Calixarene-mediated liquid-membrane transport of choline conjugates

Article abstract of DOI:10.1002/ejoc.201400025

A series of supramolecular calixarenes efficiently transport distinct molecular species through a liquid membrane when attached to a receptor-complementary choline handle. Calix[6]arene hexacarboxylic acid was highly effective at transporting different target molecules against a pH gradient. Both carboxylic-and phosphonic-acid-functionalized calix[4]arenes effect transport without requiring a pH or ion gradient. NMR binding studies, two-phase solvent extraction, and three-phase transport experiments reveal the necessary and subtle parameters to effect the transport of molecules attached to a choline "handle". On the other hand, rescorin[4]arene cavitands, which have similar guest recognition profiles, did not transport guest molecules. These developments reveal new approaches towards attempting synthetic-receptor-mediated selective small-molecule transport in vesicular and cellular systems.

Full text of DOI:10.1002/ejoc.201400025

Job/Unit: O40025
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FULL PAPER
DOI: 10.1002/ejoc.201400025
Calixarene-Mediated Liquid-Membrane Transport of Choline Conjugates
Birendra Babu Adhikari,[a] Ayu Fujii,[a] and Michael P. Schramm*[a]
Keywords: Host-guest systems / Molecular recognition / Cavitands / Calixarenes / Liquid-membrane transport / Choline
conjugates
A series of supramolecular calixarenes efficiently transport
distinct molecular species through a liquid membrane when
attached to a receptor-complementary choline handle. Calix-
[6]arene hexacarboxylic acid was highly effective at trans-
porting different target molecules against a pH gradient.
Both carboxylic- and phosphonic-acid-functionalized ca-
lix[4]arenes effect transport without requiring a pH or ion
gradient. NMR binding studies, two-phase solvent extrac-
tion, and three-phase transport experiments reveal the nec-
essary and subtle parameters to effect the transport of mol-
ecules attached to a choline “handle”. On the other hand,
rescorin[4]arene cavitands, which have similar guest re-
cognition profiles, did not transport guest molecules. These
developments reveal new approaches towards attempting
synthetic-receptor-mediated selective small-molecule trans-
port in vesicular and cellular systems.
tin–choline guest. This non-covalent assembly of guest-in-
host-in-monolayer could then sequester NeutrAvidin.[7] We
have demonstrated that such assemblies also function as
pH-selective switches, influencing the orientation of the
guest in the host.[8] We know too that fluorescently tagged
cavitands distribute regularly in giant unilamellar vesicles
(GUVs), and that these vesicles remain viable and non-per-
meable to their surroundings.[9] New hosts have emerged
that are capable of self-micelle formation and compartmen-
talization of payloads.[10] Resorcinarenes have also formed
ion channels in lipid environments.[11] This body of work
has encouraged us to apply these results to cellular systems;
it has been shown that resorcinarene cavitands can promote
the endocytosis of fluorescein, so long as a complementary
choline handle is attached.[12] The observation that selective
endocytosis is possible is quite remarkable – a non-
covalent supramolecular host–guest assembly crosses a cell
membrane!
Several different calixarene species have shown successful
binding with choline and tetraalkyl ammonium species in a
variety of solvents based on cation–π or cation–anion at-
traction.[13] Alkyl ammoniums (RNH3+) bind with tert-
butyl calix[4]arenes, but the larger tetraalkyl ammoniums
(NR4+) require a polarizable functional group on the host’s
upper rim (as seen with sulfonated derivatives),[14] a larger
internal cavity (e.g., a calix[5 or 6]arene), or a combination
of these two features.[15] New work in the area using sulfon-
ated derivatives that recognize trimethyllysines and disrupt
protein–protein interactions is of great relevance and inter-
est to us.[16] Other arrangements have also been successful
in recognizing ammoniums, including functionalization of
the lower rim with carboxylates. Phosphonate-ester-func-
tionalized cavitands similar to 6 attracted our attention, as
they have been used when embedded in lipid membranes
Introduction
The machinery of the human organism presents many
challenges that must be addressed if the transport of several
classes of molecules is to be achieved as part of the effort
to treat disease. These challenges have prompted a response
in the chemical community to develop new methods of drug
delivery.[1] The design of effective drug-transport systems
hinges on optimizing a daunting array of features such as
solubility, directed transport, biocompatibility, and the re-
lease of the encapsulated drug. Several successful strategies
have relied on the synthesis of drug–polymer conjugates or
the embedment of drugs in polymer, vesicle, or micelle car-
riers.[2] However, these approaches have had limited success
in several crucial applications, most notably in transport ac-
ross the blood–brain barrier.[3]
In 2007, we reported that hydrophobic resorcin[4]arene
cavitands could function in an aqueous micellar environ-
ment as supramolecular hosts for small organic mol-
ecules.[4] This behavior was special, as these types of mate-
rials rarely adopt a functional conformation in water – in-
stead they precipitate/dimerize into flat non-functional con-
formations to avoid unfavorable solvent interactions. Func-
tionalization of the host can circumvent this constraint,[5]
and many applications exist for these materials.[6] Since this
report, several advances have been made, including embed-
ding cavitands into lipid monolayers to give a surface that
can selectively sequester choline conjugates, including a bio-
[a] Department of Chemistry and Biochemistry, California State
University Long Beach,
1250 Bellflower Blvd., Long Beach, CA 90840, USA
E-mail: michael.schramm@csulb.edu
http://schrammlab.wordpress.com
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201400025.
