Langmuir 1990,6, 497-505
497
Bolaform Amphiphiles with a Rigid Hydrophobic Bixin Core in Surface Monolayers and Lipid Membranes Jurgen-Hinrich Fuhrhop,’ Matthias Krull, Andrea Schulz, and Dietmar Mobiust Institut fur Organische Chemie der Freien Universitat Berlin, Takustrasse 3, 1000 Berlin 33, West Germany Received June 14,1989. I n Final Form: August 17, 1989 Several bolaform amphiphiles (bolaamphiphiles)with a rigid polyene as hydrophobic core have been prepared from bixin. Concentrated solutions or precipitates tend to the formation of colorless polymers in the dark. A phenylenediamine-bridged dimer with carboxylate head groups dissolved in DPPC vesicle membranes promotes migration of borohydride and dithionite ions through the membrane. Entrapped indigodisulfonic acid is reduced to two different “leucoindigo”dyes. Surface monolayers of bolaamphiphiles with a cis-bixin Chromophore show characteristic plateau regions beginning at approximately 1.0 nm2/molecule and with fully reproducible overshoots. Surface pressure/area diagrams are interpreted with the formation and reversible pileup of islands of aggregated molecules lying parallel to the water surface. Surface potential measurements and reflection spectroscopy support this conclusion. Bolaamphiphiles with an all-trans-bixin chromophore only produce steep slopes around 0.3 nm2/ molecule. Plateaus are not found. A first interpretation favors an erect position of these amphiphiles in surface aggregates, but the spectroscopic data are not unequivocal. Bixin derivates with a gluconamide head group were effectively integrated into helical micellar gluconamide fibers, which is demonstrated by induced circular dichroism in P- and M-helices. Bolaform amphiphilic (bolaamphiphilic) molecules with a hydrophobic core and two hydrophilic head groups form vesicles with a monolayer membrane,’ if the hydrophobic core is made of oligomethylene chains. These membranes can be as thin as 1.5 nm and are nevertheless very efficient barriers to ion and electron t r a n ~ p o r t . ~Sev.~ eral natural and synthetic amphiphiles with one hydrophilic and one hydrophobic edge act as ion pores in monolayer membra ne^.^" They form hydrophilic domains and water channels within the membrane. It should, however, also be possible to transport electrons over lipid membranes by reversible redox reactions of the polyene chains as observed in polyacetylene.6 We chose bixin as a possible “electron wire” because we anticipated a reversible “Michael type addition of electrons” to the activated polyene. Bixin (la) is a natural, unsymmetric bolaamphiphile’ from seeds of Bixa orellana? which is commercially available in gram q ~ a n t i t i e s .One ~ head group is a carboxylic acid, the other a carbomethoxy group. The hydrophobic chain consists of a rigid polyene with a cisconfigurated double bond toward the methyl ester end and four methyl groups at regular intervals. The nonsymmetry of the bolaamphiphile can be removed by methylation, hydrolysis, or dimerization via a diol or diamine. It can also be accentuated by one-sided condensation with bulky hydrophilic groups. Furthermore, the cis double bond is “straightened out” (trans isomerized) by iodine ‘Max-Planck-Institut fr Biophysikalische Chemie, Am FaBberg,
3400 Gttingen-Nikolausberg, West Germany. (1) Fuhrhop, J.-H.; Fritsch, D. Acc. Chem. Res. 1986,19,130-137. (2) Fuhrhop, J.-H.; Fritach, D.; Tesche, B.; Schmiady, H. J. Am. Chem. SOC. 1984,1O6,199&2001. (3) Fendler, J. H. Membrane Biomimetic Chemistry; WileyInterscience: New York, 1982. (4) Fuhrhop, J.-H.; Liman, U.; David, H. H. Angew. Chem. 1985,97, 337-338. .. ... ( 5 ) Fuhrhop, J.-H.; Liman, U.; Koesling, V. J. Am. Chem. SOC.1988, -2 IO. - - ,fiA4MA45. ---(6) Calvert, P.Nature 1987,327,371. (7) Fuhrop, J.-H.; Mathieu, J. Angew. Chem. 1984,96, 124-137. (8) Kaner, P.;Jucker, E. Carotinoide; Birkhauser-Verlag:Basel, 1948. (9) Fa.Roth, D-7500Karlsruhe 21,W. Germany.
