Synthetic Glycosphingolipids for Live-Cell Labeling

Article abstract of DOI:10.1021/acs.bioconjchem.6b00177

Glycosphingolipids are an important component of cell membranes that are involved in many biological processes. Fluorescently labeled glycosphingolipids are frequently used to gain insight into their localization. However, the attachment of a fluorophore to the glycan part or - more commonly - to the lipid part of glycosphingolipids is known to alter the biophysical properties and can perturb the biological function of the probe. Presented here is the synthesis of novel glycosphingolipid probes with mono- and disaccharide head groups and ceramide moieties containing fatty acids of varying chain length (C₄ to C₂₀). These glycosphingolipids bear an azide or an alkyne group as chemical reporter to which a fluorophore can be attached through a bioorthogonal ligation reaction. The fluorescent tag and any linker connected to it can be chosen in a flexible manner. We demonstrate the suitability of the probes by selective visualization of the plasma membrane of living cells by confocal microscopy techniques. Whereas the derivatives with the shorter fatty acids can be directly applied to HEK 293T cells, the hydrophobic glycosphingolipids with longer fatty acids can be delivered to cells using fusogenic liposomes.

Full text of DOI:10.1021/acs.bioconjchem.6b00177

Article
pubs.acs.org/bc
Synthetic Glycosphingolipids for Live-Cell Labeling
Martin Dauner,#,† Ellen Batroff,#,† Verena Bachmann,‡ Christof R. Hauck,‡ and Valentin Wittmann*,†
‡
†Department of Chemistry and Department of Biology, Konstanz Research School Chemical Biology (KoRS-CB), University of
Konstanz, 78457 Konstanz, Germany
S
* Supporting Information
ABSTRACT: Glycosphingolipids are an important component of cell membranes that are involved in many biological
processes. Fluorescently labeled glycosphingolipids are frequently used to gain insight into their localization. However, the
attachment of a fluorophore to the glycan part ormore commonlyto the lipid part of glycosphingolipids is known to alter
the biophysical properties and can perturb the biological function of the probe. Presented here is the synthesis of novel
glycosphingolipid probes with mono- and disaccharide head groups and ceramide moieties containing fatty acids of varying chain
length (C4 to C20). These glycosphingolipids bear an azide or an alkyne group as chemical reporter to which a fluorophore can be
attached through a bioorthogonal ligation reaction. The fluorescent tag and any linker connected to it can be chosen in a flexible
manner. We demonstrate the suitability of the probes by selective visualization of the plasma membrane of living cells by confocal
microscopy techniques. Whereas the derivatives with the shorter fatty acids can be directly applied to HEK 293T cells, the
hydrophobic glycosphingolipids with longer fatty acids can be delivered to cells using fusogenic liposomes.
challenging and variation of the dye is not easily possible.
Alternatively, the fluorescently labeled B subunits of bacterial
toxins, such as Cholera toxin (CtxB) or Shiga toxin (StxB), can
be used to visualize glycosphingolipids.21 These microbial
proteins selectively bind specific glycolipids but, due to their
multimeric structure, they can also influence lipid organization
by multivalent cross-linking.21−24
During the last years, several research groups including our
own25−29 made use of a chemical reporter strategy to study
glycoconjugates in living cells.30,31 In this approach, synthetic
biomolecules with a small chemical reporter group are applied
to cells, metabolically incorporated into biomolecules, and
subsequently labeled in a bioorthogonal reaction, for example,
with a fluorescent dye. This approach, however, leads to
labeling of the whole glycome including glycolipids and
glycoproteins,32 and the isolation of individual labeled
glycolipids in preparative scale is not feasible. In addition, it
has been shown that different cell lines differ in their ability to
discriminate among variant forms of a carbohydrate which may
lead to preferential incorporation into different glycolipids.33
INTRODUCTION
■
Glycosphingolipids are an important class of lipids found in cell
membranes. They play a major role in many biological
processes, such as the formation of membrane microdomains
and the interaction of pathogenic microbes or their secreted
toxins with host cells.1,2 A large number of glycosphingolipid
derivatives have been prepared to gain insight into their
biological function.3 The toolbox of available probes includes
lipids with fluorophores, photoreactive cross-linkers, and
radiolabels.4 Fluorescently labeled derivatives in particular
have been widely used to study trafficking of glycosphingolipids
and to monitor the formation of lipid microdomains.5,6 In most
of these labeled lipids, the fluorophore is ligated to the
sphingosine backbone.7 Synthetically, this can be conveniently
achieved by de-N-acylation of natural glycosphingolipids and
reacylation with a fluorescent dye.8−11 Although several of these
compounds are commercially available, their altered biophysical
properties can perturb their biological function.4,5,12−14 For
example, Sezgin et al.15 and Honigmann et al.16 showed that
size, polarity, charge, and position of the dye within a lipid
molecule can dramatically influence the biological function in
the plasma membrane as compared to its natural counterpart.
The hydrophilic glycan part has also been used for ligation with
a fluorescent dye.15,17−20 In this case, the synthesis is more
Received: April 5, 2016
Revised: May 19, 2016
© XXXX American Chemical Society
A
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Figure 1. Alkyne- and azide-labeled glucosylceramides and fluorescent dyes.
Thus, the application of synthetic lipid derivatives modified by a
reporter group that allows for flexible attachment of a probe is
desirable. This strategy34 has, for example, been adopted for the
study of phospholipids,35−38 sphingolipids,39 and lipids with a
choline headgroup.40 However, synthetically demanding
glycosphingolipids with chemical reporter groups have been
reported only rarely.41 Here, we present the synthesis of azide-
and alkyne-tagged glycosphingolipids and their visualization in
cell membranes by subsequent click reaction with various
fluorescent dyes.
Scheme 1. Synthesis of Glucosylceramide 1
RESULTS AND DISCUSSION
■
We designed two types of glucosylceramide analogs (Figure 1),
one bearing an alkyne group in the N-acyl chain (1), and the
other with an azido group in the glucose moiety (2, 3). The
latter has the advantage that the lipid part of the molecule is not
altered, a fact that might be crucial for studies concerning the
organization of cell membranes. The length of the N-acyl chain
varies in nature and can affect the biological function of the
glycolipid.42−44 Due to their better solubility, short-chain
analogs are usually used in biological applications.4 For
sufficient solubility lipid derivatives should have a logP value
similar to that of N-acetyl sphingosine.45 Based on the
estimation of logP values with the ALOGPS-2.1 software46
we expected azidoglucosylceramide 2 with an N-butanoyl
moiety to be a promising candidate for cell studies. For
comparison, derivative 3 with an N-tetradecanoyl chain was
also synthesized.
Scheme 2. Synthesis of Benzoyl-Protected Ceramide
Derivatives 27−30
The synthesis of glucosylceramide 1 is depicted in Scheme 1.
Commercially available sphingosine 7 was N-acylated with
succinimidyl ester 8, and the resulting amide 9 was protected at
the secondary hydroxy group by a sequence of silylation,
benzoylation, and desilylation. The obtained primary alcohol 12
was reacted with trichloroacetimidate 1347 to provide protected
β-glucosylceramide 14. O-Deacylation, finally, gave glucosylcer-
amide 1.
To prepare glycosphingolipids containing fatty acids of
different chain length, we protected the ceramide derivatives
15−18 at the secondary hydroxy group by a sequence similar to
that of the preparation of 12 (Scheme 2). Tritylation of the
primary alcohol followed by protection of the secondary
alcohol with benzoyl chloride and detritylation gave ceramide
derivatives 27−30. The synthesis of glucosylceramides 2 and 3
is depicted in Scheme 3. The acetylated 6-azido glucose
B
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but the overall fluorescence intensity was rather weak (not
shown). Possibly, the molecule becomes too hydrophilic after
the click reaction and is washed out to some extent, resulting in
reduced membrane staining. This finding might reflect one of
the general drawbacks of lipids that are labeled with a
hydrophilic dye in the hydrophobic part of the molecule.
Azidoglucosylceramide 2 was labeled by copper-free click
reaction with DIBO-lissamine 6 and caused selective staining of
the plasma membrane (Figure 2E, F). The same result was
obtained after labeling with commercially available DIBO-
AlexaFluor-488. In this case the fluorescent background was
even a bit lower (Figure 2G, H). Azidoglucosylceramides with
significantly longer N-acyl chains such as C10 or C14 (3)
apparently were not incorporated into the cell membrane under
these conditions (not shown), which underscores that the
solubility of glycolipids might be a limiting factor. However,
different mechanisms how glycolipids are taken up and
incorporated into cell membranes including the formation of
micelles have to be considered.58 We speculated that a bigger
hydrophilic carbohydrate moiety can compensate for longer
lipid chains. Therefore, we tested modified lactosylceramides,
which are also important natural glycosphingolipids. According
to estimations of logP values, azidolactosylceramide 48 with an
N-octanoyl moiety was expected to have a suitable polarity and,
thus, was synthesized (Scheme 4). For comparison we also
synthesized derivative 47 with an N-butanoyl moiety and 49
with an N-eicosanoyl moiety. In these compounds, the azide tag
replaces the 6′-OH group of the lactosyl moiety. Allyl lactoside
36 was selectively TBDMS-protected at this position via a
stannane59 followed by acetylation of the remaining hydroxy
groups to produce intermediate 38. After desilylation, the 6′-
OH group was mesylated and then substituted with sodium
azide. The resulting lactosyl building block 41 was converted
into trichloroacetimidate 43 and used as glycosyl donor for
reaction with ceramides 27−30 to give, after final deprotection,
azide-modified lactosylceramides 47−49.
47 and 48 were applied to HEK 293T cells and labeled with
DIBO-lissamine 6 (Figure 3A−C) at 4 °C in the same manner
as described for glucosylceramide 2. In both cases, selective
visualization of the plasma membrane with comparable
fluorescence intensity was achieved. These results demonstrate
that a lactosylceramide can be incorporated into the plasma
membrane, even when it contains an N-octanoyl chain.
Compound 49 featuring a longer N-eicosanoyl chain, however,
could not be delivered to the cells. In further experiments we
could show that the incorporation of 47 and 48 and subsequent
staining with DIBO-lissamine 6 can also be carried out at
ambient temperature leading to comparable staining (Figure
S1). We could not observe any endocytosis under these
conditions.