Eur. J. Org. Chem. 0000, 0–0
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B. B. Adhikari, A. Fujii, M. P. Schramm
FULL PAPER
for the detection of amino acids.[17] Pillar[n]arenes are an CH2Cl2 with 10% [D4]MeOH added, as evident from the
additional class of molecules with ammonium-binding upfield trimethylammonium resonance observed around
properties.[18]
–1.5 ppm (Δδ up to –4 ppm). Resorcinarene cavitands typi-
These phenomena encouraged us to examine the charac- cally bind strongly with choline handles to form slow-ex-
teristics and properties of host–guest systems that are neces- change complexes. The addition of base changed the nature
sary to effect receptor-mediated transport – something quite of the recognition event, as monitored by NMR spec-
distinct from ion-channel formation, or induction of endo- troscopy (see Supporting Information for full details). The
cytosis. Using liquid-membrane transport experiments, we binding of 9 with 4–6 was also observable by NMR ti-
have revealed some limitations in the ability of resorcinar- tration, this time by fast-exchange.
1
enes to transport small molecules, and in the process we
have also uncovered new leads.
The H NMR spectrum of upper-rim tetraphosphonic-
acid-functionalized calix[4]arene 6 with FITC-chloline 9 in
[D4]MeOH is shown in Figure 2. Guest 9 was sparingly sol-
uble in methanol, but upon the addition of the host, it im-
mediately dissolved. Very slight changes were observed in
the chemical shifts of the signals of the ethylene group of 9
when the concentration of the host was varied (seen at δ =
3.7 and 4.2 ppm); the chemical shift of the trimethylammo-
nium group changed more noticeably (δ = 3.27–3.24 ppm).
The remaining signals of the host move modestly (see Sup-
Results and Discussion
We began our work using resorcin[4]arene benzimidazole
cavitands 1–3, calixarenes 4–6, and pillar[n]arenes 7 and 8,
all of which are known to be hosts for various small-
molecule ammonium guests. Our screening partners have a
guest–fluorophore conjugate structure (9–12), where a
small-molecule handle is attached to a fluorophore payload
(Figure 1).
The ethyl-footed benzimidzaole cavitand (i.e., 1, where R
= ethyl) was our initial host of choice, as we have previously
demonstrated that this host binds aliphatic and cationic
guests in aqueous micelles, as well as in [D6]DMSO/D2O
mixtures. Additionally, titration with base resulted in en-
hanced binding in some cases, presumably due to deproton-
ation of the imidazole rings and build-up of negative charge
in the host.[4] Unfortunately, this host was insoluble and
non-functional as a binder of 9 in organic solvents or sol-
vent mixtures containing small amounts of [D4]MeOH. We
enhanced the solubility and restored the function of the
benzimidazole cavity by introducing C5 (2) or C11 (1, 3)
Figure 2. 300 MHz 1H NMR spectra in 100% [D4]MeOH (δ =
3.32 ppm) of (a) FITC-choline 9, (b) host 6, (c) 1:0.5 host:guest,
feet. For example, host 1[19] bound FITC-choline 9 in (d) 1:1 host:guest, (e) 1:2 host:guest.
Figure 1. Resorcinarene cavitands 1–3, calixarenes 4–6, and pillar[n]arene hosts 7 and 8, and fluorophore-conjugated guests 9–12 used in
this work.
