Crystal Structure and Morphology of Poly (12-dodecalactone)

The lozenge-shaped crystals with and without spiral growth have been studied by transmission electron microscopy and atomic force microscopy. Wide-ang...
0 downloads 0 Views 664KB Size
Biomacromolecules 2005, 6, 572-579

572

Crystal Structure and Morphology of Poly(12-dodecalactone)† Eunju Kim,‡,§ Hiroshi Uyama,| Yoshiharu Doi,§ Chang-Sik Ha,‡ and Tadahisa Iwata*,§ Department of Polymer Science and Engineering, Pusan National University, Pusan 609-735, Korea, Polymer Chemistry Laboratory, RIKEN Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan, and Graduate School of Engineering, Osaka University, 2-1 Yamadagaoka, Suita, Osaka 565-0871, Japan Received August 31, 2004; Revised Manuscript Received October 15, 2004

Poly(12-dodecalactone) (PDDL) crystals in the form of chain-folded lamellae were prepared by isothermal crystallization from a 1-hexanol solution. The lozenge-shaped crystals with and without spiral growth have been studied by transmission electron microscopy and atomic force microscopy. Wide-angle X-ray diffraction data, obtained from PDDL lamellae sedimented to form oriented mats and annealed solvent-cast film, were supplemented with morphological and structural data from electron microscopy. PDDL crystallizes as an orthorhombic form with a P212121 space group and lattice constants of a ) 0.746 ( 0.001 nm, b ) 0.500 ( 0.001 nm, and c (chain axis) ) 3.281 ( 0.003 nm. There are two chains per unit cell, which existed in an antiparallel arrangement. The fiber repeat distance is appropriate for an all-trans backbone conformation for the straight stems. Molecular packing of this structure has been studied in detail, taking into account both diffraction data and energy calculations. The setting angles, with respect to the a axis, were (43° for the corner and center chains according to intensity measurements and structure factor calculations. The optimized shift along the crystallographic c axis is 0.1c (0.328 nm). A final model was obtained to yield R ) 0.180 with X-ray diffraction data and R ) 0.162 with electron diffraction data. A brief comparison is also made with related polymer structures. Introduction Biodegradable polymers, regardless of whether they are synthesized by chemical or microbial methods, have been attracting considerable attention because of their potential applications in the fields related to human life such as the environmental protection and the maintenance of physical health. Basic research on the relationship between structure, morphology, and properties as well as efforts to understand the biodegradation mechanism will enable us to design and synthesize a great variety of biodegradable polymers to fulfill the demands in practical applications. The resources of biodegradable plastics and polymers are classified into four groups: bacterial polymers, plant polysaccharides, animal proteins, and chemical synthetic polymers. Among the biodegradable polymers, aliphatic polyesters, produced by biosynthetic or chemosynthetic methods, are extensively studied on solid-state structure and physical properties. Especially, short-main-chain aliphatic polyesters, such as poly([R]-3-hydroxybutyrate),1-10 poly([R]-3-hydroxyvalerate) [P(3HV)],11-15 and poly(4-hydroxybutyrate) [P(4HB)],16-21 as well as poly(β-propiolactone) (PPL),22-24 poly(L-lactic acid),25-35 poly(δ-valerolactone) (PVL),36 and poly(-caprolactone) (PCL),37-43 are extensively investigated on their crystal and molecular structure by X-ray diffraction * To whom all correspondence should be addressed. Tel.: +81-48-4679586. Fax: +81-48-462-4667. E-mail: [email protected]. † This paper was presented at the ISBP 2004 (Interational Symposium on Biological Polyesters), held in Beijing, China, August 22-28, 2004. ‡ Pusan National University. § RIKEN Institute. | Osaka University.

of oriented film and electron diffraction of solution-grown single crystals. However, the synthesis of long-chain polylactone had been difficult before using the enzymatic polymerization. Enzymatic polymerizations provide a new synthetic method for polymers whose synthesis is difficult by conventional polymerization processes.44,45 Recently, enzymatic polymerizations have been expanded to lipase-catalyzed ring-opening polymerization and copolymerization of lactones.46-51 So far, medium-size lactones (six- and seven-membered) as well as macrolides (12-, 13-, 16-, and 17-membered) were enzymatically polymerized to produce aliphatic polyesters. Macrolides possess no strain in the ring52 and, hence, show lower reactivities, for example, alkaline hydrolysis and anionic polymerization,53 compared with medium-size lactones. Interestingly, however, these macrolides showed unusual high reactivity with lipase catalyst; the polymerization of the macrolides proceeded much faster to produce the polymer of higher molecular weight, up to 2.5 × 104, than that of -caprolactone. This behavior is due to the stronger recognition of the macrolide toward lipase. Among the biodegradable polyesters, the type -(-O-(CH2)m-CO-)n-, the structures of single crystals have been resolved for PPL (m ) 2),22-24 P(4HB) (m ) 3),16-21 PVL (m ) 4),36 PCL (m ) 5),37-43 poly(11undecalactone) (PUDL, m ) 10),54 and poly(ω-pentadecalactone) (PPDL, m ) 14).55 The interesting phenomena are that while PPL (m ) 2), PVL (m ) 4), PUDL (m ) 10), and PPDL (m ) 14) have the all-trans conformation, P(4HB) (m ) 3) and PCL (m ) 5) have a 21 helix conformation in the molecular chain. It seems that the number of methylene

