Summarized in this article are the syntheses of food effective constituents catechin and flavone toward the development for chemical biology investigations. Synthesis of (-)-5,7-dideoxy-gallocatechin (21a) was accomplished by 6-endo cyclization of corresponding epoxy-phenol 18. Inspired by the finding that (-)-5,7-dideoxy-epigallocatechin gallate (21b) possessed same biological activity with natural epigallocatechin gallate (1), we designed APDOEGCg (40) as a useful probe precursor. Synthesis of APDOEGCg (40) was accomplished by cationic cyclization utilizing neighboring participation of the gallate carbonyl group. Furthermore, the synthetic APDOEGCg (40) was readily converted to fluorescein probe 42 and immunogen 45 efficiently due to its high reactivity of amine functional group. The synthetic probes were demonstrated the imaging studies and the generation of antibodies. Regioselective synthesis of methylated-EGCgs 46 and 47 was accomplished by employment with the 2-nitrobenzenesulfonyl (Ns) as a novel protecting group of phenols. Additionally utilizing the synthetic 4,4"-diMe-EGCg (54) as an authentic sample, a rapid synthesis of PET probe 55 by incorporation of 11C atom into the EGCg derivative 47 was demonstrated efficiently. Biomimetic synthesis of theaflavin (56) from catechins (3 and 59) was also accomplished by using Ns protecting group to minimize undesired side reactions of electron-rich aromatic rings. Regioselective synthesis of chafurosides A and B (70 and 71) were accomplished from the same intermediate (78a). The both flavone rings were constructed from β-diketone intermediate (72), which was readily obtained by condensation of an acyl donor (79) and ketone (78c). Utilizing our novel flavone synthetic method, practical synthesis of nobiletin (88), a polymethoxylated flavone from citrus, was accomplished in hundred gram-scale. Synthetic nobiletin was readily converted to PET prove (99) by selective demethylation and rapid incorporation of 11C atom.
Multicomponent reaction represented by Mannich reaction, Strecker reaction and Biginelli reaction has been applied to several natural product synthesis because a complexed framework of the natural product can be constructed by assembling more than three simple components at one step. It is also attractive from a medicinal chemical point of view because it enables us to supply a range of analogues to investigate a structure-activity relationship simply by changing components in the reaction. Here, we described our recent studies utilizing a multicomponent reaction in natural product-based medicinal chemistry. First topic is about total synthesis of syringolin A class of proteasome inhibitor and its structure-activity relationship study by intramolecular Ugi three-component reaction. We also synthesized cyclic peptide natural products (sandramycin, quinaldopeptin) and analogues and revealed their biological properties. Finally, design and synthesis of simplified caprazamycin analogues using aza-Prins-Ritter reaction was described.
Histone modifications such as acetylation and methylation control gene transcription independently on DNA base sequence. Such modification system, which is called “epigenetics,” plays important roles in regulating cellular functions including cell cycle, immunoresponses, and signal transduction. However, epigenetic aberrations have been found in many abnormal cells and are closely associated with various diseases such as cancer and neurodegenerative disorders. Therefore, molecular technology for controlling epigenetics by small molecules has been of interest both in chemical biology and in epigenetic therapy. To establish this molecular technology, we have studied epigenetic inhibitors focusing on histone modification enzymes, especially on histone deacetylases (HDACs) and histone lysine demethylases (KDMs). These inhibitors have been discovered based on organic chemistry methodology, i.e. C-H activation, click chemistry, targeted drug delivery, and structure-based drug design. Herein we demonstrate the molecular technology for controlling epigenetic mechanism by presenting the design, synthesis, and biological evaluation of HDAC or KDM inhibitors.
