Comblike Complexes of Bacterial Poly(Î³,d-glutamic acid) - American

Biomacromolecules 2004, 5, 144-152

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Comblike Complexes of Bacterial Poly(γ,D-glutamic acid) and Cationic Surfactants Graciela Pe´ rez-Camero, Montserrat Garcıá-Alvarez, Antxon Martıńez de Ilarduya, Carlos Ferna´ ndez, Lourdes Campos, and Sebastia´ n Munõz-Guerra* Departament d’Enginyeria Quı´mica, Universitat Polite` cnica de Catalunya, ETSEIB, Diagonal 647, 08028 Barcelona, Spain Received July 31, 2003; Revised Manuscript Received September 25, 2003

The ability of microbially produced poly(γ,D-glutamic acid) to form stable polyelectrolyte-opposite charged surfactant complexes was investigated. A sonicated sample of polyacid with a molecular weight about 105 Da and a content of D enantiomer higher than 90% was used in this study. Nearly stoichiometric complexes of poly(γ,D-glutamate) anions and alkyltrimethylammonium cations bearing linear alkyl chains with even numbers of carbon atoms from 12 up to 22 were “synthesized” by precipitation from equimolar mixtures of aqueous solutions of the two components. All complexes were found to adopt stratified supramolecular structures made of alternating layers of poly(γ,D-glutamate) and surfactant with a periodicity increasing from 3.2 up to 4.3 nm according to the length of the alkyl side chain. No definite evidence indicative of the conformation adopted by the main chain in these complexes could be afforded. In all cases, the alkyl chains are in an extended conformation and oriented normal or nearly normal to the layer planes. Polymethylene chains with more than 16 carbon atoms were partially crystallized in the complexes in a separated paraffinic phase, whereas no crystallinity was detected for shorter lengths. The crystallized paraffinic phases were found to melt reversibly at temperatures between 40 and 70 °C. This process was found to happen with a concomitant expansion-contraction that amounts between 2 and 8% of the long period of the structure but without significant alteration of the layered arrangement. Introduction Comblike polymers consisting of a stiff backbone chain with long flexible side groups are of prime interest not only on their own for their capacity to form supramolecular assemblies but also because they are promising materials for novel practical applications.1 One distinguished class of these polymer systems is that comprising helical poly(R-peptide)s bearing long linear polymethylene side chains.2 Such type of systems was extensively investigated during the past decade, and they still continue to be object of current interest for their unique combination of properties that includes a high solubility and the ability to generate periodically layered structures at the nanometric scale.3 Very recently, similar structures have been shown to occur also in poly(R-alkyl β,L-aspartate)s4 and poly(R-alkyl γ-glutamate)s,5 two families of unconventional polypeptides able to take up regularly folded conformations of R-helix type. The synthesis of alkyl poly(glutamate)s and poly(aspartate)s involves rather complicated chemical procedures that hamper the access to these systems. On the other side, it is known that comblike structures of an ionic nature can be easily formed by complexation of polyelectrolytes with oppositely charged low molecular weight amphiphilic molecules. Such complexes are spontaneously generated when aqueous solutions of the two components are mixed, and once formed, they remain stable due to favorable electrostatic * To whom correspondence should be addressed. E-mail: sebastian. [email protected].

interactions and tend to form self-assembled mesophases.6 Ponomarenko et al.7 have demonstrated that stoichiometric complexes prepared from the sodium salt of chemosynthetic poly(R,L-glutamic acid) and alkyltrimethyl ammoniun bromide surfactants are regularly arranged with the polypeptide chain remaining in the R-helical conformation. These compounds appear to be insoluble in water, but they are soluble in organic solvents of moderate polarity. In the solid state, they become organized in ordered biphasic structures with the helical polypeptide chains arranged in layers, which are regularly spaced by intercalating surfactant molecules.7 These assemblies are closely similar to that adopted by their covalent analogous poly(γ-alkyl R,L-glutamate)s.3 The generation of polymeric supramolecular structures based on noncovalent interactions is not confined to ionically complexed polymers. In very recent research, it has been shown that hydrogen-bonding connected graft copolymers can form the same type of micellar structures as their corresponding ordinary covalently bonded graft copolymers. This will make it possible to prepare hollow spheres by simply dissolution of the core in the appropriate solvent.8 In this paper, we report on the complexes synthesized from poly(γ,D-glutamic acid) and alkyltrimethylammonium bromides surfactants with the alkyl group being linear and containing even numbers of carbon atoms from 12 up to 22. These complexes will be referred to henceforth as nATMA‚ PGGA. The possibility that bacterial poly(γ-glutamic acid) may form self-assembled complexes with oppositely charged low molecular weight compounds similar to those described

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Comblike Complexes of Polymers and Surfactants

Biomacromolecules, Vol. 5, No. 1, 2004 145

for poly(R,L-glutamic acid)7 (henceforth called nATMA‚ PAGA) is not only of great conceptual interest, but also relevant to the technical development of this naturally occurring polymer. Figure 1. Chemical formula of complexes studied in this work.

