Preparation and Membrane Properties of Oxidized Ceramide

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Preparation and Membrane Properties of Oxidized Ceramide Derivatives Takaaki Matsufuji, Masanao Kinoshita, Anna Möuts, J. Peter Slotte, and Nobuaki Matsumori Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b02654 • Publication Date (Web): 12 Dec 2017 Downloaded from http://pubs.acs.org on December 20, 2017

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Preparation and Membrane Properties of Oxidized Ceramide Derivatives

Takaaki Matsufuji,1 Masanao Kinoshita,1 Anna Möuts,2 J. Peter Slotte,2 and Nobuaki Matsumori1

1

Department of Chemistry, Faculty of Science, Kyushu University, 744 Motooka, Nishi-ku,

Fukuoka 819-0395 Japan. 2

Biochemistry, Faculty of Science and Engineering, Åbo Akademi University, Turku,

Finland.

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Abstract Ceramide is a bioactive lipid with important roles in several biological processes including cell proliferation and apoptosis. Although 3-ketoceramides that contain a keto group in place of the 3-OH group of ceramide occur naturally, ceramide derivatives oxidized at the primary 1-OH group have not been identified to date. In order to evaluate how the oxidative state of the 1-OH group affects the physical properties of membranes, we prepared novel ceramide derivatives in which the 1-OH group was oxidized to a carboxylic acid (PCerCOOH) or methylester (PCerCOOMe), and examined the rigidity of their monolayers and the formation of gel domains in palmitoyloleoylphosphatidylcholine (POPC) or sphingomyelin (SM) bilayers. As a result, PCerCOOH and PCerCOOMe exhibited membrane properties similar to those of native ceramide, although the deprotonated form of PCerCOOH, PCerCOO−, exhibited markedly lower rigidity and higher miscibility with POPC and SM. This was attributed to the electrostatic repulsion of the negative charge, which hampered the formation of the ceramide-enriched gel domain. The similarities in the properties of PCerCOOMe and ceramide revealed the potential to introduce various functional groups onto PCerCOOH via ester or amide linkages; therefore, these derivatives will also provide a new strategy for developing molecular probes, such as fluorescent ceramides, and inhibitors of ceramide-related enzymes.


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INTRODUCTION The bioactive lipid, ceramide, is involved in a variety of cellular processes and diseases. Although ceramide concentrations in plasma membranes are extremely low in normal cells, they can increase under stress or in response to a variety of stimuli such as cytokines, death receptor ligands, and anti-cancer drugs,1,2 and elicit a number of different biological responses including cell proliferation and apoptosis.3–7 Although the mode-of-action of ceramide has yet to be fully resolved, previous studies have suggested that alterations in the membrane biophysical properties induced by ceramide formation may contribute to its biological responses.8–11 Considering the extremely low solubility of ceramide in aqueous media such as cellular cytosol, it is not farfetched to assume that ceramide exerts its effects on the membrane, and consequently influences the physiological state of cells. In effect, ceramide is known to affect various physical properties of bilayer membranes including thermodynamic behavior, molecular order, and lateral distribution.12–23 Ceramide is a hydrophobic molecule, and it lacks the head group typical of phospholipids or glycosphingolipids.12 Ceramide is comprised of a sphingosine backbone and an acyl chain, the former of which generally possesses two hydroxy groups at the C1 and C3 positions. Interestingly, although 3-ketoceramides that contain a ketone in place of the 3-OH group are considered to occur naturally,24 derivatives in which the primary 1-OH group is oxidized have not been identified to date. This is likely because the 1-OH group is relevant

for

biosynthetic

transformations

to

and

from

sphingomyelins

and

glycosphingolipids, and therefore is inactive or protected against oxidation in cells. Hence, in order to study how the oxidative state of the 1-OH group of ceramide affects membrane


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physical properties, we prepared novel ceramide derivatives in which the 1-OH group was oxidized to carboxyl and methoxycarbonyl groups, and examined the rigidity of their monolayers

as

well

as

segregation

of

their

gel

domains

from

palmitoyloleoylphosphatidylcholine (POPC) or sphingomyelin (SM) bilayers.

