imines Among analysis,3 form iso- many during or other after Keywords:Accepted 29 May 2009 Keywords: NMR only reveals the fractionworkup.lation of products affordedworkup. which high imine is soluble yields. in A water, reinvestigation and (ii) imines of the3 reactionform during of benzaldehyde or after (1a) and salicylalde- Keywords: Imines AvailableImines online 6 June 2009 substrates, they investigated the reaction of benzaldehyde (1a)or workup. hyde (1b) with anilineV. Saggiomo, (2) revealed U. Lüning, two important Tetrahedron factors Lett. which, 2009 explainÓ ,2009 50 the,Ó 4663-4665. Elsevier2009 putative Elsevier Ltd. contradiction: All Ltd. rights All rights (i) reserved. reserved. Imines Solvent-free reactionsSolvent-free reactions The reaction of primary amines with carbonyl compoundsNMR to only revealssalicylaldehyde the fraction of productsÓ (1b2009 which) Elsevierwith is soluble anilineLtd. in All water, rights and(2 reserved.)( (ii)Fig. imines 13).form during or after Solvent-free reactions Schiff basesKeywords:Schiff bases workup. 8 Schiff bases Water IminesWater give imines—also called Schiff bases or azomethines—is a reaction In contrast to these results,Ó Tashiro2009 Elsevier and Ltd. All co-workers rights reserved. reported Water 1 Solvent-free reactions During the previous work (Ch. 3.1), a discrepancy was found in the literature. In two which is well known. In natureSchiff it serves bases to interconvert amino excellent yields of imines 3 when they reacted aldehyde 1a or 1b Water different publications, the same imine synthesis in water has been reported with acids and a-ketoacids into one another with the help of vitamin withcompletely aniline different (2 ) yields in water—a(refs. 7 and 8 remarkable in the following publication). putative Starting contradiction. from 1. Introduction1. Introduction 2 amountsamounts of imines of imines can be can detected be detected in water. in water. Among Among many many other other B6 (pyridoxamine–pyridoxal as coenzyme in transaminases ), but Duethe same to the starting high materials relevance (benzaldehyde for DCC, or salicylaldehyde we reproduced and aniline, these Fig. 3.2) experi- 1. Introduction amounts of imines can besubstrates, detectedsubstrates, they in water. investigated they Among investigated many the reaction other the reaction of benzaldehyde of benzaldehyde (1a)or (1a)or it has also been widely used inThe organic1. reaction IntroductionThe ofchemistry reaction primary of amines primary for numerous with amines carbonyl withsubstrates, carbonyl compoundsmentsand they compounds using investigated to in our theamountssalicylaldehyde to same laboratory the salicylaldehydesolvent of reaction imines (water), (1b of can to) benzaldehyde with understandbe (1b the detected) aniline withdifference aniline in ( (1a2 water.)( )orof thisFig. ( yields2)( Among 1 discrepancy).Fig. was 1). many more other than (see 80 %Table The reaction of primary aminesgive imines—also with carbonyl called compounds Schiff bases to orsalicylaldehyde azomethines—isbetween (1b a reaction ) the with substrates,two aniline references. (2 theyIn)( contrastFig. investigated Therefore 1). to these both the reactionresults, experiments Tashiro of benzaldehyde were and reproduced co-workers (1a8)or and8 reported the purposes since the 1800s. Duegive to imines—also its reversibility, called Schiff bases the formation or azomethines—is1 a). reaction In contrast to these results,8 Tashiro and co-workers reported give imines—also called Schiff bases or azomethines—is1 a reaction In contrast to these results, Tashiro and co-workers reported which isThe wellwhich reaction known. is well of1 primaryIn known. nature aminesIn it nature serves with it to carbonyl serves interconvert to compounds interconvertresults amino to were amino carefullysalicylaldehydeexcellentexcellent analyzed yields (1b of yieldsin) withorder imines of aniline to imines 3understandwhen (2)(3 Fig.when they and 1). reacted they explain reacted aldehyde the difference. aldehyde7 1a or 1a1bor 1b of imines haswhich gained is well increasing known.1 In nature interest it serves in recent to interconvert years amino as it isexcellent yieldsFirst, of imines the NMR3 when experiment they reacted aldehyde of Lehn’s1a or 1b group (A18 ) was repeated acids andgiveacids imines—also-ketoacids and a-ketoacids called into one Schiff intoanother bases one or with another azomethines—is the with help the of a vitaminhelp reaction of vitaminwithIn contrast anilinewith aniline to (2 these) in ( water—a2 results,) in water—a Tashiro remarkable and remarkable co-workers putative putativereported contradiction. contradiction. a 1 2 one of the reactionsacids and widelya-ketoacids used into inwhich one dynamicB6 another is (pyridoxamine–pyridoxal well known.with combinatorial theIn help nature of vitamin it as serves coenzymechem- towith interconvert in aniline transaminasesinWhen our2 amino (2) inlaboratory.both water—a), experiments butexcellent remarkableDue yields The were to of the literature iminesrepeated, putative high3 relevancewhen the contradiction. experiment same they for reacteddiverting DCC, we aldehyde wasresults reproduced carried 1a wereor 1b theseobtained. out experi- using B6 (pyridoxamine–pyridoxal as coenzyme2 in transaminases ), but Due to the high relevance for DCC, we reproduced these experi- 3 B6 (pyridoxamine–pyridoxalacids asit coenzyme and hasa also-ketoacids beenin transaminases widely into one used another), in but organic withDue the chemistry help to the ofThe vitaminhigh for main numerous relevance problemwith for aniline DCC,ments was (we that2 in) inreproduced our the water—a laboratory working theseremarkable groups to experi-understand did putative not this differentiate contradiction. discrepancy between (see Table istry. When an imine is formedit has from also been an widely aldehyde used inand organic a primary chemistry fora numerous molar2 ratioments of aldehyde in our laboratory1a to to amine understand2 of this 1:3. discrepancy A 16.5 (see mMTable solution it has also been widely usedB6 (pyridoxamine–pyridoxal in organic chemistry for as numerous coenzyme in transaminasesments in our laboratory), but Due to understand to the high this relevance discrepancy for DCC, (see weTable reproduced these experi- purposes since the 1800s. Due to its reversibility,experiment the formation and workup.1). When their experiments were repeated, it became clear that amine, one moleculepurposes since of water the 1800s.purposes is liberatedit Due has since to also its the been reversibility, per 1800s. widely molecule Due used the to in formation its organic of reversibility, imine. chemistry1). the forof formation numerous aldehydements1).1a in our D laboratoryO at pD to 7.5understand gave this 5.4% discrepancy of imine (see Table3a7 . This yield of imines has gained increasing interest in recent years as it is First,2 the NMR experiment of Lehn’s group7 (A1) was repeated of imines has gainedof increasing iminespurposes has interest gainedsince the in increasing recent 1800s. years Due interest to as its it reversibility, is in recentFirst, years the the formationthe as NMR starting it is experiment materials1). First, of and Lehn’sthe the NMR groupfinal experiment products (A1)7 was were of repeated Lehn’ssimply groupnot soluble (A1) in waswater repeated giving a Consequently, the formation of iminesone of is the facilitated reactions widely when used water in dynamic combinatorialhad been chem- determinedin our laboratory. by simply The literature integrating experiment the7 was signals carried out for using alde- one of the reactionsone widely ofof the used imines reactions in dynamic3 has gained widely combinatorial increasing used in dynamic interest chem- in combinatorial recentin our years laboratory.biphasic as chem- it is The solution. literatureinFirst, our laboratory. the experiment NMR experiment The was literature carried of Lehn’s out experiment using group (A1 was) was carried repeated out using 3 istry. When an imine is formed from an aldehyde and a primary a molar ratio of aldehyde 1a to amine 2 of 1:3. A 16.5 mM solution is removed fromistry. the3 When reaction an imineistry. mixture. is formedoneWhen of fromthe anIn reactions imine many an aldehyde is widely formedexperimental and used from a primary in dynamican aldehyde pro- combinatoriala molar and ratiohyde a primary chem- of aldehyde1a andina1a molar our imineto laboratory. amine ratio3a2 ofofinaldehyde The 1:3. D literature2 AO. 16.51a mMto experiment amine solution2 of was 1:3. carried A 16.5 out mM using solution amine,3 one molecule of water is liberated per moleculeAn additional of imine. experimentof aldehyde was carried1a in out D2 O to at reinforce pD 7.5 gavethe proof 5.4% that of imine water3a was. This not yield amine,istry. one moleculeWhen an imine of water is formed is liberated from an per aldehyde molecule4 and ofa primary imine. aof molar aldehyde ratio of1a aldehydein D O1a atto pD amine 7.5 gave2 of 1:3. 5.4% A of16.5 imine mM solution3a. This yield cedures, water-removingamine, one molecule techniques of water isConsequently, or liberated reagents per the molecule formation are employed. of imine. of iminesof is aldehyde facilitated1aWhen whenin D2O water repeating at pD 7.5had gave been this 5.4% determined ofexperiment,2 imine 3a by. This simply yield we integrating did not the use signals a buffer for alde- to playing any role in that 1a synthesis. The starting materials were3a mixed without the Consequently, the formationConsequently,amine, of imines one the molecule formationis facilitated of water of when imines is liberated water is facilitated perhad molecule been when determined of waterimine. byofhad simplyaldehyde been integrating determinedin D2O the at by pD signals simply7.5 gave for integrating 5.4% alde- of imine the. signals This8 yield for alde- The synthetic chemist therefore hesitatesis removed to from have the reaction water mixture. present In manyallow experimental for a pro- comparisonhyde 1a and to imine the Tashiro3a in D2O. experiments. With a molar Consequently, the formation of imines is facilitated whenaddition water of anyhad 4solvent been determinedand leaved byunder simply vacuum. integrating Using no the solvent, signals the for alde-imines were is removed from theis reaction removed mixture.cedures, from the In water-removing many reaction experimental mixture. techniques In pro- many orhyde experimental reagents1a and are imine pro- employed.3a inhyde D2O.1a andWhen imine repeating3a in D this2O. experiment, we did not use a buffer to is removed from the reaction mixture. In4 many experimental pro-4 hyde 1a and imine 3a in D O. when he triescedures, to synthesize water-removingcedures, an imine, techniques water-removing although or reagents techniques water are employed. would or reagents be areWhen employed. repeatingobtained inthis high experiment, When yield. Water repeating we from did this thenot 2 reaction useexperiment, a buffer can be to we easily did notremoved use a by8 buffer reduced to cedures,The water-removing synthetic chemist techniques therefore or hesitates reagents to are have employed. water4 presentWhenallow repeating for a comparisonthis experiment, to the we Tashiro did not experiments. use a bufferWith to a molar The synthetic chemistThe therefore synthetic hesitates chemist5 to therefore have water hesitates present to haveallow water for a presentcomparison toallow the Tashiro for a comparison experiments. to8 theWith Tashiro a molar experiments.8 With a molar the environmentally most benignThe solvent. syntheticwhen he chemist tries to therefore synthesize hesitates an imine, to have although water waterpressure present would drivingallow be the for reaction a comparison to the product. to the Tashiro experiments.8 With a molar when he tries to synthesize an imine, although water would be 5 whenwhen he triesthe he environmentally triesto synthesize to synthesize an most imine,an benignimine, although although solvent. water water would would be be the environmentally most benign solvent.5 5 the environmentallythe environmentally most most benign benign solvent. solvent.5 2. Results and discussion 2. Results and discussion 2. Results and discussion2. Results2. Results and discussion and discussion NH R In dynamic combinatorial chemistry (DCC), water which is pro- R 2 R In dynamic combinatorial chemistry (DCC), water which is pro- NH2 NHNH2 - H O R R In dynamic combinatorial chemistryIn dynamicInduced dynamic (DCC), combinatorial by ancombinatorial imine water formation chemistry which chemistry will (DCC), is (DCC), stay pro- water in water the whichreaction which is is mixture, pro- pro- and +2NH2 2 - H2O - H O duced by an imine formationduced will by stay an in imine the reactionformation mixture, will stay and in the reaction mixture, and + + - H2 -2 HOO N duced by an imine formationduced will stay by anthe inimine question the formation reaction arises to will which mixture, stay extent in the waterandreaction can mixture, be tolerated. and Litera- + + RN 2 + H2O R + H O N the question arises to whichthe extent questionture studieswater arises can reveal to be which tolerated. surprising extent Litera- answers. water can While be tolerated. we discovered Litera- that in 2 R + H2O NN the question arises to which extent water can be tolerated. Litera- R RCHO + H+ HO2O the question arisesture studies to which reveal surprising extentture answers. water studies revealWhile can surprisingbe we discovered tolerated. answers. that Litera- While in we discoveredCHO that in CHO 2 ture studiesa specific reveal surprising dynamic combinatorial answers. While library, we discovered water can even that be in used as CHO 2 ture studies reveala specific surprising dynamic combinatorial answers.a specificthe library, While solvent, dynamic water6 weother combinatorial can discovered researchers even be library, used have that as water found in can less even promising be used as results. A2 R = H2 6 a specific dynamic6 combinatorial library, water can even be used as CHO 1a the solvent, other researchersthe solvent, have foundother less researchers promising have results. found A less promising7 1a results. R = H A 1a R = H 2 recent6 study of Lehn and co-workers shows that only small 1b R = OH 3a R = H a specific dynamicrecent combinatorial study of Lehnthe and solvent, library,recent co-workersother study water7 researchers ofshows Lehn can and that even have co-workers only found be small used less7 shows promising as that only1b results. R small= OH A 1b1a R R= OH= H 23a R = H 3a R = H 6 7 Figure 3.2: The reaction R of = aniline OH and benzaldehyde (R = H) or salicylaldehyde3b R = (R OH = the solvent, other researchersrecent have study found of lessLehn promising and co-workers results.shows A that only small R = H 1b 3b R = OH 3b R =3a OH R = H OH) produces1a the respective imine. The reaction is reversible and water can drive it back * Corresponding7 author. Fax: +49 431 880 1558. Figure 1. The reaction of aldehydes 1 with an amine3b suchR = OH as aniline (2) to give * Corresponding author. Fax: +49 431 880 1558. 1bFigure R = 1. OHThe reaction of aldehydes 1 with an amine such as aniline (2) to give recent study of* Corresponding Lehn and author. co-workers Fax: +49 431 880E-mail 1558.shows address: [email protected] that only small(U. Lüning).Figure 1. The reactionto the of starting aldehydes materials.1 imineswith an3 amineis a reversible such as process. aniline (2) to give 3a R = H E-mail address: [email protected] (U. Lüning). imines 3 is a reversible process. E-mail address: [email protected]* Corresponding(U. author. Lüning). Fax: +49 431 880 1558. imines 3 is a reversible process.Figure 1. The reaction of aldehydes 1 with an amine such3b as anilineR = OH (2) to give E-mail address:0040-4039/[email protected] - see front matter(U.2009 Lüning). Elsevier Ltd. All rights reserved. imines 3 is a reversible process. 0040-4039/$ - see front matter Ó 2009Ó Elsevier Ltd. All rights reserved. 0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tetlet.2009.05.117doi:10.1016/j.tetlet.2009.05.117 * Correspondingdoi:10.1016/j.tetlet.2009.05.117 author. Fax: +490040-4039/$ 431 880 1558. - see front matter Ó 2009 Elsevier Ltd. All rights reserved.Figure 1. The reaction of aldehydes 1 with an amine such as aniline (2) to give E-mail address: [email protected]:10.1016/j.tetlet.2009.05.117(U. Lüning). imines 3 is a reversible process.

