Synthesis and thermotropic phase behavior of four glycoglycerolipids

Article abstract of DOI:10.3390/molecules181113546

Four glycoglycerolipids with different head groups have been synthesized and their physicochemical properties studied. The lengths of the head groups from a mono-saccharide to a trisaccharide, in addition to the anomeric stereochemistry for the smaller glycoglycerolipids, have been modified. The synthesis has been optimized to avoid glycerol epimerization and to allow up-scaling. The physicochemical properties of the glycoglycerolipids were studied and a strong de-mixing of the gel-phase, depending on the head-group, was observed.

Full text of DOI:10.3390/molecules181113546

Molecules 2013, 18, 13546-13573; doi:10.3390/molecules181113546
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Thermotropic Phase Behavior of
Four Glycoglycerolipids
Wouter F. J. Hogendorf 1, Vivien Jagalski 1,†, Thomas G. Pomorski 2, Mikael Bols 1,
Marité Cárdenas 1,* and Christian M. Pedersen 1,*
1
Department of Chemistry, University of Copenhagen, Universitetsparken 5,
DK-2100 Copenhagen Ø, Denmark
2
Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40,
Frederiksberg C 1871, Denmark
†
Physicochemical characterizations were performed by this author.
* Author to whom correspondence should be addressed; E-Mail: cmp@chem.ku.dk;
Tel.: +45-3532-0190.
Received: 16 September 2013; in revised form: 15 October 2013 / Accepted: 19 October 2013 /
Published: 1 November 2013
Abstract: Four glycoglycerolipids with different head groups have been synthesized and
their physicochemical properties studied. The lengths of the head groups from a
mono-saccharide to a trisaccharide, in addition to the anomeric stereochemistry for the
smaller glycoglycerolipids, have been modified. The synthesis has been optimized to avoid
glycerol epimerization and to allow up-scaling. The physicochemical properties of the
glycoglycerolipids were studied and a strong de-mixing of the gel-phase, depending on the
head-group, was observed.
Keywords:
glycolipid
synthesis;
calorimetrics;
biomimetic
membranes;
physiological conditions
1. Introduction
Glycolipids play a central role in plants, microorganism and animals. Glycolipids consist of a
carbohydrate head group linked to a lipid, typically a diacylglycerol (glycoglycerolipid), ceramide
(glycosphingolipid) or prenol. Any given organism can vary both the lipid part and the head group of

Molecules 2013, 18
13547
the glycolipid in order to meet changes in the environmental and physiological conditions [1,2] For
example, Acholeplasma laidlawii strain A can incorporate supplied fatty acids of a fixed composition
when grown under conditions at which the cell’s endogenous fatty acid synthesis were impaired. This
organism responds by changing the composition of the polar part (the carbohydrate) of its
glycoglycerolipids in order to balance the cell membrane composition of lamellar- and non-lamellar
forming glycolipids. The balance between lamellar and non-lamellar forming glycoglycerolipids is
critical for the growth of bacterial cells [3,4]. It has been found that, in the case for A. laidlawii,
glycolipids with one carbohydrate moiety or one acyl group (i.e. monoglucosyl-diacylglycerol,
monoacylmonoglucosyldiacylglycerol and monoacyl-diglucosylglycerol) form non-lamellar structures
of the inverse type, whereas diglucosyldiacylglycerol was found to be lamellar forming [5].
Non-lamellarity is favored upon increased lipid acyl chain length or saturation. The non-lamellar
forming glycoglycerolipids are thought to be important for the formation of non-bilayer structures and
bilayers with small radius of curvature. Moreover, glycolipids have typically very high melting
temperatures (the temperature at where the main phase transition from the gel to the fluid liquid
crystalline phase occurs) as compared to other lipids due to intra hydrogen bonding among the sugar
groups [6–8].
In eukaryotes, glycolipids comprise a small but vital fraction (10%–20%) of the membrane lipids [9]
that serve important biological functions in signal transduction, as anchors for cell surface proteins in
cascade pathways in immune response and cell communication, among others [10]. Most of the
research in the glycolipid field focuses on the direct impact on signaling pathways or immunological
reactions [11]. However, the underlying molecular mechanisms of the effects caused by glycolipids on
the physicochemical properties of cellular membranes has been poorly studied so far. In part, this is
due to the difficulty of obtaining chemically well-defined purified glycolipid fractions in large
quantities, as natural isolates are heterogeneous and difficult to purify.
As an alternative approach to lipid extraction from cellular membranes, chemical synthesis of
glycolipids is an active field of research. Special focus has been given to the glycolipids found in
pathogens as these are often found to be immunogenic. Currently, lipopolysaccharides (LPSs) are
under investigation due to their great potential as vaccine components (they are also known as the
endotoxin of Gram-negative bacteria) [12]. The Gram positive counterpart—lipoteichoic acids (LTAs)
have so far received less attention and their biological roles remain mostly unclear and have been
heavily discussed for decades. One of the major problems regarding the determination of LTAs’
biological activity was found to be inappropriate purification methods leading to partly decomposed
glycolipids, or samples containing immunogenic lipoproteins. The biological role of LTA was recently
confirmed by using synthetic LTA [13,14] from Streptococcus pneumonia [15] and Staphylococcus
aureus [16,17]. Because well-defined synthetic glycolipids are required to achieve reliable biological
results, a demand for more structures and derivatives has arisen. We are currently working on the total
synthesis of LTA from Clostridium difficile. As a part of the synthesis of the glycoglycerollipid anchor
part, the general procedure for the synthesis of gentiobiosyl diacylglycerol has been optimized for
purity and up-scaling. These improvements will be discussed in the following paragraphs.
The similarity between the lipid anchor and some of the simple glycoglycerolipids found in plants
and microorganisms prompted us to investigate the effect of these smaller glycoglycerolipids, formed
by the lysis of LTA, on the physicochemical properties of model cell membranes. As a model

Molecules 2013, 18
13548
membranes system we choose a well-described combination of two glycerophospholipids:
1,2-di-myristoyl-phosphatidylcholine (DMPC) and 1,2-di-myristoyl-phosphatidylserine (DMPS) at a
biologically relevant proportion (cellular membranes usually carry a net negative charge that ranges
from 5 to 25 mol%) [9]. In order to directly study the influence of the head group on the thermotropic
properties of the lipid membrane, model compounds were synthesized with 1,2-di-myristoyl-sn-
glycerol as the lipophilic part. Thus, the contribution of the acyl chain is kept constant among the
various lipid species studied in such a way that only the carbohydrate head contribution is
systematically varied. Four head groups were chosen: α- and β-glucopyranoside (found in various
organisms [18], β-gentiobiose (found in lipid anchor from S. aureus) and 6-O-β-D-glucopyranosyl
gentiobiose (gentiotriose; corresponding to the lipid anchor from C. difficile). Vesicles were prepared
with a fixed molar composition of DMPC, DMPS and each of the four glycoglycerolipids (79, 8 and
13 mol%, respectively) and studied by a combination of differential scanning calorimetry (DSC) and
confocal scanning fluorescence microscopy (CSFM).
2. Results and Discussion
2.1. Synthesis of the Glycoglycerolipids
Despite a large number of diacylglycerol-containing glycolipids that have been synthesized through
the years, the chemistry is still not straight forward. Glycosylation of a ketal (or acetal) protected
glycerol remains an obstacle for a fast and high yielding synthesis of the common glycolipids.
Methods [19,20] have been developed, where more persistent protective groups on the glycerol
acceptor or other modifications are used, but due to their easy access and orthogonality to other
common protective groups, the ketals are still preferred. The main problem by using ketals (or acetals)
is the acid catalyzed (Lewis or Brønsted) epimerization of the glycerol part that takes place under
practically all glycosylation methods and causes difficulties in the purification [21–24]. The problem
with epimerization seems less pronounced when having neighboring group participation [25] or
ortho-esters. However, to our surprise it turned out to be a major problem when using the gentiobiose
donor 2 (Scheme 1) which is highly attractive since it can be prepared from commercially available
gentiobiose peracetate 1 in 2 simple steps [26]. The initial glycosylation results gave up to 25% of the
glycerol epimer 4, which is seen as additional signals in the NMR spectra as a doubling of the signals
1
13
in the H-NMR at 4.49–4.54 ppm and in the C-NMR two additional peaks at 34.8 and 36.4 ppm.
(see Supplementary materials). The epimer 4 could not be chromatographically separated from the
desired epimer 3 (Scheme 1). To improve the glycosylation the conditions were optimized.
Since both the donor 2 and the enantiomerically pure cyclohexylidene glycerol are readily available
building blocks, it was decided to focus the optimization on the promoter system and the purification
methods. Initial studies, using TMSOTf as the promoter in CH2Cl2, revealed that the yields were
improved by lowering the temperature from 0 to −40 °C and the epimer formation was limited as well
(from 25% to 10%). Lowering the temperature further (−60 °C) did neither improve the overall yield
and the anomeric selectivity further (Table 1). Besides the anomeric selectivity, which to some extent
is controlled by neighboring group participation and the undesired glycerol epimerization, ortho-ester
formation was observed as a major side reaction. With up to at least eight different glycosylation products

Molecules 2013, 18
13549
formed—all with similar retention times, TMSOTf was abandoned as the promoter system. Changing the
promoter to BF3⋅OEt2, which should decrease the amount of acid catalyzed epimerization [27], resulted in
similar yields and amounts of the undesired α-anomer. The epimerization was however less dominant
and the formation of orthoesters likewise. The optimal condition was found to be −35 °C with a slow
heating to 0 °C, where the reaction was quenched with triethylamine. With the lower amount of side
products formed it was possible to selectively crystallize the product out from the crude reaction
mixture, using a mixture of ethyl acetate and petroleum ether, in a very satisfying 70% yield (Table 1).
Scheme 1. Synthesis of the gentiobiosyl donor 2 and its glycosylation with
1,2-O-cyclohexylidene glycerol.
Table 1. Conditions for the gentiobiosylation of 1,2-O-isopropylidene glycerol.
T (°C)
0
Promotor
TMSOTf
TMSOTf
TMSOTf
BF3⋅OEt2
BF3⋅OEt2
BF3⋅OEt2
Yield (%) a
α-x (%) b
Epimer (%) c
63
75
5
25
10
−40→0
−60→0
−60→0
−20→−10
−35→0
5
84
5–10
5–10
5
10–15
5
84
76
70 d
10
0
0
Mixture of glycosylation products isolated by column chromatography; Estimated (from H/13C-NMR)
amount of the α-anomer; Estimated amount (1H/13C-NMR) of glycerol epimerization; Pure product 3
a
b
1
c
d
crystallized from the crude reaction mixture.
The optimized procedure paved the way for unlimited up-scaling (so far 3 can be obtained in
multiple grams). With the access to 3 secured, the acetyl groups were removed under Zemplén
conditions, and a 6'OTBDPS group selectively installed in 85% yield. As persistent protective groups
benzyls were preferred in order to achieve a one-step global deprotection at the end of the synthesis.
Removal of the cyclohexylidene protective group on 5 gave diol 6 (Scheme 2).
Scheme 2. Synthesis of gentiobiosyl building block 7.

Molecules 2013, 18
13550
Myristoylation followed by TBAF-mediated TBDPS removal gave 7 (Scheme 2). The
hydrogenolytic debenzylation of 7 was performed in a dioxane/water/AcOH mixture using palladium
black as the catalyst. The final glycolipid 15 could be purified by reverse phase silica gel column
chromatography (Scheme 3). Removal of the TBDPS group in 5 afforded the pseudotrisaccharide
acceptor 8 ready for the chain prolongation by glycosylation or, for example, introduction of a glycerol
phosphate oligomer (S. aureus LTA) (Scheme 4).
Scheme 3. Global deprotection to give the glycolipids 15 (β-diglycolipid) and 16
(β-triglycolipid).
Scheme 4. Synthesis of pseudotetrasaccharide 10.
As with the pseudotrisaccharide acceptor 8 benzyl groups were preferred as persistent protective
groups in the donor 9 [28] (Scheme 4). A 6-O-TBDPS was again installed in order to allow future
ligation to the repeating unit via a phosphate linkage for the C. difficile LTA synthesis. Since the
benzyl groups in 9 do not participate under glycosylation conditions, the anomeric selectivity relied on
the nitrile effect [29]. Therefore, the glycosylation of acceptor 8 with donor 9 was initially explored
using MeCN as the solvent, but no selectivity was observed. Changing to propionitrile, which has a lower
melting point, improved the selectivity to 1:3.5 (α:β). An attempted PhSiF3-catalyzed glycosylation [30]
proved unsuccessful as no reaction occurred (Scheme 4). Due to the difficulties in separating the anomers,
a more selective glycosylation was needed. To ensure high selectivity the benzyl protective groups were
exchanged with benzoyl moieties, which are known to be more robust than acetyl groups under (Lewis)
acidic conditions [31] Donor 12 was synthesized from per-O-acetyl glucose 11 in 6 steps with an overall
yield of 65% (see Scheme 5). The glycosylation of gentiobiosyl acceptor 8 could then be performed
uneventfully in CH2Cl2 giving 75% of the anomerically pure pseudotetrasaccharide 13 (Scheme 5).

