Synthesis and vesicle formation from novel pseudoglyceryl dimeric lipids. Evidence of formation of widely different membrane organizations with exceptional thermotropic properties

Article abstract of DOI:10.1039/a704154c

Eight new bis-cationic dimeric lipids 2a-h have been synthesized; TEM of their aqueous dispersions confirmed the vesicle formation and from the thermal, spectroscopic, DLS and XRD studies it has been revealed that they form three different kinds of membranous aggregate depending on the m-value.

Full text of DOI:10.1039/a704154c

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Synthesis and vesicle formation from novel pseudoglyceryl dimeric lipids.
Evidence of formation of widely different membrane organizations with
exceptional thermotropic properties
Santanu Bhattacharya,*† Soma De and Shaji K. George
Department of Organic Chemistry, Indian Institute of Science, Bangalore 560012, India
Eight new bis-cationic dimeric lipids 2a–h have been
synthesized; TEM of their aqueous dispersions confirmed
the vesicle formation and from the thermal, spectroscopic,
DLS and XRD studies it has been revealed that they form
three different kinds of membranous aggregate depending
on the m-value.
block, 1,2-isopropylideneglycerol, 3 was protected in the form
of a benzyl ether 4. The isopropylidene group was then removed
by treatment with HCl in aq. MeOH and the diol obtained upon
preparative column chromatography over silica gel was alkyl-
ated with n-C16H33Br (2.2 equiv.) to give 5 which upon
hydrogenolysis afforded 6 in ~ 60% yield. Compound 6 was
then converted to the corresponding bromide 7 by reaction with
PPh3–CBr4; 7 was quaternized in the presence of Me3N in EtOH
to give 1 in 80% yield. The bisquaternary systems 2a–h were
obtained from the corresponding dimethylamino derivative 8
upon refluxing with individual alkanediyl a,w-dibromides in
60–75% yields.
Dispersion of lipids 1, 2a–h in water using probe or bath
sonication afforded liposomes with a unimodal distribution of
particle diameters as revealed by DLS (Table 1). Examination
of DLS data on bath sonicated vesicles shows that vesicles from
dimeric lipids were invariably larger. Within the vesicles among
the dimeric lipid series, however, the hydrodynamic diameter
variation as a function of m-value was very complex. The
vesicular sizes were found to be the largest with lipids, 2, of
m = 3 and then decreased with increase in m-value up to
m = 12 and then went up again with m > 12. Vesicle sizes were
also found to strongly depend on the lipid concentration,
thermal history and method of vesicle preparation. Thus while
vortexing afforded mostly open lamellae and sonication gave
small spherical, closed microstructures, reverse phase evapora-
tion (REV) gave multilamellar vesicles (not shown).
While the properties of vesicular membranes derived from a
large number of ‘monomeric’ natural lipids1 as well as their
synthetic analogues2 have been examined in great detail, there
is no report in the literature that examines the properties of the
membranes prepared from ‘multimeric’ lipids. This is despite
the fact that several ‘dimeric’ and ‘multimeric’ lipid systems
occur naturally and have important biological functions. Thus,
cardiolipins (glycerol bridged dimeric phosphatidate lipids)
constitute a class of complex phospholipids occurring mainly in
the heart and skeletal muscles and show high metabolic
activity.3 Similarly, glycolipid A2 (composed of two phosphate
headgroups and seven hydrophobic chains) serves as im-
munomodulator in mediating selective recognition of toxins,
bacteria or viruses at the cell surfaces.4
Although dimeric (gemini) amphiphiles5,6 that aggregate to
form micelles are known, no analogous gemini form of lipids
has been studied. Since cationic liposomes by themselves are
receiving enormous current attention as effective non-viral gene
transfection agents,7 the resulting aggregates of dimeric lipids if
cationic might be potentially useful. Along the lines of the
above considerations, here we report the first synthesis of a
family of bis-cationic, gemini lipids 2a–h and their unusual
membrane-forming properties on the basis of a combination of
thermal, spectroscopic, dynamic light scattering (DLS), trans-
mission electron microscopic (TEM) and reflection X-ray
diffraction (XRD) methods supported by energy-minimization
studies. To place the experimental data on these novel gemini
membranes into appropriate perspective, we also include herein
the related results from the vesicles of the corresponding
‘monomeric’ (control) lipid 1.
Table 1 The hydrodynamic diameters of vesicular aggregates, main phase-
transition temperatures of vesicular 1, 2a–h and the unit-layer thicknesses of
their corresponding self-supporting cast films
Unit bilayer
Main transitionb (T/°C) thickness/Å
Lipid
DLSa
(m-value) size/nm Microcal Fluor. Pol. Obsc
Calc.d
Altogether eight new dimeric lipid systems 2a–h were
synthesized (Scheme 1). First the CH2OH group of the building
0
3
4
5
6
12
16
20
22
15
93
52
29
29
30
37
ND
52
45.2
57e
45
57
48
43
43
45
41
62
53
45.9
45
49
49
49
49
+
48.3
44.2
43.9
46.5
40.8
69.1
66.3
45.9
45.9
44.2
31.3
29.6
29.8
30.8
–
OR1
OR1
vii
NMe2Br
ii
R2O
OR2
O
O
R2O
OR2
49
49, 16.8,f 31g
49, 21.9,f 31g
49, 27f
5 R1 = Bn
6 R1 = H
7 R1 = Br
1
iii
iv
3 R1 = H
i
4 R1 = Bn
49, 29f
Br–
Br–
v
+
N
+
a Dynamic light scattering was examined with the vesicles prepared under
NMe2
N
vi
( )m
R2O
OR2
identical conditions. All the vesicles (1 3 1023 m) were prepared by bath
R2O
OR2
R2O
OR2
b
sonication (Julabo USR3) for 10 min, at 70 °C and at 35 KHz. Average
8
2a–h
deviation is ±0.5 °C for microcalorimetry; ±1.0 °C for fluorescence
polarization method. c As obtained from reflection X-ray diffraction of cast
films. d Lengths of two molecular layers of lipids as obtained from models.
e Additional minor peaks at 66.1 and 71.6 °C were also observed. f Lengths
of unit ‘mono’layer-bilayer plans of lipids as obtained from models
g Lengths of unit interdigitated and tilted bilayer plans of lipids as obtained
from models.
R2 = n-C16H33 m = 3–6, 12, 16, 20, 22
Scheme 1 Reagents and conditions: i, BnCl, KOH, C6H6, reflux, 87%; ii,
aq. HCl–MeOH, reflux, 84%; n-C16H33Br, KOH, C6H6, reflux, 58%; iii,
Pd–C/H2, 60%; iv, CBr4, PPh3, CH2Cl2, 90%; v, Me2NH–EtOH, heat,
pressure tube, 95%; vi, Br(CH2)mBr, EtOH, reflux, 60–75% (exact yields
depend on m); vii, Me3N–EtOH, heat, pressure tube, 80%
Chem. Commun., 1997
2287

