Biomacromolecules 2003, 4, 944-949
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Inclusion Complex of β-Chitin and Aliphatic Amines Yasutomo Noishiki,*,† Yoshiharu Nishiyama,† Masahisa Wada,† Shigeru Okada,‡ and Shigenori Kuga† Department of Biomaterials Science, Graduate School of Agricultural & Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan, and Laboratory of Marine Biochemistry Graduate School of Agricultural & Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan Received January 20, 2003; Revised Manuscript Received May 2, 2003
Inclusion complexation of β-chitin with linear aliphatic amines was studied by X-ray diffraction. All tested amines, C3 to C8 monoamines and C2 to C7 diamines with terminal amino groups, reversibly formed crystalline complexes with β-chitin by immersion of dry chitin in pure liquid. Complex formation caused linear increase in the 010 sheet spacing of β-chitin depending on the carbon number of amine. The complexes could be classified as type I and type II according to the increment of sheet spacing against carbon number. All monoamines formed type II complexes. In dry conditions, diamine formed a type I complex though the type of diamine complex differed for guest species in wet conditions. Based on the unit cell dimension and thermogravimetry, type II and type I are likely to correspond to guest-host (amine-chitobiose) ratios of 2:1 and 1:1, respectively. These differences seem to arise from varied interactions between functional groups of chitin and amines. Introduction Chitin is a ubiquitous polysaccharide composed of β (1f4) linked N-acetylglucosamine, typically serving as structural component of arthropodal exoskeltons and fungal cell walls. Chitin has two crystal allomorphs, R-chitin and β-chitin.1,2 R-Chitin, comprising a majority, has an orthorhombic twochain unit lattice with antiparallel arrangement;3,4 the rarer β-chitin has a monoclinic one-chain unit cell with a parallel chain arrangement.5,6 Although R-chitin has a threedimensional hydrogen bond network, β-chitin consists of well-defined sheets, lacking hydrogen bonds between them. Because of this anisotropy and the weak association of the sheets, β-chitin readily incorporates various polar molecules and forms crystalline inclusion complexes or crystallosolvates.6-8 Although the most common inclusion complex of β-chitin is hydrate,6,7 various aliphatic alcohols have been found to be included via solvation by hydrochloric acid.8 Notably, the increase in chitin sheet spacing is linearly dependent on the carbon number of guest molecule, suggesting that they are aligned perpendicular to the chitin sheet. Such complexation is remarkable for a high-molecular-weight crystalline polymer and attracts our attention for its potential usefulness in advanced materials technology. We therefore attempted to elucidate the scope of this phenomenon, i.e., what * To whom correspondence should be addressed. E-mail: noishiki@ sbp.fp.a.u-tokyo.ac.jp. Telephone: +81-3-5841-5247. Fax: +81-3-56840299. † Department of Biomaterials Science, Graduate School of Agricultural & Life Sciences, The University of Tokyo. ‡ Laboratory of Marine Biochemistry Graduate School of Agricultural & Life Sciences, The University of Tokyo.
