Tuning nanopore formation of oligocholate macrocycles by carboxylic acid dimerization in lipid membranes

Article abstract of DOI:10.1021/jo400455x

Oligocholate macrocycles self-assemble into transmembrane nanopores by the associative interactions of water molecules inside the amphiphilic macrocycles. Macrocycles functionalized with a terephthalic acid side chain displayed significantly higher transport activity for glucose across lipid bilayers than the corresponding methyl ester derivative. Changing the 1,4-substitution of the dicarboxylic acid to 1,3-substitution lowered the activity. Combining the hydrophobic interactions and the hydrogen-bond-based carboxylic acid dimerization was an effective strategy to tune the structure and activity of self-assembled nanopores in lipid membranes.

Full text of DOI:10.1021/jo400455x

Note
pubs.acs.org/joc
Tuning Nanopore Formation of Oligocholate Macrocycles by
Carboxylic Acid Dimerization in Lipid Membranes
Lakmini Widanapathirana and Yan Zhao*
Department of Chemistry, Iowa State University, Ames, Iowa 50011-3111, United States
S
* Supporting Information
ABSTRACT: Oligocholate macrocycles self-assemble into trans-
membrane nanopores by the associative interactions of water
molecules inside the amphiphilic macrocycles. Macrocycles
functionalized with a terephthalic acid “side chain” displayed
significantly higher transport activity for glucose across lipid
bilayers than the corresponding methyl ester derivative. Changing
the 1,4-substitution of the dicarboxylic acid to 1,3-substitution
lowered the activity. Combining the hydrophobic interactions and
the hydrogen-bond-based carboxylic acid dimerization was an
effective strategy to tune the structure and activity of self-assembled
nanopores in lipid membranes.
ipid membranes are the barriers that separate the inside of
La cell from its environment and the various compartments
within the cell from the cytosol. Numerous biological functions
occur at these interfaces,1 and not surprisingly, membrane
proteins account for nearly 50% of all drug targets.2 For these
reasons, understanding how molecules recognize one another
in a lipid membrane is of great importance to both biology and
chemistry. In the past decades, chemists have gained significant
understanding of how molecular recognition occurs in solution.
However, when molecules move from a homogeneous solution
into an amphiphilic, nanodimensioned, and liquid crystalline
membrane, their intermolecular interactions (including those
with the environment) change enormously and the relative
importance of different noncovalent forces often needs
recalibration.
Our group has reported amphiphilic (oligocholate)
foldamers prepared from cholic acid.3 Their steroid-derived
these applications, it is highly desirable that the pore formation
be controlled rationally. Compound 5 contains a tricholate
macrocycle and a terephthalic acid side chain. Terephthalic acid
is known to have two crystalline forms and, in both forms, the
molecules are linked together by hydrogen-bonded carboxylic
acid dimers into infinite chains.9 Our idea was that a
combination of the hydrophobic interactions (among the
entrapped water molecules inside the cholate macrocycles) and
a tunable, directional polar interactions (among the carboxylic
acids on the side chain) would allow us to control the pore
formation.10 Since the height of the cholate macrocycle is
similar to the width of a cyclohexane, the hydrogen-bonding
interactions of the terephthalic acid and the stacking of the
cholate macrocycle should be compatible geometrically.
backbone and controlled conformations make them excellent
mimics of membrane protein transporters.4 More recently, we
prepared oligocholate macrocycles (e.g., 1−2) as novel pore-
forming agents.5 The numerous inward-facing hydroxyl and
amide groups make the molecule carry a pool of water in the
interior when it enters a membrane. Because these water
molecules have a strong tendency to interact with other water
molecules instead of the lipid hydrocarbon, the macrocycles
prefer to stack over one another to form a transmembrane
nanopore. The pore formation was confirmed by the
macrocycle-induced leakage of glucose from liposomes5 and
further by fluorescently (e.g., 3−4) and isotopically labeled
analogues using fluorescence6 and solid-state NMR spectros-
copy, respectively.7
Nanopore-forming agents have numerous applications in
Received: March 5, 2013
Published: April 11, 2013
drug delivery, separation, sensing, and catalysis.8 For many of
© 2013 American Chemical Society
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Note
Scheme 1. Synthesis of Terephthalic Acid Functionalized Macrocycle 5
Compound 5 was obtained by the hydrolysis of ester 6,
which was synthesized from the linear tricholate 7 by standard
transformations (Scheme 1). The key design of the molecule
involves the incorporation of a natural L-cysteine functionalized
with a propargyl group. The terminal alkyne allowed a late-
stage installation of the terephthalic acid moiety via a
convenient click reaction. It also enables us to change the
carboxylic side chain of the macrocycle readily (vide infra).
