Stereodependent fusion and fission of vesicles: Calcium binding of synthetic gemini phospholipids containing two phosphate groups

Article abstract of DOI:10.1021/ja962303s

Three different stereoisomers of a phosphatidic acid analog bearing two phosphate groups have been synthesized from tartaric acid and erythritol. The obtained diastereomeric gemini surfactants differ in their aggregation behavior due to the different spat

Full text of DOI:10.1021/ja962303s

4338
J. Am. Chem. Soc. 1997, 119, 4338-4344
Stereodependent Fusion and Fission of Vesicles: Calcium
Binding of Synthetic Gemini Phospholipids Containing Two
Phosphate Groups
N. A. J. M. Sommerdijk, T. H. L. Hoeks, M. Synak, M. C. Feiters,
R. J. M. Nolte,* and B. Zwanenburg*
Contribution from the Department of Organic Chemistry, NSR-Center for Molecular Structure,
Design and Synthesis, UniVersity of Nijmegen, ToernooiVeld, 6525 ED Nijmegen,
The Netherlands
ReceiVed July 5, 1996. ReVised Manuscript ReceiVed March 11, 1997X
Abstract: Three different stereoisomers of a phosphatidic acid analog bearing two phosphate groups have been
synthesized from tartaric acid and erythritol. The obtained diastereomeric gemini surfactants differ in their aggregation
behavior due to the different spatial orientations of their functional groups. The most remarkable difference in
aggregation behavior is encountered when calcium ions are added to vesicle suspensions of the respective isomers.
The vesicles formed from the (S,S) and (R,R) isomer undergo fusion, whereas those of the (R,S) isomer show vesicle
fission. This remarkable behavior can be explained by a change in the molecular packing of the lipid molecules
upon the complexation with calcium ions. An analysis of physical data obtained prior to and after the addition of
the calcium ions reveals that the head groups of the diastereomeric surfactants respond differently to these ions:
those of the (S,S) isomer increase in size, whereas those of the (R,S) isomer decrease in size. This phenomenon
accounts for occurrence of fusion and fission of the vesicles, respectively.
Introduction
in vesicles of cationic surfactants,5 and that the autocatalytic
hydrolysis of oleic anhydride in oleic acid vesicles even can
yield a self-reproducing system.6
Cell fusion and fission processes are thought to proceed
through the binding of calcium ions to acidic phospholipid
molecules present in the cell membrane.1 Most of these lipids
contain only one phosphate group, and calcium binding most
probably induces dimer formation.2 These dimers form solid
domains in the fluid membrane, allowing other lipids to express
their tendency to form nonlamellar structures, leading to the
generation of a hexagonal phase as an intermediate state in the
fusion or fission process. The effect of calcium and other
divalent ions on the aggregation behavior of pure as well as
mixed native phospholipids has been studied extensively.1-3
More recently,4 vesicle fusion has been demonstrated for
synthetic phospholipids after addition of calcium ions. Cyto-
mimetic organic chemistry has shown furthermore that aggrega-
tion, fusion, fission, and budding processes can be accomplished
A study of the influence of calcium ions on vesicle systems
of surfactants containing acidic phosphate functions seems the
most direct approach to the understanding of the mechanisms
of the fusion and fission processes in living cells. In most cases,
however, the precipitation of their calcium complexes limits
the utility of pure acidic phospholipids in mimicking these
processes. In this context, the influence of calcium ions on the
aggregation behavior of the phosphatidic acid analogs 1 is of
interest. These compounds, which also exhibit carries reducing
activity,7 are structurally related to phosphatidic acid but have
two, instead of one, phosphate groups. Surfactants of this type
are called gemini surfactants and are currently receiving great
interest.8 The formation of 1:1 complexes, rather than dimers,
could be expected upon addition of calcium ions to the
aggregates of these compounds.
In this paper the synthesis of the stereoisomeric compounds
1a-c and their aggregation behavior in water is reported. It
will be shown that the addition of calcium ions strongly affects
the self-assembling properties of these surfactants and that
vesicles prepared from 1b or 1c can be used to mimic the fusion
and fission processes of cells. These compounds do not
immediately precipitate after calcium binding, due to the
presence of the two phosphate groups, and the processes of both
fusion and fission can be monitored by electron microscopy. A
X Abstract published in AdVance ACS Abstracts, May 1, 1997.
(1) (a) Gingel, D.; Ginsberg, L. In Cell Surface ReV. Poste, G., Nicolson,
G. L., Eds.; North Holland, Amsterdam, 1978; Vol. 5, pp 791-833. (b)
Cullis, P. R; de Kruijff, B. Biochim. Biophys. Acta 1979, 559, 399. (c)
Molecular mechanisms of membrane fusion; Ohki, S., Doyle, D., Flanagan,
T. D., Hui, S. W., Mayhew, E., Eds.; Plenum: New York, 1988.
(2) Ohnishi, T.; Ito, T. Biochemistry 1974, 13, 881.
(3) (a) Verkleij, A. J.; De Kruyff, B.; Ververgaert, P. H. J. Ph.; Tocanne,
J. F.; van Deenen, L. L. M. Biochim. Biophys. Acta 1974, 339, 432. (b)
Papahadjopoulos, D.; Jacobson, K.; Vail, W. J.; Poste, G. Biochim. Biophys.
Acta 1975, 394, 483. (c) Papahadjopoulos, D.; Vail, W. J.; Pangborn, W.
A.; Poste, G. Biochim. Biophys. Acta 1976, 448, 265. (d) Dluhy, R. A.;
Cameron, D. G.; Mantsch, H. H.; Mendelsohn, R. Biochemistry 1983, 22,
6318. (e) Verkleij, A. J. Biochim. Biophys. Acta 1984, 779, 43. (f)
Nakashima, N.; Ando, R.; Fukushima, H.; Kunitake, T. Chem. Lett. 1985,
1503. (g) Siegel, D. P. Biophys. J. 1986, 49, 1171. (h) Casal, H. L.; Marin,
A.; Mantsch, H. H.; Paltauf, F.; Hauser, H. Biochemistry 1987, 26, 4408.