molecular species can form crystallosolvates with β-chitin. Here we report that linear aliphatic amines are especially effective in forming inclusion complexes with β-chitin, which have characteristic differences depending on the guest species. Highly crystalline β-chitin from the extracellular spine of diatom9 was used. Experimental Section Chemicals. All reagents were of chemical grade (Wako Pure Chemicals, Tokyo). Water means distilled- or resindeionized water throughout. β-Chitin Sample. Centric diatom Thalassiosira weissflogii (CCMP 1051) obtained from Provasoli-Guillard National Center for Culture of Marine Phytoplankton was cultured in f/2 medium supplemented by L1 medium10,11 under artificial illumination and aeration. After about one month, the suspended solid in the medium was collected by centrifugation. The precipitate was successively treated with 5% KOH (room temperature, overnight), methanol (80 °C, 2 h), 0.34% NaClO2 (buffered to pH 4.0, 70 °C, 6 h), 0.1 N HCl (boiling, 1 h), and finally 1% HF (room temperature, overnight), with centrifugal rinsing with water after each step. Purified chitin sample was freeze-dried and kept in a desiccator. Yield from a 10 L culture was between 50 and 100 mg. Oriented β-Chitin Fiber. For obtaining fiber X-ray diffraction data, β-chitin fiber was prepared as follows [adapted from the paper reported by Blackwell6]: Approximately 5 mg of dry chitin was dispersed in 7 mL of water and mixed with 3 mL of 1% fibrinogen solution [Wako Chemical] in 3% aq. sodium chloride. This mixture was supplemented by several drops of concentrated aq. thrombin
10.1021/bm034024k CCC: $25.00 © 2003 American Chemical Society Published on Web 05/29/2003
Inclusion Complex of β-Chitin and Aliphatic Amines
solution, immediately spread in a glass Petri dish to form an approximately 3 mm thick layer, and allowed to stand at room temperature. When the layer formed a soft gel (after several hours, depending on temperature and amount of thrombin), it was cut into a 50 mm × 5 mm strip and slowly stretched by hands to about 3 times with continuous removal of water by contacting a filter paper. This string-like specimen was softened by immersing in 1% KOH and further stretched to 1.5-2 times. The string was cut into 20 mm pieces, and the proper number of pieces were bundled and inserted into a glass capillary of 1 mm inner diameter with open ends. Fibrinogen in the chitin specimen was removed by alternately injecting 5% KOH solution and water into the capillary several times. The specimen was finally dried in a vacuum at 120 °C. Complex Formation. Saturated Complex. Pure liquid amine was injected into the capillary by a syringe or immersing one end of the capillary in the liquid. Hexamethylenediamine and heptamethylenediamine, having a melting point higher than room temperature, were heated to about 50 °C for this treatment. After 30 min, the excess liquid was removed through wicking by filter paper, and the capillary’s ends were flame-sealed. Vacuum Dried Complex. The chitin-amine complex specimen in the open capillary was subjected to vacuumdrying at room temperature for 2 h. In some cases, additional heat drying in air was applied for removing trace of saturated complex. X-ray Fiber Diffraction. The capillary-sealed wet specimen (saturated complex) or bare bundle specimen (vacuumdried complex) was subjected to X-ray diffraction measurements by the transmitting beam from a rotating anode X-ray generator, RotaFlex RU-200BH (Rigaku) operated at 100 mA and 50 kV, using nickel-filtered Cu KR radiation (λ ) 0.15418 nm). The diffraction pattern was recorded on an imaging plate (FUJIX BAS300UR, Fuji Film) and was read with RAXIS DS3 (Rigaku). X-ray Powder Diffraction during Thermal Treatment. Approximately 30 mg of β-chitin sample was dispersed in 10 mL of water, cast on a Teflon plate at 50 °C, and vacuumdried at 120 °C. The sheet was soaked in liquid amine for 30 min at room temperature to form complex. The complex was squeezed between filter papers, vacuum-dried at 20 °C for 2 h, and subjected to X-ray diffraction measurement. Time-resolved recording of diffraction profiles at an elevated temperature was done by using a RINT 2200 goniometer equipped with a one-dimensional position sensitive proportional counter (PSPC) (Rigaku). Nickel-filtered Cu KR radiation (λ ) 0.15418 nm) generated at 38 kV and 50 mA, was collimated with a 1.0 mm φ pinhole. The film sample was placed perpendicular to the incident X-ray (θ ) 90°) in an electrically heated holder under helium atmosphere. The sample was heated stepwise from room temperature to 300 °C with 20 °C increments. The diffraction profile was recorded at each temperature with accumulation time of 5 min. Thermogravimetric Analysis (TGA). Approximately 20 mg of the vacuum-dried complex specimen, the same as
Biomacromolecules, Vol. 4, No. 4, 2003 945
Figure 1. X-ray diffraction pattern of (A) anhydrous β-chitin and (B) its n-hexylamine complex after vacuum-drying. Reflection of 010 sheet (arrowheads) shifts from d ) 0.919 nm to 1.794 nm.