Molecules 2013, 18
Scheme 5. Synthesis of donor 12 [32] and its glycosylation with pseudotrisaccharide 8.
13551
Debenzoylation was performed using 1-propanol in combination with potassium tert-butoxide in
order to better dissolve the reactant and to simplify the purification by chromatography. Benzylation
and deprotection of the cyclohexylidene followed by introduction of the myristoyl moieties to give the
fully protected glycolipid in an overall good yield and excellent purity. Desilylation liberated the
6″O—the linkage point for the repeating unit, to give 14. To obtain the fully deprotected lipid anchor,
the benzyl groups were hydrogenolyzed using palladium black in a THF/water/AcOH mixture
(Scheme 3). Lipid anchor 16 was purified by reverse phase column chromatography to give the pure
product in 81% yield.
The 1,2-di-O-myristoyl-3-O-α-D-glucopyranosyl-sn-glycerol 17 (Figure 1) was synthesized
following the procedure developed for the synthesis of the lipid anchor from S. pneumonia [33] and
1,2-di-O-myristoyl-3-O-β-D-glucopyranosyl-sn-glycerol 18 (Figure 1) was synthesized from the
peracetylated glucopyranosyl trichloroacetimidate donor following the procedure describe above for 3 [18].
Both glycoglycerolipids are also found in bacteria [34,35].
Figure 1. Glycolipids 17 (α-monoglycolipid) and 18 (β-monoglycolipid).
2.2. Thermotropic Behavior of Glycoglycerolipid and Glycerophospholipids
Small unilamellar vesicles were prepared at various compositions (Table 2) and their thermotropic
behavior was studied by differential scanning calorimetry (DSC), see Figure 2. A brief description of
the method of preparation can be found in the supporting information. DSC measurements were performed
in duplicates and ran through two cycles for up scans (10→70 °C) and down scans (70→10 °C) to
evaluate whether there was any effect on the mixing behavior of the lipids triggered by the preparation
method. The two up/down cycle scans showed no hysteresis effects, suggesting that the method of
preparation did not have a significant effect on their thermotropic behavior.

Molecules 2013, 18
Table 2. Composition of the lipid vesicle samples used for DSC studies in this work.
13552
Sample/mol%
DMPC
92
DMPS
Glycoglycerolipid
Glycerophospholipids
8
0
8
8
8
8
0
100
13
13
13
13
18
18
17
15
16
0
79
79
79
79
For glycerophospholipids and for the pure β-monoglycolipid vesicles, a single endothermic peak
occurs that corresponds to the first order gel-to-liquid disordered phase transition. The DSC
thermograms for pure DMPC, DMPS and β-monoglycolipid vesicles (Supporting Information Figure 1)
are in excellent agreement with previous literature [7,36,37]. Interestingly, the melting temperature
for 1,2-di-O-tetradecyl-3-O-methyl-β-D-glucopyranosyl-sn-glycerol, a close analogue to β-mono-
glycolipid 18, is dramatically higher (Tm = 60 °C) than for our β-monoglycolipid (Tm = 45 °C) [38]
suggesting that the distance from the sugar unit to the lipid bilayer is a key parameter for the induction
of high Tm.
Lipid mixtures can behave ideally or can have preferential interactions. For ideal mixtures, the ideal
melting temperature, Tm,ideal, can be calculated from weighted average of each lipid Tm in the mixture.
The mixing of DMPC and DMPS is ideal since Tm,experimental = 24.5 ± 0.2 °C is comparable to
Tm,ideal = 24.3 °C. Ideal mixing for DMPC and DMPS has been previously proposed in the literature [39].
A different scenario is found for the glycerophospholipids-glycoglycerolipid mixtures, for which ideal
mixing was only observed when the β-monoglycolipid 18 was used (Tm,ideal = 27 °C while
Tm,experimental = 26.5 ± 0.2 °C). The shape of the peak, though, became more asymmetric suggesting that
the phase transition was less cooperative than for DMPC-DMPS mixtures.
For the α-monoglycolipid 17, on the other hand, a dramatic non-ideal behavior was observed.
In this case, three endothermic peaks are observed suggesting three major phase transitions. The same
qualitative thermotropic behavior occurs for mixtures with β-di- or β-tri-glycolipids (15 and 16).
DSC thermograms of structurally similar glycolipids gave a single endothermic transition at around
55–60 °C [6]. Therefore, the phase transition occurring about 57 °C should correspond to a glycolipid
rich domain. The high Tm for glycolipids is typically ascribed to strong intermolecular interactions
between their headgroups [6–8.]
As mentioned above, the appearance of three endothermic peaks strongly indicates demixing in the
lipid bilayers for all glycolipid used except for β-monoglycerolipid 18. For α-monoglycolipid 17, the
first, second and third phase transition have a corresponding Tm,1st = 28, Tm,2nd = 44 and Tm,3rd = 57 °C
respectively. The main difference in their DSC thermograms for β-di- or β-tri-glycolipid containing
mixtures is a shift of the first peak of lower Tm,1st (24.5 ± 0.2 °C). For ideal glycolipid containing
mixtures at the composition shown in Table 1, the Tm,ideal should be around 28.4 °C assuming the pure
glycolipids to have a Tm,glycolipid = 58 °C.

Molecules 2013, 18
13553
Figure 2. DSC thermograms for (1) DMPS/DMPC, (2) DMPS/DMPC/β-monoglycolipid 18,
(3) DMPS/DMPC/α-monoglycolipid 17, (4) DMPS/DMPC/β-diglycolipid 15,
(5) DMPS/DMPC/β-triglycolipid 16. Vesicles at the compositions given in Table 1.
These are up-scans representative for duplicates of the same sample. Each sample was run on two up/down
scan cycles and no major differences were observed on the thermograms.

Molecules 2013, 18
13554
There are several explanations to this strong demixing behavior. On one hand, there could be a split
population of vesicles with very distinct lipid compositions. This is supported by the fact that the Tm,1st peak
is so close to the Tm,ideal (28.4 °C) implying that there might exist a population of vesicles with an ideal
behavior at least for α-monoglycolipid. The Tm for the second peak (Tm,2nd = 44 °C) and third peak
(Tm,3rd = 57 °C) could potentially correspond to vesicles enriched with DMPS (Tm,DMPS = 39.5 °C) and
glycolipids. Vesicles with distinct composition at the single molecule level have been detected
earlier [40–42].
In an alternative scenario, each vesicle could contain several domains of distinct composition. In
this case, the first peak should correspond to the main phase transition for a DMPC-enriched region.
This is supported by the fact that for mixtures containing β-di- or β-tri-glycolipids (15 and 16), the
Tm,1st shifts to lower values that are closer to the Tm,ideal (24.3 °C) assuming a domain composed only
by DMPC and DMPS. Furthermore, domain formation is strongly supported by the highly asymmetric
shape of the two peaks with highest Tm that typically imply poor cooperativity during the phase transition.
In our glycolipid containing mixtures, the strong hydrogen network formed by the glycolipids could
induce domain formation that, upon increasing temperature, could undergo several transformations
from a glycolipid rich gel phase into a smectic type phase (liquid ordered phase) and then finally into a
liquid-disordered phase. Thus, the three phase transition observed for our mixtures with glycolipids
(except for β-monoglycolipid 18) may correspond to the gel to liquid disordered melting of the DMPC
rich phase (Tm,1s), the gel to liquid ordered melting of the glycolipid rich region (Tm,2nd), and finally the
melting of this last domain into the liquid disordered phase (Tm,3rd). In this respect, glucose based
surfactants can lead to more than one melting point in a bilayer caused by the hydrogen bonding ability
of glucose head groups [43,44], and sub gel phases have also been observed for pure glycolipid
vesicles in the past [7].
Glycolipids could potentially form complexes with lipids of smaller headgroup for which the large
sugar head shadows smaller headgroups of these lipids leading to hydrophobic shielding in a
mechanism similar to so-called “umbrella effect” [45]. The shift of Tm,1st to values closer to the pure
Tm DMPC for the β-di and β-triglycolipid containing mixtures suggest there is a preferential
complexation with DMPG for these cases. Indeed, phosphatidylserine (PS) lipids promote the
incorporation of glycolipids in lipid bilayers while phosphatidylcholine (PC) inhibited their
incorporation in fibroblasts [46].
Confocal scanning fluorescence microscopy was used to image our mixed vesicles containing
β-diglycolipid 15, see Figure 3. In this case, giant unilamellar vesicles were prepared since they allow
for visualization of micron sized long-lived domains using two different lipid dyes that partition
preferentially in fluid (DiIC12) or gel (DiDC18) domains [47].
Details about the vesicle preparation are found in the experimental section and the chemical
structures of these dyes are given in the Supporting Information. The vesicles are tethered to a surface
to allow for imaging during prolonged times. However, the strength of binding to the surface was
weakened upon increasing the temperature above 37 °C and thus we could only image below and
above the peak for the first phase transition (that should correspond to the DMPC rich domain). The
images show an uneven distribution of the dyes with regions enriched with the red dye (DiDC18),
which grow in size with temperature: the uneven dye distribution indicates that domains of different
fluidity exist at both temperatures. A vesicle composed mainly of DMPC-DMPS and displaying a

Molecules 2013, 18
13555
single-phase transition should contain an uneven dye distribution at 22 °C and even dye distribution 37 °C,
since at the onset of first phase transition occurs around 21 °C and is completed at 37 °C. Thus, these
images clearly show that, at 37 °C, domains of distinct lipid composition and fluidity exist in a single
vesicle. These domains should be either enriched in (i) glycerophospholipids (green areas)—and be
probably in the liquid disordered phase—and (ii) enriched with glycoglycerolipids (red areas)—and
probably in the gel phase.
Figure 3. Confocal scanning fluorescence microscopy images for giant unilamellar
vesicles containing β-diglycolipid 15 at 22 °C (A) and 37 °C (B). Vesicles composed of
DMPC/DMPS/5 labeled with 0.1% DiI C18 (green) and 0.1% DiIC18 (red) were prepared
as described in the supporting information. Color merged images were shown.
A"
B"
Finally, the thermotropic behavior of vesicles containing β-monoglycolipid differed significantly
from the other glycolipids studied. In this case, almost ideal mixing was observed. The main structural
difference is the configuration of the sugar group, which appears in a linear configuration or pointing
straight away from the lipid bilayer occupying a smaller surface area. This conformation might impair
hydrogen bonding with other glycolipids or lipids of smaller head-group (for instance DMPS) giving
an ideal mixture in this case. This is supported by the significantly higher Tm found for the close
analogue 1,2-Di-O-tetradecyl-3-O-methyl-β-D-glucopyranosul-sn-glycerol, for which only an extra
methyl group allows for higher mobility of the headgroup. Earlier, it was suggested that differences in
Tm of glycolipids could arise from the specific conformation of the sugar head-group and their
capability to form hydrogen bonding with other lipids [48].
3. Experimental
3.1. Synthesis
All chemicals (Merck, Carbosynth, Sigma-Aldrich) were used as received and reactions were carried
out dry, under an argon atmosphere, at ambient temperature, unless stated otherwise. Dry solvents were
taken from a solvent purification system. Glassware used for water-free reactions were dried for 12 h

