Phosphatidylcholine-derived bolaamphiphiles via click chemistry

Article abstract of DOI:10.1021/ol062557a

(Chemical Equation Presented) The copper-catalyzed azide alkyne cycloaddition is employed to modify phosphatidylcholine precursors with sn-2 acyl chains containing terminal alkyne or azide groups. Although the reactions are conducted as biphasic dispersio

Full text of DOI:10.1021/ol062557a

ORGANIC
LETTERS
2007
Vol. 9, No. 2
199-202
Phosphatidylcholine-Derived
Bolaamphiphiles via Click Chemistry
Edward J. O’Neil, Kristy M. DiVittorio, and Bradley D. Smith*
Department of Chemistry & Biochemistry, 251 Nieuwland Science Hall,
UniVersity of Notre Dame, Notre Dame, Indiana 46556
smith.115@nd.edu
Received October 17, 2006
ABSTRACT
The copper-catalyzed azide alkyne cycloaddition is employed to modify phosphatidylcholine precursors with sn-2 acyl chains containing
terminal alkyne or azide groups. Although the reactions are conducted as biphasic dispersions, the yields are essentially quantitative.
Bolaamphiphiles are formed by simply clicking together two phosphatidylcholine alkyne precursors to a central bisazide scaffold. The chemistry
introduces polar 1,4-triazole units into the lipophilic region of the bilayer membrane, and the bolaamphiphiles do not form stable vesicles.
Bolaamphiphiles are amphiphilic compounds, containing two
hydrophilic headgroups connected by a hydrophobic spacer.1
One of the best-known families of naturally occurring
bolaamphiphiles are the archae lipids. A typical structure,
shown in Figure 1, has a hydrocarbon chain that is connected
it is difficult to produce these natural products in large
amounts, several groups have investigated simplified syn-
thetic mimics and found that they also have membrane-
stabilizing properties. Synthetic bolaamphiphiles with mem-
brane-spanning macrocycles,4 or unbranched hydrocarbon
chains,5 linking two phosphoglycerol headgroups are known
to form membranes with increased melting temperatures,
decreased permeation of ions, and small polar molecules and
increased ordering of the membrane components.1 In addi-
tion, Langmuir-Blodgett bilayers containing high concentra-
tions of bolaamphiphile have diminished lateral membrane
diffusion.6 Because of these membrane-stabilizing properties,
(2) (a) Yamauchi, K.; Sakamoto, Y.; Moriya, A.; Yamada, K.; Hosokawa,
T.; Higuchi, T.; Kinoshita, M. J. Am. Chem. Soc. 1990, 112, 3188-3191.
(b) Shinoda, W.; Shinoda, K.; Baba, T.; Mikami, M. Biophys. J. 2005, 89,
3195-3202.
Figure 1. Structure of archae phospholipid, L-32-Phy.
(3) Bartucci, R.; Gambacorta, A.; Gliozzi, A.; Marsh, D.; Sportelli, L.
Biochemistry 2005, 44, 15017-15023.
(4) (a) Menger, F. M.; Chen, X. Y.; Brocchini, S.; Hopkins, H. P.;
Hamilton, D. J. Am. Chem. Soc. 1993, 115, 6600-6608. (b) Menger, F.
M.; Chen, X. Y. Tetrahedron Lett. 1996, 37, 323-326.
(5) (a) Menger, F. M.; Wong, Y.-L. J. Org. Chem. 1996, 61, 7382-
7390. (b) Wang, G.; Hollingsworth, R. I. J. Org. Chem. 1999, 64, 4140-
4147. (c) Kai, T.; Sun, X.-L.; Faucher, K. M.; Apkarian, R. P.; Chaikof, E.
L. J. Org. Chem. 2005, 70, 2606-2615. (d) Kohler, K.; Forster, G.; Hauser,
A.; Dobner, B.; Heiser, U. F.; Zeithe, F.; Rechter, W.; Steiniger, F.;
Dreschsler, M.; Stettin, H.; Blume, A. J. Am. Chem. Soc. 2004, 126, 16804-
16813.
by ether linkages to two zwitterionic phosphocholine head-
groups.2 These membrane-spanning, polar lipids rigidify the
bilayer membrane and allow the thermophilic archae to
endure temperatures in excess of 80 °C and low pH.3 Because
(1) Fuhrhop, J.-H.; Wang, T. Chem. ReV. 2004, 104, 2901-2937 and
references therein.
10.1021/ol062557a CCC: $37.00
© 2007 American Chemical Society
Published on Web 12/14/2006