0743-7463/90/2406-0497$02.50/0
catalysis’O to yield isobixin (2). It has also been found that bixin is more stable against oxidative degradation than @-caroteneor other unpolar carotenoids.” Bixin therefore constitutes a valuable starting material for various bolaamphiphiles which can be Tied within lipid membranes. Its molecular length is about 3.1 nm in the alltrans configuration. Here we report on attempts to introduce “electron wires” into vesicle membranes using derivates of the natural bolaamphiphile bixin (la). Furthermore, the monolayer behavior and the induced circular dichroism of bixin derivatives in helical fibers are described. Results and Discussion A. Syntheses and Properties. Syntheses of bixinamides 3-8 were performed by activation of bixin with chloroethyl formate to form anhydride 9 and subsequent addition of the amine in a one-pot reaction. Glucosy1 ester 6d was synthesized by activation of 6b via imidazolide 6c and reaction with unprotected D-glucose. When bromoethylamine was used to amidate bixin, the intermediate cyclized to the oxazoline 4a, which could be methylated to form the oxazolium salt 4b. The oxazoline 4a gave a characteristic carotene spectrum with two bands at 470 and 500 nm; on protonation or methylation, both bands merged to one broad absorption at 498 resp. 535 nm. Similar shifts have been observed in protonated Schiff bases of retinal.I2 I t is known that oxazolium salts react with amines in a ring-opening reaction.13 Attempts with 4,4’-bipyridine, however, produced ill-defined mixtures of compounds. The bipyridinium compound 3a was accessible by alkylation of 4,4’(10) (a) Zechmeister, L.;Polg61, A. J.Am.Chem. SOC. 1943,65,15221528. (b) Zechmeister, L. Fortschr. Chem. Org. Natur. 1960,18,320330. (11) Isler, 0.; Gutmann, H.; Montavon, M.; Ruegg, R.; Ryser, G.; Zeller, P. Helu. Chim. Acta 1957,40,1242-1249. (12) Navangul, H. V.;Blatz, P. E. J.Am. Chem. SOC.1978,100,43404346. (13) Forestier, A.;Sillion, B. J. Heterocycl. Chem. 1980,17,1381. (14) Diemair, W.;Heusser, D.2. Lebensm.-Unters.-Forsch. 1963, 97,289-296.
0 1990 American Chemical Society
498 Langmuir, Vol. 6, No. 2, 1990
Fuhrhop et al.
