Synthesis, conformational analysis, and phase characterization of a versatile self-assembling monoglucosyl diacylglycerol analog

Article abstract of DOI:10.1021/ja983883r

Glycosyl diacylglycerols are excellent lipids for the formation of both bi-and monolayer lamellar systems but they are generally not commercially available, and the synthesis of optically pure glycosyl diacylglycerols inevitably involves protection and deprotection of glycerol linkers. A novel glycolipid designed to be an easily accessible structural surrogate of monoglucosyl diacylglycerol (MGDG) has been synthesized. In this molecule, glycerol is replaced with (S)-1,2,4-trihydroxybutane. Instead of the fatty acyl moieties found in MGDG, a 2,2-dialkyl-1,3-dioxolane function provides the hydrophobic moiety of the molecule. This different functionality affords chemically and physically tunable new properties in a glycolipid. These include base stability, increased mobility of the headgroup, possibilities of new packing arrangements, and the potential for use in encapsulation strategies using liposomes where a decrease in pH is used as the environmental cue for release. The acetal linkage also makes the molecule unsusceptible to degradation by phospholipase A and other esterase activities found in biological systems thus further extending their utility. The choice of the butane triol linker removes the common problem of racemization of protected glycerol by acetal and ester migration. It also affords synthetic simplicity since only one dioxolane acetal is formed on reaction of butane-1,2,4-triol with ketones, whereas in the case of glycerol, one hydroxyl group has to be selectively protected to avoid the formation of both enantiomers. 2-D NMR homonuclear and heteronuclear correlation spectroscopy together with nuclear Overhauser effect experiments and molecular mechanics calculation were used to obtain information on the headgroup orientation and on the configuration of the trialkoxybutane backbone. These supported a structure in which the alkyl chains were extended in a parallel fashion and the headgroup, although free to rotate along C2-C3 and C3-C4 of the trialkoxybutane substructure, extended away in the other direction in a manner similar to that observed in the case of MGDG. X-ray powder diffraction and optical microscopy data both supported a lamellar phase behavior of this amphiphilic molecule in water. ¹H NMR experiments monitoring the rate of acetal cleavage of this amphiphile revealed its tunable acid susceptibility. The unique structural feature, phase behavior, and controllable acid susceptibility of this glycolipid is potentially useful in many applications.

Full text of DOI:10.1021/ja983883r

J. Am. Chem. Soc. 1999, 121, 1851-1861
1851
Synthesis, Conformational Analysis, and Phase Characterization of a
Versatile Self-Assembling Monoglucosyl Diacylglycerol Analog
Jie Song and Rawle I. Hollingsworth*
Contribution from the Department of Chemistry, Michigan State UniVersity, East Lansing, Michigan 48824
ReceiVed NoVember 9, 1998
Abstract: Glycosyl diacylglycerols are excellent lipids for the formation of both bi- and monolayer lamellar
systems but they are generally not commercially available, and the synthesis of optically pure glycosyl
diacylglycerols inevitably involves protection and deprotection of glycerol linkers. A novel glycolipid designed
to be an easily accessible structural surrogate of monoglucosyl diacylglycerol (MGDG) has been synthesized.
In this molecule, glycerol is replaced with (S)-1,2,4-trihydroxybutane. Instead of the fatty acyl moieties found
in MGDG, a 2,2-dialkyl-1,3-dioxolane function provides the hydrophobic moiety of the molecule. This different
functionality affords chemically and physically tunable new properties in a glycolipid. These include base
stability, increased mobility of the headgroup, possibilities of new packing arrangements, and the potential for
use in encapsulation strategies using liposomes where a decrease in pH is used as the environmental cue for
release. The acetal linkage also makes the molecule unsusceptible to degradation by phospholipase A and
other esterase activities found in biological systems thus further extending their utility. The choice of the
butane triol linker removes the common problem of racemization of protected glycerol by acetal and ester
migration. It also affords synthetic simplicity since only one dioxolane acetal is formed on reaction of butane-
1,2,4-triol with ketones, whereas in the case of glycerol, one hydroxyl group has to be selectively protected to
avoid the formation of both enantiomers. 2-D NMR homonuclear and heteronuclear correlation spectroscopy
together with nuclear Overhauser effect experiments and molecular mechanics calculation were used to obtain
information on the headgroup orientation and on the configuration of the trialkoxybutane backbone. These
supported a structure in which the alkyl chains were extended in a parallel fashion and the headgroup, although
free to rotate along C2-C3 and C3-C4 of the trialkoxybutane substructure, extended away in the other direction
in a manner similar to that observed in the case of MGDG. X-ray powder diffraction and optical microscopy
1
data both supported a lamellar phase behavior of this amphiphilic molecule in water. H NMR experiments
monitoring the rate of acetal cleavage of this amphiphile revealed its tunable acid susceptibility. The unique
structural feature, phase behavior, and controllable acid susceptibility of this glycolipid is potentially useful in
many applications.
1. Introduction
chemical or biological processes. The correct carbohydrate
headgroup can serve as an acceptor for other glycosyl groups
either by enzymatic or traditional chemical means. Hence a
glycosylated surface that interacts in some special manner with
some biological agent or cell can be fabricated. Finally,
glycolipids that are able to form lamellar systems are excellent
models for studying templating effect and regio- and stereo-
chemistry of oligosaccharide synthesis upon ordered surfaces,
such as membranes.
One excellent example of a glycolipid with potential for use
in the applications mentioned above is the naturally occurring
compound monoglucosyl diacylglycerol (MGDG) 1. This is one
of the common glycolipid components of the membrane of
cellular systems, especially bacteria.3-5 Unfortunately, MGDG
and other double-chain glycolipids are commercially available
in only very small (milligram) quantities as isolation products
from various natural sources. Such products have a heteroge-
neous lipid chain length distribution and various alkyl chain
modifications such as unsaturation and branching. The enzy-
matic synthesis of membrane glycolipids is achievable,6 but it
Lipids are excellent molecules for use in the design and
fabrication of highly ordered 2-dimensional molecular systems.
