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Self-assembled peptide fibers from valylvaline bola-amphiphiles by a parallel β-sheet network
Biochimica et Biophysica Acta 1475 (2000) 346^352
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Self-assembled peptide ¢bers from valylvaline bola-amphiphiles by a parallel L-sheet network Masaki Kogiso *, Yuji Okada, Takeshi Hanada 1 , Kiyoshi Yase, Toshimi Shimizu National Institute of Materials and Chemical Research, 1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan Received 18 January 2000 ; received in revised form 4 May 2000; accepted 5 May 2000
Abstract A series of dipeptide-based bola-amphiphiles, bis(N-K-amide-L-valyl-L-valine) 1, n-alkane dicarboxylate (n = 4^12), have been synthesized. The bola-amphiphiles with n = 4 and 6 self-assembled to form crystalline solids in water, whereas those with n = 7^12 produced peptide fibers. FT-IR spectroscopy and X-ray diffraction patterns revealed that the peptide fibers have parallel-type L-sheet networks between the valylvaline units. FT-IR deconvolution study of carboxyl regions indicated that these crystalline solids and peptide fibers are stabilized by interlayer bifurcated and intralayer lateral hydrogen-bond networks between the end carboxylic acid groups, respectively. ß 2000 Elsevier Science B.V. All rights reserved. Keywords : Bola-amphiphile; Peptide ¢ber; L-valyl-L-valine; Self-assembly ; L-sheet; FT-IR spectroscopy
1. Introduction Recent progress in the self-assembly studies of peptide amphiphiles [1^3] has been greatly a¡ected by the pioneer work by Kunitake et al. [4,5]. For instance, dipeptide- [6], tripeptide- [7,8] and polypeptide-containing amphiphiles [9] are known to form unique, well-de¢ned self-assembled morphologies, such as helical ¢bers, rods, and vesicles. In line with this amphiphilic molecular design, peptide agents have recently been designed and synthesized in order to target the dimerization interface of HIV-1 protease [10]. On the other hand, ¢ber formation from peptide amphiphiles has also attracted much attention in terms of the similarity of L-sheet self-assemblies within amyloid ¢brils [11^13]. The creation of protein-like molecular architectures can be realized in the self-assembly of highly ordered polyPro II-like triple-helical structures from peptide amphiphiles with collagen-model headgroups and dialkyl tails [14,15]. Thus, peptide amphiphiles can provide versatile molecular surfaces and architectures favorable for the understanding of biological phenomena.
We have recently reported the sophisticated hierarchical self-assembly of organic microtubes from glycylglycine bola-form amphiphiles (bola-amphiphiles) in aqueous solutions [16^18]. To our knowledge, this ¢nding may give the ¢rst example of supramolecular ¢brous structures driven by pseudohexagonal lattice formation (polyglycine IItype structure [19]) [20,21] of peptide hydrogen-bond networks. Self-assembled morphologies formed from peptide amphiphiles are, thus, strongly dependent on the hydrogen-bonding scheme that also plays an important role in underlying the formation of protein structures. Here we describe the self-assembly of novel synthetic peptide ¢bers with nanometer-scale widths from a series of L-valyl-L-valine bola-amphiphiles 1^9 (Scheme 1). The importance and contribution of intra- or interlayer hydrogen-bond networks is discussed in connection with crystallization and the ¢ber formation of the peptide amphiphiles [22].
* Corresponding author. Fax: +81-298-614422; E-mail : [email protected] 1 Present address: The Institute of Scienti¢c and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka, 567-0047, Japan. 0304-4165 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 0 0 ) 0 0 0 8 8 - X
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Scheme 1.
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2. Materials and methods
2.3. Powder X-ray di¡ractions
2.1. Synthesis of L-valyl-L-valine bola-amphiphiles
All powder X-ray di¡raction (XRD) patterns of dried ¢bers were taken by the re£ection method on a Rigaku di¡ractometer (Type 4037) using graded d-space elliptical side-by-side multilayer optics monochromated CuKK radiation (40 kV, 30 mA) and imaging plate (R-Axis IV).
To a solution of 1,n-alkanedicarboxylic acid (2 mmol) (n = 4^12) and 1-hydroxy benzotriazole (0.674 g, 4.4 mmol) in 10 ml of DMF, a solution of 1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride (0.844 g, 4.4 mmol) in 10 ml of chloroform was added with stirring at 35³C. To the reaction mixture, a solution of L-valylL-valine benzyl ester hydrochloride (1.509 g, 4.4 mmol) and triethylamine (0.62 ml, 4.4 mmol) in 10 ml of chloroform was added after 1 h. The reaction mixture was stirred for further 12 h with the temperature being allowed to increase to room temperature. The reaction mixture was washed with 10% aqueous solution of citric acid, water, 4% aqueous solution of sodium hydrogencarbonate, and water. The organic layer was suspended and dried over anhydrous sodium sulfate. Evaporation of the solvent gave a white solid as a C-terminal protected bola-amphiphile. The benzyl group of the C-terminal was removed by hydrogenation using a Pd/C as a catalyst. The reaction mixture was evaporated to give an oil. Recrystallization from water^ethanol gave a white powder of the bolaamphiphiles 1^9. Analytical data for 1^9 are shown in Table 1.