10.1021/bm0494747 CCC: $30.25 © 2005 American Chemical Society Published on Web 12/15/2004

Crystal Structure and Morphology of PDDL

groups plays an important role in the conformation; that is, the molecular chain takes an all-trans or a 21 helix conformation when m is even or odd, respectively. Considering the number of methylene groups, poly(12dodecalactone) (PDDL, m ) 11) is adjacent with PUDL and PPDL; therefore, the molecular and crystal structure of PDDL will help us to understand the relationship of the molecular conformation with the methylene number of aliphatic polyesters. In this paper, the crystal and molecular structure of PDDL, synthesized by 13-membered macrolide, have been studied taking into account both diffraction data and energy calculations by using the Cerius2 program. Furthermore, the morphology of PDDL solution-grown single crystals was investigated by means of transmission electron microscopy (TEM) and atomic force microscopy (AFM). Experimental Section Materials. PDDL used in this study was synthesized by enzyme-catalyzed reactions in organic solvents.51 12-Dodecanolide was purchased from Aldrich. The monomer was purified by the addition of freshly activated type 4 molecular sieves. A total of 2 g of monomer and 100 mg of Novozym435 as catalyst were dissolved in 4 mL of absolutegrade toluene. The polymerization process was carried out at 70 °C for 24 h under an Ar atmosphere. The obtained polymer was dissolved in chloroform and precipitated from methanol to remove unreacted monomers and byproduct oligomers. Preparation of Single Crystals. A 0.5 mg sample (Mw ) 56 000, Mw/Mn ) 1.97) was dissolved into 5 mL of 1-hexanol at 80 °C and maintained at this temperature for 1 h. Slow cooling was then applied until the crystallization temperature, 57 °C, and the solution was kept there for 12 h. And then, the solution was cooled slowly until room temperature. The slow cooling was performed by cutting off the heating element of a silicone oil bath. Single crystals were recovered by centrifugation and repeatedly washed with methanol. X-ray Diffraction. Oriented crystal mats suitable for X-ray diffraction were prepared by sucking the crystal suspension through a 0.1 µm poly(tetrafluoroethylene) filter using a water aspirator. The wide-angle X-ray diffraction patterns were obtained at room temperature, using Ni-filtered Cu KR radiation of wavelength 0.154 18 nm, from a Rigaku RINT UltraX 18 sealed beam X-ray generator operating at 40 kV and 110 mA. The X-ray diffraction patterns were recorded using a point-collimated beam and an imaging plate holder in an evacuated camera. The X-ray diffraction pattern of the solvent-cast film annealed at 70 °C for 24 h of PDDL was recorded on an RINT 2500 operating at 40 kV and 110 mA with Ni-filtered Cu KR radiation. The scan was carried out in the θ/2θ reflection mode.6,56 The conditions of the scan are that a divergence aperture is 0.3°, receiving aperture is 0.05°, step width is 0.5°, count time is 60 s per step, and 2θ range is 2-60°. The observed intensities (Iobs) from the X-ray diffraction pattern were measured by using R-axis display software (Rigaku). The observed structure factor (Fobs) was taken as the square root of corresponding intensities.

Biomacromolecules, Vol. 6, No. 2, 2005 573 Chart 1. Definition of Atom Types in the PDDL, Used as the Basic Unit To Compute the Unit Cells