The tricholate macrocycle has a triangularly shaped internal
cavity approximately 1 nm on the side, large enough for glucose
to pass through.5 Figure 1 compares the leakage profiles of
rate. Addition of 30% cholesterol to the POPC/POPG
membrane is known to increase the hydrophobic thickness of
the membrane from 2.6 to 3.0 nm13 and decrease its fluidity.14
Although cholesterol reduces the membrane permeability of
hydrophilic molecules in general,15 cholesterol incorporation
was found to speed up the glucose transport by macrocycles 1
and 2 across POPC/POPG membranes.5 The result was
counterintuitive according to conventional reasoning but fully
consistent with the hydrophobically driven pore formation.
To our surprise, the addition of 30 mol % of cholesterol to
the POPC/POPG membranes did not enhance significantly the
glucose leakage induced by 5 (Figure 2a). Although the leakage
Figure 2. (a) Percent leakage of glucose at 30 min induced by 5 from
POPC/POPG LUVs with (dashed line) and without 30% cholesterol
(solid line). [Phospholipids] = 104 μM. (b) Percent leakage of CF
induced by 5 from POPC/POPG LUVs with (×) and without 30%
Figure 1. Percent leakage of glucose from POPC/POPG LUVs as a
⧫
◊
function of time for macrocycle 5 ( ), macrocycle 6 ( ), and linear
▲
trimer 7 ( ). [Oligocholate] = 5.0 μM. [Phospholipids] = 104 μM.
△
cholesterol ( ). [Oligocholate] = 0.5 μM. [Phospholipids] = 2.9 μM.
The leakage experiments were typically run in duplicate.
glucose-filled large unilamellar vesicles (LUVs) induced by the
terephthalic acid-functionalized macrocycle (5), its methyl ester
derivative (6), and the linear trimer (7). The leakage assay was
based on the reactions between the escaped glucose and
extravesicular enzymes that released UV-active NADPH.11
Whereas the linear trimer showed little activity over the
background leakage (averaging 6−10% at 60 min), the
macrocycles displayed higher activity. The acid derivative was
by far the most effective transporter among the three, with all
300 mM of glucose leaking out of the liposomes in <20 min in
the presence of 5 μM of 5.
Most membrane transporters function as either a carrier or a
channel/pore.12 A carrier binds and accompanies its cargo to
diffuse across the membrane. A channel or pore, on the other
hand, is relatively stationary within the membrane. One way to
distinguish pore-based transport from a carrier-based mecha-
nism is to study the effect of lipid composition on the transport
overall was still slightly higher for the cholesterol-containing
liposomes, the effect was far smaller than that observed for 1
and 2 (up to 5−7 times faster with the same level of
cholesterol, depending on the concentration of the macro-
cycle).5
Figure 1 shows that the carboxylic acids were clearly
beneficial to the glucose transport across the POPC/POPG
membranes. Could it be possible that other mechanisms (than
nanopore formation) was responsible for the faster leakage of 5
over 6? To better understand the transport mechanism, we
switched the permeant to carboxyfluorescein (CF), a molecule
too large to permeate the cyclic tricholate nanopore. CF
displays strong self-quenching above 50 mM and, thus, emits
more strongly once escaping from a liposome.16 Our previous
study suggests that CF needs to be sandwiched by two cyclic
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Note
tricholates to move across a membrane via a carrier
mechanism.17 As shown in Figure 2b, the CF leakage induced
by 5 slowed down greatly upon the inclusion of cholesterol in
the LUVs. At 60 min, the cholesterol-containing liposomes (×)
only showed less than half of the leakage found in the
cholesterol-free ones (△).
The above experiments demonstrated that cholesterol was
indeed detrimental to carrier-based transport, in agreement
with other literature work.18 The study also assured us that,
despite the small enhancement in the glucose leakage caused by
cholesterol incorporation, macrocycle 5 did NOT function as a
carrier for glucose. If we “normalize” the cholesterol effect on
the glucose transport over its (negative) impact on the CF
transport, we could still conclude that the glucose leakage
induced by 5 from the cholesterol-containing liposomes in
Figure 2a was unusually high.
