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MASARYKOVA UNIVERZITA
PŘÍRODOVĚDECKÁ FAKULTA
ÚSTAV CHEMIE
BAMBUSURILY A JEJICH
SUPRAMOLEKULÁRNÍ INTERAKCE S
ORGANICKÝMI FOSFÁTY
DIPLOMOVÁ PRÁCE
Tomáš Fiala
VEDOUCÍ PRÁCE: doc. Ing. Vladimír Šindelář, Ph.D. BRNO 2015
Bibliografický záznam
Autor: Bc. Tomáš Fiala
Přírodovědecká fakulta, Masarykova univerzita
Ústav chemie
Název práce: Bambusurily a jejich supramolekulární interakce s
organickými fosfáty
Studijní program: Chemie
Studijní obor: Organická chemie
Vedoucí práce: doc. Ing. Vladimír Šindelář, Ph.D.
Akademický rok: 2014/2015
Počet stran: 117 + 15
Klíčová slova: bambusuril; organický fosfát; rozpoznávání aniontů;
supramolekulární chemie
Bibliographic Entry
Author: Bc. Tomáš Fiala
Faculty of Science, Masaryk University
Department of Chemistry
Title of Thesis: Bambusurils and Their Supramolecular Interactions with
Organic Phosphates
Degree Program: Chemistry
Field of Study: Organic Chemistry
Supervisor: doc. Ing. Vladimír Šindelář, Ph.D.
Academic Year: 2014/2015
Number of Pages: 117 + 15
Keywords: bambusuril; organic phosphate; anion recognition;
supramolecular chemistry
Abstrakt
Tato práce zahrnuje dva projekty zaměřené na syntézu a supramolekulární
vlastnosti bambusurilů – molekul patřících mezi makrocyklické receptory aniontů.
V rámci prvního projektu byla navržena a optimalizována vylepšená
metodologie pro přípravu bambusurilů. Nové reakční podmínky byly využity pro
přípravu nového derivátu bambus[6]urilu. Tento receptor vykazuje vysokou selektivitu
pro vazbu jodidových a chloristanových aniontů ve vodných roztocích s asociačními
konstantami převyšujícími 109 M
-1. Tak vysoké asociační konstanty byly naměřeny
vůbec poprvé pro neutrální receptor aniontů ve vodném prostředí. Pomocí NMR a ITC
experimentů bylo rovněž ukázáno, že afinita nově připraveného receptoru k aniontům
sice mírně klesá v zásaditém prostředí, avšak není ovlivněna přítomností kyselin. Tato
afinita je rovněž nezávislá na povaze přítomných kationtů.
Druhý projekt se zabývá charakterizací vazebného rozhraní mezi bambus[6]urily
a organickými fosfáty. NMR titrační experimenty a metoda kontinuální variace ukázaly,
že dialkyl-fosfáty dominantně tvoří s bambusurily v roztoku komplex se stechiometrií
1:1. Z neúspěšných pokusů o syntézu [2]rotaxanu založeném na dialkyl-fosfátu, který je
provlečený bambus[6]urilem se zdá, že pozorovaný komplex není inkluzní, ale externí.
V pevné fázi byl pomocí rentgenové krystalografie identifikován komplex
bambus[6]urilu s dialkyl-fosfátem se stechiometrií 1:2.
Abstract
This work includes two projects focusing on the synthesis and supramolecular
properties of bambusurils – molecules from the group of macrocyclic anion receptors.
In the first project, an improved methodology for the preparation of bambusurils
was designed and optimized. The new conditions were used to prepare a novel
bambus[6]uril derivative. This receptor shows a high selectivity towards iodide and
perchlorate anions in aqueous solutions with association constants exceeding 109 M
-1.
Such high association constants have been observed for the first time for a neutral anion
receptor in water. NMR and ITC measurements also showed that the affinity of the
prepared receptor towards anions slightly decreases under basic conditions, whereas it is
not influenced by the presence of acid. Furthermore, the affinity is also independent on
the nature of the present cations.
The second project deals with the characterization of the bambusuril–organic
phosphate binding interface. NMR titration experiments and the method of continuous
variation showed that bambus[6]urils and dialkyl phosphates predominantly form
complexes of a 1:1 stoichiometry in solution. As a result of failed attempts to synthesize
a [2]rotaxane based on a dialkyl phosphate threaded bambus[6]uril, it seems that the
observed complex was an external rather than an inclusion one. In the solid state, a
bambusuril–dialkyl phosphate complex with a 1:2 stoichiometry has been identified by
X-ray crystallography.
Poděkování
Je příliš mnoho lidí, kterým bych chtěl poděkovat v souvislosti s mou
diplomovou prací. Proto se dopředu omlouvám těm, na které se na těchto řádcích
nedostane. Patří jim neméně velké díky za vše, co pro mě udělali.
Na prvním místě chci poděkovat svému školiteli, doc. Ing. Vladimíru
Šindelářovi, Ph.D., za možnost pracovat na nesmírně zajímavém výzkumu a za
veškerou jeho podporu a cenné rady. Děkuji také RNDr. Václavu Havlovi za plodné
vědecké diskuse, rady týkající se ITC a NMR a vše, co jsem se od něj mohl naučit.
Nemohu zapomenout na Dr. Lauru Gilberg, které děkuji za mnohé rady a pomoc, bez
kterých by tato práce nemohla vzniknout. Velké díky patří také Karolíně Salvadori a
Kamilu Maršálkovi za spolupráci na projektu nového bambusurilu – není nad to dělat
výzkum v tak příjemném kolektivu. Mé poděkování patří také Mgr. Tomáši Lízalovi za
pomoc se softwarem PyMOL při vizualizaci krystalových struktur a Mgr. Kristíně
Tomášikové za představení ATR-FTIR spektroskopie.