2.4. FT-IR spectroscopy FT-IR spectra were obtained on a JASCO FT/IR-620 spectrometer. Spectra were measured on a KBr pellet at 4 cm31 resolution after accumulation of 100 scans. Second-derivative plots and curve-¢tting analysis results were obtained by Jasco spectra manager. Tentative baseline was carried out between 1690 and 1770 cm31 . The peak resolution was achieved by non-linear least-squares analysis. During the curve-¢tting analysis, a Lorentz function was used. 3. Results 3.1. Self-assembly of the bola-amphiphiles in water The dicarboxylic L-valyl-L-valine bola-amphiphiles 1^9 are sparingly soluble in water, but become freely soluble by addition of 3 equiv. of alkaline hydroxide. The alkaline aqueous solutions of 1^9 (10 mM, 2 equiv. of sodium hydroxide) were slowly acidi¢ed by vapor di¡usion of 1^ 5% dilute acetic acid into the solutions. However, rapid acidi¢cation of the solutions with a dilute hydrochloric acid (0.1^1 M) immediately produced amorphous solids for all the bola-amphiphiles. After 1^2 weeks, the aqueous solutions of the bola-amphiphiles 1 and 3 a¡orded crystalline solids as a precipitate. The bola-amphiphiles 2 gave no visible assemblies with the naked eye. In contrast, clear hydrogels were obtainable from the aqueous solutions containing the bola-amphiphiles 4 and 6^9, whereas a turbid hydrogel was obtainable from 5. The appearance of the self-assembled structures are listed in Table 2 for 1^9. The pH ranges, at which we obtained these self-assemblies, are also summarized in Table 2. The pH titration measure-
2.2. Energy-¢ltering transmission electron microscopy (EF-TEM) EF-TEM observation was carried out to examine ¢ne ¢ber structures in hydrogels in a similar manner as that described elsewhere [23]. Unstained specimens were prepared by placing a 3-Wl drop of the dispersion on an amorphous carbon supporting ¢lm mounted on a standard TEM grid. The drop was then blotted o¡ with ¢lter paper, followed by dryness in vacuo. Each specimen was examined at 80 keV in the electron spectroscopic imaging by using an analytical electron microscope (Carl Zeiss EM 902) with Castain^Henry type electron energy ¢lter at room temperature. Zero-loss images were recorded on an imaging plate (Fuji Photo Film, FDL 5000) with a 20 eV energy windows at 3000^250 000U and digitally enlarged. Table 1 Analytical data for the bola-amphiphiles 1^9 Bola-amphiphile
Yield (%)
Mp (³C)
[K]D (³)
Elemental analysis (calculated (found)/%)
1 2 3 4 5 6 7 8 9
56 25 69 72 79 75 61 90 77
217 182 193 183 192 183 134 136 116
^a ^ 346.9 ^ 346.1 ^ 339.9 ^ ^
calcd. calcd. calcd. calcd. calcd. calcd. calcd. calcd. calcd.
a
(n = 4) (n = 5) (n = 6) (n = 7) (n = 8) (n = 9) (n = 10) (n = 11) (n = 12)
for for for for for for for for for
C26 H46 O8 N4 , C:57.54 (57.32) H:8.54 (8.61) N:10.33 (10.20) C27 H48 O8 N4 W1H2 O, C:56.42 (56.76) H:8.77 (8.71) N:9.75 (9.64) C28 H50 O8 N4 W2H2 O, C:55.42 (55.57) H:8.97 (9.10) N:9.24 (9.22) C29 H52 O8 N4 W6/5H2 O, C:57.43 (58.01) H:9.04 (9.15) N:9.24 (9.20) C30 H54 O8 N4 W1/5H2 O, C:59.81 (60.07) H:9.10 (9.25) N:9.30 (9.25) C31 H56 O8 N4 W1H2 O, C:59.02 (59.38) H:9.27 (9.29) N:8.88 (8.76) C32 H58 O8 N4 W1/2H2 O, C:60.44 (60.24) H:9.35 (9.27) N:8.81 (8.97) C33 H60 O8 N4 W1/2H2 O, C:60.99 (61.27) H:9.46 (9.49) N:8.62 (8.41) C34 H62 O8 N4 W1/2H2 O, C:61.51 (61.54) H:9.57 (9.74) N:8.49 (8.31)
Not determined.
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Fig. 1. EF-TEM images of the peptide ¢bers self-assembled from (a) 4, (b) 5, (c) 6, and (d) 7.
ment [18] of 7 revealed that the original carboxylate anion is fully protonated at this pH value. Elemental analysis of each dried self-assembly also supported the carboxylic acid forms of the amphiphiles within these self-assemblies. 3.2. EF-TEM of the hydrogels In order to clarify microscopic dimensions of the selfassemblies, we examined the hydrogels from 4^7 using EFTEM. EF-TEM is a powerful tool for observing low-contrast organic and biological samples without staining. EFTEM indicated that the bola-amphiphiles 4^7 form a large number of ¢brous assemblies with lengths of 0.1^10 Wm (Fig. 1). High-resolution EF-TEM images of the same specimens showed that these synthetic ¢bers have the minimum widths of 10^15 nm. However, the morphologies of the ¢bers slightly di¡er from each other (Table 2). The bola-amphiphile 4 forms apparently sti¡ ¢bers (Fig. 1a). In contrast, the bola-amphiphile 5 gives ribbon structures and ¢bers (Fig. 1b). The sti¡ ¢bers similar to those of 4 are also produced from the bola-amphiphile 6, whereas
apparently soft ¢bers from the bola-amphiphile 7 (Fig. 1c and d, respectively). Almost uniform widths of ¢bers are compatible with the clear appearance of the hydrogel from 4, 6, and 7. In contrast, coexistence of the ribbon and ¢ber structures would be responsible for the turbid hydrogel from 5. 3.3. XRD analysis Fig. 2 represents the powder XRD patterns obtained for the isolated and dried self-assemblies of 1 and 3^9. The XRD patterns of the dried crystalline solids from 1 and 3 indicate highly crystalline nature of the self-assemblies, displaying many sharp re£ection peaks. The ribbon structures and ¢bers from 5 also show complex XRD patterns indicative of high crystallinity. However, the absence of any re£ection peaks attributable to long-range ordering in the small-angle region suggests that each bola-amphiphile forms no crystalline multilayer structures within the self-assemblies. This ¢nding presents a striking contrast to that observed for 2-glucosamide-based bola-amphiphiles
Table 2 Self-assembling properties of the L-valyl-L-valine bola-amphiphiles 1^9 Bola-amphiphile
Appearance
Morphologies of ¢ne structuresa
pH Range
1 2 3 4 5 6 7 8 9
precipitate solution precipitate clear hydrogel turbid hydrogel clear hydrogel clear hydrogel clear hydrogel clear hydrogel
crystalline solid ^ crystalline solid sti¡ ¢ber sheet structure and ¢ber sti¡ ¢ber soft ¢ber ¢ber ¢ber
2.7^3.0 ^ 3.4^3.8 3.6^4.0 4.3^4.4 4.3^4.7 4.7^4.9 5.1^5.4 5.3^5.4
a
(n = 4) (n = 5) (n = 6) (n = 7) (n = 8) (n = 9) (n = 10) (n = 11) (n = 12)
Determined by light or electron microscopy.