Experimental Section Materials. Sodium poly(γ,D-glutamate) with a D:L enantiomeric ratio exceeding 9:1 was used in this work. It was obtained by fermentation of B. licheniformis in medium E with a Mn(II) concentration of 0.16 µM, as reported in detail elsewhere.9 A water solution of the polymer resulting from biosynthesis (Mw ) 4 × 106 Da, Mw/Mn ) 4) was subjected to irradiation with microwaves to render a final polymer of Mw ) 120 000 Da and Mw/Mn ) 1.5.10 Linear alkyltrimethylamonium cationic surfactants of general formula RN+Me3‚Br- with R ) C12H25 (dodecyl), C14H29 (meristyl), C16H33 (cetyl), and C18H37 (stearyl) were purchased from Aldrich or Merck and used as received. Those with R ) C20H41 (eicosyl) and C22H45 (docosyl) were synthesized specifically for this work according to the procedure described in the literature.11 Organic solvents were of analytical grade and used without further purification. Water used in the preparation of the complexes was distilled and deionized in a “Milli-Q” water purification system. Synthesis of Complexes. Complexes of poly(γ,D-glutamic acid) were prepared following the methodology described by Ponomarenko et al.7 for the synthesis of complexes of the same type of surfactants with poly(R,L-glutamic acid). In brief, 100 mL of an aqueous 0.01 M solution of the surfactant of choice was added to the same volume of a 0.01 M Na-PGGA solution in water under stirring, and the mixture was left to rest at room temperature. A white precipitate appeared in a few hours, which was isolated by centrifugation, washed several times with water, and dried under vacuum at 40 °C for at least 48 h. Complexes with n ) 18, 20, and 22 were obtained by mixing the surfactant and Na-PGGA solutions at 40 °C. Measurements. Densities were measured by the flotation method in aqueous glycerin solutions. FTIR spectra were recorded on a Perkin-Elmer FT-2000 instrument from films prepared by casting from chloroform solution. Oriented films for polarized IR-dichroism measurements were prepared according to Ingwall et al.12 For this, a chloroform solution containing 2.5% (w/v) of poly(ethylene oxide) and 1% of the complex under investigation was evaporated to dryness, and the resulting mixed film was stretched at room temperature. Dichroic IR spectra were recorded by using a gold wire polarizer with the sample placed at (45° with respect to the direction of the entrance slit. Dichroic ratios (|/⊥) of characteristic peaks were calculated from the absorbance measured for the parallel and perpendicular orientations of the sample to the infrared polarization vector. 1H and 13C NMR spectra were recorded on a Bruker AMX-300 NMR

instrument provided with a CP-MAS accessory for the analysis of solids. Analysis in solution was carried out with samples dissolved in CDCl3 and using TMS as the reference. Solid state 13C CP-MAS NMR spectra were recorded at 75.5 MHz in the temperature range of 20-90 °C from 200 mg powder samples that were spun at 3.9-4.0 kHz in a cylindrical ceramic rotor. All of the spectra were acquired with contact and repetition times of 2 ms and 5 s, respectively, and 1024 transients were accumulated. The spectral width was 31.2 kHz, and the number of data points was 4K. Chemical shifts were externally calibrated against the higher field peak of adamantane appearing at 29.5 ppm relative to TMS. Calorimetric measurements were performed with a Perkin-Elmer Pyris DSC instrument calibrated with indium. Sample weights of about 2-5 mg were used in a temperature range from -20 up to 250 °C at heating and cooling rates of 20 °C min-1 and under a nitrogen atmosphere. Thermogravimetric analyses were performed under an inert atmosphere with a Perkin-Elmer TGA6 thermobalance at a heating rate of 20 °C min-1. Optical microscopy was carried out in a Olympus BX51 polarizing microscope equipped with a digital camera system. Filmy samples of nATMA‚PGGA complexes were prepared from 1% (v/v) chloroform solutions, which were left to evaporate slowly between microscope cover slides. X-ray diffraction patterns were recorded on flat films in a modified Statton camera or digitalized with a reflection Siemens D-500 diffractometer provided with a scintillation counter. The Cu KR radiation of wavelength 0.1541 nm was used in both cases. Results and Discussion Synthesis and Characterization. The nATMA‚PGGA complexes with the chemical structure depicted in Figure 1 were readily formed as a white fine powder when equal volumes of aqueous solutions of Na-PGGA and the corresponding nATMA‚Br, at the same molar concentration, were mixed and the mixture was left to stay at room temperature for a few hours. Results obtained for the six complexes examined in this study are compared in Table 1. Yields were found to oscillate between 50 and 90% and attempts made to improve these figures by using an excess of surfactant were unsuccessful. Elemental analyses were in agreement with calculated values provided that a certain amount of water ranging between 10 and 15% w/w is assumed to be present in the compound. The presence of this water is usually detected in the 1H NMR spectra as a peak at around 4.30 ppm. Such water must be firmly attached to the complex because it could not be removed by drying under high vacuum for days. The solubility behavior displayed by nATMA‚PGGA complexes is in full agreement with that observed for other complexes of similar nature such as nATMA‚PAGA.7

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Pe´ rez-Camero et al.

