Synthesis and pH-dependent self-assembly of semifluorinated calix[4]arenes

Article abstract of DOI:10.1016/j.tet.2007.04.030

A new series of highly fluorinated calix[4]arene-based amphiphilic molecules was designed and synthesized. Using the calix[4]arene scaffold, four perfluorinated hyper-hydrophobic groups and four water solubilizing chains were introduced in the same molecule and also segregated in space following the scaffold directionality. Upon solubilization in aqueous solutions, these amphiphilic molecules form microscopic fluorous domains that drive the formation of various self-assembly patterns. We found that the self-assembly of these semifluorinated calix[4]arenes is dependent on external stimuli, such as changes in the polarity of the solvent or pH. As a consequence, by changing the pH of?the solutions, it is possible to shift the aggregation pattern of these molecules, by a regular change either in the shape or in the size of the?initially formed ordered aggregates. These are examples of the variety of structures and possibilities in nano-engineering offered by fluorous-phase driven molecular recognition.

Full text of DOI:10.1016/j.tet.2007.04.030

Tetrahedron 63 (2007) 5539–5547
Synthesis and pH-dependent self-assembly of semifluorinated
calix[4]arenes
Oana M. Martina and Sandro Mecozzia,b,
*
aSchool of Pharmacy, 777 Highland Avenue, University of Wisconsin, Madison, WI 53705, United States
bDepartment of Chemistry, 777 Highland Avenue, University of Wisconsin, Madison, WI 53705, United States
Received 23 September 2006; revised 25 March 2007; accepted 9 April 2007
Available online 19 April 2007
Abstract—A new series of highly fluorinated calix[4]arene-based amphiphilic molecules was designed and synthesized. Using the calix[4]arene
scaffold, four perfluorinated hyper-hydrophobic groups and four water solubilizing chains were introduced in the same molecule and also
segregated in space following the scaffold directionality. Upon solubilization in aqueous solutions, these amphiphilic molecules form micro-
scopic fluorous domains that drive the formation of various self-assembly patterns. We found that the self-assembly of these semifluorinated
calix[4]arenes is dependent on external stimuli, such as changes in the polarity of the solvent or pH. As a consequence, by changing the pH
of the solutions, it is possible to shift the aggregation pattern of these molecules, by a regular change either in the shape or in the size of
the initially formed ordered aggregates. These are examples of the variety of structures and possibilities in nano-engineering offered by
fluorous-phase driven molecular recognition.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
Fundamental knowledge about the nature and the origin of
the fluorous phase can be gained by assessing the influence
of multi-chain fluorinated amphiphiles on the resulting
aggregate architecture. The unique combination of hydro-
phobicity and lipophobicity of fluorinated chains, together
with the versatility of calix[4]arenes as molecular scaffolds
can induce a novel structural compartmentalization and
generate amphiphilic materials with new properties. We
explored the application of the fluorophobic effect in mole-
cular recognition by using macrocyclic molecules function-
alized with perfluoroalkyl chains.5 We designed a series of
semifluorinated calix[4]arenes that self-assemble in organic
and aqueous environments, forming aggregates with different
morphologies. The self-assembly process is driven by the
fluorophobic effect.6 The three-dimensional, concave, and
relatively rigid structure of the calixarenes, coupled with
the well-developed synthetic methodologies, made these
compounds attractive platforms for development of new
supramolecular entities.7 Avariety of self-assembled systems
based on the versatile calixarene scaffold have been studied,
from dimeric capsules8, to mono- and multilayers9.
Fluorination can drastically affect the physical–chemical
properties of a molecule. While even one fluorine atom
can change the hydrogen bonding properties and the lipophi-
licity of a molecule, the introduction of several fluorine
atoms tends to generate a new behavior, performance, and
potential for separation.1 Perfluorination of organic mole-
cules can generate a fluorous phase, which is at the origin
of the unusual behavior of heavily fluorinated molecules
and polymers.2 Fluoro-organic compounds have interesting
properties and are increasingly considered as potentially
useful in various areas of life sciences, particularly in phar-
maceutical research. The use of fluorinated building blocks
in peptide, protein, and RNA design can lead to novel bio-
molecules with improved stability. Fluorinated colloidal
systems are being investigated for the delivery of drugs,
including oxygen, prodrugs, genes, vaccines, and other
immunoactive agents, contrast media, and other materials.3
Due to their unique characteristics, fluorinated amphiphiles
can self-assemble in various media into stable fluorinated
colloids, generating organized nanometer-size fluorous
phases.4 Fluorinated surfactants display properties and per-
formances that cannot be attained with standard surfactants.
They are more surface-active, displaying critical micelle
concentration (CMC) values that are usually two orders of
magnitude lower than those of the hydrocarbon analogues.
