Fluorescent Polyene Ceramide Analogues as Membrane Probes

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Fluorescent polyene ceramide analogues as membrane probes Ingrid Nieves, Ibai Artetxe, Jose Luis Abad, Alicia Alonso, Jon V Busto, Lluis Fajarí, L. Ruth Montes, Jesús Sot, Antonio Delgado, and Felix M. Goni Langmuir, Just Accepted Manuscript • DOI: 10.1021/la505017x • Publication Date (Web): 06 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Fluorescent polyene ceramide analogues as membrane probes Ingrid Nieves+1, Ibai Artetxe+2, José Luis Abad1, Alicia Alonso2, Jon V. Busto2, Lluís Fajarí3,4, L. Ruth Montes2, Jesús Sot2, Antonio Delgado1,5 and Félix M. Goñi*2

1

Spanish National Research Council (CSIC), Institute of Advanced Chemistry of Catalonia (IQACCSIC), Department of Biomedicinal Chemistry, Research Unit on Bioactive Molecules (RUBAM). Jordi Girona 1826, 08034, Barcelona, Spain. 2

Unidad de Biofísica (CSIC, UPV/EHU), and Departamento de Bioquímica, Universidad del País Vasco, P.O. Box 644, 48080, Bilbao, Spain. 3

Spanish National Research Council (CSIC), Institute of Advanced Chemistry of Catalonia (IQACCSIC), Biological Chemistry and Molecular Modeling Department. Jordi Girona 1826, 08034, Barcelona, Spain. 4

Spanish National Research Council (CSIC), Center for Research and Development (CIDCSIC), EPR Unit-Magnetic Resonance Service, Jordi Girona 1826, 08034 Barcelona, Spain

5

University of Barcelona (UB), Faculty of Pharmacy, Unit of Medicinal Chemistry (CSIC Associated Unit), Avda. Joan XXIII s/n, 08028, Barcelona, Spain. *

Corresponding author. E-mail address:

+

These two authors contributed equally to this work.

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Abstract Three ceramide analogues have been synthesized, with sphingosine-like chains containing five conjugated double bonds. Pentaene I has an N-palmitoyl acyl chain, while the other two pentaenes contain also a doxyl radical, respectively at C5 (Penta5dox) and at C16 (Penta16 dox) positions of the Nacyl chain. Pentaene I maximum excitation and emission wavelengths in a phospholipid bilayer are 353 nm and 478 nm respectively. Pentaene I does not segregate from the other lipids in the way natural ceramide does, but rather mixes with them in a selective way according to the lipid phases involved. Fluorescence confocal microscopy studies show that, when lipid domains in different physical states coexist, Pentaene I emission is higher in gel than in fluid domains, and in liquid-ordered than in liquid-disordered areas. Electron paramagnetic resonance of the pentaene doxyl probes confirms that these molecules are sensitive to the physical state of the bilayer. Calorimetric and fluorescence quenching experiments suggest that the lipids under study orient themselves in lipid bilayers with their polar moieties located at the lipid-water interface. The doxyl radical in the N-acyl chain quenches the fluorescence of the pentaene group when in close proximity. Because of this property, Penta16dox can detect gel-fluid transitions in phospholipids. The availability of probes for lipids in the gel phase is important in view of novel evidence for the existence of gel microdomains in cell membranes.

Introduction Ceramides (N-acylsphingosines) are lipids involved in important metabolic regulation events, such as apoptosis control (1, 2). They are also noted by their unusual physicochemical properties when incorporated into phospholipid bilayers, e.g. lateral segregation into ceramide-rich rigid domains (3-6). The studies of lateral phase separation in bilayers have benefited from the use of fluorescence techniques, both spectroscopic (7-9) and microscopic (10-12). Nevertheless fluorescence studies of lipids suffer from the inherent limitation of requiring fluorescent probes. The probes are


