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Preparation and Membrane Distribution of Fluorescent Derivatives of Ceramide Takaaki Matsufuji, Masanao Kinoshita, and Nobuaki Matsumori Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b03176 • Publication Date (Web): 04 Jan 2019 Downloaded from http://pubs.acs.org on January 7, 2019

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Langmuir

Preparation

and

Membrane

Distribution

of

Fluorescent

Derivatives of Ceramide

Takaaki Matsufuji,1 Masanao Kinoshita,1 and Nobuaki Matsumori1

1 Department

of Chemistry, Graduate School of Science, Kyushu University, 744 Motooka,

Nishi-ku, Fukuoka 819-0395 Japan.

1

ACS Paragon Plus Environment

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Abstract Ceramide is a bioactive lipid with significant roles in several biological processes including cell proliferation, apoptosis, and raft formation. Although fluorescent derivatives of ceramide are required to probe the behaviors of ceramide in cells and cell membranes, commercial fluorescent ceramide derivatives do not reproduce the membrane behaviors of native ceramide because of the introduction of bulky fluorophores in the acyl chain. Recently, we developed novel fluorescent analogs of sphingomyelin, in which the hydrophilic fluorophores, ATTO488 and ATTO594, are attached to the polar head of sphingomyelin via a nonaethylene glycol linker and demonstrated that their partition and dynamic behaviors in bilayer membranes are similar to native sphingomyelin. In this report, by extending the concept used for the development of fluorescent analogs of sphingomyelin, we prepared novel fluorescent ceramides that exhibit membrane behaviors similar to native ceramide and succeeded in visualizing ceramide-rich membrane domains segregated from ceramide-poor domains.

2


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INTRODUCTION Ceramide (Cer) is involved in a variety of cellular processes and diseases. Although Cer is maintained at extremely low concentrations in plasma membranes of normal cells, they can increase in response to a variety of stimuli such as cytokines, death receptor ligands, and anticancer drugs,1,2 and elicit a number of different biological responses such as cell proliferation and apoptosis.3–7 Although the mechanistic link between Cer formation and such biological responses has yet to be fully elucidated, some studies have suggested that alterations in the membrane biophysical properties induced by Cer formation may contribute to its biological responses.8–11 In effect, Cer has been known to affect various physical properties of bilayer membranes including thermodynamic behavior, molecular order, and lateral distribution.12– 23

To date, several fluorescent derivatives of Cer have been prepared to visualize the dynamic behaviors and kinetics of Cer in cells. For instance, C6-NBD-Cer (NBD-Cer) and BODIPYCer,24–26 both of which have fluorescent dyes in the N-acyl chain, are frequently used for those purposes because of their commercial availability. However, experiments using artificial membranes have also shown that these fluorescent Cers do not reproduce the membrane behaviors of native Cer; NBD-Cer was not incorporated to Cer-rich gel domains but to liquid-ordered (Lo) and disordered (Ld) domains,22 and BODIPY-Cer was partitioned strongly into the Ld phase.27 This was presumably because the bulky fluorescent moieties introduced in the N-acyl chains prevent the tight chain packing in the Cer-rich gel domains, and thus those fluorescent Cers are excluded from the gel domains. Hence, to scrutinize the physicochemical membrane properties of Cer, the development of new fluorescent 3


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derivatives that reproduce the membrane dynamics of Cer is desirable. Cer lacks the head group typical of phospholipids or glycosphingolipids, and generally possesses two hydroxy groups at the C1 and C3 positions. Recently, in order to study how the oxidative state of the primary alcohol group of Cer affects membrane physical properties, we prepared Cer derivatives in which the primary alcohol group was oxidized to a carboxylic acid and methylester (PCerCOOH and PCerCOOMe, Figure 1), and examined how the oxidative state of the primary alcohol of Cer influences membrane physical properties.28 As a result, both derivatives exhibited membrane properties similar to those of native Cer. This suggests that it is possible to introduce various functional groups to PCerCOOH via an ester or amide linkage with retaining the membrane properties of Cer. We have also reported excellent fluorescent sphingomyelin (SM) analogs (488 and 594negSMs, Figure 1), in which the hydrophilic fluorophore ATTO488 or ATTO594 was attached to the polar head of SM via an oligoethylene glycol linker.29 Although commercially available fluorescent SM analogs, in which fluorophores are attached to the acyl chain, favor SM-poor disordered membrane domains, our fluorescent SMs exhibit partition and dynamic behaviors similar to native SM, enabling the visualization of the SM-rich ordered membrane domains segregated from SM-poor Ld domains in SM/dioleoylphosphatidylcholine (DOPC)/chol ternary-component giant unilamellar vesicles (GUVs).29–31 Hence, in this report, by combining the concept used for the development of fluorescent SMs with the findings from Cer derivatives PCerCOOH and PCerCOOMe, we prepared novel fluorescent analogs of Cer (594neg-PCer1 and 2, Figure 1) that mimic the membrane behaviors of native Cer. 4


