Membrane-active calixarenes: toward 'gating' transmembrane anion transport

Article abstract of DOI:10.1016/j.tet.2007.06.124

This paper describes on-going efforts to develop calixarene amides as transmembrane anion transporters. We report on the transport of Cl^- anions across phospholipid membranes as mediated by some lipophilic calixarenes, all fixed in the cone conformation. We present significant findings regarding use of these calixarenes as transmembrane Cl^- transporters: (1) the cone conformer cone-H 2a, like its 1,3-alt and paco isomers, transports Cl^- across liposomal membranes; (2) the conformation of the calixarene (paco-H 1 vs cone-H 2a) is important for modulating Cl^- transport rates; (3) the substitution pattern on the calixarene's upper rim is crucial for Cl^- transport function; and (4) at least one of the four arms of the calixarene can be left unmodified without loss of function, enabling development of a pH-sensitive anion transporter (TAC-OH 3). This last finding is useful given the interest in gating the activity of synthetic ion transporters with external stimuli.

Full text of DOI:10.1016/j.tet.2007.06.124

Tetrahedron 63 (2007) 10743–10750
Membrane-active calixarenes: toward ‘gating’
transmembrane anion transport
Oluyomi A. Okunola, Jennifer L. Seganish, Kevan J. Salimian,
*
Peter Y. Zavalij and Jeffery T. Davis
Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA
Received 4 May 2007; revised 6 June 2007; accepted 8 June 2007
Available online 20 July 2007
We dedicate this paper to Professor David N. Reinhoudt on the occasion of his 65th birthday
Abstract—This paper describes on-going efforts to develop calixarene amides as transmembrane anion transporters. We report on the trans-
port of Cl anions across phospholipid membranes as mediated by some lipophilic calixarenes, all fixed in the cone conformation. We present
ꢀ
ꢀ
significant findings regarding use of these calixarenes as transmembrane Cl transporters: (1) the cone conformer cone-H 2a, like its 1,3-alt
and paco isomers, transports Cl across liposomal membranes; (2) the conformation of the calixarene (paco-H 1 vs cone-H 2a) is important
for modulating Cl transport rates; (3) the substitution pattern on the calixarene’s upper rim is crucial for Cl transport function; and (4) at
least one of the four arms of the calixarene can be left unmodified without loss of function, enabling development of a pH-sensitive anion
transporter (TAC-OH 3). This last finding is useful given the interest in gating the activity of synthetic ion transporters with external stimuli.
Ó 2007 Elsevier Ltd. All rights reserved.
ꢀ
ꢀ
ꢀ
1
. Introduction
Understanding relationships between channel structure and
function is crucial since maintaining the proper ion balance
across cell membranes is essential to life. Knowledge of
channel structure is also important because impaired ion
transport can cause disease. Thus, cystic fibrosis is caused
by a mutation in the CFTR protein that facilitates transmem-
As illustrated in Figure 1, we describe the use of lipophilic
calixarene amides to transport Cl anions across lipid mem-
branes. These studies build on recent work where we showed
that the partial cone calix[4]arene tetrabutylamide paco-H 1
ꢀ
1
ꢀ
4
mediated transmembrane anion transport. Before describing
our new studies we first provide some relevant background
brane Cl transport. These two-fold goals of understanding
natural ion transport processes and discovering new thera-
peutics have driven the development of synthetic transmem-
ꢀ
about (1) synthetic Cl transporters and (2) calixarenes as
membrane-active ion transporters.
5
brane ion transporters.
Structures of transmembrane ion channel proteins have been
determined recently, notably for the K and Cl channels.
Historically, most activity has centered on cation transporters.
There has been, however, continuing progress in identifying
+
ꢀ
2,3
O
N
O
H
NO3-
O
O O
1
O
O
Cl-
NH
O
HN HN
paco-H 1
Figure 1. Transmembrane anion transport as mediated by the calixarene paco-H 1 (see Ref. 1).
Keywords: Calix[4]arene; Transmembrane; Anion transport.
*
Corresponding author. Tel.: +1 301 405 1845; fax: +1 301 314 9121; e-mail: jdavis@umd.edu
0
040–4020/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2007.06.124

1
0744
O. A. Okunola et al. / Tetrahedron 63 (2007) 10743–10750
1,20–22
ꢀ
‘
small molecules’ that can transport Cl anions across lipid
relatively new.
conjugates,
Our studies on calixarene-nucleoside
designed as ion pair transporters, led us to
discover that a tetrabutylamide calix[4]arene fixed in the
3
1,32
membranes. A recent review by A. P. Davis and Smith
provides an excellent discussion of the emerging field of
ꢀ
6
ꢀ
transmembrane Cl transport. Synthetic Cl transporters
include compounds that function as self-assembled channels
or as monomolecular carriers. These compounds, which can
altertransmembraneionandpHgradients, mayhave potential
applications as biochemical reagents and/or chemotherapeu-
tic agents. Tomich and colleagues used small peptides derived
from the glycine receptor protein to form Cl channels in
phospholipid bilayers. Gokel and colleagues have reported
studies on lipophilic peptides that aggregate to form Cl se-
lective ion channels. Regen synthesized a sterol analog
whose protonated polyamine side-chain functions as a trans-
membrane ‘anion slide’. Significantly, this analog restored
Cl transport in cystic fibrosis cells. The groups of A. P.
Davis and Smith have also used sterols, coined ‘cholapods’,
to transport Cl across liposomal and cell membranes.
Gin described an anion channel formed from an amino-cyclo-
dextrin. There is also interest in the prodigiosins, tripyrrole
natural products that bind HCl. Recently, the groups of
Gale, Smith, Sessler, Thompson, and J. T. Davis have shown
that prodigiosin analogs transport Cl across lipid mem-
1,3-alternate conformation forms ion channels that move
ꢀ
20
Cl across phospholipid membranes. Presumably, the
NH protons on the calixarene’s secondary amides hydrogen
bonds to Cl and help its translocation across the mem-
ꢀ
3
3
brane. X-ray structures confirmed that this 1,3-alt calix-
arene self-assembled into channel-like motifs held together
ꢀ
ꢀ
20
by hydrogen bonds to bridging Cl anions. Voltage clamp
experiments in planar lipid bilayers confirmed that this
1,3-alt calixarene tetra-amide formed discrete ion channels.
7
ꢀ
8
ꢀ
Subsequently, we investigated the structure and Cl ion
transport activity of a related isomer, the partial cone cal-
9
1
ix[4]arene tetrabutylamide paco-H 1 shown in Scheme 1.
We proposed that Cl transport mediated by paco-H 1
ꢀ
10
ꢀ
was controlled by both ligand self-association and side-
chain conformation.