After ruling out the carrier mechanism, we performed the
lipid-mixing assay to verify the integrity of the lipid bilayers. In
the lipid-mixing assay, a batch of unlabeled LUVs is mixed with
another batch labeled with 1 mol % of NBD- and rhodamine-
functionalized lipids. If the carboxylic acid-functionalized 5
destabilized the liposomes by other mechanisms (e.g.,
membrane fusion or destruction), the fluorescence resonance
energy transfer (FRET) between the fluorescent labels would
be affected.19 In our hands, even at the highest concentration
studied (5 mol %), the liposomes showed <16% mixing (Figure
3a), indicating that none of the above-mentioned membrane-
would represent a counterbalancing effect for the pore
formation. This could also be one reason why 5 was still less
effective in the glucose transport than the parent macrocycle 1
(Figure 3b).20
If indeed the carboxylic acid dimerization from the
terephthalic acid side chains and the hydrophobically driven
stacking of the cholate macrocycles were collectively respon-
sible for the high activity of 5, altering the dicarboxylic acids
should allow one to tune the transport activity. We thus
prepared a corresponding “carboxylic acid isomer”, i.e., 12,
following a similar click coupling. Unlike terephthalic acid that
forms a chain-like structure commensurate with the stacked
nanopore, 5-substituted isophthalic acid derivatives tend to
adopt cyclic hexameric structures through the carboxylic acid
dimerization.21 Since the stacked cholate macrocycles prefer a
linear alignment of the functionalized side chains, isophthalic
acid should be less than ideal.
The above postulation was confirmed in our glucose leakage
assay. The isophthalic acid-functionalized macrocycle consis-
tently underperformed its para isomer as a glucose transporter
(Figure 3b), indicating that the orientation of the carboxylic
acids was critical to the transport. The result further ruled out
“generic” effects of carboxylic acids on the membrane. If
compound 5, for example, simply causes glucose leakage by its
amphiphilicity, with the terephthalic acid acting as a hydrophilic
moiety, it is difficult to imagine that switching the 1,4-
substitution to 1,3 would have a large effect on the transport,
especially when there are numerous rotatable bonds between
the acid-containing phenyl group and the cholate macrocycle.
The importance of carboxylic acid dimerization in membrane
is also supported by the literature. When bound to lipid
membranes, fatty acids shift their pKa from ca. 4 in solution to
7.5.22 In the protonated, uncharged form, a fatty acid can
migrate into a lipid membrane and rapidly diffuse to the other
side. The half-life of the flip-flop of fatty acids in common lipid
bilayers is <10 ms.23 Thus, these acids have no difficulty
traversing the membrane, most likely because their dimerization
lowers the polarity of the carboxylic acids and make them
compatible with nonpolar environments.
In summary, carboxylic acid dimerization could be used to
rationally tune the hydrophobically driven pore formation of
cholate macrocycles. Our previous experience tells us that
molecular recognition in membrane could differ enormously
from that in solution. The aromatic donor−acceptor inter-
actions between 1,5-dialkoxynaphthalene and NDI, for
example, were found to be 1−2 orders of magnitude stronger
than the acceptor−acceptor interactions in polar solvents.24 For
the cholate macrocycles, however, 4 transported glucose more
efficiently than either 3 or the 1:1 3/4 mixture.25 The result
suggested the acceptor−acceptor interactions were more
effective at promoting the stacking of the cholate macrocycles
in lipid membranes. Another work of ours indicates that the
strong guanidinium−carboxylate salt bridge was rather
ineffective at promoting stacking of the cholate macrocycles,
due to the strong preference of these polar groups for
Figure 3. (a) Percent lipid-mixing and the size of the POPC/POPG
LUVs upon the addition of 5. [5] = 2.5 μM, [phospholipids] = 54.0
▲
μM. (b) Percent leakage of glucose at 30 min induced by 1 ( ), 5
⧫
●
( ), and 12 ( ) from POPC/POPG LUVs. [Phospholipids] = 104
μM.
disrupting processes was significant in the presence of 5. The
conclusion was also supported by dynamic light scattering
(DLS) showing nearly constant size of the liposomes after the
addition of 5 (Figure 3a).
