Langmuir 1990,6, 1295-1300
1295
Quinone-Containing Amphiphiles and Bolaform Amphiphiles That Form Redox-Active Lipid Membranes Jurgen-Hinrich Fuhrhop,’ Hartmut Hungerbuhler, and Ulrich Siggel Institut fur Organische Chemie, Freie Universitat Berlin, Takustrasse 3, 0-1000 Berlin 33, West Germany, and Max Volmer Institut der Technischen Universitat Berlin, Strasse des 17 Juni, 0-1000 Berlin, West Germany Received October 12, 1989. In Final Form: February 5, 1990 Amphiphiles containing quinone moieties were developed in order to fix electron acceptors at the outer surface or in the center of vesicle membranes. A double-chain amphiphile with sulfonated quinone head group formed stable vesicles. Sodium borohydride reduced only their outer surfaces. A single-chain bolaform amphiphile (bolaamphiphile)gave unsymmetric monolayered vesicles where all the quinone head groups were located on the outer surface. A dicationic bolaamphiphile with a non-polar quinone in the center was localized in the center of a negatively charged dihexadecyl phosphate vesicle membrane. Some similar membrane systems, where the quinone rings remained mobile, are also shortly described. It was also found that a semiquinone of a macrocyclic bolaamphiphile containing two benzoquinone moieties was long-lived in vesicular membranes.
Introduction Quinones, which are integrated into membrane proteins, play an important role as electron acceptors in lightinduced biological charge separation processes. I t is thought that rigid fixation of the quinone moiety before and after the electron transfer is important to achieve the observed fast forward and slow back reactions.’** We have been studying for some time the fixation of two-headed bolaform amphiphiles, so-called bolaamphiphiles, within monolayer and bilayer lipid membranes of synthetic vesicle^.^ Here we report on newly synthesized quinoid amphiphiles and bolaamphiphiles and attempts to localize the quinone rings either exclusively on the outer surface of the vesicle membranes, as a model for photosynthetic electron acceptors, or in the center of vesicle membranes, as a model for pool quinones.
Polar Quinones as Head Groups Amphiphiles which produce stable vesicle membranes usually contain two oligomethylene chains with a t least 12 carbon atoms each. Such amphiphiles are waterinsoluble enough to guarantee low critical concentrations of vesicle f ~ r m a t i o n .We ~ chose the lactone5 1 as starting material for double-chain amphiphiles. It was converted to the membrane-forming quinone mixture 2 by amidation with dioctadecylamine (70% yield), oxidation with tetrachlorobenzoquinone (TCQ; 70 % ), Michael addition of hydrogen sulfite at pH 4 in 2-propanol-water 1:lO (40%), and another oxidation of the resulting hydroquinone with TCQ (70%). The overall yield was only about 1596, but several grams of 2 were routinely prepared. Sonication of an aqueous suspension of 2 produced a transparent red vesicular solution. The entrapment volume was about 1?6 (pyranine),e and typical vesicle diameters ranged from 30 t o 100 nm (Figure l a ) . T h e phase (1) Deisenhofer, J.; Epp, 0.; Miki, K.; Huber, R.; Michel, H. J. Mol. Biol. 1984,180, 385. (2) Witt, H. T. Biochim. Biophys. Acta 1979,505, 355. (3) Fuhrhop, J.-H.; Fritsch, D. Acc. Chem. Res. 1986, 19, 130. (4) Fendler, J. H. Membrane Mimetic Chemistry; Wiley: New York, 1982. (5) Snell, J. M.; Weisaber er, A.
(6)Kano, K.; Fendler, J.
J. Am. Chem. SOC.1939,61, 450. 8.Biochem. Biophys. Acta 1978,509, 289. 0743-7463/90/2406-1295$02.50/0
transition’ of the bilayer membrane was 58 f 1 “C.The quinone absorption (A, = 444 nm) hardly changed during this phase transition. If t h e vesicle membrane was dissolved in Triton X-100,a hypsochromic shift to 436 nm was observed. A solution in methanol-acetone 1O:l produced an absorption peak a t 428 nm.
