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Oksana Danylyuk

SOLID-STATE COMPLEXES OF WATER-SOLUBLE

CALIXARENES WITH BIORELEVANT MOLECULES

Dissertation completed at the

Institute of Physical Chemistry

Polish Academy of Sciences

within International Ph.D Studies

Supervisor:

Dr. hab. Kinga Suwinska

Warsaw 2007


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Most importantly, I would like to sincerely thank my

supervisor Dr. Kinga Suwinska for her guidance and

support over the last years.

I would like to thank Dr. Anthony Coleman and his

group, in particular Dr. Adina Lazar, who have made

significant contribution to this work.

I would like to thank all colleagues from

Department II who have been very helpful and

accomodating.

Finally, I would like to thank Volodja and Sofijka for

being my inspiration, support and motivation.

.


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Results described in this dissertation were partially presented in the following

publications:

Papers:

1. Lazar A. N., Danylyuk O., Suwinska K., Coleman A. W. Assembly modes in thesolid state structure of the complexes of melamine mono-cations with para-

calix[4]arene sulfonic acid and calix[4]arene dihydroxyphosphonic acid. New

Journal of Chemistry, 2006, 1, 59.

2. Perret F., Bonnard V., Danylyuk O., Suwinska K., Coleman A. W.Conformational extremes in the supramolecular assemblies of para-sulfonato-

calix[8]arene.New Journal of Chemistry, 2006, 7, 987.3. Perret F., Gueret S., Danylyuk O., Suwinska K., Coleman A. W. A stepped bilayer

packing motif for para-sulphonatocalix[4]arene: The solid-state structure of the

para-sulphonatocalix[4]arenetriethylamine complex. Journal of Molecular

Structure, 2006, 797, 1.

4. Lazar A., Danylyuk O., Suwinska K., Coleman A. W. The solid-state structure ofcalix[4]arene dihydroxyphosphonic acidL-lysine complex. Journal of Molecular

Structure, 2006, 825,20.

5. Lazar A., Danylyuk O., Suwinska K., Coleman A. W. The structure of the tetra-potassium salt of calix[4]arene dihydroxyphosphonic acid. Chemistry Journal of

Moldova. General, Industrial and Ecological Chemistry, 2007, 2 (1), 98.

Conference presentations:

1. Lazar A. N., Coleman A. W., Baggetto L. G., Michaud M. H., Suwinska K.,Danylyuk O., Navaza A., Dupont N. Complexation and biology of calix[4]arenes

diphosphonates. MoldavianPolishUkrainian Symposium on Supramolecular

Chemistry, 1012 October 2005, Chisinau, R. Moldova, poster.

2. Perret F., Danylyuk O., Suwinska K., Coleman A. W. Anionic calix[8] areneschemistry. MoldavianPolishUkrainian Symposium on Supramolecular

Chemistry, 1012 October 2005, Chisinau, R. Moldova, poster.

3. Danylyuk O., Suwinska K., Lazar A. N., Perret F., Navaza A., Coleman A. W.Solid state structure of the complexes between antiseptic chlorhexidine and three


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anionic derivatives of calix[4]arenes. 48 Konwersatorium Krystalograficzne, 29

30 czerwca 2006, Wrocaw, Poland, poster.

4. Danylyuk O., Suwinska K., A. N. Lazar, Coleman A. W. Solid state complexes ofp-sulfonatocalix[n]arenes with biomolecules. XV-th Conference Physical

Methods in Coordination and Supramolecular Chemistry, September 27October

1 2006, Chisinau, R. Moldova, oral.

5. Danylyuk O., Suwinska K., A. N. Lazar, Coleman A. Solid-state assemblies ofcalix[4]arene diphosphate with biorelevant molecules. XIth International Seminar

on Inclusion Compounds. 10-15 June 2007, Kyiv, Ukraine, poster.

6. Danylyuk O., Suwinska K., A. N. Lazar, Coleman A. Solid-state assemblies ofcalix[4]arene diphosphate with biorelevant molecules. 49 Konwersatorium

Krystalograficzne. 2830 czerwca 2007, Wrocaw, Poland, poster.

7. Danylyuk O., Suwinska K., A. N. Lazar, Coleman A. Solid state complexes ofcalix[4]arene diphosphate with chlorhexidine and pilocarpine. 24 th European

Crystallographic Meeting, 2227 August 2007, Marrakech, Morocco, poster.


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TABLE OF CONTENTS

1 INTRODUCTION ............................................................................................... 9

2 LITERATURE REVIEW .................................................................................. 11

2.1 General overview of the calixarenes......................................................112.2 Water-soluble calixarenes ...................................................................... 14

2.2.1 Synthesis of water-solublep-sulfonatocalix[n]arenes and

calix[4]arene diphosphate ..........................................................15

2.2.2 Structural characterization ofp-sulfonatocalix[4]arene

complexes................................................................................... 15


complexes................................................................................... 26


complexes................................................................................... 29

2.2.5 Structural characterization of calix[4]arene diphosphate

complexes................................................................................... 32

2.2.6 Biological activity of water-soluble calixarenes........................ 35

2.2.7 Drug solubilization..................................................................... 37

2.3 Conclusions............................................................................................ 38

3 EXPERIMENTAL............................................................................................. 394 RESULTS AND DISCUSSION ........................................................................ 43

4.1 Solid-state complexes ofp-sulfonatocalix[4]arene................................ 43

4.1.1 Crystal structure ofp-sulfonatocalix[4]arene complex with

melamine.................................................................................... 43


triethylamine .............................................................................. 47


triethyltetramine......................................................................... 50


norspermidine............................................................................. 53


chlorhexidine.............................................................................. 58


tetracaine .................................................................................... 62

4.1.7 Crystal structure ofp-sulfonatocalix[4]arene complex withtamoxifen ................................................................................... 66


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piribedil ...................................................................................... 70

4.2 Solid-state complexes ofp-sulfonatocalix[6]- andp-sulfonatocalix

[8]arenes................................................................................................. 73

4.2.1 Crystal structure ofp-sulfonatocalix[6]arene complex withbis(6-amino-hexyl)amine........................................................... 73


dimethylamine............................................................................ 75

4.2.3 Crystal structure ofp-sulfonatocalix[8]arene complex with cis-

cyclo-hexanediamine ................................................................. 78


dimethylamine............................................................................ 82


butanediamine............................................................................ 85

4.3 Solid-state complexes of calix[4]arene diphosphate.............................. 88

4.3.1 Crystal structure of calix[4]arene diphosphate potassium salt... 88

4.3.2 Crystal structure of calix[4]arene diphosphate complex with

dimethylamine............................................................................ 91


melamine.................................................................................... 96


cadaverine ................................................................................ 100


L-lysine (I) ............................................................................... 103


L-lysine (II) .............................................................................. 106

4.3.7 Crystal structure of calix[4]arene diphosphate complex withchlorhexidine............................................................................ 110


pilocarpine................................................................................ 113

4.4 The role of supramolecular complementarity in the formation of

calixarene complexes with biorelevant molecules............................... 116

5 CONCLUSIONS.............................................................................................. 123

6 SUMMARY..................................................................................................... 125

7 REFERENCES................................................................................................. 127

8 APPENDIX...................................................................................................... 135


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1 INTRODUCTION

The biological world is rich in ordered assemblies of molecules. The forces

holding together these assemblies are van der Waals interactions, hydrogen bonding,

- and cation interactions, metal coordination and hydrophobic effects. Nature hasexploited these interactions in biorecognition and biomolecular organization for billions

of years. These weak, noncovalent interactions are responsible for the protein folding,

the selective transport of ions and small molecules across membranes, transduction of

signals, enzymatic reactions, and the formation of larger aggregates. They are also the

basis of one of the fastest-growing areas of research: supramolecular chemistry. The

concepts of supramolecular chemistry are derived from biology and rely on the

phenomena of molecular recognition and self-assembly: molecules (hosts) recognizecomplementary sites (functionality, geometry, size, etc.) on other molecules (guests)

and associate into larger entities, supermolecules, via weak non-covalent interactions.

Chemists, biologists, engineers, physicists, optical scientists and materials scientists are

"architects" who apply the fundamental principles involved in the biological processes

to design and fabricate artificial supramolecular systems.

The design and synthesis of water-soluble, synthetic macrocycles as artificial

receptors and biomimetic models for enzymes has been a major subject of interest in

recent years. Self-assembly of such synthetic receptors with biorelevant molecules is a

powerful tool for the understanding, modelling and mimicking of biological systems1

and developing new materials with specific properties and functions2. Along with the

cyclodextrins, crown ethers and cryptands, the calix[n]arenes are one of the most

important categories of supramolecular hosts3. Compared to the cyclodextrins, the

calix[n]arenes exhibit a high degree of steric flexibility which confers on them a large

area of applications. There exist many conformational isomers of calixarenes, and a

large number of cavities of different sizes and shapes, which can be involved in

molecular recognition processes.

It is well known that biogenic amines, amino acids, peptides, and proteins

constitute one of the most fundamental substrates in biological and artificial processes.