With azidolactosylceramide 48 we also carried out a two-step
labeling with DIBO-biotin conjugate 5052 and streptavidin-
AlexaFluor-647 resulting in a bright staining of the plasma
membrane and extremely low background fluorescence
(Figures 3D, E, and S2). In contrast, with glycosphingolipids
1, 2, or 47 two-step labeling via a biotin derivative and
streptavidin did not lead to any observable membrane staining.
An explanation for this finding might be the fact that these
glycolipids are washed out of the membrane after reaction with
DIBO-biotin 50 and binding to streptavidin due to their shorter
membrane anchor.
Scheme 3. Synthesis of Azide-Modified Glucosylceramides 2
and 3
derivative 3148 was anomerically deprotected with ethylene
diamine/acetic acid according to the procedure described by
Zhang and Kovac49
́ ̌
and converted to the α-trichloroacetimidate
33. Schmidt glycosylation50 with ceramide 27 or 29 resulted in
protected glucosylceramides 34 or 35 that were de-O-acylated
with sodium methoxide in methanol to provide glucosylcer-
amide 2 and 3, respectively.
As fluorescent dyes we used coumarin azide 451 as well as
rhodamine azide 5 (for synthesis see Supporting Information)
to label alkyne-modified glycolipid 1. Coumarin azide 4 is
fluorogenic and has the advantage to only fluoresce after
reaction of the azide with alkyne-modified 1. For visualizing
azidoglucosylceramide 2 and 3, we synthesized lissamine-
labeled dibenzocyclooctyne (DIBO-lissamine) 6 (see Support-
ing Information) which can be employed in a copper-free
labeling procedure.52
To investigate incorporation into cell membranes, glycolipids
were applied to HEK 293T cells as a solution in Hanks’
Minimum Essential Medium (HMEM) containing 1 equiv of
defatted bovine serum albumin (dfBSA)53 for 30 min at 4 °C to
prevent endocytosis. Then, cells were washed two times with
phosphate-buffered saline (PBS) and labeled with the
fluorescent dye at 4 °C. Subsequent confocal microscopy of
cells was performed at ambient temperature. The Cu(I)-
catalyzed labeling of alkyne-modified glucosylceramide 1 using
azides 4 or 5 was carried out according to the protocol of Hong
et al.,54,55 which has been shown to be compatible with living
cells. In our case, however, tris(benzyltriazolylmethyl)amine
(TBTA)56 gave superior results than the hydrophilic copper
ligand tris(3-hydroxypropyltriazolylmethyl)amin (THPTA).
Probably, the hydrophobicity of the resulting copper-TBTA
complex facilitates efficient catalysis within the lipid bilayer. 1
caused staining of the plasma as well as intracellular membranes
(Figure 2A−D). The staining pattern is similar to that obtained
with fluorescent ceramide and glucosylceramide analogs
bearing a fluorophore in the hydrophobic part57 and shows
that the lipid can reach intracellular membranes. We speculated
that an analog of 1 in which the glucose moiety is replaced with
a lactose moiety would not be able to cross the cell membrane
spontaneously due to its bigger hydrophilic headgroup. Cell
experiments with such an alkyne-labeled lactosylceramide
indeed showed that only the plasma membrane was stained,
As mentioned above, the hydrophobic glycosphingolipids 3
and 49 could not be incorporated into the plasma membrane of
C
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Figure 2. Cell experiments with labeled glucosylceramides 1 (A−D) and 2 (E−H). HEK 293T cells were treated with 5 μM 1 (A, C) or without 1
(B, D) for 30 min at 4 °C followed by labeling with 2 μM rhodamine azide 5 (A, B) or 50 μM coumarin azide 4 (C, D) in the presence of 50 μM
CuSO4, 100 μM TBTA, 1 mM aminoguanidine, and 10 mM sodium ascorbate for 30 min at 4 °C. In experiments with azidoglucosylceramide 2,
HEK 293T cells were treated with 10 μM 2 (E, G) or without 2 (F, H) for 30 min at 4 °C followed by fluorescence labeling with 2 μM DIBO-
lissamine 6 (E, F) or 2 μM DIBO-AlexaFluor-488 (G, H) for 30 min at 4 °C. Confocal microscopy was performed at r.t. Scale bar: 20 μm.
HEK 293T cells. To overcome this limitation we investigated
the use of fusogenic liposomes which have been shown to
enhance the incorporation of biomolecules.60,61 We used
fusogenic liposomes prepared of 1,2-dioleoyl-sn-glycero-3-
phosphoethanolamine (DOPE) and 1,2-dioleoyl-3-trimethyl-
ammonium-propane (DOTAP) as neutral and positively
charged lipids, respectively, and TexasRed-labeled 1,2-dihexa-
decanoyl-sn-glycero-3-phosphoethanolamine (DHPE) as lipid
with an extended conjugated π electron system that enhances
fusion efficacy.60 The TexasRed dye, furthermore, allows
monitoring of the fusion of the liposomes with the plasma
membrane. The glycolipids 3 and 49, respectively, were
embedded in fusogenic liposomes, incubated with HEK 293T
cells for 10 min at 37 °C, and subsequently labeled with DIBO-
AlexaFluor-488 for 30 min at room temperature (Figure 4).
Both glycolipids 3 (Figure 4A) and 49 (Figure 4B) show a
bright membrane staining with only minor background (Figure
4C).
CONCLUSIONS
■
In summary, we have described the synthesis of new
glycosphingolipid derivatives containing azide or alkyne
reporter groups in either the lipid or the glycan moiety.
Visualization of these glycolipids within membranes of living
cells could be achieved by subsequent labeling through a
bioorthogonal ligation reaction. Glycosphingolipids with longer
N-acyl chains could be successfully delivered to cells using
fusogenic liposomes. This strategy allows one to choose various
dyes in a flexible manner which is a useful feature since different
fluorophores can have different spectroscopic properties and
can differ with respect to size, charge, and polarity. The small
size of the azide and alkyne reporter groups enables future
studies of lipid transport, accumulation, and metabolism. Thus,
we expect our probes to offer new opportunities for studying
the biological roles of glycosphingolipids.
D
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Scheme 4. Synthesis of Azidolactosylceramides 47−49
(petroleum ether/EtOAc 1:1); 1H NMR (400 MHz, CDCl3): δ
= 2.78 (s, 4 H, 2 × NC(O)CH2), 2.60 (t, J = 7.4 Hz,
OC(O)CH2), 2.20 (td, J = 6.9, 2.6 Hz, 2 H, CH2CCH), 1.93
(t, J = 2.6 Hz, 1 H, CCH), 1.87−1.79 (m, 2 H,
C(O)CH2CH2), 1.64−1.56 (m, 2 H, CH2CH2CCH); 13C
NMR (101 MHz, CDCl3): δ = 169.3 (2 × NC(O)), 168.4
(C(O)), 83.6 (CCH), 69.1 (CCH), 30.5 (C(O)CH2),
27.4 (CH2CH2CCH), 25.7 (2 × succinimidyl-CH2), 23.6
(C(O)CH2CH2), 18.0 (CH2CCH); ESI-MS calculated [M +
H]+ = 224.1, [M + Na]+ = 246.1, found [M + H]+ = 224.2, [M
+ Na]+ = 246.2; CHN analysis calculated C 59.19, H 5.87, N
6.27, O 28.67, found C 59.91, H 6.35, N 6.53.
EXPERIMENTAL PROCEDURES
■
General. All chemicals were purchased from ABCR, Acros,
Merck, or Sigma-Aldrich. AlexaFluor reagents were purchased
from Invitrogen. Sphingosine was obtained from Biolab
Chemicals. Technical solvents were distilled prior to use. Dry
solvents were purchased from Sigma-Aldrich and Acros.
Reactions were monitored by TLC on silica gel 60 F254
(Merck) with detection by UV light (λ = 254 nm).
Additionally, treatment with cerium reagent (5 g molybdato-
phosphoric acid, 2.5 g ceric sulfate tetrahydrate, 25 mL sulfuric
acid, 225 mL water), or 15% (v/v) sulfuric acid in ethanol,
followed by gentle heating, were used for visualization.
Preparative flash column chromatography was performed on
silica gel Geduran 60 (40−60 μm, Merck). Nuclear magnetic
resonance (NMR) spectra were recorded at room temperature
on Avance III 400 and Avance III 600 instruments from Bruker.
Chemical shifts are reported relative to solvent signals (CDCl3:
δH = 7.26 ppm, δC = 77.16 ppm; DMSO-d6: δH = 2.50 ppm, δC
= 39.5 ppm; CD3OD: δH = 3.31 ppm, δC = 49.0 ppm; D2O: δH
= 4.79 ppm). Signals were assigned by first-order analysis and,
when feasible, assignments were supported by two-dimensional
(2S,3S,E)-2-Hept-6-ynamido-3-hydroxy-octadec-4-en-1-yl
β-D-glucopyranoside (1). D-Erythro-sphingosine (7, 250 mg,
0.74 mmol) was dissolved in 15 mL DCM/THF 2:1.
Succinimidyl hept-6-ynoate (8) (200 mg, 0.89 mmol) and
Et3N (311 μL, 226 mg, 2.23 mmol) were added and the
reaction was stirred for 16 h at room temperature. The mixture
was diluted with 50 mL DCM and washed with 1× 1 M HCl
solution, 2× saturated NaHCO3 solution, and 1× brine. The
organic phase was dried with MgSO4 and evaporated under
reduced pressure. The crude ceramide intermediate was
dissolved in 10 mL dry DCM and stirred with TBDMSCl
(840 μL of a 1 M solution in THF) and imidazole (95 mg, 1.4
mmol) for 24 h at room temperature. The mixture was diluted
with 20 mL DCM and washed with 1× 1 M HCl solution, 1×
saturated NaHCO3 solution, and 1× brine. The organic phase
was dried with MgSO4 and evaporated under reduced pressure.