Děkuji také Mgr. Ondřeji Šedovi, Ph.D. za provedení MALDI-TOF MS
experimentů a Mgr. Miroslavě Bitttové, Ph.D za provedení HRMS experimentů.
Speciální poděkování patří Mgr. Michalu Babiakovi za neúnavné měření krystalových
struktur a související diskuse.
V neposlední řadě musím poděkovat přátelům a rodině za podporu a kolegům z
Laboratoře supramolekulární chemie za vytvoření příjemného pracovního prostředí.
"Supramolekulární chemie je v podstatě taková chemická sociologie."
– Lukáš Mikulů
Acknowledgment
There are too many people I would like to thank in connection with my diploma
thesis. Therefore, I hereby apologize to those, who did not make it to these lines. A just
as big thank you belongs to them for everything they have done for me.
Firstly, I would like to thank my supervisor Assoc. Prof. Vladimír Šindelář,
Ph.D. for the opportunity to engage in very exciting research and for all his support and
valuable advice. I also thank RNDr. Václav Havel for fruitful scientific discussions,
advice concerning ITC and NMR and everything I could learn from him. I cannot forget
Dr. Laura Gilberg, whom I thank for all the advice and help without which this thesis
would not exist. Many thanks also belong to Karolína Salvadori and Kamil Maršálek for
collaboration on the new bambusuril project – it was very nice to do research in such
great team. My thank you also belongs to Mgr. Tomáš Lízal for help with the PyMOL
software to visualize crystal structures and Mgr. Kristína Tomášiková for introduction
to ATR-FTIR spectroscopy.
I also thank Mgr. Ondřej Šedo, Ph.D. for carrying out MALDI-TOF MS
experiments and Mgr. Miroslava Bittová, Ph.D for carrying out HRMS experiments.
Special thanks belong to Mgr. Michal Babiak for untiringly measuring my crystal
structures and related discussions.
Last but not least, I must thank all my friends and family for support and my
colleagues from the Supramolecular Chemistry Group for a friendly working
environment.
"Supramolecular chemistry is essentially a kind of chemical sociology."
– Lukáš Mikulů
Declaration
I hereby declare I wrote my diploma thesis on my own with the use of the cited
literature.
Prohlášení
Prohlašuji, že jsem svoji diplomovou práci vypracoval samostatně s využitím
informačních zdrojů, které jsou v práci citovány.
Brno, 13. května 2015 ……………………………
Tomáš Fiala
- 10 -
Contents
1 Introduction ................................................................................................................ 12
2 Theoretical Part ......................................................................................................... 13
2.1 Bambus[n]urils ...................................................................................................... 13
2.1.1 Glycoluril ........................................................................................................ 15
2.1.2 Bambusuril Derivatives .................................................................................. 16
2.1.2.1 Bambusurils Bearing Saturated Aliphatic Substituents ........................................ 16
2.1.2.2 Bambusurils Bearing Benzylic Substitution ................................................................. 17
2.1.2.3 Bambusurils Bearing Olefinic Substitution .................................................................. 18
2.1.2.4 Semithiobambusurils ..................................................................................................................... 19
2.1.3 Applications of Bambusurils .......................................................................... 20
2.1.4 Macrocycles Related to Bambusurils ............................................................. 22
2.1.4.1 Cucurbit[n]urils ................................................................................................................................. 22
2.1.4.2 Hemicucurbit[n]urils ..................................................................................................................... 22
2.2 Supramolecular Chemistry of Phosphates............................................................. 23
2.2.1 Charged Receptors ......................................................................................... 24
2.2.2 Hydrogen Bond Donor Receptors .................................................................. 27
2.2.3 Receptors Containing Metal Ions ................................................................... 31
2.2.4 Other Receptors .............................................................................................. 33
3 Results and Discussion – Project 1 – New Bambusuril Derivative ........................ 35
3.1 Background ........................................................................................................... 35
3.2 Synthesis of the Monomer..................................................................................... 36
3.3 Macrocyclization ................................................................................................... 40
3.4 Anion-Free Bambusuril 2[6] ................................................................................... 46
3.5 Application of the New Macrocyclization Protocol on Me12BU[6] Synthesis ..... 48
3.6 Binding Properties of 2[6] ...................................................................................... 50
- 11 -
3.6.1 Theory ............................................................................................................ 50
3.6.2 NMR Experiments .......................................................................................... 52
3.6.2.1 Direct Titration Experiments ................................................................................................... 54
3.6.2.2 Competition Titration Experiments .................................................................................... 56
3.6.2.3 pH Dependence of Anion Binding ...................................................................................... 58
3.6.3 ITC Experiments ............................................................................................ 59
3.6.3.1 Characterization of the 2[6]∙Cl– Complex Using ITC ............................................. 61
3.6.3.2 Influence of the Counter-Cation on Anion Binding by 2[6] in Water ......... 63
3.6.4 Comparison to Other Receptors ..................................................................... 64
3.7 Conclusion and Future Prospects .......................................................................... 68
4 Results and Discussion – Project 2 – Interactions of Bambusurils with Organic
Phosphates ......................................................................................................... 70
4.1 Background ........................................................................................................... 70
4.2 Initial Experiments ................................................................................................ 71
4.3 Syntheses Towards a Bambusuril Rotaxane ......................................................... 73
4.3.1 p-MethylBenzyl Stopper ................................................................................ 75
4.3.2 Isophthalate and Trityl Stoppers ..................................................................... 79
4.4 Crystallization Experiments .................................................................................. 80
4.5 Further Complexation Studies ............................................................................... 82
4.6 Conclusion and Future Prospects .......................................................................... 84
5 Experimental Part ...................................................................................................... 86
5.1 Used Chemicals and Instruments .......................................................................... 86
5.2 Data Treatment ...................................................................................................... 88
5.3 Synthetic Procedures ............................................................................................. 89
References .................................................................................................................... 108
List of Abbreviations .................................................................................................. 115
Supplementary Information ......................................................................................... s1
NMR Characterization of 2[6]∙NaCl ............................................................................. s1
NMR Titration Experiments ........................................................................................ s5
ITC Experiments .......................................................................................................... s9
IR Spectra ................................................................................................................... s10
- 12 -
Chapter 1
Introduction
Inorganic phosphate as well as its mono- and diesters are among the most
abundant anions in living cells. Nucleic acids are held together by a carbohydrate–
phosphate backbone, protein regulation and signal transduction is highly dependent on
phosphorylation, and the universal energy-bearing molecule in all biological systems,
adenosine triphosphate (ATP), is a phosphoester/anhydride.