bipyridine with bromoethylamine and subsequent condensation of the (2-ammonioethyl)-4,4'-bipyridinium dibromide with the mixed anhydride 9. 20
19
R'
R2
-OH
-0CH3 -0CH3 -OH
-0CH3
-0CH3
-0CH3
-OH \N*N
\=/ O -oo *$
sw0;
CHzOH -0CH,
OH -OH
OH
0
OH
OH
O H ,p$,N )-! ,
OH
0 ,
:
e
N
"
OH
OH
OH
O
H
u 6H
-0CH,
-0CH3
AH
era1 polar compounds (Figure 1) absorbing around 320 nm. A high molecular weight polymer eluted before the dead volume of the column and showed an absorption maximum at 270 nm. In contrast, bixin (la) and also methylbixin (lb) form long-lived, air- and light-stable crystals when crystallized from ethyl acetate. We also obtained colorless polymers when aqueous dispersions of 7a were ultrasonicated. Photochemical polymerizations of crystalline dipolyenes and polyenes have been reported by Hasegawa and Schmidt.18 Compounds 1-9 then served the following purposes: (i) The viologen derivative 3b was to be tried in lightinduced charge transfer. It was hoped (a) that the carotene chain with two electron-withdrawing carboxyl end groups would be relatively stable against irreversible photooxidation and (b) that an intermediate cation radical would be stabilized by interaction of the single electron with the amide group. In our hands, however, the bixin chromophore of 3b degraded rapidly in the presence of a dialkylated bipyridinium substituent, whereas 3a was stable. Lehn mentions similar problems with caroviologens."j (ii) The p-phenylenediamine-bridgeddimers 5a,b were perceived as possible electron wires through bilayer lipid membranes. Its particular attraction is based on the possibility that the electron-deficient double bonds next to the carboxyl group might react as reversible electron acceptor in a Michael-type reaction. The outcome of this idea is discussed in subsection B. (iii) Bolaamphiphiles 6b,d and 7a,b were synthesized in order to produce unsymmetric monolayers at the airwater interface. Electron-conducting monolayers of the Langmuir-Blodgett type might thus become accessible. Related monolayer work is discussed in subsection C. (iv) The bixin derivatives 8a and 8b with open-chain D- and L-gluconamide head groups should finally be integrated into helical micellar fibers of N-alkylaldonamides. This is described in subsection D.
-0CH3
40K0-CHzCH3 0
HO
2 3a showed a typical visible spectrum of monomeric bixin chromophores and was quite air-stable. Attempts to methylate the bipyridinium substituent under various conditions led to decolorization of the bixin chromophore. The same result was obtained when the methyl ester group in the 2-gluconamide 7a was hydrolyzed with methanolic KOH. The phenomenon of bixin decolorization occurred quite regularly, when products were isolated by precipitation instead of crystallization. In the case of methylbixin (lb), synthesized by methanolysis of mixed anhydride 9 and precipitation with methanol from pyridine, we tried to identify aged colorless decomposition products, which formed spontaneously in solid material at 5 "C in the dark. The UV spectrum showed weak absorption bands at 270 and 320 nm, the 'H NMR spectrum contained no signals in the olefinic region, and the mass spectrum gave ill-defined fragments at m / e 97,113, and 129 while characteristic carotene fragments at m / e 92 and 102 (ref 15) were missing. The infrared spectrum contained ester bands but no C=C stretching bands around 1600 cm-'. HPLC with a methanol-phosphoric acid gradient showed sev~
~
(15) Enzell, C. R.;Francis, G. W.; Liaaen-Jensen, S. Acta Chem. Scan. 1968,22,1054. (16) Arrhenius, T.S.;Blanchard-Desce, M.; Dvolaitzky, M.; Lehn, J.-M.; Malthete, J. Proc. Natl. Acad. Sci. USA 1986,83, 5355-5359. (17) Liittke, W.;Klessinger, M. Chem. Ber. 1964,97,2342-2357.
H3C0Y0 I
I
h
B. Bixin Dimer 5 as "Electronic Wire" in Vesicle Membranes. Derivatives la-c, 2, 5a, and 7a were soluble in vesicle membranes and produced the visible spectrum of monomeric polyene solutions (Figure 2a). Monolayer vesicles were too small to entrap a detectable volume of redox-active dye. In order to investigate electron transfer through the vesicle membrane, we entrapped indigodisulfonic acid within DPPC vesicles and reduced it either with dithionite or borohydride in the bulk solution in the presence and absence of the supposed "electron wire" 5b. In aqueous solution without vesicle, both (18) (a) Hasegawa, M. In Organic Solid Chemistry; Desiraju, G. R., Ed.; Elsevier: Amsterdam, 1987; pp 153-177. (b) Schmidt, G. M. J. Pure Appl. Chem. 1971,27,647-678.(e) Lahav, M.;Schmidt, G. M. J. Tetrahedron Lett. 1966,26,2957-2962.
Langmuir, Vol. 6, No. 2, I990 499
Bolaamphiphiles with a Bixin Core
..._
I'
I . . . .