The crude product was purified by silica column chromatog-
raphy (eluent petroleum ether/ethyl acetate 3:1). 10 was
obtained as a colorless solid (240 mg, 0.42 mmol, 60% over two
steps). Analytical data for intermediate 10: Rf = 0.29
1
1
1H, H and H, 13C correlation spectroscopy (COSY, HMBC,
NOESY, and HSQC). ESI mass spectra were recorded on an
Esquire 3000 plus instrument (Bruker Daltonics) or on an
LCMS-2020 (Shimadzu). High-resolution ESI-TOF mass
spectra were recorded on a micrOTOF II instrument (Bruker
Daltonics). Elemental analyses were performed on a vario EL
instrument from Elementar.
Synthesis of Alkyne-Labeled Glucosylceramide 1.
Succinimidyl hept-6-ynoate (8). Hept-6-ynoic acid (500 mg,
3.96 mmol) was dissolved in 20 mL dry DCM. N-
Hydroxysuccinimide (456 mg, 3.96 mmol) and DCC (817
mg, 3.96 mmol) were added and the mixture was stirred for 16
h at room temperature. The formed precipitate was filtered off.
The filtrate was evaporated to yield 8 (822 mg, 93%) as a white
solid, which was used without further purification. Rf = 0.52
1
(petroleum ether/EtOAc 3:1); H NMR (250 MHz, CDCl3):
δ = 6.24 (d, J = 7.8 Hz, 1 H, NH), 5.81−5.72 (m, 1 H, CH
CHCH2), 5.55−5.46 (m, 1 H, CHCHCH2), 4.16 (‘t’, J = 4.4
Hz, 1 H, CHOH), 3.96−3.89 (m, 2 H, CHNH, CH2OSi),
E
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Figure 3. Cell experiments with azide-labeled lactosylceramides 47 and 48. HEK 293T cells were treated with 10 μM 47 (A), 10 μM 48 (B), or
without glycolipid (C) for 30 min at 4 °C followed by labeling with 2 μM DIBO-lissamine 5. For a two-step labeling procedure, HEK 293T cells
were treated with 10 μM (D) or without 48 (E) for 30 min at 4 °C. Then 20 μM DIBO-biotin 50 was added for 30 min at 4 °C followed by labeling
with streptavidin-AlexaFluor-647 (15 min, 4 °C). Confocal microscopy was performed at r.t. Scale bar: 20 μm.
3.76−3.73 (m, 1 H, CH2OSi), 2.28−2.19 (m, 4 H, C(O)CH2,
CH2CCH), 2.07−2.02 (m, 2 H, CHCHCH2), 1.94 (t, J =
2.6 Hz, 1 H, CCH), 1.80−1.72 (m, 2 H, CH2CH2CCH),
1.61−1.53 (m, 2 H, C(O)CH2CH2), 1.38−1.21 (m, 22 H, 11×
CH2), 0.91−0.85 (m, 12 H, CH2CH3, SiC(CH3)3), 0.06 (2 s, 6
H, Si(CH3)2).
5.58−5.50 (m, 2 H, CHCHCH2, CHOBz), 4.43−4.33 (m, 1
H, CHNH), 3.83 (dd, J = 3.0, 10.2 Hz, 1 H, CH2OSi), 3.65
(dd, J = 4.1, 10.2 Hz, 1 H, CH2OSi), 2.25−2.17 (m, 4 H,
C(O)CH2, CH2CCH), 2.06−1.98 (m, 2 H, CHCHCH2),
1.93 (t, J = 2.6 Hz, 1 H, CCH), 1.78−1.72 (m, 2 H, CH2),
1.62−1.53 (m, 2 H, CH2), 1.23 (br, 22 H, 11× CH2), 0.91−
0.85 (m, 12 H, CH2CH3, SiC(CH3)3), 0.00 (s, 3 H, SiCH3),
−0.01 (s, 3 H, SiCH3).
Compound 10 (240 mg, 0.42 mmol) was dissolved in 2 mL
dry pyridine and treated with benzoyl chloride (72 μL, 87 mg,
0.62 mmol). The reaction was stirred at room temperature for
2 h, diluted with 20 mL water, and extracted three times with
diethyl ether. The combined organic layers were dried with
Na2SO4 and evaporated under reduced pressure. After
purification by silica column chromatography (eluent petro-
leum ether/ethyl acetate 8:1 to 6:1) 11 was obtained as a
slightly yellow, viscous oil (250 mg, 0.39 mmol, 95%).
Analytical data for intermediate 11: Rf = 0.42 (petroleum
Compound 11 (250 mg, 0.39 mmol) was dissolved in 2 mL
THF and treated with 40 μL HF·pyridine (approximately 70%
HF). The reaction was stirred for 16 h at room temperature,
diluted with 10 mL DCM, and washed three times with
saturated NaHCO3 solution. The organic phase was dried with
MgSO4 and evaporated under reduced pressure. After silica
column chromatography (eluent petroleum ether/ethyl acetate
3:2 to 2:3), compound 12 was obtained as a colorless solid
(102 mg, 0.19 mmol, 50%). Analytical data for intermediate 12:
1
ether/EtOAc 6:1); H NMR (250 MHz, CDCl3): δ = 8.05−
1
8.00 (m, 2 H, aromat.), 7.57−7.52 (m, 1 H, aromat.), 7.47−
7.40 (m, 2 H, aromat.), 5.84−5.79 (m, 1 H, CHCHCH2),
Rf = 0.22 (petroleum ether/EtOAc 1:1); H NMR (250 MHz,
CDCl3): δ = 8.04−8.01 (m, 2 H, aromat.), 7.62−7.56 (m, 1 H,
F
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Figure 4. Cell experiments with azide-labeled glucosylceramide 3 and 49 using fusogenic liposomes. HEK 293T cells were treated with 3 (A), 49
(B), or without glycolipid (C) in fusogenic liposomes (DOTAP/DOPE/TexasRed-DHPE 1:1:0.05) for 10 min at 37 °C followed by labeling with
DIBO-AlexaFluor-488 (2 μM, 30 min, rt). Scale bar: 30 μm.
aromat.), 7.48−7.42 (m, 2 H, aromat.), 6.19 (d, J = 8.9 Hz,
NH), 5.91−5.80 (m, 1 H, CHCHCH2), 5.64−5.49 (m, 2 H,
CHCHCH2, CHOBz), 4.33−4.22 (m, 1 H, CHNH), 3.78−
3.65 (m, 2 H, CH2OH), 2.26−2.16 (m, 4 H, C(O)CH2,
CH2CCH), 2.07−1.99 (m, 3 H, OH, CHCHCH2), 1.94
(t, J = 2.6 Hz, CCH), 1.80−1.68 (m, 2 H, CH2), 1.63−1.49
(m, 2 H, CH2), 1.40−1.18 (m, 22 H, 11× CH2), 0.94−0.84 (m,
3 H, CH3).
(m, 11 H, 3× C(O)CH3, CHCHCH2), 1.97 (s, 3 H,
C(O)CH3), 1.96−1.94 (m, 1 H, CCH), 1.82−1.70 (m, 2 H,
CH2), 1.59−1.51 (m, 2 H, CH2), 1.36−1.18 (m, 22 H, 11×
CH2), 0.87 (t, J = 6.5 Hz, 3 H, CH3); ESI-MS: calculated [M +
H]+ = 842.5, [M + Na]+ = 864.5, found [M + H]+ = 842.3, [M
+ Na]+ = 864.4. Protected glycolipid 14 (72 mg, 84 μmol) was
dissolved in 3 mL dry MeOH and treated with 42 μL of a 1 M
solution of NaOMe in MeOH. The reaction was stirred for 16
h at room temperature. After neutralization with strongly acidic
ion-exchange resin, the solvent was removed under reduced
pressure. The crude product was purified by silica column
chromatography (eluent DCM/MeOH 9:1). Glucosylceramide
1 was obtained as a colorless solid (35 mg, 60 μmol, 71%). Rf =
Ceramide derivative 12 (102 mg, 0.194 mmol) and
trichloroacetimidate 1347 (111 mg, 0.233 mmol) were dissolved
in 1 mL dry DCM. After addition of BF3·OEt2 (48 μL, 55 mg,
0.388 mmol) the mixture was stirred for 2.5 h at room
temperature. The mixture was diluted with DCM and washed
once each with 1 M HCl solution and saturated NaHCO3
solution. The organic phase was dried with MgSO4 and the
solvent was removed under reduced pressure. After purification
by silica column chromatography (eluent toluene/acetone 9:1),
14 was obtained as a colorless solid (72 mg, 84 μmol, 44%).
Analytical data for intermediate 14: Rf = 0.54 (toluene/acetone
1
0.22 (DCM/MeOH 9:1); H NMR (400 MHz, CD3OD): δ =
7.86 (d, J = 9.1 Hz, 1 H, NH), 5.70 (dt, J = 15.3, 6.7 Hz, 1 H,
CHCHCH2), 5.49−5.43 (m, 1 H, CHCHCH2), 4.26 (d, J
= 7.7 Hz, 1 H, H-1), 4.13 (dd, J = 10.1, 5.2 Hz, 1 H,
CH2OGlc), 4.08 (t, J = 7.8 Hz, 1 H), 4.03−3.97 (m, 1 H,
CHNH), 3.87 (dd, J = 11.8, 1.8 Hz, 1 H, H-6a), 3.68−3.61 (m,
2 H, CH2OGlc, H-6b), 3.40−3.34 (m, 1 H), 3.29−3.26 (m, 2
H), 3.21 (dd, J = 9.2, 7.7 Hz, 1 H), 2.23−2.17 (m, 5 H, C
CH, C(O)CH2, CHCHCH2), 2.06−2.01 (m, 2 H, CH2),
1.75−1.66 (m, 2 H, CH2), 1.57−1.49 (m, 2 H, CH2), 1.41−
1.25 (s, 22 H, 11× CH2), 0.90 (t, J = 6.7 Hz, 3 H, CH3); 13C
NMR (101 MHz, CD3OD): δ = 175.6 (C(O)), 135.0 (CH
CHCH2), 131.2 (CHCHCH2), 104.7 (C1), 84.7 (CCH),
78.0, 77.9, 75.2, 73.1, 71.6, 69.9 (CCH), 69.8 (CH2OGlc),
62.7, 54.9 (CHNH), 36.7 (C(O)CH2), 33.4 (CHCHCH2),
1
4:1); H NMR (400 MHz, CDCl3): δ = 8.02 (d, J = 7.9 Hz, 2
H, aromat.), 7.56 (t, J = 7.5 Hz, 1 H, aromat.), 7.44 (t, J = 7.5
Hz, 2 H, aromat.), 5.92−5.84 (m, 2 H, CHCHCH2, NH),
5.55 (‘t’, J = 6.9 Hz, 1 H, CHOBz), 5.47 (dd, J = 7.2, 15.1 Hz, 1
H, CHCHCH2), 5.19 (‘t’, J = 9.5 Hz, 1 H, H-3), 5.04 (‘t’, J =
9.8 Hz, H-4), 4.96 (‘t’, J = 8.8 Hz, 1 H, H-2), 4.50−4.45 (m, 2
H, CHNH, H-1), 4.13 (dd, J = 4.3, 12.2 Hz, 1 H, H-6a), 4.05−
3.97 (m, 2 H, H-6b, CH2OGlc), 3.71−3.62 (m, 2 H, CH2OGlc,
H-5), 2.21−2.18 (m, 4 H, C(O)CH2, CH2CCH), 2.06−2.00
G
DOI: 10.1021/acs.bioconjchem.6b00177
Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
Article
33.1, 30.8−30.7 (m), 30.5−30.4 (m), 29.2, 26.2, 23.8, 18.9
(CH2), 14.4 (CH3); ESI-MS: calculated [M + H]+ = 570.4, [M
+ Na]+ = 592.4, found [M + H]+ = 570.2, [M + Na]+ = 592.2;
HR-ESI-MS: calculated [M + H]+ = 570.40004, found [M +
H]+ = 570.39922.