$)sJN l2 Xy = HH :; YX = SOg SO3
0
Addition of a large excess of sodium borohydride to the vesicular solution of 2 reduced 60-7096 of the quinone head groups to hydroquinones; addition of Triton X-100led to immediate quantitative reduction (Figure 2). Borohydride obviously does not diffuse through lipid membranes, and the average diameter (d& of vesicles should be about 40 nm. The high percentage of head-group reduction suggests the presence of small unilamellar vesicles. This is n o t in agreement with t h e findings on electron micrographs (Figure l a ) , which show large, irregular vesicles. Less than 50 5% reduction would be expected for such membrane structures. We therefore assume that a considerable part of compound 2 is present as micelles or “oil droplets”. Figure l a does indeed show several particles which are considerably smaller than the usual lowest limit of 25 nm for vesicles. Another possibility for production of polar quinone head groups consists in the Michael addition of thiols with charged substituents and subsequent reoxidation of the obtained hydroquinone.8 This procedure was applied to synthesize the bolaamphiphile 3, which contains a large succinic monoamide head group on one end and a small carboxyl head group on t h e other. We anticipated formation of unsymmetric vesicle membranes: all the large (7) Trluble, H. Naturwissenschaften 1971, 58, 277. (8) Fieser, L. F.; Turner, R. B. J. Am. Chem. SOC.1947,69, 2335.
0 1990 American Chemical Society
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1296 Langmuir, Val. 6, No. 7, 1990
w&qw-cm ca
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can.4-lmide)
c m 0
aL" I CI
0
(b)
(dl
((1)
(C)
Figure 1. Electron micrographs of vesicles made from pure quinoid amphiphiles by sonication at pH 7-9. Negatively stained with uranyl acetate: (a) the double-chain quinonesulfonate 2 (BLM);(b)the single-chain bolaamphiphile 3 (MLM); (e) the anthraquinone bolaamphiphile 4b (MLM). Containing monolayer ribbons: (d) the macrocyclic bolaamphiphile 7 (MLM).
Figure 2. Spectroscopicchanges accompanying the reduction of a vesicular solution (see Figure la) of the double-chain quinoneamphiphile2(1.5mL;l ~ 3 ~ M ) w i t h 2 0 ~ L oMf 0NaRH,: .1 b. 2Os:c. fiOs.d.900s:e.addition of200pL of IC, Triton N.100.
I
ZM
300
350
Ua
450
Iml
I
Figure 3. Quantitative reduction of the vesicular solution (see Figure lb) of the asymmetric bolaamphiphile3 (-) with an exof borohydride in Tris buffer, pH 8, to give the hydroquinone (- - .).
head groups should be on the outer surface and all the small carboxylate groups at the inside. Such arrangement has so far been described only in one case, namely, an unsymmetrically substituted m a c r ~ l i d e .The ~ yellow aqueous solution of 3 entrapped 0.5% of pyranine, showed vesicles in electron micrographs (Figure lb), and was quantitatively reduced to the hydroquinone by borohydride (Figure 3). Since this reduction occurred within a few seconds and since the vesicles proved to be impermeable for pyranine dyes, we conclude that the large quinone head groups are exclusively located on the outer surfaces of the vesicles. (91(a1 Fuhrhop.J.-H.;Mathieu. J. J Chem. SN.. Chem. Commun. 19RJ. 144. (bt Fuhrhop. J.-H.; David. H.H.:Mathmu. .I.: Liman, U.: Winter. H.d.; Rwkema. E.J . Am. ChPm. Soc. 19% IM. 17%.
.cio. I
x = o 4a X i N-I1 4L!