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supposed to mimic the behavior of natural lipids, even if sometimes, as e.g. in the widely used diphenylhexatriene, structural similarities are remote. In the case of ceramide analogues, molecules labeled with 6-[N-7-nitrobenz-2-oxa-1,3diazol-4-yl)amino] (NBD), or 4,4-difluoro-4-bora-3a, 4a-diaza-s-indacene (BODIPY) have been used, even if the bulky fluorescent moieties are likely to provide the molecules with properties different from those of the natural lipid, e.g. a ceramide containing NBD in the acyl chain was found to segregate laterally with the ceramidepoor domains, rather than with the rigid, ceramide-rich ones (13). In an effort to provide fluorescent ceramide analogues that behave as closely as possible to the natural lipid we have prepared a molecule that mimics ceramide, with a pentaene structure in the wouldbe sphingosine moiety (Figure 1, Pentaene I). Polyene derivatives are known as a good example of fluorescent lipids (6-8, 14). Two other novel molecules have been prepared that contain in addition a 4,4-dimethyl-3-oxazolinidyloxy (doxyl) radical in the N-acyl chain, at positions 5 and 16 respectively (Figure 1, Penta5dox and Penta16dox). The doxyl radical is likely to quench the pentaene fluorescence when both the acyl and sphingosine-like chains are in close proximity, thus these doxyl-containing molecules were designed in order to test the intramolecular dynamics of these ceramide analogues. The biophysical properties of these novel molecules make them useful tools in the study of sphingolipids in membranes.

Figure 1. Structure of the pentaene probes and of palmitoyl ceramide.


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Experimental Materials Commercial starting materials for the synthesis, 4aminobutyric acid, 5doxylstearic acid and 16doxylstearic acid were purchased from SigmaAldrich or TCI Europe (16doxylstearic acid). Solvents were distilled prior to use and, if required, dried by standard

methods.

phosphatidylcholine

Dioleoyl (DSPC),

phosphatidylcholine egg

phosphatidylcholine

(DOPC),

distearoyl

(ePC),

dipalmitoyl

phosphatidylcholine (DPPC), egg sphingomyelin (eSM), and cholesterol (Chol) were purchased from Avanti Polar Lipids (Alabaster, AL). The lipophilic fluorescent probe DiO (3,3’-dioctadecyloxacarbocyanine perchlorate) was purchased from Molecular Probes (Eugene, OR). Stock solutions were prepared by dissolving pure lipids and (when required) DiO in chloroform/methanol (2:1 v/v) and stored at -20 ºC. The pentaene fluorophores used in this work (Figure 1), due to their labile nature, were dissolved in THF (tetrahydrofuran) stabilized with BHT (butylated hydroxytoluene) and stored at -80 ºC. The buffer solution used in the biophysical studies was HEPES 50 mM, pH 7.4.

Synthesis For general data and synthesis of intermediates, see the Supporting Information

Esterification of 4alkylamidobutanoic acids 4ac with polyene alcohol 2 A solution of EDC (8 mg, 42 µmol) in DCM (600 µL) is added dropwise over a solution of the starting acid 4ac (30 µmol) in DCM (400 µL). After 30 min stirring at RT, the mixture is cooled to 0 ºC and treated with 100 µL of a 0.35 M solution of 2 in THF (equivalent to 6 mg, 35 µmol) followed by the addition of solid DMAP


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(4diemethylaminopyridine, 3.4 mg, 28 µmol). The resulting reaction mixture was allowed to stir at RT for 5 h and next quenched with H2O (1 mL) and extracted with DCM (3 x 3 mL). The combined organic phases were dried and concentrated to give crude esters, which were flash chromatographed to afford the final compounds (See Supporting Information for a complete characterization of the polyene probes).

Fluorescence spectroscopy Lipid vesicles for spectrofluorometric assays were prepared by mixing the desired lipids and fluorophores and evaporating the solvent under a stream of nitrogen. The sample was then kept under high vacuum for 90 min to remove any residual solvent. The resulting lipid film was hydrated by addition of the buffer solution at a temperature above the lipid main phase transition temperature (65º C for DSPC, 45º C for all other samples), followed by vigorous vortex mixing. In the experiments with multilamellar vesicles (MLV) the samples were then sonicated for 10 min in a bath sonicator at the same temperature. When unilamellar vesicles (LUVs) were required, the hydrated samples were subjected to 10 freeze-thaw cycles and extruded through polycarbonate filters, 0.1 m pore diameter. Fluorescence measurements were performed in liposome suspensions 0.3 mM in lipid with 0.3 mol% of the polyene probes using a QuantaMaster 40 spectrofluorometer (Photon Technology International, Lawrenceville, NJ) (λex = 353 nm, λem = 474 nm and a cut-off filter at 385 nm). The measurements were done under continuous stirring and at a constant temperature of 23 ºC. For experiments involving temperature ramps, scanning from 20 to 50 ºC at a heating rate of 0.8 ºC/min was performed.