mixture of pyridine, THF and DCM (1:1:1). The mixture was
stirred for 36 h at room temperature. The solvent was removed
under reduced pressure and the crude product was purified by
silica chromatography (eluent petroleum ether/ethyl acetate
2:1). 19 was obtained as a colorless solid (275 mg, 0.44 mmol,
1
Synthesis of Azidoglucosylceramide 2. 2,3,4-Tri-O-
acetyl-6-azido-6-deoxy-D-glucopyranose (32). Glucose deriv-
ative 3148 was subjected to anomeric deprotection following
84%). Rf = 0.41 (petroleum ether/EtOAc 1:1); H NMR (400
MHz, CDCl3): δ = 7.35−7.32 (m, 6 H, aromat.), 7.26−7.16
(m, 9 H, aromat.), 6.03 (d, J = 8.0 Hz, 1 H, NH), 5.61−5.53
(m, 1 H, CHCHCH2), 5.22−5.17 (m, 1 H, CHCHCH2),
4.14−4.09 (m, 1 H, CHOH), 4.03−3.98 (m, 1 H, CHNH),
3.32 (dd, J = 9.7, 3.7 Hz, 1 H, CH2OTrt), 3.24 (dd, J = 9.7, 4.0
Hz, 1 H, CH2OTrt), 2.13 (t, J = 7.4 Hz, 2 H, C(O)CH2),
1.88−1.81 (m, 2 H, CHCHCH2), 1.66−1.57 (m, 2 H,
C(O)CH2CH2), 1.25−1.14 (m, 22 H, 11x CH2), 0.91 (t, J =
7.4 Hz, 3 H, butyl-CH3), 0.81 (t, J = 6.8 Hz, 3 H, sphingosine-
CH3); 13C NMR (101 MHz, CDCl3): δ = 173.3 (C(O)), 143.4
(C aromat.), 133.6 (CHCHCH2), 128.8 (CHCHCH2),
128.6, 128.1, 127.4 (3x C aromat.), 87.5 (CPh3), 74.5 (COH),
63.2 (COTrt), 53.4 (CNH), 38.9 (C(O)CH2), 32.3 (CH
CHCH2), 32.1, 29.83 (br), 29.79, 29.76, 29.65, 29.5, 29.4, 29.2,
22.8 (9× CH2), 19.3 (C(O)CH2CH2), 14.3, 14.0 (2× CH3);
ESI-MS: calculated [M + H]+ = 612.4, [M + Na]+ = 634.4,
found [M + H]+ = 612.6, [M + Na]+ = 634.5.
the procedure described by Zhang and Kovac 49
Ethylenedi-
́
.
̌
amine (352 μL, 315 mg, 5.25 mmol) was dissolved in 60 mL
THF. Acetic acid (355 μL, 372 mg, 6.2 mmol) was added
dropwise. 31 (1.78 g, 4.77 mmol) was added and the mixture
was stirred for 3 days at room temperature. The solvent was
removed under reduced pressure. The remaining material was
dissolved in 100 mL DCM and extracted twice with 50 mL
each of 1 M HCl solution, saturated NaHCO3 solution, and
brine. The organic phase was dried with MgSO4 and the solvent
was removed under reduced pressure. The crude product was
purified by silica column chromatography (eluent petroleum
ether/ethyl acetate 2:1 to 1:1). 32 was obtained as a colorless
solid (1.35 g, 4.07 mmol, 85%; mixture of isomers α/β = 3:1).
1
Rf = 0.35 (petroleum ether/EtOAc 1:1); H NMR (400 MHz,
CDCl3): δ = 5.48 (‘t’, J = 9.8 Hz, 1 H, H-3α), 5.43 (d, J = 3.6
Hz, 1 H, H-1α), 5.19 (‘t’, J = 9.5 Hz, 1 H, H-3β), 5.01−4.93 (m,
2 H, H-4β, H-4α), 4.88−4.83 (m, 1 H, H-2β), 4.83 (dd, J = 3.6,
10.2 Hz, 1 H, H-2α, 4.73 (d, J = 8.5 Hz, 1 H, H-1β), 4.19 (ddd, J
= 3.1, 5.8, 9.9 Hz, 1 H, H-5α), 3.67 (ddd, J = 3.0, 5.9, 9.8 Hz, 1
H, H-5β), 3.35−3.32 (m, 2 H, H-6a/bβ), 3.30−3.24 (m, 2 H, H-
6a/bα), 2.04 (m, 6 H, 2× C(O)CH3), 1.99 (m, 6 H, 2×
C(O)CH3), 1.97 (m, 6 H, 2× C(O)CH3); 13C NMR (101
MHz, CDCl3): δ = 171.5, 170.7 (2× C(O)β), 170.4, 170.3,
169.9 (3× C(O)α), 169.7 (C(O)β), 95.5 (C1β), 90.1 (C1α),
73.20 (C5β), 73.17 (C2β), 72.4 (C3β), 71.3 (C2α), 69.87, 69.82
(C4α, C3α), 69.7 (C4β), 68.4 (C5α), 51.2 (C6α), 51.1 (C6β),
(2S,3R,E)-2-Butyramido-1-(trityloxy)octadec-4-en-3-yl
benzoate (23). 275 mg (0.44 mmol) ceramide 19 and 124 mg
(0.88 mmol) benzoyl chloride were dissolved in 5 mL toluene/
pyridine 4:1. The reaction was stirred for 16 h at room
temperature. The solvent was removed under reduced pressure.
The remaining material was dissolved in a small amount of
DCM and was washed with 2× 1 M HCl solution and 2×
saturated NaHCO3 solution. The organic phase was dried with
MgSO4 and evaporated under reduced pressure. The crude
product was purified by silica chromatography (eluent:
petroleum ether/ethyl acetate 4:1). 23 was obtained as a
colorless solid (307 mg, 0.43 mmol, 97%). Rf = 0.59 (petroleum
β
α
21.1 (CH3 ), 20.75, 20.73, 20.69 (3× CH3 ), 20.65, 20.64 (2×
CH3 ); ESI-MS: calculated [M + H]+ = 332.1, found [M + H]+
β
1
ether/EtOAc 1:1); H NMR (400 MHz, CDCl3): δ = 8.18−
= 332.2.
8.16 (m, 3 H, aromat.), 7.95−7.93 (m, 2 H, aromat.), 7.40−
7.37 (m, 6 H, aromat.), 7.23−7.17 (m, 9 H, aromat.), 5.92−
5.85 (m, 1 H, CHCHCH2), 5.73−5.67 (m, 2 H, CHOBz,
NH), 5.48−5.42 (m, 1 H, CHCHCH2), 4.53−4.47 (m, 1 H,
CHNH), 3.45 (dd, J = 9.5, 3.5 Hz, 1 H, CH2OTrt), 3.20 (dd, J
= 9.5, 4.1 Hz, 1 H, CH2OTrt), 2.09 (t, J = 7.2 Hz, 2 H,
C(O)CH2), 2.02−1.97 (m, 2 H, CHCHCH2), 1.64−1.57
(m, 2 H, C(O)CH2CH2), 1.33−1.18 (m, 22 H, 11× CH2), 0.92
(t, J = 7.4 Hz, 3 H, butyl-CH3), 0.88 (t, J = 6.9 Hz, 3 H,
sphingosine-CH3); 13C NMR (101 MHz, CDCl3): δ = 172.4,
162.5 (2× C(O)), 143.6 (C aromat.), 137.4 (CHCHCH2),
134.7, 133.1, 130.7, 129.8 (m), 129.0, 128.7, 128.5, 128.1,
128.0, 127.2 (C aromat.), 125.2 (CHCHCH2), 87.0 (CPh3),
74.5 (COBz), 60.5 (COTrt), 51.2 (CNH), 39.0 (C(O)CH2),
32.1, 29.81 (m), 29.80, 29.7, 29.6, 29.5, 29.4, 29.0, 22.8, 21.2
(CH2), 19.3 (C(O)CH2CH2), 14.3, 13.9 (2× CH3).
