Bolaamphiphiles promote phospholipid translocation across vesicle membranes

Article abstract of DOI:10.1021/ja0619253

A series of membrane-spanning bolaamphiphiles (molecules with two hydrophilic end groups connected by a hydrophobic linker) were prepared by a modular synthetic method and evaluated for their abilities to affect the dynamics of a surrounding bilayer membr

Full text of DOI:10.1021/ja0619253

Published on Web 06/22/2006
Bolaamphiphiles Promote Phospholipid Translocation Across
Vesicle Membranes
Christopher C. Forbes, Kristy M. DiVittorio, and Bradley D. Smith*
Contribution from the Department of Chemistry and Biochemistry and the Walther Cancer
Research Center, UniVersity of Notre Dame, Notre Dame, Indiana 46556
Received March 21, 2006; E-mail: smith.115@nd.edu
Abstract: A series of membrane-spanning bolaamphiphiles (molecules with two hydrophilic end groups
connected by a hydrophobic linker) were prepared by a modular synthetic method and evaluated for their
abilities to affect the dynamics of a surrounding bilayer membrane. The goal was to determine if the
bolaamphiphiles promote the translocation of phospholipids across vesicle membranes. The bolaamphiphiles
were incorporated at low levels (up to 5 mol %) in vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-
3-phosphocholine (POPC). Inward translocation assays were performed using fluorescent, NBD-labeled
phospholipid probes with phosphocholine (PC) or phosphoglycerol (PG) headgroups. The membrane-
spanning bolaamphiphiles promote the translocation of both phospholipid probes in the order PG > PC,
whereas shorter bolaamphiphiles (structures that must adopt a U-shape and keep both end groups in the
same leaflet of the membrane), and regular amphiphiles with one hydrophilic end group, are inactive. These
results are an exception to the rule-of-thumb that membrane-spanning bolaamphiphiles are inherently
membrane-stabilizing molecules that inhibit all types of membrane transport.
Introduction
to ascertain if and how the bolaamphiphile alters the molecular
dynamics of the surrounding bilayer. Any effect will, of course,
Bolaamphiphiles are surface-active molecules with two
hydrophilic end groups connected by a hydrophobic spacer that
has one, two, or three alkyl chains. Bolaamphiphiles often self-
assemble in water to form monolayer lipid membranes that are
more tightly packed and less permeable than analogous bilayer
membranes.1-3 For example, the membranes of archaebacteria
are composed of approximately 90% tetraether isoprenoid-based
bolaamphiphiles, and as a result, they are unusually stable at
high temperature and low pH.4,5 Bolaamphiphiles are particularly
promising building blocks for the fabrication of highly stable,
supported lipid membranes to be used in various membrane
mimetic devices.6
The molecular assembly under consideration here is not to a
membrane composed predominantly of bolaamphiphiles but
rather a fluid-phase bilayer membrane composed primarily of
the common phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-
phosphocholine (POPC), with a low concentration (up to 5 mol
%) of added bolaamphiphile. The general goal of this work is
depend on the bolaamphiphile’s exact molecular structure. For
example, a number of bolaamphiphiles have been designed to
act as ion channels or membrane disruptors. These compounds
have specific structural elements such as internal hydrophilic
residues that promote a significant transport or disruption
process.1,7 Much less attention has been paid to the ability of
structurally simple bolaamphiles to alter bilayer membrane
structure and function.8 We are particularly interested in the
dynamic process of phospholipid translocation or flip-flop. In
contrast to lateral membrane diffusion, which is rapid, trans-
location of a labeled phospholipid across a bilayer membrane
is quite slow with a typical half-life in vesicles of many hours
or even days (Figure 1).9 The activation barrier is due to the
energetic penalty for moving the polar phospholipid headgroup
through the lipophilic interior of the membrane, although acyl
chain structure is also a significant factor.9,10 Recently, we and
other, have demonstrated that phospholipid translocation can
be accelerated by a number of low molecular weight organic
(1) (a) Fuhrhop, J.-H.; Wang, T. Chem. ReV. 2004, 104, 2901-2937 and
references therein.
(2) Boalaamphiphiles self-assemble into a range of morphologies; for a
discussion, see: Ko¨hler, K.; Forster, G.; Hauser, A.; Dobner, B.; Heiser,
U. F.; Ziethe, F.; Richter, W.; Steiniger, F.; Drechsler, M.; Stettin, H.;
Blume, A. J. Am. Chem. Soc. 2004, 126, 16804-16813 and references
therein.
(3) Bolaamphiphiles can even form stable membranes in pure ethanol. Wang,
X.; Liang, Y. J. Colloid Interface Sci. 2001, 233, 364-366.
(4) De Rosa, M.; Gambacorta, A.; Gliozzi, A. Microbiol. ReV. 1986, 50, 70-
80.
(5) Fan, Q.; Relini, A.; Cassinadri, D.; Gambacorta, A.; Gliozzi, A. Biochim.
Biophys. Acta 1995, 1240, 83-88.
(6) Kai, T.; Sun, X.-L.; Faucher, K. M.; Apkarian, R. P.; Chaikof, E. L. J.
Org. Chem. 2005, 70, 2606-2615. 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) Gu, Q.; Zou, A.; Yuan, C.; Guo, R. J. Colloid Interface Sci. 2003, 266,
442-447.
(9) For recent measurements of translocation rates with unlabeled phospholipids,
see: Liu, J.; Conboy, J. C. Biophys. J. 2005, 89, 2522-2532.
(10) Phospholipid translocation is disfavored enthalpically, but it can be favored
entropically, presumably due to the release of solvating water molecules.
Homan, R.; Pownall, H. J. Biochim. Biophys. Acta 1988, 938, 155-166.
9
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J. AM. CHEM. SOC. 2006, 128, 9211-9218
9211

A R T I C L E S
Forbes et al.
Scheme 1. Membrane-Spanning Bolaamphiphiles 1-4 and
Shorter Control Compounds 5 and 6
Figure 1. Definitions for phospholipid translocation.
Figure 2. Representation of the “slip-pop” mechanism for phospholipid
flip-flop. The inherent wobbling motion of a single-pass, membrane-
spanning helical peptide causes local defects in the headgroup region of
the bilayer, which allows neighboring phospholipids to slip into the
hydrophobic center of the membrane and subsequently pop outside.19
compounds, including molecules that induce membrane pores,11
or form hydrogen bonded complexes with the phospholipid
headgroup.12-15 Membrane adsorbed polymers can also disrupt
packing and induce phospholipid translocation.14
The specific goal of this study was to determine if structurally
simple bolaamphiphiles promote the translocation of phospho-
lipids across vesicle membranes. At first glance, this idea may
seem to be counterintuitive because, as stated above, bolaam-
phiphiles are generally considered as inhibitors of membrane
transport.1,8,16-18 Nonetheless, our curiosity was raised by a
recent study by the group of de Kruijff and co-workers who
observed that hydrophobic, single-pass, membrane-spanning
helical peptides promote phospholipid flip-flop in vesicle
membranes.19 They rationalize their results in terms of a “slip-
pop” mechanism (Figure 2), which proposes that the inherent
wobbling motion of the peptide causes local perturbations in
the headgroup region of the membrane, allowing the polar
headgroups of the adjacent phospholipids to slip into the
hydrophobic center and, subsequently, pop out on either side.
