Visualization of sialyl LewisX glycosphingolipid microdomains in model membranes as selectin recognition motifs using a fluorescence label

Article abstract of DOI:10.1016/j.carres.2008.07.004

Selectin-induced leukocyte rolling along the endothelial surface is an essential step in the cellular immune response. For efficient recognition, the relevant carbohydrate epitope sialyl Lewis^X (sLe^X; α-Neup5Ac-(2→3)-β-Galp-(1→4)-[α-Fucp-(1→3)]GlcpNAc) has to be arranged in clusters. We describe the synthesis of the sLe^X-glycosphingolipid (sLe^X-GSL) with a NBD fluorescence label in the tail region, which allows the direct visualization of sLe^X-GSL microdomains to very low concentrations (0.01 mol %) in various planar phosphocholine matrices by fluorescence microscopy. Cell rolling experiments of E-selectin expressing cells along these membranes confirmed that the fluorescence analog behaves similar to the naturally occuring sLe^X-GSL. This is direct evidence for recent hypotheses on multivalent sLe^X binding as molecular basis for selectin-mediated cell rolling.

Full text of DOI:10.1016/j.carres.2008.07.004

Carbohydrate Research 343 (2008) 2361–2368
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Visualization of sialyl LewisX glycosphingolipid microdomains in model
membranes as selectin recognition motifs using a fluorescence label
b
c
a
b
Christian Gege a, , Gabriele Schumacher , Ulrich Rothe , Richard R. Schmidt , Gerd Bendas
*
a Department of Chemistry, Universität Konstanz, Box M725, D-78457 Konstanz, Germany
b Department of Pharmacy, Rheinische Friedrich Wilhelms Universität Bonn, An der Immenburg 4, D-53121 Bonn, Germany
c Department of Physiological Chemistry, Martin Luther Universität Halle, Hollystrasse 1, D-06097 Halle, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Selectin-induced leukocyte rolling along the endothelial surface is an essential step in the cellular
immune response. For efficient recognition, the relevant carbohydrate epitope sialyl LewisX (sLeX;
-Neu-
p5Ac-(2?3)-b-Galp-(1?4)-[ -Fucp-(1?3)]GlcpNAc) has to be arranged in clusters. We describe the syn-
Received 21 May 2008
Received in revised form 2 July 2008
Accepted 3 July 2008
a
a
thesis of the sLeX-glycosphingolipid (sLeX-GSL) with a NBD fluorescence label in the tail region, which
allows the direct visualization of sLeX-GSL microdomains to very low concentrations (0.01 mol %) in var-
ious planar phosphocholine matrices by fluorescence microscopy. Cell rolling experiments of E-selectin
expressing cells along these membranes confirmed that the fluorescence analog behaves similar to the
naturally occuring sLeX-GSL. This is direct evidence for recent hypotheses on multivalent sLeX binding
as molecular basis for selectin-mediated cell rolling.
Available online 11 July 2008
Keywords:
Sialyl LewisX antigen
Glycolipid
NBD
Ó 2008 Elsevier Ltd. All rights reserved.
Microdomain
Fluorescence labeling
Selectin
1. Introduction
when arranged in lateral clusters11 in model membranes.12 The
essential role of clustering sLeX lipids to create selectin recognition
Glycolipids play an important role in various cell adhesion
events, examples of which include fertilization, embryonic devel-
opment, metastasis, myelin compaction, sponge cell aggregation,
and immunological responses.1–4 In the latter case, the selectins,
a family of three transmembrane glycoproteins (E-, L-, and P-selec-
tin), trigger the migration of leukocytes from the bloodstream to
specific sites of inflammation or injury by supporting their tether-
ing and rolling along the vessel wall.5 The underlying principle of
rolling is the fast kinetics of selectin interaction with their carbo-
hydrate ligands, which facilitates the rapid association and dissoci-
ation of bonds induced by the impact of shear force. The common
minimal binding epitope of the selectins is the sialyl LewisX
motifs was interpreted with respect to multivalent binding and
resulting increased binding avidity.13 Close spacing between
binding epitopes increase local ligand density and thereby achieve
sufficiently high rates of association with the selectin, and it also
facilitates local rebinding of the selectin to a neighboring ligand,
once it is dissociated from its previous carbohydrate ligand. Several
other features, for example, epitope flexibility and accessibility or
cluster size were investigated in our group and correlated with
selectin-dependent cell rolling.14–17 The cell rolling is balanced
by sLeX concentrations and could be achieved at about 0.01 mol %
sLeX-GSL 1 arranged in clusters. Although we could confirm sLeX
clustering at higher concentrations (>1 mol %) in dependence on
membrane matrix lipids by considering the phase separation of
fluorescence markers,12 a direct visualization of the sLeX lipid
appearance at the rolling-relevant concentrations was not possible.
Therefore, a direct fluorescence labeling of the sLeX-GSL was
aspired to.