Fluorescence confocal and multiphoton microscopy


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Giant unilamellar vesicles (GUV) were prepared following the electroformation method described previously (17), using a home-made chamber that allows direct visualization under the microscope. Stock solutions of the lipid mixtures under study were prepared to a final concentration of 0.2 mM in chloroform/methanol (2:1 v/v), plus 0.3 mol% DiO and 2 mol% of the desired polyene probe. 3 l of the desired stock solution were added to the surface of platinum electrodes and the solvent was removed under vacuum for 90 min. Electroformation was then performed using a wave generator (TG330 function generator; Thurlby Thandar Instruments, Huntingdon, UK). The buffer solution used for electroformation was first preheated above the lipid main phase transition temperature. GUV attached to the platinum electrodes were visualized under an inverted confocal microscope with a high-efficiency spectral detector (Leica TCS SP5; Leica Microsystems, Manheim, Germany) and a two-photon excitation mode (MaiTai HP DS laser; Spectra Physics, Mountain View, CA). A 63X water immersion, N.A. 1.2 objective was used and the images were collected and analyzed with the LAS AF software. DiO was excited at 488 nm using an argon laser and its emission was collected in the 500-600 nm channel with the pinhole set at 1 Airy unit. Polyene probes were excited using two-photon excitation mode at 706 nm and their emission was collected in the 467-499 nm range with the pinhole completely opened. Control GUV with only DiO were used to confirm that DiO emission did not significantly contribute to the signal observed on the polyene channel. Two independent experiments were done for each sample, and within each experiment duplicates or triplicates of the samples were prepared. During visualization, several images were taken at different places of the platinum wire in order to ensure that the sample did not display significant heterogeneity. 6 ACS Paragon Plus Environment

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In GUV exhibiting lateral phase separation, detected through differential fluorescence intensities, the intensity ratios between the different domains were calculated measuring local intensities with the Leica LAS AF software.

Differential scanning calorimetry All measurements were performed in a VP-DSC high-sensitivity scanning microcalorimeter (MicroCal, Northampton, MA). MLVs to a final concentration of 1 mM were prepared as described above. Both the samples and buffer solutions were fully degassed before loading into the appropriate cell. Three heating scans were performed for each sample at 45 C/h; after the first scan, successive ones always yielded superimposable thermograms. The final lipid concentration, determined by a lipid phosphorus assay, and data from the third scan were used to obtain normalized thermograms. The data were processed using the software ORIGIN (MicroCal) provided with the calorimeter.

Quantum yield measurements Absorption and fluorescence spectra were registered in a SpectraMax M5 spectrophotometer using 1-cm path length quartz cuvette. 9,10Diphenylanthracene (9,10-DPA) was obtained from Sigma-Aldrich, and used without further purification. Ethanol (absolute for analysis) was deoxygenated thoroughly by argonbubbling prior to use. The fluorescence quantum yield of photoexcited fluorescence (ФF) is defined as the ratio of number of emitted photons vs number of absorbed photons. Quantum yields were calculated by measuring the integrated emission area of the fluorescent spectra, related to the area measured for 9,10-DPA in ethanol after excitation at 346 nm (ΦF = 0.95) (18). Quantum yields for the aminobutyric polyene products were then calculated


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using equation 1 below (19) where

and

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are the quantum yield of the sample

and that of the standard, respectively. Fs and Fref represent the area of fluorescent emission in units of photons; ηs and ηref are the refractive indices of the solvent. βs and βref are the correction absorption factors, β=1-10-A where A= absorbance.

[Eq. 1] In order to minimize reabsorption effects, the solutions for quantum yield measurements were prepared such that the optical density was generally about A=0.04 at λex=346 nm, but was never higher than 0.05 for our 1-cm path length. The absorption spectra of polyene probes showed characteristic structured shapes between 270 and 360 nm in ethanol. This profile is consistent with that of other pentaene probes described in the literature. Quantum yields calculated by this method are reliable to ± 10% (20, 21). Electron Paramagnetic Resonance (EPR measurements) The EPR measurements were carried out on an EPR / ESR Bruker EMX spectrometer, equipped with an X-band (~ 9 GHz) EMX X Premium microwave bridge, a 10" ER073 magnet with a 12 KW ER083 source power and an ER4102ST standard cavity. The spectra were recorded in the following operating conditions: microwave power (MP) 20 mW, modulation amplitude of magnetic field (MA) 0.5 G, modulation frequency (MF) 100 kHz, time constant (TC) 20.48 ms, conversion time (CT) 100 ms, sweep time (ST) 102.40 s, field width (FS) 100 G, gain (RG) 5.02 x 104, and a resolution of 1024 points. Processing was carried out using Bruker WIN-EPR System (v 2.22 Rev. 12) software. The measurements were carried out at room temperature in a capillary quartz tube with