They also observed that single-pass helices promote phospho-
lipid translocation more effectively than multiple-pass helices.19
Thus, we were interested in seeing if a bolaamphiphile, with a
(11) (a) Fattal, E.; Nir, S.; Parente, R. A.; Szoka, F. C. Biochemistry 1994, 33,
6721-6731. (b) Matsuzaki, K.; Murase, O.; Fujii, N.; Miyajima, K.
Biochemistry 1996, 35, 11361-11368.
similar structural shape as a single-pass helix,20 could also
promote phospholipid translocation. We report the design and
synthesis of a series of amphiphiles, 1-6, (Scheme 1) which
are related by having the same phosphodiester end groups. The
structures of bolaamphiphiles 1-4 are sufficiently long (30-
32 heavy atoms) to span a vesicle membrane composed of
POPC, whereas 5 and 6 are shorter control compounds. We
find that the membrane-spanning bolaamphiphiles are indeed
able to promote the translocation of fluorescently labeled
(12) Smith, B. D.; Lambert, T. N. Chem. Commun. 2003, 2261-2268.
(13) Boon, J. M.; Lambert, T. N.; Sisson, A. L.; Davis, A. P.; Smith, B. D. J.
Am. Chem. Soc. 2003, 125, 8195-8201.
(14) Boon, J. M.; Smith, B. D. Med. Res. ReV. 2002, 22, 251-281.
(15) Shukla, R.; Sasaki, Y.; Krchnak, V.; Smith, B. D. J. Comb. Chem. 2004,
6, 703-709.
(16) Kalinowski, S.; Lotowski, Z.; Morzycki, J. W. Cell. Mol. Biol. Lett. 2000,
5, 107-118.
(17) Wang, G. J.; Hollingsworth, R. I. J. Org. Chem. 1999, 64, 4140-4147.
(18) Yan, Y.; Huang, J.; Li, Z.; Ma, J.; Fu, H.; Ye, J. J. Phys. Chem. B 2003,
107, 1479-1482.
(19) Kol, M. A.; de Kroon, A. I. P. M.; Killian, J. A.; de Kruijff, B. Biochemistry
2004, 43, 2673-2681; Kol, M. A.; van Laak, A. N. C.; Rijkers, D. T. S.;
Killian, J. A.; de Kroon, A. I. P. M.; de Kruijff, B. Biochemistry 2003, 42,
231-237; Kol, M. A.; de Kroon, A. I. P. M.; Rijkers, D. T. S.; Killian, J.
A.; de Kruijff, B. Biochemistry 2001, 40, 10500-10506.
(20) A single-pass helical peptide is thicker than the single-chain bolaamphiphiles
described in this paper, but their shapes are similar in that they are both
relatively narrow rods that bisect the membrane and wobble.
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9212 J. AM. CHEM. SOC. VOL. 128, NO. 28, 2006

Bolaamphiphiles Promote Phospholipid Translocation
A R T I C L E S
Scheme 4: Molecular Structure of POPC and the Fluorescent
Scheme 2. Synthesis of the Phosphotriester Building Blocks
Probes NBD-PG and NBD-PC
7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)/dithionite quenching
assay, which uses a phospholipid probe containing an NBD
group in one of its acyl chains (all phospholipid structures are
presented in Scheme 4).14 Exo-labeled vesicles were prepared
by addition of a small aliquot of NBD-lipid (0.5 mol % of
total phospholipid) in ethanol to a dispersion of vesicles
composed of a mixture POPC and bolaamphiphile. The NBD-
lipid readily inserts into the outer monolayer of the fluid-phase
vesicles. Upon treatment with sodium dithionite (Na2S2O4), the
NBD fluorescence is quenched due to reduction of the NBD
nitro group. Vesicle membranes are effectively impermeable
to dithionite; therefore, only NBD-phospholipid that is located
in the outer leaflet is chemically quenched. At any given time,
the fraction of probe located in the outer monolayer can be
determined from the drop in fluorescence intensity when a
portion of the vesicles are subjected to dithionite quenching.
Scheme 3. General Synthetic Strategy for Compounds 1-4
All translocation measurements were conducted in 5 mM
TES/100 mM NaCl buffer at pH 7.4 and 25 °C with unilamellar
vesicles that were prepared by repeated extrusion through
polycarbonate filters with 100-nm pores. The inward translo-
cation of NBD-PG or NBD-PC across vesicle membranes
composed of POPC and 4 or 5 mol % of amphiphiles 1-6 is
shown in Figures 3 and 4. The experiments start with an initial
value of 100% exo NBD-lipid and progress to an equilibrium
value of around 60% exo NBD-lipid.13 The half-lives for these
equilibration processes are listed in Table 1, along with the
longer half-lives that were observed when the vesicles also
contained 30% cholesterol. Inward translocation of NBD-PS
was also investigated with the more active amphiphiles, but no
significant translocation was observed (data not shown). In the
case of active bolaamphiphiles 1 and 3, the rate of NBD-PC
translocation was measured over the rather narrow range of 2-6
mol % and a linear dependency was observed (uncertainty in
the translocation rate measurement increases substantially
outside of this concentration range). The fact that the NBD
probes become protected from the externally added dithionite
is strong evidence that vesicles do not allow passage of
hydrophilic ions into the vesicles. This conclusion was inde-
pendently confirmed by a carboxyfluorescein leakage experi-
ment. The water-soluble fluorescent marker 5(6)-carboxyfluo-
rescein was encapsulated inside vesicles composed of 95%
POPC and 5% bolaamphiphile 2, and no significant leakage
was observed over several hours.
phospholipids across surface differentiated vesicle membranes,
and the biological implications of our results are discussed.
Results
Synthesis. A modular synthetic strategy was employed to
prepare the bolaamphiphiles. The six-carbon and twelve-carbon
phosphotriester N-Boc-amines 9a and 9b were prepared as
shown in Scheme 2. The N-Boc groups were removed by
treatment with TFA, but prior to the next synthetic step, the
trifluoroacetate counteranions were exchanged for chloride to
prevent side reactions. The ammonium chloride salts were
treated with the appropriate bis-acid chloride to give the
appropriate phosphotriester compounds 1PNP-4PNP (shown
generically as 10 in Scheme 3), and subsequent hydrolysis with
LiOH produced the phosphodiesters 1, 3, and 4. The six-carbon
control bolaamphiphile 5 was prepared by an analogous
pathway. The Leigh-type rotaxane 2 was made by treating the
precursor thread compound 1PNP with 5-tert-butylisophthaloyl
chloride and p-xylylenediamine.21 The rotaxane product, phos-
photriester 2PNP, was hydrolyzed to give the rotaxane phos-
phodiester, 2.
Phospholipid Translocation and Leakage Assays. Phos-
pholipid translocation was monitored via the well-established
(21) Gatti, F. G.; Leigh, D. A.; Nepogodiev, S. A.; Slawin, A. M. Z.; Teat, S.
J.; Wong, J. K. Y. J. Am. Chem. Soc. 2001, 123, 5983-5989.
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A R T I C L E S
Forbes et al.
