Analytical methods in supramolecular chemistry

Historically, the development ofsupramolecular chemistry certainly depended on the development of analyticalmethods which could solve the questions associated to the complex architecture

Analytical Methods inSupramolecularChemistry

Edited byChristoph Schalley

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Analytical Methods in Supramolecular Chemistry

Edited by

Christoph Schalley

The Editor

Prof Dr Christoph A Schalley

Freie Universita¨t Berlin

Inst f Chemie u Biochemie

to be free of errors Readers are advised to keep

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Cover Design Adam Design, Weinheim Typesetting Asco Typesetters, Hong Kong Printing Strauss GmbH, Mo¨rlenbach Binding Litges & Dopf GmbH, Heppenheim ISBN: 978-3-527-31505-5

Keiji Hirose

Franz P Schmidtchen

Michael Kogej and Christoph A Schalley

5.2.1.1 Matrix-assisted Laser Desorption/Ionization (MALDI) 106

Yoram Cohen, Liat Avram, Tamar Evan-Salem and Limor Frish

6.3.1 The Basic Pulse Sequence 164

Bernard Valeur, Ma´rio Nuno Berberan-Santos and Monique M Martin

Supramolecular Complexes from Spectrophotometric or

7.3.5.2 Excitation Energy Transfer in a Self-assembled Zinc Porphyrin–Free Base

Marie Urbanova´ and Petr Malonˇ

8.5 Examples of Vibrational Circular Dichroism Applications 283

Kari Rissanen

B A Hermann

10.2.2.3 Single Molecule Force Spectroscopy – Force-Distance

10.3.2 Exemplary Results on Smaller Molecules 371

Stefan Matile and Naomi Sakai

Barbara Kirchner and Markus Reiher

12.2.5 How to Make the Connection to Experiment? 439

Supramolecular Chemistry, conceptually founded as a research field in its own inthe 1960s, is a rapidly growing field at the borderline of several disciplines such asbio(organic) chemistry, material sciences, and certainly the classical chemistrytopics, i.e (in)organic and physical chemistry Historically, the development ofsupramolecular chemistry certainly depended on the development of analyticalmethods which could solve the questions associated to the complex architecturesheld together by noncovalent bonds and those arising from weak intermolecularbonding and the highly dynamic features of many supramolecular species How-ever, not only supramolecular chemistry benefited from the methodological devel-opment Vice versa, the problems faced by supramolecular chemists led to specificmethodological solutions and thus mediated their development to a great extent.Several excellent textbooks on Supramolecular Chemistry exist, starting withFritz Vo¨gtle’s seminal best-seller ‘‘Supramolekulare Chemie’’ [1], which was trans-lated into several languages and thus found a broad international readership notonly among experts in the field, but also among chemistry students Other authors:Jean-Marie Lehn [2], Jerry Atwood and Johnathan Steed [3], and most recently Kat-suhiko Ariga and Toyoki Kunitake [4], have provided expertly written textbooks.These textbooks focus on and are organized along the chemistry involved, but donot focus much on the methods utilized to study this chemistry – with one notableexception: Hans-Jo¨rg Schneider’s and Anatoly Yatsimirski’s fine introduction intothe ‘‘Principles and Methods in Supramolecular Chemistry’’ [5] The present bookaims at a more in-depth description of different methods utilized in this branch

of chemical research

Clearly, a choice had to be made as to which of the many methods availabletoday should be included This choice is likely biased to some extent by the editor’sown preferences and a reader might arrive at the conclusion that another choicewould have been better Some chapters deal with methods of fundamental impor-tance For example, Chapter 2 provides a practical guide to the determination ofbinding constants by NMR and UV methods and thus covers an aspect imminentlyimportant to the field, which deals with noncovalent binding and weak interac-tions Similar arguments hold for the next two chapters on isothermal titration cal-orimetry and extraction methods The following chapters on mass spectrometry,diffusion-ordered NMR spectroscopy, photochemistry, and circular dichroism do

not primarily provide an in-depth introduction into these so-to-say classical ods, but focus on exciting new achievements in the context of SupramolecularChemistry Chapters 9 and 10 discuss methods used to address quite complex ar-chitectures, for example generated by crystal engineering and through surface at-tachment and self-assembly processes at surfaces The functional aspect of manysupramolecular systems appears most pronounced in Chapter 11 which introducesmethods for the characterization of membrane channels The book terminateswith a discussion of the contributions that theory can make to SupramolecularChemistry, a field which adds valuable insight, although it is often believed to bevery limited due to the sheer size of the complexes and architectures under study.Each of the chapters introduces the reader to a particular method However, thereader will probably need to have at least some basic knowledge of supramolecularchemistry itself Although the book begins with a short introductory chapter to pro-vide some necessary background, it is impossible to give a concise and comprehen-sive overview after more than four decades of quick growth in the field In thatsense, it aims at an already somewhat experienced readership.

meth-I am grateful to all authors of the individual chapters for their excellent tions to the book Particularly, I would like to thank Dr Steffen Pauly from Wiley-VCH for his great help in preparing the final manuscript and his guidancethrough the production process It was great joy to assemble this book and I sin-cerely hope that it is fun to read

References

1 First German text: F Vo¨gtle,

Supramolekulare Chemie, Teubner,

Stuttgart 1989 First English text:

F Vo ¨ gtle, Supramolecular Chemistry:

An Introduction, Wiley, Chichester

1991.

2 J.-M Lehn, Supramolecular Chemistry

– Concepts and Perspectives, Verlag

Chemie, Weinheim 1995.

3 J W Steed, J L Atwood, molecular Chemistry, Wiley, New York 2000.

Supra-4 K Ariga, T Kunitake, Supramolecuar Chemistry – Fundamentals and Applications, Springer, Berlin 2003.