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crystalline solids and peptide ¢bers. The N^H stretching band and amide I band of the crystalline solid from 3 are comprised of multiple bands. The former appears at 3306 and 3336 cm31 , and the latter at 1615, 1639, and 1665 cm31 , both suggesting the formation of two or three types of hydrogen-bond networks in the crystalline solid. In contrast, the amide bands of the peptide ¢bers display only a single peak. The N^H bands appear around 3290^3300 cm31 , while the amide I and II bands around 1640 and 1550 cm31 , respectively. These amide bands regions of the bola-amphiphiles 4^7 are compatible of the formation of L-sheet networks within the peptide ¢bers [25]. It should be noted that the absence of a weak shoulder around 1690 cm31 is indicative of a parallel-type L-sheet network. The amide I and II band frequencies for the peptide ¢bers from 4^7 were found to depend on the even^odd carbon numbers of the oligomethylene spacers (Fig. 3). The amide I bands of the odd-numbered bola-amphiphiles 4 and 6 showed relatively high frequencies than that of the even-numbered bola-amphiphiles 5 and 7. On the contrary, the amide II band had an opposite tendency against the spacer carbon numbers. Such an even^odd alternating feature can also be seen for the observed self-assembled morphologies of 4^7 (Fig. 1 and Table 2). Thus, the present ¢nding indicates the formation of the relatively weak amide hydrogen-bond network within the ¢bers from the odd carbon number. In addition, it should be noted that the interpeptide hydrogen-bond interaction determines the morphology as compared with the van der Waals interaction between the spacer chains. The observed
Fig. 2. X-ray di¡raction patterns of the dried crystalline solids from (a) 1 and (b) 3, and the dried peptide ¢bers from (c) 4, (d) 5, (e) 6, (f) 7, (g) 8, and (h) 9.
[23]. On the other hand, a single broad re£ection peak around d = 0.47 nm appeared for the peptide ¢bers from 4 and 6^9. This structural periodicity is well compatible with an interchain distance expected for the L-sheet structure [24]. 3.4. FT-IR spectroscopy FT-IR spectra were measured for the dried crystalline solids from 3 and the dried peptide ¢bers from 4^7 on a KBr pellet. The IR characteristic bands are summarized in Table 3. The CH2 bands of the crystalline solid and the peptide ¢bers are almost identical. The CH2 antisymmetric and symmetric bands of 3^7 appear at 2930^2935 and 2856^2860 cm31 , respectively. These values are relatively higher than those of the oligomethylene chains having high trans conformational proportion. Therefore, the oligomethylene spacers are in a highly disordered state. The IR band characteristics of amide groups di¡er between the
Fig. 3. Dependence of the amide I and II bands on the chain length (n) of the bola-amphiphiles 4^7.
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Fig. 4. FT-IR spectra in the carboxyl regions of the self-assemblies formed from (a) 3, (b) 4, (c) 5, (d) 6, and (e) 7. Left: curve-¢tting results. Right: second-derivative plots.
even^odd e¡ect may stem from the di¡erence in layer stacking mode (polytype) [26], which is also responsible for the same e¡ect on the ¢ber morphology of 1-glucosamide bola-amphiphiles [27]. In this case the polytype will be independent of the conformational state of the spacer chains. 3.5. Curve-¢tting analysis The CNO stretching band observed for the carboxyl groups also indicated di¡erent features between the crys-
Fig. 5. Schematic illustration of two types of carboxyl interactions within the self-assemblies. (a) Interlayer dimer-forming and (b) intralayer, lateral hydrogen bonds.
talline solid and the peptide ¢bers. No CNO stretching bands attributable to the carboxylate anion groups appeared around 1600 cm31 . This ¢nding will coincide with the fact that the original anions of the bola-amphiphiles are fully protonated within the self-assemblies during aging. A single sharp band appears at 1713 cm31 for
Table 3 FT-IR bands observed for the crystalline solid of 3 and the peptide ¢bers of 4^6 Bola-amphiphile
3 (n = 6)
4 (n = 7)
5 (n = 8)
6 (n = 9)
7 (n = 10)
NH stretching
3336 3306 2935 (2860) 1713
3299
3289
3296
3296
2934 2859 1719
2931 2857 1719
2930 2856 1725
1643
2930 2856 1724 1709 1639
1642
1640
1545 1468
1548 1466
1547 1467
1549 1467
CH2 antisymmetric stretching CH2 symmetric stretching CNO stretching for COOH Amide Ia
Amide IIb CH2 scissoring a b
1665 1639 1615 1539 1468
Mainly attributable to the CNO stretching vibration. Mainly attributable to the N^H deformation vibration.