Molecules 2013, 18
13556
at 120 °C before use. Column chromatography was performed on ROCC silica gel 60 (0.040–0.063 mm).
TLC analysis was conducted on HPTLC aluminium sheets (Merck, silica gel 60, F245). Compounds were
visualized by UV absorption (245 nm), by spraying with 20% H2SO4 in ethanol or with a solution of
(NH4)6Mo7O24•4H2O 25 g/L and (NH4)4Ce(SO4)4•2H2O 10 g/L, in 10% aqueous H2SO4 followed by
20
charring at +/− 150 °C. Optical rotation measurements ([α]D ) were performed on a Perkin Elmer 341
polarimeter (Sodium D-line, λ = 589 nm) with a concentration of 10 mg/ml (c = 1), unless stated otherwise.
13
1H- and C-NMR spectra were recorded with a Bruker 500 MHz spectrometer (500 and 125 MHz
respectively). Chemical shifts (δ) are reported in ppm relative to the residual solvent signal of either
1
13
1
CDCl3 (δ = 7.26 for H-NMR and 77.0 for C-NMR) or CD3OD (δ = 3.31 for H-NMR and 49.0 for
13C-NMR). NMR assignments were based on COSY and HSQC NMR experiments. High-resolution
mass spectral (HRMS) data were obtained on an electrospray (ESI) mass spectrometer analyzing
time-of-flight (Q-TOF instrument from Micromass).
Gentiobiosyl Derivative 3
To
a
solution of gentiobiosyl trichloroacetimidate
2
(7.04 g, 9.01 mmol) and
1,2-O-cyclohexylidene-sn-glycerol (3.11 g, 18.0 mmol) in CH2Cl2 (75 mL) was added a batch of
freshly activated powdered MS3Å and the resulting suspension stirred for 15 min. The mixture was cooled
to −35 °C and a catalytic amount of boron trifluoride diethyl etherate (55.6 µL, 64 mg, 0.45 mmol) was
added. The reaction was allowed to stir for 90 min while slowly warming up to 0 °C. Triethylamine
(0.5 mL) was added and the slurry filtered over Celite. The filtrate was concentrated under reduced
pressure and the residue crystallized from a mixture of EtOAc (150) and PE (500 mL), giving
20
pseudotrisaccharide 3 (4.97 g, 6.29 mmol, 70%) as a white solid. Mp.: 190 °C; [α]D (CHCl3): −15.8;
1H-NMR: δ = 1.33–1.43 (m, 2H, CH2 cyclohexylidene), 1.53–1.63 (m, 8H, 4 × CH2 cyclohexylidene),
1.98–2.10 (7 × s, 21H, 7 × CH3 acetyl), 3.59–3.71 (m, 4H, H-5, H-5', 2 × H-6), 3.78 (dd, 1H, J = 5.9 Hz,
8.3 Hz, CHH glycerol), 3.83–3.88 (m, 2H, CH2 glycerol), 3.99 (dd, 1H, J = 6.4 Hz, 8.2 Hz, CHH
glycerol), 4.12 (dd, 1H, J = 2.3 Hz, 12.3 Hz, H-6'), 4.21–4.24 (m, 1H, CH glycerol), 4.27 (dd, 1H,
J = 4.8 Hz, 12.4 Hz, H-6'), 4.57–4.61 (2 × d, 2H, J = 8.0 Hz, J = 8.0 Hz, H-1, H-1'), 4.87–5.01 (m, 3H,
H-2, H-2', H-4), 5.07 (t, 1H, J = 9.7 Hz, H-4'), 5.15–5.21 (m, 2H, H-3, H-3'); 13C-NMR: δ = 20.6–20.7
(7 × CH3 acetyl), 23.8, 24.0, 25.1, 34.6, 36.2 (5 × CH2 cyclohexylidene), 61.8 (C-6'), 65.8 (CH2
glycerol), 68.1 (CH2 glycerol), 68.3 (C-4'), 68.9 (C-6), 69.1 (C-4), 71.1, 71.2 (C-2, C-2'), 72.0 (C-5'),
72.8 (C-3, C-3'), 73.3 (C-5), 73.9 (CH glycerol), 100.7, 100.8 (C-1, C-1'), 109.9 (Cq cyclohexylidene),
169.2, 169.3, 169.4, 169.6, 170.2, 170.2, 170.6 (7 × Cq acetyl); HRMS: C35H50O20 + Na+ requires
813.2788, found 813.2785.

Molecules 2013, 18
13557
Conversion of 3 to 5
Intermediate A
To a solution of compound 3 (5.31 g, 6.72 mmol) in MeOH (67 mL) was added sodium methoxide
(25% solution in MeOH, 0.50 mL) after which the mixture was stirred for 1 h. The reaction was
quenched with amberlite H+ and filtered. The filtrate was concentrated in vacuo giving intermediate A
20
1
(3.33 g, 6.71 mmol, 100%) as an amorphous off-white solid. [α]D (MeOH): −24.1; H-NMR
(CD3OD): δ = 1.37–1.46 (m, 2H, CH2 cyclohexylidene), 1.56–1.68 (m, 8H, 4 × CH2 cyclohexylidene),
3.18–3.25 (m, 2H, H-2, H-2'), 3.28–3.39 (m, 5H, H-3, H-3', H-4, H-4', H-5'), 3.45–3.49 (m, 1H, H-5),
3.62 (dd, 1H, J = 6.0 Hz, 10.6 Hz, CHH glycerol), 3.68 (dd, 1H, J = 5.3 Hz, 11.9 Hz, H-6'), 3.77–3.84
(m, 2H, H-6, CHH glycerol), 3.86–3.94 (m, 2H, H-6', CHH glycerol), 4.08 (dd, 1H, J = 6.4 Hz, 8.4 Hz,
CHH glycerol), 4.16 (dd, 1H, J = 2.0 Hz, 11.5 Hz, H-6), 4.32 (d, 1H, J = 7.8 Hz, H-1), 4.33–4.37
13
(m, 1H, CH glycerol), 4.38 (d, 1H, J = 7.8 Hz, H-1'); C-NMR (CD3OD): δ = 24.8, 25.0, 26.2, 35.9,
37.5 (5 × CH2 cyclohexylidene), 62.7 (C-6'), 67.4 (CH2 glycerol), 69.9 (C-6), 71.4, 71.6 (C-4, C-4'),
71.6 (CH2 glycerol), 75.0, 75.1 (C-2, C-2'), 75.5 (CH glycerol), 77.0 (C-5'), 77.8 (C-5), 78.0 (C-3.
C-3'), 104.7, 104.9 (C-1, C-1'), 111.1 (Cq cyclohexylidene); HRMS: C21H36O13 + Na+ requires
519.2048, found 519.2053.
Intermediate B
To a solution of intermediate A (3.15 g, 6.34 mmol) and imidazole (1.73 g, 25.4 mmol)
in DMF (49 mL) was added TBDPS-Cl (2.44 mL, 2.62 g, 9.52 mmol) after which the mixture was
allowed to stir for 1 h. At this point a second batch of TBDPS-Cl (0.33 mL, 0.35g, 1.2 mmol) was
added and the reaction stirred for 1h before it was quenched with MeOH (5 mL). The mixture was
diluted with EtOAc (300 mL) and washed with water (150 mL). The aqueous layer was extracted once
with EtOAc (50 mL) and the combined organic layers washed with water (250 mL) and brine (250 mL).
The organic layer was dried with Na2SO4, filtered, and concentrated under reduced pressure.
Purification of the residue by column chromatography (acetone/DCM/MeOH, 1/1/0 → 2/2/1) afforded
20
mono-silylated intermediate B (3.95 g, 5.37 mmol, 85%) as a colourless oil. [α]D (MeOH): −21.9;
1H-NMR (CD3OD): δ = 1.06 (s, 9H, tBu), 1.37–1.43 (m, 2H, CH2 cyclohexylidene), 1.55–1.62 (m, 8H,
4 × CH2 cyclohexylidene), 3.22–3.28 (m, 2H, H-2, H-2'), 3.35–3.45 (m, 5H, H-3, H-3', H-4, H-4', H-5'),
3.47–3.51 (m, 1H, H-5), 3.61 (dd, 1H, J = 6.0 Hz, 10.5 Hz, CHH glycerol), 3.76–3.82 (m, 2H, H-6,
CHH glycerol), 3.89 (dd, 1H, J = 5.1 Hz, 11.1 Hz, H-6'), 3.93 (dd, 1H, J = 5.5 Hz, 10.5 Hz, CHH

Molecules 2013, 18
13558
glycerol), 4.01–4.07 (m, 2H, H-6', CHH glycerol), 4.21 (dd, 1H, J = 2.0 Hz, 11.4 Hz, H-6), 4.30–4.35
(m, 2H, H-1, CH glycerol), 4.41 (d, 1H, J = 7.8 Hz, H-1'), 7.39–7.44 (m, 6H, Harom), 7.74–7.77 (m, 4H,
Harom); 13C-NMR (CD3OD): δ = 20.2 (Cq tBu), 24.8, 25.0, 26.2 (3 × CH2 cyclohexylidene), 27.4
(3 × CH3 tBu), 35.9, 37.5 (2 × CH2 cyclohexylidene), 64.7 (C-6'), 67.4 (CH2 glycerol), 69.4 (C-6),
71.2, 71.4 (C-4, C-4'), 71.7 (CH2 glycerol), 75.0, 75.1 (C-2, C-2'), 75.5 (CH glycerol), 77.0 (C-5), 77.8
(C-5'), 78.2, 78.2 (C-3. C-3'), 104.7, 104.7 (C-1, C-1'), 111.0 (Cq cyclohexylidene), 128.8, 130.7, 130.8
(CHarom), 134.7, 134.9 (2 × Cq Ph), 136.7, 136.8, 136.9 (CHarom); HRMS: C37H54O13Si + Na+ requires
757.3226, found 757.3224.
Gentiobiosyl Derivative 5
To a cooled (0 °C) solution of intermediate B (3.88 g, 5.28 mmol) in DMF (35 mL) were added
benzyl bromide (5.65 mL, 8.13 g, 47.5 mmol) and sodium hydride (40% dispersion in mineral oil, 1.90 g,
47.5 mmol). After stirring for 1 h at rt the mixture was heated to 40 °C and allowed to stir at this
temperature for 75 min before the reaction was quenched with MeOH (5 mL). The reaction was diluted
with Et2O (150 mL) and washed with water (2 × 75 mL) and brine (75 mL). The organic layer was
dried (Na2SO4), filtered and the volatiles removed under reduced pressure. Purification of the residue by
column chromatography (Et2O/PE, 0/1 → 1/3) gave hexabenzylated derivative 5 (5.33 g, 4.18 mmol, 79%)
20
1
as a pale yellow oil. [α]D (CHCl3): +10.4; H-NMR: δ = 1.05 (s, 9H, t-Bu), 1.33–1.40 (m, 2H, CH2
cyclohexylidene), 1.51–1.58 (m, 8H, 4 × CH2 cyclohexylidene), 3.31 (dt, J = 2.8 Hz, 9.7 Hz, H-5'),
3.41–3.52 (m, 4H, H-2, H-2', H-4, CHH glycerol), 3.56–3.60 (m, 1H, H-5), 3.61–3.71
(m, 4H, H-3, H-3', H-6, CHH glycerol), 3.76 (t, 1H, J = 9.4 Hz, H-4'), 3.89–3.95 (m, 4H, 2 × H-6',
2 × CHH glycerol), 4.15–4.19 (m, 1H, CH glycerol), 4.23 (dd, 1H, J = 1.8 Hz, 11.4 Hz, H-6), 4.39
(d, 1H, J = 7.8 Hz, H-1), 4.47 (d, 1H, J = 7.8 Hz, H-1'), 4.54 (d, 1H, J = 11.2 Hz, CHH Bn), 4.68–4.83
(m, 6H, 3 × CH2 Bn), 4.88–4.95 (m, 4H, 2 × CH2 Bn), 5.01 (d, 1H, J = 11.2 Hz, CHH Bn), 7.16–7.42
13
(m, 36H, Harom), 7.69–7.72 (m, 2H, Harom), 7.76–7.78 (m, 2H, Harom); C-NMR: δ = 19.3 (Cq tBu),
23.8, 24.0, 25.1 (3 × CH2 cyclohexylidene), 26.8 (3 × CH3 tBu), 34.8, 36.4 (2 × CH2 cyclohexylidene),
62.7 (C-6'), 66.3 (CH2 glycerol), 68.2 (C-6), 70.3 (CH2 glycerol), 73.9 (CH glycerol), 74.8, 74.8, 74.9, 75.1
(4 × CH2 Bn), 75.2 (C-5), 75.6 (CH2 Bn), 75.7 (C-5’), 75.9 (CH2 Bn), 77.6 (C-4'), 78.1 (C-4), 82.1 (C-2),
82.4 (C-2'), 84.6, 84.9 (C-3, C-3'), 103.7 (C-1), 104.0 (C-1'), 109.9 (Cq cyclohexylidene), 127.4–128.4
(CHarom), 129.6, 129.6 (CHarom), 133.1, 133.6 (2 × Cq Ph), 135.6, 135.9 (CHarom), 138.1, 138.3, 138.4,
138.5, 138.6, 138.6 (6 × Cq Bn); HRMS: C79H90O13Si + Na+ requires 1297.6043, found 1297.6042.