By combining the properties of amphiphiles with those of
thermotropic liquid crystals, an interesting combination
between order and mobility was achieved, which provides con-
trol of the aggregate architectures. The first member of this
series, the calix[4]arene tetraamine 1, was found to self-
assemble in regularly ordered patterns in solvents as different
as water, chloroform, and perfluorohexane.10 This molecule
was also found to exhibit glassy liquid crystalline behavior at
room temperature, therefore it proved that liquid crystalline
* Corresponding author. Tel.: +1 608 262 7810; fax: +1 608 262 5345;
e-mail: smecozzi@wisc.edu
0040–4020/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2007.04.030

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O. M. Martin, S. Mecozzi / Tetrahedron 63 (2007) 5539–5547
CF3
(CF2)7
water solubilizing groups at the lower rim of the calix[4]arene
CF3
(CF2)7
(CH2)3
CF3
(CF2)7
CF3
(CF2)7
scaffold. The hydrophilic part of the molecule is composed
of a oligo(ethylene oxide) linker and a variously charged
group. By grafting four highly fluorinated chains on the
calix[4]arene scaffold, the amphiphilic character of these
molecules is highly amplified. Formation of a fluorous phase
in aqueous environments induces the self-assembly of these
highly fluorinated molecules. Intercalation of a short alkyl
chain between the calixarene scaffold and the rigid fluorous
chains imparts increased flexibility to the fluorophilic
moiety of the molecule, making possible the formation of
several alternative self-assembled structures.
(CH2)3
(CH2)3
(CH2)3
OR OR
OR
O
OR
H
N
NH2
1
2
3
R=
R=
R=
3
O
H
OH
N
O
O
O
P
OH
3
O
O
-
-
CO2
CO2
H
N
N
The modular design of the molecules allowed the synthesis
of semifluorinated calix[4]arenes with different charged
groups, such as free amines, phosphates, and aminodiace-
tates. The amine functionalities allow up to four positive
charges on the molecules, while the phosphates can allow
up to eight negative charges. The aminodiacetate group
was chosen to study the effect of manipulating the number
of charges by pH control. In this case, positively charged,
negatively charged or zwitterionic molecules can be ob-
tained, depending on the pH of the medium. We found that
the resulting self-assembled structures are dependent on
the pH of the solution as the number and nature of the
charges on the external surface of the aggregates dictate their
final shape and size.
3
O
Figure 1. Chemical structure of the newly synthesized semifluorinated
amphiphilic calix[4]arenes.
materials based on fluorinated calix[4]arenes can be de-
signed by combining the bulky and stiff perfluorooctyl
chains with the rigid calix[4]arene core.
This work has now been extended to include amphiphilic
semifluorinated calix[4]arenes that are characterized by
either a large number of negative charges to ensure higher
aqueous solubility or functionalized with groups that are
responsive to the solution pH. The new compounds that we
have designed and synthesized are the calix[4]arene tetra-
phosphate 2 and the calix[4]arene octacarboxylate 3. These
compounds were designed to improve the aqueous solubility
of these materials and to study the influence of pH on the
self-assembly patterns. The solubility of calixarenes in water
is very limited and in order to become water soluble, these
compounds usually need to be functionalized with either
groups containing charged functionalities or with neutral
but highly hydrophilic moieties (Fig. 1).11
The synthesis of the perfluorooctyl functionalized calix[4]-
arene and calix[4]arene tetraamine 1 were reported else-
where.5 The perfluorooctyl chains at the upper rim were
introduced by a radical addition reaction to p-allyl-calix[4]-
arene with perfluorooctyl iodide, followed by hydrodeiodi-
nation. After installing the four carboxylate groups at the
lower rim, the calix[4]arene tetracarboxylic acid was cou-
pled with the corresponding ethylene glycol amine using
standard peptide chemistry. A similar approach was used
for the synthesis of the calix[4]arene tetraphosphate 2
and calix[4]arene octacarboxylate 3. The corresponding
oligo(ethylene oxide) amines were synthesized starting
from known intermediates, as shown in Schemes 1 and 2.
The 9-fluorenylmethyl (Fmoc) protecting group12 was chosen,
in light of its orthogonality with the benzylphosphate
group13. The dibenzylphosphate group was introduced
2. Results and discussion
2.1. Synthesis
Calix[4]arene-based amphiphiles 2 and 3 were synthesized
by attaching semifluorinated chains at the upper rim, and
PPh3/H2O, THF
>99%
FmocCl
80%
HO
O
N3
HO
O
NH2
O
O
O
O
4
5
1) iPr2NP(OBn)2/5-Me-tetrazole
H
2) m-CPBA
92%
HO
O
N
O
O
O
O
6
O
P
OBn
H
N
BnO
O
O
O
O
O
O
7
Scheme 1. Synthesis of the benzylphosphate oligo(ethylene oxide) amine.

O. M. Martin, S. Mecozzi / Tetrahedron 63 (2007) 5539–5547
5541
BrCH2CO2Et
N3 K2CO3
97%
EtO2C
EtO2C
H2N
O
N
O
N3
O
O
O
O
8
9
EtO2C
EtO2C
PPh3/H2O, THF
77%
N
O
NH2
O
O
10
Scheme 2. Synthesis of the aminodiacetate-oligo(ethylene oxide) amine.
with dibenzyl diisopropyl phosphoramidite followed by
oxidation14, as shown in Scheme 1. The monoazide 4 was
converted to the monoamine 5 by a Staudinger reduction
with triphenylphosphine in quantitative yield.15 The mono-
amine was further protected with Fmoc (80% yield) and the
benzyl phosphate groups were introduced using diisopropyl
dibenzyl phosphoramidite and 5-methyltetrazole, followed
by m-CPBA oxidation with a 92% yield for two steps.