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Table 4 Peak tops from curve-¢tting and second-derivative results Bola-amphiphile
Peak top (cm31 ) From curve-¢tting results
3 4 5 6 7 a
(n = 6) (n = 7) (n = 8) (n = 9) (n = 10)
1739 1744 1739 1740 1741
(20)a (21) (19) (33) (20)
1724 1723 1723 1721 1724
From second-derivative plots (35) (59) (57) (55) (64)
1710 1712 1708 1710 1710
(45) (19) (25) (12) (16)
1741 1745 1738 1753 1741
1725 1718 1722 1733 1725
1711 ^ 1708 1710 1711
Area ratio of the peak (%).
the crystalline solid of 3, whereas broad or split bands around 1720 cm31 for the peptide ¢bers of 4^7 (Table 3). Fig. 4 shows FT-IR spectra in the CNO stretching regions for the carboxyl groups (left) and their secondderivative plots (right). The second-derivative plots revealed the existence of at least two or three IR bands in this region. Curve-¢tting analyses indicated that the carboxyl bands are comprised of three independent bands, which appear around 1739^1744, 1721^1724, and 1708^ 1712 cm31 , respectively. The wavenumbers and area ratios of the calculated peaks are summarized in Table 4. The IR band around 1740 cm31 can be assigned to the CNO stretching vibration of non-hydrogen-bonded carboxyl groups, while the IR band around 1720 cm31 that of the laterally hydrogen-bonded carboxyl groups [28,29]. In addition, the IR band around 1710 cm31 can be ascribable to the carboxyl groups forming an acid^acid dimer. Fig. 5 represents a schematic illustration of two types of carboxyl interactions within the self-assemblies. Each carboxyl region of 4^7 can be divided into three bands, even though the area ratios of three IR bands are remarkably di¡erent from each other. The interlayer dimer-forming hydrogen bonds of the carboxyl groups proved to be predominant in the crystalline solid for 3 (Fig. 5a). On the contrary, the intralayer, lateral hydrogen bonds signi¢cantly contribute to the stabilization of the peptide ¢bers from 4^7. The bola-amphiphiles 1 and 3 with relatively short n-alkylene spacers would require stronger interlayer interactions in order to compensate for the decrease in hydrophobic interactions.
the connecting oligomethylene spacers. According to the even^odd alternating frequencies of the amide I and II IR bands, the bola-amphiphile 5 having a longer spacer than 4 should form relatively stronger hydrogen bonds between
4. Discussion The bola-amphiphiles 1^9 having a simple L-valyl-L-valine unit at both ends produced a variety of self-assemblies in water. The bola-amphiphiles 1 and 3 form microcrystalline solids. On the other hand, synthetic peptide ¢bers were obtained from the bola-amphiphiles 4^9 with a longer hydrophobic spacer. The longer n-alkylene spacers have a tendency to induce a liquid crystalline mesophase for sugar-based bola-amphiphiles [30]. Their XRD patterns strongly support the low crystallinity of the peptide ¢bers. The di¡erence in the ¢ber morphology seems to depend on the length and even^odd carbon number of
Fig. 6. A possible molecular arrangement and hydrogen-bond networks within the peptide ¢bers.
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the amide groups. Therefore, the more-crystalline ribbon structures will appear together with the ¢bers from the bola-amphiphile 5. In addition, the characteristics of the spacers also have a remarkable in£uence on the contribution degree of the inter- or intralayer carboxyl interactions. The intralayer carboxyl interactions are responsible for the unidirectional growth of the peptide ¢bers from 4, 6, and 8. On the other hand, lamellar monolayered sheets self-assemble through the interlayer interactions to form 3D crystalline solid from 1 and 3, and also to form multilayer ribbon structures from 5. FT-IR spectroscopy revealed that the parallel L-sheet networks between the valylvaline residues stabilize the synthetic peptide ¢bers. XRD peaks around d = 0.47 nm also support the L-sheet structure. In addition, their relatively strong re£ection indicated a high-crystalline nature of the L-sheet networks within the low-crystalline peptide ¢bers. Silk ¢broins take the L-sheet networks all over the sequence of tandem (Gly-Ala-Gly-Ala-Gly-Ser)n repeats since they possessed non-bulky side chains [31]. Polyglycine forms two distinct conformations types I and II, which are stabilized by a L-sheet network and a hexagonal network, respectively [19,32]. On the other hand, glycylglycine bola-amphiphiles form only hexagonally packed polyglycine II-type network both in ¢brous microtubes [16,18] and thin-platelet crystals [20,21]. Though dipeptide units have potential to take L-sheet, hexagonal, and random coil conformations, bulky isopropyl side chains of the valylvaline residues would enforce the L-sheet conformation rather than more closely-packed hexagonal lattice. Hydrophobic interactions between the oligomethylene spacers also facilitate the interpeptide hydrogen-bond formation. A possible molecular arrangement and hydrogenbond networks within the peptide ¢bers is shown in Fig. 6. Valylvaline residues form the parallel-type L-pleats-sheets at both sides of the bola-amphiphiles. The stacking of the L-pleats-sheets and the hydrophobic interactions between the oligomethylene spacers may result in the formation of the peptide ¢bers. Acknowledgements This work was supported by NEDO for the project on Technology for Novel High-Functional Materials in Industrial Science and Technology Frontier Program, AIST, MITI.