Fluorescent analogs of naturally occurring lipids are widely
used in investigations dealing with biophysical aspects of mem-
branes, for example, lateral mobility or phase separation.18 Fluo-
rescence microscopy is a powerful tool to study microdomains in
supported lipid bilayers, which offer advantages over spherical
model membranes in terms of lipid asymmetry or staining of one
monolayer.19
(sLeX) tetrasaccharide
a-Neup5Ac-(2?3)-b-Galp-(1?4)-[a-Fucp-
(1?3)]GlcpNAc, which has become a prominent target for biolog-
ical studies.6 An important natural occurrence of this epitope is at
the terminal end of glycosphingolipids, wherein a lactose residue
serves as spacer to the ceramide moiety (Fig. 1).7 It was shown that
this sLeX-glycosphingolipid (sLeX-GSL) 1, which synthesis was
described recently,8–10 mediates selectin-dependent cell rolling
* Corresponding author at present address: X2-Pharma GmbH, Im Neuenheimer
Feld 584, D-69120 Heidelberg, Germany. Tel.: +49 6221 7390181.
E-mail address: christian.gege@web.de (C. Gege).
0008-6215/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2008.07.004

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C. Gege et al. / Carbohydrate Research 343 (2008) 2361–2368
Figure 1. Structure of the naturally occurring sialyl LewisX glycosphingolipid 18–10 and the synthesized fluorescence labeled analog 2 with NBD label in the membrane
anchor.
Among a variety of fluorescence labels,20 the 7-nitrobenz-
2-oxa-1,3-diazol-4-yl (NBD) group is the most widely used
fluorescent lipid analog in membrane studies because it is easily
chemically introduced, uncharged at neutral pH, and is highly fluo-
rescent at low concentrations in membranes.21,22 However, to
avoid any misleading results, the labeled lipid should resemble
its natural counterpart as closely as possible23 and it is crucial to
evaluate critically the reliability of the incorporated modification
and correlate the findings with additional experiments, for exam-
ple, with atomic force microscopy.24 To maintain close similarity
to the natural sLeX-GSL 1, we decided to attach the fluorophore
via an amide linkage to the terminal end of the ceramide moiety
to give compound 2 (Fig. 1). With a long dodecyl (C12) spacer,
the derivative should display a similar hydrophobicity as the natu-
ral counterpart, although it cannot be ruled out that even in this
position the NBD label is looping up to the surface of the model
membrane.21
Compound 2 was incorporated at rolling-relevant concentra-
tions into different lipid matrices, the lateral distribution was ana-
lyzed by fluorescence microscopy and related to cell rolling along
the membranes. The distribution of 2 could be imaged up to con-
centrations of 0.01 mol % in the membranes. The data confirm
the recent hypothesis of multivalent sLeX binding of selectins as
a functional prerequisite for cell rolling.12,15,17,25
ride stacking30,31 can be explained by an hydrophobic interaction
of the apolar sugar face with the phenyl group. The small coupling
constants of orthoester 6 indicate that the a-ring is not present in
the chair conformation as shown in Figure 2 but rather in the half-
chair conformation.
The fluorescence label was introduced into the alkyl chain by
reaction of 4-chloro-7-nitrobenzofurazan with 12-aminododeca-
noic acid in aqueous NaHCO3 at 50 °C as previously described for
a similar derivative32 to give 12-[N-(7-nitrobenz-2-oxa-1,3-dia-
zol-4-yl)amino]dodecanoic acid 7. Reduction of the azido group
in compound 5 with H2S in aqueous pyridine for 3 days, followed
by coupling of acid 7 with 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide hydrochloride (EDCI) afforded the protected target
compound 8 in high yield. Final removal of all O-acyl protecting
groups and saponification of the methyl ester furnished the target
sLeX-GSL 2, which was isolated as its highly hygroscopic triethyl-
ammonium salt after chromatography with CHCl3–MeOH–H2O–
NEt3 as eluent and lyophilization.
In recent studies, we could show that sLeX-GSL 1 is able to
mediate rolling of E-selectin expressing CHO cells when incorpo-
rated into a 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)
matrix. Cell adhesion and rolling is sensitively controlled by
sLeX-GSL concentration. Rolling occurred at around 0.01 mol %
sLeX-GSL 1, while cells tended to firmly stick at the membrane at
higher concentrations and lost membrane contacts at lower sLeX-
densities than 0.01 mol %. sLeX-GSL tended to separate from the
matrix and was arranged in clusters, as it was illustrated by fluo-
rescence microscopy for bilayers with 10 mol % and 1 mol % sLeX-
GSL 1.12 As the lateral lipid distribution was investigated by the
addition of 1 mol % 1,2-dipalmitoyl-sn-glycero-3-phosphoethanol-
amine-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) (NBD-PE) and de-
tected by its accumulation in the sLeX-clusters, this technique is
limited by the amount of NBD-PE and thus, rolling-relevant sLeX
concentrations could not be visualized.
For a direct correlation of ligand function and lateral appear-
ance, 0.1 mol % and 0.01 mol % of the NBD-labeled sLeX-GSL deriv-
ative 2 was incorporated into a DSPC bilayer. As illustrated by
confocal laser scanning microscopy (LSM) in Figure 3, sLeX-GSL 2
is strongly separated from the matrix and appears in a clustered
formation. Whereas the membrane with 0.01 mol % 2 displays a
very homogenous distribution of small clusters in a submicron
dimension, the tenfold higher concentration of 2 tends to form
higher aggregates. A rough correlation of fluorescent areas and
concentration of 2 could suggest a certain miscibility of 2 with
DSPC and thus the formation of mixed clusters.