They find many applications ranging from the preparation of
biocompatible films and drug delivery vehicles to biosensor
design and nanotechonology.1 Phospholipids and simple alkyl
species with polar headgroups have been extensively studied
in this context. Glycolipids, especially those with two hydro-
carbon chains, also have great potential in the preparation of
self-assembled lamellar systems. Molecules with single alkyl
chains tend to form micelles. Glycolipids are typically uncharged
(unlike phospholipids), and this not only gives them more
chemical flexibility from the standpoint of synthesis and
solubility, but also makes them better candidates in applications
where charged species would have undesired properties or lead
to complications. For instance, it is known that as drug delivery
vehicles, charged vesicles are usually more toxic than neutral
ones.2 Moreover, glycolipids have several hydroxyl groups
giving them great potential for use in applications that require
surface modification. If a surface is modified with a glycolipid
array, the headgroups can be halogenated, acylated, alkylated,
oxidized, phosphorylated, or modified by a whole spectrum of
(3) Quinn, P. J.; Williams, W. P. Prog. Biophys. Mol. Biol. 1978, 34,
109-179.
(4) Boggus, J. M. Can. J. Biochem. 1980, 58, 755-770.
(5) de Kruijff, B.; Demel, R. A.; van Deenen, L. L. M. Biochim. Biophys.
Acta 1979, 553, 331-347.
(1) Liposomes: from Physics to Applications; Lasic, D. D., Ed.;
Elsevier: Amsterdam, 1993.
(2) Mayhew, E.; Papahadjopoulos, D. In Liposomes; Ostro, M. J., Ed.;
Marcel Dekker: New York, 1983; pp 289-341.
(6) Dahlqvist, A.; Andersson, S.; Wieslander, A. Biochim. Biophys. Acta
1992, 1105, 131-140.
10.1021/ja983883r CCC: $18.00 © 1999 American Chemical Society
Published on Web 02/17/1999

1852 J. Am. Chem. Soc., Vol. 121, No. 9, 1999
Song and Hollingsworth
Scheme 1. Synthesis of Compound 2a
would be very expensive especially if large quantities are
needed. The chemical synthesis is therefore the solution to large-
scale production of homogeneous products. However, this is
not trivial, either. This is especially due to the inevitable problem
of the enantioselective protection and deprotection of the
glycerol linkers in order to prepare optically pure glycosyl
diacylglycerols.7-10 Simple structural surrogates of glycolipids
such as MGDG 1 are not known. A simple, efficient route to
glycolipids that closely match them and meanwhile have
versatile chemical and physical properties that enable broad
applications is therefore highly desirable. Such molecules should
have two alkyl chains, and the carbohydrate headgroup should
be linked to the hydrocarbon chains via a substructure that
closely approximates the glycerol moiety of typical glycolipids.
The orientation of the carbohydrate headgroup relative to the
hydrocarbon chains should also be conserved. The glycolipid
surrogate should pack and form stable lamellar systems. The
new functionality introduced should have useful chemical
properties that lend themselves to practical applications. In
addition to all of these, the new lipid should be optically pure
and easy to assemble in only a few steps. In this work, we
describe the synthesis and properties of the glycolipid 2, which
bears a close resemblance to MGDG 1 in terms of its ability to
form lamellar system, its headgroup orientation, and linker
flexibility. Its preparation is simple and requires only three key
steps. This new glycolipid, however, also has several new
physical and chemical properties that lend themselves to a
variety of important applications, including the preparation of
base and lipase stable liposomes and liposomes that can be
cleaved below a specific pH.
a Reagents, conditions and yields: (a) NaH, THF, 50 °C, 24 h; (b)
6 N HCl, THF, r.t., 24 h, 80% (for 2 steps); (c) 3 equiv of TMOF,
MeOH-THF (1:2, v/v), Cat. pTSA, Reflux, 12 h; (d) 1.5 equiv of
(S)-1,2,4-butanetriol, DMF-THF (1:1, v/v), Reflux, 24 h, 69% (for 2
steps); (e) 0.4 equiv of HgO, 2 equiv of HgCN2, PhH-CH3NO2 (1:1,
v/v), 65 °C, 12 h, 58%; (f) K2CO3 (s), MeOH, r.t., 6 h, Quantitative.
TMOF ) Trimethyl Orthoformate; pTSA ) p-toluenesulfonic acid.
areas of the body, such as primary tumors or sites of inflam-
mation and infection,11 where pH is lower than the normal
physiological value. Another potential application is the con-
trolled release of drugs to the stomach to fight ulcers and buffer
acidity. In the latter application, once the pH drops, more
vesicles would be cleaved releasing the buffering component
at a greater rate. The resulting increase in pH would stop further
hydrolysis and release until the pH drops again. This property
may also be useful where cleavable surfactants under mild acidic
conditions are desired. The resulting (S)-2,2-dihexadecyl-4-
hydroxyethyl-1,3-dioxolane was then â-linked to a glucose
moiety to give glycolipid 2.
The synthetic route is summarized in Scheme 1. A 33-carbon
symmetric ketone 3 was prepared by using methyl methylthi-
omethyl sulfoxide (MMTS) as the carbonyl donor.12 MMTS
was dialkylated with hexadecyl bromide, and direct treatment
of the crude dialkylated intermediate with hydrochloric acid in
tetrahydrofuran gave symmetric ketone 3 in 80% overall yield.
Ketone 3 was converted to dimethoxy acetal and then trans-
acetalized with (S)-1,2,4-butanetriol. The more stable acetal, the
1,3-dioxolane 4 (as opposed to the six-membered cyclic acetal),
was obtained in 69% yield from ketone 3. The coupling of the
chiral acetal linker 4 to R-acetobromoglucose in the presence
of mercury(II) salts13 gave stereospecifically the desired â-ano-
mer 5 in 58% yield, which after quantitative deacetylation
afforded target molecule 2.
This synthetic route is short and efficient. No protection of
the triol linker is needed prior to the acetalization as is the case
with the glycerol linker where acetal migration would give the
enantiomeric dioxolane leading to racemization. Syntheses
employing chiral isopropylidene acetals of glycerol require
special steps to avoid this problem.8 The deprotection of the
peracetylated glycolipid is also much more straightforward
compared to the same reaction in the synthesis of diacyl
glyceroglycolipids14 since no selective deacetylation condition
for preserving the long hydrocarbon chains needs to be
employed.