At this point, it seems reasonable to conclude that (a) the
carboxylic acids in macrocycle 5 were beneficial to the glucose
transport across the membrane and (b) the transport bore
essentially all the important hallmarks of the hydrophobically
driven pore-forming mechanism. The only “abnormality” was
the smaller enhancement of glucose transport upon cholesterol
inclusion in the membrane. The observation, however, is not
difficult to understand from the viewpoint of polarity.
Cholesterol increases the hydrophobicity of the membrane.13
Although the stronger environmental hydrophobicity facilitates
the stacking of cholate macrocycles,5 it probably lowers the
solubility of polar compounds including the terephthalic acid-
containing 5. Even if the carboxylic acid dimer may be stronger
in the more hydrophobic, cholesterol-containing membrane,
the overall lower concentration of 5 within the membrane
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The Journal of Organic Chemistry
Note
membrane surface.10 This work shows that carboxylic acid side
chains can be used to regulate the stacking of cholate
macrocycles effectively. As chemists become interested in
creating functional structures in lipid membranes, the
carboxylic acid dimer26 may be a particularly useful motif for
supramolecular construction.27
over silica gel, using 12:1 CH2Cl2/CH3OH as the eluent to give an
ivory powder (51 mg, 70%). H NMR (400 MHz, CDCl3/CD3OD =
1:1, δ): 8.24−8.07 (br, 4 H), 4.53 (br, 1H), 3.97 (s, 3H), 3.93 (br,
3H), 3.79 (br, 3H), 3.73 (s, 3H), 3.43 (br 3H), 2.35−0.78 (a series of
m), 0.66 (m, 9H). 13C NMR (100 MHz, CDCl3/CD3OD = 1:1, δ):
176.6, 175.4, 170.5, 165.7, 136.7, 134.9, 131.9, 128.0, 88.1, 76.8, 73.5,
68.7, 63.2, 53.5, 42.9, 42.8, 42.6, 42.4, 40.2, 40.0, 36.9, 36.5, 36.3, 35.9,
1
35.5, 35.2, 32.0, 28.1, 27.6, 27.2, 26.3, 23.7, 23.1, 17.7, 12.9. ESI MS
+
EXPERIMENTAL SECTION
(m/z): [M + H]
calcd for C88H134N7O14S 1544.9704, found
■
1544.9699.
The preparation of LUVs, the procedures for the leakage assays, and
the lipid mixing assay were reported previously.5 Compound 7,28
compound 8,29 methyl 2-azidoterephthlate,30 and 5-azidoisophthalic
acid30 were synthesized according to literature procedures.
Compound 5. Compound 6 (37 mg, 0.024 mmol) was dissolved
in MeOH (1 mL), and a solution of 2 M LiOH (0.2 mL, 0.40 mmol)
was added. The reaction was stirred at room temperature and
monitored by TLC. After the hydrolysis was complete, the organic
solvent was removed by rotary evaporation. After the addition of a
dilute HCl solution (50 mL, 0.05 M), the precipitate formed was
collected by centrifugation, washed with water, and dried in vacuo to
Compound 9. The carboxylic acid of 728 (450 mg, 0.37 mmol), 8
(83 mg, 0.48 mmol), 1-hydroxybenzotriazole (HOBt, 89 mg, 0.66
mmol), and (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium
hexafluorophosphate (BOP, 292 mg, 0.66 mmol) were dissolved in
anhydrous DMF (2 mL). N,N-Diisopropylethylamine (DIPEA, 0.51
mL, 2.93 mmol) was added. The mixture was allowed to react in a
microwave reactor at 100 °C for 1 h and monitored by TLC. When
the reaction was complete, the mixture was cooled to room
temperature and poured into a dilute HCl solution (0.05 M, 250
mL). The precipitate was collected, dried, and purified by column
chromatography over silica gel, using 12:1 CHCl3/CH3OH as the
1
give a white powder (22 mg, 61%). H NMR (400 MHz, CDCl3/
CD3OD = 1:1, δ): 8.24−8.05 (br, 4 H), 4.47 (br, 1H), 3.93 (br, 3H),
3.79 (br, 3H), 3.50 (br, 3H), 2.32−0.77 (a series of m), 0.67 (m, 9H).
