A Remarkably Simple Class of Imidazolium-Based Lipids and Their Biological Properties

Article abstract of DOI:10.1002/chem.201502333

A series of imidazolium salts bearing two alkyl chains in the backbone of the imidazolium core were synthesized, resembling the structure of lipids. Their antibacterial activity and cytotoxicity were evaluated using Gram-positive and Gram-negative bacteria and eukaryotic cell lines including tumor cells. It is shown that the length of alkyl chains in the backbone is vital for the antibiofilm activities of these lipid-mimicking components. In addition to their biological activity, their surface activity and their membrane interactions are shown by film balance and quartz crystal microbalance (QCM) measurements. The structure-activity relationship indicates that the distinctive chemical structure contributes considerably to the biological activities of this novel class of lipids. Lipids! A series of imidazolium salts bearing two alkyl chains in the backbone were synthesized, resembling the structure of lipids. The biological activity resulting from their surface activity and membrane interaction are shown (see figure), which were determined by the alkyl chain length.

Full text of DOI:10.1002/chem.201502333

DOI: 10.1002/chem.201502333
Communication
&
Biomimetic Synthesis
A Remarkably Simple Class of Imidazolium-Based Lipids and Their
Biological Properties
[
a]
[b]
[b]
[a]
[c]
Da Wang, Christian Richter, Andreas Rühling, Patrick Drücker, Daniel Siegmund,
[c]
[b]
[a]
Nils Metzler-Nolte, Frank Glorius,* and Hans-Joachim Galla*
Abstract: A series of imidazolium salts bearing two alkyl
chains in the backbone of the imidazolium core were syn-
thesized, resembling the structure of lipids. Their antibac-
terial activity and cytotoxicity were evaluated using Gram-
positive and Gram-negative bacteria and eukaryotic cell
lines including tumor cells. It is shown that the length of
alkyl chains in the backbone is vital for the antibiofilm ac-
tivities of these lipid-mimicking components. In addition
to their biological activity, their surface activity and their
membrane interactions are shown by film balance and
quartz crystal microbalance (QCM) measurements. The
structure–activity relationship indicates that the distinctive
chemical structure contributes considerably to the biologi-
cal activities of this novel class of lipids.
Since the isolation of the first free N-heterocyclic carbene
[
1]
(
NHC) in 1991, NHCs have become a versatile and frequently
[2]
used tool in organic chemistry. Despite the great success of
NHCs, comparably little attention has been paid to their salts,
Figure 1. Comparison of established and new surfactants.
[3]
except in the field of ionic liquids. Based on our work of sta-
[4]
bilizing palladium nanoparticles with NHCs, which contain
long alkyl chains in the backbone, we realized the structural
compliance of the NHC salts with cationic surfactants such as
the quaternary ammonium salt N-[1-(2,3-dioleyloxy)propyl]-
phosphocholine (DPPC; Figure 1), we have now developed
NHC salts with long alkyl chains in position 4 and 5 to investi-
gate the biological activities including their antitumor and anti-
microbial activities.
[
5]
N,N,N-trimethylammonium chloride (DOTMA) (Figure 1). Due
As shown in Scheme 1, dimethylimidazolium iodide (IMe·HI)
derivatives with three different alkyl chain lengths in the back-
bone (2a–c) were synthesized according to an approach re-
to their significant bioactivity, NHC-M complexes have attract-
[
6]
ed considerable attention for antitumor applications. In par-
ticular, NHC derivatives with long alkyl chains in position 1 and
[
4]
ported in the literature. Cytotoxicity measurements using C6
glioma cells cloned originally from a rat glial tumor were car-
ried out using the lactate dehydrogenase (LDH) release assay
(Figure 2), which is particularly well suited to monitor the in-
tegrity of the cell membrane. Figure 2 demonstrates that the
three compounds exhibit significant antitumor activity poten-
tially due to membrane disruption or membrane structural dis-
turbance. EC50 values of three different cell lines including epi-
thelial MDCK cells and NIH3T3 fibroblasts are given in Table 1.