Figure 3. Change in the fraction of NBD-PG in the membrane outer
monolayer (% Exo NBD-PG). Inward translocation induced at t ) 0 min
by adding 0.125 µM of NBD-PG to POPC vesicles (25 µM) that also
contained (Top) 5 mol % of 1(9), 2(2), or 6(b); (Bottom) 4 mol % of
3(9), 4(2), or 5(b).
Figure 4. Change in the fraction of NBD-PC in the membrane outer
monolayer (% Exo NBD-PC). Inward translocation induced at t ) 0 min
by adding 0.125 µM of NBD-PC to POPC vesicles (25 µM) that also
contained (Top) 5 mol % of 1(9), 2(2), or 6(b); (Bottom) 4 mol % of
3(9), 4(2), or 5(b).
Discussion
Table 1. Half-Life (t1/2) for NBD-PG and NBD-PC Translocation
Across Vesicles Containing Amphiphiles 1-6
Factors that were considered when designing the bolaam-
phiphiles for this study were synthetic accessibility, modular
variation of the building blocks, and limited flexibility in the
center of linking chain. In the case of 1-4, the end groups are
t1 2 (min)a
/
vesicle composition
NBD
−
PC
NBD−PG
POPC:1 (95:5)
POPC:2 (95:5)
POPC:3 (96:4)
POPC:4 (96:4)
POPC:5 (96:4)
POPC:6 (95:5)
POPC:Chol:1 (66:29:5)
POPC:Chol:2 (66:29:5)
15
12
120
25
40
15
15
monoanionic phosphodiesters (with terminal diphenylethanes22,23
)
that are connected by dodecyl chains to a central dicarbonyl
core. The two most likely orientations of a bolaamphiphile in a
bilayer membrane are illustrated in Figure 5. Depending on the
length and flexibility of the linking chain, the bolaamphiphile
can extend completely across the membrane or otherwise form
a U-shaped conformation in a single membrane leaflet. Previous
work by Moss and co-workers has shown that bolaamphiphiles
with linking chains that have been stiffened by the inclusion of
central aromatic rings strongly prefer extended, membrane-
spanning conformations.24 Furthermore, the translocation rate
for stiffened bolaamphiphiles is extremely slow. Therefore,
30
>120
>120
>120
>120
>120
>120
90
>120
a 5 mM TES, 100 mM NaCl, pH 7.4, 25 °C; estimated uncertainty is
(33%.
(22) A phosphodiester end group with a single phenyl ring is not large enough
to prevent unthreading of the macrocycle/thread components in rotaxane
2.
(23) Polar aromatic groups such as the side chains of tryptophan and tyrosine
are known to prefer the interfacial regions of a bilayer membrane, but phenyl
groups tend to bury into the membrane interior. Stahelin, R. V.; Cho, W.
Biochemistry 2001, 40, 472-4678. Nonetheless, preorganized hosts with
multiple aromatic rings are known to have moderate affinities for the
quaternary ammonium cation in the phosphocholine headgroup. Lambert,
T. N.; Smith, B. D. Coord. Chem. ReV. 2003, 240, 129-141. Therefore, it
is hard to predict the preferred membrane locations of the diphenylethane
end groups in these bolaamphiphiles.
Figure 5. A graphical representation of a bilayer membrane containing a
U-shape bolaamphiphile and an extended membrane-spanning bolaam-
phiphile.
(24) Moss, R. A.; Li, J. M. J. Am. Chem. Soc. 1992, 114, 9227-9229. For
additional discussion of the factors controlling translocation of bolam-
phiphiles, see: Moss, R. A.; Li, G.; Li, J.-M. J. Am. Chem. Soc. 1994,
116, 805-806.
bolaamphiphiles 1-3 with their rigid central regions are
expected to adopt extended, transmembrane orientations.
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Bolaamphiphiles Promote Phospholipid Translocation
A R T I C L E S
The data in Figures 3 and 4 clearly show that bolaamphiphiles
1-4 can greatly accelerate inward phospholipid translocation
across POPC membranes,25 whereas the control amphiphile 6,
with only one phosphodiester end group, is unable to induce
any significant translocation. Why are the bolaamphiphiles so
effective at promoting phosphoplipid translocation?26 In the case
of bolaamphiphile, 1, we considered the possibility that the
central fumaramide core may promote translocation by either
forming transient hydrogen-bonded complexes with the phos-
pholipid headgroups or self-associating to form phase-separated
microdomains. Evidence against the first process is the efficient
translocation induced by bolaamphiphiles 2 and 3, which are
analogues of 1 except that they are much less likely to form
intermolecular hydrogen bonds in the middle of the membrane.
The bolaamphiphile 2 is a rotaxane with an amide-containing
macrocycle that encapsulates and protects the central fumara-
mide core. It is well-established that the macrocycle forms
strong, internal hydrogen bonds with the fumaramide carbonyl
oxygens,21 and this co-conformational preference was confirmed
in the case of 2 by inspection of its 1H NMR spectrum in organic
solvent (the highly upfield chemical shift for the fumaramide
olefin hydrogens indicates that they are located deep inside the
macrocycle). Thus, the surrounding macrocycle in bolaam-
phiphile 2 prevents any hydrogen-bonded self-association of the
fumaramide core. A similar situation occurs with bolaamphiphile
3, which contains a 2,5-dimethoxyterephthalamide as the central
core. This aromatic group forms strong, internal hydrogen bonds
that will stiffen the central region of the bolaamphiphile and
lower the propensity for intermolecular assocation.27 The fact
that both 2 and 3 promote the translocation of NBD-PG and
NBD-PC almost as well as bolaamphiphile 1, whereas am-
phiphile 6 is completely ineffective, indicates that intermolecular
hydrogen bonding in the middle of the membrane is not a
necessary structural feature for accelerated translocation.26
Evidence against the second process, that flip-flop is accelerated
by the presence of phase-separated microdomains,28 was gained
by measuring the translocation of NBD-PC across vesicle
membranes composed of POPC:DPPC (55:45). This membrane
composition is known to form a heterogeneous mixture of fluid
and gel phase microdomains;29 however, the rate of inward
translocation of NBD-PC was as slow as that observed with
pure POPC vesicles.
Another translocation pathway that must be considered is a
small population of U-shaped orientations that are very effective
at disturbing membrane integrity and promoting flip-flop.30,31
To explore the likelihood of this translocation mechanism,
bolaamphiphiles 4 and 5 were prepared and evaluated. These
two bolaamphiphiles have 2,6-pyridine dicarboxamide units as
the central cores. This aromatic group is well-known to form
internal hydrogen bonds, with both amide NH residues directed
toward the pyridine nitrogen.32 Thus, these bolaamphiphiles are
more likely to form a U-shaped conformation; however, an
extended membrane-spanning orientation is still possible with
version 4 and its two dodecyl chains (Scheme 1). The shorter
version, 5, has two hexyl chains and cannot span a bilayer;
therefore, it must adopt a U-shaped orientation in one of the
membrane leaflets. The data in Figures 3 and 4 show that the
longer bolaamphiphile 4 is an efficient translocator, whereas
the shorter version 5 is inactive. This latter result is evidence
against the hypothesis that a bolaamphiphile in a U-shape
orientation is especially active.