bolaamphiphiles are attractive building blocks for membrane
assembly in various drug delivery and biotechnology ap-
plications. Conversely, many bolaamphiphiles have been
designed to act as membrane disruptors and ion transporters.7
Typically, these molecular designs incorporate polar func-
tionality into the central region of a spacer that is otherwise
hydrophobic. Whatever the specific functional objective, the
bolaamphiphile synthesis is always a major technical chal-
lenge, especially if the molecule contains sensitive function-
ality such as that found in the headgroups of the common,
naturally occurring phospholipids.
yields are essentially quantitative which greatly facilitates
purification of the bolaamphiphile products.
The synthesis starts with the lysophosphatidylcholine 1,
which is easily prepared in gram quantities by an enzymatic
cleavage of the sn-2 chain of dipalmitoylphosphatidylcholine
(DPPC) using bee venom phospholipase A2 (Scheme 1).13
Scheme 1. Synthesis of Phosphatidylcholine Derivatives
Our recent interest in the effects of bolaamphiphiles on
membrane structure and function8 has lead us to develop a
method of producing them in large amounts from readily
available starting materials. An obvious family of potential
building blocks that have preinstalled polar headgroups are
the lysophospholipids, with lysophophosphatidylcholine as
the archetype example. Acylation of the sn-2 hydroxyl in
lysophophosphatidylcholine with a suitably functionalized
acyl chain gives a phosphatidylcholine precursor that can
be dimerized to produce a bolaamphiphile. The chemistry
to produce symmetric couplings includes olefin metathesis9
and Glaser oxidation,4a whereas asymmetric cross-couplings
have been achieved using the Wittig reaction, nucleophilic
substitution, conjugate addition, and the Diels-Alder
reaction.5c,6 The latter reactions have also been conducted
in preformed liposomal membranes. All of these synthetic
procedures have drawbacks: either the yields are not
extremely high or the chemistry is not compatible with all
types of biomolecular functional groups. In an effort to
improve the synthesis, we have investigated the [3+2] azide
alkyne cycloaddition, a member of a larger class of reactions
known as Huisgen 1,3-dipolar cycloadditions.10,11 The cop-
per-catalyzed azide alkyne cycloaddition has emerged in
recent years as the most popular reaction in the series known
as click chemistry.12 The chemistry is compatible with a
broad array of biological functionalities, and it is finding
increasing employment in many areas of chemical technol-
ogy. We report here that click chemistry is compatible with
the phosphatidylcholine headgroup and that the coupling
Acylation of the newly generated secondary alcohol was
achieved in high yield and without acyl chain migration by
employing the conditions described by Hajdu and co-
workers.14 Briefly, a suspension of compound 1 was soni-
cated for 5 h over glass beads with a 5-fold excess of either
12-azidododecanoic acid or undecynoic acid, DCC, and
DMAP. The reaction is believed to take place on the glass
surface. By adding more glass surface area to the flask, the
reaction time is drastically reduced from days to hours. Upon
the completion of the reaction, the DMAP was removed
using Dowex 8X ion-exchange resin. The product was
purified by column chromatography to give phosphatidyl-
choline derivatives 2 and 3 in 88% and 91% yield,
respectively.
(6) Halter, M.; Nogata, Y.; Dannenberger, O.; Sasaki, T.; Vogel, V.
Langmuir 2004, 20, 2416-2423.
(7) Examples of bolaamphiphiles as ion channels include: Cameron, L.
M.; Fyles, T. M.; Hu, C.-W. J. Org. Chem. 2002, 67, 1548-1553. Goto,
C.; Yamamura, M.; Satake, A.; Kobuke, Y. J. Am. Chem. Soc. 2001, 123,
12152-12159. Leevy, W. M.; Huettner, J. E.; Pajewski, R.; Schlesinger,
P. H.; Gokel, G. W. J. Am. Chem. Soc. 2004, 126, 15747-15753. Gokel,
G. W.; Murillo, O. Acc. Chem. Res. 1996, 29, 425-432 and references
therein. For bolaamphiphiles as membrane disruptors, see: Naka, K.;
Sadownik, A.; Regen, S. L. J. Am. Chem. Soc. 1993, 115, 2278-2286.
(8) Forbes, C. C.; DiVittorio, K. M.; Smith, B. D. J. Am. Chem. Soc.
2006, 128, 9211-9218.
(9) (a) Patwardhan, A. P.; Thompson, D. H. Langmuir 2000, 16, 10340-
10350. (b) Meglio, C. D.; Rananavare, S. B.; Svenson, S.; Thompson, D.
H. Langmuir 2000, 16, 128-133.
(10) Huisgen, R. Angew. Chem., Int. Ed. 1963, 565-632.
(11) For a more recent review on 1,3-dipolar cycloadditions: Gothelf,
K. V.; Jorgenson, K. A. Chem. ReV. 1998, 98, 863-909.
(12) (a) Lewis, W. G.; Green, L. G.; Grynszpan, F.; Radic, Z.; Carlier,
P. R.; Taylor, P.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed.
2002, 41, 1053-1057. (b) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.;
Sharpless, K. B. Angew. Chem., Int. Ed. 2002, 41, 2596-2599. (c) Kolb,
H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40,
2004-2021.
The azide derivative 2 and the terminal alkyne 3 react
smoothly under the aqueous solvent conditions described by
(13) Pluckthun, A.; Dennis, E. A. J. Phys. Chem. 1981, 85, 678-683.
(14) Rosseto, R.; Hajdu, J. Tetrahedron Lett. 2005, 46, 2941-2944.
200
Org. Lett., Vol. 9, No. 2, 2007