5 H.-J Schneider, A Yatsimirsky, Principles and Methods in Supramolecu- lar Chemistry, Wiley, Chichester 2000.

List of Contributors

Dipl.-Ing (FH) Bianca Antonioli

Technische Universita¨t Dresden

Prof Ma´rio Nuno Berberan-Santos

Instituto Superior Te´cnico

Centro de Quı´mica-Fı´sica Molecular

1049-001 Lisboa

Portugal

Prof Yoram Cohen

Tel Aviv University

Tel Aviv Israel

Dr Kerstin Gloe Technische Universita¨t Dresden Fachrichtung Chemie und Lebensmittelchemie Bergstr 66

01069 Dresden Germany Prof Dr Karsten Gloe Technische Universita¨t Dresden Fachrichtung Chemie und Lebensmittelchemie Bergstr 66

01069 Dresden Germany Prof Dr B A Hermann LMU Munich, Walther-Meissner-Institute of the Bavarian Academy of Science

Center for Nano Science (CeNS) Walther-Meissner-Str 8

85748 Garching Germany Prof Keiji Hirose Osaka University Division of Frontier Materials Science Department of Materials Engineering Science Graduate School of Engineering Science 1-3 Machikaneyama Toyonaka

Osaka 560-8531 Japan

Dipl.-Ing Stefanie Juran

Forschungszentrum Rossendorf e.V.

Institut fu¨r Radiopharmazie

Academy of Sciences of the Czech Republic

Institute of Organic Chemistry and

Ecole Normale Supe´rieure

UMR CNRS-ENS 8640, De´partement de

Prof Dr Markus Reiher

ETH Zurich, Honggerberg Campus HCI

Laboratorium fu¨r Physikalische Chemie

30, quai Ernest Ansermet

1211 Geneva Switzerland Prof Dr Christoph A Schalley Freie Universita¨t Berlin Inst fu¨r Chemie und Biochemie Takustr 3

14195 Berlin Germany Prof Dr Franz P Schmidtchen Technical University of Munich Department of Chemistry Lichtenbergstr 4

85747 Garching Germany

Dr Holger Stephan Forschungszentrum Rossendorf e.V Institut fu¨r Radiopharmazie Bautzner Landstrasse 128

01328 Dresden Germany Prof Marie Urbanova´

Institute of Chemical Technology Department of Physics and Measurement Technicka´ 5

166 28 Praha 6 Czech Republic Prof Bernard Valeur Conservatoire National des Arts et Me´tiers Laboratoire de Chimie Ge´ne´rale

292 rue Saint-Martin

75141 Paris Cedex France

and ENS-Cachan UMR CNRS 8531 Laboratoire de Photophysique et Photochimie Supramole´culaires et Macromole´culaires De´partement de Chimie

61 Avenue du Pre´sident Wilson

94235 Cachan Cedex France

Introduction

Christoph A Schalley

1.1

Some Historical Remarks on Supramolecular Chemistry

The fundaments of Supramolecular Chemistry date back to the late 19th century,when some of the most basic concepts for this research area were developed Inparticular, the idea of coordination chemistry was formulated by Alfred Werner(1893) [1], the lock-and-key concept was introduced by Emil Fischer (1894) [2],and Villiers and Hebd discovered cyclodextrins, the first host molecules (1891) [3]

A few years later, Paul Ehrlich devised the concept of receptors in his Studies onImmunity (1906) [4] by stating that any molecule can only have an effect on thehuman body, if it is bound (‘‘Corpora non agunt nisi fixata’’) Several of these con-cepts were refined and modified later Just to provide one example, Daniel Kosh-land formulated the induced fit concept (1958) for binding events to biomoleculeswhich undergo conformational changes in the binding event [5] The induced fitmodel provides a more dynamic view of the binding event, compared with therather static key-lock principle and is thus more easily able to explain phenomenasuch as cooperativity Even the German word for ‘‘Supramolecule’’ appeared in theliterature as early as 1937, when Wolf and his coworkers introduced the term

‘‘U¨ bermoleku¨l’’ to describe the intermolecular interaction of coordinatively rated species such as the dimers of carboxylic acids [6]

satu-The question immediately arising from this brief overview on the beginnings ofsupramolecular chemistry is: Why hasn’t it been recognized earlier as a researcharea in its own right? Why did it take more than 40 years from the introduction

of the term ‘‘U¨ bermoleku¨l’’ to Lehn’s definition of supramolecular chemistry [7]

as the ‘‘chemistry of molecular assemblies and of the intermolecular bond’’ [8]?There are at least two answers The first relates to the perception of the scientistsinvolved in this area As long as chemistry accepts the paradigm that properties ofmolecules are properties of the molecules themselves, while the interactions withthe environment are small and – to a first approximation – negligible, there is noroom for supramolecular chemistry as an independent field of research Althoughsolvent effects were already known quite early, this paradigm formed the basis ofthe thinking of chemists for a long time However, with an increasing number of

examples of the importance of the environment for the properties of a molecule, aparadigm shift occurred in the late 1960s Chemists started to appreciate that theirexperiments almost always provided data about molecules in a particular environ-ment It became clear that the surroundings almost always have a non-negligibleeffect Consequently, the intermolecular interactions became the focus of researchand a new area was born With this in mind, chemists were suddenly able to thinkabout noncovalent forces, molecular recognition, templation, self-assembly andmany other aspects into which supramolecular chemistry meanwhile diversified.The second answer is not less important, although somewhat more technical innature Supramolecules are often weakly bound and highly dynamic Based on in-termolecular interactions, complex architectures can be generated, often with long-range order All these features need specialized experimental methods, many ofwhich still had to be developed in the early days of supramolecular chemistry Asobserved quite often, the progress in a certain research area – here supramolecularchemistry – depends on the development of suitable methods An emerging newmethod on the other hand leads to further progress in this research field, since itopens new possibilities for the experimenters It is this second answer whichprompted us to assemble the present book in order to provide information on thecurrent status of the methods used in supramolecular chemistry It also shows howdiverse the methodological basis is, on which supramolecular chemists rely.