2. Results and discussion
We previously reported the synthesis of the sLeX hexasaccha-
ride donor 326 through a convergent synthetic route and with this
trichloroacetimidate in hand, the standard ‘azidosphingosine
glycosylation procedure’27 for glycosphingolipid synthesis was
employed (Scheme 1). Thus, reaction of donor 3 with (2S,3R,4E)-
2-azido-3-(benzoyloxy)-4-octadecen-1-ol 428 and 0.4 equiv
trimethylsilyl trifluoromethansulfonate (TMSOTf) as catalyst
afforded derivative 5 after 5 h in 56% yield. The b-configuration
of the newly formed glycosidic linkage was confirmed by NMR
(J1a,2a = 7.8 Hz, 13C NMR: C-1a d 101.15 ppm). Interestingly, with
the 2a-O-benzoyl group the corresponding orthoester 6 was
formed first, which could be isolated almost exclusively after 1 h.
This orthoester intermediate then rearranged29 in 4 h to the
desired glycoside 5 in good yield, which could be conveniently
monitored by thin-layer chromatography. Figure 2 shows an over-
lay of the carbon–proton shift-correlation heteronuclear multiple
quantum coherence (HMQC) spectra of b-glycoside 5 and orthoes-
ter 6, which clearly shows the changes in the chemical shift of both
compounds. Remarkably, only the diastereomer of orthoester 6
was formed, wherein the phenyl ring is located beneath the glu-
cose moiety, which could be confirmed by corresponding nuclear
Overhauser effects (NOEs). This favorable aromatic ring-to-saccha-
Both membranes were used as substrates for the cell rolling
investigation. CHO-E cells strongly adhered at the membrane
containing 0.1 mol % of 2, while
a dominant fraction of
investigated cells (74%) rolled along the membrane with

C. Gege et al. / Carbohydrate Research 343 (2008) 2361–2368
2363
Scheme 1. Synthesis of the fluorescence labeled sialyl LewisX glycosphingolipid 2.
0.01 mol % of 2 at a mean velocity of about 8
l
m/s. This is slightly
m/s), but
late well with the cell rolling, this confirms the hypothesis that
sLeX clustering is a prerequisite for selectin binding with respect
to avidity increase.
slower than rolling along the non-labeled sLeX-GSL (12
l
in the typical rolling velocity range of E-selectin-dominated
interactions.
Taking sLeX clustering as a functional prerequisite for selectin
recognition and cell rolling, the ligand density within the clusters
appears to be a sensitive parameter. An absolute segregation of
sLeX lipids from a matrix was recently shown to result in pure
ligand clusters of high selectin binding affinity, which mediated
strong cell binding but avoided cell rolling.17
These findings are in total agreement with our recent data and
confirm on one hand that the coupling of the NBD moiety onto the
sLeX-GSL did not influence the binding function, and on the other
hand that the concentration dependency is maintained. We cannot
exclude that the NBD moiety is looping back to the membrane sur-
face, as postulated by Chattopadhyay for acyl label NBD lipids,21
although this appears unlikely due to the lateral compression
and transfer of the films. However, if looping would have occurred
it has no influence on the binding function.
To investigate the impact of diluting sLeX within a clustered
arrangement, we incorporated 0.1 mol % and 0.01 mol % of 2 in a
mixed matrix consisting of DSPC–POPC 95:5. As 2 displayed nearly
homogeneous distribution within POPC (Fig. 4), and POPC tends to
separate from the DSPC phase, we expected bigger clusters of low-
er sLeX density. This assumption was confirmed, considering the
images in Figure 5. A clustering of 2 is clearly evident at both con-
centrations, the bright areas are much bigger than in the pure DSPC
phase (Fig. 3).
To further confirm the dependency of cell rolling on sLeX
clustering, 2 was incorporated into a 1-palmitoyl-2-oleoyl-sn-
glycero-3-phosphocholine (POPC) matrix, as illustrated in Figure
4 for 0.1 mol % and 0.01 mol %.
In contrast to the DSPC bilayers, a clear separation of 2 is not
evident, especially when considering the higher concentration.
The differences between the DSPC and POPC bilayers were more
striking detecting the cell rolling behavior. Whereas only a few
CHO-E cells adhered at the 0.1 mol % containing membrane, nei-
ther cell binding nor rolling could be detected at the POPC bilayer
with 0.01 mol % of 2, which is identical to our earlier findings with
the sLeX-GSL.12 Taking the restrictions of resolution in fluorescence
microscopy in account, we cannot follow separation characteristics
up to the molecular range and so, cannot totally exclude a certain
sLex-GSL separation from the POPC matrix. However, as the find-
ings on lateral separations between 0.1 and 0.01 mol % of 2 corre-
CHO-E cells displayed a firm adhesion onto the mixed matrix
containing 0.1 mol % of 2, while a dominant fraction of the cells
(78%) rolled with velocity of about 12 lm/s along the membrane
with 0.01 mol % labeled sLeX-GSL. This is a further indication on
the essential role of clustered epitopes for rolling, which is
additionally controlled and balanced by the epitope density
within the clusters. The identical behavior of the NBD-labeled
sLeX-GSL 2 compared to the non-labeled glycolipid 1 is summa-
rized in Table 1.