Table 1. Poly(γ,D-glutamic acid)-Surfactant Complexes nATMA‚PGGA: Synthesis Results R(CH3)3N+ R

complex

yield (%)

compositiona

C (%)

H (%)

N (%)

F (g mL-1)

-nC12H25

12ATMA‚PGGAc

56

1.0:1

14ATMA‚PGGAc

62

1.1:1

-nC16H33

16ATMA‚PGGAd

67

1.1:1

-nC18H37

18ATMA‚PGGAd

57

1.2:1

-nC20H41

20ATMA‚PGGA

89

1.3:1

-nC22H45

22ATMA‚PGGA

54

1.3:1

11.03 (11.29) 10.77 (11.49) 10.90 (11.66) 10.48 (11.84) 12.02 (11.93) 11.76 (12.02)

6.77 (6.82) 6.44 (6.38) 5.10 (6.00) 4.70 (5.88) 4.75 (5.18) 4.82 (5.09)

1.00

-nC14H29

57.41 (58.50) 60.38 (60.24) 61.39 (61.77) 59.53 (58.88) 62.10 (62.18) 65.57 (65.41)

elemental analysisb

1.03 1.02 1.06 1.01 1.02

a Ratio of ATMA to PGGA in the complex, as estimated by 1H NMR. b In brackets, values calculated for the observed compositions and assuming 5c or 3d molecules of absorbed water per repeating unit.

Table 2. Infrared Frequencies (cm-1) for nATMA‚PGGA Complexes and Other Related Compoundsa

Na-PGGA 12ATMA‚PGGA (|/⊥)b 18ATMA‚PGGA (|/⊥)b 20ATMA‚PGGA 22ATMA‚PGGA PAAG-12(|/⊥)c 12ATMA‚PAGAd

amide A

amide I

amide II

COO-

3450s 3385m (0.90) 3385m (0.95) 3374m 3321m 3298m (1.59) 3300m

1630m 1647m (0.85) 1637m (0.93) 1636m 1635m 1643m (1.17) 1653m

1450m 1559,1542w (1.22) 1561,1544w (1.67) 1573w 1573w 1538m (0.72) 1549w

1590s 1600m (1.00) 1590m (1.00) 1591m 1591m 1742s (1.10) 1590m

a Approximate relative intensities denoted as s ) strong, m ) medium, w ) weak. b Dichroic ratio in brackets estimated from parallel and perpendicular polarized spectra. c Data for the helical alkyl ester poly(R-dodecyl-γ,D,L-glutamate) taken from ref 5b. d Data for the helical dodecyl-trimethylammoniumpoly(R,L-glutamic acid) complex taken from ref 7a.

nATMA‚PGGA complexes are soluble in solvents such as methanol, ethanol, TFE, DMSO, or NMP. An interesting feature in this regard is that they appear to be readily soluble in chloroform, whereas they cannot be dissolved by water, which is exactly the opposite of the behavior showed by NaPGGA. Evaporation of the nATMA‚PGGA chloroform solutions rendered flexible films that were transparent for n ) 12, 14, and 16 but that displayed a milky appearance for higher values of n. The composition and constitution of nATMA‚PGGA complexes were checked by both FTIR and 1H/13C NMR. Infrared bands characteristic of the polyamide chain with the carboxyl groups in the ionized state along with those arising from the characteristic groups of the surfactant cation moiety were present in the spectra recorded from these complexes (Table 2). Results consistent with infrared data were obtained in the NMR analysis. The 1H and 13C NMR spectra of 18ATMA‚ PGGA are depicted in Figure 2 for illustrative purposes. The chemical shifts observed for the six complexes are listed in Table 3, where similar NMR data for the complex made from L-pyroglutamic acid and the octadecyl tetramethylammonium cation (18ATMA‚PyGA) have been included for comparison. The peak areas measured for main chain and alkyl side chain protons in the 1H NMR spectra provided an accurate estimation of the ionic composition of the complexes. These results are given in Table 1 where it is seen that the cationto-anion ratio increases from 1.0 to 1.3 with the length of the alkyl side group. Such an excess of surfactant in the complex was found even when defective amounts of cations in the initial mixture were used. On the other side, it is