Staudinger reduction conditions17 to give 1-bis(ethoxy-
carbonylmethyl)amino-11-amino-3,6,9-trioxaundecane 10
in 75% overall yield.
Standard carbodiimide conditions with 1-[3-(dimethyl-
amino)propyl]-3-ethylcarbodiimide hydrochloride (EDCI)
and 1-hydroxybenzotriazole hydrate (HOBt) for amide
bond formation, were used for coupling tetraacid 11 with
the corresponding free amines (Scheme 3).
The aminodiacetate functionalized oligo(ethylene oxide)
amine was synthesized by treating amino-azide 8 with ethyl
bromoacetate.16 The resulting azide was subjected to the
Deprotection of both the phosphate and aminodiacetate
calix[4]arenes yielded the target amphiphilic semifluorinated
C8F17
C8F17
C8F17
C8F17
C8F17
C8F17
C8F17
C8F17
C8F17
C8F17
C8F17
C8F17
7
Piperidine
EDCI, HOBt
H2, Pd/C
EtOH
41%
O
62%
O
O
O
O
HN
O
O
O
O
O
HN
O
O
O
O
O
O
O
O
HO
O
O
O
O
O
NH
NH
HN
HN
O
HO
HN
HN
OH
OH
O
O
11
O
O
O
3
O
3
O
O
3
3
3
3
3
3
10
O
P
OH
HO
O
P
OBn
BnO
O
O
O
O
O
O
P
HO
HO
EDCI, HOBt 60%
O
O
HO
HO
BnO
BnO
HO
O
P
BnO
O
O
HO
P
BnO
P
BnO
O
P
P
O
O
BnO
2
12
C8F17
C8F17
C8F17
C8F17
C8F17
C8F17
C8F17
C8F17
NaOH, MeOH/H2O
55%
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
HN
O
NH
NH
HN
HN
O
HN
HN
NH
O
O
O
O
O
O
O
3
3
3
3
3
3
3
3
N
N
-
CO2 Na+
-
CO2Et
N
N
N
N
+Na-O2C
EtO2C
N
N
CO2 Na+
-
CO2 Na+
CO2Et
+Na-O2C
EtO2C
CO2Et
CO2 Na+
-
EtO2C
CO2Et
+Na-O2C
-
CO2Et
CO2 Na+
13
3
Scheme 3. Synthesis of the amphiphilic semifluorinated calix[4]arenes 2 and 3.

5542
O. M. Martin, S. Mecozzi / Tetrahedron 63 (2007) 5539–5547
calix[4]arenes 2 and 3. The Fmoc-dibenzylphosphate amine
7 was deprotected with piperidine in dimethylformamide
and then immediately added to the activated carboxylic
acid without isolation, in the presence of the coupling re-
agents. Finally, the dibenzylphosphate groups in the pro-
tected product 12 were cleaved by hydrogenolysis.18 The
ethyl ester groups in compound 13 were subjected to base
hydrolysis with sodium hydroxide to give the calix[4]arene
octacarboxylate 3.
manner for both the DLS and the TEM studies. The critical
aggregation concentrations were not determined because
even at the lowest possible concentrations detected by
DLS, the presence of non-aggregated molecules was not de-
tected. The solubility of the calix[4]arene octacarboxylate 3
does not change much with pH, while the solubility of the
tetraphosphate 2 decreases slightly going from pH 12 to
pH 4.1. This is correlated with a reduction in the number
of negative charges on the molecules from eight to four.
2.2. Self-assembly studies
The following buffer systems were prepared and used to dis-
solve the samples: hydrochloric acid buffer (pH 1.2), phta-
late buffer (pH 3), acetate buffer (pH 4.1), phosphate
buffers (pH 5.8, 7.4, and 12), and borate buffer (pH 8.6).
The viscosities and refractive indexes for each buffer were
measured separately and used as parameters for the DLS
measurements. The population distributions obtained for
the calix[4]arene tetraphosphate solutions are similar for
all the buffers that were examined. Three distributions
were determined for each solution. For example, at pH 4.1
three distributions were observed, centered at 24 nm (6%
of the aggregates), 98 nm (45%), and 265 nm (49%). By an-
alyzing the corresponding electron micrograph (Fig. 2), it
appears that small spherical micelles are formed, with sizes
of 15–20 nm, which further cluster together into larger ag-
gregates, which represent the other population distributions
in the DLS profiles. In basic buffers this negatively charged
calix[4]arene derivative self-assembles into spherical mi-
celles with an average diameter of 15–20 nm (Fig. 2a and
b). These micelles can further cluster into larger aggregates.
The self-assembly behavior of this new class of semifluori-
nated calix[4]arene amphiphiles was studied using a combi-
nation of dynamic light scattering (DLS) and transmission
electron microscopy (TEM). The two calix[4]arene deriva-
tives 2 and 3 were used to explore the influence of pH
changes on the aggregate architecture. Both these molecules
exhibit improved water solubility compared to the calix[4]-
arene tetraamine 1, which is a direct consequence of the
higher number of charges on each molecule. The tetraphos-
phate 2 has eight negative charges in basic buffers, but still
maintains four negative charges at pH 4.1, which allow for
its dissolution in the acetate buffer. This behavior is a conse-
quence of the pKa values of a phosphate group, which are
known to be 1.60 and 6.62 for ethyl phosphate.19 Solutions
of the above two calix[4]arene derivatives at different pHs
were prepared by sonication of a thin film of compound 2
or 3 in the corresponding aqueous buffer. All the solutions
had a concentration of 100 mM and were prepared in a similar
Figure 2. Transmission electron micrographs of calix[4]arene tetraphosphate [2]¼100 mM. (a) Borate buffer of pH 8.6 (the scale bar represents 50 nm). (b)
Borate buffer of pH 8.6 (the scale bar represents 100 nm). (c) Acetate buffer of pH 4.1 (the scale bar represents 100 nm).

