Addressable Cholesterol Analogs for Live Imaging of Cellular Membranes

Article abstract of DOI:10.1016/j.chembiol.2018.04.015

Cholesterol is an essential component of most biological membranes and serves important functions in controlling membrane integrity, organization, and signaling. However, probes to follow the dynamic distribution of cholesterol in live cells are scarce and so far show only limited applicability. Herein, we addressed this problem by synthesizing and characterizing a class of versatile and clickable cholesterol-based imidazolium salts. We show that these cholesterol analogs faithfully mimic the biophysical properties of natural cholesterol in phospholipid mono- and bilayers, and that they integrate into the plasma membrane of cultured and primary human cells. The membrane-incorporated cholesterol analogs can be specifically labeled by click chemistry and visualized in live-cell imaging experiments that show a distribution and behavior comparable with that of endogenous membrane cholesterol. These results indicate that the cholesterol analogs can be used to reveal the dynamic distribution of cholesterol in live cells. Cholesterol is an important component of biological membranes, but probes recording its dynamic intracellular distribution are scarce. Rakers et al. developed cholesterol-derived imidazolium salts mimicking properties of natural cholesterol. Following specific labeling via click chemistry, one of the cholesterol analogs was shown to incorporate into cellular membranes equivalent to endogenous cholesterol.

Full text of DOI:10.1016/j.chembiol.2018.04.015

Article
Addressable Cholesterol Analogs for Live Imaging of
Cellular Membranes
Graphical Abstract
Authors
Lena Rakers, David Grill,
Anna L.L. Matos, ...,
Hans-Joachim Galla, Volker Gerke,
Frank Glorius
Correspondence
gallah@wwu.de (H.-J.G.),
gerke@wwu.de (V.G.),
glorius@wwu.de (F.G.)
In Brief
Cholesterol is an important component of
biological membranes, but probes
recording its dynamic intracellular
distribution are scarce. Rakers et al.
developed cholesterol-derived
imidazolium salts mimicking properties of
natural cholesterol. Following specific
labeling via click chemistry, one of the
cholesterol analogs was shown to
incorporate into cellular membranes
equivalent to endogenous cholesterol.
Highlights
d
d
d
d
We describe the synthesis of different cholesterol-based
imidazolium salts
These cholesterol analogs mimic the properties of natural
cholesterol
The cholesterol analogs show a cholesterol-like behavior
in cells
The dynamic distribution of cellular cholesterol can be
assessed with these analogs
Rakers et al., 2018, Cell Chemical Biology 25, 1–10
August 16, 2018 ª 2018 Elsevier Ltd.
https://doi.org/10.1016/j.chembiol.2018.04.015

Please cite this article in press as: Rakers et al., Addressable Cholesterol Analogs for Live Imaging of Cellular Membranes, Cell Chemical Biology
(2018), https://doi.org/10.1016/j.chembiol.2018.04.015
Cell Chemical Biology
Article
Addressable Cholesterol Analogs for Live
Imaging of Cellular Membranes
2
3
3
1
4
Lena Rakers,1,5 David Grill,2,5 Anna L.L. Matos, Stephanie Wulff, Da Wang, Jonas Borgel, Martin Korsgen,
¨
¨
Heinrich F. Arlinghaus,4 Hans-Joachim Galla,3, Volker Gerke, and Frank Glorius
2,
1,6,
*
*
Institute of Organic Chemistry, University of Munster, Corrensstrasse 40, D-48149 Munster, Germany
*
1
€
€
2
€
Institute of Medical Biochemistry, Center for Molecular Biology of Inflammation, University of Munster, Von-Esmarch-Strasse 56,
€
D-48149 Munster, Germany
3
€
€
Institute of Biochemistry, University of Munster, Wilhelm-Klemm-Strasse 2, D-48149 Munster, Germany
4
€
Institute of Physics, University of Munster, Wilhelm-Klemm-Strasse 10, D-48149 Munster, Germany
€
5These authors contributed equally
6Lead Contact
*Correspondence: gallah@wwu.de (H.-J.G.), gerke@wwu.de (V.G.), glorius@wwu.de (F.G.)
https://doi.org/10.1016/j.chembiol.2018.04.015
SUMMARY
However, this approach is limited to intrinsically fluorescent mol-
ecules or requires the attachment of a fluorescent moiety to the
Cholesterol is an essential component of most bio- molecule of interest. As cholesterol does not show an intrinsic
fluorescence, modifications of the cholesterol body introducing
a fluorescent moiety or cholesterol binding molecules, which
logical membranes and serves important functions
in controlling membrane integrity, organization, and
signaling. However, probes to follow the dynamic
distribution of cholesterol in live cells are scarce
and so far show only limited applicability. Herein,
we addressed this problem by synthesizing and
characterizing a class of versatile and clickable
cholesterol-based imidazolium salts. We show that
these cholesterol analogs faithfully mimic the bio-
physical properties of natural cholesterol in phos-
bear a fluorescent moiety, were developed to characterize its
steady-state distribution (Gimpl and Gehrig-Burger, 2007; Max-
€
field and Wustner, 2012). These include cholesterol binding
agents such as filipin and perfringolysin O (Lopes et al., 2004;
Palmer, 2001), chemically relatedsteroidssuch as dehydroergos-
terol, which are intrinsically fluorescent (Rogers et al., 1979; Bar
€
et al., 1989; Wustner, 2007), and fluorescently labeled cholesterol
derivatives such as those containing an azi group as a fluorescent
moiety at the double bond (Thiele et al., 2000) or BODIPY coupled
pholipid mono- and bilayers, and that they integrate to the end of the alkyl chain (Li et al., 2006; Ho¨ ltta-Vuori et al.,
2008). However, the visualization of cholesterol organization
and dynamics in live cells using these markers has been
into the plasma membrane of cultured and primary
human cells. The membrane-incorporated choles-
terol analogs can be specifically labeled by click
chemistry and visualized in live-cell imaging experi-
ments that show a distribution and behavior compa-
rable with that of endogenous membrane choles-
terol. These results indicate that the cholesterol
analogs can be used to reveal the dynamic distribu-
hampered by the poor delivery of the labeled derivatives intocells,
the prerequisite for cell fixation, the relatively weak staining and
resolution, and the perturbation of the native membrane organiza-
tion, e.g., because of less efficient packing (Sezgin et al., 2017).
Therefore, our aim was to design a cholesterol-based system
that can be efficiently labeled, incorporates into cellular mem-
branes without disturbance of the bilayer, remains stable, and
tion of cholesterol in live cells.
can eventually be employed to follow the cholesterol distribution
in live cells. To achieve this, we turned to ionic liquids that have
recently gained considerable attention because of their specific
interactions with biomolecules such as phospholipids or proteins
and their potential applicability in medicinal chemistry (Benedetto
INTRODUCTION
As a fundamental membrane component of higher eukaryotes, and Ballone, 2016; Egorova et al., 2017; Benedetto, 2017). One
cholesterol is not only necessary for the regulation of membrane class of these liquids is based on imidazolium salts that show
fluidity and the generation of a semipermeable barrier, but also high stability, are amenable to diverse structural variations and
participates in several signaling processes and membrane-traf- therefore adaption of specific characteristics, and can function
ficking events (Ikonen, 2008; Brown and London, 2000; Simons as precursors for N-heterocyclic carbenes, e.g., as ligands in
and Ikonen, 2000; Cooper, 1978). Moreover, perturbation of transition-metal catalysis (Hopkinson et al., 2014; De Fremont
cholesterol homeostasis is observed in many human diseases et al., 2009; Hermann, 2002). Moreover, the use of imidazolium
including Niemann-Pick disease type C, Tangier disease, and salts in biological environments (Benedetto and Ballone, 2016;
atherosclerosis (Maxfield and Tabas, 2005). To analyze the role Egorova et al., 2017; Benedetto, 2017; Riduan and Zhang,
of cholesterol in the different membrane-related processes it is 2013), e.g., imidazolium salt-based artificial anion transporters
of central importance to assess its dynamic distribution in living (Elie et al., 2011, 2013; Gravel and Schmitzer, 2015) and long-
cells. Here, fluorescence microscopy can serve as a useful tool. alkyl chain-containing imidazolium salts as phospholipid analogs
Cell Chemical Biology 25, 1–10, August 16, 2018 ª 2018 Elsevier Ltd.
1

Please cite this article in press as: Rakers et al., Addressable Cholesterol Analogs for Live Imaging of Cellular Membranes, Cell Chemical Biology
(2018), https://doi.org/10.1016/j.chembiol.2018.04.015
Figure 1. Preparation of Two Types of
Cholesterol-like Imidazolium Salts (Zhang
et al., 2006).
Because the hydroxyl group and the
double bond are replaced in these
compounds, we first carried out a series
of biophysical experiments to analyze
whether the imidazolium salts synthe-
sized show a cholesterol-like behavior.
As the effects of cholesterol on the fluidity
and packing of phospholipid-containing
bilayers have been described in many bio-
physical studies, we compared the prop-
erties of type I and type II compounds
to those of natural cholesterol following
incorporation into 1,2-dipalmitoyl-sn-
glycero-3-phosphocholine (DPPC) mem-
branes using differential scanning calo-
rimetry (DSC), fluorescence polarization,
and filmbalance measurements. The con-
centration of the cholesterol additive was
€
€
(Wang et al., 2015a, 2015b, 2016; Ruhling et al., 2017; Drucker varied between 0 and 30 mol%. Cholesterol is known to affect the
et al., 2017), demonstrates their applicability for cellular studies. phase transition temperature of DPPC liposomes in a concentra-
Based on these results, we now designed and synthesized imida- tion-dependent manner, with an increase of the cholesterol
zolium salts that are based on cholesterol and validated their amount leading to a broadening of the transition temperature pro-
cholesterol-like properties by film balance, calorimetry, and fluo- file and a shift to slightly lower transition temperatures (McMullen
rescence polarization measurements. One member of this class et al., 1993; Demetzos, 2008; Benesch et al., 2011). Furthermore,
of cholesterol mimetics was further examined in different cultured at concentrations of more than 30% cholesterol, the phase tran-
cells revealing its cholesterol-like behavior in a natural membrane sition disappears. A similar behavior is observed for the choles-
environment. Following site-specific labeling with fluorescent terol analogs (Figures S1A and S1B), with 2a being the compound
dyes, this cholesterol-like imidazolium salt can be used to visu- showing the highest analogy to cholesterol (Figure 2A). However,
alize the dynamic distribution of cholesterol in live cells.
it should be noted that the phase transition temperature is shifted
to slightly lower temperatures in the case of all imidazolium salts,
compared with natural cholesterol, which is probably caused by
the more spacious polar group replacing the original OH group.