2
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Calixarene Mediated Liquid-Membrane Transport
porting Information for other host–guest results). Pillar[n] Table 1. Extraction of FITC-choline 9 with 1 with variable base
concentration.
arenes have little to no solubility in MeOH. Dansyl choline
12 was far more soluble in chloroform/methanol mixtures
than FITC-choline 9. Thus we combined pillar[5]arene 7
with dansyl choline 12 in chloroform/methanol, and no
binding was observed (Figure 3, a, d, e). The larger internal
volume of pillar[6]arene 8 accepts trimethylammoniums;
broadening and upfield shift (δ = 3.0 ppm) are observed
(Figure 3, a, b). Jobs plot analyses for pillar[6]arene 8 with
dansyl choline 12 and calix[4]arene tetraphosphonic acid 6
with FITC-choline 9 are consistent with 1:1 binding models.
Tetra acid 6 is probably engaging in ion–ion interactions,
while pillarene 8, which lacks acids, binds through cation–
π interactions.
Alkali volume
[%][a]
Extraction
[%]
Extraction
(control) [%]
Extraction
efficiency [%][b]
0
2
5
10
15
20
0.3
5.2
7.4
9.9
13.1
14.9
1.0
1.0
2.7
1.9
2.0
2.1
0
2.1
2.3
4.0
5.6
6.4
[a] Resorcinarene-based receptor 1 (100 μm) in CH2Cl2, guest 9
(50 μm) was dissolved in water containing the amount stated (%
v/v) of NaOH (1 m), stirred for 24 h at 160 rpm. The resulting aque-
ous phase was compared to a UV/Vis calibration curve after di-
lution. The results are representative of multiple runs. See Exp.
Sect. for full details. [b] Calculated as (extraction – extraction con-
trol)ϫ[guest]/[host].
able extraction. As previously reported, base deprotonates
the acidic benzimidazole protons (pKa = 12.8), introducing
a negative charge and enhancing the complementary elec-
trostatic interaction between the positively charged guest
handle and the negatively charged cavitand.[4] In this in-
stance, it seems that the C11 feet of 1 provide a superior
platform to extract 9 into an organic phase compared to 2
with its shorter C5 feet. We also carried out these extraction
experiments with host 1 and FITC-adamantane 10. Under
exactly the same experimental conditions, no extraction of
adamantane-handled FITC from the aqueous phase into
the organic phase occurred (results not shown), which sug-
gests that the extraction of FITC-choline under alkaline
conditions is in part due to an ionic interaction of the chol-
ine handle with the negatively charged cavity. This is some-
what surprising as the adamantyl handle is a good guest for
cavitands like 1–3, and it is also more hydrophobic. Control
Figure 3. 300 MHz 1H NMR spectra in CDCl3 with 20% [D4]-
MeOH (δ = 3.25 ppm) of (a) guest dansyl choline 12, (b) 1:1 host
8:guest 12, (c) host 8, (d) 1:1 host 7:guest 12, (e) host 7.
At this stage, it was impossible to evaluate Ka values for
hosts 4–8 in either the same solvent system as one another
or with the same guest, due to severe solvent incompatibili-
ties. Thus, we used NMR spectroscopy as a simple tool to
evaluate host–guest binding in solution for a variety of rep-
resentative guest–dye conjugates under the best conditions
to obtain NMR spectroscopic data for each combination.
To better understand the necessary properties for transport,
we turned our attention to two-phase extraction experi-
ments.
Table 2. Extraction of FITC-choline 9 with cavitands 1–3 in the
presence of base.
Host[a] Guest 9 con- Extraction Extraction
Extraction
efficiency
[%][b]
centration
[%]
(control)
[%]
[μm]
We began our two-phase extraction experiments with our
hosts dissolved in CH2Cl2 and water-soluble guests. We first
varied the concentration of NaOH in the aqueous layer, and
measured the extraction efficiency between 9 and host 1
(Table 1), using 50 μm of 9 and 100 μm of 1. Enhanced ex-
traction efficiency was observed when NaOH (1 m) was
added to the aqueous phase (10% v/v or more).