The family of calix[n]arenes is deeply involved in molecular recognition of these

compounds4, especially in the understanding of specific biomolecular interactions

which play a key role in modern supramolecular chemistry5. Water-soluble calixarenes

are of interest in building up systems that mimic natural biological processes through

the presence of hydrophobic pockets which can bind apolar guests. Moreover, they have


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2 LITERATURE REVIEW

2.1 General overview of the calixarenesCalix[n]arenes are cyclic oligomers of phenol, that can adopt various

conformations and form hydrophobic cavities. They are simple to prepare in high yields

from inexpensive starting compounds and easy to modify. Calixarenes can be decorated

with a wide variety of functional groups on the aromatic rings and/or the O-centres of

the phenolic groups, the so-called upper (or wide) and lower (or narrow) rims of the

calixarenes, respectively (Fig. 1b). In addition to their inclusion capability, qualifying

them as potential nanoscale containers, the variety of properties of functionalized

calixarenes coupled with their low cost and non-toxicity may allow their exploitation

through multidisciplary areas of research as catalysts7, extractants8, semi-conductor

materials9, switchable systems for data storage10,11 and sensors of bioactive

compounds12,13.

Fig. 1. (a) Calyx krater with the death of Aktaion; (b) representation of calix[4]arene in coneconformation.

Calixarenes have a long history, stretching back to 1872 when Adolph von

Bayer published the first results concerning the products obtained from the reaction of

phenol with formaldehyde. But he was unable to isolate the product and determine its

structure. In 1942 Alois Zinke, a professor of chemistry at the University of Graz in

Austria and his co-worker Erich Ziegler discovered that the base-induced reactions of

p-alkylphenols with formaldehyde yields cyclic oligomers. 30 years later C. David

Gutsche and co-workers14 reinterpreted the Zinke results and developed methods for

synthesizing each of the three major cyclic oligomers in good and reproducible yields. It

was also Gutsche who first proposed the name calixarenes for these molecules since

the shape of the molecule resembled the Calyx krater vases of ancient Greece (Fig. 1a).

The first crystal structure of the calix[4]arene was determined by Andreetti and co-

workers from the University of Parma and published in 197915.

Upper rim

Lower rim

a b


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The simplified nomenclature of the calixarenes uses [n] to identify the number

of phenolic units in the macrocycle, thus calix[4]arene contains four units. The nature

and positions of substituents are given by sequential numeration and the appropriate

term for the function is placed before the term calix[n]arene. Hydroxyl substituents

follows again sequential numeration and are placed after the name calix[n]arene. Forexample, the cyclic tetramer derived from p-tert-butylphenol is called p-tert-

butylcalix[4]arene, but its systematic name is 5,11,17,23-tetra-tert-butylcalix[4]arene-

25,26,27,28-tetrol.

Calix[n]arenes are readily available with n equal to 4, 6 and 8 and less readily

with n equal to 5 and 7, recently 9 and 10. Furthermore a large series of calixarenes with

hetero atoms, different substituents and aromatic groups different from benzene exist.

Fig. 2. Four main conformations of the calix[4]arenes: (a) cone, (b) partial cone, (c) 1,2-alternate

and (d) 1,3-alternate.

Another aspect of calixarene nomenclature relates to the conformations these

molecules can adopt. It was recognized by Cornforth16 that a calix[4]arene can exist in

four conformations, which were later named by Gutsche17 as cone, partial cone,

1,2-alternate and 1,3-alternate with idealized structures having C4v, Cs, C2h and D2d

symmetry, respectively (Fig. 2). The nomenclature refers to the orientation of the arene

rings with respect to one another. In the cone conformation, all arenes point up and form

a cone-like structure, whereas in partial cone three arenes point up and one points down.

Frequently, however, the actual molecule posseses conformations and symmetries

differing from the ideal as the result of torsional changes in the arene rings orientation.

For example, the cone conformation often assumes a pinched or flattened cone structure

in which one pair of arene rings becomes almost parallel while the other pair splays

outwards. Proton nuclear magnetic resonance (NMR) measurements of several

calixarenes in solution show that they mainly exist in the cone conformation, but they

are conformationally mobile at room temperature18. Some control on the calixarene

conformation can be achieved by introducing bulky substituents on the upper or lower

a b c d


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rim to inhibit rotation. The flexibility of the calixarenes can be also controlled by

crystallization which allows a desired conformation to be fixed in the solid state.

The calix[6]arene can exist in eight up-down conformations and numerous

others in which one or more of the aryl rings projects outward from the average plane of

the molecule, depending on the solvent, the nature of the complexed guest and on thefunctionalizations on the lower rim. However two conformations show higher stability,

the 1,2,3-alternate or double partial cone19,20 (also known as hinged) in which three

adjacent oxygen atoms lie on the one side and the other three on the other side of the

molecule or a pinched cone21,22 (winged) conformation in which all of the oxygen lie

on the same side of the molecule and two opposite methylene groups point to the center

of the bowl.

The much larger annulus of the calix[8]arenes, the possibility of 16 up-downconformations, and the numerous other conformations in which one or more aryl rings

project out would appear to make these more flexible and complicated than

calix[4]arenes. The calyx shape of the molecule completely disappears in most

calix[8]arenes and a new conformation which has the architecture of a pleated loop is

observed23 (Fig. 3). The undulating pleated loop structure allows maximal hydrogen

bonding with the OH groups lying above and below the average plane of the molecule.

Fig. 3. Pleated loop conformation ofp-tert-butyl-calix[8]arene24.

The cone conformation of calixarenes is flexible in its ability to accommodate

various guests. Their conformational properties help in ligating guests which can be

even much larger compared with the host molecule. Studies on inclusion properties 25,26

of calixarenes have shown that, in the presence of guest molecules, appropriatelyfunctionalized calixarene derivatives can self-assemble to form dimeric units, host

capsules, suitable to include these small organic molecules.


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Calixarenes possessing both hydrophobic and hydrophilic groups at the opposite

rims are usually suitable for the formation of bilayer arrangements involving head-to-

head and tail-to-tail interactions. For example, many bilayer structures have been

reported forp-tert-butyl-calix[4]arenes27, in which the presence of hydrophilic phenolic

rim and hydrophobic core and upper rim has resulted in the antiparallel alignment ofadjacent calixarene molecules.

2.2 Water-soluble calixarenesThe parent calixarenes are totally insoluble in aqueous solutions, and this property

is the major problem for the calixarene use in biopharmaceutical applications. Different

methods have been developed to obtain water-soluble calixarenes28. Generally to induce

aqueous solubility, the calixarenes need to be functionalized with groups containing

positive or negative charges, or with neutral but highly hydrophilic groups. Functions

such as carboxylates, phosphates, sulfonates or ammonium groups are used for such

modification Three possible sites for the functionalization exist: at the phenolic

functions,para-position to the phenolic groups and at the methylene bridges.

The tetracarboxylic acid ofp-tert-butyl-calix[4]arene, introduced by Ungaro and

co-workers in 1984 was the first example of water-soluble calixarene29. In the sameyear Shinkai reported the preparation of p-sulfonatocalix[6]arene30. Following the

preparation of the sulfonated tetramer and octamer31, other anionic water-soluble

derivatives containing nitro32, phosphonic acid33, and carboxyl34 functions appeared.

Water-soluble calix[n]arenes are a widely investigated class of compounds

becoming increasingly important in the field of supramolecular chemistry. They offer

interesting inclusion properties and a wide range of metal coordination complexes both

in solution and in the solid state. The property of water-soluble calix[n]arenes to forminclusion complexes with different guest species in water, opened a new direction of

applications. The guest molecules range from inorganic ions, through small organic

molecules to amino acids, hormones, peptides and neurotransmitters35. The molecular

recognition properties of these amphiphiles are of interest in nanochemistry where they

have potential applications in building up new synthetic materials36,37, and in medicinal

applications which include drug delivery, and sensing38. Water soluble calixarenes have

been shown to be useful as new surfactants to water-solubilise biomolecules such as

carotenoids39.


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2.2.1 Synthesis of water-solublep-sulfonatocalix[n]arenes and calix[4]arenediphosphate

The synthesis ofp-sulfonatocalix[n]arenes (n = 4, 6, 8) can be simply achieved

in two methods: the most common is sulfonation of p-H-calix[n]arenes as first

described by Shinkai30,31 and there is also direct ipso-sulfonation of the p-tert-butyl-

calix[n]arenes (Fig. 4). Direct ipso-sulfonation is simpler than the method of Shinkai

but the reaction is often perturbed by incomplete substitution and is used only for the

obtaining of calix[8]arene derivatives because of the low solubility of

p-H-calix[8]arene.

OH OH OHHO OH OH OHHO

AlCl3

toluene

H2SO4

100C

OH H OHHO

SO3HSO3H

HO3S

H2SO4

80C

SO3H

OH OH OHHO

SO3HSO3H

HO3S SO3H

Fig. 4. Synthesis ofp-sulfonatocalix[n]arenes.