2,3,4-Tri-O-acetyl-6-azido-6-deoxy-α-D-glucopyranosyl tri-
chloroacetimidate (33). 32 (1.2 g, 3.6 mmol) and trichloro-
acetonitrile (4.33 mL, 6.24 g, 43.2 mmol) were dissolved in 10
mL dry DCM at room temperature and treated with 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU, 135 μL, 138 mg, 0.91
mmol). TLC showed complete reaction after 5 min. The
mixture was filtered over a small portion of silica gel. The
filtrate was evaporated under reduced pressure. The remaining
material was purified by silica column chromatography (eluent:
petroleum ether/ethyl acetate 3:2). 33 was obtained as a
slightly yellowish solid (1.29 g, 2.91 mmol, 81%). Rf = 0.47
(petroleum ether/EtOAc 1:1); 1H NMR (400 MHz, CDCl3): δ
= 8.72 (s, 1 H, NH), 6.58 (d, J = 3.7 Hz, 1 H, H-1), 5.55 (‘t’, J =
9.8 Hz, 1 H, H-3), 5.16−5.09 (m, 2 H, H-2, H-4), 4.18 (ddd, J
= 2.7, 5.5, 10.1 Hz, 1 H, H-5), 3.40 (dd, J = 2.7, 13.6 Hz, 1 H,
H-6a), 3.32 (dd, J = 5.5, 13.6 Hz, 1 H, H-6b), 2.05 (s, 3 H,
C(O)CH3), 2.03 (s, 3 H, C(O)CH3), 2.01 (s, 3 H, C(O)CH3);
13C NMR (101 MHz, CDCl3): δ = 170.1, 170.0, 169.6 (3×
C(O)), 160.8 (CNH), 92.8 (C1), 90.8 (CCl3), 71.3 (C5),
69.83 (C3), 69.82, 69.1 (C2, C4), 50.8 (C6), 20.8, 20.7, 20.6
(3× C(O)CH3); ESI-MS: calculated [M + Na]+ = 497.0, found
[M + Na]+ = 496.9; CHN analysis: calculated C 35.35, H 3.60,
N 11.78, O 26.91, Cl 22.36, found, C 34.55, H 4.02, N 11.51.
N-((2S,3R,E)-3-Hydroxy-1-(trityloxy)octadec-4-en-2-yl)-
butyramide (19). 200 mg (0.52 mmol) ceramide 15 and 174
mg (0.63 mmol) trityl chloride were dissolved in 3 mL of a
(2S,3R,E)-2-Butyramido-1-hydroxyoctadec-4-en-3-yl ben-
zoate (27). 307 mg (0.43 mmol) ceramide 23 were dissolved
in a mixture of 1 mL dry toluene and 80 μL dry MeOH, cooled
to 0 °C and treated with 158 μL (182 mg, 1.28 mmol) BF3·
OEt2. After stirring the mixture for 20 min at 0 °C, 20 mL
DCM were added and the mixture was extracted once with
saturated NaHCO3 solution. The organic phase was dried with
MgSO4 and the solvent was removed under reduced pressure.
The remaining material was purified by silica chromatography
(eluent petroleum ether/ethyl acetate 1:1, then 2:3). 27 was
obtained as a colorless solid (132 mg, 0.28 mmol, 65%). Rf =
H
DOI: 10.1021/acs.bioconjchem.6b00177
Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
Article
0.15 (petroleum ether/EtOAc 1:1); 1H NMR (400 MHz,
CDCl3): δ = 8.04−8.02 (m, 2 H, aromat.), 7.61−7.56 (m, 1 H,
aromat.), 7.47−7.43 (m, 2 H, aromat.), 6.13 (d, J = 8.7 Hz, 1 H,
NH), 5.85 (dt, J = 14.6, 6.7 Hz, 1 H, CHCHCH2), 5.64−
5.56 (m, 1 H, CHCHCH2), 5.57−5.51 (m, 1 H, CHOBz),
4.32−4.26 (m, 1 H, CHNH), 3.74 (dd, J = 12.0, 3.8 Hz, 1 H,
CH2OH), 3.69 (dd, J = 12.0, 3.2 Hz, 1 H, CH2OH), 2.74 (br s,
1 H, OH), 2.21−2.13 (m, 2 H, C(O)CH2), 2.06−2.01 (m, 2 H,
CHCHCH2), 1.69−1.60 (m, 2 H, C(O)CH2CH2), 1.38−
1.19 (m, 22 H, 11× CH2), 0.93 (t, J = 7.4 Hz, 3 H, butyl-CH3),
0.87 (t, J = 6.8 Hz, 3 H, sphingosine-CH3); 13C NMR (101
MHz, CDCl3): δ = 173.4, 166.7 (2× C(O)), 137.7 (CH
CHCH2), 133.6, 129.9, 128.7 (3× C aromat.), 125.0 (CH
CHCH2), 74.8 (CHOBz), 62.0 (CH2OH), 53.5 (CHNH), 38.9
(C(O)CH2), 32.4 (CHCHCH2), 32.1, 29.81, 29.80, 29.7,
29.6, 29.5, 29.3, 29.0 (CH2), 22.8 (sphingosine-CH3), 19.2
(butyl-CH3); ESI-MS: calculated [M + H]+ = 474.4, [M + Na]+
= 496.4, found [M + H]+ = 474.7, [M + Na]+ = 496.6.
14.8, 6.8 Hz, 1 H, CHCHCH2), 5.45 (dd, J = 15.5, 6.1 Hz, 1
H, CHCHCH2), 4.35 (d, J = 7.5 Hz, 1 H, H-1), 4.21−4.15
(m, 2 H, CHNH, CHOH), 4.02−3.98 (m, 1 H, CH2OGlc),
3.76−3.71 (m, 1 H, CH2OGlc), 3.63−3.33 (m, 9 H, H-2, H-3,
H-4, H-5, H-6a/b, OH), 2.22 (t, J = 7.3 Hz, C(O)CH2), 2.07−
1.98 (m, 2 H, CHCHCH2), 1.69−1.60 (m, 2 H, C(O)-
CH2CH2), 1.39−1.19 (m, 22 H, 11× CH2), 0.95 (t, J = 7.4 Hz,
3 H, butyl-CH3), 0.88 (t, J = 6.8 Hz, 3 H, sphingosine-CH3);
13C NMR (101 MHz, CDCl3): δ = 174.8 (C(O)), 135.0
(CHCHCH2), 128.2 (CHCHCH2), 103.0 (C1), 76.4,
75.7, 73.5 (C2), 73.3 (CHNHCHOH), 71.2, 69.3 (CH2OGlc),
53.4 (CHNH), 51.7 (C6), 38.8 (C(O)CH2), 32.5 (CH
CHCH2), 32.1, 29.87, 29.82, 29.7, 29.5, 29.3, 22.8, 19.4 (CH2),
14.3, 13.9 (2× CH3); ESI-MS: calculated [M + H]+ = 557.4,
[M + Na]+ = 579.4, [M-H]− = 555.4 found [M + H]+ = 557.2,
[M + Na]+ = 579.2, [M-H]− = 555.2; HR-ESI-MS: calculated
[M + H]+ = 557.39088; found [M + H]+ = 557.38981.
For synthesis and analytical data of azidoglucosylceramide 3,
see Supporting Information.
(2S,3S,E)-2-Butyramido-3-(benzoyloxy)-octadec-4-en-1-yl
2,3,4-tetra-O-acetyl-6-azido-6-deoxy-β-D-glucopyranoside
(34). Ceramide 27 (132 mg, 0.28 mmol) and trichloroacetimi-
date 33 (159 mg, 0.33 mmol) were dissolved in 1 mL dry DCM
and cooled to 0 °C. After addition of BF3·OEt2 (68 μL, 79 mg,
0.56 mmol), the reaction was stirred for 2 h at 0 °C, diluted
with 20 mL DCM and washed with 10 mL saturated NaHCO3
solution. The organic phase was dried with MgSO4 and
evaporated under reduced pressure. The crude product was
purified by silica column chromatography (eluent toluene/
acetone 9:1 to 4:1). 34 was obtained as a colorless solid (125
Synthesis of Azidolactosylceramide 47. Allyl 2,3,4-tri-
O-acetyl-6-O-tert-butyldimethylsilyl-β-D-galactopyranosyl-(1
→ 4)-2,3,6-tri-O-acetyl-β-D-glucopyranoside (38). To obtain a
lactose derivative with an orthogonal protecting group at the 6′-
position, a procedure reported by Glen et al.59 was followed
with modifications. Allyl lactoside 3662 (12 g, 31.4 mmol) and
dibutyltin(IV) oxide (7.97 g, 32 mmol) were dissolved in 700
mL dry MeOH and refluxed for 4 h. The mixture was
evaporated under reduced pressure and dried thoroughly at
vacuum. 500 mL dry THF and tert-butyldimethylsilyl chloride
(5.06 g, 33.4 mmol) were added and the mixture was stirred for
3 days at room temperature. Approximately 20 mL of the
solution was taken out and evaporated. The obtained material
was subjected to silica column chromatography (eluent DCM/
MeOH 9:1), which caused slow decomposition of the product.
However, enough of 37 was obtained to be characterized by
NMR. The major part of the silylation reaction was treated with
200 mL each of acetic anhydride and pyridine and stirred for 16
h at room temperature. The solvent was removed under
reduced pressure and the remaining material was purified by
silica chromatography (eluent petroleum ether/ethyl acetate
3:2 to 1:1). 38 was obtained as a colorless solid (14.6 g, 20.7
mmol, 66%). Analytical data of allyl 6-O-tert-butyldimethylsilyl-
β-D-galactopyranosyl-(1 → 4)-β-D-glucopyranoside (37): Rf =
1
mg, 0.16 mmol, 57%). Rf = 0.45 (toluene/acetone 4:1); H
NMR (400 MHz, CDCl3): δ = 8.10 (d, J = 7.3 Hz, 2 H,
aromat.), 7.63 (′t′, J = 7.4 Hz, 1 H, aromat.), 7.51 (′t′, J = 7.7
Hz, 2 H, aromat.), 5.97−5.87 (m, 2 H, CHCHCH2, NH),
5.61 (′t′, J = 6.8 Hz, 1 H, CHOBz), 5.54 (dd, J = 15.2, 7.4 Hz, 1
H, CHCHCH2), 5.25 (′t′, J = 9.5 Hz, 1 H, H-3), 5.03−4.98
(m, 2 H, H-2, H-4), 4.60−4.53 (m, 1 H, CHNH), 4.59 (d, J =
7.9 Hz, 1 H, H-1), 4.12 (dd, J = 10.0, 4.5 Hz, 1 H, CH2OGlc),
3.78 (dd, J = 10.1, 4.1 Hz, 1 H, CH2OGlc), 3.72 (ddd, J = 9.7,
6.7, 2.8 Hz, 1 H, H-5), 3.31 (dd, J = 13.4, 6.7 Hz, 1 H, H-6a),
3.24 (dd, J = 13.4, 2.8 Hz, 1 H, H-6b), 2.24−2.18 (m, 2 H,
C(O)CH2), 2.11−2.06 (m, 11 H, CHCHCH2, 3× C(O)-
CH3), 1.74−1.65 (m, 2 H, C(O)CH2CH2), 1.42−1.27 (m, 22
H, 11× CH2), 1.00 (t, J = 7.4 Hz, 3 H, butyl-CH3), 0.94 (t, J =
6.8 Hz, 3 H, sphingosine-CH3); 13C NMR (101 MHz, CDCl3):
δ = 170.3, 169.7, 169.6, 165.6, 163.8 (5× C(O)), 137.6 (CH
CHCH2), 133.2, 130.3, 129.8, 129.1 (4× C aromat.), 124.6
(CHCHCH2), 100.4 (C1), 74.6 (COBz), 73.7 (C5), 72.5
(C3), 71.4, 69.6 (C2, C4), 67.5 (CH2OGlc), 51.0 (C6), 50.9
(CNH), 38.8 (C(O)CH2), 32.4 (CHCHCH2), 32.0, 29.8−
29.7 (m), 29.6, 29.5, 29.3, 29.0, 22.8, 20.7, 19.2 (CH2), 14.2
(sphingosine-CH3), 13.8 (butyl-CH3); ESI-MS: calculated [M
+ Na]+ = 808.4, found [M + Na]+ = 808.6.