Under neutral conditions, no guest extraction was ob-
served for this combination of host and guest. We therefore
adopted a basified aqueous source phase with 10% (v/v) of
NaOH (1.0 m) to compare several resorcinarene cavitands
and their ability to extract 9. Tetrabenzimidazole cavitands
1 and 2 showed some extraction of 9, whereas ester 3 gave
insignificant extraction (Table 2). It is notable that host 1
showed demonstrable extraction efficiency (7%) with 1 or
2 equiv. of guest present. In a large proportion of literature
reports, the concentration of the guest must be of the order
1
2
3
10
25
50
100
200
10
25
50
100
200
10
25
50
100
200
14.7
13.3
9.9
7.4
3.8
17.0
13.3
6.4
6.9
1.7
0.0
1.4
0.7
2.0
2.4
0.9
1.7
0.0
0.2
0.0
2.2
1.8
3.0
1.5
0.0
2.1
1.8
3.0
1.5
1.2
3.1
4.0
7.4
7.3
1.7
2.8
2.2
3.9
0.3
0
0
0
0
4.0
3.7
[a] Resorcinarene-based receptors (100 μm) in CH2Cl2; guest 9 was
dissolved in water containing 10% (v/v) NaOH (1 m), stirred for
24 h at 160 rpm. The resulting aqueous phase was measured using
a UV/Vis calibration curve after dilution. See Exp. Sect. for full
details. [b] Calculated as (extraction – extraction control)ϫ[guest]/
of 30–50 times higher than that of the host to effect measur- [host].
Eur. J. Org. Chem. 0000, 0–0
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B. B. Adhikari, A. Fujii, M. P. Schramm
FULL PAPER
experiments revealed that in the absence of a host, 9 and work. We noted some precipitation in these experiments at
10 showed similar % extraction into CH2Cl2. Thus, these the source/organic interface and attributed this to the host.
guests have similar partition coefficients between CH2Cl2 Thus, addition of THF to help redissolve the host was tried.
and water, but different affinities for the hosts.
In a last attempt, pH buffering and changing the guest to
The extraction of NBD-choline 11 (200 μm) from pH 7 the more hydrophobic NBD–choline conjugate 11 also
water was best achieved using host 1 (100 μm), which gave failed. Repetition with a higher guest concentration or a
a modest extraction efficiency of 5.2%. NBD-choline had a longer duration also failed. Host 3, which was unsuccessful
much higher partition into the organic layer under the con- in extraction experiments, did not work either.
trol conditions (without a host), as its fluorophore is more
Under these conditions, the organic phase was evapo-
hydrophobic than that of 9. Hosts 2 and 3 were ineffective rated, the residue was resuspended in water, and the fluores-
at extraction (results not shown), and the addition of base cence was measured. This gave similar results to our extrac-
did not resolve this issue. Thus, variation in fluorophore tion experiments, so it is clear that the guest was partition-
composition did not have a marked effect on extraction by ing into the organic layer, never to escape. The reason for
1 so long as the choline handle remained constant.
the failure of these experiments to effect any measurable
Having completed these initial experiments, we at- transport, despite the high binding affinity that these hosts
tempted to study the transport of FITC-choline 9 across a show for choline or adamantane as measured by NMR
liquid membrane using a U-tube apparatus. As some ex- spectroscopy, is unclear. The precipitation of the host at the
traction ability had been seen with a 1:1 host:guest ratio, interface, along with the presence of extracted guest in the
we began with 100 μm of host in the organic phase and organic phase, leaves some unanswered questions. Other
100 μm of guest in the aqueous source phase. Under a vari- guest “handles” might overcome this problem, or perhaps
ety of conditions, guests never appeared in the receiving deep cavitands with different upper-rim functionalization
phase (Table 3). Under both basic (necessary for extraction might overcome these problems. Good binding does not nec-
efficiency) and neutral conditions, transport was unsuccess- essarily lead to transport, so we next examined calixarene
ful. We next acidified the receiving phase with the hope that hosts 4–6, which NMR spectroscopy showed to be weak
this would allow ion exchange and pH-driven cotransport binders of choline-handle guests.
(antiport) and/or protonation of the host, resulting in re-
We used a host concentration of 500 μm and varied the
lease of the guest due to a decreased binding affinity. This guest concentration. Table 4 summarizes the results of ex-
too was unsuccessful. Trimethylammonium chloride salts traction of FITC-choline. Calix[4]arene phosphonic acid 6
were tried, with the hope that they would facilitate release is expected to ionize and bind trimethylammoniums at the
by exchange at the receiving interface, but this did not upper rim, and calix[4]arene 4, with carboxylates at the
lower rim, directs ammonium binding there. Calix[4]arene
phosphonic acid 6 showed better extraction efficiency than
Table 3. Transport experiments with cavitands 1 and 3 and fluoro-
resorcianarenes in two-phase extraction experiments under
neutral aqueous phase conditions (Table 4). Calixarene 4,
on the other hand, showed poor extraction efficiency that
did not improve with guest concentration under neutral
conditions.
phore conjugates in a liquid-membrane U-tube apparatus.