Calix[4]arene containing two dihydroxyphosphoryl groups on the lower rim

have been synthesized by consecutive treatment of easily accessible diethoxyphosphoryl

derivative with bromotrimethylsilane and methanol40 (Fig. 5).

O OH OH O

P

EtO O

OEt

P

OEtO

EtO

O OH OH O

P

HO O

OH

P

OHO

HO

bromotrimethylsilane

methanolOH OH OHHO

diethylchlorophosphate

chloroform

Fig. 5. Synthesis of calix[4]arene diphosphate.

2.2.2 Structural characterization ofp-sulfonatocalix[4]arene complexes

Anionic p-sulfonatocalix[n]arenes possess the highest known solubility in

aqueous solutions among the water-soluble calixarene derivatives and are able to

complex a wide range of inorganic and organic cations. p-Sulfonatocalix[4]arene has

been found to have a solubility at least as great as 0.1 M.

sulfonationipso-sulfonation


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Fig. 6. (a) Cone and (b) 1,3-alternate conformations ofp-sulfonatocalix[4]arene.

The smallest and most conformationally constrained p-sulfonatocalixarene,

tetramer C4S, often adopts a cone conformation with hydrophilic upper and lower rims,

while the cavity and outer midsection are strongly hydrophobic (Fig. 6a). The cone

conformation is stabilized by intramolecular hydrogen bonding among OH groups.

p-Sulfonatocalixarene with its cone-shaped hydrophobic cavity serves as a molecular

container for small organic molecules and ions, biologically significant amino acids and

nucleotide bases, globular shaped organic molecules and transition metal complexes.

Some X-ray crystal structures ofp-sulfonatocalix[4]arene show41,42 that water molecule

inside the calixarene cavity is hydrogen-bonded to the cloud of the aromatic nuclei, a

motif which may be commonly encountered in biological systems (the hydrophobic

pockets of proteins).

Anionicp-sulfonatocalix[4]arene has shown a great capacity to generate a wide

range of structural motifs. Most commonly, p-sulfonatocalix[4]arenes are assembled in

an antiparallel (up-down) fashion with sulfonate groups covering the surfaces of the

bilayers which are separated by a hydrophilic layer containing the guest molecules and

water. The ability of these molecules to intercalate cations in the hydrophilic layersbetween bilayers ofp-sulfonatocalix[4]arenes led Atwood and Coleman43 to name these

systems organic clays. In a recent review of the biochemistry of

p-sulfonatocalixarenes, Coleman and co-workers44 stated that the decision to liken the

solid state structures adopted by their hydrated alkali metal salts to organic clays, rather

than phospholipid bilayers, may have led us away from the study of the biochemistry

of these molecules. While the span of the calixarene bilayers, at 14 , is of the order

observed in smectite and vermiculite (15 ) it is much less than the 40 or so observedfor biological membranes. However, the ability to form bilayers indicates that

a b


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calixarenes, extended through upper or lower rim derivatization, have the potential to

insert themselves in biological membranes to form permanent channels45.

Subsequent investigations on the solid state supramolecular complexes of

p-sulfonatocalix[4]arene revealed the prevalence of bilayer structural motif (Fig. 7) and

perturbations thereof. The solid state structures of the supramolecular complexesbetween p-sulfonatocalix[4]arene and a variety of organic cations, both aliphatic and

aromatic, with different chain length and charge have been reported.

Fig. 7. Structure of the sodium salt ofp-sulfonatocalix[4]arene showing the typical bilayer motif.

The complexation of quaternary ammonium cations by synthetic receptors has

attracted extensive attention recently, especially after the discovery that

neurotransmitter acetylcholine is bound to acetylcholine esterase thanks to cation-

interactions. It was demonstrated that the N(CH3)3+ group of acetylcholine is held not by

negatively charged anionic groups of the enzyme, but rather by aromatic residues

creating a suitable -capsule for binding of the quaternary ammonium salt by cation

interactions46, thus highlighting the crucial role of weak cation- interactions in the

recognition processes47.

The complex of p-sulfonatocalix[4]arenes with the tetramethylammonium

cation48 shows the stoichiometry 1:5. The calixarene exists in the form of pentanion

with one deprotonated phenolic group. One of the tetramethylammonium cations is

embedded into the calixarene cavity with one methyl group situated deeply inside the

cavity. The distances between the carbon atom of the included methyl group to the

centroids of the four aromatic rings are longer than those typically found for C-H

aromatic hydrogen bonding. It is suggested that the position of the cation is stabilized

by strong electrostatic interactions with the electron-rich sulfonato groups. The


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remaining four tetramethylammonium cations and crystallization water molecules are

situated in the region between the typical calixarene bilayer.

In the complex with the trimethylanilinium cation the aromatic moiety of the

guest is included into calixarene cavity49 (Fig. 8). The ammonium cation is bound to an

electrostatic pocket formed by two neighboring sulfonate groups. The three aromatichydrogens of trimethylanilinium are in contact with the benzene -system. The phenyl

ring of the trimethylanilinium cation is significantly inclined in the cavity. This

conformation is favored to attain the maximal multipoint interaction with

p-sulfonatocalix[4]arene through both hydrophobic and electrostatic forces. The

complex features the typical clay-like bilayer structure, that is, the calixarene rings form

two hydrophobic layers, and the sulfonate groups cover the surface of the bilayer. The

ammonium cation of the guest is placed on the surface.

Fig. 8. Inclusion complex of C4S with trimethylanilinium cation.

The complex ofp-sulfonatocalix[4]arene with choline reveals the inclusion of

the guest cation in the macrocyclic cavity of the C4S50. There are two independent

complexes with different guest conformation in the asymmetric unit. In the first

complex the choline is in an extended conformation while in the second one is

disordered (s.o.f. = 0.75 and 0.25) and adopts two folded conformations. In bothcomplexes the choline quaternary ammonium cation inserts its positively charged N

terminal group inside the cavity of the receptor. The linear molecule emerges from the

cavity with its hydroxyl pointing between two sulfonates framing a window. This

contrasts with the structure of C4S with trimethylanilinium cation where the phenyl

group is located in the cavity49. The very short contacts observed between the choline

atoms and those of the calixarene groups are in line with strong interactions with the

substrate and high stability of the complex in the solution.

Leverd reported structural study on the three C4S complexes with

ethylenediamine of different stoichiometry and charge balance41. In the first complex


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water is included into the macrocyclic cavity and two ethylenediammonium dications

are complexed outside the cavity of the calixarene. If 2.5 equivalents of

ethylenediamine are added to C4S, water is expelled from the cavity by the

deprotonation of one of the phenolic groups of the macrocycle and replaced by

ethylenediammonium dication to form an inclusion complex. The asymmetric unitcomprises two independent molecules of the calixarenes that differ from one another in

the way that the guest resides in the cavity. Both incorporated ethylenediammonium

dications are stabilized in their positions by donating six hydrogen bonds to sulfonate

groups of the C4S or to water molecules. Interestingly, the addition of 3 equivalents of

ethylenediamine leads to the proton exchange reaction between the diamine and the

diammonium dication and therefore to the formation of monocation which can compete

with the dication for the inclusion inside the cavity. In the resulting mixed complex themonocation is included in the cavity ofp-sulfonatocalix[4]arene, while dications are

situated outside the cavity. Four independent calixarenes are present in the crystal

lattice, in each of which the monocation is bound in the same way. The NH3 end of the

molecule points towards the exterior of the cavity, forming strong hydrogen bonds with

oxygen atoms of either water molecules or sulfonate groups, while the other NH2 end

is embedded in the cavity and interacts with the aromatic core by N-H weak

hydrogen bond. Ncentroid distances range from 3.449(4) to 3.603(4) . Such amine

interactions with aromatic faces are a matter of importance in the folding and

recognition of biological polymers51.

The complexes of p-sulfonatocalix[4]arene with linear 1,4-butanediamine

(putrescine), 1,5-pentanediamine (cadaverine), 1,5,10-triazadecane (spermidine)52 and

cyclic cis-1,2-cyclohexanediamine show the partial inclusion of the guest molecule

within the host aromatic cavity. In all these structures, the organic cation is held inside

the cavity by ammonium-sulfonate hydrogen bonds and alkyl-aromatic hydrophobic

interactions. In the complexes with cadaverine and putrescine, two

p-sulfonatocalix[4]arene molecules of the opposing layers form a capsule, which

contains two diammonium guest molecules. In the case of much longer spermidine and

bulky cyclohexanediamine molecules, no capsules are observed due to lateral

displacement of one layer of the complex with respect to the other. This results in one

sulfonate group of calixarene of one layer being positioned above the cavity of

calixarene of the opposing layer. The spermidine trication extends outside the

p-sulfonatocalix[4]arene cavity and forms hydrogen bond to an adjacent

p-sulfonatocalix[4]arene of the same layer.