ꢀ
11
1
2
ꢀ
In this paper, we report on comparative Cl transport activity
for four calixarene amides all fixed in the cone conforma-
1
3
tion. We present significant findings regarding the use of
these calixarenes as transmembrane Cl transporters: (1)
ꢀ
ꢀ
branes, settingthestage forfurtherdevelopment ofthesecom-
pounds. Recently, we and others have demonstrated that
simple isophthalamides can function as efficient transmem-
the cone conformer cone-H 2a (R¼H), like its 1,3-alt and
1
4–17
ꢀ
paco isomers, transports Cl across liposomal membranes;
(2) the conformation of the calixarene scaffold (paco-H 1
ꢀ
18,19
ꢀ
brane transporters of Cl .
vs cone-H 2a) is important for modulating Cl transport
rates; (3) the substitution pattern on the calixarene’s upper
rim is crucial for Cl transport function; and (4) at least
ꢀ
Some of our group’s contributions in the anion transport field
A review by
1
,20–22
have used the calix[4]arene scaffold.
one of the four arms of the calixarene can be left unmodified
without loss of function, enabling the development of a
pH-sensitive anion transporter (TAC-OH 3). This last find-
ing is timely given the interest in gating the activity of syn-
Cragg and Iqbal nicely describes previous studies that
have used calixarene-type macrocycle as transmembrane
cation transporters, as channels or as mobile carriers.
2
3
2
4
25
34
Kobuke and colleagues, and Gokel and co-workers,
thetic ion transporters by using external stimuli. There is
have described formation of synthetic cation channels using
lipophilic calixarenes. Recently, Carreira et al. discovered
a calixarene-amphotericin conjugate that forms K selective
also an interesting parallel between this ‘small molecule’
and the ClC chloride transporter protein, as it has been pro-
posed that anion transport by ClC is gated by charge–charge
+
2
6
ꢀ
channels. Various calixarenes also function as mobile
cation carriers. Thus, Beer and colleagues have described
resorcin[4]arene-crown analogs that are efficient transmem-
repulsions between Cl and negatively-charged glutamate
residues within the anion channel.
3
5
+
27
brane carriers of K . Jin has also shown that a calix[4]-
arene-crown-5 analog selectively transports K across
+
2. Results and discussion
2
8
planar bilayers. Izzo and colleagues have recently de-
scribed calix[4]arene-cholic acid conjugates that can move
both H and K cations across synthetic liposomes.
2.1. Rationale for studying calixarenes 1–4
+
+
29
The compounds that we studied are shown in Scheme 1. We
previously reported that paco-H 1 transports Cl across li-
posomal membranes. Herein, we use paco-H 1 as a standard
3
0
ꢀ
While calixarenes are known to bind anions in solution,
their use as membrane-active anion transporters is
1
O
N
O
R R
H
R
R
O
O
O
O
O O
O O
O O
O O
O
HO
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
NH
NH
NH
NH
HN HN
HN HN
HN HN
HN HN
NH
O
paco-H 1
cone-H (R=H) 2a
TAC-OH 3
TAC-OEster 4
cone-tBu (R=tBu) 2b
ꢀ
Scheme 1. Compounds studied for their transmembrane Cl transport activity.

O. A. Okunola et al. / Tetrahedron 63 (2007) 10743–10750
10745
ꢀ
for comparing the Cl transport activity of the cone calixar-
enes 2–4. Compounds 2a (cone-H) and 2b (cone-tBu) are
fixed in the cone conformation and feature four n-butyl sec-
ondary amides on their lower rim. They differ in the substi-
tution pattern on their upper rims. In previous work with
paco-H 1 we found that substitution of the calixarene’s up-
a.
OH
HO
OH
OH
OH
OH HO
OH
ꢀ
7
8
per rim with tert-butyl groups led to loss of Cl transport ac-
tivity. In this study we set out to determine if this same
1
b.
phenomenon held true for the cone conformers 2a (R¼H)
and 2b (R¼tBu). The other two analogs, phenol 3 (TAC-
OH) and ester 4 (TAC-OEster), were made so that we could
determine the functional effect of either removing or replac-
ing a single n-butyl amide on the macrocycle’s lower rim.
Replacement of a secondary amide with another functional
group (as in 4) without complete loss of activity might pro-
vide the opportunity for appending other anion-sensitive
groups to the calixarene. This might allow development of
O
O
O O
c.
O O
HO
O
O
O
O
O
O
O
NH
O
NH
HN HN
O
HN HN
ꢀ
new Cl sensors and transporters. We also reasoned that
deprotonation of the phenolic proton on TAC-OH 3 might
ꢀ
TAC-OEster 4
TAC-OH 3
enable formation of a pH-sensitive switch for gating Cl
transport across lipid membranes.
Scheme 3. Synthesis of tris-N-butylamido calixarenes 3 and 4. (a) AlCl
ꢁ
3
,
, BaO, DMF, 40 C; and
PhOH, PhCH
3
, rt; (b) BrCH
Bu, Cs CO
2
CONHBu, Ba(OH)
ꢁ
2
2
.2. Synthesis of calixarenes 2–4
(c) BrCH
2
CO
2
2
3
, DMF, 70 C.
The synthesis of compounds 1–4 was straightforward. Calix-
arenes 1 and 2b (R¼tBu) were prepared using published
1
H NMR data, showing two sets of doublets with identical
integration between d 3.2 and 4.5 ppm, corresponding to
two AB systems. One AB system consists of doublets at
d 4.27 and 3.35 ppm with J¼13.5 Hz. The second AB sys-
1
,36
methods.
As shown in Scheme 2, cone-H 2a was syn-
3
7
thesized from the corresponding tetra-ester 5 via (i) hydro-
lysis to the tetra-acid 6; (ii) acid chloride activation; and (iii)
amide bond formation.
tem for the other four ArCH Ar protons in TAC-OH 3 con-
2
sists of doublets at d 4.19 and 3.43 ppm with J¼14.4 Hz. The
40
The tris-N-butylamido phenol analog TAC-OH 3 was pre-
tri-substituted TAC-OH 3 displays two pairs of doublets
because of the asymmetry at its lower rim. The cone con-
3
8
pared from calix[4]arene 8, by alkylation with 2-bromo-
N-butylacetamide (Scheme 3). The trisubstituted derivative
TAC-OH 3 was separated from other alkylation products
by chromatography. Further alkylation of TAC-OH 3 with
n-butyl-2-bromoacetate gave ester 4.
formation for TAC-OEster 4 was confirmed by the presence
of a pair of doublets. The AB system for the ArCH Ar pro-
2
tons in 4 consists of the doublets at d 4.44 and 3.26 ppm with
J¼14.0 Hz.
2
.3. Characterization
In addition to spectroscopic analysis, cone-H 2a and
TAC-OEster 4 were characterized as cone conformers by
1
13
41,42
Compounds 2a, 2b, 3, and 4 were characterized by H,
C
X-ray crystallography.
