Hiroyuki Tonami

Peroxidase-catalyzed oxidative polymerization of m-substituted phenol derivatives

Peroxidase-catalyzed oxidative polymerization of m-substituted phenols has been performed in a mixture of a water-miscible organic solvent and buffer at room temperature under air to give a new class of polyphenols. The catalysts used were horseradish and soybean peroxidases (HRP and SBP, respectively). In the polymerization of m-cresol using HRP as the catalyst, effects of an organic solvent, buffer pH, and their mixed ratio have been systematically investigated with respect to the polymer yield, solubility, and molecular weight. The HRP-catalyzed polymerization of m-cresol in an equivolume mixture of methanol and phosphate buffer (pH 7) produces the polymer in a high yield, which is readily soluble in polar solvents such as methanol, acetone, N,N-dimethylformamide, and dimethyl sulfoxide. The polymer was estimated to consist of a mixture of phenylene and oxyphenylene units from NMR and IR analyses as well as titration of the residual phenolic moiety of the polymer. The polymerization behavior of the m-substituted monomers greatly depends on the enzyme type. In using SBP as a catalyst, the polymer yield increases as a function of the bulkiness of the substituent, whereas the opposite tendency was observed in case of HRP catalysis. The relationships between the monomer substituent and the polymerization behavior are discussed in terms of the HOMO level of the monomer and the substituent volume.

Development of Nanofiber-Covered Stents Using Electrospinning: In Vitro and Acute Phase In Vivo Experiments

There are some technical difficulties in treating for a broad necked aneurysm and a higher incidence of recurrence. Because of these drawbacks, more innovative techniques for superior endovascular reconstructive treatment are required. We developed a novel covered stent employing electrospinning to deposit fine polyurethane (PU) fibers onto stents. An in vitro water leak test was designed and applied prior to animal testing to estimate the performance of covered stents and to determine the appropriate amount of PU fibers on a stent. Two tenths of a milligram of PU fibers proved to be sufficient to prevent water leakage. Then, the efficacy of the covered stents to that of bare stents was compared using 10 rabbits in which model aneurysms had been formed at the right common carotid artery by the elastase method. Angiographic evaluation on day 1 posttreatment (acute phase) revealed complete occlusion of the aneurysms and the patency of the parent arteries in animals treated with covered stents. At 10 days poststenting (subacute phase), the aneurysm neck was completely covered with neointimal layer as shown by scanning electron microscopic examination. The PU-covered stent holds promise as a device for treating cerebral aneurysms.

Cell orientation and regulation of cell–cell communication in human mesenchymal stem cells on different patterns of electrospun fibers

Cell behavior can be manipulated by the topography of the culture surface. In this study, we examined the intercellular communication and osteogenic differentiation of mesenchymal stem cells (MSCs) grown on electrospun fibers with different orientations and densities. Human bone marrow-derived MSCs (hMSCs) were seeded on poly(ε-caprolactone) (PCL) electrospun scaffolds composed of aligned (1D) or cross-aligned (2D) fibers (1.0-1.2 µm diameter) with high, medium, or low fiber densities. It was found that cells preferred to adhere onto electrospun PCL fibers rather than on the flat substrate. The immunofluorescence staining showed that the expression of vinculin, a focal adhesion protein, was limited to the periphery and the two extremities of aligned cells on the edge of the fibers. Electron microscopy showed that cells extended their lamellipodia across the adjacent fibers and proliferated along the direction of fibers. Cells grown on 1D fibrous scaffolds at all fiber densities had an obvious alignment. On 2D fibers, a higher degree of cell alignment was observed at the higher fiber density. On 1D scaffolds, the gap junction intercellular communication (GJIC) quantified by the lucifer yellow dye transfer assay was significantly promoted in the aligned cells in the direction parallel to the fibers but was abolished in the direction perpendicular to the fibers. The expression of osteogenic marker genes (RUNX2, ALP, and OCN) was significantly enhanced in seven days by culture on 1D but not 2D fibers. It was thus proposed that the promoted osteogenic differentiation of hMSCs may be associated with the fiber-guided and directional induction of GJIC.