2. Results and Discussion
Design and Synthesis of Glycolipid 2. To maintain a close
resemblance to the glycerol moiety of MGDG 1, an optically
pure (S)-1,2,4-butanetriol was chosen as the chiral linker
connecting the sugar headgroup and the hydrocarbon tails. Two
16-carbon hydrocarbon chains were attached to the linker via
an acetal linkage. Acetals are stable to base and this would allow
the de-esterification of the per-acetylated sugar at the end of
the synthesis without fear of losing the hydrophobic chains in
the aglycon. In the case of the typical acyl glycerols, this
deprotection is problematic because of the risk of cleaving the
fatty acyl groups. The acetal function should also give some
extra ordering to the headgroup once the lamellar array is formed
because of its rigidity. Another advantage beyond these is that
the acetal linkage cannot be degraded by the esterolytic lipase
activities (especially phospholipase A type activities) that are
present in all cellular systems. This is a problem with conven-
tional liposomes and leads to leakage. Finally, the acid-sensitive
acetal linkage could be utilized as a way to obtain sudden or
controlled release in response to a pH cue if this glycolipid is
incorporated in the construction of liposomes. Such liposomes
would be clinically useful if they enable drugs to be targeted to
The melting point ranges for both compounds 5 (158.0-164.0
°C) and 2 (140-145.5 °C) were quite broad and they both went
through a waxy phase before finally turning into clear liquid.
(7) Gent, P. A.; Gigg, R. J. Chem. Soc., Perkin Trans I 1975, 364-370.
(8) van Boeckel, C. A. A.; van Boom, J. H. Tetrahedron Lett. 1980, 21,
3705-3708.
(11) Yatvin, M. B.; Kreutz, W.; Horwitz, B. A.; Shinitzky, M. Science
1980, 210, 1253-1255.
(12) Schill, G.; Jones, P. R. Synthesis 1974, 117-118.
(13) Weker, N.; Benning, H. Chem. Phys. Lipids 1982, 31, 325-329.
(14) Kaplun A. P.; Shvets, V. I.; Evstigneeva, R. P. J. Org. Chem. USSR
(Engl. Transl.) 1978, 236.
(9) van Boeckel, C. A. A.; van Boom, J. H. Tetrahedron 1985, 41, 4545-
4555.
(10) Ohta, N.; Achiwa, K. Chem. Pharm. Bull. 1991, 5, 1337-1339.

Synthesis and Study of a Versatile Self-Assembling Lipid
J. Am. Chem. Soc., Vol. 121, No. 9, 1999 1853
This behavior is expected for such glycolipids since they form
liquid crystalline phases just above the melting point.
θ1 and θ2 (θ1 ) H4a-C4-C3-C2; θ2 ) C4-C3-C2-C1), and
they were measured as -173.8° and 78.0° in this lowest energy
conformer.
Conformational Analysis. When lamellar systems crystallize,
they often form thin leaflets at best. This makes X-ray structure
determination difficult because of the lack of information in
one dimension. A variety of physical methods then have to be
employed to gain information on the conformation and packing
arrangement.15 The average solution conformation, packing, and
phase behavior of glycolipid 2 were characterized by a
combination of NMR spectroscopy, molecular mechanics (MM)
calculations, polarized-light microscopy, X-ray diffraction, and
differential scanning calorimetry (DSC).
To get well-resolved 1H and 13C NMR spectra of glycolipid
2, a solvent system of 2:1 CD3OD:CDCl3 was used to balance
between the substrate solubility and the solvent polarity. This
ensured the free tumbling of the substrate in order to obtain
acceptable spectral resolution.16 A trace of pyridine was also
added to stabilize the acetal linkage. All of the 13C and 1H NMR
signals of compound 2 were assigned unequivocally with the
aid of double quantum filtered J-correlated spectroscopy (DQF-
COSY) and gradient 1H-13C heteronuclear multiquantum
coherence spectroscopy (1H-13C HMQC) experiments (Figure
1).
The solution 3-dimensional structure of glycolipid 2 was
determined by nuclear Overhauser effect spectroscopy (NOESY)
experiments and molecular mechanics calculations. The nOe
volumes and the calculated internuclear distances are listed in
Table 1. The nOe volume and the known distance between sugar
ring protons 1′ and 5′ were taken as references. Strong nOes
between protons 1a/1b/2 and protons â/â′ were also observed
(Figure 2), although the volumes could not be accurately
measured because of the overlapping of the four â/â′ protons.
Semiquantitative information could be obtained for these. The
residual acetone peak also masked nOe information of protons
3a and 3b. Despite the severe overlapping of the hydrocarbon
methylene signals at 1.18 ppm, we still observed nOe peaks
between â/â′ protons and γ/γ′ protons on the downfield edge
of the large methylene peak envelope. Although this information
cannot be used quantitatively, it provides very important
evidence for the conclusion that the two chains are parallel.
This (in addition to several other lines of information) allows
the distinction between the proposed conformation and one in
which the two alkyl chains are extended away from each other.
Molecular mechanics calculation with use of distance con-
straints listed in Table 1 gave the lowest energy conformer
shown in Figure 3, in which the two hydrocarbon chains stay
close together, with a cis turn at the beginning of each chain.
This suggests that the gain from van der Waals interaction by
packing two lipid chains close together is greater than the
penalty for having the cis turns. The full length of this molecule
was measured as 31 Å. The torsion angles φ and æ (φ ) H1′-
C1′-O-C4; æ ) C1′-O-C4-C3), which describe the relative
orientation of the sugar headgroup, were 51.4° and 166.8°,
respectively, in this lowest energy conformer in which the
glucose ring extends away from and along the same line as the
hydrocarbon chains. These values matched very well with those
obtained from MGDG conformational studies (φ ) 47°; æ )
169°).17-19 This demonstrates that the orientation of the sugar
headgroup relative to the hydrocarbon chains is well conserved
from MGDG to its structural surrogate 2. The torsion angles
describing the 4-carbon linker conformation were defined as
Admittedly, in this study we did not take into account the
surface interactions of this molecular ensemble, and also no
solvent box was included during the calculation. Some recent
studies of headgroup orientations of isotope-labeled glycolipids
at lipid bilayer interfaces incorporated a membrane interaction
term to the potential energy function in computer models. These
studies offered important insight into the degree and nature of
molecular motions at the surface of membranes.20-24 While the
results indicated more restricted motions of the carbohydrate
ring in some of these systems, the average orientation of the
headgroup is similar to those obtained by more simplified studies
such as this one, where axial symmetry was assumed.
To gain insight into the relative flexibility about the various
dihedrals of the headgroup linker region of molecule 2, grid
searches of dihedral angles φ-æ and θ1-θ2 were performed.