In conclusion we described the synthesis of a fluorescently-
labeled sLeX-GSL 2 to investigate sLeX-microdomains in supported

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C. Gege et al. / Carbohydrate Research 343 (2008) 2361–2368
Figure 2. Overlay of the HMQC spectra of b-glycoside 5 (black) and orthoester 6 (red). The large change of the relevant chemical shift signals indicates that the a-ring
conformation has changed: in 5 this ring occupies a chair conformation, whereas in 6 it is fixed in a half-chair conformation.
planar bilayers by fluorescence microscopy and their impact
on selectin recognition. These microdomains were visible in very
low concentrations (up to 0.01 mol %) in DSPC and DSPC–POPC
95:5 matrices. It was confirmed in dynamic flow experiments
that the fluorescence analog 2 resemble its natural counterpart
1 very closely, showing that labeling the tail region with NBD
Figure 3. Confocal laser scanning microscope images of transferred lipid films containing 0.1 mol % (left) and 0.01 mol % (right) of the NBD-labeled sLeX-GSL 2 in a DSPC
matrix. The images illustrate the separation of the fluorescently-labeled glycolipid from the lipid matrix and tendency to form aggregates at higher concentrations.

C. Gege et al. / Carbohydrate Research 343 (2008) 2361–2368
2365
Figure 4. Confocal laser scanning microscopic images of 0.1 mol % (left) and 0.01 mol % (right) NBD-labeled sLeX-GSL 2 in a POPC matrix (100-fold magnification). No phase
separation tendency of 2 was observed under the experimental conditions indicating a homogeneous distribution of the glycolipid within the POPC matrix.
Figure 5. Lateral distribution of 0.1 mol % (left) and 0.01 mol % (right) of the fluorescently-labeled glycolipid 2 in a ternary mixture with a DSPC–POPC 95:5 matrix film. The
images were obtained by confocal laser scanning microscopy and representative for all investigated areas.
Table 1
Characterization of the binding and rolling behavior of CHO-E cells onto different matrices containing the non-labeled (1) or NBD-labeled (2) sLeX-GSL
Ligand
DSPC
POPC
DSPC–POPC 95:5
0.1 mol % 2
Cell adhesion (86.5%)
Cell adhesion (90.1%)
Cell adhesion (17.7%)
Cell adhesion (24.8%)
Detachment
Cell adhesion (83.4%)
Cell adhesion (88.9%)
0.1 mol % 112
0.01 mol % 2
0.01 mol % 112
Cell rolling (8
lm/s)
Cell rolling (12
Cell rolling (12
l
l
m/s)
m/s)
Cell rolling (12
lm/s)
Detachment
Data are means of at least four identical experiments.
does not influence the formation and shape of sLeX-GSL
microdomains.
3.2. Chemical synthesis—general methods
Solvents were purified according to the standard procedures.
Flash chromatography was performed on J.T. Baker Silica Gel 60
(40–63 lm) at a pressure of 0.4 bar. TLC was performed on Merck
Silica Gel glass plates HPTLC 60F254; compounds were visualized
by treatment with a solution of (NH4)6Mo7O24ꢀ4H2O (20 g) and
Ce(SO4)2 (0.4 g) in 10% sulfuric acid (400 mL) and heating at
150 °C. Optical rotations were measured on a Perkin–Elmer polar-
imeter 241 in a 1 dm cell at 22 °C. NMR measurements were
recorded on a Bruker AC250 Cryospec or a Bruker DRX600 spec-
trometer—TMS was used as internal standard. The carbohydrate
3. Experimental
3.1. Materials
1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1-pal-
mitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were pur-
chased from Sigma (Deisenhofen, Germany). The purity of these
lipids was analyzed by HPTLC and regarded as greater than 99%.
They were used without further purification.

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C. Gege et al. / Carbohydrate Research 343 (2008) 2361–2368
monomers were assigned in alphabetical order, beginning from the
aglycon, based in part on HMQC measurements. Target molecule 2
was measured at 303 K in a 320 mM solution of sodiumdodecyl
sulfate-d25 (SDS) in 0.5 mL D2O with 3-(trimethylsilyl)propionic
acid sodium salt-d4 as internal standard.33 MALDI-mass spectra
were recorded on a Kratos Kompact MALDI I instrument using a
2,5-dihydroxybenzoic acid matrix.