above, was subjected to thermogravimetry by a TGD-9600 (Ulvac, Tokyo) in a platinum holder under nitrogen flow with a heating rate of 5 °C/min. Results All tested amines, C3 to C8 monoamines and C2 to C7 diamines, formed complexes with β-chitin. The complexes could be reverted to free β-chitin by water washing followed by moderate heating. Figure 1 shows the change in the diffraction pattern by complexation with n-hexylamine. The innermost equatorial reflection arising from β-chitin’s 010 sheet shifted significantly toward a lower angle (arrowheads). The 010 sheet spacing of complexes showed characteristic dependence on the carbon atom number of the guest molecule. The plots fell roughly on two lines, I and II (Figure 2). Lines I and II presumably correspond to two types of complexes, denoted as type I and type II in the following. For either type, the sheet spacing has a linear dependence on the carbon atom number. Table 1 summarizes the situation of complex
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Figure 2. Sheet spacing of the complex plotted against the carbon number of the guest. Table 1. Type of Complex for Series of Monoamines and Diamines
C no.
guest
complex type, wet
3 4 5 6 7 8 2 3 4 5 6 7
propylamine butylamine pentylamine hexylamine heptylamine octylamine ethylenediamine trimethylenediamine tetramethylenediamine pentamethylenediamine hexamethylenediamine heptamethylenediamine
II II II II II II II II I I II I
a
complex type after 2 h vac. dry
boiling point of pure amine, °C
free chitin II∼free chitina II∼free chitina II II II I + II I + II I I I + II I
49 78 104 129 155 179 117 135 158 180 205 224
The guest amine was removed gradually by elongated evacuation.
formation. All monoamines formed type II complexes under wet conditions. The type II complexes of higher amines (n ) 6-8) remained nearly the same after 2 h vacuumdrying, but those of lower amines (n ) 3-5) gave mixtures of type II complex and anhydrous β-chitin. Prolonged evacuation caused gradual removal of the guest. On the other hand, the type of diamine complex was dependent on guest species and specimen’s condition (wet or vacuum/heat dried. See Figure 3 and Table 1). The chitin sheet spacing of type I complex is definitely smaller than that of the type II complex of monoamine with the same carbon number (Figure 2). This suggests that the mode of packing between the sheets is significantly different for type I and type II. The unit cell parameters of the amine complexes were determined from X-ray diffraction data for selected cases (Table 2). The length of the b axis, corresponding to the sheet spacing, is significantly large in all complexes compared to that of anhydrous β-chitin, whereas other parameters remained nearly the same. The type II complexes, i.e., the vacuum-dried n-hexylamine complex and the wet ethylenediamine complex, can be assigned to one-chain monoclinic unit cells, similarly to anhydrous β-chitin. The vacuum-dried ethylenediamine complex (type I) required either two-chain
Figure 3. X-ray diffraction pattern of β-chitin-ethylenediamine complexes. A: type II (wet). B: Type I (dried by heating at 120 °C).