13C NMR (100 MHz, CDCl3/CD3OD = 1:1, δ): 177.3, 176.1, 171.6,
166.7, 166.4, 137.4, 135.6, 132.8,132.6,132.2, 128.7, 89.1, 77.4,
74.7,74.2,69.5, 54.2, 47.7, 47.6, 47.5, 47.4, 43.4, 43.2, 43.0, 41.0,
40.8, 40.7, 37.5, 37.2, 37.0,36.2,36.0,36.2, 35.9, 34.4,33.7, 33.3,
32.7,32.6, 31.1, 29.6, 28.3, 28.0, 27.9, 24.4, 23.9, 23.8, 18.4, 13.7,
13.5. ESI MS (m/z): [M + H] + calcd for C86H130N7O14S 1516.9391,
found 1516.9350.
Compound 12. The same procedure as in the synthesis of
compound 5 was followed to give 12 as an off-white powder (66%).
1H NMR (400 MHz, CDCl3/CD3OD = 1:1, δ): 8.22−8.07 (br, 4 H),
4.45 (br, 1H), 3.95 (br, 3H), 3.79 (br, 3H), 3.49 (br, 3H), 2.31−
0.76(a series of m), 0.68 (m, 9H). 13C NMR (100 MHz, CDCl3/
CD3OD = 1:1, δ): 177.3, 176.1, 171.6, 166.7, 166.4, 137.4, 135.6,
132.8,132.6,132.2, 128.7, 89.1, 77.4, 74.7,74.2,69.5, 54.2, 47.7, 47.6,
47.5, 47.4, 43.4, 43.2, 43.0, 41.0, 40.8, 40.7, 37.5, 37.2,
37.0,36.2,36.0,36.2, 35.9, 34.4,33.7, 33.3, 32.7,32.6, 31.1, 29.6, 28.3,
28.0, 27.9, 24.4, 23.9, 23.8, 18.4, 13.7, 13.5. ESI MS (m/z): [M − H] +
calcd for C86H128N7O14S 1514.9245, found 1514.9229.
1
eluent to give a light brown powder (272 mg, 55%). H NMR (400
MHz, CDCl3/CD3OD = 1:1, δ): 4.70 (br, 1H), 3.93 (br, 3H), 3.79
(br, 3H), 3.73 (s, 3H), 3.50 (br, 2H), 3.25 (s, 1H), 3.17 (m, 2H), 2.95
(m, 1H), 2.93 (m, 1H), 2.43 (t, 1H), 2.38−1.0 (a series of m), 0.66 (s,
9H). 13C NMR (100 MHz, CDCl3/CD3OD = 1:1, δ): 176.0, 174.8,
172.2, 79.7, 73.3, 72.4, 68.5, 62.0, 52.7, 52.3, 47.6, 46.7, 42.4, 39.9,
36.5, 36.2, 35.2, 34.0, 33.4, 32.4, 28.8, 27.1, 23.6, 23.0, 19.7, 17.6, 12.9.
ESI MS (m/z): [M + Na]+ calcd for C79H126N6NaO11S 1389.9098,
found 1390.9079.
Compound 10. A solution of compound 9 (155 mg, 0.110 mmol)
and triphenylphosphine (60 mg, 0.230 mmol) in methanol (4 mL)
was heated to reflux overnight. After the solvent was removed by
rotary evaporation, the residue was purified by column chromatog-
raphy over silica gel using 10:1 CH2Cl2/MeOH and then 8:1:0.1
CH2Cl2/MeOH/Et3N as the eluents to give an off-white powder (109
1
mg, 72%). H NMR (400 MHz, CDCl3/CD3OD = 1:1, δ): 3.95 (br,
ASSOCIATED CONTENT
* Supporting Information
■
3H), 3.79 (br, 3H), 3.74(s, 3H), 3.50 (br, 2H), 3.17 (m, 2H), 2.88 (m,
2H), 2.44 (t, 1H), 2.40−0.77 (a series of m), 0.66 (s, 9H). 13C NMR
(100 MHz, CDCl3/CD3OD = 1:1, δ): 176.0, 175.0, 172.4, 79.9, 73.4,
72.6, 68.4, 63.0, 59.4, 53.2, 51.5, 47.7, 46.9, 46.5, 42.7, 40.2, 36.9, 35.6,
34.0, 32.6, 28.8, 28.3, 27.3, 23.9, 23.0, 17.6, 12.9, 9.3,7.7. ESI MS (m/
z): [M + H]+ calcd for C79H129N4O11S 1341.9499, found 1341.9411.