RESULTS AND DISCUSSION Preparation of oxidized ceramide derivatives. In this study, we prepared oxidized ceramide derivatives bearing carboxylic acid (PCerCOOH) or methylester (PCerCOOMe) via oxidation of the 1-OH group of palmitoylceramide (PCer). In order to oxidize the 1-OH group of PCer without oxidizing the 3-OH group, we adopted TEMPO oxidation, which converted the primary alcohol into the aldehyde (Scheme 1). The obtained aldehyde was then subjected to Pinnick oxidation, which yielded PCerCOOH. To evaluate the effect of a negative charge stemming from the carboxylic acid, PCerCOOMe was also prepared via treatment of PCerCOOH with trimethylsilyldiazomethane (TMS-CHN2).

Scheme 1. Preparation of PCerCOOH and PCerCOOMe from palmitoylceramide (PCer)


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π-A isotherm experiments with ceramide derivatives. First, we investigated the

molecular properties of the ceramide derivatives via monolayer experiments. Figure 1 shows the π-A isotherms of monolayers of PCer and its oxidized derivatives on the subphases of water and Tris buffer (pH 7.4) at 25 °C. Based on these isotherms and Eq. 1 (see Experimental Section), the areal compressional modulus Cs−1, which corresponds to membrane rigidity, was estimated at the surface pressure of 30 mN/m (Table 1). Consequently, the PCerCOOH monolayer on the water subphase showed a significantly decreased rigidity as compared to the other two, while the rigidities of the PCer and PCerCOOMe monolayers were relatively similar. We speculated that a fraction of PCerCOOH was deprotonated to PCerCOO− on the water subphase, and that the resulting electrostatic repulsion hampered tight packing. This was more clearly illustrated with the Tris buffer subphase (pH 7.4), in which PCerCOOH should be mostly deprotonated; PCerCOOH showed a markedly decreased Cs−1-values from 224 mN/m to 170 mN/m, whereas the Cs−1-values of the other two samples were less sensitive to the subphase change from water to Tris buffer (Table 1). These results suggest the critical influence of electrostatic repulsion on the lipid packing.


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Figure 1. π-A isotherms of (a) PCer, (b) PCerCOOH, and (c) PCerCOOMe monolayers at 25 °C. Solid and dashed curves show the π-A isotherms on water and Tris (pH 7.4), respectively.


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Table 1. Membrane rigidity of monolayers Sample (n=3)

Cs−1-value (mN/m)* Water

Tris (pH 7.4)

PCer

393 ± 33

422 ± 6

PCerCOOH

224 ± 16

170 ± 10

PCerCOOMe

310 ± 19

300 ± 18

* Obtained at the surface pressure of 30 mN/m. Errors represent standard deviation

Gel-phase-onset concentration in POPC bilayers. The monomer miscibility of PCer in unsaturated phospholipid bilayers is generally poor,25 and saturated ceramides tend to segregate laterally into gel phases at fairly low ceramide concentrations.18,19,21,26 To measure the gel-phase-onset concentration of the ceramide derivatives, we evaluated the fluorescence lifetime of trans-parinaric acid (tPA) as a function of PCer concentration as well as those of its derivatives in POPC bilayers. The fluorescence lifetime of tPA is sensitive to the formation of ordered and gel phases.27–30 The intensity-based average lifetime of tPA fluorescence is shown in Figure 2. The gel-phase-onset concentration of PCerCOOMe in POPC bilayers was ~7.5 mol% both in water and Tris buffer (pH 7.4), which was slightly higher than that of PCer in POPC. Compared to PCer in POPC, PCerCOOH showed a similar gel-phase-onset concentration (~5 mol%), but higher in pH 7.4 buffer (~20 mol%). This difference was attributed to the formation of PCerCOO−, which hampered the formation of the gel phase via the electrostatic intermolecular repulsion. On the other hand, the effect of the formation of PCerCOO− is more pronounced on this analysis than on the above π-A isotherm analysis. Considering that the lifetime 7 ACS Paragon Plus Environment

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analysis was performed in POPC bilayers, a possible higher miscibility of PCerCOO− in POPC might further retard the gel-phase formation of PCerCOOH in Tris buffer.