Tetrahedron Letters 50 (2009) 4663–4665

Contents lists available at ScienceDirect

Tetrahedron Letters

journal homepage: www.elsevier.com/locate/tetlet

On the formation of imines in water—a comparison

Vittorio Saggiomo, Ulrich Lüning *

Otto-Diels-Institut für Organische Chemie, Christian-Albrechts-Universität zu Kiel, Olshausenstr. 40, D-24098 Kiel, Germany article info abstract

Article history: The reaction of aniline with aryl aldehydes in water has been investigated in the past, but contradictory Received 13 March 2009 results have been published. While only small amounts of imines 3 were detected by NMR analysis, iso- Accepted 29 May 2009 lation afforded high imine yields. A reinvestigation of the reaction of benzaldehyde (1a) and salicylalde- Available online 6 June 2009 hyde (1b) with aniline (2) revealed two important factors which explain the putative contradiction: (i) NMR only reveals the fraction of products which is soluble in water, and (ii) imines 3 form during or after Keywords: workup. Imines Ó 2009 Elsevier Ltd. All rights reserved. Solvent-free reactions Schiff bases Water

1. Introduction amounts of imines can be detected in water. Among many other substrates, they investigated the reaction of benzaldehyde (1a)or The reaction of primary amines with carbonyl compounds to salicylaldehyde (1b) with aniline (2)(Fig. 1). give imines—also called Schiff bases or azomethines—is a reaction In contrast to these results, Tashiro and co-workers8 reported which is well known.1 In nature it serves to interconvert amino excellent yields of imines 3 when they reacted aldehyde 1a or 1b acids and a-ketoacids into one another with the help of vitamin with aniline (2) in water—a remarkable putative contradiction. B6 (pyridoxamine–pyridoxal as coenzyme in transaminases2), but Due to the high relevance for DCC, we reproduced these experi- it has also been widely used in organic chemistry for numerous ments in our laboratory to understand this discrepancy (see Table purposes since the 1800s. Due to its reversibility, the formation 1). of imines has gained increasing interest in recent years as it is First, the NMR experiment of Lehn’s group (A1)7 was repeated one of the reactions widely used in dynamic combinatorial chem- in our laboratory. The literature experiment was carried out using istry.3 When an imine is formed from an aldehyde and a primary a molar ratio of aldehyde 1a to amine 2 of 1:3. A 16.5 mM solution amine, one molecule of water is liberated per molecule of imine. of aldehyde 1a in D2O at pD 7.5 gave 5.4% of imine 3a. This yield Consequently, the formation of imines is facilitated when water had been determined by simply integrating the signals for alde- is removed from the reaction mixture. In many experimental pro- hyde 1a and imine 3a in D2O. cedures, water-removing techniques or reagents are employed.4 When repeating this experiment, we did not use a buffer to The synthetic chemist therefore hesitates to have water present allow for a comparison to the Tashiro experiments.8 With a molar when he tries to synthesize an imine, although water would be the environmentally most benign solvent.5

2. Results and discussion R In dynamic combinatorial chemistry (DCC), water which is pro- NH2 - H O duced by an imine formation will stay in the reaction mixture, and + 2 N the question arises to which extent water can be tolerated. Litera- R + H2O ture studies reveal surprising answers. While we discovered that in CHO a speciﬁc dynamic combinatorial library, water can even be used as 2 the solvent,6 other researchers have found less promising results. A 1a R = H recent study of Lehn and co-workers7 shows that only small 1b R = OH 3a R = H 3b R = OH

* Corresponding author. Fax: +49 431 880 1558. Figure 1. The reaction of aldehydes 1 with an amine such as aniline (2) to give E-mail address: [email protected] (U. Lüning). imines 3 is a reversible process.

4664 V. Saggiomo, U. Lüning / Tetrahedron Letters 50 (2009) 4663–4665

Table 1 Reaction conditions, analysis conditions, and product ratios for the imine formation between aldehydes 1a–b and aniline (2)

Experiment Aldehyde Reaction condition Analysis Ratio 1:3

A1 1a D2O NMR 96:4

A2 1a H2O NMR after workup and vacuum 5:95 A3 1a No solvent, vacuum NMR 5:95

B1 1b D2O NMR 88:12 a B2 1b H2O NMR after workup and vacuum 3:97 B3 1b No solvent, vacuum NMR 3:97b

a Yield after recrystallization: 85%. b Yield after recrystallization: 83%. ratio of aldehyde 1a to amine 2 of 1:1 at 10 mM for each starting A second imine-forming experiment has also been investigated 7,8 material in D2O, it became obvious that both starting materials in both references in water: the reaction between salicylalde- which are liquids were not soluble in deuterated water, and the hyde (1b) and aniline (2). Also in this case, the yields reported resulting mixture was non-homogeneous. Also pure aldehyde 1a for the formation of imine 3b in water are completely different. and pure amine 2 form two layers with water, showing that the In the NMR experiment of Lehn et al.,7 the reported yield of imine solubility of these compounds is low in water. Neither heating of 3b was only 14% while in the experiment of Tashiro et al.,8 the the NMR tube nor application of ultrasound resulted in the forma- same imine 3b was obtained in 87% yield. Suspecting the same ef- tion of a single layer. Either the solution remained biphasic or it be- fects during the reaction and workup as in experiments A1–A3 [1a came emulsion like. When a 1H NMR was recorded, it resulted in with aniline (2)], this reaction was also carried out in the three dif- the same ratio of imine 3a to aldehyde 1a as reported by Lehn.7 ferent ways as described above. It must be noted that when analyzing the water layer alone by NMR experiment B1 was carried out under the same conditions recording its NMR spectrum, only a small amount of the starting as experiment A1, and it did not show drastic changes in the ratio material was analyzed. The major part of the material is in the of ﬁnal imine 3b to aldehyde 1b compared to the ratio reported by other layer and the NMR does not analyze it. The experiment Lehn.7 Although salicylaldehyde (1b) is a little bit more soluble in was repeated with 3 equiv of aniline (2) without any improvement water than benzaldehyde (1a), the solution once more was bipha- of the solubility or the percentage of imine 3a dissolved in water. sic, and, as in experiment A1, neither heating nor sonication im-

When calcium chloride (CaCl2) or hydrochloric acid (HCl) was proved the solubility of the starting materials. Experiment B2 added to the emulsion, everything dissolved completely and the was repeated in the same way as experiment A2 was carried out. solution finally was clear. However the Lewis acid and the In this particular case, after workup, when the crude oil was sub- Brønsted acid simply solubilized aldehyde 1a and amine 2 but their jected to vacuum, only a small amount of solid appeared. But after addition had no effect on the formation of imine 3a, whose concen- 12 h in vacuo, the product was almost completely solid. Recrystal- tration remained low. No imine could be detected when HCl was lization from n-hexane gave pure imine 3b as yellow needles in used, and when CaCl2 was used the amount of imine 3a was less 85% yield. than 5%. In the last experiment (B3),10 salicylaldehyde (1b) and aniline In contrast, Tashiro and co-workers were able to isolate imines (2) were simply mixed together in a ratio of 1:1, and then the reac- 3 in good yields. The authors used a 0.6 M solution of aldehyde 1a tion was evacuated for 12 h. The result was the same as in exper- and amine 2. The starting materials 1a and 2 were stirred in water iment B2. After recrystallization from n-hexane, 83% of the final vigorously for 3 h. Then, the products were extracted with dichlo- imine 3b was recovered. Thus this experiment also shows that romethane, dried, and analyzed by NMR. The yield of imine 3a was water as a solvent does not play any role during the formation of found to be 97%.8 the imine. The liquid aldehyde and the liquid amine react with When this reaction was repeated (A2), again the low solubility each other to give the respective imine 3b in the absence of of the starting materials in water could be observed. Vigorous stir- solvent. ring only provoked the reaction mixture to become emulsion like. Analogous to the reference, the reaction was stopped after 3 h, and 3. Conclusion the reaction mixture was extracted with dichloromethane, dried over MgSO4, and the solvent was evaporated in vacuo. In order to The scope of this work was to elucidate the contradictory re- record the NMR and to remove the remaining solvent traces, the sults presented in two different and recent papers. Both papers vessel containing the oily product was evacuated using an oil contain reproducible experiments and their goal is not focused pump. A few seconds after the vacuum was established, the oily on the synthesis of a single imine but on (a) a general imine syn- product solidified and heat was developed. This was the first clue thesis in the presence of water8 and (b) the relationship between that the imine-forming reaction takes place when the two starting the structure and stability of imine formation in aqueous solution.7 components are in concentrated contact with each other after the However, when the same starting materials were used (1a, 1b, and workup. Water does not play any role in the reaction as the starting 2), drastically varying yields were found for the respective imines aldehyde 1a and amine 2 as well as the final product are only 3a and 3b. slightly soluble in water. Then after the workup, when the two re- The putative contradiction in the two sets of experiments lies in agents are concentrated, they react. The oil pump vacuum simply the fact that the reaction only presumably takes place in water and helps in the formation of imine 3a by removing the water formed that the ‘water results’ are compared. But on the contrary, the reac- in the imine condensation. tion takes place best in the absence of solvent. To prove the latter hypothesis, aldehyde 1a and amine 2 were The solubilities of the starting materials, and those of the final 9 mixed without any solvent in experiment A3. After a few seconds, products, play an extremely important role in the dynamical com- a solid formed and the reaction was exothermic. The crude product binatorial chemistry. Even if the starting building blocks are well was kept in vacuo for 10 min. Subsequent NMR analysis afforded soluble in water but the final product is not, this can drive a reac- the same yield as experiment A2 (95%). tion to the latter product, into a thermodynamic trap. In order to 22 3 Results and Discussion ______

V. Saggiomo, U. Lüning / Tetrahedron Letters 50 (2009) 4663–4665 4665 have a good and reproducible distribution of products in a library, Cousins, G. R. L.; Sanders, J. K. M.; Stoddart, J. F.. Angew. Chem. 2002, 114, 938– all the components should be soluble and, by this, be free to inter- 993; . Angew. Chem., Int. Ed. 2002, 41, 898–952; (d) Storm, O.; Lüning, U. Chem. Eur. J. 2002, 8, 793–798. act with one another. 4. (a) Carey, F. C.; Sundberg, R. J. Advanced Organic Chemistry, 4th ed.; Springer: New York, 2006. p. 460; (b) Gattermann, L. Die Praxis des organischen Chemiker Acknowledgment (Gattermann; Wieland), neu bearb. von Wieland, T.; Sucrow, W. de Gruyter, 43. Aufl., Berlin, New York, 1982. 5. (a) Horvath, I. T.; Anastas, P. T. Chem. Rev. 2007, 107, 2167–2168; (b) Li, C.-J.; We are grateful for the support provided by the Marie Curie Re- Chan, T.-H. Comprehensive Organic Reactions in Aqueous Media, 2nd ed.; Wiley: search Training Network MRTN-CT-2006-035614 Dynamic Combi- New York, 2007; (c) Organic Synthesis in Water; Grieco, P. A., Ed.; Blackie Academic & Professional: London, 1998. natorial Chemistry (DCC). 6. Saggiomo, V.; Lüning, U. Eur. J. Org. Chem. 2008, 25, 4329–4333. 7. Godoy-Alcantar, C.; Yatsimirsky, A. K.; Lehn, J.-M. J. Phys. Org. Chem. 2005, 18, References and notes 979–985. 8. Simion, A.; Simion, C.; Kanda, T.; Nagashima, S.; Mitoma, Y.; Yamada, T.; Mimura, K.; Tashiro, M. J. Chem. Soc., Perkin Trans. 1 2001, 2071–2078. 1. (a) Patai, S. The Chemistry of the Carbon–Nitrogen Double Bond; Wiley: London, 9. Experiment A3: 1.0 mL (10 mmol) of benzaldehyde (1a) and 910 lL (10 mmol) 1970; (b) Patai, S. The Chemistry of the Amino Group; Wiley: London, 1968; (c) of aniline (2) were mixed in a 5 mL flask. The crude oil was left under vacuum Patai, S. The Chemistry of the Carbonyl Group; Wiley: London, 1966; (d) Layer, R. 4 (4 10À mbar) for 10 min, until all the oil became solid. Then, 10 mg of the W. Chem. Rev. 1963, 63, 489–510; (e) Goering, B. K. Ph.D. Dissertation, Cornell Â crude product11 was analyzed by 1H NMR. Ratio imine 3a/aldehyde 1a: 95:5. University, 1995. 10. Experiment B3: 966 lL (10.0 mmol) of salicylaldehyde (1b) and 911 lL 2. Aminotransferases and transaminases form enzyme class 2.6.1. For the (10.0 mmol) of aniline (2) were mixed, and the reaction mixture was left mechanisms of transaminases via imines see: (a) Meister, A. Biochemistry of 4 under vacuum (4 10À mbar) for 12 h. Then, 10 mg of the crude product was the amino acids; Academic Press: New York, 1957; (b) Frey, P. A.; Hegeman, A. Â analyzed by 1H NMR. Ratio imine 3b/aldehyde 1b: 97:3. Recrystallization from D. Enzymatic reaction mechanisms; Oxford University Press, 2007. p. 148ff; For a n-hexane gave the final product11 in 83% yield. recent review, see (c) Toney, M. D. Arch. Biochem. Biophys. 2005, 433, 279–287. 11. N-Benzylideneaniline (3a): SDBS No.: 2145, CAS No.: 538.51.2; 3. (a) Corbett, P. T.; Leclaire, J.; Vial, L.; West, K. R.; Wietor, J.-L.; Sanders, J. K. M.; o-(phenyliminomethyl)phenol (3b): SDBS No.: 7178, CAS No.: 779-84-0. Otto, S. Chem. Rev. 2006, 106, 3652–3711; (b) Meyer, C. D.; Joiner, C. S.; Stoddart, J. F. Chem. Soc. Rev. 2007, 36, 1705–1723; (c) Rowan, S. J.; Cantrill, S. J.; At this point, with knowledge on stability of this imino macrocycle in water, its calcium 3 Results and Discussion 23 complex______property and the biphasic layer experiment, there was another experiment to be carried out. A carrier3.3 Transport experiment of Calciumin a bulk Ions membrane through system, a Bulk startingMembrane from by a UseDCL of and a Dynamic not from a purified carrier should give us precious information on the question if a DCL could be used Combinatorial Library to screen directly a function, and not only a single product. TheV. fluxSaggiomo, of the carrier U. Lüning, is directly Chem. correlated Commun. to, 2009 the ,binding 3711-3713. constant of the carrier with the guest, and this latter is related to the free Gibbs enthalpy. A bulk membrane experiment was set up in the following way: the diamine and calcium ions were dissolved in the source phase, the dialdehyde was dissolved in the organic phase,A carrier and experimentthe receiver in aphase bulk membranewas pure systemsolvent was(Figure set up2). toAfter study one if aweek, Dynamic imino macrocycleCombinatorial 5 and Library calcium (DCL) ions can werebe used found to screen in the directly receiver a function,phase. hereThis transport.phase was screened by NMR, ESI-MS, and a calcium titration test. The blank experiment with only oneA bulkof the membrane two building was blocks used. The(diamine diamine or dialdehyde)and calcium reveals ions were that dissolved none of inthe the two transportsaqueous calcium source phaseinto the (Figure receiver 3.3, phase. a), the Also dialdehyde using thewas two dissolved building in blocks the organic without calciumphase in(Figure the source 3.3, b), phase and thereveals, receiver as phaseknown was by purethe biphasic water (Figure experiment, 3.3, c). thatAfter the macrocycle is almost not synthesized at all. Evenone if week,the yield imino of macrocyclemacrocycle andand calciumcalcium transportedions were found through in the the receiver bulk membrane phase. This is low (5-7%)phase this was simple screened experiment by NMR, shows ESI-MS, that a DCL and can a calciumbe useful titration to screen test. a product The blank with a function.experiment In this with field, calcium different ions goals and onlycould one be of reached the two using building this blocks kind of (diamine experiment. or Important questions where a carrier plays an significant role may be answered by such a DCCdialdehyde) approach reveals in the thatfields none of ofcleaning the two waste transports water calcium from toxicinto themetal receiver ions, phase.or more importantUsing the to screen two building a carrier blocks for drug without delivery calcium. in the source phase reveals that the In currentmacrocycle experiments, is almost synthesesnot synthesized of more at lipophilicall. dialdehydes are carried out to check in this way if the yield of calcium transport can be improved. This is the first proof of a macrocyclic carrier amplified directly by a DCL.