observed that 13C signals arising from the polypeptide counterpart appear displaced upfield in the complex and that the magnitude of the displacement relative to their respective parent anions is similar for both nATMA‚PGGA and 18ATMA‚PyGA. The thermal stability of the complexes was evaluated previously to carry out the DSC experiments in order to ensure the reliability of these studies. As representatives for the whole family, TGA traces of 12ATMA‚PGGA and 18ATMA‚PGGA are reproduced in Figure 3. The complexes were found to be stable up to temperatures well above 200 °C with maximum decomposition rates taking place near 300 °C. According to earlier studies carried out on the thermal degradation of poly(γ-glutamate)s,14 the decomposition of these complexes is thought to occur through a back-biting depolymerization mechanism of the polypeptide chain to pyroglutamic acid, a process that is encompassed in this case by the destruction of the ionic complex. As it is illustrated in Figure 4, the DSC analysis revealed significant differences in the thermal behavior of nATMA‚ PGGA complexes according to n. The DSC traces recorded from 12 and 14ATMA‚PGGA complexes did not show any thermal transition below the onset of degradation, which happens in the proximity of 250 °C. On the contrary, the thermograms obtained from 16, 18, 20, and 22ATMA‚PGGA exhibited well-defined melting peaks at increasing temperatures from 40 up to 70 °C, which were partially reproducible on cooling and reheating. It should be noted that melting of alkyltetramethylammonium bromides takes place within the range 100-150 °C and that DSC of Na-PGGA does not show any heating exchange peak up before degradation.



Figure 2. (a) 1H and (b) Table 3.

13C

NMR spectra of 18ATMA‚PGGA recorded at 25 °C. (/) peak of water.

and 1H NMR Chemical Shifts (δ, ppm) for Complexes of PGGA and PyGA with Alkyltrimethylammonium Cations

PGGA-complexes Na-PGGAa 12ATMA‚PGGAb 14ATMA‚PGGAb 16ATMA‚PGGAb 18ATMA‚PGGAb 20ATMA‚PGGAb 22ATMA‚PGGAb PyGA-complex Na-PyGAa 18ATMA‚PyGAb a

13C

CONH

COO-

RCH

13C

13C

13C

1H

13C

1H

13C

1H

181.3 175.8 175.7 176.1 175.8 176.3 171.1

177.9 173.0 173.0 173.2 173.0 173.5 173.3

57.8 54.3 54.3 54.6 54.6 54.5 54.5

4.0 4.2 4.2 4.2 4.2 4.2 4.2

35.2 32.8 32.9 32.9 32.9 32.8 32.9

2.2 2.4-1.7 2.4-1.7 2.4-1.7 2.4-1.7 2.4-1.7 2.4-1.7

30.6 28.9 29.0 29.0 29.0 29.0 28.9

1.9-1.8 2.4-1.7 2.4-1.7 2.4-1.7 2.4-1.7 2.4-1.7 2.4-1.7

184.3 178.4

182.8 176.5

60.8 58.1

4.1 4.1

32.2 25.8

2.3 2.3

27.8 22.6

2.4-1.9 2.4-2.2

CH

βCH

2

2

Spectra recorded in D2O/NaDO. b Spectra recorded in CDCl3.

The thermal parameters, melting temperatures, and associated enthalpies of the whole series of nATMA‚PGGA are compared in Table 4. By analogy to thermal data reported for analogous nATMA-PAGA complexes7 as well as for comblike poly(β-aspartate)s4 and poly(γ-glutamate)s,5b the endothermic peak is interpreted as due to the fusion of the polymethylene side chain, which was able to crystallize for a number of carbon atoms higher than 14. The enthalpy of the peak measured for the 18ATMA‚PGGA complex is about

4 kcal mol-1, whereas the fusion heat reported for the crystal to liquid transition of linear alkanes is between 1 and 0.7 kcal mol-1 per CH2 depending on whether the crystal structure is triclinic or hexagonal.13 It can be inferred therefore that only about 25-40% of the octadecyl side chain is crystallized at temperatures below 51 °C. Such degree of crystallinity is significantly higher than that found for the 18ATMA‚PAGA complex, which is around 10%,7 but much closer to that reported for comblike polypeptides with

148


Figure 3. TGA traces of 12ATMA and 18ATMA‚PGGA complexes.