EC50 values clearly increase with increasing chain length. Anti-
microbial activities have been determined and the minimal in-
hibitory concentration (MIC) on both Gram (À) and (+) bacte-
ria are given in Table 1 showing the same tendency. It is further
remarkable that the MIC is considerably higher for the Gram
(À) bacteria, which are surrounded by two cellular membranes,
the outer lipopolysaccharide carrying membrane as well as the
inner cytoplasmic membrane separated by the periplasm.
3
display antibiofilm activities attributed to their surface activi-
ty derived from the surfactant-like structure. Inspired by the
structure of natural lipids, such as 1,2-dipalmitoyl-sn-glycerol-3-
+
[
a] D. Wang, Dr. P. Drücker, Prof. Dr. H.-J. Galla
Institut für Biochemie, Westfälische Wilhelms-Universität Münster
Wilhelm-Klemm-Straße 2, 48149 Münster (Germany)
E-mail: gallah@uni-muenster.de
+
[
b] Dr. C. Richter, A. Rühling, Prof. Dr. F. Glorius
Organisch Chemisches Institut, Westfälische Wilhelms-Universität Münster
Corrensstraße 40, 48149 Münster (Germany)
E-mail: glorius@uni-muenster.de
[
c] D. Siegmund, Prof. Dr. N. Metzler-Nolte
Institut für Bioinorganische Chemie, Ruhr-Universität Bochum
Universitätsstraße 150, 44801 Bochum (Germany)
+
[
] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201502333.
Chem. Eur. J. 2015, 21, 15123 – 15126
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ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Communication
Scheme 1. Synthesis of NHC salts 2a–c from aldehydes 1a–c.
Figure 3. Phase-contrast images of C6 glioma cells: A) untreated; B) treated
by 100 mm 2b for 24 h; C) treated by 1% Triton X-100 for 10 min.
NHC salts, we have to take into account the lipid-like structure
of 2a–c which correlates with their antitumor and antimicrobi-
al activities. We assume that NHC salts bearing long alkyl
chains in the backbone are able to behave as natural lipids,
forming a stable mixed film with membrane lipids and thus
may insert into the biological membranes of both pro- and eu-
karyotes. For shorter chains, however, we have to assume a de-
tergent-like membrane disruption and/or intracellular effects.
To verify our above-mentioned hypothesis, the membrane-
mimicking property of 2a–c was investigated by film balance
measurements (Figure 4A). DPPC, a saturated electroneutral
zwitterionic phospholipid that mimics the plasma membrane is
shown for comparison. For the pure compounds 2a–c spread
at the air–water interface of the film balance, the surface activi-
ty is strongly dependent on chain length. The pressure–area
isotherms of compound 2c with the longest chains displays
a continuously increasing surface pressure under compression
and the surface activity is closely related to the lipid DPPC. The
medium chain length NHC salt 2b also forms a monolayer,
however, compared to 2c, with an increased surface pressure
(reduced surface tension) at the expanded state. It is important
to note that under compression (small area) 2b does not
reach the high surface pressure of compound 2c or even of
DPPC. Short chain 2a is completely unable to form a stable
film and the surface pressure is zero all over the whole area
range of the film balance. This surface behavior is also support-
ed by a surface-activity study determining the critical micelle
concentration (CMC) of 2a–c (Figure 4B) using the film balance
again. It is clearly shown that the CMC values of 2a–c decrease
in the order of C -IMe-HI (65.9 mm)>C -IMe-HI (8.5 mm)>C -
Figure 2. Cytotoxicity of 2a–c in% effect versus the concentration of 2a–
c for the C6 glioma cell line (n=8).
Table 1. MIC and EC50 values of the NHC salts 2a–c.
NHC
EC50 [mm]
MIC [mm]
A. baumannii
[
a]
[b]
salts C6 glioma MDCK II
NIH3T3
S. aureus
2
2
2
a
b
c
2.3Æ0.50 6.7Æ0.82 4.7Æ0.76 38.1
10.5Æ0.25 8.9Æ0.81 5.0Æ0.47 60.1
1.19
1.88
–
[
c]
[c]
28.1Æ0.74 15.9Æ0.51 7.7Æ0.20
–
[
a] Acinetobacter baumannii DSM 30007: Gram (À). [b] Staphylococcus
aureus DSM 20231: Gram (+). [c] Non-active against bacteria.