PCerCOOMe H2O PCerCOOMe Tris (pH 7,4) PCerCOOH H2O PCerCOOH Tris (pH 7,4) PCer H2O

30

20

Average lifetime (ns)

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10

0

10

20

30

40

50

Ceramide mol%

Figure 2. tPA ﬂuorescence lifetime analysis in POPC bilayers containing increasing concentrations of PCer and its derivatives. The experimental temperature was 23 °C. Each value represents the mean ± SD for n = 3-5 (except PCerCOOH in buffer, n=2).

Observation of GUVs in mixture with POPC. To further examine the gel phase separation of the ceramide derivatives in POPC bilayers, fluorescence microscopy was used to observe giant unilamellar vesicles (GUVs) composed of POPC and the ceramide derivatives. To visualize the liquid-disorderd (Ld) domains comprised largely of POPC, Bodipy-PC was added to the GUVs as an Ld-domain marker at 0.2 mol%. As a result, approximately half of the GUVs phase-separated at 5 mol% of PCerCOOMe or 8 ACS Paragon Plus Environment

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PCerCOOH (Figure 3bc), revealing a slightly weaker gel phase formation for these two derivatives in comparison to PCer, which underwent a complete phase-separation at the same concentration (Figure 3a). Next, in order to elucidate the effect of the acid-dissociation of PCerCOOH, we added 0.1 mM NaOH upon preparation of GUVs, because it is not impossible, but difficult to prepare GUVs via electroformation31 in highly-concentrated electrolytic solutions such as Tris buffer. As a result, the phase-separation largely disappeared even at 10 mol% of PCerCOOH (Figure 3d), which was consistent with the above-mentioned π-A isotherm and lifetime data.


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Figure 3. Fluorescence microscopy images of GUVs composed of POPC and PCer (a), PCerCOOMe (b), or PCerCOOH (c,d) at 5 or 10 mol%. The GUVs contained 0.2 mol% Bodipy-PC, which was localized in the liquid-disordered (POPC-rich) domains. Scale bar indicates 10 µm. (d) GUVs were prepared with 0.1 mM NaOH to determine the effect of deprotonation of PCerCOOH.

Observation of GUV bilayers prepared from palmitoylsphingomyelin. Although ceramide and SM are both sphingolipids, they are immiscible and ceramide tends to 10 ACS Paragon Plus Environment

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segregate laterally into gel phases. This lateral segregation and the resulting formation of ceramide-rich gel domains likely occur in biological membranes where sphingomyelinases hydrolyze SM to ceramide.32,33 Thus, we further observed the gel phase formation of the ceramide derivatives in a mixture with palmitoylsphingomyelin (PSM) using GUVs.34 Since we recently developed ATTO-labeled fluorescent-SMs that completely reproduce the membrane properties of SM,35 we added ATTO488-labeled SM to the GUVs and evaluated the phase separation between ceramide-rich (PSM-poor) and ceramide-poor (PSM-rich) domains, the latter of which was stained with ATTO488-labeled SM. PCer and its derivatives showed a phase-separation in water even at 2.5 mol% (Figure 4a-c), further confirming the gel phase formation of these derivatives in SM bilayers. In contrast, alkaline solutions significantly weakened the gel phase formation of PCerCOOH (Figure 4d), which was also consistent with the notion that the negatively-charged carboxylate anion hampered the assembly of the ceramide derivative due to electrostatic repulsive forces.