We also used fluorescence polarization as another method to
analyze how phase separation behavior and stiffness of lipids is
RESULTS
Synthesis and Biophysical Characterization of Two
Types of Cholesterol-Based Imidazolium Salts
The facile synthesis of imidazolium salts enables the generation affected by the cholesterol-like additives (Borenstain and Bare-
of a high diversity of molecules with the possibility to incorporate nholz, 1993). Therefore, a dye (1,6-diphenyl-1,3,5-hexatriene)
up to five different substituents on the five-membered hetero- was incorporated into liposomes containing DPPC and the
cycle. Starting with cholesterol, two straightforward possibilities corresponding cholesterol compound, and the polarization was
exist to link this moiety to an imidazolium core (Figure 1): either measured for three different concentrations of the cholesterols.
the steroidal body is attached to one of the nitrogen atoms At room temperature the fluorescence polarization is not affected
(type I) or it is placed in the backbone of the five-membered by increasing concentrations of natural cholesterol, whereas
ring (type II). In both cases, the hydroxyl group of cholesterol is an increase in polarization is observed upon cholesterol incorpo-
replaced by the imidazolium core, generating different flexibil- ration at 47ꢀC (Figures 2C and 2D), which is in accordance
ities between the cholesterol moiety and the imidazolium ring with the literature (Bhattacharya and Haldar, 2000). The same
in the type I and type II compounds. Moreover, to simplify the behavior is observed for compound 2a, showing no effect on
synthesis the double bond was hydrogenated in the first step. the fluorescence polarization at room temperature, but inducing
All synthetic steps were carried out in the absence of toxic com- an increase at 47ꢀC. In contrast, incorporation of the other two
ponents to exclude any toxic contamination of the products. compounds (type I, 1a and 1b) results in a slight decrease of po-
Different substituents on the nitrogen atoms were chosen. In larization at room temperature and constant values at 47ꢀC (Fig-
the case of type I molecules, a small group (methyl, 1a) and a ures 2C and 2D). In addition to analyzing the cholesterol-like
flexible group (benzyl, 1b) were introduced as the second nitro- behavior in phospholipid bilayers, we also carried out monolayer
gen substituent, whereas only a small group (methyl, 2a) was studies using film balance measurements (Figures S1J–S1L ). In
selected for the type II imidazolium salts.
binary DPPC/cholesterol films, an increase of relative cholesterol
2
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Please cite this article in press as: Rakers et al., Addressable Cholesterol Analogs for Live Imaging of Cellular Membranes, Cell Chemical Biology
(2018), https://doi.org/10.1016/j.chembiol.2018.04.015
Figure 2. The Cholesterol-Based Imidazolium Salt (Compound 2a) Shows Cholesterol-like Properties
Differential scanning calorimetry (DSC) endotherms of large unilamellar vesicles (LUVs) composed of binary mixtures of DPPC with varying concentrations (0–30
mol%) of compound 2a (A) and cholesterol (B) in pure water. Fluorescence polarization at 450 nm of LUVs containing DPPC, diphenylhexatriene (DPH) and
varying concentrations of cholesterol or its analogs recorded at room temperature (C) or 47ꢀC (D). All experiments were performed at least three times.
concentration is known to lead to lower pressures at the same way that would render the compound accessible for modular
area per molecule and a disappearance of the plateau indicating click chemistry without disturbing the membrane integration.
a condensing effect (Miyoshi and Kato, 2015). A similar behavior Therefore, the azide was attached via a short alkyl chain to the
is again observed for compound 2a. While low concentrations C2 position of the imidazolium core, ensuring that a clicked
of compound 2a seem to trigger a minor weakening of the film fluorophore will most likely not affect membrane integration of
(higher surface pressure at the same area per molecule), higher the cholesterol moiety. Two different lengths of the alkyl chains
concentrations lead to more stable monolayers. The monolayer were synthesized, yielding systems 2b and 2c (Figure 3).
stability of DPPC mixtures with both 1a and 1b remains almost
Both, 2b and 2c were first characterized by DSC, fluorescence
unaffected for concentrations of up to 5 mol%. Thus, 2a appears polarization, and film balance measurements that indicated no
to be the most suitable compound for cellular experiments, significant change compared with the biophysical properties of
because its behavior in mono- and bilayers shows high similar- system 2a, i.e., an integration into DPPC membranes compara-
ities to that of natural cholesterol.
ble with that of natural cholesterol (Figures S1D, S1E, S1H, S1I,
S1M, and S1N). As the film balance measurements of compound
2c showed slightly better cholesterol-like properties than com-
pound 2b, 2c was chosen for live-cell experiments. Any toxic
The Cholesterol-Based Imidazolium Salts as
Addressable Cholesterol Mimetics in Cells
Given the cholesterol-like properties observed in the biophysical or lytic effects of the cholesterol-like imidazolium salts were
analyses, we next employed the cholesterol-based imidazolium analyzed by cytotoxicity assays employing compounds 2a and
salts in cellular studies with the aim to develop a system that 2c at concentrations ranging from 0 to 100 mM on cultured
could be used to follow the dynamic distribution of cholesterol HeLa and primary human endothelial cells (human umbilical
within cells. Compound 2a was chosen for these studies as its vein endothelial cells [HUVECs]). Lactate dehydrogenase release
behavior proved to be closest to natural cholesterol (see above). as an indicator of cell lysis (Bacon et al., 1990) was only observed
Because the system lacked intrinsic fluorescence, a prerequisite at concentrations exceeding 10 mM (Figures S2A and S2B).
for live-cell imaging, we chose to integrate an azide group in a Therefore, concentrations of 2–5 mM were chosen in all future
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(2018), https://doi.org/10.1016/j.chembiol.2018.04.015
Figure 3. Click reaction of Compounds 2b
and 2c with a Dibenzocyclooctyne-Based
Fluorophore
The imidazolium salts were generated by attach-
ment of a short alkyl chain to the C2 position of
system 2a.
In a next step, we performed labeling
reactions with live HeLa cells and
HUVECs, again using compound 2c.
Two principle sets of experiments were
chosen, one employing a cell surface
cell experiments. These concentrations also had no effect on cell click reaction and the second using compound 2c pre-labeled
viability and morphology (Figure S2C).
with Alexa Fluor 555 before addition to the cells. In the first
Next, we employed a label-free method to determine whether case, cells were incubated with 2c for 90 min at 37ꢀC to guar-
the cholesterol-based imidazolium salts are correctly incorpo- antee efficient plasma membrane incorporation. The fluorophore
rated into the plasma membrane. Therefore, cryogenic time- was then added at 4ꢀC to prevent internalization by fluid-phase
of-flight secondary ion mass spectrometry (cryo-ToF-SIMS) endocytosis and the click reaction was allowed to proceed for
measurements (Draude et al., 2015) were performed. HeLa cells 20 min. Figure 5B shows that this treatment leads to an efficient
cryo-fixed after incubation with the imidazolium salts were sub- and specific labeling of the plasma membrane of both, HeLa
jected to ToF-SIMS analysis to reveal the distribution of the cells and HUVECs.
compounds based on their mass signature. Compounds 2a
The second approach yields a comparable result. When HeLa
and 2c were chosen for this analysis, also to assess whether or HUVECs were treated with pre-clicked compound 2c for
attachment of the alkyl chain and azide moiety (2c) alters any 20 min at 4ꢀC, analysis by confocal microscopy reveals a spe-
membrane incorporation of the non-functionalized compound cific label incorporation into the plasma membrane (Figure 5C).
(2a). Figure 4 shows that compound 2c is mainly located in It should be noted that some labeled compound 2c is also pre-
the plasma membrane. Only small amounts are detectable in sent in internal structures, both after surface click reaction and
the cytosolic region most likely reflecting a presence in intracel- in the case of applying the pre-clicked derivative. This internal
lular organelles such as endosomes. This is in line with the fraction increases with time of incubation at room temperature
natural cholesterol distribution and is also observed for com- and most likely reflects an internalization into endosomes as
pound 2a (Figure S3 ).
the Alexa Fluor 555-2c structures colocalize with endosomal
Given this distribution, we chose compound 2c for further live- markers (Figure S4B ). Together, the live-cell labeling experi-
cell imaging experiments to visualize the membrane incorpora- ments indicate that compound 2c is incorporated into the
tion and cellular uptake of this cholesterol analog. Site-specific plasma membrane of cultured cells thus mimicking the behavior
labeling with two different fluorophores (click-iT dibenzocy- of natural cholesterol and that following membrane integration it
clooctyne [DIBO] Alexa Fluor 555 or click-iT DIBO-Alexa Fluor is accessible to cell surface click chemistry.
488) was achieved by copper-free click chemistry. In a first set
To further analyze the cholesterol analogy of the substance 2c,
of experiments, we concentrated on the plasma membrane inte- we recorded potential changes in the localization of 2c
gration. Therefore, giant plasma membrane vesicles (GPMVs) upon alteration of the normal cellular cholesterol distribution.
were isolated from HUVECs that had been pretreated with com- First, we employed the drug U18666A, a membrane-permeable
pound 2c for 90 min. GPMVs that can be chemically induced by amino-steroid that inhibits the endosomal cholesterol export
treatment with paraformaldehyde (25 mM) and DTT (2 mM) in a protein Niemann-Pick C1 (NPC1) (Lu et al., 2015), and thereby
calcium-containing buffer reflect to a large extent the lipid leads to an aberrant accumulation of cholesterol in endosomal
and protein composition of the plasma membrane but lack an structures and its depletion from the plasma membrane (Sobo
underlying cytoskeleton (Scott, 1976; Fridriksson et al., 1999). et al., 2007). To analyze whether natural cholesterol and com-
Following isolation, the GPMVs were subjected to click reaction pound 2c are similarly affected by this drug, HUVECs containing
with Alexa Fluor 555, and labeling of the vesicles was analyzed 2c in the plasma membrane were subjected to click labeling with
by confocal fluorescence microscopy. Figure 5A demonstrates Alexa Fluor 555 before or after treatment with U18666A and then
the successful covalent binding of the fluorophore to GPMVs analyzed by fluorescence microscopy. Figure 6A shows that the
prepared from cells treated with compound 2c that was not distribution of fluorophore-labeled compound 2c is markedly
observed when the non-clickable compound 2a was used. affected after incubating the cells with U18666A. U18666A treat-
This is in line with our cryo-ToF-SIMS data and indicates that ment for 6 and 24 hr both cause an accumulation of the com-
the cholesterol-mimicking imidazolium salt had been success- pound in the enlarged endosomes. Whereas almost all 2c is pre-
fully incorporated into the plasma membrane and that the azide sent in endosomes at 24 hr, small amounts of compound 2c can
group is accessible for click chemistry in this membrane bound still be found in the plasma membrane at 6 hr of treatment (Fig-
form. No indication of background fluorescence in the lumen ure S4A ). To compare this distribution to that of natural cellular
of the GPMV was detectable confirming that the fluorophore cholesterol, the cells were also fixed and stained with filipin
does not cross the bilayer.
following the respective U18666A treatments. This shows the
4
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(2018), https://doi.org/10.1016/j.chembiol.2018.04.015
Figure 4. Cryo-ToF-SIMS Measurements of
HeLa Cells Incubated with the Cholesterol
Analog 2c
(A) Intensity distribution images of various sub-
stances: 2c; an unspecific organic fragment
(C3H5+); overlay of the two signals for identifying
the distribution of compound 2c; head group
fragment of phosphocholines (C5H15PNO4+) from
PC and/or SM; overlay of Na+ and K+ signals.
(B) Depth distribution of the substances: red, 2c;
blue, two different phospholipid references; green,
unspecific organic fragment (C3H5+); orange, po-
tassium. All experiments were performed at least
five times.
terol. Treatment of the cells with U18666A
prior to Alexa Fluor 555 labeling by click
chemistry leads to an almost complete
inhibition of labeling (Figure 6B). This
shows that the plasma membrane pool of
compound 2c is efficiently reduced by
U18666A treatment, again mimicking the
behavior of cellular cholesterol.
To further analyze the cholesterol anal-
ogy of compound 2c we concentrated
on the plasma membrane-resident frac-
tion and assessed whether its distribution
within the membrane can be affected
by treatments altering the distribution of
plasma membrane cholesterol. The pen-
tameric B subunit of cholera toxin (CTB)
was chosen as a means to cluster choles-
terol that is enriched in membrane micro-
domains containing GM1 gangliosides,
the receptors of CTB (Antes et al., 1992).
Figure 6C shows that fluorophore-labeled
2cco-clusterswithCTB, mostlikelyreflect-
inganassociationwithGM1-containingmi-
crodomains typically also observed for nat-
ural cholesterol (Lingwood et al., 2008).
