Imaging of enzyme activity by scanning electrochemical microscope equipped with a feedback control for substrate-probe distance

The enzymatic activity of diaphorase (Dp) immobilized on a solid substrate was characterized using a scanning electrochemical microscope (SECM) with shear force feedback to control the substrate-probe distance. The shear force between the substrate and the probe was monitored with a tuning fork-type quartz crystal and used as the feedback control to set the microelectrode probe close to the substrate surface. The sensitivity and the contrast of the SECM image were improved in the constant distance mode (distance, 50 nm) with the shear force feedback compared to the image in the constant height mode without the feedback. By using this system, the SECM and topographic images of the immobilized diaphorase were simultaneously measured. The microelectrode tip used in this study was ground aslant like a syringe needle in order to obtain the shaper topographic images. This shape was also effective for avoiding the interference during the diffusion of the enzyme substrates.     

Facile syntheses of 3-spiro- and 3,6-bridged 2,5-piperazinediones

The intramolecular addition of hydroxyl group of 3-(3-hydroxyl)propylidene-2,5-piperazinedione to 3-position and the same substitution of 3-(3-hydroxy)propyl derivative to 6-position gave the corresponding 3-spiro- and 3,6-bridged 2,5-piperazinediones, respectively.     

Carbon-Fluorine Bondings of Fluorinated Fullerene and Graphite

Carbon-fluorine bondings of fluorinated fullerenes and fluorine-graphite intercalation compound C_xF were investigated in detail on the basis of XPS data and the potential model using the charge distribution calculated by semiempirical method. It has been confirmed by the present study that two peaks in the C_1s spectra observed for fluorinated fullerenes are assigned to carbon atoms bonded to fluorine atoms and those unbound to fluorine atoms, and the small difference in charges and Madelung potentials of fluorine atoms in different circumstances well explains the single peak in F_1s spectra of fluorinated fullerenes. In the calculated structures of 1,3-C₆₀F₂ and 1,2-C₆₀F_x (x = 2?6) used as the models of C_xF, three kinds of carbon-fluorine bondings were observed corresponding to nearly ionic, semicovalent and covalent C? F bondings. The calculated result supports that the bi-intercalation structure of stage 1 C_xF consists of nearly ionic and semi-covalent fluorines.     

Ruthenium-catalyzed propargylic substitution reactions of propargylic alcohols with oxygen-, nitrogen-, and phosphorus-centered nucleophiles

The scope and limitations of the ruthenium-catalyzed propargylic substitution reaction of propargylic alcohols with heteroatom-centered nucleophiles are presented. Oxygen-, nitrogen-, and phosphorus-centered nucleophiles such as alcohols, amines, amides, and phosphine oxide are available for this catalytic reaction. Only the thiolate-bridged diruthenium complexes can work as catalysts for this reaction. Results of some stoichiometric and catalytic reactions indicate that the catalytic propargylic substitution reaction proceeds via an allenylidene complex formed in situ, whereby the attack of nucleophiles to the allenylidene C(gamma) atom is a key step. Investigation of the relative rate constants for the reaction of propargylic alcohols with several para-substituted anilines reveals that the attack of anilines on the allenylidene C(gamma) atom is not involved in the rate-determining step and rather the acidity of conjugated anilines of an alkynyl complex, which is formed after the attack of aniline on the C(gamma) atom, is considered to be the most important factor to determine the rate of this catalytic reaction. The key point to promote this catalytic reaction by using the thiolate-bridged diruthenium complexes is considered to be the ease of the ligand exchange step between a vinylidene ligand on the diruthenium complexes and another propargylic alcohol in the catalytic cycle. The reason why only the thiolate-bridged diruthenium complexes promote the ligand exchange step more easily with respect to other monoruthenium complexes in this catalytic reaction should be that one Ru moiety, which is not involved in the allenylidene formation, works as an electron pool or a mobile ligand to another Ru site. The catalytic procedure presented here provides a versatile, direct, and one-step method for propargylic substitution of propargylic alcohols in contrast to the so far well-known stoichiometric and stepwise Nicholas reaction.     

New hydroxyl protecting groups of a safety-catch type removable by reductive acidolysis.

New hydroxyl protecting groups of a safety-catch type, i.e., 4-methylsulfinylbenzyl-oxycarbonyl (Msz) group for Tyr and 4-methylsulfinylbenzyl (Msob) ether for Thr, have been developed. O-Msz and O-Msob groups are stable under both acidic and basic conditions and can be removed by a one-pot reaction involving reductive acidolysis using tetrachlorosilane-trifluoroacetic acid (TFA)-scavengers. Using these new protecting groups, a 17 residue-peptide, gamma-endorphin, was successfully synthesized by the efficient solid phase method.     