Molecules 2013, 18
13559
Gentiobiosyl Derivative 6
A solution of gentiobiosyl derivative 5 (223 mg, 0.175 mmol) in a mixture of acetic acid (5.5 mL),
toluene (1.0 mL) and water (1.5 mL) was stirred for 3 h at 90 °C. After cooling down to rt the mixture
was diluted with EtOAc (50 mL) and washed with water (2 × 25 mL), sat. aq. NaHCO3 (2 × 25 mL)
and brine (25 mL). The organic layer was dried with Na2SO4, filtered and concentrated in vacuo, after
which the residue was purified by column chromatography (Et2O/PE, 1/3 → 1/0) giving diol 6 (161 mg,
20
1
0.135 mmol, 77%) as a colourless oil. [α]D (CHCl3): +10.3; H-NMR: δ = 1.05 (s, 9H, t-Bu), 3.33 (dt,
J = 2.7 Hz, 9.6 Hz, H-5’), 3.39–3.44 (m, 3H, H-2, H-4, CHH glycerol), 3.48–3.52 (m, 1H, H-2'), 3.55 (dd,
1H, J = 4.0 Hz, 11.4 Hz, CHH glycerol), 3.59–3.77 (m, 8H, H-3, H-3', H-4', H-5, H-6, CH glycerol,
2 × CHH glycerol), 3.91–3.94 (m, 2H, 2 × H-6'), 4.19–4.22 (m, 1H, H-6), 4.38 (d, 1H, J = 7.8 Hz,
H-1), 4.47 (d, 1H, J = 7.8 Hz, H-1'), 4.53 (d, 1H, J = 11.2 Hz, CHH Bn), 4.67 (d, 1H, J = 10.8 Hz,
CHH Bn), 4.71–4.93 (m, 9H, 4 × CH2 Bn, CHH Bn), 4.97 (d, 1H, J = 11.0 Hz, CHH Bn) 7.16–7.40
13
(m, 36H, Harom), 7.68–7.70 (m, 2H, Harom), 7.73–7.76 (m, 2H, Harom); C-NMR): δ = 19.3 (Cq tBu),
26.8 (3 × CH3 tBu), 62.7 (C-6'), 63.5 (CH2 glycerol), 68.3 (C-6), 70.6 (CH glycerol), 73.2 (CH2
glycerol), 74.9, 74.9, 75.1, 75.1 (4 × CH2 Bn), 75.2 (C-5), 75.7 (CH2 Bn), 75.7 (C-5'), 75.8 (CH2 Bn),
77.7 (C-4'), 78.2 (C-4), 82.1 (C-2), 82.5 (C-2'), 84.6, 84.7 (C-3, C-3'), 103.9, 103.9 (C-1, C-1'),
127.6–128.1 (CHarom), 128.3–128.4 (CHarom), 129.6, 129.6 (CHarom), 133.1, 133.6 (2 × Cq Ph), 135.5,
135.9 (CHarom), 137.9, 138.2, 138.2, 138.4, 138.4, 138.5 (6 × Cq Bn); HRMS: C73H82O13Si + Na+
requires 1217.5417, found 1217.5424.
Conversion of 6 to 7
Intermediate C
To a solution of diol 6 (149 mg, 0.125 mmol) and pyridine (0.101 mL, 99.2 mg, 1.25 mmol) in
DCM (2.5 mL) were added myristoyl chloride (0.101 mL, 91.7 mg, 0.372 mmol) and a catalytic
amount of 4-DMAP, respectively. After stirring for 5 h, methanol (1.0 mL) was added and after 10 min
of stirring the volatiles were removed under reduced pressure. The residue was taken up in EtOAc (25 mL)
and washed with aqueous 1M HCl (10 mL), sat. aq. NaHCO3 (10 mL) and brine (10 mL). The organic
layer was dried (Na2SO4), filtered, and concentrated in vacuo. The residue was purified by column

Molecules 2013, 18
13560
chromatography (Et2O/PE/Et3N, 0/1/0.01 → 1/4/0.05), giving intermediate C (160 mg, 99.0 µmol, 79%) as
20
a colourless oil. [α]D (CHCl3): +12.3; 1H-NMR): δ = 0.91 (t, 6H, J = 6.8 Hz, 2 × CH3 myristoyl), 1.08
(s, 9H, t-Bu), 1.21–1.37 (m, 40H, 20 × CH2 myristoyl), 1.53–1.61 (m, 4H, 2 × CH2 myristoyl),
2.20–2.29 (m, 4H, 2 × CH2 myristoyl), 3.32–3.36 (m, 1H, H-5'), 3.43–3.46 (m, 1H, H-2), 3.47–3.62
(m, 4H, H-2', H-4, H-5, CHH glycerol), 3.63–3.73 (m, 3H, H-3, H-3', H-6), 3.80 (t, 1H, J = 9.4 Hz,
H-4'), 3.93–3.97 (m, 2H, 2 × H-6'), 4.00 (dd, 1H, J = 4.6 Hz, 10.8 Hz, CHH glycerol), 4.16 (dd, 1H,
J = 7.1 Hz, 11.9 Hz, CHH glycerol), 4.23–4.28 (m, 2H, H-6, CHH glycerol), 4.37 (d, 1H, J = 7.8 Hz,
H-1), 4.46 (d, 1H, J = 7.8 Hz, H-1'), 4.57 (d, 1H, J = 11.2 Hz, CHH Bn), 4.69–4.87 (m, 6H, 3 × CH2
Bn), 4.91–4.98 (m, 4H, 2 × CH2 Bn), 5.04 (d, 1H, J = 11.2 Hz, CHH Bn), 5.15–5.19 (m, 1H, CH
13
glycerol), 7.17–7.44 (m, 36H, Harom), 7.71–7.74 (m, 2H, Harom), 7.77–7.81 (m, 2H, Harom); C-NMR):
δ = 14.1 (2 × CH3 myristoyl), 19.3 (Cq tBu), 22.7 (2 × CH2 myristoyl), 24.8, 24.9 (2 × CH2 myristoyl),
26.8 (3 × CH3 tBu), 29.1–29.7 (16 × CH2 myristoyl), 31.9 (2 × CH2 myristoyl, 34.0, 34.2 (2 × CH2
myristoyl), 62.7, 62.7 (C-6', CH2 glycerol), 68.1 (CH2 glycerol), 68.3 (C-6), 69.8 (CH glycerol), 74.7,
74.8, 74.8 (3 × CH2 Bn), 75.0 (C-5), 75.1, 75.6 (2 × CH2 Bn), 75.7 (C-5'), 75.9 (CH2 Bn), 77.6 (C-4'),
78.0 (C-4), 81.9 (C-2), 82.3 (C-2'), 84.5, 84.8 (C-3, C-3'), 103.8, 104.0 (C-1, C-1'), 127.5–128.4
(CHarom), 129.5, 129.6 (CHarom), 133.1, 133.6 (2 × Cq Ph), 135.5, 135.9 (CHarom), 138.1, 138.3, 138.3,
138.5, 138.5, 138.6 (6 × Cq Bn), 172.9, 173.2 (2 × Cq myristoyl).
Protected Glycolipid 7
To a solution of intermediate C (142 mg, 88.0 µmol) in THF (1.8 mL) was added TBAF (1.0 M
solution in THF, 0.26 mL, 0.26 mmol). After stirring overnight at rt, the volatiles were removed under
reduced pressure after which the residue was purified by column chromatography (Et2O/PE, 1/4 → 3/2).
20
Semiprotected glycolipid 7 (105 mg, 76.4 µmol, 87%) was obtained as a colourless oil. [α]D (CHCl3):
1
+14.0; H-NMR: δ = 0.89 (t, 6H, J = 7.0 Hz, 2 × CH3 myristoyl), 1.23–1.33 (m, 40H, 20 × CH2
myristoyl), 1.53–1.61 (m, 4H, 2 × CH2 myristoyl), 2.10 (t, 1H, J = 6.4 Hz, 6'-OH), 2.20–2.29 (m, 4H,
2 × CH2 myristoyl), 3.35 (ddd, 1H, J = 2.7 Hz, 4.8 Hz, 9.6 Hz, H-5'), 3.39–3.58(m, 6H, H-2, H-2', H-4,
H-4', H-5, CHH glycerol), 3.63–3.73 (m, 4H, H-3, H-3', H-6, H-6'), 3.83–3.88 (m, 1H, H-6'), 3.94 (dd,
1H, J = 4.6 Hz, 10.9 Hz, CHH glycerol), 4.10 (dd, 1H, J = 1.4 Hz, 11.3 Hz, H-6), 4.16 (dd, 1H, J = 7.0 Hz,
12.0 Hz, CHH glycerol), 4.27 (dd, 1H, J = 3.5 Hz, 12.0 Hz, CHH glycerol), 4.33 (d, 1H, J = 7.8 Hz, H-1),
4.47 (d, 1H, J = 7.8 Hz, H-1'), 4.55 (d, 1H, J = 11.1 Hz, CHH Bn), 4.65 (d, 1H, J = 11.0 Hz, CHH Bn),
4.69 (d, 1H, J = 11.1 Hz, CHH Bn) , 4.75–4.80 (m, 3H, CH2 Bn, CHH Bn), 4.82 (d, 1H, J = 10.9 Hz,
CHH Bn), 4.87 (d, 1H, J = 11.0 Hz, CHH Bn), 4.91–4.96 (m, 4H, 2 × CH2 Bn), 5.15–5.19 (m, 1H, CH
glycerol), 7.21–7.37 (m, 30H, Harom); 13C-NMR: δ = 14.1 (2 × CH3 myristoyl), 22.7 (2 × CH2 myristoyl),
24.9, 24.9 (2 × CH2 myristoyl), 29.1–29.7 (16 × CH2 myristoyl), 31.9 (2 × CH2 myristoyl, 34.1, 34.2
(2 × CH2 myristoyl), 62.0 (C-6'), 62.7 (CH2 glycerol), 68.0 (CH2 glycerol), 68.8 (C-6), 69.9

Molecules 2013, 18
13561
(CH glycerol), 74.7, 74.8, 74.9 (3 × CH2 Bn), 74.9 (C-5), 75.0 (CH2 Bn), 75.1 (C-5'), 75.7 (2 × CH2 Bn),
77.6 (C-4'), 77.8 (C-4), 81.9 (C-2), 82.1 (C-2'), 84.5, 84.6 (C-3, C-3'), 103.8 (C-1), 103.9 (C-1'),
127.6–128.4 (CHarom), 137.9, 138.0, 138.3, 138.3, 138.4, 138.5 (6 × Cq Bn), 173.0, 173.3 (2 × Cq
myristoyl); HRMS: C85H116O15 + Na+ requires 1399.8206, found 1399.8209.
Gentiobiosyl Derivative 8
To a solution of silyl ether 5 (2.84 g, 2.23 mmol) in THF (23 mL) was added TBAF (1.0 M solution
in THF, 5.6 mL, 5.6 mmol) and the mixture stirred for 2 h before a second batch of TBAF (1.0 M
solution in THF, 3.3 mL, 3.3 mmol) was added. After stirring overnight at rt, the volatiles were
removed under reduced pressure and the residue was taken up in Et2O (100 mL) and washed with
water (2 × 50 mL) and brine (50 mL). The organic layer was dried with Na2SO4, filtered, and
concentrated in vacuo, after which the crude product was crystallized from MeOH, giving alcohol 8
20
(2.14 g, 2.06 mmol, 93%) as an amorphous white solid. M.p.: 104–105 °C; [α]D (CHCl3): +13.9;
1H-NMR: δ = 1.28–1.43 (m, 2H, CH2 cyclohexylidene), 1.50–1.60 (m, 8H, 4 × CH2 cyclohexylidene),
2.13 (bs, 1H, 6'-OH), 3.33 (ddd, J = 2.6 Hz, 4.7 Hz, 9.5 Hz, H-5'), 3.40–3.48 (m, 4H, H-2, H-2', H-4,
CHH glycerol), 3.50–3.56 (m, 2H, H-4', H-5), 3.60–3.75 (m, 5H, H-3, H-3', H-6, H-6', CHH glycerol),
3.81–3.86 (m, 1H, H-6'), 3.89 (dd, 1H, J = 4.9 Hz, 10.4 Hz, CHH glycerol), 3.93 (dd, 1H, J = 6.3 Hz,
8.2 Hz, CHH glycerol), 4.04–4.07 (m, 1H, H-6), 4.17–4.22 (m, 1H, CH glycerol), 4.35 (d, 1H, J = 7.8 Hz,
H-1), 4.48 (d, 1H, J = 7.8 Hz, H-1'), 4.53 (d, 1H, J = 11.1 Hz, CHH Bn), 4.63 (d, 1H, J = 11.0 Hz,
CHH Bn), 4.68 (d, 1H, J = 11.1 Hz, CHH Bn), 4.72–4.85 (m, 6H, 2 × CH2 Bn, CHH Bn), 4.89–4.94
(m, 4H, 2 × CH2 Bn), 7.18–7.39 (m, 30H, Harom); 13C-NMR: δ = 23.7, 23.9, 25.1 (3 × CH2
cyclo-hexylidene), 34.8, 36.4 (2 × CH2 cyclohexylidene), 62.0 (C-6’), 66.4 (CH2 glycerol), 68.9 (C-6),
70.3 (CH2 glycerol), 73.8 (CH glycerol), 74.7, 74.8, 74.9, 75.0 (4 × CH2 Bn), 75.0 (C-5), 75.1 (C-5'),
75.6, 75.6 (2 × CH2 Bn), 77.5 (C-4'), 77.9 (C-4), 82.0 (C-2), 82.1 (C-2'), 84.6, 84.6 (C-3, C-3'), 103.6
(C-1), 104.0 (C-1'), 109.9 (Cq cyclohexylidene), 127.6–128.4 (CHarom), 137.9, 138.0, 138.3, 138.3,
138.4, 138.5 (6 × Cq Bn); HRMS: C63H72O13 + Na+ requires 1059.4865, found 1059.4878.
Gentiotriosyl Derivative 13

