Gb3 Glycosphingolipids with Fluorescent Oligoene Fatty Acids: Synthesis and Phase Behavior in Model Membranes

Article abstract of DOI:10.1002/cbic.201700414

Glycosphingolipids are involved in a number of physiological and pathophysiological processes, and they serve as receptors for a variety of bacterial toxins and viruses. To investigate their function in lipid membranes, fluorescently labeled glycosphingolipids are highly desirable. Herein, a synthetic route to access Gb₃ glycosphingolipids with fluorescently labeled fatty acids, consisting of pentaene and hexaene moieties either at the terminus or in the middle of the acyl chain, has been developed. The fluorescent properties of the Gb₃ derivatives were investigated in small unilamellar vesicles composed of a raft-like mixture. Phase-separated giant unilamellar vesicles (GUVs) allowed the quantification of the apparent partitioning coefficients of the Gb₃ compounds by means of confocal fluorescence laser scanning microscopy. The determined partition coefficients demonstrate that the Gb₃ derivatives are preferentially localized in the liquid-disordered (l_d) phase. To analyze whether the compounds behave like their physiological counterparts, Cy3-labeled (Cy: cyanine) Shiga toxin B subunits (STxB) were specifically bound to Gb₃-doped GUVs. However, the protein was favorably localized in the l_d phase, in contrast to results reported for STxB bound to naturally occurring Gb₃, which is discussed in terms of the packing density of the lipids in the liquid-ordered (l_o) phase.

Full text of DOI:10.1002/cbic.201700414

DOI: 10.1002/cbic.201700414
Full Papers
Gb3 Glycosphingolipids with Fluorescent Oligoene Fatty
Acids: Synthesis and Phase Behavior in Model Membranes
Lukas J. Patalag+,[a] Jeremias Sibold+,[b] Ole M. Schꢀtte,[b] Claudia Steinem,*[b] and
Daniel B. Werz*[a]
Glycosphingolipids are involved in a number of physiological
and pathophysiological processes, and they serve as receptors
for a variety of bacterial toxins and viruses. To investigate their
function in lipid membranes, fluorescently labeled glyco-
sphingolipids are highly desirable. Herein, a synthetic route to
access Gb3 glycosphingolipids with fluorescently labeled fatty
acids, consisting of pentaene and hexaene moieties either at
the terminus or in the middle of the acyl chain, has been de-
veloped. The fluorescent properties of the Gb3 derivatives
were investigated in small unilamellar vesicles composed of a
raft-like mixture. Phase-separated giant unilamellar vesicles
(GUVs) allowed the quantification of the apparent partitioning
coefficients of the Gb3 compounds by means of confocal fluo-
rescence laser scanning microscopy. The determined partition
coefficients demonstrate that the Gb3 derivatives are preferen-
tially localized in the liquid-disordered (ld) phase. To analyze
whether the compounds behave like their physiological coun-
terparts, Cy3-labeled (Cy: cyanine) Shiga toxin B subunits
(STxB) were specifically bound to Gb3-doped GUVs. However,
the protein was favorably localized in the ld phase, in contrast
to results reported for STxB bound to naturally occurring Gb3,
which is discussed in terms of the packing density of the lipids
in the liquid-ordered (lo) phase.
Introduction
Eukaryotic plasma membranes of animals comprise a large
number of lipids, including glycerophospholipids, cholesterol
(Chol), and sphingomyelin (SM). In addition, the outer leaflet of
the plasma membrane harbors glycosphingolipids, which can
serve as receptor molecules for neighboring cells and are in-
volved in the formation of raft domains through interactions
with Chol.[1,2] Glycosphingolipids are also specific attachment
sites for a number of bacteria, viruses, and proteins.[3] Among
them, bacterial toxins are known to bind to gangliosides or
globosides. For example, cholera toxin uses the ganglioside
GM1 to enter the cell,[4] whereas Shiga toxin (STx) binds specifi-
cally to the globoside Gb3.[5–7] Cholera toxin, a typical AB5 pro-
tein, is produced by Vibrio cholerae and has been widely used
as a marker for raft-like liquid-ordered (lo) domains in phase-
separated membranes.[2,8] The bacterial STx is produced by Shi-
gella dysenteriae and by enterohemorrhagic strains of Escheri-
chia coli. With the AB5 structure, it harbors five B subunits
(STxB) capable of binding to the trisaccharidic carbohydrate
structure of Gb3.[5,9–11] Clustering of Gb3 upon STxB binding has
been shown to induce invaginations as the first step of inter-
nalization of the toxin into the cell.[6] Although STxB can serve
as a marker to localize Gb3 in the membrane, virtually nothing
is known about the distribution of Gb3, its dynamics, and inter-
action with other lipid components in the absence of bound
STxB. To localize glycosphingolipids in membranes by optical
microscopy, a fluorescent label is required. Several attempts
have been made to label the gangliosides GM1 and GM3.[8,12–15]
The fluorescent probes were attached to either the fatty acid
side chain[16] or the sugar chains.[14,17] However, if characterized
in detail, the behavior of these ganglioside derivatives were
quite different from those of the native molecules. In particular,
derivatives with bulky fluorophores in the hydrophobic part of
the membrane do not partition into the lo phase as expect-
ed.[13] The same holds true for the commercially available fatty
acid-labeled (nitrobenzoxadiazole (NBD)-labeled) Gb3. Ganglio-
sides tagged with hydrophilic fluorophores are capable of par-
titioning into the lo phase.[14] However, their binding properties
to viruses and proteins, including toxins, might be hampered
due to the bulky fluorophore at the head group.