Bolaamphiphiles Which Fix a Quinone Moiety i n t h e Center of a Host Membrane The anthraquinone bolaamphiphiles 4a,b with positively charged pyridinium end groups were synthesized as possible "pool quinones" with appropriate redox properties. At first we tested 4a,b membrane-forming amphiphiles. Sonication of its aqueous suspensions indeed gave vesicles and monolayer ribbons (Figure IC) in the case of the diamide 4b, whereas the diester 4a partially hydrolyzed. We therefore only worked with the water-stable diamide. Since the pure aqueous suspensions provied to be short-lived, we had to work with host membranes. Negatively charged dihexadecylphosphate (DHP) and positively charged dimethyldioctadecylammonium bromide (DODAB) vesicles10 were prepared in the presence of 1-3% of 4b. If more than 306 of 4b was added to 5 X M vesicular solutions of DHP or DODAB, the anthraquinone would partially precipitate. Reduction with dithionite anions gave the hydroquinone of 4b in both DHP and DODAB vesicles. In DHP vesicles, a slow pseudo-first-order reaction with TI^ = 41 s (Figure 4a) was observed; in DODAB, the reduction was complete within a few seconds. The reduction was almost quantitatively reversible with molecular oxygen. T h e membrane activity of dithionite is well-known a n d presumably depends on the formation of the S02-radical and its protonation product." Borohydride, on the other hand, has no effect on 4b in DHP vesicles (Figure 4b) or any other anionic vesicles (2 vesicles, tartaric acid amides, etc.). In cationic DODAB vesicles, borohydride first produced the oxanthrone anionL2-" by addition of one hydride anion (Figure 4c, compound 1). This reaction occurred to about 80% within 10 s, when our experimental setup allowed the first spectroscopic measurements. This means that most of the anthraquinone diffuses rapidly to the vesicular surface. The reaction is complete within about 2 min and is followed by a rearrangement" to t h e hydroquinone d i a n i ~ n , ' ~which ' ~ takes about 5 min (Figure 4c,compound 2). Since the pk. value of comparable anthrahydroquinones lies above ll,I4 the local pH a t the vesicular surface must be much higher than 9.5, the hulk pH. The monoamide 5 was reduced by borohydride under identical conditions. In DODAB vesicles, it was fully reduced to the hydroquinone dianion within 70 8, which is only about 2 times faster than the bolaamphiphile 4b. Furthermore, the monoamide 5 was also reduced by borohydride to the (10) Lukae. S.; Perovie, A. J. Colloid Interface Sei. 1985,103.586. (IO) (11) Ulrieh, E. L.; Girvin. M. E.; Cramer. W. A.: Markley. J. L.
Biochemistry 1985,24, Biochemzstrv 1985.24.2501. (12) cristh. (12) Cristol. S. j J..A&. Ace. Chem. R Res. ~ S 1971,4.393. 1971,4. . 393. (13)Carlson, S. A.; Hercules. D. M.A w l . Chem. 1913,45,1794. (14) Bredereck, K.;Sommermann.F.; Diamsntoglau, M. Chem. Ber. 1969,102,1053. (15) Broadbent, A. D.: Zollinger. H.Helu. Chim. Acto 1964,47,2140. (16) Clark, K. P.;Stonehill,H.I. J. Chem. Soe., Faraday Trans.I 1972, 68. 1616. (17) Hocking. M.9.: Bolker, H.1.; Fleming. B. 1. Con. J. Chem. 1980, 58. 1983.
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T h e anthraquinone 4b was also dissolved in t h e negatively charged vesicle membrane made of the quinone amphiphile 2. I t was anticipated that the electron transfer from the bulk solution to the membrane's center might perhaps be catalyzed by quinoid head groups. This, however, was not the case. The anthraquinone was again inaccessible to borohydride, even if the benzoquinone moieties on the vesicle's outer surface were quantitatively reduced to the corresponding hydroquinones. We also attempted fixation of dianionic bolaamphiphiles containing a central quinone moiety in vesicle membranes. The hydroquinone dicarboxylate 6 was synthesized and dissolved in water a t pH 9 (Am= = 295 nm). Oxidation with potassium persulfate gave the quinone (Am= = 248 nm). A relatively intense broad band at 370 nm was also observed, which may indicate some quinhydrone.le The low solubility of 6 and its derivatives in water a n d vesicle membranes prevented further investigations. 0
0.80
i-
h
I ZM
380
320
440
0
260
320
380
440 fNn]
(c> Figure 4. (a) Redox titration of the anthraquinone 4b (-) (5 X 10-6 M)in anionic DHP vesicles (5 X 10-3 M)with dithionite under nitrogen to anthrahydroquinone (- - - -). The insert gives the half-logarithmicplot of the slope of the 358-nm band vs time. (b)No reduction of4b is observed in DHP vesicles,if 50-fold excess of sodium borohydride is used as reductant. (c) 4b dissolved in cationic DODAB vesicles (-) is first reduced to the oxanthrone anion (1) and then to the hydroquinone dianion (2) (- - -) by borohydride.
anthrahydroquinone in negatively charged DHP vesicles. The total reaction time was about 2 min. From these results, we conclude that the anthraquinone 4b within a dicationic bolaamphiphile is only fixed in t h e center of t h e bilayer lipid membrane, if t h e membrane bears a negative surface charge. The inertness of the anthraquinone moiety against borohydride as evidenced by Figure 4b is not caused by t h e lower concentration of borohydride on DHP-membrane surfaces but by the immobility of the anthraquinone ring, which is held in position by its amphiphilic substituents.