1.2

The Noncovalent Bond: A Brief Overview

Before going into detail with respect to the analytical methods that are applied

in contemporary supramolecular chemistry, this brief introduction to some basicconcepts and research topics within supramolecular chemistry is intended to pro-vide the reader with some background Of course, it is not possible to give a com-prehensive overview It is not even achievable to review the last 40 or so years ofsupramolecular research in a concise manner For a more in-depth discussion, thereader is thus referred to some excellent text books on supramolecular chemistry[7]

Noncovalent bonds range from coordinative bonds with a strength of severalhundreds of kJ mol1 to weak van der Waals interactions worth only a few

kJ mol1 They can be divided in to several different classes Attractive or repulsiveinteractions are found, when two (partial) charges interact either with opposite po-larity (attraction) or the same polarity (repulsion) Ion–ion interactions are stron-gest with bond energies in the range of ca 100 to 350 kJ mol1 The distance be-tween the charges and the extent of delocalization over a part of a molecule or eventhe whole molecule have an effect on the strength of the interaction Consequently,the minimization of the distance between two oppositely charged ions will be a ge-ometric factor, when it comes to the structure of the supramolecular aggregate –even though there is no particular directionality in the ion–ion interaction Interac-tions between ions and dipoles are somewhat weaker (ca 50–200 kJ mol1) Here,

the orientation of the dipole with respect to the charge is important A typical ample for such an ion–dipole complex is the interaction of alkali metal ions withcrown ethers Other coordination complexes with transition metal ions as the coresare often used in supramolecular assembly Here, the dative bond has a greater co-valent contribution, which makes it difficult to clearly draw the line between supra-molecular and molecular chemistry Even weaker than ion–dipole forces (5–50

ex-kJ mol1) are the interactions between two dipoles Again, the relative orientation

of the two interacting dipoles plays an important role

Hydrogen bonding [9] is pivotal in biochemistry (e.g in the formation of doublestranded DNA and protein folding) and was also greatly employed in artificialsupramolecules One reason is that many host–guest complexes have been studied

in noncompetitive solvents where the hydrogen bonds can become quite strong.Another, maybe equally important reason is the directionality of the hydrogenbond which allows the chemist to control the geometry of the complexes and to de-sign precisely complementary hosts for a given guest (see below) One should dis-tinguish between strong hydrogen bonds with binding energies in the range of 60–

120 kJ mol1and heteroatom–heteroatom distances between 2.2 and 2.5 A˚, ate hydrogen bonds (15–60 kJ mol1; 2.5–3.2 A˚), and weak hydrogen bonds withbinding energies below ca 15 kJ mol1 and long donor–acceptor distances of up

moder-to 4 A˚ This classification is also expressed in the fact that strong hydrogen bondshave a major covalent contribution, while moderate and weak ones are mainly elec-trostatic in nature Also, the range of possible hydrogen bond angles is narrow instrong H bonds (175–180) so that there is excellent spatial control here, whilemoderate (130–180) and weak (90–150) hydrogen bonds are more flexible Fur-thermore, one should always make a difference between hydrogen bonding be-tween neutral molecules and charged hydrogen bonds The latter ones are usuallysignificantly stronger For example, the FaH  F hydrogen bond has a bond en-ergy of ca 160 kJ mol1and thus is the strongest hydrogen bond known

Noncovalent forces also involve p-systems, which can noncovalently bind to ons or other p-systems The cation-p interaction [10] amounts to ca 5–80 kJ mol1and plays an important role in biomolecules Aromatic rings such as benzene bear

cati-a qucati-adrupole moment with cati-a pcati-articati-ally positive s-sccati-affold cati-and cati-a pcati-articati-ally negcati-ativep-cloud above and below the ring plane Consequently, alkali metal and other cati-ons can form an attractive interaction when located above the center of the aro-matic ring The gas-phase binding energy of a Kþcation to benzene (80 kJ mol1)

is higher than that of a single water molecule to the same cation (75 kJ mol1).Consequently, one may ask why potassium salts don’t dissolve in benzene Oneanswer is that the cation is stabilized by more than one or two water molecules inwater and the sum of the binding energies is thus higher than that of a Kþsolvated

by two or three benzenes Another oft forgotten, but important point is the tion of the corresponding anion Water is able to solvate anions by forming hydro-gen bonds In benzene such an interaction is not feasible Again, we touch thetopic discussed in the beginning: the effects of the environment

solva-p-systems can also interact favorably with other solva-p-systems The interactionsusually summarized with the term p-stacking are, however, quite complex Two

similarly electron-rich or electron-poor p-systems (e.g benzene as a prototype) tendnot to interact in a perfect face-to-face manner [11], because the two partially nega-tive p-clouds would repulse each other Two options exist to avoid this repulsion: inthe crystal, benzene forms a herringbone-packing Each benzene molecule is thuspositioned with respect to its next neighbors in an edge-to-face orientation Thiscauses an attractive interaction between the negative p-cloud of one benzene withthe positive s-scaffold of the other Larger aromatic molecules, for example por-phyrins, may well crystallize in a face-to-face orientation However, they reducethe repulsive forces by shifting sideways The picture changes significantly, whentwo aromatics interact one of which is electron-rich (prototypically a hydroqui-none), one electron-deficient (prototypically a quinone) These two molecules canthen undergo charge transfer interactions which can be quite strong and usuallycan be identified by a charge-transfer band in the UV/vis spectrum.