(i) Casal, H. L.; Mantsch, H. H.; Hauser, H. Biochemistry 1987, 26, 7395.
(j) Casal, H. L.; Mantsch, H. H.; Hauser, H. Biochim. Biophys. Acta 1989,
982, 228.
(5) (a) Rupert, L. A. M.; Engberts, J. B. F. N.; Hoekstra, D. J. Am. Chem.
Soc. 1986, 107, 2628. (b) Rupert, L. A. M.; Engberts, J. B. F. N.; Hoekstra,
D. J. Am. Chem. Soc. 1986, 108, 3920. (c) Menger, F. M.; Balachander, N.
J. Am. Chem. Soc. 1992, 114, 5862. (d) Menger, F. M.; Gabrielson, K. J.
Am. Chem. Soc. 1994, 11, 1567. (e) Menger, F. M.; Gabrielson, K. D Angew.
Chem. 1995, 107, 2260.
(6) Wick, R.; Wade, P.; Luisi, P. L. J. Am. Chem. Soc. 1995, 117, 1435.
(7) Borggreven, J. M. P. M.; Hoeks, T. H. L.; Driessens, F. C. M.;
Zwanenburg, B. Caries Res. 1992, 26, 84.
(4) (a) Rupert, L. A. M.; van Breemen, J. F. L.; van Bruggen, E. F. J.;
Engberts, J. B. F. N. Hoekstra, D. J. Membrane Biol. 1987, 95, 225. (b)
Wagenaar, A.; Streefland, L.; Hoekstra, D.; Engberts, J. B. F. N. J. Phys.
Org. Chem. 1992, 5, 451. (c) Streefland, L. Wagenaar, A. Hoekstra, D.
Engberts, J. B. F. N. Langmuir 1993, 9, 219.
(8) (a)Menger, F. M.; Littau, C. A. J. Am. Chem. Soc. 1993, 115, 10083.
(b) Karaborni, S.; Esselink, K.; Hilbers, P. A. J.; Smit, B.; Kartha¨user, J.;
van Os, N. M.; Zana, R. Science 1994, 266, 254.
S0002-7863(96)02303-7 CCC: $14.00 © 1997 American Chemical Society

Stereodependent Fusion and Fission of Vesicles
J. Am. Chem. Soc., Vol. 119, No. 19, 1997 4339
Scheme 1
Scheme 3
Scheme 2
dimethoxypropane (Scheme 2).14 During the reaction of 2c with
sodium iodide in butanone concurrent trans-acetalization oc-
curred, probably due to the presence of traces of iodine.15 The
two diastereomers of the corresponding isobutylidene derivative
3c were not purified but were directly subjected to hydrolysis
to give 4c in 94% overall yield.
Acylation of diiodides 4 was performed in 95-97% yield
with fatty acid chlorides in pyridine using N,N-dimethylami-
nopyridine (DMAP) as the catalyst (Scheme 3). Reaction of
diiodo esters 5 with silver dibenzyl phosphate in refluxing
toluene afforded the corresponding phosphotriesters in good
yields.16 Catalytic hydrogenolysis and subsequent ion exchange
gave the respective tetrasodium salts 1 which, after lyophiliza-
tion, were isolated as white amorphous powders.17
discussion of the mechanisms of these processes based on the
shape and physical properties of the aggregates before and after
addition of the calcium ions is presented.
Aggregation Behavior and Calcium Binding. All three
stereoisomers of 1 formed planar bilayers after dispersion in
water of pH ) 7.0 by vortexing at 70 °C, as was demonstrated
by electron microscopy (not shown).18 Since 1a and 1b are
enantiomers and have therefore identical physical properties
(apart from their optical properties), only 1b and 1c were used
in aggregation experiments described below.
Sonication of an aqueous 0.1% (w/w) dispersion of the (S,S)
isomer 1b led to the formation of very small unilamellar vesicles
with diameters of 15-25 nm (Figure 1a). When calcium ions
were added to the sonicated dispersion of 1b, fusion of the
vesicles occurred, resulting in larger vesicles with diameters of
50-100 nm (Figure 1).19 This process was monitored by
preparing samples for electron microscopy at different stages
after the addition of the calcium ions. It can be deduced from
Results and Discussion
Synthesis. Compounds 1 were synthesized by acylation and
subsequent phosphorylation of the respective secondary diiodo
alcohols in order to avoid both acyl migration9 and cyclic
phosphate formation.10 Diiodobutanediols 4 were prepared from
the corresponding 2,3-isopropylidene-1,4-ditosylates 2 (Schemes
1 and 2).
The optically active ditosylates 2a,b were obtained from D-
(-)- and L(+)-tartaric acid, respectively, using literature pro-
cedures (Scheme 1).11-13 The conversion of 2a,b into the
corresponding diiodides 3a,b using sodium iodide in butanone
proceeded smoothly, and mild hydrolysis with trifluoroacetic
acid in aqueous methanol afforded the diols 4a,b in 97% overall
yield.
(14) Direct iodination of 1,4-ditosyl erythritol led to the formation of
1-iodo-3,4-buten-2-ol (see, also: House, H. O.; Ro, R. S. J. Am. Chem.
Soc. 1958, 80, 182. Cristol, S. J.; Rademacher, L. E. J. Am. Chem. Soc.
1959, 81, 1600.)
(15) (a) Szarek, W. A.; Zamojski, A.; Tiwari, K. M.; Ison, E. R.
Tetrahedron Lett. 1986, 3827. (b) Verhart, C. G. J.; Caris, B. M. G.;
Zwanenburg, B.; Chittenden, G. J. F. Rec. TraV. Chim. Pays-Bas 1992,
110, 348.