I
The presumably stiffer macrolide 7 was then prepared and investigated. Such macrolides are available in good yields because the a,w-diolsused as starting materials tend to aggregate and thereby preform the macrocycle. In our case, the dimerization of the commercial hydroquinone diol 8 was, however, hindered by the methyl substituents, and we obtained the small macrocycle 9 as the major product. The corresponding large macrocycle 10 was only obtained in 13% yield (Figure 5). The macrolide 7 was found to be very soluble and did not hydrolyzegb in DHP and DODAB vesicles. In both vesicles, it could be reduced by sodium borohydride to the hydroquinone a t p H 7. T h e carboxylate groups are obviously not acidic enough to fixate the bolaamphiphile. Protonation may occur, and the quinone then diffuses to the vesicle surface. Interestingly, it was found that the macrolide 7 formed vesicles by itself on sonication (electron micrograph, see Figure Id). In these "pure" vesicles, the quinone moiety could be readily reduced to the hydroquinone with dithionite at pH 7 (Figure 6a) or borohydride, but reoxidation with persulfate did not occur (Figure 6b). Reduction with dithionite or irradiation with visible light a t pH 12 produced the semiquinone anion radical (Amax = 320,370, 411, and 436 nm19), which was stable for several hours under nitrogen gas (Figure 6c). The equilibrium 2Q'- = Q A2- lies on the left side.20 It is usually found that in alkaline solutions the quinone is rapidly and irreversibly
+
(18)(a) Staab, H. A.; Rebafka, W. Chem. Ber. 1977, 110. (b) Staab, H. A.; Herz, H. P. Angew. Chem. 1977,89. (c) Staab, H. A.; Whlmg, A,; Krieger, C. Liebigs Ann. Chem. 1981, 1052. (d) Staab, H. A.; Starker, B.; Krieger, C. Chem. Ber. 1983,116,3831. (19) Harada, Y.; Inokuchi, H. Mol. Phys. 1964,8, 265. (20)Diebler, H.; Eigen, M.; Matthies, P. 2.Naturforschung 1961,16E, 629.
Fuhrhop et al.
1298 Langmuir, Vol. 6, No. 7, 1990 B OH
HO
OH
HO
s
OH
HO
e
Lp
Figure 5. Molecular model for the precursor formation of macrocycle 7. In the most probable conformation of diol 8, the long side chain will avoid the hydroxyl groups, and the methyl groups will be gauche. 8 will approach the diacid intermediate, made of diol 8 and maleic anhydride, only with difficulty. Intramolecular transesterification (path B)will become more important than the double esterification (paths A(1) and A@)) with another diol 8. The products are 9 and precursor 10, with half the yield of 9. destroyed by hydroxyl anion^.^^^^^ In our case, the vesicle membrane and the steric hindrance of the methyl group obviously protects the quinone against hydroxyl ion attack, and the semiquinone is long-lived. The ESR spectrum of the semiquinone of 8 dissolved in sodium hydroxide (pH 12) consists of a simple triplet (Figure 6d, curve 2; a = 5.72 MHz), which is caused by the two identical benzene protons. In the vesicular solution of the semiquinone of 7, one observed in addition to this triplet six signals (Figure 6d, curve 1). The origin of the additional hyperfine splittings was evaluated by ENDOR s p e c t r o ~ c o p y(Figure ~~ 6e). The original line pair is split into three pairs (a = 6.54,5.64, and 4.96 MHz). This should indicate an unsymmetrical ion pair splitting by sodium cati0n.~4There was no hydrogen bridge involved since no temperature or deuterium exchange effect occurred. An additional smaller coupling (a1 = 2.14 MHz) is possibly due t o the methylene groups, which apparently have no freedom of rotation.25 It was also shown by computer simulation that only one of the four methylene protons coupled with the semiquinone's single electron.