DISCUSSION
Cholesterol is an essential component of
biological membranes involved in a variety
typical U18666A-induced accumulation of cholesterol in endo- of cellular processes, which range from signal transduction and
somes with filipin fully co-localizing with Alexa Fluor 555-labeled membrane trafficking to the regulation of bacterial and viral
compound 2c (Figure 6A).
infections. Membrane cholesterol mediates its effects by choles-
The plasma membrane integration and U18666A-induced re- terol binding proteins, cholesteroylation of proteins and, in
localization of fluorescently labeled compound 2c was next quan- particular, by changing properties of the lipid bilayer, e.g., mem-
tifiedby relativefluorescencemeasurements. Therefore, HUVECs brane stiffness, permeability, and domain formation (Lingwood
were loaded with compound 2c for 90 min and then incubated for et al., 2011; Schwarzer et al., 2014; Xie et al., 2016). Choles-
0, 24, or 48 hr before labeling the plasma membrane-resident pool terol-rich nano- and microdomains are of particular relevance
of 2c with Alexa Fluor 555 by bio-orthogonal click reaction. Fig- as they have been implicated in the control of many of the pro-
ure 6B shows that the relative cellular fluorescence is only mini- cesses mentioned above (Lingwood and Simons, 2010). Such
mally reduced following incubation periods of 24 or 48 hr. This in- membrane microdomains are thought to be highly dynamic in
dicates that the majority of compound 2c resides in the plasma nature, responding to both extracellular and intracellular signals.
membrane and thus is accessible for click chemistry for extended Despite the importance of cholesterol in these membrane-
periods of time, in line with the prime localization of natural choles- related events, the visualization of its distribution in live cells,
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(2018), https://doi.org/10.1016/j.chembiol.2018.04.015
Figure 5. Compound 2c Is Incorporated into the Plasma Membrane of Different Cultured Cells
The cartoon on top of each image shows the respective order of addition with ‘‘incubation’’ indicating the treatment with compound 2c. Click-iT DIBO-Alexa Fluor
555 was used as the fluorophore.
(A) GPMVs were isolated from HUVECs incubated with (top) compound 2c or with (bottom) compound 2a and subjected to the click reaction prior to analysis by
fluorescence microscopy.
(B) HeLa cells (top) and HUVECs (bottom) were incubated with unlabeled compound 2c before adding the fluorophore.
(C) HeLa cells (top) and HUVECs (bottom) were incubated with pre-labeled compound 2c and then subjected to fluorescence microscopy. In each panel,
differential interference contrast (DIC) images are included to visualize the vesicles and cells. Scale bars, 20 mm.
and hence the formation and dynamics of cholesterol-rich do- DSC, fluorescence polarization and film balance measurements
mains, have been hampered by limitations in directly or indirectly were used to determine whether the compounds generated
labeling cholesterol in live cells (Sezgin et al., 2017). Therefore, here display cholesterol-like membrane integration and proper-
we aimed to generate a cholesterol analog that would permit ties. In these studies DPPC was chosen as a well characterized
the visualization of cellular cholesterol in a dynamic fashion model membranepermittinga comparisonof basic membrane in-
without perturbation of the existing membrane.
teractions of our different cholesterol derivatives to those of
We chose imidazolium salts as basis of our compounds as they cholesterol in this system. An important aim here was to identify
are known for their high stability and straightforward synthesis. among the synthetic cholesterol-like derivatives the compound
Starting with cholesterol, we succeeded in synthesizing three most closely resembling natural cholesterol that could then be
different molecules varying in the way the cholesterol is con- employed in the cell biological experiments. DSC recordings of
nected to the imidazolium core and in the remaining substituents DPPC/cholesterol large unilamellar vesicles (LUVs) show that
on the nitrogen(s) (see Figure 1). Biophysical analyses involving cholesterol induces
a broadening of the phase transition
6
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Figure 6. The Cellular Localization of Compound 2c Is Affected by Alteration of the Cholesterol Distribution in a Manner Similar to that of
Natural Cholesterol.
(A and B) Late endosomal accumulation of cholesterol induced by treatment with U1866A. (A) HUVECs were loaded with compound 2c and then subjected to
click chemistry with Click-iT DIBO-Alexa Fluor 555 before (top) or after (bottom) a 24 hr treatment with U18666A. Cells were then fixed and stained with filipin. Note
the accumulation of compound 2c together with natural cholesterol (stained with filipin) in late endosomal structures (upper panels); no labeling of 2c is observed
after the U1866A treatment, indicative of efficient removal of the compound from the plasma membrane (lower panels). Scale bars, 20 mm. (B) Quantification of
labeling efficiency. The relative cellular fluorescence of Alexa Fluor 555 was measured for cells subjected to the click chemistry treatment at 0, 24, or 48 hr after
addition of compound 2c or for cells treated with compound 2c for 24 hr followed by a 24 hr treatment with U18666A before the click reaction. Mean and standard
deviations were obtained from three (n = 3) independent experiments.
(C) Clustering of GM1 containing microdomains affects the plasma membrane distribution of compound 2c. HeLa cells were incubated with unlabeled
component 2c before click reaction with Click-iT DIBO-Alexa Fluor 488. GM1 microdomain clustering was induced by adding pentameric cholera toxin B (CTB)
coupled to Alexa 594 as described in the STAR Methods. Scale bars, 30 mm (in the region highlighted by a white box in the merge image) and 2 mm (shown at a
higher magnification on the right). Note the co-clustering of CTB and 2c.
temperature and a minor shift to lower transition temperatures more pronounced for compound 2a compared with 1a and 1b.
indicative of a slight destabilization of the LUV lipid bilayer mem- Presumably, the more rigid structure of 2a leads to an orientation
brane at higher amounts of cholesterol. A similar but slightly more of the steroidal body in the bilayer that is analogous to that of nat-
pronounced effect is observed for the cholesterol analogs 1a, 1b, ural cholesterol. Compounds 1a and 1b are more flexible and thus
and 2a, with 2a showing the least deviation from the cholesterol could affect the lipid packing, and thus rigidity, of the bilayer
behavior and, similar to natural cholesterol, a destabilization of slightly differently. On the other hand, integration of 1a and 1b
the LUV bilayer only at higher concentrations. Likewise, fluores- into DPPC monolayers, as probed by film balance measure-
cence polarization measurements used to probe the fluidity of ments, appears to be similar to that of cholesterol, possibly
the membrane show properties equivalent to cholesterol (see Fig- because the more flexible link between cholesterol and the imida-
ure 2). Higher values of the polarization indicate a rigidification zolium is tolerated during compression of the monolayer. Com-
of the membrane that is observed for natural cholesterol only at pounds 1a and 1b differ from one another by the bulkiness of
elevated temperatures. Similar to cholesterol, compound 2a the nitrogen substituent, methyl versus benzyl, and the somewhat
does not affect the fluidity of the bilayer at room temperature, weaker integration of 1b compared with 1a is most likely due to a
but induces a rigidification of the membrane at temperatures higher steric demand of the benzyl head group.
above the natural phase transition of DPPC (> 42ꢀC). Compounds
The comparative biophysical analyses identified compound
1a and 1b, on the other hand, lead to a slight decrease of the po- 2a as the one most closely resembling the behavior of natural
larization indicativeof membranefluidizationat roomtemperature cholesterol in the DPPC model membrane system, and thus
and no significant rigidification at 47ꢀC. Thus, while all choles- enabled the selection of this derivative for the cellular studies.
terol-based imidazolium salts synthesized here show properties It should be noted, however, that employing mono- or bilayers
reminiscent of natural cholesterol, the analogy to cholesterol is with one type of zwitterionic lipid (DPPC) only allows the
Cell Chemical Biology 25, 1–10, August 16, 2018
7

Please cite this article in press as: Rakers et al., Addressable Cholesterol Analogs for Live Imaging of Cellular Membranes, Cell Chemical Biology
(2018), https://doi.org/10.1016/j.chembiol.2018.04.015
conclusion that the synthetic cholesterol-like imidazolium salts U18666A-treated cells, unlabeled 2c is likely to follow the
behave similarly to natural cholesterol with respect to membrane same relocation as it is depleted from the plasma membrane
insertion and disturbance in this type of model membrane. and not accessible for labeling by click reaction. Second, clus-
Cellular membranes also contain negatively charged lipids that tering of plasma membrane GM1 gangliosides by treatment
show a heterogeneous lateral organization and are enriched in with pentameric CTB, which is known to induce a co-clustering
the cytoplasmic leaflets. Properties of more complex bilayers of membrane cholesterol residing in GM1-rich domains, also re-
containing these negatively charged anionic lipids, including sults in a clustering of fluorescently labeled compound 2c into
their lateral organization, could be affected by the positive nature the same domains. Thus, in line with the biophysical character-
of the cholesterol-like imidazolium salts. This should be as- izations, experiments using an established cell line (HeLa) as
sessed in future detailed analyses of the phase behavior of het- well as primary human cells (HUVECs) suggest that the choles-
erogeneous model membranes containing the cholesterol deriv- terol-based imidazolium salts synthesized here show choles-
atives that are beyond the scope of the present work. However, it terol-like properties and can be used as cholesterol mimetics
should be noted that our cell biological experiments reveal an in live-cell imaging approaches. Moreover, as the azide group
integration of the cholesterol-like imidazolium salts into the present in 2c is available for modular click chemistry, other
plasma membrane of live cells, and a partitioning into cholera ligands can be introduced including those that could interfere
toxin-induced microdomains that are known to also contain with the incorporation into tightly packed membrane microdo-
the natural cholesterol. This strongly suggests a cholesterol- mains and thus disrupt such domains. Future studies have to
like behavior of our imidazolium salts also in complex mem- reveal whether the accordingly modified imidazolium salts
branes characterized by the presence of anionic lipids.
can indeed be used to interfere with the formation and function
As the application of the cholesterol-based imidazolium salts in of cholesterol-rich membrane domains, e.g., in the course of
live-cell imaging requires a fluorescent group, we decided to cellular signaling.
attach an azide moiety linked via an alkyl chain to the C2 position
In conclusion, we were able to synthesize and characterize
of the imidazolium core of compound 2a (see Figure 3). This strat- three cholesterol-based imidazolium salts and validate their
egy was chosen to prevent steric repulsion effects of an attached cholesterol-mimicking properties. The more rigid compound 2a
fluorophore with the phospholipid head groups thus enabling a showed the highest resemblance to normal cholesterol and
stable integration as in the case of 2a. Varying the lengths of was chosen for further modification to incorporate an azide
the alkyl chain identified a C5 linker as the most preferably moiety group at the C2 position via an alkyl linker. This allowed the
yielding compound 2c whose biophysical characteristics also attachment of a fluorophore by copper-free click chemistry
resembled those of natural cholesterol. Similar to cholesterol, and thereby enabled the live-cell visualization of this compound
compounds 2a and 2c also incorporated into the plasma mem- most likely reflecting the dynamic distribution of cellular choles-
brane of cultured human cells. For HeLa cells, this was verified terol. Thus, we generated a system that is able to mimic natural
by direct cryo-ToF-SIMS measurements of quick-frozen cells cholesterol and can be labeled on-demand without perturbation
treated with the imidazolium salts. In situ mass spectrometry of the bilayer.
was used to determine the cellular distribution of unlabeled imi-
dazolium salts that could be identified by specific ionization prod- SIGNIFICANCE
ucts and related to the localization of cellular membranes. The
overall distribution of 2a and 2c identified by cryo-ToF-SIMS Cholesterol is an essential component of most biological
analysis corresponded to that of natural cellular cholesterol, membranes and serves important cellular functions
with the majority residing in the plasma membrane. Some intra- including the regulation of membrane dynamics and traf-
cellular structures were also identified as harboring compounds ficking. To assess the role of cholesterol in cellular pro-
2a and 2c. Most likely, these correspond to endosomes and/or cesses it is of central relevance to follow its dynamic distri-
Golgi membranes that are known to contain significant amounts bution in live cells. However, so far the tools to record the
of cholesterol. However, the spatial resolution of the cryo-ToF- localization of cholesterol in live cells, e.g., by fluorescence
SIMS analysis is not sufficient for the unambiguous assignment microscopy, are scarce and limited in their applicability.
of the mass profiles to specific intracellular organelles.
