0 Copyright 1991 American Chemical Society
The ACS Joumal of
Surfaces and Colloids NOVEMBER 1991 VOLUME 7, NUMBER 11
Letters Dynamics of a Bolaamphiphilic Lipid in a Bilayer Liposome Robert A. Moss,' Tsunehisa Fujita, and Yukihisa Okumura Department of Chemistry, Rutgers, The State University of New Jersey, New Brunswick, New Jersey 08903 Received June 23,1991. In Final Form: August 6, 1991 The "hemimacrocyclic", bolaamphiphilic, functional ammonium ion lipid, 1,l'-dotriacontamethylenoxybis(2-hexadecyloxypropane-3-Q),where Q is -N,N-dimethyl-N-[4-nitro-l-(benzoyloxy)-3-benzylJammonium bromide, 5-F, was synthesized. Liposomes of 5-F and the (nonfunctional)monopolar lipid, 1,2dihexadecyloxy-3-propane-N,N,N-trimethylammonium bromide, 4-NF (1:14),were created by sonication. These small (d 300 A), gel phase (T,= 39 "C) liposomes were surface differentiated by exovesicular glutathione at an exovesicular/endovesicularpH gradient of 814. The differentiation kinetics indicated that -70% of the functional groups of 5-F had been in exovesicular locations, whereas -30% occupied endovesicular sites. Endovesicular/exovesicularflip-flop reequilibration of the differentiated 5-F 4-NF liposomes occurred with t l p 5 min at 40 "C, similar to the behavior of conventional 4-F/4-NF ilayer liposomes. (4-Fis 1,2-dihexadecyloxypropane-3-Q.)The results suggest that at least 40% of the 5-F lipid molecules adopt U-plan conformations in the 4-NF liposomes; uniformly extended, bilayer-bridging conformations for the 5-F bolaamphiphiles are excluded.
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phiphilic lipids and the "hemimacrocyclic" bolaamphiphiles, such as 1, can aggregate to monolayer lipid membranes (MLM), where each lipid molecule spans the entire membrane.1a~c*d~2c-f This contrasts with the behavior of more conventional, monopolar, twin-tailed lipids that form bilayer aggregates with a head-tail/tail-head arrangement. Because the unusual molecular geometries and resulting aggregate architecture of the archaebacterial lipids might be related to the organisms' high-temperature viability, relevant model studies were initiated. In particular, Yamauchi et al. synthesized several lipids related to 1, e.g., 2, where P i3 a phosphocholine residue.laIc Upon soni(1) (a) Yamauchi, K.;Sakamoto,Y.;Moriya,A.;Yamada,K.;Hosokawa,cation lipid 2 afforded sheetlike MLM, rather than closed T.; Higuchi, T.; Kinoshita, M. J. Am. Chem. SOC. 1990,112,3188. (b) liposomes, although the latter could be obtained from Stewart, L. C.; Katea, M.; Ekiel, I. H.; Smith, C. P. Chem. Phys. Lipids mixtures of 2 and cholesterol or dipalmitoylphosphatidyl 1990, 54, 115. (c) Yamauchi, K.; Moriya, A.; Kinoshita, M. Biochim. Biodws. Acta 1989,1003.151. (d) De Rosa, M.: Gambacorta, A. J. Mol. ~holine.~ B i d . i985,182, 131. We have been interested in the relation between lipid ( 2 ) (a) Fuhrhop, J.-H.; Krull, M.; Schulz, A.; MBbius, D. Langmuir molecular architecture and lipid dynamics, particularly 1990.6.497. Ib) Yamauchi. K.: Yamamoh. I.: Kinoshita. M. Biochim.
Thermophilic archaebacteria that thrive at 50-80 "C are a source of unusual lipids;' an example, 1, features two glyceryl units, held together by an a,w-diether-bonded biphantanyl alkyl chain. The glyceryl units carry additional, unconnected phytanyl ethers at the Cz and C1f positions and polar head groups (P)that include phosphoethanolamine residues.lc There are also closely related archaebacterial lipids where the C2/C1 isoprenoid residues are so that the lipid molecule is a maclinked covalently,la~c~d rocycle that tethers two polar head groups, Le., a macrocyclic "bolaamphiphile".2 Both macrocyclic bolaam-
Bioihys. Acta'lS88,938,61; (c) Fuhrhop, J.-H.;Fritach, D. Acc. Chem. Res. 1986,19, 130. (d) Fuhrhop, J.-H.; Mathieu, J. Angew. Chem., Int. Ed. E n d . 1984.23.100. (e) FuhrhoD. J.-H.: Mathieu. J. J. Chem. SOC.. Chem. b n m u n . 1983,144.' (f') Okaiiata, Y.f Kunitake, T. J . Am. Chem: SOC.1979,101, 5231.