The above structure/activity results are consistent with the
idea that phospholipid translocaton is strongly promoted by
bolaamphiphiles in a transmembrane orientation. Evidence that
this occurs by some sort of slip-pop mechanism is the fact that
the translocase activities of compounds 1 and 2 are greatly
attenuated when the vesicles also include 29 mol % cholesterol
(Table 1). The added cholesterol is known to increase order in
the acyl chains and raise lateral pressure which is expected to
decrease bolaamphiphile wobbling and inhibit the slip-pop
pathway.19,31 The generality of this finding needs to be tested
with further studies of alternative bolaamphiphile structures,
especially compounds with different types of hydrophilic end
groups and numbers of linking chains.33
In summary, membrane-spanning bolaamphiphiles 1-4 are
able to promote the translocation of monoanionic NBD-PG
and to a lesser extent the translocation of zwitterionic NBD-
PC across vesicle membranes.25 The data are consistent with
the slip-pop model for facilitated phospholipid translocation by
membrane-spanning bolaamphiphilic structures.19,31 The mem-
brane disruption effect is subtle and does not create defects that
are large enough to allow the passage of small polar molecules.
Our results are an exception to the rule of thumb that membrane-
spanning bolaamphiphiles are inherently membrane stabilizing
molecules that inhibit all types of membrane transport.8,18
An intriguing, ongoing question in biomembrane research is
the mechanism for accelerated phospholipid flip-flop across
(25) The half-lives listed in Table 1 indicate that the monoanionic probe NBD-
PG is always translocated faster than the zwitterionic NBD-PC, a
preference that has been noted in previous studies.13,19 It is not clear if the
NBD-PG translocates as an anion or as the neutral acid. Recent work
indicates that organic anions may be more permeable than predicted by
the pH-partition hypothesis. See: Thomae, A. V.; Wunderli-Allenspach,
H.; Kra¨mer, S. D. Biophys. J. 2005, 89, 1802-1811.
(26) Previous studies by our group have evaluated small organic molecules that
were specifically designed to promote phospholipid flip-flop. These
compounds form hydrogen bonded complexes with the polar headgroup
of the phospholipid and the complexes diffuse across the membrane. With
these mobile transport carriers, the half-lives for NBD-PC and NBD-PG
translocation are around 4-30 min and <1 min, respectively. Compared
to the bolaamphiphiles reported here, these mobile transport carriers
transport NBD-PC equally as well, but they transport NBD-PG almost
10 times better.
(30) Bolaamphiphiles that adopt U-shaped conformations tend to disrupt the
membrane so much that salt transport occurs. See, for example: Fyles, T.
M.; Zeng, B. J. Org. Chem. 1998, 63, 8337-8345; Fuhrhop, J. H.; Krull,
M.; Schulz, A.; Mo¨bius, D. Langmuir 1990, 6, 497-505.
(31) Another bolaamphiphile motion that may promote phospholipid flip-flop
is the interconversion between transmembrane and U-shaped bolaamphiphile
orientation (Figure 5). Even if this bolaamphiphile interconversion process
is quite slow, it may induce a local defect that lives long enough to permit
the translocation of many phospholipids. Because transverse phospholipid
diffusion is very rapid, a small but long-lived local defect may be an
effective short-circuit point for large amounts of rapid transmembrane flip-
flop.
(27) (a) Wu, Z.-Q.; Jiang, X.-K.; Zhu, S.-Z.; Li, Z.-T. Org. Lett. 2004, 6, 229-
232. (b) Parra, R. D.; Zeng, H.; Zhu, J.; Zheng, C.; Zeng, X. C.; Gong, B.
Chem.-Eur. J. 2001, 7, 4352-4357. (c) Ernst, J. T.; Becerril, J.; Park, H.
S.; Yin, H.; Hamilton, A. D. Angew. Chem., Int. Ed. 2003, 42, 535-539.
(d) Yin, H.; Lee, G.; Sedey, K. A.; Rodriguez, J. M.; Wang, H.-G.; Sebti,
S. M.; Hamilton, A. D. J. Am. Chem. Soc. 2005, 127, 5463-5468.
(28) The passive permeation of small molecules through a bilayer increases when
gel and fluid domains coexist, apparently due to membrane defects at the
phase boundaries. Clerc, S. G.; Thompson, T. E. Biophys. J. 1995, 68,
2333-2341.
(32) Affeld, A.; Hu¨bner, G. M.; Seel, C.; Schalley, C. A. Eur. J. Org. Chem.
2001, 2877-2890.
(33) Membrane additives that increase the lateral pressure at the membrane
surface are known to increase the rate of phospholipid translocation
(Bootsveld, A.; Degenhardt, R.; Kamp, D.; Haest, C. W. M. Mol. Membr.
Biol. 2004, 21, 315-322). Thus, it is possible that bolaamphiphiles with
large hydrophilic end groups (compared to the cross-sectional area of the
linking chain) may lower the barrier for translocation. However, the
inactivity of controls 5 and 6 suggests that this is not a dominant factor in
the present case.
(29) Shoemaker, S. D.; Vanderlick, T. K. Biophys. J. 2003, 84, 998-1009.
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A R T I C L E S
Forbes et al.
Vesicle Leakage Assay. A solution of 2 (5 mol %) and POPC (100
mol %) in CHCl3:MeOH was evaporated and dried in vacuo for 1 h. A
solution of 10 mM vesicles was made by rehydration at room
temperature with 5(6)-carboxyfluorescein (CF) buffer (50 mM CF, 5
mM TES, 100 mM NaCl, pH 7.4). The vesicles were frozen and thawed
10 times and then extruded 29 times through a 19-mm polycarbonate
Nucleopore filter with 100-nm diameter pores. Unencapsulated dye was
removed from the vesicles by separation via a Sephadex-G50 column
in TES buffer. The fluorescence intensity of a 3-mL sample of vesicles
(25 µM in TES buffer) was monitored for 400 s then Triton X-100 (20
µL of 20% v:v) was added to lyse the vesicles.
Synthesis.37 N-Boc-12-aminododecanoic acid. 12-Aminodode-
canoic acid (1.00 g, 4.65 mmol) was suspended in 15.0 mL of methanol
with 0.775 mL (5.58 mmol) of triethylamine and 1.01 g (4.65 mmol)
of dissolved di-tert-butyl-dicarbonate and heated under a reflux
condenser to 60 °C overnight. The solvent was evaporated on a rotary
evaporator, and the residue was redissolved in ethyl acetate. The mixture
was then washed with 0.25 M HCl (2×), dried, and filtered, and the
solvent evaporated to give a white solid. N-Boc-12-aminododecanoic
acid (90%) was purified by recrystallization in hexanes. δH(500 MHz;
CDCl3; Me4Si) 11.67 (1H, s, COOH), 4.55 (1H, s, Boc NH), 3.10 (2H,
quar, J ) 6 Hz, Dodec), 2.34 (2H, t, J ) 7 Hz, Dodec), 1.62 (2H,
quin, J ) 7, Dodec), 1.44 (11H, s, Boc + Dodec), 1.27 (16H, s, Dodec).