[a] L. J. Patalag,+ Prof. Dr. D. B. Werz
TU Braunschweig, Institut fꢀr Organische Chemie
Hagenring 30, 38106 Braunschweig (Germany)
E-mail: d.werz@tu-braunschweig.de
[b] J. Sibold,+ Dr. O. M. Schꢀtte, Prof. Dr. C. Steinem
Georg-August-Universitꢁt Gçttingen
Institut fꢀr Organische und Biomolekulare Chemie
Tammannstrasse 2, 37077 Gçttingen (Germany)
E-mail: claudia.steinem@chemie.uni-goettingen.de
To design a fluorescently labeled Gb3 molecule able to parti-
tion into the lo phase and serve as a receptor for proteins, such
as STxB, a fatty acid labeled structure is desirable. The envi-
sioned fatty acid should fit into the tightly packed lo phase.
GoÇi and co-workers showed that polyene ceramide analogues
could serve as membrane probes.[18] They designed a polyene
ceramide analogue termed pentaene I, which appeared to par-
[+] These authors contributed equally to this work.
Supporting information and the ORCID identification numbers for the
authors of this article can be found under https://doi.org/10.1002/
cbic.201700414: detailed experimental procedures, synthetic methods,
chemical compound information, and supplementary results for Dye731-
DOPE.
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tition into the gel phase of a membrane, and the fluorescence
emission of this molecule was more intense in the gel than
that in the fluid phase. However, in contrast to this finding, the
group concluded from differential calorimetry data that pen-
taene I partitioned preferentially in the fluid phase.
such as NBD,[21] pyrene,[22] boron-dipyrromethene (BODIPY),[23]
boron complexes of iminopyrrolide ligands (BOIMPY),[24] or
benzothiadiazole,[25] but prepared fatty acids consisting of pen-
taene and hexaene moieties either at the terminus or in the
middle of the acyl chain.[19] Their slim shape and hydrophobic
scaffold should only disturb the membrane integrity to a
minor extent.[26] However, these molecular entities proved to
be rather unstable in the presence of both air and strong laser
beams. In addition, an absorption far below l=400 nm ap-
pears to be ill-suited to standard fluorescence microscopy. To
increase the stability of the fluorescent core structure and
improve the advantageous spectroscopic properties without
largely increasing the structural bulkiness, we designed two
new fluorescent fatty acids with either a phenyl (1) or a thieno-
thienyl residue (2) in conjugation to an oligoene system
(Scheme 1). Both aryl moieties extend the p system and are
expected to red-shift the absorption and emission spectra.
We commenced our synthesis with benzaldehyde (3), which
was subjected to a Horner–Wadsworth–Emmons reaction by
using allyl phosphonate 4, leading to unsaturated ester 5 in
79% yield (Scheme 2). The phosphonate allows the introduc-
Herein, we have taken this general approach and designed
fluorescently labeled Gb3 molecules with fatty acids consisting
of pentaene and hexaene moieties either at the terminus or in
the middle of the acyl chain,[19] with the hypothesis that they
can partition into the lo phase of phase-separated lipid bilayers
and are still able to bind the protein STxB.
Results and Discussion
Syntheses of glycosphingolipids with fluorescent fatty acids
Chemical synthesis of Gb3 glycosphingolipids enabled us to
access two glycoconjugates, 1 and 2, differing in their fatty
acid part (Scheme 1). In order not to alter the membrane struc-
ture and dynamics to a major extent, we did not consider fatty
acids with embedded polar and/or bulky fluorescent moieties,
Scheme 1. Gb3 glycoconjugates 1 and 2 with fluorescent fatty acids based on terminal oligoene systems.