covalently linked linear octadecyl side chains which is near to 50%.3a,4a,5b The amount of crystallized methylenes in the other “crystallizable” members of the series can be analogously inferred from their respective melting enthalpies given in Table 4. Supramolecular Structure. When chloroform-cast films of the complexes were examined under the polarizing optical microscope they showed birefringent textures varying according to the length of the alkyl side chain. Some selected pictures are shown in Figure 5. Fanlike, mosaic, and batonnet textures characteristic of smectic mesophases displaying nonplanar orientations were observed for 12ATMA‚PGGA and 14ATMA‚PGGA. Grainy textures of great complexity and difficult interpretation were found for 16ATMA‚PGGA and 18ATMA‚PGGA. Conversely, spherulitic structures characteristic of semicrystalline material were invariably present in the films obtained from 20ATMA‚PGGA and 22ATMA‚PGGA. These observations are consistent with the fact that lower members have the side polymethylene chain in the liquid state, whereas it is extensively crystallized in higher members. The structure of these films was then investigated by X-ray diffraction. The powder diffraction patterns of all complexes contained in the low-angle region a strong reflection with a Bragg spacing increasing from 3.2 to 4.3 nm for n increasing from 12 to 22. A second reflection displaying much lower intensity, and with an associated spacing of half of the value of the former, can be also observed in most of cases. On the other hand, the wide-angle region of the patterns invariably exhibited a broad halo centered on 0.45 nm, which, in the case of 18, 20, and 22ATMA‚PGGA, was accompanied by a sharp intense reflection with a spacing of 0.42 nm. X-ray diffraction patterns of stretched films of the complexes contained the same spacings as the powder patterns and revealed that wide and small angle reflections come from periodical structures that must be oriented normal or nearly normal to each other. The X-ray diffraction pattern of oriented 18ATMA‚PGGA is reproduced in Figure 6a, and all of the spacings observed for the whole nATMA‚PGGA series are compared in Table 5. In analogy to the series of nATMA-PAGA complexes studied by Ponomarenko et al.,7 the patterns obtained here are interpreted on the basis of a biphasic lamellar structure


consisting of alternating layers of poly(γ,D-glutamate) and surfactant. In 12 and 14 complexes, the packing of the polymethylene side chains exhibits only a short range order characterized by the broad reflection at 0.45 nm associated with the average lateral distance between aligned chains in the uncrystallized state. On the contrary, a hexagonal packing with a0 ) 0.48 nm (sharp refection at 0.42 nm) seems to be adopted by the paraffinic phase in complexes 18, 20, and 22ATMA‚PGGA. This has been confirmed by electron diffraction of films casted from chloroform on a water surface, which afforded a well-defined hexagonal pattern with a spacing of 0.42 nm (Figure 6b). According to the amphiphilic nature of the complexes, this pattern is interpreted as arising from a homeotropic texture oriented with the hexagonally packed alkyl chains pointing to the electron diffraction beam, i.e. normal to the film surface, which is the plane where the main chains are lying. It should be noted that alkyltrimethylammonium bromides crystallize in a monoclinic lattice.15 A monoclinic lattice is also the structure present in the paraffinic phase of comblike poly(γ-alkyl R,L-glutamate)s,3a whereas a hexagonal packing has been observed for both poly(R-alkyl β,L-aspartate)s4a and poly(R-alkyl γ,L-glutamate)s.5b Although it is known that the monoclinic packing of alkyl side chains in poly(alkyl olefin)s is more energetically efficient than the hexagonal packing,1 it is still unclear what can be the factor determining the mode of packing in comblike polypeptides. The strong peak appearing in the low-angle region of the X-ray diffraction patterns arises from the lamellar period (L0) of the structure. When L0 is plotted against the number of carbon atoms contained in the alkyl side chain, a straight line with a slope of 0.11 was obtained (Figure 7). The steady increase of 0.11 nm per CH2 is compatible with an arrangement of the alkyl side chains in a nearly extended conformation oriented normal to the lamellar surface and more or less interdigitized. This behavior would be similar to that displayed by nATMA‚PAGA complexes although the slope of the line is slightly different in each case indicating that the extent of the interdigitation does not follow exactly the same pattern in the two cases. Furthermore, the fact that L0 values measured for 12 and 14 fit well in the straight line of the series in spite that the side chain in these complexes is in the uncrystallized state indicates that the conformation of the alkyl group must be nearly extended in these two members too. A parallel straight line displaced along the ordinates was obtained for the nATMA‚Br salts according to what should be expected from the space occupied by the bromide anions as compared to the anionic polyglutamate chain. Furthermore, the same data observed for covalently bonded poly(R-alkyl γ,L-glutamate)s5b have been also plotted to illustrate the structural similitude that exists between both series. An essential feature of the structure of polypeptidesurfactant complexes is the conformation adopted by the polypeptide main chain. In nATMA‚PAGA complexes, the main chain is reported to be in the R-helix conformation, which is the usual arrangement adopted by poly(R-glutamic acid) and their esters. It was many years ago when Rydon16 put forward a helix intramolecularly stabilized by hydrogen bonds similar to the R-helix as the most probable conforma-



Figure 4. DSC traces of nATMA‚PGGA complexes. Table 4. Thermal Behavior of nATMA‚PGGA

n Tm (°C) ∆H (kcal mol-1) % CH2 cryst T0d (°C)a b Tm d (°C)

12

268 288

14

16

18

20

22

268 289

36 2.0 13 245 270

50 3.9 22 278 302

62 5.7 29 246 275

66 8.2 37 245 255, 300

a Onset decomposition temperature. b Decomposition temperature corresponding to the maximum of the derivative curve.