O. M. Martin, S. Mecozzi / Tetrahedron 63 (2007) 5539–5547
5543
When the number of charges is decreased to four, at pH 4.1,
the aggregation behavior is less regular and the aggregates
do not have a constant size or shape, as observed in
Figure 2c.
values determined for alkyl-N-iminodiacetic acids, the pos-
sible charged states of the calix[4]arene octacarboxylate are
presented in Figure 3.
This pattern of acidic constants suggests that a polymer
structure with hydrogen bonds is maintained in the aggre-
gates and that such bonds can exist in aqueous systems if
they are supported by a strong and rigid backbone structure,
as for example, the bilayers of well-organized long interdig-
itating alkyl chains.20 This rigid structure also exists in the
calix[4]arene octacarboxylate 3 and is formed by the combi-
nation of the calix[4]arene core and the bulky perfluorooctyl
chains on the upper rim. The opposite part of the molecule,
however, is more flexible due to the presence of the hydrated
oligo(ethylene oxide) units, therefore this system is more
complex than the n-alkyl-N-iminodicarboxylates. Based on
the evidence that short strong hydrogen bonds form in the
aggregates of long chain n-alkyl-N-iminodiacetic acids,20 a
similar behavior can be envisioned for the calix[4]octacarb-
oxylate-based assemblies. The formation of these hydrogen
bonds could explain the fluctuation in the size of the micellar
aggregates with pH, as observed by TEM and DLS.
Most of the sample at pH 4.1 is assembled into bilayer struc-
tures and a small fraction is still forming micelles and other
small aggregates. Therefore, a decrease in the number of
negative charges on the molecules leads, as expected, to a
decreased ability to form spherical micelles for the calix[4]-
arene tetraphosphate 2. For the calix[4]arene octacarboxy-
late 3, the patterns are more complex and more difficult to
interpret due to the various charged states this molecule
can adopt. The acidic properties of monomeric alkyl-N-
iminodiacetic acids in water are known to be in the expected
ranges with pKa values of about 1.7, 2.3, and 10.3.20 How-
ever, n-octadecyl-N-iminodiacetic acid that is present as
aggregates in water, was found to display very acidic prop-
erties of the first proton and a substantially weakened acidity
of the second proton (pKa ¼5:5ꢀ7:5, depending on the
2
ionic strength) and pKa ¼9:5ꢀ10:5.20 Based on these pKa
3
TEM analysis (Fig. 4) suggests that the octacarboxylate 3
exhibits a similar behavior to the tetraphosphate 2, forming
small spherical micelles, which cluster together into larger
aggregates. Thesize of themajority of these micelleschanges
with the pH, from 17–20 nm at acidic pH (1.2, 3, and 4.1) to
40–60 nm at pH 8.6. This change in size can be correlated
with a change in the charged state of the molecules.
H
H
H
N+
N+
N+
N
-O2C
CO2
-O2C
CO2
-
-
-
HO2C
CO2H
HO2C
CO2
pH < 1.5
1.5 < pH < 5.5
5.5 < pH < 10
pH > 10
Figure 3. Various charged states of the iminodiacetic group in calix[4]arene
octacarboxylate 3.
Figure 4. Transmission electron micrographs of calix[4]arene octacarboxylate [3]¼100 mM, the scale bar represents 100 nm. (a) HCl buffer of pH 1.2. (b) Ace-
tate buffer of pH 4.1. (c) Borate buffer of pH 8.6. (d) Phosphate buffer of pH 12.

5544
O. M. Martin, S. Mecozzi / Tetrahedron 63 (2007) 5539–5547
With the increase in pH, the charged state of the molecule
changes from four positive charges (at pH 1.2) to a zwitter-
ionic molecule with overall neutral charge (at pH 3 and 4.1),
to a total of four negative charges at pH 7.4 and 8.6. This
determines an increase in the size of the micelles that are
formed, probably due to the decreased ionic repulsion or
an increased ionic attraction between the positive and nega-
tive charges of adjacent similarly charged functional groups.
This explanation is consistent with the pKa values published
for the long chain n-alkyl-N-iminodiacetic acids. The second
carboxylic acid proton participates in the hydrogen bonds
and, as a consequence of this interaction, its pKa is five times
less than the analogue monomers with short alkyl chains
aggregates were consistently present in all the buffers:
bilayers and micelles. The bilayers seem to remain un-
changed and are more abundant at extremely acidic and
extremely basic pHs, compared to the more neutral samples.
The aggregates are held together by the fluorophobic effect
and also by hydrogen bonds within the surfaces of the aggre-
gates containing aminodiacetate groups. There seems to be
a fine balance between the hydration of the aggregate and
the formation of the fluorous phase with the perfluorooctyl
chains. Due to the unusual strength of fluorophobic effect
in polar environments, the hydration of the aggregate sur-
faces is not sufficiently strong to break these assemblies
into monomers at any pH.