Meso compound 2c was obtained (52%) from erythritol by
selective tosylation of the primary hydroxyl groups and subse-
quent protection of the secondary hydroxyl groups using 2,2-
(9) (a) Fischer, E. Ber. 1920, 53, 1621. (b) Doerschuk, A. P. J. Am.
Chem. Soc. 1952, 74, 4202. (c) van Lohuizen, O. E.; Verkade, P. E. Recl.
TraV. Chim. Pays-Bas 1960, 79, 133. (d) Serdavich, B. J. Am. Oil Chem.
Soc. 1967, 44, 1.
(10) (a) Brown, D. M.; Magrath, D. I.; Todd, A. R. J. Chem. Soc. 1955,
4396. (b) de Rooy, J. F. M.; Wille-Hazeleger, G.; Burgers, P. M. J.; Van
Boom, J. H. Nucleic Acids Res. 1979, 6, 2237.
(16) Zervas, L. Naturwiss. 1939, 27, 317.
(17) The enantiomeric purity of 1a,b was checked by 1H-NMR analysis
of the corresponding methyl esters using Eu(hfc)3 as a chiral shift reagent.
Methylation was accomplished by reaction of the free acid of 1a,b with
diazomethane. Enantiomeric splitting of the methyl signals revealed an
optical purity of at least 95%.
(11) Carmack, M.; Kelly, C. J. J. Org. Chem. 1968, 33, 2171.
(12) Seebach, D.; Hungerbu¨hler, E. Modern Synthetic Methods; Sheffold,
R., Ed.; Otto Salle Verlag, Frankfort/Munich 1980; Vol. 2, p 155.
(13) Rubin, L. J.; Lardy, H. A.; Fischer, H. O. L. J. Am. Chem. Soc.
1952, 74, 425.
(18) Sommerdijk, N. A. J. M.; Feiters, M. C.; Nolte, R. J. M.;
Zwanenburg, B. Recl. TraV. Chim. Pays-Bas 1994, 113, 194.
(19) Control experiments were performed using 1a. These experiments
revealed that this compound also exhibits vesicle fusion upon addition of
calcium ions.

4340 J. Am. Chem. Soc., Vol. 119, No. 19, 1997
Sommerdijk et al.
Figure 1. Electron micrographs of samples containing vesicles of 1b
at different time intervals after the addition of CaCl2: (a) freeze fracture
and (b-f) negative staining. (a) Before addition, (b) immediately after
addition, (c-d) after 20 min, and (e-f) after 2 h. Bar represents 100 nm.
Figure 2. Electron micrographs of negatively stained samples contain-
ing vesicles of 1c at different time intervals after the addition of
CaCl2: (a) before addition, (b) immediately after addition, (c) after 1
min, (d) after 3 min, (e) after 30 min, and (f) after 3 days. Bar represents
250 nm.
Table 1. DSC Data Obtained from Aqueous 1% (w/w)
Dispersions of 1, Before and After Addition of a Calcium Chloride
Solution (See Experimental Section)
compd
Ta (°C)
∆Η (J/g)
∆S (J/g‚K)
1b f 1b/Ca2+
1c f 1c/Ca2+
8 f 42
38 f 41
88 f 10
66 f 3
0.31 f 0.03
0.21 f 0.01
a Onset temperature.
Figure 1b that clustering of the vesicles occurs immediately after
addition of the ions. The subsequent fusion process was trapped
in different stages, showing both the incorporation of various
small vesicles into a larger one (Figure 1c) and the simultaneous
fusion of several small vesicles (Figure 1d,e). In contrast to
other systems,5c the clustering of the vesicles is probably not
the rate limiting factor, since after two hours the fusion process
was still not complete (Figure 1f).
DSC experiments showed that the addition of calcium ions
caused an increase in the gel to liquid-crystalline phase
transition temperature (Table 1). The transition entropy,
calculated from these data, decreased remarkably from 0.3
J/(g‚K) to 0.03 J/(g‚K), indicating drastic changes in the
organization of the bilayer.
Figure 3. Monolayer isotherms of 1b (left) and 1c (right) on (a, c)
pure water and on (b, d) a 1.0 mM CaCl2 subphase (T ) 20 °C).
2 Å according to powder diffraction data, indicating an
intercalation of the hydrocarbon chains, similar to the structures
formed from 1b. The addition of CaCl2 led only to a minor
shift of the phase transition temperature, but a distinct decrease
in transition entropy was observed (Table 1). A bilayer
thickness of 66 ( 1 Å was calculated from powder diffraction
data, which suggests a reorganization of the lamellae resulting
in a normal nonintercalated bilayer structure.
Monolayer Experiments. Monolayer experiments were then
performed with 1 in order to gain insight into the effect of
calcium binding on the size of the polar head groups of these
surfactants. Meso compound 1c, after being spread on an
aqueous subphase, occupied a smaller area than (S,S) isomer
1b (Figure 3a and 3c). The Π-A diagram of 1b showed a
plateau starting at 45 Å2 per molecule and after further
compression again an increase in surface pressure. This plateau
may be interpreted as the result of a collapse of the monolayer
since the minimum area to which two alkyl chains can be
compressed amounts to approximately 38 Å2. Brewster angle
microscopy20 did not give evidence for a collapse in this region;
however, a continued densification of the surface was observed.
This might be explained by a gradual formation of a bilayer,
which is supported by the increase in surface pressure upon
further compression and the observation of a collapse at an area
of approximately 20 Å2 per molecule.
Cast films of 0.1% (w/w) dispersions of the (S,S) isomer 1b
were found to consist of intercalated bilayers with a thickness
of 38 ( 2 Å, as determined from the higher order reflections in
wide angle powder diffraction patterns.18 A different diffraction
pattern was obtained after addition of calcium ions, but analysis
of the data indicated the same bilayer thickness (d ) 38 ( 2
Å).