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The solid state complex ofp-sulfonatocalix[4]arene with L-lysine shows the

stoichiometry 1:253, in contrast to the situation in solution, where 1:1 complexation is

observed54. There are two independent calixarenes and four independent lysine

molecules in the structure. For all the L-lysine molecules, the - and -amino groups

show contacts indicative of N-HO hydrogen bonds with oxygen atoms on thesulfonate groups of the calixarenes, but they display different types of interactions. Of

the four independent lysine molecules, three are found within the hydrophilic layer

separating the typical p-sulfonatocalix[4]arene bilayer, while the remaining molecule

spans this bilayer in a manner resembling biomolecules traversing a lipid bilayer (Fig.

9a).

Fig. 9. (a) Solid-state packing of C4S complex with lysine showing the lysine spanning the bilayerand (b) zigzag bilayer of the C4S complex with arginine.

For the complex with D-arginine the planar bilayer ofp-sulfonatocalix[4]arene is

replaced by a zigzag bilayer arrangement (Fig. 9b).The four crystallographically

independent molecules of C4S form a cage which accomodates six independent

arginines, each with different chain conformation55. The structure contains a water

channel diagonal to a zigzag bilayer of the host.

For alanine, histidine and phenylalanine, racemic pairs of molecules are

confined in capsules comprised of two p-sulfonatocalix[4]arenes56 (Fig. 10a). For

(S)-serine two chiral molecules are also confined in such capsules57. The (S)-alanine

and (S)-histidine form independent 1:1 complexes (Fig. 10b). For (S)-tyrosine, a

-stacked chiral pair of isomers is encapsulated by p-sulfonatocalix[4]arene. A feature

of all amino acid complexes is that the polar groups of the amino acids point away from

the calixarene cavity. For all these complexes the classical bilayer motif of

p-sulfonatocalix[4]arene is observed.

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Fig. 10. (a) Molecular capsule formed by two molecules of C4S containing racemic pair of histidine

molecules and (b) open inclusion complex of C4S with (S)-histidine.

The first p-sulfonatocalix[4]arenenucleic acid base complex structure is the

adenine complex with the stoichiometry 1:4 reported by Atwood et al.58. The complex

crystallizes with a water molecule exhibiting aromatic hydrogen bonding embedded

within the hydrophobic calixarene cavity. The adeninium cations form a hydrogen

bonded array in a layer external to the cavity. Two different bilayers exist in the

structure; one of calixarene anions, where the sulfonato groups are directed into the

hydrophilic layer, and the other of adeninium cations, where the protonated nitrogen is

directed into the hydrophilic layer. These two bilayers are separated in part by thin

layers of water molecules.

Raston et al.59 recently reported structures of supramolecular complexes of

nucleic bases (guanine, cytosine) and related compounds (benzimidazole and

2-hydroxybenzimidazole) with p-sulfonatocalix[4]arene. For all the complexes, the

nucleic acid bases are found both exo and endo to the cavity ofp-sulfonatocalix[4]arene

anions. In the case of guanine the guest dication perched above the cavity of C4S in the

hydrophilic area of the overall bilayer structure. The guanine dication is above and

almost coplanar with the plane defined by the four SO3- groups of each calixarene and is

involved in N-HO hydrogen bonds to oxygens on the sulfonato groups and

crystallization water molecules. In the mixed complex with guanine and cytosine, one

cytosine cation is located within the C4S cavity and another cytosine along with a

guanine dication situated outside the cavity. The endo cavity cytosine cations lie

perpendicular to the base plane of phenolic oxygens of the C4S cavity with their polar

groups radiating outwards and are hydrogen bonded to two sulfonato groups of the C4S.

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The other cytosine and guanine cations -stack with the aromatic rings on the

p-sulfonatocalix[4]arene anions. The presence of both guanine and cytosine exo to the

calixarene cavity disrupts the usual bilayer structure formation, but the overall structure

has the cavities of the C4S oriented in opposite directions.

For the complex C4S with 2-hydroxybenzimidazole a pair of -stacked2-hydroxybenzimidazole cations is encapsulated by a pair ofp-sulfonatocalix[4]arene

anions approximating as molecular capsule. Another 2-hydroxybenzimidazole cation is

located in the hydrophobic layer outside the calixarene cavity. The benzimidazole

complex crystallizes with a pair of -stacked benzimidazole cations encapsulated by

C4S anions, like that for 2-hydroxybenzimidazole. The molecular capsule has the endo

cavity benzimidazole cation hydrogen bonded to each of the calixarenes.

Solution and solid state studies on the complexation of three isomeric4'-(pyridyl)-terpyridines by p-sulfonatocalix[4]arene has recently been reported60. The

terpyridines are widely used as supramolecular tectons in molecular architectures and

are gaining promise in medicinal chemistry as possible anticancer and antimicrobial

agents61. For the complex p-sulfonatocalix[4]arene4 -(2-pyridyl)-terpyridine the

stoichiometry is 2:3. The asymmetric unit consists of two calixarene-terpyridine motifs

separated by another molecule of terpyridine (Fig. 11). Three of the four pyridine rings

in each terpyridine molecule are protonated, with the exception being the nitrogen atom

on the central pyridine ring. Both calixarenes adopt the expected pinched cone

conformation, with the cavity in each being occupied by the pyridine ring of terpyridine

molecule. The calixarenes are arranged in an updown fashion, and are separated by

molecules of 4'-(pyridyl)-terpyridine which are -stacked to two calixarenes from each

of the motifs. The bilayer arrangement incorporating molecules other than calixarenes is

unusual and has only been reported in a few instances for positively charged guest

molecules53,62.

The inclusion complex p-sulfonatocalix[4]arene4'-(3'-pyridyl)-terpyridine

crystallizes with the 1:1 C4Sterpyridine ratio. The terpyridine molecule is included

within the cavity of C4S via the terminal 4'-(2-pyridyl) ring. Pairs of calixarene

terpyridine motifs related by an inversion centre are molecular-capsule like in

arrangement shrouding in part two terpyridine molecules. Such molecular capsules have

been noted for smaller pairs of amino acids and related molecules where the shrouding

is more effective56,59.


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Fig. 11. The asymmetric unit of C4S complex with 4 -(2-pyridyl)-terpyridine.

In the case of 4'-(4-pyridyl)-terpyridine, a molecular capsule is formed in which

two calixarenes encapsulate one terpyridine guest molecule, with the terminal pyridine

ring being exo to the calixarene cavities. As for the previous structures, the arm of the

terpyridine in the cavity is associated with C-H interactions between the protons on

pyridine ring and the 1,3-pair of calixarene phenyl rings. A salient feature of the

structure is a slippage within the bilayer arrangement in respect to the hydrophilic and

hydrophobic domains giving rise to an overall corrugated bilayer arrangement, and the

calixarenes are unusually well separated from each other through bilayer intercalation of

terpyridine molecules. Stacking interactions are a dominant feature in each of the

inclusion complex structures, either for terpyridines in the calixarene cavities, or those

included in bilayers.

Many of the p-sulfonatocalix[4]arene capsules take on the form of Russian

Matryoshka dolls63, 64, i. e. the inclusion complexes of inclusion complexes which are

unusual examples of second-sphere supramolecular complexation (host-within-host).

These complexes are mainly formed at low pH (


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18-crown-6 molecule. It is coordinated to all six oxygens of the crown ether and to the

oxygen atom of one sulfonate group of the C4s molecule. The 18-crown-6 is cupped in

the bowl of the calixarene66 (Fig. 12b).

Fig. 12. (a) Russian doll superanionic capsule of C4S with 18-crown-6 and sodium cation; (b)

ferris wheel structure of C4S complex with 18-crown-6 and lanthanum (III) cation.

It has also been reported that p-sulfonatocalix[4]arene can generate such

structure variations as 2D coordination polimers67 and helical arrays68. Atwood and co-

workers69 have shown that C4S form large molecular assemblies of spectacular

nanometer-scale spheroids or helical tubules. These giant spheroids adopt either

icosahedral, or cuboctahedral geometries and have been touted as inorganic virus

mimics due to their large internal volumes and similar geometries. For the complex of

p-sulfonatocalix[4]arene with pirydineN-oxide in the presence of lanthanide(III) nitrate

in the ration of 2:2:1, the hydrophobic regions of the calixarenes are aligned, and

assembled in an up-up radially symmetric fashion along the surface of a sphere in the

contrast to up-down fashion when forming a bilayer. The spheroidal array consists of 12calixarenes arranged at the vertices of an icosahedron (Fig. 13a). The polar outer-shell

surface of the sphere consists of the sulfonate head groups of calixarenes and bears a

total charge of -48, the polar inner-shell surface of the nano-sphere comprises 48

phenolic hydroxyl groups, 12 of which are deprotonated. The cavities of the calixarenes

are situated just below the polar surface of the sphere and constitute a series of

hydrophobic pockets. Twelve pyridineN-oxide molecules penetrate the polar surface of

the sphere and are bound within the hydrophobic pockets via

-stacking interactions.Their oxygen atoms extend outwards and coordinate to La3+ ions above the sphere

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surface. In the same system the helical tubular structure may be obtained when the ratio

of the starting components is of 2:8:1. The calixarene molecules are arranged along the

surface of a cylinder that is analogous to the spherical assembly in that there is a polar

core, a hydrophobic mid-region constituting the tube, and a polar outer shell. However,

the organic shell is no longer composed purely of calixarene molecules, but containspyridine N-oxide molecules intercalated between the aromatic rings of adjacent

macrocycles.