Figure 2 shows the X-ray crystal
1
NMR spectroscopy and ESI-MS analysis. The H NMR
spectra of 2a, 2b, 3, and 4 indicated that each analog existed
as a cone conformer in solution, as shown by characteristic
AB coupling for bridging ArCH Ar protons. Calixarenes
2
that are tetra-substituted at their lower rim and fixed in
the cone conformation usually display a pair of doublets
between d 3.2 and 4.5 ppm for diastereotopic ArCH Ar pro-
2
tons. This was true for 2a and 2b. The cone conformation
for the trisubstituted TAC-OH 3 was also consistent with the
structures for tetra-amide cone-H 2a and tri-amide TAC-
OEster 4. In the solid-state, both compounds adopt a
4
3
‘pinched-cone’ conformation with C2v symmetry. Both
compounds also feature two intramolecular hydrogen bonds
between two amide NH groups and neighboring carbonyl
oxygens. This conformation, if maintained in solution,
would leave additional amide NH protons in cone-H 2a
(two free NH groups) and TAC-OEster 4 (one free NH
3
9
ꢀ
group) available for hydrogen bonding to Cl anion.
a.
b.
O
O
O
O O
O O
O O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
NH
EtO EtO EtO
HO HO HO
HN HN
NH
OEt
OH
5
6
cone-H 2a
2
, Et N, CH Cl , rt.
Scheme 2. Synthesis of cone-H 2a. (a) KOH aq, MeOH, THF, rt; (b) 1. SOCl
2
, benzene, reflux, 2. BuNH
2
3
2

10746
O. A. Okunola et al. / Tetrahedron 63 (2007) 10743–10750
with egg-yolk phosphatidylcholine (EYPC) liposomes con-
taining lucigenin and 100 mM NaNO /10 mM sodium phos-
3
ꢀ
phate buffer (pH 6.4). We then introduced Cl to the
extravesicular buffer by adding an aliquot of a NaCl solution
in 10 mM sodium phosphate (pH 6.4). The resulting Cl
concentration gradient across the membrane was relieved
by ligand-mediated transport of Cl into the liposomes, re-
sulting in the quenching of lucigenin’s fluorescence. Intra-
vesicular Cl anion concentration was calculated from the
dye’s fluorescence using the Stern–Volmer constant deter-
mined under the assay conditions.
ꢀ
ꢀ
ꢀ
cone-H 2a
TAC-OEster 4
Figure 2. X-ray crystal structures of cone-H 2a (left) and TAC-OEster 4
right) showing the ‘pinched-cone’ conformations for both compounds.
(
Intramolecular H-bonds between secondary amide NH protons and neigh-
boring carbonyls are indicated by the dotted lines. The butyl side chains
are removed for clarity. Arrows point to the ‘free’ amide NH groups.
L
2.5.1. Calixarene conformation attenuates Cl transport
rate. Figure 4 shows that both paco-H 1 and cone-H 2a
ꢀ
transport Cl across the EYPC liposomes. It appears that
the paco-H 1 conformer is about twice as efficient as the
cone isomer 2a when administered at the same concentration
L
2
.4. NMR titration study shows Cl binding to TAC-OH 3
(2:100 ligand to lipid). Despite its lower activity relative to
paco-H 1 the calixarene cone-H 2a is still an active trans-
membrane Cl transporter. This result encouraged us to con-
tinue our studies on the related cone analogs cone-tBu 2b,
TAC-OH 3, and TAC-OEster 4, as described below.
Compounds 2–4, which contain either three or four second-
ary amide NH groups on their lower rim, all bind Cl weakly
in non-polar organic solvents. Thus, titration of solutions
of these calixarenes with tetrabutylammonium chloride
ꢀ
ꢀ
(
TBACl) in CD Cl typically showed a downfield shift of
2 2
the secondary amide NH protons and, assuming a 1:1 bind-
ing stochiometery, gave binding constants on the order of
1
1
8
3
8
3
2
ꢀ
1
paco-H 1
K ¼10–30 M . For example, representative NMR titration
a
ꢀ
data for interaction of Cl with TAC-OH 3 are shown in Fig-
ure 3. Upon addition of TBACl to a solution of TAC-OH 3
cone-H 2a
in CD Cl we observed downfield shifts for the two ‘outer’
2
2
amide NH protons (Dd¼0.40 ppm, labeled in blue), and
for the ‘central’ amide NH proton (Dd¼0.14 ppm, labeled
in red). The association constant for binding TBACl by
Blank
ꢀ
1
TAC-OH 3 was determined to be K ¼24.1ꢂ5.1 M in
a
4
4
CD Cl , which is similar to the value that we previously
2
2
0
50
100
150
time, s
200
250
300
-
ꢀ
obtained for binding of Cl by the secondary amide NH
groups in paco-H 1 (K ¼28.7ꢂ17 M ).
ꢀ
1 1
a
Figure 4. Chloride transport across EYPC liposomes containing lucigenin
in a 100 mM NaNO /10 mM sodium phosphate buffer (pH 6.4). The Cl
3
ꢀ
concentration was determined from lucigenin’s fluorescence. Compounds
and 2a were added to give a 2 mol % ligand to lipid ratio. At t¼15 s,
0
.0 eq
1
NaCl was added to give an external Cl concentration of 24 mM. Lucigenin
ꢀ
0
1
.5 eq
.0 eq
ꢀ
O
O O
fluorescence was converted to Cl concentration using the Stern–Volmer
constant determined under the assay conditions. The traces shown are the
average of three trials.
HO
O
O
O
NH
2.0 eq
HN HN
L
5
1
.0 eq
2.5.2. Upper-rim substitution pattern is crucial for Cl
transport. To investigate the influence of upper-rim substi-
tution on Cl transport activity, the substituted analog
0.0 eq
ꢀ
TAC-OH 3
8
.00
7.50
ppm (t1)
7.00
cone-tBu 2b was tested in this lucigenin assay. As shown
in Figure 5, cone-tBu 2b, unlike its analog cone-H 2a,
was essentially inactive toward transmembrane Cl trans-
port under these standard conditions. This result is consistent
with our previous studies wherein we found that substitution
of a paco calixarene with tBu groups resulted in complete
loss of anion transport. Again, the substitution pattern on
the upper rim of the calixarene tetra-amide is clearly critical
for Cl transmembrane transport.
ꢀ
1
Figure 3. H NMR stack plot showing titration of TAC-OH 3 with TBACl.
Blue lines (–) mark the changes in chemical shift for the two terminal NH
protons, while the red lines (–) mark the changes in chemical shift for the
central NH proton.