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Figure 4. Fluorescence microscopy images of GUVs composed of PSM and PCer (a), PCerCOOMe (b), or PCerCOOH (c,d) at 2.5, 5, 10, or 15 mol%. (d) GUVs were prepared with 0.1 mM NaOH to determine the effect of deprotonation of PCerCOOH. The GUVs contained 0.2 mol% ATTO488-SM, which localized in SM-rich domains. Bars indicate 10 µm.

Phase transition of the membranes composed of PSM and ceramide derivatives Finally, we examined the gel-fluid transition of PSM membranes containing 10 mol% of the ceramide derivatives using steady-state fluorescent anisotropy experiments (Figure 5). Thermograms show that the phase transition started at ca. 40 °C, which corresponds to the 12 ACS Paragon Plus Environment

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gel-fluid transition temperature of PSM,34 and ended at 50~60 °C, which indicates the melting of the ceramide-rich gel domains. It was found that PCerCOOH and PCerCOOMe in PSM have comparable or slightly lower phase transition temperatures than PCer in PSM, which is also consistent with the aforementioned data. As expected, the phase transition of PCer or PCerCOOMe in PSM was not affected by the pH change (Figure 5ab), whereas PCerCOOH in PSM showed a slightly lower phase transition temperature in 0.1 mM NaOH solution (dashed curve in Figure 5c) than in water (solid curve in Figure 5c) by 2 °C. However, in comparison with the large effect of the alkaline condition on the phase-separation of the PSM/PCerCOOH membrane as shown in Figure 4, the reduction in the gel-fluid transition temperature of the PSM/PCerCOOH membrane in alkaline solution was unexpectedly small. A possible explanation is that, although the negative charge in PCerCOO− hampers the formation of the ceramide-rich gel domains by repulsive force between PCerCOO− molecules, the negative charge can electrostatically interact with PSM, which has a positive charge on the choline group, thus resulting in the minimal reduction in the thermodynamic stability of the PSM-PCerCOO− mixed gel phase. Further examination of thermodynamic behaviors of these ceramide derivatives is currently under way.


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Figure 5. Steady-state fluorescent anisotropy of diphenylhexatriene (DPH) in PSM membranes containing 10 mol% PCer (a), PCerCOOMe (b), and PCerCOOH (c). Measurements were performed in water (solid curves) or in 0.1 mM NaOH (dashed curves). Arrows indicate the start and end points of gel-fluid transitions.

CONCLUSIONS


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We prepared two new derivatives of PCer, in which the primary alcohol group was oxidized into carboxylic acid and ester groups, and found that their membrane properties, namely the monolayer rigidity and formability of the gel phases in POPC and SM bilayers, reproduced those of PCer to a considerable extent. In contrast, PCerCOO−, which prevails in pH7.4 buffer or in NaOH solutions, exhibited markedly decreased rigidity and higher miscibility with POPC and SM. This could be attributed to the electrostatic repulsion of the negative charge, which hampered the formation of ceramide-enriched gel domains. On the other hand, the effect of negatively charged PCerCOO− on the gel-fluid transition of PSM membranes was smaller than expected, which could be explained by the electrostatic interaction with the positively charged choline group in PSM, thus preventing the thermodynamic stability of PSM/PCerCOO− membrane from being largely decreased. As mentioned in the introduction, since oxidized derivatives of the ceramide primary alcohol have not been isolated from natural sources or chemically synthesized, this study sheds new light on structure-function relationships of ceramide. In addition, the similar bilayer properties of PCerCOOMe and PCer suggest that it is possible to introduce various functional groups to PCerCOOH via ester or amide linkage with retaining the membrane properties of PCer. In fact, we have already synthesized fluorescent ceramide derivatives as one of the applications of these ceramide derivatives by connecting fluorophores to PCerCOOH via an amide bond, and succeeded in the visualization of ceramide-enriched domains with a fluorescence microscope (to be published in due course). Accordingly, these derivatives have potential for the development


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of molecular probes such as fluorescent-labeled ceramides to explore the behavior of ceramide in cells. The deregulation of ceramide is thought to be involved in many diseases, including Alzheimer’s disease,36 cancer,37,38 and irritable bowel syndrome.39 Therefore, inhibitors of ceramide synthesis are expected to be useful in determining the importance of ceramide regulation in these diseases.40 Because various functional groups can be introduced into the ceramide derivatives reported here, these derivatives will also provide a new strategy for developing inhibitors of ceramide-related enzymes such as ceramide synthase.