CHCl3); Rf = 0.52 (2:3 toluene–acetone); 1H NMR (600 MHz, CDCl3):
d 0.88 (t, 3H; CH3), 1.18 (d, J5,6 = 6.5 Hz, 3H; 6d-CH3), 1.18–1.32 (m,
22H; 11CH2), 1.68 (dd, J3,4 = Jgem = 12.4 Hz 1H; 3f-Ha), 1.85–2.20
(m, 56H; 18COCH3, CH@CHCH2), 2.58 (dd, J3,4 = 4.3, Jgem = 12.5 Hz,
1H; 3f-He), 3.22 (br s, 1H; 2c-H), 3.29–5.88 [m, 50H; HMQC: 3.30
(10-H), 3.37 (10-H), 3.46 (5c-H), 3.55 (5b-H), 3.63 (6f-H), 3.64 (4a-
H), 3.64 (5a-H), 3.68 (3b-H), 3.81 (20-H), 3.84 (5e-H), 3.86
(COOCH3), 3.86 (4c-H), 3.93 (6b-H), 4.03 (60b-H), 4.03 (6c-H),
4.04 (5f-H), 4.06 (6a-H), 4.09 (9f-H), 4.11 (60a-H), 4.21 (3c-H),
4.22 (6e-H), 4.26 (90f-H), 4.38 (60e-H), 4.40 (1b-H), 4.52 (3e-H),
4.53 (2a-H), 4.77 (1e-H), 4.79 (60c-H), 4.88 (2e-H), 4.88 (4f-H),
4.90 (1c-H), 4.95 (2d-H), 4.96 (4e-H), 5.00 (5d-H), 5.03 (Nf-H),
5.08 (2b-H), 5.20 (3d-H), 5.31 (4d-H), 5.32 (Nc-H), 5.33 (4b-H),
5.34 (1d-H), 5.44 (7f-H), 5.45 (40-H), 5.51 (8f-H), 5.53 (30-H), 5.59
(3a-H), 5.84 (1a-H), 5.85 (50-H)], 7.33–7.99 (m, 10H; 2C6H5); 13C
NMR (151 MHz, CDCl3, excerpt): d 15.83 (6d-C), 37.41 (3f-C),
49.12 (5f-C), 53.17 (OCH3), 58.3 (2c-C), 61.00 (6c-C), 61.37 (6e-C),
61.37 (6b-C), 61.71 (9f-C), 63.07 (6a-C), 63.32 (10-C), 63.65 (20-C),
64.09 (5d-C), 66.59 (7f-C), 67.31 (5a-C), 67.36 (4e-C), 67.58 (8f-
C), 68.01 (3d-C), 68.86 (2d-C), 69.04 (4b-C), 69.42 (4f-C), 69.82
(2e-C), 69.88 (3a-C), 70.77 (2b-C), 70.94 (5e-C), 71.17 (5b-C),
71.40 (3e-C), 71.56 (4d-C), 71.94 (6f-C), 72.37 (2a-C), 72.37 (3c-
C), 73.10 (5c-C), 74.28 (4c-C), 74.48 (30-C), 75.97 (3b-C), 76.61
(2d-C), 75.52 (4a-C), 75.96 (3b-C), 76.61 (4a-C), 95.28 (1d-C),
96.84 (2f-C), 97.36 (1a-C), 99.50 (1c-C), 99.92 (1e-C), 102.15 (1b-
C), 122.76 (40-C), 138.85 (50-C), 167.85 (1f-C). Anal. Calcd for
3.3. O-(Methyl-5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
D
-glycero-
tri-O-acetyl-b-
-fucopyranosyl)-(1?3)]-(2-acetamido-6-O-acetyl-2-deoxy-b-
glucopyranosyl)-(1?3)-(2,4,6-tri-O-acetyl-b- -galacto-
pyranosyl)-(1?4)-3,6-di-O-acetyl-2-O-benzoyl-b- -gluco-
a
-
D
-galacto-2-nonulopyranosylonate)-(2?3)-(2,4,6-
D
-galactopyranosyl)-(1?4)-[(2,3,4-tri-O-acetyl-a-
L
D-
D
D
pyranosyl-(1?1)-(2S,3R,4E)-2-azido-3-benzoyl-4-octadecen-
1,3-diol (5)
A solution of sialyl LewisX trichloroacetimidate 326 (450 mg,
216 mol) and (2S,3R,4E)-2-azido-3-(benzoyloxy)-4-octadecen-1-
ol 428 (243 mg, 566
mol) in dry CH2Cl2 (5 mL) was stirred with
molecular sieves AW-300 for 30 min at rt. Then TMSOTf (15.6 L,
86 mol, 0.4 equiv) was added under argon. After 5 h the ini-
l
l
l
l
tially-formed orthoester 6 had rearranged to the desired b-glyco-
side and the solution was neutralized with NEt3, filtrated, and
concentrated. Flash chromatography (2:1 to 3:2 toluene–acetone)
of the residue gave 5 (285 mg, 56%) as a colorless foam (the excess
of 4 can be recovered). [
a
]
ꢁ15.5 (c 1, CHCl3); Rf = 0.56 (2:3 tolu-
C