Fig. 13. Schematic representation of nanometre scale spheroids based on p-sulfonatocalix[4]arene:(a) twelve C4S molecules in the presence ofpirydineN-oxide and lanthanide(III) nitrate assembleat the vertices of an icosahedron (shown by green faces within spheroid), (b) twelve C4S moleculesin the presence of 18-crown-6 and praseodymium (III) nitrate assemble at the vertices of a

cuboctahedron (shown by red faces within spheroid)64

.

The replacement of pyridine N-oxide by 18-crown-6 and lanthanum nitrate for

praseodymium (III) (or neodymium(III) or samarium (III) nitrate) in the above

mentioned ternary system C4SguestLn results in a remarkably dissimilar spheroidal

array consisting of 12 calixarenes arranged at the vertices of a cuboctahedron70 (Fig.

13b). Praseodymium (III) ions are complexed by 18-crown-6 together with two trans-

water molecules, and form the core of upper rim to upper rim molecular capsule, similar

to the trans-aqua 18-crown-6 complex of sodium65. In the extended structure these

Russian dolls are arranged in the form of cuboctahedra. This arrangement results in

the formation of pores in the spheroid shell which are occupied by water molecules.

The internal volumes of the icosahedron and cuboctahedron differ by approximately

30% in favour of the cuboctahedron. Thus, changing the nature of the guest in the

hydrophobic cavity of C4S (pyridineN-oxide to 18-crown-6) results in the expansion of

the spheroid. In addition, the formation of pores in the outer shell of the spheroid

associated with this change allows the tentative analogy to viral mimicry of the cowpea

chloritic mottle virus that has been shown to behave in a similar manner under

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particular pH control, a system that can be used to trap molecular material for study

within the virion shell71. The cuboctahedral arrangement appears to allow

communication from the hydrophilic interior of the spheroid through the hydrophobic

channels/pores to exo-hydrophilic region via the presence of water molecules within the

voids.There is also one example of the p-sulfonatocalix[4]arene in 1,3-alternate

conformation (Fig. 6b), stabilized as its bipyridinium salt72. The calixarene is

surrounded by eight 4,4'-bipyridinium cations. Four of these are situated about the

4-fold axis while the remaining four protrude into the small clefts beside the sulfonate

headgroups. The former interact with the C4S by means of relatively strong C-HO

hydrogen bonds and face to face aromatic - interactions while the latter form strong

N-HO hydrogen bonds to the oxygens of the sulfonate groups. It is worth mentioningthat this solid-state complex is completely insoluble in water, this is probably due to

intricate network of non-covalent interactions that provide a high degree of stability to

the crystal lattice, thereby compensating for the energy involved in disruption of the

C4S cone conformation .

2.2.3 Structural characterization ofp-sulfonatocalix[6]arene complexes

The supramolecular solid-state chemistry ofp-sulfonatocalix[6]arene (C6S) is

less documented, in contrast to C4S. p-Sulfonatocalix[6]arene has larger cavity and is

more conformationally flexible than its calix[4]analogue, and can act as ditopic receptor

to various guest molecules. p-Sulfonatocalix[6]arenes typically adopt an up-down

double partial cone (1,2,3-alternate) conformation73,74 (Fig. 14a). Of the thirteen C6S

based supramolecular structures found in the Cambridge Crystallographic Data Base

three show the calixarene in the up-up double cone conformation75,76,77

(Fig. 14b).

Fig. 14. Conformations ofp-sulfonatocalix[6]arene: (a) up-downdouble partial cone and (b) up-updouble cone.

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The transition- and lanthanide-metal supramolecular chemistry of

p-sulfonatocalix[6]arene shows its great potential in the formation of different

supramolecular architectures that deviate from the typical bilayer arrangement formed

with C6S. The versatility in complex formation is clearly seen in the system C6S

pyridine N-oxidemetal ion. In the presence of nickel(II) ions the calixarene in thedouble partial cone conformation acts as a ditopic receptor to two pyridine N-oxide

molecules with hexaaquanickle(II) cations residing in the hydrophilic regions within the

bilayer78 (Fig. 15a). The bilayer arrangement is similar to that seen for the calixarene in

both the corresponding sodium salt and parent sulfonic acid and assembles with the

similar-stacking distances79.

Replacement of nickel(II) from the above system with lanthanide(III) metal

results in the formation of the complex where there is one pyridine N-oxide moleculebound to one metal center79. Although a bilayer is present within the extended structure,

it is quite different from the previous one and can be described as a corrugated bilayer

(Fig. 15b). Examination of other lanthanide metal ytterbium(III) affords a hydrogen-

bonded array of a C6Spyridine N-oxideytterbium complex which has varied

coordination modes within the extended structure74. There are two independent

Fig. 15. (a) Bilayer arrangement of C6S molecules in the C6SpyridineN-oxidenickel complex and(b) corrugated bilayers of C6S in the C6SpyridineN-oxidelanthanum complex.

supramolecular tectons, one of which has two octacoordinate ytterbium ions bound to

opposing sulfonate groups of the calixarene with one pyridine N-oxide ligand also

bound to the metal center while residing in the partial cone of the C6S. The second

supramolecular tecton consists of one calixarene, again with two lanthanide metal

centers coordinated to opposing sulfonate groups within the calixarene, but with two

pyridine N-oxide ligands. Of the two pyridine N-oxide molecules coordinated to

ytterbium ions, one resides in one partial cone of the calixarene, while the other resides

in the partial cone of the nearest identical tecton.

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Raston and co-workers76 have shown that some control on the C6S conformation

can be achieved in the presence of varied stoichiometric amounts of 18-crown-6 and

selected lanthanide(III) chlorides. The calixarene may adopt an up-up double cone

conformation or centrosymmetric up-down double partial cone conformation,

depending on the ratio of the guest used and the nature of the lanthanide metal. In thepresence of smaller lanthanides, complex has a double molecular capsule arrangement

with two p-sulfonatocalix[6]arenes shrouding two 18-crown-6 molecules (Fig. 16a),

while for the larger lanthanides ferris wheel arrangement is observed (Fig. 16b). The

two complexes have the calixarenes in the elusive up-up double cone conformation.

In the presence of large excess of 18-crown-6 the C6S adopts the double partial cone

conformation with the calixarene acting as divergent receptor towards disc-shaped

crown ether molecules.

Fig. 16. (a) Molecular capsule of two C6S in up-up conformation shrouding two 18-crown-6molecules and (b) C6S in up-down conformation acting as divergent receptor towards 18-crown-6molecules.

The bis-molecular capsule motif is observed for the C6S complex with

tetraphenylphosphonium cations77. The two calixarenes in the pinched up-up

double cone conformation encapsulate tetraphenylphosphonium cation pair. Some of

the phenyl groups of the tetraphenylphosphonium cations reside in the pseudo calix[3]

cavities of the calix[6]arenes. The interaction of the cations with the calixarenes

involves C-H contacts from the aryl para hydrogens of the guests to the aromatic

rings of the calixarenes. The remaining aryl group from each of the included cations

protrudes out of the capsule into the hydrophilic domain. In the extended structure the

capsules are well separated from each other, in contrast to the usual bilayer arrangement

where the calixarenes are -stacked.

The complexation of L-leucine by p-sulfonatocalix[6]arene has been reported

recently75. The C6SL-leucine stoichiometry is 1:6. The calixarene in the double cone

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andp-H-calix[8]arene86. The charge of the calixarene is -10 (with two calixarene base

hydroxyl groups deprotonated). In this conformation C8S molecule has four grooves

on either side of the macrocycle and each groove is occupied by a 4,4'-dipyridine-N,N'-

dioxide molecule (Fig. 18). The guest molecules, some being europium bound and some

non-coordinated, interact with the calixarene by a series of -stacking and C-Hinteractions. Clearly 4,4'-dipyridine-N,N'-dioxide is an excellent choice of guest for C8S

as it fits well with the large host when it is in the pleated loop conformation. The

calixarene acts as a linking unit in a complex 3D wavy brick wall coordination

polymer.

Fig. 18. Complex of thep-sulfonatocalix[8]arene with 4,4'-dipyridine-N,N'-dioxide.