Compound 11. Compound 10 was hydrolyzed by standard
procedures using 10 equiv of LiOH.28 The hydrolyzed product (50
mg, 0.038 mmol), BOP (84 mg, 0.190 mmol), and HOBT (26 mg,
0.190 mmol) were dissolved in DMF (30 mL), followed by the
addition of DIPEA (66 μL, 0.381 mmol). The mixture was allowed to
react in a microwave reactor at 100 °C for 1 h, cooled to room
temperature, and poured into a dilute HCl solution (0.05 M, 100 mL).
The precipitate was collected, dried, and purified by purified by
column chromatography over silica gel using 8:1 CH2Cl2/CH3OH as
the eluent to give an ivory powder (30 mg, 60%). 1H NMR (400 MHz,
CDCl3/CD3OD = 1:1, δ): 4.48 (br, 1H), 3.93 (br, 3H), 3.79 (br, 3H),
3.52 (br, 3H), 3.09 (m, 1H), 2.88 (m, 1H), 2.440 (t, 1H), 2.34−0.74
(a series of m), 0.69 (s, 9H). 13C NMR (100 MHz, CDCl3/CD3OD =
1:1, δ): 176.1, 74.3, 73.1, 69.4, 48.1, 47.8, 47.5, 43.2,43.0, 40.9, 37.5,
36.6, 36.2, 35.9, 34.5, 33.7, 33.3, 32.7, 31.0, 29.6, 28.8, 25.5,28.2,
28.1,27.9, 24.5, 23.9, 20.6, 18.3,13.8, 13.6, 9.9. ESI MS (m/z): [M +
Na]+ calcd for C78H124N4O10SNa 1331.8936, found 1331.8909.
Compound 6. Compound 11 (62 mg, 0.047 mmol), methyl 2-
azidoterephthlate (13 mg, 0.062 mmol), CuSO4·5H2O (24 mg, 0.095
mmol), and sodium ascorbate (38 mg, 0.189 mmol) were dissolved in
a 2:1:1 mixture of THF/methanol/water (0.8 mL) and stirred at 40 °C
overnight. The reaction mixture was concentrated by rotary
evaporation and poured into water (50 mL). The precipitate was
collected, dried, and purified by purified by column chromatography
S
General experimental methods and the NMR data for the key
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: 515-294-5845. Fax: 515-294-0105. E-mail: zhaoy@
iastate.edu.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank NSF (DMR-1005515) for supporting the research.
■
REFERENCES
■
(1) Yeagle, P. The Structure of Biological Membranes, 2nd ed.; CRC
Press: Boca Raton, 2005.
(2) (a) Hopkins, A. L.; Groom, C. R. Nat. Rev. Drug Discovery 2002,
1, 727. (b) Lundstrom, K. Curr. Protein Pept. Sci. 2006, 7, 465.
(3) (a) Zhao, Y.; Zhong, Z.; Ryu, E.-H. J. Am. Chem. Soc. 2007, 129,
218. (b) Cho, H.; Zhong, Z.; Zhao, Y. Tetrahedron 2009, 65, 7311.
(4) (a) Zhang, S.; Zhao, Y. Chem.Eur. J. 2011, 17, 12444. (b) Cho,
H.; Zhao, Y. J. Am. Chem. Soc. 2010, 132, 9890.
(5) Cho, H.; Widanapathirana, L.; Zhao, Y. J. Am. Chem. Soc. 2011,
133, 141.
(6) Widanapathirana, L.; Zhao, Y. Langmuir 2012, 28, 8165.
4613
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The Journal of Organic Chemistry
Note
(7) Wang, T.; Widanapathirana, L.; Zhao, Y.; Hong, M. Langmuir
2012, 28, 17071.
(8) (a) Granja, J. R.; Ghadiri, M. R. J. Am. Chem. Soc. 1994, 116,
10785. (b) Sakai, N.; Mareda, J.; Matile, S. Acc. Chem. Res. 2005, 38,
79. (c) Das, G.; Talukdar, P.; Matile, S. Science 2002, 298, 1600.
(d) Sakai, N.; Sorde, N.; Matile, S. J. Am. Chem. Soc. 2003, 125, 7776.
(e) Ma, L.; Melegari, M.; Colombini, M.; Davis, J. T. J. Am. Chem. Soc.