Density Measurement. The solvent-cast film of PDDL was crystallized at 70 °C for 24 h for density measurement. The density of the films was measured at 25 °C by the flotation method in NaI aqueous solutions of increasing concentration. TEM. Drops of crystal suspension were deposited on carbon-coated grids, allowed to dry, and then shadowed with Pt-Pd alloy. For electron diffraction purposes, the dried crystals were used directly without further treatments. These grids were observed with a JEM-2000FX II electron microscope operated at an acceleration voltage of 120 kV for electron diffraction and for the imaging of shadowed crystals. Calibration of the electron diffraction patterns was done after depositing the crystals on gold-coated grids. Electron diffraction diagrams and images were recorded on Mitsubishi MEM and Kodak 4489 films, respectively, developed for 4 min with GEKKOL and Kodak D19 developer, respectively. The observed intensities (Iobs) of electron reflections recorded on Fuji imaging plates were measured by using R-axis display software (Rigaku). The observed structure factor (Fobs) was taken as the square root of corresponding intensities. Nomenclature. A representation of the atomic labeling scheme is shown in Chart 1. The carbon number of PDDL is started from the carbonyl carbon. Model Building and Packing Analysis. The software package Cerius2, version 4.2 (Accelrys, Inc.), was used in the structural modeling and diffraction simulations. The basic strategy was to determine the molecular conformation of the PDDL chain and the molecular packing arrangement within the unit cell. After the initial model building stage, a combination of energy minimization and simulations of diffraction patterns was used. This ensured that the model was stereochemically sound and that the simulated diffraction patterns were in good agreement with experimental data. In the computer-simulated X-ray diffraction patterns, the degree of arcing and intensity were chosen to match the experimental electron and X-ray diffraction patterns as closely as possible. AFM. The lamellar thickness of single crystals of PDDL was investigated on the basis of AFM. AFM was performed with a SPA400/SPI3800N (Seiko Instruments, Inc., Japan) in a dynamic force microscope mode. A rectangular silicon cantilever (Si-DF3, 450 µm in length, resonant frequency of ca. 14 kHz, stiffness of ca. 0.15 N/m) was applied in all experiments. All measurements were carried out in ambient conditions. The resulting images were flattened and planefit using Seiko Instruments software. Molecular Weight Measurement. The molecular weights of PDDL were measured by gel permeation chromatography (GPC) at 40 °C consisting of a Shimadzu 10A GPC system and a 6A refractive index detector with joint columns of Shodex K-80M and K-802 (each 4.6 × 300 mm). Chloroform was used as an eluent at a flow rate of 0.8 mL/min.

574

Biomacromolecules, Vol. 6, No. 2, 2005

Kim et al.

Figure 3. X-ray diffraction patterns of PDDL of annealed solventcast film. Table 1. Crystallographic Data for PDDL Deduced from the Electron Diffraction Diagram of the Single Crystals Figure 1. Selected-area (hk0) electron diffraction taken with the beam normal to the PDDL lamellar single crystal’s surface.

hk0a

dobs (nm)

dcalc (nm)

|Fobs|b

|Fcalc|

110 200 210 020 120 310 220 400 320 130 230 420 510 330

0.421 0.380 0.300 0.250 0.237 0.224 0.208 0.187 0.176 0.162 0.151 0.149 0.142 0.137

0.415 0.373 0.299 0.250 0.237 0.223 0.208 0.187 0.176 0.163 0.152 0.150 0.143 0.138

258.5 214.5 20.5 121.2 25.1 86.3 70.3 63.7 20.8 40.9 20.9 28.7 27.8 19.6

241.4 211.4 62.5 122.2 38.6 82.0 70.3 67.3 58.2 56.5 37.4 24.0 32.9 20.6

a Indexed in terms of an orthorhombic unit cell with parameters a ) 0.746 nm, b ) 0.500 nm, and γ ) 90°. b Fobs ) (Iobs)1/2, ∑Iobs ) ∑Icalc.

Figure 2. Wide-angle X-ray diffraction pattern obtained from oriented, sedimented single-crystal mats of PDDL. The inset shows the first and second order in the low-angle X-ray diffraction region.

The number- and weight-average molecular weights (Mn and Mw) were calculated by using a Shimadzu Chromatopac C-R7A plus equipped with a GPC program. The molecular weight was obtained with polystyrene standards of low polydispersities. Results and Discussion Space Group and Unit Cell Determination. The unit cell parameters were determined through the electron diffraction diagram of single crystals prepared from 1-hexanol (Figure 1), X-ray diffraction diagram of the single-crystal mat (Figure 2), and X-ray diffraction pattern of the crystallized film (Figure 3). The electron diffraction diagram contains 14 independent diffraction spots mirrored in the four quadrants, defined by the two orthogonal axes a* and b*. We believe

this diffraction pattern is the weighted hk0 reciprocal lattice; that is, the chains are normal to the crystal surface. Along these two axes, systematic absences occur at every odd reflection. Thus, this diffraction pattern suggests that the structure projected on the ab plane has the p2gg symmetry. On calibration of the electron diffraction spots, the diffraction pattern can be indexed in terms of the rectangular lattice with the parameters a ) 0.746 nm, b ) 0.500 nm, and γ ) 90°. A comparison of the observed and calculated d spacings in the electron diffraction diagram is shown in Table 1. The X-ray diffraction diagram obtained from an oriented, sedimented mat of PDDL single crystals prepared from 1-hexanol is shown in Figure 2, and the observed diffraction spacings are listed in Table 2. The equatorial reflections (hk0) are indexed on a rectangular net with a ) 0.746 nm, b ) 0.500 nm, and γ ) 90°, which are identical with the results obtained from electron diffractogram of an individual lamellar crystal; that is, the straight-stem segments are orthogonal to the lamellar surface. The meridional 002 and 004 signals are inset in Figure 2. A meridional diffraction signal occurs at a spacing of 1.641 nm, and other hkl diffraction signals indicate that they lie on a layer line with this spacing. Because the X-ray diffraction diagram presents only even diffraction lines along the meridian and the electron diffrac-

