Takenori Tomohiro

Platinum (II) complex with cyclometalating 2-phenylpyridine ligand showing high cytotoxicity against cisplatin-resistant cell.

A platinum (II) mononuclear complex with two kinds of 2-phenylpyridine which coordinate as cyclometalated and non-chelated ligands shows high cytotoxicity against cisplatin-resistant mouse sarcoma 180 cell in comparison with its related complexes.

Synthesis of a novel lipopeptide with α-melanocyte-stimulating hormone peptide ligand and its effect on liposome stability

Introduction of liposomes into target cells is important for drug delivery systems. For this purpose, the surface of the liposome is equipped with ligand peptides, which may bind to specific receptors on the cell membrane. An artificial novel lipopeptide (MSH-C4A2) containing the α-melanocyte-stimulating hormone (α-MSH) sequence and two long alkyl chains was designed and synthesized, and the liposome, composed of egg phosphatidylcholine (EPC) and MSH-C4A2, was prepared. The stability of the liposome was estimated by measuring calcein leakage from the liposome inner phase. The stability of the liposome decreased upon addition of MSH-A4C2, which seemed to be attributable to the amphiphilic property of the peptide moiety (α-MSH) of MSH-A2C4. The stability was, however, recovered fairly well upon addition of cholesterol (Ch) or phosphatidylglycerol (PG). It was concluded therefore that the ternary system, MSH-C4A2/Ch/EPC or MSH-C4A2/PG/EPC, is suitable for preparing the functional liposome.

Synthesis of Diazirinyl Photoprobe Carrying a Novel Cleavable Biotin

Photoaffinity labeling is a powerful tool for the identification of receptor proteins and their binding sites. The major drawback of this method, however, is the reliability of the labeling and the purity of labeled peptides after purification from a large number of unlabeled fragments. The recent development of diazirine-based biotinylated probes provides an efficient solution to this problem. In these probes, the diazirine photophore contributes to the formation of a stable cross-link that greatly improves the efficiency. The application of an N-acetylglucosamine photoprobe carrying biotinylated diazirine provided the first information regarding acceptor-site peptides of b-1,4-galactosyltransferase. We have also developed a novel method for the one-step introduction of a biotinylated diazirine photophore into unprotected carbohydrate ligands. Since biotin–avidin binding is essentially irreversible (Kd = 10 15 m), several approaches have been investigated to achieve efficient recovery of biotinylated products from an immobilized avidin matrix. Although the use of monomeric avidin contributes to the isolation of biotinylated products because of the lower affinity to biotin (Kd = 10 8 m), it requires a high

Preparation of functional liposomes with peptide ligands and their binding to cell membranes

Two novel lipopeptides, which have the peptide ligands [α-melanocyte stimulating hormone (α-MSH)] sequence and repeated [Gly-Arg-Gly-Asp-Se (GRGDS) sequence[, are designed, synthesized by the solid-phase method, and introduced into liposome membranes by the freeze-thaw method. These liposomes bearing the peptide ligands on their surface are expected to bind to cell membranes. We have confirmed that the lipopeptides are introduced into liposome membranes almost quantitatively, while such a high degree of incorporation has not been accomplished in conventional methods. In this respect, the present method is superior to prepare surface-modified liposomes that are applicable to drug carriers and so on. We have also confirmed by using immunoelectron microscopy that the peptide ligands are actually located in an aqueous phase. It has been shown by flow cytometry that the liposome bearing α-MSH peptide ligand binds to B16 cells and the liposome bearing the repeated GRGDS sequence binds to NIH3T3 cells.

Photochemical construction of coumarin fluorophore on affinity-anchored protein.

A simple and unique construction of a coumarin fluorophore on a prey protein has been achieved by sequential photoreactions using a nonfluorescent bait protein. The bait protein was first modified with a novel diazirine-based photo-cross-linker containing an o-hydroxycinnamoyl unit. The strategy involves two wavelength-dependent photoreactions: photolysis of the diazirine group and E to Z photoisomerization of the cinnamoyl group. The cross-linked interacting partners were cleaved by the consecutive steps of photoisomerization and thermal lactonization accompanied with the creation of a coumarin derivative on the prey protein as a fluorescent tag. Finally, the methodology was successfully applied for coumarin labeling of antilysozymes expressed on living B cells by using photoactivatable lysozymes.