Figure 1. ((If something is called a ﬁgure it shall have a legend ))

Figure 3.3:2. Schematic Schematic representationrepresentation of theof the bulk DCC membrane/DCL carrier experiment experiment. Starting material and detected ﬁnal macrocycle on top. Transport of calcium ions from (a) water source phase to (c) water receiver phase passing through (b) organic bulk membrane. 24 3 Results and Discussion ______

COMMUNICATION www.rsc.org/chemcomm | ChemComm

Transport of calcium ions through a bulk membrane by use of a dynamic combinatorial libraryw z Vittorio Saggiomo and Ulrich Lu¨ ning*

Received (in Cambridge, UK) 10th February 2009, Accepted 15th May 2009 First published as an Advance Article on the web 28th May 2009 DOI: 10.1039/b902847a

In a bulk membrane transport experiment, a dynamic template ion in Dynamic Combinatorial Chemistry can select combinatorial library (DCL) has been used to transport calcium its own carrier from the DCL and is transported through a ions; the calcium ions amplify the formation of a macrocyclic (bulk) membrane. carrier which results in transport. Before the transport experiments were carried out, bi-phasic behaviour of the DCL made from diamine 1, dialdehyde 28 Dynamic Combinatorial Chemistry (DCC)1 exploits the and calcium ions (Fig. 2) was investigated in a two-phase reversibility of chemical reactions. The resulting mixtures are system (water and chloroform or dichloromethane).9 called dynamic combinatorial libraries (DCLs). At first glance, Dissolving equimolar amounts of diamine 1 in the aqueous reversibility seems to complicate the chemistry as all products phase and dialdehyde 2 in the organic phase gave a DCL in a DCL may interconvert into new ones. By addition of with a distribution of final products in both layers. Using suitable template molecules, specific members of the DCL can deuterated solvents, the time dependence of the DCL be amplified if the resulting complex is more stable than composition was monitored by means of NMR. The system uncomplexed members of the DCL or complexes of other was not stirred or mixed. Every 48 h, 600 mL of each layer were members with this template.2,3 In a classical DCC experiment taken and analyzed by 1H-NMR. (Fig. 1, solid arrows), the next task is to stop the equilibration Experiment A without any template ions (Fig. 3) showed of the DCL and to isolate the desired amplified product. Then after one week many imine peaks in the organic phase it can be used for its function, for instance as a receptor or a (7.4–7.8 ppm for the pyridine protons and 8.4–8.6 ppm for carrier in a transport experiment. the imine protons). The large number of signals can be Over the past years, a large number of reversible reactions attributed to the presence of different cyclic or linear oligo- have been used in DCC. Classical DCLs frequently use and polymers (see ESI for the overlapped NMR spectra). z disulfide, hydrazone or imine chemistry, and quenching may For comparison, two blank experiments were carried out be performed by a change of pH (sulfide or hydrazone with the bi-phasic set-up: (i) Diamine 1 was dissolved in water exchange) or by reduction (imines). but no dialdehyde 2 was dissolved in the organic phase. After In this work we have investigated a ‘‘short-cut’’: the DCL one week, only a small amount (from 2% to 4%) of diamine 1 was directly used to screen for a function (Fig. 1, dashed was detected in the organic phase (determination by weight arrow).4 Transport mediated by carrier molecules has broad after evaporation of the solvent). (ii) In a second experiment, scientific interest: e.g. decontamination of wastewater, the dialdehyde 2 was dissolved in the organic layer but no diamine understanding of biological processes, drug delivery. Previous 1 was present in the water layer. In this case, no dialdehyde 2 experiments carried out by Morrow’s group5 show that a could be found in the aqueous phase after one week. Due to its library formed by the condensation of different aldehydes with basicity, diamine 1 is largely protonated and stays in the water amines (or hydrazides) can extract certain transition metal layer but some diamine 1 will always diffuse into the organic ions from water into an organic solvent in which the building layer. In contrast, dialdehyde 2 stays mostly in the organic blocks are dissolved. Our approach adds one more step. We phase. Thus a reaction between diamine 1 and dialdehyde 2 have investigated whether a DCL can transport calcium ions probably takes place in the organic phase or at the interphase. from an aqueous source phase to an aqueous receiver phase separated by organic solvent (bulk membrane). Starting from a well known host–guest system6,7 with an imino macrocycle and calcium ions, we carried out transport experiments using a DCL and not a purified carrier. The main question was: ‘‘Can a DCL built from amines and dialdehydes be used to transport particular ions?’’ In this work, we present experiments showing that a metal ion which can act as a

Otto-Diels-Institut fu¨r Organische Chemie, Olshausenstr. 40, D-24098 Kiel, Germany. E-mail: [email protected]; Fax: +49-431-880-1558; Tel: +49-431-880-2450 w Dynamic Combinatorial Chemistry, Part 3. For Part 2, see ref. 7. Electronic supplementary information (ESI) available: Experimental section,z 1H-NMR, ESI-MS and transport experiment. See DOI: Fig. 1 Flowchart of a general DCC experiment in solid arrows. 10.1039/b902847a The experiment described here is indicated by a dashed arrow.

c This journal is The Royal Society of Chemistry 2009 Chem. Commun., 2009, 3711–3713 | 3711 3 Results and Discussion 25 ______

Fig. 5 Experiment C. Building blocks (in the frame) and detected products in the transport experiment. (a) Source water phase, (b) bulk

membrane CD2Cl2, (c) aqueous receiver phase.

the formation and stabilization of macrocycle 3 and thus the Fig. 2 Building blocks and their cartoon representation. presence of calcium ions changes the DCL and amplifies the calcium complex 3 Ca2+. Á The transport ability of the DCL was tested with a bulk membrane10 experiment (experiment C, Fig. 5) which was set up in the following way: diamine 1 and calcium ions were dissolved in the source water phase, dialdehyde 2 was dissolved in the bulk membrane organic phase, and the receiver phase was pure water. All three components were used in equimolar amounts. After one week, the three phases were monitored by means Fig. 3 Experiment A. Building blocks on the left and detected of 1H-NMR.11 No NMR signals of the starting building products on the right. (a) Aqueous phase, (b) organic phase. blocks 1 and 2 nor of the final product 3 are observed in the ‘‘residue solvent region’’ of the spectra. In order not to disturb the DCL by removing solvent the spectra were recorded directly in the non-deuterated solvents. The analysis of the water source phase and the organic phase was straightforward. The results are in agreement with experiment B. The major part (ca. 60%) of macrocycle 3 was found in the source water phase, as well as polymers being found in the organic phase. But the product in the water receiver phase was too dilute to record good NMR spectra directly from the receiving solution. Fig. 4 Experiment B. Building blocks on the left and detected For this reason the solvent was removed by a flow of 12 products on the right. (a) Aqueous phase, (b) organic phase. nitrogen. The residue was dissolved in deuterated methanol and a 1H-NMR spectrum was recorded. A 5–7% yield of In experiment B (Fig. 4), an equimolar amount of calcium macrocycle 3 was found in the receiving phase. This solution chloride (CaCl2) (with respect to diamine 1 and dialdehyde 2) was also analyzed by ESI-MS, which showed the signal for the was dissolved in the aqueous phase. protonated molecule (M + H+), the molecular peak plus a After one week, macrocycle 3 was detected in the aqueous potassium ion (M + K+) and, more importantly, the phase with a yield of 62–65%. Surprisingly, even in presence of molecular peak plus a calcium ion (M + Ca2+/2). Although an organic layer, macrocycle 3 complexes calcium and goes in the ESI-MS it is quite common to find molecular peaks plus preferentially into the water layer. potassium and sodium ions, originating from the environment, Also for experiment B, two different blank experiments were it is usually impossible to detect the molecular peak plus a monitored: (i) without dialdehyde 2 in the organic layer, calcium ion if calcium is not present in the solution. Thus, the diamine 1, initially dissolved in water in presence of calcium ESI-MS proves that both macrocycle and calcium ions were ions, was found in the organic layer in 2–4% yield after one present in the water receiver phase. Furthermore, a titration week. (ii) In the absence of diamine 1, dialdehyde 2 was method was used to analyze the calcium ions in the receiving s dissolved in the organic layer, and CaCl2 in the aqueous phase. The Aquamerck calcium test (for details, see ESI ) is 1 z phase. After one week, 1–2% of dialdehyde 2 was found able to detect calcium ions in a range from 2–200 mg LÀ , and in the aqueous phase in its hydrate form (monitored by it also determined 5–7% of calcium in the receiver phase. 1H-NMR7). Here, calcium chloride seems to facilitate the Control experiments showed that with calcium chloride hydration of the aromatic aldehyde 2. alone, or calcium chloride with only one of the two building Next, the transport of calcium ions by the DCL derived blocks (diamine 1 or dialdehyde 2), there was no transport from diamine 1 and dialdehyde 2 was investigated. The at all. difference from standard transport experiments is the fact that Our transport experiments prove that a DCL can be directly no carrier exists in the beginning. Even after mixing of diamine used for a function. Not a pre-selected and isolated carrier has 1 with dialdehyde 2, macrocycle 3 which is supposed to be able been used for transport but carrier 3 was amplified from a to carry calcium ions across a lipophilic membrane only exists DCL and acted as a carrier through a bulk liquid membrane! in low concentration. But calcium ions act as a template for These results open new opportunities in many different fields.

c 3712 | Chem. Commun., 2009, 3711–3713 This journal is The Royal Society of Chemistry 2009 26 3 Results and Discussion ______

Cleaning of waste/toxic water from dangerous heavy metal 9390–9392; P. T. Corbett, J. K. M. Sanders and S. Otto, ions mediated by a DCL could be very fascinating for Chem.–Eur. J., 2008, 14, 2153–2166; K. Severin, Chem.–Eur. J., 2004, 10, 2565–2580. environmental chemistry. Another important field is drug 4 Few examples of direct use of a DCL and not a purified 13 delivery in the cell; the use of a DCL instead of a purified product are present in the literature: R. Larsson, Z. Pei and product is of easy access and could give immediate results if a O. Ramstro¨ m, Angew. Chem., 2004, 116, 3802–3804; R. Larsson, product of the library is amplified by the drug and if it acts as a Z. Pei and O. Ramstro¨ m, Angew. Chem., Int. Ed., 2004, 43, 3716–3718; A. Buryak and K. Severin, Angew. Chem., 2005, 117, carrier into the cell. A conceptually similar set of observations 8149–8152; A. Buryak and K. Severin, Angew. Chem., Int. Ed., using different chemistry is reported by the Cambridge group 2005, 44, 7935–7938; A. Buryak and K. Severin, J. Comb. in an accompanying paper.14 Chem., 2006, 8, 540–543. Nowadays the use of a chemical library We thank the EU for its support through the Marie Curie instead of a single compound is growing in importance (system chemistry): R. F. Ludlow and S. Otto, Chem. Soc. Rev., Research Training Network MRTN-CT-2006-035614 2008, 37, 101–108. Dynamic Combinatorial Chemistry (DCC), and for providing 5 D. M. Epstein, S. Choudhary, M. R. Churchill, K. M. Keil, the forum for the Cambridge and Kiel groups to exchange A. V. Eliseev and J. R. Morrow, Inorg. Chem., 2001, 40, 1591–1596; S. Choudhary and J. R. Morrow, Angew. Chem., insights and results. We thank M. Pittelkow and J. K. M. 2002, 114, 4270–4272; S. Choudhary and J. R. Morrow, Angew. Sanders for helpful discussions, and C. Goeschen and Chem., Int. Ed., 2002, 41, 4096–4098. R. Herges for lending us the transport equipment. 6 O. Storm and U. Lu¨ ning, Chem.–Eur. J., 2002, 8, 793–798. 7 V. Saggiomo and U. Lu¨ ning, Eur. J. Org. Chem., 2008, 4329–4333. Notes and references 8 Diamine 1 and dialdehyde 2 were synthesized according to: U. Lu¨ ning, R. Baumstark, K. Peters and H. G. v. Schnering, 1 P. T. Corbett, J. Leclaire, L. Vial, K. R. West, J.-L. Wietor, Liebigs Ann. Chem., 1990, 129–143. J. K. M. Sanders and S. Otto, Chem. Rev., 2006, 106, 3652–3711; 9 R. Pe´ rez-Ferna´ ndez, M. Pittelkow, A. M. Belenguer and C. D. Meyer, C. S. Joiner and J. F. Stoddart, Chem. Soc. Rev., J. K. M. Sanders, Chem. Commun., 2008, 1738–1740. 2007, 36, 1705–1723; S. J. Rowan, S. J. Cantrill, G. R. L. Cousins, 10 J. H. Moore and R. S. Schechter, AIChE J., 1973, 19, 736–741; J. K. M. Sanders and J. F. Stoddart, Angew. Chem., 2002, 114, H. C. Visser, D. N. Reinhoudt and F. de Jong, Chem. Soc. Rev., 938–993; S. J. Rowan, S. J. Cantrill, G. R. L. Cousins, J. K. M. 1994, 75–81; P. Breccia, M. Van Gool, R. Pe´ rez-Ferna´ ndez, Sanders and J. F. Stoddart, Angew. Chem., Int. Ed., 2002, 41, S. Martı´ n-Santamaria, F. Gago, P. Prados and J. de Mendoza, 898–952; I. Huc and J.-M. Lehn, Proc. Natl. Acad. Sci. U. S. A., J. Am. Chem. Soc., 2003, 125, 8270–8284. 1997, 94, 2106–2110; P. A. Brady and J. K. M. Sanders, J. Chem. 11 Typically in this kind of experiment, a conductivity meter is used. Soc., Perkin Trans. 1, 1997, 21, 3237–3253; S. Ladame, Org. Here, the transport of the calcium ions could not be followed by Biomol. Chem., 2008, 6, 219–226. conductivity because diamine 1 passes through the bulk membrane 2 Some examples for amplification in disulfide, hydrazone and imine and reaches the receiver phase. There, its protonated form 1 H+ libraries: S. Otto, E. L. R. Furlan and J. K. M. Sanders, Science, contributes to the conductivity increase, hiding the calciumÁ 2002, 297, 590–593; M. S. Voshell, J. S. Lee and R. M. Gagne, conductivity in a non predictable way. J. Am. Chem. Soc., 2006, 128, 12422–12423; K. Ziach and 12 Note: it is important to remove the solvent without heating since J. Jurczak, Org. Lett., 2008, 5159–5162. this facilitates the hydrolysis of the imino linkage. 3 It is important to note that the addition of templates affects the 13 W. A. Ritschel, Handbook of Basic Pharmacokinetics, Drug whole system. The resulting DCL will be that with the lowest total Intelligence, Hamilton, 1976. free enthalpy and thus the most stable member need not exist in 14 R. Pe´ rez-Ferna´ ndez, M. Pittelkow, A. M. Belenguer, L. A. Lane, highest concentration. Some examples: P. T. Corbett, C. V. Robinson and J. K. M. Sanders, Chem. Commun., 2009, J. K. M. Sanders and S. Otto, J. Am. Chem. Soc., 2005, 127, DOI: 10.1039/b902842k.

c This journal is The Royal Society of Chemistry 2009 Chem. Commun., 2009, 3711–3713 | 3713 3 Results and Discussion 27 ______

Supplementary Material (ESI) for Chemical Communications This journal is (c) The Royal Society of Chemistry 2009

Supplementary Information For:

Transport of calcium ions through a bulk membrane by use of a dynamic combinatorial library

Vittorio Saggiomo, Ulrich Lüning*

Otto-Diels-Institut für Organische Chemie, Olshausenstr. 40, D-24098 Kiel, Germany. Fax: +49-431-880-1558; Tel:+49-431-880-2450; E-mail: [email protected]

Contents page

General remarks 2

Experiment A (bi-phasic, no calcium ions) 3 - experimental procedure 3 - schematic set-up (Figure 1) 3 - 1H-NMR (Figure 2) 4

Experiment B (bi-phasic, with calcium ions) 5 - experimental procedure 5 - schematic set-up (Figure 3) 5 - 1H-NMR (Figure 4) 6

Experiment C (carrier experiment) 7 - experimental procedure 7 - schematic set-up (Figure 5) 7 - glassware (Figure 6) 8 - 1H-NMR (Figure 7) 9 - ESI-MS (Figure 8) 10 - Colorimetric titration 11 28 3 Results and Discussion ______

Supplementary Material (ESI) for Chemical Communications This journal is (c) The Royal Society of Chemistry 2009

General remarks

TraceSELECT® Water (Fluka) and Aquamerck® Calcium Test (Merck) were obtained commercially. 4-Methoxypyridine-2,6-dicarbaldehyde 1 and 3,6,9-trioxa-1,11- undecanediamine 1 were synthesized according to literature. NMR spectra were recorded with Bruker DRX 500 or AV 600 instruments. ESI mass spectra were recorded with an Applied Biosystem Mariner Spectrometry Workstation. All the glassware used for the experiments were first washed with ultra-pure water (purified by mean of ELSA Purelab Plus) and then washed with TraceSELECT® Water to prevent any contamination by calcium ions which are always present in distilled water. All experiments were repeated at least three times each. For a complete characterization of 14-methoxy-6,9,12-trioxa-3,15- diaza-1(2,6)-pyridinahexadecacyclophan-2,15-diene see 2.