Biomacromolecules, Vol. 6, No. 2, 2005 575

Crystal Structure and Morphology of PDDL Table 2. Crystallographic Data for PDDL Deduced from the X-ray Diffraction Pattern of the Sedimented Mat of the Lamellar Single Crystals

hkla

dobs (nm)

dcalc (nm)

hkla

dobs (nm)

dcalc (nm)

110 200 210 310 220 400 410

0.416 0.370 0.284 0.225 0.207 0.191 0.172

0.415 0.373 0.299 0.223 0.208 0.187 0.175

002 004 1,2,10 1,2,12 1,2,14 1,2,18

1.611 0.796 0.193 0.180 0.172 0.151

1.641 0.820 0.192 0.179 0.167 0.145

a Indexed in terms of an orthorhombic unit cell with parameters a ) 0.746 nm, b ) 0.500 nm, and c (fiber axis) ) 3.281 nm.

Table 3. Crystallographic Data for PDDL Deduced from the X-ray Diagram of the Annealed Solvent-Cast Film

hkla

dobs (nm)

dcalc (nm)

|Fobs|

|Fcalc|

004 006 013 110 200 114 214 216 020 120 310 400 3,1,13 130

0.824 0.548 0.458 0.416 0.374 0.371 0.281 0.263 0.250 0.237 0.222 0.188 0.167 0.163

0.820 0.547 0.455 0.415 0.373 0.371 0.281 0.262 0.250 0.237 0.223 0.187 0.167 0.163

31.6 33.2 37.4 432.3 234.5 38.7 24.5 34.6 40.0 34.6 37.4 30.0

16.5 35.2 43.0 395.0 213.7 38.6 17.6 29.1 78.8 35.8 73.2 32.0 23.2 34.0

} 33.2

Indexed in terms of an orthorhombic unit cell with parameters a ) 0.746 nm, b ) 0.500 nm, and c (fiber axis) ) 3.281 nm.

Figure 4. Transmission electron micrographs after shadowing with Pt-Pd alloy of PDDL lamellar single crystals: (A) multilayer lozengeshaped crystals and (B) lozenge-shaped crystals with screw dislocation.

tion pattern is consistent with p2gg symmetry, it can be concluded that PDDL crystals have the P212121 space group. The wide-angle X-ray diffraction pattern obtained from a PDDL crystallized film is shown in Figure 3. It is characterized by two strong reflections at 2θ equal to 21.35 and 23.80° corresponding to d ) 0.416 and 0.374 nm indexed by (110) and (200), respectively. There is a close similarity between the X-ray diffraction pattern of PDDL and those of PPL,24 PVL,36 PCL,37,38 and PUDL.54 The observed d spacings in the X-ray diffraction pattern are listed in Table 3. On the basis of the careful measurements of X-ray and electron diffraction patterns, the unit cell dimensions were calculated by a least-squares procedure. The X-ray diffraction pattern has a lower resolution, only 0.163 nm, than the electron diffraction diagram that displays diffraction spots down to 0.137 nm spacing. PDDL crystallizes as an orthorhombic form with a P212121 space group and lattice constants of a ) 0.746 ( 0.001 nm, b ) 0.500 ( 0.001 nm, and c (chain axis) ) 3.281 ( 0.003 nm. The calculated density for this unit cell is 1.075 g/cm3, which is fairly close to the observed density of 1.042 g/cm3. There are two chains in one unit cell, packed in an antiparallel arrangement, where each chain contains two residues. The refined a and b lattice parameters of PDDL (a ) 0.746 nm and b ) 0.500 nm) are very close to those of PVL (a ) 0.502 nm and b ) 0.747 nm),36 PUDL (a ) 0.743 nm and b ) 0.499 nm),54 PPDL (a ) 0.749 nm and b ) 0.503 nm)55 and only slightly larger than those of PPL (a ) 0.700 nm

and b ) 0.490 nm),24 indicating that the lateral cell expansion due to the CdO groups does not depend on the number of methylene units in the chain. On the other hand, the chain length of the 2-residue unit increased with an increase in the number of methylene units in the chain, such as 0.986 nm for PPL (m ) 2),24 1.199 nm for P(4HB) (m ) 3),21 1.484 nm for PVL (m ) 4),35 1.757 nm for PCL (m ) 5),38 3.038 nm for PUDL (m ) 10),53 3.281 nm for PDDL (m ) 11, in this paper), and 4.000 nm for PPDL (m ) 14),54 because of the molecular chain having an almost planar zigzag conformation. Chain-Folded Solution-Grown Single Crystals. Typical preparations of lamellar single crystals grown from 1-hexanol are shown in Figure 4. Lamella crystals occur as platelets of lozenge shape with dimensions of around 5 µm for the a axis and 3-4 µm for the b axis. The crystallographic a and b axes were determined by the triple exposure of selectedarea electron diffraction and the normal and selected-area images. One kind of lamellar crystals shown in Figure 4A was observed as multilayer platelets without screw dislocation, while other lamellar crystals were contained many layers from screw dislocation (Figure 4B). In general, the crystals taper to a point, often with a half angle of approximately 36° relative to the long axis of the crystals. There seems to be a correlation between this value and an angle of 36° between the crystallographic diagonal {110} planes and the a axis on the unit cell.

a

576

Biomacromolecules, Vol. 6, No. 2, 2005

Kim et al.