0
5
10
*- ,.
,"mom
~.~
6
I",,"l
Figure 1. HPLC elution profile of aged, decolorized methylbixin (lb) crystals (RP 18; methanol-phosphoric acid gradient). reductants produced "leucoindigos" with different spectra. The dithionite reduction product absorbed at 402 nm, whereas the borohydride reduction product showed two bands at 370 and 475 nm (Figure 2d). Attempts to identify the structure of these two leucoindigos failed. In contrast to textbook notations, the strhctures of leucoindigos are unknown. Spectra have, to our knowledge, only been interpreted with the aid of stable model compounds." Our attempts to isolate any of the dithionite or borohydride reduction products also failed. We therefore use the absorption bands only as indications for the effectiveness of the reagents. If reduction of the entrapped indigodisulfonic acid took place by electron transfer through the membrane, one would predict a single reduction product. If the reductant itself passed the membrane, both should produce the different leucoindigo products described above. In order to test the ability of the bixin chromophore to take up electrons, we also investigated its chemical reactivity under various conditions. The bixin derivatives la, 2, and 7a as well as 5a,b did not react with borohydride or dithionite in aqueous methanol. Attempts to reduce bixin (la) or the dimer 5b electrochemically also failed in both methanolic and DMSO solutions. Both compounds proved to be inert against polar reductants. Only in Triton X-100 micellar solution slow, irreversible decay of the visible absorption and new bands at 410 and 388 nm were observed. This is probably caused by a basecatalyzed polymerization as described above rather than by a reduction. The product was not reoxidizable to a bixin chromophore. The assumption of a "Michael addition of an electron" does, therefore, not hold. Nevertheless, we introduced the bridged dimer into DPPC vesicle membranes and probed for electron transfer. In blank experiments with DPPC vesicles and entrapped indigodisulfonic acid, reduction of the entrapped indigo dye by external dithionite or borohydride was very low. If 1% of phenylenediamine-bridged bixin dimer 5b was added to the vesicle membrane, between 80% and 90% of the entrapped indigo carmine was reduced within less than 1 min (Figure 2b,c). These experiments were fully reproducible several times. Immediate mixing with air reoxidized the leucoindigo. About 70% of the original indigo carmine absorption was recovered. The absorption spectra of leucoindigo again differed with both reductants. In the case of borohydride bands at 370 and 475 nm and in the case of dithionite, a band at 402 nm was observed (Figure 2b,c). The rigid bixin dimer 5b obviously introduces kinks into the flexible DPPC membrane, to allow migration of ions through the membrane. Evidence for a stable U-form conformation of 5b, as shown in Figure 3, comes from the behavior of surface monolayers (see subsection C). A reversible uptake of an electron in the ground state obviously does not take place. No attempts to photoreduce the entrapped dye with extemal reductants have been made, since the dark reaction occurred too fast to be eliminated by the techniques available to us.
I
"
m
Eo,
*I
m
Figure 2. (a-c) UV-vis spectra of DPPC vesicles containing bixin chromophoreswithin the membranes and (b,c) entrapped indigo carmine. (d) Indigo carmine and ita leuco derivativesin water.
Figure 3. Hypothetical arrangement of the bridged diamide 5b in a vesicle membrane, which could produce a water channel for ion transport.
C. Surface Monolayers. Carotene monolayers have been described for retinoic acid,'g retinal,m and canthaxantin.2' The 'head groups" of the polyene amphiphiles have been single carboxyl, hydroxyl, or formyl groups. The reported isotherms su ested an extrapolated molecular area of about 0.5 nm /molecule and a collapse a t 0.4 nm2/molecule. Retinal monolayers collapse a t 15 mN/ m; canthaxanthin is compressible up to 60 mN/m. Here we describe monolayers of "bola"-carotenoids with chargd carboxyl, polyhydroxy, and neutral head groups. It was hoped that some of these amphiphiles would produce monolayers with erected chromophores corresponding to molecular areas around 0.254.30 nm2/molecule. Characteristic data of the monolayers are summarized in Table I. Preparation of monolayers from the dicarboxylic acid le and the viologen derivative 3a failed. These compounds were too soluble in water and/or too insoluble in volatile organic solvents.