Molecules 2013, 18
13562
A solution of alcohol 8 (104 mg, 100 µmol) and donor 12 (96.3 mg, 110 µmol) in DCM (2.0 mL),
containing freshly activated powdered MS3Å, was cooled to −35 °C. Boron trifluoride diethyl etherate
(1.2 µL, 10 µmol) was added and the mixture was allowed to stir 2 h while slowly warming to 0 °C.
Et3N (0.10 mL) was added, the mixture filtered over Celite and the volatiles removed under reduced
pressure. The residue was purified by column chromatography (Et2O/PE, 1/9 → 1/1), giving
20
1
pseudotetrasaccharide 13 (132 mg, 75 µmol, 75%) as a colourless oil. [α]D (CHCl3): +3.9; H-NMR:
δ = 1.03 (s, 9H, t-Bu), 1.23–1.40 (m, 2H, CH2 cyclohexylidene), 1.50–1.61 (m, 8H, 4 × CH2
cyclohexylidene), 3.35 (dd, 1H, J = 7.8 Hz, 9.1 Hz, H-2'), 3.38–3.44 (m, 6H, H-2, H-4, H-4', H-5, H-5',
CHH glycerol), 3.52–3.56 (m, 1H, H-3'), 3.59–3.63 (m, 2H, H-3, H-6'), 3.70 (dd, 1H, J = 6.2 Hz, 8.2
Hz, CHH glycerol), 3.72–3.80 (m, 2H, H-5″, H-6), 3.85–3.87 (m, 2H, 2 × H-6″), 3.91–3.95 (m, 2H,
2 × CHH glycerol), 4.06–4.10 (m, 1H, H-6'), 4.17–4.25 (m, 2H, H-6, CH glycerol), 4.31
(d, 1H, J = 7.8 Hz, H-1), 4.39–4.43 (m, 2H, H-1', CHH Bn), 4.48 (d, 1H, J = 11.1 Hz, CHH Bn), 4.57
(d, 1H, J = 11.2 Hz, CHH Bn), 4.66–4.73 (m, 4H, 2 × CH2 Bn), 4.76 (d, 1H, J = 11.0 Hz, CHH Bn),
4.84 (d, 1H, J = 7.9 Hz, H-1″), 4.87 (d, 1H, J = 11.0 Hz, CHH Bn), 4.90–4.94 (m, 3H, CH2 Bn, CHH Bn),
5.57 (dd, 1H, J = 7.9 Hz, 9.7 Hz, H-2″), 5.62 (t, 1H, J = 9.7 Hz, H-4″), 5.81 (t, 1H, J = 9.7 Hz, H-3″),
7.10–7.13 (m, 2H, Harom), 7.16–7.41 (m, 42H, Harom), 7.51–7.54 (m, 1H, Harom), 7.57–7.59 (m, 2H, Harom),
13
7.70–7.72 (m, 2H, Harom), 7.81–7.87 (m, 4H, Harom), 7.89–7.92 (m, 2H, Harom); C-NMR: δ = 19.1
(Cq tBu), 23.8, 24.0, 25.1 (3 × CH2 cyclohexylidene), 26.6 (3 × CH3 tBu), 34.8, 36.4 (2 × CH2 cyclo-
hexylidene), 62.8 (C-6″), 66.3 (CH2 glycerol), 68.0, 68.0 (C-6, C-6'), 69.2 (C-4″), 70.2 (CH2 glycerol),
72.1 (C-2″), 73.3 (C-3″), 73.9 (CH glycerol), 74.3 (C-5'), 74.5, 74.7, 74.8, 74.9 (4 × CH2 Bn), 75.2,
75.3 (C-5, C-5″), 75.5, 75.5 (2 × CH2 Bn), 77.4 (C-4'), 78.0 (C-4), 82.0 (C-2'), 82.0 (C-2), 84.6 (C-3),
84.7 (C-3'), 101.3 (C-1″), 103.5 (C-1), 103.9 (C-1'), 109.9 (Cq cyclohexylidene), 127.5–128.4 (CHarom),
128.9, 129.2, 129.4 (3 × Cq Bz), 129.6–129.8 (CHarom), 133.0 (Cq Ph), 133.0 (CHarom), 133.0 (Cq Ph),
133.1, 133.2 (CHarom), 135.5, 135.6 (CHarom), 138.1, 138.1, 138.4, 138.5, 138.5, 138.6 (6 × Cq Bn),
164.9, 164.9, 165.9 (3 × Cq Bz); HRMS: C106H112O21Si + Na+ requires 1771.7358, found 1771.7367.
Conversion of 13 to 14
Intermediate D
To a solution of gentiotriosyl derivative 13 (840 mg, 0.480 mmol) in 1-propanol (10 mL) was added
a catalytic amount of KOtBu after which the reaction was allowed to stir at rt for 2 h. Amberlite H+
resin was then added to the reaction until the pH was neutral and the resulting mixture filtered and
concentrated under reduced pressure. Purification of the residue by column chromatography
20
(EtOAc/PE, 1/9 → 1/2) gave intermediate D (600 mg, 0.417 mmol, 87%) as a colourless oil. [α]D
1
(CHCl3): +3.2; H-NMR: δ = 1.06 (s, 9H, t-Bu), 1.28–1.42 (m, 2H, CH2 cyclohexylidene), 1.50–1.61

Molecules 2013, 18
13563
(m, 8H, 4 × CH2 cyclohexylidene), 3.30 (dd, 1H, J = 8.0 Hz, 9.2 Hz, H-2″), 3.37–3.51 (m, 8H, H-2,
H-2', H-3″, H-4, H-4', H-5', H-5″, CHH glycerol), 3.54–3.66 (m, 5H, H-3, H-3', H-4″, H-5, H-6'),
3.72–3.78 (m, 2H, H-6, CHH glycerol), 3.87–3.93 (m, 2H, 2 × H-6″), 3.93–4.00 (m, 2H,
2 × CHH glycerol), 4.04–4.09 (m, 2H, H-6, H-6'), 4.26–4.30 (m, 2H, H-1″, CH glycerol), 4.39 (d, 1H,
J = 7.8 Hz, H-1), 4.47 (d, 1H, J = 7.8 Hz, H-1'), 4.52 (d, 1H, J = 11.1 Hz, CHH Bn), 4.59 (d, 1H,
J = 11.1 Hz, CHH Bn), 4.68 (d, 1H, J = 11.1 Hz, CHH Bn), 4.73–4.79 (m, 4H, 2 × CH2 Bn), 4.84
(d, 1H, J = 11.0 Hz, CHH Bn), 4.89–4.94 (m, 4H, 2 × CH2 Bn), 7.18–7.43 (m, 36H, Harom), 7.69–7.72
(m, 4H, Harom); 13C-NMR: δ = 19.2 (Cq tBu), 23.8, 24.0, 25.1 (3 × CH2 cyclohexylidene), 26.8 (3 × CH3
tBu), 34.8, 36.5 (2 × CH2 cyclohexylidene), 64.7 (C-6″), 66.5 (CH2 glycerol), 66.9 (C-6'), 68.9 (C-6), 70.4
(CH2 glycerol), 71.9 (C-4″), 72.4 (C-2″), 73.8 (CH glycerol), 74.2 (C-5’), 74.8 (CH2 Bn), 74.8 (C-5″), 74.8
(CH2 Bn), 74.9 (C-5), 75.0, 75.0, 75.7, 75.7 (4 × CH2 Bn), 76.1 (C-3″), 77.9 (C-4'), 78.0 (C-4), 82.0
(C-2'), 82.1 (C-2), 84.6, 84.6 (C-3, C-3'), 102.9 (C-1″), 103.6 (C-1), 103.9 (C-1'), 110.0
(Cq cyclohexylidene), 127.6–128.4 (CHarom), 129.8, 129.8 (CHarom), 132.8 (Cq Ph), 132.9 (Cq Ph), 135.6,
135.6 (CHarom), 137.9, 137.9, 138.2, 138.3, 138.4, 138.5 (6 × Cq Bn); HRMS: C85H100O18Si + Na+
requires 1459.6571, found 1459.6587.
Intermediate E
To a cooled (0 °C) solution of intermediate D (1.64 g, 1.14 mmol) in DMF (23 mL) were added
benzyl bromide (0.814 mL, 1.17 g, 6.85 mmol) and sodium hydride (40% dispersion in mineral oil,
274 mg, 6.84 mmol), respectively. The mixture was stirred for 2 h, while slowly warming to room
temperature, before methanol (2.0 mL) was added. After stirring for 15 min, water (50 mL) was added
and the mixture extracted with Et2O (100 mL). The organic layer was washed with water (50 mL) and
brine (50 mL) after which it was dried (Na2SO4), filtered, and concentrated in vacuo. Purification of the
residue by column chromatography (Et2O/PE, 0/1 → 1/1) gave intermediate E (1.61 g, 0.943 mmol, 83%)
20
1
as a colourless oil. [α]D (CHCl3): +9.6; H-NMR: δ = 1.06 (s, 9H, t-Bu), 1.28–1.41 (m, 2H, CH2
cyclohexylidene), 1.50–1.61 (m, 8H, 4 × CH2 cyclohexylidene), 3.27–3.31 (m, 2H, H-4, H-5″),
3.36–3.41 (m, 2H, H-2', CHH glycerol), 3.45–3.49 (m, 2H, H-2″, H-5), 3.52–3.73 (m, 9H, H-2, H-3,
H-3', H-3″, H-4, H-5', H-6, H-6', CHH glycerol), 3.78 (t, 1H, J = 9.3 Hz, H-4″), 3.89–3.94 (m, 4H,
2 × H-6″, 2 × CHH glycerol), 4.14–4.20 (m, 2H, H-6, CH glycerol), 4.25–4.29 (m, 1H, H-6'), 4.28
(d, 1H, J = 7.8 Hz, H-1'), 4.36 (d, 1H, J = 10.9 Hz, CHH Bn), 4.41 (d, 1H, J = 7.7 Hz, H-1), 4.56
(d, 1H, J = 11.4 Hz, CHH Bn), 4.59 (d, 1H, J = 7.9 Hz, H-1″), 4.63–4.81 (m, 9H, 4 × CH2 Bn, CHH Bn),
4.87–4.97 (m, 6H, 3 × CH2 Bn), 5.06 (d, 1H, J = 11.2 Hz, CHH Bn), 7.12–7.42 (m, 51H, Harom),
13
7.69–7.73 (m, 2H, Harom), 7.75–7.80 (m, 2H, Harom); C-NMR: δ = 19.3 (Cq tBu), 23.8, 24.0, 25.1
(3 × CH2 cyclohexylidene), 26.8 (3 × CH3 tBu), 34.9, 36.4 (2 × CH2 cyclohexylidene), 62.6 (C-6″),
66.3 (CH2 glycerol), 68.4, 68.4 (C-6, C-6'), 70.2 (CH2 glycerol), 73.9 (CH glycerol), 74.6 (C-5'), 74.6,