δC(125 MHz; CDCl3; CDCl3) 179.7, 156.2, 79.3, 41.9, 40.8, 34.3, 30.2,
29.63, 29.55, 29.45, 29.38, 29.22, 28.6, 27.0, 24.9.
biogenic membranes (i.e., membranes that contain the enzymes
responsible for phospholipid biosynthesis) such as the bacterial
cytoplasmic membrane and the mammalian endoplasmic reticu-
lum.19,34 Because phospholipid biosynthesis occurs only on one
face of the membrane, there has to be a process to translocate
the newly formed phospholipid and allow the membrane to
grow. Indeed, phospholipid translocation across biogenic mem-
branes is known to be rapid, and for some time, researchers
have tried to identify the associated translocase protein(s). In
the case of the endoplasmic reticulum, there is evidence for an
energy-independent, bidirectional translocase that is equally
effective with a range of phospholipid headgroups, however,
the specific protein(s) has not yet been identified.35 An alterative
theory, proposed by de Kruijff and co-workers states that
phospholipid translocation across biogenic membranes does not
require a dedicated translocase because it can be can be
promoted by simple single-pass helical peptides.19 Our results
agree with the model that simple, membrane-spanning bolaam-
phiphilic structures can promote phospholipid translocation. The
bolaamphiphiles reported here are about an order of magnitude
less efficient than a single-pass helical peptide, but both systems
show the same headgroup selectivity, that is, the translocation
of monoanionic PG is faster than zwitterionic PC.25 Because
the fraction of PG in bacterial membranes (15-70%) is much
higher than in mammalian membranes (<2%), it appears that
the slip-pop mechanism is more likely to be kinetically
significant in bacterial membranes.36
N-Boc-12-aminododecanol. To a solution of N-Boc-12-aminodode-
canoic acid (4.22 g, 13.4 mmol) in 100 mL of dry THF was added
Red-Al (12.3 mL, 40.2 mmol-65% weight in toluene) via a syringe
over 30 min. Because of the extensive precipitation, a mechanical stirrer
was used. The reaction was stirred for 30 min after the precipitate
dissolved and then quenched with saturated sodium sulfate and stirred
for another 1 h. The solids formed during the quenching process were
then filtered, and the solution was concentrated to an oil on a rotary
evaporator, redissolved in ethyl acetate, and washed with water, dried,
filtered, and evaporated to give the desired product (3.39 g, 84%) as a
white solid which was recrystallized in toluene and used without further
purification. δH(500 MHz; CDCl3; Me4Si) 4.55 (1H, s, Boc NH), 3.64
(2H, t, J ) 7 Hz, Dodec), 3.10 (2H, quar, J ) 7 Hz, Dodec), 1.57 (2H,
quin, J ) 7 Hz, Dodec), 1.44 (11H, s, Boc + Dodec), 1.26 (16H, m,
Dodec). δC(125 MHz; CDCl3; CDCl3) 156.2, 79.2, 63.2, 51.3, 40.8,
33.0, 30.2, 29.75, 29.71, 29.60, 29.46, 28.6, 27.0, 25.9.
Experimental Section
Phospholipid Translocation Assay. POPC (1-palmitoyl-2-oleoyl-
sn-glycero-3-phosphocholine), NBD-PC (1-palmitoyl-2-[6-[(7-nitro-
2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-sn-glycero-3-phosphocho-
line), and NBD-PG (1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-
4-yl)amino]hexanoyl]-sn-glycero-3-[phospho-rac-(1-glycerol)] (ammonium
salt)) were purchased from Avanti Polar Lipids. Cholesterol was
purchased from Sigma-Aldrich. An appropriate mixture of POPC (in
CHCl3), cholesterol (in CHCl3), and/or bolaamphiphile (in 7:3 CHCl3:
CH3OH) was dried in vacuo for 1 h. A stock solution of 10 mM vesicles
was made by rehydration at room temperature with TES buffer (5 mM
TES, 100 mM NaCl, pH 7.4). Multilamellar vesicles were extruded to
form unilamellar vesicles with a Basic LiposoFast device purchased
from Avestin, Incorporated. The vesicles were extruded 29 times
through a 19-mm polycarbonate Nucleopore filter with 100-nm diameter
pores. An aliquot of NBD-phosopholipid (NBD-PC or NBD-PG in
140 µL of 200 proof ethanol, final concentration 0.125 µM) was added
to the vesicles (45 mL, 25 µM) in TES buffer. Samples (3.0 mL) were
withdrawn over time and assayed in the following way for % exo
NBD-phospholipid. At assay time t ) 50 s, sodium dithionite (180
µL of 60 mM solution in 1 M Tris, pH ∼10) was added and at t ) 180
s, Triton X-100 (20 µL of 20% v/v) was added. The amount of total
NBD-PX that was translocated to the inside of the vesicles was
calculated by the following equation: % Exo NBD-phospholipid )
((Fi - Ff)/Fi)*100, where Fi and Ff are the fluorescence intensities
(ex: 460 nm, em: 530 nm) just prior to the additions of sodium
dithionite and Triton X-100. The half-lives reported are the averages
of 2 or 3 independent experiments.
N-Boc-6-aminohexanol. 6-Aminohexanol (1.00 g, 8.50 mmol) was
dissolved in 5.00 mL of THF and di-tert-butyl-dicarboxylate (1.86 g,
8.50 mmol) was added. The solution was allowed to stir at room
temperature for 3 h, and solvent was removed with a rotary evaporator
to give the desired product (99%) as a colorless oil, which crystallized
after 2 weeks. The compound was used without further purification.
δH(500 MHz; CDCl3; Me4Si) 4.65 (1H, s, Boc NH), 3.62 (2H, t, J )
7 Hz, Hexyl), 3.12 (2H, quar, J ) 7 Hz, Hexyl), 2.09 (1H, s, OH),
1.57 (2H, quin, J ) 7 Hz, Hexyl), 1.49 (2H, quin, J ) 7 Hz, Hexyl),
1.44 (9H, s, Boc), 1.36 (4H, m, Hexyl). δC(125 MHz; CDCl3; CDCl3)
156.3, 79.2, 62.7, 40.5, 32.7, 30.2, 28.6, 26.5, 25.4.
2,5-Dimethoxyterephthaloyl Dichloride. This compound (a white
solid) was synthesized by a literature method.38 δH(500 MHz; CDCl3;
Me4Si) 7.55 (2H, s, Ph), 3.96 (6H, s, Me). δC(125 MHz; CDCl3; CDCl3)
163.8, 152.0, 128.3, 116.6, 57.0.
12-(tert-Butylcarbamoyl)dodecyl-4-nitrophenyl-2,2-diphenyleth-
yl Phosphate (9a). To a stirred solution of tris(p-nitrophenyl) phosphate
(200.0 mg; 0.433 mmol) and 2,2-diphenyl ethanol (85.8 mg; 0.433
mmol) in 3 mL of dry chloroform (stabilized with amylenes) was added
DBU (68 µL; 0.455 mmol), and the reaction was allowed to stir for 3
(34) Gummadi, S. N.; Kumar, K. S. Cell. Mol. Biol. Lett. 2005, 10, 101-121.
(35) Vishwakarma, R. A.; Vehring, S.; Mehta, A.; Sinha, A.; Pomorski, T.;
Herrmann, A.; Menon, A. K. Org. Biomol. Chem. 2005, 3, 1275-1283.