Conclusion and Outlook Membrane-insolule reductants such as borohydride can be used t o convert symmetric vesicle membrane with quinoid head groups into unsymmetric membranes with hydroquinones a t the outer and quinones at the inner vesicle membrane surfaces. Such membranes are stable but cannot be used in light-induced transmembrane charge (21)Siggel,U.; Hungerbaler, H.; Fuhrhop, J.-H. J . Chim. Phys. 1987, 84,1055. (22) Fuhrhop, J.-H.; Krull, M.; Schulz, A.; Mobius, D. Langmuir 1990, 6,497-505. (23) Kurreck, H.; Kirste, B.; Lubitz, W. Angew. Chem. 1984,96,171. (24) Lucken, E. A. C. J . Am. Chem. SOC.1964,86,4234. (25) Das, M. R.; Conner, H. D.:Leniart, D. S.: Freed. J. H. J.Am.Chem. SOC.1970, 92, 2258
separation, since t h e photochemically oxidized chromophore on the outer surface would immediately be reduced. The bolaamphiphile which gives membranes with an electron-accepting outer surface and an inert inner surface, e.g., 3, are considered as useful starting material for charge separating membranes. We shall now try to fix positively charged porphyrins at the inner membrane surface and to measure its electron-transfer rates to the membrane's surface. Several variations of t h e bolaamphiphile membranes using different head groups and counterions (one-sided precipitations) can be envisioned. Of particular interest is the possibility of introducing various synthetic pores into the ultrathin monolayered membranes3 and using the monolayered vesicle t o entrap redox-active colloids.26 Related to pool quinones, we have described already the light-induced charge transfer between cationic porphyrins dissolved in the bulk water volume and the cationic anthraquinone bolaamphiphile 4b in cationic DODAB and anionic DHP vesicles.21 The half-life of the excited porphyrin triplet state was 200 p s in the case of DHP vesicles and 70 pus in the case of DODAB vesicles, although selfquenching of the porphyrin triplet and other side reactions should be more important on the negatively charged DHPvesicle surface, which adsorbed the porphyrins much tighter than the DODAB vesicles did. The difference in redox quenching rates, although not very important, is in qualitative agreement with our assumption of quinone fixation, as deduced from the inaccessibility to borohydride. Further work using DHP and anionic porphyrins with a hydrophobic chain is in progress. We shall also "activate" the membrane with polyene bolaamphiphiles based on the natural product bixin, which we described recently.22 Another interesting possibility comes from t h e unexpected longevity of a charged, highly reducing species in hydrophobic membranes, namely, the semiquinone of the macrocycle 7. This entity will be very useful in tests for the efficiency of ion-conducting pores.
Experimental Section Materials. Elemental analyses and spectroscopic data (UVvis, IR, 1H NMR, 13C NMR, MS) of new compounds were alway satisfactory and according to expectations. They are fully published in a Ph.D. thesis27 and given here occasionally. Important UV-vis spectra are reproduced in the text. Extinction coefficients are, however, not indicated there, because they were found to be badly reproducible (120%) in vesicular solutions. Sodium 1-[(2-(Dioctadecylamino)-2-oxoethyl)thio]-3,6-
dioxo-1,4-cyclohexadiene-4(5)-sulfonate(2). The precursor 1 (6-hydroxy-4-thiachroman-2-one)6 was obtained from 20 g of mercaptoacetic acid (0.217 M) in 20 mL of ethanol by slow addition of 1.23 M benzoquinone in acetone at 60 OC. The product was distilled at 160 "C and 20 Torr. The solid distillate 1 was recrystallized twice from toluene: yield 3.1 g (46%); mp 171 "C. Next 1.5 g of lactone 1 (8.62 mmol) and 4.5 g of dioctadecylamine (8.62 mmol) were refluxed in 70 mL of absolute toluene for 7 h. The solvent was removed and the residue recrystallized twice by addition of acetonitrile to a refluxing dichloromethane solution: yield of 2-[ (2,5-dihydroxyphenyl)thiol-NJV-dioctadecylacetamide:4.5 g (74%);mp 50 O C . This hydroquinone (2.9 g) was oxidized with 1.0 g of chloranil in 200 mL of chloroform at 40 "C to yield the corresponding quinone. It was chromatographed on silica gel (CH2C12/CaHsOH = 100: 1): yield 2.1 g; mp 53 "C. The quinone w a then ~ sulfonated with sodium disulfite in water/2-propanol (1O:l) under nitrogen. In(26) Fuhrhop, J.-H.; Henne, B. Stable Manganite Colloids Coated by a Bolaamphiphile Lipid Membrane. In preparation. (27) Hungerbuhler, H. Ph.D. Thesis, FU Berlin, 1989.