tion for poly(γ-glutamic acid) in the un-ionized state, and a recent molecular mechanics study has confirmed the correctness of this initial proposal.17 On the other side, different helical conformations, all of them based on the occurrence of intramolecular H bonds, have been described for several esters of PGGA,18,19 as well as for several oligo(γ-peptide)s with an heterogeneous composition in amino acids.20 In line with all of these antecedents, we looked for evidence in support of the occurrence of a helical structure in nATMA‚PGGA complexes. The fact that these complexes are soluble in chloroform suggests that hydrogen bonds should be intramolecularly set in a regularly folded arrangement. Unfortunately, no clear sign of the conformation adopted by the main chain is reflected in the X-ray diffraction patterns recorded from the complexes, most likely due to the masking effect produced by the strong scattering arising from the layered structure. The weak scattering of ∼0.5 nm, occasionally detected in the fiber diffraction patterns of nATMA‚PGGA, is the only piece of evidence detected in this regard. This weak reflection could be interpreted as arising from the pitch of a helix of the type reported for poly(γ-glutamate)s.18 Nevertheless, polarized infrared spectroscopy, which has proven to be an efficient technique for the characterization of helical conformations in polypeptides,12,21 afforded contradictory results. The infrared spectra

of an oriented film of 12ATMA‚PGGA recorded with the polarization vector parallel and perpendicular to the stretching direction are compared in Figure 8, and the dichroic ratios measured for the most characteristic bands appearing in the spectra of 12 and 18ATMA‚PGGA complexes are given in Table 2. Although absorbance differences between parallel and perpendicular spectra are weak, it is possible to detect that both amide A and amide I bands display perpendicular dichroism. This result is contrary to the presence of any arrangement of R-helix type but more consistent with the occurrence of an extended conformation with intermolecular hydrogen bonds running normal to the stretching direction. It is noticed however that the distance between successive carboxylate groups in fully extended PGGA (∼0.6 nm) largely exceeds the interchain distance of the hexagonally packed polymethylene chains (∼0.48 nm). It should be expected therefore that the polypeptide backbone is somewhat folded in order to accommodate the paraffinic phase. This distortion of the fully extended conformation could explain the weak dichroism signal that is observed for these complexes. Alkyl Side Chain Melting: Structural Changes. The changes taking place in the layered structure of nATMA‚ PGGA complexes by effect of heating were finally examined. As described above, DSC data revealed the occurrence of a reversible heat exchange process in the 40-70 °C range for members with n g 16, which is clearly associated to the melting-crystallization of the alkyl side chains. The process was followed by powder X-ray diffractometry, which revealed that the sharp reflection at 0.42 nm defining the paraffinic hexagonal crystal phase disappeared upon heating above the respective melting temperatures, and reappeared upon cooling. A broad peak at 0.45 nm characteristic of the average intermolecular distance of a disordered arrangement

150



Figure 5. Polarizing optical micrographs of nATMA‚PGGA. (a) 12ATMA‚PGGA, mosaic texture. (b) 12ATMA‚PGGA, fanlike texture. (c) 14ATMA‚ PGGA, batonnet texture. (d) Spherulites of 20ATMA‚PGGA. Table 5. X-ray Diffraction Spacings (nm) of nATMA‚PGGA Complexesa side chain

lamella

nATMA‚PGGA

20 °C

80 °C

20 °C

80 °C

12ATMA‚PGGA 12ATMA‚Br 14ATMA‚PGGA 14ATMA‚Br 16ATMA‚PGGA 16ATMA‚Br 18ATMA‚PGGA 18ATMA‚Br 20ATMA‚PGGA 20ATMA‚Br 22ATMA‚PGGA 22ATMA‚Br Na‚PGGA

0.45w (br) 0.39, 0.35 0.45m (br) 0.39, 0.35 0.45m (br) 0.39, 0.35 0.42m 0.39, 0.35 0.42m 0.39, 0.35 0.42m 0.39, 0.35

0.45 (br)

3.20s, 1.63 vw 2.10, 1.05 3.50s 2.28, 1.14 3.70s 2.60, 1.30 3.85s, 1.92m 2.70, 1.35 4.15s, 1.92m 2.70, 1.35 4.30s, 1.92m 2.70, 1.35

3.20

0.45 (br) 0.45 (br) 0.45 (br) 0.45 (br) 0.45 (br)

3.50 3.85 4.15 4.25 4.40

a Intensities visually estimated and denoted as s ) strong, m ) medium, w ) weak, vw ) very weak, br ) broad.