1
L
2
.5. Transmembrane Cl transport results
ꢀ
ꢀ
Compounds 1–4 were tested for transmembrane Cl trans-
port activity using assays previously reported by our
group.
lucigenin, were subjected to a Cl gradient in the extravesic-
ular solution. Lucigenin is a dye whose fluorescence is
quenched upon binding Cl . In a typical experiment (in
2.5.3. Influence of lower-rim modification on transport:
chloride transport by phenol 3 and ester 4. The transmem-
brane Cl transport activity by the trisubstituted amide
calixarenes, phenol 3 and ester 4, was also assessed using
the standard lucigenin assay. As shown in Figure 6, the tris-
N-butylamido TAC-OH, 3 transports Cl ions across EYPC
liposomal membranes at pH 6.4 even more efficiently than
1
,22
ꢀ
Liposomes loaded with the Cl sensitive dye,
ꢀ
ꢀ
ꢀ
45
ꢀ
1
00 mM NaNO /10 mM sodium phosphate buffer (pH
3
6
.4)) we incubated calixarenes 1–4 (2:100 ligand/lipid)

O. A. Okunola et al. / Tetrahedron 63 (2007) 10743–10750
10747
OH group on TAC-OH 3 should introduce unfavorable elec-
trostatic interactions between the calixarene and Cl ions.
1
1
8
3
8
3
2
ꢀ
Such electrostatic repulsions should inhibit anion binding
and reduce the efficacy of TAC-OH 3 as an anion trans-
porter. Second, TAC-OH 3 has to integrate into the mem-
brane to enable transport. The deprotonated TAC-OH 3
would undoubtedly have a lower permeability to the lipo-
philic membrane when it is negatively charged.
cone-H 2a
cone-tBu 2b
Blank
ꢀ
On the other hand, transmembrane Cl transport by cone-H
2a should not be as sensitive to pH since 2a does not have
0
50
100
150
time, s
200
250
300
-
sufficiently acidic protons to be significantly deprotonated
at pH 7–10. The transport function of TAC-OH 3 and
cone-H 2a was monitored as a function of extravesicular
pH using EYPC liposomes loaded with the Cl dye, lucige-
nin (Fig. 7).
Figure 5. Chloride transport across EYPC liposomes containing lucigenin
in a 100 mM NaNO /10 mM sodium phosphate buffer (pH 6.4). The Cl
3
concentration was determined from lucigenin’s fluorescence. Compounds
ꢀ
ꢀ
2
a and 2b were added to give a 2 mol % ligand to lipid ratio. At t¼15 s,
ꢀ
NaCl was added to give an external Cl concentration of 24 mM. Lucigenin
fluorescence was converted to Cl concentration using the Stern–Volmer
ꢀ
constant determined under the assay conditions. The traces shown are the
average of three trials.
ꢀ
As shown in Figure 7a, the rate of Cl transport mediated by
TAC-OH 3 clearly decreased as the extravesicular solution
ꢀ
became more basic. In marked contrast, the Cl transport
rate by cone-H 2a was constant over the same pH range
1
8
3
8
3
2
TAC-OH 3
cone-H 2a
1
O
O O
HO
O
TAC-OEster 4
Blank
O
O
NH
HN HN
pH
(a)
7
6
6
.4
.4
7
0
50
100
150
time, s
200
250
300
-
5
TAC-OH 3
8.0
4
9.0
Figure 6. Chloride transport across EYPC liposomes containing lucigenin
in a 100 mM NaNO /10 mM sodium phosphate buffer (pH 6.4). The Cl
3
concentration was determined from lucigenin’s fluorescence. Compounds
3
ꢀ
2
Blank
2
a, 3, and 4 were added to give a 2 mol % ligand to lipid ratio. At t¼15 s,
1
ꢀ
NaCl was added to give an external Cl concentration of 24 mM. Lucigenin
fluorescence was converted to Cl concentration using the Stern–Volmer
ꢀ
0
0
50
100
150
time, s
200
250
300
constant determined under the assay conditions. The traces shown are the
average of three trials.
-1
does the tetra-amide cone-H 2a. This result, indicating that
replacement of one of the secondary amide side chains with
an acidic OH group could be functionally tolerated, promp-
ted us to study TAC-OH 3 as a pH-sensitive transporter (see
Section 2.5.4). The data in Figure 6 also show that ester 4 is
O
O
O
(b)
O
O
7
6
5
4
3
2
1
0
O
O
O
NH
HN HN
NH
ꢀ
less efficient as a Cl transporter than tetra-amide cone-H
a. However, while substitution of one amide side-chain
pH
2
6.4, 7.4,
ꢀ
with an ester resulted in reduction in Cl flux across the
membrane, definite transport activity remained for TAC-
OEster 4 above background. Thus, we are hopeful that sub-
stitution on the calixarene cone’s lower rim with an ester or
ether sidechain containing an anion-sensitive reporter may
cone-H 2a
8.0, 9.0
Blank
ꢀ
eventually be useful for the development of Cl anion sen-
0
50
100
150
time, s
200
250
300
-1
sors. For now, we decided to focus on TAC-OH 3 as a means
for developing a compound whose transmembrane transport
activity can be switched on and off by changing pH.
ꢀ
Figure 7. Transmembrane Cl transport as a function of pH: (a) with
TAC-OH 3 and (b) with cone-H 2a. Experiments were done using EYPC
3
liposomes with lucigenin (1 mM) in a 100 mM NaNO /10 mM sodium
L
2.5.4. Modulating Cl transmembrane transport by pH.
We reasoned that the phenolic OH on the unsubstituted arene
phosphate buffer at various pHs (6.4, 7.4, 8.0, and 9.0). Compounds 2a
and 3 were added to give a 2 mol % ligand to lipid ratio. At t¼15 s, NaCl
ꢀ
ꢀ
solution was added to give an external Cl concentration of 24 mM. Luci-
genin fluorescence was converted to Cl concentration using the Stern–
of TAC-OH 3 afforded the potential to develop a pH-tunable
Cl transporter. For example, a trimethylated calix[4]arene
with a free phenol has a pK ¼12.5. Deprotonation of the
ꢀ
Volmer constant determined under the assay conditions. The traces shown
are the average of three trials.
4
6
a

10748
O. A. Okunola et al. / Tetrahedron 63 (2007) 10743–10750
between 6.4 and 9 (Fig. 7b). The data are consistent with
a negative charge in TAC-OH 3 acting to inhibit transmem-
brane transport of the Cl anion, by limiting anion binding
and/or partitioning of the calixarene into the liposomal
membrane. Control experiments with cone-H 2a support
this conclusion.
in vacuo for 1 h and then dissolved in 20 mL of CH Cl .
2 2
N-Butylamine (1.32 mL, 16 equiv) was added dropwise to
the stirring solution at rt. Triethylamine (1.2 mL, 10 equiv)
was then added dropwise and the reaction stirred under N2
for 12 h at rt. The solvent was evaporated and the resulting
solid partitioned between CHCl and H O. The organic layer
ꢀ
3
2
was dried with sodium sulfate and evaporated. Pure 2a
332 mg, 44.5% yield from the tetra-ester) was obtained
after silica gel chromatography with 3% MeOH–CH Cl .