108H147N5O52ꢀ1/2H2O (2356.36): C, 55.05; H, 6.33; N, 2.97. Found:
D
ene–acetone); 1H NMR (600 MHz, CDCl3): d 0.88 (t, 3H; CH3), 1.18
(d, J5,6 = 6.5 Hz, 3H; 6d-CH3), 1.18–1.33 (m, 22H; 11CH2), 1.68 (m,
1H; 3f-Ha), 1.85–2.20 (m, 56H; 18COCH3, CH@CHCH2), 2.58 (dd,
J3,4 = 4.3, Jgem = 12.4 Hz, 1H; 3f-He), 3.18 (br s, 1H; 2c-H), 3.31–
4.66 [m, 27H; HMQC: 3.45 (5c-H), 3.50 (10-H), 3.63 (6f-H), 3.68
(5a-H), 3.70 (3b-H), 3.75 (5b-H), 3.83 (4a-H), 3.84 (5e-H), 3.85
(COOCH3), 3.87 (4c-H), 3.88 (20-H), 3.93 (10-H), 4.02 (6b-H, 60-b-
H), 4.03 (6c-H), 4.03 (5f-H), 4.08 (9f-H), 4.14 (6a-H), 4.21 (3c-H),
4.22 (6e-H), 4.24 (90f-H), 4.37 (1b-H), 4.38 (90e-H), 4.48 (60a-H),
4.52 (3e-H)], 4.68 (d, J1,2 = 7.8 Hz, 1H; 1a-H), 4.77–5.84 [m, 22H;
HMQC: 4.77 (1e-H), 4.81 (60c-H), 4.88 (4f-H), 4.89 (1c-H), 4.89
(2e-H), 4.94 (2d-H), 4.95 (4e-H), 4.97 (2b-H), 5.00 (5d-H), 5.10
(Nf-H), 5.19 (2a-H), 5.19 (3d-H), 5.31 (4d-H), 5.32 (1d-H), 5.33
(4b-H), 5.36 (3a-H), 5.39 (Nc-H), 5.43 (7f-H), 5.44 (30-H), 5.47 (40-
H), 5.51 (8f-H), 5.83 (50-H)], 7.40–8.00 (m, 10H; 2C6H5); 13C NMR
(151 MHz, CDCl3, excerpt): d 15.79 (6d-C), 37.38 (3f-C), 49.06 (5f-
C), 53.13 (OCH3), 58.3 (2c-C), 60.89 (6c-C), 61.34 (6e-C), 61.49
(6b-C), 61.64 (9f-C), 62.00 (6a-C), 64.07 (5d-C), 64.18 (20-C),
66.57 (7f-C), 67.34 (4e-C), 67.52 (8f-C), 67.94 (3d-C), 68.83 (2d-
C), 68.98 (4b-C), 69.21 (10-C), 69.41 (4f-C), 69.85 (2e-C), 70.92
(5e-C), 71.15 (2b-C), 71.24 (5b-C), 71.35 (3e-C), 71.53 (4d-C),
71.65 (2a-C), 71.99 (6f-C), 72.19 (3a-C), 72.35 (3c-C), 72.97 (5c-
C), 73.11 (5a-C), 74.41 (30-C), 74.23 (4c-C), 75.52 (4a-C), 75.96
(3b-C), 95.27 (1d-C), 96.81 (2f-C), 99.40 (1c-C), 99.88 (1e-C),
100.71 (1b-C), 101.15 (1a-C), 122.82 (40-C), 138.66 (50-C), 167.83
(1f-C). Anal. Calcd for C108H147N5O52ꢀH2O (2365.37): C, 54.84; H,
6.35; N, 2.96. Found: C, 54.73; H, 6.13; N, 2.73.
C, 55.03; H, 6.47; N, 2.55. MALDI-MS (positive mode, THF): m/z
1960.7 [Mꢁazidosphingosine+Na]+, 2373.0 [M+Na]+.
3.5. 12-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-
yl)amino]dodecanoic acid (7)
To a suspension of 12-aminododecanoic acid (2.61 g, 12.1
mmol) and NaHCO3 (2.0 g, 24 mmol) in H2O (15 mL) were added
4-chloro-7-nitrobenzofurazane (2.41 g, 12.1 mmol) and MeOH
(100 mL). The mixture was stirred at 50 °C for 1.5 h and then
cooled to rt, neutralized with 10% hydrochloric acid, and evapo-
rated under reduced pressure. The product, which precipitated
after adding H2O, was filtered and absorbed on silica. Flash chro-
matography (1:0 to 9:1 CHCl3–MeOH) afforded 7 (2.93 g, 64%) as
brown crystals. Mp 94 °C; Rf = 0.44 (9:1 CHCl3–MeOH); 1H NMR
(250 MHz, CDCl3): d 1.29–1.87 (m, 18H; 9CH2), 2.35 (t, Jvic = 7.4 Hz,
2H; CH2CO), 3.49 (q, 2H; CH2N), 6.18 (d, J5,6 = 8.7 Hz, 1H; 500-H),
6.38 (br t, 1H; NH), 8.49 (d, 1H; 600-H). Anal. Calcd for
C
18H26N4O5ꢀ1/4H2O (382.93): C, 56.46; H, 6.97; N, 14.63. Found:
C, 56.40; H, 6.72; N, 14.68.