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Self-assembled peptide ¢bers from valylvaline bola-amphiphiles by a parallel L-sheet network Masaki Kogiso *, Yuji Okada, Takeshi Hanada 1 , Kiyoshi Yase, Toshimi Shimizu National Institute of Materials and Chemical Research, 1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan Received 18 January 2000 ; received in revised form 4 May 2000; accepted 5 May 2000
Abstract A series of dipeptide-based bola-amphiphiles, bis(N-K-amide-L-valyl-L-valine) 1, n-alkane dicarboxylate (n = 4^12), have been synthesized. The bola-amphiphiles with n = 4 and 6 self-assembled to form crystalline solids in water, whereas those with n = 7^12 produced peptide fibers. FT-IR spectroscopy and X-ray diffraction patterns revealed that the peptide fibers have parallel-type L-sheet networks between the valylvaline units. FT-IR deconvolution study of carboxyl regions indicated that these crystalline solids and peptide fibers are stabilized by interlayer bifurcated and intralayer lateral hydrogen-bond networks between the end carboxylic acid groups, respectively. ß 2000 Elsevier Science B.V. All rights reserved. Keywords : Bola-amphiphile; Peptide ¢ber; L-valyl-L-valine; Self-assembly ; L-sheet; FT-IR spectroscopy
1. Introduction Recent progress in the self-assembly studies of peptide amphiphiles [1^3] has been greatly a¡ected by the pioneer work by Kunitake et al. [4,5]. For instance, dipeptide- [6], tripeptide- [7,8] and polypeptide-containing amphiphiles [9] are known to form unique, well-de¢ned self-assembled morphologies, such as helical ¢bers, rods, and vesicles. In line with this amphiphilic molecular design, peptide agents have recently been designed and synthesized in order to target the dimerization interface of HIV-1 protease [10]. On the other hand, ¢ber formation from peptide amphiphiles has also attracted much attention in terms of the similarity of L-sheet self-assemblies within amyloid ¢brils [11^13]. The creation of protein-like molecular architectures can be realized in the self-assembly of highly ordered polyPro II-like triple-helical structures from peptide amphiphiles with collagen-model headgroups and dialkyl tails [14,15]. Thus, peptide amphiphiles can provide versatile molecular surfaces and architectures favorable for the understanding of biological phenomena.
We have recently reported the sophisticated hierarchical self-assembly of organic microtubes from glycylglycine bola-form amphiphiles (bola-amphiphiles) in aqueous solutions [16^18]. To our knowledge, this ¢nding may give the ¢rst example of supramolecular ¢brous structures driven by pseudohexagonal lattice formation (polyglycine IItype structure [19]) [20,21] of peptide hydrogen-bond networks. Self-assembled morphologies formed from peptide amphiphiles are, thus, strongly dependent on the hydrogen-bonding scheme that also plays an important role in underlying the formation of protein structures. Here we describe the self-assembly of novel synthetic peptide ¢bers with nanometer-scale widths from a series of L-valyl-L-valine bola-amphiphiles 1^9 (Scheme 1). The importance and contribution of intra- or interlayer hydrogen-bond networks is discussed in connection with crystallization and the ¢ber formation of the peptide amphiphiles [22].
* Corresponding author. Fax: +81-298-614422; E-mail : [email protected] 1 Present address: The Institute of Scienti¢c and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka, 567-0047, Japan. 0304-4165 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 0 0 ) 0 0 0 8 8 - X
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Scheme 1.
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2. Materials and methods
2.3. Powder X-ray di¡ractions
2.1. Synthesis of L-valyl-L-valine bola-amphiphiles
All powder X-ray di¡raction (XRD) patterns of dried ¢bers were taken by the re£ection method on a Rigaku di¡ractometer (Type 4037) using graded d-space elliptical side-by-side multilayer optics monochromated CuKK radiation (40 kV, 30 mA) and imaging plate (R-Axis IV).
To a solution of 1,n-alkanedicarboxylic acid (2 mmol) (n = 4^12) and 1-hydroxy benzotriazole (0.674 g, 4.4 mmol) in 10 ml of DMF, a solution of 1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride (0.844 g, 4.4 mmol) in 10 ml of chloroform was added with stirring at 35³C. To the reaction mixture, a solution of L-valylL-valine benzyl ester hydrochloride (1.509 g, 4.4 mmol) and triethylamine (0.62 ml, 4.4 mmol) in 10 ml of chloroform was added after 1 h. The reaction mixture was stirred for further 12 h with the temperature being allowed to increase to room temperature. The reaction mixture was washed with 10% aqueous solution of citric acid, water, 4% aqueous solution of sodium hydrogencarbonate, and water. The organic layer was suspended and dried over anhydrous sodium sulfate. Evaporation of the solvent gave a white solid as a C-terminal protected bola-amphiphile. The benzyl group of the C-terminal was removed by hydrogenation using a Pd/C as a catalyst. The reaction mixture was evaporated to give an oil. Recrystallization from water^ethanol gave a white powder of the bolaamphiphiles 1^9. Analytical data for 1^9 are shown in Table 1.