Host[a] Guest Source phase Receiving phase
Transport
1
1
1
1
1
1
9
9
9
9
9
9
MQ water
MQ water
no
no
no
no
no
no
NaOH (0.1 m) NaOH (0.1 m)
NaOH (0.1 m) MQ water
NaOH (0.1 m) HCl (0.1 m)
NaOH (0.1 m) NaCl (0.1 mm)
NaOH (0.1 m) THF (0.1 mm in
MQ water)
Table 4. Extraction of FITC-choline 9 with different calixarenes 6
and 4 in the absence of base.
Host[a]
Guest 9 con- Extraction [%]
centration
Extraction
(control) [%]
Extraction
efficiency
[%][b]
1
9
NaOH (0.1 m) n-propyltrimethyl-
ammonium chloride
no
[μm]
(0.1 mm in MQ
water)
6
10
25
50
100
200
10
25
50
100
200
37.4Ϯ2.3
20.1Ϯ1.8
24.9Ϯ3.8
33.2Ϯ4.5
43.4Ϯ4.1
21.1Ϯ2.5
21.6Ϯ2.8
25.7Ϯ3.6
10.6Ϯ1.4
6.8Ϯ1.3
0.1
0.1
1.0
1.2
1.1
0.1
0.1
1.0
1.2
1.1
0.7
1.0
2.4
6.4
16.9
0.4
1.1
2.5
1.9
2.3
1
1
1
1
1
9
pH 13.26
HEPES
pH 13.26
HEPES
pH 5.94 HEPES
pH 5.94 HEPES
MQ water
no
no
no
no
no
11
9
MQ water
4
(500 μm)
9
NaOH (0.1 m) NaOH (0.1 m)
NaOH (0.1 m) HCl (0.1 m)
(500 μm)
9
(500 μm)
[a] Calixarene-based receptors (500 μm) in CH2Cl2; guest 9 dis-
solved in water, stirred for 24 h at 160 rpm. The resulting aqueous
phase was measured using a UV/Vis calibration curve after di-
lution, and the results are reported as an average of duplicate runs
with maximum deviation from the mean. See Exp. Sect. for full
details. [b] Calculated as (extraction – extraction control)ϫ[guest]/
[host].
3
3
9
11
MQ water
MQ water
MQ water
MQ water
no
no
[a] Resorcinarene-based receptors (100 μm) in CH2Cl2; guest con-
centration was 100 μm unless otherwise noted. After stirring for
24 h, aliquots were removed and analyzed for guest concentration
by UV/Vis spectroscopy.
4
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Calixarene Mediated Liquid-Membrane Transport
We again used the three-phase experiment to evaluate Table 5. Transport flux for different guests mediated by 6 at neutral
pH.
guest transport mediated by 6. FITC-choline 9 and NBD-
Guest[a]
Transport flux
Control
choline 11 were selected due to their different hydropho-
bicities and tendencies to partition into organic layers.
Equal volumes from the source and receiving phases were
[μmolcm–2 min–1]
[μmolcm–2 min–1]
FITC-choline 9
8.9Ϯ0.2ϫ10–6
0.00
drawn at 24-hour intervals, and were analyzed by UV/Vis FITC-adamantane 10 7.3Ϯ0.2ϫ10–7
4.36ϫ10–7
9.61ϫ10–6
5.20ϫ10–5[b]
1.54ϫ10–5
not measured[d]
NBD-choline 11
Dansyl choline 12
carboxyfluorescein[c]
NBD chloride
5.6Ϯ0.2ϫ10–5
4.6Ϯ0.1ϫ10–5[b]
1.6Ϯ0.1ϫ10–5
2.0Ϯ0.1ϫ10–8
spectroscopy. To our delight, the appearance of substrate in
the receiving phase increased with time for both substrates
(Figure 4). The receptor efficiently transports both sub-
strates across the bulk liquid membrane. The hydrophobic
NBD-choline 11 (1.0 mm source phase, 23% transported)
was transported more efficiently than FITC-choline
(0.5 mm source phase, 11% transported). NBD-choline had
some background transport in the absence of the calixarene
(5%), whereas FITC-choline had no inherent transport, due
to its inability to partition into the organic phase. Thus the
transport of FITC-choline is more significant.
