Dynamic analytical chemistry: A trial study of the interaction of fluorogenic reagents with living chinese hamster ovary cells

The concept of dynamic analytical chemistry, which deals with the qualitative localization or the quantitative transportation of a known or unknown component or its conversion into a product within living cells, is introduced. The concept was tested using cultured Chinese hamster ovary cells in a phosphate-buffered saline medium in the presence of fluorogenic reagents for amines and thiols. The phenomenon was observed and recorded with a fluorescence microscope equipped with a SIT camera. Among the reagents, 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) added to the culture medium was first trapped at the surface of the cells and then passed inside the cells to react with the mitochondria to give yellow fluorescence, o-Phthalaldehyde and 4-aminosulphonyl-7-fluoro-2, 1,3-benzoxadiazole reacted with the cells to give a blue florescence at certain sites inside the cells. One of the two major components labelled with NBD-F appeared to be phosphatidylethanolamine, a component of the plasma membrane. Its identification and quantitation were effected by conventional analytical techniques, such as thin-layer and liquid chromatography, following enzymatic hydrolysis. The fate of the N-NBD-phosphatidylethanolamine is discussed.     

The first example of direct cyclopropylation of arylamines at the 2-position with magnesium cyclopropylidenes

Treatment of magnesium cyclopropylidenes, which were generated from 1-chlorocyclopropyl phenyl sulfoxides with isopropylmagnesium chloride in THF at −78 °C, with N-lithio arylamines gave arylamines cyclopropylated at the 2-position in moderate to good yields. Use of the magnesium cyclopropylidenes having at least one substituent was found to be essential to this cyclopropylation. This procedure offers a novel synthesis of arylamines having a cyclopropane ring at the 2-position directly from arylamines.     

Synthesis of diaminopimelic acid containing peptidoglycan fragments and tracheal cytotoxin (TCT) and investigation of their biological functions

Bacterial cell wall peptidoglycan (PGN) is a potent immunostimulator and immune adjuvant. The PGN of Gram-negative bacteria and some Gram-positive bacteria contain meso-diaminopimelic acid (meso-DAP), and we have recently shown that the intracellular protein Nod1 is a PGN receptor and recognizes DAP-containing peptides. In this study, we achieved the synthesis of DAP-containing PGN fragments, including the first chemical synthesis of tracheal cytotoxin (TCT), GlcNAc-(beta1-4)-(anhydro)MurNAc-L-Ala-gamma-D-Glu-meso-DAP-D-Ala, and a repeating-unit of DAP-type PGN, GlcNAc-(beta1-4)-MurNAc-L-Ala-gamma-D-Glu-meso-DAP-D-Ala. For the synthesis of PGN fragments, we first established a new synthetic method for an orthogonally protected meso-DAP derivative, and then we constructed the glycopeptide structures. The ability of these fragments to stimulate human Nod1, as well as differences in Nod1 recognition of the variety of synthesized ligand structures were examined. The results showed that the substitution of the N terminus of iE-DAP is necessary for stronger Nod1 recognition, but the structure of the substituent seems not to be strictly recognized. The importance of the carboxyl group at the 2-position of DAP for human Nod1 stimulation was also shown.     

Chiral Biomineralization: Mirror‐Imaged Helical Growth of Calcite with Chiral Phosphoserine Copolypeptides

The O‐phospho‐L ‐serine [Ser(P)] containing peptides and proteins play an important role in controlling the morphology of biominerals. The poly[Ser(P)] and copoly[Ser(P)^xAsp^y] affect the calcium carbonate (CaCO₃) morphology and polymorph. The CaCO₃ helical structures were obtained in the presence of copoly[Ser(P)⁷⁵Asp²⁵]. When the L ‐copolymer was used as an additive, a clockwise P twisted spiral morphology was formed. On the other hand, when using D ‐copolymer, a counterclockwise M twisted spiral morphology was obtained.

Optical micrographs of chiral morphologies of CaCO₃ in the presence of a) L ‐copolymer and b) D ‐copolymer.     