2.4. FT-IR spectroscopy FT-IR spectra were obtained on a JASCO FT/IR-620 spectrometer. Spectra were measured on a KBr pellet at 4 cm31 resolution after accumulation of 100 scans. Second-derivative plots and curve-¢tting analysis results were obtained by Jasco spectra manager. Tentative baseline was carried out between 1690 and 1770 cm31 . The peak resolution was achieved by non-linear least-squares analysis. During the curve-¢tting analysis, a Lorentz function was used. 3. Results 3.1. Self-assembly of the bola-amphiphiles in water The dicarboxylic L-valyl-L-valine bola-amphiphiles 1^9 are sparingly soluble in water, but become freely soluble by addition of 3 equiv. of alkaline hydroxide. The alkaline aqueous solutions of 1^9 (10 mM, 2 equiv. of sodium hydroxide) were slowly acidi¢ed by vapor di¡usion of 1^ 5% dilute acetic acid into the solutions. However, rapid acidi¢cation of the solutions with a dilute hydrochloric acid (0.1^1 M) immediately produced amorphous solids for all the bola-amphiphiles. After 1^2 weeks, the aqueous solutions of the bola-amphiphiles 1 and 3 a¡orded crystalline solids as a precipitate. The bola-amphiphiles 2 gave no visible assemblies with the naked eye. In contrast, clear hydrogels were obtainable from the aqueous solutions containing the bola-amphiphiles 4 and 6^9, whereas a turbid hydrogel was obtainable from 5. The appearance of the self-assembled structures are listed in Table 2 for 1^9. The pH ranges, at which we obtained these self-assemblies, are also summarized in Table 2. The pH titration measure-
2.2. Energy-¢ltering transmission electron microscopy (EF-TEM) EF-TEM observation was carried out to examine ¢ne ¢ber structures in hydrogels in a similar manner as that described elsewhere [23]. Unstained specimens were prepared by placing a 3-Wl drop of the dispersion on an amorphous carbon supporting ¢lm mounted on a standard TEM grid. The drop was then blotted o¡ with ¢lter paper, followed by dryness in vacuo. Each specimen was examined at 80 keV in the electron spectroscopic imaging by using an analytical electron microscope (Carl Zeiss EM 902) with Castain^Henry type electron energy ¢lter at room temperature. Zero-loss images were recorded on an imaging plate (Fuji Photo Film, FDL 5000) with a 20 eV energy windows at 3000^250 000U and digitally enlarged. Table 1 Analytical data for the bola-amphiphiles 1^9 Bola-amphiphile
Yield (%)
Mp (³C)
[K]D (³)
Elemental analysis (calculated (found)/%)
1 2 3 4 5 6 7 8 9
56 25 69 72 79 75 61 90 77
217 182 193 183 192 183 134 136 116
^a ^ 346.9 ^ 346.1 ^ 339.9 ^ ^
calcd. calcd. calcd. calcd. calcd. calcd. calcd. calcd. calcd.
a
(n = 4) (n = 5) (n = 6) (n = 7) (n = 8) (n = 9) (n = 10) (n = 11) (n = 12)
for for for for for for for for for
C26 H46 O8 N4 , C:57.54 (57.32) H:8.54 (8.61) N:10.33 (10.20) C27 H48 O8 N4 W1H2 O, C:56.42 (56.76) H:8.77 (8.71) N:9.75 (9.64) C28 H50 O8 N4 W2H2 O, C:55.42 (55.57) H:8.97 (9.10) N:9.24 (9.22) C29 H52 O8 N4 W6/5H2 O, C:57.43 (58.01) H:9.04 (9.15) N:9.24 (9.20) C30 H54 O8 N4 W1/5H2 O, C:59.81 (60.07) H:9.10 (9.25) N:9.30 (9.25) C31 H56 O8 N4 W1H2 O, C:59.02 (59.38) H:9.27 (9.29) N:8.88 (8.76) C32 H58 O8 N4 W1/2H2 O, C:60.44 (60.24) H:9.35 (9.27) N:8.81 (8.97) C33 H60 O8 N4 W1/2H2 O, C:60.99 (61.27) H:9.46 (9.49) N:8.62 (8.41) C34 H62 O8 N4 W1/2H2 O, C:61.51 (61.54) H:9.57 (9.74) N:8.49 (8.31)
Not determined.
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Fig. 1. EF-TEM images of the peptide ¢bers self-assembled from (a) 4, (b) 5, (c) 6, and (d) 7.
ment [18] of 7 revealed that the original carboxylate anion is fully protonated at this pH value. Elemental analysis of each dried self-assembly also supported the carboxylic acid forms of the amphiphiles within these self-assemblies. 3.2. EF-TEM of the hydrogels In order to clarify microscopic dimensions of the selfassemblies, we examined the hydrogels from 4^7 using EFTEM. EF-TEM is a powerful tool for observing low-contrast organic and biological samples without staining. EFTEM indicated that the bola-amphiphiles 4^7 form a large number of ¢brous assemblies with lengths of 0.1^10 Wm (Fig. 1). High-resolution EF-TEM images of the same specimens showed that these synthetic ¢bers have the minimum widths of 10^15 nm. However, the morphologies of the ¢bers slightly di¡er from each other (Table 2). The bola-amphiphile 4 forms apparently sti¡ ¢bers (Fig. 1a). In contrast, the bola-amphiphile 5 gives ribbon structures and ¢bers (Fig. 1b). The sti¡ ¢bers similar to those of 4 are also produced from the bola-amphiphile 6, whereas
apparently soft ¢bers from the bola-amphiphile 7 (Fig. 1c and d, respectively). Almost uniform widths of ¢bers are compatible with the clear appearance of the hydrogel from 4, 6, and 7. In contrast, coexistence of the ribbon and ¢ber structures would be responsible for the turbid hydrogel from 5. 3.3. XRD analysis Fig. 2 represents the powder XRD patterns obtained for the isolated and dried self-assemblies of 1 and 3^9. The XRD patterns of the dried crystalline solids from 1 and 3 indicate highly crystalline nature of the self-assemblies, displaying many sharp re£ection peaks. The ribbon structures and ¢bers from 5 also show complex XRD patterns indicative of high crystallinity. However, the absence of any re£ection peaks attributable to long-range ordering in the small-angle region suggests that each bola-amphiphile forms no crystalline multilayer structures within the self-assemblies. This ¢nding presents a striking contrast to that observed for 2-glucosamide-based bola-amphiphiles
Table 2 Self-assembling properties of the L-valyl-L-valine bola-amphiphiles 1^9 Bola-amphiphile
Appearance
Morphologies of ¢ne structuresa
pH Range
1 2 3 4 5 6 7 8 9
precipitate solution precipitate clear hydrogel turbid hydrogel clear hydrogel clear hydrogel clear hydrogel clear hydrogel
crystalline solid ^ crystalline solid sti¡ ¢ber sheet structure and ¢ber sti¡ ¢ber soft ¢ber ¢ber ¢ber
2.7^3.0 ^ 3.4^3.8 3.6^4.0 4.3^4.4 4.3^4.7 4.7^4.9 5.1^5.4 5.3^5.4
a
(n = 4) (n = 5) (n = 6) (n = 7) (n = 8) (n = 9) (n = 10) (n = 11) (n = 12)
Determined by light or electron microscopy.