(36) That is not to say that all translocase proteins in the bacterial membrane
are single-pass helical structures. For example, the wzxE translocase is a
large protein with multiple transmembrane domains. Rick, P. D.; Barr, K.;
Sankaran, K.; Kajimura, J.; Rush, J. S.; Waechter, C. J. J. Biol. Chem.
2003, 278, 16534-16542.
(37) The NMR data includes magnetic field strength, solvent, and chemical shift
reference. The 13C NMR signals are quoted to two decimal places only in
spectral regions that are crowded.
(38) Sotzing, G. A.; Reynolds, J. R.; Katritzky, A. R.; Soloducho, J.; Belyakov,
S.; Musgrave, R. Macromolecules 1996, 29, 1679-1684.
9
9216 J. AM. CHEM. SOC. VOL. 128, NO. 28, 2006

Bolaamphiphiles Promote Phospholipid Translocation
A R T I C L E S
h. The reaction was then concentrated to an oil using a rotary evaporator
and the residue was redissolved in 5.00 mL of dry toluene. N-Boc-12-
aminododecanol powder (131 mg; 0.433 mmol) was added to the new
solution followed by another 68 µL of DBU, and the reaction was
allowed to stir overnight. The mixture was then diluted with ethyl
acetate and washed with 0.25 M HCl. Compound 9a (60%) was purified
as a clear viscous oil by column chromatography using chloroform as
the eluent. δH(300 MHz; CDCl3; Me4Si) 8.08 (2H, d, J ) 9 Hz, PNP),
7.22 (10H, m, DPE), 7.11 (2H, d, J ) 9 Hz, PNP), 4.67 (2H, t, J ) 7
Hz, DPE), 4.59 (1H, s-br, Boc NH), 4.38 (1H, t, J ) 8 Hz, DPE), 3.90
(2H, quar, J ) 7 Hz, Dodec), 3.07 (2H, quar, J ) 7 Hz, Dodec), 1.58
(2H, quin, J ) 7 Hz, Dodec), 1.42 (11H, s, Boc + Dodec), 1.22 (16
Hz, s-br, Dodec). δC(75 MHz; CDCl3; CDCl3) 156.1, 155.4 (d, J ) 7
Hz), 144.6, 140.1, 128.8, 128.3, 127.2, 125.6, 120.5, 79.0, 70.7 (d, J
) 7 Hz), 69.3 (d, J ) 7 Hz), 51.3, 40.7, 30.2, 30.1, 29.59, 29.56, 29.52,
29.36, 29.08, 28.52, 26.9, 25.3
Dodec), 1.37 (8H, m, Dodec), 1.24 (24H, m, Dodec). δC(125 MHz;
CDCl3; CDCl3) 164.3, 155.5 (d, J ) 7 Hz), 151.6, 144.7, 140.1, 128.9,
128.3, 127.3, 125.7, 124.7, 120.6, 115.4, 70.7 (d, J ) 7 Hz), 69.3 (d,
J ) 7 Hz), 56.7, 51.42, 51.37, 40.1, 30.2, 30.2, 29.73, 29.67, 29.63,
29.5, 29.2, 27.3, 25.4.
Bis-N,N-(p-nitrophenyl-2,2-diphenylethyl-12-aminododecyl phos-
phate)-2,6-pyridine Dicarbamide (4PNP). The product was a white
solid (78%). δH(500 MHz; CDCl3; Me4Si) 8.33 (2H, d, J ) 8 Hz, Py),
8.18 (2H, t, J ) 6 Hz, Py NH), 8.11 (4H, d, J ) 9 Hz, PNP), 7.98 (1H,
t, J ) 8 Hz, Py), 7.25 (20H, m, Ph), 7.11 (4H, d, J ) 9 Hz, PNP), 4.69
(4H, t, J ) 7 Hz, DPE), 4.40 (2H, t, J ) 7 Hz, DPE), 3.98 (4H, quar,
J ) 7 Hz, Dodec), 3.41 (4H, quar, J ) 7 Hz, Dodec), 1.59 (8H, m,
Dodec), 1.24 (32H, m, Dodec).
Bis-N,N-(p-nitrophenyl-2,2-diphenylethyl-6-aminohexyl phosphate)-
2,6-pyridine Dicarbamide (5PNP). The product was a white solid (70%).
δH(500 MHz; CDCl3; Me4Si) 8.34 (2H, d, J ) 8 Hz, Py NH), 8.18
(2H, t, J ) 6 Hz, Py), 8.10 (4H, d, J ) 9 Hz, PNP), 8.00 (1H, t, J )
8 Hz, Py), 7.27 (8H, m, Ph), 7.22 (12H, m, Ph), 7.12 (4H, d, J ) 9 Hz,
PNP), 4.69 (4H, t, J ) 7 Hz, DPE), 4.40 (2H, t, J ) 7 Hz, DPE), 3.98
(4H, quar, J ) 7 Hz, Hexyl), 3.41 (4H, quar, J ) 7 Hz, Hexyl), 1.58
(8H, m, Dodec), 1.29 (8H, m, Hexyl). δC(125 MHz; CDCl3; CDCl3)
163.7, 155.3 (d, J ) 6 Hz), 149.0, 144.6, 139.9, 139.0, 128.8, 128.2,
127.2, 125.6, 125.0, 120.5, 70.7 (d, J ) 6 Hz), 68.9 (d, J ) 6 Hz),
51.3, 39.5, 29.9, 29.8, 26.3, 24.9.
Bis-N,N-(2,2-diphenylethyl-12-aminododecyl phosphate)-fuma-
ramide (1). Compound 1PNP (200 mg, 0.161 mmol) was dissolved in
10.0 mL of DMSO, and 1.0 mL of 1.0 M LiOH solution was added.