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40-500 spectra accumulation. Samples from LUVs were prepared as described above and transferred to a capillary quartz tube. The rotational correlation time τC, the parameter that can be used to measure the motion of the nitroxide radical for fast movement (weakly immobilized nitroxide), was calculated from the following equation (22):

τ = 6,5 × 10 ∆H

+

− 2

[Eq. 2]

where h0, h+1 and h-1 are the peak-to-peak heights of the 0, +1 and -1 transitions (central, low field and high field lines) in the first derivative EPR spectrum and ∆H0 is the linewidth (in G) of the 0 transition (central line). In the case of slow tumbling nitroxide group (strongly immobilized), the parameter τC was determined using the following approximate expression (23, 24):

τ = a 1 −

!"# $$

&

%

[Eq. 3]

Where Amax is one-half the separation of the outer hyperfine extrema and Azz is the rigid limit value for the same quantity, with a and b values of 5.4 x 10-10 and -1.36 for LUVePC Penta5dox and LUVDOPC Penta5dox and 1.9 x 10-9 and 1.05 for LUVDPPC Penta5dox and LUV-DSPC Penta5dox, respectively, corresponding to a Lorentzian linewidth of 3 G in the first case and a linewidth of 8 G in the second group of compounds, for Brownian diffusion. (ref 23, p. 84). An empirical order parameter S, obtained from the EPR spectra, was used to measure the molecular motion of spin probe in the liposome membranes. This parameter is a measure of the anisotropy or random motion. The order parameter can be obtained from the experimental values of Amax and the isotropic coupling constant (aN) and the 9 ACS Paragon Plus Environment

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maximum anisotropic coupling constant in the immobilized limit (AZZ), as a relation between the observed anisotropy and the maximum anisotropy, according the following equation (48, 49):

S=

!"# ()

[Eq. 4]

** ()

For S = 0, the motion is isotropic. The deviation of this value involves anisotropic molecular motion. Other parameters used in EPR measurements: The parameter g is the Landé factor. The effective value of g varies with the electronic structure of the molecule leading to a deviation of the g value of the free electron (ge = 2.0023), due to the spin-orbit coupling. The value of g is characteristic of the nucleus where the unpaired electron resides. The g factor is defined as:

+=

,-

[Eq. 5]

./ 0

Where γ is the experimental microwave frequency in GHz and B0 is the magnetic field value at the centre of the spectrum in Tessla. This value is obtained directly by the EPR programme. ∆Hpp is the EPR signal line width, measured as the field variation between the minimum and the maximum of the line in the first derivative spectrum in Gauss (G). aN is the nitrogen isotropic coupling constant in Gauss. Results Synthesis of the probes


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The design of the probes was based on the combination of a conjugated pentaene group as fluorophore (20, 25-28) and a doxyl carboxylic acid as a fluorescence quencher. The conformationally flexible butyric acid was used as a scaffold to append the above moieties, as indicated in Scheme 1. The fluorescent probes were obtained by condensation of hydrochloride 1 with carboxylic acids 3ac to give amidoesters 4ac. Successive hydrolysis with LiOH in THF and condensation of the resulting acid with polyene alcohol (29) afforded the required probes. Polyene alcohol 2 was obtained, in turn, from HornerEmmons condensation of all-E (2,4,6,8)decatetraenal with triethyl phosphonoacetate, followed by DIBALH reduction of the intermediate ethyl decatetraenoate (15).

Scheme 1: Synthesis of the fluorescent probes. a) 3ac, EDC, HOBt in DCM; b) LiOH in THFH2O, overall yields: 85% (4a), 65% (4b), 80% (4c); c) all (E)dodeca-2,4,6,8,10-pentaen-

1-ol (2), DMAP in DCM, Penta-5dox (40%), Penta-16dox (40%), Pentane I (30%). Fluorescence of pentaene probes. The first step in the characterization of the pentaene probes was the measurement of the excitation and emission spectra in various environments. Both excitation and emission spectra of the three probes varied when dissolved in different organic solvents, absorption and emission intensities being lowest in hexane and highest in chloroform