The synthesis of cholesterol-derived imidazolium salts con-
Compound 2c could be specifically modified at the terminal taining an azide group for click chemistry addresses this
azide group by copper-free click chemistry following incorpora- problem. When incorporated into model membranes, the
tion into the plasma membrane of HeLa cells and HUVECs. addressable cholesterol mimetics show biophysical proper-
Hence, cell surface click chemistry can be carried out with the ties very similar to those of natural cholesterol. Importantly,
cholesterol-based imidazolium salts integrated into the plasma the cholesterol-based imidazolium salts are integrated
membrane. Furthermore, this approach enabled us to fluores- into cellular membranes, can be specifically labeled with
cently label a cholesterol-like compound and visualize its different fluorophors when residing in the plasma mem-
localization in live cells. Several alterations known to affect the brane of cultured cells and faithfully mimic the behavior of
distribution of cellular cholesterol reveal an equivalent behavior cellular cholesterol. Thus, the cholesterol analogs intro-
of compound 2c. First, pharmacological inhibition of the duced here represent valuable tools to monitor the dynamic
endosomal cholesterol exporter NPC1, which leads to an accu- distribution of cellular cholesterol in live cells. Moreover,
mulation of cholesterol in late endosomes, has a similar effect due to the availability for click chemistry, the cholesterol-
on the cholesterol-based imidazolium salts. While fluorescently like imidazolium salts integrated into the cell membrane
labeled 2c is directly seen accumulating in late endosomes of can not only be modified by fluorophores, but are available
8
Cell Chemical Biology 25, 1–10, August 16, 2018

Please cite this article in press as: Rakers et al., Addressable Cholesterol Analogs for Live Imaging of Cellular Membranes, Cell Chemical Biology
(2018), https://doi.org/10.1016/j.chembiol.2018.04.015
Received: December 20, 2017
to any molecule bearing an alkyne that can be introduced as
Revised: February 22, 2018
a functional tool for future cholesterol studies.
Accepted: April 13, 2018
Published: May 24, 2018
STAR+METHODS
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10 Cell Chemical Biology 25, 1–10, August 16, 2018

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(2018), https://doi.org/10.1016/j.chembiol.2018.04.015
STAR+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE
Chemicals, Peptides, and Recombinant Proteins
Cholesterol
SOURCE
IDENTIFIER
Alfa Aesar
Cat #A11470
Cat #850355
Cat #D3793
DPPC
Avanti Polar Lipids Inc.
Molecular Probes
Sigma Aldrich
BODIPY-PC
DPH
Cat #208000
Cat #C10405
Cat #C10406
Cat # BML-S200-0010
Cat. # F4767
Click-iT-DIBO Alexa Fluorꢁ 488
Click-iT-DIBO Alexa Fluorꢁ 555
U18666A
Thermo Fisher Scientific
Thermo Fisher Scientific
Enzo Inc.
Filipin III from Streptomyces filipinensis
Critical Commercial Assays
Vybrantꢁ Alexa Fluorꢀ 594 Lipid Raft Labeling Kit
Sigma Aldrich
Thermo Fisher Scientific
Cat #V34405
Experimental Models: Cell Lines
HeLa (CCL-2ꢀ)
ATCCꢁ
Cat #CCL-2ꢀ
Human umbilical vein endothelial cells (HUVECs)
Software and Algorithms
ChemDraw
PromoCell
Cat #C-12203
PerkinElmer Informatics
ImageJ
http://www.cambridgesoft.com/software/overview.aspx
Fiji
https://imagej.net/Fiji
http://mestrelab.com/
https://www.originlab.com/
N/A
MestReNova
Mestrelab Research
OriginLab
Originꢁ 9.1
SurfaceLab 6.4
ION-TOF
ZEN black edition (ZEN 2.3)
Zeiss
https://www.zeiss.de/mikroskopie/downloads/
zen.html#service-packs
CONTACT FOR REAGENT AND RESOURCE SHARING
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact,
Frank Glorius (glorius@wwu.de).
EXPERIMENTAL MODEL AND SUBJECT DETAILS
Cell Lines
HeLa (CCL-2ꢀ) cells (human female origin; established cell line, no information on age of the human subject available; see Key
Resources Table) were cultured at 37ꢀC and 7% CO2 for up to 10 passages in high glucose Dulbecco’s Modified Eagle’s Medium
(DMEM, 4500 mg/L glucose, Sigma-Aldrich) supplemented with 10% (v/v) fetal calf serum (FCS, Biochrom), 1% L-glutamine (Lonza),
100 U/mL penicillin (Lonza) and 100 mg/mL streptomycin (Lonza). Primary human umbilical vein endothelial cells (HUVECs, human
female origin; commercially obtained cells from mixed donors, no information on age of the human subjects available; see Key
Resources Table) were cultured at 37ꢀC and 5% CO2 for up to 4 passages on CellBIND plates (Corning) in 1:1 mixture of Endothelial
Cell Growth Medium 2 including supplement (ECGM2, PromoCell) with M199 (Biochrom) supplemented with 10% FCS (Sigma),
30 mg/mL gentamycin (CytoGen), 15 ng/mL amphotericin B (Biochrom) and 100 I:E. Heparin (Sigma Aldrich).
METHOD DETAILS
Chemical Synthesis
The reactions were performed in flame-dried glassware under an atmosphere of dry argon. Dry solvents (dichloromethane, tetrahy-
drofuran) were distilled in a continuous still under an atmosphere of dry argon; anhydrous ethanol and DMSO were purchased from
Sigma Aldrich and stored over molecular sieves under argon; dry ethylamine was purchased from Sigma Aldrich, distilled and stored
Cell Chemical Biology 25, 1–10.e1–e12, August 16, 2018 e1

Please cite this article in press as: Rakers et al., Addressable Cholesterol Analogs for Live Imaging of Cellular Membranes, Cell Chemical Biology
(2018), https://doi.org/10.1016/j.chembiol.2018.04.015
under argon. Solvents for extraction and purification were technical grade and flash-distilled prior to use. All chemicals were obtained
from Acros, Sigma Aldrich or Alfa Aesar and used as received. Reactions were monitored by thin layer chromatography on
pre-coated aluminum-backed plates (Merck Kieselgel 60 with fluorescent indicator UV254). Flash column chromatography was per-
formed with silica gel (0.040–0.063 mm, Kieselgel 60, Merck), applying head pressure by means of low pressure argon line (0.1–0.3
atmospheres). Brine denotes a saturated aq. solution of sodium chloride. Infrared spectra were recorded on a Varian Associated
FT-IR 3100 Excalibur with ATR unit. The wave numbers (n) of recorded IR-signals are quoted in cmꢁ1 1H and 13C-NMR spectra
.
were recorded in CDCl3 or in the solvent indicated using Bruker ARX-300, AV-300 or AV-400 spectrometers. Data are expressed
as chemical shifts in parts per million (ppm) relative to residual chloroform and CDCl3 (1H d7.28, 13C d77.2, respectively; likewise
for other solvents where applicable) as internal standard on the d scale. 1H coupling constants J are given in Hz and rounded to
the nearest 0.1 Hz. GC-MS chromatograms were recorded on an Agilent Technologies 7890A GC-system equipped with an Agilent
5975C VL MSD (EI) and a HP-5MS column with helium as carrier gas; the major signals are quoted as ratio of m/z in Daltons; ESI mass
spectra were obtained using a Bruker Daltonics MicroTof and the instrument was calibrated using sodium formicates clusters.
Synthesis of the Compounds 1a and 1b
Scheme 1. Synthesis route for type I cholesterol-based imidazolium salts.
Cholestanol (3) (Hamilton et al., 2012).
Cholesterol (9.7 g, 25.0 mmol, 1 eq.) was suspended in ethanol (730 mL), palladium on charcoal (10% Pd, 47% H2O; 1.2 g,
0.5 mmol, 0.02 eq.) was added and the flask equipped with a balloon of hydrogen. The reaction was stirred at 60ꢀC for five days,
the reaction mixture then filtered over a pad of celite and concentrated. Without any further purification the product could be obtained
as a white solid (9.63 g, 24.8 mmol, 99%).
1H-NMR (300 MHz, CDCl3): d (ppm) = 3.64 - 3.52 (m, 1H), 1.99 - 1.91 (m, 1H), 1.87 - 0.93 (m, 30H), 0.89 (d, J = 6.5 Hz, 3H), 0.85 (dd,
J = 6.6, 1.3 Hz, 6H), 0.79 (s, 3H), 0.64 (s, 3H), 0.62 - 0.54 (m, 1H); 13C{1H}-NMR (75 MHz, CDCl3): d (ppm) = 71.5, 56.6. 56.4, 54.5, 45.0,
42.7, 40.2, 39.7, 38.4, 37.2, 36.3, 36.0, 35.6, 36.0, 32.2, 31.7, 28.9, 28.4, 28.1, 24.4, 24.0, 23.0, 22.7, 21.4, 18.8, 12.5, 12.2; ESI-MS:
m/z calculated [(C27H48O)2Na]+: 799.7303, found: 799.7300.
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3b-Methansulfonyloxycholestane (4) (Sugandhi et al., 2007).
Cholestanol (1.94 g, 5.0 mmol, 1 eq.) was added to a flame-dried Schlenk tube and dissolved in dry CH2Cl2 (25 mL) and dry triethyl-
amine (970 mL, 7.0 mmol, 1.4 eq.). The solution was cooled to 0ꢀC before mesyl chloride (460 mL, 6.0 mmol, 1.2 eq.) was added drop-
wise. The solution was stirred at 0ꢀC for 30 min. The organic layer was washed with water (15 mL), hydrochloric acid (2M, 15 mL),
water (15 mL), saturated, aqueous NaHCO3 solution (15 mL) and water (15 mL) sequentially, dried with MgSO4 and concentrated.
Purification with column chromatography (n-pentane/ethylacetate = 7/1 / 5/1) resulted in the product as a white solid (2.15 g,
4.6 mmol, 92%).
1H-NMR (300 MHz, CDCl3): d (ppm) = 4.67 - 4.54 (m, 1H), 2.99 (s, 3H), 2.02 -1.92 (m, 2H), 1.85 - 1.41 (m, 9H), 1.40 - 0.94 (m, 19H),
0.91 - 0.80 (m, 13H), 0.64 (s, 3H); 13C{1H}-NMR (75 MHz, CDCl3): d (ppm) = 82.4, 56.5, 56.4, 54.2, 45.0, 42.7, 40.0, 39.6, 39.0, 36.9,
36.3, 35.9, 35.5, 35.4, 35.3, 32.0, 28.8, 28.6, 28.4, 28.1, 24.3, 24.0, 23.0, 22.7, 21.3, 18.8, 12.3, 12.2; ESI-MS: m/z calculated
[C28H50O3SNa]+: 489.3378, found: 489.3371; ATR-FTIR (cmꢁ1): 2932, 2866, 2847, 1466, 1354, 1331, 1173, 1134, 968, 934, 907,
864, 837, 756; RF (n-pentane/ethylacetate = 5/1): 0.4 (KMnO4).
IMechol , HOMs (1a).
Compound 4 (606 mg, 1.3 mmol, 1 eq.) and 1-methylimidazole (1.0 mL, 13.0 mmol, 10 eq.) were added to a flame-dried Schlenk
tube and stirred at 80ꢀC for 18 h. After purification by column chromatography (CH2Cl2/methanol = 10/0 / 9/1) the product was
obtained as an off-white solid (514 mg, 0.94 mmol, 72%).