(Fig. 3).20 Furthermore, the pKa value was found to be de-
2
pendent on the ionic strength of the medium with the acidity
increasing with increasing ionic strength. The acidic pro-
perties of the ammonium group did not seem to be much
influenced by the aggregation behavior of the n-alkyl-N-
iminodiacetic acids.20
By changing the pH of the solutions, it is possible to shift the
aggregation pattern of these molecules, for example, to de-
termine an increase in the size of the micelles or to change
their shape. These semifluorinated calix[4]arene materials
respond to external stimuli, such as changes in the polarity
of the solvent or pH. Therefore it is conceivable that solvent
dependence of fluorous-phase driven self-assembly could be
used in sensing devices and as a molecular tool to organize
complex self-assembling structures. In conclusion, signifi-
cant changes in the aggregate architecture with pH were
observed for the two amphiphilic calix[4]arene derivatives
2 and 3. Although these changes are not kinetically very
fast and there is not a complete shift from one aggregation
form to another, the properties of these molecules represent
an important advance toward the design of smart nano-
materials that can respond to external stimuli.
A very important change in the morphology of the calix[4]-
arene octacarboxylate aggregates is the appearance of
cylindrical micelles in a basic environment. At pH 12, these
appear to be the majority of the smaller aggregates (Fig. 4d).
In phosphate buffer at pH 12, a minor fraction of the sample
is still self-assembled into spherical micelles and bilayers,
but the main components of the mixture are the cylindrical
micelles. Even at pH 8.6 there are a few cylindrically shaped
aggregates present, meaning that the change is slow, starts at
pH 8.6 and is not completed at pH 12. The overall influence
of the pH is much stronger for the calix[4]arene octacarb-
oxylate than for the other derivatives, due to the various
charged states of this molecule.
4. Experimental section
4.1. General
The following trends were observed for the calix[4]arene
octacarboxylate 3: (1) the micelle size increases with
increasing pH, (2) the shape of the micelles changes from
spherical to cylindrical with increasing pH, and (3) bilayer
formation is favored by extreme acidic and extreme basic
pHs and less favored in a more neutral environment.
4.1.1. 1-(Amino)-11-hydroxy-3,6,9-trioxaundecane, 5. A
solution of 1-azido-11-hydroxy-3,6,9-trioxaundecane (5 g,
22.83 mmol) in 125 mL anhydrous tetrahydrofuran under
an argon flow was cooled to 0 ꢁC. Triphenylphosphine
(6.59 g, 25.11 mmol) was added and the mixture was
allowed to slowly warm to room temperature, after which
was stirred for 2 d. A small volume of water (1 mL) was
added to hydrolyze the intermediate phosphorus adduct. The
reaction mixture was stirred at room temperature overnight
and then at 40 ꢁC for 6 h. After diluting with water, the sol-
vent was removed in vacuo and 0.2 N HCl was added until
pH 3 was reached. The suspension was washed three times
with toluene and the aqueous solution was concentrated
down to a colorless gum. Purification by flash chromato-
graphy (gradient from 0 to 20% methanol in dichloro-
methane) yielded 5 g (95%) product as a colorless oil. MS
(ESI) m/z [M+H] calcd for C8H19NO4 194.1392, found
194.1390. 1H NMR (CDCl3) d: 3.20–3.30 (br s, 2H), 3.6–4.0
(m, 12H), 4.38–4.41 (br s, 2H), 8.15–8.25 (br s, 2H). 13C
NMR (CDCl3) d: 40.1, 61.1, 67.1, 69.8, 70.0, 70.3, 70.4, 72.5.
3. Conclusions
Newsemifluorinatedamphiphilescomposedofacalix[4]arene
scaffold functionalized with linear fluorinated chains at
the upper rim and hydrophilic groups at the lower rim
were synthesized. Using the unique calix[4]arene scaffold,
four fluorophilic (hyper-hydrophobic) groups and four water
solubilizing chains can be introduced in the same molecule
and also segregated in space by the scaffold directionality.
The self-assembly properties of these novel semifluorinated
calix[4]arenes were studied in solvents of varying polarity
and different aqueous buffers. These molecules form micro-
scopic fluorous domains that drive the formation of polarity
and pH-dependent self-assembly patterns. These are exam-
ples of the variety of structures and possibilities offered by
fluorous-phase driven recognition.