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for the three probes (Figure S1). Absorption and emission intensities of all three probes in chloroform increased linearly with probe concentration, at least between 0.3 and 2.7 µM (Figure S2). A fluorescence quantum yield for Pentaene I of 0.060 in ethanol (relative to 9,10DPA in ethanol) was determined, as described above. Similarly, quantum yields of 0.031 and 0.048 for Penta5dox and Penta16dox, respectively, were also measured. Fluorescence excitation and emission spectra of the three probes incorporated in pure lipid bilayers of different fluidities are shown in Figure 2. In DPPC (Figure 2B, B´) Pentaene I showed a maximum excitation at 353 nm and a maximum emission at 478 nm. In general the maxima remained fairly constant for the three probes and the four lipid compositions. The absolute intensities (not shown) of Pentaene I were very similar, within ± 10 %, in all four lipids. Moreover with lipids in the gel phase (Figure 2 A´, B´) the doxyl-containing probes exhibited clearly lower fluorescent emissions than Pentaene I, due to doxyl quenching, while this was less clear for probes in fluid bilayers (Figure 2 C´, D´).


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Figure 2. Fluorescence excitation (left panels) and emission (right panels) spectra for probes Penta16dox (red) and Penta5dox (green), normalized to Pentaene I (blue). Lipid bilayers were composed of: DSPC (A and A’), DPPC (B and B’), ePC (C and C’), DOPC (D and D’).

Quenching by the doxyl group of Penta16dox showed a high dependency on the fluidity of the bilayer: the degree of quenching reached 50% in gel liposomes but only 20 % in fluid liposomes (Figure 3). In contrast, the quenching ability of Penta5dox exhibited a more gradual sensitivity to the bilayer composition, from about 30-35 % quenching in liposomes made of DSPC to < 10 % in ePC bilayers (Figure 3). The data are consistent with a tighter packing of the probes in the rigid bilayers, that reduces the conformational mobility of the doxylstearate chain and favors the spatial proximity of the doxyl radical to the polyene system.


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Figure 3. Quenching of pentaene fluorescence emission intensity in liposomes of varying compositions: DSPC (1), DPPC (2), ePC (3), DOPC (4). The degree of quenching was calculated as the relative decrease of the emission intensity of Penta16dox (empty bars) and Penta5dox (full bars) as compared to the emission intensity of Pentaene I. A, effect of lipid composition; B, effect of the doxyl probe.

Absorption and emission intensities for all three probes in lipid bilayers were also dosedependent, but the dependence was not linear, at variance with the observations in chloroform (Figure S2). Spectra of probes at different concentrations in DPPC are shown in Figure S3. Fluorescence of Pentaene I increases by 2.4-fold when probe concentration varies from 0.1 to 0.3 mol % and by 1.8-fold when concentration increases from 0.3 to 0.9 mol %. Fluorescence emission intensities of the doxyl probes also increase non-linearly with concentration. Three examples of the behavior of the three pentaene probes in bilayers composed of lipid mixtures are summarized in Figure S4 and S5. The mixture eSM:pCer (1:1) at 23º C will be predominantly in the gel phase (10) and the spectra resembled very much those in DPPC or DSPC, also in the gel phase (Figure 2 A, B). The DOPC:DSPC (1:1 mol ratio) mixture gives rise to a coexistence of gel and fluid domains (30), the spectra were similar to those in pure DOPC (Figure 2). A third example is constituted by the ternary mixture DOPC:eSM:Chol (2:1:1) in which fluid-ordered and fluid-disordered domains coexist (30). In this mixture Penta-5-dox did not show any quenching capacity. Quenching values for the spectra in Figure S4 are summarized in Figure S5. The results are similar to those found in Figure 3 for pure lipids, in the sense that quenching was more efficient with Penta-16-dox than with Penta-5-dox.


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Differential scanning calorimetry Incorporation of Pentaene I into phospholipid bilayers was tested by differential scanning calorimetry. DPPC bilayers were used as host for the polyene lipid. Pure DPPC exhibits when fully hydrated a main endothermic gel fluid transition centered at about 41 ºC and a smaller pre-transition at about 33 ºC. This behavior was also observed in our DPPC samples (Figure 4). Pentaene I however did not exhibit any thermal signal in the temperature range under study (10-100 ºC) as expected for a polyunsaturated lipid. A mixture of DPPC and Pentaene I (85:15 molar ratio) gave rise to a single major endotherm, centered at 40.7 ºC. The pre-transition endotherm does not appear in the mixture. The width at half-height of the main transition was 1.80 ºC vs. 0.69 ºC for the pure DPPC. The main transition enthalpy hardly changed, 13,000 cal/mol for DPPC vs. 12,900 cal/mol for DPPC + Pentaene I. The conclusion of the DSC studies is that Pentaene I behaves in the same way as other amphipathic, unsaturated lipids, mixing well with phospholipids in bilayer form (31, 32).