Figure 5. AFM images of PDDL lamellar single crystals and line profile data trace along a white line: (A) pyramidal-like lozenge-shaped crystals and (B) lozenge-shaped crystals with screw dislocation.

AFM images of the lozenge-shaped single crystals without and with screw dislocation are shown in parts A and B of Figure 5, respectively. The investigation of numerous lamellar single crystals demonstrated that screw dislocation patterns of both right- and left-handedness occur randomly as shown in Figure 5B. Until now, it was believed that the main chirality of the molecular chain is involved in the determination of the handedness of screw dislocations in lamellar crystals. Recently, Iwata and Doi15 and Saracovan et al.14 concluded that the handedness of screw dislocations were not affected by the main-chain chirality on the basis of the observation of single crystals of (R)- and (S)-poly(epichlorohydrine), (R)- and (S)-poly(propylene oxide), and P(3HV). In the case of PDDL, the molecular chain has no chirality, as is the case for poly(ethylene succinate)57 and PUDL54 that have spiral growths originating from screw dislocations with both left- and right-handed forms. These results support our hypothesis that the mainchain chirality does not affect the direction of screw dislocation. The lamellar thickness of PDDL single crystals is about 10-12 nm despite the different morphologies. Taking the length of the all-trans conformation of PDDL molecules and molecular weight into consideration, the average contour length of our PDDL chains was estimated to be 460 nm, and, therefore, the molecular chains of PDDL must be folded within the lamella. Lozenge-shaped morphology is identical with those of P(3HV),15 P(4HB),21 polyethylene,58 n-alkane,59

poly(3,3-bis(chloromethyl oxacyclobutane)),60 and so forth. On the basis of the previous reports for the chain-folding direction of lozenge-shaped single crystals, the chain-folding mainly occurs along the {110} planes. Chain-Packing Analysis into the Unit Cell. The refinement of the structure was undertaken in stages and involved detailed stereochemical packing calculations, coupled with matching of the intensity values, from both electron diffraction and X-ray diffraction patterns. In the first instance, the molecular models were constructed by using the following bond lengths and bond angles: O1sC1 ) 0.135 nm, C1sC2 ) 0.151 nm, C1dO2 ) 0.123 nm, CnsCn+1 ) 0.154 nm (n ) 2-11), C12sO1 ) 0.148 nm, ∠O1sC1dO2 ) 125.0°, ∠O1sC1sC2 ) 114.0°, ∠O2dC1sC2 ) 121.0°, ∠C1sC2sC3 ) 113.0°, ∠C11sC12sO1 ) 115.0°, ∠C12sO1sC1 ) 116.0°, and ∠CnsCn+1sCn+2 ) 112.5° (n ) 2-10). When the chains were kept in an all-trans conformation, the end-to-end distance of two residues is 3.295 nm, which is almost the same as the observed fiber repeat distance (3.281 ( 0.003 nm). Accordingly, the alltrans conformation was selected for determining the setting angles for the intensity distribution given by the electron diffraction hk0 data (Table 1). Packing of polymer chains in the crystal including setting angle orientations, and the relative shift of molecules in the unit cell are often studied taking into account both experimental diffraction data and energy calculation. A reliability factor (R-factor, R ) ∑||Fobs| - |Fcalc||/∑|Fobs|) study was

Crystal Structure and Morphology of PDDL

Biomacromolecules, Vol. 6, No. 2, 2005 577

Figure 6. Reliability factor (R-factor) and energy as a function of the setting angles with respect to the a axis. The observed intensities of the electron diffraction diagram as listed in Table 1 were used to calculate the R-factor.