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lipids with m = 20 and 22 gave broader P vs. T variation, the
mid-points of which were lower than the Tm values obtained
using microcalorimetry. Notably again the microviscosities (ca.
3.5–4.0 poise) of the vesicular aggregates of lipids with
m = 20–22 in their gel states were considerably lower than that
of the lipids with lower m-values (ca. 10–11 poise) again
suggesting gross differences in their suprastructural organiza-
tions.
49 Å
45–46 Å
(a) Untilted bilayers
(b) Slightly tilted bilayers
Molecular mechanics calculation of the energy-minimized
conformations of the dimeric lipids using DISCOVER (IN-
SIGHT II) suggested that the packing in 2a and 2b, where
Me2N+ headgroups are separated by three and four CH2 units
respectively, is much tighter than that in lipids with higher
m-values. This should lead to a progressive decrease in Tm as
m-value increases from 3 to 10. As the m-value in lipid, 2,
reaches !12, this situation changes when the (CH2)12 connector
in 2e ‘loops’ into the vesicle interior to avoid ‘undesirable’
contacts with the bulk water. At m = 16, the spacer chain should
adopt an even more folded, wicket-like conformation further
impairing the bilayer packing.‡ The greater extent of spacer
chain looping into the membrane interior should disturb ‘intra’-
gemini lipid packing at m = 16 even further [Fig. 1(d)]. This is
consistent with the observation of lower Tm value with vesicular
2f than that with 2e. As the m-value surpasses 16 in 2, the
suprastructural organizations in the membranes change again.
This is because at m ! 20, the length of spacer may now span
as a ‘monolayer’ giving rise to ‘mono’layer-bilayer hybrid type
of suprastructures. Thus, strikingly, when m-value reaches !20,
spacer chains probably hyperextend to produce (absolutely
unrelated to the ones with lower m-values) ‘monolayer’ type
organizations reminiscent of the assemblies seen in the
bolaamphiphilic membranes [Fig. 1(f)].9
45–46 Å
31 Å
(c) Tilted bilayers
(d) Interdigitated and tilted bilayers
17 Å (m = 12)
22 Å (m = 16)
27 Å (m = 20)
29 Å (m = 22)
(e) 'Mono'layer–bilayer hybrids
(f ) Interdigitated 'mono'layer–bilayer hybrids
Fig. 1 Schematic representation of possible bilayer forming motifs with
gemini lipids as a function of m-value
The sonicated dispersions of different lipids (7 mm) were
then converted to regular self-supporting films by casting on
glass slides as described.8 Reflection X-ray diffraction (Scintag
XDS-2000) of these cast films gave the long spacings from
individual lipid films as given in Table 1. Comparison of the
corresponding molecular lengths of the gemini lipid units as
determined from CPK models suggests the formation of slightly
tilted bilayer arrangement [Fig. 1(b)] with 1 and with gemini
lipids of m-values 3, 4, 5 and 6. But with 2e, m = 12, the
experimentally determined XRD data on the unit layer thick-
nesses cannot be explained by tilting alone suggesting the
presence of additional interdigitations [Fig. 1(d)]. The alter-
native plan (e) is energetically unfavorable with the lipophilic
chains protruding into the polar headgroup regions. To explain
the reflection XRD data of 2g and 2h, we suggest a kind of
monolayer–bilayer hybrid organization as shown in Fig. 1(f).
Independent examination of the thermotropic properties of
the vesicles of 1 and 2a–h by DSC, microcalorimetry and
fluorescence methods showed that the changes in m-value in 2
profoundly influence their gel-to-liquid crystalline phase-
transition temperatures (Table 1). Vortexing of 60 mm of either
