Laboratory Introduction
Research Content

Chemistry of Natural Products

Loboratory Introduction

 While biopolymers such as nucleic acids and proteins are essential to life and thus ubiquitous to living organisms, natural products, defined here as biological substances of low molecular weights, are more diverse among biological phyla and their habitats; their major biological role being regarded as communicative mediators among cells within a multicellular individuals, or among different individuals of the same or different biological species within a given ecological environment. From this point of view, this laboratory focuses on natural products isolated from marine organisms, composing their ecological communities with chemical communication via the aqueous mediums, and aims its research efforts at ultimate elucidation of molecular mechanisms concerning physiological and ecological activity of marine natural products by way of chemical strategies. Namely, independent evaluations are pursued with regard to molecular recognition between small molecules and their target cellular components, leading to functional activation of the latter, by spectroscopic and other analytical methods. Chemical modification is actively employed here as well to elucidate structural requirement for each of the above steps in a particular biological activity, and to attach analytical markers such as fluorophores, photo-reactive units, and/or radio- or stable isotopes. Representative subjects from respective phases of research are following.

Research Content

1. Food chain and biological defense of marine toxin

 kadaic acid (OA) is a marine polyether cytotoxin, which was isolated from the marine sponge, Halichondria okadai. OA is a potent inhibitor of protein serine/threonine phosphatases (PP) 1 and 2A and the phosphatase inhibition mechanism has been thoroughly investigated. However, it is not clear why H. okadai accumulate toxic OA in its body. Generally marine sponges are a rich resource of symbiotic microorganisms and therefore OA presumably originates from them. The dinoflagellate, Prorocentrum lima produces OA and is considered to be a suspicious real producer of OA. OA may play important roles in the ecological system surrounding H. okadai. In the course of understanding chemical defense of H. okadai and accumulation mechanism of OA, target molecules of OA were investigated. A major isoform among okadaic acid binding proteins (OABP 2.1) which is a 22 kDa protein containing 189 amino acid residues that specifically binds to OA was isolated and cloned from H. okadai. Proteins that are highly homological (>40%) to OABP and the role of OABP2.1 except binding ability to OA are not known. The structural and functional analysis of OABP2.1 would elucidate the mechanism of chemical defense of marine sponges by accumulation of marine natural products including OA.

Okadaic acid

Sugiyama, N.; Konoki, K.; Tachibana, K. Isolation and Characterization of Okadaic Acid Binding Proteins from the Marine Sponge Halichondria okadai. Biochemistry 2007, 46, 11410-11420.

2. Biosynthesis of Ladder-Frame Polycyclic Ethers

 The structural class of ladder-frame polycyclic ether molecules, exemplified by ciguatoxin isolated as the major causative molecule of ciguatera, a fish poisoning caused by regional and temporal blooming of a benthic dinoflagellate at coral reef coasts, exhibit potent biological activities. Aiming to investigate their structural origin and mode of actions, our laboratory is attempting to elucidate their biosynthetic pathway and ether ring formation. In the course of theses effort, stable isotope (13C and 18O) incorporation experiment on yessotoxin, a ladder-frame polycyclic ether has been under progress. In addition, A monocyclic ether alkaloid, brevisamide was isolated from the dinoflagellate Karenia brevis that produces a variety of ladder-frame polyethers. Its proposed biosynthetic intermediate comprising a linear backbone with an E-olefin functionality was synthesized for biosynthetic studies on the marine ladder-frame polyethers.

Brevisamide(Top), Yessotoxin(Bottom)

Murata, M.; Izumikawa, M.; Tachibana, K.; Fujita, T.; Naoki, H. Labeling pattern of okadaic acid from 18O2 and [18O2]acetate elucidated by collisionally induced dissociation tandem mass spectrometry,J. Am. Chem. Soc. 1998, 120, 147-151.

M. Izumikawa, M. Murata, K. Tachibana, T. Fujita, H. Naoki. 18O-Labeling Pattern of Okadaic Acid from H218O in Dinoflagellate Prorocentrum lima Elucidated by Tandem Mass Spectrometry,Eur. J. Biochem. 2000, 267, 5197-5183.

Shirai, T.; Kuranaga, T.; Wright, J. L. C.; Baden, D. G.; Satake M.; Tachibana K. Synthesis of a proposed biosynthetic intermediate of a marine cyclic ether brevisamide for study on biosynthesis of marine ladder-frame polyethers,Tetrahedron Lett. 2010, 51, 1394-1396.

3. Chemical Synthesis of Ladder-Frame Polycyclic Ethers

 Aiming to investigate on generality and specificity of affinity to transmembranal helical segments of membrane-bound proteins based on their molecular lengths in common corresponding to the thickness of biological lipid bilayer membrane, this laboratory was attenpting total synthesis of ladder-frame polycyclic ether molecules. In the course of this effort, a synthetic protocol was devised to connect oligocyclic pieces for elongation of the ladder, where hydroboration of a 2-methylenated cyclic enol ether, followed by in situ Suzuki-Miyaura coupling, are utilized. Consequently using this protocol, total synthesis of gambierol, brevisamide, and brevisin, toxic metabolite of the same dinoflagellate, was accomplished.