On the weak end of noncovalent interactions, we find van der Waals forces (<5

kJ mol1) which arise from the interaction of an electron cloud polarized by cent nuclei Van der Waals forces are a superposition of attractive dispersion inter-actions, which decrease with the distance r in a r6 dependence, and exchange re-pulsion decreasing with r12

adja-A particular case, finally, which perfectly demonstrates the influence of the ronment, is the hydrophobic effect which relies on the minimization of the ener-getically unfavorable surface between polar/protic and unpolar/aprotic molecules.Hydrophobic effects play an important role in guest binding by cyclodextrins, forexample Water molecules residing inside the unpolar cavity cannot interact withthe cavity wall strongly If they are replaced by an unpolar guest, their interactionwith other water molecules outside the cavity is much stronger, resulting in a gain

envi-in enthalpy for the whole system In addition to these enthalpic contributions, tropy changes contribute, when several water molecules are replaced by one guestmolecule, because the total number of translationally free molecules increases.There are more noncovalent interactions which cannot all be introduced here.Forces between multipoles have been expertly reviewed recently [12] Also, weakinteractions exist between nitrogen and halogen atoms [13], and dihydrogenbridges [14] can be formed between metal hydrides and hydrogen bond donors Fi-nally, close packing in crystals is an important force in crystallization and crystalengineering The present introductory chapter will not discuss these, but ratherfocus on the most important ones mentioned above

en-1.3

Basic Concepts in Supramolecular Chemistry

The following sections discuss some fundamental concepts in supramolecularchemistry The list is certainly not comprehensive and the reader is referred to text-books for a broader scope of examples However, the selection reveals that supra-molecular research developed from its heart, i.e the examination and understand-ing of the noncovalent bond, to more advanced topics which make use of that

knowledge to build large, complex architecture, to understand the action of lecules, to implement function into molecular devices such as sensors, to controlmechanical movement, to passively and actively transport molecules, and to usesupramolecules as catalysts.

biomo-Clearly, molecular recognition processes are the prototypical supramolecular actions on which the other aspects are based Without molecular recognition, thereare no template effects, no self-assembly, and certainly no self-replication In con-trast to opinions sometimes encountered among chemists from other areas, supra-molecular chemistry did not come to a halt with the examination of hosts andguests and their interactions Sophisticated molecular devices are available whichnot only are based on, but go far beyond mere molecular recognition

re-1.3.1

Molecular Recognition: Molecular Complementarity

After these remarks, the first question is: What is a good receptor for a given strate? How can we design a suitable host which binds a guest with specificity? Ac-cording to Fischer’s lock-and-key model, complementarity is the most importantfactor Most often, it is not one noncovalent interaction alone which provideshost–guest binding within a more or less competing environment, but the additive

sub-or even cooperative action of multiple interactions The msub-ore complementary thebinding sites of the host to those of the guest, the higher the binding energy Thisrefers not only to individual noncovalent bonds, but to the whole shape and thewhole electrostatic surface of both molecules involved in the binding event Selec-tive binding is thus a combination of excellent steric fit with a good match of thecharge distributions of guest surface and the hosts cavity and a suitable spatial ar-rangement of, for example, hydrogen bond donors and acceptors, thus maximizingthe attractive and minimizing the repulsive forces between host and guest

Cation recognition developed quickly early on, due to the combination of theoften rather well-defined coordination geometry of most cationic species and theusually higher achievable binding energies coming from ion–dipole interactions.Actually, many of the basic concepts in supramolecular chemistry have been de-rived from studies in cation recognition The design of neutral hosts for neutralguests and in particular anion recognition [15] are still a challenge nowadays.1.3.2

Chelate Effects and Preorganization: Entropy Factors

A binding event in which one complex forms from two molecules is entropicallydisfavored The entropic costs need to be paid from the reaction enthalpy releasedupon host–guest binding However, strategies exist which can reduce these costs to

a minimum

One approach is to incorporate more than one binding site in one host molecule.When the first bond is formed, the entropic costs of combining two molecules aretaken care of The second and all following binding events between the same two

partners will not suffer from this effect again and thus contribute more to the freeenthalpy of binding This effect is called the chelate effect and has long beenknown from coordination chemistry, where ethylene diamine or 2,20-bipyridine li-gands easily replace ammonia or pyridine in a transition metal complex Bidentatebinding generates rings and the chelate effect depends on their sizes Optimal arefive membered rings as formed by the ethylene diamine or bipyridine ligands dis-cussed above Smaller rings suffer from ring strain, larger rings need a higher de-gree of conformational fixation compared with their open-chain forms and are thusentropically disfavored The latter argument can be refined If the same number ofbinding sites are incorporated in a macrocycle or even macrobicycle, guest bindingwill again become more favorable, because each cyclization reduces the conforma-tional flexibility for the free host and thus the entropic costs stemming from con-formational fixation during guest binding These effects have entered the literature

as the macrocyclic and macrobicyclic effect Donald Cram developed these ideasinto the preorganization principle [16] A host which is designed to display thebinding sites in a conformationally fixed way, perfectly complementary to theguest’s needs, will bind significantly more strongly than a floppy host which needs

to be rigidified in the binding event This becomes strikingly clear, if one comparesconformationally flexible 18-crown-6 with the spherand shown in Fig 1.1 whichdisplays the six oxygen donor atoms in a preorganized manner The alkali bindingconstants of the two host molecules differ by factors up to 1010!

While discussing entropic effects, it should not be forgotten that examples existfor enthalpically disfavored, entropy-driven host–guest binding This is possible, ifthe free host contains more than one solvent molecule as the guests, which uponguest binding are replaced by one large guest as discussed for cyclodextrins above

In this case, a host–solvent complex releases more molecules than it binds and theoverall reaction benefits entropically from the increase in particle number

Fig 1.1 Preorganization does matter A comparison of 18-crown-6

and the spherand on the right with respect to alkali metal ion binding

reveals that the spherand has an up to 10 orders of magnitude higher

binding constant.