[a] 6 (500 μm) in CH2Cl2; guests (500 μm) in water, stirred for 144 h
at 400 rpm. The aqueous phase was measured by UV/Vis spec-
troscopy and compared to standard calibration curves after appro-
priate dilution. The results are reported as an average of duplicate
runs with maximum deviation from the mean. See Exp. Sect. and
Supporting Information for full details. [b] For guest 12, the flux
was less than the control experiment; we determined that 6 extracts
and retains the difference in the organic layer. [c] Carboxyfluores-
cein was used at 250 μm due to solubility issues at higher concentra-
tions. [d] The transport rate with host 6 was so low that the control
experiment was superfluous.
FITC-choline 9 had the most significant enhancement in
transport based on a host–guest interaction. The thesis that
a common choline handle can be used to transport hydro-
philic and hydrophobic payloads has been demonstrated.
Dansyl choline 12 had significant flux in the absence of 6,
and we determined that the decrease in flux when calixarene
6 was present was due to 6 acting more strongly as an ex-
traction agent than a transporter. The difference in flux was
resolved by evaporation of the organic layer, resuspension
in water, and UV/Vis measurement where 12 was found.
Neither carboxyfluorescein nor NBD chloride was trans-
ported more in the presence of 6 than in its absence (i.e.,
in the control experiment), confirming that transport is a
function the specific host–guest interaction of the choline
handle.
The transport efficiency of calixarene carboxylic acids 4
and 5 towards FITC-choline and NBD-choline differed sig-
nificantly from that of phosphonic acid 6. The ion–ion in-
teraction now takes place at the lower rim of the calixarene.
Calixarene tetracarboxylic acid 4 transported 9 into the re-
ceiving phase at similar rates, independent of the pH of the
receiving phase (Table 6). NBD-choline 11 had an ac-
Figure 4. Transport of different substrates with calix[4]arene tetra-
phosphonic acid receptor 6 in a U-tube experiment. Source phase:
MQ water (4 mL), NBD-choline 11 (1 mm) and FITC-choline 9*
(0.5 mm); organic phase: CH2Cl2 (10 mL), 6 (0.5 mm); receiving
phase: MQ water; stirring speed = 400 rpm, room temperature, re-
sults are reported as the average of duplicate trials. For FITC-chol-
ine, the maximum deviation from the mean was Ϯ0.3% (not
shown), and for NBD-choline, it was Ϯ0.8% (not shown). (* guest
precipitation occurred at the interface when 1 mm solution was
used.).
Table 6. Transport flux for 9 and 11, mediated by calixarenes 4 and
5, as a function of pH.
Host[a] Guest Receiving
phase
Transport flux
Control
[μmolcm–2 min–1] [μmolcm–2 min–1]
U-tube transport experiments were repeated using dif-
ferent substrates, and transport flux was measured
(Table 5). The efficiency depends on the type of comple-
mentary guest handle and on the nature of the fluorophore.
As can be seen, the rate of transport (flux/control) is FITC-
choline 9 ϾϾϾ NBD-choline 11 Ͼ FITC-adamantane 10
Ͼ dansyl choline 12. Comparing FITC-choline and FITC-
adamantane, the substrate with the quaternary ammonium
guest handle was transported more than 12 times faster
than the substrate with the adamantane handle. The ionic
phosphate–ammonium interaction plays a key role for
transport in this system.
4
9
MQ water
0.1 m HCl
MQ water
0.1 m HCl
MQ water
0.1 m HCl
MQ water
0.1 m HCl
1.6ϫ10–5
1.2ϫ10–5[b]
3.9ϫ10–5
9.4ϫ10–5
0
0
0
9
11
11
9
9
11
11
1.3ϫ10–5
1.3ϫ10–5
0
5
1.9ϫ10–6[b]
3.1ϫ10–6
2.6ϫ10–4
0
1.3ϫ10–5
1.3ϫ10–5
[a] Calixarene-based receptors (500 μm) in CH2Cl2; guests were dis-
solved in water, stirred for 96 h at 400 rpm. The receiving phase
was measured using a UV/Vis calibration curve after dilution. [b] A
separate calibration curve was used for the acidified receiving phase
due to changes in the absorbance of 9.
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celerated flux when the receiving phase was acidified.
Transport requires the presence of ionizable groups, because
the corresponding tetraester of 4 gave no transport of 9
(data not shown). Calixarene 4 exists in a well-defined con-
formation, and it appears that this has some advantages for
transport because when calix[6]arene hexacarboxylic acid 5
was evaluated for the transport of 9 under neutral condi-
tions, no transport was observed. Acidification of the re-
ceiving phase restored modest transport.