1H-NMR (300 MHz, CDCl3): d (ppm) = 10.01 (s, 1H), 7.40 (s, 1H), 7.33 (s, 1H), 4.56 (bs, 1H), 4.11 (s, 3H), 2.75 (s, 3H), 2.37 - 2.28
(m, 1H), 2.17 - 1.42 (m, 10H), 1.40 - 0.90 (m, 19H), 0.89 - 0.81 (m, 13H), 0.61 (s, 3H); 13C{1H}-NMR (75 MHz, CDCl3): d (ppm) = 138.3,
123.3, 120.6, 56.4, 56.3, 56.2, 54.1, 42.6, 40.0, 39.9, 39.7, 39.6, 36.8, 36.2, 36.0, 35.9, 35.3, 32.8, 32.3, 31.6, 28.3, 28.1, 28.0, 25.0,
24.2, 24.0, 22.9, 22.7, 20.8, 18.7, 12.1, 11.9; ESI-MS: m/z calculated [C31H53N2]+: 453.4203, found: 453.4200; ATR-FTIR (cm-1):
3395, 3140, 3075, 2936, 2851, 2234, 1570, 1451, 1377, 1335, 1312, 1188, 1142, 1042, 953, 907, 799, 768, 729, 629, 606;
RF (Dichlormethan/Methanol = 9/1) = 0.06 (KMnO4).
IBnchol , HOMs (1b).
Compound 4 (233 mg, 0.5 mmol, 1 eq.) and 1-benzylimidazole (791 mg, 5.0 mmol, 10 eq.) were added to a flame-dried Schlenk
tube and stirred at 80ꢀC for 18 h. After purification by column chromatography (CH2Cl2/methanol = 10/0 / 9/1) the product was
yielded as a white solid (220 mg, 0.36 mmol, 72%).
1H-NMR (300 MHz, CDCl3): d (ppm) = 10.45 (s, 1H), 7.51 - 7.44 (m, 2H), 7.40 – 7.34 (m, 3H), 7.27 (d, J = 1.9 Hz, 1H),
7.19 (d, J = 1.9 Hz, 1H), 5.62 (s, 2H), 4.58 (bs, 1H), 2.79 (s, 3H), 2.40 - 2.28 (m, 1H), 2.16 - 1.42 (m, 10H), 1.37 - 0.92 (m, 19H),
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0.88 - 0.79 (m, 13H), 0.62 (s, 3H); 13C{1H}-NMR (75 MHz, CDCl3): d (ppm) = 138.0, 133.5, 132.8, 129.5, 129.5, 129.3, 121.4, 120.7,
110.1, 87.4, 56.4, 56.3, 54.2, 53.5, 42.6, 40.1, 39.9, 39.7, 39.6, 36.2, 36.0, 35.9, 35.4, 32.8, 32.3, 31.7, 28.3, 28.1, 28.0, 25.1, 24.2,
24.0, 22.9, 22.7, 20.8, 18.8, 12.1, 11.9; ESI-MS: m/z calculated [C37H57N2]+: 529.4516, found: 521.4511; ATR-FTIR (cmꢁ1): 3125,
3040, 2936, 2866, 2234, 1555, 1454, 1373, 1335, 1254, 1192, 1138, 1076, 1042, 907, 826, 768, 729, 640, 610; RF (Dichlormethan/
Methanol = 9/1) = 0.11 (KMnO4).
Synthesis of Compound 2a
Scheme 2. Synthesis route for the type II cholesterol-based imidazolium salts.
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Cholestanone (5) (Parikh and Doering, 1967).
Cholestanol (9.62 g, 24.7 mmol, 1 eq.) was added to a flame-dried Schlenk flask and suspended in a mixture of dry CH2Cl2 (100 mL)
and dry DMSO (19.3 mL, 271.7 mmol, 11 eq.). Dry trimethylamine (17.2 mL, 123.5 mmol, 5 eq.) and sulfur trioxide-pyridine complex
(7.9 g, 49.2 mmol, 2 eq.) were added at 0ꢀC and the mixture was stirred at room temperature for 3 h. To maintain a full conversion,
additional trioxide-pyridine complex was added (4.0 g, 24.7 mmol, 1 eq.) and the mixture stirred at room temperature for additional
2 h. The solution was diluted with CH2Cl2 (50 mL), the organic layer was washed with hydrochloric acid (1 M, 50 mL), saturated,
aqueous NaHCO3 solution (50 mL) and water (50 mL) sequentially, and the organic layer was dried with MgSO4 and concentrated.
The residue was purified by column chromatography (n-pentane/ethylacetate = 95/5) to yield the product as a white solid (7.78 g,
20.1 mmol, 81%).
1H-NMR (400 MHz, CDCl3): d (ppm) = 2.44 - 2.19 (m, 3H), 2.11 - 1.94 (m, 3H), 1.88 - 0.93 (m, 28H), 0.90 (d, J = 6.5 Hz, 3H), 0.86 (dd,
J = 6.6, 1.6 Hz, 6H), 0.67 (s, 3H); 13C{1H}-NMR (101 MHz, CDCl3): d (ppm) = 212.3, 56.4, 56.4, 53.9, 46.8, 44.9, 42.7, 40.0, 39.6, 38.7,
38.3, 36.3, 35.9, 35.8, 35.5, 31.9, 29.1, 28.4, 28.1, 24.4, 24.0, 23.0, 22.7, 21.6, 18.8, 12.2, 11.6; ESI-MS: m/z calculated [C27H46ONa]+:
409.3441, found: 409.3429; RF (n-pentane/ethylacetate = 95/5): 0.38 (KMnO4).
2a-Bromocholestane-3-One (6) (Bonifazi et al., 2014).
Cholestanone (6.5 g, 16.8 mmol, 1 eq.) was suspended in acetic acid (198 mL). A solution of bromine (950 mL, 18.5 mmol, 1.1 eq.),
hydrobromic acid (48%ww; a few drops) and acetic acid (13.8 mL) was added dropwise. Precipitation occurs and the suspension
was stirred for 1 h at room temperature. Afterwards, water (280 mL) was added, the residue filtered off and recrystallized
(ethanol/acetone = 5/1) to yield the product as a white solid (5.73 g, 12.4 mmol, 74%).
1H-NMR (400 MHz, CDCl3): d (ppm) = 4.75 (dd, J = 13.4, 6.3 Hz, 1H), 2.63 (dd, J = 12.9, 6.3 Hz, 1H), 2.47 – 2.35 (m, 2H), 1.99
(dt, J = 12.8, 3.5 Hz, 1H), 1.88 – 1.76 (m, 2H), 1.74 – 1.66 (m, 1H), 1.64 – 1.49 (m, 5H), 1.40 – 1.06 (m, 18H), 1.03 – 0.97 (m, 2H),
0.90 (d, J = 6.5 Hz, 3H), 0.86 (dd, J = 6.6, 1.9 Hz, 6H), 0.67 (s, 3H); 13C{1H}-NMR (101 MHz, CDCl3) : d (ppm) = 201.4, 56.3, 56.2,
54.7, 53.8, 51.9, 47.6, 44.1, 42.7, 39.8, 39.6, 39.1, 36.3, 35.9, 35.0, 31.6, 28.6, 28.3, 28.2, 24.3, 24.0, 23.0, 22.7, 21.7, 18.8, 12.3,
12.2; ESI-MS: m/z calculated [C27H45BrONa]+: 487.2546, found: 487.2548.
2a-Hydroxycholestan-3-One (7) (Poza et al., 2010).
´
´
Following a modified procedure by RODRIGUEZ und JIMENEZ et al., 2a-Bromcholestan-3-one (4.1 g, 8.9 mmol, 1 eq.), potassium
carbonate (22.7 g, 164.0 mmol, 18.5 eq.), acetone (463 mL, 6300.0 mmol, 710 eq.) and water (113 mL, 6300.0 mmol, 710 eq.)
were added to a flask and stirred at 60ꢀC for 18 h. The solution was diluted with water (600 mL), extracted with chloroform (3x500
mL), the combined organic layers dried with MgSO4 and concentrated. After purification by column chromatography (n-pentane/
ethylacetate = 7/1) the product was obtained as a white solid (2.2 g, 5.5 mmol, 62%).
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1H-NMR (600 MHz, CD2Cl2): d (ppm) = d 4.21 (dd, J = 12.3, 7.1 Hz, 1H), 3.41 (bs, 1H), 2.50 – 2.34 (m, 2H), 2.28 – 2.12 (m, 2H), 2.02
(dt, J = 12.7, 3.4 Hz, 1H), 1.88 – 1.81 (m, 1H), 1.72 (dt, J = 13.2, 3.3 Hz, 1H), 1.64 - 1.50 (m, 4H), 1.46 – 1.22 (m, 9H), 1.20 – 1.05 (m, 8H),
1.05 – 0.97 (m, 3H), 0.93 (d, J = 6.7 Hz, 3H), 0.88 (dq, J = 6.4, 2.0 Hz, 6H), 0.83 – 0.74 (m, 1H), 0.70 (d, J = 2.4 Hz, 3H); 13C{1H}-NMR
(151 MHz, CD2Cl2): d (ppm) = 211.7, 73.3, 56.8, 56.7, 54.4, 49.1, 49.0, 43.1, 43.0, 40.4, 40.0, 37.6, 36.7, 36.3, 35.2, 32.3, 29.1, 28.8,
28.6, 24.7, 24.4, 23.1, 22.9, 22.2, 19.0, 13.1, 12.4; ESI-MS: m/z calculated [C27H46O2Na]+: 425.3396, found: 425.3384; ATR-FTIR
(cmꢁ1): 2932, 2866, 2851, 1717, 1466, 1443, 1381, 1366, 1335, 1288, 1258, 1188, 1173, 1153, 1115, 1088, 1061, 1026, 991, 953,
930, 907, 810, 729, 652, 617; Rf (n-pentane/ethylacetate = 7/1): 0.37 (KMnO4).
Dicholestanone (8) (Ivonin and Lapandin, 2005).
2a-Hydroxycholestan-3-one (1.27 g, 3.2 mmol, 1 eq.) was suspended in EtOH (33 mL) and a solution of KOH (10 M, 3.1 mL) was
added dropwise. The suspension was stirred at room temperature for 3 d open to air. The resulting solid was filtered-off, washed
generously with EtOAc and the filtrate was concentrated. Water (100 mL) and CHCl3 (100 mL) were added and the aqueous layer
extracted with CHCl3 (3x100 mL). The combined organic layers were dried with MgSO4, concentrated and the resulting yellowish
solid (767 mg, 1.9 mmol, 60%) was used for the next step without further purification.
ESI-MS: m/z calculated [C27H44O2K]+: 439.2973, found: 439.2972.
Cholestane Imidazole (9) (Richter et al., 2014).
Dicholestanone (428 mg, 1.1 mmol, 1 eq.), paraformaldehyde (38 mg, 1.3 mmol, 1.2 eq.), ammonium acetate (987 mg, 2.6 mmol,
2.4 eq.), dry EtOH (29 mL) and HOAc (a few drops) were added to a flame-dried Schlenk tube under argon and stirred at 110ꢀC for
18 h. The reaction mixture was quenched by addition of saturated, aqueous NaHCO3 solution and extracted with CH2Cl2 (3x70 mL).
The combined organic layers were dried with MgSO4, concentrated and purified by column chromatography (CH2Cl2/methanol =
10/0 / 9/1) to yield the product as a yellowish solid (142 mg, 0.34 mmol, 32%).