The grid search results are shown in Figure 4. It is clearly shown
that there is a relatively restricted conformational preference at
the anomeric position whereas the 4-carbon linker employs a
large degree of flexibility. In Figure 4A, there is essentially only
one major energy minimum and the center of the energy contour
matches the measured φ and æ of the MM calculated structure.
However, in Figure 4B, there are many energy minima, which
indicates that these very closely populated conformations all
significantly contribute to the averaged solution conformation.
Such regional conformational flexibility is consistent with the
observations of the glycerol linker flexibility in natural gly-
cerolipids17,19 and is also expected on adding one more carbon
to the glycerol linker.
The flexibility of the linker region was confirmed by an
analysis of the coupling constants for the protons in the 1H NMR
spectrum of that region. Despite their complex chiral environ-
ments, the simple splitting patterns of the 7 spins indicated a
mutual conformationally averaged coupling of 6.6 Hz for all
of the vicinal pairs. Protons 1a and 1b appeared as triplets;
protons 2, 3a, and 3b appeared as quintets; protons 4a and 4b
2
appeared as doublets of doublet. The JHH(geminal) coupling
constants were 2J1a-1b ) 9 Hz, 2J4a-4b ) 8.4 Hz, and 2J3a-3b
)
6.6 Hz. Using these values, a spin simulation of this 7-spin
system was carried out and the result is shown in Figure 5. The
excellent match of the simulated spectrum with the original
spectrum proved the assignments of the coupling constants to
be correct.
Characterization of Phase Behavior. To further explore the
packing and phase behavior of compound 2, especially in water,
differential scanning calorimetry (DSC), X-ray diffraction, and
optical microscopy data were collected.
(17) Lee, J. Ph.D Dissertation, Michigan State University, 1998, Chapter
VI.
(18) Hauser, H.; Pascher, I.; Pearson, R. H.; Sundell, S. Biochim. Biophys.
Acta 1981, 650, 21-51.
(19) Hauser, H.; Pascher, I.; Sundell, S. In Molecular Description of
Biological Membranes by Computer Aided Conformational Analysis;
Brasseur, R., Ed.; CRC Press: Boca Raton, FL, 1990; Vol. 1, pp 267-
284.
(20) Sanders, C. R., II; Prestegard, J. H. J. Am. Chem. Soc. 1991, 113,
1987-1996.
(21) Ram, P.; Kim, E.; Thomson, D. S.; Howard, K. P.; Prestegard, J.
H. Biophys. J. 1992, 63, 1530-1535.
(22) Sanders, C. R., II; Prestegard, J. H. J. Am. Chem. Soc. 1992, 114,
7096-7107.
(15) Hauser, H.; Poupart, G. In The Structure of Biological Membranes;
Yeagle, P. L., Ed.; CRC Press: Boca Raton, FL, 1992; pp 3-72.
(16) Wang, Y.; Hollingsworth, R. I. Anal. Biochem. 1995, 225, 242-
251.
(23) Howard, K. P.; Prestegard, J. H. J. Am. Chem. Soc. 1995, 117,
5031-5040.
(24) Winsborrow, B. G.; Brisson, J.-R.; Smith, I. C. P.; Jarrell, H. C.
Biophys. J. 1992, 63, 428-437.

1854 J. Am. Chem. Soc., Vol. 121, No. 9, 1999
Song and Hollingsworth
1
Figure 1. DQF-COSY (top) and H-13C HMQC spectra of compound 2.
Table 1. Distances Calculated from NOESY Experiment for
Compound 2
liquid crystalline state. This is lower than major phase transition
temperatures of most natural dialkyl or diacylglycerol phos-
pholipids and glycolipids,25 but consistent with the reported
phase transition temperature of some acetal-based double chain
vesicles.26 One can take advantage of this property using local
heating to induce preferential release of entrapped species from
liposomes. It was proposed that local hyperthermia could
proton pairs
nOe volumes
distances in Å
1′-5′
1′-4a
1′-4b
2-4b
229519
137267
240664
68272
2.51
2.73
2.49
3.07
(25) Lewis, R. N. A. H.; McElhaney, R. N. In The Structure of Biological
Membranes; Yeagle, P. L., Ed.; CRC Press: Boca Raton, FL, 1992; pp
73-155.
(26) Jaeger, D. A.; Jamrozik, J.; Golich, T. G.; Clennan, M. W.;
Mohebalian, J. J. Am. Chem. Soc. 1989, 111, 3001-3006.
A transition at 33.7 °C was observed in a DSC scan of
compound 2 (in water) from 10 to 98 °C (Figure 6), which may
correspond to a transition from the gel-state to the less ordered

Synthesis and Study of a Versatile Self-Assembling Lipid
J. Am. Chem. Soc., Vol. 121, No. 9, 1999 1855
Figure 2. NOESY spectrum of compound 2. A mixing time of 500 ms was used.
X-ray powder diffraction of the dry sample showed a very
strong and sharp low-angle reflection corresponding to a d
spacing of 40.16 Å and a medium reflection corresponding to
47.50 Å, which unequivocally revealed the long-range order in
this system. They are about 1.4 times the full length of the
calculated structure of compound 2, which suggests the forma-
tion of interdigitated bimolecular layers in the solid phase.28
The two different values may correspond to (probably slightly)
different molecular packing. Reflections at 19.81 and 9.78 Å
corresponded to the second- and third-order reflections of the
one at 40.16 Å. There was also a peak corresponding to twice
the 40.16 Å separation. These all indicated a very high lamellar
organization.29 A wide-angle reflection corresponding to a d
spacing of 4.41 Å, which is characteristic of the hydrocarbon
chain separation in smectic mesophases,30 was also observed.
The X-ray diffraction pattern of fully hydrated compound 2
(>50% water content) in a capillary showed a strong reflection
corresponding to a 63.10 Å d spacing. This is twice the
calculated length of one molecule and corresponds to a solvated
bilayer structure, considering that the layer spacing would be
reduced due to the tilt of the molecule. Its second- and third-
order reflections at 31.00 and 15.51 Å were also observed. This
again was consistent with the X-ray diffraction pattern of the
1-D smectic phase, and distinguished itself from phases such
as 2-D hexagonal, 3-D cubic, nematic, etc.29 Two wide angle
reflections were observed, which corresponded to 4.25 and 3.31
(27) Yatvin, M. B.; Weinstein, J. N.; Dennis, W. H.; Blumenthal, R.