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Experiment A (bi-phasic, no calcium ions): In a test tube, 8.2 mg (0.050 mmol) of 4-methoxypyridine-2,6-dicarbaldehyde (2) was dissolved in 5 mL of CDCl3 (or CD2Cl2). A solution of 9.6 mg (0.050 mmol) of 3,6,9-trioxa-

1,11-undecanediamine (1) in 5 mL of D2O was added to the organic phase to form the biphasic system. The test tube was closed with a stopper and the air was exchanged three times by nitrogen. Every 48 h, 600 µL of both layers were taken and the respective 1H- NMR spectra were recorded. A well defined amount of a standard solution of dimethyl terephthalate (DMT) was added into each organic solution to follow the evolution of the experiment.

Figure 1: Schematic set-up of experiment A. The colored half circles symbolize the diamine and dialdehyde building blocks 1, 2 and the respective products.

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1 Figure 2. H-NMR (500 MHz, 298 K). a), c) and e) CDCl3; b), d) and f) D2O. S = internal standard (DMT). Peaks are labeled with symbols as defined in Figure 1.

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Experiment B (bi-phasic, with calcium ions): In a test tube, 8.2 mg (0.050 mmol) of 4-methoxypyridine-2,6-dicarbaldehyde (2) was dissolved in 5 mL of CDCl3 (or CD2Cl2). A solution of 9.6 mg (0.050 mmol) of 3,6,9-trioxa-

1,11-undecanediamine (1) in 5 mL of D2O was added to the organic phase to form the biphasic system. To the water layer, 5.5 mg (0.050 mmol) of CaCl2 was added. The test tube was closed with a stopper and the air was exchanged three times by nitrogen. Every 48 h, 600 µL of both layers were taken and the respective 1H-NMR spectra were recorded. A well defined amount of a standard solution of dimethyl terephthalate (DMT) was added into each organic solution to follow the evolution of the experiment. 14-Methoxy-6,9,12-trioxa- 3,15-diaza-1(2,6)-pyridinahexadecacyclophan-2,15-diene in the water phase: 62-65% yield.

Figure 3: Schematic set-up of experiment B. The colored half circles shall symbolize the diamine and dialdehyde building blocks 1, 2 and the respective products (macrocycle 3 and polymers). Calcium ions are shown as orange circles.

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1 Figure 4. H-NMR (500 MHz, 298 K). a), c) and e) CDCl3; b), d) and f) D2O. S = internal standard (DMT). Peaks are labeled with symbols as defined in Figure 1 or 3.

! " 3 Results and Discussion 33 ______

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Experiment C (carrier experiment):

Water source phase: 20 mL of Fluka TraceSELECT® Water, 49 mg (0.30 mmol) of 3,6,9- trioxa-1,11-undecanediamine (1) and 33 mg (0.30 mmol) of CaCl2.

Bulk membrane: 60 mL of CH2Cl2, 49 mg (0.30 mmol) of 4-methoxypyridine-2,6- dicarbaldehyde (2).

Water receiver phase: 20 mL of Fluka TraceSELECT® Water. Each phase was left under gentle stirring for 1 week. After 1 week, the three phases were analyzed by 1H-NMR. 550 µl of the water source phase and 550 µl of the organic phase were taken. 50 µl of deuterated solvent (D2O for the water phase and CD2Cl2 for the organic phase) were added and the 1H-NMR spectra were recorded. The solvent of the aqueous receiver phase was evaporated by a flux of nitrogen. Then the residue was 1 dissolved in CD3OD and a H-NMR spectrum was recorded. The aqueous receiver phase was also analyzed by means of ESI-MS and with a titration method (Aquamerck® Calcium- Test). Macrocycle 3 and calcium ions were found in the receiver phase with 5 - 7% of yield.

Figure 5: Schematic set-up of experiment C. For symbols, see Figure 1 or 3.

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Figure 6: Glassware used in experiment C. All three compartments were gently stirred by magnetic stirring bars.

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1 Figure 7. H-NMR (500 MHz, 298 K). a) source phase H2O + 10 % D2O; b) bulk membrane

CH2Cl2 + 10 % CD2Cl2; c) receiver phase evaporated, CD3OD. Solvent residue peaks are removed for clarity. For symbols, see Figure 1 or 3.

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Figure 8. ESI-MS of the receiving phase.

! "# 3 Results and Discussion 37 ______

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Colorimetric Titration 5 ml were taken from the aqueous receiver phase. 10 drops of Ca-1 solution (Aquamerck® Calcium-Test) were added to this solution followed by a small amount of indicator Ca-2 (Aquamerck® Calcium-Test). The solution was stirred and titrated drop by drop using a titration pipette with a solution of Ca-3 (Aquamerck® Calcium-Test). When the color changed from red-violet to blue-violet the titration was complete. From the volume of the Ca-3 solution used, the amount of calcium was calculated to be 99 mg/L (1.98 mg/20 mL receiver phase, 6 %) which is in agreement with the NMR determined 5 - 7 % of calcium complex carried from the source to the receiver phase.

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! "" 38 3 Results and Discussion ______

3.4 Ion Transport across Membranes Facilitated by a Dynamic Combinatorial Library

V. Saggiomo, C. Goeschen, R. Herges, R. Quesada, U. Lüning, Eur. J. Org. Chem 2010, DOI: 10.1002/ejoc.201000038.

After the bulk membrane experiment (Ch. 3.3), another synthetic membrane and a larger library were tested. Starting from a DCL of three building blocks (one diamine and two dialdehydes) and calcium ion as template, two macrocycles were formed due to their calcium complexing abilities and one showed a better transport activity than the other. This methodology has a double screening activity: In the first screening, two different macrocycles are amplified from a DCL, during the second screening the membrane itself chooses the best carrier between the two macrocycles (Fig. 3.4). A Supported Liquid Membrane (SLM) was used as synthetic membrane and some preliminary experiments with liposomes were carried out. This also shows the versatility of the methodology that could be applied to other membranes. Catrin Goeschen from Rainer Hergesʼ group in Kiel helped with the set-up of the first SLM experiment and with the data collection. Roberto Quesada (University of Burgos) helped in the set-up of the liposome experiments.

Figure 3.4: Schematic representation of the experiment. Starting from a DCL consisting of calcium ions, one diamine and two dialdehydes, two macrocycles templated by calcium ions are formed, and one of them transports the ions through a Supported Liquid Membrane (SLM). FULL PAPER 3 Results and Discussion 39 ______DOI: 10.1002/chem.200((will be filled in by the editorial staff))

Ion Transport Across Membranes Facilitated by a Dynamic Combinatorial Library ū

Vittorio Saggiomo,a Catrin Goeschen,a Rainer Herges,a Roberto Quesada,b Ulrich Lüning*,a

Dedication ((optional))

complexes (5•Ca2+) efficiently transports pentoxypyridine-2,6-dicarbaldehyde (4) is Abstract: A dynamic combinatorial library calcium ions due to its better balance described, too, and first transport (DCL) consisting of dialdehydes and between lipo- and hydrophilicity. The experiments with liposomes are discussed. diamines has been used to facilitate special set-up of a DCL combined with a transport of calcium ions across a supported SLM directly finds suitable carriers for ion Keywords: transport • dynamic liquid membrane (SLM). In a dual selection transport starting from diamine and combinatorial chemistry • imine • process, the calcium ions first select dialdehyde building blocks 1, 2 and 4 alone. macrocycles • supported liquid matching macrocycles 3 or 5 from the DCL. The synthesis of the new, more lipophilic 4- membrane • liposomes Second, only one of the macrocycle-calcium

(aqueous) source phase, it must be bound efficiently, the resulting Introduction host-guest complex must be soluble in the membrane, and finally the guest must be released into the aqueous receiving phase. Compartmentalization by membranes is most important for the Therefore, a fine balance between thermodynamic binding parameters and kinetic uptake and release rates must be found. This existence of life. In the cell, numerous compounds must be transported from one location to another across membranes. 1 Nature usually requires an extensive optimization work. One way to simplify selection and optimization processes is the has developed several tools: from channels which allow diffusion all 5 use of dynamic combinatorial chemistry (DCC). In the beginning the way to specific carriers. Supramolecular chemistry has investigated these different approaches, too. 2 Especially molecular of DCC, the search of an optimal host for a given guest was the recognition can be exploited also for the development of a selective central focus, and many dynamic combinatorial libraries (DCLs) transporting system. In a classical experiment, a host is chosen have been investigated in the search of a good host for a given guest. which may bind the specific guest to be transported across a All DCC experiments have in common that the interaction of the membrane. As a model for the biological membrane either bulk guest with a host stabilizes a particular host, and thus the dynamic organic layers, 3 or polymer supported thin liquid membranes 4 have equilibrium of several potential host molecules changes the been used. The guest must be recognized and taken from the composition of the DCL and forms the most stable system. A good match between a host and a guest can be used for several purposes, e. g. for analysis (sensoring), for purification (extraction), or for transport. These applications have now come into focus of DCC. 6 7 [a] V. Saggiomo, M. Sc., Dr. C. Goeschen, Prof. Dr. R. Herges, Prof. Dr. U. Lüning Besides sensoring and extraction, first transport experiments 8, 9 Otto-Diels-Institut für Organische Chemie across bulk liquid membranes have been described. Here, we like Christian-Albrechts-Universität zu Kiel to present the first example of a transport across a supported liquid Olshausenstr. 40, D-24098 Kiel, Germany Fax: (+)49-431-880-1558 membranes (SLM) by a dynamic combinatorial library, and some E-mail: [email protected] experiments showing the selection of the most efficient transporter by its own guest. Moreover we present first preliminary transport [b] Dr. R. Quesada experiment using liposomes as cell membrane mimics. Departamento de Química Universidad de Burgos E-09001 Burgos, Spain Results and Discussion

ū Dynamic Combinatorial Chemistry, Part 4. For Part 3, see ref. 8. In previous work, 8 we showed that a DCL formed by a reversible Supporting information for this article is available on the WWW under reaction between diamine 1 and dialdehyde 2 can be used to effect http://www.chemeurj.org/ or from the author. calcium transport across a bulk membrane. When the building blocks react in presence of calcium ions in the water source phase,

40 3 Results and Discussion ______macrocycle 3 is formed, along with linear oligo- and polymers, and it transports calcium ions from the water source phase to the water receiver phase. 8 The good water solubility of complex 3•Ca2+ 10 is supposed to be the weakness of its carrier activity. In fact, most of the complex was found in the water source phase. 8 First, we repeated the same experiment using a supported liquid membrane 11 (SLM) (Figure 1, building blocks 1 and 2). In an SLM experiment, the water source phase is separated from the water receiver phase by a polymeric membrane (polypropylene) soaked with organic solvent (o-nitrophenyl octyl ether, NPOE). The solvent polarity of NPOE was determined using a solvatochromic dye. 12 It N N was found to be ET = 0.33, slightly different from the ET = 0.31 of dichloromethane found in literature 12 and less polar than that of N dichloromethane (distilled but not dry) used in our laboratory (ET = 0.34). 13

Figure 2. Transport of calcium ions through a SLM using dialdehyde 2 or 4 and diamine 1. The conductivity in the receiver phase is plotted against time. In all experiments, the water source phase consisted of: 10 mM solution of

CaCl2 with: a) 0.1 mmol of 1 and 0.1 mmol of 4; b) 0.1 mmol of 1 and 0.1 mmol of 2; c) calculated sum of conductivities of 1 (0.1 mmol) and 2 (0.1 mmol) from two separate experiments in which they were dissolved separately in absence of the other respective building block; d) no addition of building blocks.

The latter was reacted with 1-pentanol in presence of a catalytic amount of sulphuric acid to give 4-pentoxy substituted diester 7. After reduction with sodium borohydride, alcohol 8 was obtained. This alcohol is a bench stable product and can be stored at room temperature without any decomposition. A fast oxidation with selenium dioxide gives the desired dialdehyde 4 ready to be used Figure 1. Building Blocks (1, 2 and 4), detected products (3 and 5) used and their cartoon representations. Set-up of the transport experiment across a supported liquid membrane (SLM) facilitated by a dynamical combinatorial library (DCL).

The two water phases were gently stirred and the conductivity in the receiver phase was recorded by means of a conductivity meter. Calcium chloride was chosen for its low solubility in organic solvents with respect to other salts (e. g. calcium nitrates or picrates are, thanks to the counter ions, more lipophilic and thus more soluble in organic media). 14 Due to the lipophobicity of calcium chloride, the control experiment with a solution of calcium chloride in the source phase showed that hardly any calcium ions pass across the membrane (Figure 2d, conductivity after 48 h = 0.25 µS cm-1). On the contrary, when the two building blocks 1 and 2 were dissolved, equilibrated for one hour and then used as source phase, after a simple purification. the formed imino macrocycle 3 was able to transport calcium Scheme 1. Synthesis of dialdehyde 4 and its cartoon representation (conductivity after 48 h = 1.02 µS cm-1, Figure 2b). In two separate control experiments, the building blocks were dissolved separately in absence of the other respective building block in the calcium The two dialdehydes 2 and 4 differ only in the pentyl chain in 4- solution (source phase). Although the two building blocks interact position of the pyridine. Thus the formation of the imino macrocycle with the supported liquid membrane carrying calcium in the receiver 5 (Figure 3) in presence of calcium ions as template and its phase, the sum of their single transport activities is less than that of complexing ability should be only marginally different from imino the macrocycle 3 (conductivity after 48 h = 0.86 µS cm-1, Figure macrocycle 3. 8,10 As proof of the formation of the imino macrocycle 2c). 5, the DCL composed of diamine 1 and dialdehyde 4 was screened In order to improve the transport activity of the macrocycle, a by the mean of 1H-NMR in presence and absence of calcium lipophilic dialdehyde was synthesized. In three steps, dialdehyde 4 chloride (using CD3OD and H2O/D2O as solvent). When calcium is was obtained from commercially available chelidamic acid (6) present, only one peak in the imine region (8.64 ppm) and one in the (Scheme 1). pyridine region (7.55 ppm) could be detected in the NMR spectra

3 Results and Discussion 41 ______

(see Supplementary Information). 15 Subsequently, the above In a control experiment, in which the solution containing the two described SLM experiment was repeated using dialdehyde 4 instead macrocycles 3 and 5 was not used as a source phase, the ratio of 2. Figure 2a clearly shows that the calcium transport activity of between the two macrocycles remains constant over time [Figure 4a macrocycle 5 is approximately six times better than that of (O)]. The shape of the curve (+) in the source phase is similar to the macrocycle 3 (conductivity after 48 h = 5.70 µS cm-1) . The shape of shape of the curve of conductivity in the receiver phase (as example this curve (Figure 2a) is not linear as those of the previous transport see Figure 2a). Thus two different detection methods, NMR in the experiments (Figure 2b – 2d), however the non-linearity of transport source phase for macrocycles and conductivity in the receiver phase curves has been observed before and has been discussed and for the calcium complexes, prove the same fact: macrocycle 5 leaves explained as a decomplexation rate-limited transport. 16, 17 the source phase carrying calcium to the receiver phase. Starting from a DCL of three building blocks and one template ion, two macrocycles are selected and amplified from the mixture of macrocycles, oligomers and polymers due to their calcium complex stabilities and one shows a better transport activity than the other.