4.1.2. 1-(Fluorenylmethoxycarbonylamino)-11-hydroxy-
3,6,9-trioxaundecane, 6. To a solution of 1-amino-11-hy-
droxy-3,6,9-trioxaundecane (2.6 g, 11.3 mmol) in dioxane,
18 mL 10% aqueous sodium bicarbonate was added, fol-
lowed by cooling to 0 ꢁC. A solution of 3.3 g (12.46 mmol)
FmocCl in dioxane was slowly added, followed by stirring
The calix[4]arene octacarboxylate 3 and calix[4]arene
tetraphosphate 2 exhibit increased water solubility than
calix[4]arene tetraammonium 1 and form a variety of aggre-
gates depending on the pH of the medium. Two types of

O. M. Martin, S. Mecozzi / Tetrahedron 63 (2007) 5539–5547
5545
at 0 ꢁC for 4 h and room temperature overnight. The reaction
mixture was diluted with water and then extracted with di-
chloromethane. The Fmoc-protected amine was obtained
as a yellow oil (4.1 g, 87% yield) after purification by flash
chromatography using ethyl acetate/petroleum ether 2:1,
followed by ethyl acetate. MALDI-FTMS: [M+Na]+ calcd
for C23H29NO6Na 438.20, found 438.188. 1H NMR
(CDCl3) d: 3.25–3.4 (m, 2H), 3.45–3.75 (m, 14H), 4.25 (t,
1H, J¼6.8 Hz), 4.45 (d, 2H, J¼6.8 Hz), 6.25 (br s, 1H),
7.25–7.35 (m, 2H), 7.38–7.42 (m, 2H), 7.63 (d, 2H,
J¼7.6 Hz), 7.75 (d, 2H, J¼7.6 Hz). 13C NMR (CDCl3) d:
41.1, 47.6, 61.7, 66.6, 70.3, 70.6, 70.8, 72.8, 120.1, 125.3,
127.2, 127.8, 141.5, 144.3, 157.0.
4.1.5. 1-Bis(ethoxycarbonylmethyl)amino-11-amino-
3,6,9-trioxaundecane, 10. A solution of 0.42 g (1.06 mmol)
1-bis(ethoxycarbonylmethyl)amino-11-azido-3,6,9-trioxaun-
decane in anhydrous tetrahydrofuran was cooled to 0 ꢁC.
Triphenylphosphine (0.31 g, 1.16 mmol) was added, after
which the mixture was allowed to attain room temperature
and was stirred for 2 d. After addition of 50 mL (2.8 mmol)
water, the solution was stirred at room temperature for 24 h
and then at 40 ꢁC for 6 h. After diluting with water and re-
moval of the tetrahydrofuran in vacuo, the pH was adjusted
to 3 with 0.2 N HCl and the resulting suspension was washed
with toluene. The aqueous solution was concentrated down to
a colorless oil, which was purified by flash chromatography
(10–20% methanol in dichloromethane) to yield 0.3 g product
(77%). MS (ESI) m/z [M+H] calcd for C16H33N2O7 365.2288,
found 365.2283. 1H NMR (CDCl3) d: 1.27 (t, 6H, J¼7.2 Hz),
2.92 (t, 2H, J¼5.6 Hz), 3.18 (m, 2H), 3.48–3.78 (m, 16H),
3.94 (m, 2H), 4.13–4.18 (q, 4H, J¼7.2 Hz). 13C NMR
(CDCl3) d: 14.2, 40.5, 54.8, 55.3, 61.2, 67.1, 67.5, 69.8,
69.9, 70.1, 70.2, 171.4.
4.1.3. 1-(Fluorenylmethoxycarbonylamino)-11-dibenzyl-
phosphate-3,6,9-trioxaundecane, 7. To a stirred mixture
of 1-(fluorenylmethoxycarbonylamino)-11-hydroxy-3,6,9-
trioxaundecane (0.159 g, 0.24 mmol) and 5-methyltetrazole
(0.04 g, 0.48 mmol) in anhydrous acetonitrile, was added di-
benzyl diisopropyl phosphoramidite (0.095 mL, 0.29 mmol).
The mixture was stirred at room temperature under argon
for 3 h, and then poured into saturated aqueous sodium bi-
carbonate. After filtering the resulting white precipitate,
the filtrate was diluted with ethyl acetate, washed with brine,
dried over anhydrous magnesium sulfate, and concentrated
to give 209 mg yellow oil, which was dried under vacuum
and used in the next step without purification. To an ice-
cooled solution of the above phosphate intermediate in anhy-
drous dichloromethane 0.059 g (0.265 mmol) m-chloro-
peroxibenzoic acid was added and the stirring was continued
at 0 ꢁC for 1 h. The reaction mixture was then treated with
saturated aqueous sodium thiosulfate and extracted with
dichloromethane. The organic layer was successively
washed with water, saturated sodium bicarbonate, and brine,
before being dried over anhydrous magnesium sulfate.
Purification by preparative TLC in ethyl acetate gave
150 mg colorless oil (92%). MALDI-FTMS: [M+Na]+ calcd
for C37H42NO2PNa 698.26, found 698.245. 1H NMR
(CDCl3) d: 3.25–3.4 (m, 2H), 3.45–3.75 (m, 12H), 4.15
(m, 2H), 4.25 (t, 1H, J¼6.8 Hz), 4.45 (d, 2H, J¼6.8 Hz),
5.0–5.1(m, 4H), 6.25 (br s, 1H), 7.25–7.35(m, 2H), 7.38–
7.42 (m, 2H), 7.63 (d, 2H, J¼7.6 Hz), 7.75 (d, 2H,
J¼7.6 Hz). 13C NMR (CDCl3) d: 41.2, 47.5, 66.8, 67.0,
69.4, 69.5, 70.1, 70.2, 70.3, 70.5, 70.8, 70.8, 120.2, 125.3,
127.3, 127.9, 128.2, 128.7, 128.8, 136.1, 141.5, 144.2.