9
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(19) Weitzel,G.;Fretzdorff,A.-M.;HeUei,S.Hoppe-Seyler'sZ.Physid.Chem. 1952,290,32-41. (20) Robert, S.: Tancrhde, P.; Sal-, C.; Lebland, R.M. Bwchim.
Biophys. Acta 1983, 730,211-225. (21) Diarra. A,; Hotchandmi, S.; Max, J. J.; Leblanc, R. M. J. Chem. Soc., Faraday Trow. 2 1986,82,2211-2231.
500 Langmuir, Vol. 6, No. 2, 1990
Fuhrhop et al.
Table I. Characteristic Data of Monolayers A, nm2/molecule
la lb 2 5a 5b 6b 6d 7a a
1.00 1.04 0.29 0.80 1.20 0.37 0.43 1.10
e 0.87 0.91 0.17
-0.50 ==0.65 0.24 0.29 0.90
AV, mV
Ap, D
690 690 700 450 500 500 390
1.64 1.60 0.45 1.00 1.47 0.39 0.40
nd"
nd
nd = not determined.
Results are presented in terms of surface-pressure area isotherms and simultaneously recorded surface potentials. Dimethyl Ester lb, Monomethyl Ester la, and Glucosamide 7a. The unsymmetric bixin (la) and methylbixin (lb) produced similar isotherms with plateaus characteristic of bolaamphiphiles. A first low-pressure slope extrapolates to 1.0 nm2/molecule at zero pressure and corresponds to the area of a bixin molecule lying on the water surface with the methylated side. If the methyl groups were parallel to the water surface, a molecular area of about 1.7 nm2/molecule would be occupied. The temperature dependence of both plateaus is unusual: the plateau pressure decreases with increasing temperature. From this temperature dependence, energies for a phase transition of about 40 kJ/mol are calculated22in both cases. The dimethyl ester lb produces characteristic and fully reproducible overshoots at high temperatures, which first broaden out and then disappear at lower temperatures. At 8 "C, the plateau is finally established at the point of inflection of the overshoot (Figures 4 and 5). Both slopes show high compressibilities ( K = 0.022 m/mN). The high pressure slopes extrapolate to 0.37 nm2/molecule at zero pressure. A similar behavior in respect to the plateau and overshoot has been observed for 1,16-bis(acryloyloxy)hexadecane diesters23and some synthetic polypeptide^.^^ The decrease of surface pressure and the simultaneous formation of crystalline islands have been interpreted with a film collapse. It seems plausible that folded and backfolded polymer chains2' behave similar to aggregates of stiff polyene bolaamphiphiles. In both cases, the monolayers of stiff, horizontally oriented molecules are not compressible by vertical folding or uplifting. Decrease of plateau pressure with increasing temperature, but no overshoot, has also been observed with monolayers of a stearic acid derivative bearing an oxazolidine oxide ring on C-12.2e This was explained with a highly temperature dependent hydration of the weakly hydrophilic oxazolidine ring. A change of oligomethylene chain conformations from bent to erect could then be triggered by increasing temperatures. In our case, a similar explanation could apply, if one replaces "bent conformation" by "horizontal Orientation". The constant surface potential over the whole range of the plateau (Figure 4), however, rather suggests that the orientation of the bolaamphiphile does not change (see below, compare with Figure 8). Upon compression to the point of inflection at (22) Glazer,J.; Alexander, A. E. Trans. Faraday SOC.1951,47,401409. (23) Dubault, A.; Casagrande, C.; Veyssie, M.; Caille, A.; Zuckermann, M. J. J . Colloid Interface Sci. 1978,64, 290-299. (24) Malcolm, B. R. J . Colloid Interface Sci. 1985, 104, 520-529. (25) Bovey, F. A. In Macromolecules; Bovey, F. A., Winslow, F. H., Eds.; Academic Press: New York, 1979; pp 317-337. (26) Cadenhead, D. A.; Moller-Landau,F. J. Colloid Interface Sci. 1974,49,131-134.