Sharpless and co-workers12 to produce the 1,4-triazole
products 4 and 5 in 95-97% yield. The high yields are quite
remarkable because the reaction mixtures are thick biphasic
dispersions from start to finish. The same reactions were
repeated using a copper wire as a catalyst instead of copper
sulfate/sodium ascorbate, and the products 4 and 5 formed
in the same very high yield. Thus, click chemistry is an
effective way to conjugate molecular cargo to the end of a
phospholipid acyl chain.15
Scheme 2. Synthesis of Bolaamphiphiles
To use this chemistry to form bolaamphiphilic structures,
various organic bisazides were synthesized from the corre-
sponding dibromides.16 Briefly, the dibromides were allowed
to react with NaN3 in DMSO for 1-4 h. The reaction was
quenched with water, and the bisazide products were
extracted into ether in 95-98% yield (Table 1) and used
without further purification.
Table 1. Synthesis of Bisazides
destroyed all of the NBD-PC fluorescence, indicating that
none of the probe was protected from the external added
quencher. Typically, about half of the NBD-PC probe is
protected if it is incorporated into both sides of enclosed
vesicle membranes.17 Finally, we analyzed aqueous disper-
sions of bolaamphiphiles 6-8 by transmission electron
microscopy and observed that they form fibers and sheets
(Figure 2).18
To form bolaamphiphiles, 2 equiv of the terminal alkyne
3 was stirred at room temperature with 1 equiv of the
corresponding bisazide along with copper sulfate/sodium
ascorbate in water. The biphasic reactions were complete
after 30 h, and the corresponding bolaamphiphile products,
6-8, were isolated by simple extraction in >95% yield
(Scheme 2).
The membrane assembly properties of bolaamphiphiles
6-8 were evaluated in a number of different ways. First,
thin films composed of 100% bolaamphiphile were dispersed
in aqueous solutions of the fluorescent dye, carboxyfluores-
cein. Various dispersion methods were tried (freeze-thaw,
sonication), but there was no evidence that the carboxyfluo-
rescein could be encapsulated inside stable vesicles. A similar
conclusion was reached after incorporation of the amphiphilic
fluorescent probe NBD-PC. Sonicated dispersions of the
bolaamphiphile with preincorporated NBD-PC were treated
with the chemical quencher sodium dithionite,17 which
Figure 2. Transmission electron microscopy of bolaamphiphiles
6-8. In each case, the scale bar is 200 nm.
The data suggest that bolaamphiphiles 6-8 do not favor
vesicle formation, a result that is not surprising considering
the propensity of bolaamphiphiles to form planar membranes5d
and the polar nature of the two 1,4-triazole units. The
molecular dipole of a 1,4-triazole is larger than an amide
bond,19 and it is unlikely that a 1,4-triazole will prefer the
(15) Click chemistry has recently been used to conjugate molecular cargo
to the headgroup of phospholipids: (a) Musiol, H.-J.; Dong, S.; Kaiser,
M.; Bausinger, R.; Zumbusch, A.; Bertsch, U.; Moroder, L. Chem. Bio.
Chem. 2005, 6, 625-628. (b) Cavalli, S.; Tipton, A. R.; Overhand, M.;
Kros, A. Chem. Commun. 2006, 3193-3195. (c) Hassane, F. S.; Frisch,
B.; Schuber, F. Bioconjugate Chem. 2006, 17, 849-854.
(16) Alvarez, S. G.; Alvarez, M. T. Synthesis 1997, 413-414.
(17) Jiang, H.; O’Neil, E. J.; DiVittorio, K. M.; Smith, B. D. Org. Lett.
2005, 7, 3013-3017.
(18) Differential scanning calorimetry measurements were made in water.
Cooling at a scan rate of 1° C/min produced a transition temperature of
40.2 ( 0.5 °C for a sample of para-isomer 6 (2.5 mg/mL) and no detectable
transition temperature between 2 and 90 °C for meta-isomer 7 under the
same conditions.
Org. Lett., Vol. 9, No. 2, 2007
201