3.6. O-(Methyl-5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
D
-glycero-
tri-O-acetyl-b-
L-fucopyranosyl)-(1?3)]-(2-acetamido-6-O-acetyl-2-desoxy-b-
-glucopyranosyl)-(1?3)-(2,4,6-tri-O-acetyl-b- -galacto-
pyranosyl)-(1?4)-3,6-di-O-acetyl-2-O-benzoyl-b- -gluco-
a
-D
-galacto-2-nonulopyranosylonate)-(2?3)-(2,4,6-
D
-galactopyranosyl)-(1?4)-[(2,3,4-tri-O-acetyl-a-
D
D
D
pyranosyl-(1?1)-(2S,3R,4E)-3-benzoyl-2-{12-[N-(7-nitrobenz-
2-oxa-1,3-diazol-4-yl)amino]dodecanamido}-4-octadecen-
1,3-diol (8)
3.4. O-(Methyl-5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
D
-glycero-
tri-O-acetyl-b-
-fucopyranosyl)-(1?3)]-(2-acetamido-6-O-acetyl-2-deoxy-b-
glucopyranosyl)-(1?3)-(2,4,6-tri-O-acetyl-b- -galacto-
pyranosyl)-(1?4)-3,6-di-O-acetyl-2-O-benzoyl-b- -gluco-
a
-
D
-galacto-2-nonulopyranosylonate)-(2?3)-(2,4,6-
D
-galactopyranosyl)-(1?4)-[(2,3,4-tri-O-acetyl-a-
L
D
-
A solution of 5 (82 mg, 35 lmol) in 4:1 pyridine–H2O (25 mL)
D
was saturated with hydrogen sulfide at 0 °C for 30 min and then
stirred at rt for 3 d, concentrated, and coevaporated with toluene
(3ꢂ). The residue was diluted in dry CH2Cl2 (10 mL), and 7
D
pyranose- 1,2-[(2S,3R,4E)-2-azido-3-(benzoyloxy)-4-octadec-
1-yl-orthobenzoate] (6)
(26 mg, 69 lmol) and EDCI (33 mg, 174 lmol) were added. After
16 h the mixture was diluted with H2O (10 mL) and extracted with
CH2Cl2 (3 ꢂ 30 mL). The combined organic layers were dried
(Na2SO4), filtered, and concentrated. Flash chromatography (3:2
If the reaction described above was neutralized after 1 h, the
orthoester 6 was isolated as the main product. [
ꢁ19.1 (c 1,
a]
D

C. Gege et al. / Carbohydrate Research 343 (2008) 2361–2368
2367
toluene–acetone) of the residue, followed by lyophilization from
dioxane, afforded 8 (68 mg, 73%) as a yellow amorphous solid.
immediately used for experiments in the flow chamber or for the
visualization with LSM.
[
a
]
ꢁ23.1 (c 1, CHCl3); Rf = 0.21 (3:2 toluene–acetone) versus
D
0.26 for 5; 1H NMR (600 MHz, CDCl3): d 0.87 (t, 3H; CH3), 1.15–
2.20 (m, 102H; 6d-CH3, 3f-Ha, 22CH2, 18COCH3), 2.58 (dd,
J3,4 = 4.3, Jgem = 12.4 Hz, 1H; 3f-He), 3.18 (br s, 1H; 2c-H), 3.38–
5.51 [m, 52H; 1a-H, 2a-H, 3a-H, 4a-H, 5a-H, 6a-H, 60a-H, 1b-H,
2b-H, 3b-H, 4b-H, 5b-H, 6b-H, 60b-H, 1c-H, 3c-H, 4c-H, 5c-H, 6c-
H, 60c-H, 1d-H, 2d-H, 3d-H, 4d-H, 5d-H, 1e-H, 2e-H, 3e-H, 4e-H,
5e-H, 6e-H, 60e-H, 4f-H, 5f-H, 6f-H, 7f-H, 8f-H, 9f-H, 90f-H, COOCH3,
2 10-H, 20-H, 30-H, 40-H, 50-H, CH2N, 2 NH], 5.78 (d, J = 9.3, 1H; NH),
6.18 (d, J = 8.6 Hz, 1H; 500-H), 6.38 (br s, 1H; NH), 7.39–8.03 (m,
10H; 2C6H5), 8.50 (d, J = 8.6 Hz, 1H; 600-H). MALDI-MS (positive
mode, THF): m/z 2705.0 [M+Na]+.
3.9. Cell cultivation
E-Selectin-transfected CHO-cells (CHO-E-cells) of mice were
grown in MEM-a media containing 10% fetal calf serum, 2 mM
L-glutamine, and 100 nM penicillin–streptomycin. Flasks seeded
with 5 ꢂ 104 CHO-E cells were incubated at 37 °C in 5% CO2 for
3–4 d to near confluence. After trypsinization for 3 min with
0.25% trypsin–EDTA, the cell suspension was transferred to slowly
rotating plastic tubes. The cells remained in suspension for up to
4 h. Within this time, the rolling experiments were performed in
the flow chamber.
3.10. Fluorescence microscopy and laminar flow experiments
3.7. O-(Triethylammonia-5-acetamido-3,5-dideoxy-
-galacto-2-nonulo-pyranosylonate)-(2?3)-(b-
galactopyranosyl)-(1?4)-[( -fucopyranosyl)-(1?3)]-(2-
acetamido-2-deoxy-b- -glucopyranosyl)-(1?3)-(b-
galactopyranosyl)-(1?4)-b- -glucopyranosyl-(1?1)-(2S,3R,4E)-
D-glycero-
a-
D
D-
The support-fixed bilayers were inserted into a parallel plate
flow chamber as described in detail in our previous investiga-
tions.12 The flow apparatus was mounted onto the inverted
fluorescent microscope Axiovert 135 of a laser scanning micro-
scope (LSM 410 invert, Carl Zeiss). The bilayers and the lateral
distribution of 2 were analyzed at 100ꢂ magnification (objective
plan-Apochromat 100/1,40 oil) after excitation with a 488 nm
argon laser, emission was evaluated with a cut-off of 530 nm.
The presented images were selected as representative from at least
six different areas of each film, experiments were repeated at least
three times. The images were not normalized with respect to
intensity. However, all experimental parameters (exposure time
of 2 s, pinhole, gain and off-set) were kept constant for the images.