In the presence of tetraphenylphosphonium and trivalent ytterbium cations,

p-sulfonatocalix[8]arene forms large 2D porous structure87. The asymmetric unit

comprises two calixarenes, nine tetraphenylphosphonium cations, a chloride anion and

disordered ytterbium cations with partial occupancies. Charge neutrality for the systemimplies that some of the sulfonate groups are protonated. In this complex the

calix[8]arene adopts an unusual conformation (Fig. 19a) where three neighboring

phenyl rings adopt a pseudo partial cone, which is occupied by a phenyl ring of one

guest tetraphenylphosphonium cation. The basic structural motif consists of two

calixarenes shrouding three tetraphenylphosphonium cations in a skewed molecular

capsule arrangement which are interspersed between an envelope of

tetraphenylphosphonium cations (Fig. 19b). Two of the included guests have C-H

interactions with a pseudo calix[3]arene cavity of the C8S. The structure is


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complicated, consisting of pseudo molecular capsules of C8S forming an overall

layered structure where the calixarenes are in bilayer arrangement. There are two kinds

of criss-crossed large channels filled with water molecules and aquated ytterbium

cations forming 2D porous structure.

p-Sulfonatocalix[8]arene has been shown to form chalice-like cavity for thesolid state inclusion of coordination complex tris(phenantroline)cobalt(III)

([Co(phen)3]3+) in the presence of trivalent ytterbium cations88. The encapsulation of

globular shaped [Co(phen)3]3+ cation results in a disruption of the calixarene hydrogen

bond network associated with the phenolic moieties, and the wrapping of the flexible

Fig. 19. (a) Unusual conformation of C8S showing pseudo calix[3]arene cavity, (b) skewed

molecular capsule of two C8S molecules shrouding three tetraphenylphosphonium cations.

host around the guest (Fig. 20). The guest cation is held within the calixarene cavity by

O-H and C-H interactions. Additionally, two cations are associated externally to

each supermolecule. The guest cations are held distant from each other through

encapsulation by three calixarenes, with each calixarene acting as a heterotritopic

receptor for [Co(phen)3]3+ cation. The calixarene is best described as a distorted version

of the pleated loop conformation, with the phenolic hydrogen bond network breaking

such that a C2 symmetrical chalice is formed that is similar to that found for a bismuth

cluster complex of a p-tert-butylcalix[8]arene89. The lower rim of the chalice is

stitched together through the two hydrogen bonding interactions between the sulfonate

group from one ring and phenolic oxygen atom from another ring in alternate

conformation. Hydrogen bonding interactions between phenolic units are absent. The

preorganisation requirement of the calixarene in forming the complex results in the

formation of large diameter, negatively charged channels within the crystal lattice.

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Fig. 20. (a) Distorted pleated loop conformation of C8S; (b) inclusion of [Co(phen)3]3+ within the

C8S cavity.

It would appear that size or/and shape of the guest has the capacity to influence

the shape of thep-sulfonatocalix[8]arene cavity adopted in the solid state. The mutually

induced fit in host-guest complexes between conformationally flexible

p-sulfonatocalix[8]arene and photolabile cholinergic ligands has been observed in

solution90. Both the host and the guests adapted to each other and selected the higher

energy but correct geometric conformers so that the guest could fit favorably into the

cavity of the host to give ditopic binding of both the aromatic ring and the cationic

ammonium moiety of the guest (Fig. 21). This system with mutually induced fit

represents an original example of adaptive supramolecular biomimetic chemistry.

Fig. 21. Adaptive supramolecular system of C8S with flexible guest molecule.

2.2.5 Structural characterization of calix[4]arene diphosphate complexes

Calix[4]arene diphosphate (C4diP) is a water-soluble calix[4]arene derivative

with two polar functions on the lower rim. Due to the anionic character of these

functional substituents, this calixarene generates astonishing architectures in the solid-

state complexes with aliphatic and heterocyclic amines.

a b

guest host

complex


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Fig. 22. Typical dimeric structural motif of C4diP.

In the solid state calix[4]arene dihydroxyphosphonic acid exists in the flattened

cone conformation as an included head to head dimer (Fig. 22)91. Intramolecular

hydrogen bonding between the unsubstituted and substituted phenolic groups rigidifies

the cone conformation of C4diP. The dimeric motif described by the interpenetrating of

aromatic rings of facing calixarenes is present in all the reported structures.

In the complex with propandiamine calixarene building block is present as an

included head to head dimer, held together by intermolecular - interactions92. One

terminal ammonium function of the propandiamine is hydrogen bonded to two

phosphonate groups of the same C4diP and water molecule, and the second one ishydrogen bonded to three phosphonate groups of three other calixarenes. C4diP dimers

are assembled into hexameric tube forming an aqua-channel structure (Fig. 23a). While

both ethanol and water molecules are present in the structure, the channel itself shows

total selectivity for water. Ethanol molecules are situated external to the channel and are

hydrogen bonded to phosphonate P=O groups. Disordered water molecules are located

along the channel. The assembly shows strong structural analogy to the aquaporin water

channel

93

, making the structure a potent mimetic of biological membrane transportchannels.

The crystal structure of the C4diP complex with 1,6-diaminohexane shows

encapsulation of a calixarene dimer within a three-dimensional matrix formed by the

diamine molecules94. Two phosphonate groups of each calixarene act as a type of

molecular tweezers for two ammonium groups of the guest, one of which is held in

place by direct hydrogen bonding to both phosphonate moieties and the second by a

bridging water molecule and a directly bound phosphonate moiety. Eight diammonium

cations form the walls of a hydrophobic cavity that is filled by calixarene dimer. The


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Fig. 23. (a) Top view of the water channel along the [111] direction in the complex withpropandiamine; (b) molecular packing of C4diP complex with phenantroline.

head to head embedded calixarene dimers are held together by face to face -stacking.

The mismatch between the size of the dimer filling the cavity and the extended length of

1,6-diaminohexane is compensated by chain folding in the diamine, allowing the

ammonium-ammonium distances to match the dimensions of the calixarene molecule.

In the case of the heterocyclic diamine phenanthroline, a face-to-face aromatic

stacked dimer of 1,10-phenantroline is held within a cage of eight hydrogen bonded

calix[4]arene dihydroxyphosphonic acid molecules by face to face aromatic interactions

and hydrogen bonding95. The central ring of the each phenanthroline molecule

undergoes face to face stacking with an aromatic ring of the calixarene. The molecules

of phenanthroline are displaced with face to face stacking between the central ring and

the non-protonated heterocycle. The trapped 1,10-phenanthroline is involved in a

hydrogen bond via its protonated nitrogen atom and the deprotonated hydroxyl of the

phosphonic acid. Multiple aromatic-aromatic interactions are present in the cage. The

crystal packing shows planes of dimers of C4diP perpendicular to the c crystal axis, Fig.

23b. These planes are held together by network of hydrogen bonds.

Recently Lazar et al.96 reported a structural study on the calix[4]arene

dihydroxyphosphonic acid complexes with bipyridyl ligands: 2,2'-bipyridine,

4,4'-bipyridine and 1,2-bipyridylethane. For the three complexes, the overall structure is

based on layers of calixarene dimers alternated with hydrated layers of bipyridil

molecules. All the complexes present - interactions between the biryridyl moieties

and the calix[4]arene dihydroxyphosphonic acid. In the case of 4,4'-bipyridine and

1,2-bipyridylethane nitrogen atoms of the bipyridil ligands are protonated, while for the

complex with 2,2'-bipyridine nitrogen atoms are probably not protonated. The structure

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of the complex C4diP2,2'-bipyridine shows a simple intercalated monolayer of

2,2'-bipyridine parallel to the dimeric layer of the calixarenes. The compact network of

hydrogen bonds formed between the water molecules and the hydroxyl groups of the

phosphonic acid generates a hydrophilic cage for each bipyridine molecule. A major

feature of the structure is the face to face aromatic-aromatic interactions betweenmolecules of C4diP and 2,2'-bipyridine.

In the case of 4,4'-bipyridine the position of the nitrogen atoms on the bipyridyl

skeleton is favorable for hydrogen bond bridging between two molecules of

calix[4]arene dihydroxyphosphonic acid to afford an extended solid-state network. The

bridge is nonsymmetric with one nitrogen atom involved in direct hydrogen bonding to

a phosphonate group and the other one, hydrogen bonded to a bridging water molecule.

In the structure there is a competitive interplay between hydrogen bonding, aromatic-aromatic interactions and steric effects generated by the constrained geometry of

4,4'-bipyridine. The network formed by the bridging molecules of 4,4'-bipyridine may

best be described as a 1D ladder structure with the rungs formed by the dimers of

C4diP.

The complex of calix[4]arene dihydroxyphosphonic acid with more flexible

1,2-bipyridylethane shows strong similarities in the general nature of packing with the

4,4'-bipyridine, both having the 1D ladder network, however, the details demonstrates

the relaxation of the structure comparing with 4,4'-bipyridine complex. In the structure,

both aromatic rings of 1,2-bipyridylethane are involved in face to face stacking

interactions with aromatic rings of other molecules of 1,2-bipyridylethane. The

hydrogen bonding type interactions between C4diP and 1,2-bipyridylethane show the

same alternation as observed in the structure C4diP4,4'-bipyridine. At one end of the

molecule 1,2-bipyridylethane, one of the pyridinium groups is also involved in

hydrogen bonding to a water molecule, but here the water molecule forms hydrogen

bonfs with two opposing phosphonate groups.

2.2.6 Biological activity of water-soluble calixarenes

In spite of extensive early work of the biological properties of the p-sulfonated

calixarenes, it is only very recently that interest in their biomedical potential has come

to the fore again. This considerable interest in water-soluble calixarenes for biological

and medical applications has resulted in many patents.


