Preparation of Amino-cyclosophoraoses from the Neutral Cyclosophoraoses Isolated fromRhizobium leguminosarum bv. trifolii

Abstract

Cyclosophoraoses are a class of unbranched cyclic oligosaccharides composed of β-1,2-D-glucans varying in size from 17 to 40 in a neutral or anionic form. They were originally found in fast growing soil bacteria, Agrobacterium and Rhizobium species, as intraor extra-oligosaccharides. Cyclosophoraoses are synthesized in the cytosol and transported to the periplasmic space where they play an important role in regulating the osmolarity in response to external osmotic shock. They are also known to be involved in the initial stage of root-nodule formation of Rhizobium species during nitrogen fixation. Throughout this interaction, cyclosophoraoses are suspected to be involved in complexation with various plant flavonoids. Thus, much attention has been focused not only on their biological functions but also on their potential ability to form inclusion complexes with other molecules. Several reports have shown that neutral cyclosophoraoses or anionic cyclosophoraoses have good potential as a host molecule in various inclusion complexation technologies such as a solubility enhancer and a chiral selector. In addition, the investigations into the chemical modifications of neutral cyclosophoraoses have been concerned with modifying their binding behaviors, e.g., carboxymethylated and sulfated cyclosophoraoses which were successfully used as a solubility enhancer and chiral selector, respectively. Their modifications are of particular importance for the investigation at the frontier of various research fields ranging from supramolecular chemistry to analytical techniques. In this study, for further application of cyclosophoraoses, neutral cyclosophoraoses were modified with tosyl, azide and amino groups through the chemical derivatization, and their modified structures were confirmed by nuclear magnetic resonance (NMR) and Fourier transform infrared (FTIR) spectroscopy. Isolation, purification and structural analyses of neutral cyclosophoraoses were carried out as described previously. Purified neutral cyclosophoraoses were separated with an Rf value of 0.125 on thin layer chromatography (TLC, ethanol : butanol : water = 5 : 5 : 4, v/v/v). Through matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry, we confirmed that the ring sizes of the neutral cyclosophoraoses ranged from degree of polymerization (DP) 17 to 23 (data not shown). Although the exact three-dimensional structure of cyclosophoraoses is not known, several NMR studies and molecular dynamics simulations have provided molecular models with flexible glycosidic linkage backbones. The cyclosophoraoses seem to have narrower cavity sizes than those expected from their bulky ring sizes (Figure 1A). The amino-cyclosophoraoses were obtained through three steps, tosylation (tosyl-cyclosophoraoses), azidation (azidocyclosophoraoses) and amination (Figure 1B). First, the hydroxyl groups of neutral cyclosophoraoses were subjected to chemical modification with p-toluenesulfonyl chloride and the reaction was monitored on TLC. The Rf value of the purified tosyl-cyclosophoraoses was 0.241 (ethanol : butanol : water = 5 : 5 : 4, v/v/v). The tosyl-cyclosophoraoses was synthesized in a 25 percent yield. Next, the azido-cyclosophoraoses were obtained from tosyl-cyclosophoraoses which were treated with NaN3 in water (90 percent yield). And then the azido-cyclosophoraoses and triphenylphos-