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RESULTS Preparation of fluorescent Cer derivatives. As mentioned above, we reported fluorescent SMs (Figure 1) that behave quite similarly to their native counterparts, in terms of partitioning into artificial raft-related membrane domains.29 Our strategy for their development was to attach hydrophilic fluorophores to the SM headgroup and to place it some distance away from the SM headgroup toward the bulk aqueous phase via a hydrophilic nonaethylene glycol (neg) linker. Based on this strategy, we designed and prepared two kinds of fluorescent Cer analogs, 594neg-PCer1 and 2 (Figure 1). First, 594neg-PCer1 was derivatized from PCerCOOH via amidation of the carboxyl group. The other fluorescent ceramide was derived from the esterification of the primary alcohol group of Cer. OH

O

HO

C13H27 C13H27

HN

HO

O

PCerCOOH

O ATTO 488 ATTO 594

O

N H

9

N N N

O

PCerCOOMe O

N

O

P

OH

O O

O O

O O

N H

9

N H

N N N

O

ATTO 594

N H

O 9

O

N N N

C13H27 C13H27 O

O

H N

OH

HN

594neg-PCer1 O

C13H27 C13H27

HN

488, 594neg-SM

ATTO 594

C13H27 C13H27

HN

O

Palmitoylceramide (PCer)

OH

O

C13H27 C13H27

HN

O

O

OH

OH O

O

C13H27 C13H27

HN

594neg-PCer2

O

Figure 1. Structures of palmitoylceramide (PCer) and its oxidized derivatives (PCerCOOH and PCerCOOMe), recently reported fluorescent-labeled SMs (488 and 5


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594neg-SMs), and newly developed fluorescent derivatives of Cer (594neg-PCer1 and 2).

For preparation of 594neg-PCer1, we first condensed PCerCOOH28 with propargylamine to introduce an alkyne group, and the resultant alkyne derivative (PropargylamidePCer) was subjected to Huisgen cyclization reaction32 with ATTO594-neg-azide29 to afford the objective compound (Scheme 1). O

NH2

OH

HO

C13H27 C13H27

HN

O

EDC•HCl, DMAP

N H

CHCl2, rt, 18 h

ATTO 594

C13H27 C13H27

HN

O PropargylamidePCer

O PCerCOOH

O

OH

O

H 2N

+

N3

O

O

9

Et3N

O N O

O

O

N H

ATTO 594

DMF, rt, 20 h

9

N3

ATTO594-neg-azide t-BuOH/H2O (4:1), rt, 5 d

CuSO4 sodium ascorbate O O ATTO 594

O

N H

O 9

O

N N N

N H

OH C13H27 C13H27

HN O

594neg-PCer1

Scheme 1. Preparation of 594neg-PCer1.

Next, 594neg-PCer2 was prepared via esterification of the primary alcohol of Cer with 4oxo-4-(prop-2-yn-1-ylamino)butanoic acid that was prepared from propargylamine and succinic anhydride, followed by the same Huisgen reaction as above (Scheme 2).

6


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2

O

1

N H

3

4

2 3

OH 1

OH

O

4-oxo-4-(prop-2-yn-1ylamino)butanoic acid

+

HO

C13H27 C13H27 CH Cl , 30°C, 3 h 2 2

HN

O

H N

EDC•HCl, DMAP

O O

O PCer

C13H27 C13H27

HN O PropargylesterPCer

+

O CH2Cl2, rt, 1 h

DMAP

OH

O

N H

ATTO 594

9

N3

O

ATTO594-neg-azide NH2

+ O

O

CuSO4 sodium ascorbate

O O ATTO 594

N H

O 9

O

N N N

t-BuOH/H2O (4:1), rt, 5 d

O

H N

OH O

O 594neg-PCer2

C13H27 C13H27

HN O

Scheme 2. Preparation of 594neg-PCer2.