Cooperativity and Multivalency

Cooperativity and multivalency are phenomena arising in molecular recognition athosts with more than one binding site In order to avoid misunderstandings, oneshould clearly distinguish the two terms Cooperativity describes the influence ofbinding a guest at the host’s binding site A on the second binding step occurring

at site B of the same host Cooperativity can be positive, which means that bindingstrength of the second guest is increased by the first one and the sum of both bind-ing energies is more than twice the binding energy of the first guest Cooperativitycan also be negative, if the first binding event decreases the binding of the secondguest Many examples for cooperativity are known from biochemistry, the mostprominent one certainly oxygen binding at hemoglobin [17] This protein is a

a2b2 tetramer with four oxygen binding hemes as the prosthetic groups, one ineach subunit Upon binding the first oxygen molecule to one of the heme groups,conformational changes are induced in the protein tertiary structure which also af-fect the other subunits and prepare them for binding oxygen more readily Fromthis example, it becomes clear that cooperativity does not necessarily rely on inter-actions between a multivalent host and a multivalent guest, but that there may well

be mechanisms to transmit the information of the first binding event to the secondone, even if both are monovalent interactions The concept of cooperativity hasbeen applied to supramolecular chemistry and was recently discussed in the con-text of self-assembly [18] (see below)

Conceptually related to the chelate effect, multivalency [19] describes the uniquethermodynamic features arising from binding a host and a guest each equippedwith more than one binding site Although sometimes not used in a stringentway in the chemical literature, one should use the term ‘‘multivalency’’ only forthose host–guest complexes, in which the dissociation into free host and guest re-quires at least the cleavage of two recognition sites The concept of multivalencyhas been introduced to adequately describe the properties of biomolecules [20].For example, selectivity and high binding strengths in recognition processes atcell surfaces usually require the interaction of multivalent receptors and substrates.Due to the complexity of many biological systems, limitations exist for a detailedanalysis of the thermochemistry and kinetics of multivalent interactions betweenbiomolecules For example, the monovalent interaction is usually unknown andthus, a direct comparison between the mono- and multivalent interaction is oftennot feasible The sometimes surprisingly strong increase of binding energythrough multivalency is thus not fully understood in terms of enthalpy and en-tropy

Recently, this concept was applied convincingly to artificial supramolecules Theexamination of artificial, designable, and less complex multivalent systems pro-vides an approach which easily permits analysis of the thermodynamic and kineticeffects in great detail As an example, the binding of a divalent calixarene ligandbearing two adamantane endgroups on each arm binds more strongly to a cyclo-dextrin by a factor of 260 compared with the monovalent interaction – a much

higher increase than expected for merely additive interactions If offered many clodextrin hosts on a surface, the binding constant again increases by 3 orders ofmagnitude [21] Another example is shown in Fig 1.2 [22] A three-armed guest iscapable of forming a triply threaded pseudorotaxane with the tris-crown derivative.Attachment of stoppers at the ends of each arm prevents deslippage of the axlecomponents The trivalent interaction increases the yield of the synthesis throughfavorable entropic contributions At the same time, the function of a ‘‘molecularelevator’’ is implemented: depending on protonation and deprotonation of the dia-lkyl amines, the crown ethers move back and forth between two different stationsalong the axle.

cy-1.3.4

Self-assembly and Self-organization

Self-assembly [23] is a strategy used by supramolecular chemists to reduce the forts required for the generation of complex structures and architectures Instead

ef-of tedious multistep covalent syntheses, simple building blocks are programmedwith the suitably positioned binding sites and upon mixing the right subunits,they spontaneously assemble without any additional contribution from the chem-ist Several requirements must be met: (i) the building blocks must be mobile, butthis requirement is almost always fulfilled with molecules in solution due to Brow-nian motion; (ii) the individual components must bear the appropriate informationwritten into their geometrical and electronic structure during synthesis to providethe correct binding sites at the right places Since their mutual recognition re-quires specificity, self-assembly is a matter of well pre-organized building blocks(see above); (iii) the bonds between different components must be reversiblyformed This means that the final aggregate is generated thermodynamically con-

Fig 1.2 Molecular elevator synthesized by utilizing multivalency The

position of the wheel component can be controlled by protonation/

deprotonation.

trolled under equilibrium conditions This aspect is important, because kineticallycontrolled processes do not have the potential for error correction and thus usuallylead to mixtures The reversibility of self-assembly processes also results in quitedynamic aggregates prone to exchange reactions of their building blocks.

Self-assembly is ubiquitous in nature [24] and often occurs on several hierarchylevels simultaneously in order to generate functional systems For example, theshell-forming protein building blocks of the tobacco mosaic virus [25] need to foldinto the correct tertiary protein structure before they can be organized around atemplating RNA strand All these processes are mediated by noncovalent forceswhich guide the formation of secondary structure elements on the lowest hierarchylevel These form the tertiary structure on the next level which displays the neces-sary binding sites for the assembly of the virus from a total of 2131 building blocks

to occur as programmed on the highest level Other examples for hierarchical assembly are multienzyme complexes, the formation of cell membranes with allthe receptors, ion channels, or other functional entities embedded into them, ormolecular motors such as ATP synthase Self-assembly is thus an efficient strategy

self-to create complexity and – self-together with it – function in nature

Self-assembly has also been applied to numerous different classes of complexes

in supramolecular chemistry [26] Since we cannot discuss them all here, Fig 1.3shows only one example of a capsule reversibly formed from two identical self-complementary monomers which are bound to each other by hydrogen bonding

Fig 1.3 Self-assembling ‘‘softball’’ Right: Computer model of the

softball bearing the hydroquinone spacer (side chains are omitted).

Box (left): Different monomers which form dimers with cavities of

volumes between 187 and 313 A˚ 3 depending on the spacer length Left:

A selection of good guest molecules which can occupy the cavity inside

the capsule.

[27] The two monomers can encapsulate guests in the interior cavity of the sule Even more than one guest can be encapsulated, and reactions can be cata-lyzed inside.

cap-Another term which is often used in the literature as synonymous with assembly is self-organization However, again, we should be precise with respect

self-to the meaning of the terms we use One suggestion for definitions would be self-todistinguish processes which lead to the thermodynamic minimum and thus lead

to chemical equilibria These processes should be called self-assembly processes

On the other hand is the broad variety of spontaneous organization which occursfar away from the thermodynamic equilibrium Many processes in living organ-isms are examples for self-organization in this sense The major difference be-tween self-assembly and self-organization is that self-assembly occurs even in aclosed system while self-organization can be characterized as a steady state inwhich a system remains without falling to the thermodynamic minimum, becauseenergy is constantly flowing through it This definition has the advantage that itmakes a clear difference between the two terms This advantage however comes atthe price that it is experimentally difficult to determine which is which by simplecriteria