Figure 6. (a) X-ray diffraction pattern of 18ATMA‚PGGA. The stretching direction is vertical. (b) ED of a film of 18ATMA‚PGGA taken with the electron diffraction beam normal to the film surface. Note that the scale is different for each picture.

of chains, roughly aligned in parallel, was the only scattering perceived at high temperatures. The diffraction profiles recorded from 18ATMA‚PGGA at temperatures increasing

from 20 up to 150 °C are compared in Figure 9. The weak sharp peak appearing at 0.39 nm probably arises from contaminating 18ATMA‚Br surfactant that has crystallized in a separated phase. Dissemination of this material would occur upon melting which avoids its recrystallization upon cooling. Melting of the paraffinic phase would imply a trans-togauche transition in the dihedrals of the polymethylene segments included in the crystalline core of the structure. It is well documented that structural changes in comblike polymers induced by thermal effects can be followed by 13C CP-MAS NMR spectroscopy.22 The changes taking place in the 13C NMR spectrum of 18ATMA‚PGGA upon heating and cooling are shown in Figure 10. At 20 °C, the peak arising from the inner methylenes of the alkyl side chain in


Figure 7. Interlayer distance of the biphasic structure as a function of the number of carbon atoms in the alkyl side chain in PGGA complexes, surfactant salts, and in the poly(R-alkyl γ,D-glutamate)s (PAADG-n) (ref 5b).

Figure 8. Infrared spectra of 18ATMA‚PGGA complexes recorded with the polarization vector parallel (dotted line) and perpendicular (solid line) to the orientation axis.

Figure 9. Thermodiffractograms of 18ATMA‚PGGA complex at the indicated temperatures.

the trans conformation appeared at 32 ppm, whereas at 90 °C, the peak is upfield displaced to 30 ppm as it should be expected for a polymethylene chain undergoing a fast transition between the trans and gauche conformers. At intermediate temperatures, both peaks are present due to the


Figure 10. 13C CP-MAS NMR spectra of 18ATMA‚PGGA at the indicated temperatures.

Figure 11. 13C CP-MAS NMR spectra of nATMA‚PGGA complexes recorded at 20 °C.

broad temperature range of the melting process. Upon cooling to 20 °C, the initial spectrum is fully recovered. This pattern of behavior is similar to that described for the A-B transition happening in comblike poly(R-alkyl β,L-aspartate)s4a and poly(R-alkyl γ-glutamate)s5b by effect of heating. On the other hand, comparison of the 13C CP-MAS NMR spectra at 20 °C for the whole series of nATMA‚PGGA (Figure 11) revealed differences in the conformation of the alkyl side chain according to its length in agreement with DSC and X-ray diffraction data. Chains with n e 16 show the inner methylenes resonance at a chemical shift of 30 ppm consistent with a fast equilibrium of trans-gauche conformers characteristic of the molten state, whereas a trans peak at lower field is observed for higher values of n. Melting-crystallization of the paraffinic phase in comblike polypeptides is known to entail a variation in the long period of the structure. The extent and sign of such a variation depend on the length of the alkyl side chain and the nature of the main polypeptide chain. Thus, a contraction of about 10% was reported to be associated with the A-B transition occurring in poly(R-alkyl β,L-aspartate)s,4a whereas either an expansion or a contraction of comparable magnitude was

152


observed for such a transition in poly(R-alkyl γ-glutamate)s depending on the enantiomeric composition of the polypeptide.5b Specifically, a contraction of ∼6% of the layer period was reported to take place in poly(R-octadecyl γ,D-glutamate) upon side chain melting.5b In the case of nATMA‚PGGA, the diffractograms at temperatures below and above the melting temperature (Figure 9) showed a displacement of the small-angle peak indicating that side chain melting in these complexes involves an expansion of the structure. The specific changes in the long period measured for every member of the series are compared in Table 5. The difference in the dimensional response to heating observed for these complexes with respect to that showed by the corresponding covalently bonded comblike systems is not readily explainable. Further investigation by simulation and dynamic molecular methods is under course to elucidate this striking difference in the thermal behavior. Concluding Remarks This work shows that ionically bonded comblike poly(γ-glutamate)s can be easily prepared by complexation of biosynthetic poly(γ-glutamic acid) with cationic surfactants containing linear alkyl groups. The complexes appear to be thermally stable up to 250 °C. The composition of the complexes is nearly stoichiometric indicating that ionic charges of opposite sign should be cooperatively canceled. The arrangement adopted by these complexes in the solid state is the biphasic layered structure described for long linear alkyl esters of poly(R-glutamate)s, poly(γ-glutamate)s, and poly(β-aspartate)s. In this case also, the alkyl side chains were able to crystallize provided that they achieve a critical length, but the main polypeptide chain seems to abandon the helical conformation usually adopted by the polyacid and their esters. The dependence of the structural parameters on chain length, and the structural changes provoked by heating, follow the same pattern as in covalent comblike polypeptides. However, the sign of the dimensional change taking place upon melting is opposite to what should be expected from the enantiomeric composition of the PGGA used in this work. The relative small effort required for the preparation of these complexes and the possibility of splitting them by modifying either the polarity or the pH of the medium are unique advantages that make them specially attractive as nanostructured materials for advanced applications. Acknowledgment. Financial support for this work was provided by MCYT with Grant PB-99-0490. One of the