Figure 3. Macrocycle 5 and its cartoon representation.

In addition to the conductivity measurements, the presence of calcium ions in the receiver phase has also been verified using a titration method (Aquamerck® Calcium Test). Even near the detection limit (2 mg L-1), this test reveals calcium ions in the receiver phase. A control experiment with dialdehyde 4 alone dissolved in the calcium source solution was not possible due to its low solubility in water. However when the dialdehyde 4 was dissolved in NPOE it did not show any carrier activity. Figure 4. a) Ratio of 3/5 vs. time (+) calculated from the integration of the Experiments using magnesium chloride and barium chloride were pyridine peaks at 7.35 and 7.32 ppm. (O): Control experiment (see text). b) 1H-NMR of water source phase. c) Expanded section of peaks at 7.35 and also carried out. A 10 mM solution of magnesium chloride was not 7.32 over time. able to dissolve the building blocks and it was impossible to use the milky solution as source phase. When a 10 mM solution of barium After these results, we carried out some preliminary experiments in chloride was used instead, the solution became clear and an increase order to explore the possibility of screening for a carrier mediated of conductivity in the receiver phase could be measured. In this by a DCL with a biologically relevant membrane. In this regard, case, the conductivity after 48 h was: 2.33 µS cm-1. This is lower liposomes are widely used as cell membrane mimics. Our approach than when calcium is the ion in the source phase showing a small was to use unilamellar phospholipid vesicles (1-palmitoyl-2-oleoyl- preference of imino macrocycle 5 to transport calcium over barium. 18, 19 sn-glycero-3-phosphocholine, POPC) loaded with sodium chloride

and dispersed in a sodium nitrate solution. Incorporation and In another experiment, a dynamic library composed of dialdehydes movement of macrocycle 5 through the phospholipid membrane 2 and 4 (4 mM each) and two equivalents of diamine 1 was could promote counterion transport driven by the concentration generated in a 12.5 mM solution of calcium chloride. The formed gradient in a symport mechanism. Chloride release from the interior DCL was used as source phase in a DCC-SLM experiment. The of the vesicles to the external medium can be easily monitored by receiver phase, monitored by the use of a conductivity meter, using a chloride selective electrode. 22 showed a flux comparable to the above experiment with only calcium carrying macrocycle 5. In intervals, the source phase was screened by 1H-NMR by analyzing 550 µL samples of the source Briefly, POPC vesicles (100 nm mean diameter) loaded with NaCl phase to which 50 µL of D2O were added. The water residue peak in (488mM, 5 mM phosphate buffer, pH=7.2) were suspended in a the NMR spectra was removed during acquisition using arbitrary NaNO3 solution (488mM, 5 mM phosphate buffer, pH=7.2) for a waveforms and pulsed field gradient. 21 In the resulting spectra, two final lipid concentration of 1 mM. To this dispersion, 5 mol% well distinguishable peaks are partially overlapping in the imine (calculated as building blocks/POPC) of the dynamic combinatorial region, and two well separated peaks for the pyridine hydrogen library composed of diamine 1 and dialdehyde 4 previously atoms could be easily assigned to the respective macrocycles 3 (7.35 equilibrated in a solution of Ca(NO3)2 was added, and the chloride ppm) and 5 (7.32 ppm) (Figure 4b). At different times, the ratio ion efflux was monitored by a chloride ion selective electrode (ISE) between 3 and 5 could be calculated from the integration of the over time. After ten minutes, the vesicles were lysed by addition of respective peaks (Figure 4c). In Figure 4a, the ratio 3/5 is plotted detergent and the final reading of the electrode was used to calibrate versus time showing that macrocycle 5 is leaving the source phase. 100% release of chloride. For comparison purposes, control

4242 3 Results and Discussion ______

2+ 2+ experiments using similar amounts of Ca(NO3)2 solution without second, the systems selects complex 5•Ca over complex 3•Ca for any of the library components or just one them, either 1 or 4, were the transport process due to 5's better balance of lipo- and carried out. The results obtained are shown in figure 5. hydrophilicity. Complex 5•Ca2+ bears a long lipophilic chain but is still water soluble and is able to pass through the NPOE membrane thus transporting calcium ions. In future experiments, pyridine Addition of dialdehyde 4 or diamine 1 (Figure 5, 4 = •, 1 = Ƒ) dialdehydes such as 2 or 4 shall be mixed with diamines of varying induced a fast initial chloride efflux which quickly reached a length to find suitable carriers for different cations in this kind of plateau. This result could be due to a small initial detergent effect double selection experiment. The DCC-SLM experiment described exerted by these compounds. Nevertheless, the plateau shows that here could also be used to analyze other complex DCL mixtures. after the initial phase, the remaining liposomes are stable in the The fittest species will cross the membrane and can then easily be presence of the starting materials for the DCL. Addition of both detected in the receiver phase, separated from all other components compounds 1 and 4, i. e. the library/carrier, results in a small yet of the library. constant chloride efflux from the liposomes (Figure 5, +). This Preliminary experiments show that this methodology can also be efflux is more than two times faster than the blank experiment in used to screen the capability of carriers to cross phospholipid which only the same aliquot (40 µL) of Ca(NO3)2 solution was membranes. This is the first experiment on the way to use a DCL added (Figure 5, O). Although the overall chloride efflux observed directly in a biomimetic system. The methodology of selecting a in these experiments is quite limited it must be stressed that carrier from a dynamic combinatorial library therefore is very macrocycle 5 has no affinity at all for anions therefore an efficient versatile: we started by screening its carrier ability with a bulk chloride transport was not expected. On the other hand, it seems membrane 8 that has a thickness of centimeters, next we moved to a clear that addition of the carrier induces a significantly faster supported liquid membrane possessing a thickness of millimeters chloride efflux compared to the control experiments. This result and with the last experiment we used liposomes which possess a shows promise for the development of dynamic combinatorial thickness of nanometers. libraries for ion pair transporters and the use of this approach in biomimetic environments. Experimental Section

Remarks

TraceSELECT® Water was obtained commercially from Fluka and was used for the source phase solution as well as for the receiver phase in the transport experiment. o- Nitrophenyl octyl ether (NPOE) was obtained from Aldrich and was purified as described below before use in transport experiments. Polypropylene Accurel® PP was obtained from AkzoNobel. Aquamerck® Calcium Test was obtained from Merck; for its use see ref. 8. 3,6,9-Trioxa-1,11-undecanediamine (1) and 4-methoxypyridine-2,6- dicarbaldehyde (2) were synthesized according to literature. 23 Chelidamic acid (6) was synthesized using standard procedures or obtained from Aldrich. For a complete characterization of the imino macrocycle 3 see ref. 10. The conductimeter used was LF 340 from WTW with automatic data storage (15 min) and non-linear compensation of the temperature set to 20 °C. During the transport experiments, the temperature varied from 19 °C to 21 °C. The probe was LR 325/01 (WTW) with a cell constant of 0.100 cm-1. The molar conductance of calcium chloride is 271.56 · 10-4 m2 S mol-1, and 279.82 · 10-4 m2 S mol-1 for barium chloride. 20 The glassware (quartz) used for the transport experiment had a half cell volume of 25 mL, a section of 8.553 · 10-4 m2 and is described in the Supplementary Information. NMR spectra were recorded with Bruker DRX 500 or AV 600 instruments. Assignments are supported by COSY, HSQC and HMBC. All chemical shifts are referenced to TMS or to the residual proton or carbon 1 13 signal of the solvent (CD3OD, 3.35 ppm H, 49.0 ppm C). Mass spectra were recorded Figure 5. Chloride efflux from 100 nm POPC liposomes promoted by with a Finnigan MAT 8200 or MAT 8230. ESI mass spectra were recorded with an addition of 40 µL of Ca(NO3)2 solution (488 mM, O); 5 mol% (per POPC) of Applied Biosystems Mariner Spectrometry Workstation. IR spectra were recorded with dialdehyde 4 in 10 µL of DMSO (•); 5 mol% (per POPC) of diamine 1 in 40 a Perkin-Elmer Paragon 1000, equipped with an ATR unit. Elemental analyses were

µL of Ca(NO3)2 solution (488 mM, Ƒ); 5 mol% (per POPC) of the dynamic carried out with a EuroEA 3000 Elemental Analyzer from Euro Vector. combinatorial library composed of diamine 1 and dialdehyde 4 in 40 µL of Ca(NO3)2 solution (488 mM, +). The liposomes (1 mM POPC) contained Transport Experiments: NaCl (488 mM) and were immersed in NaNO3 (488 mM) in a 5 mM phosphate buffer (pH 7.2). 100 % efflux was always achieved by destroying ® All glassware were washed with bidistilled water and rinsed with TraceSELECT the liposomes upon addition of detergent. The straight lines through the data Water before use. The experiments were repeated at least 2 times, also with different points are only drawn to guide the reader’s eye. concentrations in the source phase (10 or 12.5 mM calcium chloride) proving the reproducibility of the experiments. Conclusion Membrane: Commercial NPOE was purified by a short column chromatography (1:5 NPOE / silica gel (w/w), dichloromethane). The solvent was removed under reduced The concept of using a DCL to find a matching carrier for a particle pressure and the oily residue was left under vacuum in an ultrasonic bath for 12 h before ® to be transported across a membrane (here: calcium ions) has been use. A square 4.5 · 4.5 cm of micropore polypropylene polymer Accurel PP was cut and soaked with 300 mg of NPOE. The membrane was left under vacuum in a proven here. From a DCL consisting of building blocks of varying desiccator for 12 h before use. lipo- and hydrophilicity, calcium ions select their proper host (5) for transport across a supported liquid membrane. In contrast to most Source phase: In 25 mL of 10 mM calcium chloride solution (TraceSELECT® Water) DCC experiments, here the amplification process is dual: first were dissolved 100 µmol of dialdehyde (2 or 4) and 19.1 mg (100 µmol) of 3,6,9- calcium ions amplify the formation of matching macrocycles 3 and trioxa-1,11-undecanediamine (1). The solution was vigorously stirred until complete dissolution of the building blocks. When the solution became clear (from 30 min to 2 h 5 from the dynamic mixture of oligomers, polymers and hosts, then depending on the dialdehyde) it was used as source phase in the experiment.

3 Results and Discussion 43 ______

® Receiver phase: 25 mL of TraceSELECT Water, blank conductivity between 1 and 1.5 CHCl3/MeOH 96:4 (v/v). Alcohol 8 was obtained pure as a white solid. 424 mg (1.88 µS. mmol), 92 % yield. Rf [CHCl3/MeOH 96:4] = 0.20, m. p. 82-83 °C. ESI-MS: calcd. for + + C12H19NO3: 225.14; found: 226.14 (M+H ), 248.12 (M+Na ). Elem. anal.: calcd. for DCL-transport experiment: The membrane was prepared as described above. C12H19NO3: C 63.98, H 8.50, N 6.22; found: C 64.18, H 8.86, N 6.04. IR: ν Ҋ = 3341 -1 1 (OH), 2973 (alkyl), 1597, 1496 (arom.) cm . H-NMR (500 MHz, 298 K, CDCl3,TMS): į = 6.63 (s, 2H, Py), 4.62 (s, 4H, CH2-OH), 3.95 (t, J = 6.5 Hz, 2H, O-CH2), 3.65 (br, Source phase: In 25 mL of 12.5 mM calcium chloride solution (TraceSELECT® Water) 2H, OH), 1.72 (quint, J = 7.2 Hz, 2H, O-CH-CH2-CH2), 1.39-1.28 (m, 4H, CH2-CH2- were dissolved 16.5 mg (100 µmol) of 4-methoxypyridine-2,6-dicarbaldehyde (2), 22.1 13 CH3), 0.86 (t, J = 7.1 Hz, 3H, CH3) ppm; C-NMR (125 MHz, 298 K, CDCl3, TMS) į mg (100 µmol) of 4-pentoxy-2,6-dicarbaldehyde (4) and 38.2 mg (200 µmol) of 3,6,9- = 165.77 (C-4-Py), 159.09 (C-2,6-Py), 104.67 (C-3,5-Py), 67.31 (O-CH2), 63.26 (CH2- trioxa-1,11-undecanediamine (1). The solution was vigorously stirred until complete OH), 27.52 (O-CH2-CH2), 27.00 (O-CH2-CH2-CH2), 21.34 (CH2-CH3), 12.95 (CH3) dissolution of the building blocks. When the solution became clear it was used as source ppm. phase in the experiment. A 550 µL aliquot from the source phase was taken and mixed 1 with 50 µL of D2O. The resulting solution was put in a NMR tube and H-NMR spectra were recorded at different times as the control experiment. At different times, 550 µL 4-Pentoxypyridine-2,6-dicarbaldehyde (4): 424 mg (1.88 mmol) of 4- aliquots were taken from both phases (source and receiver), the 550 µL aliquot from the pentoxypyridine-2,6-bismethanol (8) was dissolved in 10 mL of dry dioxane and 207 1 mg (1.88 mmol) of SeO2 was added. The solution was left refluxing for 6 h while source phase was mixed with 50 µL of D2O, put in a NMR tube and the H-NMR spectra were recorded. stirring. Then the heterogenous solution was passed through a pad of celite and eluted with a small portion of dioxane. The solution was recovered and the solvent was removed under reduced pressure. The crude product was purified by column The receiver phase was set up as described above. chromatography [silica gel, cyclohexane/ethyl acetate 1:1 (v/v)] giving a slightly yellow

solid, 376 mg (1.70 mmol), 90 % yield. Rf [cyclohexane/ethyl acetate 1:1] = 0.25, m. p. + + Preparation of phospholipid vesicles: 67-68 °C. ESI-MS: calcd. for C12H15NO3+Na : 244.09; found: 244.09 (M+Na ). Elem. anal.: calcd. for C12H15NO3: C 65.14, H 6.83, N 6.33; found: C 64.99, H 7.04, N 6.10. -1 1 A solution of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC, Genzyme) in IR: ν Ҋ = 2931 (alkyl), 1704 (C=O), 1591, 1448 (arom.) cm . H-NMR (500 MHz, 298 K, chloroform (20 mg/mL) was evaporated forming a lipid film. This latter was dried CDCl3, TMS): į = 10.11 (s, 2H, CHO), 7.63 (s, 2H, Py), 4.14 (t, J = 6.5 Hz, 2H, O- under vacuum for 12 h. The lipid film was rehydrated with a solution of sodium CH2), 1.88-1.82 (m, 2H, O-CH2-CH2), 1.47-1.39 (m, 4H, CH2-CH2-CH3), 0.94 (t, J = 13 chloride (488 mM of NaCl and 5 mM of phosphate buffer, pH 7.2) and shaken by 7.0 Hz, 3H, CH3) ppm; C-NMR (125 MHz, 298 K, CDCl3, TMS) į = 192.46 (CHO), vortex. The suspension was then subjected to nine freeze-thaw cycles and 29 extrusions 167.14 (C-4-Py), 154.72 (C-2,6-Py), 111.50 (C-3,5-Py), 69.34 (O-CH2), 28.37 (O-CH2- through a 100 nm polycarbonate Nucleopore membrane using a LiposoFast Basic CH2), 27.94 (O-CH2-CH2-CH2), 22.32 (CH2-CH3), 13.95 (CH3) ppm. extruder (Avestin) in order to obtain unilamellar vesicles with a mean diameter of 100 4 nm. Finally, the suspension was dialysed against a NaNO3 solution (488 mM NaNO3 1 -Pentoxy-6,9,12-trioxa-3,15-diaza-1(2,6)-pyridinahexadecacyclophan-2,15-diene and 5 mM phosphate buffer, pH 7.2) to remove unencapsulated NaCl. (5): In 10 mL of CD3OD were dissolved 11.0 mg (50 µmol) of 4-pentoxypyridine-2,6- dicarbaldehyde (4), 11.5 mg (60 µmol) of 3,6,9-trioxa-1,11-undecanediamine (1) and

