A. agallochaより単離した化合物について我々が構築した蛍光プレートアッセイ7)によりHes1二量体形成阻害作用を評価したところ，化合物1と3にHes1二量体形成阻害作用を有することが示唆された．一方，化合物1の4’位の置換基がメトキシ基からヒドロキシ基になった化合物2の活性が弱かったことから，4’位の置換基が活性に重要であると考えられる．さらに，化合物1と3のフラボノイド部 (6, 7) は同方法にて活
化合物1の平面構造は、各種スペクトル解析からheronamide C (2)の8 位デオキシ体8-deoxyheronamide C (8-dHC)と決定した (図1)。Heronamide類とよく似た構造を持つBE-14106 (図6)の生合成経路を参考にすると、8-dHC (1)はheronamide C (2)の生合成前駆体であり、シトクロムP450により酸化を受けることで化合物2へと変換されると考えられる3。また、heronamide C (2)は酸化と環化反応によりheronamide A (3)へと変換されると推測されている2。以上より、化合物1-3の8, 9, 19位の立体化学は保存されていると予想し、8-dHC (1)の立体化学を化合物2, 3との比較解析から決定することにした。しかしスペクトル解析の過程
A. nigerは工業的に有機酸の製造に用いられている麹菌の一種であり、既にそのゲノム解読が完了している1。そのゲノム中には33個のポリケタイド合成酵素(PKS)、15個の非リボゾーム性ペプチド合成酵素(NRPS)および9つのPKS-NRPS hybrid型酵素遺伝子(HPN)を有する3。我々は、コンピューター上にてこれら天然物生合成遺伝子クラスターを詳細に解析し、新規天然物生合成を担う遺伝子クラスターを推測した。具体的には、まず機能未知の生合成遺伝子クラスターを抽出し、Basic Local Alignment Search Tool (BLAST)検索等により各構成遺伝子の機能を予測した。これらの情報と、既知天然物の生合成経路の文献情報から、一部の生合成遺伝子クラスターについてはその生合成産物が予測可能であった。一方、我々の研究対象はその生合成産物が予測不可能な生合成遺伝子クラスターであり、本クラスターはこれまでに発見されていない天然物（新規化合物）、あるいは生合成経路が明らかになっていない天然物を生合成する可能性を秘めていた。続いて選抜した数十種の生合成遺伝子クラスターについて、様々な培養条件にて得たmRNAをもとに、半定量RT-PCR法によりその転写量の解析を行った。その結果、い
Peptidoglycan (PGN) is a component of bacterial cell wall consisting of glycans and peptide chains forming a three-dimensional mesh-like structure outside the plasma membrane. PGN has been known as a stimulating component of innate immune system. PGN activates sensor proteins, nucleotide-binding oligomerization domain protein 1 (Nod1) and 2 (Nod2), which belong to Nod-like receptor (NLR) family,1-3 one of the major pathogen-recognizing receptor (PRR) families. Peptidoglycan recognition proteins (PGRP) are the other important protein families that recognize PGN. In addition, various kinds of enzymes and lectins have been proven to recognize PGN. However, the comprehensive analysis of the substrate structures of recognizing proteins has been not really conducted, because of the lack of pure PGN fragments. Herein, we report the chemical synthesis of the PGN fragment library in order to analyze various ligand/protein interactions. The PGN-fragments microarray was also developed for the rapid and quantitative analysis of the interactions, leading to understanding of the defense system against infection of bacteria.
1. Synthesis of PGN fragments and their glycan sequence-dependent Nod2 activation
Our previous studies revealed that MDP (MurNAc-L-Ala-γ-D-isoGln) showed the most potent activity in Nod2 stimulation. We also found that the activity was decreased as the glycan chain length and peptide chain length increased by using synthesized tetrasaccharide and octasaccharide fragments that contain GlcNAc-MurNAc (GM) repeating units (Figure 1, 2a~2d).4-6 These fragments with GM units are considered to be produced by the lysozyme in host organisms which cleaves 1,4-β-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in PGN. On the other hand, PGN fragments having more than two MurNAc-GlcNAc (MG) repeating units, which are expected to be produced by bacterial N-acetylglucosaminidase and release to the environment, had not been synthesized. Thus, we synthesized the library of PGN fragments (Figure 1) to explore their biological functions, especially the Nod2 activation.7
1-1. Synthetic strategy of PGN fragments
The syntheses of disaccharide analogues were carried out via the intermediate 3 as illustrated in scheme 1. Introduction of appropriate peptides to the liberated carboxylic acid 6 and hydrogenation gave the disaccharide fragments 1a, 1c and 1e.