Microscopic observation of fluorescent Cers in binary-component GUVs. To examine the phase behaviors of these fluorescent Cers, we observed their distribution in phaseseparated GUVs using fluorescence microscopy. First, we prepared GUVs composed of a binary mixture of palmitoylsphingomyelin (PSM) and palmitoylceramide (PCer) (95:5, mole ratio), which are known to be immiscible and to undergo a phase-separation between Cerrich (SM-poor) and Cer-poor (SM-rich) gel domains.33 The GUVs contained 0.2 mol% of 594neg-PCers and 488neg-SM, the latter of which functions as a marker of SM-rich domains.29 As a result, the membrane domains stained by 594neg-PCers were clearly different from those by 488neg-SM (Figure 2a, top and middle), thus demonstrating that 594neg-PCer1 and 2 have selectivity toward Cer-rich gel domains. The preference of 594negPCers for Cer-rich gel domains was also observed in the GUVs composed of PSM and PCer at 90:10, 85:15, and 80:20 mole ratio (Figures S1-S3). On the other hand, commercial NBD7


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Cer, which has an NBD group in the acyl chain, was shown not to be distributed to Cer-rich gel domains because its distribution was the same as that of TexasRed-DPPE, which is preferentially localized in more disordered domains (Figure 2a, bottom). Similarly,

we

observed

GUVs

composed

of

a

binary

mixture

of

palmitoyloleoylphosphatidylcholine (POPC) and PCer (95:5, mole ratio), which are known to undergo a phase-separation between Cer-rich gel and Cer-poor (POPC-rich) Ld domains.18,34,35 BODIPY-PC was used as a Ld marker.27 As expected, 594neg-PCers were selectively localized in the Cer-rich gel domains (Figure 2b). In the present and following figures, we show data only for 594neg-PCer1 because both 594neg-PCers exhibited identical phase behaviors. These observations clearly demonstrated that both fluorescent Cers, 594neg-PCer1 and 2, prefer Cer-rich gel domains in the binary-component membrane systems.

8


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Figure 2. Fluorescence microscopy images of binary-component GUVs that underwent phase separation between Cer-rich and Cer-poor domains. (a) PSM/PCer (95:5, mole ratio) GUVs containing 0.2 mol% 594neg-Cers and 0.2 mol% 488neg-SM (top and middle), or 1 mol% NBD-Cer and 0.2 mol% TexasRed-DPPE (bottom). (b) POPC/PCer (95:5, mole ratio) GUVs containing 0.2 mol% 594neg-PCer1 and BODIPY-PC (Ld marker). Bar = 10 μm. The brightness and contrast were adjusted for clarity.

Microscopic observation of fluorescent Cers in ternary- and quaternary-component 9


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GUVs. Next, we examined the localization of the fluorescent Cers in the GUVs that undergo a phase separation between the Lo and Ld domains. The GUVs were composed of a ternary mixture of PSM, dioleolylphosphatidylcholine (DOPC), and cholesterol (Chol) (1:1:1, mole ratio) (Figure 3a) or a mixture of PSM, POPC, and Chol (2:2:1, mole ratio) (Figure 3b).36-41 It was expected that 594neg-PCers preferentially localize to Lo domains because Cer was considered to prefer membranes with higher chain order. The results confirmed that 594negPCers prefer the Lo domains in both of the ternary membrane systems because they showed a different distribution from BODIPY-PC,27 which is an Ld marker (Figure 3a, top). Meanwhile, NBD-Cer was almost homogeneously distributed to the GUVs (Figure 3a bottom), although TexasRed-DPPE indicated that the GUVs underwent Lo/Ld phaseseparation.27 This was consistent with the previous report where NBD-Cer localizes both in Lo and Ld domains.22

10


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Figure 3. Fluorescence microscopy images of ternary-component GUVs that underwent phase separation between Lo and Ld domains. (a) PSM/DOPC/Chol (1:1:1, mole ratio) GUVs containing 0.2 mol% 594neg-Cer1 and BODIPY-PC (Ld marker), or 1 mol% NBD-Cer and 0.2 mol% TexasRed-DPPE (Ld marker). (b) POPC/POPC/Chol (2:2:1, mole ratio) GUVs containing 0.2 mol% 594neg-PCer1 and BODIPY-PC (Ld marker). Bar = 10 μm. The brightness and contrast were adjusted for clarity.