(3) The monopolar additives are believed to provide "spacers"situated in the "outer" region of the membrane, between the hemimacrocyclic bipolar lipid molecules, that enable the perturbed MLM to m u m e the curvature required for liposome formation.'c*f
0743-7463/91/2407-2415$Q2.50/0 0 1991 American Chemical Society
2416 Langmuir, Vol. 7, No.11, 1991 P-
Letters
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1,2-0-(dibutyl)stannylene-3-O-benzyl-sn-glycerol (71.6 A 3-fold excess of 7, and 1,32-dibromodotriacontane,'~10were then heated in a sealed tube (120-130 "C, 20 h) to give 4, R n.CIeH33 3. Phy = OCOPh 67 % of the l,l'-C32-linked lipid 8.1cJ1 Cetyl bromide etherNonfunctional (NF) series, G = H ; Functional (F) series, G = ification of 8 (NaH/THF, then cetyl bromide, reflux, 60 h) thengave the 2,2'-dihexadecylether, 9, that was purified NO2 by silica gel chromatography (78%).lC Finally, 9 was debenzylated (1atm H2,10% Pd/C, MeOH/THF, 1.5 h) to transbilayer migration, or "flip-flop", in liposomal aggreafford diol 10 in 88% yield (recrystallized with MeOH), g a t e ~ .After ~ chemical differentiation of the outer from identical with Yamauchi's material.lcJ2 the inner head groups of ammonium ion bilayer lipNext, diol 10 was converted to the dibromide 11 (CBr4, o s o m e ~ ,we ~ ~monitored the half-times for flip-flopPhaP, 25 "C, 48 h, recrystallized with EtOAc, mp 35-37 mediated reequilibration of the lipid bilayers, relating the "C, 97 %),andthence to the bis(dimethy1amino)derivative ease of flip-flop to such molecular features as alkyl chain 12 (MezNH/EtOH, 80-90 "C, sealed tube, 18 h, recryshead group structure,4calkyl chain-head group tallized with EtOH/EtOAc, mp 32-34 "C, 88%).Diamine connecting functions,4cand alkyl chain u n ~ a t u r a t i o n . ~ ~ 12 was converted into the desired 5-F or 5-NF by quatWith specific reference to archaebacterial lipids, we benernization with either 4-nitro-3-(bromomethyl)phenyl prepared the bisphytanyl, pseudoglyceryl lipids 3.5 Bizoate13[THF, 25 "C, 6 d, precipitated with ether, recryslayer liposomes constructed from 1:lO 3-F/3-NF blends tallized with 20:l EtOAc/CH2C12,87%, mp 115-116 "C, featured a very low gel-liquid crystal transition temper-25.9" (c = 10, CHC13)] or MeBr (EtzO, 25 "C, 18 ature (?', < 10 "C) and very rapid lipid flip-flop (t1p < 1 h, recrystallized with 4:l EtOAc/CH2Cl2,90%, mp 103min at 12 "C) compared to liposomes drived from the cor104 "C, -22.3' (C = 10, CHCL)]. Both 5-F and responding, unbranched hexadecyl ethereal lipids, 4-F/ 5-NF gave appropriate microanalyses (C, H, N, Br) and 4-NF, where T, = 37 "C, and no flip-flop was observed 'H NMR spectra.14 after 20 min of incubation at 25 "C (t1p 2-4 min at 40 Properties of Liposomes. Covesicles of 1:14 5-F and The contrasting properties of liposomal3 and 4 were 4-NF were prepared by incubating the surfactant blend attributed to disruptive effects of the phytanyl residues' at 50 "C in 10 mM aqueous KC1, adjusted to pH 4 with methyl groups on lipid chain packing and motional HC1, followed by sonication for 5 min (immersion probe, behavior within the liposome.l*~~*~ (6) Yamauchi, K.; Une, F.; Tabata, S.; Kinoehita, M. J. Chem. Soc., Now we turn our attention to the architecture exemPerkin Tram. 1 1986,765. plified by the hemimacrocyclic, bolaamphiphilic, archae(7) Starting with eicosane (Cm)diacid chloride, and followingthe fourbacterial lipid model 2, that readily forms MLM lamellae.lc step chain lengthening procedure of Hiinig,Bwe quantitatively obtained 7,26-dotriacontanedione-1,32-dioic acid. This w a ~esterified (SOClz, Might a similar lipid bridge the bilayers of liposomal4-NF? MeOH), converted to the bistoeylhydrazone diester (TsNHNH?, MeOH, And, thus extended across the bilayer, would the bolaamrefl., 5 h), and then directly reduced (71%) to the 7,26-dimethylene phiphile exhibit restrained flip-flop relative to the conderivative with NaBK4.8 JAH reduction of the diester10 afforded 73% of 1,32-dotriacontanediol (mp 98 "C, lit.10 mp 94 "C). Finally, the diol ventional twin-tailed analogue 4? To answer these queswaa converted to the desired 1,32-dibromodotriacontane(47% aqueous tions, we prepared bolaamphiphiles 5-F and 5-NF (Scheme HBr, 140-160 "C, sealed tube, 7 h, 89%). I), modeled after Yamauchi's lipid, 2,lC but bearing (8) Hihig, S.; Liicke, E.; Brenninger, W. In Organic Syntheses Collectiue Volume 6; Baumgarten, H. E., Ed.; Wiley: New York, 1973; pseudoglyceryl ammonium ion head groups analogous to pp 533f. those of 4-F and 4-NF. Here we describe the surprising (9) Caglioti,L. In Organic Syntheses Collectiue Volume 6;Noland, W. E., Ed.; Wiley: New York, 1988;pp 62f. dynamic properties of 5-F/4-NF and 5-F/5-NF liposomes. (10) Furukawa, A.; Shoji, H.; Nakaya, T.; Imoto,M. Makromol. Chem. Preparation of Lipids. Lipids 4-F and 4-NF were 1987,188,265. available from a previous study.& The preparations of (11)If the reaction temperature exceeda 140 "C, the isomeric 1,2'-Ca2 lipid forms as an impurity. 5-F and 5-NF followed Yamauchi's methodology for lipid (12) The 200-MHz 1H NMR spectrum, mp (60-63 "C), and optical 2 until the last several steps; cf. Scheme I. Thus, D-manrotation ([a]+ -8.1" (c = 10, CHCl3)) were in good agreement with the nitol was converted in five steps and 42% overall yield to literature descriptions.1' CH-
I
OPhy
CH-
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20 min at 30 "C, 5 min at 35 "C, 2 min at 40 "C, and