Chemoenzymatic synthesis of a poly(hydroquinone)

Chemoenzymatic synthesis of a poly(hydroquinone) was achieved by enzymatic oxidative polymerization of 4-hydroxyphenyl benzoate, followed by alkaline hydrolysis of the resulting polymer. The polymerization of 4-hydroxyphenyl benzoate was performed using a peroxidase and hydrogen peroxide as catalyst and oxidizing agent, respectively, in an aqueous organic solvent. Soybean peroxidase afforded the polymer in good yields. IR analysis of the polymer showed the formation of the polymer consisting of a mixture of phenylene and oxyphenylene units. By alkaline hydrolysis of the resulting polymer, benzoate moiety was completely removed to give poly(hydroquinone).

Regio‐ and chemo‐selective polymerization of phenols catalyzed by oxidoreductase enzyme and its model complexes

Oxidative polymerizations of phenol derivatives have been performed using an oxidoreductase enzyme and its model complexes as catalyst to produce new functional polymers. Soluble polyphenols were synthesized using peroxidase catalyst in an aqueous methanol. Enzymatic polymerization of syringic acid involved elimination of carbon dioxide and hydrogen from the monomer to give poly(1,4-oxyphenylene) (PPO). Tyrosinase-model complexes catalyzed highly regioselective oxidative polymerization of a 2,6-unsubstituted phenol, 4-phenoxyphenol, to produce unsubstituted PPO showing crystallinity with a melting point. Chemoselective polymerization of phenols having an unsaturated group took place through peroxidase catalysis, yielding crosslinkable polyphenols.

PEROXIDASE-CATALYZED POLYMERIZATION OF FLUORINE-CONTAINING PHENOLS

Peroxidase-catalyzed oxidative polymerization of fluorine-containing phenols has been performed in a mixture of a water-miscible organic solvent and buffer at room temperature under air. The monomers used were 2,6-difluorophenol, 3- and 4-fluorophenols. In the polymerization of 2,6-diflurophenol catalyzed by horseradish peroxidase (HRP), effects of an organic solvent, buffer pH, and their mixed ratio have been systematically investigated with respect to the polymer yield and molecular weight. The resulting polymer was soluble in common polar organic solvents and showed good water repellent property. From NMR and IR data, it was supposed that the polymer was of mainly 2,6-difluoro-1,4-oxyphenylene unit. Elemental analysis showed that the elimination of a small amount of the fluorine atom took place during the polymerization. HRP catalysis induced the polymerization of 3- and 4-fluorophenols, yielding a new class of fluorine-containing polyphenols.

Enzymatic polymerization of m-substituted phenols in the presence of 2,6-di-O-methyl-β-cyclodextrin in water

Abstract Enzymatic polymerization of m-substituted phenols has been achieved in the presence of 2,6-di-O-methyl-β-cyclodextrin (DM-β-CD) in a buffer. A watersoluble complex of the monomer and DM-β-CD was formed and polymerization was performed by peroxidase catalyst to give the polymer in high yields. Formation of the inclusion complex was confirmed by NOESY NMR. The association constant was determined by NMR and found to depend on the monomer structure.

Application of laser to measurement of cyclic contractile movement of cultured myotubes

Laser technique has been applied to measure cyclic contractile movement of cultured myotubes in vitro. The designed measurement system includes light source (helium neon, 632.8 nm wave length), charge-coupled devise cameras and detectors. Cyclic contraction of myotubes cultured from C2C12 (mouse myoblast) was generated by cyclic electric pulses (amplitude < 60 V, 1 ms width) through electrodes of platinum wire dipped in the medium. The spectrum of fluctuating intensity of the transmitted laser beam through the myotubes, which periodically repeated contraction and relaxation, was analyzed. The results show that the designed laser system is effective to detect frequency of cyclic contractile movement of myotubes between 0.5 Hz and 5 Hz.