hydrophobic core of a bilayer membrane, unless it is
anchored by a highly lipophilic tail.20 Indeed, the more likely
expectation with bolaamphiphiles 6-8 is that they should
be vesicle-destabilizing molecules. This hypothesis was tested
by measuring the ability of each bolaamphiphile to induce
leakage from preformed vesicles. Experimentally, this was
achieved by encapsulating carboxyfluorescein (50 mM)
inside vesicles composed of 1-palmitoyl-2-oleoylphosphati-
dylcholine (POPC) and cholesterol in a 8:2 ratio (25 µM
total lipid concentration). This water-soluble dye is self-
quenching, and fluorescence intensity increases as it leaks
from vesicles into the external solution. In separate experi-
ments, 20 µL aliquots of the bolaamphiphiles in ethanol were
added to 3 mL dispersions of vesicles. At low bolaamphiphile
concentrations of 1 µM, there was only very slow dye
leakage over many hours, but as shown in Figure 3, there
para-isomer 6 induced large amounts of leakage, whereas
the corresponding meta-isomer 7 had no measurable mem-
brane disruption effect. It is important to realize that these
two isomers of MW 1508 are highly flexible molecules that
differ in atom connectivity at one location by only a few
angstroms. Indeed, the structures are so similar that it is very
difficult to rationalize why there is such a large difference
in their membrane disruption abilities. Experiments with
amphiphiles 4 and 5 and bolaamphiphile 8 produced the same
result as 7, that is, no leakage. Thus, it appears that the
bolaamphiphile para-isomer 6 has an unusual preference to
adopt a molecular conformation or a multimeric aggregate
that disrupts the integrity of the vesicle membrane.21
In conclusion, we report that the copper-catalyzed azide
alkyne cycloaddition, which is increasingly referred to as
click chemistry, is a reaction that is compatible with the
functionality in a phosphatidylcholine headgroup and that
molecular cargo can be readily attached to the end of a
phosphatidylcholine acyl chain. Furthermore, two phospho-
lipids can be clicked to a bifunctional core molecule to
produce bolaamphiphiles in large scale, high yield, and high
purity. The synthetic processes described here should enable
the conjugation of phosphatidylcholine to a wide range of
solid supports22 and solution-state scaffolds.23 However, the
chemistry introduces polar 1,4-triazole units into the lipo-
philic region of the phospholipid structure, which may
produce molecules that disrupt the packing integrity of
bilayer membranes, as seen with one of the bolaamphiphiles
described here.
Acknowledgment. We thank William E. Archer for
assistance with the electron microscopy. This work was
supported by the National Institutes of Health.
Figure 3. Leakage induced upon addition of bolaamphiphile to
vesicles containing carboxyfluorescein (50 mM). To 25 µM of 8:2
POPC/cholesterol vesicles in TES buffer (5 mM TES, 100 mM
NaCl, pH 7.4) was added 10 µM of para-isomer 6 (9) or meta-
isomer 7 (×) at 50 s, and the vesicles were lysed by addition of
Triton X-100 at 1150 s. Repeating the experiment with amphiphiles
4, 5, and 8 yielded the same result as 7, that is, no leakage.
Supporting Information Available: Synthetic proce-
dures, spectroscopic data, and experimental procedures. This
material is available free of charge via the Internet at
http://pubs.acs.org.
OL062557A
(21) The leakage induced by 6 is not due to the sample addition procedure
because the other amphiphiles have no effect under the same addition
conditions. Furthermore, preincorporation of 6 at 10 µM prior to vesicle
formation produces extremely leaky vesicles, which suggests that insertion
of 6 into the vesicle membrane is not the process that causes the leakage.
(22) Rozkiewicz, D. I.; Janczewski, D.; Verboom, W.; Ravoo, B. J.;
Reinhoudt, D. N. Angew. Chem., Int. Ed. 2006, 45, 5292-5296.
(23) Lin, P.-C.; Ueng, S.-H.; Tseng, M.-C.; Ko, J.-L.; Huang, K.-T.; Yu,
S.-C.; Adak, A. K.; Chen, Y.-J.; Lin, C.-C. Angew. Chem., Int. Ed. 2006,
45, 4286-4290.
was a remarkable difference in leakage rates when the
bolaamphiphile concentration was raised to 10 µM. After
an induction period of several minutes, the bolaamphiphile
(19) Kolb, H. C.; Sharpless, K. B. Drug DiscoVery Today 2003, 24,
1128-1137.
(20) Menger, F. M.; Keiper, J. S.; Cran, K. L. J. Am. Chem. Soc. 2002,
124, 11842-11843.
202
Org. Lett., Vol. 9, No. 2, 2007