Biomacromolecules 2005, 6, 2362-2364
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Notes Complexation of r-Chitin with Aliphatic Amines Yasutomo Noishiki,* Yoshiharu Nishiyama, Masahisa Wada, and Shigenori Kuga Department of Biomaterials Sciences, Graduate School of Agricultural & Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan Received January 19, 2005 Revised Manuscript Received April 2, 2005
Introduction Chitin, the β-(1f4)-linked poly(N-acetylglucosamine), is a widely occurring polysaccharide in arthropodal exoskeltons and fungal cell walls.1 Of the two native crystal allomorphs, the rarer β-chitin is known to undergo characteristic intercalation by various polar molecules.2-7 This behavior has been considered to arise from the high anisotropy in interaction between chitin chains in β-chitin, i.e., the lack of hydrogen bonding along the b axis.8 Although β-chitin occurs only in limited species including vestimentiferan animals and certain diatoms, R-chitin is readily available from crab and shrimp shells.1 Therefore, possibility of complexation of R-chitin attracts attention from industrial point of view. Such complexes are expected to have characteristic nanometer-order structures leading to novel composite materials, reaction field for polymerization, or drug delivery systems based on chitin’s high biocompatibility and biodegradability. Thus, we here studied the intercalation of R-chitin by aliphatic amines, which have been found particularly strong complexing agents for β-chitin.5 Experimental Section Samples. Crab tendon, composed of uniaxially orientated R-chitin, was obtained from Alaskan crab (Paralithodes camtschaticus) or Queen crab (Chionoecetes opilio) legs. The tendon specimen was purified by soaking in 5% KOH and then in 0.2 N HCl at room temperature, each overnight. After washing with water and drying, the sample was swollen in liquid amine for 1-2 days at room temperature for monoamines, and at room temperature or 50 °C for diamines. (Actual complex formation seemed to be complete in several hours.) Excess liquid was removed with filter paper and the sample was sealed in the glass capillary (wet sample) or vacuumdried at room temperature for 2 h (dry sample). C2-C8 diamines and C3-C5 monoamines were tested as guest. All chemicals were of reagent grade from Wako Pure Chemicals. X-ray Fiber Diffraction. The specimen was subjected to X-ray diffraction measurement by transmitting beam from * To whom correspondence should be addressed. Telephone: +81-35841-5247. Fax: +81-3-5684-0299. E-mail: noishiki@ sbp.fp.a.u-tokyo.ac.jp.
Figure 1. X-ray diffraction patterns of (A) R-chitin, and (B) R-chitinEDA complex.
a rotating anode generator, RotaFlex RU-200BH (Rigaku) operated at 50 kV and 100 mA, by using nickel-filtered Cu KR radiation (λ ) 0.15418 nm). The diffraction pattern was recorded on an imaging plate (FUJIX BAS-IP SR127, Fuji Film) and scanned by a RAXIS DS3 (Rigaku). X-ray Diffraction during Thermal Treatment. X-ray diffraction of the R-chitin-ethylenediamine complex (dry) was measured at elevated temperatures by a RINT 2200 goniometer equipped with one-dimensional position sensitive proportional counter (PSPC) (Rigaku). Nickel-filtered Cu KR radiation generated at 38 kV and 50 mA was collimated with a 1.0 mm-wide pinhole. The sample was heated stepwise under helium atmosphere from room temperature to 300 °C with 20 °C increments. The diffraction profile was recorded at each temperature with accumulation time of 5 min.
10.1021/bm0500446 CCC: $30.25 © 2005 American Chemical Society Published on Web 05/05/2005
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Figure 2. Schematic drawing of the c-axis projection of the R-chitin-EDA complex.
Results and Discussion r-Chitin-Ethylenediamine Complex. The R-chitinethylenediamine (EDA) complex was obtained by immersing R-chitin in liquid EDA. Differently from β-chitin-EDA complexes,5 the X-ray diffraction patterns of the R-chitinEDA complexes in wet and dry conditions were almost the same. Figure 1 shows the X-ray diffraction pattern of the R-chitin-EDA complex (dry sample) together with that of free R-chitin. Based on this diagram, the complex has a twochain unit cell, with a ) 0.473 nm, b ) 2.271 nm, c ) 1.029 nm, R ) β ) γ ) 90°, nearly the same as free R-chitin except for b length. (R-chitin: orthorhombic, P212121, a ) 0.474 nm, b ) 1.886 nm, and c ) 1.032 nm.9) The 002 reflection was much more intense than the 004 reflection (data not shown), suggesting that this complex has no c/4 stagger as in cellulose Iβ. The pattern had systematic absence of h00, 0k0 and 00l reflections for odd h, k, and l. Though we cannot discuss symmetry of the unit cell without full determination of the crystal structure, the R-chitin-EDA complex apparently has a similar structure to that of R-chitin. The anisotropic change of the unit cell by complexation suggests that this complex retains the stacking of pyranose rings parallel to ac plane as in the original R-chitin9 and that the chitin sheets are separated by the guest molecules along the b axis. Figure 2 shows a schematic drawing of the c-axis projection of the R-chitin-EDA complex. In complexation, d020 corresponding to the sheet spacing increases retaining the sheet structure. This structure is quite similar to those of β-chitin-amine complexes5 and the celluloseamine complex.10-14 The R-chitin-EDA complex easily reverted to free R-chitin by soaking in water similarly to the β-chitin-amine complexes. Thermal Stability and Stoichiometry of the r-ChitinEDA Complex. The stability of the R-chitin-EDA complex was examined by change in X-ray diffraction profiles during stepwise heating of the complex (Figure 3; 20 °C increment, 5 min holding for measurement at each step). The diffraction profile remained the same up to 260 °C and then gradually reverted to that of free R-chitin at between 260 and 300 °C. Compared with EDA’s boiling point, 117 °C, the stability of the complex is remarkable. This stability is greater than
Figure 3. X-ray diffraction profiles of the R-chitin-EDA complex during heating.