1H-NMR (400 MHz, CDCl3): d (ppm) = 7.65 (s, 1H), 7.51 (bs, 1H), 2.59 (d, J = 15.5 Hz, 1H), 2.51 (dd, J = 15.5, 4.8 Hz, 1H), 2.27 – 2.16
(m, 2H), 2.01 (dt, J = 12.8, 3.4 Hz, 1H), 1.88 – 1.76 (m, 1H), 1.70 (dt, J = 10.4, 3.3 Hz, 1H), 1.62 – 0.94 (m, 22H), 0.91 (d, J = 6.4 Hz, 3H),
0.86 (d, J = 6.6, 1.8 Hz, 6H), 0.75 (s, 3H), 0.67 (s, 3H); 13C{1H}-NMR (101 MHz, CDCl3): d (ppm) = 132.3, 128.5, 127.7, 56.4, 56.3, 53.8,
42.6, 42.5, 40.0, 39.5, 36.9, 36.3, 36.2, 35.8, 35.6, 31.7, 29.0, 28.2, 28.0, 26.7, 24.3, 23.9, 22.8, 22.6, 21.3, 18.7, 12.0, 11.8; ESI-MS:
m/z calculated [C28H46N2H]+: 411.3734, found: 411.3721; ATR-FTIR (cmꢁ1): 3094, 2928, 2866, 2847, 1655, 1616, 1466, 1447, 1381,
1335, 1335, 1273, 1169, 1099, 1026, 961, 799, 737, 629, 610; Rf (CH2Cl2/methanol = 9/1): 0.13 (KMnO4).
Chol-IMe , HI (2a) (Richter et al., 2014).
Cholestane imidazole (142 mg, 0.34 mmol, 1 eq.), K2CO3 (122 mg, 0.88 mmol, 2.5 eq.), methyl iodide (109 mg, 1.75 mmol, 5.0 eq.)
and dry THF (3 mL) were added to a flame-dried Schlenk tube under argon and stirred at 90ꢀC for 18 h. The solution was purified by
column chromatography (CH2Cl2/methanol = 10/0 / 9/1) to yield the product as a brownish solid (130 mg, 0.23 mmol, 68%).
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1H-NMR (400 MHz, CDCl3): d (ppm) = 9.97 (s, 1H), 3.86 (d, J = 5.4 Hz, 6H), 2.58 – 2.44 (m, 2H), 2.20 (d, J = 14.8 Hz, 1H), 2.04
(dt, J = 12.8, 3.5 Hz, 1H), 1.88 – 1.79 (m, 1H), 1.74 – 0.97 (m, 24H), 0.91 (d, J = 6.5 Hz, 3H), 0.86 (dd, J = 6.6, 1.9 Hz, 6H), 0.79
(s, 3H), 0.67 (s, 3H); 13C{1H}-NMR (101 MHz, CDCl3): d (ppm) = 136.1, 129.6, 128.5, 56.3, 56.2, 53.4, 42.5, 41.6, 39.7, 39.6, 36.8,
36.2, 35.9, 35.5, 33.8, 33.8, 33.8, 31.3, 28.7, 28.3, 28.1, 24.3, 24.2, 23.9, 22.9, 22.7, 21.5, 18.8, 12.3, 12.1; ESI-MS: m/z calculated
[C30H51N2]+: 439.4047, found: 439.4056; ATR-FTIR (cmꢁ1): 3295, 3140, 2928, 2866, 2851, 1767, 1717, 1647, 1566, 1447, 1377,
1304, 1261, 1203, 1157, 1096, 1076, 1030, 953, 802, 733, 698, 610; Rf (CH2Cl2/methanol = 9/1): 0.53 (KMnO4).
Synthesis of the compounds 2b and 2c
Scheme 3. Synthesis route of azide-containing imidazolium salts.
4-Azidobutan-1-ol (10a) (Ye et al., 2011).
4-chlorobutan-1-ol (85%, 290 mg, 2.5 mmol, 1 eq.), NaN3 (370 mg, 6.0 mmol, 2.4 eq.) and H2O (2.8 mL) were added to a Schlenk
tube under air and stirred at 100ꢀC for 24 h. The solution was diluted with Et2O (5 mL) and brine (5 mL) and the aqueous layer extracted
with Et2O (3x7 mL), dried with MgSO4 and concentrated. The residue was purified by column chromatography (Et2O) to yield the
product as a clear liquid (141 mg, 1.2 mmol, 48%).
Due to volatility the solvent could not be removed completely. Therefore, solvent signals are visible in NMR.
1H-NMR (300 MHz, CDCl3): d (ppm) = 3.69 (t, J = 6.0 Hz, 2H), 3.33 (t, J = 6.4 Hz, 2H), 1.75 – 1.58 (m, 5H); 13C{1H}-NMR (75 MHz,
CDCl3): d (ppm) = 62.4, 51.4, 29.9, 25.5; ATR-FTIR (cmꢁ1): 3401, 2941, 2867, 2360, 2342, 2100, 2029, 2047, 1971, 1456, 1281, 1219,
1114, 1038, 994, 914, 744; Rf (Et2O): 0.67 (KMnO4).
6-Azidohexan-1-ol (10b) (Ye et al., 2011).
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6-chlorohexan-1-ol (335 mg, 2.5 mmol, 1 eq.), NaN3 (370 mg, 6.0 mmol, 2.4 eq.) and H2O (2.8 mL) were added to a Schlenk tube
under air and stirred at 100ꢀC for 24 h. The solution was diluted with Et2O (5 mL) and brine (5 mL) and the aqueous layer extracted with
Et2O (3x7 mL), dried with MgSO4 and concentrated. The residue was purified by column chromatography (Et2O) to yield the product
as a clear liquid (329 mg, 2.3 mmol, 92%).
Due to volatility the solvent could not be removed completely. Therefore, solvent signals are visible in NMR.
1H-NMR (300 MHz, CDCl3): d (ppm) = 3.65 (t, J = 6.5 Hz, 2H), 3.27 (t, J = 6.9 Hz, 2H), 1.70 – 1.49 (m, 5H), 1.47 – 1.32 (m, 4H);
13C{1H}-NMR (75 MHz, CDCl3): d (ppm) = 62.9, 51.5, 32.6, 28.9, 26.6, 25.4; ATR-FTIR (cmꢁ1): 3333, 2936, 2859, 2087, 1454,
1350, 1285, 1258, 1157, 1057, 1038, 1011, 972, 895, 876, 818; Rf (Et2O): 0.83 (KMnO4).
4-Azidobutan-1-al (11a).
4-azidobutan-1-ol (110 mg, 0.96 mmol, 1 eq.) was suspended in a mixture of dry CH2Cl2 (3.7 mL) and dry DMSO (755 mL, 10.6
mmol, 11 eq.) in a flame-dried Schlenk tube. Dry trimethylamine (665 mL, 4.8 mmol, 5 eq.) and sulfur trioxide-pyridine complex
(306 mg, 1.9 mmol, 2 eq.) were added and the mixture was stirred at room temperature for 3 h. To maintain a full conversion, addi-
tional trioxide-pyridine complex was added (153 mg, 0.96 mmol, 1 eq.) and the mixture stirred at room temperature for additional 2 h.
The solution was diluted with CH2Cl2 (10 mL), the organic layer was washed with hydrochloric acid (1 M, 10 mL), saturated, aqueous
NaHCO3 solution (10 mL) and dest. water (10 mL) sequentially, and the organic layer was dried with MgSO4 and concentrated.
The compound was purified by column chromatography (n-pentane/Et2O = 5/3) to yield the product as a clear liquid (107 mg,
0.95 mmol, 99%).
Due to volatility the solvent could not be removed completely. Therefore, solvent signals are visible in NMR.
1H-NMR (300 MHz, CDCl3): d (ppm) = 9.80 (t, J = 1.1 Hz, 1H), 3.36 (t, J = 6.6 Hz, 2H), 2.58 (td, J = 7.0, 1.1 Hz, 2H), 1.91 (p, J = 6.9 Hz,
2H); 13C{1H}-NMR (75 MHz, CDCl3): d (ppm) = 201.1, 50.7, 40.9, 21.6; ATR-FTIR (cmꢁ1): 2941, 2837, 2731, 2095, 1722, 1459, 1413,
1391, 1352, 1284, 1256, 1099, 950, 914, 744; Rf (n-pentane/Et2O = 5/3): 0.43 (KMnO4).
6-Azidohexan-1-al (11b).
6-azidohexan-1-ol (326 mg, 2.3 mmol, 1 eq.) was suspended in a mixture of dry CH2Cl2 (9.1 mL) and dry DMSO (1.8 mL, 25.1 mmol,
11 eq.) in a flame-dried Schlenk tube. Dry trimethylamine (1.6 mL, 11.4 mmol, 5 eq.) and sulfur trioxide-pyridine complex (726 mg,
4.6 mmol, 2 eq.) were added and the mixture was stirred at room temperature for 3 h. To maintain a full conversion, additional trioxide-
pyridine complex was added (362 mg, 2.3 mmol, 1 eq.) and the mixture stirred at room temperature for additional 2 h. The solution
was diluted with CH2Cl2 (20 mL), the organic layer was washed with hydrochloric acid (1 M, 20 mL), saturated, aqueous NaHCO3
solution (20 mL) and dest. water (20 mL) sequentially, and the organic layer was dried with MgSO4 and concentrated. The compound
was purified by column chromatography (n-pentane/Et2O = 5/3) to yield the product as a clear liquid (300 mg, 2.1 mmol, 91%).
Due to volatility the solvent could not be removed completely. Therefore, solvent signals are visible in NMR.
1H-NMR (300 MHz, CDCl3): d (ppm) = 9.77 (t, J = 1.6 Hz, 1H), 3.27 (t, J = 6.8 Hz, 2H), 2.46 (td, J = 7.3, 1.6 Hz, 2H), 1.76 – 1.53 (m, 4H),
1.48 – 1.34 (m, 2H); 13C{1H}-NMR (75 MHz, CDCl3): d (ppm) = 202.4, 51.3, 43.8, 28.8, 26.4, 21.7; ESI-MS: m/z calculated
[C6H11N3ONa]+: 164.0794, found: 164.0797; ATR-FTIR (cmꢁ1): 2939, 2865, 2829, 2725, 2360, 2331, 2094, 1724, 1457, 1412,
1391, 1351, 1260, 914, 743, 676; Rf (n-pentane/Et2O = 5/3): 0.45 (KMnO4).
Cholestane C3N3 Imidazole (12a).
Dicholestanone (325 mg, 0.81 mmol, 1 eq.), 4-azidobutan-1-ol (110 mg, 0.97 mmol, 1.2 eq.), ammonium acetate (149 mg,
1.94 mmol, 2.4 eq.), dry EtOH (22 mL) and HOAc (a few drops) were added to a flame-dried Schlenk tube under argon and stirred
at 110ꢀC for 18 h. The reaction mixture was quenched by addition of saturated, aqueous NaHCO3 solution and extracted with
CH2Cl2 (3x50 mL). The combined organic layers were dried with MgSO4, concentrated and purified by column chromatography
(CH2Cl2/methanol = 10/0 / 9/1) to yield the product as a yellowish solid (144 mg, 0.29 mmol, 36%).