Since the structurally related QACs were reported to target
mainly the plasma membrane of yeast followed by loss of per-
[7]
meability of the membrane, our NHC salts are proposed to
follow an analogous mechanism lysing the membrane lipids
which then results in cell death. To give an observable evi-
dence of the lytic activity of our compounds, phase-contrast
images of C6 glioma cells after an LDH assay of 2b were re-
corded by wide-field microscopy (Figure 3).
7
11
15
IMe-HI (1.4 mm), revealing that the NHC salts with longer alkyl
chains are more efficiently absorbed to the air–water interface
forming stable lipid-like assemblies due to their increased hy-
[
9]
Figure 3A shows clearly the intact cell membranes for the
untreated tumor cells, however, after treatment with a 100 mm
solution of 2b for 24 h we could only identify the nuclei of C6
glioma cells, whereas the tumor cell membranes are hardly
visible (Figure 3B). Treatment with the lysis reagent Triton X-
drophobicity favoring micelle formation in water. Thus, as ex-
pected, a positive correlation was found between the surface
activity and chain length: C -IMe-HI>C -IMe-HI@C -IMe-HI.
15
11
7
For single-chain 1-alkyl-3-methylimidazolium salts a logarith-
mic relationship between the CMC and the number of carbon
[
10]
1
00 for 10 min was used as a control experiment (Figure 3C),
atoms is reported. Similarly compounds 2a–c show a linear
and the results imply that our NHC salt 2b exhibits lytic activi-
ty in the tested tumor cells.
decrease of the log(CMC) with increasing chain length, fitting
[8,10]
to the corresponding published mathematical model.
In agreement with our results, the antitumor and antimicro-
bial activities of NHC salts with alkyl side chains in position 1
and 3 have been intensively investigated. However, in contrast
to our finding with this new class of NHC salts so far, a positive
Based on the investigations on surface activities of com-
pounds 2a–c, additional film balance experiments of mixed
monolayers of DPPC/compounds 2a–c were performed (Sup-
porting Information, Figure S1). We found that 2c with the lon-
gest alkyl chains and DPPC form an ordered and stable mixed
monolayer due to the strong van der Waals interaction result-
[8]
correlation with chain length was previously found. In order
to clarify the underlying mechanism of the bioactivity of our
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Communication
Figure 5. Membrane interaction and lytic activity of IMe-NHC salts 2a–c.
POPC/POPS (4:1) liposomes were tethered on a modified solid support by
À1
incubation of: 1: streptavidin, 2: approximately 0.5 mgmL , 200 nm POPC/
POPS (4:1) liposomes; and 3: washing with TBS buffer; 4: incubation of
0.1 mm 2a–c; 5: washing with TBS buffer; 6: osmotic gradient induced by
ultrapure water. Note the difference in liposome stability in comparison to
the control, especially for 2b, showing immediate loss of the liposome layer
mass.
c with the tethered vesicles was followed by live monitoring of
the frequency shift (DF=F(t)ÀF ), which is proportional to the
0
[
13]
adsorbed mass to the sensor surface.
The mass of immobilized liposomes corresponds to a fre-
quency shift of 320–340 Hz (Figure 5, arrow 2). The liposome
layer was not considerably affected by washing with pure TBS
buffer, which mimics cellular salt and pH conditions (arrow 3).
Treatment of this vesicle layer with 0.1 mM dispersion of com-
pound 2a generates only a very small shift, indicating almost
no interaction with the liposomes. Compound 2b induces an
additional shift of approximately 200 Hz relative to the mass of
bound liposomes indicating a strong binding. Compound 2c
leads to a shift of approximately 450 Hz corresponding to
a gain of 134% in mass and thus showing an even more in-
tense binding (arrow 4). Subsequent treatment again with
pure buffer (arrow 5) revealed an irreversible membrane incor-
poration process of 2b and 2c, but not of 2a. Application of
an osmotic gradient to the liposome layer thereafter revealed
a strong difference in membrane stability after NHC salt treat-
Figure 4. The surface pressure–area (p–A) isotherms of 2a–c at the air–water
interface taken at 293 K and determination of their CMC. A) Isotherms for
surface-pressure versus area in pure water (pH 5.6) at 293 K. The isotherm of
the pure lipid DPPC is given for comparison. B) Mean surface tension values
calculated from measured surface pressure taken at different concentrations.