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crystalline solids and peptide ¢bers. The N^H stretching band and amide I band of the crystalline solid from 3 are comprised of multiple bands. The former appears at 3306 and 3336 cm31 , and the latter at 1615, 1639, and 1665 cm31 , both suggesting the formation of two or three types of hydrogen-bond networks in the crystalline solid. In contrast, the amide bands of the peptide ¢bers display only a single peak. The N^H bands appear around 3290^3300 cm31 , while the amide I and II bands around 1640 and 1550 cm31 , respectively. These amide bands regions of the bola-amphiphiles 4^7 are compatible of the formation of L-sheet networks within the peptide ¢bers [25]. It should be noted that the absence of a weak shoulder around 1690 cm31 is indicative of a parallel-type L-sheet network. The amide I and II band frequencies for the peptide ¢bers from 4^7 were found to depend on the even^odd carbon numbers of the oligomethylene spacers (Fig. 3). The amide I bands of the odd-numbered bola-amphiphiles 4 and 6 showed relatively high frequencies than that of the even-numbered bola-amphiphiles 5 and 7. On the contrary, the amide II band had an opposite tendency against the spacer carbon numbers. Such an even^odd alternating feature can also be seen for the observed self-assembled morphologies of 4^7 (Fig. 1 and Table 2). Thus, the present ¢nding indicates the formation of the relatively weak amide hydrogen-bond network within the ¢bers from the odd carbon number. In addition, it should be noted that the interpeptide hydrogen-bond interaction determines the morphology as compared with the van der Waals interaction between the spacer chains. The observed
Fig. 2. X-ray di¡raction patterns of the dried crystalline solids from (a) 1 and (b) 3, and the dried peptide ¢bers from (c) 4, (d) 5, (e) 6, (f) 7, (g) 8, and (h) 9.
[23]. On the other hand, a single broad re£ection peak around d = 0.47 nm appeared for the peptide ¢bers from 4 and 6^9. This structural periodicity is well compatible with an interchain distance expected for the L-sheet structure [24]. 3.4. FT-IR spectroscopy FT-IR spectra were measured for the dried crystalline solids from 3 and the dried peptide ¢bers from 4^7 on a KBr pellet. The IR characteristic bands are summarized in Table 3. The CH2 bands of the crystalline solid and the peptide ¢bers are almost identical. The CH2 antisymmetric and symmetric bands of 3^7 appear at 2930^2935 and 2856^2860 cm31 , respectively. These values are relatively higher than those of the oligomethylene chains having high trans conformational proportion. Therefore, the oligomethylene spacers are in a highly disordered state. The IR band characteristics of amide groups di¡er between the
Fig. 3. Dependence of the amide I and II bands on the chain length (n) of the bola-amphiphiles 4^7.
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Fig. 4. FT-IR spectra in the carboxyl regions of the self-assemblies formed from (a) 3, (b) 4, (c) 5, (d) 6, and (e) 7. Left: curve-¢tting results. Right: second-derivative plots.
even^odd e¡ect may stem from the di¡erence in layer stacking mode (polytype) [26], which is also responsible for the same e¡ect on the ¢ber morphology of 1-glucosamide bola-amphiphiles [27]. In this case the polytype will be independent of the conformational state of the spacer chains. 3.5. Curve-¢tting analysis The CNO stretching band observed for the carboxyl groups also indicated di¡erent features between the crys-
Fig. 5. Schematic illustration of two types of carboxyl interactions within the self-assemblies. (a) Interlayer dimer-forming and (b) intralayer, lateral hydrogen bonds.
talline solid and the peptide ¢bers. No CNO stretching bands attributable to the carboxylate anion groups appeared around 1600 cm31 . This ¢nding will coincide with the fact that the original anions of the bola-amphiphiles are fully protonated within the self-assemblies during aging. A single sharp band appears at 1713 cm31 for
Table 3 FT-IR bands observed for the crystalline solid of 3 and the peptide ¢bers of 4^6 Bola-amphiphile
3 (n = 6)
4 (n = 7)
5 (n = 8)
6 (n = 9)
7 (n = 10)
NH stretching
3336 3306 2935 (2860) 1713
3299
3289
3296
3296
2934 2859 1719
2931 2857 1719
2930 2856 1725
1643
2930 2856 1724 1709 1639
1642
1640
1545 1468
1548 1466
1547 1467
1549 1467
CH2 antisymmetric stretching CH2 symmetric stretching CNO stretching for COOH Amide Ia
Amide IIb CH2 scissoring a b
1665 1639 1615 1539 1468
Mainly attributable to the CNO stretching vibration. Mainly attributable to the N^H deformation vibration.