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Please cite this article in press as: Rakers et al., Addressable Cholesterol Analogs for Live Imaging of Cellular Membranes, Cell Chemical Biology
(2018), https://doi.org/10.1016/j.chembiol.2018.04.015
1H-NMR (400 MHz, CDCl3): d (ppm) = 7.26 (bs, 1H), 3.36 (t, J = 6.6 Hz, 2H), 2.98 (t, J = 7.6 Hz, 2H), 2.65 – 2.48 (m, 2H), 2.28 – 2.15
(m, 2H), 2.08 (p, J = 7.0 Hz, 2H), 2.03 – 1.95 (m, 1H), 1.86 – 1.77 (m, 1H), 1.73 – 1.66 (m, 1H), 1.63 – 0.95 (m, 22H), 0.90 (d, J = 6.4 Hz, 3H),
0.85 (dd, J = 6.6, 1.9 Hz, 6H), 0.75 (s, 3H), 0.66 (s, 3H); 13C{1H}-NMR (101 MHz, CDCl3): d (ppm) = 144.9, 127.5, 126.4, 56.4, 56.4, 53.8,
50.6, 42.6, 42.4, 40.0, 39.6, 36.9, 36.3, 35.9, 35.7, 35.6, 31.7, 29.0, 28.3, 28.1, 27.5, 26.0, 24.4, 24.2, 23.9, 23.0, 22.7, 21.4, 18.9, 12.1,
11.9; ESI-MS: m/z calculated [C31H51N5H]+: 494.4217, found: 494.4218; ATR-FTIR (cmꢁ1): 3043, 2930, 2867, 2360, 2342, 2094, 1667,
1466, 1382, 1301, 1269, 1027, 895, 781, 734, 701, 630, 560; Rf (CH2Cl2/methanol = 9/1): 0.17 (KMnO4).
Cholestane C5N3 Imidazole (12b).
Dicholestanone (571 mg, 1.4 mmol, 1 eq.), 6-azidohexan-1-ol (240 mg, 1.7 mmol, 1.2 eq.), ammonium acetate (262 mg, 1.94 mmol,
2.4 eq.), dry EtOH (39 mL) and HOAc (a few drops) were added to a flame-dried Schlenk tube under argon and stirred at 110ꢀC for
18 h. The reaction mixture was quenched by addition of saturated aqueous NaHCO3 solution and extracted with CH2Cl2 (3x70 mL).
The combined organic layers were dried with MgSO4, concentrated and purified by column chromatography (CH2Cl2/methanol =
10/0 / 9/1) to yield the product as a yellowish solid (222 mg, 0.43 mmol, 31%).
1H-NMR (300 MHz, CDCl3): d (ppm) = 3.25 (t, J = 6.8 Hz, 2H), 2.99 (t, J = 7.7 Hz, 2H), 2.73 – 2.52 (m, 2H), 2.31 – 2.15 (m, 2H),
2.03 – 1.95 (m, 1H), 1.92 – 1.83 (m, 2H), 1.72 – 0.99 (m, 29H), 0.90 (d, J = 6.4 Hz, 3H), 0.85 (dd, J = 6.6, 1.4 Hz, 6H), 0.75 (s, 3H),
0.66 (s, 3H); 13C{1H}-NMR (75 MHz, CDCl3): d (ppm) = 145.7, 126.6, 125.5, 56.3, 53.7, 51.2, 42.5, 42.1, 39.9, 39.6, 36.8, 36.3,
35.9, 35.6, 34.9, 31.6, 28.9, 28.4, 28.3, 28.3, 28.1, 27.5, 26.1, 25.9, 25.3, 24.3, 23.9, 23.0, 22.7, 21.4, 18.9, 12.1, 11.9; ESI-MS:
m/z calculated [C33H55N5H]+: 522.4530, found: 522.4643; ATR-FTIR (cmꢁ1): 3358, 2969, 2935, 2870, 2359, 2332, 2098, 1694,
1559, 382, 1160, 1129, 1047, 951, 816, 753, 710, 635; Rf (CH2Cl2/methanol = 9/1): 0.29 (KMnO4).
Chol-IMe C3N3 HI (2b).
Compound 12a (135 mg, 0.27 mmol, 1 eq.), K2CO3 (44 mg, 0.32 mmol, 1.2 eq.), methyl iodide (84 mL, 1.35 mmol, 5 eq.) and dry THF
(2.6 mL) were added to a flame-dried Schlenk tube under argon and stirred at 90ꢀC for 18 h. The resulting mixture was purified by
column chromatography (CH2Cl2/methanol = 10/0 / 9/1) to yield the product as a yellowish solid (151 mg, 0.23 mmol, 85%).
1H-NMR (400 MHz, CDCl3): d (ppm) = 3.75 (d, J = 8.1 Hz, 6H), 3.58 (t, J = 6.0 Hz, 2H), 3.33 – 3.26 (m, 1H), 2.58 – 2.46 (m, 2H),
2.28 – 2.13 (m, 2H), 2.07 – 1.98 (m, 3H), 1.88 – 1.77 (m, 1H), 1.71 – 0.97 (m, 24H), 0.90 (d, J = 6.4 Hz, 3H), 0.85 (dd, J = 6.6,
1.9 Hz, 6H), 0.82 (s, 3H), 0.67 (s, 3H); 13C{1H}-NMR (101 MHz, CDCl3): d (ppm) = 144.7, 128.9, 127.9, 56.3, 56.2, 53.3, 50.4, 42.5,
41.6, 39.8, 39.6, 37.0, 36.2, 35.9, 35.5, 34.2, 33.1, 33.0, 31.3, 28.6, 28.3, 28.1, 26.6, 26.6, 24.7, 24.3, 23.9, 22.9, 22.7, 21.5, 18.8,
12.5, 12.1; ESI-MS: m/z calculated [C33H56N5]+: 522.4530, found: 522.4541; ATR-FTIR (cmꢁ1): 2929, 2867, 2360, 2093, 1660,
1529, 1382, 1382, 1257, 1086, 1028, 819, 601; Rf (CH2Cl2/methanol = 9/1): 0.60 (KMnO4).
Chol-IMe C5N3 HI (2c).
Cell Chemical Biology 25, 1–10.e1–e12, August 16, 2018 e9

Please cite this article in press as: Rakers et al., Addressable Cholesterol Analogs for Live Imaging of Cellular Membranes, Cell Chemical Biology
(2018), https://doi.org/10.1016/j.chembiol.2018.04.015
Compound 12b (222 mg, 0.43 mmol, 1 eq.), K2CO3 (72 mg, 0.52 mmol, 1.2 eq.), methyl iodide (134 mL, 2.15 mmol, 5 eq.) and dry
THF (4.2 mL) were added to a flame-dried Schlenk tube under argon and stirred at 90ꢀC for 18 h. The resulting mixture was purified by
column chromatography (CH2Cl2/methanol = 10/0 / 9/1) to yield the product as a yellowish solid (209 mg, 0.31 mmol, 72%).
1H-NMR (300 MHz, CDCl3): d (ppm) = 3.73 (d, J = 6.1 Hz, 6H), 3.33 (t, J = 6.4 Hz, 2H), 3.21 (t, J = 7.8 Hz, 2H), 2.60 – 2.45 (m, 2H),
2.29 – 2.10 (m, 2H), 2.08 – 1.96 (m, 1H), 1.91 – 1.77 (m, 1H), 1.71 – 1.00 (m, 29H), 0.91 (d, J = 6.4 Hz, 3H), 0.86 (dd, J = 6.6, 1.4 Hz, 6H),
0.82 (s, 3H), 0.67 (s, 3H); 13C{1H}-NMR (75 MHz, CDCl3): d (ppm) = 145.6, 128.6, 127.6, 56.3, 56.2, 53.3, 51.2, 42.5, 41.7, 39.8, 39.6,
37.0, 36.2, 35.9, 35.5, 34.2, 32.9, 32.8, 31.4, 28.6, 28.6, 28.3, 28.1, 26.9, 26.6, 24.9, 24.7, 24.3, 23.9, 23.0, 22.7, 21.5, 18.8, 12.5, 12.1;
ESI-MS: m/z calculated [C35H60N5]+: 550.4843, found: 550.4863; ATR-FTIR (cmꢁ1): 2928, 2866, 2362, 2092, 1660, 1529, 1467,
1444, 1382, 1289, 1256, 1087, 1028, 955, 819, 654, 601, 559; Rf (CH2Cl2/methanol = 9/1): 0.43 (KMnO4).
Materials for the Biophysical Studies
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA), 2-(4,4-
Difluoro-5-methyl-4-bora-3a, 4a-diazas-indacene-3-dod-ecanoyl)-1- hexadecanoyl-sn-glycero-3-phosphocholine (BODIPY-PC)
was obtained from Molecular Probes (Eugene, OR, USA) and 1,6-Diphenyl-1,3,5-hexatriene (DPH) from Sigma-Aldrich. All com-
pounds were used without further purification. Chloroform was of high-pressure liquid chromatography grade and was purchased
from Roth (Karlsruhe, Germany). The water used in the experiments was purified by means of a multi-cartridge system (MilliPore,
Billerica, MA, USA).
Differential Scanning Calorimetry (DSC) Measurements
Stock solutions of DPPC and the imidazolium salts were prepared in chloroform with a concentration of 2.725 mM. Mixed solutions
were prepared at the desired molar ratio to yield a total concentration of 2.725 mM for each mixture. For the large unilamellar vesicle
(LUV) preparation, chloroform was evaporated under a stream of nitrogen at 50ꢀC and then dried in high vacuum at 60ꢀC for 3 h to
remove the residual solvent. The resulting thin film was hydrated in pure water (pH 5.6) at 50ꢀC for 10 min and re-suspended by vor-
texing for 1 min. This procedure was repeated three times to yield multi-lamellar vesicles (MLVs). The MLVs were extruded 10 times
through a 400 nm polycarbonate-membrane by using an extrusion device (Liposo-fast, Avestin, Inc. 9) and stored at 60ꢀC for 10 min.
This process was repeated, so that the samples were extruded in total 30 times. The resulting LUVs were used for the DSC measure-
ments (MacDonald et al., 1991). All DSC measurements were carried out by using a computer-controlled Setaram micro DSC Ⅲ dif-
ferential scanning calorimeter equipped to perform ascending temperature mode operation (Setaram, Caluire France). The sample
chamber and the reference chamber were filled with 800 mL samples of LUVs and pure water (pH 5.6), respectively. The temperature
of both chambers was controlled by a programmable water bath. A scan rate of 30ꢀC/h was used for heating scans in the temperature
range between 15ꢀC and 60ꢀC. The main phase transition temperature was taken at the maximum of the main phase transition peak.
All experiments were performed at least three times.
Fluorescence Polarization Measurements
LUVs were prepared as described for the DSC measurements with 1 mol% of 1,6-diphenyl-1,3,5-hexatriene (DPH) added to the mix-
tures. All polarization measurements were performed using a spectrofluorometer (Fluoro Max-4, Horiba Scientific) equipped with
standard polarization accessories, a circulating water bath and the analysis software FluorEssence. 100 mL samples were added
to a quartz cuvette, excited at 360 nm and the emission was recorded at 450 nm. The polarization was calculated with the equation
Iparallel ꢁ Iperpendicular
P =
Iparallel + Iperpendicular
with Iparallel and Iperpendicular representing the intensity of parallel and perpendicularly polarized fluorescent light, respectively. The
intensities were recorded at room temperature and at 47ꢀC for each sample. All experiments were performed at least three times.
Langmuir Film Balance Measurements
Stock solutions containing DPPC and the different imidazolium salts were prepared in chloroform at equimolar concentrations of
1 mM and were kept at ꢁ20ꢀC for long term storage. Mixed solutions in various molar ratios were prepared from the respective stock
solutions, giving a total concentration of 1 mM for each mixture. All surface pressure vs. area per molecule (p-A) measurements were
performed at 293 K on pure water (MilliQ quality, pH 5.6) by an analytical Wilhelmy film balance (Riegler and Kirstein, Berlin, Germany)
with 144 cm2 operational area. The trough of the film balance was filled with 72 mL pure water, and then 15 mL of the solutions were
spread onto the water subphase using a HamiltonTM syringe, precise to 0.25 mL. The solvent was allowed to evaporate within 10 min.
Afterwards the monolayer was compressed at a constant speed of 3.3 cm2/min by a moveable barrier of the film balance and a p-A
isotherm was recorded. Each isotherm was repeated at least three times to obtain reproducible results.