Ruthenium-catalyzed propargylic substitution reaction of propargylic alcohols with thiols: a general synthetic route to propargylic sulfides

A novel cationic methanethiolate-bridged diruthenium complex [Cp*RuCl(mu2-SMe)2RuCp*(OH2)]OTf (1e) has been disclosed to promote the catalytic propargylic substitution reaction of propargylic alcohols bearing not only terminal alkyne group but also internal alkyne group with thiols. It is noteworthy that neutral thiolate-bridged diruthenium complexes (1a-1c), which were known to promote the propargylic substitution reactions of propargylic alcohols bearing a terminal alkyne group with various heteroatom- and carbon-centered nucleophiles, did not work at all. The catalytic reaction described here provides a general and environmentally friendly preparative method for propargylic sulfides, which are quite useful intermediates in organic synthesis, directly from the corresponding propargylic alcohols and thiols.     

Structural regulation of a peptide-conjugated graft copolymer: a simple model for amyloid formation

The self-assembly of peptides and proteins into beta-sheet-rich high-order structures has attracted much attention as a result of the characteristic nanostructure of these assemblies and because of their association with neurodegenerative diseases. Here we report the structural and conformational properties of a peptide-conjugated graft copolymer, poly(gamma-methyl-L-glutamate) grafted polyallylamine (1) in a water-2,2,2-trifluoroethanol solution as a simple model for amyloid formation. Atomic force microscopy revealed that the globular peptide 1 self-assembles into nonbranching fibrils that are about 4 nm in height under certain conditions. These fibrils are rich in beta-sheets and, similar to authentic amyloid fibrils, bind the amyloidophilic dye Congo red. The secondary and quaternary structures of the peptide 1 can be controlled by manipulating the pH, solution composition, and salt concentration; this indicates that the three-dimensional packing arrangement of peptide chains is the key factor for such fibril formation. Furthermore, the addition of carboxylic acid-terminated poly(ethylene glycol), which interacts with both of amino groups of 1 and hydrophobic PMLG chains, was found to obviously inhibit the alpha-to-beta structural transition for non-assembled peptide 1 and to partially cause a beta-to-alpha structural transition against the 1-assembly in the beta-sheet form. These findings demonstrate that the amyloid fibril formation is not restricted to specific protein sequences but rather is a generic property of peptides. The ability to control the assembled structure of the peptide should provide useful information not only for understanding the amyloid fibril formation, but also for developing novel peptide-based material with well-defined nanostructures.     

Functional monomers and polymers 159 synthesis and photochemical reactions of polymers containing thymine photodimer units in the main chain

Polyamides containing thymine photodimer units in the main chain were synthesized, and their photolysis by ultraviolet irradiation below 260 nm were studied in film state. Photodimers of thymine derivatives were obtained by photochemical reaction of the carboxylic acid derivatives of thymine in aqueous solution irradiated above 270 nm. An attempt was made to resolve the isomers of the photodimers, and the two kinds of cis isomers [cis–syn(head to head), and cis–anti(head to tail)] were isolated successfully. The polyamides were prepared by condensation of the photodimers with diamine using an activated ester method. The photodissociation of the thymine photodimer in the polymer main chain caused the breakage of the polymer chains, leading to the production of oligomers and dimer compounds containing thymine bases at the ends of the molecule. The dissociation rate of the polymer did not depend on the kind of the thymine photodimer which was in the main chain of the polymer.     

Excitation-energy migration in self-assembled cyclic zinc(II)-porphyrin arrays: a close mimicry of a natural light-harvesting system

The excitation-energy-hopping (EEH) times within two-dimensional cyclic zinc(II)-porphyrin arrays 5 and 6, which were prepared by intermolecular coordination and ring-closing metathesis reaction of olefins, were deduced by modeling the EEH process based on the anisotropy depolarization as well as the exciton-exciton annihilation dynamics. Assuming the number of energy-hopping sites N = 5 and 6, the two different experimental observables, that is, anisotropy depolarization and exciton-excition annihilation times, consistently give the EEH times of 8.0 +/- 0.5 and 5.3 +/- 0.6 ps through the 1,3-phenylene linkages of 5 and 6, respectively. Accordingly, the self-assembled cyclic porphyrin arrays have proven to be well-defined two-dimensional models for natural light-harvesting complexes.