EXPERIMENTAL SECTION General for synthesis. Palmitoylceramide (PCer) was purchased from Avanti Polar Lipids. Other chemicals and solvents were purchased from Nacalai Tesque, TCI, and Wako Pure Chemical Industries, Inc. and were used without further purification. Thin layer chromatography (TLC) was performed on Merck pre-coated silica gel 60 F-254 plates and 1

was visualized by UV irradiation (254 nm) or staining with phosphomolybdic acid. H NMR spectra were obtained on a JEOL ECA 600 (600 MHz) spectrometer. High resolution mass spectra (HRMS) were acquired on a Bruker micrOTOF II ESI-TOF mass spectrometer. Synthesis of PCerCOOH. To a solution of PCer (10.1 mg, 18.8 µmol) in CH2Cl2 (4.0 mL) were added a solution of TEMPO in CH2Cl2 (0.5 M, 7.52 µL, 3.76 µmol) and iodobenzene diacetate (10.9 mg, 33.8 µmol). The reaction mixture was stirred at room temperature for 8 h and then quenched with a saturated aqueous solution of Na2S2O3 and 16 ACS Paragon Plus Environment

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extracted with CHCl3. The organic layer was washed with a saturated aqueous solution of NaHCO3, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to give the crude aldehyde (10.0 mg, Rf = 0.49 [10/1 CHCl3/MeOH, v/v]) as a white solid that was used without further purification. To a solution of the aldehyde (10.0 mg, 18.7 µmol) in THF/t-BuOH/H2O (4:2:1, v/v, 5.0 mL) were added 2-methyl-2-butene (20 µL, 188 µmol), an aqueous solution of NaH2PO4 (2.0 M, 47.0 µL, 94.0 µmol) and an aqueous solution of NaClO2 (1.0 M, 56.4 µL, 56.4 µmol). The reaction mixture was stirred at room temperature for 25 h, and then diluted with H2O. The resulting mixture was extracted with ethyl acetate, and the organic layer was concentrated in vacuo to give the crude product. Purification by silica gel column chromatography (40/1 CHCl3/MeOH, v/v, containing 1 % AcOH) afforded PCerCOOH 1

(6.7 mg, 64 %) as a white solid; Rf = 0.17 (10/1 CHCl3/MeOH, v/v), H-NMR (600 MHz, CD3OD) δ 5.74 (dt, J = 15.0, 7.2 Hz, 1H), 5.49 (dd, J = 15.6, 7.2 Hz, 1H), 4.46 (d, J = 6.9 Hz, 1H), 4.32 (t, J = 6.9 Hz, 1H), 2.24 (t, J = 7.6 Hz, 2H), 2.04 (q, J = 7.1 Hz, 2H), 1.65-1.55 (m, 2H), 1.43-1.20 (m, 48H), 0.90 (t, J = 6.9 Hz, 6H), HRMS (m/z): [M+Na]

+

calcd. for C34H65NNaO4, 574.4806; found 574.4800. Synthesis of PCerCOOMe. To a solution of PCerCOOH (6.7 mg, 12.1 µmol) in CH2Cl2/MeOH (1:1, v/v, 2.0 mL) was added trimethylsilyldiazomethane (10% in hexane, 49.0 µL, 29.4 µmol). The reaction mixture was stirred at room temperature for 1 h, and then quenched with acetic acid and concentrated in vacuo to give the crude product. Purification by silica gel column chromatography (80/1 CHCl3/MeOH) afforded PCerCOOMe (6.0 mg, 1

88 %) as a white solid; Rf = 0.63 (20/1 CHCl3/MeOH, v/v), H-NMR (600 MHz, CD3OD) δ 17 ACS Paragon Plus Environment