Adhesion experiments were performed at 25 °C in a tempera-
ture-controlled environment to maintain the lateral structure of
a
-L
D
D-
D
2-{12-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-
dodecanamido}-4-octadecen-1,3-diol (2)
To a solution of 8 (65 mg, 24
lmol) in dry MeOH (15 mL) was
added NaOMe (20 mg, 370 mol). The mixture was stirred for 2 d
l
at rt, then water (0.3 mL) was added and stirred for 3 h. After neu-
tralization with Amberlite IR-120 (H+) the mixture was filtered and
adsorbed on silica gel. Flash chromatography (70:30:4:1 to
65:35:7.5:1 CHCl3–MeOH–H2O–NEt3) afforded 2 (46.6 mg, 93%)
as yellow, highly hygroscopic amorphous solid after lyophilization
from water. Rf = 0.36 (65:35:7.5:1 CHCl3–MeOH–H2O–NEt3); 1H
NMR (600 MHz, D2O + SDS):
d 0.80 (s, 3H; CH3), 1.15 (d,
the model membrane. MEM-a was used as flow medium at a shear
J5,6 = 6.4 Hz, 3H; 6d-CH3), 1.19–1.90 (m, 50H; 20CH2, 3f-Ha,
N(CH2CH3)3), 2.01 (s, 6H; 2COCH3), 2.20 (s, NCOCH2), 2.74 (dd,
1H; 3f-He), 3.19 (q, 6H; N(CH2Me)3), 3.29–4.15 (m, 40H; 2a-H,
3a-H, 4a-H, 5a-H, 2 6a-H, 2b-H, 3b-H, 4b-H, 5b-H, 26b-H, 2c-H,
3c-H, 4c-H, 5c-H, 2 6c-H, 2d-H, 3d-H, 4d-H, 2e-H, 3e-H, 4e-H, 5e-
H, 2 6e-H, 4f-H, 5f-H, 6f-H, 7f-H, 8f-H, 2 9f-H, 2 10-H, 20-H, 30-H,
NCH2), 4.42 (d, J1,2 = 7.8 Hz, 1H; 1b-H), 4.48 (d, J1,2 = 7.9 Hz, 1H;
1a-H), 4.51 (d, J1,2 = 7.8 Hz, 1H; 1e-H), 4.70 (1c-H im HDO-Signal),
4.80 (q, J5,6 = 6.8 Hz, 1H; 5d-H), 5.10 (d, J1,2 = 4.0 Hz, 1H; 1d-H),
5.38 (m, 1H; 40-H), 5.73 (m, 1H; 50-H), 6.30 (br s, 1H; 500-H), 8.53
(m, 1H; 600-H). Anal. Calcd for C85H146N8O38ꢀ10H2O (2068.27): C,
49.36; H, 8.09; N, 5.42. Found: C, 49.21; H, 7.95; N, 5.42.
rate of about 200 sꢁ1, driven by hydrostatic pressure. For the flow
experiments, 106 fluorescently marked CHO-E cells (Calcein AM,
Molecular Probes, Leiden, The Netherlands) in 100 lL medium
were injected into the streaming medium. The flow was stopped
to allow interaction of the cells with the supported membrane.
After 5 min, shear force was applied and the adhesion behavior
of the cells was monitored by a sequence of images taken every
two seconds. To characterize the cell movement, 50–150 cells
within an area of 630 ꢂ 630
lm were analyzed throughout a 20 s
period. Only those cells which adhered to the membrane without
contact to other adhered cells were counted and analyzed.
Alternatively, images were monitored capturing 25 frames per
second with a CCD-camera (CSC 795). The video sequences were
analyzed by application of a specific software (Imagoquant Multi-
track-AVI-2, Mediquant, Halle, Germany) resulting in a detailed
analysis of cell number and rolling velocity. For the presented data,
experiments were repeated at least four times.
3.8. Preparation of supported planar bilayers
Supported planar bilayers were prepared using the Langmuir-
Blodgett technique as described recently.12,17 Briefly, microscope
slides (glass, diameter of 18 mm, 0.2 mm thickness) were used as
transparent supports and incubated in concd H2SO4–H2O2 mixture
(7:3) at 80 °C for 30 min under ultrasonic conditions followed by
rinsing with ultra-pure water for 30 min. To increase the density
of silanol groups at the surface, a cleaning procedure with NH3–
H2O2–H2O (1:1:5) was performed before final rinse with ultra-pure
water and drying of the slides. To form supported bilayers, mono-
chlorodimethyloctadecyl-silane (Sigma, Deisenhofen, Germany)
was bound covalently to the surface of the slides at 50 °C for
30 min, resulting in a hydrophobic monolayer. A film of the indi-
cated matrix lipid containing the desired concentration of 2 was
pre-formed on the Langmuir trough, equilibrated for about
30 min to guarantee a stable film pressure and subsequently trans-
ferred to the hydrophobic glass support at a lateral pressure of
38 mN/m and a speed of 0.5 mm/min. The transfer ratios were
between 0.95 and 1. Freshly prepared supported bilayers were
Acknowledgments
This work was supported by the Deutsche Forschungsgemeins-
chaft and the Fonds der Chemischen Industrie. We are grateful to
Anke Friemel (University Konstanz) and Professor Armin Geyer
(University Marburg) for the structural assignments.
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