Sonication of aqueous dispersions of meso compound 1c led
to the formation of unilamellar vesicles with a diameter of 50-
100 nm (Figure 2). After addition of calcium ions, fission of
the vesicles occurred, leading to the formation of much smaller
vesicles with diameters of 10-25 nm. This process was found
to be relatively fast, compared with the fusion process described
above, being complete after 20 min. During this process,
irregular curved structures were formed, from which small
vesicles were blebbing off. These vesicles transformed into
tubular structures with a length of several micrometers and a
diameter of approximately 10-20 nm (Figure 2f) on being set
aside for several days.
Information about the most favorable spatial orientation of
the respective substituents in the various conformations of
compounds 1b and 1c can be obtained from an analysis of their
Dispersions prepared from 1c displayed a gel to liquid-
crystalline phase transition at 38 °C in DSC experiments (Table
1). These vesicles contained bilayers with a thickness of 38 (
(20) (a) He´non, S.; Meunier, J. ReV. Sci. Intrum. 1991, 62, 936. (b) Ho¨nig,
D.; Mo¨bius, D. J. Phys. Chem. 1991, 95, 4590.

Stereodependent Fusion and Fission of Vesicles
J. Am. Chem. Soc., Vol. 119, No. 19, 1997 4341
Figure 4. Newman projections of 1b (i, ii, and iii) and 1c (iV, V, and
Vi).
Newman projections (Figure 4). The conformations i and iV,
in which the ester groups are positioned anti, can be neglected,
since they would be unfavorable in terms of hydrocarbon chain
organization. Thus, for (S,S)-1b the remaining conformations
may have either a gauche (ii) or an anti (iii) orientation of the
phosphate groups. The latter, although more sterically demand-
ing, will be more favorable, since it will have the lowest degree
of electrostatic repulsion. For the (R,S) isomer 1c both V and
Vi will lead to a gauche orientation of the phosphate groups.
This explains the relatively small molecular area occupied by
this molecule as compared to 1b. Both conformations V and Vi
of 1c lead to an overall “L”-shape for the molecule, whereas
the anti conformation iii of 1b leads to a “T”-shape.
The isotherms of 1b and 1c revealed a remarkable difference
in the response toward the addition of calcium ions (Figure 3b
and d). When a subphase containing 1.0 mM calcium chloride
was used instead of water, the molecular area of (R,S) isomer
1c decreased, whereas the (S,S) isomer 1b showed a surprising
increase in area per molecule.
Figure 5. Proposed bilayer structures in the case of (A) 1b and (B)
1c after addition of calcium ions. Intermolecular complexation of
calcium ions (A) gives rise to a more stretched out conformation of
the head groups, whereas intramolecular complexation (B) of calcium
ions gives rise to a more compact conformation.
These findings can be explained by the binding of a calcium
ion to two phosphate groups of the same molecule in the case
of 1c (Figure 5b), leading to a more compact conformation of
the molecule, and the intermolecular binding to two phosphate
groups of different molecules in the case of 1b (Figure 5a).
The latter will lead to a polymeric type of structure giving rise
to a more stretched-out conformation with a larger molecular
area.
These differences in behavior upon binding of calcium ions
may be rationalized by considering again the differences in
geometric orientation of the phosphate groups in these two
diastereomeric compounds. The meso compound 1c will be a
better chelating agent than 1b, due to its preference for gauche
conformations as discussed above. Compound 1b prefers a
conformation iii in which the two phosphate groups are
antiperiplanar.
III) and merging of the two inner halves will produce a new
intercalated bilayer (Figure 6C). Cleavage of this bilayer
eventually yields the new vesicle (Figure 6D).
Chelate formation decreases the molecular area of 1c, leaving
insufficient space to accommodate four hydrocarbon chains. This
will destabilize the intercalated structure of the original bilayer
(Figure 7). Transformation to a normal lamellar structure will,
because of the conical shape of the molecules, lead to the
formation of strongly curved bilayer fragments which are
thermodynamically unstable (Figure 7B). Smaller vesicles with
a lower degree of hydrocarbon chain packing are formed by
the blebbing off of these fragments (Figure 7C-E). These
vesicles will adopt a thermodynamically more stable organiza-
tion upon standing, resulting in the formation of tubular
structures.21
In the case of compound 1b, intermolecular complexation
will cause an increase in molecular area as the molecules have
to adopt a more stretched-out conformation. Since no change
in the thickness of the bilayer was observed, it must be
concluded that this process leads to a lower degree of hydro-
carbon chain packing.22 This disorder in the bilayer interior
will render the aggregates more fluid and will prevent the
precipitation of the complexes. With compound 1c the forma-
tion of a calcium complex will lead to a smaller spatial demand
Discussion of the Mechanisms of Fusion and Fission. The
physical data obtained from the experiments described above
can also be used to rationalize the difference in response during
addition of calcium ions to vesicle dispersions of the respective
diastereomers. The assumed formation of a polymeric type of
complex (Figure 5a) will stabilize the membrane structure of
1b, allowing larger aggregates to be formed. A reduction in
surface charge will decrease electrostatic repulsion between the
vesicles. Aggregation (Figure 6B-I) may then lead to the
formation of intervesicular calcium complexes (Figure 6B-II).
Subsequent withdrawal of the outer bilayer halves (Figure 6B-
(21) These events have been observed in reversed order for the fusion
of ammonium surfactants. See ref 5d.

4342 J. Am. Chem. Soc., Vol. 119, No. 19, 1997
Sommerdijk et al.
Figure 6. Schematic representation of a possible mechanism for the fusion of vesicles of 1b after addition of Ca2+ ions: (A) intercalated bilayer
before addition of calcium ions, (B-I) clustering of the vesicles after reduction of the repulsive forces, (B-II) formation of intervesicular complexes,
(B-III) redrawal of the outer bilayer halves, (C) formation of a new intercalated bilayer, and (D) the merging of the vesicles by cleavage of the new
bilayer.
