새로운 공정에 의한 유리 아민을 가진 저분자량 수용성 키토산의 제조와

분광학적 특성

장 미 경, 나 재 운*

순천대학교 공과대학 고분자공학과

jwnah@sunchon.ac.kr

The Spectroscopic Characterization and Preparation of Low Molecular and Water Soluble Chitosan with Free-amine group by Novel Method

Mi-Kyeong Jang, Jae-Woon Nah*

Department of Polymer Science and Engineering, Sunchon National University,

Jeonnam 540-742, Korea

jwnah@sunchon.ac.kr

Abstract

Low molecular and water-soluble chitosan (LMWSC) with a free-amine group was prepared by the novel salts removal method described in this study. A molecular weight (Mw) and degree of deacetylation (DDA) of LMWSC were determined by viscometer and Kina titration, resulting in 18,579 Daltons and 93 % DDA, respectively. In the FT-IR, 1H-NMR, and 13C-NMR spectra, the absorption band by carboxyl group derived from lactic acid and the impurities formed in the enzymatic process disappeared or were significantly lower than that of the control chitosan. Also, by the 1H-NMR, and 13C-NMR spectra, it was identified that the empirical value for the area ratio of proton and carbon corresponds nearly to its theoretical values. Through MALDI-TOF mass spectrum analysis, the difference in the two adjacent peaks was calculated 161.

Introduction

Chitosan, the principal derivative of chitin derived from crab shell or squid pen, has been extensively investigated for several decades as a biomaterial industrial applications as well as the pharmaceutical field. In its structure, chitosan is similar to cellulose, but different in that it has an amine group instead of a hydroxyl group at the C-2 position within the glucose unit. Because of this, chitosan has specific properties, such as chelation applied to areas of health care, water quality improvement, pharmaceutics, affinity for protein, inhibition of tumor cells, and inhibition of microbial cell growth[1]. However, in spite of this desirable characteristic, its actual use is limited because of its poor solubility in water. Since chitosan dissolves only in water containing acetic acid, and its use as a solvent is considered another limitations for actual use due to that bioactive agents such as peptide or protein drug, genetic material, and anticancer drugs may be affected by acetic acid.

To solve the solubility problem of chitosan, many investigators have developed water-soluble chitosan. But, most of this research has been limited to modification with water soluble derivatives, such as poly(ethylene glycol)[2], N-carboxymethylation[3], sulfonation[4], quaternarization[5-7], N- and O-hydroxyalkylation[8].

In this study, we attempted to prepare water-soluble chitosan having a free-amine group at the C-2 position, and its identification, using FT-IR, 1H-NMR, 13C-NMR and MALDI-TOF mass spectrometers. Until now the structural analysis for chitosan has been discovered through X-ray diffraction[9] to determine its crystalline structure. Also, FT-IR[10], NMR[11] spectra and elemental analysis[12] have been used to measure the DDA (the degree of deacetylation) of chitosan. However, the structure of pure chitosan having a free amine group has not been determined as yet. From the results of this study, it was determined through FT-IR, 1H-NMR, 13C-NMR and MALDI-TOF mass spectrum analyses that pure water-soluble chitosan with a free-amine group was obtained by the novel salts removal method described in this study.

Experimental

Chitosan with a molecular weight of 22 kDa and DDA of 72 % was supplied from KITTOLIFE Co., Seoul, Korea. Triethylamine (TEA) with 99 % purity was purchased from Aldrich Co. and used without further purification. Various solvents were used without further purification as a reagent grade and double distilled water was used in all of the experiments. The preparation of LMWSC was progressed by that chitosan oligosaccharide prepared by enzymolysis was dissolved in phosphate buffered saline. TEA was slowly dropped in chitosan solution and stirred. After reaction, acetone was added and this mixture was harvested by centrifuge. To obtain a pure product, this process was repeated four times and then air-dried. The product was then treated with dilute HCl aqueous solutions, again for two hours. After the two hours, acetone was added to this solution following the above procedure repeated 5 times, and then air-dried. To remove the residue HCl solution and impurities, the product was dissolved in double distilled water, and then passed through the column. The resulting aqueous solution was lyophilized, yielding water-soluble chitosan having a free-amine group.

The characterization of LMWSC was determined by spectroscopic method as follows: The DDA determination by Kina titration method[13]. To calculate the average molecular weight, relative viscosity was measured using viscometer (Viscotek, Y501C, U.S.A). FT-IR (Shimadzu, FT-IR 8700), 1H-NMR (Bruker, DRX-500MHz), 13C-NMR (Bruker, DRX-500MHz) and MALDI-TOF mass spectrometers (Shimadzu, AXIMA-CFR).

