Chanho Kwon

Enantiomeric separation of some flavanones using shinorhizobial linear octasaccharides in CE

Succinoglycan, a shinorhizobial exopolysaccharide produced by Shinorhizobium meliloti, is composed of an octasaccharide subunit. S. meliloti produces both high‐molecular‐weight and low‐molecular‐weight (Mr<10 000) succinoglycans that consisted of monomer, dimer, or trimer of an octasaccharide unit. We isolated and purified the monomer among low‐molecular‐weight succinoglycans and used this microbial linear octasaccharide as a novel chiral additive for enantiomeric separation of some flavanones such as homoeriodictyol, hesperetin, naringenin, and isosakuranetin in CE. Throughout the present investigation, we firstly used noncyclic oligosaccharides for the chiral separation of flavanones. We also found that successful enantioseparation of four flavanones depends on the presence of succinate substituents of the linear monomeric octasaccharide in CE, suggesting that succinylation of succinoglycan monomer is decisive for the effective enantiomeric separation.

Chiral separation and discrimination of catechin by sinorhizobial octasaccharides in capillary electrophoresis and 13C NMR spectroscopy.

Succinoglycan, a sinorhizobial exopolysaccharide produced by Sinorhizobium meliloti, is composed of an octasaccharide subunit. S. meliloti produces both high-molecular-weight and low-molecular-weight (M(r)<10,000) succinoglycans which consist of monomers, dimers, or trimers. Succinoglycan monomers were isolated and further purified in the monomer series (M1, M2, and M3) by the degree of succinylation. We used sinorhizobial octasaccharides (M1, M2, and M3) as chiral additives in capillary electrophoresis (CE) for chiral separation of catechin and also as chiral shift reagents with (13)C NMR spectroscopy for chiral discrimination of catechin. Chiral separation of catechin took place when sinorhizobial octasaccharides (M2 and M3) were added to the background electrolyte (BGE) in CE. NMR signal splittings were also observed in the interactions of sinorhizobial octasaccharides with the enantiomers of catechin. Both chiral separation and discrimination of catechin depend on the presence of succinate substituents of the linear monomeric octasaccharide in CE and NMR spectroscopy, suggesting that succinylation of sinorhizobial octasaccharide is decisive for the effective chiral separation and discrimination of catechin.

Development of protein-cage-based delivery nanoplatforms by polyvalently displaying β-cyclodextrins on the surface of ferritins through copper(I)-catalyzed azide/alkyne cycloaddition.

Protein cages are spherical hollow macromolecules that are attractive platforms for the construction of nanoscale cargo delivery vehicles. Human heavy-chain ferritin (HHFn) is modified genetically to control the number and position of functional groups per cage. 24 β-CDs are conjugated precisely to the modified HHFn in specific locations through thiol-maleimide Michael-type addition followed by copper(I)-catalyzed azide/alkyne cycloaddition (CuAAC). The resulting human ferritins displaying β-CDs (β-CD-C90 HHFn) can form inclusion complexes with FITC-AD, which can slowly release the guest molecule reversibly in a buffer solution via non-covalent β-CD/AD interactions. β-CD-C90 HHFn can potentially be used as delivery vehicles for insoluble drugs.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric behavior of succinoglycan monomers, dimers, and trimers isolated from Sinorhizobium meliloti 1021

Low-molecular-weight (LMW) succinoglycans (monomers, dimers, and trimers) were isolated from Sinorhizobium meliloti 1021 and have been firstly investigated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) using 2,4,6-trihydroxyacetophenone (THAP) as an optimal matrix in the negative ion mode. The main fractions of LMW succinoglycans contain molecules assembled of octasaccharide subunits. MALDI-TOF mass spectra of the LMW succinoglycan monomers, the dimers, and the trimers showed the daughter ions resulting from the losses of the terminal galactose residues at the reducing ends, clearly indicating that the galactosyl linkages are more labile than the other glucosyl linkages. Furthermore, the losses of the acetyl groups as substituents rather than the succinyl and pyruvyl ester linkages by prompt fragmentation primarily occurred during MALDI-TOF analysis, suggesting the greater instability of acetyl linkages compared to pyruvyl and succinyl linkages.

Stereoisomeric separation of some flavanones using highly succinate-substituted α-cyclosophoro-octadecaoses as chiral additives in capillary electrophoresis.

α-Cyclosophoro-octadecaoses (α-C18), produced by Rhodobacter sphaeroides, are mostly homogeneous in size with 18 glucose units per ring as the predominant form. α-C18s are linked by β-(1→4)-linkages and one α-(1→6)-linkage and are also known to be highly substituted by acetyl (0-2 per mol) and/or succinoyl groups (1-7 per mol). We isolated and purified α-C18 and successfully used it in capillary electrophoresis (CE) as a chiral additive for the separation of five flavanones and flavanone-7-O-glycosides, including naringenin, hesperetin, eriodictyol, homoeriodictyol, isosakuranetin, and hesperidin. Throughout the CE experiment with unsubstituted α-C18 (uα-C18) obtained after alkaline treatment of the isolated α-C18, we found that successful chiral separation critically depends on the presence of succinate substituents attached to α-C18 in CE, suggesting that succinoylation of α-C18 is decisive for effective stereoisomeric separation.