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Hwang et al.97 patented a method of treatment of infection by enveloped viruses

such as HIV, herpes simplex and influenza viruses, with calix[n]arenes having polar

substitutent: sulfonate, carboxylate or phosphate groups. The calix[n]arenes binds

selectively to viral envelop protein and this binding blocks virus attachment to

infectable cells, thereby inhibiting virus infectivity.p-Sulfonatocalix[n]arenes posses suitable antimicrobial activity against fungal

and bacterial microorganisms98. C4S, C6S and C8S were found to exhibit antimicrobial

activity against Corynebacterium, Fusarium solani f. sp. mori (F.s-26), the fungal

strainsRosellinia necatrix [R-8], and Colletotrichum dematium [C.d.8901].

Atwood99 demonstrated the activity of different p-sulfonatocalix[n]arene

derivatives as chloride channel blockers. This study shown that the inhibition of the

ionic channels increases with the increasing size of the macrocycle. Chloride channelmodulators may serve as effective pharmaceutical for treating respiratory,

cardiovascular and gastrointestinal disorders.

Droogmans100 investigated the inhibition of volume-regulated anion channel

present on cultured endothelial cells which allows the passage of ions depending on the

membrane potential, using p-sulfonatocalix[n]arenes and their derivatives. The

p-sulfonatocalixarenes induced a fast inhibition at positive potentials but were

ineffective at negative potentials. At small positive potentials, p-sulfonatocalix[4]arene

was a more effective inhibitor than p-sulfonatocalix[6]arene and calix[8]arene, which

became more effective at more positive potentials.

p-Sulfonatocalix[n]arenes and their derivatives have been shown to posses anti-

thrombotic activity101. The conformation flexibility and the number of SO3 groups of

p-sulfonatocalix[n]arenes resemble chemical structure of anticoagulant heparin. The

anti-thrombotic activity of the p-sulfonatocalix[n]arene derivatives is believed to

proceed by a heparinoid like inhibitory effect on protease activity in the coagulation

cascade. Pinhal et al102found that p-sulfonatocalix[8]arene stimulates the synthesis of

heparan sulfate proteoglycan secreted by rabbit and human endothelial cells in culture.

p-Sulfonatocalix[n]arenes have been shown to have potential in the diagnosis of

prion-based diseases103, such as bovine spongiform encephalopathy BSE in cattle (so

called mad caw disease), scrappy in sheep, chronic wasting disease in deer and variant

Creutzfeldt-Jacob disease in humans.

Coleman et al.104,105 investigated p-sulfonatocalix[n]arene complexation by

bovine serum albumin (BSA), an arginine and lysine rich protein, using electrospray

mass spectrometry, dynamic light scattering and atomic force microscopy. For anionic


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p-sulfonatocalixarenes one strong and two weak binding sites were found on the protein

surface. The strength of the interactions between the calixarene and BSA is inversely

proportional to the size of macrocyclic ring: n = 4 > n = 6 >> n = 8.

Although the calixarenes are currently not approved for use in medicines to date

they have shown neither toxicity nor immune responses106,107

. These results providepromising signs that p-sulfonatocalixarenes could be useful in the field of

biopharmaceutical applications.

2.2.7 Drug solubilization

One way to increase the aqueous solubility of drugs is to use complexing agents

to form host-guest complexes. Among the complexing agents available, cyclodextrins

are most widely used in drug formulations108. Alternative complexing agents are

calixarenes, widely studied for their inclusion properties towards different guest

molecules.

Yang has recently investigated the solubilizing effect of

p-sulfonatocalix[n]arenes on the poorly water soluble drugs, nifedipine109 (an important

calcium channel blocker that is used extensively for the clinical management of a

number of cardiovascular diseases), niclosamide110

(an anthelmintic drug that is activeagainst most tapeworms) and furosemide111 (a high ceiling diuretic). The results show

that the size of the p-sulfonatocalix[n]arene, the pH of solubility medium, and the

concentration of the calix[n]arenes significantly changed solubility of the drug. In the

case of nifedipine the p-sulfonatocalix[8]arene improved the solubility the most, while

in the presence ofp-sulfonatocalix[6]arene the solubility of nifedipine was decreased.

Interestingly, for niclosamide the greatest increase in the aqueous solubility was

observed whenp-sulfonatocalix[6]arene was added.p-Sulfonatocalix[6]arene improvedthe solubility of furosemide the most followed by p-sulfonatocalix[8]arene, while

p-sulfonatocalix[4]arene increased the solubility of furosemide the least. The authors

suggest that the dimension of calix[6]arene is most optimal for the niclosamide and

furosemide molecules because host-sizeselectivity does exist in host-guest type

complexation with calixarenes112. The increase in drug solubility afforded by the

calixarenes was most probably the result of the incorporation of the non-polar portions

of the drug molecule into the non-polar cavities of the calixarenes similar to drug-

cyclodextrin complexes113. The other possible mechanisms involved in the


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complexation between p-sulfonatocalix[n]arenes and drugs may be hydrogen bonding

and electron donor-acceptor interactions.

The effects of the cavity size ofp-sulfonatocalix[n]arenes and pH on the stability

and fluorescent properties of berberine, a clinically important natural isoquinoline

alkaloid, have recently been studied114

. The weakest binding was observed for C4S andthe strongest for C8S. The substantial increase of the association constants with the

receptor size originates from the growing strength of the - electronic interaction

between the host and guest aromatic rings. The high flexibility of C8S and its

comparable size to berberine permit strong host-guest interaction. The authors

demonstrated that this particularly strong binding to p-sulfonatocalix[8]arene, which

leads to a significant fluorescence intensity increase, can be applied to detect even trace

amount of berberine.

2.3 ConclusionsThe use of water-soluble anionic calixarenes in supramolecular chemistry and

for the complexation of organic cations has grown considerably since the initial work of

Shinkai on the binding and structure of the p-sulfonatocalix[4]arene complex with the

trimethylanilinium cation. Water-soluble anionic calixarenes are capable of complexingsmall biologically active molecules, amino acids and proteins. The solid-state

complexes ofp-sulfonatocalix[4]arenes and calix[4]arene diphosphate studied thus far

show a remarkable diversity of structure types: from traditional bilayers through zigzag

bilayers, Russian doll capsules, Ferris wheel complexes to aqua-channel hexagonal

structures for C4diP, and nanospheroidal and tubular assemblies for C4S. The larger

p-sulfonatocalix[6]arene and the corresponding octamer show a great potential for the

assembly of complicated superstructures in the solid state.The field of biopharmaceutical application of anionic calixarenes by their

complexation to cationic groups present in biomacromolecules is just starting, but it can

be expected to be of great importance in the future.


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3 EXPERIMENTAL

In this chapter the procedures for data collecting and processing, structure

solution and refinement, and crystal structure analyses are described.

There are several stages in which we can influence the final quality of structure

model. One of them is selecting a good quality crystal of pure composition, appropriate

size, and regular shape and with no large internal imperfections, such as cracks or

twinning. The size of the crystal should be around 0.2-0.3 mm for organic compounds.

For compounds containing heavier atoms it is recommended to use smaller crystals to

reduce absorption effects.

All complexes described along this dissertation were synthesized and most of

them crystallized by the group of Dr. Anthony W. Coleman from the Institute of

Biology and Chemistry of Protines in Lyon. Crystals were obtained by the slow

diffusion of solvents or slow evaporation from water-alcohol (methanol or ethanol)

systems. A few complexes were crystallized from DMF-alcohol mixtures.

Single crystals suitable for X-ray crystallographic analysis were selected

following examination under a microscope. The crystals were mounted on glass fibers

with a small quantity of glue, because the amount of glue in the X-ray beam influences

background considerably. Some of the crystals were scooped up in a tiny loop made of

nylon and attached to a solid rod. The last stage in the selection was crystal pre-

scanning on the diffractometer, i.e. making a few quick exposures and checking the

images visually on spot size, shape and distribution. All measurements were performed

at low temperature (100 K) in order to prevent crystal decomposition, reduce thermal

vibrations, enhance signal-to-noise ratio and reduce dynamic disorder.

When a crystal is mounted and exposed to an intense beam of X-rays, it scatters

the X-rays into a pattern of spots or reflections. The relative intensities of these spotsprovide the information to determine the arrangement of molecules within the crystal in

atomic detail.

Single-crystal X-ray diffraction data for all compounds described along this

dissertation, were collected on a Nonius KappaCCD diffractometer using MoK

radiation ( = 0.71073 ). The first stage of the measurement was the determination of

the unit cell on the base of ten frames made by crystal rotation for ten degrees (scan

angle 1 per frame). Having the unit cell parameters and point group symmetry it waspossible to calculate the strategy for data collecting, i.e. the number of frames grouped


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in sets together with the time needed per degree of rotation to get a dataset with

reflections upto a specific diffraction angle. The raw data collected from the diffraction

experiment were integrated and scaled in order to obtain the indexed reflections with a

consistent intensity scale. Data collecting and processing were carried out using the

programs Collect115

, HKL2000116

, maXus117

. The structures were solved by directmethods using the programs SHELXS-97118, SIR92119, SIR97120 and refined by full

matrix least squares using the program SHELXL-97118.