5.5 mg (50 µmol) of CaCl2. After 12 h, the solution was controlled by NMR, ESI and Ion selective electrode transport assays: + + HR-MS. ESI-MS: calcd for C20H31N3O4+Na : 400.21, found: 400.22 (M+Na ). HR-MS (EI, 70 eV): calcd for C20H31N3O4: 377.23145, found 377.23146; calcd for Unilamellar vesicles (POPC, 100 nm mean diameter) prepared as described above were 13 1 C19 CH31N3O4: 378.23480, found: 378.23545. H-NMR (500 MHz, 298 K, CD3OD): į suspended in a solution of NaNO3 (488 mM, and phosphate buffer, 5 mM, pH 7.2) for a = 8.64 (t, J = 1.3 Hz, 2H, CH=N), 7.55 (s, 2H, Py), 4.31 (t, J = 6.4, 2H, (4-O-CH2-CH2), final POPC concentration of 1 mM. The dynamic combinatorial library composed of 4.05 (t, J = 4.6, 4H, CH=N-CH2), 3.96-3.90 (m, 12H, CH=N-CH2-CH2 and O-CH2-CH2- dialdehyde 4 and diamine 1 (6.25 mM each) was equilibrated for 12 h in a solution of O), 1.94-1.89 (m, 2H, 4-O-CH2-CH2), 1.56-1.44 (m, 4H, CH2-CH2-CH3), 1.00 (t, J = 7.2 Ca(NO3)2 (488 mM, phosphate buffer, pH 7.2, 5 mM) before use. 40 µL of this solution 13 Hz, 3H, CH3) ppm; C-NMR (125 MHz, 298 K, CD3OD): į = 170.47 (C-2,6-Py), was added to 5 mL of the vesicle suspension (resulting in 5 mol% of library to POPC) 164.70 (CH=N), 154.40 (C-4-Py), 115.46 (C-3,5-Py), 72.52, 71.39, 79.83, 70.03 (CH2- and the chloride ion release was monitored using an Accumet chloride selective O-CH2 and CH=N-CH2), 58.25 (Py-O-CH2), 29.55 (Py-O-CH2-CH2), 29.13 (Py-O-CH2- electrode. After 10 min, the vesicles were lysed by addition of detergent (Triton-X) to CH2-CH2), 23.40 (CH2-CH3), 14.32 (CH3) ppm. release all chloride ions (by definition giving 100% conductivity). The blank experiments with the Ca(NO3)2 solution and diamine 1 were carried out in the same way. Due to the low solubility of dialdehyde 4, it was dissolved (25 mM) in DMSO and 10 µL od this solution (corresponding to 5 mol% relative to POPC) was added to the vesicle suspension (5 mL). The experiment was repeated three times and the average of Acknowledgments the chloride efflux was plotted over time. We are grateful for support by the Marie Curie Research Training Network MRTN-CT- Syntheses: 2006-035614 Dynamic Combinatorial Chemistry (DCC). We acknowledge Dr. Frank Sönnichsen for his help in NMR spectroscopy. Dipentyl 4-pentoxypyridine-2,6-dicarboxylate (7): 500 mg (3.62 mmol) of chelidamic acid (4) was dissolved in 30 mL of 1-pentanol, and 0.2 mL of concentrated sulphuric acid was added. The solution was attached to a Dean-Stark trap and left refluxing for 4 h while stirring. Then the solvent was removed under reduced pressure, [1] a) R. MacKinnon, Angew. Chem. 2004, 116, 4363-4376; Angew. Chem., Int. Ed. the residue was dissolved in chloroform and washed with water (2 x 20 mL). The 2004, 43, 4265-4277; b) P. Agre Angew. Chem. 2004, 116, 4363-4376; Angew. organic phase was dried with MgSO . Chloroform was removed under reduced pressure 4 Chem., Int. Ed. 2004, 43, 4278-4290; c) Membrane transport, (Eds: S. L. and the oily residue was purified by column chromatography (silica gel, chloroform) Bonting and J. J. H. H. M. de Pont), North-Holland Biomedical Press, giving 869 mg (2.21 mmol, 61 %) of the final product 5 as a colorless oil. Rf [CHCl3] = + Amsterdam, 1981. 0.12. CI-MS [Isobutane] = calcd. for C22H35NO5: 393.25; found : 394.0 (M+H ). Elem. Anal. = calcd. for C22H35NO5: C 67.15, H 8.96, N 3.56; found : C 66.98, H 9.24, N 3.51. [2] A. L. Sisson, M. R. Shah, S. Bhosale, S. Matile, Chem. Soc. Rev., 2006, 35, IR: ν Ҋ = 2953 (alkyl), 1716 (ester), 1591, 1442 (arom.) cm-1. 1H-NMR (500 MHz, 298 K, 1269-1286. CDCl3, TMS): į = 7.74 (s, 2H, Py), 4.39 (t, J = 7 Hz, 4H, COO-CH2), 4.12 (t, J = 6.5 [3] Early description of a respective experimental set-up: J.-P. Behr, J.-M. Lehn, J. Hz, 4H, O-CH2), 1.87-1.80 (m, 6H, O-CH2-CH2 and COO-CH2-CH2), 1.48-1.36 (m, 13 Am. Chem. Soc., 1973, 95, 6108-6110. 12H, CH2-CH2-CH3), 0.96-0.91 (m, 9H, CH3) ppm; C-NMR (125 MHz, 298 K, CDCl , TMS): į = 166.98 (C-4-Py), 164.89 (C-2,6-Py), 150.21 (COO), 114.22 (C-3,5- 3 [4] a) Early description of a SLM with NPOE: T. B. Stolwijk, E. J. R. Sudhölter, D. Py), 68.99 (OCH ), 66.39 (COOCH ), 28.45, 28.24, 28.04, 27.99, 22.36, 22.34 (CH - 2 2 2 N. Reinhoudt, J. Am. Chem. Soc. 1987, 109, 7042-7047; b) A. Casnati, A. CH -CH ), 13.96 (CH ) ppm. 2 2 3 Pochini, R. Ungaro, C. Bocchi, F. Ugozzoli, R. J. M. Egberink, H. Struijk, R. Lugtenberg, F. de Jong, D. N. Reinhoudt, Chem. Eur. J., 1996, 2, 436-445. 4-Pentoxypyridine-2,6-bismethanol (8): 800 mg (2.05 mmol) of dipentyl 4- pentoxypyridine-2,6-dicarboxylate (7) was dissolved in 15 mL of dry methanol. 1.5 g [5] a) P. T. Corbett, J. Leclaire, L. Vial, K. R. West, J.-L. Wietor, J. K. M. Sanders, S. Otto, Chem. Rev., 2006, 106, 3652-3711; b) C. D. Meyer, C. S. Joiner, J. F. (41 mmol) of NaBH4 was slowly added to the solution at 0 °C. When the hydride was completely dissolved, the reaction was left under reflux for 4 h. Then at room Stoddart, Chem. Soc. Rev., 2007, 36, 1705-1723; c) S. J. Rowan, S. J. Cantrill, G. R. L. Cousins, J. K. M. Sanders, J. F. Stoddart, Angew. Chem. Int. Ed., 2002, temperature, 5 mL of a saturate aqueous solution of Na2CO3 was added and the solution was refluxed for additional 30 min. The solvent was removed under reduced pressure, 41, 898–952; d) O. Storm, U. Lüning, Chem. Eur. J., 2002, 8, 793-798. the residue was dissolved in CHCl and washed with water (2 x 15 mL). The organic 3 [6] A. Buryak, K. Severin, J. Comb. Chem., 2006, 8, 540-543. phase was dried with MgSO4 and the solvent was removed under reduced pressure. The crude product was purified by column chromatography, using silica gel and eluting with

44 3 Results and Discussion ______

[7] D. M. Epstein, S. Choudhary, M. R. Churchill, K. M. Keil, A. V. Eliseev, J. R. different calcium concentrations in the two water phases (10 and 0 mM Morrow, Inorg. Chem., 2001, 40, 1591-1596. initially).

[8] V. Saggiomo, U. Lüning, Chem. Commun., 2009, 25, 3711-3713. [18] The difference in binding of a barium or a calcium ion may be rationalized by the fact that, in the crystal structure, the calcium ion is located in the plane of the [9] R. Pérez-Fernandez, M. Pittelkow, A. M. Belenguer, L. A. Lane, C. V. macrocycle, while barium is displaced. The barium complex is isostructural with Robinson, J. K. M. Sanders, Chem. Commun., 2009, 25, 3708-3710. the strontium complex: D. E. Fenton, D. H. Cook, J. Chem. Soc., Chem. Commun., 1978, 279-280. [10] V. Saggiomo, U. Lüning, Eur. J. Org. Chem., 2008, 4329-4333. [19] The influence of BaCl on conductivity is comparable with that one of CaCl . [11] a) H. C. Visser, D. N. Reinhoudt, F. de Jong, Chem. Soc. Rev., 1994, 75-81; b) 2 2 The molar conductivities are 279.82 and 271.56 · 10-4 m2 S mol-1 respectively, 20 N. M. Kocherginsky, Q. Yang, L. Seelam, Sep. Pur. Technol., 2007, 53, 171- and this cannot explain the big difference measured in the receiver phase. 177. [20] D. R. Lide, Handbook of Chemistry and Physics, 80th edition, CRC Press, Boca [12] C. Reichardt, Chem. Rev., 1994, 94, 2319-2358. Raton, 1999-2000.

[13] ET(30) values are based on the solvatochromic pyridinium N-phenolate betaine N [21] T. L. Hwand, A. J. Shaka, J. Magn. Reson. Ser. A., 1995, 112, 275-279. dye as probe molecule. ET is the normalized value using water and tetramethylsilane as extreme polar and nonpolar reference solvents, respectively. [22] J. T. Davis, P. A. Gale, O. A. Okunola, P. Prados, J. C. Iglesia-Sánchez, T. Torroba, R. Quesada, Nature Chem., 2009, 138-144. [14] Y. Marcus, Ion Solvation, Wiley & Sons, New York, 1985. [23] U. Lüning, R. Baumstark, K. Peters, H. G. v. Schnering, Liebigs. Ann. Chem., [15] Macrocycle 5 was characterized in solution by 1H-, 13C-NMR, ESI-MS and 1990, 129-143. HRMS.

[16] E. G. Reichwein-Buitenhuis, H. C. Visser, F. de Jong, D. N. Reinhoudt, J. Am. Chem. Soc., 1995, 117, 3913-3921. Received: ((will be filled in by the editorial staff)) Revised: ((will be filled in by the editorial staff)) [17] It should be noted that this experiment is a proof of concept and it was not our Published online: ((will be filled in by the editorial staff)) goal to optimize the supported liquid membrane experiment. For this reason, the calcium chloride solution was 10 mM instead of 1 M, usually used as source phase in this kind of experiment. Moreover we did not use any “strip solution” as receiver phase. The use of an acidic receiver phase (strip solution) improves drastically the flux of ions through the membrane, as the driving force in this case is the difference in pH. In our experiment, the only driving force are the

3 Results and Discussion 45 ______

Supplementary Information For:

Ion Transport Across Membranes Facilitated by a Dynamic Combinatorial Library

Vittorio Saggiomo,a Catrin Goeschen,a Rainer Herges,a Roberto Quesada, b Ulrich Lüning*,a

[a] Otto-Diels-Institut für Organische Chemie, Olshausenstr. 40, D-24098 Kiel, Germany. Fax: +49-431-880-1558; Tel: +49-431-880-2450; E-mail: [email protected]

[b] Departamento de Quìmica, Universidad de Burgos, E-09001 Burgos, Spain.

!! Contents!! ! ! ! ! ! ! ! ! page! ! ! Glassware !! ! ! ! ! ! ! ! 2

! Dipentyl 4-pentoxypyridine-2,6-dicarboxylate (7)!! ! 3 !!1H-NMR!! ! ! ! ! ! ! 3 !!13C-NMR!! ! ! ! ! ! ! 4 ! ! 4-Pentoxypyridine-1,6-bismethanol (8)!! ! ! ! 5 !!1H-NMR!! ! ! ! ! ! ! 5 !!13C-NMR!! ! ! ! ! ! ! 6

! 4-Pentoxypyridine-2,6-dicarbaldehyde (4)!! ! ! 7 !!1H-NMR!! ! ! ! ! ! ! 7 !!13C-NMR!! ! ! ! ! ! ! 8 ! ! 14-Pentoxy-6,9,12-trioxa-3,15-diaza-1(2,6)- ! pyridinahexadecacyclophan-2,15-diene (5)!! ! ! 9 !!1H-NMR!! ! ! ! ! ! ! 9 !!13C-NMR!! ! ! ! ! ! ! 10 !!COSY!! ! ! ! ! ! ! ! 11

! DCL of 4 and 1 in presence and absence of CaCl2!! ! 12 !!1H-NMR!! ! ! ! ! ! ! 12

! DCC-SLM - Source Phase!! ! ! ! ! ! 13 !!1H-NMR ratio/time!! ! ! ! ! ! 13

1 46 3 Results and Discussion ______

polymer containing liquid membrane

water source phase

water receiver phase

Figure 1: Glassware (quartz) used and set-up for the Dynamic Combinatorial Chemistry - Supported Liquid Membrane experiment (DCC-SLM)

2 3 Results and Discussion 47 ______

Me O

RO OR N O O Me R =

1 Figure 2: H-NMR (500 MHz, 298 K, CDCl3,TMS) of product 7

3 48 3 Results and Discussion ______

Me O

RO OR N O O Me R =

13 Figure 3: C-NMR (125 MHz, 298 K, CDCl3, TMS) of product 7

4 3 Results and Discussion 49 ______

Me O

N OH OH

1 Figure 4: H-NMR (500 MHz, 298 K, CDCl3,TMS) of product 8

5 50 3 Results and Discussion ______

Me O

N OH OH

13 Figure 5: C-NMR (125 MHz, 298 K, CDCl3, TMS) of product 8

6 3 Results and Discussion 51 ______

Me O

N O O

1 Figure 6: H-NMR (500 MHz, 298 K, CDCl3,TMS) of product 4

7 52 3 Results and Discussion ______

Me O

N O O

13 Figure 7: C-NMR (125 MHz, 298 K, CDCl3, TMS) of product 4

8 3 Results and Discussion 53 ______

Me O

N N N Ca2+ O O O

water

free diamine 1

1 Figure 8: H-NMR (500 MHz, 298 K, CDCl3,TMS) of product 5 in the DCL formed by dialdehyde 4 and diamine 1 in presence of CaCl2

9 54 3 Results and Discussion ______

Me O

N N N Ca2+ O O O

free diamine 1

13 Figure 9: C-NMR (125 MHz, 298 K, CDCl3, TMS) of product 5 in the DCL formed by dialdehyde 4 and diamine 1 in presence of CaCl2

10 3 Results and Discussion 55 ______

Me O

H N N N H Ca2+ O O O

Figure 10: COSY (expansion) NMR (500 MHz, 298 K, CDCl3,TMS) of product 5 in the DCL formed by dialdehyde 4 and diamine 1 in presence of CaCl2

11 56 3 Results and Discussion ______

H2O+D2O, CaCl2

CD3OD, CaCl2

CD3OD, no CaCl2

Figure 11: 1H-NMR (500 MHz, 298 K), DCL formed by reacting diamine 1 and dialdehyde 4 in presence or absence of CaCl2 in different solvents.