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5.74 (dt, J = 15.6, 7.2 Hz, 1H), 5.47 (dd, J = 15.3, 7.8 Hz, 1H), 4.49 (d, J = 6.9 Hz, 1H), 4.26 (t, J = 7.6 Hz, 1H), 3.71 (s, 3H), 2.22 (t, J = 7.6 Hz, 2H), 2.10-1.99 (m, 2H), 1.64-1.53 +

(m, 2H), 1.44-1.22 (m, 48H), 0.90 (t, J = 6.9 Hz, 6H), HRMS (m/z): [M+H] calcd. for C35H68NO4, 566.5143; found 566.5134. Surface pressure vs. molecular area isotherm (π-A isotherm) measurements. π-A isotherm measurements were conducted on a computer-controlled Langmuir film balance (USI System, Fukuoka, Japan) calibrated using stearic acid. The Langmuir trough (100 × 290 mm2) was filled with ultrapure water produced by a Milli-Q System (Millipore Corp., Tokyo, Japan) or Tis buffer. Then, an aliquot (30 µL) of the lipid mixture solution (CHCl3/MeOH = 4:1, v/v, 1 mg/mL) was spread onto the subphase with a glass micropipette (Drummond Scientific Company, Pennsylvania, USA) to form the lipid monolayer. The monolayer was compressed at a rate of 10 mm2/s, following an initial delay period of 10 min for evaporation of the organic solvents. The subphase temperature was kept at 25.0 ± 0.1 °C. The measurements were repeated at least 3 times per sample to obtain reliable isotherms. Areal compressibility (Cs) at a given surface pressure of π was calculated using the following equation:

Cs = −

1  ∂Amean    , Amean  ∂π  π

(Eq. 1)

where Amean is the experimentally obtained mean molecular area. In the present study, compressibility (Cs) was expressed in terms of areal compressional modulus (Cs−1).


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Time-resolved fluorescence spectroscopy. The fluorescence lifetime of tPA was measured in multilamellar vesicles prepared as described previously.41 The samples from stock solutions were first dispensed into glass tubes to desired conentration and composition. Then the ceramides were exposed to 1 % acetic acid in MeOH for a few minutes, after which time the solvent was evaporated to yield a dry lipid film in the glass tube. This treatment converted PCerCOOH into a fully protonated form. The lipids were subsequently hydrated for 1 h at 70 °C followed by bath sonication for 5 min at 70 °C. The total lipid concentration was 0.2 mM in argon-purged pure water or Tris buffer (10 mM Tris, 140 mM NaCl, pH 7.4) as solvent. tPA was present at 1 mol %. The fluorescence decays

were

recorded

at

the

indicated

temperatures

with

a

FluoTime

100

spectro-fluorimeter, with a PicoHarp300E time-correlated single-photon-counting module (PicoQuant, Berlin, Germany). A 297-nm LED laser source (PLS300, PicoQuant) was used to excite tPA, and the emission was collected through a long-pass filter with a 395 nm cutoff. The samples were kept under constant stirring during the measurements. Data were analyzed using FluoFit Pro-software obtained from PicoQuant. GUV preparation and fluorescence observations. Chicken egg SM, PCer, and POPC were purchased from Avanti Polar Lipids (Alabaster, AL). PSM was purified from egg SM using HPLC (Cosmosil 5C18-AR-II column 20 × 250 mm, Nacalai Tesque, Kyoto, Japan). The lipids were dissolved in CHCl3/MeOH (4:1, v/v) at a concentration of 1 mg/mL or 10 mg/mL and stored at −20 °C until use. A fluorescent probe, Bodipy-PC, was purchased from Molecular Probe (Eugene, OR). This probe was dissolved in CHCl3/MeOH (4:1, v/v) at a concentration of 50 µg/mL and stored in the dark at −20 °C until use.