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Table 4 Peak tops from curve-¢tting and second-derivative results Bola-amphiphile
Peak top (cm31 ) From curve-¢tting results
3 4 5 6 7 a
(n = 6) (n = 7) (n = 8) (n = 9) (n = 10)
1739 1744 1739 1740 1741
(20)a (21) (19) (33) (20)
1724 1723 1723 1721 1724
From second-derivative plots (35) (59) (57) (55) (64)
1710 1712 1708 1710 1710
(45) (19) (25) (12) (16)
1741 1745 1738 1753 1741
1725 1718 1722 1733 1725
1711 ^ 1708 1710 1711
Area ratio of the peak (%).
the crystalline solid of 3, whereas broad or split bands around 1720 cm31 for the peptide ¢bers of 4^7 (Table 3). Fig. 4 shows FT-IR spectra in the CNO stretching regions for the carboxyl groups (left) and their secondderivative plots (right). The second-derivative plots revealed the existence of at least two or three IR bands in this region. Curve-¢tting analyses indicated that the carboxyl bands are comprised of three independent bands, which appear around 1739^1744, 1721^1724, and 1708^ 1712 cm31 , respectively. The wavenumbers and area ratios of the calculated peaks are summarized in Table 4. The IR band around 1740 cm31 can be assigned to the CNO stretching vibration of non-hydrogen-bonded carboxyl groups, while the IR band around 1720 cm31 that of the laterally hydrogen-bonded carboxyl groups [28,29]. In addition, the IR band around 1710 cm31 can be ascribable to the carboxyl groups forming an acid^acid dimer. Fig. 5 represents a schematic illustration of two types of carboxyl interactions within the self-assemblies. Each carboxyl region of 4^7 can be divided into three bands, even though the area ratios of three IR bands are remarkably di¡erent from each other. The interlayer dimer-forming hydrogen bonds of the carboxyl groups proved to be predominant in the crystalline solid for 3 (Fig. 5a). On the contrary, the intralayer, lateral hydrogen bonds signi¢cantly contribute to the stabilization of the peptide ¢bers from 4^7. The bola-amphiphiles 1 and 3 with relatively short n-alkylene spacers would require stronger interlayer interactions in order to compensate for the decrease in hydrophobic interactions.
the connecting oligomethylene spacers. According to the even^odd alternating frequencies of the amide I and II IR bands, the bola-amphiphile 5 having a longer spacer than 4 should form relatively stronger hydrogen bonds between
4. Discussion The bola-amphiphiles 1^9 having a simple L-valyl-L-valine unit at both ends produced a variety of self-assemblies in water. The bola-amphiphiles 1 and 3 form microcrystalline solids. On the other hand, synthetic peptide ¢bers were obtained from the bola-amphiphiles 4^9 with a longer hydrophobic spacer. The longer n-alkylene spacers have a tendency to induce a liquid crystalline mesophase for sugar-based bola-amphiphiles [30]. Their XRD patterns strongly support the low crystallinity of the peptide ¢bers. The di¡erence in the ¢ber morphology seems to depend on the length and even^odd carbon number of
Fig. 6. A possible molecular arrangement and hydrogen-bond networks within the peptide ¢bers.
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the amide groups. Therefore, the more-crystalline ribbon structures will appear together with the ¢bers from the bola-amphiphile 5. In addition, the characteristics of the spacers also have a remarkable in£uence on the contribution degree of the inter- or intralayer carboxyl interactions. The intralayer carboxyl interactions are responsible for the unidirectional growth of the peptide ¢bers from 4, 6, and 8. On the other hand, lamellar monolayered sheets self-assemble through the interlayer interactions to form 3D crystalline solid from 1 and 3, and also to form multilayer ribbon structures from 5. FT-IR spectroscopy revealed that the parallel L-sheet networks between the valylvaline residues stabilize the synthetic peptide ¢bers. XRD peaks around d = 0.47 nm also support the L-sheet structure. In addition, their relatively strong re£ection indicated a high-crystalline nature of the L-sheet networks within the low-crystalline peptide ¢bers. Silk ¢broins take the L-sheet networks all over the sequence of tandem (Gly-Ala-Gly-Ala-Gly-Ser)n repeats since they possessed non-bulky side chains [31]. Polyglycine forms two distinct conformations types I and II, which are stabilized by a L-sheet network and a hexagonal network, respectively [19,32]. On the other hand, glycylglycine bola-amphiphiles form only hexagonally packed polyglycine II-type network both in ¢brous microtubes [16,18] and thin-platelet crystals [20,21]. Though dipeptide units have potential to take L-sheet, hexagonal, and random coil conformations, bulky isopropyl side chains of the valylvaline residues would enforce the L-sheet conformation rather than more closely-packed hexagonal lattice. Hydrophobic interactions between the oligomethylene spacers also facilitate the interpeptide hydrogen-bond formation. A possible molecular arrangement and hydrogenbond networks within the peptide ¢bers is shown in Fig. 6. Valylvaline residues form the parallel-type L-pleats-sheets at both sides of the bola-amphiphiles. The stacking of the L-pleats-sheets and the hydrophobic interactions between the oligomethylene spacers may result in the formation of the peptide ¢bers. Acknowledgements This work was supported by NEDO for the project on Technology for Novel High-Functional Materials in Industrial Science and Technology Frontier Program, AIST, MITI.
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