4.1.6. Calix[4]arene tetrabenzylphosphate, 12. To an
anhydrous tetrahydrofuran solution of 5,11,17,23-
tetra-(heptadecafluoro-undecyl)-25,26,27,28-calix[4]arene
tetraacetic acid (100 mg, 0.04 mmol), 50.61 mg 1-[3-(dimeth-
ylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDCI)
and 35.67 mg (0.264 mmol) 1-hydroxybenzotriazole hy-
drate (HOBt) were added, followed by anhydrous dimethyl-
formamide dropwise until all the solids dissolved. The
reaction mixture was stirred at room temperature under an
argon atmosphere for 4 h. Fmoc-amine 7 of 162.168 mg
(0.24 mmol) was dissolved in 2 mL anhydrous dimethyl-
formamide, 26 mL (22.5 mg, 0.264 mmol) piperidine was
added, and the mixture was stirred at room temperature for
6 h, before being added to the first solution of activated
acid. Stirring was continued for 7 d at room temperature.
After removing all the solvents under an air stream, the
residue was taken up in dichloromethane and washed with
water, dried over anhydrous magnesium sulfate, and concen-
trated down to a yellow solid. Benzyl protected product of
106 mg was obtained (62% yield after flash chromato-
graphy). MALDI-FTMS [M+Na]+ calcd for C168H172
-
F68N4O36P4Na 4259.96, found 4260.319. 1H NMR
(CDCl3) d: 1.65–1.8 (br m, 8H), 1.9–2.1 (br m, 8H), 2.2–
2.4 (br m, 8H), 3.1–3.25 (d, 4H, J¼8.5 Hz), 3.4–3.7 (m,
64H), 4.05–4.2 (m, 8H), 4.5–4.65 (d, 4H, J¼8.5 Hz),
4.95–5.1 (m, 16H), 6.2–6.6 (br s, 8H), 7.25–7.3 (s, 40H),
7.9 (s, 4H). 19F NMR (CDCl3) d: ꢀ81.5 (t, 12F), ꢀ114.5
(m, 8F), ꢀ122.5 to ꢀ122.7 (m, 24F), ꢀ123.5 to ꢀ123.9
(m, 16F), ꢀ127.0 (m, 8F). 13C NMR (CDCl3) d: 22.3,
29.9, 39.2, 66.9, 69.4, 69.5, 70.0, 70.1, 70.3, 70.6, 70.7,
128.2, 128.7, 128.8, 136.0, 136.1.
4.1.4. 1-Bis(ethoxycarbonylmethyl)amino-11-azido-3,6,9-
trioxaundecane, 9. To a solution of 1.55 g (7.11 mmol)
1-amino-11-azido-3,6,9-trioxaundecane in 50 mL anhydrous
acetonitrile, 1.96 g (14 .2 mmol) anhydrous potassium car-
bonate was added and the suspension stirred at room temper-
ature for 3 h before adding 1.6 mL (14.2 mmol) 98% ethyl
bromoacetate. Stirring was continued at room temperature
for 2 d. After removal of the solvent, the solid residue was
partitioned between ethyl acetate and water. The organic
layer was washed with water, dried over anhydrous magne-
sium sulfate, and concentrated down to a yellow oil, which
was purified by flash chromatography (ethyl acetate/petro-
4.1.7. Calix[4]arene tetraphosphoric acid, 2. Benzyl
protected phosphate 12 of 160 mg was dissolved in 15 mL
absolute ethanol. Excess Pd/C was added and a hydrogen
balloon was adapted to the flask. The hydrogenation was
conducted overnight, after which the mixture was filtered
through Celite and concentrated down under an air stream.
White solid of 55 mg was obtained (41% yield). MALDI-
FTMS [MꢀH]ꢀ calcd for C112H123F68N4O36P4 3516.97,
found 3516.547, [MꢀNa]ꢀ calcd for C112H116F68-
1
leum ether 2:1) to give 2.23 g product (86%). H NMR
(CDCl3) d: 1.26 (t, 6H, J¼7.2 Hz), 2.97 (t, 2H, J¼5.6 Hz),
3.40 (t, 2H, J¼5.3 Hz), 3.59–3.69 (m, 16H), 4.13–4.18 (q,
4H, J¼7.2 Hz). 13C NMR (CDCl3) d: 14.4, 50.8, 53.8,
56.0, 60.6, 70.2, 70.5, 70.8, 171.5.
1
N4Na7O36P4 3670.83, found 3670.349. H NMR (CD3OD)

5546
O. M. Martin, S. Mecozzi / Tetrahedron 63 (2007) 5539–5547
d: 1.65–1.7 (br m, 8H), 1.9–2.1 (br m, 8H), 2.2–2.4 (br m,
8H), 3.1–3.25 (d, 4H), 3.4–3.7 (m, 64H), 3.9–4.0 (m, 8H),
4.5–4.65 (m, 4H), 4.95–5.1 (m, 16H), 6.4–6.6 (br s, 8H). 19F
NMR (CD3OD) d: ꢀ83.1 (t, 12F), ꢀ115.4 (m, 8F), ꢀ123.3
to ꢀ123.5 (m, 24F), ꢀ124.4 (m, 16F), ꢀ128.0 (m, 8F).