Figure 7. Schematic representation of a possible mechanism for the fission of vesicles of 1c after the addition of calcium ions: (A) intercalated
bilayer before addition of calcium ions, (B) decrease of the head group size causes a transformation to a highly curved, nonintercalated bilayer, (C)
reduced electrostatic repulsion alows contact of the head groups from different parts of the inner bilayer half, (D) head groups of the molecules will
redirect to the aqueous phase which, by merging of the bilayers, (E) leads to the blebbing off of a small vesicle.
of the lipophilic part since the alkyl chains are no longer
intercalated and hence to the formation of aggregates with a
low hydrocarbon chain organization in the interior of the
bilayer.22 No precipitation of the complex is observed initially,
but precipitation occurs after several days when the vesicles
have transformed into the more stable tubular structures.
conformational preferences compound 1c forms a 1:1 chelate,
which leads to a destabilization of the intercalated bilayer. A
transition to a normal lamellar structure restores the bilayer but
requires a highly curved surface, which leads to the fission of
the vesicles.
Due to the large head group size of the described phospholipid
molecules, which arises from the introduction of a second
phosphate group, the calcium complexes show a poor hydro-
carbon chain packing. As a result of this, the lamellar structures,
formed upon dispersion in water, do not immediately precipitate
upon addition of calcium chloride as observed in the case of
phosphatidic acids. This enabled the monitoring of the processes
of both vesicle fusion and vesicle fission. It was possible to
present mechanistic models for the shape of the molecular
assemblies during these processes based on the physical data
of the aggregates prior to and after addition of calcium ions.
Conclusion
The experiments described above, reveal that these new
gemini surfactants may serve as interesting models for biomem-
branes. The introduction of an extra phosphate group has a
dramatic effect on both the aggregation behavior and on the
calcium binding properties of the phosphatidic acids. The
increase in the size of the head group leads to the formation of
intercalated bilayers from the three different stereoisomers of
1. The difference in orientation of the phosphate groups in the
meso and the (S,S) diastereomers of 1 leads to a remarkably
different response toward calcium ions. Apparently (S,S)
compound 1b is not able to form a stable 1:1 chelate for steric
reasons but preferentially forms a linear polymeric complex.
This eventually leads to fusion of the vesicles. Due to
The described vesicular systems show that a small alteration
in the orientation of functional groups can give rise to a big
change in response to external signals. These experiments
demonstrate furthermore that the observed changes in lipid
morphologies can be brought about without the action of
complex mechanisms involving intermolecular or intercellular
interactions but simply by altering the conformational properties
of the molecules involved. These insights may be useful in
understanding cell fusion and fission processes which are
believed to be initiated by similar calcium induced alterations
in head group size and the consequent changes in the confor-
mational characteristics of the molecules involved.
(22) The reflection in the 4.0-4.5 Å region represents the side of a cell
in the hexagonal lattice in which the hydrocarbon chains are packed. For
both compounds a broadening in this reflection is observed after addition
of calcium ions. This is indicative of a distorted hydrocarbon chain packing,
see: (a) Gulik-Krzywicki, T.; Rivas, E.; Luzzati, V. J. Mol. Biol. 1967, 27,
303. (b) Tardieu, A.; Luzzati, V. J. Mol. Biol. 1973, 75, 711. (c) Ranck, J.
L.; Mathieu, L.; Sadler, D. M.; Tardieu, A.; Gulik-Krzywicki,; T. Luzzati,
V. J. Mol. Biol. 1974, 85, 249. This decrease in organization in the interior
of the membrane is also in agreement with the change in transition entropy
deduced from the DSC data (Table 1).

Stereodependent Fusion and Fission of Vesicles
J. Am. Chem. Soc., Vol. 119, No. 19, 1997 4343
under reflux for 48 h. The reaction mixture was filtered, and the filtrate
was concentrated in vacuo. The residue was dissolved in ethyl acetate
and washed with aqueous 10% NaS2O3 and brine. After drying over
MgSO4 and evaporation of the solvent, a diastereomeric mixture of 3c
was obtained to which trifluoroacetic acid/methanol/water (4:1:8, v/v/
v) was added. The reaction mixture was stirred for 48 h at room
temperature and then poured into ethyl acetate. The organic layer was
washed with an aqueous 5% solution of Na2CO3 and brine, dried over
MgSO4, and concentrated. After crystallization from chloroform, 4c
was obtained in a yield of 94%. Mp 129-131 °C. Anal.
(C4H8O2I2): C, H, Calcd: 14.05, 2.36. Found: 14.43, 2.43. IR (KBr,
cm-1): ν 3250 (OH), 2910, 2850 (C-H). 1H-NMR (CDCl3/CD3OD,
ppm): δ 3.25 (m, 2H, CH), 3.50 (m, 4H, CH2). MS (EI+, m/z): 342
(M+), 215 (M - I).
Experimental Section
General Methods. 1H-NMR spectra were recorded on a Bruker
AM-400 spectrometer using tetramethylsilane as the internal standard.
Proton-decoupled 81 MHz 31P and 50 MHz 13C NMR spectra were
obtained using a Bruker WM-200 spectrometer. Chemical shifts are
reported relative to trimethyl phosphate and CDCl3, respectively. IR
spectra were recorded with a Perkin Elmer 298 spectrometer. Mass
spectra were recorded on a VG 7070 E spectrometer. Optical rotations
were measured on a Perkin Elmer 241 polarimeter. Melting points
were determined using a Reichert Thermopan microscope and are
uncorrected. In chromatographic procedures silica gel 60H (Merck art.
no. 7736) or silica gel 60 silanated (Merck art. no. 7719) were used as
the stationary phase. For ion exchange Dowex 50 W × 2 in Na+-
form (Fluka AG, art. no. 44465) was used. Thin layer chromatograms
(2R,3R)-1,4-Iodobutane-2,3-diyl Dioctadecanoate, 5a. Stearoyl
chloride (3.5 g, 11.5 mmol) in dry ether was added at 0 °C to a solution
of 4a (0.85 g, 2.5 mmol) in dry diethyl ether (34 mL) and dry pyridine
(4.3 mL). After standing for 24 h at room temperature, the reaction
mixture was poured into ice water and extracted with diethyl ether.