Figure 4. DSC of aqueous dispersions of DPPC, Pentaene I and DPPC + 15 mol% Pentaene I.

Pentaene I in microscopy studies. 15 ACS Paragon Plus Environment

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Multiphoton/confocal microscopy was used to examine GUV composed of DOPC:eSM:Chol:Pentaene I (50:25:25:2, molar ratio). The mixture exhibits lateral separation of liquid-disordered (Ld) and liquid-ordered (Lo) domains, respectively bright and dark when the vesicles are stained with DiO (Figure 5, left-hand panel). When the fluorescence of Pentaene I is examined (Figure 5, middle panel) the Lo domains are preferentially stained, suggesting a high partition of the polyene into this kind of domains, although a higher fluorescence yield in the ordered domains cannot be discarded. A quantitative study of the average intensity ratio of Pentaene I-stained Lo/Ld domains gave a value of 3.9 ± 1.8 (112 vesicles). Moreover, the probes showed a photoselection process. This occurs because the extent of excitation of a fluorophore usually depends on the orientation of its transition moment relative to the plane of polarization of the exciting light. Therefore, when the fluorophore keeps a fixed orientation (as it happens with lipids in an ordered bilayer) the excitation efficiency will depend on the location of the probe along the liposome. That is the reason why dark areas can be seen in the Lo domains in the pentaene channel, always located along an imaginary north-south axis (Figure 5, right-hand panel blue arrows). The disordered domains, on the contrary, do not exhibit such dark areas. In bilayers consisting of mixtures of phospholipid:ceramide lateral phase separation occurs of ceramide-rich and –poor domains (10, 12, 13), the latter being preferentially stained by DiO (Figure 6). Pentaene I however appears to distribute rather evenly over the vesicle (Figure 6), with the result that when both DiO and Pentaene I are excited the ceramide-rich domains appear stained by the polyene fluorescent probe. Furthermore when gel and fluid domains are formed in mixtures of saturated and unsaturated phospholipids (Figure 6, bottom panels), Pentaene I appears to emit more intensely in domains in the gel phase, with a measured intensity ratio of 2.6 ± 0.8 for the gel/fluid domains (27 vesicles).


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Figure 5. Staining of lipid domains by Pentaene I. Representative confocal/multiphoton equatorial sections of GUVs of DOPC:eSM:Chol (2:1:1) containing DiO and Pentaene I are shown. Blue arrows show the effect of photoselection on Lo domains. Scale bar = 10 m.

Figure 6. Staining of lipid domains by Pentaene I. Representative confocal/multiphoton images of GUVs of different compositions containing DiO and Pentaene I are shown (equatorial sections). Scale bar = 10 m.

Electron paramagnetic resonance (EPR) of pentaene doxyl probes EPR spectra provide valuable information about the environment of the spin label in a lipidic environment (33-35). The dynamic parameters of pentaene spin probes Penta5dox and Penta16dox, both in solution and in LUVs, were studied on the basis of parameters τ (rotational correlation time), 2Amax and S (empirical order parameter). As the rigidity of the membrane increased and the mobility of the nitroxide radical


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diminished (from Penta16dox to Penta5dox), the profile of the EPR spectrum became more anisotropic (Figure 7). In general, the behavior of the Penta5dox probe was less dependent on the lipid environment, showing in all cases an anisotropic profile.

Figure 7. EPR spectrum of the polyene doxyl probes in tetrahydrofurane (THF) or in LUV with the lipids indicated on top. Spectra recorded at 20 C (293K) unless indicated otherwise. Through a quantitative epr calculation, the concentration of probes in the liposomes showed values ranging from 25 to 75% of the theoretical concentration (15 mol %, see Supporting Information, Table S1).