Organic agents that interact with phosphorylated molecules with high selectivity
are of increasing interest to medicinal chemists and chemical biologists. The ability to
manipulate with phosphate-dependent processes in living cells, including replication,
gene expression, enzyme regulation and cellular energetics is the first step to successful
diagnostics and therapeutics.
This work aims to investigate bambus[n]urils (BU[n]s), a family of macrocyclic
compounds, as potential binding agents for organic phosphate. BU[n]s were previously
shown to bind various anions with high affinity and selectivity. However, no research
has been done on the supramolecular chemistry of these macrocycles with organic
phosphates.
Another project included in this thesis deals with the synthesis of a novel
bambusuril derivative with high solubility in aqueous solutions. The presented
macrocycle is the first neutral member of the BU[n] family which is able to bind anions
in water with high affinity in the full pH range.
- 13 -
Chapter 2
Theoretical Part
2.1 Bambus[n]urils
In 2010, Masaryk University in Brno became the birthplace of a novel
macrocyclic molecule with unique properties.[1]
Sindelar et al. named the compound
bambus[6]uril due to its resemblance to the bambuseae tribe of plants (Figure 1). Later,
the trivial name was modified to dodecamethylbambus[6]uril (Me12BU[6]) to reflect the
alkyl substitution.[2]
Since then, a whole family of macrocycles has arisen from this
initial discovery, comprising numerous members with different properties and potential
applications.[3,4]
Figure 1: a) Structural formula of bambus[6]uril; b) the bambus[6]uril
calotte model resembles the bamboo plant (cover picture of Angew. Chem.
Int. Ed., Vol. 49, Issue 13).[1]
- 14 -
Bambus[n]urils are synthesized by a Mannich-type[5]
polycondensation reaction
of formaldehyde and the corresponding glycoluril (Scheme 1). The reaction is carried
out with an acid catalyst and a suitable anionic template. Bambusurils containing four
and six glycoluril units have been prepared so far.[1,3,4]
Scheme 1: Synthesis of bambus[n]urils; n = 4, 6.[1,3,4]
The most unique property of bambus[n]urils is that the macrocycles containing
six monomer units bind anions with high affinity and selectivity.[2,4,6,7]
Most anions
form 1:1 inclusion complexes with BU[6]. The bound anion is located in the center of
the macrocycle's cavity (Figure 2).[8]
Twelve C–H X– hydrogen bonds (X
– = anion)
stabilize the guest inside the macrocycle.*
Figure 2: Calculated structure of the Me12BU[6]∙Cl– complex, top view.
The chloride anion (green) is located inside the cavity of the macrocycle and
is stabilized by twelve C-H Cl- interactions (red dots). Color code for other
atoms: carbon – orange, nitrogen – blue, oxygen – red, hydrogen – white.[8]
* The term 'methine protons' will be used to address the hydrogen atoms pointing to the center of the
cavity throughout this thesis. Although it is not the correct IUPAC name for this type of hydrogen atoms,
it is commonly used in bambusuril chemistry.
n
N N
O
N NR R
O
NH NH
O
N NR R
O
H2CO or PFA
H+, template
- 15 -
2.1.1 Glycoluril
The main constituent of the bambusuril macrocycle is glycoluril, a
heterobicyclic molecule with a rigid, bent structure (Figure 3). Generally,
bambus[n]urils are composed of n glycoluril units joined together with n methylene
bridges.[1]
Figure 3: a) Structural formula and numbering* of glycoluril; b) bent
structure of glycoluril.
Substituted glycolurils are required for the synthesis of bambus[n]urils.[1]
Glycoluril contains multiple substitution sites which may be essentially divided into
three types (Figure 4a) – N-substitution (positions 2, 4, 6 and 8), C-substitution
(positions 1 and 5) and O-substitution (carbonyl oxygen atoms may be replaced with
other heteroatoms, e.g. sulfur).[9]
Additionally, the carbonyl oxygen atoms may be
alkylated in their corresponding tautomeric forms (Figure 4b).[10]
Not all substitution patterns are compatible with the formation of
bambus[n]urils. One pair of ureic nitrogen atoms needs to remain unsubstituted to
enable the attachment of methylene bridges. The other pair, on the other hand, must be
alkylated to block the condensation with formaldehyde. C-substitution at positions 1
and 5 is forbidden due to steric effects.[1,2]
Partial substitution of carbonyl oxygen for
sulfur atoms has been reported.[11]
The general structure of a substituted glycoluril
suitable for bambusuril synthesis is given in Figure 4c.