Finally, we examined the phase selectivity of the fluorescent Cers in a more complicated quaternary mixture of PSM/DOPC/Chol/PCer (1:1:1:0.3), which is known to undergo a ternary phase separation among Lo, Ld, and Cer-rich gel domains.42 The GUVs contained 0.2 mol% 594neg-PCer1 and 1.0 mol% NBD-Cer. Figure 4 shows representative florescence 11


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images of the GUVs, in which NBD-Cer and 594neg-PCer1 have complementary distribution patterns. Considering that NBD-Cer tends to be distributed in both Lo and Ld domains as shown above, this result suggests that 594neg-PCers are preferentially localized in Cer-rich gel domains even in such a complicated membrane system, whereas NBD-Cer was excluded from the gel domain (Figure 4, top). The result shown in the center panels of Figure 4 is consistent with a previous report that showed the formation of small-sized Cer-rich gel domains and the exclusion of NBD-Cer from the domains in GUVs composed of PC:PE:SM:Chol (1:1:1:1, mole ratio) containing 10 mol% Cer.22 We also observed that the distribution area of NBD-Cer was larger than the area stained by an Ld-domain marker, TexasRed-DPPE (Figure 4, bottom), supporting the notion that NBD-Cer was distributed both to the Ld and Lo domains in this quaternary-component membrane system. The regions indicated by yellow arrows (Figure 4) are considered as Lo domains, because the regions are darker than Cer-rich domains (indicated by white arrows) but brighter than the remaining domains (should be Ld domains).

Figure 4. Fluorescence microscopy images of quaternary-component GUVs that underwent 12


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phase separation among Lo, Ld, and Cer-rich gel domains. PSM/DOPC/Chol/PCer (1:1:1:0.3) GUVs containing 0.2 mol% 594neg-PCer or TexasRed-DPPE (Ld marker) and 1.0 mol% NBD-Cer. White arrows indicate Cer-rich gel domains. Bar = 10 μm. The brightness and contrast were adjusted for clarity.

DISCUSSION In this study, based on our proposed design strategy for fluorescent lipids29, we designed and prepared novel fluorescent Cers, 594neg-PCer1 and 2, in which a hydrophilic nonaethylene glycol (neg) linker is expected to serve to keep the fluorophore away from the hydrophobic region of the parent lipid to which the fluorophore is linked. The fluorescent Cers were demonstrated to have a preference for Cer-rich membrane domains in artificial membrane systems. In particular, Figure 4 suggests that these probes can be used to visualize the formation and collapse of Cer-rich domains in lipid rafts. The partition of 594neg-PCers between membrane domains was summarized in Table S1, which demonstrates that the derivatives can reproduce the membrane behaviors of native Cer to a considerable extent. In contrast, commercially available NBD-Cer does not show the preference toward Cer-rich domains. This is probably because the bulky NBD moiety introduced in the N-acyl chain prevents the chain packing in the Cer-rich gel domains, and thus NBD-Cer is excluded from the gel domains. This notion is consistent with our previous observation for fluorescent SMs29; although commercially available fluorescent SM analogs, in which fluorophores are attached to the acyl chain, favor SM-poor disordered membrane domains, 488 and 594negSMs (Figure 1) exhibit partition and dynamic behaviors similar to native SM. 13


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Since Cer possesses the primary alcohol that can function as both a hydrogen bond donor and acceptor, we first expected that 594neg-PCer1 is superior to 594neg-PCer2 in terms of reproducing the membrane behaviors of native Cer, because in 594neg-PCer1, the primary alcohol is substituted by the amide group that can act as both a hydrogen bond donor and acceptor, while 594neg-PCer2 possesses an ester group that works only as a hydrogen bond acceptor. However, no major difference in the distribution behaviors was observed between 594neg-PCer1 and 2 in the membrane systems tested. We previously reported that PCerCOOMe (Figure 1), which lacks the propensity for donating a hydrogen bond at the Cer C1 position, also forms Cer-rich domains,28 suggesting that the function as a hydrogen bond acceptor at the Cer primary alcohol group may be enough for forming Cer-rich domains. If this is the case, the nonaethylene glycol linker may also act as the acceptor of interlipid hydrogen bonds and serve to retain the interlipid interactions necessary for the preference for Cer-rich domains. Taking into account the fact that NBD-Cer, which has a fluorescent dye in the alkyl chain, lost preference toward Cer-rich domains, the intermolecular hydrogen bonds associated with the Cer primary alcohol may be relatively less important as a determinant factor for membrane distribution of Cer in comparison with the van der Waals molecular interactions among the alkyl chains. As mention in the introduction, Cer has important roles in several biological processes, including cell proliferation, apoptosis, and raft formation, 3–7 and the presence of the primary alcohol in Cer is assumed to be indispensable for some of the processes, such as its metabolism. In this respect, our fluorescent derivatives, in which the primary alcohol group was lost, may have limited applicability as a molecular probe to investigate the cell functions 14