Scheme 2. Synthesis of pentaene fatty acid 9 with a phenyl substituent at the terminus. a) 4, lithium bis(trimethylsilyl)amide (LiHMDS; 79%); b) diisobutyl alu-
minium hydride (DIBAL); c) MnO2 (74%); d) 4, nBuLi (24%); e) DIBAL; f) MnO2 (55%); g) LiHMDS, nBuLi (20%); h) I2 (cat.).
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tion of two E-configured C=C double bonds in a one-pot pro-
cess. Reduction to the alcohol and subsequent oxidation with
manganese dioxide led to aldehyde 6.
jected to a formal carbonylation by using DMF as a carbonyl
source to afford 12 in 70% yield (Scheme 3).
For the formation of the unsaturated diene, Horner–Wads-
worth–Emmons reagent 4 was employed.[27] The emerging
ester 13 was transformed via an alcohol into the correspond-
ing aldehyde 14, which proved to be the crucial intermediate
for elongation to the final fatty acid through a Wittig re-
action.[28] Catalytic amounts of iodine allowed isomerization to
all-E-system 15.
To obtain the desired glycolipids 1 and 2, perbenzoylated
globotriaosyl trichloroacetimidate 16 was submitted to a gly-
cosylation reaction with azide-protected sphingosine alcohol
17 (Scheme 4).[10,11] Under the influence of boron trifluoride as
a Lewis acid, protected trisaccharide 18 was obtained in 41%
yield. Staudinger reduction, subsequent amide formation by
using our novel fatty acids, and saponification under Zemplꢂn
conditions led to glycosphingolipids 1 and 2.
This species is converted into tetraene ester 7 with phos-
phonate 4. The respective aldehyde 8 was again obtained by a
reduction/oxidation protocol. To install the fifth double bond
of the pentaene system, a Wittig reaction proved to be the
method of choice. A mixture of the corresponding (E/Z) iso-
mers was obtained in 20% yield. Treatment with catalytic
amounts of iodine triggered a gentle isomerization to give the
desired all-E isomer 9. Due to the enlarged p system, the ab-
sorption maximum of 9 is red-shifted by about 35 nm (from
l=350 to 385 nm) in comparison to a non-arylated counter-
part.
The synthetic route to thienothienyl-modified fatty acid 15
resembles that previously described for 9. First, thienothio-
phene-2-carbaldehyde (10) was engaged in a Wittig reaction
to give isopropylidene-equipped product 11, which was sub-
Scheme 3. Synthesis of thienothienyl-modified fatty acid 15. a) iPrP+Ph3 Iꢀ, WS2; b) NaHMDS (95%); c) lithium tetramethylpiperidide; d) DMF (70%); e) 4;
f) nBuLi (82%); g) DIBAL; h) MnO2 (36%); i) Ph3P+(CH2)9COOH Brꢀ, WS3; j) nBuLi; k) I2 (cat.) (67%).
Scheme 4. Assembly of Gb3 glycosphingolipids 1 and 2 with fluorescent fatty acids. BF3·OEt2, 4 ꢃ MS, CH2Cl2/hexane (1:1) (41%); b) PPh3, benzene/H2O; c) 1-
[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), N,N-diisopropylethylamine (DIPEA), 9 or 14;
d) NaOMe, CH3OH.
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Scheme 5. Preparation of fluorescent Dy731-DOPE (20). The fluorophore is attached through a long PEG chain to the respective lipid. a) NEt3, MeCN, 3 days
(dark); b) DOPE (2 equiv), DIPEA (4 equiv), HATU (1.5 equiv), DMF/CHCl3, 358C, 14 h (65% over 2 steps).
Apart from the fluorescent labeling of the glycolipids, 1,2-di-
(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE)
also had to be labeled by another dye. Therefore, we attached
the fluorophore Dy731 through amide coupling to a long
poly(ethylene glycol) (PEG) linker (n=24). Emerging product
19 was further conjugated to DOPE to afford 20 (Scheme 5).