Molecules 2013, 18
13564
74.7, 74.8, 74.8, 74.9, 75.1 (6 × CH2 Bn), 75.4 (C-5″), 75.5, 75.7 (2 × CH2 Bn), 75.7 (C-5), 75.8 (CH2
Bn), 77.6 (C-4″), 78.0 (C-4), 78.2 (C-4'), 82.0 (C-2'), 82.2 (C-2″), 82.3 (C-2), 84.5, 84.8, 84.9
(C-3, C-3', C-3″), 103.5 (C-1'), 104.1 (C-1, C-1″), 109.9 (Cq cyclohexylidene), 127.5–128.4 (CHarom),
129.6, 129.6 (CHarom), 133.1 (Cq Ph), 133.7 (Cq Ph), 135.5, 135.9 (CHarom), 137.9–138.6 (9 × Cq Bn);
HRMS: C106H118O18Si + Na+ requires 1729.7980, found 1729.7984.
Intermediate F
A solution of intermediate E (1.46 g, 0.855 mmol) in a mixture of acetic acid (34 mL), toluene (8.6 mL)
and water (8.6 mL) was stirred for 3 h at 90 °C. After cooling down to rt the mixture was diluted with
EtOAc (250 mL) and washed with water (2 × 100 mL), sat. aq. NaHCO3 (2 × 100 mL) and brine (100 mL).
The organic layer was dried with Na2SO4, filtered and concentrated in vacuo, after which the residue
was purified by column chromatography (Et2O/PE, 1/9 → 2/1) giving semiprotected intermediate F
20
1
(1.20 g, 0.737 mmol, 86%) as a colourless oil. [α]D (CHCl3): +8.1; H-NMR: δ = 1.06 (s, 9H, t-Bu),
3.31–3.79 (m, 19H, H-2, H-2', H-2″, H-3, H-3', H-3″, H-4, H-4', H-4″, H-5, H-5', H-5″, H-6, H-6',
2 × CH2 glycerol, CH glycerol), 3.92–3.94 (m, 2H, 2 × H-6″), 4.16 (dd, 1H, J = 1.6 Hz, 11.6 Hz, H-6),
4.24–4.27 (m, 1H, H-6'), 4.29 (d, 1H, J = 7.8 Hz, H-1'), 4.36 (d, 1H, J = 10.9 Hz, CHH Bn), 4.47–4.51 (m,
2H, H-1, H-1″), 4.55 (d, 1H, J = 11.3 Hz, CHH Bn), 4.61 (d, 1H, J = 10.9 Hz, CHH Bn), 4.68–4.95 (m,
14H, 7 × CH2 Bn), 5.04 (d, 1H, J = 11.2 Hz, CHH Bn), 7.09–7.41 (m, 51H, Harom), 7.69–7.72 (m, 2H,
13
Harom), 7.75–7.79 (m, 2H, Harom); C-NMR: δ = 19.3 (Cq tBu), 26.8 (3 × CH3 tBu), 62.7 (C-6″), 63.4
(CH2 glycerol), 68.2, 68.3 (C-6, C-6'), 70.7 (CH glycerol), 72.8 (CH2 glycerol), 74.8, 74.9, 74.9
(3 × CH2 Bn), 74.9, 75.0 (C-5, C-5'), 75.0, 75.0, 75.2 (3 × CH2 Bn), 75.5 (C-5″), 75.6, 75.6, 75.8 (3 ×
CH2 Bn), 77.7, 78.0, 78.3 (C-4, C-4', C-4″), 81.9, 82.1, 82.5 (C-2, C-2', C-2″), 84.5, 84.6, 84.7 (C-3, C-3',
C-3″), 103.7, 104.0, 104.0 (C-1, C-1', C-1″), 127.5–128.4 (CHarom), 129.6, 129.6 (CHarom), 133.1 (Cq
Ph), 133.6 (Cq Ph), 135.5, 135.9 (CHarom), 137.9–138.6 (9 × Cq Bn); HRMS: C100H110O18Si + Na+
requires 1649.7354, found 1649.7360.
Intermediate G

Molecules 2013, 18
13565
To a solution of intermediate F (260 mg, 0.160 mmol) and pyridine (0.129 mL, 127 mg, 1.60 mmol)
in DCM (3.2 mL) were added myristoyl chloride (0.130 mL, 118 mg, 0.478 mmol) and a catalytic
amount of 4-DMAP, respectively. After stirring for 3 h, methanol (1.0 mL) was added and after 10 min
of stirring the volatiles were removed under reduced pressure. The residue was taken up in EtOAc (25 mL)
and washed with aqueous 1 M HCl (10 mL), sat. aq. NaHCO3 (10 mL) and brine (10 mL). The organic
layer was dried (Na2SO4), filtered, and concentrated in vacuo. The residue was purified by column
chromatography (Et2O/PE/Et3N, 0/1/0.01 → 1/1/0.02), giving intermediate G (293 mg, 0.143 mmol,
20
1
90%) as a colourless oil. [α]D (CHCl3): +10.7; H-NMR: δ = 0.88 (t, 6H, J = 6.9 Hz, 2 × CH3
myristoyl), 1.04 (s, 9H, t-Bu), 1.19–1.29 (m, 40H, 20 × CH2 myristoyl), 1.51–1.58 (m, 4H, 2 × CH2
myristoyl), 2.18–2.26 (m, 4H, 2 × CH2 myristoyl), 3.25–3.68 (m, 11H, H-2, H-2', H-2″, H-3, H-3', H-4,
H-4', H-5, H-5', H-5″, CHH glycerol), 3.63–3.68 (m, 3H, H-3″, H-6, H-6'), 3.76 (t, 1H,
J = 9.3 Hz, H-4″), 3.90–3.93 (m, 3H, CHH glycerol, 2 × H-6″), 4.11 (dd, 1H, J = 7.3 Hz, 11.9 Hz,
CHH glycerol), 4.16 (m, 1H, H-6), 4.20–4.27 (m, 3H, H-1', H-6', CHH glycerol), 4.36 (d, 1H, J = 11.0 Hz,
CHH Bn), 4.39 (d, 1H, J = 7.7 Hz, H-1), 4.51 (d, 1H, J = 7.8 Hz, H-1″), 4.54 (d, 1H, J = 11.4 Hz,
CHH Bn), 4.60–4.79 (m, 9H, 4 × CH2 Bn, CHH Bn), 4.85–4.96 (m, 6H, 3 × CH2 Bn), 5.04 (d, 1H,
J = 11.2 Hz, CHH Bn), 5.12–5.15 (m, 1H, CH glycerol), 7.11–7.41 (m, 51H, Harom), 7.69–7.71 (m, 2H,
Harom), 7.74–7.77 (m, 2H, Harom); 13C-NMR: δ = 14.1 (2 × CH3 myristoyl), 19.3 (Cq tBu), 22.7
(2 × CH2 myristoyl), 24.9, 24.9 (2 × CH2 myristoyl), 26.8 (3 × CH3 tBu), 29.1–29.7 (16 × CH2
myristoyl), 31.9 (2 × CH2 myristoyl, 34.1, 34.3 (2 × CH2 myristoyl), 62.6 (C-6″), 62.8 (CH2 glycerol),
68.0 (CH2 glycerol) 68.4, 68.5 (C-6, C-6'), 69.8 (CH glycerol), 74.6 (CH2 Bn), 74.6 (C-5'), 74.7, 74.8,
74.9, 74.9, 75.1 (5 × CH2 Bn), 75.2 (C-5), 75.5, 75.6 (2 × CH2 Bn), 75.7 (C-5″), 75.8 (CH2 Bn), 77.6
(C-4″), 78.0, 78.1 (C-4, C-4'), 81.8, 82.1, 82.3 (C-2, C-2', C-2″), 84.4, 84.8, 84.8 (C-3, C-3', C-3″),
103.6, 104.1, 104.2 (C-1, C-1', C-1″), 127.5–128.4 (CHarom), 129.6, 129.6 (CHarom), 133.1 (Cq Ph),
133.6 (Cq Ph), 135.5, 135.9 (CHarom), 138.0–138.6 (9 × Cq Bn), 172.9, 173.3 (2 × Cq myristoyl);
HRMS: C128H162O20Si + Na+ requires 2071.1354, found 2071.1370.
Gentiotriosyl Derivative 14
To a solution of intermediate G (274 mg, 0.134 mmol) in THF (2.7 mL) was added TBAF (1.0 M
solution in THF, 0.40 mL, 0.40 mmol). After stirring overnight at rt, the volatiles were removed under
reduced pressure after which the residue was purified by column chromatography (EtOAc/PE, 1/9 → 1/2).
Semiprotected gentiotriosyl glycolipid 14 (205 mg, 0.113 mmol, 85%) was obtained as a colourless oil.
20
1
[α]D (CHCl3): +12.5; H-NMR: δ = 0.89 (t, 6H, J = 7.0 Hz, 2 × CH3 myristoyl), 1.19–1.34 (m, 40H,
20 × CH2 myristoyl), 1.51–1.59 (m, 4H, 2 × CH2 myristoyl), 2.17–2.25 (m, 4H, 2 × CH2 myristoyl),
3.26–3.69 (m, 15H, H-2, H-2', H-2″, H-3, H-3', H-3″, H-4, H-4', H-4″, H-5, H-5', H-5″, H-6, H-6″,

Molecules 2013, 18
13566
CHH glycerol), 3.74 (dd, 1H J = 5.2 Hz, 11.1 Hz, H-6'), 3.83 (dd, 1H, J = 2.6 Hz, 11.9 Hz, H-6″), 3.92
(dd, 1H, J = 4.6 Hz, 10.9 Hz, CHH glycerol), 4.06–4.15 (m, 3H, H-6, H-6', CHH glycerol),
4.23–4.27 (m, 2H, H-1, CHH glycerol), 4.39–4.44 (m, 2H, H-1', CHH Bn), 4.46 (d, 1H, J = 7.8 Hz, H-1″),
4.55 (d, 1H, J = 11.2 Hz, CHH Bn), 4.62–4.68 (m, 3H, CH2 Bn, CHH Bn), 4.71–4.79 (m, 6H, 3 × CH2 Bn),
4.84 (d, 1H, J = 11.0 Hz, CHH Bn), 4.87–4.96 (m, 6H, 3 × CH2 Bn), 5.13–5.17 (m, 1H, CH glycerol),
7.16–7.36 (m, 45H, Harom); 13C-NMR: δ = 14.1 (2 × CH3 myristoyl), 22.7 (2 × CH2 myristoyl), 24.9, 24.9
(2 × CH2 myristoyl), 29.1–29.7 (16 × CH2 myristoyl), 31.9 (2 × CH2 myristoyl, 34.1, 34.3 (2 × CH2
myristoyl), 62.0 (C-6″), 62.8 CH2 glycerol), 68.0 (CH2 glycerol), 68.7 (C-6), 69.3 (C-6'), 69.8 (CH
glycerol), 74.6 (C-5'), 74.6, 74.8, 74.8, 74.9, 74.9 (5 × CH2 Bn), 75.0 (C-5), 75.0 (CH2 Bn), 75.2 (C-5″),
75.6, 75.6, 75.7 (3 × CH2 Bn), 77.6, 78.0, 78.0 (C-4, C-4', C-4″), 81.8, 82.1, 82.1 (C-2, C-2', C-2″), 84.5,
84.5, 84.8 (C-3, C-3', C-3″), 103.5, 104.0, 104.2 (C-1, C-1', C-1″), 127.5–128.4 (CHarom), 137.9–138.5
(9 × Cq Bn), 172.9, 173.3 (2 × Cq myristoyl); HRMS: C112H144O20 + Na+ requires 1832.0143, found
1832.0149.
Glycolipid 15
A solution of compound 7 (16.9 mg, 12.3 µmol) in a mixture of 1,4-dioxane/water/acetic acid
(5/1/0.05, 2.0 mL) was purged with argon. Palladium black (~5 mg, ~50 µmol) was added and the
reaction was stirred under hydrogen atmosphere for 2 days. The mixture was filtered over Celite and
concentrated under reduced pressure. Purification of the residue by reversed phase column
chromatography (C8 silica, H2O/iPrOH, 4/1 → 1/9) gave glycolipid 15 (4.9 mg, 5.9 µmol, 48%) as an
amorphous white solid. 1H-NMR (CD3OD): δ = 0.90 (t, 6H, J = 7.0 Hz, 2 × CH3 myristoyl), 1.26–1.38
(m, 40H, 20 × CH2 myristoyl), 1.57–1.64 (m, 4H, 2 × CH2 myristoyl), 2.31–2.35 (m, 4H, 2 × CH2
myristoyl), 3.16–3.20 (m, 1H, H-2), 3.22 (dd, 1H, J = 7.9 Hz, 9.2 Hz, H-2'), 3.26–3.30 (m, 2H, H-4',
H-5'), 3.32–3.37 (m, 3H, H-3, H-3', H-4), 3.44–3.48 (m, 1H, H-5), 3.67 (dd, 1H, J = 5.4 Hz, 11.8 Hz,
H-6'), 3.74–3.78 (m, 2H, H-6, CHH glycerol), 3.87 (dd, 1H, J = 1.8 Hz, 11.9 Hz, H-6'), 3.99 (dd, 1H,
J = 5.5 Hz, 11.0 Hz, CHH glycerol), 4.16 (dd, 1H, J = 2.0 Hz, 11.5 Hz, H-6), 4.22 (dd, 1H,
J = 6.9 Hz, 12.1 Hz, CHH glycerol), 4.28 (d, 1H, J = 7.8 Hz, H-1), 4.37 (d, 1H, J = 7.8 Hz, H-1'), 4.43
13
(dd, 1H, J = 3.0 Hz, 12.1 Hz, CHH glycerol), 5.26–5.30 (m, 1H, CH glycerol); C-NMR (CD3OD):
δ = 14.5 (2 × CH3 myristoyl), 23.8 (2 × CH2 myristoyl), 26.1, 26.1 (2 × CH2 myristoyl), 30.2–30.8
(16 × CH2 myristoyl), 33.1 (2 × CH2 myristoyl, 35.0, 35.2 (2 × CH2 myristoyl), 62.8 (C-6'), 64.0 CH2
glycerol), 68.9 (CH2 glycerol), 70.0 (C-6), 71.5 (C-4), 71.6 (C-4'), 71.8 (CH glycerol), 74.9 (C-2), 75.1
(C-2'), 77.1 (C-5), 77.8 (C-5'), 78.0, 78.0 (C-3, C-3'), 104.7 (C-1), 104.9 (C-1'), 174.9, 175.1 (2 × Cq
myristoyl); HRMS: C43H80O15 + Na+ requires 859.5389, found 859.5378.