The organic layer was washed with aqueous 2 N H2SO4 and with a
saturated solution of Na2S2O3, dried over MgSO4 and concentrated.
The product 5a crystallized from acetonitrile and was isolated as a white
solid in 97% yield. Mp 54-56 °C.25 [R]D20) +5.3° (CH2Cl2, c 1.0).
IR (KBr, cm-1): ν 2950, 2910, 2850 (C-H), 1740 (CdO). 1H-NMR
(CDCl3, ppm): δ 0.88 (t, 6H, CH3, J ) 6.7 Hz), 1.25 (m, 56H,
CH2(CH2)14CH3), 1.56 (m, 4H, C(O)CH2CH2), 2,31 (t, 4H, C(O)CH2,
J ) 7.5 Hz), 3.22 (d, 4H, CH2I, J ) 5.8 Hz), 4.66 (m, 2H, CH). 13C-
NMR {H}(CDCl3, ppm): δ 9.3 CH2I, 13.9-34.11 (C-stearoyl), 72.0
(CH), 172.7 (CdO). MS (FB+, m/z): 875 (M + 1), 747 (M - I).
were run on glass supported silica gel 60 plates (0.25 mm-layer, F254
,
Merck art. no. 5715). The molybdate23 and Knight and Young24 sprays
were used as location reagents. Solvents were dried and distilled, when
appropriate, using the following methods: diethyl ether, benzene, and
dichloromethane were distilled from calcium hydride; toluene was
distilled from sodium, pyridine was distilled from sodium hydroxide,
and THF was distilled from lithium aluminium hydride. All other
solvents used were of analytical grade.
1,4-Ditosyl Erythritol. Erythritol (4 g, 33 mmol) was dissolved in
pyridine (80 mL) and cooled to -20 °C. A solution of tosyl chloride
(12.5 g, 30 mmol) in dry dichloromethane (40 mL) was added dropwise
over a period of 8 h. The reaction mixture was stirred for another 16
h while warming to room temperature. After concentration in vacuo
dichloromethane was added to the mixture, and the organic layer was
washed with aqueous 1 M sulfuric acid, water, and brine. After drying
over MgSO4 and evaporation of the solvent, the mixture was subjected
to column chromatography (silica gel, ethyl acetate/hexane 1:1, v/v).
After crystallization from chloroform, 1,4-ditosyl erythritol was obtained
in 55% yield (mp 88 °C). IR (KBr, cm-1): ν 3500 (O-H), 3020 (C-H
arom), 2980, 2920, 2850 (C-H alkyl), 1600 (SdO). 1H-NMR (CDCl3,
ppm): δ 1.60 (br.s, 2H, OH), 2.45 (s, 6H, CH3C6H5) 4.05 (m, 4H,
CH2), 4.65 (m, 2H, CH), 7.25 (d, 4H, H3 and H5 arom, J ) 5 Hz),
7.80 (d, 4H, H2 and H6 arom, J ) 5 Hz).
1,4-Ditosyl-2,3-O-isopropylidene Erythritol, 2c. To a suspension
of 1,4-ditosyl erythritol (1.0 g, 2.39 mmol) in 2,2-dimethoxypropane
(10 mL) was then added p-toluenesulfonic acid (45 mg, 2.4 mmol),
and the mixture was stirred for 16 h at room temperature. A saturated
aqueous solution of NaHCO3 was added, and the mixture was extracted
with ethyl acetate. The organic layer was washed with water and brine,
dried over MgSO4, and concentrated in vacuo. Crystallization from
ethyl acetate gave pure 2c in 94% yield (Mp 127-129 °C) Anal.
(C21H26S2O8) C, H, S Calcd: 52.60, 5.57, 13.63. Found: 52.60, 5.37,
13.50. IR (KBr, cm-1): ν 3020 (C-H arom), 2980, 2920, 2850 (C-H
alkyl), 1600 (SdO). 1H-NMR (CDCl3, ppm): δ 1.30 (s, 3H, CH3),
1.32 (s, 3H, CH3), 2.45 (s, 6H, CH3Tos) 4.05 (m, 4H, CH2), 4.30 (m,
2H, CH), 7.25 (d 4H, H3 and H5 arom, J ) 5 Hz,), 7.80 (d, 4H, H2
and H6 arom, J ) 5 Hz). MS (EI, m/z): 470 (M+), 285 (M -
CH2OTos).
(2S,3S)-1,4-Diiodobutane-2,3-diyl Dioctadecanoate, 5b. Com-
pound 5b was synthesized starting from 4b using the same procedure
as described for compound 5a. A white solid was obtained in 96%
20
yield. Mp 54-56 °C.25 [R]D ) -6.8° (CH2Cl2, c 1.0).
(2S,3R)-1,4-Diiodobutane-2,3-diyl Dioctadecanoate, 5c. Com-
pound 5c was synthesized starting from 4c using the same procedure
as described for compound 5a. A white solid was obtained in 90%
yield. Mp 79-81 °C.25 Anal. (C40H76O4I2): C, H, Calcd: 54.92, 8.76.
Found: 55.47, 8.79. IR (KBr, cm-1): ν 2950, 2910, 2840 (C-H),
1740 (CdO). 1H-NMR (CDCl3, ppm): δ 0.88 (t, 6H, CH3, J ) 6.7
Hz), 1.25 (m, 56H CH2(CH2)14CH3), 1.66 (m, 4H, C(O)CH2CH2), 2,37
(t, 4H, C(O)CH2, J ) 7.5 Hz), 3.30 (dd, AA′XX′YY′, 2H, CH2I), 3.30
(dd, AA′XX′YY′, 2H, CH2I), 4.86 (m, AA′XX′YY′, 2H, CH). MS (FB+,
m/z): 897 (M + Na+), 747 (M - I).