An Isotope‐Coded Fluorogenic Cross‐Linker for High‐Performance Target Identification Based on Photoaffinity Labeling

A photoaffinity labeling (PAL)-based method for the rapid identification of target proteins is presented in which a high-performance chemical tag, an isotope-coded fluorescent tag (IsoFT), can be attached to the interacting site by irradiation. Labeled peptides can be easily distinguished among numerous proteolytic digests by sequential detection with highly sensitive fluorescence spectroscopy and mass spectrometry. Subsequent MS/MS analysis provides amino acid sequence information with a higher depth of coverage. The combination of PAL and heterogeneous target-selecting techniques significantly reduces the amount of time and protein required for identification. An additional photocleavable moiety successfully accelerated proteomic analysis using cell lysate. This method is a widely applicable approach for the rapid and accurate identification of interacting proteins.

A Simple and Efficient Photoaffinity Method for Proteomics of GTP-Binding Proteins

The interaction between GTP and proteins mediates important signal-transduction cascades, including hormone and small GTPase signaling pathways. GTP-binding proteins are part of a superfamily that comprises more than 100 proteins. The identification and dynamic analysis of GTP–protein interactions of diverse GTP analogues have been mainly performed by using spectroscopic and radioisotopic immunoassays. However, the properties and functions of a number of GTP-binding proteins are still unknown because of the difficulty in analyzing a single target protein in the presence of numerous proteins. 7] To achieve functional proteomic analysis of GTP-binding proteins, efficient separation of a family of GTP-binding proteins would be highly desirable. Photoreactive GTP analogues are powerful probes for covalent labeling of target proteins. 10] In particular, this technique allows direct introduction of a functional group into the binding site of a protein for its subsequent detection and isolation. Many azido-GTP derivatives have been developed for both detection of GTP-binding proteins and identification of nucleotide-binding sites. While many azido-GTP analogues are commercially available, they are chemically labile under reducing conditions, including in the presence of dithiothreitol (DTT). In addition, the covalent bonds formed with nitrene are often unstable during identification of the target protein and binding sites. This limits the utility of the nitrene method for proteomic analysis. Although diazirine has been recognized to be a promising photoreactive group for improving the azide photoprobe family, the poor accessibility of diazirine photophore has hampered its wide application in photoaffinity techniques. Recently, we developed an efficient method for preparing diazirine analogues. Here we describe the development of a simple but multifunctional photoreactive GTP analogue that possesses a diazirine moiety, and an efficient photoaffinity-based approach to proteomics of GTP-binging proteins. The multifunctional nature of the new GTP analogue is illustrated in Scheme 1. Besides a photoreactive diazirine moiety (site A), phosphate groups for metal chelation (site B) and a cleavable phosphorothioate linkage (site C) were designed to increase the efficiency of photoaffinity labeling. Modifications of the guanine base or phosphates group have previously been used successfully for the introduction of a photoreactive group. Among these approaches, modification of the gphosphate group has several advantages over other methods. GTP analogues modified at the phosphate group induce resistance to GTPases, which increases labeling efficiency, especially when using enzymatic reactions, since the complex remains intact. In addition, a g modification, for example, with fluorescent analogues, was easily prepared from 5’-(g-thio)triphosphate. Isolation of labeled proteins is crucial, especially when dealing with a large number of proteins. In this case, ACHTUNGTRENNUNGlabeled proteins should concurrently retain the phosphate groups of the probe (site B). Thereby, purification methods for phosphorylated proteins, for example, by using Fe–IMAC (immobilized metal ion affinity chromatography), can be utilized to isolate the labeled protein. An additional advantage of this approach is the cleavable P S bond (site C) which generates a SH group at the protein binding site. This can be selectively modified for tagging of target proteins and therefore used for analyzing the dynamic molecular interactions on the binding site. In this study, we used biotinylation for detection of labeled proteins. Although conventional azido-GTP probes must be meticulously manipulated for detection by using radioisotopes, chemiluminescent detection based on biotin–avidin interactions are easier to handle. Thus, the new GTP probe was designed to enable: 1) one-step synthesis without protection, 2) rapid cross-linking by irradiation of light beyond the absorption of most proteins (~350 nm), 25] 3) a radioisotope-free approach for the detection of target proteins, and 4) a conventional “fishing-out” method suitable for the proteomic effort ACHTUNGTRENNUNGdirected at GTP-binding proteins. Since the phosphorothioate triester of RNA has been reported to be more prone to hydrolysis, the cleavage ability of the P S bond of the GTP probe was first tested by hydrolysis in alkaline solution and monitored by using HPLC (Figure 1). The probe eluted at 16.0 min (Figure 1A) and two new peaks appeared at 7.7 and 8.0 min after hydrolysis in phosphate [a] M. Kaneda, S. Masuda, Dr. T. Tomohiro, Prof. Dr. Y. Hatanaka Graduate School of Medicine and Pharmaceutical Sciences University of Toyama 2630 Sugitani, Toyama 930-0194 (Japan) Fax: (+81)76-434-7515 E-mail : yasu@pha.u-toyama.ac.jp Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author. Scheme 1. The multifunctional nature of the diazirine-carrying GTP photoaffinity probe. Site A: photoreactive diazirine moiety; site B: phosphate groups for metal chelation; site C: cleavable phosphorothioate linkage.