* There are two ways how to number the glycoluril molecule – the heterocyclic approach and the bicyclic
approach. Our group has agreed to consistently use the latter which is also used throughout this thesis.
NH2 3
1
NH4
5
NH8 7
NH6
O
O
N N
N N
O
O
HH
HH
HH
a) b)
- 16 -
Figure 4: a) Substitution sites on glycoluril; b) substituted glycoluril in its
tautomeric form; c) substituted glycoluril suitable for bambusuril synthesis;
R = alkyl, alkenyl, aryl; X = O, S.[9,10]
2.1.2 Bambusuril Derivatives
In the five year history of the bambusuril family, numerous derivatives were
prepared. The new macrocycles generally aimed: (i) to solve the initial solubility issues,
(ii) to increase the efficiency of preparation and (iii) to add new functionalities to the
compound.
2.1.2.1 Bambusurils Bearing Saturated Aliphatic Substituents
Dodecamethylbambus[6]uril (Me12BU[6], Figure 5) was the first macrocycle of
the family to be prepared.[1]
The macrocycle was synthesized by stirring
2,4-dimethylglycoluril and paraformaldehyde (PFA) in 5.4 M aq. HCl at room
temperature (r.t.) for 24 h. Although the new host molecule possessed unique binding
properties, there were also major disadvantages limiting possible applications of the
macrocyclic compound. Me12BU[6] was insoluble in all single-component solvents.
Millimolar solutions could only be prepared in some solvent mixtures, e.g. MeOH–
CHCl3 or H2O–MeCN. Moreover, the macrocyclization reaction provided the cyclic
hexamer as a complex with HCl. The acid could not be removed by heating at 170 °C in
vacuo for several hours or washing with excess of water or methanol. Eventually, an
indirect procedure was designed for the preparation of anion-free Me12BU[6]. As the
macrocycle is highly selective for iodide over chloride, treatment with HI provided the
Me12BU[6]∙HI complex.[6]
Finally, oxidation of iodide with hydrogen peroxide
(Scheme 2) led to the desired product.
N N
N N
X
X
R4
R2
R6
R5
R1
R
N N
N N
O
O
R2
R1
R5
R6
R
R
8
a) b) c)
N N
NH NH
X
O
R4
R2
- 17 -
Scheme 2: Preparation of anion-free Me12BU[6].[6]
Dodecapropylbambus[6]uril (Pr12BU[6] , Figure 5) was prepared shortly after
Me12BU[6].[2]
The synthesis aimed to verify that the macrocyclization conditions used
to prepare the methylated macrocycle were robust and could enable the preparation of
other bambusuril derivatives. With minor adjustments (heating at 100 °C and shortening
the reaction time to 75 min), the macrocycle containing six dipropylglycoluril units was
successfully prepared.
Octapropylbambus[4]uril (Pr8BU[4] , Figure 5) was prepared much later by
Heck et al.[3]
by reduction of an allyl-substituted macrocycle (see Chapter 2.1.2.3 for
details).
Figure 5: Bambusurils bearing saturated aliphatic substituents.[1–3]
2.1.2.2 Bambusurils Bearing Benzylic Substitution
The protocols for synthesizing the bambusurils mentioned in Chapter 2.1.2.1
(using aq. HCl as a solvent) could not be used for the preparation of derivatives with
larger organic substituents, mainly for solubility reasons. Havel et al.[2]
developed new
reaction conditions for preparing bambusurils bearing benzyl groups (Figure 6).
Heating 2,4-dibenzylglycoluril in chloroform or toluene with p-toluenesulfonic acid as a
catalyst provided octabenzylbambus[4]uril (Bn8BU[4]) – the very first cyclic tetramer
of the bambusuril family. To prepare the six-membered macrocycle,
tetrabutylammonium chloride or iodide had to be used as a template in addition to the
conditions used for Bn8BU[4] synthesis. Anion-free Bn12BU[6] was obtained using two
different strategies. Firstly, a modification of the protocol used for the methylated
6
N N
O
N N
O
n
N N
O
N N
O
n = 4, 6
Me12BU[6] Pr2nBU[n]
- 18 -
analog was implemented. The process was successful when hydrogen peroxide was
substituted with peroxoacetic acid. Secondly, it was also possible to remove the anion
from the macrocycle's cavity by washing the chloride complex with hot acetonitrile.
However, this procedure was lower yielding compared to the oxidation protocol.
Yawer et al.[4]
have prepared a bambusuril macrocycle bearing
4-(methoxycarbonyl)benzyl substituents (Figure 6) using Havel's[2]
protocol. This
derivative could be further hydrolyzed to give the 4-carboxybenzylated bambusuril with
remarkable affinity to anions in neutral and basic aqueous solutions.
Research on bambusurils in Sindelar's group has led to significant progress in
the synthesis of new derivatives. Most recently, new macrocycles bearing 2- and
4-nitrobenzyl groups (Figure 6) have been prepared.[12,13]
Figure 6: Bambusurils bearing benzylic substituents.[2,4,12,13]
2.1.2.3 Bambusurils Bearing Olefinic Substitution
An improved protocol for fast and high-yielding synthesis of bambus[n]urils was
presented by Heck and coworkers.[3]
By utilizing microwave-assisted synthetic
techniques, they were able to improve the yields of existing bambusurils, as well as
prepare new allyl-substituted analogs – (Allyl)8BU[4] and (Allyl)12BU[6] (Figure 7).