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1 31
125
145
163
a
(d) (e) Figure 6. (a) Electronic spectrum of (1)a 10-3 M vesicular solution (see Figure Id) of the pure macrocyclic bolaamphiphile 7; (2) reduction with 10 pL of 0.05 M dithionite at pH 7; (3) further reduction with 20 pL of 0.05 M dithionite. (b) No oxidation of the hydroquinone occurs with a 50-fold excess of potassium peroxodisulfate. (c) The same reduction at pH 12: a stable semiquinone radical is formed. (d) ESR spectra of the radicals of (1)a vesicular solution of 7 and (2) a monomeric solution of 8. (e) ENDOR spectrum of the vesicular solution of 7 with the hyperfine splitting constants al to u4 (see discussion). soluble salts were filtered off, and the solution was evaporated to dryness. The residue was recrystallized to give 510 mg (42%) of sodium l-[(2-(dioctadecylamino)-2-oxoethyl)thio]-2,5dihydroxybenzene-3(4)-sulfonate. This product (600 mg) was again oxidized in 60 mL of 2-propanol with 280 mg of chloranil at 80 "C. When this mixture cooled to room temperature, a red precipitate was formed which was chromatographed on silica gel = 4:l): 420 mg (70%)of 2 was isoat 1.1 bar (CH&l&H60H lated; m p 164-168 OC. Elemental analysis of C44H7sNNaOsSrH20 (822.23). Calcd: C, 64.27; H, 9.81; N, 1.70. Found: C, 64.03; H, 9.68; N, 2.32. 2-[ (2,4,5-Trimethyl-3,6-dioxo-cyclohexa-1,4-dien-l-y1)thiol-N-( 11-carboxyundecy1)succinamic Acid (3). Product 3 is in fact a complex mixture of four enantiomers and regioisomersat the carbon atoms bearing the sulfur atom. Both the 1-and 4-amides (the 1and 4 positions in the structure of 3) are present in about equal amounts in the product. These regioisomers 3 and 3a have, however, also been separated (see below). T h e racemic m i x t u r e s were n o t resolved. Mercaptosuccinic acid (1.0 g (6.66 mmol)) and 2.0 g of trimethylbenzoquinone (13.32 mmol) were dissolved in 60 mL of ethanol and stirred at room temperature for 10 h. The solution was then refluxed for another 3 h, and 8.0 g of FeC1~6Hz0in 20 mL of water was added after 2 h. The excess of trimethylbenzoquinone was removed by steam distillation, and the residual solution was half-saturated with sodium chloride and extracted with ether. The solvents were removed, and the residue was chromatographed at 1.1bar on silica gel (CHzC12/CH&OOH = 100: 8): yield 1.3 g (66 % ) of [ (2,4,5-trimethyl-3,6-dioxo-cyclohexa1,4-dienyl)thio]succinicacid; mp 147 OC. This product was converted quantitatively to the corresponding succinyl anhydride by refluxing in acetyl chloride, mp 127-128 "C. The anhydride (500 mg (1.78 mmol)) was then dissolved in 50 mL of dry dioxane and refluxed for 1h with 385 mg of 12-aminododecanoic acid (1.78 mmol). Chromatography on silica gel at 1.1 bar (CHzClz/CH&OOH = 1O:l) gave 355 mg (40%) of the 1-amide (title compound 3; mp 107-110 "C) and 390 mg (44%) of the 4-amide 3a (mp 94-99 "C). The only major difference in
the 1H NMR spectra was a chemical shift difference for the doublet for the methine proton next to the sulfur atom. It was at 6 = 4.18 ppm for 3 and 4.50 for 3a. 64(( 1l-Pyridinioundecyl)carbonyl)amino]-9,10-anthraquinone Perchlorate (5) and 2,6-Bis[ (( 1l-pyridinioundecyl)carbonyl)amino]-9,lO-anthraquinonePerchlorate (4b). 2-Aminoanthraquinone (1.0 g, 4.48 mmol) was dissolved in 60 mL of dry pyridine, mixed with 1.27 g (4.48 mmol) of 11bromoundecanoyl chloride, stirred for 3 h at room temperature, and refluxed for another 4 h. The solvent was removed, and the residue was redissolved in hot methanol and filtered. The methanol solution was evaporated to 50 mL and diluted with 50 mL of hot water. Lithium perchlorate (476 mg, 4.48 mmol) was added and the precipitate filtered. It was recrystallized from methanol to yield 1.82 g (71%) of 5: mp 169 "C. The corresponding double salt with one chloride and one perchlorate anion per two cations of 5 was obtained by heating the perchlorate in 50% methanol with amberlite CG 400 (Cl- form). To obtain 4b, exactly the same procedure was followed, but 4.20 mmol of 2,6-diaminoanthraquinoneand 9.50 mmol of 11bromoundecanoyl chloride were used: yield 2.5 g (63%)of 4b; mp 247-249 "C. The differences between 4b and 5 were reproduced in elemental analyses as well as in the integrals of the lH NMR spectra. The ester 4a was obtained by an identical procedure as for 4b, but 2,6-dihydroxyanthraquinonewas used, and chromatography on silica gel at 1.1 bar (CHC13/CHaOH/CH&OOH = 1:2: 1)was necessary: yield 42%; mp 155-157 OC. 2,5-Bis[N-(11-carboxyundecyl)carbamoyl]hydroquinone (6). 2,5-Dihydroxy-1,4-benzenediaceticacid was obtained by literature methods.28 I t was converted to the corresponding bis-lactone by sublimation at 235 OC in vacuo. The product was crystallized from acetonitrile: mp 282 OC.27 This dilactone (1.35 g, 7.1 mmol) and 3.06 g of 12-aminododecanoic acid (14.2 mmol) were refluxed in 80 mL of dry dioxane (28) Wood, J. H.;Colburn, C. S.,Jr., Cox, L.;Garland, H.C. J. Am. Chem. SOC.1944, 66,1540.