(
3
. Summary
2
2
1
ꢁ
H NMR (CDCl , 25 C) d: 7.34 (br s, 4H, CONHCH ),
3
2
In a recent review on the use of calixarenes to effect trans-
membrane ion transport, Cragg and Iqbal suggested that
charge–charge interactions might be used as a strategy for
6
.62–6.59 (m, 12H, ArH), 4.50 (d, J¼13.9 Hz, 4H,
2 2
ArCH Ar), 4.43 (s, 8H, ArOCH CO), 3.34 (q, J¼6.6 Hz,
8
1
H, CONHCH ), 3.24 (d, J¼14.0 Hz, 4H, ArCH Ar),
2
3
2
2
gating transport. The studies reported in this paper, and
.59–1.52 (m, 8H, NHCH CH ), 1.37–1.29 (m, 8H,
2 2
1
7
in another recent paper from our labs, suggest that electro-
static repulsion can be used to turn on and off transmem-
brane transport of Cl anion. This regulatory mechanism,
observed with these ‘small molecule’ transporters, is analo-
gous to the proposal that the self-assembled ClC protein
channel makes use of negatively-charged Glu residues to
gate Cl transport. It may also be possible to modulate an-
ion transport rates by incorporating a positive charge, such as
a quaternary ammonium group, into the calixarene frame-
work. Finally, it is possible that a lipophilic compound
such as the deprotonated TAC-OH 3 may be a candidate
for transmembrane cation transport. Studies are under way
to determine these issues.
NH(CH ) CH ), 0.91 (t, J¼7.3 Hz, 12H, NH(CH ) CH ).
2
2
2
2 3
3
1
3
ꢁ
23.7, 74.5, 39.6, 32.0, 31.4, 20.6, 14.2. Mass calculated
C NMR (CDCl , 25 C) d: 170.0, 156.3, 134.7, 129.3,
3
ꢀ
1
for C H N O : 876.504. Mass found (+)-ESI-MS:
5
2 68 4 8
+
+
8
77.513 (M+H ); 899.477 (M+Na ).
ꢀ
35
4.1.1.2. 25,26,27,28-Tetrakis(hydroxy)calix[4]arene
8). A slurry of p-tert-butylcalix[4]arene 7 (1.03 g,
3
8
(
1
.59 mmol) and phenol (0.842 g, 8.94 mmol) in toluene
(
(
30 mL) was stirred under N2 for 0.5 h. Then, AlCl3
1.29 g, 9.69 mmol) was added and the mixture stirred for
2
days at rt. The reaction was quenched with 0.2 N HCl
(
(
35 mL), the solvent was removed in vacuo and MeOH
7 mL) added at which point a precipitate formed. The pre-
4
. Experimental section
cipitate was filtered to yield crude 8, which was purified by
recrystallization (2% MeOH–CHCl ) to give 8 (0.337 g,
3
ꢁ
1
4
.1. General
50%) as transparent crystals. Mp 297–300 C. H NMR
ꢁ
(
CDCl , 25 C) d: 10.20 (s, 4H, ArOH), 7.06 (d, J¼7.6 Hz,
3
1
H NMR spectra were recorded on a Bruker DRX-400 or
a Bruker Avance 400 instrument operating at 400.13 MHz.
C NMR spectra were recorded on a Bruker DRX-400 in-
strument operating at 100.52 MHz. Electrospray ionization
mass spectra were recorded on a JEOL AccuTOF-CS instru-
ment. Deuterated solvents were purchased from Cambridge
Isotope. Chemicals and solvents were purchased from
Sigma, Aldrich, Fisher, Fluka or Acros. Lucigenin was pur-
chased from Molecular Probes and EYPC was purchased
from Avanti Polar Lipids.
8H, ArH), 6.73 (t, J¼7.6 Hz, 4H, ArH), 4.27 (s, 4H,
1
3
2 2 3
ArCH Ar), 3.55 (s, 4H, ArCH Ar). C NMR (CDCl ,
25 C) d: 149.2, 129.3, 128.8, 122.7, 32.2. Mass calculated
1
3
ꢁ
C H O : 424.167. Mass found ESI-MS: 425.229 (M+H );
463.051 (M+K ).
+
2
8 24 4
+
4.1.1.3. 25-Hydroxy-26,27,28-tris(2-butylamidometh-
oxy)calix[4]arene (TAC-OH 3). A solution of 8 (0.804 g,
1.89 mmol), Ba(OH)2 (1.79 g, 5.67 mmol), and BaO
ꢁ
(1.69 g, 11.0 mmol) in DMF (15 mL) was stirred at 40 C
for 1 h under N . A solution of 2-bromo-N-butylacetamide
2
1
36
4
.1.1. Synthetic procedures. Calixarenes 1 and 2b were
(1.10 g, 5.67 mmol) in DMF (5 mL) was added dropwise
and the mixture stirred for 4 h. The mixture was diluted
with CH Cl (60 mL), washed with 0.2 N HCl (3ꢃ30 mL)
prepared as reported from the corresponding paco or cone
tetra-esters by: (i) hydrolysis to the tetra-acid; (ii) activation
to the acid chloride; and (iii) amide bond formation.