Results and Discussion

Among the various biomaterials, chitosan has been widely investigated in the pharmaceutical and food industries[14-15]. In spite of the specific properties of chitosan, its application is limited because of its poor solubility. To solve this problem, many investigators have modified the chitosan with water-soluble groups or salts. However, chitosan of these forms presents disadvantages in that its strong positive charge decreases and it becomes harmful to human body. In this study, we prepared water-soluble chitosan with a free-amine group, without the use of acetic acid by the novel salt removal method. The reaction scheme for the synthesis of LMWSC is shown in Scheme 1. Also, the spectroscopic characters that have not been revealed until now were quantitatively analyzed by FT-IR, 1H-NMR, 13C-NMR, and MALDI-TOF mass spectrum analyses. A molecular weight (Mw) and degree of deacetylation (DDA) of LMWSC were determined by viscometer and Kina titration, resulting in 18,579 Daltons and 93 % DDA, respectively. In the FT-IR, 1H-NMR, and 13C-NMR spectra, the absorption band by carboxyl group derived from lactic acid and the impurities formed in the enzymatic process disappeared or were significantly lower than that of the control chitosan. Also, by the 1H-NMR, and 13C-NMR spectra, it was identified that the empirical value for the area ratio of proton and carbon corresponds nearly to its theoretical values. Through MALDI-TOF mass spectrum analysis, the difference in the two adjacent peaks was calculated 161.

Conclusion

To apply in the pharmaceutical field, we prepared a pure sample of LMWSC without the use of acetic acid and with a free amine group by the novel method developed in our laboratory. The Mw and DDA of LMWSC were calculated by the viscometer and Kina titration methods, respectively. The structural character of LMWSC was also measured using FT-IR, 1H-NMR, 13C-NMR, and MALDI-TOF mass spectrometer analyses. The Mw of LMWSC was measured at 18,579 Daltons, lower than that of control chitosan, because the impurities and salts were removed by the novel method developed in our laboratory. The DDA, at 93 %, was higher than that of control chitosan, 72 % DDA. Because the free-amine group of LMWSC increase due to removal the impurity or salts at the C-2 position of the glucose amine unit. Within the FT-IR spectrum of LMWSC, the carboxyl group absorption band derived from lactic acid and impurities formed in the enzyme degradation process disappeared or significantly decreased. In the 1H-NMR and 13C-NMR spectra of LMWSC, it was identified that the peak derived from lactic acid disappeared, while the peaks of the various impurities decreased noticeably. Also, by the 1H-NMR, and 13C-NMR spectra, it was identified that the empirical value for the area ratio of proton and carbon corresponds nearly to its theoretical values. Through MALDI-TOF mass spectrum analysis, the difference of the two adjacent peaks was calculated as 161. This means that it corresponds with the Mw of the glucosamine unit. Consequently, LMWSC prepared in this study was quantitatively determined and it was noted that LMWSC does not introduce water-soluble derivatives into the amine group at the C-2 position.

Scheme 1. Synthetic pathway to prepare LMWSC with free-amine group

References

1. Mattheus, F. A. Goosen: Applications of chitin and chitosan. Ed. Technomic, USA (1997).

2. Tatsuro, O., Hidetoshi, N. and Yuichi, O.: Polymer, 39, 5171(1998).

3. Muzzarelli, R. A. A., Tanfani, F. and Emanuelli, M.: Carbohydr. Res., 107, 199(1982).

4. Focher, B., Massoll, A., Torri, G., Gervasini, A. and Morazzoni, F.: Makromol. chem., 187, 2609(1986).

5. Kokufuta, E., Hirai, Y. and Nakamura, I.: Makromol. chem., 182, 1715(1981).

6. Murata, J., Ohya, Y. and Ouchi, T.: Carbohydr. Polym, 29, 69(1996).

7. Muzzarelli, R. A. A. and Tanfani, F.: Carbohydr. Polym, 5, 297(1985).

8. Yamada, H. and Imoto, T.: Carbohydr. Res., 92, 160(1981).

9. Kenji, O., Keiichi, N., Takashi, M., Toshifumi, Y. and Kozo, O.: Macromolecules, 30, 5849(1997).

10. Brugnerotto, J., Lizardi, J., Goycoolea, F. M., Arguelles-Monal, W., Desbrieres, J. and Rinaudo, M.: Polymer, 42, 3569(2001).

11. Brugnerotto, J., Desbrieres, J., Heux, L., Mazeau, K. and Rinaudo, M.: Macromol. Symp, 168, 1(2001).

12. Roberts GAF.: Chitin chemistry: Macmillan: New York(1992).

13. Kina, K., Tamura, K. and Ishibashi, N.: Jpn. Anal, 23, 1082(1974).

14. Lisbeth I.: Pharmaceutical Research, 15, 1326(1998).

15. Shigehiro H.: Polymer International, 48, 732(1999).

Acknowledgements

We acknowledge financial supports from KITTOLIFE Co., Seoul, Korea.