12 3 Results and Discussion 57 ______

time time

c) d)

control experiment experiment

Figure 12: DCC-SLM experiment, source phase mixture of dialdehydes 2, 4, diamine 1 1 1 1 and CaCl2, H-NMR (600 MHz, 298 K, H2O + 10 % D2O). a) H-NMR of the mixture; b) H- NMR, expanded section; c) control experiment (no transport) and, d) time dependent transport experiment.

13 58 3 Results and Discussion ______

3.5 The First Supramolecular Ion Triplet Complex J. Eckelmann V. Saggiomo, F. Sönnichsen, U. Lüning, 2010, submitted.

This experiment was done in prevision of possible future use of polytopic carrier macrocycles able to complex both ion and counter-ion. In this collaboration, Jens Eckelmann provided the macrocycle shown in Figure 3.5. This neutral macrocycle displays three binding sites (tritopic macrocycle): four amide NHs in two different isophthalamide residues, well known to complex anions, and four oxygen atoms in the diethylene glycol part able to complex cations. The capacity of this macrocycle to extract alkaline and alkaline earth metal chlorides into an organic solvent was screened. Regarding alkaline metal chlorides, this host was capable to bind and extract into chloroform selectively LiCl over NaCl and KCl, and

CaCl2 over MgCl2 and BaCl2 regarding alkaline earth metal chlorides. Other proofs of the binding were obtained by the use of ESI-MS and NOESY experiments (thanks to the support of Frank Sönnichsen). This, to the best of our knowledge, is the ﬁrst supramolecular ion triplet complex.

Figure 3.5: Schematic representation of the ion triplet complex with calcium chloride. 3 Results and Discussion 59 ______

CREATED USING THE RSC COMMUNICATION TEMPLATE (VER. 3.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS ARTICLE TYPE www.rsc.org/xxxxxx | XXXXXXXX

The first supramolecular ion triplet complex

Jens Eckelmann, Vittorio Saggiomo, Frank D. Sönnichsen and Ulrich Lüning*

Received (in XXX, XXX) Xth XXXXXXXXX 200X, Accepted Xth XXXXXXXXX 200X First published on the web Xth XXXXXXXXX 200X

5 DOI: 10.1039/b000000x

A neutral tritopic macrocycle 1 was obtained by condensation of NO a diacid dichloride 2 with a diamine 3. 1 contains three binding 2 sites: two for anions by hydrogen bonding and one for cations by a O O ether oxygen atoms. The selective binding of LiCl and CaCl2 has b 10 been studied by NMR and MS techniques. 1 is the first host to NH HN . c form a supramolecular complex with an ion triplet: 1 CaCl2. d Cl- In supramolecular chemistry, macrocycles have always been O O e widely used to complex different organic and inorganic Ca2+ guests.1 For a long time, chemists focused their attention on O O 15 the complexation of cations and anions, with the latter task - being more challenging due to the large size, different shapes Cl 2 and the high polarisability of anions. Nowadays, the NH HN importance of targeting ion-pairs of salts as guests is growing.3 In this field, ditopic hosts are usually synthesized as O O 20 neutral compounds, in which the ditopic nature of the receptor

allows to bind both cation and anion in adjacent binding units NO 2 in close contact.4 With the matching cation for the contact pair formation in the host-guest complex, it is possible to enhance 1 the binding of a particular anion or vice versa.5 Fig. 1 Calcium dichloride complex with macrocycle 1 and hydrogen labeling scheme. For structural studies, see supporting information. 25 Although this field is growing and many different ditopic molecules have been synthesized in the last five years, there is 55 Additionally it has two nitro groups that can be further still a lack of hosts for complexing alkaline earth metal modified, e. g. by reduction. The neutral macrocycle 1 halides. In order to reach this goal, chemists must move from 1 possesses D2h symmetry giving rise to a simple H-NMR ditopic hosts to tritopic hosts. In these, two binding units for spectrum: two triplets for the NH and Hd protons, a dublet for 30 anions and one for a cation comprise the sites needed for the 6 Ha, a broad singlet for the Hb and a singlet for the He protons. complexation of an alkaline earth metal halide as ion triplet. , 1 7 60 The easily interpretable and clean H-NMR spectra were used In this work, we present the neutral tritopic host 1 that is to investigate the capability of this tritopic macrocycle 1 to capable to bind an alkaline earth metal salt, and binds calcium bind salts as contact ions, and to detect their extraction from dichloride as an ion triplet with high selectivity. To the best of solid into an organic solvent. A stock solution (2.5 mM) of 1 35 our knowledge, the formation of complex 1·CaCl2 is the first in CDCl3 with 5% of DMSO-d6 was allowed to stand over an description of a supramolecular ion triplet complex for 8 65 excess of powdered alkaline and alkaline earth metal chlorides alkaline earth metal salts (figure 1). in different NMR tubes. After 12 h, 1H-NMR spectra were The synthesis of macrocycle 1 is straightforward and starts recorded and analyzed for differences in chemically induced from two building blocks: diacid dichloride 2 and diamine 3 shifts (CIS) between the free macrocycle 1 and the complexes 40 (see scheme 1). In order to obtain and study macrocycles of (Figure 2). different sizes, we used a combinatorial approach which gave 70 When comparing the solutions containing the alkaline the [2+2]-macrocycle 1 (figure 1), together with [1+1]-, chlorides LiCl, NaCl and KCl, the NMR spectra clearly show [3+3]-, [4+4]-macrocycles and linear products which are not that macrocycle 1 is capable of binding lithium chloride described here. After a simple one step condensation of diacid selectively over other alkaline metal chlorides, as the 1H- 45 dichloride 2 and diamine 3 in the presence of triethylamine, NMR spectra do not show any change when sodium chloride the [2+2]-macrocycle 1 could be isolated from the resulting 75 or potassium chloride are used. In the case of lithium chloride, mixture by chromatography (scheme 1). a significant CIS of NH (-0.88 ppm) and Hb (-0.65 ppm) was Macrocycle 1 displays three binding sites (tritopic detected (Figure 2, LiCl). macrocycle): four NH amides in two different isophthalamide 9 The large downfield shift of almost 1 ppm of the amide proton 50 residues, well known to complex anions, and four oxygen is indicative of the formation of hydrogen bonds to the atoms in the diethylene glycol part able to complex cations. 10 80 chloride anion (NH···Cl) in presence of DMSO.

This journal is © The Royal Society of Chemistry [year] Journal Name, [year], [vol], 00–00 | 1 60 3 Results and Discussion ______

NO2 NH downfield shift (-0.90 ppm) is observed only when NO2 calcium dichloride is used (Figure 2, CaCl2). The calcium O O dichloride complex (1·CaCl2) shows proton shifts similar to the lithium chloride complex (1·LiCl). Only H is shifted more O e O NH HN 30 upfield (+0.06 ppm) with respect to 1·LiCl which reflects a Cl Cl different side chain orientation. Due to different sizes of 2 magnesium, calcium and barium, macrocycle 1 is able to O O NEt3 complex selectively calcium dichloride as a contact ion- THF triplet. To the best of our knowledge, this is the first neutral O O 35 macrocycle that binds an alkaline earth metal dihalide as O O contact ion triplet.13 Mass analyses confirm the strong complexing ability of NH2 H2N NH HN macrocycle 1 for LiCl and CaCl2 (see Supplementary 3 O O Information). When ESI-MS spectra (negative ion mode) were 40 recorded directly from the NMR solutions with either lithium chloride or calcium dichloride, it was possible to detect a NO 2 single peak as 1+Cl- (m/z=681.20). The ESI-MS scan of 1 1·CaCl2 in the positive mode showed two peaks attributable to 2+ + Scheme 1 Synthetic scheme for the formation of macrocycle 1. complex 1·CaCl2: (1+Ca )/2 (m/z=343.10) and 1+CaCl 45 (m/z=721.14). On the other hand, the ESI spectra of the lithium chloride complex 1·LiCl showed only the protonated peak 1+H+, but for this complex the MALDI-TOF spectrum revealed 1+Li+ (m/z=653.31). To obtain further insight into the complexation of the salts by 50 macrocycle 1, detailed NOESY experiments were carried 14 out. A stock solution of 1 in CDCl3 (ca. 2.5 mM) with 7% of DMSO-d615 was used to record 2D-NOESY spectra in three different NMR tubes: in the absence of salt, in the presence of excess of powdered lithium chloride, and in the presence of 55 excess of powdered calcium dichloride. Table 1 Average proton-proton distances16 (in Å) in macrocycle 1 based on NOESY experiments. For proton labels see Figure 1.

1 1·LiCl 1·CaCl2

Hb-NH 2.6 2.85 2.5 Ha-NH 2.7 4.5 4.1 NH-Hc 3.1 3.3 2.95 Fig. 2 Expanded sections of 1H-NMR spectra (500 MHz, 298 K, NH-Hd 3.5 3.85 3.4 5 CDCl /DMSO-d6 95:5) of 1 in the presence of different salts. For proton 3 Hb-Hc 4.3 4.8 4.3 assignments see Figure 1. Hb-Hd 4.0 5.0 4.7 Ha-Hc 4.1 4.6 4.1 A small difference in the chemical shifts of Ha (-0.08 ppm), Ha-Hd 3.9 - - not involved in the binding, was also detected. In the Table 1 compiles the proton-proton distances as calculated diethylene glycol chains, upfield CIS for He (+0.04 ppm) and from the NOESY measurements. When the obtained distances 10 downfield CIS for Hd (-0.05 ppm) were observed. Due to the 60 for the free macrocycle 1 are compared to those in the 11 key importance of lithium salts as drug in different diseases, complexes, significant differences are apparent. In pure

it is an interesting goal to develop lithium receptors and macrocycle 1, the average proton-proton distances of Ha-NH 12 sensors. The challenge is to bind it selectively over and Hb-NH were found to be quite similar. The NH proton is competing ions such as sodium. Macrocycle 1 achieves this at the same average distance from Ha and Hb, indicating that 15 task in an organic solvent complexing selectively LiCl as 65 the benzene ring is capable of rotating around the Ar-CO

contact ion pair over NaCl. bond. Thus in about half of the population, NH is close to Ha, Additionally, alkaline earth metal dichlorides (MgCl2, CaCl2, and in the other half, NH is close to Hb, resulting in almost BaCl2) were screened applying the same methodology. identical average distances of 2.6 Å (Hb-NH) and 2.7 Å (Ha- Indeed, macrocycle 1 is able to form an ion triplet complex NH) in the free macrocycle. 20 1·CaCl2, but remarkably only with the calcium salt. The 70 The distances Ha-NH and Hb-NH respond differently to the distinct differences in CIS for MgCl2, CaCl2 and BaCl2 show a addition of salts, i. e. the binding of chloride anions by strong selectivity of macrocycle 1 for calcium dichloride over hydrogen bonding, as they become different in both the 1·LiCl

magnesium and barium dichloride. Although in presence of and 1·CaCl2 complexes. The average proton-proton distance magnesium and barium dichloride, the NH signal shows a between NH and Ha increased considerably, thus on average 25 modest CIS (-0.14 and -0.24 ppm, respectively), a prominent

2 | Journal Name, [year], [vol], 00–00 This journal is © The Royal Society of Chemistry [year] 3 Results and Discussion 61 ______

3 Ha moved away from NH, while at the same time the apparent B. D. Smith, “Ion-Pair Recognition By Ditopic Receptors” in Macrocyclic Chemistry: Current Trends and Future, (eds.: K. Gloe, NH-Hb distance is shortened. Both facts can be explained by the binding of the NH protons to the chloride anions. In fact, B. Antonioli), Kluwer, London, 2005, pp 137-152. 4 Some recent examples of ditopic receptors: K. Salorinne, T.-R. Tero, the complex must have been rigidified upon complexation and K. Riikonen, M. Nissinen, Org. Biomol. Chem., 2009, DOI: 5 the two protons (NH and Ha) are now further away from one 10.1039/b911389d; N. Bernier, S. Carvalho, F. Li, R. Delgado, V. another, while at the same time Hb spends more time in close Félix, J. Org. Chem., 2009, 74, 4819-4827; M. D. Lankshear, I. M. proximity to the NH proton. It is interesting to note that also Dudley, K.-M. Chan, A. R. Cowley, S. M. Santos, V. Félix, P. D. Beer, Chem. Eur. J., 2008, 14, 2248-2263; M. D. Lankshear, A. R. the distances between H and H increase upon complexation b d Cowley, P. D. Beer, Chem. Commun., 2006, 6, 612-614; J. M. from 4 Å to 5 Å (1·LiCl) and 4.7 Å (1·CaCl2), respectively. Mahoney, K. A. Stucker, H. Jiang, I. Carmichael, N. R. Brinkmann, 10 Finally, we carried out first orientational experiments to A. M. Beatty, B. C. Noll, B. D. Smith, J. Am. Chem. Soc., 2005, 127, quantify the formation of the supramolecular complexes of 1. 2922-2928. J. M. Mahoney, A. M. Beatty, B. D. Smith, Inorg. Chem., 2004, 43, 7617-7621. The salts are insoluble in the solvents used for the NMR 5 17 J. M. Mahoney, A. M. Beatty, B. D. Smith, J. Am. Chem. Soc., 2001, investigations, especially CaCl2. But with a mixture of 123, 5847-5848; S. Kubik, J. Am. Chem. Soc., 1999, 121, 5846-5855. anhydrous calcium perchlorate and tetrabutylammonium 6 There is a discussion on how to name an ionic aggregate such as 15 chloride in CHCl3/DMSO (93:7), we were able to carry out CaCl2: triple ion or ion triplet. We have chosen ion triplet to highlight isothermal titration calorimetry (ITC) experiments with the fact that three ions are bound as a unit, and we have not chosen triple ion to avoid the impression that there is a remaining charge receptor 1. The results must not be overinterpreted due to the 7 (see ). 1·CaCl2 is the first neutral supramolecular ion triplet complex. insolubility problems and also to the fact that the mixture 7 G. V. Oshovsky, D. N. Reinhoudt, W. Verboom, J. Am. Chem. Soc. changes the ionic strength. Nevertheless, binding constants for 2006, 128, 5270-5278. 3 4 8 20 1:1 complexes in the range of 10 to 10 in CHCl3/DMSO With a dichloride of a more electrophilic transition metal (palladium), a structurally related complex has been described: B. A. Blight, J. A. could be determined for CaCl2 (by use of a Wisner, M. C. Jennings, Chem. Commun. 2006, 4593-4595. Ca(ClO4)2/nBu4NCl mixture), and for LiCl (by use of a 9 P. V. Santacroce, J. T. Davis, M. E. Light, P. A. Gale, J. C. Iglesias- Li(ClO4)/nBu4NCl mixture) and nBu4NCl. Sánchez, P. Prados, R. Quesada, J. Am. Chem. Soc., 2007, 129, 1886- In conclusion, a facile and accessible synthesis of macrocycle 1887; K. Kavallieratos, B. A. Moyer, Chem. Commun., 2001, 1620-1621; 25 1 has been described. The ability of 1 to complex LiCl and A. Szumna, J. Jurczak, Eur. J. Org. Chem., 2001, 4031-4039; K. 1 Kavallieratos, C. M. Bertao, R. H. Crabtree, J. Org. Chem., 1999, 64, CaCl2 was proven by means of H-NMR and mass analyses. In 1675-1683. solution, its conformational change in the presence of guests 10 M. J. Deetz, M. Shang, B. D. Smith, J. Am. Chem. Soc., 2000, 122, was analyzed and described using NOESY experiments. All 6201-6207. these experiments concentrate on the fact that the neutral 11 N. J. Birch, Chem. Rev, 1999, 99, 2659-2682; C. J. Phiel, C.A. 30 macrocycle 1 complexes calcium dichloride in its ion-triplet Wilson, V. M.-Y. Lee, P. S. Klein, Nature, 2003, 423, 435-439; H. R. form. This ion-triplet receptor should be useful in different Pilcher, Nature, 2003, 425, 118-120. 12 S. Rochat, Z. Grote, K. Severin, Org. Biomol. Chem., 2009, 7, 1147- applications such as selective extraction from solid mixtures 1153. (industrial application), membrane transport (calcium is an 13 To prove the chloride complexing abilities of macrocycle 1, essential element for biological life, and chloride tetrabutylammonium chloride (TBACl) was used as salt. And even in 35 concentration controls several processes in the cell), this case, the shifts of the protons are comparable with 1·LiCl and chemosensing, homogeneous catalysis and phase transfer 1·CaCl2 complexes. Nevertheless, the NH protons are shifted more when CaCl2 or LiCl was used instead of TBACl (naked chloride). catalysis. In our laboratory the screening of some of these The CIS of the ethylene glycol protons are more similar to the CIS of application is work in progress. 1·CaCl2 than to that of 1·LiCl. Acknowledgement 14 Unfortunately, up to now it was not possible to obtain a single crystal 40 We thank the EU for its support through the Marie Curie of 1·CaCl2. Besides, if the complexation shall be exploited in Research Training Network MRTN-CT-2006-035614 applications such as for instance transport it must be studied in solution anyway. Dynamic Combinatorial Chemistry (DCC). Eva Mucke's help 15 In comparison to the first binding experiments, a slightly higher with the calculations is gratefully acknowledged. concentration of DMSO was used in order to ensure a homogeneous organic phase, needed to record reliable NOESY spectra. 16 Notes and references 2D-NOESY experiments with varying mixing times were used to analyze the conformation and mobility of the free macrocycle and the 45 Otto-Diels-Institut für Organische Chemie, Christian-Albrechts- tritopic complexes. Using a two-spin approximation and two known Universität zu Kiel, Olshausenstr. 40, D-24098 Kiel, Germany. Tel.: +49 distances as a reference, average interproton distances were 431 880-2450 Fax: +49 431 880-1558 E-mail: [email protected] calculated from all observed NOE intensities after peak integration as † Electronic Supplementary Information (ESI) available: Experimental described in supplementary materials. In a mobile structure, the and spectroscopic details of 1. NMR and MS complexation studies. distances are physically meaningless i. e. don’t describe a single 50 Calculations. See DOI: 10.1039/b000000x/ conformation, their differences upon ion binding however indicate