Figure 7. Reliability factor (R-factor) as a function of the changed position along the c axis. The observed intensities of the X-ray diffraction as listed in Table 3 were used to calculate the R-factor.

carried out to determine the most favorable setting angle. For this purpose, the setting angle, θ, with respect to the a axis, was varied from 0 to 180° in steps of 10°, and the R-factor and energy were evaluated. Figure 6 shows the R-factor against electron diffraction data and energy as a function of the setting angle θ with respect to the a axis. The most favorable arrangement corresponded to θ ) 40 and 140° as for the R-factor. Accordingly, the R-factor and setting angle map was recomputed using finer grid steps, 1°, from 35 to 45°. The results showed that the most favorable arrangement was 43° with respect to the a axis, as shown in Figure 9A. A comparison of the observed and calculated structure factors is given in Table 1, and as a test for the agreement between the observed and calculated intensities, the calculated reliable index R was 0.162. The translation along the molecular axis was carried out against X-ray diffraction intensity data. The shift parameter along the molecular axis was changed from 0 to 0.5c (c ) 3.281 nm) at the rate of 0.025c (as shown in Figure 7). Because the unit cell has twofold screw symmetry along the a and b axes and two antiparallel chains exist in it, this translation of up to 0.5c covers all translation positions. The optimized shift value along the molecular axis was 0.10c. A comparison of the observed and calculated structure factors

Figure 8. (A) Calculated weighted hk0 reciprocal lattice pattern from the chain-folded PDDL lamellar crystal and (B) computer-simulated wide-angle X-ray diffraction pattern of PDDL lamellar single-crystal mats.

is given in Table 3, and as a test for the agreement between the observed and the calculated intensities, the calculated reliable index R was 0.180. Little particular improvement in the goodness of fit was obtained by the introduction of perturbations into the alltrans backbone. Certainly, the measured fiber repeat distance of 3.281 ( 0.003 nm matched the value found from the modeling of the all-trans conformation, though PDDL has a 21 helix conformation determined by the X-ray fiber diagram. Therefore, it suggests that the backbone of PDDL has the 21 helix conformation close to all-trans. In fact, it was confirmed by the Cerius2 program that the torsion angle could be controlled in the region of 180 ( 3°, close to all-trans. An all-trans, fully extended P(4HB) (m ) 3) chain with a 2-residue unit would be 1.240 nm, as reported by Pazur et al.18 The P(4HB) crystal with a fiber repeat of 1.199 nm does not involve the fully extended chain. It is 3.3% shorter

578

Biomacromolecules, Vol. 6, No. 2, 2005

Kim et al. Table 4. Fractional Atomic Coordinates of PDDL atom

x

y

z

O1 O2 C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12

-0.0362 +0.1561 +0.0355 -0.0454 +0.0386 -0.0454 +0.0386 -0.0455 +0.0386 -0.0455 +0.0385 -0.0455 +0.0385 -0.0455

+0.0502 -0.2169 -0.0494 +0.0632 -0.0535 +0.0632 -0.0536 +0.0632 -0.0536 +0.0632 -0.0536 +0.0632 -0.0536 +0.0632

+0.5368 +0.5728 +0.5713 +0.6095 +0.6484 +0.6873 +0.7262 +0.7651 +0.8040 +0.8429 +0.8817 +0.9206 +0.9595 +0.9984

the molecular chain might be avoided by the slight deviation ((3°) from all-trans. A final model was obtained which yielded R ) 0.180 with X-ray diffraction data and R ) 0.162 with electron diffraction data. The lists of observed and calculated structure factors with the final model are presented in Tables 1 and 3 for electron and X-ray diffraction data, respectively. The calculated weighted reciprocal hk0 lattice, shown in Figure 8A, matches the equivalent experimental pattern obtained from an individual lamellar crystal in Figure 1. The computersimulated X-ray diffraction pattern is shown in Figure 8B, which can be compared with the experimental X-ray diffraction pattern shown in Figure 2. The list of the fractional coordinates is presented in Table 4, and the packing of the chain in the crystal is shown in the projection in Figure 9. The repetitive segment in the chain-folded lamellae can, therefore, be defined as follows: from the lamellar core center of a (unit cell) corner chain through a fold, through an antiparallel center chain, through a second fold, and back to the center of another corner chain. We know a priori that chain-folding must occur within the diagonal {110} planes. Conclusion Figure 9. Projection of the PDDL structure in the ab plane (A), in the ac plane (B), and in the bc plane (C).

than that expected for the all-trans planar structure; therefore, the molecule is slightly twisted in the unit cell.21 While an all-trans, fully extended PCL (m ) 5) chain with a 2-residue unit is 1.757 nm, the PCL crystal with nonplanar conformation (21 helix conformation) has the length of 1.705 nm,38 which is the 3.0% shorter value than that of the all-trans. In the case of PDDL (m ) 11), when the chains were kept in the all-trans conformation, the backbone of PDDL has 3.295 nm. The fiber repeat distance of PDDL obtained from the X-ray fiber diagram is 3.281 nm, which is only 0.4% shorter than that of the all-trans conformation. Accordingly, PDDL has an almost all-trans conformation despite the X-ray fiber diagram indicating a 21 helix conformation. When the number of methylene groups (m) is odd, the fiber repeat consists of 2-residues caused by the crystallographic reason, and the 21 helix conformation is energetically the most stable structure. However, because the long-chain polymer such as PDDL has many dihedral angles, the energetic distortion in