that of the Type I β-chitin-EDA complex, which decomposed at 240-260 °C.5 This behavior allowed determination of host-guest ratio of the complex, based on its weight after heating at 120 °C for 1 h in a vacuum to remove excess EDA. The amount of included EDA was 15.0% (w/w based on complex), and the chitobiose:EDA ratio was determined as 1:1.04. This value can be interpreted as a stoichiometric ratio of 1:1. On the other hand, the host-guest ratio was calculated as 1:0.82 from the unit cell dimension described above and the density of liquid EDA (0.892 g/mL). The discrepancy between the two values could arise from the use of the liquid density in the latter, since the packing state of EDA molecules in the complex can be different from that in liquid. Complexation of r-Chitin with Higher Alkylamines. In the case of β-chitin, complexing ability has been found to be significantly different for monoamines and diamines.5 The situation was the same for R-chitin. Of tested monoamines (C3-C5), only propylamine gave complexation, incompletely at that. The increase in d020 of the R-chitinpropylamine complex was nearly the same as the corresponding sheet spacing increment of the β-chitin-propylamine
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Figure 5. Increase in sheet spacing of diamine complexes against carbon number of guest molecule. (b) R-Chitin-diamine complex. (4) β-Chitin-diamine complex (type I).
Figure 4. Equatorial X-ray diffraction profiles of R-chitin-diamine complexes.
complex, suggesting similarity in the structures. The R-chitinpropylamine complex, however, seemed more stable than the β-chitin-propylamine complex, since the former was not decomposed by evacuation at room temperature, whereas the latter was. On the other hand, R-chitin formed complexes with terminal diamines having up to seven carbon atoms. Figure 4 shows the equatorial X-ray diffraction profiles. The 020 reflection shifted to lower angles with increase in the chain length of guest, whereas the 110 reflection (around 20°) stayed at almost the same position. This means that these R-chitin-diamine complexes also have anisotropic swelling along the b axis by insertion of guest molecules between the chitin sheets as in the R-chitin-EDA complex and β-chitin-amine complexes. R-Chitin did not form a complex with octamethylenediamine. Therefore, the upper limit of diamines’ main chain size for complexation was C7 in the present condition. Similarly to the case of β-chitin, all of the complexes reverted readily to free R-chitin by immersion in water. Complexation with higher diamines (C5-C7) was incomplete, leaving a small peak of the original 020 reflection. Thus, the complexation of R-chitin was more difficult than β-chitin.5 This is probably because the R-chitin structure is thermodynamically more stable by the presence of inter-sheet hydrogen bonds,9 which do not exist in β-chitin.8 Figure 5 shows d020 (sheet spacing) of the complexes plotted against carbon number of guest diamine. Change in d020 was not observed between the wet and vacuum-dried
samples. Linear dependence of the spacing on carbon number is evident, roughly agreeing with that of β-chitin’s type I complexes.6 The latter was suggested to have host(chitobiose unit):guest ratio of 1:1, with diamine molecules packed between chitin’s sheets with certain inclination.5 While information about the host-guest ratio of the R-chitindiamine complexes other than EDA is lacking, they could also have host-guest ratio of 1:1 similarly to the R-chitinEDA complex, based on the similarity in crystallographic behaviors. Good agreement between R- and β-chitin seen in Figure 5 suggests that both complexes have similar arrangements of the guest between the chitin’s molecular sheets. References and Notes (1) Blackwell, J. In Cellulose and other natural polymer systems, Brown, R. M., Jr., Ed.; Plenum Press: New York, 1982; pp 403-428. (2) Blackwell, J. Biopolymers 1969, 7, 281-298. (3) Saito, Y., Kumagai H., Wada M., Kuga S. Biomacromolecules 2002, 3, 407-410. (4) Ro¨ssle, M.; Flot, D.; Engel, J.; Burghammer, M.; Riekel, C.; Chanzy, H. Biomacromolecules 2003, 4, 981-986. (5) Saito, Y.; Okano, T.; Putaux, J.-L.; Gaill, F.; Chanzy, H. In AdVances in chitin science; Domard, A., Roberts, G. A. F., Vårum, K. M., Eds.; Jacques Andre´ Publishers: Lyon, France, 1997; Vol. II, pp 507-512. (6) Noishiki, Y.; Nishiyama, Y.; Wada, M.; Okada, S.; Kuga, S. Biomacromolecules 2003, 4, 944-949. (7) Noishiki, Y.; Kuga, S.; Wada, M.; Hori, K.; Nishiyama, Y. Macromolecules 2004, 37, 6839-6842. (8) Gardner, K. H.; Blackwell, J. Biopolymers 1975, 14, 1581-1595. (9) Minke, R.; Blackwell, J. J. Mol. Biol. 1978, 120, 167-181. (10) Trogus, C.; Hess, K. Z. Phys. Chem. 1931, B14, 387-395. (11) Barry, A. J.; Peterson, F. C.; King, A. J. J. Am. Chem. Soc. 1936, 58, 333-337. (12) Davis, W. E.; Barry, A. J.; Peterson, F. C.; King, A. J. J. Am. Chem. Soc. 1943, 65, 1294-1299. (13) Creely, J. J.; Wade, R. H. J. Polym. Sci. Polym. Lett. Ed. 1978, 16, 291-295. (14) Lee, D. M.; Burnfield, K. E., Blackwell, J. Biopolymers 1984, 23, 111-126.
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