51-hydroxyCTX3C

Gambierol

Brevisin

佐々木 誠, 井上将行. ポリエーテル系天然物の化学合成:新しい中員環エーテル構築法とエーテル環連結法の開発.有機合成化学協会誌, 2001(3), 193-200.

Takakura, H.; Sasaki, M.; Honda, S.; Tachibana, K. Progress toward the Total Synthesis of Ciguatoxins: A Convergent Synthesis of the FGHIJKLM Ring Fragmentm,Org. Lett. 2002, 4, 2771-2774.

Fuwa, H.; Kainuma, N.; Tachibana, K.; Sasaki, M. Total Synthesis of (-)-Gambierol, J. Am. Chem. Soc. 2002, 124, 14893-14992.

Kuranaga, T.; Shirai, T.; Baden, D. G.; Wright, J. L. C.; Satake, M.; Tachibana, K. Total Synthesis and Structural Confirmation of Brevisamide, a New Marine Cyclic Ether Alkaloid from the Dinoflagellate Karenia brevis,Org. Lett. 2009, 11, 217-220.

Ohtani, N.; Tsutsumi, R.; Kuranaga, T.; Shirai, T.; Wright, J. L. C.; Baden, D. G.; Satake M.; Tachibana K. Synthesis of the ABC Ring Fragment of Brevisin, a New Dinoflagellate Polycyclic Ether,Heterocycles 2010, 80, 825-830.

Tsutsumi, R.; Kuranaga, T.; Wright, J. L. C.; Baden, D. G.; Ito, E.; Satake, M.; Tachibana, K. An improved synthesis of (-)-brevisamide, a marine monocyclic ether amide of dinoflagellate origin,Tetrahedron 2010, 66, in press.

4. Mode of Action of Ladder-Frame Polycyclic Ethers

 The function of living biological systems (cells, cell organelles and whole organisms) is essentially connected to the occurrence and structure of fluid biomembranes. Biomembranes act as highly selective permeability barriers separating the contents of the cell from its environment. Moreover, membranes provide membrane proteins with a suitable environment for exhibiting their functions, and they control a variety of indispensable processes for life, such as passive and active transport. It is generally considered that target molecules of ladder-frame polycyclic ethers are membrane proteins. We are attempting to elucidate the mode of action and/or detailed interaction mechanism of these bioactive polycyclic ethers. Design and preparation of membrane peoteins and bioactive polycyclic ethers as ligands have been investigated. Subtraction of target transmembranal peptide and stable isotope labeling were done, followed by reconstitution with model membrane system. Finally observation between the transmembranal peptide and its ligand is currently under progress.

Shida, T.; Tachibana, K. Dynamic NMR study on the trans-fused eight-membered ether ring model representing G ring of brevetoxin A,Tetrahedron Lett. 2005, 46, 1855-1858.

Sasaki, M; Tachibana, K. Design and synthesis of simplified polycyclic ethers and evaluation of their interaction with an a-helical peptide as a model of target proteins,Tetrahedron Lett. 2007, 48, 3181-3186.

Niitsu, A.; Harada, M.; Yamagaki, T.; Tachibana, K. Conformations of 3-carboxylic esters essential for neurotoxicity in veratrum alkaloids are loosely restricted and fluctuate,Bioorg. Med. Chem. 2008, 16, 3025-3031.

5. Bicelles as model membranes

 To evaluate bicelles as a model membrane system, we examined the morphological changes of bicelles induced by the membrane lytic peptide melittin. 31P NMR and dynamic light scattering experiments showed that melittin irreversibly disrupted the disk-shaped structure of bicelles, and that the disrupted bicelles form giant spherical assemblies above the gel-to-liquid crystalline transition temperature (Tm= 24 °C). The melittin-induced disruption of bicelles was suppressed by the addition of cholesterol, suggesting that cholesterol effectively improved the stability of the bicelle membranes in the same manner as vesicles. Furthermore, the packing of lipid chains in bicelles was 5 ~ 9% less than that in mixed micelles at temperatures between 298 ~ 318 K. This reduction of packing that accompanied the formation of bicelles changed the spectroscopic character of reconstituted bacteriorhodopsin, as indicated by static absorption measurements.

Bicelle

Sasaki, H.; Fukuzawa, S.; Kikuchi, J.; Yokoyama, S.; Hirota, H.; Tachibana, K. Cholesterol doping induced enhanced stability of bicelles,Langmuir 2003, 19, 9841-9844.

Sasaki, H.; Araki, M.; Fukuzawa, S.; Tachibana, K. The packing of lipid chains changes the character of bacteriorhodopsin reconstituted in a model membrane,Bioorg. Med. Chem. Lett. 2003, 13, 3582-3585.