(2R,3R)-Di(octadecanoyloxy)butane-1,4-diyl Bis(dibenzylphos-
phate), 6a. To a solution of 5a (2.7 g, 3.0 mmol) in dry toluene (40
mL) was added silver dibenzyl phosphate16 (6.6 mmol) while stirring
under argon. The mixture was heated under reflux for 4 h with
exclusion of light. The suspension was filtered over Hyflo, and the
filtrate was concentrated under reduced pressure. After flash chroma-
tography (silicagel, hexane/ethyl acetate ) 7/3, v/v) the product 6a
was obtained as a colorless oil and crystallized from its acetonitrile
solution (yield 85%). Mp 44-45 °C; [R]D20 ) -1.69° (CH2Cl2, c 1.0).
IR (KBr, cm-1): ν 3090, 3060, 3030 (C-H aryl), 2920, 2840 (C-H),
1745 (CdO), 1285 (PdO), 1010 (P-O-C). 1H-NMR (CDCl3, ppm):
δ 0.88 (t, 6H, CH3, J ) 6.7 Hz), 1.25 (m, 56H CH2(CH2)14CH3), 1.56
(m, 4H, C(O)CH2CH2), 2.31 (t, 4H, C(O)CH2 J ) 7.5 Hz), 4.12 (m,
4H, CHCH2O), 5.07 (dd, 8H, CH2Bz, JP-H ) 8.4 Hz, JH-H ) 1.0 Hz)
5.28 (m, 2H, CH), 7.37 (s, 20H, C6H5). 31P-NMR {H}(CDCl3, ppm):
δ -3.06. 13C-NMR {H}(acetone D6, ppm): δ 14.9-35.1 (C-stearoyl),
66.6 (CH2O, JP-C ) 5.5 Hz), 70.5 (CH2Bz), 71.2 (CH), 128.2, 129.8,
129.9, 137.6, 138.1 (C6H5), 173.6 (CdO). MS (FB+, m/z): 897 (M -
HOP(O)(OBz)2).
(2R,3R)-1,4-Diiodobutane-2,3-diol, 4a. Compound 3a12 (2.75 g,
7.2 mmol) was added to a mixture of trifluoroacetic acid/methanol/
water (4:1:8, v/v/v). The reaction mixture was stirred for 5 min at
room temperature and then poured into ethyl acetate. The organic layer
was washed with a 5% solution of sodium carbonate and brine, dried
over MgSO4, and concentrated. Product 4a was purified by sublimation
(T ) 110 °C, P ) 3 mmHg) and obtained in a yield of 97%. Mp
20
104-105 °C; [R]D ) -7.4° (methanol, c 1.0). IR (KBr, cm-1): ν
3230 (OH), 2950, 2910, 2920 (C-H). 1H-NMR (CDCl3, ppm): δ 2.56
(s, 2H, OH), 3.33 (m, 4H, CH2I), 3.93 (m, 2H, CH). MS(CI+, m/z):
343 (M + 1), 215 (M - I).
(2S,3S)-Di(octadecanoyloxy)butane-1,4-diyl Bis(dibenzylphos-
phate), 6b. Compound 6b was synthesized starting from 5b using the
same procedure as described for compound 6a. Mp 44-45 °C.
Identical spectroscopic data were obtained as described for 6a, except
(2S,3S)-1,4-Diiodobutane-2,3-diol, 4b. Compound 4b was syn-
thesized starting from 3b12 by using the same procedure as described
for compound 4a. A white solid was obtained in 96% yield. Mp 105
20
20
°C; [R]D ) +7.6° (methanol, c 1.0).
for the optical rotation: [R]D ) +1.58° (CH2Cl2, c 1.0).
(2R,3S)-1,4-Diiodobutane-2,3-diol, 4c. A solution of 2c (870 mg,
(25) This melting point range suggests the presence of impurities. They
could not be removed despite several attempts to obtain analytically pure
material. It was then decided to purify the compound at a later stage in the
synthetic sequence.
2.5 mmol) and NaI (1.49 g, 9.3 mmol) in butanone (200 mL) was heated
(23) Dittmer, J. C. Lester, R. L. J. Lipid Res. 1964, 5, 126.
(24) Knight, R. H.; Young, L. Biochem. J. 1981, 70, 111.

4344 J. Am. Chem. Soc., Vol. 119, No. 19, 1997
Sommerdijk et al.
(2S,3R)-Di(octadecanoyloxy)butane-1,4-diyl Bis(dibenzylphos-
phate), 6c. Compound 6c was synthesized in 51% yield, starting from
5c by the same procedure as described for compound 5a. Mp 53-54
°C. Anal. (C68H104O12P2) C, H, Calcd: 69.48 8.92. Found: 68.99,
8.77. IR (KBr, cm-1): ν 3090, 3060, 3030 (C-H aryl), 2940, 2910,
2840 (C-H), 1725 (CdO), 1250 (PdO), 1020 (P-O-C). 1H-NMR
(CDCl3, ppm): δ 0.88 (t, 6H, CH3, J ) 6.7 Hz), 1.25 (m, 56H
CH2(CH2)14CH3), 1.53 (m, 4H, C(O)CH2CH2), 2,22 (t, 4H, C(O)CH2,
J ) 7.5 Hz), 4.04 (m, 2H, 2CHCHaHbO), 4.16 (m, 2H, 2CHCHaHbO)
4.99 (m, 8H, CH2Bz) 5.18 (br.s, 2H, CH), 7.37 (s, 20H, C6H5). MS
(FB+, m/z): 897 (M - HOP(O)(OBz)2).