The broad EPR spectra structure of Penta5dox derivatives indicates that the probes move slowly in an anisotropic environment. The parameters of choice to study the spin dynamics in rigid systems (τC, 2Amax and S) indicate that the probes are moving in a partially ordered structure, with a mobility between the slow and rigid limit movement (Table 1). These parameters indicate an increasing immobilization in the chain segments closer to the lipid polar group in the order ePC DOPC < DPPC < DSPC. In the latter two cases the probe appears to be almost fully immobilized. Table 1. EPR parameters for probe Penta5dox in different membrane systems


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System

τC (s)

S

2Amax (G)

LUV-ePC-Penta5dox

0.58 x 10-8

0.69

53.94

LUV-DOPC-Penta5dox

0.62 x 10-8

0.70

54.48

LUV-DPPC-Penta5dox

5.42 x 10-8

0.93

62.73

LUV-DSPC-Penta5dox

8.83 x 10-8

0.95

63.73

τC: correlation time, a measure of the motion of the nitoxide radical. S: empiric order parameter. It is measured from the EPR spectrum. It indicates the anisotropy or the randomness of the motion. Amax is one-half the separation of the outer hyperfine extrema

Moreover, the shape of the Penta16dox spectra indicates that the probes are moving relatively fast. In this case, the use of τC, ∆H0 and h-1 are diagnostic and the observed values indicate that the spin probes are tumbling in structures with intermediate mobility. Both the τC and the central line width (∆H0) values, or the high- field line amplitude (h-1) are consistent with the different rigidity and polar environment in liposomes made of ePC, DOPC, DPPC and DSPC, respectively (Table 2). Table 2. EPR parameters for probe Penta16dox in different membrane systems

System

h-1

ΔH0 (G)

τC

LUV-ePC-Penta16dox

1717

2.09

1.04 x 10-9

LUV-DOPC-Penta16dox

1477

2.09

1.23 x 10-9

LUV-DPPC-Penta16dox

845.4

3.52

4.74 x 10-9

LUV-DSPC-Penta16dox

209.2

5.97

1.76 x 10-8

ΔH0 is the central line width h-1 is the high field line amplitude 19 ACS Paragon Plus Environment

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Pentaene doxyl probes in the study of phospholipid gel-fluid transitions. The observation that pentaene fluorescence of Penta16dox was quenched more efficiently in gel than in fluid bilayers (Figure 3) suggested the possibility of using Penta16dox as a probe to detect gel-fluid transitions in aqueous phospholipid dispersions. As shown in Figure 8 (top curve) when the probe was incorporated into DPPC vesicles and quenching was recorded as a function of temperature two abrupt decreases, marked by arrows, were observed at ~ 33 ºC and 42 ºC, i.e. respectively the pre-transition and the main gel-fluid transition temperatures of DPPC (36). However with ePC, that does not undergo any phase transition in the temperature range under study, Penta16dox quenching did not exhibit any discontinuity (Figure 8, bottom curve). Thus, this probe appears to be suitable for the detection of this kind of thermotropic transitions in aqueous phospholipid systems.

Figure 8. Quenching of the pentaene emission intensity of Penta16dox as a function of temperature in liposomes made of DPPC (top) or ePC (bottom). Representative curves of at least three separate experiments are shown. Heating rate: 0.8 C/min.


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Discussion Fluorescence arising from natural polyene systems has been known for decades. Sklar et al. (37) identified a naturally occurring fluorescent fatty acid with a conjugated system of four double bonds, parinaric acid, that has found important applications in the physical chemistry of lipids (38, 39). More recently Souto et al. (25) synthesized conjugated pentaenoic acids by a Wittig olefination reaction, and then Kuerschner et al. (40) included a pentaene fatty acid into a membrane lipid, sphingomyelin, that could be incorporated into cells. Following this line of experimental research, the present paper describes the synthesis and biophysical properties of three pentaene lipids that can be of use in the study of cell membrane properties. They are structural analogues of Npalmitoylsphingosine, or palmitoylceramide, with two differences, namely the lack of sphingosine C1(CH2OH) and the oxo instead of a hydroxyl group in C3. The overall effect is a decrease in the polarity of the headgroup. The conjugated double bonds are located in the sphingosine moiety (Figure 1). Moreover two of the novel lipids (Penta5dox and Penta16dox) contain a doxyl radical in the Nacyl chain. The radical is intended to quench the pentaene fluorescence when in close proximity to the sphingosine chain (41). An important property of any membrane probe is its distribution among the various lamellar phases (gel, liquid-ordered, liquid-disordered) that can make it more or less useful in physico-chemical studies of lipid mixtures. Most bulky lipid substituents, e.g. Bodipy-lipids, NBD-lipids, etc. tend to partition preferentially into the liquid-disordered phases (42, 43). Parinaric acid however is known to partition into gel phases. The DSC data (Figure 4) suggest that Pentaene I partitions preferentially in fluid over gel phases, since it causes a decrease in the gel-fluid transition temperature of DPPC. However the data in Figure 6 (DOPC:DSPC mixture) indicate that the fluorescence emission of Pentaene I is more intense in the gel than in the fluid domains. 21 ACS Paragon Plus Environment