1
0.14 (DCM/MeOH 9:1); H NMR (400 MHz, CD3OD): δ =
6.02−5.90 (m, 1 H, CH2CHCH2), 5.37−5.29 (m, 1 H,
CH2CHCH2), 5.18−5.14 (m, 1 H, CH2CHCH2), 4.37 (d,
J = 7.5 Hz, 1 H, anomeric H), 4.40−4.31 (m, 1 H, CH2CH
CH2), 4.33 (d, J = 7.8 Hz, 1 H, anomeric H), 4.18−4.10 (m, 1
H, CH2CHCH2), 3.89−3.76 (m, 5 H), 3.63−3.47 (m, 6 H),
3.42−3.37 (m, 1 H), 0.92 (s, 9 H, SiC(CH3)3), 0.11 (s, 6 H, 2×
SiCH3); 13C NMR (101 MHz, CD3OD): δ = 135.8 (CH
CH2), 117.6 (CHCH2), 105.3, 103.4 (C1, C1′), 80.9, 76.9,
76.51, 76.45, 75.0, 74.9, 72.6, 71.2, 69.8, 63.1, 62.0 (C2−C6,
C2′-C6′), 26.6 (SiC(CH3)3), 22.0 (SiC(CH3)3), −5.1, −5.2
(2× SiCH3). Analytical data of allyl 2,3,4-tri-O-acetyl-6-O-tert-
butyldimethylsilyl-β-D-galactopyranosyl-(1 → 4)-2,3,6-tri-O-
acetyl-β-D-glucopyranoside (38): Rf = 0.39 (petroleum ether/
EtOAc 1:1); 1H NMR (400 MHz, CDCl3): δ = 5.86−5.76 (m,
1 H, CH2CHCH2), 5.42 (‘d’, J = 3.2 Hz, 1 H, H-4′), 5.26−
5.20 (m, 1 H, CH2CHCH2), 5.17 (‘t’, J = 9.3 Hz, 1 H, H-3),
5.19−5.15 (m, 1 H, CH2CHCH2), 5.06 (dd, J = 10.4, 7.8 Hz,
1 H, H-2′), 4.96 (dd, J = 10.4, 3.4 Hz, 1 H, H-3′), 4.89 (dd, J =
9.5, 8.0 Hz, 1 H, H-2), 4.49 (d, J = 8.0 Hz, 1 H, H-1), 4.48−
(2S,3S,E)-2-Butyramido-3-hydroxy-octadec-4-en-1-yl 6-
azido-6-deoxy-β-D-glucopyranoside (2). Glycolipid 34 (125
mg, 0.16 mmol) was dissolved in 1 mL dry MeOH and treated
with 48 μL of a 0.5 M solution of NaOMe in MeOH. The
reaction was stirred for 90 min at room temperature and then
neutralized with strongly acidic ion-exchange resin. The solvent
was removed under reduced pressure and the remaining
material was purified by silica column chromatography (eluent
DCM/MeOH 9:1). 2 was obtained as a colorless solid (47 mg,
1
84 μmol, 53%). Rf = 0.20 (DCM/MeOH 9:1); H NMR (400
MHz, CDCl3): δ = 6.54 (d, J = 7.8 Hz, 1 H, NH), 5.75 (dt, J =
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4.43 (m, 1 H, H-6a), 4.44 (d, J = 7.7 Hz, 1 H, H-1′), 4.28 (ddt,
J = 13.2, 4.9, 1.5 Hz, 1 H, OCH2CHCH2), 4.13−4.01 (m, 2
H, H-6b, OCH2CHCH2), 3.79 (‘t’, J = 9.5 Hz, 1 H, H-4),
3.69−3.62 (m, 2 H, H-5′, H-6a′), 3.59−3.51 (m, 2 H, H-5, H-
6b′), 2.10 (s, 3 H, C(O)CH3), 2.10 (s, 3 H, C(O)CH3), 2.02
(s, 3 H, C(O)CH3), 2.02 (s, 3 H, C(O)CH3), 2.01 (s, 3 H,
C(O)CH3), 1.94 (s, 3 H, C(O)CH3), 0.83 (s, 9 H,
SiC(CH3)3), 0.00 (s, 3 H, SiCH3), −0.02 (s, 3 H, SiCH3);
13C NMR (101 MHz, CDCl3): δ = 170.6, 170.3, 170.1, 169.9,
169.8, 169.4 (6× C(O)CH3), 133.4 (CH2CHCH2), 117.7
(CH2CHCH2), 101.1 (C1′), 99.4 (C1), 76.1 (C4), 73.6
(C5′), 73.1 (C3), 72.7 (C5), 71.8 (C2), 71.4 (C3′), 70.1
(CH2CHCH2), 69.6 (C2′), 66.7 (C4′), 62.1 (C6), 60.1
(C6′), 25.8 (SiC(CH3)3), 21.0, 20.9, 20.81, 20.77, 20.75, 20.66,
(6× C(O)CH3), 18.2 (SiC(CH3)3), −5.56, −5.64 (2× SiCH3);
ESI-MS: calculated [M + H]+ = 749.3, [M + Na]+ = 771.3,
found [M + H]+ = 749.1, [M + Na]+ = 771.1; CHN analysis:
calculated C 52.93, H 7.00, O 36.32, Si 3.75, found C 52.69, H
7.01.
Allyl 2,3,4-tri-O-acetyl-β-D-galactopyranosyl-(1 → 4)-
2,3,6-tri-O-acetyl-β-D-glucopyranoside (39). Lactoside 38
(14.8 g, 20.9 mmol) was dissolved in 200 mL dry THF and
treated with 11 mL HF·pyridine (approximately 70% HF). The
reaction was stirred for 3 h at room temperature and then
neutralized with solid NaHCO3 until gas formation ceased.
Solids were filtered off and the filtrate was evaporated under
reduced pressure. The remaining material was purified by silica
column chromatography (eluent petroleum ether/ethyl acetate
1:2 to 1:3). 39 was obtained as a colorless solid (7.58 g, 11.9
mmol, 57%). Rf = 0.17 (petroleum ether/EtOAc 1:2); 1H NMR
(400 MHz, CDCl3): δ = 5.86−5.77 (m, 1 H, CH2CHCH2),
5.33 (‘d’, J = 3.4 Hz, 1 H, H-4′), 5.26−5.21 (m, 1 H, CH2CH
CH2), 5.20−5.15 (m, 2 H, CH2CHCH2, H-3), 5.11 (dd, J =
10.4, 7.8 Hz, 1 H, H-2′), 4.97 (dd, J = 10.4, 3.4 Hz, 1 H, H-3′),
4.90 (dd, J = 9.4, 7.9 Hz, 1 H, H-2), 4.52 (d, J = 6.0 Hz, 1 H, H-
1), 4.50 (d, J = 6.0 Hz, 1 H, H-1′), 4.50−4.46 (m, 1 H, H-6a),
4.30−4.25 (m, 1 H, CH2CHCH2), 4.08−4.03 (m, 2 H, H-6b,
CH2CHCH2), 3.83 (‘t’, J = 9.3 Hz, 1 H, H-4), 3.72−3.65 (m,
2 H, H-5′, H-6′a), 3.59 (ddd, J = 9.7, 5.3, 2.2 Hz, 1 H, H-5),
3.51−3.44 (m, 1 H, H-6′b), 2.58 (br, 1 H, OH), 2.14 (s, 3 H,
C(O)CH3), 2.10 (s, 3 H, C(O)CH3), 2.03 (s, 3 H, C(O)CH3),
2.03 (s, 3 H, C(O)CH3), 2.02 (s, 3 H, C(O)CH3), 1.96 (s, 3 H,
C(O)CH3); 13C NMR (101 MHz, CDCl3): δ = 171.1, 170.6,
170.3, 170.2, 169.8, 169.3 (6× C(O)CH3), 133.4 (CH2CH
CH2), 117.7 (CH2CHCH2), 101.2 (C1), 99.3 (C1′), 76.2
(C4), 74.1 (C5′), 73.5 (C3), 72.6 (C5), 71.8 (C2), 71.1 (C3′),
70.1 (CH2CHCH2), 69.6 (C2′), 67.7 (C4′), 62.2 (C6), 60.8
(C6′), 21.0, 20.98, 20.82, 20.78, 20.74, 20.66 (6× C(O)CH3);
ESI-MS: calculated [M + H]+ = 635.2, [M + Na]+ = 657.2, [M
+ K]+ = 673.2, found [M + H]+ = 635.2, [M + Na]+ = 657.0,
[M + K]+ = 673.0; CHN analysis: calculated C 51.10, H 6.04,
O 42.86, found C 50.82, H 6.35.
reduced pressure. 40 was obtained as a colorless solid, which
was used without further purification (3.46 g, 4.85 mmol, 88%).
