Langmuir 1992,8, 1509-1510
1509
Stabilization and Facilitated Formation of a /3-Structure Polypeptide by a Poly(L-glutamic acid)-Functionalized Monolayer on Water Nobuyuki Higashi, Mutsuhiro Shimoguchi, and Masazo Niwa' Department of Applied Chemistry, Faculty of Engineering, Doshisha University, Kamikyo-ku, Kyoto 602, Japan Received February 24, 1992
In this communication, we describe unprecedented conformational changes of poly(L-glutamic acid) (PLGA) assembling at the air-water interface. On the basis of the results from spreading experiments and circular dichroism measurements for the freshly prepared amphiphile, which consists of a PLGA segment (polymerization degree, 40) and two long alkyl chains, it was found that the stable &structure of PLGA could be produced specificallyby assembling the PLGA segment at the air-water interface and even under an acidic pH, ambient temperature. The conformation of the PLGA segment could be readily controlled by changing the monolayer phase. Ionic polypeptides such as poly(L-glutamicacid) (PLGA) have been widely known to show conformational transitions (helix-random coil) induced by changing pH, ion strength, solvent composition, and temperature, as well as by the addition of chemical substrates.' Only a few reports have been appeared, however, on formation of the @-structurePLGA.2*3Generally, the @-structureformation would require a particular, harsh condition (e.g., high temperature, mechanical stress, etc.) even if PLGA can exist in @-conformation. Thus, such a conformation for PLGA may not be primarily so stable. Many fibrous structural proteins have been shown to exist in the @-structure.In addition, other proteins such as enzyme have regions in the @-structure. Thus the importance of studying model @-structuresis obvious. We now report conformational changes, in particular, formation of a stable @-structure,of PLGA assembling at the air-water interface. Surface monolayers are a useful tool for ordering molecules two-dimensionally. By the use of it, water-insoluble poly(a-amino acids) have been spread on water and revealed their conformational properties on the basis of surface pressure (+area ( A ) isotherm^.^ Recently, water-soluble biopolymers, e.g., protein? and synthetic polymers6y7were organized through their specific binding to the monolayers from bulk aqueous phase. Amphiphiles of polyelectrolytes or hydrophilic polymers connected covalently with long alkyl chains could be also aligned a t the aipwater interface.8 These polymer assemblieshave provided characteristics uniquely different from those in homogeneous media. As part of our research into the polymer assemblies, we have prepared a doublechain amphiphile containing a PLGA segment (1) and describe ita conformational behavior on water in this communication. A similar amphiphilic compound has (1) Fasman, G. D. In Poly-a-Amino Acids; Fasman, G. D., Ed.; Marcel Dekker: New York, 1967;p 499. (2)Itoh, K.; Foxman, B. M.;Fasman, G. D.Biopo1ymers 1976,15,419. (3)Lenormant, H.; Baudras, A.; Blout, E. R. J.Am. Chem. SOC. 1958, BO, 6191. (4)(a)Malcolm, B. R. Polymer 1966,7,595. (b) Malcolm, B. R. h o c . R. SOC.London, Ser. A 1968,305,363. (5)Ahlers, M.; MiiUer, W.; Reichert, A.; Ringsdorf, H.; Venzmer, J. Angew. Chem., Znt. Ed. Engl. 1990,29,1269. (6)Shimomura, M.; Kunitake, T. Thin Solid Films 1985, 132,243. (7)(a) Niwa, M.; Mukai, A.; Higashi, N. Langmuir 1990,6,1432.(b) Niwa, M.; Mukai, A.; Higash, N. Macromolecules 1991,24,3314. (8)(a) Higashi, N.; Shiba, H.; Niwa, M. Macromolecules 1989,22,4650. (b) Higashi, N.;Siba, H.; Niwa, M. Macromolecules 1990,23,5297.