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Moreover Pentaene I

emits more intensely from liquid-ordered than from liquid-

disordered phases (Figure 5). The intensity of Pentaene I emission appears to depend not only on the partition coefficient but also on the microenvironmental effects on the quantum yield. In mixtures containing natural ceramide, Pentaene I partitions into both the ceramide-rich and -poor domains (Figure 6). This is at variance with the case of NBD-ceramide, containing a bulky fluorescent moiety in the Nacyl chain, that cannot partition into domains enriched in natural ceramide (13). The fact that Pentaene I labels the gel domains can be particularly useful in view of the putative presence of gel microdomains in live cell membranes (44, 45). The twotailed overall structure of Pentaene I is more similar to that of a membrane lipid than that of parinaric acid, and this is an additional advantage to mimic the behavior of cell lipids. Pentaene I, unlike natural ceramides (10), does not segregate laterally into highly enriched domains. On the contrary, it rather distributes along the whole extension of the bilayer, albeit staining the different domains with different intensities (Figures 5, 6). Ceramide is thought to form very rigid domains through a network of hydrogen bonds (4, 5). However, the analogues described here lack one OH group at C1 and have a carbonyl group instead of a hydroxyl group in C3 of the sphingosine moiety, so that the H-bonding network is severely perturbed. This relatively low level of intermolecular Hbonding allows the even mixing of Pentaene I in ceramide-rich and –poor domains, as discussed above. It is interesting in this respect that a whole group of natural ceramides have recently been discovered in mammalian cells that lack the OH group at C1 and exhibit consequently unusual biophysical properties (46). Polar lipids spontaneously organize in bilayers because of their amphipathic character. Strictly non-polar lipids tend to occupy the bilayer hydrophobic matrix. The ceramide analogues described in the present paper appear to occur at least partly with their polar 22 ACS Paragon Plus Environment

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moieties at the lipid-water interface. This is supported by the DSC data (Figure 4) that show a loss of the pre-transition and a moderate widening of the DPPC main transition in the presence of 15 mol % Pentaene I. This is the sort of effect found when DPPC is mixed with unsaturated phospholipids at 10-20 mol % concentrations (45), while nonpolar lipids that locate between the monolayers without a preferred orientation have a much smaller effect on the DSC thermograms (47). Also in favour of an orientation normal to the membrane plane is the detection of gel-fluid phase transitions by Penta16dox (Figure 8). Doxyl quenching of the pentaene fluorescence requires the sphingosine and acyl chains of the molecule to be very close to each other, and this would be achieved most easily through a phospholipid-like orientation in the bilayer. In conlusion, we propose Pentaene I, Penta-5-dox, and Penta-16-dox as useful probes in fluorescence microscopy, fluorescence spectroscopy, and EPR membrane studies. Pentaene I may be particularly useful in the observation of highly-ordered bilayers by confocal microscopy, while the doxyl-containing probes report on gel-fluid lipid transitions.

Acknowledgments This work was supported in part by funds from the Spanish Ministry of Economy (BFU 2012-36241 to F.M.G., BFU 2011-28566 to A.A.), the Basque Government (IT 849-13 to F.M.G., IT 838-13 to A.A.), the Fundación Biofisica Bizkaia, and grant 2009SGR1072 (Agència de Gestió d’Ajuts Universitaris i de Recerca de la Generalitat de Catalunya) to RUBAM. I.N. was supported by a JAEpredoc fellowship (CSIC) and I.A. by a FPI fellowship (UPV/EHU). We also thank Mrs. Avencia Díez from the EPR service of the IQACCSIC for recording the EPR spectra.


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Supporting information Supporting information available: details on the synthesis of the polyene probes, five additional figures with the fluorescence emission spectra of the pentaene probes under various conditions, and NMR spectra for various compounds described in this paper. This material is available free of charge via the Internet at http://pubs.asc.org/.

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Fluorescent Polyene Ceramide Analogues as Membrane Probes

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