Electrochemical reduction of carbon dioxide catalysed by macrocyclic Fe4S4 iron–sulphur clusters

Electrochemical fixation of CO2 into formate was efficiently carried out by using Fe4S4 cubane clusters bearing a 36-membered methylene backbone in dimethylformamide with initial current efficiencies of 40 and 23%, respectively, for t-butyl and benzyl type macrocyclic Fe–S derivatives; these derivatives are several times more efficient than conventional clusters bearing small thiolate ligands.

Preparation of Fe4S4 iron-sulphur protein analogues with hydrophobic macrocyclic tetrathiol ligand anchored to a 38-membered cyclophane type skeleton

Abstract The preparation of Fe 4 S 4 cubane type active site analogues for iron-sulphur proteins in which the active core is surrounded by an intramolecular hydrophobic domain formed by a 38-membered ring consisting of a cyclophane skeleton is described. An efficient synthesis of the macrocyclic tetrathiol ligands, bis[ N , N- bis(4-mercaptobenzoyl)- N , N ′-octamethylene- 4,4′-diaminodiphenylmethane] ( 3a ), bis- N , N ′-bis {4-(mercaptomethyl)benzoyl}- N , N ′-octamethylene -4,4′-diaminodiphenylmethane]( 3b ) and bis[ N , N ′-bis(3-mercapto-3-methylbutanoyl0)- N , N ′-octamethylene N , N ′- octamethylene-4,4′-diaminodiphenylmethane] ( 3c ) is achieved. Reaction of the cyclic tetrathiol ligand 3 with [Fe 4 S 4 (SBu t ) 4 ] 2- ( 1c ) afforded [Fe 4 S 4 {cycl- (XN- p -C 6 a-H 4 - p -CH 2 -C 6 H 4 -XN[CH 2 ] 8 ) 2 }] 2- [X = p - SC 6 H 4 CO( 2a ), p -SCH 2 C 6 H 4 CO( 2b ), SC(CH 3 ) 2− CH 2 CO( 2c )]. Thus the new clusters embedded in the cyclophane environment are obtained in good yields (70–90%) as black powders with melting points >300°C. They dissolve in DMF, DMSO and propylene carbonate, but are hardly soluble in most common organic solvents and water.

Electron spin resonance of Pt(III) in anticancer platinum pyrimidine green

Abstract The electron spin resonance (ESR) of Pt(III) in platinum pyrimidine green is investigated. The spectrum shows a typical powder pattern of uniaxial symmetry and g ∥ and g ⊥ are determined to be 1.991 and 2.413, respectively. It is concluded that the spectrum derives essentially from the 5d z 2 -like hole state of Pt(III) (5d 7 ). The hyperfine structure of the spectrum suggests that the unpaired spin is not localized on one platinum center but has a wide orbital extended over four platinum atoms. The molecular structure is expected to resemble that of α-pyridone blue.