Science 1978, 202, 1290.
(28) van Doren, H. A.; Wingert, L. M. Mol. Cryst. Liq. Cryst. 1991,
198, 381-389.
Figure 3. Favorable conformation of compound 2 obtained by
molecular mechanics calculation.
(29) Seddon, J. M. In Handbook of Liquid Crystals; Demus, D., Goodby,
J., Gray, G. W., Spiess, H.-W., Vill, V., Eds.; Wiley-VCH: Weinheim,
New York, Chichester, Brisbane, Singapore, Toronto, 1998; Vol. 1; pp 635-
679.
(30) McIntosh, T. J. In Molecular Description of Biological Membranes
by Computer Aided Conformational Analysis; Brasseur, R., Ed.; CRC
Press: Boca Raton, FL, 1990; Vol. 1, pp 241-265.
preferentially release drugs from liposomes in the heated area.27
For this purpose, glycolipid 2 may be mixed with other lipids
to construct liposomes exhibiting gel to liquid crystalline phase
transitions at a temperature slightly above body temperature and
thus attainable by local hyperthermia.

1856 J. Am. Chem. Soc., Vol. 121, No. 9, 1999
Song and Hollingsworth
Figure 4. Grid search plots of compound 2. (A) DIH 1 ) φ ) H1′-C1′-O-C4; DIH 2 ) æ ) C1′-O-C4-C3; (B) DIH 1 ) θ1 ) H4a-C4-
C3-C2; DIH 2 ) θ2 ) C4-C3-C2-C1.
Figure 5. Spin simulation of the chiral linker part of compound 2: (A) Simulated spectrum and (B) original spectrum.
Å. The shorter distance corresponds to lipid chains that are very
tightly packed, and indicates the presence of highly crystalline
domains imbedded in a more fluid, but ordered, lamellar matrix.
number of units with multiple layers or larger radii. Either one
of these will result in a reduction of light scattering.
It is known that amphiphilic carbohydrates form thermotropic
and lyotropic liquid crystals if the molecules have an appropriate
shape.31 Smectic Ad phases are well established for amphiphilic
carbohydrates with one aliphatic chain.32 However, the texture
of polarized light micrographs of amphiphilic carbohydrates with
the complexity similar to molecule 2 have never been reported.
Tilted phases of chiral molecules, which possess permanent
polarizations and are thus ferroeletric, are of particular interest
because of their high potentials in electrooptic applications. One
characteristic of chiral smectic C phases is the spiral or
The concentration range at which compound 2 starts to
associate and form vesicles or other supramolecular systems
was examined by measuring its light-scattering behavior at
different concentration levels in water. In the range of 10-14 to
10-6 mM, a periodic rise and fall was observed when the
absorbance was plotted against concentration. Three distinct
maxima appeared within this range. The appearance of several
such sudden changes instead of one is expected, since different
types and sized vesicles would be formed in solutions at different
concentrations. For instance, as the concentration is increased,
at some critical value, the particles might fuse to form a smaller
(31) Jeffrey, G. A. Mol. Cryst. Liq. Cryst. 1990, 185, 209-213.
(32) Goodby, J. W. Mol. Cryst. Liq. Cryst. 1984, 110, 205-219.

Synthesis and Study of a Versatile Self-Assembling Lipid
J. Am. Chem. Soc., Vol. 121, No. 9, 1999 1857
concentrated DCl (pD slightly lower than 3) was added, the
acetal linkage was slowly cleaved with cleavage being com-
pleted within 5 h. The spectra (Figure 9C) clearly showed this
process. As the reaction proceeded, the released long chain
symmetric ketone precipitated out of the alcohol solution and
their signals in the upfield region significantly decreased, while
the anomeric proton and the linker proton signals were upfield
shifted. When the DCl concentration was increased to 1% (pD
around 1), the solution turned cloudy immediately once
compound 2 was dissolved, and by the time the first NMR
acquisition was recorded the reaction was already completed.
The above results demonstrated that compound 2’s acid
susceptibility allows one to set specific conditions under which
the rate and extent of cleavage can be controlled. It is quite
stable in weak organic acids such as acetic acid (its Ka is lower
in ethanol than in water), while labile in strong inorganic acids
such as DCl in a controllable fashion. There is a lot of room
for screening appropriate conditions, such as choice of acids,
temperature, etc., to control the kinetics of this acetal cleavage
for various practical applications. In addition to these, another
level of control can be exerted by modifying the structure and/
or environment of the carbohydrate group. For instance, the net
charge on the carbohydrate group can be altered by preparing
the liposomes in borate buffers. Under these conditions carbo-
hydrates form negatively charged cyclic borate esters that buffer
the surface of the liposomes. The pH required for cleavage under
such circumstances would be appreciably lower. The number
of borate groups attached to the carbohydrate ring can be altered
by using sugars with different configurations.34
Figure 6. Differential scanning calorimetry thermogram of compound
2. Scanning rate was 60 °C/h.
concentric circular defects that are observed on the free surface.33
The tilt of the molecular director away from the surface normal
results in the possible motion or placement of the molecule along
a cone, the axis of which is the layer normal. It is the offsetting
of the alignment of molecules from layer to layer that leads to
this spiral defect that is characterized by a winding spiral
morphology. The polarized-light micrograph of hydrated com-
pound 2 showed a texture characteristic of smectic C phase
(Figure 7A), which further agreed with the proposed lamellar
structure. Phase contrast micrographs (Figure 7B) and confocal
reflection micrographs (Figure 7C) clearly showed the spiral
defects with their fine layered features placed in the character-
istic spiral fashion. The presence of these defects again indicated
a tightly packed lamellar structure. A lamellar structure is
obligatory for producing these effects. 3-D reconstruction of
the z-sectioning confocal reflection micrographs using 10 scans
along the z-axis with a growth step of 500 nm revealed the
conical shape of the dislocations and the complementary concave
shapes of their bases (Figure 8). This is consistent with an
upward dislocation of the layers.