2
2
and H O (3ꢃ30 mL), and dried over MgSO . The solvent
2
4
was removed in vacuo to give a yellow residue. TAC-OH
3 (0.376 g, 26%) was separated by silica chromatography
(1:1 EtOAc–hexanes) to give translucent flakes. Mp 121–
4.1.1.1. 25,26,27,28-Tetrakis(2-butylamidomethoxy)-
calix[4]arene (cone-H 2a). Cone 25,26,27,28-tetrakis-(ethyl-
acetylmethoxy)calix[4]arene 5 (275 mg, 0.36 mmol) and
mL of 45% aqueous KOH were stirred in 6 mL of metha-
nol/THF (1:1) at rt for 14 h. The solvent was evaporated
under reduced pressure. The resulting solid was dissolved
in a minimum amount of water, acidified with 6 N HCl,
and the water removed under reduced pressure. The material
was dissolved in acetone, solid KCl was removed by filtra-
tion and acetone was then evaporated to give the tetra-acid
as a white solid. The acid chloride was prepared by stirring
a solution of the calixarene tetra-acid 6 (565 mg, 0.85 mmol)
3
7
ꢁ
1
ꢁ
123 C. H NMR (CDCl , 25 C) d: 7.98 (t, J¼5.4 Hz, 1H,
3
1
CONHCH ), 7.27 (d, J¼7.8 Hz, 2H, ArH), 7.16 (d, J¼
2
7.2 Hz, 2H, ArH), 7.13 (t, J¼7.8 Hz, 1H, ArH), 7.01 (t,
J¼5.8 Hz, 2H, CONHCH ), 6.88 (t, J¼7.5 Hz, 1H, ArH),
2
6.47 (d, J¼7.4 Hz, 2H, ArH), 6.44 (td, J¼7.5, 1.8 Hz, 2H
ArH), 6.35 (dd, J¼7.3, 1.8 Hz, 2H ArH), 4.39 (s, 2H, Ar-
OCH CO), 4.36 (d, J¼14.9 Hz, 2H, ArOCH CO), 4.27 (d,
2
2
J¼13.6 Hz, 2H, ArCH Ar), 4.19 (d, J¼14.5 Hz, 2H,
2
ArCH Ar), 4.15 (d, J¼14.8 Hz, 2H, ArOCH CO), 3.75 (s,
2
2
1H, ArOH), 3.43 (d, J¼14.4 Hz, 2H, ArCH Ar), 3.35 (d, J¼
2
in 20 mL of benzene with 5.1 mL of SOCl . The reaction
2
13.5 Hz, 2H, ArCH Ar), 3.28–3.49 (m, 6H, NHCH CH ),
2
2
2
mixture was heated at reflux for 2.5 h and then concentrated
under reduced pressure. The sticky brown solid was dried
1.49–1.64 (m, 6H, NHCH CH ), 1.33–1.43 (m, 4H,
2 2
NH(CH ) CH ), 1.24–1.33 (m, 2H, NH(CH ) CH ), 0.93
2
2
2
2 2
2

O. A. Okunola et al. / Tetrahedron 63 (2007) 10743–10750
10749
(
NH(CH ) CH ). C NMR (CDCl , 25 C) d: 169.1, 168.4,
t, J¼7.3 Hz, 6H, NH(CH ) CH ), 0.75 (t, J¼7.3 Hz, 3H,
6.4, 100 mM NaNO ) to give a solution that is 0.5 mM in
3
2
3
3
1
3
ꢁ
lipid. Compounds 1–4 were added to give a 2:100 ligand
to lipid ratio. To the cuvette containing the EYPC-trans-
porter mixture was added 20 mL of 2.425 M NaCl solution
through an injection port, after 15 s, to give an external
2
3
3
3
1
1
3
55.1, 153.6, 152.5, 136.5, 132.9, 132.8, 131.4, 130.8,
29.4, 129.3, 128.7, 125.4, 125.0, 121.2, 75.1, 74.7, 40.2,
9.6, 32.2, 31.6, 31.2, 30.6, 20.8, 20.6, 14.2, 14.0. Mass cal-
ꢀ
ꢀ
culated for C H N O : 763.420. Mass found (+)-ESI-MS:
4
Cl concentration of 24 mM. Intravesicular Cl concentra-
tion was monitored as a function of lucigenin fluorescence.
The fluorescence of lucigenin was monitored at 372 nm
and emission at 503 nm for 300 s. The cuvettes were kept
6 57 3 7
+
+
7
64.477 (M+H ); 786.462 (M+Na ).
4
.1.1.4. 25-(2-Butoxycarbonylmethoxy)-26,27,28-
ꢁ
tris(2-butylamidomethoxy)calix[4]arene (TAC-OEster 4).
A solution of TAC-OH 3 (0.100 g, 0.13 mmol) and
Cs CO (0.43 g, 13.1 mmol) in DMF (6 mL) was stirred at
at 25 C during the experiment with a constant water temper-
ature bath. After 270 s, 40 mL of 10% Triton-X detergent
was added to lyse the liposomes. Experiments were done
in triplicate and all traces reported are the average of the
three trials. Lucigenin fluorescence was converted to
chloride concentration using the Stern–Volmer constant de-
2
3
ꢁ
0 C for 10 min under N gas. A solution of butyl 2-bro-
2
7
moacetate (0.255 g, 1.31 mmol) in DMF (4 mL) was added
dropwise and the mixture stirred. After 24 h, the reaction
mixture was cooled to rt, and then quenched with 1.0 M
HCl (10 mL). The aqueous mixture was extracted with
CH Cl (3ꢃ10 mL) and the resulting organic layer was
1
,22
termined under the assay conditions.
constant was determined by taking the slope of a plot of
The Stern–Volmer
f0/f versus chloride concentration.
2
2
washed with distilled H O (4ꢃ15 mL), dried over MgSO ,
2
4
and the solvent was removed in vacuo. The residue was
subsequently purified by silica gel chromatography (1:1
EtOAc–Hexanes) to give compound 4 as a white solid. Yield
Acknowledgements
1
ꢁ
5%; H NMR (CDCl , 25 C) d: 7.91 (t, 2H, CONHCH ),
3 2
4
6
2
We thank the Office of Basic Energy Sciences, U.S. Depart-
ment of Energy for financial support.
.88–6.77 (m, 7H, ArH, CONHCH ), 6.74 (t, J¼7.5 Hz,
2
H, ArH), 6.32 (d, J¼7.5 Hz, 4H, ArH), 4.77 (s, 2H,
ArOCH CO ), 4.59 (d, J¼14.0 Hz, 2H, ArOCH CONH),
2
2
2
4
.44 (d, J¼14.0 Hz, 4H, ArCH Ar), 4.44 (s, 2H ArOCH -
2
2
CONH), 4.31 (d, J¼14.6 Hz, 2H, ArOCH CONH), 4.13 (t,
References and notes
2
J¼6.8 Hz, 2H, CO CH ), 3.45–3.34 (m, 6H, NHCH ),
2
2
2
3
6
1
.26 (dd, J¼14.0, 4.5 Hz, 4H, ArCH Ar), 1.65–1.57 (m,
1. Seganish, J. L.; Santacroce, P. V.; Salimian, K. J.; Fettinger,
J. C.; Zavalij, P.; Davis, J. T. Angew. Chem., Int. Ed. 2006,
45, 3334–3338.
2
H, NHCH CH ), 1.51–1.45 (m, 2H, CO CH CH ), 1.42–
2
2
2
2
2
.32 (m, 6H, NH(CH ) CH ), 1.31–1.24 (m, 2H,
2
2
2
CO (CH ) CH ), 0.93 (t, J¼7.4 Hz, 6H, NH(CH ) CH ),
2. MacKinnon, R. Angew. Chem., Int. Ed. 2004, 43, 4265–4277.
3. Dutzler, R.; Campbell, E. B.; Cadene, M.; Chait, B. T.;
MacKinnon, R. Nature 2003, 415, 287–294.
4. Sheppard, D. N.; Rich, D. P.; Ostedgaard, L. S.; Gregory, R. J.;
Smith, A. E.; Welsh, M. J. Nature 1993, 362, 160–164.
5. For a recent review on synthetic pores and channels, see:
Sisson, A. L.; Shah, M. R.; Bhosale, S.; Matile, S. Chem.
Soc. Rev. 2006, 35, 1269–1286.
2
2 2
2
2 3
3
0
3
1
1
3
2
.92 (t, J¼7.3 Hz, 3H, NH(CH ) CH ), 0.90 (t, J¼7.7 Hz,
ꢁ
2 2 3 3 3
2
3
3
1
3
H, CO (CH ) CH ). C NMR (CDCl , 25 C) d: 171.6,
69.6, 169.5, 155.3, 135.5, 135.3, 133.8, 133.5, 129.9,
29.0, 128.7, 123.9, 123.6, 123.5, 78.1, 74.6, 71.9, 65.4,
9.7, 39.5, 39.4, 32.3, 32.1, 31.4, 31.3, 31.0, 30.1, 20.7,
0.5, 19.5, 14.3, 14.2, 14.1. Mass calculated for
C H N O : 877.488. Mass found (+)-ESI-MS: 878.507
5
2 67 3 9
+
+
2+
(
M+H ); 900.480 (M+Na ); 1014.549 (M+Ba ).