Reaction was stirred for 1 h, and the DMSO solution was added to 50
mL of HCl (0.25 M). The pure product immediately precipitated out
of solution as a white solid, was filtered, and was washed with water
to give a yield of 50%. δH(500 MHz; CDCl3/CD3OD 1:1; Me4Si) 7.26
(20H, m, Ph), 6.81 (2H, s, Fu CH), 4.51 (4H, t, J ) 7 Hz, DPE), 4.37
(2H, t, obscured by solvent peak, DPE), 3.75 (4H, quar, J ) 7 Hz,
Dodec), 3.28 (4H, t, J ) 7 Hz, Dodec), 1.54 (4H, m, Dodec), 1.25
(36H, m, Dodec). δC(125 MHz; CDCl3/CD3OD 1:1; CDCl3) 165.3,
140.7, 132.5, 128.5, 128.2, 126.8, 69.0 (d, J ) 7 Hz), 67.4 (d, J ) 7
Hz), 51.2, 39.8, 30.1, 30.0, 29.44, 29.40, 29.22, 29.04, 28.99, 26.9,
25.3. FAB-MS (NBA matrix): m/z 1004 [M + H]+.
Bis-N,N-(2,2-diphenylethyl-12-aminododecyl phosphate)-2,5-
dimethoxyterephthalamide (3). Compound 3PNP (200 mg, 0.148 mmol)
was dissolved in 10 mL of DMF, and 1.0 mL of 1.0 M LiOH was
added. The reaction was allowed to stir for 1 h, and compound 3 was
purified by diluting the reaction mixture with water and washing with
chloroform, separating and acidifying the organic layer, and then drying
on a high vacuum line (65%). δH(500 MHz; CDCl3; Me4Si) 8.09 (2H,
t, J ) 6 Hz, Tera NH), 7.88 (2H, s, Tera CH), 7.23 (20H, m, Ph), 4.47
(4H, t, J ) 7 Hz, DPE), 4.34 (2H, t, J ) 7 Hz, DPE), 3.96 (6H, s, Tera
Me), 3.72 (4H, quar, J ) 7 Hz, Dodec), 3.47 (4H, quar, J ) 7 Hz,
Dodec), 1.61 (4H, quin, J ) 7 Hz, Dodec), 1.47 (4H, m, Dodec), 1.25
(32H, m, Dodec). δC(125 MHz; CDCl3; CDCl3) 164.4, 151.6, 140.9,
128.7, 128.5, 127.0, 124.7, 115.5, 69.2, 67.7, 56.8, 51.4, 51.3, 40.0,
30.23, 30.17, 29.50, 29.46, 29.28, 29.14, 27.1, 25.4. FAB-MS (NBA
matrix): m/z 1114 [M + H]+.
Bis-N,N-(2,2-diphenylethyl-12-aminododecyl phosphate)-2,6-py-
ridine Dicarboxamide (4). Compound 4PNP (300 mg, 0.231 mmol)
was dissolved in 10 mL of DMF, and 1.0 mL of 1.0 M LiOH was
added. The reaction was allowed to stir for 1 h, then 1 mL of 2.5 M
HCl was added, and the resulting solution was concentrated on a rotary
evaporator. The residue was washed with water (2×) and then washed
with anhydrous diethyl ether (3x) to give 4 (50%). δH(500 MHz; CDCl3/
CD3OD 3:1; Me4Si) 8.28 (2H, d, J ) 8 Hz, Py), 8.01 (1H, t, J ) 8 Hz,
Py), 7.22 (20H, m, Ph), 4.51 (4H, t, J ) 8 Hz, DPE), 4.37 (2H, t, J )
8 Hz, DPE), 3.75 (4H, quar, J ) 7 Hz, Dodec), 3.44 (4H, t, J ) 7 Hz,
Dodec), 1.66 (4H, quin, J ) 8 Hz, Dodec), 1.53 (4H, quin, J ) 7 Hz,
Dodec), 1.35 (8H, m, Dodec), 1.24 (24H, m, Dodec). δC(125 MHz;
CDCl3/CD3OD 3:1; CDCl3) 164.2, 148.8, 140.7, 138.9, 128.4, 128.1,
6-(tert-Butylcarbamoyl)hexyl-2,2-diphenylethyl-4-nitrophenyl phos-
phate (9b). To a stirred solution of tris(p-nitrophenyl) phosphate (200
mg; 0.433 mmol) and 2,2-diphenyl ethanol (85.8 mg; 0.433 mmol) in
3 mL of dry chloroform (stabilized with amylenes) was added DBU
(68 µL; 0.455 mmol), and the reaction was allowed to stir for 3 h. The
reaction was then concentrated to an oil using a rotary evaporator and
the residue was redissolved in dry toluene. N-Boc-6-aminohexanol (94.0
mg; 0.433 mmol) was added to the new solution followed by another
68 µL of DBU and reaction was allowed to stir overnight. The mixture
was then diluted with ethyl acetate and washed with acid. The product
(50%) was purified as a clear viscous oil by column chromatography
using chloroform as the eluent. δH(500 MHz; CDCl3; Me4Si) 8.08 (2H,
d, J ) 9 Hz, PNP), 7.25 (10H, m, Ph), 7.11 (2H, d, J ) 9 Hz, PNP),
4.95 (1H, t, J ) 7 Hz, PNP), 4.68 (2H, t, J ) 7 Hz, Boc NH), 4.38
(1H, t, J ) 7 Hz, DPE), 3.97 (2H, quar, J ) 7 Hz, Hexyl), 3.15 (2H,
quar, J ) 7 Hz, Hexyl), 1.55 (4H, m, Hexyl) 1.41 (9H, s, Boc), 1.29
(4H, m, Hexyl).
General Procedure for the Synthesis of Compounds 1PNP-5PNP
.
The appropriate Boc-protected amine was dissolved in the minimal
amount of dichloromethane and then diluted with trifluoroacetic acid
to double the volume. The solution was stirred for 20 min after gas
evolution stopped, and the solvent was removed with a rotary
evaporator. The residue was then dissolved in methanol with 0.5 mL
of added concentrated HCl and evaporated (achieving ion exchange).
The residue was then evaporated twice with toluene and dissolved in
dry chloroform. Added to this mixture was the corresponding bis-acid
chloride (0.5 molar equivalents). Triethylamine (two molar equivalents
with respect to the amine) was first diluted with chloroform and added
to the reaction with a dropping funnel over 1 h. The reaction was stirred
for 2 h after the last addition of base, and the product was purified by
chromatography (all 5 compounds required 0-3% methanol in dichlo-
romethane as the eluent).
Bis-N,N-(p-nitrophenyl-2,2-diphenylethyl-12-aminododecyl phos-
phate)-fumaramide (1PNP). The product was a white solid (70%). δH-
(500 MHz; CDCl3; Me4Si) 8.10 (4H, d, J ) 9 Hz, PNP), 7.45 (2H, t,
J ) 6 Hz, Fu NH), 7.23 (20H, m, Ph), 7.18 (2H, s, Fu CH), 7.12 (4H,
d, J ) 9 Hz, PNP), 4.69 (4H, t, J ) 7 Hz, DPE), 4.40 (2H, t, J ) 7 Hz,
DPE), 3.99 (4H, quar, J ) 7 Hz, Dodec), 3.33 (4H, quar, J ) 7 Hz,
Dodec), 1.58 (8H, quin, J ) 7 Hz, Dodec), 1.23 (32H, m, Dodec).
δC(125 MHz; CDCl3; CDCl3) 164.9, 155.4 (d, J ) 7 Hz), 144.6, 140.1,
133.3, 128.8, 128.3, 127.2, 125.6, 120.6, 70.7 (d, 7 Hz), 69.3 (d, 7
Hz), 51.4, 40.0, 30.16, 30.11, 29.65, 29.62, 29.55, 29.51, 29.46, 29.1,
27.2, 25.4.
Bis-N,N-(p-nitrophenyl-2,2-diphenylethyl-12-aminododecyl phos-
phate)-2,5-dimethoxy-terephthalamide (3PNP). The product was a
white solid (75%). δH(500 MHz; CDCl3; Me4Si) 8.11 (4H, d, J ) 9
Hz, PNP), 8.10 (2H, br s, Tera NH), 7.89 (2H, s, Tera Ph), 7.27 (20H,
m, Ph), 7.13 (4H, d, J ) 9 Hz, PNP), 4.69 (4H, t, J ) 7 Hz, DPE),
4.40 (2H, t, J ) 7 Hz, DPE), 4.00 (4H, quar, J ) 7 Hz, Dodec), 3.99
(6H, s, Tera Me), 3.46 (4H, quar, J ) 7 Hz, Dodec), 1.61 (8H, m,
9
J. AM. CHEM. SOC. VOL. 128, NO. 28, 2006 9217

A R T I C L E S
Forbes et al.