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tanium tip but in a sonicated steam bath. The titanium dust in the presence of a drop of pyridine for 5 h. After standing would otherwise reduce the quinones. overnight, the precipitate was filtered and heated with 0.05 M HCl to 80 "C and again filtered. The solid was redissolved in Gel Filtration. Sephadex G 50 was pretreated for 24 h with hot methanol and cooled. The precipitate was then recrystalthe aqueous eluent; Sephacryl S 1000 was used directly but lized from methanol: yield 3.5 g of hydroquinone 6;mp 173 "C. flushed with water before use. Vesicular solutions (500pL) were The corresponding quinone was obtained by oxidation with applied to 1 X 20 cm columns. Typical BLM vesicles apchloranil in methanol and recrystallization from acetic acid: peared after 4-5 mL of eluate volume, MLM vesicles after 6-8 yield 88%;mp 150 "C. mL. Monomeric dyes, e.g., pyranine, appeared typically after 12 [ (2,2,17,17,19,19,34,34-Octamethyl-12,1~,8,1 1,182,186,25,28mL of elution volume. octaoxo-7,12,24,29-tetraoxa-l,l8-di( 1,4)benzenacyclotetraElectron Microscopy. An Elmiskop 1139, Siemens transtriacontaphane-9,26(27)-diyl)dithio]bis[acetic acid] (7) and mission electron microscope at 40 000-fold magnification was Side Product 9. Commercial (AGFA, Leverkusen) ethyl-2,5used. Copper/palladium grids (Polaron) with collodium/ dihydroxy-6,6,6'bf-tetramethyl-l,4-benzenedipentanoate (3.0 g) carbon films were used. A drop of the vesicular solution was was reduced to the diol 8 (2,5-dihydroxy-t,e,cf,tf-tetramethyl- sucked off the grid with filter paper down to a thin film, and a 1,4-benzenedipentanol)with lithium aluminum hydride in THF. drop of a 1% solution of uranyl acetate was added. This drop Crystallization occurred in toluene, mp 131 "C. Diol 8 (5 g, was again removed with filter paper and the resulting staining 14.38mmol) and 2.9 g of maleic anhydride (29.6 mmol) were refilm dried in a dust-free box. fluxed for 6 h in 200 mL of dry toluene. Another 600 mL of tolReductions and Kinetic Measurements. Oxygen was reuene containing 2.5 g of toluenesulfonic acid monohydrate and moved either in a nitrogen stream or, if the solutions were foam5.0 g of diol 8 was added and refluxed for 3 h with a water conM) and glucose oxidase plus ing, by addition of glucose denser. The solvent was removed and the residual oil chroperoxidase. Borohydride and dithionite solutions were always matographed at 1.1 bar on silica gel (CHC&/CH&OOH = 30: prepared freshly and were never older than 5 min. The refer1). The first fraction contained the small macrolide containing ence cuvette contained vesicular solutions of the same or simione maleic diester unit, which led to the amphiphile 9 (see belar turbidity as the probe. low): yield 3.2 g (25%);mp 158-159 "C. The second fraction Chromatography. Silica gel Woelm 32-63 was used at a nicontained the desired precursor of the large macrolide 10, nametrogen pressure of 100 mbar. ly, two diol molecules which are esterified with maleic acid on both ends: yield 1.65 g (13%);mp 165-170 "C. The above maAcknowledgment. We thank Priv.