of 1 and 2a–h in water afforded lameller gels which gave well-
resolved, nearly reversible peaks as melting transitions (Tm)
(Perkin Elmer DSC 7). Microcalorimetry (MC-2) of 1.0–2.5
mm sonicated vesicular specimens of either of the lipids
reproduced the peaks due to Tm. Relative to 1, there is a
substantial increase in the gel-to-liquid crystalline transition
temperature of the membranes derived from dimeric 2a. In
addition, unlike the membranes that are assembled from 1,
which show a sharp endothermic main transition at 45 °C, those
made from 2a exhibit a more complex melting pattern. For
lipids with m ! 16, the melting transition profiles appeared
remarkably broadened (less cooperative), while for lipids with
m @ 12, the main transitions were quite sharp. Thus although
the cooperativities of melting transitions with 2 (m = 20–22)
were severely reduced, alternative membrane organizational
features allow broad but higher melting temperatures (see
below). The latter could be a consequence of perturbation
within the headgroup and/or direct interaction of the spacer
chain with the hydrocarbon chain regions.
Since the freedom of motion of four independent hydrocar-
bon chains is critical to their properties at the membrane level,
the present study clearly demonstrates that the interconnection
of the ‘monomeric’ lipid by a polymethylene segment into a
‘dimeric’ lipid brings about ramifications far exceeding the
seemingly trivial structural modification at the lipid level. These
findings further emphasize the need for newer designs of
synthetic lipid structures to expand the understanding of their
behaviour upon membrane formation.
Footnotes and References
* E-mail: sb@orgchem.iisc.ernet.in
† Also at the Chemical Biology Unit, Jawaharlal Nehru Centre for
Advanced Scientific Research, Bangalore 560064, India.
‡ This is consistent with the observations of E. Alami, G. Beinert, P. Marie
and R. Zana, Langmuir, 1993, 9, 1465.
1 J. L. Slater and C.-H. Huang, in The Structure of Biological Membranes,
ed. P. Yeagle, CRC Press, Baca Raton, FL, 1992, esp. pp. 175–210.
2 T. Kunitake, Angew. Chem., Int. Ed. Engl., 1992, 31, 709; H. Ringsdorf,
B. Schlarb and J. Venzmer, ibid., 1988, 27, 113.
3 W. Hu¨bner, H. H. Mantsch and M. Kates, Biochim. Biophys. Acta, 1991,
1066, 166; S. M. Grunner and M. K. Jain, ibid., 1985, 818, 352.
4 O. Lockhoff, Angew. Chem., Int. Ed. Engl., 1991, 30, 1611.
5 F. M. Menger and C. A. Littau, J. Am. Chem. Soc., 1991, 113, 1451.
6 S. De, V. K. Aswal, P. S. Goyal and S. Bhattacharya, J. Phys. Chem.,
1996, 100, 11 664; S. Bhattacharya and S. De, J. Chem. Soc., Chem.
Commun., 1995, 651.
7 S. Bhattacharya and S. S. Mandal, Biochim. Biophys. Acta, 1997, 1323,
29; S. Bhattacharya and S. Haldar, ibid., 1996, 1283, 21; Langmuir, 1995,
11, 4748; Y. Xu and F. C. Szoka, Biochemistry, 1996, 35, 5616;
M. S. Spector and J. M. Schnur, Science, 1997, 275, 791; J. O. Ra¨dler,
I. Koltover, T. Salditt and C. R. Safinya, ibid., 1997, 275, 810.
8 S. Bhattacharya and S. De, Chem. Commun., 1996, 1283; N. Kimizaka,
T. Kawasaki and T. Kunitake, J. Am. Chem. Soc., 1993, 115, 4387.
9 J.-H. Fuhrhop and R. Bach, in Advances in Supramolecular Chemistry,
ed. G. W. Gokel, JAI, Greenwich, CT, 1992, vol. 2, pp. 25–63.
The fluorescence polarizations (P) due to the vesicle doped
1,6-diphenylhexa-1,3,5,-triene at various temperatures were
then determined. The temperatures related to the break in P vs.
T plot for the vesicles with m = 0–16 were similar to the Tm
values obtained by calorimetric methods (Table 1). But the
Received in Cambridge, UK, 16th June 1997; 7/04154C
2288
Chem. Commun., 1997