Unless specified, all non-hydrogen atoms were refined anisotropically.

Hydrogen atoms were placed in geometrically calculated positions and refined as riding

atoms. Hydrogen atoms of methyl and hydroxyl groups were refined in geometric

positions for which the calculated sum of the electron density is the highest (rotating

group refinement). The torsion angles were then refined during the least-squaresrefinement. All hydrogen atoms were refined isotropically with temperature factors 1.2

times those of their bonded atoms (1.5 times for methyl and hydroxyl groups). Where

possible, the hydrogen atoms of amino groups were located on Fourier difference maps

and refined with positional parameters. In the other case hydrogen atoms were placed in

positions for which the calculated sum of the electron density is the highest. Where

possible, hydrogen atoms of water molecules were located in the differential Fourier

electron density maps. In the case of bad diffraction data and disorder the DFIX and

DANG geometrical restraints were applied in order to retain the guest molecules

geometry. Disordered atoms were refined with isotropic displacement parameters. All

crystallographic calculations were conducted with the WINGX121 program package.

Program PARST122 was used for calculating geometrical parameters (planes and

angles) from the results of crystal structure analyses. Non-covalent interactions of -,

Donor-H and C-HAcceptor type were calculated using the program PLATON123.

The geometric parameters of non-covalent interactions and hydrogen bonds are

presented in the form of tables in the Appendix. All the figures were prepared using the

X-Seed124 interface. Thermal ellipsoids were drawn at 50% probability level. Hydrogen

atoms that do not take part in hydrogen bonding and disordered atoms with lower

occupancy were omitted for clarity.

A consistent numbering scheme was used for the calixarene molecules in all

structures. The calixarene molecule was divided into four functional groups (phenyl

group with substituents and methylene group) carrying the labels A, B, C and D. Where

there is more than one calixarene molecule in the asymmetric unit the labels are

augmented with the numbers 1 and 2 for the second and third crystallographically


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independent molecules, respectively. For the large C6S and C8S molecules the

asymmetric unit comprises only half of the host molecule, so the phenolic rings carry

the labels A, B, C (and D for C8S). Labels for the guest molecules carry the letters X,

Y, Z and W.


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4 RESULTS AND DISCUSSION

4.1 Solid-state complexes of p-sulfonatocalix[4]arene

4.1.1 Crystal structure ofp-sulfonatocalix[4]arene complex with melamine

Melamine (1,3,5-triazine-2,4,6-triamine) (Fig. 24) has been widely used as

precursor for supramolecular motifs such as cyclic rosettes125, tapes126 and ribbons127.

Melamine is a metabolite of cyromazine, a pesticide. It is formed in the body of

mammals who have ingested cyromazine It was also reported that cyromazine is

converted to melamine in plants128. Melamine is used combined with formaldehyde to

produce melamine resin, a very durable thermosetting plastic, and melamine foam, a

polymeric cleaning product. Melamine is also used to make fertilizers. Melamine

derivatives of arsenical drugs are potentially important in the treatment of African

trypanosomiasis.

In the complex p-sulfonatocalix[4]arenemelamine the asymmetric unit (Fig.

25) comprises one molecule of tetraanionic C4S, four melamine monocations (named as

W, X, Y and Z) and fifteen water molecules from which six have partial occupancies.

Charge equilibrium in the structure is realized by protonation of one aromatic nitrogen

atom of each melamine molecule. Crystallographic data for the complex of C4S with

melamine are presented in Table 1.

Fig. 24. Melamine monocation.

The solid-state structure of the complex is generated by bilayer motif of

p-sulfonatocalix[4]arenes which are held together by - interactions and by hydrogen

bond between phenolic and sulfonate groups of the neighboring calixarene molecule

(Fig. 26).

In Fig. 27, the complete network of hydrogen bonds generated by melamine

monocations is presented. Normally, melamine has three centres for non-covalent

linking each of which can donate two hydrogen bonds from each amine groups and

accepts one on each aromatic nitrogen. However, in the current structure the protonation


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Table 1. Crystal data and structure refinement for C4Smelamine.

Molecular formula C28H20O16S44C3N6H712.5H2OFormula weight 1467.41Temperature 100(2) KWavelength 0.71073 Crystal system Triclinic

Space group PUnit cell dimensions a = 11.1991(6) = 75.286(4).

b = 15.293(1) = 86.453(4).c = 19.008(1) = 82.786(3).

Volume 3122.3(3) 3Z 2Density (calculated) 1.561 Mgm-3Absorption coefficient 0.258 mm-1F(000) 1532Crystal size 0.20 0.05 0.03 mm3

range for data collection 2.72 to 22.02.Index ranges -11 h 11, -16 k 16, -19 l 19Reflections collected 27163Independent reflections 7510 [Rint = 0.068]Completeness to = 22.02 97.8 %Absorption correction NoneRefinement method Full-matrix least-squares on F

2

Data / restraints / parameters 7510 / 0 / 896

Goodness-of-fit on F2 1.08

Final R indices [I > 2(I)] R = 0.069, wR = 0.148

R indices (all data) R = 0.106, wR = 0.160Extinction coefficient 0.0022(4)Largest diff. peak and hole 0.54 and -0.32 e-3

Fig. 25. Asymmetric unit of the C4S complex with melamine.


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of one aromatic nitrogen atom occurs and it becomes the hydrogen bond donor. In the

complex there are five pairs of hydrogen bonds between neighboring melamine

monocations and four quintets of hydrogen bonds with solvent molecules or sulfonate

groups of the calixarenes (for each melamine monocation). One of the melamine cations

(Z) forms hydrogen bonds with four water molecules and only one with the sulfonategroup, all the three other melamine monocations (X, Y and W) are hydrogen bonded

with two solvent molecules and with three sulfonate groups. It is worth to note that

protonated nitrogen binds only to water molecules.

Fig. 26. View of the molecular packing along the a axis showing planar bilayers of

p-sulfonatocalix[4]arenes.

Fig. 27. View along b axis of the network of hydrogen bonds encountered by melaminemonocations.

The melamine monocations are arranged into linear chains and interact by

-stacking (distances between centroids of melamine molecules vary from 3.783(3) to

ZZ W

YX

X


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3.915(3) , while distances between melamine planes vary from 3.377(3) to

3.433(3) ).

Fig. 28. Sheets of melamine monocations formed by linear chains of-stacked aromatic rings.

The structure of the complex ofp-sulfonatocalix[4]arene with melamine shows a

wide variety of interactions. The anionic groups of the calixarene play significant role in

the diversity of the non-covalent interactions between melamine monocations and their

environment.


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4.1.2 Crystal structure ofp-sulfonatocalix[4]arene complex withtriethylamine

The stoichiometry of the complex is 1:2:1:3 for p-sulfonatocalix[4]arene,

triethylamine, acetone and water, respectively. Crystallographic data for the complex ofC4S with triethylamine are presented in Table 2.

p-Sulfonatocalix[4]arene is present in the flattened cone conformation with a C2v

symmetry (cone angles between the opposite aromatic rings are 44.30(8) and

100.10(7)). Hydrogen bonds occur between the four phenolic hydroxyl groups at the

lower rim (O-HO distances of 2.770(3), 2.697(3), 2.734(3) and 2.706(3) ). Oxygen

atoms of two sulfonate groups and acetone molecule are disordered over two positions.

Fig. 29. Triethylamine cation

Triethylamine molecule appears in the form of cation (Fig. 29) and the host

molecule is in the form of anion. To satisfy charge balance, C4S in the complex should

possess two protonated sulfonate groups. Unfortunately, it was not possible to locate all

hydrogen atoms from the Fourier difference map for this to be clarified.

Triethylammonium cations are complexed in two different ways (Fig. 30). Both

are complexed via strong hydrogen bond between protonated nitrogen and sulfonate

oxygen atom of the calixarene (2.753(3) and 2.738(5) for guest molecules X and Y,respectively), but whereas triethylammonium cation Y is located inside the C4S with

one ethyl group pointing into aromatic cavity and showing C-H interaction of

3.638(5) with one of the aromatic ring of the C4S, triethylammonium cation X is

complexed outside the cavity also showing C-H interaction of 3.540(4) with one

of the aromatic ring of the neighboring C4S. There are also some C-H O hydrogen

bonds between ethyl groups of the guest molecules and sulfonate oxygens of the

calixarene.


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Fig. 30. View of the asymmetric unit of the C4S complex with triethylammonium.

The complex between p-sulfonatocalix[4]arene and triethylammonium cations

shows a novel stepped bilayer solid-state packing motif. As compared to typical bilayer

or zigzag bilayer structures of C4S in which - interaction are often observed between

adjacent calixarene molecules, in this case no such interactions are observed. Water

molecules are involved in hydrogen bonding with sulfonate groups of C4S molecules

and stabilize crystal packing. A range of C-HO interactions between methylenic

bridges and sulfonate oxygens, and between aromatic carbons and sulfonate oxygens of

the

phd-danylyuk (ll)

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