3. Conclusion
An optically pure glycolipid 2 containing a (S)-2,2-dihexa-
decyl-1,3-dioxolane ring and designed as a structural surrogate
of typical glycolipids such as MGDG was successfully synthe-
sized in a short route with satisfactory yields. Various physical
measurements and conformational studies revealed that this
molecule forms the stable lamellar system and the sugar
headgroup orientation, relative to the hydrocarbon chains, is very
close to that in MGDG. The 4-carbon linker has a considerable
amount of conformational flexibility, which is similar to the
naturally occurring glycerol linker in MGDG and is also to be
expected on adding one more carbon to the glycerol linker.
Hence, this synthetic molecule may serve as a good substitute
for the expensive naturally occurring glycolipids in a broad range
of studies involving highly ordered 2-dimensional molecular
systems. Moreover, the unique structural feature and phase
behavior of this molecule opens a large variety of new potential
applications. This amphiphile’s relatively low phase transition
temperature (33.7 °C), its glycosidic linkage, and its acid-
sensitive acetal function make it a good candidate in the
construction and engineering of liposomes with triggered release
control mediated by physically or chemically induced phase
transitions, such as thermal triggering, enzymatic triggering (e.g.
by glucosidases), or pH triggering. These are all important tools
in solving drug delivery problems. The fact that the polar
headgroup of this amphiphile is glucose and the butane triol
linker is a close analogue of glycerol also makes this molecule
a promising candidate in preparing biocompatible films. The
nature of the carbohydrate headgroup can be easily altered
without significantly changing the short synthetic route. There-
fore, it offers an excellent model for studying the stereo- and
regiochemistry of oligosaccharide synthesis upon ordered
Acid Susceptibility. To get an idea of how sensitive
1
compound 2 is toward acidic environments, H NMR spectro-
scopy experiments were carried out to monitor the rate of acetal
cleavage in different acidic solutions. These experiments were
carried out in d6-ethanol since it was the most polar protic
solvent in which the solubility was high enough to afford the
many measurements to be taken in a reasonable time. As
references, compound 2’s stability in pure ethanol was assessed
at temperatures varying from 25 to 55 °C. The spectra (Figure
9A) clearly shows that it is stable under these conditions. Thirty-
seven degrees centigrade was chosen as the temperature for all
acid susceptibility tests of compound 2 for the consideration of
its potential application in pH-controlled drug release systems
with the understanding that the rate of solvolysis in ethanol could
be scaled to a rate in water. Different concentrations of d4-acetic
acid (from 1% to 20%) were added to the NMR sample and
the reactions were monitored up to 14 h. No acetal cleavage
was detected even at acid concentration as high as 20% (Figure
9B). The only change observed was the slight broadening of
the sugar ring proton signals, which is expected since the
increase of solvent polarity upon the addition of acid would
force the glycolipids to pack more tightly and therefore the sugar
headgroup mobility would decrease. However, when strong acid
DCl was used, compound 2 became very sensitive. When 0.01%
(33) Bouligand, Y. In Handbook of Liquid Crystals; Demus, D., Goodby,
J., Gray, G. W., Spiess, H.-W., Vill, V., Eds.; Wiley-VCH: Weinheim,
New York, Chichester, Brisbane, Singapore, Toronto, 1998; Vol. 1, pp 406-
453.
(34) Davidson, E. A. In Carbohydrate Chemistry; Holt, Rinehart and
Winston, Inc.: New York, 1967; pp 105-115.

1858 J. Am. Chem. Soc., Vol. 121, No. 9, 1999
Song and Hollingsworth
Figure 7. The optical texture of compound 2 observed under laser scanning microscopy: (A) polarized-light micrograghs; (B) phase contrast
micrograghs; and (C) confocal reflection micrographs.
performed on Aldrich silica gel (60 Å 200-400 mesh). Yields refer to
chromatographically and spectroscopically (1H NMR) homogeneous
materials.
surfaces, using such glycolipids with different carbohydrate
headgroups. Such glycolipids serve as both reactants (the
carbohydrate headgroups) and ordered templates (the hydro-
phobic anchors) with versatile chemically and physically tunable
properties. This part of the work is currently ongoing and the
results will be reported in due course. Finally, this work
describes the facile preparation of a molecule that forms chiral
smectic C-type phases. Such chiral smectics are very important
in technology applications of liquid crystals because of their
ferroelectric properties. They are, however, not easily accessible.
NMR spectra were recorded on a Varian 300, 500, or 600 MHz
VXR spectrometer at ambient temperature unless otherwise specified.
Chemical shifts are reported relative to the residue solvent peak unless
otherwise specified. Optical rotations were measured with a Perkin
polarimeter at 589 nm. High-resolution mass spectra (HRMS) were
recorded on a JEOL HX-110-HF spectrometer using Fast Atom
Bombardment (FAB) conditions and an N-benzyl alcohol (NBA) matrix.
Melting points were measured on a Fisher-Johns melting point
apparatus.
4. Experimental Section
Synthesis: Compound 3. A mixture of methyl methylthiomethyl
sulfoxide (3.2 mL, 30 mmol), hexadecyl bromide (21.1 mL, 67 mmol),
sodium hydride (60% dispersion in oil, 10 g), and freshly distilled
General Techniques. All reagents used were reagent grade. Reaction
temperatures were measured externally. Flash chromatography was

Synthesis and Study of a Versatile Self-Assembling Lipid
J. Am. Chem. Soc., Vol. 121, No. 9, 1999 1859
(60 mL), and nitromethane (60 mL) was stirred at 65 °C under nitrogen
for 12 h. Solvent was then evaporated and dichloromethane (80 mL)
was added. The majority of mercury salts were filtered out on a Celite
pad. After dichloromethane was evaporated, the residue was subjected
to column chromatography purification (silica gel, 1.8% methanol in
benzene, Rf ) 0.50). Pure compound 5 (1.3 g) was obtained (58%).
1
[R]D -5.2° (c 0.79, CH2Cl2); H NMR (300 MHz, CDCl3 with trace
d5-Py) δ 5.16 (1H, t, J ) 9.6 Hz), 5.05 (1H, t, J ) 9.6 Hz), 4.94 (1H,
dd, J ) 9.6, 8.1 Hz), 4.46 (1H, d, J ) 8.1 Hz), 4.24 (1H, dd, J ) 12.1,
4.8 Hz), 4.10 (1H, dd, J ) 12.1, 2.4 Hz), 4.00 (2H, m), 3.90 (1H, m),
3.66 (1H, m), 3.60 (1H, m), 3.43 (1H, t, J ) 7.5 Hz), 2.08 (2H, m),
2.05 (3H, s), 2.01 (3H, s), 1.99 (3H, s), 1.97 (3H, s), 1.52 (4H, m),
1.22 (m), 0.84 (6H, t, J ) 6.6 Hz); 13C NMR (75 MHz, CDCl3 with
trace d5-Py) δ 170.80, 170.24, 169.35, 169.17, 111.97, 100.58, 73.62,
72.76, 71.74, 71.15, 69.81, 68.28, 66.84, 61.84, 37.71, 37.32, 33.21,
31.87, 29.90, 29.65, 29.31, 23.96, 23.71, 22.64, 20.70, 20.57, 14.08;
FAB-HRMS (NBA), calcd C51H93O12 [M+H]+ 897.6667, found
897.6644.