Cell Culture and Microscopy
For all live cell labeling experiments, Hela or HUVECs were seeded on a chambered glass bottom coverslips with 2 or 8 wells (ibidi).
Fixation experiments were performed on glass coverslips in a 24 well cell culture plate. For the cryogenic Time-of-Flight Secondary
Ion Mass Spectrometry (cryo-ToF-SIMS) measurements, cells were seeded on silicon wafers in a 24-well plate. Cytotoxicity tests
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Please cite this article in press as: Rakers et al., Addressable Cholesterol Analogs for Live Imaging of Cellular Membranes, Cell Chemical Biology
(2018), https://doi.org/10.1016/j.chembiol.2018.04.015
were performed in 96-well plates. For seeding of HUVECs, all bottom dishes or slides surfaces were coated with collagen. Cell
density was adjusted to reach approx. 70 to 80% confluency at the time of imaging.
Cell imaging was performed utilizing a LSM 780 confocal laser scanning microscope (CLSM, Carl Zeiss) equipped with Argon-
Ion laser (LASOS), a Chameleon Vision tunable NLO laser (Coherent) and a Plan-Apochromat x 63 /1.4 oil immersion differential inter-
ference contrast objective lenses (Carl Zeiss).
Lactate Dehydrogenase (LDH) Assay of Compounds 2a and 2c
Cytotoxicity of compounds 2a and 2c on HUVECs and HeLa cells was assessed by measuring the lactate dehydrogenase (LDH)
activity released from damaged cells. The cells were seeded in 96-well plates with a density of 1500 cells/well and cultured for
2 d. Subsequently, the culture medium was replaced with assay medium containing different concentration, ranging from 0 to
100 mM, of compounds 2a or 2c added from their stock solution of 10 mM in DMSO. The plates were left in the incubator for 24 h
at 37ꢀC. The maximal LDH activity that represents the control values was determined by addition of 1% Triton X-100 for 10 min
to completely lyse the cells. After transfer of the cell supernatant to a new 96-well plate, they were mixed in a volume ratio of 1:1
with reaction mixtures containing the tetrazolium salt, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium (INT), followed
by incubation for exact 30 min at 37ꢀC, protected from light. The reaction was stopped by addition of HCl (1 N) to each well. The
values of relative toxicity (percentage of LDH activity as compared to the maximal value obtained after Triton treatment) were calcu-
lated after measuring the absorbance at 492 nm on a Microplate Reader. The concentration-response curve obtained was fitted to a
growth-sigmoidal function. LDH assays were carried out for each sample in sextuplicate (n = 6).
Absorbance 492 ðsampleÞ ꢁ Absorbance 492 ðlow controlÞ
Cytotoxicity ð%Þ =
ꢂ 100
Absorbance 492ðhigh controlÞ ꢁ Absorbance 492ðlow controlÞ
Cryo-ToF-SIMS Measurements of 2a and 2c
Subconfluent 2a or 2c treated HeLa cells were prepared using a silicon wafer sandwich preparation technique, which can achieve
sub-micrometer resolution with cryo-ToF-SIMS analysis. Cells were seeded on cleaned silicon wafers (7 x 9 mm2) and adjusted to
reach approximately 80% confluency at the time of freezing. Polystyrene beads (diameter 10 mm) in DMEM were added to the sub-
confluent cell layer prior to covering with a second wafer (6 x 9 mm2). The polystyrene beads act as spacers to protect the cells from
being damaged by the sandwich process. Thereafter, the packed silicon sandwich was plunged into liquid propane (minimizing cell
damage rates for the frozen cells). To prepare the sandwich for cyro-ToF-SIMS analysis, the frozen silicon sandwich wafers were
pulled apart in the instrument resulting in freeze-fracturing of the cells. Using this procedure, most of the outer cell membrane is strip-
ped off and both, the residual cell and the stripped-off membrane can be analyzed.
The measurements were performed using a house-made cryo-ToF-SIMS instrument that is largely comparable to the commercial
€
ToF-SIMS V instrument (ION-TOF, Munster, Germany). For analysis, a pulsed 30 kV Bi liquid metal ion source (Bi-LMIS, ION-TOF)
with 0.05 pA pulsed current was employed. Bi3+ cluster ions were used as primary ions (PI) for analyzing and imaging the sample
surface using the fast imaging mode of the Bi-LMIS with a pulse width of 100 ns. All measurements were performed for positive sec-
ondary ions. Sputtering was accomplished with an Ar gas cluster ion beam (Ar-GCIB, ION-TOF) using 20 keV singly charged Ar ion
clusters (Ar2200+). The average sputter current was about 1.7 nA. The primary ion current for both the Bi-LMIS and Ar-GCIB was
measured in pulsed mode on a Faraday cup and averaged for a period of 60 s to reduce statistical uncertainties.
Mass spectra, images, and 3D reconstructions were generated using SurfaceLab 6.4 software (ION-TOF). Lateral images were
recorded with a pixel density of 256 3 256 by rastering the primary ion beam over an area of about 100 3 100 mm2. To avoid shad-
owing effects, the area sputtered was larger by at least nine times than the area analyzed. The analysed area was located in the center
of the sputter crater. Cryo-ToF-SIMS measurements were performed in five (n = 5) independent experiments.
Cell Surface Click-reaction
Bioorthogonal click reaction on the cellular membrane surface of Hela cells or HUVECs treated with substance 2c were performed in
a copper free manner with Click-iT-DIBO Alexa Fluorꢁ 555 or click-iT DIBO Alexa Fluorꢁ 488. Therefore, preincubated cells were
washed four times with DMEM (for Hela) or mixed ECGM2 and M199 media (for HUVECs) and cooled down for 20 min on ice in
the same medium including 20 mM HEPES (pH 7.4). Labeling via click reaction was performed by adding 10 mM Click-iT DIBO-Alexa
Fluor 555 in the same medium for 20 min on ice, followed by washing the cells four times with PBS++ and two times with the respective
growth medium including HEPES. Imaging of the cells was performed employing a LSM 780 confocal laser scanning microscope
(CLSM, Carl Zeiss). Surface click-reactions were confirmed in twelve (n = 12, HUVEC) and six (n = 6, HeLa) independently performed
experiments.
Uptake of Pre-clicked Compounds
Hela or HUVECs were cooled down on ice for 20 min and then treated for an additional 20 min on ice with 5 mM of a pre-clicked 1:1
mixture of the cholesterol derivate 2c and click-iT-DIBO-Alexa Fluor 555, followed by four times washing with PBS++ and two times
with the respective growth medium including HEPES. Live cells imaging was performed as above. Uptake experiments with the
pre-clicked compounds were repeated at least ten (n = 10) times for both cell lines.
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Please cite this article in press as: Rakers et al., Addressable Cholesterol Analogs for Live Imaging of Cellular Membranes, Cell Chemical Biology
(2018), https://doi.org/10.1016/j.chembiol.2018.04.015
GMPVs
GPMVs were isolated from HUVECs after incubation with the compounds 2a or 2c by chemically induced cell blebbing with 25 mM
paraformaldehyde and 2 mM dithiothreitol (DTT) in a calcium-containing GPMV buffer (150 mM NaCl, 10 mM HEPES and 2 mM CaCl2
(pH 7.4) for 2 h at 37ꢀC (Sezgin et al., 2012). After harvest of the GPMVs the labeling was performed via a click reaction with 10 mM
click-iT-DIBO-Alexa Fluor 555 alkyne by incubation at room temperature (25ꢀC) for 20 min. The fluorescence of the vesicles was
measured after diluting the GPMV solution with the GPMVs Buffer (1:4) and loading the vesicles into a BSA-coated 8 well m-Slide
glass bottom (ibidi). The respective GMPV isolations were performed in four (n = 4) independent experiments.
U18666A Treatment
Cultured HUVECs were treated with 2 mg/mL U18666A (Enzo Life Sciences Inc.) at 37ꢀC for 6 or 24 h after or before the cells were
labeled via a bio-orthogonal surface click reaction with Click-iT DIBO-Alexa Fluor 555.
Filipin Staining
Cells were fixed in 4% paraformaldehyde in PBS on ice for 20 min. The cells were then washed with PBS and blocked with 2% BSA
for 30 min. For visualization of cholesterol, fixed cells were incubated with 0.25 mg/mL filipin (complex from Streptomyces filipinensis,
Sigma-Aldrich) for 30 min, washed with PBS and mounted in Mowiol.
U18666A treatment and Filipin staining were performed in at least three independent experiments.
Cell Viability Studies
For the cellular uptake and incorporation into the plasma membrane of HeLa cells or HUVECs the cells were treated with the synthe-
sized and purified compounds 2a or 2c at a final concentration of 5 mM for 90 min at 37ꢀC and 5% or 7% CO2 in the culture growth
medium DMEM (for uptake in HeLa) or in a 1:1 mixture of ECGM2 and M199 (for the uptake in HUVECs). To characterize cell
morphology and proliferation, HUVECs and Hela cells were analyzed 24, 48, 72 and 96 h after cellular uptake of compounds 2a
or 2c (90 min, 5 mM) by phase contrast microscopy. Viability studies were carried out in three (n = 3) independent experiments.
Fluorescence Spectroscopy of Labeled Cells
Fluorescence spectroscopy of surface labeled HUVECs in suspension (in PBS) was performed in a fluorescence spectrometer
(Eppendorf Biospectrometer fluorescence). Cells were first incubated with substance 2c for 90 min. Thereafter, the cells were
washed four times with medium and subjected to surface labeling with Click-it DIBO-Alexa 555, either directly or after 24 h or
48 h post-incubation with substance 2c. Cells were then washed four times with PBS++ and two times with PBS before the labeled
adherent cells were removed from the culture substrate by treatment with trypsin/EDTA solution for a short time at 37ꢀC. Trypsiniza-
tion was stopped with PBS supplemented 10% FCS. In a control experiment the cells were treated with 2 mg/ml U18666A for 24 h
prior to labeling and trypsinization. The amount of the cells in suspension was calculated using an automated Cell Counter (Bio rad):
Cells were then diluted with PBS before the fluorescent signal was measured. Non-treated cells were used as a blank-control.
Mean and standard deviations were obtained from three (n = 3) independent experiments.
Cholera Toxin Experiment
Cells were subjected to reaction with Alexa Fluor 594-conjugated cholera toxin subunit B (CTB) using the Vybrantꢁ Lipid Raft
Labeling Kit according to the manufacturer’s protocol. HUVECs were washed three times with ice-cold PBS and incubated with
the conjugated CTB for 10 min at 4ꢀC. Thereafter, cells were washed three times with ice-cold PBS and crosslinking of the CTB
labeled lipid microdomains was achieved with an anti-CTB antibody for 15 min at 4ꢀC. CTB-mediated lipid clustering was carried
out in six (n = 6) independent experiments.
Endosome Markers
To analyze whether Alexa 555-labeled compound 2c is present in endosomal membranes following extended incubation times, cells
were transfected as described before (Knop et al., 2004) with Rab7-GFP, a known marker of late endosomes (Rink et al., 2005). 24 h
post transfection, cells were treated with compound 2c for 90 min and then subjected to click chemistry with Alexa 555. Cells
were incubated for another 40 min at room temperature and then subjected to analysis by confocal fluorescence microscopy. All
experiments were performed at least three times.
QUANTIFICATION AND STATISTICAL ANALYSIS
Quantitative data (fluorescence intensity) of the fluorescence spectroscopy measurements were expressed as mean and standard
deviations of at least three independent replicate experiments (n R 3) using Origin 9.1. Mean and standard deviation of the LDH assay
were calculated from six (n = 6) independent experiments using Origin 9.1. The response curves were fitted to a growth sigmoidal
function employing Origin 9.1. The goodness-of-fit of the regression was approved by the R2 value.
e12 Cell Chemical Biology 25, 1–10.e1–e12, August 16, 2018