been used in terms of aggregation morphology in an aqueous bilayer state. C18H37
N' CB t H',
CH,CH2CH,NH~Co-~~-~~~~
$4 FH2
1 (n=40)
COOH
The PLGA amphiphile, 1,l0was prepared by polymerization of y-benzyl L-glutamate N-carboxylic anhydride (BLG-NCAll) initiated with the primary amino group of 3-(N,ZV-dioctadecylamino)propylamine(DOPA)12and then by removal of benzyl groups. The resulting amphiphile consisted of two long alkyl chains (C1BH37) as the hydrophobic part and the PLGA segment as the hydrophilic part whose polymerization degree ( n ) was 40. The monolayers were obtained by spreading a benzene solution of 1 (about 1mg mL-l) on purified water (Milli-Q system, Millipore Ltd.). Thirty minutes after spreading, the monolayer was compressed up to a certain surface pressure and then expanded continuously with a rate of 1.20 cm2 s-l.13 Wilhelmy's plate method and a Tefloncoated trough with a microprocessor-controlled film balance, FSD-50 (San-Esu Keisoku, Ltd., Fukuoka) with a precision of 0.01 mN m-l were used for surface pressure measurements. (9) (a) Yamada, K.; Ihara, H.; Ide, T.; Fukumoto, T.; Hirayama, C. Chem. Lett. 1984,1713. (b) Ihara, H.; Fukumoto, T.; Hirayama, C.; Yamada, K. Polym. Commun. 1986,27,282. (10)A chloroform solution (140mL) of BLG-NCA (3.1 g, 11.8 "01) was mixed with a chloroform solution (10mL) of DOPA12 (0.28 g, 0.5 "01) at room temperature, and the mixture was stirred for 1h. Then the reaction mixture was poured into a large excess of cold hexane, and the precipitated product was filtered off and dried. The number-average degree of polymerization (n)of the PLGA segment waa determined by lH NMR; the average number of amino acid residues was calculated by using the area ratio of the signal of o-CHs of the alkyl chains to that of benzyl CH2 in poly(y-benzyl L-glutamate) segment, aaauming the quantitative reaction of the primary amino group of DOPA. Finally, the polymer thus obtained was treated with HBr/CHaCOOH to remove the benzyl group, giving 1. (11)Fuller, W.D.; Verlander, M. S.; Goodman, M. Biopolymers 1976, 15,1869. (12)DOPA was prepared as follows. First, 3-(N,iV-dioctadecylamino)propanenitrile was synthesized by Michael addition of dioctadecylamine with acrylonitrile, and then the product 3-(N,iV-dioctadecylamino)propanenitrile thus obtained was reduced with LiAlHl to give DOPA. The structure of the final product was confirmed by lH NMR and elemental analysis. (13)Below this value (1.20 cm2 the effect of compression or expansion rate on the monolayer area was within experimental error.
0743-7463/92/2408-1509$03.00/00 1992 American Chemical Society
Letters
1510 Langmuir, Vol. 8, No. 6,1992 I
(+)-
=-E
- \
\
L
1
2
Area (nm2
3
4
molecule")
Figure 1. Surface pressure-area isotherms for the compressionexpansion cycle of (a) 1 and (b) DOPA on water at 20 "C,pH 3.9 and compressed or expanded at 1.20 cm2s?.