-Doz. Dr. H. Kirste leic acid macrolide8 (500 mg each) were dissolved in 30 mL of and Dip1.-Chem. Niethammer for measurements and 2-propanol and refluxed for 2 h with 2 mL of an aqueous 0.71 interpretations of ESR and ENDOR spectra. Financial M solution of mercaptoacetic acid (1.42 mmol) and 50 WLof pipsupport from the Deutsche Forschungsgemeinschaft (SFB eridine. The mixture was then neutralized with HCl and evap312 "Vectorial Membrane Processes"), the Forderungorated to dryness. The residue was dissolved in ethyl acetate skommission of the Freie Universitit Berlin, and the Fands and filtered, the solvent was removed, and the residue was crystallized from toluene. The yield of the hydroquinone macder Chemischen Industrie is gratefully acknowledged. rolide precursor of 7 was 450 mg (74%))with no melting point Registry No. 1, 29001-22-7;2 (Y= SO3), 126823-79-8;2 (X (hygroscopic). The bolaamphiphilic quinone macrolide 7 was = SO3), 126823-86-7;3,126823-80-1;3a, 126823-87-8;4a, 126847obtained by oxidation with FeC13.6H20 in acetc acid: yield 99-2; 4b, 126823-88-9;5, 126823-81-2;6,126823-82-3; 7, 126824(c) 256.5 (28 700); MS 74%; mp 52 "C; UV-vis (CH3OH) ,A 23-5;8,126823-83-4;9,126823-84-5;10,126823-85-6;DHP, 2197(FAB;DMSO-glycerol, X.) m/z 1015(100%;(M-H)-) 1H NMR 63-9; DODAB, 3700-67-2; benzoquinone, 106-51-4; dioctadecy8H HI, ~ (CDC13, 270 MHz, ppm) 6 1.13 (m, O C H Z C H ~ C H ~ C lamine, 112-99-2;2- [ (2,5-dihydroxyphenyl)thio]-N,iV-dioctade1.22 (s,CH&, 24 H), 1.57 (m, OCH&H2CH2,8 HI, 1.76 (m, CH2~ Hz, 3 J ~ x cylacetamide, 126823-89-0;2- [(3,6-diox~l,4-cyclohexadienyl)thio]CH&CH3,8 H), 2.67 (dd, COCHAHBCHX,2 J= 17 Nfl-dioctadecylacetamide, 12684800-8;1-[(2-(dioctadecylamino)= 6 Hz, 2 H), 2.93 (dd, COCHAHBCHX,2 J= 17 ~ Hz, ~ 'JBX = 2-oxoethyl)thio]-2,5-dihydroxybenzene-3-sulfonate, 126823-9010 Hz, 2 H); 3.34 (d, SCHDCHECO,~ J D =E 16 Hz, 2 H); 3.53 (d, 3; mercaptosuccinic acid, 70-49-5; trimethylbenzoquinone, 935~ 2 H), 3.80 (dd, COCHAHBCHX, SCHDHECO,2 5 = ~16 Hz, 92-2; [ (2,4,5-trimethyl-3,6~oxocyclohexa-l,4dienyl)thio]succinic 3 J u = 6 Hz, 3 J m = 10 Hz, 2 H), 4.06 (m, OCHzCH2,8 H),6.47 acid, 126823-91-4; [ (2,4,5-trimethyl-3,6-dioxocyclohexa-l,4(s, Chin-H, 4 H), 8.75 (8, COOH, 2 H). dienyl)thio]succinic anhydride, 126848-01-9; 12-aminododeMethods. Vesicle Preparation. The membrane-forming canoic acid, 693-57-2; 2-aminoanthraquinone, 117-79-3; 11host amphiphiles and hydroquinones were dissolved in methabromoundecanoyl chloride, 15949-84-5; 2,6-diaminoanthraquinol, and the sovlent was removed in a flash evaporator. Water none, 131-14-6; 2,6-dihydroxyanthraquinone,84-60-6; 2,5was added and the mixture left standing for about 10 min at 80 dihydroxy-1,4-benzenediaceticacid, 5488-16-4; 2,bdihydroxy"C. Ultrasonication (W 220 F, Heat System; 20 W) at 60-65 "C 1,4-benzenediacetic dianhydride, 30272-74-3; 2,5-dioxo-3,6for 20 min produced slightly opaque vesicle suspensions, which cyclohexadienyldiaceticacid, 126848-02-0;ethyl-2,5-dihydroxywere centrifuged at 3000 rpm to remove titanium particles. Ve6,6,6',6'-tetramethyl-1,4-benzenedipentanoate,85224-86-8. sicular solutions of pure quinones were not produced with a ti-