2008, 130, 2938. (f) Chen, L.; Si, W.; Zhang, L.; Tang, G.; Li, Z.-T.;
Hou, J.-L. J. Am. Chem. Soc. 2013, 135, 2152.
(9) (a) Bailey, M.; Brown, C. J. Acta Crystallogr. 1967, 22, 387.
(b) Sledz, M.; Janczak, J.; Kubiak, R. J. Mol. Struct. 2001, 595, 77.
(10) Widanapathirana, L.; Li, X.; Zhao, Y. Org. Biomol. Chem. 2012,
10, 5077.
(11) Kinsky, S. C.; Haxby, J. A.; Zopf, D. A.; Alving, C. R.; Kinsky, C.
B. Biochemistry 1969, 8, 4149.
(12) Stein, W. D. Carriers and Pumps: An Introduction to Membrane
Transport; Academic Press: San Diego, 1990.
(13) Nezil, F. A.; Bloom, M. Biophys. J. 1992, 61, 1176.
(14) Holthuis, J. C. M.; van Meer, G.; Huitema, K. Mol. Membr. Biol.
2003, 20, 231.
(15) (a) Demel, R. A.; Bruckdor, K. R.; van Deene, L. L. Biochim.
Biophys. Acta 1972, 255, 321. (b) Papahadjopoulos, D.; Nir, S.; Ohki,
S. Biochim. Biophys. Acta 1972, 266, 561.
(16) New, R. R. C. Liposomes: A Practical Approach; IRL Press:
Oxford, 1990.
(17) Cho, H.; Zhao, Y. Langmuir 2011, 27, 4936.
(18) Davis, J. T.; Okunola, O.; Quesada, R. Chem. Soc. Rev. 2010, 39,
3843.
(19) Struck, D. K.; Hoekstra, D.; Pagano, R. E. Biochemistry 1981, 20,
4093.
(20) Another reason was the flexibility in the macrocycle introduced
by the inserted L-cysteine. Our earlier work indicates that rigidity in
the macrocycle facilitates the pore formation: Widanapathirana, L.;
Zhao, Y. J. Org. Chem. 2012, 77, 4679.
(21) (a) Yang, J.; Marendaz, J. L.; Geib, S. J.; Hamilton, A. D.
Tetrahedron Lett. 1994, 35, 3665. (b) Zimmerman, S. C.; Zeng, F. W.;
Reichert, D. E. C.; Kolotuchin, S. V. Science 1996, 271, 1095.
(22) James A, H. In Carbon-13 NMR Spectroscopy of Biological
Systems; Academic Press: San Diego, 1995; p 117.
(23) (a) Kamp, F.; Hamilton, J. A. Proc. Natl. Acad. Sci. U.S.A. 1992,
89, 11367. (b) Kamp, F.; Zakim, D.; Zhang, F.; Noy, N.; Hamilton, J.
A. Biochemistry 1995, 34, 11928. (c) Hamilton, J. A.; Guo, W.; Kamp,
F. Mol. Cell. Biochem. 2002, 239, 17.
(24) Cubberley, M. S.; Iverson, B. L. J. Am. Chem. Soc. 2001, 123,
7560.
(25) Widanapathirana, L.; Zhao, Y. J. Org. Chem. 2012, 77, 4679.
(26) In CCl4 and heptane, the dimerization constant of carboxylic
acids is 103−104 M−1, equivalent to 4−5 kcal/mol in binding free
energy. See: Wenograd, J.; Spurr, R. A. J. Am. Chem. Soc. 1957, 79,
5844. Goodman, D. S. J. Am. Chem. Soc. 1958, 80, 3887. Since the
hydrophobic core of the membrane essentially comprises hydrocarbon,
a similar binding free energy is expected.
(27) Boccalon, M.; Iengo, E.; Tecilla, P. J. Am. Chem. Soc. 2012, 134,
20310.
(28) Zhao, Y.; Zhong, Z. J. Am. Chem. Soc. 2005, 127, 17894.
(29) Struthers, H.; Spingler, B.; Mindt, T. L.; Schibli, R. Chem.Eur.
J. 2008, 14, 6173.
(30) (a) Morris, W.; Taylor, R. E.; Dybowski, C.; Yaghi, O. M.;
Garcia-Garibay, M. A. J. Mol. Struct. 2011, 1004, 94. (b) Pokhodylo, N.
T.; Matiychuk, V. S. J. Heterocycl. Chem. 2010, 47, 415.
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