different pH by sonication of a thin film, obtained by evap-
oration of a methanol solution. The final concentrations
were 100 mM and all solutions were filtered through
a 450 nm microfilter before the measurements. For each
sample, DLS measurements were carried out for 5 cycles
of 10 min each or 10 cycles of 5 min each and were proved
to be reproducible. The viscosities and refractive indexes for
the aqueous buffers were measured. A Cannon-Manning
Semi-micro viscometer size 25 (range 0.5–2 cSt) was used,
equilibrated at 25 ꢁC in a water bath, to determine the kine-
matic viscosity as: kinematic viscosity (cSt)¼timeꢂ
0.002029 cSt/s. The dynamic viscosity was obtained using
the equation: dynamic viscosity (cP)¼kinematic viscosity
(cSt)ꢂr (g/mL), where 0.00029 is the viscometer constant
at 25 ꢁC and r is the density of the buffer, which was mea-
sured by weighing 1 mL of buffer. Both the density and
the viscosity measurements were done in triplicate and
average values were used for further calculations.
4.1.8. Calix[4]arene tetrakis(imino-diethylcarboxylate),
13. To an anhydrous tetrahydrofuran solution (5 mL) under
an argon atmosphere of 5,11,17,23-tetra(heptadecafluoro-
undecyl)-25,26,27,28-calix[4]arene tetraacetic acid (168 mg,
0.067 mmol), 77.06 mg (0.402 mmol) 1-[3-(dimethyl-
amino)propyl]-3-ethylcarbodiimide hydrochloride (EDCI),
and 54.3 mg (0.402 mmol) 1-hydroxybenzotriazole hydrate
(HOBt) were added, followed by anhydrous dimethylform-
amide dropwise until all the solids dissolved. After stirring
at room temperature for 3 h, a dimethylformamide solution
of 1-bis(ethoxycarbonylmethyl)amino-11-amino-3,6,9-tri-
oxaundecane 10 (175.03 mg, 0.402 mmol) was added and
stirring was continued at room temperature for 6 d. After
removal of all solvents in vacuo, the residue was taken up
in dichloromethane and purified by flash chromatography
(5% methanol in dichloromethane) to give 159 mg product
(60% yield). Final purification was carried out by prepara-
4.3. Transmission electron microscopy (TEM)
Samples were prepared as described above for the DLS
study deposited on pioloform powder grids, negatively
stained with methylamine tungstate and analyzed on a
Philips CM120 electron microscope operating at 80 kV.
˚
tive HPLC on a Jordi Gel DVB 500 A column, using a gradi-
ent of 90–100% acetonitrile/water over 30 min (retention
time¼7–8 min). MALDI-FTMS [M+Na]+ calcd for
1
C144H172F68N8O36Na 3904.08, found 3904.448. H NMR
(CDCl3) d: 1.2–1.3 (t, 24H, J¼7.2 Hz), 1.6–1.8 (br m, 8H),
1.9–2.1 (br m, 8H), 2.2–2.4 (br m, 4H), 2.9–3.0 (br m, 4H),
3.15–3.25 (d, 4H, J¼14 Hz), 3.4–3.65 (m, 73H), 4.15–4.2
(q, 12H, J¼7.2 Hz), 4.65–4.70 (br m, 12H), 6.2–6.6 (br s,
2H). 19F NMR (CDCl3) d: ꢀ81.6 (t, 12F), ꢀ114.8 (m, 4F),
ꢀ122.6 to ꢀ122.7 (m, 21F), ꢀ123.5 to ꢀ124.1 (m, 12F),
ꢀ127.0 (s, 8F).
Acknowledgements
We thank Prof. Sam Gellman for useful discussions. This
work was supported by the National Science Foundation
(CHE 0518112) and the University of Wisconsin-Madison.
Supplementary data
4.1.9. Calix[4]arene octacarboxylate, 3. Starting material
of 43 mg (3–30) was dissolved in 2 mL methanol, 0.5 mL
2 M NaOH was added, followed by an additional 1.5 mL
water to dissolve all the solids. The mixture was stirred at
room temperature for 3 d. After removal of the methanol,
the water was lyophilized off to give a white-yellow solid
Volume-weighed NICOMP distribution analysis for solu-
tions of calix[4]arene tetraphosphate and calix[4]arene octa-
carboxylate. Supplementary data associated with this article
can be found in the online version, at doi:10.1016/j.tet.2007.
04.030.
1
(yield >99%). The product was characterized by H NMR
and 19F NMR in CD3OD. The signals are broad, indicating
self-assembly, but the peaks can be easily identified. Al-
though the product is very soluble in D2O, NMR could not
be used for characterization due to the strong self-assembly
process. For example, in the 19F NMR in D2O only one very
broad peak was observed in the CF3 region and no peaks in
the CF2 region. MALDI-FTMS for the free octacarboxylic
acid: [M+Na]+ calcd for C128H140F68N8O36Na 3681.40,
found 3682.018. 1H NMR (CD3OD) d: 1.2–1.4 (br m,
25H), 1.7–1.8 (br m, 5H), 1.9–2.2 (br m, 6H), 3.4–3.7
(m, 79H), 3.9–4.0 (m, 8H), 4.5–4.65 (br m, 16H), 6.4–6.6
(br s, 7H). 19F NMR (CD3OD) d: ꢀ83.1 (t, 12F), ꢀ115.6 (m,
5F), ꢀ123.3 to ꢀ123.5 (m, 20F), ꢀ124.4 (m, 11F), ꢀ128.0
(m, 8F).
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