1.3.5

Template Effects

One way to control the outcome of a reaction is templating Like in the scopic world, a chemical template organizes reaction partners and thus allows thechemist to control their reactivity to achieve the formation of a desired product.However, it is almost impossible to give a concise definition of the term ‘‘template’’[28] Templates span the whole range from biochemistry with its complex appara-tus for DNA replication [29] to the formation of structured inorganic materials [30]

macro-to the templated synthesis of macrocycles [31] macro-to the preparation of supramolecularcatalysts [32] – just to name a few examples Nevertheless, all these have in com-mon that a template must serve different purposes: (i) it organizes reaction part-ners for the formation of a desired product whose synthesis cannot be achieved inthe absence of the template Thus, a template controls reactivity and producesform; (ii) the template needs to bind to the reaction partners Molecular recogni-tion is thus a necessary prerequisite for template syntheses and the binding sites

of the components must be complementary to each other Usually, binding is due

to noncovalent bonds, although examples for covalent templates exist; (iii) the trol of reactivity and the recognition of the reaction partners imply information to

con-be programmed into the template which is transferred to the product of the tion

reac-There are different ways to categorize templates One could for example try todistinguish template effects according to the (non-)covalent interactions involved.This classification remains ambiguous for templates operating through differentforces at the same time A maybe better way to classify templates relates to theirtopography The early templated crown ether syntheses utilized alkali metal ions

around which macrocycles form with size selectivity [33] Such templates are vex, because of their convex surface mediating the template effect In contrast, areceptor binding two molecules which react inside a cavity is concave This is truefor many templates leading to mechanically interlocked species One of the mostprominent natural templates, i.e single-stranded DNA, could be called a lineartemplate according to this classification Finally, a surface on which moleculesself-assemble into an ordered array [34] may be considered as a planar template.[35].

con-Although there certainly is some overlap, one should distinguish between a tant, a template, and a catalyst [36] A strict definition would stress that the tem-plate must be removable after a successful reaction, while a reactant at least inpart remains in the product However, these definitions become blurred For exam-ple, the synthesis of rotaxanes, catenanes, and knots [37] often relies on macro-cycles which in their cavity bind an axle component in a pseudorotaxane fashion.Thus, the macrocycle acts as the template which organizes the axle in a threadedgeometry Ring closure of the axle or the attachment of stoppers lead to catenanes

reac-or rotaxanes, respectively The macrocyclic template finally becomes part of theproduct according to the strict definition would be considered as a reactant ratherthan a template Nevertheless, this view on the synthesis of interlocked molecules– one out of many examples is shown in Fig 1.4 – neglects the organization of thetwo pseudorotaxane components which is essential for the formation of the me-chanical bond Thus, these syntheses are widely accepted as template-mediated inthe chemical literature, although the use of removable transition metal ions for thesynthesis of mechanically interlocked molecules [38] is probably the only true tem-plate synthesis for interlocked molecules in the strict sense It is similarly difficult

to separate templates from catalysts: on one hand, many templates do not promotecatalytic reactions, because the template does not generate turnover They need to

be used in stoichiometric amounts and have to be separated from the product Onthe other hand, some catalysts do not organize the reactants in space but ratherchange their intrinsic reactivity as for example encountered in general acid orbase catalysis Thus, they cannot be regarded as templates These are the clear-cutcases However, mixed forms exist, where a template is bound reversibly to theproduct or where a catalyst organizes the reactants with respect to their geometry

We therefore put forward a more abstract view of what a template is and consider atemplate as the sum of all connections between the species reacting with eachother which are involved in geometrically controlling the reactivity in the desiredway It is the array of interactions and their spatial arrangement that count

1.3.6

Self-replication and Supramolecular Catalysis

While multivalency, self-assembly, and template effects provide strategies aiming

at generating more and more complex architectures, supramolecular chemistrycan also be utilized for controlling reactivity and even catalyzing reactions Closelyrelated to organocatalysis, supramolecular catalysts [39] accelerate reactions by

lowering the barriers The principles by which they fulfil the task are very different.Increasing the local concentration of the reactands by encapsulation is one exam-ple (see Fig 1.3 above), increasing the intrinsic reactivity of carbonyl compoundsthrough hydrogen bonding [40] is another and many more exist.

Originating from the question how the living organisms came into existence,self-replication is a special, but certainly intriguing case of supramolecular catal-ysis If one thinks about the complex ribosome, which nowadays transscribes ge-netic information stored in nucleic acids into proteins, which then become in-volved in the duplication of DNA, it is immediately clear that this apparatus ismuch too complex to self-organize accidentially at the beginning of life Instead,much simpler mechanisms must have existed in the early world In order to find

an answer, several research groups provided evidence that short DNA oligomersare indeed able to self-replicate in the presence of the appropriate template [41]

Fig 1.4 Anion-templated rotaxane synthesis The axle center piece is

threaded through the macrocycle’s cavity by hydrogen bonding.

Stopper attachment to both axle ends traps the wheel on the axle.

Inset: hydroquinone-based center piece which can also be used, but

with lower efficiency.

Later, suitable a-helical peptides have been shown to self-replicate as well [42] Inthe context of supramolecular chemistry most interesting are however organicminimal-replicators [43] which are not based on biomolecules Figure 1.5 shows

an example for a minimal self-replicating system, which operates even in a selective way One given enantiomer of the template catalyzes its own formation,while the other enantiomer is by and large suppressed

chiro-1.3.7

Molecular Devices and Machines: Implementing Function

Early supramolecular chemistry certainly focussed on the noncovalent bond andthe beauty of structures which can be generated employing it This is certainly thecase for topologically interesting molecules such as rotaxanes, catenanes, knotanesand Borromean rings [37] It also holds for the generation of self-assembling cap-sules, helicates [44], or metallo-supramolecular tetrahedra, octahedra and the like[45] However, the focus has shifted in contemporary supramolecular chemistry to-wards the implementation of function into noncovalent architectures The scope offunction is broad and ranges from light-induced energy and electron transfer pro-cesses [46] and molecular wires [47] to switches [48], molecular ‘‘motors’’ [46], anddevices for the active pH-driven transport of molecules through membranes This