The C=C double bonds allowed for further transformations of the bambusuril
derivatives. Isomerization of the allyl groups to prop-1-enyl was achieved by refluxing
the BU[4] derivative in toluene with the Hoveyda-Grubbs II catalyst. The same
derivative was also hydrogenated to provide octapropylbambus[4]uril (Pr8BU[4]) – a
new aliphatic derivative as only Pr12BU[6] was previously prepared by Svec and
coworkers.[2]
Perhaps the most interesting bambusuril analog was prepared by cross
metathesis of (Allyl)8BU[4] with hex-1-ene. The reaction provided the very first
n
N N
O
N N
O
RR
R = H (Bn2nBU[n]),
o-NO2, p-NO2,
p-COOMe, p-COOH
n = 4, 6
- 19 -
asymmetrically substituted bambusuril containing seven allyl groups and one hept-2-
enyl group.
Figure 7: Bambusurils bearing olefinic substituents.[3]
2.1.2.4 Semithiobambusurils
The first bambusuril derivatives with carbonyl oxygens partially substituted for
another heteroatom were presented by Reany et al.[11]
The semithiobambusurils –
Me8semithioBU[4] and Me12semithioBU[6] (Figure 8) – were prepared adopting
Heck's[3]
protocol. Not surprisingly, the cyclic hexamer showed high affinity to anions
just like the all-oxygen analogs. However, the selectivity was rather poor. The
association constants of the halide complexes in DMSO-d6 were all in the range of only
one order of magnitude.
Figure 8: Semithiobambusurils.[11]
Semithiobambusurils carry a structural motive – the thiocarbonyl moiety –
which endows them with affinity to thiophilic metals.[11]
Me8semithioBU[4] formed 1:4
complexes with PdII and Hg
II salts in chloroform and dimethyl sulfoxide. Single crystals
of a coordination polymer of the macrocycle with HgCl2 were obtained (Figure 9).
n
N N
O
N NR R
O 3
N N
O
CH2+
N N
O
N N
O
N N
O
R = ; n = 4, 6
((Allyl)2nBU[n])
R = ; n = 4
n
N N
O
N N
S
n = 4, 6
Me2nsemithioBU[n]
- 20 -
Figure 9: Crystal structure of the Me8semithioBU[4]∙HgCl2 coordination
polymer.[11]
Color code: structures in the capped sticks model –
Me8semithioBU[4]; structures in the spacefill model – Hg (grey) and Cl
(green).
2.1.3 Applications of Bambusurils
The unique binding properties of bambus[6]urils make these macrocycles
potential anion sensors, sorbents or ion-exchange agents. If properly designed,
bambusurils could also function as drug delivery systems, selective contrast or imaging
agents. However, this family of macrocyclic molecules is very young and thus on the
beginning of its path to being explored. No bambusuril is even commercially available
yet. Nevertheless, we can track two papers where first attempts to apply bambusurils are
made.[14,15]
Notably, the first such paper deals with detecting and removing residual water
from chloroform.[14]
Bn12BU[6] was shown to form 1:2 complexes with benzoates and
tosylates which are mediated by a single water molecule (Figure 10). The water
molecule occupied the center of the macrocycle's cavity while the anions closed its
portals from both sides. In the absence of water, the same host and guests formed only
weakly bound 1:1 complexes. The water-mediated 2:1 complexes with
tetrabutylammonium benzoate and p-toluenesulfonate were insoluble in chloroform and
thus crystallized instantly from the solvent in the presence of water. On the other hand,
no crystallization was observed in the absence of water. Therefore, residual water could
be removed this way from predried CHCl3. A suitably substituted guest –
tetrabutylammonium 3,5-bis(benzyloxy)benzoate – formed a soluble water-mediated
1:2 complex with Bn12BU[6] in chloroform. This system could enable the monitoring of
water content in CDCl3 by 1H NMR spectroscopy.
- 21 -
Figure 10: Crystal structure of the water-mediated complex of Bn12BU[6]
with two benzoate anions.[14]
Recently, two bambusuril derivatives were reported to function as sensors for
qualitative and quantitative analysis of complex mixtures of inorganic anions in aqueous
media.[15]
Complexes of Bn12BU[6] with nine different anions could be distinguished by
1H NMR. The macrocycle's methine protons provided a unique signal for each of the
anions in a D2O–DMSO-d6 mixture (Figure 11). Moreover, quantitative analysis of a
mixture of up to five anions in submillimolar concentrations was possible in the
presence of an internal standard. The carboxybenzyl-substituted bambusuril could be
used in a similar manner for quantification of anion mixtures with concentrations as low
as 0.1 μM in buffered D2O.
Figure 11: 1H NMR (500 MHz, 5 % D2O–DMSO-d6) of a solution of
Bn12BU[6] and nine inorganic anions. The methine protons of each
BU∙anion complex are represented by a separate signal. The chemical shifts
of the respective complexes are given in parentheses.[15]
- 22 -
2.1.4 Macrocycles Related to Bambusurils
2.1.4.1 Cucurbit[n]urils
Bambus[n]urils were by far not the first glycoluril based macrocyclic
compounds. As early as in 1905, Behrend et al.[16]
prepared a condensate of glycoluril
and formaldehyde which later turned out to be a cyclic oligomer.[17]
The compound was
named cucurbit[n]uril (CB[n]) due to its resemblance to the pumpkin, a member of the
cucurbitaceae family of plants (Figure 12a, for reviews see ref.[18–20]
).