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GUVs composed of PSM, ceramide, and POPC were prepared using the electroformation method developed by Angelova and Dimitrov.31 Briefly, an aliquot (10 µL) of the solution (1 mg/mL) of SM/ceramide or POPC/ceramide (85:15, 90:10, 95:5, and 97.5:2.5 mol/mol) containing 0.2 mol% Bodipy-PC was spread on the surface of the electrodes (platinum wires with 100 µm diameter), and dried under vacuum for at least 16 h. The electrode surface was coated with the thin lipid film. Then, parallel aligned electrodes were placed into ~400 µL of Milli-Q water or a 0.1 mM aqueous solution of NaOH sandwiched between two cover glasses (24 mm × 60 mm, 0.12–0.17 mm thickness) using an open-square shaped rubber spacer (1 mm thickness). The samples were incubated at 70 °C for 90 min and a low-frequency alternating current (AC) (sinusoidal wave function, 10 Vpp, 10 Hz (for 60 min) and 1 Hz (for 30 min)) was applied with a function generator (20 MHz function/arbitrary waveform function generator, Agilent, Santa Clara CA). After sample preparation, the GUVs were left to equilibrate at room temperature for at least 1 h. Fluorescence observations were conducted using a fluorescence microscope (BZ-X700, Keyence, Osaka, Japan) with an air objective lens (CFI Plan Apo 60×, Nikon, Tokyo, Japan). The excitation/detection wavelengths (470/525 nm) were selected using the dichroic mirror OP-87763 (Keyence, Kyoto, Japan). In order to provide clearer images of the phase separation, the brightness and contrast were adjusted using Adobe Photoshop CS6 (Adobe Systems Inc., San Jose, CA.). Steady-state fluorescence anisotropy. Multilamellar vesicles used for steady-state diphenylhexatriene (DPH) anisotropy measurements were prepared by mixing 200 nmol of total lipids (PSM/PCer derivatives, 90:10 mol/mol) with 2.0 nmol of DPH in chloroform.


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The lipid solutions were dried under a stream of nitrogen gas for 5 min and vacuum for at least 2 h. The dry lipid films were hydrated by adding 2.0 mL of Milli-Q water or 0.1 mM NaOH at 65 °C for 30 min. Finally, the samples were vortex mixed and sonicated for 10 min in a water bath sonicator AS22GTU Ultrasonic Cleaner (AS ONE Corp., Osaka, Japan). The samples were cooled to room temperature before the fluorescence measurements. Steady-state DPH anisotropy was measured using a FP-8300 Spectrofluorometer with FDP-837 Automatic Polarizer unit (JASCO Corp., Tokyo, Japan). The excitation wavelength was set to 350 nm, and the emission was measured at 452 nm. The samples were constantly stirred, and the temperature was controlled by an ETC-815 Peltier Thermostatted Cell Holder (JASCO Corp., Tokyo, Japan) from 20 to 60 °C at a rate of 2 °C/min. The operation of the instrument and the data analyses were performed with Spectra Manager software (JASCO Corp., Tokyo, Japan). The measurements were repeated three times per sample.

AUTHOR INFORMATION *Corresponding author; [email protected]

Author Contributions T.K. synthesized the derivatives, and T.K. and M.K. performed the membrane experiments and wrote manuscript. J.P.S. and A.M. performed fluorescent lifetime experiments. N.M. designed and organize the present study and wrote manuscript.


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Notes The authors declare no competing financial interest.

Acknowledgements We thank Prof. Tohru Oishi for letting us use the mass spectrometer. This work was supported in part by Grants-in-Aid for scientific research from the Japan Society for the Promotion of Science Kiban B (15H03121), Grants-in-Aid for Innovative Areas from the Japan Society for the Promotion of Science (26102527 and 16H00773), and the Lipid Active Structure Project supported by Exploratory Research for Advanced Technology Organization of the Japan Science and Technology Agency. The Slotte laboratory was supported by grants from the Jane and Aatos Erkko Foundation, and the Sigrid Juselous Foundation.

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TOC


Preparation and Membrane Properties of Oxidized Ceramide

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