“Click reaction”, epitomized by copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), is an emerging method for conjugating molecules efficiently in broad fields, including materials chemistry and chemical biology. A copper-free variant, strain-promoted azide-alkyne cycloaddition (SPAAC), using functionalized cyclooctyne derivatives that spontaneously react with azides, have enabled the chemical modification of azido-incorporated biomolecules in cultured cells and in living animals, greatly expanding the utility of click chemistry. A variety of cyclooctyne derivatives bearing functional moieties, such as fluorescent or biotinyl groups, have been developed and some of them have become commercially available. However, on-demand synthesis is required for those with a new functionality, which is not always easy because of the high reactivity of the strained alkyne moiety. To conjugate two azide molecules easily, we have developed strain-promoted “double-click” reaction using Sondheimer diyne that bears two strained alkyne moieties. This novel convergent method capable of conjugating three molecules spontaneously has allowed for facile modification of an azido-biomolecule with a small reporter azido-molecule. We have also developed a transient protection method of cyclooctynes from cycloaddition with an azide via 1：1 complexation with a cationic copper(I) salt. Protection of a cyclooctyne bearing a terminal alkyne with a copper salt enabled the selective copper-catalyzed click conjugation with an azide at the terminal alkyne moiety, facilitating easy preparation of cyclooctyne derivatives.
Trametinib is a new anti-cancer drug that was developed by a cell-based phenotypic screening for the accumulation of CDK inhibitor p15INK4b and the growth inhibition in human colorectal cancer cell line HT-29 cells. Therefore, its molecular mechanism remained unknown at the beginning of the development process. To address this issue, we used a chemical biology approach. As a result, we identified MEK1/2 kinase as a molecular target by compound-immobilized affinity chromatography. Trametinib directly binds to MEK1 and MEK2, and allosterically inhibits their kinase activities. It was also shown that trametinib suppresses the phosphorylation status of MEK and ERK in HT-29 cells. We further confirmed that trametinib preferentially inhibits the growth of cancer cell lines harboring BRAF mutation and observed significant antitumor activity in a tumor xenograft model. In 2013, trametinib was approved as a first-in-class MEK inhibitor by the U.S. Food and Drug Administration for the treatment of metastatic melanoma with BRAF V600E or V600K mutations.
We are aiming to provide various biopharmaceuticals through naturally-derived glycosylation technologies and to enhance human health. In fact, it was difficult to control functions of biopharmaceuticals by applying biotechnology. However, the time is coming to control them by applying chemical approach together. We, GlyTech, can provide our leading edge glycosylation technology in biopharmaceutical industry and as a consequence, we can propose the new de-facto standard for biopharmaceutical.
The development of a chemical reaction for the detection of one epigenetic modification in a long nucleic acid is a chemically and biologically challenging research subject. Here we review three topics on chemical detection of methylation/demethylation process in nucleic acids; (1) sequence-specific osmium oxidation of DNA 5-methylcytosine; (2) DNA 5-hydroxymethylcytosine-specific oxidation with peroxotungstate; (3) capturing activated RNA demethylase complexes with specific RNA m6A recognition. These novel concepts of methylation/demethylation-specific reactions, based on straightforward chemical approaches, expand the range of methods available for the analysis of epigenetic modifications in nucleic acids.
8-Quinolinol (8-hydroxyquioline, 8HQ) attracts much attention in material sciences and bio-related sciences, because of their simple structures, strong metal coordination properties, fluorescence properties, and extensive possibility of functionalization. In this review, five applications of 8HQ are described. The first application is 8HQ-linked macrocyclic tetraamines as fluorescent probes of zinc ions in living cell. The second and third sections provide the applications of 8HQ to inhibitors against dinuclear zinc hydrolase and to radioprotectors that can inhibit apoptosis of radiosensitive normal cells during radiotherapy. In the third section, finding of photochemical S-O bond cleavage reaction of 8HQ sulfonates (8HQS) under aqueous conditions, its mechanistic analysis and applications to photocleavable chemical linkers are described. Finally, the photophysical properties of iridium(III) complexes having 8HQ or its analogs will be presented. This knowledge will be useful in design and synthesis of useful molecules containing 8HQ units, in bioorganic chemistry, chemical biology, pharmaceutical science, material science, and other related scientific fields.