126.7, 124.6, 69.0 (d, J ) 7 Hz), 67.3 (d, J ) 7 Hz), 51.2, 39.6, 30.1,
30.0, 29.53, 29.48, 29.42, 29.40, 29.31, 29.0, 27.0, 25.3. FAB-MS (NBA
matrix): m/z 1055 [M+H]+, 1077 [M + Na]+.
using a dual syringe pump a chloroform solution of p-xylylene diamine
and a chloroform solution of 5-tert-butyl-isophthaloyl dichloride. The
resulting precipitate was filtered through Celite, and the solvent was
evaporated using a rotary evaporator. The product was purified by
chromatography using 3% methanol in dichloromethane as the mobile
phase to give 2PNP as a white solid (15%). δH(300 MHz; CDCl3; Me4-
Si) 8.45 (2H, s, Wh Iso), 8.21 (4H, s, Wh Iso), 8.06 (4H, d, J ) 9 Hz,
PNP), 7.69 (4H, t, J ) 5 Hz, Wh NH), 7.25 (20H, m, Ph), 7.11 (4H,
d, J ) 9 Hz, PNP), 7.05 (8H, s, Wh Xy), 6.40 (2H, t, J ) 5 Hz, Th
NH), 5.72 (2H, s, Th alkene), 4.67 (4H, t, J ) 7 Hz, DPE), 4.47, (8H,
d, J ) 5 Hz, Wh Benz), 4.38 (2H, t, J ) 7 Hz, DPE), 3.98 (4H, quar,
J ) 7 Hz, Dodec), 3.14 (4H, quar, J ) 7 Hz, Dodec), 1.51 (8H, m,
Dodec), 1.37 (18H, s, t-Bu), 1.22 (32H, m, Dodec).
Bis-N,N-(2,2-diphenylethyl-6-aminohexyl phosphate)-2,6-pyridine
Dicarbamide (5). Compound 5PNP (200 mg, 0.177 mmol) was dissolved
in 10 mL of DMF, and 1.0 mL of 1.0 M LiOH solution was added.
The solution was allowed to stir at room temperature for 1 h and was
then concentrated on a rotary evaporator. The residue was dissolved
in the minimum amount of water, and the bis-lithium phosphate salt
of the product was driven out of solution with acetone. The mother
liquor was decanted, and the remaining oily residue was dissolved in
methanol with 3 drops of concentrated HCl solution, and the solution
evaporated to dryness. The product 5 was selectively extracted into
1:1 methanol/chloroform and regained as an off-white solid after
filtration and solvent evaporation (40%). δH(500 MHz; CD3OD; CHD2-
OD) 8.34 (2H, d, J ) 7 Hz, Py), 8.21 (1H, t, J ) 7 Hz, Py), 7.25
(20H, m, Ph), 4.45 (4H, t, J ) 7 Hz, DPE), 4.33 (2H, t, J ) 7 Hz,
DPE), 3.75 (4H, quar, J ) 6 Hz, Hexy), 3.50 (4H, t, J ) 6 Hz, Hexyl),
1.67 (4H, s, Hexyl), 1.55 (4H, s, Hexyl), 1.37 (8H, s, Hexyl). δC(125
MHz; CD3OD; CD3OD) 166.4, 150.3, 142.4, 129.7, 129.5, 128.2, 128.0,
125.7, 70.4, 68.4, 52.8 (d, J ) 7 Hz), 40.9, 31.1, 30.4, 27.4, 26.2. FAB-
MS (NBA matrix): m/z 925 [M + K]+.
12-(tert-Butylcarbamoyl)dodecyl-2,2-diphenylethyl Phosphate (6).
Compound 6PNP was dissolved in 10 mL of DMF, and 1.0 mL of 1.0
M LiOH was added. The reaction was allowed to stir for 1 h and was
then concentrated on a rotary evaporator. The residue was dissolved
in ethyl acetate and washed with 0.25 M NaOH (3×) and then with
0.25 M HCl (2×). The organic layer was dried with magnesium sulfate,
filtered, and evaporated to give compound 6 as a clear oil (65%). δH-
(300 MHz; CDCl3; Me4Si) 7.76 (1H, br s, Phos OH), 7.20 (10H, m,
Ph), 4.46 (2H, t, J ) 7 Hz, DPE), 4.35, (1H, t, J ) 7 Hz, DPE), 3.70
(2H, quar, J ) 7 Hz, Dodec), 3.09 (2H, t, J ) 7 Hz, Dodec), 1.47 (4H,
m, Dodec), 1.44 (9H, s, Dodec), 1.22 (16H, m, Dodec). δC(75 MHz;
CDCl3; CDCl3) 141.1, 128.7, 128.5, 126.9, 126.2, 69.1, 67.5, 51.4, 51.3,
40.8, 30.4, 30.3, 30.2, 29.7, 29.5, 29.4, 28.6, 27.0, 25.6, 25.1. FAB-
MS (NBA matrix): m/z 460 [M - Boc + H]+.
Rotaxane (2). Compound 2PNP (100 mg, 0.053 mmol) was dissolved
in 10 mL of DMSO, and 1.0 mL of 1.0 M LiOH solution was added.
The reaction was stirred for 1 h, and the DMSO solution was added to
50 mL of HCl (0.25 M). The product, compound 2, precipitated out of
solution as a white solid, was filtered, and was washed with water (3×)
(50%). Yield ) 50% 1H NMR δH(500 MHz, 3:1 CDCl3/CD3OD) 8.60
(s, 2H, Wh Iso), 8.25 (s, 4H, Wh Iso), 7.26 (m, 16H, Ph), 7.20 (m, 4H,
Ph), 7.04 (s, 8H, Wh Xy), 5.68 (s, 2H, Th alkene), 4.49 (t, 4H, J ) 7
Hz, DPE), 4.45 (s, 8H, Wh CH2), 4.36 (t, 2H, J ) 7 Hz, DPE), 3.73
(quar, 4H, J ) 7 Hz, Dodec), 3.11 (t, 4H, J ) 7 Hz, Dodec), 1.51 (m,
4H, Dodec), 1.43 (s, 18H, t-Bu), 1.26 (m, 36H, Dodec). δC(125 MHz,
CDCl3/CD3OD 3:1) δ 167.6, 165.6, 152.8, 140.9, 136.9, 133.4, 129.7,
128.9, 128.5, 128.4, 128.2, 126.7, 122.2, 68.8, 67.1, 51.3, 43.8, 39.8,
35.1, 31.1, 30.2, 30.1, 29.6, 29.5, 29.45, 29.44, 29.28, 29.09, 27.0, 25.4.
FAB-MS (NBA matrix): m/z 1647 [M + H]+, 1669 [M + Na]+.
Acknowledgment. This work was supported by the NIH and
the University of Notre Dame.
Supporting Information Available: 1H NMR spectra of
synthetic amphiphiles 1-6. This material is available free of
charge via the Internet at http://pubs.acs.org.
Rotaxane 2PNP. To a solution of 1PNP and 10 equiv triethylamine in
dry chloroform (stabilized with amylenes) was added simultaneously
JA0619253
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9218 J. AM. CHEM. SOC. VOL. 128, NO. 28, 2006