Figure 1shows the F A isotherm for the compressionexpansion cycle of 1 at pH 3.9, 20 "C. A considerable difference is observed between the compression curve and that for expansion. On the compression isotherm, at low surface pressure, as expected, molecules lie on the water surface, occupying a large area. With increasing surface pressure, they begin to associate and orient their hydrophobic tails and/or polymer segments. Following a mesophase, there is a transition to a condensed phase at a transition pressure of ca. 30 mN m-l. On the other hand, in the expansion process the P A curve causes the disappearance of such a meso-phase and provides a condensed phase even at low surface pressure. When the same cycling experiment on the r A isotherm was undertaken for DOPA without the PLGA segment, no hysteresis effect was observed. These results strongly suggest that a tight aggregation of the PLGA segments occurs within the 1 monolayer upon compression to the condensed phase and, consequently, brought about the compression-expansion hysteresis. Subsequently, we employed circular dichromism (CD) spectroscopy to reveal Conformational behaviors. A CD spectrum (JASCO 5-720) of an LB (LangmuirBlodgett) film14J5transferred at pH 3.9 and a surface pressure of 15 mN m-l, a t which the monolayer is in a liquid analogue phase, exhibits the two negative bands at 222 and 208 nm (Figure 2.1), indicating the formation of a-helical conformation, in a similar manner to pure PLGA in acidic pH16and in the film.17 The same CD pattern is also observed for the LB film transferred at 25 mN m-l in a meso-phase (Figure 2.2). When aggregation of ahelical PLGA progresses in acidic pH, CD spectra have been found to show red shifting of the 222-nm band toward 225 nm and progressive flattening of the 208- and 193-nm bands.18 In our monolayer, however, no appreciable variation of the a-helix CD pattern is observed at least in liquid and meso-phases, suggesting a relatively weak interaction between the PLGA segments of 1 in these monolayer phases. In fact, when we examined the *-A (14) LB films of 1 were transferred in the vertical mode at various surface pressures (15-35 mN m-1) and a transfer rate of 10mm min-l onto quartz plates. The transfer ratio (+15%) was 1both in the down-stroke and in the up-stroke mode. (15) The CD spectrum of the LB film was measured by putting the quartz attaching the film with four layers into a quartz cell (path length 10 mm) filled with a buffer solution of a prescribed pH. (16) Holzwarth, G.;Doty, P. J. Am. Chem. SOC. 1965,87, 218. (17) Fasmann, G.D.;Hoving, H.; Timasheff, S. N. Biochemistry 1970, 9, 3316. (18) Maeda, H.; Kato, H.; Ikeda, S. Biopolymers 1984, 23, 1333.
(-)I,
, 200
W 1
220
I
240
A.(nm)
Figure 2. Circular dichroism spectra of the LB f i h s of 1, transferred at a prescribed surface pressure, at pH 3.9, 20 OC. Numbers correspond to those in Figure 1.
isotherm for a shorter compression-expansion cycle, Le., the monolayer was compressed up to a surface pressure of 25 mN m-l and then expanded, no hysteresis loop appeared (not shown here). On the other hand, in a condensed monolayer phase, for example, at a surface pressure of 35 mN m-l, the CD spectrum of the LB film gives a trough a t 217 nm, a crossover at 207 nm, and a positive peak at 195nm (Figure 2.3). This spectral feature is very similar to that of the @-formpolypeptides.8J9 Thus, the PLGA segment in the present monolayer is considered to be mainly in the 8structure, although minor existence of a-helixand random coil may not be excluded. As is known, the &structure of polypeptides derives from intermolecular interactions between them through hydrogen bonding, while the intramolecular hydrogen bonding causesprincipally a-helical conformation. Thus, by compressing the monolayer to a condensed phase, the PLGA segments aggregate tightly and then the intramolecular hydrogen bonding is likely to be rearranged to the intermolecular one, which induced the conformational change of PLGA segments from ahelix to @-structure. Further, such a conformational change corresponds well to the phase change in the monolayer based on the F A isotherm (Figure 1). The CD spectrum (Figure 2.4) of the LB filmtransferred at a surface pressure of 20 mN m-l in the expansion process exhibits the same &structure pattern as the condensed LB film (prepared at 35 mN m-9 dose, as is expected from the hysteresis loop of Figure 1. This implies that once the /%structure is formed in condensed phases, it cannot be readily transformed to other conformations by expanding the molecular area. The CD spectrum of the LB film transferred at a condensed phase (35 mN m-l) showed again the @-structurepattern even when the film was exposed to acidic and alkalic solutions for 2 days. In conclusion, the stable @-structureof PLGA can be produced by assembling the PLGA segment connected with long alkyl chains at the air-water interface. a-Helical PLGA has been found to be converted to the @-structure in a gel state2 and in other media,18 although such a transformation must require a considerably high temperature and the presence of some chemical substrates. It is noteworthy, however,that the stable @-structureformation was observed for surface monolayer and under a mild condition. (19) Greenfield, N.;Fasman,G. D.Biochemistry 1969,8, 4108.
