trans-2-Aminocyclohexanol as a pH-sensitive conformational switch in lipid amphiphiles

Article abstract of DOI:10.1039/b807704e

Protonation-induced conformational change of lipid tails is reported as a novel strategy to render pH-sensitive lipid amphiphiles and lipid colloids. The Royal Society of Chemistry.

Full text of DOI:10.1039/b807704e

View Article Online / Journal Homepage / Table of Contents for this issue
COMMUNICATION
www.rsc.org/chemcomm | ChemComm
trans-2-Aminocyclohexanol as a pH-sensitive conformational switch in
lipid amphiphileswz
Barbora Brazdova,a Ningrong Zhang,b Vyacheslav V. Samoshin*a and Xin Guo*b
Received (in Cambridge, UK) 7th May 2008, Accepted 18th June 2008
First published as an Advance Article on the web 5th August 2008
DOI: 10.1039/b807704e
Protonation-induced conformational change of lipid tails is
reported as a novel strategy to render pH-sensitive lipid amphi-
philes and lipid colloids.
Based on our earlier studies on TACHs,6,7 we anticipated that
both trans-dodecyloxycarbonyl groups of 1 would adopt the
equatorial conformation while the morpholine and the hydroxyl
groups would be both in the axial conformation (1A in Scheme 1).
Upon protonation, the axial-to-equatorial switch of the morpho-
line and the hydroxyl groups would force both ester groups into
axial positions (1BH+), thereby drastically increasing the separa-
tion of the lipid tails. In liposomes comprising 1, this would
loosen the packing of the lipid tails in the bilayer, leading to
leakage and even to the collapse of the liposomes. To estimate the
effect of such conformational change, the stereoisomer 2
(Scheme 1) with two dodecyloxycarbonyl groups in the cis-
configuration was also studied for comparison. We expected this
isomer to be always predominantly in a conformation with three
substituents in the equatorial position (2B), and to show little or
no conformational change upon protonation (2BH+, Scheme 1).
Lipids 1 and 2 were prepared (Schemes S1, S2z) using the
approach developed previously.7 The conformer populations
(nA, nB) in the fast equilibrium [1A] $ [1B + 1BH+]
(Scheme 1) were estimated from an averaged signal width
(W = SJHH) measured as the distance between terminal peaks
Conformationally controlled molecular switches are central
devices of molecular machines1 and provide a new and pro-
mising approach to materials with controllable properties.
However, the concept of conformational switches has not
yet been applied to the design of triggerable lipid amphiphiles.
The potential advantage here is that the effect of the con-
formational switch in one molecule of lipid amphiphile can be
amplified in its colloidal assemblies, such as liposomes.
Acid-sensitive lipid amphiphiles have drawn much interest
as building blocks of colloidal drug and gene carriers that
release therapeutic payloads specifically at low-pH target sites,
such as solid tumors and inflamed tissues.2 Although the
reported acid-sensitive lipid amphiphiles have versatile chemi-
cal structures, their design falls into only two categories: (1)
change of the hydrodynamic radius of the lipid headgroups by
protonation with concomitant phase changes of the lipid
bilayer,3 and more recently, (2) hydrolysis of acid-labile
lipids.4
1
of a multiplet in H NMR: Wobserved = WAnA + WBnB.7 We
used mainly the signal of H4, geminal to the hydroxyl group
(Scheme 1), which was usually better resolved and located in a
region apart from other signals. The parameter WB was assumed
Since the resistance of lipid bilayers against diffusion mostly
relies on the stacking of the hydrophobic lipid tails,5 a change
of conformation of the lipid tails could also be exploited to
trigger liposomes. Such consideration turned our attention to
trans-2-aminocyclohexanols (TACHs), the protonation of
which results in a strong hydrogen bond between the amino
and the hydroxyl groups, forcing both these groups to adopt
an equatorial conformation.6,7 Further, this change can be
mechanically transmitted by the cyclohexane ring to induce
conformational changes of its remote substituents.6–9 Pre-
viously, TACH-based pH-sensitive conformational switches
were used to control the affinity of a crown ether and a podand
to potassium ion.7 Here we present TACH derivatives 1 and 2
(Scheme 1) as the first attempt to design conformationally
switchable lipid amphiphiles and their pH-sensitive liposomes.
a Department of Chemistry, University of the Pacific, Stockton,
CA 95211, USA. E-mail: vsamoshin@pacific.edu;
Fax: +1 209-946-2607; Tel: +1 209-946-2921
b Department of Pharmaceutics and Medicinal Chemistry, TLJ School
of Pharmacy and Health Sciences, University of the Pacific,
Stockton, CA 95211, USA. E-mail: xguo@pacific.edu;
Fax: +1 209-946-2410; Tel: +1 209-946-2321
w Dedicated to Professor Ernest L. Eliel.
z Electronic supplementary information (ESI) available: Experimental
details of lipid syntheses, NMR studies, liposome preparation and
liposome characterizations. See DOI: 10.1039/b807704e
Scheme 1 pH-triggered conformational change of trans-2-amino-
cyclohexanol-derived lipids.