Compound 2. Anhydrous potassium carbonate (2 g) was added to
a methanol solution (10 mL) of compound 5 (0.67 g, 0.75 mmol). The
mixture was stirred at room temperature for 6 h before it was filtered.
The clear filtrate was neutralized with acetic acid and then passed
through a Celite pad. After removing solvent, compound 2 was obtained
in quantitative yield. [R]D -8.0° (c 1.82, CH2Cl2); 1H NMR (600 MHz,
CDCl3:CD3OD 2:1 with trace d5-Py, chemical shifts relative to TMS
signal) δ 4.19 (H1′, d, J ) 7.8 Hz), 4.13 (H2, m), 4.00 (H1a, t, J ) 7.8
Hz), 3.90 (H4a, m), 3.76 (H6a′, dd, J ) 12.2, 2.2 Hz), 3.68 (H6b′, dd, J
) 12.2, 4.8 Hz), 3.60 (H4b, m), 3.47 (H1b, t, J ) 7.8 Hz), 3.32 (H3′ and
H4′, m), 3.19 (H5′, m), 3.15 (H2′, m), 1.86 (H3a, m), 1.77 (H3b, m), 1.50
(Hâ/â′, m), 1.18 (CH2’s, m), 0.79 (CH3’s, t, J ) 6.6 Hz); 13C NMR
(125 MHz, CDCl3:CD3OD 2:1 with trace d5-Py) δ 111.89 (CR), 102.51
(C1), 76.01 (C3′), 75.80 (C5′), 73.42 (C2), 73.16 (C2′), 69.74 (C4′), 69.36
(C1), 66.24 (C4), 61.12 (C6′), 37.16 (Câ), 36.69 (Câ′), 33.20 (C3), 31.43
(Cγ/Cγ′), 29.40, 29.18, 29.09, 28.86, 23.48, 23.22 (Cδ), 23.14 (Cδ′), 22.16
(Cꢀ/Cꢀ′), 13.38 (CH3’s); FAB-HRMS (NBA), calcd C43H84O8K [M +
K]+ 767.5803, found 767.5804.
Conformational Analysis: Two-Dimensional (2-D) NMR Experi-
ments. All NMR spectra of compound 2 were obtained in a solvent
system consisting of chloroform/methanol/pyridine in the ratio of 2/1/
trace. All 2-D spectra were measured at 600 MHz. Double quantum
filtered J-correlated spectroscopy (DQF-COSY), gradient 1H-13C
heteronuclear multiquantum coherence spectroscopy (1H-13C HMQC),
and phase sensitive nuclear Overhauser effect spectroscopy (NOESY)
experiments were performed by using a total of 256 real data sets, with
32 acquisition transients each and a relaxation delay of 1.8 s between
transients. Mixing times of 300, 400, and 500 ms were used in the
NOESY experiments. Volume integrations of cross-peaks were per-
formed with the standard VARIAN software. A mixing time of 500
ms was used to calculate nOe cross-peak volumes since it gave the
best signal-to-noise ratio.
Figure 8. 3-D reconstruction with use of confocal reflection images
from 10 sequential laser confocal microscopy scans through compound
2. The overall depth of the reconstructed image is 5 µm. Note the
conical shape of the dislocations and the complimentary concave shapes
of their bases (see edges) consistent with an upward dislocation of the
layers.
tetrahydrofuran (200 mL) was stirred under nitrogen at 50 °C for 24 h.
After the mixture was cooled, the reaction was quenched with
methanol-water and extracted with ether. The ether layer was washed
with water, then dried over sodium sulfate and concentrated. The residue
was stirred at room temperature for 24 h with tetrahydrofuran (200
mL) and 6 N hydrochloric acid (200 mL). The reaction mixture was
then diluted with water and ether. The ether layer was washed with
sodium bicarbonate solution and water, then dried over sodium sulfate
and concentrated. The residue was crystallized from ethanol to give
11.5 g of 3 (overall 80%). Mp 80.0-82.0 °C [lit.35 mp 79.7-81.7 °C];
1H NMR (300 MHz, CDCl3) δ 2.36 (4H, t, J ) 7.4 Hz), 1.54 (4H, m),
1.23 (m), 0.86 (6H, t, J ) 6.6 Hz); 13C NMR (75 MHz, CDCl3) δ
42.82, 31.93, 29.70, 29.66, 29.62, 29.49, 29.42, 29.37, 29.27, 23.89,
22.69, 14.14 (signal corresponding to carbonyl carbon was not
observed); FAB-HRMS (NBA), calcd C33H67O [M + H]+ 479.5192,
found 479.5201.
Compound 4. A mixture of compound 3 (4.78 g, 10 mmol),
trimethyl orthoformate (3.3 mL, 30 mmol), p-toluenesulfonic acid
monohydrate (0.1 g), methanol (25 mL), and tetrahydrofuran (50 mL)
was refluxed for 12 h. After the reaction was quenched by triethylamine,
the mixture was poured into sodium bicarbonate-ice water and
extracted with petroleum ether. The ether layer was dried over sodium
sulfate and concentrated. The identity of the dimethoxy acetal inter-
mediate was confirmed by the presence of a signal at δ 3.10 (6H, s,
OCH3) in 1H NMR (300 MHz, CDCl3 with trace d5-Py) and the signals
at δ 103.91 (quaternary carbon) and δ 47.58 (OCH3) in 13C NMR (75
MHz, CDCl3 with trace d5-Py). The residue was dissolved in anhydrous
N,N-dimethylformamide (50 mL) and tetrahydrofuran (50 mL) with
(S)-1,2,4-butanetriol (1.6 g, 15 mmol). The mixture was refluxed for
24 h. The reaction was then quenched by triethylamine. The mixture
was poured into sodium bicarbonate-ice water and extracted with
petroleum ether. The ether layer was dried over sodium sulfate,
concentrated, and stored with a trace of pyridine added. The yield was
3.9 g (69% from compound 3). 1H NMR (300 MHz, CDCl3 with trace
d5-Py) δ 4.19 (1H, m), 4.06 (1H, m), 3.77 (2H, t, J ) 5.7 Hz), 3.50
(1H, t, J ) 8.0 Hz), 1.78 (2H, m), 1.55 (4H, m), 1.22 (m), 0.84 (6H,
t, J ) 6.5 Hz); 13C NMR (75 MHz, CDCl3 with trace d5-Py) δ 112.46,
75.31, 69.90, 60.56, 37.71, 37.24, 35.45, 31.87, 29.89, 29.65, 29.57,
29.31, 23.93, 23.72, 22.64, 14.08; FAB-HRMS (NBA), calcd C37H75O3
[M + H]+ 567.5716, found 567.5730.