1 J. W. Steed and J. L. Atwood, Supramolecular Chemistry, 2nd ed., changes in the combination of short distances and population of the Wiley, New York, 2009. conformations present. 17 2 F. P. Schmidtchen, M. Berger, Chem. Rev., 1997, 97, 1609-1646; A. CaCl2 and LiCl are insoluble in the solvent mixture used and are Bianchi, K. Bowman-James, E. García-España in Supramolecular extracted into these solvents only by 1. Therefore, a titration with Chemistry of Anions, Wiley-VCH, New York, 1997; A. J. Evans, P. portions of the solid salt into a solution of host 1 cannot be D. Beer, Dalton Trans., 2003, 4451-4456; M. T. Reetz, C. M. interpreted when excess salt is added. For this reason, the preliminary Niemeyer, K. Harms, Angew. Chem., Int. Ed. Engl., 1991, 30, 1472- binding studies were done with salts which are soluble in the solvent 1474. mixture: Ca(ClO4)2, Li(ClO4) and nBu4NCl.

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4 Summary and Perspective

4.1 Summary Dynamic Combinatorial Chemistry (DCC) is a powerful tool to discover and synthesize novel hosts in one step. In a Dynamic Combinatorial Library (DCL), all the members of the library, due to reversible reactions, are in equilibrium one with another. When a guest template is added, the equilibrium is shifted toward the host that better binds it. Subsequently, freezing the equilibration allows the puriﬁcation of the host. Thus in one pot it is possible to obtain a host for a given guest (Fig. 4.1).

OMe OMe

N H2N O NH2 CaCl2 N + N N 3 2+ O O Ca O O O

Figure 4.1: From two building blocks (left) a dynamic combinatorial library is set up (center). All the members of the library are in equilibrium with one another. The addition of a guest template shifts the equilibrium to the host that better binds it (right).

Using a DCL directly for a function is a remarkable step forward. This methodology allow to screen in one step not only for a product, but for a product useful for an application. In this thesis the proof of concept of using a DCL directly for screening a given function is demonstrated. The transport of ions mediated by a carrier ampliﬁed from a DCL was tested and successfully achieved. A carrier macrocycle was formed directly from a DCL composed of imines using calcium ions as a template. Then the macrocycle transported calcium ions from a water source phase to a water receiver phase passing through different organic synthetic membranes. Proved for the ﬁrst time, this goal was achieved in a series of experiments. First of all the possibility of using a library composed of imines in water was studied. Usually imines are quickly hydrolyzed in presence of small amounts of water. However the hydrolysis is one of the possible pathway for the reversibility of the imine formation. Therefore it was interesting to test the stability of various imines in 4 Summary and Perspective 83 ______the presence of increasing amounts of water. Moreover the presence of a template ion on this stability was screened (Ch. 3.1).

During this work, a discrepancy was found in the literature. In two different publications the same imine synthesis in water was described with two completely different yields. Repeating the experiments and using another reaction condition, it was possible to understand and explain the discrepancy. This discrepancy lies in the fact that the starting materials and the product were not soluble in water. Therefore it was important to differentiate between reaction and workup (Ch. 3.2). Before starting the transport experiments, the same DCL of chapter 3.1 was screened in a biphasic system. At last, the possibility of amplifying a macrocyclic carrier directly in a bulk membrane experiment was proven. A carrier was amplified from the DCL and the template was subsequently transported from the aqueous source phase to the aqueous receiver phase passing trough an organic solvent bulk membrane. The one step generation of a carrier from a library was successfully achieved for the first time (Ch. 3.3). A forward development was realized setting up a double screening methodology using a larger DCL. In the first screening, the template selects and amplifies two similar macrocycles from the DCL, and in a second screening, the membrane, here a supported liquid membrane, selects the macrocycle with the best carrier activity. Thus in only one step the best carrier for a given guest was amplified and selected (Fig. 4.2). The proof of concept of a transport mediated by a DCL was also tested using liposomes as mimic of a biological membrane (Ch. 3.4).

Me O

N Me N N O Ca2+ O O O N O O

Figure 4.2: Double screening methodology. From three building blocks (left) a Dynamic Combinatorial Library is set up. In a first screening, the addition of a guest template amplifies two related hosts that better bind it (center). In a second screening the membrane itself (pear colour) selects the host with better carrier activity (right). Equilibrium arrows between the components of the library are omitted for clarity. For the cartoons representation of the other used molecules see figure 4.1. 84 4 Summary and Perspective ______

Next development must deal with the transport of counter-ions. When cations are transported, one way to keep the electroneutrality of the system, is transporting anions simultaneously. In order to achieve this goal, a politopic macrocycle is necessary. In such a macrocycle, the ion and the counter-ion are both partially shielded from the organic solvent. In a novel study, a macrocycle was synthesized and its ability to bind calcium chloride as an ion triplet was proven. This is the ﬁrst description of a ion triplet complex (Ch. 3.5).

In a few words, a Dynamic Combinatorial Library can be used to screen for a carrier. The evidences of this proof of concept are shown in this thesis. This one step methodology is extremely flexible, and it can be used with different synthetic membranes. In fact its application was successfully tested using membranes with centimetres thickness (bulk membrane) to the nanometers thickness of liposomes. Moreover the presented methodology is, in principle, adaptable to all reversible reaction. This latter point is indeed interesting for synthesizing novel and diverse carriers in a one step fashion. There is a great interest and a wide application of carriers in various field. This thesis has achieved a big step forward for the application of Dynamic Combinatorial Chemistry. Using the novel described methodologies, the screening of carriers can be done in a simple and fast way. The direct use of a library instead of a single purified product is the connection point between DCC and Systems Chemistry. Therefore this thesis stands between two new and emerging fields.

4.2 Perspective The concept of using a DCL to screen for a carrier was proven. However the route to achieve a better performance of the methodology is still long. Using larger libraries composed of a larger number of building blocks with different lipophicility will improve drastically the potential of the screening. Naturally when more products are screened, the possibilities of finding a carrier with high performance are higher. The results of this thesis work now open also two new interesting tasks: Testing the methodology with cell membranes and the possibility of screening for a polytopic host by the use of ion pairs or triplets as guests. The methodology of screening for a carrier was used with different synthetic membranes (bulk membrane, supported liquid membranes and liposomes). The logical continuation of the experiments presented in this thesis should be to use various cells. Using different cell lines it should be possible, in principle, to screen and find the best carrier for each type of cell membrane. Moreover the building 4 Summary and Perspective ! 85 ______blocks can be modified using fluorescent groups. With those groups it should be easy to follow the movement and the position of the carrier in different cells. Using various drugs and a library composed of fluorescent building blocks, the formation of the carrier and the cellular uptake of the drug could be also screened. Using contact pair or triplet ions as template, a Dynamic Combinatorial Library should be used to screen for macrocycles that bind ions as contact pairs or triplets. The use of ion pairs or triplets as template in DCC is still not exploited. 86 5 References ______

5 References

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5 J. W. Steed, D. R. Turner, K. J. Wallace, Core concepts in Supramolecular Chemistry and Nanochemistry, Wiley-VCH, Weinheim, 2007.

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7 J. F. Stoddart, Chem. Soc. Rev., 2009, 38, 1802-1820.

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12 P. A. Brady, J. K. M. Sanders, J. Chem. Soc., Perkin Trans. 1, 1997, 21, 3237-3252.

13 One example of a Dynamic Combinatorial Library based on imines studied in our laboratory: O. Storm, U. Lüning, Chem. Eur. J., 2002, 8, 793-798.

14 One example of a Dynamic Combinatorial Library of pseudo-peptide hydazone macrocycles: G. R. L. Cousins, S. A. Poulsen, J. K. M. Sanders, Chem. Commun., 1999, 1575-1577.

15 Report on selection and ampliﬁcation of hosts from a DCL of macrocyclic disulﬁdes: S. Otto, E. L. R. Furlan, J. K. M. Sanders, Science, 2002, 297, 590-593.

16 S. Lüthje, Ph.D. Thesis, Christian-Albrechts-Universität zu Kiel, 2006.

17 D. Stoltenberg, Ph.D. Thesis, Christian-Albrechts-Universität zu Kiel, 2010.

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23 Borch reductive amination by NaBH3CN: R. F. Borch, M. D. Bernstein, H. D. Durst, J. Am. Chem. Soc., 1971, 93, 2897-2904.

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27 N. K. Terret, Combinatorial Chemistry, Oxford University Press, Oxford, 1998.

28 K. C. Gupta, A. K. Sutar, Coord. Chem. Rev., 2008, 252, 1420-1450.

29 V. C. Gibson, C. Redshaw, G. A. Solan, Chem. Rev., 2007, 107, 1745-1776.

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31 N. E. Borisova, M. D. Reshetova, Y. A. Ustynyuk, Chem. Rev., 2007, 107, 46-79.

32 D. M. Epstein, S. Choudhary, M. R. Churchill, K. M. Keil, A. V. Eliseev, J. R. Morrow, Inorg. Chem., 2001, 40, 1591-1596.

33 J.-B. Lin, X.-N. Xu, X-K. Jiang, Z. -T. Li, J. Org. Chem., 2008, 73, 9403-9410.

34 A. Herrmann, N. Giuseppone, J.-M. Lehn, Chem. Eur. J., 2009, 15, 117-124.

35 A. González-Álvarez, I. Alfonso, F. López-Ortiz, A. Aguirre, S. García-Granda, V. Gotor, Eur. J. Org. Chem., 2004, 1117-1127.

36 A. González-Álvarez, I. Alfonso, V. Gotor, Chem. Commun., 2006, 2224-2226.

37 S. Ladame, Org. Biomol. Chem., 2008, 6, 219-226.

38 Catalysis used as self-screen for substrates from a Dynamic Combinatorial Library: R. Larsson, Z. Pei, O. Ramström, Angew. Chem., 2004, 116, 3802-3804; Angew. Chem. Int. Ed., 2004, 43, 3716-3718.

39 “Dynamic Combinatorial Libraries of Dye Complexes as Sensors”: A. Buryak, K. Severin, Angew. Chem., 2005, 117, 8149-8152; Angew. Chem. Int. Ed., 2005, 44, 7935-7938.

40 “Dynamic Combinatorial Libraries of Metal-Dye Complexes as Flexible Sensors for Tripeptides”: A. Buryak, K. Severin, J. Comb. Chem., 2006, 8, 540–543.

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45 It is impossible to give an comprehensive overview of transport in all of its facets without writing a book. However it will be helpful to remember that: Passive transport does not require energy. The movement of molecules or ions across the membrane is due to a concentration or electrochemical gradient. The ﬂux is achieved by simple diffusion, facilitated diffusion by the help of carriers or channels, and by osmosis. Active transport, on the contrary, is the movement of molecules or ions against a concentration or electrochemical gradient and it requires energy.

46 Reprint with permission of Prof. Tom Flyes (University of Victoria), “Membrane Transport”, XIII International Symposium on Supramolecular Chemistry, Workshop on Membrane Transport, 2004. Eidesstattliche Erklärung

Hiermit erkläre ich, Vittorio Saggiomo, an Eides statt, dass ich die vorliegende Dissertation selbständig und nur mit den angegebenen Hilfsmitteln angefertigt habe. Die Arbeit ist nach Inhalt und Form, abgesehen von der Beratung durch meinen Betreuer, durch mich eigenständig erarbeitet und verfasst worden. Die Arbeit entstand unter Einhaltung der Regeln guter wissenschaftlicher Praxis der Deutschen Forschungsgemeinschaft. Weder die gesamte Arbeit noch Teile davon wurden von mir an anderer Stelle im Rahmen eines Prüfungsverfahrens eingereicht. Dies ist mein erster Promotionsversuch.

Kiel, 04.03.2010

Vittorio Saggiomo Lebenslauf

Persönliche Daten

Vor- und Zuname: !! ! Vittorio Saggiomo Geburtsdatum und -ort: !! 31.07.1980, Napoli Nationalität: !! ! ! Italian Anschrift:!! ! ! Schauenburgerstr. 64, 24118 Kiel Email:!! ! ! ! [email protected]

Ausbildung

11/ 1998 - 03/ 2007!! ! Master in Chemistry, Federico II Università !!!!!di Napoli

03/ 2007!! ! ! Master Thesis supervised by Prof. Daniela !!!!!Montesarchio: “Novel Amphiphilic Cyclic !!!!!Oligosaccharides: Synthesis and !!!!!Self-Aggregation Properties” seit 05/ 2007 !!! Early Stage Researcher in the EU !!!!!Marie Curie Initial Training Network !!!!!“Dynamic Combinatorial Chemistry” !!!!!supervised by Prof. Dr. Ulrich Lüning in Kiel

Kiel, 04.03.2010