ꢀc
This journal is The Royal Society of Chemistry 2008
4774 | Chem. Commun., 2008, 4774–4776

View Article Online
to equal WBH+ = Wobserved in the presence of excess acid
(25.3 Hz for 1, 26.1 Hz for 2), and WA was estimated as 9 Hz
based on reported data for the related conformationally biased
cyclohexanes.7
hydroxyl group. Further, the originally well-resolved signals of
H1 (d 2.98; W = 22.3 Hz) and H2 (d 3.09; W = 23.1 Hz) in 1
became narrow and unresolved, and both shifted downfield to
d 3.4 upon the drop of pH (Fig. S1–S4z). This attests for an
axial-to-equatorial switch of H1 and H2, and thus for the
equatorial-to-axial switch of the ester groups of the lipid tails.
Neither 1 nor 2 dissolves in water, which precluded their
direct NMR characterizations in aqueous media. Neverthe-
less, 1 should undergo a similar pH-triggered conformational
change at a water–bilayer interface as we found in highly
polar, protic methanol.
To characterize the pH-triggered conformational change,
the lipids 1 and 2 were dissolved in CD3OD, and the changes
1
of their H NMR spectra (300 MHz) were monitored during
titration of the solution with trifluoroacetic acid (TFA).
As expected, lipid 2 was B95% in conformation 2B (WH4
=
25.1 Hz), and this bias slightly increased in excess acid to
B100% of 2BH+ (WH4 = 26.1 Hz) (Fig. 1). The H4 chemical
shift changed from d 3.53 at pH 8.3 (no acid added) to d 3.82
below pH 2.5 (Fig. 2). The H5 signal of 2 moved downfield
from d 2.33 (WH5 = 25.3 Hz) to d 3.3 (unresolved), indicating
deshielding caused by protonation/deuteration of the morpho-
line nitrogen.
As a first attempt to test the capacity of the lipid tail
conformational change to trigger lipid colloids, we prepared
liposome comprising 25 mol% 1, and 75 mol% 1-palmitoyl-2-
oleoyl-syn-glycero-3-phosphocholine (POPC),
a common
phospholipid that favors the formation of a lipid lamellar
phase. As a monounsaturated lipid, POPC facilitates the
mixing with both saturated and unsaturated lipids.10 The
liposomes contained fluorescent dyes, 8-aminonaphthalene-
1,3,6-trisulfonic acid, disodium salt (ANTS) and p-xylene-
bis-pyridinium bromide (DPX), so that liposome permeation
could be monitored by dye release, which de-quenches their
fluorescence.10,11 In a pH 7.4 buffer, the 1–POPC liposome
preparation did not show substantial content leakage for more
than 1 h at 37 1C. Upon exposure to mildly acidic media
(pH 5.5), 1–POPC liposome quickly released B50% of the
contents in 10 min.