Synthesis of selenium nanowires morphologically directed by Shinorhizobial oligosaccharides

Shinorhizobial cyclosophoraose (cyclic beta-(1-->2)-glucan) or succinoglycan monomer (SGM 2), which has one acetyl, pyruvyl, and succinyl group, functions as a morphology-directing agent for the synthesis of pure trigonal selenium nanowires by using ascorbic acid (vitamin C) as the reducing agent. The synthesis was achieved in water at room temperature. Under these experimental conditions, the diameters of the as-prepared Se nanowires were varied in the range of 34-120 nm by cyclosophoraose and of 33-66 nm by SGM 2, in which the nanowires were characterized using X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and Raman spectroscopy. Through this study, we propose that Shinorhizobial cyclic and linear oligosaccharides have morphologically directing functions for the synthesis of single-crystalline selenium nanowires by green chemical methods.

Low-energy collision-activated dissociation electrospray ionization tandem mass spectrometric analysis of Sinorhizobial succinoglycan monomers.

Succinoglycan monomers (M1, M2, and M3) are octasaccharides with acetyl, pyruvyl, and/or succinyl groups as substituents derived from Sinorhizobium meliloti 1021. The dissociation patterns of the octasaccharides caused by low-energy collision-activated dissociation (CAD) were investigated using triple quadrupole tandem mass spectrometry (MS) equipped with an electrospray ionization (ESI) source with increasing collision energy (CE) in negative ion mode. None of the succinoglycan monomers were fragmented at a CE of -25eV. When the CE was applied to -50 or -70eV, the loss of the terminal Gal residue and/or the succinyl group of the monomers was observed in the product ion scan mode. Interestingly, the acetyl and the pyruvyl groups in the succinoglycan monomers were not lost even when a CE of -70eV was applied, indicating that the substituents are more stable than the succinyl group in the octasaccharides.

Biotinylation of the rhizobial cyclic β-glucans and succinoglycans crucial for symbiosis with legumes

The cyclic β-glucans and succinoglycans produced by rhizobia are required for nodulation during symbiosis with legume hosts. However, only gene deletion analyses have been used to investigate their biological importance. For future studies on the physiological activity of those during symbiosis, biochemical methods need to be developed with separate carbohydrate compounds. Here, we isolated and purified rhizobial cellular carbohydrates using various chromatographic methods. Purified cyclic β-glucans, cyclosophoraoses, were monofunctionalized with biotin using the following three steps: tosylation, azidation, and amination. The mono-6-amino-cyclosophoraoses were linked with biotinamidohexanoic acid N-hydroxysuccinimide ester. Succinoglycans and monomers were tagged with biotinamidocaproyl hydrazide at the reducing sugar via reductive amination. The resulting biotinylated rhizobial carbohydrates were characterized by Fourier transform infrared and nuclear magnetic resonance spectroscopy, matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy, and electrospray ionization mass spectrometry. The resulting neoglycoconjugates can be used as solid probes to study putative plant receptors and for non-invasive imaging for in vivo tracing.

Cholesterol reduction from milk using β-cyclodextrin immobilized on glass.

β-Cyclodextrin (β-CD) was converted into β-CD-undecenyl ether by chemical modification and subsequently covalently attached to a glass surface. The functionalized glass surface was characterized by static water contact angle and x-ray photoelectron spectroscopy. Both techniques confirmed that an excellent monolayer of β-CD was formed on the glass surface. The β-CD solid surface was used to reduce cholesterol levels in milk. In 4h, 73.6% of the cholesterol was extracted at 25°C with shaking at 170rpm. This is the highest value ever reported for milk using β-CD immobilized on a solid surface. The same surface was repeatedly used for 10 cycles and maintained its efficiency with 72±2% cholesterol reduction observed in all the cycles. X-ray photoelectron spectroscopy analysis completed after 5 and 10 cycles of cholesterol reduction showed that the β-CD on the glass surface was not degraded. The high efficiency and long-term stability of the functional monolayer was attributed to the specific structure of β-CD, which is composed of a relatively low number of functional groups and long spacer chain lengths that provide great flexibility.

Proton conduction in biopolymer exopolysaccharide succinoglycan

Protonic currents play a vital role in electrical signalling in living systems. It has been suggested that succinoglycan plays a specific role in alfalfa root nodule development, presumably acting as the signaling molecules. In this regard, charge transport and proton dynamics in the biopolymer exopolysaccharide succinoglycan have been studied by means of electrical measurements and nuclear magnetic resonance (NMR) spectroscopy. In particular, a dielectric dispersion in the system has revealed that the electrical conduction is protonic rather electronic. Besides, our laboratory- and rotating-frame 1H NMR measurements have elucidated the nature of the protonic conduction, activation of the protonic motion being associated with a glass transition.