The glycoluril units in CB[n]s are connected by two rows of methylene
bridges.[17]
This arrangement makes the macrocycles very rigid, as the glycoluril units
have no rotational degrees of freedom within the molecule. Until today, various CB[n]s
(n = 5, 6, 7, 8, 10, 14)[21–24]
as well as numerous derivatives have been prepared.[25–28]
The macrocyclic molecules are suitable for binding cationic and neutral guests with
very high association constants (up to 1017
M-1
)[29]
but suffer from low solubility in both
water and organic solvents. For many years, CB[n]s have been showing a great potential
for various applications including catalysis,[30]
selective sensing,[31]
drug delivery[32]
and
constructing supramolecular machines.[33,34]
Figure 12: a) Cucurbit[6]uril; b) hemicucurbit[6]uril; the number 6 denotes
the number of monomer units of the cyclic oligomers.[1]
2.1.4.2 Hemicucurbit[n]urils
Inspired by the chemistry of cucurbiturils, Miyahara et al.[35]
used ethyleneurea –
formally a "half glycoluril" – to prepare a novel family of macrocyclic host molecules –
- 23 -
hemicucurbit[n]urils (HmCB[n]s, n = 6 or 12). These compounds had very different
properties than CB[n]s. They were significantly more soluble in organic solvents
compared to CB[n]s and adopted an 'alternate' conformation in the solid phase
(Figure 12b). The cyclic hexamer preferentially formed supramolecular complexes with
certain anions,[36]
although interactions with cations were reported as well.[37]
Furthermore, HmCB[6] was shown to function as a supramolecular catalyst.[38,39]
Since their discovery, several HmCB[n] derivatives have been prepared.[40–43]
In
fact, even bambus[n]urils can be considered derivatives of hemicucurbituril. Both
families of macrocycles share a structural motif, adopt the same conformation and have
similar supramolecular properties, especially affinity to anionic guests.
2.2 Supramolecular Chemistry of Phosphates
Phosphates and phosphorylated compounds are amongst the most abundant
anionic species in living organisms. All nucleic acids are essentially phosphodiesters[44]
and as much as 30 % of all proteins undergo phosphorylation as a posttranslational
modification.[45,46]
Even small organic molecules are phosphorylated in living cells as a
standard process in metabolic and signalling pathways.[47]
Apart from inorganic
phosphate and its esters, phosphoric anhydrides (e.g. 1,3-bisphosphoglycerate or ATP)
and imides (e.g. creatine phosphate) are very common among biomolecules.
Organic phosphates are present in our world in many shapes and sizes. Organic
and supramolecular chemists have been seeking to build very selective receptors with
high affinity to phosphorylated compounds for more than 100 years (for reviews, see
ref.[48–53]
). However, phosphates and their esters are very challenging supramolecular
guests for mainly two reasons.
Firstly, the hydration free energies (ΔGhyd) of inorganic phosphate species are
highly negative (-465, -1789 and -2765 kJ mol-1
for H2PO4–, HPO4
2– and PO4
3–
respectively) compared to other anions (e.g. for ClO4- ΔGhyd = -205 kJ mol
-1).
[54,55]
Therefore, polar solvents compete strongly with all possible receptors for these specific
guests.
- 24 -
Secondly, phosphate binding is highly dependent on the protonation state of the
anion (Scheme 3).[56]
Dihydrogen and monohydrogen phosphates comprise both
hydrogen bond donor and hydrogen bond acceptor groups. Therefore, simple receptors
with one type of convergent binding sites cannot be selective for these species.
Moreover, protonated phosphates were reported to form hydrogen bonded aggregates in
less polar solvents.[57,58]
It is worth noting that in water at neutral pH, H2PO4- and
HPO42-
coexist in almost equimolar ratio. Therefore, efficient binding in these
conditions requires receptors capable of encapsulating both species or shifting the
monohydrogen–dihydrogen phosphate equilibrium.
Scheme 3: Protolytic equilibria of phosphoric acid and its esters;
a) inorganic phosphate;[56]
b) monoalkyl phosphates; c) dialkyl phosphates
(the pK values of phosphate esters are typical values for small organic
derivatives taken from ref.[59]
).
Hundreds of various phosphate receptors have been synthesized in the history of
supramolecular chemistry. It is impossible to review all of them in this thesis.
Therefore, the most widely used binding motifs are discussed in the following
subchapters and several specific examples are given.
2.2.1 Charged Receptors
Cationic hosts tend to be most efficient for anion binding in general due to
strong electrostatic interactions.[60]
Receptors of phosphates are no exception. The first
H3PO4 H2PO4- HPO4
2- PO43-
pK2 = 7.20 pK3 = 10.9
a)
b) O
OH
OH
O
P
R
O
OH
O-
O
P
R
O
O-
O-
O
P
R
c) O
OH
O
O
P
R
R
O
O-
O
O
P
R
R
pK1 = 0.8 to 3.6 pK2 = 3.4 to 7.1
pK = 1.3 to 1.7
pK1 = 2.12
- 25 -
organic phosphate binding molecules were based on polyammonium species. The linear
spermine (Figure 13a) and spermidine molecules (Figure 13b), and cyclic azacrowns
(Figure 13c) showed moderate to high affinity to pyrophosphate and nucleoside
phosphates in near-neutral aqueous solutions (Kass up to 103 M
-1 for acyclic and 10
6 M
-1
for cyclic receptors).[61–64]
The major drawback of these receptors is their strong
dependence on pH. The protonation degree of polyamines, as well as their affinity to
anions is strongly dependent on the acidity of the solution. Even in slightly basic
conditions, these host molecules convert to the free amine form and lose their anion
binding properties completely.