The signal width of H4 in 1 increased from 13.2 Hz to
25.3 Hz (Fig. 1), indicating a sharp conformational switch
from 1A (B75% in original solution) to 1BH+ (B100% in
excess acid). The H5 signal of 1 (W = 13.7 Hz at pH 8.1; no
acid added) became unresolved upon decrease of the apparent
pH below 5, and migrated from d 2.23 downfield to d 3.3
below pH 3.5 (Fig. 2). In contrast, the multiplet H4 shifted
upfield from d 4.00 to d 3.90 despite of the deshielding effect of
the morpholinium group. Since axial protons typically give a
signal at higher field than the otherwise equivalent equatorial
protons, this observation supports an equatorial-to-axial
change of H4, and thus the axial-to-equatorial change of the
Next we elected to test whether 1 can introduce pH-sensitivity
to liposomes grafted with polyethylene glycol (PEG), a hydro-
philic polymer commonly used to deter immune detection
of liposomal drug/gene delivery systems by inhibiting their
inter;actions with serum proteins.12 We constructed a liposome
comprising 5 mol% of a PEG–lipid conjugate, N-Palmitoyl-
sphingosine-1- [succinyl- (methoxypolyethyleneglycol)2000]
(mPEG2000-Ceramide), and 24 mol% 1 or 2, together with
71 mol% POPC. The mPEG2000-Ceramide–1–POPC lipo-
some preparation was stable in a pH 7.4 buffer for more than
8 months when stored at 4 1C and for more than 48 h at 37 1C
(Table S1z). Upon exposure to mildly acidic media (pH 5.5),
mPEG2000-Ceramide–1–POPC liposome quickly released
most of the contents (480% in 20 min) (Fig. 3). In compar-
ison, the analogous mPEG2000-Ceramide–2–POPC liposome
released much less content (o30%) at pH 5.5 (Fig. 3), which
probably occurred through the increase of the headgroup
Fig. 1 pH-dependent change of the H4 signal width (W = SJHH) in
the H NMR spectra of 1 and 2 in CD3OD.
1
Fig. 2 pH-dependent change of the chemical shift of H4 and H5 in
1H NMR of 1 and 2 in CD3OD.
Fig. 3 pH-dependent leakage of mPEG2000-Ceramide–1–POPC
liposomes (1) and mPEG2000-Ceramide–2–POPC liposomes (2).
ꢀc
This journal is The Royal Society of Chemistry 2008
Chem. Commun., 2008, 4774–4776 | 4775

View Article Online
radius3 and/or the change of interactions between the lipid
headgroups13 upon protonation of the morpholine group.
Acid-induced lipid degradation can be excluded as a contri-
butor to the observed leakages for the following reasons. First,
lipids 1 and 2 were found to be stable overnight at a pH as low
as 1 during our NMR analyses (Fig. 1 and 2). Secondly, our
prior HPLC and liposome leakage studies11,14 have shown
that the ester groups in diacyl glyceride-based phospholipids
and PEG–lipid conjugates do not undergo noticeable chemical
degradation in mildly acidic media (pH 5.5) within the time
span of the liposome leakage assays (B1 h, Fig. 3).
lever. Lipid amphiphiles derived from trans-2-aminocyclohex-
anol can serve as versatile pH-sensitive molecular switches in
drug and gene delivery systems (e.g. as helper lipids15,16). We
are investigating the physicochemical mechanisms of the
TACH-induced liposome leakage (possible involvement of
raft formation or La - HII phase changes, etc.) and will
report the findings in due course.
This work was supported by SAAGrants (X.G., V.V.S.) and
Eberhardt Research Fellowship (V.V.S.) of University of the
Pacific. We thank Dr Roshanak Rahimian for the use of the
fluorometer. We thank the reviewers for insightful comments.
Fig. 2 shows that the morpholine group of 2 has virtually
identical basicity compared with that of 1. Lipid 2 differs from
lipid 1 only in the configuration of one lipid tail substituent,
and for this reason was unable to substantially change its
conformation after protonation (Scheme 1, Fig. 1). The stu-
dies on the control liposome mPEG2000-Ceramide–2–POPC
allowed the observation of all possible effects of the proto-
nation of the morpholine group (change of headgroup radius,
change of hydrogen bonding, etc.) on the liposome perme-
ability, except for the effects caused by the change of con-
formation. Therefore, the much larger and faster leakage of
mPEG2000-Ceramide–1–POPC liposome than mPEG2000-
Ceramide–2–POPC liposome at pH 5.5 can be attributed to
the pH-triggered conformational change of the lipid tails in 1.
It is interesting that the leakage of mPEG2000-Ceramide–
2–POPC liposome at pH 5.5 plateaued at about 30% (Fig. 3).
One possible reason would be that the membrane destabiliza-
tion by the protonation of 2 was so limited that the lipid
bilayer managed to reorganize into a slightly different lamellar
phase to retain most of the liposome content. In contrast, the
drastic conformational change of the lipid tails of 1 would
induce extensive reorganization of the membrane of
mPEG2000-Ceramide–1–POPC liposome and thus a faster
and almost complete liposome leakage.
Notes and references
1 Molecular Switches, ed. B. Feringa, Wiley-VCH, Weinheim, 2001;
E. R. Kay, D. A. Leigh and F. Zerbetto, Angew. Chem., Int. Ed.,
2007, 46, 72; D. Zehm, W. Fudickar and T. Linker, Angew. Chem.,
Int. Ed., 2007, 46, 7689.