The crystal structure of PDDL has been determined by the analysis of X-ray and electron diffraction patterns of the annealed film and single-crystal specimens. The unit cell of PDDL is orthorhombic with a P212121 space group and lattice constants of a ) 0.746 ( 0.001 nm, b ) 0.500 ( 0.001 nm, and c (chain axis) ) 3.281 ( 0.003 nm. Lamellar single crystals are grown in 1-hexanol were lozenge-shaped. The {110} planes were the main growth planes of single crystals, and the average direction of chain-folding was parallel to these growth planes. The density supports a model containing two antiparallel chain segments, which have an all-trans conformation, in the unit cell. The refinement of the structure was undertaken in stages and involved detailed stereochemical packing calculations, coupled with matching of the intensity values, from both electron diffraction and X-ray diffraction patterns by the computer simulation program Cerius2. The setting angles, with respect to the a axis, were (43° for the corner and center chains, respectively. A final model yielded R ) 0.180 with X-ray diffraction data and R ) 0.162 with electron diffraction data.

Crystal Structure and Morphology of PDDL

Acknowledgment. E.K. was a recipient of a 2-year fellowship of Joint Graduate School Program of RIKEN Institute, Japan. This work has been supported by a grant for Ecomolecular Science Research to RIKEN Institute and a Grant-in-Aid for Young Scientists (A) No. 15685009 (2003) from the Ministry of Education, Culture, Sports, Science and Technology, Japan, to T.I. C.-S. H. thanks the National Research Laboratory Program, Korea. References and Notes (1) Lemoignei, M. Bull. Soc. Chim. Biol. 1926, 8, 770. (2) Lundgren, D. G.; Alper, R.; Schnaitman, C.; Marchessault, R. H. J. Bacteriol. 1965, 89, 245. (3) Okamura, K.; Marchessault, R. H. In Conformation of Biopolymer; Ramachandran, G. N., Ed.; Academic Press: London, 1967; Vol. 2, pp 709-720. (4) Cornibert, J.; Marchessault, R. H. J. Mol. Biol. 1972, 71, 735. (5) Yokouchi, M.; Chatani, Y.; Tadokoro, H.; Teranishi, K.; Tani, H. Polymer 1973, 14, 267. (6) Bruckner, S.; Meille, S. V.; Malpezzi, L.; Cesaro, C.; Navarini, L.; Tombolini, R. Macromolecules 1988, 21, 967. (7) Orts, W. J.; Marchessault, R. H.; Bluhm, T. L.; Hamer, G. K. Macromolecules 1990, 23, 5368. (8) Birley, C.; Briddon, J.; Sykes, K. E.; Barker, P. A.; Organ, S. J.; Barham, P. J. J. Mater. Sci. 1995, 30, 663. (9) Aoyagi, Y.; Doi, Y.; Iwata, T. Polym. Degrad. Stab. 2003, 79, 217. (10) Iwata, T.; Aoyagi, Y.; Fujita, M.; Yamane, H.; Doi, Y.; Suzuki, Y.; Takeuchi, A.; Uesugi, K. Macromol. Rapid Commun. 2004, 25, 1100. (11) Yokouchi, M.; Chatani, Y.; Tadokoro, H.; Tani, H. Polym. J. 1974, 6, 248. (12) Marchessault, R. H.; Morikawa, H.; Revol, J.-F.; Bluhm, T. L. Macromolecules 1984, 17, 1882. (13) Marchessault, R. H.; Debzi, E. M.; Revol, J. F.; Steinbuchel, A. Can. J. Microbiol. 1995, 41 (Suppl. 1), 297. (14) Saracovan, I.; Cox, J. K.; Revol, J.-F.; Manley, R. St. J.; Brown, G. R. Macromolecules 1999, 32, 717. (15) Iwata, T.; Doi, Y. Macromolecules 2000, 33, 5559. (16) Mitomo, H.; Kobayashi, S.; Morishita, N.; Doi, Y. Polym. Prepr., Jpn 1995, 44, 3156. (17) Kobayashi, S.; Kogure, K.; Mitomo, H.; Doi, Y. Polym. Prepr., Jpn 1998, 47, 968. (18) Pazur, R. J.; Raymond, S.; Hocking, P. J.; Marchessault, R. H. Polymer 1998, 39, 3065. (19) Nakamura, K.; Yoshie, N.; Sakurai, M.; Inoue, Y. Polymer 1994, 35, 193. (20) Su, F.; Iwata, T.; Sudesh, K.; Doi, Y. Polymer 2001, 42, 8915. (21) Su, F.; Iwata, T.; Tanaka, F.; Doi, Y. Macromolecules 2003, 36, 6401. (22) Wasai, T.; Saegusa, T.; Furukawa, J. Chem. Soc. Jpn., Ind. Chem. Sect. 1964, 67, 601. (23) Suehiro, K.; Chatani, Y.; Tadokoro, H. Polym. J. 1974, 7, 352. (24) Furuhashi, Y.; Iwata, T.; Sikorski, P.; Atkins, E.; Doi, Y. Macromolecules 2000, 33, 9432. (25) De Santis, P.; Kovacs, A. J. Biopolymers 1968, 6, 299.