Fig 1.5 A minimal self-replicating system In the presence of

template A, the two reactands on the left are organised in a way

suitable for a 1,3-dipolar cycloaddition reaction The pyridineamide part

of the template recognizes the acid substituent in the reactand, while

the second reactand is recognized by the carboxylic acid incorporated

in the template Particularly interesting is the fact that template A

favors its own formation, while the other stereoisomer B is formed only

in low amounts.

area is too broad to give a satisfying introduction here and thus, the reader is ferred to the literature cited.

re-1.4

Conclusions: Diverse Methods for a Diverse Research Area

The admittedly short and simplified considerations above make clear that one aim

of supramolecular chemistry is to mimic natural processes The above sections liberately chose examples from biochemistry as well as the multitude of artificialsupramolecules in order to point to the relations which exist between the twofields Understanding the details of noncovalent binding is much more difficult in

de-a complex biomolecule, de-and thus simple model systems provide the bde-asis for de-amore profound analysis However, supramolecular chemistry goes beyond merelycreating model systems for naturally occurring species In contrast to biomole-cules, supramolecular chemistry can utilize the whole range of conditions achiev-able, for example with respect to the use of organic solvents, in which many bio-molecules would lose their integrity, because they are designed for an aqueoussurrounding Higher or lower temperatures or different pressures can also be ap-plied Supramolecules may even find their applications under conditions wherebiomolecules would not have the necessary long-term stability The implementa-tion of function also aims at new functions which are not realized in nature Inparticular, the latter two aspects lead us to the second research area to which supra-molecular chemistry contributes significantly: material sciences Self-assembly, forexample, is a strategy to create long-range order and has even been applied to par-ticles on a micro- to millimeter scale [49]

If one thinks about function, in particular switches, logic gates, and molecularwires, it becomes clear that supramolecular chemistry is also about informationprocessing However, it is not only its potentially upcoming use in microelec-tronics: information processing begins at a much more fundamental level Tem-plates transfer spatial information between molecules; in order to achieve correctlyself-assembling species, the building blocks of the assembly need to be pro-grammed with the appropriate binding sites Information transfer and informationprocessing already starts at the molecular level

A view back on the last few decades makes perfectly clear that supramolecularchemistry has become a highly diverse field which requires the interdisciplinaryuse of a huge variety of methods to answer the scientific questions addressed Di-versity however is not the only challenge for the methods that are needed Thecomplexity of the architectures meanwhile realized requires sophisticated structureanalysis tools The highly dynamic features of supramolecules need kinetic meth-ods able to address many different time scales Gathering evidence for the func-tions implemented is impossible without a sound methodological basis Finally,the wish to image and influence single molecules led to the application of scan-ning probe microscopy to supramolecular systems The present book intends totake this into account and to provide an overview on methods used in supramolec-ular chemistry – even though it is probably not possible to be comprehensive

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Definitions and Roles in ref [28e],

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23, 319; b) D A Leigh, P J Lusby,

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The Binding Constants and Binding Energies

Generally, the formation of a complex between a host and a guest is a basic andimportant process in supramolecular chemistry Selectivity in the complexation is

an important property in determining the molecular recognition ability of the hostmolecule that discriminates among different guest species The ratio of the bind-ing constants of the corresponding complexations is usually treated as a measure

of selectivity Because binding constants have been used as a basic criterion for theevaluation of the host–guest complexation process, a binding constant (K) has to

be determined for the quantitative analysis of a complex formation [1, 2, 3] In dition to the ratio of binding constants, the temperature dependence of selectivity

ad-is also important to gain insight into the origin of supramolecular functions deed, some supramolecular systems have been reported to show a large influence

In-of temperature on selectivity [4] including temperature dependent inversion In-ofenantioselectivity [5] Therefore, thermodynamic parameters [enthalpy (DH), en-tropy (DS) and Gibbs free energy (DG)] are more suitable criteria in order to ex-press the molecular recognition abilities

Through Eqs (2.1) and (2.2), the thermodynamic parameters, binding constant,and temperature (T) are related to each other as described in Fig 2.1 and Eq (2.3),the van’t Hoff equation Theoretically, the determination of binding constants atdifferent temperature offers these thermodynamic parameters from the slope andthe intercept of line in Fig 2.1 The binding energy (DG) at a distinct temperature

is calculated using the obtainedDH, DS and T according to Eq (2.2), the Gibbs–Helmholtz equation Therefore, the important point in the quantitative analysis ofhost–guest complexation is how to determine the binding constant with high reliability

DG ¼ DH  TDS ð2:2Þ) ln K ¼  DH

R 1

Tþ DS

2.1.2

A General View on the Determination of Binding Constants

A general way to determine the binding constant is based on a simple bindingequilibrium model, i.e Eq (2.4) The terms binding constant, equilibrium con-stant, and stability constant are synonymous with each other The activity coeffi-cients are generally unknown and the stability constant K, based on the concentra-tions, is usually employed Judging from this situation, the question of the activitycoefficients of the solutes is disregarded here in order to simplify the discussion.Nevertheless, it should be remembered that this point is not always insignificant.The basic equations for the host–guest complexation are the following four Eqs.(2.4)–(2.7)

with H: host; G: guest; C: complex (Ha Gb)

a, b: stoichiometry as shown in Eq (2.4)

[H]0: initial (total) concentration of host molecule

[G]0: initial (total) concentration of guest molecule

[H], [G], [C]: equilibrium concentrations of host, guest, and complex, respectively

Fig 2.1 Van’t Hoff plot, i.e the correlation of DH, DS, K and

temperature according to the van’t Hoff equation.

Equation (2.8) is derived from Eqs (2.5)Ờ(2.7).

đơH0 a  ơCỡa đơG0 b  ơCỡb đ2:8ỡThe parameters can be classified as follows:

K, a, b: Constants (a and b are integers larger than or equal to 1.)