Rf = 0.40 (petroleum ether/EtOAc 1:2); H NMR (400 MHz,
1
CDCl3): δ = 5.86−5.77 (m, 1 H, CH2CHCH2), 5.38 (dd, J =
3.6, 0.7 Hz, 1 H, H-4′), 5.26−5.21 (m, 1 H, CH2CHCH2),
5.21−5.15 (m, 2 H, CH2CHCH2, H-3), 5.09 (dd, J = 10.4,
7.8 Hz, 1 H, H-2′), 4.96 (dd, J = 10.4, 3.4 Hz, 1 H, H-3′), 4.88
(dd, J = 9.4, 7.9 Hz, 1 H, H-2), 4.53 (d, J = 8.1 Hz, 1 H, H-1′),
4.51 (d, J = 8.1 Hz, 1 H, H-1), 4.49−4.45 (m, 1 H, H-6a),
4.29−4.25 (m, 1 H, CH2CHCH2), 4.23−4.14 (m, 2 H, H-
6′a/b), 4.09−4.02 (m, 2 H, H-6b, CH2CHCH2), 3.97−3.94
(m, 1 H, H-5′), 3.82 (‘t’, J = 9.5 Hz, 1 H, H-4), 3.59 (ddd, J =
9.8, 5.0, 2.0 Hz, 1 H, H-5), 3.05 (s, 3 H, SO2CH3), 2.14 (s, 3 H,
C(O)CH3), 2.10 (s, 3 H, C(O)CH3), 2.04 (s, 3 H, C(O)CH3),
2.03 (s, 3 H, C(O)CH3), 2.02 (s, 3 H, C(O)CH3), 1.94 (s, 3 H,
C(O)CH3); 13C NMR (101 MHz, CDCl3): δ = 170.5, 170.2,
170.1, 169.9, 169.7, 169.2 (6× C(O)CH3), 133.4 (CH2CH
CH2), 117.7 (CH2CHCH2), 100.9 (C1′), 99.3 (C1), 76.2
(C4), 73.0 (C3), 72.6 (C5), 71.8 (C2), 70.9 (C5′), 70.8 (C3′),
70.1 (CH2CHCH2), 69.1 (C2′), 66.6 (C4′), 64.8 (C6′), 62.1
(C6), 37.8 (SO2CH3), 21.0 (2×), 20.8, 20.712, 20.706, 20.6
(6× C(O)CH3); ESI-MS: calculated [M + H]+ = 713.2, [M +
Na]+ = 735.2, found [M + H]+ = 713.1, [M + Na]+ = 735.0;
CHN analysis: calculated: C 47.19, H 5.66, O 42.65, S 4.50,
found: C 45.53, H 5.66, S 4.76.
Allyl 2,3,4,6-tri-O-acetyl-6-azido-6-deoxy-β-D-galactopyra-
nosyl-(1 → 4)-2,3,6-tri-O-acetyl-β-D-glucopyranoside (41).
Lactoside 40 (2.8 g, 3.93 mmol) was dissolved in 30 mL dry
DMF and treated with NaN3 (2.54 g, 39 mmol). The resulting
suspension was stirred for 16 h at 85 °C. Solids were filtered off
and the filtrate was evaporated under reduced pressure. The
remaining material was dissolved in 100 mL DCM and washed
twice with 100 mL each of saturated NaHCO3 solution and
brine. The organic phase was dried with MgSO4 and evaporated
under reduced pressure. After silica column chromatography
(eluent petroleum ether/ethyl acetate 1:1 to 2:3), 41 was
obtained as a colorless solid (1.93 g, 2.93 mmol, 74%). Rf =
0.36 (petroleum ether/EtOAc 1:2); 1H NMR (400 MHz,
CDCl3): δ = 5.86−5.76 (m, 1 H, CH2CHCH2), 5.32 (dd, J =
3.4, 0.9 Hz, 1 H, H-4′), 5.26−5.20 (m, 1 H, CH2CHCH2),
5.19−5.16 (m, 2 H, CH2CHCH2, H-3), 5.07 (dd, J = 10.4,
7.8 Hz, 1 H, H-2′), 4.92 (dd, J = 10.3, 3.5 Hz, H-3′), 4.89 (dd, J
= 9.5, 7.9 Hz, 1 H, H-2), 4.51−4.47 (m, 3 H, H-1, H-1′, H-6a),
4.30−4.25 (m, 1 H, CH2CHCH2), 4.10−4.02 (m, 2 H, H-6b,
CH2CHCH2), 3.84 (′t′, J = 9.5 Hz, 1 H, H-4), 3.73−3.70
(m, 1 H, H-5′), 3.58 (ddd, J = 9.9, 5.0, 2.1 Hz, 1 H, H-5), 3.43
(dd, J = 12.7, 7.2 Hz, 1 H, H-6a′), 3.23 (dd, J = 12.7, 5.9 Hz, 1
H, H-6b′), 2.14 (s, 3 H, C(O)CH3), 2.09 (s, 3 H, C(O)CH3),
2.03 (s, 3 H, C(O)CH3), 2.01 (s, 3 H, C(O)CH3), 2.00 (s, 3 H,
C(O)CH3), 1.94 (s, 3 H, C(O)CH3); 13C NMR (101 MHz,
CDCl3): δ = 170.5, 170.2, 170.1, 169.9, 169.7, 169.1 (6×
C(O)), 133.4 (CH2CHCH2), 117.7 (CH2CHCH2), 100.7
(C1), 99.4 (C1′), 75.7 (C4), 72.8 (C3), 72.7 (C5), 72.2 (C5′),
71.7 (C2), 71.0 (C3′), 70.1 (CH2CHCH2), 69.2 (C2′), 67.5
(C4′), 62.0 (C6), 50.2 (C6′), 21.0, 20.9, 20.8, 20.7 (2×), 20.6
(6× C(O)CH3); ESI-MS: calculated [M + H]+ = 660.2, [M +
Na]+ = 682.2, [M + K]+ = 698.2, found [M + H]+ = 660.1, [M
+ Na]+ = 682.0, [M + K]+ = 698.0; CHN analysis: calculated C
49.16, H 5.65, N 6.37, O 38.81; found C 49.21, H 5.81, N 5.94.
2,3,4-Tri-O-acetyl-6-azido-6-deoxy-β-D-galactopyranosyl-
(1 → 4)-2,3,6-tri-O-acetyl-α/β-D-glucopyranose (42). Bis-
(methyldiphenylphosphine)cyclooctadiene-iridium(I) hexa-
fluorophosphate (30 mg, 0.035 mmol) was suspended in 10
Allyl 2,3,4-tri-O-acetyl-6-O-methylsulfonyl-β-D-galactopyr-
anosyl-(1 → 4)-2,3,6-tri-O-acetyl-β-D-glucopyranoside (40).
Lactoside 39 (3.5 g, 5.52 mmol) was dissolved in 50 mL dry
pyridine and cooled to 0 °C. Mesyl chloride (643 μL, 8.27
mmol) and 4-(dimethylamino)pyridine (122 mg, 1 mmol)
were added and the mixture was stirred for 16 h, while warming
to room temperature. The solvent was removed under reduced
pressure. The remaining material was dissolved in 200 mL
DCM and washed 3× with 50 mL each of 1 M HCl solution,
saturated NaHCO3 solution, and once with 50 mL brine. The
organic phase was dried with MgSO4 and evaporated under
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mL of dry THF and bubbled with hydrogen gas for 15 min at
room temperature while stirring. The metal complex dissolved
and the solution was added to a solution of lactoside 41 (1.5 g,
1.9 mmol) in 50 mL dry THF. The reaction was stirred for 60
min at room temperature. Water (5 mL) and N-bromosuccin-
imide (405 mg, 2.28 mmol) were added. After 1 min TLC
showed complete reaction. The mixture was diluted with 200
mL DCM and washed with 3× 50 mL saturated NaHCO3
solution and 1× 50 mL brine. The organic phase was dried with
MgSO4 and evaporated under reduced pressure. The crude
product was purified by silica column chromatography (eluent
petroleum ether/ethyl acetate 1:2 to 1:3). 42 was obtained as a
colorless solid (1.17 g, 1.89 mmol, quantitative; mixture of
isomers α/β = 2:1). Rf = 0.50 (petroleum ether/EtOAc 1:3);
1H NMR (400 MHz, CDCl3): δ = 5.51 (‘t’, J = 9.7 Hz, 1 H, H-
3α), 5.37 (‘t’, J = 3.3 Hz, 1 H, H-1α), 5.33 (d, J = 2.8 Hz, 1.5 H,
H-4′α/β), 5.23 (‘t’, J = 9.4 Hz, 0.5 H, H-3β), 5.09 (dd, J = 10.4,
7.8 Hz, 1 H, H-2′α), 5.08 (dd, J = 10.4, 7.7 Hz, 0.5 H, H-2′β),
4.96 (dd, J = 10.4, 3.4 Hz, 1.5 H, H-3′α/β), 4.81−4.76 (m, 1.5
H, H-2α/β), 4.71 (‘t’, J = 7.8 Hz, 0.5 H, H-1β), 4.54−4.47 (m, 3
H, H-6aα/β, H-1′α/β), 4.17 (ddd, J = 10.0, 4.0, 1.8 Hz, 1 H, H-
CDCl3): δ = 170.4, 170.2, 170.1 (2×), 169.5, 169.2 (6× C(O)),
161.1 (CNH), 100.8 (C-1′), 93.1 (C-1), 90.9 (CCl3), 75.2
(C-4), 72.5 (C-5′), 71.2 (C-5, C-3′), 70.1 (C-2), 69.6 (C-3),
69.3 (C-2′), 67.7 (C-4′), 61.6 (C-6), 50.4 (C-6′), 21.1, 20.9,
20.80, 20.75, 20.62, 20.60 (6× C(O)CH3); ESI-MS: calculated
[M + H]+ = 763.1, [M + Na]+ = 785.1, found [M + H]+ =
762.9, [M + Na]+ = 784.6; CHN analysis: calculated C 40.88,
H 4.35, N 7.33, O 13.92, Cl 13.92, found C 41.23, H 4.80, N
7.00.
For glycosylation and deacylation to 47 as well as synthesis
and analytical data of 48 and 49, see Supporting Information.