monoclinic or one-chain triclinic unit cell. Although complete data is yet to be determined, the unit cell type seems to follow this classification according to the type of complex. Because the stoichiometry and stability are of primary importance for understanding the nature of complexation, we studied thermal decomposition of these complexes by X-ray diffraction and thermogravimetry. Figure 4 shows the change in the X-ray diffraction profile of n-hexylamine complex during stepwise heating. The innermost reflection (2θ ) 4.92°, corresponding to d spacing of 1.79 nm) of the complex disappeared between 140 and 180 °C (cf. boiling point of n-hexylamine, 129 °C), and the pattern finally became that of anhydrous β-chitin. Other diffraction peaks also underwent slight and gradual changes starting from below 80 °C. Figure 5 shows the powder X-ray diffraction profiles of the ethylenediamine complex for the same treatment. In this case, the starting complex was already a mixture of type I and type II, because it had been subjected to vacuum-drying (for avoiding damage to the diffraction apparatus). The change shows gradual conversion from a type I-type II mixture to pure type I at between 20 and 180 °C (cf. boiling
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Inclusion Complex of β-Chitin and Aliphatic Amines
Table 2. Unit Cell Parameters and the Number of Guest Molecule in Complexes of n-Hexylamine and Ethylenediamine guest mol. per chitobiosyl residuesa sample
a, nm
b, nm
c, nm
γ, deg
Nv
Ntg
anhydrous β-chitin hexylamine complex (type II) ethylenediamine complex (type II) ethylenediamine complex (type I)b
0.485 0.475 0.479 0.476
0.926 1.807 1.450 1.183
1.038 1.029 1.037 1.024
97.1 96.8 96.7 90.6
0 1.89 2.27 1.03
0 1.98 ND ND
a Nv, from unit cell size; Ntg, from thermogravimetry one-chain triclinic.
b
Approximated as one-chain monoclinic. The actual system may be two-chain monoclinic or
Figure 5. Change in the X-ray diffraction profile of the β-chitinethylenediamine complex during heating. Figure 4. Change in the X-ray diffraction profile of the β-chitinn-hexylamine complex during heating.
point of ethylenediamine, 117 °C) and then to anhydrous β-chitin at between 220 and 280 °C. Although the pyrolytic weight loss of pure chitin in TGA is a one-step process occurring at between 200 and 360 °C (Figure 6A), that of the amine complexes showed characteristic stepwise weight losses, apparently showing removal of the guest by vaporization. The n-hexylamine complex (type II) lost approximately 33% of initial weight at between 20 and 200 °C (Figure 6B). The latter temperature agrees well with that causing complete disappearance of the complex in X-ray diffraction (Figure 4). Because pyrolysis
of chitin is negligible at this temperature, the 33% weight loss is ascribed to the thermal release of guest molecules, leading to a chitobiose:amine ratio of 1:1.98. Similar analysis was not possible for the TGA curve of the ethylenediamine complex, because the starting material was a type I-type II mixture, and also the final removal of guest was overlapped with pyrolysis of chitin. Stoichiometric estimation is possible also from the volume of complexes calculated from the unit cell dimension (Table 2). By assuming that the density of guest molecules in the complex is the same as that of bulk liquid, the number of guest molecules per chitobiose unit was 1.89 for n-hexylamine (type II), giving reasonable agreement with that from
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Figure 7. Possible arrangement of guest molecule. A. Type II (nhexylamine). B. Type I (tetramethylendiamine). Antiparallel arrangement of monoamine is tentative. Figure 6. Thermogravimetry diagrams of the chitin complex; A. β-chitin, B. n-hexylamine complex, C. ethylenediamine complex.