When guest 11 was studied with calix[6]arene hexacarb-
oxylic acid 5 using a neutral receiving phase, less of the
guest was evident in the receiving phase than under control
conditions – the organic phase became highly colored, indi-
cating efficient extraction without transport. Over time,
precipitation occurred at the source phase interface. Meas-
uring as percent transported, 17% of source substrate 11
was extracted into the organic phase under neutral condi-
tions, but only 8% was transported to the receiving phase.
The remaining 9% was found in the organic layer upon
evaporation and resuspension in water. Upon acidification
of the receiving phase, the flux improved 84-fold. Com-
pared to a control experiment without 5, the flux was 20
times higher. Additionally, the decrease in source-phase ab-
sorbance resulted in a quantitative appearance of 11 in the
receiving phase (21%), so guest partitioning into the or-
ganic phase under acidic conditions became negligible.
We examined pillar[6]arene 8, a neutral host that demon-
strated binding affinity for the choline handle with guest
12, but showed no transporting ability under a variety of
conditions. In light of the NMR binding experiments (Fig-
ure 3), it is clear that ion–ion interactions are the key fea-
ture of hosts 4–6 that facilitate transport.
Figure 5. Charge balance for the extraction of guest 9 and trans-
port by 6.
A complete picture of transport by calixarene 6 first in-
volves extraction of the cationic guest into the organic layer.
This must be accompanied by charge balance – either chol-
ine is cotransported with Cl–, or ionization of one of the
phosphonic acids takes place, resulting in an ion–ion inter-
action and sequestration into the organic phase (Figure 5).
It is also possible that the guest ionizes if it contains a FITC
moiety (i.e., 9, 10) – but since NBD, which lacks an ioniz-
able group, is transported, this is not a necessary condition,
and the host is probably playing the dominant role. Deliv-
ery into a receiving phase would require a similar charge
balance, and one possibility is that phosphonate anion be-
comes protonated, and the choline handle is balanced by
hydroxide. We estimate the pKa of this type of phosphonate
monoester to be 2–3, thus ionization at the source phase is
assured. We have not ruled out the alternative cotransport
of Cl–, but our results with 4 and 5 strongly support the
hypothesis of host ionization. Charge neutrality is main-
tained by host ionization with the ammonium group, and
the inability of the corresponding esters to transport guests
is consistent with this analysis.
role in the need for a proton gradient. Efficient transport
for calix[4]arene tetraphosphonic acid 6 occurred at neutral
pH. The pH had a small effect for calix[4]arenecarboxylic
acid 4. Calix[6]arene 5, however, required a pH gradient,
the introduction of which resulted in a complete change in
When calix[6]arenecarboxylic acid 5 is used with guest
11 extraction is possible under neutral conditions, but not
transport, unless there is a pH gradient large enough to
protonate the corresponding acetate anion (Figure 6). It ap-
Figure 6. Acidification of the receiving phase with HCl accelerates
pears that the conformational differences play a significant transport of 11 by 5 over 84-fold.
6
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Calixarene Mediated Liquid-Membrane Transport
a UV/Vis spectrophotometer to determine the fraction transported
into the organic phase. The experiments were conducted in dupli-
cate, and the results presented are averages. Control experiments
without a host molecule were carried out.
behavior of the system. Indeed, ion–ion interactions must
be at work as the corresponding hexaester did not show
extraction or transport of NBD-choline 11. Thus, it is un-
likely that Cl– cotransport is operating, and that this can
resolve the issue of charge balance – ionizable groups are
essential here too.
U-tube Transport Experiments: These experiments were carried out
using a U-shaped glass tube as shown in Figure 7. The organic
phase consisted of a solution of the cavitand in CH2Cl2 (10 mL;
100 μm for resorcinarene-based cavitands, and 500 μm for calixar-
ene-based cavitands). The source phase consisted of an aqueous
solution (4 mL) of a guest molecule in either MilliQ water, NaOH
(0.1 m), or HEPES buffer solution (pH 13.26). The receiving phase
was either pure MilliQ water or HCl (0.1 m). For solutions with a
competing guest, MilliQ water or HEPES buffer solution (pH 5.94)
was used. The guest concentration in the source phase was 0.5 mm,
except in an experiment to determine the percentage transport of
NBD-choline as a function of time, in which case the guest concen-
tration was 1.0 mm. The U-tube’s upper ends were sealed, and the
experiments were started by stirring the organic phase with a mag-
netic stirring bar at 400 rpm. In experiments to determine the
transport flux, samples from the source phase and the receiving
phase were drawn after 144 h, and analyzed with a UV/Vis spectro-
photometer after adequate dilution with MilliQ water. In experi-
ments to determine the percentage transport of a guest molecule
as a function of time, equal volumes of samples (250 μL) were
drawn from the source phase and the receiving phase at 24 h inter-
vals, and analyzed with a UV/Vis spectrophotometer after dilution.