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of Cer. However, at least in terms of membrane behaviors, our fluorescent probes mimic Cer much better than commercially available fluorescent Cers, such as NBD-Cer and BODIPYCer, both of which are known to be excluded from Cer-rich membrane domains.22,27 The lack of the primary alcohol may provide another potential merit by conferring immunity to metabolic conversions to other sphingolipids upon observation of living cells, which would make it possible to observe the membrane behaviors of Cer itself for a prolonged period. In fact, NBD-Cer, which retains the primary alcohol, is known to be metabolized to sphingolipids upon incubation with living cells.24 In these contexts, these derivatives will be of significant use as new molecular probes to investigate the Cer dynamics in biological membranes and to visualize the formation and collapse of Cer-rich domains in lipid rafts.

EXPERIMENTAL SECTION General. Chicken egg SM, PCer, DOPC, and POPC were purchased from Avanti Polar Lipids (Alabaster, AL). The compounds 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-sindacene-3-dodecanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (BODIPY-PC), 1,2dihexadecanoyl-sn-glycero-3-phosphoethanolamine (TexasRed-DPPE),

and

triethylammonium

salt

6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-

yl)amino)hexanoyl)sphingosine (NBC-Cer) were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). PCerCOOH, ATTO594-neg-azide, and 488neg-SM were synthesized as previously described.28,29 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 15


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silica gel 60 F-254 plates and was visualized by UV irradiation (254 nm) or staining with phosphomolybdic acid. The 1H 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 PropargylamidePCer. To a solution of PCerCOOH (1.3 mg, 2.36 μmol) in CH2Cl2 (0.50 mL) were added CH2Cl2 solutions of propargylamine (1.0 M, 3.54 μL, 3.54 μmol), DMAP (1.0 M, 2.36 μL, 2.36 μmol), and EDC-HCl (250 mM, 28.4 μL, 7.10 μmol). After being stirred at room temperature for 17 h, the reaction was quenched with an aqueous solution of NaHCO3 and the mixture was extracted with CHCl3. The organic layer was washed with a saturated aqueous solution of NaHCO3, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. Purification by silica gel column chromatography (CHCl3/MeOH 40:1, v/v) afforded the objective compound (500 μg, 84.9 nmol, 36 %) as a white solid; Rf = 0.53 (silica gel, CHCl3/MeOH 10:1, v/v), 1H NMR (600 MHz, CDCl3): δ 7.06 (t, J = 4.8 Hz, 1H), 6.41 (d, J = 7.8 Hz, 1H), 5.76 (dt, J = 15.0, 6.6 Hz, 1H), 5.50 (dd, J = 15.3, 6.3 Hz, 1H), 4.69 (d, J = 5.4 Hz, 1H), 4.19 (t, J = 6.0 Hz, 1H), 4.04-3.98 (m, 2H), 2.23-2.15 (m, 3H), 2.03−1.99 (m, 2H), 1.38−1.18 (m, 48H), 0.86 (t, J = 6.9 Hz, 6H), HRMS (m/z): [M+Na]+ calcd for C37H68N2NaO3, 611.5122; found, 611.5120. Synthesis of 594neg-PCer1. To a solution of the ATTO594-neg-azide (1.0 mg, 0.76 μmol) in t-BuOH/H2O (4:1, v/v, 500 μL) were added an aqueous solution of CuSO4 (0.19 M, 4.00 μL, 760 nmol), an aqueous solution of sodium L-ascorbate (0.38 M, 4.00 μL, 1.52 μmol), and a MeOH solution of PropargylamidePCer (1.0 mg, 1.70 μmol). The reaction mixture was stirred at room temperature for 5 days, and then the solvent was removed by evaporation. 16


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The residue was purified by silica gel column chromatography (CHCl3/MeOH/H2O 40:10:1, v/v/v), followed by gel permeation chromatography (TOYOPEARL HW-40F, MeOH), to afford 594neg-PCer1 (146 μg, 76.7 nmol, 10 %) as a blue solid; Rf = 0.51 (silica gel, CHCl3/MeOH/H2O

20:10:1,

v/v/v),

HRMS

(m/z):