AV
3
2
- 600
8
Y
b!
-500 50 - 400
40 -
-300 30 -
20-
-0
10-
0 ) 0
I
I
20
40
I
I
I
120
100 6o
140
I
I
160
180
A ;12/moleculel
Figure 4. Surface pressure/area and surface potential/area isotherms of bixin (la), methylbixin (lb), and glucosamide 7a (2' = 23 "C). Both diagrams for la indicate a stable horizontal orientation as sketched.
I 50 40 -
a)
h
3z
E
30-
v
Y
9,lc
20 -
29,lC
10 -
8,2C
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...........
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~
I 10
0
0
10
20
30
50
40
A
60
70
80
90
100 110 120
(A*/molecde)
Figure 5. Temperature dependence of the plateau pressure of bixin (la) (a) and its methyl ester ( l b ) (b).
0.88 nm2/molecule, the pressure remains constant, when the barrier is stopped. The monolayers remain stable and their x / A characteristics upon compression and expansion are fully reversible. At smaller molecular surface areas, the surface pressure decreases slightly. When com-
Langmuir, Vol. 6, No. 2, 1990 501
Boluamphiphiles with a Bixin Core
0,90 , pression is continued, the surface pressure will immediately rise to the same value as that observed on slow, 0,75 continuous compression. Changes in surface pressure must therefore be ascribed to rearrangements of the twodimensional monolayer. e 0,60 The glucosamide 7a with a cis-bixin hydrophobic core 0,45 produced monolayers with a plateau from 0.90 to 0.40 nm2/molecule but with a less constant pressure in the C e 0,30 plateau region. Crystals, which were placed on a clean water surface, 0,15 did not show any detectable equilibrium spreading pressure (ESP);27in other words, the polyene bulk crystals 0 are extraordinarily stable. Within the plateau region of 340 400 450 500 550 600 the * / A isotherm, red stripes are observed in front of wavelength [nml the barrier, indicating density gradients which may occur in rigid films.24 An increasing horizontal deflection of the Wilhelmy-plate during compression in the plateau region also indicates the stiffness of these films. This makes the final pressure value of the high-pressure slope dependent on the system of film balance used. With a Wilhelmy balance, it amounts to 45 mN/m, while 65 mN/ m was observed with a Langmuir balance. Measurements of the surface potential at 1.10 nm2/ molecule yielded a maximal value of the dipole momentz7of 1.6 D. In comparison to the values observed on isobixin (2) this is in agreement with molecules lying flat with two head groups anchored to the water surface. For the dimethyl ester lb, the same dipole moment of 1.6 D was determined. Since the bixin amphiphiles contain chromophores, it is possible to investigate the monolayers by reflection s p e c t r o ~ c o p y .The ~ ~ spectrum of the reflectivity change, AR,with respect to the clean water surface recorded with normal incidence showed a blue-shifted bixin spectrum 0,06 A i with a maximum at 430 nm (Figure 6). Reflectivity e with an angle of incidence of 45” using polarized light showed a maximum at 438 nm with a AR,:AR, ratio 8 of 0.21. The spectra and their angle dependence corre3 2 0,04spond to aggregated chromophores with their transition moments parallel to the water surfa~e.~’”~ 2 Following Fresnel’s equation, AR1/’is proportional to 0,oz the surface layer thickness at wavelengths where the chromophore has no a b ~ o r p t i o n . ~Reflectivity , measure0 , 0 1 2 3 4 5 6 7 8 9 10 ments of bixin monolayers at 600 nm, assuming a refractime [minl tive index of 1.55,31yielded values from