Molecules 2013, 18
Glycolipid 16
13567
A solution of gentiotriosyl derivative 14 (46.8 mg, 25.9 µmol) in a mixture of THF/water/AcOH
(5/1/0.05, 5.6 mL) was purged with argon. Palladium black (~5 mg, ~50 µmol) was added and the
reaction was stirred under hydrogen atmosphere (1 bar) for 2 days. The mixture was filtered over
Celite and concentrated under reduced pressure. Purification of the residue by reversed phase column
chromatography (C8 silica, H2O/iPrOH, 4/1 → 1/9) gave glycolipid 16 (21.0 mg, 21.0 µmol, 81%) as an
amorphous white solid. 1H-NMR (CD3OD): δ = 0.90 (t, 6H, J = 7.0 Hz, 2 × CH3 myristoyl), 1.26–1.37 (m,
40H, 20 × CH2 myristoyl), 1.56–1.65 (m, 4H, 2 × CH2 myristoyl), 2.30–2.36 (m, 4H, 2 × CH2
myristoyl), 3.17–3.38 (m, 10H, H-2, H-2', H-2″, H-3, H-3', H-3″, H-4, H-4', H-4″, H-5″), 3.45–3.52
(m, 2H, H-5, H-5'), 3.68 (dd, 1H, J = 5.3 Hz, 11.9 Hz, H-6″), 3.74–3.80 (m, 3H, H-6, H-6', CHH
glycerol), 3.87 (dd, 1H, J = 1.9 Hz, 11.9 Hz, H-6″), 4.00 (dd, 1H, J = 5.5 Hz, 11.0 Hz, CHH glycerol),
4.13–4.18 (m, 2H, H-6, H-6'), 4.23 (dd, 1H, J = 7.0 Hz, 12.1 Hz, CHH glycerol), 4.30 (d, 1H, J = 7.8 Hz,
H-1), 4.36–4.38 (m, 2H, H-1', H-1″), 4.44 (dd, 1H, J = 2.9 Hz, 12.1 Hz, CHH glycerol), 5.26–5.31 (m, 1H,
CH glycerol); 13C-NMR (CD3OD): δ = 14.5 (2 × CH3 myristoyl), 23.8 (2 × CH2 myristoyl), 26.1, 26.1
(2 × CH2 myristoyl), 30.2–30.8 (16 × CH2 myristoyl), 33.1 (2 × CH2 myristoyl, 35.0, 35.2 (2 × CH2
myristoyl), 62.7 (C-6″), 64.1 CH2 glycerol), 68.9 (CH2 glycerol), 70.0, 70.6 (C-6, C-6'), 71.5, 71.6, 71.7,
71.8 (C-4, C-4', C-4″, CH glycerol), 74.9, 75.1, 75.1 (C-2, C-2', C-2″), 77.0, 77.0 (C-5, C-5'), 77.8, 77.9,
78.0, 78.0 (C-3, C-3', C-3″, C-5’″), 104.5, 104.9, 105.1 (C-1, C-1', C-1″), 174.9, 175.1 (2 × Cq myristoyl);
HRMS: C49H90O20 + Na+ requires 1021.5918, found 1021.5899.
3.2. Physical Chemical Characterization
General
DMPC, DMPS and DOPE-Biotin (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-cap biotinyl)
were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA) and used without further
purification. The fluorescent dyes DiDC18 (1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine-
4-chlorobenzenesulfonate) and DiIC12 (1,1'-didodecyl-3,3,3',3'-tetramethylindodicarbocyanineperchlorate)
were likewise used as received from Invitrogen (Paisley, UK). Unless stated otherwise, all other
chemicals and reagents were purchased from Sigma-Aldrich A/S (Copenhagen, Denmark). Milli-Q
quality water was used in all preparations.

Molecules 2013, 18
13568
3.3. Methods
Small Unilamellar Vesicles were prepared from hydrating lipid films using PBS buffer (10 mM
phosphate buffer supplemented with 100 mM NaCl, pH 7.4) to a lipid concentration of 1 mg mL−1.
The vesicles were extruded through 100 nm polycarbonate filters using the Mini-Extruder system
(Avanti Polar Lipids, Inc.). This method provides a high degree of unilamellar structures and long-term
stability what makes them suitable for calorimetric measurements [49]. To achieve an even distribution
of the lipids the extruder was heated to 52 °C ensuring DMPC and DMPS in their molten state. Each
sample ran through two cycles of up scans (10–70 °C) and down scans (70–10 °C) to evaluate whether
there was any effect on the mixing behavior of the lipids triggered by the preparation method. These
scans showed no hysteresis effects suggesting that the method of preparation did not have a significant
effect on their thermotropic behavior.
Giant Unilamellar Vesicles were prepared by gentle hydration of lipid films according to previous
protocols [42]. Briefly, lipids in chloroform were mixed in glass tubes to achieve the indicated lipid
composition. The lipid dyes DiD C(18), DiI C(12) (Invitrogen) were added to a concentration of
0.5 mol% for fluorescence imaging. Additionally, the vesicles contained 0.5 mol% DOPE-Biotin
(Avanti Polar Lipids, Inc.) to tether the vesicles on the surface of the microscope slides. Then, a thin
lipid film was prepared by drop-by-drop addition of the mixture to small Teflon cups. The remaining
chloroform was removed by storing the Teflon cups in a vacuum-chamber for 1 h. Lipids were rehydrated
in D-sorbitol solution (46.1 g L−1 in PBS) to a lipid concentration of 0.2 mg mL−1. After one night of
incubation at 50 °C all vesicles were stored at 4 °C and used within the same day.
Microscope chambers were functionalized by carefully cleaning glass coverslips in successive
washes in Helmanex (2%), MilliQ water and ethanol. For surface functionalization, 1 g L−1
BSA–biotin:BSA (1:10, weight ratio) was added to the surface and incubated at ambient temperature
for 10 min. After five times gentle washing with PBS, streptavidin (0.025 g L−1 in PBS) was added
and likewise incubated for 10 min followed by five times washing with PBS. After surface
functionalization, giant unilamellar vesicles were added to the microscopy chamber to a final constant
lipid concentration of 0.01 g L−1 (~14 µM) and allowed to stabilize for at least 30 min before the
measurement. The total volume of the microscope chamber was kept to ~200 µL.
Differential Scanning Calorimetry (DSC) was performed in a MicroCalTM VP-DSC system
(GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and data were processed using Origin7
(Origin Lab, Northampton, MA, USA). Briefly, two up/down scan cycles were performed between 10 and
70 °C at a rate of 1 °C per 1.5 min, and duplicates were obtained for all samples studied. Prior to
vesicle addition, the chambers were cleaned with plenty of 5% SDS solution followed by water, 1%
pepsin solution, water and pure ethanol dried under vacuum. Then, 700 µL of the sample was filled in
one chamber and buffer (PBS, 7.4 pH) as reference was filled in the other. The final lipid concentration
was 1 mg/mL. Each sample ran through two scan cycles consisting of up scans (10–70 °C) and down
scans (70–10 °C). The scan rate was set on 1 °C per 1.5 min. The experiments were taken out with the
MicroCalTMVP-DSC system and the data processed with VPViewerTM2000 (GE Healthcare,
Massachusetts, USA) as well as with Origin7 (OriginLab).
Microscopy imaging was performed on an inverted confocal microscope TCS SP5 (Leica, Wetzlar,
Germany). In order to avoid crosstalk, DiD-C18 was excited with a laser and recorded using the filter

Molecules 2013, 18
13569
650/50 nm while DiI-C12 was excited using a laser and recorded using the filter at 480/50 nm. The
signal was maximized setting the argon laser intensity to 19%, the acousto-Optic Tunable Filter for
Attota to 21% and the PhotoMultiplier Voltage to 537.4 V. These settings were kept fixed during the
various experiments in order to be able to perform fluorescence intensity comparison.
3.4. Fluorescence Microscopy Imaging
All glass slides were cleaned thoroughly due to sonication in Hellmannex for 1h. After sonication
they were rinsed several times with water and ethanol. Additionally they were cleaned by etching and
stored in 99% ethanol after. The glass slides were functionalyzed with 1 g/L BSA-Biotin: BSA (1:10)
in PBS for 10 minutes. Five steps of washing with PBS followed before the slides were incubated with
0.25 g/L Streptavidin in PBS for another 10 min. After another five washing steps with PBS the GUVs
were added to the surface with a final lipid concentration of 0.01 mg/L. The incubation time for the
GUVs to settle to the glass amounted 30 min before they were analyzed. The samples were analyzed
with the inverted Confocal Leica TCS SP5 and the images processed with the software Leica LAS AF
(Leica Microsystems CMS, Mannheim, Germany).
4. Conclusions
The synthesis of glycoglycerolipids from ready available starting materials has been investigated
and an improved procedure for the synthesis of gentiobiosyl glycolipids on larger scales has been
investigated. The amount of side products formed in the glycosylation of cyclohexylidene protected
glycerol have been limited by optimizing the reaction conditions. Conditions for the crystallization of
the glycosylation product from the crude reaction mixture were developed and tedious chromatography
can therefore be avoided allowing for up scaling. Prolonging the “head group” was performed by a
selective deprotection of the 6'O-TBDPS group followed by glycosylation with benzoylated donor 12
giving the pseudotetrasaccharide in high yield and purity, with a 6″O-TBDPS protective group
installed for further prolongation or attachment to a repeating unit via a phosphate linkage as it is
found in LTA from C. difficile.
Our DSC/microscopy results suggest that glycolipids induce strong de-mixing in lipid mixtures and
that the demixing ability depends on the conformation of the head-group as determined by the type of
glycosidic bond and the number of sugar residues in the glycolipid headgroup. Indeed, glycolipids are
believed to form lipid clusters for maximum efficiency of their biological roles that include
participation in signaling pathways and in receptor recognition [50,51]. Our results suggest that the
larger the number of sugars in the glycolipid headgroup, the stronger the demixing induced and that some
preferential association occurs in which DMPC is excluded from the glycolipid rich regions domains.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/18/11/13546/s1.