[M+-2H+2Na]+

calcd

for

C100H158N9Na2O22S2, 1947.0753; found, 1947.0750. Synthesis of 4-oxo-4-(prop-2-yn-1-ylamino)butanoic acid. To a solution of succinic anhydride (150 mg, 1.50 mmol) and DMAP (154 mg, 1.26 mmol) in CH2Cl2 (5.0 mL) was added propargylamine (80.6 μL, 1.26 mmol). The reaction mixture was stirred at room temperature for 1 h, and then extracted with 10 % aqueous NaHCO3. The combined water layer was acidified with 1 M HCl and extracted with ethylacetate. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to give the objective compound (100.6 mg, 648 μmol, 52 %) as a white solid; Rf = 0.61 (silica gel, CHCl3/MeOH/AcOH 100:100:1, v/v/v); 1H NMR (600 MHz, CDCl3) δ 5.85 (1H, br), 4.05 (2H, q, J = 5.4, 2.4 Hz), 2.71 (2H, t, J = 7.2 Hz), 2.51 (2H, t, J = 6.6 Hz), 2.23 (1H, t, J = 2.4Hz). Synthesis of PropargylesterPCer. To a solution of PCer (13.9 mg, 7.25 μmol) in CH2Cl2 (2.0 mL) were added 4-oxo-4-(prop-2-yn-1-ylamino)butanoic acid (1.4 mg, 8.70 μmol), DMAP (1.1 mg, 8.70 μmol), and EDC-HCl (8.3 mg, 43.5 μmol). After being stirred at room temperature for 3 h, the reaction was quenched with an aqueous solution of NaHCO3 and the mixture was extracted with CHCl3. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. Purification by silica gel column chromatography (hexane/ethyl acetate 1:2, v/v) afforded the objective compound (3.0 mg, 4.42 μmol, 61 %) 17


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as a white solid; Rf = 0.31 (silica gel, hexane/ethyl acetate 1:2, v/v); 1H NMR (600 MHz, CDCl3) δ 6.12 (1H, d, J = 7.8 Hz), 6.02 (1H, t, J = 3.0 Hz), 5.73 (1H, dt, J = 15.6, 6.0 Hz), 5.46 (1H, dd, J = 15.0, 7.2 Hz), 4.34-4.25 (2H, m), 4.19-4.13 (2H, m), 4.07-3.98 (2H, m), 2.72 (1H, d, J = 4.8 Hz), 2.68-2.56 (1H, m), 2.50 (1H, t, J = 6.2 Hz), 2.21-2.17 (2H, m), 2.01 (2H, q, J = 7.1 Hz), 1.62-1.57 (2H, m), 1.36-1.17 (46H, m), 0.86 (6H, t, J = 6.9 Hz); HRMS (m/z): [M+Na]+ calcd for C41H74N2NaO5, 697.5490; found, 697.5487. Synthesis of 594neg-PCer2. To a solution of the ATTO594-neg-azide (0.50 mg, 0.38 μmol) in t-BuOH/H2O (4:1, v/v, 500 μL) were added an aqueous solution of CuSO4 (0.19 M, 2.00 μL, 0.38 μmol), an aqueous solution of sodium L-ascorbate (0.38 M 2.00 μL, 0.76 μmol), and a MeOH solution of PropargylesterPCer (760 mM, 200 μL, 1.52 μmol). The reaction mixture was stirred at room temperature for 5 days, and then the solvent was removed by evaporation. The residue was purified by silica gel column chromatography (CHCl3/MeOH/H2O 40:10:1, v/v/v), followed by gel permeation chromatography (TOYOPEARL HW-40F, MeOH), to afford 594neg-PCer2 (62 μg, 31.2 nmol, 8 %) as a blue solid; Rf = 0.66 (silica gel, CHCl3/MeOH/H2O 20:10:1, v/v/v), HRMS (m/z): [M+-2H+3Na]2+ calcd for C104H164N9Na2O24S2, 1028.5523; found, 1028.5544. GUV preparation and fluorescence observations. PSM was purified from egg SM by 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 and stored at −20 °C until use. Fluorescent probes were 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. GUVs were prepared using the electroformation method developed by Angelova and 18


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Dimitrov.43 Briefly, an aliquot (10 μL) of the lipid solution (1 mg/mL) containing fluorescent probes 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 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). To provide clearer images of the phase separation, the brightness and contrast were adjusted using Adobe Photoshop CS6 (Adobe Systems Inc., San Jose, CA).

AUTHOR INFORMATION *Corresponding author; [email protected]

Author Contributions T.M. synthesized the derivatives, and T.M. and M.K. performed the membrane 19


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experiments. N.M. designed and organize the present study and wrote the manuscript.

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.

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