gravimetry, 1.98. The same calculation for ethylenediamine gave 2.27 for type II and 1.03 for type I. Discussion The complexation behavior of β-chitin with amines seems analogous to that of linear aliphatic alcohols.8 Although the alcohol complexes were formed via HCl complex of β-chitin, our amine complexes were formed readily by direct immersion of anhydrous chitin in liquid amines. Though we used 30 min immersion, the complex formation seemed instantaneous. This ease of complexation is probably due to the strong interaction of amino groups with chitin molecules. Inclusion of alcohols and amines both caused increases in the chitin sheet spacing roughly proportional to the carbon number of guest molecules. In fact, there is good correspondence between the extended chain length of the guest and the increase in sheet spacing, ∆ d, for type II amine complexes. For n-hexylamine, e.g., C6N, the chain length of 0.80 nm (center to center) is close to the observed ∆d of 0.88 nm. This feature leads to a model in which extended alkyl chains are oriented perpendicular to the chitin sheet (Figure 7A). The sheet spacing of type I complex, on the other hand, is approximately 1/2∼2/3 of that of type II complex with the guest having the same carbon number. This
feature leads to a smaller number of guest molecules, i.e., a guest-host ratio of 1:1 based on ∆d and liquid density of amines. Accordingly, the mode of molecular packing must be significantly different from that of type II. We propose a model in which linear amine molecules are packed obliquely as in Figure 7B. This arrangement seems somewhat similar to that of ethylenediamine in the cellulose complex reported by Lee et al.,13 but the mode of interaction with the host polymer can be different for cellulose and chitin because of the acetamide group in the latter. The occurrence of two types of complexes depending on the guest species and treatment condition is a new aspect of chitin complexation. Because the chitin molecule has a bulky acetamide group on C2 of each glucopyranoside moiety, the packing of the guest molecules would be determined by balances of hydrogen bonding between hydroxyl groups, amide groups, guests’ amino groups, and van der Waals forces between alkyl groups. Conclusions The characteristic complexation behavior of β-chitin has been known for a while, but its study has been hampered by scarcity of sample. By using a cultured diatom specimen, we explored new aspects of β-chitin’s complexation including the strong action of aliphatic amines and the occurrence of two types of complexes. So far, we studied the complex
Inclusion Complex of β-Chitin and Aliphatic Amines
formation by direct immersion of chitin in pure liquids. There is indication that even those species not forming complexes by direct immersion can form complexes by using proper intermediates. Cellulose, chitin’s close analogue and more abundant biopolymer, is also known to form inclusion complexes with amines,12-14 a phenomenon related to industrial processes such as solvation or textile processing. Exploration of the reversible inclusion of small molecules by β-chitin is expected to give opportunities for novel materials and industrial processes. Acknowledgment. We thank Dr. Y. Saito (University of Tokyo) for useful discussion and advice. References and Notes (1) Blackwell, J. In Cellulose and other natural polymer systems; Brown, R. M., Jr., Ed.; Plenum Press: New York, 1982; p 403. (2) Rudall, K. M. AdV. Insect Physiol. 1963, 1, 257. (3) Minke, R.; Blackwell, J. J. Mol. Biol. 1978, 120, 167.
Biomacromolecules, Vol. 4, No. 4, 2003 949 (4) Saito, Y.; Okano T.; Chanzy H.; Sugiyama J. J. Struct. Biol. 1995, 114, 218. (5) Gardner, K. H.; Blackwell, J. Biopolymers 1975, 14, 1581. (6) Blackwell, J. Biopolymers 1969, 7, 281. (7) Saito, Y.; Kumagai H.; Wada M.; Kuga S. Biomacromolecules 2002, 3, 407. (8) Saito, Y.; Okano, T.; Putaux, J.-L.; Gaill, F.; Chanzy, H. In AdVances in chitin science; Domard, A., Roberts, G. A. F., Varum, K. M., Eds.; Jacques Andre Publishers: Lyon, France, 1997; Vol. II, p 507. (9) Blackwell, J.; Parker, K. D.; Rundall, K. M. J. Mol. Biol. 1967, 28, 383. (10) Guillard, R. R. L.; Ryther, J. H. Can. J. Microbiol. 1962, 8, 229. (11) The recipes were listed in the website of Provasoli-Guillard National Center for Culture of Marine Phytoplankton following address: http:// b250.bigelow.org/ (12) Davis, W. E.; Barry, A. J.; Peterson, F. C.; King, A. J. J. Am. Chem. Soc. 1943, 65, 1294. (13) Lee, D. M.; Burnfield, K. E.; Blackwell, J. Biopolymers 1984, 23, 111. (14) Creely, J. J.; Wade, R. H. J. Polym. Sci., Polym. Lett. Ed. 1978, 16, 291.
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