Hideki Onagi

Molecular fibers and wires in solid-state and solution self-assemblies of cyclodextrin [2]rotaxanes

Cyclodextrin [2]rotaxanes have been prepared by coupling dimethylanilines with dicarboxylic acids using DMT-MM, in aqueous solutions of alpha-cyclodextrin, and the example illustrated shows unusual fluorescence emission and other spectroscopic behavior characteristic of the formation of molecular wires in solution, similar to the fibers observed in the solid state.

Synthesis and conformational analysis of an α-cyclodextrin [2]-rotaxane

An α-cyclodextrin [2]-rotaxane has been prepared in 10% yield, by threading α-cyclodextrin (α-CD) with (E)-4,4′-diaminostilbene in aqueous solution and capping the included guest through reaction with 2,4,6-trinitrobenzene-1-sulfonate. 1D 1H NMR spectroscopy and DQCOSY and ROESY experiments show that the α-CD rotates freely around the axle of the rotaxane, but is localised over the olefinic moiety of the stilbene. The pKa values of the α-CD [2]-rotaxane were found to be 9.3 and 9.6, which are attributable to deprotonations of the (E)-4,4′-bis(2,4,6-trinitrophenylamino)stilbene moiety. NMR experiments show that these deprotonations do not perturb the conformation of the rotaxane.

Prohormone-substrate peptide sequence recognition by peptidylglycine α-amidating monooxygenase and its reflection in increased glycolate inhibitor potency.

The interactions of nineteen peptide substrates and fifteen analogous peptidomimetic glycolate inhibitors with human peptidylglycine α-amidating monooxygenase (PAM) have been investigated. The substrates and inhibitors are the prohormones of calcitonin and oxytocin and their analogues. PAM both secreted into the medium by and extracted from DMS53 small lung carcinoma cells has been studied. The results show that recognition of the prooxytocin and procalcitonin peptide sequences by the enzyme extends more than four and five amino acid residues, respectively, from their C-termini. This substrate sequence recognition is mirrored by increased inhibitor potency with increased peptide length in the glycolate peptidomimetics. Substitution of the C-terminal penultimate glycine and proline residues of prooxytocin and procalcitonin and their analogues with phenylalanine increases the enzyme binding affinity. However, this changes the binding mode from one that depends on peptide sequence recognition to another primarily determined by the phenylalanine moiety, for both the substrates and analogous glycolate inhibitors.

Hyperthermophilic Carbamate Kinase Stability and Anabolic In Vitro Activity at Alkaline pH

ABSTRACT Carbamate kinases catalyze the conversion of carbamate to carbamoyl phosphate, which is readily transformed into other compounds. Carbamate forms spontaneously from ammonia and carbon dioxide in aqueous solutions, so the kinases have potential for sequestrative utilization of the latter compounds. Here, we compare seven carbamate kinases from mesophilic, thermophilic, and hyperthermophilic sources. In addition to the known enzymes from Enterococcus faecalis and Pyrococcus furiosus, the previously unreported enzymes from the hyperthermophiles Thermococcus sibiricus and Thermococcus barophilus, the thermophiles Fervidobacterium nodosum and Thermosipho melanesiensis, and the mesophile Clostridium tetani were all expressed recombinantly, each in high yield. Only the clostridial enzyme did not show catalysis. In direct assays of carbamate kinase activity, the three hyperthermophilic enzymes display higher specific activities at elevated temperatures, greater stability, and remarkable substrate turnover at alkaline pH (9.9 to 11.4). Thermococcus barophilus and Thermococcus sibiricus carbamate kinases were found to be the most active when the enzymes were tested at 80°C, and maintained activity over broad temperature and pH ranges. These robust thermococcal enzymes therefore represent ideal candidates for biotechnological applications involving aqueous ammonia solutions, since nonbuffered 0.0001 to 1.0 M solutions have pH values of approximately 9.8 to 11.8. As proof of concept, here we also show that carbamoyl phosphate produced by the Thermococcus barophilus kinase is efficiently converted in situ to carbamoyl aspartate by aspartate transcarbamoylase from the same source organism. Using acetyl phosphate to simultaneously recycle the kinase cofactor ATP, at pH 9.9 carbamoyl aspartate is produced in high yield and directly from solutions of ammonia, carbon dioxide, and aspartate. IMPORTANCE Much of the nitrogen in animal wastes and used in fertilizers is commonly lost as ammonia in water runoff, from which it must be removed to prevent downstream pollution and evolution of nitrogenous greenhouse gases. Since carbamate kinases transform ammonia and carbon dioxide to carbamoyl phosphate via carbamate, and carbamoyl phosphate may be converted into other valuable compounds, the kinases provide a route for useful sequestration of ammonia, as well as of carbon dioxide, another greenhouse gas. At the same time, recycling the ammonia in chemical synthesis reduces the need for its energy-intensive production. However, robust catalysts are required for such biotransformations. Here we show that carbamate kinases from hyperthermophilic archaea display remarkable stability and high catalytic activity across broad ranges of pH and temperature, making them promising candidates for biotechnological applications. We also show that carbamoyl phosphate produced by the kinases may be efficiently used to produce carbamoyl aspartate.

A Cyclodextrin-Based Photoresponsive Molecular Gate that Functions Independently of Either Solvent or Potentially Competitive Guests

The photoinduced interconversion between cinnamido-substituted cyclodextrins constitutes a gating switch through which the substituent moves to open or block access to the cyclodextrin cavity. Most unusually for a cyclodextrin-based device, the operation of this gate is solvent-independent and unaffected by potentially competitive guests. It occurs in MeOH and DMSO, as well as in water. This contrasts with other cyclodextrin inclusion phenomena that are usually driven by hydrophobic effects and limited to aqueous media.

Detection of Biosynthetic Precursors, Discovery of Glycosylated Forms, and Homeostasis of Calcitonin in Human Cancer Cells

The peptide hormone calcitonin is intimately connected with human cancer development and proliferation. Its biosynthesis is reasoned to proceed via glycine-, α-hydroxyglycine-, glycyllysine-, and glycyllysyllysine-extended precursors; however, as a result of the limitations of current analytical methods, until now, there has been no procedure capable of detecting these individual species in cell or tissue samples. Therefore, their presence and dynamics in cancer had not been established. Here, we report the first methodology for the separation, detection, and quantification of calcitonin and each of its precursors in human cancer cells. We also report the discovery and characterization of O-glycosylated calcitonin and its analogous biosynthetic precursors. Through direct and simultaneous analysis of the glycosylated and nonglycosylated species, we interrogate the hormone biosynthesis. This shows that the cellular calcitonin level is maintained to mitigate effects of biosynthetic enzyme inhibitors that substantially change the proportions of calcitonin-related species released into the culture medium.