The site-directed reaction with high efficiency and specificity to nucleic acids has become of the great interests, as such modification may be possible to induce point mutation of a genetic code and to apply the labeling for a target nucleic acid. In this study, we have developed the new reactive probes for the selective reactions to duplex DNA containing an abasic site. We designed three kinds of probes, which consist of 2-amino-6-vinylpurine (AVP) as a reactive moiety and peptides, acridine, and Hoechst as a binding moiety with high affinity to duplex DNA. We expected that AVP derivatives might form hydrogen bonds with target nucleobases at the opposite an abasic site in DNA. In the three kinds of probes, Hoechst-AVP probe exhibited high selectivity and efficient reactivity to thymine at the site opposite an abasic site in DNA. These studies provide the proof-of concept that AVP derivatives conjugated with binding molecules to nucleic acids might form hydrogen bonds with target bases in the hydrophobic pocket and lead to the selective alkylation. Now we are going to investigate the new reactive probes for the other hydrophobic pockets.
Our laboratory has been discovering unique synthetic molecules that modulate biological process. As a part of the driving force behind our chemical biological studies, diverse, designed, and unique chemical libraries have greatly contributed. This paper highlights our recent chemical biological study that was initiated with a unique chemical library of electrophiles. Transient receptor potential ankyrin 1 (TRPA1) is a chemosensor that is activated through covalent modification of multiple cysteines with a wide range of electrophilic compound. It has been a long-standing puzzle how the modification of multiple cysteine residues leads to the activation of TRPA1. Screening of 1657 electrophiles identified JT010, a highly potent agonist selective for TRPA1. In an effort to validate its mode of action, we noted JT010 opens the TRPA1 channel by covalently and selectively binding to Cys621. The results suggest that a single modification of Cys621 is sufficient to lead the channel to open state.
Fluorescence imaging is one of the most powerful techniques for visualization of the temporal and spatial biological events in living cells. Many fluorescent probes have been developed so far and contributed greatly to biological and medical researches. For biological applications, xanthene dyes have many favorable characteristics, such as high water solubility and high fluorescence quantum yield, and have been utilized as the fluorescent core for a large number of fluorescent probes. In this manuscript, we introduce a novel red fluorescein analogue, TokyoMagenta (TM), in which the O atom at the 10 position of the xanthene moiety is replaced with a Si atom, and the absorption and emission wavelengths of TM are about 90 nm longer than those of fluorescein. These silicon-substituted xanthene dyes would be useful for multicolor imaging. Nowadays, we think these xanthene dyes have been becoming one of practical fluorophores for biological researches.
Chemical protein modification is a powerful technique not only for elucidating protein functions but also for providing new tools to explore complicated biological processes in live cells. The progress of modern organic chemistry and bio-orthogonal chemistry has enabled the synthesis of artificial probe-protein conjugates with a site-specific and target-selective manner in crude environments. With the advent of ligand-directed chemistry, it is now possible to directly modify “endogenous” proteins of interest in live cells and even in animals. Furthermore, combining chemistry-based protein labeling with the latest analytical methods facilitates to comprehensive studies of proteomes of complex biological samples. In this review, we focus the recent studies in synthetic organic chemistry for protein labeling. We survey the methods for both target-selective labeling and global labeling of proteins, and describe these applications toward in situ engineering and analysis of proteins in their native habitats. Current limitations and future directions of this research area are also discussed.
Synthesis of a focused library (FL) is an efficient method to develop novel compounds regulating functions of specific enzymes. Compounds in a FL are composed of a common core structure with different building blocks. Herein, our design and synthesis of FLs focusing on selective inhibitors of dual-specificity protein phosphatases (DSPs) is summarized. A first generation FL having an acidic core structure extracted from a natural product, RK-682, does not contain a highly selective inhibitor for DSPs, and showed very weak activity at the cellular level, possibly due to poor cell membrane permeability. Upon building the second FL, the property of the core structure was modified from acidic to neutral. Construction of a second-generation FL (RE derivatives) having the enamine derivative of 3-acyltetronic acid as the core structure resulted in dramatic improvement of cell membrane permeability and inhibitory selectivity. As a result, VHR-selective RE12 and CDC25A/B-selective RE44 were discovered. Replacement of the side chain in RE12 afforded RE176, which showed more potent anti-proliferative activity against HeLa cells. Core structure modification from acidic to neutral also changed the mode of action of inhibitors. RE derivatives showed a non-competitive inhibition profile and interacts with a pocket adjacent to the active site of CDC25s.