Figure 13: Polyammonium receptors in their free amine forms;
a) spermine; b) spermidine; c) example of an azacrown macrocycle –
[18]aneN6.
Polyammonium receptors may be used in the full range of pH if the possibility
to undergo deprotonation is eliminated. Full alkylation of nitrogen atoms in quaternary
ammonium salts gives these molecules an inherent positive charge even under basic
conditions. However, a major disadvantage of these receptors is the lack of hydrogen
bond donor groups.[65]
An example of such a receptor is given in Figure 14.[66]
The
macrocycle contains four quaternary anilinium moieties. The structure is able to bind
nucleoside phosphates by a combination of electrostatic interactions, hydrophobic
interactions and π-π stacking.[67]
NH2 NHNH NH2
NH2 NHNH2
NH
NH
N
H NH
NH
N
H
a)
b) c)
- 26 -
Figure 14: An example of a quaternary ammonium receptor for organic
phosphates.[66]
Another typical charged group used in the design of phosphate receptors is the
guanidinium moiety (Figure 15a). The cation provides electrostatic stabilization, it is a
donor of two parallel hydrogen bonds and is significantly less acidic than the
ammonium group (pKa = 13.6 compared to 9.2 of NH4+).
[68] Many receptors bearing this
group have been synthesized. Figure 15b presents an example of a macrocycle
containing guanidinium moieties which selectively binds inorganic phosphate in
water.[69]
Figure 15: a) Guanidinium; b) a guanidinium based macrocycle.[69]
More recently, extensive research has been done on the binding properties of
imidazolium-group-bearing hosts. This five-membered heterocyclic cation can bind
anions by electrostatic interactions and C–H X– hydrogen bonds (X
– = anion).
[70] The
groups of Kim and Yoon[71,72]
prepared numbers of linear and macrocyclic imidazolium
receptors with fluorescent properties (examples of such receptors are given in
N+
N+
N+
N+
NH NH
NH
N
H
N
H
NH
NH2+
NH2+
NH2+
NH2NH2
NH2+
a) b)
- 27 -
Figure 16). The compounds enabled selective sensing of various phosphates by
fluorimetry.
Figure 16: Imidazolium receptors; a) an imidazolium macrocycle with high
selectivity for dihydrogen phosphate in acetonitrile;[71]
b) a combined
imidazolium/ammonium sensor for guanosine triphosphate (GTP) in
aqueous solution.[72]
Many other cationic groups were reported to interact with phosphates and their
organic derivatives. Receptors containing pyridinium, 1,2,3-triazolium and other
moieties were prepared.[73,74]
However, their detailed description exceeds the scope of
this thesis.
2.2.2 Hydrogen Bond Donor Receptors
Even receptors lacking intrinsic positive charge may show high affinity to
anions. Careful selection and arrangement of hydrogen-bond-donating groups can
provide host molecules with exceptional binding properties.[60]
The amide group is the
most widely used one in receptor design.[75]
Macrocyclic amides proved to be
particularly potent receptors of inorganic phosphate.[76,77]
Very stable complexes of
dihydrogen phosphate with macrocyclic amides having 2:1 stoichiometry were reported
(Figure 17).[78]
These assemblies were stabilized by phosphate–phosphate hydrogen
bonding, as already mentioned in Chapter 2.2.
N N+
N+ N
N
N+
N+
N
N+
N+
a) b)
- 28 -
Figure 17: An amide receptor for inorganic phosphate; a) structural formula; b) crystal
structure of the 1:2 complex with H2PO4– (taken from ref.
[52], originally published in
ref.[78]
).
1,5-Diphenylglycoluril was used by Kang and coworkers[79,80]
as the basis for
the construction of phosphate binding clefts (Figure 18). Although these receptors were
not very selective, they bound dihydrogen phosphate in acetonitrile with binding
constants of up to 7.5 104 M
-1. Moreover, the receptor in Fig. 18a could easily be used
as a UV/VIS sensor due to the nitrophenyl moiety. In a similar manner, the naphthyl
groups of the glycoluril host in Fig. 18b enabled the fluorimetric detection of anions.
- 29 -
Figure 18: Glycoluril-based clefts; a) UV/VIS active anion receptor;[79]
b) fluorescent anion receptor.[80]
Urea or thiourea are common binding motifs in a large group of phosphate
receptors. These specific amides can form a pair of parallel hydrogen bonds in a similar
manner as the guanidinium moiety.[81]
A wide range of both cyclic and acyclic
(thio)urea derived receptors have been prepared. An interesting family of tripodal
receptors was developed by Alcázar and coworkers[82]
(Figure 19). The
tris(2-aminoethyl)amine based hosts bound PO43–
with Kass of up to 1.1 104 M
-1 in
DMSO (Fig. 19a) and 1.0 102 M
-1 in water (Fig. 19c).
Figure 19: Tripodal phosphate receptors.[82]
N N
N N
O
O
NHO
NO2
NHO
NO2
PhPh
a)
N N
N N
O
O
NHO
NHO
PhPh
NHO
NHO
b)
N
NH NH
NHNHR R
X X
NH
NH
R
X
a) X = O; R = Ph
b) X = S; R = Ph
c) X = O; R =
d) X = O; R = C6F5
e) X = O; R =
COO-
NO2
- 30 -
Nucleoside-phosphate-binding dendrimers bearing urea groups were synthesized
by Vögtle et al.[83]
Extraction of adenosine monophopshate, diphosphate and
triphosphate (AMP, ADP and ATP) from neutral or slightly acidic water into
chloroform was achieved with the 2nd
and 3rd
generation dendrimers (Figure 20).