Cell Growth Conditions. HEK 293T cells were grown in
Dulbecco’s Modified Eagle’s Medium supplemented with 10%
calf serum (DMEM + 10% CS). Mouse embryonic fibroblasts
(MEFs) were cultured in DMEM with 10% fetal calf serum
supplemented with nonessential amino acids and pyruvate on
gelatin-coated culture dishes (0.1% gelatin in PBS). All cells
were incubated at 37 °C under a water saturated atmosphere
with 5% carbon dioxide and subcultured every 2−3 days.
Application of Glycolipids. Glycolipids were dissolved
and stored in a mixture of chloroform/MeOH 19:1, yielding
concentrations of typically 2−10 mM. 100 μL of these solutions
were transferred into a 2 mL glass vial and evaporated in a
nitrogen stream, followed by drying under vacuum. The
remaining solid was dissolved in a defined amount of EtOH,
yielding concentrations of typically 1−5 mM. A small amount
of this solution was diluted with 0.34 mg mL−1 dfBSA (68 kDa)
in HMEM to obtain a concentration of 1−10 μM glycolipid.63
The mixture was vortexed vigorously and added to cell cultures.
For observation by fluorescence microscopy, 2 × 105 HEK
293T cells were seeded in 3.5 cm μ-dishes (ibidi GmbH) with a
glass surface that had been coated with 1 μg mL−1 fibronectin
and 10 μg mL−1 poly(L-lysine) in PBS for 1 h at 37 °C. Cells
were grown for 16 h, washed with PBS, and incubated with 2
mL glycolipid solution for 30 min at 4 °C. Then the cells were
washed twice with PBS and labeled by click reactions.
Application of Glycolipids Using Fusogenic Lip-
osomes. The preparation of fusogenic liposomes was
performed as described.61 The lipids DOPE and DOTAP
(1:1) were dissolved in 500 μL DCM/ethanol (20:1) in a total
concentration of 1 mg mL−1. 5 mol % of the azide-labeled
glucosylceramide 3 or lactosylceramide 49 together with 5 mol
% TexasRed-DHPE were added to the solution. The solvent
was removed under reduced pressure for 2 h. The residue was
diluted in 125 μL HEPES buffer (pH 7.4) to the final lipid
concentration of 2.1 mg/mL and was vortexed for 2 min. The
suspension was further sonicated for 10 min and stored at 4 °C
before use. For incubation HEK 293T cells were treated with
75 μL of the suspension containing glycolipids 35 or 49 or
without modified glycolipid in 10 mL DMEM + 10% FCS for
10 min at 37 °C.
5α), 4.13−4.08 (m, 1.5 H, H-6bα/β), 3.87−3.79 (m, 2 H, H-4α/β
,
OHβ), 3.76−3.73 (m, 1.5 H, H-5′α/β), 3.65 (ddd, J = 10.2, 5.1,
2.0 Hz, 0.5 H, H-5β), 3.49−3.42 (m, 2.5 H, H-6a′α/β, OHα),
3.24 (dd, J = 12.8, 5.4 Hz, 1.5 H, H-6b′α/β), 2.15 (s, 4.5 H,
α
C(O)CH3αβ/β), 2.12 (s, 3 H, C(O)CH3 ), 2.10 (s, 1.5 H,
α
β
C(O)CH3 ), 2.06 (br s, 9 H, 2× C(O)CH3 , 2× C(O)CH3 ),
2.03 (s, 4.5 H, C(O)CH3α/β), 1.95 (s, 4.5 H, C(O)CH3α/β); 13C
NMR (101 MHz, CDCl3): δ = 171.0 (C(O)β), 170.67
(C(O)α), 170.65 (C(O)β), 170.4, 170.27 (2× C(O)α), 170.22
(C(O)β), 170.19 (C(O)α), 170.14 (C(O)β), 169.7 (br s,
C(O)α/β), 169.19 (C(O)β), 169.16 (C(O)α), 100.7 (C1′β),
100.6 (C1′α), 95.5 (C1β), 90.2 (C1α), 75.6 (2x, C4α/β), 73.6
(C2β), 73.1 (C5β), 72.3 (2×, C5′α/β), 72.1 (C3β), 71.5 (C2α),
71.1 (C3′α), 71.0 (C3′β), 69.6 (C3α), 69.3 (C2′α), 69.2 (C2′β),
68.4 (C5α), 67.70 (C4′α), 67.66 (C4′β), 62.1 (C6β), 62.0
(C6α), 50.36 (C6′α/β), 21.1−20.6 (m, 6× C(O)CH3α/β); ESI-
MS: calculated [M + H]+ = 620.2, [M + Na]+ = 642.2, [M +
K]+ = 658.2, found [M + H]+ = 620.1, [M + Na]+ = 642.0, [M
+ K]+ = 658.0; CHN analysis: calculated C 46.53, H 5.37, N
6.78, O 41.32; found C 46.18, H 5.59, N 6.22.
2,3,4-Tri-O-acetyl-6-azido-6-deoxy-β-D-galactopyranosyl-
(1 → 4)-2,3,6-tri-O-acetyl-α-D-glucopyranosyl trichloroaceti-
midate (43). Lactose derivative 42 (1.15 g, 1.86 mmol) was
dissolved in 15 mL dry DCM and cooled to 0 °C.
Trichloroacetonitrile (933 μL, 9.3 mmol) and DBU (28 μL,
0.2 mmol) were added and the resulting mixture was stirred for
30 min at 0 °C. The solution was filtered through a short plug
of silica gel and evaporated under reduced pressure. The crude
product was purified by silica column chromatography (eluent
petroleum ether/ethyl acetate 1:1 to 2:3). 43 was obtained as a
colorless solid (1.19 g, 1.55 mmol, 83%). Rf = 0.55 (petroleum
ether/EtOAc 1:1); 1H NMR (400 MHz, CDCl3): δ = 8.65 (s, 1
H, NH), 6.50 (d, J = 3.8 Hz, 1 H, H-1), 5.56 (‘t’, J = 9.7 Hz, 1
H, H-3), 5.35−5.33 (m, 1 H, H-4′), 5.11 (dd, J = 10.3, 7.9 Hz,
1 H, H-2′), 5.06 (dd, J = 10.1, 3.8 Hz, 1 H, H-2), 4.97 (dd, J =
10.3, 3.4 Hz, 1 H, H-3′), 4.55 (d, J = 7.9 Hz, 1 H, H-1′), 4.52−
4.47 (m, 1 H, H-6a), 4.15−4.08 (m, 2 H, H-5, H-6b), 3.93 (′t′,
J = 9.6 Hz, 1 H, H-4), 3.77−3.73 (m, 1 H, H-5′), 3.47 (dd, J =
12.9, 7.5 Hz, 1 H, H-6a′), 3.25 (dd, J = 12.9, 5.3 Hz, 1 H, H-
6b′), 2.16 (s, 3 H, C(O)CH3), 2.11 (s, 3 H, C(O)CH3), 2.09
(s, 3 H, C(O)CH3), 2.04 (s, 3 H, C(O)CH3), 2.00 (s, 3 H,
C(O)CH3), 1.96 (s, 3 H, C(O)CH3); 13C NMR (101 MHz,
Fluorescence Labeling. For copper-catalyzed click reac-
tions,54 a mixture of the following components was prepared
immediately before the labeling experiments (volume for one
cell culture dish): 2 mL PBS, 50 μM CuSO4 (stock solution 50
mM in H2O), 100 μM TBTA56 (stock solution 200 mM in
DMSO), 1 mM aminoguanidine (stock solution 100 mM in
PBS), desired amount of fluorescent dye (stock solutions in
DMSO), 10 mM sodium ascorbate (stock solution 2.5 M in
H2O). The solution was applied to HEK 293T cells in culture
dishes and incubated for 30 min at 4 °C. Cells were washed
twice with PBS, supplied with DMEM + 10% CS, and analyzed
by fluorescence microscopy. For copper-free labeling with
K
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Article
(5) Maier, O., Oberle, V., and Hoekstra, D. (2002) Fluorescent lipid
probes: some properties and applications (a review). Chem. Phys.
Lipids 116, 3−18.
cyclooctyne derivatives, cells were incubated with a solution of
2 μM DIBO-lissamine (6) (stock solution 5 mM in DMSO), 2
μM DIBO-AlexaFluor-488 (stock solution 2 mM in DMSO),
or 20 μM DIBO-biotin 50 (stock solution 50 mM in DMSO)
in PBS for 30−60 min at 4 °C or room temperature. Cells were
washed twice with PBS, supplied with DMEM + 10% CS and
analyzed by fluorescence microscopy. Cells which had been
labeled with DIBO-biotin 50 were incubated with streptavidin-
AlexaFluor-647 (stock solution 1 mg/mL in PBS; dilution
1:500 in PBS) for 15 min at 4 °C. Cells were washed twice with
PBS, supplied with DMEM + 10% CS, and analyzed by
fluorescence microscopy. Samples were observed with a TCS
SP5 confocal laser scanning microscope (Leica) using a 63×,
1.4 NA PLAPO oil-immersion objective (Figures 2 and 3) or
LSM 780 (Zeiss) equipped with a 63×, 1.4 NA oil immersion
objective and a GAsP detector array for spectral imaging
(Figures 4 and S2). Fluorescence signals of labeled specimens
were serially recorded with appropriate excitation wavelengths
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.bioconj-
chem.6b00177.
■
S
Experimental procedures and analytical data of remaining
compounds; additional structures, additional confocal
microscopy images of modified glycosphingolipids;
analysis by flow cytometry (PDF)
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and Schwille, P. (2012) Partitioning, diffusion, and ligand binding of
raft lipid analogs in model and cellular plasma membranes. Biochim.
Biophys. Acta, Biomembr. 1818, 1777−1784.
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(2013) STED microscopy detects and quantifies liquid phase
separation in lipid membranes using a new far-red emitting fluorescent
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AUTHOR INFORMATION
Corresponding Author
*E-mail: mail@valentin-wittmann.de. Phone: +49-7531-88-
4572. Fax: +49-7531-88-4573.
■
Author Contributions
#Martin Dauner and Ellen Batroff contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the Deutsche Forschungsgemein-
schaft (SFB 969), the University of Konstanz, the Konstanz
Research School Chemical Biology, and the Zukunftskolleg of
the University of Konstanz. We thank the Bioimaging Center of
the University of Konstanz for providing the fluorescence
microscopy instrumentation.
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