This highly amphiphilic conjugate was purified by reversed-
phase column chromatography (RP-18).
taining a pentaene,[18] the excitation spectra are slightly red-
shifted owing to the extended p system. The fluorescence
emission spectrum of compound 1 is broad, ranging from l=
400 to 650 nm with a fine structure with three maxima at l=
410, 434, and 458 nm. Compound 2 also shows a broad fluo-
rescence emission spectrum in the range of 400–650 nm with
one maximum at lꢁ440 nm, which is very similar to previous-
ly reported pentaene systems.[18]
Excitation and emission spectra of compounds 1
and 2
First, the optical properties of compounds 1 and 2
reconstituted into small unilamellar vesicles (SUVs)
composed of 1,2-dioleoyl-sn-glycero-3-phosphocho-
line (DOPC)/SM/Chol/Gb3 (40:35:20:5) were analyzed
(Figure 1). The excitation spectrum of 1 shows two
characteristic bands at l=347 and 366 nm and a
shoulder at l=330 nm, which is typical for the vibra-
tional resolution of polyene chromophores.[29] Simi-
Figure 1. Excitation (red) and emission (green) spectra of the fatty acid labeled Gb3-spe-
cies 1 (A) and 2 (B) in SUVs composed of DOPC/SM/Chol/Gb3 (40:35:20:5). The fluores-
cence excitation spectra were measured from l=300 to 445 nm, with lem =465 nm for
both Gb3 species. Fluorescence emission was excited at A) lex =348 nm, recorded from
l=358 to 669 nm, and at B) lex =390 nm, recorded from l=400 to 669 nm.
larly, two excitation bands are found for 2; however,
these are shifted to longer wavelengths (l=369 and
391 nm) as a result of the thiophene moiety in the
chain. Compared with a sphingosine-like chain con-
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Fluorescence emission spectra in phase-separated lipid bi-
layers
suited to quantify the partition of the Gb3 species in phase-
separated lipid bilayers. A fluorescent dye that partitions pref-
erentially into the ld phase with an absorption spectrum shifted
to longer wavelengths that does not overlap with the emission
spectra of the Gb3 species is required. Thus, we synthesized
Dy731-DOPE, which had an absorption maximum of l=
732 nm. SUVs composed of DOPC/SM/Chol/Gb3/Dy731-DOPE
(39.5:35:20:5:0.5) show the fluorescence emission spectra de-
picted in Figure 2 (green lines). Only a very small FRET peak at
lꢁ760 nm is observed. FRET does not significantly decrease
the emission of the donor, that is, compounds 1 and 2, respec-
tively, which makes this dye suited for further investigations to
quantify the partition of the two compounds in phase-separat-
ed GUVs.
In phase-separated liquid-ordered (lo)/liquid-disordered (ld)
membranes, glycosphingolipids are discussed to preferentially
partition into the lo phase. To investigate the partition behavior
of the fluorescent Gb3 species 1 and 2 in phase-separated lipid
bilayers, again a lipid mixture composed of DOPC/SM/Chol/Gb3
(40:35:20:5) was used; this is known to phase separate.[10,11,30]
For a quantitative analysis of the partition of the Gb3 species in
giant unilamellar vesicles (GUVs), as described below, the ld
phase needs to be unambiguously assigned; this is achieved
by using an ld phase marker. To first investigate whether the
synthesized fluorescent Gb3 species were compatible with an ld
phase marker, SUVs composed of the given lipid mixture were
prepared, in which DOPC (0.5 mol%) was replaced by a fluo-
rescent dye that partitioned into the ld phase. In the first at-
tempt, sulforhodamine-1,2-dihexanoyl-sn-glycero-3-phospho-
ethanolamine (Texas Red-DHPE) was chosen.[13,31] The resulting
fluorescence spectra (Figure 2, red lines) clearly indicate that
FRET between the fluorescent Gb3 species and the Texas Red
dye occurs as a result of an overlap of the emission spectra of
compounds 1 and 2 and the absorption spectrum of Texas
Red. From this result, we conclude that Texas Red-DHPE is not
Partition of fluorescent Gb3 in phase-separated GUVs
The most straightforward approach to study lipid domains is
to use GUVs doped with lipid-like dyes that partition specifical-
ly in either the lo or ld phase.[13,31] We took fluorescence micro-
graphs of GUVs composed of DOPC/SM/Chol/Gb3/Dy731-DOPE
(39.5:35:20:5:0.5) to quantify the partition of the fluorescent
Gb3 species in these phase-separated membranes (Figure 3).