Sasaki, R.; Sasaki, H.; Fukuzawa, S.; Kikuchi, J.; Hirota, H.; Tachibana, K. Thermal analyses of phospholipid mixtures by differential scanning calorimetry and effect of doping with a bolaform amphiphile,Bull. Chem. Soc. Jpn. 2007, 80, 1208-1216.

6. Biological Function of Norzoanthamine

 Norzoanthamine, a zoanthamine type alkaloid from colonial zoanthid Zanthus sp. suppresses the decrease of bone weight and bone strength in osteoporosis-model mouse. Norzoanthamine accelerates the formation of a collagen-hydroxyapatite composite and enhances collagen release from an immobilized matrix vesicle model. Norzoanthamine recognizes a peptide chain non-specifically and stabilizes its secondary structure and collagen has polyvalent binding sites for norzoanthamine. This collagen-norzoanthamine supramolecular association is considered to be one of the most significant modes of action for enhancement of bone formation. High concentrations of norzoanthamine are present in the epidermal tissue of Zoanthus sp., as determined using MALDI imaging mass spectrometry and HPLC quantification. Sodium dodecylsulfate polyacrylamide gel electrophoresis experiments indicated that norzoanthamine increases the resistance of collagen to damage from UV light, probably not by absorbing UV light as a sun-block, but by strengthening collagen itself. This is the possible function of norzoanthamine in Zoanthus sp. Zoanthus sp. contains extremely high content of norzoanthamine (>0.1 %, wet weight).

Zoanthus sp.

Norzoanrthamine

Kinugawa, M.; Fukuzawa, S.; Tachibana, K. Skeletal protein protection: the mode of action of an anti-osteoporotic marine alkaloid, norzoanthamine,J. Bone Miner. Metab. 2009, 27, 303-314.

Genji, T.; Fukuzawa, S.; Tachibana, K.Distribution and possible function of the marine alkaloid, norzoanthamine, in the zoanthid Zoanthus sp. using MALDI imaging mass spectrometry,Mar. Biotechnol. 2010, 12, 81-87.

7. Site-specific Functionalization of Proteins

 Recent advances in organic chemistry enabled organic reactions in aqueous media with high yield under mild condition. This chemical evolution has promoted chemical modification of proteins, which contributes to protein engineering and chemical biology research. An Escherichia coli suppressor tRNAPhe (tRNAPheCUA) was mis-acylated with 4-iodo-L-phenylalanine by the A294G mutant of E.coli phenylalanyl-tRNA synthetase (G294-PheRS) at a high magnesium ion concentration. The pre-acylated tRNA was added to an E.coli cell-free system to synthesize a Ras protein containing 4-iodo-L-phenylalanine residue at specific target position. Then, a new carbon-carbon bond has been regioselectively introduced into a target position (position 32 or 174) of the Ras protein by two types of organopalladium reactions (Mizoroki-Heck and Sonogashira reactions). Site-specific biotinylations of the Ras protein were confirmed by Western blot and LC-MS/MS.

 N-terminal glycine specific labeling of proteins by Pictet-Spengler reaction in combination with transamination reaction using pyridoxal-5-phosphate (PLP) under physiological condition has been demonstrated. Horse heart myoglobin, a 153 amino acid residue heme-binding protein containing N-terminal glycine residue was oxidized by PLP, and the resultant aldehyde was coupled with tryptophan analogues in phosphate buffer (pH 6.5) at 37°C to give corresponding 1,2,3,4-tetrahydro-?-carboline derivatives. Achromobacter protease I (Lys-C) liberated peptides were purified by ODS-HPLC to give N-terminal peptide (K1-peptide; the region from residue 1 to K16), which was analyzed by MALDI postsource decay (PSD) fragmentation. The ion peaks observed in the MALDI-PSD spectrum were assigned along the K1-peptide containing ?-carboline skeleton, not 1,2,3,4-tetrahydro-?-carboline skeleton. A possible reason for this phenomenon is that the 1,2,3,4-tetrahydro-?-carboline skeleton might be spontaneously dehydrated during HPLC purification and laser desorption ionization.  The circular dichroism (CD) spectral profiles of the modified myoglobin and the wild-type myoglobin were superimposable, which result showed that the tertiary structure of myoglobin was not altered during the reaction. Furthermore, modification of myoglobin was confirmed by SDS-PAGE and Western blot with no sign of decomposition.

Regioselective Carbon-Carbon Bond Formation in Proteins by Palladium-catalysis; New Protein Chemistry by Organometallic Chemistry,ChemBioChem 2006, 7, 134.

A New Protein Engineering Approach Combining Chemistry and Biology, Part I; Site-specific Incorporation of 4-iodo-L-phenylalanine in vitro Using Mis-acylated Suppressor tRNAPhe. ChemBioChem 2006, 7, 1577.

Site-specific Functionalization of Proteins by Organopalladium Reactions,ChemBioChem2007, 8, 232.

Sasaki, T.; Kodama, K.; Suzuki, H.; Fukuzawa, S.; Tachibana, K. N-terminal Labeling of Proteins by the Pictet-Spengler Reaction,Bioorg. Med. Chem. Lett. 2008, 18, 4550-4555.

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