1H NMR Spin Simulation. Protons 1a, 1b, 4a, 4b, 2, 3a, and 3b of
compound 2 were described as a 7-spin system ABCDEXY. Spin
simulation of this spin system was carried out with VARIAN standard
spin simulation software.
Molecular Mechanics (MM) Calculations and Grid Search. MM
calculations were performed on a Silicon Graphics 4D310 computer
using the DREIDING force fields36 implemented in the BIOGRAF
(Molecular simulations Inc., Waltham, MA 02154) program. The default
parameters given in this program for the carbohydrate rings were used
without modification since they had been validated earlier.37 The MM
calculations were performed in vacuo. Calculated nOe distance
constraints were included as harmonic restraints with a force constant
of 10 000 kcal‚mol-1‚Å-2
.
Grid search was performed by using the sequential search mode in
the BIOGRAF program, with a 10° step growth search from 0° to 360°
for each defined dihedral angle.
Packing/Phase Behavior Characterization: Differential Scanning
Calorimetry (DSC). DSC measurements were made on a Microcal-2
Compound 5. A mixture of compound 4 (1.4 g, 2.5 mmol),
acetobromo-R-D-glucose (1.5 g, 3.6 mmol), mercury(II) oxide (0.22 g,
1 mmol), mercury(II) cyanide (1.3 g, 5 mmol), freshly distilled benzene
(36) Mayo, S. L.; Olafson, B. D.; Goddard, W. A. J. Phys. Chem. 1990,
94, 8897-8909.
(37) Wang, Y.; Hollingsworth, R. I. Biochemistry 1996, 35, 5647-5654.
(35) Kosak, A. I.; Swinehart, J. S. J. Org. Chem. 1960, 25, 222-225.

1860 J. Am. Chem. Soc., Vol. 121, No. 9, 1999
Song and Hollingsworth
Figure 9. 1H NMR spectra of compound 2 in d6-ethanol with different acidities: (A) spectra obtained in pure d6-ethanol with different temperatures;
(B) spectra obtained in 20% d4-acetic acid solution over time; and (C) spectra obtained in 0.01% DCl solution over time.
ultrasensitive scanning calorimeter with a scanning rate of 60 °C/h.
Approximately 12 mg of compound 2 was suspended in 1.5 mL of
water and 5 scans were recorded from 10 to 98 °C.
X-ray Powder Diffraction. The X-ray diffraction spectra were
recorded on a one-dimensional scanning detector with a Rigaku rotating
anode X-ray generator (Cu KR, 40 kV, 100 mA). Compound 2 (2.6

Synthesis and Study of a Versatile Self-Assembling Lipid
J. Am. Chem. Soc., Vol. 121, No. 9, 1999 1861
mg) in dichloromethane was deposited on a microscope slide and air-
dried. Its X-ray diffraction pattern was obtained in the reflection mode
(DS ) 1/6°, SS ) 1/6°, RS ) 0.3 mm, RSm ) 0.45 mm; scan rate )
0.4°/min; 2θ ) 0.5-45°). The gel form of compound 2 in water (10
mg/50 µL) was placed into a 0.7 mm glass capillary (Charles Super
Co.). The sealed capillary was heated at 70 °C for 2 h to ensure full
hydration prior to recording its diffraction pattern in the transmittance
mode (DS ) 0.05°, SS ) 1/6°, RS ) 0.3 mm, RSm ) 0.45 mm; scan
rate ) 0.3°/min; 2θ ) 0.2-45°). Both diffraction patterns were recorded
at room temperature.
Measuring Association by Light Scattering. Compound 2 (12.73
mg) was uniformly suspended into 2 mL of well-stirred distilled water.
A 200 µL sample of this solution (8.74 mM) was transferred into one
well of an ELISA plate, then 100 µL was removed and placed into the
adjacent well containing 100 µL of water. The process of serial 50%
dilution was continued to give a total of 60 wells. The ELISA plate
was equilibrated for 4 h at room temperature before light scattering
measurements were performed by taking absorbance readings on a Bio-
Tek Instruments EL-307 ELISA plate reader with a long-wavelength
filter.
by using a Zeiss LSM210 laser scanning microscope equipped with a
488 nm Ar laser. 40× air objective lens were used. Compound 2
(predissolved in dichloromethane) was deposited on a carefully cleaned
microscope slide in a thin film and dried in a vacuum oven. It was
then slowly hydrated in a closed chamber of water at 60 °C for days.
The hydrated sample was then covered by a cover slip and kept under
100% humidity till the microscope observations.
Acid Susceptibility. d6-Ethanol, d4-acetic acid, and d-hydrochloric
acid (37% in D2O) were used as solvent and acids. All spectra were
recorded on a 600 MHz VXR spectrometer. Compound 2 was dissolved
in various acidic solutions right before NMR experiments and reaction
time was recorded since then. Compound 2 in pure ethanol was
monitored at 25, 37, 45, and 55 °C. Compound 2 in 1%, 10%, and
20% d4-acetic acid-d6-ethanol solutions was monitored at 37 °C.
Compound 2 in 0.01% and 1% DCl-d6-ethanol solutions was
monitored at 37 °C.
Acknowledgment. This work was supported by the Michi-
gan State University Research Excellence Fund.
Laser Scanning Microscopy. Polarized-light microscopy, phase
contrast microscopy, and confocal reflection images were visualized
JA983883R