Biomacromolecules, Vol. 6, No. 2, 2005 579 (26) Fischer, E. W.; Sterzel, H. J.; Wegner, G. Kolloid Z. Z. Polym. 1973, 251, 980. (27) Kalb, B.; Pennings, A. J. Polymer 1980, 21, 607. (28) Eling, B.; Gogolewski, S.; Pennings, A. J. Polymer 1982, 23, 1587. (29) Hoogsteen, W.; Postema, A. R.; Pennings, A. J.; ten Brinke, G.; Zugenmaier, P. Macromolecules 1990, 23, 634. (30) Kobayashi, J.; Asahi, T.; Ichiki, M.; Oikawa, A.; Suzuki, H.; Watanabe, T.; Fukada, E.; Shikinami, Y. J. Appl. Phys. 1995, 77, 2957. (31) Miyata, T.; Masuko, T. Polymer 1997, 38, 4003. (32) Iwata, T.; Doi, Y. Macromolecules 1998, 31, 2461. (33) Cartier, L.; Okihara, T.; Ikada, Y.; Tsuji, H.; Puiggali, J.; Lotz, B. Polymer 2000, 41, 8909. (34) Puiggali, J.; Ikada, Y.; Tsuji, H.; Cartier, L.; Okihara, T.; Lots, B. Polymer 2000, 41, 8921. (35) Sasaki, S.; Asakura, T. Macromolecules 2003, 36, 8385. (36) Furuhashi, Y.; Sikorski, P.; Atkins, E.; Iwata, T.; Doi. Y. J. Polym. Sci., Part B: Polym. Phys. 2001, 39, 2622. (37) Bittiger, H.; Marchessault, R. H. Acta Crystallogr., Sect. B 1970, 26, 1923. (38) Chatani, Y.; Okita, Y.; Tadokoro, H.; Yamashita, Y. Polym. J. 1970, 1, 555. (39) Hu, H.; Dorset, D. L. Macromolecules 1990, 23, 4604. (40) Brisse, F.; Marchessault, R. H. In Fiber Diffraction Methods; French, A. D., Gardner, K. H., Eds.; ACS Symposium Series 141; American Chemical Society: Washington, DC, 1980; p 267. (41) Dorset, D. L. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 5499. (42) Dorset, D. L. Polymer 1997, 38, 247. (43) Iwata, T.; Doi, Y. Polym. Int. 2002, 51, 852. (44) Kobayashi, S.; Shoda, S.; Uyama, H. AdV. Polym. Sci. 1995, 121, 1. (45) Ritter, H. Trends Polym. Sci. 1993, 1, 171. (46) Knani, D.; Gutman, A. L.; Kohn, D. H. J. Polym. Sci., Polym. Chem. Ed. 1993, 31, 1221. (47) Uyama, H.; Kobayashi, S. Chem. Lett. 1993, 1149. (48) MacDonald, R. T.; Pulapura, S. K.; Svirkin, Y. Y.; Gross, R. A.; Kaplan, D. L.; Akkara, J. A.; Swift, G.; Wolk, S. Macromolecules 1995, 28, 73. (49) Uyama, H.; Takeya, K.; Kobayashi, S. Proc. Jpn. Acad., Ser. B 1993, 69, 203. (50) Uyama, H.; Takeya, K.; Kobayashi, S. Bull. Chem. Soc. Jpn. 1995, 68, 56. (51) Uyama, H.; Takeya, K.; Hoshi, N.; Kobayashi, S. Macromolecules 1995, 28, 7046. (52) Huisgen, R.; Ott, H. Tetrahedron 1959, 6, 253. (53) Nomura, R.; Ueno, A.; Endo, T. Macromolecules 1994, 27, 620. (54) Kim, E.; Uyama, H.; Doi, Y.; Ha, C.-S.; Iwata, T. Macromocules 2004, 37, 7258. (55) Gazzano, M.; Malta, V.; Focarete, M. L.; Scandola, M.; Gross, R. A. J. Polym. Sci., Part B: Polym. Phys. 2003, 41, 1009. (56) Pazur, R. J.; Hocking, P. J.; Raymond, S.; Marchessault, R. H. Macromolecules 1998, 31, 6585. (57) Iwata, T.; Doi, Y.; Isono, K.; Yoshida, Y. Macromolecules 2001, 34, 7343. (58) Keller, A.; Willmouth, F. H. J. Polym. Sci., Part A-2 1970, 8, 1443. (59) Dorset, D. L. Acta Crystallogr., Sect. A 1976, 32, 207. (60) Geil, P. H. Polymer 1963, 4, 404.

BM0494747