Each measurement was performed in triplicate.
ing from their identical C15 alkyl chains. Furthermore, the posi-
tively charged imidazolium head groups are thought to lead to
an electrostatic interaction with the negative charge at the
DPPC phosphate group arranging the molecules in a more or-
[11]
dered manner at the air–water interface. In contrast, due to
the weakened van der Waals interaction of the shorter C11
alkyl chains, 2b has a poorer ability to align with DPPC result-
ing in a looser mixed monolayer formation. Compound 2a
with much shorter chains compared to the guest lipid only
slightly interacts with DPPC according to its extremely low sur-
face activity.
À1
ment. A gradient of at least approximately 240 mOsmL , gen-
erated by rinsing with ultrapure water, showed a strong mem-
brane destabilization and lytic activity after the prior treatment
only with compound 2b (arrow 6). In this case, the mass of the
liposome layer was reduced to only 1.6% of the original mass,
demonstrating a severe loss of liposomes. Prior incubation of
2a generated a relative mass of 111 %, which also is similar to
the relative extent of 104% seen in the POPC/POPS (4:1) lipo-
some control (Supporting Information, Figure S2). The latter
shows a typical osmotic swelling of tethered liposomes, which
remain intact. Thus, 2a has no major effect on the stability of
liposome membranes. The longest alkyl chain derivative, 2c,
showed a loss of mass, but this is still 183% of the relative
original liposome mass and hence it is only partially washed
out of the membrane without affecting its stability. Thus, it can
To gain a deeper understanding whether the NHC salts with
long alkyl chains do interact with membrane lipids, a bilayer
mimicking model was used which more closely resembles the
natural membrane system due to the unsaturated alkyl chains
and an additional negative charge in one of the compounds.
To distinguish between lytic processes and membrane inser-
tion we performed QCM measurements. POPC/POPS (palmi-
toyl-oleoyl-PC; palmitoyl-oleoyl-phosphatidyl-serine; 4:1) lipo-
somes were tethered on a sensor surface via the biotin–strep-
tavidin interaction (Figure 5, streptavidin self-assembly,
arrow 1; liposome attachment arrow 2). The model is well de-
[12]
scribed by Patel et al. The interaction of compounds 2a–
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Communication
be concluded that compound 2a is neither binding nor affect-
ing membrane stability, 2b is binding and highly lytic and
component 2c does bind and intercalate almost irreversibly
without affecting membrane stability.
Acknowledgements
The help of Stephanie Wulff (WWU) with the film balance mea-
surement is greatly acknowledged. Generous financial support
for this project was provided by the Deutsche Forschungsge-
meinschaft (SFB 858 and Leibniz award).
According to the QCM analysis on artificial membranes the
interactions between NHC salts and the cellular membranes
can be described as a two-stage process. Firstly, the NHC salt
molecules are absorbed to the cell membrane. In this step, the
micelle formation ability of the NHC salts plays an important
role. The longer the alkyl chain length is, the more readily mi-
celles are formed. Secondly, the hydrophobic mismatch in van
der Waals interaction between alkyl chains determines the ca-
pability to intercalate within the membrane. The long chain
derivative can easily intercalate into the membrane without
considerably affecting its stability. The shorter component 2b
can intercalate but the hydrophobic mismatch already desta-
bilizes the membrane. If the alkyl chain mismatch exceeds
a certain threshold value, no membrane intercalation is possi-
ble as shown for the shortest chain component.
Keywords: antitumor agents · cytotoxicity · lipids · NHC salts ·
surface activity
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[
2
Received: June 16, 2015
Published online on September 2, 2015
[8a–c]
salts previously reported.
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ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim