J Korean Soc Appl Biol Chem (2014) 57(3), 319−321 DOI 10.1007/s13765-013-4312-1
Online ISSN 2234-344X Print ISSN 1738-2203
SHORT COMMUNICATION
Immunological Activities of Cationic Methylan Derivatives Jung-Kul Lee · In-Won Kim · Tae-Su Kim · Joon-Ho Choi · Jung-Hoe Kim · Si-Hyung Park
Received: 26 December 2013 / Accepted: 21 April 2014 / Published Online: 30 June 2014 © The Korean Society for Applied Biological Chemistry and Springer 2014
Abstract Methylan polysaccharide was aminated to add dialkylaminoethyl and free amino groups at hydroxyl sites in the methylan backbone, and these derivatives were quaternized to produce pH-independent cationic polyelectrolytes. The immunological activities of the resulting methylan derivatives were investigated. Diethylaminoethyl (DEAE)-methylan derivatives inhibited the classical pathway of the complement system in a dose-dependent way. Quaternized DEAE-methylan exhibited the highest anticomplementary activities among the all derivatives. Anticomplementary activities increased significantly as the cationic charge of the methylan derivatives increased via aminoderivatization followed by quaternization, indicating that there is an electrostatic interaction between the methylan derivatives and the negatively charged functional residues on the cell. Keywords aminoderivatization · anticomplementary · cationic polysaccharide · immunopotentiating
Polysaccharides, such as lentinan, pachymaran, schizophyllan, and mannan, have been shown to have various immunostimulative and antitumor activities (Nishimura et al., 1984; J.-K. Lee · I.-W. Kim · T.-S. Kim Department of Chemical Engineering, Konkuk University, HwayangDong, Kwangjin-Ku, Seoul 110-521, Republic of Korea J.-H. Choi Department of Food Science and Biotechnology, Wonkwang University, Iksan-city, JeonBuk 570-749, Republic of Korea J.-H. Kim () Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 441-744, Republic of Korea E-mail:
[email protected] S.-H. Park () College of Natural Sciences, Mokpo National University, Muan, Jeonnam 534-729, Republic of Korea E-mail:
[email protected]
Chihara, 1986). In particular, lentinan and schizophyllan have been used as immunotherapeutic agents (Chihara, 1986). The human complement system plays an important role in the host defense system against foreign invasive organisms such as bacteria, fungi, and viruses. The complement system consists of over 20 serum proteins, which are activated by a cascade mechanism of the classical pathway (CP) or alternative pathway (Alexander et al., 1988). Activation of the system may contribute to or evoke pathologic reaction in a variety of inflammatory and degenerative diseases, for example, various hemolytic anemias, dermatological diseases, rheumatoid arthritis, respiratory distress syndrome, and microbial infections (Alexander et al., 1988; Strunk et al., 1988). Therefore, the modulation of complement activity can be important. Polysaccharides with anti-inflammatory properties are considered as good sources for some regulators of the complement system. Methylobacterium organophilum was found to accumulate methylan, a high-viscosity polysaccharide, extracellularly. It produces methylan using methanol as the sole carbon and energy source under specific culture conditions (Lebeault et al., 1991; Kim et al., 1996; Lee et al., 1998). The sugar components of methylan were identified by gas chromatography as glucose:galactose:mannose at the ratio of 2:3:2. Its molecular mass was estimated to be 2×106 Da by gel permeation chromatography and light scattering (Kim et al., 1996). When ingested, polysaccharide has fewer side effects than synthetic drugs, and it is also environmentally friendly. However, the effect of methylan itself is relatively weak compared to those of synthetic drugs. Therefore, in the present study, we increased the positive charge density of methylan by diethylaminoethylation and reductive amination followed by quaternization, and the anticomplementary activities of these methylan derivatives were investigated. The use of such materials as vaccine adjuvants could open up a new area of immunopotentiating substances. Methylan was diethylaminoethylated (Fig. 1). The hydroxyl group was replaced by a diethylaminoethyl group, while the polymeric structure was maintained to give diethylaminoethyl (DEAE)methylan with degree of substitution (DS) of 0.7 (1.85 mmol/g). The DS of DEAE-methylan was obtained from the elemental
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J Korean Soc Appl Biol Chem (2014) 57(3), 319−321
Fig. 1 Amination of methylan followed by quaternization.
Fig. 3 IR spectra of the methylan derivatives. A: Methylan, B: TosylMethylan, C: Azido-Methylan, D: quaternized Amino-Methylan.
Fig. 2 Diethylaminoethylation of methylan followed by quaternization.
analysis and the peak ratios between anomeric protons and the methyl protons of the DEAE groups using the 1H-NMR method (Katsura et al., 1992). Substitution at C-6 was in accordance with Katsura et al. (1992), and this was further confirmed by a model reaction with glucose. NH2-methylan derivatives were prepared (Fig. 2). Tosylation of methylan followed by azidation and then reduction afforded NH2methylan with a DS of 0.44 from the elemental analysis. IR spectra of methylan derivatives (Fig. 3). Tosylated methylan showed absorptions of aromatic C=C at 1,732 and 1,534 cm−1 and S=O stretching vibrations at 1287 and 1,176 cm−1. Treatment of tosyl-methylan with sodium azide afforded azidated methylan with a DS of 0.46. The IR spectrum of this product showed the characteristic absorption of an azido group (N=N+=N−) at 2112 cm−1. Reduction of azido-methylan with LiAlH4 afforded aminated methylan, which did not show any absorption of an azido group
by IR spectroscopy. The hydroxyl group at C-6 was believed to be replaced by NH2 as suggested by Whistler and Hirase (1961). DEAE-methylan and NH2-methylan were quaternized with methyl iodide. The completion of quaternization was verified by the ninhydrin test, which is used to detect unquaternized amino groups (Sarin et al., 1981). Characteristic of quaternary amines peaks between 1,070 and 900 cm−1 and at 3,030 cm−1 were observed by FT-IR spectroscopy. They were absent in the unquaternized DEAE-methylan and NH2-methylan. In the 1H-NMR spectra, the quaternized DEAE-methylan showed characteristic peaks at ~δ1.32, caused by the methyl protons of the quaternized DEAE groups (Fig. 4). DEAE-methylan derivatives significantly inhibited CP of the complement system. Quaternized DEAE-methylan showed a more pronounced complement inhibition than DEAE-methylan, as measured by the diminished hemolysis of sensitized EA in the presence of complement factors. DEAE-methylan and qNH2methylan were moderately active, and NH2-methylan was weakly active. In order to determine whether or not the anticomplementary activity was dose-dependent, compounds were diluted sequentially with the corresponding buffer solution. Fig. 5 illustrates the dose-
J Korean Soc Appl Biol Chem (2014) 57(3), 319−321
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Fig. 5 Anticomplementary activities of the methylan derivatives on classical pathway. Sheep red blood cells (5.0×108 cells/mL), hemolysin (1/100 dilution) and serum (1/95 dilution) were used for standard hemolysis. Symbols: □ methylan, ■ NH2-methylan, ▼ quaternized NH2-methylan, ○ DEAE-methylan, ● quaternized DEAE-methylan. Each value represents the mean of triplicate measurements and varied from the mean by not more than 15%.
Fig. 4 1H NMR spectra (500 MHz) of the methylan derivatives in D2O. A: DEAE-methylan, B: quaternized DEAE-methylan.
dependent profile of the effects on the CP of complement. The inhibition of hemolysis by quaternized DEAE-methylan was 22% at 4 mg/mL and was rapidly increased in the range 4-16 mg/mL, then came up to 75% at 16 mg/mL. quaternized DEAE-methylan, and DEAE-methylan inhibited hemolysis completely at concentrations of ca. 32 and 190 mg/mL, and partially at 4 and 16 mg/mL, respectively. In conclusion, DEAE-methylan derivatives inhibited the CP of the complement system in a dose-dependent manner. Quaternized DEAE-methylan exhibited the highest anticomplementary activities among the all derivatives, indicating the importance of electrostatic interaction. According to the above observations on methylan derivative-induced suppressive activity against complement system, methylan derivatives are immunopotentiating. The higher order structure of active methylan derivatives and the details of their mechanisms of action in the host are now under investigation. Acknowledgment This paper was written as part of Konkuk University’s research support program for its faculty on sabbatical leave in 2012.
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