Figure 20: Phosphate-binding dendrimer bearing urea groups (2nd
generation depicted); R = hexyl, octyl or dodecyl.[83]
Receptors based on pyrrole and related heterocycles as hydrogen bond donors
have been widely researched as well.[84]
Jeong et al.[85]
prepared phosphate-binding
macrocycles bearing two biindole moieties (Figure 21). The rigid planar cycle was able
to bind dihydrogen phosphate with Kass of up to 106 M
-1 in acetonitrile. However, other
anions were bound with similar affinity, thus the selectivity for phosphates was rather
poor.
Figure 21: Example of a biindole macrocyclic anion receptor.[85]
NN
N
N
NH
NH
NH
R
O
O
NHR
NH
O
NHR
NHO
NHR
N
N
NH
NH
NH
R
O
O
NHR
NH
O
NHR
NH O
NHR
N
H
N
H
N
H
N
H
- 31 -
Several structural motifs were combined to produce a selective phosphate
receptor by Kataev and coworkers.[86]
Indole, amide and guanidinium groups work in
concert to selectively bind dihydrogen phosphate (Kass = 106 M
-1) over pyrophosphate
(Kass = 103 M
-1) and other anions in MeOH–water mixtures (Figure 22).
Figure 22: Kataev's selective phosphate receptor.[86]
2.2.3 Receptors Containing Metal Ions
Synthetic chemists often seek inspiration in biological systems. Numerous
proteins use metal cations to bind organic and inorganic phosphates.[87–89]
Consequently,
artificial phosphate receptors containing transition metals emerged as well. Most of
these receptors directly use the Lewis-acidic properties of metals to bind phosphate
anions. It is then a matter of definition whether these complexes held together by
coordination covalent bonds are considered supramolecular structures.
An interesting combination of metal coordination and hydrogen bonding was
used for selective sensing of dimethyl phosphate by Kim et al.[90]
An octahedral CoIII
complex selectively bound fluoride and dimethyl phosphate anions over other halides
with the aid of extra hydrogen bonding from the aminopicolinate ligand (Figure 23).
NHNH
NHOO
NH
NH NHNH2+
NH2 NH2
NH2+
- 32 -
Figure 23: CoIII
receptor binding a dimethyl phosphate anion; a) structural
formula; b) crystal structure (taken from ref.[52]
, originally published in
ref.[90]
).
Azacrown macrocycles (already mentioned in Chapter 2.2.1 as charged
receptors) gave birth to a large family of metallic phosphate-binding receptors. The
cyclic polyamines form mono- and dinuclear complexes with transition metals capable
of binding phosphates and their esters.[91–98]
As an example, Ren and coworkers[99]
prepared a dicopper-[18]aneN6 complex which can selectively bind phosphate
monoesters (Figure 24).
Figure 24: Dicopper-[18]aneN6; a) structural formula; b) crystal structure
of the complex with two glycerol-2-phosphate anions.[99]
In anion receptor chemistry, transition metals do not necessarily have to play the
role of electron pair acceptors. They can be used as non-interacting reporters of the
binding process. Ferrocene is widely used as an electrochemical probe. Sessler et
- 33 -
al.[100,101]
demonstrated the change of electrochemical behavior of ansa-ferrocene
receptors (Figure 25a) upon complexation of dihydrogen phosphate.
Another common reporter group is the fluorescent [Ru(bipy)3]2+
complex. Beer
and coworkers[102]
synthesized a number of receptors bearing the Ru–bipy motif (an
example is given in Figure 25b) with a 10-fold selectivity to dihydrogen phosphate over
chloride in DMSO. The formation of the receptor–anion complexes could be followed
by fluorimetry, as well as cyclic voltammetry and 1H NMR.
Figure 25: Phosphate receptors with metallic reporter groups; a) an
ansa-ferrocene receptor (X = CH2OCH2 or (CH2OCH2)2);[101]
b) a Ru–bipy
receptor.[102]
2.2.4 Other Receptors
Many other phosphate receptors have been developed which cannot be included
in any of the previously discussed categories. Especially, receptors based on the
"traditional" macrocycles, like calixarenes and cyclodextrines fall within this group.
The calix[6]arene scaffold was utilized by Gupta et al.[103]
for the preparation of
a monohydrogen phosphate ion-selective electrode. The macrocycle derivatized with
carbamoylmethyl groups (Figure 26a) was highly selective for HPO42–
and had a broad
working concentration range (10-5
to 10-1
M).
Fe
N
H
N
H
N
H
O
O
N
H
X
a) b)
Ru2+
N
N
N
N
N
N
N
H
N
H
O
O
O
O
- 34 -
An example of cyclodextrin-based phosphate receptors is the work of Eliseev
and Schneider.[104,105]
Ammonium derived cyclodextrins (Figure 26b) were equipped
with a positively charged binding site while maintaining a hydrophobic cavity. These
properties predetermined the macrocycles for selective binding of nucleoside
phosphates.
Figure 26: Macrocyclic receptors for phosphates; a) a calix[6]arene
derivative used for the preparation of an ion-selective electrode;[103]
b) a
β-cyclodextrin derivative for selective binding of nucleoside phosphates.[104]
O
H
HH
H
OH
H OH
O
NH2+
7O
O NH2
CH266
7
a) b)