2 S. Simoes, J. N. Moreira, C. Fonseca, N. Duzgunes and M. C.
de Lima, Adv. Drug Delivery Rev., 2004, 56, 947; T. M. Allen and
P. R. Cullis, Science, 2004, 303, 1818.
3 D. C. Drummond, M. Zignani and J. Leroux, Prog. Lipid Res.,
2000, 39, 409; H. Karanth and R. S. Murthy, J. Pharm. Pharmacol.,
2007, 59, 469.
4 X. Guo and F. C. Szoka Jr, Acc. Chem. Res., 2003, 36, 335; J. van
den Bossche, J. Shin, P. Shum and D. H. Thompson, J. Controlled
Release, 2006, 116, e1.
5 T. X. Xiang and B. D. Anderson, Adv. Drug Delivery Rev., 2006,
58, 1357.
6 V. V. Samoshin, Mini–Rev. Org. Chem., 2005, 2, 225.
7 V. V. Samoshin, V. A. Chertkov, D. E. Gremyachinskiy, E. K.
Dobretsova, A. K. Shestakova and L. P. Vatlina, Tetrahedron Lett.,
2004, 45, 7823; V. V. Samoshin, B. Brazdova, V. A. Chertkov, D. E.
Gremyachinskiy, A. K. Shestakova, E. K. Dobretsova, L. P.
Vatlina, J. Yuan and H.-J. Schneider, ARKIVOC, 2005, (iv), 129.
´ ´
8 A. M. Costero, J. P. Villarroya, S. Gil, R. Martınez-Manez and
P. Gavina, C. R. Chim., 2004, 7, 15; A. M. Costero, M. Colera,
P. Gavina and S. Gil, Chem. Commun., 2006, 761.
9 R. Krauss, H.-G. Weinig, M. Seydack, J. Bendig and U. Koert,
Angew. Chem., Int. Ed., 2000, 39, 1835; M. Karle, D. Bockelmann,
D. Schumann, C. Griesinger and U. Koert, Angew. Chem., Int. Ed.,
When incubated in 75 vol% fetal bovine serum at 37 1C, the
mPEG2000-Ceramide–1–POPC liposome released the encap-
sulated dyes by slow kinetics (B30% in 12 h, Fig. S7z) that
was virtually identical to that of the pH-insensitive control
liposomes comprising 5 mol% mPEG2000-Ceramide and
95 mol% POPC. Since the clearance of liposomes by the
immune system is initiated by liposome-serum protein inter-
actions,12 this observation suggests that pH-sensitive lipids
based on lipid tail conformational change can be incorporated
into PEG-coated liposomal drug/gene delivery systems with-
out jeopardizing their ability to evade immune detection.
In summary, we have reported an unprecedented strategy to
render triggerable lipid amphiphiles and lipid colloids: the
protonation-induced conformational change of lipid tails
using the trans-2-aminocyclohexanol group as a molecular
2003, 42, 4546; M.-E. Juarez Garcia, U. Frohlich and U. Koert,
Eur. J. Org. Chem., 2007, 1991.
10 X. Guo, J. A. MacKay and F. C. Szoka, Biophys. J., 2003, 84,
1784.
11 H. Ellens, J. Bentz and F. C. Szoka, Biochemistry, 1984, 23, 1532;
Z. Huang, X. Guo, W. Li, J. A. MacKay and F. C. Szoka, J. Am.
Chem. Soc., 2006, 128, 60.
12 J. M. Harris and R. B. Chess, Nat. Rev. Drug Discovery, 2003
2, 214.
13 J. M. Boggs, Biochim. Biophys. Acta, 1987, 906, 353.
14 H. Chen, H. Zhang, C. M. McCallum, F. C. Szoka and X. Guo,
J. Med. Chem., 2007, 50, 4269; X. Guo and F. C. Szoka, Biocon-
jugate Chem., 2001, 12, 291.
15 C. A. H. Prata, Y. Li, D. Luo, T. J. McIntosh, P. Barthelemy and
M. W. Grinstaff, Chem. Commun., 2008, 1566.
16 G. Rethore, T. Montier, T. Le Gall, P. Delepine, S. Cammas-
´ ´ ´
¨
Marion, L. Lemiegre, P. Lehn and T. Benvegnu, Chem. Commun.,
2007, 2054.
ꢀc
This journal is The Royal Society of Chemistry 2008
4776 | Chem. Commun., 2008, 4774–4776