Molecules 2013, 18
13570
Acknowledgments
We thank FTP for financial support, Kell Mortensen for access to the Differential Scanning
Calorimeter and the Center for Advanced Bioimaging (CAB) Denmark, University of Copenhagen for
access to the confocal microscope.
Conflict of Interest
The authors declare no conflict of interest.
References and Notes
1. Rilfors, L.; Lindblom, G. Regulation of lipid composition in biological membranes—Biophysical
studies of lipids and lipid synthesizing enzymes. Colloid. Surface. B 2002, 26, 112–124.
2. Lindblom, G.; Oräddd, G.; Rilfors, L.; Morein, S. Regulation of lipid composition in
acholeplasmalaidlawii and escherichia coli membranes:ꢀ NMR studies of lipid lateral diffusion at
different growth temperatures. Biochemisty 2002, 41, 11512–11515.
3. Joshi, A.S.; Thompson, M.N.; Fei, N.; Hütterman, M.; Greenberg, M.L. Cardiolipin and
mitochondrial phosphatidylethanolamine have overlapping functions in mitochondrial fusion in
Saccharomyces cerevisiae. J. Biol. Chem. 2012, 287, 17589–17597.
4. Rietveld, A.G.; Koorengevel, M.C.; De Kruijff, B. Non-bilayer lipids are required for efficient
protein transport across the plasma membrane of Escherichia coli. EMBO J. 1995, 14, 5506–5513.
5. Larsson, K.; Puang-Ngern, S. The aqueous system of monogalactosyl diglycerides and
digalactosyl diglycerides—Significance to the structure of the thylakoid membrane. In Advances
in the Biochemistry and Physiology of Plant Lipids; Appelqvist, L.Å., Liljenberg, C., Eds.;
Elsevier/North Holland Biomedical Press: Amsterdam, The Netherlands, 1979; pp. 27–33.
6. Endo, T.; Inoue, K.; Nojima, S. Physical properties and barrier functions of synthetic
glyceroglycolipids. J. Biochem. 1982, 92, 953–960.
7. Mannock, D.A.; Lewis, R.N.A.H.; Sen, A.; McElhaney, R.N. The physical properties of
glycosyldiacylglycerols. Calorimetric studies of a homologous series of 1,2-di-O-acyl-3-O-(beta-
D-glucopyranosyl)-sn-glycerols. Biochemistry 1988, 27, 6852–6859.
8. Hashim, R.; Mirzadeh, S.M.; Heidelberg, T.; Minamikawa, H.; Yoshiaki, T.; Sugimur, A.
A reevaluation of the epimeric and anomeric relationship of glucosides and galactosides in
thermotropic liquid crystal self-assemblies. Carbohydr. Res. 2011, 346, 2948–2956.
9. Van Meer, G. Dynamic Transbilayer Lipid Asymmetry. Cold Spring Harb. Perspect. Biol. 2011,
3, A004671.
10. Finean, J.B.; Michell, R.H. Membrane Structure; Elsevier/North Holland Biomedical Press:
Amsterdam, The Netherlands, 1981.
11. Neurath, A.R.; Hartzell, R.W.; Rubin, B.A. Solubilization and some properties of the erythrocyte
receptor for adenovirus type 7 haemagglutinin. Nature 1969, 221, 1069–1071.
12. Kusumoto, S.; Fukase, K.; Fujimoto, Y. Chemical synthesis of bacterial lipid A. In Microbial
Glycobiology; Moran, A.P., Ed.; Elsevier: Amsterdam, The Netherlands, 2009; pp. 415–427.

Molecules 2013, 18
13571
13. Pedersen, C.M.; Bols, M.; Qiao, Y. Total synthesis of biologically active lipoteichoic acids.
ARKIVOC 2013, 2, 249–275.
14. Pedersen, C.M.; Schmidt, R.R. Chemical synthesis of lipoteichoic acids and derivatives. In
Microbial Glycobiology; Moran, A.P., Ed.; Elsevier: Amsterdam, The Netherlands, 2009;
pp. 455–476.
15. Pedersen, C.M.; Figueroa-Perez, I.; Lindner, B.; Ulmer, A.J.; Zähringer, U.; Schmidt, R.R. Total
synthesis of lipoteichoic acid of streptococcus pneumonia. Angew. Chem. Int. Ed. 2010, 49,
2585–2590.
16. Stadelmaier, A.; Morath, S.; Hartung, T.; Schmidt, R.R. Synthesis of the first fully active
lipoteichoic acid. Angew. Chem. Int. Ed. 2003, 42, 916–920.
17. Zähringer, U.; Pedersen, C.M.; Qiao, Y.; Schmidt, R.R. Chemical synthesis of bacterial lipoteichoic
acids: An insight on its biological significance. Org. Biomol. Chem. 2011, 9, 2040–2052.
18. Cateni, F.; Bonivento, P.; Procida, G.; Zacchigna, M.; Favretto, L.G.; Scialino, G.; Banfi, E.
Chemoenzymatic synthesis and antimicrobial activity evaluation of monoglucosyl diglycerides.
Bioorg. Med. Chem. 2007, 15, 815–826.
19. Mannock, D.A.; Lewis, R.N.A.H.; McElhaney, R.N. An improved procedure for the preparation
of 1,2-di-O-acyl-3-O-(β-D-glucopyranosyl)-sn-glycerols. Chem. Phys. Lipids 1987, 43, 113–127.
20. Nishida, Y.; Shingu, Y.; Mengfei, Y.; Fukuda, K.; Dohi, H.; Matsuda, S.; Matsuda, K. An easy
α-glycosylation methodology for the synthesis and stereochemistry of mycoplasma α-glycolipid
antigens Beilstein. J. Org. Chem. 2012, 8, 629–639.
21. Examples Of Glycerol Epimerization Under Glycosylation Conditions: Shvets, V.I.;
Bashkatova, A.I.; Evstigneeva, R.P. Synthesis of glycosyl diglycerides. Chem. Phys. Lipids 1973,
10, 267–285.
22. Gordon, D.M.; Danishefsky, S.J. Synthesis of a cyanobacterial sulfolipid: Confirmation of its
structure, stereochemistry and anti-HIV-1 activity. J. Am. Chem. Soc. 1992, 114, 659–663.
23. Imai, H.; Oishi, T.; Kikuchi, T.; Hirama, M. Concise Synthesis of 3-O-(2-O-α-D-Glucopyranosyl-
6-O-acyl-α-D-glucopyranosyl)-1,2-di-O-acyl-sn-glycerols. Tetrahedron 2000, 56, 8451–8459.
24. Shingu, Y.; Nishida, Y.; Dohi, H.; Matsuda, K.; Kobayashi, K. Convenient access to halide
ion-catalyzed a-glycosylation free from noxious fumes at the donor synthesis.
J. Carbohyd. Chem. 2002, 21, 605–611.
25. Manzo, E.; Ciavatta, M.L.; Pagano, D.; Fontana, A. An efficient and versatile chemical synthesis
of bioactive glyco-glycerolipids. Tetrahedron Lett. 2012, 53, 879–881.
26. Blattner, R.; Furneaux, R.H.; Pakulski, Z. 1,3-Dideoxynojirimycin-3-yl glycosides of β-(1→3)-
and β-(1→6)-linked gluco-oligosaccharides. Carbohydr. Res. 2006, 341, 2115–2125.
27. Figueroa-Perez, I. Synthesis of fragments and one analogue of S. Aureus LTA. Investigations
toward the synthesis of S. Pneumoniae LTA. PhD Thesis, University of Konstanz, Germany, 2007.
28. Xu, W.; Springfield, S.; Koh, J.T. Highly efficient synthesis of 1-thioglycosides in solution and
solid phase using iminophosphorane bases. Carbohydr. Res. 2000, 325, 169–176.
29. Schmidt, R.R.; Behrendt, M.; Toepfer, A. Nitriles as solvents in glycosylation reactions:
Highly selective β-glycoside synthesis. Synlett 1990, 11, 694–696.
30. Kumar, A.; Geng, Y.; Schmidt, R.R. Silicon fluorides for acid-base catalysis in glycosidations.
Adv. Synth. Catal. 2012, 354, 1489–1499.

Molecules 2013, 18
13572
31. Whitfield and coworkers have used 2-O-benzoyl protective groups in the synthesis of archaeal-
derived glycolipid adjuvants. The optimized conditions gave high yields and -selectivity; see:
Whitfield, D.M.; Yu, S.H.; Dicaire, C.J. Sprott, G.D. Development of new glycosylation
methodologies for the synthesis of archaeal-derived glycolipid adjuvants. Carbohydr. Res. 2010,
345, 214–229.
32. Uriel, C.; Gomez, A.M.; López, J.C.; Fraser-Reid, B. Some aspects of selectivity in the reaction of
glycosyl donors. J. Carbohyd. Chem. 2005, 24, 665–675.
33. Pedersen, C.M.; Figueroa-Perez, I.; Boruwa, J.; Lindner, B.; Ulmer, A.J.; Zähringer, U.;
Schmidt, R.R. Synthesis of the core structure of the lipoteichoic acid of streptococcus pneumonia.
Chem. Eur. J. 2010, 16, 12627–12641.
34. Ishizuka, I.; Yamakawa, T. Glycoglycerolipids. In New Comprehensive Biochemistry–
Glycolipids; Neuberger, A., Van Deenen, L.L.M., Eds.; Elsevier: Amsterdam, The Netherlands,
1985; Volume 10, pp. 101–197.
35. Kates, M. Glyco-, Phosphoglyco- and sulfoglycoglycerolipids of bacteria. In Handbook of Lipid
Research–Glycolipids, Phosphoglycolipids, and Sulfoglycolipids; Kates, M., Ed.; Plenum Press:
New York, NY, USA, 1990; Volume 6, pp. 1–122.
36. Harroun, T.A.; Koslowsky, M.; Nieh, M.-P.; DeLannoy, C.-F.; Raghunathan, V.A.; Katsaras, J.
Comprehensive examination of mesophases formed by DMPC and DHPC mixtures. Langmuir
2005, 21, 5356–5361.
37. Hauser, H.; Shipley, G. Interactions of divalent cations with phosphatidylserine bilayer
membranes. Biochemistry 1984, 23, 34–41.
38. Trouard, T.P.; Mannock, D.A.; Lindblom, G.; Rilfors, L.; Akiyama, M.; Mcelhaney, R.N.
Thermotropic phase properties of 1,2-di-O-tetradecyl-3-O-(3-O-methyl- beta-D-glucopyranosyl)-
sn-glycerol. Biophys. J. 1994, 67, 1090–1100.
39. Roux, M.; Neumann, J.-M. Deuterium NMR study of head-group deuterated phosphatidylserine
in pure and binary phospholipid bilayers. Interactions with monovalent cations Na+ and Li+.
FEBS Lett. 1986, 199, 33–38.
40. Larsen, J.; Hatzakis, N.S.; Stamou, D. Observation of inhomogeneity in the lipid composition of
individual nanoscale liposomes. J. Am. Chem. Soc. 2011, 133, 10685–10687.
41. Åkesson, A.; Lundgaard, C.V.; Ehrlich, N.; Pomorski, T.G.; Stamou, D.; Cárdenas, M.
Composition and structure of mixed phospholipid supported bilayers formed by POPC and DPPC.
Soft Matter 2012, 8, 8972–8980.
42. Ruggeri, F.; Akesson, A.; Chapuis, P.Y.; Nielsen , C.A.S.; Monopol, M.P.; Dawson, K.A.;
Pomorski, T.G.; Cardenas, M. The dendrimer impact on vesicles can be tuned based on the lipid
bilayer charge and the presence of albumin. Soft Matter 2013, 9, 8862–8870.
43. Sengupta, P.; Baird, B.; Holowka, D. Lipid rafts, Fluid/fluid phase separation, and their relevance
to plasma membrane structure and function. Semin. Cell. Dev. Biol. 2007, 18, 583–590.
44. Boyd, B.J.; Drummond, C.J.; Krodkiewska, I.; Grieser, F. How chain length, Headgroup
polymerization, and anomeric configuration govern the thermotropic and lyotropic liquid
crystalline phase behavior and the air-water interfacial adsorption of glucose-based surfactants.
Langmuir 2000, 16, 7359–7367.

Molecules 2013, 18
13573
45. Huang J.; Feigenson, G.W. A microscopic interaction model of maximum solubility of cholesterol
in lipid bilayers. Biophys. J. 1999, 76, 2142–2157.
46. Takuro, K.; Nobue, S.; Ikuo, G.; Yoshigoroa, K.; Makoto, O.; Gosuke, S.; Mitsuru, T.
Incorporation and degradation of GM1 ganglioside and asialoGm1 ganglioside in cultured
fibroblasts from normal individuals and patients with β-galactosidase deficiency. Biochim.
Biophys. Acta 1985, 31, 456–464.
47. Veath, S.L.; Keller, S.L. Separation of liquid phases in giant vesicles of ternary mixtures of
phospholipids and cholesterol. Biophys. J. 2003, 85, 3074–3083.
48. Yahi, N.; Aulas, A. Fantini, J. How Cholesterol Constrains Glycolipid Conformation for Optimal
Recognition of Alzheimer's β Amyloid Peptide (Aβ1-40). PLoS One 2010, 5, e9079.
49. Martin, D.K. Nanobiotechnology of Biomimetic Membranes; Springer: New York, NY, USA, 2007.
50. Harder, T.; Simons, K. Clusters of glycolipid and glycosylphosphatidylinositol-anchored proteins
in lymphoid cells: Accumulation of actin regulated by local tyrosine phosphorylation. Eur. J.
Immunol. 1999, 29, 556–562.
51. Varki, A. Glycobiology and medicine: An introduction. Glycolipids 1993, 3, 97–130.
Sample Availability: Samples of the compounds are available from the authors.
© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).