authors (G.P.C.) acknowledges the fellowship given by AECI. References and Notes (1) Plate´, N. A.; Shibaev, V. P. J. Polym. Sci., Macromol. ReV. 1974, 8, 117. (2) Loos, K.; Munõz-Guerra, S. Microstructure and Crystallization of rigid coil comblike polymers and block copolymers. In Supramolecular Polymers; Ciferri, A., Ed.; Marcel Dekker: New York, 2000. (3) (a) Watanabe, J.; Ono, H.; Uematsu, I.; Abe, A. Macromolecules 1985, 18, 2141. (b) Iizuka, E.; Abe, K.; Hanabusa, K.; Shirai, H. In Current Topics in Polymer Science; Ottembrite, R. M., Ed.; Carl Hanser Verlag: Munich, 1987. (c) Tsujita, Y.; Watanabe, T.; Takizawa, A.; Kinoshita, T. Polymer 1991, 32, 569. (d) Daly, W. H.; Poche´, D.; Negulescu, I. Prog. Polym. Sci. 1994, 19, 79. (4) (a) Lo´pez-Carrasquero, F.; Montserrat, S.; Martıńez de Ilarduya, A.; Munõz-Guerra, S. Macromolecules 1995, 28, 5535. (b) Munõz Guerra, S.; Lo´pez-Carrasquero, F.; Alemań, C.; Morillo, M.; Castelletto, V.; Hamley, I. AdV. Mater. 2002, 14, 203. (5) (a) Morillo, M.; Martıńez de Ilarduya, A.; Munõz-Guerra, S. Macromolecules 2001, 34, 7868. (b) Morillo, M.; Alla, A.; Martıńez de Ilarduya, A.; Munõz-Guerra, S. Macromolecules 2003, 36, 7567. (6) Thu¨nemann, A. F. Prog. Polym. Sci. 2002, 27, 1473. (7) (a) Ponomarenko, E. A.; Waddon, A. J.; Bakeev, K. N.; Tirrell, D. A.; MacKnight, J. Macromolecules 1996, 29, 4340. (b) Ponomarenko, E. A.; Waddon, A. J.; Tirrell, D. A.; MacKnight, J. Langmuir 1996, 12, 2169. (8) (a) Wang, M.; Zhang, G.; Chen, D.; Jiang, M.; Liu, S. Macromolecules 2001, 34, 7172. (b) Borfanou, K.; Topouza, D.; Sakellaniou, G.; Pispas, S. J. Polym. Sci., Polym Chem. 2003, 41, 2454. (9) Gross, R. A. Bacterial Poly(γ-glutamic acid). In Biopolymers from Renewable Resources; Kaplan, D. L., Ed.; Springer: Berlin, 1998; p 195. (b) Pe´rez-Camero, G.; Melis-Viotti, J.; Bou, J.; Congregado, F.; Munõz-Guerra, S. Polym. Preprints 1998, 39 (2), 138. (10) Pe´rez-Camero, G.; Congregado, F.; Bou, J.; Munõz-Guerra, S. J. Biotechnol. Bioeng. 1999, 63, 110. (11) Hendrix, W. T.; von Rosenberg, J. L. J. Am. Chem. Soc. 1976, 98, 4850. (12) Ingwald, R. T.; Gilon, C.; Goodman, M. J. Am. Chem. Soc. 1975, 97, 4356. (13) Broadhurst, M. G. J. Res. Natl. Bur. Stand. 1962, 66A, 241. (14) Melis, J. I.; Morillo, M.; Martıńez de Ilarduya, A.; Munõz-Guerra, S. Polymer 2001, 42, 9319. (15) Szulzewsky, K.; Schulz, B.; Vollardt, D. Cryst. Res. Technol. 1983, 18, 1003. (16) Rydon, H. N. J. Chem. Soc. 1964, 1328. (17) Zanuy, D.; Alemań, C.; Munõz-Guerra, S. Int. J. Biol. Macromol. 1998, 2591. (18) Puiggalı´, J.; Munõz-Guerra, S.; Rodrı´guez-Galań, A.; Alegre, C.; Subirana, J. A. Makromol. Chem., Macromol. Symp. 1988, 20/21, 167. (19) Melis, J.; Zanuy, D.; Alemań, C.; Garcıá-Alvarez, M.; Munõz-Guerra, S. Macromolecules 2002, 35, 8774. (20) Hintermann, T.; Gademann, K.; Jaun, B.; Seebach, D. HelV. Chim. Acta 1998, 81, 983. (21) Lo´pez-Carrasquero, F.; Alemań, C.; Munõz-Guerra, S. Biopolymers 1994, 36, 263. (22) Mathias, L. J. Polym. Commun. 1988, 29, 352.

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Comblike Complexes of Bacterial Poly(Î³,d-glutamic acid) - American

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