The dye Dy731-DOPE is almost exclusively localized in the ld
phase, as expected (Figure 3B and D, Figure S23 in
the Supporting Information). However, compound 1
appears to be more homogeneously distributed
among the lo and ld phase (Figure 3A). As a first esti-
mate, we determined the fraction of Gb3 species
partitioning into the lo phase from intensity line pro-
files of confocal images (Figure 3). The fluorescence
intensities of the lo and ld phase, Il and Il , respec-
o
d
tively, were determined from the peaks of the line
scans, for which the two different phases were iden-
tified by the ld phase marker Dy731-DOPE.[13] The ap-
parent partitioning coefficient (%lo) is calculated ac-
cording to Equation (1):
Figure 2. Fluorescence spectra of A) 1 (lex =348 nm) and B) 2 (lex =390 nm). Blue lines:
fluorescence spectra obtained from vesicles composed of DOPC/SM/Chol/Gb3 species
(40:35:20:5); red lines: fluorescence spectra obtained from vesicles composed of DOPC/
SM/Chol/Gb3 species/Texas Red-DHPE (39.5:35:20:5:0.5); green lines: fluorescence spectra
obtained from vesicles composed of DOPC/SM/Chol/Gb3 species/Dy731-DOPE
(39.5:35:20:5:0.5).
Il ꢂ 100
o
% lo ¼
ð1Þ
Il þ Il
o
d
Figure 3. Confocal fluorescence images of GUVs composed of DOPC/SM/Chol/Gb3/Dy731-DOPE (39.5:35:20:5:0.5). A) Fluorescence of 1 and B) the correspond-
ing fluorescence of Dy731-DOPE. C) Fluorescence of 2 and D) the corresponding fluorescence of Dy731-DOPE. Line scans, which were used for the analysis,
are highlighted in yellow and the intensity profiles from these line scans are shown in the bottom row.
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Equation (1) neglects that the fluorescent yield could be slight-
ly different in the two phases.[13] For compound 1, the appar-
ent partitioning coefficient was determined to be %lo =(45ꢃ
22)% (n=2175), which indicated that the fluorescent Gb3
species was almost equally distributed between the lo and ld
phase. For compound 2, the apparent partitioning coefficient
was %lo =(24ꢃ10)% (n=2003); this demonstrated that this
Gb3 species was preferentially localized in the ld phase.
Yet, not much is known about how Gb3 distributes between
the lo and ld phase prior to binding of a protein, such as STx.
However, it is well established that, upon binding of STxB sub-
units to Gb3-doped phase-separated GUVs, Gb3 preferentially
partitions into the lo phase.[6,32] Thus, we analyzed whether the
fluorescent Gb3 species were recruited into the lo phase upon
binding of STxB.
To further prove that, indeed STxB has been bound to the
Gb3 species, Cy3-labeled (Cy: cyanine) STxB was used. For both
fluorescent Gb3 species, STxB binds to the ld phase (Figure 5),
in contrast to results obtained for natural (Figure S24)[6,32]
or synthetic[10,11] unlabeled Gb3 species. This result clearly
indicates that the fluorescent Gb3 species do not behave like
natural Gb3 molecules, owing to the altered fatty acid struc-
ture.
It is known that sphingolipids containing BODIPY-FL or NBD
as fluorophores in the fatty acid chain prevent their incorpora-
tion into the lo phase, owing to bulkiness inserted into the
hydrophobic part of the membrane.[12,13,15,31] Moreover, it has
been shown that GM1, which is the natural receptor lipid of
cholera toxin, partitions and binds cholera toxin exclusively in
the ld phase, if the acyl chain is labeled with BODIPY-FL, in con-
trast to what has been reported for unlabeled GM1.[12,13,33]
However, the pentane moiety used in 1 was expected to fit
into the lo-phase structure because the length of the fatty acid
matches that of the naturally occurring Gb3 molecules, with
fatty acid chain lengths of 24 carbon atoms, while the aromatic
ring is attached at the very end. Our results, however, clearly
indicate that the pentaene structure, with the terminal aromat-
ic ring, is apparently already sufficiently bulky to prevent close
packing of the Gb3 molecules in the lo phase upon STxB bind-
ing. The same holds true for 2, which is even largely excluded
from the lo phase. In the absence and in the presence of
bound STxB, the thiophene moiety, which is positioned further
Partition of Gb3 in the presence of bound STxB
GUVs
composed
of
DOPC/SM/Chol/Gb3/Dy731-DOPE
(39.5:35:20:5:0.5) were incubated with 60 nm unlabeled STxB
(Figure 4). Again, for compound 1, an apparent partitioning co-
efficient was determined to be %lo =(41ꢃ21)% (n=2103);
this clearly demonstrates that the receptor lipid Gb3 does not
change its partition behavior upon STxB binding. The same
result was obtained for compound 2, with an apparent parti-
tioning coefficient of %lo =(24ꢃ11) % (n=2329).