Two sets of experiments were conducted, and the data presented
are the average values. Control experiments using pure CH2Cl2 as
the organic phase were carried out.
Conclusions
Calix[6]arenecarboxylic acids have previously been
shown to transport cationic amino acids[20] through liquid
membranes, and more interestingly multiple calixarenes fa-
cilitate the transport of polycationic cytochrome C.[21] Un-
der acidic conditions, calix[6]arene hexacarboxylic acid 5
transports 4.7 times more guest 9 than 6. More interesting
to us is the ability of 4 and 6 to carry out transport under
neutral conditions. For tetracarboxylic acid 4, perhaps the
well-defined conformation imposed by the calix[4]arene
ring has some advantages for proton exchange at the receiv-
ing phase – or simply the overall energetics of host–guest
interaction are just right. Tetraphosphonic acid 6, substi-
tuted at the upper rim, also has a well-defined conforma-
tion, and may also allow some limited cation–π interactions
to take place. In addition, the lower pKa compared to car-
boxylic acids would clearly result in faster deprotonation at
the source interface, but the host is nevertheless likely to
be reprotonated at the receiving phase. Alternatively, Cl–
cotransport could be at work, or there could be some solu-
bility advantages to a rigid calix[4]arene scaffold or solubil-
ity advantages to phosponic– monoesters in the organic
phase. These issues remain to be resolved. The conforma-
tion of calix[6]arene hexacarboxylic acid 5 is ill defined in
organic solvents, yet with an acidified receiving phase, it has
a transport flux an order of magnitude higher than 4 or 6.
The ease of ionization of these hosts helps us to understand
why resorcinarene cavitands 1–3 failed to transport. These
cavitands have ionizable protons, as demonstrated in pre-
vious reports, but either they are not readily ionized under
the current conditions, or perhaps their binding is too
strong – giving rise to extraction, but not delivery. The vari-
ety of payloads we have reported is small, but the principle
of host–guest conjugate transport has been demonstrated.
A variety of choline–drug conjugates are currently being
examined to better understand the limits to this approach.
The application of these results in vesicle and cellular sys-
tems will be reported in the sequel.
Figure 7. U-shaped glass dimensions and experimental parameters.
Supporting Information (see footnote on the first page of this arti-
cle): Complete details for all experiments. Synthesis and characteri-
zation of all new compounds is described with NMR and MS data.
Experimental Section
Acknowledgments
Extraction Experiments: These experiments were carried out in
20 mL glass vials. The organic phase consisted of a solution of the
receptor in CH2Cl2 (100 μm for resorcinarene-based receptors, and
500 μm for calixarene-based receptors), and the aqueous phase con-
sisted of a solution of guests in MilliQ water. The guest concentra-
tion was varied from 10–200 μm. Equal volumes (2 mL each) of
organic and aqueous phases were mixed in the vial, and the mixture
was stirred at 160 rpm for 24 h. After cessation of stirring and
phase separation, samples from aqueous phase were analyzed with
This project was supported by the National Cancer Institute (grant
number SC2CA167636). The content is solely the responsibility of
the authors and does not necessarily represent the views of the
National Cancer Institute or the National Institutes of Health.
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Calixarene Mediated Liquid-Membrane Transport
Host–Guest Transport
B. B. Adhikari, A. Fujii,
M. P. Schramm* ............................... 1–9
Calixarene-Mediated
Liquid-Membrane
Transport of Choline Conjugates
Keywords: Host-guest systems / Molecular
recognition / Cavitands / Calixarenes /
Liquid-membrane transport / Choline con-
jugates
Ionizable calixarenes emerge as effective
transporters of choline-conjugated guests –
with or without a pH gradient. A series of
transport distinct molecular payloads
through a liquid membrane when attached
to a receptor-complementary handle.
supramolecular calixarenes
efficiently
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