6. Davis, A. P.; Sheppard, D. N.; Smith, B. D. Chem. Soc. Rev.
2
007, 36, 348–357.
4
.1.2. Preparation of liposomes. Large unilamellar vesicles
7. Broughman, J. R.; Shank, L. P.; Takeguchi, W.; Schultz, B. D.;
Iwamoto, T.; Mitchell, K. E.; Tomich, J. M. Biochemistry 2002,
41, 7350–7358.
were prepared using egg-yolk phosphatidylcholine (EYPC)
lipid. EYPC (60 mg) was dissolved in 5 mL of chloroform/
methanol. The solution was evaporated under reduced pres-
sure to give a thin film that was dried in vacuo overnight. The
lipid film was then hydrated with appropriate phosphate
buffer solution containing 1 mM lucigenin dye (10 mM
sodium phosphate, pH 6.4; 100 mM NaCl) to give a
8. (a) Schlesinger, P. H.; Ferdani, R.; Liu, J.; Pajewska, J.;
Pajewki, R.; Saito, M.; Shabany, H.; Gokel, G. W. J. Am.
Chem. Soc. 2002, 124, 1848–1849; (b) You, L.; Ferdani, R.;
Pajewski, R.; Gokel, G. W. Chem. Commun. 2006, 603–605.
9. (a) Sadownik, A.; Deng, G.; Janout, V.; Regen, S. L. J. Am.
Chem. Soc. 1995, 117, 6138–6139; (b) Otto, S.; Osifchin, M.;
Regen, S. L. J. Am. Chem. Soc. 1999, 121, 7276–7277.
10. Jiang, C.; Lee, E. R.; Lane, M. B.; Xiao, Y.-F.; Harris, D. J.;
Cheng, S. H. Am. J. Physiol. 2001, 281, L1164–L1172.
11. (a) Koulov, A. V.; Lambert, T. N.; Shukla, R.; Jain, M.; Bood,
J. M.; Smith, B. D.; Li, H. Y.; Sheppard, D. N.; Joos, J.-B.;
Clare, J. P.; Davis, A. P. Angew. Chem., Int. Ed. 2003, 42,
4931–4933; (b) Clare, J. P.; Ayling, A. J.; Joos, J.-B.; Sisson,
A. L.; Magro, G.; Perez-Payan, M. N.; Lambert, T. N.;
Shukla, R.; Smith, B. D.; Davis, A. P. J. Am. Chem. Soc.
2005, 127, 10739–10746.
6
0 mg/mL solution of lipid. After 10 freeze/thaw cycles,
the liposomes were extruded through a 100 nm polycarbon-
ate membrane 21 times at rt using a high-pressure mini-
extruder (Avanti). The resulting liposome solution was
passed through a Sephadex (G-25) column to remove excess
dye (eluant, sodium phosphate buffer, pH 6.4, 100 mM
NaNO ). The isolated liposomes were diluted to give a con-
3
centration of 25 mM in EYPC, assuming 100% retention of
lipid during gel filtration.
4.1.2.1. Chloride transport assay in liposomes. In
a typical experiment, 50 mL of the stock EYPC liposomes
was diluted into 2 mL of 10 mM sodium phosphate (pH
12. Madhavan, N.; Robert, E. C.; Gin, M. S. Angew. Chem., Int. Ed.
2005, 44, 7584–7587.

10750
O. A. Okunola et al. / Tetrahedron 63 (2007) 10743–10750
1
1
3. Furstner, A. Angew. Chem., Int. Ed. 2003, 42, 3582–3603.
4. Gale, P. A.; Light, M. E.; McNally, B.; Navakhum, K.;
36. (a) Chang, S. K.; Kwon, S. K.; Cho, I. Chem. Lett. 1987, 947–
948; (b) Nomura, E.; Takagaki, M.; Nakaoka, C.; Uchida, M.;
Tanaguchi, H. J. Org. Chem. 1999, 64, 3151–3156.
37. Arnaud-Neu, F.; Collins, E. M.; Deasy, M.; Ferguson, G.;
Harris, S. J.; Kaitner, B.; Lough, A. J.; McKervey, A.;
Marques, E.; Ruhl, B. L.; Schwing-Weill, M. J.; Seward,
E. M. J. Am. Chem. Soc. 1989, 111, 8681–8691.
Sliwinski, K. I.; Smith, B. D. Chem. Commun. 2005, 3773–
3775.
1
5. Sessler, J. L.; Eller, L. R.; Cho, W.-S.; Nicolaou, S.; Aguilar,
A.; Lee, J. T.; Lynch, V. M.; Magda, D. J. Angew. Chem., Int.
Ed. 2005, 45, 5989–5992.
1
1
6. Seganish, J. L.; Davis, J. T. Chem. Commun. 2005, 5781–5783.
7. S ꢀa ez D ꢀı az, R. I.; Regourd, J.; Santacroce, P. V.; Davis,
J. T.; Jakeman, D. L.; Thompson, A. Chem. Commun.
38. (a) Gutsche, C. D.; Lin, L.-G. Tetrahedron 1986, 42, 1633–
1640; (b) Percec, V.; Bera, T. K.; De, B. B.; Sanai, Y.; Smith,
J.; Holerca, M. N.; Barboiu, B.; Grubbs, R. B.; Fr ꢀe chet,
J. M. J. J. Org. Chem. 2001, 66, 2104–2117.
2007. doi:10.1039/b701919j
1
8. Santacroce, P. V.; Davis, J. T.; Light, M. E.; Gale, P. A.; Iglesia-
Sanchez, J. C.; Prados, P.; Quesada, R. J. Am. Chem. Soc. 2007,
39. (a) Iwamoto, K.; Araki, K.; Shinkai, S. J. Org. Chem. 1991, 56,
4955–4962; (b) Ungaro, R. Calixarenes in Action; Mandolini,
L., Ungaro, R., Eds.; Imperial College: London, UK, 2000;
pp 5–10 and references therein.
40. The NMR data for TAC-OH 3 closely matches that of a related
triamido phenolic cone calixarene previously reported by
Vicens and colleagues: Abidi, R.; Oueslati, I.; Amri, H.;
Thuery, P.; Nierlich, M.; Asfari, Z.; Vicens, J. Tetrahedron
Lett. 2001, 1685–1689.
129, 1886–1887.
1
2
9. Li, X.; Shen, B.; Yao, X.-Q.; Yang, D. J. Am. Chem. Soc. 2007.
doi:10.1021/ja071961h
0. Sidorov, V.; Kotch, F. W.; Abdrakhmanova, G.; Mizani, R.;
Fettinger, J. C.; Davis, J. T. J. Am. Chem. Soc. 2002, 124,
2267–2278.
