Selective Hydrogenation and Transfer Hydrogenation for Post-Functional Synthesis of Trifluoromethylphenyl Diazirine Derivatives for Photoaffinity Labeling

Elucidation of protein functions on the basis of structure–activity relationships can reveal the mechanisms of homeostasis functions in life and is one of the greatest interests of scientists. In the human body, many proteins are activated and/or inactivated by ligands to maintain homeostasis. Understanding the mechanism of molecular interactions between small bioactive ligands and proteins is an important step in rational drug design and discovery. ! Photoaffinity labeling, which is one of the most familiar approaches for chemical biology analysis, was initiated using diazocarbonyl derivatives in 1962 (Singh et al., 1962). Many researchers have subsequently tried to establish alternative approaches for the direct identification of target proteins for the bioactive small ligands. These approaches are based on the affinity between the ligand and the target protein (Figure 1). Several reviews are published for the recent applications of photoaffinity labeling (Tomohiro et al., 2005; Hashimoto & Hatanaka, 2008). To archive photoaffinity labeling, researchers have to prepare photoaffinity labeling ligands. The native ligands must be modified by photoreactive compounds (photophores) by organic synthesi

Anti-allergic activity of Glycopeptide isolated from Perilla frutescens BRITTON

The anti-allergic activity of Perilla glycopeptide (Pe-GP) isolated from dried Perilla frutescens BRH-TON leaves was investigated. Pe-GP was found to be effective in inhibiting histamine release from IgE-sensitized rat peritoneal mast cells induced by a specific antigen. The histamine release induced by several substances such as concanavalin A, compound 48/80, mastoparan, and ionophore A23187, was also found to be inhibited by Pe-OP. These results suggest that Pe-GP mainly interrupts a pathway of histamine release after calcium influx. Anti-allergic activity of Pe-GP was investigated by ear-swelling response in mice that had been passively sensitized with IgE via intravenous injection. Pe-GP, injected intraperitoneally before exposure to the antigen, was found to inhibit the allergic response in a dosedependent manner (5-100 mg/kg). Pe-GP shows promise as an anti-allergic substance for medical use as well as in health foods. シソ(Perilla frutescens B_)葉熱水抽出液から分離精製されたシソ糖ペプチド(Pe-GP,6kDa)の抗アレルギー活性を検討した。Pe-GPは,ラット腹腔内より単離してIgEで感作したマスト細胞において,特異抗原で刺激したときに誘発されるヒスタミン遊離反応を抑制した。同様にPe-GPはconcanavalin A,compound48/80,mastoparan,ionophore A23187などの誘発するヒスタミン遊離反応も抑制した。これらの結果からPe-GPはヒスタミン遊離機構のうちカルシウム動員以降の経路を主として抑えると考えられる。Pe-GPの生体内における抗アレルギー活性は,IgEを静注して受動感作したマウスの耳介浮腫反応により検討した。抗原塗布前にあらかじめ腹腔内に投与しておくと,Pe-GPは5-100mg/mlの範囲で濃度依存的に浮腫を抑制し抗アレルギー活性を示した。Pe-GPの抗アレルギー活性を有する医薬品としての開発が,健康食品と同様,期待される

Behavioral impairment in SHATI/NAT8L knockout mice via dysfunction of myelination development

We have identified SHATI/NAT8L in the brain of mice treated with methamphetamine. Recently, it has been reported that SHATI is N-acetyltransferase 8-like protein (NAT8L) that produces N-acetylaspatate (NAA) from aspartate and acetyl-CoA. We have generated SHATI/NAT8L knockout (Shati−/−) mouse which demonstrates behavioral deficits that are not rescued by single NAA supplementation, although the reason for which is still not clarified. It is possible that the developmental impairment results from deletion of SHATI/NAT8L in the mouse brain, because NAA is involved in myelination through lipid synthesis in oligodendrocytes. However, it remains unclear whether SHATI/NAT8L is involved in brain development. In this study, we found that the expression of Shati/Nat8l mRNA was increased with brain development in mice, while there was a reduction in the myelin basic protein (MBP) level in the prefrontal cortex of juvenile, but not adult, Shati−/− mice. Next, we found that deletion of SHATI/NAT8L induces several behavioral deficits in mice, and that glyceryltriacetate (GTA) treatment ameliorates the behavioral impairments and normalizes the reduced protein level of MBP in juvenile Shati−/− mice. These findings suggest that SHATI/NAT8L is involved in myelination in the juvenile mouse brain via supplementation of acetate derived from NAA. Thus, reduction of SHATI/NAT8L induces developmental neuronal dysfunction