Sodium (2R,3R)-Di(octadecanoyloxy)butane-1,4-diyl Bisphos-
phate, 1a. The tetrabenzyl ester 6a (500 mg, 0.6 mmol), dissolved in
ethanol (40 mL), was subjected to hydrogenolysis for 0.5 h with Pd-
(C) as the catalyst. The catalyst was filtered off using a small RP-2
column, and the filtrate was concentrated in vacuo. The residue was
dissolved in distilled water with the aid of an aqueous 0.05 N solution
of NaOH (36 mL), treated with Dowex 50WX20 (sodium form), and
lyophilized. The product 1a was purified by reversed phase chroma-
tography (RP 2, methanol/water), lyophilized, and subsequently dried
over P2O5 at 90 °C for 8 h. The yield was 61%. Rf ) 0.48 (BuOH/
AcOH/H2O ) 4/1/1). Mp > 200 °C dec. Anal.26 (C40H76P2O12Na4‚
21/2H2O): C, H, Calcd 50.66, 8.62. Found: 50.68, 8.85. IR (KBr,
cm-1); ν 2950, 2910, 2860 (C-H), 1735 (CdO), 1200 (PdO). 31P-
NMR{H}(D2O, ppm): δ -2.27.
Electron Microscopy. Freeze-fractured samples were prepared by
bringing a drop of the dispersion onto a golden microscope grid (150
mesh), placed between two copper plates and fixated in supercooled
liquid nitrogen. Sample holders were placed in a Balzers Freeze
Etching System BAF 400 D at 10-7 Torr and heated to -105 °C. After
fracturing, the samples were etched for 1 min (∆T ) 20°), shaded with
Pt (layer thickness 2 nm), and covered with carbon (layer thickness 20
nm). Replicas were allowed to warm up to room temperature, left on
20% chromic acid for 16 h and rinsed with water. Negatively stained
samples were prepared by bringing a drop of the dispersion onto a
Formvar coated copper grid, kept at 4 °C. After 2 min the excess of
dispersion was removed, and the sample was stained with a 2% (w/w)
uranyl acetate solution.
After preparation the grids were allowed to dry and studied under a
Philips TEM 201 microscope (60 kV).
DSC. Thermograms were recorded using a Perkin & Elmer DSC7
instrument. Samples were prepared as described above. Typically 50
µL was transferred into a stainless steel large volume pan (75 µL) and
left overnight. After incubation for 15 min at 0 °C, heating runs were
recorded from 0-100 °C.
Powder Diffraction. Samples were prepared in a desiccator, by
placing a drop of the suspension on a silicon single crystal. Subsequent
evacuation caused the freezing of the sample, and after sublimation of
the ice the sample was placed in a Philips PW1710 diffractometer
equipped with a Cu LFF X-ray tube operating at 40 kV and 55 mA.
Experiments were carried out between 3 and 60°, using a step width
of 0.005°.
Monolayer Experiments. Isotherms were recorded on a thermo-
stated, home-built trough (140 × 210 mm). The surface pressure was
measured using Wilhelmy plates mounted on a Trans-Tek transducer
(Connecticut USA). On the subphase 150 µL of a chloroform solution
(0.3-0.5 mg/mL) of the surfactant was spread and allowed to evaporate
for 10 min. The rate of compression was 7.0 cm2/min. The surface
of compressed monolayers was studied with a Brewster Angle
Microscope (NFT BAM-1) equipped with a 10 mW He-Ne laser with
a beam diameter of 0.68 mm operating at 632.8 nm. Reflections were
detected using a CCD camera, and images were recorded on a Panasonic
superVHS video recorder.
Sodium (2S,3S)-Di(octadecanoyloxy)butane-1,4-diyl Bisphos-
phate, 1b. Compound 1b was synthesized starting from 6b using the
same procedure as described for compound 1a and obtained as an
amorphous powder in 63% yield. Mp > 200 °C dec. Anal.26
(C40H76P2O12Na4‚11/2H2O): C, H, Calcd 51.64, 8.57. Found: 50.53
8.43. Spectroscopic data were identical to those given for 1a.
Sodium (2R,3S)-Di(octadecanoyloxy)butane-1,4-diyl Bisphos-
phate, 1c. Compound 1c was synthesized starting from 6c using the
same procedure as described for compound 1a. The product was
obtained as an amorphous powder in 97% yield (Rf ) 0.40; BuOH/
AcOH/H2O
) 4/1/1, v/v/v). Mp >
200 °C dec. Anal.26
(C40H76P2O12Na4‚2H2O): C, H, Calcd 51.15, 8.59. Found: 50.63, 8.35.
IR (KBr, cm-1): ν 2950, 2910, 2860 (C-H), 1735 (CdO), 1200
(PdO). 31P-NMR{H}(D2O, ppm): δ -2.85.
Sample Preparation. Typically 1.0 µmol of a surfactant was
suspended in 1.0 mL of 2 mM PIPES (piperazine-N,N′-bis[2-ethane-
sulfonic acid] buffer of pH ) 7.0 by sonication at 70 °C in a bath type
sonicator (Transsonic digital; level 9, degas) for 1 h. Calcium ion
complexes were prepared by adding 1.0 mL of a PIPES buffer
containing 1.0 mM CaCl2 at 70 °C to a freshly prepared vesicle
dispersion of the surfactant. These samples were kept at 70 °C for 2
h and then allowed to cool to room temperature. The samples were
left overnight at room temperature before use, except when the fusion
and fission of vesicles were monitored.
Acknowledgment. The authors wish to thank H. P. M.
Geurts (Department of Electron Microscopy, University of
Nijmegen) for assistance in performing electron microscopy
experiments and Professor A. E. Beezer (University of Kent)
for helpful discussions.
Supporting Information Available: Electron micrographs
of 1a and powder diffraction patterns of agregates of 1b and
1c, before and after addition of calcium ions(3 pages). See any
current masthead page for ordering and Internet access
instructions.
(26) Due to the hygroscopic character of these compounds varying
amounts of water of crystallization had to be taken into account. The purity
of these compounds was further checked by HPLC (Partisil SAX column,
UV detection at 215 nm, eluent: 0.1 M KH2PO4/H3PO4 buffer of pH ) 3).
JA962303S