Incorporation and visualization of azido-functionalized: N -oleoyl serinol in Jurkat cells, mouse brain astrocytes, 3T3 fibroblasts and human brain microvascular endothelial cells

Article abstract of DOI:10.1039/c6cc02879a

The synthesis and biological evaluation of azido-N-oleoyl serinol is reported. It mimicks biofunctional lipid ceramides and has shown to be capable of click reactions for cell membrane imaging in Jurkat and human brain microvascular endothelial cells.

Full text of DOI:10.1039/c6cc02879a

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Incorporation and visualization of azido-functionalized N-oleoyl
serinol in Jurkat cells, mouse brain astrocytes, 3T3 fibroblasts and
human brain microvascular endothelial cells
T. Waltera†, L. Collenburgb†, L. Japtokc, B. Kleuserc, S. Schneider-Schauliesb, N. Müllerb, J. Becamd,
A. Schubert-Unkmeird, J.N. Konge, E. Biebericheand J. Seibel a*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
The synthesis and biological evaluation of azido-N-oleoyl serinol is
reported. It mimicks biofunctional lipid ceramides and has shown
to be capable of click reactions for cell membrane imaging in
Jurkat and human brain microvascular endothelial cells.
Ceramide
1 regulates physiological functions including
apoptosis, cell-growth, -differentiation, -migration and
adhesion and has been implicated1 in cancer,
neurodegeneration, diabetes, microbial pathogenesis, obesity,
and inflammation.2, 3 Bieberich et al. described N-oleoyl serinol
Scheme 1 Synthesis of azide modified R N-oleoyl serinol 3a from L-serine methyl ester
hydrochloride 4a or analogue with D-serine methyl ester hydrochloride 4b. Reagents
and conditions: a) NEt3, CH2Cl2, rt, 24 h, 84 % (6a), 83 % (6b). b) DIBAL-H, thf, 0 °C -> rt,
24 h, 74 % (7a), 94 % (7b). c) MsCl, NEt3, DCM, 0 °C, 1h. d) dmf, 60 °C, 16 h 80 % over
2
(S18) as a ceramide mimicking molecule (see Figure 1).4, 5 The two steps (8a), 86 % over two steps (8b). e) 4 N HCl, 107 °C, 16 h. f) Oleic acid, DCC,
NHS, DIPEA, dmf, rt, 24 h 47 % over two steps (3a), 73 % over two steps (3b).
validity of this suggestion is reflected in the fact that it induces
apoptosis in rapidly dividing neuroblastoma cells, but not in
biological studies of 3a and 3b azido analogues of S18.
Ceramide analogs with fluorescent tags proved to be useful for
the study of dynamics and visualizing membrane architecture.
However, the suitability of such lipids is limited to its ability to
behave similar to its unmodified counterpart. Bioorthogonal
chemical reporter strategies have adressed this issue and
successfully applied, i.e. the alkyne-azide “click” chemistry. In
this context, we aimed to apply click chemistry to N-oleoyl
serinol mimicking ceramide by introducing an azide group and
examining if substitution of one serinol- hydroxyl group by an
azide will preserve its biological capabilities. For the synthesis
resting or differentiated cells.4 Also,
2 activates atypical
protein kinase C (aPKC)4 and induces formation of aPKC
associated complexes with polarity proteins in stem cell
experiments.6 The similarity of S18 to ceramide is also
represented by the fact that a highly specific antibody against
ceramide, also developed by Bieberich et al., binds to S18.7
aPKC has been also evaluated successfully as a target for small
molecule drug candidates against pancreatic cancer cells.8
Here we present preliminary data on the synthesis and
of
3
we used a modified protocol of Hawkins et al.9, 10 with an
enantiomer excess (ee) of 92 for 3a and 95 for 3b. For detailed
Information of the synthesis see Scheme 1 and supporting
Informations.
Recently the Golgi apparatus has been imaged using a clicked
ceramide analogue by 3D confocal and STED microscopy in
HeLa cells.11 Building on those studies, we first examined if the
Fig. 1 Structure of C16-Ceramide 1, N-oleoyl serinol 2 and its azide-modified analogs
3a, 3b used in this work.
azido-analogues 3a and 3b of N-oleoyl serinol
2 (S18) shows
cell toxicity in Jurkat T cells. Compound 3b proved to be almost
non toxic at a concentration of 25 µM (Fig. S1 and S2),
comparable to the Golgi marker NBD-C6-ceramide.12 We next
aimed to establish if 3b would be incorporated into the target
T
cells. DIBO488 dye was coupled under copper-free
conditions to 3b 5 min following feeding of Jurkat cells and its
accumulation at the plasma membrane was analyzed over
time by flow cytometry.
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Fig. 2 Jurkat T cells were incubated with 25 µM 3b (left panels) or 2 (right panels) in
HBSS for 30 min at RT. After washing, the cells were exposed to 20 µM DIBO 488 dye
and the click reaction was performed for 5 min at RT (upper panels) or for 60 min at
37 °C (lower panels). Cells were washed three times with HBSS and kept at rt until
microscopic analysis.
Fig 3 HBMEC were treated with 25 µM 3b (left panels) in RPMI medium for 5 min at RT
or were left untreated (right panels). After washing, the cells were exposed to 25 µM
DIBO 488 alkyne and click reaction was performed for 5 min at RT (upper panels) or for
60 min at 37 °C (lower panels). Cells were washed three times with PBS and kept at RT
until microscopic analysis.
GM130, a marker for cis-Golgi (see Figure S7). In a third
approach we used human brain microvascular endothelial cells
(HBMEC) as serinol acceptors. HBMEC are unique EC
representatives as they have continuous intercellular tight
junctions (TJs), which control movement of molecules through
the EC layer. The EC-pericyte interactions lead to induction of
TJ formation. Under inflammatory conditions, pericytes
stimulate immune response by production of cytokines.14 Also