Lateral thermal expansion of chitin crystals

Measurements of the thermal expansion coefficients (TECs) of chitin crystals in the lateral direction are reported. We investigated highly crystalline α chitin from the Paralithodes tendon and an anhydrous form of β chitin from a Lamellibrachia tube from room temperature to 250 °C, using X‐ray diffraction at selected temperatures in the heating process. For α chitin, the TECs of the a and b axes were α_a = 6.0 × 10⁻⁵ °C⁻¹ and α_b = 5.7 × 10⁻⁵ °C⁻¹, indicating an isotropic thermal expansion in the lateral direction. However, the anhydrous β chitin exhibited an anisotropic thermal expansion in the lateral direction. The TEC of the a axis was constant at α_a = 4.0 × 10⁻⁵ °C⁻¹, but the TEC of the b axis increased linearly from room temperature to 250 °C, with α_b = 3.0–14.6 × 10⁻⁵ °C⁻¹. These differences in the lateral thermal expansion behaviors of the α chitin and the anhydrous β chitin are due to their different intermolecular hydrogen bonding systems. © 2000 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 39: 168–174, 2001     

Recent Progress on Cyclic Nitrenoid Precursors in Transition-Metal-Catalyzed Nitrene-Transfer Reactions

Nitrene-transfer reactions are powerful synthetic tools for the direct incorporation of nitrogen atoms into organic molecules. The discovery of novel nitrene-transfer reactions has been dominantly supported not only by improvements in transition-metal catalysts but also by the employment of novel precursors of nitrenoids. Since pioneering work involving the use of organic azides and iminoiodinanes as practical synthetic tools for nitrogen-containing compounds was reported, a new approach using various N-heterocycles containing strain energy or a weak bond has emerged. In this review, we briefly summarize the history of nitrene-transfer chemistry from the viewpoint of its precursors. In particular, the use of N-heterocycles such as 2H-azirines, 1,4,2-dioxazol-5-ones, 1,2,4-oxadiazol-5-ones, isoxazol-5(4H)-ones, and isoxazoles is comprehensively described, showing the recent remarkable progress in this chemistry.     

Reactivity of Hydroxy‐ and Aquo(hydroxy)‐λ3‐iodane–Crown Ether Complexes

We have designed a series of hydroxy(aryl)‐λ³‐iodane–[18]crown‐6 complexes, prepared from the corresponding iodosylbenzene derivatives and superacids in the presence of [18]crown‐6, and have investigated their reactivities in aqueous media. These activated iodosylbenzene monomers are all non‐hygroscopic shelf‐storable reagents, but they maintain high oxidizing ability in water. The complexes are effective for the oxidation of phenols, sulfides, olefins, silyl enol ethers, and alkyl(trifluoro)borates under mild conditions. Furthermore, hydroxy‐λ³‐iodane–[18]crown‐6 complexes serve as efficient progenitors for the synthesis of diaryl‐, vinyl‐, and alkynyl‐λ³‐iodanes in water. Other less polar organic solvents, such as methanol, acetonitrile, and dichloromethane, are also usable in some cases.     

Enzymatic Synthesis of a DNA Triblock Copolymer that is Composed of Natural and Unnatural Nucleotides

DNA molecules have come under the spotlight as potential templates for the fabrication of nanoscale products, such as molecular‐scale electronic or photonic devices. Herein, we report an enhanced approach for the synthesis of oligoblock copolymer‐type DNA by using the Klenow fragment exonuclease minus of E. coli DNA polymerase I (KF^?) in a multi‐step reaction with natural and unnatural nucleotides. First, we confirmed the applicability of unnatural nucleotides with 7‐deaza‐nucleosides—which was expected because they were non‐metalized nucleotides—on the unique polymerization process known as the “strand‐slippage model”. Because the length of the DNA sequence could be controlled by tuning the reaction time, analogous to a living polymerization reaction on this process, stepwise polymerization provided DNA block copolymers with natural and unnatural bases. AFM images showed that this DNA block copolymer could be metalized sequence‐selectively. This approach could expand the utility of DNA as a template.     

Spherulite morphology studies of N‐alkyl chitosan films cast from formic acid solutions

The films of N‐ethyl chitosan were prepared via the solution‐casting technique with formic acid as a solvent. The solutions with different concentrations (35 and 40 wt %) were prepared previously from dilute solution (1 wt %) via evaporating process. The crystalline morphology of these films was investigated by means of polarized optical microscopy and scanning electron microscopy. Normal spherulites with a low growth rate formed in the casting films. The different morphologies of spherulite appeared in the films cast from the solutions with different concentrations. After further crystallizing for a few days, the spherulites were decorated by thousands of needlelike extended‐chain crystals, which had a typical size of ～50 μm (length) × 2～5 μm (width) × 1～2 μm (height) in the central part of the spherulite, but a typical size of ～5 μm (lenght)× 1～2 μm (width) × 1～2 μm (height) in the fringe part of the spherulite. The real concentration for crystallization was determined to be 65–82 wt%. Thus, the crystallization actually appeared in supersaturated solution. © 2003 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 41: 2033–2038, 2003