Figure 4. Confocal fluorescence images of GUVs composed of DOPC/SM/Chol/Gb3/Dy731-DOPE (39.5:35:20:5:0.5) with 60 nm STxB in solution. A) Fluorescence
of 1 and B) the corresponding fluorescence of Dy731-DOPE. C) Fluorescence of 2 and D) the corresponding fluorescence of Dy731-DOPE. Line scans, which
were used for the analysis, are highlighted in yellow and the intensity profiles from these line scans are shown in the bottom row.
Figure 5. Confocal fluorescence images of GUVs composed of DOPC/SM/Chol/Gb3/Dy731-DOPE (39.5:35:20:5:0.5) with 60 nm Cy3-labeled STxB in solution.
A) Fluorescence of Cy3-STxB bound to 1 and B) the corresponding fluorescence of Dy731-DOPE. C) Fluorescence of Cy3-STxB bound to 2 and D) the corre-
sponding fluorescence of Dy731-DOPE.
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Fluorescence microscopy: Fluorescence images were recorded
with a confocal laser scanning microscope (FV 1200; Olympus,
Hamburg, Germany) equipped with a water immersion objective
(UPlanSApo N 60ꢄ/1.20 NA; Olympus, Hamburg, Germany). The
fluorescence of both Gb3 species was excited at l=405 nm and
detected at l=440–540 nm. The excitation wavelength for Cy3-
STxB was l=561 nm and fluorescence was detected from l=575
to 620 nm. Dy731-DOPE was excited with the laser line at l=
635 nm and emission was recorded from l=655 to 755 nm.
to the center of the lipid monolayer, is already too bulky to be
placed in the lo phase.
Conclusion
Two new fluorescently labeled Gb3 globosides were synthe-
sized, containing pentaene or hexaene moieties that allowed
localization of Gb3 molecules in phase-separated lipid bilayers.
Because the molecules are labeled with fatty acids, binding of
STx to the head group of the receptor lipid is preserved. How-
ever, the phase behavior of the fatty acid labeled Gb3 species
is greatly influenced, as shown by STxB binding, which binds
to the ld phase and not to the lo phase, as known from natural-
ly occurring Gb3-containing membranes. This finding strongly
suggests that even small changes in the packing density in the
lo phase can largely alter the phase behavior of the glycosphin-
golipids. To combine a fluorescence label with a glycosphingo-
lipid that retains its natural phase behavior, a more promising
approach would be to use head group labeled lipids with a
long spacer and attached to a site at the head group of the
glycosphingolipid not involved in protein binding.
Acknowledgements
We thank the DFG (SFB 803, project A05) for financial support.
Conflict of Interest
The authors declare no conflict of interest.
Keywords: fluorescence spectroscopy
membranes · proteins · Shiga toxin · vesicles
·
glycolipids
·
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Experimental Section
Materials: DOPC and SM from porcine brain were purchased from
Avanti Polar Lipids. Chol and Texas Red-DHPE were obtained from
Sigma–Aldrich. STxB and Cy3-labeled Shiga toxin B subunits (Cy3-
STxB) were purified as described previously.[20]
SUVs: SUVs either composed of DOPC/SM/Chol/Gb3 (40:35:20:5) or
DOPC/SM/Chol/Gb3/ld marker (39.5:35:20:5:0.5) were prepared by
extrusion. A solution (200 mL) of lipid in chloroform (0.5 mgmLꢀ1
was dried at the bottom of glass test tubes at 558C in a stream of
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Manuscript received: August 1, 2017
Version of record online: && &&, 0000
&
ChemBioChem 2017, 18, 1 – 9
www.chembiochem.org
8
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ÝÝ These are not the final page numbers!

FULL PAPERS
Get in place! Fluorescent labels can
alter the phase behavior of lipids con-
siderably. The synthetic route to fatty
acid-labeled Gb3 derivatives that parti-
tion in the liquid-disordered phase,
even after binding of Shiga toxin B sub-
units, is described.
L. J. Patalag, J. Sibold, O. M. Schꢀtte,
C. Steinem,* D. B. Werz*
&& – &&
Gb3 Glycosphingolipids with
Fluorescent Oligoene Fatty Acids:
Synthesis and Phase Behavior in
Model Membranes
ChemBioChem 2017, 18, 1 – 9
www.chembiochem.org
9
ꢁ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