Lactam類, Pyridone類, ならびに関連化合物74種の抗腫瘍性について

Seventy-four compounds related to lactams and pyridones were synthesized and their anti-tumor activity was examined using Ehrlich carcinoma cells. 4-(2-Piperidyl) butyric acid hydrochloride was found to have some anti-tumor effect on the solid type of Ehrlich carcinoma cells, but no other substances were found to be effective. Correlation between the anti-tumor activity and structure of the tested compounds still remains uncertain

Preparation of recombinant lysosomal cysteine protease, cathepsin K in insect cells

軟骨細胞に局在するシステインプロテアーゼであるカテプシンKは,骨の差異九州に関わる酵素である。カテプシンKの阻害剤は骨粗鬆症の治療薬の候補になり得る。スクリーニングや酵素の機能を調べるためには組み換えカテプシンKを大量に作成することが必要である。我々はカイコ幼虫を用いた発現系が分泌型酵素であるカテプシンKの発現に適していると考えた。C末端にmycタグを付けたカテプシンK遺伝子をカイコ用の発現ベクターに挿入し,限界希釈法で組み換えウイルスを得た。このウイルスをカイコ培養細胞に感染させてカテプシンKの発現場所を調べたところ,細胞内で発現していた。細胞外への分泌が観察されなかったのでカイコ幼虫での発現をあきらめ,培養細胞からの精製を試みた。DEAE-セファロースカラム,Mono-Sカラム,抗myc抗体アフィニティーカラムで順次精製した結果,非活性を細胞可溶性画分の約300倍に高めることに成功した。Cathepsin K, which is localized in osteoclasts, plays an important role of bone resorption. Selective inhibitors of cathepsin K therefore can be promising therapeutic candidates for the treatment of diseases such as osteoporosis. Recombinant cathepsin K protein is necessary for screening the therapeutic candidates and elucidating the enzymatic functions because the amount of native cathepsin K is very low. Silkworm is one of the most attractive hosts for large-scale production of eukaryotic secretory proteins. The gene for fusion cathepsin K protein with myc-tag at the C-terminus was inserted into expression vector for silkworm and recombinant viruses were isolated by end-point dilution assay. Cathepsin K was expressed in the cell lysate, but not in the medium, and consequently we gave up the idea of cathepsin K expression in the silkworm. Thus, we tried to isolate cathepsin K protein from the cultured cells, and purified it by sequential operations with DEAE-Sepharose column, Mono-S column, and anti-myc antibody column. The purification fold of cathepsin K activity increased approximately 300 times in comparison with that in soluble fraction of cell lysate

A novel biotinylated diazirinyl ceramide analogue for photoaffinity labeling

A novel photoreactive ceramide analogue, which contains (3-trifluoromethyl) phenyldiazirinyl lipid and biotinylated sphingosine, was synthesized. The probe was recognized as an antigen by anti-ceramide antibody and as a substrate for sphingolipid ceramide N-deacylase. These results indicate that the probe may be useful as a photoaffinity-biotinylating agent in sphingolipid studies

Recent Progress in Diazirine-Based Photoaffinity Labeling

Based on the recent development of molecular biology, the investigation of biofunctional machinery at their ligand accepting interface has become one of the challenging and important subject of post-genome field. The technique of photoaffinity labeling has become increasingly appreciated as a powerful methodology for the purpose. This micro review focused synthesis of trifluoromethylphenyldiazirine, which is one of the most promising photophore, and its application to the biomolecules

Selective Hydrogenation of Alkene in (3-Trifluoromethyl) Phenyldiazirine Photophor with Wilkinson's Catalyst for Photoaffinity Labeling

Selective hydrogenation of carbon-carbon double bond in the presence of nitrogen-nitrogen double bond in (3-trifluoromethyl) phenyldiazirine achieved with Wilkinson's catalyst. Photoaffinity labeling is a powerful method in the study of biological structures and functions. 1, 2) It is suitable for the analysis of biological interactions because it is based on the affinity of the ligand moiety. Various photophors, such as phenyldiazirine, arylazide and benzophenone, were used. Comparative irradiation studies of these three photophors in living cells suggested that a carbene precursor, (3-trifluoromethyl) phenyldiazirine, is the most promising, 3-6) but complicated synthesis of the diazirinyl three-membered ring prevented the application of the photophor. Post-functional synthesis of TPD derivatives is one of problems to be solved. 7-10) It is well known that catalytic transfer hydrogenation followed by Wittig reaction is a useful method for general purpose carbon elongation, but the synthetic route does not apply for diazirinyl compounds, because it was reported that the diazirinyl nitrogen-nitrogen double bond was not tolerated under a hydrogen atmosphere in the presence of Pd-C for more than 1 h. 11, 12) Although the selective hydrogenation of alkene in diazirinyl and other photophors containing photo ligand in the presence of Pd/C was reported very recently, the hydrogenation of diazirinyl derivatives is low yield compared with other photophors due to the presence of the homogeneous N=N double bond. 13) Furthermore, the applications of the method were not reported