2
2
2
2
1. Sidorov, V.; Kotch, F. W.; Lam, Y.; Kuebler, J. L.; Davis, J. T.
J. Am. Chem. Soc. 2003, 125, 2840–2841.
2. Seganish, J. L.; Fettinger, J. C.; Davis, J. T. Supramolecular
Chem. 2006, 18, 257–264.
3. Iqbal, K. S. J.; Cragg, P. J. J. Chem. Soc. Dalton Trans. 2007,
6–32.
4. (a) Tanaka, Y.; Kobuke, Y.; Sokabe, M. Angew. Chem., Int. Ed.
995, 34, 693–694; (b) Yoshino, N.; Satake, A.; Kobuke, Y.
41. Crystal data for cone-H 2a: C H N O , M 877.10,
5
2
68
4
8
r
˚
˚
space group Aba2, a¼13.207(5) A, b¼18.897(8) A, c¼
3 3
˚ ˚
20.305(8) A, V¼5000.0(3) A , Z¼4, rcalcd¼1.165 g/cm , Mo
ꢀ
1
Ka 0.071 mm . Data were collected on a Bruker SMART
1000 CCD diffractometer at 233(2) K. The structure refined
to convergence [D/sꢄ0.001] with R(F)¼5.81%, wR(F2)¼
11.14%, GOF¼1.017 for all 4378 unique reflections.
Crystallographic data for the structure reported in this paper
have been deposited with the Cambridge Crystallographic
Data Centre as supplementary publication # CCDC-649435.
Copies of the data can be obtained free of charge on application
to CCDC, 12 Union Road, Cambridge CB21EZ, UK. E-mail:
deposit@ccdc.cam.ac.uk.
2
1
Angew. Chem., Int. Ed. 2001, 40, 457–459; (c) Chen, W. H.;
Nishikawa, M.; Tan, S. D.; Yamamura, M.; Satake, A.;
Kobuke, Y. Chem. Commun. 2004, 872–873.
2
2
2
5. de Mendoza, J.; Cuevas, F.; Prados, P.; Meadows, E. S.; Gokel,
G. W. Angew. Chem., Int. Ed. 1998, 37, 1534–1537.
6. Paquet, V.; Zumbuehl, A.; Carreira, E. M. Bioconjugate Chem.
2006, 17, 1460–1463.
42. Crystal data for TAC-OEster 4: C H N O , M 878.09,
5
2
67
3
3
9
r
7. Wright, A. J.; Matthews, S. E.; Fischer, W. B.; Beer, P. D.
Chem.—Eur. J. 2001, 7, 3474–3481.
crystal dimensions 0.35ꢃ0.16ꢃ0.12 mm , monoclinic,
˚ ˚
space group P21/c, a¼13.6215(18) A, b¼19.208(2) A, c¼
3
3
˚
˚
2
2
8. Jin, T. J. Chem. Soc., Perkin Trans. 2 2002, 151–154.
9. Maulucci, N.; De Riccardis, F.; Botta, C. B.; Casapullo, A.;
Cressina, E.; Fregonese, M.; Tecilla, P.; Izzo, I. Chem.
Commun. 2005, 1354–1356.
0. For recent reviews on calixarenes as anion receptors, see: (a)
Lhotak, P. Top. Curr. Chem. 2005, 255, 65–95; (b) Matthews,
S. E.; Beer, P. D. Supramolecular Chem. 2005, 17, 411–435.
1. Sidorov, V.; Kotch, F. W.; El-Kouedi, M.; Davis, J. T. Chem.
Commun. 2000, 2369–2370.
19.424(3) A, V¼4984.0(11) A , Z¼4, r
¼1.170 g/cm ,
calcd
ꢀ
1
Mo Ka 0.071 mm . Data were collected on a Bruker
SMART 1000 CCD diffractometer at 240(2) K. The structure
refined to convergence [D/sꢄ0.001] with R(F)¼5.53%,
wR(F2)¼12.91%, GOF¼0.814 for all 5489 unique reflections.
Crystallographic data for the structure reported in this paper
have been deposited with the Cambridge Crystallographic
Data Centre as supplementary publication # CCDC-649436.
Copies of the data can be obtained free of charge on application
to CCDC, 12 Union Road, Cambridge CB21EZ, UK. E-mail:
deposit@ccdc.cam.ac.uk.
3
3
3
2. Kotch, F. W.; Sidorov, V.; Lam, Y. F.; Kayser, K. J.; Li, H.;
Kaucher, M. S.; Davis, J. T. J. Am. Chem. Soc. 2003, 125,
1
5140–15150.
43. The crystal structures of cone-H 2a and TAC-OEster 4 are
similar to a structure previously reported for a tetra-substituted
secondary ethyl-amide with t-Bu groups on its upper rim:
Arnaud-Neu, F.; Barboso, S.; Fanni, S.; Schwing-Weill,
M.-J.; McKee, V.; McKervey, M. A. Ind. Eng. Chem. Res.
2000, 39, 3489–3492.
44. The NMR association constant for TAC-OH 3 was determined,
assuming a 1:1 binding stoichiometry, using the program
‘Associate 1.6’ from the F. N. Diederich group at ETH.
45. (a) Biwersi, J.; Tulk, B.; Verkman, A. S. Anal. Biochem. 1994,
219, 139–143; (b) McNally, B. A.; Koulov, A. V.; Smith,
B. D.; Joos, J.-B.; Davis, A. P. Chem. Commun. 2005,
1087–1088.
3
3. Neutral amides are ligands for coordinating anions: (a) Beer,
P. D.; Gale, P. A. Angew. Chem., Int. Ed. 2001, 40, 487–516;
(b) Kang, S. O.; Begum, R. A.; Bowman-James, K. Angew.
Chem., Int. Ed. 2001, 45, 7882–7894.
3
4. For some references on stimuli-responsive transporters, see: (a)
Fyles, T. M.; Loock, D.; Zhou, X. J. Am. Chem. Soc. 1998, 120,
2997–3003; (b) Goto, C.; Yamamura, M.; Satake, A.; Kobuke,
Y. J. Am. Chem. Soc. 2001, 123, 12152–12159; (c) Gorteau, V.;
Perret, F.; Bollot, G.; Mareda, J.; Lazar, A. N.; Coleman, A. W.;
Tran, D.-H.; Sakai, N.; Matile, S. J. Am. Chem. Soc. 2004, 126,
13592–13593; (d) Chen, W. H.; Regen, S. L. J. Am. Chem. Soc.
2005, 127, 6538–6539.
3
5. Dutzler, R.; Campbell, E. B.; MacKinnon, R. Science 2003,
00, 108–112.
46. Araki, K.; Iwamoto, K.; Shinkai, S.; Matsuda, T. Bull. Chem.
Soc. Jpn. 1990, 63, 3480–3485.
3