[H]0, [G]0: Variables which can be set up as experimental conditions

[H], [G], [C]: Variables dependent on the equilibrium

2.1.3

Guideline for Experiments

From Eq (2.8) and the classification of its parameters, we can deduce the followingguideline for experiments to determine the binding constant When [C] is obtained

at the equilibrium in which a and b are known, K can be derived directly according

to the Eq (2.8) from the experimental conditions, [H]0 and [G]0 Consequently,

in order to determine the binding constants, the following four steps have to becarried out:

 determination of stoichiometry, namely, a and b

 evaluation of complex concentration, [C]

 setting up the concentration conditions, [H]0and [G]0

 data treatment.

The following sections deal with the principle and also the practical issues essary for an understanding and performance of the above four points in thisorder

Vari-In order to determine the stoichiometry by the Continuous Variation Method,the following four points have to be considered and carried out:

 keeping the sum of [H]0and [G]0constant (a)

is obtained from the x-coordinate at the maximum

in JobỖs curve (Fig 2.2), where the y-axis is [C] and x-axis is ơH0

đơH0ợ ơG0ỡ.For the comprehension of the theoretical background of the Continuous Varia-tion Method, the required equations are (2.4)Ờ(2.7) and (2.9)Ờ(2.11)

fđa  x  a  yỡa đa  b  y  a  xỡbg

) K  đa  b  y  a  xỡb đa  x  a  yỡaỬ y đ2:14ỡ

Fig 2.2 Determination of stoichiometry đa; bỡ from the x-coordinate

at the maximum of the curve in JobỖs Plot.

The Eq (2.14) is then differentiated, and thedy

dxis substituted by zero Then thex-coordinate at the maximum in the curve is obtained

K  ½ða  b  y  a  xÞb fða  x  a  yÞag0

þ fða  b  y  a  xÞbg0 ða  x  a  yÞa ¼dy

dxby zero yields:

K  ½ða  b  y  a  xÞb a  ða  x  a  yÞa1 a

þ b  ða  b  y  a  xÞb1 ðaÞ  ða  x  a  yÞa ¼ 0

Division by K  ða  b  y  a  xÞb1 ða  x  a  yÞa1 a produces

a  ða  b  y  a  xÞ  b  ða  x  a  yÞ ¼ 0

Equation (2.15) means that a

a þ bis the x-coordinate at the maximum

stoichi-ða ¼ b ¼ 1Þ In the case of 1:2 complexation, the maximum is at x ¼ 0:333

The practically important point here is the following: Even if the concentration

of complex ([C]) could not be measured directly, the [C] ( y-axis) can be replacedwith a parameter proportional to [C] Then the same x-coordinate and with it thesame stoichiometry is obtained at the maximum as that in Job’s plot This meansthe stoichiometry can even then be determined even when [C] cannot be obtained

directly The practical question for each individual experiment is then how to ify the y-coordinate.

mod-Depending on the selected experiment, a suitable observable property should beselected and the complex concentration [C] should be replaced by it in JobỖs plot Inthe following, UV/vis spectroscopy is discussed as a representative example.The concentrations and absorbances of each species are related as the followingEqs (2.16)Ờ(2.18) in the case of investigation by means of UV/vis spectroscopy Theobserved absorbance is expressed as Eq (2.19) and Fig 2.3 The length of the opti-cal cell is fixed here to 1 cm as a premise The definitions of the abbreviations aregiven below The definitions of other abbreviations (a, b, [H]0, [G]0, [H], [G], [C])are the same as described before

AhỬ eh ơH Ử eh đơH0 a  ơCỡ đ2:16ỡ

Ag Ử eg ơG Ử eg đơG0 b  ơCỡ đ2:17ỡ

Aobs: observed absorbance

Ah, Ag, Ac: absorbances of host, guest, and complex respectively

eh, eg, ec: molar absorptivities of host, guest, and complex respectively

Equation (2.19) is transformed to Eq (2.20) by using Eqs (2.16)Ờ(2.18)

AobsỬ eh đơH0 a  ơCỡ ợ eg đơG0 b  ơCỡ ợ ec ơC

) Aobs eh ơH0 eg ơG0Ử đec a  eh b  egỡ  ơC đ2:20ỡEquation (2.20) shows that Aobs eh ơH0 eg ơG0 is proportional to [C] be-cause đec a  eh b  egỡ is constant The molar absorptivities eh, eg can be deter-mined from independent measurements using the pure host and the pure guest,respectively The concentrations [H], [G], are known because they are the experi-

Fig 2.3 Representative UV/vis spectra to show correlation of

observed spectra and each component.

mental conditions set up by the experimenter Consequently, ðAobs eh ½H0

eg ½G0Þ is determined from the experiments by means of UV/vis spectroscopy.The stoichiometry is determined from the x-coordinate at the maximum in thecurve which might be called a modified Job’s plot where ðAobs eh ½H0 eg ½G0Þ

is plotted as the y-coordinate instead of [C] An actual example of this modifiedJob’s plot is shown in Fig 2.4 The x-coordinate at the maximum in the curve is0.5 This supports the 1:1 host–guest complexation To get a better feeling of thepractical experiment, a spreadsheet for the Continuous Variation Method is at-tached as Appendix 2.1 [9]

2.2.2

Evaluation of Complex Concentration

When the observed property is the complex concentration ([C]) at equilibrium self, there is no difficulty But the actual complex concentration cannot be observeddirectly in most cases Thus, the question how to evaluate [C] is an important prac-tical issue The practical way depends on the property that can be observed in eachexperiment In this section, two typical cases for the evaluation of the complex con-centration at the equilibrium by UV/vis spectroscopic methods are discussed

it-Case 1: the absorption bands of host, guest and complex overlap From Eq (2.20),the following Eq (2.21) is derived

½C ¼Aobs eh ½H0 eg ½G0

Fig 2.4 Modified Job’s Plot for complexation of host and guest by

UV/vis spectroscopy:  observed; -: calculated.