it has been shown that Neisseria meningitidis, which targets
brain ECs, induces the formation of ceramide-enriched
platforms on HBMEC to favor bacterial uptake. 15, 16
While NBD-C6-ceramide included as control only slightly
peaked after 25 min, this was more pronounced for 3b which
continued to accumulate at the cell surface (see Figure 2). As
revealed by mass spectrometry analyses, 70 % (pmol) of 3b
were incorporated into Jurkat cells over time. In contrast to
that seen in Jurkat cells, incorporation of 3b into unstimulated
primary T cells was very low. The differential uptake was also
observed upon supplementation of the labeling reaction by F-
pluronic, which generally increased uptake of the azide-lipids.
To analyze whether its incorporation pattern would be
compatible with that of ceramide, subcellular distribution of
3b was monitored over time in living cells. 3b efficiently
accumulated at the plasma membrane and in the Golgi-
compartment. Thus 3b behaves similar to the Golgi marker
NBD-C6-ceramide indicating that it might truly represent a
ceramide. Incorporation of the click-labeled compounds
appeared to be specific as labeling intensities were dose-
dependent and unconjugated, but not conjugated dye diffused
into the cytosol. In addition to staining the plasma membrane,
clicked 3b accumulated in an intracellular compartment also
targeted by the Golgi-marker NBD-C6-ceramide (see Figure S6
One of the fundamental biological functions of ceramides is
18
that they can induce apoptotic cell death.17, HBMEC were
incubated with unmodified N-oleoyl serinol
2 and the
enantiomeric enriched compounds 3a and 3b and cell death
were determined. Annexin V binding and propidium iodide (PI)
staining enabled to discriminate between early apoptotic, late
apoptotic and necrotic cell death. Treatment of HBMEC with
25 µM for 1 h did not result in any toxic effects (Figure S2A),
however, incubation overnight (16 h) with 10 µM or 50 µM
resulted in an increase of late apoptotic and in particular
and S7). Although both ceramide
insert into membranes in the similar way, they can have very
different biological functions.
1 and azido N-oleoyl serinol
3
In addition to colocalization studies with NBD-C6 ceramide, we
also tested colocalization of 3b with intracellular structures
labeled with an antibody raised against ceramide. We used
primary cultured astrocytes because they were previously
shown to enrich ceramide in Golgi and other vesicular
compartments13. Figure 4 shows that there the anti-ceramide
Fig 4 Primary cultured astrocytes were incubated with 5 µM each 3a and b for 1 h,
washed with PBS, and then click reaction performed with 5 µM Alexa 546 DIBO alkyne
for 20 min at RT. After washing, cells were fixed with p-formaldehyde/glutaraldehyde
and permeabilized under mild conditions (0.2 % Triton X-100/PBS, 5 min, RT).
Immuncytochemistry and co-labeling was performed with anti-ceramide rabbit IgG and
Alexa 488 anti-rabbit IgG.
antibody labeled compartments that were also positive for 3b
.
Consistent with previous results obtained with anti-ceramide
antibody and NBD-C6 ceramide labeling of the Golgi, these
compartments partially colabeled with an antibody against
2 | Chemm. Commun., 2016, 00, 1-3
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necrotic cells (Figure S2B). 3a and 3b were comparable with that more than half of the mitotic glia cells and neurons die
unmodified N-oleoyl serinol They are not toxic below during fetal brain development by apoptosis. The stained
concentrations of 50 µ (see Figure S2). Bieberich et al. ceramide analogue can be localized. Thus it might be a helpful
observed that N-oleoyl serinol induces apoptosis only in tool to investigate sphingosine-1-phosphate and ceramide,
rapidly dividing neuroblastoma cells. Here also induced and their metabolites roles in endothelial barrier function,
2.
M
2
3
apoptosis at similar concentrations. 3b incorporation in which may be a further advancement in this field.
HBMEC was efficiently (see Figure 3). While 3b is found in the This study was funded through the Deutsche
membrane after 5 min exclusively, after 60 min it is also Forschungsgemeinschaft (RU2123) and NIH grants R01
transported in the Golgi apparatus (see Figure 2, lower panels). R01AG034389 and R56NS095215 (to E.B.)
We hypothesized that
3 may thus be useful in evaluation of
PKCζ-mediated signal cascades. To test this hypothesis we
analyzed potential interaction of the ceramide analog with
PKCζ. We expressed the fusion protein human PKCζ-GFP in 3T3
fibroblasts and performed incubation and click reaction with a
Notes and references
1
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b. Figure 5 shows that GFP fluorescence as
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In summary we have demonstrated the incorporation and
visualization of azido-functionalized N-oleoyl serinol by
bioorthogonal click chemistry in Jurkat cells and human brain
microvascular endothelial cells. We have also shown that
apoptosis of the HBMEC can be titrated. It should be noted
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Fig 5 3T3 fibroblasts were transfected with human PKCζ-GFP and 24 h post-
transfection, incubated for 1 h with 5 µM each 3a and 3b. Click reaction was performed
with 5 µM Alexa 546 DIBO alkyne for 20 min at RT. After washing, cells were fixed with
p-formaldehyde/glutaraldehyde and permeabilized under mild conditions (0.2 % Triton
X-100/PBS, 5 min, RT). Immuncytochemistry and co-labeling was performed with anti-
PKCζ rabbit IgG and Alexa 647 anti-rabbit IgG.
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