Tetrasaccharide 9 was then synthesized by using 3 as common synthetic intermediate for both glycosyl donor and the acceptor (Scheme 2). Disaccharide donors 7a and 7b were prepared via cleavage of the allyl glycoside and subsequent conversion to the imidate forms (7a: trichloroacetimidate, 7b: N-phenyltrifluoroacetimidate). For the preparation of the tetrasaccharide 9, trichloroacetimidate 7a was first used as the glycosyl donor to couple with the glycosyl acceptor 8. However, the glycosylation between 7a and 8 in the presence of TMSOTf gave the desired tetrasaccharide 9 only in 16% yield, accompanied with 62% yield of recovered 8. The low yield was probably due to the low reactivity of 4-OH group of the disaccharide acceptor 8 caused by steric hindrance of 3-O-lactyl moiety in muramic acid moiety. We then used the N-phenyltrifluoroacetimidate 7b as the glycosyl donor which has similar high reactivity but improved stability in comparison to the corresponding trichloroacetimidates.8Excess acceptor 8 was used (ratio of donor : acceptor was 1 : 1.5) in order to promote the reaction. The yield of glycosylation was dramatically improved to give tetrasaccharide 9 in 61% yield. The glycan backbone 10 was then coupled with peptides to give the corresponding protected intermediates. All benzyl and benzylidene groups
Scheme 1 Retrosynthetic analysis of gymnocin-A (1)
逆合成戦略の基盤となるのが、オキシラニルアニオンを用いた[X+2+Y]型の収束的合成法である(Scheme 2)。エポキシスルホンから発生させたオキシラニルアニオンIをトリフラートIIに求核置換させてカップリング体IIIとし、環化反応を経て6員環ケトンIVとする。次いで環拡大反応に付してVとし、還元的エーテル化すると新たな連結体VIが得られる。本手法の特徴は、連結部に新たに生成する環サイズを、IVの環拡大反応の回数によって変えられる柔軟性にあり、6-6員環であるIJ環部、6-7員環であるBC, DE, GH, LM環部の合成に適用できる。
Scheme 2 Convergent synthesis method of polycyclic ethers using oxiranyl anions
In the era of ever improving living status, lifestyle disease such as diabetes and obesity has become one of the major health problems for people living in developed countries. These health problems are often associated with increased adipocyte differentiation or accumulation of lipid. In effort to provide new lead compound that can improve the symptoms of lifestyle disease, we focused on species from the Meliaceae family due to their various significant biological activities. We subsequently identified Chisocheton ceramicus bark extracts to possess in vitroanti-lipid droplets accumulation (LDA) activity on pre-adipocyte cell line. Following research led to identification of 12 limonoids, ceramicines A-L (Fig. 1) 1-4. Their relative structures were elucidated by using 1D and 2D NMR data.
Structure Elucidation of Ceramicine B from C. ceramicus Bark
Ceramicine B (2) was isolated as a colorless amorphous solid with molecular formula, C26H32O4as determined by high resolution-electrospray ionization-time of flight-mass spectra. The planar structure was elucidated with 1D and 2D NMR data (Figure 2). Thus, 2 was established as a new limonoid with cyclopenta[α]phenanthren ring system with a β-furyl ring at C-17 and a tetrahydofuran ring. The relative stereochemistry of 2 was elucidated by NOESY correlations as shown in computer-generated 3D rendering (Figure 2). NOESY correlations of H-6/Hb-28, H3-19, and H3-30, H-7/H-15, and H-12/H-17 together with 3J proton coupling constants (3JH-5/H-6 = 12.4 Hz and 3JH-6/H-7 = 3.8 Hz) suggested that H-6, H-7, and H-17 adopts β-configuration. While NOESY correlation of H-5/H-9 indicates both H-5 and H-9 adopt α-configuration.
Absolute Configuration of Ceramicine B
The absolute configuration of 2, a major limonoid from C. ceramicus, was elucidated by X-ray crystallography and CD spectrum of p-Br-benzoyl derivative results (Fig. 3). The CD spectrum of p-Br-benzoyl derivative of 2indicated that the mutual relationship of dipole moment of the benzoate and α,β-unsaturated ketone chromophores was oriented in a counter clockwise manner, indicating absolute configuration at C-7 as S configured. Further X-ray crystallography analysis results confirmed the total structure of 2 including its absolute structure [Flack parameter, c = -0.0(2)].
Ceramicines with Anti-Lipid Droplets Accumulation Activity
The LDA activities were examined on murine pre-adipocyte cell line, MC3T3-G2/PA6, and measured via nile red lipid droplets staining. Results showed that ceramicine B was demonstrated as most potent (IC50= 1.8 μM) among the ceramicine compound series 5. The potency prompted us to further investigate the structural-activity relationship of ceramicines. Nine derivatives based on the potent ceramicine B were synthesized, and the summaries of ceramicines and its derivatives SAR were shown at Fig. 4.